key: cord- - qo krxv authors: wilcox, bruce a.; gubler, duane j. title: disease ecology and the global emergence of zoonotic pathogens date: - - journal: environ health prev med doi: . /bf sha: doc_id: cord_uid: qo krxv the incidence and frequency of epidemic transmission of zoonotic diseases, both known and newly recognized, has increased dramatically in the past years. it is thought that this dramatic disease emergence is primarily the result of the social, demographic, and environmental transformation that has occurred globally since world war ii. however, the causal linkages have not been elucidated. investigating emerging zoonotic pathogens as an ecological phenomenon can provide significant insights as to why some of these pathogens have jumped species and caused major epidemics in humans. a review of concepts and theory from biological ecology and of causal factors in disease emergence previously described suggests a general model of global zoonotic disease emergence. the model links demographic and societal factors to land use and land cover change whose associated ecological factors help explain disease emergence. the scale and magnitude of these changes are more significant than those associated with climate change, the effects of which are largely not yet understood. unfortunately, the complex character and non-linear behavior of the human-natural systems in which host-pathogen systems are embedded makes specific incidences of disease emergence or epidemics inherently difficult to predict. employing a complex systems analytical approach, however, may show how a few key ecological variables and system properties, including the adaptive capacity of institutions, explains the emergence of infectious diseases and how an integrated, multi-level approach to zoonotic disease control can reduce risk. the growing problem of globally emerging infectious diseases (eids) has prompted a substantial effort by the biomedical research establishment to identify the causes and recommend action. as reported in the most recent of a series of volumes ( ), a main finding is that the current episode of global infectious disease emergence is the result of a convergence of factors involving complex interactions among numerous variables. this includes genetic, biological, social, economic, political, ecological, and physical environmental factors, and calls for an interdisciplinary research agenda. it is also concluded that "human development and large scale social phenomena are closely associated to infectious disease threats at a global level," which points to the need for research focused on "social and ecological factors affecting infectious disease emergence" ( ) . the phenomenon of globally emerging infectious diseases requires understanding biological systems in the broadest sense and dealing with their extraordinary complexity. this includes processes operating at the level of transmission and evolution of a pathogen within and among host species and humans. it extends to and includes processes involving ecosystems and regional environmental change occurring on a global scale ( ) . in fact the scale and magnitude of anthropogenic activity has reached a point of virtual co-dominance with natural processes of energy and material flows globally ( ) . understanding these kinds of processes traditionally has been the domain of classical ecology, or natural history, plus systems ecology ( , ) . adding to this are recent ecological perspectives and models applied at the molecular, cellular level, and organismal levels ( ) , and others addressing the complexity, multiple variables, cross-scale influences, and dynamic behavior at the level of natural ecosystems ( ) , and social-ecological systems ( ) . along with the research at the organismal level and below, that aimed at the level of social-ecological (coupled humannatural systems) is critical to the development of the comprehensive scientific framework necessary for understanding zoonotic infectious disease emergence in particular. not only does this new area address the dynamic behavior of complex, large scale systems, but also bridges theory from the traditionally separate biological and social science disciplines, thus contributing to the interdisciplinary research agenda also called for in the above reports. the purpose of this paper is to consider how regional and global zoonotic disease emergence trends might be explained on the basis of current thought in biological ecology including the very recent developments new to the field of infectious disease ecology. here we draw on ecological science as broadly defined as a basis for identifying causal mechanisms of zoonotic disease emergence, the ultimate goal being to enhance disease prevention and control programs. several authors have categorized causal factors of infectious disease emergence, including explicitly citing 'ecological' ones involving land use change ( ) ( ) ( ) ( ) or 'land use drivers' ( ) , human movement ( , ) , encroachment and wildlife translocation ( , ) , rapid transport ( , ) and climate change ( , ) . most recently, the institute of medicine ( ) described these along with others ( categories of factors in all) and a model stating the major categories of factors have historically converged to bring about the current global emerging infectious disease crisis. ecological factors are described as one of four major categories of factors that have converged with social, political, and economic factors; genetic and biological factors; and physical environmental factors. we take a different approach to understanding the interaction of the above factors and their causal relationships by focusing on disease emergence as an ecological-evolutionary phenomenon influenced by human factors. our interest is in how human factors interact with natural processes and, in particular, how mechanisms operating at levels meaningful to understanding pathogen transmission and evolution can result in regional and ultimately global phenomena (i.e., regional endemism, epidemics, or global pandemics). as a first step we distinguish between the two broad categories of human factors, 'demographic and societal' and 'disease intervention and policy' suggested previously by gubler ( ) ( table ) . this categorization differentiates between factors associated with specific kinds of environments or ecosystems and those involving biological and policy factors not so associated. however, both can be described as part of a single ecological framework involving interaction of systems of essentially natural versus human design, respectively. our focus is on the first category from the standpoint of how disease emergence is explained by ecological concepts and principles. this includes some relatively new models and theory not previously used to explain the current trend in increasing emerging infectious diseases. we present a general model of zoonotic disease emergence on this basis. we also discuss recent explanations based on complexity theory for how human behavior and ecosystems interact to contribute to disease emergence. classical ecology, or natural history, has been the basis and mainstay of infectious disease research since its origins with 'koch's postulates' and subsequent development of microbiology and zoonotic disease epidemiology during the th and th centuries ( , ) . throughout much of its early history, zoonotic disease research involved this descriptive, empiricallybased ecology: identifying the life cycle, transmission, incidental and natural hosts of pathogens, along with demographic, life history, dispersal, and habitat attributes of reservoirs and vectors. a substantial theoretical dimension has developed, beginning with ross's mathematical analysis of malaria transmission ( ) and extending to anderson and may's ( ) recent synthesis infectious diseases of humans. although essential to designing effective prevention and control programs, empirical field based, disease ecology has been neglected in recent years ( ) . fortunately, theoretical disease ecology, stimulated largely by the work of anderson, may and others has flourished and led to a significant syntheses involving application of ecological-evolutionary biology to the study of infectious diseases ( , ) . parallel to this, systems ecology has begun to extend its domain by applying complexity theory to emerging infections with at least initial suggestions of its implications ( ) . this development in particular, along with observations from several decades of applications of systems ecology to natural resources and economic development ( , ( ) ( ) ( ) , have resulted in important insights of significant potential in understanding zoonotic disease emergence as a cross-scale process. this area uses complex systems theory applied to coupled, human-natural systems to explain how processes such as local phenomena can result in a cascade of effects ultimately reaching global proportions. the finding suggests this crossscale behavior is controlled by relatively few variables, and is mitigated by social and ecological resilience. the loss of this resilience in ecological systems is observed to lead inevitably to unpredictable events or the 'surprise' characteristic of complex systems generally. this combination of socialecological systems and resilience theory helps explain the unpredictability of disease emergence events. it represents another potentially useful area of application to understanding emerging infectious diseases along with those areas generally considered within the domain of ecology mentioned above: population ecology and genetics, community ecology, and systems ecology. population ecology, genetics and disease emergence of particular relevance to disease emergence is the explanation provided by theoretical population biology, already mentioned, of how host (including human) population size determines whether or not a pathogen can persist in a population. the accumulated findings demonstrate thresholds exist, depending on the type of pathogen and host population, below which a pathogen cannot be sustained. considered in light of the exponentially increasing size of human and domestic host and vector populations in the world, the breaching of thresholds of pathogen persistence can explain much of the increase in emerging infectious diseases. this can be explained as follows. although zoonotic disease emergence is not entirely a tropical phenomenon, it is mostly associated with tropical developing regions undergoing the most rapid population growth and ecological changes. prior to the post-wwii economic era, most regional ecosystems in the tropics consisted of relatively scattered human settlements, and only a few large cities (> , ) ( ) . these were separated by large expanses of cropland and pastureland and relatively undisturbed forest. since then, in what has been the most rapid period of large scale ecological transformation in human history, the pattern has essentially reversed ( ) . the once scattered settlements and few large cities have coalesced into expansive megacities and surrounding periurban settlements with only remnant patches of undisturbed forest remaining in a sea of cropland, scrub, and ecologically degraded lands. dacca, bangladesh, which grew from a population size of , to million from to , is one of many examples. thus the existence of population density-dependent thresholds for disease emergence is particularly relevant ( , ) . this explains the abrupt transitions of urban diseases between non-persistence to endemic and endemic to epidemic behavior as population densities of susceptible humans, hosts, and vectors reach critical densities. the classic illustration is that of measles which, given its particular transmission rate, requires human settlements with population sizes in excess (> , ) of what historically existed in most pre-industrial states and geographically isolated populations even today ( ). thus, for example, many infectious diseases endemic on continents have not become established on islands despite their occasional introduction and the occurrence of local outbreaks. the same mathematical ecology that explains why measles and virtually all diseases have threshold densities, explains the much lower thresholds existing for vector-borne diseases such as arboviruses ( ) . particularly noteworthy is the theoretical demonstration that the pathogen 'reproductive rate' increases with the square of vector population density. this indicates threshold densities can fast be breached as domestic and peridomestic hosts and vectors expand (or re-expand) their geographic ranges (once they are introduced or re-introduced) and increase their densities. this helps explain the explosive re-emergence of dengue and dengue hemorrhagic fever in the american tropics as vector populations responded to relaxed controls and new breeding habitats associated with urbanization ( ) . this phenomenon can be likened to the gradual build-up of 'dead and down' wood across a forested landscape with a history of fire suppression. the build-up of fuel, like that of host or vector populations, becomes an 'accident waiting to happen' when a single ignition event in one locality, similar to a single infection event, spreads to the entire region. another consequence of the dramatically increased densities of humans, host reservoirs, and vectors is the increased number of pathogen genomes. the resulting increased levels of genetic variability can accelerate microbial adaptation, including evolution of pathogenesis, and antimicrobial resistance. genetic variability increases with population size and density through a variety mechanisms including mutation. the probability of producing more virulent variants not only increases with host population size but also with crowding and co-mingling of different host species ( ) . in general, parasite (pathogen)-host relations naturally constitute a co-adaptive/ evolutionary 'dance' along the pathogenicity threshold, which is likely to be crossed with greater frequency due to unnatural anthropogenic disturbances ( ) independent of increasing population sizes and pathogen genetic diversity. the study of ecological communities and the 'community ecology' theory it has yielded includes a number of principles and mechanisms that describe how human disturbances as well as natural environmental variation can contribute to any of the above population level factors ( ) . there are a number of implications to zoonotic disease emergence, although most have not yet been described in terms of disease emergence or in the medical, public health, or zoonotic disease literature. of critical significance from this area of ecology is the general principle of community assembly. research has demonstrated that communities of arranged predicably in terms of 'assembly rules' ( ) . this order, in terms of the spatial distribution, composition and the abundance of each species in an ecological community, is affected by interspecific interactions (predation, competition, and parasitism). density independent factors (e.g., weather, natural catastrophes) play an important, but a more ephemeral role in most ecosystems. the process of community assembly (and disassembly) has been particularly well demonstrated through studies of insular ecosystems, the so-called 'species area relationship,' and the phenomenon of faunal collapse ( ) ( ) ( ) . a principal outcome and the ecological significance of this body of research first described in terms of the process of 'habitat fragmentation', has been identified as the principal mechanism by which human land and resource use contributes to species extinction ( ) . although initially debated, a significant amount of evidence has since accumulated resulting in its general acceptance and applicability, particularly to tropical forest ecosystems ( , , ) . the critical significance of habitat fragmentation and related human disturbances to disease emergence stems from it contribution to the disassembly of orderly natural communities. for example, human activities such deforestation, use of pesticides, and various forms of pollution often result in the loss of predators. in fact, carnivorous mammals typically are the first species to disappear following forest fragmentation. their local extinction represents the loss of 'top down' natural control in ecological communities. this can in turn result is an increase in abundance, even 'hyper-abundance' ( ) , of species such as rodents and biting insects. community disassembly and the resulting loss of natural population control mechanisms for such species generally is associated with the conversion of natural landscapes to urban and agriculture landscapes. broad spectrum pesticide use and habitat simplification, along with habitat fragmentation, are important contributing factors. the reduction in species diversity can contribute to the phenomenon of 'ecological release' in remaining species, whose predators, competitor and parasites are reduced in numbers or eliminated. some of these may be already serving as zoonotic reservoirs or vectors. if so, ecological release may result in their proliferation. this helps explain why many emerging zoonotic diseases occur in areas where settlement and agricultural expansion recently have encroached into tropical forests. a similar phenomenon, associated with the regrowth of forests in developed regions, can lead to zoonotic disease emergence. the pattern of reforestation in eastern north america during the past half-century resulted in increased habitat favored by forest edge adapted species such as whitetail deer and white-footed mice. white-tailed deer, the principle host of the adult tick ixodes scapulari, reinvaded the area and with few predators and competitors the population exploded. this pattern of ecological change arguably has been a major factor in the emergence of lyme disease in this region. an extension of the concept of 'ecological release' has been suggested to explain the invasive species phenomenon in which super-successful introduced (alien) species have escaped from their natural complement of parasites ( ) , as well as predators and competitors. this may explain in large part our most successful invasive domestic species, rattus and aedes species, which are hosts and vectors of some of our current and historically most problematic urban zoonotic diseases. in sum, zoonotic infectious disease emergence can be explained in part as a consequence of the disruption of natural ecological communities, and the breakdown of naturally existing predator-prey, competitive, and host-parasite relationships that tend regulate and stabilize species' abundance. this can occur through the use of non-selective pesticides through changes in land use and land cover that affect the distribution and abundance of species. it should be noted that the disassembly of natural ecological communities that results from habitat fragmentation is a protracted process compared to exponential decay. depending on the extent of habitat loss and other factors such as the sizes, shapes and spatial relationships of remaining fragments, the process may require decades, centuries or longer as communities 'relax' toward a new equilibrium ( , , ) . it follows that the frequency of disease emergence can be expected to follow a similar path: highest rates at first, followed by gradual decline as an ecosystem 'stabilizes'. the application of systems thinking to ecological communities centers on the ecosystem concept and has been the basis of significant research activity in ecological science since the 's ( ) . much of the focus has been on describing and understanding energy and nutrient fluxes across different kinds of ecosystems, and more recently emphasizing the human dimensions of global environmental change ( ) . although global scale ecosystem change has been considered in reference to human and infectious diseases ( , ) , no systematic framework has yet been presented in this regard. the evidence presented has been anecdotal. however, the development and application of systems theory independently of and largely outside the realm of mainstream systems ecology ( ) shows significant promise in providing a basis for more systematic interpretation of the role of ecosystem change in disease emergence. recent thinking draws in particular on the application of the theory of 'complex adaptive systems' to ecological systems in which ecosystems are represented as scaled, self-organizing, far from equilibrium, evolutionary, and non-linear ( ) . organization, diversity, and resilience are important 'emerging' properties of complex systems-often equated with a 'healthy' state. 'surprise', or qualitative disagreements of system behavior with a priori expectations, is another property ( ) . their association with loss of system resilience and increased vulnerability applies both to increasing inflexible social systems 'managing' and attempting to 'control' natural variables (i.e., vectors, pathogens) and that of the ecosystems whose component variables (e.g., vector and pathogen populations) are targeted for management or control. alteration of natural disturbance regimes (via control of natural variation via flood control projects for example) reduces resilience, while secondary disturbance events (wildfires, storms, floods, and earthquakes) precipitate events caused by crossscale influences (e.g., a thunderstorm igniting a fire locally that spreads regionally as a result of fuel build-up from years of artificial fire suppression). regional environmental change such as that associated with population growth development often does not accommodate the need to maintain resilience ( ) . such ecosystems have been described as 'over-connected' or 'brittle' and, as stated above, 'accidents waiting to happen' ( , ) . floods, often associated with waterborne disease outbreaks, occur more frequently and with greater severity as a result of lost resilience in natural systems due to attempts to 'control' (in contrast to manage) natural variation. conversion of upland forests to plantations or cropping systems for more 'efficient' natural resource production is another, as is agriculture generally. both result in a reduction of heterogeneity in ecosystems that tend to 'buffer' against disease outbreaks, which can spread more readily across the more uniform landscape including immunologically vulnerable domesticated species. gunderson et al. ( , ) describe this as general pattern of institutional behavior. it begins with the targeting of a natural variable for control, followed by increasing institutional efficiency and inflexibility in the control methods used. as the target variable and ecosystem changes and initial signs of failure are ignored, the ultimate result is a crisis. this 'pathology of regional resource and ecosystem management' and is apparently applicable to infectious disease management as well. the above body of ecological theory and observations involving specific emerging infectious disease cases suggests a causal schema that links ecological phenomena on the scale of pathogen transmission and evolution to regional and global transformations. this is represented by figure , focusing on the role of demographic and societal factors in disease emergence (shown in table ). this schema is constructed using the generally adopted view of human-environment interaction in which the impact of human population and technology is taken as the driving force, or ultimate cause. clark et al. ( ) elaborate on this in their seminal treatment on global and regional change. here, the combination of population, technological capacity, and sociocultural organization act as the system drivers of regional environmental change. these and 'mitigating' forces are in turn influenced by 'human behavior', referring to patterns of actions and the rationales giving rise to them. broadly speaking, these forces and their affects on ecosystems represent the 'ecological factors' in social-ecological systems, while human behavior represents the 'social factors' for the purposes of discussion. this schema represents zoonotic disease emergence from the perspective of the ecosystems within a regional environment, the large scale processes involved, and the associated ecological effects and processes involved in disease emergence. thus, referring to table , the factors under 'demographic and societal changes' can be identified in reference to particular ecosystems and processes (i.e., urban and urbanization, agriculture and agricultural intensification, forest and forest alteration), and associated factors operating on regional and global scales. these are unprecedented population growth, unplanned and uncontrolled urbanization, non-biodegradable packaging of consumer goods, and modern transportation, all contributing to conditions that promote pathogen transmission and persistence. for zoonotic diseases, which by definition involve the jumping of a pathogen from its natural host to humans, and in some instances extension of its host cycle to include humans, conditions can be described for this simple schema as follows. the likelihood or frequency of transmission events change when the natural host or pathogen changes, humans change, or the ecosystem supporting both changes. thus, fundamentally the processes influencing transmission of zoonotic pathogens can be described as a consequence of one or a combination of three possible kinds of change: expansion of the habitat or geographic range of a host, of a pathogen or both; expansion of human's habitat or geographic range; or change in the habitat or ecosystem occupied by both humans and the natural host. evolutionary adaptation of pathogens is omitted from the figure. however, it can be assumed any factor contributing to increased likelihood of transmission, as well as increased population size of hosts and pathogens will contribute to the potential that new, increasingly pathogenic, infectious, antimicrobial resistant variants will emerge. anthropogenic climate change, while not incorporated in the diagram, can be considered to potentially contribute to disease emergence through its contribution to habitat alteration. our review of ecological theory and the resulting complex model described here demonstrates the limitation of classical disease ecology and natural history. for example, without incorporation of population genetic theory in ecology as 'evolutionary ecology', along with the concepts of pathogen spillover and cross-scale ecosystem dynamics, classical disease ecology cannot explain recent emergence events like those involving hiv/aids, sars, avian influenza, e. coli : , dengue, malaria, west nile virus, and nipah virus, among others. the resurgence of vector populations, having acquired pesticide resistance and lost predators and competitors as natural controls after a regimen of inappropriate pesticide use, similarly is explained by modern ecology and evolutionary biology. this has direct application to control and intervention policies and practices. for example, it points to the need to adopt control strategies that integrate landscape and habitat management with careful rotation of targeted chemical and biological agents ( ) . developing such strategies requires detailed field studies, built on traditional natural history and classical disease ecology, but supplemented with advanced molecular techniques, as well as ecological research that takes into account the community and systems level dimensions of a particular pathogen-host complex. the continual expansion of human populations since prehistoric times, and particularly since the advent of settled agriculture with its associated domesticated animals exposing humans to their pathogens as well as those spilling over from wild animal populations, has incrementally added to the pathogen load through successive invasions by different organisms over time ( , ) . well established principles of population ecology, applied via mathematical epidemiology as anderson and may and others so aptly have done, readily explain why, ceteris paribus, infectious disease incidence should generally be increasing with human population size, as it has in the world's poorest and most populous regions. yet in spite of what are in general commonly understood principles, and warning signs that went unheeded in the 's and 's ( , ), biomedical and public health institutions were unprepared for the recent surge in emerging infectious diseases ( , , , ) . not until the 's, prompted in part by the hiv/aids pandemic and the failure of 'quick-fix' solutions based on drugs, vaccines, and pesticides ( ) , did the biomedical science and public health communities begin to launch a significant response. in light of the complexity of the factors involved, this lack of preparedness should perhaps not be surprising. as explained by the infectious disease ecology described here, zoonotic disease emergence involves biological processes operating on the scale of molecules and cells to that of coupled, regional scale human-natural systems. the latter involve political economic factors and policies driving regional environmental change, spreading geographically across the globe. it is this process of globalization-its ecological underpinnings and consequences-to which the current eid global trend of zoonotic disease emergance can be largely attributed. lack of awareness of the ecological implications of fig. diagram depicting infectious disease ecology causal schema. the aggregate variable at the top of the diagram, representing population and consumption, along with mitigating socio-cultural attributes, is the driver or 'forcing function' responsible for land use and land cover change characteristic of a particular region. the result is varying degrees (and types) of urbanization, agricultural intensification (including food production and distribution), and alteration of natural habitat. these changes at the level of landscapes and habitat produce conditions influencing the ecological and evolutionary dynamics of vector and host species and vector/host-pathogen dynamics. in turn, these conditions facilitate the geographic expansion, novel appearance, or increased epidemic activity of a disease. regional environmental transformations, and of their synergy with the ecological and evolutionary consequences of inadequate or inappropriate policies or methods of vector control and disease prevention, have unwittingly promoted disease emergence. the 'ecological' changes taking place as a result of public health agencies' actions (or inaction) involve pathogen biology and are small scale in time and space: selection for vector pesticide and antimicrobial resistance. however, the cumulative effect of micro-scale processes involving pathogen adaptation and host range expansion (or re-expanding) can ultimately produce regional and even global consequences. however, policy action and inaction outside the domain of biomedical or public health agencies has produced ecological changes at a historically unprecedented rate and scale. urbanization, agricultural and food production intensification, alteration of natural habitat, along with concomitant loss of ecosystem functions, have transformed entire regional ecosystems during the past years. the role of urbanization cannot be underestimated. the direct land use changes associated with urbanization effectively concentrate human, animal reservoir and vector populations at unprecedented densities. but urbanization also is strongly tied to socio-economic and cultural factors, along with human migration dynamics, affecting agricultural and food production intensification, as well as rural and natural ecosystems. in the case of food production and distribution, dramatically increased contact with and transport of wildlife (e.g., bush meat trade in africa), and increased ruralurban transport and concentration of wild species for the exotic food market (e.g., civet cats in guangdong, china), is another contributor to increasing disease emergence. it can be assumed these impacts on wildlife populations also contribute to changes in the composition of ecological communities on a regional scale ( ) , and often result in a hyper-abundance of small mammal species low in the food chain, which are likely to serve as human disease reservoirs. similar effects of ecological disruption appear to generally apply to invertebrate commu- whether a disease 'jumps' to the regional population and the global human population scale is determined by quite different processes. these depend on demographic and transport patterns related to processes, like urbanization, influencing pathogen transmission and behavior operating on the regional and global scales. physical environmental processes such as climate variability (on the right side of the figure), which are episodic by nature, include short term and small scale variations in the form of seasonal storms (e.g., monsoons), for example, as well as larger time and space scale variations. these include, for example, decadal and regional scale changes in weather patterns such as el niño. these climate changes and weather events can precipitate floods and droughts. these act as cross-scale mediators that directly affect disease reservoir and vector populations, or pathogens (e.g, dispersal via flood waters). they cause the disease to jump from a smaller demographic unit to a larger one (e.g., from a single village to a district). human factors such as land use and land cover changes (table and fig. ) produce ecosystem changes contributing or magnifying the cross-scale processes. for example, urbanization and deforestation increase the magnitude of floods and droughts resulting from natural or anthropogenic climate variation. nities ( ) , the kinds of organisms most commonly serving as human disease vectors. the estimated acceleration of natural species extinction rates by orders of magnitude over nonanthropogenic extinction rates ( ) gives an indication of the scale and magnitude of change in natural communities, particularly in the tropics. in these regional ecosystems, the original extent of tropical forest has declined to a fraction of its original area with concomitant affects on biodiversity ( ) . large scale agricultural expansion (a common proximate cause of tropical deforestation) has decelerated and all but ended in many regions. this suggests that further contributions from what historically has been a main factor in natural habitat alteration, ecosystem disturbance and biodiversity loss, will decline. yet agricultural intensification has replaced expansion with technologies that further impact native biodiversity ( ) . the reduction in plant species richness that accompanies agricultural intensification leads to changes in the community composition of the pest complex-herbivorous insects, their natural enemies (predators and parasites), and the microbial community attacking crops ( ) . the schema described here demonstrates how regional environmental change, involving ecological dynamics such as demographic or landscape transformations on the scale of decades, interact with change on the scale of host-parasite/ pathogen dynamics. how these cross-scale mechanisms produce regional or global scale disease emergence patterns is beyond the realm of conventional epidemiology, or analytical approaches generally. this may explain why such changes either are not apparent or their implications in terms of disease emergence are a low priority to biomedical and public health agencies. such cross-scale processes are however characteristic of ecosystems, whose dynamics involve interaction of variables and operate on vastly different time and space scales, involving natural processes that are discontinuously distributed as shown in figure . change in such systems is difficult to understand employing conventional analytic approaches alone, but require new thinking and analytical methods drawn from complex systems theory. the discontinuous character of the processes or variables makes their interaction intuitively improbable except for the mediating effect of 'cross-scale influences' as illustrated in figure . examples include the eruption of hantavirus in the american southwest, thought be associated with weather events precipitated by el nino southern oscillation. storms events are similarly episodic, and represent a discontinuous form of variation transporting pathogens via flood waters in domestic and natural environments. on the other hand, massive environmental events, like the recent southeast asian tsunami, are potentially capable of producing epidemics across an entire region, and may episodically extinguish some zoonotic diseases by temporarily destroying reservoir and vector populations and habitat. the roles of resilience and vulnerability in determining the severity of such events, both in terms of social systems and ecological systems is another critical aspect of social-ecological systems behavior ( ) . the recent worldwide upsurge of zoonotic infectious diseases, involving the resurgence of a growing number of diseases previously believed under control or the emergence of newly recognized diseases, has been attributed to a list of global factors characterized in terms ranging from microbial adaptation and land use to changing ecosystems, breakdown of public health, and poverty ( , ) . the categorization provided by gubler ( ) , elaborated here based on an expanded view of infectious disease ecology, provides the basis for a schema describing the causal relations involving factors previously identified, along with hypothesized mechanisms. the complicated nature of this problem, which obviously entails numerous interacting variables operating on different time and space scales pose a significant challenge to biomedical science and epidemiological research as well as public health intervention. however, the current trend of increasing global emerging infectious diseases is linked with another issue of 'global governance', sustainable development, with which disease control and prevent strategies must be integrated. here too, the problems of politically stability, population growth, unplanned urbanization and economic development, income disparity, and environmental degradation, are all integral to the solution. this interdisciplinary imperative challenges what historically has been an increasing disciplinarily focus in infectious disease research. greater investment in research has succeeded in revealing more detail about diseases within specific disciplines. yet this arguably has been at the cost of greater disintegration rather than integration among disciplines. the division is widest between the genetic and biological aspects of disease and health on the one hand and the social, economic, political, and physical environmental factors on the other. the disciplinary distinctions within infectious disease research of course belie the true transdisciplinary nature of this and most problems in the global heath and environment arena ( ). the above disciplinary divisions largely reflect the gap between disciplines focusing on systems at or below and above the level of the organism or species. the former involve disciplines addressing the genetics, pathogenesis, or immune response within particular organisms (humans, vectors of pathogens), for example, or disease as a statistical phenomena at the species population level (the mainstay of epidemiology). the latter deal with social or ecological phenomena, essentially representing higher levels of biological organization, within which the organism-level processes operate but that also involve many other variables or processes operating on a wide range of biological scales (from genes to the biosphere) characterized by cross-scale influences, and interactions at multiple societal and ecological levels. it follows that for intervention to be globally effective, in addition to rebuilding public health infrastructure based on the comprehensive view of infectious disease ecology presented here, at least three elements are required: . a multilevel ecosystem approach, involving cross-scale integration. . incorporated ecological theory and data for the specific disease system, . local scale intervention using a participatory approach that matches pathogen management with sustainable 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change: research pathways for the next decade ecosystem change and public health: a global perspective systems thinking, systems practice adaptive dancing: interactions between social resilience and ecological crises plagues and peoples human frontiers, environments and disease emerging infectious diseases: a cdc perspective a global perspective on habitat disturbance and tropical rainforest mammals species richness-decomposition relationships depend on species dominance extinction rates agricultural intensification and ecosystem properties biodiversity and ecosystem processes in tropical forests emerging infections: microbial threats to health in the united states transdisciplinarity: recreating integrated knowledge. mcgill-queen's university press we thank shannon bennett, kristin duin, mayee wong, and yuko chiba for assisting with the research for this paper and its critical review. key: cord- - qioku authors: rehman, zaib ur.; meng, chunchun; sun, yingjie; mahrose, khalid m.; umar, sajid; ding, chan; munir, muhammad title: pathobiology of avian avulavirus : special focus on waterfowl date: - - journal: vet res doi: . /s - - -x sha: doc_id: cord_uid: qioku avian avulaviruses serotype (abbreviated as apmv- for the historical name avian paramyxovirus ) are capable of infecting a wide spectrum of avian species with variable clinical symptoms and outcomes. ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending upon the virus strain and host species. the emergence of new virulent genotypes from global epizootics, and the year-to-year genomic changes in low and high virulence apmv- imply that distinct genotypes of apmv- are simultaneously evolving at different geographic locations across the globe. this vast genomic diversity may be favoured by large variety of avian species susceptibility to apmv- infection, and by the availability of highly mobile wild birds. it has long been considered that waterfowls are not sensitive to apmv- and are unable to show any clinical signs, however, outbreaks from the ′s contradict these concepts. the apmv- isolates are increasingly reported from the waterfowl. waterfowl have strong innate immune responses, which minimize the impact of virus infection, however, are unable to prevent the viral shedding. numerous apmv- are carried by domestic waterfowl intermingling with terrestrial poultry. therefore, commercial ducks and geese should be vaccinated against apmv- to minimize the virus shedding and for the prevention the transmission. genetic diversity within apmv- demonstrates the need for continual monitoring of viral evolution and periodic updates of vaccine seed-strains to achieve efficient control and eradication of apmv- in waterfowls. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. newcastle disease (nd) is one of the most devastating and commonly prevalent diseases in the poultry industry, around the world. owing to immense economic losses, world organization for animal health has categorized the disease as "notifiable" [ ] . the disease outbreaks are enormous and the host spectrum is broad, thus making nd as one of the primary limiting factor in the development of the poultry industry, especially in the developing countries [ , ] . it is caused by the avian avulavirus (apmv- ), which belongs to the avulavirus genus within paramyxoviridae family. all apmv- strains can be classified into velogenic (highly virulent), mesogenic (intermediate virulent) and lentogenic (non-virulent) based on the intracerebral pathogenicity index (icpi) in day old specific pathogen free (spf) chickens [ , ] additional file references un-cleaved fusion protein cleavage site. apmv- is considered to be virulent, if these amino acids are basic in nature with a phenylalanine at position , and having icpi value of . in day old chicks [ , ] . apmv- is a single-stranded, negative-sense, non-segmented and enveloped rna virus with genome length of . kb. apmv- genome encodes for six co-linear genes that translate into six proteins and two non-structural proteins. structural proteins include nucleoprotein (np), matrix (m), fusion (f), hemagglutinin-neuraminidase (hn), phosphoprotein (p) and large rna-dependent rna-polymerase (l). the rna editing of the p gene can result in the expression of v and w only in the virus infected cells [ ] [ ] [ ] [ ] . virulence of apmv- varies and depends upon the host species, and chicken and turkey are more susceptible than ducks and geese. generally, waterfowls were considered to be the natural reservoir for apmv- [ ] ; commonly for lentogenic apmv- [ ] . traditional view on the resistance of waterfowl against apmv- has been challenged since the report of continuous outbreaks in different provinces of china in goose ( ) [ , ] and ducks ( ) [ ] . number of clinical nd outbreaks are increasing in the waterfowls [ ] [ ] [ ] [ ] . outbreaks of nd in ducks and geese indicate that these are not only the carrier, but also show clinical outcome of the disease. factors that led to the change in the pathogenic spectrum of apmv- in waterfowl remain elusive. investigation of the molecular mechanisms of increased pathogenicity of apmv- in waterfowls would provide foundations in designing any control strategies for the disease, as well as to improve health and welfare standards of the waterfowl. the purpose of this review is to analyze our current understanding on the host-spectrum, molecular pathobiology of apmv- in waterfowls, and host immune responses that may play crucial roles in the disease prevention and control. the class i apmv- isolate was reported in , however, currently these isolates are frequently being reported. recent epidemiological studies direct that class i apmv- are common in domestic waterfowl. all viruses belong to class i are avirulent except js -a and a b strains [ , ] which were generated by experimental consecutive passages through chicken. amongst all genotypes of class ii, genotype i and ii are the most prevalent genotypes in the waterfowl and have been isolated from many countries (summarized in table ). waterfowl-origin isolates belonging to genotype vii of apmv- are constantly increasing especially in china, republic of korea, and taiwan [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these emerging outbreaks are increasing the global burden of apmv- and causes heavy economic losses [ ] . genotype iv and v have not been isolated from the waterfowl (table ) . collectively, these epidemiological studies clearly demonstrate the susceptibility of waterfowls for apmv- and their possible roles in the epizootiology of viruses. most of our understanding on the surveillance of apmv- in wild birds came from epidemiological studies on avian influenza viruses. thus, it is required to design apmv- dedicated studies to effectively assess the true prevalence of the virus in wild birds. it is essential to understand and establish the foundations to devise control strategies, especially in wild-birds populated and highly vulnerable commercial poultry areas. waterfowls are less susceptible to apmv- compared to chickens, such as ducks and geese. a key reason for less susceptibility of waterfowl to apmv- is the presence of retinoic acid-inducible gene i (rig-i). rig-i is absent in the chicken [ ] whereas it is present in ducks and goose ( figure ) [ , ] . rig-i and melanoma differentiationassociated gene (mda ) are the foremost part of retinoic acid inducible gene-like receptors (rlrs) (figure ), which senses the cytoplasmic rna [ ] . these sensors can detect the nucleic acids of negative sense rna such as apmv- and influenza viruses [ , ] , resulting in the production of the ifn type i and iii, cytokines, chemokines and expression of the antiviral genes [ ] . there is a vital role of the rlrs in the recognition of the viruses and antiviral immune responses in macrophages, fibroblast, and dendritic cells [ , ] . the v protein of apmv- blocks the "downstream" signaling pathway by interacting with mda resulting in blockage of strong antiviral response of ifn-β in chicken, which provide the benefit to waterfowl via rig-i pathway [ , ] . positive correlation exist between the resistance to apmv- infection and expression of the antiviral genes including rig-i, irf , irf and ifn-β [ ] which was further confirmed in a study demonstrating increased expression of rig-i ultimately leading to decreased apmv- load in vitro as well as in vivo [ ] . apmv- infection increases the expression of interleukin (il) il- β, tumor necrosis factor-α-like factor and interferon (ifn)-β in duck embryo fibroblast (def) cells. distinct innate immune responses of the waterfowl against apmv- may reason the resistance of waterfowls to these infections [ ] . a higher level of innate immune genes expression has been observed in chicken embryo fibroblast (cef) compared to the def cells [ ] . an experimental study reveals that apmv- stimulate a strong and intense expression of the ifn-β in ducks compared to chickens [ ] , and also up-regulate the ifn-β, ifn-regulatory factor (irf- ), and decreases the virus titer in goose lung and air-sac postinfection [ ] . these observations dictate that the strong innate immune responses is a plausible reasons for less susceptibility of duck [ ] and geese because its strong protective effects have been revealed to decrease the virus titer in goose-transfected cell [ ] , which may not be compensated by the ifn-α [ ] . recently, yang et al. [ ] have demonstrated that overexpression of ′- ′-oligoadenylate synthetase-like gene lessens the replication of apmv- in goose cells. in conclusion, waterfowls have diverse innate immunity components, which possibly increase their resistance to the apmv- [ ] . besides the strong innate immune responses, waterfowl are generally considered long-term carrier of apmv- and disease outbreaks have been reported since [ ] [ ] [ ] , and were confirmed by follow up experimental studies. clinically and naturally infected ducks and geese with apmv- show clinical signs such as elevated body temperature, excessively excreted oral mucus, dried cloaca, watery, greenish-white diarrhea, vain attempts of eating and drinking, listlessness, anorexia, crouch, eyelid edema and emaciation [ , , , ] . ducks may show up to % decrease in egg production, % morbidity and % mortality [ , ] however the mortality in ducks varies with the different breeds, virus strain and dose of virus [ ] . some birds also show weakness of legs and wing along with unilateral or bilateral incomplete paralysis and the effects of this paralysis increases with progression of the disease [ ] . duck and geese also show the neurological signs such as muscular trembles, muscular dis-coordination, circling, and twisting of head and neck [ , , ] . these clinical signs disappear according to infection status; mildly affected recover sooner and severely affected birds may recover after days of infection [ , ] . apmv- infected ducks and geese show the gross lesions on the immune organs such as bursa, spleen, thymus, mild to severe tracheitis, kidney enlargement, necrosis of pancreas, congestion on the meninx and in the brain and diffuse brain edema, focal hemorrhages in the mucosa of the proventriculus and intestine (especially duodenum and upper part of jejunum) [ , , , ] . bursal atrophy, hemorrhagic thymus and splenomegaly with white necrotic spots were found in the apmv- infected geese and duck [ , ] . these lesions and histopathological changes may be due to higher viral loads, multi-systemic distribution of the virus in these immune organs [ ] . as these immune organs are the reservoir of immune cells, and their destruction may lead to low antibody titer and other infections. experimental infection studies are necessary to determine the virulence and pathogenesis, in different bird types, age, species, intervention strategies, evaluation, comparison of vaccines etc. different scientists propose diverse reasons for experimental infection studies and the design of the experimental infection studies varies greatly with the above-mentioned factors. experimental infection studies on the pathogenesis, infection route, most susceptible age, bird line and immune responses are limited in waterfowl and available information is summarized in additional file and are briefly discussed below. kang et al. [ ] have studied the immune related gene expression of chicken and duck embryonic fibroblast (cef, def), by infecting them with apmv- of moderate virulent strain nh- and highly virulent strain ss- . upregulated expression of the toll like receptor (tlr) , tlr , il- β, il- , ifn-α, ifn-γ, mhc-i and mhc-ii were observed both in cef and def, however, these expression levels were higher in cef (mechanism is described briefly in figure ). peking duck infection at week of age with nh- and ss- via intranasal route showed the systemic replication of the virus into small intestine, cecal tonsils, brain, lung, bursa of fabricius, thymus, and spleen. this study also demonstrated the increased expression of the tlr , tlr , rig-i, mda , il- β, il- , il- , il- , ifn-α, ifn-β, ifn-γ in lungs compared to thymus. furthermore, the higher expression level of tlr , tlr , il- β, il- , il- , ifn-α, ifn-γ and mhc ii were induced by nh- than ss- in the lungs. whereas, the expression of the il- and ifn-β in lung as well as thymus was higher for ss- group [ ] . similarly, a study of the zhong et al. [ ] has demonstrated the upregulation of the viperin (an ifn stimulated gene) in the defs and also in the spleen, kidneys, liver, brain, and blood of changbai ducks infected with g apmv- through intranasal or intramuscular route. experimental infection of geese with genotype vii apmv- upregulate the expression of tlr - , , , and , avian β-defensin - , , , and , il- , il- , il- β, and ifn-γ, and mhc class i in different tissues [ ] . intranasal inoculation of the japanese commercial ducks and chicken males with artificially made apmv- class i virus a b results in the higher ifn-β in the duck compared to chicken. this study also demonstrated that replication, distribution, tissue damage and apoptosis were more in the immune organs of the chicken compared to duck [ ] . intramuscular infection of different ducks (mallard, gaoyou, shaoxing, jinding, shanma, and peking ducks) with jsd strain showed that different strains vary in the susceptibility to the disease [ ] . mallard are the most susceptible and peking ducks are the most resistant species of birds. infection of the gaoyou duck at , , , , and with different routes indicate that their susceptibility to disease and virus shedding decreases with the age and birds seldom die after infection through the natural route [ ] . experimental co-infection of ducks with apmv- , and low or high pathogenic avian influenza virus (lpaiv and hpaiv) indicate that it decreases the virus shedding and transmission to the naïve ducks by contact [ ] . duck after immunization with inactivated vaccine of apmv- , and challenge with the same live virulent kenyan apmv- resulted in the development of more antibody titer than the unchallenged birds [ ] . experimental infection of geese with virulent apmv- genotype viid and goose origin apmv- showed the extensive replication of geese in the immune organs which correlated with the clinical signs and lesions [ , ] and also it transmission to spf chickens [ ] . geese and chicken were vaccinated, then infected with goose-origin apmv- /na- and chickenorigin apmv- /f e , and f e viruses (additional file ). geese are more resistant to f e virus after vaccination. results indicate that na- vaccine provides a better protection in the form of less morbidity, less mortality and less virus shedding after challenge [ ] . although several natural outbreaks and experimental infections of apmv- in waterfowl had been reported. however, it remain to be clarified whether it cause the disease in waterfowl [ ] . inconsistent results in the infection of apmv- in waterfowl are due to apmv- strain, dose and rout of inoculation, breed, and maternal antibody titer (additional file ). ducks are more resistant to infection through natural route than the geese [ ] . in conclusion, these studies indicate that different viruses affect the immune organs and innate immune genes in diverse mechanisms. waterfowls are more susceptible to apmv- infection at an early age, and through natural route results in less damage to immune organs. more comprehensive and detailed studies are warranted for the control of nd in waterfowl. waterfowl are naturally infected with large number of the viruses which are avirulent and do not cause the diseases in domesticated poultry. waterfowl are commonly considered to be the natural host as well as carrier of apmv- [ ] . apmv- isolates from the waterfowl are generally lentogenic or potentially pathogenic [ ] , and may be transmitted to the avian species, leading to increase attention for their role in the transmission/spread and emergence of nd [ ] [ ] [ ] . there are increasing concerns about the increased virulence from the lentogenic to mesogenic to virulent pathotypes upon cyclic replication in poultry. these lentogenic isolates may converted to pathogenic viruses through serial passage in susceptible birds [ , , ] and one such isolate have already been documented to be the causative agent of the outbreak in the ireland [ ] , but disappeared quickly. all the class i apmv- ′s had been isolated from the waterfowl, indicating them the natural carrier of these viruses (table ) . live bird market (lbm) epidemiological study in united states indicated that from avirulent viruses, % belong to class i and % belongs to class ii [ ] . there is close phylogenetic relationship between the avirulent viruses isolated from the lbm and waterfowl, indicating that apmv- ′s may be transmitted from the waterfowl to the domestic poultry [ , ] . transmission of apmv- may occur through different routes like, ingestion of the contaminated feed, water air, contaminated feces, animals, humans, contaminated eggs etc. [ ] . mostly domestic waterfowl are reared in semi-closed areas, where they may have the contact with the wild birds and domestic poultry. therefore, it provides the best natural environment for the spread of apmv- ′s [ , ] . waterfowl and shorebirds are not only the host, but are also infected by the apmv- and these viruses can also cause the disease in domesticated poultry [ , , ] . many virulent isolates from the domestic poultry cannot cause disease in waterfowl [ , ] . most prevalent virulent genotype vii causing the endemics in china, japan, korea [ , , ] are co circulating into the ducks and chicken [ ] . this coevolution was further confirmed when the results of huang et al. [ ] declared that some of the circulating apmv- had the multiple homologous genomes from chicken, ducks and geese. so, there is dire need to modernize the housing system of waterfowl according to biosecurity point of view to prevent their contact with terrestrial poultry and wild birds. otherwise, number of virulent [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and avirulent [ , , , ] isolates from the waterfowl may easily transmit to commercial poultry farm. apmv- infected waterfowls shed the virus for an extended period of time [ ] whereas, the infected chickens clears themselves rapidly and shed virus for short duration [ ] . this prolonged virus shedding may facilitate the transmission, persistence and evolved to get some point mutation in the virus [ ] . major issue that should be concerned for virologists is the high evolution rate of some class i apmv- isolates from waterfowls. pathogenicity of some class i apmv- isolates are constantly increasing and they are naturally converted to low virulent from avirulent viruses (unpublished findings from our lab). this was confirmed by the results of the meng et al. [ ] and shengqing et al. [ ] in which they artificially develop the virulent viruses through serial passage in the air sac and brain of chicken, from avirulent viruses of waterfowl origin. although the conditions provided in these studies are not naturally existing but recovery of the virus from the air sacs after challenged via nasal or ocular route indicate the possibility of this mechanism. in conclusion waterfowl plays a vital role in the transmission and re-emergence of nd in terrestrial poultry. it is recommended that rearing facilities of the waterfowl should be separated from commercial poultry. cumulating evidences indicate that genetic resistance to apmv- exists in various breeds of waterfowl [ ] . comprehensive studies are needed to determine the genetic variability against apmv- in different breeds of waterfowls. genetic resistance of more susceptible breeds can be improved by including the resistant birds in the breeding programs of the commercial waterfowl [ ] . apmv- resistant breeds should be used to produce highly efficient transgenic poultry birds [ ] . although constant outbreaks of nd have been reported in waterfowl from china and other east asian countries, however, vaccination against nd in waterfowl is still a matter of debate. there are two consortia of scientists; one favors the vaccination and, others think that domestic waterfowl should not be vaccinated. these veterinarians have their own views, scientist, in the favors of vaccination argue, that it will decrease the chances of outbreaks and lessons the virus shedding. but, scientists, which are against vaccination argue that it will increase the virus burden on the birds, consequently increase the variation rate. the annual rate of change of virulent viruses could be as high as ten times the rate of change of low virulence apmv- , suggesting that other selective pressures such as vaccination may accelerate the rate of evolution of virulent viruses [ , ] . they claim that the rearing conditions of waterfowl could not be changed in future because they require water for breeding. therefore, it is not plausible to prevent their contact with wild birds, resulting in transfer of the wild bird viruses to domestic waterfowls. secondly, they claim vaccine will develop the antibody titers that will interact with the virus in the future exposures. these antibody responses may force the viruses for evolution. our viewpoint supports the group of scientists, which claims that birds should be vaccinated. because, it had already been established that apmv- cause the disease in waterfowl [ , , , ] and vaccination prevent the chances of the outbreak. second most important reason is the prolonged virus shedding by non-vaccinated birds, which may transfer to other poultry species and cause heavy economic losses. theory, that vaccination will increase the virus burden is not very interesting in case of waterfowl because, without vaccination we have to face the same consequences of the high evolution rate. waterfowls are naturally infected with large and diverse groups of viruses [ ] . development of the vaccines for waterfowl may require strains of waterfowl origin [ ] because some lentogenic vaccines phylogenetically are far away than the infectious virus thus may provide partial protection to waterfowl [ ] . vaccine should also be killed because live vaccine virus replicates in the body and leads to shedding of the virus. virus shedding of live vaccine may interact with the other apmv- 's present in the environment or birds and results in recombination and future evolution. waterfowls are not only the carrier but also susceptible to apmv- . strong innate immune responses may attribute to the less susceptibility of nd to waterfowl. viral shedding by waterfowl for prolonged duration may increase the transmission, evolution and emergence of new viral strains. a number of apmv- isolates from the waterfowl are reported with high mutation rate, which is an alarming matter and may cause the endemics in future. pathogenicity of nd is affected by different factors such as type, dose, and inoculation route of the virus, and species and age of birds. further studies are needed to explore the mechanism, and its intervention to prevent the virus shedding by the waterfowl for a long period. continuous studies are also required to monitor the apmv- in the waterfowl, which may be the future threat to commercial poultry industry. studies on the humoral immune responses of waterfowl are crucial to develop better intervention strategies. it is recommended that rearing facilities of the waterfowl including ducks and geese should be separated from chicken and turkey flocks to prevent the virus transmission. additional file . experimental infection studies of ducks and geese with avian avulavirus . table summarizes temporal, geographic, and host distribution of avian paramyxovirus (newcastle disease virus) infectivity and pathogenicity of newcastle disease virus strains of different 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disease virus strain na- isolated from geese in china genetic diversity of newcastle disease viruses isolated from domestic poultry species in eastern china during complete genome sequences of new emerging newcastle disease virus strains isolated from china the authors declare that they have no competing interests.authors' contributions zur, cm and cd developed the idea; zur and cm collected the data, wrote and revised the manuscript; cm, ys, kmm and su helped in collection of data and contributed to writing the manuscript; mm helped in the illustrations; mm and cd critically revised the manuscript. all authors read and approved the final manuscript. key: cord- -f hjy authors: morgan, b. paul; harris, claire l. title: complement, a target for therapy in inflammatory and degenerative diseases date: - - journal: nat rev drug discov doi: . /nrd sha: doc_id: cord_uid: f hjy the complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. in this review, we describe the history, current landscape and future directions for anti-complement therapies. supplementary information: the online version of this article (doi: . /nrd ) contains supplementary material, which is available to authorized users. the complement system includes more than component proteins, regulators and receptors, present in plasma and on cells, which collaborate to provide defence against infection and to clear toxic materials. complement is a key part of innate immunity and a modulator of the adaptive immune response; inherited deficiencies in complement proteins predispose individuals to bacterial infection and/or immune complex disease. the powerful cell-targeting and cell-killing properties of complement can be turned against self, a scenario that is prevented by the abundant expression of regulator proteins. in health, these regulator proteins maintain a fragile truce in which complement is continuously activated in what is known as tickover. bacteria or other foreign surfaces lacking regulator proteins provoke massive activation and amplification of complement, generating activation products that label the invader, attract and activate phagocytic cells and directly cause lytic pathogen killing. complement comprises a cluster of activation pathways that are triggered in various ways and progress through several amplifying enzymes to converge on a final common cell-killing pathway , . surface-bound antibodies trigger the classical pathway, bacterial sugars initiate the lectin pathway and amplification is provided by the alternative pathway, regardless of initiating route (fig. ) . fluid-phase regulators prevent uncontrolled activation in plasma, whereas membranebound regulators restrict activation on cells. the protective (and pathological) effects of complement are mediated by its activation products -active fragments or complexes of complement proteins. c , the most abundant complement protein, has numerous activities; some c fragments (for example, c b, ic b and c dg) can covalently bind to targets and act as flags to instruct phagocytes to bind to and engulf the targets, or they can enhance humoral immunity by facilitating b cell activation. the small c a fragment, among its many roles, can attract and activate phagocytes. downstream of c , the terminal pathway releases the proinflammatory fragment c a and assembles a pore in target membranesthe membrane attack complex (mac) -that effects the lysis of bacteria and other activating cells. the list of diseases in which complement has a role, either primary or secondary to other triggers, is long and growing (table ) . genetic deficiencies in complement components are relatively rare and associated with infections and immune complex diseases (box ) ; however, excessive or dysregulated complement activation contributes to many inflammatory, autoimmune and degenerative diseases, and is a much larger problem , . in a minority of these diseases, genetic changes, mutations or polymorphisms in complement proteins, regulators or receptors are the direct cause of dysregulation and disease; in most of these diseases, complement is not the primary cause but is recruited in response to tissue damage and serves to amplify and exacerbate inflammation and injury. crucially, a small but growing group of conditions are now widely recognized as diseases caused by complement dysregulation, presenting obvious targets for complement-inhibiting drugs , . lessons from figure | targets for inhibition in the complement pathway. the figure shows a highly simplified view of the complement system and highlights the targets for pathway inhibition. activation is triggered through classical (antibody) or lectin (sugar) pathways that rapidly converge to form a complement c -cleaving enzyme (c convertase), c b a. the alternative pathway can be independently activated to generate its own c convertase (c bbb) but, more importantly, amplifies activation regardless of trigger. c fragments, both soluble and surface-attached, engage specific receptors on expressing cells to mediate key activities. the c convertase, formed by the recruitment of an additional c b into the c convertase, cleaves c to release a small fragment, c a, which binds to receptors on expressing cells to mediate activation events. formation of c b initiates the membrane attack pathway; sequential recruitment of components c , c , c and c creates a pore in the target membrane -the membrane attack complex (mac) -that can activate or kill the targeted cell. the complement system presents many targets for inhibition with drugs. in the activation pathways these include the initiating complexes and enzymes, the initiators of the alternative pathway loop and the c convertases. in the c -c axis, potential targets include the individual components (for example, c and c ), the activation fragments (for example, c a and c a) and the c convertases. in the terminal pathway, agents might target individual components (such as c , c or c ) or intermediates (such as c b and c b ), block the functional mac pore or inhibit the downstream signalling events that mediate cell activation or destruction. proteins and protein fragments that coat pathogens and dead or dying host cells to label them for recognition and elimination by phagocytes. the principle complement opsonins are c b and ic b. disease-associated polymorphisms and highly penetrant disease-causing mutations in complement proteins have pinpointed the pathways and components driving pathology and guided the development of therapies for the numerous disease indications in which complement has an important role (table ) . the merits of anti-complement therapy in preclinical models of disease have been explored in hundreds of papers; the earliest examples date back more than years and describe the use of cobra venom factor (cvf) to inhibit disease in animal models , . despite this long history, few drugs have entered clinical trials, and fewer still have progressed beyond phase i. the recent interest in complement and disease has been fuelled by a number of factors -the demonstration of genetic associations that firmly link complement to common diseases was a key finding, but the biggest factor has been the success of a single drug, eculizumab (soliris; alexion pharmaceuticals), in an orphan application: treatment of paroxysmal nocturnal haemoglobinuria (pnh) (boxes , ) . pnh was among the first diseases to be treated with anti-complement therapeutics (box ) ; however, pnh is rare, and the identification of a much more common disease with complement dysregulation at its root was required to catapult complement into the spotlight. agerelated macular degeneration (amd) is the commonest cause of blindness in the western world; a perfect storm of genetic, pathological and clinical evidence has demonstrated beyond doubt that complement dysregulation is a crucial driver of inflammation and tissue damage in amd and has catalysed an explosion of interest in controlling complement , . as a consequence, what began as a small industry targeting pnh and a few other orphan diseases has, over the past decade, grown into a major endeavour involving numerous companies ranging from small biotechnology companies to large pharmaceutical organizations. as the field has grown, it has become increasingly clear that there will be no 'one size fits all' solution to complement therapies; agents that are effective in one disease might do nothing in, or even exacerbate, another. rational design and selection of therapies will require in-depth understanding of the way complement works in each disease. in this review, we consider the diverse disease targets, the many intervention points, the challenges associated with drugging these targets, and the multiple approaches to inhibition that are emerging as complement moves into the clinical mainstream. below, before exploring pathology, we briefly revisit physiology. complement is a key part of innate immunity because it interacts with many other processes; for any therapy that seeks to block or modulate complement, the potential effects on these physiological roles must be considered to avoid iatrogenic injury. patients deficient in complement proteins usually present with recurrent bacterial infections. c is a crucial source of opsonins, which label bacteria for removal by phagocytes; deficiency of c is rare and always marked by severe recurrent infections . patients deficient in classical pathway or lectin pathway proteins (c , c , mannose-binding lectin (mbl), mblassociated serine protease (masp ) and masp ) may also present with recurrent bacterial infections, and deficiencies of alternative pathway components or the positive regulator properdin also predispose to infection, although these deficiencies are associated with a strong bias towards gram-negative bacterial infections. deficiencies in terminal pathway proteins specifically predispose to infections with neisseria species, typically those causing meningococcal meningitis or sepsis, as a consequence of the peculiar sensitivity of these pathogens to lysis by the mac. protection against immune complexes. immune complexes comprise antibodies bound to target antigens (cell debris) in a multi-molecular complex. unhindered, immune complexes grow by acquisition and aggregation, eventually becoming large, insoluble aggregates that lodge in capillary beds and trigger inflammation and tissue damage. in healthy individuals, immune complexes activate the classical pathway and become coated with c and fragments of c and c ; these proteins mask antigens in the immune complex and disrupt the lattice, thereby limiting growth of the aggregates and simultaneously providing ligands for the receptors (complement receptor type (cr )) on erythrocytes, which sequester immune complexes, and for the receptors (cr , cr and cr ) on phagocytic cells, which engulf and destroy immune complexes . patients with deficiencies in classical pathway components (in particular c and c ) usually present with an immune complex disease that closely resembles systemic lupus erythematosus (sle), itself a disease of complement dysregulation . priming adaptive immunity. the role of complement in stimulating the adaptive immune response was first demonstrated in complement-depleted mice years ago . antigens coated with complement activation fragments provoke markedly greater antibody responses than do those that are uncoated; the c dg fragment is crucial to this effect, ligating cr on b cells to deliver a powerful co-stimulatory signal that reduces the threshold for b cell receptor triggering and increases the amplitude of the response . the smaller fragments of complement activation, c a and c a, enhance the ability of antigen presenting cells (apcs) to present antigens and stimulate t cell proliferation; together, complement products in the inflammatory microenvironment markedly influence t cell activation and the balance between effector and regulatory t cells . other physiological roles. many other roles of complement continue to emerge, including directing haematopoietic stem cells to marrow niches, regulating triglyceride uptake and storage in adipose tissue and the regeneration of damaged tissue after injury and during wound healing . each role in itself could warrant a review; therefore, here we merely highlight that these many roles may be affected by complement inhibitors. anti-complement drugs. anti-complement drugs have the potential to affect each and all of the physiological roles discussed above (fig. ) . it is inevitable that a drug that blocks any of the complement pathways will increase the risk of infections, either non-selectively or for certain groups of organisms. any drug that stops activation of the classical pathway will affect the clearance of immune complexes and apoptotic cells. inhibition of the activation pathways may disrupt an individual's capacity to mount an adaptive immune response, although therapeutic inhibition later in life is less detrimental as adaptive immunity is established and developed in older individuals. anti-complement drugs may also disrupt normal lipid metabolism or interfere with the healing and resolution of injuries. all of these potentially damaging effects need to be considered when deciding whether and when to use an anti-complement drug and which part of the system to target. with appropriate prophylactic measures such as immunization and antibiotic therapy, and with careful consideration of the target, most of these effects can be managed. a limiting factor in the use of anti-complement drugs has been an understandable concern regarding the harmful consequences of blocking the important physiological effects of complement described above, which are graphically illustrated in individuals with complement deficiencies. deficiencies in the classical pathway prevent efficient clearance of immune complexes and apoptotic cells, leading to a high penetrance of lupus ( % penetrance in individuals with a c q deficiency and % penetrance in individuals with a c deficiency) , whereas individuals who are deficient in c or components of the alternative pathway or the terminal pathway have recurrent infections that can be life-threatening . although these risks can be managed with good clinical care, a guiding principle for any drug should be to cause as little disruption of physiological roles as possible, particularly if treatment is to be continued in the long term. researchers have devised several strategies to inhibit complement without risking iatrogenic injury: complement can be inhibited late in the pathway, wherein the only infection risk is from neisseria species; complement can be inhibited transiently, which is sufficient for acute conditions, with minimal risk of immune complex disease; or therapy can be delivered directly to disease sites such that systemic inhibition is avoided. these strategies have spawned drugs that have entered the clinic, in some instances with dramatic effect. the c -specific monoclonal antibody (mab) eculizumab prevents mac formation; opsonization is thus unaffected by eculizumab treatment and the risk of meningococcal infection can be mitigated through vaccination and antibiotics. the c blocker compstatin and an antibody antigen-binding fragment (fab) directed against factor d (fd), administered intravitreally, have undergone clinical trials for amd , . the compstatin derivative apl- (apellis pharmaceuticals; a long half-life form) has just entered phase i trials for add-on to standard-of-care in pnh; it will be interesting to see how systemic administration affects the risk of infection. chimeric agents, comprising a complement ligand-targeting modality linked to a complement inhibitor, localize activity at the disease site. this concept has shown promise in murine models and has been developed as a therapy for pnh , . for any anti-complement drug, short-term inhibition for acute indications in a hospital setting is unlikely to incur serious risk of immune complex disease, and infection risk can be managed with tailored antibiotic cover. specific issues for complement-targeted drugs dosing. experience gained from the first few drugs entering the clinic has added knowledge and identified issues that are specific to complement-targeted therapies. one major issue is dosing; most complement proteins are abundant in plasma and turn over rapidly, so adequate dosing of an inhibitor can be challenging. for example, c is present in plasma at ~ g per l, and ~ - % of total plasma c (~ - mg) is consumed and replaced daily simply through the normal tickover process . it is therefore obvious that large doses of complement-targeting drugs and frequent administration will be needed to block complement at the level of c . dosing is an issue for current drugs; the cinryze (a plasma-derived c inhibitor; shire pharmaceuticals) dose for prophylaxis in hereditary post-infection hus clinical and therapeutic *the table comprises an incomplete list of the many diseases in which complement has a role; the diseases are grouped by process, although these groupings are porous and many diseases fit more than one category. ‡ clinical indicates evidence from human studies including biomarker and pathological findings; genetic indicates evidence from gene linkage studies; models indicates preclinical evidence of complement involvement from animal models; and therapeutic indicates evidence from use of a therapeutic (in humans or in animal models). ahus, atypical haemolytic uremic syndrome; anca, anti-neutrophil cytoplasmic antibody; ards, adult respiratory distress syndrome; copd, chronic obstructive pulmonary disease; iga, immunoglobulin a; mpgn, membranoproliferative glomerulonephropathy; pnh, paroxysmal nocturnal haemoglobinuria; ttp, thrombotic thrombocytopenic purpura. , , whereas in atypical haemolytic uremic syndrome (ahus) the maintenance dose is , mg every weeks. these huge and frequent doses contrast starkly with agents targeting cytokines, which are released de novo in disease and at much lower levels; for example, the tumour necrosis factor (tnf)-specific mab adalimumab (humira; abbvie) is effective in rheumatoid arthritis at a dose of mg every weeks . the plasma c concentration is ~ mg per l and the turnover rate is ~ hours ; as a consequence, even with such high doses, breakthrough activity can occur in some patients treated with eculizumab and monitoring of complement activation in plasma is required . alexion have recently launched two new agents that are in phase i trials (alxn and alxn ; 'next-generation' eculizumab molecules) that were probably developed to overcome some of the issues with eculizumab. c is not limiting in the complement cascade; therefore, inhibition at the c stage requires near-complete blockade or depletion of c ; these threshold kinetics necessitate large doses to achieve an effect. approaches to circumvent the problem of high levels of protein turnover have been described, using drugs that only bind to activated forms of complement proteins, which are present at much lower concentrations in plasma , ; these approaches show potential but have not been tested in humans. apart from the problems arising from the sheer abundance of complement proteins, dosing is complicated because plasma complement levels vary widely in the population and because many are acute phase reactants, with synthesis increasing markedly in inflammation, which sometimes causes plasma levels to rise even in the face of increased consumption . the plasma concentration of fd is relatively low (~ mg per l) but it is turned over extremely rapidly (~ . mg per kg per day) ; it can therefore be predicted that large amounts of a drug will be needed to effectively inhibit fd in vivo. studies in nonhuman primates support this -a mg per kg intravenous dose of a fd-specific fab (lampalizumab, formerly called fcfd s, roche/genentech) inhibited the alternative complement pathway for only hours; in that time the plasma levels of fd increased -fold, which is probably reflective of the retention of fd-lampalizumab complexes in the plasma . lampalizumab, administered intravitreally, is currently in phase iii trials in amd. owing to their chemical nature, small-molecule anticomplement agents tend to have short half-lives in vivo. this is not necessarily a limitation, as full coverage can often be achieved through repeated dosing in situations of long-term therapy. daily subcutaneous dosing is manageable and is likely to be acceptable for patients with life-threatening diseases; oral bioavailability can often be achieved with these agents, which is a key advantage. in some conditions -such as traumatic brain injury, myocardial infarction, transplantation, stroke and other ischaemia-reperfusion injuries -transient complement inhibition for a few hours may be sufficient to prevent tissue damage. in these cases, small molecules have an added benefit in that once dosing is ceased, rapid clearance leads box | genetics dictate the risk of complement-mediated injury complement comprises a large number of interacting components and regulators, and common genetic variation in these proteins dictates whether a given individual possesses a complement system that is more or less active. we have reviewed this complotype concept previously , and here we focus on its relevance to complementmediated diseases and therapy. put crudely, individuals who have inherited a more active complotype are better able to resist infection but are at greater risk of complement-driven inflammation. some variant proteins directly affect the activity of the amplification loop (for example, complement c r g (c -r g) and factor b r q (fb-r q)) , and this is the primary reason for disease association, whereas others influence control local to the disease site, and risk is thus disease-or tissue-specific. this concept is well-illustrated by considering age-related macular degeneration (amd): the most important genetic risk factor in amd is a common polymorphism in the alternative pathway regulator fh that changes a single amino acid in the middle of the molecule (y h). the risk of developing amd is at least threefold greater in his-variant homozygotes than in tyr homozygotes , . the y h variant influences the localization of fh to retinal ligands, whereas other complement polymorphisms (including those in fb and c ) influence amd risk by increasing complement alternative pathway activity [ ] [ ] [ ] ; common variants in just these three proteins together alter amd risk up to -fold. in deciding whether and how to inhibit complement for therapy in an individual, the complotype should be an important consideration. mutations in complement components and regulators can also affect complement dysregulation and disease risk. again, fh provides the best example. fh is a large, elongated molecule comprising a chain of short consensus repeats. the th short consensus repeat (scr ) is a hotspot for mutations that may be single amino acid substitutions, premature stops or more complicated changes; many of these mutations are strong risk factors for another disease of complement dysregulation, atypical haemolytic uremic syndrome (ahus), which is typified by thrombosis, haemolysis and renal injury , . onset of ahus typically occurs in childhood or young adulthood and the frequency and severity of episodes is highly variable, with some patients having an indolent course requiring no therapy and others rapidly progressing to end-stage renal failure. all of the mutations in the scr domain of fh that are associated with ahus disrupt the capacity of fh to bind to and regulate complement on cells and other surfaces; regulation in the fluid phase is unaffected. numerous other complement protein mutations have been described and shown to influence the risk of ahus and/or other diseases in which complement plays a part. many of these are coding mutations that change the amino acid sequence and affect protein activity, others are non-coding mutations and probably affect the biosynthesis and expression of the protein; some mutations trigger fh-specific autoantibody production . an understanding of the influence of polymorphisms and mutations in complement proteins on complement activity in a particular disease is essential for the intelligent design and selection of therapeutic approaches. the figure summarises the effect of gain-of-function and loss-of-function polymorphisms and mutations in complement proteins and regulators that alter the risk of disease. to speedy recovery of complement activity, restoring its infection-fighting and tissue-repairing properties. when developing small-molecule drugs for specific targets, their mode of action is crucial and target tractability becomes a key consideration; in some instances, the small size of the drugs may become a limitation, for example in blocking protein-protein interactions (see below). when a drug with a longer half-life is needed, agents such as antibodies come to the fore. alternatively, small molecules may be modified to increase their mass by pegylation or by coupling to domains that extend their half-life in vivo: for example, albumin-binding or antibody crystallisable fragment (fc) domains , . in some diseases, complete inhibition of complement may not be needed for efficacy; agents such as soluble cr (scr ; also known as tp , celldex therapeutics) that downmodulate the complement enzymes through dynamic activities (such as accelerating their decay or increasing cofactor availability) can mediate dosedependent partial inhibition of complement rather than the sharp threshold effect typical of blocking antibodies , . such agents may enable therapy at scalable doses and in a safe and effective manner; however, the fact that even large biological agents such as scr have relatively short half-lives will affect the choice of indication. getting to where the action is. many pharmaceutical companies are now focused on the development and manufacture of biologics because of their historical success, good economic return and low rate of attrition. as a consequence, most anti-complement agents developed to date are biologics -either antibodies or recombinant proteins -that deliver inhibitory activity. essential to all drugs is their ability to get to the sites of disease; some complement-mediated diseases manifest in the vasculature (for example, pnh and vasculitis), whereas in other diseases (such as amd, neuromyelitis optica and arthritis) complement attacks tissues at sites remote from systemic circulation. the ability of systemically-administered drugs to diffuse to the site of tissue damage and inflammation will thus be essential for treatment success. the liver produces most of the complement components found in the circulation, and if diffusion of circulating components to disease sites drives inflammation and disease then systemic depletion or blocking of specific complement components may ameliorate the pathology. however, locally produced complement is known to have key roles in some indications. for example, in renal allograft rejection, locally produced and activated c drives the t cell response and immune rejection ; in this case, systemic anti-c therapy will be ineffective unless the drug gets into the tissues. similar challenges exist in organs protected by tight barriers, such as the nervous system and the eye. in some diseases these barriers become damaged and leaky with disease progression, but it is likely that complement activation contributes to the early, pre-inflammatory stages of eye and brain diseases, so to be of value complement --inhibiting drugs are required in the relevant organ early in the disease when the barriers are still intact. assuming that they have good bioavailability, small-molecule drugs may have advantages over biologics in these contexts because of their higher tissue penetrance; however, of the numerous small-molecule anti-complement agents that have been developed and tested in humans, none has yet progressed to the market [ ] [ ] [ ] . getting in the way. complement is a good target for biologics because the cascade involves multiple proteinprotein interactions and conformational changes, exposing large surfaces that lend themselves to large blocking box | the development of eculizumab for the treatment of paroxysmal nocturnal haemoglobinuria the first description of functional blocking of complement c with a monoclonal antibody (mab) dates back almost years; a c -specific mab, bb . , generated in c -deficient mice, blocked haemolysis in mouse serum . bb . provided a powerful proof of principle for inhibition of the membrane attack complex (mac) that was effective in numerous mouse models ; bb . also provided a rationale for the development of blocking mabs specific to human c . one such mab, g . , was selected by alexion, humanized and further manipulated to generate fab (antigen-binding) and scfv (single-chain variable) fragments , . by , humanized g . , termed eculizumab, was in early clinical trials in a range of joint, kidney and skin conditions ; however, its remarkable efficacy in paroxysmal nocturnal haemoglobinuria (pnh) focused efforts in that area . pnh is a clonal haematological disorder that involves chronic intravascular haemolysis, with complement lysis of glycosylphosphatidylinositol (gpi)-anchor-deficient erythrocytes as its principal feature. patients with pnh are anaemic with resultant lethargy and at risk of thrombosis. prior to the use of eculizumab, treatment involved frequent blood transfusions and symptomatic support. eculizumab transformed the outlook for patients with pnh, preventing haemolysis, reversing anaemia and removing transfusion dependence in most patients, thereby improving quality of life . eculizumab therapy is effective over the long term (> years), and substantially reduces mortality . the only other disease for which eculizumab is currently approved by the us food and drug administration (fda) is atypical haemolytic uremic syndrome (ahus). as with pnh, there were no specific therapies for this potentially catastrophic disease and the evidence implicating complement was overwhelming. in patients with ahus, eculizumab treatment increased platelet number and improved renal function over the course of year and some patients were no longer dependent on dialysis . numerous other clinical trials in various conditions are in progress, and these are summarized in table . a major concern of governing bodies in the united kingdom and elsewhere is cost; at the time of writing, standard pnh maintenance therapy ( mg eculizumab delivered intravenously every weeks) costs approximately uk£ , per patient per year; therefore, even for a rare disease with only ~ cases in the united kingdom, the estimated cost to the national health service (nhs) is £ million per year. a concise and up-to-date summary of the clinical data can be found on the electronic medicines compendium website (see further information). it should be noted that an eculizumab biosimilar is already being developed by epirus biopharmaceuticals. drugs . by contrast, for a small molecule to block a protein-protein interaction it must target crucial sites and specific surface features -for example, charged or hydrophobic pockets . the wealth of structural information now available in the field, including snapshots of convertase enzymes and mac precursor complexes captured in active conformations, unmasks the precise nature of these protein-protein interactions and identifies sites that are key to the interaction that can be targeted with small molecules or biologicals using structure-based drug design , . alternatively, the expansive small-molecule libraries that are now ubiquitous in pharmaceutical companies have been successfully screened to reveal agents that bind to and inhibit interactions within the cascade through competitive or allosteric mechanisms , . small peptides selected from such libraries for binding components or inhibiting complexes are now numerous in the literature; for example, the cyclic peptide compstatin, which blocks c from binding to the convertase, has already entered clinical trials , . target validation in models. validation in rodent models of disease can be challenging or even misleading, and care needs to be taken to ensure that the disease mechanism in the chosen model is the same as that in the human disease. to illustrate the differences in complement systems between mice and humans, consider the receptors for c fragments. in humans, cr and cr are encoded by separate genes and have different roles: cr binds to c dg and enhances humoral immunity whereas cr acts as a cofactor for the fi-mediated cleavage of c b and has a key role in transporting immune complexes . in mice, cr and cr are generated from the splicing of one gene and have limited expression; the main cofactor for c b cleavage and generation of c dg in the mouse is cr -related gene/ protein y (crry), a broadly expressed protein absent in humans . therefore, mice and humans respond to c fragments through different receptors and with different outcomes that will probably alter the effects of c -targeted drugs. nevertheless, in many models, disease mechanism and response to complement inhibition translate well and proof-of-principle is readily obtained. with current efforts to reduce animal use in scientific research, initiatives are emerging to better validate drug targets at an early stage of development without the use of animals, for example, using technologies such as microfluidic organs-on-chips . what does the current toolbox contain? before describing specific tools, we first provide some guiding principles and suggestions for the classification of anti-complement agents. there are several discrete ways in which an agent might act to influence complement (fig. ). an agent might inhibit progression in the activation or terminal pathways, act as an antagonist of an activation product, or cause activation and consumption of complement. another broad distinction can be drawn between those drugs that inhibit complement systemically and those that are directed to specific sites of pathology to localize their inhibitory activity. anti-complement drugs can also be divided into categories based upon their molecular characteristics: purified or recombinant forms of naturally occurring human regulators; modified protein copies or mimics of natural regulators; copies of pathogen-derived regulators; mabs or fragments of mabs; naturally occurring or synthetic small-molecule inhibitors; or antisense or similar nucleic acid constructs. finally, anti-complement agents can be grouped according to the part of the system they target, a convention that we follow in this review. targeting the binding and assembly of the initiating complex. initiation of classical or lectin pathway activation involves the recognition of an activating surface; in the classical pathway this activation occurs through a surfacebound antibody that ligates c q, whereas in the lectin pathway this occurs via specific sugars on the surface that bind to mbl or collectins (fig. ) . agents that block these events would switch off the respective activation pathways at the very first step, an attractive prospect given the amplifying nature of the complement system . a potential classical pathway inhibitor might block c q-binding sites on the antibody or the antibody-binding sites on c q. a lectin pathway inhibitor might mask surface mbl-binding sugars or the sugar-binding sites on mbl. in either case, the agent would need to bind the target with higher affinity than the physiological ligand to compete effectively. alternatively, an agent could disrupt or prevent the assembly of the multi-molecular complex, which comprises the recognition unit (c q and mbl in the classical and lectin pathways, respectively) and the associated enzymes (c r and c s in the classical pathway and masp and masp in the lectin pathway) that are essential for pathway activation . both complexes circulate preassembled in plasma, so any agent targeting the complex would need to dislodge the bound enzyme to be effective; surprisingly, the c s-and masp -targeting mabs from true north and omeros, respectively (described below), both achieve this effect. another approach to inhibiting activation of the complexes would be to develop a molecular lock that restricts the conformational changes in c q or mbl that occur upon ligand binding and are essential for activation of the associated enzymes. such approaches have been used successfully to develop small-molecule inhibitors of chemokine receptor activation , , but have not yet been reported for c or the mbl-masp complex. several c q function-blocking mabs have been reported and were recently shown to be effective in a murine model of the antibody-mediated demyelinating disease, neuromyelitis optica . numerous peptide blockers of antibody-c q interactions have been described, derived from the sequences of c q binding sites in the antibody, c q receptors or c q itself, or selected from libraries. one such peptide blocker, termed peptide j, is a powerful inhibitor of the classical activation pathway in multiple species ; however, there have been no further reports of this agent. other peptides have been described that interfere with the binding of c r and c s in the c complex, but none has progressed beyond in vitro studies. the binding of mbl to specific sugar residues on surfaces presents a potential drug target, but no agents acting in this manner are reported. a recent publication describes a virus-derived peptide, peptide inhibitor of complement c (pic ), that binds to the collagenous regions in both c q and mbl and inhibits both the lectin and classical pathways in vitro and in vivo in rodents . targeting the enzymes of the initiating complexes. the enzymatic activity of the classical or lectin pathwayinitiating complexes is provided by the serine proteases c s and masp , respectively. in the c complex, immunoglobulin binding induces conformational changes in c q, triggering auto-activation of c r that in turn cleaves and activates c s in the same complex. the precise steps involved in activation of the mbl-masp complex are less clear but probably involve a similar multi-step process . the serine protease enzymes in these complexes are potential targets for pharmacological inhibition. both the pharmaceutical industry and nature itself have invested heavily in the development of serine protease inhibitors (spis), and numerous spi drugs have been made, of differing specificities and compositions, to target the myriad serine proteases essential for digestion, haemostasis, immunity, reproduction and many other physiological and pathological processes. the biggest problem in designing an spi drug to inhibit a particular protease is specificity -almost all spi drugs have some 'off-target' effects that can limit their use. even nature struggles -the sole natural inhibitor of c r and c s is an spi, c inhibitor (c inh; also known as serping ), but c inh also controls masps and proteases in the coagulation and kinin systems. indeed, deficiency of c inh leads to dysregulation in all these proteolytic cascades that, in aggregate, cause the disease hae , . c inh removes activated c r and c s from c q to form a stable complex in which c inh itself is cleaved and inactivated. c inh has a long history of use as a drug; indeed, it can claim to be the 'first-in-man' complement drug and the first of the natural inhibitors to be recognized as a potential therapeutic. more than years ago, patients with hae were shown to be deficient in c inh and to respond to plasma replacement, provoking efforts to purify c inh for treatment of acute attacks , . remarkable successes in these early studies rapidly led to the adoption of plasma-derived c inh as the standard of care for acute episodes in hae. the obvious advantage of this approach is that a plasma-derived molecule should be low risk (once infectious agents are eliminated) and non-immunogenic. the widespread availability of industrial-scale protein-fractionation facilities -established for the production of immunoglobulins, albumin and other plasma products -made this a viable approach for purifying c inh. unfortunately, concerns about viral transmission in plasma-derived proteins have led to its removal from the clinic in several countries, including the united states. recent methodology improvements and new approaches to production of c inh have changed this unsatisfactory situation; two ultra-pure plasmaderived c inh products, cinryze and berinert (csl behring) are now approved by the us food and drug administration (fda) and other regulatory agencies. some companies invested in better methods of purifying plasma c inh, whereas others set about making it. a fulllength recombinant c inh was first reported a decade ago. this agent, termed ruconest (salix pharmaceuticals), has been approved for therapy of acute attacks in europe and, very recently (july ), also in the united states. early administration of c inh reduces the duration and severity of acute attacks, findings confirmed in the large impact study of over , attacks . its use in prophylaxis is more controversial; a subset of hae patients have frequent and severe attacks despite therapy with danazol (an anabolic steroid that increases c inh synthesis) and/or ε-aminocaproic acid (a protease inhibitor) or cannot tolerate these agents. regular intravenous administration of c inh (every - days) to restore plasma levels is an effective therapy in this group , ; however, in the united states it is now becoming accepted practice to treat all hae patients in this manner, a practice limited elsewhere by cost and lack of evidence base . berinert has been approved for on-demand, patientadministered therapy in the united states and europe. given its long history and known safety and efficacy in hae, it is surprising that c inh has not been more widely used in other conditions characterized by complement dysregulation. there are a number of published studies, mostly small, demonstrating that administration of c inh reduces mortality in sepsis, and in some studies the effect is startling , . other studies have reported positive effects of c inh in myocardial infarction and in ischaemia-reperfusion injuries in both animal models and humans, but no large-scale clinical trials in these other applications have been published , . several broad-spectrum protease inhibitors already in clinical use have c r, c s and masps among their targets; perhaps the best example of these is nafamostat mesilate (also known as futhan or fut- ), a small-molecule blocking protein-protein interactions offers numerous targets for drugs in the complement system; approaches may involve blocking the formation of an essential complex or receptor-ligand interaction, preventing a key conformational change or inhibiting a crucial enzymatic cleavage. such activities underpin many of the current and evolving therapeutics, particularly the monoclonal antibody (mab)-based agents. several of the mabs that target c convertase work in this way; either they prevent the complex from forming at all or they lock it in an inactive conformation. similarly, the staphylococcus aureus-derived inhibitor scin (staphylococcal complement inhibitor) holds the convertase in an inactive state . the c -specific mab eculizumab binds to c remote from the cleavage site, at a site in the α-chain that prevents c from binding to c convertase , , . human experimental proof was recently provided: a c polymorphism, present in ~ . % of the japanese population, caused a single residue change (r h) in the putative eculizumab binding site and rendered the protein refractory to inhibition . in r h heterozygotes treated with eculizumab, half of the circulating c remained active and paroxysmal nocturnal haemoglobinuria (pnh) therapy was ineffective. blocking protein-protein interactions using small molecules has attracted less interest because of the perceived difficulties of successfully interfering with large protein-protein interfaces ; for example, the footprint of the factor h regulatory domains on the c convertase extends over an enormous , Å (ref. ). nevertheless, some success has been achieved with peptides or other small-molecule inhibitors; the best example is that of compstatin -a amino acid cyclic peptide that inhibits c -which, over the course of almost two decades, has been modified into a highly potent high-affinity inhibitor of c cleavage , , [ ] [ ] [ ] . compstatin binds to native c and prevents its interaction with the c convertase, thus blocking further complement activation. in vivo testing of compstatin has been limited by its specificity for human and primate c ; in primates, compstatin reduced drusen load and inhibited disease in an age-related macular degeneration (amd) model . alcon (a novartis company) has taken a compstatin analogue, pot- (al- a), through phase i trials; phase ii trials in amd, which started in , are yet to report any results (see the alcon website in further information) . for their compstatin analogue, amy- , amyndas has gained european medicines agency (ema) and us food and drug administration (fda) orphan drug status for use in pnh. apellis pharmaceuticals are developing a compstatin derivative in an inhaled short-acting form (apl- ) for the treatment of asthma and chronic obstructive pulmonary disease (copd) (currently in phase i as a disease modifying therapy for copd). this derivative is also being developed in a long-acting form (apl- ) for the treatment of haemolytic disorders and amd; apl- is currently in phase i for pnh as an add-on to eculizumab therapy (clinicaltrials.gov identifier: nct ), and a phase i trial has recently started for apl- as an intraocular therapy for amd (nct ). protease inhibitor used to treat disseminated intravascular coagulation and acute pancreatitis that affects multiple plasma protease systems . however, the many off-target effects and resultant toxicity of nafamostat mesilate make it a poor candidate for regulating complement in vivo. other small-molecule inhibitors of c s of varying degrees of specificity and strength have been described, including c s-inh- (basf pharma) and bcx- (biocryst pharmaceuticals), both no longer in development, and recently a family of biphenylsulphonyl thiophene derivatives that, when pegylated, displayed strong c s inhibition and promising pharmacokinetics . modern methods of structure-based design offer the prospect of much more selective smallmolecule inhibitors of the initiating complex enzymes, as demonstrated for other proteases . indeed, the 'directed evolution' of a sunflower-derived peptide, trypsin inhibitor , created specific inhibitors of masp and masp that await testing in vivo . an inhibitory mab against c r and c s or masps might address the specificity issue that limits the clinical use of small-molecule agents. a mab against c s, tnt (true north therapeutics), prevented erythrocyte haemolysis in an ex vivo study of autoimmune haemolytic anaemia ; a humanized analogue of this mab, tnt (true north therapeutics), is being fast-tracked to clinical trials in this disease. a recombinant mab fab was recently described that bound c s with nanomolar affinity and potently inhibited classical pathway activation, but so far this has only been tested in vitro . a masp -blocking human mab (oms ), which is an effective and long-lived (up to a week) lectinpathway inhibitor that is delivered intravenously or subcutaneously, was recently awarded us fda orphan drug status for use in ahus and other thrombotic angiopathies and is currently in phase ii/iii trials (nct ). a recent (august ) omeros press release described positive outcomes in ahus from this trial. targeting the c convertases. the c -cleaving enzymes of the classical, lectin and alternative pathways, c b a and c bbb, are assembled in quite different ways despite their functional and structural similarities (fig. ) . in the classical and lectin pathways, c is cleaved by activated c s (or masp ) in the surface-bound c (or mbl-masp) complex; the large c b fragment binds to adjacent surfaces via a nucleophilic attack on a thioester group, captures c and presents it for cleavage by c s or masp as above. the c b a complex is the c convertase of the classical and lectin pathways. c a is a serine protease that cleaves c to form c a and the opsonin c b; c a can also cleave c (when properly presented) to form c a and c b. these two complement proteins are the sole substrates of the c a serine protease. assembly of the alternative pathway c convertase requires c b, generated through classical and lectin pathway activation or from tickover activation on surfaces. c b binds to fb, triggering major conformational changes that expose fb to cleavage by fd, a unique plasma serine protease that has fb as its sole substrate. the c bbb complex is the c convertase used in the alternative pathway; the bb fragment, generated from fb cleavage, is a serine protease that cleaves c and c in precisely the same manner as c a in the classical pathway. the key amplifying role of the alternative pathway makes it a very attractive source of drug targets that can be targeted in a variety of ways (fig. ) . it is obvious from the above description that the c convertases present a dizzying array of opportunities for regulation. agents might target the assembly of one or more of the complexes, preventing complex formation and/or causing complexes to break up. they might target one or more of the serine proteases responsible for formation of the convertase (c r, c s, masp or fd), convertase activity (c a or bb) or convertase regulation (fi). alternatively, they might target the substrates of these enzymes, c or c , by blocking binding to or cleavage by the convertase or acting as molecular locks to prevent conformational changes essential for activation. agents might also target active fragments (c a, c a, c b or c b) to block their downstream effects. indeed, nature has adopted some of these strategies to keep complement in check. the convertases are naturally labile and break up in a matter of minutes; regulatory proteins that accelerate this decay exist both in plasma and on membranes, and they function by binding to the complex and displacing the enzyme. some of these regulatory proteins remain bound after decay and act as cofactors for the further cleavage and irreversible inactivation of c b or c b by the plasma serine protease fi. using the endogenous c convertase regulators as drugs or leads. the plasma regulators of the c convertases were obvious targets for drug development given the success of c inh. to date, none of the other plasma regulators has entered the clinic despite a large amount of research and preclinical activity. in vitro studies more than years ago showed that modest (around % above baseline) increases in the concentrations of the complement regulators fh and fi markedly reduced plasma alternative-pathway activity, provoking the suggestion that augmentation of these proteins might be of therapeutic benefit ; however, large (and probably frequent) doses will be required to substantially alter the levels of these abundant proteins. despite this, there has been considerable recent interest in using plasmaderived fh as a therapy, particularly in those diseases strongly linked to fh polymorphisms and mutations. deficiency of fh is rare and always associated with severe c dysregulation in plasma or on cell surfaces that leads to a range of renal diseases, including c glomerulopathy and ahus; fh deficiency has been successfully treated with plasma exchange, strongly suggesting that fh would be an effective therapy in these diseases . indeed, in fh-deficient mice, therapy with human fh reversed renal injury . amd is strongly linked with the fh y h polymorphism, and this has provoked intense interest in giving at-risk individuals the protective y form of fh to prevent or treat amd. several companies established programmes to produce plasmaderived or recombinant fh with this as the primary aim, but none of these programmes survive. of note, some evidence from mouse models and human studies suggest that fh would need to be administered intra-ocularly in amd to control complement dysregulation locally and alter the course of disease , . in one study, instead of expressing full-length fh for use in therapy, this large molecule was stripped down to its key functional units and re-assembled to create a minimized fh, mini fh, which comprised four amino-terminal, complement-regulatory short consensus repeats and two carboxy-terminal, surface-binding short consensus repeats joined by a short linker. mini fh proved to be an effective complement inhibitor in the fluid phase and on surfaces in multiple in vitro assays . , and it is now in preclinical development for therapy of pnh (amy- ; see the amyndas website). other approaches aimed at targeting fh activity to surfaces are described in later sections. c bp, the fluid-phase regulator of the classical pathway c convertase, is a complex, oligomeric molecule that to date has received little attention in terms of therapeutic applications. c bp deficiencies are extremely rare, restricted to a single case report . in mouse models of arthritis, administration of plasma-derived c bp reduced severity of disease , but no human studies have been described. fi is the key enzyme for regulation of the c convertases and deficiency causes complement consumption and consequent susceptibility to bacterial infections. individuals with an fi deficiency present with repeated infections that may be life-threatening, and even heterozygotes may have an increased infection risk . plasma therapy to replace fi would, by analogy with fh deficiency, be expected to help, although a single small study found no clinical benefit . a recent report showed that fi supplementation reduced complement activity in sera from individuals with more active complotypes, provoking the suggestion that fi therapy would reduce the risk of developing diseases linked to complement dysregulation . properdin is a plasma protein that binds to and stabilizes the alternative pathway convertase; in other words, it acts as a positive regulator . properdin may also act as a pattern-recognition molecule, binding targets and initiating activation of the alternative pathway. deficiency of properdin (x-linked) predisposes individuals to infections by bacteria, particularly neisseria species. supplementation of properdin in individuals who have a deficiency would be expected to reduce infection risk. a recent study suggested a broader role for properdin therapy -low-dose recombinant properdin markedly reduced susceptibility to bacterial infection in a mouse model, raising the possibility that supplementation might be effective in sepsis and other severe infections in humans . the stabilizing role of properdin on the alternative pathway c convertase means that blocking this positive regulator may be therapeutic in inflammatory disease. a mab targeting properdin (nm ) and a small-molecule properdin antagonist have been reported by novelmed therapeutics for use in pnh and rheumatoid arthritis, respectively (see the novelmed therapeutics website). using the membrane c convertase regulators as drugs or leads. the membrane regulators of the c convertases present a different challenge compared to the plasma regulators in that they cannot readily be purified for testing as potential drugs. the only way to test whether a membrane regulator might work as a therapeutic is to make a recombinant soluble form. the first of these, described in , was a soluble recombinant form of cr (known as scr or tp ) comprising the entire extracellular sequence of thirty short consensus repeats (> , amino acids). the protein was a powerful inhibitor of both classical and alternative pathway convertases in vitro in human and rodent sera; pretreatment with scr protected against reperfusion injury in a rat model of myocardial infarction . over the ensuing two decades, this agent provided an excellent proof-of-principle for complement inhibition in diverse models of human disease, effective in a wide range of disease models and in rodents, pigs and primates. after initial development work by smithkline beecham, avant immunotherapeutics took scr into clinical trials in adult respiratory distress syndrome, acute lung injury and cardiopulmonary bypass; although some positive results were described , there were also many failures, notably in large sepsis trials, and scr has not progressed for these indications. more recently, guided by success in animal models, scr entered the amplification (c b feedback) loop is a positive-feedback cycle that consumes complement c to generate more enzyme and activation products; if unregulated, it cycles until all available c is consumed. tight regulation is provided in the plasma by enzymes and cofactors that remove the c fragment c b from the feedback cycle for breakdown into smaller fragments. as a consequence, the c b feedback cycle normally operates at a very low rate (tickover). the balance between activation and regulation is disturbed in disease; a healthy balance can be restored by providing extra control (for example, increasing regulation, such as that provided by the complement regulatory protein factor h (fh), thereby increasing 'feed out' from the amplification loop) or by preventing the formation of c b in the feedback cycle (for example, by blocking convertase enzyme, thereby decreasing 'feed in' to the amplification loop). agents that target amplification of complement can have major therapeutic effects. ba, non-catalytic fragment of fb; c b-fh, complex between c b and fh; cr , complement receptor type ; ic b-fh, complex between inactive c b and fh. adapted with permission from ref. , elsevier. clinical trials in dense deposit disease (cdx- ; celldex therapeutics); regrettably, this study terminated in because of recruitment issues. numerous modified forms of scr have been created to improve its therapeutic potential and/or simplify its production. tp incorporated multiple oligosaccharide sialyl lewis x epitopes to bind to selectins, which are upregulated on endothelia at inflammatory sites. this simple change improved efficacy compared to unmodified scr in rat ischaemia-reperfusion models . the minimum complement regulatory unit from cr was known to be the three n-terminal short consensus repeats. a drug that comprised these three short consensus repeats attached to a nonspecific membrane-binding motif (apt- ; also known as mirococept; adprotech) proved remarkably effective as a complement inhibitor when delivered locally or systemically in rodent and pig models , . the agent was inexpensive to produce and proved safe in human studies. the mrc-funded empirikal trial (uk clinical research network (ukcrn) identifier: ), which is due to commence in , tests the effect of mirococept pretreatment of donor kidneys on their survival and function post-transplant. although recombinant soluble forms of the other membrane c convertase regulators, cd and cd , have been produced and tested in animal models, most have stalled at the early preclinical stage. the exception was a chimeric cd -cd molecule (cab- ; also known as mln- ; millenium pharmaceuticals) that entered phase i trials in cardiopulmonary bypass but was not progressed. there are eight protease targets that could be used for convertase regulation. the classical and lectin pathway initiating-complex enzymes (c r, c s, masp and masp ) are discussed above. generation of the alternative pathway convertase requires fd, and deficiency of fd results in the absence of alternative pathway activity and marked susceptibility to bacterial infections . the alternative pathway has a crucial role as an amplifier regardless of the initiating trigger, making it an attractive target for therapeutic modulation and bringing fd centre stage. the first fd-targeted therapeutic to reach the clinic, lampalizumab, is a humanized immunoglobulin g (igg ) fab fragment that inhibits fd activity by binding to the exosite to prevent substrate binding . a published phase i study of intraocular administration in patients with a severe form of amd termed geographic atrophy (ga) demonstrated safety . phase ii results of monthly treatments, announced but unpublished (mahalo study), describe a % reduction in ga area compared to controls at months; these startling results were even better in patients selected based on an undisclosed fi genetic biomarker, who showed a % reduction at months. phase iii trials of therapy ( mg intravitreal every - weeks) in patients with ga are ongoing. several broad-spectrum small-molecule protease inhibitors, including dichloroisocoumarin, bind to and inhibit fd; structures obtained from these complexes have identified unique properties of fd that offer the prospect of designing specific inhibitors , . novartis recently reported the evolution of small-molecule inhibitors of fd that are active in the nanomolar range . very recently, achillion have announced the development of orally active, potent and specific small-molecule inhibitors of fd that they propose for therapy in pnh. it remains to be seen whether the high natural turnover of fd will limit the use of these small molecules in humans. a number of mabs or mab fragments have been described that inhibit fb cleavage in a variety of ways. one of these, termed mab , has been developed as a therapeutic by taligen ; ta is a fab fragment of mab that blocks formation of the alternative pathway convertase. taligen was acquired by alexion in for us$ m, and the agent is currently under going humanization for further use in humans, with amd and asthma as primary targets. another fb-specific antibody, bikaciomab (nm ; novelmed), is also in development for use in amd, and small molecule inhibitors of fb have been reported (in novartis patents) although there is no evidence yet of their use in preclinical models of disease. isis pharmaceuticals have described the use of fb antisense oligonucleotides for therapy of murine lupus nephritis, but progression of this technique to humans has not been reported . the functional homologue of fb in the classical pathway is c ; despite a more limited role in pathology, c a-specific mabs that block the classical pathway c convertase have been developed and patented by tanox (now part of genentech; patent ref. wo a ) ; no trials are reported on these interesting drugs. an intriguing agent, a linear peptide (rh d) derived from a schistosoma species protein, bound c and inhibited c convertase formation, but has not been further developed to date . individual convertase components and regulators can be targeted in various ways to influence complement activity: components may be depleted in the plasma by increasing their consumption or decreasing their synthesis; regulator concentrations may be increased by supplementation or an increase in synthesis; components and/or regulators may be prevented from undergoing essential conformational changes or enzymatic cleavage; or components may be rendered more susceptible to spontaneous or accelerated decay. one example of altering consumption is illustrated by cvf, a c b analogue present in cobra venom that, when injected into mammals, binds to fb to form a stable c convertase that is resistant to normal regulatory processes and thus activates the system to exhaustion. this activity of cobra venom was recognized over a century ago, and purified cvf was first used as a tool to render the complement system of rodents deficient almost years ago, making it the first anti-complement 'therapeutic' (refs , ) . although the empiric use of cobra venom in therapy has a long history in everything from cancer to cosmetics, cvf as a drug has severe limitations: first because it is immunogenic, which renders repeat injections ineffective, and second because rapid and complete activation of complement by cvf generates high concentrations of c a, causing circulating neutrophils to activate and aggregate in the lungs and other organs . it is also unclear whether cvf can deplete complement locally in tissues. attempts to address immunogenicity through humanization of cvf have created a hybrid that is % identical to human c ; small stretches of cvf-derived sequences in the c α-chain are sufficient to retain cvf-like complement consumption . although immunogenicity may be low, concerns over the toxic effects of continuous consumption of complement by cvf ensure that use will be restricted to acute conditions. supplementation of the plasma convertase regulators offers therapeutic possibilities above and beyond the restoration of plasma levels in rare cases of deficiency. systemic modulation of the cascade using endogenous soluble regulators (or soluble versions of membraneassociated control proteins) is likely to cause downmodulation rather than complete blockade and effect therapy in a manner that is more measured. efforts to modulate complement by administering extra fh or fi are described above. however, the amounts of protein needed to raise plasma levels sufficiently to affect convertase activity may be prohibitive; the feasibility of this approach will require careful dose prediction, and dosing will probably be disease-specific and perhaps individualspecific. gene-therapy approaches to increase local synthesis are also now attracting attention -for example, the introduction of a construct that increases the production of fh or another complement regulator in the retina might prove effective in amd, a disease in which other gene-therapy approaches have already been effective in models and have reached phase i/ii clinical trials . decreased synthesis is the aim of a variety of antisense and related agents targeting complement components either systemically or locally, and these agents are the subject of a number of published patents. however, to date these strategies have only been tested in vitro and (for a minority) in animal models; for example, c -targeted antisense oligonucleotides markedly reduced hepatic synthesis and plasma c levels, and ameliorated disease in a mouse model of traumatic brain injury . c antisense is described in a number of patents over the past years but has not progressed in vivo. alnylam described an rna interference (rnai) molecule targeting c , aln-cc , that reduced plasma c levels by > % when administered monthly by subcutaneous injection to non-human primates; this agent, developed for therapy of pnh, is now in phase i/ii trials (as announced in a recent alnylam press release). targeting the anaphylactic peptides. the polypeptides c a and c a, released owing to the action of the c and c convertase enzymes, are biologically active, and they bind specific receptors on phagocytes and many other cell types to exert effects. the range of cell targets and activities is large and reviewed well elsewhere , but their pro-inflammatory activities are particularly relevant to disease. c a is a powerful inflammatory trigger implicated in many diseases, whereas c a exhibits a complex mix of pro-and anti-inflammatory activities . both c a and c a (and their metabolites) bind to seven-transmembrane-domain g-protein-coupled receptors (gpcrs), which are members of the large cytokine and chemokine receptor family. similar to other gpcrs, those that bind c a and c a are attractive drug targets; many specific small-molecule blockers have been developed for cytokine receptors, although few have been developed for chemokine receptors, which more closely resemble the c a anaphylatoxin chemotactic receptor (c ar) and c ar (ref. ). dompé pharmaceutical have described small-molecule c ar blockers and claim potential for therapy of a wide panel of diseases; however, no trials are yet reported. perhaps the most studied c ar blocker is the cyclic hexapeptide pmx , invented by taylor and woodruff and developed first by peptech and then arana therapeutics . this agent, modelled on the c terminus of c a, showed high specificity and nanomolar affinity for c ar and worked across many species; this latter property enabled the testing of pmx in a broad sweep of rodent models of inflammatory disease, and in many of these models this peptide had good efficacy. a number of phase i and iia clinical trials with pmx were initiated in rheumatoid arthritis, osteoarthritis, psoriasis and amd; the molecule proved safe, but all trials were terminated because of poor efficacy. one possible reason for these failures was the poor oral bioavailability and tissue penetrance of pmx (ref. ) . a modified version of pmx , pmx , shows improvements in these parameters and may re-surface in future trials, perhaps in combination with other agents. an orally active small molecule that blocks c ar has also been developed by chemocentryx; this agent, ccx- , has completed phase ii studies in anti-neutrophil cytoplasmic antibody (anca) vasculitis, in which it was successful in enabling the reduction and elimination of high-dose corticosteroids and demonstrated improvement in renal health parameters. in , ccx- was granted orphan drug status in both anca-associated vasculitides and ahus. emerging evidence for the role of c a as a 'modulator' of the c a-driven inflammatory response highlights the potential for c ar agonism in acute neutrophil-driven pathologies such as ischaemia-reperfusion injury , . an alternative approach, again by analogy to the chemokine and cytokine field, is the use of blockerseither antibodies or others -that bind to c a or c a and prevent receptor engagement. cytokine-specific antibodies and decoy receptors have evolved into blockbuster drugs. inflarx have developed a blocking mab against c a (ifx- ) that is currently in a phase ii trial for early sepsis and septic shock. noxxon described a series of l-rna aptamers (spiegelmer; nox-d to nox-d ) that block c a; these agents were effective in models of sepsis and transplant rejection , , but have not yet progressed to humans. all of the above suggests that pharmacological blockade of c a and/or c a activities might be relatively straightforward; however, such an approach will only work in those situations in which these molecules are the major drivers of pathology, such as in neutrophilmediated disorders including anca-vasculitis, chronic obstructive pulmonary disease (copd) and lupus. targeting c . as described above, c is activated to create two moieties with distinct activities. the small fragment, c a, is a powerful chemoattractant, and therapeutic blockade of its activities is described above. the larger fragment, c b, forms the foundation of the mac. assembly of the mac involves the nonenzymatic aggregation of the five terminal pathway components (c b and c -c ) into a pore that inserts into and through the cell membrane. c b, while still attached to the convertase, binds sequentially to c and c ; the c b complex is released from the convertase and binds to the membrane via a labile hydrophobic site. c and multiple copies of c are recruited in turn to build the pore. mac activity is naturally regulated by inefficiencies built into the assembly system and by regulators policing the membrane to block complexes that reach the surface. the hydrophobic binding site in c b is intrinsically unstable, decaying by interaction with water in a fraction of a second. additionally, there are several proteins in the plasma that bind to the mac to further restrict its capacity to bind to the membrane: clusterin and vitronectin (also known as s protein) are multifunctional proteins that, among their many roles, inhibit mac assembly in this way, and c is an efficient inhibitor if it binds to c b in the fluid phase. once bound to the membrane, c b is protected from these plasma inhibitors and recruits c and c to form the mac. the last line of defence is the membrane regulator cd , a small glycosylphosphatidylinositol (gpi)anchored protein that binds to the forming complex and prevents the completion of the mac pore. opportunities for therapeutic inhibition are legion. the first obvious target is the first mac component, c b. cutting off the supply of c b by targeting the c and c convertases will inhibit mac production; alternatively, c itself can be targeted to render it resistant to cleavage, which is the mode of action of the c monoclonal eculizumab, the most successful complement-specific drug to date. the success of eculizumab (box ) has triggered interest in other ways of preventing c cleavage. novartis have taken a c -specific mab, lfg , into phase ii trials of intraocular therapy in advanced amd (completed but unpublished) and, more recently, in choroiditis and panuveitis . in august , novartis announced phase i trials to assess the safety and efficacy of lfg in patients with pnh (nct ). a recombinant human c -specific mab fragment, mubodina (adienne pharma & biotech), that prevents cleavage of c has undergone early-stage trials in ahus and other renal diseases, although no development has been reported recently. ornithodoros moubata complement inhibitor (omci), a small ( kda) lipocalin present in the saliva of soft ticks, is a potent c inhibitor that probably protects the tick from complement in its blood meal . recombinant omci (coversin; volution immuno pharmaceuticals) blocked c in numerous species and inhibited disease in rodent models , . the drug bound c at a site remote from the convertase cleavage site, suggesting that it functioned by either blocking the interaction of c with the convertase or preventing conformational change essential for cleavage. coversin completed phase i trials in and is in development for pnh and other mac-driven diseases. sobi is a c -blocker that is based on affibody technology and developed by swedish orphan biovitrum. inclusion of an albumin-binding motif extends the plasma halflife of sobi . this small (~ kda) protein, derived using phage display, avidly binds to c and prevents cleavage; the simple structure of sobi makes synthesis easy and inexpensive. sobi is currently in a phase i trial, which was recently listed as terminated (nct ). various non-biologic approaches have also been developed and are either soon to be tested or already in clinical trials. ophthotech have developed a c -specific aptamer (arc , also known as zimura) that, when given intraocularly in a phase iia study in combination with an antibody targeting vascular endothelial growth factor (vegf), improved outcomes in patients with wet amd. the rnai approach taken by alnylam is described above; at the american society of haematology (ash) meeting, alnylam reported effective knockdown in non-human primates using aln-cc (see further information), and phase i trials are now underway with pnh as the primary indication. interim phase i data demonstrate substantial knock-down of c in singleascending-dose studies in humans (as announced in a recent alnylam press release). rapharma have developed a cyclic peptide that binds to c and prevents cleavage and activation; promising preclinical data were reported at ash (see further information), and phase i studies are proposed for the near future. as with many of the c -targeting agents reported here, the primary indication is likely to be pnh. the crowded 'landscape' in the c space (fig. ) clearly illustrates the activity generated by the success of eculizumab and the desire to block the activities of this key component in the cascade. targeting the terminal pathway: beyond c . blocking c cleavage by any of the above strategies will stop the production of c a as well as the mac and this may have important consequences if continued long term. to specifically target the mac, agents with activities downstream of c cleavage are needed. agents that mimic the ability of the natural inhibitors clusterin and vitronectin (and c ) to compete for the membrane binding site on c b might further reduce the efficiency of membrane attack, whereas mimics of cd that block the final steps of mac assembly might be effective blockers of lytic pore formation. soluble recombinant cd (scd ) has been generated and shown to inhibit lysis in vitro; however, in vivo, scd binds to plasma proteins, markedly reducing its inhibitory effects . recombinant cd , attached to the same membranebinding motif as in mirococept, has been generated and shown to protect pnh erythrocytes from lysis ex vivo and ameliorate arthritis development in a rat model when administered intra-articularly at the time of disease induction , . alternatively, any one of the terminal pathway components, each of which is essential for figure | progress of complement therapeutics towards clinical use. the figure graphically illustrates a current 'snapshot' of the different areas of complement that are being targeted and the progress of the drugs -both marketed and en route to approval. it is a rapidly moving field and, inevitably, some compounds will progress, others will fail and new drugs will emerge to take their positions in the landscape in the coming months and years. one certainty is that this already crowded field will become much more so in the near future. mac formation, could be targeted. regenesance bv has targeted c with both blocking c -specific mab and antisense approaches, and the latter was effective in animal models . regenesance also described a smallmolecule inhibitor of c in development for guillain-barré syndrome, but further development was not reported. a recent publication describes a small molecule (aurin tricarboxylic acid) that inhibits mac assembly by blocking c binding; the agent prevented the lysis of pnh erythrocytes by serum ex vivo . localizing therapeutics to sites of pathology inhibition of systemic complement activity is a major concern with most anti-complement drugs as it renders the recipient susceptible to infection. delivery of the drug specifically to the site of disease will not only reduce risk but also enhance efficacy. at its simplest, local delivery could mean direct administration to the site of disease -into the inflamed joint, eye or brain, for example. more interesting are the many modifications that have been made to ensure the homing of an agent where needed. modification of scr to favour its accumulation on inflamed endothelia (tp ) increased efficacy in models , and addition of a membrane-binding tag to a truncated form of scr (mirococept) converted an inactive fragment into a viable therapeutic , . similarly, a recombinant truncated fh comprising the minimal active site (short consensus repeats - ) is essentially inactive in vivo, but adding in the fh membrane-binding short consensus repeats creates a powerful inhibitor (amy- ) , . localising anti-complement agents to sites of complement activation has attracted abundant attention. homing of the drug not only avoids systemic inhibition but also enhances efficacy by concentrating the drug in one site and by placing active sites in the correct orientation to inactivate target enzymes. these approaches tend to focus on the homing of agents that have regulatory functions, such as decay acceleration or cofactor activity, rather than the blocking activities typical of antibodies. it is important that the drug that is delivered to the site is able to 'recycle' and release its target once inactivated, otherwise the target surface will rapidly become saturated with useless agent. more than years ago, we described a prodrug approach whereby recombinant forms of natural regulators were generated as fc fusion proteins in which regulatory activity was absent because of steric hindrance. engineered enzyme-cleavage sites enabled the release of active agent by matrix metalloproteinases and related enzymes that are abundant at inflammatory sites . similar strategies have proved successful in anticancer therapy . complement fragments bind covalently to tissues at sites of activation and provide a potential ligand for localizing an anti-complement drug where needed. a hybrid molecule comprising the rodent c convertase regulator crry coupled to the c dg-binding short consensus repeats of cr provided proof-of-principle of this approach; the hybrid molecule localized to sites of pathology and was -fold more active compared to crry alone in mouse models of ischaemia-reperfusion . the same group also described a cr -fh hybrid protein that was effective in several murine models ; this provided the impetus to create a human cr -fh hybrid, tt , which is currently under development by alexion for therapy of pnh (alxn and alxn ) , . potential advantages of this agent over eculizumab include a reduced risk of infection, a lower dose requirement and protection of pnh erythrocytes from c deposition and the resultant extravascular haemolysis. progress towards the clinic has been slow, perhaps because of commercial rather than scientific considerations. a phase i clinical trial was initiated (nct ), although no data have been reported. looking to the future although the many roles of complement in driving pathology in diverse diseases have long been recognized, the capacity to treat these diseases by controlling complement activation has lagged behind. today, we boast just two anti-complement drugs that are being marketed, approved only in rare orphan diseases (hae, pnh and ahus), which is a rather disappointing outcome given the investments made by researchers and companies. however, the future looks much brighter; new therapeutics are entering the clinic, targeting different parts of the complement system, with different half-lives, different routes of administration and each with its own range of activities and side effects (fig. ) . importantly, although the emphasis on orphan applications was pivotal in getting the first agents to the market, many of the new agents target common acute and chronic inflammatory diseases, for example amd, arthritis and cardiovascular disease. hurdles to the commercialization of complement therapeutics still exist, including cost, routes of administration and infection risk. short-duration complement inhibition, particularly in a controlled clinical environment, carries the least risk, and anti-complement drugs will probably first become mainstream for acute indications, such as reperfusion injuries. for chronic conditions, cost, ease of administration and safety may be substantial issues. cost will become less of an issue as increased competition (the emergence of small-molecule inhibitors) and increased use should, theoretically, bring prices down rapidly. the experience of chronic therapy with agents that block complement is still relatively short-term, and it is possible that the elimination of molecules, such as c a, may affect other activities of this multi-functional molecule, such as modulation of adaptive immunity, in the long term . however, eculizumab was approved for use in pnh in and for ahus in and, although the long-term consequences of c blockade remain uncertain, it is encouraging that after almost a decade of use no substantial adverse effects have been noted in carefully monitored and compliant recipients. frequent intravenous administration is not clinically acceptable for chronic use, and so agents will need either very long half-lives or to be delivered by routes not requiring medical intervention (for example, subcutaneous or oral). it may be that a therapy could be titrated to give partial inhibition of complement, sufficient to inhibit disease but sparing key protective functions. alternatively, a drug may be engineered to deliver complement inhibition where it is needed at the site of disease. inevitably, different diseases will require different approaches to complement inhibition -some may be entirely mac-driven, others c a-dependent, and most involve a complicated interplay between the various complement products and other inflammatory mediators. selecting the right agent or agents will be crucial to success in trials, and here a comprehensive understanding of the mechanisms by which complement drives pathology in the target disease is a sine qua non. biomarkers that reflect complement activation in fluids and tissues can inform the selection of the right drug for the right patient, demonstrating engagement of the target and efficacy of the drug, and monitoring response to therapy. a lack of good biomarkers often handicaps early stage trials and simple readouts of effectiveness are crucial. in some cases this is obvious; for example, blockade of mac formation can be demonstrated in serum through simple haemolytic assays. when activation fragments such as c a, c a, c d, bb and terminal complement complex (tcc) are present in the disease their levels can be monitored to demonstrate response to therapy and confirm target engagement. in some diseases, such as pnh, there is an obvious clinical readout of target engagement -eculizumab treatment prevents intravascular haemolysis in pnh. however, for diseases restricted to specific sites, for example, the retina, central nervous system or kidney glomerulus, plasma complement biomarkers may not reflect the response to therapy and are poor tools for assessing target engagement. there is thus a need for effective, non-invasive methods to detect complement activation and deposition within affected tissues without resorting to biopsy. methods for imaging complement activation in vivo may fill this need; agents comprising the c -binding domain of cr have been used to detect c fragments deposited in the kidney by magnetic resonance imaging in mouse models . antibodies against c dg have also been developed as imaging agents and shown to identify retinal lesions in a mouse model of amd . further development of the capacity to image complement activation in vivo will inform the conduct of future clinical trials by providing measurable outcomes and early proof of target engagement. membrane complement regulatory proteins are complement deficiencies really rare? overview on prevalence, clinical importance and modern diagnostic approach complement dysregulation and disease: from genes and proteins to diagnostics and drugs a comprehensive and current review of the roles of complement in renal disease paroxysmal nocturnal hemoglobinuria and other complement-mediated hematological disorders paroxysmal nocturnal hemoglobinuria complement activation and inhibition in experimental models of arthritis age-related macular degenerationemerging pathogenetic and therapeutic concepts therapeutic targeting of the complement system in age-related macular degeneration: a review complement and immune complexes complement deficiencies and systemic lupus erythematosus role of complement in induction of antibody production in vivo. effect of cobra factor and other c reactive agents on thymus-dependent and thymus-independent antibody responses regulation of humoral immunity by complement complement modulation of t cell immune responses during homeostasis and disease complement -a key system for immune surveillance and homeostasis complement deficiencies in humans and animals: links to autoimmunity complement deficiency states and associated infections suppression of drusen formation by compstatin, a peptide inhibitor of complement c activation, on cynomolgus monkey with early-onset macular degeneration a phase ia dose-escalation study of the anti-factor d monoclonal antibody fragment fcfd s in patients with geographic atrophy complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for the complement receptor / factor h fusion protein tt protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and c fragment a clear description of a novel targeted complement inhibitor with potential for improved therapy determination of the half-life of c in patients and its relation to the presence of c breakdown products and/or circulating immune complexes discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria a useful overview of the developmental history of eculizumab use of eculizumab for atypical haemolytic uraemic syndrome and c glomerulopathies pharmacokinetics and concentrationeffect relationship of adalimumab in rheumatoid arthritis metabolism of the fifth component of complement, and its relation to metabolism of the third component, in patients with complement activation dynamics of complement activation in ahus and how to monitor eculizumab therapy structural and functional analysis of a c b specific antibody that selectively inhibits the alternative pathway of complement a humanized antibody that regulates the alternative pathway convertase: potential for therapy of renal disease associated with nephritic factors metabolism of complement factor d in renal failure complement inhibition in cynomolgus monkeys by anti-factor d antigen-binding fragment for the treatment of an advanced form of dry age-related macular degeneration emerging pegylated drugs fc based cytokines: prospects for engineering superior therapeutics phase i trial of the recombinant soluble complement receptor in acute lung injury and acute respiratory distress syndrome an early clinical trial of the first recombinant complement regulator soluble human complement receptor limits ischemic damage in cardiac surgery patients at high risk requiring cardiopulmonary bypass the role of complement in the early immune response to transplantation serine protease inhibitor nafamostat given before reperfusion reduces inflammatory myocardial injury by complement and neutrophil inhibition inhibition of the serine proteases of the complement system tnt , an inhibitor of the serine protease c s, prevents complement activation induced by cold agglutinins a study targeting classical pathway activation as a potential therapy for cold agglutinin disease structure-based inhibition of protein-protein interactions structure-activity relationships for substrate-based inhibitors of human complement factor b structures of c b in complex with factors b and d give insight into complement convertase formation selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (masp)- and - : significant contribution of masp to lectin pathway activation species dependence 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mouse models of neuromyelitis optica specific inhibition of the classical complement pathway by c q binding peptides a novel peptide inhibitor of classical and lectin complement activation including abo incompatibility peptide inhibitor of complement c , a novel suppressor of classical pathway activation: mechanistic studies and clinical potential replacement therapy in hereditary angioedema: successful treatment of acute episodes of angioedema with partly purified c inhibitor therapeutic management of hereditary angioedema due to c inhibitor deficiency genetically determined heterogeneity of the c esterase inhibitor in patients with hereditary angioneurotic edema c esterase inhibitor concentrate in hereditary angioedema attacks -final results of the i.m.p.a.c.t. study long-term prophylaxis with c inhibitor (c inh) concentrate in patients with recurrent angioedema caused by hereditary and acquired c inhibitor deficiency escalating doses of c esterase inhibitor (cinryze) for prophylaxis in patients with hereditary angioedema an evidence based therapeutic approach to hereditary and acquired angioedema c inhibitor in patients with severe sepsis and septic shock: beneficial effect on renal dysfunction c esterase inhibitor infusion increases survival rates for patients with sepsis rd the effect of c inhibitor on myocardial ischemia and reperfusion injury the effect of c inhibitor on intestinal ischemia and reperfusion injury design and synthesis of biphenylsulphonyl thiophene carbxamide inhibitors of the complement component c s translational success stories: development of direct thrombin inhibitors monospecific inhibitors show that both mannan-binding lectin-associated serine protease (masp ) and - are essential for lectin pathway activation and reveal structural plasticity of masp antibody-mediates inhibition of human c s and the classical complement pathway the modulation of the alternative pathway of complement in c deficient human serum by changes in concentration of the component and control proteins hemolytic uremic syndrome: a factor h mutation (e stop) causes defective complement control at the surface of endothelial cells treatment with human complement factor h rapidly reverses renal complement deposition in factor h deficient mice intravitreal human complement factor h in a rat model of laser-induced choroidal neovascularisation age-related macular degeneration and modification of systemic complement factor h production through liver transplantation rational engineering of a minimized immune inhibitor with unique tripletargeting properties an engineered construct combining complement regulatory and surfacerecognition domains represents a minimal-size functional factor h c binding protein deficiency in a patient with atypical behçet's disease c b binding protein (c bp) inhibits development of experimental arthritis in mice recurrent infections in partial complement factor i deficiency: evaluation of three generations of a brazilian family three cases of factor i deficiency: the effect of treatment with plasma complotype affects the extent of downregulation by factor i of the c b feedback cycle in vitro properdin in complement activation and tissue injury low-dose recombinant properdin provides substantial protection against streptococcus pneumoniae and neisseria meningitidis infection soluble human complement receptor type : in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis tp is superior to tp in reducing ischemia/reperfusion injury in rat lung grafts therapeutic efficacy of a novel membrane-targeted complement regulator in antigeninduced arthritis in the rat attenuation of myocardial reperfusion injury in pigs by mirococept, a membrane-targeted complement inhibitor derived from human cr deficient alternative complement pathway activation due to factor d deficiency by novel mutations in the complement factor d gene in a family with meningococcal infections inhibiting alternative pathway complement activation by targeting the factor d exosite structure of , dichloro isocoumarin-inhibited factor d a lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space inhibitors of factor d may provide a treatment for age-related macular degeneration therapeutic inhibition of the alternative complement pathway attenuates chronic eae inhibition of the alternative complement pathway by antisense oligonucleotides targeting complement factor b improves lupus nephritis in mice inhibition of complement by recombinant sh-crit-ed analogues decomplementation par un facteur extrait du venin de cobra the effects of a cobra venom factor and ethyl palmitate on the prolongation of survival of heterologous erythrocytes intravascular activation of complement and acute lung injury. dependency on neutrophils and toxic oxygen metabolites humanized cobra venom factor: activity and therapeutic efficacy in preclinical disease models gene therapy in age-related macular degeneration and hereditary macular disorders inhibition of the membrane attack complex of the complement system reduces secondary neuroaxonal loss and promotes neurologic recovery after traumatic brain injury in mice the role of the anaphylatoxins in health and disease distinct roles of the anaphylatoxins c a and c a in dendritic cell-mediated allergic asthma chemokine receptors as therapeutic targets: why aren't there more drugs? therapeutic activity of c a receptor antagonists in a rat model of neurodegeneration blocking the receptor for c a in patients with rheumatoid arthritis does not reduce synovial inflammation the receptor for complement component c a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization a novel c a neutralizing mirrorimage (l-)aptamer prevents organ failure and improves survival in experimental sepsis targeting complement component a promotes vascular integrity and limits airway remodeling advances in the management of macular degeneration. f prime rep complement inhibitor of c activation from the soft tick ornithodoros moubata structure of and influence of a tick complement inhibitor on human complement component in vivo characterization and therapeutic efficacy of a c specific inhibitor from the soft tick ornithodoros moubata high-density lipoproteins can act as carriers of glycophosphoinositol lipid anchored cd in human plasma generation of a recombinant, membrane-targeted form of the complement regulator cd ; activity in vitro and in vivo protection of erythrocytes from human complement-mediated lysis by membrane-targeted recombinant soluble cd : a new approach to pnh therapy aurin tricarboxylic acid protects against red blood cell hemolysis in patients with paroxysmal nocturnal hemoglobinemia generation of anti-complement 'prodrugs': cleavable reagents for specific delivery of complement regulators to disease sites antibody-directed enzyme prodrug therapy (adept) for cancer targeted complement inhibition by c d recognition ameliorates tissue injury without apparent increase in susceptibility to infection targeted complement inhibitors protect against posttransplant cardiac ischemia and reperfusion injury and reveal an important role for the alternative pathway of complement activation design and development of tt , a novel c d targeted c /c convertase inhibitor for treatment of human complement alternative pathway-mediated diseases describe novel reagents that enable imaging of complement activation in vivo noninvasive detection of complement activation through radiologic imaging the complotype: dictating risk for inflammation and infection describes the development of the concept of the complotype as predictor of disease risk common polymorphisms in c , factor b, and factor h collaborate to determine systemic complement activity and disease risk a common haplotype in the complement regulatory gene factor h (hf /cfh) predisposes individuals to age-related macular degeneration complement factor h genetic variant and age-related macular degeneration: effect size, modifiers and relationship to disease subtype variation in factor b (bf) and complement component (c ) genes is associated with age-related macular degeneration complement c variant and the risk of age-related macular degeneration age-related macular degeneration atypical hemolytic uremic syndrome mutations in factor h reduce binding affinity to c b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome association of factor h autoantibodies with deletions of cfhr , cfhr , cfhr , and with mutations in cfh, cfi, cd , and c in patients with atypical hemolytic uremic syndrome important description of link between anti fh autoantibodies, deletions in fh-related (fhr) proteins and ahus generation of a monoclonal antibody to mouse c ; application in an elisa assay for detection of anti c antibodies anti c monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease inhibition of complement activity by humanized anti c antibody and single-chain fv adis international limited eculizumab: g . , h g . , long-acting anti c monoclonal antibody g , long-acting anti c monoclonal antibody g . eculizumab (alexion) effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria multicenter phase study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal haemoglobinuria terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome a molecular insight into complement inhibition by the staphylococcal complement inhibitor family genetic variants in c and poor response to eculizumab protein-protein interaction inhibitors get into the groove structure of complement fragment c b-factor h and implications for host protection by complement regulators inhibition of complement by a c binding peptide isolated from a phage-displayed random peptide library new analogs of the clinical complement inhibitor compstatin with subnanomolar affinity and enhanced pharmacokinetic properties compstatin: a c targeted complement inhibitor reaching its prime for bedside intervention update on current and future novel therapies for dry age-related macular degeneration the amplification loop of the complement pathways pharmacokinetics of plasmaderived c esterase inhibitor after subcutaneous versus intravenous administration in subjects with mild or moderate hereditary angioedema: the passion study a phase i study of recombinant human c inhibitor in asymptomatic patients with hereditary angioedema recombinant human c esterase inhibitor relieves symptoms of hereditary angioedema attacks: phase , randomized, placebo-controlled trial spectrum of complement-mediated thrombotic microangiopathies: pathogenetic insights identifying novel treatment approaches eculizumab in aqp igg-positive relapsing neuromyelitis optica spectrum disorders: an open-label pilot study complement in multiple sclerosis: its role in disease and potential as a biomarker systemic complement profiling in multiple sclerosis as a biomarker of disease state the role of complement and complement regulators in mediating motor nerve terminal injury in murine models of guillain-barré syndrome emerging drugs for guillain-barré syndrome the membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice a randomized, double-blind, placebo-controlled phase ii study of eculizumab in patients with refractory generalized myasthenia gravis dramatic effects of eculizumab in a child with diffuse proliferative lupus nephritis resistant to conventional therapy the genetics and immunobiology of iga nephropathy eculizumab treatment for rescue of renal function in iga nephropathy rheumatoid arthritis and the complement system association of genetic variants of mannan-binding (mbl) lectin gene, mbl levels and function in ulcerative colitis and crohn's disease regulation of the alternative pathway of complement modulates injury and immunity in a chronic model of dss-induced colitis long-term response of refractory primary cold agglutinin disease to eculizumab therapy complement and cutaneous autoimmune blistering diseases complement and the antiphospholipid syndrome eculizumab prevents recurrent antiphospholipid antibody syndrome and enables successful renal transplantation association of c and cfb polymorphisms with anterior uveitis pathogenesis of anca-associated vasculitis: new possibilities for intervention the membrane attack complex of complement drives the progression of atherosclerosis in apolipoprotein e knockout mice the complement system in human cardiometabolic disease label-free quantitative proteomic analysis reveals dysfunction of complement pathway in peripheral blood of schizophrenia patients: evidence for the immune hypothesis of schizophrenia the complement control-related genes csmd and csmd associate to schizophrenia a complex role for complement in allergic asthma complement components as potential therapeutic targets for asthma treatment complement factors c a, c a, and c a in chronic obstructive pulmonary disease and asthma role of complement in a murine model of peanut-induced anaphylaxis peanuts can contribute to anaphylactic shock by activating complement role of complement in multiorgan failure double blockade of cd and complement c abolishes the cytokine storm and improves morbidity and survival in polymicrobial sepsis in mice experimental cerebral malaria: the murine model provides crucial insight into the role of complement imunomodulation in psoriatic arthritis: focus on cellular and molecular pathways complement system in psoriatic arthritis: a useful marker in response prediction and monitoring of anti-tnf treatment potential therapeutic targets for idiopathic inflammatory myopathies immunosuppressant and immunomodulatory treatment for dermatomyositis and polymyositis identification of a central role for complement in osteoarthritis a thorough analysis in mouse models implicating complement in degenerative joint disease brief report: carboxypeptidase b serves as a protective mediator in osteoarthritis innate immunity and the etiology of late-onset alzheimer's disease inflammation in alzheimer disease a brief review of the basic science and clinical literature this paper describes a novel role of complement in synaptic pruning relevant to development and disease diabetic angiopathy, the complement system and the tumor necrosis factor superfamily complement involvement in neovascular ocular diseases soluble form of membrane attack complex independently predicts mortality and cardiovascular events in patients with st elevation myocardial infarction treated with primary percutaneous coronary intervention targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia c inhibitor protects from brain ischemia-reperfusion injury by combined antiinflammatory and antithrombotic mechanisms the role of complement in trauma and fracture healing the complement system in ischemiareperfusion injuries targeted complement inhibition as a promising strategy for preventing inflammatory complications in hemodialysis therapeutic c inhibitor cp abrogates complement activation induced by modern hemodialysis filters stec-hus, atypical hus and ttp are all diseases of complement activation systemic complement inhibition with eculizumab for geographic atrophy in age-related macular degeneration: the complete study terminal complement inhibition decreases antibody-mediated rejection in sensitized renal transplant recipients epirus biopharmaceuticals: www.epirusbiopharma.com ifx- omeros press release the authors thank k. miller of glaxosmithkline, for her thoughtful review and discussion of the manuscript. the authors declare competing interests: see web version for details. key: cord- - dbqu r authors: al-helou, georges; anklesaria, zafia; kohlwes, jeffrey; ahari, jalil; dhaliwal, gurpreet title: when the illness goes off script—an exercise in clinical reasoning date: - - journal: j gen intern med doi: . /s - - - sha: doc_id: cord_uid: dbqu r nan i n this series, a clinician extemporaneously discusses the diagnostic approach (regular text) to sequentially presented clinical information (bold). additional commentary on the diagnostic reasoning process (italics) is integrated throughout the discussion. a year-old man with hypothyroidism presented to the emergency department with fever, malaise, and a nonproductive cough for days. he denied headaches, nausea, abdominal, back, or chest pain. his only medication was levothyroxine. he lived in california, but was visiting the northeast united states when he became ill. he denied any recent travel outside the country. he denied tobacco use and illicit drug use; he occasionally drank alcohol. he was in a monogamous heterosexual relationship and was employed in the film industry. the patient is a middle-aged man with hypothyroidism presenting with acute fever and cough. acute fever and cough are usually caused by a viral or bacterial upper or lower respiratory tract infection. his recent travel to the northeast united states brings to mind tickborne infections, although most such pathogens do not cause pneumonitis. cough can be seen with anaplasmosis, erlichiosis, babesiosis, and the pneumonic form of tularemia. his residence in california puts him at risk for pulmonary coccidiomycosis. illness scripts are an adaptation from cognitive psychology's script theory. in script theory, individuals use their prior knowledge to contextualize new experiences. the clinician triggers the illness scripts of familiar diseases such as viral or bacterial respiratory infections and tick-borne infections to compare to his problem representation. he uses geographic clues to consider less fitting conditions, but will likely only give them serious thought if the emerging data set deviates from his more common scripts. in the emergency department, the temperature was . degrees celsius, heart rate was beats per minute, blood pressure was / mmhg, respiratory rate was breaths per minute, and his oxygen saturation was % on liters of nasal cannula oxygen. he was in moderate distress. there were several cm firm, mobile, non-tender cervical lymph nodes and a cm left axillary lymph node. he had crackles in the bilateral mid-lung fields. heart, abdominal, and neurologic examinations were normal. there was a diffuse non-blanching maculo-papular rash on the trunk with petechiae ( fig. ) . he has signs of systemic inflammation which may arise from infection, autoimmunity, or malignancy. scattered lymphadenopathy can be explained by all three categories of disease, but sepsis is the acute concern. his cough, bilateral crackles, and hypoxia signal a diffuse parenchymal lung process; most commonly this will be a bacterial pneumonia. fever and rash is often caused by viral exanthems such as epstein barr virus (ebv), cytomegalovirus (cmv), and acute human immunodeficiency virus (hiv); while each causes lymphadenopathy and fever, primary pulmonary disease is unusual. acute influenza frequently leads to pneumonia, but rash is not characteristic. bacterial infections like endocarditis, meningococcemia, and rocky mountain spotted fever all warrant early consideration with fever and a diffuse petechial rash, although he does not have any epidemiologic risk factors for these conditions. a bacterial pneumonia with bacteremia could cause cutaneous vasculitis, but scattered lymphadenopathy is not part of that picture. while disseminated endemic fungal infections can cause fever, rash, lymphadenopathy, and pulmonary disease, an acute presentation with widespread skin lesions would be more likely in an immunocompromised patient. the rash does not have the typical appearance or pain of stevens-johnson syndrome/toxic epidermal necrolysis, but environmental and medication exposures should be queried nonetheless. sarcoidosis affects each of the involved organs in this case, but is usually more indolent. vasculitis and other autoimmune conditions like systemic lupus erythematosus (sle) and antineutrophil cytoplasmic antibody (anca)-associated diseases are possible but less likely. the same could be said for malignancy, which rarely presents with this degree of acuity, save for aggressive hematologic cancers. using sirs physiology as a starting point, the discussant builds an analytic framework around three categories (infection, autoimmunity, malignancy). the clinician outlines a broad differential diagnosis after an early focus on lifethreatening illnesses (pneumosepsis). this broad framework creates the scaffolding to access appropriate illness scripts as the case unfolds. rapid improvement with antibiotics will support the common disease (e.g., pneumosepsis). if the patient gets sicker, the clinician has already considered other diagnostic possibilities, which will facilitate the evaluation of a broad range of potential illness scripts. laboratory testing revealed a white blood cell count of /μl ( % neutrophils, % bands, % lymphocytes, and % monocytes), hemoglobin of . g/dl, and platelets of , /μl. sodium was mmol/l, blood urea nitrogen mg/dl, and creatinine . mg/dl. the rest of the electrolytes were normal. international normalized ratio (inr) was . , partial thromboplastin time (ptt) . seconds, albumin . g/dl, total protein . g/dl, total bilirubin . mg/dl, direct bilirubin . mg/dl, aspartate transaminase u/l, alanine transaminase u/l, alkaline phosphatase u/l. arterial blood gas showed ph of . , partial pressure of co of mmhg, and partial pressure of o of mmhg on l of oxygen by nasal cannula. chest x-ray showed bilateral hilar fullness with clear lung fields. the combination of thrombocytopenia and coagulopathy points to either liver dysfunction or disseminated intravascular coagulation (dic). the patient has liver function test abnormalities and hypoalbuminemia, but lacks risk factors or physical exam findings of chronic liver disease. the low albumin is likely a negative acute phase reactant phenomenon. monocytosis is non-specific. it can be a prominent feature of myeloid disorders, particularly chronic myelomonocytic leukemia. the rash, modest hepatitis, and pancytopenia could signal early leukemic infiltration of the skin, liver, and bone marrow. acute promyelocytic leukemia is the form of leukemia most associated with dic. the bilateral hilar fullness implies hilar lymphadenopathy, which can be seen in sarcoidosis, pulmonary infections such as tuberculosis or fungal pneumonia, and malignancy (including lymphoma). he has clear lung fields on chest x-ray with marked hypoxia, which always raises the question of pulmonary embolus; however, the bilateral mid-lung crackles suggests a parenchymal disorder that has yet to appear on imaging studies. the summary of the patient's acute problems includes fever, hypotension, hypoxia, lymphadenopathy, disseminated rash, leukopenia, thrombocytopenia, acute kidney injury, mild hepatitis, and probable dic. sepsis remains the leading concern, but non-bacterial infections, acute hematologic malignancy, and sarcoidosis are important contenders. as the amount of data increases, the clinician triggers many more illness scripts. none are a perfect match, so he discusses the pros and cons of each possibility to prioritize the differential diagnosis. typical clinical reasoning is often built upon the problem representation (pr), a one-line synopsis of the important characteristics of a case. this is followed by accessing illness scripts that match the important information from the pr. in this complex case, the clinician reverses the standard process by accessing many illness scripts and then summarizes the case again to refocus the differential diagnosis. restatement of the pr is a useful strategy in complex cases to focus on the key clinical elements and hone the differential diagnosis as new information becomes available. computed tomography (ct) of the chest revealed prominent mediastinal lymph nodes, right middle lobe bronchial wall thickening, and nonspecific nodular interstitial ground-glass opacities in both bases (fig. ) . the intra-thoracic lymphadenopathy, bronchial wall thickening, and interstitial lung disease is compatible with sarcoidosis. although a virus is more likely than bacteria to cause a rash, hepatitis, or pancytopenia, these are not common features of the common lower respiratory tract viral pathogens. lymphangitic spread of a lymphoma, leukemia, or carcinoma is plausible. lymph node biopsy (axillary or intra-thoracic) will be informative. intravenous fluid, vancomycin, meropenem, doxycycline, and azithromycin were administered. the patient remained febrile and developed worsening respiratory failure requiring mechanical ventilation. a repeat chest x-ray showed bilateral diffuse infiltrates consistent with acute respiratory distress syndrome (ards). norepinephrine, epinephrine, and vasopressin were administered for hypotension. he became anuric and continuous veno-venous hemodialysis was initiated. he began to bleed from his venipuncture sites and developed ischemia of his fingers and toes. labs revealed worsening thrombocytopenia (platelets , /ul) and neutropenia (absolute neutrophil count of /ul). his inr was . , ptt was seconds, and fibrinogen was mg/dl (reference range: - mg/dl). blood, sputum, and urine cultures grew no organisms. a respiratory viral panel, hiv antibody test, and urine antigen tests for legionella and streptococcus were negative. the rapid deterioration of his hemodynamics, respiratory status, kidney function, bone marrow, and coagulation system indicates a severe systemic inflammatory response. he is appropriately being treated for sepsis, but extensive laboratory data does not implicate a pathogen. with travel to the northeast us, i would still consider tick-borne pathogens that can occasionally cause fulminant sepsis, including anaplasmosis, erlichiosis, and babesiosis. the first two should have been treated by the doxycycline. babesiosis is diagnosed by microscopy, serology, or polymerase chain reaction, but is less likely given the normal hemoglobin. the lack of documented infection warrants examination of non-infectious processes that mimic severe sepsis. rapidly progressive hematologic malignancies such as acute leukemia and lymphoma (including cutaneous t cell lymphomas) can present with fevers and multiorgan failure. aggressive forms of mastocytosis may be characterized by widespread organ invasion, cutaneous involvement, and hypotension, but the characteristic flushing and gastrointestinal complaints are absent. in the autoimmune spectrum, sarcoidosis is less likely because of acuity and extent of multiorgan failure. however, those same features make catastrophic antiphospholipid syndrome and anca-associated disorders plausible. although his deterioration is likely a progression of the underlying disease, we must also consider if treatments might be responsible. for instance, could his shock be an anaphylactic reaction to one of his multiple antibiotics? as the patient is deteriorating, the clinician returns to the less common conditions he considered earlier in the context of sirs. this represents clinical bpre-planning^and enables the clinician to take the next steps quickly when initial therapies fail. as his clinical thinking evolved from pneumosepsis to pseudosepsis, he considers rarer diseases in an efficient yet constrained manner that focuses testing rather than using a shotgun approach. blood smears showed rare schistocytes but no evidence of malaria, ehrlichia, or babesiosis. an ehrlichia polymerase chain reaction and a rickettsia antibody panel were negative. a serum lactate dehydrogenase (ldh) was u/l, and serum ferritin was ng/ml. a ct of the abdomen revealed mesenteric and retroperitoneal lymphadenopathy and mild splenomegaly. high dose vasopressor medications were continued, and stress dose steroids were added empirically due to refractory hypotension. he was subsequently weaned off vasopressors but remained intubated and febrile. his rash spontaneously resolved on icu day . this information has not radically shifted the differential diagnosis or the relevant categories of disease. it is hard to know if steroids can be credited for the restoration of hemodynamic stability given simultaneous treatment with antibiotics. the problem representation is now fever, hypotension (resolved), diffuse pulmonary infiltrates, widespread lymphadenopathy, disseminated rash (resolved), leukopenia, thrombocytopenia, dic, severe aki, and mild hepatitis with no evident infection. in the absence of evidence of infection or autoimmunity, malignancy becomes the leading concern. acute promyelocytic leukemia (apl) is an important consideration with his dic, but the absence of peripheral blasts is unusual. given the widespread lymphadenopathy, elevated ldh, and mild splenomegaly, an aggressive lymphoma is probable. metastatic carcinoma of unknown primary could also cause lymphadenopathy and invade multiple organs. hemophagocytic lymphohistiocytosis (hlh) is characterized by multi-organ involvement, cytopenias, splenomegaly, and fevers. since these findings are seen in many other conditions, testing for criterion-based data that are more specific to hlh, including elevated il r level, hypertriglyceridemia, low nk activity, or a bone marrow biopsy demonstrating hemophagocytosis, is often necessary to make the diagnosis. hlh in adults is usually secondary to infection, autoimmunity, or malignancy. often the distinction between aggressive lymphoma and hlh is blurred, but i suspect his condition resides somewhere along that spectrum. the patient fulfilled five diagnostic criteria for hlh: fevers, splenomegaly, cytopenias, hyperferritinemia, and hypofibrinogenemia or hypertriglyceridemia ( mg/dl); the interleukin- receptor level (soluble cd ) was also elevated at , pg/ml (normal range - pg/ml). (table ) the diffuse lymphadenopathy raised suspicion for autoimmunity or malignancy as an underlying etiology of his hlh. an axillary lymph node biopsy showed cells that were markedly positive for cd a and s staining (fig. ) , consistent with langerhans cell histiocytosis (lch). there was no evidence of a lymphoma or hemophagocytosis, and cultures were negative. there was no evidence of histiocytosis elsewhere in the body; there were no lytic bony lesions, pulmonary nodules or cysts, or findings of central nervous system disease (e.g., diabetes insipidus). he was diagnosed with lymphadenopathic lch which precipitated an inflammatory reaction culminating in hlh. the patient was treated with etoposide and dexamethasone for hlh and defervesced within a few days. his organ failure and coagulopathy improved, although he sustained digital necrosis (presumably from dic and vasopressor treatment) requiring amputations of six fingers. he required prolonged mechanical ventilation. he was discharged to a rehabilitation facility where he regained strength over the next months and then returned home. he has returned to his job, and no relapse has occurred in the months since discharge. this patient had one rare disease-hemophagocytic lymphohistiocytosis (hlh)-caused by another rare disease, langerhans cell histiocytosis (lch). it is impossible to have an illness script for every bzebra^diagnosis. a better strategy is to have intimate knowledge of common diseases in order to easily recognize deviations from these familiar scripts. it is the mismatch between common illness scripts (pleural) and a patient's presentation that prompts diagnosticians to consider rare diseases. clinicians typically have well-developed illness scripts for common conditions. for example, pneumonia would typically be conceptualized as a febrile illness with a cough and chest xray infiltrate that improves with antibiotics. with experience, clinicians see many variations of pneumonia, including cases that lack fever or even chest x-ray infiltrates. experience also allows the clinician to recognize an unexpected data point as more than a variation, but instead as a deviation that invites consideration of other diagnoses. for example, unilateral hilar lymphadenopathy can be seen in lobar pneumonia, but symmetric, bilateral hilar lymphadenopathy is too unusual for pneumonia and will invite consideration of sarcoidosis, which can also present as a fever, cough, and infiltrate. power (a, b) . these cells stained positive for s (c) and cd a (d) to diagnose a rare disorder, a clinician does not need to know its complete illness script. the key is to know when to think about rare conditions. in this case, an early or presumptive diagnosis of hlh would have been inappropriate due to the extremely low base-rate of this disease. the clinicians knew the illness script for pneumosepsis, patiently worked under this assumption, and ultimately recognized deviations from this script. the inflammatory response was not controlled with antibiotics and no source of infection was identified, so attention turned from sepsis to sepsis mimickers. rare diseases such as hlh were considered only after the discussant found irreconcilable mismatches with the illness scripts of more common conditions. this process of identifying incongruities prompted the clinicians to request consultations and perform additional diagnostic tests. solving challenging cases is ultimately a test of clinicians' knowledge of common diseases, not rare ones. when clinicians know the illness scripts of common diseases well enough to recognize telltale deviations from the norm, they can trigger the consideration of rare conditions and request help from colleagues and other resources that will ultimately lead to a diagnosis. ) hlh is a rare syndrome of marked immune activation that can mimic sepsis. hlh may be triggered by infection, neoplasms, autoimmune disorders, or genetic mutations. ) lch is a clonal proliferation of mononuclear phagocytic cells (histiocytes). lch can be local and asymptomatic, or it can involve multiple organs. it can present as sirs and even shock. , the most common sites of involvement are the skin, bones, lungs, liver, spleen, teeth, gums, and central nervous system (manifesting as diabetes insipidus). - ) reports have linked lch and hlh. the pathogenesis is still unknown, but it is suspected that macrophage activation through t-cell activation and cytokine release plays an important role in the pathophysiology. a -year-old woman with abdominal pain a -year-old woman with abdominal pain and fever how i treat hemophagocytic lymphohistiocytosis lymph node involvement by langerhans cell histiocytosis: a clinicopathologic and immunohistochemical study of cases adult haemophagocytic syndrome report from the international registry of the histiocyte society langerhans cell histiocytosis-clinical and epidemiological aspects cell-specific gene expression in langerhans cell histiocytosis lesions reveals a distinct profile compared with epidermal langerhans cells macrophage activation and hemophagocytic syndrome in langerhans cell histiocytosis: report of cases key: cord- -mowj wyl authors: zhou, xuezhong; lei, lei; liu, jun; halu, arda; zhang, yingying; li, bing; guo, zhili; liu, guangming; sun, changkai; loscalzo, joseph; sharma, amitabh; wang, zhong title: a systems approach to refine disease taxonomy by integrating phenotypic and molecular networks date: - - journal: ebiomedicine doi: . /j.ebiom. . . sha: doc_id: cord_uid: mowj wyl the international classification of diseases (icd) relies on clinical features and lags behind the current understanding of the molecular specificity of disease pathobiology, necessitating approaches that incorporate growing biomedical data for classifying diseases to meet the needs of precision medicine. our analysis revealed that the heterogeneous molecular diversity of disease chapters and the blurred boundary between disease categories in icd should be further investigated. here, we propose a new classification of diseases (ncd) by developing an algorithm that predicts the additional categories of a disease by integrating multiple networks consisting of disease phenotypes and their molecular profiles. with statistical validations from phenotype-genotype associations and interactome networks, we demonstrate that ncd improves disease specificity owing to its overlapping categories and polyhierarchical structure. furthermore, ncd captures the molecular diversity of diseases and defines clearer boundaries in terms of both phenotypic similarity and molecular associations, establishing a rational strategy to reform disease taxonomy. disease taxonomy plays an important role in defining the diagnosis, treatment, and mechanisms of human diseases. the principle of the current clinical disease taxonomies, in particular the international classification of diseases (icd), goes back to the work of william farr in the nineteenth century and is primarily derived from the differentiation of clinical features (e.g. symptoms and micro-examination of diseased tissues and cells) (council et al., ) . despite its extensive clinical use, this classification system lacks the depth required for precision medicine with the limitations of its rigid hierarchical structure and, moreover, it does not exploit the rapidly expanding molecular insights of disease phenotypes. for example, many diseases (e.g. cancer, chronic inflammatory diseases) in the current disease taxonomies have either high genetic heterogeneity (bianchini et al., ; mcclellan and king, ) or manifestation diversity (arostegui et al., ; jeste and geschwind, ; mannino, ) , which give little basis for tailoring treatment to a patient's pathophysiology. furthermore, disease comorbidities (hu et al., ; lee et al., ; hidalgo et al., ) , temporal disease trajectories (jensen et al., ) in clinical populations, various molecular relationships between disease-associated cellular components and their connections in the interactome (blair et al., ; goh et al., ; barabasi et al., ; rzhetsky et al., ; zhou et al., ) , and many successful drug repurposing cases (li and jones, ; chong and sullivan jr., ; ashburn and thor, ; wu et al., ; evans et al., ) altogether demonstrate the vague boundary between different diseases in current disease taxonomies. moreover, the deep understanding of diseases based on the advances in disease biology, bioinformatics, and multi-omics data necessitates the reclassification of disease taxonomy (mirnezami et al., ) . in the past decade, efforts to reclassify diseases based on molecular insights have increased with studies related to molecular-based disease subtyping in different disease conditions, such as acute leukemias (golub et al., ; alizadeh et al., ) , colorectal cancer (dienstmann et al., ) , oesophageal carcinoma (cancer genome atlas research et al., ) , pancreatic cancer (bailey et al., ) , cancer metastasis (chuang et al., ) , neurodegenerative disorders (mann et al., ) , autoimmunity disorders (ahmad et al., ) , multiple cancer types across tissues of origin (hoadley et al., ) , and a network-based stratification method for cancer subtyping (hofree et al., ) . further insights will arise from integrating all types of biomedical data with a single framework to exploit diseasedisease relationships. data integration methods that utilize multiple types of data, including ontological and omics data, have been used to classify and refine disease relationships (gligorijevic and przulj, ; menche et al., ; gligorijevic et al., ) . despite these efforts, the development of a molecular-based disease taxonomy that links molecular networks and pathophenotypes still remains challenging hofmann-apitius et al., ; jameson and longo, ) . here, we aim to refine a widely used clinical disease classification scheme, the icd. to achieve this, we first quantify the category similarity among the icd chapters using ontology-based similarity measures and investigate the molecular connections of disease pairs in the same icd chapters. furthermore, we seek the correlation between category and molecular similarity, and check for the heterogeneity of molecular specificity and correlated boundary between categories in icd taxonomy. finally, we construct a new classification of diseases (ncd) with overlapping structures. the aim is to provide clear boundaries between distinct diseases belonging to different categories using a new disease classification scheme ( fig. & fig. s ). fig. . overview of the new disease taxonomy construction and validation. a. similarity calculation between the disease pairs in icd taxonomy, including the calculation of ) category similarity; ) phenotype similarity (based on icd-mesh term mapping) and ) molecular profile similarities (based on icd-umls term mapping) of disease pairs in icd; b. module or community annotations of disease association network by chapters in icd or ncd. we generate disease association network, in which nodes represent diseases and the link weights represent their corresponding phenotype or molecule profile similarities. the module annotations of the disease network correspond to icd chapters or ncd categories; c. construction of integrated disease network (idn) and generation of ncd. the links of idn are fused from the multiple similarities (e.g. phenotype similarity and shared gene similarity). based on idn, ncd is generated by community detection algorithms with overlapping disease members; d. quality evaluation and validation of icd and ncd. the molecular specificity (or inverse molecular diversity) and network modularity are used for evaluation and comparison of the quality of two disease taxonomies. furthermore, we validate the robustness of ncd with two independent phenotype-genotype association datasets, namely gwas and phewas. in this work, large curation efforts are performed to generate the related data sources (details see supplementary materials (sm) section ). we obtained the updated text version of icd- -cm ( ) and extracted the list of icd codes with their hierarchical structures. while we recognize the improvements of the currently used icd- over icd- , nevertheless, we chose to use icd- -cm as the adoption of icd- has been slow in the united states (butler, ) and since it was still being widely used at the time of the data collection for this paper (blair et al., ; wang et al., ) . furthermore, although icd- does have more codes than icd- -cm, the structure is kept almost the same. we obtained the high-quality phenotype-genotype (disease-gene) associations from disease connect database ( version) (liu et al., ) , leaving out the less reliable text mining entries and focusing only on genome-wide association study (gwas), online mendelian inheritance in man (omim) and differential expression evidence types, and manually mapped those diseases in unified medical language system (umls) codes to icd and mesh codes (sm section . ). to calculate the molecular network and phenotype characteristics related to disease phenotypes, a high-quality subset of human proteinprotein interactions was filtered from string v . (franceschini et al., ) using the score threshold at ≥ , as well as a wellestablished disease-phenotype (disease-symptom) association dataset (i.e. disease network with symptom similarity, hsdn) derived from pubmed bibliographic records and the gene ontology annotations from ncbi gene database are adopted. to ensure the results are not biased by computational predictions in the string database, we replicated the classification pipeline with manually curated ppi networks , which rely only on physical protein interactions with experimental support, and found that the results are robust (sm section . ). in addition, to validate the robustness of our results from independent data sources, we filtered the gwas and phenome wide association studies (phewas) data from university of california santa cruz (ucsc) genome browser (tyner et al., ) and phewas catalog (denny et al., ) respectively, and performed additional icd mapping task to prepare the data for validation analysis. the gwas evidence of the diseaseconnect database, which we used to build the disease associations, comes from the national human genome research institute (nhgri) gwas catalog (welter et al., ) , whereas for validation, we used the ucsc-gwas genome browser. we have ensured that the gwas data used to build the networks and to validate them have a very small overlap (sm section ). here, we systematically evaluated the consistency of disease categories in icd taxonomy from both clinical phenotype and molecular profiles (details are in sm section ). we investigated the quality of icd disease taxonomy by evaluating the correlation between the closeness of disease pairs in the disease taxonomy and the underlying molecular connections (and symptom phenotype similarities) between disease pairs. for example, if two disease pairs have close positions (e.g. have a low level common parent disease) in the disease taxonomy, then we would expect that those disease pairs might have common genes or shared protein-protein interactions or similar phenotypes. we calculated the category similarity between disease pairs using a widely used semantic similarity measure (i.e. lin measure using information content) (lin, ; pesquita et al., ) to represent the closeness of disease pairs located in the icd taxonomy. information theoretic measures such as information content have been used in the context of icd- -cm previously (dahlem et al., ) . the category similarity measure takes as input two concepts c and c and outputs a numeric measure of similarity. if two icd codes have a very specific common parent code in the taxonomic tree structure, then the category similarity would be~ . the molecular and phenotype similarity between disease pairs are calculated by evaluating the shared genes and their go annotations, molecular network similarities, and shared phenotypes by established similarity measures (e.g. cosine measure and jaccard measure). in particular, to propose a more robust representation of molecular network profiles of diseases, we partitioned the string network into topological modules (data s ) and used them to construct the relevant module vectors of diseases using odds ratio (or) as weighting measure. for example, an icd disease code would be represented with a -dimensional vector, which has a value of w ij if its related gene is in a module or otherwise. suppose we have n genes in total and m i genes of a module i. now for a disease d j with n j genes, which has k ij overlapping genes with the module i, we calculated the value of w ij as the following equation, we used the cosine measure to calculate the molecular module similarity between disease pairs after the molecular module vector (i.e. or weighting) of each disease was constructed. furthermore, as icd taxonomy proposes a framework for organizing the diseases, it is expected that there should overlapping molecular interactions or phenotype relationships between the diseases of the same chapters than those of the different chapters. thus, we assumed that when we collapse the icd chapters as the module annotations, such that all the diseases in one chapter would be considered as members of a same module, the modularity of the disease association networks, i.e. the disease networks with molecular or phenotype associations as links, would reflect the quality of icd disease taxonomy. this means that the higher the modularity, the higher the quality of the icd chapters as a disease category framework. to evaluate the quality of community structures in complex network, the modularity measure (newman, ) was proposed to quantify the extent to which the connection in communities is above the random expectation in the whole network. let a network have m edges and a vw be an element of the adjacency matrix of the network. suppose the vertices in the network are divided into communities such that vertex v belongs to community c v . then the modularity q is defined as: where the function δ(i,j) is if i = j and otherwise, and k v is the degree of vertex v. the value of the modularity lies in the range [− / , ]. it is positive if the number of edges within groups exceeds the number expected on the basis of chance. otherwise, it would be negative. we use it to measure the consistency of disease categories (icd chapter or ncd) as an annotation of topological module (or community) structures within disease networks. we hypothesize that if a disease category framework captures the molecular or phenotypic profiles of diseases, then there would be more links existing between the disease members in a category than random expectation. as a quantification of the molecular diversity (or the inverse specificity) of a disease, we calculated the maximum betweenness of diseaserelated genes in the ppi network (data s ). betweenness (freeman, ) is a widely used centrality measure to quantify how many shortest paths run through a given node. in particular, bridging nodes that connect disparate components of the network often have a high betweenness. the betweenness centrality of a node v is given by: where n st (v) denotes the number of shortest paths from s to t that pass through v and g st is the total number of shortest paths from s to t. we will adopt the convention that nstðvÞ g st ¼ if both n st (v) and g st are zero. we assume the molecular diversity of diseases would largely lie on the related genes with maximum betweenness. for example, to quantify the molecular diversity (in terms of maximum betweenness) of alzheimer's disease (ad), we calculated all the betweenness values for the ad-related genes, such as app, apoe, tnf and nos . finally, we considered the molecular diversity of ad as . e- since we found that app has the maximum betweenness of . e- among those genes (see fig. s a ). in fact, this kind of measurement has been successfully used in a previous study to evaluate the diversity of diseases, which indicated that the diversity of disease manifestations has a strong positive correlation with the molecular diversity of diseases. for disease taxonomy with good quality, we would expect it to have its lowest level diseases (the leaf nodes in the tree-structure disease taxonomy) with similar molecular diversities. we calculated the edge density to quantify the molecular interactions between icd chapters. to further detect the significant interactions between diseases in different chapters, we find an approach to obtain the diseases that have significant interactions with diseases in chapters other than their own. given a disease d i for investigation, we evaluate whether the proportion of interactions (i.e. edge density) of d i to the disease set d c k of a chapter c k is significantly larger than the average proportion of interactions between the diseases in c k (fig. s ) . we use binomial test to filter the significant interacting disease-chapter pairs, in which the edge density of the disease to the chapter is significantly higher than the average edge density of the diseases in the corresponding chapter (details are in sm section ). the results showing positive correlations between category similarity and molecular similarity, and the high molecular diversity of many diseases imply that it would be possible to predict the multi-category map for each disease using its underlying molecular connections. to demonstrate a pilot method for multiple disease category prediction by integrating molecular module and shared gene similarities, we provided a novel algorithm to generate the possible associated additional disease categories for a given disease with the corresponding molecular association scores. (details are in sm section , fig. s ). in this algorithm, we integrated the correlation between category similarity and module similarity with significant disease-chapter associations (which are based on the shared gene similarity) to predict the additional chapters for a given disease. we divide the disease pairs in the same chapter to three subsets, which correspond to those pairs with shared root parents, shared second-level intermediate parents and shared third-level intermediate parents, respectively, to help predict to what degree a pair of diseases would be located closely in the disease taxonomy. the principle of the algorithm adheres to the positive correlation between category similarity (or the closeness of position of the disease pairs in icd disease taxonomy) and molecular profile similarity of disease pairs, which means that strong molecular profile similarity between disease pairs would indicate close locations of them in the disease taxonomy. to ensure detecting the significant disease-chapter associations, we next filtered the predicted disease-chapter associations with positive association scores by the significant disease-chapter interactions based on shared genes. to integrate disease associations derived from both molecular and phenotype features, we performed several sequential analytical steps to generate a highly reliable disease network with strict filtering criterions of the disease links (details are in sm section ). firstly, we generated three disease association networks: disease network with module similarity (msdn) with , links and nodes, disease network with shared genes (sgdn) with , links and nodes, and disease network with symptom similarity (hsdn) with , , links and nodes (fig. s & table s ) according to shared genes, shared phenotypes and molecular module similarity, respectively. to reduce the possible noise and bias of disease related data sources, we applied a multi-scale backbone algorithm (serrano et al., ) to obtain high reliable disease links (with significantly high weights than the random expectations) from the three disease networks. we finally obtained , , and , high reliable links for msdn, sgdn and hsdn, respectively and retained most nodes ( for msdn, for sgdn and for hsdn) of these networks. to further reduce the possible weak associations (the disease pairs with high module similarity but no direct protein interactions) derived from module similarity, we calculated the minimum length of the shortest paths (mspls) between each disease pairs and used it as a filtering criterion (with mspl≤ ) for msdn, which resulted in a more biological meaningful subset of msdn with , links and diseases. sgdn would capture strong associations between disease pairs if they have high degree of shared genes even their related genes are not forming functional modules. however, msdn would give high weights for disease links if the disease pairs have similar co-locations on the topological modules of molecular network even they have no shared genes. therefore, msdn and sgdn are actually two complementary molecular association evidences for disease pairs and we finally obtained the union of the subset of msdn and sgdn as the molecular association disease network (madn), which contains , links and nodes with the weights derived from the two original networks. next, we adopted a highly strict criterion to obtain an integrated disease network (idn) from the fusion of madn and hsdn links, which contains , disease links and nodes. finding the overlapping disease categories could be transformed to the task of detecting the overlapping communities (i.e. modules) from the idn. bigclam (yang and leskovec, ) is a state-of-the-art overlapping community detection algorithm based on a variant of nonnegative matrix factorization, which achieves near linear running time and comparable high quality community results. we used the bigclam algorithm, which is packaged in snap complex network software (http://snap.stanford.edu/snap/) to automatically detect overlapping communities from idn network. finally, we obtained overlapping disease communities with distinct icd disease codes. these disease subcategories contain different numbers of icd codes, ranging from to (fig. s & data s ). to obtain a top-level category framework of diseases corresponding to the chapters in icd taxonomy, we calculated the overlapping degree of the disease sub-categories by using jaccard similarity to measure the common number of diseases held by two given disease categories. this generated a disease category network with links representing shared icd codes (a link is established if two disease categories share at least an icd code and the weights of links correspond to the jaccard similarity) and nodes representing disease categories. after that, we clustered the disease sub-categories additionally by a widely used non-overlapping community detection algorithm (considering the link weight and setting the resolution parameter as . ) into top-level categories (which corresponds to the number of original chapter-level categories in icd, which we named as new chapters, ncs) using the shared icd codes (fig. s c & data s ). the modularity of these top-level categories (this makes a good comparable partition with icd chapters) in the network of sub-categories is . , which means a rather good partition of the network. these ncs contain different numbers of sub-categories ranging from to or of diseases ranging from to (fig. s c & data s ), covering diseases from all of the chapters of icd taxonomy (table s ) . these ncs would still contain overlapping disease codes since the disease subcategories have overlapping disease codes. therefore, ncs with disease sub-categories form a disease taxonomy consisting of two hierarchical levels with polyhierarchical categories although with a limited number ( ) of disease members. to validate the robustness of ncd, we obtained two external phenotype-genotype data sources (i.e. ucsc-gwas and phewas catalog), which have not been integrated yet for generating ncd for further investigation. by measuring whether the disease members in the subcategories in ncd tend to incorporate the associations of shared genes from these two data sources, we would be able to validate the quality of ncd. if the diseases in such ncd sub-categories would tend to involve shared genes, then the diseases would be more likely associated with one another than other diseases. to test this hypothesis, we obtained the overlapping disease codes (odc) in both ncd and the two external phenotype-genotype association databases and evaluate the degree of these odc disease links in each ncd sub-category when considering two diseases linked if they share common genes. in detail, we firstly obtained the common disease codes involved in both ncd and ucsc-gwas or phewas database. then we generated a disease network with shared genes derived from the two datasets, in which two diseases linked if they shared at least one common gene. after that for each ncd sub-category, we generated a complete disease network with the odc diseases in it and overlaid the network on the disease network with shared genes. finally, the overlapping percentage of disease links would be calculated for evaluating the degree of molecular associations involved in diseases in each ncd sub-categories (details are in sm section , figs. s -s ). we use r . . as the main statistical tool in our work. the comparison of two percentages was calculated by binomial test or chi-squared test. wilcoxon rank sum test was used for compare two independent list of values (e.g. two types of molecular diversities and two groups of mspls). all the correlations between two variables were calculated by pearson's product moment correlation coefficient. due to the incompleteness and bias of disease-related data (i.e. disease-gene associations and disease-symptom associations), we need to distinguish the information from the background noise. therefore, for comparison with random expectation, we reshuffle ( random permutations) the symptom features and the related genes of each disease using the fisher-yates method (fisher and yates, ) . the calculations from random permutations were used for the correlation between category similarity and molecular similarity, as well as phenotype similarity. in addition, this was used for detection of the disease categories with high molecule diversity. we curated distinct icd disease codes (table s ) from the level tree structure of , icd- -cm codes, as well as high confidence protein-protein interactions consisting of , nodes and , edges (franceschini et al., ) . we compiled , distinct diseasegene associations between distinct diseases in umls codes and , genes reported in the diseaseconnect database (liu et al., ) (fig. s ) . next, by manually mapping the diseaseconnect identifiers to icd codes, we obtained , disease-gene records involving distinct icd codes and , genes (figs. s - and data s ). to evaluate the closeness of two diseases in the icd tree structure, we applied an established semantic similarity algorithm named category similarity (see methods, sm section . ). this similarity measure is based on the information content, which quantifies the specificity of a term and can be applied to any categorization scheme that has a rooted tree structure, including the icd- -cm disease classification. we then created a disease network comprising nodes (representing icd codes) and , edges. the edge weight reflects the category similarity values and higher values reflect higher similarity between diseases whose code positions are adjacent in the icd tree. the category similarity distribution showed that most disease pairs ( , , . %) had similarities between . and . (fig. s a) . disease pairs within this range mostly belong to different disease subcategories in the same chapter, such as diseases of other endocrine glands and disorders of thyroid gland. for example, type diabetes (icd: . ) and simple goiter (icd: . ), which are in icd chapter , have a category similarity of . . however, there do exist disease pairs with high category similarities, such as type diabetes (icd: . ) and type diabetes (icd: . ) with a category similarity . . overall, this measure indicates the capability of icd in bringing together similar diseases in its tree structure, and the overrepresentation of lower similarity scores is indicative of its limitations in doing so. while the icd classification was derived from clinical manifestations (including symptoms and signs) and does not necessarily reflect the connections among the molecular components of diseases, it is informative to quantify to what extent it carries molecular information. we investigated the correlations of category similarity of disease pairs with ) the degree of shared genes and shared clinical phenotypes, ) go term (cell component, molecular function, biology process) similarity (mistry and pavlidis, ) , and ) topological similarity (i.e., minimum shortest path length and molecular module similarity) among them (methods, sm section . ). we found that close disease codes (disease pairs with a high category similarity) actually have higher clinical phenotype similarity (methods, sm section . ), which adheres to the construction principle of icd taxonomy based on symptom phenotypes (fig. s b , pcc = . , % ci = [ . , . ], p = . e- ). furthermore, we observed strong correlations between higher category similarity bins for molecular profiles, compared to lower category similarity bins (fig. s c-i and table s . see methods, sm section for detailed information). in particular, we observed that in addition to the strongly positive correlations, the percentage overlap of disease pairs with shared genes was generally larger than the random controls ( fig. s c and d, see methods, sm section . ). the top disease pairs with the largest number of shared genes are all from chapter , which consists of cancer types. this might reflect the fact that cancers are the most studied and complex disease phenotypes involving various gene mutations (table s , see methods, sm section for detailed information). overall, these findings indicate that diseases in the same icd chapter tend to have a higher degree of shared genes, and the closer their positions in the icd tree, the higher is the degree of shared genes. we measured the maximum betweenness of disease-related genes in the protein-protein interaction (ppi) network to quantify the molecular diversity (the inverse of specificity) of each disease, as described previously ) (see methods, sm section ). a high maximum betweenness indicates a high molecular diversity. for example, c c c c c c c c c c c c c c the molecular diversity of alzheimer's disease could be represented by the maximum betweenness of its related genes (i.e., the betweenness of the app gene) in the ppi network (fig. s a) . we observed that the molecular diversity of diseases in the icd taxonomy is heterogeneous, with molecular diversity values varying from − to − with a median value of . e- ( fig. a and data s ). the top two disease chapters with the highest median molecular diversity were chapter ( . e- ) and chapter ( . e- ) (fig. b) . furthermore, we found that neoplasm (chapter ) and infectious disease (chapter ) categories tended to have higher molecular diversity compared to their complementary categories (neoplasms vs. non-neoplasms p b . e- , infectious diseases vs. non-infectious diseases p = . e- , fig. s b-c) and random controls. we also found that disease categories annotated as "other/unspecified" categories (sm section . ) had higher molecular diversity compared to disease categories with specific conditions (p = . e- , fig. s d , data s ; see sm section . ) and its random control. these results indicate that the diseases in neoplasms, infectious diseases, and "other/unspecified diseases" categories should be further investigated for molecular subtypes. a detailed discussion of disease cases is offered in sm section , data s & tables s -s . in the current icd taxonomy, we observed many instances where there exists a significant number of links between diseases in different chapters, comparable to the number of links between diseases within the same chapter (table s & fig. d , see methods & sm section ). for example, strong shared-gene relationships were detected between respiratory diseases (chapter ) and mental, behavioral, and neurodevelopmental disorders (chapter ) ( fig. c-d , more examples shown in sm section , tables s - ). in addition, by calculating the shared molecular connections between diseases in the context of chapters, we could detect diseases with a significant number of shared genes with diseases other than those in their own chapters (data s & sm section ). to further quantify the molecular boundaries between the disease categories in icd disease taxonomy, we evaluated the modularity, a structural measure of the tendency of the network to form close-knit communities (see methods, sm section . ), generated by either shared molecular profiles or shared phenotypes. when we mapped icd chapters as grouping annotations on the disease networks filtered by with appropriate weight thresholds, and calculated their modularity, we observed very low modularity values (fig. e) . since modularity is a widely used measure to validate the quality of partitions/module structures in complex networks, this means that the grouping of icd chapters does not agree with the natural topological groupings of their corresponding molecular networks (disease modules). this finding gives strong evidence for the blurred disease boundaries of the icd taxonomy, possibly arising from the complexity of the underlying molecular mechanisms, in particular the possible overlap of their respective subnetworks, or disease modules, in the interactome. furthermore, although the modularity of disease networks with shared phenotypes (similarity ≥ . ) is slightly positive, the weak correlation (pcc = . , p-value = . ) between phenotypic similarity and category similarity of disease pairs in each chapter (fig. f) indicates that icd taxonomy does not adequately incorporate phenotype similarity knowledge into disease category structures. these observations indicate that the strict tree structures in the icd taxonomy wherein terms can only have one lineage (cimino, ) may be inefficient for disease classification given the contemporary knowledge of disease pathobiology, and should therefore be refined to be polyhierarchical in structure. it has been proposed that if two disease modules overlap in the molecular interaction network, local perturbations in one disease might disrupt the biological pathways in the other disease, resulting in shared pathobiological characteristics . we observed a strong positive correlation between category similarity and module similarity (see methods, sm section . ) of diseases, indicating that two diseases with higher module similarity would be more closely localized in the disease category (fig. a, here, we utilize the module similarity between disease pairs to predict the categories of similar diseases. in particular, we determine the taxonomic closeness (sm section . ) of each given disease pair to predict the additional categories of diseases, by applying heuristic rules incorporating the positive correlation between category similarity and module similarity (see methods, sm section . ). in particular, using the , disease pairs with positive module similarity (data s ), we generated predicted additional category results for out of disease codes ( . %) in which each disease code had~ categories on average (data s & ). we found that the number of predicted categories positively correlated with the molecular diversity of the original disease codes (fig. c , pcc = . , % ci = [ . , . ], p b . e- ; for external validations see sm . ). this indicates that diseases with multiple pathogenic pathways could be captured by polyhierarchical mapping. for example, the diseases in chapter (i.e. diseases of the respiratory system) have been predicted to belong to over five additional chapters, such as neoplasms, infectious diseases, and diseases of the skin and subcutaneous tissue (fig. d) , which is consistent with the heterogeneous pathogenesis of copd and asthma (grainge et al., ; sharma et al., ) . a detailed discussion on the polyhierarchial map of the mental disorders is offered in sm section . (fig. s ) . furthermore, we found that the predicted category framework, which is based only on molecular module similarity, also had higher phenotype similarity than diseases with shared root codes in the original icd chapters (see sm section . , median: . vs. . ; mean: . vs . ; p b . e- , fig. e ). this observation helps to establish that the predicted category results are of higher quality than icd with respect to their phenotype homogeneity. to extend and redefine disease concepts by discovering additional categories of a disease, we generated a novel disease taxonomy by constructing an integrated disease network (idn) with: (a) shared clinical phenotypes including shared symptoms; (b) shared molecular profiles including (i) shared genes and molecular module similarity and (ii) shortest path lengths in the ppi network, based on a systematic integration process to filter out possible false positive associations (see methods, sm section , fig. s and fig. s a) , which includes diseases and , links (data s ). next, we applied high performance community detection algorithms to identify overlapping community structures in the idn fig. . lack of molecular specificity in icd taxonomy and the blurred boundary between disease categories in icd taxonomy. a. the distribution of molecular diversity of icd diseases; b. the boxplot of molecular diversity of icd chapters (ordered by median values); c. the disease network with shared genes in which the diseases belong to chapter and chapter . the icd codes , , in chapter have dense relationships to the icd codes in chapter ; d. the disease category network with shared genes. the nodes indicate the disease chapters and the weights of edges represent the edge densities between disease chapter pairs; the nodes with same color are considered as a chapter cluster, which is detected by community detection algorithm; e. modularity of disease networks with chapter as module annotations; f. the correlation between category similarity and phenotype similarity of icd chapters. (see methods, sm section and fig. s a ). in particular, we first used bigclam (see methods) since this method is able to detect overlapping communities whereby a disease can belong to multiple communities, in line with our main premise of creating a molecular based flexible disease classification. this resulted in disease subcategories with overlapping diseases as members ( fig. s a and data s ), which included distinct diseases from the icd taxonomy. these disease sub-categories contain different numbers of icd codes, ranging from to (fig. s ) , therefore, they represent different levels of disease categories similar to icd chapters and their sub-categories. to further develop a more unified view of the disease category quality, we used the established bgll method (blondel et al., ) , which detects non-overlapping communities, to cluster these subcategories further into non-overlapping, distinct parts, such that these represent the new chapter-level categories (called new chapters, or ncs) using the shared icd codes (see methods, sm section . , fig. s b ). overall, this clustering order effectively ensures distinct top-level categories that have overlapping subcategories. the resulting ncs contain different numbers of sub-categories ranging from to , or of diseases ranging from to (fig. s c) . we denote the ncs together with their disease sub-categories as our new overlapping disease classification (ncd). each of the resulting ncs reflects the shared features of integrative molecular and phenotypic profiles (sm section . ; fig. s c & table s , data s - ). for example, nc could be denoted as the "limbic system development-vision disorders-related diseases" since the most enriched ppi module (p = . e- , relevance ratio = . ) of its constituent diseases was mainly related to biological process; "limbic system development" (p = . e- ), and . % ( / ) of diseases in nc shared the phenotype, "vision disorders" (p = . e- ) (tables s -s ). to confirm the phenotypic and molecular cohesiveness of our overlapping disease categories, we compared the modularity of ncd with that of the icd taxonomy. we found that the ncs consistently have much higher modularity than the original icd chapters for all types of disease association networks (sm section . ; fig. a-h, fig. s ). this finding indicates that the phenotypic and molecular links between the diseases of a category in ncd are much denser compared to icd taxonomy. furthermore, to ensure that the performance of ncd is indeed due to the combined effect of the molecular and phenotypic profiles, we performed a controlled experiment where we determined the new disease categories based on molecular-based networks and phenotype-based networks only by running the entire analytical pipeline and applying the same category prediction algorithm (sm section . ). we found that ncd outperforms both molecular-based categories and phenotype-based categories in capturing the gene similarity, go term similarity and phenotypic similarity (figs. s -s ). this suggests the importance of integrating both clinical phenotypes and molecular profiles to obtain a high-quality disease taxonomy. furthermore, we found that the minimum shortest path lengths in the ppi network between disease pairs that belong to the same ncd categories had a larger percentage of low values (i.e., [ , ]) compared to icd (fig. i, . % vs . %, p b . e- ; sm section . ). this result indicates that diseases within an ncd category have a significantly higher degree of shared genes (or shorter path lengths) in comparison to diseases within a category in icd. on the other hand, the mspls between disease pairs in different ncd categories had a significantly lower percentage of low values than those in the icd ( . % vs . %, p b . e- , fig. i & fig. s polyhierarchical map of the disease codes in chapter , indicating that the disease codes in chapter have significant associations with two disease category clusters: ) chapter (infectious disease) and chapter (neoplasms); ) chapter (endocrine, nutritional and metabolic diseases and immunity disorders), chapter (mental disorders), chapter (nervous diseases), chapter (digestive diseases), chapter (skin and subcutaneous disease) and chapter (musculoskeletal system and connective tissue diseases); e. the boxplots of phenotype similarity of predicted disease pairs and original icd disease pairs in the same top-level chapters (p b . e- , wilcoxon test). which indicates a lower degree of shared genes (or shorter path lengths) between diseases from different categories in ncd than in icd. these findings demonstrate that our ncd framework has clearer boundaries between distinct diseases belonging to different categories than those in the original icd disease taxonomy. moreover, to validate the robustness of ncd predictions, we calculated the degree of associations in terms of network density among the diseases in each sub-category of ncd. to this end, we investigated the overlaps with the disease pairs connected by shared genes using two independent phenotype-genotype association databases, gwas and phewas (see methods, sm section . , . & ). we found that for the sub-categories in ncd, network density was significantly higher compared to random controls (gwas: p-value = . e- , fig. j ; phewas:p-value = . e- , fig. k ). this means that the diseases in the sub-categories in ncd would tend to have shared genes. for example, the new chapter: nc in ncd, including sub-categories and icd diseases (belonging to eight icd chapters), is enriched with respiratory and airway diseases (e.g. copd and asthma). we obtained overlapped diseases from the gwas database, which have a high degree of shared genes with the diseases in each sub-category of the nc (fig. a) . in particular, the sub-categories, such as nc .m (p-value = . e- ), nc .m (p-value = . e- ) and nc .m (p-value = . e- ) have significantly higher density than those of the whole gwas disease network (fig. s ) . furthermore, we found that the overlapping subcategories of the ncd are able to differentiate between different components (i.e. asthma/allergy vs. copd) of the same broad group of diseases (i.e. respiratory diseases) (see fig. s for a detailed example). indeed, in the nc disease chapter chiefly containing respiratory diseases, the two sub-categories, namely nc .m and nc .m , overlap in the underlying molecular interaction network while still containing the respective disease (asthma and copd, respectively) genes separately (fig. a) . a detailed discussion is offered in sm section (with results in data s - , tables s - & figs. s -s ). in addition, in ncd, a disease can be classified into multiple categories, and the number of categories of a disease positively correlates with its molecular diversity (fig. l , pcc = . , % ci = [ . , . ], pvalue b . e- ; external validations in sm . ). for example, we reclassified neoplastic diseases into multiple categories due to their high molecular diversity. two hundred and fifty-eight neoplastic diseases in our ncd were divided into sub-categories and ncs (figs. s & s ) . thirty-nine out of sub-categories ( . %) were enriched with "neoplasm" diseases (data s , p-value = . e- ). there were mainly ncs (i.e., nc , nc , nc , and nc ) containing these sub-categories and "neoplasm" disease codes (fig. b) , where . % ( / ) of the neoplastic diseases were classified into n sub-category, ranging from to (data s & table s ). the neoplasm with the highest molecular diversity, "malignant neoplasm of connective and other soft tissue" (icd: ; molecular diversity: . ), was reclassified into sub-categories, and "malignant neoplasms of thyroid gland" (icd: ; molecular diversity: . ) was assigned to sub-categories. furthermore, related diseases had been reclassified together in ncd, such as the well-known diseasecorrelations among h. pylori infection (icd: . ), stomach cancer (icd: ), and duodenal ulcer (icd: ) or peptic ulcer (icd: ) (fig. b) (sitas, ; graham, ) . more interestingly, some diseases, like viral hepatitis c (icds: arthritis (icds: / . ), each from different chapters in icd taxonomy, were classified together into a unique ncd sub-category (nc .m ) since % ( / ) of these diseases share a ppi module related to immune response (sm section . , fig. c , data s - , tables s - ). in addition, diseases originally in the same icd chapter, such as viral pneumonia (icd: ) and influenza (icd: ) from respiratory system-related diseases (chapter ), were reclassified into different categories in the ncd (nc , nc ). influenza shared more phenotype profiles with "episodic mood disorders" (icd: ) in nc .m , rather than viral pneumonia in nc (fig. s & data s ) , which is in accordance with recent epidemiological studies between episodic mood disorders and influenza (okusaga et al., ; canetta et al., ) , and, furthermore, we also found that influenza shared some molecular profiles with "episodic mood disorders" (icd: ) in nc .m (fig. s , data s ). these findings suggest that ncd offers a promising integrative framework incorporating both clinical phenotypes and molecular profiles for disease taxonomy that has very practical implications for the precise investigation of disease subtyping and etiologies. given the molecular network mechanisms (barabasi et al., ; zanzoni et al., ) , genetic pleiotropy (solovieff et al., ) , as well as complicated genotype-phenotype associations underlying diseases, the establishment of a molecular-based disease taxonomy with clear boundaries is essential but challenging. from the molecular network perspective, we first investigated the utility, shortcomings, and inconsistencies of icd- -cm, the established disease taxonomy for clinical settings. we found that there exist a considerable number (~ % of our investigated diseases) of diseases, for example, cancer and infectious diseases, that have diverse molecular network mechanisms and tend to interact more with diseases from other chapters. it is also these molecularly diverse diseases that mainly contribute to the blurred boundary of icd disease taxonomy (see methods, sm section & ). upon exploring the molecular diversity and cross-chapter interactions between diseases, we propose a novel disease classification system based on the integration of the clinical and molecular profiles of diseases. in particular, we integrate disease networks taking into account molecular and phenotypic connectivity among diseases, predict the multiple disease categories that diseases belong to, and finally validate the biological cohesiveness of our ncd by network topological measures such as modularity and shortest path length. our findings indicate that although general correlations exist between disease closeness in icd taxonomy and underlying molecular profiles, icd still displays significant limitations with regard to the heterogeneity of molecular diversity and clear category boundaries. in our ncd, a disease with a high molecular diversity tends to be classified into multiple disease categories, which indicates that there exist more disease subtypes for that disease. for example, "malignant neoplasm of the pancreas" was reclassified into sub-categories and ncs, which is consistent with a recent study wherein phenotypic subtypes of pancreatic cancer were enriched for distinct molecular mechanisms (bailey et al., ) . therefore, we believe that the new disease classification system may help facilitate precise clinical diagnosis and correct prognosis (jameson and longo, ) , and does so in alignment with refined molecular network diagnostics. furthermore, the molecular network underpinnings and overlapping disease categories of ncd provide a credible relationship map between diseases and disease categories that may radically transform our current understanding of diseases and relevant treatment paradigms. on the one hand, our approach accurately links diseases with all possible underlying mechanisms in the molecular interaction network. on the other hand, it presents a promising approach to the identification of targeted drugs for the treatment of related diseases. for example, breast cancer and influenza (both in nc .m ) may share potential drug targets (park, ) . as another example, metformin, widely prescribed to treat metabolic syndrome (in nc .m ), could alter the gut microbiome composition and function, improve gut microbial dysbiosis (forslund et al., ; cabreiro et al., ) , and also prevent colorectal cancer (also in nc .m ) through microbiomeinfluenced immune response modification (nakatsu et al., ) . here, it is important to note that while a considerable number of diseases have a strong environmental component, our main focus has been the many diverse molecular determinants. in the future, additional environmental factors such as epigenetic changes can be added into the data integration scheme to further refine the classification. there exist several potential limitations of this work. although we have aimed to address the possible confounders by constructing random controls and using external evaluations, the incompleteness and bias incorporated in the integrated data sources are likely to influence the generalization of our results. for example, diseaseconnect yields an incomplete disease-gene database: icd diseases could be mapped (table s ) , leading to only diseases included in the ncd. furthermore, as with other studies that rely on literature-based and ontological knowledge, investigation bias remains an issue, where the molecular mechanisms (e.g. related genes and their interactions) of some diseases (e.g. cancer) being more intensively studied than others may influence the results . we expect the results of similar works to be more refined in the future as biomedical datasets become more complete. the incorporation of more comprehensive disease-gene data sources, such as diseases (pletscher-frankild et al., ) and malacards (rappaport et al., ) , could result in an improved study. while we have chosen to keep individual external gene expression datasets outside the scope of this study since gene expression is highly tissue-and cell-type dependent, it presents an interesting future direction and could potentially improve the quality of the resulting disease categories if exhaustive lists of tissue-specific expression datasets are used in dedicated studies. in addition, our ncd merely delivers a two-level taxonomy framework without elaborated hierarchical structures in the same disease categories, which could be further refined or optimized through methods like hierarchical clustering algorithms (murtagh and contreras, ) and systematic posteriori ontology engineering method (gessler et al., ) . finally, high-quality ontologies, such as the human phenotype ontology (kohler et al., ) and disease ontology (kibbe et al., ) , can be used for external validation, or for further integration to obtain more robust and extensive ncds. in this big-data era, the dramatically increasing multi-omics databases, as well as clinical data from electronic health records (ehr) involving phenotypic, therapeutic and environmental factors information (jensen et al., ) , should also be incorporated into the new disease taxonomy refinement for patient stratification and disease treatment. at this point, a realistic assumption is that the translation of this classification to the clinic will need some time. that said, while fig. . biological insights of new disease taxonomy. a. the new chapter containing airway diseases (nc ) consists of sub-categories and icd diseases belonging to icd chapters. the subcategories overlap in the underlying molecular interaction network, while still separately including the disease genes (asthma and copd, respectively) that characterize each subcategory; b. the disease network of neoplasms in ncd. the sub-categories significantly representing neoplasms are divided into ncs (g ). helicobacter pylori [h. pylori] ( . ), malignant neoplasm of stomach ( ), duodenal ulcer ( ), peptic ulcer, site unspecified ( ), which have significant relationships, are clearly clustered into a subcategory (nc . m ) (g ); c. a sub-category (nc .m ) in ncd, which includes diseases from different icd chapters with shared molecular mechanism and phenotypes. fifty percent ( / = %) of the diseases in nc .m share a ppi module, the biological function of which is enriched with immune system response, while over % ( / = . %) of the shared common phenotype of this module is "pain". the icd is originally made "by clinicians for clinicians", it is now widely used by biomedical researchers as well to gain a deeper understanding of human diseases. we therefore believe that researchers will be the first and direct beneficiaries of our approach. in conclusion, our study provides valuable insights into the polyhierarchical network-based disease classification beyond the traditional tree structure. our integrated disease network approach is sufficiently powerful to elucidate the tangled underpinnings of human diseases and uncover distinct disease boundaries. our work may provide a new framework for the disease taxonomy reform based on bigdata fusion, so as to generate further the robust infrastructure needed for precision medicine. supplementary data to this article can be found online at https://doi. org/ . /j.ebiom. . . . the work was supported by national natural science foundation of china ( , and ), national science and technology major project for new drugs research and development of china ( zx - , zx - - ), national key r&d project ( yfc ), and the fundamental research funds for the central public welfare research institutes (zz , jbm , dut zd , dut zd , dut zd ). we also acknowledge the support by national institutes of health (nih) grants p - hg -cegs, mapgen grant (u hl ) and p hl , u hl , p hl , r hl , p hl - and rc hl . the funders had no role in study design, data 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( ) on helicobacter pylori and stomach cancer risk pleiotropy in complex traits: challenges and strategies the ucsc genome browser database: update classification of common human diseases derived from shared genetic and environmental determinants the nhgri gwas catalog, a curated resource of snp-trait associations an ancient, unified mechanism for metformin growth inhibition in c. elegans and cancer overlapping community detection at scale: a nonnegative matrix factorization approach a network medicine approach to human disease human symptoms-disease network the authors declare that they do not have any competing financial interests. key: cord- -yvh fzxt authors: nabarro, david; wannous, chadia title: the links between public and ecosystem health in light of the recent ebola outbreaks and pandemic emergence date: - - journal: ecohealth doi: . /s - - -y sha: doc_id: cord_uid: yvh fzxt nan the ebola outbreak in west africa is an unprecedented public health emergency of international concern. it is the largest and most complex ebola outbreak on record, having resulted in over , cases, and an estimated case fatality proportion of %. the ebola outbreak took a significant economic and social toll on west african nations beyond human death and illness. it has devastated families and communities, compromised essential services including health and education, weakened economies and isolated affected populations. the outbreak shows that we are still not ready to deal with infectious disease emergencies. it also demonstrates the importance of preparedness and building resilient health systems through the integration of disaster risk management into health care provision at all levels. ebola exploits fragile and overstretched health systems with limited public health capabilities. therefore, we must prioritize strengthening the health institutions, develop their infrastructure, and support the training of the health workforce. this is not the first time that a novel or re-emerging virus has tested the ability of national, regional and global community to predict and prevent a pandemic and to respond to its impact. in fact no pandemic so far has been predicted before it emerges. this is a testament to our vulnerability to zoonotic health threats. drivers of disease emergence of most pandemics, including hiv/aids, sars, and pandemic influenza, are associated with ecological, behavioural, or socioeconomic changes (morse et al. ) . the increasing demand for land, water, and energy due to human population growth and density and economic development has led to deforestation and associated biodiversity loss, climate change, imbalances in agricultural and food supply systems. these drive the emergence of new diseases, while increasing international travel and trade including the transport of live animals, food items, plants; coupled with our own fragile health systems facilitate their spread and increase their impact. most pandemics originate in animals. more than % of eids are zoonotic , and zoonoses of wildlife origin have dominated pandemics of the past years (morse et al. ) . changes in the overall pattern of pathogen-host-environment interactions and in key interfaces that promote contact among humans, livestock, and wildlife underlie disease emergence. increasing environmental change alters the dynamics of pathogen transmission within these groups, leading to increased pathogen spill-over between species, and the widespread geographic redistribution of pathogens, vectors and hosts. examples of drivers include bush or wild meat hunting and consump-tion, mass rearing of animals and use or misuse of antimicrobials and vaccines. changes in climate, weather conditions such as temperature and humidity also affect pathogen environmental survival and transmission, compounding the potential for spillover to lead to pandemic spread. against this background, several mitigation and control strategies can be adopted to predict, prevent and also respond in a timely and effective way to pandemic zoonoses. first a global strategy to coordinate pandemic preparedness and response is needed. the increasing frequency of pathogen emergence suggests that this should be a priority. lessons learned from previous efforts at pandemic preparedness and response indicate that this is extremely complex and hard to implement through conventional sector support programmes. hence the emergence of the one health approach, which refers to the collaborative multidisciplinary, cross-sectoral work by public and animal health agencies, and the environmental sector to reduce the risk of disease emergence across their interfaces. cross-sectoral approaches are needed to gain sufficient momentum in controlling emerging disease because there are constant challenges to adopting them. systems need to be put in place that make the one health vision come to life, and these require careful investment, and time, to make them work. the time used to implement these programs must be used well-to build trust, innovate, learn lessons, and establish sustainable ways of working together. adequate resources are needed to make this happen: if the funds needed for cross-sectoral collaboration are not available, collaboration will not materialize. oie, fao and who as part of the tripartite agreement and the world bank have supported countries to work across sectors with the aim of building systems that are strong and resilient and that can respond to a range of health emergencies. as a result of these efforts, more countries are exploring the one health approach and are implementing it in myriad creative ways. secondly, increasing capacity to analyse and predict disease emergence, and conduct surveillance in livestock, wildlife and people at the key interfaces where diseases emerge are of utmost importance. enhancing innovation in molecular diagnostics and geo-spatial modeling is vital to pre-empt pandemic zoonotic disease emergence through early identification of novel pathogens in high risk hotspots for disease emergence. such important work has been pioneered by researchers in international livestock research institute (ilri) and ecohealth alliance. for example global hotspots for emerging infectious diseases that originate in wildlife have been mapped (morse el al. ) . given that most pandemics have mainly originated in wildlife, these maps identify hotspots where the next pandemic is most likely to originate and thus facilitate targeted surveillance, which supports prevention and control efforts at source. the who research and development blueprint is a global strategy and preparedness plan to ensure that targeted r&d can strengthen emergency response by bringing medical technologies to patients during epidemics. the blueprint aims to reduce the time between the declaration of an international public health emergency and the availability of effective tests, vaccines, and medicines that can be used to save lives and avert crisis. the blueprint will be presented to member states at the next world health assembly in may . a whole-of-government and a whole-of-society approach, with communities at the centre of preparedness and response to infectious disease emergencies, led by strong political commitment of governments, is essential. in the current ebola outbreak, this has involved presidents recognizing that the response needs to include all sectors as well as politicians and civil society. integration of social sciences research, particularly that focused on human behavior, is pivotal to understanding the origins and evolution of pandemics (janes et al. ) . in the ebola outbreak in west africa, fear and panic led to distrust and may have exacerbated the spread of the disease and its subsequent impact on social cohesion. moreover, the reaction to returning frontline health care workers from west africa which in some cases included imposing isolation and quarantine on asymptomatic health care workers, as well as often sensational coverage by media and its use by politicians, have further damaged the response to the outbreak by delaying needed deployment of personnel. this is not new. all of the above happened in our response to the emergence of aids in the s. public messaging needs to address the uncertainties surrounding the emergence of infectious diseases and promote evidencebased practices. lessons learned during previous pandemics prove that it is difficult to be precise in communication, and that intelligent solutions are needed to deal with uncertainty in our relation with the public. it also points to the importance of engaging with communities and mobilizing societies. simple precautionary behavioral measures are often very effective in reducing exposure to pathogens and the risk of disease transmission. efforts to reduce occupational exposure could be targeted in hotspots of emerging infectious diseases at high-risk animal-human interfaces though standard sanitation and biosafety precautions. this has proved successful in reducing the risk of h n infection in for example livestock workers and food handlers in many agricultural settings (ssematimba et al. ) . finally, infectious disease emergencies put a considerable burden on health workers. in the ebola outbreak not only were doctors and nurses affected, but also ambulance drivers, hospital cleaners, and burial team members. there have been a total of confirmed health worker infections reported from guinea, liberia, and sierra leone since the start of the ebola outbreak, with reported deaths (who, june ) . this points to the challenges in implementing infection prevention and control measures, including the development of infection control protocols, training staff, and obtaining supplies. it remains crucial that we boost recovery efforts in the affected countries, underpinned by building more resilient health systems that are better able to prevent and respond to all hazards. this will also require concerted efforts towards governance and economic development. in addition to the horrific mortality, the ebola outbreak has seriously undermined economic activities and livelihoods throughout west africa. in liberia real gdp growth for , which was projected to be around . per cent was revised below per cent in (world bank ). the ebola outbreak hit three sectors of the economy-agriculture, services, and manufacturing-particularly hard, with these accounting for nearly per cent of the employed labour force in (republic of liberia, april ). critical to recovery will be addressing the unique needs of ebola survivors, including in protecting survivors from stigma and human rights violations and enabling their full reintegration into their communities. comprehensive support is needed for ebola survivors that include medical and psychosocial consequences of the disease. there is also an urgent need to address the risk of sexual transmission of the virus so that the continued transmission of the virus is interrupted, and the risk of reintroduction of the virus in areas where transmission has previously been eliminated is prevented (who ). pathogens will continue to emerge with potential spill-over and even rare events of human to-human transmission, posing the threat of a pandemic. emerging infectious diseases and pandemic threats remain a major global concern. responding to outbreaks and pandemics costs lives and livelihoods, and preventative approaches are urgently needed that are built on resilient systems at the human, animal and ecologies interfaces. it is essential to develop the capacity to anticipate, predict and prevent dangerous pathogen from spilling-over to humans causing pandemic threats. zoonotic diseases should therefore be priority for risk assessment and reduction and management efforts. collaborative multidisciplinary, cross-sectoral work is needed by environmental, public health and animal health agencies to reduce the risk of diseases at their interfaces. this calls for investment in each sector to build their capacities for prevention and control and to coordinate and sustain their collaboration. ecology of zoonoses: natural and unnatural histories prediction and prevention of the next pandemic zoonosis emerging infectious diseases: the role of social sciences avian influenza transmission risks: analysis of biosecurity measures and contact structure in dutch poultry farming. preventive veterinary medicine update on the economic impact of the interim guidance: clinical care for survivors of ebola virus disease public and ecosystem health links: ebola case study key: cord- - sp uo authors: hughes, j. m. title: emerging infectious diseases: the public’s view of the problem and what should be expected from the public health community date: journal: infectious diseases from nature: mechanisms of viral emergence and persistence doi: . / - - - _ sha: doc_id: cord_uid: sp uo nan the public's view of major threats to health, as with other contemporary issues, is largely influenced by the media. as new health-related information is released from the clinical and research communities, it is translated for and disseminated to the public through a variety of mechanisms. in the past, healthcare providers served as the primary source of health-related information for patients. today, however, an unprecedented interest in health issues has led to intense media coverage of medical developments. moreover, the internet has given interested individuals rapid access to virtually unlimited sources of information. because of this symbiotic relationship between public interest and media attention, the actual impact or severity of a public health problem can be disproportionate to the amount of media coverage it receives, creating a climate of unnecessary fear and obscuring important health messages [ ] . in , the centers for disease control and prevention (cdc) released a series of reports describing ten great public health achievements in the united states during the th century [ ] . the topics were chosen based on their impact on reducing death, illness, and disability in the united states, and include advances such as vaccinations, improved maternal and child health, safer and healthier food, fluoridation of drinking water, and safer workplaces. also among this list is control of infectious diseases, resulting from improvements in sanitation, access to clean water, and the development and use of effective vaccines and antibiotics. so dramatic were these advancements that by the middle of the th century infectious diseases were no longer viewed as major public health threats in the united states and in many other developed countries. this false sense of security was short lived, however, as newly recognized and reemerging diseases continued to appear, many of which produced devastating consequences -most notably hiv/aids. advances against infectious diseases have not been universal. worldwide, infectious diseases continue to be a leading cause of death, profoundly impacting the developing world. the world health organization (who) estimates that nearly million ( %) of the approximately million deaths that occurred throughout the world in were caused by microbial agents [ ] (table ) . leading the list are lower respiratory infections, responsible for . million deaths per year, followed by hiv/aids ( . million), diarrhea ( . million), tuberculosis ( . million), and malaria ( . million) [ ] . the true burden of death from infectious diseases, however, is much higher since underreporting remains a major factor, particularly in the developing world. moreover, many deaths associated with infections are not categorized as infection related (e.g., deaths from cancers caused by infectious agents). despite the continued dramatic impact of these global killers, they receive very limited media attention -having become commonplace compared to the new and exotic. one of the reasons for this disparity has been the actual increase in the number of emerging and reemerging infections that have surfaced during the last years (box ). examples include newly recognized diseases such as hantavirus pulmonary syndrome, new variant creutzfeldt-jakob disease, and nipah and hendra viral diseases, the introduction and spread of west nile virus infection in north america, and intermittent outbreaks of ebola hemorrhagic fever in parts of africa. other major concerns include the increasing problems created by antimicrobial resistance and the continued threat of bioterrorism. in alone, a newly recognized coronavirus spread across five continents sickening more than , people and causing deaths from a new disease designated severe acute respiratory syndrome (sars) [ ] , the exotic animal trade resulted in the first cases of human monkeypox in the western hemisphere [ ] , and highly pathogenic strains of avian influenza virus killed humans and devastated the poultry industry in parts of asia [ ] -further heightening fears of pandemic influenza. this continual onslaught of newly identified and reemerging infectious diseases, along with increased concerns on the part of policymakers, the media, and an interested public, has created a new public health perspective and a heightened sense of vulnerability regarding infectious diseases. experiences with both naturally occurring and intentionally caused diseases have clearly demonstrated that infectious diseases can have severe consequences beyond public health, impacting national security and the global economy. local outbreaks are no longer considered limited threats but rather sentinel events capable of having much wider and potentially catastrophic implications. as a result, rapid and collaborative responses to infectious disease outbreaks have become both essential and expected. in , the institute of medicine published a report highlighting the increasing risks to public health posed by emerging microbial threats [ ] . the report, microbial threats to health: emergence, detection, and response, serves as an update to the institute's landmark report on emerging infections [ ] , which issued a strong caution against complacency toward infectious diseases and called for a rebuilding of the nation's public health system. the new report categorizes the spectrum of microbial threats into five areas: the global burden of aids, tuberculosis, and malaria; antimicrobial-resistant infections; vectorborne and zoonotic diseases; chronic diseases with infectious etiology; and microbes intentionally used for harm. the report also describes more than a dozen factors -human, biological, social, and environmental -that can work alone or in combination to produce a global microbial threat. examples of these factors include human demographics, behavior, and susceptibility to infection; changes in technology, industry, travel, and commerce; changing ecosystems and microbial hosts; and social and political factors such as poverty and other inequities, lack of political will, and the consequences of war and terrorism. as if a portent, the release of the iom report in march coincided with the outbreak of sars. the disease would prove to be an archetype of a global microbial threat, spreading rapidly as a result of international travel and requiring an international response to stop its spread. although the earliest notification about the illness came on february , , through a report posted on the program for monitoring emerging diseases, or "promed" [ ], the disease had been occurring in southern china since november -spreading largely to hospital workers who had treated affected patients. the global outbreak began on february , , when a guangdong physician, traveling while ill, spent one night in a hong kong hotel. although the exact modes of transmission are unknown, this individual would infect more than a dozen other hotel guests and visitors, many of whom served as index patients for major outbreaks in hong kong, singapore, vietnam, and canada [ ] (fig. ) . in singapore, more than of the country's sars cases were linked to a single individual who became infected at the hong kong hotel [ ] . much has been learned from these recent outbreaks of emerging infectious diseases, especially sars (box ). despite its tragic health consequences and strong social, economic, and political impact, sars was fortunately not the feared "big one," appearing to spread primarily by droplets during close contact. the sars outbreak uncovered both strengths and weaknesses in global disease detection and response efforts and can therefore serve as a strong warning as well as an opportunity to prepare for future threats [ ] . sars clearly showed the unpredictability of emerging infectious threats and the vulnerability of even the most developed nations. the virus did not respond to treatment, and no vaccine was available. the use of strict isolation and quarantine precautions -some involving tens of thousands of individuals -proved the best means of stopping the epidemic. box . improving preparedness and response: lessons learned from recent outbreaks -strengthening existing and developing new national and international partnerships -training and educating a multidisciplinary workforce -ensuring "full use" of investments -encouraging transparency and political will -fostering a global commitment to address inequities -developing and implementing preparedness plans and research agendas -proactively communicating with health professionals, the media, and the public while the first line of defense in controlling an outbreak remains strong national surveillance systems that can readily detect outbreaks, the sars experience highlighted the importance of global disease detection efforts [ ] . the same interconnected world that enables microbes to rapidly cross borders can also work to effectively stop their spread, providing an opportunity for establishing surveillance systems that can approach real time. for sars, the internationally coordinated response led by who allowed clinical, research, and public health experts around the world to exchange information on the new disease as quickly as it evolved. part of this effort included the who collaborative multi-center research project on sars diagnosis, a network involving more than a dozen laboratories and countries. in less than a month, three of these laboratories determined the cause of the illness -a previously unrecognized coronavirus. also playing a major role in the response was who's global outbreak and response network (goarn), a surveillance and response system of more than organizations worldwide. although goarn responds to dozens of outbreaks in developing countries each year, the sars outbreak represented its first response to an internationally spreading illness [ ] . among goarn's most visible partners are the national influenza centers (http://www.who.int/csr/disease/influenza/ centres /en/). established in the s, this expansive network of more than institutions in over countries is responsible for tracking influenza viruses to guide vaccine development and to recognize variants that may be capable of producing a pandemic. another message clearly indicated from recent emerging and reemerging infectious diseases is the need to strengthen existing and establish new linkages between the human and animal health communities. the majority of pathogens implicated in recent outbreaks, as well as most of those identified as potential bioterrorism agents, are vector-borne or zoonotic microbes, many of which have crossed the species barrier from animals to humans [ , ] (box ). continued urbanization and other environmental and human demographic changes suggest that this emergence of new zoonotic diseases will likely continue, requiring a corresponding convergence of highly trained human and animal health experts to effectively address them. ensuring that these experts have the capacity to respond to a broad range of infectious threats requires recruitment efforts and training programs across a variety of disciplines including clinical, laboratory, epidemiologic, and behavioral research. national and international collaborations among a skilled workforce are critical for improving global disease detection and ensuring an effective response. such investments in human resources must also be met with improvements in research facilities and capacities. the benefits of such efforts can be substantial, extending beyond national borders and allowing for a "dual" or "full" use of resources. in the united states, investments made to strengthen national bioterrorism preparedness and response efforts over the past several years have improved overall preparedness for public health threats. an example is the laboratory response network (lrn), a network of public and private laboratories established in by the centers for disease control and prevention (cdc) to respond quickly to acts of chemical and biological terrorism, emerging infectious diseases, and other emergencies. in , the lrn provided valuable diagnostic services for sars, monkeypox, and avian influenza, in addition to daily monitoring of potential bioterrorist agents. the critical importance of transparency and political will in controlling infectious diseases was also evident during the sars outbreak. china's months-long delay in reporting the outbreak not only prevented efforts to contain the epidemic locally but also proved most costly for its own region. in contrast was vietnam, one of the earliest countries affected by the outbreak and the first to contain it [ ] . dr. carlo urbani, an infectious disease physician working in hanoi for who, recognized the unusual severity of the disease and quickly instituted infection control precautions, sadly too late to prevent his exposure to the infection that would cause his death. dr. urbani's prompt recognition along with vietnam's commitment and global cooperation effectively limited the spread of sars in vietnam. china ultimately demonstrated one of the most extraordinary acts of political will in addressing the epidemic when more than , construction workers built a -bed hospital in approximately one week. the importance of political will in addressing infectious diseases continues to be demonstrated most directly by its absence -an all too frequent obstacle to eradication efforts for vaccine-preventable diseases such as polio and measles. closely tied to political will is a commitment on the part of high income countries to help address inequities -the social, economic, and health disparities that contribute to the spread of infectious diseases [ , ] . in , at the united nations millennium summit, representatives from nearly u.n. member states resolved to help end human poverty and its ramifications. termed the "millennium development goals," this agreement requires countries to increase their efforts to address inadequate income; lack of food, clean water, and health care; substandard education; gender inequality; and environmental degradation. the goals also call for renewed commitment in addressing the disproportionate impact of infectious diseases on many of the world's poorest regions.a more recent undertaking is "the grand challenges in global health" initiative, funded by the bill and melinda gates foundation and administered by the foundation for the national institutes of health. this initiative was established in to help develop solutions to critical problems that perpetuate the spread of disease in the developing world. such international undertakings directed toward the diseases causing the greatest morbidity and mortality in the developing world should be priorities for wealthier countries. in addition to meeting enormous humanitarian needs, efforts to address these daunting global killers can help remove major obstacles to economic growth and development, thereby strengthening public health infrastructures and disease detection capacities worldwide. perhaps most evident during the sars outbreak was the crucial need for rapid dissemination of accurate information -both for the medical and scientific experts confronting the epidemic and for a concerned public. during the sars epidemic, the availability of electronic communications enabled networks of laboratory scientists, clinicians, and public health experts to share information and rapidly generate a scientific basis for public health action against a novel disease [ ] -a major step toward lessening the health consequences of the outbreak. these extraordinary efforts and swift actions, however, did not prevent the severe social and economic ramifications that resulted from sars. these consequences, largely generated by the fears and perceptions of a vulnerable public, highlight the critical need to communicate timely and accurate information in the face of scientific uncertainty. proactive communications directed at health professionals can enhance the ability of those on the front lines to detect the unusual -e.g., test results or patient symptoms that could signal the occurrence of a new health threat. similarly, proactive and open communication between public health officials and policymakers is essential for sound public health action. finally, proactive communications through public health websites and with the media can help ensure broad dissemination of timely and accurate risk information to members of the public that can enable them to make important decisions in protecting their health. perceived threats and real killers ten great public health achievements -united states the world health report : changing history. world health organization the severe acute respiratory syndrome the detection of monkeypox in humans in the western hemisphere outbreak news: avian influenza a (h n ) lederberg j (eds) for the committee on emerging microbial threats to health in the st century, board on global health, institute of medicine ( ) microbial threats to health: emergence, detection, and response the committee on emerging microbial threats to health, division of health sciences policy, division of international health, institute of medicine ( ) emerging infections: microbial threats to health in the united states update: outbreak of severe acute respiratory syndrome -worldwide severe acute respiratory syndrome -singapore the international response to the outbreak of sars in population biology of emerging and re-emerging pathogens severe acute respiratory syndrome (sars) -multicountry outbreak -update global health improvement and who: shaping the future the sars response -building and assessing an evidence-based approach to future global microbial threats key: cord- -qm kalyt authors: chowdhury, fazle rabbi; ibrahim, quazi shihab uddin; bari, md. shafiqul; alam, m. m. jahangir; dunachie, susanna j.; rodriguez-morales, alfonso j.; patwary, md. ismail title: the association between temperature, rainfall and humidity with common climate-sensitive infectious diseases in bangladesh date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: qm kalyt bangladesh is one of the world’s most vulnerable countries for climate change. this observational study examined the association of temperature, humidity and rainfall with six common climate-sensitive infectious diseases in adults (malaria, diarrheal disease, enteric fever, encephalitis, pneumonia and bacterial meningitis) in northeastern bangladesh. subjects admitted to the adult medicine ward of a tertiary referral hospital in sylhet, bangladesh from to with a diagnosis of one of the six chosen climate-sensitive infectious diseases were enrolled in the study. climate-related data were collected from the bangladesh meteorological institute. disease incidence was then analyzed against mean temperature, humidity and average rainfall for the sylhet region. statistical significance was determined using mann-whitney test, chi-square test and anova testing. patients were enrolled ( % male, % female, ratio . : ). all six diseases showed highly significant (p = . ) rises in incidence between the study years ( cases) and ( cases), compared with no significant rise in overall all-cause hospital admissions in the same period (p = . ). the highest number of malaria ( ), diarrhea ( ) and pneumonia ( ) cases occurred during the rainy season. on the other hand, the maximum number of enteric fever ( ), encephalitis ( ) and meningitis ( ) cases occurred during autumn, which follows the rainy season. a positive (p = . ) correlation was observed between increased temperature and the incidence of malaria, enteric fever and diarrhea, and a negative correlation with encephalitis, meningitis and pneumonia. higher humidity correlated (p = . ) with a higher number of cases of malaria and diarrhea, but inversely correlated with meningitis and encephalitis. higher incidences of encephalitis and meningitis occurred while there was low rainfall. incidences of diarrhea, malaria and enteric fever, increased with rainfall, and then gradually decreased. the findings support a relationship between weather patterns and disease incidence, and provide essential baseline data for future large prospective studies. a a a a a global warming is not a myth, rather a reality [ , ] , and the impact of climate change is multidimensional on health. the world health organization (who) recently reported an estimated . million deaths each year due to unhealthy environments, particularly climate change and pollution [ ] . the report also identifies diarrheal diseases, respiratory infections and malaria in the top ten causes of environment related deaths [ ] . climate change influences the emergence and re-emergence of many infectious diseases [ , , , ] . dengue fever, tickborne diseases, diarrheal disease, enteric fever, viral encephalitis, respiratory tract infections, and meningitis are much increased in recent times [ ] [ ] [ ] [ ] ] . in addition, emerging and reemerging diseases such as chikungunya and zika, have also been linked to climate change influences, and have been proposed as partially responsible for autochnous transmission in places not traditionally endemic for such diseases [ , ] . analysis of global temperature data led the intergovernmental panel for climate change (ipcc) to the conclusion that the average global temperature over land and ocean surfaces has risen by . ˚c in the period from to [ ] . they predicted that global surface temperature will increase by . ˚c compared to the year by the end of the st century ( - ) [ ] . climate change is predicted to directly influence zoonotic infectious disease transmission by changing the geographic range of a vector [ ] . altered climatic conditions may increase vector biting rate and the reproduction rate of the vector and shorten the pathogen incubation period [ ] [ ] [ ] [ ] . climate-related increases in sea surface temperature and sea levels can lead to higher incidence of waterborne infectious diseases [ , , ] . as a low-income country, bangladesh itself plays very little role in the process of global warming, but becomes one of the most seriously affected victims of climate change. bangladesh is the biggest delta and contains the second largest river basin in the world [ ] . the majority of the land is low and flat, and only % lies over one meter above the mean sea surface [ ] . because of monsoon weather and the presence of the bay of bengal in the south, extreme weather events like flood and cyclone are common [ , ] . this unique geographic and topographic location makes it reportedly the most vulnerable country to climate change effects [ ] . lack of resilience and adaptive capacity, dense population and poverty make the situation worse [ ] . unfortunately, very few studies on the relationship between various environmental variables and trends of infectious disease incidence have been performed so far in bangladesh, although there are reports of some infections increasing sporadically in different regions of the country [ ] [ ] [ ] . climate change and health related studies are so far mainly reported from developed countries, but studies from vulnerable countries are still meagre [ , ] . furthermore, published studies typically only focus on a single disease. this study examined six infectious diseases based on clinical syndromes and laboratory support (malaria, enteric fever, encephalitis, diarrheal disease, pneumonia and meningitis) to offer a broader scope on the trend of these infectious diseases and their possible relation to climate change in bangladesh. we chose these six diseases based on the reports of ipcc ( ) and who ( ) where they were listed as climate-sensitive infectious diseases important for asia [ ] [ ] [ ] . the main objective of the study was to see the burden of the six climate sensitive diseases over five years and to analyze the possible relationship of them with common climatic variables. the findings will be of interest to public health experts and policy makers to stimulate effective measures to combat infectious diseases and related epidemics in bangladesh and in other vulnerable countries. sylhet divison in northeast bangladesh, an area of approximately , km with a population of around million. all case files of the adult medicine ward from to were enrolled from the hospital archive. the diagnosis of the six studied diseases were confirmed based on the combination of clinical and relevant laboratory diagnosis. incomplete files, patients discharged on risk bonds, patients who died within hours (due to insufficient time to reach a diagnosis) and absconded patients were excluded from the study. square p< . ) whereas encephalitis occurred more frequently in females in females ( % vs. %, chi square p< . ) ( table ). the highest percentage ( %; ) of disease over the five years studied occurred in the th year ( ). there was a trend towards increasing number of cases of the six infectious diseases studied over the five years ( fig a) and there is a significant difference (p = . ) between the total number of cases in ( ) and ( ). all six infectious diseases individually increased in number over the five years, especially pneumonia which rose from cases in to cases in . this increase does not appear to be due to increased numbers of patients using the hospital overall. in comparison, the number of admissions for all causes to the hospital and to the medicine department over three of the years of study from to (data unavailable for and ) did not change (mann-whitney u test p = . , fig b) . the highest number of cases occurred in autumn ( ; %) followed by rainy season ( ; %), winter ( ; %) and summer ( ; %). pneumonia ( ; %) was the most common disease of the six, and malaria ( ; %) was the least prevalent disease. individually, the highest number of malaria ( ), diarrhea ( ) and pneumonia ( ) cases occurred in rainy season. on the other hand, the maximum number of enteric fever ( ), encephalitis ( ) and meningitis ( ) cases occurred during autumn (fig ) . the mean temperature in our study region during - was . ˚c, and there was no significant difference in the number of cases occurring below or above that level (table ). in , the average temperature was more than ˚c in eight out of twelve months (fig ) . there was a significant positive correlation between increasing temperature and the incidence of malaria (p = . ), and a trend for positive correlation between increasing temperature and the incidence of diarrhea (p = . ). there was a significant negative correlation between increasing temperature and incidence of encephalitis (p = . ), meningitis (p = . ) and pneumonia (p = . ). table shows the association of the six diseases with the average humidity of the studied period of to . we analysed the incidence of each disease in each of three groups of humidity recordings by month: less than . %, . - . % and more than . % based on the mean humidity of the study site. less than . percent humidity was associated with the highest percentage ( ; %; p = < . ) of all the six studied diseases (table ) . average humidity of . - . % and > . % was associated with % ( ) and % ( ) disease respectively. however, in , the average humidity was ! . % in nine out of twelve months (fig ) . higher humidity was correlated with a higher number of cases of malaria (p = . ), enteric fever (p = . ) and diarrhea (p = . ), but inversely correlated with meningitis (p = . ), encephalitis (p = . ) and pneumonia (p = . ). the average rainfall by month was divided into three groups, less than mm, - mm and more than mm in table . the highest percentage ( ; . %) of all the six diseases studied were related with less than mm of average rainfall. % ( ) malaria, % ( ) enteric fever, % ( ) diarrhea, % ( ) encephalitis, % ( ) pneumonia and % ( ) meningitis fell under this category (chi-square p = . ). average rainfall of - mm and > mm was associated with % ( ) and % ( ) of the total disease burden respectively. analyzing the data of , in six out of twelve months, there were mm average rainfall (fig ) . higher incidences of encephalitis (p = . ) and meningitis (p = . ) happened while there was low rainfall. incidences of diarrhea (p = . ), malaria (p = . ), pneumonia (p = . ) and enteric fever (p = . ) increased with rainfall, and then gradually decreased. ( - ) . the highest number of malaria, diarrhea and pneumonia cases occurred in rainy season, whilst the maximum number of enteric fever, encephalitis and meningitis recorded during autumn. student t test was applied to obtain the level of significance. https://doi.org/ . /journal.pone. .g table . association of disease with the temperature of five years ( - ). temperature previous studies in bangladesh have either focused on a single disease or relied on people's perception of climate change and infectious disease. to our knowledge, this study is the first observational study which specifically focuses on individual infectious diseases and explores the relationship between each disease and three common weather variables. the male to female ratio in this study was . : . although there are no published data, it is generally believed that more males come into hospital in bangladesh because traditionally females wait at home with their disease until complications develop [ , ] . it is likely that women get less opportunity to come into hospital and have less access to medical care. there was no significant difference by gender in the frequency of each individual disease except pneumonia and encephalitis, which significantly affected a greater proportion of the male and female population respectively. the increase in pneumonia cases seen in males may be because men have more occupational exposure to conditions that increase the chance of receiving a diagnosis of pneumonia such as dust, fumes, smoking etc. all six diseases showed a significant (p = . ) rise in incidence between the study years and . the number of cases more than doubled for all diseases except enteric fever, which also showed significant increases. in contrast, during this same period the total number of admissions to the medical ward and to in the hospital showed no significant rise. we propose that the rise in frequency of the studied diseases could be explained by the influence of weather changes. it is important to note that during this period no epidemics of the studied diseases happened in the sylhet region. the total impression is consistent with previous reports. in a review of the literature, the incidence of pyogenic meningitis, encephalitis and dengue was predicted to be greatly influenced and increased by global warming in the coming years [ ] . w.h.o reported dengue, viral encephalitis, diarrheal disease, enteric fever, pneumonia and meningitis as most sensitive to climate factors, and predicted a huge rise of cases in tropical countries [ ] . climate change cell, bangladesh reported that, from to there were , cases of malaria in bangladesh, but from to it increased to , ( % increased incidence) [ ] . although after the introduction of artemisinin treatment and government and other partner organization lead massive drive for malaria elimination, the cases decreased to . / population in bangladesh [ ] . the same report revealed an increasing trend for diarrheal diseases, kala-azar and skin diseases in three districts (drought-prone rajshahi, flood-prone manikganj and salinity-dense satkhira) of bangladesh between to [ ] . the report also described a positive correlation between rainfall and diarrheal and skin disease in rajshahi and satkhira, and a negative correlation of diarrheal disease with temperature [ ] . another focus group discussion (fgd) based study reported an increased number of diarrheal diseases, typhoid and skin problems after the cyclone sidr and aila in southern part (barguna and khulna) of the country [ ] . we found the highest number of malaria ( ), diarrhea ( ) and pneumonia ( ) cases occurred during the rainy season. the findings are consistent with other national and international studies. highest cases of falciparum malaria were found in north-eastern india during higher humidity was correlated with a higher number of cases of malaria, enteric fever and diarrhea, but inversely correlated with meningitis, encephalitis and pneumonia. two-way anova test was applied to obtain the level of significance. https://doi.org/ . /journal.pone. .g temperature, rainfall, humidity and climate-sensitive infectious diseases in bangladesh the rainy season [ ] . in the chittagong hill tract districts of bangladesh, where malaria is most endemic, the frequency of cases was highest in rainy season [ ] . an increased incidence of malaria in north-west of india has been suggested through computational modelling [ ] . studies in africa revealed mixed results, with the highest number of malaria cases during the rainy season in mali, but most cases during autumn in northern ghana [ , ] . according to the ipcc report, respiratory infections also follow a seasonal pattern [ ] . in tropical settings, where most deaths due to pneumonia occur, the incidence of lower respiratory tract illness in children is generally increased during rainy season and it supports our findings [ ] . a thai study of viral pneumonia reported the highest number of cases in rainy season [ ] , in line with our findings. this pattern of increased pneumonia cases during rainy season in tropical countries contrasts with the well described increase in pneumonia seen during colder months in temperate climates [ , ] . for diarrheal disease, our findings are supported by previous reports of increased incidence during the rainy season in taiwan ( ) and bangladesh ( ) . we found a large number of enteric fever cases ( ; %) occurred in rainy season and autumn. this agrees with earlier studies in both dhaka, bangladesh ( ) where the highest number ( %) of enteric fever occurred during monsoon period [ ] ,and a cambodian study [ ] but no relationship between incidence of enteric fever and season was seen in kenya [ ] . in our study, most meningitis ( ; %) and encephalitis ( ; %) occurred during autumn and winter. this finding is partly consistent with other studies done in africa. meningitis epidemics in west africa occurred during the coolest season [ ] . a recent time series analysis over countries found that bacterial meningitis season peaks during the winter months, [ ] , similar to our findings. a major causative organism of meningitis (neisseria meningitides) was found to be high and active during dry periods in the presence of dust and were then washed away with rainfall, so as the case frequency fell down [ , ] . however, in our study, we found an almost equal number of meningitis cases in rainy season compared to other seasons. regional findings of seasonality in terms of encephalitis are also supportive of our findings. highest number of encephalitis cases in rainy period (august-september) were seen in nepal [ ] . in india, the incidence of japanese encephalitis was also highest during august to november (rainy and early winter) with a peak in october [ ] . another study from china also reported similar findings [ ] . the current rate of increase of the annual minimum temperature (by . ˚c) is higher than that of the annual maximum temperature (by . ˚c) in bangladesh [ ] . the annual average rainfall is increasing by . mm per decade outside the usual rainy period, while rainfall during the season is decreasing by . mm per decade [ , ] . in our study, we found a positive correlation between the incidences of malaria, enteric fever and diarrhea with increasing temperature. these results are similar to other national and international findings. a previous study in chittagong, bangladesh showed increased malaria cases with increasing temperature table . association of disease with the rainfall of study years ( - ). temperature, rainfall, humidity and climate-sensitive infectious diseases in bangladesh [ ] . studies have shown a positive relationship between increased malaria and increasing temperature [ , ] . studies in south asia and south america (venezuela and columbia) have documented the association between malaria outbreaks and the el nino southern oscillation (enso) cycle [ ] . a significant increase in malaria cases with increasing temperature was seen in rwanda and uganda [ , ] , but this was not seen in studies done in the african highland region [ , ] . increased temperature allows faster replication of mosquito incidences of diarrhea, malaria, pneumonia and enteric fever increased with rainfall. two-way anova test was applied to obtain the level of significance. https://doi.org/ . /journal.pone. .g temperature, rainfall, humidity and climate-sensitive infectious diseases in bangladesh populations [ , ] . higher temperatures also change human behavior, for example more outdoor activities may be undertaken which further increases the risk of exposure [ ] . diarrheal cases including dysentery have been found to be higher in high temperature in bangladesh, taiwan and china [ ] [ ] [ ] . a study in dhaka, bangladesh showed an increase number of enteric fever cases with an increase temperature which supports our findings [ ] , as does a similar study from southern australia [ ] . furthermore, a strong linear association has been noted between temperature and notification of salmonellosis globally [ ] . temperature affects the transmission of food-borne disease in various ways. the temperature directly affects the rate of replication of bacterial and protozoan pathogens and the survival of enteroviruses in the environment [ , ] . in addition, these variations may also have a significant impact on the environmental reservoirs of infection as well as human behavior [ ] . moreover, both salmonella and cholera bacteria, for example, proliferate more rapidly at higher temperatures; salmonella in animal gut and food, cholera in water [ , ] . our study shows an inverse relationship between encephalitis, meningitis and pneumonia with temperature. the pneumonia findings are consistent with a study done in china, but not with another study conducted in spain [ , ] . for encephalitis and meningitis our findings differ to other studies done in india, china and niger where they found increased evidence of cases and vector during hot environment [ , , ] .viral encephalitis cases in sweden have reportedly increased in response to a succession of warmer winters over the past two decades [ ] . this inverse relationship with temperature in our study merits further exploration. in recent decades, more heatwaves have been reported in south asia, which also bring a change in humidity [ ] . eighteen heatwaves were reported in india between and [ ] . our study found a higher trend of malaria, enteric fever and diarrhea cases with higher humidity. the findings are further supported by studies done in chittagong, bangladesh and china with similar trend [ , ] . relative humidity influences biological and feeding behavior of mosquitoes [ ] . at higher humidity, mosquitoes generally survive for longer and disperse further [ , ] . higher humidity also affects the rate of replication of bacterial and protozoan pathogens and their survival in the environment [ ] . we did not find any influence of humidity on enteric fever and pneumonia, in line with other studies [ , ] . the incidence of pneumonia, encephalitis and meningitis is inversely related to humidity in this study. in india an inverse association was seen between the number of culex mosquitoes (the vector for japanese encephalitis) and humidity [ ] , although in china, higher cases of encephalitis was seen with higher humidity [ ] . these mixed findings may reflect differences in etiology of encephalitis and bacterial meningitis, as well as differences in case definitions between study sites. these could also explained by diagnostic limitations, and in some cases the meningoencephalitis syndrome may be caused by bacteria favoring dry and dusty weather. heavy rainfall can have a diverse range of effects on disease. for example, in tropical and subtropical regions with crowding and poverty, heavy rainfall and flooding may trigger behavioral changes such as increased contact between people and distribution of pathogens in floodwater, leading to outbreaks of diarrhea [ ] . we found that the incidence of diarrhea, malaria, enteric fever and pneumonia increased with rainfall. for diarrhea, our findings are consistent with other national and international studies [ ] [ ] [ ] . a positive relationship between increased rainfall and the incidence of enteric fever was also found in dhaka, bangladesh and southern australia [ , ] . one possible explanation is, heavy rainfall may affect the frequency and level of contamination of drinking water and hence the spread waterborne infections [ , ] . area with existing high burdens of infectious disease and poor sanitary infrastructure often experience increased rates of diarrheal diseases after heavy rainfall [ ] . our findings in terms of malaria are also similar to other national and regional studies [ , ] . rainfall plays an important role in the transmission of malaria, as mosquitoes need water to support the larval and pupal stages of development [ , ] . a study also found higher deaths from acute lower respiratory tract illness during rainy season in a pediatric population [ ] . in - , the highest incidences of encephalitis and meningitis occurred while there was very low rainfall. our findings are supported by other reports in the case of meningitis [ , ] . nevertheless, researchers found the opposite for japanese encephalitis, where the vector replicates and transmits more disease with high rainfall [ , ] . we acknowledge a limitation of this study is the relatively short timeframe. extending our study duration to ten years would have allowed greater power to detect differences, but we did not have the resources to do this. another limitation is that due to the retrospective design of the study with recruitment from the hospital archive, we relied on the diagnosis in the patient notes, which in some instances may be wrong. we tried to overcome this limitation by setting case definitions for enrollment in the study and included only the cases who survived for more than hours, where there was more clinical information to make an informed diagnosis. the death rate in the first hours due to these infections (bacterial meningitis- . %; encephalitis . % and all-cause mortality . %) during that period was low and unlikely to influence our results [ ] . due to limitations of funding and time, we conducted this study in only one hospital of northeastern region of bangladesh. further studies in other regions of bangladesh are highly desirable to represent the situation across the country. this study reported the influence of temperature, humidity and rainfall on six climate sensitive infectious diseases in the northeastern region of bangladesh. weather and climate extremes affect all sectors of the health, economy and development. the findings can be used as a baseline to launch a large cohort study throughout the country in future. this study is pivotal in giving direction to our public health experts, clinicians and other policy makers to update and change future strategies to combat the burden of climate-related health events in bangladesh and other countries. climate change synthesis report summary for policy makers climate change : impacts, adaptation and vulnerability cambridge: inter governmental panel on climate change an estimated . million deaths each year are attributable to unhealthy environments world health organization impact of climate change on health the socio-ecology of zoonotic infections. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases health and climate change: a roadmap for applied research el nino and climate change-contributing factors in the dispersal of zika virus in the americas? autochthonous chikungunya transmission and extreme climate events in southern france climate changes, environment and infection: facts, scenarios and growing awareness 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adelaide climate change and local public health in the united states: preparedness, programs and perceptions of local public health department directors gender difference in treatment seeking behaviors of tuberculosis cases in rural communities of bangladesh. the southeast asian journal of tropical medicine and public health rural women's access to health care in bangladesh: swimming against the tide? social work in public health reducing the impact of climate change using climate to predict disease outbreaks: a review reduction in malaria prevalence and increase in malaria awareness in endemic districts of bangladesh climate change impact: the experience of the coastal areas of bangladesh affected by cyclones sidr and aila rainfall and malaria transmission in north-eastern india relationship between climate change and incidence of malaria in chittagong hill tracts forcing versus feedback: epidemic malaria and monsoon rains in northwest india season, fever prevalence and pyrogenic threshold for malaria disease definition in an endemic area of mali seasonal profiles of malaria infection, anaemia, and bednet use among age groups and communities in northern ghana childhood pneumonia: a neglected, climate-sensitive disease? incidence, seasonality and mortality associated with influenza pneumonia in thailand do seasonal changes and climate influence the etiology of community acquired pneumonia? what is the seasonal distribution of community acquired pneumonia over time? a systematic review typhoid fever and its association with environmental factors in the dhaka metropolitan area of bangladesh: a spatial and time-series approach the molecular and spatial epidemiology of typhoid fever in rural cambodia population-based incidence of typhoid fever in an urban informal settlement and a rural area in kenya: implications for typhoid vaccine use in africa climate drives the meningitis epidemics onset in west africa seasonal dynamics of bacterial meningitis: a timeseries analysis environmental risk and meningitis epidemics in africa dust clouds and spread of infection japanese encephalitis in hill and mountain districts a long-term study on vector abundance & seasonal prevalence in relation to the occurrence of japanese encephalitis in gorakhpur district weather variables and japanese encephalitis in the metropolitan area of jinan city temperature and precipitation projections over bangladesh and the upstream ganges, brahmaputra and meghna systems knowledge and perception about climate change and human health: findings from a baseline survey among vulnerable communities in bangladesh trends in extreme rainfall events of bangladesh theoretical and applied climatology how malaria models relate temperature to malaria transmission optimal temperature for malaria transmission is dramatically lower than previously predicted climate change and human health: present and future risks highland malaria in uganda: prospective analysis of an epidemic associated with el nino climatic warming and increased malaria incidence in rwanda climate change and the resurgence of malaria in the east african highlands climatic suitability for malaria transmission in africa, - climate anomalies and epidemics in south america at the end of the colonial period modeling the impact of climate variability on diarrhea-associated diseases in taiwan association between climate variability and hospital visits for non-cholera diarrhoea in bangladesh: effects and vulnerable groups climate variations and bacillary dysentery in northern and southern cities of china climate variations and salmonellosis transmission in adelaide, south australia: a comparison between regression models weather and the transmission of bacillary dysentery in jinan, northern china: a time-series analysis weather: driving force behind the transmission of severe acute respiratory syndrome in china? the effects of climatic factors on the distribution and abundance of japanese encephalitis vectors in kurnool district of andhra pradesh, india severe heat waves over the indian subcontinent in , in perspective of global climate current science potential impact of macroclimatic variability on the epidemiology of giardiasis in three provinces of cuba ministry of health and family welfare (mohfw): sylhet m. a.g.osmani medical college hospital we thank the staff of the hospital archive and administration departments of somch for their cooperation. we are also grateful to the nurses and doctors of all the units of medicine department for their support. key: cord- - uptbat authors: evermann, james f.; eriks, inge s. title: diagnostic medicine: the challenge of differentiating infection from disease and making sense for the veterinary clinician date: - - journal: adv vet med doi: . /s - ( ) - sha: doc_id: cord_uid: uptbat nan diagnostic medicine has taken on a new, broader meaning in the s and reflects an expansion of clinical investigation from the diagnosis of disease to include detection of infection (evermann, ) . this leads to an entirely new perspective on how veterinary clinicians and diagnosticians view laboratory tests and how test results are interpreted. one must consider not only the specificity and sensitivity of the test, but the predictive value of the test, which relates directly to the clinical utility of the result (jacobson, ) . the definitive diagnosis of infectious diseases relies on a combination of clinical symptoms, history, and laboratory analyses of ante-mortem and/or postmortem specimens (evermann, ) . disease diagnosis has customarily used diagnostic assays for early recognition of disease and rapid implementation of therapy in an individual animal basis, and when appropriate use of corrective management (segregation, culling, vaccination, etc.) on a population basis. the detection of infection during preclinical stages has become more important as one considers the consequence of long-term infections that have prolonged incubation periods and inapparent transmission to susceptible animals in the population. this includes lifethreatening diseases, such as feline infectious peritonitis (fip), rickettsial and ehrlichial diseases and canine herpesvirus (chv) infections. of equal, if not more so, importance for the early detection of infection has been the increased recognition of zoonotic infections, such as rabies virus, salmonella typhimurium dt , and escherichia coli o :h (evans and davies, ; slutsker et al., ; smith, ) . together with the necessity to detect infections earlier during the preclinical stages, there has been a remarkable expansion in the availability of assays that can detect infectious microorganisms in low quantity. this increased sensitivity has been primarily through the detection of nucleic acid sequences after amplification by polymerase chain reaction (pcr, relman and persing, ) . pcr can allow not only early detection of infection, but rapid speciation of organisms as well as strain typing for epidemiologic analyses (fredricks and relman, ; mcdade and anderson, ) . the assessment of preclinical infections allows the veterinarian the opportunity to determine the relative risk of disease occurring, and to take preventive steps to reduce or eliminate the risks depending on the consequences of the disease in the animal and/or human (if zoonotic) population. this chapter focuses on one main issue, and that is differentiating the detection of infection from diagnosis of disease. in the course of differentiating infection from disease three questions will be addressed: ( ) how early do we want to detect infection? ( ) what are the consequences of the results? ( ) where are we heading with veterinary diagnostics? historically, the primary aim of the diagnostic laboratory was to assist the veterinarian in the diagnosis of disease. this is presented in fig. . this type of approach initially ignored the origin of the causative microorganisms and focused on the accurate diagnosis of the disease agent. an example of this type of approach was the testing of cats that were clinically ill for feline leukemia virus (felv). if tested positive, they were segregated or euthanized. further examples include fip, chv, johne's disease (mycobacterium paratuberculosis), and the mucosal disease form of bovine viral diarrhea (bvd) virus. expanded use of diagnostic results by the veterinarian and client allowed for some corrective management steps to be taken. these included the use of vaccination when available or segregation and culling to reduce the source of the infection in the population. based on this latter principle, the reduction of the source of the infection, a different approach has been taken. one may consider this an epidemiologic view of the disease process (susser and susser, a) . with a combination of more sensitive diagnostic assays, the veterinarian's concern to know the state of the preclinical infection, econom- ic incentives to minimize disease by effectively controlling the infection, and concern over potential zoonotic diseases, laboratory diagnosis has taken on a different strategy. this is presented in fig. . the primary emphasis in this scheme is to view animals preclinical and determine the disease risk and/or zoonotic potential of the infection. this has been the approach for some retroviral infections (evermann and jackson, ; knowles, ) and bacterial infections with public health concerns, such as e. coli o :h and salmonella spp. infections (evans and davies, ; firstenberg-eden and sullivan, ; mcdonough and simpson, ) . the ultimate goal of the assessment of preclinical testing is to initiate a preventive management type of control. this type of approach places more emphasis on early testing and management of infected animals rather than on diseased animals. with the shift in emphasis to preclinical testing, the knowledge of the ecology of the infectious microorganism has become very important in our overall understanding of how successful the control program may be (susser and susser, b) . the control of infections with a low degree of transmissibility and narrow host range, such as caprine arthritis-encephalitis (cae) virus, is much more realistic than the control of diseases with a wide host range, such as chlamydia and salmonella spp., or those agents spread by arthropod vectors, such as arboviruses and rickettsia (gregory and schaffner, ; hewinson et al., ; knowles, ; raoult and roux, ; saluzzo and dodet, ) . the ecology of infection provides the veterinarian with vital information with which to make decisions. the ecology of six different agents is listed in table i . the ecology of infection is divided into infection rate, attack rate (progress to become clinical), and mortality rate. it can be seen that the infection rate usually exceeds the attack rate and mortality rate in the majority of cases. exceptions to this generalization are the mucosal disease form of bvd that occur in cattle that are tolerant to bvd and persistently infected (pi), and rabies infections in mammals (innocent et al., ; smith, ) . another way to view the ecology of an infection is demonstrated in fig. . the schematic allows the veterinarian to readily use a graphic approach with clients to explain the differences between infection and disease. rabies virus is used as an example of a microorganism with a low infection rate, but high mortality. (this figure would be different if one were to diagram the ecology of rabies in bats, the natural reservoir for rabies in the united states.) the cae virus is used as an example of an infection in goats with a high infection rate, but lower attack rates, and much lower fatality rates (fig. ) . with retroviruses, such as cae virus, bovine leukosis virus (blv), and equine infectious anemia (eia), the ecology can also be subdivided into progressor (progress onto clinical signs and/or fatality) or nonprogressor (remains clinically normal, but infected and potentially contagious). with persistent bacterial infections, such as salmonella and many members of the rickettsiaciae, the ecology can be subdivided into clinical disease leading to mortality or clinical disease leading to a chronic carrier state. this chronic carrier state can then be further subdivided into inapparent infections with constant shredding and inapparent infections with intermittent shedding. with potentially zoonotic diseases, such as salmonella, rickettsioses, and psittacosis (chlamydia psittici), the ability to shed or transmit the organism into the environment or vectors becomes particularly relevant (evans and davies, ; gregory and schaffner, ; raoult and roux, ) . early detection of infection is now feasible with a number of microorganisms affecting animals. the detection may take the form of specifically identifying the nucleic acid of the infectious agent, such as bovine herpesvirus- in semen samples, blv provirus is selected blood cells populations, and foodborne bacteria in dairy products (batt, ; masri et al., ; xie et al., ) . although this form of early microbial detection is preclinical at this time, with further research it may be determined that these "subclinical infections" are actually causing alterations in cell structure and function leading to endocrine imbalances and decreased productivity. this form of disease has been referred to a "lesionless pathology," and will be the subject of further research (de la torre and oldstone, ) . early detection of infection may take a "back door" approach by analyzing the host animal's genetic predisposition to infection and disease. this interesting approach has already been used in order to control the prion disease, scrapie (o'rourke et al., ) . sheep with a unique chromosome are highly susceptible to progress onto scrapie, an irreversible disease. animals that are bred for genetic resistance to infection and/or disease will be major factors in disease control in the future (gavora, ; malo and skamene, ) . the utilization of genetic testing is essential for some infections, such as the retroviruses, which serve to activate cellular oncogenes and promote disease. identifying these cellular oncogenes would be a major step in controlling retroviral-induced diseases (wiedemann et al., ) . it will be essential to clearly define what the diagnostic assay is detecting so that the veterinarian may utilize the information appropriately. figure a graphically presents the use of thresholds to differentiate subclinical infection from clinical manifestations of the disease. figure b shows five potential diagnostic assays, each with varying levels of sensitivity. it would be critical to understand the differences between a test with high sensitivity, which detects subclinical infection and a test with lesser sensitivity, but more accurately diagnoses disease. this question becomes more difficult the more one employs preclinical testing in preventive medicine programs (clementi et al., ; jacobson and romatowski, ; smith, ) . the predictive value of a positive result may be high when an animal is clinical, such as a cat with fip. however, with early testing the problems of detecting cross-reacting viruses (feline enteric coronaviruses) increases, as does the question of whether the preclinical result accurately identifies an animal that is just infected or will progress onto disease (evermann et al., ; foley et al., ) . in infections such as eia, the consequences of infection are just as severe as the horse that has clinical signs of eia. this is because the infection is regulated by the u.s. department of agriculture and all seropositive horses and mules are required to be reported, regardless of their health status. assays for early detection of eia infection have been reported to detect viral rna in plasma samples as early as hours after infection (langemeier et al., ) . similarly in bovine tuberculosis, caused bymycobacterium bovis, the consequences of a positive test result can be economically devastating due to stringent government regulations. this becomes particularly problematic because many tests currently available may cross-react with other mycobacterial species (essey and koller, ; o'reilly and daborn, ) . to determine what consequences the test results will have on the animal and the owner it is important to ask five key questions (table ii) . is the infection and/or disease of economic concern, such as eia or m. bovis; is the infection and/or disease of zoonotic concern, such as e. coli :h ; where is the microbial agent when not causing disease, such as with rabies reservoirs in bat populations; what are the contributing factors to the infection and/or disease process, such as pregnancy for chv and other herpesviruses; and what factors can animal owners/veterinarians/public health personnel control to minimize or eliminate the risk of infection and/or the disease process? table iii lists some of the consequences of the infection and/or disease process. these range from no sale, as with a goat that is cae seropositive, to euthanasia if a horse or mule is tested eia seropositive. v. where are we heading with veterinary diagnostics? veterinary diagnostics, like their human counterparts, are already directing efforts toward more sensitive assays, which are capable of detecting infections very early (within hours of initial infection); subclinical infections that are the result of persistent infections acquired during gestation and masked by immune tolerance; latent infections due to herpesviruses and retroviruses; and infections that pose a public health risk (barrett et al., ; burr, ; clarke, ; de la torre and oldstone, ; rodriquez, ) . the evolution of diseases and the emergence of newly recognized pathogens have placed considerable pressure on new diagnostic technologies. the newer assays will assist in tracking the emerging infections, as well as linking causal association with disease to a firm cause and effect of the disease (bryan et al., ; holtzman et al., ; hoet and haufroid, ; lipstich et al., ; mcdade and anderson, ; poland et al., ) . . is the infection and/or disease of economic concern? . is the infection and/or disease of zoonotic concern? . where is the microbial agent when not causing disease (microbial ecology)? . what are the contributing factors to the infection and/or disease process? . what factors can animal owners/veterinarians/public health personnel control to minimize or eliminate the risk of infection/disease process ? table iii immune function (immune competence), ( ) assays to monitor genetic resistance/susceptibility, ( ) assays to monitor infections, ( ) assays to diagnose disease and monitor response to treatment, and ( ) assays to track emerging infections. as infectious agents continue to evolve, disease expression will change, resulting in the necessity to develop new diagnostic assays (susser and susser, b; wilson, ) . nucleic acid amplification technologies: application to disease diagnosis molecular diagnostics for dairy-borne pathogens emerging infectious diseases in the united states: improved surveillance, a requisite for prevention detection of canine herpesvirus in a wide range of tissues using polymerase chain reaction paratuberculosis and molecular biology quantitative molecular methods in virology anatomy of viral persistence: mechanisms of persistence and associated disease status of bovine tuberculosis in north america case control study of multiple-resistant salmonella typhimurium dt infections in cattle in great britain laboratory diagnosis of viral and rickettsial infections laboratory diagnosis of viral and rickettsial infections laboratory diagnostic tests for retroviral infections in dairy and beef cattle feline infectious peritonitis ez coli rapid detection system: a rapid method for the detection of escherichia coli o in meat and other foods risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus sequence-based identification of microbial pathogens: a reconsideration of koch's postulates resistance of livestock to viruses: mechanisms and strategies for genetic engineering detection of chlamydia psittici dna in avian clinical samples by polymerase chain reaction biological monitoring: state of the art predictive genetic testing: from basic research to clinical practice the use of a mass-action model to validate the output from a stochastic simulation model of bovine viral diarrhea virus spread in a closed dairy herd. prey principles of validation of diagnostic assays for infectious diseases assessing the validity of serodiagnostic test results. semin laboratory diagnostic tests for retrovirus infections of small ruminants detection of equine infectious anemia viral rna in plasma samples from recently infected and long-term inapparent carrier animals by pcr the evolution of virulence in pathogens with vertical and horizontal transmission genetic control of host resistance to infection rapid detection of bovine herpesvirus in the semen of infected bulls by a nested polymerase chain reaction assay molecular epidemiology: applications of nucleic acid amplification and sequence analysis diagnosing emerging bacterial infections: salmonellosis, campylobacteriosis, clostridial toxicosis, and helicobacteriosis. semin the epidemiology of mycobacterium bovis infection in animals and man: a review prp genotypes and experimental scrapie in orally inoculated suffolk sheep in the united states two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus rickettsioses as paradigms of new or emerging infectious diseases genotypic methods for microbiol identification detection of animal pathogens by using the polymerase chain reaction (pcr) factors in the emergence of arbivirus diseases escherichia coli o :h diarrhea in the united states: clinical and epidemiologic features new aspects of rabies with emphasis on epidemiology, diagnosis and prevention of the disease in the united states evaluation of diagnostic tests choosing a future for epidemiology: i. eras and paradigms choosing a future for epidemiology: ii. from black box to chinese boxes and eco-epidemiology chromosome rearrangement, oncogene activation and other clinical events in cancer. their use in molecular diagnostics disease in evolution: introduction detection of proviral dna of bovine leukemia virus in cattle by a combination of in situ hybridization and the polymerase chain reaction no sale public health risk early cull regulatory quarantine shedding of microbial agent to susceptible animals in the population segregation of animal euthanasia of animal(s)the future of veterinary diagnostics is now. there are at least five directions to be pursued (table iv) , none of which is new, but continuing to evolve as the needs mandate the detection of infection earlier and the diagnosis of disease at a manageable stage (wilson, ) . these five directions are the development of ( ) assays to monitor . assays to monitor immune function (immune competence) foal check calf failure of passive transfer llama/alpaca immunoglobulin status cmi response . assays to monitor genetic resistance/genetic susceptibility cellular receptors cellular oncogenes cellular prion proteins . assays to monitor infections in the environment in asymptomatic vectors (potential transmissibility) in asymptomatic carriers (potential shedders) . assays to diagnose disease prognosis monitor response to treatment via cytokines (il- , il- , etc.) . assays to track emerging infections culture invitro conserved pcr disease potential develop new detection assays develop new vaccines key: cord- -btcdjmfp authors: nan title: key note and state of the art lectures date: - - journal: ann hematol doi: . /s - - - sha: doc_id: cord_uid: btcdjmfp nan non-hodgkin's lymphoma (nhl) is a hematologic malignancy for which a rather dramatic rise in incidence has been noted during the past decade. nhl is a heterogeneous condition in every respect. there is a wide range of histologic, immunologic, molecular and clinical expression of this condition with marked differences in response to therapy and survival. various forms of combination chemotherapy and radiation have been proven to be successful for the different types of nhl, however, on average only one-half of the patient population with disseminated disease enters a durable remission [ ] . during the past years, many investigators have explored various forms of high dose therapy followed by either autologous or allogeneic hematopoietic cell transplantation. this review will present some of the original data and provide new information from more recently published clinical trials. the first group of patients treated with high dose combination therapy followed by autologous hct dates back to . in a report from the national cancer institute, nhl patients were described of whom four currently are alive and well more than years after hct [ ] . this encouraging observation was followed by a large number of clinical trials, mostly designed as feasibility trials or phase ii studies. a comparison between high dose therapy and autologous hct versus standard dose chemotherapy was reported in and demonstrated a significant advantage with respect to overall survival and disease-free survival in favor of transplantation [ ] . during the past decade, patients with nhl who had high risk features have been transplanted electively during their first clinical remission in order to avoid an early relapse. these extended phase ii trials have yielded promising results. a prospective comparison showing the advantage of high dose sequential therapy versus continued standard dose therapy was reported five years ago [ ] . the leading cause for treatment failure after autologous hct for nhl is the relapse of the underlying disease. a number of strategies to improve the outcome of autologous hct can be pursued (see table ). in this presentation, i will show several examples which indicate the importance of some of the treatment strategies mentioned in table [ ] . table . opportunities to improve the outcome of autologous hematopoietic cell transplantation • timing of autologous hct with respect to remission status • selection of myeloablative doses and combinations of drugs with or without irradiation • optimizing the graft, i.e., removal of clonogenic tumors without loss of activity for hematopoietic reconstitution • post-autologous hct to consolidate remission (cytokines, monoclonal antibodies, vaccines, cells, involved-field radiation) the timing of high dose therapy and autologous hct can have a major influence upon the treatment result. in a prospective phase ii trial, we treated patients with high dose therapy and autologous bone marrow transplantation for follicular lymphoma in first complete or partial remission [ ] . seventeen patients were in first complete remission and patients were in partial remission. the median age of this patient population was years ranging from to years. the preparatory regimen consisted of fractionated total body irradiation ( , cgy), etoposide ( mg/kg) and cy-clophosphamide ( mg/kg). all patients received autologous bone marrow cells which were purged with a set of monoclonal antibodies directed against cd , cd , cd and cd with complement lysis. an intent-to-treat analysis is shown in figure . at the time of analysis, the surviving patients had been followed for - years. overall survival was %, event-free survival % and the relapse rate was %. the causes for treatment failure included one early death due to septicemia, two patients died from acute leukemia and three patients succumbed to lymphoma. the excellent long-term survival demonstrated in this study indicates that the natural course of the underlying disease has been markedly altered by the autologous hct approach. a case-matched comparison from the stanford nhl data base indicates a significant survival advantage compared to patients not undergoing transplantation. conversely, patients with follicular lymphoma who proceed to transplantation have a reduced chance for long-term survival. four groups of investigators at boston, london, omaha and stanford have evaluated autologous hct recipients in four independent trials. four to eight years following autologous hct, overall survival ranged from - % and freedom from relapse ranged from - %. the event of a relapse of follicular nhl clearly reduces the chance for disease-free survival. a prospective clinical trial in follicular lymphoma has been completed recently in europe. in this study, patients with this type of lymphoma were treated with cytoreductive therapy to minimal disease and were then randomized to receive either high dose radiochemotherapy and autologous hct or interferon. the data of this trial are currently being prepared for publication. a preliminary review indicates a statistical advantage in progression-free survival in favor of transplantation (w. hiddemann, personal communication, ). other efforts to overcome the relapse problem include the use of maximum tolerated regimens with or without total body irradiation. more recently, radiolabeled monoclonal antibodies have been introduced and these interesting efforts have yielded promising results [ ] . another approach to reduce the post-transplant relapse rate is the employment of monoclonal antibodies directed against epitopes expressed on lymphoma cells. in an exploratory trial, we have administered the antibody directed against the cd epitope to autologous hct recipients whose lymphoma cells expressed cd [ ] . these patients had a median age of years ranging from to years. following our standardized transplant approach, an intent-to-treat analysis shows that of patients are alive and are in continued complete remission with a follow-up period ranging from . to . years. a comparison with similar autologous hct recipients with nhl who had not been treated with the monoclonal antibody after transplantation indicates that figure : overall survival, event-free survival and relapse following autologous bone marrow transplantation in patients with follicular lymphoma during their first complete or first partial chemotherapy-induced remissions this new post-transplant therapy has contributed positively to the treatment outcome. clearly, a prospective randomized study is needed to confirm our promising observation. such a trial is being performed under the auspices of the eastern oncology group (ecog trial ). in , a first group of patients ( with nhl and three with hodgkin's disease) was described indicating that extended disease-free long-term survival can be attained with high dose therapy followed by allogeneic transplantation utilizing hematopoietic cells obtained either from fully or closely matched related donors [ ] . the treatment outcome was greatly influenced by the remission status of the transplant recipient at the time when preparation for transplantation was begun, age of the recipient, compatibility with the respective donor and incidence of post-transplant complications. the leading cause for treatment failure remains graft-versus-host disease (gvhd) with associated infectious complications, other toxicities or relapse of the underlying disease. figure illustrates our institutional experience with patients (seven children/adolescents and adults). the median age of the patient population was years ranging from to years. median follow-up in continued complete remission of survivors is in excess of six years ranging from six months to more than years. overall survival is % with relapse causing treatment failure in % of allogeneic hct recipients. the remaining % of patients succumbed to complications such as graft-versus-host disease, infections or veno-occlusive disease. this limited experience is nevertheless representative for the observations made in other trials including results reported from the international bone marrow transplant registry. without question, non-relapse mortality (mostly due to gvhd) is a major obstacle to success and alternative technologies need to be explored. very promising data have recently been reported related to the use of intensity-reduced regimens followed by allogeneic hct [ ] . it can be expected that this novel concept which relies mainly on a graft-versus-lymphoma effect will lead to an increase in the successful use of allogeneic hct while the procedure-related mortality is dramatically decreased as compared to high dose therapy regimens. the transplant physician who evaluates and counsels a patient with nhl concerning the type of transplantation is faced with a serious dilemma. because of the heterogeneity of the underlying disease, it has so far not been possible to come to a clear recommendation which of the types of hct should be pursued for any given patient, autologous or allogeneic hct. the earliest comparison was reported years ago and indicated equivalence in outcome [ ] . since then, another comparative studies have been described in the peer reviewed literature. in these trials, , al-lograft recipients were compared to , autograft recipients [ ] . differences in histology, remission status, source of autologous or allogeneic cells (marrow versus peripheral blood, related versus unrelated donor) and post-transplant management make even a meta-analysis relatively meaningless. one can only conclude that autologous hct is associated with a post-transplant relapse rate in the order of - % while allogeneic hct is complicated mostly by gvhd and related toxicities resulting in equivalent outcomes for patients with nhl. a collaborative trial is currently planned by the bone marrow transplant clinical trials network in the united states to address this important question which should help to facilitate the proper choice for transplant candidates. for the time being, most centers would utilize hct if a suitably matched donor is available and to offer autologous hct in the absence of a compatible donor. mortality after hematopoietic stem cell transplantation (hsct) could be reduced to approximately % in hla-identical intrafamiliary pediatric transplantations and ranges between and % in hla-different, intrafamiliary as well as hla-unrelated transplantations. in contrast, pulmonary complications are a leading cause of mortality after hsct and contribute markedly to the morbidity [ ] . the pathophysiology of these diseases is poorly understood and their classification is highly descriptive. this overview is focussed on the pediatric age group and ( ) elucidates the circumstances which render recipients at increased risk of non-infectious pulmonary complications, ( ) describes the diseases and alterations which are encountered, and ( ) discusses diagnostic and therapeutic consequences. the number of hematopoetic stem cell transplantations has increased in recent years [ ] . statistically, pulmonary complications with respiratory insufficiency account for as much as % of the mortality, depending on the patients' age [ , , , , ] . furthermore, respiratory insufficiency is included under diagnoses such as graft-versus-host-disease (gvhd) or multi organ insufficiency. the largest retrospective analysis of italian children after allogeneic stem cell transplantation demonstrated a pulmonary mortality of % [ ] . case reports as well as retrospective or prospective analyses of different patient cohorts demonstrate between % and % incidence of irreversible pulmonary function loss after hsct [ , , , , , ] . the incidence of late-onset non infectious pulmonary complications (lonipc) is estimated to be approximately %. in one study out of patients suffered such complications three months after allogeneic hsct [ , ] . there has been no improvement over the past decade ( ) . risk factors are intrinsic to the procedure (autologous vs. allogeneic transplantation with rejection by residual host t-cells, gvhd), to the immunosuppression (total body irradiation, cyclophosphamide and busulfan, both leading to interstitial pulmonary fibrosis), to the underlying disease (sickle-cell anemia with recurrent episodes of chest syndrome and pulmonary infarction, severe combined immunodeficiency with recurrent episodes of pneumonia), to the treatment of the underlying disease (irradiation, resection) and the hyperimmune status (influenced by the histocompatibility between donor and host, e.g. donor t-cells reacting to hlaantigens). in one series of children undergoing transplantation for hematologic diseases, % had pulmonary function abnormalities before the procedure ( ) . although the topic of this review is non-infectious pulmonary complications, it is impossible to completely distinguish these from infectious ones, as the latter may trigger hyperimmune reactions, lead to vascular alterations, enhance antigen presentation and fibroblast activity, attract neutrophils and eosinophils with release of their toxic products and disturb the level of immunotolerance. in contrast, augmented immunosuppression in the course of interstitial lung disease enhances the susceptibility for infections leading to additional damage. the extent of pulmonary mortality and morbidity, the degree and the spectrum of pulmonary diseases in children as well as the relevance of possible monocausal or multifactorial etiologic risk factors (gvh-disease, irradiation, chemotherapy especially with cyclophosphamide, bleomycin, busulfane, melphalane) is still classified insufficiently [ , , , , ] . the introduction of fractional irradiation schedules has led to a reduction of the incidence of interstitial pneumonia [ ] . latent viral infections, gvhd and cytotoxic factors are not well defined in their pathogenetic role and s data about the influence of fungi is limited [ , , ] . histologic evaluations of lung tissue in gvhd-patients show epithelial and interstitial lesions typical for gvhd of the skin and the intestine [ ]. there is some evidence for an active role of cmv in bronchiolitis obliterans (bo) as well as in interstitial pneumonias. since the detection of cmv in bronchoalveolar lavage (bal) can be taken as an indicator of invasive infection [ , , ] , screening for cmv in bal and blood has been suggested as a basis for rigorous treatment [ , , , , ] . the same may hold true for other viruses such as adeno-, ebv, influenza, parainfluenza virus, hhv- and rsv ( , ) . it is usual to separate pulmonary complications up to day (early) from those after day (late), although for some risks the division is arbitrary. if one follows lung function parameters over time in clinically asyptomatic patients after hsct for childhood leukemia [ ] , abnormalities can be observed in more than % of patients with predominance of a restrictive pattern months after transplantation (possibly due to conditioning). lung function abnormalities tend to persist in patients with advanced disease states before transplantation but decrease by % in those with transplantation at early stages of childhood leukemia, suggesting a summation of adverse effects. toxic pulmonary reactions may occur within days after irradiation. clinically overt non-infectious pulmonary complications in the neutropenic (early) phase after transplantation occur in the form of engraftment-syndrome, idiopathic pneumonia syndrome (ips) or diffuse alveolar hemorrhagesyndrome (dah). furthermore the lung can be part of a systemic vascular process (capillary-leak-syndrome, thrombotic-thrombocytopenic purpura or cytokine-syndrome). late pulmonary sequelae manifest as bronchiolitis obliterans (bo), bronchiolitis obliterans organizing pneumonia (boop) or fibrosis. there are several distinct cellular systems involved in the maintenance of pulmonary integrity and, accordingly, in the disease process: (a) alveolar cells or pneumocytes are a source of surfactant and contribute to metabolism within the lung as well as to lung defence. (b) bronchial epithelial cells contribute to the clearance of bacteria and are important barriers against invasion. (c) vascular endothelial cells are essential for regulating gas exchange, fluid homeostasis (selectins, endothelin), and the migration of blood cells. (d) fibroblasts are sources of metalloproteinases and contribute to the repair (and fibrotic changes) of lung tissue. (e) dendritic cells act as antigen-presenting-cells. (f) t-cells target pulmonary structures in gvhd or autoimmunity. (g) neutrophils are the leading cells in bacterial defense but are also sources of proteases with strong elastolytic activity. (h) recently a role has been proposed for alveolar macrophages in disease processes after hsct ( ) . it is beyond the scope of this review to evalute the literature on the spectrum of immune mechanisms which include direct and indirect allorecognition as well as the activation of chemokines and lymphokines. pulmonary diagnostics include lung function testing (bedside with portable spirometers, bodyplethysmography), capillary blood gas analysis, non-invasive transcutaneous assessment of oxygen saturation, exercise tests, bronchoalveolar lavage (bal), transbronchial (tbb) or open lung biopsy, ultrafast or thin section computed tomography (ct) ( , , , , , , ) . material obtained should be evaluated by the most sensitive and specific microbiological and pathological techniques ( ) . in the early phase, infectious complications are the leading differential diagnoses, predominantly by gram-positive and gram-negative bacteria of the endogenous flora. in prolonged neutropenia, invasive fungal infections pose considerable diagnostic and therapeutic problems. secondly, infections by cytomegaly, ebv, adeno-, respiratory-syncytial, parainfluenza and influenza virus, hhv- , as well as by chlamydia, mycoplasms, legionella, mycobacteria, toxo-plasma, and pneumocystis-carinii should be considered. infections by herpes virus have reached their nadir days post hsct. however, all viral, bacterial and fungal infections can also occur at a later time point depending on the state of immunity, donor selection, and preparation of the transplant. furthermore, gvhd and its therapy play a pathogenetic role in the development of so-called noninfectious pulmonary complications. the treatment or prophylaxis of infectious diseases may have equally profound impact on the pattern of non-infectious lung diseases observed ( ). among non-infectious pulmonary complications in the early phase, a group of diffuse interstitial pneumonias is responsible for high mortality. some of these alterations were defined at the nihlbi workshop in bethesda as "idiopathic pneumonia syndrome" (ips) [ ] . immediately after transplantation the so-called "engraftment syndrome" or "capillary-leak syndrome" play an important role ( ) , which has to be distiguished from diffuse alveolar damage (dad) or the idiopathic pneumonia syndrome. the latter ones are considered primarily as forms of ards because their time course, their histopathologic morphological criteria and their clinical appearance closely resemble this complicationwhich, despite various causes, generally has an unfavourable prognosis [ , ] . the incidence of ips after allogeneic hsct has been reported as % [ ] or % [ ] for all ages, and is certainly lower for all donors among children. the mortality of this complication is high, despite intensive care treatment including mechanical ventilation ( ) . according to a retrospective analysis of hsct patients, mortality is estimated to be around % [ ] . essential diagnostic criteria are ( ) indicators of diffuse alveolar damage (multilobular infiltrates in thorax x-ray or disturbance of gas transfer, a restrictive ventilatory defect, clinical signs of pneumonia) and ( ) the exclusion of active infection of lower airways by bronchoalveolar lavage (bal) or transbronchial biopsy (tbb). patients with severe acute graft versus host disease (gvhd grade iii-iv) have a largely increased risk of developing ips. it has not been clear whether ips after transplantation represents t-cell-mediated lung damage (e.g. "acute gvhd of the lung") or is triggered by occult viral infections (e.g. adeno or hhv- ), or can be considered as a consequence of a multiple organ failure during generalised activation of inflammatory mediators (analogous to the multifactorial ards of non-transplanted patients) in acute gvhd, in the course of viral infections or sepsis. there is no specific therapy for the ips to date. therefore diffuse infiltrates on x-ray after transplantation have to be subject to a thorough work-up. if they do not respond to diuretics (to exclude pulmonary edema), they have to be evaluated radialogically, treated empirically for a short time and/or evaluated invasively depending upon the radiological appearance before or shortly after the start of antibiotics. diagnostic criteria for "capillary leak syndrome", which also occurs in the early phase after transplantation, are inadequate weight gain and disturbance of gas transfer. therapy with c -esterase inhibitor is currently under evaluation ( ) . diffuse alveolar hemorrhage syndrome (dah) is another early "non-infectious" pulmonary complication (day + until + ). its occurrence has been described predominantly after autologous transplantation [ , ] . mortality of this disorder ranges from - %. nonspecific symptoms such as progredient dyspnoea, dry cough, fever, hypoxia and diffuse lobular consolidation in xray are considered typical. hemoptoe is rare. early diagnosis (bal) is important since high-dose corticosteroids can then be applied efficiently [ ] . because of its high mortality, the late complication "bronchiolitis obliterans" (bo) stands -together with non-classifiable interstitial pneumonia, -in the first line of differential diagnoses for non-infectious pulmonary complications after hsct. not unlike bo after lung transplantation, the etiology remains unclear. lymphocyt-ic bronchitis, alloreactivity after pathologic expression of antigens due to stress (viral, radiation, medications), malnutrition of the bronchial wall, vascular changes, occult aspirations -are all considered to be causative events ( ) . histologically the disease resembles bo in patients after heart-lung and lung transplantation; it is interpreted as chronic rejection (prevalence > %) with high mortality ( %) [ ] . the first description of bo after allogeneic hematopoetic stem cell transplantation dates from [ ] . since then this complication has been described after all types of transplantation, including cord blood cell transplantation [ ] . in adults one assumes a prevalence of bronchiolitis obliterans after hematopoetic stem cell transplantation in a range between - % [ , ] . in children, incidences of up to % have been reported [ ] . if unrecognised, this late complication tends to be lethal, since the therapeutic effect of corticosteroids in advanced stages is poor. the speed of progression for bo and the ideal time for therapeutic intervention are unknown. bo is frequently associated with chronic gvhd [ , ] and they occur in combination in up to % of adult patients [ ] and up to % in children with gvhd [ ] . the clinical symptoms are nonspecific with expiratory wheeze, cough, dyspnoea and hypoxia upon exertion. typical hr-ct-signs of the thorax for bo are peripheral hypovascularity, enlargement of segmental-and subsegmental bronchi, fixed hyperinflation ("mosaic pattern"), and deformed peripheral vessels [ , , , , , ] . it is debatable, however, whether these signs are also typical for bo after hematopoietic stem cell transplantation. some of these signs can only be interpreted correctly if sequential examinations have been performed. furthermore, ct studies in children are rare. in bal differential cytology the relative number of neutrophils is elevated [ ] . the definite diagnosis of bronchiolitis obliterans can frequently only be achieved by open lung biopsy. the histopathological finding is bronchiolar inflammation with luminar scarring, fibrous tissue and obliteration of the small bronchioli without occluding the alveolar ducts. in lung function the small airways are affected first (hyperinflation) and there is exercise limitation without hypoxia. the involvement of vessels can vary. therapeutically, systemic glucocorticosteroids, cyclophosphamide, azathioprine, chloroquine, glucocorticosteroid pulses, cyclosporine, anti-thymocyte globulin, tacrolimus, methotrexate, total lymphoid irradiation, thalidomide, clofazimine, extracorporal photopheresis, humanized monoclonal anti-il- receptor antibodies are applied in bo without convincing success. furthermore, there have been no controlled studies of any of the above-mentioned medications. as a number of interstitial diseases in childhood have shown a favourable response [ , , ] , our approach is to try to detect the disease at an incipient stage and use methylprednisolone pulse therapy as a first-line treatment ( ) . in about - % of cases, at least a temporary response can be seen. unfortunately, the time window for intervention is small in most cases and the speed of progression extremely variable, rendering evaluation of therapeutic success difficult. complications of bo are pneumothorax, pneumomediastinum, bronchiectases, hypercapnia and pulmonary hypertension with cardiac failure. the "bronchiolitis obliterans organizing peumonia" (boop), which occurs more rarely, was defined by epler [ ] . the etiology remains unclear. in the literature boop has been described in autoimmune diseases, after medication,as well as with viral and bacterial infection and as idiopathic boop [ ] . lately a number of cases have been published after hsct with gvhd [ , , , ] . there are also reports on early and fulminant courses [ , , ] . the clinical picture is non-specific, with cough, dyspnea, and hypoxia. lung function shows a restrictive pattern and impairied diffusion capacity. histopathology demonstrates granulation -and fibrous tissue in terminal bronchioles reaching into alveoli -as well as interstitial lymphocytic inflammation. cellular profiles of bal show high lymphocyte counts, with low cd /cd -ratio (results from adults) [ ] . characteristic hr-ct-patterns in boop are ground-glass opacification and nodular shadows [ ] . typical bilateral, focal, predominantly peripheral infiltrates can be detected earlier using hr-ct rather than by x-ray of the thorax [ , ] . in the literature (including patients after hsct) the beneficial effect of treatment with glucocorticosteroids is emphasized beginning at an early stage and continuing for - months ( , , , , , ) . bronchiolitis obliterans organizing pneumonia (boop) must be separated diagnostically from "lymphocytic interstitial pneumonia (lip)", "diffuse alveolar damage" (dad) and "lung fibrosis" as well as a nsid (nonspecific interstitial disease) and pulmonary veno-occlusive disease ( , , , ) . these diseases tend to occur after hematologic diseases, infections and therapeutical interventions (radiation, chemotherapy). some of them are termed as late onset non specific interstitial lung disease (lonsild). prognosis varies greatly. treatment modalities range from antioxidants to supportive care (oxygen) ( ) . with severe progression, the only chance for survival may be lung transplantation. post transplant lymphoproliferative disease (ptlpd) can manifest itself in the lung and can occur any time after transplantation. frequency varies depending on the kind of transplantation, intensity and mode of immunosuppression, ebv-status of donor and recipient, and bone marrow reconstitution. although rarely confined to the lung, the occurrence of this lesion together with other infections (like aspergillus) or gvhd makes diagnosis difficult, usually requiring an invasive approach. the who classification is not applied uniquely. treatment modalities range from lowering immunosuppression to chemotherapy, using anti cd antibodies to antiviral therapy, depending, among other factors, on histology, ebv-status, extent of the disease, and clinical course. lung transplantation has been applied as ultima ratio in patients after an interval of up to years after hsct, mostly after pulmonary complications involving gvhd. the preferred surgical technique in most cases has been double lung transplant. reported survival is similar to transplantation for other causes -with one patient surviving more than seven years [ ] . out of this series, one patient developed boop, a condition rarely seen after lung transplantation. a girl with a lobar transplant from the same living donor as her hematopoetic stem cells is the only known person without immunosuppression after lung transplantation [ ] . generally, prognosis after lung transplantation is a % survival rate after years. pulmonary complications after hematopoetic stem cell transplantation are poorly understood and treatment is unsatisfactory. there is no standardised pulmonary follow-up for children after hematopoetic stem cell transplantation with disappointing rates of pulmonary function measurements [ ] . on the other hand, the spectrum of pulmonary complications is limited and the time sequence more or less known. diagnostically it would seem essential to follow a scheduled plan of pulmonary surveillance measures including body plethysmography, assessment of gas exchange and exercise capacity as well as screening for infections. this should make it possible to gain insight into the pathogenesis of the diseases and examine whether bronchiolitis obliterans can be prevented by therapeutical measures at an early stage. as therapeutical options are limited in advanced bo, prophylaxis and preemptive therapy may be crucial. as a prerequisite, risk factors should be carefully studied and causal strategies developed in a multicenter approach. ( ) the treatment of inborn metabolic diseases such as lysosomal hydrolase deficiencies [ , ] and peroxisomal disorders such as adrenoleukodystrophy (ald) [ , ] has been limited to symptomatic supportive care. intravenous enzyme replacement has been largely ineffective, especially in storage diseases with central nervous system (cns) manifestations. allogeneic hematopoietic cell transplantation (hct) with related or unrelated bone marrow cells, peripheral blood progenitor cells, or placental (umbilical cord) blood cells in these conditions repopulates recipient hematopoietic and lymphoid cells with metabolically normal donor-derived cells and thus provides a self-renewing source of hydrolase. in addition, hct effectively repopulates mononuclear phagocytic cells, including macrophages, hepatic kupffer cells, and pulmonary alveolar macrophages, in which substrate often accumulates in storage diseases. as discussed below, the repopulation of microglia with donor-derived cells after allogeneic hct is a potentially important therapeutic mechanism of allogeneic hct in patients with cns manifestations of metabolic diseases. intercellular transport of lysosomal hydrolase from normal donor cells occurs by both receptor-mediated endocytosis and direct transfer of enzyme from adjacent cells [ ] . the heterogeneity of receptor-mediated endocytosis systems limits the extent of hydrolase uptake among various cell types and tissues [ , ] . direct intercellular transfer of hydrolase occurs independent of specific receptors but requires cell-to-cell contact and participation of adhesion molecules such as lfa- and - , icam- , - , and - , and cd (the sheep erythrocyte receptor on t lymphocytes) [ , ] . unlike lysosomal storage diseases, ald is due to a defective protein component of the peroxisomal membrane that is neither secreted from normal cells nor transferred between cells, leading to defective oxidation and elevated plasma levels of very long chain fatty acids (vlcfas) [ ] . co-culture with normal cells does not improve vlcfa oxidation by ald fibroblasts, indicating that improvement of ald after hct is likely due to repopulation by metabolically normal cells instead of intracellular transfer of molecules from donor cells. other beneficial effects of allogeneic hct in ald include normalization of plasma vlcfa levels and decreased perivascular inflammation. microglia are the mononuclear phagocytic cells in the cns [ , ] and account for approximately % to % of non-neuronal cells in the brain. activated microglia, also referred to as cns macrophages, are involved in antigen presentation and responses to inflammation, infection, or cns injury [ ] . microglia are derived from hematopoietic precursors that normally enter the brain from the peripheral circulation during embryonic and early postnatal life but not in adulthood [ ] . in rodent hct recipients, donor-derived cells can be identified throughout the cns [ ] and over time completely repopulate the microglial compartment [ , , ] . in feline β-mannosidosis, these donor cells effectively transfer lysosomal hydrolase to recipient neurons in vivo [ ] . after hct, donor-derived cells also differentiate into other non-neuronal cns cell populations such as astrocytes (also referred to as macroglia) [ , ] . postmortem studies confirm that donor mononuclear cells are also present throughout the brains of human allogeneic hct recipients [ ] . the kinetics of repopulation of microglia after hct is slower than that observed with other mononuclear phagocytes like pulmonary alveolar macrophages and hepatic kupffer cells [ , ] . in humans, post-hct repopulation with donor-derived microglia requires approximately year, which may explain the ineffectiveness of allogeneic hct in stabilizing or pre-venting neurological deterioration in some rapidly progressive storage diseases [ , ] . the availability of spontaneously occurring heritable animal models and the development of transgenic "knockout" lysosomal hydrolase deficient animals allows preclinical evaluation of the biochemical, physiological and clinical effects of allogeneic hct in storage diseases. the most extensive studies of hct in preclinical storage disease models have been in canine α-l-iduronidase deficiency (a model of mucopolysaccharidosis [mps] ih, or hurler syndrome) [ , , ] , murine β-galactosidase deficiency (a model of mps vii, or sly syndrome) [ , , ] , and murine galactosylceramidase deficiency (the "twitcher" mouse, a model of the sphingolipidosis globoid cell leukodystrophy [gcl]). we have extensively studied hct in the twitcher murine model [ ] , which most closely resembles krabbe disease, the early infantile form of human gcl. hct leads to prolonged survival, clinical improvement, attenuation of hindlimb paralysis [ , ] , remyelination in peripheral nerves and cns [ ] , and stabilization of motor nerve conduction velocities [ ] in presymptomatic twitcher mice but is of no benefit in symptomatic animals. after hct, galactosylceramidase is present in the cns and non-neural tissues [ , ] , and levels of the toxic metabolite psychosine (galactosylsphingosine) [ ] , are significantly decreased in the cns [ ] . postnatal hct is not curative in murine gcl, most likely because repopulation of the cns and peripheral nervous system with donor cells does not occur rapidly enough to stabilize the progressive demyelination. murine gcl may be an important model for critical evaluation of the effects of intrauterine cellular transplantation for infantile-onset sphingolipidosis. several recent reports have summarized the results of allogeneic hct for storage diseases and ald [ , , ] . this review will focus on current concepts of allogeneic hct for mps ih (hurler syndrome), globoid cell leukodystrophy and ceramidase deficiency (farber disease). the prognosis of untreated mps ih (α-l-iduronidase deficiency) is poor, with a median survival of . years and with very few children surviving into the second decade of life [ ] . in contrast, survival after hct in children with mps ih is % after bone marrow transplantation (bmt) from hla-identical siblings, % after bmt from hla-mismatched relatives, and % after bmt from unrelated donors [ , ] . hepatosplenomegaly, joint mobility, and upper airway obstruction in mps ih resolve within months to a year after hct. corneal clouding stabilizes or slowly resolves [ ] , and visual acuity may improve even without regression of corneal clouding. unfortunately hct does not correct the skeletal manifestations (dysostosis multiplex) of mps ih, which are due to metabolic dysfunction in chondrocytes and osteoblasts that arise from mesenchymal precursors. the orthopaedic complications of mps ih require ongoing evaluation and surgical management even after hct [ ] . mental retardation is a hallmark of untreated mps ih [ ] , and both age and neurodevelopmental status are important determinants of outcome after hct. when carried out in patients under age years, hct preserves neurocognitive function and prevents or reverses increased intracranial pressure. this favorable outcome is in part due to the fact that the developmental quotient (dq) falls below in most children with mps ih after age years [ ] . in general, children with mps ih with normal intelligence before hct maintain that level of cognitive functioning after transplant [ , , ] , but those with significant neurocognitive impairment (e.g., dq below ) at hct have progressive deterioration and do not benefit from the procedure. globoid cell leukodystrophy (galactosylceramidase deficiency) is one of the sphingolipidoses, which are characterized by demyelin-ation of the cns and/or peripheral nervous system because of deficiencies in specific acid hydrolases involved in the metabolism of sphingomyelin, gangliosides and cerebrosides [ ] . characteristic globoid cells, derived from microglia or cns macrophages and containing periodic acid-schiff (pas)-positive myelin breakdown material, are present in the brains, spinal cord and peripheral nerves. interestingly, the widespread demyelination in gcl is due to accumulation of psychosine (galactosylsphingosine), which is derived from the substrate galactosylceramide and is toxic to both oligodendroglia and schwann cells [ ] . the four clinical phenotypes of human gcl vary in clinical manifestations and tempo of disease progression. patients with early (age of onset, to months) or late (age of onset, months to years) infantile gcl have profound psychomotor retardation, failure to thrive, spasticity, seizures, optic atrophy and cortical blindness [ ] . juvenile gcl affects children from to years of age, with insidious onset and progression of visual loss, lower extremity spasticity, and in some patients dementia. adult gcl occurs in patients over age years and is characterized by slowly progressive difficulty in walking, long-tract signs, asymmetric weakness of the extremities, and difficulties with coordination and balance, but intellect is generally unaffected [ ] . allogeneic hct is not curative in symptomatic early infantile gcl (krabbe disease), in which neurodegeneration persists despite post-transplant biochemical improvement [ , ] , analogous to observations in the preclinical studies of hct in murine gcl. although very limited experience suggests that presymptomatic infants with infantile gcl may benefit from hct [ ] , early identification of the affected infant and of a suitable hct donor are obvious practical challenges to this therapeutic strategy. the role for allogeneic hct is more firmly established in patients with presymptomatic or minimally symptomatic juvenile or adult gcl, in whom hct leads to stabilization and gradual improvement in clinical, neurological and neurocognitive status [ , , ] . of the six phenotypes of ceramidase deficiency [ ] , at least % of patients have the classic infantile form, or farber disease phenotype. affected infants have painful joint swelling, multiple painful subcutaneous nodules, hoarseness, swallowing difficulties, and failure to thrive [ ] . microscopic examination of the nodules shows granulomata containing ceramide-laden macrophages. granulomata in the aerodigestive tract cause hoarseness and deglutition problems. hepatomegaly, recurrent pulmonary infections and respiratory difficulties are common and due in large part to organ infiltration by ceramide-laden macrophages. infants with farber disease die with progressive and profound psychomotor retardation at a mean age of to months [ ] . two infants with farber disease received allogeneic bmt at months [ ] and months [ ] of age, respectively. subcutaneous nodules, joint pain and hoarseness regressed within months after transplant in both patients. the older patient had progressive neurodegeneration and died months after bmt. the younger patient, who had mild developmental delay and slight hypotonia at bmt, had progressive psychomotor retardation and graft failure, with a developmental age of months at chronological age months. despite loss of donor cell engraftment, levels of ceramidase in the peripheral blood leukocytes remained in the donor heterozygous range, suggesting ongoing production of ceramidase by non-circulating donor cells and hydrolase uptake by recipient blood cells [ ] . these limited observations indicate that hct does not stabilize the rapid neurological deterioration in classic farber disease. among new approaches for treatment of metabolic disorders by cellular transplantation, intrauterine hct may be worthy of exploration, especially in the rapidly progressive infantile forms in which postnatal hct is not effective. the major limitation to this approach is the low levels of donor cell engraftment, which may not provide sufficient hydrolase or metabolically active cells to correct the underlying biochemical defect [ ] . allogeneic hct using reduced-intensity nonmyeloablative preparative regimens may be considered in some indolent forms of storage diseases, providing gradual donor cell engraftment without the risk of aplasia and other short-and long-term toxicities associated with intensive marrow-lethal preparative regimens [ , ] . the use of these regimens is not feasible in more aggressive forms of storage diseases. insertion of genes for specific lysosomal hydrolases or for the ald protein into autologous hematopoietic stem cells (hscs) and transplantation of these cells is theoretically very attractive but remains at the level of preclinical investigation at this time because of ongoing challenges such as efficient introduction and integration of the exogenous gene in truly primitive hscs and sustained, consistent high-level expression of the hydrolase in these cells and their progeny [ ] . a potentially exciting approach for storage diseases with skeletal manifestations (dysostosis multiplex) is the co-transplantation of mesenchymal stem cells (mscs), which may differentiate into chondrocytes and osteoblasts [ , ] . although clearly not a clinical therapeutic option at this time, intracerebral injection of mscs may favorably affect the neuropathological and biochemical abnormalities in a murine model of acid sphingomyelinase deficiency (niemann-pick disease types a and b) [ ] . clinical experience for more than two decades has shown that allogeneic hct may benefit some but not all patients with inherited metabolic diseases. the hct procedure is most effective in presymptomatic patients and those with indolent forms of storage diseases but is ineffective in those with overt neurological symptoms or aggressive neonatal or infantile forms. hct alone does not correct skeletal dysplasia in mpss and may not prevent development or progression of the peripheral neuropathy in sphingolipidoses and ald. decisions regarding hct in patients with storage diseases should be made by investigators knowledgeable about these diseases, with judicious use of laboratory and 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hematologic diseases outcome of displacement bone marrow transplantation in farber's disease: a report of a case genetic disorders of lipid, glucoprotein, and mucopolysaccharide metabolism the twitcher mouse: a model for krabbe disease and for experimental therapies nerve conduction studies in the twitcher mouse (murine globoid cell leukodystrophy) male donor-derived cells in the brains of female sex-mismatched bone marrow transplant recipients: a ychromosome specific in situ hybridization study bone marrow transplantation corrects the enzyme defect in neurons of the central nervous system in a lysosomal storage disease the mucopolysaccharidoses longterm outcome of hurler syndrome following bone marrow transplantation reversal of pathology in murine mucopolysaccharidosis type vii by somatic cell gene transfer stem cell transplantation in lysosomal storage diseases prolonged survival and remyelination after hematopoietic cell transplantation in the twitcher mouse (murine globoid cell leukodystrophy) hematopoietic cell transplantation after administration of high-dose busulfan in murine globoid cell leukodystrophy (the twitcher mouse) repopulation by donor-derived macrophages in the murine central nervous system (cns) after congenic bone marrow transplantation (bmt): a quantitative study hematopoietic cell transplantation in the twitcher mouse. the effects of pretransplant conditioning with graded doses of busulfan bone marrow transplantation for infantile ceramidase deficiency (farber disease) key: cord- -o zfilcl authors: laidler, matthew r.; thomas, ann; baumbach, joan; kirley, pam daily; meek, james; aragon, deborah; morin, craig; ryan, patricia a.; schaffner, william; zansky, shelley m.; chaves, sandra s. title: statin treatment and mortality: propensity score-matched analyses of – and – laboratory-confirmed influenza hospitalizations date: - - journal: open forum infect dis doi: . /ofid/ofv sha: doc_id: cord_uid: o zfilcl background. annual influenza epidemics are responsible for substantial morbidity and mortality. the use of immunomodulatory agents such as statins to target host inflammatory responses in influenza virus infection has been suggested as an adjunct treatment, especially during pandemics, when antiviral quantities are limited or vaccine production can be delayed. methods. we used population-based, influenza hospitalization surveillance data, propensity score-matched analysis, and cox regression to determine whether there was an association between mortality (within days of a positive influenza test) and statin treatment among hospitalized cohorts from influenza seasons (october , to april , and september , to april , ). results. hazard ratios for death within the -day follow-up period were . ( % confidence interval [ci], . –. ) for a matched sample from the – season and . ( % ci, . – . ) for a matched sample from the pandemic. conclusions. the analysis suggests a protective effect against death from influenza among patients hospitalized in – but not during the pandemic. sensitivity analysis indicates the findings for – may be influenced by unmeasured confounders. this analysis does not support using statins as an adjunct treatment for preventing death among persons hospitalized for influenza. . deaths have been estimated to range between ( ) ( ) and ( ) ( ) annually [ ] . influenza-associated mortality in pandemic years can be higher still and typically shifts toward younger age groups [ ] . this result was especially evident during the pandemic because cross-protective immunity to the pandemic strain was present among older adults [ ] . during the recent influenza pandemic, % of the deaths occurred in persons < years of age, with children and young adults and middle-aged adults having rates of hospitalization and death to times and to times greater, respectively, than estimates from the years - [ ] . the best available strategy to prevent and control influenza is through influenza vaccination. in the united states, all persons ≥ months of age are recommended to receive influenza vaccine annually. however, influenza vaccine effectiveness can vary from season to season, depending on the match between the vaccine influenza strains and the circulating strains and host factors. in addition, during influenza pandemics, the development and deployment of an influenza vaccine may be delayed. influenza antiviral therapy for persons with severe influenza illness or who are at risk for complications is an important adjunct intervention to the influenza vaccination program, reducing morbidity and mortality during seasonal or pandemic influenza [ ] [ ] [ ] [ ] [ ] [ ] . nonetheless, there is always the possibility of widespread circulation of an influenza virus strain resistant to available antiviral agents, and, in a pandemic situation, the risk of antiviral shortages is ever present. the use of immune-modulating drugs, particularly statins, has been postulated as an additional tool for the treatment and prophylaxis of influenza, especially in countries where influenza vaccine and antiviral agents are not readily available [ , ] . statins have wide-ranging down-regulatory effects on inflammatory and immune mechanisms [ ] [ ] [ ] , and there is some evidence that statin treatment may beneficially alter the clinical course of some infectious diseases [ ] [ ] [ ] [ ] . to date, no randomized clinical trials have been conducted to address whether statins could reduce complications of influenza, although some observational studies have suggested protective effects [ ] [ ] [ ] [ ] . a study by vandermeer et al [ ] , using data from a populationbased influenza surveillance system, found a protective effect of statin use on mortality among patients hospitalized with laboratory-confirmed influenza during the - influenza season. nonetheless, due to the observational nature of the study, biases could potentially explain this association, even after controlling for confounders. we sought to repeat the - influenza season analysis of vandermeer et al [ ] and to analyze the influenza a (h n ) pandemic data from the same surveillance platform to study the possible association between influenza-associated mortality and statin treatment. in this analysis, we take into account potential treatment indication biases through the use of propensity score-matched analysis. this study was conducted with data from the centers for disease control and prevention's (cdc) emerging infections program (eip) influenza hospitalization surveillance. the eip influenza hospitalization surveillance system collects data on persons hospitalized with laboratory-confirmed influenza from october through april of the following year, because influenza typically circulates in the fall to spring months in the northern hemisphere. the exception to this was the influenza pandemic, in which hospitalization data were collected from september , through april , . the eip network comprises selected counties in us states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, new york, oregon, and tennessee) and includes a catchment area of approximately million people. cases were identified through active surveillance from reports from hospitals and review of infection control logs or hospital laboratory lists. ascertainment of cases was based on laboratory testing ordered by attending healthcare providers for clinical purposes. cases included patients ( ) ≥ years of age, ( ) residing within the eip catchment area, ( ) admitted to a catchment area hospital, and ( ) admitted within days of a positive influenza test either by viral culture, real-time reverse transcription polymerase chain reaction, immunofluorescence antibody staining (indirect or direct), rapid influenza diagnostic test, or any test of unknown type recorded in the medical chart. patients possibly infected with influenza virus during hospitalization ( positive influenza test > days after admission) were excluded as case subjects. demographic, epidemiologic, and clinical information were collected from chart reviews. influenza vaccination status was determined from the medical chart, primary care provider, or via phone interview (of patient or proxy). patients were considered vaccinated for influenza if a vaccine had been administered > weeks before hospitalization, regardless of whether the patient had seasonal vaccine, h n monovalent vaccine (for the pandemic), both seasonal and monovalent vaccine, or unknown vaccine type. if antivirals were administered at any point during the course of illness, a patient was considered treated. age was categorized into groups ( - , - , - , - , and ≥ years). race and ethnicity were determined by chart review or by self-report during patient interviews for vaccination status information. race was categorized into groups (white, black, and other), and ethnicity was categorized as hispanic or non-hispanic. race and ethnicity were analyzed separately. underlying health conditions of interest included asthma, chronic cardiovascular disease (excluding hypertension), chronic metabolic disease, renal disease, chronic lung disease, immunosuppressive disorders (including cancer diagnosis in the months before hospital admission), seizure disorders, history of lymphoma or leukemia, blood disorders, neuromuscular disorders, obesity, and cognitive dysfunction. we combined all underlying chronic disease variables other than cardiovascular disease, chronic metabolic disease, chronic lung disease, renal disease, and asthma into a variable for "other chronic diseases." height and weight were collected during the pandemic but not during the - influenza season. height and weight were used to determine body mass index (bmi), which was used to categorize patients as underweight (bmi < . ), normal weight (bmi . - . ), overweight (bmi . - . ), obese (bmi . - . ), or morbidly obese (bmi ≥ ). the exposure of interest was statin treatment, either before or during hospitalization, which was determined from hospital records. data on statin dose or frequency of administration were not collected. death within days of a positive influenza test was the outcome of interest. mortality after hospital discharge was determined by linkage of hospitalization data with the social security death index (ssdi) by state. linkage was done using registry plus™ link plus (version . ), a probabilistic record linkage program. mortality during hospitalization was determined through chart review and data linkage with ssdi data. this study was submitted for review and approved by the institutional review boards serving the cdc and participating states. propensity scores were used to predict the probability of treatment with statins. the use of propensity scores in observational studies facilitates similar distributions of baseline characteristics between treated and untreated groups, reducing potential treatment selection bias [ ] . logistic regression models were iteratively assessed to determine the balance of covariate proportions between statin treatment groups in the subsequent matched samples. the best and final model was the one that balanced covariates between treatment groups, as determined by standardized differences < . . the final logistic regression model for the - matched sample included age, sex, race, ethnicity, cardiovascular disease, chronic metabolic disease, chronic lung disease, renal disease, asthma, and vaccination status as covariates. the final logistic regression model for the - matched sample included age; sex; race; cardiovascular disease; chronic metabolic disease; chronic lung disease; renal disease; weight category; long-term care residence; vaccination status; and interaction terms for age and race, age and sex, and age and cardiovascular disease. after calculation of propensity scores, a greedy matching algorithm was used to identify untreated patient for each treated patient in the respective matched samples [ ] . the χ test was used to assess differences in characteristics between the statin treatment groups in the unmatched cohort. categorical variables were transformed to indicator variables to facilitate assessment of balance of covariates between statin treatment groups after matching. standardized differences [ ] were used to evaluate measured baseline covariate distributions between statin treatment groups in the matched cohorts. mcnemar's test for matched pairs was used to assess the difference in proportion of deaths between treated and untreated groups. we used cox proportional hazards models with robust standard errors, stratified on matched pairs, to determine the effect of statin treatment on mortality within days of a positive influenza test. we used the method described by rosenbaum [ ] for survival outcomes for determining the sensitivity of point estimates to hidden bias. we conducted statistical analysis using sas software (version . ; sas institute, cary, nc), and openepi (version . . ) was used for post hoc sample size calculation [ ] . table shows demographic and clinical characteristics of treatment groups before matching, for both the - influenza season (statin treatment group n = , nontreatment group n = ) and the pandemic (statin treatment group n = , nontreatment group n = ) cohorts. in the - cohort, the statin treatment group was older, with a greater proportion of males, a greater proportion of whites, and a higher prevalence of chronic medical conditions (with the exception of asthma and "other chronic diseases") compared with the nontreatment group. in the pandemic cohort, the statin treatment group compared with the nontreatment group was older, with a greater proportion of males and whites. the statin treatment group had a higher prevalence of cardiovascular disease, chronic metabolic disease, chronic lung disease, and renal disease, but not of other chronic conditions. the statin treatment group also had a greater proportion of persons considered obese and morbidly obese. influenza vaccination was more prevalent among those in the statin treatment group, for both cohorts. there was no significant difference between the proportion of those treated with antivirals for either cohort. after matching on propensity score (table ) , both the - and pandemic matched samples were balanced on treatment groups. for all covariates, the standardized differences after matching were < . for both cohorts. there were pairs in the - sample. for pairs, the treated case subject died within days, but the untreated case subject did not. there were pairs in which the untreated case subject died within days but the treated subject did not. for pair, both the treated and untreated case subjects died, and for pairs, neither case subject died. the results of mcnemar's test indicate that the -day mortality rates between the treated ( . %) and untreated ( . %) groups were significantly different (p < . ). there were pairs in the - cohort sample. for pairs, the treated case subject died within days but the untreated case subject did not. there were pairs in which the untreated case subject died within days but the treated subject did not. for pairs, both the treated and untreated case subjects died, and for pairs, neither case subject died. the results of mcnemar's test indicate that the -day mortality rates between the treated ( . %) and untreated ( . %) groups were not significantly different (p = . ). cox proportional hazards models with robust standard errors were fit to the matched samples, stratified on matched pairs. the sole predictor variable for the models was statin treatment. we tested post hoc whether logistic regression on the unmatched pandemic cohort would have resulted in a significant point estimate for an effect of statins on mortality. we used a logistic model with backward deletion (and all first-order covariates). because statin treatment was not a covariate selected through stepwise selection, we forced statin treatment into the final model. in a final model that included age, sex, and renal disease as covariates, the adjusted odds ratio (or) for statins was not significant (or = . ; % ci, . - . ). a sensitivity parameter and corresponding bounds were calculated for the observed point estimate from the - matched sample. the results (gamma = . ; maximum p value = . ) indicate that the point estimate is sensitive to hidden bias. an unmeasured confounder that could explain a % difference in the odds of statin treatment between groups could explain the observed association between statin use and mortality. sensitivity analysis for the point estimate for - data was not calculated due to the lack of an observed significant effect. in this analysis, using data from a population-based laboratoryconfirmed influenza hospitalization surveillance platform, we evaluated the effects of statin use over influenza seasons. we found that statins had no statistically significant effect on mortality during the pandemic season, but there was a significant statin effect on reducing mortality during the - b other chronic diseases is a combination of any underlying chronic illnesses mentioned in patient medical records other than cardiovascular disease, chronic metabolic disease, chronic lung disease, renal disease, asthma, and obesity. c any antivirals administered during the course of illness. influenza season. the differential impact of statin treatment on mortality between seasons could be due to differences in circulating strains, because the predominant influenza a virus subtype in - season was h n , whereas influenza a(h n )pdm virus predominated during the pandemic period. age group-specific attack rates among different influenza subtypes can vary, and differences in immune histories by age could have an impact on the different results between the - season and the pandemic. statins have been found to modulate anti-inflammatory effects [ ] ; whether the difference in apparent efficacy of statins between the seasons could be explained by variation in age-specific immune histories or variation in the degree of cytokine dysregulation caused by the different influenza virus subtypes is an area for additional investigation. however, on further examination, the latter association was measurably sensitive to bias and therefore could reflect omissions of covariate measurement rather than an actual relationship between statin treatment and death after influenzaassociated hospitalization. data from the - season was previously analyzed using a multivariable logistic regression model [ ] . results from that analysis showed that after controlling for demographic characteristics, underlying medical conditions, vaccination, and antiviral treatment, the use of statins reduced the odds of death (adjusted or, . ; % ci, . -. ). however, a limitation of observational studies is the lack of random treatment assignment, which can result in sizeable differences in the distribution of covariates between treatment groups. we sought to balance the covariates between the statin-treated and untreated groups by using propensity score analysis. nonetheless, we also found a protective effect of statins on death among laboratoryconfirmed influenza patients hospitalized during the - season. questions persist about the efficacy of statin medications in reducing severe complications of influenza because we showed that our findings could be driven by unmeasured biases. four other studies that looked at statin use and influenza outcomes also found equivocal results. two of them examined the impact of statins in reducing severe disease in adults hospitalized with laboratory-confirmed influenza during the h n pandemic [ , ] . neither found a significant association between use of statins and severe disease (classified as either intensive care unit admission or death), although both were potentially hampered by small sample size. two studies evaluating statin use during multiple influenza seasons in the decade before the pandemic found a protective effect on influenza mortality, although of the studies found only a modest ( % reduction in deaths from pneumonia) effect [ , ] . these latter studies relied entirely on administrative claims data; patients identified with influenza were not laboratory-confirmed, raising concern for misclassification of outcome, and may have been additionally biased by misclassification of exposure, because both studies abstracted data on previous history of statin use but not actual use at the time of the influenza hospitalization. information about whether there is benefit to initiating statin use in patients without other indications for statins at the time of influenza diagnosis would certainly be of clinical and public health interest. in this context, randomized controlled trials (rct) that address initiation of statin treatment in patients with influenza would offer clear advantages. recent rct data have been pessimistic regarding the role of statins in modulating inflammatory responses in infectious disease processes. one rct observed no clinical effect of statins on -day mortality among patients with ventilator-associated pneumonia [ ] ; another showed that statins did not improve clinical outcomes among patients with acute respiratory disease syndrome associated with sepsis [ ] . in contrast, a recent in vitro study showed that statin treatment can protect host cells against influenzainduced inflammation by reducing the production of tumor necrosis factor-α, interleukin- , and interferon-γ, and therefore inhibit influenza a virus replication [ ] . further studies to evaluate the effect of immunomodulatory agents in reducing influenza-related complications may still be warranted, but they may be better suited for settings where these drugs are not used widely. this study has several limitations. we could not determine whether a patient's statin treatment continued throughout the -day follow-up period because we only had medical data for the period of time the patient was hospitalized. the length of exposure to statins before hospitalization was not measured nor was the dose or frequency of statin use before or during hospitalization, which could have a possible effect on the outcome. in addition, identification of death after hospital discharge via data linkage between the ssdi and eip data could result in an underdetection of deaths; the probabilistic linkage algorithm used is highly accurate, but it is not a match-merge of data by, for example, a unique identification number. however, there is no reason to expect that either mortality status would be misclassified or that deaths would be undetected as a result of treatment status or any particular covariate. emerging infections program influenza surveillance sites do not collect detailed data on socioeconomic status or baseline functional status, and data on bmi were not collected during the - influenza season. certainly, an unmeasured covariate such as insurance coverage could hypothetically increase the likelihood of statin treatment prehospitalization while simultaneously reducing the likelihood of death after hospitalization. healthy user bias may certainly apply to the current study as well as most studies cited above [ ] [ ] [ ] [ ] ] . patients being treated with statin medication have been found to have better access to preventive services such as screening services and vaccinations, and therefore they may be healthier at baseline than patients not taking statins [ ] . moreover, those with limited access to care would be more likely to be hospitalized later in the course of illness, and they would have reduced opportunities to respond to medical interventions. during an influenza pandemic, we may have very few tools to prevent and treat influenza virus infection and therefore reduce mortality, because vaccine development can occur very late in the course of the pandemic and antivirals may be in limited quantity and of unknown effectiveness to a novel strain. our study results do not find a definite protective effect of statins on influenzaassociated death. promotion of the use of statins as part of public health pandemic preparedness or for an individual patient's benefit is not warranted based on current available data. influenza illness and hospitalizations averted by influenza vaccination in the united states influenza-associated hospitalizations in the united states estimates of deaths associated with seasonal influenza -united states influenza and the winter increase in mortality in the united states, - cross-reactive antibody responses to the pandemic h n influenza virus global mortality estimates for the influenza pandemic from the glamor project: a modeling study oseltamivir and risk of lower respiratory tract complications in patients with flu symptoms: a meta-analysis of eleven randomized clinical trials outcomes of adults hospitalised with severe influenza antiviral therapy and outcomes of influenza requiring hospitalization in ontario treatment with neuraminidase inhibitors for critically ill patients with influenza a (h n )pdm impact of neuraminidase inhibitor treatment on outcomes of public health importance during the - influenza a(h n ) pandemic: a systematic review and meta-analysis in hospitalized patients patients hospitalized with laboratory-confirmed influenza during the - influenza season: exploring disease severity by virus type and subtype pandemic influenza: a potential role for statins in treatment and prophylaxis how will physicians respond to the next influenza pandemic? beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide statins in the intensive care unit do statins have a role in preventing or treating sepsis? the effect of statin therapy on infection-related mortality in patients with atherosclerotic diseases prior statin therapy is associated with a decreased rate of severe sepsis statins and sepsis in patients with cardiovascular disease: a population-based cohort analysis statins for the prevention and treatment of infections: a systematic review and meta-analysis pre-admission statin use and inhospital severity of pandemic influenza a(h n ) disease association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study influenza and copd mortality protection as pleiotropic, dose-dependent effects of statins influenza morbidity and mortality in elderly patients receiving statins: a cohort study an introduction to propensity score methods for reducing the effects of confounding in observational studies performing a :n case-control match on propensity score observational studies openepi: open source epidemiologic statistics for public health statins as a newly recognized type of immunomodulator effect of immunomodulatory therapies in patients with pandemic influenza a (h n ) complicated by pneumonia effect of statin therapy on mortality in patients with ventilator-associated pneumonia: a randomized clinical trial rosuvastatin for sepsis-associated acute respiratory distress syndrome protective effect of fluvastatin on influenza virus infection association between statins and mortality adherence to lipidlowering therapy and the use of preventive health services: an investigation of the healthy user effect we thank the following personnel: erin parker, mph potential conflicts of interest. w. s. reports personal fees from merck and personal fees from sanofi-pasteur during the conduct of the study; j. m. reports grants from the cdc during the conduct of the study; p. r. reports grants from the cdc during the conduct of the study; and s. m. z. reports grants from the cdc during the conduct of the study. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -cpl zf f authors: provoost, judith; valour, florent; gamondes, delphine; roux, sandrine; freymond, nathalie; perrot, emilie; souquet, pierre-jean; kiakouama-maleka, lize; chidiac, christian; lina, gérard; dumitrescu, oana; sénéchal, agathe; ader, florence title: a retrospective study of factors associated with treatment decision for nontuberculous mycobacterial lung disease in adults without altered systemic immunity date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: cpl zf f background: nontuberculous mycobacteria (ntm) lung diseases are increasingly recognized as chronic opportunistic infections, occurring in individuals with a wide variety of underlying conditions. in the absence of systemic immunodeficiency, decision of ntm lung disease treatment must relies on a careful risk/benefit assessment, given the requirement of long-term administration of multidrug therapies supported by limited evidence. the primary objective was to identify the factors associated with anti-ntm treatment initiation. clinical and radiological outcome upon treatment were studied. methods: this retrospective, single center study ( – , months) addressed the criteria supporting treatment decision among adults with ntm lung disease without systemic immunodeficiency at our institution, with the assigned goal to harmonize the practice. all patients matched the current international definitions of ntm lung disease according to the american thoracic society criteria. factors associated with anti-ntm treatment were investigated by conditional logistic regression. clinical and radiological outcomes of treated and untreated ntm-disease cases were examined. mortality rate was assessed. an expert radiologist conducted a blinded computed tomography (ct)-scan review of the treated and untreated patients. results: among cases of ntm lung diseases, ( %) received anti-ntm treatment. in univariate analysis, a body mass index (bmi) < kg/m( ) (odds ratio (or), . [ % confidence interval (ci) . – . ]; p = . ), hemoptysis (or, . [ % ci . – . ]; p = . ), excavation(s) (or, . [ % ci . – . ], p = . ), prior anti-ntm treatment (or, . [ % ci . – . ]; p = . ), aspergillus spp. co-infection (or, . [ % ci . – . ]; p = . ) were associated with treatment initiation. in multivariate analysis, aspergillus spp. co-infection was the only independent determinant of treatment initiation (or, . [ % ci . – . ]; p = . ). twenty-one ( %) patients received ≥ anti-ntm drugs. median treatment duration and follow-up were . (interquartile range [iqr], . – . ) weeks and . (iqr, . – . ) months, respectively. regarding radiological outcome, ct-scans were reviewed, showing similar rates of regression or stabilization in treated and untreated patients. overall mortality rate was not different in treated and untreated patients. conclusion: the most relevant variable associated with anti-ntm treatment initiation was aspergillus spp. co-infection. radiological regression or stabilization of pulmonary lesions was not different between the treated and untreated patients. nontuberculous mycobacteria (ntm) are ubiquitous bacteria of environmental origin including a widely diverse number of species (> ), some of which cause disease in humans [ ] [ ] [ ] . prevalence of ntm lung diseases is unexpectedly increasing in industrialized countries, as consistently uncovered by recent studies [ , ] . the key issue remains to determine whether ntm are the true and single promoter of an evolving lung disease or chronic airway colonizers, among others. to standardize the diagnosis of ntm lung disease, the guidelines for ntm diagnosis of the american thoracic society (ats)/infectious disease society of america (idsa) and the british thoracic society (bts) require isolation and growth of the same ntm strain on at least two separate samples from the patient [ , ] . human host and pathogenic ntm relationship is still poorly understood, as ntm virulence is highly variable from one species to another. ntm lung disease is strongly associated with pre-existing pulmonary conditions such as chronic obstructive pulmonary disease (copd), cystic fibrosis, idiopathic bronchiectasis, prior active tuberculosis or pneumoconiosis [ ] . it is also frequently associated with genetic or acquired systemic immune deficiency such as defects in the pathways of inflammatory cytokines interleukin (il)- , tumor necrosis factor (tnf)-α or interferon (ifn)-γ, immunosuppressive treatments (including anti-tnf-α therapy or corticosteroids), solid-organ transplantation, or acquired immune deficiency syndrome (aids)/human immunodeficiency virus (hiv) infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, it may also occur in individuals without recognized severe immune local or systemic deficiency. in the absence of patent predisposition, ntm diseases are overrepresented among the specific morphotype of slender women with a low body fat [ ] . treatment decision for ntm lung disease is challenging. there is debate as to which patients should benefit the most from treatment according to medical background, comorbidities, clinical status, radiologic features and causal ntm strain. assessment of clinical, microbiologic, and radiologic response to treatment is not standardized as well. we focused on ntm lung diseases in adults without systemic immunodeficiency that met the ats criteria guidelines. based on the comparison of a group of treated and untreated patients, the primary objective was to identify the factors associated with physician decision of initiating anti-ntm treatment. secondary objectives were to study the outcome upon treatment and to propose a standardized evaluation for the diagnosis and decision making to treatment of ntm lung diseases in adults without systemic immunodeficiency. we conducted a retrospective, observational, single-center study between january and february ( months) among adults (≥ year-old) without systemic immunosuppression presenting ntm lung disease. exclusion criteria were hiv infection, cystic fibrosis, primary ciliary dyskinesia, active malignant disease, solid-organ transplantation or ongoing immunosuppressant treatments such as tnf inhibitor or high-dose corticosteroid (≥ mg/kg more than days). case identification was based on cross-referencing the databases of the mycobacteria laboratory and the departments of infectious and pulmonary diseases. patients eligible for inclusion in the ntm lung disease cohort were those who matched the criteria previously defined by the ats/idsa and the bts guidelines with the minimum requirement of clinical and microbiologic following criteria: (i) pulmonary symptoms associated with multifocal bronchiectasis with multiple small nodules on computed tomography (ct)-scan; (ii) proper exclusion of other diagnoses; (iii) ntm-positive culture results from at least two separate expectorated sputum samples or a ntm-positive culture result from at least one bronchial wash or lavage [ , ] . patients' characteristics at diagnosis were collected in order to perform analysis on selected variables: demographics; history of predisposing factors; underlying pulmonary diseases; comorbidities; pulmonary function testing; respiratory bacterial or mycological co-infection(s), which definition was similar to ntm criteria, namely positive culture isolation of the same species from at least two separate expectorated sputum samples or a positive culture result from at least one bronchial wash or lavage; immunologic status; nutritional status; clinical features; microbiologic assessment through identification of ntm species on positive ntm cultures and sample culture conversions; radiologic features on high-resolution ct-scans (fibrocavitary disease or nodular/bronchiectasis disease); prior treatment for ntm lung disease, treatment combination and duration; outcome. because of the retrospective observational nature of the study and the lack of any modification in patients' management, the need for informed consent was waived with the authorization of the ethics committee of lyon university hospital (comité d'ethique, hospices civils de lyon), which approved the study under the number - . an independent expert chest radiologist, blinded to the patient information, retrospectively reviewed the ct-scans performed without injection of intravenous contrast media, assigned in random order at diagnosis and six to months after treatment or during the follow up of the untreated patients. the number and size of cavity(ies) and their wall thickness were evaluated in the lung window setting. nodular opacity(ies) (≥ mm), cluster(s) of small nodules (≤ mm), the tree-in-bud pattern, the presence of bronchiectasis in any of the lobes or multifocal bronchiectasis were evaluated. based on the number and size of the lesions, the expert classified the lesions as improved, stable or worsening. the primary endpoint was to identify the factors significantly involved in the decision of initiating anti-ntm treatment by patient referent physician. secondary endpoints were the assessment of clinical and radiological outcomes upon anti-ntm treatment in comparison with no treatment. based on these findings, a standardized appraisal was proposed to assist diagnosis management and treatment decision for ntm lung diseases in adults without altered systemic immunity. descriptive data were used to estimate the frequencies of the study variables. there were expressed as count (percentage, %) for dichotomous variables and as medians (interquartile range [iqr]) for continuous values. the number of missing values was excluded from the denominator. non-parametric statistical methods fisher exact test, χ test and mann-whitney u test were used to compare groups, where appropriate. the probability of treatment initiation over time was evaluated by kaplan-meier survival curve analysis, with group comparison using the log-rank (mantel-cox) test. stepwise binary logistic regression analysis was used to assess the determinants for treatment initiation, expressed as odd ratios (or) with % confidence intervals ( % ci). after checking the variables for interactions, variables with medical meaning and with p-values obtained in the univariate analysis of < . were included in the final multivariate model. a value of p < . was considered significant. all analyses were performed using spss software version . (spss. chicago. il). out of patients eligible to ats/idsa ntm lung disease criteria, patients were included in the study, of who ( %) received and ( %) did not receive anti-ntm treatment (fig. ). the median age was (interquartile range [iqr], - ) year-old with a male/ female ratio of . . etiologic ntm agents were mycobacterium avium (n = , . %), m. chimaerae (n = , . %), m. xenopii (n = , . %), m. intracellulare (n = , . %), m. simiae (n = , . %), m. kansasii (n = , %), and m. abscessus (n = , %), with three patients having ≥ concomitant ntm lung diseases. importantly, the evidence of ntm lung disease has led to diagnose six underlying chronic lung diseases, of which a genetically documented cystic fibrosis in a year-old women. on descriptive analysis, patient's characteristics did not significantly differ, to the exception of a lower bmi (p = . ) and a higher number of previously known ntm lung disease (p = . ) in treated versus untreated patients (table ) . notable percentages of missing data at diagnosis have to be acknowledged for active tobacco smoking ( . %, n = ), respiratory functional testing ( . %, n = ) with a very few patients having a -min walk test, baseline arterial blood oxygenation levels ( . %, n = ), ct-scan follow up within months after diagnosis in untreated patients ( %, n = ). factors leading the patient referent physician to initiate anti-ntm treatment were assessed using bivariate analysis. table ). the probability of treatment initiation over time according to the presence or not of targeted variables was investigated. the probability was significantly higher in case of bmi < vs. > kg/m (p = . ), of hemoptysis vs. no hemoptysis (p = . ), of aspergillus spp. co-infection vs. no co-infection (p = . ), of pulmonary excavation(s) vs. no excavation (p = . ) (fig. a, b, c and d, respectively) . (table ) . finally, all-cause mortality was not different between treated and untreated groups, although lost to follow-up was high in the untreated group (n = , . %). regarding the four patients that deceased (n = in the treated group and n = in the untreated group), the cause of mortality was linked to the underlying diseases rather than ntm-related mortality. in the present study, treated patients were characterized by bmi < kg/m , presence of hemoptysis and excavation(s), aspergillus spp. co-infection, and prior anti-ntm treatment. aspergillus spp. co-infection was the only independent factor associated with treatment initiation. a single study from five english centers has recently addressed the factors that influence anti-ntm treatment initiation using similar retrospective design of treated and untreated cohort comparison inclusion criteria which allowed non-aids/hiv immunosuppressed patients ( and % in the treated and untreated subsets, respectively) to be evaluated. in the multivariate analysis, patients had increased odds of anti-ntm treatment in case of cavitation on ct imaging, night sweats and weight loss [ ] . here, three out of four criteria are part of guideline criteria leading to decision of anti-ntm treatment. they all reflect a degree of severity of ntm lung disease linked with progression of an impaired respiratory condition. the current problematic of ntm lung diseases shifts from distinguishing colonization from infection toward differentiating stable, poorly active vs. progressive active ntm disease, the latter being responsible for further structural lung damage(s). the risk/benefit analysis includes prescribing recommended long-term multidrug regimens with concerns over suboptimal cure rates and frequently reported drug-related side effects. to support these arguments, others have already shown that physician ats/idsa guideline [ ] . another study conducted in france has shown that among a cohort of ntm lung diseases, only ( . %) received appropriate treatment matching ats/idsa guidelines [ ] . inappropriate prescriptions were mostly related to shorter treatment duration ( months or less) and/or off-recommendation regimen, particularly those excluding macrolide from the combination or those using a single-drug macrolide regimen [ , ] . it has to be acknowledged that guidelines specify that treatment for mac-associated lung disease in hiv-negative individuals can be a three-times-weekly drug regimen upon culture conversion while on therapy for year, which may favor treatment compliance [ , , ] . patients with bronchiectasis and ntm lung disease have a higher prevalence of being sensitized to aspergillus than patients with ntm-free bronchiectasis [ ] . allergic airway manifestations in response to aspergillus are termed aspergillus-related lung diseases with a spectrum going from aspergillus-induced hypersensitivity to the severe allergic bronchopulmonary aspergillosis (abpa) [ ] . by itself, the coexistence of ntm and aspergillus in lung airway justifies the need for testing aspergillus serology, total serum immunoglobulin (ig)e and aspergillus-specific ige levels as well as mycological direct examination and culture of sputum or bronchial aspirates for presence of filamentous fungi in the diagnosis algorithm of patients with ntm lung diseases. active co-infections with ntm and aspergillus spp. have also been described, in which patients with ntm lung disease develop chronic forms of pulmonary aspergillosis which definitions and management have been revisited in recent updated guidelines [ ] [ ] [ ] [ ] . radiographic improvement may be hampered by concomitant lung disease and the limited potential for resolution of consolidated radiologic abnormalities. a previous study has investigated radiologic response to treatment showing consistent results with those found in the present study. although anti-ntm treatment led to an improvement or stabilization of lesions for a majority of patients, these modifications were not significantly different from the untreated group who went through ct-scan follow up indicating that anti-ntm treatment did not lead to radiological abnormalities reversion [ ] . the present study has strengths and limitations. the strength is the study of the largest cohort so far of ntm lung diseases in patients without systemic immunodeficiency with exhaustive data collection and blinded radiological assessment. we acknowledge the biases that contribute to mitigate conclusions from the study such as being conducted in a single center, the important differences in physician's management resulting in lack of consistency in treatment decision making, the number of missing data. in addition, the ct-scans were not performed at fixed intervals, particularly in the untreated subset of patients. finally, treatment duration and outcome criteria were not standardized, which prevent to properly assess treatment efficacy. useful consensus definitions for key outcome parameters to be used in the treatment of ntm lung diseases have been released very recently, which should harmonize data collection regarding ntm treatment [ ] . future researches are necessary to better define criteria associated with progressive active ntm disease in the immunocompetent setting. concretely, this preliminary study has led to implement in our institution a standardized appraisal for the diagnosis of ntm lung diseases in this particular setting ( table ). the aim is to provide a future basis for the development of a diagnosis scoring system supporting anti-ntm treatment decision. future studies should focus as well on the most relevant ct imaging variables associated with response to treatment over time that may be applied in future clinical trials to assess treatment outcome. in summary, the main factors supporting anti-ntm treatment decision in immunocompetent were low bmi, hemoptysis, lung excavation(s), prior anti-ntm treatment and aspergillus pp. co-infection, the latter being the only independent factor. anti-ntm treatment did not achieve radiological abnormalities reversion, as pulmonary lesions assessment showed no difference between the treated and the untreated patients. a diagnosis of ntm lung disease in an immunocompetent patient requires investigating the presence of a chronic pulmonary underlying disease. pulmonary disease caused by non-tuberculous mycobacteria nontuberculous mycobacterial lung disease: the top ten essentials leveraging advances in tuberculosis diagnosis and treatment to address nontuberculous mycobacterial disease pulmonary nontuberculous mycobacterial disease prevalence and clinical features: an emerging public health disease an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases british thoracic society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (ntm-pd) pulmonary disease by nontuberculous mycobacteria-clinical management, unmet needs and future perspectives patients with nontuberculous mycobacterial lung disease exhibit unique body and immune phenotypes factors which influence treatment initiation for pulmonary non-tuberculous mycobacterium infection in hiv negative patients; a multicentre observational study lack of adherence to evidence-based treatment guidelines for nontuberculous mycobacterial lung disease are guidelines on the management of non-tuberculous mycobacteria lung infections respected and what are the consequences for patients? a french retrospective study from early results (at months) with intermittent clarithromycin including regimens for lung disease due to mycobacterium avium complex intermittent antibiotic therapy for nodular bronchiectatic mycobacterium avium complex lung disease nontuberculous mycobacterial disease and aspergillus-related lung disease in bronchiectasis nontuberculous mycobacterial lung infection complicated by chronic necrotising pulmonary aspergillosis chronic necrotizing pulmonary aspergillosis as a complication of pulmonary mycobacterium avium complex disease chronic pulmonary aspergillosisrationale and clinical guidelines for diagnosis and management treatment outcome definitions in nontuberculous mycobacterial pulmonary disease: an ntm-net consensus statement the authors gratefully acknowledge pr gilles devouassoux for helpful insights. no external funding was received for this study. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions jp contributed to conception and design of the study, acquisition of the data, interpretation of the data, drafted the manuscript and approved the final version; fv carried out the statistical analysis, participated in revision of the paper for important intellectual content, and approved the final version; dg carried out the blinded review of ct-scans with the examination of each imagery, she participated in revision of the paper for important intellectual content, and approved the final version; gl and od are in charge of the mycobacteria laboratory and have provided ntm strain identification and drug susceptibility tests. they participated in revision of the paper for important intellectual content, and approved the final version; sr, nf, ep, pjs, lkm, and cc contributed to acquisition of the data, revision of the paper for important intellectual content, and approved the final version; fa and as are the project initiators, contributed to conception and design of the study, drafted the manuscript and approved the final version. fa coordinated the project until the submission of the article. all authors read and approved the final manuscript. the ethics committee of lyon university hospital (comité d'ethique, hospices civils de lyon) approved the study under the number - . because of the retrospective observational nature of the study and the lack of any modification in patients' management, the need for informed consent was waived with the authorization of the ethics committee of lyon university hospital (comité d'ethique, hospices civils de lyon), which approved the study under the number - . not applicable. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. no writing assistance was utilized in the production of this manuscript. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.author details département de pneumologie, hospices civils de lyon, lyon, france. key: cord- -lt is is authors: preston, nicholas d.; daszak, peter; colwell, rita r. title: the human environment interface: applying ecosystem concepts to health date: - - journal: one health: the human-animal-environment interfaces in emerging infectious diseases doi: . / _ _ sha: doc_id: cord_uid: lt is is one health approaches have tended to focus on closer collaboration among veterinarians and medical professionals, but remain unclear about how ecological approaches could be applied or how they might benefit public health and disease control. in this chapter, we review ecological concepts, and discuss their relevance to health, with an emphasis on emerging infectious diseases (eids). despite the fact that most eids originate in wildlife, few studies account for the population, community, or ecosystem ecology of the host, reservoir, or vector. the dimensions of ecological approaches to public health that we propose in this chapter are, in essence, networks of population dynamics, community structure, and ecosystem matrices incorporating concepts of complexity, resilience, and biogeochemical processes. definitions of one health have varied among different authors and institutions, but a defining central tenet is that a one health approach brings a holistic understanding of health. this broader view includes human medicine, veterinary medicine, and an understanding of the ecological context of health (which we call 'ecohealth'). to date, one health approaches have tended to focus on closer collaboration among veterinarians and medical professionals, but remain unclear about how ecological approaches could be applied or how they might benefit public health and disease control. in this chapter, we review ecological concepts, and discuss their relevance to health, with an emphasis on infectious diseases, notably emerging infectious diseases (eids). nearly two-thirds of eids are zoonotic, and a majority of those ( %) originate in wildlife (anderson et al. ; cleaveland et al. ; daszak ) . diseases are considered 'emerging' if they are identified as occurring in a new geographic area, expanding their incidence rapidly, displaying novel genetic code, or moving into humans for the first time. the most important are pandemics, those that become established in humans and spread internationally. pandemics tend to be zoonotic, foodborne, or antimicrobial resistant pathogens, and their emergence and spread is overwhelmingly a result of changes in human demography (e.g. travel, population growth), behavior (e.g. drug use), economic activity (e.g. agricultural intensification), or anthropogenic changes to the environment (e.g. land-use change, climate change) (weiss and mcmichael ; jones et al. ) . the interactions among these underlying drivers and the dynamics of pathogens in wildlife, livestock, and people are a key focus of studies of the ecology of infectious diseases. ecology emerged from natural history and rose to prominence as a scientific discipline in the late nineteenth century as the 'study of the interactions of organisms with their environment' (haeckel ) . while originally a descriptive science, the theories of adaptation, evolution, and speciation rapidly became central to the field, and led to increasingly analytical approaches (lawton ) . over the past few decades, ecologists have analyzed data from field observations, laboratory studies, and large-scale field experiments to describe the structure and dynamics of populations, their interactions within communities, and the complexity of ecosystems. in this chapter, we build on the work of wilcox and jessop ( ) and last ( ) , adding an ecosystem network perspective to describe how ecological approaches can be focused on infectious diseases. we focus on three components: population, community, and ecosystem ecology. population ecology is the study of the population dynamics of a species with relevant metrics of density, natality, mortality, immigration, and emigration (hall ; murray ) . population dynamics are generated through competition, predation, parasitism, and the distribution of species. community ecology describes the clustering of populations of species into communities and the processes that dictate composition and diversity. pertinent community metrics are similarity, continuity, species, and genetic diversity. ecosystem ecology is the study of biotic and abiotic components of ecological systems, their biophysical interactions, and the flow of energy and materials (lindeman ; odum ; cook ) . the metrics for ecosystems include state, rates, and productivity. ecosystems provide the framework for organization of species and resource compartments, and modulate rates and dynamics of functions, processes, and services. in modern ecological approaches, a network perspective can be used to describe interactions among ecosystem components, with populations referred to as nodes, links between nodes in a community as edges, and the overall environment and abiotic components as an ecosystem matrix (fig. ) . this framework complements a shift in ecosystem thinking from structures and hierarchies, to networks and webs. while the concepts in this chapter are presented in the context of wildlife and emerging infectious diseases, they are generalizable to diverse ecosystem interactions. traditional views of ecosystems were focused on the concept of directional succession, whereby ecosystems developed along a predictable pathway to a climax system (e.g. mature deciduous forest) (clements ; gleason ) . in reality, ecosystems are dynamic and complex aggregations of communities continually adapting to internal and external influences. rarely are they stable or at fig. diagram illustrating the ecosystem components described in the text: a node is a population of organisms; edges are links between nodes in a community; and the overall environment including abiotic components is the ecosystem matrix. nodes are structured vertically into trophic levels and horizontally along an environmental gradient equilibrium, as described by static representations of food chains, trophic guilds, and species dominance. moreover, they demonstrate non-equilibrium dynamics in a mosaic of patches shifting among stable states when disturbed. the complex structure of ecosystems has long frustrated efforts to forecast and predict their behavior, yielding models of resilience, complexity, and chaos. ecosystems: abundance, structure, and flow historically, public health has focused on the dynamics and structure of human populations-only a single node within the context of global ecosystems. holistically, human health can best be considered in the context of other organisms within a network of populations, communities, and ecosystem interactions. infectious diseases within a one health context require an additional dimension, namely the natural environment as the habitat of the disease agents themselves, examples of which include water borne diseases. population ecology focuses on the dynamics of an individual species in a defined area, where the malthusian growth model is a central theory. however, wildlife populations are not static, nor is their growth linear. moreover, they display complex cycles and populations evolve from interactions, including competition, predation, herbivory, and mutualism, while demonstrating stochastic dynamics and lagged responses to disturbance. because knowledge of wildlife populations still is incomplete, there are many species for which historic data are lacking or routine monitoring not yet possible. furthermore, coverage varies across geographic regions, taxonomic groups, size, abundance, and economic or social values. despite the fact that most eids originate in wildlife, few studies account for the population dynamics of the host, reservoir, or vector, in contrast to studies of human populations and demographics. this uncertainty concerning wildlife health presents a threat both to domestic animals and human populations. at a minimum, those infectious disease agents closely linked to human health should be identified and subjected to intense study, e.g., those that incubate and spread disease or provide ecological services such as disease regulation and/or vaccine discovery. identifying these key species helps set priorities for routine surveillance, as well as uncovering as yet unknown species that present a threat or cure. fluctuations in host and vector abundance engender a variable risk distribution for disease transmission. some species are unique in their proximity to human physiology (e.g. primates and wild pigs) or in their expansive ranges (e.g. birds and bats), posing unique threat as integrators, spreaders, and laboratories for recombination and mutation of disease pathogens (daszak ) . wildlife population ecology can be employed to improve global health models, but within limitation. populations are difficult to define and species-based definitions are generally inadequate. those that are naturally or artificially isolated often exhibit distinct behaviors and present differential risks (levins ). populations are a continuum, where factors such as age, sex, and size can influence risk of disease transmission, especially where distribution of the agent is not uniform. thus, it is simplistic at best to consider population dynamics in isolation from the community structure and ecosystem matrix. community ecology describes an assemblage of nodes and their interactions, or edges. the contributions of individual populations can be characterized by employing network metrics, an example of which would be a high degree of connectivity that identifies critical, keystone nodes influencing the structure of the system. it should be noted that communities can demonstrate both equilibrium and non-equilibrium dynamics. characterizing biodiversity is fundamental to community ecology. it is also one of the more widely reported and popular concepts. biodiversity varies across spatial scales and describes both intraspecific or genetic diversity of a node, as well as diversity of nodes described in terms of richness, abundance, and evenness (bisby ; jost ; whittaker ) . food webs represent a central concept in ecology, being employed to model community structure as complex hierarchies of nodes (lindeman ; elton ; forbes ; hairston et al. ). inter-node interactions (edges) among consumers and resources form the backbone of food-web networks and the nodes can be structured into trophic levels, or functional groups, such as top predators (borrvall and ebenman ; finke and denno ) , mesopredators (elmhagen and rushton ), herbivores, and primary producers. edges are generally unidirectional, but can flip during the life history of an organism when lower trophic levels prey on juveniles of higher trophic levels. single trophic food webs are the simplest (tilman ), but few real-world examples exhibit those dynamics, with multi-trophic perspectives more realistic, albeit complex (cohen ; deangelis ; polis and winemiller ) . predator-prey relationships are dominant in representations of the structure of trophic hierarchies, along with the influences of co-evolution, mutualism, autotrophy, herbivory, competition, genetics, and speciation. food webs are structured from top and bottom. top-down control of food webs can occur via predation and resource consumption by consumers, influencing community size structure. at the same time, bottom-up mechanisms operate via abundance, availability, and edibility of primary producers (autotrophs) and secondary producers (herbivores). structural dynamics of a system, i.e., arrangement of nodes and edges, can influence the magnitude and variability of community response to disturbance. endogenous (internal) pressure from one node can reorganize the entire system. food webs are often portrayed with linear connections among trophic levels; however, responses to exogenous (external) disturbance can expose complex nonlinear dynamics and feedback loops. unlike characterizations such as a balance of nature or tree of life, it is apparent from food-web manipulations that ecological networks are complex systems encompassing hierarchies, webs, nested systems, cycles, and flows (carpenter and kitchell ; scheffer and carpenter ) . when ecology is incorporated into public health endeavors, the scope is frequently limited to distribution and abundance of individual nodes. studying the population dynamics of disease hosts and vectors clearly is important if zoonotic disease emergence is to be understood, but populations need to be studied in the context of edges defining their interactions with other nodes. indeed, a community approach to disease emergence can reveal important nodes and interactions that differ from those identified in population analyses. for example, some nodes, such as keystone species, may be disproportionally important to the system due to strong connectivity or high centrality. superspreaders are highly connected and rapidly disseminate disease through a network. identifying and monitoring the keystone species, superspreaders, and nodes that regulate host and vector abundance is important in disease prevention and control. trophic cascades regulate host abundance when changes at one trophic level cascade through the food web. for example, when a predator population collapses, regulation of the disease is reduced if the disease host or vector is thereby released from control by predation. removing predators directly relieves pressure on prey abundance and may also alter physiological stress, behavior (bakker et al. ) , and morphology (werner and peacor ) of their prey. clearly, both host and predator require monitoring in such circumstances. inter-species competition affects abundance, evolution, diversity, and pathogenicity of a disease agent. these processes can be tightly coupled to their pathogen hosts and, in turn, the community dynamics of the system. hence, the invasion of an exotic species, triggered by wildlife trade, transportation, or climate change for example, could cause food webs to reorganize thereby altering the probability of disease emergence. on one hand, the introduction of a species like the tiger mosquito (aedes albopictus), which is an aggressive disease vector, can alter the conditional (binary) probability of contracting certain vector-borne diseases. on the other hand, invasions by suboptimal hosts can 'dilute' disease risk. invasions can also introduce boom and bust dynamics, destabilizing systems and tipping native populations into irreversible alternate states. invasive species also diverge genetically from their original populations through isolation and founder effects, contributing to ''waves'' of disease occurrence. spatiotemporal variance in food webs is particularly acute for migratory populations, where resource consumption changes with habitat and the effect on nodes in one system can be transferred to another. in effect, migration provides a unique opportunity for populations and communities to exchange pathogens. in these scenarios, mapping distributed food webs could help identify pathways for disease transmission. food web and community network analyses introduce a high degree of complexity to mathematical and statistical models of systems. furthermore, it is difficult to determine accurately the trophic position of individual nodes in food web models. while advances in stable isotope analysis, fatty acids, and ecological stoichiometry help determine trophic position relative to other nodes in the community, as well as composition of diet; isotopic measurements often have location-specific limitations, whereby values are relative to local autotrophic production in the system as influenced by external subsidies. thus, it is difficult to draw meaningful comparisons among food webs. what is required is a method that generalizes models and captures topological position and functional importance of networks without a food web-specific bias (olff et al. ). by using food-web manipulations, it has been possible to demonstrate biogeochemical processes play an important role in structuring communities (carpenter and kitchell ; scheffer and carpenter ) . so, although community ecology considers both nodes and the edges that connect them, these systems must ultimately be studied in the context of their environment or ecosystem matrix. ecosystem ecology encompasses biophysical mechanisms regulating ecosystem metabolism across both biotic and abiotic compartments, this includes ecological function, physiological processes, populations and communities, resource availability, nutrient cycling, and connections among systems. the connections, flows, and cycles affecting the life history of an organism are highlighted, including materials of composition and their life cycle. traversing networks that incorporate abiotic pathways may help map these connections, a useful example of which is the carbon cycle, with biotic and abiotic compartments through which carbon can flow, sequester, or transform. the ecosystem matrix is a spatiotemporal mosaic that provides background structure for ecosystem networks. it is a complex system with unpredictable dynamics, including bidirectional relationships among organisms that extract, modify, and release resources into their surroundings. the physical-chemical conditions that surround an organism regulate metabolism as they consume resources and generate waste (begon et al. ) . ultimately, resource flows influence system dynamics of populations and communities. biogeochemistry describes the flow of matter, such as nutrients and toxins, through an ecosystem matrix, including processes such as decomposition and decay. it spans biotic and abiotic compartments through biologically mediated chemical cycling of nutrients. microorganisms play a critical role in the availability of resources by decomposing waste and processing mineral components, essentially driving nutrient cycles in ecosystems. temperature, salinity, ph, and redox generate gradients regulating distribution of organisms and their metabolism, in effect the availability of resources (schlesinger ) . availability of resources, notably nutrients, is related to population dynamics, e.g., the life cycle of organisms, and community structure, such as food webs. phosphorus, for example, is a commonly limiting nutrient in freshwater lakes that constrains productivity. while phosphorus can be introduced through external subsidies, the ecological community can influence availability of the resource internally, thereby altering community composition. certain zooplankton, for example, sequester phosphorous for their reproductive needs to the extent that they limit growth and abundance of competing species. these competitive interactions will ultimately affect water quality and physical characteristics, such as transparency and temperature profiles (elser et al. ) . physical-chemical conditions of the matrix drive enzymatic processes and affect habitat suitability and niche structure in ecosystems. olff et al. ( ) proposed an additional horizontal ecological-stoichiometry axis to supplement the vertical trophic axis in food webs (fig. ) . these frameworks build upon research in marine systems (azam et al. ) and terrestrial systems (bardgett ; wardle ) that emphasize a 'dual foundation' for food webs based on both organotrophs and autotrophs. the landscape provides the physical structure for the ecosystem matrix, including habitat niches for organisms. physical connectivity (e.g., wildlife corridors) can dictate the distribution and dispersal of organisms. as chemicals transition among media such as water, the atmosphere, and land they are modified in ways that alter their availability. landscapes support a mosaic of abiotic conditions that determine the phase space of abiotic resources, including chemical state and suitability for uptake. ecosystem engineering is the process whereby organisms influence the biophysical feedback mechanisms that structure their habitat. this can fundamentally alter ecosystem function from local to global scales (e.g., beaver dams to forest respiration). ecosystem engineers influence the matrix in which they live, rendering it more or less habitable for themselves and their competitors (jones et al. ; wright and jones ) . in australia, for example, a rabbit fence was built to confine expansion of invasive rabbits, altering patterns of herbivory that, in turn, affected evapotranspiration and regional precipitation. ultimately, this altered the microclimate and suitability of the environment for multiple organisms and processes (lyons et al. ) . feedback loops and cycles add complexity and nonlinearity to the system. they can lead to emergence of alternate stable states, with abrupt tipping points, where shifts to alternate regimes modify function and introduce chaos (scheffer and carpenter ; carpenter et al. ; huisman and weissing ; van de koppel et al. rietkerk ). ecosystem processes influence human health directly via interaction with toxins and nutrients, and indirectly via regulation of disease cycles and intensity. bioaccumulation of toxins throughout food webs poses a health threat, an example of which is dichlorodiphenyltrichloroethane (ddt), effective in controlling disease vectors but endangering animal and human health by its bioconcentration. ecosystems provide services such as sequestering toxins in wetlands and sediments, but these processes often are fragile and their disruption results in system-wide impacts. nutrient enrichment, or eutrophication, of lakes has been directly correlated with prevalence of aquatic disease agents (johnson and carpenter ) . regulation of disease is an indirect ecosystem service. however, perturbations of ecological systems can alter the regulatory process and unleash novel pathogens, demonstrated vividly by lyme disease and the dilution effect (ostfeld and keesing ) . in today's world, the biosphere is undergoing unprecedented anthropogenic ecosystem engineering, ranging from land conversion to ecological simplification and extensive biogeochemical change. the impact of these alterations spans ecosystem nodes, edges, and pathways with profound ramifications for ecosystem services and resilience. as the modified ecosystems and regimes emerge, their potential to impact human health must be understood. investigators have developed hotspot maps to characterize risk of disease emergence (jones et al. ) and threats to biodiversity (mittermeier et al. ). however, coupled socio-ecological models of risk have yet to be developed. as a network evolves, dynamic risk mandates continuous adaptive iterations to monitor emerging threats. the major drivers are direct anthropogenic land-use change, e.g., deforestation, agricultural expansion, habitat destruction, and complex indirect feedback from anthropogenic impact affecting biogeochemical cycles, e.g., nutrient cycles and climate change. the phase space for ecosystems evolves as communities are restructured. emerging systems are unstable and exhibit complex non-equilibrium dynamics and alternate states. getting ahead of an epidemic curve (fig. ) requires more than simply aggregating concepts of populations, communities, or ecosystems (schoener ). the changing network properties of the system must be monitored, along with indicators of resilience and leading indicators of collapse, if how a major disturbance is propagated or dampened through the system is to be understood. failing to comprehend the emerging topology of coupled socio-ecological systems presents a challenge of knightian uncertainty, where risk is immeasurable, and panarchy, where cause and effect are disproportionate. in these instances, disturbances can become amplified through emerging network dynamics. changes like habitat destruction and land-use/land-cover change affect the physical structure of the environmental matrix and have profound impacts on ecosystems. they jeopardize critical services, e.g., disease regulation and other, as yet unknown, ecosystem services. for example, minor disturbances from deforestation in the peruvian amazon exposed frontier effects, whereby cases of infectious disease peaked with human encroachment, but re-stabilized as humans and pathogens adapted (olson ) . it is difficult to anticipate consequences of ecosystem encroachment since the dynamics are highly variable and outcomes unpredictable. however, in this case the system exhibited altered contact and transmission rates, as well as improved habitat for malaria vectors. genetic diversity dictates adaptability. we should anticipate widespread physiological, morphological, and behavioral adaptations with land-use change, and inevitable consequences for disease emergence. geography and landscapes have long been known to play a critical role in disease, indeed the earliest disease maps by finke and humboldt date from the early victorian period (tylianakis et al. ). hence, we can anticipate that landscape changes will fundamentally alter existing ecosystem networks. changes at the landscape level, such as isolation due to habitat fragmentation, counter the trend of more highly connected systems. however, an increase in isolated systems may lead to increased genetic drift and introduce new vulnerabilities from founder effects and genetic bottlenecks. these refugia and biological corridors become hotspots for disease transmission as organisms are crowded out of the human landscape and stressed by reduced resource availability. the green revolution brought widespread alterations to global biogeochemistry. accompanying changes in agricultural practice altered the agrarian landscape-an important habitat in terms of both surface area and productivity. in this context, biogeochemistry is particularly relevant to health, given anthropogenic modification of global processes. following world war ii, the industrial efficiencies of bomb factories were adapted to production of agricultural fertilizers. as a consequence, ecological stoichiometry was radically altered. in geological time, this is a short-term experiment and it is not yet clear what the long-term implications will be for global-scale ecosystem processes. indeed, the fertilizers manufactured are typically nutrients that limit productivity. hence it is inevitable that these will impact abundance and distribution of organisms, including disease hosts and vectors. in , researchers convened by the wildlife conservation society (wcs) coined the term ''one world-one health,'' at a time of increasing global interest in connections between emerging infectious diseases and environmental stewardship. what has become the one health movement calls for interdisciplinary and crosssectoral approaches to disease prevention, surveillance, monitoring, control, and mitigation, as well as environmental conservation. the goal of improving lives, with integrated health approaches, has been embraced by veterinary, medical, public health, agricultural, and environmental health organizations in the one health initiative. this movement has helped integrate ideas from environmental, veterinary, and agricultural science with public health, and has been successful in bringing broader attention to socio-economic influences on human and animal health. ecohealth emerged in the s from an interest in connecting ecosystems and health through the original work of the international development research council (idrc) (lebel ) . the ecohealth community has since grown to include researchers from a broad range of disciplines, all of whom share an interest in the intersection of ecology and health. humans must be included in ecohealth models and wildlife in one health models. otherwise, our understanding of disease risk cannot be complete. conceptual and mathematical models from the social sciences and public health can usefully be combined with those developed for agriculture and ecology. thus, the coupled socio-ecological models will allow characterization of emerging systems, with the challenge of capturing non-linear complex behaviors. in conclusion, the dimensions of ecological approaches to public health that we propose in this chapter are, in essence, networks of population dynamics, community structure, and ecosystem matrices incorporating concepts of complexity, resilience, and biogeochemical processes. case studies disease emergence can strongly impact the abundance and diversity of wildlife populations. the dynamics of wolf and moose populations on isle royale is a classic case study. the single predator-prey dynamic is unique in its simplicity and is one of the longest studied. the system has never achieved equilibrium and cannot be explained either by top-down control of moose abundance by wolf predation or bottom-up control of wolf abundance by moose availability (vucetich et al. ) . moreover, the system exhibits both influences, with episodic disturbances from disease and climate. the introduction of parvovirus by a domestic dog caused the wolf population to crash in . subsequently, the moose population exploded which impacted balsam fir, their winter food. consequently, in the moose population crashed during a harsh winter. moose are mega-herbivores (owen-smith ) that grow sufficiently large to escape predation from wolves, so wolves are only able to prey on the young and infirm. the moose are vulnerable to ticks, which contributes to poor body condition and makes them more vulnerable to wolf predation. ultimately, the dynamics of an invasive disease agent influenced community structure, as did predation, resource availability, parasitism, abiotic conditions, and genetic diversity. these events challenged the certainty of predictive models of population dynamics and community structure. this case study illustrates the difficulty of modeling eids in relation to ecosystem dynamics. correlation of the incidence and intensity of cholera, primarily a waterborne disease, with environmental parameters, e.g., temperature, salinity, nutrients, conductivity, and other factors, including rainfall, extreme weather events, and with access or lack of access of the populace to safe water and sanitation has been studied by many investigators over the past years. the observation of colwell and huq ( ) that the causative agent of cholera, vibrio cholerae, is a commensal of zooplankton, predominantly copepods, led to examination of the annual incidence of cholera in bangladesh. controlling factors were determined to be water temperature and salinity, but also relationship to the annual cycle of plankton (colwell ) . the annual bimodal peaks of cholera in bangladesh (spring and fall) correlated with plankton blooms in the spring and fall, with copepods proving to be a vector for v. cholerae (de magny et al. ) . further studies, employing satellite remote sensing to monitor chlorophyll, sea surface temperature, and sea surface height in the bay of bengal, provided useful models of the relationship of cholera and climate (lobitz et al. ) . refinement of the models and detailed analyses of the river system of the ganges delta led to further and more detailed characterization of the drivers of the spring and fall cholera outbreaks, namely rainfall, river height and flow, and salinity (jutla et al. ) . cholera, and very likely other waterborne diseases, can be tracked to their environmental source (jutla et al. ) . thus, ecology of the v. cholerae proved to be key in understanding incidence of the disease (colwell et al. ; lipp et al. ) . based on ecology and evolution of v. cholerae, predicting cholera incidence in various regions of the world is promising. in fact, preliminary results demonstrate effectiveness of regional hydroclimatology combined with satellite data for cholera prediction models for coastal regions in south asia and sub-saharan africa, providing lead time to strengthen intervention efforts before the seasonal outbreaks of cholera occur in these endemic regions. the role of wildlife and livestock in the transmission of infectious agents to humans has been recognized for decades (karesh et al. ). zoonoses such as rabies remain endemic in wildlife and continue to spillover to people as they have done for probably centuries. however, the importance of wildlife from which pathogens are transmitted has become critical in the era of eids. the majority of eids are zoonotic and originate in wildlife (jones et al. ). pathogens such as nipah virus (niv), sars coronavirus, and ebola virus originate in wildlife species from tropical or subtropical regions, where human population density is high, and rapid changes to the environment drive increasing risk of spillover. the role of ecology in understanding patterns of zoonotic disease emergence is significant and ecologists need to be integrated into one health efforts. traditional epidemiological investigations of emerging zoonoses focus on the network of human cases affected by an eid, tracing back to origins and examining risk behavior. unfortunately, studies tend to view the role of wildlife as a risk factor for spillover and rarely involve detailed studies of wildlife population dynamics. for example, fruit bats were identified as the reservoir of nipah virus (niv) in malaysia and are, therefore, a risk factor for its emergence elsewhere. in malaysia, niv first emerged in pig farms close to fruit bat habitats. it was hypothesized that the intensive nature of the farms were the trigger for its emergence (chua et al. ). an alternative hypothesis was that bats brought the virus into the country from nearby sumatra following forest fires there during a severe el nino event (chua et al. ) . a collaborative group including wildlife biologists, veterinarians, virologists, mathematical modelers, physicians, and epidemiologists collected and analyzed data on the hunting of bats, pig population dynamics at the index farm, large-scale movement of fruit bats and the capacity of the virus to survive in urine, saliva, and fruit juices (pulliam et al. ) . this work was able to demonstrate that the continued presence of bats in the index farm region, and the particular dynamics of intensive production allowed the virus to invade the pig farm, produce a partially immune population of pigs, then re-invade to create a long-term exposure of pig workers, and the large-scale outbreak observed (pulliam et al. ) . early epidemiological studies of the emergence of niv in bangladesh identified drinking of date palm sap as a risk factors, and suggested that this might be due to contamination of the collecting pots by fruit bats (luby et al. ) . subsequent investigations involved wildlife biologists who used infrared cameras to confirm contamination in the field (khan et al. ) , and conducted longitudinal surveillance of bat populations to examine whether seasonal patterns exist that could be used to estimate risk. these studies demonstrate the value of analyzing wildlife reservoir ecology in tandem with epidemiological and specific disease investigations. such an approach will become increasingly important, given the disproportionate rise in eids originating from wildlife over the last few decades (jones et al. ) . emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers the ecological role of water-column microbes in the sea experimental manipulation of predation risk and food quality: effect on grazing behaviour in a central-place foraging herbivore ecology: individuals, populations and communities. blackwell science, oxford bisby fa ( ) characterization of biodiversity early onset of secondary extinctions in ecological communities following the loss of top predators leading indicators of trophic cascades nipah virus: a recently emergent deadly paramyxovirus anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence plant succession; an analysis of the development of vegetation. carnegie institution of washington, washington cohen je ( ) food webs and niche space environmental reservoir of vibrio cholerae the causative agent of cholera vibrio cholerae, vibrio parahaemolyticus, and other vibrios: occurrence and distribution in chesapeake bay raymond lindeman and the trophic-dynamic concept in ecology emerging infectious diseases: bridging the gap between humans and wildlife environmental signatures associated with cholera epidemics trophic control of mesopredators in terrestrial ecosystems: topdown or bottom-up? stoichiometric constraints on food-web dynamics: a whole-lake experiment on the canadian shield predator diversity and the functioning of ecosystems: the role of intraguild predation in dampening trophic cascades the lake as a microcosm the individualistic concept of the plant association ueber die fossilen medusen der jura-zeit community structure, population control, and competition an assessment of several of the historically most influential theoretical models used in ecology and of the data provided in their support biodiversity of plankton by species oscillations and chaos influence of eutrophication on disease in aquatic ecosystems: patterns, processes, and predictions organisms as ecosystem engineers global trends in emerging infectious diseases partitioning diversity into independent alpha and beta components tracking cholera in coastal regions using satellite observations the ecology of zoonoses: their natural and unnatural histories use of infrared camera to understand bats' access to date palm sap: implications for preventing nipah virus transmission are there general laws in ecology health: an ecosystem approach. international development research centre, ottawa levins, r ( ) evolution in changing environments effects of global climate on infectious disease: the cholera model climate and infectious disease: use of remote sensing for detection of vibrio cholerae by indirect measurement foodborne transmission of nipah virus land-atmosphere interaction in a semiarid region: the bunny fence experiment hotspots: earth's biologically richest and most endangered terrestrial ecoregions. conservation international, cemex sa de cv, agrupación sierra madre, mexico city murray bg ( ) can the population regulation controversy be buried and forgotten the strategy of ecosystem development parallel ecological networks in ecosystems deforestation and malaria in mâncio lima county biodiversity and disease risk: the case of lyme disease megaherbivores: the influence of very large body size on ecology agricultural intensification, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis self-organized patchiness and catastrophic shifts in ecosystems catastrophic regime shifts in ecosystems: linking theory to observation mechanistic approaches to community ecology: a new reductionism? habitat modification alters the structure of tropical host-parasitoid food webs do alternate stable states occur in natural ecosystems? evidence from a tidal flat scale dependent feedback and regular spatial patterns in young mussel beds predicting prey population dynamics from kill rate, predation rate and predator-prey ratios in three wolf-ungulate systems communities and ecosystems: linking the aboveground and belowground components a review of trait-mediated indirect interactions in ecological communities evolution and measurement of species diversity in: frumkin h (ed) environmental health: from global to local the concept of organisms as ecosystem engineers ten years on: progress, limitations, and challenges acknowledgments we thank alexa frank (ecohealth alliance) and norma brinkley (university of maryland) for invaluable assistance in the preparation of this chapter.the human environment interface key: cord- -rje bnph authors: ballas, samir k. title: sickle cell anaemia: progress in pathogenesis and treatment date: - - journal: drugs doi: . / - - sha: doc_id: cord_uid: rje bnph the phenotypic expression of sickle cell anaemia varies greatly among patients and longitudinally in the same patient. it influences all aspects of the life of affected individuals including social interactions, intimate relationships, family relations, peer interactions, education, employment, spirituality and religiosity. the clinical manifestations of sickle cell anaemia are protean and fall into three major categories: (i) anaemia and its sequelae; (ii) pain and related issues; and (iii) organ failure including infection. recent studies on the pathogenesis of sickle cell anaemia have centred on the sequence of events that occur between polymerisation of deoxy haemoglobin (hb) s and vaso-occlusion. cellular dehydration, inflammatory response and reperfusion injury seem to be important pathophysiological mechanisms. management of sickle cell anaemia continues to be primarily palliative in nature, including supportive, symptomatic and preventative approaches to therapy. empowerment and education are the major aspects of supportive care. symptomatic management includes pain management, blood transfusion and treatment of organ failure. pain managment should follow certain priniciples that include assessment, individualisation of therapy and proper utilisation of opioid and non-opioid analgesics in order to acheive adequate pain relief. blood selected for transfusion should be leuko-reduced and phenotypically matched for the c, e and kell antigens. exchange transfusion is indicated in patients who are transfused chronically in order to prevent or delay the onset of iron-overload. acute chest syndrome is the most common form of organ failure and its management should be agressive, including adequate ventilation, multiple antibacterials and simple or exchange blood transfusion depending on its severity. preventitive therapy includes prophylactic penicillin in infants and children, blood transfusion (preferably exchange transfusion) in patients with stroke, and hydroxyurea in patients with frequent acute painful episodes. bone marrow and cord blood transplantation have been successful modalities of curative therapy in selected children with sickle cell anaemia. newer approaches to preventative therapy include cellular rehydration with agents that inhibit the gardos channel or the kcl co-transport channel. curative gene therapy continues to be investigational at the level of the test tube and transgenic mouse models. tomatic management includes pain management, blood transfusion and treatment of organ failure. pain managment should follow certain priniciples that include assessment, individualisation of therapy and proper utilisation of opioid and nonopioid analgesics in order to acheive adequate pain relief. blood selected for transfusion should be leuko-reduced and phenotypically matched for the c, e and kell antigens. exchange transfusion is indicated in patients who are transfused chronically in order to prevent or delay the onset of iron-overload. acute chest syndrome is the most common form of organ failure and its management should be agressive, including adequate ventilation, multiple antibacterials and simple or exchange blood transfusion depending on its severity. preventitive therapy includes prophylactic penicillin in infants and children, blood transfusion (preferably exchange transfusion) in patients with stroke, and hydroxyurea in patients with frequent acute painful episodes. bone marrow and cord blood transplantation have been successful modalities of curative therapy in selected children with sickle cell anaemia. newer approaches to preventative therapy include cellular rehydration with agents that inhibit the gardos channel or the kcl co-transport channel. curative gene therapy continues to be investigational at the level of the test tube and transgenic mouse models. although sickle cell anaemia has been referred to as the 'first molecular disease' [ ] and paved the way to modern molecular biology, its management has lagged behind other subsequently described molecular disorders. for decades the management of sickle cell pain, the hallmark of sickle cell anaemia, was limited to bed rest, hydration and inadequate analgesia. lack of understanding of the nature and pathophysiology of the pain associated with sickle cell anaemia hampered rational approaches of therapy and had an adverse effect on the quality of life of an already compromised health status of affected patients. fortunately, during the last decade, advances in the field ushered significant changes in the attitude of care providers towards sickle cell anaemia. most important among these has been the finding that treatment of patients with sickle cell anaemia with hydroxyurea [ ] [ ] [ ] had a significant salutary effect on the clinical picture as shown in table i. moreover, long-term follow-up of adult patients with sickle cell anaemia showed that hydroxyurea therapy is associated with reduced mortality. [ ] this fact rekindled interest in sickle cell anaemia and opened the gates for revitalised basic and clinical research on various aspects of sickle cell anaemia. we are amid a renaissance in the field where sickle cell anaemia is no longer regarded as an unmanageable disorder, and effective treatment and cure are achievable goals in the near future. this paper reviews recent advances in the pathogenesis and treatment of sickle cell anaemia. sickle cell anaemia is a hereditary disorder of haemoglobin (hb) where the sickle gene is inherited, homozygously, from both parents. the sickle mutation is the result of a single base change (gat → gtt) in the sixth codon of exon of the β-globin gene responsible for the synthesis of the β-globin polypeptide of the hb molecule (α β ). this change, in turn, results in replacement of the normal glutamic acid with valine at position of the β-globin chain and the formation of sickle hb. [ , ] in a sense, the mutation is akin to a typographical table i . beneficial effects of hydroxyurea in patients with sickle cell anaemia decreases the frequency of acute painful episodes decreases the incidence of acute chest syndrome decreases the blood transfusion requirement decreases morbidity and mortality error where a change of one letter of a keyword of a manuscript ('punctuation mutation') corrupts the meaning of the intended message. typographical errors in articles, however, are episodic and have a transient effect that is effaced with time. the sickle mutation, on the hand, is permanent and often afflicts a life with pain, disability and morbid existence. the most important pathophysiological event in sickle cell anaemia that explains most of its clinical manifestations is vascular occlusion which may involve both the micro-and macrovasculature. [ ] [ ] [ ] [ ] factors that culminate in vascular occlusion are listed in table ii. [ , [ ] [ ] [ ] [ ] the primary process that leads to vascular occlusion is the polymerisation of sickle hb upon deoxygenation which, in turn, results in distortion of the shape of red blood cells (rbcs), cellular dehydration, and decreased deformability and stickiness of rbc that promotes their adhesion to vascular endothelium. progress in the pathogenesis of vascular occlusion pertains to cellular dehydration and adhesion to endothelial cells described in sections . . and . . , respectively. cellular dehydration is secondary to loss of k + and water. two major transport mechanisms seem to play a significant role in cellular dehydration. the first mechanism is the kcl co-transport pathway activated by acidification and cell swelling. [ ] [ ] [ ] this pathway is most active in reticulocytes and is a feature of low-density sickle cells (reversibly sickled cells -rsc). reticulocyte dehydration appears to contribute to the generation of dense sickle cells directly without going through repetitive cycles of oxygenation-deoxygenation. [ ] the second transport system that mediates cellular dehydration is the ca + -activated potassium channel or the gardos pathway, which seems to be activated by ca + reflux-induced deoxygenation. [ ] [ ] [ ] [ ] [ ] [ ] although much of the intracellular ca + in sickle cells is sequestered in endocytic ves-icles, [ ] [ ] [ ] transient reflux of ca + during deoxygenation-induced sickling seems to be responsible for stimulating the gardos pathway. unlike the kcl co-transport, the gardos pathway seems to be most active in the dense fraction of sickle cell anaemia rbcs. however, in most patients both transport systems are operative. it should be noted, however, that the exact mechanism by which polymerisation of sickle hb leads to cellular dehydration is not fully delineated to date. further research in this area may refine current approaches to molecular therapy. adhesion of sickle rbc to vascular endothelium appears to be a pathophysiological contributor to vaso-occlusion. sickle rbc adhere to cultured endothelial cells in vitro under both static and dynamic conditions whereas normal cells do not. [ ] [ ] [ ] [ ] these findings suggest that sickle rbc have sticky surfaces that promote their attachment to monolayers of cultured endothelial cells. these in vitro observations have been documented to also occur in ex vivo perfusion studies in rats [ ] and transgenic mice. [ ] both cellular and plasma factors have been reported to affect adhesion of sickle rbc to vascular endothelium. thus, young deformable sickle rbc appear to be more adherent to vascular endothelium than are dense, rigid, irreversibly sickled cells. [ , , ] two receptors, α β and cd are present on sickle cell anaemia rbc. the former has been shown to play a role in the adherence of sickle rbc to endothelial cells via vascular cell adhesion molecule (vcam)- . [ , ] plasma factors that enhance adhesion include fibrinogen, factor viii, fibronectin, hyperosmolality, von willebrand factor, thrombospondin and microparticles from activated platelets. [ , , , [ ] [ ] [ ] [ ] thrombospondin released from activated platelets bridges the gap between endothelial cells and sickle rbc by binding to cd receptors on the former and cd receptor or sulfated glycan on the latter. [ , ] matsui et al., [ ] have recently reported that when endothelial cells are activated, p-selectin, a glue-like molecule, moves from their intracellular environment to their outer surface where it binds to sickle cells. this is a novel finding that sheds new light on the pathogenesis of vaso-occlusion. previous studies (vide infra) showed that activated p-selectin triggers clotting in platelets and helps leucocytes to adhere to endothelial cells, and they assumed that rbc did not bind directly to pselectin. matsui et al. [ ] presented the first evidence that p-selectin binds to normal rbc and, to a greater extent, to sickle rbc. this finding suggests that inhibition of p-selectin should be considered as a novel approach for the treatment of acute sickle cell painful episodes. adherence of sickle rbc to vascular endothelium results in intimal hyperplasia in larger vessels that may lead to vascular occlusion and tissue infarction. [ , ] hebbel et al. [ ] reported strong correlation between the degree of adhesion of sickle cell anaemia rbc to endothelial cells in vitro and the severity of the disease in patients with sickle cell anaemia or other variants of sickle cell disease. these interesting findings, however, await documentation by others. hypofibronectinaemia seems also to be related to disease severity, the lower the level of plasma fibronectin, the more severe the disease. [ ] recent in vivo studies in transgenic mice suggest that vascular occlusion results in the creation of an inflammatory state. [ , ] the sequence of events seems to be as follows: (i) reticulocytes carrying the α β receptor adhere to endothelial cells; (ii) this is followed by logjam where there is propagation of occlusion caused by the accumulation of rigid deoxygenated mature rbc proximal to the site of adhesion; (iii) the obstruction eventually clears leading to reperfusion and its associated injury; and (iv) a new cycle of adhesion starts thus creating a vicious cycle of occlusion and reperfusion. evidence of reperfusion injury includes: (i) inflammatory response in the vascular bed of the transgenic mouse with increased leucocyte rolling, adhesion and emigration after hours of mild hypoxia followed by reperfusion; (ii) local production of free radicals; and (iii) the complete inhibition of (i) and (ii) after the infusion of a monoclonal murine anti-p-selectin antibody, but not an anti-reselectin antibody, before reoxygenation. [ ] the implication of this sequence of events is that restoration of oxygen to ischaemic tissue results in the generation of free radicals associated with inflammatory endothelial and tissue injury. further evidence of the importance of adhesion in the pathogenesis of sickle cell anaemia was provided by kaul et al. [ ] who investigated the ability of two murine monoclonal antibodies (mab) to inhibit sickle rbc-endothelium interactions induced by platelet activity factor (paf). the mab used were e and lm- . the former mab ( e) inhibits both α v β and glycoprotein (gp) iib/iiia that bind primarily to fibrinogen and von willebrand factor. lm- , on the other hand, selectively inhibits α v β . infusion of washed sickle cell anaemia rbc in the ex vivo mesocecum of the rat, pretreated with paf, with or without a control antibody resulted in extensive adhesion of sickle rbc in venules associated with post-capillary blockage. pre-treatment of the vasculature with either e or lm- , but not with control antibody, inhibited the adhesion of sickle rbc in postcapillary venules. whether the same sequence of events occurs in patients with sickle cell anaemia or not awaits carefully designed clinical trials with antibodies that inhibit the interaction of sickle rbc and endothelial cells. major concerns of similar trials in humans were raised by hebbel. [ ] these include: (i) the risk associated with the inhibiting effect of mab e on platelet function with an unpredictable net clinical effect (i.e. thrombosis versus bleeding); and (ii) to be effective, these antibodies have to be administered before the rbc adhere to the vessel wall (i.e., pre-treatment before the onset of a vaso-occlusive event) a scenario that is unpredictable in patients with sickle cell anaemia. available data suggest that treatment with these mab after the onset of vaso-occlusion may not be effective. the abnormally high base-line leucocyte count both in patients with sickle cell anaemia and in the sickle transgenic mouse seems to be a marker of this proposed chronic inflammatory state of sickle cell anaemia. [ , ] other factors that influence vaso-occlusion in sickle cell anaemia pertain to the α-genotype, βhaplotype, total hb level and fetal hb (hb f) levels. sickle cell anaemia can be divided into subcategories depending on the α-genotypes and β-haplotypes. [ ] [ ] [ ] about % of patients with sickle cell anaemia have normal α-genotypes (β s /β s , αα/αα), % have one α gene deleted (β s /β s , -α/αα) and the remaining % have two α genes deleted (β s /β s , -α/-α). the effect of α gene deletion on the clinical picture of sickle cell syndromes is controversial. generally speaking, α gene deletion is associated with milder anaemia, [ ] and hence fewer complications associated with severe anaemia and less blood transfusion. the increased haemoglobin level associated with α gene deletion, however, increases the blood viscosity, which is often accompanied by increased frequency of painful crises [ , ] and vaso-occlusive episodes such as avascular necrosis. [ , ] the effect of α gene deletion on the clinical picture is best illustrated in patients with sickle cell anaemia with two α gene deletions (β s /β s , -α/-α). table iii lists the unique features of this type of sickle cell anaemia. [ ] [ ] [ ] [ ] noteworthy is that hemoglobin a (hb a ) is elevated in sickle cell anaemia with two α gene deletions, a finding that confuses this diagnosis with s-β-thalassemia that is typically also associated with elevated hb a levels. this clinical picture, family history, haematological data and molecular diagnostics can differentiate the two diagnoses. [ ] β-haplotypes refer to the nucleotide sequence ′ and ′ to the sickle gene. three major types have been described in africans and african-americans. [ ] these are the senegalese (sen), benin (ben) and central african republic (car) haplotypes. the significance of these haplotypes pertains to their effect on hb f production. it has been established that the higher the hb f level, the milder is the sickle cell anaemia. [ ] the sen haplotype, especially in the homozygous state, is associated with relatively high hb f levels and, hence, milder disease. [ , ] however, these conclusions are based on population data and may not apply to an individual patient. sickle rbc from patients with a high level of hb f seem to be less adherent to vascular endothe- [ ] found that paediatric sickle cell anaemia patients with high levels of f cells had a concomitant decrease in the number of cd +, very late antigen (vla) + and cd + erythrocytes, and hence, less adherent rbc. moreover, hb f seems to affect the exposure of phosphatidylserine on the surface of rbc and coagulation activation. in vivo cycles of sickling/ unsickling with resulting membrane changes and microvesicle formation are one factor responsible for phosphatidylserine exposure. [ ] phosphatidylserine-exposing rbc in the transgenic sickle mouse [ ] were found to have shortened red cell survival. children with sickle cell anaemia and high hb f levels were reported to have less phosphatidylserine-exposing rbc and, hence, milder haemolytic anaemia suggesting a possibly milder clinical picture. [ ] other factors that may effect the severity of sickle cell anaemia include gene modifiers (epistatic genes) that may affect the phenotypic expression of the sickle mutation. styles et al., [ ] reported that specific human leucocyte antigen (hla) alleles may influence the risk of stroke in sickle cell anaemia. adekile et al. [ ] found that the c→t mutation of the methyltetrahydrofolate reduction gene is relatively frequent among kuwaiti patients with sickle cell anaemia, but did not find any correlation with disease severity or prevalence of avascular necrosis. the role of epistatic genes in modifying the phenotypic expression is currently an active area of research. future research findings may enable us to decipher the intricate pathophysiology of sickle cell anaemia and its complications, and usher in newer therapeutic venues. in addition to the factors discussed in section . , there is growing evidence that psychosocial and environmental factors may precipitate vasoocclusion and affect the frequency and severity of painful episodes. physical stress, trauma, dehydration and infections are such known factors. the clinical management of the majority of patients with sickle cell anaemia is primarily palliative in nature. palliative care is the total comprehensive care of patients whose disease is not responsive to curative therapy. [ ] it targets the numerous complications of sickle cell anaemia during the life of a patient from childhood through adulthood. the major goal of palliative care is the achievement of the best quality of life of patients and their families. palliation in sickle cell anaemia includes: (i) general supportive care; and (ii) targeted symptomatic management of complications. supportive care pertains to empowering the patients to live with their disease and be authorities on its manifestations that apply to them individually. this includes the following: • education about sickle cell anaemia, its genetic basis, inheritance and family counselling • adherence to regular schedule of medical follow-up • avoidance of situations that exert an adverse effect on their disease and adoption of those activities of daily living that are beneficial to them • knowing their rights and responsibilities as patients with sickle cell anaemia when dealing with care providers, medical facilities and the workplace • whenever possible, participation in local support groups and communication with community leaders and advocates. sickle cell pain is unique and like other types of pain is a complex human experience that is strongly affected not only by pathophysiological factors, but also by psychological, social, cultural and spiritual factors. the pain is the result of tissue damage generated by the sickling process and occlusion of the microvasculature. tissue damage, in turn, releases several mediators of inflammation that ini-tiate a painful stimulus that is transmitted along a-δ and c peripheral nerve fibres to the dorsal horn of the spinal cord. from there the stimulus crosses to the contralateral side and ascends along the spinothalamic tracts to the thalamus, which in turn, sends the message to the brain where the stimulus is perceived as pain. other concurrent processes may affect pain perception. one process pertains to descending fibres from the midbrain to the dorsal horn that inhibits the transmission of painful stimuli via endogenous endorphins. another process pertains to communications between the thalamus, the reticular formation and the limbic system, which together modulate the emotional response to pain that may enhance or inhibit the intensity of pain perception. the unique features of sickle cell pain are listed in table iv. it is primarily a nociceptive type of pain, i.e. the result of tissue damage. however, it could be or could have a neuropathic component. [ ] the latter is characterised by burning sensation, tingling and numbness. thus, it is important to conduct a thorough history and physical examination to determine whether sickle cell pain is associated with a neuropathic component or not. this is an important aspect of the treatment because the management of neuropathic pain utilises special approaches as will be discussed in this section. rational and effective management of sickle cell pain includes: (i) thorough assessment; (ii) utilisation of both non-pharmacological and pharmacological agents; and (iii) a comprehensive plan for disposition and longitudinal follow-up. assessment is the most important initial step in effective pain management. it should be conducted before and periodically after the administration of analgesics. [ ] [ ] [ ] assessment relies heavily on patient self report. other factors in the process of assessment should include the presence or absence of other complications of the disease, family member reports and vital signs. the patient self report should include multidimensional scales describing the intensity, quality, location, distribution, onset, duration, mood, sedation, pain relief and factors that aggravate or relieve pain. the intensity of pain can be assessed by using any of several available scales such as the visual analogue scale, verbal, numerical or wong-baker faces scale for children. it is important, however, to stick to one scale and use it routinely so that both the patient and provider become familiar with it and its significance to a certain patient on an individual basis. nociceptive sickle cell pain, typically, is sharp and/or throbbing in nature. pain that is burning, shooting, lancinating or tingling in nature suggests the presence of a neuropathic component of pain that entails the utilisation of certain adjuvants as will be discussed in this section. [ , ] initial pain assessment establishes a baseline against which the effectiveness of analgesics in achieving pain relief can be compared. subsequent assessment allows increasing the dose of analgesics to achieve desirable pain relief, tapering the dose of analgesics as the painful episode resolves, identification of adverse effects of therapy or the emergence of complications of the disease which allow intervention to modify the treatment plan as needed. non-pharmacological management of pain includes cutaneous stimulation [transcutaneous electrical nerve stimulation (tens), heat, cold and vibration] distraction, relaxation, massage, music, topical agents: lidocaine/prilocaine; capsaicin tramadol corticosteroids guided imagery, self-hypnosis, self-motivation, acupuncture and biofeedback. although there are no well-controlled clinical trials on the efficacy of these modalities on the management of sickle cell pain, there are many anecdotal reports of their efficacy in pain management. pharmacological management of pain includes three major classes of compounds: non-opioids, opioids and adjuvants. [ , ] a major difference between non-opioids and opioids is that the former have a ceiling effect which refers to a dose above which there is no additive analgesic effect. [ ] nonopioids (table v) include paracetamol (acetaminophen), non-steroidal anti-inflammatories (nsaids), topical agents, tramadol and corticosteroids. paracetamol has analgesic and antipyretic effects, but no anti-inflammatory component. [ ] the daily total adult dose must not exceed g in to divided doses. [ ] high dosages damage the liver and could be fatal. the daily dose should be decreased in the presence of liver disease. the daily dose of combination medications (medications that contain paracetamol plus an opioid) must be controlled so that the g limit of paracetamol is met. nsaids (table vi) include non-selective cyclooxygenase (cox) inhibitors, and selective and partially selective cox- inhibitors. [ ] [ ] [ ] nsaids have an anti-inflammatory effect in addition to their analgesic and antipyretic potential. they act primarily at the level of nociceptors where pain impulses originate and, hence, are often referred to as peripherally acting analgesics. they exert their analgesic effect by decreasing the synthesis of prostaglandins by inhibiting cox enzymes, [ ] thus, decreasing or abolishing the sensitisation of nociceptors by prostanoids. the traditional non-selective nsaids inhibit both the housekeeping cox- and the inducible cox- enzymes. selective nsaids inhibit only the cox- enzyme and spare cox- , which is needed to produce physiological levels of prostaglandins. nsaids have potentially serious systemic adverse effects. they include gastropathy, nephropathy and haemostatic defects. they should not be administered to patients with renal disease or with history of peptic ulcer disease. it is advisable not to administer them continuously to patients with sickle cell disease for more than days. moreover, certain nsaids are associated with idiosyncratic (non-prostaglandin-mediated) reactions as shown in table vii. most recent among these is immune thrombocytopenia resulting from sensitivity to metabolites of naproxen and paracetamol. [ ] the antibodies described were mostly specific for gp iib/iiia and less often to gp ib/ix/v. cox- inhibitors are associated with significantly less gastrointestinal and haemostatic adverse effects [ , ] than the non-selective nsaids, but their effect on renal function seems to be the same. [ , ] the concomitant administration of ketorolac with opioids was reported to exert an additional analgesic effect and decrease the amount of opioids consumed for the treatment of acute painful episodes. [ ] tramadol [ ] is a synthetic centrally acting analgesic not chemically related to opioids. it acts as a weak agonist with preferential affinity to the μ receptors. moreover, it inhibits neuronal re-uptake of both serotonin and norepinephrine, and stimulates the release of serotonin. thus, functionally, it has properties of an opioid and an antidepressant. the initial enthusiasm that this drug is not associated with clinically significant respiratory depression or addiction potential [ ] waned after reports indicating that seizures may be an adverse effect and an increasing abuse potential. currently, tramadol is not a scheduled drug and it seems to be as effective as paracetamol with mg codeine with the added advantage of a tricyclic antidepres-sant-like effect. tramadol may be administered by the oral or parenteral route, and it is available in slow-release form. only the oral form is approved for marketing in the us at the present. anecdotally, tramadol seems to be effective in the management of mild or moderately severe pain in some patients with sickle cell anaemia. opioid analgesics [ ] have fewer systemic adverse effects than nsaids but their use in sickle cell disease is often associated with many myths about drug-seeking behaviour and addiction. there are four major classes of opioids: agonists, partial agonists, mixed agonists-antagonists, and antagonists (table viii) . traditionally, opioid antagonists have been regarded as having no analgesic effect and their use is primarily limited to counteract the depressive effects of opioid agonists. recently, however, [ ] there have been reports showing that small doses of antagonists in combination with agonists seem to enhance the analgesic effect, and prevent or delay tolerance to opioid agonists. should this approach be proven by controlled trials, it would be a novel tool in the management of pain. opioid agonists are most often used in the management of sickle cell pain, especially in adults. they decrease or modify the perception of pain at the level of the central nervous system (cns). they exert their effect by binding to μ, κ, and to a lesser extent, δ receptors. [ ] opioid agonists can be administered via several routes (orally, subcutaneously, intramuscularly, intravenously, transdermally, etc.) and methods, including continuous intravenous drip, patient-controlled analgesia (pca) pump or intermittent injection. pethidine (meperidine), morphine and hydromorphone are the major opioid analgesics used in the treatment of severe pain in the emergency department (ed) and hospital. long-acting opioids, such as the oxycodone controlled-release (cr) formulation oxy-contin ® (oxycodone cr) and morphine cr, are useful in the management of chronic pain and in combination with short-acting opioids for breakthrough pain. adverse effects of opioid analgesics include itching, nausea, vomiting, sedation and respiratory depression. seizures may be associated with op-ioids, especially with the prolonged use of pethidine, in some patients. tolerance and physical dependence occur in some patients, but addiction is rare. [ ] as a group, opioid analgesics have no ceiling effect (with the possible exception of codeine) and hence, the only limiting factor for dosage is adverse effects. severe sedation and respiratory depression are the most important adverse effects. hospitalised patients receiving opioid analgesics on a regular basis should be monitored for their respiratory rate and sedation level. a respiratory rate less than per min and/or severe sedation justifies missing, decreasing or delaying the dose, or discontinuing the opioid in question until the depressive effects disappear. opioid analgesics should be used carefully in patients with impaired ventilation, asthma, increased intracranial pressure and liver failure. the dosage of pethidine and morphine should be adjusted in the presence of renal failure. they are also histaminergic and histamine release may trigger bronchospasm or initiate an allergic reaction. morphine is the most histaminergic of all opioids. [ ] the presence of paracetamol in combination with codeine or oxycodone limits the daily dose that can be safely used so that the maximum allowable dosage of paracetamol is not exceeded. the use of paracetamol in conjunction with monoamine oxidase inhibitors may cause a severe adverse reaction characterised by excitation, hyperpyrexia, convulsions and death. [ ] the co-administration of antipsychotics with pethidine may cause neuromuscular disorders including akathisia, dystonia, tardive dyskinesia and neuroleptic malignant syndrome. [ ] adjuvants include antihistamines, antidepressants, benzodiazepines and anti-convulsants. these are heterogeneous compounds that potentiate the analgesic effect of opioids, ameliorate their adverse effects and have their own mild analgesic effect. the most commonly used adjuvants in the management of sickle cell pain are listed in table ix. the role of selective serotonin reuptake inhibitors (ssri) in sickle cell anaemia is not clear at the present. adjuvants must be used with care and patients should be monitored carefully when receiving them. adjuvants also have adverse effects, some of which precipitate or worsen some of the manifestations of sickle cell anaemia as is discussed later in this section. [ ] acute painful episodes of mild or moderate severity are usually treated at home using a combination of non-pharmacological and pharmacological modalities. home treatment of pain usually follows the three step analgesic ladder proposed by the world health organisation (who). [ ] mild pain is treated with non-pharmacologic agents alone or in combination with a non-opioid. more severe pain entails the addition of an opioid ± an adjuvant. data from the multi-center study of hydroxyurea (msh) [ , ] in sickle cell anaemia showed that a oxycodone/paracetamol formulation was the opioid most often used for the home treatment of pain. [ ] however, this report was before the advent of the new formulations of opioids such as oxycodone cr. whether oxycodone/paracetamol continues to be the first in this scenario of pain management remains to be seen. severe acute sickle cell painful episodes are usually treated in a medical facility using paren-teral analgesics. progress in this area pertains to the advent of day hospitals where patients are promptly evaluated by a team of experts in the management of sickle cell pain without exposure to the delay that is common in hospital emergency rooms. [ ] available data in the literature show that management of patients with severe acute painful episodes in such facilities, especially those that operate on a -hour basis, reduce the frequency of hospital admissions. these findings should encourage other metropolitan hospitals in cities with a large population of african-americans to follow suit by establishing acute care facilities specifically designed for patients with sickle cell disease. the establishment of such facilities nationwide may, in turn, verify the cost-saving potential of this approach to healthcare. data from the msh showed that the parenteral opioid most often used in the management of acute sickle cell painful episodes in the ed or hospital was pethidine. [ ] again, this was in and since then, there have been many anecdotal reports from many hospitals of switching to other opioids than pethidine, but detailed studies to confirm this are not available to date. patients with chronic sickle cell pain and those with very frequent acute painful episodes are best managed with a combination of long-acting opioids and a short-acting opioid for breakthrough pain. again, there are anecdotal reports that this approach decreased the frequency of admissions to the ed and/or hospital, but data to confirm this are not available to date. oxycondone cr seems to be unique in that it has both an immediate analgesic effect and a delayed long-acting effect. these properties made oxycodone cr popular among drug abusers who learned to remove the mesh and release high-dose of pure oxycodone that has immediate 'euphoric' effect. [ ] care providers should exert caution in prescribing oxycodone cr as well as other opioids and keep records of assessment and plans of management of their patients. most patients usually tolerate the chronic anaemia of sickle cell disease. sickle hb has decreased promethazine oxygen affinity and, hence, is efficient in delivering oxygen to tissues. the two major objectives of blood transfusion in sickle cell anaemia are; (i) improvement of the oxygen carrying capacity of blood; and (ii) dilution of circulating sickled rbc in order to improve microvascular perfusion. specific indications for blood transfusion are listed in table x. [ ] [ ] [ ] the goal of exchange transfusion is to increase or maintain a hb level of about g% and to decrease the level of sickle hb to < %. [ , ] in patients with stroke undergoing exchange transfusion to prevent recurrence of a cerebrovascular accident, a level of sickle hb < % seems to be acceptable. [ , ] however, there are no controlled studies to show which target of sickle hb is better in relief of the acute symptoms and prevention of recurrence. more blood units may be needed to decrease sickle hb to < % and thus, more exposure of the patient to donor blood. on the other hand, decreasing sickle hb to < % only may increase the fre-quency of blood exchanges required to keep sickle hb at this level. blood selected for transfusion for patients with sickle cell anaemia should meet the criteria listed in table xi. because the majority of patients with sickle cell anaemia are africans or african-americans and because they receive blood given by caucasians, there is a high incidence of allo-immunisation in transfused patients with sickle cell anaemia. [ ] the most prevalent allo-antibodies in patients with sickle cell anaemia include anti-c, -e and -k. thus, the use of phenotypically matched blood, at least for these antigens, is highly recom- mended. [ ] some blood banks implemented programs to increase communication between the african-american community and medical facilities to ensure the presence of a blood supply from african-american donors directed for patients with sickle cell anaemia in order to reduce the incidence of allo-immunisation to those antigens that are prevalent in african-americans. however, there are no data to show the desirable outcome, if any, of this practice. one concern about this practice (designated donations from african-americans to patients with sickle cell anaemia) is that it may increase the incidence of transfusion-related graft versus host disease unless such blood is routinely irradiated. some of the complications seen in patients with sickle cell anaemia are secondary to therapeutic interventions. table xii lists some of these complications. patients transfused before , when a reliable second generation screening test for hepatitis c virus (hcv) was introduced in blood banks, are at a higher risk for transmission of hcv. approximately to % of adult patients who received a blood transfusion before are positive for hcv antibody. these patients should be followed-up regularly and the levels of hepatic enzymes monitored periodically. increase in the enzyme level beyond what is expected in sickle cell disease is an indication for a liver biopsy. if the latter shows evidence of hepatitis, specific therapy with a combination of interferon and ribavirin [ ] would be considered. patients with sickle cell anaemia taking ribavirin should be monitored carefully because ribavirin may worsen their haemolytic anaemia. [ ] the transfusion of leucocyte-reduced components decreases the chances for allo-immunisation and febrile transfusion reactions. the transfusion of phenotypically identical rbc also minimises the chances for allo-immunisation and haemolytic reactions. iron overload is best monitored by periodic determination of serum ferritin levels in frequently transfused patients. [ ] serum ferritin levels > μg/l in the steady state are suggestive of iron overload. to confirm the diagnosis of iron overload, a liver biopsy is indicated in order to quantitate the amount of iron per gram of tissue. hepatic iron concentration > mg/g liver dry weight is diagnostic of iron overload and is an indication for chelation therapy with deferoxamine. prophylaxis: prophylactic oral penicillin (or a macrolide if there is sensitivity to penicillin) should be given to infants and children with sickle cell disease for a minimum of years. [ ] all patients with sickle cell disease should receive the polyvalent ( -valent) pneumococcal polysaccharide ( ps) vaccine starting at age years and every to years thereafter. [ ] the heptavalent pneumococcal conjugate vaccine (pc v ) should be given to all children at , , and months of age. patients with sickle cell disease should also receive the pc v vaccine at age and years. other recommended vaccinations, in addition to routine childhood vaccines, include haemophilus influenza type b conjugate vaccine at ages , and months, influenza vaccine annually, and hepatitis b vaccine at birth or the first visit of children and adults who have no serological evidence of previous exposure to the hepatitis b virus. immunosuppression: sickle cell anaemia has an unusual relationship to certain infectious agents. individuals with sickle trait are resistant to infection by plasmodium faliciparum but patients with sickle cell anaemia are susceptible. individuals with fy(a-b-) red cells are resistant to infection by other types of malarial parasites. several acquired abnormalities render patients with sickle cell disease immunocompromised and hence susceptible to a number of infections that are a major cause of mortality and morbidity. these include immunomodulation secondary to blood transfusion and iron overload. [ ] the increased susceptibility of patients to infection with polysaccharide-encapsulated bacteria (streptococcus pneumoniae and h. influenzae) is secondary to absence of splenic function. cellular immunity may be compromised by transfusion-related iron overload and abnormalities in b cell immunity may explain antigen processing defects. infections due to escherichia coli are usually associated with urinary tract infection (uti) in adult patients. patients with sickle cell anaemia are susceptible to osteomyelitis secondary to salmonella typhimurium in addition to the usual causes of bacterial osteomyelitis such as staphylococcus aureus. [ ] the susceptibility to infection by salmonella may reflect the ability of this organism to flourish in partially necrotic bone. management: treatment options for infections include penicillin, cephalosporins, macrolides, tetracyclines, fluoroquinolones, aminoglycosides and cotrimoxazole (trimethoprim/sulfamethoxazole) [table xiii]. the choice of antibacterial depends on the possible pathogen, the possibility of resistance, the severity of the infection and the characteristics of the patient such as age, allergies and concomitant complications. bacterial resistance is a serious and rapidly increasing worldwide problem. pneumococci continue to be the most important organisms causing pneumonia (especially community acquired pneumonia) and also play a very important role in other infections such as sinusitis, otitis media and meningitis. [ ] approximately to % of s. pneumoniae strains are resistant to penicillin. highly penicillin-resistant strains of s. pneumoniae are also resistant to amoxicillin/clavulanic acid and cephalosporins. approximately % of these strains are resistant to macrolides and % to tetracyclines. resistance to fluoroquinolones is currently low. moreover, approximately to % of h. influenzae strains are resistant to penicillin. neurological complications occur in % of patients with sickle cell disease and are more common in sickle cell anaemia than in other sickle cell syndromes (hb sc disease, s-β thalassemia). cerebral infarction (figure ) is more frequent in children, whereas intracerebral haemorrhage is more prevalent in adults. microaneurysms (commonly referred to as moya moya) involving fragile dilated vessels that develop as compensatory collateral circulation around areas of infarction seem to be responsible for haemorrhage in adults (figure ). unlike other vascular beds, large vessels rather than microvessels seem to be the site of occlusion with consequent infarction. approximately twothirds of children with cerebral infarction (who are not transfused) may develop further ischaemic events within years [ , ] a major breakthrough in the management of cns complications in children has been the prevention of stroke in patients at risk for this complication. [ , ] specifically blood transfusion prevented the occurrence of stroke in children who had abnormal results on transcranial doppler ultra-sonography (tcd). blood velocity of cm/sec or more in either the internal carotid or middle cerebral artery is abnormal (normal < cm/sec) and is an indication for blood transfusion or exchange transfusion. the appropriate therapy for a child with cerebral infarction due to vaso-occlusion or an asymptomatic patient with abnormal transcranial doppler studies is exchange transfusion or hypertransfusion to maintain the sickle hb level below %. red cell transfusions are usually continued for a minimum of years after which transfusion therapy is individualised. whether chronic transfusion therapy for adults with cerebral infarction secondary to vaso-occlusion is indicated or not remains unknown. similarly, the appropriate treatment of an adult patient with cerebral haemorrhage has yet to be determined. a thorough search for aneurysms should be made and surgical intervention considered. ware et al. [ ] have recently suggested that some children with sickle cell disease and stroke may discontinue chronic transfusion and use hydroxyurea therapy to prevent stroke recurrence. this approach is desirable because it prevents or decreases the risks associated with blood transfusion mentioned above. more data, however, are needed to justify this modality of therapy. other risk factors for stroke in children include bacterial meningitis, family history of a sibling with stroke, severe acute chest syndrome, acute anaemic episodes, recurrent episodes of seizure, impaired cognitive skills, lack of α-gene deletion and the car β s -haplotype. one study suggested that high homocysteine levels may be a risk factor for cerebrovascular accidents in patients with sickle cell anaemia. [ ] seizures in sickle cell disease may be secondary to an epileptic focus as a result of infarction or to treatment with large doses of pethidine, or they may be idiopathic. antiepileptic therapy is recommended for patients with abnormal electroencephalograms. charache et al. [ ] introduced the term acute chest syndrome to define acute episodes of fever, chest pain, increased leukocytosis and pulmonary infiltrates in adult patients with sickle cell anaemia, most of whom probably had pulmonary infarction. with time, the definition of acute chest syndrome has expanded to include hypoxemia, cough, shortness of breath, wheezing, chills and worsening anaemia. [ ] moreover, the current definition of acute chest syndrome stresses that the infiltrates must be 'new'. the signs and symptoms of acute chest syndrome vary from very mild to very severe and even life-threatening. another feature of acute chest syndrome that is not included in the definition is the presence of blister cells in peripheral blood. [ ] acute chest syndrome is second to acute painful episodes as the most common cause of hospitalisation of patients with sickle cell disease and also the most common complication of surgery and anaesthesia. [ ] acute chest syndrome is the most common cause of death and is closely associated with acute painful episodes especially in adults. [ , ] although acute chest syndrome is usually self-limited and resolves with treatment, it can be associated with respiratory failure with a mortality rate of about . % in children and . % in adults. [ , ] risk factors for developing acute chest syndrome are listed in table xiv. [ ] the incidence of acute chest syndrome is highest in sickle cell anaemia, followed by s-β -thalassemia, hb sc disease and s-β + -thalassemia in decreasing order of frequency. the single most important preventive factor is a high level of hb f due either to endogenous genetic factors or to exogenous induction with drugs such as hydroxyurea. [ , [ ] [ ] [ ] [ ] the mean corpuscular volume (mcv) of rbcs, platelet count and α-thalassemia bear no relation to acute chest syndrome. [ ] aetiologies of acute chest syndrome include infection, especially communityacquired pneumonia, pulmonary infarction as a result of in situ sickling, fat-bone marrow embolism or pulmonary embolism. infection is commonly caused by chlamydia, mycoplasma, respiratory syncytial virus, coagulase-positive s. aureus, s. pneumoniae, mycoplasma hominis, parvovirus and rhinovirus in decreasing order of frequency. [ ] adhesion of sickled rbc to endothelial cells of small or medium-sized pulmonary vessels may result in occlusion of microvascular flow and consequent pulmonary infarction. [ , , ] this sequence of events is supported by dynamic imaging studies but confirmatory clinical data are not available. pulmonary thromboembolism is uncommon as a cause of acute chest syndrome despite the presence of a hypercoagulable state in sickle cell disease. [ ] it seems that this hypercoagulable state plays a more important role in stimulating cellular adhesion and activating the inflammatory system than in initiating the thrombotic cascade. [ , ] pulmonary fat-bone marrow embolism (figure ) in patients with sickle cell anaemia appears to be more common than previously thought. [ , ] the characteristic clinical picture is that of severe bone pain, usually in long bones, followed by dyspnea, hypoxia and fever. tissue infarction of the bone marrow within the long bones seems to generate a source of fat and necrotic tissue that has been demonstrated on autopsy. at the same time, serum levels of secretory phospholipase a , (spla ), an inflammatory mediator, increase in patients with acute chest syndrome [ ] and liberate free fatty acids from membrane phospholipids of damaged tissue that are believed to cause damage to pulmonary endothelium culminating in a leak syndrome which, if severe, may be similar to adult respiratory distress syndrome (ards). an elevated level of spla is both a marker and probably a predictor of acute chest syndrome. diagnostic work-up of acute chest syndrome should include serial chest radiographs, induced deep sputum and blood cultures, monitoring arterial blood gases, monitoring haemoglobin level, ventilation and perfusion scans, and ruling out thrombophlebitis in the pelvis or lower extremities. the diagnosis of fat embolism entails the identification of fat-laden macrophages in induced deep sputum, or better bronchoalveolar lavage fluid obtained by bronchoscopy. [ , ] blister cells have been described in the peripheral blood of patients with sickle cell disease and acute chest syndrome. [ ] management of acute chest syndrome includes oxygen, incentive spirometry, antibacterials, simple blood transfusion or exchange transfusion, judicious use of analgesics, careful hydration and possibly vasodilators. incentive spirometry prevents splinting and atelectasis, and may actually prevent acute chest syndrome in patients who have rib infarction. [ ] intravenous antibacterials are indicated since it is difficult to rule out pneumonia or infected lung infarcts. a combination of a third generation cephalosporin and a macrolide or a fluoroquinolone (table xiii) should be used to cover typical and atypical pathogens. simple transfusion or exchange transfusion is indicated in patients with worsening respiratory function. the beneficial effects of blood transfusion may not be due simply to decreasing the proportion of sickled rbc and other mechanisms may be involved. these include: (i) an immunomodulatory mechanism by which inflammatory cytokines [interleukin(il)- , in particular] bind to the duffy antigen present on transfused rbcs, but often absent on rbcs of african-americans; [ ] and (ii) the albumin that is present in transfused units or used in blood exchange may bind free fatty acids, thus neutralising their damaging effect on the pulmonary endothelium. although intravenous corticosteroids in children with acute chest syndrome may be beneficial, [ ] their use in adults with acute chest syndrome is controversial. huang et al. [ ] reported two adult patients with sickle cell disease whose clinical picture deteriorated and was complicated by worsening pain, fat embolism and coma after corticosteroid therapy. adults, unlike children, have more adipose tissue that may hypertrophy with corticosteroids, increasing the chances of fat embolisation. moreover, corticosteroids may induce or worsen avascular necrosis, which is more common in adults than in children. excessive use of opioid analgesics may precipitate acute chest syndrome because of the depres- sive effect on respiration. recent reports [ ] recommend the use of nsaids. this recommendation should be considered carefully. opioids have a few systemic adverse effects, and careful monitoring of their use ensures their safety. they should be discontinued if the respiratory rate is ≤ per minute and their adverse effects can be quickly reversed with opioid antagonists. nsaids, on the other hand, have considerable systemic adverse effects that may not be readily obvious. nsaids decrease the levels of prostaglandins and prostacyclin, prostanoids that are essential in modulating the vascular tone of smooth muscle and renal blood flow. thus, nsaids may worsen the clinical picture of acute chest syndrome as a result of vasocontrictive effects. preliminary reports on the use of nitric oxide (no), a vasodilator, in patients with sickle cell disease support a possible role of this agent in the management of acute chest syndrome in the future. [ ] other vasodilators such as prostacyclin and calcium channel antagonists have not been reported in the management of acute chest syndrome. another recent investigational approach to treat acute chest syndrome includes the use of purified poloxamer , which is a non-ionic surfactant. it is hypothesised that this agent reduces blood viscosity, prevents adhesion of rbcs to vascular endothelium and improves microvascular blood flow. [ , ] because acute chest syndrome is relatively frequent in patients with sickle cell anaemia, and in view of the need to monitor arterial blood gases in its management, it is important to establish baseline blood gases and pulmonary function tests for all patients. these determinations will be of value in evaluating patients who present with acute onset of pulmonary signs and symptoms. sickle cell anaemia is associated with numerous renal complications that span a spectrum from hyposthenuria to end-stage renal failure (table xv) . [ ] [ ] [ ] [ ] uti is usually caused by e. coli and is more common in females than in males. the increased frequency of uti in sickle cell anaemia may relate to renal infarction or to immunodeficiency. the hypoxic, acidotic and hypertonic environment of the renal medulla causes sickling of the rbc in the vasa recta and leads to infarction of the renal medulla, hyposthenuria and haematuria (gross or microscopic). inability to acidify the urine after an acid load can also occur. these renal tubular defects (haematuria, hyposthenuria) occur not only in patients homozygous for the sickle gene, but also in patients who are heterozygous (for example, as, sc, sd and so). management of haematuria in a patient with sickle cell anaemia follows conservative guidelines. strict bed rest alone results in spontaneous remission in most patients. in a few instances, gross haematuria may be severe enough to warrant blood transfusion or exchange transfusion. nephrectomy should be avoided. the use of aminocaproic acid and desmopressin may be effective in controlling haematuria. renal medullary carci- noma should be ruled out in all patients with gross haematuria. potassium excretion is also impaired and episodes of hyperchloraemic acidosis have been reported. in vitro haemolysis of collected blood samples kept in the lab at room temperature for some time before analysis may explain the spurious hyperkalaemia in some patients with sickle cell anaemia. occasionally, hyperkalaemia is reported in association with type renal tubular acidosis, but renal insufficiency is present in the majority of these patients. papillary necrosis may be more common in hb sc disease. [ ] hyperuricaemia in patients with sickle cell anaemia is the result of both increased bone marrow activity with consequent enhanced urate production secondary to purine metabolism and an acquired decreased renal tubular clearance of urate. gout has been described in a few patients. allopurinol may be indicated to lower serum urate levels. nephrotic syndrome, with or without hypertension, occurs frequently. microscopic haematuria, proteinuria, hypertension and the nephrotic syndrome are markers of incipient end-stage renal failure. proteinuria occurs in % of patients with sickle cell anaemia and elevated serum creatinine levels in approximately %. proteinuria causes congestion of tubular endothelium as a result of tubular uptake of protein. congestion induces growth factors which, in turn, lead to proliferation of fibroblasts that culminate in renal fibrosis and renal failure. the pathological lesion is usually glomerular enlargement and peripheral focal segmental glomerulosclerosis. treatment with enalapril (an angiotensin-converting enzyme inhibitor) seems to reduce the degree of proteinuria in patients with sickle cell anaemia suggesting that capillary hypertension may be a pathogenic factor in sickle cell nephropathy. once chronic renal failure sets in, patients require long-term haemodialysis and are candidates for kidney transplantation. [ , , ] priapism occurs when sickle cells congest the copora and prevent emptying of blood from the penis. it can result from tricorporal involvement (both of the corpora cavernosa and the corpus spongiosum) or bicorporal involvement (both corpora cavernosa). the latter is more common, especially in children, and is not regularly associated with impotence. there are two major clinical presentations of priapism: acute and chronic. [ ] the acute presentation is characterised by a prolonged painful erection that persists beyond several hours, responds poorly to exchange transfusion and frequently requires surgical intervention. acute priapism may be followed by complete or partial impotence. the chronic form of priapism is characterised by repetitive, reversible, painful erections called 'stuttering' priapism. it usually occurs after intercourse or it may awaken patients early in the morning. stuttering priapism responds well to diazepam or pseudoephedrine. patients who become impotent may benefit from psychological counselling and the insertion of penile implants. a practical and relatively simple approach to manage outpatients with priapism has been recently reported. [ ] specifically, aspiration of the corpora cavernosa followed by irrigation with a dilute epinephrine solution was effective in producing detumescence in most patients. patients who do not respond to this approach are potential candidates for exchange transfusion and/or surgery. [ ] leg ulcers leg ulceration is a painful and sometimes disabling complication of sickle cell anaemia that occurs in to % of adult patients. severe pain may necessitate the use of opioid analgesics. leg ulcers are more common in males and older patients, and less common in patients with α-gene deletion, high total hb level or high levels of hb f. [ ] leg ulcers seem to be more common in patients who are also carriers of the car β-gene cluster haplotype. [ ] treatment of leg ulcers includes wound care using wet to dry dressings soaked in saline or burrow's solution. with good localised treatment, many ulcers heal within a few months. leg ulcers that persist beyond months may require blood transfusion or skin grafting, although results of the latter treatment have been disappointing. because leg ulcers may recur after minimal trauma, protec-tive legging with non-elastic (special velcro) lower-extremity orthoses with ankle straps, to be worn during working hours, appears to be an effective preventive measure. [ ] principles of management of leg ulcers include education, protection, infection control, debridement and compression bandages. efficacy of blood transfusion/exchange transfusion, hyperbaric oxygen and skin grafting is anecdotal in nature. osteomyelitis may complicate chronic leg ulcers, especially those associated with deep wounds, and it is advisable to rule out this complication with a bone scan or magnetic resonance imaging (mri), with gadolinium as needed. to date, there has been no controlled trial of the treatment of sickle cell leg ulcers to identify the best approach to management. the relationship between leg ulcers in patients with sickle cell anaemia and hydroxyurea is not clear. early reports [ ] showed that hydroxyurea seems to have a salutary effect on leg ulcers. recent reports [ ] indicated that hydroxyurea used in the treatment of myeloproliferative disorders is associated with increased incidence of leg ulcers. to date, there is no evidence whether hydroxyurea is beneficial or harmful in the management of leg ulcers in patients with sickle cell anaemia. ferster and colleagues [ ] recently reported their experience with a group of children and young adults with sickle cell disease treated with hydroxyurea for a median follow-up of . years. leg ulcers did not complicate the clinical picture of these patients. recent advances in the management of leg ulcers include the topical application of a plateletderived growth factor prepared either autologously or by recombinant technology, and the use of cultured skin grafts. the use of newly described semipermeable polymeric membrane dressing may promote healing. [ ] to date, there are no data about these new modalities in the management of leg ulcers in patients with sickle cell anaemia. avascular necrosis (also called ischaemic necrosis or osteonecrosis) is the most commonly observed complication of sickle cell disease in adults. although it tends to be most severe and disabling in the hip area, it is a generalised bone disorder in that the femoral and humeral heads as well as the vertebral bodies may be equally affected. the limited terminal arterial blood supply and the paucity of collateral circulation make these three areas especially vulnerable to sickling and subsequent bone damage. patients with sickle cell anaemia and α-gene deletion have a higher incidence of avascular necrosis because the relatively high hematocrit increases blood viscosity and thus, enhances microvasculopathy in the aforementioned anatomic sites. [ , ] the mcv and serum aspartate aminotransferase (ast) levels are negatively correlated with avascular necrosis. [ ] medical treatment of avascular necrosis is symptomatic and includes providing non-opioid and/or opioid analgesics for pain relief, as well as minimal weight bearing. advanced forms of the disease require total bone replacement. core decompression (figure ) in the management of avascular necrosis appears to be effective if done in the early stages of avascular necrosis. [ ] results of hip arthroplasty in patients with sickle cell anaemia are not as encouraging as results of arthroplasty performed for an arthritic hip. [ ] placement of an internal prosthesis may be difficult owing to the presence of hard sclerotic bone in patients with sickle cell anaemia. other problems associated with hip arthroplasty in these patients include an increased incidence of infection, [ , ] a failure rate of about % and a high morbidity due to loosening of both cemented and uncemented prosthesis. recent techniques of arthroplasty may improve the life expectancy of hip prostheses. chronic hyperbilirubinaemia, cholelithiasis and gall bladder disease are common in patients with sickle cell anaemia. at least two-thirds of patients with sickle cell anaemia have hepatomegaly and % have cholelithiasis. about % of patients with cholelithiasis undergo cholecystectomy either prophylactically or after an episode of acute calculus cholecystitis. most cholecystectomies are currently performed by laparoscopy, a much simpler procedure than laparotomy, and are associated with less morbidity. [ , ] a genetic basis for the hyperbilirubinaemia pertains to mutations in udp-glucoronyl transferase (ugt a), the enzyme that catalyses bilirubin glucoronidation. it seems that genetic polymorphism of the ugt a enzyme affects the metabolism of bilirubin. the bilirubin level as well as gallstone formation appear to be significantly higher in patients with the / genotype compared with the / genotypes of the enzyme. [ ] similar findings were reported in patients with hb e-thalassemia. [ ] hepatic crisis (also called sickle cell intrahepatic cholestasis) is manifested by the sudden onset of right upper quadrant pain, progressive hepatomegaly, increasing bilirubin levels (mostly indirect), and prolongation of prothrombin and partial thromboplastin times. [ ] the levels of liver enzymes [γ-glutamyl transpeptidase (γgt) and alanine amino transferase(alt)] are also increased but not to those levels seen in acute viral hepatitis. hepatic crises vary in severity from mi-nor episodes to severe life-threatening situations. total blood exchange is a recommended form of therapy. blood exchange is indicated if the total bilirubin level increases progressively to values greater than g/l. at that level, the prothrombin time values are usually prolonged. blood exchanged should be total in nature, that is, remove whole blood and replace it with red cells and fresh frozen plasma in order to correct the coagulation abnormality. it is the hope of patients with sickle cell disease and their families that there will be, in the near future, therapy that will either cure or markedly alter the natural history of this disease. with better understanding of the pathology of sickle cell disease and co-ordination of multiple therapies that attack different pathological mechanisms of the disease, this goal seems likely. table xvi lists current approaches that have the potential to ameliorate or cure sickle cell disease. although the long term safety and efficacy of these novel therapies have not been well studied, there is good potential for them to reach phase iii clinical trials in the near future. allogeneric bone marrow transplantation (bmt) as a treatment for sickle cell anaemia was first used in europe for patients from africa on the assumption that the risks of disease in their countries of origin justified the hazards of transplantation. [ ] these patients did well and since then additional [ , ] children and adolescents younger than years of age who have severe complications (stroke, recurrent acute chest syndrome or refractory pain) and have an hla-matched donor available were the best candidates for transplantation. about % of patients with sickle cell anaemia met these requirements. about patients with sickle cell anaemia have undergone bmt compared with more than patients with β-thalassemia. more than % of the patients with sickle cell anaemia survived, to % had event-free survival and % graft rejection. neurological complications (seizures or intracranial bleeding) were common in the first transplant recipients. careful control of blood counts, blood pressure and anticonvulsantdrug prophylaxis may forestall these complications. follow-up is still short and the full extent of toxicity is unknown. whether transplantation can reverse established organ damage is also not known, but early reports from europe suggest some improvement in chronic lung, bone and cns disease. [ ] data from the us, however, showed that bmt does not reverse the progression of neurological events. successful umbilical cord blood transplantation from related and unrelated donors in children with sickle cell anaemia seems to be possible. [ ] [ ] [ ] a recent report of two patients with high risk sickle cell disease, publicised by the news media, achieved cure by using unrelated umbilical cord blood cell transplantation (ucbct). [ ] both patients were reported to be alive with donor haematopoietic engraftment and without new manifestations of sickle cell disease at . and . months, respectively, after ucbct. gene therapy, in simple terms, is the introduction of new genes into healthy or abnormal cells either in vitro or in vivo. gene therapy in sickle cell anaemia is limited at present to investigational laboratory procedures and the use of transgenic mouse models to determine the most effective and safest method of altering the genetic information in haematopoietic stem cells. research in this area has advanced at a faster rate than previously expected and gene therapy may be available for trial in selected patients with sickle cell disease in the near future. [ ] the goal of preventive therapy is to ameliorate the clinical picture of sickle cell disease in general, and to decrease the frequency and severity of acute painful episodes in particular. for many years the major goal of primary therapy for sickle cell disease was to identify an antisickling agent that would prevent or reverse the polymerisation of sickle hb in rbcs. although the search for beneficial antisickling compounds continues, the promising approach to prevent the polymerisation of sickle hb has been the use of compounds that increase the production of hb f. the status of these attempts is as follows. induction of hb f: high levels of hb f have a beneficial effect in patients with sickle cell anaemia. platt et al. [ ] has shown that there is a significant inverse correlation between the frequency of painful crises and hb f levels greater than %, i.e. the higher hb f, the milder the disease. hb f interferes with the polymerisation of sickle hb and, the higher (and the more pancellular) it is, the lower the intracellular concentration of sickle hb. however, there are exceptions to this rule in that there are patients with high hb f levels and severe disease and vice versa. agents that have been shown to increase the level of hb f in humans are listed in table xvii. among these, currently hydroxyurea as monotherapy seems to be the least toxic and most effective. [ ] [ ] [ ] moreover, the only drug studied for efficacy in a relatively large scale, placebo-controlled, randomised clinical trial is hydroxyurea. hydroxyurea: is a cell-cycle specific cytotoxic agent that inhibits ribonucleotide reductase. the molecular mechanism(s) by which hydroxyurea increases the production of hb f is(are) unknown. possible mechanisms include perturbations in cellular kinetics and/or recovery from cytotoxicity, recruitment of early erythroid progenitors and recruitment of primitive erythroid progenitors (bfu-e) that lead to production of hb f-containing reticulocytes (f-reticulocytes). long-term hydroxyurea therapy with the maximum tolerated dose (mean dose . ml/kg) with respect to myelosuppression, raises hb f by as much as to % (mean . %, range . to . %). in the randomised, placebo-controlled, doubleblind msh study, among adult patients with sickle cell anaemia with three or more painful crises per year, hydroxyurea resulted in a significant (p < . ) reduction in the incidence of painful crises, acute chest syndrome and transfusion requirement. [ , ] there was no difference between the placebo and hydroxyurea arms in the incidence of death, stroke and hepatic sequestration. maximum tolerated doses of hydroxyurea were not required to reduce the incidence of painful episodes. although an increase in hb f seems to be the obvious and logical explanation for the salutary effects of hydroxyurea, other reasons for its beneficial effects include changes in rbc volume, cellular hydration, the cell membrane and a direct effect on endothelial cells adverse effects of hydroxyurea are listed in table xii. toxic effects are dose-and time-dependent. careful monitoring of blood counts every weeks after starting hydroxyurea can prevent these. later the frequency of monitoring of blood counts and blood chemistries can be decreased to once every to months once the patient is in a stable condition and receiving an acceptable maintenance dose. anaemia is a rare toxic effect of hydroxyurea and, in fact in the msh study, most patients who took hydroxyurea experienced an increase in their hb levels. the idiosyncratic effects of hydroxyurea occur in some patients but not others. however, the incidence of these effects was similar between the placebo and hydroxyurea in the msh study. [ ] in animal studies, hydroxyurea had carcinogenic and teratogenic effects. [ ] [ ] [ ] to date, however, no carcinogenic effect has been reported in patients with polycythemia vera and erythrocytosis due to congenital heart disease treated with hydroxyurea. [ , ] the following limitations should be considered when using hydroxyurea to prevent painful crises in patients with sickle cell disease. firstly, hydroxyurea was approved in the us by the food and drug administration for the prevention of crises. secondly, the long-term effects of hydroxyurea in patients with sickle cell disease are not known, and finally, some patients do not respond to hydroxyurea. methods to identify these non-responders are being studied in order to improve the selection process for hydroxyurea therapy. in some patients combining hydroxyurea with other agents that augment hb f production may be indicated. short chain fatty acids: the role of butyrate analogues as potential inducers of hb f synthesis was based on the observation that infants who have high plasma levels of γ-aminobutyric acid (gaba) in the presence of maternal diabetes mellitus do not undergo the normal fetal-to-adult haemoglobin switch. this led to the discovery that butyric acid, sodium butyrate, and gaba inhibit the normal progress of haemoglobin switching in developing animal models and stimulate the production of hb f in adult animals. butyrate seems to exert its effect through sequences near the transcriptional start site to induce the activity of the human γ-globin gene promoter. a small phase i/ii study where three patients with sickle cell anaemia and three patients with β-thalassemia were treated with intravenous arginine butyrate showed significant and rapid increase in fetal globin synthesis to levels that can ameliorate the clinical picture of these disorders. [ ] the demonstration of specific neuropathological lesions in baboons receiving extended infusions of arginine butyrate at -fold the human dose, however, raised safety concerns about the use of butyrate in humans. [ ] the prolonged use of arginine butyrate is limited because it has to be given intravenously and because it is rapidly metabolised by the liver. this generated efforts to find oral butyrate analogues for the induction of hb f. isobutyramide, a butyrate derivative, has been produced as an oral alternative and has been shown to increase hb f production. [ ] sodium phenylbutyrate, an analogue of butyric acid, is an investigational drug currently undergoing investigation in a phase iii trial for the treatment of patients with inherited disorders of the urea cycle. non-anaemic patients receiving sodium phenylbutyrate were found to have high levels of hb f. [ ] because butyric acid and its analogues are short chain fatty acids, other compounds that belong to this category were considered as potential inducers of hb f. non-anaemic patients receiving valproic acid for epilepsy had increased levels of hb f. [ ] with the exception of arginine butyrate, the analogues mentioned in this section (isobutyramide, phenylbutyrate, phenylacetate and valproic acid) are available in oral form. together, available data suggest that short chain fatty acids may play a role in the primary treatment of sickle cell disease by increasing hb f production. however, their precise role, either alone or in combination with other agents, awaits controlled phase iii clinical trials. erythropoietin: hematopoietic growth factors such as il- , colony-stimulating factor (csf) and granulocyte-monocyte csf can augment hb f lev-els in erythroid cell cultures and in experimental animals. [ ] however, none of these have been reported for this purpose in clinical trials. recombinant human erythropoietin (rhuepo) has been shown to increase hb f levels in erythroid cell cultures, and in non-anaemic baboons and macaques. the molecular mechanism by which rhuepo augments hb f levels seems to be the result of recruitment of f positive progenitor cells, primarily cfu-e derived from an influx of the more primitive bfu-e compartment. [ ] clinical trials in which high doses of rhuepo along with iron supplementation were given to patients with sickle cell anaemia showed an increase in the percentage of freticulocytes and hb f. [ ] extreme caution must be exercised in giving growth factors to patients with sickle cell anaemia. the administration of g-csf to three patients with sickle cell disease caused severe pain and multiorgan failure with a fatal outcome in one. [ ] [ ] [ ] the administration of erythropoietin to patients with sickle cell disease on a regular basis without transfusion will increase the production of sickle hb and the hb level to g% or higher that, together, are associated with increased blood viscosity which, in turn, may accelerate vaso-occlusion. polymerisation of deoxy sickle hb results in cellular dehydration which, in turn, increases the intracellular concentration of sickle hb that leads to further polymerisation, thus, creating a vicious cycle. major mechanisms by which water is lost from sickle cells include the ca + -activated potassium channel (gardos channel) and the kcl cotransport channel. activation of these channels results in k + and water loss from sickle erythrocytes with consequent dehydration. a decrease in the intracellular concentration of sickle hb, even small decreases, can slow the polymerisation of sickle hb to a point where rbcs can exit from the capillaries (decreased transit time) before the sickle hb polymerises (increased delay time for polymerisation). hydroxyurea achieves this goal by decreasing the effective concentration of sickle hb and diluting it with hb f, which does not participate in polymerisation. another approach to inhibit polymerisation is to rehydrate sickle rbcs and restore their normal water content. [ , ] recently, a selective approach to specifically rehydrate sickle rbcs by inhibiting the gardos pathway has been tried by using oral clotrimazole. [ ] in five patients treated with mg/ kg/day of clotrimazole, the rbc gardos channel was inhibited, cell k + content increased, rbcs were rehydrated, and a very modest increase in haemoglobin levels was noted. the effects of clotrimazole on cellular rehydration, however, were very modest compared with those seen in hydroxyurea. nevertheless, the advent of clotrimazole offers a novel and different therapeutic approach for the treatment of sickle cell disease. it warrants a larger long-term clinical trial to determine its efficacy in the primary treatment of sickle cell disease. furthermore, a combination of hydroxyurea and clotrimazole may ensue in an additive beneficial effect in ameliorating the clinical picture of sickle cell disease. similar results were found by the use of oral magnesium, [ ] which inhibits the kcl co-transport channel. these include no, [ ] anti-adhesion molecules, [ ] the surfactant poloxamer- , [ ] levocarnitine, [ ] arginine, [ ] zileuton, a -lipoxygenase inhibitor, [ ] green tea, [ , ] aged garlic, [ , ] and herbal extracts. [ ] some of these agents are being used on an investigational basis. there are anecdotal reports of success in a few patients using some of these agents. the efficacy of any of these agents, however, awaits proof by phase iii doubleblind, placebo-controlled trials. in recent years, there have been considerable advances in understanding the pathogenesis of sickle cell anaemia. although management of sickle cell anaemia continues to be primarily palliative in nature, there have been promising preventative and curative approaches to therapy. pain management should be individualised and coupled with the proper utilisation of opioid and non-opioid analgesics in order to acheive adequate pain relief. early recognition and treatment of organ failure minimises morbidity and improves outcome. the use of hydroxyurea decreases the morbidity and mortality of sickle cell disease. cure is possible in selected children with bone marrow or cord blood transplantation. future research seems to focus on 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causing sickle cell crisis fatal sickle cell crisis after granulocyte colony-stimulating factor administration granulocyte colony-stimulating factor-inducted sickle cell crisis and multiorgan dysfunction in a patient with compound heterozygous sickle cell b+ thalassemia inhibition of ca +-dependent k+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease pulmonary hypertension in sickle cell disease and treatment with l-carritine arginine therapy: a new treatment for pulmonary hypertension in sickle cell disease zileuton: a potential new treatment approach for acute chest syndrome sickle cell anemia: a potential nutritional approach for a molecular disease green tea extract and aged garlic extract inhibit potassium chloride co-transport in sickle cells in vitro, but vitamin-c and vitamin-e do not hydration of sickle erythrocytes using a herbal extract (pfaffia paniculata) in-vitro department of medicine, cardeza foundation for hematologic research supported in part by the sickle cell program of the commonwealth of pennsylvania, harrisburg, pennsylvania, usa. there are no potential conflicts of interest that are relevant to the contents of this manuscript. key: cord- - tewne a authors: tago, damian; hammitt, james k.; thomas, alban; raboisson, didier title: the impact of farmers’ strategic behavior on the spread of animal infectious diseases date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: tewne a one of the main strategies to control the spread of infectious animal diseases is the implementation of movement restrictions. this paper shows a loss in efficiency of the movement restriction policy (mrp) when behavioral responses of farmers are taken into account. incorporating the strategic behavior of farmers in an epidemiologic model reveals that the mrp can trigger premature animal sales by farms at high risk of becoming infected that significantly reduce the efficacy of the policy. the results are validated in a parameterized network via monte carlo simulations and measures to mitigate the loss of efficiency of the mrp are discussed. financial aid to farmers can be justified by public health concerns, not only for equity. this paper contributes to developing an interdisciplinary analytical framework regarding the expansion of infectious diseases combining economic and epidemiologic dimensions. movement restriction policies (mrps) are one of the most popular strategies implemented within and between countries to fight the spread of infectious animal diseases in europe and worldwide [ ] . their efficiency has been demonstrated in the field, often in combination with other measures. in europe, following the council directive / /eec, whenever an infectious disease is detected three zones are delimited over which different restrictions on animal movements are implemented. scale-free networks [ ] , which characterize the structure of many real-world networks, provide a theoretical basis for designing strategies for controlling infectious diseases in cattle and other livestock. applications have been developed recently for animal [ ] and human diseases [ ] and for viral computer infections [ ] . the main characteristic of scale-free networks is the existence of highly connected nodes, i.e. with a degree that greatly exceeds the average such that the degree distribution (the frequency distribution of the number of connections of each node) is fat-tailed. these nodes are called hubs and their high connectivity is the main reason why a disease, technology, or fashion can spread much more quickly through a scale-free network than through a random network [ ] . the idea behind control strategies such as the mrp and vaccination [ ] is that removing infected nodes or immunizing susceptible ones are efficient mechanisms to fight the spread of a disease. it has been found that in fat-tailed degree distribution networks, random immunization (for example vaccinating a proportion of nodes chosen randomly) is not effective in stopping the spread of a disease since for many diseases immunization of %- % of the population is required to halt epidemics [ ] . on the other hand, targeted immunization of the most highly connected nodes (hubs) is very effective, but requires global information on the network [ ] . nevertheless, strategies that use only partial information have been found to efficiently reduce the spread of diseases [ ] . new efforts regarding the traceability of infected animals, such as the construction of databases recording every movement of cattle in a country [ ] , make implementation of the mrp more efficient since they allow tracking the origin of an infection and improving the efficiency of emergency procedures. the removal or isolation of infected nodes will prevent the disease from continuing to spread but this strategy is effective only under demanding conditions. for example, surveillance protocols must be sufficiently efficient that infections are detected almost immediately, or farmers must detect and quickly report the presence of an infectious disease. the time between contagiousness of an animal and its detection depends on the disease, and early detection may not occur when clinical signs are not easily visible. even active-surveillance programs may not detect contagious animals quickly enough when tests are imperfect. moreover, farmers may be slow to report possible infection, in particular when immobilization of their animals can be associated with very large costs [ ] . a great advantage of the mrp is that it can be implemented to control an infectious disease as soon as the disease is detected, whereas strategies based on vaccination require time to develop, produce, distribute, and administer the vaccine to large populations. the efficiency of the mrp also depends on the disease to be controlled. even if detection and isolation of infected farms are perfectly efficient, if the disease is transmitted by insects or other vectors the mrp cannot by itself assure that the epidemic will be halted, as observed during the btv- epizootic [ ] . studies of human-disease epidemics have recently begun to incorporate human reactions [ ] . most of these studies point to the existence of preventive responses that can reduce the spread of the infection, such as the voluntary use of vaccines or facemasks that reduce the risk of being infected [ ] . in the field of animal diseases, the human factor has received less attention. however, the role of biosecurity measures on the spread of animal diseases has been analyzed taking into account the externalities and strategic behavior of farmers [ , ] . among english and welsh cattle farmers, it has been shown that behavioral changes have an impact on the implementation of disease-control programs [ ] . in asian countries, misbehavior by farmers (lack of perfect compliance with regulation) and the emergence of underground poultry markets have been recognized as possible consequences of implementing control strategies [ ] , but their impacts have not been quantified. in the model we propose, changes in farmers' behavior induced by implementation of the mrp can make the policy ineffective under certain conditions. this is due to a mismatch between the time that the mrp is announced in a country (when an infectious disease is detected) and the time that immobilization is implemented in a specific region of that country. this result holds even under the assumption of perfect compliance by farmers (no misbehavior). it can be explained by reasoning similar to that of the so-called "green paradox", which states that some environmental policies oriented to slow global warming act as an announced expropriation of fossil fuels, inducing the owners of these resources to accelerate extraction, which accelerates global warming [ ] . hence possible induced changes in farmers' behavior should be considered when designing control strategies for infectious diseases. the remainder of the paper is organized as follows. first, a susceptible-infected epidemiological model at the farm level is described to analyze diseases that can be transmitted through both the cattle trade network and the geographical network. second, a two-period economic decision model is presented to understand how the mrp can change the strategic behavior of farmers located close to the restriction zone. the conditions that trigger anticipatory sales are established and the effect of the mrp on the spread of an infectious disease is analyzed, with and without anticipation effects. the results are presented, the implications for anticipatory sales are discussed, and a corrective mechanism is proposed. finally the conclusions are presented. the epidemiological model national cattle trade networks have been characterized by previous studies, which find that they have a fat-tailed degree distribution [ , ] . the degree of a node represents the number of connections (edges) that this node has with the rest of the network. in networks with fattailed degree distributions, highly connected nodes (hubs) are more frequent than in random networks; the distribution of degrees is heavy-tailed with power-law behavior of the form p(k)~k −φ with φ , where p(k) is the density function evaluated for degree "k". to study the spread of an infectious disease under different scenarios a fat-tailed degree distribution network is constructed following the characteristics of the french cattle trade network described by rautureau et al. [ ] . the trade network was constructed using the complex networks package [ ] with a power-law degree exponent φ = . [ ] . to mimic the french cattle network (table ) , the nodes were ranked by degree and the top . % were classified as markets, the next . % as dealers, and the remaining . % as farms. each group (farms, dealers, and markets) is characterized by a probability of selling derived from the french cattle network and modeled as a random variable with a uniform distribution in order to capture some of the heterogeneity (table ) . a node is connected to the trade network in each period when it sells animals, which is simulated as the outcome of a bernoulli trial using the corresponding probability of selling. rewiring of the network takes place at each time period. we extend the standard susceptible-infected (si) epidemiological model to incorporate features that are essential for our analysis: the efficiency with which infectious disease is detected and the existence of multiple channels of infection. in our framework, each node represents an agent involved in the cattle trade (farm, dealer, or market) and each agent can be in one of two states: susceptible (s) or infected (i). if detection is imperfect, there are two types of infected nodes: detected (i d ) and non-detected (i nd ), so the total number of infected nodes is i = i d + i nd . to adapt the model to vector-borne diseases an additional transmission channel must be considered. with vector-borne diseases, the infection can propagate through (a) commercial flows and (b) geographical proximity to infected nodes. the incorporation of both channels has been highlighted as a desirable feature for realistic epidemiologic models [ ] . an additional geographic network capturing the risk of vector-transmitted infection of nodes located close to infected ones has to be taken into account. an overview of the baseline model for a susceptible node exposed to infection is illustrated in fig . at each time period there are stages. in the first stage the trade of cattle takes place. at this stage all the market decisions are taken, animals are traded, and susceptible nodes can be exposed to infection through the arrival of infected animals. second, there is the infection stage in which susceptible nodes exposed to infection (i.e. directly connected to an infectious node) can change from the s-state to the i-state with probability λ. the probability of a node becoming infected depends on the number of infectious neighbors and can be computed as -( -λ) n , where n is the number of infectious neighbors. after the infection stage the disease is detected with probability γ, which is equal to if detection is perfect. finally the control stage takes place, in which movement-restriction policies are implemented in two steps: first, the i dnodes are isolated with probability α; second, direct neighbors at the geographic network of infected and controlled nodes are isolated. table summarizes the main parameters involved in the transition between states. the transmission rate λ depends on the disease and may also depend on other factors such as cattle density [ ] and environmental factors [ ] , which affect vector capacity (ability of vectors to acquire and transmit pathogens). the detection rate γ depends on two factors: the characteristics of the disease and the efficiency of the detection tools. the former is associated with the length of the incubation period or the rate of subclinical cases while the latter may depend on the sensitivity of available tests. the parameter α reflects the efficiency of the authorities in implementing the mrp. notice that the mrp isolates the nodes detected as infected as well as their geographic neighbors, which are considered to be those nodes directly linked through the geographic network. this simulates establishing a protection zone around the infected perimeter in which no infected animals have been detected but movements are restricted. the geographic network is simplified to a square lattice in which each node is connected to its immediate neighbors, so there are no hubs in the network. both networks have the same number of nodes and are associated through a one-to-one mapping that randomly assigns the at the infection stage, the node changes to infected status with probability λ; otherwise it retains the s-status during the whole period. at the detection stage, an infected node is detected with probability γ and adopts the i d -status; otherwise it takes the i nd -status. when disease is detected the node is removed from the network with probability α. nodes with i nd -status and those with i d -status that are not removed (i dnc ) remain in the network until the next time period when they are again at risk of being detected or removed. nodes can be removed from the network if they are infected, detected, and effectively controlled (i dc at control stage i), or if they enter a protection zone triggered when any direct neighbor in the geographic network is infected and controlled (c at control stage ii). node's location, which remains fixed throughout the simulation. the infection is introduced by infecting one random node in the first period and the spread of the infection is summarized by the number of infected nodes up to time periods. since the probability of selling was derived for weekly cattle trade networks, a time period can be interpreted as a week in this analysis. for each scenario explored, simulations were performed using matlab (mathworks). the immobilization of animals may impose costs on producers whose profits rely on the movement of their animals [ , ] . to illustrate the implications of the mrp on the behavior of farmers, we consider a simple two-period economic model where a seller of cattle has to choose when to sell his animals. consider a risk neutral agent who has to choose between selling or not selling in each period (t = { , }). at any time period t, if the farmer decides to sell he receives: where: y t is the farmer's income, p t is the market price of cattle, and w t is the total weight of the animals that the farmer sells in period t. if the farmer decides to keep his animals and wait for the second period, y = -c, where c represents the costs associated with keeping the animals for an additional period such as feeding and labor costs. to simplify the problem, assume there is no market risk or price trend such that p = p = p, and the animals gain weight according to the growth parameter d (i.e. w = w à ( + d), where d represents the one-period weight gain percentage). in case the farmer decides not to sell at t = nor at t = , he receives an option value v, which is smaller than the revenue he would receive by selling at t = (i.e. v < p à w à ( + d)). in the context of beef weaned calves (young animals that are sold to fattening units), if animals become too heavy/old, farmers may face a lower price, which can be represented by a penalty p> on the option value (v = p à w à ( + d) à ( − p)). this penalty does not represent fluctuations of market prices (which are fixed in our model) but the fact that if a farmer does not sell at the right moment, the animal losses some of the characteristics valued by buyers. for example, the cost of transporting beef weaned calves increases as the animals become heavier, which makes fattening units less prone to buy these animals. the present study focuses on the most interesting case, where market conditions are such that the gain from waiting one period is larger than the cost of keeping the animals (i.e. c < p à w à d), making the strategy of waiting at t = and selling at t = strictly dominant. usually, beef weaned calves producers face this type of situation and feeding costs play an important role in their decision. if an infectious disease is detected at t = , a farmer sufficiently close to the infected zone will face the risk that the restricted zone (rz) will expand to include his location by the next period (with probability q). if at t = the farmer is located in the rz he will not be able to sell. hence a farmer that does not expect any compensation in case of falling into the rz will decide to anticipate and sell prematurely if: solving for q yields if the gain from trading in the first period is larger than the payoff from keeping the animals and being unable to sell them in the second period (i.e., [v − c] < p à w ) the existence of q ( , ) is assured. to incorporate the anticipatory behavior into the si model, define nr it as farm i's set of neighbors in the geographic network that are located in the rz at period t, and b q p à w à dÀc p à w ð þdÞÀv as the threshold for q above which anticipatory selling is an optimal choice. we assume i such that nr it ¼ ;; i.e. farms geographically adjacent to a farm in the rz perceive the probability of being in the rz in the next period to be high enough such that it is optimal to sell in anticipation (fig ) . therefore, anticipatory sales affect the rewiring process, which modifies the spread of the disease. a monetary transfer to farmers in the rz is a simple mechanism to avoid (or reduce) the anticipatory behavior of farmers at risk of entering the rz. the size of the monetary transfer (denoted by t) that makes farmers with probability q it of entering the rz in the next period indifferent between anticipating and waiting can be derived from: therefore: the size of the monetary transfer was calibrated taking information on prices, weight evolution of animals, and production costs, using data corresponding to the resurgence of the french btv- epidemic in . the monetary transfer is expressed as a function of the subjective probability of being immobilized in the next week. the main parameters of the economic model are summarized in s table, including the values used to derive the monetary transfer required to avoid anticipatory sales. the efficiency of the mrp was quantified by an index defined as the proportional increase in the time-weighted number of uninfected nodes associated with a policy, compared with no policy. this represents the proportional increase in the area above the curve of accumulated infected nodes due to the policy (s fig). the index ranges from to infinity, where is associated with a policy with no benefits in terms of slowing the disease spreading. a rank-correlation analysis was performed to explore how the characteristics and location of the initial case of infection contribute to variability in the results [ ] . in this type of analysis, the degree of statistical dependence between two variables that follow different distributions is quantified and used to identify which factors are the most relevant to the spreading of a disease. the correlations explored are between the accumulated number of infected nodes at a specific time period (the period was chosen such that the average fraction of nodes infected was %) and three different variables that characterize the early periods of infection: the degree (number of edges) and closeness (defined as the inverse of farness, which is the sum of the distances from the selected node to the rest of the nodes in the network) of the initially infected node and the time period at which the first infection of a hub (dealer or market) takes place. results focus on the accumulated number of infected nodes over time after the introduction of a disease. in the absence of control strategies, a % infection rate leads to % of nodes being infected within approximately weeks (fig , red line) . in the case of non-vector-borne diseases, i.e. when the transmission channel is restricted to the trade network, the mrp is an effective control strategy. if the detection tools are perfect it can immediately stop the spread of the disease (fig , green line) . in cases where the disease can be transmitted through both geographic and trade networks (as in the case of vector-borne diseases), the outbreak cannot be contained by movement restrictions alone even if the detection and control rates are % since the geographic channel cannot be closed by the mrp (fig , blue line) . however, the mrp helps to slow the disease spreading. when a disease is difficult to detect (γ close to ) the benefits of the mrp are severely dampened (s fig). when both the geographic and trade networks are considered, a disease with a % infection rate infects half of the nodes around periods , , and for detection rates equal to %, %, and %, respectively. when the strategic behavior of agents is incorporated the outcome of the mrp changes significantly. the anticipation effect increases the speed of infection, and for a low detection rate, γ = %, the benefits of the mrp are severely reduced (fig ) . as the detection rate increases, the anticipation effects become less relevant since infected farms are removed from the network before they can sell. although there exists some overlap in the results of the simulations under different scenarios, the mean trajectory of infected nodes under the mrp with anticipation effects is significantly higher than in the case without anticipation. such overlapping disappears when the % most extreme outcomes are omitted from the analysis (s fig). the effect of anticipation anticipatory behavior-connectivity of a susceptible node. a susceptible node can be located in the rz or the unscathed zone (uz). if located in the rz, the node is unable to sell (probability of selling = pr = ) and has no effect on the dissemination of the disease. a node located in the uz can face two situations: a) the node is not connected to any node located in the rz so it will decide to sell or not according to pr = p s , which is associated with its type (farm, dealer, market; see table ); b) the node is connected to a node located in the rz so q i;t > b q, which triggers anticipatory sales (pr = ). doi: . /journal.pone. .g can be assessed using the efficiency index, which decreases from . without anticipation effects to . when anticipation effects are included. the rank correlation coefficients, whose range is [- , ], are small and positive for the degree and closeness of the initially infected node (between . and . ), and large and negative for the time at which the first hub is infected (between - . and - . , see fig ) . finally, the size of the monetary transfer required to prevent anticipatory selling is derived for two option values that correspond to different penalties ( % and %) on the value of the animal, considering that too old/heavy animals the efficiency of the mrp is significantly reduced when facing a vector-borne disease because the policy cannot control the spread by vectors. however, the mrp restricts the transmission of the disease to the geographical channel. because the geographic network has no hubs, the rate of spreading is significantly slower than through the commercial network. geographic networks can be important even for diseases that are not spread by vectors. for example, it has been found that in the case of diseases transmitted through close animal-to-animal contact, such as foot-and-mouth disease, the plumes of the virus can be dispersed over long distances [ ] . therefore, considering both the geographic and trade networks can be important for some non-vector-borne diseases. factors that limit the speed of detecting infections are an obstacle for implementation of the mrp. the mrp becomes significantly less efficient when infected nodes that have not been detected spread the disease through both trade and geographic networks. the distribution of the time between the infection and control events has previously been highlighted as a crucial ingredient to identify for effectively managing disease spreading [ ] . factors that can limit the rate of detection include a high rate of subclinical cases, long incubation periods, and poor detection tools. the detection period can also be lengthened by the absence of active surveillance activities or lack of declaration in cases of clinical suspicion. for cow-calf systems or for dry dairy cows, farmers may easily miss some moderate clinical signs on some animals and not identify cases until clinical signs have become severe. note that in the present study, changes in the efficiency of implementation of the control policy (α) have analogous results to changes in the detection rate (γ), since the effective removal of the infected nodes depends on the product αà γ. the introduction of strategic behavior of farmers into the analysis significantly reduces the efficiency of the mrp. according to our efficiency index, the anticipatory behavior of farmers facilitates spreading of the disease and reduces the efficiency of the policy by %. when the mrp is implemented, farmers that perceive a high risk of entering the restriction zone will prefer to sell prematurely to avoid the immobilization costs. this increase in the connectivity of the network accelerates the dissemination of disease. in the case of vector-borne diseases, the holdings located close to the rz have a higher risk of becoming infected. if the rate of subclinical cases is high or the incubation period is long, infection at farms outside the rz may be unnoticed and may be transmitted through premature selling of undetected but infected animals. although the mrp by itself is unable to stop the spreading of a vector-borne disease, it can be very useful for reducing the speed of spreading, especially when waiting for the availability of vaccines or for the seasonal inactivity of vectors. the fat-tailed degree distribution of the cattle trade network plays an important role in accelerating the rate of disease spreading. anticipatory sales can infect hubs, which can quickly infect many other nodes. if the trade network were homogenous, hubs would not exist and the effect of anticipatory sales on the spread of diseases would be smaller. the assumption that all farmers having a neighbor (through the geographic network) in the rz perceive that the risk of becoming immobilized in the next period is sufficiently high to induce them to sell their animals (eq ( )) is strong. if this assumption is relaxed, there would be fewer anticipatory sales and the disease would spread more slowly. moreover, when an infected node is detected and isolated, its neighboring nodes are isolated too. therefore, the way the restriction zone is defined has an impact on the results. if the mrp were modified to include not only direct neighbors but the neighbors of neighbors as well ( nd degree connections), the set of nodes that sell prematurely would change. in particular, at early stages of an epidemic, the number of nodes engaging in anticipatory selling (i.e. those at the frontier of the rz) is increasing in the size of the rz and decreases as the entire territory becomes rz. but if there is a correlation between the risk of infection and the geographic proximity to infected nodes (as is the case of vector-borne diseases), by increasing the rz, the nodes located at the frontier are less likely to be infected so they have a smaller effect on disease spreading even if they sell prematurely. the rank-correlation analysis shows that the longer the hubs remain uninfected, the slower the epidemic will spread (fig ) . this result is consistent with the recommendation of immunizing hubs as quickly as possible to avoid the expansion of a disease [ ] . this relationship is stronger when no policy control takes place (r = - . ) than when the mrp is implemented (r = - . with anticipation, r = - . without anticipation). on the one hand, the role of hubs as amplifiers of disease spreading is reduced through the mrp, but on the other hand, complementary strategies to control the spread of infection focused on the immunization of hubs will be less efficient compared to the situation without mrp. this trade-off between the benefits of implementing the mrp and the decrease in efficiency of complementary strategies (focused on the immunization of hubs) should be taken into account when designing an integrated control strategy. the risk imposed by the anticipatory behavior is not a consequence of farmers violating the rules; anticipation effects can be important even in contexts where farmers report new infections promptly and comply with the movement restrictions. the key element in this model is that detection is imperfect (because the tools are limited or the latent or subclinical period of the disease is long) so a farm could sell infected animals without knowing it. a monetary transfer conditional on entering the rz is proposed as a simple mechanism to avoid the change in behavior of farmers affected by the policy. this monetary transfer must be credible and announced in advance so that farmers know of it when considering anticipatory selling. in practice, the monetary transfer can be directed to a specific type of farmer since not all farmers have incentives to sell prematurely. for example, the mrp affects farms specialized in dairy products to a lesser extent than farms specialized in the production of young beef weaned calves, which are sold to fattening units as intermediate products. recognizing this fact, during the - bluetongue outbreak the french government provided financial aid only to farmers who generated at least % of their revenue by the sale of livestock (c-dgpei/ sdepa/c - ) and to the producers of young beef weaned calves (ns-dgpei/sdepa/ n - ). the monetary transfer to farmers located in the rz reduces the costs associated with being immobilized and therefore discourages premature sales. the size of the monetary transfer for risk-neutral farmers is given by eq ( ) . when the risk of falling into the rz exceeds a threshold, (q>b q), the monetary transfer that should be offered in the case of getting into the rz is positive (it can be shown that the transfer is increasing and concave in q). in eq ( ), the first element of the monetary transfer pÃw q it À ðv À cÞ h i represents the gain from selling prematurely adjusted by the risk of falling into the rz compared with waiting and being unable to sell in the second period. the second element ( Àq it q it à ½p à w à ð þ dÞ À c) represents the discount due to the uncertainty of the event. this discount is given by the odds of not getting into the rz in the next period ( Àq it q it ) that multiplies the gain from selling in the discount goes to zero for farms with a high risk of getting in the rz (q it ! ). in this case the government should offer full compensation if the farmer gets in the rz to avoid the anticipation. the rationale for this discount is that the government benefits from the incentives of the seller to wait and sell in the second period (which is the dominant strategy) and therefore does not need to offer a transfer equal to the full cost of immobilization. in the past the rationale for providing financial aid to farmers affected by the mrp was compensatory. when anticipation effects are a risk factor for spreading the disease, giving financial aid to farmers facing movement restrictions becomes an issue of public health and the arguments in favor of this measure are stronger. additional measures that help in the control of infectious diseases can be classified in three categories: measures oriented to reduce the transmission rate such as vaccination or the use of insecticides in the case of vector-borne diseases; measures oriented to increase the detection rate such as the adoption of new technologies (e.g., thermal scanners) and the implementation of preventive protocols; and measures oriented to detect high-risk zones to target surveillance. the third type of measure is possible only with the collection of accurate data and the construction of reliable models. the european effort of constructing national databases to register all the cattle movements is a significant advance and could be extended to other species. network analysis of the spread of disease that incorporates human responses to policy provides new insight into the efficacy of alternative control measures. extensions of this analysis to risk-averse agents and multiple periods should be useful. the current study considers both geographic and commercial networks as potential channels of disease transmission and highlights the relevance of incorporating economic and behavioral elements to evaluate control strategies. for simplicity, we assumed the same transmission probability in both networks. in reality, the transmission probability can differ between transmission paths (geographic or trading) and can change over time. efforts to identify temporal and spatial risk factors of disease spreading associated with the landscape and meteorological conditions should be included in the analysis. the mrp is the main strategy to control the spread of infectious diseases when vaccination is not available (as is the case for newly discovered serotypes of diseases). arguments for implementing mrp are weaker for vector-borne than other diseases because this additional transmission channel reduces the mrp's efficiency. however, it must be taken into consideration that even in the worst-case scenario (vector-borne disease with high rate of subclinical cases) the mrp helps to slow the spreading. this can be very useful in some contexts, such as when the period of inactivity of the vectors (cold months) is approaching, when a vaccine is in the process of development, or even when it is necessary to wait for the vaccines to be dispatched, injected, or boosted (when two injections are needed). for each disease, the costs and the benefits of the mrp should be analyzed in order to take the best possible decision. the strategic behavior of agents can be an important element to consider when analyzing the efficiency of control strategies. the mrp produces incentives that may yield behavioral responses that reduce its effectiveness. we have shown how the strategic behavior of farmers can reduce the efficiency of mrp in controlling the spread of infection. this loss in efficiency should be considered in order to produce more-accurate estimates of the expected benefits of the mrp. the inclusion of complementary measures among emergency protocols related to infectious diseases can increase the efficacy of these policies. by including in the emergency protocols the financial aid for farmers in the rz, farmers know in advance that they will be compensated in case of entering the rz and the anticipatory behavior may be avoided. the monetary transfer required to avoid anticipatory sales by farmers is a function of the subjective probability of being in the rz in the next period (q) for different option values (v = pÃw Ã( +d)Ã( -p), where p is the penalty associated with less desirable animals). the model is calibrated using french data for august th , and estimates for the weight evolution of a charolais calf: p = . eur/kglwt; w = kg; d = / ; c = . eur. (tif) s table. parameters for the economic model. (docx) the effects of the bse outbreak in the united states on the beef and cattle industry emergence of scaling in random networks where diseases and networks collide: lessons to be learnt from a study of the foot-and-mouth disease epidemic spread of a novel influenza a (h n ) virus via global airline transportation epidemic spreading in scale-free networks velocity and hierarchical spread of epidemic outbreaks in scale-free networks immunization of complex networks infectious diseases of humans: dynamics and control network robustness and fragility: percolation on random graphs efficient immunization strategies for computer networks and populations identification and registration of animals in the european union risk factors of poultry outbreaks and human cases of h n avian influenza virus infection in west java province, indonesia why did bluetongue spread the way it did? environmental factors influencing the velocity of bluetongue virus serotype epizootic wave in france capturing human behaviour on the existence of a threshold for preventive behavioral responses to suppress epidemic spreading strategic behavior and spread of animal infectious diseases plos one | behavioral incentives, equilibrium endemic disease, and health management policy for farmed animals biosecurity and spread of an infectious animal disease perceptions, circumstances and motivators that influence implementation of zoonotic control programs on cattle farms wet markets-a continuing source of severe acute respiratory syndrome and influenza? the lancet public policies against global warming: a supply side approach. int tax and public finance vulnerability of animal trade networks to the spread of infectious diseases: a methodological approach applied to evaluation and emergency control strategies in cattle network analysis of italian cattle trade patterns and evaluation of risks for potential disease spread available: www.levmuchnick.net/ content/networks/complexnetworkspackage.html modelling bluetongue virus transmission between farms using animal and vector movements did vaccination slow the spread of bluetongue in france? minimization of the impact of aujeszky's disease outbreaks in the netherlands: a conceptual framework cost assessment of the movement restriction policy in france during the bluetongue virus episode (btv- ) spearman rank correlation forecasting the airborne spread of foot-and-mouth disease the foot-and-mouth epidemic in great britain: pattern of spread and impact of interventions epidemic spreading in a variety of scale free networks conceived and designed the experiments: dt. analyzed the data: dt jkh at dr. wrote the paper: dt jkh at dr. key: cord- - va yyit authors: nan title: its asm date: - - journal: ir j med sci doi: . /s - - -z sha: doc_id: cord_uid: va yyit nan it is my great pleasure to welcome you to limerick, host city to the irish thoracic society annual scientific meeting. we look forward to a very exciting program, offering the best of original research and state of the art guest lectures against the backdrop of warmth and conviviality for which the meeting is known. thank you to all those who submitted abstracts and case studies this year-we received over for presentation, reflecting the high quality and innovative work taking place in research centres throughout the island and further afield. i would also like to thank the abstract review committee and judges for their time and expertise in what is never an easy task due to the increasingly high standard of submissions received. special features of the meeting include guest lectures on 'asthma genomics and the respiratory biome', 'use of niv in acute respiratory failure' and 'the role of nasal electrophysiology in the difficult cf diagnosis' as well as a symposium on 'chronic respiratory failure'. i am delighted to welcome distinguished guest speakers from the uk and usa who will share their expertise on these topics. welcome also to the patient and professional organisations represented. networking and sharing information on the wealth of activities taking place across the respiratory healthcare community has become an integral part of the meeting. i would like to extend a particular welcome to the exhibitors and sponsors of this year's meeting. we are very grateful for their continued support, without which the meeting would not be possible. we present the case of an -year-old gentleman who had an inflammatory neuropathy secondary to a thymoma. he underwent a vats thymectomy, the first case in ireland. the surgery involved a cm transverse incision above the supra-sternal notch, partial mobilization of the thymus, and -port placement with full thymic mobilization and excision. the advantages of a vats approach include lower blood loss ( vs. mls), lower post-operative ventilation rates ( . vs. . %), shorter lengths of hospitalisation ( . vs. . days), and improved cosmesis. the patient made an excellent recovery, discharged to home on post-operative day . we report a case of pleural giant b cell lymphoma in a year old man induced by immunosuppressive therapy for rheumatoid arthritis (ra). despite adequate control of his ra with anti-tnf treatment, this was substituted with leflunomide and prednisolone after a myocardial infarction. he subsequently developed recurrent bloody exudative pleural effusions containing a lymphomatous cell population. prolonged remission was obtained with the anti cd antibody therapy, rituximab. although the attribution of lymphoma with anti-tnf therapy and immunosuppressive treatment remains controversial [ ] [ ] , the association is more than spurious [ ] . despite a low risk of lymphoma from biological therapies, continued vigilance is warranted. pericardial atresia is a rare cardiothoracic malformation. it is mostly diagnosed incidentally, on surgery, or autopsy. it usually has benign symptoms, but herniation of the heart through a partial defect can be fatal. we describe an unusual case of years old zimbabwean lady who presented with severe headache, syncope, bradycardia and orthostatic hypotension. she was also noted to have elevated d-dimers and was hypoxic on room air. a chest x-ray reported cardiomegaly. a ct pulmonary angiogram demonstrated no embolism, but did show features consistent with absence of the pericardium. further cardiac mri confirmed this finding in addition to left ward rotation of heart into left thoracic cavity and four chamber dilatation. a review of the available literature on clinical presentation, diagnostic assessment and therapeutic options is presented. and soft tissue, including primitive neuroectodermal tumors (pnets). it responds relatively well to a combination of surgical resection, chemotherapy and radiation therapy. we describe a years old polish girl who presented with year history of right scapular, paraspinal and anterior chest wall pain. imaging confirmed . . cm mixed solid and cystic mass in right lung apex invading into supraclavicular muscles and neural foramen. the biopsy and immunotyping revealed small round ''blue cells'' soft tissue tumour equivalent of ees/pnet. she had neoadjuvant chemotherapy followed by surgical resection of the tumour with a good result and further planned to continue chemotherapy and evaluate treatment response. a year old man with known kartagener's associated bronchiectasis presented with increased cough and sputum production associated with pyrexia. radiologically, he had a right lower lobe infiltrate but sputum culture and urinary legionella antigen were negative. however, he failed to improve on a penicillin. bronchoscopy was performed and lavage specimens grew legionella pneumophila serogroup . he recovered following treatment with levofloxacin. there was no history of foreign travel but he did sleep in a camper van. there are known subgroups of legionella pneumophila and only serogroup is detected by urinary antigen. culture remains the gold standard. a year old lady was referred to the haematology service with a relapse of follicular lymphoma grade a. following salvage chemotherapy and pre transplant conditioning with alemtuzumab, she had a non myeloablative allogenic stem cell transplant. as part of the investigation for recurrent lower respiratory tract infections a bronchoscopy revealed a large exophytic tumour occluding the anterior orifice of the left upper lobe. biopsy and culture confirmed mycobacterium kansasii. the patient had an excellent clinical, microbiological and radiological response to treatment with resolution of the tumour at bronchoscopy. our case demonstrates an unusual presentation of mycobacterium kansasii. atypical mycobacterial infections although rare must be considered in this patient group. a -year-old woman presented in symptomatic hypercapnic respiratory failure. proximal muscle and neck flexor weakness was noted on examination. pulmonary function testing revealed restrictive physiology. creatinine kinase and serological testing were normal and acid-maltase deficiency was excluded. deltoid muscle biopsy was consistent with myofibrillar myopathy. myofibrillar myopathies are rare, encompassing a heterogenous group of sporadic and familial neuromuscular disorders characterised by slowly progressive muscular weakness. cardiac and respiratory muscles are involved in only a small subset of people. our patient has improved with non-invasive ventilation. further molecular genetic analysis is pending. a -year-old lady presented with dysphagia. she had a right lower lobe pneumonectomy for a t nomo non-small cell bronchial carcinoma years previously. a computed tomography scan of the thorax showed oesophageal dilatation, accounting for the presenting complaint, and an incidental thrombus in the pulmonary artery stump (pas). the formation of a pas thrombus is a common radiological finding following pneumonectomy [ ] . there is no strong evidence to suggest anticoagulation is beneficial in this group of patients. as no respiratory symptoms were present, anticoagulation could not be justified. a -month interval scan showed no thrombus propagation and the patient remained asymptomatic. department of respiratory medicine, beaumont hospital, dublin , ireland a year old gentleman presented to the respiratory clinic with a / history of progressive dyspnoea on exertion associated with cough. he reported longstanding lower limb weakness, progressing to involve his upper limbs. arterial blood gas demonstrated partially compensated hypercapnoeic respiratory failure whilst pfts showed a classic restrictive pattern. electromyography was abnormal and a muscle biopsy confirmed a diagnosis of nemaline myopathy. he was commenced on nocturnal non-invasive ventilation and his respiratory function improved significantly. nemaline rod myopathy is a rare congenital myopathy that typically presents in childhood. presentation in adulthood with respiratory difficulties is highly unusual. jo- positive polymyositis is an inflammatory myopathy and commonly presents with progressive muscle weakness and basal predominant interstitial lung disease. this is a case report of two women with different clinical presentations of jo- positive polymyositis who had bibasal interstitial lung disease on high resolution ct thorax. they both had worsening pulmonary function studies and one of them required icu admission and intubation for management of severe respiratory distress. they both responded to different regimens of iv cyclophosphamide and long term oral immunosuppressants. these cases illustrate the respiratory manifestations of jo- positive polymyositis and its treatment. year old male with a recent history of travelling abroad presented with week history of dry cough and day history of progressive sob on exertion and one episode of haemoptysis. cxr showed rul consolidation and rll effusion, ctpa was negative for pe. subsequently he developed recurrent spikes of temperature and septic screen was negative. he had three different antibiotics regime with no improvement. thereafter multiple blood cultures, vasculitic, typical and atypical microorganism, hiv, tb screen and bronchial washings came back as negative. repeat ctpa and serial cxr confirmed persistent bilateral consolidation. on day of admission he developed new systolic murmur, a-fib and type respiratory failure. tte and subsequent toe confirmed severe mr with mitral valve vegetation and prolapse treated with mitral valve replacement. dublin , ireland years old male admitted year ago with mesenteric ischaemia diagnosed& nstemi, had emergency small bowel resection with right hemicolectomy. admission has been complicated over year by wound infection, multiple pneumonias, multiple line infections, multiple admissions to itu, later diagnosed with short bowel syndrome, and c difficile colitis; and thrombosis of superficial venous system. due to difficult iv access and low albumin. recently admitted to itu deteriorated due to acute electrolyte disturbance. had transhepatic line (tipss) inserted by ir due to difficult iv access and low albumin. day became acutely sob, cxr complete opacification of right lung. chest drain inserted had drained litres of milky fluid, tubogram confirmed leak through diaphragm into pleura. a. mohamed, r. smyth, j.j. gilmartin university college hospital galway, co galway, ireland a year old man presented with a year history of progressive dyspnea. physical examination revealed a right pleural effusion. a chest radiograph and ct scan confirmed the presence of an effusion with thoracic lymphadenopathy and large mesenteric and para-aortic masses. a pleural aspiration was performed and litre of milky white fluid was removed. pleural fluid analysis noted a protein concentration of g/l and a triglyceride level of mg/dl. cytology revealed a wbc count of /ml with % of these being mononuclear. subsequent lymph node and bone marrow biopsy confirmed the diagnosis of chylothorax with small lymphocytic lymphoma. a -year-old gentleman presented with sub-acute onset of dyspnea associated with diffuse bilateral infiltrates on chest radiograph. his respiratory failure worsened despite broad spectrum antibiotics and he required intubation. he had recent type diabetes mellitus that was well controlled on oral hypoglycaemics. bronchial washings were positive for pcp on silver stain. despite extensive testing a cause for immunological deficiency could not be identified. he developed ards related fibrosis and died. pcp is the most common opportunistic infection in aids patients [ ] , it has been rarely reported in previously well, immunocompetent patients. an year old non-smoking female presented to the er with a week history of progressive dyspnoea and cough. the patient also recounted difficulty reclining at night to the point where she slept in a sitting position. examination revealed mild expiratory wheeze and hypoxemia on room air. initial clinical impression was of an infective exacerbation of late onset asthmatic bronchitis. cxr demonstrated a large hiatus hernia. pulmonary function studies showed a mixed obstructive and restrictive pattern. ct thorax revealed large diaphragmatic hernia with evidence of compression of the main bronchi. bronchoscopy showed dynamic airway collapse with complete obstruction of the left and right main stem bronchi and distal trachea on coughing. this represents an unusual cause of dynamic airway collapse and the imaging and literature are reviewed. a year old lifelong non-smoker with asthma presented to er with a days history of worsening breathlessness. on examination he was unable to complete sentences and reduced air entry through out chest.cxr showed right middle lobe consolidation. he had been treated as infective exacerbation of asthma. but unfortunately he deteriorated later on that day progressing to respiratory and circulatory failure and subsequently to disseminated intravascular coagulation. although having best of icu care he passed away the following day. all of his investigations including blood culture, atypical pneumonia screen, h n serology were came back negative. his post mortem examination hasn't revealed any cause other than right sided pneumonia contributing to his death. this represents a shocking but mysterious case of pneumonia as the cause of death. a year old gentleman was referred to the rapid access lung cancer service with a week history of progressive sob and new pleural effusion. of note, he had recently presented to his general practitioner with two soft tissue lesions on the right flank. on examination, axillary lymphadenopathy was palpable with a noted interval increase in the size of the cutaneous lesions. trucut biopsy of the skin lesion and pleural aspiration revealed a histological diagnosis of mantle cell lymphoma. subsequent radiology showed extensive systemic disease and chemotherapy was offered. our case illustrates the extranodal involvement that can occur in mantle cell lymphoma, however, patients rarely present with initial cutaneous involvement as in our case. a year old gentleman was referred to our lung cancer clinic with abnormal chest x-ray and palpable supraclavicular node. a staging ct thorax suggested lung primary with mediastinal and supraclavicular nodes. a biopsy of the palpable node showed hodgkin's disease (hd). a ct-guided biopsy of the lung lesion favoured lung primary and mediastinal nodal sampling revealed hd. he proceeded to lobectomy for excision of the primary lung lesion and the pathology returned a non hodgkins lymphoma. this case illustrates a presentation of dual pathologies: both hodgkins and non hodgkins lymphoma in the same patient mimicking locally advanced lung cancer. a -year-old man presented with a -month history of back pain and cough. he had a normal chest radiograph and was reassured. subsequent haemoptysis in this patient prompted ct imaging which showed a large left lower lobe mass that was concealed by the cardiac shadow. further mri imaging revealed extensive spinal metastases and thus stage iv disease. we wish to present a case series of five patients with benignappearing single plane chest radiography. further imaging revealed advanced stage lung cancer in these patients. these cases illustrate limitations of single plane radiography and identify anatomical areas where tumours may be missed such as behind the heart. they emphasise the importance of obtaining lateral films and ct imaging in patients with a reassuring single plane chest radiograph where clinical suspicion persists. a year old latvian security guard with a background of relapsing polychondritis and recurrent escherichia coli lrti's, represented to hospital with recurrent haemoptysis and pyrexia of unknown origin. hrct thorax revealed innumerable randomly distributed pulmonary micronodularity. bronchoscopy and bal were unremarkable. fresh sputum cytology confirmed the diagnosis of pulmonary nematodes consistent with strongyloides stercoralis. the patient was treated with high dose ivermectin, resulting in complete resolution of the pulmonary micronodularity. this case report discusses the diagnostic criteria for relapsing polychondritis, the international issues of pulmonary nematodes and the importance of understanding the three radiological subtypes of pulmonary micronodularity (random, centrilobular and peri-lymphatic). a year old gentleman with severe autism spectrum disorder (asd) presented acutely to the emergency department with behavioural disturbance. his prior history was notable for a right lower lobe infiltrate on chest radiography identified year prior to admission. his behavioural disturbance manifested by self harm, and had previously precluded in-hospital investigation. ct thorax confirmed right lower lobe consolidation and flexible bronchoscopy identified a . cm tree branch within a segmental bronchus of the right lower lobe. this case highlights the importance of flexible bronchoscopy and the difficulties of access to appropriate care for those patients with asd. a year old male was admitted with pleuritic chest pain after knee replacement. routine bloods, including liver function tests were normal. a chest radiograph showed right upper quadrant calcification. ct pulmonary angiography was normal but a ct abdomen showed an area of calcification in segment of the liver with low attenuation centrally consistent with a hydatid cyst. hydatid disease is caused by ingestion of the dog tapeworm echinococcus granulosus. it is uncommon within ireland. treatment includes monitoring of chronic cysts, medical therapy with antibiotics in combination with either surgery of percutaneous drainage. a year old female smoker was admitted with right sided pleuritic chest pain. ct thorax showed extensive right lung consolidation and bronchiectasis. bronchoscopy showed unexpected thickening and nodularity of the upper trachea. biopsies confirmed tracheal amyloidosis and immunohistochemical staining of the deposits was negative for serum amyloid a protein, transthyretin, and kappa and lambda immunoglobulin light chains, indicating amyloid of non-aa type. there was no evidence of amyloid at any other location. patients with tracheobronchial amyloidosis may be asymptomatic or present with dyspnoea, cough, haemoptysis or recurrent pneumonia. department of respiratory medicine, beaumont hospital, dublin , ireland a -year old male presented to beaumont hospital with dyspnoea, cough and haemoptysis due to alveolar haemorrhage, requiring icu admission for high frequency oscillatory ventilation and iv cyclophosphamide. originally diagnosed with a pauci-immune vasculitis, a lung biopsy confirmed a diagnosis of pulmonary capillaritis requiring escalating treatment with pulsed iv steroids and immunosuppression. while initially clinically stable, he has had multiple icu admissions, and during an exacerbation presents with symptoms of dyspnoea, a dropping haemoglobin and bilateral infiltrates on chest radiograph. he is now managed by an adult respiratory service with specialist advice, with iv steroids, iv ig and rituximab. we discuss a minimally invasive approach with video assisted thoracoscopic surgery (vats) in performing thymectomy procedures for cases of myasthenia gravis. a year old female was referred with a background history of myasthenia gravis. despite optimal medical therapy m.n. still experiences persistent symptoms and occasional hospitilization for iv immunoglobulins. a ct thorax showed an enlarged thymus. using a three port vats technique the thymectomy was completed and she was discharged days post operation. this case illustrates the possible benefits of minimally invasive approach to thymectomy and avoidance of sternotomy for cases of myasthenia gravis. we report the case of a y.o man who presented with classical pancoast syndrome symptoms caused by a large apical ewing's sarcoma of the chest wall. neoadjuvant chemotherapy localized the tumour to the first rib. complete enbloc surgical resection of the mm residual mass (entire first rib, partial second rib and sublobar lung) was accomplished by a combined anterior hemiclamshell and posterior approach. ypt . the c nerve root was spared and this led to a complete resolution of symptoms. we report the case of an y.o. who underwent curative resection by right sided lobectomy for a pt an adenocarcinoma. she developed this uncommon syndrome of intracardiac shunting of blood immediately post extubation. this syndrome causes profound dyspnoea as a result of arterial hypoxia which is accentuated by the upright position and relieved by recumbency. echocardiography based on clinical suspicion was diagnostic. this was successfully treated by the placement of an occluder device the patent foramen ovale. the case of a year old male smoker with a week history of haemoptysis, night sweats and dyspnoea on exertion is presented. chest radiography confirmed a right upper lobe thick-walled cavity with adjacent nodularity. both transbronchial and percutaneous biopsy sampling was non-diagnostic and microbiological testing was negative. following an episode of large volume haemoptysis he underwent emergency lobectomy. final histopathologic examination confirmed an anaplastic lymphoma kinase (alk)-negative pulmonary anaplastic lymphoma, an extremely rare cause of pulmonary cavitation. we report a g d homozygote: fev % predicted, oxygendependent with recurrent exacerbations ( in months) awaiting transplant assessment, started on ivacaftor through a named patient program. within weeks transplant assessment was deferred, fev had increased to % predicted, continuous oxygen was discontinued, sweat chloride had fallen from to mmol/l and exacerbation rate decreased to in months prospectively. a year old lady presented with headache and cough. neuroimaging raised the possibilitiy of cns mycobacterial infection. pansensitive mycobacterium tuberculosis was cultured from bronchoalveolar lavage. the patient experienced a type hypersensitivity reaction following the first dose of intravenous rifampicin. the patient was commenced therefore on iv amikacin, oral moxifloxacin and ethambutol. intravenous desensitisation to rifampicin was carried out using a day protocol. this was successful allowing the patient to return to oral therapy for the duration of her treatment. ipf and panca-positivity predating vasculitis is known but not widely appreciated. we describe two cases of ipf, initially anca-negative who became anca-positive with associated vasculitis. a -year-old, house-wife diagnosed with ipf in was anca-negative. in she was more breathless with borderlinepositive panca (repeat negative). in , she developed acute mononeuritis multiplex, with a highly positive panca, responsive to immunosuppression. ipf remained stable throughout. a -year-old male, with established ipf (anca-negative), presented acutely with alveolar haemorrhage, renal failure and now panca-positive, responsive to plasma exchange, haemodialysis and immunosuppression. these cases support the rationale for serial anca measurements in ipf. bronchoscopy in a year old female months after radical chemoradiotherapy and oesophageal stent insertion demonstrated stent erosion into the proximal trachea with recurrent oesophageal scc obstructing the carina. a mm covered ultraflex tracheal stent was deployed, with an oesophageal stent telescoped proximally into the displaced oesophageal stent. imaging out ruled a leak facilitating oral intake. six weeks later, staged cryotherapy and stenting of the carinal obstruction was successfully performed. cork university hospital, wilton, cork, ireland, mercy university hospital, cork a -year-old male was reviewed with increasing shortness of breath, hoarseness and stridor. his past medical history was remarkable for supraglottic amyloidosis. these lesions were thermally ablated in . he was followed up routinely. a ct thorax and subsequent bronchoscopy and biopsy was undertaken. at bronchoscopy he was found to have two large nodular protrusions that were biopsied. pulmonary amyloidosis is rare and manifestations include tracheobronchial infiltration, parenchymal infiltration (amyloidomas) persistent pleural effusions and pulmonary hypertension. symptoms of tracheobronchial amyloidosis can include hoarseness, stridor, dyspnoea and overt airway obstruction [ ] . treatment involves invasive bronchoscopic therapies such argon photocoagulation (apc) and occasionally surgery [ ] . we present two mushroom workers with bird fancier's lung. workers presented with progressive dyspnoea, cough and sweats, with features of hypersensitivity pneumonitis on hrct, pfts and bal/tbbx. serological studies to aspergillus fumigatus, saccharopolyspora rectivirgula, thermophilic actinomyces were negative but positive for avian proteins. workplace process analysis revealed chicken litter as fundamental in mushroom compost production. both workers received corticosteroids with symptomatic and radiological improvement. workplace relocation resulted in complete resolution of symptoms in one worker. the second worker remains exposed, wearing appropriate ppe with ongoing medical surveillance. detailed workplace analysis may be required in proper diagnosis of work-related respiratory diseases. wheeze is a continuous musical sound that lasts longer than ms [ ] . upper airway obstruction is commonly misdiagnosed as asthma. we describe four cases presenting with upper airway obstruction of different aetiologies. a -year-old female was referred with 'poorly controlled asthma'. inspiratory stridor was noted on physical exam and spirometric flow volume loops showed variable extrathoracic airway obstruction. laryngobronchoscopy confirmed paradoxical vocal cord movement. ir j med sci ( ) (suppl ):s -s a -year-old female was referred with 'poorly controlled asthma'. physical exam revealed inspiratory stridor and spirometric flow volume loops showed fixed upper airway obstruction. larynogbronchoscopy revealed subglottic stenosis. a -year-old male with a -pack-year history of smoking was referred with worsening wheeze. physical exam and investigations revealed subtotal tracheal compression secondary to a retrosternal goitre which was successfully resected with resolution of symptoms. a -year-old male was referred with wheeze, fatigue and intermittent apnoea while sleeping on his left side. physical exam revealed inspiratory stridor and spirometric flow volume loops showed fixed upper airway obstruction. ct thorax and bronchoscopy revealed a congenital double aortic arch, splitting to come around the trachea (see fig. ) causing tracheal compression. the four cases show the importance of considering misdiagnosed upper airway obstruction in the assessment of wheeze. a year male smoker with gastro oesophageal adenocarcinoma developed dyspnoea on treatment with epirubicin, oxaliplatin, and capecitabine, raynaud's phenomenon was noted and hrct showed bilateral interstitial infiltrates. scl antibodies were positive and thoracoscopic lung biopsy confirmed scleroderma-associated interstitial lung disease. patients receiving pneumotoxic agents should be assessed thoroughly for other causes before a diagnosis of drug-induced interstitial lung disease is considered. mid-western regional hospital limerick, limerick, ireland a year old woman presented with recurrent lower respiratory tract infections and weight loss of over a stone over the last months. she was a smoker of pack year. ct showed diffusely abnormalities in both lungs. there was ill-defined pulmonary nodules with small cavitating lung lesions. specimens taken from vats showed numerous airway-centred and airway-destructive nodules composed of eosinophils and large histiocytes with vesicular chromatin that are cd a positive. this is consistent with langerhans cell histiocytosis. she was advised on smoking cessation which she adhered to. follow-up cxr showed complete resolution of the multi-focal interstitial infiltrates several studies reported relationship between anca-associated vasculitides and malignancies (pre-existing or developed during patients follow up), bringing to discussion the putative role of tumor antigen in driving the auto-immune response. we describe the case of years old male with e-cadherin genetic mutation, mandibular cementoma, horse shoe kidney, alopecia and nails dystrophy who presented with haemoptysis and bilateral pulmonary infiltrates years after his first asthma diagnosis. bronchial washings demonstrated pigment laden cells consistent with alveolar haemorrhages. serological tests showed positive c-anca. there was no renal involvement. he was successfully treated with high dose of corticosteroids. we present an interesting case of endobronchial tb in patient who lacked normal host immunity. a gentleman ( years) presented with haemoptysis on a background of ulcerative colitis managed with mesalanine and azathioprine. bronchoscopy demonstrated a mass partially occluding the left main bronchus. pan-sensitive mtb was isolated and he was treated for months. four months following presentation an endobronchial resection was performed. however, repeat bronchoscopy following nine months of treatment demonstrated a persistent lesion in the left main bronchus and a new lesion in the trachea. the recurring lesion was characterised by the persistent afb, that were stainable, but did not grow. conclusion: the immune derangement, characteristic of inflammatory bowel disease patients, contributed to his aberrant persisting host response to dead organism. [ ] . in these two cases dipnech was diagnosed along side carcinoid tumour via lobectomy and mediastinal lymph node sampling. one patient was symptomatic for at least months prior to diagnosis complaining of episodic wheeze, flushing and diarrhoea. this patient was treated with a somatostatin analogue post operatively. dipnech has rarely been described and its prognosis and management have varied from several different case reports [ ] . these two cases highlight, that although a rare entity, it needs to be considered in cases of symptomatic cough and wheeze with radiological findings suggestive of discrete pulmonary nodules. in march , the first case of a subsequent outbreak of pansensitive mycobacterium tuberculosis, beijing strain, was diagnosed in a man from an irish prison. last year, the first patients in this sequence were presented. herein we discuss eight further cases connected to this outbreak. all cases, but one, are males of european origin aged between and years. clinical presentations ranged from classical symptoms of night sweats and weight loss to acute abdomen. four of the pulmonary tb cases had, on initial presentation, acid fast bacilli positive sputum on microscopy and culture. the radiological disease was primarily consolidation or cavitating lesion in the upper to mid zones. more than a year later, this case series continues to highlight the ongoing and mounting difficulties of managing this irish prison outbreak. a year-old irish male presented with a month history of dyspnoea, unsteady gait, numbness over both flanks and weight loss. he attended months earlier at another centre with swollen painful right testes. cxr demonstrated bilateral hilar lymphadenopathy, and rightsided pleural effusion. ct tap revealed marked mediastinal lymphadenopathy with right sided pleural effusion, mild splenomegaly, ill-defined right testes, and bilateral inguinal lymphadenopathy. serum ldh and ace were elevated. thoracentesis revealed serosanguinous exudative fluid. transbronchial and inguinal lymph node biopsy revealed evidence of non-caseating granulomas. mri spine revealed abnormal cystic areas throughout thoracic and lumbar spine. a diagnosis of sarcoidosis with multisystem involvement was made and high dose steroids were commenced with good clinical response. pleural and spinal cord sarcoidosis is a rare and interesting presentation. histology showed chronic inflammatory change and ulceration of the bronchi with no evidence of vasculitis or granuloma. no malignancy was evident. pulmonary changes similar to this have been described in patients with uc. alpha- antitrypsin deficiency (aatd) is an autosomal co-dominant genetic disorder associated with a substantially increased risk for the development of chronic obstructive pulmonary disease (copd) and liver disease. ats/ers guidelines recommend testing of all individuals with copd and poorly controlled asthma. the objective of the study was to investigate the diagnostic experiences of zz aatd individuals in ireland. a total of zz aatd individuals completed a questionnaire at an alpha- clinic in relation to their diagnostic experiences and clinical presentation. the mean age of symptom onset was . ± . years (range . - ); mean age of diagnosis was . ± . years (range . - ). the interval between onset of symptoms and aatd diagnosis was years. the smoking history analysis revealed % were past smokers, % never smokers and % were currently smoking. for the past smokers cohort % stopped smoking within the first months of a diagnosis of aatd; % stopped smoking after the first months of a diagnosis of aatd and % had stopped smoking prior to a diagnosis of aatd. our results further underline the need for increased awareness and early detection of symptomatic aatd individuals in the irish population, especially among the copd population. aat deficiency (aatd) results from mutations in the serpina gene, classically presenting with early-onset emphysema and/or liver disease. the most common mutation causing aatd is the z mutation, with the s mutation weakly associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. ats/ers guidelines advocate screening all copd, poorlycontrolled asthma, and cryptogenic liver disease patients, as well as first degree relatives of known aatd patients. over , individuals have been screened to date following ats/ ers guidelines in the national targeted detection programme. sequencing of the serpina gene was performed to identify rare mutations. we have identified zz, sz, ss, mz, ms, and over individuals with clinically significant rare phenotypes (e.g. iz, fz, is, null, mmalton). this yields gene frequencies of . and . for s and z, respectively, in this targeted population. a number of rare and novel serpina mutations have also been identified. our results underline the need for increased awareness and early detection of aatd. all copd patients should be tested for aatd as per ats/ers guidelines. our data demonstrates that aatd in ireland is not a rare disease but a disease that is rarely diagnosed. rationale: alpha- antitrypsin (aat) deficiency (aatd) is genetic disease that results in low levels of aat and predisposes individuals to developing chronic obstructive pulmonary disease (copd). the z-allele is responsible for [ % cases of aatd. key studies have demonstrated that excessive infiltration of neutrophils into the lung and neutrophil derived proteins play a pathological role in aatd lung disease. in particular a key cytokine associated with copd disease progression is tnf-alpha which is also the cause of many problems associated with autoimmune diseases. the aim of this study was to determine if there is a novel autoimmune element driving inflammation in aatd and examine aat impact on this. methods: plasma and neutrophils were isolated from mm controls, asymptomatic zz aatd and aatd patients receiving augmentation therapy. tnf-alpha was quantified by a sandwich elisa. evaluation of neutrophil degranulation was carried out by western blot analysis of neutrophil supernatants for markers of tertiary (mmp- ) and secondary granules (lactoferrin) and via flow cytometry. autoantibodies against neutrophil granule proteins were quantified in plasma by elisa. results: our results demonstrate that there are high levels of tnfalpha in aatd plasma compared to controls (p = . ). in vitro, tnf-alpha caused an increase in the rate of degranulation of tertiary and secondary granules from zz aatd neutrophils compared to mm cells (p \ . ). analysis of autoantibodies against major neutrophil granule proteins revealed a high titer of anti-lactoferrin igg autoantibodies present in patients with zz aatd. treatment of aatd patients with aat augmentation therapy resulted in a decrease in the plasma levels of neutrophil granule proteins while also reducing the titer of anti-lactoferrin igg autoantibodies (p \ . ). conclusion: this study has uncovered that tnf-alpha inflammatory signaling pathway can result in the development of an autoimmune element in aatd. furthermore it highlights aat therapy can impact on neutrophil degranulation and thereby reduce development of novel anti-lactoferrin autoantibodies. chronic hypoxia (ch) exposure induces diaphragmatic remodelling similar to chronic obstructive pulmonary disease (copd). elucidating the underlying mechanisms may inform therapeutic strategies to combat muscle dysfunction in copd. this study investigates the effects of ch on redox homeostasis in mouse diaphragm muscle. c bl j mice were exposed to one and weeks of ch ( % f i o ) or normoxia. following treatment, excised muscles were homogenised and incubated with carbonyl-or thiol-reactive fluorophores before gel electrophoresis and fluorescence scanning. optical density (od) of fluorescence was normalised to total protein, determined by colloidal coomassie staining. a nine-fold increase in free thiol groups was observed after week of ch ( . ± . vs. . ± . ; mean od ± sem, ch v normoxia, n = per group; p = . , student's unpaired t test), while there was a significant decrease after weeks. a significant increase in carbonylation was observed after weeks of ch ( . ± . vs. . ± . ; mean od ± sem, ch vs. normoxia, n = per group; p \ . ). we have demonstrated that despite a reduction in oxygen tension and a large, initial increase in free thiol groups, weeks of ch significantly increases oxidative stress in mouse diaphragm muscle. changes in redox homeostasis are likely to affect redox-malleable proteins that are central to muscle performance. a- antitrypsin (aat) is a -kda glycosylated-protein synthesised in the liver which functions as a serine protease inhibitor. the pizz variant is associated with early onset emphysema. we hypothesize that a difference in the number of isoforms and the n-glycosylation pattern of pimm and pizz aat protein exists. the aim of this study was to compare the isoform composition of aat from pimm controls with that of pizz individuals. aat from pimm and pizz individuals was extracted and purified from plasma using alpha- -antitrypsin select-affinity chromatography medium. isoelectric-focussing of purified aat was performed followed by d-page. gels were stained with coomassie brilliant blue and were immuno-blotted for aat. eight and six isoforms of pimm and pizz-aat were identified by d-page, respectively. densitometric analysis demonstrated higher protein expression of pimm-aat compared to pizz-aat. the pi range of pimm-aat was . - . and the range for pizz-aat was . - . . the pizz-aat demonstrated a . - . pi cathodal shift of all bands, supporting the hypothesis that the pizz-aat protein is differentially n-glycosylated. this study confirms the presence of multiple isoforms of pimm-aat and demonstrates at least different isoforms of pizz-aat. this requires further investigation to establish differences in n-glycan groups and possible functional consequences. understand the therapeutic potential of alpha- antitrypsin alpha- antitrypsin (aat) is a glycosylated protease inhibitor found in human plasma. aat deficiency predisposes individuals to early onset emphysema and treatment currently consists of weekly intravenous infusions of purified plasma aat. although aat has previously been shown to exert anti-inflammatory properties by binding interleukin- and apolipoprotein b- , the latter being implicating in atherogenesis, hypothesized that aat may have multiple binding partners and that these complexes are involved in additional regulatory and anti-inflammatory pathways. the aim of this study is to identify all proteins that interact with aat as it circulates throughout the body in order to fully understand its therapeutic potential. to examine aat's interaction with potential linker proteins, permeation chromatography of plasma through su-perose / gl was performed. protein profiles were visualized by coommassie blue staining of sds-page gels and western blotting. immuno-bands were quantified by densitometry. aat eluted with molecular masses of approximately and kda indicating multiple binding partners, with the remainder eluting at the predicted molecular mass of kda. protein identification by mass spectrometry (lc ms/ms) is required to identity novel binding partners. identification of all proteins that bind to aat will progress our understanding of the molecular mechanisms by which aat regulates inflammation. ultimately identification of new functions of aat may be utilised to develop novel treatment options for chronic inflammatory diseases including cystic fibrosis, copd and severe asthma. no potential conflict of interest is reported. transthoracic echocardiography, neurocognitive/psychological assessments were also performed. on follow-up, all patients had normal renal function. two of seven had reduced respiratory and cardiac functions. had slightly reduced neurocognitive functions and had decreased feeling of well-being, and from depression. in this small follow up study of a cohort of severe h n patients, there was good recovery. given the initial severity of their respiratory decline i.e., requiring ventilation, this group did not seem to suffer severe chronic respiratory functional limitation. cryptogenic organising pneumonina (cop) is a disease of unknown cause, which can occur in the context of connective tissue disease. a more aggressive variant termed rapidly-progressive cop follows a fulminant course, leading to respiratory failure, and has high mortality [ ] . a -year-old gentleman with a history of dermatomyositis presented with dry cough and dyspnoea on exertion of -months duration. computed tomography (ct) of the chest revealed subpleural patchy ground glass opacities involving both lungs. bronchoalveolar lavage (bal) showed benign bronchial epithelial cells with % macrophages, % neutrophils, % lymphocytes and % eosinophils. staphylococcus and hemophilus were isolated on culture. broad-spectrum antibiotics and steroids were commenced. there was evident improvement clinically, and on pulmonary function tests, however, no significant change on ct chest neccesitated vats biopsy.histopathologic findings were consistent with cop. h after vats biopsy patient developed acute onset dyspnoea with respiratory failure requiring mechanical ventilation and intensivecare-unit admission. ct chest (see fig. ) showed diffuse worsening of ground glass appearance and left lung consolidation. despite best efforts to rescuscitate the patient he passed away h later. this represent a fatal case of rapidly progressive cop resistant to steroids. rapid deterioration resulted in poor prognosis with no lea way of trial of immunosupressive therapy. over a week period thirty seven patients were admitted by nchds to cork university hospital with clinical diagnosis of community acquired pneumonia. an audit was performed on the choice of antibiotic by nchd in comparison to the local antimicrobial guidelines based on a patients curb- score. this audit also compared the admitting nchd's interpretation of a chest x-ray and this was compared to the formal report given by the radiology department. the audit highlighted that antimicrobial prescribing in adherence with local guidelines weakened with an increasing curb- score. in total . % of patients were prescribed antibiotics according to the local guidelines. this fell to . % for a curb- score of / . approximately % of nchds incorrectly diagnosed an infiltrate on chest x-ray which was later refuted by the formal radiology report. antibiotic resistance is a growing global concern and adherence to selected local and regional guidelines for prescription of antibiotics is paramount in reducing the spread of resistance pathogenic bacteria [ ] . reference: rapid administration of guidelines-compliant empiric antibiotic therapy in emergency department (ed) can reduce mortality in patients admitted with community-acquired pneumonia (cap) [ ] . a -week prospective audit was conducted in january to assess adherence to local guidelines and use of curb- score in treatment of such patients. all patients admitted via ed during acute medical take with symptoms and signs of chest infection and new localising radiological shadowing were included. there were relevant admissions ( males, age - years, mean . ). curb- was calculated in ( . %) patients. seven patients ( . %) received antibiotics according to guidelines. in patients without curb- estimation adherence to guidelines was . % vs . % in those with curb- . the results of the audit were presented at hospital grand rounds and new copies of guidelines were circulated to each ward and ed. a laminated copy was placed in a prominent position in ed. a week prospective re-audit was conducted in april ( admissions, males, age - , mean . years) and demonstrated an increase in appropriate antimicrobial prescribing to . % vs . % in the original audit. appropriate antibiotics prescribing was again higher when curb- was calculated. medicine is an evolving field with increased pressure to produce an optimally functioning health care system within budget constraints. there is a current vogue of specialisation, with medical practitioners moving further from general medicine, to condition specific delivery of services. our key concern was whether the case mix breakdown warranted further stratification of the general respiratory outpatient clinic based on conditions. data of patients attending a respiratory clinic was collected for year. the primary diagnosis was coded using international classification of diseases ( th revision) coding scheme and analysed using a statistical analysis package (spss). asthma (n = , . %), copd (n = , . %) and sarcoidosis (n = , . %) accounted for . % of patients and the remainder of the top fifteen conditions were all respiratory in nature. . % (n = ) of attendances were for non-respiratory diseases as the clinic also provides follow-up for general medical patients post hospital admission. the gender mix was male (n = ) . %; female (n = ) . %. the mean age was . years (sd = . ). the difference in mean age for asthma ( . sd . ) and copd ( . sd . ) was significant at . (p = . ). the above analysis reveals a strong case for the creation of three specialist outpatient clinics, for asthma, copd and sarcoidosis. the single breath method to measure diffusion capacity requires a subject to inspire a gas mixture followed by a s (s) breath hold. we sought to determine if breath hold time reduction had a significant effect on measured lung diffusion for carbon monoxide (dlco) values. forced spirometry and co diffusion by the single breath method (dlcosb) were performed in duplicate with breath hold for s, s and s in controls (fev ± . % predicted), severe copd patients (fev . ± . % predicted), and patients with interstitial lung disease (ild) (fev . ± . % predicted). there was no significant difference between dlcosb and dlco(va) measured at , and s in the control (p = . ) and interstitial lung disease (ild) groups (p = . ). however, there was a significant difference between dlcosb (p = . ) and dlco(va) (p = . ) measured at , and s in the chronic obstructive pulmonary disease (copd) group. in the presence of severe airway obstruction the dlco decreases with breath hold time reduction. however, in healthy controls and patients with ild, there was no significant change in the dlco when breath hold time is reduced from to s. this could allow for a reduction in breath hold time when measuring the dlco in patients with advanced ild who are unable to breath hold for s. current guidelines recommend mechanical insufflation-exsufflation (mi-e) for airway clearance [ ] . the aim of this study was to determine the current use of mi-e in neurological conditions by physiotherapists in the uk. a questionnaire was sent to relevant members of the exercise has been shown to improve quality of life in respiratory patients. through exercise, pulmonary rehabilitation operated on the concept of encouraging people with chronic obstructive pulmonary disease (copd) to improve their exercise capacity and subsequently reducing the incidence of copd exacerbation and admissions to hospital. methods: this cross sectional study was conducted between january and february at the mid-western regional hospital, limerick, ireland. all patients attending our respiratory clinic over a period of weeks were invited to complete a questionnaire on arrival at the clinic. findings: the total number of participants was seventy-eight. asthma was the most frequently listed respiratory illness (n = ).fifty-two patients said they exercised ( . % of the study population). higher levels of exercise participation were seen in the younger age groups (p = . introduction: respiratory diseases, largely represented by copd, are the third most common cause of acute hospital admission.our aim was to audit the prescribing habits of inhaled, nebulised medication and oxygen by doctors in a general hospital. methods: all adult patients admitted medically with chronic respiratory diseases that were on inhaled or nebulised medication were included prospectively (jan to june ) in this study. a proforma was used to collect data from the patients. results: there were patients ( % male). the mean age was . patients ( %) had a diagnosis of copd and the others had asthma and pulmonary fibrosis. seven patients ( %) were currently smoking. four patients ( %) were on long term oxygen treatment. in only % of patients the correct dose of inhaler was prescribed. patients ( %) had the correct inhaler device charted. none of the patients had their inhaler technique checked on admission. in only % the dose of nebulised medication was charted. patients were given oxygen of which only was prescribed ( %). conclusion: this audit proves that our prescribing habits of inhaled, nebulised drugs and oxygen are not good. we intend to present this data to our colleagues and reaudit again. adherence to inhaled medications is difficult to assess. a prospective, observational study on patient inhaler usage while in hospital was carried out. the hypothesis was as inhalers are left at the bedside and not administered directly, doses are being missed. a device was designed that makes an acoustic record each time an inhaler was used. the drug prescription sheets on medical wards were screened to identify patients who were prescribed fluticasone/salmeterol via diskus. patients were then approached and asked to participate. the devices were analysed by two independent investigators. doses were classified as early if \ h and late if [ h were between doses. errors were classified as not priming the device correctly, blowing into the device, insufficient inhalation, inadequate breath hold. among patients, taking doses, ( %) doses were taken too early, while separately ( %) doses were missed. in addition, patients blew into the inhaler ( %) times and inadequately inhaled or failed to hold breath ( %). overall, ( %) had an error either in timing or technique. none of these irregularities were documented in the drug prescription sheet. in conclusion, administration of inhalers should be directly supervised by staff, documented and an action plan for patients that are unable to use inhalers be drawn up. results: patients participated in the audit. % knew the names of their respiratory medication, and % knew the general indication for their respiratory medication. % understood the specific indication for their preventer inhaler, % for their reliever inhaler, and % for the combination inhaler. % of patients knew the correct frequency of use of their inhaler, with % of patients demonstrating adequate inhaler technique. % of participants had previously being reviewed and educated by a respiratory nurse. conclusion: a significant percentage of respiratory patients lack adequate knowledge of their respiratory medication; this is despite a majority having previously been educated on this medication. recommendations: ongoing education and regular assessment of respiratory patients needs to occur. ir j med sci ( ) hcws are a cohort at risk of mycobacterium tb infection. qft-g, a lab based assay, is free of the bias and errors of tst placement or reading with the need for - h follow-up for interpretation eliminated. . % of our cohort had a false positive tst, which calls the sensitivity of this test into question. qft-g has been proven to approach % sensitivity [ ] and % specificity [ ] and is therefore a suitable replacement for tst in hcw screening. while there has been an increased recognition of non-tuberculous mycobacteria (ntm) as a clinical problem, much of this experience has come from specialised population such as cystic fibrosis patients. we evaluated our experience in a general respiratory service. positive non tuberculous mycobacterial culture results in st. vincent's university hospital from january to july were reviewed. patients with a known diagnosis of cystic fibrosis were excluded. fifty-six patients were identified with positive cultures for nontuberculous mycobacteria. thirty-eight isolates were from the respiratory tract, of which fourteen samples were sputum samples, thirteen samples were from both bronchial lavage and sputum and nine samples were bronchial lavage. the medical records of thirteen patients (seven female, six male) with probable disease were reviewed. the most frequent isolated ntm in our institution are mycobacterium avium (n = ). two patients had mycobacterium szulgai. mean age was . ± . years; all except one patient had underlying respiratory disease. five of the thirteen patients received some treatment for ntm but only three completed a full course due to intolerability to the medications and also side-effects. our studies confirm that ntm primarily affects patients with chronic lung disease and that the treatment for this disorder is poorly tolerated. lymphopenia in active mycobacterium tuberculosis (mtb) infection is a common and well-documented finding. rather than being an epiphenomenon, this effect likely contributes to pathogen persistence in the host and the lack of a meaningful response during chronic mtb infection. our study was designed to determine the baseline and post-treatment values of total lymphocyte count and its subsets in hiv-negative patients diagnosed with active pulmonary mtb. we prospectively recruited hiv-negative patients diagnosed with pulmonary mtb infection over a -month period. pre and post treatment analysis of total lymphocyte count and its subsets were performed at baseline and after months of tb chemotherapy. a control group comprising of patients with community acquired pneumonia also had pre-treatment lymphocyte counts performed. ten patients with active mtb infection and seven comparable controls were recruited over a -month period. baseline total lymphocyte count was lower in the study group ( . ± . ) compared to control ( . ± . ). treatment was associated with significant improvements in total lymphocyte, b-cells, cd , cd (p \ . ) and nk cell (p \ . ) counts. recovery of total lymphocyte count in the control group was not significant (p = . ). our study demonstrates treatment of active mtb in hiv-negative patients is associated with significant improvements in total lymphocyte count and its major subsets. pleural tuberculosis is a diagnostic challenge. ada is a biomarker that has been proposed to diagnose tuberculous pleurisy but not routinely used [ ] . we aim to evaluate the sensitivity and specificity of ada in the diagnosis of tuberculous effusions and to improve current practice. we prospectively examined ada levels from patients with pleural effusions and followed the clinical course to establish the final diagnosis via culture, histology and clinical diagnosis. data were analysed using mann-whitney u test. there were cases of tuberculous effusions with mean ada levels of ± . iu/l (ci . - iu/l) while the mean ada of non-tuberculous effusions were . ± iu/l (ci . - . iu/l, p value = . *). true positive rate was / and true negative rate was / . false positive rate was / while false negative rate / . if iu/l is taken as cut value, the specificity is % and the sensitivity is %. we describe four cases of tb occurring during pregnancy and post partum. in all cases the women were non-nationals with a mean age of years. in two cases the tb presented as vertebral osteomyelitis, in one case tb lymphadenitis and in one case as miliary pulmonary tb. the diagnosis and treatment of tb in pregnancy presents many challenges. the cases discussed highlight some of the complexities which we encountered. these included dealing with language and cultural barriers, multi-disciplinary management of tb osteomyelitis in a pregnant woman; involving close collaboration with the obstetric and orthopaedic teams and managing adverse effects of anti-tuberculous medications in the pregnant patient. most samples tested showed no evidence of mycobacteria, indicating need for improved case selection prior to testing. median time to zn testing is as recommended by guidelines, though there is room for improvement. in an unselected population, likelihood of negative results is high after weeks of negative cultures. however, clinical suspicion should remain high until culture negativity is declared at weeks, particularly when there is a high clinical probability of mycobacterial disease. background: a number of patients with bronchiectasis have a middle lobe/lingula predominant radiological pattern. other than an association with non-tuberculous mycobacteria there is a paucity of published data on this subgroup of patients. methods: we retrospectively analysed data from all patients with non-cf bronchiectasis who underwent bronchoscopy with bal in a university hospital over a month period. radiological features, demographic data and microbiology were reviewed. results: patients with bronchiectasis were assessed. ( %) had predominant middle lobe/lingular bronchiectasis. in this group ( %) patients were females compared to ( %) (p = . ) of other patients and mean age was ± compared to ± years (p = . ). bal microbiology in the middle lobe/lingular group revealed no growth in ( %), h influenzae in ( %), s aureus in ( %), mac in ( %) and other ( %). conclusion: middle lobe/lingula predominant bronchiectasis is a common radiological pattern particularly in females and only a minority have ntm infection. cystic fibrosis (cf) is characterised by neutrophil-dominated airway inflammation, in part attributable to the potent chemotactic agent leukotriene b (ltb ). the aim of this study was to investigate the ability of exogenous alpha- antitrypsin (aat) to inhibit ltb signaling. the biological consequence of the described aat induced inhibition was investigated at the level of neutrophil released proteolytic enzymes. circulating neutrophils isolated from healthy control volunteers (n = ) were stimulated with ltb ( - nm/ ) in the presence and absence of aat ( . - . lm) for increasing increments of time ( , , and min). the level of degranulated proteins in surrounding supernatants was determined by western blot analysis. the ability of aat to bind ltb was assessed specrophometrically with uv spectra recorded on a jenway spectrophotometer at °c. our in vitro data has shown that levels of degranulated mpo, ll- and mmp- (markers of primary, secondary and tertiary granule release, respectively) were significantly decreased in the presence of aat (p \ . ). the mechanism of inhibition involved direct binding of aat to ltb as reduced vibrational fine structure of the ltb / aat uv absorbance spectrum indicated complexation of the two molecules. the results of this study indicate that aat can inhibit ltb signaling thereby reducing the proteolytic activity of neutrophils and propose aat aerosolized augmentation therapy as an effective treatment for ltb associated pulmonary diseases including cystic fibrosis and severe asthma. the modified-shuttle-walk-test (mswt) is increasingly used in cystic fibrosis (cf) patients. however, few studies have correlated mswt with severity of disease or assessed the prognostic value of these tests. the aim of this study was to see if a correlation existed between mswt and forced expiratory volume in -s (fev ) and/or cf-able score. a total of mswt assessments were analysed. correlations (spearman) among fev , cf-able-score, percentage predicted distance travelled and percentage predicted distance travelled to desaturation were calculated. nine out of mswt showed desaturation. the mean distance travelled was , m; . % of predicted, and mean distance to desaturation was . m; . % predicted. there was a significant correlation between distance travelled and fev (r = . /p \ . ) ir j med sci ( ) (suppl ):s -s and inverse correlation with cf-able-score (r = - . /p \ . ). there was a significant but poor correlation between distance to desaturation with fev (r = . /p \ . ) and cf-able-score (r = - . /p = . ). however, the presence of desaturation during testing did not correlate with fev (r = - . /p = . ) or correlate significantly with cf-able-score (r = . /p = . ). in conclusion there is significant correlation between total distance walked and both fev and cf-able-score, however, the absence of a correlation with the presence of desaturation during testing highlights the usefulness of mswt as a possible independent predictive measure, with further study needed. prolonged antibiotic therapy for cystic fibrosis (cf) exacerbations leads to increased picc (peripheral inserted central catheter) use. consequently incidence of venous thromboembolism (vte) has risen. rates of picc induced thrombosis in adults are . % [ ] . we aimed to ascertain prevalence of picc induced thrombosis in adult cf patients. a retrospective review of radiology was conducted on patients who had picc insertion for antibiotics for cf exacerbations from january to december . we analyzed patients with confirmed vte on doppler ultrasound and recorded patient demographics, size of picc and site of insertion. piccs were inserted, ( . %) had vte. ( . %) were female and ( . %) were male with symptomatic vte, presenting with arm swelling and pain. further complications were ( . %) with superior vena-cava syndrome, ( . %) with pulmonary embolism. of these, were treated with months anticoagulation therapy, and were anticoagulated for months once repeat doppler ultrasound confirmed no thrombosis. our rate of vte was . %, lower than in existing studies. piccinduced thrombosis depends on the population studied, as well as acquired thrombophilia secondary to inflammation, or deficiencies of anticoagulant proteins (protein c and s) due to vitamin k deficiency and/or liver dysfunction. studies have documented very poor real-life adherence to nebulised antibiotic therapies [ ] . no data exists on real-life experience with inhaled antibiotics. consecutive adult cf patients commencing inhaled antibiotic therapy (tip) were recruited over a -month period. a questionnaire recording safety, efficacy, lung function and adherence at time of recruitment, assessed traditional nebulised treatment (tis) versus new inhaled therapy (tip) at , and months. wilcoxons rank test and paired sample t-tests were employed for statistical analysis. patients have been enrolled to date. patient died (unrelated to the drug). patient received a lung transplant. / ( %) discontinued tip; due to cough/bronchospasm and due to refractory oral candidiasis. / ( %) were intolerant of tis prior to enrolment, with / ( %) subsequently tolerating tip. there was a significant increase in mean adherence score from . in the tis group to . in the tip group (p value . ). there was no significant difference in cough, lung function, or adverse events between the groups. in a real-life clinical setting with new inhaled antibiotic therapy (tip) we demonstrate, improved tolerability, adherence, lower discontinuation rates and stable clinical phenotype. also subgroup analysis supports a trial of this in those who failed traditional nebulised treatment. aim to assess the clinical utility of a hand-held nno analyser to differentiate between pcd, respiratory disease and healthy subjects. clinically stable patients were recruited over a -month period. each subject completed compatible pcd phenotype proforma, nno analysis (niox mino Ò ), and one nasal brushing for electron microscopy (em) analysis. nno was measured using passive sampling at a flow rate of ml/s during tidal breathing. em images will be reviewed internally and externally at an international centre of excellence (unc chapel hill). independent t-tests were used to compare mean nno values between groups. subjects were recruited (n = pcd, n = cf, n = non-cf/ non-pcd bronchiectasis, n = copd, n = healthy subjects). mean nno levels (ppb ± sd) were ± . (pcd), ± (cf), ± (non-cf/non-pcd bronchiectasis), ± (copd) and ± (healthy control). although nno levels were reduced in pcd when compared to copd (p = . ) and healthy subjects (p \ . ), there was no statistically significant difference between nno levels in pcd and cf (p value . ). results of em analysis are pending. in this study, the hand-held niox mino Ò nno analyser distinguished patients with pcd and cf from patients with copd and healthy subjects but not cf from pcd. ir j med sci ( ) studies suggest that incorrect usage of inhalers impacts negatively on asthma control. the aim of this study was to evaluate inhaler technique and symptom control in patients with severe asthma. patients referred to a newly established clinic in cork university hospital were consecutively recruited over a month period. inhaler technique was assessed using a validated scoring system and instruction on correct usage given if scores were suboptimal. patients completed a validated asthma control questionnaire (acq) and asthma quality of life questionnaire (aqlq). at a follow-up clinic months later technique was reassessed and acq repeated. results at baseline and follow-up were compared using standard statistical methods. patients were recruited (female = %), and / were followed up. mean[sd] fev % predicted at baseline = . % ( . ). % of patients were classified as incorrect inhaler users initially, decreasing to % at follow up, indicating a significant improvement in inhaler usage post-training (p = . ). acq scores improved significantly from median (range) . ( . - . ) to . ( - . ), p = . . the aqlq results indicated that patients' qol is moderately affected by asthma; median (range) score of . ( . - . ). this study demonstrates the importance of formally assessing inhaler technique in patients with severe, long-standing asthma as part of their clinical review. asthma is a chronic airway disease characterized by airway inflammation, bronchial hyperresponsiveness and airflow obstruction. patients with persistent symptoms despite maximum treatment as per gina guidelines are considered to have severe persistent asthma. omalizumab is a recombinant humanized monoclonal antibody licensed as an add-on therapy in these patients. the aim of this study is to assess the clinical benefit amongst responders to omalizumab therapy at a tertiary referral centre. this was a retrospective audit assessing the effects on asthma control, frequency of exacerbation and hospitalisation rates over months before and after therapy. the study included responders ( females). there was a reduction in exacerbation and hospitalization rates following initiation of omalizumab, and %, respectively (p value . ). the number of exacerbations decreased from . ± . to . ± . and the mean number of admissions from . ± . to . ± . over the study duration (p \ . ). there was % reduction in the weekly need for rescue salbutamol with mean of . ± . puffs to . ± . puffs after omalizumab (p \ . ). seventy-nine percent of patients were able to reduce their maintenance oral corticosteroid. in summary, responders to omalizumab therapy are less likely to experience an asthma exacerbation and hospitalisation. they were also more likely to reduce maintenance corticosteroid therapy as well as the need for rescue reliever therapy. these data suggest that omalizumab has proven effective in improving health outcomes for a cohort of carefully selected patients with severe allergic asthma in ireland. bronchial thermoplasty (bt) is a bronchoscopic procedure aimed at reducing the mass of airway smooth muscle and attenuating bronchoconstriction in severe asthmatic patients failing medical therapy. we report our experience with the first four patients treated with bt. between december and august , four patients with severe asthma per gina guidelines, underwent three sessions of bronchial thermoplasty, weeks apart. stringent entry criteria were required, including ongoing symptoms despite optimal medical management with the use of ics and laba's. two patients had a limited response to omalizumab. thus far, four patients met study entry criteria. three females and male. the mean age was years (sd . ). the frequency of severe asthma exacerbations was - per year. the mean fev and fev /fvc prior to procedure was . and . %, respectively. the mean fev and fev /fvc after the procedure was . and . %, respectively. patients reported a subjective strong improvement in quality of life post bt with more symptom-free days and less use of rescue inhalers. this emerging data relating to bt in an irish population is consistent with international data sets. bronchial thermoplasty is an additional treatment option for patients with severe asthma. approximately - % of patients with asthma have gastrooesophageal reflux (gor) and it has been postulated that this may worsen asthma severity. this study was undertaken to examine the incidence of gor in an irish steroid-dependent severe asthma cohort. patients with severe asthma were recruited into this descriptive study from the severe asthma clinic in cork university hospital. our cohorts mean age was years. the mean (sd) fev was . ( . ) l ( % predicted). the mean time from asthma diagnosis was . ( . ) years with the patients being steroid dependent on oral steroid therapy for mean . ( . ) years with a mean dose of . mg prednisolone. ( %) reported symptoms of gor; with being concomitantly treated with proton pump inhibitor. ten patients had undergone a barium swallow with five demonstrating gor radiological evidence. a further patient previously had undergone fundoplicative surgery. there was no association between gor and cumulative systemic steroid dose or fev in a subgroup analysis. in our study of steroid dependent asthmatics, % of those formally assessed were found to have evidence of gor on barium swallow, which is consistent with reported research [ ] . the incidence of gor did not depend on cumulative steroid exposure or fev . introduction: incorrect inhaler usage is a significant problem in asthma management, resulting in poor control of asthma symptoms. the ability of patients to correctly use their inhaler might be directly linked to inhaler technique education. education may result in better inhalation technique, improved compliance and asthma control. the economic burden of asthma is very substantial and is one of the highest among chronic diseases. in the united states of america, approximately - billion dollars is wasted because of inhaler misuse per year (fink, ) . research question: ''what is the impact of a nurse-led education programme in promoting compliance with inhaler use in patients with asthma''. methodology: this is a quantitative study engaging a quasi-experimental pre-test and post-test design. a cohort of patients who met the inclusion criteria were recruited from the out-patient department over a period of six months. during each stage, the patient was asked to demonstrate how they take their inhaler. any errors in technique were identified and rectified. their demonstration was measured through observation and the use of an inhaler proficiency schedule (ips). the participant was also asked a series of specific questions in relation to their condition, confidence level with self-administration of their inhaler, and adherence to prescribed frequency of use. the findings in this study show that inhaler education improves technique, promotes compliance and increases participant confidence levels in taking an inhaler, and as a result asthma symptoms improve. it also emerged that participants believed they were taking their inhaler correctly and so assumed that education drives were not targeted at them. % ( ) felt that the service provider had assisted in their success with therapy. overall, respondents use their devices as prescribed, keep in contact with their hospital department and service provider, and most feel their quality of life has improved as a result of starting treatment with cpap. obstructive sleep apnoea syndrome (osas) is characterized by repetitive upper airway (ua) obstruction during sleep. alcohol consumption increases osas severity by diminishing ua muscle tone, aggravating snoring and osas-related daytime symptoms. we hypothesized that behavioural adaptation could lead to reduced alcohol consumption in subjects with more severe osas. the influence of anthropometric, social and demographic variables, along with osas severity on alcohol consumption among subjects undergoing inpatient sleep studies was examined. regression analyses were utilised to identify independent predictors of alcohol consumption, and generate odds ratios (aor) for excessive alcohol consumption by osas severity. subjects were assessed; . % were female, . % in paid employment, and . % married. . % had no osas [apnoeahypopnoea index (ahi) \ ] and . % severe osas (ahi [ ). alcohol consumption was . (± . ) u/week, with . % exceeding recommended limits. stepwise regression revealed male gender and employment status, but not ahi, as independent predictors of increased alcohol use. no difference in adjusted mean alcohol intake by osas severity class was observed. severe osas patients tended towards increased odds of excess alcohol consumption compared to those without (aor . ; % ci . - . ; p = . ). increasing osas severity is not associated with lower alcohol consumption; rather, the reverse may be more likely. in our sleep clinic, ess is a poor predictor of ahi, sacs did not perform as expected, and a combined measure is of limited utility. following polysomnography diagnosis, untreated osas cases were assessed. cpap compliant subjects were re-assessed * weeks later. body composition was assessed by bio-electrical impedence analysis. sensewear armband Ò (swa) measured free-living ee. swa data was included if average weartime was [ %. subjects ( male) (mean age, . years) were included. restrained-eating score was inversely associated with osas severity ( . , . , . in mild, moderate and severe, respectively). conversely, both uncontrolled eating score ( . , . , . ) and emotional eating score ( . , . , . ) were positively associated with osas severity. bmi (p = . ) and fat % (p = . ) were significantly higher in severe versus mild osas. cognitive-restraint was inversely associated with bmi and fat %, whereas both uncontrolledand emotional-eating were positively associated with these parameters. among this sample, more severe disease was associated with adverse eating behaviors. nutritional counseling targeting specific eating behaviors may be beneficial in osas. significant osa (ahi [ ) was present in % of the clinical group and % of the screened group. no correlation of clinical significance was proven between ahi and ess in either group. ess provides useful information on subjective sleepiness but this study might suggest that it is not a reliable predictor of the presence of osa or its severity. decreased energy expenditure (ee) contributes to overweight. we investigated free-living ee and body composition in obstructive sleep apnoea syndrome (osas). following polysomnography (psg) diagnosis, untreated osas cases wore the sensewear armband Ò (swa) for * days, including weekend days. swa quantifies free-living ee and physical activity (pa). data was included if weartime was [ %. body composition was assessed with bio-electrical impedance analysis. upper airway muscle dysfunction is implicated in the pathophysiology of obstructive sleep apnoea syndrome (osas). pharyngeal dilator muscle inotropes may serve as adjunct therapies. we hypothesized that tempol, a superoxide scavenger, would increase sternohyoid muscle power under conditions of oxidative stress (hypoxia). excised sternohyoid muscles from adult male wistar rats, were connected to a dual-mode force transducer, between stimulating electrodes, in a bath of krebs solution at °c, in either high oxygen (control) or low oxygen (hypoxia) ± mm tempol. stress and shortening were measured in muscles contracting from zero up to isometric load under tetanic conditions. peak power was determined. sternohyoid peak power was . ± . and . ± . w/cm in control and hypoxic conditions (drug-free), respectively, and . ± . and . ± . w/cm in control and hypoxic conditions (+tempol), respectively. two-way anova revealed that hypoxia (p \ . ) and tempol (p = . ) were significant factors without drug-gas interaction. tempol increased the power-load relationship over the early ( - %) portion of the load step test and this was significant under hypoxic conditions. we conclude that tempol increases sternohyoid muscle power under control and hypoxic conditions. our results suggest that antioxidant therapy may be useful in the treatment of osas and other muscle weakness disorders. r. lee obstructive sleep apnoea syndrome (osas) is reported as common among ipf patients [ ] . we determined the prevalence of the disorder in a cohort of ipf patients not on long term oxygen therapy and medically stable, excluding patients with active coronary disease and diabetes mellitus. patients with ipf patients attending a specialized clinic underwent overnight polysomnography following a night of acclimatization. a quality of life questionnaire (sf- ) and epworth sleepiness score (ess) were also completed. statistical analysis was by student-t and man-whitney u-testing. of the patients were male and mean age was . ± . (sd). % were current or ex-smoker. only % of the patients received steroids at some time in their treatment. patients had significant sleep-disordered breathing (sdb) based on the standard definition of ahi c /h but only were sleepy (ess c ), thus having osas. bmi correlated positively with ahi (r = . , p = . ). bmi was . ± . kg/m but higher in the osas/sdb group (p = . ). no difference in quality of life was evident between those with or without sdb or osas. we conclude that sdb and osas are as prevalent in ipf as a similar general population and bmi is the principal predictor of ahi in these patients. studies to date reveal wide variability ( - %) in the prevalence of fungus in the cf airway using culture-based methods. this study profiles the fungal microbiota of the cf airway using high-throughput-sequencing, and correlates this with standard culture-based methods and clinical phenotype. clinically stable adult cf patients were prospectively recruited, donating one or more sputum samples. culture-based methods were employed at time of sampling. high-throughput bar-coded sequencing targeting the internal transcribed spacer (its) and small sub-unit (ssu) regions was used to profile the fungal microbiota, with subsequent sequencing on a genome sequencer flx platform. baseline fev % predicted, genotype, gender, bmi and pseudomonas status, were recorded by retrospective review of medical notes. in a total of samples, culture-based methods detected fungus (aspergillus spp. and candida spp. only) in patients. highthroughput-sequencing identified rich fungal communities in greater than % of the patient sputum samples, with over % of the species found not detected by culture. fungi detected included c. albicans, c. dubliniensis saccharomyces cerevisiae, malassezia spp., fuscoporia ferrea, fusarium culmorum, acremonium strictum, thanatephorus cucumeris and cladosporium spp. a comparison of patient status with diversity and species richness of fungal microbiota identified that lower fungal diversity associates with decreased lung function. aim: to identify common anaerobes and their proteases and assess their ability to cleave natural host innate human antiproteases such as alpha one antitrypsin (aat). method: we prospectively recruited patients at our site in beaumont hospital. we obtained both sputum and bronchoalveolar lavage fluid (balf) at both stable and pre and post exacerbation timepoints. all samples were processed using both anaerobic bacteriologic techniques and s r rna sequencing methods. supernatants from p. melaninogenica were cultured in luria-bertani broth (lb) broth, (sigma l - tab) and basal anaerobic media (bam) broth under strict anaerobic conditions in an anaerobic cabinet (davidson & hardy). protease production was measured using sensolyte red protease assay (anaspec). this assay measures matrix metalloproteinase (mmp) activity in broth. the days with highest protease production were recorded. native aat was incubated for selected time points with supernatant and cleavage products visualised by sds-page electrophoresis and western blotting analysis using specific antibodies raised against the antiproteases. results: using sputum and broncheoalvelar lavage from patients with cf, prevotella species accounts for % of anaerobic samples identified from our group and p melaninogenica is the most common anaerobe grown from this group. the sensolyte red protease assay showed p. melaninogenica cultures produced the highest levels of active proteases on day , and . the western blot analysis demonstrated that when day and supernatants were incubated with aat, this antiprotease was degraded to give a distinct cleavage pattern. conclusion: this study is examining for the first time the pathogenicity of anaerobic bacteria found in cf lung and shows that the proteases produced by anaerobic bacteria are destroying host defense mechanisms and that this may impact other natural host innate antiproteases in the cf lung and play a role in inflammation. cf is a genetic disease with a high prevalence in ireland. in cf lungs chronic bacterial infection contributes to progressive respiratory failure. in particular, pseudomonas aeruginosa (pa), forms biofilms in the lungs which significantly contributes to antibiotic resistance. we have previously published on the importance of mif as a key inflammatory mediator in cf [ , ] . building on this work, we hypothesised that mif enhances biofilm formation in the cf lung contributing to enhanced antibiotic resistance. using in vitro biofilm formation methods and qpcr we examined the effects of mif ( ng/ml) on the growth, antibiotic resistance and gene expression of pa (strain pao ). our results to date have shown that mif significantly enhances biofilm formation of the pao strain of pa (p \ . ). in addition we have found a significant earlier induction of specific quorum sensing genes in response to mif. mif in pa cultures is associated with significantly less bacterial killing following antibiotic treatment. this raises the possibility of mif as an adjunct therapy with antibiotics by significantly this supports our hypothesis of mif inhibitors as an adjunct therapy improving the antibacterial effectiveness of antibiotics. macrophage migration inhibitory factor (mif) was one of the first cytokines to be discovered. mif is produced by a wide variety of tumours and is thought to play an important role in tumour progression. mif possesses a unique enzymatic activity linked to this role in cancer. to investigate this further we designed and evaluated a panel of small molecular weight inhibitors of mif and looked at their ability to block mif activity in vitro and in vivo. the small molecules were found to specifically inhibit the enzyme activity of mif when co-incubated with recombinant mif and its substrate. the inhibitors also significantly reduced cellular proliferation induced by treatment with recombinant mif (proliferation reduced by [ %, p \ . ) and significantly inhibited lpsinduced tnf-a production (tnf-a reduced by [ %, p \ . ). in vivo, the inhibitors were found to reduce tumour growth in a subcutaneous model of lewis cell carcinoma (tumour volume reduced by [ %, p \ . ). here we present data describing a number of novel small molecular weight inhibitors of mif found to be effective in vitro and in vivo. these inhibitors have the potential to be developed for therapeutic use in a cancer setting. idiopathic pulmonary fibrosis (ipf) is a progressive disease characterized by fibrosis. il- is a proinflammatory cytokine that has been shown to play a role in many fibrotic diseases including ipf. il- also induces the expression of, and binds to, one of its receptors, il- ra , which has been thought to function as a non-signaling decoy receptor. the cxc chemokine receptor (cxcr ) and its ligands-cxcl , cxcl , and cxcl -have been implicated in vascular remodeling and fibroblast motility during the development of the disease. in this study, cultured pulmonary fibroblasts from wild type and cxcr -deficient mice were treated with various cytokines, and the expression levels of il- ra and cxcr were measured. we demonstrate for the first time the expression of cxcr in cultured pulmonary fibroblasts from mice. also, il- was shown to downregulate basal and ligand-induced cxcr expression in fibroblasts. using wild-type and cxcr -deficient animals, cxcr was found to be necessary for the il- mediated upregulation of il- ra , and blocking cxcr significantly reduced the basal expression of il- ra . manipulation of the cxcr -mediated regulation of il- ra or the il- mediated downregulation of cxcr may represent novel therapeutic modalities in cases of acute lung injury or chronic inflammation that may progress to fibrosis. epithelial cell to mesenchymal transition (emt), whereby epithelial cells undergo transition to a mesenchymal phenotype, giving rise to fibroblasts and myofibroblasts has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (ipf). alveolar epithelial cells (aec) are recognised to undergo emt in response to various stimuli including transforming growth factor-b (tgf-b ). comparison of gene expression, migration and chemokine secretion in normal and transitioned aec with primary pulmonary fibroblasts derived from normal and ipf patients was performed. a cells underwent h (h) serum starvation followed by h treatment with tgf-b ng/ml. total rna was extracted from a cells and primary human pulmonary normal and ipf fibroblasts. changes in expression of a panel of tgf-b target genes was determined by real time polymerase chain reaction array with subsequent validation. migration studies of the various cell types in response to serum and enzyme-linked immunosorbent assay (elisa) of cxcl , cxcl and il- levels in cell supernatants were performed. transitioned aec assumed a mesenchymal phenotype, exhibiting a marked reduction in differential gene expression when compared to fibroblasts. migration in response to serum by transitioned aec was increased significantly compared to normal or ipf fibroblasts as was production of cxcl and cxcl . background: aatd disease is a hereditary disorder leading to the development of emphysema. our group has published first evidence of upr activation within the endoplasmic reticulum (er) of zz monocytes. here we study mirna regulation of upr in healthy and emphysematous zz monocytes. method: monocytes mirnas were profiled using nanostring technologies. mirdip portal and kegg database identified mirna targets and gene networks. transfections with nm anti-mir were performed using siport-neofx. mrna, mirna and protein were measured by qrt-pcr, taqman mirna assay and western blotting. results: sixty mirnas were differentially expressed in zz versus mm monocytes. mir- a- p is overexpressed by [ -fold and predicted to target multiple genes which are enriched for pathways in the er stress response. emphysematous versus healthy zz patients have decreased mir- a- p expression. mir- a- p inhibition increased expression of two arms of the upr; grp and atf . conclusion: mirnas are differentially expressed in zz monocytes and may play a role in the upr. mir- a- p, predicted to target upr genes, is overexpressed in healthy zz monocytes, and negatively regulates the upr. emphysematous zz patients may have lost this protective mirna regulation leading to increased er stress in monocytes, contributing to the inflammatory milieu of aatd lung disease. . alpha- antitrypsin augmentation therapy is associated with decreased neutrophil adam- activity, plasma tnf-a levels and normalisation of neutrophil apoptosis alpha- antitrypsin deficiency (aatd) is characterised by neutrophil driven lung destruction and early emphysema in a low alpha- antitrypsin (aat) and high neutrophil elastase (ne) environment in the lungs of affected individuals. timely and effective neutrophil programmed cell death is essential for the resolution of inflammation and we have previously shown that neutrophils apoptosis is accelerated in aatd individuals. endoplasmic reticulum (er) stress is associated with the release of the pro-apoptotic cytokine tnf-a and, on the cell surface the activity of the sheddase adam- leads to the release of tnf-a from its membrane bound to its soluble form. the aim of our study was to determine if aat augmentation therapy can normalise the accelerated neutrophil apoptosis seen in aatd through inhibition of adam- activity and resultant tnf-a release. neutrophils were isolated from aatd individuals receiving aat augmentation therapy pre and post treatment. the kinetics of apoptosis were measured by caspase- cleavage utilising western blotting and cd b expression by facs analysis. adam- activity measured using a fluorogenic peptide substrate. plasma tnf-a levels were measured by elisa. er stress was determined using the er stress marker grp- . adam- activity was increased in individuals with aatd. in addition, adam- activity, plasma tnf-a levels and caspase- cleavage were reduced after augmentation therapy (p \ . ). cd b expression was increased after therapy indicating normalisation of apoptosis. grp- expression was unchanged. from our data we have demonstrated that aat augmentation therapy can normalise neutrophil apoptosis by ameliorating adam- activity and resultant tnf-a release. the observed normalisation of neutrophil apoptosis may lead to reduced inflammation and a reduction in recurrent infections which characterises patients with aatd. to identify lung-selective mirnas, extracted rna was probed to mirna microarrays (mra- ; , human mirnas), and results confirmed by taqman analysis. in silico analysis using targetscan and microrna.org identified genes targeted by identified mirnas. using a subtractive mirna strategy, lung-selective hypoxic responsive mirnas were identified (anova p \ . ); including mir- a- p and mir- . in silico analysis predicted that mir- a- p targets erythropoietin, which has a well-documented role to play in endothelial repair and angiogenesis. furthermore, mir- targets cullin , which has previously been shown to stabilize hypoxia-inducible factor-a and promote angiogenesis. we conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-selective alterations in mirna expression. mir- a- p and mir- may play important roles in pulmonary vascular remodelling and angiogenesis. further studies of these mirnas may uncover novel treatment strategies for hypoxic lung disease. this research project is funded by science foundation ireland. to assess whether mal plays a role in killing of intracellular mtb, we infected murine wild-type and mal knockout macrophages with virulent (h rv) mtb. we found that mal deficient cells were unable to kill mycobacteria. human macrophage cell lines transfected with sirna against mal showed the same deficiency in killing mycobacteria. we then proceeded to evaluate key macrophage mechanisms of killing mycobacteria. we found that phagolysosomal maturation and autophagy were mal-dependent in murine and human macrophages. pro-inflammatory cytokine production was also mal dependent. we then sought to determine the effect of the common mal s l polymorphism on macrophage responses to mtb. primary bone marrow derived macrophages from mice with the sl and ll polymorphisms displayed impaired mycobactericidal activity and phagolysosomal maturation. mal plays a critical role in determining macrophage responses to mtb through a pathway culminating in phagolysosomal maturation and killing of intracellular bacteria. asthma has been linked to the vitamin d deficient (vdd) state. we investigated whether vdd was associated with impaired lung function and inflammation. patients with respiratory symptoms (asthmatic and non-asthmatic) underwent spirometry and had serum analyzed for total immunoglobulin e (ige), high sensitive c-reactive protein (hscrp), eosinophil cationic protein (ecp), and -hydroxyvitamin d we examined caucasians (mean age years; mean bmi kg/m , mean fev = . % predicted). mean (oh)d was . nmol/l. % of recruits were vdd, % were insufficient, while only % were vitamin d sufficient. vitamin d levels were positively associated with fev (r = . , p = . ). % of patients with airway obstruction (fev \ % predicted) were vdd. ecp, hscrp and ige were non-significantly elevated in the vdd state compared to sufficiency. however, all patients with ige doctors' education about inhaled respiratory medication is extremely important in management of copd and asthma. while exercise-induced oxygen desaturation is a widely used clinical measure in ipf, data on sleep-related desaturation are lacking. we compared gas exchange during sleep and exercise in a cohort of ipf patients attending a specialized clinic not on long term oxygen therapy and medically stable, excluding patients with active coronary disease and diabetes mellitus. ipf patients underwent overnight polysomnography, including transcutaneous carbon dioxide (p tc co ) measurement, after a night of acclimatization. cardiopulmonary exercise testing was performed by incremental cycle ergometer. pulmonary function and awake arterial blood gases were also measured. statistical analysis included student-t and man-whitney u-testing. patients had significant sleep-disordered breathing (sdb) based on an apnoea-hypopnoea index (ahi) [ . fev was . ± . % (sd) and diffusion (dlco) . ± . % predicted. pao was . ± . kpa and paco . ± . kpa. p tc co rose by . ± . kpa during sleep (p \ . ) consistent with hypoventilation. the minimum oxygen saturation during sleep was lower than exercise ( ± . vs. . ± %), p = . and the fall in oxygen saturation was also greater during sleep ( . ± . vs. . ± . , p \ . ). we conclude that ipf patients desaturate more during sleep than exercise and suggest that nocturnal oxymetry be considered part of the clinical assessment of such patients. diabetes mellitus (t dm) causes increased risk of cardiovascular death, while glycosylated hemoglobin (hba c) level predicts longterm cardiovascular mortality in non-diabetics. while obstructive sleep apnoea syndrome (osas) is associated with adverse cardiometabolic outcomes, it remains unclear if this effect is independent of obesity and other confounders. we examined the relationship of osas severity with t dm prevalence and hba c levels in a large european population. subjects attending university-affiliated sleep laboratories across countries were prospectively assessed. all underwent overnight sleep studies, with bloods drawn to assess glycaemic health. the relationship of osas severity with t dm prevalence, and hba c levels in non-diabetics was examined with regression models adjusting for confounding factors, including obesity. , subjects were assessed, . % male, . % obese, and % with an apnoea-hypopnoea index (ahi)[ events/h. following adjustment for confounding factors, moderate and severe osas remained significant predictors of t dm (adjusted odds ratio . ; %ci . - . ; p = . ). in non-diabetics ahi (standardized b . ; p \ . ), and mean spo (standardized b - . ; p \ . ) were significant independent predictors of elevated hba c levels. osas severity and nocturnal hypoxaemia predict both prevalent t dm and hba c levels even after rigorous adjustment for confounding variables including obesity, which may contribute to excess mortality in osas populations. background: portable devices that determine tst may act as an adjunct to level diagnostic tests for osa. the swa is such and measures tst using a proprietary algorithm. calculation of tst could improve the accuracy of a level diagnostic device. aim: correlation of tst by swa and npsg, in a population with and without sleep apnoea. consecutive patients undergoing npsg because of a suspicion of osa wore an swa on the same night. patients were stratified by the presence and severity of osa. correlation coefficient for tst were determined between swa and npsg for all subjects and in the osa subgroups. results: the prevalence of a normal psg, mild moderate and severe osa was ( . %), ( . %), ( . %) and ( . %) of subjects. and the respective correlation coefficients were r = . , . , . and . . clinically important differences are presented with bland-altman plots (figs. , ) . correlation of tst between the two methods was weakest in those with severe osa. conclusion: the determination of tst by swa in a population with severe osa is likely to be unreliable. npsg remains the gold standard for determination of tst. the relationship between lung cancer and pulmonary fibrosis remains poorly understood. the aim of this study was to conduct a descriptive analysis of clinical data collected from a cuh cohort of patients with both ild and lung cancer. a database of patients with a histological diagnosis of lung cancer between august and december was reviewed. patients with established ild on ct scan were identified. data from clinical notes and radiology patterns were reviewed and analysed. the male to female ratio was . : . all were smokers. % of carcinomas in these patients were non-small cell lung cancer (nsclc). % of patients had usual interstitial pneumonia pattern, % had non-specific fibrosis, and % had asbestosis. the overall median survival was months (sem . ; % ci . to . ). median survival for patients with early stage disease who underwent surgery (n = ) was months, followed by those who received chemo-radiotherapy ( months), those who received no intervention ( months), and those who received radiotherapy alone ( months). survival for patients with lung cancer and ild was lower than published figures for patients with lung cancer alone. surgical candidates had the best survival though the survival benefit was very modest, while patients who received no intervention or radiotherapy alone faired very poorly. pleural ultrasound has a number of advantages over traditional imaging modalities with regard to visualisation of pleural pathology, in particular, pleural effusions. these include portability, the absence of radiation, dynamic imaging as well as increased sensitivity versus computed tomography scans in terms of differentiating between pleural fluid, thickening and masses [ , ] . we present a case series of patients who underwent pleural ultrasound under the care of respiratory physicians trained in this technique in our hospital from january to august . in total, ultrasound scans were carried out in the month period on a total of patients using a portable ultrasound machine. the average patient age was . years (range - years) and % were male. based on ultrasound findings, the physician proceeded directly to aspiration on occasions ( %) and a total of chest drains were inserted ( %). of those that were aspirated, the vast majority were exudative in nature (n = , . %). ( %) of the aspirates were due to malignant effusions. no procedure-related complications occurred. this case series highlights that imaging at the bedside is a feasible and, with the proper training, very safe method for managing pleural effusions. this audit shows that the vast majority of patients with mesothelioma are male with a poor prognosis regardless of therapeutic approach. approximately % die in the hospital/hospice setting. finally, more patients with mesothelioma should be considered for clinical trials. aat deficiency (aatd) results from mutations in the serpina gene, classically presenting with copd and liver disease. the most common mutation causing aatd is the z mutation, with the s mutation weakly associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. ats/ers guidelines advocate screening all copd, poorly-controlled asthma, and cryptogenic liver disease patients, as well as relatives of known aatd individuals. over , individuals have been screened following ats/ers guidelines as part of the national aatd targeted detection programme. rare and novel mutations were identified by dna sequencing of the serpina gene. a number of rare serpina mutations including i, f, x christchurch , z bristol , and m malton were identified. the i mutation (arg cys) was present at a relatively high frequency ( . ) with over cases identified. the f mutation (arg cys) was found in cases. in addition, novel null mutations were identified, q dublin and q cork. current testing of suspected aatd cases is often limited and can miss rare and novel clinically significant serpina mutations. our findings underline the need for a comprehensive diagnostic work up of all patients with low aat levels including phenotyping, genotyping and if necessary, dna sequencing of the serpina gene. we previously observed that weaning-failure patients experience increased intensity of dyspnea (ajrccm ; :a ). we also observed that patients reported different qualitative sensations suggesting that more than one mechanism may contribute to dyspnea. the purpose of this study is to determine whether dyspnea experienced in weaning-failure patients is related to changes in pco or increase in respiratory effort or both. methods: tracheostomized patients who were being weaned from prolonged mechanical ventilation at a specialized facility were enrolled. dyspnea, transdiaphragmatic pressure-time product (ptpdi), minute ventilation, and transcutaneous pco (ptcco ) were measured during a -h trial of spontaneous breathing. patients who developed respiratory distress during the trial were considered weaning failures. patients who tolerated the trial and continued to breathe unassisted for at least h after the trial without signs of distress were considered weaning successes. results: patients were studied; were women; age, + (se) years; duration of ventilation before the study, + days. fourteen patients were weaning successes; patients were weaning failures. in the failures, dyspnea score increased from . + . at the start to . + . at the end of the trial (p \ . ). the increase in dyspnea in the failures was accompanied by increases in minute ventilation (p \ . ) and ptcco (p \ . ); ptpdi, an index of patient effort, remained unchanged during the trial. in the successes, dyspnea, minute ventilation and ptcco did not increase during the trial; ptpdi, however, decreased from the start to the end of the trial (p = . ). these findings suggest that an increase in pco , a major driver of minute ventilation, contributes to an increase in dyspnea during weaning failure. that the increase in dyspnea in the failures was not accompanied by an increase in ptpdi together with the successes exhibiting a decrease in ptpdi without any change in dyspnea suggests that effort is not a major determinant of dyspnea during weaning failure. conclusion: dyspnea increases in weaning-failure patients but not in weaning-success patients and the increase in dyspnea is accompanied by increase in pco and minute ventilation but not by an increase in respiratory effort. background: respiratory disease constitutes one of ireland's greatest public health challenges. patients with respiratory disease are often undiagnosed despite symptoms and risk factors for lung disease. the purpose of this study was to determine the prevalence of respiratory symptoms and disease in a targeted population screening program. study design: subjects were asked to complete a questionnaire on respiratory symptoms and risk factors as part of well-publicised free spirometry testing on world spirometry day. multiple linear regression analysis was performed to identify factors contributing to variation in population fev . logistic regression was used to identify predictors of airflow obstruction (fev /fvc \ %) followed by predictive model development and roc curve analysis to determine model diagnostic accuracy. results: ten centers throughout ireland participated in the study. analysis was limited to an initial discovery cohort of patients ( % female; age = years (range - ); fev = % predicted(range - %). factors associated with reduced population ir j med sci ( ) (suppl ):s -s fev (% predicted) were smoking history, male gender, lower educational status and history of existing lung disease. in those with no history of lung disease (n = ), % had abnormal spirometry with % demonstrating airflow obstruction. predictors of airflow obstruction were age, presence of cough and number of pack-years of smoking. presence of cough, age [ years and exposure [ pack years were associated with highest sensitivity and specificity for identifying airflow obstruction although predictive ability was only fair (roc auc = . ). conclusions: demographic and socioeconomic factors influence lung health in ireland. undiagnosed respiratory disease is common, particularly airflow obstruction and targeted screening is justified to identify patients with respiratory disease early. bronchiolitis affects one-third of babies in their first year of life. half of those hospitalised will have persistent cough and wheeze. to map this epidemic, we investigated all bronchiolitis admissions to tallaght hospital in the last years. this will aid future planning of the service and provide an insight into the epidemic in the irish population. from until , , children were admitted to tallaght hospital due to bronchiolitis. we analysed these on the basis of time of year of admission, length of stay, gender and age and compared them to national and international data. the busiest month was december, with . % of admissions. however, there was a significant increase in the incidence of bronchiolitis in the early spring of and (more than doubled) compared to previous years. the average length of stay is . days, male sex had % dominance and average age was . weeks, in keeping with international data. there has been in a significant shift in the timing and incidence of bronchiolitis in tallaght hospital in the last years. we explored the reasons for this, with special attention to rsv incidence, possible climate causes, vaccine programs and exposure risk. we identified children, males and females. mean weight was . kg (range . - kg). mean age was . months (range days- months). stridor was the commonest presenting symptom %. diagnosis was confirmed by micro-laryngobroncoscopy and supplemented by ct in %. % had complete tracheal ring stenosis and % had concurrent cardiac anomalies. two patients had bronchus suis. extracorporeal life support (ecls) was utilized in one patient preoperatively. cardio pulmonary bypass (cpb) or ecls was utilised for the repair. laryngeal release was required in / patients. patients underwent end-end anastomosis, slide and double slide tracheoplasty. a polydiaxanone suture was used for all anastomosis. mean (cpb) time was . min (range - min). mean cross clamp time was . min (range - min). mean length of ventilation was days (range . - days). mean icu length of stay was . days (range - days). there were two hospital mortalities. one patient only required re-intervention with balloon dilation. % were symptom free on a mean follow up of . months (range weeks- years). distal tracheal stenosis can be managed effectively utilizing cpb that also allows concurrent correction of congenital heart anomalies. mayo general hospital, midlands regional hospital background and aim: attempting to reduce unnecessary attendances of well patients at outpatient clinics is prudent. this study evaluated the asthma control test (act)t and respiratory proforma, with feedback through mobile texts, in children with asthma, to determine attendance at clinic or not. methods: patients between and years with a diagnosis of asthma were eligible for inclusion. the parent was surveyed, by post, weeks prior to the clinic date and asked to complete the asthma control test (act) and a respiratory proforma which assessed uacs symptoms, medication usage inclusive of intensification episodes and medical concerns. mobile telephone numbers were requested. parents mailed their responses in a supplied stamped envelope supplied. respondents were divided into two categories (a) act score greater than and a non concerning respiratory proforma, who were texted not to attend the clinic but supplied with another outpatient appointment and (b) the remainder were texted to attend the clinic. results: over clinics the parents of eligible children were surveyed. one hundred and forty-one ( %) replied of whom ( %) were well and did not attend the clinic but rebooked. of who attended, had new symptoms of uacs and had pneumonia. of who did not reply, forgot to reply, came to clinic with completed questionnaires, had good control. thirty-five did not attend the clinic of whom were discharged to the family doctor. conclusion: asthma care through postal survey with mobile text feedback is an option in the outpatient setting. background and aims: asthma is common in paediatrics with the most difficult to manage being those less than years. this study evaluated the impact of a nurse delivered education program developed for parents of children under years in terms of knowledge gained and parental empowerment. methods: twenty parents of children age - years were invited to attend five h educational sessions which related to asthma pathophysiology, signs and symptoms, clinical assessment and medication use. a specific educational program was developed. prior to enrollment each parent was administered two surveys; ( ) a previously tested asthma questionnaire containing statements, and ( ) a survey of parental concerns related to asthma. one month after the program was completed parents asthma knowledge and perceptions of empowerment were reassessed. results: while parents were enrolled data sets for were available for analysis. the pre-intervention mean knowledge level was . ( %) (range - ) and post knowledge level was mean . ( %) (range - , paired t test 
). the parental survey identified asthma recognition and poor coping skills as major themes which the educational program addressed. conclusion: a targeted asthma educational program improves parental knowledge and enhances parental empowerment. written action plans (w.a.p.) are recommended in international guidelines for the management of asthma [ ] . despite this, uptake remains poor [ ] . a qualitative prospective study of parents of children attending the paediatric asthma out-patient clinic at cork university hospital was performed to examine if; ( ) written action plans are valued by parents. ( ) they assist in recognition of symptoms. ( ) parents commence appropriate treatment at home and identify when to seek medical advice as a result of w.a.p. ( ) parents feel assured by possession of w.a.p. thirty parents of children aged - years were interviewed by the paediatric asthma nurse specialist to assess level of asthma control, knowledge of treatment and level of concern. parents were provided with a colour coded w.a.p. and all aspects of treatment were discussed. a follow up telephone interview was performed months later. in the pre intervention group only / felt they had enough information to manage their child's asthma; this increased to / post intervention (p \ . ). / knew the location of their w.a.p.s. there were no incorrect responses regarding dose/frequency of medication. / subjects had dropped a level of concern regarding their child's asthma (p \ . ). with sufficient written information and education, the anxiety and concern that many parents undergo while managing a child with asthma, can be reduced. eight patients ( male) underwent respiratory (ecls). there were -preterm, -term neonates, -infant and -child. indications included, congenital diaphragmatic hernia- , bronchiolitis- , primary pulmonary hypertension- , pertussis- and complete tracheal ring stenosis- . % of patients were transferred to sweden or uk. eight children ( males) underwent (ecpr) runs, with a mean age of . years (range weeks- years). / had underlying congenital heart disease, of which had univentricular pathology. mean conventional (cpr) time before initiation of (ecpr) was min (range - min). in the respiratory (ecls) cohort mortality was . %. the only survivor was treated in ireland. in the (ecpr) cohort our survival rate of % exceeded the international extracorporeal life support organization published results of %. all ecpr patients were treated in ireland. currently there is no funding for pediatric respiratory (ecls) in ireland. patients are being treated abroad at significant expense, family inconvenience and mortality. these results would suggest a change in health policy is overdue! recent evidence has confirmed a high prevalence of bronchiectasis and impaired lung function in school aged children with cf despite little in the way of symptoms of lung disease in this group in preschool years. if we are to significantly improve long term outcomes in cf we must gain a greater understanding of lung disease in the preschool years and intervene earlier with disease modifying treatments before irreversible lung disease occurs. the key to understanding early lung disease in greater detail lies in the design of robust, comprehensive, well powered longitudinal studies. shield cf was established in with these requirements in mind and is a framework through which we can start to answer some important questions. shield cf is centred around the annual cf bal surveillance programmes in our institutions. currently the shield cf programme includes: • bal-immediately processed, aliquoted and biobanked • whole blood-immediately processed, aliquoted and biobanked • oropharyngeal swab-processed for rna extraction • clinical information-baseline and ongoing information related to lung health entered onto online database from individual centres. in the future we plan to include: • lung function measured by lung clearance index (lci) • lung structure determined by chest ct to date patients have been recruited with a total of samples (n = cf, n = control). shield cf has contributed samples to different international multicentre studies. table below summarises key baseline findings within shield cf to date. within the next year shield cf will incorporate preschool children between the three centres. aim of our study was to audit hospitalization for copd exacerbations with respect to patient characteristics, diagnosis and standards of care. all patients admitted to roscommon hospital with a diagnosis of aecopd from july st to december st were included. medical notes were reviewed for data collection. patients were included in the study. there was a frequent failure to objectively confirm the diagnosis of copd by spirometry. only ( . %) patients had spirometry performed at any stage. patients were current smokers. inhaler technique was assessed in only patients. ( %) patients received chest physiotherapy. out of current smokers had documented smoking cessation advice, and received smoking cessation pharmacotherapy. ( %) patients were treated with antibiotics. management of aecopd in our hospital is frequently suboptimal, and may be managed better with respiratory physician involvement. there should be more frequent spirometric confirmation of diagnosis, more conservative use of antibiotics, better screening for ltot and improvement in smoking cessation service. many copd patients return for review at the respiratory outpatient department when clinically stable. they are often reviewed by less experienced nchds who may lack the knowledge and confidence to discharge them. furthermore many beneficial clinical and lifestyle interventions are not commenced. through a series of pdsas, a checklist was developed and implemented to ensure that copd patients received appropriate interventions and highlight which patients could be safely discharged to the care of their general practitioner. this combined a checklist of criteria for optimisation of copd patient management devised in the respiratory department as well as both the validated copd assessment test (cat) and modified medical research council dyspnoea scale. discharged patients had a cat score of b and no significant outstanding treatment modifications. the checklist was completed in copd patients of whom ( %) were suitable for discharge. interventions such as optimised pharmacological therapy, assessment of inhaler technique and vaccination education were not instituted in , and % of patients, respectively. we conclude that an opd discharge checklist is an appropriate intervention to improve quality of care for patients with copd and facilitate the discharge of stable patients from a respiratory opd. pulmonary rehabilitation (pr) is a multidisciplinary approach to improving the exercise capacity and symptoms of people with copd and ild. however, compliance is often suboptimal. this study investigated whether the education and literacy level of patients may affect attendance and completion of pr. patients were divided into two groups based on diagnosis; copd or ild. nine factors were studied: sex, age, baseline activity level, education, literacy, social isolation, transport to programme, oxygen requirements and anxiety and depression scores. completion of pr was defined as attending [ % of the classes. our findings demonstrated that % of copd patients failed the literacy test compared to % of ild patients. % of copd patients had only primary level education in contrast to % of ild patients. % of copd patients completed third level education compared to % of ild patients. % of copd patients travelled to pr in their own car in contrast to % of ild patients. % of copd patients got public transport to pr compared to % of ild patients. % of copd patients and % of ild patients completed pr. although there were significant differences in educational achievements between groups, this did not affect their compliance in completing pr. osteoporosis has not been fully evaluated in copd patients. the development of osteoporosis among copd patients is multifactorial. the objectives of this study were: ( ) to explore the prevalence of osteoporosis among copd patients, ( ) to observe any correlations between t-scores and different disease related variables. copd patients attending respiratory clinics were randomly assigned for dexa scanning. pts were excluded because of the coexistence asthma, age [ years and early menopause. the mean age of the studied patients was (range - ) years, and % were female. mean fev of these patients was %. seventy-two percent had osteoporosis, and % had osteopenia. mean t-score was - . (male - . , female - . ). t-score was noted to have positive correlation with age (r = . , p = . ), but no correlation with bmi (r = . , p = . ) and fev (r = - . , p = . ) was noted. statistical difference in t-score was observed between patients with normal/reduced mobility vs. poor mobility (p = . ) but no difference was observed among patients with steroids inhalers alone (over the last years) or in combinations with oral steroids (p = . ). very high prevalence of osteoporosis was noted among our cohort of copd patients. surprisingly, there was no association of bmd with fev , bmi and corticosteroid exposure. the aim of this study was to determine the benefits of standardised reassessment of long term oxygen therapy (ltot) patients. long-term oxygen therapy (ltot) is the treatment proven to improve survival in chronic obstructive pulmonary disease (copd) patients with chronic respiratory failure. this study was prompted by an absence of any formal or regular assessment of ltot after initial prescription. the patient's oxygen requirements were assessed carrying out abg analysis after the patient had been taken off oxygen for thirty minutes. the patient then participated in a six minute walk test ( mwt) to determine the need for ambulatory oxygen. of the patients contacted attended for ltot reassessment. % patients no longer met the criteria for ltot i.e. pao [ . kpa. % required a decrease in their static oxygen requirements. . % required an increase in their ambulatory oxygen requirements. . % needed a prescription for ambulatory oxygen. % had a sub therapeutic oxygen prescription. in total % of participants required a change to their oxygen prescription. current procedures for the assessment of ltot result in a large proportion of recipients not having appropriate prescription. there is a need for the initiation of standardised regular reassessment of all ltot patients. with increased ambulatory treatment of copd exacerbation, there is a need for opd assessment of blood gases and ph. we looked at the correlation between arterial blood gas (abg) and capillary blood gas (cbg) in a stable population of opd patients with copd. patients attending for oxygen assessment had a capillary blood sample taken from the fingertip pulp. this was analysed using the epoc Ò point of care analysis system. an arterial blood sample was obtained from the radial artery and analysed using a radiometer blood analyser. patients attended for oxygen assessment. we used pearson's correlation to compare the results of abg and cbg. it showed a moderately high correlation of pco at % (p = . ). a correlation of % with po (p = . ). the hco and base excess correlations were extremely high at % (p = . ) and % (p = . ), respectively. independent sample t tests were used to look at the agreement between abg and cbg values. it showed that cbg could be useful in predicting ph, hco and base excess but not very useful clinically in predicting pco and po . cbg could be used in the opd setting to monitor blood ph and may be useful in copd outreach assessments. identifying lung disease early is very important and can be done with spirometry testing. early detection of these diseases can greatly improve outcomes and quality of life for patients. members of the public were given the opportunity to have their lung function tested for free, as part of the world spirometry day campaign. spirometry was performed by respiratory scientists. access to smoking cessation advice, inhaler technique and the benefits of exercise to maintain healthy lungs were also provided. patients were tested. % (n = ) were male and % (n = ) were female. % (n = ) were under years. % (n = ) were [ years. % (n = ) reported that they had a previous lung function test. % (n = ) reported they never had a lung function test performed. % (n = ) did not answer. % (n = ) of those previously tested reported a history of known lung disease. % (n = ) who were never tested reported a history of lung disease. % (n = ) were non-smokers, % (n = ) were current smokers, % (n = ) were ex-smokers and % (n = ) unknown smoking status. % (n = ) achieved normal spirometry. % (n = ) results were abnormal. % (n = ) results were unreliable. of the abnormal results; % (n = ) demonstrated obstructive lung disease, % (n = ) restrictive lung disease & % (n = ) mixed lung disease. implementing and evaluating palliative care responses for patients with advanced respiratory disease. this multi-site action research project used mixed methods data collection strategies including qualitative interviews with patients and families, expert focus groups and quantitative methods such as education surveys, evaluation and chart audits. study findings demonstrate that palliative care interventions can be implemented within respiratory services for those patients with advanced disease. the study methodology of action research ensured that all key stakeholders in the service delivery across several sites contributed to sustainable organisational change which delivered measurable care improvements for patients throughout the research process. interventions that have developed include; shared training and education across sites, multi-disciplinary team meetings, pulmonary rehabilitation session on coping, death reviews and the development of a respiratory palliative care pathway for patients. palliative care interventions are feasible within care delivery models for patients with advanced respiratory disease. referral links and a care pathway between the hospital and hospice settings are at the cornerstone of this care delivery model. half of all patients admitted with copd in ireland are either dead ( %) or re-admitted to hospital ( %) within days [ ] . variation in the management of copd patients may contribute to this. implementation of care pathways has been suggested to improve outcomes such as mortality, admission rates and length of stay. the acmb was introduced as a sticker in the healthcare record in feb . a cross-sectional audit reviewed staff practice prebundle implementation, post-bundle and following an educational drive. % of patients in the pre-bundle group received nebulised bronchodilators within min of presentation, in comparison to[ % in the post-education group. prescription of oral corticosteroids improved with a corresponding decrease in patients receiving none. there is ongoing use of intravenous corticosteroids ([ %). the use of intravenous antibiotics was unchanged at approximately % although over % of patients received an antibiotic recommended in the acmb. % of pre-bundle patients received an abg, % within min; following the educational drive this increased to % with % within the timeframe. further education may be required to decrease the frequency of intravenous medications along with examination of barriers to managing patients within the acmb timeframe. the recent ers audit of irish patients with exacerbations of copd was the first nationwide assessment of survival and readmission rates for this patient cohort. it showed an average length of stay of days, a day readmission rate of % and mortality rate of . %. our copd outreach provides supported or pre-emptive discharge as an alternative to hospital treatment for copd exacerbations. we undertook an audit of patients treated by the outreach team over years to compare rates of these values with those from the ers audit. among patients who died in this period ( %) died in the day period from discharge and ( %) were readmitted. of those that died, % were readmitted within days and % died in hospital within a short period of readmission. of overall readmissions ( ) % were from early discharge programme, ( %) assisted and ( %) prevent readmissions. of note % of prevent readmission patients died within days. the median length of stay was days. these data indicate that survival and readmission rates are the same whether patients are treated in hospital or by outreach teams, however, with shorter lengths of stay the later is associated with substantively lower costs. future research will need to focus on identifying the factors that contribute to the high readmission rates. the central purpose of pulmonary rehabilitation is to reduce morbidity by improving functional capacity through exercise. it is still unknown if improvements in functional capacity are maintained in the long-term and lead to increased physical activity levels. the hypothesis of this study was that pulmonary rehabilitation would lead to a sustained increase in standard outcome measures and in daily physical activity. a prospective study of subjects with copd was performed, registered at clinicaltrials.gov (clinical trial number nct ). the primary outcome was a maintained improvement in standard outcome measures with a secondary aim of an increase in daily physical activity. a convenient sample of the cohort (n = ) was re-evaluated at a third time point at year. a weeks hospital based outpatient pulmonary rehabilitation programme led to a significant reduction in total energy expenditure (p \ . ) and breathlessness (borg, p \ . ) and improved exer- these findings show that while pulmonary rehabilitation increased exercise capacity this was not transmitted into increased daily physical activity. alternative methods to alter/affect behavioural change may need to be addressed. the national clinical programme for chronic obstructive pulmonary disease (copd) aims to improve quality, access and cost of care for patients with copd. pulmonary rehabilitation (pr) has been proven to meet these aims. the purpose of this study was to audit pr throughout ireland. hospitals and all local health areas (lha) were surveyed about prp in their catchment areas -ongoing programmes, length of and numbers on waiting lists, and enrolments in prp in the first months of . those without access to prp were asked about barriers to setting up such programmes. descriptive statistics were used. all hospitals (n = ) and lha (n = ) responded re access. % of hospitals (n = ) and % of lha (n = ) have access to pr. hospital settings have mean waiting lists . months( , ), mean numbers waiting ( , ) and mean number enrolled ( , ), (respondents = ). respondents (n = ) from the community showed waiting lists of and months, numbers waiting and and numbers enrolled and . 'black spots' with no access were identified. barriers that may be amenable to intervention include increased support for primary care teams, facilitation of appropriate referrals and the development of spirometry services in the community. patients were selected as per gold guidelines. the duration of the study was weeks. four primary care centres were selected, one each in longford, athlone, tullamore and mountmellick. one spirometry clinic was held every week on a rotational basis with the intention of accommodating patients per clinic. it was free service and intended for patients who had not previously undergone spirometry testing. the gp practise booked patients directly into the spirometry clinic. a total of patients were booked across the spirometry clinics. of the patients booked, % did not attend and % cancelled their appointment. out of the clinics utilized all available slots with one clinic utilizing only % of available slots. a total of patients were tested. out of these, had normal spirometry, had gold stage mild copd, had stage moderate copd, had stage severe copd and had stage very severe copd, had possible restrictive lung disease and unreliable data. success and efficiency of this type of service is heavily dependent on spirometry clinics being gp driven. a number of advantages, disadvantages and recommendations arose out of this study. . an audit of steroid and antibiotic therapy for acute exacerbations of copd in a cork hospital sought to determine compliance with these guidelines in the mercy university hospital, cork. a retrospective review was conducted on patients admitted with exacerbations of copd from st june to st august . forty-six patients attended the ed with exacerbations of copd. thirty-three charts were available for review. of the patients, ( . %) were prescribed iv hydrocortisone, despite being able to take oral prednisolone, ( . %) were prescribed oral prednisolone and ( . %) were not prescribed steroids. eight patients were prescribed co-amoxiclav alone, patient was prescribed clarithromycin alone and no patients were prescribed doxycycline alone. four were treated with piperacillin/tazobactam, patient with piperacillin/tazobactam and linezolid, patients with co-amoxiclav and clarithromycin, one patient with moxifloxacin, patients with ciproxin and for patients, antibiotics were not prescribed. of the patients prescribed co-amoxiclav and clarithromycin, had infiltrates on cxr. the prescription of antibiotics and steroids in the muh in patients with acute exacerbations of copd did not meet guidelines as per the national copd acute management bundle. though niv significantly improves outcomes in acute copd patient care, there is no compulsory niv training programme for nchds. we assessed nchd's knowledge of the use of niv in copd using a questionnaire, based on bts guidelines, with reliability assessment in a subset of nchds. experience level, knowledge of niv contraindications and settings were examined. questionnaires were completed. inter-rater agreement was good (kappa . , se . , % ci . - . ). % had previously worked in respiratory medicine. % had commenced patients on niv and/or titrated settings on call. % had received training in the use of niv. % adjusted niv settings incorrectly for hypercapneic acidemia. % used epap incorrectly. those with respiratory work experience had greater niv titration experience (p = . ) and superior knowledge of niv settings (p = . ). though % of interns had previously titrated settings, their confidence tended to be lower (p = . ) and their knowledge of niv settings was significantly inferior to more experienced doctors (p = . ). though niv is widely used by nchds of all grades, the present study shows training and knowledge deficiencies, especially among interns and those not in a respiratory post. there is a need for structured training in this key skillset among nchds. table . conclusions: approximately one-third of the patients in who presented to the ralc were classified as asymptomatic. approximately / of asymptomatic and only / of symptomatic patients had early stage cancer ( and ) which was a statistically significant difference. there was no difference in smoking history gender, tissue type and ecog status at presentation between symptomatic and asymptomatic patients. lung cancer is the most common cause of cancer death in ireland. a recent large screening trial demonstrated a % survival advantage with low-dose ct over chest x-ray [ ] . however, the false positive rate of . % for screening detected nodules is a major drawback. a lung cancer biomarker would lead to improved specificity, reduced costs and a reduction in unnecessary procedures for patients with ct-detected pulmonary nodules. biomarkers would also be useful for monitoring response to therapy or disease progression and may reveal the molecular mechanisms underlying cancer development. proteomics represents an important tool for identifying novel cancer biomarkers. recent advances in mass spectrometry allow rapid and accurate analysis of several thousand proteins in a single study [ ] . we prospectively recruited patients presenting to beaumont hospital rapid access lung clinic between april and july . paired bronchoalveolar lavage (bal) and serum samples were obtained. patients were subsequently grouped after clinical and mdt follow-up into ( ) benign, ( ) possible (for surveillance), and ( ) confirmed lung cancer. samples were then analysed by orbitrap mass spectrometry and candidate lung cancer biomarkers identified based on the clinical and histological characterisation. principal components analysis (pca) of the peptides found to be differentially expressed between control bal and cancer bal was performed to determine any outliers in the data and also to identify group clustering as per diagnosis. leave-one-out cross validation (loocv) of a protein plasma combination demonstrated an accuracy of * . % (auc: . ) for distinguishing benign conditions of the lung from cancer (sclc/ nsclc) (fig. ) . further analysis and validation is underway. trapped lung after malignant pleural fluid drainage is a contra-indication to talc pleurodesis. vats identifying patients amenable to talc may be prohibited by co-morbidities. initial experience with a miniinvasive alternative is described in three patients, two transferred with debilitating dyspnoea and previous multiple admissions for drainage procedures. ultrasound was performed and pleurx Ó insertion under la (single in patient, bilateral in ), with palliation of symptoms facilitating discharge in all three. a rapid access lung cancer clinic (rac) with radiological support was recently introduced aiming to both decrease the time to diagnosis of patients, whether negative or positive, and also to decide the most appropriate investigation for each patient with on-site radiologist support. in this observational study, we used a historical cohort of the last fifty red flag patients assessed at the respiratory clinic before the introduction of the rac, and compared it with the first thirty-five patients seen at the rac. time to diagnosis and the number of invasive investigations [bronchoscopy/fine needle aspiration (fna)] were compared as outcome measures. continuous variables were compared using mann-whitney u test and categorical variables using fischer's exact test. patients were similar in terms of demographics. there was a statistically significant reduction in the time to diagnosis (p \ . , mann-whitney u), whether negative or positive, following the introduction of the rac (fig. ) . there was also a statistically significant reduction in the number of bronchoscopies (p \ . , fischer's exact test) carried out after the introduction of the rac. these results demonstrate that the rac with radiological support decreases both the time to diagnosis and the number of invasive investigations that may not have yielded a diagnosis. tbna has been widely available for sampling mediastinal lymph nodes (ln) for over two decades. unfortunately, blind tbna has low sensitivity and limited access to only certain ln stations. ebus-tbna has revolutionised ln sampling demonstrated in many prospective multicentre trails. over a -month period, patients with evidence of lymphadenopathy on chest ct, underwent ebus-tbna. procedures were performed on patients ( males, mean age of . years). diagnostic yield, and sensitivity were calculated by reviewing clinical notes, radiological imaging, cytology, transbronchial/endobronchial biopsy, bal, and mediastinoscopy reports, and redo ebus-tbna reports from another centre. in % of procedures, ln were sampled. . % of ebus-tbna samples were diagnostic ( cases for sarcoid, and for lung cancer). diagnostic yield was compared in st . -month period versus nd and returned versus . %. sensitivities for sarcoidosis and lung cancer were calculated at and . %, respectively. review of current data shows diagnostic yield and sensitivities varies significantly, however, our results were below current published standards. radiologically guided lung biopsy is a relatively safe procedure to reach a histological diagnosis for suspicious lung lesions. we audited the performance of university hospital galway against the bts guidelines. this was a cross-sectional study. all patients who had radiologically guided percutaneous lung biopsies between january and october were included. primary outcome was the ability to reach a histological diagnosis. secondary outcome was development of pneumothorax or other complications. biopsies were performed. were males and were females. mean age was . years. mean lesion size was . ± . cm. the procedure was done under ct-guidance in patients ( . %), fluoroscopy in patients ( . %) and ultrasound in patients ( . %). the overall diagnostic rate for benign and malignant causes was . %. malignancy was diagnosed on biopsies ( %). sensitivity for detection of malignancy for lesions [ cm in size was ( . %). the procedure was complicated by pneumothorax in patients ( . %). only patients ( . %) required chest tube insertion. we achieved a higher diagnostic rate than the level set by the bts but pneumothorax rate was slightly higher. this could be because the majority of our samples were taken using large bore cutting needles. sarcoidosis is a systemic granulomatous disease of unknown aetiology that primarily affects the lung. several reports have suggested that analysis of cd +/cd + lymphocytes can be used to differentiate sarcoidosis from other causes of interstitial lung disease. the aim of this study was to evaluate the diagnostic utility of bal fluid cd /cd ratio in diagnosing pulmonary sarcoidosis. this was a retrospective cohort study. study population included all patients who had bal fluid obtained during fibre-optic bronchoscopy in university hospital galway between november and june . outcome variable was cd +/cd + ratio as calculated on flow-cytometry performed on bal fluid. patients got bal fluid analysed. ( . %) were found suitable for analysis by flow-cytometry. had a transbronchial biopsy performed. out of patients with histological evidence of sarcoidosis, only patients had bal fluid suitable for analysis. ( . %) of them had a cd /cd ratio higher than . . sensitivity of cd /cd ratio [ . in diagnosing pulmonary sarcoidosis was % with a specificity of %. positive predictive value was % and a negative predictive value was . %. the distribution of cd /cd ratios in patients with biopsy-proven sarcoidosis suggests that substitution of bronchoalveolar lavage cellular analysis for transbronchial biopsy is not advisable. we previously reported that smoking cessation (sc) services are available but lacked uniformity or consistency countrywide [ ] . we found that ( %) of service providers (sp) were collecting and analysing data on pregnancy status but % did not analyse it and % did not collect any data on pregnancy. we aimed to look at outcomes of sc in pregnancy, provide evidence based data to improve services for this vulnerable population and aid their evaluation and effectiveness. using a census of all known smoking cessation programmes throughout ireland [ ] , sps were asked to pilot a treatment database for a month period. many different data were entered including pregnancy status and treatment outcomes. the data were returned to tfri for analysis using a statistical package spss. a database was piloted over a month period by sps. a convenience sample of , patients was recruited while attending sc service throughout ireland and their data were entered into the database. a total of pregnant women attended the smoking cessation services during this period which represents . % of the females treated. they achieved a quit rate of . % at weeks and . % at months compared with . and . % of the rest of the female population treated in the same time period. the poor outcomes may be a result of the paucity of services in ireland for this population group. the findings will facilitate planning and delivery of effective sc programmes to pregnant women ensuring equity in service provision. maternal smoking in pregnancy is an important risk factor for low birth weight (lbw) (\ , g) and prematurity (\ weeks gestation) [ ] . the purpose of this study was to examine smoking rates and prevalence of associated adverse birth outcomes in the coombe women and infants university hospital, dublin over a year period - . a cross-sectional observational study was conducted using routinely collected data from the euroking k maternity system from january -december (n = , ). smoking prevalence declined significantly from . to . % over the period. rates in teenage mothers remained very high ( . % in ). smoking prevalence was almost twice as high in mothers of lbw compared to normal birthweight babies, one and a half times higher in mothers of preterm babies compared to full term and more than twice as high in mothers of small for gestational age (sga) babies compared to non sga. a statistically significant decline was seen in the prevalence of sga babies in the period. no statistically significant change was seen in the prevalence of lbw or preterm babies. prevalence rates in pregnancy are high in ireland compared with other developed countries. increased focussed efforts are needed to reduce smoking rates. smoking-related diseases account for over , admissions per year in ireland. % of all smokers will die of smoking-related disease, and smokers on average die years younger than non-smokers. the aim of this project was to assess attitudes and beliefs in smokers and ex-smokers. patients who were smokers or ex-smokers were interviewed using a standardised questionnaire. candidates were recruited from respiratory clinics and general-inpatient wards in beaumont hospital. data regarding underlying medical history was acquired from medical records. patients were included, ex-smokers and smokers. there were no differences in gender or medical history. ex-smokers had a mean age of . years compared to . years in smokers (p = . ). smokers had longer smoking history; . years compared to . years (p = . ). smokers reported lower expectations regarding the benefits of smoking-cessation than the exsmokers; only % believed there would be short-term health benefit and only % believed quitting was worthwhile compared to . and . %, respectively, in the ex-smoking group (p = . /p = . ) there is significant variation amongst smokers and ex-smokers regarding attitudes to smoking-cessation; this is despite receiving the same smoking-cessation advice and having low fagerstrom-scores. of note current-smokers were younger but had longer smoking histories and smoked from an earlier age. results: a total of patients have been referred, . % (n = ) male and . % (n = ) females. the mean age at referral was . years. . % of patients admitted to being current smokers, of these . % were male and . % were female. . % had a previous history of smoking . % of patients were referred from their gp, . % were referred from another consultant based with the mwrh and . % from another centre. a total of patients referred to the clinic subsequently had a diagnosis of cancer. patients had a diagnosis of lung cancer (adenocarcinoma = , scc = , other nsclc = and small cell = ). patients were diagnosed with a malignancy other then lung cancer, the most common being lymphoma, or metastatic lung disease. male patients accounted for . % (n = ) and female . % (n = ) of those diagnosed with cancer, with a mean age across both genders of . years. conclusions: the results of the audit would suggest that the ralc clinic is in line with national trends as regards smoking prevalence and age. the referral pathway needs to be the focus for the future facilitated by early referral from gp's. this will enable the ralc clinic to prioritise prompt investigations and treatments to optimise survival. sarcoidosis. we aimed to assess the diagnostic accuracy of ebus- [ ] . twenty ipf patients attending a specialized clinic were recruited. their blood samples were collected. full pulmonary function and awake arterial blood gas, high resolution computed tomography of chest (hrct) were performed. ccl was quantified using a enzyme-linked immunosorbent assay. ccl levels are presented as median ± interquartile range. correlations were analysed using pearson and simple regression. median concentration of ccl was higher in ipf patients [ , . pg/ml ( , . - , . )] compared to healthy controls [ , pg/ml ( , - , ), p = . ] [ ] . ccl levels were inversely correlated with diffusing capacity for carbon monoxide (dlco) (r = - . , p = . )* and awake carbon dioxide (co ) tension (r = - . , p = . *). positive correlations were observed between a-a gradient and ccl concentration (r = . , p = . *) and co rise during sleep (r = . , p = . *) but no relationship between ccl and fibrosis score, mean reticulation score or mean ground glass score on hrct. ccl levels correlate with physiological marker of fibrosis. we found significant correlation between ccl and markers of daytime hyperventilation and nocturnal hypoventilation. ccl may be a useful marker of disease activity in ipf. were reviewed. patients with normal cxr who had ctpa performed were included in study. in patients who had no pe but incidental findings (n = ), further review was done to see if these findings would account for presentation. in patients with normal cxr and negative ctpa, an alternative diagnosis directed by incidental findings was able to account for presentation in . % (n = ) of cases. of these, respiratory causes accounted for % (n = ), with pneumonia being the commonest overall cause at . %. other causes included atelectasis %, emphysema %, effusion %, collapse %, lesion % and pneumothorax %. ctpa is able to offer an alternative diagnosis in patients who had initial normal cxr and negative ct pa. our study is suggestive of high incidence of pneumonia in patients scanned for suspected pe. ctpa is the investigation of choice for suspected pulmonary embolism (pe). determining pretest probability for risk of pe using risk assessment scores and d-dimer is an important step in diagnosis. to establish positive predictive value of clinical and lab-based risk assessment for diagnosis of pe and to determine whether or not there was an overuse of ctpa in our service. a retrospective audit identified patients (n = ) who had ctpa performed from january -march . patients were classified as being low, intermediate or high risk for pe, based on wells criteria [ ] . cases were reviewed to establish if pretest assessments were used correctly in predicting probability of pe and need for ctpa. in high risk group (wells score [ ) (n = ), there were diagnosed pes (ppv . %). in intermediate risk group (wells score c b ) (n = ), there were pes (ppv . %). in low risk group (wells score b ) (n = ) there were pes (npv . %). in our sample, wells scores had reasonable predictive value for diagnosis of pe in high risk patients. positive predictive value in intermediate group was significantly lower and ctpa may be overused in this group. negative predictive value for low risk group was very high. therapeutic thoracentesis: the role of ultrasound and pleural manometry ultrasound-guided thoracentesis the utilization of endobronchial ultrasound for sampling of primary lung we performed a retrospective audit of mesothelioma patients diagnosed in the whsct between and and compared our practice against guidelines. patients ( male, female) were diagnosed-mean age . years, range - years. asbestos exposure was documented in patients ( . %). patients ( %) had right-sided chest disease with ( %) having left-sided disease /hospice. patients are still alive. all patients were known to a lung cancer specialist nurse pilcher alfred hospital intensive care, melbourne, australia we examined the use of bronchoscopy in a bed icu/hdu over a month period. our aim was to establish the number of procedures done, the indications and complications. we also performed an audit of procedure documentation and set-up. procedures were identified over the period in question. % patients were male and ages ranged from to with a mean of years. various indications were documented with infection ( %), tracheostomy insertion ( %) and difficulties with oxygenation ( %) being the most common interstitial lung disease and pulmonary vascular disease chairs d. o'callaghan, mater misericordiae university hospital we now characterize potential mechanisms associated this b cell phenotype in sarcoidosis. serum was collected from treatment-naive, sarcoidosis patients and control patients. b cell activating factor (baff; r&d systems) and soluble (s) cd (ebioscience) levels were measured by elisa. expression of b cells in sarcoidosis granulomas were assessed using standard immunohistochemistry. all subjects signed an informed consent prior to participation. b cells were detected in sarcoid granulomas, but using serial sections no cd positive b cells were identified with respect to reduced cd b cells in sarcoidosis, we found no evidence that these cells are sequestered in sarcoid granulomas and that the key critical b cell cytokine, baff is appropriately elevated. scd is increased raising the possibility that the phenotype in this population reflects shedding of cd from the surface of memory b cells the diagnosis of sarcoidosis required the presence of non-caseating granulomata with negative ziehl-neelson stain and fungal stain plus negative culture for mycobacterium tuberculosis (tb), other bacteria and fungi. results: patients were tested. / ( . %) had positive tblb. / had negative tblb ( . %). of the negative tblb group, / ( %) had positive ebb. tb cultures on all tblb biopsy samples were negative and bal was negative for bacterial and fungal growth in all cases. no patient suffered complications secondary to the procedure. conclusion: tblb remains a useful diagnostic test in the diagnosis of sarcoidosis serum fibroblastic growth factor- in acute sarcoidosis p other serum biochemical markers measured included calcium, phosphate, h urinary calcium, parathyroid hormone (ipth) and -hydoroxy vitamin d . results: subjects were male and were female. mean (sd) ipth was . ( ) ng/l, serum calcium . ( . ) mmol/l, serum phosphate . ( . ) mmol/l, serum -hydroxy vitamin d ( . ) nmol/l, and h urinary calcium excretion was . ( . , . ) mmol/l. fgf- was detectible only in those patients who also had hypercalciuria and was elevated in all those with hypercalcaemia. after adjusting for covariates using stepwise multivariate linear regression fgf- was independently associated with serum calcium conclusions: this study describes the distribution and determinants of serum fgf- in acute sarcoidosis for the first time. evidence is accumulating that fgf- may have a pathogenic role in adverse cardiovascular outcome, whether this applies to patients with sarcoidosis and normal kidney function merits further investigation convex probe endoscopic and endobronchial ultrasound (eus/ebus) for the diagnosis of sarcoidosis ebus and eus guided lymph node aspiration is a minimally invasive procedure widely used for diagnosis and staging of lung cancer. there has been increasing interest in utilising these modalities for the diagnosis of benign conditions including pirfenidone is an emerging therapy for limited ipf mcnicholas department of respiratory medicine, st. vincent's university hospital, dublin idiopathic pulmonary fibrosis (ipf) patients have reduced exercise capacity [ ], which correlates with parameters of pulmonary function and arterial oxygen tension resting pulmonary function testing and arterial blood gases were recorded. statistical analyses included student-t and mann-whitney u testing. maximal work load was reduced at . ± . % predicted corrected for sex, age and height. vo max was moderately reduced at . ± . % predicted. mean pre and post exercise borg score was and . a positive correlation was observed between vo max and fvc (r = exercise testing in the evaluation of diffuse interstitial lung disease pathophysiology of activity limitation in patients with interstitial lung disease diagnostic serum biomarkers to distinguish idiopathic pulmonary fibrosis from scleroderma-associated lung disease and healthy controls b. kennedy , p. branagan cork idiopathic pulmonary fibrosis (ipf) may be difficult to differentiate from scleroderma-associated interstitial lung disease potential biomarkers were measured by elisa or biochip immunoassay. differences between groups were calculated by unpaired t-tests. data are presented as mean ± sem. mean serum levels of kl- ( ± vs. . ± ng/ml elevated serum sp-d may help differentiate ipf from ssc-ild and healthy controls. the elevated serum sp-d in ipf compared to ssc-ild may reflect increased leakage from pulmonary capillaries arising from more extensive alveolar injury bone marrow and peripheral blood derived cd + cells as agents of tissue remodelling in idiopathic pulmonary fibrosis b cd + cells originate in bone marrow, circulate and then migrate to tissue where they may differentiate into macrophages. we investigated extrapulmonary cd + cells as a source of mediators of tissue remodelling in ipf. peripheral blood (pb) and bone marrow (bm) were obtained from /ml, p = . ) cd + cells. ipfs and controls did not differ significantly in ccl , ccl , tgf-b , ccl , mmp , mmp or timp expression in either pb or bm cd + cells. ipfs and controls do not demonstrate any quantitative differences in the expression of mediators of tissue remodelling in pb or bm cd + cells. nevertheless ccl as an indicator of pulmonary fibrotic activity in idiopathic interstitial pneumonias and systemic sclerosis circulating levels of the chemokine ccl but not cxcl are elevated and correlate with disease activity in rheumatoid arthritis diffusing capacity of carbon monoxide (dlco) are the physiologic characteristics mostly correlated with hrct findings. high fibrosis score and extent of the reticulation and honeycombing on ct are associated with increased the risk of death [ ]. we aim to investigate the correlations between radiological and physiological features of ipf. patients with ipf were recruited from our database. full pulmonary function testing and awake arterial blood gas were performed. survival data were collected after a period of follow up. hrct was scored by a core radiologist on-site. data was analysed using linear regression and spearman correlation high-resolution computed tomography in idiopathic pulmonary fibrosis: diagnosis and prognosis ct features of lung disease in patients with systemic sclerosis: comparison with idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia incidental abnormalities found on negative ct pulmonary angiograms performed on patients for suspected acute pulmonary embolism and with normal chest x-ray primary chest wall tumours and lung cancer invasion of the chest wall are the usual indications for chest wall resection and reconstruction. we evaluated all patients who underwent chest wall resection ± reconstruction in our unit from february to july . patients were identified. reconstruction was completed using either a composite of marlex mesh and methyl-metacrylate or a goretex sheet. reconstruction of the chest wall prevents post operative complications and restores the respiratory dynamics by avoiding paradoxical or harmful movements. in the majority of cases, it was possible to approximate the soft tissue over the reconstruction. in selected cases, our plastic surgical colleagues used a variety of flaps to approximate the defect.we looked at mean length of stay, histological types, morbidity and survival. there were no intra-operative mortalities. day mortality was . %.chest wall resection is not a common procedure but may be performed in high volume centres with low morbidity and mortality. in those with primary chest wall tumours, a wide resection margin is associated with low recurrence rates. in patients with nsclc (t ) involving the chest wall, en bloc lung and chest wall resection has a proven curative benefit with excellent year survival. fibreoptic bronchoscopy is considered a safe diagnostic tool [ ] . it is suggested, however, that post-bronchoscopy complication rate increases with age [ ] . we decided to study the complication rate and the outcomes of bronchoscopy in patients over years in our institution. a retrospective review of case notes of patients over years who underwent bronchoscopy between september and november was performed. data on complications experienced during and after bronchoscopy and the influence of the results on subsequent management were collated and analysed.ninety-six patients were included. mean age was . years (sd . ). thirty subjects ( . %) had a documented lung disease. fifty-nine patients ( . %) were current or ex-smokers. indications for bronchoscopy were; to evaluate for malignancy ( . %) and to evaluate for tb ( . %). post bronchoscopy complications were noted in eight ( . %) cases including hypoxia ( . %), infection ( . %), tachycardia ( %) haemoptysis ( %) and pneumothorax ( %). six patients required post bronchoscopy treatment for complications. malignancy was diagnosed in twenty cases ( . %) and infection was detected in six ( . %). as a result of bronchoscopy, management was altered in fifty-one patients.in conclusion, bronchoscopy is relatively safe and has good diagnostic utility in patients aged more than eighty years. the introduction of an ebus-tbna program more than halved the numbers of endobronchial brushings without a statistically significant change in the number of combined bals and washings or biopsies. we plan to investigate the impact of ebus-tbna on the diagnostic yield of brushings biopsies bal and washings. the use of fibreoptic bronchoscopy in an intensive care unit tbna and eus-fna in patients with suspected sarcoidosis after its implementation at a university hospital. methods: we retrospectively analysed data on all patients with suspected sarcoidosis who underwent ebus/eus since the start of the service. sensitivity and diagnostic yield were calculated based on cytology results and further invasive diagnostics for negative samples. resuts: over months patients ( males females) with a mean age of ± were assessed. % had stage i disease. underwent ebus and eus. lymph nodes (lns) were sampled in total with a mean of . /patient. diagnostic yield was . % with patients diagnosed with sarcoidosis and patient with tuberculosis. there were false negatives yielding a sensitivity for detecting sarcoidosis of %. tumour necrosis factor alpha (tnf-a) inhibitors have had a huge impact on the treatment of inflammatory arthritis, inflammatory bowel disease and psoriasis. there is also much interest in the use of tnf-a inhibitors in the treatment of refractory sarcoidosis [ ] . paradoxically sarcoidosis in response to tnf-a inhibitors is increasingly recognised with over cases reported to date [ ] . we report a series of three cases of sarcoidosis that developed on tnf-a inhibitors.a year old lady with a history of severe crohn's disease developed a right upper lobe consolidation and respiratory failure following two doses of adalimumab. investigations for tuberculosis were negative. a ct guided biopsy of a persistent right upper lobe infiltrate confirmed non caseating granulomas consistent with sarcoidosis.a year old gentleman on etanercept for ankylosing spondylitis presented with dyspnoea and a dry cough. cxr showed diffuse fibrotic change. ct findings and a transbronchial biopsy showing multinucleated giant cells led to a diagnosis of sarcoidosis.a year old man on adalimumab for rheumatoid arthritis was admitted with cough and purulent sputum. cxr and ct thorax confirmed mediastinal and hilar adenopathy. investigations were negative for tuberculosis. endobronchial ultrasound guided needle aspiration showed non caseating granulomas consistent with sarcoidosis.sarcoidosis in response to tnf-a inhibitors is a rare but increasingly recognised condition. following exclusion of tuberculosis a high index of clinical suspicion is needed to prevent a delay in diagnosis. the distribution of cd /cd ratios in patients with biopsy-proven sarcoidosis suggests that substitution of bronchoalveolar lavage cellular analysis for transbronchial biopsy is not advisable. pirfenidone has been approved for the treatment of mild to moderate ipf in europe. pirfenidone regulates the activity of tgf-b and tnfa. we evaluated a single centre experience with pirfenidone.a retrospective cohort design was used to study ipf patients prescribed pirfenidone. a titrating dose of pirfenidone was commenced on patients with an fvc[ % predicted and dlco[ %. primary outcome was change in percentage predicted forced vital capacity (fvc). secondary outcome was change in percentage predicted transfer factor (dlco). symptomatic patients were prescribed pirfendione. the mean age was . years. patient died due to an exacerbation of ipf, others discontinued pirfenidone secondary to adverse-events. patients reached target dose. subjects continued pirfenidone at a reduced dose. participants reported side effects likely related to pirfenidone. the most commonly reported side effects were gastrointestinal disturbance and photosensitivity. no significant decline in fvc or dlco was noted in patients who continued pirfenidone in . weeks follow up. key: cord- -fj j authors: morin, b. r.; kinzig, a. p.; levin, s. a.; perrings, c. a. title: economic incentives in the socially optimal management of infectious disease: when [formula: see text] is not enough date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: fj j does society benefit from encouraging or discouraging private infectious disease-risk mitigation? private individuals routinely mitigate infectious disease risks through the adoption of a range of precautions, from vaccination to changes in their contact with others. such precautions have epidemiological consequences. private disease-risk mitigation generally reduces both peak prevalence of symptomatic infection and the number of people who fall ill. at the same time, however, it can prolong an epidemic. a reduction in prevalence is socially beneficial. prolongation of an epidemic is not. we find that for a large class of infectious diseases, private risk mitigation is socially suboptimal—either too low or too high. the social optimum requires either more or less private mitigation. since private mitigation effort depends on the cost of mitigation and the cost of illness, interventions that change either of these costs may be used to alter mitigation decisions. we model the potential for instruments that affect the cost of illness to yield net social benefits. we find that where a disease is not very infectious or the duration of illness is short, it may be socially optimal to promote private mitigation effort by increasing the cost of illness. by contrast, where a disease is highly infectious or long lasting, it may be optimal to discourage private mitigation by reducing the cost of disease. society would prefer a shorter, more intense, epidemic to a longer, less intense epidemic. there is, however, a region in parameter space where the relationship is more complicated. for moderately infectious diseases with medium infectious periods, the social optimum depends on interactions between prevalence and duration. basic reproduction numbers are not sufficient to predict the social optimum. during an epidemic, there are several options available to private individuals interested in managing infectious disease risk. these include self-quarantine, vaccination, voluntary travel restrictions, contact reduction, prophylaxis, and preferential mixing (philipson ; geoffard and philipson ; philipson ; taylor and ampt ; brownstein et al. ; ferguson et al. ; germann et al. ; hunter ; rao et al. ; curtis et al. ; fenichel et al. ) . the choice of which measures to use frequently depends on costs-the cost of illness and the cost of risk mitigation. the cost of illness is determined by the likelihood that people will become infected if they make contact with an infectious individual, together with the cost of treatment and loss of earnings if they do become infected. the cost of risk mitigation is the resources sacrificed to reduce risk by some amount. diseases that are perceived to be highly infectious and life-threatening warrant greater private risk mitigation effort than diseases that are perceived to be only moderately infectious and with few symptoms (sanchez ; leroy et al. ; team ). diseases that are novel, and therefore uncertain in their effects, warrant greater private risk mitigation effort than diseases that are more familiar (taylor et al. ; presanis et al. ; rubin et al. ). it follows that private risk mitigation efforts might be expected to evolve over the course of an epidemic as information improves. a number of studies have, for example, tracked changes in the efforts made by private individuals to reduce disease risk during the a/h n epidemic bayham et al. ; springborn et al. ) . the problem addressed in this paper is that disease risk mitigation by one individual affects the well-being of both that individual and others. people vaccinating themselves against seasonal influenza, for example, reduce the likelihood that they will become infected, but also reduce the likelihood that they will become infectious. vaccination, in such cases, is said to be an impure public good. it confers benefits on the individual, but also on those with whom the individual interacts (sandler et al. ) . on the other hand, people avoiding infection now by sequestering themselves may simply delay the moment at which they become infected and infectious and hence may unwittingly prolong an epidemic . private diseaserisk mitigation, in this case, is an impure public bad. in providing benefits to the individual, it imposes costs on society. because people cannot capture any wider benefits they confer, and are not confronted by any wider costs they impose, they have little incentive to take those benefits and costs into account when making their decisions. the public good (bad) is therefore under-provided (over-provided). the general response of public health authorities to the under-provision of private disease risk mitigation is the adoption of social distancing measures that include quarantines, travel restrictions, and school closures. these are not, however, the only options available. since private disease risk mitigation efforts reflect the costs of illness and illness avoidance, interventions that change those costs are also among the options available to public health authorities . in this paper, we consider policy instruments that close the gap between the socially and privately optimal levels of disease risk mitigation. such policy instruments align the socially and privately efficient solutions by aligning the social and private cost of diseaserisk mitigation. we assume private disease-risk mitigation strategies similar to morin et al. ( ) and consider a control that operates on the cost of illness. this has parallels with the health belief model originally developed in the s, which supposes that private health-risk mitigation depends on beliefs about the likelihood and consequences of contracting disease, the efficacy and costs of the proposed action, and the decision makers' capacity to implement the action (rosenstock ) . we note that risk is used here in the economic sense. it is the product of the probability that an event will occur and the cost of the event if it does occur. the two most common approaches to the management of disease risk are mitigation and adaptation. mitigation implies an action that reduces the probability that the individual will fall ill. adaptation implies an action that has no effect on the probability that the individual will fall ill, but reduces the cost of illness. the probability that an infection will occur, cbp si (/(t)), depends on three things: contact volume c, the probability that contact results in infection b, and the probability that contacts are with infectious individuals p si (/(t)), a function of mitigation effort /(t). we treat the first two of these parametrically, although we acknowledge that they may be affected by, respectively, strategies to quarantine infectious individuals or to reduce activity levels , and strategies such as prophylaxis or vaccination (weycker et al. ) . we focus instead on the probability that contact will be made with infectious individuals. it was shown in fenichel et al. ( ) that despite travel restrictions during the h n swine flu outbreak, individuals did not cancel travel plans (reduce contact volume) so much as alter the timing of their travel. in previous work, we have shown the equivalence of private strategies targeting c or p si ) and have identified the social costs and benefits of private strategies that alter disease trajectories (morin et al. ) . in this paper, we study the impact of changes in the private cost of illness on mitigation effort, and show how diseases with the same r can trigger very different interventions, depending on which parameter drives the basic reproduction number. the paper is structured as follows. we first describe a model of preferential mixing based on observable disease states. we then detail two economic decision models, one private and one public, and show how they are coupled. since the models are not tractable enough to yield analyt-ical solutions, we solve both numerically in order to show the epidemiological effects of public taxes or subsidies on private cost of illness. this is followed by a discussion. our modeling approach builds on existing affinity-based mixing compartment models where compartments represent different disease states (busenberg and castillo-chavez ; blythe et al. ; castillo-chavez et al. ; fenichel et al. ; morin et al. ) . we suppose that individuals mix preferentially, conditional on their own disease state and the (observable) disease states of others. the only information available to individuals is the expression of symptoms in either themselves or others. as in , the resulting mixing strategy depends on the relative costs of illness and illness avoidance. this framework has been shown to provide the most mathematically general solution to the problem of who mixes with whom under the assumptions of symmetric contacts (blythe et al. ) . in what follows, individuals are defined only by their health state, although they could just as easily be grouped according to various shared attributes such as economic status, cultural or ethnic identity, geographical location, age, or disease awareness. we suppose that all individuals who do not show symptoms-because they have either not become ill, are asymptomatically ill or have recovered from illness-are treated equally by others. the use of the affinity framework allows three factors to determine the volume of contact between groups of like individuals: ( ) the size of each group, ( ) the nominal activity level or disease-free contact rate of each group and ) the relative affinity/disaffinity between groups. in what follows, we treat the affinity/disaffinity between groups as endogenous to the epidemiological system. susceptible individuals, or at least individuals who believe themselves to be susceptible, choose the people with whom they mix in order to alter the probability that they will encounter infectious individuals and subsequently become ill themselves. in the most general case, people who believe themselves to be susceptible at some time are taken to include all those who have been free of symptoms up to that time. this includes those who are actually susceptible, those who are asymptomatically infectious and those who are recovered but have never had symptoms. we hold the nominal level of activity (the contact rate) constant throughout the course of the epidemic and take it to be equal for all individuals. this makes it possible to consider only the effect of changes in mixing preferences. see fenichel et al. ( ) for a treatment that selects the volume of contacts, and for an analysis of the conditions under which choice of contact rates and avoidance effort are equivalent strategies. the main dif-ference between strategies is that while choice of contact rates allows complete isolation, choice of avoidance effort does not. to illustrate the approach, we first focus on a susceptible-infectious-recovered (sir) model-but note that we will be reporting results for a range of other models including susceptible-exposed or latent-infectious-recovered (seir), one-path and two-path susceptible-asymptomatical infectious-infectious-recovered (one-path sair, two-path sair). in the sir case, only susceptible individuals are free of symptoms. the disease dynamics are summarized in three differential equations: as is standard with the sir model, we let c be the nominal contact volume of all individuals. p si (/(t)) is the conditional probability that a contact made by a susceptible individual, committing /(t) effort to avoiding infection at time t, is with an infectious individual, and c is the rate at which an individual recovers and becomes immune. i t ð Þ and r t ð Þ are, respectively, the numbers of infectious and recovered (immune) individuals. the conditional probability that an individual in the ith disease state encounters an individual in the jth disease state is given by the elements of a time-dependent mixing matrix, p(t) = (p ij (t)), that is taken to satisfy three axioms economic incentives in the socially optimal management (busenberg and castillo-chavez ; blythe et al. ; castillo-chavez et al. ): . p ij ; for all i; j fs; e; a; i; rg . p j fs;e;a;i;rg p ij ¼ ; for all i fs; e; a; i; rg; . iðtÞp ij ¼ jðtÞp ji ; for all i; j s; e; a; i; r f g : these three axioms imply that, collectively, susceptible individuals have the same expectation of encountering infected individuals as infected individuals have of encountering susceptible individuals. it has been shown that the unique solution to these mixing axioms is given by where mðtÞ ¼ sðtÞm s ðtÞ þ iðtÞm i ðtÞ þ rðtÞm r ðtÞ; and uðtÞ ¼ ð/ ij ðtÞÞ is a symmetric affinity matrix, in this case . the main difference between the approach here and previous use of affinity models is that we treat the elements / ij (t) of the affinity matrix are private choice variables. they may be interpreted as the effort that the representative individual in disease state i makes to avoid individuals in disease state j, if / ij (t) < , or to associate with individuals in disease state j, if / ij (t) > . if the representative individual in every disease state i makes no effort to avoid individuals in disease state j, and vice versa, then / ij (t) = . we then have classic proportionate mixing. we take zero elements in the affinity matrix to be evidence of 'avoidance-neutrality.' that is, they show the representative individual to be neutral about a pairing event with someone from another disease class. by contrast, negative (positive) elements reflect the desire of the representative individual in one disease state to avoid (seek out) individuals in other disease state. avoidance, / ij (t) < , can result from individuals in both states wishing to avoid one another; individuals in one state wishing to avoid individuals in other states who may be neutral to the pairing; or individuals in one state wishing to avoid individuals in other states more strongly than those individuals favor the pairing. similarly, engagement results from individuals in both states favoring the pairing; individuals in one state seeking out individuals in other states who may be neutral to the pairing; or individuals in one state wishing to engage with individuals in other states more strongly than those individuals wish to avoid the pairing. this is a similar measure to that used in models of assortative mating (karlin ) and selective mixing (hyman and li ) and is a form of a contact kernel (gurarie and ovaskainen ) . the elements of the affinity matrix, / ij (t), describe what the representative individual in each health state wants. what they actually get depends both on the preferences of others in the population and on the relative size of all health classes. more particularly, the elements of the mixing matrix p = (p ij ) depend both on the proportion of the population in each disease state and on the affinity matrix. they describe the conditional probabilities that an individual of disease state i contacts someone in disease state j. in what follows, we focus on individuals who believe themselves to be susceptible (who have been symptom free up to that point) and assume that they maximize the net present value of the contacts they make, taking into account the cost of illness and illness avoidance, by choosing the effort to commit to preferential mixing: the elements of u t ð Þ. formally, the decision problem for individuals who believe themselves to be susceptible, collectively labeled x, is to choose the level of mitigation effort, / xi (t), in order to maximize the difference between the benefit of not being symptomatic, b, and the cost of mitigation effort, c(/ xi (t)), given the weight they place on future well-being (the discount rate d) and their planning horizon, t. if susceptible individuals are averse to mixing with symp-tomatic (infectious or otherwise) individuals in the sir model, and if all others are neutral, u t ð Þ has the structure: in which represents neutrality and / si (t) < represents the effort susceptible individuals make to avoid mixing with infectious individuals at time t ). this defines we may then write the mixing matrix of conditional probabilities as each figure shows the prevalence with public health authority intervention, i pha (t), and with only the private mitigation effort, i priv (t), for various b. the present value difference (pvd) in these curves is shown along with the intervention level of the public health authority. positive pvd, corresponding to an early reduction in incidence that is not offset by the future ''fatter'' tail, is analogous to a tax on the cost of illness. negative pvd was always found to include an early increase infection and is analogous to a subsidy on the cost of illness. the magnitude of intervention is directly related to the magnitude of the pvd. as shown in morin et al. ( ) , we note that mitigation effort is restricted to the range: with proportionate mixing resulting from applying effort. p si (t) = i(t) is the maximum probability of contact and private quarantine of infectious individuals at the left endpoint, p si (t) = is the minimum probability of contact. from an economic perspective, private quarantine implies that the marginal cost of illness is greater than the marginal cost of illness avoidance for all levels of illness avoidance effort. we assume that a forward-looking representative individual, who believes himself/herself to be susceptible, seeks to mitigate disease risks by avoiding those who are obviously (symptomatically) infectious. we suppose that individuals may belong to any one of the following epidemiological states at a given time: (s)usceptible to the disease, (e)xposed or latently infected being asymptomatic and noninfectious, (a)symptomatically infectious, (i)nfectious with symptoms, or (r)ecovered and immune to the disease. individuals who believe themselves to be susceptible may include those in states s, e, and a. we further assume the motivation for selective mixing is the desire to avoid the costs of illness only. we do not allow individuals to behave altruistically. we also assume that individuals who know themselves to be infected have no incentive to avoid others. only susceptible people (or those who believe themselves to be susceptible) react to disease risk. this includes all individuals in health classes x ¼ s; e; a; or r a (recovered from asymptomatic infection). because all individuals who react to disease risk consider themselves to be equally susceptible to the disease, their mixing decisions are both identical and symmetric [i.e., all / xi (t) = / ix (t) are equal to one another and all other entries in u t ð Þ are ]. formally, these individuals screen contacts by choosing the elements / ij (t) of the matrix uðtÞ, i, j [ {s, e, a, i, r a } so as to maximize the difference between the expected benefits of contact and the expected cost of illness and illness avoidance, given their current health state. the benefits of contact range from the satisfaction to be had from purely social engagement to the financial gains to be had from market transactions with others. for simplicity, we assume that the benefits of a contact are financial gains and that they are the same for individuals in all health states, b i ¼ b; i s; e; a; i; r f g . the cost of illness generally includes both forgone earnings, lost wages, and the cost of healthcare. for simplicity, we take the cost of illness to be the cost of treatment. the cost of illness avoidance is simply the cost of the effort made to avoid contact with people who are ill: the cost of choosing / ii t ð Þ; i s; e; a; r f g . the net benefits of contacts with others by an individual in the ith health state at time t thus comprise the difference between the benefit of contacts made in that health state, b i , and the cost of disease and disease risk mitigation, table ). all individuals within a particular disease class are assumed to behave in the same way. we define u i ð/ ij ðtÞÞ ¼ b i À c i ð/ ij ðtÞ; iðtÞÞ to be the net benefits of contact to the representative individual in health state i at time t. we define e v j t þ s; / ij t þ s ð Þ to be the individual's expected value function from time t + s forward, where the probability that the individual will be in health state j in the future depends on the disease recovered from symptomatic infection, immune no b r susceptible, exposed, asymptomatically infectious, and recovered (from asymptomatic infection) individuals all choose to mitigate risk as if they were susceptible and thus carry the cost of mitigation. dynamics and their mixing strategy while in health state i. the decision problem for the representative susceptible individual in health state i may be expressed via the hamilton-jacobi-bellman equation: where s is a short interval of time. this is subject to the dynamics of the disease: eq. ( ), table the h-j-b equation identifies the problem solved by the representative individual in state i: to maximize the expected net value of current and future contacts by choosing the extent to which they mix with individuals in other health classes. following fenichel et al. ( ) , we assume that individuals form their expectations adaptively. the value function v i (t, h(t)) is defined recursively as the sum of the current net benefits of contact in health state i given the information available at time t plus the discounted stream of expected net benefits over the remaining horizon. this expectation is conditional on the effects of disease risk mitigation decisions on the probability of transitioning between health states. more particularly, we assume individuals observe the state of an epidemic at time t and make a forecast for the epidemiological trajectory over the interval s. their mixing strategies are then adapted over time as they make new observations on the state of the epidemic. we assume that individuals make the simplest forecast-that all disease classes are constant over the interval s, but that they adapt to new data as it emerges. the representative individual will increase effort to avoid infection up to the point where the marginal cost of illness avoidance is just offset by the marginal benefits it yields-the avoided cost of illness. efforts to avoid infection will be increasing in the cost of illness and decreasing in the cost of illness avoidance. in models without risk mitigation, disease dynamics may be completely characterized from initial conditions. with risk mitigation, the evolution of the epidemic reflects feedback between the cost of disease and disease avoidance on the one hand, and averting behavior on the other [see (fenichel and horan ; horan et al. ) for further discussion]. to solve the problem, we take a discrete time counterpart to eq. ( ) and solve numerically using techniques similar to those in fenichel et al. ( ) . specifically, we solve the adaptive expectation problem using a method we call cast-recast. at each time, the individual solves the h-j-b equation using backwards induction from their time horizon ( days) to the present, while supposing that there is no change to the state variables over the time horizon. having determined the optimal mitigation effort, / ij (t), the individual commits that effort until the next time step (day). that is, their mitigation effort is held constant for time step. the ordinary differential equations describing the disease dynamics are advanced, and the process is repeated. note that the private forecast used has little effect on the optimal outcomes so long as the forecast period, the time interval s, is short relative to the disease dynamics. if the epidemic evolves rapidly relative to the period over which the individual commits to a fixed level of risk mitigation, then the assumption that the disease states are constant may induce errors. in previous work using this method morin et al. morin et al. , morin et al. , , we found a smooth response-the decisions made by individuals were much the same from day to day. since the epidemic evolves on a timescale of weeks, this gives us confidence in the cast-recast method for the private problem. in the discussion, we outline when the results from such a method may favor more rapid transmission of epidemiological state variables, and when individual behavior choices need to be more ''agile'' in order to match timescales with the disease spread. the choice of / ij (t) maximizes the private net benefits of contact for the individual over the course of an epidemic, given the private cost of illness and illness avoidance. however, as was shown in morin et al. ( ) this may well be publically suboptimal, depending on the social rate of discount or the social cost of illness. the social cost of illness is the sum of the costs borne by all infected and symptomatic individuals, together with the cost of disease avoidance by all others. in a real system, it would also include the infection risk borne by healthcare workers, but we do not address that here. of the many intervention options open to public health authorities-quarantine, social distancing measures such as school closures, vaccination campaigns and so on-we focus on instruments that change risk mitigation by changing the private cost of illness. in doing this, we follow a literature that integrates epidemiology and economics to explore ways in which economic behavior affects disease spread . this literature has concentrated on the economic causes and epidemiological consequences of peoples' contact decisions hammer , ; barrett and hoel ; funk et al. ; fenichel et al. ; funk et al. ; springborn et al. ). by treating the economic factors behind contact and mixing decisions as central elements in disease transmission, the approach opens up a new set of disease management options. in what follows, we suppose that the public health authority is able to use an economic policy instrument to alter the private cost of illness. we consider the instrument, r d , d sir, seir, one path sair, two path sair f g see tables and , which may be interpreted as a disease-specific tax or a subsidy on the cost of illness, c i i; r d ð Þ := c i þ r d ð Þ. if r d > (illness is 'taxed'), the private cost of illness is increased. enforced, uncompensated sick 'leave' would be an example of this. we expect this to increase disease-risk mitigation effort and hence the illness avoidance costs carried by individuals. if r d < (illness is 'subsidized'), the private cost of illness is reduced. subsidized health insurance schemes would be an example of this. we expect this to reduce disease-risk mitigation effort, and with it the illness avoidance costs carried by reactive individuals. by changing the privately optimal level of disease-risk mitigation, it is possible to change overall disease dynamics. there are many ways in which interventions change the private cost of illness in real-world conditions, ranging from direct subsidies or taxes on drugs and treatment, through health insurance costs and coverage, to statutory obligations on sick leave. we suppose that r d can be applied in a way that proportionately reduces or increases the relative private cost of illness. this would be consistent with, for example, mandatory insurance cover for a specific proportion of potentially forgone earnings. our baseline case assumes that the policy instrument is revenue neutral. if r d < (illness is subsidized), the cost is met by a levy on all income from contacts. if r d > (illness is taxed), the revenue is returned as a tax benefit on all income from contacts. since taxes and subsidies both potentially impose an efficiency cost in the form of a deadweight loss of consumer and producer surplus, we include a proxy for this in the optimization problem. more particularly, we include a term that specifies any deadweight loss as a proportion of the cost of taxes or subsidies. the public health authority's problem for an sir disease thus takes the form: subject to the disease dynamics described by the relevant compartmental epidemiological model and to the private decision problem described in eq. ( ). that is, the public health authority selects r d so as to maximize the net benefits of risky contacts to society-where society is the sum of all individuals in all health classes. the final term in the public health authority's problem is our proxy for the deadweight loss associated with taxes or subsidies on the cost of illness. a [ [ , ] is the proportion of the cost of the intervention that is recovered. to solve these two problems, we maximize the integral in eq. ( ) over the entire epidemic by solving the complete private problem for each ''guess'' of r d . this was implemented using matlab's fminbnd function. we investigated the effect of a subsidy/tax on the cost of illness on private disease risk mitigation in the key epidemiological classes across four compartmental models: sir, seir, and two sair models, a one-path progression and a two-path progression. within the one-path model, susceptible individuals are first asymptomatically infectious and then progress to symptomatically infectious and then immune. in the two-path model, a susceptible individual becomes either asymptomatically or symptomatically infectious and then recovers. individuals who recover from asymptomatic infection (r a ) are expected to behave as if they are susceptible. individuals who recover from symptomatic infection (r i ) are not. because we have assumed no heterogeneity aside from health status, the use of a disease-specific instrument, r d , has the potential to be efficient. if the population were heterogeneous, however, we would expect to need more targeted interventions. we did not consider models with reentry to the susceptible class, e.g., sis, sirs, and other cyclical models. this is for two reasons. first, each of these models is capable of endemic levels of infection. this, combined with the fact that people may experience reinfection, would require individuals to form expectations (possess memory) with respect to the impact of different avoidance strategies. second, numerical simulations of these models reveal very broad oscillations that confound comparison with single outbreak models. there are no entries (births) or removals (deaths) from the system (see table for example diseases listed for each model). each model considered here is therefore a so-called single outbreak model; the population is not only kept at a fixed number, but it is also closed to the introduction of new individuals. we modeled the dynamics of the epidemic types in table using ordinary differential equations (see table ). this has two main implications for disease dynamics: (a) once nonzero, the state variables will never again be zero in finite time, and (b) in an infinitely small amount of time ''mass'' will move into each compartment as long as the transition rates are nonzero. these affect the interpretation to be given of the point at which an epidemic is ''over.'' it is feasible that extreme risk mitigation early in the course of the epidemic could wipe out the infection within a population. however, within the differential equation framework, as soon as mitigation weakens, coupled with the fact that there is a nonzero infectious population with potentially a very large susceptible population, the infection will again spread, potentially causing additional peaks. the instantaneous transfer of individuals from one compartment to another also serves to induce reaction timing that may not conform to data. in recognition of this, we supposed that the differential equations represent an expectation of outcomes over a population divided between three health classes. in previous work (morin et al. , , we demonstrated that private disease-risk mitigation reduces peak symptomatic infection levels and the total number of people who experience symptomatic infection while prolonging the epidemic. we seek to understand how public health interventions aimed at minimizing the cost of disease and disease avoidance affect individual risk mitigation decisions. our measure of mitigation effort is a relative one. it is the proportion of maximum mitigation effort undertaken, where maximum effort is defined as that which results in p si (t) = . this enables us to compare effort across diseases. the background against which public health authority intervention occurs is that private mitigation effort is increasing in infectiousness (b), infectious duration ( = c ), and the share of the public cost of intervention recovered by the public health authority (a). in other words, private mitigation effort is increasing in the cost of illness. we find that in all cases peak effort occurs early in the epidemic-within the first days for our parameters-and that action is taken sooner, the greater the severity of the disease. our results on the socially optimal response this induces for different diseases follow. these results are driven by the assumptions we make about the cost of illness (equal to the benefit gained from being healthy) relative to the cost of mitigation. specifically, we assume that the cost of mitigation is low relative to the cost of illness. to explore the sensitivity of the optimal intervention strategy of the public health authority to the severity of disease, we varied b and c over intervals so that b=c : ; : ½ ; and a in steps of . from to , with baseline values of a = , b ¼ : , and c = / . while the socially optimal public health authority intervention decision can be viewed as a function of the disease's basic reproduction number r = b/c, the two components of r (infectiousness and duration) have rather different effects on the public health authority intervention. nor is the optimal social response monotonic in either case. r (b) tests the sensitivity of health interventions to the impact of infectiousness on r and tests the sensitivity of health interventions to the impact of disease duration on r . for r (b) [ [ . , . ] , the optimal public health authority intervention involves an increase in the private cost of illness-a 'tax' on illness that will stimulate higher levels of private disease risk mitigation. as infectiousness falls, the optimal public health authority incentive to mitigate first rises and then falls, the turning point being determined by parameters describing both the cost of illness and the cost of illness avoidance. the optimal tax in this case reaches a maximum of % for r (b) = . . for diseases where infectiousness is either very low (r (b) < . ) or very high (i.e., r (b) > . ), the optimal public health authority intervention involves a reduction in the cost of illness-a 'subsidy' on illness that lowers private diseaserisk mitigation effort (fig. ) . the duration of illness has a slightly different effect. note that small r (c) values are indicative of diseases of short duration. for r (c) = . , for instance, the illness is symptomatic for less than days. we found that for diseases of moderate to short duration, r (c) < . , it would generally be optimal for the public health authority to stimulate an increase in private risk mitigation by raising the private cost of illness-by taxing illness. on the other hand, for diseases of longer duration, r (c) > . , we found that it would always be optimal for the public health authority to reduce private risk mitigation by lowering the private cost of illness. given the assumed cost of illness and mitigation effort, private individuals will overreact to the risk of diseases of long duration and high infectiousness and will underreact to diseases of short duration and low infectiousness. to see the trade-off between infectiousness and duration, we considered the range of (b, c) that generates r [ . , . ]. figures and show the parameter combinations that leave the public health authority indifferent between intervening or not over this range of values, or that favor intervention to increase or decrease privately optimal disease risk mitigation. while infectiousness and duration trade off against each other, the relation is not linear. where private individuals take excessive precautions, pha interventions will generally discourage private risk mitigation for highly infectious diseases of long duration. this changes, however, if the infectiousness of diseases of long duration is very low or the duration of highly infectious diseases is very short. symmetrically, pha interventions will generally encourage private risk mitigation for less infectious diseases of short duration. once again, though, this changes if the infectiousness of diseases of short duration is very high or the duration of less infectious diseases is very long. the socially optimal intervention will also dependintuitively-on the degree to which the susceptible and infected individuals carry the cost of public health authority interventions. to capture this, we included a cost recovery parameter, a, and explored the sensitivity of the optimal intervention strategy of the pha to variation in a (fig. ) . the proportion of the cost of a subsidy, on the cost of illness, recovered through taxation (or the proportion of tax revenues returned to the wider population) is given by a. specifically, we varied a in steps of . from to with a baseline value of a = . for the baseline values for infectiousness and duration, b ¼ : and c = / , we found that the greater the value of a, the less one needs to subsidize the cost of illness in order to align private and social optima. the reason is that the cost of illness is affected both by the instrument itself and by cost recovery. as the rate of cost recovery increases, the cost of illness rises along with the privately optimal level of risk mitigation. indeed, for a ! . the public health authority switches from discouraging to encouraging private risk mitigationfrom a subsidy to a tax. the inclusion of a class of individuals who have unknowingly contracted the illness but are not a danger to others (they are asymptomatic and noninfectious) changes the economics of the problem in important ways. on the one hand, it unnecessarily increases the aggregate cost of mitigation since exposed individuals continue to mitigate, even though they there is no need. on the other hand, by driving up p se and reducing p si , exposed individuals reduce infection rates below those that would occur if only susceptible individuals mitigated risk. that is, the risk mitigation undertaken by exposed individuals confers an external benefit on society. the result is that public health authority intervention in the seir case increases private risk mitigation for all but the most severe diseases (i.e., for all r . ) (fig. ) . we also note that the basic reproduction number for the seir model is identical to that for the sir model and does not involve the latent period. over reasonable latent periods from to days, we found an increase in the 'tax' rate applied by the public health authority, but no switching or nonlinearities in behavioral response. the big differences between the sir and seir cases are that the introduction of a latency period slows the spread of disease, while the mitigation by exposed individuals reduces the rate of new infections. interestingly, when we decomposed the marginal benefit of public health authority intervention by the infectiousness and duration of disease, we found the marginal benefit of intervention to be increasing in the duration of disease, but decreasing in infectiousness (fig. ) . as b increases and causes r (b) to pass . , we found the marginal benefits of public health authority intervention to be monotonically decreasing. this is because @ / si @r d @b < : however, as duration of disease increases, causing r c ð Þ to increase, we found the marginal benefits of pha intervention to rise monotonically after approximately . . private disease-risk mitigation in the sir case reduces peak prevalence but prolongs the epidemic at higher levels than would have occurred in the absence of mitigation. in the seir case, by contrast, private disease risk mitigation reduces peak prevalence and prolongs the epidemic, but at lower levels than would have occurred in the absence of mitigation. moreover, the longer the duration of the disease the greater the social net benefits this offers. the one-path sair model introduces an asymptomatically infectious stage between susceptible and symptomatically infectious. these individuals now undertake risk mitigation because they do not know they're infected. unlike the seir case, however, this is potentially harmful to the population. while @p si @/ si < , it is also true that @p sa @/ si [ . we may assume that the two classes have different levels of infectiousness. the net effect of private disease risk mitigation is therefore given by the combined impact of two things: the marginal reduction in the infection rate due to the avoidance of infected people and the marginal increase in infections due to susceptible-asymptomatic contact. the marginal effect of risk mitigation through mixing is @p si @/ si b i and the marginal effect of susceptible-asymptomatic contact is @p sa private risk mitigation reduces prevalence regardless of the actions of asymptomatically infec- it has the opposite effect. whether the public health authority seeks to increase or decrease private disease risk mitigation effort depends on the net effect. given our baseline parameters, if b a < À : b i þ : then the public health authority will seek to increase private mitigation, and if b a [ À : b i þ : , it will seek to reduce private mitigation. the strength of intervention increases with the distance of (b i , b a ) from the line b a ¼ À : b i þ : (which is roughly analogous with the condition that the basic reproduction number is ). in the two-path sair model, asymptomatically infectious individuals can spread disease due to their (redundant) risk mitigation actions. as in the seir model, those who've recovered from asymptomatic infection undertake mitigation that does not benefit them, but may offer a benefit to susceptible individuals by reducing their chance of making infectious contacts. in some sense, the r a class becomes like a vaccinated class of potentially highly connected individuals. they therefore dilute the contact pool for susceptible individuals. we find the interesting effect that whether to tax or subsidize is sensitive only to the recruitment into the i class. if the process favors the generation of symptomatic infection, then the pha intervenes with a tax on illness. if the process favors the generation of asymptomatic infection, then the pha intervenes with a subsidy. a process favorably generates asymptomatic infection when b a p sa p aa þ b i p si À p ii ð Þ[b a p sa À p aa ð Þ þ b i p si p ii where p xx is the probability that an infected person of type-x creates another individual of type-x when they spread infection. therefore, adjustments will be made to the cost of where @/ si @r d cancels out from both sides. if p aa ; p ii < , then the marginal condition is never met and no mitigation will occur. likewise, if both p aa ; p ii [ then mitigation will occur indefinitely until p si = . if one is less than / with the other greater, then the level of mitigation is unclear because it now depends on the relative strengths of infection and the sensitivities of pair probabilities to mitigation. when private disease-risk mitigation has general epidemiological consequences, the optimal level of mitigation should be determined by reference to the costs and benefits to society at large. because disease-risk mitigation changes the characteristics of an epidemic, it changes the social costs and benefits of disease. if private mitigation reduces prevalence, for example, it confers benefits on society. if it increases the duration of an epidemic, on the other hand, it imposes costs. since the private and social calculus of the costs and benefits of mitigation are different-individuals calculate only the costs and benefits to themselves, society calculates the costs and benefits to all-we expect the privately and socially optimal level of disease risk mitigation to differ. we have argued that individuals base their mitigation decisions on the expected net benefits of particular actions. very similar arguments have long been made in the medical literature. the health belief model, for example, was developed to explain why people undertook (or abstained from) private disease-risk mitigation (champion and skinner ) . the model, originally developed in the s, assumed that individuals engaged in health-risk mitigation to reduce the threat of illness based on their beliefs about four things: the likelihood of contracting disease; the consequences of falling ill (symptoms, loss of work wages, loss of personal interactions); the efficacy and costs of the proposed action; and their capacity to undertake the action adequately. it also assumed that individuals responded to incentives-'cues to action' that signaled susceptibility, severity, costs and the like. the perceived benefits and costs of alternative behaviors determined which behaviors would be undertaken (rosenstock ) . in the context of this paper, the perceived susceptibility of an individual at time t , conditional on a mitigation effort /(t), is given by r t t exp Àbp si / s ð Þ ð Þ ð Þ ds, and the expected cost of illness is the daily forgone income (adjusted by public health authority intervention) multiplied by the expected duration of illness, c ill ( + r d )/c. while we only allow a single risk mitigation choice, affinity-based mitigation, we allow it to vary from ''doing nothing'' (/(t) = ) where x(t) is the total population of susceptible individuals). as in the framework of the health belief model, we expect that mitigation effort applied will be increased up to the point at which its cost is just offset by the benefits, in terms of the reduced probability of illness. applications of the health belief model to disease-risk mitigation in the swine flu outbreak found that the framework adequately segmented the respondent populations into those who vaccinated and those who did not (janz and becker ) . they found that perceived susceptibility, benefits, and barriers were strongly correlated with vaccine-seeking behavior and that these results were strengthened by the fact that, even if it was unsuccessful, the vaccine alleviated the symptoms of illness (aho ; cummings et al. ; larson et al. ; rundall and wheeler ; janz and becker ) . the central proposition of this paper is that since disease-risk mitigation is a function of the private cost of disease, it can be managed through changes in the private cost of disease. whether private disease-risk mitigation is above or below the socially optimal level of mitigation, there exist interventions that have the potential to align private choices with the interests of society. our numerical results on the difference between the privately and socially optimal mitigation reflect the specific assumptions made about the relative cost of disease and disease avoidance. these were selected to illustrate the range of potential responses and the sensitivity of responses to disease characteristics. given our cost assumptions, we find that for sir diseases individuals confronted with highly infectious illnesses of long duration overreact to the associated risks. they mitigate more than is socially optimal. conversely, individuals confronted with short illnesses of low infectiousness underreact. for seir diseases, on the other hand, the privately optimal level of disease-risk mitigation is less than the socially optimal level for almost all parameter values. the gap between privately and socially optimal investments in risk mitigation is partly due to the fact that private individuals base their risk mitigation decisions on the observed health state of others-whether or not others are symptom-free. this is a source of error that is greater in some cases (seir, two-path sair) than others (sir, onepath sair). it is also due to the epidemiological effects of actions that influence when a susceptible person becomes infected in the course of an epidemic. one of the main external effects of private disease-risk mitigation is the prolongation of epidemics and with it the cost of diseaserisk mitigation. we therefore considered interventions that ran in both directions: subsidies on the cost of illness (e.g., health insurance) that discourage mitigation and taxes (e.g., uncompensated mandatory days off or other such penalties) that encourage mitigation. it can, by the way, be shown that subsidies or taxes on the cost of mitigation rather than the cost of illness have similar effects. for any adjustment to the cost of illness, there exists an adjustment to the cost of mitigation that produces the same private response. it does not follow, however, that the two instruments are equally efficient. adjusting the cost of illness alters 'severity' in the language of the health belief model while adjusting the cost of mitigation alters 'barriers.' which is the more cost-effective in any real application would depend on the effort needed to achieve the desired epidemiological effect and the relative cost of effort in each case. within the sir model, each individual has perfect information on their infection state and all relevant information on others (infectious or not). the other three models each introduce error due to the existence of asymptomatic classes. they also introduce changes in disease dynamics that affect the socially optimal level of risk mitigation. whether it is socially optimal to intervene in ways that increase or decrease private disease risk mitigation depends on the effect of mitigation on the time profile of infections. the point has already been made that mitigation reduces peak prevalence but also prolongs epidemics. when the additional cost of the longer (and potentially fatter) tail of an epidemic outweighs the benefits offered by a reduction in peak prevalence, the pha will intervene to reduce private risk mitigation-and vice versa (as shown in fig. ) .the advantages of instruments that operate on the cost of illness are that they can be put in place in advance of an outbreak and that they operate automatically. diseasespecific insurance or treatment costs, for example, can be established in advance but only influence private decisions when an outbreak occurs. we have not considered particular instruments in this paper and note only that the effect of each depends on how it would impact the relative costs and benefits of mitigation actions. there is a range of cost-based instruments that could be applied to diseases whose properties are well understood. the instruments that should be applied to specific diseases are those expected to cost effectively close the gap between socially and privately optimal disease risk mitigation. while we consider only policy instruments determined in advance of an epidemic-i.e., that correspond to the known properties of known diseases-we acknowledge that this would not be reasonable in the case of novel or emerging infectious diseases. there is less scope for the use of incentives of this type in such cases, but it would be worth considering how the choice of r d might evolve during the course of an epidemic in future work. finally, it is worth noting that any intervention of this kind in a real-epidemiological-economic environmental would have distributional consequences. it would make some people better off and some people worse off. we have abstracted from any distributional consequences in this paper by treating all individuals in each disease class as homogeneous, and by assuming that the payoff to contact is the same for everybody. implementation of a policy instrument of this kind in a real system would, however, need to take account of the distributional goals of the society concerned. infectious diseases are a comparatively small part of the burden of disease in highincome countries, but still the largest part of the disease burden in low-income countries. this is precisely where distributional issues are of greatest concern and where individuals are least able to bear the cost of disease-risk avoidance. participation of senior citizens in the swine flu inoculation program: an analysis of health belief model variables in preventive health behavior measured 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on aggregate contact patterns and resulting epidemic spread the social benefits of private infectious disease-risk mitigation merging economics and epidemiology to improve the prediction and management of infectious disease private vaccination and public health: an empirical examination for us measles economic epidemiology and infectious diseases the severity of pandemic h n influenza in the united states adherence to self-quarantine recommendations during an outbreak of norovirus infection the health belief model and preventive health behavior public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey factors associated with utilization of the swine flu vaccination program among senior citizens in tompkins county ebola viruses a conceptual framework for understanding global and transnational public goods for health optimal random exploration for trade-related non-indigenous accounting for behavioral responses during a flu epidemic using home television viewing risk factors for human disease emergence travelling smarter down under: policies for voluntary travel behaviour change in australia ebola virus disease in west africa-the first months of the epidemic and forward projections population-wide benefits of routine vaccination of children against influenza this study was made possible by grant # r gm - from the national institute of general medical sciences (nigms) at the national institutes of health and contract hshqdc- -c- from the science and technology directorate, department of homeland security. the contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of dhs or nigms. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. it was also funded by nsf grant as part of the joint nsf-nih-usda ecology and evolution of infectious diseases program. economic incentives in the socially optimal management key: cord- -upwe cpj authors: sullivan, kathleen e.; bassiri, hamid; bousfiha, ahmed a.; costa-carvalho, beatriz t.; freeman, alexandra f.; hagin, david; lau, yu l.; lionakis, michail s.; moreira, ileana; pinto, jorge a.; de moraes-pinto, m. isabel; rawat, amit; reda, shereen m.; reyes, saul oswaldo lugo; seppänen, mikko; tang, mimi l. k. title: emerging infections and pertinent infections related to travel for patients with primary immunodeficiencies date: - - journal: j clin immunol doi: . /s - - - sha: doc_id: cord_uid: upwe cpj in today’s global economy and affordable vacation travel, it is increasingly important that visitors to another country and their physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population thwaites and day (n engl j med : - , ). this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population [ ] . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. emerging infectious diseases are a result of a convergence of numerous factors and comprise complex interactions among multiple variables. some of those factors are human movement, land use change, encroachment and wildlife translocation, rapid transport, and climate change. several studies demonstrate that for a pathogen to persist in a population, a minimal host population size that is specific for the type of pathogen and host population. of particular relevance to emerging infections is the pattern of rapid population growth. in the tropics, before wwii, most regional ecosystems consisted of few large cities and scattered human settlements separated by large areas of cropland, pastureland, or undisturbed forest. today, the pattern is the opposite: many large cities have developed with only patches of undisturbed forest or grassland. domestic vectors have therefore expanded their population with increasing urbanization and this markedly impacts the interactions between vectors and pathogens [ ] . human activities such as deforestation, use of pesticides, pollution, etc. lead to the loss of predators that naturally regulate rodent and insect populations. this contributes to emerging zoonotic diseases and explains why they occur more frequently in areas recently settled. in today's global economy and affordable vacation travel, it is increasingly important that visitors to another country and their the southern, with a reduction in the number of cold days per year, changes in rainfall (more winter precipitation and summer droughts) [ ] , and together these changes increase the risk of several vector-borne diseases in new areas. climate change involves not only global warming but also changes in precipitation, wind, humidity, and the location and frequency of extreme weather events like floods, droughts, or heat waves. changes in climate produce changes in pathogens, vectors, hosts, and their living environment. increases in precipitation can increase mosquitoes for example, but heavy rainfalls may cause flooding that temporarily eliminates larval habitats and decreases mosquitoes. flooding may force rodents to look for new habitats in houses and increase the opportunities of vector-human interactions, as occurs for example in the case of epidemic leptospirosis. humidity is another very important factor of climate change in the development of vector-borne diseases. mosquitoes and ticks do not survive well in dry conditions. therefore, weather impacts infectious pathogen distribution in complex ways that are not predictable by the forecast. extreme weather events can precipitate outbreaks of infection. an increase in the frequency and intensity of natural disasters like hurricanes and tsunamis, in relation to the el niño/southern oscillation phenomenon, may result in more flooded grasslands, which favor the breeding of aedes and culex mosquitoes [ ] , and impact water sanitation fostering outbreaks such as cholera. flooded areas can displace rodents leading to plague. tornados and other severe weather can stir up soil leading to infections with soil fungi leading to episodic outbreaks of invasive fungal disease such as mucormycosis such as apophysomyces trapeziformis [ ] . malaria is a common disease that can vary dramatically depending on weather, and extreme weather can alter the very landscape, providing new bodies of water to support larval development. if the melting of glaciers and the polar ice caps bring coastal cities underwater, or if overpopulation and waste cause drinkable water shortages in certain regions of the world, we can expect mass migrations. these could change the patterns of infection and drive outbreaks. migrants traversing tropical forests, or feeding with meat from game or carcasses, are but two scenarios that could be envisioned for the emergence of zoonotic infectious diseases [ ] . several predictive models have been developed to evaluate the impact of climate change on the emerging infectious diseases: climex, dymex, miasma models. nevertheless, it remains difficult to predict when and where pathogen behavior will deviate from its typical pattern. climate change primarily affects vector-borne diseases by increasing rates of reproduction and biting and by shortening the incubation period of the pathogen they carry. ticks have gained spread from the mediterranean basin to northern and eastern europe, as well as appearing at higher altitudes. increased survival, density, and activity have also been reported following shorter, milder winters. climate change has also resulted in more days of activity per year for mosquitoes. as temperatures rise, more parasites are viable within regions ranging from the mediterranean and tropical zones, up to the balkans, russia, scandinavia, and the uk. for some ticks and fleas, temperatures over °c with relative humidity of over % are optimal for their proliferation and activity throughout the year [ , , ] . for example, dengue fever is usually limited to a tropical latitude and a low altitude, since mosquito eggs and larvae lose viability with sustained low temperatures. during unusually warm summers, however, dengue has been reported as high up as m above sea level. warmer temperatures also result in smaller adult mosquitoes, which need to bite more frequently to feed themselves and be able to lay eggs, thus increasing the rate of transmission [ ] . in contrast, the incidence of malaria has followed mixed patterns of increase and decrease along recent decades, and computer models have failed to predict the spread. the explanation for this is, in part, that climate change also results in diminished survival of the vectors (warming over °c affects the survival of both parasites and vectors), and in part, that the effect of climate change is non-linear and complex [ ] . the frequency of emerging vector-borne infections varies per changes in land use, human activity, intervention maneuvers to eradicate the vector or prevent transmission to humans, drug treatment, and vaccines. both ecologic and economic changes may bring together rodents and humans. hunting activities may change vector distribution and large-scale animal movements can impact disease distribution. impoverishment of cities and overcrowding in slums, but also reforestation, golf club development, and the urbanization of rural suburbs facilitate exposure to ticks and rodents [ ] . many patients with pidd require immunoglobulin replacement. immunoglobulin products have been demonstrated to improve outcomes in hepatitis a and measles [ , ] . at least some neutralizing antibody is present directed to rsv and group b streptococci [ , ] . this raises three distinct issues for patients: ( ) difficulty in the diagnosis of infections in travelers because locally produced immunoglobulin may have antibody titers to local infections that are not typical for other countries, ( ) safety concerns about locally produced immunoglobulin, if the patient resides in the location long enough to require immunoglobulin from a local provider, ( ) the decision to use locally produced immunoglobulin products to provide superior prevention of infection compared to the patient's usual product. there are limited data on each of these subjects. serologic diagnostic testing in patients on immunoglobulin therapy will be addressed below in terms of issues related to lack of specific antibody produced by the patient (potentially) after infection. the converse can also be an issue. patients arriving from countries with significant occurrences of infections unusual in their current country may have igg antibodies to those infections simply through their immunoglobulin product and not reflecting a true infectious event in the patient. this can lead to diagnostic confusion when serologic testing demonstrates the presence of antibodies due to the infusion product. patients will often ask if immunoglobulin products from other countries are held to the same rigorous standards as their home country. today, commercially produced immunoglobulin is safe and tightly regulated. all commercially produced immunoglobulin around the world has one of the time-tested viral inactivation procedures such as nanofiltration, caprylate absorption, pasteurization, solvent/detergent, vapor heating, and low ph treatment. these procedures uniformly inactivate enveloped viruses. many emerging viruses are specifically tested for their ability to withstand the purification process. much has been learned since the transmission of hepatitis c viruses through immunoglobulin products in the early s [ ] . nevertheless, vigilance is important. in , counterfeit immunoglobulin was identified. therefore, patients should ensure that they receive only brand name products while traveling. the subject of whether a patient's interests would be best served by using a local immunoglobulin product, with antibodies to pathogens that are prevalent in the community, or have their home physician ship their usual product, for which the patient has a known tolerance is hotly debated. patients with a history of intolerance to immunoglobulin products should not switch unless necessary. however, there are compelling reasons to consider a locally produced product when patients are in a foreign country for an extended period. it is known that antibodies to west nile virus have tracked with the distribution of the virus as it has emerged in several areas [ , ] . titers in products using donors from the usa have higher neutralizing titers to west nile virus than those using donors from europe, although there is a -fold difference in titers between lots from the usa [ ] . similarly, protective antibodies to concerning pathogens may be optimal in locally produced immunoglobulin. it is critical to inquire where the plasma source is derived. in most countries, the utilized immunoglobulin is produced in europe or the usa. having a different name does not ensure that the plasma pool comes from a different country. most lots of immunoglobulin are produced from to , plasma donors. the infrastructure to support such an endeavor is not easy to establish in each geographic area. serologic testing is commonly used for the diagnosis of infection. this approach relies upon detection or quantitation of antibodies made by the host against specific pathogens. the presence of igm antibodies against a specific pathogen indicates recent infection, while igg antibodies against a specific pathogen indicate past infection. importantly, serologic testing can only be applied for the diagnosis of infection if the host can mount a specific antibody response to the pathogen. conversely, serology cannot be relied upon to diagnose infection in the setting of immune deficiency where there is impairment of the specific antibody response, such as in the case of primary antibody deficiencies, cellular immune deficiencies, combined immune deficiencies, and other secondary immune deficiencies affecting t and/or b cell function. in these situations, the causative pathogen must be identified by alternate means such as culture of the organism, antigen detection, or molecular approaches (nucleic acid hybridization, nucleic acid sequencing, or oligonucleotide probe arrays). molecular approaches are particularly relevant for the diagnosis of infection in patients with pidd. signal and target amplification techniques can be coupled with nucleic acid hybridization or probe assays to allow detection of pathogen dna or rna that is present in very small amounts in clinical samples. in patients with pidd, vaccines could play an important role in preventing infections with vaccine-preventable diseases. even pidd patients may generate some protective responses [ , ] . the decision to immunize such patients or not depends on the type and severity of pidd as well as the type of vaccine to be administered (live or inactivated) ( table ). in general, inactivated vaccines are safe for pidd patients while immunization with live attenuated vaccines is a known hazard to patients with serious immunodeficiencies of t cell, b cell, and phagocytic cell origin (table ). in less severe pidd, the vaccine can induce adequate protection as in healthy individuals or the efficacy may be reduced [ ] [ ] [ ] . of note, immunoglobulin replacement therapy induces passive immune protection to some vaccine-preventable infections, such as measles, mumps, rubella (mmr), and varicella. in addition, live viral vaccines have greatly reduced efficacy while on immunoglobulin replacement. therefore, vaccine administration in patients receiving regular immunoglobulin replacement treatment should be withheld until at least to months (depending on dose) after cessation of such treatment, if cessation and vaccination are safe. in addition, pidd patients who have received hematopoietic stem cell transplantation but have incomplete immune reconstitution or are under immunosuppression should not receive live attenuated vaccines. in general, the decision of administering live viral vaccines should be made by clinical immunology experts [ ] . in developing countries where polio is still endemic and oral polio vaccine is essential for eradicating the disease, it is of utmost importance that all pidd patients and family members should not receive live oral polio (opv) because of the reported prolonged excretion of the virus for months and even years [ ] . in addition, vaccine-associated paralytic polio is a real risk for some with pidd. these patients and family members should receive inactivated polio vaccine (ipv) instead of opv. similarly, the hazards of administering bacillus calmette-guerin (bcg) vaccine to pidd patients have been documented. in a series of bcg, vaccinated severe combined immunodeficiency (scid) patients from centers in countries, % of scid patients developed disseminated bcg infection and had the worst outcome [ ] . patients with chronic granulomatous disease vaccinated with bcg also developed local and disseminated bcg infection. recently, vaccine strains of rubella virus were found to be associated with skin granulomas in pidd patients [ ] [ ] [ ] . siblings and household contacts of patients with suspected or diagnosed pidds should receive all the national immunization scheduled vaccines. ipv should be substituted for op in families where an antibody-deficient patient exists. yearly influenza vaccination of family members is recommended in order to reduce the risk of household-social transmission [ , , ] . diseases where routine vaccination has reduced incidence can occasionally experience a resurgence in times of economic hardship with reduced attention to vaccination. diphtheria is currently seen in venezuela for this reason. war and disruption of health infrastructure are other common reasons for resurgence in vaccine-preventable diseases. in other settings, antivaccination sentiment has led to outbreaks of diphtheria, measles, and mumps. an additional consideration is that not all countries provide the same level of vaccination, and therefore, vaccine-preventable illness can still be seen regionally. these outbreaks represent a significant risk to patients with pidd. a universal consideration for patients with pidd is the concern about antibiotic resistance, which varies dramatically around the world. for certain high impact infections, the emerging antibiotic resistance patterns will be discussed below. antimicrobial resistance occurs naturally, but misuse and overuse of antimicrobials are accelerating this process. in nearly every country, antibiotics are overused and misused in people and animals leading to antibiotic resistance in every country. key resistance patterns to common bacteria include emergence of carbapenem-resistant klebsiella pneumoniae around the world with high frequency of resistance (due to different mechanisms) in the mediterranean, with greece, italy, and turkey having endemic spread of this pathogen [ ] . carbapenem-resistant strains among other genera of enterobacteriaceae have also been recognized. they are particularly common in greece, but have been found widely distributed [ ] . the new delhi metallo-beta-lactamasemediated resistance, which is endemic in the indian subcontinent but becoming increasingly spread worldwide, is a growing concern [ , ] . as a common cause of urinary tract infections, colistin is the only recourse when carbapenemresistant enterobacteriaceae, and colistin resistance has recently emerged in small outbreaks throughout the world [ ] . in these cases, the infection is essentially untreatable. fluoroquinolone-resistant escherichia coli, a common cause of urinary tract infections, now accounts for over half of the isolates in some asian countries [ , ] . t he emergence of resistance to antibiotics in grampositive pathogens has become a major international problem as there are fewer, or even sometimes no effective, antimicrobial agents available for infections caused by these bacteria. methicillin-resistant staphylococcus aureus is common in many countries and in fact has spawned a nomenclature recognized by the general public: mrsa. several studies have reported resistance to the newer antimicrobial agents like linezolid, vancomycin, teicoplanin, and daptomycin [ ] . thus far, these isolates appear to be uncommon and have been found in < % of isolates in brazil, china, ireland, and italy, with nearly undetectable rates elsewhere. vancomycinresistant enterococcus (vre) is growing in frequency and can now be a cause of primary bacteremia in immunocompromised individuals [ ] . a key message is that antibiotic resistance is increasing (generally) and travelers should be alerted to resistance to commonly encountered organisms, and if antibiotic prophylaxis is required, their prophylaxis is adjusted. neisseria gonorrhoeae is a specific organism for which resistance has become especially problematic. it has progressively developed resistance to virtually all antimicrobial agents since introduction of sulfonamides in mid- s. the current treatment guidelines recommend dual antimicrobial therapy (ceftriaxone - mg × plus azithromycin - g × ) as first-line regimen [ , ] . although dual therapy is very effective, development of concomitant ceftriaxone and azithromycin resistance is likely to emerge [ ] . the risk of untreatable n. gonorrhoeae demands better global antimicrobial surveillance system, clinical trials on combined therapy of existing drugs as well as novel agents in monotherapy, and development of a gonococcal vaccine. for pidd patients, guidance on barrier methods for the prevention of sexually transmitted diseases should be a part of any pre-travel counseling. mycobacteria tuberculosis (mtb) is an age-old pathogen with emerging resistance. drug-resistant tb, fueled by the hiv epidemic, is a global threat. in , who estimated , new cases of multidrug-resistant tb (mdr-tb) and an additional , new cases of rifampin-resistant tb (rr-tb) who would also require mdr-tb treatment. treatment of mdr-tb and mycobacterium bovis disease is beyond the scope of this text and reader is referred to recent who mdr treatment guidelines [ ] . regions of the world with > % mdr tb include regions of azerbaijan, kazakhstan, russia, uzbekistan, china, georgia, and eastern europe. extensively drug-resistant tb (xdr tb) refers to mtb resistant to isoniazid, rifampin, any fluoroquinolone and at least one second-line drug. xdr tb has been reported in countries. on average, % of patients with mdr tb have xdr tb. as is true for all types of mtb, xdr tb is contagious and small outbreaks related to person-person transmission have been reported. non-tuberculous mycobacteria (ntm) cause significant systemic infections in patients with defects of the il- / ifnγ/stat axis as well as in gata deficiency and can cause significant pulmonary infections in pidd patients with bronchiectasis. compared to tb, ntm is acquired from the environment and not from person-to-person transmission; therefore, acquisition of antibiotic-resistant strains is less common. however, in these individuals with pidd, ntm disease is often chronic and can be difficult to eradicate, and resistance can then easily develop during therapy. using combination of antibiotics is essential, and consultation with those familiar with treatment of treatment refractory ntm disease is recommended. aspergillus species are ubiquitous inhaled molds with worldwide distribution that cause opportunistic infections in immunocompromised patients [ ] . aspergillosis also occurs in pidds associated with quantitative and/or qualitative phagocyte defects; it develops most frequently in chronic granulomatous disease (cgd) patients (prevalence,~ %), while it is seen less often in patients with gata deficiency, card deficiency, and congenital neutropenia syndromes [ , ] . upon inhalation, aspergillus species cause invasive pulmonary disease in susceptible hosts with the exception of card deficiency, where aspergillosis has a predilection for extrapulmonary tissues with sparing of the lungs [ ] . diagnosis is established by fungal culture and/or histopathology showing acute-angle septate hyphae and/or detection of galactomannan, an aspergillus cell wall component released during invasive infection, in serum or bronchoalveolar lavage fluid [ ] . while azole-susceptible aspergillus fumigatus is still the most common infecting species in pidd patients, the emergence of azole-resistant a. fumigatus and nonfumigatus aspergillus species underscores the importance of a high index of suspicion in patients who do not respond to front-line voriconazole treatment [ ] . the advent of fungal molecular diagnostics has demonstrated that patients with pidds are more prone to infections by uncommon low-virulence aspergillus species with intrinsic resistance to azole antifungal agents that do not infect patients with iatrogenic immunosuppression. these primarily include aspergillus viridinutans, aspergillus tanneri, and neosartorya udagawae, which pose major diagnostic and therapeutic challenges due to their impaired sporulation and propensity for contiguous and distant tissue spread, respectively. in addition, acquired azole resistance in a. fumigatus can be seen in patients on long-term exposure to azole drugs used as treatment and/or prophylaxis [ ] . azole resistance in these strains is predominantly caused by point mutations in the lanosterol α-demethylase gene that encodes the cyp a protein, the primary target of azole drugs. importantly, infection by azole-resistant a. fumigatus strains without prior exposure of patients to azole antifungals has recently emerged as an important global health concern due to the widespread use of sterol demethylase inhibitor fungicides in agriculture that results in cross-resistance with the triazole antifungals used in clinical practice [ ] [ ] [ ] . fungicide-driven azole-resistant environmental aspergillus strains were first observed in the netherlands in and have since then been documented in other parts of europe, south and north america, the middle east, australia, africa, and asia. the prevalence of these azole-resistant aspergillus strains among clinical aspergillus strains recovered from patients in european countries was reported to be . %, while alarmingly in some areas > % of recovered strains exhibited azole resistance [ ] . the emergence of such aspergillus environmental strains poses serious threats to the treatment of immunosuppressed patients. mortality rates as high as % have been seen due to delays in diagnosis and suboptimal treatment with azole antifungals [ ] . although no prospective data exist for the treatment of patients with such resistant fungi, the use of amphotericin b-and echinocandin-based regimens are preferred over azoles [ ] . in the absence of azoles, the lack of alternative oral antifungal agents is particularly challenging for pidd patients such as those with cgd who require long-term suppressive antifungal treatment. candida species are commensal yeast fungi that colonize the mucosal surfaces of~ % of healthy individuals [ ] . when perturbations in immunity and/or microbiota occur, candida causes opportunistic mucosal or systemic infections that depend on clearly segregated immune responses for host defense. specifically, t cells of the th program are critical for mucosal and phagocytes for systemic immunity [ ] . indeed, a proportion of patients with cgd and complete myeloperoxidase deficiency develop systemic, but not mucosal candidiasis [ ] , whereas patients with monogenic syndromes of chronic mucocutaneous candidiasis due to mutations in the il- signaling pathway (il ra, il rc, il f, act ) or in other genes that adversely affect th differentiation (rorc, stat , stat , aire, dock , stk , irf ) do not develop systemic candidiasis. the only known pidd to date that results in combined mucosal and systemic candida infection susceptibility is card deficiency. systemic candidiasis in card -deficient patients has a predilection for the central nervous system, associated with brain-specific impaired recruitment and effector function of neutrophils [ ] [ ] [ ] . diagnosis of candida infections is established by culture. azole-susceptible candida albicans is still the most common infecting species in pidd patients; however, emergence of azole-resistant c. albicans is not uncommon during chronic azole use, making long-term therapy challenging due to lack of alternative oral antifungal treatment options [ ] . beyond c. albicans, non-albicans candida species can rarely infect pidd patients, some of which have intrinsic resistance to azole antifungals, including candida glabrata and candida krusei [ ] . most recently, candida auris has emerged as a global public health concern due to its resistance to antifungal drugs, high virulence potential, propensity for health careassociated horizontal transmission and outbreaks in health care settings, persistence in the human skin and hospital environment, inherent resilience to antiseptics, and misidentification by routine biochemical methods as other yeasts (most often candida haemulonii, but also candida famata, rhodotorula glutinis, or saccharomyces cerevisiae). c. auris was first recovered from the ear canal of a patient in japan in and has since then been reported to cause life-threatening infections and hospital outbreaks in europe, asia, africa, the middle east, and south and north america [ ] [ ] [ ] [ ] . most of the reported strains of c. auris have intrinsic resistance to fluconazole and other triazole antifungal agents, while a significant proportion of strains has elevated minimum inhibitory concentrations to amphotericin b and echinocandins, with some strains reportedly resistant to all three classes of azoles, polyenes, and echinocandins [ ] . avoidance of azole antifungals is important in c. auris-infected patients, and echinocandinor amphotericin b-based regimens are preferred, guided by strain-specific in vitro susceptibility patterns. this section on vector-borne infections is a major focus of this review because the infections are often more severe in immunocompromised individuals and because there are mitigation strategies that should be considered even in the absence of defined medical treatments for infection. prevention of mosquito bites depends somewhat on the endemic species but there are generalizations. the use of a mosquito repellant such as deet, oil of lemon eucalyptus, ir , or picaridin is as important as using long sleeves and long pants while in an affected area. deet and picaridin are safe in pregnancy, and some data support their greater efficacy [ ] . air conditioning and fans tend to keep mosquitoes away but netting at night is essential in mosquito-prone areas. light-colored clothing is less attractive to mosquitoes than dark clothing, and scented detergents and use of dryer sheets tend to attract mosquitoes, hence should be avoided. aedes species prefer to bite indoors and thrive in urban areas with small puddles of water. they bite most frequently around dawn and dusk. anopheles species have very similar behaviors. culex mosquitoes, in contrast, bite primarily at night. tick and fly bite prevention is focused on physical and chemical prevention. for ticks, physical inspection for biting ticks should also be incorporated. arthropod-borne viruses (arboviruses) are transmitted to humans through the bites of infected insects: mosquitoes, ticks, sand flies, or midges. some arboviruses can be transmitted through blood transfusion, organ transplantation, perinatal transmission, consumption of unpasteurized dairy products, or breastfeeding. there are > arboviruses causing human disease. most arboviral infections are asymptomatic. infectious manifestations range from mild febrile illness to severe encephalitis. arboviral infections are often categorized into two primary groups: neuroinvasive disease and non-neuroinvasive disease. tables and list the encephalitigenic viruses and the febrile/hemorrhagic disease causing viruses. in this section, we will highlight west nile virus, the most common of the encephalitogenic arboviruses. west nile virus is a single-stranded mosquito-borne rna virus of the family flaviviridae. the natural transmission cycle of the virus occurs in culex mosquitoes and birds. humans and horses are dead-end hosts for the virus. the most common mode of transmission to humans is through the bite of infected mosquitoes. other less common modes of transmission include blood transfusions, organ transplantations, occupational exposure in laboratories and mother-to-child transmission during pregnancy, childbirth, and breastfeeding. west nile virus has been diagnosed in > people in the usa with slightly more than half having neuroinvasive disease. since , > , people in the usa have been infected. it is also common in africa, europe, and asia [ ] . infection with west nile virus is asymptomatic in most individuals [ , ] . the incubation period lasts for to days. however, it can be significantly longer in immunocompromised hosts. clinical manifestations following infection develop in - % of those infected and include fever, headache, myalgia, arthralgia, vomiting, and rash. severe neuroinvasive disease leading to meningitis, encephalitis, and flaccid paralysis develops in less than % of infected individuals. the overall case fatality is approximately % which is a disproportionately high mortality in patients with encephalitis and myelitis. diagnosis of west nile virus rests on demonstration of specific antibody responses especially specific igm antibodies in the serum or csf of infected individuals by enzyme immunoassays. plaque reduction neutralization tests can differentiate cross-reactive antibodies. detection of virus in csf, blood, or tissue specimens by culture or pcr is particularly useful in immunosuppressed individuals who may have impaired serological responses. west nile virus has been reported in the context of both primary and secondary immunodeficiency. severe neurological manifestations have been reported in hiv-positive individuals, individuals receiving immunosuppressive therapy including rituximab, and individuals with pidd. infection with wnv has been reported in individuals with common variable immunodeficiency, idiopathic cd lymphopenia, gata deficiency, and a case of probable good's syndrome [ ] [ ] [ ] . individuals with antibody defects, neutropenias, and impaired t cell responses are potentially at an increased risk of severe manifestations of wnv disease including severe neurological involvement. this section highlights the four important non-neuroinvasive arboviruses based on current geographical distribution: dengue, yellow fever, zika, and chikungunya (table ) . approximately countries/territories have reported local transmission for both chikungunya and dengue viruses [ ] . dengue is due to infection with one of four dengue virus serotypes, transmitted by a mosquito (typically aedes aegypti). this febrile illness affects all ages with symptoms appearing - days after the infective bite. symptoms range from mild to high fever, with severe headache, musculoskeletal pain, and rash. severe dengue (also known as dengue hemorrhagic fever) occurs in . - % of cases and is characterized by fever, abdominal pain, persistent vomiting, bleeding, and breathing difficulty and is a potentially lethal complication [ ] . paradoxically, the main risk factor for dengue hemorrhagic fever is pre-existing antibodies. early clinical diagnosis and comprehensive management by experienced clinicians increase survival. dengue is ubiquitous throughout the tropics with the highest infection rates in the americas and asia. dengue is now endemic in countries, causing up to million infections a year and , deaths, mainly among children. over half of the world's population inhabits areas at risk for dengue infection [ ] . the presence of a. aegypti in over countries potentially puts almost the whole world at risk of becoming infected with this virus. pcr is widely used as serologic methods to diagnose dengue. immunity to one serotype does not confer protection against the other three serotypes, and heterologous antibody may be a risk factor for hemorrhagic dengue [ ] . the natural history of dengue has been studied in hiv patients where hiv did not appear to increase severity. there have been no reports of patients with pidd having dengue; however, dengue infection after solid transplantation has been reported [ ] [ ] [ ] [ ] with some patients having severe complications suggesting that t cell compromise in pidd could be a risk for severe disease. there are no antiviral medications utilized for dengue virus. care of patients with hemorrhagic disease requires meticulous approach to fluids and coagulation status. one dengue vaccine has been registered in several countries (cyd-tdv) for individuals from to (or ) years old. it is a live attenuated recombinant tetravalent vaccine with backbone of the attenuated yellow fever d virus genome with the prm and e genes that encode the proteins from the four wild-type dengue viruses. the who has suggested its use in regions where seroprevalence of dengue virus of any serotype is % or greater, but has not recommended it to hiv-infected, immunocompromised individuals, nor pregnant or lactating women [ ] . most people infected with the yellow fever virus have no illness. symptoms of yellow fever include sudden onset of fever, chills, headache, musculoskeletal pain, nausea, vomiting, fatigue, and weakness. the incubation period is typically - days, and symptoms may appear after return from travel. most people improve after the initial presentation, but % of cases progress to develop a more severe form of the disease, usually after a day of presumed recovery. the severe form is characterized by high fever, jaundice, bleeding, and eventually shock and failure of multiple organs [ ] . yellow fever virus is an rna virus that belongs to the genus flavivirus. it is transmitted from mosquitoes after biting an infected primate. it is widely distributed in the equatorial tropics [ ] . aedes species of mosquitoes are primarily responsible for transmission. large epidemics of yellow fever occur when the infection enters heavily populated areas with a high mosquito density and where most people have little or no immunity. west africa has undergone a large-scale vaccination campaign with impressive results and yellow fever is now uncommon in west africa [ ] . serologic testing for yellow fever is the diagnostic standard. pcr can be performed on tissue samples. there are no published studies of yellow fever in immunocompromised people, but the elderly, very young, people with autoimmune disease, or who are post-thymectomy are at risk from the attenuated vaccine strain. thus, it seems likely that any immunodeficiency would be associated with more severe wildtype disease. currently, no specific antiviral drug for yellow fever exists. treatment of dehydration, liver and kidney failure, and fever improves outcomes. the yellow fever vaccine is highly effective; however, immunodeficient patients should not receive it. infection with zika virus is often asymptomatic. it represents a mild infection for those who have any symptoms [ ] . the zika virus has been detected in urine, semen, and saliva of infected individuals, and transmission from transfusion and sexual relations has been reported. it is also detectable in breast milk, but breastfeeding-associated transmission has not been reported so far [ ] [ ] [ ] . contact with highly infectious body fluids from patients with severe zika infection has also been suggested as a possible mode of transmission [ ] . of tremendous importance is the presence of prolonged shedding of zika virus in a congenitally infected newborn [ ] . the main public health risk of zika virus is microcephaly in newborns from infected mothers [ ] . zika virus is capable of infecting human neural progenitor cells in vitro. infection results in disruption of cell cycle, increased cell death, and attenuated neuron growth [ ] . zika is not thought to be a major risk for people with pidd (based on the experience with hiv patients, but our recognition of zika is very recent. there is no known specific treatment for zika; however, there is an important effort to develop a vaccine. chikungunya fever is an acute febrile illness caused by the alphavirus, chikungunya virus. the incubation period is usually - days after the bite of an infected aedes mosquito. there is abrupt onset of high fever, and the fevers can be biphasic [ , ] . severe polyarthraligias develop after the onset of fever. the joint pains can affect any joint, but the pattern is usually symmetric and a true acute arthritis is not uncommon. the proportion of infected people with rash has varied across studies. when seen, the rash appears after the fever as a truncal maculopapular type of rash [ ] . cervical adenopathy is another common feature of infection. death is uncommon in chikungunya, but serious complications such as myocarditis have been seen. over half of the patients report continued joint symptoms year after acute illness and % have long-term joint symptoms [ ] . chikungunya originated in central/east africa. in forests, the virus circulates in aedes mosquitoes and non-human primates. in urban centers, the virus circulates between humans and mosquitoes similar to the pattern of dengue. there have been periodic urban outbreaks in asia and africa since with an acceleration in spread since [ ] . an important consideration is the periodic outbreaks with high attack rates in naïve populations. areas at risk currently are east africa, central africa, la reunion, india, and southeast asia. diagnostic testing utilizes pcr or serology. the threat to immunodeficient patients is not entirely clear. there are a few provocative cases where the immunocompromised appears to have been associated with fewer joint symptoms, but there were two patients, medically immune compromised, who had very severe disease [ , ] . this suggests that the presentation may be atypical and the course may be severe in immunodeficient people. treatment is supportive, although chloroquine, acyclovir, ribavirin, interferon-ɑ, and steroids have limited preclinical data to support clinical trials. babesia microti (the main species in the usa) infection can be asymptomatic, but many people develop fever, chills, headache, myalgias, anorexia, nausea, or fatigue [ ] . babesiosis often causes hemolytic anemia. b. microti is spread by ixodes scapularis ticks in the usa and babesia divergens (the main species in europe) is spread by ixodes ricinus. symptoms begin - weeks after a bite from an infected tick with b. divergens having a higher mortality rate and greater symptomatology compared to b. microti. the main geographic areas involved are the coastal eastern usa and cattle breeding areas throughout europe. the diagnosis is usually by inspection of a blood smear or through serology. a pcr test has just been developed. immunodeficiency, asplenia, and older age are recognized risk factors for severe disease and even death [ ] [ ] [ ] . thus, congenital asplenia would be considered a major risk for severe disease. a combination of atovaquone and azithromycin is generally used for therapy, although clindamycin and quinine have been used with success. patients with severe illness have been treated with exchange transfusions. five different types of plasmodium (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae, and plasmodium knowelsi) infect humans. malaria is transmitted primarily by female anopheles mosquitoes. symptoms vary depending on the type of plasmodium involved but usually include high fever, chills, and headache. in some cases, the illness can progress to severe anemia, kidney and respiratory failure, and death. the incubation period typically ranges from to days for p. falciparum, to days for p. vivax and p. ovale, and to days for p. malariae. in p. vivax and p. ovale infections, relapses can occur months or even years without symptoms. p. vivax and p. ovale have dormant liver stage parasites that must be specifically eradicated through medical therapy. malaria has been a global health concern throughout history and is a leading cause of death and disease across many tropical and subtropical countries. over the last years, new control measures have reduced malaria by over half [ ] . the democratic republic of the congo and nigeria account for over % of the estimated total of malaria deaths globally. high rates of malaria are seen in india as well. nevertheless, malaria exists in most tropical regions of the americas, africa, and asia [ ] . the diagnosis of malaria depends on the demonstration of parasites in the blood, usually by microscopy. the threat to immunodeficient patients is not entirely clear, but patients with hiv seem to have no additional burden of disease other than an increase in placental malaria, suggesting that t cells are not central to the defense of malaria [ , ] . asplenia is a known risk factor for severe malaria [ ] . antibodies appear to be both protective and pathologic [ , ] . treatment and prophylaxis depend on the region of the world because the parasites and resistance are highly variable and highly dynamic. therefore, it is best to consult an infectious disease specialist familiar with the prophylaxis before travel and for treatment of acute cases. leishmaniasis is due to infection with an obligate macrophage intracellular protozoa of the genus leishmania. it causes a spectrum of disease ranging from a cutaneous ulcer to mucosal disease and the most severe form, visceral leishmaniasis (vl). the liver, spleen, and bone marrow are major sites of parasite growth and disease pathology in vl [ ] . purely cutaneous leishmaniasis is most often caused by leishmania major, leishmania. tropica, leishmania aethiopica, leishmania infantum, and parasites belonging to the leishmania mexicana complex, the leishmania braziliensis complex, and the leishmania guyanensis complex. mucocutaneous disease is most often due to l. braziliensis complex, leishmania panamensis, leishmania amazonensis, and rarely by leishmania guyanensis. vl is most often caused by leishmania donovani and leishmania infantum (previously l. chagasi) [ ] . cutaneous leishmaniasis can have many variations but is most often an ulcer that develops after an indolent papule. the incubation period ranges from weeks to months. the ulcer usually heals within months to years, and there can be mild adenopathy. mucocutaneous leishmaniasis follows a cutaneous ulcer and is only caused by l. braziliensis parasites. oral and respiratory mucosa are most often involved with granulomatous lesions that may be extremely destructive. vl is associated with fever, lymphadenopathy, hepatosplenomegaly, wasting, hypoalbuminemia, and pancytopenia. this picture evolves over months to years. there can be secondary immune deficiency due to the pancytopenia. the epidemiology has changed dramatically and has been impacted by climate change [ ] . the sand flies that spread the parasite are affected by temperature and rainfall. in most endemic regions, leishmania has a patchy distribution due to micro-ecologic factors. poverty has been demonstrated to be a major risk factor for leishmaniasis [ ] . it has been estimated that up to half a million new cases of vl occur every year, but the majority are in resource-poor countries such as bangladesh, nepal, india, sudan, ethiopia, and brazil. emergence of resistance to antimony-based drugs has also led to a major resurgence of disease. the primary reservoir for leishmania is forest rodents, but dogs are increasingly important. the growing spread of leishmania is due to a combination of factors, and now countries have reported cases. immunodeficient patients are more susceptible to infection, and relapse occurs more frequently [ ] . the risk of developing vl is estimated to be between and times higher in hiv-infected than in non-hiv-infected individuals [ ] , and these patients have higher rates of treatment failure with the illness often taking a prolonged chronic course and higher mortality rates [ ] . a similar picture has been seen in patients with vl-infected post-kidney transplantation [ ] . dendritic cells, t cells, and the generation of reactive oxygen species have been shown to be essential for parasite control [ ] [ ] [ ] . pidd with impaired il- production have been associated with severe disease [ ] . a patient with cd l deficiency, associated with poor il- production, had chronic leishmania and died in spite of aggressive treatment. vl has been reported in cgd patients [ ] . six cgd patients were infected by leishmania, and they developed hemophagocytic syndrome with a poor outcome for one of them [ ] . the diagnosis of leishmaniasis is usually by visual inspection for parasites. immunofluorescence microscopy, direct agglutination, skin test, and pcr have been used. treatment is long-term and difficult. emerging resistance to first-line treatment is increasingly problematic. pentavalent antimonials are the mainstay of treatment in most countries, but liposomal amphotericin is widely used where resistance occurs. newer drugs with more favorable side effect profiles have been used in certain geographic settings: miltefosine, paromycin, and sitamaquine. rickettsiae are small gram-negative bacteria. they are obligate intracellular parasites, and the primary target in humans appears to be endothelial cells with subsequent thrombosis and clinical presentation of vasculitis [ ] . the rickettsiaceae family, originally defined by non-specific phenotypic characteristics, was reclassified into different strains and subspecies based on gene sequencing and genetic phylogeny ( table ). the clinical presentation of rickettsial disease can vary, but the classic triad of fever, rash, and headache still provides major clues for the diagnosis [ ] [ ] [ ] . however, rash is not an obligatory sign, and the incidence of rash can range between % for rickettsia conorii infection,~ % for rickettsia rickettsii, % for rickettsia africae, and less than % in the case of anaplasma phagocytophilum infection. therefore, fever in patients with exposure to a potential vector should raise a concern for a rickettsial disease, especially if there is also evidence of rash, inoculation eschar, or localized lymphadenopathy. additional supporting laboratory findings can include neutropenia, thrombocytopenia, and increase in liver transaminases. the geographic distributions of rickettsioses and ehrlichioses are mostly dependent on their vector distribution [ ] . as such, louse-borne and flea-borne are worldwide, reflecting the worldwide distribution of lice and fleas, with a tendency to parasite poor people in cold places and, characteristically, during wars. ticks, on the other hand, depend on their environment and most do not have a worldwide distribution. with the exception of the dog tick, vector for r. conorii in asia and north africa, r. rickettsii in the usa, rickettsia massiiae and erhlichia canis worldwide, most other tick-borne diseases are restricted to areas of the world correlating with the distribution of their vector [ ] . for that reason, it should be anticipated that climate and environmental changes will affect vector distribution and its reservoir host and, hence, the geography and epidemiology of tick-borne diseases [ , , ] . diagnosis presents a challenge, as it is extremely difficult to grow these organisms in culture. immunohistochemistry and pcr can be helpful. the severity of rickettsial disease varies with the causative agent and the host. r. rickettsii, rickettsia prowazekii, and orienta tsutsugamushi are considered most pathogenic. as for host factors, although severe and fatal cases have been described in healthy immunocompetent hosts [ , ] , there is evidence to suggest that children under the age of [ ] and immunocompromised hosts either secondary to hematologic malignancies, immunosuppressant treatment for organ transplantation, or hiv infection are at a greater risk to develop more severe disease with higher case fatality rates [ , ] . all rickettsiaceae are intracellular pathogens, and one could expect an increased risk for severe disease in pidds with abnormal t cell function. five to days of doxycycline is the preferred treatment for non-pregnant adults and children. treatment should not be delayed while awaiting diagnostic testing [ ] and can be given to children despite a minimal risk for dental staining. alternative treatments include azithromycin for mild disease [ ] and chloramphenicol for pregnant women. anaplasma is an intracellular bacterium that infects wild and domestic mammals, including man. a. phagocytophilum was formerly known as human granulocytotropic ehrlighiosis but is now known as human granulocytotropic anaplasmosis [ ] . e. chaffeensis infects monocytes and causes human monocytic ehrlichiosis [ ] . anaplasma and ehrlichia have historically cycled within non-human enzootic hosts, and man has become infected through increasing interactions with the environment. ehrlichia and anaplasma are transmitted by ixodes species of ticks, and their ranges include the eastern usa, south central usa, and scattered regions of europe, as far north as sweden. these infections have not been seen in humans in the southern hemisphere, but there are reports of organisms being identified [ ] . a less common mode of transmission is through transfusions. the symptoms of ehrlichia and anaplasma infections are similar [ ] . abrupt onset of an influenza-like illness occurs about days after a tick bite. ehrlichia can cause a mild rash ( % of adults and % of children), but rash is uncommon in anaplasma infections. highly suggestive laboratory features are leukopenia and thrombocytopenia. mortality in the general populations appears to be < %, but icu admission is not uncommon. hemophagocytosis has been described with anaplasma [ ] and ehrlichia [ ] . both infections are more severe in any setting of immune compromised, including asplenia [ , ] . the diagnosis is typically made by pcr, and doxycycline is the recommended treatment. intracellular inclusions can be seen on cbc smears (more often in anaplasma than ehrlichia). an uncommon but well described facet of these infections is that the tick vector can also transmit borrelia burgdorferi and babesia microti, and simultaneous infection with multiple organisms can occur. c. burnetii is a highly pleomorphic gram-negative coccobacillus and the causative agent of q fever. q fever is a zoonosis, and the most common reservoirs are cattle, sheep, and goats but many other animals can be infected by c. burnetii [ , ] . when infected, these domestic animals can shed the organism in urine, feces, milk, and especially birth products. the pathogen survives within the phagolysosome of host cells, and a spore stage has been described. this spore stage explains the ability of c. burnetii to survive in unfavorable environmental conditions, and it can be an environmental risk for months to years after shedding from an infected animal. q fever is considered an occupational disease affecting people with direct contact with infected animals; however, indirect contact through exposure to contaminated animal products has also been described to cause disease outbreaks. humans are infected by inhalation of contaminated aerosols. following an average incubation period of days, infected patients can present with severe headache, fever, chills, fatigue, and myalgia. other signs and symptoms depend on the organs involved. in contrast to rickettsial diseases described above, rash rarely occurs in the early stages of the disease. c. burnetii can cause a range of clinical symptoms. a self-limited febrile illness is probably the most common form of q fever. pneumonia, either atypical or severe, is also common and can be a part of acute q fever syndrome. in contrast, a variety of manifestations can be recognized in chronic q fever, including endocarditis, endovascular infection, osteomyelitis, hepatitis, interstitial pulmonary fibrosis, prolonged fever, and purpuric vasculitic rash. q fever diagnosis is based on serologic testing with indirect immunofluorescence being the best for differentiating between acute and chronic q fever (high antiphase i antigen titer). the treatment of choice for acute q fever is doxycycline, with co-trimoxazole, chloramphenicol, or rifampin being an accepted alternative. there is no agreement on the treatment for q fever endocarditis, and a combination of doxycycline with either fluoroquinolone or hydroxychloroquine is recommended. there is also controversy regarding the duration of treatment, ranging from years to indefinite treatment. old evidence suggests that q fever is more common in immunocompromised patients. a french study showed higher incidence of antibodies to c. burnetii in hiv positive compared to hiv-negative patients ( . vs . %). in addition, out of hospitalized patients were hiv positive ( . %), suggesting a more frequent symptomatic disease [ ] . a smaller similar study performed in central africa failed to show increased incidence of seropositivity in hivpositive patients [ ] . two case reports describe severe disease in immunocompromised patients. the first was a case of fatal q fever disease in an -year-old male with cgd [ ] . the patient was treated with broad spectrum antibiotics, but without coverage for q fever. the second case was a -yearold asplenic male who presented with fever, jaundice, and encephalopathy and was diagnosed with acute q fever [ ] . the patient was successfully treated, but the two case reports could suggest susceptibility in cases of phagocytic disorders. the bartonellaceae are fastidious, facultative intracellular gram-negative bacilli (table ). most species infect primarily non-human animals, and in most cases, human are considered incidental hosts. the documented common human pathogens include bartonella bacilliformis, bartonella henselae, and bartonella quintana, and it is believed that humans are the primary mammalian reservoir of b. quintana. infection occurs through inoculation of bartonella-infected arthropod feces into breaks in the skin. the epidemiology of bartonella infection in humans follows the distribution of the vector. as such, infection with b. bacilliformis follows the distribution of the sand fly vector (lutzomya) and is confined to the andes mountain in peru, ecuador, and colombia at heights of between and m. the human body louse pediculus humanus is the vector of b. quintana, which explains the global distribution of this pathogen and worldwide outbreaks of trench fever, mostly in conditions of poor sanitation and upon exposure to body lice. trench fever was responsible for over a million infections during world war i. fever, either abrupt or indolent in onset, with a maculopapular rash, conjunctivitis, headache, myalgias (most often affecting legs), and splenomegaly was described. urban b. quintana infections occur most often among homeless and have a distinct clinical picture with fever as the most common manifestation. endocarditis occurs in many [ ] . bartonella henselae is globally endemic, and domestic cats are a major reservoir. the major arthropod vector of b. henselae is the cat flea, which is responsible for cat-to-cat transmission. human infection, called cat scratch disease, is assumed to involve inoculation of bartonella-infected flea feces into the skin during a cat scratch. b. henselae causes primarily adenopathy and neurologic symptoms [ ] . b. bacilliformis causes a condition with two phases: the acute phase with fever, anemia, and transient immunosuppression followed by nodular dermal eruption [ ] . recently appreciated are the ongoing systemic features during the eruptive phase such as arthralgias, adenopathy, and anorexia [ ] . diagnosis of bartonella-associated diseases can be achieved by direct examination of clinical material, bacteriologic culture methods, serologic and immunocytochemical studies, pcr-based assay, or combination of these methods. bartonella infection can present differently in immunocompromised hosts [ ] . in addition to higher prevalence of bartonella infection in hiv patients [ ] , both b. quintana and b. henselae can induce neovascular proliferation which might involve the skin, lymph nodes, and a variety of internal organs including the liver, spleen, bone, brain, lung, bowels, etc. these neovascular lesions, known as bacillary angiomatosis/peliosis (ba/bp), were initially described in hiv-infected patients with advanced disease and was later described in other immunocompromised hosts secondary to immunosuppressant treatment for solid organ transplantation or hematologic malignancy [ ] [ ] [ ] [ ] . cutaneous ba lesions can vary in presentation and can be subcutaneous, dermal nodules, single or multiple papule that can be flesh colored, red, or purple. lesions may ulcerate and bleed. they can change in number and size (millimeters to centimeters; few to hundreds) and can involve mucosal surfaces or deeper soft tissues. similar variation can be seen with visceral involvement. histologically, lesions consist of lobular proliferation of small blood vessels and neutrophilic predominant cell infiltration. the term bacilliary peliosis is used to describe bloodfilled cystic spaces mostly involving the liver, spleen, and lymph node. pathogenic bacteria can be isolated from vascular lesions. while both pathogens were associated with cutaneous lesions, only b. henselae was associated with visceral bp [ ] . based on hiv literature, it is reasonable to expect an unusual presentation of bartonella infection especially in pidds involving t cell dysfunction. bartonella infection was described as a cause for hepatitis in a single cd l deficiency patient [ ] . in addition, since cases of granulomatous disease due to bartonella infection [ ] [ ] [ ] have been described, it should be considered in the differential diagnosis of pidd with granulomatous inflammation. borrelia spp. the genus borrelia belongs to the spirochaetaceae family. it includes b. burgdorferi which causes lyme disease and species that cause relapsing fever. the latter are further divided into tick-borne species and louse-borne species. louse-borne relapsing fever (lbrf) is caused only by borrelia recurrentis and is spread by human body louse. the disease was epidemic in the early twentieth century, and it is estimated that more than , died of lbrf during world war ii. with sanitation improvement lbrf is now found only in the horn of africa and among homeless people in europe. more recently, cases of lbrf in refugees and migrants were described [ , ] . tick-borne relapsing fever (tbrf) is caused by a group of pathogens which are maintained by and survive in softticks (orinthodoros genus). each tbrf borrelia species depends on one specific species of soft-body tick. except for australia and antarctica, tbrf species can be found in all continents. the animal reservoir includes small animals and rodents. since the spirochetes persist in the tick salivary gland, disease transmission occurs when humans intrude the tick's environment. tick bites are painless, and history of a tick bite is often missing. therefore, a history of potential exposure can be valuable. the major clinical symptom is relapsing fever. after a median incubation period of days, patients present with febrile episode that can last - days, followed by afebrile period of - days. patients with tbrf can have up to febrile relapses, while lbrf is usually associated with only one relapse. other symptoms include myalgia, arthralgia headaches, and vomiting, and physical findings include lymphadenopathy and splenomegaly with rash occurring only in third of the patients. a range of neurologic complications as well as systemic inflammatory response syndrome also have been described [ ] . diagnosis is based on identifying the spirochetes on blood smear. sensitivity of blood smear is higher during febrile period (about %), and a negative blood smear does not exclude rf. in lbrf, the spirochete load can be low and specific stains can be helpful. other diagnostic methods include serologic testing, pcr, and mouse inoculation. doxycycline, tetracycline, and penicillin are the preferred treatment, with most patients treated with - days of doxycycline. jarisch-herxheimer reactions with high fever and leukopenia occur in half of the patients following the first antibiotic dose and can develop into a severe reaction with hypotension, respiratory distress, and death [ ] . without treatment, tbrf carries a mortality rate of up to % with even higher % mortality rate for untreated lbrf. two cases of meningoencephalitis with borrelia miyamotoi in heavily treated immunocompromised patients have been described [ , ] . lyme borreliosis is the most common vector-borne disease in the northern hemisphere caused by a group of at least genospecies. lyme disease is a multisystem illness affecting the skin, joints, nervous system, and heart. human infection is caused mainly by three species: b. burgdorferi is the most common cause in north america but also found in europe, and borrelia afzelii and borrelia garinii which cause the disease in europe and asia. emerging infections in the mid-western usa with borrelia mayonii cause a condition similar to lyme disease. most tick species do not carry borrelia species. the vectors of all borrelia species are the ixodid tick species; this includes the deer tick, i. scapularis, in the northeast and midwest of the usa, ixodes pacificus in the west, the sheep tick, ixodes ricinus, in europe and the taiga tick, ixodes persulcatus, in asia. the ixodid tick demonstrates a complex vector ecology with preferences for different hosts in different geographical regions and at different stages of its development. more than different species, including deer, rodents, birds, and reptiles, have been described. infection rates also show seasonal variation with highest rates during lyme disease follows several stages starting with localized disease at the site of inoculation, followed by dissemination stage and, later, persistent infection [ ] . however, an individual patient can show highly variable disease progression with different patterns of organ involvement and disease severity. erythema migrans (em) is often seen at the site of the tick bite after - days of incubation. regional lymphadenopathy can be seen. secondary skin lesions represent hematogenous dissemination. at this stage, constitutional symptoms of general fatigue, fever and headaches, migratory musculoskeletal pain, conjunctivitis, and cardiac involvement occur. in total, % of untreated patients can develop frank neurologic manifestations of meningitis, encephalitis, and variable forms of neuritis with fluctuating symptoms. persistence can occur in untreated (on inadequately) patients. antibiotic refractory arthritis is well described. however, even without treatment, intermittent or persistent attacks usually resolve completely within several years. co-infection with a. phagocytophilum and b. microti can cause diagnostic confusion [ , ] . the diagnosis of lyme disease is established based on clinical symptoms, history of potential exposure, and serologic studies. although positive culture can confirm the diagnosis, it can usually be obtained only from early em lesions. pcr testing is superior to cultures and can be performed on joint fluid samples [ ] . cdc recommendations for the diagnosis of lyme disease are based on serology which might be impossible in pidd patients with abnormal humoral response. cdc guidelines require both an elisa (or comparable test) to be positive and a western blot ( out of bands ( , , or kd) on the igm or out of bands on the igg ( , , , , , , , , , kd) . most lyme manifestations can be treated with oral antibiotics, while patients with neurologic abnormalities and some patient with lyme arthritis require intravenous therapy [ ] . doxycycline is the treatment of choice for early and disseminated disease, with amoxicillin as the second-line choice. jarisch-herxheimer-like reactions can appear in the first h in about % of the patients. for patients with clear neurologic symptoms, - weeks of iv ceftriaxone is the most commonly used therapy. few cases of neuroborreliosis and hiv have been described with a good response to treatment. descriptions of lyme disease in pidd patients are lacking. zoonoses are infectious diseases that pass between animals and humans and span the spectrum of pathogens including viruses, bacteria, fungi, and parasites. zoonoses are very common and range from mild such as certain forms of tinea to lifethreatening infections such as rabies. some of the zoonoses that are vector-borne will be covered in other sections. risk mitigation strategies for zoonoses include patient education, proactive advice about risk scenarios, and avoidance of infected animals. several zoonoses are associated with contact with mammals such as rodents or domestic farm animals through direct contact or through contact with their feces. for instance, hantavirus infections are often associated with exposures to mouse droppings when staying in cabins in the western usa. occupational exposures can occur with buffalopox or parapoxvirus (causing orf infection) through direct contact with buffalo and goats/sheep, respectively [ ] [ ] [ ] [ ] . in general, there are very few cases of pidd with zoonotic infections acquired from mammals. however, there are a few special considerations. for instance, lymphocytic choriomeningitis virus is acquired through exposure to house mice primarily, with hamsters being a less common source of infection. both domestic and wild mice can carry the infection without exhibiting symptoms. although infection is rare, there have been severe cases in patients with t/ nk cell dysfunction, such as a case in xlp and cases in solid organ transplant recipients [ ] . therefore, in patients with severe t/nk defects, consideration should be given to whether small rodents are appropriate household pets. tularemia is a disease of animals and humans caused by the bacterium francisella tularensis. rabbits, hares, and rodents are the main reservoirs. humans become infected through direct contact, ingestion of contaminated water, or inhalation of organisms. ticks and deer flies can also transmit the disease through bites. fever is universal, but other features depend on the mode of transmission. a patient with cgd had a complex course suggesting myeloid defects are a risk for more severe disease [ ] . rabies is an almost universally fatal infection caused by contact with oral secretions from infected mammals, typically raccoons, bats, or foxes, and there is no suggestion that pidd or immune compromised modifies the prognosis. for individuals with high-risk exposures, such as those working with wildlife or traveling in endemic areas, pre-exposure prophylaxis is given with vaccination, and if an exposure occurs, rabiesspecific immunoglobulin is provided as well as vaccination. however, for those with humoral immunodeficiencies who cannot respond to the typical pre-exposure vaccination, there needs to be counsel on the additional risk without vaccination. in table , several of the bacterial and viral zoonoses are summarized with their typical endemic areas, which is somewhat limited by diagnostic abilities and reporting, as well as the typical clinical scenarios, known cases in immunodeficiency and an approach to diagnosis and therapy. nipah virus causes a range of infectious phenotypes ranging from asymptomatic infection to acute respiratory distress and encephalitis. nipah virus was identified in on pig farms in malaysia, leading to identification of human cases, including human deaths and the culling of one million pigs [ ] . the natural host is the fruit bat: pteropodidae pteropus. symptoms of infection from the malaysian outbreak were primarily encephalitic in humans, but later, outbreaks have caused respiratory illness, increasing the likelihood of human-to-human transmission. fever, headache, cough, abdominal pain, nausea, vomiting, weakness, problems with swallowing, and blurred vision were common. seizures were seen in % and coma in %. relapses of encephalitis have been described [ ] . increasing infections due to nipah virus is thought to be due to an increasing overlap between bat habitats and pig sties in malaysia. all outbreaks thus far have been in india, bangladesh, or malaysia. the diagnosis of nipah virus relies on pcr of fluid samples, serology in convalescent samples, and immunofluorescence of tissue. there have been no infections of immune compromised patients reported. therapy is largely supportive, although preliminary reports of ribavirin use have been encouraging. a vaccine is under development. severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) are two zoonotic coronaviruses. the sars pandemic in - resulted in reported cases in countries. no further sars cases were reported after the pandemic except isolated cases linked to laboratory accidents. patients usually presented with fever and respiratory symptoms, but occasionally had diarrhea and vomiting. about - % of sars patients required mechanical ventilation, with a case fatality rate of about % [ ] [ ] [ ] . mers was first noted in saudi arabia in , and countries around the arabian peninsula are now endemic for mers-cov. patients usually present with fever, cough, chills, sore throat, myalgia, and arthralgia rapidly progressing to pneumonia with over % of patients requiring intensive care. about one-third of patients present with diarrhea and vomiting, and acute renal impairment is a striking feature of mers. risk factors for poor outcome include diabetes, hypertension, and renal and lung disease. cases have been exported to at least countries with travel occasionally causing cluster of secondary outbreaks. one such example is the mers-cov outbreak involving patients in south korea, and the median incubation period was estimated to be days with a range of to days [ ] . at the end of , there were confirmed mers with a % mortality rate [ ] [ ] [ ] . bats are the natural reservoirs of both sars-cov and mers-cov. sars-cov crossed the species barrier into palm civets and other animals in live animal markets in china, which then infected human, while a mers-cov ancestral virus crossed species barrier into dromedary camels. abundant circulation of mers-cov in camels results in continuous zoonotic transmission of this virus to human, while sars-cov was not found to circulate in the intermediate reservoirs, explaining sars being a one-off outbreak and mers a continuing zoonotic disease [ ] . aerosolgenerating procedures such as intubation were associated with increased viral transmission of both covs resulting in nosocomial outbreaks [ ] . super-spreaders are responsible for large and prolonged outbreaks [ ] . the diagnosis for sars and mers include both serological tests and pcr assays that can quantify viral loads [ ] . functional genetic polymorphisms leading to low serum mannose binding lectin (mbl) are associated with susceptibility to but not severity of sars in both southern and northern chinese [ ] [ ] [ ] . mbl was shown to bind to sars-cov and inhibit the infectivity [ ] , suggesting its role as first-line defense against sars-cov. although no patients with primary immunodeficiency infected with sars-cov or mers-cov were identified, likely due to the limited number of such infections, patients with t cell defect and type interferon pathway defects could suffer a more severe disease course [ , ] . virus-based and host-based treatment strategies are largely experimental with uncertain benefits. ribavirin, type interferons, small molecules, and monoclonal antibodies that block covs entry have been explored [ ] . passive immunotherapy and multiple candidate vaccines have been tested in various animal models. convalescent plasma immunotherapy has been considered, but clinical trials are lacking in mers [ ] , while for sars a systematic review concluded convalescent serum may reduce mortality and appear safe [ ] . the filoviridae family contains three known genera, the ebolaviruses, marburgviruses, and cuevavirus. ebolavirus and marburgvirus cause hemorrhagic fever syndromes in primates and humans, with high fatality rates. cuevavirus infects only bats. the ebolavirus genus contains five species, with two of the species (zaire ebolavirus and sudan ebolavirus) being responsible for the majority of cases of human disease, while marburgviruses contain two species (marburg virus and ravn virus). filoviruses are capable of replicating in a number of cell types (with the exception of neurons and lymphocytes). upon entry into the body of the host (via breaks in the skin, parenterally, or through mucosal surfaces), filoviruses employ a variety of mechanisms to evade the activity of the immune system [ ] . the incubation period (interval from infection to onset of symptoms) varies from to days. symptoms begin abruptly, with high fever, severe headache, malaise, myalgia, diarrhea, nausea, and vomiting. a rash can occur between and days after onset of symptoms. hemorrhagic manifestations occur between and days, and fatal cases usually have some form of active bleeding. in an outbreak setting, the symptoms are unmistakable but confusion with malaria can occur early in the disease or in sporadic cases. since their original descriptions in and , respectively, for marburg and ebola, there have been a number of sporadic cases and several major outbreaks. the largest marburg virus outbreak occurred in angola in (with a fatality rate of > %), while the largest ebola epidemic happened between and in west africa (sierra leone, guinea, and liberia) and claiming over , lives (fatality rate > %). although not definitively proven in the case of ebola, bats are believed to be the natural animal reservoir for these viruses [ , ] . these viruses are transmitted via contact with blood or body fluids from an infected host; notably, certain body fluids can harbor virus for weeks to months after resolution of disease. given the recent outbreak in west africa, there has been renewed interest in understanding the pathogenesis of filovirus infections and possible therapies. literature regarding how the pathogenesis of disease may be altered in patients with pidd is lacking. however, the assumption is that in the absence of an intact cellular and/or humoral immune response, the patient with a pidd may be at increased risk of mortality in the setting where mortality is already high. these viruses induce apoptosis of lymphocytes and macrophages, and there is therefore a profound secondary immune compromised [ , ] . filoviruses can be detected in multiple body fluids via pcr. although practiced for decades, a study in guinea in failed to show a decrease in mortality among patients receiving convalescent plasma from previously infected donors [ ] . a number of additional compounds (e.g., tkm-ebola, bcx , and gs- ) and biologics (zmapp) have been shown to offer protection in animal models of ebola, but to date, no controlled and appropriately powered clinical trials have addressed their efficacy in humans. finally, a number of vaccines for ebola are undergoing clinical studies (including four in phase iii trials). importantly, in late , the rvsv-zebov vaccine was shown to have displayed high efficacy in protecting immunized adults during the guinea ebola outbreak, and the data also suggested that the vaccine may even offer bherd immunity^to unimmunized persons in proximity to recipients of the vaccine [ , ] . hepatitis e virus is a single-strand rna virus of the hepeviridae family. it is an important zoonotic disease in asia and africa, and fecal-oral spread is the usual route of transmission [ ] . handling of pig or boar meat is a risk factor, and - % of pig livers sold in grocery stores in japan and the usa are infected [ , ] . swine represent the major reservoir, although antibodies to the virus have been found in many species [ ] . the incubation period is weeks to months, and viremia disappears with the onset of symptoms. the mortality rate is - % and can reach % in pregnancy [ ] . acute hepatitis usually resolves but can lead to liver failure in severe cases. patients with hepatitis e posttransplant have had severe courses in some cases [ ] . in immune compromised patients, the course can become chronic [ ] [ ] [ ] . in these cases, cirrhosis develops. the diagnosis is by serology or pcr, and the treatment is supportive. prevention modalities for infections transmitted by humans are conceptually different than infection prevention for vector-borne infections. hand hygiene is extremely important, and avoidance of clearly infected people can be helpful. recognition of infections with fecal-oral transmission and the importance of water purity are critical for patients with pidd. in contrast, infections transmitted by aerosols require prevention strategies related to droplet precautions. in outbreak scenarios, if the risk to the patient is high, specific chemoprophylaxis may be considered. influenza viruses type a and b cause annual epidemic influenza, while type c causes sporadic mild influenza-like illness. patients present with sudden onset of fever, chills, and myalgia, followed by sore throat and cough. other less common features include diarrhea, acute myositis, and encephalopathy [ , ] . co-infection with bacteria such as pneumococci results in more severe disease [ , ] . influenza pandemics occur yearly around the world. influenza viruses infect to % of the global population, resulting in~ , deaths annually [ ] . the viruses circulate in asia continuously and seed the temperate zones, beginning with oceania, north america, and europe, then later seeding into south america [ ] . diagnosis of influenza includes direct/ indirect immunofluorescent antibody staining for antigens in nasopharyngeal aspirates and pcr. a patient with compound heterozygous null mutations of the gene encoding irf , a transcription factor for amplifying ifn-α/β, was reported to have life-threatening influenza during primary infection [ ] . fatal influenza-associated encephalopathy in both chinese and japanese children has been reported to be associated with genetic variants of thermolabile carnitine palmitoyltransferase ii [ ] . patients with scid will have prolonged viral shedding [ ] . severe pandemic influenza a virus (h n ) infection has been associated with igg and igg subclass deficiency [ , ] . in addition, influenza infection can be more severe in pidd patients with underlying lung disease, such as bronchiectasis, and antibiotic coverage of chronic colonizing bacteria (such as pseudomonas) in this setting may be helpful. inactivated seasonal influenza vaccine should be given to pidd patients even those with humoral deficiencies as their t cell response to influenza could be normal and offer protective immunity against severe influenza [ , ] . antiviral drugs include neuraminidase inhibitors (oseltamivir and zanamir) and adamantanes (amantadine and rimantadine), but resistance to adamantanes is widespread. measles is a single-stranded, negative-sense, enveloped (nonsegmented) rna virus of the genus morbillivirus. measles is highly communicable, transmitted by droplets, and less commonly by airborne spread. patients present with fever, cough, coryza conjunctivitis rash, and koplik spots. complications include pneumonia, acute encephalitis, and subacute sclerosing panencephalitis (sspe) [ ] . diagnosis of measles includes serological tests, virus isolation, and pcr. in an outbreak, the clinical features may be sufficient for diagnosis. measles vaccine has caused severe measles in children with stat and ifn-α/β receptor deficiency [ , ] , demonstrating the importance of type interferon pathway in controlling measles. immune compromised of nearly any type is associated with severe disease and higher mortality [ ] . t cell deficiency states are the most strongly associated with the development of giant cell pneumonia and inclusion body encephalitis, the most feared complications of measles. sspe is a slow encephalitis due to persistence of replication defective measles virus in the cns. it is most frequently seen when young infants are infected with measles and - years later, sspe becomes evident. there are case reports supporting the immune compromised as increasing the risk of sspe [ ] . treatment of sspe with ribavirin has shown some improvement, but the prognosis in general with sspe is very grave. patients with cgd have defective memory b cell compartment, resulting in lower measle-specific antibody levels and antibody-secreting cell numbers, but severe disease has not been reported [ ] . pidd patients may harbor the virus latently for longer than usual, leading to complications at the time of transplant [ ] . specific antiviral therapy is lacking, but ribavirin has been given to severely ill and immunocompromised children. for measles post-exposure prophylaxis, intravenous immunoglobulin (ivig) is recommended for severely immunocompromised patients without evidence of measles immunity [ ] . this would likely include patients with scid and hypogammaglobulinemia who are not yet on regular ivig. measles vaccine, given in a two-dose regimen, has brought down incidence enormously worldwide and the who is planning for eradication globally. enteroviruses (evs) are among the most common viruses infecting humans worldwide. evs are small non-enveloped, single-stranded rna viruses of the picornaviridae family. human evs are categorized into seven species that include hundreds of serotypes, such as polioviruses (pv), coxsackie viruses a, and b (cv-a and b), echoviruses, and human rhinoviruses (hrvs). of these species, many important serotypes are known to infect human such as pv - , cv-a , cv-b , ev-a , ev-d , and hrv (table ) . non-polio enteroviruses (npevs) have a worldwide distribution. infants and young children have higher incidence of infection and a more severe course of illness than adults. the mode of transmission is mainly through fecal-oral and respiratory routes. infection occurs all around the year in tropical and subtropical regions, while in temperate climates the peak incidence of infection is during summer and fall months [ ] . npevs are associated with diverse clinical manifestations ranging from mild febrile illness to severe, potentially fatal conditions. most cases are asymptomatic or have mild symptoms including fever with or without rash; symptoms of hand, foot, and mouth disease; herpangina; acute hemorrhagic conjunctivitis; upper respiratory infection; and gastroenteritis. more severe symptoms occur in infants and young children [ , ] . acute flaccid paralysis [ ] , neonatal enteroviral sepsis [ ] , myocarditis/pericarditis [ , ] , hepatitis, pancreatitis, pneumonia, and atypical hemolytic uremic syndrome [ ] are severe manifestations seen in immunocompetent people. chronic infections have been seen in immunocompromised patients [ ] . each virus may produce one or more of the aforementioned manifestations; however, some serotypes are often associated with particular features ( table ) . the definitive diagnosis of npev infection relies on pcr or virus isolation from the cerebrospinal fluid, blood, stools, urine, or throat swab [ , ] . treatment of npevs is mainly supportive since most infections are self-limited. high doses of intravenous immunoglobulin (ivig) are recommended in patients with severe symptoms. the efficacy of some new antiviral drugs (pleconaril, vapendavir, and pocapavir) is still under investigation [ ] . no vaccine has been licensed yet for npevs. however, phase clinical trials of inactivated monovalent ev-a vaccines manufactured in china showed high efficacy against ev-a in infants and young children [ ] . patients with a variety of pidds are unusually susceptible to ev [ ] . the most susceptible groups are patients with primary antibody deficiency such as xla, cvid, and hyper-igm syndrome (higms) as well as those having scid and major histocompatibility class ii deficiency [ , ] . the most severe form of infection has been described in patients with xla due to the profound deficiency of immunoglobulins essential for viral neutralization during infection. affected patients usually present with indolent but relentlessly progressive non-necrotizing meningoencephalitis. regression of cognitive skills, flaccid quadriplegia, and deafness has been described. the reported non-neurologic presentations in xla include septicemia, dermatomyositis-like disease, hepatitis, and/or arthritis [ , ] . the incidence of npev meningoencephalitis in large registries of xla cases is - % [ ] . unpublished data from the kuwait national pidd registry, which includes pidd patients, showed that nine patients had documented npev infections and two died from these infections. the two deaths were seen in scid patients (personal communication with prof. waleed al-herz, md). in addition, npev meningoencephalitis and/or septicemia were reported in few cases with either primary b cell deficiency such as b cell linker (blink) protein deficiency [ ] or acquired b cell deficiency following the administration of anti-cd (rituximab) [ , ] . in all reports, better outcome was attributed to the early administration of high doses of ivig during npev viremia [ ] . npev infection in pidd diseases remains a major threat to patients. also, the possible prolonged viral excretion and the emergence of resistant strains runs the risk of spreading infection to the surrounding community. oral polio vaccine (opv) consists of a mixture of three live attenuated poliovirus serotypes. opv induces production of neutralizing antibodies against all three serotypes, in addition to a local intestinal immune response. opv can result in vaccine-associated paralysis (vap) secondary to reversion of the vaccine strain to the neurovirulent wild-type strain. an example for such an event was demonstrated by the - outbreak in the dominican republic and haiti [ ] , believed to be driven at least in part by undervaccination of the population, which allowed the spread of the reverted vaccine strain [ ] . although rare, patients with pidd have a higher risk to develop vap. reports have shown that pidd patients with antibody deficiency can have prolonged viral replication which can persist for years and therefore theoretically increase the risk for a spontaneous reversion within the immunodeficient host [ ] [ ] [ ] [ ] . cases of vap were shown in patients with antibody deficiency and combined immunodeficiency syndromes [ , , ] . therefore, opv is contraindicated in patients with pidd, and this contraindication extends to their household contacts [ ] . beyond the obvious risk for the pidd patient, prolonged virus shedding also increase the risk for spreading vaccine-derived paralytic strain in the general population. bacterial infections have molded human behavior and altered societies over human history. today, largely ignored due to antibiotic susceptibility, they continue to cause misery and disease around the world. three infections are highlighted, and additional commonly encountered infections are listed in table . pertussis is a respiratory infection caused by bordetella pertussis that begins after a -to -day incubation period as a minor upper respiratory infection that progresses with cough. initially intermittent, it evolves into paroxysmal coughing spells usually followed by vomiting in infants and young children. it lasts to weeks and can have many complications such as syncope, weight loss, rib fracture, and pneumonia. infants under months are more severely affected, developing pneumonia, pulmonary hypertension, hypoxia, subdural bleeding, and seizures. death can occur, especially in young infants [ , ] . adults typically have a prolonged cough with fewer complications [ ] . it is transmitted via aerosolized droplets during close contact. people are most contagious during the catarrhal stage and the first half of the paroxysmal phase, totaling to weeks [ ] . the introduction of whole-cell pertussis vaccine (dpt) in the s in the usa reduced the incidence of the disease from , cases to around cases per year in the s. a resurgence in was associated with the substitution of the whole-cell vaccine by the acellular pertussis vaccine (dtap) [ ] . new strategies such as boosters with acellular pertussis for adolescents and adults with tdap and use of tdap during pregnancy seem to be effective in partially reducing the incidence of the disease [ ] ; however, pertussis cases in the usa remain higher than the s. the lack of persistence of antibody in the adult population means adults not only represent a reservoir for the disease but also do not provide sufficient titers to immunoglobulin products prepared from adult plasma pools. a relatively recent requirement in some countries is vaccination of adults every years to maintain immunity. this should, over time, improve titers in immunoglobulin products. culture of specimens obtained by nasopharyngeal swabs is the gold standard of laboratory diagnosis due to the % specificity, but polymerase chain reaction (pcr) is gaining prominence due to its higher sensitivity and speed of results; serodiagnosis can be used in the late stages of the disease [ ] . filamentous hemagglutinin (fha) and pertussis toxin (pt) antibodies were detected at peak measurements in pidd patients on regular ivig, although some of them had pt antibodies below the protective level as trough measurements [ ] . severe pertussis cases have not been reported in pidd patients, but severe disease has been seen in malignancies [ ] . antimicrobials such as azithromycin, erythromycin, and clarithromycin, if given during the catarrhal stage, may ameliorate the disease and shorten the contagious period. to avoid cases of pertussis, it is also worth emphasizing the importance of good vaccine coverage rate among the whole population, but especially among healthcare workers and family members of patients with pidd. neisseria meningitidis the onset of neisserial meningitis is associated with sore throat, headache, drowsiness, fever, irritability, and neck stiffness [ , ] . purpuric lesions are very characteristic. this pathogen can also present with sepsis which has a % mortality rate as opposed to % mortality with a meningitic presentation. this bacterium can also cause a chronic condition referred to as chronic meningococcemia. this condition is characterized by intermittent fevers lasting week to - months [ ] . a non-purpuric rash is common which may evolve into purpura. arthritis, similar to that seen with gonococcus, is common. meningococcal disease primarily affects children under years of age. n. meningitidis is a global pathogen [ ] . there are serogroups, but the majority of invasive meningococcal infections are caused by organisms from the a, b, c, x, y, or w serogroups. the annual number of invasive disease cases worldwide is estimated to be at least . million, with , deaths related to invasive meningococcal disease. serogroups b and c are responsible for most infections in europe. serogroup a has historically been the major organism in africa; mass vaccination has led to some improvement, but the emergence of group x disease is worrisome. the hajj in the middle east has seen epidemics of w- , and vaccination is now required for hajj travelers. b and c serogroups are the most common through the americas. n. meningitidis cases occur at a rate of about case per , people throughout the world [ ] , but across the sahel of africa and in china, epidemics can lead to case rates of per , [ ] . the bacterium is a natural human commensal, with carriage rates of about %. diagnosis can be by clinical examination in epidemics or by gram stain and culture. complement-deficient individuals have an increased risk of neisserial disease, but not necessarily increased mortality. hiv is associated with increased disease, suggesting that t cells are also important for defense. thirdgeneration cephalosporins are typically used for treatment. penicillin, ampicillin, aztreonam, and chloramphenicol are alternatives. there is great inter-individual variability in the development of tb disease. roughly, % of infected individuals develop clinical disease within years of infection (mostly during childhood). about % became latently infected without clinical disease, and the remaining to % develop pulmonary tb later in life, either from reactivation of latent infection or reinfection. molecular epidemiology studies from high burden areas suggest more disease results from recent transmission than from reactivation of latent tb, particularly in people living with hiv [ ] . acquired or inherited host factors may at least partially account for the variable disease course, resulting in increased susceptibility to mycobacteria infections [ ] . pidd associated with tb and ntm infections include t cell deficiencies, gata deficiency, cgd, anhidrotic ectodermal dysplasia with immunodeficiency, x-linked (xl) recessive cd ligand deficiency, autosomal recessive (ar) stat deficiency, ar irf deficiency, and ar tyk deficiency. in addition, a group of disorders with a strong susceptibility to ntm, named mendelian susceptibility to mycobacterial diseases (msmd), have been recognized since the s. these are rare inborn errors of ifn-γ signaling pathway that present with isolated predisposition to infections caused by weakly virulent mycobacteria such as bcg vaccine and environmental ntm, in otherwise healthy patients. the genetic defects involve impairment in the production of interferons (ar il rβ , ar il p , autosomal dominant (ad) irf , ar isg , xl recessive nemo) or response to interferons (ifn-γr, ad stat , ad irf , cgd) [ ] . an acquired form exits: adults with ntm infection in thailand and taiwan were found to have high-titer anti-interferongamma antibody [ ] . these individuals from southeast asia were found to have hla-drb * / : and dqb * : / : . patients suspected of having pulmonary tb should have acid-fast bacilli (afb) smear microscopy and culture performed in three sputum samples. pcr for mtb can be performed [ ] . the use of rapid tests facilitates early diagnosis, and the who has recently recommended their use. the only recommended rapid test for detection of tb with and without rifampicin resistance is the xpert mtb/rif assay (cepheid, sunnyvale, ca). the who recommends the xpert test for those suspected of having drug-resistant tb or in hiv; however, culture is still the mainstay and is not replaced by the xpert test. tb skin testing (mantoux testing) uses a purified protein derivative injected under the skin. its advantages are that it can be used for large-scale screening and it is cost effective. skin testing does have several disadvantages when used as a diagnostic test. reading the induration requires training and immunodeficiencies can alter the magnitude of the induration. immunosuppressed patients (hiv, organ transplant) are considered positive if the induration is ≥ mm. some immunodeficiencies may completely ablate the response. other causes of false-negative tests are malnutrition, concurrent infection, recent live viral vaccine administration, renal failure, malignancy, medical stress, very elderly, young infants, or with a very recent infection with mtb. conversely, the results may be falsely positive if bcg has been administered. interferon gamma release assays can be used in any setting where skin testing would be done but are considered superior in settings where the patient has had bcg vaccination and, in some cases, where skin testing has been sown to have high false-negative rates. in general, tb treatment for patients with impaired immune response, including pidd, hiv infection, and immunosuppressive therapy, is based on the standard -month regimen consisting of a -month intensive phase of isoniazid, rifampin, pyrazinamide, and ethambutol, followed by months of isoniazid and rifampin. decisions regarding treatment duration can be individualized, taking into account disease severity, organs involved, and response to treatment. significant pharmacological interaction can occur between rifampin-based mtb regimens and immunosuppressive drugs, such as calcineurin inhibitors or rapamycin, requiring strict monitoring of drug plasma concentrations [ ] . therapy for ntm is complex with highly variable drug resistance patterns and a need for biological augmentation to effectively clear the organism. aspergillus fumigatus (see above), cryptococcus gattii, histoplasma capsulatum, coccidioides immitis (or c. posadasii), blastomyces dermatitidis, paracoccidioides brasiliensis, emmonsia pasteuriana, and penicillium (talaromyces) marneffei are environmental fungi that are endemic in certain parts of the world (table ). with the exception of penicillium marneffei and emmonsia pasteuriana that only cause disease in profoundly immune compromised individuals, these fungi can cause infection in healthy individuals, ranging from mild, self-limited pulmonary disease to infection that requires antifungal therapy for eradication. on the other hand, patients with acquired defects in cell-mediated immunity such as those infected with hiv, and patients with specific monogenic disorders, particularly those involving the il- / ifn-γ/stat signaling pathways, scid, and gata depends on underlying state of immunosuppression and magnitude of environmental exposure icl idiopathic cd lymphocytopenia, aids acquired immune deficiency syndrome, stat signal transducer and activator of transcription , gata gata binding protein , scid severe combined immunodeficiency, cvid common variable immune deficiency, pidd primary immunodeficiency, il rb , interleukin- receptor subunit beta , ifngr gamma interferon receptor deficiency, are at high risk of developing life-threatening disseminated infections by these endemic fungi following environmental exposure [ , [ ] [ ] [ ] [ ] [ ] . diagnosis relies on culture of the corresponding fungus, histopathological demonstration of the fungus-specific characteristic morphologies, and/or surrogate serological and fungal antigen tests. treatment of clinical disease (as opposed to colonization) typically involves an initial induction phase with amphotericin b, followed by long-term azole maintenance therapy and secondary prophylaxis, and prognosis varies significantly depending on the fungal pathogen and underlying pidd. melioidosis is caused by b. pseudomallei, a gram-negative bacteria found in soil and water, in tropical climates of southeast asia and northern australia [ , ] . melioidosis is an emerging, potentially fatal disease ( % mortality). b. pseudomallei can be transmitted by inhalation, ingestion, or direct contact (through open skin) with contaminated soil or water. animals (sheep, goats, swine, horses, cats, dogs, and cattle) are also susceptible to infection and cases of zoonotic transmission through direct contact of skin lesions with infected animal meat or milk have been described [ ] . b. pseudomallei infections are endemic in northern australia and southeast asia. approximately % of reported infections occur during the rainy seasons. cases have also been reported in south pacific, africa, india, and the middle east. in temperate areas, infection is extremely rare and is predominantly imported by travellers or immigrants [ ] . the incubation period of melioidosis is variable from day to years, although common symptoms develop between and weeks after exposure. melioidosis presents most frequently in adults - years of age, but can occur in all ages, with one study reporting % of cases occurred in children [ ] . in australia, the average annual incidence in - was reported as . cases per , people [ ] . the incidence in indigenous australians was higher at . cases per , . a case-cluster in an australian community was associated with post-cyclonic flooding. a recent review suggests that b. pseudomallei is increasingly prevalent in the americas, with a mortality rate of % [ ] . infection in healthy individuals is uncommon, and more than % of cases occur in the setting of underlying conditions such as chronic renal disease, diabetes, chronic lung disease, and alcoholism. a recent review of melioidosis in travelers found that % of cases were acquired in thailand. symptoms usually started at days (range - days) after leaving the endemic area. traveller infections were less often associated with predisposing risk factors ( . %), diabetes mellitus being the most common ( %). melioidosis in travelers had lower mortality ( %) than infection in autochthonous cases in southeast asia [ ] . pneumonia (~ - %) is the most common presentation in adults. there is usually high fever, headache, anorexia, and myalgia. b. pseudomallei infection may also present as localized skin infection, septicemia, or disseminated infection. localized infection results in an ulcer, nodule, or skin abscess. this usually occurs from the bacterium breaching through a break in the skin. patients with renal disease and diabetes are more susceptible to sepsis. in disseminated infection, abscesses may develop in the liver, spleen, lung, and prostate. in children, primary cutaneous melioidosis is the commonest presentation ( %). bacteremia is less common in children than in adults, but brainstem encephalitis has been reported [ ] . difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes [ ] . diagnosis of melioidosis is primarily by isolation of the organism. identification of b. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. although various serological tests have been developed, they are generally unstandardized bin house^assays with low sensitivities and specificities. pcr assays have been applied to clinical and environmental specimens but are not widely available and sensitivity remains to be evaluated. cases of melioidosis have been reported in patients with acquired or inherited immune deficiency. melioidosis was the presenting complaint in several patients with cgd. bacteremic melioidosis was recently reported in two patients with prolonged neutropenia, who succumbed despite appropriate antibiotics [ ] . it is likely that there is increased susceptibility in situations where innate or adaptive immunity is compromised. treatment is with intravenous antimicrobial therapy for - days, followed by - months of oral antimicrobial therapy. intravenous therapy with ceftazidime or meropenem is usually effective. oral therapy may continue with trimethoprim-sulfamethoxazole or doxycycline. free-living amoebas (fla) are protozoa found worldwide that do not require hosts to survive. fla do not employ vectors for transmission and are not well adapted to parasitism in humans. however, there are four genera/species that can cause human disease: naegleria (n. fowleri), acanthamoeba (multiple species), balamuthia (b. mandrillaris), and sappinia (s. pedata). all of these amoebae are capable of inducing cns disease in humans, but acanthamoeba species also cause various extra-cns infections, especially in immunocompromised hosts. the fla that are pathogenic in humans are reviewed below. naegleria are a diverse group of fla flagellate protozoans with a large number of distinct species. only one species, n. fowleri, has been shown to cause infection in humans. n. fowleri has a multi-stage life cycle with amoeboid and trophozoite-infective forms as well as a cyst form [ ] . n. fowleri is found commonly in warm freshwater around the world including lakes, rivers, and hot springs. humans become can become infected when swimming or diving in contaminated water. in rare circumstances, infections have also been attributed to exposure from contaminated tap water sources when utilized for religious cleansing of the nose or irrigation of the sinuses. thus, tap water should not be used for nasal and sinus irrigation. it is not possible to become infected from drinking contaminated water or from contact with an infected person, and the amoeba is not found in salt water. after entry to the nasal cavity, the amoeba travels through the cribiform plate to the olfactory bulbs and migrates to the cerebellum, resulting in primary amoebic meningoencephalitis (pam), a rapidly fatal brain infection characterized by the destruction of brain tissue. in its initial presentation, pam can mimic bacterial meningitis, further delaying accurate diagnosis and initiation of therapies that may save the patient. overall, n. fowleri infections are rare. worldwide, most cases are reported in the usa, australia, and europe; however, in developing countries, it is suspected that a large number of cases go unreported. between and , there were only infections reported in the usa with of the cases attributed to contaminated recreational water, infections following nasal irrigation with contaminated tap water, and case where a person was infected following use of a backyard slipn-slide utilizing contaminated tap water [ ] . the fatality rate associated with n. fowleri infection is over %, and between and , only of the infected persons in the usa have survived infection. initial symptoms of pam start to days after infection and can include headache, fever, nausea, or vomiting [ ] . progressive symptoms can include stiff neck, confusion, lack of attention, loss of balance, seizures, and hallucinations. cardiac arrhythmias have also been observed. the infection progresses rapidly after initial onset and causes death within to days after exposure (mean of . days). since infection often progresses rapidly to death, there is often insufficient time to mount a robust immune response. however, both the innate (neutrophils, macrophages, and complement system) and the adaptive (both t and b cells) arms of the immune system have been shown to participate in the immune response to n. fowleri [ ] . patients with pam have csf with elevated pressure that is often cloudy or hemorrhagic, with neutrophil-predominant pleiocytosis, elevated protein levels, and very low glucose. wet mounts from centrifuged csf will show motile mono-nucleated trophozoites measuring~ - μm in size. additionally, trophozoites can be identified with giemsa and wright stains of csf smears combined with an enflagellation test [ ] . confirmation can be achieved via a variety of timeconsuming methods including an immunofluorescence assay [ ] , culture of csf [ ] , or pcr-based methods [ ] . the optimal therapy for n. fowleri pam is still debated. the use of intravenous amphotericin b and fluconazole followed by oral administration of rifampin resulted in survival of a year-old child with pam [ ] . another child was shown to survive following intravenous and intrathecal amphotericin b and miconazole as well as oral rifampin [ ] . most recently, an adolescent girl was successfully treated with the combination of azithromycin, rifampin, fluconazole, and miltefosine [ ] . prevention is critical for this highly fatal infection and warning pidd patients not to use tap water for nasal irrigation is important. since its original description in , over cases of b. mandrillaris infections have been described worldwidewith most cases occurring in south america and the usa. balamuthia are found in soil, and acquisition of disease has been associated with agricultural activities, dirt-biking, gardening, and swimming in contaminated water sources. b. mandrillaris is thought to enter the body of the host through breaks in the skin and or via inhalation. the organism is believed to access the cns through hematogenous spread, resulting in a chronic, insidious, but often fatal granulomatous amoebic encephalitis (gae), which has been documented in both immunocompetent and immunocompromised hosts [ , ] . the incubation period from exposure to development of clinical symptoms is not well established and experts believe that this may occur between months and years. finally, an alternative mode of transmission via solid organ transplantation has also gained attention [ ] [ ] [ ] . in many cases, gae is diagnosed post-mortem, due to delayed diagnosis or unawareness of the clinical entity. following infection by b. mandrillaris, two clinical patterns of presentation have been described. in the first pattern, patients initially develop a skin lesion that may resemble a painless plaque that may evolve into subcutaneous nodules and rarely ulcerations [ ] . these patients may develop neurologic manifestations weeks to months later. histopathologic examination of these lesions typically reveals granulomatous reactions in the reticular dermis, associated with lymphocytic and plasma cell infiltrates as well as multinucleated giant cells, without distinct epidermal changes. skin lesions will harbor trophozoites, but these are scarce and often easily overlooked as they resemble histiocytes. it is believed that early diagnosis of b. mandrillaris infections in those presenting with skin lesions may prevent subsequent development of cns disease, but there have also been cases in which patients presenting with skin lesions have progressed to developing gae despite treatment. in the second pattern, patients present with cns involvement without a previously recognized skin lesion. patients presenting with gae may initially display fever, malaise, headache, nausea and vomiting, and frank lethargy. later, these symptoms evolve into visual abnormalities, cranial nerve palsies, seizures, focal paresis; as intracranial pressure builds, coma, and eventually death with tonsilar or uncal herniation ensue within - weeks [ ] . upon infection with b. mandrillaris, brain endothelial cells produce the proinflammatory cytokine il- , thereby initiating an inflammatory response [ ] . moreover, the amoebic trophozoites infiltrate blood vessel walls. degradative enzymes, vessel wall infiltration, and the host inflammatory responses result in tissue necrosis and infarctions in the cerebral hemispheres, cerebellum, and the brainstem. in a mouse model of b. mandrillaris infection, cd + t cells were shown to be protective [ ] , suggesting that patients with lowered number or dysfunction in cd + t cells may be more susceptible to disease by this amoeba. however, b. mandrillaris infections have been described in a variety of human hosts [ ] , ranging from the young, healthy, and presumably immunocompetent to the elderly, and those with hiv, chronic corticosteroid exposure, on post-transplant immunosuppression and even patients with cvid. as such, further research is necessary to fully delineate the susceptibility of pidd patients. in patients who develop the characteristic skin lesions, recognition, testing, and treatment for b. mandrillaris may prevent subsequent gae. as such, obtaining tissue and looking for granulomas and trophozoites is quite helpful. skin biopsies can be stained via immunofluorescence or immunoperoxidase techniques to identify b. mandrillaris [ ] . additionally, a pcr technique that identifies mitochondrial s ribosomal rna from b. mandrillaris is also available through the cdc [ ] . in patients in whom the diagnosis is confirmed via skin biopsy, wide resection and medical treatment appears to prevent development of cns disease in at least a proportion of patients. in patients presenting with cns involvement, lumbar punctures reveal csf with lymphocytic pleiocytosis, low-to-normal glucose, and mildly to significantly elevated protein levels. trophozoites are not typically found in the csf, but pcr analysis may be performed. ct or mr imaging may show multiple nodules with ring enhancement; some of these nodules may also contain focal areas of hemorrhage. biopsies of brain tissues typically reveal granulomas and foamy macrophages and multinucleated giant cells surrounded by lymphocytic infiltrates. additionally, there will be areas of necrosis filled with neutrophils, multinucleated giant cells, and lymphocytes, with balamuthia trophozoites and cysts interspersed with macrophages [ ] . as with the skin biopsies, immunofluorescent and immunoperoxidase stains may aid diagnosis and should be performed. unfortunately, the optimal medical management of cns disease is unknown. in the usa, a few patients have been successfully treated with a combination of fluconazole, flucytosine, pentamidine, a macrolide antibiotic (either clarithromycin or azithromycin), and one of the following agents: liposomal amphotericin b, miltefosine, sulfadiazine, or thoridazine [ ] [ ] [ ] ; others in peru have been treated successfully with fluconazole (or itraconazole), albendazole, and miltefosine [ ] . based on these case reports, most experts recommend treatment with a combination of medications (along with partial or complete resection of nodules) for a prolonged period of time to prevent further deterioration and death [ ] [ ] [ ] [ ] . acanthamoeba spp. the genus acanthamoeba contains at least morphologically distinct species that live in a diverse array of habitats, including soil, salt, brackish, and fresh water. acanthamoeba spp. have also been found in humidifiers, heating and cooling unit components, jacuzzis, hot water tanks, bathrooms and drains, eye wash stations and dentistry irrigation systems, and more. acanthamoeba spp. have been isolated from reptiles, birds, and other non-human mammals, suggesting a broad distribution in the environment. acanthamoeba trophozoites feed on bacteria, but have also recently been shown to harbor a number of bacteria (including legionella and burkholderia spp., e. coli, listeria monocytogenes, vibrio cholerae, mycobacteria spp., chlamydophila, and others) and at least one virus (mimivirus) as endosymbionts. acanthamoeba infections in humans can present in a variety of ways. of primary importance are cns infections. like b. mandrillaris, acanthamoeba spp. can induce gae (described above). there is a high predilection for gae in those with hiv/aids, patients on chemotherapy, and those receiving broad spectrum antibiotics [ ] . acanthamoeba are rarely found in csf, but some case reports indicate isolation of amoebae by culturing csf on bacterized agar plates. similar to gae seen with b. mandrillaris, cns histopathology may reveal edema, multiple areas of necrosis and hemorrhage, and occasional findings of angitis and blood vessel cuffing by inflammatory cells, as well as occasional trophozoites or cysts. cns disease treatment is not standardized, but several patients have been successfully treated with pentamidine, fluconazole, flucytosine, sulfadiazine, as well as miltefosine. acanthamoeba can rarely cause cutaneous infections; these lesions, like gae, are also predominantly seen in immunocompromised hosts. these lesions can start as nodules or papules on the lower extremities and develop into necrotic ulcers. histopathologic examination may reveal granulomatous dermal lesions in immunocompetent hosts, with histiocytes, as well as neutrophils and plasmacytes; trophozoites are typically visible [ , ] . the optimal management of cutaneous disease is not known, but typically involves combinational therapy with topical (e.g., chlorhexidine, gluconate, or ketoconazole) and systemic (miltefosine, sulfadiazine, flucytosine, liposomal amphotericin b, azole antifungals, etc.) drugs. additionally, nasopharyngeal and sinus infections have been seen in people with severe compromise in immunity [ , ] . patients typically present with purulent nasal discharge, and examination may reveal erosion of the nasal septum. nasopharyngeal disease can present concomitantly with cutaneous disease. treatment of nasopharyngeal or sinus disease is difficult and involves surgical debridement and combinations of systemic drugs. disseminated disease is also seen in immunocompromised hosts and typically involves concomitant pulmonary and cutaneous disease in the presence or absence of cns infection. keratitis readily occurs in immunocompetent hosts-with the major risk factor being contact lens wearing without proper adherence to recommended cleansing protocols. this infection less commonly presents as a result of direct inoculation with trauma. one of the most common reasons for contact lens wearers to acquire disease is due to the use of non-sterile tap water in preparing contact lens saline solutions [ ] , although contaminated solutions from manufacturers have also been identified. patients will have pain and photophobia. physical exam reveals conjunctival injection and epithelial abnormalities (including pseudodendritic lesions) and stromal infiltrates [ ] . the proper diagnosis can be made by staining corneal scrapings with calcofluor or wright-giemsa stains and examined by confocal microscopy, culture, or pcr analysis. prompt therapy with a combination of polyhexamethylene biguanide (or biguanide-chlorhexidine) and propamidine or hexamidine [ , ] is indicated, but misdiagnosis and delayed therapy are common. more severe cases may also require debridement. the use of topical steroids before administration of combinational therapy may result in worse outcomes and should be avoided; however, if scleritis ensues, it may be necessary to use immunosuppressants to reduce the need for enucleation. severe and/or refractory cases may result in the need for cornea transplantation. phenotypes seen in pidd like hsv- and candida [ ] . patients with ifnar deficiency seem highly susceptible to cns disease caused by mmr vaccine, an otherwise extremely rare phenomenon [ ] . recently, a case of noroviral cns disease was described associated with a novel, yet unpublished pidd, suggesting that some pidds may lead to susceptibility of the cns to viruses that normally do not exhibit neurotropism (casanova jl, personal communication) . this again favors metagenomic approaches in the study of cns sequelae in pidd patients. in pidds, the cns is also more vulnerable to virally induced immunodysregulation [ ] . conditions like primary hemophagocytic lymphohistiocytosis (hlh) may present as isolated cns disease or relapse only in the cns [ ] [ ] [ ] . almost % all human malignancies are associated with chronic infections by hbv, hcv, hpv, ebv, hhv /kshv, htlv-i, hiv- , hiv- , jcv, merkel cell polyomavirus (mcpv), helicobacter pylori, schistosomes, or liver flukes [ ] . accordingly, pidd patients' malignancies are often associated with chronic infections. mcpv-associated merkel cell carcinoma has now been described in gata and tmc (ever ) deficiencies as well as other forms of pidd [ , [ ] [ ] [ ] [ ] [ ] . large follow-up cohorts are needed to refute or confirm associations between novel pidds and malignancies, such as hyperactivating pik cd and ovarian dysgerminoma or gata deficiency and leiomyosarcoma [ , ] . recently, hymenolepis nana was found to have driven malignant transformation in an hiv patient. likely, other novel pidd-and pathogen-associated malignancies will be found in the future by those with an open and inquisitive mind [ ] . understanding the specific infection susceptibility for each pidd allows not only a better understanding of host defense, but also allows the clinician to collaborate with the microbiology laboratory to make definitive diagnoses and provide the best therapy. reviewing all of the infections for each pidd is not within the scope of this article, but there are several infections that are unique for specific pidds and require special attention from the microbiology laboratory. three examples are provided below. g. bethesdensis is a gram-negative bacterium that was identified to cause disease in patients with cgd in [ ] . g. bethesdensis is a member of the methylotroph group of bacteria, which are able to use single-carbon organic compounds as their only source of energy. they are widespread in the environment, but are rare human pathogens, and infections with g. bethesdensis have been limited thus far to patients with cgd. the organism was first detected in an adult patient with indolent and recurrent necrotizing lymphadenitis [ ] . subsequently, g. bethesdensis was isolated from nine patients with cgd, primarily causing lymphadenitis, but there have been two fatalities [ ] . treatment has been most effective with intravenous ceftriaxone. the microbiology laboratory should be alerted when there is concern for g. bethesdensis infection to allow for proper culture media. charcoal yeast extract (cye) agar and lowenstein jensen (lj) media are appropriate culture media. mycoplasma and ureaplasma spp. as molecular techniques are becoming more widely used to detect pathogens, the spectrum of infections that were previously only detected through serologic assays and research laboratories will increase. this is important especially for patients with pidd who have unique susceptibility to infection and may not have the ability to mount a serologic response. examples of infections in this group are those caused by mycoplasma and ureaplasma [ , ] . these pathogens have been known to cause osteoarticular infections for those with antibody deficiency, such as xla and cvid. recently, mycoplasma orale, typically an oral commensal, has been isolated from two patients with defects in the activated pi k delta syndrome, as chronic lymphadenitis in one and chronic splenic abscess in the other (sm holland personal communication). defects in pi kcd and pi kr are frequently associated with hypogammaglobulinemia and therefore would fit in the pattern of mycoplasma infections in those with humoral immunodeficiency. mycoplasma orale has also previously been reported as causing bone disease in a patient with cvid [ ] . in patients with xla, helicobacter, camplyobacter, and the related flexispira bacteria that are typically isolated to the gi tract can disseminate and often lead to chronic bacteremia, ulcers, and bone infections [ ] [ ] [ ] . xla patients have higher susceptibility than other humoral pidd and are thought to be due to the role that igm is playing in controlling the dissemination of these pathogens and potentially iga in providing mucosal immunity. these bacteria can be fastidious to grow, and therefore, when there is suspicion, identification needs collaboration with the microbiology laboratory. for instance, the blood culture media may allow growth (although with a longer incubation period), but then the organisms may need molecular techniques for identification, such as s sequencing, as they will not grow on the agar plates. treatment is often difficult, requiring combination antimicrobials for prolonged periods (such as year), and relapse is common. this review provides an important perspective for practicing immunologists, namely that we are a part of a global community as are our patients. this overview of emerging infections and infectious concerns for travelers serves as a foundation for practical considerations for clinicians and patients. using prevalence data, an estimation of the number of infected patients with pidd (table ) can be developed [ , , [ ] [ ] [ ] [ ] . thus, the concerns addressed in this review are not theoretical but impact a considerable number of patients already. the landscape of emerging infections is by its nature highly dynamic. during the preparation of this manuscript, a mumps outbreak in the usa occurred, a new bunyavirus outbreak causing an hlh picture was reported (severe fever with thrombocytopenia syndrome), a new outbreak of the hantavirus seoul virus occurred, and an enlarging demographic of the h n influenza virus was reported. this review lists several resources, many of which are updated in real time to support efforts to provide 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patient with gata deficiency: a novel association with primary immunodeficiency spontaneous regression of merkel cell carcinoma developed in a patient with epidermodysplasia verruciformis merkel cell polyomavirus detection in a patient with familial epidermodysplasia verruciformis merkel cell polyomavirus-positive merkel cell carcinoma in a patient with epidermodysplasia verruciformis merkel cell polyomavirus in merkel cell carcinoma from a brazilian epidermodysplasia verruciformis patient association of gata deficiency with severe primary epstein-barr virus (ebv) infection and ebv-associated cancers malignant transformation of hymenolepis nana in a human host granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family acetobacteraceae methylotroph infections and chronic granulomatous disease osteoarticular infectious complications in patients with primary immunodeficiencies increased susceptibility to mycoplasma infection in patients with hypogammaglobulinemia disseminated mycoplasma orale infection in a patient with common variable immunodeficiency syndrome relapsing campylobacter jejuni systemic infections in a child with x-linked agammaglobulinemia bacteremia and skin/bone infections in two patients with x-linked agammaglobulinemia caused by an unusual organism related to flexispira/helicobacter species successful approach to treatment of helicobacter bilis infection in x-linked agammaglobulinemia prevalence and morbidity of primary immunodeficiency diseases, united states epidemiological characteristics of spotted fever in israel over years complications of bacille calmette-guerin (bcg) vaccination and immunotherapy and their management acknowledgements the authors would like to thank thomas krell for information regarding ivig safety and david peden for recognizing global warming as central to medical knowledge. conflict of interest the authors declared that they have no conflict of interest. pidds display wide genetic and phenotypic heterogeneity [ ] . similar disease phenotypes may be caused by multiple genes, while patients' phenotypes caused by the same gene and even by the same mutations vary between individuals. importantly, after a novel pidd has been described, subsequent reports often reveal a wider variation in associated infections and cellular findings, often without clear genotype-phenotype correlations [ ] [ ] [ ] [ ] [ ] . variation may be caused by mechanisms such as other contributing genes or geographical variation in infectious exposures. geographic differences seem most pronounced in intracellular and often chronic infections. while the numbers of described pidd patients increase, at first seemingly rarely pidd-associated infections turn out to be found in a significant subset of pidd patients [ ] [ ] [ ] . for example, patients with cd l deficiency living in endemic areas display susceptibility to bartonellosis and paracoccidioidomycosis, infections not described in european and us cohorts [ , , ] .often, an infectious phenotype previously only described in secondary immunodeficiencies may reveal the possibility of an underlying primary immunodeficiency [ , , ] . increasing numbers of genetic defects causing early-onset, severe, and recurrent susceptibility to commonly circulating pathogens like pneumococci, tuberculosis, herpes simplex, and influenza viruses as well as endemic protozoans like trypanosomes and fungi are being recognized, and thus, infections with unusual pathogens require a high index of suspicion for pidd [ ] . in contrast, pidds may also manifest as suspected infection but sterile inflammation. for example, in inflammatory lesions like granulomas and necrotizing fasciitis where no clear pathogens are found, one needs to rule out aberrant host responses due to pidd [ ] .chronic viral and fungal infections may also display novel phenotypes never or rarely seen in secondary immunodefic i e n c i e s . i n f e c t i o n s l i k e d e r m a t o p h y t o s i s a n d phaeohyphomycosis deeply infiltrating the skin and lymph nodes, occasionally extending to bones and central nervous system (cns) as well as predisposition to primary cns candidiasis and extrapulmonary aspergillus slowly revealed the full phenotypic spectrum of card deficiency [ , , ] . chronic skin ulcers caused by hsv- and severe molluscum contagiosum suggest dock deficiency or gain-of-function mutations of stat [ , ] . chronic mucocutaneous candidiasis has revealed a large group of monogenic diseases (il ra, il rc, il f, stat (gof), rorx, act ), which may also be associated with recurrent bacterial infections or syndromic features [ ] . while novel diseases by newly described viruses are being discovered, one needs awareness to suspect these in pidd patients [ ] .interestingly, most novel forms of infectious disease in pidd patients have been described either in easily accessible sites like the skin or in immunologically privileged, normally sterile sites like the cns. this suggests that with the increasing use of invasive sampling and sensitive metagenomic approaches, we might find more novel infectious phenotypes. pathogens highly suggestive of certain pidds, like chronic enteroviral cns infections in xla patients are reviewed above. in hypomorphic mutations, cns seems to be especially vulnerable to chronically active and/or recurrent novel infectious bsmoldering^focal encephalitis lesions by pathogens key: cord- -bufbjdmw authors: clement, annick; nathan, nadia; epaud, ralph; fauroux, brigitte; corvol, harriet title: interstitial lung diseases in children date: - - journal: orphanet j rare dis doi: . / - - - sha: doc_id: cord_uid: bufbjdmw interstitial lung disease (ild) in infants and children comprises a large spectrum of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. these disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. typical features of ild include dyspnea, diffuse infiltrates on chest radiographs, and abnormal pulmonary function tests with restrictive ventilatory defect and/or impaired gas exchange. many pathological situations can impair gas exchange and, therefore, may contribute to progressive lung damage and ild. consequently, diagnosis approach needs to be structured with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. several classifications for ild have been proposed but none is entirely satisfactory especially in children. the present article reviews current concepts of pathophysiological mechanisms, etiology and diagnostic approaches, as well as therapeutic strategies. the following diagnostic grouping is used to discuss the various causes of pediatric ild: ) exposure-related ild; ) systemic disease-associated ild; ) alveolar structure disorder-associated ild; and ) ild specific to infancy. therapeutic options include mainly anti-inflammatory, immunosuppressive, and/or anti-fibrotic drugs. the outcome is highly variable with a mortality rate around %. an overall favorable response to corticosteroid therapy is observed in around % of cases, often associated with sequelae such as limited exercise tolerance or the need for long-term oxygen therapy. interstitial lung disease (ild) in infants and children represents a heterogeneous group of respiratory disorders that are mostly chronic and associated with high morbidity and mortality (around %) [ , ] . these disorders are characterized by inflammatory and fibrotic changes that affect alveolar walls. typical features of ild include the presence of diffuse infiltrates on chest radiograph, and abnormal pulmonary function tests with evidence of a restrictive ventilatory defect (in older children) and/or impaired gas exchange [ ] . there have been many different approaches to the classification of ild, with major shifts based on clinical investigation, improvement in chest imaging, and collaboration with pathologists. in , katzenstein and myers proposed four histopathologically distinct subgroups of idiopathic interstitial pneumonias: usual interstitial pneumonia (uip), desquamative interstitial pneumonia (dip) and a closely related pattern termed respiratory bronchiolitis-associated ild, acute interstitial pneumonia (formerly hamman-rich syndrome), and non specific interstitial pneumonia (nsip) [ ] . in , an international multidisciplinary consensus classification of idiopathic interstitial pneumonias was proposed by the american thoracic society (ats)/european respiratory society (ers) [ ] . this classification defined a set of histologic pattern that provided the basis for clinico-radiologic-pathologic diagnosis, with the final pathologic diagnosis being made after careful correlation with clinical and radiologic features. however, as discussed in several reports, the classification schemes of adult ild are not satisfactory for the pediatric cases which seem to comprise a broader spectrum of disorders with a more variable clinical course [ ] . in addition, pediatric histologic patterns often do not resemble pathologic features of lung tissues from adults and some forms are only observed in children younger than years. among the proposed classifications for pediatric ild, one strategy frequently used is to separate the primary pulmonary disorders and the systemic disorders with pulmonary involvement. recently, an additional group has been introduced which is based on the concept that some pediatric ild are observed more frequently in infants, while others are more specific to older children. the last ers monography on ild provided a chapter on pediatric classification which is based on a clear distinction between children aged - years and children over years-old [ ] . indeed the stage of lung development and maturation should be taken into consideration when approaching a diagnosis of pediatric ild. in this view, a new term "diffuse lung disease" has recently been introduced that comprises a diverse spectrum of lung disorders with impaired gas exchange and diffuse infiltrates by imaging. these disorders, more prevalent in young children, include diffuse developmental disorders, lung growth abnormalities, neuroendocrine cell hyperplasia and pulmonary interstitial glycogenosis, surfactant dysfunction disorders, disorders related to systemic diseases, disorders of immunocompromised host, and disorders of normal host caused by various insults such as aspiration syndrome or infections [ ] . some diseases are mostly observed in older children such as systemic diseases, idiopathic disorders as described in adults (dip, uip, nsip and lymphoid interstitial pneumonia (lip)), unclassifiable ild and also infectious disorders [ ] . it is important to point out that the pathologic processes underlying the so-called diffuse lung diseases involve not only the alveolar structure but also the distal part of the small airways and the conducting zone, i.e. the terminal bronchioles. terminal bronchioles are lined with a simple cuboidal epithelium containing clara cells, basal cells and a limited number of ciliated cells. clara cells secrete nonsticky proteinaceous compounds to maintain the airway in the smallest bronchioles, which constitute the quiet zone between the conducting and the respiratory lung zones [ ] . the terminal bronchioles are surrounded by a spiral of smooth muscle. each of the terminal bronchioles divides to form respiratory bronchioles which contain a small number of alveoli. consequently, the term of diffuse lung disease refers to disorders that can affect both the distal part of the conducting and the respiratory lung zones, and include ild as well as pathological processes leading to obstruction/ obliteration of small airways [ ] . therefore, diffuse lung diseases encompass a broader group of diseases than ild which refers to disorders that affect the respiratory function of the lung and consequently the pulmonary structure responsible of the diffusion of gases between blood and air (i.e. the alveolar epithelium, the interstitium, and the pulmonary capillary endothelium). the present review focuses on ild in immunocompetent children, and excludes pulmonary consequences of previous lung injury in situations of chronic aspiration syndromes, resolving acute respiratory distress syndrome, and bronchopulmonary dysplasia. an estimated prevalence of . per million has been reported by dinwiddie and coworkers through a national survey of chronic ild in immunocompetent children in the united kingdom and ireland over a three year period ( ) ( ) ( ) ( ) [ ] . this prevalence is certainly under-estimated due to the lack of standardized definitions and the absence of organized reporting systems. from the limited published data composed mainly of case reports and small series, it seems that pediatric ild occurs more frequently in the younger age and in boys [ ] . in addition, nearly % of cases appear to be familial [ ] . the understanding of the mechanisms underlying the development and progression of ild remains elusive [ , ] . indeed, for a long time, chronic ild and pulmonary fibrosis were believed to result mainly from chronic inflammation following an initial injury to the alveolar epithelial lining [ , ] . in cases of limited injury, it was thought that the reparative attempt could reverse the trend toward fibrosis. by contrast, in situations of continuing injury, the repair process driven by inflammatory molecules produced by the local cells will result in scarring and structural changes. therefore, by targeting the inflammatory response, the belief was that fibrosis could be prevented or controlled. this theory explains the large use of anti-inflammatory therapy with, however, limited clinical efficacy. based on clinical and experimental observations, a new paradigm has progressively emerged with the alveolar epithelium being viewed as a key actor in the development of ild [ ] [ ] [ ] . following injury, alveolar epithelial cells (aec) may actively participate in the restoration of a normal alveolar architecture through a coordinated process of re-epithelialization, or in the development of fibrosis through a process known as epithelial-mesenchymal transition (emt) [ ] . complex networks orchestrate emt leading to changes in cell architecture and behaviour, loss of epithelial characteristics and gain of mesenchymal properties. the reasons for epithelial cell loss and inappropriate re-epithelialisation are still debated, but ongoing apoptosis is believed to be a key component in the progression of the disorder [ ] . prolonged denudation of the basement membrane contributes to altered interactions and cross-talk between aecs and mesenchymal cells, resulting in profound modifications of cell functions with imbalanced production of oxidants, proteases, and polypeptide mediators including cytokines and growth factors such as transforming growth factor (tgf)-β and endothelin (et)- . a consequence is the perpetuation of a vicious cycle with tgf-β promoting epithelial cell apoptosis, which in turn increases the local production of tgf-β [ ] . et- is also considered to be an important actor, based on the current knowledge of its numerous functions including fibroblast and smooth muscle cell mitogen, and stimulant of collagen synthesis [ , ] . recent studies showed that et- is produced by aec, and could induce alveolar emt via stimulation of endogenous tgf-β production. ild may be caused by myriad etiologies with differing prognoses and natural history. indeed, multiple factors may injure the alveolar epithelium and initiate the development of ild [ ] . the initiating injury can be introduced through the airways and the circulation, or can occur as a result of sensitization. consequently, the mechanisms underlying disease progression will be influenced by the causative event as well as by the host and the environment. these mechanisms are developed through interactions of multiple pathways, which include apoptotic pathways, developmental pathways, and endoplasmic reticulum (er) associated pathways ( figure ). apotosis plays a central role in lung remodeling associated with ild [ ] . an important molecule in the events associated with epithelial cell apoptosis is tgf-β, which is overexpressed in ild. downstream events linked to upregulation of tgf-β include modifications in the expression of various components of the cell cycle machinery, mainly the cyclin-dependent kinases (cdk) system that plays an essential role in ensuring proper cell cycle progression. recently, much work has been focused on the protein p cip , a member of the cdk inhibitor family. this protein promotes cell cycle arrest to apoptosis in cases of cellular dna damage. interestingly, upregulation of p cip has been reported in the lung tissues of patients with pulmonary fibrosis, primarily in hyperplastic alveolar epithelial cells [ ] the increased expression of p cip can favour the process of epithelial cell apoptosis. apoptotic cells can also produce tgf-β. a consequence would be the perpetuation of a vicious cycle with tgf-β promoting epithelial cell apoptosis, which in turn increases the local production of tgf-β. recently, it has been suggested that genes associated with lung development and embryonic pathways could be involved in aberrant epithelium-mesenchymal crosstalk and epithelial plasticity, and could therefore participate in the development of chronic ild. selman and coworkers reported that lung fibrosis is characterized by enrichment for genes associated with cell adhesion, extracellular matrix, smooth muscle differentiations, and genes associated with lung development [ ] [ ] [ ] [ ] . during emt in the embryonic period, cells undergo a switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype [ ] . molecular processes governing emt are induced by a cooperation of receptor tyrosine kinases or oncogenic ras (rtk/ras) pathway and tgf-β signaling [ ] . recently, additional pathways and effectors have been reported to play a role in the induction of emt, such as wnt//β-catenin, notch and sonic hedgehog signalling [ ] . recent reports strongly suggest that the er stress may represent an important mechanism of the altered repair process observed in the alveolar epithelium of fibrotic lung [ ] . situations associated with abnormal regulation of the cascade of events leading to the formation of mature protein result in either misfolding or mistargeting of the protein. these events trigger induction of intracellular aggregate formation and er stress, which can lead to cell death through apoptosis and autophagic pathways [ , ] . several stimuli including oxidant-antioxidant imbalance, viral proteins, inflammatory molecules, nutrient deprivation may induce er stress [ , ] ( figure ). among the cytoprotective mechanisms available are the er chaperones such as binding immunoglobulin protein (bip). interestingly, mutant bip mice have been reported to die within several hours of birth from respiratory failure due to impaired secretion of pulmonary surfactant by type aec. in these animals, expression of surfactant protein (sp)-c was reduced and the lamellar bodies were malformed, indicating that bip figure alveolar structure disorder-associated ild and er stress. the (endoplasmic reticulum) er and its protein maturation machinery allow the synthesis of mature secretory and membrane proteins with specific folded conformation. in situations of stress induced by genetic mutations or environmental factors, unfolded or misfolded proteins are retained in the er and induce a defence mechanism called the er stress response. the induction of er chaperones is critical to increase the er folding capacity allowing the production of correctly folded protein. when this defence mechanism is impaired, the misfolded proteins can either be degraded by the proteasome or form protein aggregates. protein aggregates are toxic and can cause conformational diseases. within the alveolar epithelium, misfolding of sp-c could trigger induction of intra-cellular aggregate formation and er stress, with consequently development of alveolar structure disorder-associated ild and conformational disease. plays a critical role in the biosynthesis of surfactant [ ] . several recent reports suggest the possible implication of er stress in ild, with activation of stress response markers in fibrotic lung tissues. it is now well established that surfactant dysfunction plays an important role in the development and progression of ild. pulmonary surfactant is a multimolecular complex constituted of phospholipids and proteins secreted by type aec into the alveolar space. it assures alveolar stability by reducing surface tension along the epithelial lining and this role involves mainly the lipids and the specific hydrophobic sp, sp-b and sp-c. other important players in surfactant metabolism include the atp-binding cassette, sub-family a, member (abca ) and the thyroid transcription factor (ttf- ). surfactant deficiency can be induced by a number of primitive and secondary mechanisms. among them are genetic defects with mutations in sp-b gene (sftpb) as well as genes coding for sp-c (sftpc), abca , and ttf- [ ] [ ] [ ] . more than sftpb (located on chromosome ) mutations have been identified among patients with a congenital deficiency in sp-b. for sftpc located on chromosome , at least mutations have been described, localized primarily in the coohterminal brichos domain [ , ] . a proposed function of the brichos domain is a chaperone-like activity, which could prevent misfolding and aggregation of the parent protein. alterations in the brichos domain could therefore lead to diseases through mechanisms related to abnormal protein processing and cell toxicity [ ] . recently, several studies have also documented the role of abca deficiency in ild. abca functions in the transport of surfactant lipids into lamellar bodies and is required to maintain pulmonary surfactant phospholipid homeostasis. another contributor of ild is ttf- (nk homeobox ) dysfunction. ttf- is a critical regulator of transcription for the surfactant protein sp-b and sp-c. it is encoded by a gene located on chromosome q and is composed of three exon and two introns [ ] . it is expressed in the thyroid, brain and lung. stem cell dysfunction represents a new domain of investigation. alveolar epithelium regeneration and repair requires activation and proliferation of tissue-resident (progenitor) cells and their differentiation to replace the damaged cells [ ] . however, unlike cancer cells, stem cells are not immortal and display decreasing telomere length with aging [ ] . telomere shortening has been documented to be associated with reduced capacity for stem cell renewal, and decreased activity of telomerase, the polymerase responsible for telomere maintenance. the stem cells of the alveolar epithelium are the type aec, and expression of telomerase has been documented in these cells [ ] . experimental studies have also indicated that telomerase is expressed mainly during lung development with a peak expression before birth followed by a decrease to nearly undetectable levels in mature alveolar epithelium. interestingly, telomerase expression and activity could be reinduced in normal quiescent type aec exposed to oxidative stress [ ] . the current understanding is that a population of type aec may have the capacity to survive injury through telomerase activation, and consequently may be responsible for repopulation of the damaged alveolar epithelium. on the basis of reports of pulmonary disorders in dyskeratosis congenita (a rare hereditary disease of poor telomere maintenance), recent and exciting findings have documented mutations in the telomerase gene in familial idiopathic pulmonary fibrosis [ ] . in addition, it is likely that environmental factors such as inflammation, oxidative stress, or virus infection may modify telomerase activity and account for the development of organ-specific disease associated with telomerase dysfunction. in this view, new data in chronic respiratory diseases support the concept that alveolar stem cell dysfunction may play an important role in the rate of progression or severity in ild [ ] . the question whether telomerase mutations or telomere dysfunction may be implicated in pediatric ild needs to be addressed in prospective studies, one possible tool being determination of telomere length in circulating leukocytes. the frequency of lung fibrotic disorders is much lower in children than in adults. some clinical situations have features certainly unique to children, but many of these diseases overlap their adult counterparts with the primary event being injury and damage of the alveolar epithelium [ , ] . yet, the overall outcome and prognosis of the diseases in children are thought to be less severe than in adult patients. in addition, pediatric ild is more responsive to therapeutic strategies than adult ild [ ] . these differences may be explained by the types of initial injury, which may not be similar due to changes in the host environment. another explanation is the modifications of the process of wound healing with age. comparison of the response to injury in foetal and adult skin shows clear differences [ ] . skin wound healing in the foetus is characterized by complete regeneration of the skin and the absence of scar formation. progressively with age, the skin looses the capacity to regenerate the original tissue architecture with the result being scar formation that extends outside the wound bed. the process of healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide growth factors [ ] . the slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory factors. in a study on the role of aging in the development of cardiac fibrosis in the rabbit, differences in the cascade of events leading to myocardial remodeling were observed, with mainly the presence of more myofibroblasts synthesising collagen and expressing high levels of tgf-β in older animals [ ] . a study of growth factors involved in skin wound healing in young and aged mice also showed age-dependent changes. expression of all the fibroblast growth factors was diminished in aged mice, even in healthy skin. in addition, the post-wound regulation of expression of these factors and of tgf-β was less pronounced and slower than in young mice. these findings are in agreement with data observed in muscle that indicated significant alterations in the tgf-β production with age [ , ] . other potential mechanism is linked to the observation that injury in adult tissues does in certain circumstances stimulate tissue regeneration, depending on the presence of small subsets of primitive stem cells. stem cells are the self-renewing, primitive, undifferentiated, multipotent source of multiple cell lineages [ ] . while such cells are critical for development and growth through childhood, residual pools of adult stem cells are hypothesized to be the source of the frequently limited tissue regeneration and repair that occurs in adults [ ] . unlike embryonic stem cells, adult stem cells are not immortal, and show decreasing telomere length with increasing age. the naturally limited replacement capacity of such endogenous stem cell pools may occur via exhaustion of the stem cell pool or arise as a consequence of inherited or acquired mutations that alter proper stem cell function [ ] . the limited life span of cells may result from replicative senescence in response to various stresses including dna damage, oxidants, and telomere erosion [ ] . all these forms of injury have been documented in the lung from adult patients with ild. the prevalence of children ild is higher in the younger patients: more than % of patients are less than years at diagnosis, as recorded by the recent ers task force. % have parental consanguinity and nearly % of case siblings were affected by similar diseases. there is a male predominance with a sex ratio of . . the presenting clinical manifestations are often subtle and nonspecific. the onset of symptoms is, in most cases, insidious and many children may have had symptoms for years before the diagnosis of ild is confirmed. however, the majority of patients has symptoms for less than one year at the time of initial evaluation. the clinical manifestations vary from asymptomatic presentation with radiological features suggestive of ild to more characteristic presence of respiratory symptoms and signs such as cough, tachypnea and exercise intolerance [ , ] . these varying presentations are also reflected in the report published by fan et al. who systematically evaluated the clinical symptoms and physical findings of consecutive children with ild [ ] . common symptoms at presentation included cough, dyspnea, tachypnea and chest wall retraction, exercise limitation and frequent respiratory infections. cough is observed in almost % of the patients, is normally non-productive and does not disturb sleep. tachypnea is observed in % of patients and is usually the earliest and most common respiratory symptom. unexplained fever is also reported in almost one third of infants. failure to thrive ( %), tiring during feeding and weight loss are also common symptoms, mainly in young patients. although a history of wheezing may be elicited in almost % of the patients, wheezing is documented by physical examination in only % of the cases. the frequent clinical findings are inspiratory crackles ( %), tachypnea and retraction. in a child with a normal birth history, these are strongly suggestive of ild. other findings associated with an advanced stage of lung disease include finger clubbing ( %) and cyanosis during exercise or at rest ( %) [ , ] . during physical examination it is essential to check the presence of associated non-respiratory symptoms such as joint disease, cutaneous rashes, and recurrent fever suggestive of collagen-vascular disorders. details should also be obtained on precipiting factors such as feeding history, infections, or exposure to dust and drugs. in addition, information on relatives or siblings with similar lung conditions should be gathered. plain radiographs are usually performed in a child suspected of ild at first presentation, but the information provided is often limited and the key chest imaging tool for diagnosis is the high resolution computed tomography (hrct), which can visualize the parenchymal structure to the level of the secondary pulmonary lobule. hrct technique for ild diagnosis has been extensively discussed [ ] [ ] [ ] . to optimise spatial resolution, there is a general agreement to use thin sections, the smallest field of view and a sharp resolution algorithm. the most common hrct feature of ild is widespread ground-glass attenuation. intralobular lines, irregular interlobular septal thickening and honeycombing are less common findings. large subpleural air cysts in the upper lobes adjacent to areas of ground-glass opacities have been also reported in young children with ild. these cysts are interpreted as paraseptal or irregular emphysema. hrct is useful for ild diagnosis and selection of lung area to be biopsied. it is proposed that it also may contribute to monitor disease activity and/or severity. however, evaluation is still needed to support a role of hrct as a follow up tool in pediatric patients. pulmonary function testing (pft) techniques are well established in children and adolescents. however, children aged - years represent a real challenge in pulmonary function assessment as they cannot be sedated and find it difficult to cooperate with all respiratory manoeuvres. in , an ats and ers statement on pft in preschool children summarized the current knowledge on the pft techniques suitable for young children [ , ] . although pft does not provide specific information, it represents a useful investigation for both the diagnosis and the management of ild [ ] . generally, in ild, pulmonary function abnormalities reflect a restrictive ventilatory defect with reduced lung compliance and decreased lung volumes [ ] [ ] [ ] . vital capacity (vc) is variably diminished; the decrease in total lung capacity (tlc) in general is relatively less than in vc. functional residual capacity (frc) is also reduced but relatively less than vc and tlc, and residual volume (rv) is generally preserved; thus the ratios of frc/tlc and rv/tlc are often increased. airway involvement is observed only in a minority of patients. lung diffusing capacity of carbon monoxide (dlco) or transfer factor (tlco) is often markedly reduced and may be abnormal before any radiological findings. however, dlco corrected for lung volume may also be normal in many children. hypoxemia as defined by a reduced resting arterial oxygen saturation (sao ) or a reduced resting arterial oxygen tension is often present. hypercarbia occurs only late in the disease course. during exercise the above described dysfunctions become even more pronounced. thus, gas exchange during exercise might be a more consistent and sensitive indicator of early disease [ ] . bronchoalveolar lavage (bal) usefully provides specimens for cytological examination, microbial cultures, and molecular analysis. besides infections, bal can be of diagnostic value in several situations. in the context of pulmonary alveolar proteinosis, bal abnormalities are characterized by milky appearance fluid, abundant proteinaceous periodic acid schiff positive material, and presence of foamy alveolar macrophages (am) [ ] . bal can also be diagnostic for pulmonary alveolar haemorrhage [ ] . this diagnostic is easy when the bal fluid has a bloody or pink color, but its gross appearance may be normal. microscopic analysis may then be of value by documenting the presence of red blood cells in am or haemosiderin laden am [ ] . among other situations, the diagnosis of langerhans cell histiocytosis can be performed with the use of the monoclonal antibodies revealing the presence of cd a positive cells (in more than % of the bal cells) [ ] . lipid disorders with lung involvement represent another indication of bal. this includes congenital lipid-storage diseases (gaucher's disease and niemann-pick disease) or chronic lipid pneumonia due to chronic aspiration [ , ] . however, in cases of aspiration syndromes, the presence of lipid laden am is sensitive but not specific [ ] . in other pathological situations, bal can usefully serve to direct further investigations. accumulation of bal t-lymphocytes with prevalence of cd + cells is suggestive of sarcoidosis, whilst prevalence of cd + cells is suggestive of hypersensitivity pneumonitis [ ] . also, an increase in bal eosinophils suggests pulmonary infiltrates associated with eosinophilia syndromes [ ] . depending of the underlying diseases, a number of cellular and molecular investigations can be proposed including the studies of various surfactant components, phospholipids and apoproteins [ ] . with increasing recognition of the different patterns of ild and their clinical significance, histological investigation has become increasingly important. depending on disorder presentation, biopsy may concern more accessible organs than the lung such as the skin or the liver in sarcoidosis. histological evaluation of lung tissue usually represents the final step in a series of diagnostic approaches. different methods may be used to obtain lung tissue. the major difference between individual methods lies mainly in balancing invasiveness against the potential for obtaining adequate and sufficient tissue for diagnosis. the techniques of choice are open lung biopsy and video assisted thoracoscopy biopsy. in children, open lung biopsy usually provides sufficient tissue with few complications related directly to the biopsy procedure [ ] . video assisted thoracoscopy biopsy is an alternative to open lung biopsy, and it has been shown that the procedure can be safely performed, even in small children [ ] . the place of other methods like transbronchial lung biopsy and percutaneous needle lung biopsy in appropriate diagnosis of pediatric patients with ild has to be established [ ] [ ] [ ] . the lung histological patterns that can be observed in ild have been reviewed by the ats/ers [ ] . in children, they include mainly: dip, nsip, and lip. dip is characterized by airspaces filled with am, thickened alveolar septa, scattered mixed inflammatory cells and minimal fibrosis. many alveolar spaces are lined by hyperplastic type aec. recently, association with surfactant disorders has been reported [ , [ ] [ ] [ ] . nsip encompasses a broad spectrum of abnormalities with varying degrees of alveolar wall inflammation or fibrosis. the cellular pattern of nsip is characterized by mild to moderate interstitial chronic inflammation and type aec hyperplasia in inflammation areas. it has been reported in a variety of underlying conditions including connective tissues diseases and surfactant disorders. lip features include a marked diffuse infiltrate of mature lymphocytes, plasma cells and histiocytes within the pulmonary interstitium, particularly the alveolar walls. they are often associated with either connective tissues disorders or immunodeficiency states, both congenital and acquired [ ] . another pattern described mainly in adults is diffuse alveolar damage (dad), which includes diffuse homogeneous thickening of alveolar interstitial walls with myofibroblast accumulation, prominent type aec hyperplasia and atypia, and hyaline membranes containing surfactant proteins and cellular debris [ ] . usual interstitial pneumonia (uip) is rare in children [ ] . it is characterized by severe remodeling of the alveolar structure with heterogeneous appearance consisting of contiguous areas of normal lung, dense scarring, and bronchiolar abnormal proliferation. interstitial inflammation is usually mild to moderate. histologic patterns of ild unique to infancy are described below. laboratory tests are used to exclude a number of respiratory diseases in childhood that does not typically present with ild such as chronic aspiration syndromes, resolving acute respiratory distress syndrome, tuberculosis, cystic fibrosis, bronchopulmonary dysplasia and diffuse pulmonary disease such as cystic fibrosis. laboratory tests also verify the absence of immunodeficiencies [ ] . when these conditions have been eliminated, the spectrum of investigations that should be performed for the diagnostic approach will be guided by the history and clinical presentation in each individual child. these investigations are discussed below for the various disorders. in addition, an increasing number of blood and bal biomarkers for evaluation of disease severity and progression is currently investigated. the studied molecules include various cytokines and chemokines, surfactant protein d, krebs von den lungen- antigen (kl- ), matrix metalloproteinases mmp and mmp and defensins [ ] [ ] [ ] [ ] . a large number of pathological situations can impair gas exchange and contribute to progressive lung damage and ild. consequently, diagnosis approaches need to be organized by cause, with a clinical evaluation requiring a careful history paying attention to exposures and systemic diseases. indeed, in a number of pathological situations, no final diagnosis is proposed and the conclusion reported by the physician in charge of the patient is ild of unknown cause. however, information from recent studies highlights the concept that lung insults caused by substances from the environment or in the context of systemic diseases are largely under-estimated and should be more often discussed considered in the diagnostic process. based on this consideration, the following diagnostic grouping for pediatric ild can be considered ) exposure-related ild; ) systemic disease-associated ild; ) alveolar structure disorder-associated ild; and ) ild specific to infancy. accordingly, a step-by-step etiological diagnostic approach is required and is summarized in figure . once the diagnosis of ild is established on clinical, radiological, and functional findings, a careful history should be obtained for potential exposure-related diseases leading to discuss the need for specific serum antibodies against offending antigens. the following step focuses on the search for systemic disease associated ild, oriented by the presence of clinical and functional extra-pulmonary manifestations. in such situations, additional investigations should include specific serum antibodies and possibly tissue biopsies in organs other than the lung. finally, elimination of these groups of causes with a lung restricted expression of the disease allows discussing the potential interest of a lung biopsy. exposure-related disease refers to diseases caused by a sufficient level of exposure (dose) to components with target organ contact, and subsequent biologic changes and clinical expression. many agents have been associated with pulmonary complications of various types including ild. the adult literature has provided extensive lists of candidate molecules [ ] . in children, the potential involvement of these molecules is not similar as the environmental conditions and the use of therapeutic drugs differ. it is important to point out that exposure-related diseases are certainly under-estimated in the pediatric age. one reason is linked to the fact that the diagnosis is less often discussed than in adults as pediatricians and other child health care providers do not usually have the expertise necessary to take an environmental history. in this review, the most frequent causes of exposure-related ild are discussed. hypersensitivity pneumonitis (hp) is a cell-mediated immune reaction to inhaled antigens in susceptible persons [ , ] . in children, hp is often associated with exposure to antigens in the home environment as well as with certain hobbies. the most frequent types of hp include bird fancier's diseases, humidifier lung diseases, and chemical lung diseases. bird fancier's diseases are induced by exposure to birds with the antigens being glycoproteins in avian droppings, and on feathers. importantly, respiratory symptoms in exposed patients who have only one pet bird at home should raise the suspicion of hp [ ] . humidifier lung diseases (air conditioner lung, misting fountain lung, basement lung diseases) are caused mainly by free-living amoeba and nematodes, as well as bacteria and fungi. chemical lung diseases can be induced by various inorganic antigens such as those from vaporized paints and plastics. low-molecular-weight chemicals may react with proteins in the airways, thus forming complete antigens. once exposure history is obtained, additional information is required and includes biologic tests allowing measurements of environmental contaminants and interpretation of the results by environmental medicine experts. as hp is believed to be an adult disease, children are often diagnosed at the chronic stage of the disease resulting of a long-term exposure to low levels of inhaled antigens. children can develop subtle interstitial inflammatory reactions in the lung without noticeable symptoms for months [ ] . clinical features in the classic form include non productive cough, dyspnea, malaise, asthenia and occasional cyanosis [ ] . lung function abnormalities are not specific and appear similar to changes observed in other ild. hrct abnormalities vary from ground glass attenuation predominantly in the mid-upper zone to nodular opacities with signs of figure search for ild etiology in children. ild is defined by the presence of diffuse infiltrates on chest radiographs or chest high resolution computed tomography, and abnormal pulmonary function tests with evidence of a restrictive ventilatory defect (in older children) and/or impaired gas exchange. the search for etiology requires a systematic step-by-step diagnostic strategy for identifying: exposure-related ild; systemic disease-associated ild; alveolar structure disorder-associated ild; and ild specific to infancy. air-trapping [ , , ] . laboratory tests focus mainly on the search for serum-precipitating igg antibodies against the offending antigen [ ] . however, the presence of these antibodies is considered to be of questionable clinical relevance for diagnosis, as it is observed in up to % of serum samples of exposed but asymptomatic individuals. bal cell profile study typically shows an increase in total cell count with a remarkable elevation in the percentage of lymphocytes often over % with a decreased cd /cd ratio [ , ] . however, in contrast to studies in adults, the cd /cd ratio could be within the normal range for children [ ] . histopathologic evaluation of lung tissue is usually not necessary for the diagnosis of hp. at the present time, there is no diagnostic test that is pathognomonic for hp, and only significant predictors of hp are identified. the most significant diagnostic tool is a detailed environmental exposure history. other diagnostic features include: positive precipitating antibodies to the offending antigen; recurrent episodes of symptoms; symptoms occurring - h after exposure; occurrence of diffuse parenchymal lung disease by lung function and hrct; bal abnormalities with lymphocytic alveolitis and increased cd + t cells. medication, drug, radiation and tobacco exposure drugs used in inflammatory or cancer pediatric diseases can cause ild. they include anti-inflammatory agents (e.g. aspirin, etanercept), immunosuppressive and chemotherapeutic agents (e.g. azathioprine, methotrexate, cyclophosphamide), antibiotics, cardiovascular agents, and, for teenagers, illicit drugs [ , ] . there are no distinct clinical, radiographic or pathologic patterns, and the diagnosis is usually made when a patient is exposed to medication known to result in lung disease, with a timing of exposure appropriate for disease development and elimination of other causes of ild. treatment relies on avoidance of further exposure and corticosteroids in markedly impaired patients. exposure to therapeutic radiation in the management of pediatric cancer may also results in ild. patients presenting within months of therapy generally have radiographic abnormalities with ground glass patterns in both radiation-exposed and unexposed tissue [ ] . the association between tobacco use and ild is less well appreciated than the relation with chronic obstructive pulmonary disease (copd). in addition, pediatric patients do not usually have a significant smoking history to develop respiratory disorders [ ] . connective tissues disorders (ctd) are a heterogeneous group of immunologically mediated inflammatory diseases. their origins are multifactorial with genetic, constitutional and environmental elements contributing to their development. ctd refers to any disease that has the connective tissues of the body as a primary target of pathology. the connectives tissues are composed of two major structural proteins, elastin and collagen, with different types of collagen proteins in each tissue [ ] . many ctd feature abnormal immune system activity associated with inflammation. pulmonary manifestations of ctd may include both vascular and interstitial components. from recent reports, the incidence of ild in the context of ctd appears to be higher than previously appreciated [ , ] . importantly, ild may precede the development of clinically obvious ctd, sometimes by months or years. table provides information on suggestive clinical and serological features in selected conditions. the main disorders to be considered in childhood are rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. the other include sjögren syndrome, dermatomyositis and polymyositis, ankylosing spondylitis, and mixed connective tissue disease. rheumatoid arthritis rheumatoid arthritis (ra) is an inflammatory disorder defined by its characteristic diarthroidal joint involvement. it is the most common ctd in children, but pulmonary involvement is less frequent than in adults. genetic and environmental factors seem to be important contributors of disease progression, with influence of sex (more frequent in male), presence of two copies of the hla-drb "shared epitope" (hla-dr se) and anticyclic citrullinated peptide antibody (anti-ccp), and possibly tobacco exposure [ , ] .almost % of patients with ra have specific serologic abnormalities several years before the onset of joint symptoms, and the findings of elevated serum levels of igm rheumatoid factor or anti-ccp is associated with a high risk for the development of ra [ ] . systemic sclerosis systemic sclerosis (ssc) is characterized by a progressive dermatologic abnormality [ ] . its etiology remains unknown; it is believed to be a complex disease in which interactions between environmental, auto-immune, and genetic factors result in various disease phenotypes [ ] . although it is a rare disease in childhood, the diagnosis is based on skin disease. cardiopulmonary complications are common and have been associated with death in young patients. almost all patients with ssc have serum antinuclear antibodies. the other autoantibody markers are listed in table . recently, the presence of anti-dna topoisomerase ii autoantibody has been reported to be a key factor in the development of ild, in association with class ii mhc status (hla-dr , hla-dpbi) [ ] . systemic lupus erythematosus systemic lupus erythematosus (sle) is an auto-immune disorder characterized by the involvement and dysfunction of multiple organ systems. the mechanisms of tissue injury involve autoantibody production and immunocomplex formation leading to an inflammatory process. diverse clinical phenotypes are observed, including a variety of mucocutaneous lesions, non erosive arthropathy, renal disease (glomerulonephritis and interstitial nephritis), lung disease, pericarditis, and a spectrum of neurologic disorders. laboratory abnormalities are characterized by the presence of antibodies reactive to nuclear (ana) and cytoplasmic antigens. pulmonary vasculitis are observed in vasculitic syndromes that preferentially affect small vessels (arterioles, venules, and capillaries). they include the anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis (wegener's granulomatosis, churg-strauss syndrome, and microscopic polyangitis) that share histologic similarities without immune deposits; anti-glomerular basement membrane (gbm) disease; henoch-schönlein purpura and cryoglobulinemia vasculitis. vasculitic syndromes that affect large/medium vessels (such as kawasaki's disease, polyarteritis nodosa) only occasionally affect the lung [ ] . wegener's granulomatosis wegener's granulomatosis (wg) is a rare disease of uncertain cause. it seems to affect children as much as adults with an increasing reported incidence around . cases/million/year, mostly in teenagers with a reported median age of . years ( - years) [ , ] . it is characterized by inflammation in a variety of tissues including blood vessels (vasculitis). wg primarily affects the upper respiratory tract, lung, and kidneys. the diagnosis is based on the combination of symptoms and a biopsy of affected tissue with necrotising granulomatous vasculitis in the absence of an infectious etiology. the diagnosis is further supported by positive blood tests for cytoplasmic-staining (c)-anca pr type [ ] . churg-strauss syndrome churg-strauss syndrome (css) is a granulomatous small-vessel vasculitis. the cause of this allergic angiitis and granulomatosis is not known, but autoimmunity is evident with the presence of hypergammaglobulinemia, increased levels of immunoglobulin e (ige), and perinuclear-staining (p)-anca. the diagnosis relies on biopsy evidence for vasculitis and at least criteria among the following: moderate to severe asthma, blood eosinophilia (at least %), and nonfixed pulmonary infiltrates with extravascular eosinophils on biopsy [ ] . twenty-nine pediatric cases have been reported so far in the literature, with lung involvement in % of [ ] . anti-glomerular basement membrane disease goodpasture syndrome is a rare disease that involves rapidly progressive kidney failure along with lung disease and is characterized by the deposition of anti-gbm antibodies. several cases have been reported in the pediatric literature. the autoantibodies mediate tissue injury by binding to their reactive epitopes in the basement membranes. this binding can be visualized as the linear deposition of immunoglobulin along the glomerular basement membrane. the principle component of the basement membrane is type iv collagen which can be expressed as different chains, from alpha to alpha . the goodpasture antigen has been localized to the carboxyl terminus of the noncollagenous domain of the alpha chain of type iv collagen. the anti-gbm antibody can usually be found in serum [ ] . strong evidence exists that genetics play an important role. patients with goodpasture disease have an increased incidence of hla-drb compared to control populations [ ] . granulomatous disorders are characterized by the presence of granulomas defined as a focal, compact collection of inflammatory cells in which mononuclear cells predominate. granulomas form as a result of tissue injury by a wide variety of agents including microorganisms, antigens, chemical, drugs and other irritants. in other situations including sarcoidosis, the etiologic factors remain to be determined. sarcoidosis sarcoidosis is a chronic inflammatory disease in which granulomatous lesions can develop in many organs, mainly the lung. its cause remains obscure, and most likely involves environmental and host factors [ ] . the current concept is that a still unknown stimulus activates quiescent t cells and macrophages leading to recruitment and activation of mononuclear cells, with, as a consequence, granuloma formation, alveolitis, and in some cases interstitial lung fibrosis [ ] . sarcoidosis is relatively uncommon among children. its diagnosis is based on a combination of suggestive clinical features, with histologically-documented noncaseating granuloma, in the absence of other known causes of granuloma formation [ ] . the incidence and prevalence of sarcoidosis are reported to be influenced by age, race and geographic localization [ ] . although the youngest patients reported were infants and -months old, most of the cases in children occur in preadolescents and adolescents. from the national patient registry on patients with sarcoidosis in denmark during the period - , patients with a confirmed diagnosis were ≤ years of age [ ] . the calculated incidence was . per . person-years. in children ≤ years of age, the incidence was . ; it increased gradually to . in children aged - years. marked racial differences in the incidence and prevalence of sarcoidosis have been reported by many authors [ ] . various reports in the literature also indicate that race and ethnicity affect both the patterns of organ involvement and disease severity. in a follow-up study we have conducted in children with pulmonary sarcoidosis, children were black [ ] . also the number of organs involved was higher in the black than in the caucasian children. clinical manifestations in sarcoidosis are the consequences of local tissue infiltration with sarcoid granuloma. therefore, disease expression depends on the organ or system involved and a variety of symptoms and physical findings can be observed [ ] . the modes of presentation include non-specific constitutional symptoms, alone or associated with symptoms related to specific organ involvement. in the report of children with sarcoidosis in denmark, the most common non specific symptoms were asthenia, weight loss, and fever [ ] . clinical findings mainly include respiratory manifestations, lymphadenopathy, skin lesions, ocular and central nervous system abnormalities. the most common radiographic findings are hilar lymph node enlargements, with or without lung changes. lung function abnormalities are frequently observed in children with restrictive pulmonary pattern and abnormal diffusing capacity [ ] . other investigations such as bal documenting a lymphocytic alveolitis with increased cd /cd ratio, and elevated serum angiotensin-converting enzyme may provide additional evidence of sarcoidosis [ ] . other granulomatous disorders in children a number of pathological situations are associated with granulomatous disorders defined by the presence of non-caseating granuloma in biopsied tissues. infections are the main causes of other granulomatous diseases, and are in some cases related to disorders of neutrophil function such as chronic granulomatous disease (cgd) [ ] . most children with cgd present with recurrent bacterial and fungal infections. the most frequently encountered pathogens are staphylococcus aureus, aspergillus, burkholderia cepacia, and enteric gram negative bacteria [ ] . the most prominent pulmonary lesions include an extensive infiltration of the lung parenchyma and hilar adenopathy. in some situations, a homogeneous distribution of small granulomatous lesions can occur, with a radiological appearance of miliary tuberculosis. the other granulomatous diseases can be seen in other described diseases, such as immune disorders (including crohn's disease and histiocytosis x), hs pneumonitis, vasculitis disorders or neoplasms. lysosomal diseases gaucher's disease is an autosomal recessive disease and the most common of the lysosomal storage diseases. it is caused by a genetic deficency of the enzyme lysosomal gluco-cerebrosidase that catalyses the breakdown of glucocerebroside, a cell membrane constituent of red and white blood cells. the consequence is an accumulation of glucocerebroside in reticuloendothelial cells, leading to excessive deposition of fatty material in the spleen, liver, kidneys, lung, brain and bone marrow. pulmonary expression is mainly characterized by physiologic involvement (reduction in lung the diffusion capacity and the functional residual volume). lung imaging may show interstitial changes [ ] . niemann-pick diseases are genetic diseases primarily due to deficiency of sphingomyelinase resulting in the accumulation of sphingomyelin within lysosomes in the macrophage-monocyte phagocyte system, mainly the brain, spleen, liver, lung, and bone marrow. histology demonstrates lipid laden macrophages in the marrow, as well as "sea-blue histiocytes" on pathology. the infantile form with a dominant neurologic expression is rapidly fatal. in older patients, cases of ild have been reported [ ] . hermansky-pudlak syndrome is a heterogeneous group of autosomal recessive disorders associated with accumulation of a ceroid-like substance in lysosomes of a variety of tissues. it is characterized by albinism, bleeding tendency associated to poor platelet aggregation and systemic complications associated to lysosomal dysfunction. a chronic inflammatory process may explain the progressive development of ild and fibrosis [ ] . familial hypercalcemia with hypocalciuria familial hypercalcemia with hypocalciuria is caused by autosomal dominant loss-of-function mutations in the gene encoding the calcium-sensing receptor (casr), a g-protein coupled membrane receptor expressed in many tissues [ ] . loss-of-function mutations in casr impair the feedback inhibition of parathyroid hormone secretion in response to a rise in the blood calcium concentration. the result is hypercalcemia associated with inappropriately normal or mildly elevated levels of parathyroid hormone. in the kidneys, mutations in casr prevent the feedback inhibition of calcium reabsorption in situation of hypercalcemia, leading to relative hypocalciuria. respiratory symptoms are usually mild and associated with reduction in the lung diffusion capacity. lung histology indicates the presence of foreign body giant cells and mononuclear cells infiltrating the alveolar interstitium, without circumscribed granulomas. langerhans'-cell histiocytosis is part of the histiocytosis syndromes, which are characterized by an abnormal proliferation of langerhans' cells [ ] . the langerhans cells are differentiated cells of monocyte-macrophage lineage that function as antigen-presenting cells. the origin of the expanded population of langerhans' cells is unknown; in adults, the only consistent epidemiologic association is with cigarette smoking. these cells may form tumors, which may affect various parts of the body. most cases of pediatric langerhans'-cell histiocytosis are observed in children between ages and years, with usually bone involvement ( %) including the skull. the tumors produce a punched-out appearance on bone x-ray, and can cause fracture without apparent traumatism. langerhans'-cell histiocytosis can also affects various organs including the lung [ ] . children with pulmonary langerhans'-cell histiocytosis present in a variety of ways. they can be asymptomatic or present common symptoms such as nonproductive cough and dyspnea. hrct of the chest is a useful and sensitive tool for the diagnosis. indeed, the combination of diffuse, irregularly shaped cystic spaces with small peribronchiolar nodular opacities, predominantly in the middle and upper lobe, is highly suggestive of pulmonary langerhans'-cell histiocytosis [ ] . other abnormalities include ground-glass attenuation. the presence of increased numbers of langerhans' cells in bal fluid (identified by staining with antibodies against cd a) with a proportion greater than percent is also strongly suggestive of pulmonary langerhans'-cell histiocytosis. histologically, the cellular lesions forms nodules containing a mixed population of cells with variable numbers of langerhans' cells, eosinophils, lymphocytes, plasma cells, fibroblasts, and pigmented alveolar macrophages. several forms of ild have been reported to occur with inflammatory bowel diseases (crohn's disease) and celiac disease [ ] . primary biliary cirrhosis and chronic hepatitis have also been reported to be associated with parenchymal lung dysfunction [ , ] . in addition, there are reports on ild in association with neurocutaneous disorders (tuberous sclerosis, neurofibromatosis, ataxia-telangiectasia) and amyloidosis [ ] . depending on the causes, the components of the alveolar structure (the epithelium and the alveolar space, the interstitium, and the pulmonary capillary endothelium) can be involved differently and can serve as primary targets of the underlying pathological processes. based on history, clinical presentation, bal data, and, most important, on information from lung tissue studies, the disorders can be gathered in groups according to predominant structural targets (figure ). the disorders affecting primarily the alveolar epithelium and the alveolar space share common histopathological description, with preserved pulmonary architecture, hyperplasia of type aec, interstitial infiltrates composed of immuno/inflammatory cells and scattered myofibroblasts, and the alveolar space filled with either immuno/inflammatory cells, desquamated materials, or components derived from surfactant lipid and protein complex. in the coming years, it is likely that the list of disorders will expand rapidly with the availability of specific tissue markers. currently, the following grouping can be proposed: infections, surfactant disorders, and eosinophilic lung diseases. infections the role of infection, mainly viral, in the development and progression of ild is sustained by a number of human and experimental reports. from recent knowledge, it is strongly suggested that latent viral infections may be involved in the pathogenesis of ild, through targeting of the alveolar epithelium. the main virus implicated include adenovirus, members of human herpes virus family (epstein-barrr virus and cytomegalovirus), and respiratory syncitial virus [ ] . number of other viruses can also be involved such as influenza a, hepatitis c, or even human immunodeficiency virus (hiv) in immunocompetent children [ ] [ ] [ ] [ ] . human adenovirus being predominantly respiratory pathogens, adenovirus infections can cause a variety of pulmonary symptoms and can persist for long periods of time. several studies in adult patients have indicated that the adenovirus gene product e a could be detected figure alveolar structure disorder-associated ild. depending on the causes, the alveolar structure components can be involved differently and serve as primary targets of the underlying pathological processes. based on history, clinical presentation, bal and lung tissue information, the disorders can be gathered in groups according to the predominant alveolar targets: epithelium, vascular or interstitial components. in lung tissues by in situ hybridization in up to % of cases of idiopathic pulmonary fibrosis. the causative role of the virus in the initiation of the disease remains uncertain, but it may be an important factor in its progression as treatment with corticosteroids may make patients more susceptible to adenovirus infection or reactivation from latency. e a has been shown to increase the production of tgf-β and to induce lung epithelial cells to express mesenchymal markers, thereby contributing to remodeling of the alveolar structure [ ] . isolation of the virus from the throat and serologic studies are diagnostic supportive, but the diagnosis is confirmed by the detection of the virus in lung tissues. epstein-barrr virus (ebv) and cytomegalovirus (cmv) are widespread pathogens that share the characteristic ability of herpesviruses to remain latent within the body over long periods. in mice, the control of herpesviruses replication have also been reported to be associated with the arrest of lung fibrosis [ ] . ebv is present in all populations, infecting more than % of individuals within the first decades of life. infection by cmv is reported in % of individuals aged and older and more than % of aged individuals have antibodies against cmv. in addition, cmv is also the virus most frequently transmitted to a developing fetus. most healthy people who are infected by ebv and cmv after birth have no symptoms, but infection is important to certain high-risk groups of infants and immunocompromised individuals. several studies in the adult literature have reported an increased incidence of ebv and cmv infection in patients with pulmonary fibrosis, associated with virus dna-positive lung tissue biopsies in several cases [ ] . however, so far, no evidence of causal relationship between viruses and pulmonary fibrosis has been provided. respiratory syncytial virus (rsv) is the most common cause of viral lower respiratory tract infection. it affects people of all ages, and can cause severe disease in infants, in older immunodeficient children and the elderly. an intriguing feature of rsv infection is the susceptibility of previously infected individuals to reinfection with antigenically closely related viruses or the identical virus strain. recently, increased interest has been focused on the contribution of persistent rsv in several chronic lung diseases including chronic obstructive pulmonary disease [ ] . the role of rsv in the physiopathology of theses disorders as well as and the mechanisms of its persistence remain to be elucidated [ ] . interestingly, in a recent work on the histopathology of untreated human rsv infection, the presence of the virus in aec has been documented [ ] . from these various data, a role of rsv in the development of ild needs to be investigated. immunostaining with rsv-specific antibodies of tissues from lung biopsy should be proposed. among the other pathogens, chlamydophila pneumoniae and mycoplasma pneumoniae are currently drawing increasing consideration. they are frequent causes of community acquired pneumonia in children. before the age of years, almost % of children have had chlamydophila pneumoniae infection based on serological studies [ ] . these pathogens are intracellular organisms that primarily infect respiratory epithelial cells and alveolar macrophages and have the propensity to persist within several cell types such as macrophages. they are well known to cause a wide variety of respiratory manifestations, with possible progression towards diffuse parenchymal diseases associated with interstitial infiltrates on chest imaging and reduction in the lung diffusion capacity [ ] . regarding legionella pneumophilia infection, progression towards ild has been infrequently reported in adult patients. results from recent studies provided evidence that viruses can infect the alveolar epithelium and may be documented in lung tissues from patients using virus dna detection and immunohistochemistry. a number of specific antibodies are currently available and should prompt to investigate the presence of the above cited viruses in the lung tissues from children with ild. surfactant disorders surfactant disorders include mainly genetic surfactant protein disorders and pulmonary alveolar proteinosis the deficiency in sp-b is a rare autosomal recessive condition known to be responsible for lethal neonatal respiratory distress. rare survivals have been described in partial deficiencies [ , ] . the sftpc mutation i t (c. t > c) is the more prevalent mutation. others are described in only one family. the phenotype associated with sftpc mutations is extremely heterogeneous leading from neonatal fatal respiratory failure to children and adults chronic respiratory disease with ild [ ] . recessive mutations in the abca gene were first attributed to fatal respiratory failure in term neonates but are increasingly being recognized as a cause of ild in older children and young adults. over abca mutations have been identified in neonates with respiratory failure and in older children with ild [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mutations in the ttf- gene are associated with "brainlung-thyroid syndrome" which combines congenital hypothyroidism, neurological symptoms (hypotonia, chorea), and ild of variable intensity [ ] [ ] [ ] [ ] [ ] [ ] [ ] . so far, few mutations have been reported, mostly in exon [ , ] . pulmonary alveolar proteinosis (pap) is a rare lung disorder characterized by alveolar filling with floccular material derived from surfactant phospholipids and protein components. pap is described as primary or secondary to lung infections, hematologic malignancies, and inhalation of mineral dusts. recently, the importance of granulocyte/macrophage colony-stimulating factor (gm-csf) in the pathogenesis of pap has been documented in experimental models and in humans. gm-csf signaling is required for pulmonary alveolar macrophage catabolism of surfactant. in pap, disruption of gm-csf signaling has been shown, and is usually caused by neutralizing autoantibodies to gm-csf. therefore, the emerging concept is that pap is an autoimmune disorder resulting in macrophage and neutrophil dysfunction. in a recent report, it has been reported that gm-csf autoantibodies are normally present in healthy individuals, but at lower levels than in pap patients [ ] . in addition, in vitro experiments indicated that these autoantibodies reduce gm-csp signaling similarly in healthy individuals and in pap patients. at levels above a critical threshold, gm-csf autoantibodies are associated with multiple impaired gm-csf dependent myeloid function [ ] . several cases of genetic defects in the common beta chain for the gm-csf receptor have been documented [ ] . eosinophilic lung diseases eosinophilic lung diseases constitute a diverse group of disorders of various origins. the diagnosis is suggested by the presence of pulmonary infiltrates on chest imaging and peripheral eosinophilia. it is confirmed by the presence of increased amounts of eosinophils in bal and/or lung tissue eosinophilia. in this section, eosinophilic vasculitis will not be discussed (see chapter . . ). the search for an etiology includes a combination of clinical and laboratory investigations. eosinophilic lung diseases of known cause in children include mainly allergic bronchopulmonary aspergillosis, parasitic infections and drug reactions. eosinophilic lung diseases of unknown cause comprise loeffler syndrome (characterized by migrating pulmonary opacities), acute eosinophilic pneumonia, and chronic eosinophilic pneumonia [ , ] . the idiopathic hyper-eosinophilic syndrome is a rare disorder observed mainly in adults; it is characterized by prolonged eosinophilia and a multiorgan system dysfunction due to eosinophil infiltration with pulmonary involvement documented in almost half of the patients [ , ] . alveolar capillary dysplasia and pulmonary capillary hemangiomatosis the pulmonary capillaries form a dense sheet-like meshwork composed of short interconnected capillary segments. the capillary meshes are wrapped over the alveoli, with only a single sheet of capillaries between adjacent alveoli on the same alveolar duct. impaired development of this vascular network can be caused by genetic defects, prematurity or injury. aberrant angiogenesis documented in pediatric patients include mainly alveolar capillary dysplasia, and pulmonary capillary hemangiomatosis [ ] . alveolar capillary dysplasia is a rare disorder, presenting with persistent pulmonary hypertension of the newborn [ ] . the strongest diagnostic features are poor capillary apposition and density, allied with medial arterial hypertrophy and misalignment of pulmonary vessels [ ] . pulmonary capillary hemangiomatosis is also a rare disease that is characterized by proliferation of capillary-sized vessels within the alveolar walls of the lung [ ] . intimal thickening and medial hypertrophy of the small muscular pulmonary arteries are present resulting in elevated pulmonary vascular resistance. most cases appear sporadic. chest imaging shows nodular pulmonary infiltrates and septal lines. a definitive diagnosis can be made only by histologic examination. interestingly, capillary proliferation in the alveolar wall has been reported in hereditary haemorrhagic telangiectasia [ ] . lymphatic disorders alveolar structure formation is characterized by refinement of the gas exchange unit and functional adaptation of endothelial cells into vessels including pulmonary lymphatics. the pulmonary lymphatic network promotes efficient gas exchange through maintaining interstitial fluid balance. lymphatic disorders can be classified as primary or secondary. congenital errors of lymphatic development can lead to primary pulmonary lymphatic disorders that include lymphangiomas and lymphangiomatosis, lymphangiectasis, and lymphatic dysplasia syndrome [ , ] . lymphangiomas are focal proliferations of well differentiated lymphatic tissue, and lymphangiomatosis describes the presence of multiple lymphangiomas. most of these disorders are discovered in fetuses or during the early postnatal period. lymphangiectasis is characterized by pathologic dilation of lymphatics. the term "lymphatic dysplasia syndrome" includes congenital chylothorax, and the yellow nail syndrome (a triad of idiopathic pleural effusions, lymphedema, and dystrophic nails) [ ] . secondary forms of lymphatic disorders result from a variety of processes such as chronic airway inflammation that impair lymph drainage and increase lymph production [ ] . diffuse alveolar hemorrhage syndromes diffuse alveolar hemorrhage (dah) syndromes are caused by the disruption of alveolar-capillary basement membrane as a consequence of injury to the alveolar septal capillaries, and less commonly to the arterioles and veinules. the hallmarks are intra-alveolar accumulation of red blood cells, fibrin, and hemosiderin-laden macrophages. it is important to point out that approximately one third of patients with dah do not manifest hemoptysis, and bal can be extremely helpful if this entity is suspected by showing the presence of siderophages or red blood cells within the alveoli. dah can be observed in association with systemic findings or without evidence of associated diseases. in children, situations of dah in the context of other disorders are reported in several forms of vasculitis discussed above. other disorders that can also be accompanied by dah include pulmonary hypertension and congenital heart diseases, pulmonary veino-occlusive disease, arteriovenous malformations and hereditary haemorrhagic telangiectasia, coagulation disorders, and celiac disease [ ] . in the absence of systemic findings, isolated pulmonary capillaritis should be discussed with the search for positivity of the antiglomerular basement membrane antibody with linear deposits in the lung tissue biopsy as well as suggestive serologic features such as p-anca antibodies [ ] . idiopathic pulmonary hemosiderosis is a diagnosis of exclusion based on patient presentation with acute, subacute, or recurrent dah, on the results of lung biopsy showing evidence of 'bland' pulmonary hemorrhage (ie, without capillaritis or vasculitis), and after exclusion of the conditions listed above [ ] . in this situation, red blood cells leak into the alveolar space without evidence of damage and/or inflammation of the alveolar capillaries. in addition, the diagnosis of idiopathic pulmonary hemosiderosis can only be considered after exclusion of diseases induced by environmental factors such as pesticide and cow's milk (heiner's syndrome) [ ] . this syndrome is a hypersensitivity disease that affects primarily infants, and is caused by antibodies to cow's milk proteins. the diagnosis is supported by positive milk precipitin test and rapid improvement of symptoms and pulmonary infiltrates on chest imaging after exclusion of milk proteins. in the resolution phase of tissue injury, elimination of mesenchymal cells and recruited inflammatory cells is essential for restoration of normal cellular homeostasis. dysregulated repair process in ild is associated with accumulation and dysfunction of interstitial fibroblasts [ ] . in the coming years, it is likely that progress in the understanding of the mechanisms involved in the impaired myofibroblast apoptosis as well as evasion of these cells from immune surveillance will open new areas of investigations and will provide support for the characterization of disorders that affect primarily the alveolar interstitial components in pediatric ild. indeed, recently, distinct intrinsic differences in gene expression pathways has been reported between control and lung fibrosis myofibroblasts which suggests that ild myofibroblasts are pathological cells with fundamental changes [ ] . in the context of ild, pulmonary interstitial glycogenosis, neuroendocrine cell hyperplasia, and chronic pneumonitis in infancy have been reported to be exclusively observed in very young children [ ] . pulmonary interstitial glycogenosis (pig) is a non lethal disease, reported in neonates with respiratory distress syndrome developed shortly after birth [ , ] . very few cases are described so far but it seems to have a male preponderance [ ] . the histological hallmark of pulmonary interstitial glycogenosis is the accumulation of monoparticulate glycogen in the interstitial cells on lung biopsy. it is thought to represent a maturation defect of interstitial cells that leads them to accumulate glycogen within their cytoplasm [ , ] . it is discussed that pig could meet "chronic pneumonitis in infancy" as this remains a generalized term [ ] . as well, pig could be considered as a premature lung disease, but more than half of published cases were in term infants [ , , ] . the long term consequences in these infants need to be ascertained. neuroendocrine cell hyperplasia of infancy (nchi) is also a non lethal disease characterized by tachypnea without respiratory failure. the human airway epithelium contains highly specialized pulmonary neuroendocrine cells (pnec) system. it's function remains unknown but is hypothysed to act in modulation of fetal lung growth and in post-natal stem cell condition [ ] . the pnec system permits synthesis and release of serotonin and neuropeptides such as bombesin [ ] . as normal bombesin levels decrease after mid-gestation, its overexpression in nchi could be attributed to a nonregression of neuroendocrine cells [ ] . clinical presentation is typically a respiratory distress in post-natal young infant (mean age . months in a large serie, but cases in older children have been reported [ ] . hrct shows patchy centrally ground-glass opacifications and air trapping [ ] . on lung biopsy, the histological abnormality is hyperplasia of neuroendocrine cells within bronchioles documented by bombesin immunohistochemistry. the follow-up reveals in some cases the persistence of tachypnea and oxygen requirement for several months. usually, there is a good prognosis [ , , , ] . chronic pneumonitis in infancy was first described by katzenstein et al. [ ] . the clinical and radiologic features are similar to those observed in other forms of ild. specific histologic abnormalities include diffuse thickening alveolar septa, hyperplasia of type aec, and presence of primitive mesenchymal cells within the alveolar septa. in some cases, foci of pulmonary proteinosis-like material have been observed in air spaces. the prognosis has been reported to be poor with a high mortality rate. other disorders associated with pulmonary development and growth abnormalities encompass a broader spectrum of respiratory manifestations and are more adequately integrated in the classification of diffuse lung diseases [ ] . management of children with ild includes administration of oxygen for chronic hypoxaemia, and maintenance of nutrition with an adequate energy intake, immunization with influenza vaccine on an annual basis is recommended along with other routine immunizations against major respiratory pathogens [ ] . in addition, aggressive treatment of intercurrent infections and strict avoidance of tobacco smoke and other air pollutants are strongly recommended. a very few children do not require any treatment and recover spontaneously. in the majority of cases, treatment with immunosuppressive, anti-inflammatory, or anti-fibrotic drugs is required for weeks, months or even years [ , , ] . various drugs discussed below can be used, but no guidelines for treatment of ild in children have been proposed so far. the major reason is the very limited number of pediatric patients available for a prospective clinical trial. in addition, controlled studies with a placebo arm are unacceptable because of the poor prognosis of untreated cases and the reported efficacy of anti-inflammatory therapies in a number of pediatric ild. at the present time, the main therapeutic strategy is based on the concept that suppressing inflammation may most likely prevent progression to fibrosis. among the anti-inflammatory agents used in pediatric ild, steroids are the preferred choice, administered orally and/or intravenously. this has been well illustrated by the results of the ers task force on pediatric ild [ ] . oral prednisolone is most commonly administered at a dose of - mg/kg/day [ ] . children with significant disease are best treated with pulsed methylprednisolone at least initially [ , ] . this is usually given at a dose of - mg/kg/day for days consecutively at monthly intervals. the minimum number of cycles recommended is but treatment may need to be continued for a longer period of months or more depending on response. when the disease is under control, the dosage of methylprednisolone can be reduced or the time between cycles can be spaced out. the disease may then be controlled with oral prednisolone preferably given as an alternate day regime. in few cases oral prednisolone is used from the beginning simultaneously with intravenous methylprednisolone but this is only recommended in those with very severe disease. methylprednisolone may be effective when other forms of steroids administration fail without significant side effects. an alternative to steroids is hydroxychloroquine with a recommended dose of - mg/kg/day. individual case reports have described a response to hydroxychloroquine even in the presence of steroid resistance [ , , ] . some groups have proposed to base the decision as to which agent to use on the lung biopsy findings, with a preference for steroids in case of large amount of desquamation and inflammation and for hydroxychloroquine if increased amounts of collagen representing pre-fibrotic change are found. however, as documented in the ers task force on pediatric ild, the preferred choice between steroids or hydroxychoroquine in children is highly dependent on the expertise of the center in charge of the patient, and does not seem to be oriented by the histopathological pattern [ ] . in case of severe disease, steroids and hydroxychloroquine may be associated. in situations of inefficiency of steroids and hydroxychloroquine, other immunosuppressive or cytotoxic agents such as azathioprine, cyclophosphamide, cyclosporine, or methotrexate may be used. these treatments have been used mainly in situation of autoimmune disorders. promising therapeutic options include macrolides. indeed, these antibiotics have been shown to display a number of anti-inflammatory and immunomodulatory actions. although the mechanisms and cellular targets specific to macrolide activity remain to be elucidated, beneficial effects in several chronic lung diseases including chronic obstructive pulmonary diseases (copd) and cystic fibrosis have been reported [ , ] . of interest is the ability of macrolides to accumulate in host cells including epithelial cells and phagocytes. in a recent report, a favorable response to treatment with clarithromycine has been described in an adult patient with dip [ ] . other new therapeutic strategies currently proposed in adult patients target fibrogenic cytokines. the th cytokine interferon-γ has an antifibrotic potential through suppression of th fibrogenic functions. antagonists to tgf-β include pirfenidone and decorin. the use of molecules directed against tnf-α such as the soluble tnf-α receptor agent etanercept is also under investigation. to date, there are no reports on the use of these novel therapies in pediatric ild. finally, in the coming years, it is likely that an expanding number of molecules aimed at favoring alveolar surface regeneration and repair through activation and proliferation of tissue-resident (progenitor) cells will come out. depending on the underlying diseases, several specific treatment strategies needs to be considered. these include whole lung lavage for pulmonary alveolar proteinosis, which has been reported to be effective by removing the material from the alveolar space [ ] . gm-csf has also been shown of interest in this disease [ ] . other strategies such as interferon-α for pulmonary haemangiomatosis are effective [ ] . in recent years, lung transplantation has emerged as a viable option in children of all ages, even in young infants, and lung or heart-lung transplantation may be offered as an ultimate therapy for end-stage ild [ ] . the outcome and survival do not seem to be different from those reported in conditions others than ild, although comparisons are difficult to establish due to the limited number of reported cases. response to treatment and outcome can be evaluated in children based on several criteria such as decrease in cough and dyspnea, increase in oxygenation at rest and sleep, and changes in pulmonary function tests [ , ] . improvement on thoracic hrct may also be seen, but tends to occur over a much longer period of time. reports in pediatric ild have not shown a good correlation between histological findings and outcome. some children with relatively severe fibrosis on lung biopsy make good progress, whereas others with mild desquamation have a poor outcome. this is probably due to the variable severity of the disease in different parts of the lung especially in relation to the particular area biopsied, despite hrct guidance. overall a favorable response to corticosteroid therapy can be expected in - % of cases, although significant sequelae such as limited exercise tolerance or the need for long-term oxygen therapy are often observed. reported mortality rates are around %. the outcome for infants is more variable [ , ] . pediatric ild comprises a large spectrum of disorders, with compelling evidence that some of these disorders are observed more frequently in infants, while others are more specific to older children. ongoing basic research will provide new insights into the molecular basis of ild pathogenesis (including genetic factors causing familial disease) in children, and is expected to identify important preclinical markers of disease, pathways of disease regulation, and novel potential targets for therapeutic intervention. for the future, there is a strong need for international collaboration which will allow collecting sufficiently large cohorts of patients with specific entities in order to perform proper therapeutic trials. as a prerequisite, however, a clear and standardised classification of the histopathology of the underlying conditions is critical. such multicenter trials will help to reduce the still considerable morbidity and mortality in children with ild. abbreviations (ards): acute respiratory distress syndrome; (aec): alveolar epithelial cells ankylosing spondylitis; (ab): antibodie; (anti-ccp): anticyclic citrullinated peptide; (anti-gbm): antiglomerular basement membrane anca): anti-neutrophil cytoplasmic antibody; (ana): antinuclear antibodies u -rnp): anti-u -ribonucleoprotein; (sao ): arterial oxygen saturation binding immunoglobulin protein; (bal): bronchoalveolar lavage; (casr): calciumsensing receptor; (cgd): chronic granulomatous disease; (copd): chronic obstructive pulmonary disease; (css): churg-strauss syndrome; (ctd): connective tissue disorders; (cmv): cytomegalovirus; (c): cytoplasmicstaining; (dip): desquamative interstitial pneumonia; (dad): diffuse alveolar damage; (dah): diffuse alveolar hemorrhage; (dlco): diffusing capacity of the lung for carbon monoxide functional residual capacity; (sftpb): gene coding for sp-b; (sftpc): gene coding for sp-c; (gm-csf): granulocyte/ macrophage colony-stimulating factor human immunodeficiency virus; (hp): hypersensitivity pneumonitis; (ig): immunoglobulin; (ild): interstitial lung disease lymphocytic interstitial pneumonia; (mmp): metalloproteinases; (mpa): microscopic polyangiitis; (mctd): mixed connective tissue disease; (nchi): neuroendocrine cell hyperplasia of infancy; (nsip): non-specific interstitial pneumonia pulmonary function testing; (pig): pulmonary interstitial glycogenosis pulmonary neuroendocrine cells; (rv): residual volume; (rsv): respiratory syncitial virus; (ra): rheumatoid arthritis; (rnp): ribonucleoprotein; (srp): signal recognition particle systemic lupus erythematosus; (ssc): systemic sclerosis; (ttf- ): thyroid transcription factor ; (tlc): total lung capacity; (tlco): transfer factor of the lung for carbon monoxide; (tgf): transforming growth factor; (uip): usual interstitial pneumonia idiopathic interstitial pneumonitis in children: a national survey in the united kingdom and ireland factors influencing survival in children with chronic interstitial lung disease diffuse parenchymal lung disease. cape town: karger idiopathic pulmonary fibrosis: clinical relevance of pathologic classification european respiratory society international multidisciplinary consensus. classification of the idiopathic interstitial pneumonias pediatric interstitial lung disease: children are not small adults paediatric interstitial lung disease diffuse lung disease in young children: application of a novel classification scheme task force on chronic interstitial lung 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interstitial lung diseases in children this work was supported by inserm, université pierre et marie curie-paris , paris, assistance publique-hopitaux de paris, ministère de la santé (centre de référence des maladies respiratoires rares), and comité de soutien de belleherbe. the authors would like to especially thank malika malhoul, delphine michon, alexandra blondel, aurore coulomb and hubert ducou le pointe for all of their effort towards the creation of the reference center for rare lung diseases. authors' contributions ac and nn contributed equally to this work and should be considered as joint first authors. ac, nn and hc drafted the review. re and bf have been involved in revising critically the review. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -fy yg xh authors: popper, helmut h. title: interstitial lung diseases—can pathologists arrive at an etiology-based diagnosis? a critical update date: - - journal: virchows arch doi: . /s - - - sha: doc_id: cord_uid: fy yg xh interstitial lung diseases (ild) encompass a group of diseases with a wide range of etiologies and a variety of tissue reactions within the lung. in many instances, a careful evaluation of the tissue reactions will result in a specific diagnosis or at least in a narrow range of differentials, which will assist the clinician to arrive at a definite diagnosis, when combining our interpretation with the clinical presentation of the patient and high-resolution computed tomography. in this review, we will exclude granulomatous pneumonias as well as vascular diseases (primary arterial pulmonary hypertension and vasculitis); however, pulmonary hypertension as a complication of interstitial processes will be mentioned. few entities of pneumoconiosis presenting as an interstitial process will be included, whereas those with granulomatous reactions will be excluded. drug reactions will be touched on within interstitial pneumonias, but will not be a major focus. in contrast to the present-day preferred descriptive pattern recognition, it is the author’s strong belief that pathologists should always try to dig out the etiology from a tissue specimen and not being satisfied with just a pattern description. it is the difference of sorting tissue reactions into boxes by their main pattern, without recognizing minor or minute reactions, which sometimes will guide one to the correct etiology-oriented interpretation. in the author’s personal perspective, tissue reactions can even be sorted by their timeliness, and therefore, ordered by the time of appearance, providing an insight into the pathogenesis and course of a disease. also, underlying immune mechanisms will be discussed briefly as far as they are essential to understand the disease. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. interstitial lung diseases (ild) are characterized by a diffuse infiltration of both lungs, usually evaluated by highresolution computed tomography (hrct) scan. the types of infiltrating cells are not predefined, so this can be inflammatory as well as tumor cells. tumorous infiltration will be excluded, since there are too many entities, not only carcinomas but also lymphomas and certain sarcomas such as angiosarcomas. therefore, our main focus will be on interstitial pneumonias (ip). many classifications on ip have been published over the last four decades, and our understanding of these processes has improved stepwise and resulted in a refined schema, which can be used to sort these diseases accordingly. however, there are still many problems, making the use of this classification problematic to pathologists, not specifically dealing with lung pathology. originally, liebow [ ] proposed a classification based on morphological descriptions, with the following entities: usual interstitial pneumonia (uip), bronchiolitis obliterans-interstitial pneumonia (bip), diffuse alveolar damage (dad, also acute interstitial pneumonia [aip] , clinically corresponding to electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory distress syndrome [ards]), lymphocytic interstitial pneumonia (lip), desquamative interstitial pneumonia (dip), and giant cell interstitial pneumonia (gip). katzenstein's updates from and [ , ] were the next major step, adding nonspecific interstitial pneumonia (nsip) to the list of uip, dip, bip, and aip/dad and removing lip and gip because an etiology could be assigned to them. later on, muller and colby showed a radiologic-pathologic correlation and used the previously created name bronchiolitis obliterans-organizing pneumonia (boop) [ , ] instead of bip. when these entities were combined with clinical data, it was apparent that there was a major difference between uip and the "rest": patients with uip had a worse prognosis and most of them died within years after diagnosis [ ] . another intention to separate idiopathic interstitial pneumonias (iip) from those with known cause was to provide prognostic and therapeutic information to the clinicians: no response of patients with uip/idiopathic pulmonary fibrosis (ipf) towards corticosteroids and immunosuppressive drugs and dismal prognosis, whereas responsiveness of patients with nsip to corticosteroids and immunosuppressive drugs and a better prognosis. the next step happened, when uip and the fibrosing variant of nsip were compared to each other, showing that the initial difference vanished especially when evaluated for years survival [ ] . then, ip were classified into idiopathic and those with known etiology: dip and respiratory bronchiolitis with or without interstitial lung disease (rbild) were excluded from idiopathic because, in both entities, cigarette smoking was identified as the main cause of the disorder. lip was also skipped, probably because of a clearly defined etiology in almost all cases, either lymphoma, allergic, or autoimmune diseases. gip was skipped, since it either is induced by hard metal inhalation or viral infection (measles, respiratory syncytial virus [rsv] , and others) [ , ] . what makes the present-day classification complicated is the combination of radiology, pathology, and pulmonology, resulting in provisional diagnoses or divergent names for pathology and clinics. in addition, the final diagnosis needs to be discussed between clinicians, radiologists and pathologists. this also changed the general view: many clinicians suppose they are the only ones being able to make the diagnosis of ipf, and pathologists in the classification committee have accepted it. there are examples which support such a perspective: organizing pneumonia (op) has a wide variety of etiologic causes, and the idiopathic form cryptogenic organizing pneumonia (cop) needs exclusion of all other causes, which on several occasions can be done by pathologists, but in other cases only by combining morphology with clinical information. furthermore, radiology has gained a major impact on the diagnosis of ild, which resulted in decreasing numbers of patients for whom a pathologic diagnosis is required. it cannot be neglected that pathologists have also contributed to this situation: there are colleagues who are happy to make the diagnosis of, e.g., uip and do not further look for features which would allow the differentiation between uip/ipf versus other causes of uip, e.g., collagen vascular diseases (cvd). based on recommendations from a joint committee established by the european respiratory society (ers) and american thoracic society (ats) pathologists, radiologists, and pulmonologists proposed a new classification and also a diagnostic algorithm for ild [ , ] (table ; fig. ). in this review, i will discuss iip following the schema shown in fig. . when discussing ip with known etiology, i will group these diseases according to their etiologic basis. therefore, some of the morphologic patterns of idiopathic ip will be recapitulated. finally, ild with various etiologies will be discussed at the end of this review. granulomatous pneumonias including extrinsic allergic alveolitis/hypersensitivity pneumonia (eaa/hp) will be excluded because this alone would fill a review on its own. however, in some of the diseases to be discussed, granulomas do appear and, therefore, will be explained briefly. this review is based on the personal experience of the author being responsible for the diagnostic workup at the medical university of graz, but also acting as a consultant for many central european hospitals, which allowed me to set up a huge lung and pleura biobank. in addition, several published articles and reviews in this area including published classifications by the ats/ers joint committees on ild are critically reviewed. usual interstitial pneumonia/idiopathic pulmonary fibrosis uip/ipf is a chronic progressive fibrosing disease of the lung, which leads to death of the patient usually within - years after the diagnosis is made. it affects predominantly patients in their fourth to fifth decade of life; however, lesions may occur much earlier and remain undetected until acute interstitial pneumonia (aip) diffuse alveolar damage (dad) they will cause impaired lung function by their increasing number-uip/ipf is seen more often in younger-aged patients, probably due to increased awareness. characteristically, lesions are found in both lower lobes with a predominance of subpleural regions. the involvement of both lobes is most often symmetrical. by hrct, different features can be seen: fibrotic and scar lesions, ground glass areas, and honeycombing. cystic lesions, consolidations, and scars are found on gross cut surface. the pleura usually show multiple retractions, giving the surface a cobblestone appearance, but pleuritis is not seen. the histological hallmarks are fibroblastic foci, scars and diffuse fibrosis, honeycomb areas, and uninvolved areas in between (figs. and ; suppl. fig. ). the cause and the etiology of ipf/uip are not well understood. there is a working hypothesis, which can explain some of the features. the disease starts with an as yet unidentified epithelial injury causing apoptosis of pneumocytes [ ] [ ] [ ] [ ] . inflammatory signals released by the dying pneumocytes cause transformation and proliferation of fibroblasts and myofibroblasts in a myxoid stroma and repair [ ] (so-called fibroblastic focus). genetic abnormalities may underlie these apoptotic responses: in the recent years, research in familial forms of ipf has highlighted the importance of surfactant apoproteins in maintaining homeostasis between injury and repair and that mutations in the surfactant apoprotein c gene might be causally related to the development of familial ipf [ ] . in these familial ipf, mutations in genes encoding surfactant apoprotein c and a increases endoplasmic stress reactions in pneumocytes type ii, and in addition, mutations in the telomerase genes tert and terc are responsible for telomere shortening, probably decreasing the pool of peripheral lung stem cells and thus impairing repair and regeneration [ ] . this later defects are also found in sporadic ipf cases. therefore, inhalation of any kind of toxic material from the environment might cause an overwhelming oxygen stress reaction leading to increased apoptosis of pneumocytes and impaired regeneration [ ] . this fits quite well into the epidemiology of ipf patients: the majority are smokers, and some have a history of environmental dust exposure [ , ] . there is also evidence of epithelialmesenchymal transition (emt) of pneumocytes into myofibroblasts (fig. ) , and also scattered bone marrow-derived mesenchymal stem cells seem to move into these foci [ ] [ ] [ ] . these foci undergo maturation with collagen deposition, and finally, the process results in fibrosis of alveolar septa and bronchiolar walls [ ] . this in turn causes obstruction of the terminal airways resulting in cystic destruction of the remaining peripheral lobules, giving rise to honeycombing and remodeling of the lung parenchyma [ , , ] . the process develops stepwise, which means there are lung lobules not affected yet looking normal, whereas others are destroyed or even completely lost to fibrosis and scarring. this is meant by the term "timely heterogeneity." in the author's experience, a diagnosis of uip/ipf can be established in some cases even without clinical information when the following features are given: fibroblastic foci, timely heterogeneity (involved and uninvolved peripheral lobules), cystic and fibrotic destruction resulting in honeycombing, and most importantly, the absence of inflammatory infiltrates in areas of fibroblastic foci, absence of granulomas, or features of other interstitial inflammation. let us briefly characterize the main morphologic features, since this still causes confusion and misunderstanding: the fibroblastic focus lies within the walls of alveolar and interlobular septa, as well as bronchioles. they do not project into the alveolar lumen. in early stages, they are composed of myofibroblasts and fibroblasts in an immature myxoid matrix. this matrix will stain for immature collagen and reticulin fibers. the overlaying surface is either denuded (no pneumocytes) or can show pneumocyte regeneration with a lot of reactive changes of the nuclei, even epithelial giant cells can be present (figs. and a ). when the focus get's older, mature collagen appears and the cells look more like fibrocytes. the overlaying epithelium looks reactive and usually has a type ii or bronchiolar cell appearance. the honeycomb lesion was originally defined by radiologists as a single or multicystic lesion within a fibrotic lung area [ ] . given the differences in resolution between hrct and histology, there is a substantial difference in size between the two. pathologically, a so-called honeycomb lesion is a cystic lung lesion involving a secondary lobule. this lobule has lost most of the peripheral alveoli, shows a cystic central area composed of bronchioles and centroacinar structures, covered by a cuboidal and cylindrical epithelium, resembling bronchiolar epithelium and transformed pneumocytes type ii (figs. b and ). in some cases, a pseudostratified squamous-looking epithelium can be present. the cyst walls are fibrotic and often merge with scarred lung tissue or large fibrotic areas involving sometimes a subsegment of the lung. within the lumen, mucus can accumulate, and in late stage, this can be the starting point for secondary infection and bronchopneumonia, causing death of the patient. i prefer the term lobular cystic lung remodeling, instead of honeycombing, because of the size differences between hrct and microscopy (suppl. fig. ). the areas of fibrosis and scarring and the uninvolved lung tissue (heterogeneity) do not need an explanation. but what about inflammation? from what we understand presently, ipf/uip is not an immune-driven or classic emt in a fibroblastic focus: immunohistochemistry using double stain for ttf (brown, nuclear) and smooth muscle actin (red, cytoplasmic) in the same cells. magnification, × fig. cystic remodeling of lobules, the histologic correlate to ct scan of honeycombing; bar, μm; compare the size of the cystic lesion to a ct lesion inflammatory disease. therefore, we do not expect inflammatory cells within the fibroblastic foci. if lymphocytes appear in numbers (> /hpf) within a fibroblast focus, this should raise the possibility of an underlying immune reaction. the appearance of granulocytes within these foci should prompt the search of remnants of hyaline membranes because this may represent organizing dad. the ats/ers recommends that a panel of experts composed of pulmonologists, radiologists, and pathologists (clinical-radiological-pathological [crp]) should make the diagnosis of ipf. the clinical presentation and course, the hrct picture, and the pathologic pattern of uip should be combined. in some cases, the diagnosis of ipf can be based on clinical and ct findings alone. whenever pathologic evaluation is involved, a diagnosis of uip is mandatory for the diagnosis of ipf. acute exacerbation of uip/ipf is clinically characterized by rapid worsening of the patient's symptoms, with severe hypoxia most often requiring mechanical ventilation and oxygen supply. many patients will die under this condition. histologically, two types of acute exacerbations can be seen when examining autopsy cases: secondary infection with infectious pneumonia in the background of uip or multiple fibroblastic foci and severe fibrosis leaving not much lung parenchyma for ventilation. in these latter cases, there is usually severe lung edema present. if a viral infection is present, the histological pattern is dad [ ] overlaying uip; if bacterial or fungal infection causes exacerbation, a purulent bronchopneumonia is found. besides, in ipf, a uip pattern can occur in many other diseases, such as autoimmune diseases, allergic diseases, toxic inhalation, drug-induced pneumonias, and many more. this still causes a lot of confusion because the term uip is not used uniformly: some authors use uip strictly in the sense of ipf, others do not care about etiology and simply diagnose uip as a pattern, and a third group discerns uip and uip-like tissue reactions. the same happens with clinicians: most think that a uip diagnosis already means ipf and are confused to learn that uip can present in chronic eaa as well as drug reactions for example. we will discuss these later on. nsip is a diffuse ip, characterized by loose lymphocytic, macrophagocytic, and histiocytic cell infiltration within alveolar septa combined with mild fibrosis. there is no timely heterogeneity, meaning that the lesions seem to have appeared at the same time. hyperplasia of the bronchus-associated lymphoid tissue (balt) is usually not present [ , ] . the lung architecture is preserved in contrast to uip, and cystic destruction is absent. two forms are discerned, which in some cases might represent timely sequences of the disease: the cellular and fibrotic types (figs. and ). both behave differently; the cellular type has a better prognosis, whereas the fibrotic variant is more close to uip [ ] . in the etiologic background, nsip is most often associated with autoimmune diseases, especially with cvd [ ] [ ] [ ] [ ] [ ] . an association with druginduced pneumonia and also with allergic diseases such as eaa/hp has also been reported [ , ] . only those cases without an identifiable etiology are labeled as idiopathic nsip. however, the morphologic pattern is identical; therefore, in most instances, idiopathic nsip remains a clinical diagnosis. clinically, nsip shows diffuse infiltrations, corresponding to ground glass opacities on hrct. symptoms as in the other ild are quite unspecific. many patients with nsip will respond to corticosteroid and/or immunosuppressive drug treatment, and also spontaneous resolution of the disease has been reported [ , ] . cop is a diagnosis of exclusion, based on the morphology of op (formerly boop). on hrct, op/cop shows a pattern with combinations of ground glass opacities and consolidations and the almost diagnostic tree-in-bud pattern, sometimes also reticulonodular pattern [ ] . in rare cases, the consolidation can mimic a tumor [ ] . histologically, the hallmark of op is an intra-alveolar granulation tissue, the so-called masson body (figs. and ). it consists of proliferating fibroblasts and myofibroblasts with inflammatory cells like neutrophils, lymphocytes, histiocytes, and macrophages. few hemosiderin-laden macrophages are often present. the granulation tissue can start from the wall of bronchi, bronchioli, and alveoli. there is usually a defect of the epithelial layer and also the basal lamina. fibroblasts and myofibroblasts grow into the defect; however, in contrast to normal repair, the granulation tissue does not stop but continuously grows into the airspaces, filling these completely or incompletely. in later stages, pneumocytes will grow over these granulation tissue plugs and, therefore, a slit-like airspace can be formed (fig. ) [ ] . the amount of inflammatory cells within the granulation tissue depends on the cause of op. the morphologic pattern of op has a very wide range of etiologies. it can occur as a post-infectious process, in inactive or resolving stages of autoimmune disease/cvd and vasculitis, in toxin inhalation, in drug-induced lung diseases, in chronic inflammatory bowel diseases, or idiopathic, which is cop [ ] [ ] [ ] [ ] . in some cases of op, the etiologic cause can be determined, for example, by viral inclusion bodies in postviral op ( fig. ) or by endothelial cell reactions in druginduced op. in some cases, an additional pathologic tissue reaction besides op can also point to the underlying etiology. so what are the diagnostic features? & granulation tissue growing into bronchi, bronchioles and alveoli, usually with remnants of inflammatory cells. & fibrotic occlusion of whole lobules or remaining slitlike spaces covered by pneumocytes. & a mixture of inflammatory cells within these granulation tissue plugs depending on the cause of previous damage. if looking for the etiology, one should also closely investigate the small blood vessels and the regenerating pneumocytes: viral inclusion bodies might be still visible, scattered neutrophilic granulocytes can be found in the granulation tissue in cases of bacterial or fungal infection, and eosinophils might be seen pointing to a previous drug-induced pneumonia. in virus-induced pneumonias, another feature can be found, even after several months: single transformed pneumocytes showing atypical nuclei and a homogenously stained smudged chromatin pattern (suppl. fig. ). in drug-induced and metabolic as well as in autoimmune diseases, the vascular walls can show various structural changes, making an etiology-based diagnosis probable: eccentric vasculopathy with scattered lymphocytes and without endothelial damage might point to deposition of idiotypic-anti-idiotypic immune complexes (without complement activation; fig. ; suppl. fig. ), endothelial damage with fibrosis and repair can point towards druginduced damage (suppl. fig. ). cop as a crp diagnosis is a diagnosis of exclusion: if all possibly underlying diseases are excluded, cop can be diagnosed. this has some importance, since cop responds well to corticosteroid treatment. clinically, aip (also ards) is characterized by acute onset of severe hypoxia, with the radiological appearance of white lung. histologically, there is edema and fibrinous exudate, widened edematous alveolar septa. later on, hyaline membranes are formed (dad). inflammatory infiltrates are usually scarce. depending on the cause of dad, neutrophilic and/or eosinophilic granulocytes can be found in bacterial, toxic, or drug-induced dad or scattered lymphocytes are seen in viral and rickettsial infections [ , ] (fig. ). inflammatory infiltrates may be even absent such as in various kinds of shock. rarely, cases of "idiopathic aip" have been reported. probably, some of these cases represent cases of undiagnosed systemic lupus erythematosus (sle) or drug toxicity. in the author's experience, in all cases sent for consultation and primarily labeled as idiopathic dad, an etiology could finally be established. so it might be questioned, if idiopathic dad does exist. hamman and rich described an ip with fulminant course, leading to death in their six cases within months. in the authors' description, there was no hyaline membrane mentioned, but a proliferation of fibroblasts. since the tissues from these cases were all lost, this disease cannot be reconstructed and remains an enigma [ ] . the sequence of events in dad is largely dependent on the cause: toxic metabolites of drugs or released collagenase and elastase from necrotizing pancreatitis will cause endothelial damage, followed by leakage of the small peripheral blood vessels. this causes edema, followed by pneumocyte cell death (hypoxia). serum proteins will pass into the alveolar lumina, coagulate there, and by the breathing movements are compressed into hyaline membranes. in case of airborne disease, e.g., infection or inhaled toxins, pneumocytes type i die followed by type ii. the basement membrane is either preserved or destroyed (especially in viral infection). this again causes leakage of capillaries, edema with/ without bleeding, protein extravasation into the alveoli, and finally, formation of hyaline membranes. the lethality of dad is still high despite improvements which have been made in the past decade. in some cases, the progression of the disease might be blocked by antiprotease treatment [ ] . in more recent time, extracorporeal oxygenation or nitric oxide treatment has shown some benefit. if the patient survives the acute phase, dad will be organized, which is essentially an op-type of lesion, but is most often labeled as organizing dad: granulation tissue grows into the alveoli and hyaline membranes are incorporated into the plugs. they can be demonstrated several weeks after the initial injury. if a tissue biopsy or an autopsy specimen is available early on in the course of the disease, the etiology might be elucidated. in viral infection, inclusion bodies can be seen, later followed by atypical proliferation and transformation of pneumocytes type ii. in contrast to atypical pneumocyte hyperplasia (aah), these atypical cells are single, do not form a continuous layer along the alveolar wall, and usually show bizarre nuclei and nucleoli. rickettsial infection results in less pronounced proliferation of pneumocytes. in shock-and drug-induced dad, the endothelia will undergo apoptosis and necrosis, and fibrin cloths might be seen in capillaries. in these cases, the alveolar septa are widened and edematous. inflammatory cells are scarce or absent. in later stages of drug-induced pneumonia, scattered eosinophils are encountered-their function being completely unknown in these cases. what are the characteristics of dad? & edematous fluid accumulation in alveoli and in the interstitium (depending on the time course), & fibrin cloths in alveoli with/without hyaline membranes, & scarce inflammatory infiltrates (neutrophils and/or lymphocytes, etiology-dependent), & minor diagnostic but etiologically important features are damage of pneumocytes, endothelial cells, fibrin thrombi in small blood vessels, and regeneration±atypia. lip almost vanished from the literature in the last years. the major problem is the separation from nsip. when nsip was described, it was never clearly separated from lip [ ] . when comparing my own cases and reports from the literature, it becomes evident that differences do exist: in lip, the lymphocytic and plasmocytic infiltration is dense, hyperplasia of the balt is common, and within lymph follicles, germinal centers are usually present [ ] . the infiltration in lip is more diffuse, architectural distortion is common, and scarring does occur. histiocytic and monocytic cellular infiltrations are much less pronounced compared to nsip. lymphoepithelial lesions do occur similar to lymphomas, in some entities aggressively infiltrating and destroying the epithelium and in other cases no epithelial disruption does occur. in contrast to nsip, the architecture of the peripheral lung is remodeled, especially in later stages (figs. and ). the organization phase is often characterized by op. within the etiologic spectrum, similar diseases are found as in nsip: autoimmune diseases especially cvd, allergic diseases such as eaa (acute and subacute), allergic drug reactions, and in children, different types of immunodeficiency (t cell defect, nk cell defect). the most important differential diagnosis, however, are extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (malt/balt) type and lymphomatoid granulomatosis type i. in all cases, the clonality has to be evaluated and a lymphoma needs to be excluded by proof of multiclonality. also, it is important to exclude posttransplant lymphoproliferative disease [ ] , which can present in a similar pattern (large lymphoid cells usually epstein-barr virus-positive). however, it should be taken into account that some of the autoimmune diseases have a high propensity of developing non-hodgkin lymphomas later in the course [ ] . within the autoimmune diseases, sjøgren's disease (sjs) most often presents with the lip pattern [ , ] . what are the characteristics of lip? & diffuse dense lymphoplasmocytic infiltrates in alveolar septa and bronchial/bronchiolar walls. in some cases, the lymphocytic infiltration can form concentric rows encasing capillaries and venules; & hyperplasia of balt with well-formed follicular centers; & focal fibrosis and scarring with distortion of the peripheral lung architecture; & lymphoepithelial lesions; & eccentric sclerosis of vessels walls with narrowing of lumina. this is usually a sign of deposition of immune complexes in the vessel walls and should prompt the search for diseases associated with the production of autoantibodies, such as sjs and systemic sclerosis (ssc). in autoimmune-induced ild, many different factors come together: a wide variety of immune reactions can cause a wide variety of tissue reactions, for example, circulating autoantibodies either capable or devoid of complement activation, circulating immune complexes including large insoluble immune complexes formed by idiotypic-antiidiotypic antibody networks, activation of coagulation, metabolism of proinflammatory substances, involvement of different types of leukocytes, and not the least, drugs given for the relieve of symptoms. these drugs themselves can cause toxic or inflammatory side effects. each of the different diseases will induce a different reaction pattern, and this pattern will be modified during the course of the disease. therefore, we cannot expect a single reaction or pattern, but a complex picture composed of new and old lesions, resolving lesions, and acute exacerbations of the disease. it will always be of help to know the mechanisms and underlying pathogenesis of each disease to interpret the histological picture in its presented form. since we have already extensively discussed the different ip, we will focus now on the modifications induced by the autoimmune diseases. rheumatoid lung disease ip in rheumatoid arthritis (rha) is not uncommon; however, it is often complicated by additional drug reactions, which can look like rha-induced pneumonia. most often, nsip is associated with this disease [ , ] , but these reported studies have in common a selection bias: they looked up the incidence of rha in cases presenting with nsip. the diversity of reaction patterns is much better reflected in studies looking up patients with rha and lung involvement [ ] . uip was more common in this study. in our own experience (stacher et al., submitted), most often, a mixture of reaction patterns occurred, such as uip combined with dense lymphocytic infiltrates or lip and uip combined with op or nsip. if features of dad occur, one should discuss drug reactions with the clinicians because many immunosuppressive drugs given in rha and other cvd can cause dad [ , , , ] . not uncommonly, granulomas are present too. these are most often foreign body-type granulomas with giant cells, sometimes classical rheumatoid granulomas with palisading histiocytes, and rarely epitheloid cell granulomas [ ] . in classical rheumatoid granulomas, it is essential to exclude infectious organisms, since patients receiving immunosuppressive drugs like methotrexate and leflunomide are prone to acquire infections [ ] [ ] [ ] (figs. and ). this is even more important with the new "biologicals." the etiology of rha is still an enigma. genetic variants for several immune regulators such as toll receptors and interleukins may form the basis for the susceptibility to adversely react against antigens and immune complexes trapped in cartilages and by that induce an inflammatory reaction, resulting in cartilage damage [ , ] . regulatory t cells either deficient or functionally impaired might also play an important role in rha. in addition to the genetic basis for the disease, the autoimmune reaction might be triggered by streptococcal infections, and in that respect, probably mimicry of proteins of the organism might come into play. in acute lupus manifestation, which is more often autopsied and rarely seen as surgical material, hemorrhagic pneumonia, infarcts, or dad or all mixed are found [ ] [ ] [ ] . most probably, the extent of any of these reactions depend on the extent of intravascular death of neutrophils attacked by lupus autoantibodies: low numbers of dying neutrophils might release less toxic enzymes and, therefore, cause focal endothelial cell death, interstitial edema, proteins leaking out into alveolar spaces, and finally, dad with hyaline membrane formation. in case of massive neutrophilic cell death, there might be massive leakage of vessel walls and hemorrhage will occur. in later stages, dad will be organized, so op is another feature found in systemic lupus. since the disease affects the coagulation cascade, lung infarction is a common feature of sle. i personally have seen, in addition, perivascular amyloidosis in a classical active sle case. the occurrence of pulmonary hypertension is less well understood [ , ] . the etiology of sle is also not well understood. autoantibodies directed against granulocytic enzymes have been shown to be secondary effects and not the cause of sle. in a recent study in familial forms of sle, a null mutation in the dnase l gene has been found. this finding confirms the critical role of impaired clearance of degraded dna in sle as a probable cause [ ] , a finding also seen in adult sle [ ] . however, given the wide range of autoantibodies found in sle, there might be much more autoimmune mechanisms involved than anticipated [ , ] . ssc usually presents with a mixture of tissue reactions, dependent on the immune phenomena present at the time of biopsy. most often, a uip or nsip pattern is found, accompanied by lymphocytic infiltration of lip type and a hyperplasia of balt (suppl. fig. ). germinal centers are less common compared to sjøgren's syndrome. ill-formed granulomas composed of histiocytic and/or epitheloid cells can be seen (fig. ) . the distribution pattern of the ip is irregular, involving peripheral as well as midzone areas of the lung. this is clinically helpful in separating it from uip/ ipf. another feature helpful in the diagnosis is a vasculopathy. medium-and small-sized arteries show a thickening of the intima and media. within the thickened vessel wall, there is a myxoid change of the matrix. a few lymphocytes can be seen, however, no endothelial necrosis or any other sign of vasculitis. these changes can possibly be best interpreted as a consequence of immune complex deposition (fig. ). functionally, these vascular changes will cause pulmonary hypertension, which is common in ssc [ , ] . genetic studies provided some new insights into ssc. interleukins, especially il- , has an impact on lung fibrosis in ssc patients [ ] . also, transforming growth factor-beta (tgf-β) and connective tissue growth factor (ctgf) received attention as essential factors in the pathogenesis of ssc. ctgf mrna expression was observed in the fibrotic lesions, serum ctgf concentrations were significantly elevated, and correlated with skin sclerosis and lung fibrosis. in an animal model, tgf-β induced subcutaneous fibrosis and subsequent ctgf application caused persistent fibrosis. based on these data, the authors of this study hypothesized that tgf-β induces fibrosis in the early stage, whereas ctgf acts to maintain tissue fibrosis [ ] . in another study, fibrillin has been investigated. it has been demonstrated that caveolin- and fibrillin- influence storage and regulation of tgf-β and other cytokines and fibrillin- mutations might be responsible for a congenital form of scleroderma called stiff skin syndrome [ , ] . in addition to tissueresident fibroblasts, bone marrow-derived fibroblasts and endothelial and epithelial cells undergoing emt are also under the control of fibrillin. gain-of-function and loss-of-function abnormalities of these mediators may account for the characteristic activated phenotype of ssc fibroblasts [ ] . the impaired expression of the nuclear orphan receptor ppar-γ in ssc seems to play an important role in causing uncontrolled progression of fibrosis through impaired control of fibroblast activation and differentiation [ ] . dermatomyositis rarely affects the lung. if lung involvement is present, various forms and combinations of pneumonias can occur. uip and nsip are the most common alterations; however, histiocytic and ill-formed epitheloid cell granulomas can be encountered in some cases in my personal experience. lymphocytic infiltrates are quite common, most often exceeding what is seen in nsip and better matched by lip. vasculopathy is rare. the serosa is usually involved too; however, this is also common in other cvd. polyserositis in contrast to the other cvd cannot be diagnosed on tissues, since these serosal infiltrations are unspecific and occur in a multitude of diseases. sjøgren's disease sjs is another multisystemic collagen vascular or autoimmune disease. it affects predominantly the mucosa of salivary and lacrimal glands, but can also similarly affect the lung. the main finding is an aggressive lymphocytic infiltration into the epithelial lining of bronchi and bronchioles and a diffuse infiltration of the alveolar walls (lip). in the bronchi/bronchioles, lymphoepithelial lesions occur, similar to what is seen in marginal zone lymphomas. the epithelial layer is disrupted, which later on is repaired and can present as op (fig. ) . the lymphocytic infiltration is polyclonal and composed of t and b lymphocytes. lymphollicles are well formed and will show activated germinal centers. other types of ip can be associated to lip, even uip can occur, usually in the form of uip-lip mixed pattern. as in ssc, interleukins play an important role in sjs too. il- overexpressing transgenic mice developed bronchial and alveolar abnormalities such as lymphocytic infiltrates around the bronchi, cell proliferation in the alveolar septa, and increased interstitial and alveolar macrophages, strikingly similar to those found in the lungs of sjøgren's patients. there were also fourfold higher numbers of natural killer cells. a new mouse model highlights the role of il- in the initiation of sjøgren's syndrome [ ] . rangel-moreno studied the hyperplasia of balt in patients with pulmonary involvement in rha and sjøgren's syndrome. increased expression of cxcl and ccl , as well as b cell-activating factor of the tnf family (baff), icos ligand, and lymphotoxin, correlated with balt hyperplasia. the presence of balt hyperplasia correlated also with tissue damage in the lungs of these patients [ ] . in a recent investigation, genes related to one of the major symptoms of sjøgren's syndrome have been identified, namely, the immunological attack of salivary and lacrimal glands resulting in the loss of acinar cell tissue and function, leading to stomatitis sicca and keratoconjunctivitis sicca. one gene lying on chromosome (autoimmune exocrinopathy [aec ]) and the second on chromosome (autoimmune exocrinopathy [aec ]) have been shown to be necessary and sufficient to replicate sjs-like disease in c bl/ mice. aec lies distal to the centromere. this chromosomal region contains several sets of genes known to correlate with various immunopathological features of sjs. one gene in particular, tumor necrosis factor (ligand) superfamily member (or ox ligand), encoding a product whose biological functions correlate with both physiological homeostasis and immune regulations, could be a potential candidate sjs susceptibility gene [ ] . although many open questions remain, this mouse model opens the way to better understand this autoimmune disease and also will serve to study the mechanisms, which are responsible for the common development into malt lymphomas. mixed cvd cannot be diagnosed with certainty. the combination of features of two different cvd makes it almost impossible to come up with an etiologic suggestion or proposal. depending on the features of the single cvd, morphologic mixtures can be found. for example, mixed sjøgren-lupus cvd can either have dominant features of sjs or systemic lupus [ ] . however, general features suggestive of cvd are usually present: a combination of different features of ip with lymphocytic infiltrations, hemorrhage, etc. so when to think about lung affected by autoimmune diseases? & any combination of ip; & any kind of ip with a high proportion of lymphocytes; & any combination of an ip with other inflammatory reactions not fitting within ips, such as a combination of uip/ nsip or lip with epitheloid or histiocytic granulomas; & any kind of ip with unusual vasculopathy (not sclerosis!) and/or alveolar hemorrhage. goodpasture's syndrome is an autoimmune disease not related to cvd. the cause has been identified recently: by mimicry against bacterial proteins, cross-reacting autoantibodies are formed, which bind to the α- chain of collagen iv causing disruption and hemorrhage by complement activation (most often the alternate pathway) [ , ] . there are still unexplained features such as why collagen iv in glomerular, alveolar, and alveolar capillary basement membranes is attacked, but not those in other organs. the major finding is alveolar hemorrhage without infiltrating leukocytes. later on, macrophages with hemosiderin can be seen within alveoli and septa (fig. ) . depending on the duration of the disease, the final result is septal fibrosis [ ] . other autoimmune diseases affecting the lung ip can be encountered in behcet disease and kikuchi disease, whereas in whipple's disease, granulomatous pneumonia with histiocytes and macrophages is the prominent feature, similar to what is seen in the small bowel [ ] . igg -related sclerosis is an autoimmune disease involving many organs such as the pancreas, mediastinum, and retroperitoneal space. hypergammaglobulinemia and deposition of igg is seen in affected tissues. in the lung, the disease usually presents with extensive lymphoplasmocytic infiltration (suppl. fig. ), very often simulating inflammatory pseudotumor (plasmocytic variant); however, in this disease, increased numbers of igg -positive plasma cells are found (≥ % of igg-positive cells), not seen in inflammatory pseudotumor (myofibroblastic tumor) [ ] . this inflammation typically results in sclerosis of the lung tissue [ ] . phospholipid autoantibody-mediated lung disease is another rare disease not only affecting the lung but also other organs [ ] . in the affected lung, the major finding is alveolar hemorrhage or hemorrhagic pneumonia and dad (suppl. fig. ), followed by an op in a later stage. the cause is unknown, and the morphologic findings are not specific. the disease needs the proof of phospholipid autoantibodies in the serum. airway-centered interstitial fibrosis (acif) is a new, recently described ild mainly affecting patients with a history of environmental exposure to toxic or allergic substances. also, cocaine abuse was found in one [ ] . the morphology is characterized by fibrosis along the small bronchi extending into the peripheral lung following a lobular distribution. in some cases, fibroblastic foci can occur, however, always associated with this distribution pattern. cystic lung remodeling is absent; instead, a whole lobule or subsegment is destroyed by fibrosis. metaplastic epithelium is common in the affected lobules and also hyperplasia of smooth muscle cells (muscular cirrhosis; fig. ). the disease rapidly progresses, and in the reported series, almost half of the patients died of disease. corticosteroid treatment was effective in some patients. here, we will focus on several diseases with quite different morphological features, but all of them related to cigarette smoke exposure, most often in young-aged heavy smokers. combinations of these diseases are currently more often seen, probably due to the increased use of hrct followed by lung biopsy because of the wide variety of differential diagnosis, even including malignant disease. langerhans cell histiocytosis (lhch, histiocytosis x, eosinophilic granuloma) is caused by excessive inhalation of tobacco smoke. it occurs predominantly in young-aged people. it has been postulated that tobacco plant antigens still present within tobacco smoke (incomplete combustion) might cause this accumulation and proliferation of langerhans cells, which are part of the antigen-presenting reticulum cell population [ ] [ ] [ ] . so the continuous exposure of langerhans cells to plant proteins in susceptible persons might cause proliferation of these cells to keep with the increasing amount of antigens. langerhans cells proliferate within bronchial mucosa as well as in the peripheral lung. in bronchi, this proliferation causes necrosis of the mucosa, occlusion of the lumen, and finally, scar tissue [ ] (fig. ; suppl. fig. ). langerhans cell proliferation is accompanied by an infiltration of eosinophils, hence the old name eosinophilic granuloma. these eosinophils are attracted by cytokines such as interleukin secreted by the langerhans cells [ ] . eosinophilic granulocytes might also act cytotoxic in cooperation with langerhans cells releasing eosinophilic basic proteins and destroy the epithelium. the granulomas undergo regression especially in patients with smoking cessation. the resulting scar has a star-like appearance and is surrounded by bronchiolectasis and emphysema blebs. on ct scan, this results in a characteristic picture (starry sky). an underlying genetic abnormality has recently been identified [ ] [ ] [ ] . mutations in the braf oncogene has resulted that lhch is now regarded as a tumor. however, several issues remain to be solved before this view can be accepted: lung cases are most often induced by smoking and, in many patients, will undergo regression in case of smoking cessation, which is unlikely in a tumor [ ] . braf mutations were not found in all cases. on the other hand, there exists a tumorous form, characterized by a multiorgan involvement, seen in children and young adults and not related to smoking. so probably, we might be confronted with two different diseases. so far, the reactive form cannot be discerned from the tumor form other than by involvement of at least two organ systems [ , ] . further investigations hopefully will increase our knowledge about this disease. in the differential diagnosis, lhch has to be separated from other histiocytosis or reticulum cell proliferations by their positive staining for cd a and langerin [ , ] , whereas the positivity for s protein is not specific. respiratory bronchiolitis-interstitial lung disease rbild is a common disease in heavy smokers. rb can be seen in many cigarette smokers with lung carcinoma, if the lung tissue adjacent to the tumor is sampled and investigated. rb can also be seen combined with lhch [ ] . an accumulation of alveolar macrophages within the respiratory bronchioles and the adjacent centrilobular region of the lung lobules characterize rbild. the macrophages usually contain dirty brownishyellow fine granular pigment (fig. ) . ultrastructurally, this pigment represents phagolysosomes filled with tobacco waste [ ] [ ] [ ] . functionally, this macrophage accumulation obstructs the terminal bronchioles and impairs airflow, resulting in distension of alveoli and eventually rupture of septa. some authors use the diagnosis of rb only in those cases, where no other pathology is present, others this author included follow the morphology and accept rb diagnosis also in heavy smokers with lung carcinoma in whom rb is also present. the argument is that rb is a disease of smokers, and there is no good argument why smokers are not allowed to have more than one disease, e.g., rb, carcinoma, lhch. however, this results in different statistical figures: if rb diagnosis is only accepted presenting as a singular disease, it is rare; if diagnosed by its morphological features regardless of other smoking induced diseases, it is a common disease, present in many patients with lung cancer. rb and rbild in my opinion are subsequent stages of the same disease. in the early stages, accumulation of alveolar macrophages is concentrated within bronchioles. if tobacco smoke exposure goes on, more and more areas of the centroacinar region of alveoli are occupied by these cells, resulting in radiological ground glass opacities, now clinically called rbild. the term dip was created by liebow in [ ] , long before immunohistochemistry was invented. liebow misinterpreted the cells accumulating within the alveoli as pneumocytes type ii, therefore the term desquamative. by immunohistochemistry, these cells were identified as macrophages [ , , ] . therefore, the name macrophagocytic pneumonia would have been more appropriate. by definition, dip is characterized by an accumulation of pigmented smoker macrophages within alveoli, completely obscuring the peripheral airspaces. no infiltration of bronchioles is present (suppl. fig. ). fibrosis of alveolar septa, if present, is mild. dip can radiologically simulate a tumor with ground glass opacity, not uncommonly misdiagnosed as adenocarcinoma in situ [ ] . smoking-induced interstitial fibrosis (srif) and rbassociated ild might represent the same diseases characterized by rb and a paucicellular eosinophilic collagenous thickening of alveolar septa with a subpleural distribution [ , ] . in some areas, the disease resembles fibrotic nsip, but the typical association with tobacco smoking points to this underlying etiology. in looking up several cases of rb, we also recognized similar reactions as described by s. yousem and a.l. katzenstein, but in addition, also cases showing fibroblastic foci associated with emphysema blebs and fibrosis (fig. ) . in these cases, also rb could be seen in different areas. in contrast to uip, there were no honeycomb lesions and almost all lobules showed changes of centrilobular emphysema. some of these patients were clinically diagnosed as having chronic obstructive pulmonary disease (copd); in others, the lesions were found incidentally because of pneumothorax. so this might represent another form of smoking-induced lung fibrosis, probably resulting from the release of toxic enzymes from macrophages and subsequent alveolar septa destruction and repair. acute interstitial pneumonia/diffuse alveolar damage many different agents can cause aip/dad. classical ones are viral infections, but toxic inhalation, drug reactions, and all variants of shock reactions will also present with this morphologic picture. the features have been described above, so we need to focus only on specific changes pointing to a specific etiology. in viral infection, the most characteristic feature is the viral inclusion body, which can present either as nice large inclusion bodies (cytomegalovirus [cmv], respiratory syncytial virus [rsv]) or by red-violet-stained nucleic acids forming illdefined speckles in nuclei and/or cytoplasm (adenovirus) [ ] . typically, the infected cell shows enlargement, an atypical large bizarre nucleus, and an accentuated nuclear membrane due to increased nucleic acid traffic induced by the virus. these cellular features can last for several months. in contrast to preneoplastic lesions in viral infection, the atypical cells are single cells being grouped together with otherwise normal-looking pneumocytes. giant cell interstitial pneumonia (see also under pneumoconiosis) gip has a quite narrow etiologic spectrum either being caused by hard metal dust or by viral infection. the former will be discussed later. several viruses can cause gip, the classical one being measles virus. however, in contrast to pneumoconiosis in infections, the giant cells are mixed epithelial as well as macrophagocytic. the epithelial giant cells (hecht cells) are transformed pneumocytes type ii in whom nuclear division was not followed by cell division, giving rise to multinucleation [ ] . the additional features are identical to dad as described above. especially within the epithelial cells, viral inclusion bodies can be found (suppl. fig. ). besides measles, rsv can also present, with this picture predominantly in children [ ] . we have already described op under the idiopathic ilds, so we have only to focus on other causes of op. op can have a great variety of causes. in many cases, this is a post-infectious organization of a purulent bacterial bronchopneumonia, when for several reasons the exudate could not be completely degraded and, therefore, has to be organized by granulation tissue. also, in viral infections, organizing dad is morphologically identical to op, as discussed previously. in other cases, op is a form of organization of an autoimmune disease, usually in the inactive phase. the resolution phase of toxic inhalation is usually in the form of op, and drug-induced lung toxicity is also often organized the same way (suppl. fig. ). chronic pneumonia of infancy originally, surfactant-related ip with alveolar proteinosis were included into chronic pneumonia of infancy (cpi); however, since the different causes of alveolar proteinosis were discovered, it has been excluded. therefore, cpi has been reduced to those pediatric interstitial diseases with unknown cause. it is now quite rare. cpi is characterized by an infiltration of lymphocytes and macrophages/histiocytes in the alveolar septa, accumulation of debris within the alveoli, and hyperplasia of type ii pneumocytes (suppl. fig. ), all causing thickening of the septa and impaired gas exchange. cpi predominantly occurs in newborn or small children [ , ] . in many instances, a careful investigation of the biopsies might uncover underlying infectious diseases, such as wilson-mikity syndrome and infections caused by respirotropic viruses, chlamydiae, or uroplasms [ ] ; another cause might be gastroesophageal reflux [ ] . in rare instances, a metabolic disease, interstitial glycogenosis, might be the cause of cpi [ , ] . however, it should be taken into account that, although the clinical symptoms in affected children are severe, the density of the inflammatory cells is much less compared to pneumonias in adults. surfactant-related interstitial pneumonias-alveolar proteinosis alveolar proteinosis occurs in two forms, either as a genetically inherited disease or as an acquired disease of adults [ ] [ ] [ ] [ ] [ ] . it is characterized by an accumulation of surfactant lipids and proteins within the alveoli, in severe cases completely obstructing the peripheral parenchyma and causing severe dyspnea. morphologically, there is eosinophilic material within the alveoli sometimes mixed with debris and, in some cases, accompanied by an inflammatory infiltration of the alveolar walls. in the inherited form, the underlying genetic defect is either a mutation in the surfactant apoprotein genes b or c or a mutation in the transporter protein gene abca [ , ] . this results in the production of immature surfactant or an accumulation of surfactant within giant lamellar bodies. this material is secreted but does not function as mature surfactant. the result is alveolar collapse, followed by apoptosis of pneumocytes, infiltration of inflammatory cells (lymphocytes, macrophages), and sometimes also formation of hyaline membranes or fibrin cloths within alveoli. the histological picture simulates but never completely imitates irds (suppl. fig. ). in the acquired form (predominantly adults), different defects of the degradation cascade of surfactant do occur. the most common is a deficiency of granulocyte macrophage colony-stimulating factor (gm-csf) caused by autoantibodies against this protein [ , , , ] . gm-csf is necessary for the uptake and subsequent degradation of surfactant by alveolar macrophages. the hallmark is an accumulation of surfactant material within alveoli. there is usually no inflammatory infiltrate present (fig. a) . the diagnosis can be even easily made by bronchoalveolar lavage (bal): the recovered fluid looks milky. in later stages, the disease can get chronic. in addition to the accumulation of surfactant lipids and proteins, there is a diffuse interstitial fibrosis, which can cause death of the patient (fig. b) . sometimes, alveolar proteinosis can be induced by tuberculosis, acute silicosis, and other diseases; the mechanisms in these are still unclear. although some authors prefer to include idiopathic neuroendocrine hyperplasia of infancy (inhi) into pediatric ild, i do not follow this line because inhi does not present with diffuse interstitial infiltrations but rather with an increase of neuroendocrine cells within bronchi and bronchioles and less pronounced neuroendocrine bodies in the periphery [ ] . idiopathic pulmonary hemosiderosis is an interstitial disease usually found in children, but also young adults. the characteristic clinical feature is recurrent alveolar hemorrhage and morphologically diffuse lymphocytic and histiocytic/macrophage infiltrations in both lungs. the etiology is largely unknown. many patients develop iron deficiency anemia due to recurrent bleeding. there is usually a high mortality rate. a degradation of elastic fibers followed by an incrustation by iron-containing proteins (prussian blue-positive) can be seen on histologic examination and is a diagnostic feature. usually, foreign body giant cells are found in the areas of ongoing destruction of the elastic fibers ingesting the fibers together with the iron coat [ ] (suppl. fig. ). in later stages, interstitial fibrosis results. vasculitis, granulomatous inflammation, or immunoglobulin deposits are absent. corticosteroids alone or in combination with other immunosuppressive agents may be effective for either exacerbations or maintenance therapy of idiopathic pulmonary hemosiderosis [ ] . lymphangioleiomyomatosis lymphangioleiomyomatosis (lam) is an inherited disease, based on the dysfunction of tsc genes. tsc coding for hamartin and tsc coding for tuberin are both required for controlling the expression and regulation of mtor and the mtor complexes torc / . both proteins form a complex, which activates camp kinase, which subsequently inactivates mtor. in lam, tsc is more often mutated then tsc [ ] [ ] [ ] [ ] . tuberin is thus not functional and does not form heterodimers with hamartin, and torc / is constantly activated. tsc mutation can be a germline mutation as in tuberous sclerosis syndrome or mutated somatically, called by some authors as "form fruste" [ ] . the second allele is most often mutated somatically. the type of mutation also dictates the extent of syndromes and tumors associated with this mutation. patients carrying the germline mutation present with genetic instability causing several somatic mutations and, therefore, in addition to lam, can present with different other benign tumors. previously, lung transplantation was one of the few choices of treatment; since the discovery of the function of both tsc proteins, a clinical trial with anti-mtor therapy has been started and seems to be effective [ ] [ ] [ ] . interestingly, in rare cases, with lung transplantation, a recurrence of the disease occurred from circulating lam cells, repopulating the transplanted lungs [ ] [ ] [ ] [ ] [ ] . lam is characterized by a proliferation of immature myoblasts in the periphery of the lung and in lymph nodes. in addition, perivascular epitheloid cells (pec) also proliferate (probably derived from pericytic stem cells). the proportion of myoblasts and pec can vary considerably: in some cases of lam, pec are numerous, in other cases scarce. the cells together form microscopic nodules in the bronchiolar mucosa, along lymphatics and arteries, and in alveolar septa, causing lymphatic obstruction and rupture (chylothorax) and also bronchial obstruction and cystic lung destruction. these myoblasts show immature myofilaments, are not ordered in parallel as normal smooth muscle cells, and also have a more epitheloid appearance. nuclei are round and larger than in regular smooth muscle cells. most importantly, these cells proliferate in locations where no muscle cell proliferation occurs (suppl. fig. ). by immunohistochemistry, the myoblasts express smooth muscle actin, and a few also desmin, whereas the pec express hmb [ , ] . hermansky-pudlak syndrome is an autosomal recessive disease, which results in the inability of several cells to form intracellular vesicles. this can affect the ability to form melanosomes (resulting in albinism), platelet granules (inducing bleeding diathesis), and also phagolysosomes in macrophages and lamellar bodies in type ii pneumocytes [ ] . to date, seven forms are known, but only two of them (types i and iv) constantly affect the lung, causing phagocytosis defects with accumulation of macrophages, lamellar body defects in type ii pneumocytes with disturbed surfactant release and macrophage foam cell changes, inflammation, and finally, lung fibrosis [ ] [ ] [ ] [ ] . the characteristic morphologic changes are hyperplasia of pneumocytes type ii, giant lamellar bodies (large pas-positive cytoplasmic granules), accumulation of foamy macrophages within the alveoli, lymphocytic interstitial infiltration and lung fibrosis, sometimes as op with intra-alveolar granulation tissue, and also as interstitial fibrosis with myofibroblastic foci, identical to those in uip ( fig. ; suppl. fig. ). even both types of fibrosis op and uip can be present concomitantly in one patient. in contrast to uip/ipf, honeycomb lesions are scarce, and usually, the lung is diffusely involved, leaving not much uninvolved tissue in between (no timely heterogeneity). in contrast to uip/ipf, the involvement of the lung is not symmetric, fibrosis can occur peripherally as well as centrally, and is always patchy. ultrastructurally, there are no well-formed lamellar bodies within pneumocytes type ii. within macrophages, lysosomes are enlarged and irregularly contoured, while phagolysosomes are absent (no fusion of lysosomes with phagosomes). erdheim-chester disease is a rare systemic histiocytosis (non-langerhans dendritic cells) that may present with pulmonary symptoms. the condition seems to be nonfamilial and typically affects middle-aged adults. radiographic and pathologic changes in the long bones are diagnostic, but may mimic multisystemic lhch [ , ] . patients often present with extraskeletal manifestations. advanced pulmonary lesions are associated with extensive fibrosis that may lead to cardiorespiratory failure [ ] . in rare instances, there are diffuse dense infiltrations by histiocytes accompanied by lymphocytes and plasma cells. these histiocytes are negative for langerhans cell markers (cd a, langerin) and markers for follicular and interdigitating dendritic cells (cd , cd ), but can be positive for s protein (much less intensive staining compare to interdigitating dendritic cells) and are usually positive for cd , cd , and lysozyme (suppl. fig. ). mutation within the braf gene has been found in few cases (personal communication). histiocytes can express pdgfrα and pdgfrβ, which might be used as therapeutic targets. diffuse panbronchiolitis (dpb) was first described in patients from southeast asia. it is characterized by an accumulation of macrophages within bronchioles and a lymphocytic infiltration within the bronchiolar walls. hyperplasia of balt does occur and follicle centers might be present, usually related to recurrent infections [ ] . in contrast to lip, the infiltration is always concentrated along bronchi and bronchioles. dpb might be difficult to separate from other forms of bronchiolitis. however, there are some features which are of help: in dpb, the obstructive lesions are confined to the respiratory bronchioli; chronic parasinusitis is common; follicular bronchiolitis is a common finding [ ] (fig. ) . the cause is a phenotypic variation in the hla system involving hla-b , hla-a , and hla-drb * / [ ] , which leads to susceptibility to otherwise nonpathogenic bacterial infection in immunocompetent children. children with this hla type need to be treated with erythromycin every time a respiratory tract infection occurs. in nontreated children, the disease will cause secondary destruction of the peripheral lung with cyst formation and fibrosis. initially, dpb was mainly identified in children of asian descent; however, this disease has also been diagnosed in caucasians [ ] . but it should be mentioned that the diagnosis should be confirmed by morphologic analysis because of similarity with other forms of bronchiolitis, seen by hrct [ , ] . amyloidosis is a metabolic disease characterized by the deposition of amyloid in lung tissues. amyloidosis is presently regarded as a protein misfolding disease [ , ] . normally, four proteins are folded into a complex with the help of chaperones. many different forms of amyloid proteins have been identified. misfolding can occur in several different compositions of amyloid [ ] . most common are amyloid a and p; however, microglobulin β, transthyretin, and others can also be seen in pulmonary amyloidosis. the reason for amyloid deposition can be a tumor; one of the best-known examples is plasmocytoma. in this case, amyloid is formed out of immunoglobulins secreted by the tumor cells. chronic inflammation such as cvd can also cause amyloid deposition [ , ] . amyloid deposition can occur as a tumor-like deposit, either along the bronchial tree or within the lung periphery (nodular form), or it can be diffuse perivascular and interstitial (suppl. fig. ). amyloid stains red by eosin stain, orange by the congo red stain, or can be demonstrated by immunohistochemistry using specific antibodies for the different components [ ] . if a congo red stain is applied, the tissue section should be examined under polarized light, where the stain exhibits a green birefringence. amyloid deposits very often will cause a tissue reaction with foreign body giant cells forming granulomas, and also calcification and metaplastic bone formation can occur. the major differential diagnosis to amyloidosis is igg -mediated fibrosis and hyalinosis; however, the dense lymphoplasmocytic infiltrations will lead to the correct diagnosis. we will not discuss tracheobronchopathia chondroosteoplastica because this is a localized process in the trachea and large bronchi. although pulmonary ossification would also qualify to be included into metabolic diseases, it is usually a very focal disease and thus does not fit into diffuse interstitial diseases. however, diffuse metabolic calcification and microlithiasis are diffuse interstitial processes and fall herein. metabolic/metastatic pulmonary calcification diffuse calcification of alveolar septa can occur in patients with hyperparathyroidism and hypercalcemia [ ] [ ] [ ] [ ] . most often, the kidneys and the stomach, which functionally are also involved in ion exchange, can be affected. in the lungs, calcium deposits are seen along the alveolar septa, forming a network completely outlining the septa (fig. ). there is no inflammatory reaction towards the calcium deposits in contrast to microlithiasis. treatment needs to be focused on the underlying disease; no specific treatment for the calcification does exist. microlithiasis is a diffuse lung disease characterized by a deposition of microliths in alveolar septa and lumina with a foreign body giant cell reaction (fig. ) . the giant cells phagocytose the microliths. large foreign body cell granulomas can be formed. the microliths usually show a center which can be calcified. the etiology was unknown until recently: first, familial cases were described [ ] , and finally, a candidate gene, slc a , that encodes a type iib sodium phosphate cotransporter was found to be mutated in all six patients being investigated. slc a is specifically expressed in type ii pneumocytes, and the mutation abolishes the normal protein function [ , ] . there exist no specific therapy; in severe cases, lung transplantation can be considered [ , ] . microlithiasis should be clearly separated from lung tissue with alveoliths, i.e., concentric lamellae of proteins around a crystalline center, lying within alveolar lumina without any tissue reaction. eosinophilic lung diseases all together are rare. lhch has already been discussed. we will not discuss bronchial asthma, although it can present with severe eosinophilic infiltrations along the bronchi and bronchioles, but much less in the alveoli. we also exclude churg-strauss syndrome because it would require a discussion of systemic vasculitis. allergic bronchopulmonary mycosis (formerly allergic bronchopulmonary aspergillosis) is not in our focus, since the majority of cases present as a specific form of bronchitis (mucoid impaction) and less frequently as bronchocentric granulomatosis. there are rare forms of allergic bronchopulmonary mycosis presenting as eosinophilic pneumonia, however, not showing any specific feature other then the forms discussed below. eosinophilia is most often induced by the release of interleukins and [ , ] . it can vary quite remarkably in the different diseases, less pronounced in lhch, whereas massive in eosinophilic pneumonias. by bal, a tentative diagnosis can be made: eosinophil counts in bal usually are between and % in lhch, whereas in the eosinophilic pneumonias, it usually exceeds %. in parasitic diseases, sometimes the parasites (larvae) might be seen in bal. acute eosinophilic pneumonia (aep) is characterized by an acute onset with dyspnea and diffuse infiltrations by eosinophils in both lungs. blood eosinophilia can be present, especially in loeff ler's syndrome and in parasitic infections. on histology, the alveoli and the alveolar and bronchiolar walls are stuffed by eosinophils and macrophages. etiologically, parasitic infection or hypersensitivity reaction for drugs is the cause; however, an idiopathic form also does exist. an infection by helminthes will usually cause mild symptoms, whereas in filarial infections (tropic eosinophilia), the symptoms can be severe (high fever, cough, wheezing, peripheral eosinophilia; suppl. fig. ) [ , ] . in very rare cases, an allergic reaction for fragments of fungi can present with aep (allergic bronchopulmonary mycosis). many drugs can cause eosinophilia and pneumonia such as beclomethasone, bleomycin, carbamazepine, chlorpromazine, chlorpropamide, cromolyn, dilantin, gold salts, nonsteroidal anti-inflammatory drugs, naproxen, nitrofurantoin, penicillin, phenothiazine, propylthiouracil, phenylbutazone, sulfonamide, tetracycline, and clarithromycin, so a proper investigation of treatment protocols is advised (more details are found on www.pneumotox.com). loeffler's syndrome is a self-limiting condition, resolving usually within month. on ct scan, migratory pulmonary infiltrates can be seen, accompanied by a peripheral blood eosinophilia. in some cases, specific causes such as chronic eosinophilic leukemia can be identified; however, in other cases, no underlying etiology can be identified. in acute idiopathic eosinophilic pneumonia, there is acute febrile illness usually of week duration, with myalgia, chest pain, and hypoxemic respiratory failure. on ct scan, alveolar and interstitial infiltrates are seen; in bal, eosinophils increase over %, however, there is no blood eosinophilia. patients rapidly respond to steroid therapy. no cause can be identified. chronic eosinophilic pneumonia is a serious disease that requires a specific treatment. most often affected are middleaged patients, sometimes also young atopic women (suppl. fig. ). the disease starts with an insidious onset with progressive respiratory symptoms. a history of asthma is present in - % of cases. some cases can be attributed to drug toxicity (ampicillin, bleomycin, nitrofurantoin, penicillin, streptomycin, tetracycline, and others); in others, hypersensitivity to fungi has been shown. some cases represent chronic infection with parasites. single cases have been described where chronic eosinophilic pneumonia was associated with cocaine or nickel carbonyl vapor inhalation. the characteristic histologic picture shows dense eosinophilic infiltrations accompanied by macrophages and lymphocytes. there can be eosinophilic abscesses. fibroblast proliferations can be seen focally, which finally results in interstitial fibrosis. hypereosinophilic syndrome is a rare disease of unknown cause [ ] . it is characterized by increased eosinophilic infiltrations in multiple organs (mainly in the heart and central nervous system, rarely in the lungs). fatal cases have been reported mainly caused by restrictive cardiomyopathy. environmentally induced diffuse interstitial lung diseases (excluding conventional pneumoconiosis) there are two forms of gip, one caused by infection such as measles virus (see above), while the other form caused by inhalation of hard metal dust. hard metal is an alloy composed of cobalt, chromium, tungsten, titanium, and a variety of other metals. in experiments, it has been shown that cobalt is the most toxic compound within this alloy, can easily dissolve out, and will subsequently induce giant cell formation of macrophages [ ] [ ] [ ] (fig. ) . following the deposition of metal dust the disease starts with respiratory bronchiolitis; giant cells loaded with compound from this alloy are formed early on. the macrophage accumulation spreads further to the periphery, and finally macrophages and giant cells can be found in alveoli, septa, and peribronchiolar. also dad can be seen in some cases. the disease can present with acute illness, shortness of breath, and severe hypoxia. if the patient survives the acute onset, peribronchial/peribronchiolar fibrosis and fibrosis of alveolar septa results [ ] [ ] [ ] . large scars are usually absent. fig. gip in hard metal disease. note the accumulation of foreign body giant cells containing a dirty brown-black material, in this case identified as tungsten and titanium. many other components of the hard metal (cobalt, nickel, and chromium) alloy can dissolve easily and are, therefore, dissolved by fixation and embedding procedures, so mainly tungsten and titanium compounds remain and can be detected by energy-dispersive x-ray spectroscopy. h&e; magnification, × asbestosis asbestosis is another diffuse fibrosing lung disease, which can present with uip or op morphology. in the early phase, it starts with bronchiolitis and peribronchiolar pneumonia. later on, fibrosis with uip morphology can be the predominant feature. in contrast to uip/ipf, the fibrosis is focal, asymmetric in distribution, and early on can involve central portions of the lung. the diagnostic clue is the demonstration of asbestos bodies together with lung fibrosis. drug-induced ild present with a variety of tissue reaction, most of them already discussed in previous chapters. a common presentation is dad as an acute reaction, often followed by organizing dad, where hyaline membranes can still be recognized, and finally, ending as op and lung fibrosis. other drugs induce nsip-like tissue reactions. the major problem in interpreting drug reaction in the lung is our limited understanding of drug metabolism. some drugs will induce toxic injury of endothelial cells, thus the blood barrier is leaking, and proteins can enter the interstitium and finally the alveolar lumen. here, these proteins will form complexes and, by the action of respiration, hyaline membranes will form. in addition, the exudate from the capillaries will cause a transient edema and this is followed by hypoxia affecting the pneumocytes. this damage will contribute to dad development. later on, op can result. other drugs act on the immune system, forming fig. comparison of different types of fibroblast/ myofibroblast proliferations; a, b fibroblastic focus in uip by h&e and movat stains; by movat stain, the immature collagen fibers are stained green, whereas mature collagen (usually collagen ) stains yellow. c early granulation tissue in op, here bronchiolitis obliterans, d later stage of op; note the proliferating immature blood vessels, quite characteristic in these early phase op. different types of fibrosis in emphysemaassociated lung fibrosis: e scarring with entrapped emphysema blebs and f fibroblastic focus within the wall of an emphysema bleb. magnification; bars, , , , , , and μm, respectively immune complexes either because immunogenic by itself or by complexing with endogenous proteins like a hapten. in these cases, an nsip or lip pattern can result. in these cases, scattered eosinophils are regularly found. granulomas are rarely formed, usually pointing to an underlying immune mechanism. since our understanding is so limited and no systematic experimental investigation has been performed, we still need to rely on databases, summarizing all described drug reactions in a systematic way (www.pneumotox.com) (fig. and suppl. fig. ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the 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toxicity of pure cobalt powder and cobalt-tungsten carbide mixture in rat experimental research into the pathogenesis of cobalt/hard metal lung disease pathologic spectrum and lung dust burden in giant cell interstitial pneumonia (hard metal disease/cobalt pneumonitis): review of cases metal-induced diffuse lung disease is giant cell interstitial pneumonitis synonymous with hard metal lung disease? oxidant-induced lung injury in anticancer therapy naphthalene-induced respiratory tract toxicity: metabolic mechanisms of toxicity pulmonary metabolism of foreign compounds: its role in metabolic activation lung injury: cell-specific bioactivation/deactivation of circulating pneumotoxins cryptogenic organizing pneumonitis during oxaliplatin chemotherapy for colorectal cancer: case report fatal pulmonary toxicity after a single dose of cyclophosphamide pathology of drug-induced lung disease key: cord- - l slziv authors: kraetsch, hans-georg; rascu, astrid; kalden, joachim richard; manger, bernhard title: verlauf und prognostische parameter bei still-syndrom des erwachsenen: eigene erfahrungen und literaturübersicht date: journal: med klin (munich) doi: . /bf sha: doc_id: cord_uid: l slziv □ patients and methods: ten patients with adult onset of still’s disease (aosd) were examined one to nine years after the established diagnosis. clinical symptoms, laboratory parameters and the outcome of the cases are presented and compared to international literature and to yamagushi’s in proposed diagnostic criteria. nine patients were reexamined in our out-patient clinic. the chart of one additional patient, who died month after the initial symptoms was also available for data analysis. retrospectively, it was investigated whether any parameters were predictive for a chronic or severe form of the disease. □ results: one patient died month after the diagnosis was established due to a secondary haemophagozytic syndrome. one patient developed a chronic form of the disease, whereas patients had a chronic-remitting form. six patients presented a self-limiting, shorter than month lasting course of aosd with a restitutio ad integrum. all patients fulfilled the diagnostic criteria of yamagushi et al. three of patients developed a chronic form of aosd, compared to up to % of the patients reported by others. the patient who died was significantly older ( years) than the average age ( , years) of all patients. interestingly, he did not present still’s rash or lymphadenopathy, but rather developed a secondary hemophagocytic syndrome with an excessive hyperferritinaemia. □ conclusion: predicting parameters for a chronic course of the disease could not be found. each patient’s diagnosis retrospectively could be confirmed using the yamagushi’s diagnostic criteria. thus, these criteria appear helpful in the difficult diagnostic process of this disease. beschfieb bywaters erwachsene patienten mit symptomen ~ihnlich denen einer juvenilen chronischen polyarthritis, die frª als ,,still's disease" bezeichnet wurde [ ] . intemational gilt daher heute die bezeichnung ,,adult onset of still's disease" (aosd). die erkrankung ist weltweit verbreitet; vorwiegend sind junge erwachsene zwischen und jahren betroffen. frauen und m~inner erkranken zu gleichen teilen, eine famili~ire h iufung wurde bisher nicht festgestellt [ ] . die fiitiologie der erkrankung ist unldar. bei mehreren patienten wurden rubellaviren isoliert, in einzelfiillen ebstein-barr-, zytomegalie-, mumps-, parainfluenza-, koxsackievirus, yersinia enterocolitica und mycoplasma pneuinoniae [ , , , . eine eindeutige infekti se ursache konnte jedoch bisher nicht bewiesen werden. eile genetische pr~idisposition wurde ron mehreren autoren beobachtet, einheitliche daten existieren hierzu bis heute nicht [ ] . es sind sowohl selbsdimitierende, intemfittierende als auch chronische bis hin zu t dlichen verl~iufen besch¡ worden [ , t ] . monozyldische verl~iufe erstrecken sich in der fzegel ª einen zeitraum von einem jahr. bei den intemlittierenden verlfiufen kommt es gelegentlich zu kompletten remissionen, um dann wieder mit den typischen symptomen zu exazerbieren, meistens weniger stark als zu beginn der erkrankung und ª einen kª zeitraum als in der ersten phase. ungefahr bis % der bekannten f~ille weisen einen chronischen verlaufauf, etwa bis % sind selbsdimitierend oder intemaittierend [ ] . eine schwangerschaft scheint die erkrankungsaktivit~it ira gegensatz zu anderen entzª rheumatischen erkrankungen nicht zu beeinflussen [l ]. letale verlfiu~~ sind selten. bisher sind letale krankheitsver-l~iufe bei aosd ver ffendicht worden. h~iufigste todesursachen waren hierbei infektionen, amyloidose oder leberversagen [ ] . auberdem k nnen ards, herzversagen, status epilepticus, dic, thrombotisch-thrombozytopenische purpura und sekund~ires himophagozytisches syndrom [ , , , ] chart ofone additional patient, who died month after the initial symptoms was also available for data analysis. retrospectively, it was investigated whe'ther any parameters were predictive for a chronic or severe forro of the disease. results: one patient died month after the diagnosis was established due to a secondary haemophagozytic syndrome. one patient developed a chronic form of the disease, whereas patienta hada chronic-remitting form. six patients presented a self-limiting, shorter than month lasting course ofaosd with a restitutio ad integrum. all patienta fulfilled the diagnostic criteria ofyamagushi et al. three of parienta developed a chronic form ofaosd, c£ to up to % of the patienta reported by others. the patient who died was significan@ older ( years) than the average age ( , years) ofall parienta. interestingly, he did not present still's rash or lymphadenopathy, but rather developed a secondary hemophagocytic syndrome with an excessive hypeffer¡ : conelusion: predicting parameters for a chronic course of the disease could not be found. each patient's diagnosis retrospectively could be confirmed using the yamagushi's diagnostic criteria. thus, these criteria appear helpful in the difrcult diagnostic process ofthis disease. rey words: aosd adult onset of still's disease prognostic critefia course of disease outcome review of the literature case reports med. klin ;d zum zeitpunkt der diagnosestellung. [ , , [ ] [ ] [ ] [ ] ] . es sind vorwiegend junge erwachsene betroflen, obwohl auch ~iltere menschen an aosd erkranken k nnen [ ] . typisch ftir das aosd ist das auftreten von hohem fieber (> ~ in der ikegel mit einer fieberzacke gegen abend oder nachts, woraufsich die temperatur wieder normalisiert. in % der eille sinkt das fieber allerdings nicht auf normale temperaturen [ ] . aue zehn patienten boten dieses symptom. sieben unserer patienten zeigten das charaktefistische lachsrosafarbene, makul~ire oder maku-lopapul~ire exanthem [ , ] . es tritt meist gleichzeitig nfit den abendlichen fieberspitzen auf. betroffen sind vor allem der stamm und die proximalen extremiditen (abbildung , patient nr. ), gelegentlich aber auch das gesicht [ ] . bei einem d¡ der f~iuejucken die hauteffloreszenzen, so dab es leicht zu verwechslungen mit einer medikamenten-auergie kommen kann. der hautausschlag tritt gelegendich auch an stellen mechanischer irritation auf. abbildung zeigt dieses koebner-ph~inomen bei einem unserer patienten (nr. ). ein viertel der patienten in der literatur [ ] und zwei ron unseren zehn patienten beklagten haarausfau. arthralgien sind nahezu immer vorhanden. alle zehn ron uns behandelten patienten gaben arthralgien an. die intensidit der gelenkbeschwerden korreliert dabei oft mit zunehmendern fieber. nur % der patienten zeigen in ihrem krankheitsverlauf keine gelenkbeteiligung [ ] . betroffen waren bei unseren paª sowohl grobe gelenke, wie hª knie-und sprunggelenke, als auch kleine gelenke, zum beispiel metakarpalund metatarsalgelenke. gelegentlich bestehen mildere gelenkbeschwerden, die durch andere, dramatischere klinische probleme ª werden. das muster des gelenkbefalls ~hnelt zum teil dem der chronischen polyarthriª wobeijedoch auffallend h~iufig auch die distalen interphalangealgelenke betroflen sind [ , ] [ ] . in vielen f~illen wird eine zervikale lymphadenopathie beobachtet. sie wird meist begleitet von einer exsudativen phanryngitis [ ] . dieses oft schon zu beginn der erkrankung stehende symptom wurde bei acht unserer patienten beobachtet. die hffufig auftretende hepatosplenomegalie ist ffir ge-w hnlich nur mild ausgeprfigt. in einzelfiillen wurde von schwerer leberbe-teili~mg berichtet. gelegentlich treten schmerzen im abdominalbereich auf,, die teilweise in ihrer intensitst bis zum bild des ,,akuten abdomens" gehen k nnen [ , ] . eine schwere hepatische beteili~mg trat bei unserem sp~iter verstorbenen patienten ira p,.ahmen eines hffmophagozytischen syndroms auf[ ], auch dies schon als seltene und lebensbedrohliche komplikation des aosd bekannt. pleurale schmerzen gehen zum teil mit pleura-und perikardergª einher. nicht ungew hnlich ist eine aseptische pneumonitis [ ] . selten kommt es zur ausbildung eines ar.ds, chronisch obstruktiver lungenerkrankung, perikardtamponade oder myokarditis [ , , ] [ , , . hn i'zoutinelabor zeigt sich die ffir systemische entzª prozesse typische konstellation (zahlen fª unser patientengt, t in klammern): regelhaft eine beschleunigte bks ( /li)), % der hille in der literatur wiesen erh hte thrombozytenzahlen ( / ), fiber % eine entziindungsaniimie ( /li)) und ª % eine leukozytose auf ( / ). bei % der patienten sind erh hte transaminasenwerte festzustellen ( / ), die allerdings zum teil auch als nebeneffekt der jeweiligen therapie, zum beispiel mit nichtsteroidalen antiphlonstika, gelten mª ira gegensatz zu anderen entzª chen erkrankungen zeigen patienten mit aosd zum teil extrem gesteigerte ferritinwerte ( / l ) [ , ] . ob dieser parameter eine aussage ª den verlaufder erkrankung zulfilk, mufl durch weitere beobachtungen gekl~irt werden. zirkulierende immunkomplexe werden bei bis zu einem drittel der patienten gefunden [ ] . zu beginn der erkrankung zeigen sich im fz ntgenbild lediglich unspezifische ver~indemngen wie weichteilschwellung und gelenknahe entkalkungen, bei chronischem verlaufeine verminderung der knorpeldicke, erosionen und zystische verfinderungen (siehe abbildung ). eine verschmfflerung des gelenkspaltes in karpometakarpaloder interkarpalgelenken mit sp~iterer kn cherner ankylosierung gilt als eine ftir das aosd charakteristische radiologische verffnderung [ ] , die in dieser forro bei unseren patienten nicht zu beobachten war. die differentialdiagnose des aosd ist schwierig. zu beginn bestehen hfiufig lediglich ein reduzierter allgemeinzustand mit gewichtsverlust, die cha-rakte¡ abends auftretenden fieberschª ohne nachweis eines infekti sen oder malignen geschehens, begleitet von einer aseptischen pharyngitis. der ,,still's r.ash" kann zu verwechslungen mit einer medikamentenallergie fª insbesondere dann, wenn kurz zuvor eine probatorische antibiotische therapie eingeleitet wurde. da die diagnose aosd eine aus-schlubdiagnose ist und die symptome an cine viruserkrankung erinnern, mul eine austiihrliche diagnostik vor einleitung einer immunsuppressiven therapie erfolgen. zur etablierung der diagnose haben sich fª unser krankengut die kriterien von yamagushi et al. als hilfi'eich erwiesen. als prfidiktoren ffir eine schlechtere prognose haben sich eine fr he polyarthritis sowie eine arthritis der proximalen gelenke erwiesen. f~ille mit diesen manifestationen zeigen hfiufiger einen chronischen verlauf mit spiiter einsetzender kemission, wie auch die auswertung der datcn unserer patienten zeigen konnte. die erstmanifestation in der kindheit, die beteiligung des htiftgelenks sowie die einnahme von corticosteroiden ª zwei jahre scheinen ncgative prognostische faktoren zu sein. ob h heres alter und exzessiv gesteigerte ferritinwerte -bei unserem sp~iter verstorbenen j'ahrigen patienten lag der fer¡ etwa vier w ochen vor dem tod bei maximal ng/ml [ -signifikant mit einem er-h hten fzisiko assoziiert sin& mul durch weitere untersuchungen gekl~irt werden. die fehlende dermale manifestation bei diesem patienten stimmt mit beobachtungen aus der literatur ª ein, nach denen ein fehlendes exanthem mit einer schtechteren prognose korreliert ist. darª hinaus war bei diesem patienten die ansonsten hfiufig auftretende lymphadenopathie nicht zu beobachten. vergleicht man klinisches bild und laborchemische ver~inderungen be| unseren patienten mit chronischem und chronisch-intermittierendem verlauf mit entsprechenden befunden der patienten mit akutem verlauf, so lassen sich keine weiteren typischen, ffir einen bestimmten verlauf pr~idisponierenden parameter entdecken. festgestellt werden kann lediglich, dafl alle drei patienten mit insgesamt l~ngerem verlaufinitial eine arth¡ aufwiesen, aber nur drei von sechs patienten mit akutem, selbstlimitierendem verlaufdieses symptom zeigten. schon in anderen untersuchungen konnte das frª vorliegen einer arthritis der groben gelenke oder einer polyarth¡ als prognostisch ungª faktor ausgemacht werden [ , ] . literatur. rr carpal alterations in adult-onset still's disease.juvenile chronic art hritis and adult onset rheumatoid arthritis: comparative study cerebral involvement in adult onset still's disease. clin, p-heum. (i still's disease in the adult. ann. rheum. l)is l)ecaux: hyperferritinemia in adult onset still's disease and the hemophagocytic syndrorne cooperstein: adult-onset still's disease. clinical course and outcome. arthr. and l.kheum fulminant hepatic failure in adult onset stil|'s diease adult~onsr still's disease: twenty-year follo,v up and further studies of patients with active disease dieppe: lkheumatology'. mosby-year book europe limited adult onset still's disease: report oftwo cases and literatur review still's disease associated with co~ackie infection and haemophagocytic syndrome adult onset still's disease and pregnancy sensorineural hearing loss in adult onset still's disease cardiac tamponade: ah unusual feature ofadults onset still's disease adult stin's disease and inflammatory myositis davson: stil|'s disease and the virus-associated haemophagocytic syndrome aduk still's disease: keview of cases flora the literature adult still's disease: a multicenter survey ofjapenese patients adult still's disr manifr disease course and ou tr in patients adultonset stili's disease. experience in patients and literatute review with emphasis on organ failure myocarditis in adult still's disease evaluation ofserum fer¡ asa marker for adult still's disease activity vasquez: still's disease in a -year old man semm fcr¡ and isofer¡ are tools for diagnosis of active adult still's disease adult still's disease and mesangial glomerulonephfitis, lkeport oftwo cases adult-onset still's disease; clinical and laboratory features, treatment and progress of tases, quart adult onset still's disease and viral infections prdiminary cfiteria for classification ofadult still's disease key: cord- -mktbkn u authors: harris, mallory j.; hay, simon i.; drake, john m. title: early warning signals of malaria resurgence in kericho, kenya date: - - journal: biol lett doi: . /rsbl. . sha: doc_id: cord_uid: mktbkn u campaigns to eliminate infectious diseases could be greatly aided by methods for providing early warning signals of resurgence. theory predicts that as a disease transmission system undergoes a transition from stability at the disease-free equilibrium to sustained transmission, it will exhibit characteristic behaviours known as critical slowing down, referring to the speed at which fluctuations in the number of cases are dampened, for instance the extinction of a local transmission chain after infection from an imported case. these phenomena include increases in several summary statistics, including lag- autocorrelation, variance and the first difference of variance. here, we report the first empirical test of this prediction during the resurgence of malaria in kericho, kenya. for summary statistics, we measured the approach to criticality in a rolling window to quantify the size of effect and directions. nine of the statistics increased as predicted and variance, the first difference of variance, autocovariance, lag- autocorrelation and decay time returned early warning signals of critical slowing down based on permutation tests. these results show that time series of disease incidence collected through ordinary surveillance activities may exhibit characteristic signatures prior to an outbreak, a phenomenon that may be quite general among infectious disease systems. despite modern advances in disease control, the world health organization reports that nearly one-third of deaths in developing countries are due to infectious diseases [ ] . so far, only one human global disease eradication campaign has been successful (smallpox). especially in low-resource settings, premature abandonment of disease elimination programmes as other health priorities compete for limited resources may result in reestablishment, even when the disease is on the brink of elimination. for instance, in sri lanka, malaria incidence climbed from to cases between and [ , ] . indeed, malaria resurgence occurred repeatedly throughout the last century. a systematic review identified resurgence events worldwide between and , attributed largely to the weakening of malaria control programmes, increasing the potential for transmission and evolution of drug and insecticide resistance [ ] . exactly those conditions are found for other diseases now on the cusp of global eradication (e.g. polio [ ] , dracunculiasis [ ] and human africa trypanosomiasis [ ] ). it follows that effective disease elimination might be guided by techniques for differentiating between efforts that will lead to stable disease elimination and scenarios where cases are diminished without a corresponding shift in the system's stability. few methods are available that are appropriate to the relevant problems of limited data and non-stationary disease dynamics at these transition points. forecasting methods that depend directly on raw case incidence have been proposed for infectious diseases, but techniques such as the moving epidemic method, which establishes thresholds for identifying the start of an epidemic, rely on regular, seasonal trends and are therefore not applicable for predicting the resurgence of vector-borne diseases [ ] . recent theory suggests that critical transitions in infectious disease systems may be detected using nonparametric statistical methods [ , ] . resurgence is a type of critical transition, wherein the stable state of the disease system shifts from the disease-free equilibrium (where cases are due to imported infections and subcritical stuttering chains of local transmission) to self-sustained transmission. in systems where the critical transition is driven by a gradual shift in underlying disease parameters toward conditions favouring disease transmission, any outbreak is preceded both by an approach to criticality, which ends when transmission is exactly maintained by an effective reproduction number of one, and a delay as susceptible individuals accumulate in the population [ ] . during the approach to criticality, the transmission system is expected to exhibit slower returns to mean (i.e. longer and longer chains of subcritical transmission) following the introduction of cases, which is a manifestation of critical slowing down [ ] . the mathematical theory of critical slowing down has recently been worked out in detail for a number of specific disease systems [ ] [ ] [ ] ] . general predictions of the theory are that cases will exhibit an increase in several summary statistics (table ) as the critical point is approached [ , , ] . these early warning signals would precede the increase in cases at the beginning of the supercritical period that triggers an alert in the moving epidemic method [ ] . this paper reports the first test of this theory on real rather than simulated data for a vector-borne disease. here, we show the evidence of critical slowing down in a time series of monthly plasmodium falciparum malaria case incidence leading up to a resurgence in kericho, kenya. the april resurgence event has been attributed to the parasite's development of resistance to chloroquine, the anti-malarial drug administered to workers as a control strategy on these plantations [ ] . thus, the proposed mechanism of the disease's return is a gradual underlying process--the increasing frequency of resistance in the population--rather than a sudden event like importation. in five of the indicators, we detect increases that serve as early warning signals of malaria resurgence during the approach to criticality. theory suggests this phenomenon may be a broadly applicable one. data were obtained from two relatively isolated tea plantations for through . the company-operated hospital provided case numbers based on stained blood smears from patients with suspected malaria [ , ] . malaria reached epidemic levels in these areas during world war ii, carried by soldiers returning from ethiopia. in march , a combination of proguanil prophylaxis and ddt house spraying brought malaria incidence down, and transmission was mostly controlled via the antimalarial medication chloroquine until the april outbreak, when a spike in the number of cases exceeded all prior months by two and a half fold (figure ) [ ] . operationally, we therefore define april as the boundary between subcritical and supercritical transmission. it is known that chloroquine remained fully effective against p. falciparum through december [ ] . thus, we considered december to april to be the period during which the system approached criticality. we performed a sensitivity analysis to determine the effect of alternative endpoints for this window (electronic supplementary material, files s and s ). the theory of critical slowing down developed by o'regan et al. [ ] predicts that the indicators of critical slowing down should have increased during this time (table ) . table . names and equations for summary statistics used in these analyses. the correlation coefficient and p-value corresponding to testing between december and april is also given for each indicator. royalsocietypublishing.org/journal/rsbl biol. lett. : to test this prediction, from december to april , we calculated each of these leading indicators over a rolling window (i.e. the statistics were repeatedly calculated within a fixed size temporal subsample of the data, shifting the window by one month for each computation) (table ). to better distinguish the signal of critical slowing down from background noise and periodic trends, we preprocessed the raw case data by detrending with a gaussian kernel. the early warning signal itself was calculated so that for a moving window centred at time index i, a corresponding kernel weight w ij was assigned to the value of the statistic (mean, variance, etc.) at index j and then normalized so that n i ¼ p j w ij . thus, for the time i and statistic f, the rolling window estimator m i was defined as where the only tuning parameter is the bandwidth b = (here set to months, exceeding the period of malaria's seasonality to reduce any influence of seasonality on the overall trend). a sensitivity analysis shows that statistical results are relatively insensitive to the choice of bandwidth (electronic supplementary material, file s ). all analyses were conducted in r using the spaero package [ ] , a freely available package that provides a wide range of methods for investigating critical slowing down in time series. data and code for reproducing analyses reported in this paper are available online at https://github.com/mjharris / kericho-ews.git. the performance of each indicator as an early warning signal was then assessed by performing significance tests on the rolling window statistics as follows. first, we determined the trend in each indicator over time by calculating kendall's correlation coefficient, τ, between the calculated rolling window statistic at each time and the time index i. there are other statistics one could use (e.g. spearman's correlation coefficient), but the use of kendall's τ has become standard in the literature on anticipating critical transitions and our results are robust to choice of correlation coefficient (electronic supplementary material, file s ) [ ] [ ] [ ] [ ] . the sign of τ indicates the direction of change, while the magnitude corresponds to the strength of the temporal trend. unlike simulation studies, where an unlimited number of examples are available to investigate detectability, analysis based on data requires a means to assign statistical significance without access to replication. we used the following permutation approach to assess the significance of the estimated correlations. we first generated permutations of case incidence during the approach to criticality (december to april ), thus preserving the general features of the original data (e.g. amplitudes of fluctuations) while eliminating underlying patterns of serial dependence. we then performed rolling window analyses on the permuted data to generate a null distribution of correlation coefficients for each indicator. the false positive rates of the tests were estimated by finding the proportion of these permutations with a value for τ more extreme than that calculated for the kericho data. the resulting p-values quantify the reliability of their corresponding statistics as early warning signals. in an applied surveillance setting, time series data would be updated regularly. to reflect this, we first included only data collected at least months prior to the critical transition (april ) and repeatedly added an additional month of data, recalculating p-values for each month. the test values for all indicators except for kurtosis and skewness lie in the right tails of their null distributions, consistent with the prediction that critical slowing down results in an increase in these statistics (electronic supplementary material, file s ). visually, it appears that most of the indicators exhibit intermittent periods of both upward and downward trend. the first difference of variance was the first indicator to return a low p-value (about months prior to the critical transition), although the p-values for this indicator increased after a few months, remaining high for the remainder of the study (figure ). the p-values for increases in decay time and lag- autocorrelation both dropped rapidly months prior to the critical transition. the p-values for increases in autocovariance and variance also rapidly decreased months prior to the critical transition and dropped below . fifteen and four months prior to the critical transition, respectively. these results show that simple summary statistics may serve as early warning signals of disease resurgence. nine of the statistical indicators increased during the approach to criticality, as predicted by the theory of critical slowing down, although only the increases in half of these indicators corresponded to low p-values (below . ) at any point in the months preceding the critical transition. the first difference of variance was the first statistic to return early warning signals, followed by autocorrelation and decay time, then autocovariance, then variance. in the months immediately preceding the critical transition, the p-values for increases in the latter four declined sharply. this suggests that certain royalsocietypublishing.org/journal/rsbl biol. lett. : indicators may be more reliable early warning signals of critical slowing down at different times during the approach to criticality. the finding that variance and lag- autocorrelation are relatively robust early warning indicators is consistent with findings in simulation studies [ , , ] and in other (non-disease) natural systems [ , ] . our conclusion is that the statistical signatures of critical slowing down may be detected in ordinary surveillance data prior to disease resurgence and therefore should be incorporated into monitoring programmes and decision support for proactive response. the statistical features of the lag between the critical transition and disease resurgence, particularly in vector-borne disease systems, are not yet well understood [ ] . there are qualifications to this conclusion. our methods assume that the system is forced through a critical transition due to some slow-moving underlying mechanism, causing one or more disease transmission parameters to vary over time. the kericho example meets these conditions, drawing from a nearly closed system without major human immigration, and undergoing a critical transition due to the gradual development of parasite resistance to chloroquine, with drug-resistant parasites becoming prevalent in the areas of kenya starting in the early s [ ] . vector-borne disease resurgence provoked by imported cases or a sudden change in the efficacy or administration of control measures is not expected to be detected in this manner. however, the accumulation of insecticide and pesticide resistance remains leading causes of malaria resurgence [ ] . moreover, in many areas, slow environmental forcing may drive additional changes in transmission, for instance as climate change shifts the geographic region suitable for malaria transmission [ ] . furthermore, this framework could be used to predict a critical transition toward elimination, provided that the system undergoes a gradual approach to criticality, for instance through increasing insecticide and bed net usage [ , ] . thus, we think monitoring programmes based on critical slowing down might be used to inform intervention strategies by helping to distinguish between true changes in system stability and a temporary decrease in cases. although this study provides evidence that rolling window statistics may be used to anticipate disease resurgence, applications may be hampered by data limitations [ ] . for instance, under-reporting, especially failure to detect asymptomatic carriers, may corrupt disease surveillance data. in our study, since the population eligible for healthcare remained constant over the course of the time series, inaccurate case reports are unlikely to vary in any systematic way, justifying our decision to use case reporting as a measure of incidence [ ] . moreover, theoretical studies suggest that early warning indicators based on critical slowing down are surprisingly robust to imperfect reporting [ , , ] . under intervention strategies that change case recovery rate, variance is expected to be robust to underreporting, whereas autocorrelation is expected to be sensitive to under-reporting [ ] . further theoretical studies are needed on the indicators of critical slowing down, particularly decay time, in the context of vector-borne disease systems. investment in improved surveillance could alleviate the risk of costly resurgence responses. a related problem is that the models of the disease system approaching a critical transition used to identify potential early warning signals consider the total number of infectious individuals, whereas reported data (such as studied here) reflect the number of cases reported in a sampling interval (here, monthly hospital reports) [ , ] . in our case, since the resolution of the time series is a month, exceeding the initial infectious period of malaria, and the system slowly approaches resurgence, new cases are approximately equivalent to the total infectious individuals at a time point. but, such convenient alignment of case reporting and disease transmission may not be obtained in other situations. we recommend more extensive investigation of critical slowing down across infectious disease systems, particularly to identify which indicators most reliably predict critical slowing down and to analyse the effects of bandwidth and notional month of transition on early warning signal detection. additional studies could also inform the selection of detection thresholds based on p-value and correction for testing based on multiple statistics that are not independent. data requirements for such testing include regularly reported measures of incidence for tens to hundreds of infection generations, occurrence of outbreaks and a method for determining when the system reached the critical point. this last criterion could be satisfied either through parametric modelling of the data themselves or from independent information. in conclusion, the results of this study support the use of rolling window calculations of indicators of critical slowing down in disease surveillance [ , , , , ] . this study also demonstrates that early warning signals can be detected in monthly incidence data several years prior to a critical transition. the algorithmic nature of these methods points to the possibility that automated, model-independent disease forecasting systems could be developed for application directly to clinical data to assist in the detection of trends and predict disease resurgence, improving the efficiency of disease control campaigns and contributing to a welcome shift from reactive to preemptive response. for instance, we envision early warning systems that routinely monitor not only changes in the number of reported cases but also changes in these summary statistics and trigger an alert when a threshold is reached [ , , , , ] . the detection of early warning signals could be followed by efforts to more specifically identify whether underlying features of the system are shifting (e.g. the development of drug resistance). in sum, being able to predict critical transitions might make malaria control (and other vertical eradication programmes) more efficient by prompting preemptive action prior to resurgence. data accessibility. code used in the analyses and to generate figures and the corresponding data are available at https://github.com/mjharris / kericho-ews.git global, regional, and national age-sex specific mortality for causes of death, - : a systematic analysis for the global burden of disease study malaria resurgence: a systematic review and assessment of its causes resurgent vector-borne diseases as a global health problem progress toward polio eradication-worldwide slaying little dragons: the impact of the guinea worm eradication program on royalsocietypublishing.org/journal/rsbl assessing strategies against gambiense sleeping sickness through mathematical modeling influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method leading indicators of mosquito-borne disease elimination monitoring the path to the elimination of infectious diseases waiting time to infectious disease emergence critical transitions in nature and society anticipating epidemic transitions with imperfect data modelling fluctuation populations forecasting infectious disease emergence subject to seasonal forcing malaria in kenya's western highlands meteorologic influences on plasmodium falciparum malaria in the highland tea estates of kericho data from: code to accompany evidence of critical slowing down prior to malaria resurgence in kericho spaero: software for project aero. . pananos ad critical dynamics in population vaccinating behavior early warning of climate tipping points can we detect ecosystem critical transitions and signals of changing resilience from paleo-ecological records? ecosphere early warning signals of ecological transitions: methods for spatial patterns changing response to chloroquine of plasmodium falciparum in saradidi resurgence of malaria in the usambara mountains, tanzania, an epidemic of drug-resistant parasites mapping physiological suitability limits for malaria in africa under climate change. vector borne zoonotic dis disentangling reporting and disease transmission acknowledgements. the malaria inpatient data were supplied by the chief physician dr walter odonde, unilever tea kenya ltd and updated to by mr geoffrey kores, the records officer. some of the computing for this project was performed on the sherlock cluster. we would like to thank stanford university and the stanford research computing center for providing computational resources and support that contributed to these research results. key: cord- -qw e cof authors: cantas, leon; suer, kaya title: review: the important bacterial zoonoses in “one health” concept date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: qw e cof an infectious disease that is transmitted from animals to humans, sometimes by a vector, is called zoonosis. the focus of this review article is on the most common emerging and re-emerging bacterial zoonotic diseases. the role of “one health” approach, public health education, and some measures that can be taken to prevent zoonotic bacterial infections are discussed. key points: a zoonotic bacterial disease is a disease that can be very commonly transmitted between animals and humans. global climate changes, overuse of antimicrobials in medicine, more intensified farm settings, and closer interactions with animals facilitate emergence or re-emergence of bacterial zoonotic infections. the global “one health” approach, which requires interdisciplinary collaborations and communications in all aspects of health care for humans, animals, and the environment, will support public health in general. new strategies for continuous dissemination of multidisciplinary research findings related to zoonotic bacterial diseases are hence needed. zoonotic diseases are those infections that can be transmitted between animals and humans with or without vectors. there are approximately pathogens, which are known to infect humans and % of these cause zoonotic diseases ( ) . the unique dynamic interaction between the humans, animals, and pathogens, sharing the same environment should be considered within the "one health" approach, which dates back to ancient times of hippocrates ( , ) . bacterial zoonotic diseases can be transferred from animals to humans in many ways ( ): (i) the transfer may occur through animal bites and scratches ( ) ; (ii) zoonotic bacteria originating from food animals can reach people through direct fecal oral route, contaminated animal food products, improper food handling, and inadequate cooking ( ) ( ) ( ) ; (iii) farmers and animal health workers (i.e., veterinarians) are at increased risk of exposure to certain zoonotic pathogens and they may catch zoonotic bacteria; they could also become carriers of the zoonotic bacteria that can be spread to other humans in the community ( ) ; (iv) vectors, frequently arthropods, such as mosquitoes, ticks, fleas, and lice can actively or passively transmit bacterial zoonotic diseases to humans. ( ) ; (v) soil and water recourses, which are contaminated with manure contains a great variety of zoonotic bacteria, creating a great risk for zoonotic bugs and immense pool of resistance genes that are available for transfer of bacteria that cause human diseases ( , ) . bacterial zoonotic infections are one of the zoonotic diseases, which can, in particular, re-emerge after they are considered to be eradicated or under control. the development of antimicrobial resistance due to over-/misuse of antibiotics is also a globally increasing public health problem. these diseases have a negative impact on travel, commerce, and economies worldwide. in most industrialized countries, antibiotic resistant zoonotic bacterial diseases are of particular importance for at-risk groups such as young, old, pregnant, and immune-compromised individuals ( ) . almost years ago, prior to application of hygiene rules and discovery of neither vaccines nor antibiotics, some bacterial zoonotic diseases such as bovine tuberculosis, bubonic plague, and glanders caused millions of human deaths. the spread and importance of some bacterial zoonoses are currently globally increasing. that is precisely why most of the developing countries are sparing more resources for a better screening of animal products and bacterial reservoirs or vectors for an optimal preventative public health service ( ) . improvements in surveillance and diagnostics have caused increased recognition of emerging zoonotic diseases. herein, changes in our lifestyles and closer contacts with animals have escalated or caused the re-emergence of some bacterial infections. some studies lately have revealed that people have never been exposed to bacterial zoonotic infection risks as high as this before ( ) . it is probably due to closer contact with adopted small animals, which are accepted and treated as a family member in houses. on the other hand, more intensified animal farms, which have a crucial role in the food supply, are still one of the greatest sources of food-borne bacterial zoonotic pathogens in today's growing world ( , ) . people who have closer contact with large numbers of animals such as farmers, abattoir workers, zoo/pet-shop workers, and veterinarians are at a higher risk of contracting a zoonotic disease. members of the wider community are also at risk from those zoonoses that can be transmitted by family pets. the immune-suppressed people are especially at high risk for infection with zoonotic bacterial diseases. people can be either temporarily immuno-suppressed owing to pregnancy, infant age, or long-term immuno-suppressed as a result of cancer treatment or organ transplant, diabetes, alcoholism or an infectious disease (i.e., aids). this manuscript reviews the most common bacterial zoonoses and practical control measures against them. companion animals are increasingly treated as family members, and pets have many bacteria that may infect their owners. the human population of the european union (eu) was approximately million in . the number of pet owning households was estimated at around million in . the most commonly suffered zoonotic bacterial infections in humans are transmitted via animal bites and scratches. various dog breeds have been characterized for their role in killing dog bite attacks, such as pit bull breeds, malamutes, chows, rottweiler, huskies, german shepherds, and wolf hybrids ( ) ( ) ( ) . in usa, pit bull breeds accounted for almost half of the dog bite-related zoonotic infections, three times more than german shepherds ( ) . the oral cavity of healthy dogs and cats contains hundreds of different pathogenic bacteria including pasteurella sp. ( ) . only % of dog bites get infected overall compared with % in cats. there are times higher pasteurella multocida infection risks after a cat bite than a dog bite ( , ) . p. multocida infected bite wounds appear usually within h. it is estimated that approximately % of animal bites or scratches get infected in humans ( ) . bacterial culturing from pet bite infections in humans is found to be smilar to the oral microbiota of the pets. infections in dog bite wounds are usually dominated by aerobic bugs: p. multocida ( %), alpha-hemolytic streptococcus ( %), staphylococcus ( %), neisseria ( %), and corynebacterium ( %). however, following anaerobic bacteria are also isolated from infected wounds: fusobacterium nucleatum ( %), prevotella heparinolytica ( %), propionibacterium acnes ( %), prevotella intermedia ( %), and peptostreptococcus anaerobius ( %) ( ) . normal human skin bacteria or other environmental microorganisms are scarcely isolated from infected wounds in bitten person ( ) ( ) ( ) . usually, infection occurs within - h after the animal attack, with variable pain on the site of the injury. the cellulitis might be followed by discharge that contains pus, which can sometimes be foul-smelling. immuno-suppressed patients with diabetes or liver dysfunction are frequently predisposed to develop serious infections after animal bites. in those cases, they may develop bacteremia faster and pass away in a shorter period of time ( ) . a penetrating bite close to the joints and bones may cause septic arthritis and osteomyelitis. knowing the microbial composition of dental plaque biofilm formation in pets' mouth is a key factor in wound chronicity in humans ( , ) . cat-scratch disease is a clinical syndrome that has been reported in people for over years. yet, the etiological agent bartonella henselae, which was transmitted by cat scratches and bites, was only identified in ( ) . however, contact with cat saliva on broken skin or sclera can also cause bartonellosis. a person who has had a cat scratch may show papules and pustules at the site of injury (the first initial sign). the disease may progress with a chronic non-healing wound, fever (sometimes), weak regional lymph circulation, and abscession. cat owners and veterinarians are most at risk ( ) . systematic medical treatment is usually needed in people with suppressed immune systems. otherwise, encephalopathy, osteomyelitis, and granulomatous conjunctivitis might develop. horses and humans have always shared a close relationship due to recreation, sporting, and occupational reasons, for over thousands of years. in europe, the number of horses per capita remained relatively stable during the past decade. germany and great britain have the largest horse populations in the eu, whereas sweden has the highest number of horses per capita. the frequency of infected horse bite wounds is estimated to be - % in europe ( , ) . however, it has been roughly estimated that the horse bites account for as high as % of overall animal bites in turkey, which comes after dog bites ( %) ( ) . more extensive muscle damage may develop in most of the horse attacks, which is different from small animal bites. a mixture of aerobic and anaerobic organisms has been isolated from horse bites in humans, which are frequently dominated by actinobacillus lignieresii ( , ) . escherichia coli and bacteroides species have also been isolated from foul-smelling infections and pus drainage after horse bites in humans ( ) . infectious diarrhea in companion animals is caused by salmonella sp., escherichia coli, shigella sp., and campylobacter sp. can also be transmitted to people through fecal oral route. it is difficult to estimate the distribution of these ubiquitous microorganisms. but it is known that they can be isolated from many healthy animals, which can be shed in their feces for long periods of time. campylobacteriosis were the most frequently reported zoonotic bacterial diseases in among the eu member countries in humans ( ) . like many other enteropathogens, they can cause gastroenteritis (diarrhea, vomiting), headaches, and depression, sometimes even leading to death. it is obvious that raw food diets for pets dramatically increase the risk of human exposure to such zoonotic bacterial enteropathogens, which cause gastrointestinal diseases. although pet birds, also called songbirds (e.g., canaries, finches, sparrows) and psittaciformes (e.g., parrots, parakeets, budgerigars, love birds) are a small fraction of adopted pets, they are widely popular in europe and they are potential carriers of zoonotic diseases ( ) . some of them could have an important impact on human health, such as chlamydophilosis ( ) , campylobacteriosis ( ) , and salmonellosis ( ) . parrot fever (chlamydophilosis), which is caused by intracellular bacteria, chlamydia psittaci, lives within the frontiers in public health | infectious diseases respiratory system of birds. inhalation of dust, dander, and nasal secretions of infected birds is the main way of transmission to humans ( , ) . the mild to severe flu-like illnesses may develop and infected people might be misdiagnosed as influenza. there is unfortunately a lack of quantitative research into the antimicrobial susceptibility of bacterial zoonotic organisms isolated from bite/scratch wounds or companion animal associated gastroenteritis. zambori et al. ( ) revealed an increased prevalence of drug resistance in animal bite isolates from people. furthermore, methicillin-resistant staphylococcus aureus (mrsa) or extended-spectrum beta-lactamases (esbl) producing enterobacteriaceae, which are known as nosocomial infections have been frequently isolated in companion animals ( ) , including horses ( ) . it might be one of the main reasons for the rising prevalence of these potential zoonotic pathogens in human clinical samples. food producing animals in stock has reached a total of more than million (cattle, pigs, sheep, goats, and chicken) living on farms in europe (see text footnote ). it has been demonstrated that farm animals are reservoirs of many zoonotic pathogens to humans ( , ) . however, annually, a large amount of drugs are being used worldwide to sufficient quantities of food to feed a rapidly growing world human population ( ) ( ) ( ) ( ) . the farm animals consume worldwide approximately eight million kilograms of antibiotics annually ( % of which is used for non-therapeutic purposes such as growth promotion; forbidden in the eu from january , and disease prevention) compared with only approximately one million kilogram per year used in human medicine ( ) . antibiotics are routinely fed to livestock as growth promoters to increase profits and to ward off potential bacterial infections in the stressed and crowded livestock factory environment ( ) ( ) ( ) ( ) ( ) . despite large differences in methodology, most results demonstrate that not so long after the introduction of an antibiotic in veterinary practice, resistance in pathogenic zoonotic bacteria and/or the fecal flora increases. in particular, the wide-spread use of antibiotics in animals has resulted in an increased emergence of bacterial resistance to antibiotics, in zoonotic organisms such as salmonella, campylobacter, shigella, yersinia, listeria, and enterococcus genera, as well as the e. coli species. these zoonotic bacterial organisms are propagated primarily among animals and subsequently infect people ( ) ( ) ( ) ( ) . humans can be infected by contact with animals and their dung or droppings or consumption of infected food. severe diarrhea may develop, sometimes with blood in the feces. at all ages, but especially in children under years and adults over years, very serious illnesses often occur. these complications can result in loss of life or permanent kidney damage. according to the latest epidemiological trends, salmonellosis and campylobacteriosis are indicated as the most frequent food-borne bacterial zoonoses in europe. the main food sources were eggs and mixed foods ( ) . furthermore, the recent emergence of esbl-producing and carbapenemase positive enterobacteriaceae bacteria in animal production ( ) , the emergence of farm associated mrsa st (the main pig associated clone) ( ) ( ) ( ) , and of plasmid-mediated quinolone resistance in animal isolates and food products ( , ) are great threat for public health. unfortunately, these antimicrobial resistant "superbugs" are not only confined to hospital environments where antimicrobial use was high and many pathogens were prevalent. they are already widespread in the european community and animal populations that have a great hazard on public health ( , ) . the causative agent of bovine tuberculosis, mycobacterium bovis (m. bovis) has been identified worldwide. thanks to decades of disease control measures that the occurrence of the infection has been greatly reduced. but there are still hundreds of new cases of human tuberculosis reported in the usa ( ) . experience in europe and the usa, has shown that m. bovis can be controlled when it is restricted in livestock; however, eradication is almost impossible once it has spread into wildlife as follows; the european badger in the united kingdom ( ), elk in canada ( ) and white tailed deer in the usa ( ) . in the last decade, q fever caused by coxiella burnetii was one of the most devastating farm animal originated bacterial zoonotic bacteria in europe. the netherlands, in particular, has experienced several outbreaks from to following identification of a q fever outbreak on various dairy farms in . infected humans were mainly located within the intensive dairy goat farms (< km) ( ) . the infection is spread by ticks, inhalation of the organism from the placental fluids, urine, and consumption of unpasteurized milk -meat products of sheep, goats, and cattle. the clinical signs in humans might be severe flu-like syndrome that may last for months ( ) . in the eu, many vector-borne zoonotic diseases are considered as emerging infectious diseases, which either appear in a population for the first time or may have existed previously but spreading rapidly. the ecology of vector-borne zoonotic bacterial diseases is complex where climate and weather may influence the transmission cycles. milder winters, earlier start of spring or long intervals between winters cause extended seasonal tick activity and hence pathogen transmission between hosts in new regions of the world ( , ) . many vector-borne infections occurred in new regions in the past two decades, while many endemic diseases have increased in incidence ( ) . the following bacterial pathogens were most frequently identified as the causes of emerging vector-borne infections in the last decades in the eu: rickettsiae spp., anaplasma phagocytophilum, borrelia burgdorferi, bartonella spp., and francisella tularensis ( , ) . rickettsia rickettsii causes rocky mountain spotted fever and spreads to humans by ticks. the signs of this disease are fever, headache, muscle pain, and spots with very dark rash. hiking in an area with many infested ticks is a great risk factor. a tick bite of < h is usually not enough to transfer these bacteria to a person ( ) . ehrlichiosis (anaplasma phagocytophilum) and lyme disease (borrelia burgdorferi) have emerged as an important vector-borne zoonotic disease since s ( , ) . hard ticks are principal vectors, whereas small rodents are known as their natural vertebrate reservoir. a wide variety of signs including rash, joint pains, fever, enlarged lymph nodes, and some neurological signs may www.frontiersin.org develop. the trend of house buildings in woodlots where humans share the same ecology with reservoirs and vectors was found to be correlated with the increased frequency of such diseases in humans ( ) . bartonella spp. is transferred to humans via fleas, lice, and sand flies ( ) . however, recent studies have shown the importance of tick exposure in human bartonellosis ( ) . as previously mentioned elsewhere in this article, bartonellosis are usually associated with cat-scratch diseases. lately, researchers have revealed that bartonella spp. can be transmitted via cat fleas without any scratches to humans ( ) . symptoms include fever, enlarged lymph nodes (after - weeks), and a papule at the inoculation site. etiological agent of tularemia, f. tularensis, is a rare disease in europe ( ) . bacteria are usually transferred by slaughtering (hunters are at a higher risk), eating of infected hares, respiration of dust, or drinking of contaminated water ( ) . the prevalence of f. tularensis was found to be - % from dog ticks in north america ( ) . clinical symptoms depend on how the organism is acquired: erythematous papule at inoculation side within h, pneumonia (the most serious form), endotoxemia, which gives continuous fever, acute pharyngotonsillitis, mucopurulent conjunctivitis (rarest form) ( ) . among many others, brucellosis, which is not an emerging disease, has a significant impact on the endemic southern european countries with sporadic outbreaks. fortunately, the impact on humans has not increased since ( ) . however, the cross border tracing of some brucella strains isolated in germany revealed concordance with sheep isolates originating from eastern anatolian, turkey. it is a characteristic example for the global spread of such diseases, in that case most probably by turkish immigrants living in germany ( ) . plague, caused by yersinia pestis, is the most important reemergent bacterial wild rodent borne disease. the current case reports of plague are primarily limited to africa. however, it is a great potential public health hazard for europe due to increased traveler mobility or a potential bioterrorist attack ( ) . bacterial zoonoses have a major impact on global public health. both emerging and re-emerging bacterial zoonoses have gained increasing national and international attention in recent years. the closer contact with companion animals and rapid socioeconomic changes in food production system has increased the number of animal-borne bacterial zoonoses. animal bite injuries in daily human-animal contact are not surprising, especially for the school-aged children. most of these wounds are medicated by patients as first aid and not registered in health systems. in more developed countries, most of the victims with moderate to severe bite injuries will seek professional medical treatment. regardless, all bites should be treated as serious, especially if the skin is broken. prompt diagnostic and treatment can prevent wound complications. the possibility to form biofilms by previously mentioned wound microorganisms is quite high, may cause severe tissue damage and protect the bacteria from innate-immune response and antimicrobials. the most of the commercial topical agents and wound dressings are ineffective against the biofilm matrix. surgical repair (for example, co surgical laser techniques, leon cantas, personal research notes ), which is usually used to obtain a better cosmetic result might be needed to remove biofilm formed bite infections. this mechanical debridement is essential in the eradication of a wound biofilm. antimicrobials may be more effective in the treatment of the wound after debridement in the prevention of biofilm reformation. despite the use of currently optimal culturing methods, approximately % of infected wounds yield no bacterial growth. in such cases, some other fastidious pathogens, i.e., chlamydia spp., mycoplasma spp., and even viruses should be investigated. new advanced molecular diagnostic techniques are needed. prevention strategies for animal bites include close supervision of child-animal interactions, stronger animal control laws, better reporting of animal bites, and public education for better ownership of pets. regular nail trimming, routine oral examinations under annual health checks and comprehensive dental treatments of the companion animals (i.e., routine removal of the teeth tartar and plaques) by veterinarians will reduce the bacterial mass exposure to humans in case of direct contacts or animal bites. it is important to realize that enteropathogenic zoonoses may be contracted from both clinically sick and apparently healthy companion animals. feeding of pets with raw food diets is a potential source of salmonella, campylobacter, and other important bacterial zoonoses; however, some recalls of commercial pet food diets have also occurred as a result of contamination with those microorganisms. pig ear dog treats, in particular, have been implicated as an important source of salmonella infection for dogs, which can also serve as a source of infection to humans. nevertheless, it can be said that easy-to-use personal hygiene rules should be applied by companion animal owners. thorough hand washing with soap after handling of a companion animal and before eating or drinking, avoiding mouth-to-mouth contact, avoiding aerosolization of dusty fecal matter will help to prevent transmission of the zoonotic disease to humans. the animals with diarrhea should be isolated immediately and veterinary advice should be sought. the household should be cleaned with agents and kept as clean as possible. on the other hand, the prevalence of antimicrobial resistance in small animal pathogens is increasing globally due to overuse of broad spectrum antibiotics by veterinarians. there is an immediate need for worldwide smarter use of antimicrobials that have some positive effect on the recovery of animals from life threatening diseases. national veterinary antimicrobial treatment guidelines should be established by the local authorities according to the updated routine surveillance results. chronic diarrhea, dermatitis, ear and eye infections of pets caused by microbes demand longer durations of antimicrobial remedies at home. more frequent use of advanced laboratory tests, such as; feed/insect/mould allergy tests and differential diagnosis of the other relevant auto-immune disorders may help to investigate the main underlying cause of the such reactions which can be managed in various alternative treatment methods (i.e., hypoallergenic diets) rather than antibiotics solely. herein, pet specific auto-immune vaccines against allergens and auto-lactobacillales (auto-lac, leon cantas, personal research notes, - ) as dietary supplements can also be more frequently administered within the preventative veterinary practice measures. owners should be encouraged to insure their family animals to afford such costly veterinary services contradictory to the cheaper and sometimes life-long medical (i.e., antibiotic) treatment demanding options. veterinarians should also spear more time to educate the pet owners under consultations to handle infected-antimicrobial treated animals with precaution due to irreversible consequences of the antimicrobial resistance development and its spread in households. proper hand washing and use of gloves are strictly recommended while handling antimicrobial in veterinary clinics. veterinarians should prescribe broad spectrum and synthetic antimicrobials preferably after culturing with extreme precautions (i.e., dosage, dosing intervals and length of the treatment). reduced antibiotic use will hinder the development of antibiotic resistance in animal microbiota which might cause zoonotic infections in humans ( , ) . food-borne zoonoses are an important public health concern worldwide and every year a large number of people affected by diseases due to contaminated animal originated food consumption. food hygiene education of the consumers is an important competent of food-borne diseases prevention. however, main prevention of food-borne zoonoses must begin at the farm level with in the concept of "one health." herein, control of the production stress especially in intensive livestock industry, with the development of better animal health management routines (i.e., routine vaccinations, immune stimulants: pre-, probiotic feed additives) and the increased animal welfare programs, will contribute eventually to an optimal production of animal health. increased antimicrobial resistance among emerging and re-emerging farm-borne bacterial pathogens in crowded settings (i.e., poultry, pig farms) is a growing problem. restrictive antimicrobial choice with better animal welfare managements are needed to control the spread of antibiotic resistance elements. in the eu, the use of avoparcin was banned in and the use of spiramycin, tylosin, and virginiamycin for growth promotion were banned in . all other growth promoters used in feeding of food producing animals were banned from january , after a few national bans the years ahead . in the u.s., politicians are still discussing to introduce a similar ban (s- , th u.s. congress (preservation of antibiotics for medical treatment act). despite the ban on the use of all antibiotics as growth promoters in the eu and a ban on the use of quinolones as growth promoters in the poultry feed in the us medical, important antibiotics are still routinely fed to livestock prophylactically to increase profits and to ward-off potential bacterial infections in the stressed and crowded livestock and aquaculture environments in some parts of the world ( , , ) . because stress lowers the immune system function in animals, antibiotics are seen as especially useful in intensive animal confinements ( ) . the non-therapeutic use of antibiotics involves low-level exposure in feed over long periods -an ideal way to enrich resistant bacterial population ( , ) . moreover, antibiotic resistance has been detected in different aquatic environments ( ) . fish pathogenic bacteria often produce devastating infections in fish farms where dense populations of fish are intensively reared. bacterial infections in fish are regularly treated with antibiotics in medicated feed. so far, most of the fish pathogenic bacteria with a http://europa.eu history in diseased fish farms have developed drug resistance ( ) . modern fish farming relies increasingly on vaccination procedures and improved management to avoid infections ( ) . for example, the norwegian aquaculture industry has produced over one million tons farmed fish by using improved vaccines, management techniques, and only kg of antimicrobials in ( ) . vector-borne and zoonotic bacterial pathogens are a major source of emerging diseases, and since the time of hippocrates, weather and climate are linked to the incidence of such infectious diseases. complexity of epidemiology and adoptive capacity of microorganisms and the arthropods make the vector-borne disease almost impossible to eradicate. insect repellants, routine tick checks after outdoor activity in risk regions, prompt-proper tick removal, use of long sleeves and trousers (light-colored), and routine insecticide treatment of pets are recommended as general preventative measures ( ) . herein, lyme disease, tick-borne illness, is vastly underestimated over past decades and clearly the urgent prevention is needed. besides individual awareness of such vector-borne diseases, better national surveillance and reporting programs will contribute to improved the disease control strategies. clinicians have an important role in the effective management of vector-borne zoonotic diseases, with enhanced differential diagnostic skills based on clinical symptoms and rapid molecular identification techniques ( ) ( ) ( ) ( ) . most of the time, the clinicians are on the first line of detection of these epidemics due to large group of patients with novel sets of similar symptoms. increased medical networking via online databases offer a broad overview to followers with regard to changes in temporal patterns of illness in real time, which helps faster detection of new epidemics ( ) . identification and control of emergent zoonotic bacterial diseases require a "one health" approach, which demands combined efforts of physicians, veterinarians, epidemiologists, public health workers, and urban planners. collaborative international routine surveillance strategies, prompt -reliable agent identification techniques, and optimization of the treatment regiments will ensure the prevention and management of such infections. leon cantas 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in beef cattle administered selected subtherapeutic antimicrobials in a feedlot setting antibiotic resistance of gram-negative bacteria in rivers, united states antibiotic resistance associated with veterinary drug use in fish farms fish vaccination, an overview usage of antimicrobial agents and occurrence of antimicrobial resistance in norway tick-borne disease curtailing transmission of severe acute respiratory syndrome within a community and its hospital strategies for mitigating an influenza pandemic identification of a novel polyoma virus from patients with acute respiratory tract infections microbe hunting digital disease detection: harnessing the web for public health surveillance the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -bqlf fe authors: rydell-törmänen, kristina; johnson, jill r. title: the applicability of mouse models to the study of human disease date: - - journal: mouse cell culture doi: . / - - - - _ sha: doc_id: cord_uid: bqlf fe the laboratory mouse mus musculus has long been used as a model organism to test hypotheses and treatments related to understanding the mechanisms of disease in humans; however, for these experiments to be relevant, it is important to know the complex ways in which mice are similar to humans and, crucially, the ways in which they differ. in this chapter, an in-depth analysis of these similarities and differences is provided to allow researchers to use mouse models of human disease and primary cells derived from these animal models under the most appropriate and meaningful conditions. although there are considerable differences between mice and humans, particularly regarding genetics, physiology, and immunology, a more thorough understanding of these differences and their effects on the function of the whole organism will provide deeper insights into relevant disease mechanisms and potential drug targets for further clinical investigation. using specific examples of mouse models of human lung disease, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, this chapter explores the most salient features of mouse models of human disease and provides a full assessment of the advantages and limitations of these models, focusing on the relevance of disease induction and their ability to replicate critical features of human disease pathophysiology and response to treatment. the chapter concludes with a discussion on the future of using mice in medical research with regard to ethical and technological considerations. although the genetic lineages of mice and humans diverged around million years ago, these two species have evolved to live together, particularly since the development of agriculture. for millennia, mice (mus musculus) were considered to be pests due to their propensity to ravenously consume stored foodstuff (mush in ancient sanskrit means "to steal" [ ] ) and their ability to adapt to a wide range of environmental conditions. since the s, domesticated mice have been bred and kept as companion animals, and in victorian england, "fancy" mice were prized for their variations in coat color and comportment; these mouse strains were the forerunners to the strains used in the laboratory today. robert hooke performed the first recorded inquiry-driven experiments on mice in , when he investigated the effects of changes in air pressure on respiratory function [ ] . more recently, with data from the human genome project and sequencing of the mus musculus genome showing remarkable genetic homology between these species, as well as the advent of biotechnology and the development of myriad knockout and transgenic mouse strains, it is clear why the mouse has become the most ubiquitous model organism used to study human disease. in addition, their small size, rapid breeding, and ease of handling are all important advantages to scientists for practical and financial reasons. however, keeping in mind that mice are fellow vertebrates and mammals, there are ethical issues inherent to using these animals in medical research. this chapter will provide an overview of the important similarities and differences between mus musculus and homo sapiens and their relevance to the use of the mouse as a model organism and provide specific examples of the quality of mouse models used to investigate the mechanisms, pathology, and treatment of human lung diseases. we will then conclude with an assessment of the future of mice in medical research considering ethical and technological advances. as a model organism used to test hypotheses and treatments related to human disease, it is important to understand the complex ways in which mice are similar to humans, and crucially, the ways in which they differ. a clear understanding of these aspects will allow researchers to use mouse models of human disease and primary cells derived from mice under the most appropriate and meaningful conditions. in , the encyclopedia of dna elements (encode) program published a comparative analysis of the genomes of homo sapiens and mus musculus [ ] , as well as an in-depth analysis of the differences in the regulatory landscape of the genomes of these species [ ] . encode, a follow-up to the human genome project, was implemented by the national human genome research institute (nhgri) at the national institutes of health in order to develop a comprehensive catalog of protein-encoding and nonproteincoding genes and the regulatory elements that control gene expression in a number of species. this was achieved using a number of genomic approaches (e.g., rna-seq, dnase-seq, and chip-seq) to assess gene expression in over mouse cell types and tissues; the data were then compared with the human genome. overall, these studies showed that although gene expression is fairly similar between mice and humans, considerable differences were observed in the regulatory networks controlling the activity of the immune system, metabolic functions, and responses to stress, all of which have important implications when using mice to model human disease. in essence, mice and humans demonstrate genetic similarity with regulatory divergence. specifically, there is a high degree of similarity in transcription factor networks but a great deal of divergence in the cis-regulatory elements that control gene transcription in the mouse and human genomes. moreover, the chromatin landscape in cell types of similar lineages in mouse and human is both developmentally stable and evolutionarily conserved [ ] . of particular relevance regarding modeling human diseases involving the immune system, in its assessment of transcription factor networks, the mouse encode consortium revealed potentially important differences in the activity of ets in the mouse and human genome. although conserved between the two species, divergence in ets regulation may be responsible for discrepancies in the function of the immune system in mouse and human [ ] . certainly, the biological consequences of these differences in gene expression and regulation between human and mouse invite further investigation. the anatomical and physiological differences between model organisms and humans can have profound impacts on interpreting experimental results. virtually every biological process under investigation in experimental studies involves at least one anatomical structure. to aid in interpretation, many anatomy compendia have been developed for model organisms; the most useful organize anatomical entities into hierarchies representing the structure of the human body, e.g., the foundational model of anatomy developed by the structural informatics group at the university of washington [ ] . although an analysis of the myriad differences between mouse and human anatomy is beyond the scope of this chapter, a few of the most critical issues that have an impact on the interpretation of data from mouse experiments should be mentioned. the most obvious difference between mice and humans is size; the human body is about times larger than that of the mouse. size influences many aspects of biology, particularly the metabolic rate, which is correlated to body size in placental mammals through the relationship bmr ¼ Â mass ( . ), where bmr is the basal metabolic rate (in kcal/day). thus, the mouse bmr is roughly seven times faster than that of an average-sized human [ ] . this higher bmr has effects on thermoregulation, nutrient demand, and nutrient supply. as such, mice have greater amounts of metabolically active tissues (e.g., liver and kidney) and more extensive deposits of brown fat [ ] . furthermore, mice more readily produce reactive oxygen species than do humans, which is an important consideration when modeling human diseases involving the induction of oxidative stress (i.e., aging, inflammation, and neurodegeneration) [ ] . the lung provides an excellent example of the similarities and differences between human and mouse anatomy. similar to the human organ, the mouse lung is subdivided into lobes of lung parenchyma containing a branching bronchial tree and is vascularized by the pulmonary circulation originating from the right ventricle. there are a number of subtle variations in this general structure between species, i.e., the number of lobes on the right and left, the branching pattern, and the distribution of cartilage rings around the large airways, but the most important differences between the mouse and human lung are related to the organism's size (airway diameter and alveolar size are naturally much smaller in the mouse) and respiratory rate. moreover, there are important differences in the blood supply of the large airways in humans versus mice [ ] . specifically, the bronchial circulation (a branch of the high-pressure systemic circulation that arises from the aorta and intercostal arteries) supplies a miniscule proportion of the pulmonary tissue in mice (the trachea and bronchi) compared to humans; the majority of the lung parenchyma is supplied by the low-pressure, high-flow pulmonary circulation. in the mouse, these systemic blood vessels do not penetrate into the intraparenchymal airways, as they do in larger species [ ] . this difference, although subtle, has important ramifications regarding the vascular supply of lung tumors which, in humans, is primarily derived from the systemic circulation [ ] . these differences may also have profound consequences when modeling human diseases involving the lung vasculature. the adaptive immune system evolved in jawed fish about million years ago, well before the evolution of mammals and the divergence of mouse and human ancestral species [ ] . many features of the adaptive immune system, including antigen recognition, clonal selection, antibody production, and immunological tolerance, have been maintained since they first arose in early vertebrates. however, the finer details of the mouse and human immune systems differ considerably, which is not surprising since these species diverged million years ago [ ] . while some have claimed that these differences mean that research into immunological phenomena in mice is not transferable to humans, as long as these differences are understood and acknowledged, the study of mouse immune responses can continue to be relevant. research on mice has been vital to the discovery of key features of both innate and adaptive immune responses; for example, the first descriptions of the major histocompatibility complex, the t cell receptor, and antibody synthesis were derived from experiments performed on mice [ ] . the general structure of the immune system is similar in mice and humans, with similar mediators and cell types involved in rapid, innate immune responses (complement, macrophages, neutrophils, and natural killer cells) as well as adaptive immune responses informed by antigen-presenting dendritic cells and executed by b and t cells. however, due to the anatomical and physiological differences between these species as described above, divergence in key features of the immune system, such as the maintenance of memory t cells (related to the life span of the organism) and the commensal microbiota (related to the lifestyle of the organism), has arisen [ ] . similar to what has been discovered regarding the genetics of mice and humans, i.e., broad similarities in structure but considerable differences in regulation, there are a number of known discrepancies in the regulation of innate and adaptive immunity in mouse models of human disease mice versus humans, including the balance of leukocyte subsets, t cell activation and costimulation, antibody subtypes and cellular responses to antibody, th /th differentiation, and responses to pathogens (described in detail in table ). in addition to these differences in immune cell functions, the expression of specific genes involved in immune responses also differs, particularly those for toll-like receptors, defensins, nk inhibitory receptors, thy- , and many components of chemokine and cytokine signaling; additionally, differences between mouse strains are known to exist for many of these mediators [ ] . another important consideration when using mice to perform immunological research (with a view to translating these findings to human medicine) is the availability of hundreds of strains of genetically modified mice that have enabled exquisitely detailed studies on immune cell function, regulation, and trafficking. many of these strains involve the expression of inducible cre or cas that allow for targeted knockdown or overexpression of key immune function-related genes in specific cell types at specific moments in time. however, it is important to note that drift between mouse colonies has long been known to occur. in fact, a recent report described the fortuitous discovery of a point mutation in the natural cytotoxicity receptor (ncr ) gene in the c /bl cd . mouse strain, resulting in absent ncr expression. this mutation was found to have profound effects on the response of mice to viral infection, i.e., the mice were resistant to cytomegalovirus infection but more susceptible to influenza virus [ ] . this cautionary tale highlights the importance of understanding the genetic evolution of laboratory strains of mice, the effect of these genetic and immunological changes on mouse biology, and the impact on the translation of these results to human medicine. in addition to the differences between mouse and human genetics, physiology, and immunology highlighted above, several factors must also be taken into account when performing in vitro assays using isolated mouse cells and applying these findings to our understanding of human disease. particularly with regard to stem cell research, it should be noted that the telomeres of mouse cells are five-to tenfold longer than human telomeres, resulting in greater replicative capacity [ ] . there are also important differences in the regulation of pluripotency and stem cell differentiation pathways in humans and mice [ ] . moreover, there are considerable species differences in the longevity of cultured cells; for example, mouse fibroblasts are capable of spontaneous immortalization in vitro, whereas human fibroblasts become senescent and ultimately fail to thrive in culture [ ] . in summary, although there are considerable differences between mice and humans, constant improvement in the analytical techniques used to delineate these differences and their effects on whole organism and cell function have provided vital information and contributed to our understanding of both murine and human biology. experimentation employing mouse models of human disease will continue to provide key insights into relevant disease mechanisms and potential drug targets for further clinical investigation. however, several important considerations must be taken into account when selecting a mouse model of human disease, as described in the following section, using mouse models of human lung disease to illustrate this point. the two most salient features of a mouse model of human disease are the accuracy of its etiology (it employs a physiologically relevant method of disease induction) and its presentation (its ability to recapitulate the features of human disease). the relevance of any given mouse model can be judged on the basis of these two criteria, and there is considerable variation within mouse models of human disease in this regard. as a full assessment of the advantages and limitations of all currently available mouse models of human disease would be prohibitively long and complex, here we have elected to assess the accuracy of currently available models of human lung diseases, i.e., asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, focusing on the relevance of disease induction in these models and their ability to replicate critical features of human disease pathophysiology and response to treatment. the first and foremost notion when modeling human disease in mice is to acknowledge the species differences, which are significant [ ] . as described above, genetics, anatomy, physiology, and immunology differ between mice and humans, but despite these differences, mouse models of human disease are useful and necessary, as long as data interpretation is performed appropriately. an elegant example of differences between mice and humans that must be considered when designing a mouse model of human inflammatory lung disease is the key effector cell type in human asthma, i.e., mast cells. these leukocytes differ in granule composition as well as localization in the mouse and human airways [ ] . mice mostly lack mast cells in the peripheral lung [ ] , whereas humans have numerous mast cells of multiple subpopulations in the alveolar parenchyma [ ] . another example is anatomy: in contrast to humans, mice lack an extensive pulmonary circulation, which may have significant effects on leukocyte adhesion and migration, and subsequently inflammation [ ] . still, as long as these differences are taken into consideration, mouse models can be powerful tools in the discovery and exploitation of new targets for the treatment of human disease. the world health organization (who) defines asthma as a chronic disease characterized by recurrent attacks of breathlessness and wheezing, which may vary in severity and frequency from person to person. the disease is characterized by airway hyperresponsiveness, airway smooth muscle thickening, increased mucus secretion and collagen deposition, as well as prominent inflammation affecting both large and small airways [ ] . nowadays, it is recognized that asthma is not a single homogenous disease but rather several different phenotypes united by similar clinical symptoms [ , ] . only a few animal species develop asthma naturally, including cats and horses [ , ] , whereas mice do not [ ] . however, mice can be manipulated to develop a type of allergic airway inflammation, which is similar in many ways to the human disease, in response to different aeroallergens [ ] . importantly, these models are capable of recapitulating only the allergic type of human asthma and have less relevance for other types of asthma (i.e., endotypes induced by medication, obesity, and air pollution). as with many human diseases, asthma has a complex and multifaceted etiology, where environmental factors, genetic susceptibility, and microbial colonization all contribute; thus, it is important to take strain differences into consideration. generations of inbreeding have created mouse strains that differ not only in coat color and disposition but also from a physiological, immunological, and genetic perspective. different strains may be more susceptible to allergic airway inflammation or pulmonary fibrosis, whereas others are more or less resistant. choosing the right strain to model a specific disease or pathologic event is thus essential. the most widely used strains for models of allergic airway inflammation are balb/c and c bl/ . these strains differ regarding the type of immune response mounted to an inhaled allergen: c bl/ is generally considered a t h -skewed strain, whereas balb/c is regarded as a t h -skewed strain [ ] . due to their strong t h response, and subsequent development of robust asthmatic responses, balb/c has been commonly used to model asthma [ ] . however, most humans do not express such a strongly t h -skewed immune system, suggesting this strain may not be the best model of human disease; instead, c bl/ may be more suitable as immune responses in this strain are more similar to those of atopic human subjects [ ] . furthermore, as c bl/ is the most commonly used strain for the development of genetically manipulated mice, using these mice allows for very specific investigations into disease pathology; thus, this strain is increasingly used in models of human lung disease. besides the genetic differences in the mouse strains used in these models, the etiology (the method of disease induction) of commonly used models of asthma is highly variable. in humans with allergic asthma, environmental allergen exposure occurs at the airway mucosa; the immune response is coordinated in the bronchopulmonary lymph nodes, and the t cells, macrophages, and eosinophils recruited as part of this response travel to the lung where they mediate the cardinal features of asthma: airway inflammation, structural remodeling of the airway wall, and airway hyperreactivity [ ] . ideally, these features should be found in a physiologically relevant mouse model of asthma. however, for the sake of cost and convenience, early mouse models of asthma used the surrogate protein ovalbumin (ova) [ ] rather than an environmental allergen to induce an immune response, which also requires the use of a powerful t h -polarizing adjuvant such as alum delivered via the intraperitoneal route, followed by ova nebulization-a clear divergence from the etiology of human asthma [ ] . in terms of disease presentation, mice develop some hallmarks of asthma, including airway eosinophilic inflammation, goblet cell metaplasia, and increased airway smooth muscle density [ ] . after the cessation of ova exposure, most of the remodeling resolves, although some structural alterations remain up to month after the last challenge [ ] . based on these attributes, the ova model is primarily a model to investigate the initiation of inflammation, rather than the chronic progression and maintenance of inflammation [ ] . a clear advantage with the ova model is the number of studies where it is used; both the pros and cons are familiar. it is easy to find a suitable protocol, and the model is readily accessible and flexible regarding the number of sensitizations and allergen doses. the model is relatively easy to reproduce, as ova and different adjuvants are easily obtained. however, the resolution of remodeling following the cessation of allergen provocations is a disadvantage, as is the practical problem with the nebulization of an allergen-it ends up in the mouse's coat and is ingested during grooming, potentially resulting in systemic exposure (this is particularly relevant in models employing systemic, intraperitoneal sensitization). in addition, concerns have been raised against the use of adjuvants to induce the immunological response, as well as the clinical relevance of ova as an allergen, which have driven the development of more clinically relevant allergens and models [ ] . the common environmental aeroallergen house dust mite (hdm) extract is increasingly used to initiate disease in mouse models of allergic airway inflammation, as it is a common human allergen (around % of asthmatics are sensitized to hdm [ ] ) that evokes asthma attacks and other allergic responses in susceptible individuals. in addition, hdm has inherent allergenic properties, likely due to components with protease activity [ ] , so there is no need to use an adjuvant, thus improving the etiological similarity of these models with the clinical situation [ ] . in contrast to ova, prolonged exposure of hdm (up to weeks) induces asthma-like severe airway inflammation with prominent eosinophilia, severe hyperreactivity to methacholine, and robust remodeling of the airway wall [ ] , i.e., the presentation of chronic respiratory hdm exposure in mice effectively recapitulates the key features of human allergic asthma. importantly, the airway structural changes induced by chronic hdm exposure, such as increased collagen deposition, airway smooth muscle thickening, and microvascular alterations, persist for at least weeks after the cessation of hdm exposure [ ] , another commonality with human asthma in which airway remodeling is currently considered to be irreversible. thus, the advantages of using hdm as the allergen in mouse models of asthma are the clinical relevance of the allergen [ ] and the route of delivery via the respiratory tract. moreover, studies have shown that the type of inflammation and characteristics of tissue remodeling are relatively similar to those seen in human asthmatics [ , , ] . one disadvantage is the complexity of hdm extract; as a consequence of this complexity, variations exist in some components between batches, particularly regarding the content of lipopolysaccharide, so reproducibility in these studies may be problematic. with similarity to hdm, these models were developed to be as clinically relevant as possible, as many patients suffer from allergy toward cockroach allergen, molds, and other environmental irritants. a common feature of these allergens is their complex nature, as they commonly consist of a mix of different allergic epitopes and fragments. this complexity is most likely why the immunological reaction in mice is relatively similar to that seen in asthmatics [ ] . cockroach allergen (cra) is a common allergen, known to induce asthma in susceptible individuals; thus, it shares with hdm the advantage of being highly clinically relevant [ ] . cra induces peribronchial inflammation with significant eosinophilic inflammation and transient airway hyperresponsiveness, both of which can be increased by repeated administrations of the allergen [ ] . colonization of the airways with aspergillus fumigatus is the cause of allergic bronchopulmonary aspergillosis (abpa), a disease where the lungs are colonized by the fungus, but allergens from aspergillus fumigatus can also induce asthma similar to other allergens [ ] . the reaction to aspergillus allergens is robust, and often no adjuvants are needed to elicit inflammation [ ] . in addition to aspergillus, other fungi such as penicillium and alternaria can also induce asthma in humans and have been used to model disease in mice [ ] . a common difficulty with these allergens is the method of administration, as the physiological route is believed to be the inhalation of dry allergens; mimicking this route with a nebulizer introduces the risk of the animals ingesting the allergen and thus causing systemic responses [ ] . exacerbations of asthma are defined as the worsening of symptoms, prompting an adjustment in treatment, and are believed to be associated with increased inflammation in the distal airways. clinically, exacerbations are believed to be induced by infections (most common), allergen exposure, or pollutants, which can be modeled in different ways [ , ] : . infections with viruses and bacteria or exposure to proteins/ dna/rna derived from these microbes. . administration of a high dose of allergen in a previously sensitized animal. . exposure to environmental pollutants, such as diesel exhaust or ozone. modeling exacerbations adds a layer of complexity, as robust ongoing allergic airway inflammation needs to be established first, before challenge with the exacerbating agent. both the ova and hdm models are used in this respect, and in both cases chronic protocols extending for several weeks before triggering an exacerbation have been used [ ] . chronic obstructive pulmonary disease (copd) is characterized by chronic airway obstruction, in contrast to asthma where the obstruction is reversible (particularly in response to bronchodilator treatment). clinically, in copd, chronic bronchitis and emphysema can occur either separately or in combination. copd is almost always associated with either first-or secondhand tobacco smoking or in rare cases with a deficiency in the production of α antitrypsin (a serpin that prevents elastin breakdown as a result of neutrophil degranulation) [ ] . the etiology of copd is highly complex and is believed to develop after many years of smoking in combination with other known factors such as genetic susceptibility or environmental factors [ ] . in similarity to asthma, inflammation is a major component in copd, but the leukocyte profile is very different: the most prominent players in copd-related inflammation are neutrophils and, to some degree, macrophages [ ] . due to the complex etiology of copd, it is difficult to recapitulate all aspects of this disease in a single model, so in most cases, the aim is to induce copd-like lesions by exposing mice to tissue-damaging substances (usually cigarette smoke) or to mimic emphysema by the administration of tissue-degrading enzymes [ , ] . clearly, mice do not smoke cigarettes on their own, so to model copd by cigarette smoke (cs) inhalation, the mice need to be exposed to unfiltered cs in an induction chamber; moreover, in an attempt to better model the chronic aspects of copd, this needs to be performed for a prolonged period of time. mice are very tolerant to cs, but eventually (over a period of several weeks), cs induces pulmonary neutrophilic inflammation that is associated with some degree of tissue degradation and destruction [ ] . an important advantage of this model is the fact that cs is the actual irritant responsible for disease in humans, and mice develop several features similar to the clinical disease, making this model highly clinically relevant [ ] . a significant drawback is the self-limitation of the model-the pathological changes do not progress after the cessation of cs exposure [ ] . furthermore, the exposure time needed for mice to develop copd-like pathology is extensive, i.e., studies have shown that an exposure protocol of days per week for a minimum of months is needed to generate robust structural changes to the lung [ ]. the pathological image in copd is complex and varies greatly between patients, commonly encompassing chronic bronchitis and bronchiolitis, emphysema, fibrosis, and airway obstruction. although mice develop some of these symptoms when exposed to cs, they do not develop all the symptoms of human disease; thus, cs has advantages as a model but fails to mimic the complexity of the clinical situation and disease presentation [ ] . other models of copd rely on the administration of proteases (protein-degrading enzymes) that are believed to be involved in the pathology of this disease in a subset of patients, such as elastindegrading elastase. this approach mimics the emphysematous changes seen in copd, but the pathological process underlying tissue destruction is likely very different compared to the clinical situation [ ] , as very few patients show evidence of elastase dysregulation [ ] . however, if the aim of the study is to investigate the general effect of protease-induced tissue destruction and regeneration, then this is a highly relevant method [ ] . some studies on copd have also used genetically modified animals, such as mice overexpressing collagenase, which results in tissue destruction without inflammation or fibrosis with an end result fairly similar to the type of emphysema observed in copd [ ] . pulmonary fibrosis, the accumulation of fibrotic tissue within the alveolar parenchyma, is merely a symptom of disease, and the etiology of this pathology in humans varies greatly [ ] . the most enigmatic class is perhaps the idiopathic interstitial pneumonias, especially idiopathic pulmonary fibrosis (ipf). ipf is a debilitating and progressive disease with a grave prognosis, characterized by progressive fibrosis believed to reflect aberrant tissue regeneration [ ] . as the reason behind this defective repair is unknown, although a combination of immunological, genetic, and environmental factors are suspected, it is very difficult to model disease in a clinically relevant fashion [ ] . the most common method used to model pulmonary fibrosis in mice is administration of the chemotherapeutic agent bleomycin; this agent is known to cause pulmonary fibrosis in humans as well, but this may not accurately reflect the true etiology of most cases of human disease. the strain of choice is c bl/ , as it is prone to developing pulmonary fibrosis, whereas balb/c is relatively resistant, a feature believed to reflect the cytokine response following cellular stress and damage [ ] . bleomycin administration can be performed locally or systemically, producing very different results. the most common model of pulmonary fibrosis is a single intranasal or intratracheal administration of bleomycin, with analysis to weeks later. during this time, the drug causes acute tissue damage in a restricted area of the lung (where the solution ends up during administration), followed by intense inflammation in this area and subsequent fibrosis, which gradually resolves within weeks. however, if older mice are used, the fibrosis will persist longer than in younger mice, which is in accordance with clinical ipf, where the majority of the patients are years of age or older [ , ] . a great advantage of this model is how well-characterized it is. in addition, local administration is labor-effective, as only one administration is required and the result is highly reproducible. the fibrosis is robust, only affects the lungs, and the accumulation of extracellular matrix can be easily measured using standard techniques [ ] . furthermore, as it is used throughout the world, studies performed in different labs and by different groups can be compared relatively easily. unfortunately, the intense pulmonary inflammation may be lethal, and fatalities are to be expected with this model [ ] , representing an important ethical limitation. furthermore, fibrosis is heterogeneous-it develops where the bleomycin solution is deposited. the solution usually deposits within the central lung, a localization that is not in agreement with the clinical situation where fibrosis is located in the more distal regions of the lung parenchyma. in addition, the fibrosis that develops as a result of severe tissue damage is self-limiting and reversible, unlike what is observed clinically [ ] . the severe degree of tissue damage induced by bleomycin may in fact be more relevant for modeling acute lung injury (ali) or acute respiratory distress syndrome (ards). bleomycin can also be administered systemically, through intravenous or subcutaneous injection. in contrast to local administration, this route requires multiple administrations and is thus more laborintensive [ ] . some studies have described the usage of osmotic mini-pumps, where bleomycin is slowly administered over a short period of time, and then fibrosis continues to develop over subsequent weeks [ ] . irrespective of the route of delivery, systemic administration results in more homogenous fibrosis, affecting the entire lung through the pulmonary endothelium and persisting much longer than following local administration [ ] . the main advantages of systemic administration are that inflammation is limited, while the fibrosis is more apparent and displays a more distal pattern, all of which mimics the clinical situation relatively well. the multiple administrations allow for lower doses with each injection; this is less stressful to the animals and results in little to no mortality [ ] and is thus more ethically acceptable. a major disadvantage with this model is that it takes time for fibrosis to develop [ ] , which may be the reason it is used relatively scarcely, and thus the pathological development is less well-understood. in addition, as ipf is a local disease, local administration of the etiologic agent may better mimic the clinical reality [ ] . the administration of fluorescein isothiocyanate (fitc) induces focal inflammation, primarily involving mononuclear cells and neutrophils, and localizes in areas where the fitc solution is deposited [ ] . antibodies against fitc can be detected after week, and the fibrosis persists for up to months after instillation [ ] . the benefits of this model are mainly related to the persistent fibrosis that does not appear to be self-limiting, thus reflecting the clinical situation, and it is also very easy to determine which part of the lung has been exposed to fitc, as the molecule is fluorescent [ ] . it is also an advantage that both c bl/ and balb/c mice are susceptible and develop fibrosis following fitc administration [ ] . the disadvantages of this model include profound variability due to differences between batches of fitc, as well as in the method used to prepare the solution before instillation. importantly, given the characteristics of the etiologic agent used to induce this model of ipf, this model is considered a very artificial system with limited clinical relevance [ ] . adenovirus vectors have been used to overexpress the pro-fibrotic cytokine transforming growth factor (tgf)-β, which results in pulmonary fibrosis. as tgf-β overexpression in the lungs is known to be crucial in the development of fibrosis in humans [ ] , this model mimics an important feature of disease etiology. however, the delivery system has some drawbacks, as the virus itself initiates an immune response. moreover, adenoviruses display significant tropism for epithelial cells and rarely infect other cell types such as fibroblasts [ ] , which are the cells meant to be targeted in this model. as tgf-β has major effects on fibroblast biology, the main feature of this model is the effect of epitheliumderived tgf-β on fibroblasts and myofibroblasts, resulting in the deposition of ecm proteins and areas of dense fibrosis [ ] . an advantage of this model is the relatively low degree of inflammation, as well as what appears to be a direct effect on fibroblasts/ myofibroblasts [ ] , which is in accordance with the clinical situation (as we understand it today). silica administration induces a similar pathology in mouse lungs as in humans exposed to silica, and as is also observed in human silicainduced fibrosis, structural remodeling persists when administration is halted [ ] . following the administration of silica particles, fibrotic nodules develop in mouse lungs, with considerable resemblance to the human lesions that develop after exposure to mineral fibers [ ] . the fibrotic response is accompanied by a limited inflammatory response, and different pro-fibrotic cytokines such as tgf-β, platelet-derived growth factor, and il- are involved in disease development, which is in accordance with the clinical situation [ ] . another advantage is that nodules develop around silica fibers, and these fibers are easy to identify by light microscopy. the response in this model is strain-dependent, with c bl/ mice being the most susceptible. the main drawbacks are the time required to establish disease, i.e., - days, and the need for special equipment to aerosolize the silica particles. however, since the route of administration, the driving etiologic agent, and the resulting pathobiology are all similar to the characteristics of this subtype of pulmonary fibrosis [ , ] , the silica exposure model can be considered to have very good clinical relevance. what does the future hold for mouse models of human disease? medical research using experimental animals (not only mice but other animals including rats, guinea pigs, zebrafish, and fruit flies) has greatly contributed to many important scientific and medical advances in the past century and will continue to do so into the near future. these advances have contributed to the development of new medicines and treatments for human disease and have therefore played a vital role in increasing the human life span and improving quality of life. despite the acknowledged benefits of performing research using experimental animals, a number of considerations must be made before embarking on this type of research. of course, the financial aspects of conducting this type of work are an important limitation, as the costs of purchasing and housing mice can be prohibitive, especially when genetically modified mice and colony maintenance are required for the study. the practicalities of working with animals such as mice may also be an issue, as this type of work requires specialized facilities, equipment, and staff to ensure studies are carried out in a manner that is safe for both the researchers and the animals. moreover, as discussed in detail in this chapter, the relevance of the selected animal model to human disease must be carefully evaluated to ensure that these experiments provide robust results that are translatable to human health and disease. another important and demanding aspect of biomedical research using animals is the ethics of imposing pain and suffering on live animals. although there has been a considerable reduction in the numbers of animals used in research in the last years, animal research remains a vital part of biomedical research. however, no responsible scientist wants to cause unnecessary suffering in experimental animals if it can be avoided, so scientists have accepted controls on the use of animals for medical research. in the uk, this ethical framework has been enshrined in law, i.e., the animals (scientific procedures) act . this legislation requires that applications for a project license to perform research involving the use of "protected" animals (including all vertebrates and cephalopods) must be fully assessed with regard to any harm imposed on the animals. this involves a detailed examination of the proposed procedures and experiments, and the numbers and types of animal used, with robust statistical calculations to support these numbers. the planned studies are then considered in light of the potential benefits of the project. both within and outside the uk, approval for a study involving protected animals also requires an internal ethical review process, usually conducted by the research institution where the work is taking place, with the aim of promoting animal welfare by ensuring the work will be carried out in an ethical manner and that the use of animals is justified. additionally, the uk has a national animal use reduction strategy supported by the national centre for the replacement, refinement and reduction of animals in research (nc rs; london, uk). this consortium was established in to promote and develop high-quality research that takes the principles of replacement, refinement, and reduction (the rs) into account. replacement strategies often involve the use of alternative, non-protected species (e.g., zebrafish, fruit flies, flatworms) and in vitro correlates (two-dimensional cell culture or threedimensional organoids containing multiple cell types) to test hypotheses and assess the effects of therapeutic interventions. the main obstacle with studies on non-protected animals is the difficulty of accurately mimicking the complex physiological systems involved in human health and disease, as described in detail above. for example, the fruit fly drosophila melanogaster is an excellent model organism for studies on genetic diseases, aging, and pathogen-borne illnesses but may be less relevant for studies on complex lung diseases. importantly, model organisms such as fruit flies, zebrafish, and flatworms do not possess lungs, which somewhat limits the translatability of research on these animals in the field of respiratory disease. as such, it is likely that rodents will remain the model organism of choice for studies into lung disease for some time to come. there has been considerable progress recently in imitating single organs such as the liver, lung, and brain in vitro using multiple cell types and a physical scaffold. as an important advantage, these in vitro tests have replaced a large number of rodents in initial drug discovery experiments, while also speeding up the process [ ] . these studies still require further refinement and validation to establish them as suitable models for an entire organ; importantly, these in vitro organoids cannot take into account interactions between organ systems in complex, multisystem diseases such as copd. refinement involves selecting the most clinically relevant model for the disease available, informed by the discussion above on closely recapitulating the etiologic agent and disease pathobiology associated with clinical cases. another important factor is refining the management of pain. an assessment of the procedures used and the effects of the substance on the animal, as well as the degree of handling, restraint, and analgesia, are other important aspects of refinement. this standard of animal care is achieved through strict regulations and controls on how personnel are trained to carry out experiments on live animals. adequate training is an important aspect of refinement and should be reviewed and improved on an ongoing basis. moreover, refinement can be achieved by improving animal housing by environmental enrichment, e.g., providing a place for mice to hide in the cage and housing social animals such as mice in appropriate-sized groups. these simple changes can improve the physiological and behavioral status of research animals; this not only increases animal well-being but also contributes to the quality of the experimental results by reducing stress levels. the rs aspect of reduction focuses on the statistical power of experiments and by following the animal research: reporting of in vivo experiments (arrive) guidelines, originally published in plos biology in . these guidelines provide a framework to improve the reporting of research performed on live animals by maximizing the quality of the scientific data and by minimizing unnecessary studies. the arrive guidelines provide a checklist of aspects that must be considered in good quality research using live animals. the guidelines are most appropriate for comparative studies involving two or more groups of experimental animals with at least one control group, but they also apply to studies involving drug dosing in which a single animal is used as its own control (within-subject experiments). the guidelines provide recommendations on what should be considered when preparing to report on the results of experiments involving live animals, i.e., by providing a concise but thorough background on the scientific theory and why and how animals were used to test a hypothesis, a statement on ethical approvals and study design including power and sample size calculations, a clear description of the methods used to ensure repeatability, objective measurements of outcomes and adverse effects, and interpretation of the results in light of the available literature and the limitations of the study. in addition to the positive impact of the arrive guidelines on reducing the number of animals used in experiments, this checklist provides an easy-tofollow roadmap on what is required for good quality reporting of experimental results. in conclusion, the use of animals in research will continue to be an important aspect of medical research, and these procedures can be ethically justified provided the proper controls are in place. the benefits of animal research have been vital to the progress of medical science; abandoning these studies would have severe negative consequences on human health. by considering aspects such as the rs and the arrive guidelines in planning experiments involving live animals, the number of animals used and suffering of these animals for the benefit of human health can be minimized. this requires a strong regulatory framework such as that found in the uk and many other countries, as well an ongoing public debate on the advantages and limitations of animal experimentation. use of house mice in biomedical research the laboratory mouse a comparative encyclopedia of dna elements in the mouse genome principles of regulatory information conservation between mouse and human of mice and men: aligning mouse and human anatomies mouse models of human disease: an evolutionary perspective structure and composition of pulmonary 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endothelin- receptor a inhibition prevents bleomycin-induced pulmonary inflammation and fibrosis in mice (r)-resolvin d ameliorates bleomycin-induced pulmonary fibrosis in mice extracellular matrix alterations and acute inflammation; developing in parallel during early induction of pulmonary fibrosis smad signaling involved in pulmonary fibrosis and emphysema adenovector-mediated gene transfer of active transforming growth factor-beta induces prolonged severe fibrosis in rat lung the ethics of animal research. talking point on the use of animals in scientific research key: cord- - u t u authors: mao, ying; zhang, ning; zhu, bin; liu, jinlin; he, rongxin title: a descriptive analysis of the spatio-temporal distribution of intestinal infectious diseases in china date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: u t u background: intestinal infectious diseases (iids) have caused numerous deaths worldwide, particularly among children. in china, eight iids are listed as notifiable infectious diseases, including cholera, poliomyelitis, dysentery, typhoid and paratyphoid (tap), viral hepatitis a, viral hepatitis e, hand-foot-mouth disease (hfmd) and other infectious diarrhoeal diseases (oidds). the aim of the study is to analyse the spatio-temporal distribution of iids from to . methods: data on the incidence of iids from to were collected from the public health science data centre issued by the chinese center for disease control and prevention. this study applied seasonal decomposition analysis, spatial autocorrelation analysis and space-time scan analysis. plots and maps were constructed to visualize the spatio-temporal distribution of iids. results: regarding temporal analysis, the incidence of hfmd and hepatitis e showed a distinct increasing trend, while the incidence of tap, dysentery, and hepatitis a presented decreasing trends over the last decade. the incidence of oiid remained steady. summer is the season with the greatest number of cases of different iids. regarding the spatial distribution, approximately all p values for the global moran’s i from to were less than . , indicating that the incidences of the epidemics were unevenly distributed throughout the country. the high-risk areas for hfmd and oidd were located in the beijing-tianjin-tangshan (btt) region and south china. the high-risk areas for tap were located in some parts of southwest china. a higher incidence rates for dysentery and hepatitis a were observed in the btt region and some west provincial units. the high-risk areas for hepatitis e were the btt region and the yangtze river delta area. conclusions: based on our temporal and spatial analysis of iids, we identified the high-risk periods and clusters of regions for the diseases. hfmd and oidd exhibited high incidence rates, which reflected the negligence of class c diseases by the government. at the same time, the incidence rate of hepatitis e gradually surpassed hepatitis a. the authorities should pay more attention to class c diseases and hepatitis e. regardless of the various distribution patterns of iids, disease-specific, location-specific, and disease-combined interventions should be established. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. intestinal infectious diseases (iids), also known as infectious enteric diseases, are transmitted via the faecal-oral route through contaminated food, water, or fomites. the main symptoms of intestinal infectious diseases include nausea, vomiting, fever, headache, limb pain, abdominal pain, loss of appetite, systemic poisoning, diarrhoea, and other gastrointestinal symptoms, which may lead to death if not treated promptly. iids have posed public health threats and resulted in severe social and economic burdens due to the high incidence and morbidity rates, particularly in young children [ ] [ ] [ ] . several large, severe global disease outbreaks have been associated with iids, such as the outbreaks of hfmd in east and southeast asia in the early twenty-first century [ ] , the constantly high burden of cholera that persists in many african countries, and the cholera outbreaks with active cholera transmission in many sub-saharan african countries [ ] . according to the sustainable development goals (sdg), we should combat hepatitis, water-borne diseases and other communicable diseases. iids represent a significant obstacle to achieving sdg [ ] . the chinese government enacted the law of the people's republic of china on the prevention and treatment of infectious diseases and regularly reports cases of notifiable diseases to effectively monitor and control infectious diseases. all reported infectious diseases are divided into classes a, b and c in terms of severity, which is shown in additional file . in china, iids are one of the main types of infectious diseases, presenting the characters of a high morbidity rate and low mortality rate [ ] . eight iids are listed in the law, including cholera, poliomyelitis, dysentery, typhoid and paratyphoid (tap), viral hepatitis a, viral hepatitis e, hand-foot-mouth disease (hfmd) and other infectious intestinal diseases (oiids). among these diseases, cholera and poliomyelitis have been almost completely eradicated. in contrast, according to the data reported by the chinese cdc in , viral hepatitis a, viral hepatitis e, and dysentery are among the top five most severe diseases in class b. at the same time, hfmd and oidds were the most severe diseases in class c. the status of intestinal infectious diseases requires further attention. previous studies have investigated the epidemiology of different intestinal infectious diseases. first, temporal analysis of different intestinal infectious diseases was conducted. the initial efforts in conducting a temporal analysis investigated the temporal variations in the incidence rates of different diseases in different age groups and areas. for example, tian et al. analysed the temporal characteristics of hfmd and the relationship between meteorological factors and the incidence of hfmd in beijing, china. may to july is the period with peak hfmd incidence each year in beijing [ ] . xiao ( , ) ] to predict the trend in the incidence of typhoid using data collected from wuhan city in china from to [ ] . liu et al. forecasted the incidence of bacillary dysentery diseases by applying the seasonal trend model based on the moving average method, which forecast the incidence of hepatitis a in [ ] . ming et al. investigated the seasonal signals and long-term trends in a series of igs sites in china [ ] . xing et al. conducted a study examining the epidemiological characteristics in china from to , emphasizing seasonal patterns among people at different ages [ ] . temporal analyses of infectious diseases have allowed researchers to develop a wide range of research methods, namely, time series analysis and seasonal analysis. however, temporal analysis failed to reflect the overall circumstances of infectious intestinal diseases. on the other hand, the academic world gradually began to realize the importance of performing spatial analysis of infectious diseases. zhang et al. conducted a space-time scan analysis of hfmd in liaocheng city in china from to at the town level [ ] . the distribution of the identified cluster was described in the article. zheng et al. mapped the incidence rate and coefficient of determinants of hfmd to present and identify the most severely affected areas and the most influential determinants [ ] . as shown in the study by wang et al., the typhoid prevalence is spatially clustered and exhibits a gradually decreasing trend [ ] . according to ma et al., bacillary dysentery is not equally distributed across sichuan province in china [ ] . nie et al. used a spatial correlation analysis to explore the associations between selected factors and bacillary dysentery in guangxi province in china [ ] . the spatial analysis visually displayed the discrepancies among different regions, which would be able to present additional results if combined with the temporal analysis. in conclusion, temporal and spatial analyses are useful. previous studies have focused on the epidemiological characteristics of different iids. however, a comprehensive study analysing the spatial and temporal distributions of all reported iids in china is lacking. second, an overall introduction to iids in china to researchers in other countries is unavailable. therefore, this study will collect data on the basic iids and populations, use seasonal decomposition analyses to explore the temporal epidemiology, and perform spatial autocorrelation analysis and space-time scan analysis to explore the spatial epidemiology. among the eight iids, cholera and poliomyelitis have been almost completely eradicated. the number of deaths related to cholera and poliomyelitis since were and , respectively, indicating that the two diseases were less emergent. at the same time, according to the chinese classification of viral hepatitis, the hepatitis a and e should be analysed separately [ ] . overall, this study analyses the spatiotemporal distribution of six intestinal infectious diseases, namely, dysentery, typhoid and paratyphoid (tap), viral hepatitis a, viral hepatitis e, hand-foot-mouth disease (hfmd) and other infectious intestinal diseases (oiid). to better demonstrate the trends of incidence of iids, we analysed these diseases from to , which represents two equal periods: - and - . the data were collected from public health science data centre issued by chinese center for diseases control and prevention (china cdc) [ ] . the sorted data are presented in additional file and additional file . additional file displays the number of cases of iids. additional file displays the number of cases of iids by month and the morbidity of iids by month. intestinal infectious diseases are strictly monitored by the chinese cdc. the centre aims to integrate the scattered data distributed by governments, universities, research institutes and scientists. the seasonal decomposition analysis was based on the seasonal trend decomposition using loess (stl), which incorporates three components: trend, seasonal, and remainder or residual [ ] . advantages of the method include its simplicity and speed of computation, the robustness of results, and flexibility in the period of the seasonal component [ ] . in the present study, iids were analysed by performing a seasonal decomposition of the time series. this assay employed holt-winters filtering and ljung-box test to define the structure (additive or multiplicative) and seasonality (stationary or non-stationary). using an additive model, the iid results were compiled by summing three components: where z t represents the monthly incidence rates of the diseases, m t represents the trend, s t symbolizes the seasonal variation, and r t denotes the remainder. the result of seasonal decomposition analysis is displayed in a figure with the time from to on the horizontal axis and the incidence rate on the vertical axis. the concept of spatial autocorrelation was proposed by tobler in the first geography law [ ] : "everything is related to everything else, but nearest things are more related than distant things." moran's i was the tool used to measure spatial autocorrelation and consists of two types: global moran's i and local moran's i. global moran's i measures the general spatial autocorrelation and the spatial distribution of research object. local moran's i reflects the local spatial autocorrelation and the cluster regions. in the present study, spatial autocorrelation was applied to analyse the iids. global moran's i shows the overall cluster level and distribution of iids; local moran's i reveals the specific cluster regions and cluster categories and the hotspots of iids [ ] . global moran's i is an index ranging from − to . when the index was distributed around − , the overall spatial distribution displayed the dissimilarity, indicating that high cluster regions bordered on low cluster regions. when the index was approximately , a distinct spatial cluster was not observed in the studied regions. when the index was close to , the overall spatial distribution revealed the similarity, indicating that the same cluster category was bounded on another cluster category. the following equation was used to calculate the global autocorrelation: where n denotes the number of observed values, x i represents the incidence rate in province i, x j represents the incidence rate in province j, x indicates the mean value, and w ij represents a spatial weight matrix of systematic binomial distribution, which represents neighbouring relations between geographical units with n representing the total number of those units . in the present study, the data were based on regions. the value for w ij is if province i and province j are adjacent. otherwise, the value is . local moran's i avoids the weaknesses of global spatial autocorrelation by analysing the spatial autocorrelation of certain characters in local regions. the range and explanation for the local moran's i was same as the global index. the cluster results obtained from local moran's i were classified into four types: high-high cluster (hh, which indicated that the high cluster areas were surrounded by other high cluster areas), high-low cluster (hl, which indicated that the high cluster areas were surrounded by low cluster areas), low-high cluster (lh), and low-low (ll) cluster. the clusters were visualized using lisa cluster maps. the following equation was used to calculate local moran's i: where y i represents the incidence rate in province i, y j represents the incidence rate in province j, y indicates the mean value. [ ] in the present study, we analysed a long period to investigate the incidence rates of different iids. the years , , were selected to show the long-term changes. space-time scan statistics were introduced by kulldorff [ ] . the space-time scan statistic based on the discrete poisson model was applied to detect the space-time cluster of iid cases in high-risk or low-risk regions in china [ ] . the shape of space-time scanning windows is cylindrical with the geographic units and the height associated with time [ ] . the null hypothesis presumed that the window area and outside areas have the same relative risk (rr) of incidence. the difference in the incidence inside and outside the windows was evaluated by calculating the log likelihood ratio (llr): where c denotes the total number of cases, c represents the number of observed cases inside the window, and n represents the number of expected cases inside the window [ ] . the space-time analysis was applied to identify the clusters according to the llr value, including the most likely cluster, secondary cluster , secondary cluster , secondary cluster , and secondary cluster , as well as the cluster time. statistical significance was evaluated using a monte carlo simulation with , replicates and a significance level of . . for the other parameters, the maximum radius of the circular base was set to % of the total population at risk and the maximum height of the cylinder was set to % of the total study period. the method is sensitive to user-controlled parameter choices. a reliability analysis was conducted to determine the sensitivity and consistency of the results. multiple scans with different maximum sizes ranging from to % of the population were conducted. reliability was measured using the following equation: where r i denotes the reliability of different provincial units, s indicates the number of scans, and c i represents the number of high-risk areas in these scans. the range of reliability is from to points, where indicates that all scans report a place as high risk and indicates no scan reports [ ] . the reliability results were visualized by constructing a map (additional file and additional file ). the seasonal decomposition analysis were visualized using microsoft excel (version , microsoft corp, redmond, wa, usa). the seasonal decomposition analysis was performed using ibm spss statistics (version , ibm, armonk, ny, usa). geoda (version . . , github, san francisco, ca, usa) was employed to attain the global moran's i and local moran's i hierarchical maps, and lisa cluster maps, and space-time scan maps were obtained using arcgis (version . , esri inc., redlands, ca, usa). the space-time scan was analysed using satscan (version . , kulldorff and information management services, inc., boston, ma, usa). we listed all iids reported in each provincial unit in china from to . table presents a descriptive analysis of selected iids, including the average, maximum and minimum incidence from to . all provincial units were divided into east, central and west china. according to the descriptive summary, hfmd was the most severe disease, with an average incidence of . . oidd ranked second with an average incidence of . from to . the third most common disease was dysentery, with an average incidence of . . regarding the region, east china had the highest incidence rates for hfmd, oidd and hepatitis e. the west area was the area with the greatest number of tap, dysentery and hepatitis a cases. the seasonal decomposition plots contain four parts, the raw data, remainder, seasonal variation, and trend. the raw data represent the original incidence rate of certain diseases. the remainder represents the irregularity of data. seasonal variation exists when the rangeability is greater than . . the trend symbolizes the long-term variation in the data. the seasonality and variation in the trends in monthly data were distinguished from the remainder or residual using a decomposition analysis. figure shows the results of the decomposition analysis for all iids. regarding the seasonal variation, the incidence rates of hfmd and dysentery were high in summer. higher incidence rates for oidd and hepatitis a were observed in summer and autumn. hepatitis e showed a high rate in spring. a distinct seasonal variation in the incidence of tap was not observed. regarding the trend, the hfmd, oidd and hepatitis e displayed increasing trends, and the other diseases showed decreasing trends. based on the remainder, a -month stochastic variance was displayed. the spatial autocorrelation analysis was divided into global spatial autocorrelation and local spatial autocorrelation. the former was considered to represent geographical -difference in whole areas, while the latter was regarded as the difference at the regional cluster level. global spatial autocorrelation table shows the results from the global spatial autocorrelation analysis and significance test. in terms of the significance, all moran's i values for dysentery and hepatitis e reached the significance threshold, while of the values for oidd, tap, and hepatitis a achieved results, with one insignificant index each. hfmd had five significant indexes. in the global spatial autocorrelation analysis, moran's i for hfmd ranged from . to . throughout the years without an obvious increasing or decreasing trend, suggesting that the disease local spatial autocorrelation analysis beijing and shanghai were the areas with the highest incidence rates, while the areas with the lowest rates included xizang, qinghai, yunnan, and guangxi, among others. figure displays the spatial clusters of all iids, reflecting the regional variation from to . the hot spot for hfmd is located in guangdong, while cold spots were detected in some areas of the central and west provinces, such as sichuan, chongqing, hubei, guizhou, and jiangxi. in , the hot spots shifted to adjacent provinces, guizhou and jiangxi; new cold spots shifted to areas in the north province, such as gansu, jilin, xinjiang, and inner mongolia. for oidd, the hh cluster feature was observed in btt areas, while heilongjiang and jilin in northwest china showed the ll cluster feature in . throughout the ensuing years, jilin and hebei were no longer present in the original hot spots and cold spots. jiangxi showed the hh cluster character as well. for tap, hh cluster character was witnessed in yunnan, jiangxi, and guizhou throughout the -year period, while xizang, sichuan, neimenggu and xinjiang showed the character of a cold spot. in terms of dysentery, inner mongolia, beijing, and tianjin were located in hh cluster areas, while the lh cluster was located in hebei. guangxi and guangdong contained the ll cluster. in and , the cluster characters presented the same trends. beijing, tianjin, and hebei were the hh cluster areas, while the ll cluster was located in guangdong. hebei experienced a change from a cold spot to a hot spot. for hepatitis a, the hh cluster character was present in west china, including xinjiang, gansu, qinghai, xizang, sichuan, and chongqing, throughout the -year period, while the ll cluster character was located in the btt area, jiangsu and guangdong. xizang was a hot spot in and , but was a cold spot in . the hot spots for hepatitis e were mainly concentrated in the btt area and yangtze river delta area, while cold spots were located in areas of west china, jiangxi and hubei. space-time scan analysis was used to explore the cluster likelihood, the level of which was classified as the most likely cluster, secondary cluster, nd secondary cluster, rd secondary cluster and th secondary cluster. this study analysed iids in china from to . fig. and table studies of the epidemiology of different intestinal infectious diseases, particularly the temporal and spatial distributions, have played an important role in preventing infections prevention. however, previous studies have neglected to analyse the correlation between the temporal and spatial analyses. a systematic spatio-temporal analysis of iids has not been conducted in china. this study supplements data from other related research in the area of infectious intestinal diseases by performing a temporal analysis and spatial analysis and determining the correlation between them. the evidence provided insights into potential solutions to diminish the diseases. on one hand, we would like to compare the temporal analyses of the six intestinal infectious diseases. according to the incidence rates recorded from to , the temporal trends differed. regarding the absolute incidence of cases, the incidence of hfmd was higher than the other iids, and it gradually became a wide-spread disease, which is consistent with the results from the study by zhang [ ] . then, dysentery and oidd were less severe diseases. hepatitis a, hepatitis e and tap were the least severe diseases, according to the incidence rates. regarding the trend, the incidence rates of hfmd and hepatitis e showed a distinct increasing trend, consistent with the results from the study by zhu. as the epidemic of hepatitis a was controlled, the percentage of hepatitis e cases among patients with viral hepatitis and among patients with iids has increased. the mortality rate of hepatitis e has increased among infectious diseases [ ] . the trend in the incidence of oidd was almost unchangeable. dysentery and tap displayed obvious decreasing trends. the analysis of dysentery filled the research gap in the study by xie et al., which showed a decreasing trend in the incidence of dysentery from to [ ] . the results for tap were similar to the findings reported by liu [ ] , which showed a decreasing trend. the improvement in sanitation facilities and the reduction in food and water pollution likely contributed to the decreasing trend in the incidence rates of these diseases. hepatitis a exhibited a slight increasing trend in the first years, followed by a decreasing trend. this result was similar to the findings reported by zhu [ ] . in conclusion, class b diseases were prevented with high efficiency. class c diseases experienced a higher incidence rate and gradually increasing trend. regarding the seasonal changes, the incidence of hfmd and dysentery peaked in the summer, hepatitis e exhibited a high-incidence season in spring, oidd peaked in summer and a smaller peak in autumn, the peak incidence of hepatitis a occurred in summer and spring, and a distinct peak for tap was not observed. previous studies have revealed an association between the iids and seasons [ , [ ] [ ] [ ] [ ] . the occurrence of intestinal infectious diseases is related to climatic factors, such as the sunshine duration, temperature and humidity, as well as the quality of food and drinking water [ ] . due to the high temperature and humidity in summer, which are conducive to bacterial reproduction, food and water are easily contaminated. at the same time, the human immune system is relatively weak due to higher bodily exertion in summer. in conclusion, summer is the season with the highest incidence rates for iids and should receive closer attention. on the other hand, the high-risk areas for different iids were mainly located in the beijing-tianjin-tangshan (btt) region, yangtze river delta, south and west china. the former two sites are developed areas with extensive urbanization, which attract larger mobile populations characterized by low immune systems, poor living environments and living conditions, and poor health and knowledge of epidemic prevention measures. the hot spots for hfmd, oidd, dysentery and hepatitis e are located in the btt region and yangtze river delta [ ] . south china is located closer to the equator with a subtropical monsoon and tropical monsoon climate, which are characterized by high humidity, temperature, rainfall, and wind speed. iids are strongly correlated with the climatic character of south china [ ] , which is a high-risk area for hfmd, oidd, and tap. the high-risk areas for tap were located in southwest china (yunnan, guizhou, and guangxi), which are also the most likely cluster areas and hh cluster areas. the main reasons are that southwest china borders southeast asia (vietnam, laos, and myanmar), which has a high-risk population due to low sanitation. moreover, the population of southwest china has a medical history of tap and very poor climatic, geographical (karst landform) and sanitation conditions. who estimates of the global burden of foodborne diseases from farm to plate, make food safe food safety: what you should know from farm to plate, make food safe. world heal organ reg off south-east asia, indraprastha estate, mahatma gandhi marg integrated management of childhood illness: distance learning course: who epidemiologic and clinical features of non-polio enteroviral infections in northern taiwan in cholera outbreaks in africa transforming our world: the agenda for 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variables on hand, foot, and mouth disease in mainland china the emergence and outbreak of multidrug-resistant typhoid fever in china springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the national social science fund of china for its support and the chinese center for disease control and prevention for sharing the valuable data. finally, personal eating habits contribute to the incidence of tap [ ] . the west area experiences poor sanitation and a lower economy and is a high-incidence area for hepatitis a and dysentery.the strengths of this study are listed below. first, the study performed a comprehensive analysis of iids, including the temporal and spatial analyses of all reported intestinal diseases in china, to provide a complete picture of iids to other countries. second, the visualization of diseases represented a convenient method to show the distribution of diseases. however, this study had some limitations. we were unable to obtain more sophisticated results for the provincial units in china than were obtained for the county unit. regional discrepancies were also observed among provinces. furthermore, additional studies that explore the temporal and spatial distributions in smaller region units are needed. in conclusion, seasonal patterns and trends in different provincial geographical units were determined. higher incidence rates of iids were observed from may to october, which is the season with a climate characterized by heavy rain, high temperature, and high humidity. the climate zone is associated with the incidence. the highrisk areas for iids were detected in part of the border region, the south region and the region with better economic development.based on the results of our temporal and spatial analysis of iids, we identified the high-risk periods and cluster regions for the studied diseases. hfmd and oidd exhibited high incidence rates, reflecting the negligence of the government in monitoring class c diseases. at the same time, the incidence rate of hepatitis e gradually surpassed that of hepatitis a. the authorities should closely monitor class c diseases and hepatitis e. notable epidemiological trends were observed among different provinces. an effective response requires the implementation of a series of coherent and coordinated measures, which should be specific for the diseases that are endemic to a particular area. authors' contributions nz, ym conceptualized and designed the study. nz put forward the outline of the article with ym. bz, nz made data analysis, draw pictures and drafted the manuscript. jll and rxh revised the article. all authors read and approved the final manuscript. the study was funded by the major project of national social science fund of china: research on big health putting prevention first and construction of healthy china (grant number zda ). the funder had no role in the study design, data collection and analysis, interpretation of data, and writing the manuscript. all data generated or analyzed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.author details key: cord- -l f l authors: leitch, a e; duffin, r; haslett, c; rossi, a g title: relevance of granulocyte apoptosis to resolution of inflammation at the respiratory mucosa date: - - journal: mucosal immunol doi: . /mi. . sha: doc_id: cord_uid: l f l the respiratory mucosa is responsible for gas exchange and is therefore, of necessity, exposed to airborne pathogens, allergens, and foreign particles. it has evolved a multi-faceted, physical and immune defense system to ensure that in the majority of instances, potentially injurious invaders are repelled. inflammation, predominantly mediated by effector cells of the granulocyte lineage including neutrophils and eosinophils, is a form of immune defense. where inflammation proves unable to remove an inciting stimulus, chronic inflammatory disease may supervene because of the potential for tissue damage conferred by the presence of large numbers of frustrated, activated granulocytes. successful recovery from inflammatory disease and resolution of inflammation rely on the clearance of these cells. ideally, they should undergo apoptosis prior to phagocytosis by macrophage, dendritic, or epithelial cells. the outcome of inflammation can have serious sequelae for the integrity of the respiratory mucosa leading to disease. therapeutic strategies to drive resolution of inflammation may be directed at the induction of granulocyte apoptosis and the enhancement of granulocyte clearance. supplementary information: the online version of this article (doi: . /mi. . ) contains supplementary material, which is available to authorized users. the respiratory mucosa is the largest body surface area to be exposed to, and require defense from, the external environment. it is not surprising, therefore, that a robust, vigorous, and rapidly responsive immune defense system has evolved over millennia of exposure to diverse and increasingly sophisticated microbes. in addition, air-borne irritant particulate matter such as allergens, biopersistent fibers such as asbestos and combustionderived nanoparticles (e.g., diesel exhaust particles) must be overcome to maintain a high level of function. the architecture of the respiratory system provides a first layer of defense against air-borne microorganisms and particulate matter, effectively blocking the lower airways from anything greater than m in diameter. inhaled air is warmed and filtered through the nasal hair and nasopharyngeal passages before negotiating the larynx (which ordinarily prevents any intrusion from matter destined for the gastrointestinal system and allows phonation) down the large tubular trachea and into the upper bronchi. the initially large bronchial airways repeatedly bifurcate until they reach an extremely narrow gauge and form alveoli at which point gas exchange with the capillary network that lines the alveolar wall becomes possible. pseudostratified columnar-ciliated epithelium predominates in the trachea and bronchi forming the muco-ciliary escalator. any intruding microorganism or misplaced particulate matter slipping out of the air stream in the upper airways is trapped in the muco-ciliary escalator and forced up and out by the regular beating motion of the respiratory cilia. should these initial defenses be breached then an intruder must encounter an uninviting but nonspecific chemical milieu including lysozyme, endogenous antimicrobial agents, adverse ph, iga, and surfactants. should this fail then a more goaldirected system is brought on line. the respiratory mucosa has developed a sensitive and specific recognition strategy that allows it to identify microbe-specific protein patterns such as lipopolysaccharide, lipotechoic acid, formylated peptides, flagellin, and non-methylated dna. once detected, these galvanize resident alveolar macrophages and initiate a variety of proinflammatory pathways that instigate the classical elements of inflammation (calor (heat), rubor (redness), dolor (pain), tumor (swelling), and loss of function as described by celsus and (debatably) virchow) driven by increased vascular permeability leading to a proteinaceous infiltrate and leucocyte recruitment. the tolllike receptors (tlrs) mediate most of this form of recognition and though they each have a specific role, tlrs can function in unison to expand their powers of recognition. resident alveolar macrophages deal with the majority of insults that trigger this " relevance of granulocyte apoptosis to resolution of inflammation at the respiratory mucosa " review alert system but if they are overwhelmed recruited leucocytes including granulocytes lend assistance. the invading microbes are rendered highly visual to recruited granulocytes by comprehensive opsonization mediated by both complement-dependent and -independent means. they are then ingested (phagocytosed) by neutrophils or, if they should prove resistant to this because of size or learned subversion, are subjected to a chemical onslaught (exocytosis) as neutrophils forcibly externalize toxic granule substances such as lactoferrin and myeloperoxidase as well as reactive oxygen species (ros). , a subset of neutrophils (and other leucocytes potentially ) employs web-like neutrophil extracellular traps to ensnare and kill resistant organisms but must themselves die in the process. in beneficial neutrophil-dominant inflammation, the organisms or foreign particles are detected and phagocytosed by neutrophils, which then undergo an organized, nonprovocative programmed cell death (apoptosis) that promotes their own recognition and removal by macrophages or dendritic cells. at this point, the interface between the innate and adaptive immune systems occurs as macrophages, which migrate to the lympho-reticular system following ingestion of apoptotic neutrophils, act as antigen-presenting cells allowing the lymphocyte population to complete the resolution and remembrance process. the next time this particular organism is encountered a preprepared, specific response should be available to ensure it has less opportunity to make an impact. the lungs are the arena for another type of granulocyte-driven inflammatory response. eosinophils are present in larger numbers within the lungs of asthma sufferers and are recruited in greater numbers in response to sensitizing allergens such as pollen, house dust mite, and animal dander. this response has no obvious beneficial effects and it is still unclear why it should occur. the eosinophil is a useful and active defender against parasitic infection and has an armament specific to that end. it has been noted that as parasitic infections have been largely eradicated in western society, the incidence of this abnormal eosinophil response (termed allergy or atopy) has increased and it seems that eosinophilic inflammation occurs almost as an outlet for redundancy. inflammation does not always resolve neatly and, unfortunately, for diverse reasons, pulmonary inflammatory disease is one of the biggest drains on health resources in this and many other countries (respiratory disease cost in the united kingdom was £ . billion in ). , non-resolving or chronic inflammation is established when an acute inflammatory response fails to counter an instigating stimulus. in pulmonary infection, this may occur because a microorganism can subvert host defense by surviving either within inflammatory cells ( mycobacterium tuberculosis ) or inside a protective micro-environment ( pseudomonas aeruginosa ). , persistent inflammation may occur because of a recurrent stimulus like tobacco smoke (chronic obstructive pulmonary disease (copd)), allergens (asthma), or long (> m) biopersistent fibers (asbestosis). inflammation is also chronic where the host immune system malfunctions and becomes under-or overactive or misdirected as occurs in autoimmune disease (rheumatoid arthritis, systemic sclerosis), immune deficiency syndromes (chronic granulomatous disease, severe combined immunodeficiency syndrome), cystic fibrosis (cf), or adult respiratory distress syndrome / acute lung injury (ards / ali). in some cases, the etiology is unknown and subject to debate (idiopathic pulmonary fibrosis (ipf)). regardless of the cause, it is apparent that the over-recruitment, ineffective clearance, and hence accumulation and misdirected or frustrated activation of neutrophils and eosinophils lead to tissue damage and an inciting feedback loop of inflammation that perpetuates chronicity ( figure ). it is evident that granulocytes play an important role in the pathogenesis of pulmonary inflammation but other cell types such as macrophages, dendritic cells, mast cells, and lymphocytes are equally and potentially more influential in subsets of inflammatory respiratory disease. in this review, our focus will be on granulocyte research as expert reviewers have addressed the role of other cell types across the spectrum of respiratory disease and the interplay of different cell types within single diseases. our aim will be to address current understanding of granulocyte biology in relation to respiratory inflammatory diseases and prospects for driving resolution of inflammation at the respiratory mucosa to develop effective treatments for lung-based disease. pluripotent hematopoietic stem cells in the bone marrow generate the common myeloid progenitor cell. this cell differentiates into a common granulocyte progenitor cell, which when given appropriate stimulation can produce any granulocyte lineage. potential granulocyte lineages include neutrophils, eosinophils, and basophils. basophils are the least well studied and least numerous of the granulocytes, and their physiological function seems to overlap somewhat with that of eosinophils. they are probably meant to counter parasites but their relevance to western society is in the production of numerous mediators necessary for the incitement of the asthmatic response. neutrophils and eosinophils are discussed in greater detail below. granulocytes are terminally differentiated and having therefore exited the cell cycle, they remain in the g phase of growth for the duration of their lives. however, a recent report suggests that postmitotic neutrophils are capable of radical phenotypic switching to take on major characteristics of macrophages when stimulated with growth factors, whereas another study shows that mouse neutrophils can be switched to a dendritic cell phenotype following in vitro incubation with colony-stimulating factor- . , extravasated granulocytes recruited to the tissues ought to die by apoptosis whilst non-migratory, circulating granulocytes are removed by the combined efforts of the bone marrow, liver, and spleen. there is renewed interest in granulocyte life cycle and differentiation as pharmacological inhibitors of the cell-cycle machinery have been shown to promote granulocyte apoptosis. , neutrophils are - m in diameter and account for % of the circulating leucocyte population, which corresponds review to approximately . - . × / l neutrophils. they survive in the circulation for approximately - h but if compelled by inflammatory circumstance or chemical persuasion can extend their life span up to h and beyond. this population turnover requires efficient production by the bone marrow and prompt clearance by that same organ with help from the spleen and liver. neutrophils possess at least four different types of granules termed: primary (azurophilic), secondary (specific), gelatinase, and secretory; each carries a specific arsenal of toxic chemicals that the neutrophil uses against non-host elements. the neutrophil is a key effector cell at the front line of immune defense and is efficacious (and lifesaving) in the majority of instances because of its versatility. neutrophils prove to be effective phagocytes because on accomplishment of this task they are programmed to die a prompt, quiet and importantly, contained, apoptotic death. this limits the time available to internalized microorganisms and prevents hijacking of cellular controls to enable subversion (as is thought to occur when m. tuberculosis is internalized by macrophages ). this timely death also signals larger scale phagocytes such as macrophages, dendritic cells, and epithelial cells to initiate a phagocytic response. if a neutrophil is unable to phagocytose an invader then it will disgorge granule contents into the surrounding environment causing tissue damage and amplifying the inflammatory response. this should not be construed as a purely detrimental response as it serves to block potential routes of entry for invading organisms as tissue damage causes capillary shutdown and collapses lymphatics, sends a strong signal of imperiled defense, and allows time for an alternative immune strategy to be adopted. neutrophils may also employ extracellular traps to ensnare and kill resistant organisms. neutrophil extracellular trap formation involves the extrusion of fine chromatin and granulecoated tendrils that are microbicidal and fungicidal. this effort proves fatal to the neutrophil. finally, neutrophils are not just blunt effectors but also smooth operators responsible for negotiating the recruitment and education of other arms of the immune system. it is becoming appreciated that there is a false division between the innate and adaptive immune response systems and that a more realistic model involves a continual interplay between constituents of these systems. neutrophils not only alert antigen-presenting cells and lymphocytes to danger but regulate their response to it and in return receive counter regulation. appropriate resolution of an inflammatory immune response is finely balanced. figure diagram showing both resolution and failure of resolution of eosinophil-and neutrophil-dominant inflammatory processes. neutrophilia at the respiratory mucosa is resolved by apoptosis of infiltrating neutrophils and phagocytic clearance by macrophages. it fails to resolve where neutrophils are in great excess or are not efficiently cleared and undergo secondary necrosis following apoptosis. this leads to alveolar damage and destruction followed by fibrotic healing. histology on the right-hand side of the diagram shows neutrophil-dominant inflammation. eosinophil-dominant inflammation is resolved by the same mechanisms and fails to resolve for the same reasons. the effects of eosinophil-dominant inflammation in the asthmatic airway are depicted and an example of the histology demonstrated on the left-hand side of the diagram. histology was kindly provided by william wallace (pathology department, edinburgh royal infirmary). it is not surprising, given the constant exposure of the vast respiratory mucosa to threat, the fine balance of the immune response and the pivotal inflammatory role played by neutrophils, that neutrophil-dominant inflammation has been implicated in the pathophysiology of numerous inflammatory respiratory diseases including pneumonia, copd, ipf, cf, and ards. eosinophils are approximately - m in size and, under normal circumstances, account for less than % of the circulating leucocyte population. they may survive for up to h in the circulation but have the ability to extend their longevity to over a week if required. not only are they bigger than neutrophils but they also wear more flamboyant colors when stained by the romanowsky method (methylene blue and eosin), which accounts for their name. eosinophils may also be distinguished by their production of charcot -leyden crystals (manufactured from lysophospholipase, an eosinophil-derived enzyme), which are often visible in their cytoplasm. eosinophils, like neutrophils, are supplied with numerous granules though their constituents differ including major basic protein, eosinophil cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin. this array is certainly capable of tissue damage and probably evolved to combat helminthic infection. increasingly, new roles for eosinophils are being identified and it seems likely that they have a role in combating viral infection as eosinophil cationic protein and eosinophil-derived neurotoxin have been shown to degrade single-stranded rna viruses. they are said to play an early role in innate immunity by production of important cytokines such as interleukin (il)- though this is probably only physiologically important in the gastrointestinal tract. they can also modulate adaptive immunity by specific activation of t cells. eosinophils are implicated in a different spectrum of disease from neutrophils, of which the most common variety is allergic / atopic disease, which includes asthma, allergic rhinitis, and eczema. in other countries, parasitic infection is still common including schistosomiasis, dranunculoriasis, ascariasis, filariasis, and hookworm. eosinophil numbers may be increased in various cancers as well as in rare conditions such as churg -strauss syndrome, aspergillosis, and eosinophilic pneumonia. in eosinophil-dominant disease, it has been consistently demonstrated that eosinophil excess due to excessive recruitment, apoptosis avoidance, and failed clearance has a correlation with disease pathology. neutrophils and to a lesser extent eosinophils are short-lived cells, a feature that may help to limit their potential for causing damage by ensuring that they cannot be subverted by pathogens. they are explosively reactive cells and it is therefore surprising that their death is a model of contained self-restraint. apoptosis is a physiological marvel that allows cells with an incendiary cargo (granules) to package it efficiently and safely (in plasma membrane) so that the cellular environment is protected. in addition, the cell nucleus condenses and chromatin is chopped up and re-organized into packages termed nucleosomes. throughout this process, the cell membrane is retained intact though glycoprotein and phospholipid signals are displayed to attract macrophages and facilitate interaction and uptake (efferocytosis). fluorescently-tagged annexin-v is often used to label phosphatidylserine residues, which are characteristically flipped to the outer membrane of apoptotic cells thereby enabling flow cytometric identification of apoptosis. another member of this family, annexin- , has been shown to induce granulocyte apoptosis but is also released by both neutrophils and macrophages to enhance phagocytosis of apoptotic cells. , the whole process is amplified by many orders of magnitude during inflammation whether it be neutrophil or eosinophil dominant. unfortunately, apoptotic cells cannot stay that way forever, and if they are not cleared by phagocytes then they undergo secondary necrosis and all the good work is undone. large-scale granulocyte recruitment must necessarily be followed by large-scale granulocyte apoptosis and clearance by macrophages. it is extremely important that this is taken into account when plotting to drive granulocyte apoptosis to promote resolution of inflammation. , neutrophil and eosinophil apoptosis are similar but not identical processes. it is generally believed, though not universally, that there are two pathways by which apoptosis proceeds, both of which are ultimately dependent on the caspase family ( figure ). the intrinsic pathway occurs when the cell faces withdrawal of growth / survival factors, genotoxic stress, or ultraviolet irradiation. this pathway relies on proapoptotic members of the bcl- family, which escape regulation by their antiapoptotic counterparts and translocate to the mitochondria facilitating liberation of cytochrome c . the various components of the apoptosome (apaf- , cytochrome c etc.) then assemble to cleave the inactive zymogen, procaspase- , to active caspase- , which inexorably commits the cell to caspase- -mediated apoptosis. the extrinsic pathway proceeds through external cell-membrane death receptors such as the tumor necrosis factor receptor (tnfr), the fas receptor (fasr), and tnf-related apoptosis-inducing ligand receptor (trailr). ligand activation of these receptors promotes clustering of receptors and association with their internal adaptor proteins (tnfr-associated death domain protein and fas-associated death domain protein) in the lipid-raft. multiple procaspase- molecules assemble at the adaptor proteins (formation of the death-inducing signaling complex), and their physical approximation generates an autocatalytic reaction initiating the caspase cascade. the death receptor cd has also been implicated in neutrophil and eosinophil apoptosis but it is currently unclear what its physiological role is. in neutrophils the importance of caspase action in death receptor-mediated apoptosis has previously been a matter of debate but evidence of caspase-independent apoptosis has been effectively countered by work demonstrating that culture conditions and concentrations of caspase inhibitor had not been optimally utilized in these studies. to further complicate the story, it is apparent that there is a degree of cross talk between the intrinsic and extrinsic pathways. caspase- may alternatively cleave bid, which can translocate review to the mitochondrial membrane and permeabilize it allowing apoptosis to proceed through intrinsic pathway apoptotic machinery. , eosinophil apoptosis most likely occurs along approximately the same lines; however, controversy still reigns with regard to the caspase family. it has previously been stated that caspase- , - , and - have no demonstrable role in eosinophil apoptosis. subsequently, it has been suggested that perhaps caspase- plays a role and that therefore the mitochondrial pathway is of importance. this work was confirmed by observations with regard to the effects of known eosinophil apoptosis-inducing agents such as glucocorticoids, which appear to mediate their effects through mitochondrial permeabilization, with caspase activation merely a downstream effect. in contrast, fas ligation has been shown to promote eosinophil apoptosis in a caspase- -and - -dependent manner, and although mitochondrial integrity was disrupted it proved non-essential for apoptotic progression. , - support for the predominance of mitochondria-driven granulocyte apoptosis is provided by the increasing evidence that survival proteins are key determinants of neutrophil longevity. the antiapoptotic bcl- family member, mcl- , is present in both neutrophils and eosinophils, but other family members show contrasting expression. neutrophils express a , eosinophils express bcl-xl but bcl- has not been shown to be constitutively present in either cell at demonstrable levels. interestingly, however, in eosinophils, bcl- expression can be stimulated by il- . these survival proteins mediate their effects by marshaling proapoptotic bcl- family counterparts away from the mitochondrial membrane. proapoptotic bcl- family members are capable of causing permeabilization of the outer mitochondrial membrane (momp) when numbers predominate over those of their chaperones. bax is perhaps the best-characterized member of this family and appears to play a pivotal role in neutrophil apoptosis. in eosinophils, however, bax fails to be downregulated by antiapoptotic survival factors weakening its position as a key player in apoptosis. the role of mcl- in neutrophil apoptosis has received a great deal of attention and it appears that this protein is an essential component of neutrophil viability; a contention that is supported by the neutropenic phenotype of the mcl- knockout mouse in comparison with the increased apoptotic phenotype of the a knockout mouse. , granulocyte longevity is necessarily highly regulated and consequently highly variable from a matter of hours up to several days for neutrophils and extending to weeks for eosinophils. persistent markers of bacterial infection such as lipopolysaccharide, pathogen-derived molecules that act as ligands for tlr- , - and - , and inflammatory cytokines such as il- , il- , granulocyte macrophage-colony stimulating factor, and tnf can delay neutrophil apoptosis whereas resolving infection indicated by successful neutrophil phagocytosis of bacteria, removal of bacterial products, and downregulation of inflammatory cytokines will promote apoptosis. , granulocyte longevity is also extremely dependent on intracellular signaling pathways. perhaps the most important of which is that controlled by the pivotal transcription factor of inflammatory cytokines, nuclear factor (nf)-b. nf-b can be activated by lipopolysaccharide, and is known to control the survival proteins xiap (an inhibitor of caspase- , - , and - ) and bcl-xl thus enhancing neutrophil and eosinophil longevity by tangible means. pharmacological inhibitors of nf-b such as gliotoxin promote neutrophil apoptosis and, in combination with tnf-, cause dramatically enhanced apoptosis. this feature may partially explain why tnf-has opposing effects on neutrophils at early and late time points. , other important signaling pathways include the phosphoinositide -kinase pathway, which can be stimulated by granulocyte macrophagecolony stimulating factor to promote longevity by upregulation of mcl- in neutrophils and which has an emerging role in the maintenance of eosinophilic inflammation. , similarly, the extracellular signal-regulated kinase pathway is likely to be important for survival factor-mediated antiapoptotic effects at sites of inflammation. however, direct inhibition of either of these pathways will not promote granulocyte apoptosis per se . , perhaps the most interesting and clinically relevant modulation of granulocyte apoptosis is that achieved with the use of glucocorticosteroids. these drugs extend neutrophil life span but promote eosinophil apoptosis. this effect seems to correlate with their ability to upregulate mcl- in neutrophils but downregulate the same protein in eosinophils. dexamethasone has also been shown to promote macrophage phagocytosis of apoptotic neutrophils. the impact of putative inflammation-resolving agents on clearance of apoptotic neutrophils is of paramount importance as prompt removal is essential to avoid secondary necrosis and loss of toxic contents. this is perhaps why the discovery of a new class of endogenous lipid-derived proresolution agents has been so exciting. the lipoxin family produced by neutrophils and macrophages limits the recruitment of neutrophils to inflammatory sites and enhances macrophage phagocytosis of apoptotic neutrophils. this occurs as part of a natural brake on progression of inflammation and there is hope that enhancement of this pathway may provide a novel therapeutic strategy to counter inflammatory disease, a hypothesis that has already shown promise in several animal models. another exciting prospect for therapeutic modulation of granulocyte apoptosis has emerged with the discovery of active cell-cycle machinery in neutrophils, which can be inhibited to promote apoptosis. cyclin-dependent kinase inhibitor drugs promote neutrophil apoptosis and drive resolution of inflammation in animal models. given that these drugs are already in use for the treatment of cancer, it is possible that they could make the transition from bench to bedside for the management of inflammatory disease in the near future. , in normal healthy lungs, the predominant alveolar leucocyte is the macrophage, a cell that becomes resident following differentiation from a bone marrow-produced circulating precursor monocyte. alveolar macrophages occupy a unique position at the interface between inhaled air (and hence the external environment) and the circulation (in the form of the alveolar capillary network, which is closely juxtaposed to the alveolus to promote efficient gaseous exchange) and because key macrophage functions include chemotaxis, phagocytosis, and cytotoxicity they are lynch pins of immune defense. importantly, during pulmonary infective or inflammatory disease, a key role in resolution of inflammation falls to both alveolar and recruited circulating macrophages. these cells are responsible for the removal of apoptotic neutrophils and to some extent eosinophils (though epithelial cells may be equally important for eosinophil removal) by efferocytosis. in order for an apoptotic cell to be recognized by a macrophage it must display specific signals. the most obvious change in the apoptotic granulocyte ' s plasma membrane is the externalization of phosphatidylserine residues but this in itself is not sufficient to expedite phagocytosis. the search for a macrophage-docking receptor on granulocytes has been exhaustive but inconclusive and currently a combination of various glycoproteins and phospholipids is of postulated importance. these putative receptors include the recently identified tim, , stabilin- , and bai . the process of efferocytosis, literally meaning " burying the dead " , is a proresolution strategy in itself. macrophages that consume apoptotic neutrophils switch to a resolution phenotype that allows them to secrete tgf-and il- as opposed to proinflammatory cytokines such as il- , il- , and tnf-. this is in contrast to macrophages that have consumed necrotic neutrophils where the opposite is true and inflammation is actively propagated. in addition, the proresolution phenotype includes the production and enhanced responsiveness to lipoxins, protectins, and resolvins. these mediators enhance macrophage phagocytosis and promote proresolution cytokine production. there has been a tendency, certainly in clinical circles, to question the validity of inflammation-based hypotheses for various diseases based on the efficacy or lack of efficacy of glucocorticosteroid medications. given the complexity of the inflammatory response and the relative bluntness of this therapeutic tool, it is overly simplistic to make such assumptions. steroid medications have certainly been a paradigmatic therapy in the treatment of inflammatory disease but they do not and will not drive resolution of inflammation in all settings. with increased understanding of the mechanisms of the inflammatory review response and a new focus on its resolution, it is hoped that novel incisive or pleiotropic therapy combinations may be developed to address the inflammatory lung diseases discussed below. in most cases of copd ( - -antitrypsin-deficient patients are a notable exception), the respiratory mucosa is damaged by repeated exposure to inhaled toxic chemicals leading to chronic inflammation, reduced immunity, and susceptibility to respiratory infections. copd is a prevalent, largely smoking-related (though there is an increasingly recognized occupational contribution) disease in this country (in others it is related to the burning of bio-fuels), which presents with increasing breathlessness and a productive cough. it is an obstructive airways disease but unlike asthma this obstruction is irreversible with inhaled therapies. the mainstay of current treatment is with inhaled or oral corticosteroids and antibiotics when exacerbations are judged infective. (for interested readers, the refs. , , - give an overview of copd pathophysiology.) neutrophils are likely to play an important role in this condition and they are found in increased numbers throughout the respiratory tract. the highest concentrations of neutrophils are found in sputum and bronchoalveolar lavage fluid (bal) (which is perhaps representative of rapid airway-directed migration) but numbers of neutrophils are also increased in lung parenchyma and airway smooth muscle. neutrophilic inflammation is characteristic of copd exacerbations and there is a correlation between the resting burden of lung-based neutrophils and severity of disease phenotype. neutrophilic inflammation appears to be driven by the irritant force as smoking drives alveolar macrophages and epithelial cells to express increased levels of il- , a potent neutrophil chemoattractant. il- also stimulates neutrophils to release myeloperoxidase whereas tnf-and leukotriene b (also produced by epithelial cells, mast cells, and t lymphocytes) cause neutrophil activation (degranulation, reactive oxygen species production). meanwhile, macrophage phagocytic function is impaired by cigarette smoking and cannot keep pace with the increased neutrophil burden. enhanced neutrophil recruitment and activity combined with decreased macrophage phagocytosis weigh the scales heavily toward neutrophil-mediated tissue damage. copd lungs are subjected to high levels of toxic neutrophil products including reactive oxygen species, elastase, and proteinases. these toxic substances overload the capacity of native antiproteinases and antioxidants to neutralize them, which leads to damaged epithelial cilia and decreased mucociliary clearance. damage to the respiratory mucosa means that alveolar cells are replaced by goblet cells that increase mucous production. furthermore, airways are progressively remodeled by the reparative process so that they become thicker, less-efficient conductors of air. there is some controversy with regard to the longevity of neutrophils isolated from the airways of copd patients. studies have shown both enhanced longevity and no enhancement of longevity whereas peripheral blood neutrophils appear to have an increased life span in keeping with systemic inflammation. chronic obstructive pulmonary disease is notoriously resistant to glucocorticoid therapy perhaps because these drugs promote neutrophil longevity by upregulating the survival protein mcl- . it is also known that smoking promotes dysfunction of histone deacetylase - , an enzyme that is usually recruited by glucocorticoid receptors to switch off the transcription of proinflammatory genes. the small benefits attributable to steroid therapy in copd may stem from enhancement of macrophage phagocytosis of apoptotic cells. of other therapies currently in use, theophylline (a nonspecific adenosine antagonist and phosphodiesterase inhibitor) is known to restore histone deacetylase- function, reversing corticosteroid resistance and reducing il- concentrations and sputum neutrophilia. longacting agonists (originally employed to relax smooth muscle in peripheral airways) have now been shown to inhibit neutrophilic inflammation as measured by sputum or bal analysis, and they are reported to drive neutrophil apoptosis though the circumstances of this are difficult to ascertain. it is clear that potential therapies must consider targeting neutrophil recruitment as well as striving to drive neutrophil apoptosis and removal. , , , in asthma, the respiratory mucosa is hypersensitive to exposures that are comfortably processed under normal conditions and a characteristic inflammatory response ensues. it is part of a spectrum of atopic or allergic disorders, where an enhanced sensitivity to particular antigens or environmental conditions results in a disease flare. exacerbations of asthma are characterized by narrowing of the airways (bronchoconstriction), which leads to shortness of breath, wheezing, and cough. (for interested readers, these refs. , - give an overview of asthma pathophysiology.) recent work has suggested that there may be many subtle mucosal abnormalities that contribute to the pathogenesis of asthma. for example, it appears that exacerbations of asthma caused by rhinovirus may be facilitated by a defect in interferon induction, which increases susceptibility to invasive disease whereas viral exacerbations or damage caused by chronic inflammatory disease facilitate epidermal growth factor receptor signaling (a wound repair response), which enhances neutrophil function and chemotaxis. , the allergy arm of this disease is mediated by ige, which is produced by b cells in response to an initial exposure to a given antigen. ige attaches to mast cells and basophils priming them to release histamine, leukotrienes, and ils at subsequent exposures. traditionally, asthma is an eosinophil-dominant disease and eosinophils are heavily recruited by the same th cell type signaling (il- , il- , il- ) responsible for the production of ige. this process was thought to be under the direct control of il- but as anti-il- therapy has proved ineffective, it is suspected that there is a greater redundancy in the system. anti-il- therapy failed to significantly reduce eosinophil numbers in the asthmatic airway but did decrease deposition of extracellular matrix proteins suggesting a role for il- in airway remodeling. , interestingly, an il- analog (pitrakinra), which review prevents binding of il- and il- to il- receptor complex, attenuated the late-phase response (bronchoconstriction measured by reduction in forced expiratory volume in s) to allergen challenge. it is as yet unclear whether this success was due to inhibition of il- alone, il- alone, or due to combined inhibition. in addition, a role for th cells has been postulated particularly in forms of asthma where neutrophil recruitment contributes to pathogenesis. regardless of the super-intending mechanism, it is clear that a heavy burden of apoptosis-resistant eosinophils concomitantly resistant to macrophage phagocytosis, yet capable of disgorging their formidable armament onto the respiratory mucosa, have significant potential to cause tissue damage and airway remodeling. thankfully, asthma is usually responsive to glucocorticoid therapy partly because steroids are effective at promoting eosinophil apoptosis. this apoptosis may, in direct contrast to experience in neutrophils, be mediated by downregulation of mcl- . rarely, asthma may be steroid resistant and there are different theories as to how resistance might arise. first, it is clear that smokers with asthma respond poorly to steroids because of the defective action of histone deacetylase- (discussed earlier with regard to copd patients). second, it is apparent that a subset of asthmatics as well as end-stage or severe asthmatics manifests a neutrophil-dominant inflammation for reasons that are unknown. it has been postulated that these patients may represent a different disease or disease phenotype. third, it is known that airway epithelial damage results in smooth muscle hypertrophy causing significant luminal narrowing without a requirement for a significant bronchoconstrictor stimulus and finally, it is postulated that resistant asthmatics might express gene polymorphisms encoding defective caspase machinery. regardless of efficacy, the side effect profile (adrenal suppression, osteoporosis, peptic ulcer disease) of higher dose steroid therapy in the subset of resistant patients makes it less than ideal as they potentially require prolonged or maintenance treatment. it should be emphasized, however, that maintenance therapy with routine doses of inhaled steroids has established symptom control and reduced mortality in the majority of patients without these side effects becoming a problem. anti-ige therapy is showing promise in a subset of patients, and increased eosinophil apoptosis and downregulation of inflammatory cytokines have been demonstrated in peripheral blood samples taken from asthmatics treated with omalizumab (a recombinant humanized monoclonal antibody that selectively binds ige). future developments will probably include anti-il- , anti-il- , and potentially anti-th -based therapies, now that there is a rationale for their development. however, it is not always best practice to attempt a one-hit, magic-bullet cure especially in inflammatory disease where there is considerable redundancy in the cytokine system. if an agent was developed that could promote eosinophil apoptosis without detriment to macrophage phagocytosis and with a negligible side effect profile, it would be an extremely useful addition to the pharmacopoeia. idiopathic pulmonary fibrosis is a condition characterized by the devastation of the respiratory mucosa though its pathophysiology is complex. there is a great deal of debate about the relative importance of inflammation vs. aberrant wound repair in the pathogenesis of ipf. there is not sufficient space to address that debate in this article, suffice to say that there is enough evidence to justify an argument in support of a significant neutrophilderived component to the disease model. (for interested readers, these references give an overview of the pathophysiology of ipf and a flavor of the debate. , - ) the argument is that the initial and subsequent " exacerbatory " insults are acute and inflammatory even if the characteristic phenotype is conveyed by a disordered " healing " process. healing by fibrosis results in significant loss of lung architecture and vital respiratory mucosa to scarring. neutrophils are known to be present in bal from ipf patients and are also significantly increased within lung tissue. in addition, there is evidence of the toxic chemicals produced by neutrophils including myeloperoxidase, elastase, collagenases, and proteases. the importance of this chemical insult is supported by the elastase knockout mouse, which is significantly protected against lung injury and also fails to upregulate tgf-. a recent clinical paper has shown that significant neutrophilia within the bal fluid of ipf patients correlates with increased mortality. it has been argued that the terminal scarring process of ipf occurs because of a significant loss of lung architecture and that where lung architecture is preserved there is potential for reversal of the remodeling process and resolution. it has also been suggested that the fibrotic response may in a sense be driven by a persistent but ineffective proresolution of inflammation phenotype. this hypothesis is drawn from the observation that tgf-is a key proresolution molecule but is also intimately involved in fibrogenesis. in a disease model where non-resolving low-level inflammation results in sustained tgf-production, it is possible to conceive that a fibrotic healing phenotype might evolve. therapeutic strategies that drive neutrophils toward apoptosis will certainly have something to add to treatment of this disease if they can prevent or ameliorate the inflammatory insult (whether it be acute, chronic, or relapsing) that must be responsible for such extreme distortion of lung architecture. , adult respiratory distress syndrome / acute lung injury is the pulmonary component of the multi-organ dysfunction syndrome (mods) and represents a global failing of lung function in response to a specific, nonspecific, or unidentifiable stimulus. effectively, these conditions are the result of an aggressive mucosal inflammatory immune response. (the refs. , - cover the wider pathophysiology of ards / ali.). characterized by the pathological reaction termed diffuse alveolar damage, ards / ali seems to be a neutrophil dominant disease (interestingly, diffuse alveolar damage is also seen in the terminal stages of ipf). analysis of bal fluid from patients with early-stage ards demonstrates increased numbers of activated neutrophils and their numbers correlate with severity of review lung injury. indeed, persistence of bal neutrophilia at day is associated with increased mortality. the pulmonary circulation contains a large sequestered neutrophil population termed the " marginated " pool that does not normally circulate but is loosely adhered to the vessel walls. this population can be mobilized into the circulation by steroid therapy or by exercise. neutrophils are subject to slow transit through the pulmonary microvasculature where blood vessel diameters are smaller than their own. the ability of neutrophils to progress depends on their considerable properties of distensibility. in ards / ali, neutrophils are recruited early in large numbers, and an alteration in their rheological properties (they become less deformable) means that they struggle to maneuver through vessels whereas cytokine excess encourages their adhesion to vessel walls and subsequent translocation into the lung parenchyma and airways. the influx of huge numbers of neutrophils and proteinaceous inflammatory edema fluid from permeabilized vessels impairs gas exchange and makes adequate ventilation extremely difficult. neutrophil influx is driven by alveolar macrophage production of il- , and excessive levels of this cytokine have been found to be predictive of progression to ards in susceptible patients. there is no effective, specific therapy for this condition and yet some patients survive with supportive therapy alone, implying that successful resolution is possible and can be mediated by physiological mechanisms. neutrophils isolated from the bal of ards patients have enhanced longevity, are activated, and may cause insurmountable damage to the respiratory mucosa leading to healing by fibrosis and permanent scarring with loss of lung architecture and function. in experimental models where neutrophils are depleted, ali caused by endotoxin is attenuated. in other studies, enhancement of neutrophil apoptosis also reduced inflammatory parameters and tissue damage. the picture is complicated by apparent ards / ali in neutropenic human patients. it is also clear that given the extravagant neutrophil influx associated with this condition, it would likely require a dual strategy of enhancing both neutrophil apoptosis and neutrophil clearance to ensure that secondary necrosis (and tissue damage) is prevented by prompt removal. , pneumonia pneumonia is the term given to an infection within the lower respiratory tract that is sufficiently significant to result in visible changes on a chest radiograph. there are many potential causative organisms including viruses and bacteria. invading pathogens that overcome immediate host defenses are nonetheless recognized as foreign by their non-host constituents such as lipopolysaccharide or formylated peptides in their outer membranes. tlrs, cd , and g-protein coupled receptors (e.g., fpr) found on macrophages and other respiratory mucosal cells are responsible for this detection and subsequently stimulate an acute inflammatory reaction by the production of cytokines such as tnf-, il- , il- , and il- . this proinflammatory milieu prolongs neutrophil life span and promotes neutrophil activation to allow effective microbial phagocytosis and killing. , , if the inflammatory reaction is successful in containing the pathogens and they are cleared by phagocytosis or killed, then the inflammatory reaction remains localized to a single lobe or segment of the lung. the upregulation of anti-inflammatory, proresolution molecules should ensure containment of inflammation. neutrophil phagocytosis of bacteria and the induction of proresolution cytokines such as il- and tgf-promote neutrophil apoptosis and removal by macrophages. the alternative is non-resolution with enhanced neutrophil longevity and local tissue damage (resulting in abscess, empyema, or bronchiectasis) and / or loss of containment resulting in global lung inflammation characterized by ards or spread through the circulation to other organs (sepsis) with the potential for mods. streptococcal pneumonia (the pathogen responsible is streptococcus pneumoniae ) is often cited as a paradigm of resolving inflammation as despite an acute inflammatory response characterized by a massive neutrophil influx (giving the pathological appearance referred to as " red hepatization " ) it is possible for the lung to achieve complete recovery. there is, however, a sub-population of severe pneumonia that behaves aggressively or is not contained by the patient ' s immune system and antibiotic therapy resulting in admission to intensive care and potentially mods, sepsis, terminal decline, and death. antibiotics are central to the treatment of pneumonia but they are not always sufficient and it is possible that addressing acute inflammation associated with pneumonia may prove a successful adjuvant therapy. in this setting, it is unclear whether the ideal strategy is to dampen down the inflammatory response or to augment it in the hope that a supra-physiological immune reaction will prove successful in eradicating the organism responsible. glucocorticoid therapy has been used as an adjuvant to antibiotics in this kind of patient with varying success rates in different trials. the theory is that steroids should dampen inflammation through downregulation of nf-b activation and consequently the inflammatory cytokines under its direct transcriptional control. a recent study that utilized a physiological steroid dose (supra-physiological doses had been employed in previous studies) has shown a significant reduction in length of hospital stay and mortality in intensive care unit patients with severe community-acquired pneumonia. it is possible therefore that in a subgroup of patients with pneumonia, strategies to aid resolution of inflammation (in combination with antibiotic therapy) may be warranted. cystic fibrosis is the commonest inherited disease in caucasian populations affecting one in , births. it seems surprising that a defect in salt transport (the genetic abnormality affects the cystic fibrosis transmembrane regulator gene ( cftr ), which encodes a chloride channel) should be responsible for such widespread organ pathology including pancreatic insufficiency, bronchiectasis, liver dysfunction, infertility, and gut defects. mortality (on average at age in western society) is most frequently from respiratory failure caused by aggressive bronchiectasis. there are competing hypotheses as to the root cause of the repeated infective exacerbations that lead to bronchiectasis in cf. it is postulated that a relative dehydration of the respiratory table continued on following page review mucosa means that the mucociliary escalator is compromised by viscid mucus and that altered mucosal ph results in malfunction of host antimicrobial peptides. there may also be a failure of normal bacterial internalization processes and it has been suggested that an intrinsic proinflammatory phenotype is conferred by cftr malfunction. the proinflammatory phenotype theory postulates that an overburdened endosomal system encumbered by the processing of faulty cftr signals distress, which leads to activation of nf-b. production of il- is stimulated resulting in enhanced neutrophil recruitment. regardless, the net result is a failure of mucosal immunity, and a succession of bacterial infections occur (initially, staphylococcus aureus and / or haemophilus influenzae but subsequently and devastatingly p. aeruginosa , burkholderia cepacia , and stenotrophomonas maltophilia ). with bacterial colonization, innate defense is roused and a significant neutrophil influx occurs. this is usually thwarted, initially by microbial resistance and subsequently by distorted lung architecture. this results in an insurmountable accumulation of inflammatory cells and toxic damage from chemicals eluted by their necrotic carcasses. elastase, reactive oxygen species, and myeloperoxidase cause further lung damage, and the clearance of inflammatory cells is so poor that abbreviations: ards/ali, adult respiratory distress syndrome/acute lung injury; cf, cystic fi brosis; copd, chronic obstructive pulmonary disease; ipf, idiopathic pulmonary fi brosis. the lungs literally become clogged with inflammatory cell dna. eventually, there is a terminal paucity of functional gas-exchange equipment, and respiratory failure and death supervene if lung transplant is not possible. recent work on p. aeruginosa has provided an interesting insight into the mechanisms by which this organism thwarts immune defense. it was known that p. aeruginosa could effectively hide from the immune system in a protective and impenetrable bio-film. a more subtle effect mediated by pseudomonas produced pyocyanin has now been recognized. this chemical promotes neutrophil death but inhibits macrophage clearance resulting in neutrophil death by necrosis. this leads to the spillage of cathepsin g and elastase, which can cleave the important chemokine receptor cxcr on other neutrophils. this receptor would usually allow il- -mediated enhancement of neutrophil killing prowess, which enabling neutrophils to kill pseudomonas. in cf patients, neutrophils have little cxcr for the above reason and are consequently effectively crippled as well as out-maneuvered. even more detrimental, fragments of this receptor stimulate the tlr system resulting in enhanced neutrophil recruitment and prolongation of the inflammatory response. anti-inflammatory therapy in the form of non-steroidal anti-inflammatory drugs has been used to mild benefit in cf but it appears that the early introduction of a potent anti-inflammatory proresolution therapeutic strategy might be of enhanced benefit. neutrophils in cf patients are dysfunctional and ineffective at restraining or removing typical cf pathogens, so driving their apoptosis and removal might be of more benefit than allowing them to remain in the hope that they are contributing to defense against these microbes. there are many methods available for the production of relevant animal models of inflammatory disease. these include direct chemical / other injury, constitutive and inducible transgenics, viral vector delivery of relevant genes, adoptive cell transfer, and direct infection strategies. the advantage of the animal model is that it reproduces the complexity of the wider immune / inflammatory response on an organ or system-wide basis as opposed to cell-line strategies that can only examine individual cellular responses. a selection of key rodent models and their major advantages / disadvantages are shown in table . a more comprehensive discussion of this field is not possible due to space limitations but there are excellent reviews available in this area. - clearly, reliable animal models are central to the development of efficacious and safe pharmaceutical agents that drive granulocyte apoptosis and clearance to enhance resolution of inflammation. the respiratory mucosa is frequently threatened with microbial invasion because it is responsible for gas exchange. consequently, it has developed a sophisticated, sensitive surveillance system and a rapid-response immune policing arm to ensure that it usually prevails in a given interaction. in inflammatory disease, this system is thwarted or dysfunctional with devastating results. the respiratory mucosal immune and inflammatory response can be volatile and damaging for lung function and architecture. to understand respiratory inflammatory disease, it is essential to fully appreciate the biology of archetypal inflammatory cells such as granulocytes. to drive granulocyte apoptosis and enhance granulocyte clearance is a research priority that will hopefully enable amelioration or cure of prevalent (and obscure) respiratory inflammatory conditions. crofton and douglas's respiratory diseases disturbance of function (functio laesa): the legendary fi fth cardinal sign of infl ammation, added by galen to the four cardinal signs of celsus toll-like receptor (tlr)-based networks regulate neutrophilic infl ammation in respiratory disease modulation of granulocyte apoptosis can infl uence the resolution of infl ammation resolution of infl ammation: state of the art, defi nitions and terms phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation neutrophil extracellular traps kill bacteria neutrophils and immunity: challenges and opportunities the future of lung research in the uk british thoracic society . health select committee inquiry into health inequalitiessubmission from the british thoracic microbial subversion of the immune response subversion of a lysosomal pathway regulating neutrophil apoptosis by a major bacterial toxin, pyocyanin granulocyte apoptosis and its role in the resolution and control of lung infl ammation mouse neutrophilic granulocytes express mrna encoding the macrophage colony-stimulating factor receptor (csf- r) as well as many other macrophage-specifi c transcripts and can transdifferentiate into macrophages in vitro reprogramming of human postmitotic neutrophils into macrophages by growth factors life and death during hematopoietic differentiation cyclin-dependent kinase inhibitors enhance the resolution of infl ammation by promoting infl ammatory cell apoptosis interactions of pathogenic mycobacteria with host macrophages . microbes infect benefi cial suicide: why neutrophils die to make nets the eosinophil annexin and neutrophil apoptosis annexin- and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages granulocyte apoptosis in the pathogenesis and resolution of lung disease tumor necrosis factor alpha induces a caspase-independent death pathway in human neutrophils neutrophil apoptosis pathways and their modifi cations in infl ammation z-vad-fmk augmentation of tnf alpha-stimulated neutrophil apoptosis is compound specifi c and does not involve the generation of reactive oxygen species regulation of eosinophil and neutrophil apoptosis--similarities and differences molecules involved in the regulation of eosinophil apoptosis regulation of eosinophil apoptosis: transduction of survival and death signals critical role for caspases and in neutrophil but not eosinophil apoptosis il- but not interferon-gamma (ifn-gamma) inhibits eosinophil apoptosis by up-regulation of bcl- expression bcl-xl-and bax-alphamediated regulation of apoptosis of human neutrophils via caspase- the antiapoptotic protein mcl- is essential for the survival of neutrophils but not macrophages accelerated neutrophil apoptosis in mice lacking a -a, a subtype of the bcl- -related a gene the role of toll-like receptors in the regulation of neutrophil migration, activation, apoptosis nf-kappab activation is a critical regulator of human granulocyte apoptosis in vitro possible new role for nf-kappab in the resolution of infl ammation phosphoinositide- kinases critically regulate the recruitment and survival of eosinophils in vivo: importance for the resolution of allergic infl ammation tissue-and stimulus-dependent role of phosphatidylinositol -kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo granulocyte clearance by apoptosis in the resolution of infl ammation the differential effect of dexamethasone on granulocyte apoptosis involves stabilization of mcl- l in neutrophils but not in eosinophils glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of infl ammation resolution phase of infl ammation: novel endogenous anti-infl ammatory and proresolving lipid mediators and pathways novel pharmacological strategies for driving infl ammatory cell apoptosis and enhancing the resolution of infl ammation tim- and tim- glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells rapid cell corpse clearance by stabilin- , a membrane phosphatidylserine receptor bai is an engulfment receptor for apoptotic cells upstream of the elmo/dock /rac module macrophages that have ingested apoptotic cells in vitro inhibit proinfl ammatory cytokine production through autocrine/paracrine mechanisms involving tgf-beta, pge , paf alveolar macrophages in chronic obstructive pulmonary disease (copd) new molecular targets for the treatment of neutrophilic diseases pathogenesis of chronic obstructive pulmonary disease macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modifi ed by cigarette smoke and lipid peroxidation products corticosteroid resistance in chronic obstructive pulmonary disease: inactivation of histone deacetylase the neutrophil in chronic obstructive pulmonary disease pathophysiology of asthma pathophysiology of severe asthma targeting airway infl ammation in asthma: current and future therapies understanding the pathophysiology of severe asthma to generate new therapeutic opportunities the pathophysiology of asthma eosinophils and bronchial epithelial cells: new insights into the resolution of infl ammation in asthma role of defi cient type iii interferon-[lambda] production in asthma exacerbations enhancement of neutrophil function by the bronchial epithelium stimulated by epidermal growth factor anti-il- treatment reduces deposition of ecm proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics eosinophil's role remains uncertain as anti-interleukin- only partially depletes numbers in asthmatic airway effect of an interleukin- variant on late phase asthmatic response to allergen challenge in asthmatic patients: results of two phase a studies il- mrna in sputum of asthmatic patients: linking t cell driven infl ammation and granulocytic infl ux? opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes a different disease, many diseases or mild asthma gone bad? challenges of severe asthma acute exacerbations of idiopathic pulmonary fi brosis jr challenges in pulmonary fi brosis x : the nsip/uip debate state of the art. does current knowledge explain the pathogenesis of idiopathic pulmonary fi brosis?: a perspective infl ammationassociated remodelling and fi brosis in the lung -a process and an end point what differentiates normal lung repair and fi brosis? infl ammation, resolution of repair, fi brosis mice lacking neutrophil elastase are resistant to bleomycin-induced pulmonary fi brosis jr baseline bal neutrophilia predicts early mortality in idiopathic pulmonary fi brosis neutrophils and acute lung injury the pulmonary physician in critical care * : the pathogenesis of ali/ards acute respiratory distress syndrome interactions between mechanical and biological processes in acute lung injury acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management interleukin- and development of adult respiratory distress syndrome in at-risk patient groups ventilator-associated lung injury: a search for better therapeutic targets bts guidelines for the management of community acquired pneumonia in adults decreased apoptosis and increased activation of alveolar neutrophils in bacterial pneumonia neutrophils undergo apoptosis following ingestion of escherichia coli infl ammation--a new therapeutic target in pneumonia hydrocortisone infusion for severe communityacquired pneumonia: a preliminary randomized study airway infl ammation in cystic fi brosis incapacitating the immune system in cystic fi brosis transgenic animals in infl ammatory disease models cystic fi brosis mouse models murine models of asthma models of pulmonary fi brosis murine models of pulmonary fi brosis modeling infl ammation in the zebrafi sh: how a fi sh can help us understand lung disease high dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fi brosis the bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fi brosis? conditional expression of transforming growth factoralpha in adult mouse lung causes pulmonary fi brosis adenovector-mediated gene transfer of active transforming growth factor-beta induces prolonged severe fi brosis in rat lung gene transfer for cytokine functional studies in the lung: the multifunctional role of gm-csf in pulmonary infl ammation transient expression of il- beta induces acute lung injury and chronic repair leading to pulmonary fi brosis expression of a tumor-necrosis-factor-alpha transgene in murine lung causes lymphocytic and fi brosing alveolitis -a mouse model of progressive pulmonary fi brosis transgenic modeling of interleukin- in the lung animal models of asthma mechanisms of cigarette smokeinduced copd: insights from animal models the pathogenesis of copd: lessons learned from in vivo animal models transgenic and gene-targeted mice as models for chronic obstructive pulmonary disease hyperoxic acute lung injury and ventilator-induced/ associated lung injury: new insights into intracellular signaling pathways characterization of a murine model of endotoxin-induced acute lung injury systemic blood loss affects nf-kappa b regulatory mechanisms in the lungs small-volume fl uid resuscitation with hypertonic saline prevents infl ammation but not mortality in a rat model of hemorrhagic shock respiratory reovirus /l induction of diffuse alveolar damage: a model of acute respiratory distress syndrome we thank the wellcome trust (wt ) and the medical research council (g ) for their support of the work that has enabled us to contribute to this review, and our friends and colleagues within the field whose work constitutes the bulk of the information provided. key: cord- -r e t c authors: de rooij, doret; rebel, rebekka; raab, jörg; hadjichristodoulou, christos; belfroid, evelien; timen, aura title: development of a competency profile for professionals involved in infectious disease preparedness and response in the air transport public health sector date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: r e t c background: recent infectious disease outbreaks highlight the importance of competent professionals with expertise on public health preparedness and response at airports. the availability of a competency profile for this workforce supports efficient education and training. although competency profiles for infectious disease control professionals are available, none addresses the complex airport environment. therefore, the main aim of this study is to develop a competency profile for professionals involved in infectious disease preparedness and response at airports in order to stimulate and direct further education and training. methods: we developed the competency profile through the following steps: ) extraction of competencies from relevant literature, ) assessment of the profile in a national rand modified delphi study with an interdisciplinary expert group (n = ) and ) assessment of the profile in an international rand modified delphi study with an airport infectious disease management panel of ten european countries (n = ). results: we systematically studied two competency profiles on infectious disease control and three air transport guidelines on event management, and extracted relevant competencies for airports. the two rand modified delphi procedures further refined the profile, mainly by specifying a competency’s target group, the organizational level it should be present on, and the exact actions that should be mastered. the final profile, consisting of competencies, covers the whole process from infectious disease preparedness, through the response phase and the recovery at airports. conclusion: we designed a profile to support training and exercising the multidisciplinary group of professionals in infectious disease management in the airport setting, and which is ready for use in practice. the many adaptations and adjustments that were needed to develop this profile out of existing profiles and air transport guidelines suggest that other setting-specific profiles in infectious disease control are desirable. a a a a a to the best of our knowledge, there is currently no profile that describes the competencies that professionals involved in infectious disease management at airports need. therefore, this study aimed to develop such a profile that describes the competencies that a multidisciplinary group need-in addition to the basic knowledge and skills required for their profession-when called upon to prepare and respond to infectious diseases in the airport environment. we performed the systematic rand modified delphi consensus procedure [ ] to develop a competency profile for professionals involved in infectious disease preparedness and response at airports. competencies were extracted from existing competency profiles and air transport guidelines on event management and consecutively presented to a national and an international panel of professionals. both panels followed a methodologically identical path (fig ) that consisted of two rounds of data collection. first, panels assessed the relevance of competencies regarding their expertise during a digital questionnaire. next, consensus meetings were held to gather consensus on undecided competencies. the refined final competency profile based on the first national round formed the concept competency profile for the international validation. this method is commonly used for the development of competency profiles [ ] , using the collective subjective judgment of professionals [ ] . step and were performed twice in this study: first with a national expert panel and after with an international expert panel. the study population of the national panel consisted of professionals involved in infectious disease preparedness and response at schiphol airport. this airport is the only ihr designated airport in the netherlands, one of europe's largest airports and a major hub in international air traffic [ ] . the study population of the international panel consisted of professionals involved at a local or national level in infectious disease preparedness and response at major european airports. the research team aimed for two panels consisting out of - participants [ ] , resulting in a national panel of and international panel of participants. in this way, we tried to assure both enough reflection of different opinions and a setting for a safe and fruitful discussion. the panels were recruited using purposive sampling according to inclusion criteria until a representative panel was composed or time was limited for further inclusions. the research team composed an invitation letter by e-mail explaining the background, aim, and method of the study for both panels, through which professionals could confirm their participation. for the national panel, we consulted an experienced nurse from the public health service at schiphol airport to discuss our sample. we invited experienced professionals for the national round from the following disciplines: international medical advice, airport medical services, public health service, air traffic control, flow management of aircrafts and passengers, airport fire officer, continuity & crisis management, and the disaster medicine organization. international medical advice, airport medical services, and the public health service collaboratively ensure that the infectious disease policy is feasible and effective at the airport. the public health service is responsible for the public health response. at their request, international medical advice facilitates medical items on board, such as personal protective equipment, and debrief crew and ground staff of an affected aircraft. in the situation that medical care is required, the ambulance will assist passengers upon arrival. professionals from air traffic control receive warnings from the pilot in command of a possibly affected aircraft and are responsible for informing the flow management of aircrafts and passengers which adjusts logistical processes at the airport. the airport fire officer ensures coordination among various emergency services during the management of an infectious disease case. the continuity & crisis management is responsible for advising on risk, crisis, and physical safety management. disaster management organization provides training in the field of infectious disease control at the airport. the international panel was recruited by selecting professionals who participated in a european, face-to-face -day training course on infectious disease control at designated airports. this course was part of the european union (eu) joint action healthy gateways [ ] and took place on - september in belgrade, serbia. we used these professionals' selfdeclared competence on local and national level before the training to approach a variety of professionals. on the local level, a functional distinction was made between competence in preparedness planning, decision making, and implementation. we invited professionals in all competence areas and tried to include one participant per country, with countries geographically divided over europe. step -literature search and extraction of competencies. we performed a literature search to identify competency profiles and air transport guidelines on event management. the search was executed in the th week of march using pubmed and google scholar. for competency profiles, search terms were 'infectious diseases' or 'public health', and 'competency' and their synonyms. for the airport guidelines, search terms were 'infectious diseases' or 'emergency preparedness' or 'public health event', together with 'airport' or 'air travel' or 'points of entry'. in pubmed, the search terms were limited to the title or abstract. in google scholar, we searched several combinations of search terms and screened the results until hits in a row were irrelevant. the search terms, the search strategy, the screening, and application of the selection criteria are shown in s file. regarding the competency profiles, the criteria for title/abstract screening were whether studies aimed at presenting competencies, and were aimed at infectious disease preparedness or control. inclusion criteria for the full-text screening of the competency profiles were the explicit presentation of a competency set in the study, competencies aimed at graduate level or professionals in practice, applicable to infectious disease management. further criteria to select the landmark studies contained a full array of competencies, being a peer-reviewed study, and being relevant for the airport setting. regarding the airport guidelines, the criteria for title/abstract screening were documents describing infectious disease management in the air travel setting with a disease-generic approach. the full-text screening inclusion criteria were documents aimed for the airport or air travel setting, prescribing infectious disease management in terms of specific preparedness and response tasks, with a disease a-specific approach. further selection was made based on the target group at airport level, whether unique topics were prescribed compared with other included studies, and presenting a full array of the preparedness and response process. the competency profiles and air transport guidelines were matched to develop a concept competency profile, using the following steps. first, we extracted main tasks for infectious disease preparedness and control from the guidelines that cover infectious disease management at airports during preparedness, response, and recovery. then, the competency profiles and air transport guidelines were independently reviewed by two researchers (ddr & rr) to extract competencies or textual fragments that could be reformulated into a competency, and which were relevant for a task. results were compared and dissimilarities were discussed until consensus was reached. subsequently, a data reduction round took place by applying three specifying criteria. first, we further narrowed the scope to the airport environment itself instead of competencies required regionally or countrywide. in addition, we restricted competencies on hygiene to the commanding level and excluded all operational cleaning. lastly, competencies or textual fragments regarding general team competencies were excluded in the profile because the team resource management (trm) skill set is already available and includes these [ ] . the trm skill set, developed for aviation safety, covers skills such as interpersonal communication, leadership and decision making, and is complementary to our competency profile. finally, we combined overlapping or congruent competencies and fragments. the included textual fragments were reformulated into competencies according to bloom's taxonomy of education objectives [ ] . data reduction was performed by rr & ddr collaboratively, all doubts and several versions of the draft profile were discussed with other researchers (eb and at). next to specifying competencies to a task, we also specified them to a role to further enhance the logical structure and the usability of the profile. for this purpose, we adapted the canmeds roles [ ] , which are widely used in medical education, into the following roles required in infectious disease management: ( ) health expert, ( ) organizer (including policy development), ( ) scholar. roles with respect to the communicator, professional and collaborator are required in general, and are, therefore, considered general tasks. during several discussion meetings with a third researcher (at), competencies were divided over tasks and roles and overlapping competencies were removed. this first step resulted in a concept competency profile. step -the national panel: digital questionnaire & consensus meeting. the next step contained the assessment of the draft competency profile in the national panel. a digital questionnaire was built in the web-based program formdesk [ ] . it was successfully pilot-tested on comprehensibility and usability by an experienced medical trainer working at the national institute for public health and the environment, and a physician specialized in infectious diseases from the maastricht aachen airport. the digital questionnaire started with information regarding the aim and process of the study and key definitions. also, participants received supportive documents on formulating competencies, according to bloom's taxonomy of educational objectives [ ] . in addition, a privacy statement was included. the first questions were related to demographic characteristics, such as their gender, profession, the number of years in their current profession, and their experience with infectious disease preparedness and response at the airport on a -point likert scale ( = very inexperienced, = very experienced). we requested e-mail addresses for planning the consensus meeting. subsequently, the relevance of competencies in the concept competency profile was graded by the participants using the following question: 'to what extent do you consider this competency as a relevant element for infectious disease preparedness and response at airports'? scoring was done using a -point likert scale ( = totally irrelevant, = totally relevant). the introduction, privacy statement, and demographic questions were in dutch, but the questions regarding the relevance of competencies, as well as the competencies were in english. all questions on relevance were mandatory. participants could comment on individual competencies as well as suggest new ones per category. after grading each competency, participants were asked if the competency profile reflected their knowledge, skill, and attitude regarding infectious disease preparedness and response at the airport. data were collected in the three weeks following may . participants who completed the digital questionnaire were invited to the consensus meeting. participants received a personal feedback report in advance of the meeting. this report showed the group ratings of each competency, together with the participants' individual ratings. participants could therefore identify major dissimilarities within ratings beforehand. the purpose of the meeting was to reach consensus by discussing these dissimilarities face to face. the moderator asked all participants to keep an incident in mind while considering the relevance of each competency. first, uncertain competencies were discussed, then competencies proposed by participants themselves, and lastly the suggestions for the reformulations of relevant competencies. if the participants could not reach consensus, a voting round was held to either accept, textually amend or reject the competency based on the majority opinion. after discussing all competencies, participants were asked whether the refined final competency profile reflected their knowledge, skills and attitude. finally, the usability level of this profile was discussed, including any further suggestions for improvement. the national meeting was in dutch because this was the native language of all participants. the meeting took place on june at the training center of schiphol airport. an experienced moderator (at) led the discussion. step -international validation: digital questionnaire & consensus meeting. the competency profile from step functioned as the starting point for international validation. the competencies were already stated in english, but we had to translate the introduction, statements, and demographic questions into english. we asked the respondents in the introduction to keep a recent incident or outbreak in mind during the completion of the questionnaire and asked them to specify which event this was. we added numbers to the tasks to manage expectations on the size of the competency list during completion, and added a suggestion for additional competencies on data protection and privacy. except from these changes, this international study was methodologically identical to the national study. the participants were invited on august and were reminded twice by e-mail after two and four weeks. the data collection through the questionnaire ended on september . the consensus meeting took place on september in belgrade, serbia, and was moderated by an experienced moderator. data from formdesk was transferred to an excel file and checked on irregularities. first, a descriptive analysis of demographic characteristics was performed. secondly, the median rating and the amount of dispersion of ratings between participants were calculated for each competency. if the competency had a median within - range and � % of the participants scored it in the top tertile (score , or ), then the competency was marked as 'accepted'. if the competency had a median of < and < % scored in the top tertile, then the competency was marked as 'not accepted' and excluded. if the median score laid between - and < % of the participants scored in the top tertile, then the competency was marked as 'uncertain'. in the national study, competencies with scores spread over all tertiles, despite any median, were also classified as 'uncertain'. table shows the classification of the competencies by the participants' median score and level of agreement. as the digital questionnaire contained openended questions and formulated competencies by the participants, responses were grouped and coded accordingly. to support the reliability, two researchers (ddr & rr) performed the data analysis independently and compared their results. the consensus meetings were audio-taped and in outline transcribed. a distinction was made between individual opinions and contributions to the group consensus. in accordance with the general data protection regulation, no names were used in the transcript. each participant received his or her own code, making it visible which recordings belong to the same professional. the codes of each participant were kept confidential and were only accessible to two researchers (rr and ddr). two researchers (rr and ddr) performed the data coding and analysis of the consensus meeting independently. the researchers discussed dissimilarities until consensus was reached. after the analysis of the international consensus round, an official translator reviewed the competency profile on use of language. the study protocol (lci- ) was reviewed by the clinical expertise centre of the national institute for public health and the environment. based on this review, they determined that the research plan did not fall under the scope of the dutch law on medical research involving humans. all necessary precautions were taken to protect the anonymity and confidentially of the participants; in the invitation letter, participants were informed about their voluntary participation and informed that they were free to decline at any time. furthermore, the participants were informed that their responses were processed anonymously and only used for research purposes. the literature search for competency profiles resulted in unique studies included in titleand abstract screening, and studies included in the full-text screening, of which two had the highest applicability and were therefore selected. these profiles were aspher's european list of core competences for the public health professional [ ] and public health emergency preparedness-core competencies for the eu member states [ ] . the competency profile for infectious disease management at airports the literature search for airport guidelines resulted in unique documents. nine were included during the full text screening. three guidelines had the highest applicability and were therefore selected, being the handbook for the management of public health events in air transport [ ] , coordinated public health surveillance between points of entry and national health surveillance systems [ ] and the guide for public health emergency contingency planning at designated points of entry [ ] . during data extraction, fifteen tasks and competencies were selected from the source documents. after data reduction, around competencies and exactly eleven tasks were left. aggregating double competencies resulted in competencies which originated from existing competency profiles and from reformulated textual fragments from the air transport guidelines. as a result, the concept competency profile consisted of competencies divided over eleven tasks ( thirty professionals were approached for the national study by e-mail. ten were approached personally; professionals from the afo and ams were approached as a group (n = ). professionals from phs, ima, ams, aas, and dmo agreed to participate, professionals from atc, afo, and fma could not participate in the study due to time constraints. however, one participant from aas had previously worked at the afo and voluntarily stated as a note in the questionnaire to keep this experience with afo in mind while filling in the questionnaire. nine out of ten included participants that completed the questionnaire had extensive experience in infectious disease preparedness and response at schiphol airport. the input from the dmo participant was not included in the analyses since she pointed out in an e-mail to the researchers that she is solely involved in the organization of training courses and therefore unable to grade the competencies in terms of relevance. sixteen professionals from thirteen countries were approached for the international study, of which ten from ten countries (austria, cyprus, germany, italy, ireland, malta, slovenia, spain, switzerland, and poland) accepted to participate and completed the questionnaire. nine professionals (from austria, cyprus, germany, italy, ireland, slovenia, spain, switzerland, and poland) attended the consensus meeting. their self-assessed competence areas were equally represented among professionals with most representing more than one area: six from the ten professionals were involved in preparedness planning, six in decision making and six in implementation of measures at the airport level. on the national level, five of ten participants were involved with preparedness planning. table shows the demographic characteristics of the included participants. the first questionnaire round resulted in "accepted" competencies, "irrelevant" competencies, and competencies that were marked as "uncertain" (fig ) . participants provided three main reasons for their low grading of relevance: ( ) not relevant to all professionals; ( ) concerning individual risks and not aimed at public health; ( ) out of scope, primarily focused on other areas (e.g., laboratories). the inconsistency in relevance scores of uncertain competencies reflected different professions. the participants proposed additional competencies. three participants confirmed, five partly, and one denied that the competency profile reflected their competencies in infectious disease preparedness and response at airports. no irregularities were found within the data analysis. five of nine participants attended the consensus meeting. the participants from dmo, ams, and one from aas could not attend the meeting due to time constraints. the"uncertain" competencies were discussed of which three were excluded, ten were included after textual amendment, and five were included without textual amendment. textual amendments included adjusting the target group described within the competency or adapting a verb from an executive to an advisory focus. of the suggestions for additional competencies, three were included. these included competencies entailed the understanding of the logistical structure and functioning of airports, implementing contact tracing, and contacting professionals with extensive epidemiological knowledge for outbreak investigation. ten competencies that were already included were reformulated. the group reached consensus for all competencies, no voting rounds were initiated. the reflection after the consensus meeting resulted in several the competency profile for infectious disease management at airports strengths and difficulties of the profile. during the national study, participants stated that they had enjoyed discussing the profile and this had improved their understanding of roles and responsibilities of different professions involved. several participants involved in design and organization of training and exercises would use the profile either to derive training goals or as a background document for participants. suggestions for improvements were to design a better manageable length or more practical format of the profile, either by splitting it for different disciplines, or connecting competencies to specific tasks. one participant indicated that the content of the competencies had become more evident after the meeting ( table ). the questionnaire round with the international panel resulted in "accepted" competencies, seven that were marked as "uncertain", and one "irrelevant" competency. the excluded competency entailed balancing costs and results during ph response. the inconsistency in relevance scores of uncertain competencies reflected different professions. the participants proposed twelve additional competencies. seven participants confirmed, and three partly confirmed that the competency profile reflected their competencies in infectious disease disaster medicine organization (dmo) airport level-ph decision making airport level-ph preparedness planning airport level-ph measure implementation national level-ph preparedness planning the competency profile for infectious disease management at airports preparedness and response at airports. no irregularities were found within the data analysis. six of the ten participants stated to have imagined a case during completion of the questionnaire, of which all referred to evd or viral hemorrhagic fevers in general. other named diseases were seasonal influenza, tuberculosis and measles. nine out of ten participants attended the consensus meeting. during discussion of the first competency, the group initiated a voting procedure which was used during the majority of competencies. the"uncertain" competencies were discussed, of which three competencies for surveillance and risk assessment were excluded because they were not considered as tasks at the local level. three competencies were included without textual amendments, and one after further specification of the target group. the only excluded competency in the questionnaire round-on taking the costs in consideration during ph response-was decided yet to be the competency profile for infectious disease management at airports included. of the twelve suggestions for additional competencies, four were added to other existing competencies, five were declined, and three were included. these included competencies entailed knowledge of specific terminology in use, data management, and network management of key partners to assure rapid response and recovery. analysis of the discussions during and after the consensus meeting indicated that people had sometimes doubted the relevancy of certain competencies due to difference between their own functional level and the scope of the profile (airport level). also, the differences between several countries in the division of tasks and responsibilities became clear. participants would use this profile as a source for training goals at the airport or as a background document during training ( table ) . to enhance the profile in any kind, they suggested to use the profile during a future training or exercise or to discuss it with their entire team at a local airport. the final competency profile (table ) consisted of competencies that were categorized into eleven tasks: communication (c = ), collaboration (c = ), professionalism (c = ), training (c = ), contingency planning (c = ), surveillance (c = ), risk assessment (c = ), outbreak investigation (c = ), management of ill / exposed travelers (c = ), public health measures (c = ), and evaluation and recovery (c = ). the majority of competencies entailed the roles of the health expert (c = ) or organizer (c = ). four competencies involved the scholar who is needed during outbreak investigation and implementation of health measures. in this study, we developed and validated a competency profile for professionals involved in infectious disease preparedness and response at airports. to the best of our knowledge, this is the first systematically developed competency profile describing the competencies for the air travel environment. the multidisciplinary consensus procedure ensures coverage of all major aspects of preparedness, response and recovery; the international consensus procedure with experts from various countries provides content and face validity to the competency profile and increases its potential for international application. a setting-specific profile is required because of the specific requirements according to international regulations [ ] , and the numerous actors which are involved from a range of sectors "i would use it as a source of competencies that you like to address in a training or exercise or anything like that. so, if you are running a table top or live exercise at the airport, these are the aspirational competencies that you would like, not only for the public health staff but for the whole response." "i really like it actually that you make this profile especially for the point of entry. i have never seen anything like this before but it is really nice. i consider it to be a good reflection of someone being responsible for working directly and being involved in public health measures at the airport." pha = public health authority at the airport; ima = international medical advice; md = medical doctor; f = female; m = male. https://doi.org/ . /journal.pone. .t the competency profile for infectious disease management at airports table . competency profile for professionals involved in infectious disease preparedness and response at airports. communicator • understand and implement the basic principles of risk communication to airport and airline staff, travelers, the public and media. • establish trust with airport and airline staff, travelers, the public and media by using rapid communication channels and ongoing two-way communication. • understand the terminology used by different levels and organizations at the airport. professional • minimize the discomfort or distress associated with public health measures experienced by crewmembers, ground staff, and passengers. • apply relevant laws to data collection, storage, management, dissemination and use of information. • understand the importance of multidisciplinary collaboration during acute outbreak management. • be an effective team member, adopting the role necessary to contribute constructively to the accomplishment of tasks by the group. • participate in the implementation of established plans which ensure the continuity of operations. • create and manage a network of key partners in rapid response and recovery. health expert • provide training and exercises on communication within, and between, involved airport organizations and include healthcare providers in this training. • identify training needs, and plan and organize courses. • periodically practice and test the ability to make decisions in unpredictable circumstances. health expert • be familiar with job-related standards and recommended practices concerning infectious disease control of national and international aviation organizations (iata, icao and capsca). • periodically assess whether the implementation of strategies, standard operating procedures (sops) and action plans requires any changes. • before the response operation, identify which triggers will require key decisions to be made during the outbreak response (keeping in mind that triggers may need modification to fit specific situations). • before the response operation, plan for the storage and stockpiling of medical and non-medical countermeasures. • understand the logistical structure of the airport and the international context of airports and their functioning. • identify key partners and develop a common understanding of roles, resources, planning assumptions, risks or vulnerabilities, and information needs. • support core-capacity-building at the airport and understand the importance of it. • develop, test and evaluate a public health emergency contingency plan (phecp) on a periodical basis. • provide healthcare workers with clinical guidelines for emerging infections from abroad, especially those that may be carried by travelers and the severely contagious. health expert • recognize a potentially infectious disease by key symptoms and signs of events among travelers. • understand the relevance of early detection of public health threats. • understand the components of surveillance systems and how these work. • understand the roles and responsibilities of local, national and international organizations involved in infectious disease control. • be familiar with laws on the surveillance and reporting of infectious diseases at national, european union level and globally (international health regulations). (continued ) health expert • understand risk analysis frameworks, with the elements of risk assessment, risk management and risk communication. • determine when a risk assessment should be carried out, and appropriate measures taken. • perform a risk assessment and continuously review the risk assessment as further information becomes available. • interpret the diagnostic and epidemiological significance of laboratory tests reports. • collect and integrate the facts of an event, based on information from multiple sources, including the traveler, the aircraft operator, ground-based medical services for aircraft in flight (when available) or the agent responsible for the baggage or cargo. • know when case reports or clusters require further investigation, and how to initiate such investigations. health expert • conduct outbreak investigations to identify pathogens and other agents, characterize affected population groups, and sources of exposure. • use reliable systems for disseminating case definitions to standardize both the diagnosis and the reporting of case numbers (e.g. confirmed, suspected, probable, or possible, cases). • systematically generate required information about the number of travelers such as those targeted for screening, screened, referred to secondary screening, and identified as confirmed cases. • implement contact-tracing based on a careful, case-by-case, risk assessment basis, taking into account factors such as feasibility, the severity of the disease and its potential for epidemic spread, the infectivity of index patients, and the duration of the trip. • identify who is responsible at national level for receiving the information on the investigation from the local or intermediate level health authority. • maintain up-to-date and job-specific knowledge about characteristics of infectious diseases such as the reservoir, potential sources, modes of transmission, risk groups, and duration. • be able to contact professionals who have the biological, clinical, and epidemiological knowledge necessary to characterize (potentially novel) pathogens and other agents responsible for an outbreak disease. • use evidence-based methods to identify and recommend control and preventive measures to control an outbreak. health expert • provide ground-based medical support (gbms) regarding infectious disease events, including medical recommendations to manage the discovery of a suspected communicable disease during flights, to support decisions regarding medical treatment and use of on-board medications or equipment. • assess the health status and travel history of travelers arriving from, or going to, an affected region, or who have been exposed to a potential public health risk during air travel. • provide disembarking travelers with information regarding the precautions to take in the event of illness, information sources for any updates on the event and the public health authority (pha) contact information where subsequent enquiries can be made. • provide advice concerning the appropriate parking stand for an incoming affected aircraft and the order of disembarkation of passengers. • provide advice to ensure port health staff respond efficiently so as reduce the time that travelers spend on a board-affected aircraft, and identify space requirements for interviews and health assessments of arriving travelers. • provide advice on a traveler's possible transfer to a medical facility by ambulance and facilitate the rapid transport of suspected cases of an infectious disease. other than public health [ ] . as such, airports function as coherent subsystems within the broader public health system. the aspher's european list of core competences, which we used as a main source, acknowledges the challenge to apply their general public health competency profile to such systems [ ] . however, the use of competencies can be pivotal in targeted and effective training for professionals, and the evaluation of their competence, as is proven by many studies [ , , [ ] [ ] [ ] . to support the development of well-functioning infectious disease systems at airports, we made general formulations more specific and added setting-specific knowledge, skills and attitudes to end up with this airport setting-specific competency profile. we, therefore, demonstrate in this study, that it is possible to design a competency profile for airports across countries. presumably, other points of entry, such as ground-crossings or ports, would also benefit from a setting-specific profile. these have a regulated and sustainable role in prevention of cross-border spreading of diseases and face comparable challenges to airports. examples are fisheries [ ] , drilling platforms [ ] , and many other mass gatherings [ ] or other places imaginable where an international transfer of people and goods take place, and where infectious disease diseases might be introduced. during the development of the profile, we learned more about the possibilities and challenges of competencies. both during the extraction of competencies from the literature, as well as during the consensus meeting we experienced that the concise and theoretical formulations of the competencies sometimes thwart their usability. it happened to us, as well as to the participants, that competencies had to be read and reread out loud before the full meaning of a competency was fully understood. the usability of the competency profile can be enhanced in several ways. for example, the use of entrustable professional activities (epa), as used in medical education, may bridge a potential gap between the theory of competencies and practice. an epa tells whether a professional can be entrusted to carry out all critical activities of a major task [ ] . they integrate the demonstration of competence with the respective supervision level and hereby form a usable tool during education or training activities. because our profile covers competencies required by the team of professionals involved in infectious disease management, participants experienced that competencies could only be translated into practice in close collaboration with different disciplines replicating findings of previous studies [ ] . another possibility to increase the usability of this profile is to merge disciplinary competencies into functional roles at an airport. in line with recommendations from • equip relevant airport and airline staff with information regarding the public health event so that they can protect themselves and safeguard healthy travelers as required. • organize the use of public health measures underpinned by scientific evidence and expert public health opinions, so as to avoid any contradictory or unnecessary restrictions of individuals. health expert • clearly define goals and objectives of the evaluation of training, exercises or real response. • develop a formal evaluation of the response, including recommendations for prevention and mitigation for future incidents, and share the evaluation with all stakeholders when the public health event is under control or has concluded. this study's participants, the profile could be minimized to a specific group of people, for example, first responders. a third option, is to use the profile and apply it in practice. discussing the profile enhanced usability according to our participants. in addition, we lowered the barrier of translating the theoretically formulated competencies into practice by inviting our participants to think of a possible event. during the international questionnaire, people noted which event they had used during completion. remarkable is that all the events listed comprised a viral hemorrhagic fever. while the chance for an event or suspicion of a viral hemorrhagic fever is still low, it is an interesting finding that its high impact, possibly due to the evd pandemic of - and the recent endemic state in the democratic republic of the congo, affects the thoughts of preparations in europe. remarkable findings in the profile are the low number of competencies for recovery and evaluation after an event. in the airport guidelines that we used as a source, evaluation and recovery are scarcely named and hardly elaborated on [ , , ] . however, the use of after action reviews are a required action according to the ihr, and major benefits of either training or real response are made here [ , ] . it is therefore worrisome that this phase of the event is hardly elaborated upon in current landmark guidelines and competency profiles, and, consequently, is scored as a rather small topic in this study's competency profile. we cannot determine, however, whether the small number of competencies resembles the real need or resembles a lack of awareness. we suggest to critically review the competencies required for recovery and the after action review after a major event. it may seem remarkable that many competencies in our profile are knowledge or skills in comparison to attitudes. this could be the result of several aspects. first, we build upon former general profiles and focused on competencies specific for the airport setting. attitudes such as leadership, flexibility, team work or reflection are mostly general, i.e. not airport-specific. in addition, these are already widely covered in the team resource management skill set, which is additional to our profile and focusses on attitude [ ] . however, another possibility is that knowledge and skills are more concrete while attitudes remain harder to distinguish by our participants. the use of this profile in practice should indicate whether attitudes are sufficiently covered. we consider it as a unique opportunity to thoroughly and extensively develop a competency profile by performing two consecutive rand modified delphi studies, one at schiphol airport, one of europe's largest airports, and one internationally with experts from ten european countries. as in every study, we faced several challenges that we tried to cope with. first, composing a draft profile demanded a pragmatic step of combining and formulating competencies of different profiles and guidelines. diminishing the large amount of overlapping competencies with slight differences in word choice, specificity level and combinations of activities was done as systematically as possible based on thoroughly produced profiles in the international literature to present an assessable profile to the participants. another challenge was to compose expert panels with optimal representation of all involved disciplines. in the national study, we could not include all professionals involved in infectious disease management at schiphol airport. however, we assume to have caught the major perspectives, since professionals that are involved with most tasks were represented. in addition, all included participants were highly experienced in outbreak management and collaborated with the missing experts in daily practice. during the international study, we had to select participants based on their self-assessed competence regarding the subject and could not prevent a mixed group regarding experience in real practice. we therefore consider it a strength that the international participants all participated in the european training course, leading to an equally educated group in the consensus meeting. a third challenge is to design a profile that is internationally usable while it turned out that designation of tasks between national, regional or airport levels differs among countries. however, we explicitly demanded participants to look further than the specific situation at their airport, leading to a profile that forms a thorough base for training in european countries. future steps would be to test the usability and implementation of this profile in real practice by means of trainings, exercises and evaluations. it is our aim that this profile is applied at airports worldwide to facilitate a competent workforce, by integrating it in training or exercising schedules as training material or background reference for organizers. standardized and extensive use of this profile could help to standardize terminology among professionals, and contribute to better communication and coordination. subsequent development of tools such as an epa profile on a european level, or the implementation of competencies into discipline specific profiles at specific airports are possibilities. finally, we hope that this competency profile can be used as a basis to develop specific competency profiles for points of entry other than airports, or other settings important in cross-border spreading of disease. in this study, we developed an interprofessional competency profile for professionals involved in infectious disease preparedness and response at airports by means of landmark literature and expertise from professionals in eleven european countries. this profile could be considered as promising 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globalization and health the authors are thankful to all participants for sharing their knowledge and practical experience. we thank saskia van egmond for her help in selecting the best representable panel, and for her advice and enthusiasm during the project. we thank eline westland-hogenbirk for the configuration of fig ; jeannette de boer for her reflections, including on the future applications and usability of our profile; and corien swaan for her contributions before and during the international consensus meeting; and our colleagues from the eu joint action healthy gateways for the opportunity to perform our study during the training-of-trainers in belgrade. conceptualization: doret de rooij, evelien belfroid, aura timen. key: cord- -wccmeaep authors: orcutt, connie j. title: emergency and critical care of ferrets date: - - journal: vet clin north am exot anim pract doi: . /s - ( ) - sha: doc_id: cord_uid: wccmeaep ferrets are becoming increasingly popular as pets in the united states. emergency situations involving ferrets are most often caused by gastrointestinal disease, neoplasia, cardiac disease, or endocrinopathy. hospitalization and supportive care of the critically ill ferret, emergency treatment techniques, and diagnostic procedures are discussed. diseases most commonly involved in critical presentations are reviewed along with treatment protocols. localities mandate euthanasia of the animal for rabies testing? if the ferret is active yet tractable, support the body vertically under the forelimbs for examination. with more energetic ferrets or those prone to biting, scruffing and holding the patient vertically elicits relaxation and a yawning reflex. distraction is afforded by offering nutri-cal (evsco pharmaceuticals, buena, nj) or ferretone ( -in-l pet products, hauppauge, ny), but avoid sugary treats in ferrets suspected of insulinoma. fractious ferrets can be scruffed in lateral or sternal recumbency with one hand while placing the other hand cranial to the pelvis and stretching the body slightly? awareness of specific anatomic, physiologic, and behavioral characteristics of the ferret is important for accurate clinical assessment. modify the extent of the physical examination based on the ferret's status. dyspneic animals may tolerate only brief periods of handling without oxygen supplementation. bruxism, ptyalism, or pawing at the mouth most often indicates gastrointestinal discomfort or nausea resulting from hypoglycemia or other causes. hypoglycemic ferrets may also appear dazed. posterior paresis in the ferret can be a manifestation of hypoglycemia, neurologic disease, or weakness of any cause. the ferret's normal body temperature is ° to ° f. testing the hydration status of a ferret by tenting the skin can be inaccurate, and evaluating mucus membrane capillary refill time is generally a more reliable method. the normal heart rate of to beats / min often varies greatly due to the ferret's normal respiratory sinus arrhythmiay the thorax is long relative to the total body length of a ferret, and the entire area must be ausculted for murmurs or abnormal arrhythmias. splenomegaly is a common finding in many ferrets, but pronounced splenomegaly or abnormal splenic texture may indicate pathology. most pet ferrets in the united states originate from large breeding facilities in which kits are neutered at to weeks of age, and a tattoo is placed inside the right pinna. although this makes disease involving the reproductive tract less likely, it does not preclude the presence of a reproductive remnant. a swollen vulva in a female ferret may indicate adrenal disease (most commonly, especially in middle-to older-aged animals), an intact female in estrus, or the presence of an ovarian or uterine remnant. male ferrets have an os penis, and the prepuce is located on the ventral abdomen. the critically ill ferret is optimally hospitalized in a quiet area separate from dogs and cats. isolation areas must be available for pa-tients suspected of having infectious disease (for example, canine distemper virus or epizootic catarrhal enteritis). hospital personnel with influenza should avoid contact with ferrets. the ferret requiring supplemental oxygen or heat can be hospitalized in the same type of oxygen cage or incubator used for a dog or cat. alternatively, ferrets can be placed in acrylic intensive care cages designed for birds. monitor ferrets in incubators carefully to prevent hyperthermia. the normothermic, eupneic ferret hospitalized in a wellventilated cage must also be monitored closely to prevent hypothermia. placing a towel or disposable diaper in the cage discourages the ferret from expending energy while attempting to burrow under cage paper. anorectic ferrets are at risk of developing hepatic lipidosis or hypoglycemia. enteral feeding is recommended whenever possible. debilitated ferrets refusing their regular food often accept syringe feeding or soft foods offered on a tongue depressor. aid diet (hills pet products, topeka, ks) provides an easily digestible diet accepted by most ferrets. syringe feeding is dosed at - ml three to four times daily. supplements such as nutri-cal or deliver . (mead johnson, evansville, il) provide additional calories when added to syringe-fed foods, but these sources are not nutritionally complete for ferrets and should not be fed as their sole diet for extended periods. nutri-cal and other sugarcontaining formulations can cause rebound hypoglycemia in ferrets that have insulinoma and should be avoided in those animals. raw meat or eggs that may contain bacterial pathogens are also not recommended. pharyngostomy tube placement in ferrets has been described? the technique is identical to that described for cats and utilizes an to fr pediatric feeding tube. in cases of protracted diarrhea or vomiting, gastrointestinal ulceration, or resistance to syringe feeding, a total nutrient admixture (tna) has been used successfully to provide parenteral nutrition (pn) to more than ferrets in the author's clinical practice. if a veterinary practice does not have the capability to compound parenteral solutions, some human hospitals mix pn solutions in their own pharmacies and will formulate a bag of tna for a veterinarian when provided with a prescription (table ) . a mixture of lipid and dextrose provides the ferret's resting energy requirement (rebecca remillard, phd, dvm, acvn, angell memorial animal hospital, boston, ma, personal communication, november ). this mixture is supplemented with amino acids, electrolytes, water-soluble vitamins, and minerals, and fluid is added so the total solution meets daily fluid volume requirements. such all-in-one solutions can be refrigerated for at least days. a silicone elastomer or polyurethane jugular catheter (cook veterinary products, bloomington, il) is recommended for delivery of the solution by infusion pump. hospitalized ferrets eating voluntarily and without special dietary requirements are optimally provided with their regular diet. otherwise, offer a premium-quality dry cat, kitten, or ferret food . ferrets have high fat and animal protein requirements and metabolize fat more efficiently for energy than carbohydrates. if a ferret requires fasting before surgery for the evaluation of blood glucose or for radiographic examination of the gastrointestinal tract, do not withhold food for more than hours. ferrets with insulinoma are especially at risk for developing profound hypoglycemia during extended periods of fasting. the critically ill ferret should be stabilized before pursuing extensive diagnostics. however, even severely compromised ferrets usually toler-ate the sampling of a small volume of blood from a peripheral vein for estimated blood glucose (bg), total protein (tp), packed cell volume (pcv), and blood urea nitrogen (bun) measurements. use an insulin syringe with a -gauge needle for collecting small volumes of blood « . ml) from either the lateral saphenous or cephalic vein. visualization of peripheral veins is facilitated by using a / -in. penrose drain as a tourniquet. for larger volumes of blood, the jugular vein or anterior vena cava are preferred sites of venipuncture. a healthy ferret's blood volume is approximately % to % of its body weight? as much as % of the blood volume can be safely withdrawn in a healthy ferret?- determine the ferret's initial pcv and level of hydration before withdrawing substantial volumes of blood. for jugular venipuncture, use a -gauge needle on a tuberculin or -ml syringe. hold the ferret at a table's edge or in lateral recumbency as for a cat, or alternatively, wrap the ferret in a towel and restrain in dorsal recumbency? the ferret's jugular vein lies more lateral than that in a dog or cat?- venipuncture of the anterior vena cava for ferrets has been described. this procedure can be performed without sedation in a debilitated ferret. however, if the patient resists restraint or the practitioner is inexperienced with this technique, anesthesia may be required. restrain the ferret in dorsal recumbency while one assistant extends the forelimbs caudally and a second assistant stretches the body with gentle traction applied cranial to the pelvis (fig. ) . palpate a slight depression at the thoracic inlet lateral to either side of the manubrium and cranial to the first rib. approach the site at a ° angle to the skin while aiming toward the opposite hip. insert a -gauge needle attached to a -ml syringe up to the needle hub. apply negative pressure, and slowly withdraw the needle until blood begins to fill the syringe. withdraw the needle if the ferret struggles. it is not necessary to apply pressure to the venipuncture site after the sample is collected. a method for obtaining volumes of as much as . to . ml of blood from the ventral tail artery of ferrets has been described? this procedure is painful to perform in the unanesthetized patient. restrain the ferret in dorsal recumbency. insert a -gauge needle attached to a syringe along the ventral midline of the tail, to cm distal to the tail base. direct the needle at a shallow angle toward the body while gently aspirating. peripheral catheterization can be performed in the awake debilitated ferret, but more active ferrets often require anesthesia. use a -or gauge peripheral catheter for placement in the lateral saphenous or cephalic vein. to facilitate intravenous (iv) catheter placement, first puncture the skin overlying or adjacent to the vein with a -or gauge needle while being careful to avoid the vein. secure the catheter with sterile tissue glue or a tape butterfly sutured to the skin before bandaging the leg. for jugular catheterization, the author most often uses a -gauge, -in. through-the-needle catheter on a - auge needle (intracath, becton dickinson vascular access, sandy, ut). a cutdown procedure may be necessary to access the vein. ferrets often act depressed with a jugular catheter and neck bandaging in place. access to the vascular system for more than several days can be achieved with a subcutaneously placed vascular access system (vascular-access-port, access technologies, skokie, il). an intraosseous ( ) catheter can be placed in the humerus, femur, or tibia, with the femur being the site least likely to impede the ferret's movement, , use a -or -gauge, . -in. spinal needle or a -or gauge hypodermic needle with a sterile surgical wire inserted into the lumen as a stylet to prevent occlusion of the needle with a plug of bone. , placement of the catheter is done with the ferret under anesthesia unless the animal is very debilitated. alternatively, the soft tissues and the periosteum can be blocked with local anesthetic. the technique for catheter placement is the same as that described for a cat. catheters can be left in place for several days during which time prophylactic use of parenteral antibiotics is recommended? maintenance fluid requirement for the ferret is estimated at ml/kg / h ? calculate additional fluid requirements for correction of dehydration and compensation for ongoing losses following protocols used for small animals. iv or fluid delivery is recommended in severely debilitated ferrets requiring dextrose supplementation. continuous delivery of fluids using an infusion pump is recommended. alternatively, a buretrol device (baxter healthcare, deerfield, il) can be used to deliver small fluid volumes. monitor the ferret carefully for overhydration, which may first be evident by auscultation of harsh lung sounds or a heart murmur. ferrets with cardiac disease are especially at risk of overhydration. debilitated, anorectic animals may require fluids supplemented with dextrose, vitamin b, and potassium following the protocols used for small animals. in cases of protracted anorexia, a tna delivered iv can be substituted for this fluid mix. use only small volumes of heparinized saline when flushing any catheter in the ferret to prevent heparin overdosage. antibiotics and most other medications are administered at dosages used with cats on a per kilogram basis. when an indwelling catheter is not in place, use a -gauge butterfly catheter for iv medications. most medications given intravenously, with the exception of some chemotherapeutic drugs, can alternatively be administered via an catheter? limit the use of intramuscular ( m) medications in ferrets owing to their reduced muscle mass. this author prefers the quadriceps for m injections because of its increased mass and the reduced risk of iatrogenic nerve damage at this site compared with the caudal thigh. administer subcutaneous injections as for a dog or cat. oral medications are best administered in liquid form, because pill administration is difficult in ferrets. most tablets can be crushed and compounded into a suspension with a vehicle such as ora-plus (paddock laboratories, inc., minneapolis, mn), flavoring, and water in a : : ratio. refrigerate the medication and shake well before using. there is no guarantee of stability. transfusion in the ferret has been recommended when the pcv drops below % to %, depending on whether the anemia is acute or has developed gradually. blood groups have not been demonstrated in ferrets, and there is little risk of a transfusion reaction even without crossmatching and after using a variety of donors?' , estimation of the blood volume required by the recipient can be calculated as for a cat. d . . rcv desired -fcv of recipient anticoagulated blood volume (ml) = body welght (kg) x x fcv f d . . i o onor ill anhcoagu ant place a -gauge jugular catheter or a -gauge catheter for transfusion delivery. the author treats the recipient minutes before transfusion with a slow iv bolus of a short-acting corticosteroid (prednisolone sodium succinate at mg/kg or dexamethasone sodium phosphate at -to -mg/kg)y choose a clinically normal ferret (pc v at least %) as a donor. the author collects a maximum blood volume (milliliter) equivalent to . % of the donor's body weight. use a -gauge butterfly catheter on a -to -ml syringe for blood collection. an acid-citrate-dextrose (acd) solution is used as an anticoagulant. flush the needle, tubing, and syringe with acd before collection, and use . ml acd per . ml whole blood collected from the anesthetized donor ferret. whole blood can be transfused immediately with a slow bolus or with a syringe pump?' , sedation is recommended for cystocentesis in all but very debilitated ferrets, because the bladder wall is thin and can be easily lacerated if the patient struggles? use a -or -gauge needle for cystocentesis. urethral catheterization requires anesthesia even with depressed patients. most cases of urethral obstruction involve male ferrets and are the result of cystic calculi or hyperplastic prostatic-like tissue at the neck of the bladder? catheterization of the male is complicated by the jshaped os penis as well as the very small diameter of the penile urethra. place the male in dorsal recumbency, and aseptically prepare the prepuce. if the prepuce or tip of the penis is swollen, a small incision can be made in the preputial opening to exteriorize the penis. the tissue covering the os penis is very thin. a surgicalloupe aids visualization of the small penile urethral opening lying on the ventral surface of the penis several millimeters proximal to the tip of the os. a -gauge pediatric catheter with the stylet removed can be used to localize the urethral orifice (fig. ) . flush the catheter with sterile saline if resistance is met while catheterizing the urethra. in some cases, a . -fr rubber feeding tube can be used as an indwelling catheter, but this will be too large in many ferrets. the author most often uses a -or -gauge, -in. jugular catheter with the stylet removed (see the section on iv catheterization). the stylet can be left in place with the end retracted to provide support around the pelvic flexure, but exercise extreme caution to avoid perforation of the urethra. suture the catheter to the skin using tape butterflies, and tape the administration port of the catheter to a tongue depressor to prevent kinking. place a padded bandage taped to the skin, and attach the catheter to a closed urinary collection system. monitor the ferret carefully, because many animals attempt to remove the bandage and catheter. the ferret with an indwelling urinary catheter should be treated empirically with a broad spectrum antibiotic (see the section on cystitis). for catheterization of the female ferret, place the anesthetized patient in ventral recumbency, elevate the hindquarters, and aseptically prepare the vulva and perivulvar area. use a small vaginal speculum or otoscope cone to locate the urethral opening on the ventral floor of the vaginal vestivule, approximately to . cm cranial to the clitoral fossa?' use a catheter as described above for the male ferret. in some instances of pneumothorax or pleural effusion, placement of a chest tube may be required. the author has performed this technique as described for a cat by using, as a chest tube, an -fr rubber feeding tube with a kirschner wire stylet. once sutured in place, the tube is capped for intermittent suction. the author has successfully used continuous suction at to cm h -negative pressure in one case of a ferret with a continuous spontaneous pneumothorax. isoflurane provides the safest means of anesthesia in the critically ill ferret. avoid the use of injectable agents in debilitated animals. ideally, a ferret should be fasted for to hours before induction. an induction chamber is recommended, because ferrets often violently resist induction by face mask. many ferrets hypersalivate during isoflurane induction, but because this is usually a transient behavior, atropine is generally not necessary? ferrets are not prone to laryngospasm, and intubation is relatively easy with a . to . mm endotracheal tube. , deliver anesthesia via a nonrebreathing circuit. to prevent hypothermia, place the ferret on a circulating warm water heating pad for any procedure lasting more than a few minutes. administer iv fluids to debilitated patients, taking care to avoid overhydration, especially in patients suspected of having cardiac disease, administer . % to % dextrose-containing fluids to hypoglycemic ferrets during prolonged surgical procedures?' monitor anesthesia with a pulse oximeter or a doppler ultrasound blood pressure monitor. a rectal probe may increase the usefulness of the pulse oximeter in small animals , clinical signs of pain in ferrets include lethargy, anorexia, vocalization, stiff movements, reluctance to curl up in a sleeping position, and squinting? buprenorphine ( , to . mg/kg iv, m, or sc q to h) or butorphanol ( . to . mg/kg m or sc q h) is well-tolerated by ferrets ?' monitor carefully for signs of significant depression, hypothermia, or hyperthermia. if necessary, naloxone ( . mg/kg iv, m, or sc) can be administered as a reversal agent? the pcv of ferrets is high relative to that of other species, and normal values have been reported to range from % to %. the normal leukocyte range of . to x , with clinically normal ferrets often having white blood cell counts of to x , is generally lower than that of other species, there is no evidence that ferrets demonstrate a leukocytosis with stress? the ferret's normal differential parallels that of the cat. isoflurane administered for minutes has been shown to result in significant decreases in hematocrit, hemoglobin, and plasma protein as well as red and white blood cell counts. coagulation profiles have not been standardized for ferrets, but the normal mean prothrombin time has been reported to be . to seconds, in contrast to the cat and the dog, bun values in the ferret appear to be more sensitive than creatinine in the evaluation of renal status. in cases of renal failure, bun levels are often drastically elevated compared with relatively less significant increases in creatinine. , however, an elevated bun is not specific for a renal disorder. other significant clinicopathologic findings with renal disease in the ferret include hyperphosphatemia, hypocalcemia, hyperkalemia, and acidosis. the cardiac silhouette of a normal ferret may appear slightly elevated from the sternum on lateral radiographs as a result of fat accumulation surrounding the ligament extending from the heart to the sternum, and this finding alone should not be interpreted as a pneumothorax. normal ferrets should have only a small amount of gas in the gastrointestinal tract. variations from this norm may indicate an obstructive pattern or ileus. the spleen is often pronounced in radiographs of even clinically normal ferrets. cardiopulmonary resuscitation in the ferret is based on protocols published for cats. establish an airway by intubation with a . to . mm endotracheal tube, and ventilate the ferret at a rate of to breaths/min taking care not to overinflate the lungs. evaluate the cardiac rhythm with an electrocardiogram. in cases of asystole, place the ferret in lateral recumbency, and gently perform cardiac massage at a rate of /min. evaluate the femoral pulse frequently. if external massage alone does not stimulate a heart beat, administer epinephrine ( . to . mg/kg diluted with sterile water delivered intratracheally, or . mg/ kg via an intracardiac, iv, or route). establish an iv line, and administer fluids at a rate of ml/kg/h. if mechanical systole returns but the ferret is bradycardic, administer atropine at a dose of . mg/ kg intravenously or . mg/kg intratracheally. continue to monitor the electrocardiogram for treatment of specific arrhythmias. differential diagnoses for the ferret in respiratory distress include pleural effusion (cardiac disease, neoplasia, infection, heartworm disease, hypoproteinemia, metabolic disease); pulmonary edema (cardiac disease, hypoproteinemia, metabolic disease, electrical cord bite); anterior mediastinal mass; pneumonia; pneumothorax; diaphragmatic hernia; tracheal obstruction; metabolic disease (acidosis); and profound weakness (circulatory collapse, hypoglycemia, anemia). , hyperthermia or pain may also manifest as dyspnea in ferrets. the dyspneic ferret often requires oxygen supplementation before pursuing extensive diagnostics. evaluate the initial pcv, bg, tp, and bun if the ferret is insufficiently stable to collect blood for an initial complete blood count (cbq and serum chemistry. if cardiac disease is suspected, administer furosemide (see the section on cardiac disease). obtain whole body radiographs as soon as the animal tolerates further handling. perform thoracocentesis in cases of pronounced pleural effusion or pneumothorax, and submit fluid collected for cytology and a gram's stain as well as culture and sensitivity if indicated. cardiac disease is seen most frequently in middle-to older-aged ferrets. the most common form of cardiac disease in the ferret is dilated cardiomyopathy (dcm), with hypertrophic cardiomyopathy occurring less frequently. historical abnormalities include lethargy, anorexia, weight loss, exercise intolerance, and periods of dyspnea, tachypnea, or coughing. tachycardia, murmurs, irregular arrhythmias, moist rales, muffled heart and lung sounds, rear limb weakness, and hypothermia are possible physical examination findings. radiographs may reveal an enlarged cardiac silhouette, pleural effusion, pulmonary edema, ascites, hepatomegaly, or splenomegaly. electrocardiography is recommended if abnormal arrhythmias are ausculted. isoflurane anesthesia is usually needed to obtain an electrocardiogram (ecg). the ferret ecg normally contains small p waves but large r waves. echocardiography is ultimately the most informative aspect of the cardiac workup in the ferret and should be performed as soon as possible. use feline dosages on a per kilogram basis for cardiac drugs when uncertain of specific dosing in the ferreu furosemide for treatment of pulmonary edema is dosed at to mg/kg iv, sc, m, or po q to hy, although the iv route is most direct in cases of fulminant heart failure, the ferret in severe respiratory distress may only tolerate m injection. digoxin is used as a positive inotrope in cases of dcm or to depress av nodal conduction of supraventricular tachyarrhythmias at a dose of . mg/kg po q h dosed on lean body weight (estimating a normal % body fat). balanced vasodilators used to decrease afterload in cases of dcm include enalapril ( . mg/kg po q h) or captopril ( / of a . -mg tablet/ferret po q h),b, monitor the ferret closely for hypotension, decreasing the frequency of dosing to once every third day if the ferret becomes lethargic. alternatively, during the initial management of heart failure, one can use a venous vasodilator such as % nitroglycerin ointment ( / in. applied to the skin or inner pinna q to h). monitor the patient for signs of hypotension. beta-adrenergic blockers (atenolol, . mg/ferret po q h) or calcium channel antagonists (diltiazem, . to . mg/ferret po q h) are most commonly used to relax the myocardium and to improve diastolic filling in cases of hypertrophic cardiomyopathy.l , administer fluid therapy judiciously in ferrets with cardiac disease to avoid fluid overload and subsequent development of pulmonary edema. prednisone and diazoxide, medications often used to treat insulinoma, can also increase preload on the heart. lymphosarcoma (lsa) is one of the most common forms of neoplasia reported in the ferreu dyspnea can result from an anterior mediastinal mass, pleural effusion, pulmonary metastasis, or profound weakness. mediastinal masses and pleural effusion are most common in ferrets less than year of agey in young animals, acute onset of clinical signs or sudden death are typical with the rapidly progressive mediastinal form of lsa. . other clinical signs may include anorexia, weight loss, vomiting, depression, dehydration, or signs associated with secondary infections. accompanying physical examination findings may be referable to any body system but most commonly include hepatomegaly, splenomegaly, and lymphadenopathy. lo diagnosis of mediastinal lymphoma is generally made by cytologic examination of pleural fluid. ultrasound-guided aspiration of an anterior mediastinal mass can be used to collect samples for cytology.j ferrets that have mediastinal lsa have been reported by some authors to respond favorably to chemotherapeutic protocols described in the references. . . infection with the canine heartworm (dirofilaria immitis) is uncommon in ferrets, but the risk increases in heartworm endemic areas and when the ferret is housed outside. signs include lethargy, coughing, dyspnea, ascites, heart murmur, pulmonary congestion, or sudden death. . the presence of even one adult worm in the small heart of a ferret can be fatal, peripheral microfilaremia is uncommon in ferrets with dirofilariasis, so testing for the dirofilaria antigen may be inconclusive. • . antibody tests used for feline patients are species specific and are not applicable to ferrets (karen rosenthal, ms, dvm, abvp-avian, personal communication, october ). radiographic changes with dirofilariasis include cardiomegaly, pulmonary congestion, pleural effusion, and ascitesy· a peripheral eosinophilia may be evident on the cbc. the test of choice for diagnosing dirofilariasis in ferrets is echo cardiography, which images the parasite in the heart (karen rosenthal, ms, dvm, abvp-avian, personal communication, october ). in some cases, angiography has been used to confirm the presence of adult worms. treatment protocols have been reported, but results are inconsistent. • pneumothorax pneumothorax can occur as a result of trauma, primary pulmonary disease, or esophageal perforation. the author has treated two ferrets with spontaneous pneumothorax. thoracocentesis is performed using protocols described for other small animals. if pleural air continues to accumulate, perform a tube thoracostomy. in uncommon cases of severe pneumothorax, resolution can be rapid when managed by continuous underwater suction if pressure is carefully monitored. o perform diagnostics as indicated to reveal the primary pathology, and treat as indicated. pneumonia is uncommon in ferrets and is most commonly associated with severe influenza virus or canine distemper virus (cdv) infections. coughing is not as prominent a sign of pneumonia in ferrets as in dogs. clinical signs of influenza include sneezing, epiphora, serous ocular discharge, rhinitis, anorexia, lethargy, and pyrexia. , o influenza is usually mild and self-limiting in adult ferrets but can be fatal in neonates. because ferrets are susceptible to human influenza viruses, affected caretakers should avoid contact with ferrets. ferrets with cdv may initially present with clinical signs similar to influenza but go on to develop a severe crusting dermatitis on the face and other areas of the body as well as marked hyperkeratosis of the footpads. ferrets that have advanced cdv infection may also present with neurologic signs. bacterial pneumonia is occasionally seen in ferrets. pulmonary mycosis is uncommonly reported. , radiographs and a cbc are often the most helpful initial diagnostic tests in cases of pneumonia. a neutrophilic leukocytosis with a left shift may be pronounced. , o radiographs demonstrate an interstitial pattern progressing to an alveolar pattern if the pneumonia worsens. aspiration pneumonia primarily involves the dependent lung lobes. if the ferret is stable, a tracheal wash to collect samples for cytology as well as bacterial and fungal cultures is important in guiding treatment. diagnosis of cdv is generally made on the basis of clinical signs, but viral inclusion bodies may be visualized or fluorescent antibody tests performed on conjunctival or mucus membrane scrapings. treatment for influenza consists of supportive care. antihistamines used to control sneezing or to improve appetite include chlorpheniramine ( to mg / kg po q to h) or diphenhydramine ( . to . mg/ kg po q to h). , use antibiotics only if a secondary bacterial infection is suspected. euthanasia is recommended for ferrets with cdy' , antibiotic treatment of bacterial pneumonia is based on culture and sensitivity results. empirical broad-spectrum antibiotics to consider while awaiting culture results include trimethoprim / sulfonamide ( mg/kg of combined drug po qi h), cephalexin ( mg / kg po qi h), or amoxicillin / clavulanate ( mg/kg po q h). , , parenteral antibiotics used with cats more rapidly achieve therapeutic blood levels in severe cases. nebulization with gentocin may be useful in young animals. clinical signs of gastrointestinal (gi) discomfort in ferrets are often not specific: collapse; lethargy; anorexia; dehydration; nausea evident by bruxism, ptyalism, pawing at the mouth, or frequent swallowing; retching or gagging; diarrhea; or vomiting. a number of the gi diseases affecting ferrets can have similar clinical presentations. profuse diarrhea can rapidly produce severe dehydration in the ferret. in contrast to the canine patient, diarrhea in the ferret is difficult to classify as being small intestinal or large intestinal in character. differential diagnoses for diarrhea include gi foreign body or trichobezoar, dietary indiscretion, helicobacter mustelae gastritis, eosinophilic gastroenteritis or other inflammatory bowel disease, neoplasia, metabolic disease (i.e., hepatopathy), clostridial overgrowth subsequent to prolonged antibiotic administration, influenza, rotavirus (usually in very young, unweaned ferrets), edv (generally accompanied by respiratory signs and a crusting dermatitis), epizootic catarrhal enteritis ("green slime disease"), gi parasitism (i.e., coccidiosis, giardiasis), and proliferative bowel disease. , , less common entities associated with diarrhea in pet ferrets include salmonellosis, aleutian disease, and mycobacteriosis. , , frank blood in the stool may be evident with proliferative bowel disease, salmonellosis, or clostridial overgrowth. , differential diagnoses for melena in the ferret include h. mustelae gastritis, toxin ingestion, gi foreign body or trichobezoar, hemorrhaging gi polyps, neoplasia, uremic ulceration, iatrogenic causes (i.e., nonsteroidal inflammatory drug induced), and aleutian disease (uncommon in pet ferrets). , . vomiting is not as common in ferrets as in dogs or cats and must be distinguished from regurgitation. the most common differential diagnoses for true vomiting in . ferrets include gi foreign body, h. mustelae gastritis, or inflammatory gastroenteritis (including eosinophilic gastroenteritis). vomiting is not commonly seen with metabolic disease (i.e., azotemia or hepatic disease) in ferrets. regurgitation or gagging may be caused by esophageal foreign body or megaesophagus. initial treatment of dehydration and electrolyte imbalances is critical while pursuing diagnostics. evaluate the pev, bg, tp, and bun before treatment, and submit a ebe and serum chemistry. obtain initial survey radiographs. additional imaging diagnostics may include ultrasonography, endoscopy, or colonoscopy. evaluate a fecal sample wet mount and flotation as well as a fecal gram's stain. in cases of hematochezia or if the ferret is febrile, culture the stool for campylobacter jejuni or salmonella spp. rubber, latex, or cloth objects are the most common gi foreign bodies in ferrets under to years of age, whereas trichobezoars are more common in older ferrets. , , in contrast to cats, linear foreign bodies are uncommonly ingested by ferrets. , clinical signs of gi foreign bodies vary greatly. in cases of total gastric or intestinal obstruction, the ferret may present recumbent, dehydrated, and in circulatory collapse. in general, vomiting is an infrequent finding with gi foreign bodies in ferrets. , , diarrhea or melena may be a part of the history with trichobezoars or other foreign bodies. in some cases, gastric foreign bodies may not be totally obstructive and instead act as chronic ball valves, causing intermittent nausea, vomiting, melena, gagging, anorexia, and weight loss. esophageal foreign bodies, though uncommon, can elicit regurgitation, gagging, or pawing at the mouth. on physical examination, the ferret may be dehydrated with tacky mucus membranes and a delayed capillary refill time. ferrets often resist abdominal palpation or grind their teeth as a sign of gi pain, especially with small intestinal foreign bodies. a gas-or fluid-dilated stomach or bowel may be palpable, and in some cases, the foreign body may be localized as well. radiographs are often diagnostic with abnormal findings including segmental ileus, gaseous distension of the stomach, and occasionally a visible object or trichobezoar. the ferret's normal gi transit time is to hours? include the esophagus in the film for evaluation. barium studies are rarely needed to confirm the diagnosis of a gi foreign body. obtain a pcv, tp, bg, and bun as a minimum database on admission, and submit blood for a cbc and serum chemistry. with ferrets in circulatory collapse, begin fluid therapy immediately via an iv or catheter. administer a broad-spectrum antibiotic with severely compromised animals or those with melena. consider removal of a gi foreign body a surgical emergency when accompanied with signs of obstruction. an esophageal foreign body, if devoid of sharp edges, may be removable with a small diameter endoscope, or alternatively, can be pushed into the stomach where it can be retrieved by way of gastrotomy.l if surgery is not an option in cases of suspected obstruction, fluid therapy and administration of a cat hairball remedy may facilitate passage of small objects. h. mustelae naturally colonizes the stomach and the pyloric duodenum of many ferrets and may be clinically silent or can cause mild to severe gastritis, gastric and duodenal ulceration, and gastric adenocarcinoma. , , , , pathology may be exacerbated by environmental stress owing to environment or additional disease. , clinical signs include bruxism, ptyalism, abdominal pain, chronic vomiting, anorexia, weight loss, and melena, which can cause significant anemia. while clinical disease is reported to be most common in ferrets to weeks of age, this author has seen ferrets of all ages affected. assessment of the initial pcv and tp is especially important in cases of severe melena. a cbc, serum chemistry, and radiographs are part of the initial database. once the patient is stabilized, definitive diagnosis includes biopsy of the gastric mucosa via an endoscopic or surgical approach. administer fluids to correct hydration. supportive care includes parenteral nutrition in cases of severe ulcerative gastroenteritis. treatment for h. mustelae gastritis has centered on a three-drug combination: amoxicillin ( mg/kg po q h), metronidazole ( mg/kg po q h), and bismuth subsalicylate ( ml/kg po q h) administered for to weeks. , a combination of omeprazole (a proton-pump inhibitor), amoxicillin, and clarithromycin has shown a notable success rate in the eradication of helicobacter pyloru however, omeprazole capsules supplied in timed-release form should not be opened before administration, therefore dosing is difficult in the ferret. epizootic catarrhal enteritis (ece) is a highly infectious diarrheal disease that affects ferrets and that can rapidly cause severe dehydration. this syndrome has been called /i green slime disease" by the lay community because of the profuse green diarrhea that is its most pronounced clinical manifestation. however, green mucoid diarrhea can be seen in a variety of malabsorptive syndromes affecting ferrets and is not pathognomonic for ece. although the etiology of ece has not been identified, histologic findings appear to be similar to those caused by rotavirus or corona virus. ferrets most commonly affected are those recently exposed to new ferrets, that is, at fairs, pet stores, or as recent additions to the household. incubation appears to be only several days before the onset of clinical signs that may include a brief period of vomiting followed by anorexia, profuse diarrhea that often lasts from days to weeks, and subsequent pronounced dehydration. morbidity is high, but mortality is low with the most severely affected individuals being those having underlying disease. diagnosis is presumptive based on the ferret's history and clinical signs. the pcv and tp may be drastically elevated due to dehydration. profound leukopenia is generally not evident. radiographs may demonstrate segmental ileus, which can be confused with an obstructive pattern. hospitalized ferrets must be isolated from other ferrets, and personnel should be made aware of possible viral transmission by fomites. treatment consists of aggressive supportive care aimed at correcting dehydration and providing nutritional support. antibiotic use appears to be of little use unless indicated with accompanying disease. the author has noted significant improvement in persistent cases after administration of prednisone at anti-inflammatory doses. eosinophilic gastroenteritis (ege) is an uncommon disease of ferrets generally seen in animals older than months. the cause is unknown, but some authors suggest an allergic or immunologic response to foods or parasites as an initiating factor. clinical signs include vomiting, diarrhea, anorexia, melena, lethargy, and weight loss. thickened abdominalloops or enlarged mesenteric lymph nodes may be evident on palpation. ege is suspected when clinical signs are accompanied by a peripheral eosinophilia (equal to or greater than /mm ); however, not all cases demonstrate an abnormal differentia . histopathologic demonstration of an eosinophilic infiltration of intestine, lymph node, or other tissues provides the definitive diagnosisy, initiate supportive care with fluids and nutritional supplementation. although this author has successfully treated the majority of diagnosed cases with prednisone, treatment may be unreliable because the underlying cause is unknown. adjust the dose of prednisone ( . to . mg/ kg sq or po ql h) to control clinical signs. resolution of signs is most often evident within days, but treatment is recommended for an additional month before tapering the dose of corticosteroid. in some cases, treatment may be lifelong. lymphoplasmacytic gastroenteritis is a similar clinical syndrome. histopathology provides the definitive diagnosis, and treatment is similar to that for ege. primary neoplasia of the gi tract is uncommon in ferrets. lsa can affect any area of the ferret's digestive tract. o , pyloric adenocarcinoma has been associated with chronic h. mustelae-associated gastritis in the ferreu affected animals may have clinical signs similar to those seen with a gi foreign body. palpation and radiographs may demonstrate a fluid-distended stomach. lo , , definitive diagnosis is made on biopsy, but abdominal ultrasonography may aid localization of pathology. critical care is supportive until the animal is stable for abdominal exploratory. surgical resection of the pyloric mass in a ferret followed by a bilroth i gastroduodenostomy was reported to be successful in one case of gastric adenocarcinoma. lsa involving the intestine or liver typically responds poorly to chemotherapy. , , hepatopathies can present as acute or chronic conditions in the ferret. differential diagnoses include hepatic lipidosis, toxin ingestion, primary or metastatic neoplasia, bacterial infection, and inflammatory disease. other than neoplasia, primary hepatopathies appear to be rare in ferrets. hepatic lipidosis is a common result of chronic anorexia. steroid hepatopathies are very rare, and to date, vascular shunts have not been reported in the ferret. clinical signs of hepatic disease include lethargy, anorexia, weight loss, diarrhea, vomiting, melena, anemia, and icterus (in advanced stages). . the most consistent diagnostic abnormality with hepatic disease is an often drastic elevation of alanine aminotransferase (alt). . alkaline phosphatase may be elevated as well, and in severe cases, total bilirubin levels may increase. abdominal ultrasound is recommended, but this author does not encourage ultrasound-guided liver biopsy, because coagulation panels, useful to assess risk of hemorrhage, have not been standardized for ferrets. definitive diagnosis requires biopsy of the liver. although bacterial hepatitis is not commonly reported for the ferret, in cases of persistent elevation of liver enzymes, submission of tissue for culture and sensitivity is recommended. institute supportive care while determining the definitive diagnosis of hepatopathy. nutritional supplementation is important in the treatment of hepatic lipidosis. once anorexia has resolved, hepatic lipidosis commonly requires no further treatment in the ferret. in cases of suspected bacterial hepatitis, empirical antibiotic choices pending culture and sensitivity include enrofloxacin ( mg/kg po ql h) or amoxicillin ( mg/kg po ql h) in combination with metronidazole ( mg/kg po qi h). administer vitamin k at the feline dosage on a per kilogram basis in cases of suspected coagulopathy. lactulose ( . to . mg/ kg po q - h) is indicated with hepatic encephalopathy. definitive treatment of hepatic neoplasia is unrewarding unless a small portion of the liver is involved and can be resected. although acquired megaesophagus is a chronic condition in the ferret, complications of the disease can lead to a critical situation. the cause of megaesophagus in the ferret is unknown. clinical signs include lethargy, anorexia, dysphagia, regurgitation, coughing or gagging, and weight ss. aspiration pneumonia is a common complication. diagnosis relies on history, clinical signs, and radiographic evidence of a dilated esophagus. aerophagia or aspiration pneumonia may also be apparent on radiographs. an esophagram may delineate esophageal abnormalities, and fluoroscopy can illustrate abnormal esophageal motility. management of megaesophagus is generally less successful in fer-rets than in dogs, and the prognosis is very poor. motility modifiers (metoclopramide at a dose of . to mg/kg po or sc q to h, or cisapride at a dose of . mg/kg po q to h) may be useful. treat aspiration pneumonia with protocols described in the section on cardiorespiratory emergencies. rectal prolapse is not common in ferrets but can occur in some cases of proliferative bowel disease (pbd), coccidiosis, or neoplasia. , , , pbd, uncommonly seen in the author's practice, generally affects ferrets less than months of age. , clinical signs, in addition to rectal prolapse, include chronic liquid or mucoid diarrhea sometimes with frank blood, tenesmus, anorexia, and cachexia, pbd may be exacerbated by environmental stress, and affected ferrets may be more susceptible to other diseases, an affected ferret may be moderately to severely dehydrated with thickened bowel loops evident on abdominal palpation. initial diagnostics include a fecal direct wet mount and flotation, radiographs, a cbc, and serum chemistry. definitive diagnosis is made on biopsy of intestinal mucosa, rectal prolapse can initially be repaired with a pursestring suture. chloramphenicol ( mg/kg m, sc, or po q h for to days) is the treatment of choice for pbd. , resolution of the prolapse may occur spontaneously as the colon heals. coccidiosis is treated with feline protocols, urethral obstruction most commonly affects male ferrets, clinical signs include stranguria, lethargy, and anorexia. male ferrets may have a palpable mass caudodorsal to the bladder. ferrets with accompanying adrenal disease may also be pruritic with bilaterally symmetrical or diffuse alopecia. in the author's experience, the most common differential diagnoses for urinary obstruction include prostatic enlargement associated with adrenocortical disease in males, urolithiasis, and cystitis. less frequently, bladder neoplasia has also been reported as a cause of dysuria. initial treatment for urinary obstruction is urethral catheterization. place an indwelling catheter while determining the origin of the obstruction. if a urinary catheter cannot be passed, a -or -gauge needle can be used to perform cystocentesis as an emergency measure. submit urine collected for urinalysis and culture and sensitivity. a cystostomy allows catheterization of the bladder directly from the body wall if passage of a catheter via the urethra is obstructed. submit blood for a cbc and serum chemistry. place an iv catheter and begin fluid diuresis concentrating on correction of dehydration and electrolyte imbalances. empirical broad-spectrum antibiotic therapy is recommended once a urine culture has been submitted and a urinary catheter placed (refer to the section on cystitis). further diagnostics to determine the cause of the obstruction include radiographs and abdominal ultrasound. along with ultrasonographic evaluation of the bladder, areas of special interest include the urethra, periurethral region, and adrenal glands. partial or complete urethral obstruction by hypertrophied prostatic tissue accompanies some cases of adrenocortical disease in male ferrets, and prostatic disease may be the most common cause of stranguria in male ferrets over years of age. s , , androgens of adrenal gland origin are hypothesized to cause hyperplasia or cystic changes of prostatic tissue, which subsequently presses on the urethra. , in some cases, prostatic tissue may become abscessed with a resultant thick purulent discharge from the prepuce. abdominal ultrasound may reveal unilateral or bilateral adrenomegaly, but not all cases of adrenal disease can be reliably diagnosed with this method. although studies of adrenal gland ultrasonography in ferrets have been published, diagnosis of adrenomegaly may be difficult for the ultrasonographer with little experience imaging ferrets. elevated plasma androgen and estrogen levels have been shown to be useful in diagnosing adrenal disease in ferrets, s, but test results are often not available soon enough to be useful in an acute situation. removal of the diseased adrenal gland generally results in rapid dissipation of prostatic hypertrophy. , cystotomy may be necessary to remove accumulation of purulent material,b and if a prostatic abscess is involved, surgical resection of the involved tissue is recommended after a sample has been retrieved for culture and sensitivity. this author maintains an indwelling urethral catheter for at least to days after surgery. persistent obstruction by prostatic tissue may be a result of infection or neoplastic changes or may be stimulated by remaining abnormal adrenal tissue. s , the prognosis with prostatic abscessation may be poor because it is difficult to remove all affected tissue, and response to antibiotics is uncertain.s, magnesium ammonium phosphate (struvite) uroliths are the most common type of calculi reported in ferrets. the incidence of struvite urolithiasis in ferrets has been less frequent with the introduction of higher-quality, animal protein-based diets that produce a more acidic urine than do plant protein-based foods. s , diagnosis and treatment are similar to procedures used with cats and dogs. the practitioner can treat cases of severe or persistent urolithiasis with prescrotal urethrostomy using a technique similar to that described for the dog. although primary cystitis is reported to be rare in the ferret, , purulent material in the bladder can be thick enough to cause a complete urethral obstructiony what appears to be cystitis in some male ferrets may actually be discharge from a prostatic abscess. submit a urinalysis and a urinary culture and sensitivity obtained by cystocentesis. in cases of urethral obstruction, place a urinary catheter and stabilize the ferret. obtain radiographs and an abdominal ultrasound to evaluate the bladder, urethra, and adrenal glands. while awaiting culture and sensitivity results, begin treatment with a broad-spectrum antibiotic: trimethoprim/sulfonamide ( mg/kg of combined drug po q h), amoxicillin ( mg/kg po q h), or amoxicillin/ clavulanate ( mg/kg po q h). severe cases of cystitis may require treatment for months or longer. clinical signs of renal failure may be nonspecific and include collapse, depression, lethargy, dehydration, anorexia, weight loss, rear limb weakness, polydipsia or polyuria, anemia, melena, oral ulcerations, or an azotemic odor to the breathy, differential diagnoses for renal failure in the ferret include chronic interstitial nephritis, pyelonephritis, neoplasia, toxins, and glomerulonephropathy secondary to other causes such as aleutian disease (rarely seen in the clinical setting with pet ferrets). diagnostics include a cbc, serum chemistries, urinalysis, urine culture and sensitivity, and radiographs. abdominal ultrasound is helpful in evaluating renal cysts, urinary tract calculi, or abnormal renal parenchyma. an ultrasound-guided renal aspirate may yield diagnostic information. initial treatment consists of iv fluid support geared toward correction of dehydration and electrolyte imbalances. an active urinary sediment indicates the need for a broad-spectrum antibiotic while awaiting results of a urine culture. the most common cause of hypoglycemia in pet ferrets in the united states is insulinoma (pancreatic islet cell neoplasia). additional differential diagnoses for hypoglycemia include anorexia or starvation, sepsis, neoplasia, hepatopathy, and other metabolic disease. insulinoma affects ferrets ranging in age from to yearsy initial signs may be insidious and attributed by the owner to geriatric changes in their ferret. the history may include episodes of collapse with hypersalivation or extreme weakness lasting from minutes to hours that typically resolve after administration of sugar-containing solutions. other common clinical signs include depression, rear limb weakness (which may be apparent as posterior paresis or ataxia), gagging, pawing at the mouth, or a dazed appearance. occasionally severely hypoglycemic ferrets appear dyspneic. signs are often intermittent and may not be apparent on clinical presentation. although hypoglycemic seizures are the most common clinical sign of insulinoma in the dog, they are rare in ferretsy when a ferret over the age of years presents collapsed or exhibiting any of the clinical signs described, immediately evaluate the bg level. initial evaluation can be performed with glucose measurement strips or a digital glucometer. for more accurate evaluation of bg levels, submit blood in a sodium fluoride (gray top) tube. a bg glucose level less than mg/ dl with accompanying clinical signs is suspicious for insulinoma, and ferrets presenting collapsed or comatose often have bg levels lower than mg/dl. , an elevated serum insulin concentration (using an assay that has been validated for ferrets) concurrent with hypoglycemia is diagnostic for insulinoma, in cases of hypoglycemic collapse, administer a slow iv bolus of % dextrose ( . to ml) to response, the goal of treatment is stabilization and not the complete reversal of hypoglycemia. if dextrose is administered too rapidly, the tumor will be stimulated to release large amounts of insulin, resulting in rebound hypoglycemia, place an iv catheter and begin infusion of fluids supplemented with % dextrose, control rare persistent seizures with diazepam (refer to the section on seizures). administer prednisone ( . to mg/kg sq) to inhibit peripheral tissue uptake of glucose and stimulate gluconeogenesis. begin with the lowest dose needed to maintain adequate bg levels (i,e" > mg/ dl), continue to monitor clinical signs along with bg concentrations q h, and increase dextrose supplementation if necessary. once the ferret is stable, administer prednisone orally. in cases of persistent hypoglycemia, concurrently administer diazoxide, which acts to inhibit insulin release from pancreatic beta cells, beginning at the low end of the dose range ( to mg/kg po q h). once clinical signs have resolved, gradually discontinue dextrose supplementation while monitoring the bg level, and adjust medications accordingly, prednisone doses can often be lowered with concurrent administration of diazoxide. offer a meatbased, high-protein ferret or feline diet while avoiding foods high in sugar or carbohydrate. in a minority of cases, hypoglycemic seizures or collapse may recur in spite of medications once iv dextrose supplementation is discontinued, these ferrets, once stabilized, may require surgical debulking of the tumors to allow further management. ongoing medical and surgical management of insulinoma is well described in the references. , , spontaneous diabetes mellitus (dm) is uncommon in ferrets. most cases of hyperglycemia develop secondary to surgery for insulinoma. clinical signs are similar to those seen in other small animals with dm. dm is suspected when the bg concentration is consistently greater than mg/ dl in conjunction with glycosuria. in cases of ketoacidosis, ketones are detected in the urine. a low insulin concentration concurrent with hyperglycemia confirms the diagnosis of dm. for the initial treatment of ketoacidotic dm, follow protocols used with cats to stabilize metabolic derangements. tight regulation of bg levels in ferrets with dm can be difficult. one protocol suggests initiation of treatment with insulin when the bg level is consistently greater than mg/ dl. neutral protamine hagedorn (nph) insulin is administered at an empirical dose of . unit per ferret twice daily with serial bg concentrations to dictate dosagey once the ferret is stabilized with a bg concentration less than mg / dl, treatment is continued with either nph or ultralente insulin (which only requires once daily dosing). the owner is instructed to use dipsticks to check for the presence of glucose and ketones in the ferret's urine. the same dose of insulin is administered if only trace amounts of glucose are present in the urine. if no glucose is detected, insulin is not administered, and if the glucose concentration is elevated, the insulin dose is increasedy in cases of transient dm secondary to debulking of pancreatic tumors, hyperglycemia usually resolves within several days to weeks after surgery. , ferrets with ongoing hyperglycemia are often very difficult to regulate. the differential diagnoses for anemia are broadly divided on the basis of regenerative versus nonregenerative processes. differentials for regenerative anemia in ferrets include blood loss (i.e. gi bleeding, trauma, flea infestation) and hemolytic anemia potentially caused by heavy metal toxicosis or immune-mediated disease. causes of nonregenerative anemia include estrogen toxicosis of the bone marrow secondary to persistent estrus in an intact female, ovarian remnant(s), or adrenocortical disease; neoplastic infiltration of the bone marrow; or anemia of chronic disease. immune-mediated hemolytic anemia has not been identified in ferrets owing to the lack of reagents specific for ferret antibodies. aleutian disease, an immune-mediated disease caused by a parvovirus is uncommon in pet ferrets. although estrogen-induced bone marrow suppression was once a serious and common cause of nonregenerative anemia involving intact female ferrets, this form of pancytopenia is rarely seen owing to the current practice of spaying females at to weeks of age. rarely, an ovarian remnant may secrete estrogen with similar results. increased levels of estrogen may infrequently be associated with adrenocortical disease either as a result of estradiol secretion from an adrenocortical tumor or subsequent to conversion of tumor-secreted androgens to estrogen in peripheral tissues. , resultant pancytopenia has been reported, but this syndrome is uncommon in spite of markedly increased serum estrogen concentrations in some ferrets with adrenocortical disease. clinical signs observed with pancytopenia of any cause include petechiation, ecchymosis, gi bleeding, infection, and sepsis. obtain an initial pcv from the critically anemic ferret before collecting a larger volume of blood for a cbc, platelet count, reticulocyte count, and serum chemistries. avoid anterior vena cava venipuncture in cases of suspected pancytopenia. normal mean reticulocyte counts for female and male albino ferrets have been reported as . % and %, respectively. pursue further diagnostics as indicated. if the pcv is less than %, transfuse whole blood. if an catheter is used, obtain a bone marrow sample before transfusion. if an ovarian remnant is suspected, administer human chorionic gonadotropin (hcg) ( iu per ferret) to stimulate ovulation. administration of hcg has no effect on hyperestrogenism secondary to adrenocortical disease. if the pcv remains below %, the prognosis is poor, and multiple transfusions along with b complex vitamins, an anabolic steroid (stanazolol, . mg/kg po, sc q h),b iron supplementation, fluid therapy, and nutritional support may be required. , pursue surgical treatment for adrenocortical disease, a retained ovary, or bleeding gi masses once the patient is stabilized. anecdotal reports of reactions following canine distemper vaccination in ferrets describe clinical signs ranging from diarrhea, gagging, vomiting, fever, or erythematous skin to circulatory collapsey although many of these reactions happen within minutes after vaccination, less severe signs can be noted up to several hours later. in cases of postvaccination collapse, administer diphenydramine hydrochloride ( . to . mg/kg iv or im), epinephrine ( j-lg/kg iv, m, sc, or intratracheally), and a slow iv bolus of a short-acting corticosteroid ( mg/kg of prednisolone sodium succinate or to mg/kg of dexamethasone sodium phosphate).b administer iv fluids, and provide supportive care following treatment protocols for small animals. seizures are uncommon in ferrets with the most common cause arguably being insulinoma-induced hypoglycemia. l additional differential diagnoses include hypoglycemia from other causes; trauma; toxin ingestion; ens infection (including rabies or canine distemper), inflammation, or neoplasia; renal failure; hepatopathy; or other metabolic derangements. evaluate the bg level on presentation, and treat hypoglycemia as described above (refer to the section on hypoglycemia). administer diazepam ( to mg iv to effect), and treat supportively using protocols applied to other small animals. pursue diagnostics as indicated. lumbar cerebrospinal fluid tap has been described in the ferret. l gastrointestinal disease, neoplasia, cardiac disease, and endocrinopathy are among the most common syndromes affecting the ferret that presents in an emergency situation. knowledge of these and other disease processes, indicated diagnostic testing, and immediate treatment protocols are critical to provide efficient and effective care to the ferret in crisis. musculoskeletal and neurologic diseases a cluster of cases of juvenile mediastinal lymphoma in a ferret colony helicobacter muste/ae gastritis, proliferative bowel disease, and eosinophilic gastroenteritis intraosseous catheters in small mammals biology and medicine of the ferret ferrets: basic anatomy, physiology, and husbandry (of the ferret) clinical techniques in domestic ferrets stranguria in a castrated male ferret a practitioner's guide to rabbits and ferrets ferrets, rabbits, and rodents: clinical medicine and surgery. philadelphia, wb saunders medical and surgical management of esophageal foreign body in a ferret diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: cases ( - ) exotic animal formulary s. pet ownership and pet populations chemotherapeutical remission of multicentric lymphosarcoma in a ferret (mustela putorius juro) cardiac emergencies clinical and pathologic findings in ferrets with lymphoma: cases ( - ) helicobacter mustelae-associated gastric adenocarcinoma in ferrets (mustela putorius jura) gastric colonization of the ferret with helicobacter species: natural and experimental infections cystic ovarian remnant in a ferret critical care of ferrets, rabbits, and rodents ferrets, rabbits, and rodents gastrointestinal diseases of ferrets (mustela putorius juro! urogenital diseases cardiac disease in ferrets gastrointestinal diseases gastrointestinal diseases of ferrets helicobacter infection normal parameters and laboratory interpretation of disease states in the domestic ferret blood transfusions multisystemic eosinophilic complex in a ferret (mustela putorius juro) estradiol- j -secreting adrenocortical tumor in a ferret lack of detectable blood groups in domestic ferrets: implications for transfusion effect of isoflurane on hematologic variables in ferrets anesthesia, analgesia, and sedation for small mammals differential diagnoses for common clinical problems in ferrets soft tissue surgery gastrointestinal foreign body in ferrets: cases ultrasonography of adrenal glands in normal ferrets pleura and pleural space helicobacter mustelae-associated hypergastrinemia in ferrets (mustela putorius jura) basic approach to veterinary care endocrine diseases: insulinoma use of a vascular access system for administration of chemotherapeutic agents to a ferret with lymphoma pyloric adenocarcinoma in a ferret the collapsed ferret endocrine diseases: adrenal gland disease, d iabetes mellitus, and thyroid disease evaluation of plasma androgen and estrogen concentrations in ferrets with hyperadrenocorticism proceedings of the north american veterinary conference ferret respiratory disease diagnosis new therapeutics in small mammals respiratory diseases stranguria in a castrated male ferret cardiovascular diseases: cardiac disease small mammal radiology radiographic and angiographic evaluations of ferrets experimentally infected with dirofilaria immitis estrogen induced bone marrow depression in a ferret (mus tela putorius juro) with adrenal gland tumors key: cord- -tta o t authors: vlietstra, wytze j.; vos, rein; van den akker, marjan; van mulligen, erik m.; kors, jan a. title: identifying disease trajectories with predicate information from a knowledge graph date: - - journal: j biomed semantics doi: . /s - - - sha: doc_id: cord_uid: tta o t background: knowledge graphs can represent the contents of biomedical literature and databases as subject-predicate-object triples, thereby enabling comprehensive analyses that identify e.g. relationships between diseases. some diseases are often diagnosed in patients in specific temporal sequences, which are referred to as disease trajectories. here, we determine whether a sequence of two diseases forms a trajectory by leveraging the predicate information from paths between (disease) proteins in a knowledge graph. furthermore, we determine the added value of directional information of predicates for this task. to do so, we create four feature sets, based on two methods for representing indirect paths, and both with and without directional information of predicates (i.e., which protein is considered subject and which object). the added value of the directional information of predicates is quantified by comparing the classification performance of the feature sets that include or exclude it. results: our method achieved a maximum area under the roc curve of . % and . % when evaluated with two different reference sets. use of directional information of predicates significantly improved performance by . and . percentage points respectively. conclusions: our work demonstrates that predicates between proteins can be used to identify disease trajectories. using the directional information of predicates significantly improved performance over not using this information. we have recently shown that analyses that are performed on protein knowledge graphs benefit from predicate information [ ] . by using the predicates that specify the mechanisms by which proteins interact, temporal pathobiological relationships may also be identified, although this has not been demonstrated yet. a key application for such temporal analyses is the identification of disease trajectories, which are commonly occurring temporal sequences of diseases diagnosed in patients [ , ] . an example of a disease trajectory found in a study by jensen et al. [ ] is rheumatoid arthritis-precedes-heart failure, where precedes is defined as "occurs earlier in time. […]" [ ] . the occurrence of the reverse, heart failure-precedesrheumatoid arthritis, was found to occur significantly less frequently in the same study, and therefore was not classified as a trajectory. identifying relationships between diseases is an important and popular research topic for protein-protein interaction networks (see related work section). in such analyses diseases are represented by so-called disease proteins, which are proteins encoded by genes that are associated with a disease [ , ] . often cited benefits include an improved understanding of the biological mechanisms underlying disease interactions [ , , ] , and the ability to anticipate the next disease, thereby providing the knowledge necessary to improve treatment plans and interventions [ , ] . however, the temporal aspects of relationships between diseases still require further investigation. we therefore aim to automatically determine whether a given sequence of two diseases forms a trajectory. we do so by leveraging the predicate information from paths between (disease) proteins in a knowledge graph. we also determine whether there is added value in using directional information of predicates for this task. previous authors have mostly focused on identifying undirected relationships between diseases with protein networks [ ] [ ] [ ] [ ] [ ] . for example, kontou et al. created a disease-disease graph, where an edge between diseases indicated that they shared at least one disease gene [ ] . sun et al. calculated the similarity between diseases based on their shared disease proteins, shared physiological processes associated with these proteins, or the graph structures between the proteins [ ] . li and agarwal identified which biological pathways were associated with diseases via their disease proteins, and identified relationships between diseases based on the number of shared pathways [ ] . menche et al. identified so-called disease modules, which are clusters of closely interrelated disease proteins [ ] . they found that short distances between the modules of diseases were predictive for pathobiological relationships. contrary to kontou et al. , they demonstrated that sharing disease proteins is not a requirement for diseases to be related to each other. to our knowledge, bang et al. were the only ones to use a directed protein-protein interaction network to identify disease trajectories [ ] . the disease proteins of pairs of diseases were used to identify shared biomolecular pathways, after which the locations of the disease proteins within these pathways were determined. the disease with most upstream disease proteins was classified as the first within the sequence of diseases. additionally, million medicare records were used to calculate two relative risk scores for each pair of diseases, corresponding with the two possible temporal sequences of the disease pair. if the sequence determined with the protein pathways concurred with the sequence that generated the largest relative risk, that sequence was identified as a trajectory. between a total of diseases, their method suggested trajectories. these were evaluated with the biomedical literature, where further leads were found for of them. because the authors only evaluated the trajectories that were suggested by their method, it is unclear how many trajectories the method failed to identify. the ability of our method to identify disease trajectories was evaluated with two reference sets, which have identified disease trajectories by different means. the first reference set consisted of statistically-derived disease trajectories from a large retrospective study of danish hospital data, while the second set consisted of literaturevalidated disease trajectories that were based on a small prospective study of dutch general-practitioner data. the first reference set was based on a study of jensen et al. [ ] . they retrospectively identified disease trajectories from . million electronic patient records of danish hospitals based on diagnoses assigned over . years. all diagnoses in these patient records were represented as international statistical classification of diseases and related health problems th revision (icd- ) codes. jensen used the hierarchy within the icd- to aggregate all diagnoses to a high abstraction level, resulting in two-digit codes, such as "malignant neoplasm of breast" (c ) or "type diabetes mellitus" (e ). jensen derived the disease trajectories from the danish hospital data in a two-step process. first, they identified sequences of two diseases that were diagnosed within years from each other in at least patients, and which had a relative risk higher than . subsequently, the direction of each sequence had to be corroborated by a binomial test that compared the frequency of the sequence to the frequency of its reversed sequence. sequences that fulfilled both criteria were classified as disease trajectories. to represent the diseases in the jensen set on the protein level, we used the expert-annotated associations between proteins and diseases from the manually curated subset of disgenet [ ] . the unified medical language system (umls) mrconso table was used to map the icd- codes of the jensen trajectories to the umls identifiers that are used in disgenet. two diseases, "accidental poisoning by and exposure to other gases and vapours" (e ) and "influenza due to identified zoonotic or pandemic influenza virus" (j ), were lost because their icd- codes could not be mapped to a umls identifier. because only % of the high-level diseases in the jensen set were represented within disgenet, we used the "narrower" and "child" relationships from the umls mrrel table to identify subclasses of all diseases. by expanding the diseases with their subclasses, the percentage of diseases to which disease proteins could be assigned was increased to % ( of diseases). from the disease trajectories in the jensen set, there were trajectories where disease proteins could be assigned to both diseases ( %). these trajectories, which were used as positive cases in this reference set, contained of the diseases to which disease proteins could be assigned ( %). on average, diseases had disease proteins assigned to them (median: , interquartile range: - ). disease proteins were on average assigned to . diseases (median: , interquartile range: - ). a set of , non-trajectories was constructed by creating all possible sequences of the included diseases, minus the trajectories that were described by jensen. the set of non-trajectories thereby included random pairs of diseases, the reversed temporal sequences of these random pairs, as well as the reversed temporal sequences of the trajectories. in the following, we will refer to the trajectories and non-trajectories as positive and negative cases to align with common terminology in the machine learning field. the second reference set was based on a prospective cohort study on disease susceptibility by van den akker et al. [ ] . they followed a dutch cohort of patients over years, during which their general practitioner notes were examined for sequences of international classification of primary care (icpc) codes that represent chronic, permanent, and recurrent diseases. in the netherlands, each citizen is registered with a general practitioner, who acts like a gatekeeper for secondary and tertiary medical care, and is responsible for maintaining a complete medical history of the patient. a total of unique sequences of diseases were found in this cohort, containing distinct diseases. each sequence was manually evaluated using the published biomedical literature and medical handbooks. there were sequences of diseases where the literature stated that the first disease increased the susceptibility of acquiring the second disease, and sequences where no evidence of increased susceptibility was found. to maintain consistent terminology, we will refer to sequences with increased susceptibility as trajectories or positives and to sequences without increased susceptibility as non-trajectories or negatives. to assign disease proteins to these diseases we followed the same procedure as for the jensen set by using the mrconso table to map the icpc codes to umls identifiers, after which the mrrel table was used to group them with their subclasses. disease proteins could be assigned to diseases, which formed trajectories and non-trajectories. on average, diseases had disease proteins assigned to them (median: , interquartile range: - ). disease proteins were on average assigned to diseases (median: , interquartile range: - ). to determine whether our method could also identify the correct temporal sequence of the trajectories, additional non-trajectories were created by reversing the sequence of the diseases in the literature-supported trajectories (the reverse sequence of one trajectory was already included as a non-trajectory in the data from the general practitioners). the predicates between proteins were extracted from the euretos knowledge platform (ekp), a commercially available knowledge graph (http://www.euretos.com). in the ekp, information from more than knowledge sources from a wide variety of domains in the life sciences is represented as triples. the biomedical entities such as proteins, drugs, or diseases that form the subjects and objects of these triples are represented in the knowledge graph as vertices, each of which has one or more identifiers associated with it from external databases. mappings between the entities described in the different knowledge sources underlying the knowledge graph were made by matching their identifiers. the predicate and provenance of each triple are specified as part of an edge between the two vertices that represent the subject and object. the direction of the predicate goes from subject to object. the predicates in the underlying knowledge sources were matched to a standardized set of predicates. if an exact match was not available, a predicate was manually mapped. if there were no explicit predicates in a database that was used as a knowledge source, the predicates were manually derived from the database schema. a path between two vertices is defined as a sequence of triples, or possibly a single triple, connecting the vertices. the contents of the ekp are represented as documents in a nosql database, which allows them to be flexibly modelled and indexed. the ekp can be run on a reasonably-powered server, requiring an -core processor and gb of memory as a minimum. it has previously been used in pre-clinical research for drug efficacy screening [ ] , prioritizing existing drugs as repurposing candidates for autosomal dominant polycystic kidney disease [ ] , and pathway enrichment [ ] . the paths between the disease proteins were extracted from the ekp. to keep our graph comprehensible, we only extracted paths that consisted of one or two triples, i.e., paths where two disease proteins are connected by at most one intermediate protein. within this range, three scenarios for the paths between the disease proteins of two diseases a and b were distinguished (fig. .) two different methods to represent indirect paths between disease proteins were compared. the first method constructed so-called metapaths [ ] , where the sequence of predicates in an indirect path was used as single feature. the second method, which we refer to as split paths, considered each predicate in the indirect paths as a separate feature [ ] . each method was tested both with and without directional information of predicates. predicates were either considered to all be undirected, or predicates were categorized as being directed or undirected based on expert assessment (described in the assessment of predicate directionality section below), which we refer to as the mixed variation. in the overlap scenario, where the subject and the object were the same protein, predicates were always considered to be undirected. all features were binary. figure shows the four feature sets that are derived from the example shown in fig. . we also experimented with feature sets where all predicates were directed as indicated by the subject and object of the triple in the ekp. however, because some predicates are explicitly defined as being undirected, using any directional information from triples with these predicates would contradict these definitions. nonetheless, for the sake of completeness we have chosen to present these results in additional file . random forests were trained to classify the sequences of diseases as positive or negative. classification performance fig. schematic overview of the overlap, direct, and indirect scenarios that were extracted from the knowledge graph. both diseases a and disease b have three disease proteins (dp) associated with them according to the manually curated subset of disgenet. disgenet describes that dp is known to be associated with both diseases, while the knowledge graph describes that it has a "binds with" relationship to itself. dp and dp have a direct "inhibits" relationship, and dp and dp are connected through an indirect path, by an intermediate protein (ip) . the arrows between the proteins indicate which protein is the subject of the "inhibits" predicate, and which one its object. the "binds with" predicate was considered to be undirected by the experts, and therefore does not have a direction. based on the paths in the knowledge graph, four feature sets are created, based on two methods to represent indirect paths, and both with and without the directional information of predicates was measured with the area under the receiver operator characteristic curve (auc) of a -fold cross-validation experiment [ , ] . we report the mean and standard deviation of the aucs of repeated cross-validation experiments. the same folds that were used in the experiments with undirected predicates were also used in the experiments with directed predicates, after which the differences between the test folds were tested for significance with a two-sided, paired t-test. to control for the differences in prevalence and number of cases between the two reference sets, we also report the classification performance after undersampling the number of positive and negative cases in the jensen set to match those in the van den akker set. for the best performing classifiers we also report sensitivity and specificity at the probability cutoff for which the youden index (sensitivity + specificity - ) is largest [ ] . machine learning and evaluation of results were performed in r [ ] with the packages caret [ ] , ranger [ ] , and proc [ ] . three experts with a strong biomedical background and familiarity with knowledge graphs assessed the directionality of distinct predicates that were found in the extracted paths. they were provided with definitions of these predicates which were obtained from the pathway commons resource [ ] . if not available, definitions were sought through the national library of medicine [ ] , or the obo foundry [ ] . the assessors could categorize each predicate as "directed", "undirected", or "don't know". to establish directionality, a predicate had to be categorized as directed or undirected by a majority (i.e., two or three) of the assessors. predicates that contain a negation (e.g., "does not interact with") were automatically categorized the same as the corresponding predicate without negation ("interacts with"), and therefore not presented to the assessors. for some predicates the categorization was straightforward. for example, pathway commons defines the predicate "interacts with" as "this is an undirected relation between participant fig. the four feature sets that were derived from the paths between the disease proteins in fig. . all features are binary: black fields indicate a "true" value, while empty fields indicate a "false" value. for the "mixed" feature sets, the "binds with" predicate is assessed to be undirected by experts, while the "inhibits" predicate is assessed to be directed proteins of a molecular interaction. […]" , and the predicate "catalysis precedes" as "this relation defines directed interactions between proteins. […]" [ ] . six predicates did not reach a majority in the first round and were anonymously commented upon by the assessors to motivate their categorization. these comments were shared between the assessors, after which they could update their initial choice. each predicate was then categorized with a majority. table shows the predicates that were categorized as undirected by the three experts. the other predicates were categorized as directed. a complete overview of the predicates can be found in additional file . in total, distinct disease proteins were assigned to the diseases in both reference sets, three of which could not be mapped to the ekp. another ( . %) of the disease proteins were not found in any of the extracted paths. from these disease proteins, had no relationship to any other protein in the ekp. the remaining disease proteins were involved in , , direct paths and , , indirect paths, while disease proteins had paths to themselves. all paths were based on , , distinct triples, which contained , different proteins and different predicates. the overlap scenario, where the two diseases in the trajectory share at least one disease protein (scenario , the classification performance for both reference sets is shown in table . mixed metapaths performed best, achieving mean aucs of . % for the jensen set and . % for the van den akker set. overall, classification performance on the van den akker set was . to . percentage points lower than on the jensen set, while standard deviations were . to . percentage points higher. metapaths performed . to . percentage points better than split paths. the performance of the mixed feature sets was . to . percentage points higher than the undirected feature sets. all differences between mixed and undirected feature sets were significant (p-values for jensen metapaths and split paths: < . ; van den akker metapaths: . , split paths . ). to quantify how much of the difference in auc between the two reference sets could be attributed to their difference in size, the jensen set was undersampled to the same number of positive and negative cases as the van den akker set. with the exception of the mixed metapaths, performance dropped below the performance that was achieved with the van den akker set. the standard deviations also increased from . - . % to . - . %. the latter values are comparable to the standard deviations on the van den akker set. figure shows the receiver operating characteristic (roc) curves of the mixed metapath classifiers that performed best. for the jensen set, sensitivity and specificity at the maximum youden index were . % and . %, respectively, while for the van den akker set these were . % and . %. for our best classifier (mixed metapath features, trained on the jensen set), we analyzed the top- false-positive and the top- false-negative cases, searching the literature for information that might explain the errors. the results of our analysis of the false positives are shown in table . overall, the first out of the top false positives appear to be omissions from the jensen set rather than misclassifications. for two false-positive cases, potential mechanisms have been suggested, but the current evidence is inconclusive on whether those mechanisms are valid. for the remaining three false-positive cases no literature could be found, which may therefore be interesting leads for further investigation. table shows the results for the top- false negatives. for six false negatives, the second disease was likely to be caused by the treatment of the first disease. for example, the radiation that is used to treat the malignant neoplasm of the larynx may compromise the immune system around the throat and mouth, thereby increasing susceptibility to oropharyngeal candidiasis [ ] . two false-negative trajectories are likely to have mechanical causes, rather than molecular pathways. the trajectory from malignant neoplasms of the ovary to nutrient deficiency can be explained by the blocking of the intestines by the ovarian tumor, thereby blocking the entire digestive system [ ] . for four of the false-negative trajectories, no description could be found in the literature, making their assessment impossible. assessment of the three remaining false negatives is speculative. for example, the trajectory from transient ischemic attacks (tia) to vitamin b deficiencies may be an artifact of the medical record keeping. vitamin b is known to protect against tias [ ] , so what may often happen is that a vitamin b deficiency is only diagnosed after the more acute tia has been treated in an emergency room. our work demonstrates that disease trajectories can be identified with the predicates between proteins in a knowledge graph. to do so, our machine-learning based methodology needed to successfully identify both the correct pairs of diseases, as well as their correct temporal sequences. overall, representing indirect paths as metapaths performed superior as compared to representing them as split paths. using the directional information of predicates significantly improved performance over not using this information. undersampling the jensen set to the same number of positive and negative cases as the van den akker set showed that its lower performance and higher standard deviations could partially be explained by its small size. in previous work, bang et al. [ ] identified disease trajectories by calculating the relative risk between two diseases and combining this with the relative position of disease proteins in biomolecular pathways. their method is fully dependent on shared disease proteins between the two diseases, whereas our method also works when there are only (in) direct paths between disease proteins. in the jensen set, this holds for % of the trajectories. performance comparison of the methods is difficult because bang et al. only validated the disease trajectories that were suggested by their method, but did not validate the non-trajectories. thus, only the precision of their method can be calculated but no insight is provided in the number of false-negative trajectories. a final complication for the comparison between the two methods is the claim of bang et al. to discover causal relationships between diseases, rather than only temporal ones. unfortunately, they refer to an example to define causal relationships between diseases, making it difficult to pinpoint how these differ from disease trajectories. although we do not foresee direct clinical application of our work, our high performance may persuade experts to further examine the protein paths underlying some positively classified trajectories, either known or the values in the columns indicate the mean auc and its standard deviation in % of cross-validation experiments newly suggested. interpreting these protein paths might provide additional clues about the mechanism through which the first disease leads to the second. identifying and understanding these mechanisms is likely to improve prevention, prediction of disease progression, intervention, and drug development, thereby indirectly supporting clinical practice and health-care policy. for now, such detailed examinations of the protein paths were beyond the scope of this project. a downside of working on the protein level was that not all disease trajectories could be studied. more than a third of the trajectories of the jensen set, and a fifth of the van den akker set was lost because disease proteins could not be assigned to one or both of the diseases in a trajectory. even when disease proteins could be assigned to both diseases, alternative explanations were sometimes more plausible. for example, our analysis of the false-negative cases suggested that some trajectories could be explained mechanically, or were likely due to a side effect of the treatment for the first disease. to determine the true performance of our method, a validated set of trajectories that are caused by biomolecular mechanisms would be needed. alternatively, the range of trajectories that can be analyzed may be broadened by linking diseases to other types of disease information available in the ekp, e.g., information about drugs or physiological processes. the two reference sets that were used in this research were both based on patient data, but differed in many other respects. the sequences of diseases in the jensen set were classified as trajectories based on statistics calculated from years of nationwide hospital data. despite this large volume of data, our analysis of the false-positive cases showed that the set of trajectories was incomplete. the literature evaluation underlying the van den akker set ensures that such omissions are unlikely to occur there. furthermore, the negatives in the van den akker set either were observed in patients, or were reversals of literature-supported trajectories. because the negative cases in the jensen set were based on randomization, this set is likely to contain pairs of diseases that never co-occur within patients. finally, the types of diagnoses within the trajectories differ between the two reference sets. the hospital patients in the jensen set are more likely to suffer from more serious and complicated diseases than patients visiting a general practitioner in the van den akker set. on the other hand, the van den akker set only included chronic, permanent, and recurring diseases, thereby excluding diseases that are acute and rapidly treatable. only the definitions from pathway commons stated whether the predicate was directed or not. the definitions of predicates from other knowledge sources, including the national library of medicine, left room for interpretation. as a result, six predicates required a second round of assessment before a majority was achieved between the assessors. with ontologies playing increasingly important roles in data standardization and sharing [ ] , the directionality of predicates should always be clear. the relationship ontology already supports categorization of predicates as directed or undirected, which it refers to as asymmetric or symmetric predicates, but unfortunately is far from complete and did not cover the predicates in our set [ ] . a potential new application for our method would be to identify trajectories for rare and low-prevalence diseases, where insufficient patient data is available for studies such as those performed by jensen or van den akker. because our method identifies trajectories based on a protein network, it is independent of the prevalence of a disease. furthermore, many of the estimated to thousand rare diseases have well known genetic causes [ ] , making them highly suitable to be analyzed with our method. a possible extension of our work would be the identification of longer disease trajectories, e.g. the trajectories consisting of sequences of four diseases that were also described by jensen et al. [ ] . however, as far as we are aware all available knowledge-graph methods limit themselves to identifying relationships between two entities. expanding the current methods to identify longer sequences should therefore be a separate investigation. our work demonstrates that disease trajectories can be identified with the predicate information from a knowledge graph. we also demonstrate and quantify the added value of using directional information of predicates for this task. our work thereby enables the discovery of temporal relationships with predicate information from knowledge graphs. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . description and results of the directed variation feature sets. this file describes the feature sets and classification results of the variation where all predicates in the feature sets have a direction as specified by their triples in the knowledge graph. their categorization as directed or undirected by the assessors was not used in this variation. figure s shows an example of the feature sets derived from fig. , with other intervertebral disc disorders m other polyneuropathies g likely mechanical cause [ ] biological knowledge management: the emerging role of the semantic web technologies w c.org triple specification. w c.org semantic web for integrated network analysis in biomedicine a survey of current trends in computational drug repositioning systematic integration of biomedical knowledge prioritizes drugs for repurposing network biology methods integrating biological data for translational science mining genotype-phenotype associations from public knowledge sources via semantic web querying network medicine: a network-based approach to human disease exploring the human diseasome: the human disease network what do we learn from high-throughput protein interaction data? protein interactions and disease: computational approaches to uncover the etiology of diseases human diseases through the lens of network biology using predicate and provenance information from a knowledge graph for drug efficacy screening temporal disease trajectories condensed from population-wide registry data covering . million patients identifying temporal patterns in patient disease trajectories using dynamic time warping: a population-based study semmeddb: a pubmed-scale repository of biomedical semantic predications the genomic and functional characteristics of disease genes disgenet: a comprehensive platform integrating information on human disease-associated genes and variants a pathway-based view of human diseases and disease relationships predicting disease associations via biological network analysis causality modeling for directed disease network disease networks. uncovering disease-disease relationships through the incomplete interactome network analysis of genes and their association with diseases in an exploratory prospective study on multimorbidity general and disease-related susceptibility could be distinguished drug prioritization using the semantic properties of a knowledge graph transcriptional profiling and biomarker identification reveal tissue specific effects of expanded ataxin- in a spinocerebellar ataxia type mouse model the use of the area under the roc curve in the evaluation of machine learning algorithms the use of receiver operating characteristic curves in biomedical informatics index for rating diagnostic tests r: a language and environment for statistical computing building predictive models in r using the caret package ranger : a fast implementation of random forests for high dimensional data in c++ and r proc: an open-source package for r and s+ to analyze and compare roc curves pathway commons, a web resource for biological pathway data constructing a semantic predication gold standard from the biomedical literature the obo foundry: coordinated evolution of ontologies to support biomedical data integration alcohol and alzheimer's disease-does alcohol dependence contribute to betaamyloid deposition, neuroinflammation and neurodegeneration in alzheimer's disease? hypertension: a harbinger of stroke and dementia alzheimer's disease and osteoporosis therapy insight: type diabetes mellitus and the risk of lateonset alzheimer's disease hyperinsulinemia and risk of alzheimer disease sudden death in schizophrenia urinary tract infections and lupus erythematosus vestibular loss predicts poorer spatial cognition in patients with alzheimer's disease respiratory manifestations of systemic lupus erythematosus: old and new concepts sepsis and the heart calcium hypothesis of alzheimer's disease relation between therapy options for graves' disease and the course of graves' ophthalmopathy: a systematic review and meta-analysis a scarlet pimpernel for the resolution of inflammation? the role of supra-therapeutic doses of cobalamin, in the treatment of systemic inflammatory response syndrome (sirs), sepsis, severe sepsis, and septic or traumatic shock alcohol-induced psychotic disorder: a review aphakic glaucoma after congenital cataract surgery nutrition and risk of stroke malnutrition and cachexia in ovarian cancer patients: pathophysiology and management oral candida species in head and neck cancer patients treated by radiotherapy ocular associations of diabetes other than diabetic retinopathy complications of laparoscopic surgery for benign ovarian cysts spinal cord injury: association with axonal peripheral neuropathy in severely paralysed limbs the fair guiding principles for scientific data management and stewardship relations in biomedical ontologies networking for rare diseases: a necessity for europe publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank euretos b.v. for providing access to the euretos knowledge platform, and drs. anneke m. sijbers and solène grosdidier for their help in assessing the predicates. the difference that in this variation the "binds with" predicate also is directed. table s shows the classification performance of the directed feature sets along with the performances of the undirected and the mixed variations. table s shows the p-values of the two-sided paired ttests between all variations.additional file . overview of predicates that were found in the paths. this file contains table s , which shows the predicates that connect proteins in the knowledge graph and were used to construct the features. authors' contributions wv, rv, evm, and jk designed the study. wv created the feature sets, performed the error analysis, and drafted the manuscript. wv and rv performed the data analyses. mvda and rv supplied the van den akker reference set. rv, evm and jk supervised the study and critically revised the manuscript. all authors read and approved the final manuscript. no funding was received for this project. the datasets and scripts that are used in this study are available at https://github.com/wytz/diseasetrajectories ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -erigjz g authors: robertson, colin title: towards a geocomputational landscape epidemiology: surveillance, modelling, and interventions date: - - journal: geojournal doi: . /s - - - sha: doc_id: cord_uid: erigjz g the ability to explicitly represent infectious disease distributions and their risk factors over massive geographical and temporal scales has transformed how we investigate how environment impacts health. while landscape epidemiology studies have shed light on many aspects of disease distribution and risk differentials across geographies, new computational methods combined with new data sources such as citizen sensors, global spatial datasets, sensor networks, and growing availability and variety of satellite imagery offer opportunities for a more integrated approach to understanding these relationships. additionally, a large number of new modelling and mapping methods have been developed in recent years to support the adoption of these new tools. the complexity of this research context results in study-dependent solutions and prevents landscape approaches from deeper integration into operational models and tools. in this paper we consider three common research contexts for spatial epidemiology; surveillance, modelling to estimate a spatial risk distribution and the need for intervention, and evaluating interventions and improving healthcare. a framework is proposed and a categorization of existing methods is presented. a case study into leptospirosis in sri lanka provides a working example of how the different phases of the framework relate to real research problems. the new framework for geocomputational landscape epidemiology encompasses four key phases: characterizing assemblages, characterizing functions, mapping interdependencies, and examining outcomes. results from sri lanka provide evidence that the framework provides a useful way to structure and interpret analyses. the framework reported here is a new way to structure existing methods and tools of geocomputation that are increasingly relevant to researchers working on spatially explicit disease-landscape studies. while the global burden of disease has been shifting from communicable to non-communicable diseases and injuries (murray et al. ) , infectious diseases continue to causes persistent (hiv/aids; tuberculosis; malaria), and episodic (ebola in west africa) public health threats. in both cases, the environment is critical to the risks to human health and wellness posed by infectious agents (lash et al. ; weiss and mcmichael ) , environmental toxins (ard ) , access to healthcare (wang and luo ; kwan ), social disadvantage (wilkinson ) , and stress (shankardass ) . there is a pressing need to develop the theories, tools and datasets that support investigating links between the environment and health. a number of converging trends are currently aiming towards a greater role for geography in studies of environment and human health. firstly, the emergence and re-emergence of infectious pathogens over the last few decades has re-emphasized the importance of local exposures and risks, whether due to environmental or communicable transmission processes that result from complex assemblages of local and globalscale processes. secondly, spatially explicit models of infectious diseases have been developed at a range of spatial and temporal scales-from household-level models of infectious disease spread (riley ) , to global models of the factors driving the spatial distribution of zoonotic pathogens (jones et al. ) . these models require spatial datasets describing every aspect of disease risk from wind patterns to health-seeking behaviours. the use of geographic information systems (gis) is critical to the building and operation of spatially explicit disease risk models. finally, spatial modelling has been improved by the concurrent rise in sources of geographical data to support such modelling efforts across all scales (though not all diseases). the importance of the 'landscape' in emerging, zoonotic, and infectious disease literatures is widely recognized from both a substantive perspective due to greater interdependencies between populations (khan et al. ), incursion into natural areas (e.g., field et al. ) , intensification of livestock production (gerber et al. ; graham et al. ) , wildlife trade (karesh et al. ) as well as more practical considerations due to the abundant data and technologies now available (e.g., vitolo et al. ) . we focus on landscape epidemiology (le) which is interested in the interaction between features of the landscape (and their composition and configuration) with disease and risks of disease. this is distinct from the broader domain of spatial epidemiology, which includes studies of health services and health systems. despite the growing adoption of geographic tools and methodologies, their deployment in infectious disease epidemiology, modelling, and control at the implementation level has been mostly piecemeal and study-specific. lambin et al. ( ) suggested ten propositions which help to situate relationships between landscape change and infection risk, identifying key themes such as connectivity, landscape configuration and composition, the importance of examining processes and patterns at multiple spatial and temporal scales, and human behaviours. while these propositions provide a valuable guide for researchers, they do not provide aid to the use of that knowledge in applied settings. most research into landscape and disease risks assume a better understanding of infection risk will ultimately 'trickle down' to effective uses of that enhanced knowledge. recent advances in understanding of how knowledge is adopted in decision-support contexts, and the translation of knowledge to specific communities of users in application of health research and technologies has shed light on the importance of the types of knowledge and expertise required to make this transaction possible (straus et al. ) . the simple fact that knowledge exists does not necessarily mean that it will be pushed to development and application (estabrooks et al. ) . in this paper we take a different approach, by focusing on three core knowledge use contexts that broadly encompass the majority of spatial epidemiological studies. we aim to develop a single organizational framework for conducting studies of landscape and infectious diseases that situate methods and data within the broader conceptual context and thereby promote more rigorous and transparent study design and interpretation and translation of findings. need for organizing framework for landscape approaches to health the rapid expansion of methods and data available for landscape-oriented epidemiological studies has considerably transformed health-environment research over the last two decades. it is now customary to include landuse/landcover data sensed from satellites, climate data obtained from global repositories of networked weather stations (e.g., worldclim) or from satellite sensors such as the tropical rainfall monitoring mission that provides daily rainfall data for all of asia. also, these data products are increasingly available at a global scale, and as 'pre-processed' information products consumable by spatial models. for example, the worldpop project (http://www. worldpop.org.uk/) aims to provide high-resolution spatially explicit demographic data across south and central americas, asia, and africa (tatem ) . geographically granular case data aggregated over small health areas are also commonplace, both in studies in the developed and developing worlds. data describing other features of the environment such as roads, river networks, water bodies, built-up areas, health care service locations are also increasingly available in even remote parts of the world. we are on the cusp of real-time global-scale epidemiology, whereby 'feeds' describing the important features of the environment for health is updated in real-time. for diseases with immediate environmental components of risk (e.g., arboviruses, heat-related illnesses), the benefits of timely updating of spatially explicit information on risk factors provide opportunities for highly granular public health responses and data-driven policy formulations. further, diseases with longer latencies and more complex risk profiles also stand to benefit from enhanced environmental information. with this data-rich environment in machine-readable forms, and the capacity for automated model-building, it is important to structure analysis using such large, spatially explicit information sources within a broader framework for understanding disease-landscape dynamics (e.g., lambin et al. ) . citizen-based, user-generated, or volunteered data have also become increasingly prevalent, sometimes described as participatory epidemiology (brownstein et al. ). the recent cholera outbreak in haiti provides a salient example, where volunteers on the ground and on the other side of the world contributed data and expertise to the mapping of local environment in support of disease control and health programs. citizen-generated data is also increasingly used in empirical studies themselves, for example-actively in mobile health-reporting apps such as 'outbreaks near me' (freifeld et al. ) as well as passively, when web-search data are repurposed for syndromic surveillance of seasonal influenza (hulth et al. ), in support of what is often called 'epidemiological intelligence'. in landscape epidemiological research, all of these datasets can be accessed, mapped, and integrated via their geographies to derive factors affecting disease risk, and the cumulative risk distribution in space and time. the data processing methods, software, statistical tools, visualization approaches, and reporting mechanisms that drive this research vary considerably, and encompass a wide array of levels and areas of expertise. as such, the place of geocomputation-the tools and methodologies of geographic computing-within this spectrum of approaches is unclear, and more importantly, selecting the appropriate approach for a given study objective is increasingly difficult. reviews of 'gis in epidemiology' have not provided this sort of framework. many reviews have rightly focused on broad overviews of spatial analysis or gis methods and disease (cromley ; rytkönen ) , and/or their application to areas such as animal disease (freier et al. ) , and other areas such as surveillance (robertson et al. ) . the shortcomings of gis approaches to health research and specifically the disconnect between analytical methods and parameters such as neighbourhood weights matrices in disease mapping models, and their epidemiological meaning was highlighted by yang et al. ( ) , whereas fritz et al. ( ) review and compare methods for handling point event data in spatial health research. barrett et al. ( ) reviewed the scope for big data to improve understanding and tracking of health-related behaviours and outcomes. and while the emphasis in health geography over the last two decades has focused largely on social determinants (kearns ; kearns and moon ) , these approaches have bifurcated somewhat from approaches dominant in spatially oriented infectious disease epidemiology, which have remained quantitative and modelling/biomedically oriented. rather than debate the relative merits of these paradigms, it is instructive to re-examine the motivation for these studies in the first place, and perhaps contextualize these differing research frames within a common framework. recent work relating infectious disease risk to climate change has introduced ideas from adaptation research such as vulnerability and adaptive capacity that are conditioned on socioeconomic processes (kienberger and hagenlocher ) is an example of a new generation of integrative spatial studies of health and disease that may be bridging this gap. in this paper, we present a framework oriented around knowledge uses and existing tools in order to provide a methodological grounding for new integrative studies in landscape epidemiology (le). a key requirement for landscape epidemiology is the ability to handle, process, represent, and transform large amounts of data. the backbone of gisrelational database management systems-has provided the technology for this historically, although geojournal ( ) : - rarely identified explicitly in research related to le studies. however, given the increasingly granularity of satellite data, the volume of mobile sensor data, and the advent of 'big data' generally (and real-time epidemiology), these technologies are starting to become insufficient to handle the current data-rich environment. new geocomputational methods for spatial data storage and access may be required for new research in landscape epidemiology-including spatial indexes, data compression algorithms, and knowledge discovery algorithms. for example, social media data streams are by definition 'always on'-and extracting and storing a subset of these defined by space and time into a relational database for the purposes of on-line surveillance of new disease reports induces a delay in the processing chain. application programming interfaces (apis) as a way of accessing data portals algorithmically partially sidestep this issue, but these tools are by their nature reserved for researchers with advanced technical skills. consequently, the 'data processing step' is an increasingly relevant but frequently overlooked component of le studies, as data integration based on geography is often required prior to undertaking any analysis. complex processing chains that incorporate web-based geographic data repositories, cloud-based storage and analysis, and delivery of results via web-map services may encompass the computational tools of neogeography (e.g., openlayers, cartodb, d , leaflet), open data portals and cloud services (e.g., amazon ec ) and a myriad other software tools and packages. situating these methods and tools within their functional context relevant for le will provide an organizational mapping to aid understanding of this complex and rapidly evolving landscape. a key theme in the adaptation of landscape approaches to human health is the explicit recognition of the importance of multiple spatial and temporal scales in the study of health outcomes. while scale has increasingly been emphasized in the health geography literatures, much of this work has been conceptual and theoretical rather than applied. while laying the necessary theoretical foundations for a multi-scale approach to understanding health and disease in populations is an important first step, concrete tools and methodologies are required in order to operationalize these ideas for applied le. geocomputational methods are well-suited to multi-scale analysis, however there are several approaches to multi-scale analysis that can be taken, and little guidance is available to researchers aiming to take a multi-scale approach to le. characterizing features of the landscape at multiple scales is perhaps one of the most widely employed tactics, whether it is compositional factors (e.g., percent forest cover) or configurational properties (e.g., edge density, fragmentation). identifying 'zones' of scale consistency and abrupt changes in landscape properties with scale can provide insight into the appropriate spatial scale of investigation (wu ). this approach is consistent with the landscape ecology paradigm of hierarchical patch dynamics, which may provide a conceptual basis for investigating landscape influences on health in patchy landscapes. the theories, tools and data driving applied le research today have significant potential to improve health of individuals and the allocation of scarce healthcare resources. our aim in proposing the framework that follows is to more clearly organize current methods, provide a structure for research design, and more concretely link methods to knowledge translation and use. surveillance can provide health workers with the baseline knowledge required to judge when and where unusual events are taking place, which can catalyze early responses to outbreaks or changes in disease incidence. knowledge of risk factors can contribute to formulating effective interventions, and identifying areas and populations vulnerable to disease. once knowledge of landscape influences on health and disease is established, this can be applied and integrated into healthcare delivery systems in contextually appropriate ways (lai et al. ). we provide the motivation for our framework through the lens of applied le. while these use-cases are not encompassing of all studies that might employ le approaches, these categories cover the majority of applied research objectives, and as such will provide a useful lens through which to contextualize our organization of geocomputational methods applicable for le. it is worth highlighting that these categories are not mutually exclusive, and often studies and research projects aiming to take a le approach will encompass two or even three of the use-cases. surveillance: what has changed? public health surveillance is defined by the world health organization (who) as continuous, systematic collection, analysis and interpretation of healthrelated data needed for the planning, implementation, and evaluation of public health practice (who ) . a variety of objectives may drive surveillance including outbreak detection, situational awareness, and identifying long term trends, and statistical and computational methods will be specific to the system objective (robertson et al. ). additionally, a variety of information sources may be required to achieve a given public health objective targeted through surveillance (who ). the inclusion of explicit geographical information is now a key component of most actionable public health surveillance systems, including those focused on infections with major landscape components. spatial risk modelling: why are things are where they are? many spatial-epidemiological studies are interested in identifying the factors that contribute to the spatial risk distribution. most regression-type models employed through either generalized linear modelling framework or bayesian hierarchical modelling fall into this category. with respect to landscape risks, a wide variety of modelling methods are now being used such as maxent (phillips et al. ) , random forests (and related methods) (breiman et al. ) , and general additive models employing various nonlinear mappings (hastie and tibshirani ) . the elucidation of spatial risk factors in le can provide key insight into how landscape impacts risk, but often these connections are difficult to identify and confounded with sampling unit and spatial scale (e.g., modifiable areal unit problem). evaluating disease control methods has been largely the domain of mathematical modelling methods that have been developed for outbreaks of infectious diseases, and environmental heterogeneities are rarely included-often because spatially explicit data are lacking and/or relationship to environmental factors are unknown (although see a recent example of the ebola outbreak by merler et al. ) . however policy-scenario modelling in the context of disease control can be used to investigate the effect of different public health policies on health outcomes of interest (e.g., claude et al. ). the required knowledge in order to parameterize models for evaluating interventions is generally very extensive and requires understanding some degree of absolute risk (e.g., lengeler et al. ) . here we propose a functional framework for organizing analytical tasks that are commonly employed as part of le, which we are terming geocomputational landscape epidemiology (gle). geocomputation was defined in openshaw ( ) as the 'application of a computational science paradigm to study a wide range of problems in geographical and earth systems (the geo) contexts'. this definition makes special point to emphasize that the geo includes both human and physical systems, and that geocomputation is part of a wider shift towards computational science in social sciences, natural sciences, and subfields of the humanities disciplines that emphasizes mathematical models, simulation, and high-performance computing. geocomputation is not equivalent to data mining, machine learning, or computer science, but may include aspects of these disciplines (couclelis ) . the defining characteristic of geocomputation is that advanced computational tools and methods are deployed for solving complex geographical problems that would otherwise not be possible. thus the tools in geocomputation do not only provide a faster way of doing calculations, they provide a methodology for conceptualizing new research questions from a computational science perspective, and the tools to answer these questions. geocomputation may be seen as analogous or congruous to the computational science approach to social science generally, described by torrens ( ) as ''making use of computing and informatics in exploring the mechanisms that drive complex social, behavioral, and economic systems'', emphasizing themes of complexity, modelling and simulation, visualization, cyberspaces, semantics, and socio-technical systems. in the natural sciences, advanced computational methods have been deployed for modelling and simulating environmental processes for many years, often as implementations of numerical models. there is a natural fit to apply the tools of geocomputation to landscape epidemiology, which was described originally by pavlovksy ( ) as the concept of the 'natural nidality of human diseases', recognizing that the sources of human disease existed naturally in the environment, varying with climate, soils, elevation, vegetation, and other landscape components. these ideas, most evident for the zoonotic infections for which they were discovered, have also been extended to chronic disease, and recognition of environmental effects on health is now widespread. recent adaptations and extensions have been formulated as ecosystem-health, which focuses on health from an ecological perspective (waltner-toews et al. ) , one health, which emphasizes inter-relatedness of animal and human health (coker et al. ) , and wildlife health (grogan et al. ; stephen ) , which takes a similar approach to the protection and promotion of healthy wildlife populations. we present this framework as a general organizing system for studying disease from an explicitly 'landscape' approach, one which by its nature requires some sophistication in spatial analysis and geographic information handling methodology. more importantly, the framework provided in fig. outlines a knowledge-based organization of geocomputational methods. characterize the assemblage the first component of gle is called 'characterizing the assemblage' and encompasses all methods of analysis that aim to describe a pattern or relationship. this phase usually comes before other phases and often will be the aim of study itself, such as to estimate a realistic distribution of disease risk from aggregated case data. there are two basic approaches to characterizing assemblages, which correspond to the two types of properties associated with a spatial pattern: pattern composition, and pattern configuration. pattern composition includes measures that describe how much of a quantity is distributed on the map, or broadly answer the question 'what is where?' we keep this definition broad as many of the approaches used in geocomputational approaches to le are aimed at answering these types of questions. for example, cluster detection and hotspot mapping methods are examples of this, which might not normally be described as measures of composition. measures of configuration-alternatively, seek to describe the spatial configuration of quantities on the landscape. configurational measures answer the question 'how is x configured on the landscape?'. answers to these types of questions are therefore descriptions of spatial pattern, rather than locations. details about the methods within this part of the framework can be found in table . measures of composition in this context comprise all methods that aim to describe or characterize the spatial or temporal distribution of risks and/or risk factors. as this is an extremely common and broadly defined task, this encompasses many different statistical approaches, but all aim to quantify or explain the distribution. measures of configuration contrast with those of composition as they are aimed at quantifying the spatial configuration of the pattern. the methods here are used to complement compositional measures as part of a gle study, and often have direct epidemiological relevance and can be used as covariates with modelling methods. characterizing functions of a system or disease of interest with geocomputational methods differs from the above as the focus here is on dynamic elements of quantities of interest. many of the methods and approaches noted in this section are more general than geocomputation alone, but do have important or special considerations when implemented in a setting where space is explicit. functional properties include describing inputs, outputs, and connections between different functions that might combine to form a complex system. as the ideas here are very general, we have broken up the concepts into some classes, though emphasize they area all descriptive of functional characteristics, and in most cases, require that basic characterization of the underlying landscapedisease assemblage has already been done. flows: activity patterns, trade flows, animal movement, vector movement many concepts relevant for describing health in le are better described as 'flows' then factors or static measurements of landscape properties. in our framework, flows can be defined as methods, variables, and concepts that describe movement of a quantity of interest in geographic and/or parameter space. in practice, this step of analysis might be used to develop spatial variables that are used in a study aimed at the 'characterize the assemblage' step, or may be the fig. framework for geocomputational landscape epidemiology which moves from lower level complexity of a describing patterns, b describing processes that interact with those patterns, c examining how patterns and processes contribute to disease risk and healthpromoting factors, and d evaluating final information products (maps or other) and link these to research gaps and knowledge uses geojournal ( ) : - objective of analysis. examples of flows include the movement of mosquito vectors with climate change, movement of cattle to markets in a region, commuting patterns of residents of a large metropolitan area, and the flow of capital into resource extraction industries in individual countries or regions. while we don't constrain our description to physical flows (e.g., diurnal movement of people within the city), we emphasize aspects of flows that can be represented and or analyzed within a geocomputational framework. often, non-geographic flows have important properties and expressions that are embedded within geographic space. for example, the use of social network analysis in recent years has increased-in order to identify physical contact tracing and exposure/transmission opportunities, but also to model the flow of knowledge about public health-related factors such as health-promoting activities or risk perceptions within and between vulnerable communities. in complex systems, feedbacks are defined as those components of a system that engage in or foster learning and exhibit behavior. in geocomputation, learning and behavior are increasingly relevant concepts, as algorithms are developed that learn relationships across space from continuous data streams (young ). many of the dynamical models of mathematical modelling center on feedbacks between system components, and estimation of critical epidemiological parameters. while geography has traditionally been excluded or assumed away in these models, the field of spatial statistics has developed spatially explicit extensions to classical epidemiological models (riley et al. ) . however, for le the majority of approaches that are focused on representing connectivity and positive and negative feedback between system components fall into the category of conceptual modelling approaches rather than empirical/mathematical modelling. the translation of conceptual models of complex systems to workable mathematical models that can be fit to observational data is one of the most challenging aspects of gle. in practice, there is often iteration between the approaches described in tables and and those in table . feedbacks can also exist within complex systems designed to represent a health domain that are identifying high risk areas kulldorff and nagarwalla ( ) , getis and ord ( ) and anselin ( ) gravity models effect of commuting patterns on spread of an infectious disease haynes and fotheringham ( ) and balcan et al. ( ) glm in r/spss etc. bishop ( ) and gahegan ( ) programming/applicationspecific geojournal ( ) : - created as loosely coupled ensembles of simple models-which are integrated with an aim of intelligence, situational awareness, or monitoring. events in the context of gle are discrete in space and time, and have relevance for understanding how landscape influences disease dynamics. the international health regulations ( ) define an event as ''a manifestation of disease or an occurrence that creates a potential for disease'' (ihr ) . this includes infectious disease, zoonotic disease, toxic pollution events, and these threats to public health form the basis for the 'event-based surveillance' approach described as part of who's early warning and response system (who ) . events in gle therefore correspond to methods and tools that are required for the handling, processing, and analysis of information sources that provide context or early-warning for occurrences that create potential for landscape-oriented disease (see table ). while the 'phases' above have focused on a categorization of existing methods used in geocomputational approaches to le, we now turn to a phase where the functions described in part are mapped back onto the compositional and configurational patterns described in part . here, we begin to see how interacting components of the system are spatially structured, which functional components share geographic properties, and what the overall controlling contribution of landscape is to the risk profile of interest. this might be considered an integrative or meta-analysis step as this is rarely done within the context of a single study, but generally is part of a review study or systemsbased empirical analysis of existing literature. there are few formal methods available for this step, but these types of meta-analyses or systems analysis are typically depended on when major new health events such as the emergence of a new disease occurs, as they provide a holistic description of the processes that conspired to lead to the event. a good example is provided by wang and eaton ( ) which describes the overall conditions that led to the emergence of sars in southeast china in including documented human-to-human spread, independent transmission events from animal-to-human in four separate cases, and animal-to-animal transmission among palm civet cats in a market environment. an example of a technological approach to this step might be that of healthmap which provides integration of reported health events obtained from the web from all over the world (brownstein et al. ) . through scraping, geocoding, and mapping health event data in a common platform, this platform provides the capacity for 'epidemic intelligence'using space to index health events and draw common cause and connections where they otherwise might not be apparent. this also provides multi-scale exploration of the patterns of outbreaks, from the local to international. the final part of this framework is to examine the outcomes, real or simulated, from the geocomputa- na tional representation of the disease/landscape system of interest. the task of examining the outputs of a model can include visual analysis, pattern comparison measures and sensitivity analyses to changes in model parameters. additionally, this sort of integrated interpretation also considers the advances made in the nonlandscape aspects of the disease that have taken place and may provide alternate (re)-interpretations of patterns. for example, when a new disease emerges, patterns of spread and distribution will be unexplained until the natural reservoir has been isolated and genetic work completed. the objective of this level of analysis is to realize integrated insight into landscape-disease interactions and risk. what follows is a brief case study that highlights the components of the framework in relation to a zoonotic disease of global importance, leptospirosis (bharti et al. ) . the case study is not exhaustive of the framework phases, and includes a review of previously published work and new analysis of a large outbreak in in sri lanka. leptospirosis is a one of the most prevalent zoonoses in the world, affecting millions of people annually every year. the bacterial spirochaete (i.e., leptospira) that gives rise to the disease in humans have a large number of animal reservoirs, including cattle, rodents and foxes. the bacteria are passed from animal reservoirs to the environment through urine-where they can then infect humans. in sri lanka, leptospirosis is historically associated with rice agriculture. transmission occurs when open wounds or skin abrasions come into contact with urine from infected rodents (giving rise to the local name 'rat fever'). diagnostic facilities are limited and the clinical presentation is similar to hantavirus infections (gamage et al. ; sunil-chandra et al. ) , and only a fraction of suspected cases are tested. a large outbreak of suspected leptospirosis occurred in sri lanka in . the reasons for the outbreak remain speculative, but are hypothesized to be at least partially due to expanded cultivation of paddy field areas that resulted from policies aimed at increasing domestic rice production. as part of the programme 'api vavamu, rata nagamu', unused fertile lands were targeted for food production among landholders, many which had not traditionally been involved in farming. the development of this program was a direct response to the global rise in food prices that began accelerating in , with international price of rice increasing % between (kelegama . according to the central bank of sri lanka, rice paddy production increased . % between and (kelegama ) . there are many questions related to the causes of the epidemic of notified cases of leptospirosis in sri lanka in to be investigated, including (a) whether cases truly were leptospirosis or a concurrent outbreak of some other clinically indistinct outbreak as has been theorized (agampodi et al. ); whether the epidemic had strong environmental determinantseither in higher than average rainfall, flooding, or other factors, and whether the epidemic was predictable and (c) what early warning could have been forecast using available data; (d) would the epidemic have happened had the local food production policy not been in place, and finally-(e) what was the importance of variability in clinical suspicion in driving the spike in reported cases. many of these questions have geographical dimensions that warrant and/or necessitate a geocomputational approach. employing the framework outlined in this paper as a guide, we will show how we could approach some of these research questions. starting with part -in many studies where a gle approach is employed, variables related to local meteorology and lu/lc will be the first step towards characterizing the assemblage of relevance for understanding the disease distribution. in many developing countries, these variables exist but are difficult to access or not available directly in digital form, and researchers often rely on long-term normals. depending on the application, these may be suitable candidates, but for most applications and gle contexts daily meteorological observations are required (or a combination of daily and normal)-a step that can require extensive text parsing to transform the data into a format suitable for storage in a geographic database. note that an important precursor to this step is to identify the spatial and temporal scales that are relevant for the study. the key questions we will explore in this analysis relate to the relationship between rainfall and reported cases of leptospirosis during the peak of the epidemic in . as disease reporting occurs over the administrative units scribed for the ministry of health, called medical officer of health (moh) areas in sri lanka, we need to standardize both measures over these geographical units. the potential mechanistic relationships include at least those outlined in table so the actual temporal lag required to investigate the relationship between cases is a key parameter of interest, as this could be used to determine the earlywarning value of rainfall modelling. in order to transform daily rainfall records obtained from the department of meteorology into seamless rainfall maps for the entire country, spatial interpolation modelling was performed. previous comparative analysis of interpolation methods found that thin-plate smoothing splines were a generally effective method for interpolating rainfall in sri lanka (plouffe et al. ) . daily rainfall records were tabulated into monthly totals for each of the stations, and each month was interpolated using tps function in the fields package in r (nychka et al. ) . averaging interpolated total rainfall for each month over each moh area provided concurrent estimates of rainfall and reported cases of leptospirosis over equivalent geographical units. surveillance data for weekly reported cases of leptospirosis by moh area were aggregated by year, and standardized by population to produce estimates of incidence per , for the year in each geographical unit. plots of the time series of annual cases and the monthly rainfall maps were compared visually. simple cross-correlation analysis between case counts and rainfall was used to identify potential temporal lags of importance for early-warning. the monthly time series of cases reveals a large spike in cases in september (fig. ) . the timing of this spike is consistent with peaks in cases in previous years, however the magnitude is much greater and worthy of further investigation. examining the rainfall patterns over evident in fig. , the most striking pattern is the unseasonably intense and extensive rainfall that occurred in march . whether this unusually high amount of rainfall occurred was related to the spike in reported cases on leptospirosis is unclear. correlation analysis revealed both positive and negative associations between incidence and rainfall ( table ) . the highest rank monthly correlations in terms of t-statistic magnitude were july (positive), march (negative), and april (positive). the magnitudes of the correlations were not high; indicating limited predictive potential at least at the scales investigated here. direct comparison of month-tomonth correlations would provide a more robust indicator of the relationship-however power is also reduced due to the smaller numbers and smaller effect sizes. month-to-month cross correlations indicated both positive and negative associations (fig. ) . while some interesting spatial patterns emerge from this analysis, the evidence is insufficient to explain the outbreak in , as key information is missing. note that our previous modelling work of landscape factors related to clusters of high risk found associations between paddy areas, small agricultural holdings, distance to cities and distance to rivers (robertson et al. ). with a basic description of the reported leptospirosis surveillance data now in place, we can move to use this example to motivate additional analyses within the gle framework that would improve our understanding of the dynamics of risk and the causes of the outbreak. the analyses reported in the section above indicated some evidence for a relationship driving at least the endemic cycle of cases, an anomalous event in september which does not have an obvious rainfall signal, and evidence of spatial variability in the relationships, suggesting perhaps both rural agriculture risk profile and a periurban risk profile combining to produce the risk landscape in . a functional analyses would examine the flows, feedbacks, and events that would describe the movement of people, their interaction with the health care system and care-seeking behaviours, application of control mechanisms, distributions of animal reservoirs and their habitats or production chains in the case of livestock, and individual interactions with paddy fields. as these forms of data are much more difficult to obtain than those used in the previous section, a dynamic modelling approach using individual-based models would allow for exploration of the parameter space in a way that we could test alternate scenarios that might lead to the observed pattern. we may also investigate specific events that occurred in september that may explain the spike in reported cases. for example, the year saw heavy fighting as the sri lankan army engaged in an offensive military push to end the civil war with the tamil tigers (ltte) that controlled much of the territory in the north of the country. in early september, the sri lankan army captured the town of mallavi that served as an administrative centre for the ltte. further study could investigate the impact of these events on disease risk and disease reporting. examining the surveillance data, the largest number of reported cases in september was reported from the homagama area; part of the colombo district in western province. this represents a % increase over the next highest reported month in this district, which otherwise follows expected seasonal dynamics. the cause(s) of this spike remains to be investigated, and could range from a simple data entry error, misdiagnoses, to linkages with the activities associated with the end of civil conflict in the north. looking at the weekly surveillance data, the fact that these cases were relatively evenly distributed across the weeks suggest a disease-causing event did occur during this time frame. in order to obtain system-level inferences on the leptospirosis-landscape system being investigated here, several additional studies would be required. we would suggest that these 'higher-level' inferences tend to be qualitative in nature, collaborative in their genesis, and built from shared interpretations of the more quantitative approaches to analyses described earlier. this aspect of analyses, though rarely formally articulated, should constitute a significant step in gle, especially given the highly multi and interdisciplinary nature of team-based research projects including ecologists, biologists, veterinarians, geographers, and social scientists. knowledge translation activities that include workshops, videos, tutorials, and others that optimally translate scientific knowledge to user-communities and knowledge-users is a critical integrative step for realizing 'decision-support' capacity of advanced spatial and statistical modelling efforts. we have presented a framework for gle that provides a categorization of methods commonly used to investigate landscape-disease interactions, can be used to design and frame future studies, and to provide a functional mapping between knowledge uses and methods. the framework is general and extendable, and will situate stronger research design for spatially focused projects in le. additionally, the framework may be a useful educational tool for introducing newcomers to the extensive library of models and methods available for spatially explicit data. this may enhance multidisciplinary research teams working on complex disease-landscape interactions. leptospirosis outbreak in sri lanka in : lessons for assessing the global burden of disease population biology of infectious diseases: part i local indicators of spatial association-lisa trends in exposure to industrial air toxins for different racial and socioeconomic groups: a spatial and temporal examination of environmental inequality in the u.s. from spatstat: an r package for analyzing spatial point patterns multiscale mobility networks and the spatial spreading of infectious diseases big data and disease prevention: from quantified self to quantified communities bayesian analysis of space-time variation in disease risk leptospirosis: a zoonotic disease of global importance pattern recognition and machine 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leptospirosis-hantavirus co-infection in acute patients hospitalized in sri lanka: implications for a potentially worldwide underestimated problem mapping population and pathogen movements geography and computational social science web technologies for environmental big data. environmental modelling and software the ecosystem approach: complexity, uncertainty, and managing for sustainability bats, civets and the emergence of sars wildlife and emerging zoonotic diseases: the biology, circumstances and consequences of cross-species transmission assessing spatial and nonspatial factors for healthcare access: towards an integrated approach to defining health professional shortage areas image quality assessment: from error visibility to structural similarity modelling spread of foot-and-mouth disease in wild white-tailed deer and feral pig populations using a geographic-automata model and animal distributions social and environmental risk factors in the emergence of infectious diseases the epidemiological transition: from material scarcity to social disadvantage? early detection, assessment and response to acute public health events: implementation of early warning and response with a focus on event-based surveillance effects of changing scale on landscape pattern analysis: scaling relations spatializing health research: what we know and where we are heading landscape epidemiology and machine learning: a geospatial approach to modeling west nile virus risk in the united states acknowledgments the authors gratefully acknowledge the epidemiological unit of the ministry of health, government of sri lanka, for providing access to the leptospirosis surveillance data used in this paper. conflict of interest none.research involving human participants and/or animals no humans were involved in this research.informed consent no human participants were involved in this study. key: cord- -fi s x authors: andries, k.; pensaert, m.; callebaut, p. title: pathogenicity of hemagglutinating encephalomyelitis (vomiting and wasting disease) virus of pigs, using different routes of inoculation date: - - journal: j vet med b infect dis vet public health doi: . /j. - . .tb .x sha: doc_id: cord_uid: fi s x summary: forty‐eight pigs were inoculated by different routes with the vw isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. all piglets inoculated by the combined oral — nasal route ( ) or into the infraorbital nerve ( ) became sick after an incubation period of days. six of the pigs inoculated into the stomach wall, of the pigs inoculated intramuscularly and of the pigs inoculated intracerebrally became ill after an incubation period of – . days. none of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. all diseased pigs showed the vomiting and wasting syndrome. in oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. pigs inoculated by other routes were positive for virus when sick. all except one of the pigs which remained healthy had seroconverted. the site of virus replication which gives rise to the vomition could not be determined. it was concluded from the present studies that virus spread within the body occurs along nerve pathways. zusammenfassung: die pathogenität von hämagglutinierendem enzephalomyelitis‐virus (kümmern und erbrechen) bei shweinen nach infektion über vershiedene inokulationswege achtundvierzig schweine wurden übegr verschiedene inokulationswege mit dem vw ‐isolat des hämagglutinierendtn enzephalomyelitis‐virus (kümmern und erbrechen) infiziert. alle schweine, die entweder kombiniert oro‐nasal ( ) oder über den infraorbitalnerv ( ) infiziert wurden, erkrankten nach einer inkubationszeit von tagen. sechs der sieben über die magenwand inokulierten, oder intramuskulär und der intrazerebral infizierten tiere wurden nach einer inkubationszeit von – , tagen krank. bei den schweinen, die intravenös oder in die bauchhöhle bzw. direkt in den magen inokuliert wurden, kamen erkrankungsfälle nicht vor. alle erkrankten schweine zeigten das syndrom des kümmerns und erbrechens. von oro‐nasal infizierten schweinen, die während des frühstadiums der erkrankung getötet wurden, konnte das virus regelmäßig von den tonsillen und dem respirationstrakt und gelegentlich vom gewebe des pons‐medulla‐bereiches sowie aus der magenwand reisoliert werden. von schweinen, die nach infektion über andere routen erkrankten, ließ sich immer virus isolieren. alle tiere, die nicht erkrankten (mit ausnahme eines ferkels) bildeten jedoch antikörper. der ort der virusvermehrung, mit dem das erbrechen zusammenhängt, ließ sich nicht ermitteln. die ergebnisse der vorgelegten untersuchungen lassen den schluß zu, daß die virusausbreitung im körper über die nervenbahnen erfolgt. rÉsumÉ: pathogénicité du virus hémagglutinant de l'encéphalomyélite du porc (dépérissement et vomissement) après infection par différents modes d'inoculation porcs ont été infectés selon différents procédés d'inoculation avec l'isolement vw du virus hémagglutinant de l'encéphalomyélite (dépérissement et vomissement). tous les porcs infectés soit par la voie combinée oronasale ( ) soit par le nerf infraorbital ( ) tombèrent malades après une incubation de jours. des animaux infectés par la paroi stomacale, des par voie intramusculaire et des intracérébralement tombèrent malades après un temps d'incubation de – , jours. il n'y a pas eu de maladie chez les porcs inoculés par voie intraveineuse, dans l'abdomen ou directement dans l'estomac. tous les porcs malades ont présenté le syndrome de dépérissement et de vomissement. chez les animaux infectés par voie oro‐nasale et sacrifiés au début de la maladie, on a pu régulièrement réisoler le virus à partir des amygdales et de l'appareil respiratoire, parfois du tissu de la région «pons‐medulla» et de la paroi stomacale. le virus a toujours été isolé chez les porcs tombés malades après un mode d'infection différent. tous les animaux qui ne furent pas malades (à l'exception d'un porcelet) formèrent des anticorps. l'endroit de multiplication du virus lié au syndrome de vomissement n'a pas été déterminé. les résultats de ces essais permettent de conclure que la propagation du virus dans le corps se fait par voie nerveuse. resumen: la patogeneidad del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos) en el cerdo tras infección a través de vías diversas de inoculación se infectaron cuarenta y ocho cerdos a través de diferentes vías de inoculación con el aislamiento vw del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos). todos los cerdos infectados bien con arreglo al procedimiento combinado buco‐nasal ( ) o bien a través del nervio infraorbitario ( ) enfermaron tras un tiempo de incubación de días. seis de siete animales inoculados a través de la pared gástrica, de por vía intramuscular y de por vía intracerebral enfermaron tras un tiempo de incubación de – , días. no se registraron casos de enfermedad en los cerdos inoculados por vía intravenosa o en la cavidad abdominal resp. directamente en el estómago. todos los cerdos que enfermaron presentaban el síndrome de hipotrepsia y vómitos. de los cerdos infectados por vía buco‐nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tracto respiratorio y, en ocasiones, del tejido correspondiente al ámbito puente de varolio‐medula, así como de la pared gástrica. se logró siempre aislar virus de cerdos que enfermaron tras infección por otras vías. sin embargo, todos los animales que no enfermaron (excepción hecha por un lechón) produjeron anticuerpos. no se logró descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con los vómitos. los resultados de los estudios presentados permiten llegar a la conclusión de que la propagación del virus en el organismo acontece a través de las vías nerviosas. in , an infectious encephalomyelitis of viral origin was observed in pigs in canada ( ) . the clinical illness was characterised by a prodromal stage with anorexia, depression, retching and vomiting, followed by motor disorders ( ) . the virus responsible for this disease was later found to be a porcine coronavirus ( ; ) which, because of its hemagglutinating properties, was called hemagglutinating encephalomyelitis virus or hev ( ) . an infectious disease of suckling pigs, characterised by vomiting and wasting, was described in england in ( ) and later observed in other european countries ( ; ; ; ; ) . the disease, called vomiting and wasting disease, was shown to be caused by a virus antigenically and structurally similar if not identical to the hev from canada ( ; ; ). a similar coronavirus, which caused mainly vomiting and wasting symptoms in hysterectomy-derived colostrum-deprived pigs was also reported from the u.s.a. ( ). using the same field isolates mengeling was able to reproduce experimentally the two clinical entities, the encephalitic form and the vomiting and wasting form ( ) . it was, therefore, concluded that both types of the disease, are caused by the same virus. few studies have been performed on the pathogenesis of this viral infection. transmission experiments have proved that reproducible results could be obtained only if colostrum-deprived pigs were used ( ; ). clinical disease has been succesfully reproduced after inoculation of the virus by oral, intranasal, intramuscular and intracerebral routes ( ) . in other studies, the virus was reisolated with regularity from brain, respiratory tract and oronasal swabs, but only if the pigs were examined during the early stages of the disease ( ; ) . hemagglutination inhibiting and seroneutralizing antibodies were demonstrable starting at and days after inoculation respectively ( ). these antibodies are suspected to preclude virus isolation at later stages of the disease ( ; ; ). the present studies were primarily designed to determine whether a virus isolate, obtained from pigs with the vomiting and wasting syndrome only, could produce clinical signs after inoculation by different routes. at the same time, some information was obtained on the optimal conditions for virus reisolation and on the distribution of the virus in different organs particularly after oronasal inoculation. . . virus the virus strain, designated vw , was isolated in belgium in as previously described ( ) . the virus stock used represented the loth passage in primary pig kidney (ppk) cells and contained "' tcid,, per ml. the studies were performed in hysterectomy-derived-colostrum-deprived (hdcd) pigs which were individually housed in horsfall type units. they were inoculated at the age of to days using different routes of inoculation. a blood sample was withdrawn prior to inoculation to assure the absence of specific viral antibodies. sixteen piglets were inoculated by combined oral and nasal (on) route, using ml. of stock virus. in seven piglets, . to ml. of stock virus was inoculated directly into the stomach wall after laparotomy around the pyloric sphincter at four inoculation sites. five pigs were inoculated with ml. of stock virus directly into the gastric lumen after laparotomy by inserting a needle through a canula which perforated the stomach wall. in two pigs, ml. of stock virus was brought into the abdominal cavity after laparatomy. two pigs were inoculated with ml. of stock virus intravenously into the ear vein. five pigs were inoculated in the cerebral cortex, using ml. of stock virus, by perforating the skull with a needle. four pigs were inoculated into the infraorbital nerve with . ml. of stock virus by insertion of the needle into the infraorbital canal. eight pigs were inoculated with ml. of stock virus intramuscularly either in the neck muscles or in the foreleg muscles or in the hindleg muscles. all pigs were observed three times a day for clinical signs. when sick, pigs were killed at time intervals varying from one to five days after the appearance of clinical signs and different tissues were collected for virus isolation. from the on inoculated pigs, serum was collected at the time of killing for detection of hemagglutination inhibiting (hi) and seroneutralizing (sn) antibodies. the pigs which did not become sick during the present study were followed clinically until weeks after the inoculation. during the last week of the observation period, a serum sample was collected and examined for the presence of hi-antibodies. from the pigs killed at different time intervals after inoculation, the following tissues were collected for virus isolation : nasal mucosa, tonsils, lungs (apical and cardiac lobes), pyloric region of the stomach, pons and medulla combined, cerebrum, cerebellum and blood clot. from each organ, a per cent suspension (w/v) was prepared in phosphate buffered saline by homogenisation. after centrifugation at x g., the supernatant fluid was collected and . ml. inoculated in each of tubes with fully sheeted primary pig kidney (ppk) cells. after four days, hemadsorption with chicken erythrocytes was attempted on tubes. the remaining tubes were used for making a second passage. the cells and culture medium were subjected to two cycles of freezing and thawing and, after centrifugation a t x g., the supernatant was inoculated in tubes with fresh monolayers of ppk-cells. hemadsorption was performed on all tubes days later. the procedures used for the hemagglutination inhibition and seroneutralisation test were previously described ( ) . in the presentation of the results, differentiation will be made between the on inoculated pigs and the pigs inoculated by other routes. the number of pigs in the on inoculated group was sufficiently high to allow a more complete study since animals could be killed at several time intervals after the appearance of clinical signs. for the other routes of inoculation, it was the main purpose to see whether clinical illness could be obtained or not. the overall results obtained after on inoculation of the pigs are presented in table i . the individual results of these pigs are presented in table . all pigs were free of hiand sn-antibodies at the time of inoculation. it can be seen from the tables that all on inoculated pigs became ill. the incubation period varied from to days with an average of days. the earliest clinical signs were characterised by anorexia and vomition which suddenly appeared. the vomition usually started after the uptake of milk. the vomit first consisted of milk curds and changed, after a few hours, to a yellow foamy fluid. as soon as the piglets took up more milk, curds were seen again. vomition and retching movements were very regularly present during the first hours. afterwards, the frequency of vomition decreased but the appetite remained markedly reduced. the smaller pigs became emaciated and moribund after to days. the course of the disease was somewhat slower in the stronger pigs. virus could be isolated from of the pigs. as shown in table , the virus was isolated most consistently from the lungs and tonsils. it was sometimes isolated from the pons + medulla and nasal mucosa, and only in pigs from the stomach wall. virus was not isolated from the cerebrum, the cerebel- lum and the blood clot. all pigs which were killed days or later after inoculation had hi-antibodies, whereas sn-antibodies were detected starting days after inoculation. the pons + medul li a of these pigs was positive by immunofluorescence. the overall results obtained in the pigs inoculated by other routes are presented in table . none of the pigs inoculated into the stomach lumen, the abdominal cavity or the ear vein had become sick at weeks after inoculation. all these animals, except one of the pigs inoculated into the gastric lumen, had hi-antibodies at that time. a variable number of pigs became sick after inoculation by other routes. the clinical disease was not different from that observed after on inoculation. in table , the sick pigs were considered to be positive for virus when it was isolated from one or more of the collected organs, the inoculated organ not included. six of the seven pigs inoculated in the pyloric region of the stomach became sick after an average incubation period of days. from two of the six pigs, the virus was reisolated from the stomach wall only whereas, in the remaining four, the virus could be detected in other tissues also. hi-antibodies (titer ) were present in the serum of the pig without clinical signs at the end of the observation period. of the intracerebrally and intramuscularly inoculated pigs, three of the five and six of the eight respectively became sick after an average incubation period of * / days, all the diseased pigs were positive for virus. the pigs which remained healthy had built up hi-antibodies at titers varying from to at the end of the experiment. all three pigs inoculated into the infraorbital nerve became sick after an average incubation period of days and were positive for virus. the clinical disease and the results of virus isolation obtained upon combined oral and nasal (on) inoculation were similar to those previously described ( ) . the virus was most consistently reisolated from the respiratory tract which is probably the natural way of infection. seroneutralizing antibodies appear soon after the beginning of the clinical symptoms and may be a limiting factor for the further succesful isolation of the virus from organs. for the diagnosis of hemagglutinating encephalomyelitis by virus isolation, pigs should therefore be examined preferably within two days after the start of clinical signs. the negative results obtained in pigs and . , killed within this period of time, are probably due to the fact that tonsils and pons + medulla were not collected for virus isolation. the pons + medulla of these pigs was later found to be positive by immunofluorescence. the irregularity with which virus was isolated from the stomach wall and pons + medulla does not allow any conclusions to be drawn about the exact target organ, if it is supposed that virus replication in one of these organs is responsible for clinical signs. this irregularity may be due to the lack of sensitivity of the virus isolation technique. recently, it has been found that centrifugation of tissue suspensions at x g. instead of x g. can remove up to log,, tcid,, of hemagglutinating encephalomyelitis virus. in this way, virus isolation from a tissue suspension with little infectious virus could give a false negative result. it is known from electron microscopic studies that hev during intracellular replication is mainly present in cytoplasmic vesicles with a large amount of membranous material ( ). the removal of virus by centrifugation may, therefore, be due to sedimentation of particles associated with subcellular fragments. upon inoculation into the stomach lumen, clinical signs were not observed. this finding indicates that virus reaching the gastric lumen after swallowing does not initiate clinical disease. however, the seroconversion observed in of the pigs indicates that a subclinical infection has occurred. the ability to reproduce typical disease after a very short incubation period in of the pigs inoculated in the stomach wall may be significant in the light of the pathogenesis. in two of these piglets, virus was isolated from the stomach wall only. this points to the stomach wall as a possible target organ for the virus. it is, however, not excluded that the isolated virus is a residue of the inoculum rather than a result of virus replication. the first possibility is rather improbable since these pigs were killed and days after inoculation respectively. in the present experiments, not all the pigs became sick after intracerebral inoculation and the disease consisted of the vomiting and wasting syndrome. these results are somewhat different from those described by greig, who obtained clinical signs consisting of motor central nervous disorders in all inoculated pigs ( ) . this may be related to the observation that, with the present virus isolate, only the vomiting and wasting syndrome has been observed, not only in the original outbreak but also in experimental pigs. it should be mentioned, however, that mengeling was able to reproduce both clinical entities with the same virus isolate ( ) . the inability to find the virus in the cerebral cortex of on inoculated pigs may indicate that the present isolate has little or no affinity for this part of the central nervous system. it is not impossible that, in intracerebrally inoculated pigs, the appear-ance of clinical signs is determined by the ability of the virus to reach the pons + medulla, another possible target organ, from the deposition site. the findings that none of the pigs became sick after intravenous inoculation and that the virus was never isolated from the blood clot of pigs killed at different time intervals after on inoculation indicate that the target organ is not reached through viraemia. on the other hand, the ability to obtain typical disease after inoculation into the infraorbital nerve suggests that virus spread within the body occurs via nerve pathways. whether clinical signs are obtained in pigs after intramuscular inoculation may be dependent on the ability of the virus to enter into nerves. from the present studies, it was impossible to determine the exact target organ of the vw virus. the results indicate that virus replication, either in the stomach wall or in the pons + medulla or possibly in both, may be responsible for causing the vomiting and wasting syndrome in pigs. forty-eight pigs were inoculated by different routes with the vw isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. all piglets inoculated by the combined oralnasal route ( ) or into the infraorbital nerve ( ) became sick after an incubation period of days. six of the pigs inoculated into the stomach wall, of the pigs inoculated intramuscularly and of the pigs inoculated intracerebrally became ill after an incubation period of - . days. none of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. all diseased pigs showed the vomiting and wasting syndrome. in oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. pigs inoculated by other routes were positive for virus when sick. all except one of the pigs which remained healthy had seroconverted. the site of virus replication which gives rise to the vomition could not be determined. it was concluded from the present studies that virus spread within the body occurs along nerve pathways. the study was supported by the belgian research institute for industry and agriculture (instituut tot aanmoediging van het wetenschappelijk onderzoek in nijverheid en landbouw), brussels, belgium. the technical assistance of gerry van ( ) la patogeneidad del virus hemoaglutinante de la enckfalomielitis (hipotrepsia y vbmitos) en el cerdo tras infeccibn a travds de vias diversas de inoculacibn se infectaron cuarenta y ocho cerdos a travks de diferentes vias de inoculacibn con el aislamiento vw del virus hemoaglutinante de la enckfalomielitis (hipotrepsia y v mitos). todos s cerdos infectados bien con arreglo a procedimiento combinado buco-nasal ( ) o bien a travks del nervio infraorbitario ( ) enfermaron tras un tiempo de incubacidn de dias. seis de siete animales inoculados a travks de la pared glstrica, de por via intramuscular y de por via intracerebral enfermaron tras un tiempo de incuba-ci n de - , dias. no se registraron casos de enfermedad en s cerdos inoculados por via intravenosa o en la cavidad abdominal resp. directamente en el estbmago. todos s cerdos que enfermaron presentaban el sindrome de hipotrepsia y v mitos. de s cerdos infectados por via buco-nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tract respiratorio y, en ocasiones, del tejido correspondiente a lmbito puente de varolio-medula, asi como de la pared gistrica. se logr siempre aislar virus de cerdos que enfermaron tras infecci n por otras vias. sin embargo, todos s animales que no enfermaron (excep-ci n hecha por un lech n) produjeron anticuerpos. no se logr descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con s v mitos. los resultados de s estudios presentados permiten llegar a la conclusi n de que la propagaci n del virus en el organism acontece a travds de las vias nerviosas. vomiting and wasting disease in piglets isolement en france et identification du virus de la maladie du vomissement et du dcpcrissement des porcelets (coronalike virus) lesions induced by hemagglutinating enccphalomyelitis virus strain n in pigs vomiting and wasting disease in piglets a hemagglutinating virus producing encephalomyelitis in baby pigs characteristics of a coronavirus (strain n) of pigs experimentally induced infection of newborn pigs with hemagglutinating encephalomyelitis virus strain n pathogenicity of field isolants of hemagglutinating encephalomyelitis virus from neonatal pigs characteristics of a coronavirus causing vomition and wasting in pigs the size and morphology of tge and vomiting and wasting disease viruses of pifs a viral encepha omyelitis of pigs. further studies on the transmissibility of the disease in ontario vomiting and wasting disease of piglets key: cord- -sxz s kz authors: daniel givens, m.; marley, m.s.d. title: infectious causes of embryonic and fetal mortality date: - - journal: theriogenology doi: . /j.theriogenology. . . sha: doc_id: cord_uid: sxz s kz the purpose of this review is to summarize bacterial, fungal, protozoan, and viral causes of reproductive dysgenesis in cattle, sheep, goats, pigs, horses, dogs, and cats. the clinical presentations of disease due to reproductive pathogens are emphasized, with a focus on assisting development of complete lists of causes that result in abortion and infertility in these species. clinicians are encouraged to assess clinical presentation, create complete lists of differential diagnoses, obtain appropriate diagnostic samples, maximize diagnostic laboratory support, and avoid zoonotic infections resulting from reproductive pathogens of animals. the foundation of an accurate diagnosis of reproductive loss due to infectious pathogens facilitates the prudent use of immunization and biosecurity to minimize reproductive losses. the purpose of this review is to summarize infectious causes of reproductive dysgenesis that compromise viability of domestic animals prior to parturition. for sake of clarity, the embryo is defined as the part of the conceptus that gives rise to the neonate, from fertilization to completion of organogenesis (day of pregnancy in cattle). furthermore, the fetus is defined as that part of the conceptus that gives rise to the live neonate from completion of organogenesis to completion of the second stage of parturition. although more deaths occur at and around birth than between week of age and weaning, embryonic losses are usually higher than perinatal losses. notably, maternal infections during pregnancy may or may not impact fetal development. the impact on fetal development may be caused directly by infectious agents or their toxins, or indirectly by placentitis. furthermore, detection of a reproductive pathogen should not preclude further diagnostic testing, as dual infections are not uncommon. embryonic or fetal death may result in resorption, mummification, maceration, or abortion. factors that impact the outcome of embryonic and fetal death include gestational age, cause of death, and source of progesterone for pregnancy maintenance (table ) . aborted fetuses may be autolytic due to death - h before abortion. fetal mummification occurs most commonly in polytocous species (e.g. swine). bacterial infections are usually not involved in mummification; however, viruses are a common cause of mummification in pigs, dogs, and cats. fetal maceration results when abortion or parturition fails to occur following fetal death and cl regression (occasionally in bovine www.theriojournal.com available online at www.sciencedirect.com theriogenology ( ) - table infectious causes of infertility and abortion in domestic animals fungal protozoan viral trichomoniasis and vibriosis). the majority of abortions are not epizootic but sporadic, with < % of pregnant animals in a group aborting. severe maternal illness resulting in high fever (e.g. mastitis, pneumonia), hypoxia (e.g. anaplasmosis, severe anemia), or endotoxemia may result in abortion. although very few lesions are pathognomonic, gross fetal lesions may be characteristic for mycotic abortion (fresh dysmature fetus with dermatitis) and epizootic bovine abortion (fresh fetus with a nodular liver). when multiple fetuses are present in utero, infectious pathogens may result in different outcomes in different fetuses. sows physiologically terminate pregnancies comprised of less than four embryos and subsequently exhibit a regular interestrus interval. thus, litters of four or less liveborn piglets suggest embryonic or fetal death, even if stillbirths or mummified fetuses are not clinically observed. numerous bacterial, viral, protozoan and fungal pathogens have been associated with infertility and abortion in cattle. these pathogens can result in substantial economic losses, indicating the need for control measures to prevent infection or disease. leptospira spp. are zoonotic spirochetes. cattle are the maintenance hosts for leptospira interrogans serovar hardjo (type hardjoprajitno) and leptospira borgpetersenii serovar hardjo (type hardjo-bovis) and incidental hosts for serovar pomona which is maintained in swine [ ] . transmission among maintenance hosts is through contact with infected urine, milk, placental fluid, transplacentally, or venereally. transmission to an incidental host occurs via contact with an environment contaminated with infected urine. the bacteria gain access through the mucous membranes of the eyes, nose, vagina, or abraded skin. infection in pregnant animals can lead to abortion, stillbirth, or birth of weak calves. abortion following infection with serovar pomona occurs in the last trimester, whereas abortion caused by serovar hardjo occurs from months of gestation to term. abortion rates within a temporal ''outbreak'' are generally higher following infection with serovar pomona compared with hardjo. infertility is also considered to occur with serovar hardjo infections. campylobacter fetus subsp. venerealis is a gramnegative rod transmitted venereally among cattle [ ] . it might also be transmitted through contact with contaminated bedding or instruments or other infected animals. infection of the vagina, cervix, endometrium and placenta can occur. infected animals can experience signs of infertility due to early embryonic death and abortion between and months of gestation. infected cows usually clear the organism within - months. histophilus somnus is part of the normal bacterial flora of the reproductive tract, but has rarely been associated with bovine abortion [ ] . the organism might also lead to infertility by binding to the zona pellucida of embryos [ ] . transmission occurs via contact with materials contaminated by infected respiratory or vaginal discharges; the bacteria then spread hematogenously to the fetus. brucella abortus is a zoonotic, gram-negative coccobacillus, transmitted via ingestion of fetus, placenta, uterine discharge, or materials contaminated by these products [ ] . through hematogenous spread, the bacteria localize in the trophoblasts. abortions generally occur after the fifth month of gestation. retained fetal membranes and metritis often occur. listeria monocytogens is a zoonotic, gram-positive coccobacillus, transmitted through ingestion of spoiled feed or feed contaminated with infected fetus, placenta or uterine discharge [ ] . the organism spreads hematogenously to the placenta and fetus. abortion generally occurs in the last trimester. infected cows can show clinical signs of fever, weight loss, endometritis, and retained fetal membranes. although ureaplasma diversum and mycoplasma spp. are part of the normal flora of the reproductive tract, they can also cause reproductive failure [ , ] . transmission can occur through direct contact, environmental contamination with infected urine, and venereally. infection with u. diversum can lead to granular vulvitis, infertility, abortion, and birth of weak calves. mycoplasma bovigenitalium infection can cause granular vulvovaginitis, infertility, and endometritis. experimentally, mycoplasma bovis can cause endometritis, salpingitis, infertility and abortion. chlamydophila abortus and chlamydia-like waddlia chondrophila can infect cattle [ , , ] . infection with c. abortus can result in abortion ( - months of gestation) or birth of weak calves. w. chondrophila also causes abortion. transmission occurs through ingestion or inhalation of feces, urine, or contaminated discharges (feces, nasal, ocular, vulvar, uterine, placenta). the organism causes endometritis and can multiply in cotyledons. these pathogens are zoonotic and may cause abortion in women. abortions can also be caused by other bacterial pathogens that are part of the normal microflora or present in the environment [ , ] . these bacteria might include: arcanobacterium pyogenes, bacillus spp., escherichia coli, pasteurella spp., pseudomonas spp., salmonella spp., staphylococcus spp., and streptococcus spp. bacteria are able to spread hematogenously to the fetoplacental unit and cause abortion, usually late in gestation. bovine herpesvirus is an alpha herpesvirus that can lead to respiratory and genital infections, as well as abortion [ , ] . transmission occurs through contact with upper respiratory, conjunctival or genital tract mucous membranes, aborted fetuses, or through venereal transmission. abortions are most commonly associated with the respiratory form of the disease and not the genital form. cows can have fever, anorexia, red nasal mucosa, coughing, and conjunctivitis, followed by abortion in - d. abortion generally occurs between and months of gestation. infection also can result in early embryonic death [ ] . bovine herpesvirus establishes latent infections in the trigeminal and sacral ganglia; following immunosuppression, the virus can become reactivated. therefore, these animals serve as a source of infection for unexposed cattle. bovine viral diarrhea virus is a pestivirus that is transmitted transplacentally or through inhalation or ingestion of material contaminated with infected secretions [ , ] . animals with acute infection present with fever, nasal discharge, enteritis, and leukopenia. pregnant animals infected up to d of gestation can have decreased fertilization rates and embryonic death. infection between and d of gestation can result in abortion; however, fetuses that survive infection with a noncytopathic strain of bvdv between and d of gestation usually become persistently infected (pi). animals that are pi shed large amounts of bvdv and generally do not produce antibodies to bvdv. these animals can be stunted in growth or appear normal. fetal infection occurring at - d of gestation can result in congenital abnormalities, including cerebellar hypoplasia, microencephalopathy, cataracts, microopthalmia, and thymic aplasia. fetuses infected between and d of gestation are usually able to clear the virus, develop normally, and exhibit precolostral neutralizing antibodies to bvdv. bluetongue virus is an orbivirus that is transmitted by a midge (culicoides variipennis) [ ] . fetuses infected during the first d of gestation, resorb or abort. infections between and d of gestation can result in stillbirths, birth of weak calves, or birth of calves with cerebral abnormalities. infection after day of gestation does not generally have a negative effect on the fetus. epizootic bovine abortion is a disease that results in late-term abortion [ , ] . abortion does not generally occur the next year. the disease is possibly caused by an unnamed bacteria in the delta subdivision of the class proteobacteria and in the order myxococcales. the organism is transmitted by the tick ornithodoros coriaceus. the disease occurs in california, nevada, oregon, and southern idaho. akabane disease is caused by a bunyavirus [ ] . the disease occurs in australia, japan, the middle east, south africa, and turkey, and affects cattle, sheep and goats. in japan, the virus is transmitted by mosquitoes and in australia transmission occurs via the midge (culicoides brevitarsis). fetuses infected in the first trimester usually die shortly after birth or have sensory, motor and optical nerve damage. infection during the second trimester can result in arthrogryposis, neurogenic torticollis, kyphosis, and scoliosis. tritrichomonas foetus is a flagellated protozoa that causes venereal disease in cattle [ ] . infected cows can experience early embryonic death, infertility, and abortion in the first half of gestation. some cows develop postcoital pyometra. generally, the infection is cleared within d. neospora caninum is a protozoa that can cause abortion early in the second trimester [ ] . infected cows can abort repeatedly. the definitive host for the organism is the dog that ingests tissue cysts. the cow then ingests sporulated oocysts in feed, water or soil contaminated with the dog feces. tachyzoites can then be transmitted through the placenta to infect the fetus. infected cows are asymptomatic. mycotic abortions can be caused by various molds and yeasts [ , , ] . aspergillus fumigatus is the most common pathogen, with morteriella wolfii being common in the southern hemisphere. other pathogens include other aspergillus spp., absidia spp., rhizomucor pusillus, rhizopus arrhizus, pseudallescheria boydii, species of penicillium, candida, and torulopsis. the disease occurs sporadically, usually occurring when animals are housed indoors or in small pens and fed hay. the fungal pathogens might enter the respiratory or gastrointestinal tract and then gain access to the systemic circulation and spread to the placentomes. abortion generally occurs between and months of gestation. placentitits and retained placenta are frequent occurrences. other clinical signs are generally not seen. however, cows infected with morteriella wolfii can have postabortion pneumonia and death within h after abortion. many of the bacterial, viral, and protozoan causes of ovine abortion are zoonotic. thus, attendants should employ protective measures to prevent human infection. campylobacter is the most important cause of abortion in sheep in north america [ , ] . campylobacter jejuni is responsible for sporadic abortions, whereas campylobacter fetus subsp. fetus is associated with large outbreaks of recurring abortion. the zoonotic, gram-negative rods are transmitted by ingestion and shed in feces, aborted fetuses, placentae, and vaginal discharges of ewes that abort. ewes are asymptomatic and abort in the third trimester, have stillbirths or give birth to weak lambs. some ewes are persistently infected and continue to shed the organism in their feces. infection with listeria monocytogenes occurs through ingestion of the organism in poor-quality alkaline silage or pastures contaminated with feces containing the zoonotic organism [ ] . ewes can abort - d after infection. prior to aborting, ewes might show signs of fever and decreased appetite. following abortion, ewes often develop metritis. as well, listeria ivanovii has also been shown to cause abortion in sheep. abortion in sheep can be caused by brucella melitensis or rarely b. ovis [ , ] . brucella can be transmitted to the ewe through mucous membrane (vaginal, preputial, and conjunctival) contact with infected rams. ewes are generally asymptomatic, but can abort in the third trimester, have stillbirth or give birth to a weak lamb. ewes clear the bacteria within a few weeks following an abortion. b. melitensis has zoonotic potential, whereas b. ovis does not. abortion in ewes can follow infection with salmonella abortus-ovis, salmonella montevideo, or salmonella arizonae [ , ] . overcrowded conditions and shipping can predispose flocks to abortion storms. affected ewes can be asymptomatic prior to aborting, or have clinical signs of fever, depression and diarrhea. metritis and retained placenta can occur after aborting. these pathogens are zoonotic. c. abortus is responsible for causing ovine enzootic abortion [ ] . the organism is transmitted through ingestion or exposure to aborted materials, vaginal discharge, or contaminated environment. when the infected ewe becomes pregnant, the organism is hematogenously spread to the trophoblast cells and by d of gestation, has infected the cotyledons and intercotyledonary areas. infection of the fetus and abortion may result. prior to d, stillbirth or resorption can occur. if the ewe is not pregnant at the time of infection, the organism can affect the subsequent pregnancy. once abortion occurs, the immune system will prevent subsequent abortions, but the organism can continue to be shed in vaginal secretions. c. abortus can cause abortion in humans. coxiella burnetti is transmitted through inhalation, mucous membrane contact, ticks, and possibly semen [ ] . infected ewes might develop placentitis and abort late in gestation, although abortion is not as common as in does. abortion is not likely to occur the next lambing season due to development of immunity within the flock. humans can become infected when handling infected placentas or lambs, and from inhalation of contaminated dust. the clinical signs of infection in people may include prolonged illness, undulant fever, atypical pneumonia, hepatitis, myalgia, or endocarditis. tick-borne fever occurs in europe and is caused by anaplasma phagocytophilum [ ] . clinically, the disease results in fever, inappetance and abortion of sheep, cattle and goats. the bacteria causes flu-like symptoms in humans. yersinia pseudotuberculosis [ ] and yersinia enterocolitica (o serotype) [ ] have been isolated from ovine abortion cases. infection of ewes with y. pseudotuberculosis can lead to abortion, stillbirth or birth of weak or healthy lambs. infection with y. enterocolitica resulted in placentitis and abortion, with subsequent normal pregnancies. acute mesenteric lymphadenitis with fever, anorexia, vomiting, and diarrhea are common in people infected with these organisms. border disease virus is a non-zoonotic pestivirus similar to bovine viral diarrhea virus and hog cholera virus [ ] . ewes infected before days - of gestation have fetal resorption, abortion, maceration or mummification. if the fetus survives, it will be persistently infected with the virus, have potential cerebellar damage, shortened facial and long bones, and produce hair rather than wool. if infection occurs after day of gestation, abortion, birth of weak or normal lambs, or birth of antibody positive lambs can occur. no clinical signs are seen in the ewe. bluetongue virus, an orbivirus, is transmitted by a midge (culicoides variipennis) [ , ] . infected ewes can abort, have mummified fetuses or deliver lambs with congenital defects (hydranencephaly, porencephaly, cerebellar dysgenesis, skeletal deformities) [ ] . the ewe can show clinical signs of fever, lameness, oral and nasal ulcers, and swollen tongue, ears or face [ , ] . akabane virus can cause abortion, stillbirth, mummified fetuses, and congenital deformities (arthrogryposis, hydranencephaly) in sheep [ ] . cache valley virus, a bunyavirus, is transmitted by mosquitoes [ ] . infected ewes have fever, depression, and a reluctance to move. infection at - d of gestation results in early embryonic death and mummification. infection at - d of gestation causes cns and musculoskeletal deformities (arthrogryposis, brachygnathia, hydranencephaly, agenesis of the spinal cord), whereas infection at - d leads to milder musculoskeletal deformities, with nonsuppurative encephalitis and encephalomyelitis. more than one clinical syndrome can be present in a litter. ewes are infected by toxoplasma gondii through ingestion of feed contaminated with sporulated oocysts [ ] . the fetus can then be infected approximately d after ingestion of the parasite. whereas ewes are asymptomatic, infection occurring < d of gestation results in embryo resorption. infection at - d of gestation leads to maceration, mummification or abortion, whereas infection after d can result in stillbirth or birth of weak or healthy lambs. different stages of fetal involvement can be seen within the flock and within the same litter. t. gondii is a zoonotic agent that can affect nonimmune individuals. similar to ovine reproductive pathogens, caprine pathogens include many zoonotic organisms. infection with l. monocytogenes early in gestation can result in abortion, whereas infection late in gestation results in stillbirth or birth of weak kids [ ] . prior to abortion, does may have fever, decreased appetite, and reduced milk production. the organism can be shed in the milk from the aborting doe. generally, the encephalitic form of the disease does not occur simultaneously with abortions. l. monocytogenes can survive in soil and feces, and grows in poorly fermented silage. abortions are reported after grazing on boggy, high-ph soils. listeria is zoonotic and can cause neurologic disease in humans. c. abortus is the most common cause of infectious abortion in goats in the usa [ , ] . it is responsible for abortions, stillbirths, birth of weak kids, and neonatal pneumonia. the organism does not proliferate until d of gestation, at which time fetal death and abortion can occur. does can show anorexia, fever, and a bloody vaginal discharge - d before aborting. following abortion, uterine clearance of c. abortus takes months. the organism can reside in pigeons or sparrows and be transmitted via ticks or insects. the organism is shed in uterine discharge, fetus and placenta. c. abortus is zoonotic and can result in flu-like symptoms and abortion in pregnant women. brucellosis is present in africa, mexico, the middle east, india, pakistan and parts of south america [ , ] . b. melitensis is transmitted to goats through ingestion of contaminated feed or water. in pregnant does, the bacteria can infect the placenta with resultant late-term abortion. does might show clinical signs of fever, depression, weight loss, diarrhea, mastitis, lameness, and birth of weak kids. the bacteria is shed in milk, urine, feces, and for - months in vaginal discharge. the organism causes undulant fever (synonymous with malta fever, gibraltar fever, and mediterranean fever) in humans that consume unpasteurized contaminated milk or cheese. coxiella burnetti is the causative agent of q fever [ ] . this gram-negative coccobacillus causes disease in sheep, cattle, goats, and other mammals. affected animals might have no clinical signs of disease, but serve as a source of infection, or they can abort late in gestation and have stillbirths. the organism is transmitted in placenta, uterine fluids, milk, urine, feces, and aerosol droplets. transmission occurs through contact, inhalation, or possibly ingestion. as well, the organism is zoonotic and can be transmitted to humans by dogs and cats. affected individuals might have no clinical signs, or they might show fever, headaches, myalgia, coughing, fatigue, vomiting, respiratory disease, and rarely endocarditis. mycoplasma can cause late-term abortion, mastitis, vulvovaginitis, arthritis and conjunctivitis [ , , ] . following abortion, does can shed the organism in milk, amniotic fluids, and the placenta [ ] . leptospirosis can result in abortion in the last trimester, anorexia, fever, jaundice, anemia, and nervous signs in affected does [ , ] . transmission occurs through exposure to environments contaminated with infected urine. due to the zoonotic potential, humans should avoid contact with aborted tissues and infected urine. s. abortus-ovis infection of does can cause abortion, metritis, retained placenta, diarrhea, and systemic illness [ , ] . although abortion can occur throughout gestation, it is more common at the end of gestation. the bacteria are transmitted through ingestion. salmonella infections are zoonotic and can cause enteritis, abdominal pain and abortion in humans. although campylobacterosis is a common cause of abortion in sheep, it is less prominent in goats [ , ] . does may exhibit late abortions, stillbirths, birth of weak kids, and a mucopurulent vaginal discharge. the placenta is edematous, with necrosis of the cotyledons. the does do not show evidence of systemic illness, but may have diarrhea. as well, campylobacter jejuni causes mild gastroenteritis in humans. y. pseudotuberculosis is transmitted by fecal-oral route [ ] . abortion and early neonatal death have been reported in goats [ ] . the bacteria is zoonotic. akabane virus is transmitted by mosquitoes and midges (culicoides brevitarsis) [ , , ] . it occurs in australia, japan, the middle east, south africa, and argentina. nonpregnant goats do not show clinical signs, whereas pregnant does can appear healthy but abort, have stillbirths or mummified fetuses. kids have arthrogryposis and/or hydrencephaly that can result in dystocia. caprine herpesvirus is an alpha herpesvirus that can cause late-term abortion without any prior clinical signs [ ] [ ] [ ] [ ] . the virus can also cause vulvovaginitis and respiratory disease. subsequent pregnancies are not affected by the virus [ ] . like other herpesviruses, caprine herpesvirus has a latent state that can be reactivated following stress, immunosuppression or possibly estrus in does. following reactivation, the virus can be shed via the respiratory or genital route. toxoplasmosis can cause abortion, stillbirth, fetal death, fetal resorptions, birth of a weak kid, or birth of a healthy kid [ , ] . infection early in gestation ( - d) generally results in fetal resorptions or mummification, whereas infection in the last half of gestation results in asymptomatic does aborting - weeks prior to parturition. abortion occurs due to necrosis of the cotyledons. the protozoa is transmitted to cats through ingestion of infected rodents or birds. does ingest food or water that is contaminated with cat feces containing resistant oocytes; the organism then enters the bloodstream and spreads to the placenta and fetus. toxoplasmosis has zoonotic potential. sarcocystosis is caused by a cyst-forming protozoan [ ] . inoculation of pregnant does ( - d of gestation) with sporocysts of sarcocystis resulted in abortion and neonatal death [ ] . unlike reproductive pathogens in other species, porcine reproductive pathogens are predominantly viruses. this is hypothesized to be due to the density of the swine population within many production units. brucella suis is transmitted through direct contact with aborted fetuses and secretions, as well as venereally [ ] . infected gilts or sows might present with infertility. in addition, abortion can occur in the first trimester, if infection occurs at breeding, and during late gestation if infection occurs after day of gestation. the organism has zoonotic potential. swine suspected of having brucellosis should be reported to state animal health authorities. leptospirosis is caused by a gram-negative spirochete [ , ] . transmission occurs through contact of oral, nasal or ocular mucosa with contaminated urine. pigs are maintenance hosts for serogroups pomona, australis and tarassovi, whereas incidental infections occur with strains of the canicola, icterohaemorrhagiae, and grippotyphosa serogroups. acute infection of leptospirosis is generally asymptomatic. however, chronic leptospirosis may manifest as abortions, stillbirths, infertility, and birth of weak piglets. leptospirosis is an important occupational zoonosis for farmers and abbatoir staff in contact with pigs. erysipelothrix rhusiopathiae is a gram-positive rod that causes erysipelas [ ] . the bacteria is transmitted through oronasal exposure, followed by septicemia. the disease is characterized by abortion, fever, diamondshaped skin lesions, arthritis, vegetative endocarditis, and sudden death. abortion can occur at any stage of gestation, additionally, some piglets may become mummified. as well, embryo resorption can occur. the bacteria is zoonotic, causing erysipeloid in humans. although streptococci are part of the normal flora of the vagina, they have also been associated with abortion, stillbirths, vaginitis, postparturient agalactia, and early neonatal death [ , ] . chlamydiae are bacteria that can be transmitted by inhalation of aerosols, ingestion of contaminated feed, and venereally [ ] . the species involved are chlamydophila psittaci, chlamydophila pecorum, and chlamydia trachomatis. chlamydial infections can be inapparent or have clinical signs of fever, dyspnea, pneumonia, conjunctivitis, pericarditis, and polyarthritis. in addition, gilts or sows may exhibit lateterm abortions, birth of weak piglets, or mummified piglets. actinobacillus suis, actinobacillus ross, and actinobacillus equuli have been suggested to cause abortion, metritis, and decreased litter sizes [ ] . mauch and bilkei reported late-term abortions during the first and second parities due to a. suis [ ] . a. suis and a. ross are normal flora of the vagina and upper respiratory tract of healthy sows, and the bacteria can be isolated from healthy herds [ , ] . transmission of a. suis occurs via inhalation of aerosols or through abrasions in the skin and mucous membranes. infected animals might demonstrate fever, erythematous skin lesions, and anorexia. mycoplasma suis is the causative organism of eperythrozoonosis [ ] . m. suis attaches to and replicates on the red blood cell. it causes anemia, icterus, anorexia, immune suppression, and fever. furthermore, infertility, abortion, stillbirth, and birth of weak piglets can also result from infection. porcine reproductive and respiratory syndrome virus (prrsv) can be shed in nasal secretions, feces and urine; infected pigs can be long-term carriers [ ] [ ] [ ] . the virus might be spread by contact, oral transmission and aerosol transmission. experimentally, the virus is transmitted in semen [ ] . additionally, the virus can be transmitted transplacentally [ ] , most commonly in the last trimester. during the first phase of the disease, reproductive signs include abortions and irregular returns to estrus. pulmonary edema or nephritis also can result in sows. during the second phase of the disease, pregnant animals can give birth to a combination of normal or weak piglets and stillborn, autolytic, or mummified fetuses. porcine parvovirus is endemic in most herds, with most gilts and sows exhibiting active immunity against the virus [ ] . gilts that do not have immunity to porcine parvovirus before conception are at a high risk of infection and reproductive disease. pseudorabies is caused by an alpha herpesvirus [ , ] . the pig is the only natural host of pseudorabies, whereas infection in cattle, sheep, goats, dogs, and cats is fatal. transmission is primarily through direct contact with nasal and oral secretions, but can occur by aerosols, venereally, or transplacentally. like other herpesviruses, pseudorabies can establish latent infections. in naïve herds, the clinical signs might be severe. infection during the first trimester results in resorption and a return to estrus, whereas infection during the second and third trimester results in abortion, stillbirth, or birth of weak piglets. neonatal pigs may have % morbidity and mortality rates. the dam's immunity against pseudorabies determines the clinical signs seen in piglets. piglets of dams lacking immunity have nervous signs (trembling, hypersalivation, ataxia, circling, paddling) and die within - d, whereas piglets of immune dams usually do not show clinical signs. classical swine fever, previously known as hog cholera, is caused by a pestivirus [ ] . pigs are also susceptible to two other pestiviruses, bovine viral diarrhea virus and border disease virus. pigs are the only natural host of classical swine fever virus. transmission occurs through oronasal contact with infected pigs, ingestion of contaminated feed, airborne spread over short distances, indirectly by fomites, and potentially through semen [ , ] . clinical signs include fever, anorexia, conjunctivitis, diarrhea, and respiratory signs. transplacental infection can occur at any stage of gestation resulting in abortions, mummies and stillbirths. infection at - d of gestation can result in the birth of persistently viremic piglets. these piglets appear normal initially, but subsequently develop congenital tremors and lose weight. they serve as a continual reservoir of classical swine fever virus. porcine enterovirus and teschovirus are picornaviruses [ ] . transmission is through fecal-oral route, but transmission by fomites might also occur. sows and gilts may experience infertility, embryonic death, stillbirths, and mummies, without any other clinical signs. encephalomyocarditis virus is a picornavirus that is isolated from several species [ ] . in swine, the disease is primarily transmitted through feed or water contaminated by infected rodent feces, urine, or carcasses. pregnant females might not show clinical signs, or they might experience abortion, mummies, or stillbirths [ ] . porcine cytomegalovirus is a beta herpesvirus that is transmitted oronasally and congenitally [ ] . pigs > weeks of age are asymptomatic, whereas younger pigs might show coughing, nasal discharge, rhinitis, neurological signs, or death. pregnant animals can have mummies and stillbirths without showing any other clinical signs. as well, preweaning mortality can be %. rubulavirus is a paramyxovirus that causes blue eye disease in swine in mexico [ ] . transmission of the virus occurs through nose-to-nose contact or contact with fomites. infected pigs of all ages develop corneal opacity. pregnant animals show reproductive signs for approximately months. these signs consist of stillbirths, mummies, decreased number of live births, increased number of animals returning to estrus, and an increase in weaning-to-service interval. in closed herds, the disease is self-limiting. menangle virus is a paramyxovirus that has caused disease in new south wales, australia [ ] . the virus causes disease in pigs, humans and fruit bats, which are the reservoir host. transmission is suspected to be through fecal-oral or urinary-oral routes. the disease caused reproductive failure in sows [ ] . stillbirths and mummified fetuses were present, as well as stillborn piglets with congenital deformities such as arthrogryposis, brachygnathia, kyphosis, and degeneration of the brain and spinal cord. many sows had a delay in return to estrus. additionally, two farm workers were affected. they experienced fever and headaches, but fully recovered. porcine circovirus type has been associated with reproductive disease, respiratory disease, postweaning multisystemic wasting syndrome, and porcine dermatitis and nephropathy syndrome [ ] . the virus is thought to be transmitted by oronasal exposure. reproductive disease is manifest as late-term abortion, stillbirths, and mummified fetuses [ ] . japanese encephalitis virus is a mosquito-borne virus that causes disease in humans and animals [ ] . swine are reservoir hosts that allow mosquitoes to transmit the virus to humans (dead-end hosts). the disease is manifest in pregnant sows and gilts as abortions, stillbirths, mummified fetuses, and birth of weak piglets that can have hydrocephalus and subcutaneous edema. infection after - d of gestation does not appear to affect piglets. soft ticks (ornithodorus moubata and ornithodorus erraticus) are the reservoir and vector for african swine fever virus [ , ] . as well, the virus can be spread among pigs via oronasal secretions. the clinical signs of disease can vary from sudden death (with no previous clinical signs) to inapparent infection. acute infections may consist of fever, anorexia, cyanosis, hemorrhages in internal organs and skin, and abortion. toxoplasmosis occurs through ingestion of food, water or soil contaminated with sporulated oocysts or through ingestion of meat containing tissue cysts [ ] . whereas most infections are asymptomatic, abortion may occur [ , ] . additionally, piglets may be born premature, dead, weak, or die soon after birth [ ] . prevention of toxoplasmosis in pigs is important to prevent infection of humans through ingestion of undercooked pork. pathogens that cause sporadic reproductive loss in mares may be due primarily to host factors, e.g. impaired reproductive defense mechanisms. notably, other pathogens may cause disease, depending on variables of the environment (e.g. movement of horses that predisposes naïve mares to novel infections). streptococcus zooepidemicus is one of the most common bacteria isolated from mares with uterine disease [ ] . whereas the uterus of a healthy mare is more likely to clear the infection, the uterus of a mare with impaired defense mechanisms or poor genital conformation might become inflamed or develop an infection leading to infertility. as well, streptococcus zooepidemicus can cause abortion via ascension through the cervix and infection of the fetus and placenta, or possibly through a maternal systemic infection [ , ] . bacterial abortions usually occur at - months of gestation [ ] . in addition to bacterial culture, diagnosis of infection is coupled with evidence of autolysis and inflammation of the fetus and placenta. other bacteria implicated in infertility or abortion are e. coli, pseudomonas aeruginosa, klebsiella pneumoniae var genitalium, staphylococcus aureus, streptococcus equisimilis, leptospira spp., s. abortus equi, and nocardioform actinomycetes. although no longer very common, s. abortus equi has caused abortion outbreaks [ ] ; in one of these, of mares aborted between and months of gestation [ ] . taylorella equigenitalis is a gram-negative coccobacillus that causes contagious equine metritis (cem) [ ] . taylorella equigenitalis is transmitted venereally, highly contagious, and can cause a copious vulvar discharge [ ] . diagnosis is by bacteriologic culture of the organism from the clitoral fossa, clitoral sinuses, and endometrium or cervix. as well, polymerase chain reaction can be used to identify the organism. serologic tests are also available to detect antibodies against taylorella equigenitalis in the serum. most mares recover spontaneously from cem over a period of several months. imported horses used for breeding should be quarantined and extensively tested. any animals suspected of having cem should be reported to state or federal animal health authorities. equine herpesvirus can cause abortion, neonatal death, respiratory disease, and neurologic disease [ ] . the virus is transmitted through inhalation of infectious aerosol droplets, or by direct contact with infectious secretions. like other herspesviruses, equine herpesvirus persists in a latent state that can be reactivated. most abortions are sporadic and occur between months of gestation and term. the abortions can occur up to months after respiratory disease, which may have been clinical or subclinical. equine viral arteritis can cause sporadic abortions between and months of gestation [ ] . the virus is transmitted by inhalation of infectious aerosol droplets or venereally. if clinical signs of disease are apparent, they can consist of fever, hind limb edema, nasal and ocular discharge, urticarial rash, anorexia, coughing, and dyspnea [ ] . the virus can also cause uterine disease, with subsequent abortion in pregnant mares [ ] . trypanosoma equiperidum is a venereally transmitted protozoa that causes dourine [ ] . although it has been eradicated from north america and most of europe, the disease is still found in africa, south and central america, and the middle east [ ] . clinical signs include a mucopurulent vulvar discharge, fever, and cutaneous raised plaques followed by ataxia, depression, severe anemia, and death [ , ] . mortality can reach - %. strict quarantine, testing, and eradication of positive animals are recommended for control of the disease. fungal pathogens account for up to % of reported sporadic abortions [ ] . aspergillus fumigatus is the most common fungal pathogen, but mucor spp. and allescheria boydii have also been reported. infection of the uterus may occur via ascension through the cervix or hematogenously. abortions usually occur at - months of gestation, and may be associated with a purulent vulvar discharge. the affected chorioallantois is thickened, yellowish, and covered with a mucoid exudate. infectious causes of canine reproductive losses include some pathogens with a zoonotic potential, as well as viruses that can quickly spread throughout a kennel if bitches are not adequately vaccinated. brucella canis is a small, gram-negative intracellular coccobacilli. infection can cause infertility, early embryonic death, fetal resorptions, and late-term abortion [ , ] . the bitch might not show any clinical signs prior to aborting. following abortion, a serosanguinous vaginal discharge may be present for - weeks. large numbers of bacteria can be present in the aborted material and postabortion vulvar discharge; therefore, caution should be exercised to prevent other dogs from contacting the fluid or aborted tissue. whereas the zoonotic potential of b. canis is less than most brucella sp., immunosuppressed or pregnant individuals should avoid contact with aborted fluid or tissue. streptococci are gram-positive cocci that are commensal microflora of the genital tract [ ] . although the bacteria have been associated with abortion, infertility and neonatal septicemia, its isolation does not confirm the cause of the reproductive problem. campylobacter is a gram-negative curved rod. although campylobacter jejuni has been isolated from the feces of normal dogs [ ] , it has also been associated with abortions [ ] . if campylobacter is suspected, the diagnostic laboratory should be contacted to obtain instructions for sample submission and to be informed of the suspected pathogen [ ] . also, owners should be informed that the bacteria can cause enteric disease in humans. salmonella is a gram-negative bacilli that is transmitted through the gastrointestinal tract via contaminated water, food, or fomites [ ] . infection with salmonella can cause abortion, stillbirth and birth of weak puppies. it is important that owners by notified of the zoonotic potential of salmonella. . . escherichia coli e. coli is commonly isolated in cases of metritis and pyometra [ ] . it has also been isolated in the case of a partial abortion [ ] . mycoplasma and ureaplasma can be isolated from the genital tract of both healthy and infertile bitches [ ] . although these organisms have been associated with infertility, fetal resorption, abortion, stillbirth and birth of weak puppies [ ] , it is recommended that culture results be interpreted in the context of other diagnostic findings to determine if antibiotic treatment should be instituted [ ] . when submitting specimens to the diagnostic laboratory, samples should be placed in amies medium (without charcoal) or modified stuart bacterial transport medium and the laboratory informed of the suspected pathogen [ ] . canine herpesvirus can lead to abortion, stillbirth and embryonic resorptions [ ] . a pregnant bitch can become infected through direct contact with mucosal secretions (respiratory or genital) [ , ] . as well, a latent infection might be reactivated during pregnancy with resultant virus shedding. neonatal infection usually occurs at birth; however, transplacental infection can occur and lead to mummified or dead fetuses, stillbirth, or birth of weak puppies. canine distemper is caused by a morbillivirus [ ] . the virus has been shown to cause abortion, stillbirth and congenital infections in puppies [ , ] . abortion can follow systemic infection of the bitch or transplacental infection [ ] . puppies infected transplacentally can develop neurologic signs within weeks of birth [ ] . canine parvovirus type , the causative agent of minute virus of canines, can cause embryo resorptions, stillbirth, or birth of weak puppies [ ] . toxoplasmosis can cause placentitis with spread of tachyzoites to the fetus [ ] . experimental infection of bitches caused congenital infection and abortion. . . neospora caninum n. caninum has been shown experimentally to be transmitted transplacentally [ ] . neosporosis can result in early fetal death, mummification, resorption and birth of weak puppies [ ] . however, it has not been shown to cause abortion. feline reproductive loss is more commonly attributed to a viral etiology than to bacterial or protozoan pathogens. although uncommon, abortions can occur when normal vaginal flora (e. coli, staphylococcus sp., streptococcus sp.) ascend into the uterus and cause fetal infection [ ] . the queen might show clinical signs of infection, such as anorexia, pyrexia, abdominal discomfort, and vaginal discharge [ ] . feline herpesvirus is an alpha herpesvirus that causes rhinotracheitis [ , ] . experimental infection caused abortion and intrauterine fetal death [ ] ; however, the virus has not been isolated from the aborted fetal tissue [ ] . hickman reported that in a herpesvirus outbreak in a specific pathogen-free colony, only of queens pregnant at the initial time of the outbreak aborted part of her litter [ ] . however, there was a % mortality rate within week of birth among the kittens born to queens acutely infected during the perinatal period. feline immunodeficiency virus (fiv) is a lentivirus, similar to human immunodeficiency virus [ ] . the virus can be transmitted in utero resulting in abortion, stillbirth, arrested fetal development, and birth of viable, virusinfected kittens [ ] . weaver et al. reported that experimental inoculation of specific pathogen-free queens with fiv resulted in % of kittens being resorbed or having arrested fetal development [ ] . feline infectious peritonitis is caused by a coronavirus [ ] . the virus is associated with late-term abortion, stillbirths, fetal resorptions, endometritis, and high mortality in kittens the first week of life. some purebred cats have a genetic predisposition for fip (heritability of %), and thus should not be used as breeding animals [ ] . feline leukemia is a retrovirus that can lead to abortion, infertility and fetal resorptions [ ] . generally, queens are asymptomatic prior to aborting. feline panleukopenia virus is a parvovirus that can cause abortion, stillbirth, and cerebellar hypoplasia in kittens [ ] . these signs are not always associated with the classical gastrointestinal disease in the queen. although not common, t. gondii is reported to cause abortion and congenital infections [ , ] . infection can occur transplacentally or postnatally via milk [ ] . preparation is essential to maximize the potential for accurate diagnosis of reproductive pathogens. whenever possible, entire fetuses, placental tissues, and serum and urine from the dam should be sent promptly, along with a complete case history, to the diagnostic laboratory. in many cases, listing the pathogens of concern on submission forms can improve diagnostic test selection. effective communication with your diagnostic laboratory to ensure appropriate sample handling and prudent test selection can maximize your diagnostic support. it is noteworthy that appropriate precautions should be taken to avoid zoonotic infection of personnel (in clinical or diagnostic settings) with reproductive pathogens. finally, appropriate immunization or biosecurity to prevent infection often can reduce reproductive losses in domestic animals. bacterial causes of bovine infertility and abortion adherence of haemophilus somnus to bovine embryos after in vitro exposure bovine genital mycoplasmosis chlamydia-related abortions in cattle from graubunden parachlamydia spp. and related chlamydia-like organisms and bovine abortion infectious diseases causing bovine abortion and fetal loss bovine herpesvirus infection and infectious bovine rhinotracheitis viral diseases of the fetus experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy: effect on the bovine corpus luteum and conceptus reproductive consequences of infection with bovine viral diarrhea virus the geographic distribution of the putative agent of epizootic bovine abortion in the tick vector, ornithodoros coriaceus epizootic bovine abortion (foothill abortion) veterinary virology current therapy in large animal theriogenology protozoal abortion in cattle mycotic bovine abortion fungi associated with bovine abortion in the northern plains states (usa) philadephia: w.b. saunders company abortion in sheep: diagnosis and control anaplasma phagocytophilum-the most widespread tick-borne infection in animals in europe ovine abortion and stillbirth due to purulent placentitis caused by yersinia pseudotuberculosis experimental yersinia enterocolitica placentitis in sheep observations on transplacental infection with bluetongue virus in sheep infectious causes of abortion caprine abortion following exposure to mycoplasma capricolum subsp. capricolum isolation of mycoplasma mycoides, mycoides (lc variant), from two naturally aborted caprine fetuses abortion and early neonatal death of kids attributed to intrauterine yersinia pseudotuberculosis infection multiple abortions associated with caprine herpesvirus infection in a goat herd abortion and ulcerative posthitis associated with caprine herpesvirus- infection in goats in california caprine herpesvirus- abortion storm in a goat herd in quebec serologic and reproductive findings after a herpesvirus- abortion storm in goats toxoplasma gondii-induced abortion in dairy goats abortion and death in goats inoculated with sarcocystis sporocysts from coyote feces bacterial, rickettsial, protozoal, and fungal causes of infertility and abortion in swine diseases of swine streptococcus suis type ii-associated diseases in swine: observations of a one-year study miscellaneous bacterial infections actinobacillus suis, a potential cause of abortion in gilts and low parity sows exploratory study of mycoplasma suis (eperythrozoon suis) on four commercial pig farms in southern brazil porcine reproductive and respiratory syndrome virus (porcine arterivirus) viral causes of porcine reproductive failure-part i porcine reproductive and respiratory syndrome detection of porcine reproductive and respiratory syndrome virus in boar semen by pcr diseases of swine aujeszky's disease (pseudorabies) viral causes of infertility and abortion in swine classical swine fever and other pestiviruses transmission of classical swine fever virus by artificial insemination porcine enteric picornaviruses encephalomyocarditis virus reproductive failure in sows following experimental infection with a belgian emcv isolate diseases of swine rubulavirus, menangle, and nipah virus infections reproductive disease and congenital malformations caused by menangle virus in pigs porcine circovirus diseases myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus japanese encephalitis and west nile viruses afrcian swine fever coccidia and other protozoa isolated, spontaneous toxoplasma abortion in a young sow bacterial causes of subfertility and abortion in the mare abortion in mares an outbreak of abortion in mares associated with salmonella abortus equi infection sexually transmitted (venereal) diseases of horses equine herpesvirus infections equine viral arteritis equine viral arteritis pathology of maternal genital tract, placenta, and fetus in equine viral arteritis disease transmission in horses current therapy in large animal theriogenology clinical signs and diagnosis of brucella canis infection canine brucellosis: outbreaks and compliance canine pregnancy factors influencing fecal shedding of campylobacter jejuni in dogs without diarrhea abortion in the dog due to campylobacter species infectious diseases of the dog and cat partial abortion associated with genital escherichia coli infection in a bitch the genital mycoplasma and ureaplasma flora of healthy and diseased dogs infectious diseases of dogs and cats canine herpesvirus- (chv- ): clinical, serological and virological patterns in breeding colonies infectious diseases of the dog and cat clinical considerations of canine herpesvirus infection infectious diseases of the dog and cat experimental and naturally occurring transplacental transmission of canine distemper virus pathogenicity of minute virus of canines (mvc) for the canine fetus infectious diseases of dogs and cats transplacental neospora caninum infection in dogs canine and feline theriogenology infectious causes of infertility, abortion and stillbirths in cats an epizootic of feline herpesvirus, type in a large specific pathogen-free cat colony and attempts to eradicate the infection by identification and culling of carriers herpesviral abortion in domestic animals placental immunopathology and pregnancy failure in the fiv-infected cat vertical transmission of feline immunodeficiency virus the inheritance of susceptibility to feline infectious peritonitis in purebred catteries key: cord- -gdc e t authors: muramoto, osamu title: retrospective diagnosis of a famous historical figure: ontological, epistemic, and ethical considerations date: - - journal: philos ethics humanit med doi: . / - - - sha: doc_id: cord_uid: gdc e t the aim of this essay is to elaborate philosophical and ethical underpinnings of posthumous diagnosis of famous historical figures based on literary and artistic products, or commonly called retrospective diagnosis. it discusses ontological and epistemic challenges raised in the humanities and social sciences, and attempts to systematically reply to their criticisms from the viewpoint of clinical medicine, philosophy of medicine, particularly the ontology of disease and the epistemology of diagnosis, and medical ethics. the ontological challenge focuses on the doubt about the persistence of a disease over historical time, whereas the epistemic challenge disputes the inaccessibility of scientific verification of a diagnosis in the past. i argue that the critics are in error in conflating the taxonomy of disease (nosology) and the act of diagnosing a patient. medical diagnosis is fundamentally a hypothesis-construction and an explanatory device that can be generated under various degrees of uncertainty and limited amount of information. it is not an apodictic judgment (true or false) as the critics presuppose, but a probabilistic (bayesian) judgment with varying degrees of plausibility under uncertainty. in order to avoid this confusion, i propose that retrospective diagnosis of a historical figure be syndromic without identifying underlying disease, unless there is justifiable reason for such specification. moreover it should be evaluated not only from the viewpoint of medical science but also in a larger context of the scholarship of the humanities and social sciences by its overall plausibility and consistency. on the other hand, i will endorse their concerns regarding the ethics and professionalism of retrospective diagnosis, and call for the need for situating such a diagnosis in an interdisciplinary scope and the context of the scholarship of the historical figure. i will then enumerate several important caveats for interdisciplinary retrospective diagnosis using an example of the retrospective diagnosis of socrates for his life-long intermittent neurologic symptoms. finally, i will situate the present argument in a larger context of the major debate among the historians of medicine and paleopathologists, and discuss the similarities and differences. diagnosing medical conditions of a famous historical figure based on evidence found in documents, arts, and other artifacts is a small but popular genre of medical publishing [ ] . what is the diagnosis of the illness that tormented frédéric chopin for so many years? what neurological disease did friedrich nietzsche suffer from for many years before he died? these are typical questions discussed occasionally in medical journals. but this activity is not without criticisms. some serious scholars in the humanities and social sciences are not happy about this "fun escape of doctors" [ ] [ ] [ ] [ ] [ ] [ ] . first, these "hobbyist" historians are not following the methodological disciplines of historiography, literary criticism, and other relevant subject areas of the humanities and social sciences. for example, they often literally interpret the documents in translation without critically analyzing the primary source in the original language. but more importantly, as these retrospective diagnoses become more and more medically sophisticated as medical knowledge advances, these critics are increasingly skeptical about the authenticity of such highly specific and speculative diagnoses, such as alpha- antitrypsin deficiency in chopin [ ] , which became known only due to the recent advancement in medical technology. these reports often do not consider the possibility that different diseases might have existed in historical time, or the same disease might have been described through different illness experiences that are bound by a particular historical time and place. hence the critics call them "anachronistic diagnosis" [ , ] . this is an ontological challenge because it questions the existence of a certain disease in historical time. another challenge is the impossibility of verifying or falsifying the medical hypothesis of retrospective diagnosis for obvious reasons-we cannot examine and test the historical subject. this is an epistemic challenge because it questions the access to historical knowledge. and finally, another important challenge pertains to the ethics and professionalism of those clinicians who render retrospective diagnoses. they publish a diagnosis of a patient with whom they never had a physician-patient relationship, and without any consent. there is such a stark contrast to the diagnosis of their contemporary patients, which is strictly regulated and sanctioned by their professionalism and medical ethics. even if medical affairs of public figures in historical times are outside the boundaries of medical ethics and professionalism, the question remains what goals such retrospective diagnoses serve. it is often unclear how such a highly specific diagnosis would make any difference in the scholarship of the historical person in question. without such a discussion these new diagnostic labels are seen by the critics as just a hobby of those physicians who want to exercise their diagnostic acumen for fun. one critic goes so far as to advise journal editors to reject those papers for publication ( [ ] , p , also see endnote a for a qualification of this criticism). this essay intends to systematically reply to these critics from the viewpoint of clinical medicine, philosophy of medicine, and medical ethics. before going into further discussion, however, let me first clarify the concept and terminology of "retrospective diagnosis". this term has been used at least in four different ways, as shown in the table . first, in clinical medicine, it is a generic term for any diagnosis that is made retrospectively, usually based on new information that was not available in an earlier diagnosis. postmortem diagnosis based on autopsy is a prototypical retrospective diagnosis. for clinicians, a retrospective diagnosis of a recently deceased patient is routine and uncontroversial, and postmortem diagnosis based on textual evidence might be considered an extension of this practice. in psychiatry, the retrospective diagnosis of mental disorders using phenomenological and psychopathological analysis of written texts has been a branch of study called pathography (pathographie) [ ] , which was first introduced by such german neuropsychiatrists as julius möbius, ernst kretschmer, and karl jaspers over a century ago [ ] . this methodology is also well established and uncontroversial. but only recently, is the term "pathography" also used in a totally different meaning of illness narratives, particularly in the medical humanities [ ] . while the diagnostic pathography has a longer tradition and established usage, the term "retrospective diagnosis" is preferred in recent literature in order to avoid confusion with the illness narrative. medical diagnosis of historical figures based on textual evidence and other artifacts is the third category of retrospective diagnosis, which is the very subject of this essay. the fourth category of the usage of the term "retrospective diagnosis" is in the field of the history of medicine and paleopathology. the main interest of their investigation is to identify a disease that was experienced and described by the population in a historical time and place, and often diagnosed by medical professionals of that time. while the term "diagnosis" is used, the main endeavor of these medical historians and paleopathologists is the scientific identification of a historical disease, not so much of diagnosing a particular individual patient by analyzing a complex life-long history of illness. the meanings and concepts of all these four categories of retrospective diagnosis are related to each other with many overlaps, but there are also important differences which i will discuss later. in what follows, i will first analyze the criticisms of the retrospective diagnosis of famous historical figures in the realms of ontology, epistemology, and ethics/professionalism, and provide my own replies. i will show that the critics' skepticism of retrospective diagnosis regarding the ontological persistence of disease entities and the epistemic non-verification through diagnostic testing originates in the erroneous conflation of the taxonomy table different meanings and concepts of "retrospective diagnosis" of diseases (nosology) and the act of diagnosing. on the other hand, i will endorse and further develop their criticisms regarding the ethics and professionalism of retrospective diagnosis, particularly the need for clear interdisciplinary scope and goals. next, i will demonstrate these points using an example of the retrospective diagnosis of socrates for his life-long intermittent neurologic symptoms. finally, i will review the relevance of this current proposal with the ongoing discussion among the medical historians and paleopathologists. in this section, i will show important similarities and differences between the retrospective disease identification of historical diseases, and the retrospective diagnosis of a historical person. while most historians and paleopathologists seem to agree that human anatomy and physiology have not changed or evolved to a significant degree of their interest during the past several thousand years of human history, there has been a tremendous change in human environment, including sanitation, natural climate, and the evolution of other organisms such as microbes and parasites. leven ([ ] , p ) introduces the concept of "pathomorphosis" to capture this problem. based on this concern, he concludes: the entity tuberculosis itself, like nearly all other disease entities, has achieved the rank of a natural species … what then should prevent us from identifying the present disease entity tuberculosis in descriptions of the past? the intention of this article has been to show that history and its rules should prevent us from such an approach ( [ ] , p ). behind this conundrum of identifying the present tuberculosis and the past descriptions of diseases with similar manifestations, there are two kinds of philosophical questions: one is whether disease x which we recognize as tuberculosis today is the same and identical disease as "phthisis", "consumption", or whatever they called in historical time. in this conception, disease x is one and identical (caused by the same mycobacterium tuberculosis infection) but it is argued that "tuberculosis" and "phthisis" are different because they are conceptualized in a different context with different linguistic, social and cultural meanings. even if we are dealing with the identical disease x, biological responses are also different among historical patients due to differences in the immune system and other host factors. recognition of disease x by physicians is also different in different historical times due to differences in diagnostic conceptions and technologies. for all these reasons, the critics argue, it is impossible to make a retrospective diagnosis of tuberculosis. this is, however, not a question of the ontology of disease x because its existence and persistence over historical time is not questioned. what prevents retrospective diagnosis is the differences in human experience, including naming and knowledge, of disease x. because this is a form of epistemic argument, i will come back to this question in the next section. the other way to analyze this conundrum is to consider modern tuberculosis representing disease x, while historical tuberculosis representing disease x′. diseases x and x′ may be related to each other, but they are not identical, or may be clinically similar but may be different entities with different etiology and pathophysiology. if we use the above example, historical phthisis most likely included miscellaneous conditions clinically similar to tuberculosis [ ] . contemporary tuberculosis (disease x) is caused by mycobacterium tuberculosis whereas the historical tuberculosis (disease x′) might have included the conditions caused by mycobacterium bovis [ ] . in this conception, the underlying diseases themselves are different on top of the historical changes in human experience including the biological responses and classification. this is an ontological question because it questions the persistence of a disease entity itself over time. many infectious agents are known to evolve in a relatively short period of time (e.g. influenza viruses) and clinical diseases caused by these agents emerge and ebb over time. the ontological and epistemic questions are, however, closely related to each other because, in order to know exactly what disease x′ was, we still need to know how historical people experienced disease x′ from historical records. we then face the challenge of the accessibility and verifiability of our historical knowledge of disease x′. it seems that the epistemic question is pivotal to the ontological questions. one way to address the persistence of disease entities over historical time is to empirically investigate historical diseases through modern scientific techniques. this is an important area of study in paleopathology. for example, if we can test the remains of past patients, such as bones, teeth, and mummies, we might make a reasonable confirmation that the same disease existed in the past. it seems uncontroversial that the same or closely similar skeletal pathologies and skeletal diseases such as paget's disease of bone existed in the historical past ( [ ] , pp - ). moreover, by extracting ancient dna from human remains, paleopathologists can provide valuable information regarding the speciation and the identification of the strains of infectious agents. (see roberts [ ] for biomolecular identification of mycobacterium tuberculosis and haensch et al. [ ] for yersinia pestis, the agent for the black death, from archeological remains.) while the ontological challenge to retrospective diagnosis may seem to be answered by empirical studies, paleopathologists are still faced with the onerous challenge of epistemology. for example, it is emphasized that paleopathological diagnostic criteria have to derive from modern clinical medicine ( [ ] , pp - ). yet paleopathologists themselves are struggling with the challenges of understanding historical diseases within their historical context [ ] . in other words, there seems no way to discuss disease x and x′ other than by analyzing them and describing them in today's methodology and terminology in order to put them into our contemporary discourse. if so, the epistemic problem of historicizing diseases can be a hurdle against any effort to settle the ontological problem. nevertheless, i agree with the critics to the extent that it is important, whenever we discuss retrospective diagnosis, to address the question whether disease x and disease x′ are identical or different, and if different, whether the difference is ontological (difference in disease itself ) or epistemic (difference in human experience of the same disease). often times, we have no definitive answer, but i argue that the absence of evidence for the persistence of a disease is not the evidence for the absence. there is often circumstantial evidence that disease x and x′ are very close, if not identical, given the approximate continuity of human anatomy, physiology, and chemistry over historical time. we should not dismiss retrospective diagnosis altogether just because we are unable to ascertain that disease x and x′ are identical, as leven argues. a similar but somewhat different concern is expressed by karenberg [ ] . he argues that medical knowledge and nosology change over time, and gives an example of frédéric chopin's diagnoses since . this includes five medical diagnoses related to his respiratory problem, from tuberculosis to cystic fibrosis to alpha- antitrypsin deficiency, and five psychiatric diagnoses related to his mental symptoms, from "psychasthenia" to bipolar disorder. karenberg wrote that these diagnoses represent a "perfect self-referential system" within medicine, and "little appreciated by outsiders, … historians of music or biographers." "[t]he emerging concepts of disease and medical diagnoses proposed [for chopin's case] over the last years strongly correspond with research on these diseases in clinical medicine ([ ], pp - )". for historians, it is perplexing that the medical condition that a historical figure had, which is supposed to be fixed as a historical reality forever, changes so frequently in correlation with the progress of modern medicine. "how come chopin can have such a state-of-the-art modern disease which was not known in his time?" is the typical reaction. leven also echoes karenberg by saying that it is "a symptom of an anachronistic self-image of medicine" ([ ], p ). as a new conception of disease and diagnosis is introduced, clinicians are tempted to apply such a new label to a historical figure. but to historians, it does not make sense at all to put new labels to a historical event without any new historical evidence. behind this challenge is the critics' doctrine that historical facts in general, and historical diseases in particular, must be interpreted in their historical context, or historicism. retrospective diagnosis is anachronistic exactly because it tries to diagnose a disease of the past in contemporary terms. this challenge is also complex because there are two prongs. one is whether a historical fact, in this example the fact that chopin had disease x, is independent from the historian who describes it, and the other is whether a medical diagnosis, in this example the diagnosis of disease x in the patient chopin, whether it is in contemporary or historical time, is independent from diagnosticians. it seems that the critics' challenges are based on the assumption that it is an observer-independent fact that a certain disease existed in a historical person whether any historian can know and describe it or any medical professional can diagnose it. that is the historical reality that is independent of observers. were they to accept observer-dependent historical reality, they would have had no qualms about the ever-changing nature of chopin's diagnosis over the past years, because the description of his disease and diagnosis would have been all observer-dependent, always analyzed and described from the observer's viewpoint of his time, and necessarily changed over time. we might call their position ontological realism about historical facts in general, and the persistence of diseases and medical diagnoses more specifically. (for the comparison of realism and antirealism about historical facts, see murphy [ ] , and about medical diagnosis, see simon [ ] ). but antirealists have a ready reply to those realists: how do they know that there are observer-independent historical facts and diagnoses? if they assume that chopin's disease is one and fixed forever, how do they know and confirm that alleged fact? once we start analyzing chopin's disease using modern methodology and the language of historiography, paleopathology, and modern diagnostics, the result is of course observer-dependent-dependent on us modern historians and diagnosticians-and thus it is completely anachronistic. how could they possibly prove their alleged historical reality in a non-anachronistic way that is independent from our current "anachronistic" methods? is there an a-historical vantage point ("god's eye view") to view diseases of different historical times? as we analyze the critics' ontological challenges against retrospective diagnosis, it becomes clear that they are intricately entangled with the epistemic challenges, to which i will turn in the next section. assuming that a historical figure did have a real disease in the past, how do we know what disease he had? this is the central question of retrospective diagnosis. there seem to be two competing views. first, one believes not only that a certain disease existed in the reality of a historical person, but also that we should be able to know that reality in a non-anachronistic way if we were provided with an ideal access to that reality. however, we are not able to know it exactly because we have no adequate access to the historical reality. texts and artifacts are not preserved as medical data, and they should not serve as a means to access the medical reality of the past ( [ ] , p ). the second thought is that one also accepts the existence of the medical reality of the historical figure in the past, but believes that the only thing we can say about this reality is to construct or reconstruct it in our discourse through our own perception using presently available information, particularly texts and artifacts, along with state-of-the-art medical knowledge. while he may believe it is the best approximation of the historical reality, he readily admits that there is no guarantee that such a construction exactly matches the historical reality. let me call the first argument realist about retrospective diagnosis, and the second antirealist or constructivist about retrospective diagnosis. in what follows, i will reject the former, and defend the latter. the realists are pessimistic about the possibility of retrospective diagnosis of historical figures. they have to remain agnostic about such a diagnosis. thus leven wrote ( [ ] , p , emphasis added): "is it possible then to identify these past diseases with our present diseases? this … question cannot be answered with 'yes' , rather, it cannot be answered at all …". let me call this argument skeptical agnosticism. karenberg also presents a variant of leven's argument to cast the same doubt about the epistemic access to retrospective diagnosis: as a final proof (such as a pathoanatomical finding, a lab test or a genetic analysis) usually cannot be provided, it is impossible to falsify or verify a hypothesis of this kindthe "cases" of alexander the great, mozart and van gogh are excellent examples for this situation. as a matter of fact, a "may-be"diagnosis concerning a historical patient never can be ascertained in the same way as a modern patient ( [ ] , p ). let me call this the verificationist argument, which is a classical doctrine of logical positivism (empiricism) which says that a proposition can be said to be true only if it could conceivably be shown to be false, if false ( [ ] , p ). what is common between the above skeptical agnosticism and the verificationist argument is the positivist view of medical diagnosis, which takes it as something to be "identified" and "verified". as karenberg states, medical diagnosis is equated with laboratory tests and genetic tests and these tests are considered the "final proof". since these tests give a positive-or-negative answer most of the time, medical diagnosis also becomes a binary yes-or-no, or true-or-false question. disease entities are something that you have or you do not have. (the verificationist argument does not necessarily entail a binary judgment, and i am merely asserting that the critics are using these together.) leven also states ( [ ] , p ), "disease entities for modern scientific medicine seem to be biological entities." and this view is not limited to the critics of retrospective diagnosis; most people exposed to current technological medicine perceive medical diagnosis as a binary judgment of having or not having a disease. but i submit that this view is only a contemporary brainchild of modern biomedical sciences. diagnostic categories are a man-made construction, and not the product of nature itself or "biological entities" as leven asserts (see wilson [ ] ). as cunningham [ ] states, a disease or a disease concept does not exist independent from the act of diagnosing. he expresses this fact in a simple phrase: "you die of what your doctor says you die of". a diagnosis that is independent of a diagnostician does not exist even if there may be anatomical, physiological, and chemical changes that are consistent with a disease in a patient. only after a diagnostician diagnoses a patient by assigning her into a diagnostic category do we say that the disease exists in the patient. take a simple example of hypertension to illustrate this point. blood pressure reading is a continuous spectrum from low to high numbers, and the numbers themselves do not represent a disease or normalcy. it is a man-made cut-off point above which is the value for disease or positive and below which is normal or negative that determines the diagnosis of hypertension. most laboratory tests have a similar man-made cut-off point to differentiate positive or negative. even pathological and genetic tests have grey zones and human judgment is final to categorize these cases as disease or no disease. but as we become used to these diagnostic categories, we tend to believe that a diagnostic category or nosology itself, such as hypertension or coronary artery disease, is some sort of natural kind that belongs to nature itself independent from human construction. but as any medical practitioner knows, it is a man-made construction which is constantly revised and reconstructed. good examples of constantly revised diagnostic categories are icd (international classification of diseases) code and diagnostic and statistical manual of mental disorders (dsm). in the premodern era up to the th century, diagnoses that patients received were only about symptoms, and there was no concept of differential diagnosis which includes identifying disease a against b or no disease, or verifying against diagnostic standards. the preoccupation of a medical practitioner was to offer various therapies depending on symptoms [ , ] . in this respect, it is ironical that the critics of retrospective diagnosis who demand historicized diagnoses of a historical figure also demand nonhistoricist identification and verification in modern terms, which seems to be self-contradictory and anachronistic. but a more fundamental question is whether an act of medical diagnosis is all about identifying, verifying, and categorizing underlying diseases of the patient. in fact, medical diagnosis has many important aspects, among which identification and verification are only one of these. in the rest of this section, i will enumerate at least five aspects of medical diagnosis that are important and relevant to retrospective diagnosis. first of all, medical diagnosis is a process of hypothesis-making and hypothesis-adjustment. medical diagnosis is not just a "truth-finding" expedition into the reality of the sick body and mind of the patient, as natural scientists discover a hidden truth of nature. nor is it just a classification of the patient's condition into a known taxonomy, as a botanist examines and classifies a newly discovered plant. medical diagnosis is a dynamic guide for clinical decision-making with constant iteration between hypothesis-making and hypothesis-evaluation and adjustment according to the clinical reality that changes continuously and rapidly [ ] . because the clinician makes decisions under uncertainty, he has to adjust the diagnostic hypothesis on the fly according to what happens next. and this is one of the reasons why the same patient with the same condition can receive many different diagnoses with different focuses and specificity. each of these is a valid and important diagnosis at each step of medical care, and gives a different level of guidance to the next step. the second important aspect of clinical diagnosis is that it is fundamentally a probabilistic judgment (something is more likely or less likely) under uncertainty rather than an apodictic judgment (something is true or false) under certainty, as the critics understand. and the iteration of hypothesis-making and hypothesis-adjustment follows bayesian probabilistic reasoning [ , ] . in the most simplified form, bayesian reasoning is expressed as: (posterior probability) = (prior probability) × (likelihood ratio), where posterior probability is the probability of correct diagnosis after the diagnostic evaluation (hereafter "postodds") and prior probability is the probability of correct diagnosis before the diagnostic evaluation (hereafter "preodds"). likelihood ratio is the ratio of the true-positive rate divided by the false-positive rate of the evaluation. when the post-odds increases from the pre-odds, that means that the probability of the hypothesis being correct increases (or the diagnosis becomes more plausible), and thus the diagnostic evaluation is correct, that is to say the true-positive rate is greater than the false-positive rate. when the post-odds decreases, the plausibility decreases and the hypothesis is deemed less likely. the clinician must go back to the previous stage to adjust the initial hypothesis or select a different method of evaluation. by iterating this process of hypothesis-making, -evaluation, and -adjustment according to bayesian reasoning, the diagnosis can achieve a higher probability of certainty. the third aspect is that the degree of certainty of a diagnosis need not be %. there is no such thing as a % certain diagnosis, but more importantly clinicians often are satisfied with an uncertain diagnosis depending on the clinical need and context. such a diagnosis is still "correct" and fully useful in clinical reality. for example, when a physician sees a patient with upper respiratory symptoms and makes a diagnosis of "viral syndrome", he will not embark on any "truth-finding" expedition to confirm this diagnosis and find out the exact virus that is causing the patient's symptoms. it is a syndromic diagnosis without etiological confirmation. he is merely assigning the patient to a large undifferentiated diagnostic group of miscellaneous viral infections which are self-limited. this is because identification and verification have no use for clinical management, and he knows that the patients with "viral syndrome" recover spontaneously only by symptomatic treatment. on the other hand, if there is an outbreak of a certain viral infection which needs to be contained, he would probably do a test to determine the exact virus, such as sars or h n . this example shows that a clinical diagnosis and a diagnostic category to be employed are dictated by the need and the context of the patient, place, and time. as edmond murphy stated, "a major function of diagnosis is precisely to discern where and where not to look in detail". ( [ ] , p ) it is not that one diagnosis is "true" and the others are "false". the fourth aspect of medical diagnosis that is relevant to the current discussion is that the act of diagnosing is a social practice, and medical diagnosis is an explanatory device with many social implications [ ] . a clinician is not a natural scientist whose task is to uncover a hidden state of affairs of nature; she is only applying natural sciences to more pragmatic tasks of caring and treating a sick patient, explaining the condition, and prognosticating the future course of his suffering. when an ancient physician "diagnosed" a patient with epilepsy as "sacred disease", it was an explanatory device to advise the patient regarding what to do and what to expect [ ] . in the same way, when a modern neurologist diagnoses a patient with epilepsy as "mesial temporal lobe epilepsy with hippocampal sclerosis", it is also an explanatory device to advise the patient for specific treatment and prognosis that are attached to this specific diagnostic category. it is the best construction of the state of affairs at the moment of the advancement of medical knowledge but it does not represent the entire reality of the patient. that is why medical diagnosis as a construction of concepts has changed and will change any time in the history of medicine [ ] . this fact is best seen in syndromic diagnoses of functional disorders (e.g. fibromyalgia, chronic fatigue syndrome) and many psychiatric disorders. despite the lack of any confirmatory test, these syndromic diagnoses are a highly useful construction of the state of affairs of the patient. syndrome (meaning "running together" in greek) is "the aggregate of signs and symptoms associated with any morbid process, and constituting together the picture of the disease." (steadman's medical dictionary, emphasis added). syndromes do not have identifiable or verifiable underlying biological entities, yet they are a very useful explanatory device and used more and more. and most importantly for the discussion of retrospective diagnosis, syndromic diagnosis can be made from history alone and is highly successful. in one frequently cited study [ ] clinicians could reach the correct diagnosis more than % of the time by clinical history alone without examining the patient or obtaining laboratory tests. these diagnoses were mostly syndromic. the remaining % of the cases ended up with different diagnoses after examination and/or laboratory tests. but the lesson we learn from this study is that even for those diseases that have known underlying biological entity can still be diagnosed by syndromic diagnosis % of the time. and this is a very decent odds for a judgment under uncertainty. if a clinician is provided with the history of the patient's entire life with many witness accounts of symptoms in a form of biography and other historical documents, is it not unreasonable to expect the accuracy of such a syndromic diagnosis even better? the last but not the least relevant point is that medical diagnosis has several methodological dimensions. one condition can be diagnosed from many different dimensions: by clinical signs and symptoms (clinical diagnosis); by laboratory tests (laboratory diagnosis); by genetic tests (genetic diagnosis); by identifying etiology (etiological diagnosis); by pathological examination (pathological diagnosis). these methodological dimensions are not synonymous labels for the same condition. for example, aids (clinical diagnosis) and hiv infection (etiologic diagnosis) are not synonymous; a patient can have the latter without former. which methodological dimension to be used depends on the clinical context. as already mentioned, the diagnosis of "viral syndrome" (clinical diagnosis) is sufficient without etiological or laboratory diagnosis in certain clinical contexts. in fact, numerous syndromic diagnoses without identifiable underlying biological facts are being used in contemporary clinical medicine. when a clinician makes a retrospective diagnosis of a historical figure based on his works (texts and arts) and the records of behavior (biography), it is impossible to make a laboratory, pathological, or genetic diagnosis since none of these tests are available unless we also have access to some material that can be analyzed by the methods of paleopathology. and since etiological diagnoses almost invariably rely on tests, these are also impossible. that means that retrospective diagnosis makes sense only if it is a clinical diagnosis. for example, the retrospective diagnosis of "alpha- antitrypsin deficiency" for chopin does not make sense because this requires a laboratory test to measure a serum alpha- antitrypsin level, which is not available for chopin. one could still say chopin had a clinical syndrome consistent with or similar to alpha- antitrypsin deficiency. whether such a clinical diagnosis makes sense is debatable, however, given the fact that this condition is clinically very difficult to distinguish from more common and prevalent emphysema or copd. because of this inherent methodological limitation of making a retrospective diagnosis beyond clinical and syndromic diagnosis, i suggest that retrospective diagnosis be limited to these methodological dimensions. to sum up, medical diagnosis has many different aspects and dimensions and need not be "identifying" and "verifying" an underlying biological reality. skeptical agnosticism and verificationism are based on the erroneous conflation of taxonomizing the imagined natural kinds of diseases with the multifaceted social act of diagnosing a patient. when the critics realize that the taxonomy of diseases against which diagnoses are "identified" and "verified" is nothing more than a dynamic man-made construction of a given historical time, they lose support of their concept of the observer-independent medical and historical reality. so far, i have argued that it is a misguided demand on the part of the critics of retrospective diagnosis to expect any medical diagnosis to be "identifying" and "verifying" a disease in a patient. however, the critics would object to this argument as follows: retrospective diagnosis is not for a patient whom the clinician is seeing in his clinic or hospital. the patient about whom the diagnosis is rendered is a dead historical person whom the clinician never saw. therefore, many arguments from the logic of clinical diagnosis as discussed in the previous section do not apply to retrospective diagnosis. for example, there is simply no possibility of hypothesismodification in the diagnostic process of a historical patient, since no new clinical information is available, except for a rare occasion of newly discovered historical documents or artifacts. and above all, the critics would contend that as long as a retrospective diagnosis is presented as evidence for some larger proposition in the humanities and social sciences, it is necessary that such a diagnosis come with acceptable proof or verification, which is unavailable. i acknowledge that this is indeed a very serious problem, and as long as we treat retrospective diagnosis, or any medical diagnosis for that matter, as an apodictic scientific proposition, it would be very difficult to put it on the table of any scholarly discourse. (this is a common conundrum of epidemiologists who have to rely on the accuracy of clinician's diagnoses for disease identification). nevertheless, i would like to offer a new perspective to accommodate retrospective diagnosis in a broader scholarly discourse of the humanities and social sciences as well as medicine. as i discussed earlier, modern medical diagnosis pretty much follows bayesian reasoning under uncertainty. i suggest that we expand this process to a larger scope of retrospective diagnosis. under the conception of the bayesian model of probabilistic judgment, unverifiable retrospective diagnosis can be evaluated for its plausibility and coherence in a larger context. this is also nothing new in present-day medical practice; when clinicians do not have any definitive evidence to follow in order to make a clinical decision, they still follow peer reviews and expert opinions. in order to do this, they regularly present difficult cases in clinical conferences, and see if their diagnosis is supported by the peers. or sometimes, the case is presented to nationally known experts of the disease in question and their agreement can serve as the support. it is an old-fashioned way to "confirm" a diagnosis when there is no evidence to rely on, but it is still one form of bayesian decision-making because the change of plausibility from pre-odds to post-odds by increasing or decreasing the likelihood ratio is considered a confirmation or disconfirmation of the diagnosis. i suggest that retrospective diagnosis be evaluated in similar peer-review methods. currently peer review medical journals seem to do this job in terms of the soundness of diagnostic reasoning. unfortunately, however, the review does not seem as rigorous as for contemporary case reports, as retrospective diagnoses are viewed as of minor importance, which the harsh criticisms of the critics will only aggravate. while this peer review process may serve as a process of confirmation about the medical aspect of retrospective diagnosis, it is also important to address the critics' concern that such medical discussions do not contribute to the scholarship in the humanities and social sciences. in this regard, it is interesting to note that there is a commonality between medical diagnosis and historiographic theories: both are hypotheses that are confirmed or disconfirmed by evidence using probabilistic, not apodictic, judgment. in other words, both medical diagnosis and historiographic reasoning involve the iteration of hypothesismaking and hypothesis-adjustment according to the evidence available. day and radick [ ] propose two models, the bayesian and "explationist" (this term is original to day and radick) models, to capture this aspect of historiographic reasoning. let us use a hypothetical case to demonstrate how these models work to confirm or disconfirm hypotheses of retrospective diagnosis and art history. suppose a hypothetical legendary painter in his 's started painting pictures with the left side of the canvas being less detailed and more abstract. no medical record was available for this historical artist. art historians thought that this peculiar change in his painting style was due to his artistic maturation and a shift toward more abstract style and technique. then a clinician presented a hypothesis that this change was due to brain damage in his right hemisphere. (similar cases are reported by bäzner and hennerici [ ] ). what was the evidence? suppose the clinician can produce paintings painted by other painters who were known to have right hemispheric brain damage, and their paintings were very similar to the ones painted by the legendary painter in terms of neglecting the details on the left side of the pictures. or the clinician can cite the diary of the painter's wife who witnessed that the painter somehow did not pay attention to the left side of the tray when he was eating-a telltale sign of left hemineglect, which is a sign of right hemispheric damage. how do we evaluate this new hypothesis based on a retrospective diagnosis? under the bayesian model, the question is whether this evidence makes the brain-damage theory more plausible than the style-change theory. under the "explationist" model, the question is which hypothesis explains the change in his paintings better. the answer comes from further evaluation of the existing and additional evidence that the art historians can produce to support their style-change theory, and further debates will lead to more hypothesis-making and hypothesis-modification. the point i would like to make from this hypothetical case is that a retrospective diagnosis should be evaluated as a theory which competes with other non-medical theories in a larger context of the humanities and social sciences. as usual with many theories in these fields, it is pointless to expect that such a theory would render an apodictic judgment of true-or-false; most of the time, they are a probabilistic judgment evaluated on a moreor-less plausible or better-or-worse explainable basis, or coherence with other evidence. and in order for such a case report to be incorporated inside a broader context of scholarly discussion, it is important to state what theory and what question a particular case of retrospective diagnosis would address. it is interesting to note that the act of clinical diagnosis is under the intense scrutiny of medical ethics and professionalism, yet as far as making a diagnosis of a historical figure, there seems to be no such concern. is there any problem with ethics and professionalism in retrospective diagnosis? in the absence of a clinicianpatient relationship, usual principles of clinical ethics do not apply, but that should not be taken as a reason to believe that anything goes. it is important to consider, first of all, why the retrospective diagnosis is made, and what is the impact that it can make on the scholarship and to our society. the recently debated proposal to exhume chopin's heart to make a genetic diagnosis of cystic fibrosis is a prime example of ethical concern [ ] . genetic testing of paleopathological material in general carries moral risks. besides the disputed desecration of the dead, there is a potential impact on the descendents of that person. more generally, there is a concern for publicizing a medical diagnosis without consent from the patient and family. one could argue that these historical celebrities are immune from privacy protection because their lives are open to the public. furthermore because they are long dead, the subject of any potential harm no longer exists. yet, such an argument is still controversial. first, even though philosophers disagree on how a posthumous event, such as damaging a person's reputation by libel and slander, can harm the person when the person to be harmed does not exist, most philosophers are in agreement that posthumous harm does happen [ , ] . second, even though the record of one's life is open to the public, it is not clear whether the medical information extracted from historical evidence that was not known to the person or family has more weight in the protection of privacy. for example, certain descendants might object to the publication of a diagnosis that potentially tarnishes the reputation of their ancestor. third, it is also important to consider the integrity of professionalism when publishing potentially private medical information. even if there is no proven harm to the deceased celebrity and there is no legal violation of privacy, if a clinician publicizes medical information of a celebrity with a paparazzi mentality, some would see a potential of slippery-slope degradation of medical professionalism. some might ask, "if this clinician is so nonchalant about disclosing the privacy of a dead celebrity, can he do the same thing for me if i die some day?" and lastly, it is also important to remember that some dead celebrities have enthusiastic followers (e.g. certain religious figures) and intense abominators (e.g. certain dictators). it is ethically considerate to take into account the feelings of these contemporary people. for all these reasons, there should be a justifiable scholastic reason that is carefully balanced against potential ethical concerns before initiating such a medical evaluation in the absence of the patient's consent. and such a justification should come from a broader scholarly context and appropriate multidisciplinary peer review. (from this aspect, the rejection by the polish government to exhume chopin's heart seems quite appropriate [ ] ). there seem to be at least three scholarly purposes for making a retrospective diagnosis in a historical figure. first it helps understand the influence of a disease on the works (writing, arts, music etc) and behaviors (personal, social, and political conducts) of that person. this is most useful to scholars in the history of art, music, literature, philosophy, religion, and politics, among others. second, it helps understand through his works and recorded behaviors how it was like to live with the disease in that historical time. this is of interest to medical historians and those who are interested in historical illness narratives. third, it can also serve as a precious source to learn the life-long history of a particular disease through a medically reconstructed biography. this is of particular interest for clinicians dealing with certain chronic diseases. there may be more than these to justify the publication of a retrospective diagnosis, but the point here is that it is important to clarify how such a diagnosis would contribute to the scholarship of that historical person, or i would call a broader-context question. in evidence-based clinical medicine, clinicians in general are not advised to do any diagnostic workup unless the result will change the management of the patient. in such cases, certain specifications of the diagnosis are left unknown. earlier, i mentioned an example of the generic diagnosis of "viral syndrome" in which clinicians do not initiate the tests for the specification of causative viruses unless there is a particular purpose. i suggest that the same principle be applied to retrospective diagnosis. for example, how does the specification of chopin's chronic respiratory illness to several different diseases, tuberculosis, copd, cystic fibrosis, alpha- antitrypsin deficiency, make any significant difference in understanding chopin's artistic accomplishment, music style, music performance, and chopin's biography overall? [ ] . in this example, each diagnosis carries a different etiology but the syndromic presentation is similar: all present with chronic intermittent respiratory distress with cough and sputum. in such cases, it is important to clarify how the degree of diagnostic specificity is justified not only by the medical evidence but also by the scholarship of chopin. otherwise, a purely technical overspecification of retrospective diagnosis would only alienate non-medical scholars from the entire practice of retrospective diagnosis. lastly, is there any role of retrospective diagnosis with little or no scholarly implication? is it appropriate to speculate a medical diagnosis of a dead celebrity just for fun? some say it is an "irresistible," "eccentric pursuit of doctors", "fun escape for doctors" [ ] and medical professionals' "historical hobby" [ ] . nothing seems to prevent such activities, but i still maintain that the above mentioned caveats be considered when such an account is published. however, i would also argue that there is a problem of advising journal editors and reviewers to reject many of those reports of retrospective diagnosis as critics suggest [ , ] . if journal editors abide by this advice, eventually some important reports of retrospective diagnosis would fall through the cracks and become orphans without a forum for scholarly dialogue. the reason is that the editors of medical journals are not in the position to evaluate the scholarly impact of such reports in the humanities and social sciences, whereas the editors of those non-medical journals can in no way evaluate the authenticity of medical discussions in such papers. as a result, they flatly reject those papers following the critics' advice, and some important papers are not published in any journals at all. for this reason, it is important that the soundness of the medical diagnosis be evaluated in the peer review process of a medical journal, and at the same time or thereafter, the appropriateness of the historiographical methodology and the scholarly impact of the diagnosis be evaluated by the peer review process of the respective non-medical journals. in this regard, it is also strongly advisable for a clinician to team up with scholars in the respective field from the beginning of the project. by doing so, medical and non-medical peer reviews can be accomplished in parallel. in this section, i will take stock of what has been discussed so far, and come up with a list of the key elements in retrospective diagnosis that can be taken seriously by scholars of the humanities and social sciences. this list is by no means intended to claim the novelty and originality of this essay, and should be taken as a composite of the current thesis combined with the caveats that have been published by other scholars. i will use a case of socrates' peculiar symptoms of hearing a voice and trance-like episodes to demonstrate these points [ ] . the original case report was short and did not have all the important elements that i discussed above. the first point to be considered is what question is to be answered by the retrospective diagnosis ("broader-context question"). in order for a retrospective diagnosis to be incorporated in the scholarship of the humanities and social sciences, it is imperative to situate the question in the middle of an important debate. in case of socrates, there has been an ongoing controversy among ancient philosophers and classicists about the nature of socrates' "divine sign". according to plato, socrates had recurrent episodes of hearing a voice ever since childhood that commanded him to stop or refrain from certain actions. socrates said, "i have a divine or spiritual sign which meletus has ridiculed in his deposition. this began when i was a child. it is a voice, and whenever it speaks it turns me away from something i am about to do, but it never encourages me to do anything" (apology dall the quotes from plato hereafter are from [ ] ). socrates variously called this voice "divine sign", "spiritual sign", "my prophetic power", or in greek "daimonion", which visited socrates unexpectedly and abruptly irrespective of the importance of the context in which he was placed (apology d; a "[daimonion] opposed me, even in small matters "; euthydemus e- a). the duration of this episode was usually very brief, probably a few seconds to a minute at most, and it often came when he was about to initiate an action or speech, though it was quite unpredictable. the voice seemed inarticulate; socrates never attributed any specific words to the voice. the prevailing theories among ancient scholars include; ) a prima facie interpretation that it was a divine voice and the experience is religious in nature (e.g. mcpharran [ ] , p ), ) that socrates referred to his own voice of reason (e.g. nussbaum [ ] , p ), and ) that it was a sort of "hunch" or feeling of monition (e.g. vlastos [ ] ). one of the leading scholars of plato, gregory vlastos, was quoted as saying that socrates' daimonion is "the gravest of the difficulties we all have to face in our effort to make sense of socrates" ( [ ] , p ), as each theory has its serious flaws, and there has been no consensus. on one hand, some philosopher such as bertrand russell [ ] raised a question of socrates being "insane" but this was widely dismissed by other scholars. the attempt to explain socrates' behavior outside of his religion and philosophy has been an anathema for ancient philosophers and classicists in general. for example, mark joyal [ ] , referring to such attempts, wrote: "to be sure, it is in research on the divine sign that some of the low points in the history of socratic scholarship have been plumbed…" against this backdrop, a clinician can ask: "is it still possible that socrates had a certain neurological condition and a retrospective diagnosis can shed a different light on this debate?" another possible hypothesis might be: "did socrates attribute his symptoms of seizures to a god, just as the hippocratic source informs us that most laypeople those days attributed seizures in general to gods and spirits?" [ ] . as the critics repeatedly admonish [ , , , ] the textual source has to be as close as possible to the person of interest, and should be interpreted in the original language. this is a basic methodological requirement in the humanities and social sciences, but it is not easy for clinicians to fulfill. it is best achieved by collaborating with scholars of the area of interest. because socrates never wrote anything, all surviving records about his remarks and behaviors are from plato, xenophon, and aristophanes, who are all socrates' contemporaries and independently left records. the original greek texts are readily available for investigation, and this project is an interdisciplinary collaboration of a neurologist and a classicist. the relevant point in this retrospective diagnosis is that it is based on the personal observations of intelligent and observant friends of socrates'. there are several other ancient texts mentioning socrates, but these were written centuries after his death, and are believed to be less reliable than the records written by his direct contacts. still, caution is in order when we interpret plato for the description of socrates because of plato's own bias and admiration toward socrates. as discussed in the above section epistemic challenges of retrospective diagnosis, the best methodology that yields the most plausible retrospective diagnosis is syndromic; all other methodologies of diagnosis more or less require laboratory tests and are not suitable for retrospective diagnosis of historical figures. if a clinician really wants to specify the diagnosis to the level of pathological, etiological or laboratory diagnosis as opposed to syndromic diagnosis, it is more appropriate and less misleading to add a phrase such as "a clinical syndrome similar to or consistent with" before the diagnosis. in the case of socrates, the authors arrived at a syndromic diagnosis of temporal lobe epilepsy based on four independent clinical features ( [ ] , p ): ) brief, reversible, and episodic neurologic symptoms consisting of involuntary and unexpected hearing of a voice, mostly associated with action (simple partial seizure); ) intermittent episodes of diminution or arrest of responsiveness and motion with complete recovery except for various degree of memory loss (complex partial seizure); ) episodes of prolonged unresponsiveness and arrest of motion (complex partial status); ) childhood onset with relatively benign course into late adulthood. although temporal lobe epilepsy carries in its name a localizing implication of the temporal lobe, temporal lobe epilepsy as a syndromic diagnosis does not imply that the localization of pathology is definitive, and it can be used as a diagnosis without brain imaging studies or electroencephalography. the clinical syndrome of temporal lobe epilepsy can happen as a result of lesions in the frontal lobe or parietal lobe, and this syndromic diagnosis can allow such variations. if, however, the authors specified the diagnosis as "mesial temporal lobe epilepsy with hippocampal sclerosis", which is the most common type of temporal lobe epilepsy syndrome but requires an imaging study, it would have been a case of overspecification, unless the claim is qualified by the phrase "a clinical syndrome consistent with …" with justifiable reason. is there any possibility that the disease being diagnosed did not exist in the particular historical time? as discussed above in the section ontological challenges of retrospective diagnosis, this question is fundamentally an empirical question posed to medical historians and paleopathologists, but it is probably very difficult to find an answer. at least it is important to consider the possibility that the disease in question was caused by a new genetic mutation of the host or a mutation of pathogens (such as aids according to the current theory) or unique environmental factors (such as nutritional deprivation) at some point of historical time. in the case of socrates, the difference from most other cases of retrospective diagnosis is that socrates was not diagnosed as ill or abnormal in his time. therefore, the retrospective diagnosis does not raise the most common question whether disease x (temporal lobe epilepsy) and an ancient disease x′ are the same, but whether disease x existed in historical time without being recognized as a disease. it is well-known that the generalized forms of epilepsy have existed at least since the time of hippocrates [ ] , but the descriptions of non-convulsive epilepsies, such as temporal lobe epilepsy and absence epilepsy, are difficult to come by. however ancient physicians were also remarkably astute in describing non-convulsive auras of epileptics ( [ ] , p ). since most auras are often a partial seizure, or a small-scale, initial, and non-convulsive phase of larger seizures, and these could stop by themselves without developing into generalized convulsive seizures, it is logical to assume that non-convulsive forms of epilepsy also existed in historical time. in other words, given the fact that generalized convulsive seizures are just the tip of the iceberg of many other convulsive and non-convulsive forms of seizures, the presence of generalized seizure as the "sacred disease" in ancient times is consistent with the coexistence of many other forms of milder and nonconvulsive seizures in the background. as discussed above in the sections epistemic challenges of retrospective diagnosis and retrospective diagnosis as theory construction, it is important to evaluate the hypothesis on the basis of the bayesian model or the "explationist" model (more or less plausible or better explainable) rather than whether it is true or false. this can be done in two areas: medical and non-medical criticisms. hypothesis-evaluation from a medical viewpoint consists mainly in evaluating differential diagnosis. the main competing differential diagnosis for socrates would be schizophrenia with auditory hallucination. it is important to discuss, for example, the comparison between temporal lobe epilepsy and schizophrenia in terms of the nature and content of the voice that socrates heard, life-long benign history, age of onset, etc, and which diagnosis can explain these features better. in this case one could argue, for example, that a very brief, non-articulate stereotyped voice is more consistent with auditory seizures than schizophrenic auditory hallucinations; that if socrates had childhood-onset schizophrenia, it would have been almost impossible for him to maintain his physical and mental health up to age , but it would still be possible if he had a mild case of temporal lobe epilepsy. non-medical evaluation is to compare the retrospective diagnosis with the competing theories presented by scholars in ancient philosophy, classics, and history. for example, the discussion can include which theory explains better the several difficulties of the prima facie interpretation of the voice that have already been presented by socratic scholars, such as the onset in childhood, the occurrence that is unpredictable and unexplainable to socrates, the vagueness of the voice and message, and the personal nature of "god" who utters the voice. (socrates never named the source of the voice, and from every aspect it was personal to him. that seems to be one of the reasons that socrates was charged for "believing in a new god" and executed.) the proponent of retrospective diagnosis might also present a new perspective on the historical socrates. his bizarre behavior, which is well recognized by ancient scholars as 'atopia' (or strangeness), along with his intense religiosity (compared to his contemporaries, not to our current standards), might be better explained by a personality and behavioral disorder associated with temporal lobe epilepsy (this behavioral disorder is often referred to as geschwind syndrome). socrates might have been an epileptic savant who was executed because of his eccentric thoughts and behavior that were at least partially attributable to his neurologic disorder. the final element is the ethical consideration. first of all, we need to ask whether making a retrospective diagnosis is justified by its overall goal. we also need to consider any potential harm to the person of interest and his descendents and followers. as discussed above, the retrospective diagnosis of socrates was prompted by the scholarly debate outside medicine, and it remains to be seen how the socratic scholars respond. it is also interesting to note from plato's record of socrates' behavior that socrates himself was not convinced of the origin of the voice. he was often baffled by unexpected and unexplainable occurrences of the voice. particularly when he was accused of believing in a new deity and condemned to death, he was clear that he did not create the divine voice, but he was not at all convinced of its origin. it is quite possible that he would have welcomed an alternative explanation of the voice if it had been available. at any rate, there is no conceivable reason that this diagnosis could have harmed socrates, and if anything it would have benefited him. one can object to such an ethical consideration because it is a moot point for someone who has been dead for more than years, and historians in general have no qualms about revealing any reality, good or bad or ugly, of a historical figure. however, i still maintain that if it is done particularly by a practicing clinician, it is prudent at least to consider this aspect of retrospective diagnosis for the sake of professional integrity, as discussed above in the section problems of ethics and professionalism in retrospective diagnosis. the relevance to the ongoing debate among historians and paleopathologists so far i have focused my discussion on retrospective diagnosis of an individual historical person by means of biographies and other sources for clinical diagnosis. as stated in the introduction, however, the concept of retrospective diagnosis used for the diagnosis of an individual person from a life-long history has a subtle but significant difference from that used by medical historians and paleopathologists. in this section, i will briefly go over the debate among them and how it relates to the current proposal for a diagnosis of an individual. while this author, who is neither a historian nor paleopathologist, has no qualification to jump into this debate, it is only fair to explain the relevance of this essay to their debate since i have extensively quoted their works and arguments, and incorporated many of them into my discussion. as already discussed in the section ontological challenges of retrospective diagnosis, medical historians and paleopathologists are struggling among themselves with a similar challenge of identifying historical diseases without relying on anachronistic methodology in modern academic discourse. this debate is commonly called among those historians as the "cunningham debate" [ , ] . on one hand, there are historians who deny the possibility of retrospective diagnosis altogether, such as wilson [ ] and cunningham [ ] , because medical diagnosis is essentially a social construction of the time and place in history, which cannot be transferred to a different historical time. on the other hand, there are those who accept the thesis that historical diagnoses are a social construction [ ] or "social diagnosis" [ ] , but do not deny the possibility of retrospective diagnosis altogether, insofar as the investigators take necessary steps to avoid anachronistic pitfalls when interpreting historical sources. they also advocate, as i do in this paper, a team project among medical professionals and historians. karenberg ( [ ] see also endnote a ), similar to mitchell [ ] , also advocates cautious approaches to historical material without ruling out the possibility of retrospective diagnosis altogether, though he does not advocate the team approach. how does my current approach, then, relate to this debate? as i already stated in the section epistemic challenges of retrospective diagnosis, i fully agree with wilson [ ] , cunningham [ ] , and arrizabalaga [ ] for that matter, that medical diagnoses are fundamentally a social construction, and a disease or a disease concept does not exist independently from the act of diagnosing in a particular historical time and place. in this sense, i respectfully disagree with mitchell [ ] , who contrasts between historical diagnosis as "social diagnosis" on one hand, and contemporary diagnosis as "biological diagnosis" on the other. with cunningham, and against mitchell, i would submit that every diagnosis, whether historical or contemporary, including those diagnoses using modern bioscientific technologies, which mitchell is ready to classify as "biological diagnosis", is still a social construction. it is true that, generally speaking, modern diagnoses rely much more on biology than social factors, but the difference is a matter of degree, not of kinds. but more importantly, no matter how much or how little "biology" is involved in diagnosis, all diagnostic categories are man-made constructions, as i stated in great detail in the section epistemic challenges of retrospective diagnosis. in other words, all the diagnoses, modern or historical, biological or non-biological, are constructed by physicians who are acting in the midst of their society with all the influences from their social environment, including religion, culture, science, and technologies. (see endnote b for an example). and their diagnoses are constructed to suit the needs of the patient, family, and society by informing, explaining, theorizing, treating, and prognosticating what happened to the sick patient. identifying disease is only one component of this social practice, and may not be required in many cases. the act of diagnosis always has its social purposes and implications, whether ancient or modern. this is what i meant by saying that all the diagnoses are fundamentally a social practice and social construction, and i suspect that this is what cunningham meant when he wrote "turning away from diseases … and also away from disease concepts … and turning it instead toward how diagnosis happens" ( [ ] , p , emphasis original), and "you die of what your doctor says you die of" (ibid, p , emphasis original). at any rate, cunningham then argues, correctly i think, that it is impossible to translate a historical diagnosis into disease identification in contemporary terms. where i disagree with cunningham, and agree with mitchell and others, is that retrospective diagnosis is still possible. but the reason is totally different from mitchell, karenberg and others. i disagree with cunningham regarding his thesis that retrospective diagnosis is impossible exactly because i fully endorse the other part of his thesis that all medical diagnoses are essentially a social construction. it seems paradoxical, but the key to understanding this claim is my thesis that, as i argued in the section epistemic challenges of retrospective diagnosis in great length, a clinical diagnosis as a social construction is an explanatory device and a theory construction that serve many purposes in society. it can serve not only the patient and her family to receive the explanation of what is going on in her, but also serve as an explanation of what happened to a deceased patient. while i agree with cunningham that we cannot directly transfer a historical disease concept to a contemporary disease identification, i am arguing, against cunningham, that we can still use our contemporary diagnosis as a modern explanatory device and theory construction, to explain what happened to a historical individual in question, just as historians and archeologists are all using state-of-the-art technology and concepts to explain what happened in the historical world. to insist that physicians must use ancient concepts and tools to diagnose an ancient patient is as illogical as to insist that archeologists must use ancient tools and concepts to investigate their archeological sites and materials. (see also [ ] p , "… it is vital that diagnostic criteria have a secure basis which derives ultimately from clinical medicine". here i am talking about the tools and concepts of clinical diagnosis and archeological investigation, not the concepts used to interpret historical documents, which of course requires ancient or historicized concepts). by situating my current thesis in the midst of the cunningham debate among historians, it seems clear to me that my disagreement with cunningham derives from the subtle but critically important difference between retrospective diagnosis as disease identification in historical time, or the retrospective construction of historical nosology, which is the primary concern for historians and paleopathologists such as cunningham, and retrospective clinical diagnosis of a deceased individual, or retrospective diagnosis as the construction of a medical theory to explain what happened to a historical individual, which is the primary concern for those clinicians interested in retrospective diagnosis. as i have discussed in great detail, neglecting this subtle but critical difference and conflating disease identification, or nosology, and the social act of clinical diagnosis have resulted in harsh criticism against the retrospective diagnosis of historical individuals. at the same time, in order to avoid this confusion i have proposed in this essay that such a retrospective diagnosis should be limited to syndromic diagnosis without identifying disease unless there is justifiable reason for such specification. while professional historians and paleopathologists have their own debate and agenda regarding retrospective diagnosis as reviewed in the preceding section, i have focused this essay on only one type of retrospective diagnosis, that of famous historical individuals. presently, this type of retrospective diagnosis in academic publications receives two polarized evaluations [ ] : in one camp, it is dealt with as a hobby of historically minded clinicians who enjoy testing their diagnostic acumen in famous celebrities and uncovering intriguing medical secrets. these reports usually belong to an accessory section for incidental topics or letters to the editor in medical journals. in the other camp, serious scholars of the humanities and social sciences have little interest in those highly specific diagnostic labels generated by clinicians whose methodologies and wild speculations have little justification. some of these harsh critics that i have argued against are pessimistic about the possibility of retrospective diagnosis altogether, as long as it uses the modern concept of medical diagnosis. one of the aims of this essay has been to narrow the gap between these two camps and try to elevate the status of retrospective diagnosis of famous historical figures in a truly interdisciplinary arena. but in the end, the distance between me and the critics of retrospective diagnosis turns out to be much narrower than it would seem; we both agree that retrospective diagnosis has to base on historicized interpretation, and the proper methodologies of historiography should be followed. we also agree that more attention should be given to ethics and professionalism of publishing retrospective diagnosis. where i disagree with the critics is their argument that retrospective diagnosis as historicized knowledge is impossible because the modern scientific verification of historicized diagnosis is simply illogical or akin to oxymoron. i have replied to the critics that their pessimistic skepticism and obsession with the scientific verification of diagnosis is based on their conflation of the act of categorizing diseases in a system of nosology, or disease identification, and the social act of diagnosing a patient from her history, or the construction of a medical theory. instead of advising journal editors to stop publishing reports of retrospective diagnosis, they should collaborate with those interested medical specialists, exchange ideas and develop a truly interdisciplinary field of retrospective diagnosis. at the same time, i have emphasized the importance of those "hobbyist" clinicians to reach out to non-medical scholars of the relevant field by situating the retrospective diagnosis in a larger theoretical framework instead of toying with their diagnostic acumen. such a collaboration has already been proposed by some of the cautious proponents of retrospective diagnosis in the history of medicine and paleopathology [ , ] . my hope is that this essay can pave such a collaborative path in the field of retrospective diagnosis of famous historical figures. endnotes a in all fairness, karenberg [ ] does not deny the possibility of retrospective diagnosis altogether; he rejects only those approaches exemplified below, which he calls "naive retrospective diagnosis". yet it is unclear how the editors and reviewers of medical journals or any journals outside history and related fields, who are not historians, can identify which report is "naive" and hence to be rejected. they could rather interpret karenberg's advice simply as an indication that all the papers dealing with retrospective diagnosis are illegitimate. b as i quoted in the section ontological challenges of retrospective diagnosis, karenberg's work [ ] on the diagnoses proposed for chopin's conditions clearly demonstrates that the diagnostic concepts change in parallel with the advancement of modern biomedical science. in other words, contemporary physicians are bound to use diagnostic concepts and tools that are produced by the contemporary culture of modern biotechnology, just as ancient physicians are bound to use diagnostic concepts and tools that are produced by the ancient culture of sciences and religious beliefs in their society. it is not that one diagnosis is social and another is biological, but they are all socially determined. chopin's example which karenberg beautifully demonstrated, even though for a different purpose, clearly attests to this relationship. investigating the ills of long-dead celebrities retrospective diagnoses of conditions affecting historical individuals retrospective diagnosis: use and abuse in medical historiography next emperor, please! no end to retrospective diagnostics at times these ancient facts seem to lie before me like a patient on a hospital bed'-retrospective diagnosis and ancient medical history commentary. the diseases of alexander the great the long suffering of frederic chopin neuropathography: origins and methodology psychiatrische anfänge der pathographie pathography: patient narratives of illness the white plague: tuberculosis, man, and society re-emerging infections: developments in bioarchaeological contributions to understanding tuberculosis today the relationship between paleopathology and the clinical sciences distinct clones of yersinia pestis caused the black death integrating historical sources with paleopathology realism about the past handbook of the philosophy of science the logic of medicine on the history of disease concepts: the case of pleurisy identifying disease in the past: cutting the gordian knot the history of the doctor-patient relationship the art of diagnosis: medicine and the five senses collecting and analyzing data: doing and thinking a companion to epistemology diagnosing and defining disease the falling sickness: a history of epilepsy from the greeks to the beginning of modern neurology companion encyclopedia of the history of medicine relative contributions of history-taking, physical examination, and laboratory investigation to diagnosis and management of medical outpatients historiographic evidence and confirmation painting after right-hemisphere stroke -case studies of professional artists row over plan to dna test chopin's heart on harming the dead luper s: posthumous harm chopin's heart may hold secret of his death socrates and temporal lobe epilepsy: a pathographic diagnosis , years later the religion of socrates proceedings of the boston area colloquium in ancient philosophy socrates and his daimonion. correspondence among the authors. in reason and religion in socratic philosophy socrates and religious experience a history of western philosophy the divine sign did not oppose me': a problem in plato's apology? problematizing retrospective diagnosis in the history of disease retrospective diagnosis and the use of historical texts for investigating disease in the past submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the author wishes to thank lynn jansen and david barnard for their helpful comments on an earlier version of this paper. i am also indebted to the anonymous reviewers of philosophy, ethics and humanities in medicine for their constructive criticisms and valuable suggestions, which greatly helped improve this essay. the author has no competing interest.author's information osamu muramoto, md, ma, dmedsci is senior scholar at the center for ethics in health care, oregon health & science university, and a board-certified neurologist (retired). key: cord- -j qmw zy authors: nan title: chapter the need for chemotherapy and prophylaxis against viral diseases date: - - journal: perspect med virol doi: . /s - ( ) - sha: doc_id: cord_uid: j qmw zy the chapter discusses the need for chemotherapy and prophylaxis against viral diseases. it briefly mentions clinical diseases and syndromes such as influenza, respiratory tract infections, hepatitis, and arbovirus infections resulting from virus infections. viruses causing respiratory diseases, as well as many other diseases in humans are also discussed in the chapter. it describes the vaccines that are used to check the attack of different viruses as well as their cost-effectiveness. there is a list of some viruses that have been ranked according to different variables in an attempt to select a good candidate for an antiviral drug. the incidence of the viral disease is naturally an important factor, as is the severity of the disease. the incidence can be obtained for diseases being reported in accordance with local regulations, but in many cases viral diseases are not reported and the incidence has to be calculated from different surveys. also, a grading of the severity is not easy and an example is when herpesvirus infections are handled as a group, which includes both herpes encephalitis and cold sores. the greatest challenges and probably the most difficult and medically important areas for prophylaxis and therapy of viral diseases are the viruses that are rapidly changing in antigenic composition and/or viruses with animal reservoirs (influenza and arboviruses) and those forming latent infections (herpesviruses). the need for chemotherapy and prophylaxis against viral diseases marks the centenary of the first use of one of the most effective viral vaccines (rabies, pasteur, ) as well as being the famous milestone of fictional political forecasting (orwell, ) . this year period has witnessed the industrialization of countries throughout the world and, along with this social and economic development, marked changes in patterns of infectious disease both accompanying and in some cases caused by the population and social changes. excellent examples of these medical changes are shown in figs. . - . , where mortality in different age groups is compared for the two years, and in germany for infectious diseases, heart and circulation disease and neoplastic disease, respectively. a dramatic drop in mortality at all ages, but particularly in children, from infectious disease is noted between and , whereas, conversely, an increase in mortality in older persons (but not children) is noted which is caused by heart and circulation diseases and neoplasms. disease patterns are still changing today and new viral diseases are discovered regularly (witness the fevers of marburg and lassa in the last decade, and, more recently, the different viruses associated with aids). indeed there is every reason to assume that continual change in infectious diseases is to be expected in the future. even the arrival of potent antiviral compounds may not eradicate diseases such as herpes, but rather may alter the pathogenic process of the virus itself as strong selective pressures are brought to bear. a similar phenomenon has been happening with bacteria such as staphylococci or streptococci as, over the years, strong selective pressures exerted by antibiotics have led to the emergence of organisms with reduced or altered pathogenicity. in the case of malaria, wide- spread use of antimalarials on the one hand has led to a gradual and threatening build up of drug resistant strains of parasite and also the use of ddt to destroy the mosquito has led to development of resistance in the vector. therefore both eukaryotes and prokaryotes possess a dramatic ability to adapt to a changed environment, and viruses are no exception. as bacteriologists before us have done, we should ask ourselves which viral dis- eases are important causes of mortality and morbidity, and, amongst these which would be the most suitable for ultimate control or even eradication. in fact, the question is more complex than this because in different parts of the world different viral diseases are important and even the same virus (such as measles) may cause very different disease syndromes. whilst in most major european countries respiratory viruses are an important cause of morbidity and mortality, in third world countries measles, polio and diarrhoea diseases predominate in this respect. a potential for distorted programmes of control of viral disease may easily occur, with most research centred on developed countries and therefore oriented towards viral disease in those countries. an excellent example is in the area of antiviral chemotherapy. at the present time most research effort is undoubtedly in the area of herpes infections, particularly hsv- causing genital infections, and this is closely followed by the search for new compounds against influenza a virus and rhinoviruses. also, although influenza a is a pandemic virus causing mortality and morbidity throughout the world the same cannot be said for herpes viruses. although infections with herpes viruses are universal, the impact of these nevertheless pale into insignificance beside medical problems with measles, arboviruses and hepatitis a and b in third world countries. so the direction of research effort needs continuous reassessment but, more importantly, some international perspective and direction. the world health organisation has provided this perspective for the eradication of smallpox from the whole world and continues to provide direction with its integrated 'health for all by the year ' programme ( fig. . ) which calls on member states to pursue a programme aimed at attainment by all people of the world, of a level of health that will permit them to live a socially and economically produc- . piped water reaches a small community in the soloman islands: satisfying needs for water calls for a complex interlocking planning and action at many levels. c. inequality in the great cities of the world. (from 'world health', who publication.) tive life. the programme includes sections on the control of certain viral diseases causing respiratory infection and diarrhoea diseases. the who conference at alma-ata in the ussr in stressed the importance of primary health care: "essential health care must be based on practical scientifically sound and socially acceptable methods and technology made universally accessible to individuals and families in the community through their full participation and at a cost that the community and the country can afford to maintain at every stage of their develop-ment in the spirit of self reliance and self determination". this programme does not, at present, envisage eradication of any particular viral disease, as smallpox was eradicated, because this may not gain the necessary full support of all countries. individual countries might, and are, considering 'national' eradication of certain virus infections such as measles in the usa, cuba, bahamas, canada and barbados where mortality rates of less than measles death per population have now been achieved. polio is another example of a viral disease which can be eradicated nationally, but of course a 'national' eradication always means that re-introduction from outside can constantly occur. table . illustrates illness and death caused by infectious diseases (viruses, bacteria and parasites) in africa, asia and latin america. viruses are the single chief cause of mortality, amounting to many millions of persons per year. among these virus infections the worst culprits are respiratory viruses such as influenza, and measles and rotaviruses. although an estimated - million persons die of enteric infections in these three continents each year, viruses will cause only a proportion of these cases and a similar situation will be true for respiratory illness. we shall examine the methods developed for the prevention of measles, influenza, polio and rotaviruses later on (chapters , , and , respectively) but it may be mentioned here that live polio vaccines used so successfully in industrialized countries are much more difficult to apply successfully in third world countries where problems of vaccine administration, heat lability control and viral interference become very important. measles programmes are being initiated now in certain third world countries (reviewed by mckenzie, ) , but vaccines against diarrhoea diseases are in their infancy. research has been orientated towards the development of successful inactivated or live vaccines or chemoprophylactic agents against influenza for the last years with only limited success and even less success against other respiratory viruses such as respiratory syncytial virus and parainfluenza viruses. little emphasis is given in table . to mortality from arboviruses other than dengue, and from hepatitis a and b viruses. a successful yellow fever vaccine was developed in the s but is not used for mass immunization but rather for containment, whereas the development of vaccines or chemoprophylactic agents against hepatitis is only beginning and so far has only resulted in vaccines and drugs with a very limited use on a large scale because of the expense involved. rabies vaccine, on the other hand, and the topic of our introductory sentence has been a success story in developed and underdeveloped countries. cheap and effective rabies vaccines have been produced locally, with the help of the network of pasteur institutes throughout the world, to contain this terrifying disease (chapter ). in general therefore we can conclude that certain viral diseases are 'shared' between developed and underdeveloped countries (such as influenza, measles, polio and rabies) and research and technical developments in industrialized nations will hopefully be applied worldwide. an important qualification here is that new vaccines and antivirals should not be too expensive and, unfortunately, recent rabies vaccines (chapter ) and hepatitis vaccines (chapter ) are extremely costly. live attenuated polio and measles vaccines are produced very cheaply and with more attention to their delivery (chapters and ) can and should result in a dramatic decrease of polio and measles in these countries in the next decade. as we have emphasized above, deaths from infectious viral disease in industrialized nations and also in those third world countries with well organized social infrastructure (such as china in the last years) have been steadily dropping over the last years, although there is still a considerable scope for further reductions in many countries. table . shows death rates from infectious diseases in countries with 'fully developed health services' compared to those without these facilities. table . further emphasizes marked differences in mortality caused by acute respiratory infection (bacterial and viral) in children between a third world continent (africa), the usa and europe. mortality is overall times higher in africa than in the usa but nearly times higher in the - year olds. also interesting is the two fold higher mortality in children in europe compared to the usa, at least at the beginning of the s. to put mortality from infectious diseases into some perspective table . shows mortality in the usa in june from all causes. deaths from pneumonia and influenza, for example, list seventh in such an analysis. the incidence of viral dis- in summary, therefore, in the industrialized and more wealthy nations of the world infectious diseases including viral diseases play a much reduced role in causing mortality but amongst the important viruses in this respect are particularly respiratory viruses such as influenza a and b, and respiratory syncytial virus. pandemic influenza a virus, in addition, causes considerable morbidity and economic disruption. we have briefly mentioned above clinical diseases and syndromes such as influenza, respiratory tract infections, hepatitis, and arbovirus infections resulting from virus infections. but many of these disease syndromes may be caused by a multitude of viruses, making specific diagnosis without the aid of a laboratory impossible. most antiviral agents discovered to date have a very restricted range of antiviral activity and so to use these in the field it will be necessary to identify particular viruses causing a clinical syndrome rapidly and precisely. partly for this reason, methods of rapid virus diagnosis have been investigated rather intensively during the last few years and the advent of monoclonal antibodies, for example, should hasten this process. meanwhile it will be useful here to briefly summarize the range of viruses responsible for these differing clinical syndromes, and finally to list the major viruses of man in a more logical 'scientific' manner, so that the reader will not end up completely confused! a very wide range of viruses ranging from the rna-containing pleomorphic influenza viruses to the dna icosahedral adenoviruses cause respiratory infection which may be completely indistinguishable by a clinician. nevertheless some general observations are a useful guide, including the facts that rhinoviruses, echo viruses, reoviruses and coronaviruses rarely, if ever, cause lower respiratory tract infection but confine their attention to the upper respiratory tract and hence may often produce only mild respiratory illness (table . ). cytomegalovirus and varicella zoster, on the other hand, tend to produce only lower respiratory tract infections whereas influenza, measles, parainfluenza, rsv and adenovirus produce both. finally, most upper respiratory tract diseaseall ages (but not all) clinical cases of influenza are caused by influenza a and b viruses, whilst most cases of croup in infants are caused by rsv ( fig. . in chapter ). similarly, a wide range of viruses have a predilection for nervous tissue (table . ) . some viruses such as rabies, polio and certain arboviruses target upon nerve cells, but in the case of most viruses central nervous system (cns) effects result as an aftermath and complication of viral replication (e.g. mumps, herpes, rubella). certain of these disease syndromes in the cns such as rabies can be identified by clinical examination of the patient, but even polio-like paralysis can be caused by other enteroviruses (chapter ). certainly encephalitis following rash diseases such as rubella, mumps and measles presents few problems of diagnosis unless the person has been infected without a rash. " types - , and . "jc and sv -pmlviruses. types a , , -- , , , , and b - . certain rashes in their typical form can be easily diagnosed as caused by the viruses of chickenpox (varicella zoster), measles or rubella (table . ). however, diagnosis of rashes caused by certain of the enteroviruses such as echo or coxsackie a may be easily misdiagnosed as rubella. this would not be particularly important were it not for the propensity of rubella to cause foetal infections and abnormalities (congenital rubella syndrome). table . lists viruses which have been implicated in causing foetal abnormalities, abortion or postnatal infections. undoubtedly the most significant and dramatic effects on the foetus are caused by rubella virus and the many and varied deleterious effects on the developing embryo have been encompassed as the 'expanded rubella syndrome' (table . ). infection of the mother in the first week of pregnancy may result in infection and sequelae in - % of the foetuses (chapter ). herpes viruses (via infection of the cervix of the mother) may infect the foetus during delivery, whereas cytomegalovirus and vaccinia can infect the foetus in utero in the final trimester of pregnancy. increased foetal deaths or abnormalities have been noted following infection of the mother with other viruses including polio, arboviruses, measles and influenza but these effects on the foetus are more likely to be caused via constitutional upset in the mother rather than by actual infection of the foetus or foetal tissue by the virus itself. most of the above mentioned viruses cause acute infection in humans following transmission from another person or from a vector such as a mosquito or other insects. but certain viruses can afterwards establish a latent or persistent infection occurs in the presence of high levels of neutralizing antibody such vaccines may have little or no effect, unless they stimulate cell mediated immunity (chapter ). similarly, antivirals are unlikely to eliminate latent herpes viruses from the ganglia and would merely hasten the healing of a particular reactivation episode. herpes simplex venereal infections are of considerable interest to the general public, virologists and antiviral chemotherapists and data from the usa appear to suggest infections of epidemic proportions (see chapter ). data from the uk show a steady increase in hsv- genital infections but certainly not in epidemic proportions. indeed it is most useful to place this virus in a context of other venereal infections. recently published data from the uk would suggest that non-specific genital infections are increasing in incidence at least as rapidly as hsv infections. although the aetiology of non-specific genital infection is still not fully elucidated, chlamydia traclzomatis is recognized to be the commonest cause in britain, and isolation rates from the cervix of unselected women attending sexually transmitted disease clinics in britain may reach up to %. total new attendances at special (venereal disease) clinics in the united kingdom rose by . x in compared with , continuing the increase noted each year since the early s. this is less than the previous annual increase of % (which, however, was unusually large). the overall picture of sexually transmitted disease in british clinics in the past years is one of the increasing importance of new cases requiring treatment in categories other than syphilis or gonorrhoea, which now account for only % of total cases requiring treatment. the largest absolute increase in new attendances by diagnostic category in , apart from 'other conditions requiring treatment', was in non-specific genital infection: there were new attendances, an increase of ( . %) over those in , but this was about half the previous annual rise of %. there were rises in most other diagnostic categories. the number of new attendances due to herpes simplex infection increased by ( . %), those due to warts by ( . %) and those due to candidiasis by ( %). other viruses including cytomegalovirus and hepatitis b virus are transmitted in semen from infected persons, but the general significance of this is not clear. htlv-i has been implicated as a possible cause of acquired immunodeficiency syndrome (aids) in promiscuous homosexuals (see chapter ) and presumably transmission via person to person in semen might be a major factor. table . and fig. . list, rather exhaustively (but not completely), a classification of viruses causing human disease, and table . briefly summarizes information on currently used vaccines and chemoprophylactic agents against viral diseases. the epidemiology, strategy of replication and physical and antigenic structure of these viruses will be discussed as fully as possible in the ensuing chapters. however, a word of warning should be introduced here. dna technology ('genetic engineering') techniques are being introduced very rapidly indeed and are expected to revolutionize the previously used biological approaches to development of new viral vaccines. the reader can safely assume that for most viruses discussed in the following chapters, even if it is not indicated in the text, that someone is cloning the particular gene into a eukaryote or prokaryote cell. it cannot be overemphasized that with both cew vaccines and antivirals the initial discovery is often made by individuals and single groups. only during later developments are the large teams of scientists required. also, with antivirals, a new era has arrived which is seeing the first extended use in the clinic of inhibitory molecules against viral diseases, particularly herpes. so we shall undoubtedly see a plethora of new molecules each with certain biological and pharmacological advantages compared to the parent. indeed, we are witnessing this trend already with molecular derivatives of acyclovir such as dhpg and dhbg (chapter i). we have tried, in the ensuing chapters to present the reader with a review of the basic scientific knowledge and principles underlying development of vaccines and antivirals. new data should simply enhance interest in the topic and perhaps even encourage a reader to develop a vaccine or antiviral him or herself! the impact of viral diseases on society can, to some extent, be expressed in economic terms and this again illustrates the magnitude of the infectious viral disease problem. in the usa, during the period - , the annual mortality from influenza was deaths, the annual cost of treatment $ million and the annual loss of productivity $ million. the cost of less severe respiratory viral diseases, such as common cold occurring on the average more than twice yearly, is probably of the same magnitude. the distress and pain caused by recurrent labial and genital herpes infections is very large, and possibly increasing. the number of patients is difficult to express in economic terms, but an estimated number of americans are contracting genital herpes each year and approximately million episodes of labial herpes will affect the usa population each year. the impact of gastrointestinal syndromes caused by viral infection can be illustrated by an annual mortality of - million due to rotavirus infections in children in asia, africa and south america many chronic conditions are initiated in infectious diseases. hepatitis b may result in chronic infection in % of the cases, leading to chronic cirrhosis. an esti- for some communicable diseases such as rabies, although the rate of morbidity is small or non-existent, nevertheless substantial costs are incurred in surveillance, prevention and health education. also the costs for rabies extend into the agricultural sector of the community. with all communicable diseases allowance must be made for indirect costs such as production losses in the economy and these may sometimes amount to twice as much as the health service costs. indirect costs again may spread over a number of sectors of the economy, including infected animal stock or food products etc. to date very few detailed studies have been undertaken to establish the cost and none mononucleosis effectiveness of programmes for the prevention and treatment of viral diseases. moreover, these have been concerned with vaccines and no work has been published as regards specific antivirals. however, the estimated cost to develop an antiviral agent is $ - million which, in relation to common diseases such as influenza and herpes, is economically acceptable but with less frequent viral diseases can be a problem for a private company. such data as there are suggest that improved strategies of prevention of viral disease could make substantial savings and result in better health outcomes. some viral diseases such as measles and polio maintain high incidence levels unless immunization levels are constantly maintained, whereas others can be self eliminated when a threshold level of infection is reached. where the incidence of a disease changes over time, established forms of treatment may lose their justification and should be phased out or altered. the patterns of some viral diseases have changed due to medical intervention in another sphere and an example is the improvement in treatment of cancers and immunosuppression of transplant patients. this has resulted in an increasingly common situation where a successful, and often very expensive, treatment of a disease is threatened by opportunistic viral infections, mainly by latent herpes viruses. the cost of developing antiviral agents against these types of infections should be considered in the context of the total cost to manage these patients and the risk of an infection. an informal weighing of risks and benefits of immunization (e.g. for smallpox) has been carried out in some societies, like the uk, for hundreds of years. however, it is now possible to apply more precise scientific analysis to the problem. such a scientific analysis of immunization benefits in the early s led to a major alteration in national health policy in the usanamely the decision that routine smallpox vaccination should be discontinued. the last case of variola minor in the usa was in and by the risk of death from all smallpox vaccinations was per million for primary vaccinees, rising to per million for children under year of age. in addition, among primary vaccinees the combined rate of post vaccinia encephalitis and vaccinia necrosum was . per million for infants. on the other hand, the probability of a smallpox importation into the usa in was importation every years. it would probably have required smallpox importations per year to produce the same mortality which was then associated with smallpox vaccination. this is an excellent example of the direct usefulness of statistics. in the united states alone more than types of vaccines, both bacterial and viral are used and one may question how worthwhile some of these vaccines are in economic terms. cost effectiveness analysis and cost benefit analysis aggregate the net medical care costs and net health benefits from a vaccination programme and thus help to give an economic analysis. net medical care costs often include the cost of vaccine and its administration, cost of treating vaccine complications and the medical care savings due to prevention of disease. net health benefits include reduction in morbidity and mortality. also one may include the gains in productivity resulting from a reduction of absence at work. other qualitative and more difficult to cost-estimate considerations must be included to place a vaccine programme in an accurate social and medical perspective e.g. pain and anguish of illness, compensation of victims of severe vaccine reactions etc. these days, quite necessarily so, political, social, economic and medical factors are all taken into account (or at least should be by national health authorities). it is an interesting and useful exercise to see how these analyses apply to popular and seemingly useful viral vaccines (willems and sanders, ) . the salient features of this analysis which mainly refers to experience in the usa (and therefore may differ in details in european countries, for example) are presented in table . . the crucial issue for a vaccine strategy against rubella is the appropriate age of immunization. prevention of congenital rubella syndrome is the aim of this programme, because rubella itself is a mild disease scarcely worth considering if it were not for the teratogenic properties of the virus. the teratogenic effect of the virus is unique in its selectivity of action and most embryos of mothers infected during the first trimester would be affected. as an example of the community effects in the - epidemic before vaccine was introduced, therapeutic abortions were carried out and children were born with rubella syndrome in the usa alone. also excess prenatal deaths were associated with the epidemic. of the children born with congenital rubella, were deaf, deaf and blind and several thousand suffered moderate to severe mental retardation. estimated direct costs of the epidemic in the usa were $i billion, mainly ( %) as regards long term care associated with rubella syndrome. in the usa after , when vaccine became widely available, the strategy was to immunize a// children as a routine, whereas adolescents and women of childbearing age were immunized selectively. in the uk, on the other hand, mainly school girls (not boys) were immunized. the basic strategy in the usa was to displace wild virulent rubella by attenuated non-teratogenic vaccine virus. a reduced objective was aimed for in the uk and the programme to date has not been so successful. in the usa, rubella epidemics have been prevented and cases of rubella have decreased continuously in the under year age group. most cases of rubella now occur among adolescents. a study of costs and benefits found positive net benefits. in the direct cost of acute rubella for million persons was estimated at $ . million and the costs of congenital rubella syndrome in the offspring of million unprotected females was $ . million. the benefit cost ratio for vaccine to million females at years of age was : , assuming % immunization of the target population. since mumps vaccine was licensed in in the usa more than million doses h, p have been used and the incidence of mumps has dropped from rates of - per population to - per i in the late s. it may be calculated that the use of mumps vaccine for a group of a million persons would prevent cases of mumps and deaths. over a year period mumps vaccine would reduce costs by over % and the benefit cost ratio approximates to : : i . mumps vaccine in austria has a benefit cost ratio of . : and in switzerland . : (data not shown in the table) . comparable analyses appear to show that influenza vaccine is less effective in economic terms than rubella or measles vaccines but very different considerations pertain. the vaccine is recommended for special risk groups and not particularly for children. medical care costs of vaccination during - totalled $ million and l so million persons were immunized in the usa, giving a cost of $ per year of healthy life gained. moreover, the cost effectiveness of vaccination improves with increasing age of the person vaccinated (because the highest mortality occcurs in the over year age groups) so that it costs $ per vaccination for each year of healthy life gained in the under age group, to $ per vaccination in the - age group, to positive cost savings in the + age group. assuming influenza vaccine is % effective, vaccinating a million elderly persons would result in additional years of life at a net cost of $ per year of life gained. most people would agree that this is a reasonable use of medical resources. four million cases of measles occurred in the usa each year before vaccine was introduced in , with cases of encephalitis and - deaths each year. net benefits of measles immunization between and include savings of . million hospital days, million school days and cases of mental retardation. the benefit cost ratio was approximately o:i. in finland, as an example of a european country, the benefit cost ratio was . :l. however, as measles declines, marginal reductions in incidence will become increasingly costly. measles will probably be eliminated as an endemic disease in the usa during . an excellent recent example of the application of cost effectiveness of viral vaccines is being carried out in several countries at the present moment with the advent of an effective vaccine against hepatitis b (hbv) virus (szmuness et al., ) . the vaccine is rather costly because of the technical problems of purifying and inactivating antigen from human sera ($ per course of doses of vaccine) and, more important, its availability may be limited in the near future. therefore 'decision analysis' can be used to estimate likely costs and benefits of different immunization approaches in different populations at risk. a decision analysis model was constructed to compare alternatives for prevention, which were: a. immunizing all persons with no prior screening for indication of previous infec t io n b. screening all persons for indications of previous infection and then immunizing only those sero or antigen negative persons c. passive immunization of persons exposed to hbv. the estimated cost per person of hepatitis vaccination in a year period in a homosexual population with a % prevalence of hbv markers and % annual attack rate without screening and vaccinations is $ . . vaccination of all persons in this group would result in a lowering of hbv incidence from to %, costing $ . per person. however, screening followed by immunization would cost only $ . (because fewer people would be immunized). in contrast, for a group of hospital employees with relatively high exposure ( . %) and annual attack rate of %, vaccination without screening is the lowest cost strategy ($ . ). for a low risk population ( . % annual attack rate) neither vaccination nor screening followed by vaccination would result in a saving in medical care costs. indeed the net medical costs per case of hepatitis prevenfed by vaccinating the latter population would be $ . in contrast, net medical care costs per case prevented are negative when the attack rate is greater than . %. a very important caveat of the above discussion, however, is the fact that indirect costs saved by immunization, such as loss of productivity etc, are not included. if one is only interested in medical care savings then hbv vaccination should be carried out before or early during a period of unavoidable high risk as with surgical registrars, new dialysis unit patients and staff members, new prisoners, newly institutionalized mentally retarded patients, and promiscuous homosexuals. we should note that in the example given above, which is rather usa-orientated, extrapolation to conditions in europe may be questionable. in fact an excellent example of these differences has been highlighted with the current discussion about hepatitis b vaccine. for example, in greece, the prevalence of anti-hbv among health workers is - % and in medical and nursing students it was and %, respectively. screening costs around $ , whereas vaccine costs approximately $ and so it seems reasonable to vaccinate health care workers after screening, and medical and nursing students without screening. in the uk the problem of finance is very relevant because additional funds may not be made available to health authorities and so again, screening may be resorted to as a cost saving excercise. the development of an antiviral drug is a major undertaking and will require, at least for a private company, that the market for a drug is large enough to correspond to the cost and risk of development. table .i lists some viruses which have been ranked according to different variables in an attempt to select a good candidate for an antiviral drug. the incidence of the virus disease is naturally an important factor, as is the severity of the disease. the incidence can be obtained for diseases being reported in accordance with local regulations, but in many cases viral diseases are not reported and the incidence has to be calculated from different surveys. also a grading of the severity is not easy and an example is when herpesvirus infections are handled as a group, which would include both herpes encephalitis and cold sores. therefore, a rather subjective average has been used in table . . an additional important factor in deciding the targets for antiviral chemotherapy is the absence or availability of good viral vaccines and the probability of developing vaccines in the future. for viruses like rhino and influenza with many serotypes or antigenic variants, vaccine production may always be a problem especially with influenza when new antigenic types appear rapidly. in the case of hsv-i and hsv- infections the development of a successful vaccine seems unlikely when one considers that patients with frequent episodes of labial or genital herpes have high titres of neutralizing antibodies and that reinfection can occur in spite of circulating antibodies. aspects of vaccines are discussed in more detail in chapter and in connection with the different viruses. rapid diagnosis (preferably by the patient!) is of importance since an antiviral drug is likely to have a narrow spectrum of activity and the virus to have a short time period of replication. it will be necessary to know exactly which virus is causing the infection and thus which drug should be used. the self-diagnosis of recurrent diseases such as labial and genital herpes is rather easy for the patient and, from that point of view, herpesvirus infections are good targets for antiviral drugs. major points of attack when developing new antiviral drugs are viral enzymes. herpesvirus enzymes are easily accessible and well characterized, as are also influenza virus enzymes, and this feature also makes these two viruses attractive as targets for antiviral inhibitors. the use of viral enzymes as targets for antiviral drugs is discussed in chapter . in essence, we conclude from the considerations in table . that herpes and influenza should be major goals for the development of new antiviral drugs. this is also reflected in the literature where anti-herpes compounds are the most flourishing area at present. rank list of candidate viruses for the development of antiviral drugs. the listing has been made to place the virus most favourable in the development of antiviral drugs on the top of each column. the added rank numbers will then give a crude estimation of the incentive to develop an antiviral drug against each virus. the lower the number the better the target. problems easy accessible incidence with vaccine diagnosis virus enzyme aalthough not included in the six target diseases, there is a relationship between this programme and the prevention and control of diseases such as yellow fever, hepatitis, rubella and mumps. epi, expanded immunization programme; cdd, campaign against diarrhoea disease; ari, acute respiratory infections. the great need for therapy and prophylaxis of viral diseases is obvious. in recent years we have seen a swift expansion in our knowledge of the biological and chemical processes involved in viral diseases. this has made possible rational efforts to manage and prevent viral diseases, and we are now seeing the results in new vaccines and antiviral agents. the greatest challenges and probably the most difficult and medically important areas for prophylaxis and therapy of viral diseases are those viruses which are rapidly changing in antigenic composition and/or viruses with animal reservoirs (influenza and arboviruses) and also those forming latent infections (herpesviruses). the three major international co-ordinating programmes of who (table . ) include both these 'challenge' viruses and also more common viruses such as measles and polio where particular help is required in developing countries. the relative role of vaccines and antiviral drugs is difficult to predict. in cases where cheap and effective vaccines exist or can be developed as exemplified for polio, measles and hepatitis b, this is likely to be the optimum control method. in cases such as influenza, the great variability of the virus probably poses insurmountable difficulties for vaccines, and makes the antiviral drug approach more promising. finally, for viruses such as the herpesviruses, causing recurrent infections (in spite of both preexisting humoral and cell mediated immunity) vaccines seem an unlikely approach and antiherpes drugs now appear to be more promising. approaches to immunization of infants and young children against gastroenteritis due to rotaviruses target diseases for future antiviral drugs pp viral diseases in south east asia and the western pacific classification and nomenclature of viruses nouvelle communication sur la rage passive active immunization new trends and developments in vaccines cost effectiveness and cost benefit analysis of vaccines key: cord- - pfk qve authors: kaneko, naoe; kurata, mie; yamamoto, toshihiro; morikawa, shinnosuke; masumoto, junya title: the role of interleukin- in general pathology date: - - journal: inflamm regen doi: . /s - - - sha: doc_id: cord_uid: pfk qve interleukin- , an inflammatory cytokine, is considered to have diverse physiological functions and pathological significances and play an important role in health and disease. in this decade, interleukin- family members have been expanding and evidence is accumulating that highlights the importance of interleukin- in linking innate immunity with a broad spectrum of diseases beyond inflammatory diseases. in this review, we look back on the definition of “inflammation” in traditional general pathology and discuss new insights into interleukin- in view of its history and the molecular bases of diseases, as well as current progress in therapeutics. in terms of general pathology, inflammation is one of the adaptive responses to various injuries including physical, chemical, and biological factors. the roman encyclopedist a. cornelius celsus described four cardinal signs of inflammation in one concise sentence: "now the signs of an inflammation are four: redness (rubour) and swelling (tumour), with heat (calour) and pain (dolour)" [ ] . a century and a half later, galen added a fifth sign: "disturbance of function" (funcio laesa) [ ] . the classical signs of inflammation are considered to be related to cells and tissues responding to pathological cell injury caused by internal stimuli, including damage-associated products and metabolites, and external stimuli, including bacteria and viruses [ ] [ ] [ ] [ ] . the host bears the receptors that facilitate recognition of these damage-associated molecular patterns (damps) and/ or pathogen-associated molecular patterns (pamps) that are not host-derived. these receptors are termed pattern recognition receptors (prrs) [ ] . prrs directly or indirectly detect infection and/or noxious chemicals, resulting in inflammation that is coupled with the induction of immune responses and a tissue reparative component [ ] . the signal transduction triggered by these prrs leads to the acute inflammatory mediator expressions that regulate the elimination of pathogens and infected cells [ , ] . there are several known prrs: toll-like receptors (tlrs), rig-i-like receptors (rlrs), nod-like receptors (nlrs), and c-type lectin receptors (clrs). the majority of nod-like receptors such as nlrp , nlrp , nlrc , nlrp , and nlrp can interact with apoptosis-associated speck-like protein containing a caspase-recruitment domain (asc) and caspase- , and the resulting complex is a sensor of cell injury called "inflammasome", an interleukin (il)- β-processing platform that plays a crucial role in il- β maturation and secretion from cells. other pyrin-domain (pyd)-containing proteins such as aim , ifi- , and pyrin are also known to form inflammasomes. among them, nlrp inflammasomes monitor membrane integrity and pore-forming toxins, crystals, and many other noxious stimuli and are involved in il- β processing and maturation [ ] [ ] [ ] [ ] . it is now widely accepted that an inflammatory response is the extreme end of a spectrum that ranges from a homeostatic state of inflammation to a stress response and finally inflammation [ , ] . the homeostatic state of inflammation, which is not inflammation from the perspective of general pathology, was suggested to be maintained by prrs expressed in stromal and/or immune cells, detecting endogenous ligands in parenchymal cells and/or pathogens, leading to chronic inflammatory responses ranging from the basal homeostatic state to disease-causing inflammation [ , ] . in addition to several forms of inflammation including classical inflammation, homeostatic inflammation, a distinct nomenclature for low-grade inflammation, such as para-inflammation (an adaptive response against stress or malfunction) and meta-inflammation (metabolically triggered inflammation), has been proposed [ ] [ ] [ ] . as discussed above, there are various factors involved in forms of inflammation; in particular, since il- is a downstream cytokine of the sensor of cell injury, the inflammasome, it is important for regulating inflammation and tissue damage beyond inflammation [ ] . il- is a master regulator of inflammation via controlling a variety of innate immune processes [ ] . from a historical point of view, il- has a wide range of biological functions, which include acting as a leukocytic pyrogen, a mediator of fever and a leukocytic endogenous mediator, and an inducer of several components of the acute-phase response and lymphocyte-activating factor (laf) [ , ] . laf was later shown to be a macrophage-derived immune mediator acting on t-and b-lymphocytes and was designated as il- in the second international lymphokine workshop held in switzerland in [ , ] . in addition, serum blocking factors in breast cancer patients identified by the leukocyte adherence inhibition test were reported. the serum adherence-promoting factors were regulated by il- [ ] [ ] [ ] . to date, the tumor microenvironment has been characterized by dominant immunosuppression, being infiltrated by tumor immunosuppressive myeloid-derived suppressor cells (mdscs), regulatory t cells (tregs), and tumor-associated macrophages (tams) [ , ] . il- is capable of inducing the recruitment of tams and mdscs, which promote tumor development in breast cancer [ ] . currently, human sequence algorithm technologies suggest that the il- family comprises a total of members with similar or distinct biological effects [ , ] . il- α, il- β, il- ra, il- , il- , il- α, il- β, il- γ, il- ra il- , and il- have been identified and characterized (table ) [ ] . among them, il- α, il- β, il- , il- , and il- are receptor-agonistic, and il- ra, il- ra, and il- are receptor-antagonistic. il- is the only anti-inflammatory cytokine. although the function of each il- family member is now being investigated, il- is the most characterized among these members. there are two individual forms of il- , il- α and il- β, isolated from two distinct cdnas, but they are indistinguishable in terms of their biological functions [ ] . although the homology between il- α and il- β is not high ( %) in terms of amino acid sequences, il- α and il- β are structurally similar and show the same functions by sharing a common receptor, il- type receptor (il- r ), and both have the same central β-barrel along with adjoining loops [ , ] . the difference between il- α and il- β is an n-terminal extension of residues beyond the n-terminus of il- α and il- β [ ] . the molecular weight of each precursor is approximately kda, and il- α and il- β are processed by specific proteases to mature forms. the n-terminal domain of il- α contains a nuclear localization sequence (nls) and shows transcription activity [ ] . il- α is produced as a -amino acid (aa) precursor protein. for transcription of the il- α gene, transcription factor specificity protein (sp ) activates the il- α promoter activity in the ′-upstream gc box (− to − bp) [ ] and nf-κb, which is also activated by il- α itself, and stimulates the consensus promoter region (− to − bp) to induce its own gene expression and production in an autocrine manner [ ] . the precursor of il- α translocates into the nucleus to bind to chromatin and also exists in a membrane-anchored form. upon stress responses, il- α is processed by ca + -dependent protease calpain or other proteases, such as cytotoxic t-lymphocytes (ctl)/natural killer (nk)-granzyme-b, mast cell chymase, or neutrophil elastase to the c-terminal aa as mature il- α [ ] . the il- α processing separates nls from its precursor, which is not linked to secretion or cell death [ ] ; however, il- α is a key danger signal that induces inflammation on release from necrotic cells [ ] . the il- α precursor triggers il- r on resident macrophages in necrotic tissues, producing il- β as well as chemokines as post-necrotic inflammation [ ] . il- β is produced as a -aa precursor protein and processed by caspase- , which is also known as il- β-converting enzyme (ice), activated in inflammasomes, to the c-terminal aa as mature il- β [ , , , ] . the il- β precursor is also processed by other serine proteases [ ] . neutrophils derived from caspase- -deficient mice release mature il- β processed by elastase in response to lipopolysaccharide (lps) stimulation [ ] . the neutrophil proteases, such as elastase, chymases, granzyme a, cathepsin g, and proteinase- , cleave the il- β precursor into a secreted, biologically active form [ ] [ ] [ ] . these alternatively cleaved forms of functional il- β were detected in synovial fluid of a patient with inflammatory polyarthritis and gout [ ] . occasionally, massive neutrophil infiltration appeared in excess-inflammation-damaged tissues and organs, such as in septic shock or systemic inflammatory response syndrome. thus, the nlrp inflammasome-related inflammation induced by a variety of factors described above may be a target of anti-il- therapy [ ] . currently, a two-step model of the initiation of nlrp inflammasome activation is suggested. the first step mediates transcriptional and post-translational priming of nlrp (step ), and the second step is activation of inflammasomes (step ). step is the first synthesis of a biologically inactive il- β precursor by nf-κb binding to the consensus binding site (− to − bp) to transcribe the il- β gene. step is processing into mature, biologically active il- β by caspase- activated by a cytosolic activation platform called inflammasome [ , ] . the inflammasome is a large protein complex, which consists of prrs, such as nlrs, aim , rig-i or pyrin, an adaptor protein asc, and caspase- . among them, the nlrp inflammasome is a prototype inflammasome, which has been reported to be activated by a wide range of pamps and damps [ , ] . various nlrp -activating pamps have been reported, i.e., bacteria-derived rna or dna, pore-forming toxins, lethal toxins, flagellin/rod proteins, muramyl dipeptide (mdp), m protein, virus-derived rna or dna, fungus-derived β-glucans, hypha mannan, zymosan, and protozoon-derived hemozoin [ ] . nlrp -activating damps have also been reported, i.e., self-derived glucose, β-amyloid, hyaluronan, atp, cholesterol crystals, monosodium urate (msu) crystals, calcium pyrophosphate dihydrate (cppd) crystals, environment-derived alum, asbestos, silica, alloy particles, uv radiation, and skin irritants [ ] ; however, their diverse physiological and chemical signals leading to the direct activation of nlrp have not been fully elucidated. instead, efflux of potassium has been identified as the common activator of most known nlrp step signals [ , ] . the nlrp activation by potassium efflux suggested to lead nlrp -nek interaction to drive inflammasome activation [ ] [ ] [ ] . the mechanism underlying the secretion of il- β has been suggested to overlap with il- α secretion [ ] . also, mitochondrial and phagosomal reactive oxygen species (ros) have been proposed to activate the nlrp inflammasome. alternatively, non-canonical pathways of nlrp -inflammasome activation associated with proinflammatory caspases, caspase- , caspase- , and caspase- have been proposed. in this process, lps is recognized by the caspase-recruitment domain (card) of respective caspases, leading to activation [ ] [ ] [ ] [ ] . caspase- or proteases in neutrophils are also processed and activate il- β. several prrs, such as nlrp , nlrp , nlrc , pyrin, and aim , convert the assembly of the adaptor molecule asc into a high-molecular-weight complex, called the pyroptosome [ ] . then, the caspase- precursor is recruited to the pyroptosome to also form helical structures, which enable its proximity-induced proteolytical auto-activation. with caspase- precursor maturation into the active p /p heterotetramer, it cleaves the il- β precursor, leading to pyroptotic cell death. this cell death is mediated by the caspase- -dependent cleavage of gasdermin-d (gsdmd) [ ] [ ] [ ] . in turn, the mature n-terminal fragment of gsdmd translocates to the inner leaflet of the plasma membrane to form round and pore-like structures of approximately nm in diameter [ ] [ ] [ ] [ ] . tissue distributions of interleukin- il- α and il- β are expressed in a wide range of tissues and a variety of cells, especially in macrophages in lymphoid organs including the thymus, spleen, lymph nodes, peyer's patches, and bone marrow. in non-lymphoid organs, il- α and il- β are expressed in tissue macrophages in the lung, digestive tract, and liver. they are also expressed in cellular subepithelial endometrial tissue of the uterus, in the glomeruli, in outer cortical areas of the kidney, and in various specific cell types, including neutrophils, keratinocytes, epithelial and endothelial cells, lymphocytes, smooth muscle cells, and fibroblasts [ , ] . there are two cell surface il- receptors, il- r and il- type receptor (il- r ), a decoy receptor. il- binds to il- r , which requires the formation of a heterodimer with the il- type receptor (il- r ) (also known as il- racp) accompanied by adaptor il- receptor-associated kinase (irak) and myeloid differentiation primary response protein (myd ) [ ] . il- r initiates inflammatory responses when binding to the ligands il- α and il- β and has been reported to be expressed by tlymphocytes, fibroblasts, epithelial cells, and endothelial cells. il- r , which does not initiate signal transduction, is expressed in a variety of hematopoietic cells, especially in b-lymphocytes, mononuclear phagocytes, polymorphonuclear leukocytes, and bone marrow cells. notably, expression levels of il- r and il- r are different among the cell types; for example, neutrophils predominantly express il- r . as a result, much higher concentrations of il- β are required to activate neutrophils, whereas low concentrations of il- β are sufficient to activate endothelial cells. the il- r -mediated signaling pathways also differ according to the cell types [ , ] . il- r is a co-receptor for il- r , responsible for signaling after binding ligands il- α and il- β, and has been reported to be ubiquitously expressed by all cells responsive to il- . il- r b is a brain-specific isoform of il- r generated by alternative splicing, and it has been reported to be expressed in the brain, cerebellum, and spinal cord [ ] . activated il- is incapable of functioning until recognized by cell surface receptors. the complex contains a motif of gtpase activity and activates gtpase-activating protein and protein kinases [ ] [ ] [ ] . in contrast, il- r is thought to reduce the biological response to il- . the proximity of the two cytoplasmic domains of il- r and il- r is thought to initiate signal transduction by the hydrolysis of gtp. this is followed by c-jun n-terminal kinase (jnk) and p map kinase [ ] . irak and tumor necrosis factor (tnf) receptor-associated factor (traf) activate nf-κb, as well as p , jnks, extracellular signal-regulated kinases (erks), and mitogen-activated protein kinases (mapks) [ ] . the nf-κb activation pathway is dependent on the iκ-b kinase (ikk) complex, composed of ikkα, ikkβ, and nf-κb essential modulator (nemo), via associations with tak , tak , traf , and traf in the il- r -signaling pathway [ ] . these signals play important roles in both acute and chronic inflammation in various diseases [ ] . single nucleotide mutation of the cias gene results in nlrp mutation, which induces constituted inflammasome activation causing cryopyrin-associated periodic syndrome (caps). this is termed autoinflammatory disease, including familial cold autoinflammatory syndrome (fcas), muckle-wells syndrome (mws), and neonatal-onset multisystem inflammatory disease (nomid)/chronic infantile neurologic, cutaneous, and arthritis (cinca) syndrome, which leads to greater il- β secretion without any damps or pamps [ ] [ ] [ ] [ ] [ ] [ ] . the most common autoinflammatory disease is familial mediterranean fever (fmf). fmf is autosomal recessive; however, mutations in the causative mefv gene, encoding mutated pyrin, leads to active pyrin inflammasome [ ] . inflammatory diseases like those above, characterized by the enhanced secretion of il- β, include a group of autoinflammatory diseases such as nlrp autoinflammatory syndrome; hyperimmunoglobulinemia d and periodic fever syndrome (hids)/mevalonate kinase deficiency (mkd); pyogenic arthritis, pyoderma gangrenosum, and acne (papa) syndrome; pyoderma gangrenosum, acne, and suppurative hidradenitis (pash) syndrome; pyogenic arthritis, acne, pyoderma gangrenosum, and suppurative hidradenitis (papash); majeed syndrome; and tnf-receptor- -associated syndrome (traps) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . on deficiency of the il- -receptor antagonist (dira), excess il- β induces other proinflammatory cytokines and chemokines [ ] . excess stress responses disrupt body homeostasis under physiological conditions and lead to excess cytokine production. nlrp inflammasomes have also been reported to be involved in low-grade subclinical inflammation induced by chronic exposure to high levels of free fatty acids and glucose, leading to increased apoptosis and impaired insulin secretion of β-cells in obese type diabetes mellitus (t d) patients [ ] [ ] [ ] . indeed, islet amyloid polypeptide (iapp) oligomers activated nlrp inflammasomes to induce significant il- β production by infiltrating macrophages in an in vivo study [ , ] . higher concentrations of glucose activate nf-κb and il- precursors in cells [ ] . minimally oxidized low-density lipoproteins stimulate tlr , which triggers il- β expression [ , ] , and accumulations of islet amyloid polypeptides are deposited and mediate nlrp inflammasome activation in islet macrophages [ ] . another oligomer of amyloid, amyloid β, can induce il- β via nlrp inflammasomes in a process involving the phagocytosis of amyloid β in glial cells in patients with alzheimer's disease (ad) and subsequent lysosomal damage and release of cathepsin b [ ] . ros are considered to be involved in the activation of nlrp inflammasomes, and it was suggested that direct interaction between amyloidogenic peptide and nlrp could initiate nlrp inflammasome formation in a cell-free system [ , ] . both il- α and il- β gene polymorphisms have been reported to be associated with central obesity and metabolic syndrome in a population with coronary heart disease in an epidemiologic study [ ] . thus, these diseases are il- dependent cytokinopathies (interleukinoneopathies). besides the above diseases, numerous inflammatory diseases related to excess il- signaling have also been identified [ ] [ ] [ ] . for example, high il- β levels in humans and mice result in increased th -dominant immunopathology, and il- β expression was limited to macrophages and neutrophils, which account for a large proportion of the cd α cells in the cervix upon chlamydia muridarum infection [ ] . consequently, il- β promotes the differentiation of monocytes into conventional dendritic cells (dcs) and m -like macrophages and supports the proliferation of activated b-lymphocytes and their differentiation into plasma cells [ ] [ ] [ ] . il- in combination with il- promoted not only the expansion of nk cells but also cd + cd + t-lymphocytes [ ] . il- β generated by activated antigen-presenting cells (apcs) induced type immune responses, which produced ctl and led to the polarization of cd + t -lymphocytes towards t-helper cell type (th ) [ , ] . il- β plays a role in resolving acute inflammation resulting in the initiation of adaptive anti-tumor responses; however, chronic inflammatory conditions increase the risk of developing cancer [ ] . in human breast cancer, higher expression of il- β is associated with tumor invasiveness and aggressive tumor biology [ ] . expression of il- α, il- β, and their receptors in human breast cancer tissues results in the activation of a population of cells and subsequently contributes to angiogenesis, tumor proliferation, and tumor invasion in the microenvironment [ ] . in a spontaneous mmtv-pymt mouse mammary gland tumor model, mature il- β levels in primary mammary tumors and metastasis sites were significantly elevated, being associated with inflammasome activation and the infiltration of myeloid cells in tumor microenvironments. in this model, cd b + gr + and cd b + gr − myeloid cell populations were also significantly increased in both tumor tissues [ ] . il- β generated in a tissue with a tumor microenvironment dominated by tams promotes tumor growth and metastasis in breast cancer [ , ] . il- , by promoting mdscs and sustaining the immunosuppressive activity of tams, contributes to the suppression of effective adaptive anti-tumor immune responses [ ] . actually, the sphingolipid sphingosine- -phosphate (s p) on tams promotes lymphangiogenesis and lung metastasis via nlrp /il- β in mouse breast cancer model [ ] . for example, obesity induces an increase in tumor-infiltrating mdscs with activated nlrc inflammasome, leading to il- β production, which drives tumor progression through adipocyte-mediated vascular endothelial growth factor (vegf) a expression and angiogenesis [ ] . a recent report showed that il- β orchestrates tumor-promoting inflammation in patients with high-risk her -negative breast cancer who would benefit from il- -blocking therapeutics with anakinra (described later on). the report indicates that while anakinra downregulates gene expressions for il- β, il- r , il- r , and il- r , increased gene expressions of nk cells and ctls are observed [ ] . although il- has been well-characterized, il- and other il- family members have been less comprehensively investigated. il- can be processed by caspase- and proteinase- as well as il- β, to be activated [ ] [ ] [ ] . considering the pathogenesis of il- -related diseases, il- could be involved [ ] . il- was originally identified as interferon (ifn)-γ-inducing factor [ ] . il- is the most structurally related to il- β. il- is synthesized as a -kda inactivated precursor and is cleaved by caspase- to a biologically active -kda mature form [ , ] . although il- β is biologically active within the pg/ml range, il- requires - ng/ml and sometimes higher levels for in vitro activation [ , ] . since the il- precursor is expressed ubiquitously in tissues [ ] , il- signaling is thought to be regulated concentration-dependently. mature il- forms a signaling complex with the il- receptor alpha chain (il- rα) with low affinity. if the cell expresses an il- receptor β chain (il- rβ), a high affinity complex is formed like the il- r accessory chain il- r . the complex of the heterodimer recruits myd through the toll-il- receptor (tir), four iraks, and traf- , leading to the degradation of i-κb and activation of nf-κb, as that for il- signaling [ ] . il- is involved in regulation of the th response by modulating the production of ifn-γ. for example, in synergy with either il- or il- , which upregulates the expression of the il- rβ co-receptor, il- induces the production of ifn-γ by t cells [ ] . il- induces ifn-γ production by nk cells, and nk cells express ccr and produce high levels of ifn-γ [ ] . the combination of il- and il- induced high levels of ifn-γ upon hypoglycemia, intestinal inflammation, and inanition [ ] . some human autoimmune diseases are associated with the elevated production of ifn-γ and il- . autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis (ra), type- diabetes mellitus, crohn's disease and psoriasis, and graft versus host disease are thought to be mediated by il- [ ] . so far, several anti-il- therapies have been reported. an anti-il- , multicenter, randomized, single-blind, placebo-controlled, parallel-group, phase iia trial for the treatment of t d was reported whereby anti-il- monoclonal antibody, gsk , was well-tolerated; however, the anti-il- therapy did not lead to any improvements in glucose control [ ] . interleukin- binding protein (il- bp) was purified from urine by chromatography on il- beads that abolished il- induction of ifn-γ, il- , and activation of nf-κb in vitro [ ] . the il- inhibition using il- bp significantly decreased mdscs in the tumor microenvironment in a preclinical osteosarcoma mouse model [ ] . il- bp (tadekinig α®) was successful in the treatment of still's disease and nlrc -mutated autoinflammatory macrophage activation syndrome (mas), for which anti-il- treatment had failed [ , ] . several inhibitors of il- signaling have been clinically approved (fig. ) . one is a recombinant human intrinsic il- receptor antagonist (il- ra), anakinra [ ] . anakinra is the pharmaceutical name of a recombinant form of intrinsic human il- ra, a . -kda protein consisting of amino acid residues. il- ra was first reported in as a bioactive il- inhibitor of - kda in the supernatants of human monocyte culture, and it was independently identified as an il- inhibitor from the urine of febrile patients [ , ] . anakinra was the first biological drug of a selective il- r antagonist to receive approval from the us food and drug administration (fda). since anakinra is an il- receptor antagonist, it can prevent the activity of both il- α and il- β by competitively blocking their binding to il- r and il- r . anakinra has been applied for a wide range of diseases including autoinflammatory diseases, non-cancer inflammatory diseases, and malignancies [ ] . to date, no serious adverse effect of anakinra has been reported [ ] . another is rilonacept (ril on' a sept), a soluble decoy receptor (fig. ) . rilonacept is a recombinant fusion protein consisting of the extracellular portion of human il- r and il- r fused with the fc portion of human igg [ ] [ ] [ ] . rilonacept binds to both il- α and il- β with high affinity and inhibits the activity of both with a long-term inhibitory effect. rilonacept was first approved by the fda for the treatment of caps in . subcutaneous injection with a loading dose and a weekly injection of half the loading dose was administered [ ] . there are no known severe adverse effects of rilonacept due to il- signaling inhibition. these drugs could modulate the immune response. the most common side effects (> % of treated patients) are inflammation of the upper respiratory tract or sinuses, headache, and redness at the injection site [ ] . the third is canakinumab (fig. ) . canakinumab, a specific human monoclonal igg antibody targeting il- β, is intravenously or subcutaneously infused to neutralize the bioactivity of human il- β [ , ] . canakinumab does not react with il- α or il- r . therefore, canakinumab is a more specific inhibitor of il- β, expected to have no effect on il- α-dependent host defense [ ] . early clinical trials established the administration of canakinumab every weeks as safe and effective against several inflammatory diseases [ , ] . there are several agents currently undergoing clinical trials. il- α production is a very early step in the sterile inflammatory response at the center of the malignant phenotype that drives angiogenesis, tumor stromal remodeling, tumor invasiveness, metastasis, and cachexia [ , [ ] [ ] [ ] . thus, il- α may be a particularly important target for the treatment of cancer. a neutralizing true human igg κ a b d c fig. interleukin- receptors and inhibitors of il- signaling. a il- r interacts with both il- α and il- β and promotes signal transduction, together with its co-receptor il- r (il- racp). il- ra is a protein that binds to il- r but not il- r , and it is as an inhibitor of il- signaling. il- r is a decoy receptor because it lacks a cytoplasmic segment. b anakinra is a recombinant form of intrinsic human il- ra. it works as an antagonist of il- r , and it is able to inhibit both il- α and il- β. c rilonacept is a recombinant fusion protein including the extracellular protein of human il- r and il- r fused with the fc portion of human igg . it binds to both il- α and il- β with high affinity and has a long-term inhibitory effect. d canakinumab and mabp are monoclonal antibodies against il- β and il- α, respectively. they bind to and neutralize their targets specifically monoclonal antibody specific for human il- α, mabp , has been developed, and it was well-tolerated with no dose-limiting toxicities or immunogenicity [ , ] (fig. ) . mabp treatment for patients with advanced colorectal cancer in a randomized, double-blind, placebocontrolled, phase study revealed that mabp improved clinical performance in patients with advanced colorectal cancer [ ] . mabp is a promising treatment for patients with hidradenitis suppurativa not eligible for the anti-tnf-α antibody adalimumab [ ] . gevokizumab is an anti-il- β monoclonal antibody, igg , which improved glucose control and β-cell function in a diet-induced-obesity mouse model [ ] and in the presence of il- β-driven inflammatory diseases [ ] . ly is a humanized monoclonal antibody (igg ) that binds to il- β to neutralize its activity. its affinity is comparatively high ( . pmol/l). previous clinical studies evaluated not only its safety and pharmacokinetics but also its effects on ra (nct ). weekly treatment of t d patients with ly for months resulted in modest reductions in glycated hemoglobin and blood glucose [ ] . population pharmacokinetics (pk) of ly were characterized using data from t d subjects (study h c-mc-bbdk) who received weekly subcutaneous doses of ly ( . , , and mg) and ra subjects (study h c-mc-bbde) who received five weekly intravenous (iv) doses ( . - . mg/kg) [ ] . no additional study has been reported. amg is a fully human, igg monoclonal antibody that binds to human il- r , inhibiting the activity of il- α and il- β [ ] . patients with osteoarthritis received placebo or amg subcutaneously (sc, or mg) or intravenously (iv, or mg) once every weeks for weeks or received placebo or mg amg sc, once every weeks for weeks; however, there was non-significant but numerically greater improvement in pain compared with the placebo group based on womac pain scores [ ] . amg is now termed medi- which has been studied in not only osteoarthritis, but also chronic obstructive pulmonary disease. in all cases, the benefit is limited [ , ] . ebi- is a protein chimera of il- β and il- receptor antagonists (il- ra or anakinra). ebi- binds to il- r and inhibits il- signaling and has been studied for the treatment of ocular surface inflammatory diseases [ ] . since il- β is known to be processed and activated by caspase- , caspase- could be an indirect target for il- β signaling. to examine this, the highly selective caspase- inhibitor vx- was applied to a rat model of myocardial infarction (mi) and mouse model of ad [ , ] . the recombinant human il- -receptor antagonist anakinra is markedly effective against caps such as mws, fcas, and nomid/cinca. weekly rilonacept treatment markedly improved the clinical symptoms of caps and normalized the levels of saa in those at risk of developing amyloidosis [ , , , ] . in several case reports of patients with fmf, anti-il- treatment with anakinra, canakinumab, or rilonacept in colchicine-resistant patients was highly effective [ ] [ ] [ ] [ ] . it was also reported that there was a rapid and lasting response of pyoderma gangrenosum to targeted treatment with anakinra in a patient with papa syndrome [ ] . anakinra and canakinumab therapies were also reported to be effective in patients with mkd/hids [ ] . in the case of traps, although tnf-α is considered to be mainly involved in clinical manifestations, marked improvement following il- β blockade occurred [ , ] . an open-label, phase ii study was reported whereby patients with active recurrent or chronic traps ( / , %; % ci . % to . %) achieved the primary efficacy endpoint. canakinumab treatment for traps rapidly improved the median time to clinical remission to days ( % ci to days) [ ] . skin findings also promptly improved upon anakinra treatment in a patient with dira [ ] . monotherapy involving canakinumab for the treatment of fmf has been reported [ ] . a nationwide report on il- treatment of patients with fmf revealed that fmf patients ( [ %] female; mean age, . years [range, ) were included; the mean age at onset was . years (range, - ), and the mean colchicine dose was . mg/day (range, . - . ). anakinra was administered to patients, and canakinumab was administered to patients. anti-il- treatment was used in % of colchicine-resistant patients and % of amyloidosis patients. during the mean of . months of treatment (range, - ), the attack frequency per year was significantly decreased (from . to . ; p < . ), and symptoms of . % of colchicine-resistant patients with fmf were ameliorated. in this study, the complete remission rate was % and inefficacy rate was % in patients treated with anakinra, whereas the complete remission rate was % and inefficacy rate was % for patients treated with canakinumab [ ] . although the response rates were not significant (p = . and χ = . ) in the study above [ ] , in our opinion, long-acting canakinumab may be more efficacious than anakinra, considering the necessity of daily subcutaneous anakinra injection because of its short half-time clearance of less than h [ ] . there are suspected etiologies of autoinflammatory disorders, but all lack a known genetic basis. in patients with adult-onset still's disease (aosd), anakinra monotherapy is significantly effective and has become the standard therapy, especially in prednisone-resistant patients. commercially available anti-il- agents (anakinra/kineret®, canakinumab/ ilaris®, or rilonacept/arcalyst®) for patients with treatmentresistant aosd are effective. canakinumab and anakinra were also effective for patients with schnitzler syndrome, an adult-onset autoinflammatory disease characterized by focal urticaria and systemic inflammation including fever with bone and muscle pain, in the first placebo-controlled study, and several clinical trials are currently ongoing [ ] [ ] [ ] [ ] . il- blockade therapy using anakinra is successful in patients with psoriatic arthritis, ankylosing spondylitis, and ra. on the other hand, its efficacy and safety are insufficient, precluding use for patients with systemic lupus erythematosus or sjögren syndrome, and il- β inhibition using canakinumab had no effect on the decline in β-cell function after diabetes onset in patients with type diabetes mellitus resulting from autoimmune-mediated β-cell loss [ ] [ ] [ ] [ ] [ ] . as for ra, the enhanced release of other proinflammatory cytokines such as tnf-α and il- plays important roles in the inflamed synovium of ra patients [ ] . in patients for whom tnf-α blockers are contraindicated, anakinra is effective in controlling the disease activity of ra and has been licensed for treatment at a dose of mg/day by subcutaneous injection every day [ , ] . compared with anakinra, tnf inhibitors, such as the anti-tnf-α monoclonal antibody infliximab, or etanercept that fuse the tnf receptor to the end of the igg antibody, dominate the field of biologics for ra because of the sense of well-being experienced by patients within hours of treatment [ ] . tocilizumab, a humanized anti-il- receptor (il- r) monoclonal antibody, has also been shown to improve the symptoms of patients with ra [ ] . however, those agents are associated with the risk of reactivating bacterial pathogens such as tuberculosis (tb) and malignancies [ ] . notably, no cases of tb reactivation were reported in ra patients after anakinra treatment, whereas cases of tb reactivation were reported in , ra patients after tocilizumab treatment, and and cases of tb reactivation were reported in and ra patients after tnf-inhibitor treatment with golimumab and certolizumab pegol, respectively. this suggests the low risk of tb reactivation in ra patients treated with anti-il- therapy [ ] . anakinra is safe and may be associated with a dose-related survival advantage in patients with septic shock syndrome complicated by acute respiratory distress syndrome, disseminated intravascular coagulation, and renal dysfunction, and subsequent secondary hemophagocytic lymphohistiocytosis (hlh), or macrophage-activating syndrome (mas) [ , ] . for sepsis with mas, anakinra treatment led to significant improvements in hepatobiliary dysfunction and disseminated intravascular coagulation in patients ( . % anakinra vs. . % placebo) and the -day survival rate, with the hazard ratio for death being . ( . - . ; p = . ) for the treatment group on cox regression. the data included adults in the original study cohort, randomized to receive either anakinra or placebo [ ] . for metabolic syndromes il- inhibition by anakinra, rilonacept, or canakinumab is efficacious for gout patients [ ] . il- plays a role in the progression of atherosclerosis as well [ ] . in patients with a history of mi, canakinumab significantly reduced the high-sensitive serum crp concentration from the baseline, as compared with a placebo, without affecting the ldl-cholesterol concentration. a -mg dose of canakinumab resulted in a significantly reduced risk of recurrent cardiovascular events compared with a placebo [ ] . the inhibition of il- with anakinra improved glycemia and the pancreatic β-cell function and reduced systemic inflammation [ ] . although il- β inhibition with canakinumab had similar effects on major cardiovascular events among those with and without diabetes, treatment over a median period of . years did not reduce incident diabetes [ ] . the blockade of il- with anakinra improved glycemia and the β-cell secretory function and reduced markers of systemic inflammation [ ] . anakinra also prevented transthyretin extracellular deposition in the sciatic nerve in a familial amyloidotic polyneuropathy mouse model [ ] . during ischemic disease, such as mi or cerebral infarction, or tissue injury, cell death by necrosis takes place and the il- α precursor is released in sterile inflammation [ ] . when there is no time for the synthesis of il- α, il- α is ready to function as soon as it leaves the dying cell in the first few hours following tissue ischemia or injury [ ] . in fact, animal studies showed that the inhibition of il- is effective in limiting atherosclerosis and cardiovascular events and improving the symptoms of acute mi and ischemic stroke [ , ] . two pilot studies of il- inhibition in st-segment elevation mi revealed a reduced acute inflammatory response and favorable overall performance at the -month follow-up [ ] . il- β is thought to play an important role in cancer invasiveness, progression, and metastases via inflammation in the tumor microenvironment. a further analysis in the canakinumab anti-inflammatory thrombosis outcomes study (cantos), a randomized trial of the role of inhibition of il- β in atherosclerosis, revealed that anti-inflammatory therapy with canakinumab targeting the il- β innate immunity pathway could significantly reduce lung cancer mortality [ ] . moreover, treatment of patients with metastatic breast cancer-related with anakinra eliminates a systemic transcriptional signature of il- -associated inflammation in blood cells. the inflammatory signature in primary breast cancers identifies a subset of patients that could potentially benefit from il- β-targeted therapies [ ] . safety profiles of both anakinra and canakinumab were reported [ ] . in this study, several clinical and therapeutic data on patients treated with either anakinra or canakinumab were retrospectively analyzed. four hundred and seventy-five patients participated; anakinra and canakinumab were prescribed in and treatment courses, respectively. eighty-nine adverse events were recorded; ( . %) were classified as serious adverse events during a mean follow-up of . ± . months. [ ] . in addition, anakinra is applied to metastatic cancer. a trial involving metastatic colorectal cancer patients showed significantly increased survival when anakinra was added to standard chemotherapy for colorectal cancer and patients with her -negative breast cancer [ , ] . the il- blockade will reduce il -driven inflammation and immunosuppression that may contribute to the tumor metastatic table effective anti-il- therapy for inflammatory diseases autoinflammatory diseases: cryopyrin-associated periodic syndrome (caps) [ , ] familial mediterranean fever (fmf) [ ] pyogenic arthritis, pyoderma gangrenosum and acne syndrome (papa) [ ] nlrp autoinflammatory syndrome [ ] tumor necrosis factor receptor- -associated syndrome (traps) [ ] hyperimmunoglobulinemia d and periodic fever syndrome (hids)/ mevalonate kinase deficiency (mkd) [ ] deficiency of the interleukin- -receptor antagonist (dira) [ ] autoimmune diseases: psoriatic arthritis [ ] ankylosing spondylitis [ ] rheumatoid arthritis (ra) [ ] metabolic syndrome: gout [ ] atherosclerosis [ ] type diabetes mellitus (t d) [ ] amyloidosis [ ] neurodegenerative disease: alzheimer's disease (ad) [ ] infections and inflammatory responses: septic shock syndrome [ ] acute respiratory distress syndrome (ards) [ ] disseminated intravascular coagulation (dic) [ ] hemophagocytic lymphohistiocytosis (hlh) [ ] macrophage-activating syndrome (mas) [ ] ischemic diseases: myocardial infarction (mi) [ ] stroke [ ] malignant rumor: breast cancer [ ] microenvironment [ ] . the timeline of therapeutics is summarized in fig. . we described il- as an important cytokine for not only inflammation related to cell injury but also homeostasis of cells, tissues, and organs in view of the general pathology. in addition, we also described recent expanding il- signal-targeting for the treatment of diseases. once the balance of il- signaling is disrupted, it may markedly contribute to the pathogenesis of not only inflammatory disease, but also malignancies. il- -targeted biologics have been expanding, as there are no known serious adverse effects such as lymphoproliferative disorder or virus reactivation like tnf or il- -targeting therapies. therefore, il- is expected to become an attractive molecular target to treat a wide range of diseases such as autoinflammatory diseases, autoimmune diseases, infectious disease, metabolic syndromes, ischemic diseases, and malignant tumors [ , ] (table ) . availability of data and materials not applicable. the manuscript was written by nk and jm, and all authors read and approved the final manuscript. ethics approval and consent to participate not applicable. not applicable. disturbance of function (functio laesa): the legendary fifth cardinal sign of inflammation, added by galen to the four cardinal signs of celsus robbins & cotran pathologic basis of disease inflammation and metabolic disorders endoplasmic reticulum stress and the inflammatory basis of metabolic disease inflammatory mechanisms in obesity approaching the asymptote? evolution and revolution in immunology stress, inflammation, and defense of homeostasis pattern recognition receptors and inflammation editorial overview: special section: effects of endogenous immune stimulants: from a defence system against infection to a homeostatic mechanism linking metabolism with inflammation the pyrin-card protein asc is an activating adaptor for caspase- . srinivasula sm the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-β nucleotide-binding oligomerization domain-like receptors and inflammasomes in the pathogenesis of nonmicrobial inflammation and diseases regulation of inflammasome signaling homeostatic inflammation, an emerging concept homeostatic inflammation in innate immunity inflammation, metaflammation and immunometabolic disorders origin and physiological roles of inflammation it's time to redefine inflammation il- family members in the pathogenesis and treatment of metabolic disease: focus on adipose tissue inflammation and insulin resistance immunological and inflammatory functions of the interleukin- family potentiation of the t-lymphocyte response to mitogens. i. the responding cell macrophage sensitivity to endotoxin: genetic control by a single codominant gene biochemical and biological characterization of lymphocyteactivating factor (laf) produced by the murine macrophage cell line, p d interleukin : the first interleukin detection of anti-tumour cell mediated immunity and serum blocking factors in cancer patients by the leucocyte adherence inhibition test cell-mediated immunity and specific serum factors in human cancer: the leukocyte adherence inhibition test leukocyte adherence inhibition by soluble tumor antigens in breast cancer patients the inflammatory microenvironment in tumor progression: the role of tumor-associated macrophages blockage of the nlrp inflammasome by mcc improves anti-tumor immune responses in head and neck squamous cell carcinoma targeting inflammasome/il- pathways for cancer immunotherapy il- family nomenclature the interleukin- family: back to the future partial purification of human lymphocyte-activating factor (laf) by ultrafiltration and electrophoretic techniques crystallographic refinement of interleukin β at . a resolution tructure of interleukin at . -a resolution structure and function of interleukin- , based on crystallographic and modeling studies the precursor form of il- α is an intracrine proinflammatory activator of transcription the interaction with sp and reduction in the activity of histone deacetylase are critical for the constitutive gene expression of il- alpha in human melanoma cells molecular analysis of constitutive il- alpha gene expression in human melanoma cells: autocrine stimulation through nf-κb activation by endogenous il- α inflammasome activators induce interleukin- α secretion via distinct pathways with differential requirement for the protease function of caspase- identification of a key pathway required for the sterile inflammatory response triggered by dying cells the interleukin- α precursor is biologically active and is likely a key alarmin in the il- family of cytokines cloning, sequence and expression of two distinct human interleukin- complementary dnas inflammasome-independent regulation of il- -family cytokines treating inflammation by blocking interleukin- in a broad spectrum of diseases expression and alternative processing of il- in human neutrophils rapid and specific conversion of precursor interleukin β (il- β) to an active il- species by human mast cell chymase multiple biological activities of human recombinant interleukin granzyme a is an interleukin beta-converting enzyme processing of precursor interleukin β and inflammatory disease nf-κb regulates il- β transcription through a consensus nf-κb binding site and a nonconsensus cre-like site mechanisms of interleukin- β release activation and regulation of the inflammasomes mechanisms and functions of inflammasomes the inflammasome nlrs in immunity, inflammation, and associated diseases activation of the nalp inflammasome is triggered by low intracellular potassium concentration k + efflux is the common trigger of nlrp inflammasome activation by bacterial toxins and particulate matter a genome-wide crispr (clustered regularly interspaced short palindromic repeats) screen identifies nek as an essential component of nlrp inflammasome activation nek is an essential mediator of nlrp activation downstream of potassium efflux nlrp activation and mitosis are mutually exclusive events coordinated by nek , a new inflammasome component noncanonical inflammasome activation by intracellular lps independent of tlr cytoplasmic lps activates caspase- : implications in tlr -independent endotoxic shock inflammatory caspases are innate immune receptors for intracellular lps caspase- mediates non-canonical activation of the nlrp inflammasome in human myeloid cells bax/bak-induced apoptosis results in caspase- -dependent il- β maturation in macrophages cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death caspase- cleaves gasdermin d for non-canonical inflammasome signalling gasdermin d is an executor of pyroptosis and required for interleukin- β secretion gsdmd membrane pore formation constitutes the mechanism of pyroptotic cell death gsdmd p elicited by caspase- during pyroptosis forms pores in membranes poreforming activity and structural autoinhibition of the gasdermin family inflammasomeactivated gasdermin d causes pyroptosis by forming membrane pores detection of il- alpha and il- beta gene expression by in situ hybridization. tissue localization of il- mrna in the normal c bl/ mouse the interleukin- family: years of discovery mass spectrometric analysis of the endogenous type i interleukin- (il- ) receptor signaling complex formed after il- binding identifies il- racp, myd , and irak- as the stable components mouse neutrophils express the decoy type interleukin- receptor (il- r ) constitutively and in acute inflammatory conditions il- receptor type suppresses collagen-induced arthritis by inhibiting il- signal on macrophages the interleukin- receptor family interleukin- -induced activation of a protein kinase co-precipitating with the type i interleukin- receptor in t cells nf-κb activation by interleukin- (il- ) requires an il- receptor-associated protein kinase activity evidence from sequence information that the interleukin- receptor is a transmembrane gtpase interleukin- (il- ) pathway the interleukin- receptor/toll-like receptor superfamily: signal transduction during inflammation and host defense targeting of tak in inflammatory disorders and cancer tnf and map kinase signalling pathways mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and muckle-wells syndrome de novo cias mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (nomid): a new member of the expanding family of pyrinassociated autoinflammatory diseases chronic infantile neurological cutaneous and articular syndrome is caused by mutations in cias , a gene highly expressed in polymorphonuclear cells and chondrocytes neonatal-onset multisystem inflammatory disease responsive to interleukin- beta inhibition pattern of interleukin- beta secretion in response to lipopolysaccharide and atp before and after interleukin- blockade in patients with cias mutations genetic and molecular basis of inflammasomemediated disease innate immune sensing of bacterial modifications of rho gtpases by the pyrin inflammasome papa, pash and papash syndromes: pathophysiology, presentation and treatment the b . domain of pyrin, the familial mediterranean fever protein, interacts directly with caspase- to modulate il- β production pyrin binds the pstpip /cd bp protein, defining familial mediterranean fever and papa syndrome as disorders in the same pathway clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an nlrp mutation lack of isoprenoid products raises ex vivo interleukin- secretion in hyperimmunoglobulinemia d and periodic fever syndrome clinical, genetic, and therapeutic diversity in patients with severe mevalonate kinase deficiency dramatic improvement following interleukin beta blockade in tumor necrosis factor receptor- -associated syndrome (traps) resistant to anti-tnf-α therapy an autoinflammatory disease with deficiency of the interleukin- -receptor antagonist glucose-induced beta cell production of il- beta contributes to glucotoxicity in human pancreatic islets thioredoxin-interacting protein links oxidative stress to inflammasome activation free fatty acids induce a proinflammatory response in islets via the abundantly expressed interleukin- receptor i activation of the nlrp inflammasome by islet amyloid polypeptide provides a mechanism for enhanced il- β in type diabetes iapp boosts islet macrophage il- in type diabetes resident macrophages mediate islet amyloid polypeptide-induced islet il- β production and β-cell dysfunction the nalp inflammasome is involved in the innate immune response to amyloid-β iapp/amylin deposition, which is correlated with expressions of asc and il- β in β-cells of langerhans' islets, directly initiates nlrp inflammasome activation amyloid β directly interacts with nlrp to initiate inflammasome activation: identification of an intrinsic nlrp ligand in a cell-free system association of interleukin- gene polymorphisms with central obesity and metabolic syndrome in a coronary heart disease population pattern of interleukin- β secretion in response to lipopolysaccharide and atp before and after interleukin- blockade in patients with cias mutations autoinflammation: translating mechanism to therapy central delivery of iodine- -labeled cetuximab, etanercept and anakinra after perispinal injection in rats: possible implications for treating alzheimer's disease critical role for interleukin- β (il- β) during chlamydia muridarum genital infection and bacterial replication-independent secretion of il- beta in mouse macrophages negative regulation of cytokine signaling influences inflammation interleukin- β triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to t cells the role of interleukin in human b cell activation: inhibition of b cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocytic pyrogen ril -induced proliferation of human circulating nk cells and t lymphocytes: synergistic effects of il and il il- β strikingly enhances antigen-driven cd and cd t-cell responses the nlrp inflammasome in kidney disease and autoimmunity cancer-related inflammation expression of interleukin- β in human breast carcinoma the interleukin- family of cytokines and receptors in human breast cancer: implications for tumor progression cancer: an infernal triangle il- and il- regulatory pathways in cancer progression and therapy s pr on tumor-associated macrophages promotes lymphangiogenesis and metastasis via nlrp /il- β obesity-associated nlrc inflammasome activation drives breast cancer progression il receptor antagonist controls transcriptional signature of inflammation in patients with metastatic breast cancer neutrophil proteinase -mediated induction of bioactive il- secretion by human oral epithelial cells activation of interferon-gamma inducing factor mediated by interleukin- beta converting enzyme caspase- processes ifn-gamma-inducing factor and regulates lps-induced ifn-gamma production interleukin- and il- binding protein endotoxin-induced serum factor that stimulates gamma interferon production a novel role for interleukin- in adhesion molecule induction through nf-κb and phosphatidylinositol (pi) -kinase-dependent signal transduction pathways differences in signaling pathways by il- beta and il- gene expression, synthesis, and secretion of interleukin and interleukin beta are differentially regulated in human blood mononuclear cells and mouse spleen cells interferon-gammainducing factor enhances t helper cytokine production by stimulated human t cells: synergism with interleukin- for interferon-gamma production m-csf induces the expression of a membrane-bound form of il- in a subset of human monocytes differentiating in vitro toward macrophages ifn-gammadependent and -independent mechanisms in adverse effects caused by concomitant administration of il- and il- natural killer cells in human autoimmune diseases a study to investigate the efficacy and safety of an anti-interleukin- monoclonal antibody in the treatment of type diabetes mellitus interleukin- binding protein: a novel modulator of the th cytokine response inhibition of il- -mediated myeloid derived suppressor cell accumulation enhances anti-pd efficacy against osteosarcoma cancer prolonged treatment with tadekinig alfa in adult-onset still's disease life-threatening nlrc -associated hyperinflammation successfully treated with il- inhibition interleukin receptor antagonist. a new member of the interleukin family effects of immune complexes on production by human monocytes of interleukin or an interleukin inhibitor identification of a specific interleukin inhibitor in the urine of febrile patients an expanding role for interleukin- blockade from gout to cancer a pilot study to evaluate the safety and efficacy of the longacting interleukin- inhibitor rilonacept (interleukin- trap) in patients with familial cold autoinflammatory syndrome efficacy and safety of rilonacept (interleukin- trap) in patients with cryopyrinassociated periodic syndromes: results from two sequential placebocontrolled studies church ld, mcdermott mf. canakinumab, a fully-human mab against il- for the potential treatment of inflammatory disorders antiinflammatory therapy in clinical care: the cantos trial and beyond il- α secreted by colon cancer cells enhances angiogenesis: the relationship between il- α release and tumor cells' potential for liver metastasis interleukin- α secreted by pancreatic cancer cells promotes angiogenesis and its therapeutic implications il- is required for tumor invasiveness and angiogenesis mabp , a first-in-class true human antibody targeting interleukin- α in refractory cancers: an open-label, phase dose-escalation and expansion study mabp as a novel antibody treatment for advanced colorectal cancer: a randomised, double-blind, placebo-controlled, phase study mabp targeting il- α for moderate to severe hidradenitis suppurativa not eligible for adalimumab: a randomized study xoma , an anti-il- β monoclonal antibody, improves glucose control and β-cell function in the diet-induced obesity mouse model il- β inhibition in cardiovascular complications associated to diabetes mellitus double-blind, randomized study evaluating the glycemic and anti-inflammatory effects of subcutaneous ly , a neutralizing il- β antibody, in patients with type diabetes population pharmacokinetic modeling of ly after multiple intravenous and subcutaneous administrations a phase randomized, double-blind study of amg , a fully human monoclonal antibody to il- r, in patients with rheumatoid arthritis a randomized, double-blind study of amg (a fully human monoclonal antibody to il- r ) in patients with osteoarthritis of the knee a randomised, placebo-controlled trial of anti-interleukin- receptor monoclonal antibody medi in chronic obstructive pulmonary disease preclinical development of ebi- : an il- receptor- inhibitor for the topical ocular treatment of ocular surface inflammatory diseases the highly selective caspase- inhibitor vx- provides additive protection against myocardial infarction in rat hearts when combined with a platelet inhibitor caspase- inhibition alleviates cognitive impairment and neuropathology in an alzheimer's disease mouse model interleukin- -receptor antagonist in the muckle-wells syndrome prevention of cold-associated acute inflammation in familial cold autoinflammatory syndrome by interleukin- receptor antagonist the efficacy of anakinra in an adolescent with colchicine-resistant familial mediterranean fever anti-il- treatment for secondary amyloidosis in an adolescent with fmf and behçet's disease rilonacept for colchicine-resistant or -intolerant familial mediterranean fever: a randomized trial canakinumab for the treatment of autoinflammatory recurrent fever syndromes targeted treatment of pyoderma gangrenosum in papa (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome with the recombinant human interleukin- receptor antagonist anakinra efficacy of interleukin- -targeting drugs in mevalonate kinase deficiency beneficial response to interleukin receptor antagonist in traps canakinumab treatment for patients with active recurrent or chronic tnf receptor-associated periodic syndrome (traps): an open-label, phase ii study deficiency of interleukin- receptor antagonist responsive to anakinra canakinumab as monotherapy for treatment of familial mediterranean fever -first report in central and eastern europe region nationwide experience with off-label use of interleukin- targeting treatment in familial mediterranean fever patients efficacy and safety of canakinumab in patients with still's disease: exposure-response analysis of pooled systemic juvenile idiopathic arthritis data by age groups rapid responses to anakinra in patients with refractory adult-onset still's disease adult onset still's disease-the evidence that antiinterleukin- treatment is effective and well-tolerated (a comprehensive literature review) efficacy and safety of canakinumab in schnitzler syndrome: a multicenter randomized placebo-controlled study anakinra for rheumatoid arthritis: a systematic review an open-label pilot study of the efficacy and safety of anakinra in patients with psoriatic arthritis refractory to or intolerant of methotrexate open label trial of anakinra in active ankylosing spondylitis over weeks a systematic review of the off-label use of biological therapies in systemic autoimmune diseases interleukin- antagonism in type diabetes of recent onset: two multicentre, randomised, double-blind, placebo-controlled trials revolutionary change in rheumatoid arthritis management with biological therapy interleukin- in the pathogenesis and treatment of inflammatory diseases tuberculosis risk in patients treated with nonanti-tumor necrosis factor-α (tnf-α) targeted biologics and recently licensed tnf-α inhibitors: data from clinical trials and national registries blockade of tnf-α rapidly inhibits pain responses in the central nervous system study of active controlled monotherapy used for rheumatoid arthritis, an il- inhibitor (samurai): evidence of clinical and radiographic benefit from an x ray reader-blinded randomised controlled trial of tocilizumab il- ra sepsis syndrome study group. initial evaluation of human recombinant interleukin- receptor antagonist in the treatment of sepsis syndrome: a randomized, open-label, placebo-controlled multicenter trial therapeutic role of anakinra, an interleukin- receptor antagonist, in the management of secondary hemophagocytic lymphohistiocytosis/sepsis/multiple organ dysfunction/macrophage activating syndrome in critically ill children interleukin- receptor blockade is associated with reduced mortality in sepsis patients with features of macrophage activation syndrome: reanalysis of a prior phase iii trial anti-interleukin- therapy in the management of gout interleukin- β inhibition and the prevention of recurrent cardiovascular events: rationale and design of the canakinumab anti-inflammatory thrombosis outcomes study (cantos) cantos trial group. antiinflammatory therapy with canakinumab for atherosclerotic disease anti-inflammatory therapy with canakinumab for the prevention and management of diabetes interleukin- signaling pathway as a therapeutic target in transthyretin amyloidosis interleukin- -receptor antagonist in type diabetes mellitus the sterile inflammatory response differential release of chromatin-bound il- discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation blocking interleukin- as a novel therapeutic strategy for secondary prevention of cardiovascular events comparative safety of interleukin- blockade with anakinra in patients with st-segment elevation acute myocardial infarction (from the vcu-art and vcu-art pilot studies) effect of interleukin- β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomised, double-blind, placebo-controlled trial working group" of systemic autoinflammatory diseases of sir (italian society of rheumatology). safety profile of the interleukin- inhibitors anakinra and canakinumab in real-life clinical practice: a nationwide multicenter retrospective observational study fluorouracil and bevacizumab plus anakinra for patients with metastatic colorectal cancer refractory to standard therapies (irafu): a single-arm phase study interleukin- beta-a friend or foe in malignancies? blocking il- : interleukin receptor antagonist in vivo and in vitro treating inflammation by blocking interleukin- in humans all authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - bo pnl authors: scott, g. r. title: guidelines for the control of equine viral infections date: - - journal: equine vet j doi: . /j. - . .tb .x sha: doc_id: cord_uid: bo pnl summary: twelve dna viruses and forty‐three rna viruses are known to infect horses. in addition, there are three unclassified viruses and, at least, three alleged viruses infecting horses. differential diagnosis is difficult. at least twenty‐eight of the fifty‐eight viruses induce clinical disease but the range of syndromes is limited; eleven provoke respiratory symptoms and eleven cause encephalitis. thirty‐four equine viruses with a limited geographical distribution are transmitted by arthropod vectors. twenty viruses are spread by contact and their distribution, in general, is global. the vector‐transmitted virus diseases are best controlled by prophylactic vaccination. the viral contagions are not, in general, well controlled by vaccination and it is likely that prophylactic chemotherapy will become increasingly important in the future. rÉsumÉ: on sait que douze virus adn et quarante trois virus arn sont responsables d'infections chez le cheval. en outre trois virus non classés et trois autres agents de type viral au moins sont également infectants pour cette espèce. le diagnostic différentiel est difficile. vingt huit au moins des cinquante trois virus provoquent des maladies cliniques mais la gamme des syndromes est restreinte: onze virus engendrent des symptomes respiratoires, onze déterminent des encéphalites. trente quatre virus dont la distribution géographique est limitée sont transmis par des arthropodes vecteurs. vingt virus sont disséminés par contact et leur répartition est en général à l'échelle du globe. les maladies provoquées par des virus à propagation vectorielle sont mieux controlées par une vaccination préventive. les contagions virales ne sont pas, en général, efficacement controlées par la vaccination et l'on peut penser que la chimiothérapie préventive de ces affections connaitra une importance croissante à l'avenir. zusammenfassung: zwölf dns‐viren und rns‐viren vermögen das pferd zu infizieren. dazu kommen drei unklassierte viren und zum mindesten drei erreger, die angeblich viruscharakter haben. die differentialdiagnose ist schwierig. mindestens der viren verursachen klinisch manifeste krankheiten, aber die eigenart der syndrome ist limitiert; elf davon provozieren respiratorische symptome und elf rufen encephalitis hervor. equine viren benötigen als vektoren arthropoden; ihre geographische ausbreitung ist beschränkt. viren werden durch kontakt übertragen; sie werden in der regel auf der ganzen welt angetroffen. die durch vektoren übertragenen viruskrankheiten können am besten durch prophylaktische impfungen kontrolliert werden. die übrigen können im allgemeinen durch impfungen nicht gut kontrolliert werden und es scheint wahrscheinlich, dass die prophylaktische chemotherapie in zukunft an bedeutung gewinnen wird. sumario: virus equinos doce dna virus y cuarenta y tres rna virus son reconcodios como infectantes al caballo. en adicion hay tres no clasidicados virus y por lo menos tres tipos de virus muy se mejantes que afectan el caballo. el diagnostico diferencial es dificultoso. aproximadamente de los virus inducen enfermedades clinicas con y espectro de sindromes limitado. provocan sintomas respiratorios y causan encefalitis. virus equinos con limitacion en su distribucion geografica son transmitidos por vectores artropodos. veinte virus son diseminados por contacto y su distribucion en general es global. es controlada la enfermeead de estos vectiores per medio de vacunacion. los contagio del virus no son bien controlados por lo general mediante vacunacion y solo una terapia profilactica que havenido tomando aunge en el futuro. isolated from the respiratory tract of a colt with rhinitis, ceo virus (kono and kobazashi, ) isolated in japan spontaneously from cultures of equine kidney and bone marrow cells, herpes (karpas, ) isolated from the kidneys of an apparently healthy foal, equine cytomegalovirus (hsiung, fischman, fong and green, ) isolated from spontaneously degenerating equine kidney cell cultures after prolonged cultivation, australian coital exanthema virus (pascoe, spradbrow and bagust, ) isolated from lesions on a stallion, and iowa leukocyte virus (kemeny and pearson, ) isolated from the leucocytes of out of apparently normal iowa horses. the antigenic relationships of the iowa leukocyte virus to the other equine herpesviruses has yet to be established. rna viruses have either cubic, helical or unknown symmetries. there are two groups of cubic rna viruses, picornavirus and reovirus, four groups of three diseases of horses, borna disease (zwick and witte, ) , near east equine encephalomyelitis (daubney and mahlau, ) and nigerian horse staggers (porterfield, hill and morris, ) are caused by viruses as yet unclassified and it is possible that the three diseases are caused by the same virus. finally, there are at least three other alleged viral diseases, serum hepatitis (theiler, ) , grass sickness (obel, ) and virus diarrhoea (rooney, ) . in summary, viruses are known to infect horses and every year more are added to the list. at least of the viruses induce clinical disease; the other are incriminated only by the detection of specific antibodies in horse sera. the differential diagnostic problems posed by this array of viruses are formidable and highlight the need for more laboratories and more virologists. diagnosis is further confounded by the fact that eleven of the viruses provoke respiratory symptoms and eleven cause encephalitis (table iv ). in addition, equine herpes and equine arteritis viruses induce abortion; equine arteritis virus is also associated with pinkeye; herpes i and australian coital exanthema viruses cause venereal lesions; pox, grease, papular dermatitis and vesicular stomatitis viruses cause skin and mucosal lesions; equine infectious anaemia virus induces progressive weakness and anaemia; and african horse sickness virus produces oedema subcutaneously as well as in the lungs. (plummer, ) , rhino ) and aborted foal virus (bohm, ) . african horse sickness virus is believed now to be a reovirus (ozawa, ) . three myxoviruses, influenza a (heller, espmark and viriden, ) , influenza b (ditchfield, macpherson and zbitnew, ) , and influenza c and three rhabdoviruses, rabies, vesicular stomatitis and coca viruses, infect horses. serological evidence incriminates parainfluenza- and parainfluenza- viruses belonging to the paramyxovirus group. in addition, parainfluenza- virus has been isolated from horses with upper respiratory tract infections . there is possibly one coronavirus infecting horses; ditchfield ( ) isolated a virus from a thoroughbred with undifferentiated respiratory disease and found that it possessed a morphology similar to that of infectious bronchitis virus of poultry, the type-virus of the coronavirus group. there are more than arboviruses and at least infect horses. equine infectious anaemia virus probably belongs to the oncornavirus group, i.e. the rna tumour viruses. infectious arteritis virus (doll, bryons, mccollum and crow, ) , on the other hand, is known to be a rna virus but it is as yet ungrouped. recent electron microscopic studies revealed a morphology like that of the arboviruses (breese and mccollum, ) . a key factor in controlling infectious disease is a knowledge of the modes of transmission. i n general, viruses are transmitted either by arthropod vectors or by contact between sick and healthy animals. none of the dna viruses affecting horses is transmitted by vectors, eight are contagions and four are transmitted by modes as yet unknown. in contrast, rna viruses are transmitted by vectors and by contact. two of the three unclassified viruses, near east equine encephalomyelitis virus and nigerian horse virus, are transmitted by arthropods. daubney ( ) has suggested that borna disease virus is also arthropod-borne. the geographical distribution of equine viruses is closely correlated with the mode of transmission. the vector-transmitted viruses have a restricted global distribution (table v) whereas the contagions are widely distributed (table vi) . with the possible exception of african horse sickness, infections of horses by vector-transmitted viruses are accidental and play no role in the natural life cycles of the viruses concerned. cases are sporadic. eradication is impossible. one is limited, therefore, to prophylactic vaccination as being the only practical control measure. eleven of the thirty-four known vector-transmitted viruses cause disease and vaccines have been developed against six of them (table vii) . there is also a vaccine for vesicular stomatitis but it has not been used to protect horses (corrza, ) . similarly japanese b encephalitis vaccine and wesselsbron vaccine have not been used to protect horses (kodama, sasaki and tnoue, ; neitz, ) . unlike the diseases transmitted by vectors, the contagions are open to attack on several frontssegregation, deliberate exposure, vaccination, therapy. nevertheless the favoured technique is vaccination. seventeen of the twenty known viral contagions of horses cause disease and vaccines have been developed against five of them (table viii ). in addition, parainfluenza- vaccines have been developed for use in humans and cattle (potash, tytell, sweet, machlowitz, stokes, weibel, woodhour and hilleman, ; byrne, abinanti and huebner, ) . how efficient are the vaccines? in francis published a classification of human viral infections based on the duration of the infection in the host and the durability of the immunity induced. he recognised four groups; the first were diseases in which the virus persisted and immunity did not; the second were diseases in which the infection was transient and immunity was persistent; the third were the diseases in which both the francis classification furnishes a guide as to whether or not vaccines are feasible and whether or not they can be expected to be efficient. diseases falling into the first group will never be controlled by vaccines. in contrast, diseases falling into the other three groups are amenable to control by vaccination but the only efficient vaccines as measured by the durability of the immunity induced are those developed against the diseases classified in the second group. equine viral diseases occupy every niche of the francis classification (table ix) . thus equine infectious anaemia is ranked in the first group, the group of diseases for which vaccines cannot be developed. equine serum hepatitis, if it is a virus infection, probably also belongs to the first group. eleven diseases are assigned to the second group and vaccines developed against these are, or can be expected to be highly efficient inducing long-lasting sterile immunity. the viruses responsible are papilloma, the poxes, rabies and the arboviruses. six diseases fall into the third group and vaccines developed against these diseases in general, have proved disappointing. the viruses responsible are characterised by having a multiplicity of serotypes. there are, for oxyuridine, likewise, acts against smallpox and human herpes. others will follow-they will be needed! example, nine serotypes of african horse sickness virus. undoubtedly, many of the poor results from vaccination can be attributed to antigenic drift because immunity to the homologous serotype is often of long duration. these are the diseases in which the virus persists. consequently relapses are common despite the presence of antibodies. successful vaccination against the members of this group necessitates use of live attenuated viruses and it is, in effect, deliberate infection. three of the current vaccines developed against vectortransmitted viruses and three likely to be developed in the future can therefore be classed as excellent. three current vaccines are poor. two still to be developed will probably be poor. in contrast, three of the five current vaccines used against equine viral contagions are vaccines of low efficacy and, in the future, it is likely that eleven out of sixteen possible vaccines will be poor products. treatment if the forecast that most of the vaccines developed against equine viral contagions will be of low efficacy is correct then other control measures must be exploited. treatment whether by serotherapy or by chemotherapy of established viral infections is doomed to failure because the virus population curve in the infected animal reaches its peak before the onset of clinical signs. for example, in horses infected with eastern equine encephalitis the peak of the virus population curve occurs one day before the onset of fever and five days before the onset of encephalitis (byrne, french, yancey, gochenour, russell, ramsburg, brand, scheider and buescher, ) . similarly, in horses infected with influenza a/equi/ virus the peak of the virus population curve coincides with the first wave of the intermittent fever and precedes by many hours the onset of coughing (blaskovic, kapitancik, sabo, styk, vrtiak and kaplan, ) . treatment, therefore, to be successful must be prophylactically administered; it is too late to alter the course of the first clinical case but the disease can be averted in the in-contacts. the common prophylactic treatment is the administration of antiserum. unfortunately it is expensive and not without dangers, particularly, the risk of transmitting serum hepatitis. interferon is another possible natural product but it has failed to live up to its initial promise. many pharmaceutical houses are synthesising and developing anti-viral drugs but the "breakthrough" has yet to come. amantadine appears to have a selective action on influenza virus. methisazone shows promises in protecting smallpox contacts. lodode-ten diseases are assigned to the fourth group. twelve dna viruses and forty-three rna viruses are known to infect horses. in addition, there are three unclassified viruses and, at least, three alleged viruses infecting horses. at least twenty-eight of the fifty-eight viruses induce clinical disease but the range of syndromes is limited; eleven provoke respiratory symptoms and eleven cause encephalitis. thirty-four equine viruses with a limited geographical distribution are transmitted by arthropod vectors. twenty viruses are spread by contact and their distribution, in general, is global. the vector-transmitted virus diseases are best controlled by prophylactic vaccination. the viral contagions are not, in general, well controlled by vaccination and it is likely that prophylactic chemotherapy will become increasingly important in the future. differential diagnosis is difficult. le diagnostic differentiel est difficile. vingt huit au moins des cinquante trois virus provoquent des maladies cliniques mais la gamme des syndromes est restreinte : onze virus engendrent des symptomes respiratoires, onze determinent des encephalites. trente quatre virus dont la distribution geographique est limitke sont transmis par des arthropodes vecteurs. vingt virus sont disskmints par contact et leur repartition est en general i'echelle du globe. les maladies provoqukes par des virus a propagation vectorielle sont mieux controltes par une vaccination preventive. les contagions virales ne sont pas, en gtnkral, efficacement controlees par la vaccination et i'on peut penser que la chimiothkrapie preventive de ces affections connaitra une importance croissante i'avenir. mindestens der viren verursachen klinisch manifeste krankheiten, aber die eigenart der syndrome ist limitiert; elf davon provozieren respiratorische symptome und elf rufen encephalitis hervor. equine viren benotigen als vektoren arthropoden ; ihre geographische ausbreitung ist beschrankt. viren werden durch kontakt ubertragen; sie werden in der regel auf der ganzen welt angetroffen. die durch vektoren ubertragenen viruskrankheiten konnen am besten durch prophylaktische impfungen kontrolliert werden. die ubrigen konnen im allgemeinen durch impfungen nicht gut kontrolliert werden und es scheint wahrscheinlich, dass die prophylaktische chemotherapie in zukunft an bedeutung gewinnen wird. sumario virus equinos doce dna virus y cuarenta y tres rna virus son reconcodios como infectantes a caballo. en adicion hay tres no clasidicados virus y por lo menos tres tipos de virus muy se mejantes que afectan el caballo. el diagnostic diferencial es dificultoso. aproximadamente de s virus inducen enfermedades clinicas con y espectro de sindromes limitado. provocan sintomas respiratorios y causan encefalitis. virus equinos con limitacion en su distribucion geografica son transmitidos por vectores artropodos. veinte virus son diseminados por contact y su distribucion en general es global. es controlada la enfermeead de estos vectiores per medio de vacunacion. los contagio del virus no son bien controlados por lo general mediante vacunacion y solo una terapia profilactica que havenido tomando aunge en el futuro. die differentialdiagnose ist schwierig. viruses of vertebrates experimental infection of horses with a-equi /miami/ / and human a /hong kong/ / influenza viruses. i. the course of infection and virus recovery experimental immunization of cattle with myxovirus parainfluenza vaccines clinical and immunologic interrelationship among venezuelan, eastern, and western equine encephalomyelitis viruses in burros the properties and classification of two new rhinoviruses recovered from horses in upper respiratory disease in thoroughbred horses : studies of its viral etiology in the toronto area association of myxovirus parainfluenza (re ) with upper respiratory infection of horses rhinoviruses and parainfluenza viruses of horses isolation of a filterable agent causing arteritis of horses and abortion by mares. its differentiation from the equine abortion (influenza) virus. mechanisms of infection and immunity in virus diseases of man. immunological relationship between infectious cough in horses and human influenza a characterization of a cytomegalo-like virus isolated from spontaneously degenerated equine kidney culture cytopathogenic equine orphan (ceo) virus in horse kidney cell culture. . isolation and properties untersuchungen iiber die i bei pferden beobachtete hustenepidemie (hoppengartener husten) in der bundesrepublik prophylaxis of vesicular stomatitis: a field trial in guatemalan dairy cattle viral encephalitis of equines and domestic wesselsbron disease studies on grass disease. the morphological picture with special reference to the vegetative nervous system studies on the properties of african horse sickness virus an equine genital infection resembling coital exanthema associated with a virus anequine respiratory virus with enterovirus properties equine herpes viruses respiratory virus vaccines. amer. rev. resp. dis. isolation of a virus from the brain of a horse with "staggers diarrhoea in horses with particular reference to a chronic diarrhoea syndrome. acute liver atrophy and parenchymatous hepatitis in horses. th and th rep near-eastern equine erforschung der bornaschen krankheit des pferdes m. a. p. simons (epsom) opened the discussion and congratulated dr. scott on his review of the equine viruses. the urgency of a better knowledge of virus diseases had been underlined a t the recent international conference in paris. he asked dr. scott if viruses were associated with labial and oral lesions and with grease and mud fever, and he described a case of papular dermatitis in a -year-old thoroughbred and wondered if this might be a viral condition. mr. simons said that since many viruses known to infect horses did not occur normally in the british isles veterinary sanitary regulations were of the first importance and proper inspection by trained eyes was necessary. air transport was, of course, the hazard. was the use of insecticidal sprays of any value? on another aspect of epidemic control what proportion of a susceptible population was it necessary to vaccinate to halt the spread of infection? what veterinary laboratories were currently investigating virus diseases?dr. g. r. scott replied that papular dermatitis was a contagion of probably world-wide distribution. the value of insecticidal spraying to limit the spread of arbo-viruses was considerably aided by the inability of most arbo-viruses to flourish for any length of time outside their endemic home area. on control by vaccination it was generally accepted that vaccination of per cent of a population was adequate to abort a n epidemic.on the other hand there was a recent hypothesis that vaccination of only per cent was adequate to prevent measles spreading. current work on equine viruses was being carried out by robert burrows at pirbright, influenza was under study at cambridge and pox infections were under investigation at liverpool.dr. j. r. campbell (glasgow) asked for how long prophylactic treatment should be given to prevent outbreaks of virus infection.dr. scott replied that drugs might well be used in a closed community where animals could be segregated and movement stopped. drugs could be used prophylactically over a short period for a non-persistent virus. persistent viruses should be dealt with by infecting horses with live attenuated virus vaccines before they were required to hunt or race. g. n. sutherland (york) enquired if an alteration of serotype would affect the value of this year's 'flu vaccine.dr. scott agreed that it would and added that infection in an immunised animal was a good way to produce a new serotype. vaccine, if the one changed then the other must be changed.he asked why hunters acquired a better immunity than thoroughbreds.he was aware that young animals reacted more severely than older ones.dr. scott replying, did not agree that this was so. dr. g. r. scott advised periodic influenza vaccination and said that live vaccine against rhinopneumonitis and equine arteritis had been effective in producing lifelong immunity in the u.s.a. but there was some doubt if the use of such live vaccines was legal in the british isles. key: cord- -v r lr authors: vasan, aditya; friend, james title: medical devices for low- and middle-income countries: a review and directions for development date: - - journal: j med device doi: . / . sha: doc_id: cord_uid: v r lr the development of diagnostics and medical devices has historically been concentrated in high-income countries, despite a significant need to expand healthcare services to low- and middle-income countries (lmic). poor quality healthcare extends beyond lmic to underserved communities in developed countries. this paper reviews diseases and conditions that have not received much attention in the past despite imposing a significant burden on healthcare systems in these circumstances. we review the underlying mechanism of action of these conditions and current technology in use for diagnosis or surgical intervention. we aim to identify areas for technological development and review policy considerations that will enable real-world adoption. specifically, this review focuses on diseases prevalent in sub-saharan africa and south asia: melioidosis, infant and maternal mortality, schistosomiasis, and heavy metal and pesticide poisoning. our aim with this review is to identify problems facing the world that require the attention of the medical device community and provide recommendations for research directions for groups interested in this field. a formal search of peer-reviewed articles pertaining to diseases affecting low-and middle-income countries (lmic) was performed using google scholar and pubmed in order to compile this qualitative systematic review. keywords used to identify diseases and technologies included, but were not limited to, the following terms: medical devices, low-cost design, global health, low-income countries, low-and middle-income countries, pointof-care diagnostics, neglected tropical diseases (ntd), neonatal mortality. the names of specific health challenges in conjunction with other keywords were also used in the search. identified conditions were compared based upon the number of affected people, disability adjusted life years, and current research carried out to address the condition. the diseases chosen for this review are melioidosis and neglected tropical diseases such as schistosomiasis. these two diseases combined affect over , people in south asia and sub-saharan africa [ ] . this is a fraction of the number of people affected by mosquito-borne diseases [ ] , but still presents a significant healthcare burden on developing economies. in contrast to, for example, malaria or hiv, these diseases have not received notable attention nor the development of technological solutions toward their treatment. one of the key aims of this review is to expose this unmet need. a section of this review focuses on neonatal mortality, which accounts for over  deaths worldwide and is a significant economic burden due to the number of productive years of life lost [ ] . finally, a section on policy and implementation is provided to aid researchers in identifying useful areas of study. melioidosis (whitmore's disease) is an infection caused by the bacterium burkholderia pseudomallei and is prevalent in south and east asia and northern australia. in major regions where the disease is endemic, the annual incidence rates are up to cases per , people [ ] . it has been classified as a category b bioterrorism agent by the united states and recent reports indicate that the endemic areas have expanded to southern china, hong kong, taiwan, and parts of the americas [ ] . although healthcare facilities have seen significant improvements over the past few years, reports suggest that diagnosis of melioidosis is only possible in large regional referral centers. this could lead to a delay in diagnosis and treatment, and an underestimate of the number of people infected with the disease [ ] . diagnosis of melioidosis can be complicated by the presenting symptoms and their similarity to tuberculosis: abscesses in the lungs, liver, spleen, and brain stem encephalitis. the bacterium typically infects the host by inoculation onto the skin, by inhalation, or by ingestion. inoculation onto the skin can cause subcutaneous abscesses, inhalation can cause pneumonia, and the bacterium can reach internal organs such as the spleen, liver, and the lungs through the blood. the most common presentation of melioidosis is pneumonia, with over % of cases either occurring through lung abscess or due to septicemic spread. an additional challenge with melioidosis is that the bacteria, b. pseudo-mallei, is a difficult organism to kill, with studies showing that it is capable of surviving in triple distilled water for years [ ] . the disease primarily occurs during the rainy season [ ] and people who are in direct contact with wet soil are most vulnerable to the disease. predispositions such as diabetes mellitus, cirrhosis, and alcoholism, or those who are immunosuppressed increase the risk of contracting the bacterium [ ] . over % of patients diagnosed with melioidosis already have diabetes mellitus as a predisposition [ ] . this presents a major problem for south asian countries as the number of patients living with diabetes as an underlying condition is projected to increase by % between and [ ] . an additional risk factor is the age group for contracting diabetes mellitus in these countries is - , with reports showing that incidence of melioidosis peaking in this age group, and most countries of this region exhibiting aging demographics. the recommended treatment of melioidosis involves the intravenous injection of the antibiotic ceftazidime for at least days [ ] . the therapeutic response to the drug is slow and physicians tend to prematurely switch antibiotics in a considerable number of cases, with the assumption that the bacterium has developed antibiotic resistance. the mortality for a -week administration of the drug is % [ ] . although there have been developments over the past years with multiple clinical trials [ ] [ ] [ ] in treating melioidosis, the cost of the antibiotics is still high (from us$ -$ a day [ ] ), imposing a significant economic burden on developing countries. simpson et al. [ ] comment on the price of drug regimens used to treat the disease and cite lack of financial support from pharmaceutical companies for diseases prevalent in rural areas as one of the challenges in developing treatment and diagnostic solutions. melioidosis has been described as "the great imitator" of other tropical diseases such as malaria, typhoid fever, leptospirosis, and tuberculosis [ ] . the varying clinical manifestations of the disease and the multiple affected organs often lead to an incorrect diagnosis. delayed identification results in the administration of antibiotics that are usually unsuitable to treat melioidosis, which has led to high mortality rates as indicated by multiple studies [ , ] . it is critical that rapid diagnostic solutions are developed to reduce mortality rates. the most widely used method to detect b. pseudo-mallei is culturing in a modified ashdown medium with colistin [ ] , but even this technique may yield inaccurate results, especially in nonendemic areas where the clinical suspicion is low. in cases where there is a positive culture, it may be difficult for commercial systems to differentiate between b. pseudo-mallei and phenotypically similar species such as burkholderia thailandensis [ ] . recent reports highlight not only the limitations of bacterial gene amplification and detection but also the development of new screening and amplification techniques [ ] . since the sequencing of the complete genome of b. pseudo-mallei [ ] , several quantitative polymerase chain reaction (pcr) assays have been developed to identify the species [ ] . however, it is still a challenge to identify primers that can differentiate b. pseudo-mallei from closely related species. clinical laboratories have recently started adopting matrixassisted laser desorption/ionization mass spectrometry systems for the identification of bacterium [ ] [ ] [ ] [ ] , with modest clinical success alongside reports that suggest the databases used mass spectrometry require expansion to accurately identify b. pseudomallei. this technique shows great potential to be used as a rapid identification technique but has the limitation of requiring skilled personnel, expensive equipment, and the infrastructure to support them. low-cost molecular profiling and analysis has been a challenge, and advancements in this field would have a significant beneficial impact on healthcare outcomes in endemic areas. surface acoustic wave-based atomization has been proposed to simplify the sample extraction and filtration process [ ] . highfrequency surface acoustic wave-based atomization was used for the formation of micrometer scale droplets of cell lysate, and the purification step was achieved by loading the sample on a porous membrane placed in the direction of propagation of the surface acoustic waves. since the main cause of melioidosis spread is through soil during the rainy season, analyzing soil for the presence of b. pseudomallei would prevent infection and hospitalization. environmental samples are difficult to purify via pcr due to the typical presence of a diverse species of bacteria and fungi. development of better enrichment and purification techniques would enable an increase in the number of relevant dna templates for amplification to occur. an alternative to sampling environmental specimens is the analysis of relevant clinical samples-blood and sputum [ , ] . the sensitivity of pcr-based techniques is high for sputum and pus, but, by contrast, the sensitivity for blood samples is quite low [ , ] . there may be as many as , cases of melioidosis every year with an estimated , deaths [ ] . the most commonly used technique for identification, bacterial culture, is % specific but has low sensitivity. slow or incorrect diagnosis results in a significant burden on healthcare systems and can be fatal. matrix assisted laser desorption/ionization-time of flight mass spectrometry is increasingly being used as a rapid and accurate technique to identify isolates [ , ] , though significant challenges to its adoption remain but likely can be addressed by further research in this field. this section details the risks associated with poisoning due to metals, such as lead and cadmium, and associated detection techniques. additionally, we consider the effects and detection of environmental pollutants arising from intensive agricultural and industrial practices. evidence of the toxicity of lead has been documented in studies conducted as early as [ ] and studies demonstrating the adverse effects of lead on attention, childhood development, and neurological disorders were established in the s [ ] . lead poisoning has been shown to impair cognitive ability and social function [ ] . the toxic properties of lead are partially due to its ability to compete with calcium at a cellular level, for neuronal signaling [ ] and in inhibiting calcium influx into cells [ ] . the neurological effects are due to its inhibitory impact on stimulated neurotransmitter release [ , ] . at high doses, lead can inhibit myelin formation and compromises the integrity of the blood brain barrier. children are more sensitive to lead than adults due to increased hand-to-mouth activity and increased absorption in the gut. the acceptable thresholds for lead in blood have drastically changed since the s in the united states, from lg/dl to essentially recognizing that there is no safe level of lead exposure, with amounts under lg/dl having been shown to adversely impact learning [ ] . significant reduction in lead exposure in the united states has been achieved by eliminating lead in gasoline and paint, but lead contamination of water remains a problem without a commensurate level of attention [ ] . the decision in by the city of flint to change its primary source of water saw lead levels in water rise to as much as lg/dl in some cases [ ] , with lasting healthcare implications. the only medical treatment for lead exposure is chelation [ ] , but chelating drugs may not be available in lmic and in lower income regions of even wealthy countries. in any case, they are not very useful to treat chronic exposure. another heavy metal that has adverse health effects is cadmium, commonly used in the electronics industry in batteries and circuit boards. chronic exposure to cadmium results in accumulation in the kidneys that eventually leads to renal damage [ ] . cadmium can also be absorbed by plants more readily than other heavy metals, presenting another mode of uptake in regions contaminated with industrial waste. the number of people worldwide affected by heavy metal poisoning is difficult to estimate due to the varying sources of poisoning but, given that incidents like the one in flint that occurred in a developed country with safeguards in place to prevent lead poisoning, there is a need to estimate atmospheric and water-based heavy metal exposure risks across the world. the daily per-capita supply of calories has increased by over % over the last years [ ] , and as this demand increases, there has been a push toward maximizing agricultural productivity. increasing agricultural yield has been possible, in part, by the increased use of pesticides. the food and agriculture organization of the united nations defines pesticides as growth regulators and includes substances that can prevent harm to crop during or after harvest [ ] . exposure to pesticides may occur through direct means through occupational exposure or via indirect means through drinking water, dust and food. the world health organization (who) estimates that  severe pesticide poisonings occur annually and that at least , people die as a result of exposure, with % of these cases being from lmics [ ] . the effects of pesticide exposure are wide ranging: chronic diseases such as cancer and developmental disorders [ ] , immunosuppression, hormone disruption, diminished intelligence, and reproductive abnormalities [ ] . of the various classes of pesticides in use today, organophosphate pesticides have been shown to cause substantial morbidity and mortality [ ] but still account for over % of fertilizer produced [ , ] . organophosphates are readily absorbed through the skin, and the gastrointestinal and respiratory tracts. the mechanism of action is by inhibition of acetylcholnesterase, which promotes the production of the neurotransmitter acetylcholine [ ] . overstimulation of nerves due to excess acetylcholine results in autonomic dysfunction, involuntary motor movements, and respiratory depression. long-term exposure has been shown to result in carcinogenicity [ ] . many farmers in developing countries are exposed to pesticides due to unsafe storage and handling practices, a lack of protective equipment during spraying, or chronic exposure to contaminated soil and water. although there have been attempts to implement safe handling practices [ ] and reduce pesticide use, there is a need to develop protective equipment that can be deployed in developing countries to prevent exposure alongside devices that can detect the presence of harmful pesticides in the local environment. there is furthermore a need to monitor the presence of heavy metals and organophosphates in water supplies and in soil, particularly in regions of significant industrial or agricultural output. paper-based microfluidics was first reported by george whitesides' group [ ] and has the advantage of being inexpensive, readily available, and can wick biological fluids without active pumping. a recent point-of-care paper diagnostic assay for the detection of lead and mercury in water was developed by lewis et al. [ ] . the device consists of hydrophobic regions that define where the fluid travels through capillary action. the assay is designed in such a way that the time taken for the fluid to travel from one part of the chip to another is indicative of the concentration of the analyte. this is accomplished by the use of glucose oxidase or streptavidin, which inhibit flow depending on analyte concentration. it has not been determined if time-based assays provide quantitative results for a range of users. more sensitive tests to detect the presence of lead in drinking water have been developed, with one group reporting a fourfold increase in sensitivity over conventional lateral flow assays [ ] . the increased sensitivity was achieved by conjugating gold nanoparticles to a lead-specific monoclonal antibody (anti-pb(ii)-itcbe). organophosphate detection schemes have been considered due to their historical use as chemical warfare agents [ ] , though there has been a lack of development in the detection of trace amounts of organophosphates, despite knowledge they pose a significant health risk. as of , about  babies die in the neonatal period (the first weeks of life) and a similar number of babies are stillborn [ ] . a majority of these deaths are in low-income and middleincome countries in africa and southeast asia [ ] as illustrated in fig. . some estimates show that % of all deaths in children under the age of five occur within the first month of life [ ] . assessment of the cause of death is difficult due to a lack of data collection in lmics, but estimates indicate that complications arising from asphyxia, severe infections, and preterm births are the main causes of death [ ] . the who defines birth asphyxia as the clinical description of a newborn that fails to initiate or maintain regular breathing at birth [ ] . this term applies to a clinical condition and is not a specific cause of death. advanced resuscitation is needed for less than % of babies at birth and babies that typically require advanced resuscitation by intubation typically require a neonatal intensive care unit to recover [ , ] . these are typically not available in regional hospitals in lmics. basic resuscitation, however, which only requires bag-and mask-type ventilators, is easy to implement for cases where intrapartum breathing assistance is required. published guidelines vary on when to provide resuscitation [ ] , but the who recommends resuscitation in cases where the baby does not cry, breathe at all, or is gasping for s [ ] . it is crucial that these guidelines become widely known, as there is potential for adverse effects such as upper airway damage if respiratory support is provided to healthy babies [ ] . existing devices in use in low-resource settings include mucus extractors with one-way valves and rubber bulb suction devices. the key downside of these devices is that they present an infection hazard for both the neonate and the healthcare provider [ ] , alongside the potential risk of hypothermia in prolonged resuscitation of a neonate [ , ] , and asphyxia. however, asphyxia may be overcome with expanded care and improved healthcare provider training, but it is crucial that clear protocols for resuscitation be established first. congenital abnormalities [ ] likewise receive less attention than other medical conditions, especially given that they account for % of all neonatal deaths. one of the most common abnormalities in neonates is spina bifida, which results from the failure of fusion of the caudal neural tube. a large percentage of cases can be prevented by folic acid supplementation as long as the condition is diagnosed [ ] . the underlying cause of the condition is not known but it has been established that women with pregestational diabetes or a high body mass index are at an increased risk of having a child with spina bifida. the protein a-fetoprotein (afp) and ultrasound can each be used to identify fetuses that have the condition [ ] . point-of-care testing of afp would enable screening and potential early detection of spina bifida and allow the patient to seek further medical care in a primary setting. the typical values in maternal serum are under ng/ml until - weeks of gestation [ ] . a quantum-dot-based sensor for the detection of afp has been reported by yang et al. [ ] , with sensitivities to ng/ml. this test, however, relies on comparing the relative fluorescence intensities of the sample with a control, and can be difficult to interpret in the absence of optics and skilled personnel. the who compendium of medical devices and technologies for low-resource settings lists two ultrasound scanners, which are priced at us$ , and us$ , [ ] , still a significant cost in comparison to the annual budget of many hospitals in low-resource settings [ ] . ultrasound is otherwise commonly used to diagnose congenital structural defects during pregnancy. one such condition typically diagnosed using fetal ultrasound is hydrocephalus, a condition where fluid accumulates in the ventricles within the brain. the treatment for this condition involves the implant of a shunt that drains away excess cerebrospinal fluid into the abdominal cavity. the problem with this procedure irrespective of the settingwhether in an lmic or a high-income country-is that implanted shunts fail due to an obstruction caused by glial and inflammatory cells [ ] . a comparison between an inexpensive shunt developed and used in africa and one currently in use in the united states to treat the condition showed that there was no significant difference in clinical outcomes [ ] . the failure rate in the first years for implanted ventriculoperitoneal shunts remains high, at over % [ ] , indicating that there is a need to evaluate failure due to occlusion in greater detail. neglected tropical diseases are a group of thirteen bacterial and parasitic infections that affect around .  people worldwide [ ] as illustrated in fig. . they are highly prevalent in sub-saharan africa and south asia, where over % of the population lives on less than $ a day [ ] . these diseases make it difficult for people affected by them to rise out of poverty as their effects can be chronic or, in some cases, fatal. schistosomiasis is such an ntd, affecting  people in sub-saharan africa alone, accounting for % of the world's cases, and associated with increased human immunodeficiency virus transmission [ ] . about % of the population of sub-saharan africa lives near bodies of water that have been contaminated with schistosomiasis hosts [ ] , with those living near dam reservoirs at higher risk [ ] . the population most affected by schistosomiasis is children and young adults. schistosomiasis can be caused by two strains of bacterium, one of which affects the urinary tract and the second infecting the intestine. schistosoma haematobium is one of the leading causes for urinary tract infections, and around , deaths annually [ ] . schistosoma mansoni causes bowel ulceration and an estimated , deaths [ ] . adult male and female worms can live in the human host for an average of years and the disease spreads by the transmission of eggs to an intermediate snail host typically found in water bodies. the worms feed on glucose present in blood and erythrocytes and excrete waste into the blood stream. biomarkers present in waste may serve as a target for detecting schistosomiasis. the primary treatment for the disease is the drug praziquantel, which is only effective against mature worms [ ] . prolonged treatment for - weeks is required to completely cure the disease. although this drug is being employed in the field to treat schistosomiasis, the mechanism of action is still unknown and side effects include abdominal pain and passage of blood in the stool [ ] . the current diagnostic standard for schistosomiasis is the presence of viable eggs in urine or fecal matter, but the test has a low sensitivity [ ] . the three recommended detection techniques by the who are microscopy, a urine-based dipstick assay [ ] , and the kato-katz fecal examination [ ] . the kato-katz method was developed in the s and uses a cardboard cutout over a glass slide and microscopy to easily count the number of eggs in a sample and quantify the number of eggs per unit weight but this technique, like the others recommended by the who, lack sensitivity. a point-of-care lateral flow assay to detect the presence of circulating cathodic antigen was developed [ ] , addressing the sensitivity limitations of the kato-katz technique, but only for schistosoma mansoni. it did no better than the kato-katz technique when used for stool samples. recent reports on diagnosis of the disease have called for better diagnostic tools [ ] , one of which is pet (positron emission tomography) imaging. this group made use of glucose intake of the worms in vivo to detect and quantify their presence using a pet scanner. with groups starting to develop handheld pet scanners [ ] , this might present a novel research direction for groups looking to develop diagnostic technologies. pcr-based techniques have also been developed to analyze cell-free parasite deoxyribonucleic acid in human plasma [ ] , and the development of microfluidic pcr-based systems [ ] for schistosomiasis could lead to improved detection. control measures against the disease include preventative therapy using praziquantel [ ] , issued by schools screening and treating children for this and other diseases. an additional screening technique that has been attempted in the past is to monitor and eliminate snails that serve as intermediate hosts. the who in pledged to control the morbidity caused by the disease by and eliminate it as a public health problem by , leading to an increased effort in diagnosing and educating the communities most affected by the disease. this has stimulated increased funding and opportunities for novel interventions. there have been significant improvements in healthcare outcomes over the past few decades due to a combination of efforts by public and private organizations, and technological advancements in diagnostics and surgical devices. despite this, there are some limitations to adoption and implementation of technology. in some cases, the underlying disease or condition may not have a device or diagnostic solution. this is common in the case of neglected tropical diseases that are a significant health burden but have seen insufficient investment from the private sector. a point-of-care monoclonal antibody-based dipstick for urinary schistosomiasis was developed by a ghanian group in [ ] but has not been commercialized [ ] . the technology may exist, but other problems such as distribution or energy required to power the devices may not exist [ ] . oxytocin is used to reduce the risk of postpartum hemorrhage but requires refrigeration, unavailable throughout the day in resourcelimited settings. advances in packaging that could extend the shelf life of drugs and vaccines would expand access to available care in these cases. in the case of diagnostics, a more sensitive and specific test may exist but there could be reluctance to adopt it or mistrust in the results it produces due to past misdiagnosis with old technology. rapid diagnostic tests for malaria have been shown to have superior sensitivity and specificity than the current gold standard for malaria detection, microscopy [ ] . but a study found that doctors prescribed antimalarial drugs-even in the case of a negative test result [ , ] . skilled healthcare is crucial to improve surgical outcomes. for example, access to skilled surgeons for delivery is as low as % in sub-saharan africa and less than % in south asia [ ] . designing medical devices for lmic's presents unique challenges not seen in developed markets. the who estimates that % of medical equipment coming from developed countries does not work in hospitals in developing countries [ , ] due to lack of trained personnel, limitations with infrastructure, and the lack of spare parts or support for equipment. it is evident from these examples that research and development of novel diagnostic tools and devices alone is insufficient for adoption. in the context of the conditions presented in this paper, there is a need to develop an effective way to screen for schistosomiasis and melioidosis. the spread of melioidosis starts through soil in the rainy season and the presence of schistosomiasis can easily be detected in fecal matter. a technique to purify samples coupled with existing point-of-care diagnostics would stop the spread of the disease at the source. this can be extended to detect the presence of environmental contaminants such as organophosphates and heavy metals. an increase in sensitivity can be achieved if adequate processing is done prior to point-of-care diagnosis. the un's fourth millennium development goal called for a two-third reduction in neonatal mortality by . although there has been significant progress toward this goal, there are still  neonatal deaths every year, predominantly in south asia and africa. developing medical devices specifically for neonatal care and not having to repurpose devices made for adults would enable easier surgical procedures. better detection schemes for diseases such as spina bifida or hydrocephalus using low-cost ultrasound transducers would prepare doctors better for surgery. it is critical to rethink what low-cost means for such applications, especially considering the annual budget of hospitals in lmic's. frugal technologies that can be used repeatedly can withstand environmental conditions and can be used easily by healthcare workers in target countries should be prioritized. schistosomiasis (primer) clinical review: severe malaria cost effectiveness analysis of strategies for maternal and neonatal health in developing countries melioidosis the global distribution of burkholderia pseudomallei and melioidosis: an update melioidosis: the tip of the iceberg? survival of burkholderia pseudomallei in the absence of nutrients melioidosis: epidemiology, pathophysiology, and management risk factors for melioidosis and bacteremic melioidosis melioidosis global prevalence of diabetes: estimates for the year and projections for 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to detect burkholderia pseudomallei in clinical specimens from patients with suspected melioidosis performance evaluation of active melioidosis detect-lateral flow assay (amd-lfa) for diagnosis of melioidosis in endemic settings with limited resources development of a prototype lateral flow immunoassay (lfi) for the rapid diagnosis of melioidosis predicted global distribution of burkholderia pseudomallei and burden of melioidosis maldi-tof mass spectrometry: an emerging technology for microbial identification and diagnosis maldi-tof mass spectrometry tools for bacterial identification in clinical microbiology laboratory lead poisoning in children with especial reference to lead as a cause of convulsions effects of inorganic lead on behavioral and neurologic function neuropathology of heavy metal intoxication picomolar concentrations of lead stimulate brain protein kinase c lead-calcium interactions in cellular lead toxicity effects of chronic lead ingestion on social development in infant rhesus monkeys behavioral impairments produced by developmental lead exposure: evidence from primate research subclinical neuropathy at "safe" levels of lead exposure lead contamination in flint-an abject failure to protect public health flint water study lead toxicity and chelation therapy heavy metal poisoning: the effects of cadmium on the kidney per-capita food consumption public health impact of pesticides used in agriculture adverse health effects of pesticides in agrarian populations of developing countries pesticides as a cause of occupational skin diseases in farmers subclinical health effects of environmental pesticide contamination in a developing country: cholinesterase depression in children chronic central nervous system effects of acute organophosphate pesticide intoxication total fertilizer production fertilizer and pesticides organophosphate pesticide exposure and neurodegeneration health effects of chronic pesticide exposure: cancer and neurotoxicity facing hazards at work-agricultural workers and pesticide exposure in kuttanad, kerala diagnostics for the developing world: microfluidic paper-based analytical devices a prototype point-of-use assay for measuring heavy metal contamination in water using time as a quantitative readout rapid and highly sensitive detection of lead ions in drinking water based on a strip immunosensor new method for retrospective detection of exposure to organophosphorus anticholinesterases: application to alleged sarin victims of japanese terrorists million neonatal deaths: when? where? why? perinatal mortality for the year : estimates developed by who child and infant mortality infant mortality in guidelines for health care equipment donations the impact of the neonatal resuscitation program guidelines (nrpg) on the neonatal mortality in a hospital in zhuhai, china impact of education and training on neonatal resuscitation practices in teaching hospitals in india resuscitation of the newly born infant: an advisory statement from the pediatric working group of the international liaison committee on resuscitation the pros and cons of suctioning at the perineum (intrapartum) and post-delivery with and without meconium neonatal resuscitation in low-resource settings: what, who, and how to overcome challenges to scale up? the epicure study: outcomes to discharge from hospital for infants born at the threshold of viability the transparent baby bag: a shield against heat loss spina bifida screening for neural tube defects comparison of amniotic-fluid and maternal serum alpha-fetoprotein levels in the early antenatal diagnosis of spina bifida and anencephaly quantum dot-based immunochromatography test strip for rapid, quantitative and sensitive detection of alpha fetoprotein who medical devices setting healthcare priorities: a description and evaluation of the budgeting and planning process in county hospitals in kenya pathophysiology of shunt dysfunction in shunt treated hydrocephalus comparison of -year outcomes for the chhabra and codman-hakim micro precision shunt systems in uganda: a prospective study in children randomized trial of cerebrospinal fluid shunt valve design in pediatric hydrocephalus control of neglected tropical diseases neglected tropical diseases the developing world is poorer than we thought, but no less successful in the fight against poverty association between genital schistosomiasis and hiv in rural zimbabwean women geographical information system and predictive risk maps of urinary schistosomiasis in ogun state schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk the global burden of urinary bladder cancer quantification of clinical morbidity associated with schistosome infection in sub-saharan africa praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis utility of repeated praziquantel dosing in the treatment of schistosomiasis in high-risk communities in africa: a systematic review validation of a chart to estimate true schistosoma mansoni prevalences from simple egg counts the control of schistosomiasis. second report of the who expert committee a simple device for quantitative stool thick-smear technique in schistosomiasis mansoni a five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of schistosoma mansoni human schistosomiasis portable pet probes are a novel tool for intraoperative localization of tumor deposits diagnosing schistosomiasis by detection of cell-free parasite dna in human plasma analytical study of a microfluidic dna amplification chip using water cooling effect the control of schistosomiasis: report of a who expert committee applicability of a monoclonal antibody-based dipstick in diagnosis of urinary schistosomiasis in the central region of ghana stagnant health technologies in africa medical device landscape for communicable and noncommunicable diseases in low-income countries rapid diagnostic testing for malaria point-of-care diagnostics for global health rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in tanzania: randomised trial barriers for medical devices for the developing world technologies for global health key: cord- -hr smx authors: van kampen, antoine h. c.; moerland, perry d. title: taking bioinformatics to systems medicine date: - - journal: systems medicine doi: . / - - - - _ sha: doc_id: cord_uid: hr smx systems medicine promotes a range of approaches and strategies to study human health and disease at a systems level with the aim of improving the overall well-being of (healthy) individuals, and preventing, diagnosing, or curing disease. in this chapter we discuss how bioinformatics critically contributes to systems medicine. first, we explain the role of bioinformatics in the management and analysis of data. in particular we show the importance of publicly available biological and clinical repositories to support systems medicine studies. second, we discuss how the integration and analysis of multiple types of omics data through integrative bioinformatics may facilitate the determination of more predictive and robust disease signatures, lead to a better understanding of (patho)physiological molecular mechanisms, and facilitate personalized medicine. third, we focus on network analysis and discuss how gene networks can be constructed from omics data and how these networks can be decomposed into smaller modules. we discuss how the resulting modules can be used to generate experimentally testable hypotheses, provide insight into disease mechanisms, and lead to predictive models. throughout, we provide several examples demonstrating how bioinformatics contributes to systems medicine and discuss future challenges in bioinformatics that need to be addressed to enable the advancement of systems medicine. systems medicine fi nds its roots in systems biology, the scientifi c discipline that aims at a systems-level understanding of, for example, biological networks, cells, organs, organisms, and populations. it generally involves a combination of wet-lab experiments and computational (bioinformatics) approaches. systems medicine extends systems biology by focusing on the application of systems-based approaches to clinically relevant applications in order to improve patient health or the overall well-being of (healthy) individuals [ ] . systems medicine is expected to change health care practice in the coming years. it will contribute to new therapeutics through the identifi cation of novel disease genes that provide drug candidates less likely to fail in clinical studies [ , ] . it is also expected to contribute to fundamental insights into networks perturbed by disease, improved prediction of disease progression, stratifi cation of disease subtypes, personalized treatment selection, and prevention of disease. to enable systems medicine it is necessary to characterize the patient at various levels and, consequently, to collect, integrate, and analyze various types of data including not only clinical (phenotype) and molecular data, but also information about cells (e.g., disease-related alterations in organelle morphology), organs (e.g., lung impedance when studying respiratory disorders such as asthma or chronic obstructive pulmonary disease), and even social networks. the full realization of systems medicine therefore requires the integration and analysis of environmental, genetic, physiological, and molecular factors at different temporal and spatial scales, which currently is very challenging. it will require large efforts from various research communities to overcome current experimental, computational, and information management related barriers. in this chapter we show how bioinformatics is an essential part of systems medicine and discuss some of the future challenges that need to be solved. to understand the contribution of bioinformatics to systems medicine, it is helpful to consider the traditional role of bioinformatics in biomedical research, which involves basic and applied (translational) research to augment our understanding of (molecular) processes in health and disease. the term "bioinformatics" was fi rst coined by the dutch theoretical biologist paulien hogeweg in to refer to the study of information processes in biotic systems [ ] . soon, the fi eld of bioinformatics expanded and bioinformatics efforts accelerated and matured as the fi rst (whole) genome and protein sequences became available. the signifi cance of bioinformatics further increased with the development of highthroughput experimental technologies that allowed wet-lab researchers to perform large-scale measurements. these include determining whole-genome sequences (and gene variants) and genome-wide gene expression with next-generation sequencing technologies (ngs; see table for abbreviations and web links) [ ] , measuring gene expression with dna microarrays [ ] , identifying and quantifying proteins and metabolites with nmr or (lc/ gc-) ms [ ] , measuring epigenetic changes such as methylation and histone modifi cations [ ] , and so on. these, "omics" technologies, are capable of measuring the many molecular building blocks that determine our (patho)physiology. genome-wide measurements have not only signifi cantly advanced our fundamental understanding of the molecular biology of health and disease but table abbreviations and websites have also contributed to new (commercial) diagnostic and prognostic tests [ , ] and the selection and development of (personalized) treatment [ ] . nowadays, bioinformatics is therefore defi ned as "advancing the scientifi c understanding of living systems through computation" (iscb), or more inclusively as "conceptualizing biology in terms of molecules and applying 'informatics techniques' (derived from disciplines such as applied mathematics, computer science and statistics) to understand and organize the information associated with these molecules, on a large scale" [ ] . it is worth noting that solely measuring many molecular components of a biological system does not necessarily result in a deeper understanding of such a system. understanding biological function does indeed require detailed insight into the precise function of these components but, more importantly, it requires a thorough understanding of their static, temporal, and spatial interactions. these interaction networks underlie all (patho)physiological processes, and elucidation of these networks is a major task for bioinformatics and systems medicine . the developments in experimental technologies have led to challenges that require additional expertise and new skills for biomedical researchers: • information management. modern biomedical research projects typically produce large and complex omics data sets , sometimes in the order of hundreds of gigabytes to terabytes of which a large part has become available through public databases [ , ] sometimes even prior to publication (e.g., gtex, icgc, tcga). this not only contributes to knowledge dissemination but also facilitates reanalysis and metaanalysis of data, evaluation of hypotheses that were not considered by the original research group, and development and evaluation of new bioinformatics methods. the use of existing data can in some cases even make new (expensive) experiments superfl uous. alternatively, one can integrate publicly available data with data generated in-house for more comprehensive analyses, or to validate results [ ] . in addition, the obligation of making raw data available may prevent fraud and selective reporting. the management (transfer, storage, annotation, and integration) of data and associated meta-data is one of the main and increasing challenges in bioinformatics that needs attention to safeguard the progression of systems medicine. • data analysis and interpretation . bioinformatics data analysis and interpretation of omics data have become increasingly complex, not only due to the vast volumes and complexity of the data but also as a result of more challenging research ques- tions. bioinformatics covers many types of analyses including nucleotide and protein sequence analysis, elucidation of tertiary protein structures, quality control, pre-processing and statistical analysis of omics data, determination of genotypephenotype relationships, biomarker identifi cation, evolutionary analysis, analysis of gene regulation, reconstruction of biological networks, text mining of literature and electronic patient records, and analysis of imaging data. in addition, bioinformatics has developed approaches to improve experimental design of omics experiments to ensure that the maximum amount of information can be extracted from the data. many of the methods developed in these areas are of direct relevance for systems medicine as exemplifi ed in this chapter. clearly, new experimental technologies have to a large extent turned biomedical research in a data-and compute-intensive endeavor. it has been argued that production of omics data has nowadays become the "easy" part of biomedical research, whereas the real challenges currently comprise information management and bioinformatics analysis. consequently, next to the wet-lab, the computer has become one of the main tools of the biomedical researcher . bioinformatics enables and advances the management and analysis of large omics-based datasets, thereby directly and indirectly contributing to systems medicine in several ways ( fig. . quality control and pre-processing of omics data. preprocessing typically involves data cleaning (e.g., removal of failed assays) and other steps to obtain quantitative measurements that can be used in downstream data analysis. . (statistical) data analysis methods of large and complex omicsbased datasets. this includes methods for the integrative analysis of multiple omics data types (subheading ), and for the elucidation and analysis of biological networks (top-down systems medicine; subheading ). systems medicine comprises top-down and bottom-up approaches. the former represents a specifi c branch of bioinformatics, which distinguishes itself from bottom-up approaches in several ways [ , , ] . top-down approaches use omics data to obtain a holistic view of the components of a biological system and, in general, aim to construct system-wide static functional or physical interaction networks such as gene co-expression networks and protein-protein interaction networks. in contrast, bottom-up approaches aim to develop detailed mechanistic and quantitative mathematical models for sub-systems. these models describe the dynamic and nonlinear behavior of interactions between known components to understand and predict their behavior upon perturbation. however, in contrast to omics-based top-down approaches, these mechanistic models require information about chemical/physical parameters and reaction stoichiometry, which may not be available and require further (experimental) efforts. both the top-down and bottom-up approaches result in testable hypotheses and new wet-lab or in silico experiments that may lead to clinically relevant fi ndings. biomedical research and, consequently, systems medicine are increasingly confronted with the management of continuously growing volumes of molecular and clinical data, results of data analyses and in silico experiments, and mathematical models. due fig. the contribution of bioinformatics ( dark grey boxes ) to systems medicine ( black box ). (omics) experiments, patients, and public repositories provide a wide range of data that is used in bioinformatics and systems medicine studies to policies of scientifi c journals and funding agencies, omics data is often made available to the research community via public databases. in addition, a wide range of databases have been developed, of which more than are currently listed in the molecular biology database collection [ ] providing a rich source of biomedical information. biological repositories do not merely archive data and models but also serve a range of purposes in systems medicine as illustrated below from a few selected examples. the main repositories are hosted and maintained by the major bioinformatics institutes including ebi, ncbi, and sib that make a major part of the raw experimental omics data available through a number of primary databases including genbank [ ] , geo [ ] , pride [ ] , and metabolights [ ] for sequence, gene expression, ms-based proteomics, and ms-based metabolomics data, respectively. in addition, many secondary databases provide information derived from the processing of primary data, for example pathway databases (e.g., reactome [ ] , kegg [ ] ), protein sequence databases (e.g., uniprotkb [ ] ), and many others. pathway databases provide an important resource to construct mathematical models used to study and further refi ne biological systems [ , ] . other efforts focus on establishing repositories integrating information from multiple public databases. the integration of pathway databases [ - ] , and genome browsers that integrate genetic, omics, and other data with whole-genome sequences [ , ] are two examples of this. joint initiatives of the bioinformatics and systems biology communities resulted in repositories such as biomodels, which contains mathematical models of biochemical and cellular systems [ ] , recon that provides a communitydriven, consensus " metabolic reconstruction " of human metabolism suitable for computational modelling [ ] , and seek, which provides a platform designed for the management and exchange of systems biology data and models [ ] . another example of a database that may prove to be of value for systems medicine studies is malacards , an integrated and annotated compendium of about , human diseases [ ] . malacards integrates disease sources into disease cards and establishes gene-disease associations through integration with the well-known genecards databases [ , ] . integration with genecards and cross-references within malacards enables the construction of networks of related diseases revealing previously unknown interconnections among diseases, which may be used to identify drugs for off-label use. another class of repositories are (expert-curated) knowledge bases containing domain knowledge and data, which aim to provide a single point of entry for a specifi c domain. contents of these knowledge bases are often based on information extracted (either manually or by text mining) from literature or provided by domain experts [ - ] . finally, databases are used routinely in the analysis, interpretation, and validation of experimental data. for example, the gene ontology (go) provides a controlled vocabulary of terms for describing gene products, and is often used in gene set analysis to evaluate expression patterns of groups of genes instead of those of individual genes [ ] and has, for example, been applied to investigate hiv-related cognitive disorders [ ] and polycystic kidney disease [ ] . several repositories such as mir disease [ ] , peroxisomedb [ ] , and mouse genome informatics (mgi) [ ] include associations between genes and disorders, but only provide very limited phenotypic information. phenotype databases are of particular interest to systems medicine. one well-known phenotype repository is the omim database, which primarily describes single-gene (mendelian) disorders [ ] . clinvar is another example and provides an archive of reports and evidence of the relationships among medically important human variations found in patient samples and phenotypes [ ] . clinvar complements dbsnp (for singlenucleotide polymorphisms) [ ] and dbvar (for structural variations) [ ] , which both provide only minimal phenotypic information. the integration of these phenotype repositories with genetic and other molecular information will be a major aim for bioinformatics in the coming decade enabling, for example, the identifi cation of comorbidities, determination of associations between gene (mutations) and disease, and improvement of disease classifi cations [ ] . it will also advance the defi nition of the "human phenome," i.e., the set of phenotypes resulting from genetic variation in the human genome. to increase the quality and (clinical) utility of the phenotype and variant databases as an essential step towards reducing the burden of human genetic disease, the human variome project coordinates efforts in standardization, system development, and (training) infrastructure for the worldwide collection and sharing of genetic variations that affect human health [ , ] . to implement and advance systems medicine to the benefi t of patients' health, it is crucial to integrate and analyze molecular data together with de-identifi ed individual-level clinical data complementing general phenotype descriptions. patient clinical data refers to a wide variety of data including basic patient information (e.g., age, sex, ethnicity), outcomes of physical examinations, patient history, medical diagnoses, treatments, laboratory tests, pathology reports, medical images, and other clinical outcomes. inclusion of clinical data allows the stratifi cation of patient groups into more homogeneous clinical subgroups. availability of clinical data will increase the power of downstream data analysis and modeling to elucidate molecular mechanisms, and to identify molecular biomarkers that predict disease onset or progression, or which guide treatment selection. in biomedical studies clinical information is generally used as part of patient and sample selection, but some omics studies also use clinical data as part of the bioinformatics analysis (e.g., [ , ] ). however, in general, clinical data is unavailable from public resources or only provided on an aggregated level. although good reasons exist for making clinical data available (subheading . ), ethical and legal issues comprising patient and commercial confi dentiality, and technical issues are the most immediate challenges [ , ] . this potentially hampers the development of systems medicine approaches in a clinical setting since sharing and integration of clinical and nonclinical data is considered a basic requirement [ ] . biobanks [ ] such as bbmri [ ] provide a potential source of biological material and associated (clinical) data but these are, generally, not publicly accessible, although permission to access data may be requested from the biobank provider. clinical trials provide another source of clinical data for systems medicine studies, but these are generally owned by a research group or sponsor and not freely available [ ] although ongoing discussions may change this in the future ( [ ] and references therein). although clinical data is not yet available on a large scale, the bioinformatics and medical informatics communities have been very active in establishing repositories that provide clinical data. one example is the database of genotypes and phenotypes (dbgap) [ ] developed by the ncbi. study metadata, summarylevel (phenotype) data, and documents related to studies are publicly available. access to de-identifi ed individual-level (clinical) data is only granted after approval by an nih data access committee. another example is the cancer genome atlas (tcga) , which also provides individual-level molecular and clinical data through its own portal and the cancer genomics hub (cghub). clinical data from tcga is available without any restrictions but part of the lower level sequencing and microarray data can only be obtained through a formal request managed by dbgap. medical patient records provide an even richer source of phenotypic information , and has already been used to stratify patient groups, discover disease relations and comorbidity, and integrate these records with molecular data to obtain a systems-level view of phenotypes (for a review see [ ] ). on the one hand, this integration facilitates refi nement and analysis of the human phenome to, for example, identify diseases that are clinically uniform but have different underlying molecular mechanisms, or which share a pathogenetic mechanism but with different genetic cause [ ] . on the other hand, using the same data, a phenome-wide association study ( phewas ) [ ] would allow the identifi cation of unrelated phenotypes associated with specifi c shared genetic variant(s), an effect referred to as pleiotropy. moreover, it makes use of information from medical records generated in routine clinical practice and, consequently, has the potential to strengthen the link between biomedical research and clinical practice [ ] . the power of phenome analysis was demonstrated in a study involving . million patient records, not including genotype information, comprising disorders. in this study it was shown that disease phenotypes form a highly connected network suggesting a shared genetic basis [ ] . indeed, later studies that incorporated genetic data resulted in similar fi ndings and confi rmed a shared genetic basis for a number of different phenotypes. for example, a recent study identifi ed potentially pleiotropic associations through the analysis of snps that had previously been implicated by genome-wide association studies ( gwas) as mediators of human traits, and phenotypes derived from patient records of , individuals [ ] . this demonstrates that phenotypic information extracted manually or through text mining from patient records can help to more precisely defi ne (relations between) diseases. another example comprises the text mining of psychiatric patient records to discover disease correlations [ ] . here, mapping of disease genes from the omim database to information from medical records resulted in protein networks suspected to be involved in psychiatric diseases. integrative bioinformatics comprises the integrative (statistical) analysis of multiple omics data types. many studies demonstrated that using a single omics technology to measure a specifi c molecular level (e.g., dna variation, expression of genes and proteins, metabolite concentrations, epigenetic modifi cations) already provides a wealth of information that can be used for unraveling molecular mechanisms underlying disease. moreover, single-omics disease signatures which combine multiple (e.g., gene expression) markers have been constructed to differentiate between disease subtypes to support diagnosis and prognosis. however, no single technology can reveal the full complexity and details of molecular networks observed in health and disease due to the many interactions across these levels. a systems medicine strategy should ideally aim to understand the functioning of the different levels as a whole by integrating different types of omics data. this is expected to lead to biomarkers with higher predictive value, and novel disease insights that may help to prevent disease and to develop new therapeutic approaches. integrative bioinformatics can also facilitate the prioritization and characterization of genetic variants associated with complex human diseases and traits identifi ed by gwas in which hundreds of thousands to over a million snps are assayed in a large number of individuals. although such studies lack the statistical power to identify all disease-associated loci [ ] , they have been instrumental in identifying loci for many common diseases. however, it remains diffi cult to prioritize the identifi ed variants and to elucidate their effect on downstream pathways ultimately leading to disease [ ] . consequently, methods have been developed to prioritize candidate snps based on integration with other (omics) data such as gene expression, dnase hypersensitive sites, histone modifi cations, and transcription factor-binding sites [ ] . the integration of multiple omics data types is far from trivial and various approaches have been proposed [ - ] . one approach is to link different types of omics measurements through common database identifi ers. although this may seem straightforward, in practice this is complicated as a result of technical and standardization issues as well as a lack of biological consensus [ , - ] . moreover, the integration of data at the level of the central dogma of molecular biology and, for example, metabolite data is even more challenging due to the indirect relationships between genes, transcripts, and proteins on the one hand and metabolites on the other hand, precluding direct links between the database identifi ers of these molecules. statistical data integration [ ] is a second commonly applied strategy, and various approaches have been applied for the joint analysis of multiple data types (e.g., [ , ] ). one example of statistical data integration is provided by a tcga study that measured various types of omics data to characterize breast cancer [ ] . in this study breast cancer samples were subjected to whole-genome and -exome sequencing, and snp arrays to obtain information about somatic mutations, copy number variations, and chromosomal rearrangements. microarrays and rna-seq were used to determine mrna and microrna expression levels, respectively. reverse-phase protein arrays (rppa) and dna methylation arrays were used to obtain data on protein expression levels and dna methylation, respectively. simultaneous statistical analysis of different data types via a "cluster-of-clusters" approach using consensus clustering on a multi-omics data matrix revealed that four major breast cancer subtypes could be identifi ed. this showed that the intrinsic subtypes (basal, luminal a and b, her ) that had previously been determined using gene expression data only could be largely confi rmed in an integrated analysis of a large number of breast tumors. single-level omics data has extensively been used to identify disease-associated biomarkers such as genes, proteins, and metabolites. in fact, these studies led to more than , papers documenting thousands of claimed biomarkers, however, it is estimated that fewer than of these are currently used for routine clinical practice [ ] . integration of multiple omics data types is expected to result in more robust and predictive disease profi les since these better refl ect disease biology [ ] . further improvement of these profi les may be obtained through the explicit incorporation of interrelationships between various types of measurements such as microrna-mrna target, or gene methylation-microrna (based on a common target gene). this was demonstrated for the prediction of short-term and long-term survival from serous cystadenocarcinoma tcga data [ ] . according to the recent casym roadmap : "human disease can be perceived as perturbations of complex, integrated genetic, molecular and cellular networks and such complexity necessitates a new approach." [ ] . in this section we discuss how (approximations) to these networks can be constructed from omics data and how these networks can be decomposed in smaller modules. then we discuss how the resulting modules can be used to generate experimentally testable hypotheses, provide insight into disease mechanisms, lead to predictive diagnostic and prognostic models, and help to further subclassify diseases [ , ] (fig. ) network-based approaches will provide medical doctors with molecular level support to make personalized treatment decisions. in a top-down approach the aim of network reconstruction is to infer the connections between the molecules that constitute a biological network. network models can be created using a variety of mathematical and statistical techniques and data types. early approaches for network inference (also called reverse engineering ) used only gene expression data to reconstruct gene networks. here, we discern three types of gene network inference algorithms using methods based on ( ) correlation-based approaches, ( ) information-theoretic approaches, and ( ) bayesian networks [ ] . co-expression networks are an extension of commonly used clustering techniques , in which genes are connected by edges in a network if the amount of correlation of their gene expression profi les exceeds a certain value. co-expression networks have been shown to connect functionally related genes [ ] . note that connections in a co-expression network correspond to either direct (e.g., transcription factor-gene and protein-protein) or indirect (e.g., proteins participating in the same pathway) interactions. in one of the earliest examples of this approach, pair-wise correlations were calculated between gene expression profi les and the level of growth inhibition caused by thousands of tested anticancer agents, for cancer cell lines [ ] . removal of associations weaker than a certain threshold value resulted in networks consisting of highly correlated genes and agents, called relevance networks, which led to targeted hypotheses for potential single-gene determinants of chemotherapeutic susceptibility. information-theoretic approaches have been proposed in order to capture nonlinear dependencies assumed to be present in most biological systems and that cannot be captured by correlation-based distance measures . these approaches often use the concept of mutual information, a generalization of the correlation coeffi cient which quantifi es the degree of statistical (in)dependence. an example of a network inference method that is based on mutual information is aracne, which has been used to reconstruct the human b-cell gene network from a large compendium of human b-cell gene expression profi les [ ] . in order to discover regulatory interactions, aracne removes the majority of putative indirect interactions from the initial mutual information-based gene network using a theorem from information theory, the data processing inequality. this led to the identifi cation of myc as a major hub in the b-cell gene network and a number of novel myc target genes, which were experimentally validated. whether informationtheoretic approaches are more powerful in general than correlationbased approaches is still subject of debate [ ] . bayesian networks allow the description of statistical dependencies between variables in a generic way [ , ] . bayesian networks are directed acyclic networks in which the edges of the network represent conditional dependencies; that is, nodes that are not connected represent variables that are conditionally independent of each other. a major bottleneck in the reconstruction of bayesian networks is their computational complexity. moreover, bayesian networks are acyclic and cannot capture feedback loops that characterize many biological networks. when time-series rather than steady-state data is available, dynamic bayesian networks provide a richer framework in which cyclic networks can be reconstructed [ ] . gene (co-)expression data only offers a partial view on the full complexity of cellular networks. consequently, networks have also been constructed from other types of high-throughput data. for example, physical protein-protein interactions have been measured on a large scale in different organisms including human, using affi nity capture-mass spectrometry or yeast two-hybrid screens, and have been made available in public databases such as biogrid [ ] . regulatory interactions have been probed using chromatin immunoprecipitation sequencing (chip-seq) experiments, for example by the encode consortium [ ] . using probabilistic techniques , heterogeneous types of experimental evidence and prior knowledge have been integrated to construct functional association networks for human [ ] , mouse [ ] , and, most comprehensively, more than organisms in the string database [ ] . functional association networks can help predict novel pathway components, generate hypotheses for biological functions for a protein of interest, or identify disease-related genes [ ] . prior knowledge required for these approaches is, for example, available in curated biological pathway databases, and via protein associations predicted using text mining based on their cooccurrence in abstracts or even full-text articles. many more integrative network inference methods have been proposed; for a review see [ ] . the integration of gene expression data with chip data [ ] or transcription factor-binding motif data [ ] has shown to be particularly fruitful for inferring transcriptional regulatory networks. recently, li et al. [ ] described the results from a regression-based model that predicts gene expression using encode (chip-seq) and tcga data (mrna expression data complemented with copy number variation, dna methylation, and microrna expression data). this model infers the regulatory activities of expression regulators and their target genes in acute myeloid leukemia samples. eighteen key regulators were identifi ed, whose activities clustered consistently with cytogenetic risk groups. bayesian networks have also been used to integrate multiomics data. the combination of genotypic and gene expression data is particularly powerful, since dna variations represent naturally occurring perturbations that affect gene expression detected as expression quantitative trait loci ( eqtl ). cis -acting eqtls can then be used as constraints in the construction of directed bayesian networks to infer causal relationships between nodes in the network [ ] . large multi-omics datasets consisting of hundreds or sometimes even thousands of samples are available for many commonly occurring human diseases, such as most tumor types (tcga), alzheimer's disease [ ] , and obesity [ ] . however, a major bottleneck for the construction of accurate gene networks is that the number of gene networks that are compatible with the experimental data is several orders of magnitude larger still. in other words, top-down network inference is an underdetermined problem with many possible solutions that explain the data equally well and individual gene-gene interactions are characterized by a high false-positive rate [ ] . most network inference methods therefore try to constrain the number of possible solutions by making certain assumptions about the structure of the network. perhaps the most commonly used strategy to harness the complexity of the gene network inference problem is to analyze experimental data in terms of biological modules, that is, sets of genes that have strong interactions and a common function [ ] . there is considerable evidence that many biological networks are modular [ ] . module-based approaches effectively constrain the number of parameters to estimate and are in general also more robust to the noise that characterizes high-throughput omics measurements. a detailed review of module-based techniques is outside the scope of this chapter (see, for example [ ] ), but we would like to mention a few examples of successful and commonly used modular approaches. weighted gene co-expression network analysis ( wgcna) decomposes a co-expression network into modules using clustering techniques [ ] . modules can be summarized by their module eigengene, a weighted average expression profi le of all gene member of a given module. eigengenes can then be correlated with external sample traits to identify modules that are related with these traits. parikshak et al. [ ] used wgcna to extract modules from a co-expression network constructed using fetal and early postnatal brain development expression data. next, they established that several of these modules were enriched for genes and rare de novo variants implicated in autism spectrum disorder (asd). moreover, the asd-associated modules are also linked at the transcriptional level and transcription factors were found acting as putative co-regulators of asd-associated gene modules during neocortical development. wgcna can also be used when multiple omics data types are available. one example of such an approach involved the integration of transcriptomic and proteomic data from a study investigating the response to sars-cov infection in mice [ ] . in this study wgcna-based gene and protein co-expression modules were constructed and integrated to obtain module-based disease signatures. interestingly, the authors found several cases of identifi er-matched transcripts and proteins that correlated well with the phenotype, but which showed poor or anticorrelation across these two data types. moreover, the highest correlating transcripts and peptides were not the most central ones in the co-expression modules. vice versa , the transcripts and proteins that defi ned the modules were not those with the highest correlation to the phenotype. at the very least this shows that integration of omics data affects the nature of the disease signatures. identifi cation of active modules is another important integrative modular technique . here, experimental data in the form of molecular profi les is projected onto a biological network, for example a protein-protein interaction network. active modules are those subnetworks that show the largest change in expression for a subset of conditions and are likely to contain key drivers or regulators of those processes perturbed in the experiment. active modules have, for example, been used to fi nd a subnetwork that is overexpressed in a particularly aggressive lymphoma subtype [ ] and to detect signifi cantly mutated pathways [ ] . some active module approaches integrate various types of omics data. one example of such an approach is paradigm [ ] , which translates pathways into factor graphs, a class of models that belongs to the same family of models as bayesian networks, and determines sample-specifi c pathway activity from multiple functional genomic datasets. paradigm has been used in several tcga projects, for example, in the integrated analysis of urothelial bladder carcinomas [ ] . paradigm-based analysis of copy number variations and rna-seq gene expression in combination with a propagation-based network analysis algorithm revealed novel associations between mutations and gene expression levels, which subsequently resulted in the identifi cation of pathways altered in bladder cancer. the identifi cation of activating or inhibiting gene mutations in these pathways suggested new targets for treatment. moreover, this effort clearly showed the benefi ts of screening patients for the presence of specifi c mutations to enable personalized treatment strategies. often, published disease signatures cannot be replicated [ ] or provide hardly additional biological insight. also here (modular) network-based approaches have been proposed to alleviate these problems. a common characteristic of most methods is that the molecular activity of a set of genes is summarized on a per sample basis. summarized gene set scores are then used as features in prognostic and predictive models. relevant gene sets can be based on prior knowledge and correspond to canonical pathways, gene ontology categories, or sets of genes sharing common motifs in their promoter regions [ ] . gene set scores can also be determined by projecting molecular data onto a biological network and summarizing scores at the level of subnetworks for each individual sample [ ] . while promising in principle, it is still subject of debate whether gene set-based models outperform gene-based one s [ ] . the comparative analysis of networks across different species is another commonly used approach to constrain the solution space. patterns conserved across species have been shown to be more likely to be true functional interactions [ ] and to harbor useful candidates for human disease genes [ ] . many network alignment methods have been developed in the past decade to identify commonalities between networks. these methods in general combine sequence-based and topological constraints to determine the optimal alignment of two (or more) biological networks. network alignment has, for example, been applied to detect conserved patterns of protein interaction in multiple species [ , ] and to analyze the evolution of co-expression networks between humans and mice [ , ] . network alignment can also be applied to detect diverged patterns [ ] and may thus lead to a better understanding of similarities and differences between animal models and human in health and disease. information from model organisms has also been fruitfully used to identify more robust disease signatures [ - ] . sweet-cordero and co-workers [ ] used a gene signature identifi ed in a mouse model of lung adenocarcinoma to uncover an orthologous signature in human lung adenocarcinoma that was not otherwise apparent. bild et al. [ ] defi ned gene expression signatures characterizing several oncogenic pathways of human mammary epithelial cells. they showed that these signatures predicted pathway activity in mouse and human tumors. predictions of pathway activity correlated well with the sensitivity to drugs targeting those pathways and could thus serve as a guide to targeted therapies. a generic approach, pathprint, for the integration of gene expression data across different platforms and species at the level of pathways, networks, and transcriptionally regulated targets was recently described [ ] . the authors used their method to identify four stem cell-related pathways conserved between human and mouse in acute myeloid leukemia, with good prognostic value in four independent clinical studies. we reviewed a wide array of different approaches showing how networks can be used to elucidate integrated genetic, molecular, and cellular networks. however, in general no single approach will be suffi cient and combining different approaches in more complex analysis pipelines will be required. this is fi ttingly illustrated by the diggit (driver-gene inference by genetical-genomics and information theory) algorithm [ ] . in brief, diggit identities candidate master regulators from an aracne gene co-expression network integrated with copy number variations that affect gene expression. this method combines several previously developed computational approaches and was used to identify causal genetic drivers of human disease in general and glioblastoma, breast cancer, and alzheimer's disease in particular. this enabled identifi cation of klhl deletions as upstream activators of two previously established master regulators in a specifi c subtype of glioblastoma. systems medicine is one of the steps necessary to make improvements in the prevention and treatment of disease through systems approaches that will (a) elucidate (patho)physiologic mechanisms in much greater detail than currently possible, (b) produce more robust and predictive disease signatures, and (c) enable personalized treatment. in this context, we have shown that bioinformatics has a major role to play. bioinformatics will continue its role in the development, curation, integration, and maintenance of (public) biological and clinical databases to support biomedical research and systems medicine. the bioinformatics community will strengthen its activities in various standardization and curation efforts that already resulted in minimum reporting guidelines [ ] , data capture approaches [ ] , data exchange formats [ ] , and terminology standards for annotation [ ] . one challenge for the future is to remove errors and inconsistencies in data and annotation from databases and prevent new ones from being introduced [ , , - ]. an equally important challenge is to establish, improve, and integrate resources containing phenotype and clinical information. to achieve this objective it seems reasonable that bioinformatics and health informatics professionals team up [ - ] . traditionally health informatics professionals have focused on hospital information systems (e.g., patient records, pathology reports, medical images) and data exchange standards (e.g., hl ), medical terminology standards (e.g., international classifi cation of disease (icd), snomed), medical image analysis, analysis of clinical data, clinical decision support systems, and so on. while, on the other hand, bioinformatics mainly focused on molecular data, it shares many approaches and methods with health informatics. integration of these disciplines is therefore expected to benefi t systems medicine in various ways [ ] . integrative bioinformatics approaches clearly have added value for systems medicine as they provide a better understanding of biological systems, result in more robust disease markers, and prevent (biological) bias that would possibly occur from using single-omics measurements. however, such studies, and the scientifi c community in general, would benefi t from improved strategies to disseminate and share data which typically will be produced at multiple research centers (e.g., https://www.synapse.org ; [ ] ). integrative studies are expected to increasingly facilitate personalized medicine approaches such as demonstrated by chen and coworkers [ ] . in their study they presented a -month "integrative personal omics profi le" (ipop) for a single individual comprising genomic, transcriptomic, proteomic, metabolomic, and autoantibody data. from the whole-genome sequence data an elevated risk for type diabetes (t d) was detected, and subsequent monitoring of hba c and glucose levels revealed the onset of t d, despite the fact that the individual lacked many of the known non-genetic risk factors. subsequent treatment resulted in a gradual return to the normal phenotype. this shows that the genome sequence can be used to determine disease risk in a healthy individual and allows selecting and monitoring specifi c markers that provide information about the actual disease status. network-based approaches will increasingly be used to determine the genetic causes of human diseases. since the effect of a genetic variation is often tissue or cell-type specifi c, a large effort is needed in constructing cell-type-specifi c networks both in health and disease. this can be done using data already available, an approach taken by guan et al. [ ] . the authors proposed tissue-specifi c networks in mouse via their generic approach for constructing functional association networks using lowthroughput, highly reliable tissue-specifi c gene expression information as a constraint. one could also generate new datasets to facilitate the construction of tissue-specifi c networks. examples of such approaches are tcga and the genotype-tissue expression (gtex) project. the aim of gtex is to create a data resource for the systematic study of genetic variation and its effect on gene expression in more than human tissues [ ] . regardless of the way how networks are constructed, it will become more and more important to offer a centralized repository where networks from different cell types and diseases can be stored and accessed. nowadays, these networks are diffi cult to retrieve and are scattered in supplementary fi les with the original papers, links to accompanying web pages, or even not available at all. a resource similar to what the systems biology community has created with the biomodels database would be a great leap forward. there have been some initial attempts in building databases of network models, for example the cellcircuits database [ ] ( http://www.cellcircuits.org ) and the causal biological networks (cbn) database of networks related to lung disease [ ] ( http://causalbionet.com ). however, these are only small-scale initiatives and a much larger and coordinated effort is required. another main bottleneck for the successful application of network inference methods is their validation. most network inference methods to date have been applied to one or a few isolated datasets and were validated using some limited follow-up experiments, for example via gene knockdowns, using prior knowledge from databases and literature as a gold standard, or by generating simulated data from a mathematical model of the underlying network [ , ] . however, strengths and weaknesses of network inference methods across cell types, diseases, and species have hardly been assessed. notable exceptions are collaborative competitions such as the dialogue on reverse engineering assessment and methods (dream) [ ] and industrial methodology for process verifi cation (improver) [ ] . these centralized initiatives propose challenges in which individual research groups can participate and to which they can submit their predictions, which can then be independently validated by the challenge organizers. several dream challenges in the area of network inference have been organized, leading to a better insight into the strengths and weaknesses of individual methods [ ] . another important contribution of dream is that a crowd-based approach integrating predictions from multiple network inference methods was shown to give good and robust performance across diverse data sets [ ] . also in the area of systems medicine challenge-based competitions may offer a framework for independent verifi cation of model predictions. systems medicine promises a more personalized medicine that effectively exploits the growing amount of molecular and clinical data available for individual patients. solid bioinformatics approaches are of crucial importance for the success of systems medicine. however, really delivering the promises of systems medicine will require an overall change of research approach that transcends the current reductionist approach and results in a tighter integration of clinical, wet-lab laboratory, and computational groups adopting a systems-based approach. past, current, and future success of systems medicine will accelerate this change. the road from systems biology to systems medicine participatory medicine: a driving force for revolutionizing healthcare understanding drugs and diseases by systems biology the roots of bioinformatics in theoretical biology sequencing technologies -the next generation exploring the new world of the genome with dna microarrays spectroscopic and statistical techniques for information recovery in metabonomics and metabolomics next-generation technologies and data analytical approaches for epigenomics gene expression profi ling predicts clinical outcome of breast cancer diagnostic tests based on gene expression profi le in breast cancer: from background to clinical use a multigene assay to predict recurrence of tamoxifentreated, node-negative breast cancer what is bioinformatics? a proposed defi nition and overview of the fi eld the importance of biological databases in biological discovery the nucleic acids research database issue and an updated nar online molecular biology database collection reuse of public genome-wide gene expression data experimental design for gene expression microarrays learning from our gwas mistakes: from experimental design to scientifi c method effi cient experimental design and analysis strategies for the detection of differential expression using rna-sequencing impact of yeast systems biology on industrial biotechnology the nature of systems biology gene expression omnibus: microarray data storage, submission, retrieval, and analysis the proteomics identifi cations (pride) database and associated tools: status in metabolights--an open-access generalpurpose repository for metabolomics studies and associated meta-data the reactome pathway knowledgebase data, information, knowledge and principle: back to metabolism in kegg activities at the universal protein resource (uniprot) path models: large-scale generation of computational models from biochemical pathway maps precise generation of systems biology models from kegg pathways pathguide: a pathway resource list pathway commons, a web resource for biological pathway data consensus and confl ict cards for metabolic pathway databases the ucsc genome browser database: update biomodels database: a repository of mathematical models of biological processes a community-driven global reconstruction of human metabolism the seek: a platform for 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archive of relationships among sequence variation and human phenotype searching ncbi's dbsnp database dbvar and dgva: public archives for genomic structural variation using electronic patient records to discover disease correlations and stratify patient cohorts on not reinventing the wheel beyond the genomics blueprint: the th human variome project meeting comprehensive molecular characterization of urothelial bladder carcinoma open clinical trial data for all? a view from regulators clinical trial data as a public good biobanking for europe whose data set is it anyway? sharing raw data from randomized trials sharing individual participant data from clinical trials: an opinion survey regarding the establishment of a central repository ncbi's database of genotypes and phenotypes: dbgap mining electronic health records: towards better research applications and clinical care phenome connections phewas: demonstrating the feasibility of a phenome-wide scan to discover genedisease associations mining the ultimate phenome repository probing genetic overlap among complex human phenotypes systematic comparison of phenomewide association study of electronic medical record data and genome-wide association study data finding the missing heritability of complex diseases systems genetics: from gwas to disease pathways a review of post-gwas prioritization approaches when one and one gives more than two: challenges and opportunities of integrative omics the model organism as a system: integrating 'omics' data sets principles and methods of integrative genomic analyses in cancer toward interoperable bioscience data critical assessment of human metabolic pathway databases: a stepping stone for future integration the bridgedb framework: standardized access to gene, protein and metabolite identifi er mapping services integration of transcriptomics and metabonomics: improving diagnostics, biomarker identifi cation and phenotyping in ulcerative colitis a multivariate approach 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techniques. adaptive computation and machine learning inferring gene networks from time series microarray data using dynamic bayesian networks the biogrid interaction database: update architecture of the human regulatory network derived from encode data reconstruction of a functional human gene network, with an application for prioritizing positional candidate genes a genomewide functional network for the laboratory mouse string v . : protein-protein interaction networks, with increased coverage and integration advantages and limitations of current network inference methods computational discovery of gene modules and regulatory networks a semisupervised method for predicting transcription factor-gene interactions in escherichia coli regression analysis of combined gene expression regulation in acute myeloid leukemia integrating large-scale functional genomic data to dissect the complexity of yeast regulatory networks integrated systems approach identifi es genetic nodes and networks in late-onset alzheimer's disease a survey of the genetics of stomach, liver, and adipose gene expression from a morbidly obese cohort an introduction to systems biology: design principles of biological circuits from signatures to models: understanding cancer using microarrays integrative approaches for fi nding modular structure in biological networks weighted gene coexpression network analysis: state of the art integrative functional genomic analyses implicate specifi c molecular pathways and circuits in autism multi-omic network signatures of disease identifying functional modules in protein-protein interaction networks: an integrated exact approach algorithms for detecting signifi cantly mutated pathways in cancer inference of patient-specifi c pathway activities from multi-dimensional cancer genomics data using paradigm pathway-based personalized analysis of cancer network-based classifi cation of breast cancer metastasis current composite-feature classifi cation methods do not 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investigations: the mibbi project data standards for omics data: the basis of data sharing and reuse biomedical ontologies: a functional perspective pdb improvement starts with data deposition what we do not know about sequence analysis and sequence databases annotation error in public databases: misannotation of molecular function in enzyme superfamilies improving the description of metabolic networks: the tca cycle as example more than , problems with protein domain databases: transmembrane regions, signal peptides and the issue of sequence homology biomedical and health informatics in translational medicine amia board white paper: defi nition of biomedical informatics and specifi cation of core competencies for graduate education in the discipline synergy between medical informatics and bioinformatics: facilitating genomic medicine for future health care elixir: a distributed infrastructure for european biological data enabling transparent and collaborative computational analysis of tumor types within the cancer genome atlas personal omics profi ling reveals dynamic molecular and medical phenotypes tissue-specifi c functional networks for prioritizing phenotype and disease genes the genotype-tissue expression (gtex) project on crowd-verifi cation of biological networks inference and validation of predictive gene networks from biomedical literature and gene expression data verifi cation of systems biology research in the age of collaborative competition dialogue on reverse-engineering assessment and methods: the dream of highthroughput pathway inference revealing strengths and weaknesses of methods for gene network inference wisdom of crowds for robust gene network inference we would like to thank dr. aldo jongejan for his comments that improved the text. key: cord- -qbyk psh authors: kwon, somin; iba, michiyo; kim, changyoun; masliah, eliezer title: immunotherapies for aging-related neurodegenerative diseases—emerging perspectives and new targets date: - - journal: neurotherapeutics doi: . /s - - - sha: doc_id: cord_uid: qbyk psh neurological disorders such as alzheimer’s disease (ad), lewy body dementia (lbd), frontotemporal dementia (ftd), and vascular dementia (vcid) have no disease-modifying treatments to date and now constitute a dementia crisis that affects million in the usa and over million worldwide. the most common pathological hallmark of these age-related neurodegenerative diseases is the accumulation of specific proteins, including amyloid beta (aβ), tau, α-synuclein (α-syn), tar dna-binding protein (tdp ), and repeat-associated non-atg (ran) peptides, in the intra- and extracellular spaces of selected brain regions. whereas it remains controversial whether these accumulations are pathogenic or merely a byproduct of disease, the majority of therapeutic research has focused on clearing protein aggregates. immunotherapies have garnered particular attention for their ability to target specific protein strains and conformations as well as promote clearance. immunotherapies can also be neuroprotective: by neutralizing extracellular protein aggregates, they reduce spread, synaptic damage, and neuroinflammation. this review will briefly examine the current state of research in immunotherapies against the most commonly targeted proteins for age-related neurodegenerative disease: aβ, tau, and α-syn. the discussion will then turn to combinatorial strategies that enhance the effects of immunotherapy against aggregating protein, followed by new potential targets of immunotherapy such as aging-related processes. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. age-related neurodegenerative diseases such as alzheimer's disease (ad), parkinson's disease (pd), dementia with lewy bodies (dlb), frontotemporal dementia (ftd), and vascular dementia (vcid) are being increasingly classified as major public health concerns [ ] . in a rapidly aging world in which people over the age of are projected to make up a fifth of the population in just years, the prevalence of these dementias is expected to triple within that same time frame [ ] [ ] [ ] . however, there are few, if any, disease-modifying treatments to date, making dementia one of the costliest conditions to society [ ] . in response to this public health emergency, many countries have established national plans to address the lack of therapies [ , ] . in , the usa enacted the national alzheimer's project act (napa) (public law - ). the act defines "alzheimer's" as alzheimer's disease and related dementias (adrds), including ftd, dlb, and vcid. the law calls for the development of treatments to prevent or slow the rate of ad progression by the year , as well as a national plan and coordination among international bodies to fight ad on a global scale [ ] . as a result, funding for ad research via the national institute on aging (nia) has increased substantially within the last years, jumping from million dollars per year to approximately . billion. the nia and its sister institutes and centers at the national institutes of health (nih), including the national institute of neurological disorders and stroke (ninds), use milestones electronic supplementary material the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users. and recommendations from the ad summits held at the nih bethesda campus to prioritize areas of research. the most common pathological hallmark of age-related neurodegenerative diseases is the accumulation of proteinaceous deposits in the intra-and extracellular spaces of selected brain regions [ ] [ ] [ ] [ ] [ ] [ ] . these proteins have been shown to accumulate several years prior to clinically observable cognitive, behavioral, and motor symptoms [ , ] . whereas there remains some debate as to whether protein accumulation is pathogenic or merely a byproduct of disease [ ] (fig. ) , the majority of therapeutic research has been focused on clearing these aggregates [ ] [ ] [ ] [ ] . ad, dlb, pd, and ftd are thus often defined as proteinopathies of the aging population that display selective degeneration of neuronal circuitries and progressive accumulation of specific proteins such as amyloid beta (aβ), tau, α-synuclein (α-syn), tar dna-binding protein (tdp ), and repeat-associated non-atg (ran) [ , [ ] [ ] [ ] [ ] [ ] among many others (fig. ) . ad plaque and tangle formation are most frequently associated with aβ and tau, whereas the primary protein component of lewy bodies in pd and dlb is α-syn [ ] [ ] [ ] [ ] [ ] [ ] [ ] (fig. ) . ftd aggregates are generally comprised of tau, tdp- , or fused in sarcoma (fus) [ , ] , but cases with a ggggcc expansion mutation in intron of the c orf gene also present with accumulations of tdp and repeat-associated non-aug-dependent (ran) translation proteins [ ] . it must be pointed out, however, that α-syn and tdp aggregates are also commonly found in ad, as well as aβ, tau, and tdp in dlb [ ] [ ] [ ] [ ] [ ] [ ] [ ] (fig. ) . moreover, recent studies have shown significant overlap in ad and pd pathology in which a single individual over the age of can present with aggregates of several of the above proteins [ ] (fig. ). as such, simultaneously targeting multiple aggregating protein species may be more effective at treating these disorders than monotherapy [ , ] . abnormal protein accumulation reflects an imbalanced proteostasis network [ , , ] . how these protein aggregates lead to neurodegeneration is unclear but may involve synaptic dysfunction and neuroinflammation triggered by the formation of neurotoxic oligomers and the cell-to-cell propagation of oligomers, protofibrils, and fibrils [ , , , ] . given that age is the greatest risk factor for neurodegenerative disease, age-related alterations in proteostasis, inflammation, stem cell biogenesis, mitochondrial alterations, cell senescence, and dna damage/repair [ ] might also play critical roles in pathogenesis (fig. ) . understanding the role of protein homeostasis as it relates to aging could identify new drug targets and delineate reliable markers to accurately determinate a patient's prognosis and appropriate treatment options [ ] . research on disease-modifying therapies has primarily focused on reducing the accumulation and propagation of protein aggregates by decreasing synthesis and aggregation or enhancing the rate of clearance [ , ] (fig. ) , with less emphasis on targeting aging-related processes. these include gene therapy to bolster clearance and degradation (e.g., autophagy, proteolysis, lysosomal degradation) [ ] , anti-sense technology to block synthesis (e.g., tau and α-syn genes) [ ] [ ] [ ] , small molecules to decrease aggregation (e.g., fig. combined mechanisms of neurodegeneration in ad/adrd include protein accumulation and aging-related pathways. in the neurodegenerative process of ad/adrd, synaptic damage and neuroinflammation may lead to neuronal dysfunction which results in dementia and motor deficits. the leading hypothesis is that progressive accumulation of proteins such as amyloid beta (aβ), tau, α-synuclein (α-syn), tar dna-binding protein (tdp ), and repeat-associated non-atg (ran) is the primary culprit. however, aging is also likely to play a critical role in pathogenesis, either in synergy with protein accumulation or as an independent pathway. aging processes relevant to neurodegeneration include inflammation, proteostasis, dna damage, cell senescence, and mitochondrial alterations fig. overlapping protein aggregate pathology in ad/adrd. although aβ and tau are classically associated with ad, α-syn with lewy body dementia, and tdp and tau with ftd and limbic associated tdp encephalopathy (late), it is becoming increasingly evident that older individuals with dementia (+ years old) display mixed pathology inhibitors of aβ, tau, and α-syn oligomers and higher-order aggregates) [ ] [ ] [ ] [ ] , and immunotherapy to enhance clearance, degradation, and decrease aggregation (fig. ) . immunotherapies have garnered particular attention for their specificity [ , ] . this review will discuss types of immunotherapy: active vaccines, in which inactivated fragments of the pathogenic protein are directly administered to produce a long-lasting immune response, and passive immunization, in which patients are infused with antibodies against the target protein. both strategies can modulate inflammation, prevent future oligomerization and aggregation activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and promote clearance by phagocytic microglia or lysosomal degradation via the endosomal sorting complexes required for transport (escrt) pathway [ ] . other immunotherapy modalities, including t-cell modulation or harnessing cellular immunity to target neurodegeneration pathways, are discussed elsewhere [ , ] . in addition to the various mechanisms above, immunotherapy has many other advantages. its inherent specificity allows for selective targeting of specific strains and conformations with less off-target effects [ , [ ] [ ] [ ] (fig. ) . polyvalent single-chain antibodies or combinations of antibodies and vaccines may also allow for the simultaneous targeting of multiple protein aggregate species [ , [ ] [ ] [ ] [ ] (fig. ) . furthermore, immunotherapies can be neuroprotective by neutralizing extracellular protein aggregates and thereby reducing subsequent spread, synaptic damage, and neuroinflammation [ ] . the very concept of immunotherapy for ad originated from the observation that the amyloid-β (aβ) peptide accumulates extracellularly and is therefore accessible to antibodies that can recruit microglia to clear such deposits [ ] (fig. ) . it was accordingly considered unlikely that intracellular protein aggregates such as those of tau, α-syn, tdp , and ran would be good targets for immunotherapy. however, the advent of new technology to facilitate intracellular trafficking of antibodies [ ] [ ] [ ] [ ] and the discovery that these so-called intracellular proteins also exist in cell membranes and extracellular spaces have driven immunotherapy development forward for pd, dlb, and ftd [ , , , , ] . in march , years after the original publication by schenk et al. that propelled the field of immunotherapy for neurodegenerative diseases [ ] , clinical trials for the aβ antibody aducanumab were halted early for futility. one of the most promising ad therapies had become yet another failed drug to meet clinical endpoints after reaching phase iii. seven months later, however, the company sponsoring the trial surprised the world by announcing that the results of the futility analysis were premature, and that they would seek us food and drug administration approval by using a larger dataset with longer exposure times to a high dose [ ] . the news, while providing renewed hope for ad patients and families, should be received with cautious optimism [ ] . years of clinical trial failures for ad suggest that monotherapy against aggregation-prone proteins may not be enough for clinical efficacy [ ] . identifying an effective treatment for the heterogeneous group of patients affected by neurodegenerative disease may rest on an amalgamation of factors, including developing accurate and early diagnostic techniques, pursuing earlier preventive treatment, simultaneously targeting different proteins, identifying novel targets and age-related pathogenic cascades, and even reducing variability between clinical trial populations such as apoe carrier status [ , , ] . this review will briefly examine the current state of research in immunotherapies against the most commonly targeted proteins for age-related neurodegenerative disease, aβ, tau, and α-syn. the discussion will then turn to combinatorial strategies and new potential targets for future immunotherapy development. based on the amyloid cascade hypothesis, the transmembrane amyloid precursor protein (app) can undergo proteolytic cleavage by β-secretase (bace ) to produce a soluble extracellular fragment and a cell membrane-bound fragment [ ] [ ] [ ] . with its catalytic subunit presenilin, γ-secretase further cleaves the cell membrane fragment to release the amyloid-β (aβ) peptide. mutations in the genes encoding app or presenilin carry the greatest incidence of familial ad [ ] . under nonpathological conditions, there is evidence that aβ is involved in regulating synaptic function and even acting as an antimicrobial peptide to protect against infection and injury [ ] [ ] [ ] . the aβ peptide can be of varying lengths, the most prevalent being aβ . longer forms, such as aβ , are less soluble and are prone to accumulate extracellularly to form oligomers, protofibrils, fibrils, and ultimately, plaques [ , , [ ] [ ] [ ] . in , schenk et al. published the first immunotherapy for ad: an active vaccine called an- and comprised of synthetic full-length aβ with qs- adjuvant [ ] . the vaccine produced long-lasting and nearly complete clearance of aβ deposits in many patients, but had no impact on the prominent tau pathology and severe dementia [ , ] . the trial was halted after patients developed meningoencephalitis related to t-cell infiltration. successful mapping of the bcell epitope to the n-terminus of aβ [ ] [ ] [ ] and additional work on th -biased adjuvants [ ] [ ] [ ] [ ] [ ] were thus essential to the future aβ vaccine development, allowing for a robust antibody response without the potentially harmful th lymphocyte activation [ ] . vanutide cridificar (acc- ) was designed as such to include only the b-cell epitope of aβ plus qs- adjuvant [ ] . phase ii trials for acc- , however, did not reach efficacy endpoints for cognitive evaluations, volumetric brain mri, and csf biomarkers, and development was halted in [ , ] . another recent strategy has been to use mimotopes, synthetic peptides that closely resemble the target protein epitope, as active vaccines. affitope® ad by affiris (wien, austria), a -amino-acid peptide that mimics the aβ n-terminus, was regrettably also terminated in phase ii for lack of clinical efficacy [ ] . there are currently active immunizations being tested in phase ii trials. cad from novartis (basel, switzerland) fuses a qβ virus-like particle to multiple copies of the aβ nterminus (a.a. - ) [ , ] , and is being tested in homozygous apoe carriers as part of the alzheimer's prevention initiative (api) program (nct ) [ ] . similarly, aci- by ac immune (lausanne, switzerland) uses aβ - peptides anchored to the surface of a liposome and is being tested for mild-to-moderate alzheimer's disease and down's syndrome [ ] . another n-terminus vaccine, ub- , is comprised of synthetic aβ - -targeting peptides linked to helper t-cell peptide epitopes contained in a th -biased formula [ ] . after completing phase iia trials for ub- , united neuroscience (dublin, ireland) is now assessing longterm safety, tolerability, and immunogenicity in an extension study (nct ). abvac by araclon biotech s.l. (zaragoza, spain) began phase ii trials (nct ) in mild cognitive impairment (mci) last year and is the only vaccine to use the c-terminal end of aβ [ ] . the first passive immunotherapy to reach phase iii trials was bapineuzumab, the humanized murine monoclonal antibody d [ ] [ ] [ ] (table ) . bapineuzumab targets the aβ n-terminus to mediate clearance of both soluble and fibrillar forms, but did not meet clinical endpoints in phase iii and, moreover, produced amyloid-related imaging abnormalities (aria) with edema in patients that received a high dose [ ] . solanezumab, humanized murine antibody m , is specific for soluble monomeric aβ and is proposed to operate by the peripheral sink hypothesis, in which removal of aβ in the periphery also leads to a reduction in the brain by passive diffusion [ ] . multiple phase iii trials, including one in early ad patients, either failed to meet clinical efficacy or were terminated early for futility [ , ] (table ) . similarly, phase iii trials for crenezumab, human monoclonal igg against oligomeric, fibrillar, and plaque conformations of aβ, were stopped early in jan for futility [ ] . however, a phase ii prevention trial with crenezumab in p r e s e n i l i n ( p s e n ) e a m u t a t i o n c a r r i e r s (nct ) is still underway as part of the api-autosomal-dominant alzheimer's disease (adad) trial in fig. multiple mechanisms of action of immunotherapy in ad/ adrd. immunotherapy involves various mechanisms of action to target protein aggregates in the membrane and the intra-and extracellular spaces. antibodies can block propagation, trigger lysosomal clearance of proteins, reduce inflammation, and promote neuroprotection medellin, colombia [ ] ( table ). the study is expected to be completed in february with the primary outcome being change in api adad composite cognitive test total score at weeks after baseline. secondary outcomes include time to mci or dementia progression because of ad, pet assessment of fibrillar amyloid accumulation, volumetric measurements by mri, csf tau biomarker levels, and various measures of memory and neurocognitive function. aducanumab was originally slated to take its place among the failed phase iii immunotherapy candidates despite being shown to selectively reduce aβ plaques and slow cognitive decline in earlier trials [ , ] . although phase iii was halted earlier this year, the company that developed this antibody is now seeking fda approval with new analysis of a larger data set [ , ] (table ) . similarly, although development of human igg gantenerumab was halted in for futility, a new phase iii trial was initiated at a higher dose (nct ) after the open label extension demonstrated a dramatic decline in aβ deposition in participants [ ] . both gantenerumab and solanezumab are also being revived as potential preventative therapies by the dominantly inherited alzheimer network trials unit (dian-tu) in patients with ad-causing mutations (nct ) [ ] . unfortunately, topline analyses in february showed that both drugs failed to meet primary endpoints [ ] . preventative trials for sporadic ad are also underway, in which the so-called a phase iii trial (nct ) tests solanezumab at higher doses and for longer durations in asymptomatic patients with preclinical ad as defined by positive amyloid pet scans [ ] . this phase iii trial recruited over participants and will be reporting in the next couple of years. humanized murine mab ban (nct ) is the only other passive immunotherapy currently in phase iii, but has already produced very promising results in phase ii. specifically, the -month study demonstrated a statistically significant slowing in cognitive decline at the highest dose with less than % of subjects experiencing aria [ ] . the antibody is specific to soluble aβ protofibrils, which have been proposed to be the toxic species rather than the plaques themselves [ ] (table ). in conclusion, despite previous failures in phase iii immunotherapy trials targeting aβ, interest has resurged following the recent glimmer of promising results coupled to advances in the early diagnosis and biomarker accessibility of ad. the antibodies currently being tested in clinical trials target a variety of aβ species, including soluble, oligomeric, and fibrillar aβ. mechanisms of action include microglia-facilitated removal of extracellular amyloid oligomers and fibrils, blocking primary and secondary aβ nucleation, and targeting monomers and soluble aβ in the periphery that could otherwise trigger aβ accumulation in the cns. rather than for late-stage disease reversal, immunotherapy against aβ aggregates appears to be more appropriate for prevention initiatives with prolonged treatments past weeks at high doses guided by reliable biomarkers. apoe carriers exposed to high doses, however, are susceptible to complications such as aria-e and may need to be closely monitored by mri [ ] . diseases characterized by progressive α-syn accumulation in neuronal and non-neuronal cells of cortical and subcortical regions are collectively termed synucleinopathies, and include pd, dlb, multiple system atrophy (msa), and a subset of ad [ , , , ] . dlb/pd is the second most common cause of dementia and parkinsonism in the aging population after ad, affecting about million in the usa [ ] . α-syn is a presynaptic protein reported to be involved in endosomal formation and vesicle release at the synapse [ , ] . the pathological hallmarks of pd and dlb are intraneuronal occlusions called lewy bodies and lewy neurites that are composed of fibrillar α-syn along with cytoskeletal and other synaptic proteins [ ] . α-syn accumulation can also occur in glia, such as glial cytoplasmic inclusion (gcis) in msa patient oligodendrocytes or lewy body-like structures in the amygdala, hippocampus, and neocortex of ad and down syndrome patients [ ] [ ] [ ] . although astrocytes and microglia do not constitutively express α-syn, they actively uptake extracellular α-syn which can lead to glial aggregates in conditions of impaired clearance [ ] [ ] [ ] . this is supported by increasing evidence that small amounts of α-syn aggregates are released from neurons into the extracellular space and subsequently interact with glial receptors such as tolllike receptor to trigger a pro-inflammatory response [ , , [ ] [ ] [ ] [ ] . the original approach to use antibodies to target and remove amyloid plaques from the brain made intuitive sense given that aβ aggregates are found in the extracellular space [ ] . it was unclear how this method might be applied to dlb, pd, and other synucleinopathies, as the majority of the proteinaceous aggregates were thought to form within neurons of the striatonigral system, limbic areas, and deep layers of the neocortex. in the early s, however, we showed that immunization of a newly developed α-syn transgenic model (line d, pdgf-α-syn wt) with recombinant human α-syn mixed with freund adjuvant resulted in the production of high titer antibodies against c-terminal α-syn that were capable of removing α-syn aggregates in neurons and ameliorating neurodegeneration and functional deficits [ ] . through additional active and passive immunization experiments in transgenic, viral, and preformed fibril injection models [ , ] , we learned that these antibodies had multiple mechanisms of action, such as recognizing α-syn aggregates in the membrane and triggering endocytosis and clearance via autophagy, lysosomal activity, or proteasomal degradation [ ] [ ] [ ] [ ] (fig. ) . moreover, antibodies can be trafficked intracellularly with single-chain antibodies or intrabodies specifically engineered to penetrate the cell membrane via apolipoprotein b (apob), tat fusion proteins, or uptake by endogenous receptors [ ] [ ] [ ] [ ] [ ] [ ] (fig. ) . single-chain variable fragments (scfvs), for example, are designed to retain the specificity of the original full-length antibody without activating unwanted fcmediated responses [ , , ] . several studies have shown that transgenic mice injected with viral vectors encoding scfvs against aβ [ ] [ ] [ ] , α-syn [ , ] , or tau [ , ] show long-term in vivo scfv expression, improved functional deficits, and reduced pathogenic protein accumulation. intrabody technology is continuing to be improved, such as in a recent study in which chatterjee et al. ( ) enhanced the solubility of single-domain immunoglobulin fragments by engineering a polypeptide tether construct, and demonstrated its protective effects on motor function when delivered by gene therapy to a pd rodent model [ ] . these intrabodies and cell-penetrating single-chain antibodies can block aggregation and target α-syn for degradation in the lysosomes using the escrt system [ , , , ] . antibodies can also block α-syn oligomerization and fibrillation, target specific strains and isoforms, prevent cell-to-cell transmission, and facilitate clearance via microglia [ , , , ] . therefore, antibodies can target both intracellular and extracellular α-syn aggregates as they spread from cell to cell (fig. ) . this approach has been since applied to other proteinopathies with predominantly intracellular accumulations of tau, tdp [ , ] , sod [ ] [ ] [ ] , ran peptides [ ] , and huntingtin (htt) [ , ] . additional lessons from α-syn immunotherapy studies include the use of antibodies to develop blood, csf, and tissue biomarkers to monitor the effects of immunotherapy and the ability of cterminus-specific antibodies to block protease-mediated c-terminus truncation of α-syn and subsequently prevent oligomerization and transmission [ , , [ ] [ ] [ ] (fig. ) . in this context, main strategies for α-syn immunotherapy have been pursued: mimotope vaccines and antibodies against the n-or c-terminal ends of α-syn or specific conformations of oligomeric and fibrillar α-syn (fig. ) . whereas most antibodies have been developed with recombinant or synthetic αsyn monomers or aggregates, a recent and novel variant uses antibodies cloned from human healthy volunteers producing high titers of auto-antibodies against α-syn [ ] [ ] [ ] . as a result of seminal cell-free in vitro and in vivo studies, several immunotherapies are currently been tested in clinical trial for synucleinopathies. the vaccines affitope® pd a and pd a were well tolerated in phase i and produced a dosedependent immune response in patients with early msa, but plans for phase ii have yet to be disclosed [ , ] . for passive immunotherapies, prasinezumab (also known as prx or ro ) is a humanized monoclonal antibody against the c-terminus of α-syn undergoing phase ii trials in patients with early pd (nct ) [ ] ( table ) . a phase i trial for biib , a human monoclonal antibody that preferentially binds to aggregated α-syn, was recently concluded and showed favorable safety, tolerability, and pharmacokinetic profiles [ ] (table ). medi is also in phase i clinical trial testing (nct ) as an antibody that can bind both monomers and aggregates. in preclinical studies, medi was shown to readily cross the blood-brain barrier and block transmission of α-syn aggregates in a combined viral vector model [ ] (table ). in summary, antibodies against α-syn ameliorate lewy body pathology by multiple mechanisms such as promoting the clearance of intracellular α-syn and blocking the propagation of extracellular α-syn. other rising immunotherapy strategies are t-cell modulation, such as that of copaxone® [ ] , and combinatorial approaches. we have tested several of such combinations, including simultaneous administration of affitopes® against aβ and α-syn [ , ] , nanoparticles containing both recombinant human α-syn and the immunomodulatory drug rapamycin, and the anti-inflammatory drug thalidomide given alongside a single-chain antibody against oligomeric α-syn derived from human dlb/pd brains and conjugated to apob [ ] . through these studies in dlb/pd mouse models, we show that combined immunotherapy may be more effective than monotherapy. this topic will be discussed in more detail in the following sections. the main challenge in the field of synucleinopathies is the lack of reliable and accessible biomarkers and the overlapping pathology among neurodegenerative diseases. tau is a major member of the microtubule associated proteins (map) family and abundantly expressed in neurons [ ] . by binding to tubulin dimers, tau can stabilize microtubule formation and modulate cytoskeletal dynamics [ ] . the degree to which tau is phosphorylated is an important regulator of microtubule stability [ ] . nonphosphorylated forms preferentially bind to microtubules, whereas hyperphosphorylation is associated with neurofibrillary tangle (nft) formation from paired helical filaments (phf) [ , ] . intracellular nfts are a major hallmark lesion of ad and other neurodegenerative tauopathies such as ftd, cortico-basal degeneration, pick's disease, and progressive supranuclear palsy (psp) [ ] . the severity of tau pathology has been shown to correlate with the degree of cognitive impairment and neuronal loss [ ] [ ] [ ] [ ] , making the tau protein an attractive target for new immunotherapies. axon neuroscience (staré mesto, slovakia) recently completed a -month phase ii trial for active tau vaccine aadvac (nct ), announcing in september that the vaccine was safe and well-tolerated, and generated antibodies against pathological tau in over % of patients [ ] . aadvac consists of a b-cell epitope from a cysteinated -mer tau peptide conjugated to keyhole limpet hemocyanin, a carrier protein that stimulates a th immune response [ ] . based on the promising trends in cognitive improvement and decelerated accumulation of ad biomarkers in trial participants, axon neuroscience plans to move forward with the next phase of clinical trials and is also testing the vaccine for primary progressive nonfluent aphasia (nct ) [ , ] . in another active vaccine, aci- , copies of synthetic tau fragments phosphorylated at ser and ser are embedded to the surface of a liposome [ , ] . in august , ac immune announced a phase ib/iia trial in collaboration with janssen pharmaceuticals (beerse, belgium) to assess the safety, tolerability, and immunogenicity of the second generation of this vaccine, ac- . [ ] . ac immune is also conducting phase studies on the antitau passive immunotherapy semorinemab (also called ro , mtau a, and rg ) for prodromal/ m i l d a d ( n c t ) a n d m o d e r a t e a d (nct ) in partnership with roche/genentech (south san francisco, ca). other ongoing trials for passive anti-tau immunotherapies include that of zagotenemab (also in progress called ly ) in ad patients (nct ) and abbv- e (also called tilavonemab or c n- e ) for early ad (nct ). zagotenemab is the humanized mouse monoclonal antibody mc- and targets soluble tau aggregates [ ] . similarly, abbv- e is the humanized version of mouse monoclonal antibody hj . that was shown to reduce tau seeding, hyperphosphorylation, and cognitive deficits in p s transgenic mice [ , ] . although phase ii trials for abbv- e in psp patients were halted in july , favorable safety and tolerability profiles were obtained [ ] . gosuranemab (biib ), the humanized antibody against extracellular n-terminal tau from biogen (cambridge, ma), was also undergoing phase ii trials for both psp (passport) and ad (tango) [ , ] . however, biogen recently announced that topline results from the passport study failed to meet primary endpoints and that it would no longer pursue development of gosuranemab for psp and other primary tauopathies [ ] . interestingly, current preclinical studies seem to be stepping away from targeting n-terminal tau in favor of other epitopes. dc e has been recently characterized as a promising antibody that targets tau at homologous epitopes present in each microtubule binding domain repeat [ ] . dc e was shown to not only preferentially bind truncated pathological tau over physiological tau but also prevent both the formation of beta sheets and the uptake of tau into neurons via sulfated heparan proteoglycans (hspgs) [ ] . in another study, courade et al. ( ) identified an antibody against the central region of tau, dubbed "antibody d," that was effective at blocking tau seeding in vitro [ ] . albert et al. ( ) further observed that this central tau epitope antibody was more efficacious at preventing ad-like pathology and cell-to-cell transfer of tau in mice [ ] . in summary, considerable efforts have been made for over years in the development of immunotherapies for neurodegenerative disorders. antibodies targeting aβ have led the way with a number of phase iii trials and at least one that reported meeting primary outcome measures, followed closely behind by a handful of immunotherapies for α-syn and tau in phase i and ii trials. whereas accessible biomarkers are currently available for aβ-and tau-related pathologies to guide the immunotherapy trials, there is a pressing lack of such measures for synucleinopathies. the other challenge at hand is that older patients present with protein aggregates that are no longer primarily one pathologic protein species but comprised of aβ, tau, α-syn, tdp , and others, in addition to aging-related processes such as inflammation, proteotasis deficits, dna damage, mitochondrial alterations, and stem cell alterations. thus, there is a dire need to develop reliable biomarkers and powerful combinatorial therapies to address these polyproteinopathies and age-related pathophysiological alterations, as will be discussed in the following section. combination therapy is already a standard of treatment for many cancers [ ] and chronic diseases including hypertension, chf, epilepsy, and hiv [ ] . combinations of currently available treatments are already being shown to improve cognition and behavior in ad patients, such as coadministration of cholinesterase inhibitors and memantine [ , ] and enhancing memantine efficacy with beta-asarone and tenuigenin [ ] . although a few ad mouse studies have tested combinations of lipid mediators [ ] or naturally occurring dietary compounds [ ] , combinatorial therapeutics in neurodegenerative disease remains a largely underexplored field (fig. ) . monotherapy alone may be insufficient against the complex and overlapping pathophysiology of age-related neurodegenerative diseases. as such, there is a growing need to simultaneously target multiple aggregating proteins as well as modulate aging-related mechanisms that synergize with protein aggregation to trigger neurodegeneration (fig. ) . overlapping pathology has been well-documented in agerelated neurodegenerative diseases, such as the presence of lewy body-like pathology in ad [ ] [ ] [ ] [ ] [ ] [ ] . one study reported that to % of patients with pd copresent with aβ plaques and nfts [ , ] , whereas over % of dlb patients [ ] and more than % of ad patients [ ] may have overlapping and, as a result, more aggressive pathology [ ] . indeed, a growing amount of evidence suggests that this copathology directly impacts disease progression. for example, dementia in pd is associated with high levels of ad copathology [ , [ ] [ ] [ ] [ ] . several groups have also shown that α-syn can contribute to the formation of toxic aβ and tau species, as well as vice versa [ ] . moreover, as stated earlier, demented individuals over the age of present with multiple pathologies including aβ, tau, α-syn, tdp , inflammatory, and vascular alterations [ ] . as such, one therapeutic strategy may be to target the clearance of more than one pathologic protein via immunotherapy. for example, immunotherapies that generate a response against both α-syn and aβ may be more effective than either alone given that α-syn and aβ may directly interact in ad patients and app transgenic models [ ] [ ] [ ] [ ] . ad patients have been shown to display elevated levels of α-syn in the csf [ , ] , and further, aβ may promote α-syn aggregation and toxicity [ , ] . in mouse models, hippocampal neurons with α-syn accumulations were found to be more susceptible to aβ-mediated toxicity [ ] , whereas genetic depletion of α-syn prevented the degeneration of cholinergic neurons and attenuated behavioral deficits [ , ] . similarly, α-syn has been shown to directly interact with psen , which is important for the proteolytic processing of the app to yield aβ [ ] . α-syn infusion in app tg mice also blocked aβ seeding but enhanced synaptic degeneration [ , ] , potentially by blocking snare-vesicle fusion in a cooperative manner [ ] . we recently covaccinated a double transgenic mouse model of dlb (mthy -happ-and mthy -α-syn) with affitope peptides against aβ (ad ) and α-syn (pd a) [ ] . remarkably, targeting one protein often concomitantly lowered the accumulation of another, in which ad effectively reduced aβ pathology as well as that of phosphorylated tau and α-syn. this study also noted potential additive effects, particularly in alleviating behavioral deficits, suggesting that a combined immunotherapy approach may be appropriate for the heterogenous pathology of ad and dlb and other age-related neurodegenerative diseases (fig. ) . tau and α-syn were also found to colocalize in the same cellular compartments, particularly in axons [ ] . although both have been shown to form intracellular aggregations, unlike aβ, α-syn can self-aggregate whereas tau requires an aggregation-inducing agent [ ] . many studies have demonstrated that α-syn interacts with and catalyzes the oligomerization of tau [ ] [ ] [ ] [ ] [ ] . α-syn may also demonstrate - - cochaperone activity on - - targets, including tau, promoting mutual misfolding [ ] [ ] [ ] . α-syn preformed fibrils were shown to induce intracellular tau aggregation in vivo [ ] , and may be closely involved in the gsk β-mediated phosphorylation of tau in a reciprocal manner [ ] [ ] [ ] [ ] . αsyn oligomers may induce a distinctly toxic tau oligomeric strain in a cross-seeding effect [ , ] or even form coaggregates [ ] . a recent paper also reported that an age-dependent reduction in glucocerebrosidase (gcase) activity was associated with accumulations of lipid-stabilized α-syn and phosphorylated tau [ ] . as such, a combination of immunotherapies against both tau and α-syn may be especially effective for cases in which both tau and synuclein pathology are present, as it can not only remove existing aggregates and toxic species but also prevent future cross-seeding events. the ultimate approach would be to develop immunotherapies that simultaneously target aβ, tau, α-syn, tdp , and inflammation (fig. ). this could be achieved by ) combining vaccines against aβ, tau, α-syn, and tdp among other proteins; ) combining antibodies against aβ, tau, α-syn, and tdp ; ) combining passive and active immunization; or ) using multivalent single-chain antibodies that can target or more of these proteins simultaneously. another approach would be to design antibodies to recognize a conformation that is similar across multiple protein aggregate species, rather than a specific sequence. interestingly, studies in app and αsyn transgenic models have demonstrated that some antibodies against oligomeric tau are effective at reducing α-syn, whereas others have suggested that antibodies against aβ may also target tau [ , ] . moreover, a combined vaccination approach targeting aβ and tau has been shown to decrease disease pathology in tau / xfad double transgenic mice [ ] . as such, a combination of these polyfunctional antibodies is another way of maximizing the effectiveness of immunotherapy (fig. ) . combination immunotherapies for ad/adrd targeting protein aggregates and aging-related pathways. immunotherapy can also be used to target other pathogens and age-related pathogenic processes associated with aging. one such pathogen may be the herpes simplex virus (# ). aging-related pathways that can be targeted with antibodies include inflammation (e.g., tnf, tlr , inflammasome) (# ), senescent cells (e.g., dpp ) (# ), and immune surveillance cells (e.g., nk cells) (# ). immunotherapies in neurodegeneration have been traditionally developed to target protein aggregates (e.g., aβ, tau, α-syn, tdp ) (# ) alone or in combination (# ). combination therapies may involve targeting multiple protein aggregates (# ) or protein aggregates (# ) and agerelated pathways (# - ) given the aforementioned copathology, immunotherapy may also be effective in combination with gene silencing approaches. several studies have shown that α-syn and tau deletion in animal transgenic models can delay the onset of disease. for example, α-syn knockout prevented neurotoxininduced neurodegeneration in mptp and rotenone pd mouse models [ , ] . injecting modified anti-sense oligonucleotides (aso) targeting snca similarly promoted survival of th-positive neurons and ameliorated motor deficits in mice expressing wild-type or mutant human snca [ ] . another recent study found that tau deletion in a t α-syn tg mice rescued some cognitive and synaptic deficits without affecting α-syn expression or phosphorylation [ ] . gene therapy is also progressing in human neurodegenerative diseases, most notably nusinersen for the neuromuscular disorder spinal muscular atrophy [ , ] . as such, immunotherapy to clear existing aggregates and anti-sense therapy to prevent further translation of pathologic proteins may be a viable combination, particularly in early stages of the disease. as pointed out at the beginning of this section, effective combination immunotherapy should target not only the protein aggregates but also age-related pathological processes contributing to neurodegeneration, such as inflammation and cell senescence. in this regard, although this review was focused on antibody-based immunotherapy, targeting cellular immunity is another attractive approach to treat neurodegenerative disorders given its ability to reduce the protein aggregate load by targeting t cells. we developed a mixed cellular and active immunization in which α-syn and rapamycin are simultaneously delivered in an antigen-presenting celltargeting glucan microparticle (gp) vaccine system [ ] . in this case, the α-syn peptide elicits the production of antibodies against α-syn, whereas rapamycin triggered the recruitment of tregs into the cns. in turn, the tregs immunomodulate microglia and induce greater microglial clearance of α-syn aggregates and reduced neurodegeneration and inflammation in α-syn tg mice. this vaccine, collectively termed gp+rap/αsyn, was capable of triggering neuroprotective treg responses in synucleinopathy animal models, and the combined vaccine was more effective than the humoral or cellular immunization alone. these results demonstrate the promise of multifunctional vaccine approaches for the treatment of ad and dlb/ pd. another interesting and novel approach would be to trigger immune surveillance by nk cells to target senescence cells for elimination [ ] . moreover, senescent and immune cells can be targeted with specific antibodies. for example, a recent study used antibodies against the surface molecule dpp (dipeptidyl peptidase ) of senescent cells [ ] to facilitate their clearance (fig. ) . antibodies against interleukins, tumor necrosis factor (tnf), and toll-like receptors may also be effective at modulating the immune response in neurodegenerative disease [ ] [ ] [ ] [ ] . previous studies have shown that aβ, tau, and α-syn toxicity are mediated by the inflammasome [ ] [ ] [ ] . in the case of synucleinopathies, we have shown that extracellular α-syn aggregates bind to tlr to trigger neuroinflammation and neurodegeneration and that selective neutralizing antibodies against tlr were effective at blocking these effects and behavior deficits in αsyn tg animals [ , ] . in fact, tlr has been developed as an important novel target for synucleinopathies [ , ] (fig. ) . pathogens such as viruses or bacteria have been proposed to contribute to progressive aggregate formation and chronic inflammation given the antimicrobial properties of aβ and age-related impaired clearance mechanisms [ ] (fig. ). as such, directly targeting these pathogens with immunotherapy may effectively attenuate neuroinflammation, particularly in combination with immunotherapies against aggregating protein. infection with herpes simplex virus (hsv- ) in ad patients has long received attention for its association with ad pathology and decreased cognitive function [ ] [ ] [ ] [ ] , although increasing work is being performed on other viruses such as herpes zoster, epstein-barr virus, and human cytomegalovirus [ ] [ ] [ ] [ ] . bacteria of particular interest include chlamydia pneumoniae, helicobacter pylori, and porphyromonas gingivalis [ ] [ ] [ ] [ ] [ ] [ ] . for a comprehensive review of the evidence pertaining to pathogenic agents in agerelated neurodegenerative disease, please see panza et al. [ ] . in summary, we have described the need and potential directions for combinatorial immunotherapies that include active, passive, and cellular approaches against specific protein aggregates as well as age-related neurodegenerative pathways such as inflammation and cell senescence (fig. ) . these immunotherapy approaches may certainly also be amenable for combination with gene therapy (e.g., anti-sense), small molecules (e.g., autophagy modulators, antiaggregation, senolytics), and nonpharmacological (e.g., exercise, diet, training) treatments. combinatorial therapeutics will also open exciting opportunities for personalized medicine, including catering to different stages of disease. for example, immunotherapy with anti-sense as described above may be wellsuited to presymptomatic stages of disease. another early intervention may be to combine immunotherapy with means of upregulating proteostasis components such as chemical chaperones or gene therapy for bip or xbp to prevent further misfolding and aggregation [ ] [ ] [ ] [ ] . following the onset of significant pathological changes or clinical symptoms, however, polyvalent immunotherapy may be needed to address the frequent presence of multiple pathogenic proteins. combining polyvalent antibodies with stress signaling inhibitors or regenerative therapy may prevent further synaptic loss and delay progression of symptoms [ ] [ ] [ ] . furthermore, different combinations may be used to cater to not only the stage of disease but also the patient's specific symptoms, lifestyle risk factors, and genetic risk factors. for instance, given alone, the therapeutic efficacy of statins or antihypertensives for ad [ ] [ ] [ ] [ ] [ ] [ ] [ ] and pd [ ] [ ] [ ] [ ] [ ] [ ] [ ] patients remains largely inconclusive. however, a patient presenting with overlapping pathology from both ad and vcid may benefit from a combination of protein-targeting immunotherapy and such cardiovascular disease treatments. current preventative immunotherapy trials such as that of the api are also just the beginning of genetics-based treatments. many key initiatives for genetics research in neurodegenerative disease, such as the international parkinson disease genomics consortium (ipdgc), are spearheading a movement for accessible analytics tools and diverse and representative data. in the future, we may be able to use this groundwork to identify a patient's specific risk variants and ultimately design a combinatorial therapy that can address both the protein accumulation and the biological associations for those variants. let it also not be forgotten, however, that in addition to further development of these therapeutic strategies, there is a dire need for an overhaul of current policy and clinical trial practices in order to truly pursue combinatorial and personalized medicine for diseases as complex as age-related neurodegeneration. geneva: world health organization alzheimer's disease facts and figures geneva: world 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deficits in an aav-alpha-synuclein rat model of parkinson's disease virus-mediated delivery of antibody targeting tar dna-binding protein- mitigates associated neuropathology elimination of tdp- inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals single chain variable fragment antibodies block aggregation and toxicity induced by familial als-linked mutant forms of sod adeno-associated virus-mediated delivery of a recombinant single-chain antibody against misfolded superoxide dismutase for treatment of amyotrophic lateral sclerosis intracerebroventricular infusion of monoclonal antibody or its derived fab fragment against misfolded forms of sod mutant delays mortality in a mouse model of als early or latestage anti-n-terminal huntingtin intrabody gene therapy reduces pathological features in b .hdr / mice bifunctional anti-huntingtin proteasomedirected intrabodies mediate efficient degradation of mutant huntingtin exon 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burge, harriet a. title: airplanes and infectious disease date: - - journal: air quality in airplane cabins and similar enclosed spaces doi: . /b sha: doc_id: cord_uid: vxjyjql air travel is associated with crowded conditions that can facilitate the transmission of airborne infectious diseases. the risk of contracting such diseases depends on the presence of an infected person who is shedding infectious particles and sufficient exposure of a sensitive person to achieve an adequate dose to cause disease. proximity to the infectious person and the length of time spent near the person are the most important risks for contracting a disease. ventilation patterns play a lesser role in disease transmission. well-documented outbreaks of influenza, severe acute respiratory syndrome (sars), and tuberculosis have occurred. other common respiratory illnesses have probably also been spread via aircraft, but outbreaks remain unrecognized. research on the spread of infectious disease in aircraft has focused on sampling for microorganisms in air (which has little relevance), and on the development of models to predict the risks for specific diseases. commercial air transport has provided access to the world for those able to undertake such travel. in fact, . million passengers travel each year, representing passenger miles of travel [ ] . there are risks associated with all forms of travel and many of them are similar across transport types [ ] . thus, stress, accidents, and exposure to disease agents are associated with all forms of transportation. however, because of the distances that can be traversed in a relatively short time, the crowded conditions on most commercial aircraft, and the inability to "escape", concern regarding the risk of contracting infectious disease during air travel has become significant [ , ] . it is important to remember that there are other infection-associated concerns associated with air travel. historically, transport of disease vectors has been of particular concern. many studies could be cited regarding transport of mosquitoes that subsequently led to outbreaks of malaria [ ] . control of these problems has focused on disinsection of aircraft during flight [ ] . food-borne outbreaks of disease have also occurred on aircraft [ ] [ ] [ ] . these are uncommon, and control depends on appropriate food handling rather than any change in the aircraft environment. aircraft sewage also contains human pathogens that could be transported over long distances, but the risk of transmission from this source is probably low [ ] . the movement of infectious agents from one part of the world to another via aircraft is another important concern [ ] . people traveling in an infectious state may contribute to the spread of epidemics. this phenomenon is of concern for the spread of weaponized organisms that cause contagious disease. on the other hand, modeling studies have indicated that halting air travel in order to interrupt the transmission pathway even for very serious diseases such as smallpox is likely to cause greater disruption than the disease itself [ ] . the possibility that long-distance flights could temporarily damage the immune system has also been raised [ ] . such damage could lead to recurrent infections acquired on the ground after a flight. further research will be needed to document the validity of these concerns. the primary concern today appears to be person-to-person spread of contagious disease in the aircraft cabin, and that will be the focus of this chapter. contagious diseases are infections that are spread from an infected person to an uninfected sensitive person. contagious diseases can be spread by aerosols, by large droplets, by direct contact with materials or surfaces that have been contaminated by an infectious person (fomites) or by direct person-to-person contact [ ] . all of these modes of transmission could occur in a crowded aircraft cabin. aerosol-transmitted diseases are generally caused by agents that are resistant to environmental stresses, and diseases that lead to environmental release of agent-containing secretions. thus, respiratory illnesses are most commonly airborne, although many other diseases could be spread artificially or accidentally via the airborne route. examples in this latter category include hepatitis, some gastrointestinal viruses and smallpox. for a contagious disease to be transmitted from one person to another there must be an infectious person in the environment (i.e., a source for the agent). there must be an exposure pathway so that the agent can travel from the infectious person to another person, and there must be a person who is sensitive (i.e., not resistant) to the agent. thus, the risk of infectious disease transmission is related to the probability that: . there is a person infected with the agent in the environment . the infected person is actively shedding the disease agent . there is an uninterrupted pathway from the infected person to other people . there are one or more sensitive persons in the environment . these sensitive people are in the aerosol long enough for sufficient exposure to occur. in order for contagious disease transmission to occur, one or more infected individuals must be present, or have been present recently in the case of fomite transmission (fomites are inanimate objects that act as reservoirs for disease agents). the risk of disease transmission is related to the probability that one or more individuals infected with the agent will be present in the environment. the exception to this is, of course, bioterrorism, for which an inanimate reservoir may have been prepared. the probability that an infected person will be in the environment is related to the incidence of the disease in the population likely to be in the environment. thus, agents of the common cold are probably in most aircraft. during the influenza season, there is a reasonably high probability that one or more persons harboring the influenza virus will be on board. on the other hand, there is only a small probability that a person with tuberculosis will be on board any individual aircraft. in the usa, tuberculosis is not uncommon, but is present primarily in populations that do not routinely fly (e.g., prison and homeless populations) [ , ] . in other parts of the world, tb is very common, but, again, most of those with the disease do not fly. for diseases that are rare in the general population (e.g., sars, hemorrhagic fevers, plague), it is extremely unlikely that an infectious individual will be aboard any individual aircraft. not only must the infected individual be present, but the disease must be in a stage where agents are being shed into the environment in sufficient quantity that transmission could occur. for some diseases, this stage occurs before symptoms appear. for most, however, active disease must be present resulting in symptoms that lead to agent release. in the case of tb, for example, the majority of people with the disease do not shed large quantities of organisms, further reducing the probability of disease transmission. bacterial and viral aerosol particles are essentially droplet nuclei. the organisms are shed from the infected host in wet droplets containing one or more agents embedded in wet mucous secretions. once in the air, the droplets dry rapidly, shrinking to the size of the contained agents surrounded by dried secretions. these dried secretions tend to protect the organisms from environmental damage. bacteria and viruses in aerosols act as small particles, settling and/or traveling on air currents and in response to electrical charges in the same way as other small particles [ ] . true aerosol-transmitted disease agents can remain airborne, travel relatively long distances and remain infective [ , ] . distribution of the aerosols depends on air movement patterns within the space. although theoretically air movement in aircraft is in a circular motion within rows, actually there is some general transfer of air backward within the aircraft (see sect. . ). aerosols generally decay (become less concentrated) logarithmically with distance both physically and biologically. needless to say, the closer one is to the source, the greater the risk of sufficient exposure to cause illness. for diseases such as measles, where inhalation and deposition of very few virions can cause illness, the risk of contracting the disease decreases less with distance than, for example, for anthrax, where a significant number of organisms must reach an appropriate site before illness is likely. droplet-borne diseases are thought to spread up to one meter from the source patient, and hospital infection control practices reflect this belief. the droplets may impact directly in the respiratory tract, or on the hands or other skin surfaces, or even on nearby inanimate surfaces. touching contaminated surfaces could transfer organisms to the host's hands, and infection could occur with hand transfer to the respiratory tract. a study of rhinovirus colds transfer by this method, however, indicated that secretions would have to be transferred while still damp, which in the aircraft environment would be a very short time [ ] . direct contact means just what it says: touching an infected person in some way. some diseases can be transmitted with minimal contact (many common colds). others require intimate contact of a sort unlikely to occur accidentally. ventilation can only interrupt airborne infectious diseases, and then only for individuals relatively distant from the source. increasing clean air ventilation rates has been shown to reduce the incidence of upper respiratory infections in large office buildings [ , ] . high ventilation rates combined with good filtration will reduce exposure over time. in aircraft where recirculation is used, the filtration systems are probably adequate to remove the vast majority of droplet nuclei. even with good filtration and very high air exchange rates, however, ventilation cannot significantly reduce the risk of exposure for those very close to the source (e.g., sitting in the same row in an aircraft). in order to develop an infectious disease, the host must be sensitive [ ] . infections can only occur in people who do not have either natural or specific acquired immunity to the agent. there is a range of natural immunity in the population, with some people being highly resistant while others are highly sensitive, and it is not a given that any individual without specific immunity to a disease agent will become infected. the risk of illness following exposure along this immunity distribution ranges from near zero for the most resistant to very high for the most sensitive. in addition to this natural sensitivity range, acquired immunodeficiency is becoming more and more common in the population, and some of these individuals are exquisitely sensitive even to agents of relatively low virulence. for many of the common contagious childhood diseases, a large majority of the us population has naturally or artificially acquired immunity. for tuberculosis, many countries other than the us immunize for this very common disease. tb is sufficiently uncommon in the us that immunization is relatively rare. the time spent in an aerosol is another critical factor in calculating risk of a disease-causing exposure. the longer one is in the presence of an infectious aerosol, the higher the risk that a potentially infectious dose will be inhaled. the lower the aerosol concentration (or the number of agents on a surface or in each respiratory droplet) the longer one must remain in contact with the aerosol. clearly, time spent in contact with tb patients is critical in determining the risk of contracting the disease [ ] . although less well studied for other diseases, this is probably a universal phenomenon. theoretically, any contagious disease that is airborne could cause an outbreak on an aircraft, and those that are droplet borne could spread to those in adjacent seats. obviously, diseases that fit these categories and are common world wide are the most likely candidates for such spread. thus, common colds, influenza, tuberculosis, and measles have been spread on aircraft, and many cases have probably occurred that have gone unreported because of follow up failures. the rare or newly emergent diseases are much less likely to be present on aircraft. on the other hand, follow up is likely to be intensive, so that these are over-reported in relation to the more common illnesses. in addition, there is the problem of separating infections acquired in flight from those acquired at other points in travel [ ] . thus it is impossible to say whether or not a specific case of the common cold was contracted while staying away from home, on the airplane going to or from home, or, in fact, at home before travel began. given these cautions, the following is a brief discussion of some of the outbreaks that have been documented on commercial aircraft. zitter et al. evaluated the development of upper respiratory symptoms in passengers on jets with % recirculation versus % fresh air. there was no difference between the two populations. it should be noted, however, that the trips were relatively short (san francisco to denver) [ ] . given that some very common upper respiratory infections are airborne, and others produce copious droplets that could easily infect adjacent passengers, it is likely that such disease transmission does occur routinely on aircraft as it does in any other crowded environment. given the frequency of these dis-eases, it would be nearly impossible to trace an outbreak to the aircraft environment. moser et al. reported an outbreak of influenza on board a commercial aircraft grounded for three hours with no ventilation. the index case remained aboard, and the other passengers who remained on board were the most likely to develop the illness [ ] . this is a rare case where the passengers all disembarked in a relatively small city and many were seen by the same physician. if the plane had landed in new york or washington (for example) the passengers would have dispersed, and the outbreak would have gone unrecognized. given the infectiousness of the influenza virus and its airborne transmission ability, the incidence of outbreaks of this disease linked to commercial airliners has probably been underestimated. outbreaks of tuberculosis (tb) have occurred following transport of the organisms through ventilation systems in settings other than aircraft [ ] . one of the best documented cases of tb transmission aboard a commercial airliner involved a crew member with active tb. time spent with this crew member was the principal risk factor, and at least two other crew members acquired positive skin tests. frequent flying passengers may also have been affected [ ] . another well-documented case involved a multiply drug-resistant strain of mycobacterium tuberculosis, and indicated that the presence of a highly infectious person, a long flight, and close proximity to the infectious person are the primary risk factors for transmission [ ] . a study using retrospective tb testing following transport of an infected patient indicated (but did not prove) transmission to several other passengers [ ] . on the other hand, a pilot with active tb failed to infect any other of the pilots with whom he flew [ ] . although this may confirm the low risk for transmission, it may also represent a case with little shedding of infectious bacteria, or the very high ventilation rate in the cockpit compared to the passenger cabin. although generally considered droplet-borne, airborne precautions are also recommended in the clinical setting, and one building-related outbreak was determined to be airborne [ , ] . in fact, it seems clear that airborne trans-mission can occur. the first patients in the taiwan sars epidemic were closely associated with an infected person on an airplane [ ] . likewise, sars may have been introduced to france by a patient who contracted the disease in hanoi and infected others on the hanoi-paris flight [ ] . in an interview survey, olsen et al. [ ] . documented transmission of sars to airline passengers seated within the three rows in front of the index case. other studies have estimated a low risk of sars transmission on aircraft, possibly related to the stage of the illness in the sars patient, or the number of agent units being released [ ]. measles is another disease for which transmission through a ventilation system has been documented [ ] . on the other hand, one study revealed no new measles cases following a seven-hour flight with an index patient [ ] . although measles is a common disease, most people have some level of immunity, and children traveling with active infectious measles are probably rare. chicken pox transmission has not been reported related to airliner cabins, probably for the same reason that measles outbreaks are rare in this environment. for shingles, also caused by the chicken pox virus, the long delay between infection and symptoms makes tracing exposure sources nearly impossible. the centers for disease control have reported one case of bacterial meningitis acquired during travel on a commercial airliner [ ] . while this disease agent, neisseria meningitidis, is the most common cause of bacterial meningitis in the usa, it, nevertheless is a relatively rare disease, and is likely to present only a small risk for air travelers. the filoviruses ebola and marburg are able to transmit disease via the airborne route in the laboratory, but to date have not been documented to do so in the natural environment. thus, the risks are probably low for the aircraft environment. however, the possibility of their use as biological weapons remains of potential concern. h.a. burge several studies have evaluated bacterial levels in commercial airliners during flight. bacterial levels have either been lower than ground-based interiors or higher [ , ] . it is important to remember, however, that the organisms found in airliner cabin air are rarely (if ever) those likely to cause disease. the chance of actually collecting a disease agent on any individual flight is the product of the probabilities of an infectious person being on board, this person releasing infectious organisms, the samples being collected in close proximity to this person, and the organisms being identifiable using the sampling and analytical method chosen. these factors apply to other environments as well, making air sampling not the most logical approach for documenting (or monitoring) airborne infectious disease. rudnick et al. [ ] developed equations that could be used to model infection risk on aircraft. their models depend on knowing the air supply rate and having it more or less constant, both of which are achievable in aircraft. they use carbon dioxide measurements, assuming that the co is exhaled from the occupants. beggs et al. [ ] evaluated several different models and determined that gammiatoni and nucci's [ ] generalized formulation is most appropriate for ventilated spaces. ko et al. used single and sequential box models to estimate the risk of tb transmission. these authors concluded that the risk is small except for those in close contact with the infective patient [ ] . rydock et al. used tracer gas measurements and came to the same conclusion. this study also documented the minimal effect of ventilation rate on the potential for disease transmission [ ] . while contagious disease could be acquired during travel on commercial airliners, the risk of contracting anything more serious than the common cold is low. excluding persons from aircraft who are obviously suffering upper and lower respiratory symptoms (sneezing, coughing continuously) would help to prevent transmission of some diseases. however, the risks of this approach would have to be balanced with the actual risks of transmission and of the diseases' outcomes. increasing quality of filtration and/or ventilation rates is unlikely to significantly lower the risk of disease transmission in the aircraft environment where each passenger spends such a relatively short time. the risk for crew members would have to be calculated, but probably remains quite low. further research that applies some of the good infectious disease transmission models to the aircraft environment would elucidate actual risks for specific diseases [ ] . the use of molecular epidemiological techniques in tracking outbreaks would also contribute [ ] . until further studies can document a significant risk for infectious disease transmission in the aircraft environment, cost-increasing measures to reduce already minimal risks seem unwarranted. airplanes and infectious disease key: cord- -mvoq vln authors: nan title: autorenregister date: - - journal: med genet doi: . /s - - - sha: doc_id: cord_uid: mvoq vln nan complex mechanisms of dosage compensation regulate the mammalian x chromosome due to the presence of one copy in males (xy) and two in females (xx). x inactivation silences one x chromosome in females in early development, leading to specific epigenetic and structural changes. the inactive x chromosome becomes condensed and forms a bipartite structure within the nucleus, as we have shown by chromatin conformation analyses. specific long non-coding rnas are implicated in the formation of this unique structure. the inactive x chromosome is preferentially located near the lamina or the nucleolus. genes that escape x inactivation tend to be located at the periphery of the condensed inactive x chromosome. such genes are more highly expressed in females, and thus associated with sex-specific differences manifested even in early development. we have found that significant sex bias in gene expression are associated with escape from x inactivation in human tissues from normal males and females, and in tissues from individuals with sex chromosome aneuploidy, including turner or klinefelter individuals. institute for genomic medicine, columbia university medical center, new york, usa a central challenge in human disease genetics is the identification of pathogenic mutations. one key approach to distinguishing benign and pathogenic mutations is to use population genetic data to identify regions of the human genome under purifying selection. here i describe how the residual variation intolerance scoring framework has been applied to identifying pathogenic mutations in and outside protein encoding regions of the genome. next i report how these are related approaches are being used to identify pathogenic mutations in large-scale scale studies in epilepsy and other neurodevelopmental diseases. finally, i discuss how the identification of genetic causes of disease can inform treatment choices. scents indicate things, make promises, attract attention and stimulate imagination, feed anxieties and hopes: they are the salt in the atmospheric soup. we regard seeing and hearing as more important sensory functions, because they contribute more to conscious, cognitive processes of perception -but at moments of the greatest enjoyment we close our eyes and taste the scent, smell the taste. before the spirit and beauty of a person can fascinate us, our nose must become infatuated. the olfactory system in the nose acts as a window, monitoring environmental chemical information and convert chemical stimuli in electrical nerve impulses which are conducted along the olfactory sensory neuron to their glomerular target in the brain. olfactory receptors (ors) activation shows the distinguished (camp-based) transduction pathway for odorant perception. in buck and axel discovered the olfactory gene family, the largest gene family in the human genome, and postulated an exclusive expression in the olfactory epithelium. however, recent whole genome sequencing data from our and other labs show that ors have been found in every tissue of human body which was analyzed by next generation sequencing. the importance of such ectopic expression of ors is raised since the physiological function of some of ors was characterized. when identifying additional expression profiles and functions of or in non-olfactory tissue, there are limitations posed by the deorphanization of ors concerning the activated ligands and by the small number of antibodies available. in contrast to the olfactory sensory neurons which are believed to express all functional or genes (only one or type per cell), cells in non-olfactory tissues tend to express more than one individual or gene per cell. in addition, some of the signaling pathways in non-olfactory tissues seem to involve completely different components in comparison to the olfactory neurons. what is the functional role of these ectopically expressed olfactory receptors? evidences rapidly accumulate that ors participate in important cellular processes outside its primary sensorial organ where they function in odor detection and discrimination. in our lab the functional expression of the first was demonstrated in spermatozoa ( ) . in the meantime we could show the existence and function of ors in the cardiovascular system (heart, blood cells), the gastrointestinal system (small intestine, liver, pancreas), the genito-urinary system (kidney, testis, spermatozoa, prostate), the respiratory system (lung, smooth muscle cells), the skin (keratinocytes, melanocytes) and sensory organs (retina). interestingly we found a broad spectrum of important functions like cell-cell communication and recognition, tissue injury, repair and regeneration, cancer growth, progression and metastasis, nutrient sensing and muscle contraction. nevertheless the functional importance of ectopic ors is still not sufficiently understood. studies seeking to determine the function of ectopic ors are still in its infancy and require further intensive exploration. however, the potential of ors to serve a target for a wide range of clinical approaches is indeed given. this hold promises that the knowledge gained by future investigations would lead to deepen our understanding of or function in health and disease and may provide the basis for the development of applications in diagnosis and therapies in near future. enzyme replacement therapies have been developed over the last years for several of the lysosomal storage disorders (lsd's). the success of enzyme replacement therapy for gaucher disease paved the way for the development of similar treatments for the mucopolysaccharidoses, fabry and pompe disease and lately also for neuronopathic lysosomal storage disorders by intrathecal or intracerebral injections. in addition, small molecule approaches have been developed including substrate reduction therapies and chaperones, which can be used orally. while in gaucher disease enzyme as well as substrate reduction therapy results in reversibility of disease manifestations, with decreases in hepatosplenomegaly, normalization of blood counts and prevention of skeletal disease, this is unfortunately not the case for all patients affected with other lysosomal storage disorders. an important concept is the "window of opportunity for treatment" which is different for these disorders. for example, in fabry disease, early fibrosis fects are well defined, neither the specific mechanisms underlying neurological abnormalities nor the role of decreased cholesterol versus sterol precursor accumulation in disease pathogenesis have been clearly delineated. to identify cellular phenotypes and causative signaling pathways, we derived induced pluripotent stem cells (ipscs) from slos and lath subjects to model these diseases in vitro. slos subjects were known carriers of the most common dhcr mutations, including the intronic splice acceptor mutation c. - g>c and the missense mutation p.t m. while all ipscs demonstrated the expected biochemical defects due to dhcr or sc d mutations, cellular assays uncovered a defect in neural stem cell maintenance resulting in accelerated neuronal formation in slos ipscs. further molecular and biochemical analyses demonstrated inhibition of cholesterol-wnt interactions and loss of wnt/β-catenin activity mediated cellular phenotypes. however, this cellular phenotype was exclusive to slos, as lath ipscs did not exhibit a neural progenitor defect or inhibition of wnt/β-catenin activity. while this work demonstrates the utility of ipscs for modeling rare diseases and identifies signaling deficits potentially underlying slos phenotypes, questions remain regarding cellular and functional consequences, the specificity of lipid-wnt interactions, and the role of other disrupted signaling pathways in mediating developmental and functional deficits in these diseases. unpublished work using a variety of approaches will be discussed comparing the specific effects of cholesterol synthesis mutations on cell fate, functional activity, and lipid modulated signaling pathways to more precisely define the consequences of cholesterol synthesis defects and identify potential targets for patient therapy. induced pluripotent stem cell (ipsc) technology has become one of the major approaches for disease modeling since its first report in . the ability to reprogram cells from somatic into embryonic stem cell-like state and to differentiate them into desired cell types in the culture dish has allowed scientists to carry out the study of several diseases in cells such as neurons which, in the past, could not be isolated from living subjects. williams syndrome (ws), a genetic neurodevelopmental disorder where - genes are hemizygously deleted, is among those. despite cardiovascular abnormalities, its unique neurological phenotypes i. e. hypersociability is of our interest. for several decades, research on different neurological aspects of ws has been conducted in a variety of models such as patient-derived cell lines (lymphoblastoid cells and fibroblasts), post mortem tissue, and mouse models. however, the lack of physiologically relevant cell types such as neural progenitor cells (npcs) and neurons has left a critical gap in our knowledge the disease's cellular and molecular phenotypes. to fill this gap, we took the advantage of the reprogramming technology to capture the genomes of ws subjects in ipscs, which could be then differentiated into npcs and neurons, enabling evaluation of whether the captured genome with hemizygous deletion of those genes leads to relevant neuronal cellular phenotypes. dental pulp cells-derived ipscs of classical ws, rare ws and typical developing (td) subjects were neurally induced via dual-smad inhibition in order to generate npcs and neurons. we discovered that classical ws npcs exhibited increased apoptosis, and, therefore, doubling time, compared to td neurons. this could possibly contribute to the reduction in cortical surface area in classical ws individuals as assessed by magnetic resonance imaging. surprisingly, we found that rare ws npcs behaved similarly to td npcs rather than to classical ws npcs in terms of apoptosis. we confirmed that frizzled , which is deleted in the classical ws but not in our rare ws genome, is responsible for such phenotype via gain-and loss-of-function assays. moreover, classical ws neurons in general showed increased frequency of activity-dependent calcium transient compared to td neurons. finally, classical ws neurons acid alpha-oxidation, and ( .) glyoxylate detoxification. with respect to peroxisomal fatty acid oxidation peroxisomes catalyze the chain-shortening of certain fatty acids including very-long-chain fatty acids, but requires the active help of mitochondria to catalyze the degradation of acetyl-coa and the reoxidation of nadh as produced in peroxisomes. furthermore, with respect to ether phospholipid biosynthesis peroxisomes heavily rely on the endoplasmic reticulum to complete formation of ether phospholipids whereas fatty acid alpha-oxidation also requires the functional interplay between peroxisomes and mitochondria and the same is true for glyoxylate detoxification. recent evidence holds that the interaction between peroxisomes and the different subcellular organelles, including mitochondria and endoplasmic reticulum, is mediated by specific tethering protein complexes which bring organelles physically together thereby allowing metabolism to proceed smoothly. the importance of peroxisomes in metabolism is stressed by the existence of a large group of single peroxisomal enzyme deficiencies of which x-linked adrenoleukodystrophy is best known. our current state of knowledge with respect to the role of peroxisomes in metabolism and the peroxisomal enzyme deficiencies will be presented at the meeting. huntington's disease: rna-sequencing, small rna-sequencing, chip-sequencing and gwas data. department of neurology, boston university school of medicine, boston, ma , usa huntington's disease (hd) is a dominantly transmitted neurodegenerative disease of midlife onset. recently several different unbiased genome wide studies in hd have been performed. these analyses point to a variety of pathological pathways that are associated with important features of the disease, including age at onset, cag repeat size, and the extent of neuropathological involvement. genome wide association studies (gwas) have identified several regions of the genome that contain genes that are associated with the age at onset for hd. the strongest of these is located at q . for rs for which a very rare allele (maf = . %) is associated with an approximate -year younger age at onset for carriers of the minor allele. the same locus contains an independent effect for rs where a more common minor allele (maf = . %) is associated with a . year older age at onset for carriers of this allele. these single nucleotide polymorphisms are in the region of fan , mtmr and several other genes; some of which are not expressed in brain and are not likely candidates for hd modification. eqtl analysis has not resolved which gene may be implicated. other gwas implicated loci include rs at q . and rs at p . . we have sought to combine the information derived from multiple platforms to gain additional insight into the pathways that may be implicated in hd pathogenesis. in this strategy, we have performed mrna-sequencing, small rna-sequencing and chip-sequencing using the h k me mark for active transcription and the repressive mark h k me in human hd brain samples with gwas genotyping. while the striatum is most involved in hd, the extent of neurodegeneration in post-mortem tissue precludes meaningful comparison between disease and control samples, and consequently we studied prefrontal cortex (ba ). several common pathways were seen across these three platforms. mrna-sequencing and mirna-sequencing data identified altered transcriptional profiles implicating developmental pathways involving the hox genes and related homeo-box domain genes (e. g. pitx , pou f , etc.) . notably, micrornas located in hox gene clusters were among those most increased and levels of these correlate with pathological involvement in the striatum. these genes, associated with early embryonic development, are commonly silent in normal adult brain, and were among the most differentially expressed genes in hd brain. these prominent statistical effects are driven by the near total absence of expression in normal brain. pathways implicated in mrna-seq and chip-seq studies, included immune function and regulation of gene expression. these associations were very strong, indicating a large immune reactive response in the hd in the heart is related to unresponsive disease and unfortunately fibrosis may occur already in an early stage, sometimes even without prior hypertrophy. whether earlier intervention will be beneficial is largely unknown. and then: what is early? many unresolved questions exist at this stage, including the following: -what is the natural history and "point of no return" for the different lsd's? -what is the natural history and "point of no return" for subgroups of patients within one lsd's? -what are the long term complications: treatments change the phenotype rather than cure the disease -what is the influence of antibody generation on clinical effectiveness? -how do we manage the extreme costs of these products, especially in light of the many unsolved issues with respect to effectiveness? surprisingly, so far healthcare professionals, governments and industry have failed to systematically address these issues, resulting in insufficient knowledge for potentially lifesaving treatments. early conditional access, followed by a strict, transparent, independent, collaborative evaluation in addition to fair pricing should be explored. after the recent explosion in sequencing throughput, variant interpretation has quickly become the bottleneck in our effort to usher in the era of genomic medicine. while homozygosity for apparently pathogenic variants in the context of disease states is a well-established phenomenon, homozygosity can uncover many medically relevant aspects of the human variome that are difficult to study otherwise. for example, seemingly benign variants may prove pathogenic in the homozygous state. this includes variants with benign prediction using in silico tools as well as variants in dominant genes with no phenotype in carriers because they represent bona fide recessive inheritance. variants that are associated with one phenotype in compound heterozygous states may express themselves quite differently phenotypically when homozygous. furthermore, previously reported pathogenic variants can be challenged when their presence in homozygosity is associated with no abnormal phenotype, thus improving the specificity of the annotation of the morbid genome. homozygosity for lof variants is a special scenario that allows us to study naturally occurring human "knockouts", a powerful tool to study the physiological context of genes in humans. finally, homozygosity in the context of autozygosity provides a robust mapping tool that can greatly aid in the identification of relevant variants, especially those that exert their pathogenic effect in ways that defy detection by our usual algorithms. by expanding the spectrum of phenotypes that are studied, one can unlock the full potential of homozygosity to understand the medical relevance of the human variome in it its full range from embryonic lethal to essentially benign. helmholtz zentrum münchen gmbh, institut für experimentelle genetik, ingolstädter landstr. , neuherberg, germany the inheritance of epigenetic information in mammals across generations has been controversial. some reports provided initial evidence that a paternal high fat diet may propagate obesity and glucose intolerance in offspring, but potential confounders such as molecular factors present in seminal fluid, paternal-induced alterations in maternal care or transmission of microbiomes were not ruled out in these studies. we have shown in mice that a parental high fat diet (hfd) renders offspring derived via in vitro fertilization (f ) more susceptible to develop excessive overweight and type diabetes (t d) in a gender and parent-of-origin specific mode. female, but not male, offspring from obese parents became significantly more obese during a hfd challenge than female offspring from lean parents. body weight trajectories and distribution patterns of individual body weights in female offspring from one obese and one lean parent demonstrate that paternal and maternal germline propagate obesity in a roughly equitable and additive fashion, but likely different mode of action. in contrast, a more deteriorated state of hfd-induced insulin resistance was observed in both f genders, albeit predominantly inherited via the maternal germline. towards the identification of epigenetic information in sperm and oocyte from hfd and low fat diet fed parents, we are currently analyzing their transcriptome and methylome signatures. the status of this analysis will be presented. we report for the first time epigenetic inheritance of an acquired metabolic disorder via mammalian oocytes and sperms excluding confounding factors. such an epigenetic mode of inheritance may contribute to the observed pandemic increase in obesity and t d prevalence rates, especially in an environment where nutrition is abundant. brain which may be a major influence contributing to neurodegeneration. in many instances enrichment of h k me at transcription start sites was not accompanied by a corresponding increase in expression. the apparent inconsistency suggests that common regulatory mechanisms in the hd brain are disrupted and this may contribute to a complex interplay of factors contributing to the neurodegenerative process. often findings in human hd brain samples conflicted with those reported in hd transgenic mouse models, suggesting that one may wish to be cautious in interpreting the significance of either type of study in isolation. the causal pathogesis of huntington disease, new therapeutic approaches r. laufer senior vp discovery and product development global r&d, teva r&d product development mgmt. hatrufa st, netanya, israel huntington disease (hd) is an autosomal dominant neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, cognitive decline and death - years after motor onset. hd is uniquely caused by a polyglutamine encoding cag expansion in the huntingtin gene (htt), which allows for identification of pre-manifest mutation carriers as much as decades before onset and should facilitate development of disease modifying therapies. yet over years after identification of the hd mutation, available therapies offer only symptomatic relief and are fraught with side effects. development of safe small molecule therapies for hd has been hindered by difficulties identifying and validating tractable drug targets within the disorder's complex pathogenesis. teva pharmaceuticals is developing potential novel treatments based on a mechanistic understanding of disease pathways common to neurodegenerative diseases. the progress of these studies will be reviewed. rare complete gene knockouts in adult humans p. sulem statistics department, decode genetics, sturlugata , reykjavik, iceland loss-of-function mutations cause many mendelian diseases. here have create a catalog of autosomal genes that are completely knocked out in humans by rare loss-of-function mutations. we sequenced the whole genomes of over , icelanders and imputed the sequence variants identified in this set into a total of chip-genotyped and phased icelanders. of the genotyped icelanders, around % are homozygotes or compound heterozygotes for loss-of-function mutations with a minor allele frequency (maf) below % in close to genes (complete knockouts). genes that are highly expressed in the brain are less often completely knocked out than other genes. homozygous loss-of-function offspring of two heterozygous parents occurred less frequently than expected (deficit of per , transmissions for variants with maf < %, % confidence interval (ci) = - ). we are currently systematically phenotyping such human complete knock out. this phenotyping lasts hours and attempts to cover most of the observable diversity in a non-invasive and cost efficient manner. i will demonstrate how using systematic phenotyping can advance the knowledge on individual gene knockout. we use results from in-house transcriptomics, existing animal models and complementary approaches to assess the observation in human. we will also discuss the scrutiny in other population in order to detect such complete knock-out. we will exemplify the impact of founder population and consanguinity in such an odissey. early onset and severe obesity can be inherited via loss of function mutations within the melanocortin pathway of hypothalamic body weight regulation. the most prominent player in this signalling pathway is the fat cell hormone leptin. leptin gene mutations were the first to be linked to monogenic early onset obesity. after binding of leptin to leptin receptors in the arcuate nucleus of the hypothalamus the neuropeptide msh is processed from the precursor pomc and acts as a ligand at the mc receptor. mutations in the leptin receptor gene, the pomc gene and the mc receptor gene were subsequently diagnosed in further patients with extreme early onset obesity. while leptin mutation patients can be treated with recombinant leptin -as shown already in the late s -all other monogenic obesity forms are leptin resistants, and additional leptin failed to decrease body weight. only recently pomc gene deficient patients were successfully treated with the msh-analogue setmelanotide (kühnen et al. ) . common severe obesity is defined by the lack of disease causing monogenic defects. a plethora of gwas identified a large number of snps in common obesity associated with the individual bmi but only to a low amount of not more then %. however, almost all these common obese patients are characterized by high leptin levels suggesting sufficient generation of leptin in the increased fat tissue and a state of leptin resistance. several new data concerning the contribution of epigenetic and genetic variants in the pomc gene locus argue for a role of the melanocortin pathway also in common obesity and imply, therefore, a potentially new treatment option also in common obesity based on msh-analogues. genome-wide association studies have highlighted the role of genetic associations with susceptibility to common inflammatory diseases, highlighting potential new insights into disease pathogenesis and opportunities for therapy. however understanding the functional basis of these associations and delivering translational utility remains a significant challenge to the field. non-coding regulatory genetic variants are most commonly implicated in such studies. recent work highlights how such variants are also major drivers of diversity in the immune response transcriptome. this talk will discuss approaches we are taking to try and establish functional links between immune phenotype-associated regulatory genomic and epigenomic variation, and specific modulated genes and pathways. i will describe insights from the application of expression quantitative trait (eqtl) mapping to define genomic modulators of the global transcriptomic response in different primary immune cell populations and to specific innate immune stimuli in health and disease. this work highlights the extent of local and distant context-specific eqtl, enabling resolution of immunoregulatory variants and the identification of specific modulated genes involving disease associated loci. examples will be described showing how mapping trans-regulatory loci can be a powerful approach for discovery and dissection of gene networks informative for disease. i will also show how we have applied analysis of the genetics of gene expression in patients with sepsis admitted to intensive care, revealing new insights into disease pathogenesis. further progress in this area will require characterisation of associated variants in the context-specific disease relevant epigenomic landscape in which they may act, requiring careful consideration of relevant immune cell types and environmental modulators to study, to-abstracts aktuellen stellungnahme der deutschen forschungsgemeinschaft sollen humane genomsequenzierungen die möglichkeit der rückmeldung von analyseergebnissen enthalten. als orientierung für einen verantwortungsvollen umgang mit dieser frage wird auf die projektgruppe eurat verwiesen. dennoch bleibt das problem der einordnung mitteilungswürdiger ergebnisse aus forscher-und probandensicht und der bereitstellung der für aufklärungs-und rückmeldungsalgorithmen erforderlichen ressourcen. darüber hinaus ist bis heute nicht geklärt, welche kommunikativen prozesse eine ausreichende basis für ein informiertes einverständnis darstellen. diese und weitere fragen möchten wir mit frau prof. dr. med. dr. phil. eva winkler (nationales centrum für tumorerkrankungen, heidelberg), herrn dr. phil. martin langanke (theologische fakultät, universität greifswald) und herrn pd dr. phil. peter burgard (universitätskinderklinik heidelberg) kontrovers diskutieren. die organisatoren werden in die fragestellung einführen, anschließend sind impulsreferate (ca. min) der referenten vorgesehen, bevor eine debatte mit dem publikum angeregt wird. fangerau, f. söhner (düsseldorf) in den er jahren erlebte die humangenetik wie eine reihe anderer medizinischer disziplinen auch eine erhebliche institutionelle ausweitung. in den er und er jahren wurde beispielsweise an den universitäten der brd das gros der humangenetischen lehrstühle, in den ausgehenden er und er jahren auch in der ddr, eingerichtet. ging vom symposium "genetik und gesellschaft" im rahmen des marburger "forum philippinum" die initiative aus, in der ganzen bundesrepublik genetische beratungsstellen zu gründen und damit die genetische forschung (wieder) medizinisch nutzbar zu machen. in dieser phase der etablierung der humangenetik auf akademischer und praktischer ebene setzt das geplante zeitzeugenprojekt ein. es will die entwicklung der humangenetik in ihrem selbstverständnis als quer-und als längsschnittfach (pfadenhauer : ) im deutschsprachigen raum ab den er jahren mit hilfe von expertengesprächen dokumentieren und analysieren. im forschungsprojekt sollen zwei komplexe von fragestellungen bearbeitet werden: ein wissenschaftshistorischer, in dem die entwicklung und anwendung von diagnostischen und therapeutischen techniken im mittelpunkt steht, und ein sozialhistorischer, in dem es um die etablierung und den ausbau der institutionen der humangenetik sowie um den verlauf der das fach betreffenden gesellschaftlichen debatten geht. neben der gründung von instituten und der fachgesellschaft sowie der normierung der ausbildung für ärztliche und naturwissenschaftlich ausgebildete humangenetiker sollen die funktion der historischen reflexion und bearbeitung der facheigenen nationalsozialistischen vergangenheit in den er jahren für die etablierung des fachs und der schwierige institutionelle trennungsprozess von der anthropologie mit ihren wirkungen auf das selbst-und fremdbild der humangenetik analysiert werden (weingart, kroll, bayertz ) . zusätzlich zur entwicklung in der brd sollen dabei auch die entwicklung des fachs in der ddr und mögliche deutsch-deutsche kooperationen zur sprache kommen (in jena befand sich z. b. in den er jahren das zentrale referenz-institut für genetische beratung in der ddr (vogel : ) . das projekt kann sich auf zahlreiche arbeiten zur geschichte der deutschen humangenetik stützen (vgl. kröner , cottebrune cottebrune , weingart ; bennike dysmorphism carrying a pathogenic variant in the ebf gene detected by whole-exome sequencing. five missense, two nonsense, one -bp duplication, and one splice-site variant in ebf were found; the mutation occurred de novo in eight individuals, and the missense variant c. c>t [p.(arg trp) ] was inherited by two affected siblings from their healthy mother who is a mosaic. ebf belongs to the early b-cell factor family (also known as olf, coe, or o/e) and encodes a transcription factor involved in neuronal differentiation and maturation. structural assessment predicts perturbing effects of the five amino acid substitutions on dna-binding of ebf . transient expression of ebf mutant proteins in hek t cells revealed mislocalization of all but one mutant in the cytoplasm in addition to nuclear localization. by transactivation assays, all ebf mutants showed significantly reduced or no ability to activate transcription of the reporter gene under control of the cdkn a promotor that corresponds well with loose association of ebf mutants with chromatin as demonstrated by in situ subcellular fractionation experiments. finally, rna-seq and chip-seq experiments demonstrate that ebf acts as a transcriptional regulator at cis-regulatory sequences and ebf mutant had reduced function due to partial disruption of the dna-binding domain. these findings demonstrate that ebf -mediated dysregulation of gene expression has profound effects on neuronal development in humans and add ebf to the growing list of genes in which mutations cause syndromic forms of intellectual disability. step, we performed wes in further unelucidated uhs cases and identified homozygous nonsense mutations in tgm (transglutaminase ) and in tchh (trichohyalin), respectively. elucidation of the molecular outcomes of the disease causing mutations by cell culture experiments of padi and tgm and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild type proteins. by immunofluorescence analysis, we could demonstrate a diffuse homogenous cytoplasmic distribution of the wt padi , whereas in the mutants the proteins were observed to form large aggregates throughout the cytoplasm. by use of human anti-citrullinated protein autoantibodies, we could show a strong labelling in the wt whereas the staining of the mutants was barely above background. in order to demonstrate the importance of padi in hair shaft formation, we generated padi knockout mice. electron microscopy observations revealed morphological alterations in hair coat of padi knockout mice. for tgm , we performed a transglutaminase activity assay. the analysis results revealed that the wt had a significantly higher transglutaminase activity in comparison to the truncated protein. here, we report for the first time the identification of uhs causative mutations located in the three genes padi , tgm and tchh. the two enzymes responsible for posttranslational protein modifications, and their target structural protein, are all involved in hair shaft formation through their sequential interactions. these findings provide valuable information regarding the pathophysiology of uhs and contribute to a better understanding of this protein interaction cascade. this could be of further value for cosmetics and pharmaceutics industries paving the way for development of novel products. deadenylases are best known for degrading the poly(a) tail during mrna decay. the deadenylase family has expanded throughout evolution and, in mammals, consists of mg + -dependent ' end ribonucleases with mostly unknown substrate specificity. pontocerebellar hypoplasia type (pch ) is a unique recessive syndrome characterized by neurodegeneration with ambiguous genitalia (mim% ). we studied human families with pch , uncovering biallelic, loss of function mutations in toe , which encodes an unconventional deadenylase. toe -morphant zebrafish displayed mid-and hind-brain degeneration, modeling pch-like structural defects in vivo. surprisingly, we found toe associated with incompletely processed small nuclear (sn)rnas of the spliceosome, which is responsible for pre-mrna splicing. these pre-snrnas contained ' genome-encoded tails often followed by post-transcriptionally added adenosines. human cells with reduced levels of toe accumulated ' end-extended pre-snr-nas, and immuno-isolated toe complex was sufficient for ' end maturation of snrnas. our findings reveal the cause of a neurodegenerative syndrome linked to snrna maturation and uncover a key factor involved in processing of snrna ' ends. the kidney maintains acid-base homeostasis and electrolyte balance through highly specialized cells. in the distal nephron acid secretion is mediated by type a intercalated cells (a-ics) , which contain v-type at-pase-rich vesicles that fuse with the apical plasma membrane on demand. intracellular bicarbonate generated by luminal h+ secretion is removed by the basolateral anion-exchanger ae . dysfunction of type a intercalated cells results in distal renal tubular acidosis (drta) and human mutations in v-atpase subunits and ae are causative for this condition. for the ae r h mutation a dominant-negative trafficking mechanism was proposed to explain ae -associated dominant drta based on studies in mdck monolayers. to test this hypothesis in vivo and to test potential rescue strategies correcting this mistargeting defect, we have generated a r h knock-in mouse strain, which corresponds to the most common dominant drta mutation in human ae , r h. heterozygous and homozygous r h knock-in mice displayed incomplete drta characterized by compensatory upregulation of the na+/hco -cotransporter, nbcn . as expected for the r h mutation, red blood cell ae -mediated anion-exchange activity and surface polypeptide expression were unchanged. surprisingly, basolateral targeting of the mutant ae in a-ics was preserved in contrast to previous studies in mdck cells. instead, we found ae expression in a-ics strongly reduced in a r h dosage-dependent manner. additional cell culture studies in two widely used immortalized renal cell lines verified that targeting and half-life time of mutant ae protein was indeed preserved. surprisingly, atpase expression was reduced and its plasma membrane targeting upon acid challenge compromised. ultrastructural analysis revealed a loss of apical vesicles in a-ics, while we observed lysosomal inclusions and multilamellar bodies. accumulation of p -and ubiquitin-positive material in a-ics of knock-in mice suggest a defect in the degradative pathway, which may ultimately lead to loss of a-ics. highlighting the expression of ae specifically in a-ics, type b intercalated cells were unaffected. we propose that reduced basolateral anion-exchange activity in a-ics inhibits trafficking and regulation of v-type atpase, compromising luminal h+ secretion and possibly also lysosomal acidification. our findings illustrate the considerable, context-dependent complexity of ae -related kidney disease. b. vona , d. liedtke , k. rak , , r. katana , l. jürgens , pr. senthilan , i. nanda , c. neuner , mah. hofrichter , l. schnapp , j. schröder , u. zechner , s. herms , , , p. hoffmann , t. müller , m. dittrich , , o. bartsch , pm. krawitz , e. klopocki , w. shehata-dieler , mc. göpfert , t. haaf although many genes have already been identified as causing non-syndromic hearing loss (nshl), diagnostic rates of approximately % among hearing impaired patients suggest that many more genes are remaining to be identified. nshl is the most common sensory deficit that has a prevalence between one and two per newborns. furthermore, it demonstrates classic genetic heterogeneity with as many as % of coding genes in the genome anticipated to be involved in non-syndromic forms of deafness. autosomal recessive ( - %) and autosomal dominant ( - %) forms dominate inheritance patterns of deafness; however, in a small fraction of cases, x-linked deafness ( - %) can be observed. whole exome sequencing of a german family with diagnostically unresolved nshl revealed a novel missense variant predicted as pathogenic in the gene ferm and pdz domains containing protein (frmpd ) on chromosome xp . . this gene, also known as preso , was first described as a regulator of dendritic spine morphogenesis. previous screening of pathogenic cnvs in array based comparative genomic hybridization among families with heterogeneous x-linked intellectual disability (xlid) showed duplication of xp . including part of frmpd which implicated the gene in xlid. interestingly, a segregating truncating and a de novo missense mutation in frmpd have associated this gene with xlid, a phenotype not observed in our family. mouse expression studies localize frmpd to spiral ganglion neuron peripheral dendrites of the developing cochlea. in addition, we analyzed frm-pd knockdown and loss-of-function zebrafish mutants for innervation and structural defects in the otic vesicle and lateral line neuromasts. posterior lateral line neuromasts are observed with reduced axonal outgrowth that is also likely reduced in the lateral line nerve. abnormal innervation is also apparent in the otic vesicle. fluorescent neuromast labeling marked a significant reduction of overall otic vesicle and lateral line neuromasts in mutants versus wild type zebrafish. scanning electron microscopy revealed a pronounced absence of kinocilia in posterior lateral line neuromasts of frmpd -/zebrafish. furthermore, adult frmpd mutants show significantly delayed acoustically evoked behavioural responses compared to wild type fish indicating hearing impairment. investigation of transgenic drosophila insertion mutants detected a mild auditory phenotype i. e. a reduction in mechanical amplification gain and associated reduction in antennal fluctuation power. our results associate frmpd with x-linked hearing loss and suggest mutations in this gene are correlated with pleiotropic effects abstracts has also been demonstrated to be an important pathobiochemical feature in rtt. to test whether common deficits in mtor signaling could be responsible for the molecular pathogenesis underlying both syndromes, we generated and studied a novel cdkl knockout (cdkl -/y) mouse model and performed in vitro experiments in human cells. in cdkl -/y knockout mice loss of cdkl is accompanied by reduced phosphorylation levels of critical components of the mtor signaling cascade. these findings point at a regulatory role of cdkl /cdkl on mtor activity and function. to gain further insights into the possible mechanism through which cdkl /pi k interaction could regulate mtor signaling, we used hek-t cells as cellular model. following knock-down of cdkl , the amount of pi k protein was significantly reduced compared to controls. to evaluate the contribution of our findings to pathogenesis, we performed rescue experiments in cdkl knock-down hek-t cells using wild-type and patient-specific mutant cdkl constructs. further experiments are ongoing to clarify the molecular mechanism by which cdkl regulates pi k protein level in the cells. inferring expressed genes by whole-genome sequencing of plasma dna medical university graz, graz, austria, university of technology, graz, austria the analysis of cell-free dna (cfdna) in plasma represents a rapidly advancing field in medicine. cfdna consists predominantly of nucleosome-protected dna shed into the bloodstream by cells undergoing apoptosis. we performed whole-genome sequencing of plasma dna and identified two discrete regions at transcription start sites (tsss) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. by employing machine learning for gene classification, we found that the plasma dna read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. in patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. we were able to determine the expressed isoform of genes with several tsss, as confirmed by rna-seq analysis of the matching primary tumor. our analyses provide functional information about cells releasing their dna into the circulation. institute of human genetics, fau-erlangen-nürnberg, erlangen, germany, institute of medical genetics, university of zurich, schlieren, switzerland rna-splicing is an important mechanism for eukaryotic gene expression and regulation. defective splicing significantly contributes to monogenic disease in humans. indeed, the mutational space for variants affecting splicing is larger than for coding variants. several computational methods have been developed to predict a variant's effect on splicing but lack predictive value outside the canonical splice sites and do not predict aberrant transcripts. thus the plethora of dna variants generated by recent advances in "next-generation" based sequencing (ngs) can be scored for a possible splicing effect, but a laborious wet-lab based confirmation and characterization is still required. rna-seq is widely used for quantification of gene expression and can be used to detect splicing events, but is limited for this use by the variable and often low read coverage of individual congenital anomalies of the kidneys and urinary tract (cakut) are the most common cause of chronic kidney disease in children. as cakut is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new cakut causing genes. using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (lifr) gene causing instability of the mrna in a patient presenting with bilateral cakut and requiring kidney transplantation at one year of age. lifr encodes a transmembrane receptor utilized by il- family cytokines, mainly by the leukemia inhibitory factor (lif). mutational analysis of further patients with severe cakut yielded two rare heterozygous lifr missense variants predicted to be pathogenic in three unrelated patients. lifr mutants showed decreased half-life and cell membrane localization resulting in reduced lif-stimulated stat phosphorylation. lifr showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying lifr variants. additionally, a form of cryptorchidism was detected in all lifr -/mice and the patient carrying the lifr frameshift mutation. altogether, we demonstrate heterozygous novel or rare lifr mutations in . % of cakut patients, and provide evidence that lifr deficiency and deactivating lifr mutations cause highly similar anomalies of the urogenital tract in mice and humans. loss of cdkl associated with deficient mammalian target of rapamycin (mtor) signaling in mice and human cells we and other groups have shown that mutations in the x-linked cyclin-dependent kinase-like (cdkl ) gene cause a severe neurodevelopmental disorder with clinical features including intellectual disability, early-onset intractable seizures and autism, that are closely related to those present in rett syndrome (rtt) patients. rtt is caused by mutations in the x-linked mecp gene. cdkl is a serine/threonine kinase and to date knowledge about its functional roles is scarce. we searched for cdkl interacting proteins by yeast-two hybrid screens. one of the candidates identified in these screens is a subunit of the phosphatidylinositol- , -biphosphate -kinase (pi k). the results obtained in yeast could be confirmed in vitro in mammalian cells and in mouse brain by immunoprecipitation experiments and by co-localization studies. pi k phosphorylates membrane lipids which act as docking sites to recruit targets upstream of mtor and thereby regulate among major cellular processes synaptic plasticity, which is the cellular basis for learning and memory. alteration of mtor signaling gene tubes for stabilizing rna immediately after drawing the samples. the subsequent rt-pcr analysis showed that of the variants located at potential splicing sites indeed affect splicing. thus of these variants could be classified as deleterious (iarc class ), while one chek variant could not be unequivocally classified as the rt-pcr analysis identified only % of the mutant transcript indicating continued usage of the constitutive splice acceptor site. this led to the classification as a probably hypomorphic allele. the variants in cdh and mlh did not affect splicing and were classified as benign (iarc class ). none of the rare synonymous and nonsynonymous exonic variants showed any effect on splicing. in conclusion, this analysis allowed the disambiguation of out vus at potential splice sites into a definite category (either iarc class or ). this work highlights the importance of computational splicing prediction and validation using rt-pcr of peripheral blood rna to assess the pathogenicity of vus. this in turn, allows more accurate genetic counseling and clinical management of affected families. gliomas present the major group of neoplasia in the central nervous system. they typically show invasive growth and high recurrence rate and are currently not curable. idh mutations are detected in nearly % of low grade gliomas and are considered to play a key role in low grade glioma development. while it is known that idh / mutation leads to high-levels of -hydroxyglutarate ( -hg) that functions as an oncometabolite, little is known about the influence of idh / mutations on energy metabolism and metabolic reprogramming in the tumor cells. since patient derived idh mutant cells do not grow in cell culture, previous studies from our group and others used transduced cell lines that overexpress idh . in order to develop in vitro models with reduced side effects, we used crispr/ cas to introduce the idh r h mutation in a patient derived glioblastoma cell line. the edited cells expressed idh r h in western blot and expression levels of idh were comparable to the expression in wild type cells. the mutation was stable in long time culture experiments, without signs of senescence. moreover we found elevated -hg levels, proving that the idh r h neoenzymatic function is present in our cell lines. thus, we were able to edit and culture genomic idh r h mutated glioma cells for the functional analysis of the idh r h mutation for the first time without the effects of overexpression models. edited idh r h cell lines showed extended doubling times compared to wildtype cells. measurement of krebs cycle metabolites using mass spectrometry revealed elevated glutamate levels. we found enhanced atp-levels that could be a consequence of decreased atp consumption. additionally, the cells showed reduced viability compared to wildtype cells when cultivated in glycolysis inhibiting media, pointing out the enhanced dependency on glycolysis in idh r h cells. these results indicate changes in tumor cell metabolism and energy household induced by the idh r h mutation. since we and others could show that idh r h can alter nad+ and nadph levels, we tested if the idh r h mutated cells are more susceptible to selective inhibition of nad/p regenerating enzymes. esirna-silencing of nampt specifically decreased cell viability in idh r h but not wildtype cells with a concomitant increase of dead cells. in conclusion, we developed genes. thus, a standardized ngs based approach to characterize potential splice variants is lacking. hence we investigated the utility of hybridization based gene-panel enrichment and ngs of cdna. based on results of computational simulation we selected twenty rna-samples of patients with a known pathogenic splice-site variant in an inherited cancer predisposition gene. these variants were previously characterized by rt-pcr in our lab or in the literature. after rrna depletion and dna digestion we performed first and second strand cdna synthesis followed by "tagmentation"-based library preparation, targeted enrichment using the trusight cancer panel and sequencing on an illumina miseq platform. a computational pipeline was established to enable automated detection of aberrant splicing events by implementing different alignment and splice-junction detection algorithms together with filtering against control data sets. we also considered variant calling for the detection of allelic imbalance and gene-level expression analysis in this data. breast and ovarian cancer (bc/oc) predisposition has been associated with a number of high-and low-penetrance susceptibility genes. advances in sequencing technology has made multigene testing a practical option when searching for genetic variants associated with risk for bc/oc. variants of uncertain significance (vus), though, represent a major problem. we now studied patients fulfilling criteria for brca and testing using the next generation sequencing based trusight sequencing cancer panel on a miseq platform (illumina). data was analyzed after remapping with bwa to hg (grch ) using seqnext software (jsi) for variants in known high and moderate penetrance susceptibility genes (brca / , atm, chek , palb , rad c, rad d, nbn, cdh , tp , mlh , msh , msh , pms ) . besides deleterious mutations we also identified vus. of these, variants ( each in brca , brca , palb , rad c, rad d, cdh , and mlh , and in chek and mlh , respectively) affect possible splicing sites. in addition, synonymous and nonsynonymous variants outside the splicing sites ( in brca , brca and cdh , respectively, in rad d) were not reported in exome variant server or exome aggregation consortium (exac) databases, so far. no families were available to study familial segregation. for all these variants a potential effect on splicing efficiency was predicted by three different computational algorithms (bdgp: splice site prediction by neural network, netgene server and the human splice finder (hsf . ) algorithm). we took advantage that these genes are ubiquitously expressed to investigate possible effects of these variants on mrna splicing using easily accessible peripheral blood. as mrna is notoriously unstable, we used pax-abstracts was evaluated for both the individual markers and their combinations derived from multiple algorithms. pronounced demethylation of all markers was observed at baseline among cases compared to controls. risk of developing lc increased with decreasing dna methylation levels, with adjusted ors ( % ci) of . ( . - . ), . ( . - . ) , and . ( . - . ), respectively, for participants in the lowest quartile of ahrr, p . , and f rl compared to participants in the highest quartiles of each site among controls. the individual markers exhibited similar accuracy in predicting lc incidence, with aucs ranging from . to . . combination of the markers did not improve the predictive performance (auc = . ). the individual markers or their combination outperformed self-reported smoking exposure particularly in light smokers. no variation in risk prediction was identified with respect to age, follow-up time, and histological subtypes. ahrr, p . , and f rl methylation in blood dna are predictive of lc development, which might be useful for identification of risk groups for further specific lc screening, such as ct examination. over the past decades the search for disease causing variants has been focusing exclusively on the coding genome. this highly selective approach has been extremely successful however, recent data have revealed the importance of the non-coding genome in fundamental processes such as gene regulation, d chromatin folding, and pinpointed its role in disease. in this study, we systematically investigate the cis-regulatory landscape of pitx , a homeodomain transcription factor that is exclusively expressed in the hindlimb. mutations and non-coding structural variations at the pitx locus have been shown to associate with a variety of congenital limb defects including club feet, polydactyly, and arm-to-leg transformation (liebenberg syndrome). we performed in vivo enhancer reporter essays in transgenic mice and identified several limb enhancer elements at the pitx locus; surprisingly they all showed both forelimb and hindlimb activity, although pitx is never expressed in the forelimb. capture hi-c experiments revealed a hindlimb-specific chromatin-organization at the pitx locus, which enables its promoter to contact several enhancers bearing a pan-limb activity only in the hindlimb. this tissue-specific chromatin folding plays a determinant role to refine the unspecific limb regulatory landscape toward a highly controlled and hindlimb delimited transcriptional output. to gain a better understanding of the pathology of pitx associated limb defects, we used crispr/cas to generate a set of deletions and inversions in the pitx cis-regulatory landscape in mice. genetic perturbations of the regulated d chromatin conformation lead to an ectopic forelimb expression of pitx , resulting in an arm-to-leg transformation in mice and in human patients respectively. our data further highlight the role of non-coding mutations affecting chromatin folding in congenital disease and give new insights into the regulation of pitx during development and the pathomechanism of associated limb defects. hoxb , a member of the embryonic homeobox transcription regulators, has been identified as the first susceptibility gene specific for prostate cancer (prca). the founder missense mutation g e, which likely originated from finland, can be found in most populations of european ancestry. we determined the frequency of hoxb g e for the german population, assessed in a cohort of unrelated cases, each with positive family history of prca, sporadic prca cases and in controls. additional affected relatives from prca families were included to explore association with aggressive disease in subgroups with high gleason score (> ), advanced tumor stage, or psa at diagnosis > ng/ml. carriers of g e were rare in controls ( . %) and showed increased frequencies in both sporadic ( . %) and familial prca cases ( . %). estimated risks were or = . (p = . ) and or = . (p = . ), respectively. the risk effect size increased with the number of affected individuals per pedigree: or = . (p < . ) for or more, and or = . (p < . ) for or more affected men. the strongest association with clinical features was observed between g e and advanced tumor stage (or = . ; p < . ). in conclusion, the observed frequency of hoxb g e mutation carriers in our study cohort was intermediate as compared to the common prevalence in scandinavia and the rare occurrence in mixed european populations from the us. the risk estimates of hoxb g e and the stronger effect sizes in families with increasing number of affected relatives were in line with a high penetrant germline predisposition. the association between g e status and tumor stage may be of greater interest for clinical practice, but needs further validation. the absolute penetrance of the hoxb g e mutation should be investigated in further studies in order to elucidate its suitability as a genetic predictor for prca. smoking-associated dna methylation markers predict lung cancer incidence homozygous smn loss causes spinal muscular atrophy (sma), the most common lethal genetic childhood motor neuron disease. smn encodes smn, a ubiquitously expressed housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. sma individuals harbor low smn expression from one to six smn copy genes, which is insufficient to functionally compensate for smn loss. however, rarely individuals with homozygous absence of smn and only three to four smn copies are fully asymptomatic, suggesting protection through genetic modifier(s). previously, we identified plastin (pls ) overexpression as an sma protective modifier in humans and showed that smn deficit impairs endocytosis, which is rescued by pls overexpression. here, we identify reduction of the neuronal calcium sensor neurocalcin delta (ncald) as a protective sma modifier in five asymptomatic smn -deleted individuals carrying only four smn copies. we demonstrate that ncald is a ca + -dependent negative regulator of endocytosis, as ncald knockdown improves endocytosis in sma models and ameliorates pharmacologically induced endocytosis defects in zebrafish. importantly, ncald knockdown effectively ameliorates sma-associated pathological defects across species, including worm, zebrafish and mouse. in conclusion, our study identifies a previously unknown protective sma modifier in humans, demonstrates modifier impact in three different sma animal models and suggests a potential combinatorial therapeutic strategy to efficiently treat sma. since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in sma and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies. mutations affecting coding or regulatory regions of smc cause dysregulation of condensins resulting in a phenotype reminiscent of cohesinopathies cornelia de lange syndrome (cdls) is a dominantly inherited malformation syndrome caused by mutations in genes encoding subunits (smc a, smc , rad ) or regulators (nipbl, hdac ) of the cohesin complex. this dna-bound complex regulates several chromatin-related processes such as chromosome segregation, dna-damage repair, transcription and chromatin structure. the project presented initially started with two children and their mother who showed clinical features reminiscent of cdls. while various sequencing approaches failed to identify the disease-causing mutation, a kb spanning deletion co-segregating with the phenotype was identified by array-cgh. besides the last exons of cylc , encoding a sperm head protein, no other genes were affected. subsequent in-silico analyses predicted the existence of a ~ kb tissue-specific regulatory element within this region, located approximately mb distant from the next protein-coding gene smc , which encodes a subunit of the cohesin-related condensin complex. significant reduction of smc expression was verified in patient's fibroblasts by qpcr analysis. accordingly, a strong dysregulation of smc was observed in hek and sh-sy y cells deficient for the putative kb regulatory element, which was deleted by crispr/cas genome editing. reporter gene assays further highlighted the functional relevance of the identified regulatory element in regulating the smc gene promoter. interestingly, we could prove on protein as well as on mrna level that alterations in smc expression are correlated with the dysregulation of other condensin subunits such as smc in patient's samples as well as in cris-pr/cas -generated cells. in a large exome sequencing project we have identified a smc frameshift mutation in an additional family with two patients who show clinical features overlapping with those seen in our initial family. quantitative pcr analyses in fibroblasts of both subjects also showed significant reduction of smc and smc expression, which is consistent with our findings in the first family. to further investigate whether alterations in condensin gene expression are specific for the dysregulation of smc , we have decreased smc levels in different cell types by sirna. quantitative protein as well as mrna analyses revealed reduced smc /smc expression. our data show for the first time the coordinated expression of different condensin subunits and its relevance for human disease. abstracts human pedigree. cardiac valves initially form through a process called endothelial-to-mesenchymal transition (emt) then subsequently elongate and mature during early juvenile life. expression analysis throughout embryonic and postnatal stages of adamts -/-mice revealed an expression in all cardiac valves after valve formation. high resolution, digital echocardiography showed that mice without adamts expression develop dysfunctional aortic valves early in life, reminiscent of the human phenotype. notably, the expression of adamts in the valve was restricted to valvular interstitial cells and not observed in endothelial cells. functional analysis using proteomic approaches suggest that the presence of ad-amts is necessary to maintain extracellular matrix remodelling during valve development and its maturation. not only do the lof mice fully recapitulate the human phenotype, they also highlight adamts as a novel marker for valvular interstitial cells to specifically target initial post-emt processes as well as serve as an important model to understand an ageing valve phenotype in humans. exome sequencing of bipolar disorder patients with rapid cycling implicates novel candidate genes in disease development bipolar disorder (bd) is a severe neuropsychiatric disorder characterized by recurrent episodes of mania and depression. bd has a lifetime prevalence of about % and a high heritability of about %. although recent genome-wide association studies identified the first susceptibility genes contributing to disease development, the cumulative impact of common alleles with small effect may only explain around % of the phenotypic variance (lee et al. ) . in consequence, rare variants of high penetrance have been suggested to additionally contribute to bd susceptibility. in the present study we focused on bd patients with rapid cycling (rc). rc is a course specifier of bd defined as having at least four recurrent episodes of acute illness within one year. since rc showed strong evidence for familiarity, we hypothesized that bd patients with rc might represent a more defined etiological subgroup and that rare variants of high penetrance might contribute to the development of rc in bd patients. we selected unrelated bd patients with rc of german origin and performed exome sequencing using the illumina hiseq platform. for data analysis, the varbank pipeline of the cologne center for genomics was used. we filtered for rare (minor allele frequency < . %), heterozygous and non-synonymous variants that were predicted to be possibly damaging or disease causing by at least of applied prediction tools. after these filtering steps, we identified a total of different genes which harbored rare functional variants in at least three independent patients. gene set analysis for these genes using consensuspathdb revealed decker , g. nuernberg , , d. hassel , g. a. rappold , mutations in the homeobox gene shox cause shox deficiency, the most frequent monogenic cause of short stature. the clinical severity of shox deficiency varies widely, ranging from short stature without dysmorphic signs to mesomelic skeletal dysplasia (léri-weill dyschondrosteosis, lwd). in rare cases, individuals with shox deficiency are asymptomatic. to elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with five affected individuals with lwd using whole genome linkage analysis and whole exome sequencing. the variant p.phe cys of the retinoic acid catabolizing enzyme cyp c co-segregated with the shox variant p.val ala in the five affected individuals, while the shox mutant alone was present in three asymptomatic individuals. two further independent lwd cases with shox deficiency and damaging cyp c variants were identified. the identified damaging variants in cyp c affected its catabolic activity, leading to an increased level of retinoic acid. we also provide evidence that high levels of retinoic acid significantly decrease shox expression in human primary chondrocytes and zebrafish embryos. individual morpholino knock-down of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. together our findings demonstrate that shox and cyp c act in a common molecular pathway controlling limb growth and describe cyp c as the first genetic modifier for shox deficiency. heart valve dysfunction in men and mice is caused by loss of function mutations in adamts , a novel marker for valvular interstitial cells on a global perspective defects of the cardiac valves are one of the most common heart abnormalities in humans, with a substantial number of them requiring surgical intervention at least once in their life. several mechanisms have been proposed ranging from acquired to developmental causes, but thus far the majority can not be explained on the molecular level. here we report on the identification of a unique human family affected by multiple dysfunctional cardiac valves early in life. genetic screening revealed a homozygous deletion of the first eight exons in ad-amts , a novel candidate gene for valvular heart defects. to investigate its role in heart valve development, we designed a transgenic mouse model that reconstitutes the loss of function (lof) in adamts found in the statistically analyzing de novo mutations identified in > , id patients highlighted ppm d as a candidate id gene. ppm d is a type c phosphatase that functions as a negative regulator of cell stress response pathways by mediating a feedback loop of p -p signaling, thereby contributing to growth inhibition and suppression of stress induced apoptosis. we identified patients with mild-moderate id and a de novo truncating ppm d mutation. deep-phenotyping of the patients revealed in addition to id overlap for behavioural problems (adhd and anxiety disorder), hypotonia, broad based gait, facial dysmorphisms and periods of fever and vomiting. ppm d is shown to be expressed during fetal (brain) development and in the adult brain. all mutations were located in the last, or penultimate exon, suggestive of escaping nonsense-mediated mrna decay. both ppm d expression analysis and cdna sequencing in patient ebv-lcls support the presence of a stable, but truncated transcript, consistent with this hypothesis. exposure of patient's cells to ionizing radiation resulted in normal p activation suggesting that p signaling is not affected by the truncated protein. however, a cell growth disadvantage was observed. thus, we show that de novo truncating ppm d mutations in the last and penultimate exon cause syndromic id which provides novel insights in the role of cell cycle checkpoint genes in neurodevelopmental disorders. de novo truncating variants in asxl are associated with a unique and recognizable clinical phenotype harvard stem cell institute, department of stem cell and regenerative enriched pathways (q < . ) including actin cytoskeleton and calcium ion binding. subsequently we applied the residual variation intolerance score (rvis) and identified genes which were ranked among the % most intolerant genes in the genome. these genes included the previously reported genome-wide significant bd risk genes syne and mll . in addition, we identified novel, promising candidate genes which have not previously been implicated in bd development such as ryanodine receptor (ryr , affected in six patients) and huntingtin (htt, patients). both genes are ranked among the . % most intolerant genes of the genome. ryr encodes a brain expressed intracellular cation channel that mediates the rapid release of ca + from the endoplasmic reticulum, thus making it a highly plausible candidate gene for contributing to rc. abnormal expansion of a trinucleotide repeat in the htt gene causes huntington disease which is a neurodegenerative disease characterized by motor, cognitive and psychiatric symptoms. the seven most promising genes are currently being followed up by resequencing in larger cohorts of independent bd cases (including patients with rc) and controls of european ancestry using the single molecule molecular inversion probes (smmips) technology. de novo truncating mutations in the last and penultimate exon of ppm d cause a novel intellectual disability syndrome abstracts with gα. signaling properties of g protein complexes carrying mutant gβ subunits were further analyzed by their ability to couple to dopamine d r receptors by real-time bioluminescence resonance energy transfer (bret) assays. these studies revealed altered functionality of the missense mutations r g, g v, a t, p s, p l, a t, and d g but not for l f, h r, and k q. in conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in gnb as a cause of gdd and provide functional evidence for a loss-of-function mechanism underlying the disease. comprehensive phenotyping and trio-exome analysis of children with neurodevelopmental disease whole exome sequencing (wes) has been proven as a powerful analytical tool to dissect the genetic basis of human hereditary disorders. here, we report on a prospective deep phenotyping and trio-wes study of children affected by previously undiagnosed and diverse complex neuropediatric disorders. all children underwent a standardised and comprehensive clinical work-up in a single centre that included detailed clinical evaluations by pediatricians and clinical geneticists, extensive laboratory and metabolic analyses, analyses of cerebrospinal fluid, mri of the brain and eeg, followed by trio-wes analysis. this systematic approach allowed to identify a pathogenic mutation in a known disease gene in altogether children ( %) and discovered a convincing candidate disease gene in additional children ( %). taken together, this translates into a successful genetic diagnosis of up to % in this cohort. in children with mutations in a known disease gene ( / = . %) the molecular diagnosis substantially influenced the clinical management and drug treatment. we further document an expansion of the phenotype in known disease entities in individuals. the extraordinary high gene discovery rate in our cohort emphasizes the potential of trio-wes even in a clinically inhomogeneous group of individuals with likely genetic disease. however, this requires a multidisciplinary approach including deep and sometimes reverse phenotyping, research-based interpretation of trio-wes identified genetic alterations, extensive review of the literature, use of several mutation prediction and protein-modelling tools, as well as openness and exchange of data with national and international researchers and clinicians working on similar diseases. exome sequencing of pooled dna samples for large-scale screening in individuals with sporadic intellectual disability b. popp, a. ekici, s. uebe, c. thiel, j. hoyer, a. wiesener, a. reis, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germany high throughput sequencing has enabled identification of many novel disease genes and empowered diagnostic testing for heterogeneous disorders, especially for intellectual disability (id) where more than genes have been implicated. due to this extreme heterogeneity gene panels are ineffective, and expensive exome or genome sequencing is necessary. furthermore, many affected individuals have to be sequenced to confirm candidate genes and to refine the phenotypic spectrum. we now explored if pooling strategies could satisfy the need for a genome-wide, simple, cheap and fast screening technology. the asxl genes (asxl , asxl and asxl ) participate in body patterning during embryogenesis and encode for proteins that are involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. germline de novo truncating variants in asxl and asxl have been respectively implicated in causing bohring-opitz and bainbridge-ropers syndromes, resulting in overlapping features of severe intellectual disability and dysmorphic features. to our knowledge, asxl has not yet been associated with a human mendelian disorder. in this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. all six had de novo truncating variants in asxl . a careful review en abled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia and developmental disabilities. although overlapping features with bohring-opitz syndrome and bainbridge-ropers syndromes exist, features that distinguish the asxl -associated condition from asxl -and asxl -related disorders are macrocephaly, absence of growth retardation and more variability in the degree of intellectual disabilities. we were also able to demonstrate with mrna studies that these variants are likely to exert a dominant negative effect, since both alleles are expressed in blood, with the mutated asxl transcripts escaping nonsense mediated decay. in conclusion, de novo truncating variants in asxl underlie a new neurodevelopmental syndrome, with a clinically recognizable phenotype. this work expands the germline disorders that are linked to the asxl genes. functional characterization of novel gnb mutations as a rare cause of global developmental delay over the past years, prioritization strategies that combined the molecular predictors of sequence variants from exomes and genomes of patients with rare mendelian disorders with computer-readable phenotype information became a highly effective method for detecting disease-causing mutations. the drawback of phenotype-based prioritization, however, is that they require a deep and comprehensive feature description to gain good performance. but in routine diagnostics, the naming of phenotypic features varies among clinicians, and sometimes a comprehensive phenotypic overview is not possible because of missing terminology. these gaps can be reduced by including a new layer of phenotypic information using facial recognition technology to detect dysmorphic features from two-dimensional photographs. automated image analysis is in principle able to identify any deviation from the norm and to quantify it objectively. we therefore developed an approach that combines facial dysmorphology novel analysis (fdna) technology with standard phenotypic and genomic features to identify pathogenic mutations in exome data. we have started collecting data from a diverse spectrum of patients with molecularly confirmed diagnoses in a multi-center study, and we present the current results. at the time of abstract submission more than patients from over contributing institutions were evaluated and used for simulation of a training set of exomes. automated facial recognition yields the correct diagnosis amongst the first ten suggested syndromes in more than two thirds of the cases and shows a high correlation with syndrome predictions that were based on expert annotated features. hereby, we could also confirm the diagnosis in cases with only subtle facial features. consequently, we used classical machine learning approaches to integrate scores based on the image analysis, phenotypic description and exome se-after initial evaluation of available computational methods by virtual pooling of exome data or simulated reads using different pooling fractions, we decided to exome sequence individuals with sporadic id in pools of samples each. this was suggested to be the optimal combination with a % detection rate. dna was mixed in equimolar concentrations and submitted for exome sequencing. read data was aligned to the human reference, and variants were called using a ploidy of . resulting variant calls in known id genes (sysid database) were then filtered for loss-of-function (lof) variants and for missense variants that were either previously reported as pathogenic or computationally predicted to be deleterious. furthermore, we screened id candidate genes and haploinsufficiency intolerant genes for lof variants. subsequently, sanger sequencing was used to determine the individual carrying each variant in the respective pool and to test segregation in the parents. this approach resulted in the identification of pathogenic variants (assumed or confirmed de novo) in known id genes (ahdc , ankrd , atp v b , cask, chd , kcnq , kmt a, kras, med l, rit , setd , tcf , wac, zbtb ), two pathogenic variants inherited from a symptomatic or healthy parent, respectively, (zmynd , ifih ), and a homozygous variant in the recessive trappc gene. this included loss-of-function and missense variants. additionally, we identified de novo variants in candidate genes. in our id cohort this resulted in a high mutation detection rate of %. thus, detection of rare variants from exome sequenced dna pools (pool-seq) is feasible and has a high detection rate similar other screening approaches. compared to affected-only exome sequencing this method can reduce costs by more than % with only marginal increase in sanger-sequencing costs and significantly speed up wet lab work with an acceptable increase in computational complexity. in contrast to targeted sequencing methods like molecular inversion probes or hybridization-based panels, our method has the advantage of allowing flexible re-analysis of the same data for new genes. in conclusion, we established exome pool-seq as a method for large-scale, cost-efficient and flexible sequencing in highly heterogeneous but well characterized disorders like id. three years of experience with targeted next-generation sequencing of developmental delay next-generation sequencing (ngs) has opened up new possibilities especially in the search for disease-causing mutations in disorders with common clinical features but a heterogeneous genetic background. the identification of the underlying genetic defect provides a clear diagnosis for patients more and more influencing their management and occasionally even their therapy, and it is the prerequisite for prenatal or preimplantation decisions in the affected family. ngs panels are used widely in clinical settings to identify genetic causes of various monogenic disease groups, such as intellectual disability (hu et al. ) , neurodevelopmental and neuromuscular disorders, among others. however, many new challenges have been introduced both at the technical level and at the bioinformatic level, with consequences including new requirements for interpretation of results, and for genetic counseling. we report on our experience with a targeted ngs panel comprising over brain related genes (mpimg- -test) in the routine clinical diagnostics of patients with syndromic and non-sydromic forms of developmental delay as well as patients with neuromuscular disorders. patients (age - ; mean ) with syndromic (s) or non-syndromic (ns) developmental delay or with neuromuscular symptoms (nm), seen at the genetic counselling unit of our institute, were analyzed with targeted exon enrichment and ngs. chromosomal re-arrangements and copy number variations were excluded in all patients previously by conven-it was recently shown that that clonal hematopoiesis can be driven by somatic point mutations. these acquired mutations occur with normal aging in up to % of older (> y) individuals ( ) ( ) ( ) and few reports in younger individuals. here we present a targeted re-sequencing assay that combines high throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smmip) ( ) . we have now analyzed dna from healthy blood donors from different age groups ( - ; - ; - ; - and - y) , with no previous diagnosis of cancer, for somatic mutation in loci. those loci included known drivers of clonal hematopoiesis ( ) ( ) ( ) and novel or candidate loci. the improved assay allows low-frequency variant detection with variant levels down to < . %. this improved sensitivity allowed the identification of somatic mutations in a limited set of loci in > % of old individuals, but also report those mutations in individuals of the youngest age group. most prevalent mutations include known hotspot mutations in dnmt a and asxl . here we show that somatic drivers of clonal hematopoiesis are more prevalent and occur in younger individuals than previously reported. these somatic events are age-related. however, the high prevalence and their occurrence in relatively young individuals implicates their origin as a common biological process involved in normal aging. a. m. nissen, j. graf, c. rapp, m. locher, a. laner, a. benet-pagès, e. holinski-feder medizinisch genetisches zentrum -mgz, munich, germany gene dosage abnormalities account for a significant proportion of pathogenic mutations in rare genetic disease related genes. in times of next generation sequencing (ngs), a single analysis approach to detect snvs and cnvs from the same data source would be of great benefit for routine diagnostics. however, cnv detection from exon-capture ngs data has no standard methods or quality measures so far. current bioinformatics tools depend solely on read depth which is systematically biased. we developed a novel approach based on: . utilization of five independent detection tools to increase sensitivity, . different reference sets for different kits and normalization against samples from the same sequencing run to improve robustness against workflow conditions, . definition of special quality thresholds for single exon events to minimize false negatives, . identification of reliable regions by assessment of capture efficiency using a reference set of cnv negative patients to minimize false positives. a cnv quence of the patients and could predict the pathogenic mutation among the top positions in a prioritized exome in more than % of the monogenic cases in our cohort. hence, our results show that computer-assisted facial recognition is not only a promising technology that could be applied in the routine diagnostic workflow, but also a technology that allows diagnosis in cases with non-typical clinical presentation and boosts the diagnostic yield in exome studies. the added value of rapid exome sequencing in critical clinical situations for critical clinical situations, turnaround times (tats) of exome sequencing need to be fast in order to have an impact on clinical decision making. we therefore set out to develop a fast exome sequencing approach (max. days). urgent exomes are preferably sequenced as trios to enable de novo analysis and assist data interpretation. dna library preparation is performed using the sureselect qxt protocol (v , agilent), and sequencing is done on a nextseq (illumina) with a high coverage ( - x). automated file handling allows rapid bwa mapping, gatk variant calling and annotation. a total of one-hundred samples have been sequenced until now using this rapid procedure: trio's, x mother and child, x parents plus children, and single cases. six trios with known aberrations were used for experimental setup. of the remaining families, in families (possible) pathogenic snvs were identified, of which some still need further follow up, whereas families remained negative after inspection of snvs and small indels. for cnv analysis, a trio based reference-free cnv approach is still under development. preliminary data show that all control cnvs ( kb- mb) are detected correctly, and retrospective cnv analyses of the other samples identified three possibly de novo cnvs that need further follow up. shorter tats days were already beneficial for some patients, i. e. an adult male suffering from myelofibrosis and autoinflammatory symptoms. a sting-like phenotype (= stimulator of ifn genes) was suspected, with a possible involvement of the jak/stat pathway. urgent exome sequencing was performed and results were available within days. interestingly, both a somatic variant in mpl (= trombopoetine receptor > myelofibrose) and a heterozygous variant in acp (trap, known immune dysregulation disorder) were identified, both fitting to the patients phenotype. based on these results the medication of the patient was changed, resulting in a substantial improvement of the patients constitution. in conclusion, we have implemented a rapid exome sequencing workflow for urgent cases. the rapid identification of pathogenic variants already had implications on patient treatment, underlying the added value of a fast genetic diagnosis. ement insertion, however, was spliced into the mrna of nabp , leading to a frameshift mutation and a premature stop codon, potentially altering or abolishing gene function. in summary, we have shown that transposon insertions, both common variants as well as rare or de novo variants, can be detected in wes data. such insertions in coding or regulatory regions of disease-relevant genes might therefore explain some of the cases in which no pathogenic coding mutation can be identified by wes. the influence of human genetic variation on epstein-barr virus sequence diversity: a genome-to-genome approach c. hammer , , a. loetscher , , em. zdobnov , genome-wide association studies (gwas) have identified common genetic polymorphisms that associate with clinical manifestation and immune response parameters of various infections. we here present an alternative approach, using variation in the virus sequence as phenotype, which is specific by nature and unique to genomic research in infectious diseases, for genome-to-genome (g g) association studies. building on the unprecedented possibility to combine large-scale human and viral genomic data, we explored interactions between human genetic variation and viral sequence diversity in individuals infected with epstein-barr virus (ebv). the major goal is the identification of key genetic players in the evolutionary 'arms race' between pathogen and host. ebv is the pathogenic agent of infectious mononucleosis and is associated with a broad spectrum of lymphoid and epithelial malignancies, including lymphomas and nasopharyngeal carcinomas. there is also evidence for a role of ebv in the pathoetiology of multiple sclerosis. its genome is approximately kbp long and encodes around proteins, not all of which have been definitely identified or characterized. it is known that high loads of ebv are present in patients with advanced human immunodeficiency virus (hiv)-induced immunodeficiency. we therefore selected immunosuppressed patients included in the swiss hiv cohort study (shcs) with low cd + t cell counts, and quantified ebv copy number in peripheral blood mononuclear cells (pbmcs). cell samples contained more than , viral copies in total and were subjected to target isolation and subsequent enrichment using the sureselect method by agilent biotechnologies, followed by illumina whole-genome sequencing. after data processing and quality control, variable amino acids were called as binary variables, resulting in > variable positions per individual in average. the same patients also underwent genome-wide genotyping to obtain host genetic variation, followed by imputation based on the haplotype reference consortium reference panel. the association analyses are currently ongoing, and we will present the results at the conference. we use logistic regression to test for association between host single nucleotide polymorphisms (snps) and binary ebv amino acid variants. bonferroni correction is applied for multiple testing correction on the sides of both host and pathogen. stratification is taken into consideration by including principal components (pcs) for the host, and phylogenetic pcs for the virus. this project will offer a global description of the adaptive forces acting on ebv during natural infection. we have shown before for hiv that a virus genome associates much more strongly with human genetic variants than clinical endpoints. the analysis of all signals resulting from the interaction between human and viral genomes has the potential to identify novel host defense mechanisms, which could serve as future diagnostic and therapeutic targets. is called in a reliable region if at least two out of five tools are concordant for the respective cnv. the pipeline shows a sensitivity of % and a precision of %. within routine gene panel diagnostics we analyzed a total of patients indicated to have rare mendelian diseases for snv and cnvs. in patients a cnv was detected in genes associated with the respective individual phenotype. interestingly, in several cases the cnv completed the patients report as it was detected in genes with a recessive mode of inheritance where previously only a heterozygous pathogenic snv was found. overall, with the additional analysis of cnvs we increased the diagnostic yield from % (class , single nucleotide events) to %. however, there are still issues in the detection of cnvs from ngs data for routine diagnostics. cnv pipelines are very prone to errors caused by enrichment inconsistencies compared to snv detection tools. the assessment of sensitivity and specificity is difficult due to the lack of datasets to validate cnv detection pipelines. originally, the analysis of cnvs was performed mainly in patients with mental retardation disorders, resulting in a paucity of cnv data linked to other mendelian diseases. moreover, the identification of the actual size and thus the assessment of pathogenicity of a cnv is difficult, because targeted ngs gene panels do not cover all genes. in conclusion, ngs data is a suitable data source for the simultaneous detection of snvs and cnvs for clinical diagnosis; however, with the current tools it is only applicable in accurately validated regions. identification of transposon insertions in whole-exome sequencing data s. lukassen, n. Übelmesser, ab. ekici, u. hüffmeier, ct. thiel, c. zweier, a. winterpacht humangenetisches institut, erlangen, germany % of the human genome consists of transposable element derived sequences, the most abundant of which are l and alu elements, followed by endogenous retroviruses. several hundred of these elements remain active, leading to insertion frequencies of up to one in live births for alu elements and posing a threat to genome integrity. while most studies on transposons employ whole-genome sequencing (wgs) or target-enrichment based sequencing approaches, the most commonly used form of diagnostic high-throughput sequencing is currently whole-exome sequencing (wes). we were therefore interested in investigating transposon insertions in wes data as a possible source of disease causing mutations. we developed a software to call non-reference transposon insertions from single-end wes datasets by split-read mapping and analyzed exomes this way. on average, non-reference insertions were identified in each exome, with an average of . sites per patient identified in < = . % of other patients. of these rare variants, % were deemed plausible by visual inspection. automated confidence calls of the software were concordant with visual inspection in % of cases. in % of cases a plausible insertion was awarded a lower score by the algorithm and in another % not called at all. in % of cases the automated call appeared to be falsely positive, in another % at the wrong position within the same bp window. laboratory validation of convincing insertions revealed a % true positive rate, leading to an estimated specificity of %. when performing calls for reference l insertions on exomes, % ( % - %) of known elements whose flanking regions were covered by at least two reads were correctly identified, leading to a sensitivity of %. we thus estimate the average number of non-reference transposon insertions in our wes dataset to be ( - ). % and . % of sites identified were associated with alu and l elements, respectively, with the majority of calls stemming from evolutionary young transposons still assumed to be active. . % of sites were located within the cds, . % in the utrs of genes, . % spanned an intron/ exon border, . % were intronic and . % of insertions were found in intergenic regions. we then chose insertions within intronic ( ) or utr ( ) regions for further analysis. seven were not detected in the mrna. one intronic alu el-abstracts ws - novel insights into male-pattern baldness pathobiology via integration of differential hair follicle mirna and mrna expression profiles with gwas data male pattern baldness (mbp) is a highly heritable condition and the most common form of hair loss in men. the phenotype is characterized by a distinct pattern of androgen-dependent progressive hair loss from the scalp that is restricted to hair follicles (hf) in the frontal and vertex scalp area. the molecular mechanisms that underlie this characteristic pattern and the differences in androgen-sensitivity between hf subpopulations in the frontal/vertex and the occipital scalp remain however elusive. to gain novel insights into the underlying biology and contributing genes and pathways, we systematically investigated for a differential expression (de) of mirna-and mrna-genes in hf samples from the frontal and occipital scalp area of healthy male donors. array-based genome-wide mirna and mrna profiling revealed expression of mirnas and , mrnas in human hf, of which mirnas ( %) and , mr-nas ( %) showed a de between hf subpopulations. the strongest de mirnas included mir- , mir- and mir- . among the strongest de mrnas were the wnt-signaling inhibitor dkk , the protein kinase pak and the retinoid acid receptor rora. a subsequent pathway-based analysis in mirpathdb revealed that de mirnas targeted numerous interesting pathways. among them the wnt-and mtor signaling pathway which have been implicated in the control of hair follicle cycling, a mechanism that is disturbed in mpb affected hf and other plausible candidate pathways such as estrogen, thyroid hormone signaling or epidermal growth factor binding which have not yet been implicated mpb pathobiology. to yield further evidence for an involvement of de mirnas and mr-nas in the developement of mpb, we subsequently integrated our expression data with association data from a large gwas meta-analysis on mpb (n = , ). of the de mirnas and mrnas, only mirna (mir- b) and mrnas were located within mb of one of genome-wide significant mpb risk loci. notably, the analysis revealed a co-localization of de mirna, de mrna, and nominally significant association signals (p < - ) at other genomic loci, pointing towards a role of these genomic regions in mpb pathogenesis. among them a locus on chromosome q . that comprises the genes encoding the ephrin-type-b receptor (ephb ) and the prostaglandin transporter slco a . interestingly, ephrins have been shown to be regulated by androgens and to play a role in hf formation, proliferation and hair cycling. and expression of prostaglandin d , which is transported by slco a , has been found to be upregulated in balding scalp where it inhibits hair growth. in summary, our systematic analysis of differential mirna and mrna expression and the subsequent integration with genetic association data identified novel potential risk loci for mpb and numerous candidate genes and pathways that are likely to play a role in mpb pathogenesis and emphasizes the importance of data integration of large-scale omic-analyses. palaeontological genomic analyses have shown that interbreeding between anatomically modern humans and neandertals occurred in europe and asia . - years ago. approximately . - % of the modern european and asian genome consists of introgressed dna from neandertals. some of these introgressed regions have been suggested to contribute to several traits and phenotypes including major depression and other mood disorders. in order to further assess the role of neandertal ancestry in cognition and the contribution of genetic risk for psychiatric disorders, we performed genome-wide analyses of neandertal alleles in publicly available psychiatric genomics consortium (pgc) gwas summary statistics with samples sizes ranging from about to individuals for the following phenotypes: educational attainment, attention deficit hyperactivity disorder (adhd), anorexia nervosa, anxiety disorders, autism spectrum disorder, bipolar disorder, major depressive disorder and schizophrenia. we estimated the proportion of heritability explained by snps in neandertal introgressed regions using stratified ld score regression (ldsc) and two sets of previously inferred neandertal introgressed regions. in a secondary analysis, we investigated whether specific functional annotations such as 'utr, promoter regions or histone marks within neandertal regions were significantly associated with selected phenotypes. we identified a modest enrichment of heritability in neandertal introgressed regions in anorexia nervosa, autism spectrum disorder, bipolar disorder and major depressive disorder, although none of the results were statistically significant. several functional annotations, such as h k me histone marks within neandertal introgressed regions, appeared significantly enriched for snps contributing to the heritability of anorexia nervosa and autism spectrum disorder. in bipolar disorder, dnasei digital genomic footprinting regions, h k ac histone marks and super enhancer regions within neandertal regions appeared particularly enriched for heritability. on the other hand, both sets of neandertal regions were slightly depleted of snps contributing to the heritability of schizophrenia. for example, one set of neandertal regions that contained % of all analysed snps only contributed to % of the variance of risk (standard error: . ; p-value: . × - ). in comparison to the rest of the genome, neandertal introgressed regions also contributed less to the heritability of educational attainment, adhd and anxiety disorders, although these findings were not statistically significant. to our knowledge this is the first study to systematically investigate the extent to which snps attributable to neandertal introgressed regions contribute to the heritability of several psychiatric/cognitive phenotypes. we are currently increasing our power to detect snp heritability in neandertal regions by applying the ldsc method to larger pgc datasets. harbor genes involved in the complement system, high density lipoprotein metabolism or extracellular matrix homeostasis. these pathways are known for their pleiotropic role in other conditions, such as cardiovascular disease, auto-immune diseases and cancer. here we aimed to investigate the extend of overlap between the genetic risk of various complex diseases and traits and the genetic risk for amd. methods: first, we catalogued , previously published, genome-wide significant variations associated with complex diseases or traits. next, we computed a genetic score by calculating the (weighted) sum of risk increasing alleles for each disease or trait. consequently, a higher genetic score indicates that an individual has more risk/trait increasing alleles of a given disease or trait. for each score, we computed the association with late stage amd using a dataset provided by the international amd genomics consortium (iamdgc) including , late stage amd cases and , controls. we also assessed the association of each variation individually with late stage amd risk in order to identify novel disease loci with strong evidence for pleiotropy. results: nineteen genetic scores of complex diseases and traits were significantly associated with amd risk (fdr < . ). most notably, all genetic scores related to autoimmunity were elevated in amd patients (p < . × - ), while scores related to cardiovascular disease were reduced in amd patients compared to controls (p < . × - ). we also found that the genetic scores of melanoma and related malignancies were higher in amd patients (p < . × - ). in addition, out of , variants, which were used to compute the genetic scores, were significantly associated with amd (fdr < . ), implicating novel, pleiotropic loci in amd risk. conclusion: our findings demonstrate a substantial overlap between the genetic risk of complex diseases/traits and the genetic risk for amd and provide evidence for novel, pleiotropic loci associated with amd. while our findings highlight common disease pathways that may facilitate to develop multi-use drug targets, they also challenge the notion that gene/genome manipulation could be applied in general terms to eradicate risk for a defined complex disease. worldwide genetic association study of exfoliation syndrome and glaucoma identifies common genetic variants at five new susceptibility loci exfoliation syndrome (pex), a complex systemic disorder of the extracellular matrix, is the commonest cause of secondary glaucoma in aging population and thus a major cause of blindness globally, affecting - million subjects worldwide. inside a large, international collaboration project a genome-wide association study (gwas) was carried out on , pex cases and , controls, recruited from countries across six inhabited continents, with replication in a further independent , cases and , controls from countries. significant association was observed at seven loci, of which two confirmed the already known associated loci at the genetic markers mapping to loxl -and cana a-gene, five are new (p < × - ). the five new loci map to chromosomes q (rs near flt -pomp-slc a , p = . × - ), q . (rs near tmem -arhgef , p = . × - ), p (rs at agpat , p = . × - ), p (rs at rbms , p = . × - ) and q (rs near sema a, p = . × - ). to determine the pathophysiological role of the three most significantly associated loci ( q , q . , p ), we investigated the expression and localization of the six related genes (flt , pomp, slc a , tmem , arhgef and ag- male-pattern baldness (mpb) is characterized by a progressive hair loss from the frontal and vertex scalp that affects ~ % of men at the age of years. epidemiological studies have shown positive associations between mpb and coronary heart disease (chd) and related phenotypes such as blood pressure (bp), diabetes (dm) or elevated blood lipid levels. the results however vary with regard to the associated pattern of hair loss (frontal or vertex) and the assessed endpoint measures for chd. and so far no study has investigated for a shared genetic determinant between the traits. using data from the heinz nixdorf recall study (n = , males) and a large meta-analysis on mpb (n = , ), we aimed at a systematic investigation of the association between mpb and chd on (i) an epidemiological and (ii) a genetic level. , men with vertex balding showed a higher bmi (β = . kg/m ), elevated fasting triglyceride (β = . mg/dl) and lower hdl-c levels (β = - . mg/dl). to assess the genetic overlap between mpb and chd, we created a risk score (rs) from mpb lead snps (p < × - ) and tested for association with chd and related traits phenotypes. no significant associations were observed. however, an age-stratified analysis revealed a % per allele risk increase for chd (hr = . , %ci: . ; . ) and a decrease in fasting triglyceride levels (β = - . ). we next used ld score regression analysis in to test for genome-wide genetic correlation between mpb and chd. the analysis revealed no significant correlations with cardiometabolic (n = ), lipid (n = ) or metabolic traits (n = ). finally, to investigate for a genetic overlap at single loci, we compared the mpb risk loci with reported gwas signals for chd. the analysis identified seven overlapping associations between mpb and bp (n = ); qt-interval length; atrial fibrillation; sudden cardiac arrest; and dm. for the majority of loci, the direction of effect differed between mpb and chd, opposing previous epidemiological findings. positive associations were identified between mpb and diastolic bp (fgf , q . ) and sudden cardiac arrest (atf , q . ). interestingly, fgf is known to stimulate cell growth and proliferation in multiple cell types, including cardiac myocytes and hair follicle (hf) cells, and atf is a hf expressed regulator of cell growth and differentiation that has been shown to prevent foam cell formation, which suggests that fgf and atf -signaling contribute to both traits. thus, our data support an association between mpb and chd related phenotypes and suggest that mpb deserves further evaluation as an additional risk factor for chd. pleiotropic effect of genetic variants associated with complex diseases and traits in age-related macular degeneration purpose: age-related macular degeneration (amd) is the leading cause of vision loss in western societies and is caused by both environmental and genetic risk factors. with regard to the latter, several associated risk loci abstracts otyping, results of which will be presented at the conference. of note, for nonsyndromic cleft lip with/without cleft palate (nscl/p), the most frequent form of orofacial clefting, risk loci have been detected by gwas so far, with some of them reaching (nearly) genome-wide or significant p-values in samples much smaller than cases. in the imputed nscpo dataset none of the presently known nscl/p risk loci showed a p-value < - . our data so far confirm previous molecular and epidemiological findings, that nscpo is genetically distinct from nscl/ p. furthermore, the results indicate that common variants alone might not contribute to the same extent to nscpo as compared to nscl/ p. the correlation between defects at specific imprinted loci and distinct imprinting disorder (id) was accepted for a long time. however, it is now put into question because of a growing number of patients with multilocus imprinting disturbances (mlid), i. e. the aberrant methylation at more than one imprinted locus. in particular, mlid is present in individuals with silver-russell syndrome (srs) and beckwith-wiedemann syndrome (bws), and it has meanwhile turned out that patients with opposite phenotypes can share common epimutation patterns. on the other hand, mlid always occurs as mosaicism and varies in different tissues of the same individual. interestingly, the majority of mlid carriers show only one specific id phenotype, though loci of other ids are affected in addition to the one specific for the phenotype. we become aware of a growing number of patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis for referral. amongst others, we detected the srs specific icr hypomethylation in p in two of our patients referred as bws. in the first case, the icr hypomethylation was detected only in lymphocytes but was not present in buccal swab dna. the patient only had a slight asymmetry, but showed normal growth and did not exhibit any other feature compatible with bws, nor with srs. the reason for the lack of clinical features is unclear, but is comparable to the observation in monozygotic, but clinically discordant srs and bws twins. here the unaffected twin often carries the epimutation only in lymphocytes whereas the affected one shows the alteration in additional tissues. a reason might be sharing of hematopoietic stem. it can be postulated that the patient presented here is born after an (undetected) twin pregnancy with early loss of the affected twin. in the second case, the initial diagnosis of bws was made due to asymmetry, though overgrowth or other features were not present. further clinical ascertainment did not confirm this diagnosis, but growth of the patient was in the lower percentiles, in concordance with the icr hypomethylation. these cases as well as further cases in our cohort confirm that there is an urgent need to provide detailed clinical data upon requesting molecular diagnostics for imprinting disorders. in fact, the growing number of patients with unexpected results complicates the interpretation and illustrates the broad phenotypic range, but also provides further insights in the etiology of ids and setting of imprinting marks pat ) by qrt-pcr, immunohistochemical-and western-blot analysis in genotyped ocular tissues of pex and control patients. all six genes displayed moderate mrna expression in all ocular tissues analysed, with highest levels in iris, ciliary body, and retina. however, only pomp showed a trend towards reduced expression in the presence of the rs risk allele, in both pex and control patients. in general, both mrna and protein expression of pomp and tmem were significantly reduced up to % (p < . ) in anterior segment tissues in pex eyes compared to controls. no differences in mrna and protein expression were detected for the remaining genes analysed. immunofluorescence analysis showed that pomp, a proteasome maturation protein, is ubiquitously expressed in most ocular cell types and that tmem , a transmembrane protein of unknown function, is primarily localized to endothelial cells of blood vessels and aqueous outflow structures. additionally, protein staining intensities for pomp and tmem were markedly reduced in anterior segment tissues of pex eyes compared to controls and co-localized to abnormal accumulation of pex material on ocular surfaces and in blood vessel walls. thus, at least two of the newly identified loci provide new biological insights into the pathology of pex syndrome/glaucoma and highlight a role for impaired proteasome function as well as vascular and trabecular endothelial dysfunction in the disease pathogenesis. nonsyndromic cleft palate only -evidence for a limited contribution of common variants in contrast to nonsyndromic cleft lip ± palate cleft palate only (cpo) is a common congenital malformation which might occur as part of a syndrome or in an isolated form, i. e., nonsyndromic cpo (nscpo). nscpo has a prevalence of : and is considered multifactorial with genetic as well as environmental factors contributing to the disorder. in a recent study we identified the first genome-wide significant locus for nscpo which has been independently confirmed in another study. in order to discover more nscpo risk loci we performed a genome-wide imputation study with gwas data from case-parent trios with european, asian and african ancestry which was retrieved from db-gap upon approved data access. notably, this gwas dataset had not yet been imputed, and we hypothesized that we can increase power to identify novel genetic associations by increasing the marker density and follow-up of suggestive findings by independent replication. genome-wide genotypes were imputed using impute based on genomes haplotypes, and snps were selected based on info-score > . and minor allele frequency > %. the imputation did not reveal any genome-wide significant snp, however, snps at loci showed p-values < - . loci with more than two variants below this threshold (n = ) were to be replicated using the massarray system (agena bioscience). three independent samples were used: two case/control replication cohorts from central europe ( cases, controls) and yemen ( cases, controls), and one european case-parent trio replication cohort (eurocran study; trios). in a first round we genotyped snps at eleven loci. one variant, rs at chr. q , showed p < . in the replication cohort and after combining replication and gwas data, resulted in a decrease of p-value from . × - to . × - . this indicates that this locus, which includes candidate genes such as igs , a known cell-adhesion molecule with yet unknown function in craniofacial development, might harbour a common risk variant with low effect size. we are currently performing a second round of gen-we report biallelic mutations in cad, encoding an enzyme of de novo pyrimidine biosynthesis, in four patients with developmental disability, epileptic encephalopathy, anaemia, and anisopoikilocytosis. two children died after a neurodegenerative disease course. treatment of two surviving children with oral uridine led to immediate cessation of seizures in both. a four-year-old girl, who was previously in minimal conscious state, started to communicate and walk with assistance after nine weeks of treatment. a three-year-old girl likewise showed developmental progress. blood smears normalised and anaemia resolved. our findings support the efficacy of uridine supplementation rendering cad deficiency a treatable neurometabolic disorder. delineation of the grin a phenotypic spectrum alterations of the n-methyl-d-aspartate (nmda) receptor subunit glu-n a, encoded by the gene grin a, have been associated with a spectrum of neurodevelopmental, speech and epilepsy disorders. we identified previously unreported patients with heterozygous pathogenic variants in grin a, including novel variants. after re-evaluation of all published grin a cases, previously reported patients met the acmg criteria for being pathogenic or likely pathogenic. thus, we are able to collectively review genotypes and phenotypes of individuals with grin a-related disorders. we show that the known phenotypic spectrum is expanded and ranges from near-normal development to severe and unspecific encephalopathy, comprising any disorder of speech development. furthermore, some patients do not display seizures. in contrast to previous reports, gri-n a missense variants cluster within the functionally most relevant domains. we are the first to describe genotype-phenotype correlations in grin a-related disorders, where carriers of pathogenic missense variants tend to have more severe neurodevelopmental phenotypes compared to carriers of truncating variants. the most severe end of the phenotypic spectrum was found to include novel features, such as infantile spasms and arthrogryposis and was associated with pathogenic variants in the pore-forming domain of grin a. the eponymic name galloway-mowat syndrome (gamos; omim ) has been coined for the association of early-onset nephrotic glomerulopathy, microcephaly with variable brain anomalies, and facultative diaphragmatic hernia. it is supposed to be inherited as an autosomal recessive trait and clinical as well as genetic heterogeneity has been suggested. in , wdr mutations were identified as a cause of gamos, but only a few cases have been reported to date. over the last years, we have collected dna samples and clinical data from unrelated families with one or more children affected by gamos or a gamos-like syndrome (glomerulopathy plus variable anomalies of brain morphology or function as inclusion criteria), including consanguineous families. in this cohort, we performed whole exome sequencing followed by targeted analysis by sanger and ngs multigene panel resequencing. in a total of families of this cohort ( %) the probable underlying genetic defect could be identified. in affected individuals from two consanguineous families, homozygous mutations of wdr could be found (vodopiutz et al., ) . thus, this gene accounted for only % of cases of our cohort. the affected child of another family had a novel homozygous mutation in arhgdia. this gene has previously been described in three families to cause early-onset steroid-resistant nephrotic syndrome (gupta et al., ; gee et al., ) , but there is some evidence that non-specific brain anomalies may also be part of the arhgdia-associated phenotype. fourteen and three index patients from unrelated families had mutations in one autosomal (osgep) and one x-linked gene (lage ), respectively, both encoding for components of the keops protein complex that has been implicated in transcription, telomere maintenance and chromosome segregation. no human phenotype has previously been assigned to mutations in this complex. notably, eight unrelated families with an identical mutation originated from the east asian population where the carrier frequency for this allele is . . in one consanguineous family with multiple affected children the disease segregated with a homozygous mutation in the sgpl gene encoding for sphingosine- -phosphate lyase. in four families, the kidney phenotype could be attributed to mutations in genes for non-syndromic nephrosis (nphs , plce , one novel gene), while the brain phenotype was apparently independent. in conclusion, the molecular genetic findings in this cohort confirmed that gamos is exceedingly heterogeneous, and still in almost half of the patients with a gamos-like phenotype the genetic cause remained unclear. on the basis of our findings we are now able to define new biologic mechanisms that are critically involved in both, brain development and integrity of the glomerular filtration barrier. genotype phenotype correlations are emerging. finally, we demonstrate that gamos can also be inherited as an x-linked trait. abstracts taminase gene (tgm ) is mutated in the majority of patients (around %), and its gene product, tgase , is therefore primarily targeted in our approach for protein substitution. patients with arci have an impaired skin barrier function, most of them are born with a collodion membrane and suffer subsequently from varying degrees of hyperkeratosis, erythema, transepidermal water loss and infections. the disease can be life threatening neonatally but lacks a causative therapy and is still only treated symptomatically. therefore, our aim is to develop a personalised, causative therapy where the defective protein is substituted topically via a nanocarrier. therapeutic, human tgase was synthesized in hek cells and assessed by western blot and flow cytometry analysis. enzyme activity was measured by in vitro assay. tgase was then coupled to a polyglycerol-based nanogel (dpg-ng) containing the thermoresponsive linker poly(n-isopropyl)acrylamide (pnipam), stabilising the enzyme as well as adding a thermal protein release trigger at °c, which is favorable for cutaneous applications. immunocytochemical stainings for tgase on monolayered basal keratinocytes that lack tgm expression confirmed the successful uptake of extrinsic tgase into the cells. further analysis over time showed that the enzyme was no longer detectable after h and consequently led us to define a treatment schedule for the following experiments. d full thickness skin models were used as in vitro system to determine barrier function and enzyme activity after treatment with varying concentrations of the dpg-ng/tgase complex. three different sets of skin models were used for these experiments: normal models mimicking the healthy skin with an intact barrier function, models where tgm was knocked down, and models made with arci patient cells with tgm mutations. franz cell tests on treated skin models lacking intrinsic tgase confirmed the impaired barrier activity in disease models, demonstrated an improved barrier function after repeated treatments with dpg-ng/ tgase and showed restored tgase activity using an in situ assay. furthermore, first toxicity tests using mtt revealed high biocompatibility of dpg-ng/tgase after treatment of d and d cell cultures. these findings are successful steps for an advanced topical drug delivery system and are a promising approach for causative therapeutic intervention in arci. after further optimization concerning protein dosage and thorough toxicity tests, we will adapt this system also for the use with other proteins involved in arci. pigmentation disorders (pds) comprise a large group of rare and heterogeneous disorders that are mainly characterized by various coloration abnormalities affecting single parts of the body or the complete integument. the large group of pds includes the autosomal dominant inherited hyperpigmentation disorder dowling-degos disease (ddd). ddd is genetically heterogeneous, and to date causal mutations in three genes, namely krt , pofut and poglut have been identified. after exclusion of mutations in these genes, we performed exome-and sanger-sequencing in six unrelated ddd-patients/families and identified six heterozygous truncating mutations in psenen encoding the presenilin enhancer protein . on closer examination of the histological sections, we came upon a novel feature that distinguished these individuals from previous ddd-cases by the presence of follicular hyperkeratosis. to assess the functional significance of psenen mutations in ddd pathogenesis, we performed mammalian cell culture based studies and knockdown experiments of psenen homolog psenen in zebrafish larvae (zfl). knockdown of psenen in zfl resulted in a phenotype with scattered pigmentation, which mimicked human ddd. in vivo-monitoring of pigment cells in the developing zfl suggested that disturbances in melanocyte migration and differentiation underlie ddd pathogenesis. interestingly, six of the psenen mutation carriers presented with co-morbid acne inversa (ai), an inflammatory hair follicle disorder. all individuals had a history of nicotine abuse and/or obesity, which are known trigger-factors for ai. although psenen mutations have been identified in a small subset (< %) of familial ai previously and the co-manifestation of ddd and ai has been reported for decades, our study is the first to demonstrate experimentally that mutations in psenen indeed can cause co-manifestation of ddd and ai, most likely triggered by predisposing factors for ai. thus, the present report describes a clinically and histopathologically novel ddd subphenotype in psenen mutation carriers, which is associated with an increased susceptibility to ai. protein substitution therapy for autosomal recessive congenital ichthyosis (arci) overall burkitt lymphoma showed a low genomic complexity with a low number of snvs and svs. however, the integration of cnas, snvs and svs allowed us to identify recurrently affected genes, which are involved predominately in the pi( ) kinase pathway, tonic bcr signaling, and cell cycle regulation, chromatin composition and germinal center development. burkitt lymphoma (bl) is the most common mature aggressive b-cell lymphoma in childhood. the genetic hallmark of bl is a chromosomal translocation involving the myc oncogene and one of the immunoglobulin loci leading to myc deregulation. three epidemiologic variants of bl are differentiated: endemic (ebl), which occurs predominantly in equatorial africa and is associated with ebv-infection, sporadic (sbl), which occurs in westernized countries and immunodeficiency-associated. in addition, burkitt leukemia (b-al) is differentiated from bl in cases with more than % of the bone marrow cells being lymphoma cells. another rare bl-variant is myc-positive precursor b-cell acute lymphoblastic leukemia coexpressing tdt and myc (tdt+bl). finally, we recently described a myc-negative variant which shows a typical alteration on chromosome (mnbll). the aim of the present study was to examine the epigenetic landscape of these bl variants. to this end, we analyzed the dna methylation of bl ( sbl, ebl, b-al, mnbll, tdt+bl) using the humanmethylation beadchip and contrasted the findings to diffuse-large b-cell lymphoma (dlbcl) and follicular lymphoma (fl). the majority of lymphoma were recruited in the framework of the icgc mmml-seq and mmml projects. the ebl were obtained from the nci ghana burkitt project. as controls, we used public available dna methylation data from b-cell burkitt lymphoma (bl), including its leukemic variant burkitt leukemia (b-al), is the most common type of pediatric b-cell lymphoma accounting for - % of new cases. its biological hallmark is the ig-myc translocation involving myc and mostly the immunoglobulin heavy (igh) locus or more rarely one of the immunoglobulin (ig) light chain loci. at the cytogenetic level the ig-myc translocation is the sole abnormality in around % of cases. overall, bl is characterized by a low genomic complexity. the aim of the present study was to analyze the genomic and transcriptomic landscape of pediatric/adolescent burkitt lymphoma by sequencing according to the guidelines of the international cancer genome consortium. a total of samples of bl/ b-al from pediatric/adolescent patients entered this sequencing study. all patients were treated in population-based prospective clinical trials. inclusion criteria were besides availability of suitable materials, consent to participate in the study and appropriate diagnosis: age at diagnosis (≤ years), the presence of ig-myc rearrangement detected by fish and/or whole genome sequence (wgs), absence of rearrangements of bcl or bcl genes. we performed wgs of tumor and matched control as well as transcriptome sequencing of the tumor cells according to the standards of the icgc (www.icgc.org). the pathognomonic ig-myc translocation was detected in of of the cases using wgs, but was observed in all cases by fish. an igh-myc juxtaposition was detected in patients and its variants igk-myc and igl-myc in and cases, respectively. we identified two different expression patterns of myc transcripts which were associated with the translocation breakpoint location. on the one hand the canonical myc transcript and on the other hand an alternative transcript with a transcription start site before the second exon. the latter produces an mrna which contains nucleotides not included in the canonical transcript but nevertheless it encodes the identical protein. the integration of single nucleotide variants (snv) and copy number aberrations (cna) identified a total of recurrently (≥ samples) mutated genes. myc, id , tp , ccnd , smarca , arid a, fbxo , ddx x were mutated in ≥ % of samples. in / ( %) cases, the id / abstracts ed. the annotation with the chromatin segmentation data of cd + t-cells from the blueprint project revealed enrichment of changes in methylation in distinct genomic regulatory elements in t-lgl. these differentially methylated functional regions were enriched for a set of transcription factor binding sites, known to be relevant in other lymphoid neoplasms. by bioinformatic analysis of methylation data and integration with gene expression data we identified hypermethylated and hypomethylated genes (e. g. bcl b, themis, zeb , hivep ) which point to candidate pathways potentially deregulated in the pathogenesis of t-lgl. conclusion: our study identified dna methylation changes in a set of candidate genes involved in various signaling pathways, which could potentially be used for diagnosis, prognosis and may become targets for novel treatment options. burkitt lymphoma (bl) is a mature aggressive b-cell lymphoma genetically characterized by a chromosomal translocation leading to ig-myc juxtaposition. treatment of bl is usually very successful particularly in children, with a cure rate of over % even among patients with advanced stage disease. however, the prognosis of the remaining patients experiencing disease progression and/or relapse is still very poor. bl has an overall low genomic complexity, thus secondary chromosomal changes in addition to the ig-myc translocation are rare. however, genomic complexity has been associated with aggressive disease and poor prognosis in various lymphomas including bl. because little is currently known about the underlying genetics of disease progression in bl we aimed at characterizing the molecular changes and characteristics that might lead to the relapse of bl. sequential tumor biopsies from initial diagnosis (id) and follow-up were available from a total of patients ( - years at id), which were divided into two groups: five patients experienced a relapse from their initial bl diagnosed - days after id (group ). in contrast, three patients developed twice a bl, i. e. presented with bl as secondary neoplasms diagnosed - years after id (group ). dna extracted from archival formalin-fixed, paraffin-embedded (ffpe) tissue was used to analyze genome-wide copy number alterations (cna) using the oncoscan® platform (affymetrix) and mutational landscape by whole exome sequencing (wes). analysis of the cna in the paired bl samples (group ) revealed an increase in genomic complexity in / pairs as in id a mean of cna was detected in contrast to . cna in relapse samples (p = . ). of note is that in all pairs, the relapse shared almost all cna which were present in id. wes analysis of group showed similar results in all analyzed pairs. in total, . % of mutations (median number of mutations = ) were shared in id and relapse. nevertheless, a considerable amount of mutations were unique in id and relapse with a median of ( . %) and ( . %) mutations, respectively. on the other hand, mutations detected in samples populations of various differential stages. furthermore, we investigated whole-genome bisulfite sequencing (wgbs) data of sbl and b-al in comparison to germinal center b-cell populations from healthy donors to decipher differentially methylated regions (dmr). these are defined as or more cpgs differentially methylated between two groups. unsupervised dna methylation analysis of bl, fl and dlbcl revealed that all bl variants cluster apart from the non-bl cases. thus, supporting on epigenetic level that all analyzed bl samples are bl variants. multigroup comparison (σ/σ max = . , q < e - ) separated the bl variants roughly in groups: ebl, ebv-positive sbl and all other bl variants. furthermore, this analysis revealed ebl to harbor a massive hypermethylation in comparison to all other bl variants. comparison of the dna methylation using the humanmethylation beadchip data of sbl and b-al revealed cpgs to be differentially methylated (σ/σ max = . , q < . ). in contrast, using the wgbs data of the same samples a total of dmrs could be identified which were mostly located in enhancer and polycomb target regions. in conclusion, we show that all analyzed bl variants share a similar dna methylation profile. interestingly, dmrs between sbl and b-al were mainly located in enhancer and polycomb regions. in contrast, ebl showed a massive hypermethylation in comparison to the other bl variants. thus, the differences identified by dna methylation analysis can improve the understanding of the biological and clinical differences of the bl variants. dürig , introduction: t-cell large granular lymphocytic leukemia (t-lgl) is a mature t-cell leukemia which often arises in the context of autoimmune disease. genetic changes like recurrent chromosomal aberrations are rare. recent studies identified somatic stat and tnfaip mutations in t-lgl cells. however, the molecular events driving leukemogenesis remain largely unknown. objectives: the goal of our study was to characterize the epigenetic basis of t-lgl to better understand leukemogenesis and potentially identify druggable pathways or diagnostic biomarkers for t-lgl. p. johansson , , l. klein-hitpass , g. castellano , k. kentouche , f. nicolau , i. oschlies , e. carrillo-de santa pau , m. przekopowitz , a. queiros , m. seifert , a. valencia , ij. martin-subero , em. murga penas , o. ammerpohl , u. dührsen , r. küppers , j. we analyzed the dna methylome of facs sorted tumor cells of t-lgl cases in comparison to benign αβ t-cell subsets. the infinium human methylation bead chip was used for analysis. we annotated our data with the publicly available chromatin segmentation data of cd + t-cells from the ihec/blueprint project. the expression levels of selected genes were tested by reverse transcription real-time pcr. results supervised analysis of t-lgl compared to benign cd + memory cells resulted in , cpg loci significantly (q < . ) differentially methylat- krawitz , , a. knaus , , m. jäger , , r. flöttmann , t. eggermann , b. hoechsmann , h. schrezenmeier paroxysmal nocturnal hemoglobinuria (pnh) is an acquired disorder of the blood-forming system. typically, affected hematopoietic stem cells (hscs) in pnh harbor a single somatic loss-of-function mutation in the x-linked piga gene. previously, a pnh patient with a different molecular etiology has been described and herein we report three more cases of this new subgroup: a predisposing germline mutation in pigt, which is an autosomal gene of the glycosylphosphatidylinositol (gpi)-anchor synthesis pathway, is followed by a second somatic hit. by means of deep sequencing and array-cgh, we observed acquired deletions of mb to mb on chromosome q in pnh cells that include pigt as well as a region that is commonly deleted in myeloproliferative neoplasms and myelodysplastic syndromes and that is known to be differentially methylated. this results in a complete loss of expression of certain genes at this locus which is also thought to contribute to the clonal expansion. the deficiency of gpi-anchored proteins on pnh cells results in a lack of the complement regulatory proteins cd and daf/cd on the cell surface and leaves them more vulnerable to the c b- membrane attack complex. in contrast to classical pnh without any fully synthesized gpi-anchors, pigt mutations impair the transamidase that links the substrate to the anchor and thus result in an accumulation of unbound gpi molecules. this difference in the pathophysiology can also be visualized in flow cytometric analysis of peripheral blood: while cd and cd surface levels are reduced in all pnh cells, the atypical pnh cells due to a transamidase deficiency can be discriminated by a specific antibody, t mab, that binds free gpi anchors. besides the classical pnh symptoms of anemia, thrombosis, and hemolysis, patients with pigt mutations also manifest with additional autoinflammatory symptoms, such as urticaria, fever, arthralgia and meningitis, and it is hypothesized that the free gpi-anchor that accumulates in affected cells is causally related to autoinflammation. based on these findings, we propose the new entity of atypical pnh. background: the prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. genetic variants explain only a minor part of this obesity epidemics induced by physical inactivity and over nutrition. epidemiological studies in humans and animal models of di-from patients with secondary neoplasm (group ) were mostly unique to id (= , . %) whereas only . % of all mutations were shared in id and secondary neoplasm samples (= ). furthermore there were no shared cna in the corresponding samples identified by oncoscan® analysis. to sum up, the oncoscan® and wes analysis, of the paired bl group ( ) provide strong evidence for a linear clonal evolution, meaning relapses may directly evolve from the previous lymphoma clone rather than a common precursor. in contrast, results obtained for patients with secondary neoplasm (group ) showed no indication for linear but rather for divergent evolution. thus, analysis of recurrent mutations shared in id and second neoplasm samples can provide important information about disease progression and are therefore subject of ongoing analysis. y. murakami , t. hirata , s. murata , t. kinoshita , m. kawamoto , s. murase , h. yoshimura , n. kohara , n. inoue , m. osato , j. nishimura , y. ueda , y. kanakura , p. m. in runx mutated aml the number of runx mutations, loss of the wild-type allele and the number and kind of additional mutations impact on prognosis a. stengel, w. kern, m. meggendorfer, k. perglerovà, t. haferlach, c. haferlach mll munich leukemia laboratory, munich, germany, mll , praha, czech republic aml with mutated runx show a distinct pattern of cytogenetic and molecular genetic abnormalities and an adverse prognosis. we analyzed the impact of multiple runx mutations and runx wild-type (wt) loss on associated genetic alterations and survival. for this, aml cases with runx mutations (mut) were split in ( ) runx wt loss (n = ), ( ) > runx mut (n = ) and runx mut (n = ). cases were selected for mutation analyses of genes. in cases with runx mut, + was frequently found, whereas in wt loss + was the most abundant trisomy (+ : % in runx mut vs. % in wt loss, p = . ; + : % vs. %, p < . ). cases with > runx mut showed an intermediate distribution (+ : %, + : %). missense mutations were the most abundant mutation type in wt loss cases ( % vs. %, p = . ), whereas in runx mut, frameshift mutations were found more frequently ( % vs. %, p = . ). in cases with > runx mut, both were observed at similar frequencies (missense: %, frameshift: %). mutation analyses of selected cases revealed additional molecular mutations. % of cases showed at least one runx -accompying mutation (range: - ). the median of accompanying mutations was n = in the total cohort and in cases with runx- mut and > runx mut, whereas it was n = in runx wt loss. srsf ( %), asxl ( %), dnmt a ( %), idh ( %), sf b ( %), tet ( %) and bcor ( %) were revealed as most frequently mutated genes. cases with runx wt loss showed a higher frequency of asxl mut compared to the other cases ( % vs. %, p = . ), while u af mut were absent from this group ( % vs. %, p = . ). median overall survival (os) in the total cohort was months. wt loss (os: months) and > runx mut ( months) showed an adverse impact on prognosis compared to runx mut ( months; p = . and p = . , respectively). mutations in asxl and kras and the presence of ≥ additional mutations also negatively impacted os ( vs. months, p = . ; vs. months, p < . ; vs. months, p = . ). in univariate cox regression analysis runx wt loss (hr = . ; p = . ), ≥ additional mutations (hr = . ; p = . ), asxl mut (hr = . ; p = . ) and kr-asmut (hr = . ; p = . ) had an adverse impact on os. multivariate cox regression analysis revealed an independent adverse effect on os for runx wt loss (hr = . ; p = . ) and krasmut (hr = . ; p = . ). for / cases we received samples during course of the disease. in none of these cases, an evidence for a runx germline mutation was found by analyzing the mutation loads, thus all runx mutations are somatically acquired. taken together, we found strong differences between the subgroups in regard of cytogenetic and molecular genetic aberrations as well as regarding prognosis. thus, not only the presence and number of runx mutations but also the conservation of an intact runx allele as well as the number and kind of additional mutations is biologically and clinically relevant. abstracts different chromatin states, where methylation is inversely correlated with active histone marks. using the hardy-weinberg law, we estimate that there are dmrs with a maf> . . we hypothesized that cis-acting dna polymorphisms could be responsible for the inter-individual variation of the dmrs methylation levels. we genotyped . million snps in the five donors and found that / ( %) dmrs have methylation levels highly correlated (> . ) with the genotype of at least one nearby snp (± kb window). this correlation was verified in / dmrs by targeted bisulfite sequencing in monocytes from individuals used for wgbs and from additional individuals. to validate our results in a larger population and possibly find correlating snps outside the ± kb window for the remaining dmrs, we performed genome-wide association studies (gwas) using snp genotypes and illumina k cpg methylation data from blood samples of individuals from the heinz nixdorf recall study. these methylation arrays encompass only cpgs contained in of our dmrs, showing that they fail to identify a great number of potentially important regions. we certified that for these cpgs, monocyte and whole blood dmrs methylation levels were correlated, and performed a gwas with ~ , snp for each of the cpgs. for / cpgs, the correlation peak was near the cpg position. for each gwas, the snp with lowest p-value (in most cases p < e - ) was designated as lead-snp. snps in high linkage disequilibrium (r > . ) to the lead-snps were located within the corresponding dmr or bp to ~ kb from it. many regions are bound by ctcf and other transcription factors. it is likely that snps affect the binding of these factors and thus the methylation state of the region. we conclude that these inter-individual differences in dna methylation are mainly driven by genetic factors. the dystonia (dyt ) protein thap recruits the histone deacetylase hdac to mediate gene repression sektion für funktionelle genetik am institut für humangenetik, universität zu lübeck, lübeck, germany, institut für neurogenetik, universität zu lübeck, lübeck, germany dystonia describes a heterogeneous group of neurological movement disorders characterized by contractions in various muscles resulting in abnormal postures, involuntary twisting and repetitive movements. dystonia (dyt ), a primary torsions dystonia that first has an impact on cranio-cervical muscles causing problems with speaking and eating, is caused by mutations in the thap gene (thanatos-associated domain-containing apoptosis-associated protein ). thap belongs to the family of thap proteins that are characterized by the presence of an evolutionarily conserved specific dna-binding thap zinc finger motif at their n-terminus. in humans thap family members are known, designated thap to thap . interestingly, most of the dyt -causing mutations affect this thap domain. while we have previously described thap -mediated repression of specific target genes, the molecular mechanisms how thap regulates promoter activity are rather unknown. it is known, that other members of the thap family such as thap and thap interact with the histone deacetylase hdac to mediate transcriptional repression. we have performed yeast-two-hybrid and gst pulldown assays to identify a specific interaction of thap with hdac . by the use of truncated thap fragments we were able to narrow down hdac binding to the n-terminal thap-domain. for further functional characterization we have decreased hdac levels by sirna treatment or chemical inhibition and used taqman analyses to quantify the effect on thap -target genes expression. thus, a significant increase of thap -target genes expression was detected in those cells treated with hdac sirna. to further investigate whether the observed increase in gene expression is due to alterations of histone acetylation within the promoter regions we performed chromatin immunoprecipitation (chip) assays followed by qpcr using antibodies specific for different acetylated n-terminal residues of histone as markers for transcriptional active promoters. by this, we detected an increased acetylation within the promoter regions of thap target genes that are dysregulated in cells treated with decreased hdac levels. et-induced obesity indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. possible adverse paternal effects are likely transmitted by the sperm to the next generation. to prove this hypothesis, we have systematically analyzed the effects of paternal obesity on the sperm epigenome and its implications for the next generation. results: to study the possible transmission of paternal bmi effects to the next generation, methylation levels of eight paternally expressed imprinted genes (peg , peg , peg , peg , peg , peg , nespas and igf ), two maternally expressed imprinted genes (meg and h ), and the obesity related gene hif a were quantified by bisulphite pyrosequencing in sperm of donors (undergoing ivf/icsi) and fetal cord blood (fcb) of resulting offspring (conceived by ivf/icsi with the same sperm samples). hif a showed a significant positive correlation between sperm methylation and paternal bmi. this effect on the sperm epigenome was replicated in an independent cohort of sperm samples. for hif a, paternal bmi also showed a significant positive correlation with fcb methylation. on the other hand, peg /nnat exhibited a significant negative correlation between paternal bmi and fcb methylation. in contrast to pyrosequencing, deep bisulphite sequencing (dbs) allows one to study dna methylation at the single molecule level and enables us to distinguish between maternal and paternal alleles in fcb samples with an informative snp. epimutations which are defined as alleles showing > % aberrantly (de)methylated cpg sites can also be identified with dbs. upon performing dbs on sperm samples, we observed a higher epimutation rate in the high bmi ( - ) group when compared to the low bmi ( - ) group across the four studied genes (peg , hif a, h and nespas). we are presently analyzing dbs data in selected cord blood samples with an informative snp to separately quantify methylation at the paternal and maternal alleles. it is important to decipher the methylation of the paternal allele when studying whether sperm methylation alterations are transmitted to the offspring. conclusions: our results suggest that male obesity is associated with modification of the sperm dna methylome, which may affect the epigenome (in fetal cord blood) of the next generation. allele-specific dna methylation occurs at functionally different regions: ) at imprinting control elements, ) on the silent x chromosome in females and ) across the genome and probably dependent on the dna sequence in cis. the latter is termed haplotype-dependent allele-specific methylation and may contribute to inter-individual phenotypic variation. in a previous study on monocyte to macrophage differentiation, we showed that dna methylation differences between individuals were greater than between the two cell types. to study the genetic basis of these inter-individual differences in dna methylation, we analysed the methylome obtained by whole genome bisulfite sequencing (wgbs) of monocytes from five unrelated donors. for identifying differentially methylated regions (dmrs), we created two synthetic methylomes: one with the highest methylation values of each cpg in the five samples and one with the lowest methylation values. defining a dmr as a region of at least cpgs with a methylation level difference of at least . , we identified dmrs, which cover cpgs and fall into cer, respectively. we show that ns-associated rit mutants intensified signal flux through the mek-erk pathway upon growth factor stimulation. by using heterologous expression systems, we identified the p -activated kinase (pak ) as novel effector of rit . we found that rit interacts with the rho gtpases cdc and rac , both of which are crucial upstream regulators of pak . disease-causing rit mutations enhance protein-protein interactions and uncouple complex formation from growth factors. expression of both wild-type rit and its mutant forms resulted in dissolution of stress fibers and paxillin-containing focal adhesions from the cell center and increased cell movement. we conclude that rit is a potent regulator of actin dynamics, and dysregulated rac /cdc -pak signaling controlling cell adhesion and migration may be one aspect of the molecular basis of ns. medical systems biology, tu dresden, germany, institute for clinical genetics, tu dresden, germany, cancer science institute of singapore, national university of singapore, singapore, institute of molecular biology, mainz, germany telomeres are short repetitive ttaggg sequences that cap the ends of chromosomes. these stretches of dna are covered by proteins and rnas which together protect the putative double strand break from dna repair mechanisms and facilitate replication. however, telomeres shorten with every cell division due to the end replication problem. the ribonucleoprotein telomerase counteracts this process by de novo elongation of telomeric repeats but its expression is mostly confined to the germ line and stem cells. even in the latter its activity is usually not sufficient to completely prevent telomere shortening. all cancer cells are also faced with this challenge and while the majority of cancer cells rely on telomerase, approximately % of cancers ensure sufficient telomere length via the recombination-based alternative lengthening of telomeres (alt) mechanism. to better understand telomere biology we aimed to identify novel telomeric factors by systematically screening for telomere-binding proteins in cell lines from different vertebrates. here, we identified and characterized zbtb , a zinc finger protein, as a novel direct telomere-binding protein across the vertebrate lineage. zbtb is directly binding to telomeric dna in vitro and it is localizing to telomeres in vivo via one specific zinc finger domain in both telomerase-and alt-positive cancer cells. interestingly, zbtb knock-out cells have longer telomeres, suggesting that zbtb limits telomere elongation. in addition, the combination of chipseq, rnaseq and proteome analysis revealed a transcription factor activity for a small, but specific set of target genes of zbtb , linking its telomeric functions to mitochondrial metabolism. in conclusion, zbtb is a novel direct telomere binding protein with transcription factor activity that acts as negative regulator of telomere length. our data show for the first time a functional interaction of the 'dystonia protein' thap with the histone deacetylase hdac and therefore give new insights into the molecular mechanisms of thap -mediated gene repression. interestingly, previous functional studies as well as structure analyses revealed that only a subset of the dyt -causing mutations affecting the n-terminal thap domain alter thap -binding to dna. in ongoing studies we want to investigate the consequences of dyt -causing mutations on thap -hdac complex formation and its relevance in the molecular pathology of dystonia. reproductive homeobox (rhox) genes are clustered on the x chromosome and share a unique amino acid helix-turn-helix dna binding homeodomain. they were identified in several species as having important roles in reproductive tissues, notably in the testis. the human rhox cluster is composed of three genes: rhoxf and two copies of rhoxf (rhoxf a, rhoxf b) which are referred to as rhoxf / b. rhox proteins are expressed exclusively by germ cells in human testis and aberrant rhox methylation is associated with several sperm parameters. because little is known about the molecular mechanism of rhox function in humans, the aim of the study was to identify target genes of human rhox proteins and to investigate the impact of rhox mutations on protein function. using gene expression profiling, we identified genes regulated by members of the human rhox gene cluster. some genes were uniquely regulated by rhoxf or rhoxf / b, while others were regulated by both of these transcription factors. several of these regulated genes encode proteins involved in processes relevant to spermatogenesis, e. g. stress protection and cell survival. one of the target genes of rhoxf / b is rhoxf , suggesting cross-regulation to enhance transcriptional responses. the potential role of rhox in human infertility was addressed by sequencing rhox in a group of patients with severe oligozoospermia. this revealed two mutations in rhoxf (c. g>a and c. c>t) and four in rhoxf / b (- c>g, c. g>a, c. c>t and c. g>a), of which only one (c. g>a) was found in a control group of men with normal sperm concentration. functional analysis demonstrated that c. g>a and c. g>a significantly impaired the ability of rhoxf / b to regulate downstream genes. molecular modelling suggested that these mutations alter rhoxf /f b protein conformation. by combining clinical data with in vitro functional analysis, we demonstrate how the x-linked rhox gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility. colorectal cancer (crc). here, we proposed the possible molecular mechanisms responsible for crc initiation, progression and invasion using a network biology approach. materials and methods: in order to investigate the underlying crc pathogenesis, the dataset gse consisting of normal tissues, stage i, stage ii, stage iii and stage iv of crc were obtained from gene expression omnibus (geo) and further examined. the differentially expressed genes (degs) were subjected to protein-protein interaction databases and a ppi network was constructed for each crc stage. topological analysis of resulted ppi networks revealed functional hub genes and involved in crc development. furthermore, the overlap genes between four studied crc stages were determined and deeply evaluated to identify deregulat ed biological networks during crc development. a standard real-time pcr was performed to validate the in silico findings utilizing sw and ncm cell lines. results: the most important hub genes (cdk for stage i, ubc for stage ii, esr for stage iii and atxn for stage iv) and sub-networks were identified in crc stages. moreover, several novel biomarkers were also introduced for each crc stage. gene ontology (go) and signaling pathway enrichment uncovered the important roles of wnt, mapk and jak-stat signaling pathways in regulation of crc pathogenesis. functional annotation of overlap genes revealed that cell cycle regulating genes are the most highly regulated genes during crc initiation, progression and invasion. in vitro analyses confirmed deregulation of atxn and cdk , two hub genes of stage iv, in metastatic colon sw cells compared to normal colon ncm cell line. our study provides a new insight into the distinct molecular mechanisms underlying the pathogenesis of crc. the functional hub genes, sub-networks, prioritizes key pathways and novel crc biomarkers were also provided that can be useful in therapeutic programs. targeted next-generation sequencing approaches as well as next-generation whole exome sequencing are becoming more widespread in routine molecular diagnostics for patients with ataxia. however, since ngs at present is not suitable to detect (trinucleotide) repeat expansions, a pre-ngs testing for common polyglutamine expansion scas seems mandatory. but also sca subtypes caused by expansions in non-coding regions of genes like sca , sca , sca , and sca as well as other ataxias known to be associated with repeat expansions like the fragile x-associated tremor ataxia syndrome (fxtas) should be taken into account before applying ngs-based diagnostics. in order to find an optimal diagnostic strategy in future more information about the frequency and phenotypic characteristics of rare repeat expansion disorders associated with ataxia would be helpful. we therefore analyzed a cohort of patients with symptoms of cerebellar ataxia, dysarthria and other unspecific symptoms who were referred to our center for sca diagnostics and showed alleles in the normal range for the most common sca subtypes sca - , sca , sca , and sca . these patients were screened for expansions in sca , sca , sca , sca and fxtas as well as for the pathogenic hexanucleotide repeat in the c orf gene. no expanded repeats for sca , sca or sca were found in the analyzed patients. five patients with ataxia of unknown etiology showed sca cta/ctg combined alleles ( - ) that are discussed to be potentially pathogenic. one -year-old male patient with unclear dementia syndromes was diagnosed with a large ggggcc repeat expansion in c orf . and the analysis of the fmr gene identified one patient with a permutation (> cgg repeats) and seven patients poster *** = für den posterpreis nominiert preventive genetic counseling in neurogenetic disorders needs a better collaborative approach between genetic and neurology clinics -a report of four siblings with unverricht-lundborg disease: genetic counseling is the process of helping people to understand and adapt the medical, psychological and familial implications of genetic contributions to disease. for parents with a previous child or other family member with a known genetic syndrome expands options for preimplantation or prenatal diagnosis for the current or the future pregnancies. however, timely referral by health providers to genetic counselor and for discussing with couples regarding possible options is important. additionally, other factors such as personal decision making especially due to high price of some genetic services and uncertain results cause considerably delays to genetic testing. there are more than various types of inherited neurological disorders in which alterations in genes lead to an inherited condition such as huntington disease, inherited forms of alzheimer disease, ataxia, muscular dystrophies and epilepsies. the knowledge of the causative gene mutations in the affected individual is critical in the possible prenatal diagnosis in other members of the pedigree. therefore a multidisciplinary care team, including neurologist and genetic counselor for the conditions diagnosed as inherited neurological disorders is critical in prenatal setting and consideration of an effective management. here, our report of four siblings affected by a rare form of inherited epilepsy (unverricht-lundborg disease) with an autosomal recessive pattern highlights the importance of the needs for a better collaborative approach in the neurogenetic setting. in fact, the birth of four successive siblings affected by similar neurogenetics disorders in a specific family is showing the need for more attention to this important issue, especially in terms of intersectoral collaboration. poorebrahim hort: / differentially expressed transcription units). these differences in gene expression we detected did not correlate with dna methylation changes at the corresponding transcription regulatory sites. from our results we conclude that altered expression of imprinted genes indeed plays a role in tumorigenesis of germinal center derived b-cell lymphomas. however, the altered transcriptional regulation of these genes seems not to rely on the usual epigenetic mechanisms known from constitutional imprinting disorders. mf. abazari , h. bokharaie , m. asghari , v. poortahmasebi , h. askari , m. investigating the expression of genes associated with autism spectrum disorders to identify sex related differences s. berkel, a. eltokhi, g. rappold institute of human genetics, heidelberg university hospital, heidelberg, germany neurodevelopmental disorders such as autism, attention deficit and hyperactivity syndrome as well as language problems and learning difficulties have a higher prevalence in male individuals compared to females. autism is characterized by impairments in social interaction, communication deficits and restricted and repetitive behaviors. boys are more frequently affected than girls; the ratio of affected boys compared to girls is : for autism and : for asperger syndrome. in this study we aim to elucidate the reason for this gender difference by following up two hypotheses: ( ) risk genes for autism spectrum disorders (asd) might be expressed at different levels in males and females and ( ) asd risk genes might interact with sexually dimorphic pathways. first, we investigated the expression of genes associated with autism spectrum disorders, including the shank gene family, in the brain of male and female mice to identify sex-dependent differences. the rna expression levels were analyzed in five different brain regions (cortex, hippocampus, striatum, cerebellum, thalamus) at different developmental stages (e , e , p , p , p and adult) in male and female mice. we identified a sex dimorphic expression of shank and shank , but not of shank . due to the fact that early brain development is strongly influenced by sex hormones (estrogen, testosterone), we further investigated the influence of these hormones on shank expression in human neuroblastoma cells (sh-sy y) and primary mouse hippocampal neurons. a better understanding of the sex differences in the brain might help to explain the vulnerability for neuropsychiatric disorders like autism and paves the way to discover putative risk or protective factors for these disorders. imprinting defects in temple syndrome are caused by a failure in imprint establishment and/or maintenance j. beygo, c. mertel, g. gillessen-kaesbach, b. horsthemke, k. buiting institut für humangenetik, universitätsklinikum essen, universität duisburg-essen, essen, germany, institut für humangenetik, universität zu lübeck, lübeck, germany temple syndrome (ts ) is a rare imprinting disorder characterised by low birth weight and height, muscular hypotonia and feeding difficulties in the infant period, early puberty and short stature with small hands and feet and often truncal obesity. in a subset of patients with ts , the disease is caused by an imprinting defect (id) affecting the paternal allele of the imprinted region q . the id results in aberrant methylation of the three known differentially methylated regions (dmrs), the germline-derived primary dlk /meg intergenic (ig-)dmr (meg /dlk :ig-dmr), the postfertilization-derived, secondary dmr at the meg promoter (meg :tss-dmr), and the postfertilization-derived, secondary intragenic meg -dmr (meg :int -dmr). the meg /dlk :ig-dmr and the meg :tss-dmr are methylated on the paternal chromosome and hypomethylated in patients with ts and an imprinting defect. the meg :int -dmr is unmethylated on the paternal chromosome and hypermethylated in these patients. both the meg /dlk :ig-dmr and the with alleles in the grey zone ( to cgg repeats), thus suggesting that individuals with fmr repeat expansions in the gray zone may also present with neurological signs. bernhart , , , h. kretzmer , , r. wagener , c. mmml some genes are subject to the mechanism of imprinting, i. e. their expression depends on parental origin. they primarily function in the control of proliferation, fetal development and cellular differentiation. constitutional imprinting disorders are in part also associated with an increased tumor risk. loss of imprinting has been also described as somatic event in tumorigenesis. while this phenomenon has been broadly analyzed in solid tumors, data on alterations of imprinting in lymphatic neoplasms are largely missing. we analyzed the rna expression of transcription units/regions known or supposed to be subject to imprinting in two cohorts of normal b-cells and germinal center derived b-cell lymphomas. the first cohort (mmml) contains samples: burkitt lymphomas (bl), non-burkitt lymphomas (non-bl, including various subtypes like follicular and diffuse large b-cell lymphoma) and normal germinal center b-cell samples (gcbc, as controls). the second cohort (icgc mmml-seq) comprised samples with bl, non-bl and gcbc samples. gene expression was analyzed with affymetrix u a genechips in the mmml cohort and by rna sequencing in the icgc mmml-seq cohort. results of the transcriptional analyses in the icgc mmml-seq cohort were compared to the dna methylation available from a subset of the analyzed samples (kretzmer et al., nat genet, ) . of the transcription units sites, corresponding to transcription units, were present on the applied array used for the analysis of the mmml cohort. a two group comparison revealed significantly differentially expressed sites corresponding to transcription units between bl and non-bl including the plagl and peg genes. in total, and sites corresponding to and transcription units are differentially expressed between bl versus gcbc and non-bl versus gcbc, respectively. comparison of gene expression in the icgc cohort revealed differentially expressed sites corresponding to transcription units between bl and non-bl (overlap with mmml cohort: / differentially expressed transcription units), including again peg and plagl , differentially expressed sites corresponding to transcription units between bl and gcbc (overlap with mmml cohort: / differentially expressed transcription units) and differentially expressed sites corresponding to transcription units between non-bl and gcbc (overlap with mmml co-abstracts maintenance dna methylation of l promoters, spoc could function in targeting g a to l sequences. in conclusion our data implicate the epigenetic reader spoc in the suppression of line elements during germ cell development. s. bens , j. kolarova , m. kreuz , sh. characterization of the expression of the imprinted kcnk -gene in specific brain regions and the phenotypic analysis of kcnk knockout mice a kcnk /kcnk is a maternally expressed imprinted gene whose mutations are responsible for the maternally inherited birk-barel mental retardation dysmorphism syndrome. it encodes a member of the superfamily of k+channels with two pore-forming domains and is involved in the modulation of the resting membrane potential and excitability of neuronal cells. so far, only homozygous kcnk knockout mice with inactivation of both parental alleles were phenotypically characterized. these mice displayed cognitive deficits as well as a reduction of k+ leak current by %. in the light of maternal-specific imprinted expression of kcnk /kcnk and the maternal inheritance of the birk-barel mental retardation dysmorphism, a thorough phenotypic analysis of heterozygous kcnk knockout mice with inactivation of only the maternally inherited allele is also warranted. as first aim of our study, we characterized the parental allele-specific expression of kcnk in various regions of the mouse brain. quantification of allele-specific expression by pyrosequencing (quasep) method was performed for different brain areas from several developmental stages of (c bl/ xcast/ei) f hybrid mice. exclusive expression from the maternal kcnk allele was detected in the dentate gyrus, hippocampus, mesencephalon, medulla oblongata, thalamus and pons. biallelic expression with, however, a strong bias towards the maternal kcnk allele ( - % of the transcripts) was observed in the olfactory bulbs, cortex, cerebellum, striatum and olfactory tubercles. as the second aim of our study, the phenotypes of wildtype, heterozygous kcnk knockout mice with maternal inherited knockout allele (kcnk komat) and homozygous kcnk knockout mice (kcnk kohom) were comparatively examined in a behavioral test battery. due to the already known cognitive defects of kcnk kohom animals and especially the phenotype of the patients with birk-barel syndrome, it was assumed that kcnk komat and kcnk kohom animals show deficits in some of the tests. the spontaneous alternation in the y-maze test was significantly reduced by approximately - % in kcnk komat and kcnk kohom mice compared to wildtype mice indicating a clearly impaired working memory. in addition, kcnk komat and kcnk kohom mice displayed a reduced prepulse inhibition of startle response compared to wildtype mice indicating an impairment of sensomotoric gating, a process to filter out irrelevant information. acoustic startle response as a measure of anxiety levels was also significantly decreased, but only in kcnk kohom mice. our findings shall further elucidate the role of kcnk /kcnk in brain physiology and pathophysiology and open new avenues for treatment of cognitive dysfunctions in birk-barel syndrome. meg :tss-dmr act as imprinting control centres, although the meg / dlk :ig-dmr functions as an upstream regulator of the meg -dmr. so far, the function and regulation of the meg -dmr is unknown. the hypomethylation of the paternal allele in ts -id patients at the meg / dlk :ig-dmr and the meg :tss-dmr point to a failure in the establishment of the methylation imprint or to maintain the methylation imprint after fertilization. in this case, the incorrectly imprinted chromosome would be inherited from either the paternal grandfather or grandmother. to prove this assumption we are investigating the grandparental origin of the affected chromosome in our cohort of ten ts -id families by studying the parent-of-origin specific methylation of the three dmrs in combination with informative single nucleotide variants (snps). at the moment we have identified three families informative for the meg / dlk :ig-dmr, two families for the meg :tss-dmr and two families for the meg :int -dmr. so far we have obtained results in two families for the meg :tss-dmr. we found that in one case the allele harbouring the id was inherited from the paternal grandmother, but in the second case from the paternal grandfather, indicating that the id occurred after erasure of the parental methylation imprints. a complete lack of methylation observed in the majority of ts -id patients is therefore likely due to a problem in establishing methylation on the paternal chromosome, whereas in rare cases with methylation mosaicism, the id is probably due to a problem to maintain the paternal imprint after fertilization. bosch, s. lukassen, j. kaindl, j. schwarz, c. nelkenbrecher, a. herrmann, a. reichel, a. ekici, t. gramberg, t. stamminger, a. winterpacht humangenetisches institut, erlangen, germany, virologisches institut, erlangen, germany spoc /phf is a gene located on human chromosome region p . and mouse chromosome qe . the protein was first described in patients with epithelial ovarian cancer, where its expression correlated with tumour progression and reduced survival time. spoc is a reader of the epigenetic mark h k me / , dynamically associates with chromatin during mitosis and plays a role in chromosome condensation. spoc deficient mice show a pronounced hypoplasia of the testis with a progressive loss of germ cells. although loss of spoc leads to a significantly reduced chromatin condensation of the sex chromosomes in meiosis, the protein is not expressed in spermatocytes but in the undifferentiated precursor cells, the spermatogonial stem cells (sscs). here, we present chip-seq data of mouse testis tissue demonstrating that spoc strongly binds to evolutionary young l elements in undifferentiated spermatogonia. we show that in hek cells overexpression of spoc leads to repression of transposition activity of line-elements strongly indicating a role of spoc in l element suppression. the cell has developed several lines of defence against retrotransposition to maintain genomic integrity, including dna methylation. these defence mechanisms are most elaborate in spermatogonial stem cells since transposition events in these cells would have a dramatic impact on the next generation. therefore, the repression of retrotransposition is of fundamental importance for germ cell development and ultimately the quality of the gametes. moreover, we present medip results showing that the methylation levels of l sequences decrease upon spoc -knockout and demonstrate that the histone methyltransferase g a is strongly upregulated in preleptotene meiocytes of spoc -/mice. g a is expressed from spermatogonia until early meiosis where it regulates h k di-methylation and has been shown to be involved in the repression of l element in mouse spermatogonia. we are able to demonstrate that h k me levels are unaltered in spoc -/mice, suggesting a potential functional link between g a and spoc that does not affect the catalytic activity of g a. since g a can regulate de novo and medizinische genetik · lated to investigate ciliogenesis. data resulting from rnaseq experiments are analyzed by established informatics tools (tophat, cufflinks and derivatives thereof). we will show results from our work in progress and we hope to convince people to intensify rna analyses even in routine labs to uncover hidden mechanism and/or mutations impacting mrna splicing and thereby causing human disease. telomeres are located at the ends of chromosomes and have an essential role in the maintenance of genome stability. after each cell division, a small part of this specialized sequence is lost. when telomeres reach a critically reduced length, the cell either dies through apoptosis or enters a state of permanent cell cycle arrest. it has been demonstrated that telomere biology is directly linked to basic biological phenomena such as aging, tumorigenesis and maintenance of dna integrity. it is known that oxidative stress accelerates telomere shortening in cells, resulting in premature cell senescence. shorter leukocyte telomeres have been observed in type ii diabetes or degenerative disease like dementia and alzheimer disease as well in chromosomal instability syndrome, such as fanconi anemia (fa) and nijmegen breakage syndrome (nbs). any link between telomere length and inflammation has not yet been extensively studied in autoimmune diseases. accelerated length shortening might be related to autoimmune disease predisposition. yet the reasons for this shortening are likely manifold, including the individual genetic background, oxidative stress and chronic inflammation. in order to shed light on these relationships, we investigate genomic dna extracted from blood of patients diagnosed with multiple sclerosis and from patients with huntington disease. the samples were divided into age groups. stepone q-pcr was applied to detect the relative telomere length as a function of age. initially identified differences in telomere lengths still have to be confirmed in larger cohorts. background: intrauterine exposure to gestational diabetes mellitus (gdm) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. gdm-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these increased disease risks. to identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (fcbs) from gdm and control pregnancies. methods and results: using illumina's k methylation arrays and following correction for multiple testing, cpg sites ( of which are associated with genes) displayed significant methylation differences between gdm and control samples. three of four candidate genes, atp a , prkch, and slc a , from our methylation screen and one, hif a, from the literature were validated by bisulfite pyrosequencing. the gdm effect on fcb methylation was more pronounced in women with insulin-dependent gdm who had a more severe metabolic phenotype than women with dietetically treated gdm. however, the effect remained significant after adjustment for the maternal bmi and gestational week in a multivariate regression model. e. g. dekomien , , b. bellenberg , , n. trampe , , r. schneider , , c. prehn , , c. krogias , , r. kropatsch , , m. regensburger , , c. lukas , , r. gold , , human genetics, bochum, germany, ruhr-university bochum, germany, radiology, st. josef-hospital, bochum, germany, neurology, st. josef-hospital, bochum, germany, molecular neurology, erlangen, germany, university of erlangen, germany a pair of monozygotic -year-old twins suffering from hereditary spastic paraplegia (spg ) is described. patients underwent thorough clinical examination and magnetic resonance imaging (mri) and mr-spectroscopy (mrs) at tesla. genetic testing was performed by sanger sequencing and alternative splicing by rna analysis. clinically the patients presented a similar spectrum of symptoms with a higher level of disability in one of the patients. mri studies including morphometry and regional microstructural analysis by diffusion tensor imaging (dti) of the corpus callosum (cc) revealed marked thinning and corresponding increases of axial diffusivity (ad), radial diffusivity (rd) and apparent diffusion coefficient (adc) and reduction of the fractional anisotropy (fa) as compared to healthy controls in all cc sections, particularly in the anterior callosal body. there was marked supratentorial white matter reduction and to a lesser extent grey matter reduction in both patients. involvement of the cortico-spinal tracts was reflected by fa and rd alterations and cervical cord atrophy. the more strongly affected patient showed a higher degree of callosal microstructural damage and cervical cord atrophy. genetic testing of the spg gene revealed two mutations in compound heterozygous state, a known frameshift mutation as well as a novel synonymous exonic splice site mutation. this study shows similar but distinct clinical and imaging findings in monozygotic twins suffering from spg , suggesting individual downstream genetic effects. targeted next generation sequencing techniques tremendously improved our ability to identify sequence variants. however fixing disease causing mutation still lack behind because of several reasons: inappropriate gene specific data bank, insufficient prediction tools, incomplete analysis and others. in addition identified sequence variants are a mixture of severe disease causing mutations and a myriad of variants of unknown pathogenicity. in addition an unknown number of silent mutations, neutral polymorphism and sequence variants deeply buried in introns might severely influence splicing of the premature rna molecule. by solely analysis of the dna sequence this impact onto the integrity of the mrna is completely ignored. in order to catalog the mrna isoforms derived from genes of our interest we started to set up rnaseq technologies in our routine lab. to reduce the amount of data, to improve the power of analyses and to identify rare isoforms of transcripts we use targeted rnaseq to characterize the mrna molecules originating from those genes we are interested in (e. g.: hereditary breast cancer core genes ( genes), hereditary colon cancer ( genes), primary ciliary dyskinesia (pcd)( genes). genes involved in pcd offer the invaluable advantage that the respiratory epithelium where these genes are normally expressed can be sampled from the inferior turbinate of the nose by brush biopsy either from healthy probands or from patients suffering from pcd. in addition to direct preparation of rna from these cilia, cilia carrying cells or tissue can be cultured and manipu-abstracts long-range pcr and direct sequence analysis. the comparative analysis of parental haplotypes with the sequences flanking the deletion breakpoints in the patients revealed the absence of any de novo mutations in breakpoint-flanking regions of prs -mediated and prs -mediated type- nf deletions. we conclude that although nahr is a mutational mechanism causing large nf deletions, there is no evidence for a local mutagenic effect of these recombination events. hence it is unlikely that nahr underlying type- nf deletions involves error-prone translesion polymerases that would increase the de novo mutation rate in breakpoint flanking regions. furthermore, the detailed haplotype analysis of prs , a highly active nahr hotspot mediating the majority of large nf deletions, revealed that non-allelic homologous gene conversion (nahgc) between nf -repa and nf -repc, which results from non-crossover resolution of recombination intermediates, is the major driving force responsible for the haplotype diversity in this region. remarkably, the haplotype diversity patterns observed for nf -repa and nf -repc were markedly different indicating that during nahgc, nf -repa is disporportionately more often the donor sequence used to repair mismachtes in heteroduplex regions than nf -repc. we also noticed a correlation between haplotype diversity and the number of prdm a-allele binding sites suggesting that haplotype diversity and hence the nahgc rate within prs in nf-repa is regulated by prdm . heidelberg center for personalized oncology dkfz-hipo dkfz, heidelberg, germany dna methylation aberrations at differentially methylated region of imprinted genes interfere with the naturally parental-specific mono-allelic expression. that leads to a bi-allelic or absent expression of the imprinted gene, a cause of imprinting disorders (ids). we aimed at analyzing the genome wide dna methylation pattern of two patients with ids, namely transient neonatal diabetes mellitus (tndm) and multi locus imprinting disturbance (mlid), and their respective parents. the dna methylation profiles of these individuals were obtained by whole genome bisulfite sequencing (wgbs) on b cells sorted by magnetic cell isolation. the sequencing libraries were prepared as described in kretzmer et al. [ ] and sequenced on an illumina hiseq machine. wgbs data were processed with the methylctools toolkit. briefly, bisulphite-treated sequences were aligned with bwa-mem using a three-letter approach, and the methylation ratios were quantified for ~ . million out of . million cpg sites (coverage> ) genome-wide. quality control was performed to assess the quality of the dna methylation profiles and genetic fingerprinting was performed on the wgbs data confirming sample origin and family relationship. the wgbs data was further compared to already existing genome-wide human snp array . (snp array) and humanmethylation k bead array ( k) data, resulting in a good accordance with pearson's correlation coefficients > . . we detected an overall dna methylation level around % in all samples. already known dna methylation alterations, e. g. hypomethylation in plagl were validated by wgbs. by searching for differentially methylated regions (dmrs), defined as regions composed of at least five consecutive cpg loci showing a methylation difference between patient and corresponding parents above %, we identified dmrs in the mlid our study supports an association between maternal gdm and the epigenetic status of the exposed offspring. consistent with a multifactorial disease model, the observed fcb methylation changes are of small effect size but affect multiple genes/loci. the identified genes are primary candidates for transmitting gdm effects to the next generation. they also may provide useful biomarkers for the diagnosis and prognosis of adverse prenatal exposures and assessing the success of interventions during pregnancy. the nuclease hsnm b/apollo has a dual function in both dna-repair and maintenance of telomeres. as to the repair of dna interstrand crosslinks (icl), hsnm b/apollo is linked to the fanconi anemia (fa) pathway and cells depleted for hsnm b/apollo (sirna) resemble those from patients with fa. we have identified a single nucleotide polymorphism, rs , which is associated with quantitative differences in hsnm b/apollo expression (mrna). we analyze whether the differential expression relates to the degree of cellular sensitivity to the dna interstrand crosslinks inducing mutagen mitomycin c (mmc) and ionising radiation (ir), which induces, among other lesions, dna double strand breaks. all experiments are realized using lymphoblastoid cells derived from generally healthy donors. results of rt-pcr analysis of hsnm b/apollo expression and of the cell viability assays will be presented and discussed in the context of the potential usefulness of considering rs in predicting individual sensitivity to mutagens relevant in anti-cancer treatment. p-basepi- nahr events causing type- nf microdeletions are not associated with an increased mutation rate in breakpoint-flanking regions m. hillmer , , a. summerer , , v. f. mautner , , l. messiaen , , h. institute of human genetics, ulm, germany, university of ulm, ulm, germany, department of neurology, hamburg, germany, university hospital hamburg eppendorf, hamburg, germany, department of genetics, birmingham, usa, university of alabama at birmingham, birmingham, usa large deletions of the nf gene and its flanking regions are the most frequent recurrent mutations in patients with neurofibromatosis type (nf ). different types of large nf deletions have been identified which are distinguishable in terms of their size and breakpoint position. most frequent are type- nf deletions spanning . -mb and characterized by breakpoints located within the low-copy-repeats nf -repa and nf -repc which exhibit . % sequence homology within -kb. type- nf deletions are caused by non-allelic homologous recombination (nahr). two nahr hotspots have been identified termed prs and prs which encompass -kb and -kb, respectively. approximately % of all type- nf deletion breakpoints cluster within the prs and prs nahr hotspots. in this study, we analysed whether the nahr events causing type- nf deletions would be associated with an increased de novo mutation rate of sequences located in breakpoint-flanking regions. to do so, we sequenced the deletion breakpoint-flanking regions in the patients and compared these sequences with the homologous regions amplified from dna of the patients' parents who are not affected by nf . however, in the germline of these parents, the deletions were mediated by nahr and then transmitted to their offspring. the parental haplotypes within the prs or prs regions of nf -repa and nf -repc were analysed by in the alpl gene and inherited as an autosomal dominant trait can cause milder forms. so far detailed knowledge of the milder forms is lacking. patients with a mutation in the alpl gene were interviewed in a standardized questionaire concerning the different disease manifestations: teeth, bone fractures, pain of bones and muscles and quality of life. subgroups were formed with regard to the localization of the mutations in the three protein domains. patients with mutations clustering in the catalytic site of the molecule showed the most severe odontohypophosphatasia: one individual had premature primary tooth loss, % of patients showed adult tooth loss, % suffered from dental caries. the majority had the first manifestation before the age of . persons suffering from mutations in the two other domains reported a relatively high quality of live with low pain of muscles and bones. unexpectedly in all groups there was no significant difference in the portion of patients with bone fracture. conclusion: the clinical signs of dominant hpp are mostly unspecific. especially dental problems like severe adult teeth loss, an early manifestation of dental caries or enlarged pulp chambers can be a sign of odontohypophosphatasia and a dominant inherited mild hpp. mutations in the catalytic site of the alpl molecule are associated with a more severe odontohypophosphatasia. screening of non-neoplastic lymphatic tissues from children for the igh-myc fusion using a highly sensitive -color fish-assay burkitt lymphoma is a mature b-cell lymphoma which on the genetic level is characterized by the burkitt translocation t( ; )(q ;q ) juxtaposing the igh locus in q next to the myc locus in q . in a minor part of burkitt lymphomas, immunoglobulin light chain variants of the translocation result in overexpression of myc. despite being pathognomonic for burkitt lymphoma, the ig-myc juxtaposition alone is not sufficient on its own for a malignant transformation of the cell. other igh rearrangements like the igh-bcl fusion, typical for follicular lymphoma, were detected in a significant number of healthy individuals. for the igh-myc translocation, only scarce data in healthy individuals exist. this is most likely due to scattering of the breakpoints which are far more difficult to target by pcr than the igh-bcl translocation. therefore, we aimed at investigating if myc-translocation positive cells can also be detected in normal b-cell maturation. considering the epidemiology of burkitt lymphoma being the most common b-cell lymphoma in children, we focused on samples from young individuals. on the one hand, we analyzed non-neoplastic tissue specimen of bone marrow (n = ) (age range - , median age . years) and lymph nodes (n = )(age range - years, median age years). on the other hand, considering the typical clinical presentation of burkitt lymphoma, we included non-neoplastic tissue specimen containing peyer patches (n = )(age range hours- years, median age years). the specimen were analyzed using a four-color fluorescence in situ hybridisation (fish) assay with probes flanking the breakpoints on chromosomes and . in this setting, a positive result comprised the break on both chromosomes (seen as signal split for each locus) and fusion of the involved genes (leading to two different fusion signals). the assay was first validated on controls of cells with a normal male karyotype from healthy individuals as well as on five burkitt cell lines and each five ffpe embedded t( ; ) negative and positive tissues as negative and positive controls. the assay was then applied for the screening of a igh-myc fusion in the above mentioned paraffin-embedded tissues. successful hybridizations of overall , and ffpe sections from bone marrow, lymph nodes and peyer patches respectively could be obtained. trio and dmrs in the tndm trio. in the mlid trio / dmrs showed increased and dmrs decreased dna methylation in the patient's sample. of dmrs, are located in regions potentially associated with transcriptional regulation. further analysis revealed that / dmrs are associated with imprinted genes. in the tndm trio, we detected / dmrs to show hypermethylation in the patient compared to her parents and / dmrs with lower dna methylation. in these trio / dmrs are associated with regions potentially correlated to transcriptional regulation and dmrs with imprinted genes summarized, our results show that wgbs is a well suited and valid method for analyzing dna methylation. while the overall dna methylation levels does not differ between the analyzed patients and parents, a detailed analysis of smaller regions revealed the existence of respectively differentially methylated regions between the analyzed mlid and tndm patient and their parents. supported by bmbf through fkz: gm und gm and gm p-basepi- characteristic mutational profile in children of individuals exposed to ionizing radiation p. krawitz, h. manuel, a. knaus, g. hildebrandt, m. jäger, m. schubach, m. rodriguez de los santos, t. pantel, d. beule, s. mundlos, k. sperling institute for medical genetics and human genetics charite, berlin, germany the dna damaging effects of ionizing radiation are deliberately used in cancer therapy as well as feared in accidents related to nuclear technology. despite its influence on the exposed organism, irradiation was believed to have no major effect on succeeding generations, as irreparable dna damages were thought to result in cell death. recently, however, genome-wide mutation screenings in offsprings of male mice that were irradiated with high dosages showed an accumulation for certain de novo events. we therefore focused on these mutational classes in a small cohort of human individuals that were conceived while or after their fathers were exposed to high frequency radiation. in the whole genome sequences of such offsprings we could confirm de novo rates for single nucleotide variants in the order of - per base as previously reported. interestingly, however, we found de novo translocations of paternal origin as well as increased numbers of clustered de novo mutations that resemble the results from animal studies. this characteristic mutation profile might thus be used as an indicator of irradiation exposure in one of the individual's parents. from the upstream regular rb promoter. to test this hypothesis, we generated a genetic model carrying modifications in the rb promoter and in cpg using crispr/cas technology. data on the establishment of the model and first results will be presented. the gid/ctlh protein complex with its seven core protein members is conserved in all eukaryotic cells. in saccharomyces cerevisiae it functions as an ubiquitin ligase complex and regulates the metabolic switch from gluconeogenesis to glycolysis ( ) . recently, we could show that the vertebrate gid/ctlh complex also functions as an ubiquitin ligase, however substrates and exact function remain unknown ( ) . a growing number of components of the ubiquitin protein system (ups) are described to be regulators of ciliogenesis ( ). defects in such genes are considered to cause ciliopathies, genetic disorders with typical phenotypic variations in patients and model organisms ( ) . first data supports our hypothesis that the ctlh complex plays a major role in ciliogenesis, e. g. the ctlh subunit rmnd a localizes to the basal body which is a modified centriole of primary cilia in nih- t cells and rmnd knock down in xenopus laevis leads to defects in cilia formation of epidermal multiciliated cells. n. reich, m. sandbothe, r. buurman, b. schlegelberger, t. illig, b . skawran department of human genetics, hannover, germany background and aims: hepatocellular carcinoma (hcc) is characterized by genetic and epigenetic changes that lead to a deregulation of important tumor suppressors and oncogenes in a multistep process. one of these epigenetic changes is the elevated expression of histone-deacetylases (hdacs) which contribute to a transcriptional repression of certain genomic regions by remodeling the chromatin structure. thereby, not only the expression of tumor-relevant genes is affected, but also the expression of micrornas (mirnas). selected mirnas have been shown to play important roles in carcinogenesis. we aimed to identify mirnas deregulated by histone deacetylation and to understand their functional consequences in hcc tumorigenesis. methods: histone acetylation was induced by the global hdac inhibitor trichostatin a (tsa) in four hcc cell lines (hle, hlf, huh , hepg ) and two immortalized liver cell lines (thle- and thle- ) in order to identify differentially expressed mirnas and messenger rnas (mrnas) by global expression profiling. findings were validated by transfection of microrna mimics and sirna-mediated knockdown in hcc cell lines, quantitative pcr, western blotting and luciferase reporter assays. results: after hdac-inhibition, hsa-mir- - p was significantly upregulated. the mir- - p holds tumor suppressive potential and its expression is reduced in different types of tumors. one predicted target gene of mir- - p is the hepatoma-derived growth factor (hdgf). this mitogenic growth factor is highly expressed in a variety of cancers, for example in hcc, and its expression correlates with a poor prognosis, irrespective of the tumor type. hdgf is a multifunctional protein that is involved in several signaling pathways, contributing to proliferation and metastasis of cancer cells, induction of angiogenesis and inhibition of apoptosis. incubation of hcc cells with tsa or transfection with mir- - p reduced expression of hdgf. luciferase assays indicate a direct regulation of hdgf by mir- - p. moreover, expression of the death receptor fas, which is a potential downstream target of hdgf, is also regulated by the mir- - p. a translocation t( ; )(q ;q ) was not detectable in any of the investigated tissues. with the established assay we were able to provide a highly sensitive tool for the detection of the translocation t( ; )(q ;q ). however, we did not detect normal b-cells carrying this translocation. this does not exclude that such cells exist. alternatively, the growth advantage conferred by myc may promote the acquisition of secondary genetic changes. this may result in a rapid tumorigenesis, that if occurring these cells only present as full blown burkitt lymphoma. myotonic dystrophy: links to the nuclear envelope p. meinke, s. hintze, s. limmer, b. schoser friedrich-baur-institute, munich, germany myotonic dystrophies (dm) are slowly progressing multisystemic diseases with a predominant muscular dystrophy -making dm the most frequent muscular dystrophy in adulthood. dm is caused by heterozygous dna-repeat expansions in the dmpk gene (dm ) or the cnbp gene (dm ). the repeat-containing rna accumulates in ribonuclear foci and splicing factors are sequestered to these foci, resulting in abnormal regulation of alternative splicing. dm patients show overlapping phenotype presentations with progeroid laminopathies, which are caused by mutations in nuclear envelope proteins. in search for molecular signatures of this overlap, we found an enrichment of nucleoplasmic reticuli in dm and dm patient myoblasts. additional, we found an alternative splicing of the lmna gene -both effects that are associated with progeroid laminopathies. this implies possible shared pathomechanism between dm and progeroid laminopathies. retinoblastoma is a tumor of the retina occurring in young children up to the age of five. it is caused by biallelic inactivation of the tumor suppressor gene rb . we have shown that human rb is an imprinted gene and as such characterized by differential dna methylation of a cpg island (cpg ) in rb intron . cpg is not methylated on the paternal allele and acts as a promoter for the alternative rb transcript, rb -e b. it is argued that expression of rb -e b is causative of the observed skewing of regular rb expression in favor of the maternal allele. a true gametic differentially methylated region (gdmrs) is established in only one of the parental germ lines. it is supposed to be stable during early embryonic development and to be passed on to all daughter cells. we could show that cpg is free of methylation in human sperm. publicly available methylome data on oocytes revealed that cpg is fully methylated in human oocytes. these data are in agreement with cpg being a maternal methylated gdmr. we showed that the level of cpg methylation is percent in blood, as expected. however, in eight tissues of three individuals we observed a gain of methylation at cpg ranging from to percent in liver and skin, and increasing to to percent in the other tissues (heart, kidney, muscle, brain, lung and spleen). interestingly, the degree of methylation was lower in fetal tissue than in adult tissue, as determined for brain and muscle. we also observed gain of methylation at cpg in two human embryonic stem cell lines and induced pluripotent stem cells. this is consistent with the finding of complete methylation at cpg in eight different retinoblastoma cell lines. we therefore conclude that cpg is an unstable dmr. in oocytes, dna methylation of gdmrs is established by transcriptional read-through from an upstream promoter. therefore, we hypothesize that gain of dna methylation at cpg is caused by run-through transcription erozygous state (nomenclature according to hgvs; reference sequence nm_ . ). in silico-analysis by alamut (version . . ) predict the loss of the donor splice site of intron of the atm gene. cdna-analysis was performed and revealed the loss of exon of the atm by a complex activation of two kryptic splice sites. a premature stop codon was generated giving rise to a truncated protein that leads to a pathogenic variant. the results of the genetic analysis are discussed in the context of the clinical findings. identification of the underlying genetic causes of gastric cancer will give a better view of the mechanisms that contribute to the pathophysiology of the disease. gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, zentrum für diabetologie bergedorf, schwerpunktpraxis, hamburg, germany about - % of all pregnant women develop gestational diabetes mellitus (gdm) during their pregnancies and diabetes complicating pregnancy is associated with adverse maternal and perinatal outcomes, notably, risk of fetal macrosomia and neonatal hypoglycemia and development of diabetes after pregnancy. gdm is considered to result from interaction between genetic and environmental risk factors. the case of a -year old female german patient with a novel mutation in the pax gene (rare mody gene type ) as a cause of gestational diabetes mellitus is presented. we describe clinical, biochemical and genetic features of the patient, who developed gdm and gave birth to her child by cesarean section. mody genes type - were analyzed. sequencing the pax gene revealed a novel mutation in exon , pax ,c. delc, p.(leu cysfs* ); reference sequence nm_ . ), a deletion of a cytosine leading to a truncated, non-functional protein. to date, no small deletion has been detected in the pax gene. identification of the underlying genetic causes of gdm will give a better view of the mechanisms that contribute to the pathophysiology of the disease. furthermore, early identification may improve options to prevent gdm and complications for the mother and her child. the results of the genetic analysis are discussed in the context of the clinical findings. the modulation of dna methylation is highly flexible and plays an important role during cell differentiation. furthermore, the dna methylome alters considerably during aging. age related changes in the dna methylation of regulatory genes are assumed to have a major impact on carcinogenesis (teschendorff, ) . moreover, it was demonstrated that the chronological age of a human donor can be predicted with high accuracy by analyzing the dna methylation of a specific minor set of cpg loci which are aberrantly methylated during aging (horvath, ) . hence, we intended to investigate the effect of epimutations identified in different lymphoma entities in comparison with the influence of epigenetic changes in sequential b cell differentiation stages on the epigenetic age. the altered expression of the tumor suppressor mir- - p due to chromatin remodeling may play a fundamental role in hepatocarcinogenesis. we expect that histone deacetylation and putative target genes of epigenetically deregulated mir- - p can be targeted by new therapeutic agents. the microrna- family inhibits tgf-β-mediated liver cancer cell migration by targeting sox introduction: modulation of microrna expression is considered for treatment of hepatocellular carcinoma (hcc). therefore, we characterized the epigenetically regulated microrna- family (mir- a, mir- b, mir- c) with regards to its functional effects and target genes in hcc. methods: after transfection of mir- a, mir- b, and/or mir- c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. binding specificities, target genes, and regulated pathways of each microrna were identified by microarray analyses. target genes were validated by luciferase reporter assays and expression analyses in vitro. furthermore, target gene expression was analyzed in primary human hccs compared to normal liver tissue. results: tumor suppressive effects, binding specificities, target genes, and regulated pathways of mir- a and mir- b differed from those of mir- c. transfection of mir- a, mir- b, and/or mir- c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. importantly, mir- a, mir- b, and, to a lesser degree, mir- c directly targeted sox , which codes for a transcription factor involved in epithelial-mesenchymal transition and hcc metastasis, and thereby inhibited tgf-β-mediated cell migration. conclusions: this study provides detailed insights into the regulatory network of the epigenetically regulated microrna- family and, for the first time, describes distinct tumor suppressive effects and target specificities of mir- a, mir- b, and mir- c. our results indicate that particularly mir- a and mir- b may be considered for mirna replacement therapy to prevent hcc progression and metastasis. novel mutation in the atm gene and activaton of two kryptic splice sites in an year old female patient with gastric cancer gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, schwerpunktpraxis, hämatologie, onkologie und palliativmedizin, hamburg, germany, israelitisches krankenhaus, chirurgische klinik, hamburg, germany gastric cancer is a global public health concern, ranking as the third leading cause of cancer mortality. familial aggregation of gastric cancer is common in about % of the cases, and about half of these can be attributed to hereditary germline mutations. however, for most gastric cancer cases, whether genetic events contribute to cancer susceptibility remains unknown. here we present a case report of a patient with gastric cancer, a family history of breast cancer and a novel mutation leading to complex cryptic splicing in the atm gene. ngs panel sequencing and cnv/mlpa analysis of genes associated with gastric and breast cancer were performed. sequencing revealed a novel mutation in intron of the atm gene, atm,c. + g>a in an het-abstracts tions remained unidentified since positive deletion-junction pcr products could not be amplified. to identify the breakpoints of the deletions, we performed custom-designed microarray cgh analysis with a high resolution of probes located within and flanking the nahr hotspots prs and prs . the array analysis suggested that of the deletions exhibit breakpoints within prs , even although previously performed breakpoint-spanning pcrs with primers designed according to the reference sequence of the human genome have been negative in these cases. since prs exhibits high sequence diversity resulting from frequent nonallelic homologous recombination events without crossover, we surmised that haplotype diversity is responsible for the failure of the breakpoint-spanning pcrs performed with primers designed according to the reference sequence. therefore we characterized the haplotype diversity of prs in human individuals and designed deletion-junction pcr primers that facilitate the amplification of rare prs haplotypes. so far, we have identified the breakpoints of four of the type- nf deletions predicted to have been mediated by prs according to the array results. we are confident to identify further breakpoints by extending these analyses using primers suitable to amplify rare prs haplotypes. our findings indicate that the characterisation of nahr hotspots in terms of haplotype diversity is a premise to identify the breakpoints of nahr-mediated microdeletions by means of deletion-junction pcrs. p-basepi- *** array-based dna methylome analyses of primary lymphomas of the central nervous system ulm university, ulm, germany, university of cologne, cologne, germany, christian-albrechts-university kiel, kiel, germany, university hospital muenster, muenster, germany primary lymphomas of the central nervous system (pcnsl) are defined as diffuse large b-cell lymphomas (dlbcl) that are confined to the central nervous system (cns). although pcnsl cannot be distinguished from dlbcl by their morphology as well as their histology, they differ in prognostic outcome. the aim of the present study was to compare the epigenomic landscape of pcnsl and dlbcl. to this end, we analyzed the dna methylation of a total of pcnsl (cryopreserved or formalin fixed and paraffin embedded (ffpe)) using the infinium humanmethylation beadchip array (illumina) and contrasted these findings to dlbcl (kretzmer et al., ) . as controls, we used publicly available dna methylation data from a total of normal brain samples derived from different regions of the cns (gilbert et al., ; jaffe et al., ; kurscheid et al., ; mur et al., ; wockner et al., ) . after normalization of the data we performed thorough filtering and removed the random snps, all loci located on gonosomes, as well as those loci with a detection p-value > . in at least one of the samples, leading to , loci entering subsequent analyses. when comparing the dna methylation profiles of pcnsl versus dlbcl we identified differentially methylated loci (σ/σ max = . ; q < e- ). in order to remove those loci which represent a "brain signature", we compared dlbcl versus brain (σ/ σ max = . ; q < . ) based on the list of the previously identified loci. after removing this "brain signature", we ended up with loci that are differentially methylated between pcnsl and dlbcl. in a next step we wanted to make sure that the differences in methylation at these loci are not due to differences in starting material (cryopreserved versus ffpe) which is known to influence the outcome of the beadchip analysis. therefore, we compared the dna methylation profiles of five cryopreserved versus ffpe samples (derived from the same tissue samples) and identified differentially methylated loci (σ/σ max = . , q < . ). only five loci of both lists overlapped, which were subsequently removed from further analysis so that we ended up with a final list of loci which are differentially methylated between pcnsl and dlbcl. in order to analyze the biological implications of the differentially methylated loci we evaluated an enrichment of known functional groups (ku-additionally, our aim was to analyze whether entity-specific differences in the resetting of the epigenetic clock are generated during lymphomagenesis or derive from modified dna methylation in the germinal center b cells of origin. to address these issues, we performed dna methylation profiling (hum-anmethylation beadchip) of burkitt lymphoma samples (age - yrs), diffuse large b cell lymphoma samples (age - yrs) and follicular lymphoma samples (age - yrs) from the icgc mmml-seq and mmml-consortium (kretzmer et al., ) and the hematopathology section kiel as well as peripheral blood samples of healthy individuals ( - yrs) available from the same project and current publications of our group (kolarova et al., ; friemel et al., ) . in addition, we received b cell subpopulation samples ( - yrs) covering different stages of b cell differentiation that were measured in the same way (kulis et al., ; lee et al., ) . the epigenetic age of the samples was predicted using the "online age calculator" accessible at https://dnamage.genetics.ucla.edu and compared with the corresponding chronological age of the donors. in fact, the epigenetic age of peripheral blood samples of healthy donors was in high accordance with their chronological age (pearson's r . , p-value < . ) while the correlation between epigenetic and chronological age of sequential b cell differentiation stages was slightly lower (pearson's r . , p-value< . ). in contrast, the predicted epigenetic age of the burkitt lymphoma samples was significantly higher than the corresponding chronological age. this deviation may be interpreted as "epigenetic pre-aging". nevertheless, the epigenetic age of diffuse large b cell lymphomas and follicular lymphomas tended to be less affected. in conclusion, we found significant epigenetic pre-aging in burkitt lymphoma samples that seems to be induced during lymphomagenesis and does not derive from altered dna methylation patterns in the germinal center b cells of origin. moreover, no significant shift of the epigenetic age was observed for the other lymphoma entities, healthy blood samples and b cells of sequential differentiation stages. identification of type- nf deletion breakpoints mediated by rare prs haplotypes a. summerer , , m. hillmer , , v. f. mautner , , l. messiaen , , h. institute of human genetics, ulm, germany, university of ulm, ulm, germany, department of neurology, hamburg, germany, university hospital hamburg eppendorf, hamburg, germany, department of genetics, birmingham, usa, university of alabama at birmingham, birmingham, usa neurofibromatosis type (nf ) is a hereditary cancer syndrome with an incidence of in . in % of all nf patients, large deletions encompassing the nf gene and its flanking regions are causing the disease. the majority of all large nf deletions are of type- ; they encompass . -mb and are mediated by nonallelic homologous recombination (nahr) with crossover. the breakpoints of type- deletions are located within the lowcopy repeats nf -repa and nf -repc which exhibit high sequence homology to one another. previous studies suggested that type- deletion breakpoints cluster within the paralogous recombination sites prs and prs spanning -kb and -kb, respectively. in our present study, we investigated patients with type- nf deletions using long-range pcrs to detect breakpoints located within prs or prs . according to these analyses, ( %) of the breakpoints are located within prs and ( . %) in prs . however, ( . %) of the type- deletions were not positive for these deletion-junction pcrs. we surmised that some of these deletions may have breakpoints within the -kb region located between prs and prs . this -kb region also exhibits high sequence homologoy between the nf -reps which is a prerequisite for nahr. indeed, of the type- nf deletions exhibited breakpoints within this -kb region as determined by the analysis of seven overlapping deletion-junction pcrs. however, the breakpoints of dele-esophageal adenocarcinoma (ea) represents one of the most rapidly increasing cancer types in high-income countries. barrett's esophagus (be) is a premalignant precursor of ea and has an estimated prevalence of - % in the population. however, only . to . % of be patients develop ea. within an international consortium, we carried out a gwas meta-analysis in be patients, ae patients and . controls (gharahkhani et al., lancet oncology, ) . in a comprised be/ae analysis, we identified genome-wide significant risk loci, of which seven were previously unreported. the strongest associated new risk variant was identified for rs (p = . × - ), which maps within intron of the cftr gene. cftr encodes a protein that functions as a chloride channel and that is mutated in patients with cystic fibrosis (cf). mutations in cf lead to abnormal viscous secretions with altered chemical composition, resulting in dysfunction of the respiratory system and the gastrointestinal tract. the most common cf mutation is Δf , a deletion of three nucleotides in cftr that results in the loss of a single codon for phenylalanine on protein position . interestingly, cf patients show a highly increased incidence of gastroesophageal reflux, which represents the major risk factor for be and ae. in view of the phenotypic overlap for gastroesophageal reflux and cystic fibrosis, and for gastroesophageal reflux and both be and ae, combined with the association of cftr risk variants in patients with be and ae, it seems plausible that a common pathophysiological mechanism is triggered by cftr. in order to test this hypothesis, we analyzed the association of Δf in a european case-control cohort with be and ae patients. for this, we performed a genotyping assay of Δf in be patients, ae patients and controls. we could not observe a significant association (p = . ). this might be (i) due to insufficient sample power or (ii) due to the fact, that not Δf but other genetic variants at the locus might explain the underlying functional mechanism of the association. fine mapping of all genetic variation at the cftr locus and exten-lis et al., ). remarkably, cpg loci that are differentially methylated during normal b-cell maturation were significantly depleted. in turn we saw an enrichment of loci located in heterochromatin. in summary, we detected more than loci that are differentially methylated between pcnsl and dlbcl, which do not play a functional role in normal b-cell differentiation. replication study of gwas-identified genetic modifiers of age at huntington's disease onset although there is a strong correlation between cag repeat length and age at onset (ao) of motor symptoms, individual huntington disease (hd) patients may differ dramatically in onset age and disease manifestations despite similar cag repeat lengths. since the modifier variations described so far only account for a small fraction of the heritable contribution to the ao, the identification of loci and genes using genome-wide methods appears highly promising. against the background of incomplete understanding of the hd disease pathophysiology, the hypothesis-free approach of gwas offers an ideal starting point for the search of modifier genes. recently, a combined analysis of all gwa data to hd modifiers identified different loci with genome-wide significant signals for association to residual age at motor onset [gem-h. consortium]. interestingly, none of the most significant association signals and none of the trending snps in the european gwa analysis corresponded to any previously suggested candidate modifier genes. in order to be able to better assess these data, we tried to replicate the top ten associated gwas variants in a comprehensive cohort of german hd patients. we only found modest association with one of the top ranked snps (rs ), all remaining variations showed no correlation with the ao. this inconsistency highlights once again the difficulties of modifier searching in hd or any other monogenic disorder, which faces the same challenges as the genetic characterization of complex disorders. with an incidence of . - . / . malignant tumors of the thymus are a rather rare type of cancer. here we report the case of a man of german descent, who presented with a thymoma at age . in the pathological report the thymus tumor was described as an extremely unusual thymoma with partial loss of keratin and massive proliferation of myoid cells. it was subsumed to a primary thymic, partially epithelial neoplasia, resembling an uncommon b /b -thymoma. after the patient's death his widow looked for genetic advice concerning the risk of disease for her children. detailed personal and familial history brought up surprising information: thymoma was one of four cancers in our patient. he developed adenocarcinoma of the colon at age , squamous cell cancer of the nose/upper lip at years and in addition current cancer staging revealed a papillary renal cell carcinoma. according to family history his father and his uncle developed colon cancer with and years, the son of this uncle was diagnosed with colon cancer at age . this cousin of the propositus was referred to genetic counseling, because of msi-high-status and loss of mlh and psm in immunohistochemistry. he was found to have a deleterious mutation in exon of mlh gene (c. c>a, p.tyr stop) resulting in a premature termination of mlh -protein. our patient has never been tested for hnpcc. however a post mortem performed immunhistochemical examination of thymic cancer cells revealed an almost complete loss of mlh nuclear expression suggesting the presence of a mlh germline mutation and indicated hnpcc. considering the loss of mlh in tumor cells it is more than likely that the development of thymoma was the consequence of deficient dna mismatch-repair. there have been reports of rare tumors in hnpcc families in the last years (i. e. clear cell renal carcinoma and uterine sarcoma). pande et al. reported one case of thymoma in their registration of cancer occurrences in mutations carries from hnpcc families [ ] . our case emphasizes the importance of detailed family history and contributes to the discussion of widening the inclusion criteria for genetic counseling and testing for hnpcc. to this day the revised criteria of bethesda are used to identify families at risk. we propose that the established criteria have to be revised and rare tumors should be included. unknown partner genes in leukemias with rare translocations can be identified using targeted rna sequencing c. haferlach, n. nadarajah, m. meggendorfer, n. dicht, a. stengel, w. kern, t. haferlach mll, munic, germany in hematological malignancies fusion genes play an important role and function as therapeutic targets, impressively shown for e. g. bcr-abl and etv -pdgfrb. thus, the identification of fusion genes is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. the aim of this study was to test the value of targeted rna sequencing in a routine diagnostic work up. cases were selected harboring rearrangements of kmt a (n = ), runx (n = ), etv (n = ), pdgfrb (n = ), npm (n = ), rara (n = ) and jak (n = ) identified by chromosome banding (cba) and fish analyses. in none of the cases the partner gene could be identified using standard methods. targeted rna sequencing was performed using the trusight rna fusion panel (illumina, san diego, ca) consisting of probes covering genes known to be involved in gene fusions. this assay allows the capture of all targeted transcripts. sequencing was performed on nextseq (illumina, san diego, ca). analysis was performed with the rna-seq alignment app (basespace sequence hub) using star for alignment and manta for gene fusion calling with default parameters (illumina). sive functional analysis are needed to find the causal variant that explains how the cftr locus interferes with the pathomechanism of be and ae. a recent functional study indicated cftr as a tumor suppressor gene in murine and human intestinal cancer, providing further evidence for cftr as a true disease gene for be and ae. background: it has long been established that mutations in brca predispose for pancreatic adenocarcinoma with brca germline mutations identified in - % of familial pancreatic cancer cases. consequently, screening for pancreatic cancer has been recommended for mutation carriers with an affected first-degree relative since early detection has been shown to significantly improve -year survival from - % to %. for brca mutations, however, relevance in pancreatic tumorigenesis is still being discussed with several studies questioning an elevated risk of pancreatic cancer in families with brca mutations while others are suggesting that brca may also play an important role in predisposing to pancreatic cancer. clinical screening for pancreatic cancer commonly remains unavailable to brca mutation carriers and it has even been questioned whether brca should be analyzed in familial pancreatic cancer at all. clinical report: here we report on a year old woman with metastatic pancreatic cancer whose sister had died of pancreatic cancer at years of age. in this family we identified a pathogenic brca -germline mutation (brca : nm_ . :c. dupt,p.(leu phefs* )) by next-generation sequencing using a -gene panel. the index patients' tumor was available for genetic analysis and showed loss-of heterozygosity for brca . this strongly suggests the brca mutation to be causative of the pancreatic cancer development in this patient. when the family was first introduced to genetic counselling there was no evidence of breast-or ovarian cancer in any relatives. only after identification of the mutation did the index person reach out to distant family members and it was thereby revealed that a distant branch of the family had independently been counselled for hereditary breast and ovarian cancer. in this part of the family, however, there had not been any cases of pancreatic cancer. subsequent predictive testing was offered to healthy family members and further mutation carriers could be identified. two women were referred to breast cancer screening. additionally, the mutation was identified in a relative with recurrent metastatic breast cancer at the age of years. for her and the index patient parp-inhibition therapy thus became a possible further treatment option. conclusions: in conclusion we propose next-generation sequencing approaches including the analysis of brca to be used in familial pancreatic cancer. we also argue that brca mutation carriers with pancreatic cancer cases in their family should be offered the same screening program as brca mutation carriers. within the framework of a study this could allow for more precise risk stratification in the future. contralateral dcis is unknown. only the male breast carcinoma was herceptin receptor positive, all other breast carcinomas of the chek mutation carriers were her negative. among our chek positive families we noticed the association with chek mutation and female breast cancer. we observed a contralateral breast cancer, male breast cancer and other tumors in our families as well. the majority of the observed breast cancers was estrogen and progesterone receptor positive and herceptin negative. while benign uterine smooth muscle tumors are among the most frequent human symptomatic tumors, their malignant or borderline lesions are only rare findings. both lesions can show somatic copy number alterations, but their patterns differ, thus constituting helpful diagnostic tools. aimed at an advanced classification of the lesions we have performed molecular inversion probe array analyses of these tumors. besides complex patterns of genomic alterations seen in nearly all cases, two of the lesions presented with copy number neutral uniparental disomies i. e. normal copy numbers with an apparent monoallelic origin. in one case, an upd of part of the long arm of chromosome was detected in a uterine leiomyosarcoma. the tumor showed genetic heterogeneity with gains and losses. in addition, the . mb segment located at q . -q . was clearly of monoallelic origin throughout all cells investigated. all other genetic alterations were restricted only to part of the cells of the sample thus reflecting the presence of tumor cells as well as normal bystander cells which in general characterizes mutations that had arisen during tumor development. in contrast, the upd that was detected in all examined cells clearly suggests its germline occurrence. the second tumor was a leiomyoma-variant of the type with bizarre nuclei. again, besides gains and losses an apparent germline upd was found that covered a . mb segment on chromosomal segment q . . upd for even the whole arm of chromosome repeatedly has been reported not to coincide with phenotypic manifestations. nevertheless, the question arises whether or not the observed upds might be related to a familiar predisposition for uterine muscle tumors. of note, as a result of genome-wide association studies snps on q recurrently have been found to be significantly associated with fibroid development. triple negativity is an independent predictor of germline mutations in breast cancer predisposing genes breast cancer is the most common cancer in women. - % of all tumors are triple-negative breast cancers (tnbc) lacking expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor . so far, tnbc have been mainly associated with mutations in brca , although recent studies also found mutations in other breast cancer susceptibility genes. a brca / -centered perspective thus may ignore the significance of other predisposing genes, whose relevance appears obvious as dna damage repair by homologous recombination is a complex process involving many proteins. in / cases with rearrangements involving kmt a (n = ), runx (n = ), etv (n = ), pdfgrb (n = ), rara (n = ), npm (n = ) or jak (n = ) the partner genes were identified. these were in kmt a rearranged cases: mllt (n = ), mllt (n = ), itpr , flnc, asxl , dcp b, maml and arhgef . in runx translocated cases partner genes were plag (n = ), prdm , mecom, zfpm , man a , n amt , and kiaa l. prdm , mecom and zfpm have previously been described in the literature as runx partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements. the other identified partner genes have not been described so far. interestingly, prdm , mecom, zfpm and the newly identified plag are all members of the c h type zinc finger gene family. partner genes identified in etv rearranged cases were: abl , ccdc , clptm l, erg, foxo and cflar-as . wdr , zbtb , nfia and mprip were identified as partner genes of pdgfrb and rpp in an npm -translocated aml. in an all patient a jak -ppfibp fusion was identified leading to classification as a bcr-abl -like all. in an apl patient showing an ins( ; ) (q ;q q ) a zbtb -rara fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. further in a case with t( ; )(q ;q ) an irf bp -rara fusion was detected. conclusions: targeted rna sequencing was able to characterize rare gene fusions and provided the basis for the design of rt-pcr based assays for monitoring mrd. targetable genetic aberrations were identified, which were not detected by cba enabling more individualized treatment. targeted rna sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques. female carriers of a pathogenic mutation in the chek gene are reported to have a life time risk of about - % to develop breast cancer. there is evidence for increased risks for contralateral breast cancer, male breast carcinoma and other types of tumors. in addition to well-known mutation chek :c. del, other pathologic mutations are being identified in the gene due to the inclusion of the gene in most breast cancer gene panels for dna testing. between - the center for hereditary breast and ovarian cancer regensburg cares for families with pathogenic or probably pathogenic mutations in the chek gene affecting nine female patients and one male patient ( × c. del, × deletion of exon , and × variants considered as likely pathogenic: c. g> c, c. c> t, c. a> c). the mean age of diagnosis of breast cancer (both sexes) was . years (range - years). the patient with the deletion of exon was first diagnosed at years of age and developed a contralateral breast carcinoma (dcis) at years of age. the male patient was diagnosed with breast cancer at years of age and at years with a renal carcinoma. one patient was diagnosed with a papillary thyroid carcinoma at age years and developed breast cancer with years. in out of the families, breast carcinoma diagnosed with . years on average, was reported in the family history. in addition, there were additional malignancies such as prostatic carcinoma, thyroid carcinoma, colorectal cancer, gastric carcinoma, leukemia, cervical carcinoma and malignant melanoma. none of the affected family members was tested for the respective chek mutation. the tumors with an initial diagnosis at years and years were estrogen-receptor-negative and progesterone-receptor-negative. the other of the breast cancers were positive for the estrogen receptor, of the tumors were positive for the progesterone receptor. the receptor status of the abstracts it has been shown that in d culture hescs can be differentiated into neural retina containing organoids. we established this differentiation schedule and started comparative differentiation of wildtype h hescs and the rb null derivative (g , rb mt/mt ) into neural retina. during the first weeks of differentiation into neural retina organoids generated from the rb mt/mt hescs have a smaller diameter and thinner retina layer compared to wildtype organoids. however, during the time-course the mutant organoids began to catch up. thus, at later stages no difference in size and thickness could be observed anymore. comparative immunostainings of cryosections at d show no difference in expression of the markers pax and sox between the wildtype and mutant hescs. further comparative immunostainings for markers specific for neural retina like e. g. rx and vsx at d and d are ongoing and will be presented. exome sequencing identified potential causative candidate genes for unexplained cowden syndrome purpose: cowden syndrome (cs) is a cancer predisposition syndrome characterized by the occurrence of breast cancer, epithelial thyroid cancer, endometrial carcinoma and various other findings such as mucocutaneous lesions and macrocephaly. cs belongs to the pten hamartoma tumor syndrome (phts) primarily associated with germline mutations in pten. in recent years, germline mutations in additional genes (sdhb, sdhc, sdhd, pik ca, akt , sec b) have been described in few patients; however, to date, in - % of patients meeting clinical criteria for cs the underlying cause remains unclear. methods: to uncover predisposing causative genes, the exomes of clinically well characterized, mutation negative patients with suspected cs were sequenced (illumina hiseq) using leukocyte dna. assuming a monogenic disease model, the called variants were filtered for rare (minor allele frequency ≤ % for homozygous/compound heterozygous variants and ≤ . % for heterozygous variants according to dbsnp, evs, and exac), truncating (nonsense, frameshift, highly conserved splice sites), and missense germline variants (predicted to be pathogenic by at least / in-silico tools). for data analysis and variant filtering the gatk software and the cartagenia bench lab ngs software were applied. all candidate genes were included in a pathway analysis (ingenuity). in a first preliminary analysis, we focused on known cancer genes and genes interacting with pten. results: after stringent filtering steps, comparison with large datasets from population-based controls, and detailed manual inspection to exclude artifacts, genes were affected by presumed biallelic variants ( homozygous and putative compound-heterozygous), one of these is a known cancer gene (cbfa t ); in genes biallelic variants were found in - patients. heterozygous variants were found in genes in - patients, but none of these are known cancer genes. in genes, heterozygous truncating mutations occurred in only one patient, of these are cancer genes (msh , wrn, kdm a, pml) . the phenotype of the patient with a msh frameshift deletion fulfilled key features of cs (early-onset metachronous papillary thyroid cancer, breast cancer, endometrial and colorectal cancer), however, the tumor spectrum is partly compatible with lynch syndrome/ hnpcc. examination of the colorectal cancer demonstrated microsatellite instability and a loss of msh protein expression. the pathway analysis of the remaining candidate genes identified several interacting partners of pten (grhl , ehhadh, cstf ). conclusions: preliminary data indicate that exome sequencing might identify potentially relevant causative genes for cs, some of which are recurrently mutated. the present work-up consists of the inclusion of further non-cancer genes, validation of variants by sanger sequencing, testing of to determine the prevalence of mutations we performed panel-based germline mutation testing of high and low-moderate penetrance breast cancer susceptibility genes (brca , brca , atm, cdh , chek , nbn, palb , rad c, rad d and tp ) in consecutive individuals affected with tnbc unselected for age at diagnosis or breast and ovarian cancer family history. age at diagnosis ranged from to years with an average of . and a median of years. in women ( . %) we detected a pathogenic mutation, with a higher frequency ( . %) in the group manifesting cancer before years. deleterious brca mutations occurred in . % of tnbc patients, predominantly frameshifting ( / , . %). the most frequent, both among brca mutations and in total, were the founder mutations c. dupc and c. _ delag. deleterious brca mutations occurred in . % of patients, all but one (c. du-pa) being unique. while no mutations were found in cdh and tp , mutations ( %) were detected in one of the six other predisposition genes (palb , chek , atm, nbn, rad c, rad d). no individual presented more than one mutation. almost half of all deleterious mutations ( . %) were detected in very young women aged years or less. the median age at diagnosis was significantly younger for brca ( years) and brca ( . years) carriers compared to patients without a mutation (p = . e- ; mann-whitney) or compared to non-brca / mutation carriers (p = . ). in contrast, patients with non-brca / mutations were not significantly younger than mutation negative women (p = . ). interestingly, family history had an independent influence on age at diagnosis. taken as a whole, women with family history had a median age at diagnosis years earlier than those without (p = . ). this difference was lost in mutation carriers while it remained in cases without mutation. in summary, our data confirm and expand previous studies of a high frequency of germline mutations in genes associated with ineffective repair of dna damage by homologous recombination in women with tnbcs. many of these women would go untested with current restrictive criteria. in order that each patient receives therapies tailored to her genetic status, gene panel based mutation testing should be offered to all women diagnosed with tnbc, irrespective of age at diagnosis or family history. p-cancg- *** neural retina differentiation of hescs as an in vitro model for retinoblastoma d. kanber, m. hiber, d. lohmann, l. steenpass institute of human genetics, university hospital essen, university duisburg-essen, essen, germany retinoblastoma is the most common eye tumor of early childhood. inactivation of both alleles of the retinoblastoma gene (rb ) results in the development of retinoblastoma. our aim is to establish a human cell-based model for retinoblastoma. using the crispr/cas system we have generated human embryonic stem cells (hescs) carrying a mutation either on one or both rb alleles. all the detected mutations are located in exon of the rb gene and close to the splice donor site of this exon. analyses on dna, rna and protein level were performed for three mutant and one double-mutant clone. the following genotypes were identified by deep sequencing (nm_ . (rb _v )): clone c , c. _ del, heterozygous; clones c and g , c. _ del, heterozygous; clone g , c.[ _ del; c. _ dup] (complex mutation on one allele), homozygous (loss of heterozygosity). the mutations of all four clones result in a premature stop codon in exon . on rna level we detected expression of mutant rb transcripts reflecting the genotype in all clones and an additional mutant rb transcript with skipping of exon in three clones. as the heterozygous clones also showed expression of the wildtype rb transcript, rb protein (prb) could be detected for these clones (c , c , g ) by western blot analysis. however, the double-mutant clone g showed no expression of prb. so far, we have characterized heterozygous and one homozygous clone. another three double-mutant clones are under investigation. the recurrent germline missense mutation g e in the hoxb gene has been demonstrated to predispose to hereditary prostate cancer (prca), despite the underlying pathogenic mechanism is not yet understood. molecular examination of a first set of g e positive tumors sought for somatic characteristics, and suggested that oncogenic ets gene fusions may appear at unusually low frequencies as compared to the general prevalence of ets fusions in prca ( % vs approx. %). hypothesizing that hoxb could predispose to ets fusion negative prca, we have analyzed cases from three european ancestry populations (finland, germany and us) for the coincidence of hoxb g e and the most common ets fusion, tmprss :erg, in corresponding tumor samples. while the prevalence of tmprss :erg fusions was similar among the three study groups (range: . - . %), the frequency of g e genotypes differed markedly between us ( . %), german ( . %) and finnish samples ( . %). despite the expected frequency gradient among study populations, all subsamples showed a strong enrichment of g e mutation carriers among tmprss :erg fusion negative cases as compared to fusion positive cases (center adjusted or = . ; %ci = . - . ; p = . ). consistent with the previous study, the crude frequency of the tmprss :erg fusion in hoxb g e carriers was . % (range . % - . %). examination of disease characteristics highlighted age at diagnosis to be associated with tmprss :erg negative status (per year or = . , p = , ) and by trend, also with the presence of the g e germline variant (per year or = . , p = . ). within the subtype of tmprss :erg fusion negative carcinoma carriers of g e were diagnosed . years earlier as compared to non-carriers ( . ± . years versus . ± . years, p = . ). in conclusion, this study demonstrated a significant tumor subtype specific association for hoxb g e mutation carriers having a higher frequency of tmprss :erg fusion negative prca. meta-analyses from case control comparisons suggested that subtype specific risk of hoxb g e for tmprss :erg negative prca could be as high as or = . , as compared to or = . , when prca is regarded as one entity regardless of fusion status. finally, although tmprss :erg negative prca is usually known to be associated with later ages of diagnoses, hoxb mutations may indicate a subgroup of earlier onset cases within the fusion negative entity. relatives to determine the phase of assumed biallelic variants and segregation with the phenotype where applicable. with to cases in million inhabitants per year adrenocortical cancer (acc) is a rare disease. due to often late diagnosis and limited treatment options prognosis for patients are poor with a year overall survival rate of to %. though knowledge about molecular genetic events in acc increased over the last few years no reliable molecular prognostic factors, no effective targeted cancer therapy and no personalized treatment approach has emerged to date. that's why we intend to establish a reliable method to define a molecular signature of accs that could be used for a prognostic classification of adrenocortical cancers, for planning an individualized therapeutic approach and for the identification of known or potential targetable molecular events in the single patient. in a retrospective study dna from acc and matched blood samples is sequenced to detect somatic single nucleotide variants (snv), small insertions and deletions (indel) and copy number alterations (cnv). sequencing data are then compared to clinical data e. g. tumor stage, resection status, ki -index and time of progression free and overall survival to define molecular prognostic factors. target enrichment of genes that are known to be associated with different entities of cancer is performed with the human comprehensive cancer panel (qiagen) and sequenced on a nextseq (illumina). data are analysed with gensearchngs (phenosystems). znrf , a gene that was also described to be involved in the development and the progression of acc a few years ago, is sequenced separately with sanger and analysed with gensearch (phenosystems). to date tumor samples and matched blood samples from patients were analysed. one or more tumors comprise one or more snvs or small indels in of genes of the panel and in znrf . snvs and small indels are most often found in tp , ctnnb and znrf with frequencies of %, % and % respectively. in of genes cnvs -duplications and deletions -occur. cdk is duplicated in over % of the cohort. mdm gains are found in over %. one can also find three types of cn patterns: a quiet type with low number of copy alterations, a noisy one with high number of chromosomal breakages and a chromosomal one with high frequency of alterations of chromosomal arms. while no correlation between snvs and small indels and clinical outcome could be found so far, cn patterns of the accs seem to correlate with progression free survival and overall survival. patients with a noisy cn pattern have a shorter progression free and overall survival than patients with chromosomal and quiet type. though tendencies in the correlation of molecular markers and prognosis for patients suffering acc can be recognized, further samples need to be analysed to confirm the results. it is planned to sequence another tumor samples and matched blood samples for this retrospective study and to validate the results in a prospective study with another patients. expression of mir- a- p and mir- - p was analysed in serum samples by quantitative pcr. the cohort of gcnis patients consisted of patients with a solitary testicle, who had undergone orchiectomy for contralateral tgct, and patients with two testicles, one of which with gcnis, but no concurrent tgct. twenty men with non-malignant testicular disease served as controls. additionally, in situ hybridisation (ish) with a probe against mir- a- p was performed on four testicular biopsy specimens known to harbour gcnis. sequential step sections of the corresponding tissue blocks were analysed immunohistochemically, using oct antibody to visualise gcnis. the median expression value of mir- a- p in gcnis-patients was . (interquartile range [iqr] = . ) which is significantly higher than the median expression of . (iqr = . ) in controls. both of the two gcnis subgroups had significantly higher mir- a- p levels than controls, with a median expression of . (iqr = . ) and . (iqr = . ), respectively. regarding mir- - p expression, there were no significant differences between gcnis and controls. using a relative quantity of as a cut-off value, the mir- a- p was able to detect . % ( % confidence interval [ % ci] = . - . %) of gcnis, while only % ( % ci = . - . %) of the controls were positive. in the subgroup with previous tgct . % ( % ci = . - . %) of gcnis could be detected and in the subgroup without previous tumour the rate was . % ( % ci = . - . %). the detection rates for all gcnis and for both subgroups were significantly higher than for the controls. ish staining demonstrated the expression of mir- a- p in gcnis cells in two of the four cases. in conclusion, this study indicates a new and minimal-invasive way of diagnosing gcnis by measuring serum levels of mir a- p. this approach is endorsed by the demonstration of mir a- p in gcnis cells by ish staining. however, the sensitivity is still low and thus, the method certainly needs refinement possibly by applying a panel of additional mi-crornas. nonetheless, measuring serum levels of mir a- p may constitute a valuable aid in clinical assessment of men afflicted with high-risk factors of tgct. p-cancg- *** the mir- a- p is a highly specific and sensitive serum-based marker for the diagnosis and follow-up of testicular germ cell tumours testicular germ cell tumours (tgct) are a paradigm of curable malignancies. clinical management largely relies on measuring the serum biomarkers. inopportunely, the markers beta-hcg, afp and ldh are only elevated in about % of patients. therefore, micrornas of the clusters mir- - and mir- / were proposed as novel serum-based markers. we evaluated four of the candidate mirnas (mir- a- p, mir- - p, mir- - p and mir - p) with regard to their usefulness as tgct markers. overall, serum samples from tgct-patients and from controls were analysed using quantitative pcr. the first consecutive patients and controls were analysed for all four mirnas. after roc-analysis only the marker with the greatest discriminative power was studied further. the decline of mirna expression after orchiectomy was quantified in cases and in metastasized cases the marker was analysed repeatedly during the course of chemotherapy. additionally cases with relapsing disease were studied. the mir- a- p featured the highest discriminative power (area under the curve: . ; % confidence interval [ % ci]: . - . ). in the entire cohort, patients could be distinguished from controls with a sensitivity of . % ( % ci: . - . %) and a specificity of . % ( % ci: neuroendocrine tumor of the adrenal gland: an unusual manifestation of tsc c. müller-hofstede, j. horvath, b. dworniczak, p. wieacker institute of human genetics, university of münster, germany we report on a young woman asking for the recurrence risk of the neuroendocrine tumor of her mother deceased at the age of . her mother clinically presented because of therapy-resistent hypertonia, dyspnoe, progressive edema in the legs and face and a caput medusae. mri scan revealed a tumor ( × cm) in the right adrenal gland with lymph node metastases compressing the v. cava inferior and synchronous metastases in lung and liver. laboratory examinations showed highly elevated levels of cortisol and adrenocorticotropin (acth). cerebral mri was normal suggesting an ectopic acth secretion by a non-pituitary tumor. histologically, an undifferentiated, largely necrotic tumor was described so that the neuroendocrine nature of the tumor could not be proven. she died within three weeks after diagnosis. on suspicion of multiple endocrine neoplasia type we initially performed a sequence analysis of men on tumor dna by next generation sequencing without detection of a pathogenic mutation. thereupon the molecular genetic panel analysis (nf , ret, sdhb-d,tmem , tsc , tsc , vhl) uncovered the heterozygous mutation c. c>t (p.arg trp) in the gene tsc . this mutation is already described as pathogenic (hu et al. ). in the tumor dna the allele frequency of the normal allele mounted up to %, whereas the allele frequency of the mutant allele came to % pointing to a loss of heterozygosity (loh). the mutation was confirmed by sanger sequencing. taken all together, we assumed, that the mutation in the gene tsc represents a germline mutation. mutations in the suppressor genes tsc and tsc cause tuberous sclerosis, an autosomal-dominant disorder, resulting in hamartomatous tumors in the heart, brain, kidneys, skin and other organs. once in a while it is discussed whether neuroendocrine tumors (nets) represent a characteristic of tsc. there are some case reports describing nets in the context of tsc, but mainly in connection with nets of the pancreas (e. g. insulinoma) or the pituitary. to the best of our knowledge there exists only one case report of a bronchial carcinoid as a result of a germline mutation in tsc (dworakowska et al. ) and no description of net of the adrenal gland due to a mutation in tsc or tsc . ngs provides the opportunity of wide-spread testing, even post-mortem, in order to get clarification for the descendants. although in our case we could not distinguish if the mutation detected represents a germline mutation or a somatic mutation, we were able to offer a predictive testing to the daughter and other family members. we report on a rare case of net of the adrenal gland because of mutation in the gene tsc . this case illustrates that in the differential diagnosis of nets, tsc genes should also be considered. germ cell neoplasia in situ (gcnis) is the precursor lesion of testicular germ cell tumours (tgct). if detected clinically, this lesion may herald a pending tgct. unfortunately, the only way of diagnosing gcnis is by testicular biopsy and subsequent immunohistochemical examination. therefore, non-invasive methods of diagnosis are required. mirnas of the mir- - and mir- / cluster had been suggested as serum biomarkers of full-blown tgcts. we aimed to explore the utility of these mirnas for the detection of the pre-invasive stage of tgcts and we looked to the expression of two mirnas in serum samples of gcnis patients. psmc ip located on chromosome q is a putative tumor suppressor gene that encodes for the nuclear psmc interacting protein. the protein functions as coactivator of steroid hormone mediated gene expression and is important for rad and dmc -mediated homologous recombination during dna repair of double-strand breaks. recently germline variants in psmc ip, also known as gt , tbip, and hop , have been identified with low frequency in early onset familial breast and ovarian cancer (hboc) patients and in a patient with apparently sporadic early onset breast cancer. somatic variants in psmc ip are frequently observed in breast, ovarian, and fallopian tube cancers. in this study, we analyzed a cohort of brca / mutation-negative hboc (n = ) or early onset sporadic breast cancer patients (n = ) for variants in psmc ip. we identified seven different heterozygous variants in out of index patients: c.- g>a (rs ); c.- t>a (rs ); c.- a>t (rs ); c.- c>g (rs ); c. g>a p.(trp *); c. + g>c (rs ); c.* g>a. these variants were not listed or at very low frequency (< %) in the exac database. carriers of psmc ip germline variants were mostly ( / ) affected by early onset breast cancer (median age of onset years). for three out of seven different variants (c.- g>a, c. g>a, and c* a>g), a possible impact on psmc ip expression or function was observed. the stop mutation c. g>a p.(trp *) was found in two sisters, which were both diagnosed with unilateral breast cancer at age . the premature stop codon is located within the dna-binding domain of psmc ip and is predicted to induce nonsense-mediated mrna decay (nmd). remarkably, c.- g>a was already described in familial breast and ovarian cancer, and was found once in this study in a female that developed unilateral breast cancer at the age of years. the variant c.- g>a (rs ) was shown to induce a slightly, albeit significant decrease of reporter gene expression. the c. *g>a variant was identified in a woman diagnosed with unilateral breast cancer at the age of years. luciferase reporter assays indicated an impaired effect of c. *g>a on microrna binding. germline variants in psmc ip are present in breast and ovarian cancer families. whether mutated psmc ip is a new risk factor for early onset breast/ovarian cancer in families with hboc and/or apparently sporadic early onset breast cancer remains to be shown. . - . %) with this marker. in patients without metastases the mir- a- p expression declined significantly after surgery. in metastasized cases the levels dropped sharply after chemotherapy. all of the relapses had elevated mir-levels, and expression decreased upon chemotherapy. mir- a- p has significantly higher sensitivity than each one of the classical tgct markers and than a combined panel of beta-hcg, afp and ldh ( . % vs . %). in non-metastasised seminoma the mir- a- p expression depended significantly on tumour size. mir- a- p is highly sensitive and specific for tgct. it correlates with the stage of disease and with treatment effects and it therefore fulfils the prerequisites of a valuable serum-based biomarker. the significant association with tumour bulk in localised disease provides evidence for the tgct being the primary source of mir expression. the sensitivity of mir- a- p surpasses that of classical tgct markers by far, and thus it may become the new gold standard for serum diagnostics of tgct in the coming years. co-occurrence of radioulnar synostosis and amegakaryocytic thrombocytopenia (rusat) was initially described as an inherited thrombocytopenia syndrome that is caused by a mutation in hoxa . in three simplex patients, de novo missense mutations in mecom were reported as an alternative origin of the disease (rusat ). mecom, identified as a common ecotropic viral integration site (evi ) in murine myeloid leukemia, is known as a key transcriptional regulator in hematopoiesis and sporadic myeloid leukemia. we report here on a novel mecom mutation cys gly (uniprot q - ) identified by whole exome sequencing in a family with rusat, hearing impairment, hand dysmorphisms, and patellar hypoplasia in four patients spanning three generations. notably, two of four affected individuals in our family developed a myeloid malignancy. the novel mecom missense mutation cys gly affects a heavily conserved cysteine residue in c h -zinc finger motif in the c-terminal zinc finger domain of mecom. this residue is crucial for the tetrahedral coordination of a zinc ion stabilizing the zinc finger conformation and thus, is essential for dna binding of the c-terminal zinc finger domain. our findings reconfirm the causality of mecom mutations and indicate that mecom mutations also need to be considered in familial rusat patients. in addition, we report for the first time that mecom germline mutations targeting the c-terminal zinc finger domain are associated with an increased risk for myeloid malignancies. this extends the rusat -associated phenotype and proposes that mecom germline mutations can cause a genetic predisposition to myeloid malignancy. (z., b. and s., d. contributed equally to this work) many genes that harbor rare mutations which entail a medium or high risk for breast cancer (bc) belong to dna double strand repair and have been identified by linkage analysis or by sequencing of candidate genes in bc families. in addition, a considerable number of common but low risk germline variants have been found in genome wide association studies. however, these predisposing factors yet only explain a fraction of bc cases. with the intention to identify low frequency variants conferring an intermediate bc risk, we performed an association study using a candidate gene approach and testing dna repair capacity with the micronucleus test (mnt) as an additional second phenotype. rs in the slx /fancp gene showed an association with bc that was pronounced in younger cases and was confirmed in a verification cohort (combined analysis of , cases, controls: or = . ( . - . ) , p = . for cases ≤ years). genotyping additional snps and imputation revealed a specific european haplotype of ca. kb length that spans slx and adjacent genes. it is tagged by the observed mutual dependence of the two phenotypes allowed a considerably improved interpretation of the results: (i) the unknown causal variant on the haplotype can be assumed to be present mostly in cases, indicating a rare variant with a rather strong effect; (ii) using this information on the two phenotypes in the association between the mnt results and bc improved considerably the identification of the specific risk among cases (< years) who carried the haplotype. roc curves for bc depending on mnt results revealed that the stratification on carriers of the haplotype increased the auc from . (p = . ) to . (p = . ). both associations can be best explained by a risk variant carried by a fraction of the haplotypes that is enriched in early onset bc cases. slx is the only gene in the tagged region which can be functionally related to both associated phenotypes, while for the other genes no connection to bc or dna repair is reported. inherited dna repair mutations: are they modifiers of brca and brca penetrance and age at onset of hereditary breast and ovarian cancer? background: inherited mutations in brca and brca are the most common causes of hereditary breast and ovarian cancer (hboc). the risk of developing breast cancer by age in women carrying a brca mutation is - % and - % in brca carriers. however, mutations in brca and brca only explain about % of all hboc cases. the lifetime risk varies between families and even within affected individuals of the same family. the cause of this variability is unknown but it is hypothesized that additional mutations or rare variants in genes that are possibly interacting with brca / in different dna-repair pathways contribute to this phenomenon. methods: we obtained samples of patients positive for brca or brca mutations and an age-of-onset (aoo) of breast cancer below or above years of age from the german consortium for hereditary breast and ovarian cancer. panel sequencing was done to screen germline dna for mutations in genes involved in different dna-repair pathways. variants were classified into five classes according to a modified version of plon et al ( ) . only truncating mutations and known pathogenic missense mutations were considered pathogenic or likely pathogenic. results: the patient group with an early aoo ( women) had developed breast cancer at a mean age of . years (± . ) and the control group ( women) had developed breast cancer at a mean age of . years (± . ). a total of , variants were detected in all patients and of these ( . %; % ci, . %- . %) were presumed to be deleterious. mutations were found in genes other than brca and brca . mutations were mainly found in single-strand break repair (ssbr %), double-strand break repair (dsbr %) and checkpoint factors ( %). the rest were found in genes with other functions such as brca / interactors, centrosome formation, and signal transduction. the putative mutations were found in women of the control group ( . %; %ci, . %- . %) compared to women of the patient group ( . %; % ci, . %- . %). the incidence of germline mutations in dna-repair genes did not differ according to the age of onset (p = . ). prevalence of additional germline mutations in dsbr in patients ( %) was not significantly different from prevalence of dsbr mutations in controls . % (p = . ) conclusions: the preliminary results failed to show a difference in mutation load between the two cohorts of brca / carriers sorted by age of onset. larger studies are needed and may provide further insight into the role of mutation load in hboc age of onset of brca / carriers. objective: glioblastoma stem-like cells (gscs) carry stem cell features and therefore seem to be responsible for tumor initiating, maintaining, recurrence and chemo-and/or radiotherapy resisting. the current knowledge on genetic and transcriptomic characteristics of these cells especially in comparison to glioblastoma tissue is still limited. the aim of this study is to compare the genetic and genomic profile of glioblastoma tissue and gscs. thereby, differences in involved genes and affected pathways on dna level as well as on gene expression level are identified. material and methods: peripheral blood and tumor tissue were obtained from patients with glioblastoma. tumor tissue derived explant cell culture and serum-free culture were established. based on multi-parameter magnetic-activated cell sorting (macs) technique, cd and cd labeled cell subpopulations of gscs could be isolated. the tumor tissue, serum-free culture, and the isolated cell subpopulations as well as blood were analyzed by snp array and gene expression (excluding blood) in a paired design. for preliminary characterization of gscs in the serum-free culture we confirmed the stem cell features of gscs by the expression of nestin, sox , and cd (applying immunofluorescence staining). results: our results of snp array analyses showed genetic aberrations in all analyzed cellular entities (tumor tissue and cell subpopulations, e. g. gain of chromosome , loss of q . , loss of q . ->q . , and complete loss of chromosome ). furthermore, distinct genetic differences between the cell subpopulations and tumor tissue were observed (e. g. loss of chromosome and segmental uniparental disomy of p . ->p . , only in cancer stem-like cell subpopulations). in addition, we detected many possibly candidate cancer genes and pathways which may have an influence on tumorigenesis. gene expression analyses revealed strongest differences between fresh tumor tissue and serum-free culture based cells, where more than a third of investigated genes were affected. when contrasting fresh tumor tissue with stem cell marker positive serum-free cultured cells, , genes were upregulated in the stem cell marker positive cells, whereas , genes where upregulated in fresh tumor tissue. within these genes, strongest enriched pathways in stem cell marker positive cells included positive regulation of cell cycle and cancer-related pathways, whereas in fresh tumor tissue predominantly immune-related pathways were found, e. g. myeloid leukocyte activation, inflammatory response and phagocytosis. conclusion: differences between gscs and tumor tissue using snp array analyses and gene expression were detected. our results may help to get more information about the molecular pathomechanisms of glioblastoma. it still needs more investigations on the field of genetic and genomic analyses between gscs and glioblastoma tissue to identify novel potential targets for therapy development. background: fanconi anemia (fa) is a rare inherited chromosomal instability syndrome associated with bone marrow failure as well as myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). one-third of fa individuals exhibit bone marrow cytogenetic clones, notably gains of q and q and/or loss of / q, and % to % of fa patients developed mds/aml. in recent years, the application of high throughput technologies has revealed recurrent somatic mutations in genes implicated in myeloid malignancies. as additional genetic maladies facilitating mds/aml development in fa is lacking, we aimed to elucidate whether these mutations would be present in fa patients with mds/aml. methods: using illumina trusighttm myeloid sequencing panel (san diego, ca), we performed next-generation sequencing (ngs) on dna extracted from bone marrow specimens from fa mds/aml patients registered in the european working group of childhood mds. the sequencing panel targeted genes frequently mutated in hematologic neoplasms. results:. ten of the ( . %) evaluable patients had lesions ( to mutations per patient; missense, nonsense, insertion and duplication) in genes. the presence of a somatic mutation did not appear to correlate with complex karyotype or − / q. all affected genes occurred in isolation with exception of runx and kras. while of the mutations were pathogenic, were variance of unknown significance. mutations in genes involved in epigenetics (dna methylation, chromatin maintenance and cohesin complex; idh , tet , dnmt a, idh , ezh , rad and asxl ) and mutations in transcription factor genes (runx , ikzf and etv ) represented the most frequently affected genes. this was followed by mutations of genes encoding signaling molecules including the ras pathway (kras and ptpn ). altered runx was the most common lesion and occurred in individuals with aml, raeb or raeb-t. one patient with refractory anemia with ring sideroblasts (rars) had a mutation in the spliceosomal gene, sf b . conclusions: while the most common mutations encountered in sporadic cases of mds were in genes involved in rna splicing and epigenetics, these two broad categories of genes appeared to have less influence in our fa patients. most mutations were nonrecurring suggesting that there is no specific mutation pattern of these genes in fa-related mds/aml. however, runx mutations and also mutations involved in genes of the ras pathway appear to play a pathogenic role in fa mds/aml development. taken together, the data suggests that mutations in genes that cause clonal hematopoiesis in the population at large do not contribute significantly to fa hematopoietic clonal disease; however, particularly acquisition of runx and ras pathway alterations promote malignant myeloid disease progression. more extensive studies analyzing more patients are necessary to further define the secondary hits leading to fa myeloid disease. chromatin remodeling is a complex process shaping the nucleosome landscape, thereby regulating the accessibility of transcription factors to regulatory regions of target genes and ultimately managing gene expression. the swi/snf (switch/sucrose nonfermentable) complex remodels the nucleosome landscape in an atp-dependent manner and is divided into the two major subclasses brahma-associated factor (baf) and polybromo brahma-associated factor (pbaf) complex. somatic mutations in subunits of the swi/snf complex have been associated with different cancers, while germline mutations have been associated with autism spectrum disorder and the neurodevelopmental disorders coffin-siris (css) and nicolaides-baraitser syndromes (ncbrs). css is characterized by intellectual disability (id), coarsening of the face and hypoplasia or absence of the fifth finger-and/or toenails. so far, variants in five of the swi/snf subunit-encoding genes arid b, smarca , smarcb , arid a and smarce as well as variants in the transcription factor-encoding gene sox have been identified in css-affected individuals. arid is a member of the pbaf subcomplex, which until recently had not been linked to any neurodevelopmental phenotypes. in , mutations in the arid gene were associated with intellectual disability. in this study, we report on two individuals with private de novo arid frameshift mutations. both individuals present with css including id, coarsening of facial features, other recognizable facial dysmorphisms and hypoplasia of the fifth toenails. hence, this study identifies mutations in the arid gene as a novel and rare cause for css and enlarges the list of css-associated genes. the ubiquitin pathway is an enzymatic cascade including activating e , conjugating e , and ligating e enzymes, which governs protein degradation and sorting. it is crucial for many physiological processes. compromised function of members of the ubiquitin pathway leads to a wide range of human diseases, such as cancer, neurodegenerative diseases, and neurodevelopmental disorders. mutations in the thyroid hormone receptor interactor (trip ) gene (omim ), which encodes an e ligase in the ubiquitin pathway, have been associated with autism spectrum disorder (asd). in addition to autistic features, trip mutation carriers showed intellectual disability (id). more recently, trip was postulated as a novel candidate gene for intellectual disability in a meta-analysis of published id cohorts. however, detailed clinical information characterizing the phenotype of these individuals was not provided. in this study, we present seven novel individuals with private trip mutations including two splice site mutations, one nonsense mutation, three missense mutations, and one translocation case with a breakpoint in intron of the trip gene and clinically review four previously published cases. the trip mutation-positive individuals presented with mild to moderate id ( / ) or learning disability [intelligence quotient (iq) in one individual], asd ( / ) and some of them with unspecific craniofacial dysmorphism and other anomalies. in this study, we provide detailed clinical information of eleven trip mutation-positive individuals and thereby due to its heterogeneous etiology, primordial growth retardation is often a challenge for geneticists and clinicians in respect of diagnosis, therapy and prognosis. thus, pinpointing its genetic origin is required for a personalized treatment and prognosis. one syndrome mainly characterized by intrauterine and postnatal growth is silver-russell syndrome (srs), a clinically and molecularly heterogeneous disorder with a considerable overlap with other syndromes. in only % of patients with the characteristic srs phenotype the diagnosis can be confirmed molecularly, but % of cases remain without molecular diagnosis. in fact, in clinically less well characterized patients referred for diagnostic testing, the detection rate is less than %. however, systematic investigations on the contribution of mutations in genes which may be considered in the differential diagnoses of srs are still missing. we examined patients referred for molecular testing of srs but without molecular alterations associated with srs by ngs. a targeted ngs approach comprising genes implicated in the differential diagnoses of srs or suggested as srs candidate genes was performed. in patients fulfilling the criteria of srs accordingly to our recently developed clinical scoring system, disease-causing variants were found. these patients carried mutations in genes associated with bloom syndrome, mulibrey nanism, kbg syndrome, short syndromes or ig-f r-associated short stature, respectively. indeed, some of the differential diagnoses detected in our cohort have a major impact on clinical management, including cancer screening because of a high risk for tumor development. furthermore, we did not identify any pathogenic mutation in one of proposed srs candidate genes (e. g. mest, grb , copg ), thus raising the question whether these genes are indeed involved in the etiology of srs. we show that a (targeted) ngs approach is an important tool to identify the genetic cause in patients with unexplained growth retardation. furthermore, our data show (positive) clinical scoring in srs should not impede the consideration of differential diagnoses and other molecular causes. submicroscopic deletions of chromosome band p . have been reported in more than patients. common clinical features include intellectual disability/developmental delay, central obesity and behavioural difficulties. myt l became the main candidate gene for id and obesity since it is deleted or disrupted in all published patients. however, reports of deletions affecting only this gene and even more so of deleterious myt l sequence variants are very rare. to our knowledge, until now only two patients with de novo myt l point mutations have been reported. in the present study, we analysed a cohort of individuals with intellectual disability of unknown aetiology and their unaffected parents by whole exome sequencing. we identified de novo myt l sequence variants in two out of patients. patient carried a nonsense mutation (c. g>t, nm_ . ; gly *) whereas patient carried a direct splice site mutation (c. - a>g). according to prediction algorithms, both detected myt l variants are deleterious (patient : sift score , cadd score ; patient : cadd score . ). in addition, patient carried a de novo splice site variant in setd b. however, this variant is predicted to be benign (cadd score . ) as well as a known snv (rs , maf . ). a comprehensive clinical characterisation of the two patients yielded only mild or moderate intellectual disability, behavioural problems and muscular hypotonia as common clinical signs. surprisingly, obesity was only present in patient . postnatal tall stature and transient microcephaly were present in one patient each. this clinical picture is compared to the published phenotypes of patients with myt l point mutations, patients with microdeletions of only myt l and patients with larger p . deletions. with the reduced penetrance regarding obesity, the clinical picture of patients with myt l mutations is becoming more and more unspecific. the retina and anterior neural fold homeobox gene (rax) controls the embryonic eye development and is involved in human autosomal-recessive microphthalmia. so far only a few compound heterozygous mutations in rax have been described in microphthalmia patients. we report a first case of microphthalmia caused by a novel homozygous mutation in rax. the -month-old patient was born to consanguineous parents and presented with extreme microphthalmia, panhypopituitarism and developmental delay. mri of the brain showed bilateral agenesis of the anterior visual pathway and tractus opticus. ocular ultrasound confirmed bilateral anophthalmia. additionally, dysgenesis of the corpus callosum and an abnormal pituitary gland have been detected. the first child of these parents, who died shortly after birth, had also been diagnosed with bilateral anophthalmia. using panel diagnosis of the disease associated genome, we identified the homozygous pathogenic variant c. del, (p. fs) in rax. we performed a segregation analysis and confirmed that both parents are heterozygous for this variant. so far developmental delay and panhypo-expand the clinical spectrum of the trip gene in non-syndromic intellectual disability with or without asd. background: whole exome sequencing (wes) using next generation sequencing has proven to be a powerful tool in determining the underlying genetic cause of rare disorders. here, we show, that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering further disease-causing mutations. clinical report and genetic findings: we report follow-up data of a previously published consanguineous family with two children, a boy and a girl, suffering from severe encephalopathy, hypotonia, microcephaly and retinal dystrophy. wes had shown a homozygous intronic splice variant in pgap (c. - a>g;p.?) causative for the symptoms. both parents were heterozygous carrier for the pgap variant (granzow, paramasivam et al, mol cell probes ) . in the next pregnancy, the unborn child presented hydrops fetalis, omphalocele, short tubular bones and cystic kidneys. chorionic villus sampling showed the fetus to be homozygous for the pgap variant. however, neither of these symptoms fit with a pgap -associated disorder. additional wes of fetal dna and re-evaluation in the family showed a homozygous nonsense variant in ift (c.g t;p.e *) consistent with a diagnosis of mainzer-saldino syndrome (mss) which is characterised by the association of renal disease, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia, as a second diagnosis in the fetus. again, both parents were shown to be a heterozygous carrier for the ift variant. yet, as omphalocele was not accounted for by any of the identified conditions, a third genetic cause cannot entirely be excluded. alternatively, omphalocele may be a rare manifestation of mss, or be the result of a combination of both disorders. the couple opted for induced abortion. discussion: it is estimated that an individual carries multiple heterozygous variants for autosomal recessive disorders in his or her genome. especially in consanguineous families, this results in an elevated risk for children with more than one disorder. in recent publications of clinical exomes, double diagnoses have been reported in to % of investigated subjects. thus, the possibility of more than one causative gene should be carefully explored when working with wes and re-evaluation in case of additional clinical symptoms within a family should be considered. also, follow-up of families with rare genetic disorders may lead the clinical geneticist beyond the assumed single cause to multiple single gene disorders in the same family. conclusion: using wes, we have identified two independent single gene disorders in a consanguineous family demonstrating that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering multiple disease-causing mutations in one family. we present a case study using molecular cytogenetic approaches on a year old boy presenting with microcephaly-brachycephaly, macroglossia and absent speech development. the boy is the first child of healthy, consanguineous parents of pakistani origin. following an uncomplicated pregnancy, the hypotrophic newborn was delivered at weeks weighing g. in the third month of life, the baby had viral meningitis. regular pediatric follow-up revealed psychomotor delay with hypotonia. creatine kinase, lactate and fibroblast growth factor measured in serum were high. subsequent investigations at the age of months included brain mri, electroencephalogram and muscle biopsy that gave hints of a mitochondriopathy or potential neuropathy of axonal type. due to the suspicion of a complex mitochondriopathy, whole exome sequencing was performed using a sureselect human all exon kit (agilent, mb v ) on a hiseq (illumina). the analysis revealed a heterozygous microdeletion of kb on chromosome q . which was classified as an unclear variant (uv), ehmt -gen was not affected. re-examination of proband's dna using array cgh detected a larger kb heterozygous deletion in the q . region (arr[hg ] q . ( , , - , , ) x ), encompassing exon of ehmt (euchromatin histone methyltransferase ; transcript nm_ . ). haploinsufficiency of this gene results in kleefstra syndrome (omim ), a multisystem disorder due to either microdeletions in q . encompassing ehmt or intragenic point mutations. mlpa analysis (ehmt mpla-kit p ) of parental dna further indicated a de novo origin of the deletion in our proband. a similar deletion has been described previously in a case presenting with clinical features of kleefstra syndrome [ ] strengthening the importance to include the ' part of ehmt in sequencing as well as cnv screening. in summary, our study clearly shows that array cgh is a valuable complementary approach to ngs especially for poorly covered regions in ngs i. e. exon of many transcripts. we report on a three-generation family with variable manifestations including delayed and incomplete tooth eruption, early tooth loss due to short dental roots, acroosteolysis, osteoporosis, tendon ruptures, joint hypermobility, muscle weekness, glaucoma, neurological features, and psoriasis. after detection of elevated cd /siglec -expression on monocytes and an upregulation of interferon-stimulated gene transcripts, singleton-merten syndrome was diagnosed. the novel heterozygous mutation c. c>g (p.thr arg) in ifih was found in three affected family members. singleton-merten syndrome is a very rare autosomal dominant interferonopathy, so far described in not more than four families. until now, only two different gain-of-function mutations in ifih have been detected. mutations in ifih are also associated with aicardi-goutière syndrome and recently features of both conditions were found in the same family. our findings expand the mutational spectrum of singleton-merten syndrome and demonstrate the high intrafamiliar variability associated with mutations in ifih . pituitarism have not been described in association with rax mutations. therefore we conducted array comparative genomic hybridization and karyotyping in the index patient. both tests gave normal results. prenatal diagnosis by chorionic villus sampling in the next pregnancy excluded a homozygous carrier status for this rax mutation. mutations in the phf gene are associated with borjeson-forssman-lehmann syndrome (bfls), an x-linked intellectual disability disorder affecting mainly males. female carriers usually show no or mild clinical signs. however, recent studies described females with de novo phf gene defects (mutations, deletions) and severe phenotypes resembling coffin-siris syndrome [ , ] . here, we report on a girl with a maternally inherited phf mutation and a phenotype resembling those described previously in affected females. the mother had learning difficulties and mild dysmorphological features (hypertelorism, prominent forehead). when seen at age months, the proposita showed muscular hypotonia, was unable to sit and had limited head control (developmental delay months). dysmorphic features included scaphocephaly, hypertelorism, a small flat nose with anteverted nares, low set, prominent ears, a high, narrow palate, absent labia minora and linear skin pigmentation on the thighs. ophthalmologic investigation identified strabism convergens of the left eye, hyperopia and an excavated papilla with a pale optical nerve. ultrasound showed patent foramen ovale, tricuspid insufficiency and a unilateral incomplete duplication of the renal pelvis. karyotyping performed elsewhere was normal ( ,xx). results of a genome-wide snp array analysis (affymetrix cytoscan hd) were also normal. using a targeted ngs approach for syndromic and non-syndromic developmental delay encompassing over brain related genes (mpimg- -test), we identified a heterozygous lof-mutation c. c>t (p.gln *) in the phf gene (encoding phd finger protein ). in addition, a human androgen receptor (humara) assay using blood dna showed a highly skewed x-inactivation ( : ). segregation analysis indicated a maternal origin of the variant. the mother also had skewed x-inactivation in blood. her husband and other daughter tested normal for the c. c>t variant. here, we describe the first female patient with a maternally inherited phf mutation and a severe phenotype. the mild phenotype in the mother might be due to different patterns of x-inactivation or to (undetected) mosaicism. this report represents the th case of a severely affected female patient with a phf gene defect. findings support the assumption [ ] that the female phenotypes of bfls might be more common than previously estimated. toms typical for ada deficiency such as fever, livedo racemosa, abdominal colics, arthralgias, and raynaud's phenomenon were observed months later. cecr sequencing (nm_ . ) revealed two previously described pathogenic missense mutations: c. g>c, p.(gly ala) and c. g>a, p.(arg gln). compound heterozygosity was confirmed by parental analysis. to the best of our knowledge, this combination of mutations has not been described until now. the p.(arg gln) is considered as founder mutation in the dutch population, but first phenotype-genotype analyses did not allow further prediction of clinical outcomes. ada deficiency should be considered in patients with childhood stroke despite the absence of systemic inflammation and cerebral vasculitis. congenital hyperinsulinism (chi) has been described as heterogeneous entity caused by at least different genes. in , tein et al. first described a defective activity of l- -hydroxyacyl-coa dehydrogenase in a -year old patient with hypoketotic hypoglycemic encephalopathy. biochemical markers of l- -hydroxyacyl-coa dehydrogenase deficiency (schad) are high -oh-glutarate excretion in urine and c -oh-carnitine in plasma. the clinical presentation is very heterogeneous with regard to age of onset, severity of symptoms as well as response to medical treatment and leucine-sensitivity. in some patients even a near total pancreatectomy was performed. schad dependent hyperinsulinism (hhf ) is a rare autosomal recessive disorder that is caused by mutations in the gene hadh. here we describe patients from unrelated families out of a cohort of chi patients mainly from central europe. patients and are siblings from unrelated parents. the older brother manifested with hypoglycemic convulsions at the age of weeks. a subtotal pancreatectomy was performed in an outside academic hospital. in further course he developed epilepsy and has been treated with diazoxide and anticonvulsants. the nd child was born with hypoketotic hypoglycemia and chi was diagnosed in first days of life. diazoxide treatment stabilized blood glucose and both children were referred to our pediatric endocrinology at the age of years and months, respectively. mutational analysis revealed the homozygous variant c. - c>g within the region of the splice acceptor site in intron of the hadh gene in both affected children. this change is neither registered in exac nor described in the mutation databases hgmd or in the literature and was predicted to disrupt proper splicing. we then completed mutational analysis in unidentified patients of our chi cohort with diazoxide responsiveness and known or suspected consanguinity. patient was born to consanguineous parents and chi manifested in the girl at neonatal age with hypoketotic hypoglycemia. she was successfully treated with diaxozide. later, she developed convulsions and statomotoric developmental delay. the homozygous splice mutation c. + g>t in intron of hadh was detected in the child. the parents were identified as heterozygous carriers. in patient , a girl born to consanguineous turkish parents, chi manifested at the age of months with hypoglycemic seizures. she responded well to diazoxide treatment. a homozygous missense mutation (c. a>g; p.lys glu) in exon of hadh was detected in the patient and her parents were heterozygous carriers. hadh mutations in case and have been previously described in probands of turkish descent and appear to be founder mutations in the turkish population. in conclusion, we recommend hadh mutation analysis to be considered in chi children with unknown cause and known consanguineous pedigrees or originating from populations with higher prevalence of consanguinity. homozygous or compound heterozygous mutations in cecr (cat eye syndrome chromosome region, candidate ) have recently been identified to causing deficiency of adenosine deaminase (ada ; dada ) with childhood polyarteritis nodosa (pan) (omim # ). this inflammatory vasculitis affects the skin, and inner organs (predominantly kidneys and gastrointestinal tract) and also shows a high risk of ischemic stroke, brain hemorrhage as well as peripheral neuropathy. using whole-exome sequencing it was also found that the six adult patients (aged - ) described by sneddon in (sneddon syndrome, omim # ) likewise carried compound heterozygous cecr mutations. sneddon syndrome is characterized by a combination of dermatologic features (livedo racemosa) and ischemic brain infarctions. recently, clinical and genetic data of more than sixty ada patients have been reviewed and underlined the wide clinical variability in age at onset, clinical findings, outcome of neurological involvement, and additional hematological symptoms. typically, stroke has been reported to follow systemic inflammatory disease and predominantly affects posterior and central brain areas. here we describe one of the rare patients in whom acute mesencephalic stroke preceded systemic inflammation and presented as initial clinical symptom. symp- triploidy is a recurrent finding in prenatal diagnostics. in a small number of individuals, correction of triploidy has been suggested based on the finding of (mosaic) genome-wide uniparental disomy (upd). we here investigated uncultured und cultured amniotic cells (ac) and placental tissue from a fetus, in which ultrasound examination in the + th week of gestation revealed growth retardation, left diaphragmatic hernia with parts of stomach and bowel localized in the chest, dextrocardia, short nasal bone and single umbilical artery. these findings were confirmed at the + th week when the pregnancy was terminated. the pregnancy was conceived spontaneously by a -year-old mother and a -year-old father, both healthy and with uneventful family history. the parents were non-consanguineous and carry a normal karyotype. microsatellite analyses of uncultured ac obtained at initial presentation showed for chromosomes , and a pattern suggesting triploidy with only biallelic presentation. while y-chromosomal sequences were lacking the x-chromosome showed, unexpectedly a rather disomic pattern. metaphase yield on cultured ac was low but showed a mosaic karyotype ,xxx-,+ [ ]/ ,xxx[ ], which was confirmed for several chromosomes by interphase fish. remarkably, a triploid clone was cytogenetically not detected. thus, we performed further analyses using microsatellite markers, oncoscan technology and fish. these studies unraveled in uncultured ac a pattern suggestive of triploidy with the supernumerary chromosomal complement derived from a maternal isodisomy with the notable exception of the x chromosome. in cultured ac and placental tissue for all chromosomes, except x and , a diploid pattern was observed with alleles from both parents identical to those in the uncultured ac. trisomy x was confirmed in both tissues with the supernumerary chromosome x being of paternal isodisomic origin. the trisomy was seen only in cultured ac, and likely represents a pseudomosaic which nevertheless could not be proven due to insufficient yield of mitoses from the parallel cultures. finally, retrospective interphase fish on remnant uncultured ac showed two diploid clones, one disomic (approximately % of nuclei) and one trisomic ( %) for the x chromosome. the most likely explanation for the findings is a mosaicism for one diploid clone with genome-wide maternal isodisomy and a second diploid but bi-parental cell line with paternal trisomy x. given the identity of the (maternal) alleles in both clones our findings suggest that originally a triploid clone due to a maternal division error/inclusion of a polar body ii existed which underwent (erroneous) triploidy rescue resulting in one diploid biparental clone and one haploid clone of maternal origin that underwent haploid rescue resulting in genome-wide maternal isodisomy. the biparental clone with trisomy x either resulted from a sperm with two x-chromosomes or an erroneous x-duplication during trisomy rescue. since the introduction of non-invasive prenatal diagnosis (nipd, e. g. har-mony® test) in , this test is frequently demanded and routinely applied in prenatal centers and medical practices. mostly, nipd is intended to detect autosomal trisomies ( , , ), but also offers the possibility to analyze sex chromosomes. therefore, also sex chromosome aneuploidies (sca) (e. g. monosomy x (turner syndrome), triple x, xxy (klinefelter syndrome), xyy) are incidentally found. so far, in our prenatal center sca were detected in pregnancies by har-mony® test, consisting of three pregnancies with monosomy x (turner syndrome) and two pregnancies with klinefelter syndrome (xxy). triple x and xyy were detected one time each. of the three cases with suspected monosomy x, the diagnosis of turner syndrome could only be confirmed in one case. this fetus also had a hydrops at week + . for the other two fetuses, the chromosomal analysis of amniotic fluid revealed normal female karyotypes ( ,xx). in both cases with suspected klinefelter syndrome, this diagnosis could be disproved by amniocentesis (karyotype ,xy). in the pregnancies with assumed triple x and xyy, the true fetal karyotype was not further determined yet. from our experience, the rate of false positive results concerning the sex chromosome aneuploidies is noticeably higher than reported in two studies of nicolaides and hooks . this has to be strongly considered in the counselling of patients who wish to know the fetal sex by nipd. congenital myopathies and congenital muscular dystrophies: will genetic testing replace muscle biopsy in the near future? congenital myopathies and muscular dystrophies are a group of inherited neuromuscular diseases with early onset and broad genetic and histopathological overlap. the diagnostic approach has considerably changed with next generation sequencing methods available. here, we describe the diagnostic value of genetic and histological methods in a cohort of index patients and hence the efficacy of diagnostic procedures. of patients had a muscle biopsy as a first-tier approach. in of patients muscle biopsy was informative, leading to a classification in subgroups of cm or cmd. however, in only a few of these cases biopsy led to a specific diagnosis (e. g. merosin deficiency). in of patients genetic testing (candidate gene sequencing or ngs) was performed additionally to muscle biopsy as a second-tier diagnostic step, while patients of the whole cohort received genetic testing only. in almost two-thirds of these patients genetic testing identified known pathogenic or most likely pathogenic variations. these findings illustrate that genetic testing is superior to muscle biopsy in accurately diagnosing cm or cmd. in conclusion, we suggest that invasive muscle biopsy should be replaced by genetic testing as first-tier diagnostic procedure in patients with clinical signs of cm or cmd. nmd inhibition increases the amount of gaa-rna in patient's lymphocytes as well as in the cells of his parents. the residual function of the resulting protein has to be investigated. discussion and conclusion: rna analysis in lymphocytes with and without nmd inhibition is a simple method for analysing splice defects in all monogenic disorders with expression of the disease causing gene in lymphocytes. a further advantage for the patient is the use of blood cells instead of fibroblasts, because a skin biopsy can be avoided and analysis times are reduced. the exact characterization of pathogenic variants is an important aspect of diagnosis, prediction of disease severity and genetic counselling. in vitro nmd inhibition in lymphocytes of affected patients allows the characterization of splice defects. in the future successful inhibition of nmd in vitro might help to identify patients, who may profit from a therapeutic intervention with nmd inhibitors. even expression of a partial protein with low or no activity reduces the risk for the patient to develop antibodies hampering enzyme/protein replacement therapy. p-cling- q microdeletion syndrome: a family with short stature and silver-russel (srs)-like phenotype. introduction: the silver-russel syndrome (mim ), first described independently by silver and russell in , is a condition with intrauterine growth retardation, postnatal growth failure and other characteristic features, including relative macrocephaly (defined as a head circumference at birth ≥ . sd score (sds) above birth weight and/or length sds), prominent forehead, body asymmetry and feeding difficulties as recently defined in an international consensus statement. patients and methods: we report here on first degree relatives with a silver-russel syndrome phenotype who presented with prenatal-and postnatal growth retardation, feeding difficulties, a prominent forehead and a failure to thrive. additional features such as dysmorphic facial features, periodically increased sweating, and scoliosis were present in one of the family members only, whereas learning problems and cardiac arrhythmia were present in one other. none of the patients had relative macrocephaly. high resolution array-cgh was performed to screen for cncs and mlpa to confirm the array-cgh result. results: no hypo-methylation of the imprinting center on p nor uniparental disomy of chromosome and were found in the index-patients. high-resolution array-cgh identified a q . microdeletion of . mb (arr[grch q . ( , , , )× ). the heterozygous loss was confirmed by mlpa in the index patient and the other two affected family members (i. e. her brother and mother). the deletion includes the genes hmga , llph, tmbim , irak , helb, grip , and the pseudogene rpsap . conclusion: to the best of our knowledge this is the first report on familial presentation of a silver-russel syndrome due to a microdeletion in q . . none of the patients had relative macrocephaly. supporting the hypothesis by takenouchi et al. that the causative gene for relative macrocephaly resides centromeric to hmga , the region centromeric of hmga is not included in the deletion in our family. spastic ataxia of charlevoix-saguenay (sacs) is an autosomal recessive neurodegenerative disorder and is caused by homozygous or compound heterozygous mutations in the sacs gene. first symptoms of sacs are walking difficulties due to unsteady gait. further typical clinical features include spasticity, ataxia, pyramidal tract signs, nystagmus and dysarthria. here, we report on a -year-old female patient who initially presented with disturbances in motor abilities including frequent falls and high arched foot. cranial mrt was normal while nerve conduction velocity was significantly reduced. the patient's parents did not show any clinical features. since no pmp duplication was detected we performed a gene panel including genes that are associated with hereditary motor and sensory neuropathies (hmsn) and related disorders by using targeted next generation sequencing. we identified the two heterozygous stop mutations c. t>a (p.leu ter) and c. dupt (p.leu phefs* ), located at the same position in the sacs gene. sanger sequencing did not enable us to properly display that there is a transversion and a duplication of the same nucleotide at two different alleles. this exemplifies that, in contrast to sanger sequencing, ngs can illustrate both alleles separately. to conclude, this case was only resolvable by ngs which makes this method appropriate for the detection of compound heterozygous mutations, especially in the rare event when two mutations occur at the same position. background: the precise identification and characterization of genetic variants in monogenic diseases has a wide influence on diagnosis and therapy. about % of pathogenic variants are splicing variants. due to the complex mechanism of splicing regulation it is difficult to predict the effects of variants on mrna splicing. possible consequences are exon skipping, intron retention, generation of novel splice sites or the utilization of a cryptic splice site. common consequences are a frame-shift and the generation of premature termination codon. this leads to rna degradation via the nonsense mediated decay (nmd) pathway. in a patient with the clinical symptoms of non-classical infantile pompe disease and a confirmed acid alpha-glucosidase (gaa) deficiency, we detected two novel, exonic variants in the gaa gene. both base pair exchanges suggested either an amino acid exchange or a splice defect as consequences. however, conventional investigation of the leucocyte mrna of the patient and his parents was inconclusive. degradation of the respective mutated rna by nmd was suspected. we developed an approach in order to characterize novel splicing mutations in a simple and non-invasive manner. material and method: isolated blood lymphocytes from patient and his parents were cultured in standard leucocyte medium supplemented with different concentrations of the nmd inhibitors ocadaic acid, anisomycin, and wortmannin for h. cells were harvested and rna was isolated. the reverse transcribed cdna was amplified in allele specific pcrs and qpcr assays. results: compared to the non-stimulated lymphocyte controls nonsense mediated rna decay was inhibited by anisomycin. the consequences of aberrant rna splicing were detectable: the maternal mutation results in exon skipping, the paternal mutation in intron retention. furthermore vascular ehlers-danlos syndrome (type iv) is considered to be an autosomal dominant disorder caused by heterozygous mutations in col a , which are missense or splice site variants in about % of cases. we here report on a three-year-old female of non-consanguineous parents born with bilateral clubfoot as well as dysmorphic facial features, joint laxity, and mild contractures of finger joints. developmental delay became evident. after trauma at years of age she developed brain haemorrhage. mri diagnosis at this age revealed an additional frontal aneurism as well as frontoparietal polymicrogyria. we identified novel compound heterozygous col a mutations: the nonsense mutation c. c>t (p.arg *) and the c. delc (p.pro leufs* ) frameshift mutation leading to a premature stop codon. further studies showed that the mutations were inherited from each parent who had no features for ehlers-danlos syndrome type iv. only two other families have been reported so far with recessive mutations of this gene and a severe vascular phenotype and polymicrogyria. biallelic mutations of col a seem to be accompanied with a significantly worse outcome compared with heterozygous mutations and polymicrogyria is an additional phenotypic feature. here we describe five patients with epidermolytic epidermal nevi in different degrees of severity with the mosaic mutation c. c>t (p.arg cys) in krt gene. the same mutation has previously been described in patients with ei (bygum et al. ) . we analyzed dna from peripheral blood and/or skin biopsies from affected and unaffected skin with next generation sequencing (ngs) and sanger sequencing methods. using ngs we found this mutation in blood in mosaic states ranging from % to %. the mosaic could only be confirmed by sanger sequencing in the patient with the highest mosaic frequency of %. in four of our patients we investigated skin biopsies from affected and unaffected skin. it is noteworthy, that only one of four patients showed the mutation in heterozygous state of % in the affected skin, whereas the other patients presented a mosaic state also in the affected skin. to exclude a recurrent sequencing artefact at this position, we examined control patients for this mosaic mutation using ngs. in none of these patients we found the same dna change. patients with epidermolytic epidermal nevi have a higher risk to have children with a full-blown ei phenotype. our results show the importance of ngs as the method of choice to explore the molecular genetic basis of epidermolytic epidermal nevi. strikingly, all our patients carry the same mosaic mutation c. c>t in krt . we suggest that this position is a hotspot for postzygotic mutations in krt . non-syndromic hearing loss (nshl), with presently around associated genes, is one of the most genetically heterogeneous disorders constituting nearly % of genetic deafness with a predominantly recessive inheritance pattern. thirty percent of hearing loss (hl) can be connected as a part of over distinct syndromes. next generation sequencing (ngs) technologies have revolutionized pathogenic variant identification. different strategies enhance pathogenic variant detection supporting detailed hl investigation to overcome the many ambiguities associated with clinical heterogeneity. detection of the disease causing variant in correlation with the phenotype can be challenging in small families, in situations with ambiguous clinical histories and allelic heterogeneity. using a clinical and whole exome sequencing approach, we tested over probands as part of a multicentre iranian and german genetics of hl study that included probands primarily with sporadic or dominant hl in a parent-child or parent-sibling trio context. the majority of these probands were pre-screened for defects in gjb and strc. libraries were prepared using trusight one and nextera rapid capture exome enrichment and sequenced using the miseq and nextseq desktop sequencers (illumina). analysis was performed using gensearchngs and an inhouse exome analysis pipeline. around % of cases were resolved from phenotype matching and segregation analysis. interestingly, the fraction of resolved cases was much higher in our iranian cohort (> %) compared to our german cohort (> %) which may be attributed in part to increased consanguinity in the iranian families. we observed likely disease causing variants in syndrome-associated genes including eya causing branchio-oto-renal syndrome, a phenotype that was retrospectively confirmed by acquisition of additional clinical information. with few exceptions, we observed a diverse collection of affected genes in probands from our german collected cohort. contrastingly, the iranian cohort revealed frequent mutations in myo a and otof. furthermore, co-segregation of variants in myo and tecta, with expected dominant hl phenotype, was a hindrance overcome by extensive segregation testing. familial locus heterogeneity was also observed by mutations in cib and slc a segregating in different branches of the same extended pedigree. success in the identification of disease causing variants in known hl genes is contingent upon analysis strategy, clinical information and opportunity for segregation testing. the ability to retrospectively connect an already apparent syndromic phenotype to a syndrome-associated gene without prior knowledge is a powerful application of comprehensive analysis that is not restricted to nshl genes. this work provides an improved understanding of population-specific genetic epidemiology of hereditary hl and highlights the challenges in defining genetic causes in a highly heterogeneous disorder such as hl. vere disproportionate microcephaly (- , sd), corneal clouding, myopia, teeth abnormalities and dysmorphism. panel diagnostic by next generation sequencing for primary microcephaly, including all known genes for seckel syndrome, was unremarkable. microarray analysis (affymetrix® cy-toscan hd) revealed a heterozygous kb deletion, spanning the plk gene. this deletion was confirmed by mlpa (multiplex ligation-dependent probe amplification) analysis. subsequent sequence analysis of the plk gene showed a variant of unknown significance on the second allele. in silico analysis of this variant indicated a significant decrease of the relative splice efficiency at the splice donor site. rt-pcr analysis confirmed altered splicing, resulting in a predominant loss of exon of the transcript and predicting truncation of the plk protein. interestingly, a residual wild-type transcript was also detectable in patient rna, implying that this variant effects splicing only partially. by analysis of the parents, the splice variant and the large deletion were proven to be compound heterozygous. discussion: up to now, only a few patients with plk mutations have been described in the literature. the phenotype comprises primary microcephaly, primordial dwarfism and chorioretinopathy (mccrp ). to our knowledge, we describe the first case of a plk heterozygous whole gene deletion and at least partial biallelic inactivation of the gene, therefore expanding the genetic background of this disorder. furthermore, we give a detailed phenotypic description of a further individual with plk alterations. the girl does not show retinopathy so far. while generalised retinopathy was discussed to be one of the most prominent distinctive features between mccrp and primary microcephaly/seckel syndrome, we consider plk rather to be a further candidate gene pointing towards seckel syndrome. additional investigations on centriole function in patient-derived cells are in progress. pathogenic variants of mitochondrial dna cause a wide range of severe congenital disorders with maternal inheritance and a high transmission risk for female carriers. we report on eight families with an index case presenting with the common pathogenic variant m. t>g (p.leu arg) in the mt-atp gene in virtually homoplasmic form. in five families the mutation was detectable in peripheral blood from the mother in heteroplasmic form. in three families with a sporadic case of leigh syndrome the mutation was not detectable in peripheral blood (or urinary or buccal cells) from the mother, possibly indicating a de novo event. furthermore, one family presented with a de novo nonsense mutation in the gene mt-atp , which was present in peripheral blood of the index case in about % and was not detectable in the mother or the unaffected sister. two female carriers with a heteroplasmy level of % asked for prenatal testing. both pregnancies showed an apparently homoplasmic load of the mutation. mutations in lztfl (bbs ) may be associated with a severe renal phenotype huntington's disease (hd) is a rare autosomal dominant neurodegenerative disorder caused by expanded cag repeats as diagnosed via direct dna analysis. for asymptomatic individuals, predictive testing (pt) can facilitate life planning and diminish uncertainty, but it is also associated with substantial social and psychological challenges. we present a prospective case series of counselees seeking predictive hd testing at the huntington centre north-rhine westphalia (bochum, germany) between and . the international protocol including several pre-test sessions was followed throughout. the aim of this study was to prospectively follow the decision-making process of individuals at risk in our centre and explore their experiences following the decision as well as the impacts of mutation test results by means of standardized questionnaires and a semi-standardized telephone interview one year after the initial counselling session. individuals participated in at least one of the three phases of the survey, including individuals for the telephone interview. in our cohort, almost all interviewees reported a balanced emotional state one year after initial counselling, regardless of the decision for or against the test. the most important motivations for a decision in favor of pt were the ability to plan private life and to eliminate uncertainty. the most important motivations against pt were the fear of an increasing risk for others (e. g. offspring) and the fear to obtain an unfavorable htt mutation result, followed by the considered, willful decision for "wanting to not know". furthermore, we identified evidence for gender-specific aspects in decision-making in line with and expanding our previous observations. this study represents one of the few comprehensive prospective evaluations regarding decision-making and coping strategies related to predictive testing for huntington's disease. we submit that gender-related aspects should be heeded in genetic counselling during the predictive testing and counselling processes. our findings could serve as a basis for more extended prospective evaluations with higher numbers of participants and longer follow-up intervals. institute of human genetics, heidelberg, germany, department of conservative dentistry, heidelberg, germany, cegat gmbh, center for genomics and transcriptomics, tübingen, germany background: plk (polo-like kinase ) has been designated as "master regulator" of centriole assembly. complete loss of plk is lethal in mice, whereas biallelic plk mutations with some retained function have been described in a few patients with microcephaly, growth failure and retinopathy (mccrp , omim # ). this is a heterogeneous entity overlapping with mcph (primary microcephaly) and seckel syndrome. during the last years several new genes have been discovered associated with this spectrum. clinical report and genetic findings: we report on a year old female patient with intellectual disability, primordial dwarfism (- , sd), most se-abstracts linked to rare autosomal recessive diseases with poor prognosis. we then compared couples and filtered for variants present in genes overlapping in both partners. putative pathogenic variants were tested for co-segregation in affected fetuses where material was available and in unaffected siblings. out of eleven couples of mediterranean and arabian ancestry (c: , nc: ) and two non-consanguineous couples of european ancestry, we found five cases ( / , %, c: , nc: ) with both parents being heterozygous carriers of rare potentially deleterious variants in one or more overlapping genes. in four of these couples the underlying genetic cause for pre-or early postnatal child death could be established, in two of the families the diagnosis was confirmed by homozygous detection of the parental variant in the available dna of the affected child. in a consanguineous couple with pathogenic variants for a severe autosomal recessive disorder identified in both parents, the molecular diagnosis for their child that had died at months of age could not be established. out of couples in whom no causative diagnosis could be achieved consented to undergo further wes analysis. identified variants are now used for preimplantation and prenatal diagnostics in all four families in which a causative diagnosis was established. our data show that ngs based gene panel sequencing of selected genes involved in lethal autosomal recessive disorders is an effective tool for carrier screening in parents and for the identification of recessive gene defects in families that have experienced early child death and/or multiple miscarriages. k. komlósi , s. diederich , d. l. fend-guella , u. zechner , s. schweiger , o. bartsch recently an x-linked syndrome with maternally inherited or de novo mutations in taf was described with global developmental delay, intellectual disability (id), delayed speech, characteristic facial dysmorphology, generalized hypotonia and variable neurologic features (mrxs , mim: # , xlr). there have been only three publications of unrelated families, with single-nucleotide changes and with gene duplications including taf (kaya et al., ; o'rawe et al., ; hu et al., ) . we identified a german family in which two brothers ( and years) showed severe intellectual disability, absent speech and understanding, and hypotonia but different neurologic and behavioral phenotypes. besides severe id the older brother also had postnatal short stature (- sd), a severe lennox-gastaut epilepsy and a neurodegenerative course. the younger brother showed autistic behavior and lost his very limited skills at age to . years. both showed mild dysmorphic features (prominent supraorbital ridges, sagging cheeks, long philtrum, long face, thin upper lip, and high-arched palate), oropharyngeal dysphagia and generalized hypotonia. a gluteal crease with a sacral caudal remnant described as a characteristic feature was not seen in our case, and hearing impairment, microcephaly, dystonic movements or tremor were not observed either. the family history was highly suggestive of x-linked inheritance with an affected maternal uncle, a maternal aunt with multiple miscarriages, and two aunts with learning disability. since a previous analysis of known x-linked mental retardation genes had not revealed the cause in the older brother, we used a targeted ngs approach (mpimg -test: > brain related genes) for the analysis of the younger brother. following enrichment a bp paired end sequencing was carried out on an illumina miseq system with > % of target covered > -fold (hu et al, ) . only taf fitted the x-linked model and the phenotype. the unreported hemizygous sequence variant c. g>a (p.asp asn) in exon of taf was deemed pathogenic. it affected a highly conserved residue in the central "duf " domain, where / previously described mutations had clustered. segregation analysis confirmed hemizygosity in the older brother and heterozygosity in the mother with completely skewed x-inactivation ( : ). gonadism and learning disabilities. because of the delayed onset of symptoms, the diagnosis is often established during late childhood. however, in some cases renal morphological changes detected by ultrasound may resemble those usually seen in autosomal recessive polycystic kidney disease (arpkd). however, histologically bbs-kidneys differ distinctly from other polycystic disorders by cystic orientation, localisation, extension, structure and size. bbs genes have been identified to date. mutations in bbs , bbs , bbs and bbs have been found to cause an antenatal presentation of bbs that may in some aspects mimic meckel gruber syndrome (mks). there is increasing evidence that bbs, at least in some families, shows an oligogenic mode of inheritance with three mutations at two bbs loci. yet, only three patients in two families with bbs caused by mutations in lztfl (bbs ) have been reported. their diagnosis was established in childhood and all patients had mesoaxial polydactyly as a distinct manifestation. in contrast to previous lztfl cases, in our family the diagnosis of arpkd was suspected sonographically at weeks gestation (wg). pregnancy was terminated at wg. autopsy revealed postaxial polydactyly of both hands, enlarged spongy kidneys, hemivertebra t and some features of potter's sequence. histological examination of the kidneys showed multiple, not radially oriented thin walled cysts, internally lined by thickened pas-positive basement membranes and microcystic dilatation of collecting ducts. cystic changes were accentuated in the renal medulla. corticomedullar differentiation was mainly preserved. the tentative diagnosis was bbs. fetal dna was investigated using a next generation sequencing panel which included known bbs causing genes. hereby a heterozygous nonsense variant (np_ . : p.glu *) inherited from the mother and a heterozygous missense variant (p.glu lys) inherited from the father of the lztfl gene were identified. furthermore a maternally inherited heterozygous missense variant of unknown clinical significance in bbs was detected (np_ : p.pro ala). our case shows for the first time that mutations in lztfl can lead to a severe prenatal presentation of bbs due to profound renal manifestations with a kidney histology that is not considerably milder but distinct from that observed in mks. it is not clear to which extent the bbs variant may act as a disease modifier. this may challenge genetic counselling and prenatal diagnosis in a further pregnancy. furthermore our case shows that mesoaxial polydactyly is not always present in bbs patients with lztfl mutations and further studies are necessary to establish the frequency of mesoaxial polydactyly and other genotype phenotype correlations for bbs patients with lztfl mutations. p-cling- *** targeted next-generation sequencing analysis in couples at increased risk for autosomal recessive disorders k. komlósi, s. diederich, d. l. fend-guella, j. winter, o. bartsch, u. zechner, s. schweiger genetic childhood disorders leading to prenatal, neonatal or early childhood death are genetically heterogeneous. many follow autosomal recessive or x-linked modes of inheritance and bear specific challenges for genetic counselling and prenatal diagnostics. parents are carriers but unaffected and diseases are typically very rare but with recurrence risks of % in the same family. often, affected children (or fetuses) die before a genetic diagnosis can be established, post-mortem analysis and phenotype descriptions are insufficient and dna material of affected fetuses or children is not available for later analysis. a genetic diagnosis showing biallelic mutations or mutations on the x-chromosome in male fetuses or children is, however, the requirement for targeted carrier testing in parents, risk calculations, and prenatal and preimplantation diagnostics in further pregnancies. we employed targeted next-generation sequencing (ngs) for carrier screening of autosomal recessive lethal disorders in consanguineous (c) and non-consanguineous (nc) couples with one or more affected children. we searched for heterozygous variants (non-synonymous coding or splice variants as well as cnvs) in parents' dnas in a set of genes r. kropatsch , , j. preine , , jt. epplen , department of human genetics, ruhr university, bochum, germany, charcot-marie-tooth disease (cmt), also commonly called as hereditary motor sensory neuropathy, is the most common monogenetic disease of the peripheral nervous system with significant clinical and genetic heterogeneity. the main clinical manifestations of cmt include progressive distal muscle weakness and atrophy, impaired distal sensation, depressed tendon reflexes and high-arched feet. based upon electrophysiological and histopathological features cmt can be divided into predominantly demyelinating or axonal forms. an intermediate form also exists characterized by evidence of both, demyelinating and axonal, impairments. genetically cmt can be caused by mutations in over genes, including the dnm gene encoding dynamin protein, a large gtpase primarily involved in receptor-mediated endocytosis and membrane trafficking. only a small number of mutations in dnm causing cmt have been described so far. we report the case of a -year-old man presenting with backache, ataxic gait and distal muscle weakness of the lower limb considered to be a consequence of the pre-diagnosed disc prolapse years ago. over the past months bilateral progressive weakness of ankle dorsiflexion, foot drop and tingling paresthesia in stocking distribution have occurred. neurological examination disclosed depressed tendon reflexes of the upper and lower limbs. neurophysiologic investigations revealed an axonal sensible polyneuropathy with normal distal motor latencies and nerve conduction velocities. the sural nerve biopsy indicated single unmyelinated or thinly myelinated axons, loss of myelinated nerve fibers, numerous clusters of regenerating fibers without onion bulb formations suggesting an intermediate form of cmt. by using next generation sequencing (ngs) and a multi-gene panel, consisting of inherited neurological disease-associated genes, we identified a heterozygous missense mutation c. g>a, p.asp asn (rs ) in the dnm gene. this particular mutation is located in a highly conserved nucleotide region encoding the catalytic n-terminal gtpase domain. this evidence suggests a pathogenic phenotype caused by the described mutation, which is being underlined by the following facts: public genome database covering harmless variants of the human genome does not report it. additionally, the exac browser with exome sequencing data of > , unrelated individuals, lists the mutation and shows a low allele frequency of . , corresponding to one known heterozygous mutation carrier. other online prediction tools like the mutation taster, polyphen, sift and provean categorize it as pathogenic. in conclusion, the novel dnm mutation is responsible with high probability for the late-onset form of intermediate cmt of the investigated patient. heterozygous mutations in pcdh cause an x-linked female-limited form of an early infantile epilepsy (juberg-hellman syndrome). the phenotype of this syndrome is variable, ranging from benign focal epilepsy to severe, serial seizures, repeating up to more than times a day for several consecutive days. the intellectual outcome of affected patients ranges from normal to severe intellectual disability. psychiatric disturbances are frequent and manifest as autism, schizophrenia or aggressive behavior. neurological features such as ataxia may also be present. women with triple x syndrome usually show a normal physical development. cognitive deficits in vivo functional modeling of taf has already provided evidence for an effect on a neuronal phenotype. the phenotype in patients can be reminiscent of rett syndrome, but with milder regression and normal movements lacking a specific stereotypic pattern (no hand wringing). while severe neurodegeneration has been described in duplications, the present probands clearly showed developmental regression associated with a missense mutation. the analysis of further family members is pending. our case adds to the phenotypic spectrum of x-linked syndromic mental retardation type . targeted enrichment sequencing was successfully applied to identify greenberg dysplasia as cause of fatal anomalies in one fetus of a dizygotic twin pregnancy. p. m. kroisel , , b. csapo , , m. häusler , , l. michelitsch , , s. verheyen , , p. klaritsch , , k. wagner , institute of human genetics, medical university of graz, graz, austria, department of obstetrics and gynecology, gynecologist and obstetrician, weiz, austria in the first pregnancy of a year old kosovarian woman and her year old husband during the th week of gestation one of her dizygotic twins showed a severe skeletal dysplasia with all long bones extremely shortened and partially bended. the thorax was short and narrow. in addition a ventriculomegaly of the brain and an increased nuchal translucency was noticed. a very bad prognosis was expected and an achondrogenesis was suspected clinically. the other twin appeared to be normal. an amniocentesis was performed to potentially identify the genetic basis of the disorder. qf-pcr to rule out common trisomy's and cytogenetics revealed normal results and following a normal agilent k array cgh analysis as a next diagnostic step next generation sequencing by using the trusight one gene panel focusing on three genes including slc a , trip and col a was performed. since no pathogenic mutation was found by this approach, a more extended bioinformatics study was initiated. by filtering out common variants in the more than genes of the panel in our own database or in the exac-and genome databases our search was extended to genes with rare homozygous or compound heterozygous variants. by this strategy it was possible to reduce the potentially causative gene mutations dramatically and among those remaining genes for known very severe skeletal phenotypes just in the lbr gene the homozygous missense mutation c. a>g, p.asn asp was identified in our fetus. since this particular mutation is already known to be pathogenic leading to the lethal greenberg dysplasia (clayton et al., nucleus. ) the diagnosis could be achieved in the affected fetus of the pregnancy of our patient still before completion of the rd week of gestation. both parents were found to be heterozygous for this mutation in the lbr gene. recently it was shown that different mutations of the very same gene can also lead to less severe forms of bone dysplasia. the couple was informed about our results and possible consequences were discussed and offered. the couple however came to the decision not to draw any consequences. both fetuses especially the affected one were well documented sonographically including in a series of d images. in the th gestational week during a sonographic investigation the affected fetus did not show cardiac function and an oligohydramnios was found. since development of the second non affected fetus was still within the normal range, we hope the now single pregnancy will carry on normal until birth. from our finding we would propose that our chosen strategy is straightforward and can be applied in a wide range of pregnancies to identify various up to severe and fatal single gene disorders associated with sonographic anomalies within a few weeks which should provide substantial benefits for these families. after the working diagnosis ps had been established, molecular analyses regarding the recurrent akt mutation (p.glu lys) were performed by sanger sequencing in available affected tissue specimen of all three individuals. this revealed a high level of mosaic state for the akt mutation c. g>a, (p.glu lys) in affected tissues from bone and in meningiomas. re-evaluation of the ngs data from blood (individual i) confirmed the absence of that mutation in all reads, and no mutation was detected by sanger sequencing in dna from blood in individuals ii and iii. thus, a somatic mosaicism leading to a mild proteus phenotype could be confirmed as the underlying genetic cause in all three affected individuals. in conclusion, mild forms of proteus syndrome caused by the recurrent akt mutation in patients with limited regional involvement may be particularly difficult to diagnose and might be underdiagnosed. distal gne-myopathy: rare differential diagnosis of polyneuropathy here, we report a case of a -year-old patient with presumed polyneuropathy and elevated creatine kinase levels ( - u/l). clinical features included atrophic and bilateral paresis of lower legs of the frontal and rear compartment without high arched foot, while sensibility was not affected. additionally, a myopathic emg in m. tibialis anterior and a slight axonal damage in the motor neurography was detected. due to this overlapping neuromyological phenotype we performed a gene panel including genes associated with neuromuscular diseases using targeted next generation sequencing. gene panel analysis revealed the homozygous mutation c. c>t (p.arg trp) in the gne gene. this mutation is described in the literature as cause of a distal gne-myopathy and was also detected in an affected brother (ck u/l), having consanguineous parents. the current case emphasizes that a large gene panel analysis is recommended in case of an overlapping neuropathological and myopathological phenotype. c. landgraf, g. schmidt, s. morlot, b. schlegelberger, b. auber institute of human genetics, hannover medical school, hanover, germany ehlers-danlos syndrome (eds) is a heterogeneous group of connective tissue disorders. according to the villefranche classification, eds comprises six major types as well as some rare specific entities. one of these has been referred to as "eds, musculocontractural type" (mc-eds), "adducted thumb-clubfoot syndrome" or "eds, kosho type". first described in as an eds vi subtype, it recently was identified to be caused by biallelic changes in either the chst or dse genes, resulting in a loss of dermatan sulfate (ds) biosynthesis. characteristic symptoms are multiple congenital malformations such as contractures (club feet, adducted thumbs), visceral and ocular anomalies, generalized joint laxity, scoliosis, muscular hypotonia, fragile, hyperextensible and bruisable skin, as well as a typical craniofacial appearance. distinctive features include hypertelorism, down-slanting palpebral fissures, bluish sclerae, micro-corneae, short nose with hypoplastic columella and long philtrum, thin upper lip vermillion, small mouth, retrognathia, low-set and posteriorly-rotated ears. the psychomotor development is delayed. (chst ) and (dse) patients have been reported as yet. the patient, a -year-old woman using a wheelchair, had club feet, surgically corrected asd ii, muscular hypotonia, the characteristic face and hyperextensible skin with atrophic scars; particularly visible were those and learning disabilities are more common than in the general population and compared to siblings. their motor skills are likely to be somewhat impaired and coordination problems are frequent. in some patients psychological problems were described. furthermore, eeg abnormalities are occasionally observed, with clinical seizures present in up to % of patients. here we report on a -year-old girl with a ,xxx karyotype and early infantile, intractable epileptic seizures, beginning at the age of months. about three years later, she developed severe, serial seizures often related to febrile infectious diseases. in adolescence, the epileptic symptoms became less intense. she additionally showed autistic features, mental deficiencies, hypermobility of the joints and ataxia. array-cgh, fragile x-analysis as well as sanger sequencing and mlpa of the scn a and mecp genes revealed no additional abnormalities, besides the xxx karyotype. in the pcdh gene the heterozygous missense mutation c. a>g (p.asn ser) was identified, whereby the mutated allele seemed to appear in a : ratio compared to the wild type allele. this mutation has been previously described as disease causing. furthermore, three in silico prediction programs (sift, polyphen- , mutationtaster) classified the mutation as pathogenic. the patient's asymptomatic mother had a normal ,xx karyotype and was not a carrier of the pcdh mutation. pcdh -related epilepsy exhibits an unusual mode of inheritance in which only heterozygous females are affected and hemizygous males are asymptomatic carriers. random x-inactivation in the brain of females with pcdh mutations causes a cellular mosaicism, which likely accounts for the pathogenesis by altering the cell-cell-interactions ("cellular interference"). however, the precise mechanism is still unknown. hypothetically, the wide range of phenotypic expressions may be explained by partially skewed x-inactivation and thereby limitation of the cellular interference. hence, an unequal ratio of mutated to wild type cells should give a milder phenotype compared to the fifty-fifty situation. in contrast to this hypothesis, the phenotype in our patient was rather severe. nevertheless, we cannot exclude that the triple x status contributes additionally to the observed phenotypic expression. proteus syndrome (ps, omim ) is a highly variable disorder with asymmetric and disproportionate overgrowth of the body, connective tissue nevi, epidermal nevi, dysregulated adipose tissue, and vascular malformations, caused by a somatic activating akt mutation. we report on three unrelated individuals (two adults and one year old boy) who showed similar clinical findings that not fulfilled the rigorous clinical criteria for ps (biesecker, ) . beside an asymmetric hyperostosis of the skull or facial bones, all three had an ocular dermoid. two individuals developed alveolar hyperostoses and intracranial calcifying meningiomas. only one individual showed skin changes. all three had normal feet and no vascular lesions. molecular analyses in individual i performed in blood revealed normal results for array karyotyping and no relevant variant in whole exome sequencing (trio approach). introduction: incontinentia pigmenti (ip) is a rare x-linked male lethal genodermatosis that affects the neuroectodermal tissue and is always associated with a bullous rash of the skin along blashko lines in female neonates. it is caused by mutations in ikbkg which encodes the regulatory subunit of the ikb kinase complex required for nf-kb activation. ikbkg has a pseudogene (ikpkgp) with identical exons to . the most frequent ip mutation is a recurrent exon _ deletion due to non-allelic homologous recombination with the pseudogene. here, we report a novel deletion of exons and in the ikbkg transcript recognized by rna analysis. patient: we investigated a yr old girl with typical erythematous rash after birth which resolved within - m. apart from a small hyperpigmented area around the right mammilla there were no skin alterations. she had few conically shaped teeth, normal nail and hair structure, no neurological manifestation and normal intelligence. only clinical sign were repeated vitreous hemorrhage of the left eye from age m. family history was negative. methods: analyses on genomic dna extracted from blood included testing for the common ikbkg exon _ deletion by long range pcr and mlpa (p -a , mrc holland), x inactivation analysis in the androgen receptor gene as described, and massively parallel sequencing (mpseq) of the ikbkg gene (trusightone, nextseq, illumina®; data analysis with nextgene/geneticist assistant [softgenetics®] and seqnext [jsi®]; reference sequence grch , hg ). rna was extracted from cultured blood lymphocytes, and the entire ikbkg transcript (nm_ . , exons) was sanger sequenced on cdna level (the a of the start codon is in exon ); the results were analyzed with sequencepilot (jsi). result: genomic dna analyses including mlpa of ikbkg and ikbkgp specific probes in our patient did not reveal a putative mutation. there was a completely skewed x-inactivation pattern. cdna sequencing of ikbkg demonstrated skipping of exon and (r. _ del) which is predicted to cause a frame shift starting from codon p.gln , a premature stop codon amino acids downstream (p.gln glyfs* ) and complete loss of protein function. the loss of exons and is most likely due to an intronic splice variant in intron ; investigations regarding the origin of this deletion are ongoing. conclusion: the presence of the high homologous pseudogene makes sequence analysis of ikbkg challenging. we report a deletion of exons and in the ikbkg transcript that required rna analysis for its identification. due to the skewed x-inactivation and typical clinical picture causality of the detected deletion is certain. the exact genomic cause of this alteration remains to be clarified. also in the era of mpseq, rna analysis may be necessary for detection of deep intronic mutations or the study of genes with homologous pseudogenes, as shown here in the case of ikbkg. primary congenital glaucoma (pcg) and early onset glaucomas are one of the major causes of of blindness in children and young adults' worldwide. both autosomal recessive and dominant inheritance have been described resulting from bowel surgeries due to colon perforation. her older sister who died aged following an acute abdomen had club feet and the typical facial appearance, while three healthy sisters seem to be unaffected. her parents are first cousins of turkish origin and do not show eds symptoms either. the two affected sisters had been diagnosed with a syndromic disorder that could represent a rare form of eds. however, neither a confirmation of the suspected diagnosis nor a classification had yet been achieved. due to the distinctive symptom complex and the presumed autosomal recessive inheritance pattern, we strongly suspected this to be a case of mc-eds. sequencing of the chst gene (reference sequence: lrg_ ) revealed a formerly undescribed homozygous variant (c. c>t; p.pro- leu). the variant changes the highly conserved pro residue which is located in the critical '-phospho- '-adenylyl sulfate binding site and can be classified as likely pathogenic (acmg standards and guidelines, richards et al., genetics in medicine ) . the parents are heterozygous carriers of this variant, respectively. this case represents a unique entity within the umbrella term eds and illustrates the importance of clinical assessment leading to a diagnosis confirmed by genetic analysis. the underlying genetic defect in patients with mitochondrial peo is either a primary mutation of the mitochondrial genome (single, large-scale mtdna deletion or mtdna point mutation) or recessively and dominantly-inherited mutations in nuclear genes involved in mtdna maintenance leading to clonally-expanded multiple mtdna deletions in muscle. the nuclear disease genes are largely implicated in the replication and stability of mtdna, and as such a pathogenic mutation leads to secondary instability of the mitochondrial genome. causal mtdna deletions can be found in a heteroplasmic (mixture of mutated and wild type mtdna) state. however, each tissue/cell has its own biochemical threshold of mutant mtdna load which needs to be exceeded before focal respiratory chain deficiency becomes evident. to investigate this, muscle biopsies of patients with genetically-and clinically-characterized mitochondrial disease of nuclear origin ( polg, twnk, rrm b and slc a (ant )) and healthy controls were analysed using quadruple oxphos immunohistochemistry, quantifying the biochemical phenotype in individual muscle fibres of patient muscle biopsies. this technique is based on quadruple immunofluorescence to detect structural components of complexes i (ndufb ) and iv (coxi), as well as porin (a marker of mitochondrial mass) and laminin (a cell membrane marker to define the boundaries of muscle fibres). further studies on / patients ( polg, rrm b, twnk, slc a (ant )) included the correlation of the biochemical deficiency with the mtdna abnormality in individual cells, following laser microcapture and determination of the size and level of clonally-expanded mtdna deletion within fibres by real-time pcr. our preliminary data from quadruple immunocytochemical studies show that the muscle biochemical phenotype is different in patients with multiple mtdna deletions compared to other mtdna mutations; work is continuing to determine the exact size and level of clonally-expanded mtdna deletion in individual muscle fibres and correlate this with the observed biochemical defects and disease thresholds. abstracts these families, and functional studies, together with phenotype descriptions in the literature, are essential for pathogenic grading. however, several difficulties remain, such as the huge size of the ttn gene (> kb) impeding functional studies, the wide spectrum of phenotypes and variants, the still small patient cohort, and often unspecific immunohistochemical abnormalities in muscle biopsies. the clinical evaluation of ttn variants thus presents a great challenge to the field of human genetics diagnostics. compound heterozygous variants in the qars gene (omim ) have been identified in only four patients with autosomal recessive progressive microcephaly with seizures and cerebral and cerebellar atrophy (mscca), to date. these patients showed severe developmental delay, progressive primary microcephaly, intractable seizures, hypomyelination or delayed myelination, thin corpus callosum, and small cerebellar vermis on brain imaging. here we report on two unrelated girls with progressive primary microcephaly, epilepsy and brain anomalies. trio exome analysis in each of the families revealed two different combinations of compound heterozygous variants in qars. all four variants are highly conserved throughout vertebrates, not reported in any database, yet, and in silico analysis predicted the variants as possibly damaging or deleterious. the first patient was born to non-consanguineous german parents. at birth, she was too short (− . sd) and mildly microcephalic (− . sd). she developed intractable seizures within the first hour of life. her growth continued to be mildly retarded (− . sd at age years) but microcephaly was progressive (− . sd at age years). she did not achieve any of the motor or cognitive developmental milestones, she did not have eye contact. the only interaction with her surrounding was a diffuse reaction to being touched. cranial mri showed no myelination of the supratentorial region, corpus callosum agenesis, simplified gyral pattern of frontal lobes, enlarged cerebral ventricles, and normal brain stem and cerebellum. trio-exome sequencing revealed the compound heterozygous qars variants c. c>t, p.(arg- cys) and c. c>t, p.(arg *). segregation analysis by sanger sequencing confirmed the heterozygous variants in the parents and two non affected sibling of the index patient. the second patient was initially evaluated at days of age when she exhibited myoclonic seizures, intrauterine growth retardation, microcephaly, and elevated lactic acid. at birth, she was microcephalic (hc cm) and microcephaly was progressive (− . sd at age months). cranial mri suggested undersulcation. she has required a gastrostomy feeding tube. trio-exome sequencing revealed the compound heterozygous qars variants c. g>a, p.(gly ser) and c. c>t, p.(arg trp). segregation was confirmed by sanger sequencing analysis. together with the four previously described patients we conclude that compound heterozygous variants in qars are associated with a primary and progressive microcephaly, early onset of intractable seizures and severe developmental delay. brain imaging in the neonate can show simplified gyral pattern as an early characteristic feature. overlapping phenotypes are seen in patients with epileptic encephalopathy, lissencephaly and primary microcephaly. application of ngs panels or exome technology will allow for early diagnosis and further collection of patients for better delineation of the phenotype. with involvement of several genes including cyp b , foxc , pitx , myoc and pax . however, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes. to elucidate further genetic causes of these conditions we performed whole exome sequencing in a patient with pcg and retinal detachment and identified compound heterozygous variants in col a (p.met leu; p.ala- thr). targeted col a screening of additional patients detected three further heterozygous variants (p.arg *, p.gly ser and p.gly- val) in three distinct subjects: two of them were diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (oi), one patient had a diagnosis of pcg at age years. all five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main col a binding proteins: hsp and fibronectin. dominantly inherited mutations in col a are known causes of connective tissues disorders such as oi. these disorders are also associated with different ocular abnormalities, although the common pathology for both features is seldom recognized. our findings expand the role of col a mutations in different forms of early-onset glaucoma with and without signs of oi. thus, we suggest including col a mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with oi. the gene ttn encodes the largest known protein, titin, which plays a key role in structural, mechanical, developmental and regulatory functions of cardiac and skeletal muscles. accordingly, titinopathies are characterized by great clinical and genetic heterogeneity. the clinical spectrum ranges from severe phenotypes with cardiac involvement to pure myopathies at the milder end, including autosomal recessive and dominant inheritance patterns (chauveau et al. , hum mutat; : ) . next generation sequencing analysis identifies a large number of variants of unknown clinical significance; the potential clinical relevance of these variants cannot be assessed with certainty without further studies. three case reports highlight the difficulties in human genetics diagnostics concerning ttn. the first case is of a year-old woman with proximal muscle weakness, slightly elevated ck, scoliosis, and no family history. a heterozygous known pathogenic variant was identified in mex , associated with autosomal recessive congenital core myopathy combined with primary heart disease. additionally, an unknown variant was detected. both variants could be clinically relevant with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the second case is of a month-old finnish girl who presented with severe muscle hypotonia at birth and mental alertness with normal brain mri and eeg. congenital fiber-type disproportion was suspected. a homozygous frame-shift mutation in mex was identified which to our knowledge has not yet been described in the literature. this variant is likely of clinical relevance with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the third case is that of a year-old woman with suspected myofibrillar myopathy. a known pathogenic homozygous frame-shift mutation in mex was detected which is associated with autosomal recessive congenital myopathy with central nuclei. segregation analysis revealed that the healthy parents are heterozygous carriers of this variant. the clinical diagnosis of a ttn-associated disease could therefore be confirmed. ttn variants need to be assessed in combination with detailed clinical and muscle biopsy data. segregation analysis is necessary but not sufficient for the clinical grading of variants. identification of a variant in several independent families, segregation of the variant with disease phenotype in gous pathogenic smad variant c. dupt;p.(ala fs) (ncbi reference sequence nm_ . ). gastric as well as colonic cancer and polyposis was present in the paternal family history. conclusions: ts and other imprinting disorders are likely underdiagnosed, as the main clinical features (e. g. growth retardation, hypotonia) are distinct but unspecific. as exome sequencing becomes a more frequent diagnostic procedure, imprinting disorders caused by mutations in imprinting centers will presumably be diagnosed more often. methylation defects, however, will remain underdiagnosed, without a specific clinical differential diagnosis, which would guide to appropriate analysis of the methylation status. a bowel invagination in early childhood due to a single polyp can be a symptom of jps, especially in the context of a paternal history of polyposis and intestinal cancer; thus, family history should be carefully obtained. in the outpatient clinic, child psychiatrists as well as neurologists thoroughly work up phelan-mcdermid patients according to a standardized protocol by taking medical history, performing physical examination, and, if needed, organizing further supplementary examinations. in addition, a genetic analysis and hair/tissue sampling is performed. since its foundation, a steadily increasing number of so far patients from all over germany has been seen and treated. the outpatient clinic aims at facilitating and accelerating the diagnosis of phelan-mcdermid syndrome, improving medical support for affected patients of all ages, and, last but not least, fostering a better understanding of the causes and pathomechanisms leading to the symptoms of the disease. t. m. neuhann, l. neuhann, c. rapp, a. laner, a. benet-pages, e. holinski-feder medizinisch genetisches zentrum, münchen, germany congenital eye malformations, such as the microphthalmia-anophthalmia-coloboma (mac) spectrum, congenital cataracts, anterior segment dysgenesis (asd), and congenital glaucoma, affect more than : . newborns. the phenotypic spectrum of the aforementioned entities is highly variable and partially overlapping. eye malfomations are very heterogeneous; to date causative mutations have been described in more than next-generation-sequencing (ngs) technology has revolutionized genomic research and has transformed clinical diagnostics. ngs offers enormous potential for providing accurate diagnoses to individuals with previously unresolved syndromes. in the pediatric endocrine clinic, clinicians are often faced with the task of making a diagnosis in children with syndromic short stature. as there may be considerable clinical overlap between short stature syndromes, deriving a clinical diagnosis may prove challenging. furthermore, even if a clinical differential diagnosis is established, often several genes would need to be tested before a molecular diagnosis is made. as access to genetic testing is limited in algeria, we conducted a pilot study on algerian patients with syndromic short stature using a combination of two different ngs modalities, namely whole-exome-sequencing (wes) and mendeliome sequencing (trusight one sequencing panel). a molecular diagnosis could be established in / patients, making the diagnostic rate in this initial cohort %. as patients had novel mutations we could expand the mutational spectra of several genes, namely cul , npr , sos , vps b, and znf . we could thus substantiate the clinical utility of wes and the mendeliome in patients with a diverse array of syndromic short stature syndromes. chromosome harbours an imprinted locus at q . maternal uniparental disomy of chromosome , paternal deletions and paternal loss of methylation at the intergenic differentially methylated region (ig-dmr) and the somatic dmr within meg are associated with temple syndrome (ts , mim ). the phenotype of ts consists of pre-and postnatal growth retardation, early feeding problems and muscular hypotonia, joint laxity, motor developmental delay, premature puberty, and truncal obesity. juvenile polyposis syndrome (jps, mim ) is characterized by predisposition to hamartomatous polyps in the gastrointestinal (gi) tract, specifically in the stomach, small intestine, colon, and rectum, including the risk for gastrointestinal cancer. pathogenic variants in the bmpr a and smad gene are identified in about - % of affected families. we report on a family with two female children. the index patient, an -year-old girl, was diagnosed to have ts due to hypomethylation at the somatic dmr within meg with clinical features reminiscent of prader-willi syndrome in early childhood and milder clinical signs at further age (i. e. mild global development delay, muscular hypotonia, suspected central obesity, no prominent facial dysmorphisms). snp array-cgh analysis was unsuspicious and no deletion of the imprinting center was observed. thus, ts is caused by a sporadic imprinting defect in our patient. her -year-old sister was diagnosed with smad -associated jps after an episode of intestinal invagination due to a polyp, histologically diagnosed as peutz-jeghers polyp, in early infancy. sequencing identified a heterozy-abstracts coding vmat have only very recently been described as causal for brain dopamine-serotonin vesicular transport disease in two families with multiple affected children (rilstone et al., n engl j med , , - ; jacobsen et al., j inherit metab dis , , - ) . the index case presented here is a -year-old girl with severe mental retardation and a dystonic movement disorder. she is the tenth child of a consanguineous arabic couple and was initially referred to neuropaediatric examination at the age of four months due to recurrent oculogyric crises and muscular hypotonia. blood metabolic testing and cerebrospinal fluid (csf) analyses were inconclusive. notably, biogenic amines were within their normal ranges and the differential diagnosis of aromatic l-amino acid decarboxylase (aadc) deficiency could not be confirmed. conventional cytogenetics, subtelomeric screening, array-cgh and different ngs panel analyses did not identify a causative mutation. both parents and all eight living siblings are obviously unaffected. a brother with a known hypotonic movement disorder died at the age of three years due to prolonged seizures with hyperthermia and cerebral edema. by utilizing whole-exome sequencing, we identified a homozygous substitution in the slc a gene of the index case causing an amino acid change (c. c>a; p.pro his) in a conserved transmembrane domain of vesicular monoamine transporter (vmat ). homozygosity for this missense change could also be verified in a dna sample of her deceased brother. an obvious reduction in frequency of oculogyric crises was observed in our index case under therapy with pramipexole already within weeks after start of treatment. furthermore the patient shows less dystonic movements under therapy. the case presented here highlights the importance of considering brain dopamine-serotonin vesicular transport disease as differential diagnosis for early-onset extrapyramidal movement disorders combined with mental retardation even if neurotransmitters in csf are normal. for a large number of individuals with intellectual disability (id), the molecular basis of the disorder is still unknown. however, whole exome sequencing (wes) is providing more and more insights into the genetic landscape of id. in the present study, we performed trio-based wes in patients with unsolved id and additional clinical features, and identified homozygous cplx mutations in three patients with id from two unrelated families. all displayed marked developmental delay and migrating myoclonic epilepsy, and one showed a cerebellar cleft in addition. the encoded protein, complexin , is crucially involved in neuronal synaptic regulation, and homozygous cplx knockout mice have the earliest known onset of ataxia seen in a mouse model. recently, a homozygous truncating mutation in cplx was suggested to be causative for migrating epilepsy and structural brain abnormalities. id was not reported. the currently limited knowledge on cplx suggests that complete loss of complexin function may lead to a complex but variable clinical phenotype, and our findings encourage further investigations of cplx in patients with id, developmental delay and myoclonic epilepsy to unravel the phenotypic spectrum of carriers of biallelic cplx mutations. genes. due their heterogeneity, diagnostic testing for congenital eye malformations was limited in the pre-ngs era. we performed exome analysis in patients with congenital eye malformation (mac spectrum, asd, congenital cataract, congenital glaucoma). primarily, a gene panel comprising genes associated with eye malformations was evaluated. additionally the exome data was evaluated in selected patients as a second step. the panel analysis revealed pathogenic sequence variants in patients and genes (mab l , bcor, nhs, prss , cyp b , foxc , pitx , gcnt ). putatively causative sequence variants were identified additional patients. the diagnostic yield of the panel was highest in patients with non-syndromic microphthalmia/coloboma and congenital cataracts, and lowest in patients with syndromic mac spectrum (i. e. additional systemic features/malformations). ngs based panel testing is a strong diagnostic tool to determine the underlying causes of non-syndromic congenital eye malformations. due to the partially overlapping phenotypes and high heterogeneity it is more sensible to perform large gene panel analysis, as opposed to smaller single phenotype based panels. superactivity of phosphoribosyl¬pyrophosphate synthetase i (prpps) is a rare inborn error of purine metabolism that is characterized by increased levels of uric acid in blood and urine (omim ). the disorder is caused by gain-of-function mutations in the x-chromosomal gene prps . in male patients, disease manifestation is in early childhood. additional clinical characteristics include intellectual disability, hypotonia, ataxia and hearing loss. heterozygous female mutation carriers have a later age of onset and a less severe clinical course. only seven families with prps gainof-function mutations have been reported to date. we report on a -year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias and facial dysmorphisms. his mother also had hyperuricemia that was diagnosed at age years but was otherwise healthy. a novel prps missense mutation (c. g>c, p. leu phe) was detected in the proband and his mother. enzyme activity analyses confirmed superactivity of prpp synthetase. the family reported here broadens the clinical spectrum of prpps superactivity and indicates that this rare metabolic disorder is associated with a recognizable facial gestalt. homozygous and compound heterozygous mutations of the rnu at-ac gene are associated with mopd and roifman syndrome. mopd is characterized by severe microcephaly with brain malformations including abnormal gyral pattern, corpus callosum agenesis or hypoplasia, vermis hypoplasia and intracranial cysts, psychomotor retardation, short stature, skeletal dysplasia, dry skin, sparse hair, flexion contractures, round face with beaked nose and protruding eyes, and premature death with a majority of the patients who die before the age of months for unknown reasons. roifman syndrome was first described as a novel association of antibody deficiency, spondyloepiphyseal chondro-osseus dysplasia, retinal dystrophy, poor pre-and postnatal growth, cognitive delay and facial dysmorphism including long eyelashes, downslanting palpebral fissures, a long philtrum and a thin upper lip. all patients with roifman syndrome reported so far lack brain malformations. the rnu atac gene encodes a small nuclear rna (snrna), which is essential for minor intron splicing. homozygous (g. g>a, g. g>a) and compound heterozygous mutations (g. g>a;g. g>a, g. g>a;g g>a and g. c>t;g. g>a) have been described in mopd . all mutations involve the ' or ' stem loop of the u atac snrna. in contrast, all cases with roifman syndrome investigated so far showed compound heterozygous rnu atac mutations with one allele harboring a mutation in the mopd associated ' stem loop and the other allele showing a mutation in the stem ii site of the u atac snrna, which has not been involved in mopd , so far. thus, the different pattern of the mutations observed in mopd and roifman syndrome may contribute to the distinct features of both syndromes. however, our patient shows, that features of mopd , i. e., brain malformations, may also be present in patients who show roifman syndrome associated rnu atac mutations. this indicates that both syndromes may represent overlapping features of the clinical spectrum of rnu atac mutations. h. roth, h. stöhr, b. h. f. weber institute of human genetics, university of regensburg, germany introduction: inherited retinal degenerations comprise a genetically heterogeneous group of eye diseases with overlapping clinical presentations. up to now, more than genes have been associated with different forms of retinal dystrophies (rd) such as retinitis pigmentosa (rp) or cone-rod-dystrophies (crd) with mutations in and causative genes, respectively. here, we present the results from three patients with remarkable findings and discuss their implications for risk prediction and genetic counseling. methods: targeted next-generation sequencing (ngs) technology based on agilent custom designed gene panels (sureselect) has been established in our diagnostics department to identify causative mutations in a large patient cohort with approximately rd patients. high-throughput sequencing data are routinely analyzed with the clc biomedical workbench. classification of variants was based on bioinformatic analyses using alamut visual software, mutationtaster, sift and polyphen- prediction programs, allele frequencies, amino acid conservation and literature. results. ngs analysis revealed two patients with rp and one patient with crd, each of whom carry putative causative mutations in several rd genes. first, a male patient with a family history of crd, is a carrier of a nonsense mutation p.(arg ter) in rims and two likely pathogenic missense mutations in aipl (p.(tyr phe)) and guca a (p.(pro- leu)), each in a heterozygous situation. mutations in all three genes can cause adcrd. in addition, the patient carried a hemizygous nonsense mutation p.(glu *) in the x-chromosomal rpgr gene. secondly, a female patient with simplex rp was found to be homozygous for a frameshift-causing deletion p.(ser leufs* ) in the impg gene causing arrp. she also carried three heterozygous, likely pathogenic missense mutations in crx (p.(tyr cys)) causing adrp, in the x-chromosomal rpgr (p.(ala val)) and in ush a (p.(ile val)) associated m. s. reuter , a. riess , u. moog , t. a. briggs , , k. e. chandler background: disruptions of the foxp gene, encoding a forkhead transcription factor, are the first known monogenic cause of a speech and language disorder. so far, mainly chromosomal rearrangements such as translocations or larger deletions affecting foxp have been reported. intragenic deletions or convincingly pathogenic point mutations in foxp have up to date only been reported in three families. we thus aimed at a further characterization of the mutational and clinical spectrum. methods: chromosomal microarray testing, trio exome sequencing, multi gene panel sequencing and targeted sequencing of foxp were performed in individuals with variable developmental disorders, and speech and language deficits. results: we identified four different truncating mutations, two novel missense mutations within the forkhead domain and an intragenic deletion in foxp in fourteen individuals from eight unrelated families. mutations occurred de novo in four families and were inherited from an affected parent in the other four. all index patients presented with various manifestations of language and speech impairment. apart from two individuals with normal onset of speech, age of first words was between and years. articulation difficulties such as slurred speech, dyspraxia, stuttering or poor pronunciation were frequently noted. motor development was normal or only mildly delayed. mild cognitive impairment was reported for most individuals. conclusion: by identifying intragenic deletions or mutations in fourteen individuals from eight unrelated families with variable developmental delay/cognitive impairment and speech and language deficits, we considerably broaden the mutational and clinical spectrum associated with aberrations in foxp . h. rieder, f. beleggia, d. wieczorek we report on a -year-old boy with microcephaly, arachnoidal cysts, pachygyria, microgyria, and severe intellectual disability. he also had short stature including shortening and deformation of the femora, brachydactyly, and short ribs with costochondral dysplasia. he showed facial dysmorphism with narrow palpebral fissures, a short nose with a depressed nasal bridge, and a broad mouth with full lips. clinical laboratory investigations demonstrated persistently slightly elevated liver enzymes. exome sequencing revealed compound heterozygous mutations of the rnu at-ac gene, g. g>a;g. g>a, which has been described in an individual with roifman syndrome. we report on a seven-year-old girl, first child of non-consanguineous italian parents, with developmental delay, muscular hypotonia and distinctive craniofacial features (epicanthus inversus, ptosis, broad nasal bridge, mild retrognathia, low-set posteriorly rotated ears and malpositioned teeth in the mandible). because of the tentative diagnosis of blepharophimosis-ptosis-epicanthus inversus syndrome (bpes), conventional cytogenetic analysis, sanger sequencing and mlpa (multiplex ligation-dependent amplification) of foxl were initiated and showed unremarkable results. microarray-cgh revealed a kb microduplication of genetic material on q . : arr[hg ] q . ( _ )x encompassing the genes tubgcp , cyfip , nipa and nipa of maternal origin. patients with q . microduplication have been described to be affected by developmental delay, motor and/or expressive language delay, epilepsy, learning disabilities and/or behavioral problems. however, genotype phenotype correlation is complicated by incomplete penetrance. healthy and mildly affected carriers are reported in the literature. we speculate that the microduplication might contribute but does not fully explain the phenotype of our patient, in particular concerning the craniofacial features. subsequent trio whole-exome sequencing identified a de novo heterozygous mutation in setbp (c. t>a/ p.tyr *) leading to a premature stop codon and most probably resulting in a truncated and functionally impaired protein. mutations in the set binding protein gene (setbp ) on q . have been identified to cause schinzel-giedion syndrome (sgs, omim ), a rare autosomal dominant disorder characterized by postnatal growth failure, severe developmental delay, seizures, facial dysmorphism, genitourinary, skeletal, neurological, and cardiac defects. chromosomal deletions in q including setbp have been reported to cause a milder phenotype known as "autosomal dominant mental retardation- " (mrd , omim ). these observations suggest that the severe sgs phenotype might be the consequence of a gain-of-function or dominant-negative effect of the mutations and that setbp haploinsufficiency results in a different, milder phenotype. so far, the function of the set-bp protein is unknown. the presented case adds up to the yet small number of reported cases of mrd and thereby contributes to the clinical spectrum of setbp haploinsufficiency. this work was supported by "förderstiftung des uksh" (project number: _ ). the demand for genetic counseling had been constant, in germany, over many years. from to around . cases per year on the average and with minor fluctuations were reimbursed by the german sickness funds (public health insurance system; pabst and schmidtke, gendiagnostik in deutschland, bbaw, p. - , ) . in connection with the "genbin "-project, a new nationwide survey was initiated regarding with arrp. finally, in another female rp patient with no family history of rd, we detected a nonsense mutation p.(trp ter) and a likely pathogenic splice site change (c. + a>g) in the arrp gene eys, assuming compound heterozygosity. in this patient we also identified two heterozygous, likely pathogenic missense mutations in hmcn (p.(pro thr)) and cep (p.(arg cys)) underlying dominant and recessive forms of rd. in all three cases, specific mutation(s) could not be uniquely identified as causative. conclusion. results in the three rd cases emphasize that ngs can generate unexpected results that are difficult to interpret, particularly in the absence of segregation analysis and functional data on pathogenicity. the implications for genetic counselling and predictive testing will be discussed. smart qnipt study -detection of fetal trisomy based on methylation-specific quantitative real-time pcr m. sachse, s. werler, j. bonnet, u. neder, h. sperling, s. busche, s. grömminger, w. hofmann lifecodexx ag, konstanz, germany objectives: current non-invasive prenatal testing (nipt) methods for the detection of fetal trisomy (t ) are primarily based on next generation sequencing (ngs) strategies which are quite costly in clinical application and hence are limited to patients who can afford the testing. here, we describe the results of a blinded study with respect to the test accuracy of a newly developed nipt assay based on quantitative real-time pcr (qpcr) for prenatal testing of fetal trisomy (qnipt). methods: in the study maternal plasma samples were collected from , pregnant women and blinded by an independent contract research organization. after extraction of cell-free dna using qiasymphony instrument and methylation-specific digestion of dna samples a multiplex qpcr was performed. the primary qpcr data were finally evaluated with our ce marked data analysis software. results from this analysis and from confirmatory ngs testing were compared with nipt results using ngs. the study results of successfully analysed maternal plasma samples (n = ) demonstrated a positive percentage agreement (ppa; equates to sensitivity) of % (lower -sided % confidence interval of . %; n = / ) and a negative percentage agreement (npa; equates to specificity; n = / ) of % compared to ngs-based results. the negative predictive value (npv) for the novel qnipt and confirmatory ngs testing was % (lower -sided % confidence interval of . %). the average fetal fraction of the examined blood samples was . %. the qnipt assay provided reliable test results in blood samples with a fetal fraction below % and as low as . %. conclusion: our results suggest that the proprietary qnipt assay is a very reliable and robust method suitable for clinical routine in accordance with international medical associations. the assay represents a more cost-efficient solution over ngs testing and will also be able to provide results in the shortest possible time. while current nipt methods require a minimum fetal fraction of % in blood samples from singleton pregnancies, we could demonstrate in the study that our smart qnipt assay can be employed on blood samples with a fetal fraction of as low as . %. in summary, the application of smart qnipt could have the potential to become a nipt solution on a global scale for pregnant women of all ages and risk groups. further studies which aim to include the determination of trisomy and trisomy are currently underway. with respect to the developmental delay of our index patient, chromosome analysis and array-cgh were performed. a microduplication in p . (app. kb) of unknown significance and a microduplication in xq . (app. kb), which comprises the fmr -gene, were identified and shown to be of maternal origin (arr[hg ] p . ( , , , )x , xq . ( , , , )x ). fmr is associated with fragile x syndrome, which is one of the most common causes for x-linked mental retardation. cgg-trinucleotide repeat expansions in the ' untranslated region (> repeats) lead to aberrant hypermethylation of the fmr -promotor and silencing of fmr expression. in contrast, premutations ( - repeats) lead to a higher expression of fmr and cause a clinical syndrome that is characterised by late progressive cerebellar ataxia (fxtas). in line with this gain-of-function mechanism, we hypothesize that the xq . duplication, which could lead to an increased gene dosage of fmr , causes a fra(x)-/fxtas-like syndrome and explain the clinical findings in our family. vengoechea et al. described a patient with a similar duplication, who was affected by developmental retardation, epilepsy and hyperactivity. they discussed the microduplication, which arose de novo in their patient, as the cause for the boy's symptoms (vengoechea j. et al., eur j hum genet., nov; ( ): - ) . in conclusion, we assume a fmr -duplication syndrome in our family with variable expressivity and a different impact on male and female patients. to further prove this hypothesis, we are planning to perform a segregation-analysis within the whole family. background: congenital myasthenic syndromes (cms) are a genetically heterogenous group of disorders leading to weakness of skeletal muscles -especially ocular, bulbar and limb muscles -with onset mostly at birth or in early childhood. the severity of cms can vary significantly ranging from death in early childhood due to respiratory insufficiency to only mild muscle weakness in adulthood. more than genes that are highly expressed in the neuromuscular junctions are associated with cms. mutations in the chrne gene on chromosome p . are responsible for about one half of genetically solved cms cases. they can cause different subtypes of cms with either autosomal dominant or autosomal recessive inheritance. results: here, we report a -year-old boy who was born with bilateral eyelid ptosis and congenital vertical talus of the right foot that needed surgical correction. the boy displayed muscular hypotonia with a myopathic facial expression and delayed motor development. ophthalmologic examination revealed external ophthalmoplegia. a next generation sequencing based gene panel for congenital myopathies detected the homozygous frameshift mutation c. _ dup (p.leu profs* ) in the chrne gene in the boy. gene dosage analysis did not show an exonic deletion in the chrne gene. sanger sequencing confirmed the mutation in a heterozygous state in the boy's father. however, his mother did not carry the mutation in the chrne gene. conclusions: these results suggest the rare event of a (partial) paternal uniparental isodisomy of chromosome as cause of the homozygous c. _ dup (p.leu profs* ) in the chrne gene in the boy. further experiments are currently undertaken to confirm this hypothesis. the annual reimbursement frequencies of the relevant entries in the ebm fee schedule, , , and , for which only specialists in human genetics and subspecialists in medical genetics can account, from until . contrary to the findings in the earlier period the demand for genetic counseling has risen sharply: , (of a total of , ) cases in ; , ( , ) in ; , ( , ) in ; , ( , ) in ; , ( , ) in ; and , ( , ) in . we speculate that the temporal correlation of the rise of genetic counseling demand with the enactment of the german act on testing (february , ) is not coincidental. further factors that might contribute to the increase in demand are the ensuing guidelines of the german commission on genetic testing and cme activities related to attaining a qualification for genetic counseling for specialties other than human genetics. in the course of these activities the awareness for the importance of genetic counseling delivered by specialists in human genetics and subspecialists in medical genetics may have risen. acknowledgements: the "genbin "-project supported by the robert koch-institute through funding from the german federal ministry of health. we gratefully acknowledge the collaboration with dr. michael erhart, zentralinstitut der kassenärztlichen bundesvereinigung (zi-kbv), berlin, germany it is well known that duplications of down syndrome critical region (dscr) on chromosome q can cause down syndrome whereby the distinct phenotype is associated with the involved genes and the size of duplication. however, in literature are hardly any cases with mosaic duplications of dscr described. here we report on a year old boy with some clinical features of down syndrome including distinctive craniofacial dysmorphism, simian crease and sandal gap as well as delayed motor and speech development. no other organ abnormalities are known. conventional chromosome analysis showed no numerical or structural aberration whereas interphase fish analysis revealed three signals for dscr in approx. % of lymphocytes and in approx. % of buccal mucosa cells. array-cgh analysis on dna from peripheral blood confirmed a , mb duplication of chromosome q . q . . the duplication involves among others the gene dyrk a which is reported as a candidate gene for down syndrome. this case presents one of the smallest known duplications within dscr which causes even in a mosaic state a mild phenotype of down syndrome. -year-old boy was referred to our outpatient clinic due to global developmental delay mainly affecting his speech and his fine motor development. in addition, muscular hypotonia and an abnormal gait were reported by the referring paediatrician. his mother, his maternal grandmother, and numerous relatives are affected by gait ataxia. no causative mutation was detected in the maternal grandmother by means of a multi-gene panel for spinocerebellar ataxia encompassing genes. genetic testing for friedreich ataxia was also without pathological findings. cer, but the patient mother's grandfather had a cancer of unknown origin and died at the age of years. because of the suspicion of having a lynch syndrome an immunohistochemistry and microsatellite analysis have been performed on the tissue of the colorectal cancer and the hepatic metastasis. all four mmr proteins were properly expressed in immunohistochemistry in colorectal cancer. just the expression of mlh protein in the hepatic metastasis was focally weakened and inhomogeneous. the microsatellite markers bat , bat , d s (apc), d s and d s (mfd) were all stable, a a t-kras mutation was found. after performing a multi-gene panel (ngs, next-generation sequencing), a gross heterozygous deletion of exon in msh gene has been found in the cnv analysis of the ngs data. this mutation was confirmed with a mlpa and quantitative real time pcr analyses. furthermore, rna expression of msh was reduced to % in blood lymphocytes in comparison to control samples pointing to a potential role of msh loss in the patient's tumor development. we are observing more and more patients with probably pathogenic and pathogenic mutations in one of the mmr genes with normal immunohistochemistry and microsatellite analysis. therefore, we propose that the criteria for performing a molecular genetic analysis of hnpcc/lynch syndrome should be revised. exome sequencing reveals gata mutation in a patient with partial delta-storage pool deficiency and mild thrombocytopenia objectives: we report about a -year-old male patient of russian background with severe and frequent epistaxis and hematoma since infancy. he presented with mild thrombocytopenia and increased mean platelet volume. von willebrand's disease and subhemophilia had been excluded. previously, he was diagnosed with immune thrombocytopenic purpura. he never underwent elective surgery. his parents were asymptomatic. however, his -year-old daughter also suffers from severe bleeding symptoms (multiple, light red hematoma in consequence of minimal trauma). methods: whole exome sequencing (wes) was carried out for the patient, his asymptomatic wife, his symptomatic daughter and her asymptomatic -year-old brother. platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. lysates of gel-filtered platelets were analyzed for total granule p-selectin, cd and von willebrand factor (vwf) content by western blotting and for serotonin levels by elisa, respectively. results. platelet function and characterization of the patients granula suggested a delta-storage pool disease (spd). in most cases delta-spd occurs as part of a syndrome, e. g. combined with albinism, immunodeficiency or a thalassemic-like blood disorder. as the patient and his daughter did not show any conclusive phenotype, their dna was subjected to wes. exome sequencing revealed a not yet described gata -mutation close to two zinc finger domains (znf and znf ) in a highly conserved region of the gata gene in the -year old daughter (c. a>c, p.t p, heterozygous) and her father (c. a>c, p.t p, hemizygous). this mutation was absent in wildtype-controls but could also be demonstrated in the indexpatients' asymptomatic mother. only a few mutations are known to be located in this c-terminal region to date. mutations in gata may lead to different clinical presentations, depending on their location within gata (e. g. diamond-blakfan anemia (exon ), x-linked thrombocytopenia (znf ), transient myeloproliferative disorder (intron , exon , exon ) and acute megakaryoblastic leukemia (intron , exon , exon ) in case of down-syndrome). significantly increased hbf-levels (reference level: ≤ , %) in the affected family members of , % ( -year-old daugh- our proposita is a years old woman, who was transferred to our genetic counselling department for the suspicion of m. osler (hereditary hemorrhagic teleangiectasia, hht). during routine check up an anemia was diagnosed. a tumor search was initiated and unexpectedly the ct of the abdomen showed a suspect coin lesion of about . cm in diameter localized in the basal part of the right lung. further investigations revealed a pulmonary arterio-venous malformation which was hemodynamically relevant and already led to chronic right heart overload. a coil embolization was performed. retrospectively, medical history of the patient included episodes of severe epistaxis in childhood and a neurosurgical intervention for intracerebral bleeding at the age of years without permanent neurological deficits. during genetic counselling our proposita mentioned that her years old daughter also suffered from anemia due to multiple polyps of the colon. after polypectomy her hemoglobin values normalized. although histologically the polyps appeared as juvenile ones a mutation search in the apc-gene was initiated by the gastroenterologists without identifying a pathogenic mutation. combining the two pieces of information, we offered a mutation search in the smad gene and a pathogenic mutation c. c>t (p.arg cys) was found in both patients in heterozygosity. colonoscopy in the mother did not show juvenile polyps or gastrointestinal vascular malformations. vice versa, no cerebral or pulmonary arteriovenous malformations could be detected in the daughter. our family illustrates, that the same mutation within a family may phenotypically appear as different diseases. a careful taking of medical history and the knowledge of all relevant diagnostic findings (in this case e. g. the histology of the polyps) can enable the geneticist to offer a precise differential diagnosis leading to a well-directed molecular testing. to our opinion this is still relevant even in the era of ngs-based panels because the more precise the clinical diagnosis and the choice of the genes to analyse the less problems with unclassified variants will arise. the hereditary non-polyposis colon cancer (hnpcc, lynch syndrome) is caused by pathogenic germline mutations in mismatch repair genes (mlh , msh , msh and pms ) causing microsatellite instability (msi) and decreased or lost expression of the appropriate mismatch repair protein (mmr) in the immunohistochemistry (ihc) on tumor material. thus, ihc and msi testing help to identify the mmr gene, which most likely harbors a germline pathogenic variant. msi and ihc testing prior to germline analysis are specified in the s guidelines of hnpcc and if negative normally preclude further genetic analyses. here, we present a year old patient with a synchronous colonic (ceacum) and renal cancer at the age of years. at the time of the diagnosis hepatic metastases of the caecal adenocarcinoma have already been present. histologically, the colonic tumor was a poorly differentiated adenocarcinoma (pt pn) with lymphangiosis and haemangiosis carcinomatosa. the renal cancer showed histology of a moderately differentiated clear cell renal carcinoma. in the family history the year old sister and the parents are healthy. the twin sister of the patient's mother had a collateral breast cancer at the age of years and died two years later. the mother's grandparents had no canmedizinische genetik · p-cling- pitfalls in molecular genetic diagnostics a. tibelius, e. fey, k. hinderhofer institute of human genetics, heidelberg, germany probably every clinical laboratory geneticist may look back on at least one case in his career which has caused him quite a headache. this means those cases with completely unexpected and at a first glance implausible results which could be interpreted correctly only after intensive enquiry and additional testing. here, we report on three of such pitfall cases from our routine diagnostics. case : a -year-old woman, pregnant with monozygotic twins, was referred to prenatal cystic fibrosis diagnosis. she and her partner were carriers of mutations in the cftr gene ( + g>t and g x, respectively). prenatal molecular diagnosis demonstrated that both fetus had inherited only the paternal mutation. routinely, we performed maternal cell contamination analysis by comparing polymorphic microsatellite loci between the maternal and fetal dna. surprisingly, of tested microsatellite loci revealed a discrepancy between the maternal and fetal genotypes, meaning neither of both maternal alleles was present in fetal dnas. a potential confusion of samples was excluded. moreover, the presence of paternal mutation in fetal dnas indicated a correct genetic relationship between awaited children and the partner of pregnant woman. the only one plausible interpretation of the obtained result was a pregnancy by egg donation. afterwards, this suspicion was confirmed by the couple. case : we present a -year-old man with infertility resulting from azoospermia. conventional chromosomal analysis and an additional fish analysis using y probes indicated a ,xx karyotype with no detectable sry. in parallel, a molecular azf (azoospermia factors) diagnostic was performed by a standard multiplex pcr. by this method the absence of sry region and a deletion of regions azfb and azfc, was identified, explaining the observed azoospermia. interestingly, the pcr showed that the azfa region was still present in patient chromosomes, contradicting cytogenetic and fish results. thus, a complementary fish analysis was performed in order to reveal a low-grade y-mosaicism and sry material was detected in % of the cells (a result under the threshold level). based on this observation, pcr conditions for the azf diagnostic were modified and a very weak sry-specific pcr product detected. case : a molecular diagnostics for frax (fragile x syndrome) was requested for a -year-old boy with a slight delay in speech development. his brother was already molecular-genetically diagnosed as having frax. the analysis by southern blot hybridisation in the patient revealed a smear of methylated fragments characteristic for an expanded allele in the full mutation range. surprisingly, two fragments of normal length, methylated and non-methylated, could also be detected in patient's dna. a subsequent aneuploidy mlpa confirmed a supernumerary x-chromosome in the patient consistent with a klinefelter syndrome. these results were verified by an independent cytogenetic analysis. the syndrome of congenital symmetric circumferential skin creases (cscsc and cscsc ) replaces the old term michelin-tire-baby syndrome (mim ) and is characterized by congenital circumferential skin folds, primarily of the limbs, facial dysmorphism, cleft palate and intellectual disability. mutations in the β-tubulin encoding gene tubb or in the microtubule end binding family member mapre are the underlying genetic cause. ter) and , % ( -year-old indexpatient) suggested dyserythropoiesis, although thalassemic features of the blood count were lacking. conclusion: we describe a gata mutation as the cause of a delta-storage pool disorder. imbalanced x-chromosome inactivation might explain the different phenotypes of the gata mutation carriers and will be investigated through allele-quantification based on rna isolated from whole blood and from platelet rich plasma in case of the index patient and different family members. we saw three siblings (aged , , ; two female, one male) with variable signs of retinal disease. all three developed night blindness till the rd decade, furthermore near-sightedness and deterioration of the visual acuity to a various degree at age - . the clinical diagnosis was given as stargardt's disease, fundus flavimaculatus or unspecified retinal degeneration respectively. two more siblings (aged and ) and the mother (died age ) were clinically unaffected. the father (died age ) was reported to have had bad eyesight beginning in the th decade, his father similarly (no further information available). the three affected siblings have a total of five children (age - ), none of them showing clear signs of retinal disease up to now. considering the clinical diagnosis stargardt's disease we analysed the genes abca , elovl , prom and cngb by sanger sequencing. no pathogenic mutation could be detected. afterwards we performed next generation sequencing of several genes associated with retinal dystrophies and found a novel splice site mutation in the prph gene (mim # ). the mutation was confirmed in all three affected siblings by sanger sequencing. considering that mutation pathogenic we could re-diagnose our patients with patterned macular dystrophy type (mim # ). that disease is inherited in an autosomal dominant fashion, corresponding to the pattern of inheritance evident in our family. the etiology of epileptic encephalopathies, characterized by severe, early-onset seizures accompanied by developmental delay or regression, is highly heterogeneous. in recent years, next-generation sequencing approaches have led to the discovery of numerous causative genes; however the spectrum of associated phenotypes still needs to be further explored for many of these genes. we performed multi-gene panel analysis in a little boy of german non-consanguineous parents who showed severe early-onset infantile epileptic encephalopathy and almost absent neurological development. in this patient we identified a novel heterozygous missense mutation in the gabrg gene which was absent in the parents. in silico analyses strongly suggested a pathogenic relevance of this sequence variation which resides within a highly conserved region. so far, gabrg mutations have mainly been associated with milder types of epilepsy such as febrile seizures and childhood absence epilepsy. therefore, our findings extend the phenotypic spectrum associated with mutations in this gene at the severe end. referring on a case report by al marzouqi et al. ( ) , who reported on a girl with bilateral amastia in the context of skewed x-inactivation, we want to underline the importance of mlpa analyses in the case of negative sequencing of the eda gene, as whole exon duplication can be the cause of hypohidrotic ectodermal dysplasia. to our knowledge, the report of al marzouqi et al. has been the only case about this genetic alteration in the literature so far. novel pogz mutation in a patient with intellectual disability, microcephaly, strabismus and sensorineural hearing loss we report on a -year-old male patient with severe intellectual disability, microcephaly, sensorineural hearing loss, ocular abnormalities (strabismus and hyperopia), congenital heart defect (atrial septum defect and pulmonary stenosis) and minor facial abnormalities (thin upper lip, frontal upsweep). next-generation sequencing analysis revealed a novel heterozygous de novo mutation in pogz: c. _ del, p.(thr serfs* ) . the clinical problems of this patient are in accordance with the findings in the previously reported pogz mutation carriers. reports of additional patients with pogz mutations will be needed to establish detailed phenotype-genotype correlations of this novel and probably underdiagnosed syndrome. novel clinical and molecular aspects in two patients with kleefstra syndrome d. wand, b. seebauer, k. heinimann, i. filges medical genetics, university hospital, basel, schweiz the phenotype of kleefstra syndrome is clinically variable but characterized by facial dysmorphism, intellectual disability, childhood hypotonia and variably associated other malformations. haploinsufficiency of ehmt , caused by either heterozygous microdeletions in q . or sequence mutations in ehmt , has been identified to be the underlying causal mechanism. here we present two girls from two unrelated families with clinical signs of kleefstra syndrome. besides the main features such as facial dysmorphism, intellectual disability/developmental delay and childhood hypotonia, the years old girl presented with additional accelerated growth whereas the other girl, years old, showed failure to thrive. both children have no heart defect, renal or urogenital anomalies or severe respiratory infections. we identified two rare variants likely to be causal: a novel heterozygous splice site ehmt variant and a heterozygous microdeletion in chromosome q . , including exons an of the ehmt -gene . these patients broaden the spectrum of kleefstra -associated ehtm causes, contribute to novel aspects of genotype-phenotype correlations and a better understanding of the clinical variability of the disorder. here, we present two girls from two non-consanguines families showing clinical aspects of the syndrome of congenital symmetric circumferential skin creases. additional features are present in both girls. patient is the first child of healthy parents. she was born at + weeks of gestation with a weight of g, a length of cm and a head circumferences of cm. respiratory distress, a cleft palate, a heart defect (atrial septum defect), an anogenital malformation and facial dysmorphism were diagnosed after delivery, additionally to the skin creases phenotype. conventional karyotyping performed on blood lymphocyte cultures and cgh-array analysis showing normal results. patient is the second child of unrelated healthy parents. her older sister is healthy as well. because of an intrauterine growth retardation an amniocentesis with chromosomal analysis was performed, showing a normal karyotype of ,xx. patient was born at + weeks of gestation by caesarean section. the birth weight was g, the birth length was cm and the head circumferences was cm and the apgar score was / / . due to respiratory distress and hypoxia a tracheotomy was initiated. she also presented with cleft palate, feeding difficulties, a heart defect (atrial and ventricular septum defect), asplenia and facial dysmorphism. the skin phenotype was remarkably similar to that of patient with a prominent neck fold and skin creases mainly on the back part, but also at the limbs. in both children the skin folds gradually diminish over the time without any intervention like it was described for michelin-tire-baby syndrome/ cscsc and cscsc patients. for these reason, a disease-causing mutation in the genes mapre and tubb were excluded in both children. to identify a genetic cause, we performed a trio whole-exom sequencing, including the healthy parents and the affected child, in both families. a de novo stop mutation was detected in patient , while no promising results could be detected in patient . further studies, especially functional in vivo studies and analysis of further patients with a similar clinical presentation will answer the question if the above described phenotype is an expanded variant of the congenital symmetric circumferential skin creases or a unique new syndrome. clinical findings in a family with x-linked hypohidrotic ectodermal dysplasia due to a duplication of exon in the eda gene -a case report we want to summarize the phenotypic spectrum in one affected three generation family with x-linked hypohidrotic ectodermal dysplasia. we report on one strongly affected male and slightly affected female carriers, carrying a duplication of exon in the eda gene. reason for genetic assessment was the request of a female for evaluation of a possible risk for occurrence of the familial disorder in a further planned pregnancy. the woman reported an inability to breastfeed and she and her daughter showed conical teeth, a dry skin and sparse hair. the affected male showed absent deciduous teeth, hypodontia of permanent teeth, missing regulation of the temperature due to a lack of sweat glands, bilateral nipple hypoplasia, a dry and wrinkled skin, missing eye lashes, eyebrows and scalp hair and sparse body hair. the fingernails were inconspicuous, whereas the nails of the toes, particularly the nails of the hallux were yellowish and thickened. the affected male had an operation due to a gallstone and cysts were found in one kidney. there was no increased predisposition to infections. the suspected diagnosis of x-linked hypohidrotic ectodermal dysplasia was confirmed by genetic analysis of the eda gene (sequencing and mlpa analysis). a duplication of exon was detected in the affected male patient and was confirmed in the two mildly affected female relatives by realtime-pcr. terminator sequencing mix v . on an abi xl genetic analyzer (applied biosystems) and detected an insertion of bp between the exons and . we located the insertion's sequence in intron of the dmd gene and sequenced flanking sequences of gdna to find the underlying mutation causing the insertion. two hemizygous single nucleotide variants (snvs) surrounding the inserted fragment could be identified. the first variant (c. - a>c) is a common polymorphism (maf according to genomes project: . %) at the position of an existing acceptor splice site. the second variant (c. - a>g) is novel and creates a new cryptic donor splice site with high probability. these two cryptic splice sites create the formation of a bp pseudoexon which produces a frameshift and a premature stop codon (p.asp alafs*) in the dmd gene. in summary, we could genetically confirm the clinical diagnosis of a dystrophinopathy by two divs in dmd. although the insertion of the pseudoexon creates a premature stop codon the patient's clinical phenotype indicates a milder type of becker muscular dystrophy. this contradiction could be explained by the remaining existence of dmd wild type mrna most likely due to a not constantly active cryptic splice site. most interesting about the present case is the fact that a common snv facilitates the creation of a pseudoexon. this makes the region a potential hotspot for divs in the dmd gene which would be worthwhile further investigations. with more than % of all humans preferring to use their right hand, handedness is the most noticeable functional expression of cerebral lateralization in humans. however, the precise molecular mechanisms that regulate handedness and other related forms of cerebral lateralization remain largely elusive. therefore, the question which genetic, epigenetic and environmental factors contribute to human handedness is one of the central questions in research on lateral asymmetries. handedness is a complex, heritable trait, for which polygenic inheritance is assumed, meaning that a large number of genetic factors with a small additive effect contribute to the observed variance in hand preference. to date, genetic association studies have implicated only a few specific genes influencing handedness. particularly interesting is the association between the human androgen receptor (ar) gene and different aspects of handedness, since the interrelationships constitute a conceptual bridge between the theories that invoke testosterone as a factor in the development of cerebral asymmetries with theories proposing that the x chromosome contains a locus that influences the direction of hand preference. in an initial large association study in samples of healthy adults we already demonstrated that handedness in both sexes is associated with the ar cag-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. in addition, we have performed a second association study in an independently collected healthy cohort of more than test persons with comprehensive data on the handedness phenotype. we were able to replicate the association with longer cag repeats being related to a higher incidence of non-right-handedness, especially in females. since longer cag-repeat blocks have been linked to less efficient ar function, these results implicate that differences in ar signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization. dej. waschk, ac. tewes, p. wieacker, s. ledig institute of human genetics, münster, germany the mammalian female and male reproductive tracts develop from the paramesonephric ducts (müllerian ducts, md) and mesonephric ducts (wolffian ducts, wd), respectively. in the absence of testicular differentiation and anti-müllerian hormone, the wd regress and the md give rise to the fallopian tubes, uterus, cervix and the upper part of the vagina. disorders of normal md development in females can manifest as fusion anomalies of the uterus such as septate uterus, bicornuate uterus, unicornuate uterus and uterus didelphys, or more complex malformation patterns like mayer-rokitansky-küster-hauser syndrome (mrkh) or herlyn-werner-wunderlich syndrome. mrkh is characterized by congenital absence of the uterus and the upper two-thirds of the vagina in individuals with a normal female karyotype, most of whom show normal ovarian function. mrkh can further be associated with additional malformations e. g. of the kidneys and the skeletal system. herlyn-werner-wunderlich syndrome is characterized by uterus didelphys, obstructed hemivagina and ipsilateral renal agenesis. despite anomalies of the md occur quite frequently, the etiology of most cases with these disorders remains unknown. the homeodomain transcription factor emx (empty spiracles homeobox ) was found to be critical for urogenital and central nervous system development. previous studies showed that in emx mutant mice, the kidneys, ureters, gonads and genital tracts were completely missing. in order to elucidate whether mutations in emx are causative for md anomalies in humans, we performed sequence analyses of emx (genbank nm_ . ) in our study group of female patients with clinically characterized disorders of the md including patients with mrkh and cases with herlyn-werner-wunderlich syndrome. we found the heterozygous intronic mutation c. - c>a twice in this cohort. this variant has been described earlier (rs ) and is listed in the exome aggregation consortium exac variant with a minor allele frequency of . %. in silico analysis revealed no significant changes for the correct splicing of emx . we therefore consider this variant to be a benign polymorphism. we conclude that mutations in emx are not causative for disorders of the md in our cohort. a. zaum, w. kreß, a. gehrig, s. rost institute of human genetics, würzburg, germany dystrophinopathies are x-linked muscle diseases caused by mutations in the dmd gene (omim: ). due to the huge size of this gene, the detection of mutations is sometimes challenging. despite multiplex ligation-dependent probe amplification (mlpa) and sequencing of all exons, about % of patients do not show any mutations in coding regions and therefore remain without molecular diagnosis. we assume that the majority of these patients have deep intronic variations (div) which are not detectable by standard diagnostic techniques. the index patient analysed is a twelve-year-old boy who was by chance diagnosed with elevated ck levels (up to , iu/i) at eight weeks of age. today he is still able to walk without walking aids but needs assistance when climbing stairs. in , a muscle biopsy revealed complete absence of dystrophin which established the diagnosis of dmd. for the molecular diagnosis, standard diagnostics ascertained no causative mutation. therefore we decided to search for deep intronic mutations. we isolated mrna from muscle tissue of the patient and amplified overlapping cdna fragments using rt-pcr. the fragments were analysed by gel electrophoresis for size differences compared to an unaffected control. the cdna product comprising exons - revealed an augmented fragment size compared to the control and the expected product size (about bp instead of the expected bp). we sequenced the altered cdna product using bigdye recent research in psoriasis has identified pustular psoriatic manifestations as either mendelian traits or as major genetic risk factors in contrast to the numerous associated snps in classical plaque psoriasis vulgaris. autosomal recessive mutations in il rn have been identified in ~ - % of patients with generalized pustular psoriasis (gpp), a rare, severe pustular variant of psoriasis. in addition, heterozygous missense variants in card and ap s have been associated to pustular psoriasis as well and shown to be functionally relevant. in order discover how relevant those genes were in our large gpp cohort of patients recruited all over germany, we screened them for coding variants in il rn, card and ap s by sanger sequencing and for quantitative aberrations by mlpa. we identified homozygous or compound heterozygous il rn mutations in of gpp patients ( %) and single heterozygous mutations in patients ( %). the most common mutations were c. >c>t/ p.ser leu and c. c>t/ p.pro leu, present on %/ % of mutated alleles, respectively. we also identified three so far unreported mutations: c. c>a/ p.ser x, c. - delacct-tc/ p.thr _phe del and c. g>a/ p.val met. according to molecular modeling, c. c>a/ p.ser x resulted in a shortened, de-stabilized protein analogous to c. g>t/ p.glu x. the other two mutations were also predicted to result in destabilized, likely disease-relevant, loss-of-function proteins. heterozygous ap s mutations were detected in two gpp patients, both of whom carried additional homozygous or compound-heterozygous il- rn mutations. gpp patients were heterozygous carriers of rare missense variants in card ( %); of note, two of these patients carried additional mutations in il rn. our genotype-phenotype correlation revealed a similar gender distribution in carriers of il rn mutations and wildtype carriers, but a strong association between bi-allelic mutations in il rn and early age of manifestation (p = . x* - ). as in other autosomal recessively inherited mutations, the frequency of parental consanguinity was significantly higher in patients with two il rn mutations compared to non-carriers. overall, our genetic studies suggest a lower impact of variants in ap s and card in pustular psoriasis than of those in il rn. interestingly, the combination of il rn mutations with either ap s or card congenital prosopagnosia (cp), also known as developmental prosopagnosia or face blindness, describes the inability to recognize faces. cognitive functions such as intelligence as well as the sensory visual capabilities are usually not impaired but people with prosopagnosia are negatively affected in their social life because individuals with the disorder have difficulty in recognizing family members, close friends or colleagues. although the prevalence of cp is estimated at . % and it appears to run in families, the contexts, which genetic, epigenetic and environmental factors contribute to this trait, are largely unknown. therefore we started to establish a large, well-characterized cp cohort for genetic studies. we hypothesize that rare highly penetrant non-synonymous genetic variants could explain some cases of cp. as part of a larger genetic study of patients with cp, we performed family based whole-exome sequencing and targeted re-analysing on four individuals from two families with multiple affected members. by obtaining samples from affected and unaffected members of the same family, we hope to effectively identify de novo and inherited variations. variations are considered on the basis of allele frequency, mutation type, literature and mutation prediction tools, thus generating a list of candidate variations/genes for each patient that is amenable to interpretation and further analyses in the extended cohort. through this approach, we hope to identify causal variations/genes in families and isolated patients with cp. erythrokeratodermia variabilis (ekv) is a rare, autosomal dominant genetic skin disorder with a highly heterogeneous phenotype. to date, three causative genes (gjb , gjb and gja ) are described but further genetic heterogeneity is expected. card mutations are only described for psoriasis vulgaris, generalized pustular psoriasis and pityriasis rubra pilaris. for the first time, we present disease causing card mutations in patients with an "ekv-like" phenotype. it refers to one familial case with two affected individuals, with an autosomal dominant transmission from the mother to the daughter and one independent sporadic case. all patients present typical ekv symptoms. a rash of well-demarcated erythematous and scaly plaques interspersed with distinct islands of uninvolved skin or small reddish papules coalescing into large reticulated scaly plaques and palmoplantar keratoderma. to confirm the suspected diagnosis of ekv, we analyzed a custom designed multi-gene panel by next generation sequencing with genes associated to hereditary skin diseases (agilent haloplex technology). the sequencing results did not reveal any mutation in the genes gjb , gjb and gja , but we found two pathogenic mutations in card . in the familial case c. t>c p.leu pro (rs , enst . ) was detected, while the sporadic case carries c. t>c p.leu pro. we hypothesize that different genetic and environmental factors are involved in the evolution of the phenotype in patients with card mutations. our cases show that classification of unusual skin phenotypes can be challenging without genetic testing. therefore, gene panel sequencing is a cost-efficient and time-saving solution for solving difficult cases with sometimes unexpected genetic background. bipolar disorder (bd) is a complex psychiatric disorder affecting more than % of the world's population. the highly heritable disease is characterized by recurrent episodes of manic and depressive symptoms. as the cumulative impact of common alleles with small effect may only explain around % of the phenotypic variance for bd, rare variants of high penetrance have been suggested to contribute to bd susceptibility. in the present study we investigated individuals of large multiply affected families of european origin using whole exome sequencing (wes). in each family, two to five affected individuals with bd or recurrent major depression were selected for sequencing. wes was performed on the illumina hiseq platform and the varbank pipeline of the cologne center for genomics was used for data analysis. all identified variants shared within each family were filtered for a minor allele frequency < . % and potentially damaging effects predicted by at least four of five different bioinformatics tools. we identified a total of rare, segregating and functional variants implicating different genes, of which were brain expressed. subsequently, we applied the residual variation intolerance scores (rvis, petrovski et al., ) and identified genes that were ranked among the % most "intolerant" genes in the genome. gene enrichment analysis of these genes showed a significant enrichment for a total of pathways (p < . ) including neuron projection, axon development and cell adhesion. for follow up analyses, we prioritized genes that were either found in at least two unrelated families in the present study or that were previously reported in next generation sequencing or gwas studies of bd. in addition, we enclosed the genes that were predominantly driving the significant pathways in the above mentioned gene enrichment analysis. the different approaches of prioritization yielded candidate genes that are currently being followed up by resequencing in cohorts of about independent bd cases and controls of european ancestry. the candidate genes include cdh that encodes a calcium-dependent cell adhesion protein that may play an important role in the morphogenesis of neural cells during the development and maintenance of the brain. for resequencing we use the single molecule molecular inversion probes (smmips) technology that enables multiplex targeted resequencing in large cohorts. the smmips sequences were designed with an empirically variants in several patients indicated an oligogenic inheritance rather than a purely monogenic one. moreover, the oligogenic basis of this group of inflammatory diseases might currently be underestimated, as our study suggests that genetic risk factors other than il rn mutations remain to be identified in the majority of gpp patients. exome sequencing of multiply affected schizophrenia families provides new insights into the pathogenesis of the disorder schizophrenia (scz) is a multifactorial psychiatric disorder with a lifetime risk of ~ % and a heritability of about - %. analysing multiply affected families using whole exome sequencing (wes) is a very promising approach to identify new scz risk factors. in these families, individuals are affected with scz over several generations. it is likely, that in multiply affected families genetic variations with particularly strong effect co-segregate with the disorder and contribute to the development of the psychiatric symptoms. to our knowledge, the present study is the largest study analysing multiply affected scz families using wes worldwide so far. we included families with at least affected members each. from each family, - individuals were exome sequenced on an illumina hiseq and analysed using the varbank pipeline of the cologne center for genomics (http://varbank.ccg.uni-koeln.de) and the clc bio biomedical genomics workbench. we included rare (allele frequency ≤ . % in the exome aggregation consortium dataset) variants that were predicted to be pathogenic (combined annotation dependent depletion score ≥ ; cadd.gs.washington.edu), confirmed by sanger sequencing and co-segregating with the disorder. in total we identified potentially pathogenic mutations in ~ genes. a substantial proportion of these will not contribute to the pathogenesis of scz. in order to further tease out the most promising candidate genes we applied multiple strategies: (i) screening our mutations in independent patient and control cohorts through international cooperations (access to more than , scz patients), (ii) gene-based tests, (iii) pathway-and network-analyses, (iv) gene expression analyses and (v) sequencing of the candidate genes in , scz patients and , controls. analyses are ongoing and will be presented at the upcoming conference. abstracts and our evolution, we know very little about the different mutagenic processes in our germline. of particular interest are a handful of highly recurrent dnm associated with congenital disorders and/or rasopathies, that have been described as driver mutations expanding in the male germline. the mutation itself causes a change in the tyrosine kinase receptor/ras/ mapk pathway, which in turn confers the spermatogonial stem cell a proliferative advantage. selfish or driver mutations are quite common in cancer, but we still know very little about the selfish expansion in the male germline. the reason might be that mutations in the human germline are very rare, and it is rather difficulty to directly measure such rare events. most of our knowledge on germline mutagenesis comes from indirect sequence comparisons or whole genome sequencing of pedigree families, but it renders little information about individual mutagenic events. for this reason, we have adapted an ultrasensitive, next generation sequencing (uss) technology for the measurement of rare mutations to study the expansion of selfish genes in the male germline. as a proof-of-principle, we have sequenced at an extremely high coverage exon and of the fgfr gene in young and old sperm donors. we found an increased mutation frequency for the loci associated with achondroplasia and thanatophoric dysplasia ii in sperm of older donors. our results also show that we can distinguish ultra-rare mutations occurring at a frequency of one in hundred thousand wild type; thus, making this method ideal to discover potential driver dnm that might be expanding with paternal age. s. sivalingam , , a. j. forstner , , , s. herms , , , a. maaser , , c. s. reinbold , , t. andlauer , j. frank , h. dukal , d. schendel , , p. hoffmann , , , t. kircher , u. dannlowski , , a. krug , a. cichon , , , s. witt , m. rietschel , m. m. nöthen , introduction: affective disorders such as major depressive disorder (mdd) and bipolar disorder (bd) are genetically complex and heterogeneous disorders. both genetic and environmental risk factors contribute to the etiology of the diseases. however, the neurobiological correlates by which these risk factors influence the disease development are hardly understood. increasing evidence suggests that epigenetic modifications such as dna methylation have important implications on the development of psychiatric disorders including mdd and bd. several studies revealed that alterations in the dna methylation can modulate gene expression in response to the environment. to investigate this, genome-wide dna methylation analysis of female individuals from three extreme groups (genetic-, environmental risk and healthy controls) was performed. methods: for the genome-wide dna methylation analysis we selected: (i) individuals with genetic risk (at least one st degree relative with a life-time diagnosis of mdd or bd), (ii) individuals with environmental risk (maltreatment in the childhood trauma questionaire) and (iii) matched healthy controls. all individuals were of european origin. processing was done according to the manufacturer's protocol using the infinium methylationepic beadchip (illumina, san diego, ca, usa) covering more than . methylation sites at the life & brain center (bonn, germany). state-of-the-art data processing protocols, including correction for blood cell type heterogeneity, color correction, eliminating probes containing snps and cross reactive probes were used. after quality control trained design algorithm mipgen (boyle et al., ) and sequencing is currently performed on the illumina hiseq platform. our preliminary results strongly suggest that rare and highly-penetrant variants in neuronal and cell adhesion genes contribute to bd etiology. the results of resequencing of a large case/control sample will provide further evidence for an involvement of particular pathways. the use of zebrafish as model system to quantitatively assess the impact of risk variants in non-coding regions in vivo s. l. mehrem , , b. nagarajan , n. ishorst , , a. c. böhmer , , e. mangold , b. odermatt , k. u. ludwig , department of genomics, life & brain center, university of bonn, bonn, germany, institute of human genetics, university of bonn, bonn, germany, institute of anatomy, university of bonn, bonn, germany most human malformations occur early in embryonic development and are present immediately after birth. one common human birth defect is nonsyndromic cleft lip with or without cleft palate (nscl/p), affecting about in , newborns. nscl/p has a multifactorial background with a strong genetic component. recent genome-wide association studies identified several loci as risk factors for nscl/ p. notably, most of them map to non-coding regions and are expected to have a functional impact through regulatory mechanisms. given the early developmental time point of facial development and the resulting lack of accessible human tissue, follow-up analyses of risk variants are challenging. we are hypothesizing here that we might be able to quantitatively detect differences in regulatory activity between wildtype and risk variants located in predicted enhancers by using the zebrafish as model organism. the advantages of using the zebrafish are ( ) ex utero development, ( ) transparency of the fish, ( ) easy manipulation and ( ) relatively short generation times. we applied a dual-luciferase assay plasmid system which is based on a sequential measurement of two luciferases (firefly and sea pansy luciferase) in fish lysates upon injection of a single plasmid. this plasmid, which contains a minimal promoter (minp) and the enhancer region of interest, is microinjected into zebrafish eggs of one-cell stage. after three days, fish are collected, lysed and luciferase activity is measured using a luminometer. for our proof-of-principle analysis we analyzed an nscl/p risk locus on chromosome q (ludwig et al. ) . through database research one enhancer was predicted that contained two strongly associated risk variants. in vivo fluorescence analysis using egfp in zebrafish embryos revealed this enhancer to be active in craniofacial development, but qualitative differences in activity were not observed by eye. upon cloning of the enhancer in the dual-luciferase system, our injection results in vivo indicate that the system is working in principle. however, a high standard deviation between single replicate measurements was observed, probably due to variability in transfection efficiency. we therefore are planning to adapt the protocol in order to screen for positively injected fish embryos. we are currently investigating the functionality of these screening constructs in zebrafish embryos. results will be presented at the meeting. once successful, our approach represents a practical method to quantify the activity of regulatory elements in real time in vivo. this will be of particular importance in the functional follow-up of genetic findings in non-coding regions for the majority of birth defects. previously genome-wide association methods in patients with classic bladder exstrophy (cbe) found association with isl , a master control gene expressed in pericloacal mesenchyme. this study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. genotypes of markers obtained from cbe patients of australian, british, german ital-and normalization , cpg-sites were tested for genome-wide dna methylation by a linear regression model while accounting for biological as well technical covariates. results: the genome-wide dna methylation analysis of the three extreme groups revealed cpg sites (p < × - ) in the subgroup analysis "environmental risk vs. controls" and cpg sites (p < × - ) in the analysis "genetic risk vs. controls". in addition, we identified cpg sites (p < × - ) in the comparative analysis of "genetic risk vs. environmental risk". none of these cpgs showed significant differential dna methylation after correction for multiple testing. however the hierarchical clustering of the differentially methylated sites provided some evidence for differentially methylated patterns between the subgroups. discussion: our genome-wide dna methylation analysis of the extreme groups provided some evidence for differentially methylated cpg sites which unfortunately did not withstand correction for multiple testing. this may reflect at least in part that the sample size of the present study was too small to detect differentially methylated cpgs at the genome-wide level. a. tafazzoli , , t. vaitsiakhovich , l. pethukova , , , s. redler , , r. kruse , b. blaumeiser , m. böhm , g. lutz , h. wolff , , am. christiano , p. kokordelis , mm. nöthen , , rc. betz , alopecia areata (aa) is a common hair loss disorder that occurs in both sexes and all age groups. aa is thought to be a tissue-specific autoimmune disease directed against the hair follicle. both, gene-based and genome-wide association studies have identified more than susceptibility loci for aa; however, a large percentage of the overall heritable risk still awaits identification. to provide further insight into the immune related nature of aa, we and our us collaborators had each performed an immunochip-based analysis. we recently performed a meta-analysis, combining the data from both studies, and are now aiming to follow-up the best results in an additional german cohort by use of a sequenom assay to identify novel susceptibility loci. we conducted the meta-analysis by using data from the above mentioned two studies on illumina beadchip arrays including a total of , cases and , controls of central european origin. method of synthesis of regression slopes (msrs) was used for the analyses which are implemented in metainer software package. for follow-up step, we chose the most promising candidate snps. these will be examined with the sequenom massarray iplex platform in an independent aa sample comprising , cases and controls. by use of the meta-analysis combining data from the us and our sample, we identified novel loci with a suggestive p-value of pbecker-wu ≤ - (phet ≥ . ). among them, nfkb is the most significant locus (pbecker-wu = . × - ). in order to achieve genome-wide significance, we plan to follow-up the most promising snps in an independent german sample. we considered the most significant loci (lower p-becke-wu value) for the replication step. the experiments are ongoing and results will be presented at the meeting. despite the recent identification of susceptibility loci for aa, our understanding of the genetics of aa is incomplete. identification of new loci may provide novel insights into biological pathways and a better elucidation of disease pathophysiology. q . encompassing only one gene: kcnj . the duplication segregates with the phenotype in the family. kcnj belongs to the same subfamily of potassium channels as the known disease gene for cooks syndrome kcnj and both share several biological functions. recent data show that gain of function mutations in another potassium channel kcnh cause zimmermann-laband syndrome, a congenital malformation syndrome also associated with hypoplasia or aplasia of nails and terminal phalanges. therefore we propose that duplications of kcnj may also cause tissue specific misregulation resulting in digit and nail defects. taken together we show in a three generation pedigree that cooks syndrome is associated with a duplication of kcnj . our data further highlight the emerging role of potassium channels in congenital digit and limb anomalies. case report: deletion of the terminal short arm of chromosome (chromosome p deletion syndrome) without q-duplication with a familial history of a large pericentric inversion of chromosome b. gläser, n. hirt, e. botzenhart, j. fischer, m. leipoldt institut für humangenetik, universitätsklinikum freiburg, freiburg, germany we report on a male patient referred as a newborn with typical clinical features of chromosome p deletion syndrome. conventional karyotyping of lymphocyte cultures confirmed a deletion of the terminal short arm of chromosome with breakpoint in p . the size of the deletion ( mb) could be refined by microarray analysis and assigned to pos : - . parental cytogenetic investigations showed a normal karyotype in the mother whereas the father was revealed to be carrier of a large pericentric inversion of one chromosome . odd crossing-over in the inverted segment of a pericentric inversion in a parent can lead to unbalanced offspring caused by gametes with a terminal deletion of the p-arm together with a duplication of the q-arm or gametes with a duplication of the p-arm together with a deletion of the q-arm. in order to find out, whether the p-deletion in the child is the result of an independent event or if it is related to the structural chromosomal aberration of the father a microsatellite analysis is going to be performed p-cytog- a small supernumerary marker chromosome of the pericentric region of chromosome in a child with intellectual disability: case report and literature review b. hoffmann, g. gillessen-kaesbach, i. hüning institut für humangenetik, universität zu lübeck, lübeck, germany small supernumerary marker chromosomes (ssmc) are reported in . % of newborn infants. we report on a girl, which was born preterm at weeks of gestation via cesarean section due to pathological ctg. the pregnancy was complicated by gestational diabetes. she presented with muscular hypotonia, multiple hemangiomas, dysmorphic features and feeding problems. the body measurements were in normal range. the feeding problems made a tube feeding necessary until the age of four months. facial features consisted in epicanthus, high palate, broad nasal tip and broad nasal root. a brain mri showed periventricular leukomalacia and hypoplasia of corpus callosum. drug-resistant seizures with hypsarrhythmia started at the age of ten months. the affected girl was the only child of healthy non-consanguineous parents. the father also presented with a few small hemangiomas in the face. there was no history of intellectual diasability in the extended family. karyotyping showed ssmc in mosaicism. molecular characterization by array-cgh showed that the ssmc consists of pericentric chromosomal material derived from chromosome (arr [ h g ] p . p . ( , , - , , ) x ~ , q . q . ( , , - , , ) x ~ dn (grch /hg ). ian, spanish and swedish origin and , ethnically matched controls and from cbe case-parent trios from north america were analysed. only marker rs at the isl locus showed association (p = . × - ). a meta-analysis of rs of our previous and present study showed a p value of . × - . developmental biology models were used to clarify the location of isl activity in the forming urinary tract. genetic lineage analysis of isl -expressing cells by the lineage tracer mouse model showed isl -expressing cells in the urinary tract of mouse embryos at e . and distributed in the bladder at e . . expression of isl in zebrafish larvae staged hpf to hpf was detected in the developing pronephros region. our study supports isl as a major susceptibility gene for cbe and as a regulator of urinary tract development. to date, seven patients with interstitial deletions at chromosome q . -q . have been described in the literature. all patients reported had moderate to severe intellectual disability and a characteristic facial phenotype including blepharophimosis, telecanthus, epicanthus, flat malar region, and a thin upper lip vermillion. six of the seven patients had epileptic seizures. by analyzing the deletion's overlaps, two distinct critical regions have been suggested for the facial phenotype as well as for intellectual disability and seizures. here we present another patient with a de novo . mb deletion in q . -q . . the patient shows moderate mental retardation. he has an abnormal eeg, however, only one episode of clinical seizures has been observed so far. the facial gestalt resembles the typical dysmorphic features of the patients with q . -q . deletions reported previously. minor anomalies were short fingers and toes, and a single palmar crease. our report supports the assumption that deletions in q . -q . cause a distinctive and clinical recognizable microdeletion syndrome with characteristic facial features and intellectual disability. since the patient's deletion overlaps with most of the critical region for the dysmorphic phenotype but only with parts of the intellectual disability critical region, the molecular data presented here further narrow down the critical region for the intellectual disability seen in patients with q . -q . microdeletions. p-cytog- *** cooks syndrome is associated with a duplication of the potassium channel kcnj mundlos , , , d. horn , m. spielmann , , institute for medical genetics and human genetics, charité universitätsmedizin berlin, berlin, germany, max planck institute for molecular genetics, berlin, germany, northern ireland regional genetics service, belfast city hospital, belfast, ireland, berlin-brandenburg school for regenerative therapies -bsrt, berlin, germany cooks syndrome (mim ) is a rare autosomal dominant disorder classically characterized by onychodystrophy, and anonychia, with absence or hypoplasia of the distal phalanges of the hands and feet. large duplications including kcnj were shown to be causative for cooks syndrome. recently mouse studies revealed that tissue specific misregulation of kcnj , a potassium channel of the subfamily j, cause hypoplasia of nail beds and abnormal distal phalanges, thus resembling the cooks phenotype. here we report on a three generation pedigree with typical features of cooks syndrome that was negative for kcnj testing. we performed high resolution array-cgh and identified kb duplication on chromosome p-cytog- chromosome q . -q . deletion syndrome -an additional case with sensorineural hearing loss. s. leubner, c. hennig, a. junge mitteldeutscher praxisverbund humangenetik, dresden, germany the chromosome q . -q . deletion syndrome (mim # ) is a contiguous gene syndrome caused by a deletion encompassing the chromosome region q . -q . . initially, it has been described by ballif et al. ( ) . up to now, only a few cases with a microdeletion q . q . have been reported. most of them carry a microdeletion with recurrent breakpoints and similar size of about . mb. the common clinical features of cases with the chromosome q . -q . deletion syndrome comprise mild-to-moderate developmental delay, microcephaly, postnatal growth retardation, heart defects, limb anomalies, and hearing loss. we present an additional male patient with a . mb deletion of chromosome q . -q . , detected by array cgh (cytochip isca × k v . , illumina). our index patient shows typical symptoms of the chromosome q . -q . deletion syndrome like developmental retardation (in particular speech delay), a head circumference at the rd centile, postnatal growth retardation with a body height at the th centile, heart anomalies (right-sided aortic arch, patent ductus arteriosus), and sensorineural hearing loss on both sides. our data improve the characterization of the typical phenotype caused by a chromosome q . -q . deletion and reinforce the suspicion that this region might be associated with sensorineural hearing loss. partial, homozygous deletions of ahi gene causes joubert syndrome type d. meier, a. behnecke, jwg. janssen, u. moog, k. hinderhofer institute of human genetics, university hospital heidelberg, heidelberg, germany we describe a patient who is second child of consanguineous healthy parents from turkey. he was born after weeks of gestation with normal birth parameters ( g, cm, ofc cm). at the age of three months atypical eye movements became apparent. psychomotor development was delayed from the beginning, he presented with hypotonia and later developed ataxia. an abnormal breathing pattern was not noticed. an abnormal mri with hypoplasia of the cerebella vermis at the age of months and the clinical signs described above led to a clinical diagnosis of joubert syndrome. at that time no diagnostic testing was available. he returned to the outpatient clinic of the institute of human genetics at the age of years having developed retinopathy in the meantime. his height was below average (~ cm, <p ) but still within his family's range. aside from the known irregular eye movements he showed nail hypoplasia of the thumbs, and small hands and feet. he was working at a sheltered workshop. the year before, the pediatric department of the university hospital heidelberg had initiated testing of of the by then known genes causative for joubert with no mutation being found (nphp , tmem , cc d a, mks , rp-grip l, ofd- and mks ). the gene associated with joubert syndrome type (omim # ) was not included though. with the external mri not being available at that time we decided to first perform chromosome and array analyseis. the conventional chromosome analysis was without pathological findings. subsequent array analysis by affymetrix® cytoscan hd oligo/snp showed an kb homozygous deletion in q . within the ahi gene, the gene for joubert syndrome type . the deletion encompasses exon to which includes parts of the wd-repeats (tryptophan-aspartic acid (w-d) dipeptide) on the protein level. the homozygous deletion was confirmed by mlpa in the patient and both parents identified as heterozygous carriers. so far, alterations in ahi leading to joubert syndrome comprise homozygous point mutations or small heterozygous deletion/duplications in combination with a second pathogenic mutation. so we here present the first case of a large homozygous deletion in the ahi gene, being causative for joubert syndrome type . we conclude that in un-a review of the literature showed that the few patients with a ssmc of the pericentric regions of chromosome described in literature show early feeding problems, developmental delay, delay in acquired language, difficulties in social skills, difficulties in attention and activity levels or autistic-like behavior. the facial dysmorphic features were not specific. multiple hemangiomas have been reported in some cases. west syndrome or seizures have not been reported so far. the phenotype of our patient is consistent with the literature data, however there are no patients with a ssmc showing the same breakpoints reported so far. especially due to the mosaicism and the rare number of patients described in the literature, no precise clinical prognosis for this patient was possible. the generation of patient-specific induced pluripotent stem cells (ipscs) by reprogramming of adult somatic cells (e. g. skin fibroblasts) represents a novel technology for studying disease mechanisms. for generation of ipscs several reprogramming methods, including the use of integrating vectors and non-integrating vectors, have been developed. since genetic abnormalities can arise during the reprogramming process, genetic quality assurance is indispensable. the aim of this analysis was to compare the genetic stability of ipscs generated using either a dna-integrating or a dna-non-integrating reprogramming technique in order to reveal possible method-specific differences. we studied ipscs derived from patients with parkinson's disease (pd), the second most common neurodegenerative disease worldwide. to monitor the genetic stability, we investigated copy number variants (cnvs) in addition to conventional chromosome analyses in ipscs of ten pd patients and six healthy control individuals. using high-resolution chromosomal microarray analysis (cma), we monitored the formation of relevant cnvs during reprogramming to pluripotency by comparing fibroblasts and ip-scs of the respective individual. to date, fibroblast cultures of all selected probands as well as retroviral reprogrammed ipsc clones and eight sendai-viral reprogrammed ipsc clones were analyzed. aneuploidies were detected in three fibroblast cultures ( %), in five retroviral ipsc clones ( %), and in none of the sendai ipsc clones. de novo cnvs were identified in retroviral ipscs ( %) - clones showed one ( %), nine clones showed two ( %), four clones showed three ( %), and one clone showed four newly arisen cnvs ( %). in seven of eight sendai clones, de novo cnvs were detected ( %). two de novo cnvs were identified in three clones ( %), three cnvs in two clones ( %), four in one clone ( %), and even six de novo cnvs were detected in one clone ( %). additionally, a mosaic gain of the whole long arm of one chromosome was identified in one sendai clone. only of the retroviral reprogrammed clones ( %) as well as one sendai reprogrammed clone ( %) showed no newly arisen cnvs. the cnvs were between kb and . mb in length in retroviral clones and between kb and kb in sendai clones. because almost all cnvs contained genes, including genes involved in neurodevelopment and transcriptional regulation, a biological relevance seems highly probable and changes in cell phenotype cannot be excluded. since ipscs and cells derived thereof -independent of reprogramming methods -often contain aberrations not detected by standard chromosomal analysis, our findings highlight the importance of high resolution cma procedures. in spite of differences in the analyzed number of clones, our results indicate more and larger cnvs in sendai clones than in retroviral reprogrammed clones. supported by forips, bavarian research network induced pluripotent stem cells. abstracts mother's lymphocytes showed a balanced form of the aberrant karyotype found in her son. the healthy mother possesses the insertion ins( ; ) (q . ;q q ), the simultaneous resection of the q ? - q . material and generation of the additional ring chromosome with this material. during meiosis a normal chromosome , the derivative chromosome and the ring chromosome were passed on to the son leading to the unbalanced karyotype as described above. further analysis with pan-centromeric probes and immunohistochemistry with an antibody against cenp-a might confirm the existence of a neocentromere on the ring chromosome which was meiotically transmitted from the mother to the son. the mtor (mechanistic target of rapamycin) kinase is the most important regulator of local dendritic and presynaptic protein translation in the brain. it has been shown to fundamentally influence ampa receptor activity by shifting glua to glua balance and to control the synthesis of several proteins involved in synaptic function and plasticity. both, loss and gain of mtor activity lead to significant disturbances of brain homeostasis and neuronal function resulting in intellectual disability (id), epilepsy, autism and behavioural alterations. in order to analyse mechanisms of homeostatic regulation of mtor-dependent synaptic function we are using a heterozygous tsc knockout (ko) mouse model for tuberous sclerosis (ts). tsc (together with tsc ) is part of a complex that inhibits the mtor kinase. mutations in either of the two genes result in increased mtor activity and upregulated downstream signalling and are causative for autosomal dominant ts. it had been shown previously that mtor hyperactivity leads to a set point shift and significantly influences neuronal homeostasis by altering cell excitability and e/i balance in heterozygous tsc ko animals. according to these studies we hypothesize that this shift significantly leads to synaptic connectivity and plasticity as well as network alternations that result in progressive development of cognitive dysfunction, epilepsy, autism and behavioural alterations over time in ts. we have established a behaviour battery, which consists of a novel object recognition test (nort), a morris water maze test (mwm) and an evaluation of nest building and social interaction to study social behaviour and cognitive performance in heterozygous tsc ko mice in different age groups. interestingly we found that, while two months old animals do not show any alterations, three to four months old animals develop significant disturbances in social behaviour in the social interaction test and in nest building. furthermore, we could show significant changes in cognitive performance of seven to eight months old heterozygous tsc ko mice in the nort compared to three to four months old heterozygous tsc ko animals. these data show that the ts phenotype builds up on shifts of the e/i balance that develop at a very early stage. pharmacological intervention with e/i balance is a promising therapeutic strategy to prevent brain damage caused by congenital hyperexcitability and homeostatic dysfunction. clear cases of a joubert phenotype an array-cgh is recommended besides sequencing to clarify the molecular basis of the phenotype. recently, a new syndrome of intellectual disability (id) with dysmorphisms due to deletions within the chromosomal band q . harboring the tbl xr gene was described. additionally, a specific point mutation in tbl xr causes pierpont syndrome, a distinct mental retardation syndrome. we report four patients in which array-cgh analysis and real-time quantitative pcr of genomic dna revealed tbl xr microduplication. adjacent genes were not affected. the microduplication was verified as de novo by real-time pcr of parental dna in one patient. the other three cases occurred in two generations of a second, unrelated family. we compare the remarkably similar clinical findings in tbl xr microdeletion, point mutation and microduplication cases and expand the tbl xr -associated phenotypic spectrum. among others, intellectual disability, hearing loss and autism spectrum disorders are common features of tbl xr -associated diseases. our clinical observations add to the increasing evidence of tbl xr 's role in physiological brain development and simultaneously demonstrate that opposing genetic disease mechanisms affecting tbl xr can lead to similar id phenotypes. in conclusion, the observed phenotypic overlap indicates that this novel microduplication is a genomic sister-disorder to the q . microdeletion syndrome. neocentromere formation has been reported in more than small supernumerary marker chromosomes (ssmc). here we report the formation of a putative neocentromere within a supernumerary ring chromosome derived from chromosome in a five year old patient with generalized developmental delay, microcephaly and hexadactyly. chromosomal analysis (gtg-banding, resolution bands) of the boy showed an aberrant karyotype with a derivative chromosome demonstrating additional chromosome specific material inserted into the distal part of the long arm (ins( ; )(q . ;q q )), a simultaneous deletion of the chromosomal region q ? -q . on the same homolog and an additional supernumerary small marker chromosome, presumably a ring chromosome in all metaphases tested. in parallel, array-cgh was performed and revealed a gain of . mb in chromosomal region q -q . without a detectable imbalance of chromosome material within the limits of the method ( k array). fish analysis with wcp probes and telomeric probes for chromosomes and confirmed the interstitial insertion of chromosome material into the long arm of the derivative chromosome . the marker chromosome stained completely with a wcp probe but centromere probes for chromosomes and were both negative suggesting the formation of a neocentromere within region q ? q . . in total, the complex rearrangement resulted in a partial trisomy q in the boy as a presumable explanation for the phenotype. the mother has experienced one abortion and the parents have two more children who show a normal development so far. both parents were karyotyped. the analysis of the father was normal ( ,xy). konventional chromosomal analysis of the in flvcr have previously been linked to vision impairment and posterior column ataxia in humans, but not to hsan. using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the flvcr -mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans. autosomal-dominant familial hypercholesterolemia (fh, omim ) is one of the most common genetic diseases with an estimated prevalence of : to : . three major genes have been associated with the disease: low density lipoprotein receptor (ldlr), apolipoprotein b (apob) and proprotein convertase subtilisin/kexin (pcsk ). we have recently published data on the mutation spectrum in these three genes in about fh patients from germany, however, this screen for mutations was negative in about % of the cases. stap (signal transducing adaptor family member ) was recently suggested to be the fourth fh gene. we therefore screened the stap gene for disease causing variants in a group of fh patients negative for mutations in the three established fh genes by direct dna sequencing (sanger). the newly identified variants will be presented including a phenotype-genotype correlation. phenotype: we present a -year-old girl with developmental delay, craniofacial anomalies, congenital eye anomalies ( . dpt), short stature and behavioral disorder with auto-aggressions. preliminary examinations (array-cgh analysis, karyotyping, diagnostic of metabolism, eeg, cmrt, sequencing of srcap-gene) revealed normal results. methods: in course of the midas-project, we sequenced the index patient and her healthy, non-consanguineous parents on a nextseq platform (illumina, san diego, ca, usa) to perform a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed with a sureselect human all exon kit v (agilent, santa clara, ca, usa), targeting coding exons and conserved splicing sites of over . genes. the library of the index patient was sequenced with a -fold mean coverage as -bp paired-end reads. % of the target region were covered -fold or higher. data analysis and variant evaluation was done using the clc genomic workbench . (qiagen, hilden, germany) and annotations from commercial as well as public databases. results: the trio analysis of whole-exome sequencing data from the index patient and her parents revealed variants. we identified the disease-causing de novo adnp mutation c. c>t (p.arg *, adnp), resulting in the introduction of a stop codon in exon / and truncation of the corresponding protein. both of the parents did not carry this mutation. adnp is part of the atp-dependent baf chromatin-remodeling p-monog- the challenge to insert costello syndrome causing hras mutations into human keratinocytes using the crispr/cas editing technology. l. brandenstein, k. kutsche, g. rosenberger university medical center hamburg-eppendorf, hamburg, germany germline missense mutations in the hras gene cause costello syndrome, a rare developmental disorder characterized by a typical facial gestalt, postnatal growth deficiency, intellectual disability, and predisposition to malignancies as well as skeletal, cardiac and dermatological abnormalities. the molecular pathophysiology caused by heterozygous hras gain of function mutations has been analysed in various tissues and cell types, however, up to date the molecular basis for cutaneous manifestations in costello syndrome is largely unknown. to address this question in an appropriate model system, permanent human keratinocyte (hacat) cells carrying costello syndrome-associated mutations in the endogenous hras gene should be generated by using the crispr/cas technology. double strand breaks induced by cas can be repaired in two ways: the error-prone non-homologous end-joining pathway for the generation of knockout models or the homology directed repair pathway, which allows precise editing. the latter enables the introduction of specific point mutations into a cell line by using a single stranded dna (ssdna) as repair template. however, we found that this is a very rare event and its efficiency depends on various factors including used cell line, selected guide rna, length and amount of ssdna and also cas variant. nonetheless, by using cas wildtype, we could insert the disease-associated c. g>t (p.g v) mutation into genomic hras both in hacat cells ( positive clones out of ) and hek cells ( positive clones out of ). in contrast, using the cas nickase protein variant that prevents off target effects, did not result in positive clones. taken together, in months of working with crispr/ cas we gradually gained experience with many problems and pitfalls of this technology and, finally, now we are able to introduce point mutations in cell lines. next, we will use the mutant hacat cell line to gain deeper insight into the function of hras for epidermal homeostasis and its deregulation in costello syndrome. ccdc could also play a role in prenatal development of the mouse retina and brain. embryonic stages of interest comprise <e at the initial development of the eye, e after formation of nasal pits and prominent brain growth, when brain vesicles are initially visible and e where the eye and nasal brain develops. this study aims at characterizing ccdc rna and ccdc protein expression during key steps of prenatal development in the wt mouse and respective ccdc reporter gene expression in the ccdc -/model. methods: ccdc rna expression was analyzed by quantitative ccdc qrt-pcr and ccdc protein expression was determined by sds page and western blot in eye and whole brain homogenates of the mouse at selected embryonic stages (<e , e and e ). in addition, µm sagittal cryosections of mouse embryos were hematoxylin and eosin stained and analyzed for ccdc reporter gene expression by x-gal staining or antibody staining followed by light or fluorescence microscopy. results: ccdc rna and ccdc protein is detected in the wt mouse at different developmental time points in the eye and brain tissue. accordingly, ccdc -/reporter gene expression is evident at prenatal developmental stages in cryosections of whole embryos. conclusion: expression studies of ccdc gene and products in the eye and brain in wt and ccdc -/mice in different developmental embryonic stages are of crucial interest, and the role of ccdc gene and products in prenatal development has to be evaluated in detail in further studies. nn. hauer, b. popp, e. schoeller, ab. ekici, a. reis, ct. thiel institute of human genetics friedrich-alexander-universität erlangen-nürnberg, erlangen, germany the application of next generation sequencing technologies (ngs) has been extremely successful in the identification of the underlying causes of genetic disorders. whereas, most variants are located in coding regions, variants within splice-sites that might lead to alternative splicing affecting protein function or causing mrna decay are less well characterized. even for canonical splice-sites, prediction of the splicing effects for an identified variant is cumbersome. this becomes even more apparent as ngs based exome and genome sequencing tremendously increase the number of variants potentially affecting splice-sites. in our study to identify the underlying causes of idiopathic short stature by whole exome sequencing in more than patients, we observed variants in consensus splice-sites defined as up to bases from the ' and bases from the ' end of an exon. we used several computational methods (splice site finder, maxentscan, nnsplice) to calculate their potential effect on splicing. these results were often inconclusive and did not predict the definite effect on transcripts. therefore we performed rtpcr amplification of the respective regions, electrophoretic evaluation of fragments and sanger sequencing. in case of a suspected alternative splicing the evaluation of the distinct resulting isoform, though, was often hampered by the overlap of sequences. this was especially severe when multiple naturally occurring alternative transcripts were present. to overcome these issues, we established a deep sequencing approach (targeted rtpcr-seq) to identify all present isoforms in the affected patients compared to controls. for this method the rtpcr fragments were normalized to generate an equimolar mixture. libraries for patients and controls, respectively, were generated by ligation of distinct index adaptors for both amplicon pools and subsequently sequenced on an illumina miseq platform. the resulting reads were aligned and visualized as sashimi plots. observed splice-junctions in patients were compared to the controls and public databases. we found concordant results between standard rtpcr evaluation and our rtpcr-seq approach for variants (alternative splicing for variants). for these confirmed variants the specific effect was more obviously ascertained by the ngs based approach. for variant only rtpcr-seq was able to separate multiple co-occurring distinct isoforms, both in controls complex, which is involved in the regulation of gene expression. an influence on neuronal cell differentiation is postulated. mutations in the adnp gene were previously reported in patients with autism and mild to severe intellectual disability. all reported adnp mutations are characterized by de novo mutations that are clustering in exon and severely disrupting the protein-coding sequence, either by the introduction of a stop codon or a frameshift induction. by additionally considering the clinical symptoms, we were able to diagnose the helsmoortel-van der aa-syndrome (hvdas; omim id: ). discussion: the challenge of next-generation sequencing (ngs) approaches in diagnostics is no longer the technical feasibility or costs but the medical interpretation of the large sequencing data sets. besides a robust genetic analysis with focus on disease-associated genes, family anamnesis and detailed information of clinical symptoms in a standardized scheme are important for a reliable diagnosis. here, we present one of over cases that will help to establish the multiple integration of data annotation software (midas), which will enable phenotype-genotype-correlations to enhance the validity of results of genetic analysis, minimize the risk of misinterpretation and enable faster diagnosis in routine patient care. peripheral neuropathies are a group of clinically and genetically heterogeneous disorders, mutations in a large number of genes have been described so far. among patients with charcot marie tooth disease (cmt)/hereditary motor and sensory neuropathies (hmsn), a common duplication of the pmp gene on chromosome p is by far the most frequent mutation; sequence variants in the genes gjb , mpz and mfn account for a considerable proportion of patients but all other associated genetic variants are extremely rare. deletions of the pmp gene lead to hereditary neuropathy with liability to pressure palsies (hnpp) but transition between the different phenotypes may be rather fluid in some instances. next generation sequencing (ngs) has revolutionized cmt diagnostics as a large number of genes can be investigated at a time but it also leads to challenging questions. use of ngs has certainly improved the diagnostic yield in cmt diagnostics and it has repeatedly resulted in unexpected findings. broad molecular testing has already disclosed overlaps between clinical conditions and more surprising results are yet to come. here we report our recent experiences with the diagnostics of neuropathies. our results, different testing strategies, implications for clinical management and genetic counselling will be discussed and possible further steps suggested. (gerding et al., hum mol genet., ) . retinal ccdc protein expression in the wildtype (wt) mouse demonstrates highest levels shortly after birth. moreover, ccdc protein can also be detected early postnatally in mouse brain tissue. therefore, there is strong evidence that p-monog- *** individuals with hereditary trichilemmal cysts present a combination of an inherited high-risk plcd allele (including two sequence variants) and additionally acquired somatic plcd variants in the cysts. trichilemmal cysts are benign tumors derived from the outer root sheath of the hair follicle. they occur most often solitary or multiple on the scalp but can also be found on other body parts. the cysts can develop sporadically or in an autosomal dominant mode of inheritance with incomplete penetrance. in , the hereditary cyst form was mapped to the tricy locus on chromosome p -p . in a familial genome-wide linkage study. we analyzed affected individuals from different tunisian families by whole-exome sequencing. the bioinformatic evaluation revealed no rare, disease causing mutations but the sequence variant c. c>t, p.s l (maf = . , rs , enst . ) in the phospholipase c delta (plcd ) gene within the tricy locus. furthermore, all five individuals present a second variant c. a>g, p.p p (maf = . , rs ) in the same gene. plcd is a member of the phospholipase c family. the enzyme is involved in calcium-dependent intracellular signal transduction and catalyzes the hydrolysis of phosphatidylinositol , -bisphosphate into the second messenger diacylglycerol and inositol triphosphate. homozygous knockout-mice have hair defects and show aberrant skin development with increased progression of skin tumors and intradermal hair-follicle derived cysts. in humans, plcd is highly expressed in the hair follicle but so far only nonsense mutations have been described causing hereditary leukonychia totalis without any skin or hair abnormalities. a segregation analysis of plcd in a tunisian family cohort and one german family ( families, individuals, affected) showed that all affected individuals contain the same two sequence variants. based on these results, we propose that plcd is responsible for the cyst phenotype. cdna sequencing from three different cysts revealed additional acquired somatic sequence variants in plcd . we found c. c>t p.s l in two cysts and the two variants c. c>t, p.s f and c. c>t p.s f in the third one. all three somatic variants are not described in the exac database and the genomes project. allele-specific rt-pcrs were performed with cyst cdna and we could show that the somatic variants are on the same allele as the inherited variants. the acquired somatic cyst sequence variants lie within or respectively near the c domain of the plcd protein. the c domain is involved in the calcium-dependent binding to membrane-integrated phospholipids. depletion of the domain leads to decreased membrane association and protein activity. we assume that the allele with the variants c. c>t and c. a>g is a risk factor for hereditary trichilemmal cysts and that additional acquired rare sequence variants are the genetic trigger for the development of the cysts. and patients. discordant results for variants proposed a higher specificity of the rtpcr-seq. thus, we established a simple amplicon based deep sequencing approach for standard rtpcr fragments to ascertain the effects of specific splice site variants. this technique has proven to be highly scalable, fast and efficient to analyze splice-site variants. p-monog- eif s mutations associated with severe x -linked intellectual disability syndrome mehmo mehmo (mim % ), is a rare x-linked syndrome characterized by profound intellectual disability, epileptic seizures, hypogonadism, hypogenitalism, microcephaly, and obesity. in steinmüller and colleagues described a large family with mehmo syndrome with five affected males in two generations and assigned the disease locus to the short arm of chromosome x (xp . - . ). we took advantage of massively parallel sequencing in four families with mehmo syndrome, including the family reported by steinmüller et al. to identify the underlying genetic cause if this severe disorder. we here show mehmo syndrome is associated with mutations in the x chromosome gene eif s . in three families we identified a c-terminal frameshift mutation (p.ile serfs) and in an unrelated boy who is less severely affected, we identified a novel maternally inherited missense mutation (p.ser arg) in eif s . eif s encodes the gamma subunit of the eukaryotic translation initiation factor (eif ). eif is essential for eukaryotic translation initiation and regulation of the integrated stress response (isr). subsequent studies in patient fibroblasts (p.ile serfs) showed increased isr activation due to the mutation and functional assays in yeast demonstrated that the p.ile serfs mutation impairs eif gamma function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the affected males with ile serfs mutation. our results suggest that more severe mutations in eif s cause the full mehmo syndrome, while less deleterious mutations are associated with a milder form of the syndrome with only a subset of the symptoms. introduction: larger structural genomic duplications or deletions (copy number variations = cnvs) are routinely detected by array comparative genomic hybridization (acgh). while acgh has been established as a robust and effective approach for cnv screening, it remains expensive and is limited by resolution. in addition, commercial mlpa testing today allows the identification of exon deletions or duplications for a limited number of core genes. more recently, massive parallel sequencing of multi gene panels (mgps) has been introduced as a fast and cost-effective tool in routine genetic diagnostic testing to identify causal intragenic sequence alterations not only in core genes but also in those with small contribution to the respective phenotypes. the obtained mgps data may also be bioinformatically assessed to detect exon deletions or duplications within the analyzed genes panels and thus can be expected to further improve the diagnostic yield. methods: more than patients were sequenced with phenotype specific gene panels on a miseq platform (illumina) and analyzed with our bioinformatic diagnostic workflow including a quantitative data assessment using our in house java based bioinformatic script to search for gene or exon deletions. detected deletions were confirmed by an independent method (e. g. mlpa, pcr amplification of the junction fragment or linkage analysis), if available. results: we here report details for six suspected deletions in seven patients detected by our in house bioinformatic workflow: heterozygous gene deletions of pafah b , spastin or arfgef , respectively; two heterozygous cftr deletions of exon and , one homozygous partial sftpb deletion and a homozygous ispd exon deletion. the complete gene deletions of pa-fah b and spastin as well as the cftr deletion of exon and could be confirmed by mlpa. for the partial sftpb deletion both breakpoints could be precisely located within the readout, allowing determining correct deletion size and design of primers to amplify the junction fragment. the homozygous deletion of exon in the ispd gene could be confirmed by pcr and linkage analysis. discussion: diagnostic multi gene panel sequencing after nextera enrichment allows sufficient homogeneity of the obtained patient and control data per target to quantitatively search for constitutional deletions covering two or more adjacent targets. combined data assessment considering the individual clinical data will not only further increase the diagnostic yield but can also be expected to further delineate the mutational spectrum for specific phenotypes by the simultaneous detection of clinically relevant sequence variants as well as cnvs. mgps sequence assessment may also allow to gain new insights into the genomic architecture and origin of target regions and haplotypes, involved in common structural variations. c. niemietz, v. sauer, l. fleischhauer, s. guttmann, s. reinartz groba, a. zibert, h. schmidt universitätsklinikum münster, klinik für transplantationsmedizin, münster, germany various types of somatic cells have been reprogrammed to induced pluripotent stem cells (ipsc) followed by differentiation into hepatocyte-like cells (hlc). recently, cells that shed from the renal epithelial system were shown to be a suitable and convenient source for ipsc generation. in the current study, urine-derived cells (ucs) were isolated from urine donations of patients having familial amyloid polyneuropathy (fap), a neurodegenerative disease caused by mutation of the transthyretin (ttr) gene and wilson disease (wd), a genetic disorder of atp b causing copper accumulation, predominantly in liver and brain. patient-specific hlcs were differentiated in order to study disease-specific mechanisms and to investigate the efficacy of novel compounds. for isolation of renal epithelial cells, urine of fap and wd patients was processed. ucs were reprogramed into ipscs using plasmids resulting in transient expression of factors sox , oct / , klf , and c-myc. after characterization of ipsc that expressed high levels of pluripotency markers, like oct and nanog, a -step hepatocyte differentiation protocol was performed. ipscs were subjected to a treatment with growth factors (activin a, wnt a, fgf , hgf) for days. the hepatic, patient-specific character of differentiated hlcs was assessed by functional analysis, gene expression profiling, genotype analysis, and immunostainings. therapeutic oligonucleotide efficacy targeting ttr was determined by immunocytochemistry, qrt-pcr and western blot analysis. ttr-stabilizing activity of tafamidis was investigated by means of thermal shift assay and western blot analysis. copper chelation by methanobactin was determined by atomic absorption spectroscopy. reprogramming of ucs resulted in stable ipsc lines with characteristic pluripotent marker expression. differentiated hlcs showed high similarity to human hepatocytes in terms of genetic profile and functional activity. small-interfering rnas (sirnas), antisense oligonucleotides (aso) (niemietz et al. , plosone ( ) :e ), and the ttr stabilizing compound tafamidis that are currently assessed in clinical studies were studied in hlcs derived from fap patients. a novel chelator was used to determine intracellular copper accumulation in hlcs derived from wd patients (lichtmannegger et al. , jci ( ): - ). fap-specific hlcs revealed differently expression of key regulators of the protein quality control (pqc) system. our results demonstrate that ipsc derived from urine are excellently suited to study hereditary liver diseases. hlcs could be investigated in the patient-specific genetic background. the efficacy of novel compounds was assessed and individual responses were monitored. involved in gene regulation. by parent-patient trio whole-exome sequencing, we were able to characterize the unterlying genetic cause in the patient, who has undergone multiple diangostic test before without receiving a diagnosis. discussion: although for many congenital syndromic diseases the disease-associated genes are known it remains difficult for physicians to interpret the highly variable phenotypes as well as their variable nomenclature in order to request the appropriate molecular analysis. here we present one of more than cases that will help to establish a database connecting specific phenotypes, using the human phenotype ontology terms, with the each corresponding disease-causing genes (midas). novel compound heterozygous nalcn variants in two brothers with muscular hypotonia and global development delay l. segebrecht , c. weiß , m. jäger , , t. zemojtel , , , d. horn , n. ehmke , institute of medical and human genetics, charité -universitätsmedizin berlin, berlin, germany, spz dpt. pediatric neurology, charité -universitätsmedizin berlin, berlin, germany, berlin-brandenburg center for regenerative therapies -bcrt, charité -universitätsmedizin berlin, berlin, germany, institute of bioorganic chemistry, polish academy of sciences, poznań, poland, labor berlin -charité vivantes gmbh, berlin, germany, berlin institute of health, berlin, germany we report on two brothers aged two and three years, with muscular hypotonia, global development delay, abnormal respiratory rhythm, mild facial dysmorphism, recurrent respiratory infections, and failure to thrive. sequencing of disease related genes identified compound heterozygosity for two novel mutations in nalcn: c. c>a (p.phe leu) and c. + t>c. nalcn encodes a voltage-independent, non-selective cation channel, which is involved in regulation of neuronal excitability. the missense variant c. c>a affects the highly conserved amino acid position phe which is located in segment s of domain iv in the pore-forming unit of nalcn. the variant c. + t>c alters the donor splice site in intron and is predicted to cause skipping of exon , resulting in loss of function of nalcn. biallelic mutations of nalcn are associated with infantile hypotonia, psychomotor retardation and characteristic facies (ihprf ), whereas heterozygous de novo mutations cause congenital contractures of the limbs and face, muscular hypotonia, and global developmental delay. the clinical features of our patients resemble mild ihprf , caused by a biallelic missense mutation in segment s of domain iv. it has been suggested that variants in or close to s and s of the pore-forming domains lead to the above mentioned autosomal dominant condition whereas variants in other regions or loss of function mutations result in autosomal recessive inheritance. this is the first report of a mutation in a s segment, inherited in autosomal recessive manner. our findings indicate that phenotype-genotype correlations in nalcn are more complex than suggested so far. phenotype: in a young undiagnosed patient with developmental delay, intellectual disability and craniofacial dysmorphic anomalies, whole-exome sequencing (wes) identified a de novo insertion in the gene arid b, known to cause the rare congenital coffin-siris syndrome and nicolaides-baraitser syndrome, respectively. methods: as part of the midas genotype-phenotype-correlation project, we sequenced the index patient and his healthy parents on a next-seq platform (illumina, san diego, ca, usa) and performed a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed using the sureselect human all exon kit v (agilent, santa clara, ca, usa) to target most of the over . genes. the libraries were sequenced to approximately -fold mean coverage as bp paired end reads. % of the target region was covered -fold or higher. data analysis and variant evaluation was performed using the clc genomic workbench . (qiagen, hilden, germany) and annotations from commercial as well as public databases (dbsnp, hgmd, clinvar, exac). results: we identified a de novo bp insertion in the arid b gene, causing a frameshift mutation that leads to the truncated protein. arid b is part of the atp-dependent chromatin remodeling baf-complex, which is abstracts tient affected by psoriasis carried the surrogate marker snp rs -a for the psors risk variant hla-cw haplotype homozygously; and the same snp was found heterozygous in his prp-affected father. neither the pathogenic variant in card , nor the risk variants for psoriasis described above were found in the healthy mother. whole exome sequencing revealed genetic variants, predicted to have serious consequences in further genes involved in the nf-κb as well as the notch pathway. these variants either segregate with prp or are present in the psoriasis affected individual only. the presence of an individual carrying the same card mutation as his prp-affected relatives but suffering from psoriasis instead strengthens the relation between prp and psoriasis, which has been repeatedly suggested in literature. we propose a balance between familial prp and psoriasis in the family investigated in this study and present genetic variants, which might influence this balance in addition to variants in card . wilson's disease (wd) is an autosomal recessive disease resulting from copper (cu) excess due to mutations in the atp b gene coding for a cu-transporting atpase. wd pathogenesis, however, can not only be explained by gene coding mutations since phenotypes exhibit strong variations despite the same exonic dna makeup in the gene. also in several patients with clinical wd symptoms no gene coding variants are detectable. our former studies revealed decreased liver atp b mrna expression in some wd patients. this decrease was not only observed in patients with nonsense atp b mutations leading to rapid mrna decay, but also in patients with missense mutations and also in some patients with suspected wd without atp b mutations. patients with low atp b expression presented with a more fulminant disease progression. however, we could not detect mutations in the atp b promoter region (c.- to atg) in those patients. there are possibly other deregulating mechanisms responsible for decreased atp b mrna expression. up to now, atp b transcriptional regulation is only poorly characterized. it is known, that four metal responsive elements (mre a, c, d and e) are located within the atp b promoter. gene regulation through mres is often metal-dependent. liver atp b mrna expression revealed also to increase under cu addition in several species by an unknown mechanism. up to now, only one atp b transcription factor (tf), the mrea binding ku protein, is known. the aim of our work was to further analyze the regulation of the atp b gene, especially through mres. to screen for tf-mre interactions and to narrow down the binding site of tf, we performed electrophoretic mobility shift assays (emsa) by incubating nuclear extracts of the liver cell line hle with probes corresponding to atp b mrec, d and e. to identify mre-binding tf matinspector analysis was performed. identified candidate tf were coexpressed with atp b promoter-driven reporter gene to evaluate their impact on reporter gene expression. one in the reporter assay positively tested tf was validated by different emsa experiments. further it was overexpressed with and without addition of metal ions in hle to investigate the impact on endogenous atp b expression. we showed that tf mtf is able to bind to mree within the atp b promoter and significantly increases atp b promoter driven reporter gene expression. mtf binding was primarily mediated by the first three bases of the mre consensus sequence. also for mrec and mred specific protein interaction could be shown and the protein binding site was narrowed down by emsa with protein identification still pending. fur-polyglutamine diseases is the formation of the so called neuronal intranuclear inclusion bodies (nii). as ataxin- is predominantly located in the cytoplasm, the formation of protein aggregates in the nucleus require a nucleocytoplasmic shuttling of ataxin- . we already demonstrated in vivo using transgenic mouse models that the toxicity of expanded ataxin- depends on its intracellular localization: while nuclear ataxin- gave rise to a strong phenotype with a high number of protein aggregates, purely cytoplasmic ataxin , however, even with a highly expanded polyglutamine repeat ( glutamines), was not able to induce a phenotype and even did not aggregate. we further identified and characterized intracellular transport signals (two nuclear export signals, nes, and one nuclear localization signal, nls) within the coding sequence of ataxin- . therefore, it is evident that proteins involved in the nucleocytoplasmic transport machinery recognize these localization signals, control the intracellular localization of ataxin- , thereby influence the toxicity and aggregation of ataxin- and, thus, the pathogenesis of sca . we now screened a library of transport proteins in order to identify the transport protein which is critically involved in the nucleocytoplasmic shuttling of ataxin- . we indeed identified a transport protein which modifies both the formation of aggregates and the intracellular localization of ataxin . while the overexpression of this protein moved ataxin- into the nucleus, its downregulation kept it out of the nucleus. we replicated this correlation in vivo in drosophila and observed in addition to this again a clear link between the intracellular localization of ataxin- and its toxicity i. e. its ability of induce neurodegeneration and a behavioral phenotype. likewise we even confirmed in a mouse model of sca the importance of the identified transport protein as its knockout largely prevented ataxin- from aggregating and alleviated behavioral and movement deficits. understanding the mechanisms behind the intracellular transport of ataxin- could give us clues into the pathogenic functions of expanded ataxin- and ways to mediate the progression of neuronal degeneration in sca . evidence for genetic factors outside card influencing the phenotype of a family with familial pityriasis rubra pilaris and psoriasis familial pityriasis rubra pilaris (prp) is an erythematous inflammatory skin disease caused by heterozygous activating mutations in card , a known activator of the nf-κb pathway. different genetic variants within card have been associated with psoriasis. the purpose of our study was to clinically and genetically investigate affected as well as unaffected members of a family with prp in order to determine the mutation responsible for this severe skin disease in the three affected family members. a father, three of his adult children as well as the mother of one child affected by prp were investigated clinically. in addition we extracted genomic dna from the blood of each individual and performed whole exome sequencing as well as direct sequencing of single genes. clinical investigation confirmed that the father and two of his children were affected by familial prp, with the skin showing the characteristic pattern of prp, early onset and chronic course. a third child was unaffected by prp, suffered however from psoriasis. the mother of one child affected by prp showed no sign of skin disease. genetic investigation revealed a heterozygous missense mutation in exon of card , c.[ c>t], p. present in all investigated individuals with prp or psoriasis. the same mutation has been described before as being pathogenic in a different family with prp. regarding genetic variants associated with psoriasis, we found the risk alleles of three coding variants in card : rs , c. short stature is a common condition of great concern to patients and their families. in most cases it is genetic in origin but the underlying cause often remains elusive due to clinical and genetic heterogeneity. in an unbiased approach we carefully phenotyped patients and randomly selected for whole exome sequencing. sequence variants were analyzed for pathogenicity and the affected genes characterized regarding their functional relevance for growth. all patients received extensive clinical and endocrinological examinations, careful clinical genetic phenotypic evaluation followed by targeted diagnostic assessment for suspected diagnoses. we identified a known disease-cause in only % of patients, the most common causes being cnvs found in %, followed by syndromic monogenic causes in % and turner syndrome in %. whole exome sequencing identified additional mutations in known short stature associated genes ( ) in % of patients who manifested only part of the symptomatology precluding an early clinical diagnosis. here, heterozygous carriers of recessive skeletal dysplasia alleles (acan, npr ) were a surprisingly frequent cause of idiopathic short stature found in . % of cases. we next selected known short stature genes with mutations for pathway analyses of the affected proteins and found that % are involved in the main functional categories cartilage formation, chromatin modification and ras-mapk signaling. in addition we identified further strong candidate genes, of which seven had deleterious mutations in at least two families. interestingly, % of these candidate genes are involved in the main functional categories already identified for the known short stature associated genes further supporting their pathogenicity. finally, in % of the sequenced individuals our findings were of significant clinical relevance regarding preventive measures, symptomatic or even targeted treatment. besides evaluation for orthopedic or developmental issues especially screening for neoplasias (trim , ptpn , nf ), symptomatic treatment for chronic kidney disease (clcn ) and targeted treatment for severe hypertension (pde a) were of clinical relevance for the affected individuals. these results demonstrated that deep phenotyping combined with targeted genetic testing and whole exome sequencing is able to increase the diagnostic yield in short stature up to % with concomitant improvement in treatment and prevention. rigorous variant analysis considering phenotypic data further led us to the identification of further probable novel candidate genes. thermore, we found the endogenous atp b mrna expression to be significantly increased in hle after cu treatment or cu treatment and concurrent mtf overexpression. sole mtf overexpression did not alter atp b expression. we newly identified mtf to bind mree within the atp b promoter. its in vivo role in the pathogenesis of wd needs to be further elucidated. modifying genes have been identified for lung function in cystic fibrosis [ ] , disease severity [ ] and several comorbidities [ ] . within the european cf twin and sibling study, we focus on genes that modify the basic defect, assessed as defective chloride conductance in cftr-expressing epithelia, which we could describe by an association study on patient cohorts selected for informative endophenotypes among f del-cftr homozygous patients [ , , ] . as a first example, rs in fas modifies fas gene expression (p = . , data from intestinal biopsies [ ] ), cf disease phenotype (praw = . , comparing concordant mildly affected f del homozygous sib pairs and concordant severely affected sib pairs; praw = . , comparing unrelated f del homozygous index cases without residual chloride secretion by nasal potential difference measurement (npd) to patients with cftr-mediated chloride secretion; [ ] ) and alters binding affinity for the transcription factors nf-kbp , nf-kbp and hif a [ ] which govern the cellular response to infection and hypoxia. as a second example, the transcription factor ehf, derived as a positional candidate from a north american genome wide scan [ ] , is associated with npd-defined phenotypes (praw = . , comparing index cases with high response to amiloride in npd to index cases with low response to amiloride in npd; praw = . , comparing index cases without chloride secretion in intestinal current measurement (icm) to index cases with cftr-mediated residual chloride secretion in icm [ ] ) and affects the transcriptome of cf patients' intestinal biopsies in favor of a better processing of f del-cftr [ ] which we could confirm in epithelial cell lines as sirna provided against ehf results in a downregulation of mgat and mgat , both of which are key enzymes for the complex glycosylation of proteins such as cftr. these examples indicate that small, albeit carefully selected subpopulations facilitate the identification of genetic variants by an association study that can be validated in functional assays. furthermore, we suggest that while the selection of subsamples within a population with a rare disease such as cystic fibrosis results in a loss of power, findings obtained for more than one endophenotype are indicative for a true-positive finding of a modifying gene. finally, transcriptional regulation influences the cf basic defect. interference with these pathways may results in better f del-cftr maturation, leading to better cftr function in patient's tissue and thereby promoting health in cystic fibrosis. funding by: deutsches zentrum für lungenforschung dzl; mukoviszidose institut ggmbh abstracts at presentation at age years her head circumference was on the th centile, her height below the rd centile, and her weight on the rd centile. she showed a disproportional short stature and mild skeletal signs like clinodactyly of the ths fingers. minor facial dysmorphisms included an oval face, epicanthic folds, upslanting palpebral fissures and a bulbous nose. karyotype was normal and copy number variants were excluded by high-resolution cma. as no aetiological diagnosis could be made clinically we performed whole exome sequencing (wes) and detected a homozygous splice site mutation (c. + g>t) in pik c a (phosphatidylinositol- -phosphate -kinase catalytic subunit type alpha) (nm_ ). sequencing of rt-pcr products from cdna of patient's fibroblasts showed in-frame skipping of exon and , equally affecting all known isoforms. phosphatidylinositol -kinases (pi ks) are lipid kinases involved in a large set of biological processes, including membrane receptor signaling, cytoskeletal organization, and endocytic trafficking. pi kc a is ubiquitously expressed and has been proposed to play an important role in clathrin-mediated endocytosis and regulation of phosphatidylinositol -phosphate (ptdins p) levels. furthermore pik c a has been implicated in the biology of the primary cilium. the patient's distinct phenotype resembles the previously described phenotype in pik c a hypomorphic mice with pre-and postnatal growth retardation and a broad spectrum of renal abnormalities. the complete knockout of mouse pik c a showed embryonic lethality. ongoing studies on the exact consequences of the splicing defect will determine if and how much residual wild-type transcript is retained and if this is a hypomorphic variant. this case is to our knowledge the first description of a pik c a human phenotype. adaption of the crispr/cas genome editing system as a platform for the mutation of nipal as a representative of arci-associated genes in hela cells n. ballin, m-a. rauschendorf, j. fischer institute of human genetics, freiburg, germany autosomal recessive congenital ichthyosis (arci) is a rare genetic disorder with known disease-causing mutations in genes. functional implementations of identified mutations are in most cases still unknown, which is amongst others due to the limited amount of skin biopsies of arci-patients. thus, suitable cell culture models for the investigation of keratinocyte differentiation are highly needed. during the last years the prokaryotic clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated (cas) system has been turned into a potent tool in the field of genome engineering. the cas endonuclease is directed by a short guide rna (grna) to its target sequence, where it generates a dna double strand break (dsb). as cellular repair mechanisms often fail in reconstituting the original sequence, insertion/ deletion (indel) mutations occur potentially leading to a complete gene knock-out (ko). consequently, the crispr/cas system offers a simple rna-programmable tool for in vitro mutation of arci-associated genes. hence, this system was applied in hela cells to target nipa like domain containing (nipal ), the second most frequently mutated gene in arci patients. in this context, functional studies were used to validate nipal as suitable target in hela cells. successful application of the crispr/cas system lead to the generation of a new clonal hela cell line carrying a one basepair insertion in nipal exon (c. insg (ccds . ) ). this mutation was further characterized and potentially results in a complete ko of nipal . this system can now be used as a basis for targeting further arci-associated genes and for transferring the system into keratinocytes, which are the amyotrophic lateral sclerosis (als) is a late-onset progressive, neurodegenerative syndrome. most als cases are sporadic (≈ %). in familial forms, mutations in several different genes have been identified with a repeat expansion in c orf and mutations in sod being the most prevalent. no non-genetic cause of als has been identified. since there are no overt clinical or pathoanatomical differences between sporadic or familial cases, de novo mutations have been suggests in disease pathogenesis and two previous studies provided some evidence for this hypothesis. we present data of patient-parents trios from an international collaborative study. by whole exome sequencing we identified non-synonymous de novo mutations in the patients, however all of them occurred in different genes. there was no concordance between the mutated genes found in our trio set and the two earlier smaller trio studies. in silico analyses suggest that none of the here identified mutations are part of any of the previously postulated molecular pathways. also, gene-gene-interaction analyses failed to find an enrichment of interacting genes. lastly, we demonstrate that the de novo mutations in als patients in this and the two earlier studies are located in genes prone for de novo mutations in general. our results thus indicate that, in contrast to previous reports, de novo mutations do not seem to be a major contributor for als. disproportioned short stature and multiple anomalies in a patient with a homozygous pik c a mutation -a new ciliopathy? we present a year old female with disproportional growth retardation, eye and renal anomalies. she was born small for gestational age as the first child of healthy, consanguineous tunisian parents at weeks after an uneventful pregnancy ( g, cm). during her first year of life, there were severe feeding problems with regurgitation and she developed postnatal growth retardation. developmental milestones were normal. during the second year the girl developed strabismus in both eyes. later on, a cataracta polaris anterior, anomalies of the cornea, hyperopia and progressive retinal degeneration led to profound visual impairment. furthermore, a right kidney agenesis and a complex tubulopathy, as well as increased echogenicity of the single left kidney were diagnosed. r. brumm, m. schmuck, y. dinçer, s. schulz, s. wilson, i. rost, hg. klein, sh. eck center for human genetics and laboratory diagnostics dr. klein, dr. rost and colleagues, martinsried, germany next-generation sequencing may lead to a significant improvement in diagnostic yield for rare, heterogeneous disorders through the ability to simultaneously sequence all genes contributing to a certain indication at a cost and speed that is superior to traditional sequencing approaches. on the other hand, the practical implementation of ngs in a clinical diagnostic setting involves a variety of new challenges which need to be overcome. among these are the generation, analysis and storage of unprecedented amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. especially the variant interpretation emerges as current bottleneck of the diagnostic workflow. here we present midas (multiple integration of data annotation software), a central software system for data integration in a diagnostic laboratory. the goal of midas is to construct a modular software system to integrate data from laboratory information management system (lims), data from the routine sanger sequencing workflow as well as ngs sequencing results and correlate the identified variants with the patients' phenotypical features to aid in variant interpretation and accelerate reporting. the phenotype is systematically recorded using the human phenotype ontology standard nomenclature. in particular, genotype-phenotype correlations identified in one patient are made available for all other cases, to aid the interpretation and build a comprehensive knowledge base. the midas software may thus serve as central information system all diagnostic patient data. midas is implemented in java using direct database access via jdbc, and javafx as graphical user interface. its architecture is designed modular including a dynamic module loader, a user management with ldab connection and basic search functionality. according to available modules the user management and search form are adjusted; granting access for module specific views. as an advantage of this architecture, other molecular diagnostic data, such as arraycgh or mlpa results can easily be integrated by implementing new modules. midas aims to aid molecular diagnostics by simplifying and accelerating data analysis and interpretation, improving patient care. midas is being developed as part of a prospective multicentric study including clinical, diagnostic and software development partners. a grant by the bavarian ministry of economy, media, energy and technology is used to fund this effort. institute of human genetics -medical faculty -rwth aachen university, aachen, germany, izkf aachen -rwth aachen university, aachen, germany with the implementation of next generation sequencing (ngs) based assays as key tool in dna-sequencing, conventional sanger-sequencing has become a standard method to verify single nucleotide polymorphisms (snps) of interest. however, designing specific pcr-and sanger-sequencing primers has always been a rather time consuming task in terms of searching the right genomic sequence, considering known snps that could result in allele-specific amplification, and converting selected primer-sequences to upload them to web-based services. to circumvent this, we developed optimus primer, a python script, that automatically designs respective primers. the script uses the database of the ucsc genome browser to download positional information of genomic sequence (refseq gene: hg ) and common snps (snp common: hg ) in the region of interest. the genomic sequence is downloaded via the ucsc primarily affected cell type in the skin of arci patients. furthermore, it is only a small step to expand the system from generating gene ko to gene editing allowing the introduction of patient-specific mutations in non-patient derived keratinocyte cells. hence, setting-up the crispr/cas system to target an arci-associated gene is an important starting point for future studies to investigate pathogenic effects of arci-causing mutations and to understand arci pathogenesis on a molecular level. in this context, the transfer of the established protocol into keratinocytes offers the possibility to generate d cell culture models of mutations in arci-associated genes and allows in vitro investigation of their implications on the differentiation process. this system would further represent an organotypic model of arci-disease with potential application in screening and identification of chemical compounds for the complementation of existing clinical therapies. background: telomeres cap and protect chromosome ends from degradation and fusion, and are therefore essential for maintaining chromosome stability and genomic integrity. they have a length of to kb (depending on age, sex and cell populations). due to the end-replication problem there is a continuous telomere loss of to bp/cell cycle and thus throughout lifetime. in laboratory practice different methods of telomere length measurement are used to identify patients with bone marrow failure syndromes (e. g. dyskeratosis congenita, aplastic anemia), hematological diseases, or other telomeropathies. each telomere length measurement method has its advantages and disadvantages regarding material required the complexity and feasibility of the method and other parameters. methods: in this study we compared and validated four different methods for telomere length measurement, i. e. southern blot analysis, quantitative pcr (qpcr), quantitative fluorescence in situ hybridization (t/ c-fish) and flow cytometry-fish (flowfish). whenever possible, edta and/or heparin blood samples were collected from a population of healthy individuals of different age groups (newborn - years). depending on the method dna (southern blot and qpcr), metaphases (t/ c-fish) or rather vital cells (flowfish) were analyzed. results: comparison and validation of the telomere length measurement methods allowed us to calculate percentiles for all age groups. percentile curves could be used in diagnostic to identify patients with short telomeres. all methods showed acceptable accuracy, but equally imply the necessity of validation and appropriate controls in each experiment. here, flowfish was the most precise, accurate and reproducible method compared to the other methods. discussion: our study emphasizes the influence of expertise and experience that is required in order to produce robust and reliable telomere length analyses. here, we provide advice on how to choose the appropriate method in general and for individual cases to safely discriminate between natural variability and pathological telomere shortening in individual cases. in the next generation sequencing age, the strongest challenges have shifted from genotyping to handling the myriad of variants detected. whole exome sequencing yields tens of thousands of coding and non-coding variants, a number increased to millions if the whole genome is sequenced. to master this mountain of data, computer-based identification of potential disease mutations is absolutely indispensable. however, current computational strategies are usually generated by computer scientists without integrating human geneticists into software development. this often leads to tools which fulfil their main purpose but are not ideally suited to the needs of human geneticists. to assess the relevance of a suggested disease mutation, geneticists need detailed information about the effect on the protein and about the gene or protein itself. to close this gap, we have developed mutationdistiller, a web-based tool for user-driven variant prioritisation based on biological disease properties. its analysis includes the potential role of a mutated gene in pathogenesis as well as the estimated effect of a variant on gene/protein function. thus, mu-tationdistiller allows human geneticists to use every piece of information they consider relevant. unlike similar tools, its input goes beyond the human phenotype ontology and can include complete diagnoses, biological pathways, gene expression, and gene function. potentially harmful variants are identified by mutationtaster, which provides a deleteriousness score together with data on the actual effect of the variants. mutationdistiller is not restricted to non-synonymous variants but can also handle intragenic non-coding or synonymus variants. moreover, the program incorporates the known modes of inheritance of disease genes and the genotype of the queried variants (including compound heterozygosity). the output page provides all information on one site: the core component is a concise overview table of the most likely disease genes and variants. here, the most crucial information such as the variants' effect on the protein, frequencies in polymorphism databases and known diseases caused by mutations within this gene and their mode of inheritance are listed. more detailed gene and disease information and hyperlinks to external data are provided below, hence offering a comprehensive overview of the available knowledge. this allows geneticists to draw their own conclusions without any tedious collection of relevant information from the internet. a beta-version of the program is freely available at http://www.mutationtaster.org. m. jäger , , m. schubach , t. zemojtel , k. reinert , d. m. church , p. n. robinson , , institute for medical and human genetics, charité, berlin, germany, bcrt, berlin, germany, institute for bioinformatics, department of mathematics and computer science, freie universität, berlin, germany, x genomics, pleasanton ca, united states, department of mathematics and computer science, freie universität, berlin, germany, the jackson laboratory, farmington ct, united states with the grch human genome release in the genome resource consortium extended the previous linear, "golden-path" paradigm of the human genome and introduced a more graph-like model in the sense of regions with alternate loci, representing common alterations in sequence and structure. in whole-genome sequencing (wgs) these stretches of sequence are largely but not entirely identical between the primary assembly and an its corresponding alternate locus can result in multiple variant calls against regions of the primary assembly. this results in characteristic and recognizable patterns of variant calls at positions that we term alignable scaffold-discrepant positions (asdps). we developed an algorithm (asdpex) that analyzes these patterns in the structurally variable regions of the current grch genome assembly. a heuristical approach then infers whether the pattern of variant calls of a sample contains sequences from the primary assembly, an alternative locus, or their heterozygous combination at each of these regions. api and common snps are annotated. in the following step primers are picked using primer (whitehead-institute). the target sequence is either the snp-containing exon in case of exons smaller than bp, or, the genomic region flanking the snp of interest. the positional information of the snp is gathered using the mutalyzer api (lumc). the last two bases of each primer are checked for all snps in the ' end (snp : hg ). the script will be integrated into a website for free easy access and usability (www.optimus-primer.com). the only information needed is the refseq id, the exon, and the cdna position (eg: nm_ . . :c. t>g). the script optimus primer will provide the user with primers in the widely used primer format. the users are also able to download the source from our website, and to run it on a local unix system as a command line tool. mutationtaster : moving towards a comprehensive evaluation of disease causing mutations o. ebner, jm. schwarz, d. hombach, m. schuelke, d. seelow charitè universitätsmedizin, berlin, germany mutationtaster is a free and user-oriented application for comprehensible evaluation of non-synonymous and synonymous as well as non-coding dna sequence variants. as + citations show, the software has been strongly embraced by the clinical and research community. however, its capacities for assessing the functional consequences of non-coding variants are still limited. while mutationtaster is able to predict the variant's effect on major intragenic regulatory features such as splice sites or polyadenylation signals, many other potential effects of intronic or utr variants are not covered by the software yet. variants in the non-coding region of the genome are frequent and thought to cause a substantial part of yet unsolved mendelian diseases. these variants can occur either in extragenic regulatory regions (see abstract on regulationspotter) or in the untranslated regions, including introns, of a gene. unfortunately, their effects are much harder to predict than those of non-synonymous variants. therefore, only few disease mutations outside the coding sequence have hitherto been found and experimentally confirmed. to close this gap, we are advancing the current version of mutationtaster to improve the analysis of intragenic non-coding variants. we do so by implementing additional tests for variants in the ' and 'utr to determine their effect on au-rich elements, microrna binding sites and the influence of secondary structure on gene expression. moreover, we plan to shift from the analysis of exclusively monogenic to complex diseases with cancer being the first disease model to be integrated into mutationtaster . already implemented enhancements of mutationtaster comprise the improved splice site analysis using maxentscan and the integration of mitochondrial polymorphisms from the human mitochondrial genome database. the addition of links to the integrative genomics viewer and the lof-metrics of the exac browser (exome aggregation consortium) streamline the usage of the mutationtaster results and enable researchers to get a detailed view of the relevant variants and associated genomic sequences. taken together, mutationtaster will offer much better capabilities to predict the disease-causing potential of intragenic variants than its predecessor. the software is freely available at http://www.mutationtaster.org neurocure exzellenzcluster, berlin, germany, neuropädiatrie charité-universitätsmedizin, berlin, germany, medizinische genetik charité-universitätsklinikum, berlin, germany, berliner institut für gesundheitsforschung bih, berlin, germany ifications, or genome-wide interactions. all variants which have the potential to influence one or several candidate genes are presented in a graphical interface and ranked according to their predicted effect on the target gene(s). instead of giving scores, we present the potentially affected regulatory features in an intuitive graphical matrix. the software can freely be used at http://www.mutationtaster.org p-techno- genecascade -a one-stop shop for finding disease mutations d. seelow , in the last years, our genecascade software suite for the elucidation of rare diseases has seen some major extensions, mainly for the discovery of non-coding mutations. the website contains a number of tools focusing on the different steps in studying genetic disorders. all applications are web-based, have easy-to-use interfaces and are aimed directly at human geneticists, without any need to install software, use the command line or to try to explain clinical features to it specialists. we think that software should adapt to the user -not the other way around. and, unlike other web shops, its use is completely free. homozygositymapper finds disease-linked regions in consanguineous families. users can upload genotypes from snp chips as well as wes and even wgs data. we display likely disease loci in intuitive graphical interfaces. the results can also be used in our 'downstream' applications such as genedistiller, mutationtaster, or mutationdistiller to identify the actual disease mutation. genedistiller provides a user-driven way to find the best candidate gene for a genetic disorder. users can specify various aspects of the patients' phenotypes and the results are presented in a comprehensive way without the need to manually query other internet resources to collect further information relevant to asses the disease potential. mutationtaster evaluates the disease potential of non-synonymous as well as of non-coding and synonymous dna variants within genes. it does not only display a prediction but also detailed information on the variants' likely effect on mrna and protein. it can either analyse single variants as found by sanger sequencing or complete vcf files from wes or wgs projects. regulationspotter lists information about potentially regulatory dna variants outside of genes. variants are ranked according to their predicted effect on gene regulation. in addition to a score, we provide a user-friendly summary of the functional effects a variant may have. mutationdistiller combines genedistiller and mutationtaster in a single application for convenient variant and gene prioritisation. it offers human geneticists much more freedom to enter the phenotype than similar applications and provides deeper information about the variant and the gene. cnvinspector is aimed at the study of copy number variants. users can upload their patients' (or cohorts') cnvs and compare them with their own healthy controls or public data stored in our database. we include deci-pher to indicate known diseases caused by cnvs at the same position. epossum examines the effect of dna variants on transcription factor binding. as tf binding site prediction is notoriously unreliable, we also give an indication of the statistical relevance of a result. the genecascade website can be accessed at http://www.mutationtaster.org. we investigate in-house wgs datasets and found that on average . ± . of the regions correspond to an alternate locus rather than the primary assembly sequence. filtering these genomes with our algorithm identified around variant calls per genome that colocalized with asdps. our findings suggest the potential of fully incorporating the resources of graph-like genome assemblies into variant calling. our algorithm already uses the information contained in the structurally variable regions of the grch genome assembly to avoid spurious variant calls in cases where samples contain an alternate locus rather than the corresponding segment of the primary assembly. the human phenotype ontology project provides three resources, the ontology of clinical features, disease-phenotype associations and algorithms that enable analysis of data that is described using hpo. this resource is being used for computational deep phenotyping and precision medicine. it enables clinical data integration for translational research. the hpo is being increasingly adopted in software, research projects and companies world-wide. we will discuss the progress and recent developments that the hpo project has made since . this will include the expansion of hpo for common (complex) diseases, novel algorithms for phenotype-driven analysis of genomic variation, cross-species mapping of phenotype data, translation of hpo into several languages and also the addition of a more patient-friendly terminology for hpo terms. millions of patients worldwide suffer from a rare genetic disease. to date, the omim database lists more than diseases with proven or suspected mendelian basis, but the molecular cause is known for less than %. although next generation sequencing has drastically facilitated the discovery of disease mutations, a substantial fraction of whole exome sequencing (wes) projects fail to identify the causal variant. this may be due to the fact that disease-causing mutations are not always located within the coding sequence. whole genome sequencing (wgs) could solve this problem, but we are not yet readily prepared to handle the vast amount of variants which are generated by this technique, as intuitive and reliable software solutions for variant evaluation are missing. the currently existing approaches are not well-suited for a diagnostic setting, because they output a battery of different scores whose interpretation is left to the users. however, human geneticists are specialists for the assessment of symptoms of genetic diseases -not for the analysis of numerical scores indicating different likelihoods of the occurrence of regulatory elements. this is especially true when it comes to the hundreds of thousands of extragenic variants found by wgs, for which effect predictions always face a high level of uncertainty. we consider it crucial to provide geneticists with the information they need to determine the significance of a variant, not to flood them with scores whose meanings are difficult to grasp. to facilitate the interpretation of extragenic variants, we are developing regulationspotter. in contrast to other approaches such as genomiser or cadd, we integrate as much knowledge as possible in a user-friendly fashion to pinpoint the variants which are most likely to disturb the expression of candidate genes. regulationspotter includes different data on regulatory dna elements such as dna methylation, transcription factor binding sites, histone mod-the human exon in the ' end of the murine huntingtin gene. by using a novel object recognition test with a h interval between sample and test phase we have found a profound deficiency of hippocampus dependent long-term memory in heterozygous transgenics. this phenotype was detected as early as weeks of age and is complementary to deficits that we have identified in the hdhcag mouse model previously. motor deficits as well as intranuclear aggregates are described at much later stages in both of these models. we have shown previously that in hd patients, mediated through mtor signaling, translation of mrna carrying expanded cag repeats is elevated (krauss et al., ) . we have also seen that the biguanid metformin antagonizes mtor signaling in neurons in-vitro and in-vivo (kickstein et al., ) . we show here that metformin, by interfering with the mtor kinase and its opposing phosphatase, pp a, regulates local protein synthesis in the brain and is able to suppress the production of disease making protein in early hd. furthermore metformin leads to a significant improvement of movement abilities in a c-elegans model for hd and to a rescue of early cognitive symptoms in the hdhcag animal model. these data suggest that metformin is a very promising candidate for early phase treatment of hd patients. allele-specific suppression of dominant-negative bestrophin mutations a. milenkovic, lm. braun, f. grassmann, b. h. f. weber institute of human genetics, regensburg, germany purpose: retinal pigment epithelium (rpe) differentiated from human induced pluripotent stem cells (hipsc) demonstrated degradation and mislocalization of mutant bestrophin (best ) protein in autosomal dominant best disease (bd). importantly, mutated alleles revealed a dominant-negative effect leading to an impairment of volume-regulated chloride transport, the basic function of the homo-pentameric best channel. here, our study aimed for a proof-of-concept to treat bd by selectively eliminating best mutant transcripts in patient-derived hipscs prior to rpe differentiation via the crispr/cas genome editing technology. methods: adult human dermal fibroblast were obtained from skin biopsies of bd patients and reprogrammed into hipscs. single guide rna sequences (sgrna) targeting best mutations were selected by the "optimised crispr design tool" (zhang lab, mit ) . editing efficiency and specificity of designed sgrnas were tested in hek cells using an established fluorescence-based assay. after transfection of hipscs the percentage of indel formation of on-and off-targets was determined by a pcr-based crispr/cas cleavage detection kit. cas -treated stem cell populations were analyzed for pluripotency and selected for full genome sequencing before differentiation to rpe cells. results: computational design of disease-causing best variants (n k, v m, s r, q r, a v, and i del) offered at least one sgrna with predicted high quality per mutant allele. the guide sequences were cloned into the cas -expressing plasmid px and co-transfected with pcag-egxxfp plasmids containing genomic fragments of ~ bp of either the mutated or wildtype sequence to the corresponding sgrna. as targeted cas cleavage results in reconstitution of the egfp expression cassette by homology dependent repair, the efficiency and specificity of cas cleavage was evaluated on a plate reader by quantifying egfp fluorescence after h of transfection. as a result, out of the sgrnas tested showed high allele-specificity and are now used for targeted genome editing in hipscs. conclusion: so far, there is no treatment for bd although the molecular pathology of best has recently been established. our proof-of-concept study aims to determine whether haploinsufficiency of normal best protein is sufficient to fully or partly restore cellular function in cells of primary bd pathology, namely the rpe. to this end, we will determine the degree of rescue (i. e. reconstitution of volume-regulated chloride conductance) by whole-cell patch-clamp analysis. if successful, our crispr/ cas -driven approach will be useful to treat other diseases with dominant negative effects of the mutated allele. p-therap- *** metformin rescues early cognitive symptoms in the hdhcag mouse model and is therefore a promising candidate for treatment of hd patients. huntington's disease (hd) is an autosomal dominant neurodegenerative disorder that is caused by an unstable glutamine (cag) trinucleotide repeat expansion within exon of the huntingtin gene and leads to cognitive decline and affects motor abilities. in the prodromal phase of the disease patients develop mood swings, personality changes and subtle cognitive impairment. close understanding of clinical signs and molecular mechanisms behind this early stage of hd is an important step for the development of a causal therapy. we have analysed a knock-in mouse model that carries cag repeats and single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation age-related clonal hematopoiesis associated with adverse outcomes clonal hematopoiesis and bloodcancer risk inferred from blood dna sequence age-related mutations associated with clonal hematopoietic expansion and malignancies cancer spectrum in dna mismatch repair gene mutation carriers: results from a hospital based lynch syndrome registry update on kleefstra syndrome characterization of a novel transcript of the ehmt gene reveals important diagnostic implications for kleefstra syndrome national institute of child health and human development abstracts p-monog- novel mutation in hoxc expand the mutation spectrum of pure hair and nail ectodermal dysplasia abstracts in conclusion, targeted sequencing represents an effective, fast, cost-efficient and flexible method, since new candidate genes can be added easily to the panel, for the sequential analysis in chh patients. furthermore, panel sequencing alleviates the uncovering of oligogenic inheritance in genetic traits like chh abstracts p-monog- neurodegeneration in the olfactory bulb and olfactory impairment in the ccdc -/-mouse model for retinal degeneration s. schreiber , e. petrasch-parwez one hallmark of sca and other mechanisms that govern ccdc -deficient degeneration, a detailed evaluation is performed in order to reveal processes that contribute to retinal degeneration during early postnatal development and adulthood. methods: the functional role of ccdc deficiency is investigated by two independent approaches: ) gene expression profiles at p and p are analyzed in retinal tissue of cccdc -/-and wildtype mice (genechip mouse gene . st array, affymetrix) followed by quantitative real-time pcr (qrt-pcr) and ) potential retinal interaction partners of ccdc protein are identified by yeast-two hybrid screening in the wildtype mouse and further analyzed by immunohistochemistry. results: using two screening methods (rna-expression profiles and protein interaction partners), our results indicate that ) the ccdc deficient mouse model reveals early changes in retinal rna gene expression already at p and the highest number of genes differential expressed at p . most expression differences were related to genes associated with the extracellular matrix. further genes are involved in retinal degeneration, angiogenesis, transcription factors and proteolysis. ) the ccdc protein interacts with the proteins eps (epidermal growth factor receptor kinase substrate ) and mpdz (multiple pdz-domain protein). both proteins are expressed in several retinal layers of the retina, confirmed by immunohistochemistry. moreover, mutations in the mpdz gene were already identified in patients with retinitis pigmentosa/leber congenital amaurosis. conclusion: expression profiles reveal expression changes at an early time point of retinal degeneration in the ccdc -/-mouse model enabling further studies on the role of genes and processes involved in early retinal degeneration. in addition, the interaction partners of ccdc , eps and mpdz, are the basis for further studies examining the pathways of retinal degeneration in the mammalian retina including man -and possibly contribute to future studies in man and human disease. ccdc null mutation causes retinal degeneration and dysfunction of medical and molecular genetics and skeletal dysplasia multidisciplinary unit hospital unit of genetics of neurodegenerative and metabolic diseases sel- , ws - , p-compl- ws - p-cling- , p-cling- ws - , ws - , p-cling- , p-cling- , p-cling- ws - , p-cling- , p-cling- , p-monog- p-cancg- p-cling- , p-cling- , p-cling- p-cling- , p-cling- ws - , ws - , p-cling- ws - , p-cling- p-cancg- , p-cancg- , p-cling- ws - medizinische genetik · ws - , p-cling- ws - , p-cling- ddd study . p-cling- ws - , ws - , p-cling- , p-compl- ws - , p-cling- , p-cling- distelmaier f. ws - ws - , p-basepi- , p-cancg- ws - , ws - , p-cling- , p-cling- , p-cling- ws - , p-cling- , p-monog- ws - ekici ab. ws - , p-monog- sel- p-cancg- , p-techno- plen , ws - golla a dfg-workshop göpfert mc sel- , sel- haferlach c. ws - haferlach t. ws - ws - , ws - , p-compl- , p-compl- p-cancg- , p-cancg- , p-cancg- gilissen c. ws - ws - , p-basepi- , p-cling- , p-cling- p-cling- , p-cling- , p-cling- , p-cling- ws - , p-cling- p-cling- , p-cling- , p-cling- ws - , p-compl- p-monog- medizinische genetik · ws - hüffmeier u. ws - ws - , p-cling- seq consortium ws - ws - , ws - , p-compl- p-cling- , p-cling- , p-cling- , p-cling- ws - , p-cancg- ws - , ws - , ws - , p-compl- p-cancg- , p-cancg- ws - ws - , p-cling- , p-cling- , p-cling- , p-cytog- ws - , ws - , p-compl- , p-compl- , p-compl- ws - , p-cling- p-cling- , p-cling- p-cling- , p-cling- p-cling- , p-cling- , p-cling- ws - , p-cancg- ws - , p-cling- ws - , ws - , p-cancg- khor cc. ws - sel- , ws - ws - medizinische genetik · p-cling- , p-cling- , p-cling- ws - , p-compl- , p-compl- ws - , p-cancg- ws - , p-cling- meggendorfer m. ws - ws - , p-cancg- p-cling- , p-cling- , p-cling- p-cling- , p-cling- ws - , p-cancg- ws - , ws - , p-monog- ws - ws - ws - , p-cling- pasutto f. ws - nanda i. ws - nöthen mm. ws - , ws - , ws - , ws - , ws - ws - ws - , ws - , ws - , ws - , p-cling- , p-compl- , p-compl- sel- medizinische genetik · p-cancg- schnapp l. ws - ws - , p-cancg- , p-cancg- , p-cling- ws - , p-basepi- , p-cling- p-cling- , p-cling- p-cancg- , p-cling- salaverria i. ws - ws - , p-compl- ws - , ws - , ws - , p-cancg- , p-cling- , p-cling- , p-cytog- , p-monog- , p-monog- p-cancg- , p-cancg- , p-cling- , p-cling- ws - sel- , ws - , p-compl- the international gamos consortium e ws - , ws - , ws - , p-cancg- , p-monog- , p-monog- sel- , ws - ws - , p-cytog- p-cancg- , p-cling- , p-cling- p-cancg- , p-cancg- , p-cling- stengel a. ws - p-cling- , p-cling- ws - , ws - , ws - , p-cling- , p-cling- , p-cling- , p-cling- , p-cling- p-cancg- , p-cling- ws - ws - , p-cling- ws - witsch-baumgartner m. p-cling- p-cancg- p-cancg- wunderle m. ws - zechner u. ws - , ws - , p-basepi- , p-cling- , p-cling- , p-cling- ws - , p-cancg- , p-cling- edu , p-cling- , p-cling- , p-cling- , p-cling- , p-cling- , p-cling- p-cling- , p-cling- , p-cling- wiesener a. ws - , p-monog- ws - , ws - , p-cancg- , p-cling- , p-compl- , p-monog- ws - ws - , p-cancg- autorenverzeichnis zenker m. ws - , ws - , p-cling- , p-cling- dialog mit shg, p-cling- ws - , ws - , p-cling- , p-monog- ac. koller , , j. strohmaier , ku. ludwig , , fc. degenhardt , m. wulff , , d. breuer , , l. winkler , , f. neukirch , , a. maaser , , a. forstner , , s. sivalingam , , a. reif , a. ramirez , w. maier , d. rujescu , i. giegling , h. thiele , p. nürnberg , , a. fischer congenital hypogonadotropic hypogonadism (chh) is a rare and clinically and genetically heterogeneous disorder. chh is characterized by incomplete or absent puberty caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (gnrh) neurons, disturbed secretion or action of gnrh, or both. chh is often associated with anosmia and is then termed kallmann syndrome (ks), as well as with other phenotypes like unilateral kidney agenesis, skeletal abnormalities, midline malformations, and hearing loss. x-linked, autosomal-dominant, and autosomal-recessive, as well as di-and oligogenic inheritance has been described for chh. in the meantime a multitude of genes has been reported to be associated with chh. actually, in fewer than % of the chh cases the underlying genetic cause can be identified. we analyzed a total of patients with chh ( female/ male) by using targeted sequencing of chh-associated genes kal , chd , fgf , fgfr , fshb, gnrh , gnrhr, hs st , kiss , kiss r, nsmf, prok , prokr , spry , tac and tacr and identified pathogenic mutations in chh patients of our cohort. mutations were detected in kal ( patients), tacr ( patients), prokr ( patient), hs st ( patient) and gnrhr ( patient). furthermore, we found in two patients with described pathogenic mutations (one patient with prokr mutation and the other with kal mutation, respectively) additional mutations in nsmf gene and tacr gene, respectively, suggesting digenic inheritance in these cases. pain is necessary to alert us to actual or potential tissue damage. specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (hsan). although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the flvcr (feline leukemia virus subgroup c receptor ) gene, which encodes a broadly expressed heme exporter. different flvcr isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. mutations institute of human genetics, bonn, germany, the rild institute, wonford, exeter, uk introduction: ectodermal dysplasias (eds) are a large group of heterogeneous genetic disorders characterized by abnormal development in ectoderm-derived tissues and organs including skin, hair, and nails. among the eds, pure hair and nail ectodermal dysplasia (phned) is a rare genodermatosis characterized by nail dystrophy and sparse or absent hair on the scalp. materials and methods: a family of iranian origin was enrolled in this study. two children from a consanguineous marriage are affected from phned. in addition, the father has alopecia areata (aa) but does not show any nail dysplasia. the mother is unaffected. the paternal and maternal grandfathers had nail dysplasia almost similar to the siblings but did not manifest any hair loss or aa. homozygosity mapping and genedistiller analysis were performed to identify candidate genes. sanger sequencing was used to localize the mutations. results: in total, we identified homozygous regions with almost candidate genes. among these genes were also krt and hoxc , already known to be related to the phenotype of our patients. therefore, we focused our additional analyses on krt and hoxc . sanger sequencing showed a so far unknown homozygous insertion of bp in exon of hoxc . conclusion: we identified an unknown mutation for phned which expands the spectrum of mutations for phned. the hair loss of the father seems rather be due to a distinct type of hair loss, namely aa, which is quite common in the general population. however, the nail dysplasia from both grandfathers is unclear and cannot be examined anymore. it still remains unclear if the nail dysplasia in the grandfathers was due to the same mutation in hoxc or is based on a different mutation. generation of a cell culture model for epidermodysplasia verruciformis by knock-out of ev , a novel gene involved in this genodermatosis e. imahorn , m. aushev , sj. de jong , e. jouanguy , , , jl. casanova , , , ph. itin , , j. reichelt , epidermodysplasia verruciformis (ev) is a rare hereditary skin disease leading susceptibility to certain types of cutaneous human papilloma viruses, mainly β-hpv, and a high risk for development of cutaneous squamous cell carcinoma. homozygous or compound heterozygous loss of function mutations either in tmc or in tmc have been described in several ev patients, but more than / of affected families do not have mutations in one of them. a third gene (ev ) has recently been identified to be mutated in ev patients without tmc or tmc mutation. investigations on the function of this gene may elucidate pathomechanisms of ev. we aim to generate an ev deficient model cell line to study the effects of ev despite the scarcity of patient material. detection of deletions during diagnostic massive parallel ngs gene panel sequencing neuronal differentiation at a later time point. a novel mutation c. a>g, pser gly in des gene in a family with catecholamine polymorphic ventricular tachycardia s. komatsuzaki, p. villavicencio lorini, k. hoffmann institute for human genetics, university hospital halle, germany background: myofibrillar myopathy (omim ) is a heterogeneous neuromuscular disease characterized by progressive muscle weakness, partially with cardiomyopathy and/or arrhythmia. pathohistological examinations show desmin-positive protein aggregations. the mutations in des, cryab, bag , myot, ldb , flnc, fhl , and dnajb have been identified in patients with myofibrillar myopathy. desmin is a intermediate filaments and composed of non-helical n-and terminal head domain and a central a-helical rod domain. till now more than mutations myofibrillar myopathy were identified in patients with and a genotype-phenotype was reported: mutations in rod domain tend to cause neuromuscular symptoms, and mutations in head domain is associated with cardiac symptoms. the head-domain is a serine rich domain and phosphorylated by protein kinase c. almost all reported mutations in head-domain in des are at serine residues (e. g. s i, s f, s f, s f, s y, s t). these findings suggested that the serine residues in head-domain could play an important role in biological function of desmin. here we report a novel mutation c. a>g, ps g in des in a family with catecholamine polymorphic ventricular tachycardia. case: a years old patient developed a syncope in a cold winter. the cardiological investigations revealed a diagnosis of catecholamine polymorphic ventricular tachycardia and the implantation of permanent pacemaker was indicated. his older brother, father and paternal uncle suffered from arrhythmia and all of them received a permanent pacemaker implantation at around years of age. his cousin was died at age of years due to sudden cardiac arrest. we performed a genetic analysis for ryanodin-rezeptor -gen and lamin a gene and no mutation in these gene was identified. next, we performed an exome sequencing. here a novel heterozygous mutation c. a>g, ps g in des was identified. this mutation was also identified in uncle of index patient. the c. a>g, ps g in des was not reported neither in human genome project, nor in exome aggregation consortium. a mutation at serine (p.s i) was previously reported in patients with myofibrillar myopathy with cardiac symptoms. based on these findings, it was strongly supposed that the p.s g in des gene is a causative mutation for myofibrillar myopathy with arrhythmia in our patients. discussion: recent studies suggest that the mutations in des influence not only on muscle stability and myocardial force generation, but also impaired ubiquitin proteasome system, which could be caused by aggregated desmine. generally, phosphorylation of serin and threonine in head-domain of desmin is related to disassembly of filaments. therefore the p.ser- gly mutation in des could cause altered phosphorylation of desmin. the functional abnormalities caused by pser gly in des gene need to be studied. gene dosage manipulation of the chromatin organizer ctcf in the nervous system of drosophila melanogaster results in neurological and morphological phenotypes e. konrad, a. gregor, m. brech, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germanythree-dimensional organization of eukaryotic genomes is crucial for temporal and spatial regulation of gene expression. architectural proteins, like the ccctc-binding factor ctcf are responsible for establishing and maintaining this organization. ctcf is involved in virtually all chromatin regulating processes including enhancer function, insulation, alterna-for this purpose we used the crispr/cas system to delete the ev coding sequence in an immortalized keratinocyte line. after isolation by facs, single cell clones have been expanded. we screened clones for deletion of the whole gene as well as for the expression of ev . three clones showed no detectable gdna sequence or expression of the ev gene. we found only one wildtype clone without deletion of ev or alterations near the cas cut sites. all clones have been characterized by a snp-array as well as sequencing of the knockout site and the most probable offsite targets. these ev deficient keratinocyte lines are the first cell culture model for ev. it will be a valuable tool to identify cellular pathomechanisms of the disease and allow insight into the control of β-hpv in the general population. hemizygous loss of function of single x-chromosomal genes is the most frequent cause for genetic intellectual disability (id). while a long list of gene mutations have so far been described to be responsible for the disease phenotype, little is known about the underlying neuronal mechanisms. reprogramming of somatic cells into stem cells (ipscs) followed by differentiation into neuronal precursors (npcs) is an important tool for translating research allowing an understanding of network dysfunction in id patients. opitz bbb/g syndrome (os) is characterized by a number of ventral midline defects combined with learning disability, developmental delay and intellectual disability. it is caused by mutations in the x-linked mid gene, which, as we have shown previously, regulates mtor dependent local protein synthesis. in a mouse model, loss of mid function leads to significant disturbance of axonal outgrowth. we have generated ips cells from several patients with os and from one mother that carries a loss of function mutation in the mid gene. by sorting cell clones after reprogramming we have established ips cell clones from this female carrier of a bp deletion in the mid gene (c. _ delctcc) either expressing from the mutated x-chromosome or the non-mutated x-chromosome and have shown that indeed the generated ipsc-clones express either % of the mutated or % of the wildtype mid . we determined this directly using an allele-specific mid -rt-pcr and indirectly by comparing the methylation of humara-alleles. comparison of mutation expressing ipsc-clones with non-mutation expressing ipsc-clones showed that the mid mutation results in significantly smaller cells with reduced s phosphorylation supporting aberrations in the mtor/pp a signaling cascade. when differentiating ipscs into neuronal precursor cells, significantly bigger embryoid bodies (ebs) can be detected in the mutation expressing clones, while the total eb number is higher for the non-mutated mid expressing clones. when further differentiated, ebs from all ipsc-clones form neuronal rosette structures expressing beta-iii-tubulin, with a lower rosette structure count in the mutated mid expressing cells. these data clearly hint a defect in neurogenesis in cells with hemizygous mid mutations. interestingly, while ipscs stably kept the x-incativation pattern of the original fibroblast, during differentiation x-inactivation was lost leading to biallelic expression from both x-chromosomes in the npcs pointing towards an as yet unknown reactivation mechanism of the inactive x-chromosome. we are currently analyzing x-inactivation throughout the differ- mutations in the aminoacyl-trna-synthetase genes sars and wars are associated with autosomal recessive intellectual disability l intellectual disability (id) is the common feature of a very heterogeneous group of disorders, which comprises a broad variety of syndromic and non-syndromic phenotypes. here we present mutations in two aminoacyl-trna synthetases that are associated with id in two independent iranian families. in the first family, we found a missense mutation (c. g>a, p.d n) in the cytoplasmic seryl-trna synthetase (sars) gene that affects the enzymatic core domain of the protein and impairs its enzymatic activity. this probably leads to reduced trnaser concentrations in the cytoplasm. in silico analyses predicted the mutant protein to be unstable. this prediction could be experimentally substantiated by results obtained through studies with ectopic mutant sars in transfected hek t cells. in the second family, we identified a compound heterozygous genotype of the mitochondrial tryptophanyl-trna synthetase (wars ) gene, consisting of a nonsense mutation (c. dela, p.ser alafs* ), which very likely leads to nonsense-mediated mrna decay, in combination with a missense mutation (c. t>g, p.w g). the p.w g mutation affects the mitochondrial localization signal of wars , leading to mislocalization of the mutant protein. thus, when taking aimp into account, which we have recently implicated in the aetiology of id as well, there are now three genes with a role in trna-aminoacylation that are associated with this condition. hence we propose that the functional integrity of t-rnas in general is an important constituent in the development and maintenance of human cognitive functions. dravet syndrome is a rare autosomal dominant genetic disorder with early-onset epileptic encephalopathy is mainly caused by different de novo mutations of the scn a gene encoding the type subunit of voltage-gated sodium channel. whole exome sequencing(wes) enables scanning a large number of genes which not only can confirm the diagnosis but also helpful in understading for possible relationship between clinical manifestations and mutaion. the authors investigate wes in a months term boy with hypotonia and convulsion of normal and relative parent. she had uncontrolled sizure without fever, and developmental delay from months.in treatment protocol anti covulsants changes to valorate, clonazepam and stiripentol as well. a deleterious novel heterozygous splice site mutation in scn a tive splicing, imprinting, v(d)j recombination, chromatin loop formation and defining topologically associated domains (tads). recently, we identified de novo mutations in ctcf in patients with a surprisingly mild phenotype of variable developmental delay or intellectual disability, mild short stature and microcephaly, and behavioural anomalies. apart from observing brain malformations and early lethality or learning deficits in two conditional knockout mouse models, little is known about the role of ctcf in neuronal development and preservation so far. therefore, we utilized the model organism drosophila melanogaster to further explore the role of ctcf in cns development and function. similar to observations in knockout mice, complete knockout or ubiquitous knockdown of ctcf is embryonic lethal in drosophila. we therefore utilized the uas/gal system to induce tissue specific knockdown or overexpression of ctcf in the fly nervous system. we first investigated development and morphology of the larval neuromuscular junctions (nmjs), an established model for synaptic development. while pan-neuronal overexpression of ctcf showed no morphological nmj alterations, pan-neuronal knockdown resulted in fewer nmj branches than in a specific control. additionally, we observed a reduced number of active zones in a hypomorphic mutant line compared to a wildtype control. using the negative gravitaxis assay to examine gross neurological function, we found a highly significant impairment of geotaxis behavior in flies with ctcf knockdown in neurons, motoneurons and muscle and in flies with overexpression of ctcf in glia cells, muscle and motoneurons. currently we are testing learning and memory behavior with the courtship conditioning paradigm. our findings of various neurological and morphological anomalies upon manipulation of ctcf dosage in the fly nervous system emphasize the role of ctcf in nervous system development and function and provide a basis to further study the molecular mechanisms underlying cognitive dysfunction caused by ctcf-deficiency. congenital or early-onset nystagmus (cn) is characterized by involuntary eye movements and shows enormous clinical and genetic heterogeneity. cn may be an ambiguous sign of many different diseases, including retinal dysfunction/degeneration, ocular/oculocutaneous albinism, and severe central nervous system disorders, such as pelizaeus-merzbacher or pelizaeus-merzbacher-like diseases (pmld). due to enormous heterogeneity found among the diseases leading to cn, whole exome sequencing (wes) and panel-based bioinformatics was considered as an approach to rapidly identify disease-associated genetic sequence variants. we have analyzed families with cn-affected patients. herein, we present three clinically different patients who were initially affected with cn, but developed further clinical symptoms of various severities. one of these patients showed features of retinal dysfunction, including night blindness and myopia, while two other patients developed severe phenotypes including mental retardation or pmld. wes identified four genetic variants in genes associated with cn. the first patient showed a hemizygous splice-donor variant (c. + g>a) in the calcium channel voltage-dependent alpha- f subunit (cacna f) gene, the second patient carried a hemizygous variant (c. g>a, p.r h) in the ferm domain-containing protein (frmd ) gene, and the third patient showed two heterozygous variants (c. c>g, p.y * and c. t>c, p.v a) in the gap junction protein gamma- (gjc ) gene. sanger sequencing confirmed the identified variants in the index patients and verified co-segregation in several family members. our results suggest a beneficial role of wes to identify the molecular causes of cn and to rapidly confirm an initially unclear clinical diagnosis. especially, patients with rare and severe disorders (e. g. pmld) will benefit from a wes analysis performed in the early stage of the disease. the ccdc -deficient (ccdc -/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al., hum mol genet., ) . in order to determine whether ccdc gene expression might also play a role outside the retina, this study aimed at characterizing ccdc protein expression during early postnatal development of the mouse brain. furthermore, morphological and behavioral impact of ccdc deficiency in the mouse brain was analyzed. methods: ccdc protein expression was determined by sds page and western blot in whole brain homogenates and in selected brain regions of interest (olfactory bulb, hippocampus, cortex, striatum, cerebellum, brain stem) during early postnatal development and in adult wildtype (wt) mice. in addition, cryosections of the ccdc -/-olfactory epithelium and bulb (during postnatal development) and the rostral migratory stream (in adult) were analyzed for ccdc reporter gene expression by x-gal staining. selected brain regions were additionally analyzed by electron microscopy. in order to correlate anatomical with behavioral data, olfactory performance was studied in aged ccdc -/-mice compared to ccdc +/+ controls by an olfactory habituation/dishabituation test (yang and crawley, curr protoc neurosci., ) , where olfactory exploration-time during the presentation of neutral and social odors is examined. results: ccdc protein was detected throughout the early postnatal development of the wt mouse brain, decreasing after birth. amongst analyzed brain regions, highest expression of ccdc protein was detected in the olfactory bulb exhibiting similar ccdc levels to retinal expression. accordingly, ccdc reporter gene expression was demonstrated in the mature olfactory bulb glomeruli, the adjacent olfactory epithelium and along the rostral migratory stream in the ccdc -/-mouse brain. interestingly, strong ccdc reporter gene expression in glomeruli of the ccdc -/-olfactory bulb was correlated with signs of degeneration in the ccdc -/-mouse, but not in controls. the degeneration was also reflected by olfactory impairment in ccdc -/-mice, which spent significantly less time for sniffing at initial presentation of unknown, neutral odors and barely responded to social odors. conclusion: besides the retina, ccdc protein plays a crucial role in the olfactory system as shown by its expression there as well as by ccdc deficiency resulting in neurodegeneration and alteration of olfaction-related behavior in the ccdc -/-mice. as impairment of the olfactory sense in multiple neurodegenerative disorders is a common finding, the ccdc -/mouse model is not only restricted to study retinal degeneration but possibly also degeneration of the central nervous system. background: the ccdc -deficient (ccdc -/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al. ) . in order to gain insights into the molecular disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. here we report a novel homozygous missense variant (c. g>a; p.g r) in the arl b gene, which we identified by whole exome sequencing of a trio from a consanguineous family with multiple affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. the same variant was also identified in a second family. we saw a striking difference in the severity of ataxia between affected male and female individuals in both families. functional analysis demonstrated that dihydrotestosterone treatment of sh-sy y cells induced a down regulation of arl b expression. both arl b and arl b-p.g r expression rescued the cilia length and shh defects displayed by arl bhennin (null) cells, indicating that the mutation did not disrupt either arl b function. in contrast, arl b-p.g r displayed a marked loss of arl guanine nucleotide-exchange factor activity, despite retention of its gtpase activities, highlighting the correlation between its loss of function as an arl guanine nucleotide-exchange factor and joubert syndrome. s. renner, a. busch, t. bierhals, j. butter, v. kolbe, g. rosenberger institute of human genetics, university medical center hamburg-eppendorf, hamburg, germany taad (thoracic aortic aneurysm and dissection) is a heterogeneous disease that often remains silent until a life-threatening complication occurs. it belongs to the connective tissue disorders and causes % of death in industrial countries. several disease genes have been already identified; however, about % of patients with taad-associated syndromes do not show a mutation in these genes. thus, further heterogeneity is obvious. since individual risk stratification and therapeutic options highly depend on the individually mutated gene, it is very important to identify more disease genes which are aimed to be found by whole exome sequencing (wes). many of the known disease genes encode for proteins that are important for the structure and stabilization of the extracellular matrix as well as for the contraction of vascular smooth muscle cells. one central pathway is the tgf-beta signaling which functions among other proteins via the tgf-beta receptor, its ligand and its downstream target smad / . our project plan includes (i) exome sequencing both in affected individuals within families as well as in sporadic patients, (ii) filtering of raw data and prioritization of sequence variants by using a bioinformatic in house pipeline, (iii) verification of novel putative disease genes in a cohort of mutation-negative patients with taad spectrum disease and (iv) functional analyses to gain deeper insight into the pathobiology of taad. in a first round of wes analysis and variant prioritization, we identified a highly conspicuous sequence variant in three family members with taad. screening of the respective gene in a large cohort of mutation negative patients revealed another variant in two siblings with taad. structural and functional considerations strongly support deleterious effects for both identified putative pathogenic missense variants that affect a novel cell cycle-and/or apoptosis regulating protein. functional analyses did not show an involvement of this protein in tgf-beta signaling. in ongoing experiments, we focus on mutation-induced consequences on cell proliferation, cell cycle progression and apoptosis. indeed, we found inhibitory effects of the missense variants on proliferation by affecting the cell cycle key protein cdkn a. we hypothesize that dysregulation of proliferation and/ or apoptosis of specific cells, e. g. smooth muscle cells, underlies taad.abstracts for screening. positively tested compounds were reanalyzed by whole-cell patch clamp recordings and cell volume measurements. results: the halide assay revealed reproducible halide permeability across wells and, as a control, reliably detected mdckii cells overexpressing wild type best by a decrease of yfp fluorescence to % following seconds iodide stimulation. cells harboring mutant best showed % of default yfp fluorescence after the same time interval. conclusion: the current study established an assay appropriate for high and small-scale compound screening targeting best localization and function. this assay will be used to screen for compounds in mutant cells lines best -t p and best -y n for their ability to improve trafficking to the pm or correcting protein folding to enhance ion permeability. background: neuropeptide y-y receptor (y receptor), an auto-receptor of neuropeptide y (npy) and attractive guanine nucleotide (g) protein-coupled receptor target, has been implicated as a potential therapeutic target for many clinical conditions, including epileptic seizure, depression, pain, and alcoholism. in huntington's disease (hd) patients and animal models of hd, npy-expressing striatal interneurons are selective preserved and increased with advancing disease. however, the potential role of y receptor in hd pathology remains under-explored. aims: to investigate whether activation of y receptor using npy and selective y r ligands could ameliorate behavioral deficits and neuropathology in r / mouse model of hd. methods/techniques: npy and selective y receptor agonist npy - were intranasally administered to r / mice, five days in a week, beginning from weeks of age until weeks of age. in the second study, r / mice received daily intraperitoneal administration of selective non-peptide y receptor antagonist (sf- ) to selectively block y receptor. results/outcome: intranasal application of npy showed significant increase in rotarod performance compared to the saline and sf- treated r / mice (*p < . and **p < . at and weeks of age respectively, n = ). however, treatment with npy - showed a clear trend towards increased rotarod performance at weeks of age compared to the saline and sf- treated r / mice but the difference did not reach significance. also, treatment with npy and npy - showed no significant effect on body weight loss in r / mice, contrasting with previous data obtained with single intracerebroventricular (icv) injection of npy in r / mice. furthermore, intranasal application npy or npy - led to decrease in mutant huntingtin (htt) aggregation and mediated increase in dopamine-and camp regulated phosphoprotein (darpp- ) and brain derived neurotrophic factor (bdnf) levels. additionally, we found that npy and npy - attenuate microglial activation, inducible nitric oxide synthase (inos) expression, and proinflammatory cytokines production in r / mice compared to the saline and sf- treated r / mice. conclusion: taken together, our findings suggest that targeting npy-y receptor might be a potential neuroprotective therapy for hd and other neurodegenerative diseases. best of both world's: a novel, rapid capture protocol that overcomes drawbacks associated with dna fragmentation in established methods j. seggewiß, c. ruckert, p. wieacker institute of human genetics, westfaelian wilhelms-university of muenster, muenster, germany rapid capture protocols are an attractive proposition for clinical sequencing labs, as they enable quicker sample-to-sequencing turnaround times. the fragmentation of input dna for the construction of pre-capture libraries is a bottleneck in established protocols. mechanical shearing is the gold standard, but is laborious using single-tube covaris instruments; and higher-throughput instrumentation is cost-prohibitive to many smaller labs. "tagmentation"-based methods (e. g. the nextera rapid capture system from lllumina, or agilent's sureselect qxt system) employ transposases for fast ond simple library construction. however, these protocols are associated with significant sequence bias, especially with low-quality ffpe samples and are extremely sensitive to dna input -thus requiring meticulous quantification of viscous, high-molecular weight dna. here we describe a newly-developed rapid capture protocol that combines the kapa hyperplus kit (kapa biosystems) with integrated, low-bios enzymatic fragmentation, and agilent's proven sureselect xt target enrichment technology. the streamlined method follows for the preparation of high-quality, sequencing-ready libraries in one working day. the novel enzymatic fragmentation reagent does not require careful quantification of input dna, yielding reproducible fragmentation profiles optimal for capture over the fold range-tested ( - ng) the single-tube kapa hyperplus protocol results in very efficient conversion of input dna to precapture library thereby decreasing duplication rates and increasing the complexity of the library going into the modified, min sureselect fast xt hybridization protocol. our protocol represents a significant improvement for fast routine diagnostics, where robust and reproducible pipelines ore needed to support timely treatment decisions. lmj. braun, a. milenkovic, bhf. weber institute of human genetics, regensburg, germany purpose: human bestrophin- (best ) is a chloride channel controlled by ca + and cell volume and is localized at the basolateral membrane of the retinal pigment epithelium (rpe). so far, there is no therapy for the best -associated diseases, of which the most common is best vitelliforme macular dystrophies (bvmd). in this study, we developed an assay applicable for high and small-scale compound screening targeting best localization and function. methods: to assess best channel function we developed a halide assay. briefly, mdckii cell lines were established, stably expressing wildtype best or bvmd-associated best mutants together with a yellow fluorescent protein (yfp)-based halide sensor. in polarized mdckii cells, wildtype best and best -r c localize regularly at the basolateral plasma membrane (pm) while best -l m and best -y n appear significantly reduced in quantity and grossly mislocalized to cytosolic compartments. cells were stimulated with extracellular addition of iodide known to pass the pm through anion cannels and, as a consequence, intracellularly quench yfp fluorescence. variations in yfp fluorescence levels as a marker for best function were recorded in well plates by a plate reader setup. a small-scale , compound library, commercially available as spectrum collection (microsource discovery systems, gaylordsville, usa) was used key: cord- -q u hdp authors: olsavszky, victor; dosius, mihnea; vladescu, cristian; benecke, johannes title: time series analysis and forecasting with automated machine learning on a national icd- database date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: q u hdp the application of machine learning (ml) for use in generating insights and making predictions on new records continues to expand within the medical community. despite this progress to date, the application of time series analysis has remained underexplored due to complexity of the underlying techniques. in this study, we have deployed a novel ml, called automated time series (autots) machine learning, to automate data processing and the application of a multitude of models to assess which best forecasts future values. this rapid experimentation allows for and enables the selection of the most accurate model in order to perform time series predictions. by using the nation-wide icd- (international classification of diseases, tenth revision) dataset of hospitalized patients of romania, we have generated time series datasets over the period of – and performed highly accurate autots predictions for the ten deadliest diseases. forecast results for the years and were generated on a nuts (nomenclature of territorial units for statistics) regional level. this is the first study to our knowledge to perform time series forecasting of multiple diseases at a regional level using automated time series machine learning on a national icd- dataset. the deployment of autots technology can help decision makers in implementing targeted national health policies more efficiently. accurate disease forecasts can help medical organizations in taking countermeasures and advance preparedness of hospitals and the general population. recently, machine learning (ml) techniques are being increasingly implemented in the analysis of healthcare data [ ] . ml analysis can help combat diseases and improve medical systems by increasing their efficiency. particularly, deep learning, a subset of ml, has been extensively deployed over the past years due to increasing computer processing power and the availability of so-called big data sets [ , ] . deep learning (dl) algorithms are able to perform highly complex computational analysis of massive labeled and unlabeled raw data [ ] . while such dl applications have already been widely used as diagnostic tools either in disease predictions, [ ] [ ] [ ] or in clinical [ , ] or pathological image analysis [ , ] , there is limited ml deployment described for time series forecasting in the current literature [ ] . since epidemics or pandemics are known to cause not only individual, but also societal damages [ ] [ ] [ ] , the majority of disease forecast models have been created for infectious diseases [ , [ ] [ ] [ ] . analyses, in order to improve disease prevention, reduce medical costs, and allow officials to allocate resources effectively in response to public health issues. romania started using the us drg (diagnosis-related group) mechanism for hospital reporting in [ ] . switching towards an australian system, the current version adopted by romania uses the same international classification of the diseases, which made the data compatible for analysis and comparison for the entire period of time [ ] . data from the national drg database is reported monthly to the national school of public health, management and professional development (nsphmpdb) in bucharest. over a period of years, starting from until , all hospitalized patients in romania classified into a diagnosis-related group (drg) [ ] were included in the database [ ] . data was prepared from the primary national database using paxata in the datarobot platform [ ] . datasets per analyzed affliction were extracted on a regional nuts level according to the corresponding icd- codes provided by nsphmpdb (table s ). disease codes were searched and validated with the who icd- online application [ ] and the "icd- _am diagnosis and procedures list" provided by the national school of public health, management and professional development (nsphmpdb). only new hospitalized cases with targeted diseases recorded as the main diagnosis were selected and aggregated into new cases per month per nuts region. the necessary features for creating a secondary time series database are shown in the flow chart of the study selection process ( figure s ). data is not normalized during data preparation. we further enriched the dataset with the number of working days in the month and the total number of days in the month, as well as a calendar of events of public holidays (table s ). the secondary time series database is deposited online (https://www.synapse.org/#!synapse:syn ). after uploading each time series dataset onto the autots platform [ ] and selecting the appropriate forecasting target, i.e., "new cases", a time frame needed to be set to define a rolling window to derive descriptive features relative to the forecast point, i.e., the time the prediction is being made. this so-called derivation window was empirically tested using , , , , and months before the forecast point for each disease. the derivation window that produced models with the lowest gamma deviance was chosen (table ) . a forecast window (fw) defines the range of future values chosen to be predicted relative to the forecast point, called forecast distances (fds). a fd of months was used for each disease. after defining the modeling project settings and target, a model fitting procedure of preprocessing, algorithms, and postprocessing steps was performed by the autots tool ( figure s ). the autots platform simplifies model development by performing a parallel heuristic search for the best model or ensemble of models, based on both the characteristics of the data and the prediction target. during the modeling process, many independent challenger models are developed. the time series functionality works by encoding time-sensitive components (such as lags and moving averages) as features, transforming the original input dataset into a modeling dataset that can use conventional machine learning techniques. the autots tool automatically creates and selects time series features in the modeling data and automatically detects whether a project's target value is stationary. if the target is not stationary, or shows strong seasonality, it attempts to make it stationary by applying a differencing strategy prior to modeling, thus improving the accuracy and robustness of the underlying models. next, a series identifier is defined as the nuts hospital region. more precisely, a column containing the nuts region of the hospital must be identified, so that the different timepoints of a disease can be attributed to their corresponding nuts region. this tells the autots tool that there are multiple subsets of data to model and evaluate in the dataset. importantly, having multiple series allows the algorithms to learn effects that are present across the nuts regions. finally, information about the selected target variable and predictors is used to define a set of candidate blueprints for analysis; here, blueprint stands for the combination of data preprocessing steps, transformations, and machine learning algorithm. it then trains models for each blueprint and ranks them based on a validation and holdout accuracy score ( figure s ). in order to assess any model's performance, out-of-time validation (otv) is employed, which allows the selection of specific time periods to test the model stability, creating so-called data "backtests" [ ] . backtesting ensures that each algorithm is learning its parameters, or "fitting" the data, on historical examples only, and model performance is only being evaluated on unseen, "out of sample", data in a proceeding period of time in the future. the length of training data used was years for each dataset with three backtests used by the autots tool ( figure s ). the validation time period for each backtest was one year for all diseases, except diabetes, road injuries, stroke, and heart disease using eight months. across all projects, the final year of data was set aside as "holdout" data and was not evaluated until the final models were selected. the year was chosen as the holdout partition. the performances of these models are ultimately exposed, enabling the selection of the best model for the problem being addressed (table s ) . after the data had been examined by the platform, it began the modeling process. a wide variety of models are tried by the tool, including common techniques such as sarima and more modern approaches such as extreme gradient boosting. on average, different models were tried per disease ( table ). the models were evaluated according to a number of metrics, including gamma deviance, mean absolute error (mae), and mean absolute percentage error (mape). these scores were available for the first backtest, average of all backtests, and the holdout portion of the data. ultimately, the top performing models were chosen based on the score of the optimization metric chosen by the platform, either gamma deviance or root mean square error (rmse), on the holdout portion of the data. rmse is a frequently used goodness-of-fit statistic, which summarizes the discrepancy between observed values and the values expected under the model in question. it is a good measure of how accurately the model predicts the response, and it is the most important criterion for fit if the main purpose of the model is prediction. deviance is another goodness-of-fit statistic for models using the sum of squares of residuals (gamma) in ordinary least squares to cases where model-fitting is achieved by maximum likelihood. after the model was identified, it was further refined by examining permutation-based feature importance. features that contributed to the performance of the model were kept, while all other features were dropped. the model was run again using the new feature list, and if it performed better than the previous version of the model, it was selected as the final model (table s ) . to extract predictions out of the best performing model, first the model is retrained up to the last month available in the dataset, using the same hyperparameters. next, using the most recent observations as defined by the derivation window, predictions can be obtained from this updated model, which now consists of the estimated values of new cases per nuts hospital region for each of the months in the forecast window. this study was reviewed and approved by the ethics committee of the national school of public health, management and professional development (nsphmpdb) from bucharest, romania ( - . . ) and by the medical ethics committee ii of the medical faculty mannheim, heidelberg university ( - r), germany. note. after choosing the length of training data for the backtests, derivation window (dw), and the length of forecasted data (fd), models were compared and validated for each disease by the aml (automated machine learning) platform. the year was chosen as holdout, and the predicted values were compared to the actual values. model selection was based on the gamma deviance or root mean square error (rmse). other calculated estimators were r-squared, the mean absolute error (mae) and the mean absolute percentage error (mape). the total number of compared models, as well as the final selected model, are listed. these final selected models were either the avg (average) blender, the extreme gradient boosting model or the elastic-net regressor. in order to perform time series forecasting, a series of data points in time order had to be prepared for each one of the top deadliest diseases, as defined by the who [ ] . for this purpose the corresponding icd- codes for ischemic heart diseases, stroke, chronic obstructive pulmonary disease, lower respiratory infections, alzheimer's disease, lung cancer, diabetes mellitus, road injuries, diarrheal diseases, and tuberculosis (table s ) were extracted from the whole icd- data set of hospitalized patients in romania from the period - . since the aim of the study was to predict future new cases of each disease, only the icd- codes used as main diagnoses in the data set were employed. we have deliberately not included icd- codes categorized as secondary diagnoses, since physicians often tend to encode recoveries, anamnestic recalls, or unproven diagnoses in this category [ ] . hence, new cases of each disease represent the absolute count of every main diagnosis that necessitated a hospitalization episode. these disease counts were further classified into eight nuts regions to facilitate the detection of regional differences. the retrospective analysis of ischemic heart diseases revealed an obvious decline in new cases from to in all regions ( figure a) . a slight decrease in new cases was still observable after in most regions, apart from bucharest-ilfov and center, which were also the regions with the highest numbers of ischemic heart disease hospitalizations. stroke, on the other hand, showed a constant decline in almost all regions from to , with the exception of north east, a region where the stroke counts started increasing after ( figure b) . overall, bucharest-ilfov had the highest stroke case counts. unexpectedly, bucharest-ilfov revealed the lowest case counts in chronic obstructive pulmonary disease, a disease with an obvious decline in new cases in all regions ( figure c ). lower respiratory infections showed an alternating course of case counts in almost all regions ( figure d ). in this case, a decline in new cases was observable from to followed by an overall, but sinusoidal, increase in new cases after . next, alzheimer's disease counts showed a small trend upwards, with bucharest-ilfov being the region with the highest and concurrently stable case counts over the years ( figure e ). interestingly, the second highest counts of alzheimer's disease were observed in south muntenia with an obvious ascending slope. regarding lung cancer, there was a clear decline in case counts in bucharest-ilfov ( figure f ). in comparison, all other regions showed a rather small decline with at least half as many case counts compared to the bucharest-ilfov region. in case of diabetes mellitus, the counts of new cases were relatively stable over the years with bucharest-ilfov having the highest and south west oltenia the lowest numbers ( figure a) . differently, road injuries showed a clear decline from to throughout all regions with the numbers almost halving during this observation period ( figure b ). interestingly, bucharest-ilfov shared with north east the highest counts of road injuries, starting with . while diarrheal diseases also showed a stable disease count with a noteworthy increase from to ( figure c ), tuberculosis displayed a striking decline of approximately % over the whole period of years ( figure d ). most tuberculosis cases were noted north east, while the center region had the lowest counts. the year was chosen as the holdout partition ( figure ). the holdout was not part of the training data set and only served for verifying the model. therefore, every trained model predicted the monthly case counts for and was compared to the actual values. the top performing model for each disease was selected based on the optimization metric chosen by the platform, either gamma deviance or rmse (table s ) . other estimators, such as r-squared (coefficient of multiple determination for multiple regression), mean absolute error (mae, average magnitude of the errors), and mean absolute percentage error (mape, average of the unsigned percentage error), were also taken into consideration. notably, ensembles of multiple models, in the form of an average (avg) blender model, yielded the lowest mae scores in most datasets (table ) . this model takes the predictions from several input models and averages them together into a metamodel. predictions are made from each of the input models and ultimately combined. other selected models included extreme gradient boosting and elasticnet regressor. gradient boosting machines (gbms) are a generalization of freund and schapire's adaboost algorithm [ ] to handle arbitrary loss functions. gbms differ from random forests in a single major aspect: rather than fitting individual decision trees in parallel, the gbm fits each successive tree to the residual errors from all the previous trees combined. extreme gradient boosting is a very efficient, parallel version of gbm that has been heavily optimized and tweaked for faster runtimes and higher predictive accuracy. elasticnet is a linear regression model trained with l (lasso regression) and l (ridge regression) prior as regularizer. this model is useful when there are multiple features which are correlated with one another. with the exception of tuberculosis, alzheimer's diseases, diarrheal disease, and road injuries, the mape was lower than % (table ) . for the purpose of better visualization and comprehension, disease counts of the last two years in the analyzed dataset, namely, and , were plotted on a monthly basis next to the predicted counts of and (figures and ) . the overall ischemic heart diseases development of new cases seems to remain stable with low fluctuations ( figure a) . a dip in case counts was noticed during december of each year. furthermore, there are parallel curve progressions of the predicted disease counts of every region, with bucharest-ilfov showing the highest case counts. while the prediction of stroke counts also shows a certain stability, south muntenia and north east predominantly reveal the highest numbers of stroke hospitalizations ( figure b ). moreover, bucharest-ilfov showed a decline in case counts starting with . another reduction in case counts is observed with chronic obstructive pulmonary disease, especially in the north east region, when comparing the predicted years to the previous ones ( figure c ). furthermore, there is a peak in hospitalization episodes noticeable during wintertime in all regions for chronic obstructive pulmonary disease. next, lower respiratory infections will retain their strong fluctuation during the years ( figure d ). hospitalizations due to lower respiratory infections are usually high during the first three months of each year and have the lowest counts during the summer. noteworthy is a second relatively small peak occurring during october of each year. regarding alzheimer's disease, bucharest-ilfov and south muntenia have the most cases, while center, north west, west, and south east share highly similar numbers ( figure e ). here, south west oltenia remains the region with the fewest alzheimer's disease hospitalizations. another dip in counts is visible in this case during december. lung cancer has a similar disease course to alzheimer's disease ( figure a ). according to our prediction, there are no significant changes in lung cancer case counts when compared to and . moreover, the predicted case counts of diabetes mellitus are very similar to the years before ( figure b ). another distinct seasonal trend with the peak during summer and the highest counts in north east and bucharest-ilfov is seen in road injuries ( figure c ). here, south west oltenia shows the lowest fluctuations of predicted counts. interestingly, we observed a partial dependence of % to calendar dates. spring holidays are associated with higher case counts of road injuries ( figure s ) . similarly, diarrheal diseases also show seasonality, with the highest counts in summer and lowest counts in winter, mainly in november and december ( figure d ). finally, there is yearly seasonality observed with tuberculosis, including the already known reduced hospitalization cases in december ( figure e ). there is a stable decline when looking at tuberculosis from the beginning of onwards, which is continued throughout and . the regions north east and south west oltenia keep alternating in regard to the highest case counts. when compared to the current literature, this is the first study on a national icd- database to perform thorough time series forecasting on multiple diseases on a regional level using automl to select the most accurate of a multitude of models (table s ) . this is the first study to apply automated machine learning for time series forecasting on a nationwide icd- dataset. using data from all hospitalized patients from - , we were able to analyze region-specific hospitalization counts for the ten deadliest diseases in romania and perform forecasts for the years and . our findings corroborate previous studies in several important ways. cardiovascular diseases, such as ischemic heart diseases and stroke, are the leading cause of death in romania [ ] . western countries, for example, have managed to lower the mortality caused by ischemic heart diseases due to improvement of primary prevention and advances in diagnostic approaches [ ] . our retrospection of the last decade in romania echoes this statement to a certain extent as well, since the hospitalizations of ischemic heart diseases and stroke showed a continuous drop from until . our predictions do not confirm this trend but show a rather stable count for these diseases for and . since there have only been analyses on romanian macroregions [ ] , our nuts regional forecast could help decision makers identify specific regions with rising trends. given that romania has one of the highest estimated risks of developing stroke [ ] , and the number of new stroke cases is expected to double by [ ] , additional actions are mandatory in the public health sector to lower this incidence of these cardiovascular diseases. according to the who, chronic obstructive pulmonary disease cases will continue to grow in the future and become the third leading cause of death by [ ] . romania has an intermediate prevalence, calculated at around % [ , ] , and both our retrospective and forecasting analyses revealed decreasing numbers in chronic obstructive pulmonary disease hospitalizations. lower respiratory infections, on the other hand, consistently showed seasonality, with peaks in winter and lows in summertime. this is especially important for romania, since the influenza incidence is the highest among children aged - years [ ] and the lower respiratory infections mortality rate per , people for all ages is the highest in this country when compared to the whole balkan peninsula [ ] . alzheimer's disease presented a doubling in hospitalizations after , with continuously growing numbers ever since [ ] . while we also observed an upwards trend after , our forecasting results revealed steady counts for and . importantly, bucharest-ilfov and south muntenia are leading regions in alzheimer's hospitalizations in comparison to all other nuts regions. similarly, high counts were seen for bucharest-ilfov and north west with lung cancer predictions. despite several indications that the incidence of lung cancer would continue to rise in romania [ , ] , we observed a decline in hospitalization episodes. this might be due to nationally instituted antitobacco policies [ ] . we have observed a fall in diabetes mellitus cases until and predicted constant counts. it should be noted that hospitalizations episodes do not necessarily reflect the overall incidence of a disease. this could be especially true for diabetes mellitus. while icd- hospitalization case counts are relatively constant, a rise in incidence has been predicted for romania [ ] . road injuries, on the other hand, make up almost % of all injuries treated in emergencies clinics or hospitalizations [ ] . while romania has managed to reduce road mortality, it still has the most road traffic fatalities in the european union [ , ] . we even predicted the highest road-injury-related hospitalization cases in the north-east region during the summer peaks. in the case of diarrheal diseases, estimated to be the leading cause of death globally and having a declining incidence [ , ] , we only noticed a slight reduction in new cases over the period - and predicted further stable counts. finally, tuberculosis, with romania known to have the highest incidence of extensively drug-resistant tuberculosis in the european union [ ] , displayed promising declining hospitalization cases over the years. starting in , romania has made significant progress in fighting the tuberculosis epidemic by implementing nationwide prevention and management programs [ ] . this decreasing trend was supported with our forecast model. in line with other time-series analyses on tuberculosis that described a seasonality [ , , ] , we see a strong dip in the hospitalization curve in december, followed by a steep rise in the early months of the year. this dip is visible in most diseases, namely, ischemic heart disease, chronic obstructive pulmonary diseases, alzheimer's, lung cancer, diabetes mellitus, and tuberculosis, not only in the predicted months of december and , but also december and . this is attributed to a fall in hospitalization cases due to the winter holidays of saint nicholas, christmas, and new year's eve. in summary, we performed an exhaustive, time-saving analysis with a nation-wide icd- medical dataset encompassing a period of eleven years. given the fact that hospitals use different applications to collect own patient data (diagnostics, blood tests etc.), which cannot be harmonized and aggregated, the icd- dataset represents the only major, internationally used big dataset that can be employed for medical studies. by utilizing a novel automated machine learning tool, we could perform highly accurate predictions of the ten leading causes of death on a regional level for the whole country of romania. while other machine learning studies usually use one model for one disease, the deployed autots platform compared a multitude of models and allowed the selection of the most accurate one. it is noteworthy that the used dataset did not contain any outpatient, but only hospitalization records. therefore, one important limitation of our study is the predicted case counts not representing the incidence of the ten analyzed causes of death, but rather the hospitalization episodes attributed to these diseases. another selection bias could arise from inconsistencies of the primary national database, since diseases are coded by healthcare workers. some hospitals may not have the necessary resources for training professional healthcare coders, given that some diseases have long lists of potentially relevant codes, which could lead to confusion. nevertheless, the predicted changes in case counts and their geographic dynamics can help officials performing countermeasures, allocating resources, or raising public awareness through more aimed operations. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : flow chart of study selection process, figure s : model development workflow process (model blueprint), figure s : schematic representation of model development procedure for lower respiratory infections, figure s : average of hospitalizations due to road injuries on selected public holidays in romania, table s : listing of icd- codes selected for data extraction and preparation from the whole icd- data set of hospitalized patients in romania during the period - , table s : romanian public holidays, table s : exemplary listing of model performances calculated by the aml tool for lower respiratory infections, table s : exemplary summary statistics for lower respiratory infections, table s : comparison of other studies using prediction models on different diseases. the author received no specific funding for this work. the study was supported technically through pro bono software licenses. how to develop machine learning models for healthcare a guide to deep learning in healthcare deep 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and elderly population global estimates of diabetes prevalence for and projections for epidemiology of road traffic injuries treated in a large romanian emergency department in tîrgu-mureş between road safety target outcome: , fewer deaths since contributors to pedestrian distraction and risky behaviours during road crossings in romania estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the global burden of disease study quantifying risks and interventions that have affected the burden of diarrhoea among children younger than years: an analysis of the global burden of disease study european centre for disease prevention and control. tuberculosis surveillance and monitoring in europe tuberculosis remains a public health problem in romania we are grateful to victor s. olsavszky for scientific discussions. the authors declare no conflict of interest. key: cord- -t w cdr authors: nan title: royal academy of medicine in ireland date: - - journal: ir j med sci doi: . /s - - - sha: doc_id: cord_uid: t w cdr nan the tuberculin-skin-test is the most commonly used test to screen for tuberculosis worldwide. in most cases it is administered by the most junior member of the medical team. there is some anecdotal evidence to suggest that junior doctors have limited knowledge of how to administer and interpret this test correctly. the aim of this audit was to assess the proficiency of interns and senior-house-officers in st. vincent's university hospital at performing the tuberculin-skin-test and improve standards. a multiple choice questionaire was used to assess doctors' knowledge of tuberculin-skin-test administration, interpretation, alternatives and the availability and awareness of information regarding the tuberculin-skin-test within the hospital. interns and senior-house-officers were assessed. of those questioned . % correctly identified intradermal as the method of administration. . % knew to correctly assess the induration at - h, but only % knew that the induration should be measured across the forearm. only . % were aware of the information leaflet within the hospital. . % of senior-house-officers correctly identified intradermal injection as the method of administration. it is apparent that the tuberculin-skin-test is often administered and/or interpreeted incorrectly. we recommend formal teaching for junior doctors in this area, coupled with improved availability of the information leaflet. mucinous tubular and spindle cell carcinoma (mtscc) is an extremely rare type of kidney tumour that has only recently been described, with less than eighty cases in the literature. this was only recognized as a specific entity in the world health organization classification of renal cell carcinoma (rcc). mtsccs are polymorphic renal neoplasms characterized by small, elongated tubules lined by cuboidal cells with cords of spindled cells separated by pale mucinous stroma. we report the case of a -year old lady who had an incidental finding of a mass in her right kidney. the radiological features were consistent with a rcc and following a multidisciplinary team discussion she underwent a laparoscopic radical nephrectomy. macroscopic examination revealed a well circumscribed . . right lower pole mass. histologically it was composed of elongated tubules, small tubules and papillary structures with a necrotic centre. the cells demonstrated cuboidal and spindle cell morphology. histological grade was fuhrman grade . subsequent ct thorax abdomen and pelvis staged the tumour as pt b. the majority of mtsccs are indolent, and there is only one report of a distant metastasis which responded favourably to adjuvant sunitinib. to date there is no international consensus on long term surveillance of these patients. due of the favourable prognosis with this type of tumour, mtscc must be differentiated from papillary renal cell carcinoma to avoid administration of excessive adjuvant treatment to patients. this is the first recorded case of this recently classified, rare tumour in ireland. this incidental finding of solid pseudopapillary neoplasm (spn) was discovered when a -year-old female underwent a chest x-ray to investigate a wheeze. a subsequent ct abdomen revealed a cm well circumscribed mass adjacent to the tail of the pancreas. this neoplasm had reached a significant size of cm appreciable on radiological imaging and yet was asymptomatic and not palpable on physical examination. laparatomy revealed a highly haemorrhagic and calcified mass emanating from the pancreas. this was adherent to the omentum, distal pancreas and splenic vessels. distal pancreatectomy was performed with en bloc resection of the mass. repeated ct scans at , and months failed to demonstrate recurrence. solid pseudopapillary neoplasms are rare entities accounting for between . and . percent of pancreatic tumours. this neoplasm has a predilection for females under the age of . these tumours are indolent and usually reach a large size before detection. diagnosis is confirmed on histology and complete surgical excision of localised tumours is curative. we aimed to assess the prevalence of smoking among patients with vascular disease and the role of the health care profession in encouraging smoking cessation. patients who attended the vascular outpatient department were surveyed over a month period in . patients gave verbal consent to partake in the audit and the surveyor entered the responses into a standardised questionnaire response sheet. % of patients were current smokers, % ex-smokers and % had no history of smoking. % smoke over cigarettes per day and % had a smoking history spanning over years. just % of smokers who are under the care of the vascular service have been advised to give up smoking in the past by a healthcare professional. smoking has long been established as a major modifiable risk factor for the development of atherosclerosis however % of patients attending the vascular service continue to smoke. just half of patients who were offered smoking cessation advice found it was effective. therefore a system needs to be put in place where all vascular patients are advised of the benefits of smoking cessation and the manner in which information is dispensed needs to be revised. to investigate the optimum location for the teaching of procedural skills to medical students english n, o'flynn s introduction: procedural skill training is a vital component of medical education. traditionally it has been teaching hospital based however general practice rotations may provide greater opportunities than previously thought. aims: this study aimed to ascertain whether a general practice setting or a teaching hospital setting provided a better environment for acquiring procedural skills in terms of opportunity to practice and the variety of skills performed. the correlation between end of year osce results and the amount of procedural skill exposure was also looked at. methods: a cross-sectional quantitative study which included all rd year medical students at ucc was conducted. a log book listing procedural skills was made available to all students before beginning both general practice and teaching hospital rotations. students were instructed to indicate on the log when they performed any of these skills and in which location. logs were returned to medical school. data was obtained and analysis performed using spss . results: a response rate of % was achieved. . % of students performed more skills at the gp setting. . % (n = ) did not perform any skills while in a teaching hospital . skills were performed significantly more frequently in a gp setting while were performed more frequently in a teaching hospital. students who performed a high number of skills in one location were no more likely to perform a high number in the other. conclusions: students were able to take greater advantage of procedural skills opportunities in a gp setting. as this was the students first clinical year it is likely that the one-to-one teaching scenario provided them with a more suitable location to practice skills for the first time. this study also highlighted the diverse nature of procedural skills which a general practice setting can provide. accuracy of sentinel node biopsy in determining the requirement for second axillary surgeries in t -t breast cancer with retrospective application of z criteria background: lymph node status is the most important prognostic marker in breast cancer management. in tandem with breast conser-vative surgery, surgical approaches to the axilla have also become less invasive thus decreasing the morbidity associated with axillary clearance. the acosog z trial reported no difference in survival in patients undergoing sentinel lymph node biopsy (slnb) alone versus axillary lymph node dissection (alnd) in t -t tumours. our aims were to establish whether sentinel lymph node biopsy was a true representative of axillary burden. we also analysed whether retrospective application of criteria from z trial would have prevented patients undergoing second axillary surgery. methods: all patients with t -t tumours undergoing sentinel node biopsy were included in our study (n = ). analysis of our prospectively updated breast cancer database was performed. minitab version . was used to carry out statistical analysis of the data results: slnb procedures for t & t tumours were performed over a year period. patients were reported as histologically negative and were positive. of the lymph node positive group, patients progressed to axillary clearance. staging of patients remained unchanged with only patients having [ axillary lymph nodes reported as positive. patients from the slnb negative group also had an axillary clearance. of these patients had further axillary disease with patient being upstaged having [ axillary lymph nodes positive. with retrospective application of z criteria % of patients would have avoided second axillary surgery. conclusions: sentinel node biopsy is a strong indicator of axillary tumour burden. this study highlights the accuracy of sentinel lymph node biopsy in staging disease and representing overall tumour burden. flaherty ra, kelly bd, coyle d, quinlan mr, d'arcy ft, rogers e, jaffry sq we report the first case of a spontaneous right nephrocutaneous fistula (ncf) with an accompanying fistula limb communicating with the right ureter. a -year-old man presented with a groin mass, which was initially diagnosed as a hernia. he was scheduled for an inguinal hernia repair. upon incision there was extravasation of urine from the wound and the procedure was abandoned. a ct urogram identified a ncf running from the right lower pole calyx, anterior to the psoas muscle and emerging on the right groin skin with an accompanying fistula limb communicating with the right ureter. during the course of investigation it was discovered that the patient was suffering from chronic indolent calculus pyelonephritis which led to the formation of both aberrant pathways from the kidney and the ureter and that both had calculi located at their origins. the patient was first treated with a nephrostomy and ureteric stenting to relieve urinary obstruction and after this failed to resolve the fistula, was successfully treated with percutaneous nephrolithotomy for removal of the calculi and fibrin glue injection into the fistula. this case is one of only a few reported cases of spontaneous nephrocutaneous fistula and the anatomy of the fistulous tract in this case is very unusual and posed a particular challenge for surgical management. this case report further advocates the use of fibrin glue in the management of complicated ncf. this is a retrospective case study. there were six cases of ocular tuberculosis over the year period, one annually, four of whom are women, with ages ranging from to years old. two were foreign-born. all patients presented with reduced visual acuity. four developed posterior uveitis, one anterior uveitis and one panuveitis. this was also complicated by vitritis, retinal detachment and retinal vasculitis in four. the median duration of symptoms until commencement of treatment was months. all cases had a positive mantoux and one case had evidence of pulmonary tuberculosis on chest x-ray. tuberculosis was isolated in two cases. the intended duration of anti-tuberculous therapy for all patients was months. vision improved in all cases. ocular tuberculosis is rare in developed countries, with prevalence ranging from \ to %. however, it is important to be considered in all cases of uveitis. despite the use of pcr, most cases are presumptive. this leads to delayed commencement of therapy causing further complications. a high index of suspicion is required. we describe the case of a -year-old gentleman who presented to our emergency department (ed) with a very unusual complication of central venous catheterisation. this resulted in spontaneous extrusion of a retained intravenous guide wire from the base of the occiput. this has been described only once previously in the literature, but not at such a delayed time interval from insertion [ ] . this -year-old gentleman presented to the ed reporting that he felt the point of a sharp object irritate his finger in his midline occipital area. he had successfully retrieved approximately cm of a thin metal wire. he had a history of rheumatic fever and had undergone an elective aortic valve replacement years previously, necessitating central venous cannulation. he had remained asymptomatic up to this time. plain radiography of his neck revealed a short segment of wire in the posterior spinal musculature. this segment of wire (approximately cm) was removed manually with minimal force and minor manipulation. the procedure was uncomplicated and the patient was discharged shortly afterwards. retained foreign bodies may migrate slowly over many years eventually extruding from the body, without any serious complications. events such as retained or lost guide-wires are rare. this phenomenon may become more frequent with increasing complexity of medical care and with increasing use of cv catheters in the treatment of sepsis and other emergent critical conditions. physicians should be aware of the possibility of retained foreign bodies and should be somewhat re-assured by reports of simple uncomplicated removal. we present the case of a -year-old gentleman who was recently admitted with symptomatic right heart failure and new onset atrial fibrillation. our patient had been treated in the community for symptoms suggestive of ccf but had not previously been investigated. of note, he has no history of a chronic inflammatory condition and no symptoms suggestive of an underlying neoplastic process. on presentation he was also noted to have evidence of an arthropathy affecting his knees and ankles and bipedal oedema. renal function was abnormal with a urea of . and a creatinine of . urinalysis was positive for protein and h urine collection for protein is ongoing. liver enzymes were also elevated and revealed a cholestatic picture. echocardiogram showed a reduced ejection fraction of % and findings consistent with amyloidosis. biopsy of abdominal fat pad at time of writing is pending. amyloidosis refers to an uncommon group of disorders characterised by extracellular tissue deposition of a variety of proteins in an abnormal fibrillar pattern which are resistant to degradation. it can occur alone (primary) or can complicate many chronic inflammatory conditions (secondary). the major sites for clinically reported amyloid deposition are the kidneys, heart and liver. clinically patients present more often with right heart failure; pulmonary oedema is rare. amyloid infiltration results in increased echogenicity on echocardiogram and gives a ''sparkling'' appearance to the myocardium. biopsy is diagnostic. this was achieved using a retrospective review of all children receiving gh therapy (n = ) over a -year period (october -october ). of patients on gh therapy had ghd. of these, had ighd ( male) and had cphd ( male). all had appropriate work-up and follow-up. age at presentation to endocrinology was older in the ighd group (mean . years) than in those with cphd (mean . years). / children with ighd presented with short stature, compared to only with cphd; the remainder presenting with clinical features of other pituitary hormone deficiencies. the mean height centile at diagnosis was lower in the ighd group ( . th) versus the cphd group ( th). mri brain/pituitary was abnormal in the majority of patients ( / ) with cphd, compared with / with ighd. both groups responded well to treatment and height increased by one centile on average at months. all patients diagnosed with ghd at temple st had appropriate work-up and follow-up. children with ighd presented later than those with cphd, and had shorter height centiles at diagnosis. there was a strong male predominance in children presenting with ighd, which may reflect psychosocial factors. structural pituitary abnormalities were more common in those with cphd, and their clinical presentation was more varied. response to therapy was similar in both groups. background: out of hospital cardiac arrests have poor survival rates approx - %. improving outcomes in ireland have been seen in the past decade. better outcomes are seen if arrest is witnessed and when bystander basic life support was initiated. worse prognosis is seen in a rural setting due to delay in paramedic response times and in administration of advanced cardiac life support. case report: a -year-old donegal male experienced chest pain in his rural home and subsequently cardiac arrested. his spouse, whom months prior had trained in basic life support as part of a fas course contacted the 'out of hours' gp and ambulance service and commenced cpr. the gp failed to reach the house and the first ambulance broke down. on arrival of second ambulance, one person cpr had been administered for [ min. paramedics delivered dc shocks and intubated the patient. in the regional hospital pc was admitted to the intensive care unit for days being managed with acute respiratory distress syndrome (ards). transoesophageal echocardiogram on day of admission showed ef %. ct brain carried out showed no acute pathology. once stable, angiography was carried out showing multivessel disease. discussion at st james's hospital (sjh) cardio-thoracic conference resulted in plan for transfer and pci. in sjh pressure wire study of left anterior descending (lad) coronary artery was positive and stenting (drug-eluting) commenced. lad stents, left circumflex stents and right coronary artery (rca) stents. patient is currently well with no overt signs of hypoxic brain injury and is enrolled in cardiac rehabilitation programme. discussion: this is an incredible case of an out of hospital cardiac arrest. elapsed time in the chain of survival events would predict a negative outcome. however, adequate cpr was administered preventing long term brain injury and certain death. this highlights the need for a greater community-based cpr skill base. recently citalopram and escitalopram have been reported to cause dose dependent qtc prolongation. prescribing guidelines have since changed including contraindication of co-prescription with other qtc prolonging agents. domperidone is a dopamine antagonist widely used as an anti emetic. qtc prolongation and ventricular arrhythmias have also recently been highlighted with domperidone and, since november , caution advised when prescribing domperidone, particularly in patients [ years of age, or at doses [ mg/day. in this audit, we aimed to study whether information on qtc prolongation affects prescribing practice by looking at the prescription of a commonly used medical drug, with recently highlighted qtc effects, and its co-prescription with psychotropics. a list of drugs with substantial evidence for qtc prolonging effects was obtained. a kardex review was completed from acute medical and surgical; long stay and rehabilitation wards. kardexes with domperidone were reviewed for dose, age, gender and co-prescription of other qtc prolonging agents. of surveyed kardexes, % (n = ) were prescribed domperidone. % were[ years. % were on[ mg/day. coprescription with another qtc prolonging agent seen in % of cases; of these % were psychotropics, most commonly citalopram (n = ). four patients were co-prescribed [ qtc prolonging agent. qtc prolonging agents were commonly co-prescribed with domperidone, which continues to be used even in at-risk groups. psychotropics were the most likely class to be concurrently prescribed. further work in this area is necessary to inform clinical psychiatric practice and encourage responsiveness to new evidence regarding cardiac risk. the development of a mathematical model to predict the time to osteoporosis (tto) using dexa scanning background: dual-energy x-ray absorptiometry (dexa) is the gold standard used for measuring bone mineral density and such readings are currently used to predict osteoporosis and osteoporotic fractures. however, no similar prediction model has been developed to identify the time it will take to become osteoporotic based on dexa scanning. objective: the aim of this study was to develop a mathematical model to determine the tto based on two or more dexa scans with tto defined as the age at which the patient will enter the osteoporotic t-score range. methods: fifty patients who had previously undertaken five dexa scans were identified from the dexa database. t-scores were graphed against patient age using graphpad prism software. straight line curves for the most recent scans and cumulative scans were generated with the age at which the curve intersects t = - . being classed as tto. results: the mathematical model developed successfully predicted the time to osteoporosis for each patient, as well as creating a cumulative osteoporotic trend based on total dexa scans performed. additionally, if the patient was classified as osteoporotic following dexa scanning, the model also successfully predicted the time out of osteoporosis. implication: the tto provides a simple and informative parameter of dexa scanning that a patient can immediately comprehend and understand, while also providing a more simple measure to monitor response to therapy. based on the results presented tto can be incorporated into future dexa scans result summaries. further research will involve validation of this tool. an audit of clinical outcomes in transcervical resection of the endometrium compared to outpatient balloon thermablation anglim bc, von bunau g department of gynaecology, adelaide and meath children's hospital, tallaght, dublin thermablation was introduced to the coombe in november and thus far it has provided a quick and effective means of treating women with menorrhagia refractive to medical treatment. a retrospective audit was carried out over a year period in tallaght hospital from november to october . the aim of the study was to compare the efficacy of balloon thermablation compared to transcervical resection of the endometrium (tcre) with or without mirena coil insertion, in the treatment of menorrhagia. patients in total were studied, of which underwent a tcre, and of which underwent balloon thermablation. out of those who underwent a tcre had successful treatment of the menorrhagia and and weekly follow up, had continued menorrhagia which may require a future hysterectomy, however one of which was due to a large fibroid, and one patient described a reduction in menorrhagia however an increase in dysmenorrheoa. out of those who underwent thermablation were treated successfully, had continued menorrhagia to be considered for hysterectomy, had reduced bleeding but increased dysmenorrhoea and one patients symptoms had resolved however she then developed idiopathic thrombocytopenia purpura which led to a recommencement of symptoms. one can therefore conclude that there are both pros and cons to both procedures, tcre being less expensive, however it requires general anaesthesia and may require mirena insertion. thermablation is more expensive however it is a quick outpatient procedure ( min, s) and is done under local anaesthetic. akinmoluwa s, tormey s department of breast surgery, mid-western regional hospital, limerick breast pain is a common problem especially among women of reproductive age. it accounts for a great percentage of gp visits by young women. it represents a huge proportion of gp referrals to the breast clinic. the palpable effects of this include, among others, an increase in waiting time, increase in healthcare cost, stress on the limited resources and ultimately a decrease in quality of care. in this era of unfavorable economic climate, it is prudent to sanitise our healthcare systems by way of identifying and eliminating practices that have not been proven to alter the course of care. in this study, i reviewed the number of breast pain cases referred to ms tormey's breast clinic in the month of march. the objective of this study is to determine whether or not all breast pain complaints should be referred for specialist review. to achieve this objective, i reviewed all the cases of breast pain referred to the breast clinic in march. the table represents my findings. it is evident from the study that hormonal mastalgia accounts for majority of breast pain complaints in women of reproductive age while a few other cases are attributed to musculoskeletal and other benign disorders. these women, with no risk factors, only need reassurance and pain relief. they do not require specialist intervention. alrashed d introduction: anaemia is a common finding in the elderly population. it may be a sign of chronic disease, underlying malignancy, nutritional status, or blood loss. depending on the classification of anaemia, further investigations such as haematinics and endoscopy may be warranted, as replacing the haemoglobin deficit is never a definitive treatment. objective: to determine the prevalence of anaemia in a population of elderly in-patients and whether further screening was performed. methods: this was a cross-sectional review of all patients years and older under a gastroenterology, a rheumatology, and three geriatrics services at a large teaching hospital. patients' full blood counts were reviewed during their current admission. anaemic patients were then categorised based on anaemia subtype and whether haematinics were investigated. results: out of patients under the five teams, were years and older. out of of these elderly patients were anaemic. none of these subjects had microcytic anaemia during their current admission. out of of these patients had normocytic anaemia. out of of anaemic patients had macrocytic anaemia. haematinics were investigated in out of , including out of patients with normocytic anaemia and out of patients with macrocytic anaemia. one patient had abnormal haematinics after being investigated for macrocytic anaemia. conclusion: anaemia was very prevalent in the patients selected for this audit, with the normocytic subtype being the commonest. haematinics were investigated in half those patients. anglim b, murphy c aims: to determine the nature of surgical management of ovarian cysts in the adolescent and paediatric population over a year period. methods: a retrospective audit was carried out over a year period in tallaght hospital from january to december . this audit reviewed cases of ovarian cystectomy, oopherectomy and salpingooopherectomy using both a hospital online database and records of theatre procedures to identify these patients. results: a total of cases were identified. the commonest presentation was due to pelvic pain. there was a total of ovarian cystectomies, fimbrial cystectomies, oopherectomies, bilateral oopherectomy, and salpingo-oopherectomies. a total of appendicectomies were performed in conjunction with these. histology varied from functional and non functional cysts to dermoids and cystadenomas. there were a total of functional cysts, of which were hemorrhagic. there were follicular cysts, fimbrial cysts, paratubal cysts, dermoid cysts, endometrial cysts, cystadenomas, ovarian torsions and fimbrial torsions. of the total amount of procedures performed were done by a paediatric surgeon, and by a gynaecologist. notably there were fewer cases of benign histology in those procedure performed by gynaecologists. conclusions: adnexal surgery is commonly performed in adolescents and children. pathology is frequently benign. there may be a role for more conservative management. we suggest that imaging of the pelvis and tumour markers should be used more frequently in the pre-operative period. protocols may be developed for future implementation. anglim bc, crowley p day surgery is an efficient way of using hospital beds, provided patients are discharged as planned on the day of surgery. unplanned overnight stay following day surgery places an extra burden on a hospital with the busiest accident and emergency department in ireland. a retrospective audit was carried out of one years day case admissions to determine the incidence and causes of unintended or unplanned overnight stay. women were admitted as day cases over the period of st july to june th . a total of diagnostic laparoscopies, operative laparoscopies, diagnostic hysteroscopies, ninety-three operative hysteroscopies, tension free vaginal tapes (tvt) and miscellaneous minor procedure were carried out during this time period. women ( . %) were retained overnight. the main reason for overnight stay was excessive post-operative pain. additional reasons included voiding difficulties, reactions to spinal anaesthetic, asymptomatic tachycardia and the need for intravenous antibiotics. there was no evidence of inappropriate selection amongst the laparoscopies and hysteroscopies, however % of the patients undergoing tvt required admission. one can conclude from this study that most patients were appropriately selected for day case admission. patients undergoing tvt surgery should be scheduled for a h hospital stay. a vulval clinic is an ideal and efficient way of detecting patients with vulval cancer. once potential patients have been flagged by general practice clinicians or other specialities within the hospital, immediate steps can be taken to rule out malignancy. a retrospective audit was carried over a month period on a new vulval clinic which commenced in tallaght hospital on / / . the aim of the study was to determine the need for a specialised vulval clinic for detection of vulval cancer. a total of patients were referred to the four clinics which took place over this time frame. the majority of referrals were from general practice, other referrals were from dermatology, gynaecology and colposcopy clinics. the main reason for referral was vulval pruritis and pain. nine patients were referred with suspicious lesions on clinical examination. a total of biopsies were taken, two of which showed vulval intraepithelial neoplasia (vin). amongst the other biopsies were cases of lichen sclerosis and the remaining biopsies showed non specific dermatitis. one can conclude from this study that a combined dermatological-gynaecological clinic would be of benefit. in addition a . % detection rate of vin was achieved and therefore highlights the necessity of this clinic. the prevalence of renal disease in patients aged above with normal serum creatinine balasubramanian i, peters c, lyons d and o'connor m department of ageing and therapeutics, mid-western regional hospital, limerick background: the prevalence of chronic kidney disease (ckd) increases with age. older patients have lower lean muscle mass and therefore using serum creatinine alone as marker of renal function can lead to underdiagnosis of ckd. objective: the aim of this study was to review the prevalence of ckd amongst a cohort of elderly patients with normal serum creatinine. methods: doctot application on the smartphone was used to calculate egfr in a cohort of patients over years with a normal serum creatinine on admission. patients were included. this application is based on the mdrd formula (includes age, sex, ethnicity and serum creatinine). patients were then classed into the various stages of ckd. results: of the patients reviewed, had renal disease. interestingly, only had a diagnosis of renal impairment recorded in the medical notes. of the patients had stage ckd and the other had stage ckd. of the patients with renal impairment especially stage , were found to be frail females over years. this group also had a number of co-morbidities including diabetes and hypertension. conclusion: egfr is better than serum creatinine alone for assessment of renal function in the elderly. it is important not only for diagnosis but also for appropriate medical investigation and drug prescribing. as the mdrd formula excludes bmi, further research is warranted to compare measurement of egfr using mdrd formula with the cockcroft and gault equation in this older population. chronic obstructive pulmonary disease (copd) is increasingly prevalent worldwide and the main responsibility for it's prevention and management lies with general practitioners. the aim of this audit was to analyse current standards of care of copd patients in a suburbanrural general practice by examining icgp criteria and comparing results with best practice guidelines. the existing coded population of active patients with copd were telephoned and consent was obtained to ask a set of questions designed to examine certain criteria chosen from the icgp copd quick reference guide [ ] . . of the patients included in the audit (n = ), % of patients were male, the mean age was years (sd = . ) and % were general medical service (gms) patients. there was poor recording of smoking status, high uptake of influenza vaccines compared to international figures, a lower uptake of pneumococcal vaccinations and an increased need for osteoporosis prophylaxis. vaccination reminders, smoking cessation advice and information leaflets have been posted to these patients. development of protocols for coding and management have been implemented. in conclusion, general practitioners must focus on ensuring optimum managment of copd in the community. clinical audit is a useful tool to initiate change. we assessed the accuracy of continuous non-invasive haemoglobin measurement using the sphb pulse co-oximeter Ò when compared to traditional laboratory haemoglobin assessment in an outpatient antenatal population. a total of women were recruited. traditional laboratory haemoglobin samples were taken and quantified in the hospital laboratory. the sphb pulse co-oximeter Ò was calibrated and the mean of three non-invasive measurements of haemoglobin were recorded prior to venipuncture. bland-altman plots were used to determine acceptability of the new non-invasive test as a replacement for invasive testing in a clinical setting. the mean gestation at haemoglobin estimation was . ( . ) weeks. laboratory haemoglobin values ranged from . to . g/dl with a mean of . ( . ) g/dl. the range for the sphb pulse co-oximeter Ò assessment was . to . g/dl with a mean of . ( . ) g/dl. non-invasive haemoglobin measurement provides a clinically acceptable accuracy compared to traditional haemoglobin testing. pressure wound therapy (npwt). in this review we examine the role of npwt in wound healing, compare the products available to clinicians in irish hospitals and explore cost implications today. we achieved this through review of online data, peer reviewed articles regarding efficacy, collection and assessment of data from suppliers of npwt and examining the use and cost of npwt in the mater misericordiae university hospital. we summarise the mechanism of action of npwt, patient selection and indications for its use. the products available on the irish market are compared. through examination of these elements we clarify a role of npwt in management of complex wounds and identify flaws in the management of this service that are both wasteful of money and hospital services and create barriers to discharge. potential strategies to correct the issues identified are detailed, for example, funding of the product by the treating hospital rather than by local authorities in the community or selection of less costly devices in negotiation with suppliers by local health authorities. the solutions we outline will potentially have a financial benefit to the hospital, will lead to the more efficacious running of the hospital system and as such will benefit the patient. we conclude that this is a fundamental service and that there are alternative approaches to implementing use of the product in a more efficacious manner. poster q fever: questions to be answered? brandon l, bannon c, fleming c department of infectious diseases, university hospital galway q fever, an aptly named condition, describes infection with gramnegative bacteria coxiella burnetti. q denotes a question, and there are many to be answered in this rare, but not unknown, condition. take mr. m.c, a -year-old farm worker, who had an aortic valve replacement in , for congenital aortic valvular disease. he next presented to medical services in , with fevers, sweats, fatigue and weight loss. investigations at the time diagnosed autoimmune hepatitis, following liver biopsy. he commenced prednisolone and azathioprine. in , again symptomatic, he had another aortic valve replacement. post-operatively, he required weeks of antibiotics for a culture negative valvular infection. in , still on immunosuppression, he developed culture negative meningitis, requiring weeks of antibiotics. azathioprine was discontinued. a renal biopsy revealed proliferative glomerular nephritis in , carried out for macroscopic haematuria. he commenced high dose prednisolone and cyclophosphamide. throughout this time, he regularly presented to medical personnel with high fevers, up to c, present since . they responded to steroids but relapsed on doses below mg. in , the fevers were investigated with a toe, and vegetations seen on the aortic graft. he was diagnosed with culture negative bacterial endocarditis, and subsequently tested positive for q fever. this case highlights the q behind q fever, and raises important issues for medical personnel. when should we remember it? when should we test for it? and what can we do to ensure high risk populations dont slip through the cracks, as this gentleman did? previous point prevalence studies of antimicrobial use in sch have consistently produced the same conclusions and recommendations pertaining to prescribing habits, highlighting doctors' failure to meet ideal standards of antimicrobial prescription. the aim of this study was to assess antimicrobial prescribing habits from the doctors' point of view, to compare this to available prescription data and to raise awareness of the principles of prudent antimicrobial prescribing. a multiple choice questionnaire was used to examine antimicrobial prescribing habits with regard to documentation of indication, documentation of a stop/review date, awareness of local empiric guidelines and other principles of prudent antimicrobial prescribing. trainee and consultant doctors were surveyed. of those questioned, % claimed they always ensure that an indication for commencing antimicrobial treatment is documented in the patient's healthcare record. only % always document a stop/ review date when prescribing antimicrobials, while % indicated that they had failed to do this at least once in the preceding month. % of those surveyed sometimes or never consult local guidelines. when switching patients from intravenous to oral therapy, % believed oral bioavailability to be an important factor, with only % citing cost as being relevant. identifying doctors self-reporting of their deficits allows us to target appropriate interventions to these deficits. our survey identifies areas where awareness of diverted resources and safety issues could be used as a fulcrum for changing prescribing practices. we recommend formal teaching for doctors in this area, with particular emphasis on prudent prescribing and the correct use of empiric guidelines. venous thromboembolism (vte) is a cause of inpatient morbidity and mortality which may be reduced by appropriate thromboprophylaxis. it is well established that vte risk assessment and thromboprophylaxis prescribing may often be inadequate. recently it has been estimated that as many as , deaths per year due to hospital-acquired vte in england may have been prevented with appropriate prophylaxis [ ] . in the current study, a cross section of inpatients was examined to establish concordance with current evidence-based guidelines for vte prophylaxis. data was collected from inpatient charts and drug kardexes relating to patients on three medical wards. laboratory data was also obtained from the hospital it system. data relating to patient mobility was obtained from medical charts, nursing staff, observation, and the patient themselves. sixty-three medical patients and surgical patients (including one patient under obstetrics and gynaecology) were included in the study. ( . %) out of at-risk medical patients who were suitable candidates for thromboprophylaxis had sub-cutaneous heparin prescribed, whereas out of of the suitable at-risk surgical patients were prescribed thromboprophylaxis. medical patients ( . %) and surgical patients ( %) were prescribed anti-embolism compression stockings. prescribing of thromboprophylaxis is relatively thorough in this patient population although it remains less than optimal. there exists some evidence of disagreement amongst clinicians regarding the optimum vte prophylaxis strategy [ ] . implementation of hospitalspecific guidelines regarding thromboprophylaxis is recommended in keeping with recognised guidelines [ ] . ali sheikh a, chandra r, gardezi a, o'hare j mid-western regional hospital, limerick background: good documentation represents good medical practice. objectives: to assess our current standard of documentation of allergy in admission notes, synchronicity with risk alert bands, information given by the patient, documentation on the front allergy alert section of medical notes and drug kardex. methodology: we assessed five parameters i.e. drug kardex, alert band, medical and admission notes and gathered information from each patient staying in medical and surgical services. there were patients in hospital. results: % of the patients admitted under medical and surgical teams had allergy or allergies to different drugs. % had single allergy, whereas % had multiple allergies. penicillin allergy was the commonest . % followed by opioids . %. furthermore, it is found that recording of allergies was under par as % was on front page and % appeared in medical notes. more than half of allergy information was found on drug kardex %, patient knowledge % and allergy bands %. it is well documented that use of elastic compression stockings (ecs) prevents post thrombotic syndrome in patients with prior deep venous thrombosis (dvt). a % reduction in these complications has been noted, with year duration of therapy suggested [ ] . the advice given to patients, their understanding of the benefits of this therapy and adherence issues has not been documented at sligo general hospital (sgh), this research aimed to address this. a short patient questionnaire was undertaken. this consisted of demographic information, and questions regarding the advice and use of ecs. the population consisted of patients with prior dvt attending the warfarin clinic at sgh. this data collection took place from october -february . the questionnaires were collated and results identified using microsoft excel with simple statistical analysis. eleven patients were included in the study, % were not advised to wear ecs, and only % wore the ecs daily. reasons for nonadherence include; difficulty fitting, discomfort and no benefit noted. improvement in adherence could be achieved if advice was given promoting use, the benefits explained, optimal frequency/duration of use advised and correct measurement. as research strongly supports use of ecs, it is essential adherence is encouraged to reduce the risk of post-thrombotic syndrome and future dvt. opiate injecting drug use is a well-established phenomenon in inner city dublin. the complications arising from this practice affect a predominantly young cohort of patients, who under different circumstances would be expected to enjoy good health. acute infections, acute vascular issues such as pseudoaneurysm, and chronic medical conditions such as hepatitis c and hiv are well recognised and frequently encountered by medical physicians who care for these patients. we present the case of a lady in her thirties with a long history of opiate injecting drug use. approximately months prior to presentation, she underwent left sided pseudoaneurysm repair. she presented to the emergency department in a drowsy opiate induced state. on waking, she stated that she had lost some needles while injecting into her groins. plain radiology of pelvis revealed the two ''lost'' needles ( fig. ) . on closer questioning she admitted to significant manipulation of the needle injecting path and angle in the weeks prior to presentation. she had attributed this to her previous surgery and the duration of her injecting drug use. follow-up duplex sonography revealed bilateral intact femoral arteries. further surgical management was non-operative with the focus on addiction counselling and further attempts at facilitating cessation of heroin use. bilateral ''lost'' needles is an unusual complication of injecting drug use and certainly would not rank as one of the protean manifestations of such practices. the aim of the study was to investigate patients' recall of their surgery and influencing factors. a questionnaire was given to patients at outpatient follow up and surgical details were recorded. patients completed the questionnaire. the median age was years (range - ) and the median follow up was months (range - ). the extent of surgery did influence patients' recall with those having an alnd (n = , %) having significantly more accurate recall of their surgery as opposed to those who had slnb (n = , %), p = . . the presence of ongoing postoperative symptoms also significantly improved recall, p = . . almost half the patients who had slnb ( . %) could not accurately remember the extent of the surgery they had but . % were more careful of their arm or would not allow cannulation. the patient's consent process influenced patient accuracy. patients who filled the consent at both the outpatient consultation and in the hospital were significantly more accurate than those who had signed the consent at the clinic or hospital alone, p = . . patients who have minimally invasive surgery, such as slnb are not accurate at recalling their surgery. this misinformation results in confusion over the subsequent vigilance of their upper limb. the consent process may have a role in improving patient recall. introduction: waiting times can exceed days for general surgical clinics and can reach up to months in different surgical specialities. many outpatient slots are lost by patients who do not attend (dna) to their scheduled appointment. we sought to ascertain whether a reminder text message (rtm) could decrease the number of patients who dna to surgical outpatients. methods: a single text message was sent to patients days before their scheduled appointment, reminding them of the date and time of their upcoming surgical outpatient visit. this incentive was initiated in january . outpatient appointment scheduling and attendances for a single surgical team were analysed over a year period, encompassing two month periods before and after implementation of the rtm service. data was exported to spss v for statistical analysis with p \ . considered statistically significant. results: over the month period there were , scheduled outpatient appointments for the surgical service, with attending prior to the implementation of the reminder text message service and the remaining attending in the months following its implementation. the percentage of dna patients did not differ significantly ( . classically, focussed assessment with sonography in trauma (fast) addresses a yes/no binary question as to whether fluid is present in the context of trauma. fast generally concentrates on four areas: perihepatic, peri-splenic, pelvic and a sub-xiphoid view of the pericardium. we report on two patients who were the victims of trauma. both patients had normal haemodynamic parameters. in both patients, the initial fast ultrasound scan was technically negative but it exhibited other signs of intraperitoneal injury. in the first case, a young gentleman sustained a penetrating injury to his right upper quadrant area. morison's pouch (the interface between the liver and the right kidney) did not exhibit any fluid. there was, however, a thin anechoic strip around the gallbladder. ct confirmed the suspicion of peri-cholecystic fluid and this patient required urgent laparotomy and repair of his hepatobiliary injury. in the second case, a gentleman in his thirties sustained a blunt injury to his left upper quadrant. ultrasonography exhibited heterogeneous echogenicity of the spleen. this patient proceeded to have urgent laparotomy and splenectomy for this shatter-type injury. as experience with fast techniques grows, the binary question of whether intra-peritoneal fluid is present becomes more nuanced. the objective of this audit was to review the hospitals compliance with hospital guidelines, to get an overview of how fluids are being prescribed in the hospital and to produce quality improvement plans. thirty drug kardexs were chosen randomly from wards around the hospital, both medical and surgical. if a kardex was found to have no fluid prescription, an alternative kardex was chosen in its place. note was taken on whether the prescription had the patient name and hospital number, the date, name, dosage and strength of the prescription, the route of administration and the frequency and rate of administration. the main areas of non-compliance were found to be: name: only ( . %)orders out of had the name on the order medical record number: only ( . %)orders out of had the mrn on the order, and the route of administration was not present on any of the orders checked. in conclusion, this audit would suggest that there is a lack of compliance with detailing the patients name and mrn on fluid orders, that the route of administration was not written on any kardex, however the back page of each is exclusively dedicated to iv fluid prescription and also that non-approved abbreviations are being used when prescribing fluid orders. spontaneous hip fractures, or fractures without a fall have been described in up to % ( , ) of cases of hip fracture. an upsurge in such cases was recently observed in our emergency department. we present these in the form of a retrospective case series. patient is a -year-old ex intravenous drug user who presented with non-traumatic right-sided hip pain over a period of weeks. initial plain films did not reveal fracture. over week her symptoms deteriorated to the extent that she became unable to weight-bear. patient is a -year-old gentleman with increasing left sided hip pain following a seemingly innocuous fall months prior to index presentation. again initial radiographs did not reveal an abnormality. patient is an -year-old bed-bound nursing home resident with end-stage alzheimer's disease. she was noted by nursing staff to have bilateral hip symptoms post seizure. the patient was unable to mobilise independently and had not fallen out of bed at any stage. patient is a -year-old lady who presented with unilateral sacroiliac pain following a recent intensive exercise program including kickboxing week previously. in each of these cases, subsequent review and plain films demonstrated fracture and in one case bilateral fractures secondary to seizure were demonstrated. our cases highlight the need for diagnostic vigilance and a structured approach in dealing with possible radiologically occult hip fractures, even in patients with no proximate antecedent history of trauma. delerium, or acute confusional state, is a common presentation to our emergency departments, and occurs in up to % of hospitalised patients. we describe the case of acute deterioration in mental status, on a background of alzheimer's disease, with an interesting aetiology. mr k's family sought emergency medical review of days deterioration; withdrawal, somnolence and general disorientation. he is a -year old with moderate alzheimer's disease. history and initial investigations were unremarkable. he was mildly dehydrated and physical exam showed only mild truncal ataxia. further investigations to elucidate cause included lumbar puncture, mri brain and immunological and vasculitic parameters. serology revealed human immunodeficiency virus (hiv) infection with acute seroconversion pattern. a history obtained with help of his family identified several casual heterosexual partners within past year. this included a contact who may be an intravenous drug user, with involvement in commercial sex work. symptoms abated within a week of admission, following pattern of hiv viral load. he has subsequently commenced antiretroviral therapy. this case highlights several areas of interest. sexual history is often overlooked in the older patient, which can be deleterious to outcomes. trends of hiv infection in ireland include primary infection in the older person, in addition to greater longevity of people infected in earlier adult life. we would advocate opt-out testing within the emergency department, and this is currently under study in our tertiary emergency department. comparison of comorbidities in patients with pre-diabetes to those with diabetes mellitus type the management of type diabetes and its complications are well researched. the prevalence of these complications in pre-diabetes has not been researched to the same extent. there has been no research comparing the prevalence of complications in pre-diabetes and type diabetes in ireland. a cross sectional study performed on pre-diabetes and type diabetes patients, selected from the diabetes interest group database (a database of the diabetic patients in general practices in cork region) using stratified sampling for age and gender. a questionnaire was designed and completed in each practice assessing the presence of diabetes related complications in pre-diabetes and type diabetes patients. data was analyzed on spss. the prevalence of complications was determined and the chi square test performed to see is there a statistically significant difference in the prevalence of these complications between pre-diabetic and type diabetic patients. the prevalence of ischaemic heart disease and autonomic neuropathy is actually higher in pre-diabetes but the prevalence of renal disease and cerebrovascular disease is higher in type diabetes. none of these differences in prevalence are statistically significant. the prevalence of peripheral vascular disease, eye disease and peripheral neuropathy is higher in type diabetes, this difference being statistically significant. the prevalence of many of the complications in pre-diabetes is as high as in type diabetes which may have implications for the screening and management of these conditions and the related comorbidities. the both groups were evenly matched. the median age was and median homocysteine level was (range - . ). results: in group b, immediate clinical improvement was equivalent between the normal homocysteine group and treated hyhc group. median time to binary restenosis in hyhc was months and in normal homocysteine was months. p = . . secondary endpoints and all cause survival showed no significant difference. pre-treatment multivariate logistic regression for group a; depicts that hyhc is the main culprit of graft occlusion and limb loss p \ . . multivariate logistic regression for treatment group reports that corrected hyhc is no longer a significant factor of operative outcome. conclusion: patients with treated hyhc have similar outcomes compared to those with normal homocysteine. it is therefore crucial to measure homocysteine in all patients with cli and correct aggressively prior to intervention to improve outcomes. the efficacy of clinical guidelines in promoting co-prescription of bone protection with glucocorticoids among hospital doctors treating inpatients background: therapeutic glucocorticoids (gc) rapidly decrease bone mineral density, inducing a catabolic shift by promoting osteoclast differentiation and activation and by inhibiting osteocytes. current guidelines ( ) direct that bisphosphonates (bp's) and calcium carbonate , mg (ca ++ co -) with vitamin d (vit. d ) should be given at initiation of gc therapy as it is known that bone catabolism occurs early with steroid usage. we circulated these guidelines within our hospital after auditing the existing practice of the hospitals doctors and year later we sought to measure the efficacy of our intervention by completing an audit loop. methods: a cross sectional audit was performed of all adult medical and surgical inpatients in a tertiary referral centre teaching hospital. it was noted if inpatients had been prescribed gc and if concurrent anti osteoporotic medication had been prescribed. subsequent to the initial audit, guidelines promoting the use of bp's, ca ++ co and vit. d when prescribing gc's were advertised on hospital notice boards, in hospital bulletins, hospital prescribing guidelines and on the hospital website. one year after publishing the new guidelines the audit loop was completed by performing a similar cross sectional audit. results: all inpatient medical records (n = ) were reviewed in jan of whom % were female and % were older than . / ( %) inpatients were prescribed gc's. ca ++ co with vit. d was prescribed for % of patients on gc's with % also receiving bp therapy. % of patients were also receiving-post menopausal hormone replacement therapy. in nov year after guideline publication all inpatient medical records (n = ) were reviewed of whom % were female and % were older than . / ( %) inpatients were prescribed gc's. ca ++ co with vit. d was prescribed for % of patients on systemic steroids with % also receiving bp therapy. creation and circulation of hospital guidelines resulted in an improvement in the co-prescription of ca ++ co and vit. d and bp's with gc's by the order of . and respectively. however % of patients on systemic steroids received no bone protection and % received suboptimal bone protection from steroid induced osteoporosis. conclusion: publication and advertisement of current bone protection guidelines when prescribing systemic steroids resulted in a substantial but suboptimal improvement by hospital doctors in our hospital in the co-prescription of bone protecting drugs to prevent steroid induced osteoporosis. in this audit it appears that the majority of prescribers do recognise the necessity to protect bone health when a patient requires steroids. however a substantial number of patients did not receive any bone protection. it is our perception that most physicians are not aware that short courses of steroids reduce bone mineral density and therefore greater efforts must be made to enhance doctor awareness of the necessity for bone protection to be prescribed at initiation of systemic steroids. there is a trend towards longer total survival for jetflow tcvcs. these results suggest a potential advantage from using this line type, however, further study and formal cost analysis needs to be undertaken prior to changing our practice. with increasing resource restrictions, appropriate ordering of blood tests is vital for medical economic viability. this study evaluated the pattern and cost of thyroid function test (tft) requests and aimed to determine if tsh alone identifies thyroid abnormalities. a retrospective review of tfts performed on in-and-out-patients at a -bedded regional hospital was undertaken in january , evaluating the number, results and costs of tsh, t and t levels. tsh, t and t were ordered. / patients ( . %) were euthyroid. tsh abnormalities occurred in / ( . %) ( table ) only / ( . %) patients had a normal tsh despite an abnormal t or t level. / ( . %) of these patients had known thyroid disease, undergoing treatment with thyroxine or thyroidblocking medications. / ( . %) had t levels \ nmol/l outside the normal range and asymptomatic so were considered to be euthyroid. / ( . %) had a variety of diagnoses, for example, pituitary disease. tft reagents alone cost € , . ir j med sci ( ) (suppl ):s -s this study has identified that non-selective requests for t and t add little diagnostic value, except in certain circumstances like treatment of thyroid disease, in pregnancy or if pituitary disease is suspected. optimising tfts requests could save in the region of € , /per annum. tsh alone would appear to be adequate for the majority of patients. case study: neurodegenerative disorders we present a case with an unusual combination of neurodegenerative disorders. a fit and healthy -year-old man, with no history of medical or psychiatric illness deteriorated progressively over a year span, presenting initially with speech and language difficulties, followed by development of extra-pyramidal signs non responsive to levodopa. neurological permacol Ò mesh is an acellular porcine-derived dermal collagen surgical implant used in a wide variety of surgical reconstructions and repairs. we describe two cases where permacol Ò mesh was used to anchor the contents of the femoral triangle in patients undergoing radical block nodal dissection as part of the surgical management for metastatic penile squamous cell carcinoma, one of whom had an atrophied sartorius muscle due to previous infection with poliomyelitis. both patients underwent successful inguinal node dissections and femoral triangle repairs, with permacol Ò proving to be an effective means of protecting the femoral vessels in both patients despite complications related to wound healing secondary to a fixed flexion deformity in one patient. a -year-old gentleman, with a past history of vestibular schwannoma requiring a ventriculoperitoneal shunt (vps) was admitted with acute diverticulitis. his condition worsened and required a laparotomy for bowel perforation and faecal peritonitis. this case reports the successful perioperative management of the patient with a vps in situ in the setting of an emergency abdominal surgery. vps placement is an effective treatment of hydrocephalus, diverting cerebrospinal fluid (csf) into the peritoneal cavity. unfortunately, the shunt devices have a high incidence of malfunction mainly due to catheter obstruction or infection and are associated with various complications, % of which are abdominal [ ] . incidental pathology unrelated to the vp shunt can also occur such as appendicitis [ ] , endometriosis [ ] and diverticulitis as in this case. no standard current set of guidelines for perioperative management of vps exists for patients undergoing general gastrointestinal or urologic procedures with varying degrees of contamination [ ] . this case reports successful and conservative management of a patient with a vp shunt that underwent contaminated abdominal surgery. there is no consensus on the management of vps in patients undergoing elective or emergent abdominal surgery and further studies are required in this area. the use of antithrombotic therapy on management of atrial fibrillation in an irish general practice malomo k , breen n , dunne l , farrell g , bryne p ucd (university college dublin), ireland, now intern, mid-western regional hospital, limerick; general practice, dublin, ireland; pottersway medical centre, bunclody, ireland background and objective: atrial fibrillation (af) is a common cardiac arrhythmia associated with increased risk of stroke events [ ] . to assess the use of antithrombotic therapy in patients with known af attending an irish general practice (igp) and use of stratification schemes to assess their suitability for oral anticoagulant therapy. methods and subject: permission to carry out the study was sort from university-college-dublin ethics committee. there were patients with af attending the igp identified using the computerized disease coding system who international classification of disease (icd- ). thirty patients were diagnosed between / / and / / and their data from the computerized medical notes was used to calculate chads , cha ds -vasc, has-bled scores and identify antithrombotic therapy they were using. results: there were af patients. sixty-three percent (n = ) were males and % (n = ) were females (ratio . : ). twentythree percent (n = ) of patients were aged \ years, % (n = ) - years inclusive and % (n = ) =/[ years. two patients with chads score zero were on warfarin although one of them had cha ds vasc score of one. sixty-percent (n = ) were on warfarin alone, % (n = ) aspirin alone, % (n = ) warfarin plus aspirin, % (n = ) aspirin plus clopidogrel and % (n = ) on warfarin plus clopidogrel. seven patients were not on warfarin for various reasons. the has-bled score revealed patients at low risk, moderate risk and at high risk of bleeding. implications: ninety-three percent of patients were correctly managed and two patients were on warfarin with chads scores of zero. the use of evidence based management guidelines is necessary to manage patients. keywords: atrial fibrillation, chads score, cha ds vasc score, has-bled score meckel's diverticulum is the most common congenital abnormality of the gastrointestinal tract. only % of meckel's diverticulum are symptomatic [ ] . it can cause complications such as ulceration, obstruction, intussusception, haemorrhage and perforation and these complications are more common in the paediatric age group. a -year-old has a lifetime risk of . of developing a complication, this falls to zero over time [ ] . adults most commonly present with bleeding [ ] . we have a case of a -year-old male who presented with a day history of abdominal pain, constipation and anorexia. on examination he had rif tenderness, but no signs of peritonism. a provisional diagnosis of appendicitis was made. the patient was taken to theatre the next morning for laparoscopy and appendicectomy. the appendix was normal and surgery proceeded to laparotomy. an inflamed and perforated meckel's diverticulum was found. a terminal ileum resection with side to side anastomosis was performed. the patient made an uneventful recovery and was discharged to opd follow up. this case illustrates the importance of further evaluation following normal laparoscopy in the case of the ill patient. references: neonatal graves disease is a rare condition, caused by transplacental transfer of thyroid stimulating antibodies from mother to fetus. . % of pregnant women have graves disease and . % of their offspring will have overt hyperthyroidism. a further % will have biochemical thyrotoxicosis without symptoms. this is the case of a baby girl with neonatal graves disease. her antenatal course was uncomplicated until weeks gestation. at this point, her mother became clinically thyrotoxic. maternal blood tests showed an elevated free thyroxine level ( pmol/l) and positive thyroid receptor antibodies. a diagnosis of graves disease was made. she was commenced on treatment but remained thyrotoxic at the time of delivery. the baby was healthy at birth. however, thyroid function tests on day of life showed an elevated free thyroxine ( pmol/l) and thyroid receptor antibodies were positive. clinically, she remained asymptomatic and examination was normal. treatment with carbimazole was commenced and the dose titrated to maintain her euthyroid. most neonates affected by neonatal graves disease will have biochemical thyrotoxicosis but are clinically asymptomatic. the minority will be severely affected with goitre, eye signs, weight loss, tachycardia, arrhythmias and heart failure. it is a transient disorder, limited by clearance of maternal thyroid receptor antibodies and is usually self-limiting over - weeks. mortality rates of up to % are reported in untreated cases, usually from arrhythmias and heart failure. this case emphasises the importance of close monitoring of pregnant women with a history of thyroid disorders, before and during their pregnancy, as well as monitoring their babies in the neonatal period. fibreoptic bronchoscopy is considered a safe diagnostic tool [ ] . it is suggested however that post-bronchoscopy complication rate increases with age [ ] . we decided to study the complication rate and the outcomes of bronchoscopy in patients over the age of years in our institution. a retrospective review of the case notes of patients aged greater than years who underwent bronchoscopy between september and november was carried out. data on complications experienced during and after bronchoscopy and the influence of the results on subsequent management of patients were collated and analysed. ninety-six patients were included. the mean age was . years (sd . ). thirty subjects ( . %) had a documented lung disease. fifty-nine patients ( . %) were current or ex-smokers. indications for bronchoscopy were; to evaluate for malignancy ( . %) and to evaluate for tb ( . %). post bronchoscopy complications were noted in eight ( . %) cases including hypoxia ( . %), infection ( . %), tachycardia ( %) haemoptysis ( %) and pneumothorax ( %). six patients required treatment including nebulised bronchodilators ( . %), antibiotics ( . %), and oxygen therapy ( . %). malignancy was diagnosed in twenty cases ( . %). clinically significant pathogens were detected in six cases ( . %). as a result of bronchoscopy fourteen patients ( . %) had alterations to their drug therapy, three ( . %) received lung cancer treatment with curative intent, eighteen ( . %) had palliative care input, seventeen ( . %) were referred for further investigation and thirty-seven ( . %) had no change to their management. in conclusion, bronchoscopy is relatively safe and has good diagnostic utility in patients aged more than years. patient records were identified from a database of patients who underwent a spinal mri to investigate spinal metastatic disease between november and april . an analysis of the management of those diagnosed with mscc, specifically radiotherapy and/or surgical intervention was performed. three hundred and sixtyone patient records were identified with one hundred and seventy-one patients having metastatic spinal column disease. of these, thirty-four had mri evidence of metastatic spinal cord compression. radiotherapy alone was the most common therapy employed for patients with mscc. a multidisciplinary team approach was not taken in the majority of cases. a surgical opinion was sought in the minority of cases. this is not congruous with nice guidelines as a management protocol. the complexity of management decisions for metastatic spinal cord compression demands a multi-disciplinary approach. current practise in this major supra-regional cancer centre does not routinely employ this approach. a surgical opinion is sought in the minority of cases. this reflects the national trend with some centres having no spinal surgeons as staff. we recommend the establishment of a care pathway in order to comply with best evidence based practise as outlined by the nice guidelines. pet ct as a staging modality in primary cervical cancer; to establish the correlation between histological subtype and fdg- avidity of the primary lesion purpose: pet ct has become one of the mainstays of diagnostic imaging both in staging and prognosis of cervical cancer. we wanted to establish the link between fdg- uptake in the primary lesion and correlation with specific histological subtypes of cervical cancer including squamous cell carcinoma, adenocarcinoma and other rarer subtypes such as clear cell and adeno-squamous carcinoma. methods and materials: the main audit involved working out the fdg uptake in the primary lesions from the cervical cancer database of patients. the patient list was derived from a database of patients collated by the gynaecological services at sjh of all patients who received workup and treatment for cervical cancer from - . the computer system at sjh was employed for analysing pet-ct reports and histology reports. microsoft excel was used to store this information parameters and complete statistics on the data. results: the results of this study are to follow. conclusion: there is a correlation between fdg avidity and histological subtype of cervical cancer and this provides valuable information on the reliability of pet-ct findings in a specific cohort of patients with cervical cancer. we present the case of a -year-old male with a primary piriform fossa squamous cell carcinoma (scc) who attended for staging positron emission tomography/computerised tomography (pet/ ct) scan. distant to the primary lesion, focused f fluorodeoxyglucose (fdg) uptake was noted in the left iliac bone, without underlying abnormality on the accompanying ct scan. low grade uptake was also noted in subcentimetre upper mediastinal nodes, without any underlying lung parenchymal abnormality. these nodes were felt to be inflammatory or reactive in origin. though an unusual pattern for metastatic head and neck scc, the left iliac bone lesion was concerning for malignancy. thus, a percutaneous biopsy of this region was performed under image guidance. histology revealed non caseating epithelioid granulomata consistent with sarcoidosis. the patient was subsequently able to have potentially curative treatment of his head and neck primary. discussion: sarcoidosis is a chronic inflammatory multisystem condition characterised by the presence of non-caseating granulomas in affected organ tissues. it commonly affects young and middle aged adults with a slightly higher prevalence in women. the disease shows a predilection for adults under , peaking between and , with a second peak in women over [ ] . despite its unknown aetiology, it is felt that t lymphocytes play a central role in the development of sarcoidosis, as they likely propagate an excessive cellular immune reaction. it has been shown that abnormalities with the cd /cd ratio and production of t helper and (th /th ) cytokines such as interferon and tumour necrosis factor (tnf) are found in sites of disease activity [ ] . the importance of tnf in sarcoidosis is demonstrated by the efficacy of anti-tnf medications such as pentoxifylline and infliximab [ ] . it is estimated that bone lesions occur in - % of sarcoidosis patients [ ] . these figures are however based on radiographic data and are likely an underestimate as the majority of bone lesions would be asymptomatic [ ] . varying osseous manifestations of sarcoid have been described; punched out lytic lesions, lace-like destruction and subperiosteal resorption mimicking hyperparathyroidism. commonly, the small bones of the hands and feet (predominantly the middle and distal phalanges) are involved often bilaterally and symmetrically. while pulmonary involvement occurs in % of patients with sarcoidosis [ ] , bony involvement is rare without other clinical manifestations of the disorder [ ] . indeed our patient had low grade subcentimetre mediastinal nodes. the fdg avidity of sarcoid is a well documented phenomenon. indistinguishable from metastatic disease on f fdg pet scan alone it can lead to false-positive appearance of metastatic disease on pet/ ct. furthermore one-third of pet/ct positive sarcoidosis have osseous abnormalities on pet/ct the majority of which will not be evident on low dose ct [ ] . this case serves to remind us of the diagnostic limitations of f fdg pet in the differentiation of inflammatory and metastatic processes. in a patient with an unusual pattern of 'metastatic' disease tissue diagnosis is a necessity. distinct islet auto antibodies against antigens insulin, gad , ia and znt have been identified. the presence of autoantibodies has been shown to be predictive of reduced beta cell mass. international data suggests that - % of patients with newly diagnosed t dm are positive for at least one of the above antibodies. our aim is to study the prevalence of autoantibody positivity in our population of children with newly diagnosed t dm over a year period ( ) ( ) ( ) ( ) ( ) . details of all children newly diagnosed with t dm were collected using the endocrinology department database and chart review was undertaken. children diagnosed elsewhere whose care was transferred to our centre and children who had non type diabetes were excluded. one hundred and thirty-six children were diagnosed with t dm in our centre, of which ( %) presented in diabetic ketoacidosis. age at diagnosis ranged between months and . years. the male to female ratio was : . other autoimmune conditions (coeliac disease, hypothyroidism, addison's) were present in %. ninety-six percent (n = ) were tested for one of the three antibodies. % were positive for at least one antibody, % positive for two, % positive for all three antibodies. the most common antibody found was anti gad ( %). positive autoantibodies are helpful in confirming the presence of t dm and their absence in raising the possibility of monogenic diabetes. the absence of pancreatic islet autoantibodies at diagnosis can be predictive for maintained beta cell function during the years after diagnosis. maternal obesity, based on a body mass index (bmi)[ . kg/m , is associated with increased pregnancy complications. moderate exercise during pregnancy is associated with decreased complications such as pre-eclampsia [ ] and gestational diabetes mellitus [ ] and has a beneficial effect on mood with those who exercise experiencing fewer symptoms of depression and anxiety both during and after pregnancy [ ] . the purpose of this study was to determine if obese women exercise less during pregnancy. we recruited women at their convenience after a routine scan confirmed an early ongoing pregnancy. maternal height and weight were measured accurately and bmi calculated. women completed the international physical activity questionnaire. of the studied in early pregnancy, . % took no exercise, . % walked only, . % undertook moderate exercise and . % undertook vigorous exercise. of the obese women (n = ), only % reported moderate-vigorous exercise in early pregnancy compared with . % in women from the normal bmi category (n = ). also % of the obese group reported doing no exercise compared with % of those with a normal bmi. women with a bmi of or more were found to sit for an average of min per day whereas those with a normal bmi sit for min per day on average. although bmi increases with age and parity, these variables were not found to influence exercise levels in early pregnancy. exercise may be physically challenging in obese women, particularly if morbidly obese, but due to its beneficial effects it should be encouraged antenatally in all pregnant women irrespective of their bmi category. references: angiogram showed an absence of coronary artery disease and echocardiogram ruled out structural abnormality. exercise stress test showed short runs of vt in recovery. further tests included ajmaline and adrenaline challenges. cardiac mri showed right ventricular outflow tract scarring consistent with either a primary diagnosis of arvc or secondary with that of myocarditis. sarcoidosis was outruled by further laboratory and radiological means. non-sustained runs of vt on telemetry were noted and a dual chamber implantable cardiac defibrillator was placed. on discharge, medication included atenolol mg daily and patient will undergo genetic screening. follow up for the siblings included phenotyping and mri. discussion: history, presentation and pathology uncovered are consistent with a diagnosis of arvc. suspected paternal inheritance of an autosomal dominant genetic defect predisposed to the ventricular arrhythmias which at first, manifested as self-limiting palpitations however, later caused a near fatal event. long term management may include cardiac transplantation. prevalence of diagnosed atrial fibrillation in adults: national implications of rhythm management and stroke prevention: the anticoagulation and risk factors in atrial fibrillation (atria) study complications of fiberoptic bronchoscopy at a university hospital the relationship between age and process of care and patient tolerance of bronchoscopy central skeletal sarcoidosis mimicking metastatic disease sarcoidosis is a th /th multisystem disorder osseous sarcoidosis treated with tumour necrosis factor-inhibitors: case report and review of literature. spine (phila pa ) musculoskeletal manifestations of sarcoidosis multiple atypical bone involvement in sarcoidosis imaging in sarcoidosis. semin respir crit carre med f- fdg pet/ct for detecting bane and bone marrow involvement in sarcoidosis patients poster diaphragmatic rupture: delayed diagnosis and its consequences-a case report diaphragmatic rupture: a frequently missed injury in blunt thoracoabdominal trauma patients diaphragmatic rupture due to blunt trauma: sensitivity of plain chest radiographs the introduction: lymphoscintigraphy has been shown to be accurate in identifying sites of potential nodal metastases in melanoma patients. recent guidelines published by the eortc-eanm have defined specific criteria with relation to performing lymphoscintigraphy in melanoma patients. methods: the aim of this study was to audit all patients with malignant melanoma who underwent sentinel lymph node biopsy (slnbx) and lymphoscintigraphy in university college hospital galway between - . results were compared with eortc-eanm recommendations. results: melanoma patients underwent slnbx during the study period. patients had preoperative lymphoscintigraphy using intradermal injections of technetium m. sentinel nodes were identified in of patients ( . %) on lymphoscintigraphy. . % of lymphoscintigrams were reported on the same day as the procedure, . % after day and . % greater than day postop. obligatory imaging, as defined in the eortc-eanm guidelines, was obtained in % of all patients undergoing lymphoscintigraphy. no nodal uptake was reported in patients, of whom received imaging in accordance with the guidelines. the location of those melanomas with no nodal uptake was . % on the head and neck, and . % on the trunk. the overall rate of false-negative lymphatic mapping and sentinel node biopsy was . %. in patients receiving lymphoscintigraphy the false negative rate was . versus . % in patients who did not have lymphoscintigraphy. conclusion: preoperative lymphoscintigraphy is an essential adjunct in identifying the sentinel lymph node in clinically node negative melanoma patients and should adhere to eortc-eanm guidelines. conflict of interest: none. on examination, she was alert, hr , bp / . she was tachypnoeic, but reported this to be her baseline. there was a palpable, non-reducible mass in the left upper quadrant. a chest x-ray showed loops of bowel above the diaphragm. ultrasound showed an abscess in the rectus sheath, which drained mucopurulent fluid.mb opted not to have the diaphragm repaired, despite medical advice. she was readmitted weeks later with a recurrence of the abscess. her clinical condition deteriorated, with severe abdominal pain, and oxygen saturations of %. an emergency laparotomy was performed, which showed an obstructing lesion in the descending colon, with large and small bowel above the diaphragm. she had an extended right hemicolectomy, with restoration of bowel to the abdominal cavity and mesh repair of the diaphragm. histology showed an descending colon adenocarcinoma, t n m .traumatic diaphragmatic rupture is a rare problem, occurring in - % of blunt and penetrating traumas. ( ) plain films and ct scans are not always diagnostic in the acute phase, due to concomitant injuries. ( ) repair is essential once diagnosis has been reached to avoid herniation of abdominal viscera. patients with ongoing dyspnoea after blunt trauma may benefit from a repeat chest x-ray. a -year-old retired veterinary surgeon was referred to tertiary referral with a months history of a painless enlarging neck mass. clinical examination showed a right side neck mass approximately cm cm in size which extended through both anterior and posterior triangles. cervical lymphadenopathy was not appreciated and the patient was clinically euthyroid. patient was admitted under the care of the maxillofacial service, where he underwent a needle core biopsy of the neck mass. this was returned showing poorly differentiated spindle cell tumour with large pleomorphic nuclei and abundant abnormal mitoses. the immunoprofile was consistent with metastatic poorly differentiated sarcomatoid carcinoma and the differential diagnosis included origin fro the kidney, lung or thyroid.the case was discussed at the head and neck mdm and a consensus was reached that the patient as developed a sarcoma of the neck, with a level neck dissection the most appropriated intervention.intraoperatively, following the removal of the neck mass it was noted that the right lobe of the thyroid was enlarged. an intra-op fna was performed on the mass in the right lobe of the thyroid. the fna was returned showing bizarre giant cells, suggestive of malignancy. ultimately the patient underwent a total thyroidectomy but, despite surgery the patient died weeks post-operatively. using feedback from the pilot study and analysis of the preexamination consultant and registrar-led teaching schedule for students a further 'intern-led' tutorial timetable was structured. it allowed for a weekly maximum of h of teaching dependent on demand and intern availability. programme duration was weeks, january to march . group sizes were a maximum of students. tutorials were all at the patient bedside. feedback forms were distributed at the end of the programme.sixty-four tutorials were given in total. seventy feedback forms were returned. mean number of tutorials attended per student was . . students rated statements - ( -strongly disagree, -disagree, -neutral, -agree, -strongly agree). median scores were used. scoring showed improvements were made from last year in terms of level of intern preparation for tutorials and importantly, the students own subjective view of their level of preparation for forthcoming examinations. most importantly, students agreed that tutorials improved their history taking skills and strongly agreed that their examination skills improved. matching feedback from the pilot study, students strongly agreed that intern-led teaching is an appropriate adjunct to the final year programme.of the intern working in st james's hospital, participated. seventeen of these had received tutorials on the pilot programme. of the that did not participate, many had never received formal intern teaching.the feedback obtained from the pilot study was invaluable in organising and delivering this teaching programme. ongoing improvements will be made for next year based on this audit. this also highlights that the intern-teaching tool is extremely beneficial, yet largely underused. key: cord- -mygj nd authors: nan title: proceedings of the nd annual meeting of the american rheumatism association a section of the arthritis foundation june & , new york city abstracts of papers presented date: - - journal: arthritis rheum doi: . /art. sha: doc_id: cord_uid: mygj nd nan azathioprine has been shown t o be an effective agent in the treatment of active rheumatoid arthritis (ra); however, potential long-term neoplastic effects remain a serious concern. to clarify this relationship, patients with classic or definite ra being treated with azathioprine (group ) were chosen at random and studied by means of clinical, laboratory, and radiologic measures to detect potential early markers of neoplastic disease. nineteen age matched patients with ra were concurrently similarly studied as controls (group ). group had a longer disease duration ( . versus . years). the mean duration of treatment with azathioprine was . months, with a mean dose of . mg/day. histories of cigarette smoking, hormone treatment, and fertility were comparable. chest x-rays revealed comparable numbers of inflammatory and fibrotic abnormalities. there was an increased frequency of tumors in first degree relatives in group i ( versus ). hematologic studies revealed a trend to lower cell counts in group with a striking increase of marrow megaloblastosis in this group ( versus i ) . there were no differences in iron saturation or b levels. serum folate was decreased in group i ( versus ). there were neither liver function test abnormalities in group , nor evidence of malignancy in gastrointestinal x-rays when clinically indicated. no neoplastic lesions were identified on mammography in either group. four patients in group and none in group had atypical or dyskaryotic cells on urine cytology. one patient in group had an abnormal pap smear and was subsequently shown to have endometrial carcinoma, on chromosomal analysis, group i showed a greater percent of cells with aberrations, a higher aberration incidence and index, and more patients with hyperdiploidy. patients in group had a history of benign neoplasms. patients in group i had a history of i benign neoplasm prior to the initiation of azathioprine; subsequently neoplasms were diagnosed, benign and malignant ( endometrial carcinomas and carcinoma-in-situ of the cervix). in summary, group i had an increased number of cellular abnormalities, with cytologic atypia, megaloblastic bone marrows, and increased chromosomal aberrations. clinically this group had more tumors diagnosed while on treatment, particularly in females, and arising from the genitourinary tract. more long-term studies of patients on cytotoxic agents are clearly warranted. solid phase clq (sclq) radioimmunoassays. c was determined by radial immunodiffusion and antibodies to dna by binding of radiolabeled dna by the millipore filter technique. these assays were correlated with each other, general presence or absence of symptomatic disease, active renal disease, and active joint disease. to evaluate predictive capabilities, a change in cic result was correlated with a change in disease activity. change in disease activity was defined as an sle related event requiring hospitalization, a change in symptoms requiring an increase in or addition of steroids or immunosuppressive agents, or a marked improvement in symptoms such that drugs were tapered or discontinued. the fclq results did not correlate with any parameters of disease activity. a fall in c or an increase in antibodies to dna usually signaled a change in disease activity; however, many patients experienced a change in disease activity without any change in c or dna binding. the sclq assay correlated with symptoms of sle in general (p < . ), with active renal disease (p < . ), and with active arthritis (p < . ). an appropriate change in sclq determination correlated best with a significant change in disease activity (p < . ). we conclude that the sclq method of determining cic correlates best with active clinical manifestations of sle and is a useful predictor of disease activity. we have recently found that certain rheumatoid factors (rfs) cross react with dna-protein (dnp) . this type of cross reacting r f is frequently present in seropositive rheumatoid arthritis (ra), of patients, and also in order r f positive patients: rheumatoid overlap syndrome / , mixed connective tissue disease (mctd) / , other diseases / , thus far rfs reactive with dnp have not been found in systemic lupus erythernatosus (sle) / or essential mixed cryoglobulinemia / . rfs which react with dnp can give positive antinuclear antibody (ana) and le tests which can be blocked by aggregated igg or dnp. in contrast, positive ana and le tests in sle are not blocked by aggregated igg. hence rf induced ana and le tests may be considered "false" positives when these tests are used in the diagnosis of sle. in some sera these rfs could be detected only following isolation with insoluble igg or dnp, suggesting the presence of rfs complexed with antigen. by use of a monoclonal r f which cross reacts with dnf in a sensitive competitive inhibition radioimmunoassay designed for detection of immune complexes (jci : , ) , complexes larger than s by ultracentrifugation studies could be detected in several ra sera and synovial fluid and mctd sera. in some studies dnase treatment of these complexes resulted in a decrease in size. these studies suggest that complexes consisting of dnp antigen and r f may be present in the circulation in ra and mctd. the relationship, if any, of these complexes to the immunopathology in these diseases is currently under study. these studies also suggest that not all rfs may be induced by immune aggregated igg and that a re-exploration of rfs in various diseases for cross reactions with non-igc antigens is indicated, such antigen may provide clues to etiologic factors in these diseases. previous studies demonstrated that patients with rheumatoid arthritis (ra) frequently have an antibody referred to as rheumatoid arthritis precipitin (rap). this antibody was shown by the indirect immunofluorescent technique to react with a nuclear protein in human, b lymphocytes from continuous cell culture (wil,) and the antigen was termed ra associated nuclear antigen (rana) . infectivity experiments with epstein-barr virus (ebv) or other herpes viruses have suggested that rana may be associated with ebv infection; although rana shares some characteristics with eb nuclear antigen, they are probably two different antigens. in this study we have expanded our work on rana. in addition to its presence in wil, cells, it has now been found in other human, b lvmphocyte lines but was not present in t lymphocyte lines. rana was not found in extracts from normal spleen, kidney, heart, or lungs, or a spleen extract from a patient with felty's ra. this antigen was not present in normal lymphocytes stimulated with b or t cell mitogens. rana was not found in peripheral leukocytes from patients with ra. however, it was found in of extracts made from pannus layers and an extract made from nodules taken from patients with seropositive ra but was not present in extracts made from pannus layers taken from seronegative ra patients and synovium from a traumatized joint. initial physicobiochemical characterization of rana demonstrated that it was stable to heat ( oc or oc) or cold ( o to - ooc) treatments or at ph values of ph . - . . studies performed by immunoelectrophoresis indicated that rana was an anion at ph . - . . will extracts were separated on a calibrated % agarose column and the molecular weight estimated at , d. in other studies the antigen could not be detected on the surface of wil, cells. immunodiffusion studies using burkitt's lymphoma sera ( ) or nasopharyngeal carcinoma sera ( ) containing high titers of ebv antibodies to: viral capsid antigen or early antigens d or r do not react with wil, extract, suggesting these ebv antigens are not present in the extract and therefore cannot be rana. the data suggest that rana may be a new, heat stable, acidic, nuclear protein associated with ebv infection which could be important in the pathogenesis of ra. after medical workup, arthroscopy was performed on patients ( : , men: women; ages - ) with painful knees of unknown cause. arthroscopy of the knee was achieved on ambulant patients under sterile technique in an outpatient minor surgery suite utilizing a needlescope or watanabe arthroscope. biopsies were guided by visualization. postarthroscopy diagnoses are reflected in the table. in most cases the procedure allowed adequate visualization of the suprapatellar sac, articular cartilage, menisci, anterior cruciate, and synovial lining. no arthroscopic abnormality was visualized in %. nearly half of the patients had osteoarthritis including patients with chondromalacia patella. the group with osteoarthritis also included patients who had at least one torn meniscus. septic arthritis was confirmed in % by synovial biopsy including one case of tuberculosis. previous undiagnosed synovitis was confirmed but not further defined in patients. four biopsies suggested rheumatoid synovitis; the diagnosis was confirmed by subsequent clinical and laboratory course. results of arthrography ( ) were confirmed in , contradicted in , and provided additional findings in i. patients were ambulant after the procedure with minimal morbidity- hemarthroses and an acute pseudogout, all treated by arthrocentesis. the gainful knee often presents a diagnostic enigma particularly when degree of disability is a question. ar-throscopy is a safe minor surgery procedure that may reveal the underlying cause of pain and preclude the need for arthrotomy. diagnosis computer assisted tomography of the spine and/or roentgenographic transaxial lumbar tomography were performed on patients (age iz sd years) with back pain and concomitant paget's disease. clinical assessment with routine lumbosacral x-rays was performed by a rheumatologist and an orthopedic surgeon. both tomographic methods are xray techniques that transect the spinal canal and the intervertebral foramina producing a computer printout or laminogram along the axial plane of the spine. the following was found: . spinal stenosis: spondylotic osteophytes encroaching on the spinal canal in patients. three had a history of pseudointermittent claudication. lumbar x-rays supported a diagnosis of paget's disease at the involved site in . . lateral recess syndrome: spondylotic osteophytes encroaching on the intervertebral foramina in patients. all had sclerotogenous pain (nonspecific pain referred to the buttocks, groin, and/or thighs). lumbar x-rays supported a diagnosis of paget's disease at the involved site in . . pagetic pain: increased thickness of the laminae, pedicles, or bodies of the vertebrae secondary to paget's disease in subjects without alteration of the spinal canal. . spondylotic changes of articular facets without stenosis or lateral recess in ; had adjacent pagetic changes. in the assessment of back pain, axial tomography proved to be a useful tool in defining anatomic spinal lesions that were not appreciated by routine x-ray techniques. the methods were particularly helpful in outlining the zygapophyseal articulation for definition of radiographic joint space and hypertrophic changes. neural entrapment by bone due to hy-pertrophic changes was similarly defined. in several cases the procedure obviated the need for myelography. in paget's disease, determining the degree of spondj osis could guide therapy since it would predict those that might be expected to respond to suppressive therapy of their paget's disease. hla-b is a potent genetic marker for ankylosing spondylitis (as), reiter's syndrome (rs), and other spondylitic variants. this cell surface antigen is determined by the hla-b locus, one of a linear array of genes clustered on the sixth chromosome. whether hla-b is the disease-conferring gene or only linked to another nearby gene remains unclear. studies of hla-d on one side of hla-b ( map unit) have shown no associations with as or b . the hla-c locus lines . map units to the opposite side of hla-b. it was our purpose to study c locus antigens in b positive and negative patients in order to more precisely localize the disease promoting gene. eighty-eight patients ( as, rs, sacroiliitis, psoriatic spondylitis, and colitic spondylitis) were hla cwl, p < . for cw , and p < . for either cwl and cw . similar significant differences were found when both the b + patients and the b + controls were compared with the matched controls. there were no significant differences noted between b + patients and b +controls or between b patients and matched controls. the occurrence of cw and cw was similar within all groups. thus, these data confirm strong linkage disequilibrium between b , cwl, and cw which does not relate to disease. most importantly, the absence of cwl and cw in b patients provides further evidence that recombination between b and c loci is unlikely, and that disease susceptibility is intimately related to or inseparable from the b locus. control groups (n= ), % (n= ), % (n= ), % (n= ),% typed for a, b, and the c locus antigens, cwl, cw , cw , and cw . in addition, age, sex, and race matched normal controls and b positive normal controls were similarly studied and compared to those with disease. hla-b was present results of c locus typing are shown in the table. an immunofluorescent complement fixation (ifcf) test was used to determine if nuclear antigen-antibody complexes of different specificities possessed different capacities to fix complement. sera from patients with rheumatic diseases were specially selected and demonstrated to contain only antibodies to sm antigen ( patients), nuclear rnp ( ), ss-b antigen ( ), or nuclear histones ( ). column chromatography isolated-igg from these sera were reacted with sections of mouse liver to form nuclear ag-ab complexes of the different specificities described above. binding of complement components to the nuclear ag-ab complexes was determined after washing away excess serum and incubating with different sources of complement: ) normal human serum (nhs); ) nhs-egta mg, conditions which are known to block activation of the classic pathway; ) immunologically clq-depleted sera in which other complement proteins were demonstrated to be % hemolytically active. fixation of complement was determined by using fluorescein-conjugated antiserum to clq, c , properdin, factor b, c , c , and c . all sera with antibodies to sm antigen, nuclear rnp, and ss-b nuclear antigens showed fixation of all complement proteins when nhs was used as the source of complement. when the source of complement was egta-mg or clq-depleted serum, clq and c were negative by ifcf. however, complement proteins properdin, factor b, c , c , and c were positive, showing that these nuclear antigen-antibody complexes were able to fix complement proteins of the alternative pathway, without concomitant fixation of the classic pathway. the sera with antibodies to nuclear histones did not fix any complement component of either the classic or alternative pathways. these studies demonstrate two important findings. bodies of certain specificities to activate complement may have certain nuclear ag-ab complexes are capable of activating the relationship to their capacities to cause tissue damage by realternative complement pathway, independent of classic path-lease of inflammatory peptides mediated by complement actiway activation. reactions between nuclear histones and anti-vation. our results may explain why drug-induced lupus erbody, as demonstrated by the immunofluorescent technique, ythematosus, characterized by anti-histone ab, is rarely appear not to be complement-fixing reactions or at least not associated with inflammatory kidney disease. detectable by this technique. the capacity of antinuclear anti- it has been suggested that renal involvement in sle patients may depend upon qualitative immunochemical characteristics of antibodies to dna, e.g., precipitating ability, affinity, ig class or subclass, and complement fixing (cf) capacity. we used the immunofluorescent crithidia luciliae method [a-dna(cl)] to delineate some of these characteristics in sera from patients with active sle. using fitclabeled polyspecific antiserum to igg, iga, and igm, a-dna(cl) was found in of patients with active lupus nephritis (gp-i) and of patients with lupus activity without nephritis . median titer in gp-i was : and in gp-i : ; the difference in titers between these groups was significant (p < . ). c f antibodies to dna were found in patients in g p i and patients in gp-i using a double sandwich technique to detect c binding. when titer of c f activity was compared with a-dna(cl) titer, a strong correlation was found (r = . ; p < . ). igg, iga, and igm a-dna(cl) were detected with high frequency in both groups. in addition, we compared titers of a-dna(cl), which detects antibodies primarily directed against dsdna determi-nants, with dna binding capacity detected by the millipore filter (mf) method of ginsberg and keiser, in each of these groups. as antigen we employed * -labeled calf thymus dna (type i, sigma) passed through an . p, type ha millipore filter. in contrast to the cl method, levels of a-dna(mf) did not differ significantly in gps i and . when we compared a-dna(cl) titer in gp-i sera with quantitative a-dna(mf), a highly significant correlation was found (r = . , p < . ), while no correlation between these methods was evident in sera from gp-i patients. these findings suggest that: ) the presence of high titered a-dna(cl) is strongly correlated with active renal lupus; ) detection of c-fixing a-dna(cl) may reflect total amount of antibody; demonstration of c-fixing ability may generally be expected in high titered sera; ) antibodies to at least sets of constituents of the dna preparation employed in the mf method can be detected, one occurring in active lupus nephritis and correlated with antibodies detected by cl, and one in patients with active lupus without nephritis, detected minimally or not at all by the cl method. cell mediated cytotoxicity is an important pathogenic mechanism in certain rheumatic diseases. recent studies from this laboratory have demonstrated that adherent monocytes are potent killers of antibody coated (adcc) nonerythroid target cells and also kill uncoated target cells to a lesser extent. although immune complexes can inhibit lymphocyte cytotoxicity, their effect on monocyte cytotoxicity is unknown. this study compares human monocytes adcc and natural killer (nk) activities with that of lymphocytes. the effects of heat aggregated igg ( . - mg per ml) and preformed soluble keyhole limpet hemocyanin (klh)/anti-klh complexes were evaluated. monocytes and lymphocytes were separated by their differential adherence properties in plastic microtiter wells and were at least % pure. cytotoxicity was measured by "chromium released from antibody coated (adcc) and nonsensitized (nk) k cells, an established myeloid cell line. precipitated klh/anti-klh complexes formed at equivalence were the strongest inhibitors of cell mediated cytotoxicity (suppression: monocyte nk / , monocyte adcc %, lymphocyte nk o%, lymphocyte adcc %). aggregated igg also strongly inhibited both monocyte and lymphocyte nk ( % and % respectively) and also suppressed adcc to a lesser extent (monocyte %, lymphocyte %). klh/anti-klh in antigen excess had no effect on monocyte nk and suppressed lymphocyte nk by only %. although soluble complexes did not block monocyte nk, they did significantly block monocyte adcc ( %). by use of serial dilutions of complexes in aggregated igg, a dose dependent relationship was established between complexes and percent suppression of cytotoxicity. these studies reveal that both klh/anti-klh immune complexes and aggregated igg can inhibit monocyte cytotoxic activity and that, in general, these effects correlated with lymphocyte nk and adcc. significant differences between soluble immune complexes and aggregated igg were demonstrated. finally, the finding that certain immune complexes inhibit both nk and adcc would imply that fc receptors are required for both cytotoxic responses. the relatively greater inhibitory effect on nk would imply that antibody fixed to target cells potentiates attachment by the effector cell and renders it less susceptible to inhibition by immune complexes. gout is easily diagnosed from a classic clinical history and the demonstration of urate crystals in synovial fluid and/ or tophi; however the diagnosis may not be suspected in patients with an atypical history or physical examination. to ascertain the prevalence of atypical findings we are conducting a prospective study in our gouty population randomly admitted to the clinical research unit. we report the following findings in the first patients with crystal proved gout: ) % had saturnine gout " to moonshine consumption, % gave a history of heavy ethanol consumption but did not have lead intoxication, and % were obese. all patients were male, % black, % white and % american indian. the average age at the time of study was . years with a mean duration of gout of years. seventythree percent had evidence of liver disease (elevated sgot or hepatomegaly), % were hypertensive, % had elevated triglycerides, and % had abnormal glucose tolerance. ) twenty-seven percent had a polyarticular onset of gout (> joint); % subsequently had polyarticular attacks. seventythree percent had tophi at the time of the study. chronic synovitis was present in % of patients. in the patients examined the following joints of the upper extremity were involved; % m p and/or ip, % wrist, ' elbow, % shoulder. in the lower extremity the knee was involved in %, m p joints in %, and ankles in %. ) a positive latex fixation test was observed in % of patients; being > : and > : . ra latex was present in % of patients with tophi and % of patients without tophaceous deposits. in the patients with chronic synovitis % had a +ra latex, and % free of chronic synovitis were also positive. the mean age in patients with a positive test was . years and . years for those who were negative. in contrast to these negative associations, evidence of liver disease was correlated with the finding of a +ra latex. fifty percent of patients with liver disease had a +ra latex whereas it was absent in all without liver disease. we conclude that chronic synovitis and polyarticular arthritis are common in our gouty patients. in addition, rheumatoid factor is found in a significant proportion of gouty patients and it does not seem to correlate with age, tophi, or chronic synovitis. ra latex seems to correlate best with the presence of liver disease which in most instances was a concomitant of chronic ethanol abuse. a polysaccharide iden tical to a proprionibacterium acnes polysaccharide (pps) has been isolated from hyaluronidase treated rheumatoid synovial fluids and synovial leukocyte pellets using a phenol-water (westphal) extraction and sepharose b column chromatography. pps was identified by counterimmunoelectrophoresis (cie) by rabbit antibody to sonicated p acnes organisms. sensitivity by this method is approximately . ng of pps and identity of synovial and bacterial pps was shown by cie. of common aerobic and anaerobic bacteria tested, only ps aeruginosa contained a similar antigen. pps is not present in e coli or s typhosa lipopolysaccharide. pps could not be identified in synovial fluid without extraction procedures even when a more sensitive radioimmunoassay with lzsi labeled rabbit antibody was used. sixty percent of rheumatoid fluids and % of rheuma-toid synovial leukocyte pellets contained pps. only of ( %) nonrheumatoid fluids was positive. two of ( %) nonrheumatoid inflammatory synovial leukocyte pellets contained pps. antibody to pps by cie was found in % of rheumatoid sera ( p < . ), % of nonrheumatoid inflammatory arthritic sera ( p < . ), and % of normal control sera. characteristics of pps (bacterial and synovial) include sensitivity to mm periodate oxidation, resistance to rnase, dnase, pronase, and boiling. uronic acid and hexosamine account for approximately % by weight. less than . % protein is present. it appears polydisperse on sephadex g with a m w of approximately x ' to >lp. electrophoresis in . % polyacrylamide showed a single pas positive band which formed a preciptin line with anti-p acnes antibody by crossed cie. no lipid staining bands were noted. these findings are consistent with a polysaccharide antigen. the isolation of a bound antigenic bacterial polysaccharide primarily from rheumatoid effusions associated with diminished antibody suggests a pathogenetic role for pps in rheumatoid arthritis possibly as an immune complex type. an in vitro system for examining the cellular requirements necessary for initiating and regulating antinucleic acid antibody responses has been developed to facilitate a better understanding of the mechanisms responsible for triggering antibody responses to this group of antigens in murine and human sle. it has been found that spontaneously appearing anti-ssdna hemolytic plaque forming cells (pfc) can be detected when spleen cells from normal mice of different hz types (cba, dba, bdf , cfw, c /b ) or young ( weeks) autoimmune (nzb x nzw f (b/w), nzb, nzw) strains of mice are cultured under standard mishell dutton conditions in the absence of added antigen. such pfc( - /culture) which peak on day and cannot be detected prior to culture consist entirely of specific igm anti-ssdna antibody. the response, which appears to be dependent on active antibody synthesis, can be selectively abolished by specific removal of ssdna antigen binding cells, by rosette depletion. while low responses can be augmented by pokeweed mitogen, there is no obligatory requirement of serum, thymic (t) cells, or macrophages in the culture system. this in vitro response can blocked by: ) adding small quantities of ssdna (lo- opg) but not similar concentrations of dna or rna directly to culture on day ; ) pre-incubation of spleen cells with ssdna ( hour) and washing prior to culture. unblocking can be demonstrated by subsequently treating these cells with trypsin-dnase, suggesting that this inhibition may be due to a membrane receptor blockade mechanism; ) the addition to young b/w spleen cells of t cells from young ( weeks) or old (> months) syngeneic mice similarly abrogates this in vitro anti-ssdna response without effecting cell viability or the anti-srbc antibody response. on the other hand syngeneic bone marrow cells which markedly suppress the anti-srbc antibody response have no effect on the anti-ssdna response of these spleen cells. these in vitro studies suggest mechanisms that may be relevant to the normal regulation of anti-nucleic acid antibody and other autoantibody responses in vivo. the ability of old b/w t cells to exhibit inhibitory effects similar to those of young preautoimmune b/w mice on the in vitro antinucleic acid antibody response suggests that the suppressor potential of these old b/w t cells may be blocked in vivo. the ehlers-danlos syndrome (ed-s) is an uncommon, genetically determined disorder of connective tissue. of the seven clinically distinct types, a basic molecular defect has been reported in types iv, vi, and vii, all of which are inherited by a recessive mode, and appear to be related to deficiencies in the activity of specific enzymes involved in postribosomal changes. types i to i are inherited by a dominant mode and might be expected to have defects at the transcriptional level. type v is sex-linked and has been reported to result from a cross-link deficiency. we have studied patients with ed-s type i, patients with type , and with the x-linked type v. the results show that the reducible crosslinks are present and undergo the same maturation process to nonreducible cross-links as in normal skin. we were unable to confirm the absence of reducible cross-links in the x-linked type ed-s. the ability of the ed-s skin to produce apparently normal cross-linked collagen was supported by cell and tissue culture studies. transmission electron micr~scopy and optical birefringence revealed a normal ultrastructure of the collagen fibrils. at a higher morphological level of organization scanning electron microscopy demonstrated a gradual increase in fiber bundle disorder from the x-linked to the myasthenia gravis, where the fibers making up the large fiber bundles demonstrated a considerable inability to aggregate. this disorder could account for the prolonged lag phase in the stressstrain curves, while the presence of cross-links ensures that once stress is on the individual fibers the elasting modulus is normal and the skin can return to normal after hyperextension. the absence of cross-links would lead to an irreversible elongation of the fiber. the low breaking point of the fiber is therefore due to the lack of integrity of the fiber bundles and suggests that the defect lies at a higher order of organization. the cryoprecipitates (cryos) from many sle sera contain lymphocytotoxic antibody and, when used to immunize rabbits, induce antibodies to human lymphocytes, suggesting that they contain membrane fragments complexed to antibody. lymphocyte-reactive antibodies in sle sera also recognized antigens on neuronal cells. to determine whether the membrane fragments in the cryos have the antigenic determinants shared with neurons, antisera generated against sle cryos were tested in an antibody-dependent, cell-mediated cytotoxicity assay by use of cr-labeled human neuronal cells, sk-n-sh, as targets and normal human peripheral blood lymphocytes (pbl) as effectors. the anti-cryos produced . f . mean % "cr-release. two of the antisera were strongly positive at and % cytotoxicity, and others were modestly cytotoxic in the range of . to . %. the remaining antisera were not significantly different from the controls; . f . % ' -release from normal rabbit sera (nrs), and . f . % cytotoxicity from a control group consisting of non-sle anti-cryos. the antibody activity was not linked to sk-n-s h cells. the same sera reacted with human neuronal line, la-n- , and human glial lines, a-i and u-l mg. unexpectedly there was no correlation between reactivity against the neuronal cells and antibody activity against normal human pbl. antibody t o pbl was not detected in / anti-cryos with neuronal antibody, and of the antisera unreactive with sk-n-sh had anti-pbl activity. absorption of the anti-cryo reactive against both sk-n-sh and pbl with the human lymphoblast line wll removed all anti-pbl activity but not the neuronal reactivity. absorption with sk-n-sh eliminated the neuronal reactivity and also diminished the antibody to pbl. the antineuronal activity was not removed by absorptions with human platelets and human rbc. passage of the anticryos over immunoglobulin containing immunoabsorbents removed the detectable anti-immunoglobulin activity without diminishing the neuronal reactivity. thus, sle cryoproteins contain antigenic determinants shared by neuronal and glial cells but not by the major cellular elements in blood. it is not yet clear whether those antigens are present on fragments of neuronal membranes or on antigenic structures eliciting cross reactive antibodies. it is generally accepted that antibodies to double stranded (ds) dna are a feature of active sle, while antibodies to single stranded (ss) dna occur in many rheumatic diseases. the natural occurrence of antibodies to dsdna presumes an interaction with the sugar-phosphate backbone of dna, the immunogenicity of which is unproved. we have attempted t o define the preferential reaction site of dsdna antibodies on a molecular species of dsdna containing the multiple ss regions, herewith designated ds/ssdna. calf thymus dna was sheared to a molecular weight of x iw daltons. characterization by hydroxyapatite and benzyolated naphthoylated deae cellulose chromatography (j immunol : , ) indicated the dna was predominantly ds with numerous ss regions. antibodies to chromatographically pure dsdna (> % binding in a standard farr assay) were partially purified by ammonium sulfate precipitation, iodinated with lz i, and reacted with ds/ssdna. the resulting complexes were precipitated with . % polyethylene glycol (mw, ) and redissolved in . m tris-hci, ph . , . m mgci , . m nacl (the endonuclease buffer). the solubilized complexes were reacted with ss specific endonuclease from neurospora crassa ( . pg enzyme/ pg dna) and compared t o undigested complexes by velocity sedimentation in a % to % sucrose gradient run at ,ooog for i hours. undigested complexes sedimented at a position of s; after endonuclease digestion there was a dramatic decrease in the sedimentation velocity to a s position. these findings could be explained by: ) a disintegration of the dna into many smaller ds molecules still attached to labeled antibodies, ) a liberation of antibodies formerly attached to ss regions of the ds/ssdna, ) a displacement, by the endonuclease, of antibodies reactive with ss regions. to test these possibilities the procedure was repeated without labeling of the immunoglobulins, the resulting complexes were reacted with lz l dsdna in a standard farr assay. after endonuclease digestion free dna antibodies were readily detected, as shown by a -fold increase in dna binding, % to %. these findings indicate that dsdna antibodies preferentially react with ss portions of dsdna molecules. whether dsdna antibodies ever react with determinants unique to dsdna, or are merely a manifestation of high avidity binding to a very short ss region of the dsdna molecule, remains to be determined. t cell cytotoxic function in nzb mice has previously been tested utilizing target cells differing from nzb at the major histocompatibility complex (mhc) of mice, h- . in the experiments reported here, t cell cytotoxic functions of nzb mice against targets identical to nzb at the mhc were investigated. normal mice do not generate cytotoxicity under these circumstances. spleen cell suspensions of nzb mice (h- d) were cultivated for days in the presence of irradiated stimulator cells of other h- d strains (b .d , balb/c, dba/ , hw ). on the fifth day labeled target cells identical to the stimulating cells were added and the 'cr release from these targets was measured after hours. nzb effector cells exerted a highly significant specific lysis in all four of the h- d strains tested; none of these reacted against nzb. in experiments in which nzb cells were sensitized against one h- d strain (balb/c) target cells from all four h- d strains were lysed, demonstrating crossreactivity of the lytic reaction. the amount of lysis observed was dependent on the ratio of effector to target cells. the action of the effector cells was abolished by treatment with anti-thy- serum, indicating their t cell character. the capacity to respond against balb/c could be demonstrated in nzb mice as early as weeks and as late as months of age. the capacity of nzb mice to generate cytotoxic t-cells following primary in vitro sensitization against targets carrying the same major transplantation antigens as nzb establishes a qualitative difference between nzb mice and all normal strains since generation of cytotoxicity is restricted under the applied conditions to targets differing at the h- in all normal strains heretofore tested. it is suggested that the observed lack of restrictioq of the cytotoxic reaction in nzb mice is related to the autoimmune reactivity of this strain. immune deposits in the choroid plexus of patients with neuropsychiatric manifestations of systemic lupus erythematosus (sle) and nzb/nzw f, hybrid mice are thought to relate to the pathogenesis of central nervous system (cns) lupus. since previous studies did not include in their control groups patients with sle who had no neuropsychiatric manifestations, we looked for immune deposits in the choroid tissue of patients with and without cns lupus. fixed brain tissue containing choroid plexus was retrieved from sle patients and cont.rols. ependymal lining cells, choroid stroma, and cortical vessels were reviewed for deposits of igg, igm, iga, igd, ige, and kappa and lambda light chains by an immunoperoxidase technique (rabbit antihuman immunoglobulin and peroxidase-antiperoxidase). complement components were destroyed by fixation. immunoglobulins and light chains were found in the ependymal cells and/or choroid stroma of each sle patient. the pattern or intensity of immunoglobulin deposition did not distinguish those patients with and without neuropsychiatric manifestations. no staining was seen in tissue from the controls. though the choroid plexus serves a filtering function, the finding of immunoglobulin deposits in the choroid plexus cannot be correlated specifically with any of the diverse manifestations of cns lupus. other clinicopathologic correlations must be sought. multinucleate cells (heterokaryons) are frequently found in rheumatoid synovium as well as in cultures of isolated, adherent cells from this tissue. using an experimental polyethylene glycol (peg) was used to fuse cells in monolayer cultures of rabbit synovial fibroblasts. fusion began within minutes of peg treatment and continued for about hours; approximately % of the cells developed or more nuclei. in some experiments, up to % of fused cells contained or more nuclei, indicating true giant cell formation. measurement of intracelluar ( rubidium) to indicate the presence of a leaky cell membrane showed that immediately after peg treatment, intracellular ( rb+) dropped to % of control levels. it began to approach normal by minutes but did not recover fully for about hours, at completion of fusion. during the first hours after peg treatment, the treated cultures incorporated / as much ( h) thymidine as did control cultures, but incorporation of ( h) leucine into tca-precipitable radioactivity was unaffected. autoradiographic studies using ( h) thymidine revealed that peg depressed incorporation of the label into dna for at least days. secretion of proteinases from peg-treated and control cultures in serum-free dulbecco's modified eagle's medium was compared. peg-treated cultures containing multinucleate cells secreted u (cumulative) latent collagenase into medium changed every hours over days, compared with u produced by control cells during the same period ( u collagenase degraded kg collagen/hour at °c. collagenase was activated from its latent precursor form by tpck trypsin-i wg/ml, minutes, oc, followed by addition of excess soybean trypsin inhibitor.) neither peg-treated nor control cells released substantial or significantly different amounts of neutral or acid proteinases into medium during the same period. our data show that compared to controls, cultures of multinucleated cells have a decreased rate of cell replication and increased rate of collagenase production. multinucleate cells in rheumatoid synovium may amplify mechanisms for active collagenolysis. fourteen previously untreated polymyositis (pm) patients were treated with prednisone mg/day until cpk values normalized and then with mg/day for a total of weeks. they were also randomly placed on either azathioprine (a) mg/kg/day or placebo (p) in double-blinded manner. manual muscle testing, total prednisone dose required over weeks, and muscle biopsy changes were used to detect differences in the two groups. the a group ( patients) turned out to be weaker at onset (total manual muscle testing score - ) than the p group ( patients with score of - ) but improved more ( points to - ) than the p group ( points to - ). however, the difference in improvement was not statistically significant. cpks in the two groups normalized a t about the same rate and each group therefore received comparable doses of prednisone over the weeks. all followup muscle biopsies at the end of weeks showed improvement in the majority of the eight microscopic features reviewed and in total numerical score. the trend was toward greater improvement in the a group but not significantly so. changes consistent with steroid myopathy were noted in of women but only of men ( in a and in p) and of these patients were weaker at weeks. of special note is the observation that of patients with normal cpk at i weeks, still had significant inflammatory infiltrates. therefore, azathioprine plus prednisone is not dramatically better than prednisone alone in polymyositis over the initial weeks of treatment. the development of steroid myopathy in over one half the patients is not influenced by a or p and at the prednisone dosages used complicates the evaluation of changes in muscle strength; nevertheless, the trend is toward greater improvement in the azathioprine group in muscle strength and also follow-up muscle biopsy scores. surprisingly, a normal cpk does not indicate resolution of inflammation in the muscle and cannot be used reliably to indicate full control of the disease. followup is continuing. with the appreciation of the relationship between immunogenetics and infection in the pathogenesis of rs, interest in this condition is increasing. in order to determine more precisely the natural history of rs, data are presented from two distinct california communities: university (pa) and community (sb). to date, consecutive patients have been studied: (pa) and (sb). rs is defined as an asymmetric oligoarthropathy (predominantly lower extremity) accom-panied by one or more of the following symptoms: ) urethritis, ) diarrhea at onset, ) inflammatory eye disease, ) mucocutaneous manifestations consisting of balanitis, buccal ulceration, or keratodermia blennorrhagica. patients with ankylosing spondylitis, psoriatic arthropathy, or other rheumatic syndromes were excluded. nineteen ( %) of the presented with diarrhea and ( %) had at least a triad of the above features. there were more similarities than differences between sb and pa. for example, females represented % (sb) and % (pa). the mean duration of disease to date is . months (sb) and . months (pa), disease activity continuing (albeit, sometimes, episodically) in % of the sb practice and % of the pa patients. hla-b was present in % (sb) and % (pa). only % had a persistent monoarthropathy. twenty-four percent of patients had a positive family history for an inflammatory polyarthropathy. other similarities included keratodermia blennorrhagica in % (sb) and % (pa), tendinitis in % (sb) and % (pa) and heel disease in % (sb) and % (pa). sacroiliitis occurred in %, % of whom had asymmetric change. only patient with sacroiliitis was hla-b negative. the esr ( - , mean ) appeared unrelated to disease activity. aortitis was present in only (pa) patients. differences between pa and sb patients were minimal; balanitis and uveitis appearing more frequently in the former group. buccal ulceration was seen in % of the pa patients and % of the sb group. from this study we conclude: ) rs is a major chronic rheumatic disease; at least two-thirds of patients have active disease at an month followup. the prognosis for occupation requiring significant exertion should remain guarded. ) there are minimal differences between an academic and community population of rs patients. ) there were no discernible differences between disease severity in hla-b positive and negative patients. the activities of the major fragment of the third component of complement, c b, are controlled by at least two proteins, c b inactivator and b h globulin. using in vitro model systems, we have previously demonstrated that h physically binds to c b and blocks its activity. in the present report immunofluorescent examination of renal biopsies has been used to demonstrate that similar c b-blh interaction occurs in vivo. twelve biopsies from patients with systemic lupus erythematosus or acute poststreptococcal glomerulonephritis were examined. igg and complement components were found by immunofluorescence to be deposited in a granular fashion in the glomeruli of these specimens. by electron microscopy, mesangial, subendothelial, intramembranous and/or subepithelial electron dense deposits were found. a single biospy in which only c was found to be deposited, consistent with activation via the alternative pathway, was also studied, as was one patient with goodpasture's syndrome, whose biopsy contained linear deposits of proteins along the glomerular basement membrane. six specimens from patients with such nonimmune renal diseases as arteriolar nephrosclerosis and acute tubular necrosis were also examined. by use of direct immunofluorescence with anti-blh specifically shown to be free of any contaminating anti-c , deposits of b h were found in every instance ( of exams) in which c deposits were observed. this was true regardless of the underlying disease which resulted in the c deposits. the spatial distribution of p l h within the glomerulus was often congruent with the distribution of c in the same tissue, suggesting that these two proteins were intimately associated. n o deposition of b h was observed in those with nonimmune renal disease. we conclude that binding of / h to fragments of c , presumably c b, occurs in vivo during immunologic activation of the complement system, just as we had previously demonstrated it to occur in vitro. hahn et al. (ann int med , ) have shown a relationship between anti-hdz and anti-dna in patients with hdz-sle, and yamauchi et al. (j clin invest : , ) have shown immunologic cross reactivity between dna and hdz with rabbit antisera to hdz-albumin conjugates. to find if the development of antibodies to dna or dnp in patients taking hdz represents the production of anti-hdz which cross reacts with dna/dnp determinants, we followed prospectively hypertensive patients taking this drug (average dose mg/day) for one year. antibodies to hdz were measured by passive hemagglutination, to native dna by in the prospective group the major finding was development of increased levels of anti-dnp in patients, of whom also made anti-hdz. nine other patients made anti-hdz without anti-dnp. none made anti-ndna. one developed a mild hdz-sle syndrome with anti-hdz and anti-dnp. examination of sera of other patients with hdz-sle syndrome by radioimmunoassay failed to show an immunologic cross reaction between hdz and dnp although such a reaction was found in guinea pig anti-hdz-bsa serum. the results show a relationship between production of antibody to hdz and to dnp in patients taking hdz but do not support the hypothesis that anti-dnp in such patients is antibody cross reacting with hdz. they are consistent with the findings of fritzler and tan (clin res :a , ) that anti-dnp in drug-related sle reacts with the histone moiety. the relationship of the immune response to hdz to that to dnp remains unexplained. anti-idiotypic antibodies recognize antigenic determinants (idiotypes) uniquely associated with a given antibody molecule, or closely related molecules. the in vivo interaction between idiotypes and anti-idiotypic antibodies has been postulated to play a central role in the regulation of the immune system. the purpose of this study was to characterize the idiotypic antigens on three monoclonal igm anti-y-globulins and to determine if cross-reacting idiotypes were present in the sera of patients with rheumatoid arthritis (ra). anti-idiotypic antibodies against the purified igm ( k ) anti-y-globulins lay and si, and the igm (a) anti-y-globulin koh, were raised in rabbits. when analyzed by solid phase radioimmunoassay (ria), each antibody reacted only with the immunizing antigen, and not with pooled igg or other igm paraproteins lacking anti-y-globulin activity. studies with recombinant molecules reconstructed from lay or si light or heavy chains and light or heavy chains from heterologous proteins demonstrated that the idiotypic antigens were formed from a specific heavy-light chain interaction. the idiotypes were closely related to the antibody combining site, as judged by the inhibition of idiotype-anti-idiotype binding by antigen, i.e., igg, and of idiotype-antigen binding by anti-idiotype. idiotypes cross-reacting with the monoclonal igm ( k ) anti-yglobulin si, but not with the igm (a) anti-y-globulin koh, were found in increased concentration in the sera of patients with seropositive ra. the idiotype positive material, as analyzed by gel filtration, was found in the s but not the s fraction of serum. from these experiments we conclude: ) that the idiotypes on igm anti-y-globulins are associated with the antibody binding site and are the product of a specific light-heavy chain combination; ) igm rheumatoid factors in ra patients, although polyclonal, may have idiotypic antigens cross-reacting with those on monoclonal igm ( k ) anti-y-globulins; ) igg rheumatoid factors probably have different idiotypic antigens than igm rheumatoid factors and may therefore have different active sites and antibody specificities; ) anti-idiotypic antibodies may be useful for the selective suppression of anti-yglobulin production without a general depression of immune responsiveness. connective tissue activating peptides from lymphocytes (ctap-i) and platelets (ctap- ) are known to stimulate glycosaminoglycan synthesis, glycolysis, and mitogenesis in connective tissue cell cultures. direct evidence suggested that increased accumulation of cyclic amp was involved in the mechanism of action of these peptide agonists, and increased prostaglandin e synthesis was postulated on the basis of indirect evidence. in the present experiments, ctap-i and - were incubated with human and murine cells in culture, and prostaglandin e was measured by radioimmunoassay using antibody directed primarily to prostaglandin e,. both ctap-i and - markedly stimulated the elabo-ration of prostaglandin e, into culture medium, the earliest evidence of increased synthesis occurring at hours with maximal concentration found at hours. in a typical experiment, after hours incubation, buffer, ctap-i and ctap-i treated cells produced, respectively, , , and pg of prostaglandin e,/ . x cp normal human synovial cells. substantial residual stimulation persisted at least through hours. the lymphocyte factor (ctap-i) appeared to be more potent than ctap-i in stimulating prostaglandin synthesis. indomethacin these studies substantiate the postulated increase in synthesis of e series prostaglandins by human connective tissue cells on exposure of ctap-i and - , and clarify the mechanism of action of these agonists on "activated" target cells. the importance of elevated extracellular concentrations of prostaglandins is uncertain, although they may potentiate the actions of ctap-i and - . in order to examine the repair collagens produced by cells present in injured cartilage, the femoral articular surfaces of three groups of new zealand white rabbits were scarred in the following manner: superficial and deep lacerations, and drilled holes. eight weeks after surgery the rabbits were sacrificed and slices of injured articular cartilage harvested. the types of collagen produced at the site of these lesions were identified by labeling the recovered specimens with *h proline and characterized by sds gel electrophoresis, cmc chromatography, and cnbr peptide analysis. in all cases, tissue-specific type i ([ai( ) ) cartilage collagen was synthesized. histologic examination revealed the chondrocytes bordering the cartilage injured by deep laceration and drill holes responded by increased cellular activity. grossly, the drilled holes were completely filled with tissue possessing staining and morphologic characteristics similar to that of hyaline cartilage. these data strongly suggest that in the lacerative type of injury, the surrounding tissue will produce enough matrix for repair only when the subchondral bone is violated. both repaired and normal cartilage produce tissue-specific type i collagen. we recently demonstrated that, from birth on, nzb splenic b cells spontaneously secrete to times as much pentameric igm as normal b cells (j immunol : (j immunol : , . spleen cells from to week old mice have been examined in further study of this abnormality. nzb spleen suspensions contain to times as many spontaneous plaques to sheep erythrocytes and to tnp conjugated sheep erythrocytes as normal mice. there is a similar increase in the number of cells secreting igm as detected by a reverse plaque assay. after cold ethanol-acetic acid fixation, it can be shown that nzb spleen contains - cells with cytoplasmic igm compared to . - . % in normals. nzb spleen cells were sorted by flow microfluorometry (fmf) into surface ig negative cells and pools with increasing levels of surface igm or total ig. the surface ig negative fraction produced no igm but the positive pools each had the same level of igm production and cytoplasmic staining. when sorted into pools on the basis of size, however, all the igm production was found in the largest fraction. nzb spleen suspensions contain more large cells than normal spleens by coulter volume measurements and fmf reveals more large surface ig positive cells in nzb than normal spleens. when surface ig was enzymatically removed before fixation, it was found that the nzb cells with cytoplasmic ig were to s times brighter than those from normal mice. thus nzb spleen contains approximately times as many cells which synthesize and secrete igm as normal mice. each such nzb cell appears to contain and presumably secrete about times as much igm as do cytoplasmic ig positive cells in normal mouse spleen. because nzb mice spontaneously produce antibody to thymocytes, we utilized fmf at high gain to search for immunoglobulin on peripheral t cells. none could be demonstrated. after fixation, however, immunoglobulin was readily detected in nzb splenic t cells. whether this immunoglobulin is passively acquired and rapidly pinocytosed and thus not available for staining on the cell surface or endogenously synthesized by the t cell is under investigation. in an effort to define the cellular basis of abnormalities of polyclonal b cell activation previously noted in nzb mice, the surface immunoglobulin (sig) isotypes of spleen cells from young and old nzb mice were examined. after lactoperoxidase-catalyzed radioiodination, cells were lysed, the immunoglobulins bound to rabbit anti-mouse immunoglobulin and the immune complexes absorbed to s aureus. the complexes were solubilized and the cell surface immunoglobulins analyzed by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. cell surface immunoglobulin isotypes were also analyzed by staining spleen cells with fluorescein-conjugated rabbit anti-p, anti- , and anti-ig sera and examining stained cells by fluorescence microscopy or by automated cytofluorometry on a becton-dickinson facs- cell sorter. spleen cells from both young ( weeks old) and old (one year old) nzb mice were found to have markedly increased ratios of cell surface igm/igd compared to cells from balb/c control mice. the altered ratio of sig isotypes was not a consequence of increased proteolytic activity present in nzb cell suspensions since the addition of nzb cells to iodinated balb/c cells did not alter the balb/c ratio of sigm/sigd. it was not due to the presence of cytophilic antibody or autoanti-body since the sigm was found to have a sedimentation coefficient of s and was thus not of serum origin. when examined by fluorescence microscopy or by cytofluorometry, nzb spleen cells were found to have normal numbers of sigm+ and sigdf cells, indicating that the altered sigm/sigd ratio observed in the radioiodination experiments was due to abnormal densities of these isotypes on b cells rather than to the absence of an igd-bearing b cell subset. the increased sigm/sigd ratio may be a consequence of in vivo polyclonal b cell activation, since in vitro polyclonal activation of m o p e spleen cells has been shown to alter this ratio in a similar manner. since the increased ratio was noted as early as weeks, these data provide support for the concept that polyclonal b cell activation precedes the onset of autoimmune disease in nzb mice. sera from patients with systemic lupus erythematosus (sle) will bind native dna (n-dna). poly dat, a synthetic n-dna, consists of regularly recurring base pairs. this results in a restricted antigenicity but ensures that the molecules do not contain single-stranded (ss) regions. many laboratory preparations of n-dna will be contaminated with either ss dna or structures with ss regions in predominantly duplex molecules. the aim of this study was to determine a relationship between antibodies to poly dat and native dna in individual sle sera. one hundred sera from patients with sle were used in this study. antibodies to poly dat and n-dna were measured in duplicate in each serum using a millipore filter technique. poly dat was prepared from datp and *h labeled dttp in the presence of e coli dna polymerase. native ah labeled dna was extracted from hae cells after incubation with 'h thymidine. optimal concentrations of each antigen were calculated for use in the assay. each preparation was structurally analyzed by both hydroxyapatite (hap) elution studies and ethidium bromide fluorescence (ebr) to determine the presence of ss dna or ss regions within a predominantly duplex molecule. the specificity of antibodies to poly dat and n-dna was assessed by passing sera over agarose columns bound to either circular pm dna or poly dat and measuring both n-dna and poly dat antibodies in serum and eluate. neither preparation contained ss dna assessed by hap elution. poly dat was entirely duplex as assessed by ebr fluorescence but our n-dna contained % ss regions within the predominantly duplex molecules. under optimal conditions each serum showed consistently less binding to poly dat than to n-dna. a significant correlation was seen between the binding to poly dat and n-dna (r = . , p < . ). this correlation was consistent in all sera tested and no serum showed exclusive binding to only one preparation. binding to both antigens could be removed by passing a serum over an agarose column carrying either poly dat or n-dna. these results suggest that in clinical practice diagnosis and management of sle will be provided by measurement of antibodies to either poly dat or n-dna provided that the n-dna preparation is well characterized and does not contain ss dna or significant ss regions. despite its limited potential antigenic sites, poly dat provides a useful alternative to n-dna and does not possess ss regions to which other antibodies may bind with less diagnostic specificity. cultured adherent rheumatoid synovial cells (asc) produce collagenase and prostaglandins, particularly pge;. with age in culture and passage, collagenase and pge, production decrease; however, release of these substances can be stimulated up to > one hundredfold by a factor (apparent mol wt of - , ) released by cultured human peripheral blood mononuclear cells (mcf). mcf stimulation of collagenase production by asc is enhanced by addition of low concentrations of indomethacin (indo), nm; such concentrations of indo block pge, synthesis > wo even in the presence of mcf. at higher indo concentrations ( - fim) which inhibit pge, synthesis - , collagenase stimulation is usually inhibited by - %. in some asc stimulated with mcf, this inhibition by indo ( pm) is reversed by addition of small amounts of pge, ( ng/ml). addition of these small amounts of pge, results in augmentation of collagenase stimulation to levels greater than those seen when cells are stimulated with mcf alone. addition of larger amounts of pge, ( pg/ml) under these conditions tends to inhibit collagenase. asc respond to exogenous pge, with increased levels of camp. mcf also modulates this camp response: preincubation of asc with either indo or mcf plus indo augments camp response to exogenous p g b . it is possible that some of the effects of pge, on collagenase in asc are mediated through adenylate cyclase. pge, levels thus have profound effects on collagenase production by asc and modulate collagenase response to stimulation with mcf. therefore, pge, inhibition with pharmacologic agents in vivo could have beneficial or deleterious effects with regard to connective tissue destruction depending upon the type, sensitivity, and prior environment of the responding cell. ty cells, a subset of t lymphocytes, bear fc receptors for igg and have suppressor activity for antibody production after activation with igg immune complexes. levels of ty cells as well as total t lymphocytes were measured in patients with systemic lupus erythematosus (sle), with active and with inactive disease, and in normal subjects. mononuclear cells were separated on a ficoll-hypaque gradient, depleted of adherent and phagocytic cells, and pelleted with neuraminidase-treated sheep erythrocytes. rosette-forming cells were counted to determine the percentage of total t lymphocytes. rosettes were then mechanically dissociated and t cells separated from sheep erythrocytes through a ficoll-hypaque gradient at °c. purified t cells were pelleted with ox erythrocytes sensitized with rabbit igg and rosette-forming cells recorded as ty cells. a total of cells in each of three replicate assays was counted. active sle patients showed a significant decrease in total t lymphocyte percentages compared to normal subjects (p < . ), confirming previous reports. in addition, markedly depressed percentages of ty cells (p < . ) were found in patients with active disease. patients with inactive sle were not significantly different from normal subjects, both with regard to total t lymphocytes as well as ty cells. two acute sle patients who went into remission showed significant increases in both total t lymphocytes and ty cells with clinical and laboratory improvement. active sle patients show decreases both in total t lymphocytes and the ty subset of lymphocytes associated with suppressor activity. low levels of detectable ty cells in sle may reflect the presence of high levels of circulating immune complexes and/or the defect in suppressor activity reported in these patients. the - fold increase in red cell turnover in patients with sickle cell anemia results in uric acid overproduction. since uric acid overproduction in these patients begins within the first years of life, we studied patients with sickle cell anemia and normal gfr ranging in age from to to determine the natural history of serum uric acid and urate excretion in sickle cell disease. hyperuricemia (plasma urate . mg/dl) was observed in only of patients younger than age . however, urinary uric acid/creatinine ratios and hour urinary uric acid levels were elevated in these patients, indicating uric acid over-production and hyperuricosuria. thirty-seven percent of adults ( / ) were hyperuricemic (mean plasma urate . f . mg/dl); were normouricemic. of normouricemic adults studied while on a purine-free diet, of ( %) had hour urine uric acid excretion greater than mg. urate clearance in these patients was increased (cur . f . ml/min), indicating that normouricemia was maintained by hyperuricosuria. urate clearance in the hyperuricemic subjects was decreased (cur . f . ml/min) as compared to both normal subjects (cur . f . ml/min) and normouricemic adults with sickle cell disease (cur . f . ml/min). urate clear-ance appears to be the major determinant of serum uric acid concentration even in sickle cell patients with urate overproduction. responses of urate clearance to probenecid and pyrazinamide were exaggerated in the normouricemic overexcretors [pza suppressible urate clearance (psur):ss, . f . ml/min; control . f . ml/min; probenecid (pb) response; ss, . f ml/min; control, . f . mi/min] and were diminished in the hyperuricemic subjects with diminished urate clearance (psur, . f . ml/min; pb response, f . ml/min). urate clearance was correlated pah clearance. these results suggest that changes in urate clearance were secondary to changes in tubular secretion in urate. the initial response to urate overproduction in ss disease is hyperuricosuria with increased urate clearance and maintenance of normal serum urate concentration. hyperuricemia is found in adults with diminished urate clearance and is associated with other evidence of impaired renal tubular function. nephropathy is a little known complication of polymyositis. thus, we describe the features of glomerulonephritis in patients with polymyositis. the patients ( men and woman) aged - years each presented with symmetrical limb girdle weakness, widespread polyarthritis, myalgias, recurrent fever, and proteinuria. cpk, ldh, and sgot levels were elevated in all cases, and the diagnosis of polymyositis was confirmed by electromyography and muscle biopsy. although raynaud's phenomenon and sacroiliitis were each present in one case, no patient had the skin changes of dermatomyositis, and no evidence of other diffuse connective tissue disease or underlying neoplasm. ana was negative in all patients; cryoglobulins, le preparations, and asot were absent in patients tested. c complement level was decreased in one case. rheumatoid factor was negative in and latex positive ]:i in one case. blood urea nitrogen and serum creatinine were normal in each patient; however, the hour urine protein excretion ranged from . to . gm. three patients had an abnormal urine sediment and one had myoglobinuria. renal biopsies ( patients) showed a mild focal segmental mesangial increase. immunofluorescence was negative in one biopsy and mildly positive in a granular pattern for igg and igm in others. treatment of all patients with high-dose corticosteroids led to rapid clearing of the proteinuria and slower improvement of the polymyositis. a -year review in our center of polymyositis cases showed proteinuria exceeding mg/ hours in patients and abnormal urinary sediment in . none of these latter patients had underlying malignancy. a focal glomerulonephritis associated with polymyositis may be more common than is generally appreciated. although the pathogenesis of the renal lesion in these cases is unknown, it may be related to myoglobin. two enzymes of the purine nucleotide degradation pathway, adenosine deaminase and purine nucleoside phosphorylase, are associated with specific immunodeficiency syndromes. a third catabolic enzyme, '-nucleotidase, was measured on the external surface of peripheral circulating lymphocytes from patients with immunoglobulin deficiencies. all male patients with congenital agammaglobulinemia demonstrate reduced activity of lymphocyte ecto- '-nucleotidase to to % of the normal value. the mean activity is . f .o nm/hr/lob cells for congenital agammaglobulinemia as compared to the mean normal value of . * . . patients with common, variable hypogammaglobulinemia or selective iga deficiency have values within the normal range. two other lymphocyte plasma membrane ectoenzymes, atp'ase and nonspecific phosphatase, have similar values in lymphocytes from normal subjects or from subjects with congenital agammaglobulinemia. the activity of '-nucleotidase in lymphocyte lysates has similar values in normal and enzyme deficient subjects. ecto- '-nucleotidase has similar activity in both t and non-t lymphocytes in all subjects and the deficiency occurs in both these categories of cells in the affected patients. ecto- '-nucleotidase from normal and enzyme deficient subjects has a similar p h optimum'of . , is similarly inhibited by adenosine- ' , p-methylene diphosphonate, has hyperbolic kinetics and a similar michaelis constant of pm for amp. these data suggest that the reduction of lymphocyte '-nucleotidase activity is an abnormality localized to the plasma membrane in this x-linked, b-cell immunodeficiency syndrome. in vivo clearance of exogenous single-stranded dna (ssdna) is extremely rapid and occurs primarily through the liver. with higher doses of ssdna, however, both liver uptake and blood clearance approach a maximum, enabling large molecular weight ssdna to persist in the circulation (emlen, mannik; j exp med, march, ) . in the present study, we examined the effects of prior saturation of the liver with immune complexes on the clearance kinetics of ssdna. heat denatured calf thymus '*'i ssdna was chromatographed on hydroxyapatite and a defined molecular weight was obtained by gel filtration over sepharose b. immune complexes were prepared at -fold antigen excess with human serum albumin and purified rabbit anti-human serum albumin. female c bl/ j mice were injected at time with immune complexes containing mg antibodies or with buffer. at , , , , or hours, animals were given pg of ' ssdna; serial blood samples were assayed for radioactivity and clearance velocities were calculated by linear regression analysis. clearance of ssdna was slowed in the mice pretreated with immune complexes. control mice cleared ssdna at a rate of . f . pg/ml/min, while animals pretreated with immune complexes cleared the ssdna at a rate of . f . at hours (p < . ), . f . at hours (p < . ) and . f . at hours (p < . ). clearance returned to normal at and hours. the degree of suppression of ssdna clearance velocity at different times after administration of immune complexes correlated with the previously established values for the amount of immune complexes present in the liver at a given time. to examine the effects of pretreatment with immune complexes on the saturability of ssdna clearance mechanisms, , , or pg of ssdna were given hours after administration of immune complexes. with the pg dose the clearance velocity approached a maximum, but this value was less than half of the maximum clearance velocity in normal mice. these observations indicate that immune complexes alter ssdna clearance by decreasing the number of sites in the liver to which dna can bind, or by decreasing access of dna to the liver. by altering clearance kinetics, immune complexes may contribute to the persistence of dna in the circulation of patients with sle. (supported by nih grant am .) rheumatoid arthritis (ra) frequently affects the wrist, leading to pain, loss of function, and destruction of normal anatomy. surgical treatment by synovectomy alone does not correct carpal deformities and ulnar deviation; fusion of the wrist, although effective in reducing pain, severely limits function. to prevent loss of upper extremity function while simultaneously correcting deformities and relieving pain, we combined synovectomy with distal ulnar excision, correction of carpal supination-subluxation, and repair of damaged extensor tendons. this surgical stabilization approach was performed times in patients with ra. all had definite or classic ra and had not responded to aggressive physiotherapy, including splints and local and systemic medications. all had synovitis with deformity and instability. the deformity most commonly seen was carpal supination-subluxation with relative prominence of the distal ulna. the primary reasons for operation were frank ( ) or potential tendon rupture ( ), persistent synovitis with pain at rest ( ), and pain with significant deformity ( ). the mean age was years (range: - ). the wrist was exposed with preservation of the exten-sor retinaculum. following excision of the distal ulna, a synovectomy of the radiocarpal and intercarpal joints was carried out. any existing carpal dissociations were realigned, and the carpal supination-subluxation deformity was corrected by placing the wrist in pronation and reconstructing the triangular ligament using the volar capsule. extensor tendon repair and grafting were then carried out. length of followup was months with a range of - years. results were classified as good if the patient was satisfied, had only occasional pain, a stable wrist, and no recurrence of synovitis. three patients were lost to followup and of the remaining , good results were obtained in . an additional had objective improvement but experienced recurrence of synovitis and pain and an extensor lag of up to o . less satisfactory improvement was found in of the . the average loss of motion was ' , leaving patients with a combined palmar and dorsiflexion range of ". although this stabilization procedure is not considered appropriate for the severely destroyed wrist, the overall / improvement (fair and good results) justifies its consideration in preference to either synovectomy or wrist fusion alone. the destructive inflammation observed in synovia of rheumatoid joints may be largely due to autoimmune processes. antibodies to types i, , collagens have previously been detected in joint fluids of rheumatoid patients and cellassociated antibodies which react only with cartilage type i collagen can be locally detected in inflamed rheumatoid synovia. we are interested in finding evidence for cell-mediated immunity to collagen. immune lymphocytes challenged with antigen produce substances (lymphokines) that can grossly stimulate the secretion of the enzyme collagenase from macrophages. collagenase, which can degrade cartilage collagen producing cartilage destruction, is also secreted by macrophage-like cells in rheumatoid synovia and its secretion can be stimulated from synovial cells by a factor(s) produced by lymphocytes, unstimulated or stimulated with phytohemagglutinin. in the present study inflamed synovia were removed at surgery from patients with classic rheumatoid arthritis. synovial fragments were maintained in a viable state in organ culture for up t o weeks. culture media were changed every days and the collagenase secreted into the culture medium was assayed. to these cultures were added type i or type collagen in the form of diffusible immunologically active fragments prepared by cyanogen bromide cleavage of native collagen. in of rheumatoid patients, the addition of type i collagen fragments resulted in a significant increase in collagenase secretion compared with explants which had not been stimulated: artificial stimulation of lymphocytes in these explants with phytohemagglutinin also resulted in a similar increase in collagenase secretion. type i collagen peptides had no stimulatory effect. these results support the thesis that in some patients rheumatoid synovia contain immune lymphocytes which can respond t o type i collagen peptides produced by degradation of hyaline cartilage collagen. when challenged with antigen, these cells may produce factors that stimulate the secretion of collagenase from synovial cells leading to further cartilage destruction. although intraarticular corticosteroid injection has been considered a therapeutic adjunct in rheumatology for the last years, scientific proof of efficacy is still lacking. the present study was undertaken, therefore, to determine the effect of intraarticular steroids on pain in the osteoarthritic knee by use of a controlled, double-blind protocol. thirty-four patients with symptomatic osteoarthritis of the knee were included in the study. patients previously injected with local steroids were excluded. half the patients were treated with mg of triamcinolone hexacetonide injected into the knee, the other half with the same volume of placebo (vehicle without steroid). the two physician-experimenters performed the arthrocentesis and withdrew any synovial fluid, if possible. a nurse-assistant then injected either steroid or placebo through the same needle according to a predetermined, random schedule. thus, the physicians who conducted the study did not know the nature of the material each patient received. knee pain was quantified prior to, and at , , , and weeks after injection. at week the steroid group had significantly reduced knee pain compared with the pretreatment assessment. this reduction persisted through the week evaluation period. the placebo group also exhibited a significant amount of pain relief at week, but this was significantly less than that experienced by the steroid group. from through weeks there was no statistically significant difference between the groups. whether or not synovial fluid was initially aspirated did not affect results. postinjection flares occurred as often with placebo as with steroids and did not affect the subsequent course. it is concluded that intraarticular steroids reduce the pain of osteoarthritis, but such a response cannot be distinguished from a placebo effect by weeks postinjection. it has been reported previously that antibodies to histones can be demonstrated by immunofluorescence. when tissue sections are extracted with . n hci, histones are eluted from nuclei, and the extracted tissue contains only d n a devoid of nuclear proteins. histones can be reconstituted to nuclear d n a by incubation of acid-extracted tissues with purified calf thymus histones. sera containing antibodies to histones show positive antinuclear antibody (ana) staining on untreated tissue, become ana negative on acid-extracted tissue and regain ana positivity on histone-reconstituted tissue. sera from patients with drug-induced lupus erythematosus (procainamide , isoniazid , nitrofurantoin ) and patients with idiopathic (not drug-induced) systemic lupus erythematosus (sle) were studied. all drug-le patients had positive ana on control tissues but ana became completely negative on acid-extracted tissues. on histone-reconstituted tissues, / again became ana positive. in contrast, of idiopathic sle sera which were positive for ana on control tissues, only became negative and of these, again became ana positive on histone-reconstituted tissues while the other remained negative. three other sle sera showed partial reduction in ana titer but increased in titer on histone-reconstituted tissue. thus, / drug-le patients ( %) had antibodies to histones compared to / sle patients ( %). to determine the specificity of anti-histone antibodies, the h , h a-h b and h -h fractions of calf thymus histone were prepared by differential salt extraction and sephadex gel filtration and used in reconstitution experiments. it was shown that in / of the drug-induced sera antibodies were primarily against h a-h , while in the idiopathic sle sera studied, antibodies to all classes of histone were found. a further difference was that idiopathic sle sera had antibodies to native dna and to the nonhistone proteins sm and nuclear rnp whereas drug-induced le sera did not. the heterogeneity of anas in idiopathic sle and the striking prevalence of anti-histone antibodies in drug-induced le, as demonstrated by immunofluorescence, have been valuable clinically in differentiating between these two syndromes. the etiology of pss is unknown and progress toward its understanding has been hampered by the lack of a model for the disease. it has recently been reported that skin changes similar to those of scleroderma develop in some long-term survivors of bone marrow transplantation (bmt). five such subjects, who lived an average of months post-bmt, developed sclerodermatous cutaneous involvement and also underwent examination for visceral complications resembling pss. at present, are alive and have died. we compared their findings with the findings in well-characterized patients with scleroderma to ascertain the similarities and differences between these two groups. taut hidebound skin was found in all patients in both populations and selected skin biopsies were also similar. restrictive lung disease, characterized by decreased vital capacities and diffusing capacities, was found in % of the pss patients and in / ( wo) of the bmt group. cardiac disease and raynaud's phenomenon, similar to that found in the pss group, were noted in one post-bmt patient, and pathologic evidence of pss-like esophageal involvement was found in another. in both the bmt and pss groups, the responses of lymphocytes to suboptimal concentrations of pha were d e pressed, while complement levels, screening tests for autoantibodies and circulating levels of iga and igm were essentially normal. the frequency of patients with abnormal ana and dna-bindings was higher in the pss group than in the bmt group. the incidence of abnormal levels of immune complexes, igg levels, anergy, and decreased percentages of b-and t-lymphocytes was higher in the bmt patients than in those with pss. even though only months have elapsed since transplantation, these post-bmt patients have begun to develop signs and symptoms of a pss-like disease. we suggest that bmt bears closer examination as a model for pss. this study was initiated to further investigate the inhibitory effect of igm rheumatoid factor (rf) in an antibodydependent cell-mediated cytotoxicity (adcc) assay. this assay employs human peripheral blood lymphocytes as effector cells and igg or igm sensitized, cr labeled ox erythrocytes as target cells. serum and synovial fluid were obtained from patients with seropositive classic rheumatoid arthritis (ra) and heat inactivated. rf was prepared by dialyzing the sera and synovial fluid against sodium acetate buffer (ph . ), fractionating on sephadex g- with the same buffer and pooling the fractions constituting the leading side of the s peak, dialyzing against pbs and further purifying by passage over an igg-coupled sepharose b column and eluting with acid. this igm-rf was tryspin digested (enzymexubstrate ratio = : ) at °c for minutes and the reaction stopped by adding soybean trypsin inhibitor. the fragments were fractionated on biogel a m to separate undigested igm, fc,p and fab. the igm-rf, fc& and fab fragments were tested for inhibitory capacity in igm and igg induced adcc at both the effector and target cell levels. it was found that igm and igg induced adcc was inhibited by igm-rf and its tryspin digest fragments. inhibition at the effector cell level was achieved by rf fc& and fab whereas inhibition of the target cell level was achieved only by rf fab. these results indicate that r f may inhibit adcc at the effector or target cell level and thus modulate an immune response that is of potential importance in immune-complex disease. patients with seropositive rheumatoid arthritis (ra) of adult-onset were compared to control groups by utilizing two primary histocompatibility procedures, b-cell (ia) alloantigen typing and mixed lymphocyte culture (mlc) reactivity. by use of a two-stage microcytotoxicity assay with a panel of alloantisera, the frequencies of various b-cell alloantigens in patients with ra were compared to those of control populations, including patients with multiple sclerosis. three b-cell alloantisera were identified that gave particularly high frequencies of reactivity with ra patients. delineation of the specificity of these alloantisera on a panel of b-cell lines derived from individuals homozygous for mlc alleles revealed that reactions were obtained only with those lines from individuals that were positive for hla-dw , dw , or dwlo. absorption of the alloantisera with b-cell lines from dw , dw , or dwlo positive individuals eliminated the seroreactivity with all b-cell lines having any of these three de-terminants, thus demonstrating that the alloantisera react with a b-cell antigen common to cells with either of the three mlc specificities. mlc testing of the ra patients with a panel of normal homozygous cells defined in the seventh international histocompatibility workshop revealed an increase in the frequencies of hla-dw and dwlo as compared to controls ( % versus . % and % versus . %, respectively), while the frequency of hla-dw was not significantly different ( % versus . %). the individuals who were negative for the hla-d alleles dw , dw , and dwlo by conventional mlc testing yet positive for the shared b-cell alloantigenic specificity were of particular interest. the results indicate that a shared antigenic specificity exists among alleles of b-cell alloantigens that is, in turn, partially related to particular mlc alleles. thus susceptibility to ra may be a function of the inheritance of the molecules bearing this antigenic specificity. activation of mediator cells such as platelets and neutrophils plays an important role in the pathogenesis of gout. monosodium urate crystals (msu), the probable initiating agents of gouty inflammation, have been shown to stimulate suspensions of washed platelets or neutrophils. when msu crystals are coated with igg (as occurs in plasma), stimulation is markedly enhanced. these studies, using msu induced human platelet serotonin secretion as a model, examined the nature of cellular recognition mechanisms for the igg-coated msu crystal and the uncoated crystal. f(ab') fragments of specific anti fc antibody blocked and the lipopolysaccharide of s minnesota r enhanced human platelet secretion induced by igg-coated urate crystals. these agents had little effect on stimulation by uncoated crystals. this indicated that urate crystals stimulate platelets independently of fluid phase igg. urate crystals directly stimulated suspensions of washed rabbit platelets which lack fc receptors. in contrast to human cells, stimulation was blocked by igg. this again demonstrated igg-independent cell stimulation by urate crystals. calcium pyrophosphate dihydrate crystals could trigger human platelet secretion only when coated with igg. this suggests that when crystals were coated with igg, the surfacebound igg alone may be the stimulus to the cell. this was confirmed by the finding that polyvinyl pyridine-n-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated but not igg-coated urate crystals. in contrast to igg, f(ab') fragments, igm, or iga did not enhance human platelet stimulation by urate crystals. this indicates that the effect of igg on urate crystal stimulation of platelets depends on the fc region of igg. these data demonstrate that urate crystals (and potentially other surfaces or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of absorbed igg or when coated with igg, by triggering the cell via fc receptors. the correlation of serologic abnormalities, especially low serum complement and high levels of anti-dna antibody, with disease activity has been established in sle. the significance of abnormal serologic tests in the absence of active clinical disease is still unclear. this report describes a group of patients seen in the course of a prospective study of sle in whom a discordance between clinical and serologic features was apparent. these patients had persistently positive le preps and antinuclear antibody tests, low serum complements, and high levels of dna binding. the mean lymphocyte response to con a mitogen was suppressed in these patients as compared to age and sex matched controls done on the same day. these patients did not differ significantly from other patients with sle, seen during the same period, in the frequency of skin manifestations, raynaud's phenomenon, and arthritis; they showed trends toward a lower frequency of photosensitivity, serositis, mucous membrane ulcers, and neu-ropsychiatric involvement and had a statistically significant lower frequency of alopecia and nephritis. they also resembled the larger group in the presence of most laboratory abnormalities with only hypergammaglobulinemia occurring less frequently. these patients have been followed untreated for a mean of % years without evidence of clinical exacerbations of disease. thus in individual patients with sle it would seem advisable to determine whether their clinical course and laboratory abnormalities are concordant. some patients will illustrate this pattern and thus therapeutic manipulation according to changes in laboratory variables may be indicated. some patients will display clinical-laboratory discordance and could then be treated for clinical exacerbations only, irrespective of laboratory changes. followup in our patients would indicate that patients with such humoral and cellular immune abnormalities may safely be followed untreated for prolonged periods. independently and in two separate laboratories, cocultivation techniques have demonstrated the presence of viruslike particles (vlps) in rheumatoid synovial membrane cells (rsc). one technique consisted of cocultivation of long-term rsc cultures with human lung fibroblasts (wi ); the other system utilized cocultivation of freshly dissaggregated rsc with fetal rabbit synovialcell cultures. controls in both laboratories consisted of similar cocultivations using cultures derived from nonrheumatoid membranes. all cocultivations were maintained for - months without subculture, during which time small foci of heaped-up cells were observed. by electron microscopy, all the rheumatoid cultures ( ) but none of the control cultures ( ) showed vlps. these appeared as spherical bodies with a spike-like external array, seen both in sonicated negatively stained (pta) preparations, and in thin sections of harvested rsc cocultivations. these vlps were found budding from the cell membranes, and also within cytoplasmic vacuoles. their morphology resembled that of budding rna viruses, although coronaviruses and myxoparamyxoviruses may also show these characteristics in certain situations. similar particles have not been previously demonstrated in rsc. serial passage of cocultivation extracts containing vlps onto other cell types resulted in repeated transient cytopathic effects. extracts of the rsc cocultivations were injected into suckling mouse brains, resulting in a reproducible illness not observed when control extracts were similarly injected. additional studies are under way to identify the agent and to explain its presence in the rsc cocultivation systems. the mechanism responsible for leukopenia in felty's syndrome (fs) is unknown. both increased peripheral destruction of leukocytes and decreased marrow production may play a role in this process. the present study was designed t o determine whether factors in the sera of patients with fs inhibit the maturation of granulocyte precursors from human bone marrow. sera were obtained from patients with fs; patients with rheumatoid arthritis, splenomegaly, and normal white cell counts; patients with active ra, and patients with osteoarthritis. nucleated cells were isolated from human marrow, and single cell suspensions were cultured in mccoy's medium and fetal calf serum. the serum to be tested, . ml, was incubated with . ml of the cell suspension (containing x ' cells/ml) and . ml rabbit serum, and the mixture was placed in % agar. at the end of days, the number of cells giving rise to granulocyte-macrophage colonies (cfu-c) were counted. serum from a healthy adult was run simultaneously as a control. inhibition of maturation was considered to be present if the number of cfu-c in the agar incubated with the test serum was reduced by at least % as compared to healthy control serum. sera from of patients with fs caused a significant reduction in the number of cfu-c. seven of the patients with serum inhibitors had undergone splenectomy; in of these subjects the inhibitor was absent prior to splenectomy, but present after splenectomy. none of the sera from the control subjects gave a positive response. these data indicate that the sera of certain patients with fs contain a factor that inhibits human granulocyte precursors in vitro. this factor may play a role in the induction of leukopenia in fs. the presence of a serum inhibitor of granulocyte maturation may also explain the failure of some patients with fs to respond to splenectomy. this study was designed to determine-whether lymphocytotoxic antibodies (lca) in patients with sle had specificity for the ii antigen system; the ii antigens are known to be present on the surface of all formed elements in blood. sera from patients with sle were tested for lca using the standard microdroplet assay. following determination of the lca in whole sera, each serum was serially diluted twofold in saline and retested for lca. the dilution of the serum which gave a % decrease in cytotoxicity was used in the following absorption experiments. each diluted serum was absorbed threefold with either adult (rich in i antigen) or cord (rich in i antigen) red cells a t "c, and then retested for lca activity. eluates were prepared from sera incubated with either adult or cord cells a t "c, and the eluates assayed for lca. the cytotoxicity of the whole sera was % or greater in all but one serum. seven of the diluted sera showed an obvious reduction in cytotoxicity following absorption with cord cells, but minimal or n o reduction after absorption with adult erythrocytes. of the remaining sera, the lca were equally reduced by adult and cord cells in , and were unaffected by absorption in . in the sera studied, eluates prepared from cord cells showed greater lca activity than eluates from adult cells. statistical analysis confirmed that the mean cytotoxicity values in the sera after absorption with cord cells were significantly different from those obtained after absorption with adult cells (p < . ). additional studies showed that there was a strong correlation between the level of lca and the titers of cold agglutinins against cord red cells. these data indicate that the lca in most sle sera react with i antigen. cold-reactive antibodies with i specificity have been associated with hemolytic anemia and thrombocytopenia in other disease, thus, the capability of lca to react with i antigen may explain the observation that lca were found in higher frequency and greater titer in sle patients who have hematologic abnormalities. clinical and experimental observations indicate that estrogens may play a part in rheumatoid arthritis (ra) and in the regulation of immune responses. in order to delineate this relationship, the effect of oophorectomy in an experimental immune synovitis resembling ra was studied. nineteen adult female rabbits were sensitized to homologous igg and then randomly divided into groups. group i ( ) underwent oorphorectomy. group i ( ) had the same but were treated with mg of estradiol intramuscularly every other week. group i ( ) had a sham procedure; group iv ( ) were unoperated controls. an immune synovitis was then induced in the right knee by the biweekly intraarticular injection of mg of igg and the animal sacrificed after weeks. skin testing to igg was done at week intervals. serum cortisol levels were performed prior to sacrifice by use of a radioimmunoassay technique. gross and histologic assessment of synovial inflammation and cartilage changes was recorded using a "blind" grading previously reported from for no changes to + for severe involvement. synovial cathepsin d activity was analyzed by the anson technique. there were no changes in the initial positive skin tests to igg in any groups. cortisol levels in groups , , and iv were . , . , . hg% without significant differences, while in group i, the . was significantly depressed. synovial inflamation in the oophorectomy rabbits was +, compared to the unoperated and sham groups of +. the reduced synovitis in group i was reflected by a significantly lower cathepsin d level of . p moles/hour/l mg of protein compared to . and . for the other groups. however, when oophorectomy rabbits were given estrogen replacement, there was an increased synovitis rated + with a significantly elevated cathepsin d level of . . cartilage changes were rated + and appeared uniform throughout the groups. the results of this study indicate that in this model of immune synovitis, oophorectomy appeared to inhibit synovial inflammation while estrogen replacement seemed to enhance this response. whether this inhibition is by direct endocrine mechanisms or through the immune system remains to be studied. finally, since ra has a significant female predilection, these findings may be important in the pathophysiology of this disease. we investigated the role of suppressor cells in the depressed cellular immunity of patients with sarcoidosis. we have recently described a glass adherent prostaglandin producing suppressor cell that inhibits t-cell mitogenesis in phytohemagglutinin (pha) stimulated cultures of human peripheral blood mononuclear cells (pbmc) by secreting pge, (j exp med : , . increased activity of this cell is responsible for the hyporesponsiveness to pha seen in pbmc from patients with hodgkin's disease (n engl j med : , the mean response of patients with active sarcoidosis to three concentrations of pha was significantly (p < . ) less than controls. passage of the cells over glass wool resulted in a % increase in the mean response to pha in the sarcoidosis patients and a % decrease in controls. the pha response of the active sarcoidosis patients went from % of control before passage over glass wool to % of control after. addition of indomethacin, a prostaglandin synthetase inhibitor, to pha cultures increased the response of the pbmc from patients with sarcoidosis f % versus a f % ). increase in controls (mean f sem, p < . ). the sarcoidosis patients had an increased percentage of. monocytes in the peripheral blood mononucleat cell preparations ( f % monocytes for patients, f % for controls, mean f sem, p < . ) and the percent monocytes in a pbmc preparation from patients with active sarcoidosis correlated with the percent increase in pha response after glass wool passage (r = . , p < . ). thus, there appear to be at least three mechanisms operating in the depressed pha response of pbmc from patients with active sarcoidosis. first, there appears to be increased activity of the prostaglandin producing suppressor cell. second, the increased proportion of monocytes correlates with increased suppression, either via a diluting effect or perhaps through another active suppressive effect. blockade of these first two mechanisms by indomethacin and glass wool passage does not result in total restoration of the pha response of patients with active sarcoidosis, and therefore, a third, as yet unknown, factor must also contribute to the depressed pha response. the rheumatologist is aware of hand deformities in the adult. very little is written concerning the hand of the child with juvenile rheumatoid arthritis (jra). roentgenographic and clinical analysis was done, review of surgery and outline of treatment is given. five-hundred patients are under treatment. there are three basic types of onset in jra. systemic onset is similar to that described by still; polyarticular closely resembles the disease in the adult; pauciarticular involves four or less joints. jra has a much better prognosis than the adult type but tends to produce more stiffness. examination of successive children showed % had radial deviation of the rnetacarpophalangeal (mcp) joint and % bad decreased flexion. seven percent had boutonniere deformity and % had a swan-neck contracture. there was decreased flexion of the pip in % and decreased extension in %. the wrist showed % decreased extension and only % decreased flexion. a statistical review of the records and x-rays of children showed finger involvement in approximately %. wrist involvement in approximately %, and very few cases of boutonniere or swan-neck. ulnar shortening in joints measured on roentgenogram showed a range of - mm with an average of . mm. ulnar wrist deviation was seen in joints with an average of degrees and radial deviation in joints with an average of degrees. metacarpophalangeal ulnar deviation was seen in with a range of to degrees and an average of , and a mcp radial deviation also of between and degrees with an average of . degrees. there was n o ara abstracts statistical correlation between ulnar shortening, ulnar deviation, and metacarpophalangeal radial deviation, as reported in previous literature. surgery between and was rare and included collateral ligament release in patients, tenolysis on occasions, carpal tunnel release, and reconstruction of thumb mcp. terminal vasospasm required finger amputation. synovectomy of mcp and pipjoints was done and studied between and . n o recurrences were seen in late followup, but stiffness precluded continuing this procedure. the common problem of wrist flexion contracture is best managed by physical therapy and extension orthosis, and the loss of finger flexion by active exercises, occasional injection, and active splinting. we have previously demonstrated that the translational movement of glucose in pathologic synovial fluids is too rapid to be accounted for by bulk diffusion (fed proc : , . such enhanced diffusivity is a property of a n isolated . % matrix of ha as well, wherein both diffusivity and matrix structure are critically dependent on ca++ concentration (see semin arthritis rheum : - , for review). the current study examines translation movement of glucose in a i% matrix. human umbilical h a and agarose were prepared as % matrices in phosphate buffered saline at ph . . the bulk diffusion coefficients (d) for glucose and sucrose within each matrix were determined in codiffusion experiments employing a capillary method. the d for both solutes in h a was indistinguishable from that in agarose. if the matrix was prepared in % horse serum, glucose diffusivity was enhanced twofold in h a while slightly diminished in agarose. a % dialysate of serum as a solvent enhanced glucose diffusivity in ha threefold; the dialyzed protein had little effect. the en-hanced glucose diffusivity in the presence of a % dialysate was inversely dependent on solute concentration. the diffusivity of sucrose in h a was little altered by these solvent changes. in order t o gain insights into matrix structure that might underlie the enhanced glucose diffusivity, darcy numbers were determined. permeability is dependent on h a concentration, has a ph optima of . in a % matrix, and is critically dependent on ca+ + concentration rising rapidly below mm. matrix structure is highly dependent on the solvent environment particularly in the physiological range. constituents in the serum dialysate may induce a matrix configuration that supports the enhanced glucose diffusivity. the matrix configuration that exists in the presence of the dialysate is likely to be present in synovial fluid as well. this configuration can facilitate the translational movement of a nutrient such as glucose. facilitated movement may be homeostatic in inflammatory states such as rheumatoid arthritis where low synovial fluid glucose concentrations are well described. sera from patients with mctd were examined for the presence of immune complexes (ic). the diagnosis was based on serologic and clinical criteria. all patients had antibodies to extractable nuclear antigen by passive hemagglutination. titers of : , , or greater were found in patients and : ,ooo and : , in the remaining two. treatment of the antigen with rnase resulted in a , -fold or greater decrease in titer in patients and a -fold decrease in one. rheumatoid factors were present in patients and antinuclear antibodies in . all patients had a mixed rheumatic syndrome. clinical features included: raynaud's phenomenon- , swollen hands and/or sclerodactyly- , myopathy- i , pleuritis-- , malar or diffuse rash- , and arthritis- . one patient had a stable iga membranous glomerulopathy. ic were measured in sera by a raji cell radioassay (rc-ra), monoclonal rheumatoid factor radioimmunoassay (mrf-ria), and clq binding assay (clq-ba). ic were detected by at least one method in ( %) sera. the single negative serum was from a patient with clinically inactive disease. ic were found by the rc-ra in ( %) sera, by mrf-ria in ( %), and by clq-ba in ( %). mean values for each assay compared to normal controls were: . versus . pg agg.-igg equiv/ml for the rc-ra (p < . ); . versus . pg agg.-igg equiv/ml for the mrf-ria (p < . ); . % versus . % la -clq bound for the clq-ba (p < . ). ic were detected by all three assays in ( %) sera and by two methods in ( %). thus, ic were found by two or more methods in ( ) sera. only three sera failed to react in more than one assay. a different pattern of reactivity was found in rheumatoid arthritis sera where ic were detected at a higher frequency by mrf-ria ( %) and clq-ba ( %) while the frequency by rc-ra ( %) was lower. sufficient clinical data were available for of the mctd patients to permit preliminary evaluation of the relationship of disease activity and ic. changes in levels of ic appeared to parallel clinical activity in patients by rc-ra, in by mrf-ria, and in by clq-ba. these data indicating a high incidence of ic in mctd and a different pattern of reactivity from ra emphasize the wide spectrum of immune complex disease. hydroxyapatite (ha) has been found by others in synovial fluid and fluid leukocytes by electron microscopy and electron probe. we have developed a method to quantify ha in synovial fluid which avoids possible em artifacts. ("c) ehdp, (sa . pci/omole) was used in a final concentraion of nm/ml in normal serum or phosphate buffered saline (pbs) containing standard ha or to joint fluid samples; serum or joint fluids were processed under oil. after rotation x h "c, radioactivity/o.l ml was determined before and after centrifugation ( x g), and expressed as percent loss of nuclide from the supernatant. standard ha binding in serum monsodium urate or calcium pyrophosphate dihydrate (cppd) crystals failed to bind in concentrations up to . mg/ml and .o mg/ml respectively. the centrifuged pellets were washed in distilled water and dried to a spot which showed ha by x-ray diffraction. this method was sensitive to - pg ha standard. we confirm the finding of ha crystals in joint fluids, handled physiologically, in amounts equal to - pg ha standard/ml. such small amounts and the low wbc in these fluids fail to support a phlogistic role for ha crystals. patients with systemic lupus erythematosus (sle) have circulating immune complexes (ic) which are thought to be involved in disease pathogenesis. using a newly described method for in vivo evaluation of res igg fc membrane receptor function, we previously demonstrated a defect in untreated patients with active sle and related this res defect to the presence of circulating immune complexes. a serial study of the correlation of defective res fc receptor function, immune complexes, and disease activity was undertaken to further investigate the interaction of these factors. isologous yr-labeled erythrocytes were sensitized with human anti-rh(d) and their intravenous clearance determined. clearance rates (t- / ) c' levels and ic levels by z -clq precipitation were determined prior to therapy in sle patients, of whom had active disease. fifteen of had markedly prolonged clearance rates (range = to > ,ooo minutes; average clearance t- / in normal volunteers = minutes, range to minutes). the correlations of clinical activity with ) clq binding and ) prolonged clearance were statistically sig-nificant (p < . and p < . , respectively; spearman rank coefficient). in addition, elevated clq binding and depressed c levels were significantly correlated (p < . ). at the end of from to months, patients who initially had prolonged clearance were restudied. patients had been treated with either corticosteroids ( of ) or nonsteroidal, antiinflammatory drugs ( of ). clinically, of were improved; the remaining continued to do well. although a clearance defect persisted in all (t- / from to minutes), in of t- / was markedly improved, suggesting improved fc receptor function. in patients, there was an apparent correlation between increased clearance and clinical improvement. there was also a significant correlation between clinical improvement and a decrease in clq binding (p < . ). these studies underscore the interrelationships between the presence of circulating immune complexes, defects in membrane fc receptor function which might lead to continued circulation of these complexes, and disease activity in sle. we have found immune complexes in patients with lyme arthritis, an apparently tick-transmitted illness that typically begins with the skin lesion, erythema chronicum migrans (ecm). many patients subsequently develop clinical attacks of synovitis histologically like that of rheumatoid arthritis; some also develop other systemic complications (aseptic meningitis, peripheral neuropathy, or carditis). we measured circulating immune complexes (cic) serially in patients with ecm, by the l*si-clq binding assay. the table shows the first test result on each patient's serum in each of the designated categories. circulating immune complexes-an antigenic component of which may be related to the causative agent-were almost always present very early in the illness and in patients with systemic complications. in contrast, they were found less often during attacks of arthritis and still less often during remissions. quantitative levels tended to diminish with time; in individual patients they fluctuated in a dampened hemi-sinewave pattern in parallel with recurrent bouts of active disease. synovial fluid from all of patients contained immune complexes, compared to only of concomitant sera. in the positive sera the concentrations were much less than in the corresponding joint fluids. we suggest that the causative agent is introduced into the skin, where ecm is the initial clinical manifestation of a systemic, immune-mediated inflammatory disorder that often becomes localized to and propagated in synovium. thus, lyme arthritis can be considered a human model for an infectious etiology of rheumatoid arthritis. little attention has been given to analgesia, divorced from suppression of inflammation, in rheumatoid arthritis (ra), despite its importance to patients. this study was designed to determine the comparative efficacy of five often used analgetics for the pain of active ra synovitis. single doses of mg aspirin (asa), mg acetaminophen (ac), mg codeine (co), mg pentazocine (pe), mg propoxyphene (pro), and placebo (pi) were given in a randomized double blind fashion, and on a prn basis, to each of carefully selected hospitalized r a patients with painful synovitis of at least joints. all other drugs and distracting activities were prohibited during the study period and at least hours of observation required between doses of test agents. subjects recorded time of onset and duration of maximal relief, percent of relief achieved from each agent and all possible side effects, and, after taking all agents, ranked their order of preference, with as most effective. onset and duration of action of all agents were similar except ac and pi, which required longer to act and lasted a shorter time. side effects occurred significantly more frequently with pe, somewhat more often with co, but were otherwise insignificant. effectiveness was analyzed according to some of all ranks, mean percent relief and percent of subjects achieving % or greater relief for each agent. as expected, by parameters measured in this study and by cost to the patient, asa is a superior analgetic in ra. however, ac, by cost and effectiveness, is an acceptable alternative, as codeine would be except for its narcotic properties and cost. cost, side effects, and limited effectiveness restrict the usefulness of pe. pro seems no more effective than pi and of questionable usefulness in ra, except perhaps as a placebo. sum there is evidence that the inflammatory properties of crystalline material may be modified by their interaction with serum proteins. in this study, the activation of c by crystals is quantitated by two dimensional crossed immunoelectrophoresis (tdiep). in polypropylene tubes . ml aliquots of fresh human serum were mixed with . ml of buffer containing . mg of washed crystals. the tubes were incubated with agitation of oc for hour and then centrifuged at ,ooog for minutes. aliquots of the supernatants were diluted appropriately in edta-containing buffer to inhibit further conversion of c . by use of standard techniques, tdiep was performed using rabbit antisera to human c . a method was devised whereby samples could be compared simultaneously on a single plate. after staining, the combined area under the pic and bia peaks was measured using planimetry. the numerical results below are percentage points of total area under the p a peak in excess of that seen in saline controls. the coefficient of variation of the ratio of the two peaks was . for a single specimen on the same plate. the areas could be corrected to indicate milligrams of c converted. sodium urate was highly active in the electrophoretic conversion of c with a dose and time related response. activation was prominent a t concentrations of crystals seen in gouty effusions. heating the crystals at ' c for hours reduced c conversion by nine-tenths, but also altered morphologic properties of the crystal preparations. conversion was inhibited by increasing concentrations of edta and eliminated by . m edta. the edta inhibition was not seen in the presence of excess calcium. with the conditions described above, potassium urate crystals were much less active ( %) than sodium urate ( %). eight different commercially available preparations of steroids gave varying but small amounts of c activation (- t o %). it is demonstrated that different crystals and altered forms of the same crystals vary in their interaction with serum and their ability to activate complement. it is not known what properties are responsible for these phenomena. the calcium requirement suggests that the interaction of sodium urate with complement occurs at or before c . it is possible that immunoglobulin, which has a high affinity for sodium urate and is found in sodium urate crystals in vivo, is altered and initiates complement activation by the classic pathway. temporal artery pain, polymyalgia rheumatica, and blindness are well recognized manifestations of giant cell arteritis. however, the disease may often present in a less evident fashion. this is demonstrated by ( %) of our series of patients. of patients with biopsy-proved giant cell arteritis, the chief complaint was polymyalgia rheurnatica in ; temporal pain in , and loss of vision in . in the remaining patients, the predominant complaint that brought them to the physician did not suggest the diagnosis of giant cell arteritis, since headache and polymyalgia were either absent or so minimal as to escape notice. these complaints included fever of unknown origin in patients; malaise, anorexia, weight loss, and abnormal liver function tests suggesting occult malignancy in ; and unexplained anemia in . four patients presented with a neurologic syndrome; had diplopia and acute weakness of one arm. claudication was the chief complaint of patients, involving the leg in one, the arm in one, and the jaw in . all patients responded well t o steroid therapy. the diagnosis of giant cell arteritis in patients such as this who do not present in typical fashion depends on detection of a very rapid erythrocyte sedimentation rate, biopsy of a temporal artery, and the awareness that the disease may manifest itself in a variety of ways. the administration of certain drugs induces clinical and laboratory features of systemic lupus erythematosus (sle) in man but few studies of this human disease model have been undertaken. hydralazine, a useful antihypertensive agent, is one of these drugs and has acquired a "bad press" because of such observations. a prospective study of hypertensive patients with age, sex, and race matched controls was begun years ago. all subjects had complete clinical examinations; anf, d n a binding; immunoglobulin and complement levels; skin tests to a battery of antigens and lymphoprolifera-tive responses t o mitogens and antigens obtained prior t o starting hydralazine and serially throughout the study. all subjects were a n f negative at the start of treatment. fifteen of the have shown varying a n f response (in undiluted serum) but only had a titer of :lo or above, one becoming positive at months and at months. one of these subjects had clinical symptoms suggestive of sle and the drug was stopped. five subjects have shown a proliferative lymphocyte response to hydralazine and/or its metabolites (jci : , ) including the with positive anf. acetylation rates were obtained in of the hypertensive patients. thirteen were slow and fast acetylators. the a n f positive subjects were all slow acetylators as were of the with proliferative lymphocyte responses to hydralazine and its metabolites. this study t o date suggests that hydralazine in its present form may be less likely t o cause serologic and clinical sle than previously reported and that screening by acetylation rates may define a susceptible population. this and other lupus related drugs continue t o be a rich source of data relevant to lupus in humans. (supported by an niamdd grant). normal neutrophils (pmns) develop intracytoplasmic inclusions after incubation with sle sera. the resulting inclusions stain for igg, igm, and c and are believed to be immune complexes removed from the sera in vitro. such inclusions have also been identified in the circulating pmns from sle patients. the present study examined the relationship between pmn inclusions and clinical and laboratory features of sle. sera were carefully collected at " from sle patients. normal buffy coat cells were incubated in the sle sera for minutes a t ' and then centrifuged and washed. slides of the washed cells were prepared in the cytocentrifuge, stained with fitc goat anti-human igg, igm, iga, and c , and examined under ultraviolet light. the composition of these complexes was also examined with a fluorescent probe for double-stranded polynucleotides with the aid of ethidium bromide (eb). eb was added t o sera known to contain high concentrations of anti-dna antibodies and then incubated with normal pmns as before. preparations included ) no added eb, ) eb only, ) fitc anti-human igg (fianti-igg) or ) f -anti-igg and eb. inclusions containing both igg and igm correlated with clinical activity (p < . ), depressed serum complement (p = . ), cryoglobulinemia (p = . ), and anti-ndna antibodies (p < . ). igg inclusions alone were not significantly correlated with any of the parameters. c and igm appeared to be mutually exclusive, i.e. when one was present, the other was never present. eb staining inclusions (red fluorescence) suggested that polynucleotides were present. these findings suggest: ) the pmn inclusions consist of immune complexes which contain double-stranded polynucleotides, ) these complexes correlate with disease activity when igg and igm are both present, and ) such complexes may contribute to a number of granulocyte disturbances seen in patients with sle. in previous studies on preservation of articular carti-( , dpm/mg protein) permitted quantitation of the neulage performed in our laboratories, it was found that addition tral protease activity released into the media of the stored of a-tocopherol ( pg/ml) to the culture media of stored cartilage explants. employing this assay we found that acartilage resulted in improved preservation of its biochemical tocopherol resulted in significant inhibition of this activity as and biomechanical properties when compared to cartilage shown in the table. stored in absence of the vitamin. although it has been shown that a-tocopherol stabilizes lysosomal membranes in other lage were due to stabilization of lysosomal membranes, we no. control + a-tocopherol control + a-tocopherol developed a highly sensitive assay for neutral protease activity employing a biosynthetically prepared protein substrate. this five further patients with sle have been treated with plasmapheresis. six to liters of plasma per week were removed, using a haemonetics model hood cell separator. from to liters were removed in all. levels of complement components were measured by immunodiffusion and by a hemolytic assay. dna antibodies were measured by passive hemagglutination of dna coated red-cells. immune complexes were measured in vitro by the raji-cell assay. two patients were critically ill. one had seizures, pericarditis, pleurisy, and severe edema, despite treatment with mg prednisone daily for one month. the raji assay was pg/ml (normal < pg/ml). following the removal of liters of plasma her condition improved dramatically, complement levels rose to normal, and raji assay fell to - pg/ml. the second acutely ill patient had lupus cerebritis, with a level of only pg/ml on raji assay. after the removal of . liters of plasma there was no significant improvement in her clinical condition. the remaining three cases were less severely ill and had raji assays of , , and pg/ml. they showed a reduction of circulating immune complexes after each plasmapheresis, and an overall, variable fall after to weeks of treatment, but no striking clinical benefit. those cases with a high raji-assay also showed increased binding of doublestranded dna. the raji assay appears to be of value in predicting the response to plasmapheresis: patients with a very high level may show a massive and sustained reduction, with a good clinical response. in those with lower levels, the response is less predictable. plasmapheresis appears to be a valuable accessory form of therapy in the severely ill patient with acute sle. to investigate the vascular lesion in scleroderma (sd), the effect on human endothelial cell (ec, umbilical cord) growth of sera from patients with early sd was compared with healthy control sera. ec growth was monitored by tritiated thymidine chtdr) uptake and by direct cell counts. shtdr uptake of ec was inhibited up to % by of sd sera compared to the remaining sd and control sera. mean htdr uptake was cpm for control sera (range: - l), cpm for the noninhibitory sd sera ( - ). and cpm for the i i inhibitory sd sera ( - ). the sd patients with inhibitory serum activity tended toward shorter disease and symptom duration and were more likely to have peripheral edema and hypergammaglobulinemia than sd patients without inhibitory activity. furthermore, the inhibitory effect was no longer present in one patient after therapy with glucocorticoids which was associated with reduction of peripheral edema and improvement of myositis. the activity was further characterized in selected sera. sd serum completely inhibited the increase in ec htdr uptake seen with increasing normal serum concentration from to %. htdr uptake correlated with reduction in ec number ( % reduction at % sd serum concentration versus % increase with control serum) and with cytotoxicity ( % cell death versus % in control serum). these effects were not observed when the target cells were mouse t or human fibroblasts, smooth muscle cells, or peripheral monocytes, suggesting specificity of the inhibitory effect of sd sera for ec. the inhibitory effect of sd serum was heat stable ( "c, minutes), nondialyzable, present in both plasma and serum, and unaffected by mixing with control serum. when serum was fractionated on sephadex g- column, the inhibitory activity was found to elute in the position of serum albumin. sera from patients with non-sd active rheumatic diseases (ra, sle, pan, dm) showed no inhibitory effect on human ec, fibroblasts or smooth muscle cells or mouse t cells. these preliminary observations describe an ec-specific cytotoxic serum factor(s) in patients with sd. the in vivo role of this factor(s) in the pathogenesis of the proliferative vascular lesions in scleroderma is unknown. activation? activation of the complement (c) system in ra has been amply evidenced by a) detection of c and immune complexes in ra synovial leukocyte inclusions; b) depressed ch , c , and c levels as well as split products of c , c , and fb in ra synovial fluids; c) split products of c and c in ra plasma samples; and d ) hypercatabolism of c in meta-bolic turnover studies. we measured the fractional catabolic rates (fcr) of radioactive labeled c and fb in ra patients and normal controls to assess the relative importance of classical and alternative pathways of complement activation in ra. our results demonstrated: ) hypercatabolism of c in of ra subjects and inverse variation of c fcr with plasma c levels; ) hypercatabolism of f c in of ra patients; ) c fcrs disproportionately higher than fb fcrs in of ra subjects undergoing simultaneous studies; ) correlation of c and fb catabolism and homologous igg catabolism; and ) occurrence of hypercatabolism of c mostly in the extravascular space, a phenomenon previously observed by us also with the igg hypercatabolism of ra. patients with high fb fcrs had higher titers for rheumatoid factors. patients with high c fcrs had higher total joint counts and a greater number of manifestations of extraarticular disease. c and fb fcrs did not correlate with circulating immune complexes by the raji cell assay or with the sedimentation rate. the results suggest that complement activation in ra occurs primarily through the classical pathway and in the extravascular compartment, probably principally by immune complexes not detected by the raji cell assay, and with participation of the alternative pathway in some of the patients. studies from this laboratory have demonstrated that the pha and con-a responses of splenic lymphocytes from rats with adjuvant-induced disease are diminished. these diminished responses return to normal with spontaneous, corticosteroid (cs), or methotrexate (mtxtinduced remissions. the diminished pha and con-a responses are due to two types of suppressor cell: one which adheres to plastic ar glass, and another which adheres only to glass. the latter cell, which accounts for the bulk of the suppression, is more sensitive to the inhibitory effects of cs and mtx in vitro than are the mitogen-responsive lymphocytes. virazole (vzl), a synthetic nucleoside, is a noninterferon inducing antiviral agent which inhibits aid. four groups of highly inbred fisher rats were studied as follows: ) untreated rats; ) rats treated with mg of vzl given days prior to sacrifice; ) rats treated with freund's adjuvant given days prior to sacrifice; ) rats treated with freund's adjuvant followed by vzl days later. whole spleen cell suspensions were studied, as well as suspensions after removal of plastic-adherent cells by incubation in petri dishes or after removal of glass-adherent cells on columns of glass wool. each of these three types of suspension was also incubated with a range of concentrations of vzl and with either pha or con a. ah-rdr incorporation into nucleic acid was determined at hours. spleen cells from untreated rats, rats given mg of vzl days prior to sacrifice, and adjuvant-treated rats given vzl had normal pha and con a responses. the spleen cells from rats given adjuvant alone had markedly diminished pha and con-a responses due to the two types of supprcssbr cells, both of which were more sensitive to vzl in vitro than were the mitogen responsive cells. this preferential sensitivity of suppressor cells to vzl was greater than that noted with cs or mtx. two possible interpretations are: ) vzl has a more specific immunosuppressive effect on suppressor cells than cs or mtx; ) suppressor cell function is due to a virus which is inhibited by vzl. twenty-four cerebrospinal fluid (csf) samples from patients with systemic lupus erythematosus (sle) were evaluated for cyclic-gmp (c-gmp) concentration by radioimmunoassay. for comparison studies, the patients were divided into three groups based on their clinical status at the time of each lumbar puncture: those with ) active neurologic and psychologic abnormalities (group i), ) active neurologic abnormalities only (group ii), and ) psychologic abnormalities only (group ). groups i and i had mean csf c-gmp values of . nm f . (se) and . nm f . respectively, which were both significantly higher than the mean for group i ( . nm f . ) (p < . ) as well as for the mean of a previously studied group of patients with lumbosacral pain syndromes ( . nm f . ) (p < . ). other csf findings did not display this close correlation with activity of neurologic disease. the number of samples with abnormal csf leukocyte counts was significantly greater for group i ( / ) compared with that found for group ( / ) (p < o.ol).however, no significant difference was found between group i and group , for this abnormality. in sle patients significantly higher levels of csf c-gmp were found on serial sampling during times when neurologic abnormaltiies were active. neither prednisone nor psychoactive drug therapy had any demonstrable effect on csf c-gmp levels in these pa-tients. comparison of clinical markers of sle disease activity among the three groups revealed hematologic and serologic abnormalities not to be of significant value in distinguishing sle patients with active neurolgic involvement (groups i and ) from those without (group ). however, fever and, to a lesser degree, rash, proteinuria, and abnormal urinary sediment were present more frequently in groups i and i than in group . thus elevated csf c-gmp concentration may be a marker of active neurologic disease in sle. assay of csf c-g m p may therefore be helpful in the clinical assessment of sle patients with neurologic and/or psychologic abnormalities both with regard to their diagnosis and therapy. this study reports the first serial observations of c-gmp in a group of sle patients with correlation of manifestations of disease activity and extends our previous studies of cyclic nucleotides in sle. mp produces a chronic arthritis in mice with histopathologic features resembling rheumatoid arthritis. since the chronicity of arthritis appears to result in part from persistence of mp within the joints, elucidation of host mechanisms responsible for elimination of mp is of considerable importance. the role of complement in the pathogenesis of mp-induced arthritis was studied in mice congenitally deficient in the fifth component of complement (c ). in all experiments, at least . -week-old male mice per strain free of detectable mycoplasmas were inoculated intravenously with x p color change units of mp. subjective assessment of the arthritis was carried out by scoring ankles, wrists, qetacarpal, metatarsal, and digital joints on a scale of to . in a preliminary study c deficient dba/ mice and strains of normal mice: t.o., c h, cba, and c bl/ were used. in the normal strains the .arthritis peaked in severity within days and declined thereafter. in contrast the arthritis in the dba/ mice reached a plateau at days and gradually progressed in severity for as long as months postinoculation. a second study was carried out with strains of c deficient mice, i.e. dba/ , akr and a strain and normal strains: c h and c bl/ . the arthritis in the c deficient strains persisted at a high level months after inoculation despite considerable variability in the severity of the acute arthritis among the strains. the arthritis in the normal strains rapidly declined as before. at months postinoculation, mp was isolated from the joints, lungs, and spleens of c deficient mice more frequently and in larger numbers than from normal mice, consistent with the failure of cs deficient mice to eliminate mp. complement-fixing antibody to mp was detected in higher titers in c deficient strains. the results demonstrate the importance of c in the elimination of mp from the joints and organs of infected mice. moreover, the study may have clinical relevance since secondary complement deficiencies exist in a number of rheumatic diseases. it supports the concept that complement deficiency might contribute to the persistence of as yet undefined etiologic agents in human arthritidies. we have studied american black patients with primary ankylosing spondylitis (as) unassociated with ulcerative colitis, crohn's disease, psoriasis, or reiter's syndrome. all the patients were unrelated and met the rome criteria for as. sixteen ( %) of them possessed hla-b . we further studied the remaining hla-b -negative patients to see if there was an association with other b locus antigens belonging to the hla-b cross-reactive group (hla-b , b , bw and bw ). hla-b was present in of these patients ( . %) compared to of ( . %) b -negative black controls (x' with yates correction = . , p < . , relative risk = ). bw was found in one patient who also had b . bw was not tested for. antisera used to detect b , b , and bw were free of noteworthy cross-reactivity. duration of disease, age at onset of as, skeletal manifestations, prevalence of acute anterior uveitis and frequency of a positive family history for as were compared between the b -positive (gp-i) and the b -positive (gp- ) patients. duration of disease at the time of study was not significantly different in the two groups. however, mean age at onset of as differed significantly: . years in gp-i and . years in gp-i (p < . ). skeletal manifestations of the disease did not differ significantly in these groups. uveitis occurred in patients ( %) in gp-i and patients ( %) in gp-i (p = . ). a family history of as could be obtained in none of gp-i patients and ( . %) of the gp-i patients (p = . , using fisher's exact test). these data indicate that hla-b may be associated with the development of as in the b -negative black popu-lation. the b -positive black as patients are older at onset of their disease than the b -positive patients and tend to lack obvious familial aggregation of the disease. the findings suggest that american black as patients lacking b but possessing b represent a subgroup of patients with this disease. prostaglandin synthesis inhibitors (pgsi) may affect renal function in humans, especially under conditions of renal injury such as lupus nephritis. to explore this preferential effect in systemic lupus erythematosus (sle), we measured basal excretion of urinary prostaglandin e (ipge) by radioimmunoassay in female sle patients and normal women on a constant meq sodium diet. no patient or normal control received nonsteroidal antiinflammatory drugs during the study period but of patients were on maintenance low dose prednisone ( i mg/day). six of patients had biopsy-proved nephritis; had insufficient urinary findings to prompt a diagnostic biopsy. all ipge values were determined in triplicate on at least three separate -hour collections except in one patient with two such collections. sle patients had a significantly higher mean ipge excretion than normals ( . f . versus . f . ng/hour; mean f sem; p < . ). sle patients with biopsy-proved nephritis had a higher mean excretion than those without biopsy ( . f . versus . f . ng/hour) although the difference was not statistically significant. urinary sediment and general disease activity did not correlate with urinary ipge. all patients showed a decrease in creatinine clearance with pgsi administration but the percent change did not correlate with basal urinary ipge excretion. elevated urinary ipge excretion in sle may reflect renal inflammation due to immune complex nephritis and may explain the greater sensitivity of renal autoregulation to pgsi in this setting. the therapy of lupus glomerulonephritis has been evaluated in a randomized study of corticosteroid treated patients comparing bolus ivcy ( . - . mg/kg, every months), combined oral cy + az ( mg of each, daily), and prednisone alone (pr). five years after the start of the trial, patients have been randomized: ivcy ( ), cy + az ( ), and pr ( ). renal involvement at study entry has been characterized by urine sediment abnormalities ( ), impaired function (crci < ml/min) ( ), nephrotic proteinuria (lo), glomerular proliferation on biopsy ( ), and an estimated disease duration of less than one year ( ). the assigned drugs have been continued without complications in patients with a median course of months. toxicity has necessitated discontinuation of the drugs in courses; cy + az (pulmonary infection, hemorrhagic cystitis, amenorrhea) and ivcy (hepatitis). death has occurred in patients; ivcy (epiglottitis, sudden death), and pr (pulmonary embolism). the changes of renal function of the patients receiving a minimum of months of continuous therapy have been assessed by evaluating the number of patients demonstrating a reduction of function (crci) by at least % (alo) or % (a ) from baseline determinations at study entry. this interim analysis of an ongoing trial is encouraging and suggests that ivcy and cy + az may be more effective than pr in the maintenance of renal function of patients with lupus glomerulonephritis. extended follow-up will be required to determine the duration of these effects and the development of drug related complications. the mechanism(s) underlying the destruction of bone observed in chronic inflammatory diseases such as rheumatoid arthritis or osteomyelitis remain(s) to be elucidated. soluble mediators derived from immunologically activated cells may play an important role in such phenomena. we have observed that alloantigen-stimulated human mononuclear leukocytes (mnl) release a factor(s) capable of resorbing fetal rat bones in vitro. we have investigated procedures for the large-scale generation and partial characterization of this bone resorbing factor(s). human mnl obtained by leukophoresis (ibm cell separator) of two nonrelated healthy individuals (yield = - x lolo cells/donor) were cultured together. ( . x v cells/ml, hours, "c, mishell-dutton atmosphere). supernatant fluids were removed and assayed for bone resorbing activity by measurement of ca release from fetal rat bones in vitro. these supernatants effected significant bone resorption when compared to either control supernatants prepared from a single donor's cells or medium alone. indomethacin ( x to x wm) did not inhibit either the production or biological activity of this factor(s). bone resorbing activity eluted with molecules of - ,ooo daltons on sephadex g- and appeared in the breakthrough when applied to deae cellulose ( . m phosphate, . m naci, ph . ). eluate containing bone resorbing activity after sephadex g- chromatography x and deae cellulose chromatography was applied to amionic disc gels ( "c, ph . , trisglycine). a single peak of bone resorbing activity was observed, although this activity could not be attributed to a stainable protein bond. the results indicate that alloantigen stimulated human mnl elaborate a low molecular weight bone resorbing factor(s) resembling the previously described osteoclast activating factor (oaf). methods presented for the large-scale generation and partial purification of this activity should facilitate clarification of the role of this factor in immune tissue destruction. inflammatory cells may play a role in modulating connective tissue growth and function in a number of rheumatic diseases. the interaction of lymphoid and connective tissue cells was studied in vitro by exposing human dermal fibroblasts (fb) to supernatants (sn) of peripheral blood mononuclear cells (pbmc). proliferation was assessed by bh-thymidine incorporation and direct cdl counts. protein synthesis was determined by ah-proline incorporation and collagen synthesis by protease-free collagenase releasable counts. sn of both unstimulated and pha-stimulated pbmc suppressed fb growth in a dose-dependent fashion. sn suppressive activity was non-dialyzable, heat-stable, not cytotoxic, and removed by absorption of sn with fb but not with pbmc. there was suppression of protein synthesis by pbmc-sn in direct proportion to the suppression of cell number ( %), but disproportionately greater suppression of collagen synthesis ( %). culture sn of t-cell depleted pbmc were as active in suppressing fb growth as were sn of unfractionated mc ( - % suppression, mean f % for t-depleted versus - %, mean f % for unfractionated). supernatants of t-cell enriched populations had decreased activity ( - %, mean f % suppression). the growth suppression seen was due, at least in part, to stimulation of fb prostaglandin (pg) synthesis. immunoreactive pge, production by fb was increased -fold ( . versus . ng/ cells) by pbmc-sn. when inhibitors of pg synthesis (indomethacin, sodium meclofenamate) were added to fb cultures just prior to addition of pbmc-sn, the growth suppressive effect was abrogated ( - %). furthermore, exogenously added pge, (final concentration = ng/ml) resulted in suppression of fb growth comparable to that seen with pbmc-sn. thus, lymphoid cells produce soluble factors which induce fb to modulate their own growth in vitro and which alter collagen synthesis. the effector of the growth autoregulation appears to be a pg. this system provides an in vitro model for the study of lymphoid and connective tissue cell interaction which may improve the understanding of proliferative and fibrotic human disease. although there is compelling evidence to support the view that altered endocytic-lysosomal functions of synovial tissue may play a role in the joint tissue injury seen in rheuma-toid arthritis (ra), there are no data which quantitate these functions in human synovial cells (sc). therefore, we investigated the rates of pinocytosis and intracellular digestion of a soluble protein, horseradish peroxidase (hrp), in monolayers ( - days in primary culture) of sc from ra and non-ra patients. pinocytosis was linear with increasing h r p concentrations ( . - mg/ml), time ( - min), cell numbers ( - x ) and cell protein ( -lo pg; x i@ cells equivalent to loopg protein). specific activity (sa; ng h r p taken up/ pg cell protein/ hr) increased in direct proportion to cell concentration. sa of r a cultures ( - pg protein) after exposure to mg/ml h r p was f (mean f sd; n = ); an equivalent concentration of non-ra sc had a sa of f (n = ). uptake in both r a and non-ra sc was inhibited most effectively by - m potassium fluoride ( %) and °c ( %). r a sc digested % of cell bound h r p (t%) in hours, whereas t% for non-ra sc was hours. uptake and digestion of immune complexes by ra sc were also studied. insoluble hrp-anti h r p complexes formed at equivalence were added to sc at . pg hrp/ml (total protein concentration . pg/ml). uptake of complexed h r p was at a rate ( . % of this load/ pg cell protein/hr.) approximately times that for soluble h r p (at a load of mg/ ml). only % of complexed h r p was digested by hours. these studies indicate: ) human sc have a relatively high rate of pinocytosis and r a sc demonstrate the greatest rates of uptake; ) kinetics of pinocytosis in sc are primarily those of fluid phase uptake; ) the rate of pinocytosis increases with increased cell density, a relation which may be important in ra synovial tissue; ) r a sc can readily accumulate immune complexes, but their ability to digest them is much more limited than it is for soluble proteins. further quantitative studies of the mechanisms whereby sc process joint constituents, soluble antigens and immune complexes should lead to a more precise definition of the contribution of sc endocyticlysosomal functions to tissue injury in ra. this study was designed t o evaluate the effects of several nonsteroidal antiinflammatory drugs commonly used in arthritis therapy on gastric mucosa, and to evaluate the effects of agents, tolectin and motrin, in subjects with demonstrated clinical intolerance to aspirin. gastroscopy was carried out before and after all treatment schedules, and gastric mucosa was graded by the endoscopist (fl) on a scale of - +. photographs were taken and subsequently reviewed in a randomized double-blind manner by both the endoscopist (fl) and an impartial gastroenterologist (rn). remarkably consistent results were obtained: part i. forty volunteers were randomly divided into groups with subjects in each group and treated for week with either motrin ( mg/day), motrin ( mg/day), indocin ( mg/day), indocin ( mg/day), naprosyn ( mg/day), naprosyn ( mg/day), aspirin ( . gm/day), and placebo. part . five normal volunteers who had developed + hemorrhagic gastritis during a previous study after week of aspirin ( . gm/day) were given motrin ( gm/day), tolectin ( mg/day), and placebo for week in a randomized crossover manner with at least a -week washout between medications. results. part i. placebo subjects always showed the least pathology followed by motrin ( me), naprosyn ( mg), motrin ( mg), indocin (i mg), indocin ( mg), naprosyn ( mg), and aspirin ( . gm). in patients receiving naprosyn ( mg) and indocin ( mg) frank gastric ulcer was produced. differences between motrin or mg and aspirin were highly significant (p < . ) as were differences between aspirin and placebo (p < , ). differences between motrin ( mg) and indocin ( mg), and between motrin ( mg) and naprosyn ( mg) approached significance ( . < p < . ). part . of the subjects taking tolectin, had hemorrhagic gastritis of a degree comparable to that with aspirin, and had similar but less extensive changes. one subject on motrin had extensive hemorrhagic gastritis as with aspirin, and had minimal or no changes. in both parts i and , gastric mucosal biopsies, blood levels, and photographs confirmed endoscopic pathology. this study demonstrated that severe gastric mucosal hemorrhagic and ulcerative changes occur in subjects using nonsteroidal antiinflammatory drugs, and that significant differences exist between various drugs. seven patients with active gonococcal (gc) arthritis immunoelectrophoresis (cie). the diagnosis of gc arthritis were studied for the presence of gc antigen (ag) and anti-in all seven patients was made by typical clinical presentation, body (ab) in serum and synovial fluid by counter-positive local culture for ngonorrhoeae (ng) , and response to treatment. gram stain and culture of synovial fluid, as well as blood cultures, were negative in all patients. specimens were run against rabbit antisera to n g (difco) to detect gc a g and against a turbid solution of fresh isolates of n g to detect g c ab. glass slides were coated with ml of . m agarose in . m barbital buffer p h . . pairs of wells mm apart and mm in diameter were cut in the agar and filled with lop of samples-ag in the cathode well and ab in the anode well. slides were electrophoresed in barbital buffer for minutes at room temperature at mamps/slide, and examined for the presence of precipitin lines. results are shown in the table. g c a g was found in the serum of patient and in the synovial fluid of others. gc ab was detected in the serum of the remaining patients and in simultaneous synovial fluid of of these. g c ab was absent in convalescent serum in patients # and # at months. only l of patients with initial or recurrent active gc urethritis, cervicitis, or salpingitis had serum g c ab detected by cie; none had g c ag. these synovial fluid patient no. results suggest that the finding of gc a g or ab in synovial fluid o r serum by cie provides evidence for gc arthritis and may be positive when gram stain and culture are negative. cie therefore appears to be a useful test in the diagnosis of this condition. f m f frequently presents a diagnostic dilemma in childhood. its musculoskeletal manifestations may be confused with septic arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, or other rheumatic disease. although % of patients have symptom onset in childhood, diagnosis is usually delayed. we have followed patients who presented before age years. there were male and female children. the mean age of onset was . years (range - ); mean age at diagnosis was . years (range - ). there were armenians, sephardic jews, and of other ethnic origins. all had recurrent fever and peritonitis, pleuritis, or arthritis, without evidence of other rheumatic or nonrheumatic disease. all were of mediterranean ancestry. presenting complaints included fever in loo%, abdominal pain in %, chest pain in %,joint pain in %, and skin rash in %. musculoskeletal manifestations ranged from arthralgias to arthritis. the knees were involved in children, ankles in , shoulders in , sacroiliac joint in , and wrists in . both children with sacroiliac joint involvement had marked erosive changes and were initially thought to have ankylosing spondylitis. four patients had splenomegaly. n o patient had proteinuria o r other evidence of amyloidosis. all of children tested were negative for hla-b . fifteen children with frequent and severe attacks, mean age . years (range - ), were selected for colchicine therapy. patients were begun on the nearest whole tablet equivalent of .o mg/meter squared. in nonresponders, the dosage was raised to a maximum of . mg/day. six patients were noncompliant, but the remaining children have been followed for - months (mean ) on continuous colchicine therapy. the mean number of attacks for the month period prior t o initiation of therapy was . (range - ), but only . (range - ) for the most recent months of colchicine therapy (p < . ). the only side effect was transient diarrhea, which usually resolved without reduction in dosage. although the long term safety of colchicine in childhood has not been established, n o deleterious side effects have been observed. it appears that children tolerate colchicine well. colchicine therapy may allow children incapacitated by severe attacks of f m f t o assume a nearly normal life. a new amino acid, y-carboxyglutamic acid (gla), originally discovered in prothrombin where gla residues are sites for specific binding of ca++ ions, more recently has been identified in osteocalcin, a non-collagenous protein from bone matrix. based on amino acid composition and sequence of bovine bone gla-protein, osteocalcin is distinct from all other vitamin k dependent clotting proteins (factors , vii, ix, and x) synthesized in the liver. the posttranslational formation of gla, studied extensively in liver microsomes from warfarin treated rats, occurs in a vitamin k dependent enzymatic oxidative carboxylation of selected glutamic acid residues in prothrombin requiring bicarbonate ion and nadh or chemical reducing agent. to show that gla synthesis occurs in bone tissue and that the carboxylase enzyme has similar requirements in bone, microsomes were prepared from the long bones of day old chick embryos. the eggs were injected at , , and days with sodium warfarin, an inhibitor of gla synthesis, to allow for the accumulation of endogenous substrate available for specific labeling with nah cob. the microsomes separated from bone homogenates by differential centrifugation incorporated c into gla only in the presence of vitamin k . "c-gla was identified after alkaline hydrolysis of the microsomes and separation on an amino acid analyzer. further confirmation of "c-gla was obtained by acid hydrol-ysis of the putative gla component resulting in decarboxylation to glutamic acid and recovery of approximately % of the incorporated radioactivity in glutamic acid. the posttranslational enzymatic generation of specific calcium binding sites in bone suggests that the vitamin k dependent carboxylation reaction may have a possible role in regulating mineral deposition in calcified tissues. since the formation of a specific calcium binding amino acid in bone is vitamin k dependent, anomalies might be expected to occur in mineralized tissues under conditions of vitamin k deficiency or anticoagulant therapy. of significance is the finding of gla-containing proteins in ectopic calcifications (subcutaneous scleroderma and dermatomyositis plaques, atheromatous plaque) which suggests gla may be important in the formation and resorption of bone in a wide variety of disorders. a retrospective study was instituted on patients in the ucla lupus nephritis clinic in an attempt to determine the ability of three serologic indicators-specifically immune complexes (ic), anti-dna antibodies (dna-ab), and c -to predict the activity of sle renal disease as indicated by changes in hour proteinuria, serum creatinine, and creatinine clearance. we chose not to assess urinary sediment because of difficulty in quantitating terms such as "rare," "occasional,'' and "full field" as used by different observers. patients' records and serum samples from the period - were employed. the mean number of matched clinical samples per patient was with a range of - . similarly, the mean number of matched serologic samples per patient was with a range of - . "normal" daily or short term variation for each clinical and serologic parameter was assessed from paired samples taken within - days of each other. these "normal" values were then used as a baseline, variations from which could be tested for abnormality by statistical techniques. by use of simple regression analysis it was noted that ic as determined by % polyethylene glycol precipitation correlated better than either dna or c with clinical parameters. intriguingly, ic correlated with neither dna-ab or c . more importantly, when attempts were made to predict deterioration of clinical parameters - weeks after either a single abnormal serologic determination or after paired determinations of serologic parameters demonstrating significant deterioration, no test or combination of tests produced less than a % false positive rate. however, all tests were capable of predicting stability or improvement of clinical parameters with a true negative rate that averaged . % for ic, . % for c , . % for dna-ab. no combination of tests improved these rates. pyogenic infection originating within the intervertebral disc space, once thought to be uncommon, is becoming increasingly recognized. we report the clinical findings in adult patients with non-tuberculous pyogenic infection of the intervertebral disc occurring in two teaching hospitals over an month period. chills, rigors, and low grade fever were common prodromal complaints ( :ls). the acute onset of back and neck pain ( :ls) with point tenderness over the involved spinous process ( : ) most accurately indicated the diagnosis. referred abdominal pain or sciatica were less commonly seen. acute paraparesis or quadaparesis occurred in patients with epidural abscess. the sedimentation rate reflected disease activity in all patients. plain radiographs were positive in only patients. tomography was helpful in another . ""'tc pyrophosphate bone scan was positive in : cases and responsible for diagnosis in of patients with normal radiographs. overall the bone scan was positive in of cases studied. responsible organisms were cultured usually from the involved disc space ( : ). staphlococcus aureus ( ) and e. coli ( ) were the most common pathogens, although other microorganisms were responsible. no pathogen was isolated in cases. delay in diagnosis averaged weeks. previous spinal roentgen abnormalities ( ), disc surgery ( : ) , another site of active infection ( : ) , and alcoholism ( : ) were predisposing factors. intensive intravenous antibiotics followed by two months oral antibiotic therapy proved curative. ten of patients could be followed one year after therapy and were asymptomatic except for patients with pre-existing mecha-nial low back pain. none had evidence of active septic discitis. one patient died of pulmonary embolism post-discectomy. epidural abscess was the usual indication for surgery. the sedimentation rate returned to normal in all survivors. septic discitis is a common disease, but delay in diagnosis is usual despite a characteristic clinical syndrome. the sedimentation rate and bone scan are the most useful adjuncts to diagnosis. aspiration of the suspected disc space for responsible organisms is advocated. most patients are afforded excellent prognosis following judicious rest and prolonged antibiotic administration. musculoskeletal complaints of uncertain etiology have been noted in up to % of patients receiving a kidney transplant. we have studied prospectively consecutive renal transplant recipients. joint pain, stiffness, and swelling were assessed - days in every week of the postoperative period. six patients experienced joint pain affecting chiefly knees but also shoulder, ankle, and temporomandibular joints. arthralgias were variable in severity and duration, and in time of onset after transplantation. in patients pain coincided with reduction of steroid dosage and was abolished by modifying the treatment regimen. a third patient, who developed transient joint pain days after appearance of a painless effusion, had hyperparathyroidism. joint effusions, confirmed by arthrocentesis, appeared in knees of patients. mean duration from transplantation to first joint aspiration was days. effusions were generally unaccompanied by pain, except on extreme flexion, and persisted for hours to weeks. twenty-five synovial fluid samples were obtained from the knees. the mean synovial fluid volume was ml (range, - ml) and the mean cell count was /mma (range - /mms). in every case viscosity and mucin test were normal and crystals were not found on polarization microscopy of the centrifuged fluids. joint x-rays showed only soft tissue swelling. electron microscopy of three fluids revealed no hydroxyapatite-like material. synovial biopsies in patients were normal by light microscopy. no cases of aseptic necrosis have been identified. tests for serum ana, immune complexes, and rheumatoid factors were all negative. neither the arthralgias nor effusions bore any apparent relationship to the source of the donor kidney, renal function, fever, or use of anti-thymocyte globulin. no major transplant rejection episodes occurred in any of the patients with effusions or arthralgias, whereas no joint problems arose in any of patients who rejected donor kidneys in the postoperative period. thus, benign effusions are common after renal transplantation. they appear unrelated to immunologic factors or crystal deposition and may be due to transudation associated with high-dose steroid treatment. to investigate the hypothesis that infection may precipitate certain arthritis syndromes, we performed a battery of infectious agent serologic titrations on early-diagnosed arthritis patients and normal controls. forty-five antigens were employed: for common bacteria, for mycoplasma, for respiratory viruses, for other viruses, and for y enterocolitica. the arthritis patients included with sle, with ra, with jra, and with arthritis of undetermined diagnosis (aud). data were analyzed by diagnostic groups and b status. no significant difference was found among the arthritis and control groups in antibody titers to any of the common bacterial antigens or the mycoplasma species, of the respi-ratory viruses, and of the other viruses. in jra patients, adenovirus (group) and herpes simplex (types and ) virus titers were somewhat low, and in the ra and aud groups cytomegalovirus titers were low. in sle, mumps virus titers were slightly higher than in the other groups. however, in contrast to the occasional difference serologic titrations with previously mentioned antigens, highly significant differences in antibody titers to all domestic y enterocolitica antigens ( serotypes) were found among the groups. arthritis patients had consistently lower median levels than normal controls with the lowest titers found in sle, followed by jra, aud, and finally the ra group. the finnish y enterocolitica antigens (serotypes and ) showed no significant difference among groups but yielded the lowest median titers of all yersinia antigens. evidence of yersinia reactive arthritis was not found in this patient sample, and median antibody titers of the b positive and b negative arthritis patients without either rheumatoid factor positivity or the diagnosis of sle were highly comparable. the results provide no serologic evidence of infectious agent association with early-diagnosed arthritis but reveal impressive hyporeactivity to domestic y enierocoliticu serotypes which suggests decreased natural igm agglutinating antibodies to gram negative organisms as previously reported in sle patients (baum j, ziff m: j clin invest : , ) . among the various microvascular abnormalities present in the skin of patients with connective tissue diseases and demonstrable by in vivo microscopy, the most characteristic pattern of capillary abnormalities is found in patients with scleroderma (sd) (maricq hr, spencer-green g, leroy ec: skin capillary abnormalities as indicators of organ involvement in scleroderma (systemic sclerosis), raynaud's syndrome, and dermatomyositis. amer j med : - , ) . the prevalence of this sd pattern in a larger sample of patients with sd and related connective tissue diseases was studied in three separate arthritis centers and in patients with the following diagnoses: sf (so), systemic lupus erythematosus (sle, a), mixed connective tissue disease (mctd, ), and raynaud disease (rd, ) . results from widefield microscopic observations and photomicrography show sd-type capillary abnormalities present in the following numbers of patients in each diagnostic category: sd, ( %); sle, ( %); mctd, ( %); and rd, (%). tortuous capillaries were noticed in patients with sle ( %), with sd ( %). with mctd ( %), and with r d ( %). only patients (with mctd) had both a sd pattern and tortuous capillaries ( % of mctd or % of all patients). non-specific microvascular changes were present in patients with sle ( %), with sd ( %), with mctd ( %), and with rd ( %). many sle ( patients, %) and r d ( patients, %) patients showed no capillary abnormalities, as did patients with mctd ( %) but only with sd ( %). these results confirm the high frequency of sd-type capillary pattern in sd patients and separate them from patients with sle, who do not show a pathognomonic pattern by the present techniques. tortuous capillaries, frequent in sle, occur in other diseases and in normal subjects, although to a lesser degree. it is interesting to note that patients with mctd, an overlap syndrome having clinical features of both sd and sle, exhibited no specific pattern of capillary abnormalities. about half of the patients showed sd pattern, and the other half demonstrated tortuous capillaries, other non-specific changes, or no observable microvascular abnormalities. clinical criteria for classification of systemic sclerosis (ss) were derived from prospectively entered data on early-diagnosed rheumatic patients contributed by centers. cases included patients with definite or probable ss, or scleroderma in overlap. controls were patients with systemic lupus erythematosus (sle), polydermatomyositis, or raynaud's phenomenon alone. computer assisted and multivariate analysis techniques were used to construct criteria with minimal redundancy. careful definition of cutaneous involvement contributed the most powerful criteria. sclerodermatous involvement proximal to the metacarpal phalangeal or metatarsal phala-ngeal joints (ie, "proximal" scleroderma), whether in an acrosclerotic distribution (acrosclerosis), on the face or neck (scleroderma facies), or on the trunk or abdomen, was present in % of definite ss cases and in only % of combined controls. if the major criterion of proximal scleroderma was absent, ss cases could be distinguished by having at least of minor criteria, ie, sclerodactyly, digital pitting scars, pulmonary fibrosis, or large bowel sacculations. employing the major with minor criteria yielded % sensitivity in definite ss patients and % specificity in total controls, without using exclusions. when applied to an independent rheumatic patient the course of articular involvement in early rheumatoid arthritis (ra) is variable and has not been quantitatively characterized. in a prospective study of younger adult, early ra patients (negative for hla-b ), detailed articular, systemic, laboratory, and therapeutic data were collected twice yearly for a mean followup of over years. based upon the number of joints involved during followup, each patient was assigned to separate pain/tender (p/t) and swelling (sw) articular course patterns. monocyclic pattern was defined as one cycle of documented arthritis with articular remission observed for at least year; intermittent as two or more arthritis cycles, each separated by joint remission of at least months; continuing as continuous joint involvement but without persistent progression; and progressive as continued progressive involvement. the table shows numbers of cases in each pattern, mean numbers of joints involved at entry, and the mean annual change (a) in numbers of joints involved during followup, determined by regression analysis. monocyclic groups included significantly more males (p/t, p < . ; sw, p < . ), and seronegative cases (p/t, p < . ; sw, p < . ). only one woman experienced complete articular remission while progressive swelling occurred in only cases. entry joint involvement correlated with subsequent p/t and sw articular patterns (p < . and p < . , respectively). cross-sectional analysis of the numbers of joints involved at each protocol exam and longitudinal analysis of regression slopes of individual patient joint involvement both indicated trimodal distributions: monocyclic; chronic (ie, combined intermittent and continuing groups), and progressive patterns, which were independent of therapy. the data provide quantitative evidence for monocyclic, chronic, and progressive articular course patterns in early ra, with identification of significant sex and clinical correlates. in some postmortem cases of systemic lupus erythematosus (sle) associated with diffuse proliferative glomerulonephritis (dpgn), an antigen related to mammalian type c rna viral core (p ) protein is deposited in the glomerular lesions in an immune complex pattern (proc natl acad sci : , ) . now an attempt is made to support this finding by examining the glomerular immune deposits in sle-dpgn for evidence of type c virus antibody. human immunoglobulins (igs) were eluted from the glomerular immune deposits in two fractions by sequential treatment with dnase to elute anti-dna antibodies, followed by acid buffer to elute remaining antibodies. the eluted igs were then assayed by a sensitive, specific, and quantitative enzymoimmunoassay, which compares favorably with radioimmunoassay, for the detection and measurement of anti-p antibody against purified p proteins of the four chief groups of mammalian type c viruses: murine, feline, endogenous feline rd-i /endogenous primate, and simian sarcoma virus type i (ssv-i)/infectious primate virus groups. human igs which showed specific anti-p antibody activity, particularly against p antigen of the rd-i virus group and to lesser extent against p antigen of the murine and ssv-i virus groups, was eluted by acid buffer from the glomerular immune deposits in two cases of sle-dpgn that were known from previous study to contain deposits of viral p -related antigen in the same tissue lesions. these findings support the hypothesis, stemming from studies of the new zealand mouse model of sle, that subinfectious antigenic expression of a type c virus is involved in the pathogenesis of dpgn associated with sle. (this work was supported by nci, dhew grant no. male b/w mice generally die after one year of age with lupus nephritis and/or lymphoid malignancy. these mice gradually develop a severe depression of cell-mediated immunity which may contribute to the subsequent fatal autoimmune and lymphoproliferative disease. we studied t lymphocyte function in young ( - month) and old ( - month) male b/w mice in separate paired experiments. the spleen cell response to phytohemagglutinin was , cpm (geometric mean) for young mice compared with cpm for old mice (p < .oool). when spleen cells from young and old mice were mixed together, there was significant suppression of young cells by old splenocytes in all experiments ( - % suppression). this suppression is mediated by a radio-resistant, nonadherent, mononuclear leukocyte, probably a small lymphocyte. although this suppressor cell is eluted in the "t cell fraction" of a nylon wool column, it cannot be identified as a t cell because it is resistant to anti and complement treatment . in a typical experiment, unfractionated old spleen cells incorporated cpm compared t o , cpm for young spleen cells. a mixture of old and young cells incorporated , cpm, whereas the predicted incorporation for this / mixture was , cpm ( % suppression). old spleen cells from the t cell fraction of the nylon wool column were enriched for suppressor cells ( % suppression in the mixing experiment). in contrast, the b cell fraction was completely depleted of suppressor activity (actually % greater than predicted incorporation). anti and complement treatment of spleen cells from a n month old b/w mouse demonstrated % suppression by the untreated cells and % suppression by the preparation depleted of &bearing cells. these results have been consistently reproduced. there is no evidence of a suppressor cell in old mice of four normal strains (dba/ , c b / , balb/c, and nzw). we conclude that a mononuclear suppressor cell, probably a lymphocyte, contributes significantly to the severely depressed t lymphocyte function of aging b/w mice. this suppressor mechanism may participate in the pathogenesis of the lymphoproliferative and autoimmune disorder characteristic of the strain. none of the clinical and laboratory parameters presently used for the assessment of sle kidney disease is completely reliable. immunofluorescence of the urinary sediment (ifus) has been recently reported to accurately predict the rejection of renal transplants, and the purpose of this study was to evaluate its usefulness in renal sle. thirty consecutive patients who met the ara preliminary criteria for sle were included in the study. all had recent kidney biopsies and were treated with prednisone and immunosuppressors at usual doses with urinalysis, urinary light chains, c , anti-dna binding, npn, and creatinine as parameters of activity. random urine specimens were collected weekly for months and the urinary sediment was studied by direct immunofluorescence with antisera anti iga, igg, ig m, and fibrinogen degradation products. clinicai grading and fluorescence readings were done by independent observers. nine patients had normal kidney biopsies and ifus was always negative. nine patients had abnormal biopsies, were classified as inactive, and ifus was always negative. in patients with abnormal biopsies and active renal disease, ifus was always positive, even before the appearence of proteinuria, hemoglobinuria, casts, and light chains. ifus was positive to months before c dropped in patients and became negative to months before c returned to normal in patients. in the presence of residual proteinuria, ifus became negative in patients. there was no correlation be-tween the histological type and the immunofluorescence findings in the kidney biopsy with the type of immunoglobulin found in the urine. this procedure is easy to perform, not costly, and the results are available within hour. our data suggest that it reflects the changes in renal status months before the other parameters and therefore it may be a valuable method for guiding a more flexible treatment of patients with sle kidney disease. fifteen percent or less of patients with juvenile rheumatoid arthritis (jra) have positive latex fixation tests (lft); whereas, approximately % have hidden rheumatoid factor (rf) ( s igm r f in the peripheral blood probably bound with s igg and, therefore, not detectable by the standard lft). in this study, a complement-dependent hemolytic assay was used to determine the presence of hidden rf. sera of children with seronegative jra, with seropositive jra, children with connective tissue diseases, with leukemia, and normal children were separated by gel filtration at ph . to obtain the igm-containing fraction. these igm fractions were subjected to the complement-dependent hemolytic assay in which sheep erythrocytes (srbc) are coated with reduced, alkylated, and acid-treated rabbit igg anti-srbc antibody and are hemolyzed by guinea pig complement in the presence of s igm rf. thirty-seven of patients with jra, of which of polyarticular jra, of pauciarticular jra, and of systemic jra, had titers > : . none of normal controls and only of disease controls had titers > : . the median titer of all jra patients was : and healthy and disease controls, : . estimates of the significance of the differences between the median titers of jra patients and of the controls were obtained by mann-whitney analysis. they were significant at p < . . when data from patients with active disease were analyzed separately, the median titers for polyarticular jra were : , pauciarticular : , and systemic : . patients with inactive disease did not have significantly different titers from controls. the active polyarticular and pauciarticular values were significant at p < . and p < . . these results demonstrate: ( ) % of seronegative jra patients have hidden rf by this procedure; ( ) the hemolytic assay is more sensitive than the lft or sheep cell agglutination tests (scat) in determining the presence of hidden rf in jra; and ( ) activity of disease correlated with higher titers in the hemolytic assay, and the assay was superior to the lft or scat as an indicator of disease activity. this study was devised to answer in relation to articular cartilage proteoglycans (pgs) whether degradation products or products of incomplete synthesis are detectable in the earliest pathological lesions of this rabbit model of osteoarthritis. an ultramicro transport method for obtaining s value polydisperse profiles for pg aggregates (pgagg) and subunits (pgs), and miniaturized extraction, dialysis and cscl density gradient ultracentrifugation techniques have allowed study of these pgs and various pg products within the s value range of - from histologically defined sites. thirty new zealand white rabbits (weighing - . kg) were subjected to partial medial meniscectomy and killed - weeks postoperatively under nembutal anesthesia. micro-dissected medial femoral condyle samples included ) ulceration, ) mm rim surrounding the ulcer often with fibrillation, and ) cartilage from the medial and lateral femoral condyle which occasionally revealed fibrillation but usually appeared to be normal. amounts of cartilage equivalent to ) above were obtained from the contralateral (left) knee for use as a primary control and from sham operated (right) knees for use as a second control. the cartilage from rabbits was required to obtain a pool of - mg wet cartilage for each zone - above. these cartilages were diced and pgs extracted, dialysed, and partitioned. for pg extracted with . a gucl from left femoral articular cartilage of control knees, the profile shows a subunit peak with weight average sediment coefficient s; o of about and an aggregate peak extending from up to s with an average of . similar profile data on the tibia cartilage provided an average sediment coefficient of . about / of pg was in aggregated form and the rest pgs. interestingly, the undissociated pgagg revealed an s value range of - . pgs extracted without dissociation in . m gucl gave similar data. in regard to the profile of s values of pgs from small ulcers, the weight average sediment coefficent s: o of the peak was . peaks indicative of pg products with lesser values were found in pooled samples with large ulcerations. a similar study run after isolation of pgs in a dissociative csci gradient confirmed this result. in conclusion, the major abnormality detected early was a disturbance in pg aggregation, and later pgs degradation. we recently observed the development of glomerulonephritis in patients with sicca syndrome (ss) who did not fulfill the diagnostic criteria of systemic lupus erythematosus. these patients developed ss to years prior to the onset of glomerulonephritis. two were diagnosed as having membranoproliferative glomerulonephritis by light and electron microscopy, and was found to have membranous glomerulonephritis. although immunofluorescent studies were not available, all clearly had microscopic evidence of immune complex (ic) deposition. moreover c' levels were decreased in all three a t the time of onset of renal disease, and all had high levels of circulating ic as determined by the lz i clq precipitation test. these findings raised the possibility that ic plays a role in the development of certain aspects of tissue injury in ss. for these reasons patients with ss were studied for the presence of circulating immune complexes with the lasi clq precipitation test. increased ' ' ' clq binding activity (clqba) ( %) was found in ( %) patients (mean clqba = %, range - %). thirty-five patients ( %) had rheumatoid factor (rf) present in their serum. analysis by spearman rank correlation revealed a positive association between the clqba and the titer of r f as determined by the bentonite flocculation test (bft), (rho = . , p < . ). it was found that the highest positive correlation between clqba and bft titer existed for those patients with ss alone (rho = . , p < o.oos), and then decreased for those patients with ss plus extraglandular disease (rho = . , p < . ) and even more for patients with ss plus rheumatoid arthritis (rho = . , . < p < . ). there was no apparent correlation between clqba and bft titer in patients with ss plus another connective tissue disease. pretreatment of sera with . m mercaptoethanol to dissociate igm into monomers resulted in the eradication of rheumatoid factor as measured by bft but caused only slight to moderate reductions in clqba. thus circulating immune complexes exist in many patients with ss, are distinct from rheumatoid factor, and may contribute to certain aspects of tissue damage in this disease. we have recently described a high ( %), but not absolute, association of sicca syndrome (ss) with the hla-dw allelle, which is in linkage disequilibrium with hla-b (nejm : , ) . in this study we determined the blymphocyte antigens of patients ( females and males, ages to ) and normal controls. in addition to ss, of the patients have rheumatoid arthritis and have systemic lupus erythematosus. immunoglobulin-bearing lymphocytes were separated from heparinized peripheral blood on goat anti-human igg (fab) coated plastic plates and tested with antisera in complement dependent cytotoxicity tests. the antisera used were obtained from multiparous women and absorbed with pooled platelets to remove hla-a, -b, and -c antibodies. two antisera, and ags, reacted with the b-lymphocytes from all the ss patients compared to and % of the normal controls, respectively. four additional antisera ( , , , and ) reacted more frequently ( , , , and %) with the patients' b-cells than with those of controls ( , , , and %) . the remainder of the antisera tested had the same frequency of reactivity in this disease as in normals. t o determine if these antisera recognized the same or different antigens, their reactions with the lymphocytes from the normal controls were compared by the x ' test. antisera correlated with both and but not with ags or . antisera ags correlated only with . similar statistical analysis was done for the patients. the antisera and ags were excluded because of their % prevalance in ss. in the ss patient group antisera , , and were associated with each other but not with . this association can be due t o either cross-reactivity or linkage disequilibrium. family studies are in progress to determine whether ss patients can be heterozygous for and ags and, if so, whether they can be present in trans position. assuming that the b-lymphocyte antigens in humans are coded by loci of an ir region, our results suggest that two immune response genes may be involved in the pathogenesis of ss. in addition, the absolute association of and ags antigens with these patients should provide a n additional aid in diagnosis of ss. glucocorticoid administration is associated with induction of osteopenia in man and in some laboratory animals. there is debate about dose and duration required, and whether bone loss is primarily trabecular o r cortical. accordingly, adult female rabbits were divided into groups: received saline, received . mg cortisol/kg body weight (wt), and received . mg cortisol/kg wt. all received equal volumes subcutaneously once daily early in the morning. xylenol orange ( mg/kg days before kill) and tetracycline ( mg/ kg days before kill) were injected as bone markers. rabbits were given food and water ad libitum, and weights were measured every - days. bone mineral content (bmc) and width (w) were measured by photon absorptiometry before treatment and every weeks. measurement site was the humerus cm proximal to the bent elbow (all cortical bone). results are shown in the table. histopathology. the amount of bone was reduced in cortisol-treated rabbits. in the humerus this was localized to the endosteal surface. in the lumbar vertebrae the endosteal side of the ventral cortex was primarily affected with some thinning of trabeculae. calculation of rates of formation (f) and resorption (r) showed f was essentially nil in the cortisol groups. r was identical in the saline and low-dose cortisol rabbits, while r was accelerated in the . mg/kg treated rabbits. analysis of bmc and cross sections from the same site of the humerus for bone area showed a good correlation (r = . ). summary. rabbits treated with cortisol in the doses used developed generalized bone loss. this loss appears to involve more cortical than trabecular bone. bone marker studies show that the main effect is interference with f, and, with the larger dose, acceleration of r. in vivo measurement of bmc accurately reflects the progression of the bone loss. intermediate complexes are polymers of igg-rheumatoid factor which sediment between the . s and s components of normal serum. they are best identified by analytical ultracentrifugation. their presence in serum can be inferred by a double "gullwing" precipitin line of the igg on immunoelectrophoresis due to accumulation of the molecular aggregates of igg around the well. in this study we have shown they can also be detected by evaluation of the difference between the electrophoretically determined y-globulin concentration and the igg concentration determined by radial immunodiffusion (rid). molecular aggregates of igg are quantified by serum protein electrophoresis, but they are underestimated by r i d because their effective or molar concentration is low relative to the monomeric igg standard. therefore, when molecular aggregates of igg are present, the difference between the electrophoretically determined y-globulin and the igg measured by r i d is abnormally high. sixty-one consecutive blood donor sera and hypergammaglobulinemic sera from patients with diseases known not to be associated with the presence of intermediate complexes o r in which the presence of intermediate complexes was excluded by analytical ultracentrifugation made up the reference population. eleven sera with known intermediate complexes were examined. the mean and % tolerance intervals (covering % of the population with % confidence) of the reference population for the difference between the electrophoretically determined y-globulin concentration and the igg as measured by r i d was . f . g/dl. eight of i patients' sera with known intermediate complexes fell outside the upper limit. all sera which fell within the % intervals had concentrations of intermediate complexes less than . g/ dl. in addition, the degree of deviation from the reference mean showed a direct linear correlation with the level of intermediate complexes present. if the electrophoretically determined y-globulin concentration minus the igg concentra-tion by r i d is greater than .o g/dl, intermediate complexes should be presumed to be present in concentrations greater than . g/dl. synthesis of antibodies to dna (anti-dna) in systemic lupus erythematosus (sle) is postulated t o result from a lack of control mechanisms which normally suppress autoantibody production. we investigated these mechanisms by measuring anti-dna synthesis by normal and sle peripheral blood lymphocyte (pbl) populations stimulated with pokeweed mitogen (pwm). pbl isolated from patients with sle and controls were further separated into tand b-cell enriched fractions by density sedimentation of spontaneous rosettes formed by aet treated sheep red blood cells and t lymphocytes. a portion of the t-cell fraction was irradiated with rads to inactivate were cultured with normal or sle-b cells ( . x iv) at ratios these studies show that sle-b cells from some patients are capable of synthesizing anti-dna which is best demonstrated in co-culture with suppressor inactivated, irradiated t cells. normal-t cells suppress the response, whereas suppression exerted by sle-t cells is variable from patient to patient. anti-dna the antibody produced by rheumatoid arthritis (ra) synovial lymphocytes may be directed at a uniquejoint antigen inciting the inflammation, and thus may be useful to identify the antigen. we have made permanent r a synovial lymphoblastoid cell lines capable of producing unlimited quantities of antibody. r a synovial membranes were obtained surgically, finely minced in medium (rpmi , % fetal calf serum, glutamine, antibiotics), placed in mm linbro plates, infected with epstein-barr virus, incubated ( oc, air + % co,) and fed weekly. typical lymphoblastoid cell lines developed in of ( %) cultures after a mean days, range - . once established, the cell lines were put in large flasks and medium changed completely times a week. eight of the permanent lines were lost after a mean duration of days (range - ) due to bacterial contamination or poor viability. the latter was due to suboptimal growth support by specific lots of rpmi or serum; subsequently these were pretested. the remaining lymphoblastoid lines now have a mean duration of days (range - ), during which their mean volume was ml (range - ). maximum volume was . liters, but could be expanded indefinitely in liters or larger flasks on shaker platforms at low speed. mean cell doubling time was hours, range - . igg concentration in harvested supernatant medium was measured sequentially by a radioimmunoassay described previously. mean igg production by each line was , , , , and pg igg/day, decreasing slowly with time. adjusted for cell concentration, mean production was . , . , . , . , and . pg igg/iv cells/day. total production to date was . , . , . , . , and . milligrams. the culture producing the lowest amount may be producing a predominance of another immunoglobulin type. in of lines igg production tended to be significantly higher when cell concentration was lower (r = . - . ; p < . ). these ra lines produced more igg than previously reported for lymphoblastoid lines derived from other human tissues ( - bg/lp cells/day). they also produced - times more igg than our previous batch organ cultures of ra synovia. their increased igg production may be due to improved culture conditions and/or intrinsic immunologic hyperactivity of ra lymphocytes. these cultures may provide an excellent source of antibody specific for the antigen(s) inciting ra. recently we reported that partial disuse of a joint produced a defect in proteoglycan (pg) aggregation in human articular cartilage indistinguishable from that seen in osteoarthritis. the present study examines the rapidity with which defective pg organization develops after total immobilization of a limb, and the reversibility of the defect. the right hind limb of dogs was immobilized in a cast for days to weeks, at which time the animals were killed. during this period the dogs ambulated freely on legs but bore no weight on the immobilized limb. in some cases the cast was removed after weeks and the dogs then ambulated fully for up to weeks prior to sacrifice. cartilage from the distal femur of the immobilized and the contralateral control knees was cultured for hours in ham's f- nutrient mixture containing iwo fetal calf serum and s ,. pgs were extracted with m guanidinium chloride (guhci) and purified by successive cesium chloride density gradient centrifugations in . m and . m guhci, ie, under conditions favoring forma-tion of pg aggregates and disaggregation of pgs, respectively. after only days of immobilization a s , incorporation into pgs was suppressed by , and this diminution in pg synthesis persisted through weeks of immobilization. after weeks of immobilization no evidence of pg aggregation could be found. at that time pgs from the second gradient were the same size as those from the first gradient (sepharose b k., = . ) and showed no shift in their sepharose b elution profile after incubation with hyaluronic acid (ha) in vitro, indicating that pg-ha interaction had not occurred. however, week after removal of the cast, aggregates had again formed in the cartilage and were as large in hydrodynamic size as those in control cartilage. these results emphasize the importance of joint motion in maintenance of the normal organization of cartilage pgs. the pg aggregation defect which occurs with immobilization alters hydraulics of the cartilage, especially with impact loading, and may thus predispose to chondrocyte injury. in the course of examining host defenses against infection in patients with systemic lupus erythematosus (sle), we have found a previously undescribed serum inhibitor of complement (c )-derived -chemotactic activity (ctxa). serum from of patients, when activated with zymosan, failed to attract polymorphonuclear leukocytes (pmn) comparably to normal zymosan-treated serum (zts) (measured by the "leading front" method of zigmond and hirsch). incubation of normal pmn with these sera did not affect their random motility or subsequent chemotactic response to normal zts. whereas levels of c and c (measured immunochemically) were modestly low in these sera, no gross abnormalities involving alternative complement pathway activation could be detected. when preincubated with normal zts ( : ) at " for minutes, these sera caused significant inhibition ( - ooo/o) of ctxa. they also inhibited the ctxa of columnpurified c -derived peptides (from normal zts), but had no effect on the ctxa of either the synthetic peptide, n-formylmet-leu-phe or the bacterial chemotactic factor from e coli. the inhibitor in these patients' serum was heat-stable ( °c for minutes) and acted specifically on c -derived ctxa (not on pmn). mixing (without preincubation) of patient serum with normal zts failed to cause inhibition of ctxa. the inhibitor also acted reversibly; molecular sieve chromatography dissociated heat-stable inhibitory activity (a single peak with an apparent molecular weight of - , daltons) from normal amounts of c -derived ctxa in patients zts and in mixtures of normal zts incubated with patient serum. further characterization of the inhibitor has revealed it to be a basic protein (pi between and ) which can be inactivated completely by treatment with pronase. despite its effect on c derived ctxa, the inhibitor did not influence two other c derived biologic activities in zts: pmn lysosomal enzyme releasing activity and pmn aggregating activity. this heatstable inhibitor, uniquely specific for c -derived ctxa in serum from some patients with active sle, may account, in part, for increased susceptibility to infections caused by pyogenic microorganisms. the evidence that type c viruses are involved in systemic lupus erythematosus (sle) is conflicting. we tried to detect type c expression in a total of sle patients during various collaborative studies over the past four years. forty tissues ( placenta or gestational products, spleen, kidney, other) and/or cell cultures derived from them were tested a total of times using different methods. seventyfive percent of the tissues were tested by or more methods and % by or more. type c virus isolation was attempted using four distinct protocols: culture with sedimentation of sh-uridine-labeled virions, cocultivation with viral rna-dependent d n a polymerase assay, cocultivation with focus formation assay for helper virus rescue of the defective murine sarcoma virus genome (a albino), and triple cell fusion with viral polymerase assay. thirty-four tissues from patients were tested a total of times with negative results except for one type c isolate in a recent experiment. detection of both type c interspecies and primate species antigens was attempted using different radioimmunoassays in separate laboratories (g. j. todaro, h. p. charman), and indirect immunofluorescence (r. c. mellors). twenty-two tissues from patients were tested a total of times with negative results. electronmicroscopy of gestational products from patients revealed type c-like particles in placentas, but also in of normal controls (m. imamura). type c-related sequences were not found in cellular d n a from patients using hybridization to a murine type c cdna probe (g. s. aulakh). various false-positive results were also encountered in most of the studies. the virus isolate-positive patient has not yet been tested by other methods, but the patients with type c-like particles were each tested by to other methods with negative results. thus of the total tests were positive on of the tissues from patients. these combined collaborative studies are the most comprehensive yet done in sle. if type c expression is enhanced in sle, it is not regularly demonstrable using current methods. a prospective clinical, serologic and histopathologic study was performed on consecutive patients suspect for sjiigren's syndrome (ss) referred to an interdepartmental university clinic during the years - . one hundred female and male patients met at least two of the following three criteria: ) lymphocyte focus sco.re greater than one (fs > ) on labial salivary gland (lsg) biopsy, ) keratoconjunctivitis sicca (kcs), ) associated extraglandular connective tissue or lymphoproliferative disorder. of the ss patients, lsg biopsy fs > occurred in %, definite kcs in %, and associated extraglandular disease in %. rheumatoid arthritis was present in %, scleroderma in %, systemic lupus erythematosus in %, and polymyositis in %. lymphoproliferative disease or connective tissue abnormalities not fulfilling criteria for a coexisting connective tissue disease (ctd) were present in % of patients. patients with extraglandular disease and kcs almost always had a fs > ( : patients), whereas patients with extraglandular disease and a fs > did not necessarily also have kcs (only : patients). we conclude that lsg biopsy is more sensitive than kcs for detection of ss in patients with an underlying connective tissue or lymphoproliferative disorder, and may help establish a diagnosis in patients with clinically undiagnosed autoimmune or lymphoproliferative disease. moreover, lsg biopsy is far superior t o clinical symptoms or signs of salivary gland dysfunction which are not specific for ss. in fact, only % of patients with any symptoms suggestive of ss actually had the disease confirmed. local glandular disorders, anxietydepressive syndromes, and parasympatholytic drugs were common causes of oral and/or ocular complaints. our study suggests a new definition of ss as an autoimmune exocrinopathy based on the utility and diagnostic value of lsg biopsy. in view of the hla-b association and unique precipitating antibodies to nuclear antigens (ha, ss-a, ss-b) found in ss, autoimmune exocrinopathy might be considered a genetically and serologically distinct connective tissue disease related t o but separable from other connective tissue diseases. rheumatoid arthritis or a coexisting connective tissue disease occurs in only a minority of patients with autoimmune exocrinopathy and should not be a requirement for diagnosis. degradation of collagen at sites of tissue injury and inflammation is effected by the dual action of collagenase and nonspecific proteases present in neutrophils, macrophages, and other cells. monocytes eventually accumulate at such sites and as macrophages perform important phagocytic functions. mechanisms whereby monocytes are attracted to areas of in-flammatory reactions are incompletely understood, although several different chemotactic factors have been described. we have measured the chemotactic response of normal human peripheral blood monocytes to different types of human collagens and collagen degradation peptides by a modified boyden technique. chemotactic activity (ctx) expressed as monocytes per oil immersion field for various preparations tested were as follows: type i collagen ( . p m collagenase retained chemotactic activity. additional studies were undertaken with synthetic tri-and dipeptides containing amino acids common to the three different collagens. peptides containing proline or hydroxyproline (for example, gly-pro, gly-hyp, pro-hyp, gly-pro-hyp, and gly-pro-ala) were chemotactic for monocytes at concentrations ranging between lo-' and -bm. these data suggest that peptides generated as a result of degradation of collagen by collagenase and other proteases might function to chemotactically attract monocytes to sites of tissue damage and inflammation in vivo. although it is known that a small percentage of patients with sjbgren's syndrome (ss) may develop malignant lymphoma, the frequency of ss in lymphoma has not been established. therefore, consecutive untreated patients with biopsy-proven non-hodgkin's lymphoma were screened to establish the prevalence of ss. patients were defined as having ss if they exhibited objective evidence of keratoconjunctivitis sicca and xerostomia. all were assessed by history, physical examination, schirmer test, rose bengal staining of the cornea and bulbar conjunctiva, and if possible a serial salivary scintiscan, serologic studies, and a lip biopsy. of the patients seen, were identified as having ss. of these, all had a history of xerostomia and keratoconjunctivitis sicca, while had a positive schirmer test, had positive staining with rose bengal, and had a markedly abnormal salivary gland scan. three had a positive anti-nuclear antibody (ana) and anti-salivary duct antibody. of the with ss, had adequate lip biopsies. of these, were normal, and showed mild, non-specific lymphocytic infiltration, while was highly iuggestive of ss with lymphocytic infiltration and salivary gland atrophy. five of the had musculoskeletal complaints, had classic or atypical raynaud's phenomenon, and had a concurrent diffuse connective tissue disease ( had scleroderma and had probable rheumatoid arthritis). in summary, of patients screened, had ss as defined above. of these, had positive schirmer tests, had positive rose bengal staining, and had abnormal salivary scans. three had a positive ana, had anti-salivary duct antibody, and had abnormal lip biopsies. five had musculoskeletal complaints, had raynaud's phenomenon, and had a concurrent diffuse connective tissue disease. thus, ss in patients with non-hodgkin's lymphoma appears to be more common than is generally appreciated. hydroxyapatite crystals have been recently suggested as a cause of crystal induced synovitis in humans. we have performed the following studies in an attempt to develop an experimental model to further study the relation of hydroxyapatite to inflammation. articular calcification was induced in six-week-old nzw rabbits with techniques similar to those described by selye in other tissues. eight rabbits were given a single dose of , u oral vitamin d, and the next day left knees were injected with i mg feci,. right knees were injected with saline. four rabbits received intraarticular feci, without vita-min d. cartilage and synovial membrane were studied by light and electron microscopy at , , and days. synovianalysis, roentgenograms, and microradiography were also done. six other six-week-old rabbits were given vitamin d orally , u for days without any intraarticular injection. six controls were followed without vitamin d and all were killed at days for studies as above. mild synovial inflammation was seen in feci, injected joints without vitamin d. in rabbits given vitamin d there was tissue necrosis in the feci, injected joint with synovial calcification visible by light microscopy by the fifth day. calcifica-tions were all characteristic of hydroxyapatite by electron microscopy and could be seen in interstitium, occasionally on collagen fibers, and in vacuoles of synovial cells. small amounts of iron were seen in phagocytes without relation to the calcification. calcification increased over days. cartilage was not calcified except for a small surface deposit at days in one rabbit. synovial fluids had only very low leukocyte counts with predominance of mononuclear cells. joints not injected with iron were normal. the rabbits given vitamin d for days all developed round, mid-zone articular cartilage calcifications similar to those spontaneously occurring in older rabbits. there was no synovial calcification, inflammation, or joint effusion. by electronmicroscopy all calcium deposits were in the interstitium and were hydroxyapatite-like needles. many chondrocytes showed degenerative changes. thus, different patterns of articular calcification can be produced in rabbits' knees with techniques described. acute crystal associated inflammation was not demonstrated. crystals formed and sequestered in synovial or cartilage tissue appear to be tolerated without inflammation. studies of systemic lupus erythematosus (sle) families and populations suggest that a gene(s) linked to the major histocompatibility complex (mhc) influences sle. we have examined the mhc in sle families, patients, and controls by determining the cytotoxicity of antisera which detect mhcdetermined antigens expressed selectively on b lymphocytes. b lymphocytes from sle patients and controls were tested against a panel of pregnancy sera, and reaction frequencies of individual sera were compared. twenty-eight of the sle patients were also typed with a panel of hla-drelated sera from the th international histocompatibility workshop, hla-drw types assigned, and compared with workshop controls. the hla-drw types and individual serum reactivities which were increased in the sle population are shown. sle families shows that one hla haplotype is usually shared among those individuals with sle and other autoimmune abnormalities in a given family. exceptions exist, however, indicating that the haplotype itself is neither necessary nor sufficient for the expression of autoimmunity. this study demonstiates that certain mhc-related bcell alloantigens, possibly products of immune response genes, are increased in sle. family study indicates that the requirements for sle development are not limited t o the mhc, however, and probably involve additional genetic and/or environmental factors. clinical observations of increased osteoarthritis (oa) with menopause and studies of oa in experimental animals suggest that androgens worsen and estrogens ameliorate oa. further studies have demonstrated that estrogens suppress and androgens increase s incorporation into nonarticular cartilage. using a rabbit partial meniscectomy model of experimentally induced oa, we compared nontreatment ( rabbits), estradiol valerate . mg/kg intramuscularly every weeks ( rabbits), and testosterone cypionate mg/kg intramuscularly every weeks ( rabbits) on the development of oa and on proteoglycan (pg) synthesis by cartilage. animals were killed weeks post partial meniscectomy, and osteoarthritic lesions were noted. knee sections processed for histologic examination were stained with h and e and safranin- with fast green counterstain. cartilage metabolism in the groups was examined by in vitro measurement of ""so, incorporation into articular cartilage after hour incubation in dulbecco's modified eagle medium. medial and lateral components of femoral and tibia knee joint surfaces were studied separately. normal unoperated knees served as additional controls. frequency and severity of osteoarthritic lesions were the same in all groups. osteoarthritic and normal articular cartilage from estradiol-treated animals revealed statistically significant reduction in s s incorporation as compared to untreated animals. androgens had no significant effect on s incorporation. femoral s s , incorporation was uniformly greater than tibial , incorporation in all groups (p < . ). estradiol did not ameliorate nor testosterone worsen oa. both normal and osteoarthritic articular cartilage were susceptible to estradiol suppression of proteoglycan synthesis. the poor correlation between severity of oa and rate of pg synthesis may require reevaluation of the role of the latter in the oa disease process. variations in cartilage metabolism from different surfaces (femoral versus tibial) may relate to known differences in susceptibility of joints to development of oa. we have been analyzing antibody activities in sera from individuals working in various laboratories and in sera from a normal population. compared to the normal sample, the prevalence of elevated anti-dna activity was significantly greater in samples of sera from over personnel in systemic lupus erythematosus (sle) laboratories (p < . ), from laboratories involved with nucleic acids (p < . ), and from routine hospital laboratory personnel (p < . ). evidence that this anti-dna activity was due to gammaglobulin was obtained by the presence of y -d n a bound to igg in a radioautograph. in addition, differences were found in the prevalence of dna reactivity between the normal sample and the samples from the three laboratory groups when anti-human gammaglobulin was used as the precipitating agent. also, we have isolated the material from a serum containing high dna reactivity by a dna-cellulose affinity technique, and the fraction with dna binding activity contained only igg. lymphocytotoxic activity was also increased in the sera from sle laboratory personnel compared to the other laboratories and to the normal sample (p < . ), as previously reported (xiv international congress of rheumatology, abstract p. , ). immunoglobulin levels were analyzed by immunofluorescence. there was no significant difference in the igg and igm levels, but the mean iga level of the sle laboratory personnel was . f . mg/ml compared to . * . mg/ml in the normal sample ( p < . ). when sera from of the laboratory personnel with the highest anti-dna activities were compared to normal sera, additional abnormalities were found. not only was there a significant difference in the anti-dna activity (p < . ) and the iga levels ( p < . ), but also the mean igm level in the laboratory personnel ( . f . mg/ml) was significantly greater than in the normal sera ( . f . mg/ml)(p < . ). no difference in the mean igg levels was found. of additional specific antibody activities quantitated by radioimmunoassay, one of these (anti-bovine gammaglobulin) was elevated more frequently in the laboratory personnel than in the normals (p < . ). the data suggest that laboratory personnel tend to have an increased immune reactivity, particularly those working with sle sera. this condition might be due to laboratory exposure to a stimulus, causing an immune response that includes autoantibody production. in most cases, amyloid arthropathy (aa) has been associated with multiple myeloma or demonstrable paraproteinemia. various modes of presentation and laboratory features have been described. we have analyzed clinical, laboratory, and radiographic features of patients with aa seen over a -year period. none of these patients had identifiable diseases known to cause secondary amyloidosis. presenting symptoms were those of carpal tunnel syndrome ( : ) and/or swollen hands with stiffness ( : ) . periarticular tenderness of the hands and thickened palmar tendons were noted. pitting edema of the hands, at times massive, was noted in of . knee and elbow effusions were present in patients. sedimen-tation rates, rheumatoid factor, and antinuclear antibodies were normal or negative. serum and urine protein electrophoresis and immunoelectrophoresis failed to detect any paraprotein. joint fluids ( ) were noninflammatory except for one aspirated during an acute attack of pseudogout. radiographs showed degenerative changes ( : ) and chondrocalcinosis ( : ) . synovium obtained by open biopsy ( : ) revealed deposits of material which displayed metachromasia after crystal violet staining. in cases, electron microscopy was performed and revealed typical fibrils in synovium. deposits were localized to perivascular and subsynovial locations. attention is called to the presentation of aa as edema of the hands with or without median nerve compression. edematous hands and seronegative arthritis should raise the possibility of aa. these patients are also unusual in that aa was not associated with dysproteinemia and absence of a paraprotein should not discourage invasive measures to document aa. the associated chondrocalcinosis has been previously recognized in only one case report. chondrocalcinosis in of patients may be a chance occurrence in an elderly population (age range - ) or may cause aa by chronic local inflammation. antimalarial therapy for connective tissue disease has been limited by its potential retinal toxicity. the present study was undertaken to assess visual problems in patients treated with long-term hydroxychloroquine given in standard dosage. all patients who received both treatment and ophthalmologic evaluation a t albany medical college were included and consisted of patients treated for a t least year. each patient was examined a t baseline and then every months for visual acuity, central fields using a red test object, funduscopic abnormalities, accommodation, corneal deposits using a slit lamp, and keratoconjunctivitis by shirmer test. electro-oculograms (eog) were performed in patients who had received high total doses or had abnormal central fields suggesting toxicity. almost all patients received hydroxychloroquine in the maximal daily dose of mg. total dose ranged from to g (median ) and duration of treatment from to months (median ). diseases treated were rheumatoid arthritis in patients, systemic lupus erythematosus in , juvenile rheumatoid arthritis in , mixed connective tissue disease in , and other in . ophthalmologic toxicity was minimal. no patients were precluded from taking hydroxychloroquine at baseline evaluation. no corneal deposits or accommodation defects were found. three patients had abnormalities in central fields: paracentral scotomata and/or minor field restrictions. all had rheumatoid arthritis. toxicity occurred after total doses of , , and g. visual changes were completely reversible in the first patient who was the only one in the series with funduscopic changes. she has now received a total dose of g and has normal fundi, central fields, and eog. the second patient continues to have central field restriction but has had resolution of paracentral scotomata, a normal eog, and no visual complaints. the third had reversal of central field defects but had an abnormal eog when subsequently tested. we conclude that hydroxychloroquine in a dosage of mg/day is safe from significant ophthalmologic toxicity if followed by appropriate testing, and we find n o evidence from an association of increased toxicity with higher total dose or with the diagnosis of systemic lupus erythematosus. the metal-dependent neutral proteoglycanase, extracted from , grams of human articular cartilage, occurred in four electrophoretic forms. these were purified , -fold and produced single proteolytically active bands on disk electrophoresis. these forms were separated by preparative flat-bed isoelectric focusing and had approximate isoelectric points of . , . , . , and . . gel filtration and sds gel electrophoresis showed that they have an apparent molecular weight of - ,ooo and are composed of subunits of - , . gel filtration and dialysis indicated their tendency to disaggregate and reaggregate into monomers and dimers. their proteoglycanase activity passed through visking tubing and the passage continued on repeated dialysis with fresh buffer. this was prevented to a large extent by dialysis against zinc or cobalt ions. all the forms degraded the protein core of proteoglycan subunit optimally at ph . and were inhibited almost completely by addition of o-phenanthroline or passage through the chelating resin, chelex. however, they differed in their inhibition by edta, the most cationic forms being the least inhibited. also, the most cationic forms had n o activity on casein, histone, and the link proteins, indicating a relative specificity for degrading proteoglycan. these enzyme forms which are active at the ph in cartilage matrix may have an important role in proteoglycan degradation in osteoarthritis. bxsb mice (developed by dr. e. d. murphy, jackson laboratory) spontaneously evolve a lupus-like disorder similar to the disease of nzb/nzw (b/w) mice. however, in contrast to b/w mice, the bxsb disease is more severe in males who die of immune complex nephritis at a mean age of months. we have compared the formation of antibodies to dna in bxsb and b/w mice. the early appearance of autoimmunity in male bxsb mice is associated with a premature switch from igm to igg serum antibodies to dna. this is analogous t o the early switch from igm to igg antibodies in female b/w mice which is associated with accelerated disease and impending death. we have also measured the spontaneous synthesis of antibodies to dna by spleen cells cultured for hours. culture supernatants contain immunoglobulin which binds dna specifically as determined by radioimmunoprecipitation. two month old male bxsb mice produce more antibody to d n a in culture than d o age-matched female mice of bxsb, b/w, balb/c, or c b / strains. anti-dna production in culture increases with age in b/w mice. older female b/w mice subjected to prepubertal castration and treated with androgen produce significantly less anti-dna in culture as compared to sham or estrogen-treated controls (p < . ). the effect of hormone treatment on bxsb mice is currently under investigation. these results suggest that the male-dominant disease of bxsb mice is reflected in early synthesis of antibodies t o d n a which is apparent both in vivo and in vitro. female the rheumatoid nodule : clinical significance although the subcutaneous nodule (scn) is the extraarticular hallmark of rheumatoid arthritis (ra), its clinical significance has not been established. it was our thesis that the presence of scn would serve to identify the subset(s) of rheumatoid patients with systemic and highly immunoreactive disease. all patients with definite ( ) and classic ( ) rheumatoid arthritis who had an initial unit admission during the interval january through june were selected for study. primary reasons for admission included active synovitis ( %), orthopedic surgery ( %), and a miscellany ( %) of therapeutic complications and intercurrent illness. of the patients, ( %) had scn on admission or by documented history. medical record analysis per protocol revealed patients with and without scn were comparable in terms of demography, duration, and activity of ra. similarly, systemic features did not differ significantly between two groups, as shown in the table. with regard to sero-reactivity, patients with scn and the anodular group were also alike in terms of the presence of rheumatoid factors ( % versus %), antinuclear factors ( % versus %), and hypocomplementemia ( % versus %). there was a tendency only for those with scn to have higher latex test titers. thus, contrary to expectations, subcutaneous nodules cannot be relied upon to screen out those patients with rheumatoid variants and systemic disease. twenty-four patients ( women, men, mean age mg daily, versus placebo for months, while continuing years) with definite rheumatoid arthritis were entered into a baseline non-steroidal antiinflammatory therapy and/or preddouble-blind study using the immunomodulator levamisole, nisone up to mg/day. assessment criteria included articular index, grip strength, feet walking time, duration of morning stiffness, subjective pain relief, sedimentation rate, and latex fixation titers. to date, patients have completed the initial phase. eight o f on levamisole improved while the remaining patient, who did not appear to respond, flared after drug discontinuation. two patients on levamisole were excluded because of skin rash. four of on placebo appeared to improve but not to the degree seen with levamisole. the mean changes in articular index, number of swollen joints, and duration of morning stiffness were statistically significant a t months for levamisole treated patients but not for placebo (p < . ). subjective pain scale responses were significantly better among levamisole versus placebo, but n o differences were observed between the groups in grip strength or feet walking time. five levamisole treated patients converted to seronegativity after months. sedimentation rates, however, remained stable in both groups: skin rashes developed in patients on levamisole, and were discontinued; in , rash was unrelated, and continued on a lower dose. of interest, of on placebo had rashes. preliminary investigations of delayed skin hypersensitivity, lymphocyte responsiveness, phagocytic function, and cutaneous inflammatory responses failed to show any correlation with clinical responsiveness. levamisole is a potentially therapeutic agent in rheumatoid arthritis, but its mode of action is yet to be determined. since synovial fluid cells in patients with systemic lupus erythematosus (sle) have not previously been examined by electron microscope, we have studied sle joint effusions with emphasis on the occurrence of le cells and tubuloreticular structures (trs) and on correlations with clinical and light microscopic findings. all patients fulfilled at least ara preliminary criteria for sle and had antinuclear antibodies in their serum. three patients had drug-induced sle. synovial fluid volumes varied from . - cc; leukocyte counts ranged from - , but only were over , . polymorphonuclear leukocytes predominated in effusions including the one with , cells. cultures were all negative. monocytes and large sudan positive macrophages were prominent. joint fluid le cells were found in patients while had extracellular hematoxylin bodies, and had a variety of other smaller eosinophilic, hematoxyphilic, or cellular inclusions. le cells were identified by electron microscope in patients. the major and often sole visible constituent of the inclusion was a clump of short filaments about nm in diameter. these lay in vacuoles which also contained acid phosphatase. these filaments which appear to be products of nuclear chromatin also were seen extracellularly surrounding some cells and in smaller vacuoles. small acid phosphatase positive vacuoles also contained cell cytoplasmic debris, intact nucleii, erythrocyte fragments, and large amounts of finely granular protein-like material. the finely granular material was shown t o contain igg by immunoperoxidase electron microscope staining. t r s were found in synovial fluids and were predominantly in mononuclear cells with dense bodies and rough endoplasmic reticulum. they were infrequent in small lymphocytes or transformed lymphocytes with polyribosomes. two l e cells had trs. buffy coat blood cells contained trs in the cases studied. synovial fluid cell trs were seen in of inflammatory and noninflammatory synovial fluid controls but no control joint fluids had the filamentous le inclusions seen by electron microscope. n o correlation of any synovial fluid finding with therapy, effusion duration, or disease severity has been found. synovial fluids in sle contain trs, many phagocytic cells, and can have higher leukocyte counts and pmn percentages than often appreciated. le inclusions in joint fluid are composed predominantly of filaments derived from nuclear chromatin. the sera of patients fulfilling the ara criteria for systemic lupus erythematosus (sle) were studied by immunodiffusion and the modified farr technique for the presence of antibodies to the following antigens: ss-a, ss-b, rana (rap antigen), rnp, sm, scl- , and ds-dna. the incidence of antibodies to ss-b, rnp, sm, and ds-dna was similar to those previously reported. the incidence of anti-scl-l antibody was %, a finding which has not been reported pre-viously. the incidence of r a p ( %) was higher than the % previously reported. the incidence of anti-ss-a antibody was %. this antibody was found in high frequency in primary sjbgren's syndrome ( %) in the past but was not found in the small number of sle patients studied at that time. in this study where a large number of sle patients were studied and anti-ss-a antibody was present, only % had any evidence of keratoconjunctivitis sicca based on schirmer's tests. this indicates that not only is anti-ss-a an antibody "marker" for primary sjogren's syndrome, but it is relatively common in sle as well and furthermore, can exist in sle in the absence of keratoconjunctivitis sicca. serial studies were also performed on some patients. titers of anti-ss-a antibody were determined. these varied from neat to and correlated with disease activity. anti-ss-a often paralleled anti-ds-dna levels; however, anti-ss-a in some cases was an earlier predictor of clinical flares. thus, anti-ss-a antibody may be helpful not only in the management of sle patients, but with further investigation, aid in determining the pathogenesis of this disease. the phenanthridine dye ethidium bromide (eb) intercalates with native double-stranded d n a (dsdna) resulting in an enhancement of fluorescence when assayed fluorometrically. contamination of dsdna preparations by singlestranded dna (ssdna) does not affect the assay because there is no fluorescence enhancement when ssdna is added to eb. igg purified by deae column chromatography from the sera of normal volunteers: systemic lupus erythematosus (sle) sera with d n a binding activity as measured by the millipore filter (mpf) radioimmunoassay, and sle sera without d n a binding activity were tested for their ability to compete with eb for binding to dsdna. in of of the sle with mpf-dna binding activity, there was greater than inhibition of the fluorescence normally shown when eb binds dsdna. in contrast, of control igg preparations and of of the sle igg from non-dna binders had any such effect. furthermore, the decrease in fluorescence due to the inhibition of eb binding to dsdna was linear with in-creasing amounts of igg, thus allowing direct quantitation of anti-dna activity. since the binding constant of eb is known to be > x -'m, this assay measures high avidity antibodies and is not affected by non-specific, low-avidity d n a binding molecules. the specificity of anti-dna antibodies in the eb assay was shown when absorption with excess nucleoside monophosphates did not alter the inhibition of eb binding in unabsorbed samples. however, the synthetic polynucleotides poly da, poly dt, poly dc, poly da:dt, and poly dg:dc were able t o absorb dna binding activity. therefore, the eb assay has demonstrated several features which make it an important adjunct in studying the specificity and binding characteristics of anti-dna antibodies. in addition, the assay is inexpensive, is easily and quickly performed, and it does not require the use of radiolabeled substrates making it practical for further development in investigative and clinical use. degradation of collagen in tendon, bone, and cartilage by collagenase is a significant factor contributing to the morbidity of the inflammatory arthritides. although considerable information is available as to the effect of drugs and mediators of inflammation on collagenase production and activity, the mechanisms during aging in the collagen fibril causing increased resistance to collagenase degradation are poorly understood. in this study, the effect of collagen cross-linking on collagenolysis was measured with an in vitro model system consisting of purified chick calvarium collagen fibrils and lysyl oxidase, the cross-linking enzyme. chick calvatia collagen fibrils were cross-linked by incubation with lysyl oxidase for varying time. controls consisted of collagen incubated without enzyme or with lysyl oxidase plus / -aminopropionitrile, an irreversible inhibitor. the fibrils were subsequently measured for nascent cross-link content or incubated with purified rheumatoid synovial collagenase, and the rate of collagenolysis was measured. after synthesis of approximately . schiff base cross-links per collagen molecule, a ten-fold resistance to collagenase digestion was observed. inhibition of collagenase by edta prevented digestion. synthesis of collagen fibrils with higher cross-link content resulted in further resistance. these results demonstrate that the increased resistance of collagen to collagenolysis observed during fibril maturation in vivo is due to synthesis of native, unreduced schiff base cross-links. the cross-link content at which resistance to degradation develops is significantly less than that which affects fibril tensile strength in vivo. this suggests that resistance to collagenase is the earliest physiological effect of cross-linking in vivo. the rate of cross-link synthesis may be a significant factor regulating the rate of net collagen deposition in normal and pathologic states. vascular abnormalities have been shown to play an important pathogenic role in progressive systemic sclerosis (pss), especially in the group of patients who develop rapidly progressive kidney disease. as part of a study of the kidney in pss, we have also performed comprehensive investigations of coagulation parameters in order to evaluate the possibilities that: ) coagulopathy occurs in these patients; and ) coagulation abnormalities may be related to the development of renal disease in pss. seven patients with pss and normal renal function, as judged by normal blood pressure, creatinine clearance, and absence of proteinuria, were enrolled in the study. coagulation parameters studied included prothrombin and partial thromboplastin times, fibrin split products, fibrin monomer, sonoclot@ (which reflects fibrin monomer formation in recalcified whole blood), thromboelastograph (which reflects fibrin polymer formation), and platelet aggregometry. in addition, each patient underwent percutaneous renal biopsy. patients with pss showed platelet hyperreactivity t o collagen but not to other agents ( % aggregation versus control of . % p < . ). the following abnormalities, indicative of a hypercoagulable state, were found: increased rate of clotting by thromboelastograph in patients; increased rate of clotting by sonoclot in patients; and premature onset of clotting by thromboelastograph in patients. the patient with the most hypercoagulable profile has subsequently developed mild proteinuria. five patients had renal biopsy findings on light microscopy suggestive of pss kidney disease, including vascular intimal fibrosis, sclerosis, and fibrinoid necrosis. four of these were hypercoagulable by one or more tests; both patients with normal biopsies had normal coagulation profiles. these data suggest that: ) platelet-collagen interactions may be abnormal in pss; ) certain patients with pss manifest laboratory evidence of hypercoagulability; and ) hypercoagulability appears to correlate with renal biopsy abnormalities in pss patients with normal renal function. normal human mononuclear cells spontaneously lyse in vitro targets derived from cell lines of tumor origin. the natural killing of k- , a tumor cell line derived from a patient with chronic myelogenous leukemia, varies in patients with systemic lupus erythematosus (sle) ( % f %) and controls ( % f %). t o evaluate the possible role of serum factors, normal peripheral blood mononuclear cells were incubated with sle sera, normal control sera, or media alone in the natural killing assay. . x l(p ficoll-hypaque purified mononuclear cells after pretreatment with sera or media were incubated for hours with x ' s chromium ("cr) labeled k- cells. cytotoxicity was measured by the release of into the supernatant. the mean cr release in the presence of normal human sera was % f % of that with media alone. in the presence of sle sera, mean cr release was % & % of that with media alone. the magnitude of natural killing suppression by sle sera did not correlate with complement levels, ana titer, or d n a binding. the natural killing suppression by an individual sle serum did not correlate with its inhibition of the antibody dependent cell mediated cytotoxicity of chicken red cells. the factors in sle sera responsible for suppression of natural killings are non-dialyzable, precipitable with % (nh,),so,, and removable by absorption with anti-ig antibody. the factors are excluded by a g- column. the major portion of the suppressive activity was found in fractions of igm or greater density on sucrose gradients. these findings are compatible with factors in sle sera, most likely immune complexes or antibodies, capable of inhibiting the natural killing of tumor cell lines. these factors may prevent cell mediated cytotoxic destruction of malignant cells in vivo and may explain the increased association of sle and malignancy noted by canoso et af. (arthritis rheum : , ). full evaluation of any hyperuricemic patient requires these problems can be largely avoided by assessing uric acid assessment of uric acid excretion, but the standard -hour excretion per ml of glomerular filtrate. this simple, physiurine method presents many problems. ill-timed and inologically sound parameter is obtainable from single, untimed complete collections may combine with bacterial con-urine specimens by multiplying urinary uric acid by plasma tamination and crystal precipitation to cause significant errors. creatinine and dividing by urinary creatinine (all in mg/ ml). to minimize possible diurnal effects on uric acid excretion, all samples were taken in the morning. to evaluate the precision of the method, normal men collected two -hour urines. spot urine specimens with concurrent serum samples were also obtained on two separate mornings. uric acid was measured spectrophotometrically, and creatinine was measured by standard autoanalyzer technique. the coefficient of variation (sd/x) was i% for paired, quantitative -hour urine uric acid determinations, while spot, mid-morning assessments of uric acid excretion per ml of glomerular filtrate had a more satisfactory coefficient of %. since most laboratories do not employ the enzymatic spectrophotometric method, urine uric acid determinations were also performed by a colorimetric, autoanalyzer technique. these values correlated well (r = . ) with the more specific spectrophotometric findings, but were an average of % higher. samples from normal, adult men, the mean urinary excretion of uric acid in mid-morning was . f . mg/ ml (sd), while gouty men (studied between attacks and off hypouricemic drugs) had a mean of . f . mg/ ml. included in the latter group were significant overexcretors with values of . , . , . , . , and . mg of uric acid per ml of glornerular filtrate. we believe that this assessment of mid-morning serum and urine samples effectively identifies overexcretors of uric acid. in addition, it is more convenient, more physiological, and more precise than the conventional -hour method. (supported by nih grant am .) in mixed connective tissue disease (mctd), a newly described rheumatic syndrome, no comprehensive histopathologic descriptions exist. we have followed mctd children over a range of - years, (mean years); have died. three autopsies and renal and muscle biopsies were reviewed. among adults with mctd, tissues from autopsies, and kidney, muscle, and lung biopsies were reviewed. in children, proliferative vascular lesions, with intimal and medial thickening and luminal narrowing but without fibrinoid or inflammatory change, affected large vessels (aorta, coronary, renal), and small arterioles of many organs. inflammatory infiltration, predominantly plasmacytic, was marked in skeletal muscle : , liver : , salivary glands : , intestine : , and heart : . distinctive lesions of esophagus (atrophy of inner muscle layer), thymus (hyperplasia), kidney (membranous change), liver, lung, and salivary glands differed significantly from those expected in childhood systemic lupus erythematosus, polyarteritis, or scleroderma. many of these organs were not clinically involved during life. histologic estimates of numbers of t and b lymphocytes in spleen and lymph nodes, and degree of plasmacytosis (with hyperglobulinemia), differ from systemic lupus erythematosus and juvenile rheumatoid arthritis. in adults with mctd, muscle changes included: diffuse inflammatory infiltrate : (perior endomesial, and perior intravascular). by atp-ase, : i i had type i fiber predominance. on muscle immunofluorescence, : had vascular, sarcolemmal basement membrane, or granular fiber staining, with igg or igm. in kidneys there were included: mesangial proliferation , focal-local change , membranous , membrano-proliferative i , proliferative vessels , normal . lung tissue ( ) revealed: vascular proliferation , vascular medial hypertrophy , and interstitial fibrosis . in mctd, children and adults have similar lesions: inflammatory lesions may predominate early, but can occur late. immunofluorescent data suggest an immune basis for injury; the late predominance of proliferative vascular lesions suggests that vascular sclerosis is a serious complication. the findings of many significant histologic lesions, without clinical signs or symptoms, suggest that features of this multi-system disease evolve slowly, and that the full spectrum of mctd is not yet known. with ra and normal subjects. after isolation of the lymphocytes by isopycnic sedimentation, the cells were placed in tissue culture and half were infected with ebv. every days for month the supernatant fluid was removed from each culture and fresh medium was added. total igm and igm-rf secreted into the supernatant were measured by solid phase radioimmunoassay. independently the cells were examined for transformation and numerically graded. all infected cultures produced igm-rf, which correlated with a high grade of transformation. the amount of rheumatoid factor (rf) produced by ebv infected normal, but not rheumatoid, lympho- the incubation of pmns with a chemotactic factor (cf), in the absence of a gradient, prevents the cells from responding with directional migration when, after washing, they are challenged with a gradient of the same or a different chemotactic factor (deactivation and cross-deactivation). structurally unrelated cfs, some shown to bind to distinct cell receptors, can cross-deactivate, which suggests that deactivation is not due to receptor blockade. we propose that deactivation is the result of generalized microtubule assembly induced by c f incubated with the cells in the absence of a gradient, thus rendering the cells incapable of responding to a c f gradient with distinctive localized assembly, a proposed requirement of normal chemotaxis. if this scheme is correct, the simultaneous preincubation of pmns with suitable concentrations of colchicine, a microtubule disrupting agent, and a c f should protect the cell against deactivation and colchicine-induced suppression of chemotaxis. human neutrophils were preincubated, ' at oc, with colchicine to -'m); the chemotactic factors gly-his-gly (iopg) or crystalinduced chemotactic factor (ccf) pg alone; colchicine and either chemotactis factor; or hanks. cells were washed and tested for chemotactic response against the c f using a radioassay that utilizes y r labeled neutrophils. preincubation of cells with either chemotactic factor or colchicine alone resulted in a dose-dependent inhibition of chemotaxis. when cells were preincubated with both chemotactic factor (gly-his-gly iopg or ccf pg) and suitable concentrations of colchicine ( or io-o), a reversal of the inhibition of chemotaxis was noted. deactivation reappeared when the balanced ratio between colchicine and c f was altered. preincubation of c c f with colchicine had no direct effect on its chemotactic activity, and colchicine ( - m) did not alter the specific binding of radiolabeled c c f to neutrophils. additionally, both c c f and gly-his-gly induced microtubule assembly by electron microscopy. functional evidence is presented with two distinct chemotactic factors, which suggests that the basis for deactivation is overpolymerization of microtubules that prevents the pmns from responding to a chemotactic gradient with directional migration. fever is a common occurrence and frequent reason for hospitalization of patients with systemic lupus erythematosus (sle). to assess the frequency, causes, and clinical and laboratory characteristics of febrile episodes in hospitalized patients with sle, the medical records of admissions of sle patients during the year interval to were reviewed. eighty-three febrile episodes, defined as a n oral temperature oc and one blood culture drawn for evaluation of fever, occurred in patients. the febrile episodes were classified: group a-clinically active sle only ( ). group b-documented infection (l ), and group c-miscellaneous ( ). clinically active sle accompanied episodes in b and in c. treatment a t the time fever developed included steroids in , , and % and cytotoxic agents in , , and % of episodes in a, b, and c, respectively. infectious causes of fever were bacterial septicemia ( ), localized bacterial infections ( ), herpes zoster ( ), and miliary tuberculosis ( ). miscellaneous causes were procedure-related fever ( ). drug fever ( ), addisonian crisis (i), myocardial infarction (i), and unidentified ( ). the initial clinical impression was correct in of infectious episodes. two episodes of bacterial septicemia were unexpected; one occurred in a patient with concommitantly active sle. the clinical impression was correct in of episodes in a; infection ( ), acute appendicitis (i), and drug fever ( ) were suspected in the others. comparing a and b, patients' age, disease duration, and fever patterns were similar. shaking chills were more frequent with infection (p < . ) but were present in % of a. laboratory studies helpful in identifying patients with infection were leukocytosis > x ip/mm (p < . ), neutrophilia > x ip/mm (p < . ) and normal dna binding (p < , ). four deaths occurred despite appropriate therapy: in a with necrotizing lupus pneumonitis, and in b with gram negative sepsis, of whom had active sle. in summary, infection accounted for % of febrile episodes in hospitalized patients with sle. bacterial septicemia was found in % and was associated with a high mortality. the initial clinical impression was usually correct and the wbc count, absolute neutrophil count, and dna binding were helpful in distinguishing between infection and active sle. renal involvement in progressive systemic sclerosis (pss) is the most devastating form of the disease with one year mortality close to % unless hemodialysis or transplantation is successful. at present, there are no reliable predictive factors which allow us to separate those who will eventually develop renal disease from those who will not. because of this, we have studied patients with pss who were normotensive and had normal renal function (mean serum creatinine . mg%, mean creatinine clearance cc/min, mean protein excretion mg/ hours) and normal urinalysis. studies included percutaneous renal biopsies, evaluation of the renin-angiotensin system, and a cold pressor test to try to determine renal vascular reactivity. five of renal biopsies demonstrated distinct vascular lesions on light microscopy with intimal fibrosis in , hyaline intimal sclerosis in , and arteriolar fibrinoid necrosis in . electron microscopy of the vessels showed changes similar to those on light microscopy. in addition, wrinkling of the glomerular basement membrane (gbm) with expansion of the mesangium by gbm-like material was seen in of biopsies. although nonspecific, these changes are suggestive of ischemia in the kidney and are compatible with the vascular changes of pss. immunofluorescence studies revealed c in vessels in all biopsies. plasma renin activity (ppa) was performed in of patients. of the patients with abnormal biopsies, had elevated plasma renins ( . ng/ml/hr) at the time of biopsy and the fifth patient has subsequently developed significant elevation. the patients with normal biopsies had normal plasma renins ( . ng/ml/hr). cold pressor testing with pra determination resulted in a mean maximal increase of . ng/ ml/hr in those with abnormal biopsies, . ng/ml/hr in those with normal biopsies, and . ng/ml/hr in control subjects. these findings indicate that renal histologic vascular involvement may precede the onset of clinical renal disease in pss and correlates well with pra elevations and increased responsiveness of the renal vasculature. followup of these patients will determine the clinical significance of these findings. ninety-six patients with classic or definite rheumatoid arthritis were consecutively screened for the presence of early ocular manifestation of disease. each patient was followed for an average of . months and received an average of ophthalmologic exams at an average of month intervals. none of the patients demonstrated ocular lesions definitely attributable to rheumatoid arthritis. eighteen patients developed new ocular pathology consisting of chronic blepharitis, chronic iritis, corneal subepithelial defects, keratitic precipitates, keratitis, corneal abrasion, and abnormal tear film. twenty-one patients of this group and normal adult controls were screened for keratoconjunctivitis sicca. slit lamp examination, rose bengal staining, shirmer's testing, and tear lysozyme concentration were measured in each patient. tear lysozyme concentration was indirectly determined spectrophotometrically by measur-ing lysis of the dried cell walls of micrococcus lysodeikticus. patients with rheumatoid arthritis demonstrated abnormal rose bengal staining (rheumatoid arthritis, patients; control, none), significant decreased tear production (rheumatoid arthritis, . f . mm wetting shirmer strip; control, . f . mm wetting schirmer strip) p < . , and markedly increased tear lysozyme concentration (rheumatoid arthritis, . f . pg/ml; controls . f . pg/ml) p < . . thus, unlike patients with severe sjagrens syndrome who have low tear lysozyme concentrations, patients with rheumatoid arthritis demonstrate significant increases in tear lysozyme levels. this new finding, as yet unexplained, may represent the earliest lesion in the development of keratoconjunctivitis sicca in patients with rheumatoid arthritis. nzb/nzw mice develop autoimmune disease similar t o that of humans with lupus systemic erythematosus. treatment with cyclophosphamide (cp) protects against the disease but often is associated with a high incidence of tumors. tumors resulting from direct chemical carcinogenic activity increase with rising cumulative dose and time after exposure and are of diverse types. immunosuppression itself is reported to facilitate tumor growth or, perhaps, oncogene expression and has been primarily associated with sustained immunosuppression, predominantly with tumors of reticuloendothelial (re) origin. we compared tumor development in nzb/nzw mice given different cp regimens (see table) . reticuloendothelial tumors increased with daily dose but did not correlate with cumulative dose or duration of treatment. smaller numbers of other (non-re) types of neoplasms occurred in all groups. our results ) suggest that re tumor development in autoimmune disease is more dependent on continuous, high doses of cytotoxic drug than on cumulative dose or duration of treatment and ) are compatible with the possibility that re tumors result from immunosuppression or oncogene expression. no. the success of colchicine in the treatment of acute gouty arthritis is well established. however, the usefulness of this drug in the therapy of the pseudogout syndrome has been less enthusiastically regarded. in general, assessments of such therapy refer to lower success rates and somewhat inconsistent results. nevertheless the use of other antiinflammatory drugs to treat acute pseudogout is sometimes militated against by the presence of associated conditions commonly seen in the age group of these patients, including congestive heart failure, gastrointestinal disease, or neurological deficits. we have treated consecutive patients with the acute arthritis of pseudogout with a standard regimen of colchicine by the intravenous (iv) route. there were males and females, ages to , and all presented with acute mono-or oligoarticular arthritis. in all cases typical calcium pyrophosphate dihydrate (cppd) crystals were identified by red-compensated polarized light microscopy of the synovial fluid. none of the patients had elevated serum uric acid, except which was on diuretics, and synovial urate crystals were absent. synovial fluid white counts varied from to / mma with - % polymorphonuclear leukocytes; of the had radiologic evidence of chondrocalcinosis. all patients were treated within hours of the onset of the acute arthritis. colchicine was usually given at a dose of . mgm iv over a period of minutes followed by . mg iv every hours for the next - hours. total iv colchicine dosage varied from to . mgm. an excellent response occurred in - hours in of patients and in - hours in the other , with complete resolution of inflammation in all . we conclude that ) iv colchicine may provide effective and consistent therapy for the acute arthritis of pseudogout; and ) because of this, prompt response of an acute arthritis to iv colchicine remains an insufficient clinical criterion for the diagnosis of gout. the high pyrophosphate (ppi) concentrations observed in osteoarthritis (oa) and chondrocalcinosis (cc) synovial fluid (arthritis rheum : , ) suggest that ppi may be a precursory ion involved in human articular mineral formation, whether in midzone sites in cc-cappi deposition-or in "tidemark" cartilage in oa. why predominantly hydroxyapatite and often some cappi (bjelle, personal communication) are seen in oa, and cappi is deposited in cc, might hypothetically depend in part on moderate and severe ppi hydrolase(s) deficiencies. such deficiencies are postulated to be associated with the calcifying subcellular apparatus in oa and cc articular cartilage, respectively. this postulated ppi deficiency would be relative to normal calcifying growth cartilage in which ppi in mineral is present in only trace amounts (wuthier et al., calc tiss res , ). to examine this hypothesis, articular cartilage from patients with oa ( male, female, age - ), with cc ( male, female, age - ), and normal controls was assessed for various phosphohydrolase activities. cartilage was sliced in a cryostat, homogenized, and extracted in triton x- . the extract was centrifuged and the supernatant, once dialyzed, was passed through a de- column and enzyme fractions were eluted according to prevjously described techniques. although total protein and dna content were about equal from all cartilage samples, alkaline phosphatase activity was found in the following ratios: normal: cc: oa = i : : . in all samples, peaks of alkaline phosphatase activity were eluted similar to the findings of arsenis et al. in calf growth cartilage (calc tiss res , ). uniquely, with cc some of the total activity ( %) was found in the void volume of both peaks. characterization of alkaline phosphatase using various metals, inhibitors, and substrates was similar for all samples tested. however, analysis of the ratios of ppiase: alkaline phosphatase for growth cartilage (arsenis, above) versus oa and cc samples ranged from : to : respectively. in conclusion, the high ppiase to alkaline phosphatase ratio in growth cartilage compared to this ratio in cc and oa supports the view that reduced ppiase activity might play a role in ) the probable elaboration of ppi into synovial fluid in oa and cc; ) the in vitro elaboration from articular cartilage of ppi into eagle's medium in patients with oa and cc (j clin invest : , ); and ) provision of ppi ion for variable cappi crystal deposition. despite the prominence of neuropsychiatric features in systemic lupus erythematosus (sle), no satisfactory method exists for the diagnosis and monitoring of central nervous system (cns) involvement. this study describes the application of a new technique for studying both cerebral metabolism and blood flow in sle patients by using inhaled molecular -oxygen and -oxygen labeled carbon dioxide. tracer amounts of -oxygen are inhaled, and after a period of equilibration, the brain is scanned with a gammacamera. the image produced represents cerebral metabolism. the procedure is repeated using carbon -dioxide, and the resultant image represents cerebral blood flow. twenty-eight scans were performed on sle patients who had been classified as having clinically definite cns disease ( ), clinically probable cns disease ( ), or no clinical evidence of cns disease ( ). the scans, which were reported blind, were classified as normal (showing a full pattern of both cerebral blood flow and metabolism), as showing a major abnormality, or as showing a minor abnormality. the results are shown in the table. scan abnormalities were seen in of the studies, usually afecting several cortical areas. in patients in whom multiple recordings were made, improved -oxygen scan appearances correlated with clinical improvement. -oxygen brain scanning appears t o offer a highly sensitive non-invasive technique for the identification and study of cerebral involvement in sle. neutrophils stimulated at sites of inflammation release lysosomal enzymes. experimentally, a variety of agents are capable of stimulating neutrophils to release their lysosomal enzymes. neutrophil membranes contain fc receptors for igg, and thereby possess surface bound igg. in these studies we examined the hypothesis that specific antibodies could combine with surface bound igg, perturb the cell membrane, and provoke inflammatory responses of neutrophils. antisera were raised to immunoglobulins g , m, a, d, e, and isolated y chains, fc and f(ab'), of igg. these were rendered monospecific by solid phase immunoabsorbents. isolated normal human peripheral blood neutrophils were incubated with specific antisera and the release of lysosomal / -glucuronidase, a-mannosidase, and lysozyme were measured. non-lethal release of lysosomal enzymes was observed with antisera to whole igg, iga and t o y and fc of igg. the igg fraction of anti-igg antiserum also stimulated non-lethal release, thereby suggesting that the response was not complement-mediated. specific igg and iga receptors were detected on neutrophils using immunofluorescent labeled antibodies. n o such receptors were detected for igm and igd. these results indicate that inflammatory responses of neutrophils can be triggered directly by anti-immunoglobulins, and suggest one mechanism for initiation of the inflammatory response in patients with diseases characterized by the presence of antibodies to igg and other immunoglobulins. incubation of polymorphonuclear leukocytes (pmn) with immune complexes trapped in micropore or collagen membranes results in the phenomenon of "frustrated phagocytosis," that is, enhanced release of lysosomal hydrolases. w e have previously shown immune complexes irreversibly trapped in joint collagenous tissues in antigen-induced rabbit experimental arthritis and in rheumatoid arthritis. the present experiments were designed t o investigate in vitro interactions between pmn and joint collagenous tissues obtained from rabbit joints with experimental arthritis or control tissues from saline or monosodium urate crystal-injected joints. experimental and control articular cartilage samples and menisci obtained from such animals were incubated for hour with normal pmn isolated from rabbit peritoneal exudates or blood. after fixation, the tissues were examined by electron microscopy. fresh cartilage and menisci from arthritic joints showed only few damaged pmn near the articular surface. after incubation with pmn, large numbers of pmn attached to the articular surface were seen. in areas of superficial erosion, the pmn invaded the tissue several cell diameters below the lamina splendens. degranulated pmn were observed with immunoelectron microscopy in scattered areas to phagocytose amorphous material containing rabbit ig. following addition of pmn t o control tissues, only a few pmn became attached to the articular surface. in monosodium urate injected joints, after incubation with pmn, these cells were found attached to the surface in moderate numbers, but no invasion into the tissues was seen. these studies indicate that immune complexes trapped in joint collagenous tissues may induce the phenomenon of "frustrated phagocytosis" leading to enhanced release of lysosomal hydrolases. similar studies using rheumatoid joint tissues are in progress. (suppported by nih grant am .) half of the girls. intervals between onset of symptoms and diagnosis ranged between month and years (median months). twenty-eight children were white, black, native american, asian, and of other races. most frequent presenting findings were fever and malaise, musculoskeletal complaints, skin rash, and renal disease. unusual presentations included cholecystitis, isolated nephrotic syndrome, and chorea. three patients presented with isolated thrombocytopenia. serious pulmonary manifestations occurred in patients; died within months of disease onset. clinical evidence of nephritis occurred in of boys and of girls. thirty-six patients had or more renal biopsies. three patients with normal urinalyses had histologic nephritis by biopsy. therapy consisted of prednisone ( of patients) and either azathioprine or chlorambucil ( of patients). since therapy has been geared to normalizing serum hemolytic complement values as well as controlling clinical manifestations of disease. chronic renal failure developed in patients; only had had adequate therapy early in disease. one renal failure patient received a renal transplant, and are on hemodialysis. six girls and boys have died ( ' ). seven patients had active lupus at time of death with severe uncontrolled multisystem lupus ( ), pulmonary lupus ( ), central nervous system lupus (l), and renal failure ( ). one patient with active sle also had a myocardial infarction (age ) as a contributing cause of death. two patients died while in clinical remission, of a myocardial infarction (age ) and of clostridial sepsis with hemolysis. infection played a major role in deaths (fungal brain abscess, acute staphylococcal endocarditis, disseminated herpes infection, clostridial sepsis). thirty-three patients continue to have active sle requiring treatment; are in remission and are not on medications; i patient was unavailable for followup in . in patients with systemic lupus erythematosus (sle), abnormal unfractionated lymphocyte responses to phytohemagglutinin (pha) can be corrected by removal of adherent mononuclear (m) cells (arthritis rheum , ) . the present study demonstrates that this abnormal response occurs when ) autologous adherent mononuclear cells or ) cell-free supernatant fluids from autologous and allogeneic adherent cells are added to t-cell cultures, and that ) suppression does not occur when adherent cells are pre-treated with indomethacin. ficoll-hypaque (fh) separated peripheral blood mononuclear cells from normals and from patients with sle were further fractionated into t cells by passage over an antihuman f(ab), column ( % e-rosettes) and m cells by plating on glass ( % peroxidase-staining). cell number was constant in all experiments. supernatants from normal controls and from sle patients were obtained after hour culture of fh, t, and m cells in rpmi containing ab serum. normal and sle t cells were tested for pha response in the presence and absence of autologous and allogeneic m cells or cell-free su-pernatants from fh, t, or m-cells. autologous but not allogeneic m cells suppress t cell responses. cell-free supernatants derived from m cells of consecutive experiments with sle patients caused an average % suppression of normal t-cell response to pha (mean peak cpm relative to control). suppression was not due to media exhaustion. supernatants from normal m cells caused an average % suppression. supernatants from both normal and sle t cells did not suppress either autologous or allogeneic t-cell responses. when m cells of patients with sle were cultured in the presence of pg indomethacin/ml, the obtained supernatant was not inhibitory. the data indicate that poor in vitro unfractionated lymphocyte response to pha in patients with sle is mediated by a soluble indomethacin-sensitive product derived from the adherent mononuclear cells, possibly prostaglandin. since supernatants from normal adherent cells were also inhibitory when m-cell concentration was held constant, it is likely that the difference between sle and normal lymphocyte responses is quantitative rather than qualitative. renal crisis in scleroderma, with malignant hypertension and rapidly progressive renal failure, is perhaps the most ominous of clinical syndromes in the rheumatic diseases. survival with medical management has not been reported, although a few patients have survived with renal dialysis or transplantation. we report patients surviving scleroderma kidney with medical management alone for periods of , , and years. patient with malignant hypertension in the fourth year of his classic systemic sclerosis was hospitalized with blood pressure of / , generalized seizures, visual acuity decrease to virtual blindness, and striking grade hypertensive retinopathy. creatinine rose to a high of . . extraordinarily vigorous antihypertensive treatment with massive doses of drugs reduced blood pressure to low normal ranges. renal function years later is stable with a creatinine of . mg . patient developed her malignant hypertension after years of classic scleroderma, with blood pressure / , grade hypertensive retinopathy, generalized seizures, a creatinine of . , and urine protein of gms per hours. plasma renin was ng/dl. aggressive antihypertensive treatment with drugs reduced blood pressure to the low normal range, and over the following years renal function has improved to a creatinine of . . patient developed malignant hypertension after several years of classic scleroderma. blood pressure was / , creatinie rose to . , hypertensive grade eye changes were noted, and renal biopsy confirmed afferent arteriolar necrosis. hemorrhage following renal biopsy resulted in transient hypotension, with subsequent control of blood pressure in the low normal range with combination antihypertensive drug therapy. her renal function remains stable with a creatinine of . mg% after years. skin lesions improved following hypertensive control in of these patients. the common denominator in these successfully managed patients appeared to be aggressive and determined reduction of blood pressure to low normal ranges, with later reliance upon propranolol in of the patients. the percentage of patients who will respond to such management is unknown, but aggressive early treatment of hypertension appears indicated. the multi-system involvement and varied clinical presentation of systemic lupus erythematous (sle) make its treatment difficult and often controversial. to determine present clinical practices, rheumatologists and nephrologists, selected randomly from the directory of medical specialists, were surveyed for their management practices for hypothetical sle patients. responding physicians follow over patients with sle. patients were managed as follows: ( ) joint and skin involvement only: ninety-four percent of respondents chose asa ( - tabs/day) with % also using hydroxycholoroquin. ( ) early clinical nephritis: seventy-eight percent would initially treat with prednisone, usually at about mg/ kg/day but with % choosing . mg/kg. ( ) active nephritis with rising creatinine: ninety percent would initially employ prednisone, at doses approximating mg/kg, and % would also use immunosuppressives. ( ) serologicalfiare in an asymptomatic patient: forty-six percent would treat with prednisone and % would withhold treatment. (three percent demanded a renal biopsy). ( ) central nervous system sle: uniformly treated with prednisone, at least mg/kg, with % also employing immunosuppressants. ( ) late stage arotemic in-active sle: fifty-nine percent elected to treat with prednisone. ( ) prednisone taperingpractices: from a starting dose of mg/day, physicians tapered to a mean of mg/day after months and mg/day after months. thirty-three percent used a divided dose initially, and % used alternate day schedules later. ( ) flare while tapering: all physicians increased medication, with % increasing prednisone dose, % adding an immunosuppressant, and many doing both. rheumatologists and nephrologists were similar in treatment of most problems. however, nephrologists used immunosuppressive agents twice as frequently in early disease but treated late stage nephritis less vigorously than did rheumatologists. nephrologists also used fewer divided daily doses of prednisone and used alternate day schedule sooner and more frequently. treatment was highly case specific. substantive disagreement occurred when there was serological disease without clinical disease, and in treatment of end-stage nephritis. central nervous system episodes and early progressive nephritis were uniformly treated aggressively. immunosuppressive agents were employed only by a minority of respondents. antibody-dependent cell-mediated cytotoxicity (adcc) was studied in adult patients with rheumatoid arthritis (ra) and in healthy controls. three effector cell populations from the peripheral blood were studied. these included a mixed mononuclear population ( - % latex positive), a monocyte-depleted fraction (less than % latex positive), and a monocyte enriched fraction ( - % latex positive). the target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody (igg fraction); multiple effector: target ratios were studied. there was no significant difference in lymphocytotoxic antibody is found in the serum of patients with systemic lupus erythematosus (sle). the antibody is defined by its reaction with lymphocytes, but it also reacts with antigens on human brain tissue. the existence of an antibody in sle serum recognizing both brain and lymphocyte antigens prompted a search for a similar substance in the cerebrospinal fluid (csf) of sle patients, with and without central nervous system (cns) disease. csf was obtained from patients with sle in the course of evaluations for fever, headache, or manifestations of cns dysfunction. an independent clinical analysis of the sle patients and their hospital records revealed that of the had cns dysfunction attributable to sle. criteria for cns disease were one or more of the following: seizures, transverse myelitis, chorea, ataxia, hemiparesis, psychosis, or hallucinations. multiple abnormalities were present in of the patients, with l patient having , and patients having of the above abnormalities. a population of patients without rheu-matic disease who were being evaluated for a variety of neurological abnormalities served a s controls. the csf was tested in a dye-exclusion microcytotoxicity assay using peripheral blood lymphocytes from normal donors as targets. cytotoxicity was defined as greater than % killing of lymphocytes from or more of the normal donors. nine of the sle patients'had lymphocytotoxic activity in their csf compared to of controls. when lymphocytotoxicity of csf was compared with presence or absence of lupus cns disease, a positive correlation was found (x' = . , p < . ). the mean % lymphocytotoxicity in the cnfs did not correlate with the serum titer of lymphocytotoxic antibody (r = . ). cytotoxic activity to human lymphocytes has been identified in the csf of patients with cns manifestations of sle. if the cytotoxicity is cross-reactive with neuronal antigens, as is the serum antibody, it may have a pathogenetic role in cns lupus. the role of bone marrow derived cells in determining genetic variation in susceptibility to experimentally induced amyloidosis in mice was investigated. mice of the amyloid susceptible cba strain were lethally irradiated and repopulated with marrow cells from the highly resistant a strain. lethally irradiated cba mice reconstituted with syngeneic marrow served as controls. to determine the effects of irradiation per se on amyloid production, normal cba mice, as well as a strain mice that were irradiated and reconstituted with a marrow, were also studied. following weeks of daily subcutaneous casein injections, spleen sections stained with congo red were scored for the degree of amyloidosis by independent blind observers using the following point scale: grade i-a rim of amyloid around or more splenic follicles; grade -a rim of amyloid in more than % of splenic follicles; grade -a rim of amyloid in all splenic follicles; grade -diffuse splenic amyloid with bridging be-tween almost all follicles and some distortion of the splenic architecture. thirty percent of cba mice given a marrow failed to develop amyloid, with an average score of . for this entire group. in contrast, control mice given syngeneic marrow all developed significantly more amyloid, with an average score of . . non-irradiated cba mice developed a n intermediate severity of lesions. irradiated a mice remained amyloid free. the progression of amyloidosis in cba mice was significantly retarded by grafting of marrow from the resistant a strain, even though irradiation seemed to accelerate amyloid production in cba mice. the resistance of the a strain to amyloidosis was not affected by lethal irradiation. these data demonstrate that bone marrow derived cells are an important factor in the pathogenesis of amyloidosis and that the genetic difference between susceptible and resistant mouse strains lies, at least partially, in their hematopoietic tissues. the biochemical features of families with purine nucleoside phosphorylase (pnp) deficiency and t-cell immunodeficiency disease were compared. the erythrocyte enzyme from brothers in family had . % normal pnp activity. plasma urate values in these patients are . mg/dl and plasma inosine levels are . and . p m . urine samples contain uric acid and mg/gm creatinine, inosine . and . millimoles/gm creatinine, and guanosine . and . milli-moles/gm creatinine. when compared t o normal, their enzymes showed a) a -fold increase in the apparent km for inosine, b ) a diminution of the isoelectric p h from normal values of . to . to . to . , c) an inability of inosine to protect the mutant enzyme against thermal inactivation as compared to the protection of the normal enzyme, d ) a loss of the normal near optimum activity a t ph . , and ) a normal value for the stokes radius. the enzyme from a patient in family has . % of normal activity. this patient has a plasma urate of . mg/dl and a plasma inosine of w m . urine samples contain uric acid mg/gm creatinine, inosine . millimoles/gm creati-nine, and guanosine . millimoles/gm creatinine. the enzyme protein had a ) a to -fold increase in the apparent km for inosine, and b ) only minor changes in enzyme activity over a ph range compared to normal. these observations suggest that a ) the degree of abnormality in uric acid and nucleoside concentrations in the plasma and urine reflect the severity of the enzymatic deficiency, b) structural alterations of the mutant pnp proteins result from structural gene mutations and genetic heterogeneity in the disease pnp deficiency. since pnp activity was found in every human tissue assayed, the systemic involvement of pnp deficiency can be accounted for. recently we reported the finding of an antibody, pm-i , directed toward one of the nuclear acidic protein antigens (napa), in sera of patients with polymyositis. this study was undertaken to investigate the relationship between pm-i antibody, polymyositis syndromes, and other rheumatic diseases. sera were tested against nuclear extracts by immunodiffusion and counterimmunoelectrophoresis for the presence of antibodies to pm- and other napa. clinical data were then obtained and analyzed. fifty-two patients were identified who had polymyositis syndromes as defined by significant muscle weakness, elevated serum muscle enzymes, myopathic electromyogram, and muscle biopsy typical of polymyositis. of these, had polymyositis, had polymyositis-scleroderma overlap, and had derrnatomyositis. pm-i antibody was found in % of polymyositis, % of polymyositis-scleroderma, and % of dermatomyositis. polymyositis syndromes, a difference in the prevalence of the clinical characteristics was noted as shown in the table. blinded serum exchanges of sera between medical centers revealed the prevalence of the pm-i antibody to be % in childhood dermatomyositis ( of ), % in adult dermatomyositis ( of ). and % in polymyositis ( of ). patients with other rheumatic diseases were negative for pm-i antibody ( of ). we conclude that the pm-i antibody has a high specificity for polymyositis and that it may identify a subset of polymyositis patients who have other connective tissue disease manifestations. pm-i positive pm-i negative clinical characteristic n = n = the pm- antibody was found rarely ( < i % ) in lo patients other muscular disorders such as myasthenia gravis, muscular pulmonary disease dystrophy, and polymyalgia rheumatica. among patients with an association between viral infection and joint inflammation has repeatedly been reported. our recent studies showed that interferon inducer, double stranded polyinosinate-polycytidylate (poly l:c), as well as human interferon, stimulated both prostaglandin e (pge) and hyaluronic acid production by human cultured synovial fibroblasts. the present study was performed in order to evaluate further the role of interferon and poly i:c in joint inflammation. polynucleotides, mouse interferon, or phosphate buffered saline were injected intraarticularly in -week-old wistar derived male rats by methods previously described. animals were killed hours later and the synovia were removed for histological examination and determination of pge. injection of poly i:c or mouse interferon induced an inflammatory response. poly a:u was only slightly active in this respect. by contrast, pbs or single stranded polyinosinate (poly (i)) or polycytidylate (poly (c)) did not induce any significant inflammatory response. rat knee joints injected with either poly i:c ( , , or pg/joint) or interferon ( or lo p/joint), appeared macroscopically swollen and edematous and contained an increased amount of viscous fluid. microscopically inflamed synovium contained variable amounts of inflammatory cells, most of them polymorphonu-clear. synovial pge levels were increased by poly i:c, poly a:u, and interferon but not by poly (i) or poly (c). mouse interferon also induced an inflammatory response when injected in mouse knee joint. we suggest that interferon may be a mediator in the initiation of inflammation by viruses. nineteen patients with musculoskeletal complaints after jejuno-ileal bypass for morbid obesity were reviewed for evidence of connective tissue disease and immunologic abnormalities. presence of arthritis or arthralgias and extraarticular connective tissue symptoms, elevated westergren sedimentation rate (esr), and absence of other significant forms of arthritis were criteria for diagnosis of postintestinal bypass arthritis. eleven of the patients met these criteria. seven were females. six of the i had joint swelling and had only arthralgias. all had moderately elevated esr. extraarticular manifestations include erythema nodosum ( patients), pleural effusion ( patients), and carpal tunnel syndrome ( patient). synovial fluid analysis in showed mild to moderate inflammation. serum rheumatoid factor was present in patient in low titer while patients had positive ana in titers ranging from : to : . in patients the ana reverted to negative with therapy, and in the third patient the ana became negative after the bypass was reversed. four had hypercomplementemia, had elevated serum igg, and had ele-vated serum iga. none of the patients demonstrated cryoglobulins, but had soluble immune complexes by complement inhibition technique. synovial biopsy in case showed chronic inflammation and iga and c' deposition by immunofluorescence. hla typing of of the patients with post bypass arthritis showed no trend. in i patient the length of bypassed bowel was reduced, and in another the bypass was reversed. in both cases the arthritis remitted. two patients required low dose prednisone therapy for the arthritis; the others were treated effectively with nonsteroidal antiinflammatory agents. these patients had an inflammatory arthropathy often accompanied by features suggestive of connective tissue disease, such as erythema nodosum, pleural effusions, and circulating ana. although none had cryoglobulins, which were previously reported by others, had circulating immune complex. this suggests that immune mechanisms are involved in the pathogenesis of arthritis in some, if not all, of these patients. honig et a (arthritis and rheum , recently reported that patients with active systemic lupus erythematosus (sle) usually do not have markedly increased serum concentrations (> pg/ml) of c-reactive protein (crp) and that such a finding suggests the presence of superimposed infection rather than active lupus. since a semi-quantitative capillary precipitin technique was employed in that study, in which only very high crp concentrations were regarded as positive, we investigated the significance of lesser increases in serum crp concentration in patients with sle, using a quantitative radial immunodiffusion method sensitive to . pg/ml. we retrospectively determined crp concentrations in serum samples collected from a group of patients with sle over a mean period of months. thirtytwo episodes of significant increase in serum crp concentration were detected. careful review of clinical findings associated with each crp peak revealed that these episodes were associated with active lupus without infection in instances (median crp concentration pg/ml, mean . , range . - . ) in instances, elevations of serum crp concentration were attributed to proven or suspected superimposed infection or bone fracture (median pg/ml, mean . , range . - . ). three crp elevations of . , . and . pg/ml occurred at a time when there was clinical evidence of neither lupus activation nor infection. there were instances of onset or exacerbation of lupus activity at times when serum samples did not show elevated crp levels. in of these, very active disease was present; crp elevations were noted in the next samples obtained, and days later. in a third patient with both infection and lupus activity, crp levels were found elevated days later. these data indicate that moderate to marked increases in serum crp concentration may occur in the course of sle in association with activation of disease, as well as in association with infection or other cause of tissue necrosis. rarely no obvious clinical cause can be found. occurrence of serum crp elevation in patients with sle does not differentiate between lupus activity and infection. and immunologic study of the post-intestinal bypass arthritis-dermatitis syndrome medical versus surgical management of ischemic necrosis of bone in systemic lupus intestinal bypass surgery has become a successful means of effecting weight reduction in morbid obesity. as a complication of surgery, a fraction of patients develop an arthritis and tenosynovitis which may be associated with circulating cryoproteins. we have had the opportunity to study patients with post-jejunoileostomy arthritis, all of whom had, as a striking feature of the illness, a widespread dermatitis.the patients were all female. the arthritis developed from to months after surgery and predilected both large and small joints of the upper and lower-extremities. associated with the arthritis were erythematous macules, ranging in size from to mm in diameter. these lesions developed into papules over days, subsequently became pustular-vesicular, and predilected arms, legs, trunk, and face. lesions were found in different stages of evolution.cryoglobulins consisting of igg, igm, and c , c were found in of patients. immune complexes (raji cell) were found in of patients, including patient with absent cryoglobulins. synovial fluid analysis in patients revealed california wbc ranging from to with from to % pmn. immune complexes (raji cell) were found in of synovial fluids.excisional biopsies of the skin lesions were performed in patients. there were no areas of fat necrosis. small capillaries and venules were infiltrated with pmn in all cases.immunofluorescent stains revealed deposits of igg and c -c in vessel walls in of patients. fluorescein conjugated antisera against bacterial pathogens revealed no staining in patient. treatment with oral antibiotics was initiated in all patients but was not invariably successful in decreasing the dermatitis or arthritis. in patients, intravenous antibiotics were also unsuccessful, but oral prednisone was promptly followed by decreased dermatitis-arthritis. in responsive patients, the serum cryoprotein titer decreased; the kinetics of this decrease and the reappearence of the cryoprotein after stopping antibiotic-prednisone treatment was studied.post-intestinal bypass surgery may cause a systemic immune complex disease primarily involving joints and skin. each met at least sle classification criteria, had clinical criteria of disease activity, and had elevated serum dna binding ( - % by farr assay). three had low serum hemolytic complement levels ( - u). their drug regimen, including prednisone ( - mg/day), remained stable for i days prior to, during, and for days after frentizole therapy. rash ( of ), synovitis ( of ), mucosal ulcers ( of ), and pleurisy ( of ) improved during frentizole therapy. synovitis worsened in case. the only toxicity was transient sgot and sgpt elevation in cases. bone marrow and skin tests were not suppressed by the drug. frentizole had no effect on hemoglobin concentration, creatinine clearance, or urinary protein ex-response. other laboratory findings (mean f sem) are summarized in the table. these results suggest that frentizole is an active, relatively non-toxic immunoregulatory drug in sle patients. current long-term studies should define its role in the therapy of sle and other autoimmune diseases. clinical and laboratory findings of children with systemic lupus erythematosus (sle) prospectively followed since have been summarized. mean disease duration was . years (range months to years, median years). there were girls and boys, less of a female preponderance than that of most adult series. youngest age for onset of systemic complaints was years. ten of boys had disease onset prior to sexual maturation, whereas disease began after menarche in adcc activity between patient cells and control cells when either the mixed mononuclear population or monocyte-depleted population were studied as effectors. the monocyteenriched fraction from patients with ra, however, mediated a significantly increased degree of cytotoxicity (table) . enhanced cytotoxicity was more evident at low effector: target ratios and was independent of phagocytosis. adcc may be important in ra since it reflects both humoral and cell-mediated immune mechanisms. the enhanced effector function of the peripheral blood monocyte in this system may be an indication that mononuclear phagocytes are "activated" in patients with ra. the ara preliminary criteria for the classification of systemic lupus erythematosus (sle) were developed before the widespread use of tests for anti-dna antibodies and serum c levels. analysis of our data suggests that these tests should be included in the ara criteria and could replace of the present manifestations which are less specific and less sensitive than the serum c level and circulating anti-native dna antibody level (a-dna).three thousand three hundred thirty-four sera from sle patients were studied. only patients observed by us and whose sera were tested both during periods of remission and disease activity were included in this study. an average of serum samples per patient was studied. the average period of observation was . months. patients had at least of the manifestations of the ara criteria. c levels and a-dna were studied from normal individuals, patients with rheumatoid arthritis followed by us, and from patients with other diseases.two of the non-sle patients ( %) had a-dna level (expressed as % dna bound) of more than % ( %, %). thus, a a-dna of > % binding had a specificity for sle of %. eighty-two percent of the sle patients had a-dna of > % binding as their highest value-a sensitivity of %. none of the non-sle patients had a c of < mg%-a specificity of %. fifty-five percent of the sle patients had a c level of < mg% as their lowest c -a sensitivity of %.these data were analyzed using the method employed to select the manifestations of the ara criteria. the average rate of correct classification (the arithmetic mean of the sensitivity and specificity) (arcc) for a-dna of > % binding is . and for c of < mg% is . the arcc for the items which comprise the manifestations of the ara criteria varied from . to . . of these, only the presence of le cells had a arcc greater than that for a-dna. if c and a-dna data had been available at the time the ara criteria were developed, they would have been included. the arcc for the mean of the items combined for the cns manifestations and the arcc for the mean of the hematologic items were lower than those for either c or a-dna. thus, the c and the a-dna could replace these manifestations. the data suggest that the ara criteria should be revised.the significance of ischemic necrosis of bone (inb) in sle and the cause of the bony lesion are not established. in this study, patients with sle and inb have been compared to patients with sle alone; and, in those with inb, the cause of the osseous lesion has been analyzed in relation to medical versus surgical management.those clinical features of sle significantly correlated with inb include raynaud's phenomenon ( % versus %, p < o.ool), myositis ( % versus %, p < o.ol), and vasculitis ( % versus %, p < . ). the course of inb sites in the patients initially grouped into medical ( patients; sites) and surgical (i patients; sites) is shown in the table.the initial orthopedic procedure was uniformly core decompression. all patients were receiving corticosteroids. the failure of medical management alone is shown by the persistence of symptoms ( or %), x-ray progression ( or %), and need for reconstructive surgery ( or %). in stages i and of the surgical group, only sites ( %) were symptomatic, sites ( %) progressed on x-ray, and only ( %) required further surgery. none in stage i advanced.differences in followup intervals would not appear to explain these statistically significant differences.thus, in the steroid-treated sle patient, especially with raynaud's, myositis, and/or vasculitis, the risk of inb is major and the need for early diagnosis essential if progression of the bony lesion is to be retarded by orthopedic intervention. acute abdominal syndromes have been increasingly recognized as major life-threatening events in patients with rheumatic disease. early diagnosis is critical to the institution of appropriate medical/surgical management and may be impeded by the masking effects of antiinflammatory, especially steroidal, therapy. it is our purpose here to report those features, clinical and laboratory, which appear to relate specifically to intraabdominal arteritis, our major cause of an acute abdomen in those with systemic lupus erythematosus (sle) and polyarteritis (pa).during the past years, of patients admitted to the unit with sle and of patients with pa developed an acute abdomen. in i ( %) of those with sle and all with pa, the abdominal event was secondary to mesenteric arteritis. the remaining patients with sle had polyserositis ( ), pancreatitis ( i ) and, in the other patient, an undiagnosed recurrent syndrome which responded each time to increased corticosteroids alone. when patients with sle and an acute abdomen were compared to the patients without abdominal syn-dromes, the significant discriminating features in the abdominal group were increased peripheral vasculitis ( % versus %, p < . ), thrombocytopenia ( % versus %, p < o.ooos), and presence of rheumatoid factor by the latex test system ( % versus %, p < . ). of the patients with pa, peripheral neuropathy was present in all, thrombocytopenia developed concomitant with the abdominal syndrome in all, and rheumatoid factor was uniformly present in the patients tested ( : , : , i : , ). eight of those with sle and the with pa died from the abdominal catastrophe. six of the surviving sle patients represent those most recently seen.thus, in the patients with sle and pa, the presence of thrombocytopenia and circulating rheumatoid factor are poor prognostic signs and may pathogenetically predispose the patient to intraabdominal arteritis. outcome can only be favorably altered by early recognition and prompt institution of appropriate medical and/or surgical management. key: cord- -k fq b m authors: gunderman, richard b. title: medical valor in plague time: dr. benjamin rush date: - - journal: acad radiol doi: . /j.acra. . . sha: doc_id: cord_uid: k fq b m nan sylvania. when thomas jefferson was preparing lewis and clark for their epic expedition across the country, he sent them to rush to obtain necessary medical training and supplies. a staunch abolitionist, rush argued that blacks were in no way naturally inferior to whites. he also campaigned against capital punishment and promoted the education of women. one of the most prominent mental health reformers in us history, in rush published his "medical inquiries and observations upon the diseases of the mind." he deplored the conditions under which many psychiatric patients were kept and lobbied for more humane care. he promoted the engagement of mental patients in activities such as gardening and washing, and he was a strong advocate for the view that alcoholism is a disease. his medical students were so impressed by rush that they founded rush medical college in chicago, now rush university medical center, in his honor. it was rush who persuaded former political opponents john adams and thomas jefferson to rekindle their friendship. an understanding of yellow fever is necessary to understand the epidemic. it is caused by an rna virus spread by the bite of infected mosquitoes. after an incubation period of several days, most patients develop mild symptoms including fever, headache, and anorexia, recovering in less than a week. but approximately % of patients enter a second phase with recurrent fever and jaundice, from which the disease derives its name, as well as hemorrhage from the mouth, nose, and eyes, and bloody diarrhea. among such patients, mortality rates may be as high as %. those who survive generally recover completely, with the added benefit of lifelong immunity to the disease. when a female mosquito ingests the blood of an infected human or primate, the virus begins replicating in the epithelial cells of the insect's gastrointestinal tract. after they take up residence in its salivary glands, they are then transmitted the next time the mosquito bites. because mosquitoes tend to be active in warmer months, yellow fever outbreaks tend to occur in late summer. once the virus enters the bloodstream of a human victim, it begins reproducing in lymphatic organs, from which it can infect the cells of the liver. deaths are often due to the aggressiveness of the host immune response, producing a vicious cycle of escalating immune response sometimes known as "cytokine storm." more than two-dozen outbreaks of yellow fever marked the history of north america, involving, in addition to philadelphia, such cities as savannah, georgia, new orleans, louisiana, and norfolk, virginia. of special historical interest was the problem the disease presented to would-be builders of the panama canal. initial french efforts were essentially doomed by the disease, eventuating in over , deaths, and the resulting business failure incited financial turmoil in france. the us eventually succeeded in completing the canal largely through the elucidation of the role of mosquitoes in transmitting the disease, followed by successful eradication efforts. at the time of the epidemic, philadelphia was the largest city in the united states, with a population of , . it was also the us capital, and the outbreak of disease spurred efforts to move the capital to what became washington, dc. the epidemic began in august, with the deaths of two immigrants. rush, who had lived through another outbreak of the disease in , recognized what was happening and immediately alerted officials to the return of a "highly contagious as well as mortal re-mitting yellow fever." citizens were warned to avoid habits they thought might promote the disease, such as excessive exertion, and the city's streets were cleaned. as the month of august wore on, the deaths of prominent citizens, including physicians active in the fight against disease, led to increasing agitation and varying degrees of panic. hundreds of people were dying each week, and tens of thousands of people, including national leaders, chose to flee the city. samuel breck, a merchant who had newly arrived in the city, described the scene: in private families the parents, the children, the domestics lingered and died, frequently without assistance. the wealthy soon fled; the fearless or indifferent remained from choice, the poor from necessity. the inhabitants were reduced thus to one-half their number, yet the malignant action of the disease increased, so that those who were in health one day were buried the next. the burning fever occasioned paroxysms of rage which drove the patient naked from his bed to the street, and in some instances to the river, where he was drowned. insanity was often the last stage of its horrors. many physicians left the city, but rush remained. no one, rush included, had ever heard of a virus, and the role of mosquitoes in transmitting the disease was unsuspected, but some of rush's ideas helped to contain the disease. he believed that the epidemic might be traced to foul vapors and pushed for their eradication. for example, he urged that decaying food by swept from nearby docks, that sewage be disposed of in a more sanitary matter, and that improved hygiene standards be adopted. rush resisted attempts to blame recent immigrants and instead insisted that the city be cleaned up, so that future generations would not be similarly afflicted. rush, a staunch humoralist, naturally treated the febrile illness with phlebotomy and purging. he also relied heavily on mercury-containing compounds of the sort he provided lewis and clark for their expedition. describing his therapeutic approach, he wrote: i have found bleeding to be useful, not only in cases where the pulse was full and quick but where it was slow and tense. i have bled twice in many and in one acute case four times, with the happiest effect. i consider intrepidity in the use of the lancet, at present, to be necessary, as it is in the use of mercury and jalap, in this insidious and ferocious disease. rush recognized the limitations of the therapies available to him, but like many physicians during the covid- pandemic who prescribed scientifically unproven chloroquine for patients, rush believed that it was preferable to do something rather than nothing. and rush practiced what he preached. years later, when he lay dying, he insisted on being bled himself. perhaps rush's greatest contribution during the yellow fever epidemic of was his moral example. at a time when many of his colleagues were fleeing the city, rush chose to remain behind, saying, "i have resolved to stick to my principles, my practice, and my patients to the last extremity." rush did not waver in his resolve, even though three of the apprentices he had recruited died of the disease, as did his sister. and rush himself fell ill, for a time too sick to leave his house. despite seeing as many as one hundred patients a day, rush rarely failed to write to his wife of the work he was doing, sharing his prayers for his "poor patients." as a physician with vast political experience and a deep belief in the power of institutions to improve human life, rush naturally sought to engage existing organizations in the cause and played a prominent role in founding new ones. each day he commuted from his house just outside the city into the new hospital that had been constructed in response to the disease, where he served as the chief physician. believing that blacks were less susceptible to the disease, he organized groups of black women to serve as nurses, and later told his wife that the majority of his patients were cared for by "african brethren." unfortunately, however, rush was wrong, and blacks enjoyed no greater immunity. although phlebotomy and mercury-based medications may have done little to restore the health of rush's patients, and may have even hastened the demise of some, rush himself was celebrated as a hero, primarily because he stood by his patients and his city when many others were abandoning them. a judge wrote of rush, "he is become the darling of the common people and his humane fortitude and exertions will render him deservedly dear." and rush's recommendations concerning sanitation likely provided many practical benefits, undermining the conditions favorable to the propagation of multiple epidemic infectious diseases, such as cholera and malaria. rush's response to the yellow fever epidemic of serves as an inspiring example to radiology learners and educators confronting crises of their own. first, physicians exist to serve patients, and serving patients sometimes entails personal risk. second, by remaining calm and committed to the professional mission, physicians can serve as exemplars to others, helping them locate their own inner courage and resist their impulse to abandon their mission of service. finally, rush deeply understood the vital role that well-led institutions could play in meeting surmounting disaster. as a model of professional purpose and dedication, rush summoned forth the best, both from himself and those around him. medical inquires and observations upon the diseases of the mind. u.s. national library of medicine digital collections centers for disease prevention and control the path between the seas: the creation of the panama canal philadelphia under siege: the yellow fever of . pennsylvania center for the book rush family papers, yellow fever, key: cord- -svsfu fj authors: richt, j. a.; vandewoude, s.; zink, m. c.; clements, j. e.; herzog, s.; stitz, l.; rott, r.; narayan, o. title: infection with borna disease virus: molecular and immunobiological characterization of the agent date: - - journal: clin infect dis doi: . /clinids/ . . sha: doc_id: cord_uid: svsfu fj borna disease virus (bdv), which seems to be distinct from all other known viruses, exhibits a unique mechanism of pathogenesis. this review highlights several aspects of the biology of infection with this virus and summarizes the preliminary characterization of the agent. studies on bdv may help to illuminate several important areas of neurobiology, including the mechanisms regulating the replication of a new type of rna virus in the nuclei of neural cells, the neuroinvasiveness and neurotropism of such viruses, their t cell-mediated immunopathology, tolerance in newborn animals to persistent viral infection of the central nervous system, and behavioral diseases and eating disorders induced by such agents. mainly in the gray matter of the brain, and pathognomonic intranuclear joest-degen inclusion bodies in neurons and astrocytes of the frontal cortex [ , ] . mechanisms of transmission of the virus in horses and sheep are not well understood, but experimental data suggest that intranasal infection is the common route [ ] . recent seroepidemiological and virological surveys of borna disease in horses have shown that bdv-specific antibodies are present in many horses without clinical signs of the disease. since most of these animals remain clinically healthy for at least year, a long incubation period may be characteristic of the pathogenesis of borna disease [ ] . these data suggest further that bdv is more widely disseminated than was previously thought. in fact, bdv-specific antibodies have been found in clinically healthy horses in the united states (authors' unpublished results). bdv has been only partially characterized. although the agent is known to have physical and replicative properties typical of conventional viruses [ , , ] , its nucleic acid has only recently been identified as rna [ ] [ ] [ ] [ ] . bdv is highly neurovirulent in most laboratory animals and in cell cultures. it has a tropism for cells derived from the neural crest: neurons, astrocytes, and schwann cells. tissue culture cells of nonneuronal origin exhibit only low-level susceptibility to infection, but this resistance can be overcome by cocultivation with bdv-infected primary brain cultures [ ] . the virus replicates noncytopathically in infected cells and is tightly cell associated. infection with bdv is persistent; cultures produce approximately one infectious unit per cell [ ] . treatment of infected cells with interferon a/, does not influence the establishment or maintenance of persistent infection [ ] . persistently infected mdck (madin-darby canine kidney) cells are widely used in indirect immunofluorescence assays for the detection of bdv-specific antibodies in serum and csf of affected animals and humans [ , ] . the viral antigens in persistently infected cells accumulate in the nuclei (sparing the nucleoli) and in the perinuclear area of the cytoplasm and appear as punctate grains in immunofluorescence reactions. bdv particles have never been visualized in infectious material, but replication of bdv is associated with synthesis of at least three novel polypeptides with respective molecular masses of , , and - kd [ ] [ ] [ ] [ ] . antibodies to these virus-associated antigens are produced in infected animals and can be recognized readily by radioimmunoprecipitation and western blot analysis [ ] [ ] [ ] [ ] . sera from animals previously infected with bdv or immunized with bdv-specific proteins do not neutralize the infectivity of the virus [ ] [ ] [ ] . polyclonal antibodies to all three bdv proteins are found in the serum of infected animals. monoclonal antibodies (mabs) to the / -kd and -kd antigens have been obtained after immunization of mice with bdv-specific antigens [ , ] or after infection of rats [ ] . most of these antibodies react in western blot analyses exclusively with the protein used for immunization in lysates of infected cell cultures and infected rat brain. evidence for an antigenic relation between these two proteins has been found by protease digestion and tryptic fingerprint analysis [ ] . several identical peptides were revealed after digestion of the / -kd and -kd proteins. furthermore, the cross-reactivity of mabs to the purified / -kd and -kd proteins indicates that these proteins share antigenic determinants [ , ] . neither protein is glycosylated, but the -kd antigen was found to be phosphorylated at serine residues [ ] . bdv has not yet been classified taxonomically because infectious particles have never been visualized, and only recently has this agent been identified as an rna virus [ ] [ ] [ ] [ ] ] . since novel proteins were identified in bdv-infected cells and tissues, it was assumed that bdv-specific mrnas must also be present in infected material. thus the strategy used to isolate bdv-specific molecular clones was to construct subtractive cdna libraries from mrnas isolated from uninfected and bdv-infected cell cultures and brain [ , ] . no specific nucleic acid probe for bdv was available; the cdna cloning was therefore done in an expression vector, and clones were screened with mabs [ ] specific for the bdv-specific / -kd protein [ ] or with a radiolabeled subtraction probe enriched for bdv-specific sequences [ ] . the bdv-specific clones isolated in our laboratory [ , , ] and in that of another group [ , ] recognized four bdv-specific rnas of . , . , . , and . kilobases (kb), respectively, in bdv-infected rat brain; all of these rnas were enriched by polyadenylate selection [ ] . lipkin et al. [ ] reported that the largest rna species is . kb in size and is not polyadenylated. in vitro transcription and translation of the bdv-specific cdna clone isolated in our laboratory (clone b ) produced proteins of and kd that were recognized by both polyclonal and monoclonal antibodies to bdv [ , , ] (figure ). no significant similarities to any known viral or cellular sequences were detected with both the nucleotide and the amino acid sequences of clone b [ , ] . in addition, southern blot hybridization with bdv-specific cdna clones isolated by both laboratories [ , ] showed that these clones were not of cellular origin and did not hybridize to any dna species in infected material. these data and the identification of four species of rna in infected material clearly demonstrate that bdv is an rna virus [ ] [ ] [ ] [ ] ] . furthermore, since the bdv-specific rnas in infected material are sensitive to digestion with pancreatic rnase, bdv seems to be a single-stranded rna virus [ ] . oligonucleotides with negative polarity from different regions of the b clone all hybridized to the same four positivestrand rnas identified for the entire b clone [ , , ] (figure , lane b). this result indicates that all of the nucleotide sequences in the b clone are present in all of the larger rnas and thus suggests that the organization of the rnas is a nested set of overlapping rnas, as in coronaviruses [ ] [ ] [ ] . in addition, positive-polarity oligonucleotides from different regions of clone b hybridized to three bdv-specific rnas of . , . , and . kb [ , ] (figure , lane d). these rnas probably represent negative-strand complements of the positive rna species. recently, negativestrand rnas have been identified in coronavirus-infected cells; these rnas are thought to be important in replication of the subgenomic rnas [ , ] . thus, both the organization of the rnas and the presence of positive-and negativestrand rnas indicate a striking similarity of bdv to the coronavirus family, although it has not yet been resolved whether bdv is a negative-or a positive-strand rna virus. the resolution of this issue will require the isolation of bdv and the identification of the polarity of its genomic rna. bdv is infectious for a wide range of animal species, from chickens to nonhuman primates and possibly humans [ , , , ] . the clinical manifestations and outcome of experimental infection vary among animals. rabbits develop an reproduced with permission from [ ] . acute, fatal paralytic disease similar to that seen in horses within - weeks [ , ] ; tree shrews (tupaia glis) develop an unusual, nonfatal behavioral disease characterized by aberrant social interactions [ ] . several strains of rat can be infected with bdv [ ] [ ] [ ] ] , but only some of these strains develop disease [ ] . with the exact manifestation depending on the viral variant, lewis rats can develop a biphasic neurological illness characterized by an initial hyperacute aggressive phase that is followed by a passive somnolent stage, an obesity syndrome with fertility disturbances, or paralysis followed by death [ , , , ] . rhesus monkeys develop severe paralytic disease with retinopathy [ , , ] , whereas mice and hamsters show no clinical signs despite replication of bdv in the cns [ , ] . histologic studies of brains from animals with borna disease show typical meningoencephalomyelitis, with the most severe lesions in the frontal region of the cerebral cortex. no pathological abnormalities have been observed in infected mice and hamsters. tree shrews have been used extensively in studies of social interactions because of their well-defined behavioral pat-terns. sprankel et al. found that these animals, classified at the phylogenetic root of the primates, developed a long-lasting, persistent, productive infection in the cns and unique behavioral abnormalities after is inoculation with bdv [ ] . characteristic perivascular infiltration of mononuclear cells and intranuclear joest-degen inclusion bodies in the cns were observed. housing conditions influenced the reaction of these animals to infection. the behavior of all paired animals changed dramatically. these animals showed a need for increased body contact with their partners during the first few weeks after infection. further, infected animals tended to accept their partners much more quickly in grooming social interactions than did uninfected shrews. normally passive females became as aggressive as their male partners and took poor care of their offspring. in contrast, only % of the infected animals maintained in solitary cages showed clinical and behavioral changes. these animals exhibited hyperactivity, with drastic shortening of resting time, and developed eating disorders (bulimia) about weeks after infection. the hyperactive phase was followed by a hypoactive phase characterized by retirement into sleeping boxes and decreased interest in self-grooming. only some of these animals devel-retinopathy in humans. thus, in studies of these changes, rhesus monkeys were infected ic with bdv [ , , ] . the animals developed a persistent infection in the cns and produced antibodies to bdv-specific antigens in serum, csf, and aqueous humor. pathological alterations in the cns and retina consisted of infiltrations of mononuclear cells similar to those in naturally infected animals; however, no destruction of the neuronal layers of the retina was observed [ , ] . clinical signs started with apathy and anorexia - weeks after infection and progressed to severe neurological manifestations dominated by paralysis of the hind limbs. one animal splenectomized months before infection showed signs of apathy but developed no paralysis and had fewer cellular infiltrates in the cns and the retina than did other animals [ ] . these findings suggested that the pathogenesis of borna disease in primates is based on an immunopathological mechanism-an idea confirmed in studies with lewis rats and rabbits. neurotropism of bdv figure . northern blot analysis of rna from rat brain. lanes a and c contain µg of total rna from uninfected rat brain. lanes b and d contain tig of total rna from bdv-infected rat brain. lanes a and b were hybridized with a negative-sense p-labeled oligonucleotide ( ), lanes c and d with a positive-sense p-labeled oligonucleotide ( ). the blot was stripped and rehybridized with a chicken li-actin probe to ensure that equal amounts of rna were loaded in each lane. reproduced with permission from [ ] . oped more severe neurological signs, such as ataxia and partial paralysis of the limbs [ ] . the pathological changes in the brain and retina of bdvinfected animals resemble certain types of encephalitis and most studies on the pathogenesis of bdv infection have involved experimentally inoculated lewis rats. as has already been mentioned, the clinical manifestations of the infection in these animals depend on the passage history of the virus used for inoculation. thus a variety of bdv variants have been used, including strains that cause paralysis and death (similar to the disease in horses and sheep), obesity with fertility disturbances, behavioral changes, or inapparent infections. in all cases, ic inoculation of bdv into adult rats resulted in productive viral replication in the nervous system, where viral antigen was confined to astrocytes, schwann cells, and central and ganglionic neurons and their axonal-dendritic processes [ , , ] . infectivity was first detected in brain homogenates days after inoculation with - id /ml by day [ ] . this infectivity titer in the brain was maintained throughout an observation period of months (figure , top). similar data were obtained for black hooded rats, which were found to be resistant to the disease [ ] . lower levels of infectivity ( - id /ml) were usually found in spinal cord, adrenal glands, and ganglia of the autonomic nervous system. infectivity in the eyes was transient, lasting - weeks after virus appeared in the brain. loss of infectivity coincided later with degeneration and loss of retinal neurons. in immunocompetent adult rats no infectivity was found in extraneural tissue at any stage [ , ] . neither infectious virus nor viral antigens were detectable in lung, spleen, kidney, muscle, peritoneal macrophages, or blood. the same pattern of tropism was seen in cyclophosphamide-treated rats [ , ] (figure , bottom) and in athymic adult rats after ic inoculation of bdv [ ] . the lack of susceptibility of cultured macrophages and spleen fibroblasts week-old rats infected with bdv and treated with cyclophosphamide ( mg/kg) day later. the persistently high levels of infectivity in the brain and eyes and the lack of infectivity in nonneural tissues are shown. rats did not respond immunologically to the virus and did not become ill. reproduced with permission from [ ] . to infection with bdv demonstrated the strong tropism of the virus for cells of neural origin [ , ] . in rats inoculated ic or intranasally at birth, bdv replicated preferentially in the cns and the peripheral nervous system but also spread to nonneural tissues. whereas infectivity in nervous tissue was maintained at a level of - id /ml, titers in the nonneural tissues such as heart, lung, liver, spleen, kidney, pancreas, and bladder were approximately two orders of magnitude lower [ ] . infectious virus was shed in saliva, lacrimal fluid, and urine [ ] . infectivity assays and immunocytochemical assays on sequential tissue samples obtained after inoculation from infected newborn rats showed that the agent first began to replicate in the cns and then disseminated along nerve pathways to nonneural tissues. presumably, infection in the latter tissues was initiated at the point of innervation, with subsequent spread from cell to cell. no viremia was detected in these animals. the similar pattern of dissemination noted in infected adult rats treated for prolonged periods with cyclosporin a [ , ] may suggest that nonneural cells are innately susceptible to infection but that yet-unknown factors in the host prevent access of the virus to those cells. delineation of the scheme for the dissemination of bdv via neural pathways from and to the cns was based on the initial observation by krey et al. [ ] that in rabbits spread of the virus from the brain to the retina could be prevented by ablation of the optic disk. the incubation period of the disease in rats (the period between inoculation of virus at a peripheral site and onset of illness) varied significantly with the route of inoculation [ , ] . the onset of boma disease could be recognized only after the virus invaded the brain. disease appeared days after ic inoculation, - days after intranasal inoculation, and days after footpad inoculation [ ] . after intranasal infection (probably the natural route), bdv entered the neural receptors in the olfactory epithelium and migrated into the brain (figure ) [ ] . similar results were obtained with inoculation into the hind footpad of adult rats (figure ). infection and disease were prevented by sectioning of the sciatic nerve before or within day after inoculation of bdv [ ] . in contrast, neither iv inoculation of bdv weekly for weeks nor oral inoculation of the virus resulted in infection [ ] . thus bdv infection in the host resulted only after obligatory replication in nervous tissues, with subsequent dissemination to nonneural tissues via neural pathways in immunologically impaired animals. once introduced into the cns of the rat, bdv usually caused a persistent infection with continuous productive replication in the brain and spinal cord. all immunocompetent infected animals developed antibodies to bdv-specific antigens, and these antibodies coexisted with infectious virus in the cns. hyperimmune sera or csf from bdv-infected animals did not protect against infection in neutralization experiments in vivo or in vitro [ , ] . infection of newborn rats. although infection of newborn rats resulted in persistent viral replication in the cns as well as in visceral organs, these animals developed no inflammatory response or signs of borna disease. however, "luxury" functions of the cns seem to be affected, possibly as a result of morphological changes confined to the loss of neurons from the dentate gyms of the hippocampus and of some neurons from the nuclear and photoreceptive layers of the retina [ , , ] . the animals produced normal litters, and no virus was found in brain homogenates of their progeny [ ] . (however, when housed in the same cage as their infected newborns, mothers can become infected [ ] .) infected newborn animals developed bdv-specific antibodies late after infection [ , ] . infection of immunocompetent rats. infected immunocompetent rats developed severe disseminated meningoencephalitis in which the most intense inflammatory reaction was centered in the gray matter of the cerebral cortex. the onset of clinical disease coincided with the appearance of meningoencephalomyelitis rather than being a direct effect of viral replication in the brain [ , ] . the most prominent changes were dense accumulations of mononuclear cells in the perivascular spaces and throughout the neuropilcorresponding to the areas with greatest viral replication [ ] [ ] [ ] ] . immunocytochemical investigation of the composition of the inflammatory cell population during the course of infection revealed that macrophages and lymphocytes of the cd + phenotype were dominant at all stages [ , ] . cd + t cells were less frequent. b lymphocytes and plasma cells became prominent during later stages of the disease, when marked parenchymal deposition of immunoglobulin developed [ ] . the severity of the inflammatory reaction reached its max-imum between day and day , during the hyperacute phase of the disease. edema and necrosis of cellular elements in the neuropil accompanied the inflammatory changes. this phase was characterized by alertness, loss of fear, and frenzied hyperactivity. infection with an "aggressive" variant led to paralysis and death during this phase. the cell loss was most extensive in the cerebrum between the frontal and temporal cortices, and this effect finally led to hydrocephalus ex vacuo in surviving animals [ , ] . hydrocephalus progressed slowly, and, despite continuous productive replication of bdv in the brain, the inflammatory response began to decline after day , with only minimal inflammatory lesions detectable in the brain by day . there was no further loss of brain substance and no further extension of hydrocephalus after the inflammatory cells disappeared. beyond day , by which the inflammatory response had become mild, the animals became much calmer, spending most of the time in an inactive state and asleep. no pathological changes were found in the ependymal lin- ing of the ventricles or the choroid plexus throughout the study [ , ] . despite the dynamic changes in the intensity of inflammation, high levels of viral replication in the cns persisted in these animals ( figure , top) . pathological changes in the eyes were similar to those in the brain and developed subsequent to encephalitis. there was a degeneration of rods and cones and a gradual focal disappearance of neurons from the inner and outer nuclear layers [ , ] . by day most of the animals had become blind since the infection resulted in the complete destruction of the inner and outer nuclear layers of the retina. no inflammatory cells were found in the organs at late stages. viral variants differed in their ability to cause various clinical manifestations in the rat. this observation raises the intriguing possibility that bdv exhibits marked genetic variability and that different strains may cause different pathological manifestations of neurological disease. the obesity syndrome developed gradually in immunocompetent infected rats, starting - months after infection ( [ ] and s. herzog and r. rott, unpublished results). the obese rats exhibited characteristic behavioral and metabolic alterations, with significant increases in the intake of food and water and the development of high levels of triglycerides, insulin, and glucose in the blood [ ] . histologic examination revealed prominent hydrocephalus and meningoencephalitis. in long-term survivors, titers of infectious virus in the cns declined drastically and antibody levels decreased ( [ ] and s. herzog and r. rott, unpublished results). prevention of disease in rats. in gierend and ludwig [ ] reported that treatment of bdv-infected rabbits with glucocorticoids and/or cyclophosphamide resulted in a reduction in antibody production, the development of only low-intensity encephalitis, and a delayed onset of clinical disease. similar studies showed that a single ip injection of cyclophosphamide ( mg/kg), given i day before or after ic inoculation of bdv into -week-old rats, prevented the onset of borna disease. these rats did not develop antibodies to bdv, encephalitis, or clinical disease [ , ] . the level of production of virus in the cns and the neurotropism of the agent were similar in treated and untreated infected animals ( figure ) . thus a persistent, productive type of viral replication developed in the brain and eyes of cyclophosphamide-treated rats (figure , bottom) without the complications of pathological effects or clinical disease. as in infected newborn rats, neurons in the dentate gyms of the hippocampus degenerated and were replaced by glial cells. since other infected neuronal groups did not degenerate, loss of these cells may have been due to down-regulation of trophic factors. in contrast, no necrosis among other cell types in the brain occurred in these animals. similarly, infected newborn, athymic, and cyclosporin a-treated adult rats failed to develop encephalitis or disease [ , , , [ ] [ ] [ ] . these data suggested strongly that borna disease was caused by the cellular immune response to bdv in immunocompetent animals. reconstitution of bdv-specific inflammation: immunosuppressed animals. the immune basis of borna disease was confirmed in adoptive transfer experiments in which spleen or cervical lymph node cells from infected rats were transferred to infected, -week-old, cyclophosphamide-immunosuppressed virus carriers. after adoptive transfer these tolerant virus carriers developed bdv-specific antibodies, encephalitis, and behavioral disease (table ). the donor note. four-week-old lewis rats were inoculated ic with bdv; some rats were treated with cyclophosphamide ( mg/kg) hours later. animals rendered tolerant by this treatment were injected iv with various cell suspensions, as indicated, and were maintained for clinical and pathological examination. nmi t cells are described in the text; ppd t cells = purified protein derivative-specific t cells. figure . acetone-fixed astrocytes persistently infected with bdv were stained with tetrarhodamine isothiocyanate-labeled rat antibodies to bdv and fluorescein isothiocyanate-labeled rabbit antibodies to glial fibrillary acidic protein (gfap). arrows point to punctate accumulation of bdv antigens in the nuclei, and arrowheads show diffuse staining pattern of gfap in the cytoplasm. cells had no effect when injected into immunocompetent animals [ ] . further, transfer of hyperimmune bdv-specific sera into virus carriers did not cause clinical disease [ ] . with the goal of identifying the cell type responsible for inducing disease in virus carriers, a permanent t cell line, nm , was developed from the popliteal lymph node cells of lewis rats immunized in their hind feet with affinity-purified, bdv-specific / -kd protein [ , ] . the nm cells responded specifically to the / -kd bdv antigen in lymphocyte proliferation assays and did not recognize unrelated antigens. cytofluorographic studies with mabs to leukocyte differentiation antigens showed a staining pattern characteristic for cd + t cells. inhibition studies with mabs against selective restriction elements of the major histocompatibility complex (mhc) revealed that these cd + effector t cells were mhc class ii restricted [ , ] . the latter cells were functionally characterized in studies with astrocytic cultures used as antigen-presenting cells or targets and pulsed with bdv-specific / -kd protein or infected with bdv. astrocytes are known to become infected during bdv-induced encephalopathy [ , ] , and primary astrocyte cultures are susceptible to bdv infection when inoculated with infectious rat brain homogenate (figure ) . the persistent noncytopathic infection of astrocytes with bdv did not result in significant spontaneous expression of mhc class ii antigens, although such expression has been reported for other neurotropic viruses [ , ] ; however, the expression of this antigen was easily induced by incubation with recombinant interferon- (ifn-y). in lymphocyte proliferation experiments, uninfected, ifn- treated astrocytes were able to induce specific proliferation of nm cells when inoculated exogenously with the / -kd protein [ ] . when persistently bdv-infected astrocytes were used as antigen-presenting cells after induction with ifn-y, only slight specific proliferation of the nm cells was found. however, exogenous addition of the bdv-specific / -kd protein to these cultures resulted in antigen-specific proliferation [ ] . furthermore, when analyzed in a conventional cytotoxicity assay, bdv-infected astrocytes were found to be susceptible to lysis by nm t cells (j. a. richt and l. stitz, unpublished results). the bdv-specific nm i cells induced acute meningoencephalitis mainly in the gray matter of the brain and produced hyperacute paralytic disease when injected iv into immunosuppressed syngeneic virus carriers [ , ] (table i ) . the clinical signs became manifest as early as days after transfer, with development of severe apathy and somnolence; most of these animals had to be killed within hours after the onset of disease. inflammatory exudates contained a large number of cd + t cells, fewer cd + lymphocytes and b cells, and some neutrophils [ , , ] . the animals did not go through the hyperactive phase that typically occurs during regular infection or reconstitution with spleen cells. their disease was hyperacute and rapidly fatal. the fact that disease could be induced after transfer of mhc class ii-restricted, bdv-specific, cytolytic cd + t lymphocytes suggested that the lesions in borna disease may be a delayed-type hypersensitivity reaction. the mechanism for the different clinical disease that resulted from reconstitution with nm cells-as opposed to spleen and lymph node cells-is not understood. the nm cells probably caused elaboration of unusually large amounts of acute-phase proteins and cytokines, which in turn could have caused an imbalance in neurotransmitter functions in the brain [ ] . the difference in clinical disease caused by the spleen and lymph node cell suspensions may also have been due to the combination of nonselected immune cells used for transfer. carriers. the pathogenesis of borna disease has several features in common with lymphochoriomeningitis (lcm) virus infection in mice. in both cases the virus causes acute cns disease that can be prevented by immunosuppression, and this tolerance can be overcome by reconstitution with specifically sensitized cells. the similarity in the pathogenesis of the two infections applies in newborn animals. newborn mice inoculated with lcm virus become persistently infected, but their growth is severely retarded and they later die from immune complex disease [ ] . newborn rats inoculated with bdv also become persistently infected, as has already been described. however, unlike their murine lcm-infected counterparts, persistent bdv-infected rats do not become ill [ ] . in contrast to adoptive transfer into cyclophosphamidetreated virus carriers [ ] , injection of spleen cell suspensions from diseased syngeneic rats into newborn bdv carriers did not result in inflammation or in clinical boma disease [ ] . for evaluation of the role of immunologic tolerance in neonatal virus carriers, these animals were surgically joined to syngeneic normal rats via the peritoneal cavity in a manner that allowed the free flow of humoral and cellular immune elements [ ] ; this scheme followed the classical experiment of billingham et al. [ ] . the normal-counterpart rats were then inoculated with bdv and developed typical borna disease. examination of the tissues from the persistently infected chimera showed that these animals developed mild lesions in the central and peripheral nervous systems. adoptive transfer of the cd + virus-specific t cell line nm into neonatal ( -week-old) infected rats also resulted in mild transient encephalitis [ ] ; this development suggested that tolerance to the virus in newborn infected animals can be overcome by transfer of bdv-specific immune cells, although reconstitution was not as efficient as in cyclophosphamide-treated virus carriers. regulatory immune mechanisms present in neonatal infected animals, but not in adult rats rendered tolerant by cyclophosphamide, may be responsible for this unique mechanism of tolerance and resistance to the induction of disease. the behavioral changes in animals infected with bdv are somewhat reminiscent of some types of affective disorders in human beings. indeed, the serum and csf of some patients with psychiatric illnesses (including recurrent unipolar depression, bipolar affective disorders, and residual-type schizophrenia or personality disorders) contain bdv-specific antibodies [ , ] . antibodies were present in %- % of sera obtained from - , psychiatric patients from germany, the united states, and japan. the percentage of patients seropositive was highest in a region of southern germany where borna disease has been known to be endemic among horses and sheep. approximately % of , randomly collected sera from otherwise hospitalized patients from this area in which borna disease is endemic contained antibodies to bdv [ ] . examination of acutely seropositive patients by magnetic resonance imaging revealed that % had cerebral lesions in one or both hemispheres, whereas no one in a seronegative control group had such lesions [ ] . serological examination by bode et al. of patients infected with human immunodeficiency virus has shown an incidence of bdv antibodies of - % [ ] . the same investigators have reported a high incidence of bdv-specific antibodies among patients with chronic inflammatory neurological disorders such as multiple sclerosis [ ] . the bdv specificity of the human antibodies was recently reinforced by the finding that antibodies from three of seven seropositive patients recognized the -kd protein expressed by a bdv-specific cdna clone [ , ] (figure ). these data suggest either that a bdv-like infection is prevalent in the human population or that some patients with inflammatory neurological diseases have cross-reacting antibodies that recognize antigens expressing epitopes homologous with those of bdv proteins. these observations were substantiated further by attempts to isolate bdv from the csf of three seropositive patients [ ] . csf from these patients was either applied to fetal rabbit brain cells or inoculated is into rabbits, which are highly susceptible to borna disease. in cell cultures a small number of immunoreactive cell foci were found with bdv-specific antibodies - days after inoculation. however, the cells lost their antigen during subcultivation. the inoculated rabbits developed no signs of borna disease; neither lesions in the cns nor bdv-specific antigens were demonstrable in brain sections by immunohistologic techniques. nevertheless, these animals developed bdv-specific antibodies in their sera. that the brain homogenate from one rabbit was infectious for fetal rabbit brain cells was demonstrated by positive cell foci in immunofluorescence assays [ ] . however, again, the antigen disappeared during attempts to propagate the agent by subcultivation of the cells. these findings can be interpreted as typical of abortive infection. although these data strongly support the hypothesis that a bdv-like agent causes infection in humans, a defined mental disorder has not been correlated with the presence of bdv-specific antibodies. the development of such a disorder may be related to various factors, such as the genetic properties of the virus; the route of infection; and the age, immune status, and genetic makeup of the infected individual. evidence that these factors play a role in the outcome of borna disease has been found in naturally infected horses and sheep as well as in experimentally infected animals. die bornasche krankheit der pferde und schafe virologisch gesicherter ausbruch der bornaschen krankheit in einer schafherde der schweiz experimentelle untersuchungen fiber die seuchenhafte gehirn-und rackenmarksentztindung der pferde (bornasche krankheit) the cerebrospinal fluid of rabbits infected with borna disease virus handbuch der viruserkrankungen untersuchungen fiber das klinische verhalten der seuchenhaften gehirnrtickenmarksentziindung (bornaschen krankheit) des pferdes nebst angaben fiber diesbeztigliche therapeutische versuche borna disease in a sheep untersuchungen fiber die pathologische histologie, pathogenese und postmortale diagnose der seuchenhaften gehirn-rtickenmarksentziindung (bornasche krankheit) des pferdes die ausbreitung der enzephalitischen reaktion bei der bornaschen krankheit der pferde und deren beziehungen zur encephalitis epidemica, zur heine-medinschen krankheit und zur lyssa des menschen. eine vergleichend-pathologische studie axonal transport of borna disease virus along olfactory pathways in spontaneously and experimentally infected rats seroepidemiologische untersuchungen zur bornaschen krankheit (ansteckende gehirn-riickenmarkentziindung) der pferde borna disease: a persistent virus infection of the central nervous system preliminary studies on the biology of borna disease virus molecular characterization of the borna disease agent isolation and characterisation of borna disease agent cdna clones a borna virus cdna encoding a protein recognized by antibodies in humans with behavioral diseases analysis of borna disease virus-specific rnas in infected cells and tissues replication of borna disease virus in cell cultures influence of interferon on persistent infection caused by borna disease virus in vitro borna disease, a possible hazard for man? isolation and characterization of a molecular weight protein from brains and tissue cultures persistently infected with borna disease virus purification and properties of an intranuclear virus-specific antigen from tissues infected with borna disease virus atypical dissemination of the highly neurotropic borna disease virus during persistent infection in cyclosporin a-treated, immunosuppressed rats antigenetic relationship and further characterization of two major borna disease virus proteins behavioral disease in rats caused by immunopathological responses to persistent borna virus in the brain pathogenesis of borna disease in rats: immune-mediated viral ophthalmoencephalopathy causing blindness and behavioral abnormalities pathogenesis of borna disease in rats: evidence that intraaxonal spread is the major route for virus dissemination and the determinant for disease incubation molecular and immunopathological studies of borna disease virus infection in rats coronaviruses and their replication coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis minus-strand copies of replicating coronavirus mrnas contain antileaders detection of serum antibodies to borna disease virus in patients with psychiatric disorders behavior abnormalities in tree shrews (tupaia glis, diard, ) induced by boma disease virus persistent, tolerant or subacute infection in borna disease virus-infected rats studies on the genetic control of resistance of black hooded rats to borna disease untersuchungen fiber die experimentelle bornavirus-infektion bei der ratte borna disease in rhesus monkeys as a model for uveo-cerebral symptoms virus-induced pigment epithelitis in rhesus monkeys. clinical and histological findings persistent borna virus infection in adult hamsters adaptation of borna disease virus to the mouse astrocytes and schwann cells are virus-host cells in the nervous system of rats with borna disease determination of immune cells and expression of major histocompatibility complex class ii antigen in encephalitic lesions of experimental borna disease replication of borna disease virus in rats: age-dependent differences in tissue distribution effect of borna disease virus infection on athymic rats inhibition of immunemediated meningoencephalitis in persistently borna disease virusinfected rats by cyclosporine a spread of infectious virus along the optic nerve into the retina in borna disease virus-infected rabbits borna disease virus-induced meningoencephalomyelitis caused by a virus-specific cd + t cell-mediated immune reaction escape from lethal disease in rats after borna disease virus infection: survival with obesity syndrome influence of immunosuppressive treatment on borna disease in rabbits borna disease, a progressive meningoencephalomyelitis as a model for cd + t cell-mediated immunopathology in the brain viral particles induce la antigen expression on astrocytes tumor necrosis factor amplifies measles virus-mediated la induction on astrocytes narayan , oldstone mba. neurotransmitter abnormalities in borna disease southern p. virus and immune responses: lymphocytic choriomeningitis virus as a prototype model of viral pathogenesis induction of borna disease and inflammation in rats inoculated with borna disease virus as neonates quantitative studies on tissue transplantation immunity. iii. actively acquired tolerance borna disease virus. a possible etiological factor in human affective disorders? kernspintomographische befunde bei psychiatrischen patienten mit and ohne semm-antikfirper gegen das borna disease virus-specific antibodies in patients with hiv infection and with mental disorders increased incidence of borna disease virus infection in chronically diseased patients key: cord- -a q dq authors: pace, david; gauci, charmaine; barbara, christopher title: the epidemiology of invasive meningococcal disease and the utility of vaccination in malta date: - - journal: eur j clin microbiol infect dis doi: . /s - - - sha: doc_id: cord_uid: a q dq invasive meningococcal disease (imd) is a vaccine-preventable devastating infection that mainly affects infants, children and adolescents. we describe the population epidemiology of imd in malta in order to assess the potential utility of a meningococcal vaccination programme. all cases of microbiologically confirmed imd in the maltese population from to were analysed to quantify the overall and capsular-specific disease burden. mean overall crude and age-specific meningococcal incidence rates were calculated to identify the target age groups that would benefit from vaccination. over the -year study period, out of the eligible notified cases were confirmed microbiologically of which . % had septicaemia, . % had meningitis, and . % had both. the mean overall crude incidence rate was . / , population with an overall case fatality rate of . %. meningococcal capsular groups (men) b followed by c were the most prevalent with w and y appearing over the last years. infants had the highest meningococcal incidence rate of . / , followed by . / , in – year olds and . / , in – year old adolescents. the introduction of menacwy and menb vaccines on the national immunization schedule in malta would be expected to reduce the disease burden of meningococcal disease in children and adolescents in malta. invasive meningococcal disease (imd) manifests predominantly as meningitis and/or septicaemia with most affected individuals having a sudden presentation and rapid deterioration. although rare, imd affects all ages, and the brunt of the disease burden is highest in infants, children below years of age and adolescents [ , ] . compared with other age groups, there is also a relatively increased incidence of imd in the elderly who suffer the highest case fatality rates of all [ ] [ ] [ ] . despite advances in medical care, around % of individuals with imd die, and up to - % of survivors sustain permanent disabilities such as sensorineural hearing loss, neurodevelopmental problems, seizures and amputations [ ] [ ] [ ] [ ] . imd is a worldwide disease, but the epidemiology of the meningococcal capsular groups (men) is unpredictable and varies by geographical regions and over time. menb still causes the majority of imd within europe, followed by menc and more recently menw [ ] . in the usa, menb, menc and meny are each responsible for around one third of the imd cases [ ] . classically, mena was a major cause of epidemics within countries in the meningitis belt of sub-saharan africa [ ] , with recent emergence of menw, c and x disease [ , ] . nasopharyngeal carriage provides a continuous reservoir making eradication of the disease difficult. migration and international travel poses a risk of transmission of men capsular groups especially to family members within hosting countries [ ] . the introduction of meningococcal conjugate vaccines on national immunization programmes has resulted in significant reductions in the corresponding burden of imd. control of imd at a population level by meningococcal conjugate vaccines is not only a direct effect of protection of the vaccinated individuals but also is largely a result of their ability to interrupt transmission through reduction in meningococcal carriage rates, thus inducing a herd immune effect. this effect is only seen when adolescents, who are the main transmitters of the meningococcus, are included in meningococcal immunization strategies. drastic reductions in imd in all ages, including unvaccinated groups, have been observed with immunization programmes that included adolescents in catch-up or routine campaigns for menc in europe and salvador in brazil and mena in africa [ ] [ ] [ ] [ ] [ ] . in contrast, exclusive vaccination of at-risk children < years old against menw disease with a menacwy conjugate vaccine in chile and with a menc conjugate vaccine in bahia, brazil, without immunizing adolescents did not impact menw and menc disease in unvaccinated age groups, including the elderly, in the respective countries [ , ] . the recent introduction of proteinbased vaccines against menb in developed countries holds promise for control of the most prevalent meningococcal capsular group although up until now their effect is only envisaged to result from direct protection since impact on nasopharyngeal carriage has not been demonstrated [ , ] . the maltese archipelago is a small group of islands situated in the southern mediterranean area having a population of around , people, around . million tourists per year and a population density of persons per square kilometre, the highest in europe [ , ] . as yet, meningococcal vaccines have never been introduced on the national immunization schedule in malta although such vaccines are available privately for individuals wishing to protect themselves or their children against imd. we aimed to study the population epidemiology of imd in the maltese islands in order to assess the utility of a meningococcal vaccination programme. all microbiological confirmed cases of imd over an year period, from to , were collected from the bacteriology laboratory at mater dei hospital (mdh) which provides care for all patients suffering from imd. cases were included if they satisfied the european centre for disease prevention and control (ecdc) laboratory criteria for imd which consisted of (a) identification of the meningococcus through culture or molecular methods from usual sterile sites (blood, cerebrospinal fluid (csf), synovial fluid and any other usually sterile fluid) or from purpuric lesions or (b) detection of gram-negative diplococci from direct visualization of the csf or through a positive meningococcal rapid antigen screen (ras) [ ] . subsequently, isolates were classified according to the capsular group, if groupable. identification of the serogroups was performed by slide agglutination tests using specific capsular antisera (remel europe ltd., kent, uk). all isolates were sent to the public health england (phe) meningococcal reference unit (previously health protection agency meningococcal reference unit) in manchester, uk, where the capsular group was reconfirmed and phenotyping was performed by identification of the serotypes and serosubtypes with monoclonal antibodies as described by gray et al. [ ] . multilocus sequence typing (mlst) was not carried out. from to , a polymerase chain reaction assay (pcr) amplifying the specific capsular genes was performed at the phe laboratory on csf and/or blood of patients whose cultures were negative. in , a meningococcal screening pcr was introduced at the microbiological laboratory in mdh, and any positive samples were sent to the phe laboratory for capsular gene identification. meningococcal capsular gene detection was performed at mdh in . meningococcal isolates identified from non-sterile sites such as throat or nasopharyngeal swabs and sputum were excluded. notification of imd is statutory obligatory in malta. all notified cases of imd, identified through passive surveillance, were collected from the infectious disease prevention and control unit (idcu) in malta. the criteria for reporting adopted by the idcu were in accordance with the case definitions for confirmed imd set by the european union commission decisions in , and subsequently in [ , , ] which also included possible cases of imd as defined by the presence of one of the clinical criteria of meningitis, haemorrhagic rash, septic shock or septic arthritis or probable cases, as defined by the presence of clinical criteria with an epidemiological link to a case of imd, in the absence of laboratory confirmation of the infecting pathogen. cases were notified to the idcu by clinicians when a patient was clinically suspected to have imd and subsequently by the laboratory when the meningococcus was identified in clinical specimens. when microbiological results did not reveal an invasive pathogen, idcu contacted the clinicians to ascertain that the clinical picture, investigations and progress of the cases still satisfied the set case definitions of imd. these data were used to calculate the total number of imd cases per year in malta over the study period. the results of microbiological investigations of all notified cases were validated against electronic results and/or written case records. individuals with a foreign hospital number or whose laboratory results showed an alternative diagnosis were excluded from the analysis. population data were obtained from the national statistics office in malta. this study was approved by the faculty of medicine and surgery university o f m a l t a r e s e ar c h e t h i c s c om m i t t e e ( r e f n o : frecmds_ _ ). the primary objective of this study was to perform population-based descriptive statistics on imd in order to quantify the disease burden caused by the meningococcus in the maltese population. the median was used in preference to the mean to describe the age demographics as in view of the relative rarity of imd, it was expected to have a small number of cases with a wide age range. the proportion of laboratoryconfirmed imd cases was calculated per year from the total number of reported imd cases. the disease burden caused by the different meningococcal capsular groups was calculated for each year from the number of laboratory confirmed isolates. the distribution of cases was analysed according to prespecified age groups as follows: < , - , - , - , - , - , - , - and ≥ + years. a cut-off of < years was taken to indicate the paediatric population as determined by the hospital admission policy for children in mdh, malta. this enabled calculation of the age-specific incidence rates of meningococcal disease, and the corresponding % ci using a poisson regression analysis, in maltese children and identification of the target groups that would benefit from vaccination. fisher's exact test was used to analyse differences in the disease burden between the capsular groups. a p value < . for all analyses was taken as being statistically significant. the software stata was used for the analyses. a total of cases of meningococcal disease were identified over the -year study period: were notified, whilst additional cases had not been notified but were identified from the bacteriology lab from the results of their cultures. of these, individuals resident in malta were eligible to be included in the analysis, of whom . % were male ( / ). of the excluded cases, were tourists, and were found to have an alternative diagnosis on review of their electronic results or case records (either because csf was suggestive of viral meningitis in culture negative cases or a different pathogen was noted during validation of the electronic records). only . % ( / ) of cases were confirmed microbiologically, of which . % ( / ) were confirmed on culture ( fig. ). meningococcal pcr, introduced in , confirmed of the culture negative cases from to all of which were in children, although testing was performed at the request of the caring doctor rather than routinely on all suspected cases. the median age of the laboratoryconfirmed cases was . years (range, . - . years). out of the microbiologically confirmed cases of imd, . % ( / ) had confirmed septicaemia (confirmed from blood or petechial scrapings), . % ( / ) had confirmed meningitis, . % ( / ) had confirmed septicaemia and meningitis, . % had arthritis, and . % had pericarditis. *these cases were picked up by the microbiological laboratory but were not notified. pcr, polymerase chain reaction assay. ras, rapid antigen screen; men meningococcus. the variation of the annual total number of confirmed cases generally followed the variation of the total number of notified cases except for when only out of a total of notified cases ( . %) were confirmed microbiologically (fig. ) . the mean overall crude incidence rate of confirmed imd from to (table ) was . / , ( % ci, . to . ) population. although the mean crude incidence rate decreased from . / , ( % ci, . to . ) population in - to . / , ( % ci, . - . ) population from to , this was not statistically significant (difference . ; p = . ). the mean age-specific incidence rate of imd was significantly higher in infants ( . / , ; median age months; range, . - . months), - -year-old children ( . / , ; median age . years; range, . - . years) and - -yearold adolescents ( . / , , median age . years; range, . - . years) than the rest of the population (fig. ) with the highest burden being in infants. there were deaths caused by confirmed imd (table ) , of which ( . %) were in children < years old. the overall case fatality rate (cfr) was . % ( / ). in children < years of age, the highest age-specific cfr was in - -year-olds ( / ; . %), - -year-olds ( / ; . %) and - -year-olds ( / ; . %), whilst in adults, the highest risk of dying was in - -year-olds ( / ; . %) and in ≥ -year-olds ( / ; . %). the months with the highest total and similarly confirmed number of imd cases were january, february, march and august, whilst the lowest numbers were recorded in april and from october to december. no epidemics of meningococcal disease occurred during the study period. in view of the unexpected peak of imd in august, the cases of imd in tourists who became unwell whilst in malta were looked at (these had been excluded from the primary analysis). a peak in imd in tourists was also noted in august when / ( . %; median . years, range, . - . years) confirmed cases occurred. identification of the meningococcal capsule was successful in / cases, of which were isolated by culture (fig. ) . the most frequent isolated capsular groups were menb ( . %; / ) and menc ( . %; / ). capsular groups w and y and non-groupable meningococci collectively constituted a minor proportion of cases ( . %; / ), although capsular group y became prevalent from to ( . %; / cases) and capsular group w became more frequent from to ( . %; / cases) (fig. ) . menb disease was significantly more prevalent than other capsular groups in children < years of age. infants suffered the highest incidence of menb, menc and menw disease compared with all other age groups (fig. ) . meny disease was only observed in - year-olds, whilst menw affected teenagers and infants. the incidence rates of menb, c, w and y disease were not significantly different in elderly people ≥ years old. the overall mean crude incidence rate of menb in malta was . / , ( % ci, . to . ) population with an overall case fatality rate of . % ( / ). a statistically significant downward trend from . / , population, % ci . to . , over - , down to . / , population, % ci . to . , over - , (difference . ; p = . ) was observed (fig. ) . the highest disease burden of menb disease was in infants ( . / , ; % ci, . - . ) with a median age of . months, followed by - -year-olds ( . / , ; % ci: . - . ), median age . years, and - year-olds ( . / , ; % ci, . - . ), median age . years (fig. ) . only pora phenotyping was performed, with % ( / ) of the typed isolates having pora subtypes p . ,p . (table ) . % ci, . to . ), difference . , p = . (fig. ) . the mean age-specific incidence rate of menc disease was significantly higher in infants ( . / , infants; % ci, . - . ; median age . months, range, . - . months) compared with all other age groups (fig. ) . fatalities from menc disease were more common in children, with of the deaths occurring in children aged months, years and years. of the menc cases, three were confirmed by detection of the sia d gene by pcr, whilst were confirmed by culture. phenotyping of the cultured menc strains was performed on isolates, since of the meningococcal cultures did not remain viable in storage and could not be shipped to the phe laboratory in the uk for further identification ( table ) . out of the cases who succumbed to menc disease, were phenotyped, and all had the pora subtypes p . ,p . . the overall mean crude incidence rate of menw and y disease was . / , ( % ci, . - . ) each, with an associated cfr of % ( / ) and % ( / ), respectively. deaths occurred predominantly in > -year-olds although menw error bars represent % ci the average annual incidence of confirmed imd cases of . / , population ( % ci, . - . ) in malta remains significantly higher than the mean incidence reported overall from to in europe ( . / , ; % ci, . - . ; p = . ) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] and the usa ( . / , ; % ci, . - . ; p = . ) [ ] , where a significant decrease in overall imd has been observed over the last few years. the drop in menb disease observed in malta and similarly in the eu and the usa is very likely a result of natural variation in the epidemiological trends of menb. however, the reduction in menb disease has had no effect on the overall incidence of imd in malta since it was offset by the persistence of menc disease and the appearance of menw and men y disease since . in contrast, the incidence rates of menc imd in the eu were significantly reduced (from . / , , % ci, . - . in - down to . / , , % ci, . - . in - ; p = . ) as a result of the introduction of menc vaccination programmes in several european countries, many after catch-up campaigns targeting adolescents, and the introduction of routine adolescent vaccination [ , ] . in the usa, the incidence of menc and y disease was reduced through the introduction of routine adolescent menacwy vaccination since [ ] . in salvador, brazil, control of menc disease was similarly rapidly achieved within years following the introduction of a menc vaccination programme targeting children < years of age as well as - -year-old adolescents and young adults [ ] . similarly, control of a mena epidemic was achieved within years in mossala, chad, following the introduction of a mena conjugate vaccine targeting - -year-olds in [ ] . in contrast, a herd immune effect was not seen in the state of bahia, brazil (excluding salvador), when a different menc conjugate vaccination strategy that just targeted children from months to years of age was introduced [ ] . similarly, a strategy using a conjugate menacwy vaccine targeting -month to -year-old children to control menw disease in chile provided direct protection to the vaccinated group but did not result in a herd immune effect [ ] . these strategies emphasize that the success of meningococcal conjugate vaccines relies on their ability to interrupt meningococcal transmission through a decrease in meningococcal carriage, an effect that can only be achieved if adolescents are targeted in meningococcal vaccination programmes. control of menb disease is unlikely to be attained with a similar approach since menb protein-based vaccines have no effect on carriage [ ] ; however, as observed in the uk, direct protection of infants and young children can still be achieved with a modest vaccine effectiveness of . % years after an infant priming and boost schedule [ ] . discrepancies between malta and other countries could reflect geographical differences in the epidemiology of imd; however, the lack of a national meningococcal vaccination programme in malta is most likely contributing to the higher incidence rates of imd. the higher number of imd cases from january to march is similar to the seasonality of imd observed in europe and the usa [ , ] . in malta, these months of the year are the coldest months (mean - °c) with the highest values for relative humidity, which reaches around % [ ] , climatic factors that are known to be associated with a higher risk of imd [ ] . the peak in imd seen in august in malta is difficult to explain as this month is dry and is relatively less humid [ ] but is characterized by the highest number of inbound tourists, the majority of whom come from europe [ ] . inbound tourists reach a mean of , in august, reaching a proportion of % of the mean , individuals constituting the maltese population [ ] . intriguingly, a similar peak in the number of imd cases also occurs in august in tourists whose median age was . years compared with . years in maltese individuals with imd in the same month. nineteen per cent of tourists visiting malta in august are aged < years [ ] with adolescents and young adults, who have the highest rates of meningococcal carriage [ ] ; very likely to visit overcrowded places such as pubs, bars and discotheques; and human behaviour that is associated with a higher risk of meningococcal transmission [ ] [ ] [ ] [ ] . any causal association of climatic factors and imd is challenging to reach in view of the confounding effects of human behaviour and the seasonality of other infectious diseases caused by influenza and other respiratory viruses [ ] . in malta, infants suffer the highest overall rate of imd, followed by children aged - years and teenagers, similar to the age distribution of imd seen in europe and the usa [ , ] . the burden of capsular group b and c disease in malta is similarly disproportionately highest in infants but also affects - year-old children, adolescents and young adults. in european countries, the highest menb and c disease burden is similarly seen in infants (although the incidence rate reached . / , and . / , infants for menb and c, respectively, much less when compared with the mean incidence rate of . / , and . / , infants for the corresponding capsular groups in malta), with children less than years old and adolescents and young adults being more affected than other age groups [ , ] . decay of transplacentally acquired maternal menb and c antibody in infancy, subsequent lack of protective menb and c bactericidal antibody in children in the absence of a menb and c vaccination programme [ , ] and nasopharyngeal mucosal damage from concurrent respiratory tract viruses, which are more common in children < years old [ ] , possibly explain the higher risk of menb and c disease in these age groups. the increased risk among adolescents and young adults is likely a result of increased exposure from changes in social behaviour leading to enhanced transmission of the meningococcus and lack of immunity [ ] . the appearance of meny disease in adolescents and menw disease in infants reflects the recent rise of these capsular groups in europe and poses a risk of disease in both age groups [ , ] . natural immunity from asymptomatic menb and c carriage [ ] or cross-reactive immunity to other microorganisms, such as escherichia coli k which has a sialylated polysaccharide capsule that is structurally similar to that of menc [ ] and to neisseria lactamica which shares antigens found on meningococcal outer membrane proteins [ ] , could contribute to the decreased incidence of menb and c disease observed in the - -year-olds; however, there are no robust data to support this hypothesis. the rise in non-menb disease after years of age could plausibly be due to a decline in immunity from immunosenescence [ ] , but further studies are needed to ascertain this. the overall cfr of . % for cases of imd is similar to the - % reported in europe and the usa [ , ] . cfr from menb and c disease, which reached . % and . %, respectively, and which affected children predominantly, was also similar to the - % menb and c cfrs reported in the usa and europe [ , ] . although mlst typing was not done, the menb phenotype p . ,p . is known to belong to the sequence type clonal complex which is hyperendemic in europe [ ] , and the menc phenotypes c a:p . ,p . , c a:p . and c:nt:p . ,p . have all been previously identified as belonging to the hyperinvasive st- meningococcal clone in other european countries [ ] [ ] [ ] . currently, two protein-based menb vaccines are available: menb- c, (bexsero, glaxosmithkline biologicals, belgium) licenced from months of age, and a menb-fhbp vaccine (trumenba, pfizer ltd., new york) licenced from years of age [ , ] . introduction of a menb vaccine will address the most prevalent cause of imd in malta, although projections of vaccine effectiveness would require whole genome sequencing and vaccine antigen sequence typing of the menb isolates. in the past, a serotypespecific menb outer membrane vesicle vaccine, menzb, that matched the menb strain b: :p . - , causing a prolonged epidemic in new zealand (which was used in a dose schedule for mass vaccination of < year olds from to and subsequently for routine infant vaccination up to ), had an estimated effectiveness of % in children < years of age and an overall effectiveness of % [ , ] . a reduced two-dose infant schedule of the currently available menb- c vaccine at and months followed by a boost at months of age, as introduced in the uk, would be expected to result in a modest % vaccine effectiveness up to years following the last vaccination assuming high vaccine coverage and favourable vaccine sequence matching with invasive menb strains [ ] . in contrast to meningococcal conjugate vaccines, indirect protection in unvaccinated population groups from herd immunity is very unlikely to be observed since menb- c has not been shown to reduce menb carriage, or other pathogenic capsular types, in a large cohort of australian adolescents [ ] indicating that menb transmission will still occur despite vaccination. similarly, carriage data have also discouragingly not demonstrated a reduction in menb carriage following vaccination with a menb-fhbp vaccine during an outbreak [ ] . the lack of an impact on menb carriage makes the utility of catch-up vaccination campaigns against menb questionable [ , ] . protection against menb disease in infancy, early childhood and adolescence will have to rely on the direct protection offered by a menb vaccine. infant menb vaccine priming and boost schedules will still need boosting in adolescence as, similar to the antibody decline seen with conjugate menc vaccines, bactericidal antibody will not last into the teenage years [ , ] . introduction of menb vaccination at and months with a boost at months of age together with two-dose vaccination of adolescents starting from the age of years would be expected to have an impact on menb disease in malta. monovalent menc and quadrivalent menacwy vaccine formulations became available on the private market in malta in and , respectively, but were never introduced on the national schedule. a mean of conjugate vaccines against menc was given per year in children aged < years, amounting to vaccination of around % of the paediatric population per year (national immunization service, personal communication; pfizer malta, vivian corporation, personal communication). such practice of meningococcal conjugate vaccination on request results in individual protection, but considering the epidemiology of menc, w and y disease over the last years is evidently not enough to induce herd protection. control of infant menc and w disease in malta may be achieved with the introduction of a routine infant menacwy vaccination programme consisting of a single menacwy dose at months of age, followed by a month boost, a schedule which would induce protective menc bactericidal antibodies in infants and toddlers [ ] and with extrapolation also to menw. this would have potentially prevented . % ( / ) and % ( / ) of cases of invasive menc and menw disease, assuming % vaccine efficacy. an effective national menacwy conjugate vaccine strategy would also need to target adolescents, not only to target meny disease observed in - year olds in malta but also to contribute towards a desired herd immune effect and reduce the corresponding imd in non-vaccinated groups, especially in the elderly > years of age in whom . % ( / ) of imd was caused by menc, w and y. furthermore, adolescent vaccination would eventually serve the purpose of boosting those children who would have received the routine infant and toddler menacwy schedule since adequate protection against all of these capsular groups will not persist until adolescence [ , ] . alternatively, a single conjugate menacwy vaccine dose could be introduced at months of age concurrently with immunization of adolescents. such a schedule using a monovalent menc conjugate vaccine was introduced in ontario, canada, in / [ ] . although a reduction in invasive capsular group c disease was seen in vaccinated and unvaccinated age groups, the reduction in the incidence of menc disease in infants over the subsequent years was not statistically significant [ ] . in contrast, the introduction of routine mencc vaccination in in -month-old children following a catch-up vaccine campaign in -month to -year-olds in the netherlands resulted in control of invasive menc disease, even in infants [ ] . similarly, the introduction of routine infant menc conjugate vaccination in concurrently with a catch-up vaccine campaign in to -year-olds in the uk was also successful [ ] , although a menc boost was introduced at months of age in due to low vaccine efficacy rates just within years of infant vaccination [ ] . the importance of a catch-up campaign targeting adolescents and young adults to control menc disease was also observed in brazil where sole vaccination of children < years of age in the state of bahia did not result in a herd immune effect in contrast to salvador where the concurrent introduction of a catch-up campaign targeting - year-olds resulted in control of menc disease in all age groups [ ] . similarly, vaccination of -month to -yearold children with a conjugate menacwy vaccine without adolescent catch-up vaccination did not control menw disease on a population level in chile [ ] . considering such data, the introduction of a routine infant and toddler menacwy vaccination programme concurrently with the launch of a one-time menacwy vaccine catch-up campaign targeting children aged from to years old and adolescents and young adults aged from to years old in malta would not only induce direct protection of these highrisk age groups but also result in indirect protection of unvaccinated individuals from decreased menc, w and y transmission by adolescents. such induction of herd protection would translate in a reduction in imd in older unvaccinated individuals (as seen in countries introducing a menc catch-up vaccination programme), the disease burden in whom was substantial over the last years ( . %, / cases) in malta. herd immunity against menc, w and y could then be sustained through a routine menacwy vaccine dose at years of age. once the epidemiology of men disease shows that control of invasive menc and w disease in infants has been achieved (reflecting minimal menc and w transmission), which, as seen in the uk for menc, would be expected to be reached within years [ ] , then the infant menacwy dose may be dropped. this would leave a routine meningococcal conjugate vaccine schedule consisting of a single menacwy dose at months of age, which is important for priming, followed by a booster dose in adolescence. the induction of robust protection against invasive menacwy disease in adolescence would protect teenagers and young adults from possible transmission of these capsular groups from the large number of foreigners that visit malta and mix with the young population in summer. currently, the maltese national immunization schedule in early childhood consists of the following: diphtheria, tetanus, acellular pertussis, inactivated polio and hib (dtap-ipv-hib) vaccine at weeks, , and months; hepatitis b vaccine at , and months; and the measles, mumps and rubella vaccine at months and between and years of age. adolescents receive a tetanus, diphtheria and inactivated polio (td-ipv) vaccine at - years with girls being vaccinated with doses of the human papilloma virus vaccine at -< years of age. introduction of a conjugate menacwy vaccine at months and months of age with another routine dose at years would easily fit within this schedule even when a pneumococcal conjugate vaccine is eventually introduced. similarly, menb vaccination in a , infant priming and month boost in addition to two-dose vaccination starting from years of age can also be easily accommodated within the national vaccination programme. capsular groups b, c, w and y meningococcal disease are endemic in malta, a country with a relative high incidence of imd. the introduction of menacwy vaccination following a single dose in early infancy with a boost in the nd year of life and in adolescence would be expected to impact menc, w and y disease in malta. the concurrent launch of a one-time menacwy vaccine catch-up campaign in children aged between and years and adolescents and young adults aged - years would be expected to induce herd protection and help achieve a faster decline in menc, w and y disease. furthermore, a menb immunization programme consisting of a dose prime and boost menb infant vaccine schedule in addition to menb adolescent vaccination at years of age would also be projected to provide direct protection and reduce the incidence of menb, which is responsible for the highest meningococcal disease burden in malta. invasive meningococcal disease. annual epidemiological report for surveillance reports: neisseria meningitidis case fatality rates of invasive meningococcal disease by serogroup and age: a systematic review and meta-analysis investigators of the canadian immunization monitoring program outcomes of invasive meningococcal disease in adults and children in canada between and : a prospective cohort study cost impact of complications in meningococcal disease: evidence from a united states managed care population the clinical features and long-term sequelae of invasive meningococcal disease in children outcomes of invasive meningococcal serogroup b disease in children and adolescents (mosaic): a case-control study emergence and control of epidemic meningococcal meningitis in sub-saharan africa whole-genome characterization of epidemic neisseria meningitidis serogroup c and resurgence of serogroup w emergence of epidemic neisseria meningitidis serogroup x meningitis in togo and burkina faso the threat of meningococcal disease during the hajj and umrah mass gatherings: a comprehensive review effectiveness of meningococcal serogroup c conjugate vaccine years after introduction protection from routine vaccination at the age of months with meningococcal serogroup c conjugate vaccine in the netherlands impact and effectiveness of meningococcal c conjugate vaccine following its introduction in spain impact of meningococcal c conjugate vaccination programs with and without catch-up campaigns in adolescents: lessons learned from bahia impact of menafrivac in nine countries of the african meningitis belt, - : an analysis of surveillance data meningococcal invasive disease by serogroup w and use of acwy conjugate vaccines as control strategy in chile meningococcal b vaccine and meningococcal carriage in adolescents in australia acosta ; oregon meningococcal carriage team. meningococcal carriage following a vaccination campaign with menb- c and menb-fhbp in response to a universityserogroup b meningococcal disease outbreak-oregon available from www.nso.gov.mt census of population and housing the european commission. official journal of the european union. decisions. / /eu epidemiology of meningococcal disease in england and wales / to / : contribution and experiences of the meningococcal reference unit the commission of the european communities the commission of the european communities. commission decision. /iv european union invasive bacterial infections surveillance network. invasive neisseria meningitidis in europe european union invasive bacterial infections surveillance network. invasive neisseria meningitidis in europe ecdc: european centre for disease prevention and control. surveillance network for invasive neisseria meningitidis in europe - & . ecdc european centre for disease prevention and control. invasive meningococcal disease european centre for disease prevention and control. annual epidemiological report. reporting on - surveillance data european centre for disease prevention and control. annual epidemiological report -vaccine-preventable diseases -invasive bacterial diseases european centre for disease prevention and control. invasive meningococcal disease ecdc: european centre for disease prevention and control meningococcal disease in adolescents and young adults: a review of the rationale for prevention through vaccination centers for disease control and prevention. revised recommendations of the advisory committee on immunization practices to vaccinate all persons aged - years with meningococcal conjugate vaccine four years of case-based surveillance of meningitis following the introduction of menafrivac in moissala, chad: lessons learned vaccination of infants with meningococcal group b vaccine ( cmenb) in england the changing epidemiology of meningococcal disease in the united states, - the climate of malta: statistics, trends and analysis - environmental exposures and invasive meningococcal disease: an evaluation of effects on varying time scales the stonehouse survey: nasopharyngeal carriage of meningococci and neisseria lactamica united kingdom meningococcal carriage group ( ) social behavior and meningococcal carriage in british teenagers meningococcal carriage among a university student population -united states meningococcal carriage in dutch adolescents and young adults; a cross-sectional and longitudinal cohort study prevalence of meningococcal carriage in children and adolescents aged - years in chile in effectiveness and impact of a reduced infant schedule of cmenb vaccine against group b meningococcal disease in england: a national observational cohort study seroprevalence of meningococcal serogroup c bactericidal antibody in england and wales in the pre-vaccination era invasive pneumococcal and meningococcal disease: association with influenza virus and respiratory syncytial virus activity? meningococcal serogroup y disease in europe: continuation of high importance in some european regions in cross-antigenicity and immunogenicity between capsular polysaccharides of group c neisseria meningitidis and of escherichia coli k antigenic cross-reactivity between outer membrane proteins of neisseria meningitidis and commensal neisseria species b-cell responses to vaccination at the extremes of age changes in neisseria meningitidis disease epidemiology in the united states significance of meningococcal hyperinvasive clonal complexes and their influence on vaccines development genetic characterization of a new variant within the et- complex of neisseria meningitidis associated with outbreaks in various parts of the world serogroup and clonal characterization of czech invasive neisseria meningitidis strains isolated from to genotypic and phenotypic characterization of carriage and invasive disease isolates of neisseria meningitidis in finland summary of product characteristics: bexsero meningococcal group b vaccine for injection in prefilled syringe. available from www.medicines.org.uk summary of product characteristics: trumenba. available from www.medicines.org.uk effectiveness of a vaccination programme for an epidemic of meningococcal b in new zealand use of an observational cohort study to estimate the effectiveness of the new zealand group b meningococcal vaccine in children aged under years persistence of the immune response after cmenb vaccination, and the response to an additional booster dose in infants, children, adolescents, and young adults antibody persistence and immunological memory at age years after meningococcal group c conjugate vaccination in children in the united kingdom ) immunogenicity of reduced dose priming schedules of serogroup c meningococcal conjugate vaccine followed by booster at months in infants: open label randomised controlled trial persistence of the immune response at years of age following infant immunisation with investigational quadrivalent menacwy conjugate vaccine formulations antibody persistence up to years after vaccination of toddlers and children between months and years of age with a quadrivalent meningococcal acwy-tetanus toxoid conjugate vaccine epidemiology of serogroup c and y invasive meningococcal disease (imd) in ontario, - : vaccine program impact assessment is a single dose of meningococcal serogroup c conjugate vaccine sufficient for protection? experience from the netherlands meningococcal c conjugate vaccine: the experience in england and wales publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by david pace. the first draft of the manuscript was written by david pace, and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. the authors declare that they have no conflict of interest. key: cord- -lyfh ixi authors: hutber, marcus title: the use of vaccines to control disease is not a simple matter date: - - journal: vet j doi: . /j.tvjl. . . sha: doc_id: cord_uid: lyfh ixi nan the use of vaccines to control disease is not a simple matter the paper by hägglund et al. ( ) , published in this issue of the veterinary journal, is interesting for two reasons. firstly, it presents empirical data, with rationales that are subsequently open to useful debate; secondly, the authors raise some valuable generic questions concerning the effectiveness of vaccination against a variety of infectious diseases. a recent letter in the veterinary record (leslie, ) asked why vaccination is available and yet (for some animal diseases) has not been used as an implemented control measure. infectious diseases that exhibit high morbidity, heightened mortality and high transmission rates, can significantly diminish farming productivity and it has been argued that all modes of control (including vaccination) should be used to abate disease. of course, the usefulness of a vaccine is largely dependent upon its efficacy or efficiency. some vaccines are very effective (such as rinderpest), whereas others may only be partially effective (e.g. foot-and-mouth disease or avian influenza). the efficacy can also be significantly dependent upon age, reproductive status, species, mode of the transmission or aetiology, and, interestingly, the degree of the disease challenge. disease challenge can, in turn, be affected by climate (temperature and humidity), spatial and geographical barriers between affected and susceptible animals, farm management techniques on infected premises (stocking densities, the number of farm segregations, the spatial extent of segregations, etc.), and the innate immunity of individual animals and species. the use of vaccination is not simplistic. as the time period from vaccine administration to a protective boost of antibody levels can take several days, culling can be more effective as a control when disease transmission is rapid. an alternative approach is to vaccinate more regularly, but regular, prophylactic vaccination can lead to the development of subclinical disease such that occult transmission occurs between animals without the manifestation of clinical signs. this in turn can lead to endemic disease, where the infection persists and control becomes even more difficult. within regions of endemic disease, regular vaccination becomes more worthwhile, however, since the disease challenge is persistent, and the likelihood of disease-free status diminished. conversely, for non-endemic regions, a trading ban can be levied (at least for notifiable diseases) until the disease has been eradicated, or sometimes for several months afterwards. moreover, the period of the ban can be greater when vaccination has been implemented instead of culling control. the administration of vaccines is further complicated by the need to vaccinate concurrently for different diseases strains. there are also practical difficulties with vaccinal control. some vaccines remain effective for only weeks or months, so that repeated vaccine administrations could be required during any lengthy epidemics. regular surveillance of herd immunity levels become an integral requirement for vaccination programmes that deliver partial immunity in order both to monitor the efficacy of the vaccinal protection and to permit concurrent testing for subclinical disease. whilst surveillance remains practicable for intensively farmed livestock, it becomes less viable for extensively managed animals. the theoretical benefits of using vaccination remain significant. however, in practical terms, the use of vaccines is complex and they can be less effective for disease control than other measures. for all of the reasons mentioned above, it is important to be aware of research that examines the disease control measures for any given disease. hägglund et al. ( ) reported a reduced morbidity of bovine respiratory disease (brd) amongst calves using management techniques without vaccination. the paper further outlines that the breed of cattle may affect disease incidence and differential levels were indicated for charolais, simmental, blonde d'aquitaine < hereford, aberdeen angus. concurrent disease was also recognised as a significant factor in brd, and the respective prevalence of brd components was found to be parainflueneza virus (piv) > bovine coronavirus (bcov) > bovine respiratory syncytial virus (brsv). these findings should be useful in examining how to better control brd. with an ''all-in-all-out'' management system, hägglund et al. ( ) reported that free mixing (or unrestricted spatial cohabitation) of livestock did not increase disease incidence but the finding was dependent upon prior quarantine impositions being applied to any incoming livestock, combined with disease screening (for bvd), which can be compared to the livestock movement restrictions that are usually imposed for a highly contagious disease. the article by hägglund et al. ( ) indirectly highlights both quarantine and the restriction of livestock movements as effective measures for disease control and questions whether such biosecurity could enhance the impact of subsequent vaccinations presumably by maintaining and maximizing a clean susceptible pool. it is a good question. epivet limited, chesil house, shakespeare road, boyatt wood, hampshire, so sy, uk e-mail address: hutber@epivet.fsbusiness.co.uk a six-year study on respiratory viral infections in a bull testing facility eradication of fmd virus key: cord- - kcgc fx authors: warren, kimberly r.; postolache, teodor t.; groer, maureen e.; pinjari, omar; kelly, deanna l.; reynolds, mark a. title: role of chronic stress and depression in periodontal diseases date: - - journal: periodontol doi: . /prd. sha: doc_id: cord_uid: kcgc fx an extensive body of experimental and clinical evidence documents the negative impact of chronic psychological stress and depression on the immune system and health. chronic stress and depression can result in general dysregulation of the immune system, of both cellular and humoral pathways, which may contribute to pathogenic infection and concomitant periodontal tissue destruction. in general, the evidence is consistent with the hypothesis that stress can modify the host defense and progression of periodontal infections in patients susceptible to periodontitis. however, substantial evidence also indicates that these conditions can mediate risk for disease, including periodontitis, through changes in health‐related behaviors, such as oral hygiene, smoking and diet. the unequivocal interpretation of studies has also been hampered, in part, by issues related to conceptualization of stress and depression, as well as commonly associated comorbidities, such as diabetes, that can modify the onset and progression of periodontal disease. in addition, stress and depression appear to fall into a spectrum, ranging from mild to severe, involving a complex interaction of genetic background, coping strategies and environment. differences in the conceptualization of stress and depression are probably important in assessing associations with other biologic and clinical measures. future studies are necessary to clarify the complex interactions of chronic stress and depression in periodontal diseases. psychological stress refers to the emotional and physiological reactions experienced when a person confronts a life event, such as marital conflict, financial debt or death of a loved one, that exceeds his or her ability to cope effectively with the situation. stress evokes emotional and physiologic reactions and is an important modifiable risk factor for both mental and physical illnesses. a robust, and presumably causal, association exists between stressful life events and major depressive episodes ( , ) , with chronic stress being closely linked to depressive disorders. the neurobiology underlying stress and depression is thought to result from molecular and cellular abnormalities that interact with genetic and environmental factors ( ) . epidemiologic studies provide strong evidence that chronic psychosocial stress and depression increase the risk of atherosclerotic cardiovascular disease, diabetes and other systemic conditions ( ) , as well as adversely affect the course and outcome of the conditions ( , , ) . moreover, numerous studies show that stress and depression are associated with increased morbidity and mortality across a range of systemic conditions ( , , , , , ) . several pathophysiologic mechanisms may explain the association of chronic stress and depression with systemic diseases ( , , , , , ) . basic and clinical studies demonstrate that stress and depression are associated with atrophy and loss of function of limbic brain regions that control mood and depression, including the prefrontal cortex and the hippocampus ( , , , , , ) . in addition, experimental animal models suggest that chronic stress induces vascular inflammation through elevations in circulating proinflammatory cytokines ( ) . prevalence estimates of depression and stressrelated disorders in the united states vary across studies; however, a recent survey revealed that . % of adults met the crieria for current depression, with . % meeting the criteria for major depression ( ) . in addition, a national survey commissioned by the american psychological association found that % of employees perceived work as a significant source of stress and % typically felt tense or stressed during the work day ( ) . health-care expenditures associated with stress and depression are high, especially those attributable to comorbities, such as cardiovasular disease and diabetes. recent estimates suggest that nearly % of health-care costs are attributable to modifiable risk factors, such as depression, in the united states ( ) . in a study of , employees from seven organizations over an average of years, goetzel et al. ( ) found that in the category of psychosocial risks, health-care costs for employees with depression were % more expensive than for those not at risk ($ , in higher costs). similarly, the health-care costs of workers reporting high stress were . % ($ ) higher than the costs of those not reporting high stress. the purpose of this narrative review was to summarize the literature on stress and depression as it relates to periodontitis, highlighting the emerging role of neuroendocrine and neuroimmune mediators in the pathophysiology of inflammatory diseases. we reviewed english language publications from to , retrieved using the electronic database pub-med. the search terms psychiatry, psychological stress, depression, dental, periodontal disease, periodontitis, teeth, oral health, and immune function were used in the searches. central nervous system communication with both the immune and endocrine systems has given rise to the field of psychoneuroimmunology. neuroendocrine-derived peptides and hormones have long been recognized as immune modulators. early studies in animals found that stress was associated with increased susceptibility to infectious disease ( ) as well as experimental models of inflammatory disease ( ) . research in human psychoneuroimmunology has shown that immune-regulatory processes are an inextricable part of a complex network of adaptive responses ( ) . individuals experiencing stress exhibit prominent abnormalities of behavior, such as depressed mood and impaired sleep, along with dysregulation of the neuroendocrine and sympathetic nervous systemsthe latter systems are critical efferent pathways in the regulation of immunity by the brain. the pattern and magnitude of the response to stress appears to be influenced by mulitple factors, such as the duration of stress exposure (acute vs. chronic), the type of stress (physical vs. psychological) and gender, among others ( ) . multiple stress mediators, including monoamines, neuropeptides and steroid hormones, are necessary to convey the stress signal to the central nervous system and contribute to the resulting functional changes in the central nervous system. a basic understanding of the role of the limbic-hypothalamic-pitu-itary-adrenal axis and the cytokine regulatory loop are depicted graphically in fig. . complex bidirectional interactions have been established between the central nervous system and the immune system, mediated by the endocrine system. two important features of these interactions include the production of stress hormones by the limbic-hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal-medullary axis ( ) . hormones effect immune function through receptor binding and modulation of cytokines. the modulation of cytokines has been shown to feed back to the brain, producing changes in the hypothalamic-pituitaryadrenal axis, providing a mechanistic basis for common alterations in sleep patterns and depression. acute, time-limited stressors appear to result in adaptive redistribution of cells and preparation of the natural immune system for possible infection or injury, or both. chronic stressors have been associated with more global immunosuppressioncellular immunity followed by both natural and specific immunity, including t-helper (e.g. t-cell-proliferative responses) and t-helper (e.g. antibody to influenza vaccine) parameters ( ) . therefore, the adaptiveness of immune changes appears to decrease with increasing chonicity of a stressor. psychological stress clearly exerts the capacity to modify the susceptibility of animals and humans to infectious agents, influencing the onset, course and outcome of certain infectious pathologies ( ) . psychological stress is generally recognized as a potential cofactor in the pathogenesis of infectious disease. cohen et al. ( ) prospectively studied the relationship between psychological stress and the frequency of documented clinical colds among subjects intentionally exposed to respiratory viruses. after completing questionnaires assessing the degree of psychological stress, healthy subjects were given nasal drops containing one of five respiratory viruses (rhinovirus type , or ; respiratory syncytial virus; or coronavirus type e) and an additional were given saline nasal drops. stress was associated in a dose-response manner with an increased risk of acute infectious respiratory illness, and this risk was attributable to increased rates of infection rather than to an increased frequency of symptoms after infection. in a recent systematic review and meta-analysis, pedersen et al. ( ) conclude that the evidence supports the hypothesis that psychological stress is associated with increased susceptibility to upper respiratory tract infection, consistent with the potential importance of psychological factors in other infectious diseases. stress has a direct impact on the immune system , similarly to many other strategies which people use to cope with stress, whether healthy (e.g. exercise) or unhealthy (e.g. smoking). genco et al. ( ) , for example, found a higher risk of more severe periodontal attachment loss (odds ratio = . ) and alveolar bone loss (odds ratio = . ) among individuals with financial strain and an inadequate coping style, when compared with those with low levels of financial strain within the same coping group, after adjustment for age, gender and cigarette smoking. there is also evidence for psychological modulation of the immune response to viral vaccines. one of the earliest studies was conducted in medical students who were inoculated with the hepatitis b virus vaccine. both virusspecific t-cell and antibody responses were up-regulated in those students with more stress and less social support ( , ) . chronic stress associated with caregiving for a spouse with alzheimer's disease was associated with a less desirable antibody response to an influenza virus vaccine compared with matched controls ( ) . a similar study in hong kong compared caregivers of spouses with chronic conditions that impaired their activities of daily living with matched controls. participants were injected with trivalent influenza vaccine and were assessed for symptoms of stress and depression. caregivers had statistically significantly lower cell-mediated immune responses to the influenza vaccine compared with controls ( ) . other studies show that caregivers of those with chronic illnesses are at higher risk of infection and immune reaction ( , , ) . collectively, these studies and others ( ) suggest that stress and inadquate coping strategies or social support can play a role in the activation or re-activation of herpesviruses and other viral infections in the periodontium. high periodontal loads of active epstein-barr virus and cytomegalovirus tend to be associated with aggressive periodontitis, whereas latent herpesvirus infections are more common in chronic periodontitis and gingivitis ( ) . stress also alters clinical wound healing and underlying immunologically mediated processes ( ) . studies on the healing of acute experimental and surgical wounds consistently report slower wound healing in individuals with high levels of psychological stress, regardless of stressor duration ( , ( ) ( ) ( ) ) . a decrease in local pro-inflammatory cytokines in the wound bed has been postulated as one biologic mechanism ( , ) , presumably caused by the immunosuppressant effects of cortisol, norepinephrine and epinephrine ( , ) . in animal models of psychological stress-impaired cutaneous wound healing ( ) , delay in healing is also associated with deficits in bacterial clearance ( ) and susceptibility to opportunistic infection ( ) at the wound site. oral mucosal wound healing is also known to be impeded by stress ( ) . depressive symptoms also predict the rate of mucosal wound healing in healthy young adults ( ) . the mechanisms through which stress produces inflammation are complex and bidirectional (stress can produce inflammation and inflammation can produce stress). these processes involve networks of communication including genetic, neural, endocrine and immune interactions. it is clear from both animal and human studies that stress affects the immune system in multiple ways. stress increases neuroendocrine hormones, such as glucocorticoids and catecholamines. through the activation of these hormones, stress has detrimental effects on immune functions, including reduction of lymphocyte populations, lymphocyte proliferation, natural killer cell activity and antibody production and the re-activation of latent viral infections ( ) . the limbic-hypothalamic-pituitary-adrenal axis and the sympathetic nervous system are the major neural pathways activated by physical (i.e. pathogens or toxins) and psychological (i.e. major life events, abuse, or workor relationship-related factors) stressors ( , , ) . early conceptualizations accepted a global immunosuppressive action of stress, but it is now clear that both immunosuppression and immune activation occur in various types of stress states ( ) . chronic or repetitive stress, of the sort that is quite typical for those individuals with mental illness, seems to provoke a state of chronic inflammation through the activation of macrophages, dendritic cells, microglia, adipocytes and endothelium, which secrete cytokines. other effects include altered cell trafficking, natural killer cell cytotoxicity changes and alterations in the t-helper /t-helper balance, all of which could contribute to the potential for poor immunoresponsiveness to microorganisms and vaccines, susceptibility to infections, re-activation of latent viruses and delays in wound healing ( ) . the chronic inflammation occurs often in the presence of limbichypothalamic-pituitary-adrenal activation and secretion of high levels of normally immunosuppressive glucocorticoids ( ) . chronic stress acts through a variety of molecular mechanisms to produce a state of inflammation. central to the production of this state is the release of proinflammatory cytokines responding to particular signals. cytokines are signaling proteins that transmit information between immune cells and also between the immune system and the brain and endocrine system. cytokines can act in autocrine, paracrine and endocrine manners to produce their effects ( ) . the cytokines with greatest involvement in producing a stress-associated state of chronic inflammation include interleukin- beta, tumor necrosis factoralpha and interleukin- , a process that is mediated by nuclear factor-kappa b ( ) . inflammation is normally a cell and tissue response to injury or infection, with safeguards to keep inflammation from becoming unchecked and chronic ( ) . there are also systemic effects of inflammation, largely accomplished through the release of cytokines, that produce the well-known 'sickness behavior' of inflammation through actions of cytokines on the brain ( ) . the normal pathways for proinflammatory secretion are through liganding of receptors on cytokinesecreting cells. these receptors include toll-like receptors, which respond to pathogen-associated molecular patterns as well as to danger-associated molecular patterns ( ) . danger-associated molecular patterns are classified as 'alarmins' (i.e. molecules that signal cell damage) and include heat shock proteins, dna and many different products of injured tissue (e.g. high-mobility group protein b , defensins, annexins and uric acid; ). inflammation produced through the alarmin pathway is considered 'sterile' as it is not provoked by pathogens. in the stress associated with mental illness, the inflammation is of this latter sort. another important receptor-mediated pathway is through alpha-and beta-adrenergic receptors that bind norepinephrine, which may be increased in stress states through activation of the sympathoadrenal system ( ) . liganding of these receptors initiates secretion of proinflammatory cytokines through several signaltransduction pathways, one involving nuclear factorkappa b and another involving mitogen-activated protein kinases. both pathways act to increase the expression of tumor necrosis factor-alpha and interleukin- beta genes ( ) . interleukin- production is stimulated in an autocrine manner by interleukin- beta, and the major signal transduction factor is signal transducer and activator of transcription ( ) . opposing these pathways are the glucocorticoid receptors. glucocorticoid receptors are nuclear receptors that, when bound with cortisol, bind to glucocorticoid-response elements on the promotor regions of genes which code for proinflammatory (and other) molecules, inhibiting transcription ( ) . in stress, there may be activation of the sympathoadrenal system and the limbic-hypothalamic-pituitaryadrenal axis, both of which may be involved in the production of the chronically inflamed state. adrenergic mechanisms may account for many of the short-term inflammatory effects of acute stress. chronic stress, on the other hand, is more likely to be associated with a low-grade state of inflammation ( , ) . glucocorticoids inhibit the reproductive axis on multiple levels by reducing secretion of gonadotropin-releasing hormone, luteinizing hormone, follicle-stimulating hormone, testosterone, estradiol and progesterone. the latter inhibitory effects may contribute to sexual dimorphisms of stress-related behavioral syndromes and autoimmune inflammatory disorders ( ) . neurologic and behavioral characteristics of the chronically stressed or depressed state can be explained, in part, through the cytokine effects of fatigue, decreased mobility, anhedonia and depression ( ) . these states are characterized not only by higher plasma levels of proinflammatory cytokines, particularly interleukin- , but also by higher than normal levels of acute-phase reactants, the synthesis of which is stimulated in the liver by the proinflammatory cytokines. these include c-reactive protein, fibrinogen and various adhesion molecules ( ) . there is also evidence of limbic-hypothalamic-pituitary-adrenal axis activation and release of glucocorticoids in the face of ongoing inflammation ( ) . the latter is thought to take place through several mechanisms. one concept is that glucocorticoid receptors become down-regulated or impaired and insensitive or resistant to the high levels of cortisol. without a damping mechanism through the glucocorticoid pathway, inflammation persists and exerts damage to vulnerable tissues ( ) . it is also now established that a number of glucocorticoid receptor gene polymorphisms exist that could play a role in susceptibility to stressinduced inflammation and also that epigenetic mechanisms can alter the glucocorticoid receptor gene ( , ) . genetic variability in the expression of inflammatory mediators in response to stress can also contribute to differences between individuals in systemic markers of inflammation and risk of morbidity and mortality ( ) . for example, the presence of the guanine/cytosine single nucleotide polymorphism (rs ) in the promoter of the interleukin- gene, - bp upstream of the transcription start site, affects the binding of a beta-adrenergic-sensitive transcription factor, gata- . following a beta-adrenergic stimulus, only the interleukin- - g single nucleotide polymorphism leads to increased interleukin- production. in a study of spousal bereavement in older adults, schultze-florey et al. ( ) found that the interleukin- - single nucleotide polymorphism moderated the effect of bereavement on the vulnerability of an individual to higher circulating levels of inflammation. moreover, epidemiologic analysis revealed an increased -year mortality risk associated with late-life depressive symptoms that occurred solely in homozygous carriers of the gata sensitive g allele ( ) . such studies underscore the importance of genetic variability in determining the biologic effects of environmental stressors. depression: is stress the tie that binds? the connection between stress and depression is also complex and bidirectional. evidence supports the hypothesis that psychosocial stress can lead to depression in susceptible individuals, which, in turn, can be aggravated by depression. in a recent meta-analysis, howren et al. ( ) reviewed the associations between depression and the inflammatory markers c-reactive protein, interleukin- and interleukin- in circulating peripheral blood from community and clinical samples. all three inflammatory markers had a positive association with depression. the strongest association (based on clinical interviews) was found in clinically depressed patients, but the association was also significant in the community-based samples. depression was also found to be predictive of c-reactive protein and interleukin- levels in cardiac and cancer patients. interestingly, the relationship between depression and circulating levels of interleukin- became weaker with increasing age ( ) . the latter finding may reflect, in part, age-related increases in circulating levels of interleukin- , tumor necrosis factor-alpha and other inflammatory cytokines, even in healthy adults and in the absence of acute infection ( ) . in a more recent meta-analysis, hiles et al. ( ) also found a moderate and significant elevation in serum interleukin- levels in depressed compared with nondepressed groups, consistent with earlier systematic reviews ( , , , ) . however, a high heterogeneity in the effect size was present among studies. elevations in interleukin- were larger in studies of patients with major depressive disorders and no known comorbid conditions. comorbidities associated with depression were found to reduce the effect size between depressed and nondepressed groups. body mass index, for example, was shown to be a moderating factor, as reflected in smaller, but significant, positive associations between depression and serum inflammatory markers. studies evaluating the effect of antidepressants on systemic markers of inflammation provide additional evidence linking depression to elevations in markers of systemic inflammation. in a meta-analysis of studies, hannestad et al. ( ) examined whether serum levels of tumor necrosis factor-alpha, interleukin- and interleukin- beta change in response to therapy with commercially available antidepressants in patients with major depressive disorder. studies were included only if serum markers were obtained before and after treatment. the results revealed that treatment with specific serotonin re-uptake inhibitors reduced the symptoms of depression as well as the serum levels of interleukin- beta and interleukin- . a similar trend was found for levels of tumor necrosis factor-alpha in response to specific serotonin reuptake inhibitors. other antidepressants, including serotonin and norepinephrine re-uptake inhibitor and tricyclic antidepressants, reduced depressive symptoms but exerted minimal, if any, effects on serum markers of inflammation. the authors interpreted these findings to suggest that inflammatory cytokines can contribute to depressive symptoms and that antidepressants have the potenital to impede the effects of inflammatory cytokines on the brain ( ) . collectively, these findings suggest that comorbidities, such as obesity and periodontitis, which are associated with elevations in systemic markers of inflammation, have the potential to modify the severity of depression. chronic inflammatory diseases, such as periodontitis, have a complex pathogenesis and a multifactorial etiology, involving complex host-parasite interactions ( ) . (fig. ) genetic variations in genes encoding the molecular components of the host immune defense ( , ) , coupled with specific bacterial species in the subgingival plaque, set the stage for individual differences in risk for periodontitis. compared with women, men appear at greater risk for periodontitis ( , ) . basic and clinical studies document the potential for comorbidities (e.g. diabetes) and co-infections (e.g. herpesviruses) to modify the initiation and/or progression of periodontitis ( , , ) . cigarette smoking, poor nutrition, alcohol consumption and low socio-economic status also have been shown to be associated with a higher risk of periodontitis ( ) . a substantial body of evidence indicates that psychological stress ( ) and ineffective coping ( ) can influence the onset and progression of many chronic diseases, including periodontitis ( ) . early studies provided both anectoctal and clinical evidence of an association between psychosocial stress and acute necrotizing periodontal diseases, especially necrotizing ulcerative gingivitis ( , ) , as well as periodontitis ( , , ) . the nature of the association, however, was based largely on a temporal concordance between the 'stressful' event, gingival infection, and physiological measure of stress, such as urinary hydroxycorticosteriod. similarly, associations have been made between stress and depression and aggressive ('rapidly progressive') periodontitis based on psychosocial and clinical data obtained at the same time ( , ) . therefore, chronic stress and depression have been hypothesized to reduce immune responsiveness, resulting in more pathogenic infection and concomitant periodontal tissue destruction. evidence also indicates that chronic stress and depression can mediate risk and progression of periodontitis through changes in health-related behaviors, such as oral hygiene, smoking and diet ( , ) . although stress can adversely impact multiple health-related behaviors, including oral hygiene ( , ) , there is strong evidence that stress plays a contributory role in the pathophysiology of periodontitis. peruzzo et al. ( ) conducted a systematic review of the evidence on the influence of stress and psychological factors on periodontal disease. of the studies (seven case-control, six cross-sectional and one prospective clinical trial) meeting inclusion criteria for the review, the majority ( %) reported a positive relationship between stress/psychological factors and periodontal disease. another . % of the studies observed a positive relationship between some characteristics of stress and periodontal disease, demonstrating that the majority of the work published to date examining this relationship have indeed found significant associations ( ) . in a recent study not included in the systematic review, stress scores and salivary stress markers (chromogranin a, cortisol, alpha-amylase and beta-endorphin) were shown to be significantly correlated with clinical parameters of periodontal disease in adult patients with periodontitis ( ) . salivary cortisol and beta-endorphin were significantly associated with tooth loss and periodontal clinical parameters, after adjusting for stress variables. moreover, the greatest tooth loss was observed in those patients who neglected to brush their teeth during stressful periods ( ) . the results of these studies are consistent with the hypothesis that stress can modify the host defense and progression of periodontal infections in patients susceptible to periodontitis ( ) . depression has also been linked to periodontitis in clinical studies in adolescents/young adults ( , ) and adults ( , , , , , , , ) . in a casecontrol study, moss et al. ( ) found that smoking and elevated antibody titers to tannerella forsythia at baseline were associated with more severe periodontal breakdown in patients scoring high on a depression inventory. in a cross-sectional investigation, patients with rapidly progressive periondontitis showed significantly higher scores on measures of depression and loneliness compared with adults with either chronic periodontitis or without significant clinical attachment loss ( ) . genco et al. ( ) found that stress and distress manifested as depression was a significant risk indicator for more severe periodontitis in a cross-sectional study of , subjects between and years of age. johannsen et al. ( ) found that women on long-term sick leave for depression had more severe periodontitis and higher concentrations of interleukin- in gingival crevicular fluid compared with healthy controls. these clinical results are consistent with findings in an established animal model of depression. breivik et al. ( ) tested whether depression induced by olfactory bulbectomy in rats and treatment with the antidepressant serotonin re-uptake enhancer, tianeptine, could influence susceptibility to ligature-induced periodontitis. when compared with sham-operated controls, olfactory bulbectomy rats were shown to develop significantly more periodontal bone loss. tianeptine treatment significantly inhibited alveolar bone loss. however, a clomipramine-induced model of depression in lewis rats did not alter the pathogenesis of ligature-induced periodontitis ( ) . susceptibility to periodontal breakdown in response to stressful life events appears to depend, in part, on the effectiveness of a person's coping behavior ( , ) . in a cross-sectional investigation, genco et al. ( ) examined the relationship of periodontal disease and stress, distress and coping behaviors in a large population-based adult sample ( ) . this study revealed that psychosocial measures of stress (financial strain) and distress, manifest as depression, were significant risk indicators of periodontal disease severity in adults, after adjusting for gender (male), smoking, diabetes mellitus, t. forsythia and porphyromonas gingivalis ( ) . several clinical studies have failed to demonstrate an association between measures of depression and periodontal disease in community-based samples ( , , , ) . persson et al. ( ) , for example, did not find an association between depression and radiographic bone loss or periodontal probing depths ( mm or greater) in a sample of older adults. however, depression (self-reported or based on a psychometric score) was associated with a history of greater tooth loss, which may reflect greater susceptibility to periodontal destruction during periods of depression. anttila et al. ( ) reported a positive correlation between depression and edentulousness in a sample of finnish residents. it is noteworthy that depression often manifests with multiple comorbidities, including obesity and diabetes, many of which are independently associated with elevations in systemic markers of inflammation ( ) . in general, studies exploring the association between depressive symptoms and periodontitis have been based on nonpsychiatric populations without consideration of comorbidities, limiting the potential of these studies to identify an association between depression and periodontal disease. studies of serum and salivary stress-related steriods provide further evidence of an association between stress, depression, and periodontal disease. in a cross-sectional study, rosania et al. ( ) asked patients with periodontitis to complete psychometric tests and questionaires as well as to undergo clinical examinations and measurement of salivary cortisol levels ( ) . stress, depression and salivary cortisol scores were found to correlate signficantly with severity of periodontitis and the number of missing teeth, when controlling for age, family history and brushing frequency. moreover, patients who reported neglecting their oral care during stressful or depressed periods exhibited the greatest clinical attachment loss and highest number of missing teeth. in a similar study of dentate patients, ≥ years of age, mannem & chava ( ) reported that stress scores and salivary cortisol levels significantly discriminated between patients with and without periodontitis. similar associations have also been reported between serum levels of the stress-related steroids, such as cortisol and dehydroepiandrosterone-sulfate, and measures of periodontal disease ( , ) . patients with periodontitis often have higher systemic levels of c-reactive protein, interleukin- , interleukin- and tumor necrosis factor-alpha ( ) . the mechanisms underlying the relationship of psychlogical stress and depression with periodontitis probably involve a combination of factors related to alterations in behavior and neuroimmunologic function. experimental animal models provide additional evidence that stress adversely affects immune defense and susceptibility to oral infection. bailey et al. ( ) found that social stress enhances the production of interleukin- beta and tumor necrosis factor-alpha in cd b + spleen cells in response to p. gingivalis lipopolysaccharide ( ) . the latter finding suggests that stress can enhance the responsiveness and production of inflammatory cytokines by macrophages in response to an oral pathogen. macrophages from the spleens of mice exposed to this same stressor were shown to express significantly higher levels of both toll-like receptor- and toll-like receptor- ( ). breivik et al. ( ) also found that injection of lipopolysaccharide provoked a significant increase in the circulating levels of corticosterone and alterations in cytokine levels, consistent with dysregulation of the immune system, in an animal model of depression. psychological stress has been also shown to exacerbate oral infection with the opportunistic pathogen, candida albicans, in rodents ( , ) . finally, stress and depression have been shown to exert a negative effect on treatment outcomes in patients with periodontitis. elter et al. ( ) examined whether depression was predictive of the proportion of residual probing periodontal sites ( mm or greater) and tooth loss between the initial and -year post-treatment examinations in patients enrolled in a health-maintenance organization. a diagnosis of depression was documented from medical records in . % of the patients. depression was significantly associated with both poorer periodontal treatment outcome and tooth loss during the -year follow-up period. kamma & baehni ( ) found that scores on a stress inventory predicted future clinical attachment loss in patients with aggressive periodontitis in supportive periodontal care over a period of years ( ). linden et al. ( ) found that a measure of occupational stress was a significant predictor of clinical attachment loss in patients with periodontitis in regular periodontal maintenance over a period of nearly years. these findings parallel other reports ( ) indicating that psychosocial factors play a significant role in recovery from surgery and are predictive of surgical outcome. an extensive body of experimental and clinical evidence documents the negative impact of chronic psychological stress and depression on the immune system and health. brief stressors appear to suppress cellular immunity whilst preserving measures of humoral immunity; in contrast, chronic stressors generally result in dysregulation of the immune system, involving both cellular and humoral pathways ( ) . therefore, chronic stress and depression have been hypothesized to reduce immune responsiveness, resulting in a higher rate of infection with pathogenic organisms and a greater degree of periodontal tissue destruction. in general, the evidence is consistent with the hypothesis that stress can modify the host immune defense and permit the progression of periodontal infections in patients susceptible to periodontitis ( ) . however, substantial evidence also indicates that these conditions can mediate risk for disease, including periodontitis, through changes in healthrelated behaviors, such as oral hygiene, smoking and diet ( , ) . stress and depression are commonly associated with comorbidities, such as diabetes ( ) , that can modify the onset and progression of periodontal disease; however, these conditions have generally not been addressed in available studies. in addition, stress and depression appear to fall into a spectrum, ranging from mild to severe, involving a complex interaction of genetic background, coping strategies and environment. differences in the conceptualization of stress and depression are probably important in assessing associations with other biologic and clinical measures. 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a broad field of study that combines genome and transcriptome-wide studies in humans and model systems to refine our understanding of human biology and ultimately identify new ways to treat and prevent disease. the approaches to translational genomics can be broadly grouped into two methodologies, forward and reverse genomic translation. traditional (forward) genomic translation begins with model systems and aims at using unbiased genetic associations in these models to derive insight into biological mechanisms that may also be relevant in human disease. reverse genomic translation begins with observations made through human genomic studies and refines these observations through follow-up studies using model systems. the ultimate goal of these approaches is to clarify intervenable processes as targets for therapeutic development. in this review, we describe some of the approaches being taken to apply translational genomics to the study of diseases commonly encountered in the neurocritical care setting, including hemorrhagic and ischemic stroke, traumatic brain injury, subarachnoid hemorrhage, and status epilepticus, utilizing both forward and reverse genomic translational techniques. further, we highlight approaches in the field that could be applied in neurocritical care to improve our ability to identify new treatment modalities as well as to provide important information to patients about risk and prognosis. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. translational genomics represents a diverse collection of research approaches that leverage human genomics and model systems to identify new approaches to treat and prevent disease and improve healthcare ( , ) . rooted by the central dogma of dna to rna to protein, genomic research examines the entire genome concurrently, and may include analyses of dna variants in association with traits of interest as well as the impact of genomic variation on gene transcription and translation. genomic research has been enabled by technological advances to accurately and cost-effectively study variation across the genome at scale, as well as computational techniques to store and analyze genomic data quickly and efficiently ( ) . while translational research is often defined in terms of the traditional "bench to bedside" techniques that advance discoveries from model systems through biomarkers and mechanisms ultimately to clinical applications, genomic research offers a strong use-case for an alternative approach. termed "reverse translation," this approach starts with humans as the model system, utilizing genomic associations to derive new information about biological mechanisms that can be in turn studied further in vitro and in animal models for target refinement (fig. ) . both of these approaches possess advantages and drawbacks ( , ) . forward translation depends on the relevance of the model system to human disease, both in terms of the physiologic responses to disease or insult, as well as the approach taken to perturb the system. for instance, the human applicability of genomic studies of the response to traumatic brain injury (tbi) in a mouse model require that the mouse's response to tbi is analogous to a human's, and that the approach taken to pavlos myserlis and farid radmanesh contributed equally to this work. create a tbi in the mouse provokes a similar pattern of injury seen in human tbi ( ) ( ) ( ) ( ) . as such, a great deal of careful work is required to demonstrate the validity of these model systems before the results arising from them can be judged relevant to human disease. the challenges of bridging this divide are illustrated by the universal failure of neuroprotection mechanisms that reached human trials in the last several decades, essentially all of which had promising model system data in preclinical development ( ) ( ) ( ) ( ) ( ) . reverse genomic translation, in contrast, begins with humans ( fig. ). as such, there are few concerns as to the relevance of the system for discovery of biomarkers and mechanisms of disease. however, this approach carries a new series of challenges in study design and data acquisition ( ) . compared to isogenic cell lines or carefully bred animals in a controlled setting, humans are highly variable in both their environmental and genetic exposures. this is advantageous in identifying genetic susceptibility to disease risk and outcomes, but teasing out these small genetic effects from highly variable non-genetic exposures requires both careful computational techniques as well as large sample sizes. furthermore, because genomic data is both identifiable and can potentially lead to discrimination, human genomic studies require complex consent and data management procedures ( ) . in neurocritical care, the relative rarity of many of the diseases we encounter, coupled with the challenges of critical illness and surrogate consent make human genomic studies all the more difficult to execute effectively ( ) ( ) ( ) ( ) . neurointensivists routinely encounter diseases and complications for which there are a dearth of effective treatments, or even foundational knowledge of their underlying pathophysiologic mechanisms ( , ) . in this review, we will highlight some of the approaches being taken to apply translational genomics to the study of diseases commonly found in neurocritical care, utilizing both forward and reverse genomic translational techniques. further, we will highlight some of the best practices in the field that could be applied in neurocritical care to improve our ability to identify new treatment modalities as well as risk and prognosis information to patients and their families. in advance of the human genome project and the hapmap consortium, genetic studies were confined to the study of candidate genes and lower-resolution genome-wide techniques such as categorization of restriction fragment length polymorphisms (rflp), tandem repeats, and microsatellites ( ) . these genomic features enabled early efforts to perform linkage analyses in families with related traits and disorders, as well as selected populations of unrelated individuals. careful work in this arena led to validated discoveries that have survived replication in the common era, such as chromosome in late-onset alzheimer disease (ad), ultimately mapped to the apoe locus that has become a target for a great deal of genetic research in ad, as well as many other diseases including tbi and intracerebral hemorrhage (ich) ( ) ( ) ( ) ( ) . still, much of the pregwas era was characterized by candidate gene studies that suffered from low statistical power and multiple sources of confounding that led to a failure to replicate many reported associations in the gwas era that followed ( , ) . the most substantial source of confounding in candidate gene analyses is population stratification, in which differences in allele frequency due to ancestral imbalance between cases and controls introduces spurious associations (positive or negative) between genotype and trait based solely on these cryptic ancestral imbalances ( , ) . even in studies of apoe in european ancestry populations, uncontrolled variation in the percentages of individuals of northern vs. southern european ancestry between cases and controls can mask true associations between apoe and ich, for instance ( ) . the gwas era, in which variants across the genome could be reliably genotyped and mapped to a common reference template by chromosomal location, ushered in a new system of best practices that could minimize the contribution of many of the sources of confounding in describing associations between genomic variation and traits or diseases. the international hapmap consortium obtained genotypes on individuals across ancestral populations around the globe, creating a resource that described the patterns of allele frequency variation across diverse populations ( ) . with these breakthroughs and a number of landmark evolutions that followed, case/control and population-based gwas have led to the identification of over , associations with human diseases and other traits (https://www.ebi.ac.uk/gwas/). obviously there is an enormous disconnect between the discovery of genetic loci and leveraging of this information for human benefit, which is where the translational genomic work that serves as the topic of the present review becomes relevant ( ) . post-gwas, in addition to functional and translational efforts, the movement has been towards so-called "next-generation sequencing" methodologies consisting of whole exome sequencing (wes) and whole genome sequencing (wgs). using these approaches, each nucleotide in the exome or genome is ascertained with high reliability, permitting the identification of rare and de novo variants that escape detection in traditional gwas ( ) . wes captures within-gene coding variation only, offering detection of variants that may more directly impact protein structure and function than non-coding variation detected by wgs ( ) . because the coding exome is only~ % of the overall genome, it is more cost-effective than wgs, but debate continues as to which is the more appropriate tool for large-scale study of the human genome ( ) . regardless, both wes and wgs remain orders of magnitude more expensive than traditional gwas approaches at this time, and as such well-powered sequencing studies remain unreachable for many diseases in the current pricing models. less common diseases and conditions that one may find in a neurocritical care unit are doubly disadvantaged, as even larger sample sizes are required for sequencing analyses than gwas, due to the need for many observations to identify rare exonic or intronic variants associated with disease ( , ) . as pricing models improve and larger and larger community or hospital-based cohorts receive sequencing through clinical or biobanking efforts, it is hoped that even uncommon conditions such as subarachnoid hemorrhage or status epilepticus will benefit from the insights achievable through sequencing analysis, where case/control and smaller sequencing studies have shown promise ( , ) . obviously genomic research need not be limited solely to human studies. a wealth of information about disease pathogenesis and response to injury can be gleaned from model systems of human conditions using genomic and transcriptomic approaches. because animal models and isogenic tissue cultures are specifically designed to limit genetic differences between individual animals or plated cells, dna-based association tests typically do not offer insight in the same way that they do in humans. as such, many model system studies start with rna, examining how the genome responds to perturbation through the transcriptome. however, there are substantial genomic differences between model systems and humans, as coding sequences are not necessarily conserved, promoter and enhancer control of gene expression can vary, and in the case of immortalized tissue and cell-based assays, the chromosomal architecture itself can be quite different from the organism from which it was derived ( , ) . these differences can be highly relevant when determining whether observed transcriptomic and proteomic results from model systems are likely to be shared in humans. with those caveats, the dynamic nature of the transcriptome in model systems offers opportunities to assess the way in which the genome responds to noxious insults or drug exposures, and in animal models this can even be done across specific organs or tissues of interest ( ) . as one example, traumatic brain injury researchers have obtained insight into both the initial injury cascade as well as brain response to potential injury modulators such as valproate using animal models and transcriptional microarrays, in which rna expression patterns in brain tissue can be rapidly and replicably assessed across the transcriptome ( ) . using more recent technological advancements such as drop-seq, rna expression can be assessed in single cells, as has been done in individual hippocampal neurons in a mouse model of tbi ( ) . at a minimum, these elegant studies can help to identify relevant cell types important in the response to injury, highlighting testable hypotheses that may be important in human conditions, all with access to tissues and control over experimental conditions that would never be possible in human-based research. given that diseases common to the neurocritical care population so rarely afford access to brain tissue for pathologic or genomic analysis antemortem, model system genomic studies offer an important adjunct for translational research. forward genomic translation begins with model systems with the goal of using the measured associations in these models to derive insight into biological mechanisms that may also be relevant in human disease. forward translation requires wellcharacterized models that are often designed to mimic the human exposures of interest as closely as possible. this is often challenging given the natural differences between humans and many of the animals chosen to serve as models. in this section, we will highlight several model systems in current use for translational genomics relevant to neurocritical care, but the field of translational modeling in neurologic disease is suitably large to prevent an exhaustive review herein. malignant cerebral edema is a highly lethal complication of ischemic stroke, with mortality of - % ( ) . currently, hemicraniectomy is the only available option to prevent death and yet it does not address the underlying pathophysiology. hyperosmolar therapy is potentially useful as a bridge to surgery. preclinical data based on a forward translation approach has been useful in highlighting mechanisms underlying postinfarct edema as potential targets for therapeutic manipulation. the sulfonylurea receptor (sur ) is encoded by the abcc gene that is upregulated after cns injury, forming an ion channel in association with transient receptor potential melastatin (trpm ). continuous activation of this complex can lead to cytotoxic edema and neuronal cell death, which has been demonstrated in both animal and human models ( , ) . sur is also found in pancreatic beta cells, constituting the target for the oral hypoglycemic agent, glyburide. studies of rodent and porcine stroke models demonstrated that in the first few hours after an ischemic insult, both sur and trpm are upregulated ( , ) . limited case series of human postmortem specimens also demonstrated upregulation of sur in infarcted tissue ( ) . therefore, intravenous glyburide has been proposed for treatment of malignant cerebral edema. targeting sur in rat models of ischemia have consistently resulted in reduced edema and better outcomes ( ) . in particular, glyburide infusion starting h after complete middle cerebral artery occlusion resulted in decreased swelling by two thirds and % reduction in mortality ( ) . one desirable characteristic of glyburide is that it cannot penetrate intact blood-brain barrier, but that is facilitated following brain injury ( ) . the effect of glyburide for treatment of cerebral edema has also been studied in tbi with promising data obtained from animal studies ( ) . limited randomized trials in human using oral glyburide have shown promising results; however, use of oral formulation and study design limitations prohibit generalizability of results ( , ) . building on this preclinical data, the phase randomized clinical trial (games-rp) showed that the iv preparation of glyburide, glibenclamide, is associated with reduction in edema-related deaths, less midline shift, and reduced rate of nih stroke scale deterioration. however, it did not significantly affect the proportion of patients developing malignant edema ( ) . the phase charm trial, sponsored by biogen, is currently enrolling patients with large hemispheric infarction to determine whether iv glibenclamide improves -day modified rankin scale scores. if this trial proves successful, this vignette will represent a dramatic success story for the forward translation paradigm in genomic research. in the light of recent advances in revascularization therapy, the national institute of neurological disorders and stroke has supported an initiative aiming to develop neuroprotective agents to be used as adjunctive therapy to extend the time window for reperfusion and to improve long-term functional outcome. this stroke preclinical assessment network (span) supports late-stage preclinical studies of putative neuroprotectants to be administered prior to or at the time of reperfusion, with long-term outcomes and comorbidities constituting the endpoint. the goal is to determine if an intervention can improve outcome as compared to reperfusion alone and/or extend the therapeutic window for reperfusion. span directly applies to forward translation efforts in preclinical models of neuroprotection after stroke and is an outstanding opportunity to stimulate research efforts in a field more remembered for its past failures than the promise it holds for the future of therapeutic development in the area. other societies have also begun to endorse more comprehensive modeling approaches in areas with few therapeutic options with the hope of implementing a paradigm shift. for example, the neurocritical care society has initiated "curing coma" campaign with the -to -year mission to improve the understanding of the mechanisms and to ultimately develop preventative and therapeutic measures. traumatic brain injury (tbi) is among the leading causes of disability and death worldwide, particularly in the young. the type of tbi is in part determined by the attributes of mechanical forces, including objects or blasts striking the head, rapid acceleration-deceleration forces, or rotational impacts. following the primary injury, an intricate cascade of neurometabolic and physiological processes initiates that can cause secondary or additional injury ( , ) . intensive care management has improved the prognosis of tbi patients; however, specific targeted treatments informed by pathophysiology could have a tremendous impact on recovery. the period of secondary tissue injury is the window of opportunity when patients would potentially benefit from targeted interventions, given that in tbi, the primary injury cannot be intervened upon by the neurologist or intensivist. the goal of therapy is therefore to reduce secondary damage and enhance neuroplasticity. the utility of animal models of tbi primarily depends on the research question, as each model emulates specific aspects of injury and has selective advantages and disadvantages. these include biomechanics of initial injury, molecular mechanisms of tissue response, and suitability for high-throughput testing of therapeutic agents, to name a few. although phylogenetically higher species are likely more representative models for human tbi, rodent models are more commonly used given the feasibility to generate and measure outcomes, as well as ethical and financial limitations of higher-order models. table summarizes some common and representative tbi models [ table ]. in contrast with the rodent models described in table , other model systems in tbi have been selected specifically to study other aspects of the physiologic response to tbi. for example, a swine model of controlled cortical impact offers the opportunity to readily monitor systemic physiologic parameters such as tissue oxygen and acid-base status while investigating therapeutic interventions, which is argued to provide greater insight into human response to injury ( ) . translation of preclinical studies using these animal tbi models to humans is inherently challenging. differences in brain structure, including geometry, craniospinal angle, gyral complexity, and white-gray matter ratio, particularly in the rodent models, can result in different responses to trauma ( ) . the limitation of extrapolating animal studies to human is also manifested at the genetic level, as differences in gene structure, function, and expression levels may suggest genetic mechanisms that are incompletely correlated with humans. as an example, female sex may be associated with better outcome through the neuroprotective effect of progesterone in animal models, but these observations did not carry over to humans in the protect-iii trial ( , ) . variable outcome measures, including neurobehavioral functional tests, glasgow outcome scale correlates, and high-resolution mri have been used in attempts to correlate animal responses to injury with those of humans. the lack of a large cache of standardized tools further limits comparison or pooling the results of different studies that use variable models of tbi or outcome measurement. transcriptomics, a genomic technique in which global rna expression is quantified through either expression microarrays or rna sequencing, has been employed to characterize specific inflammatory states following tbi. many studies have assessed the transcriptome in the acute post-tbi interval within - days after injury, with some showing upregulation of inflammation and apoptosis genes. gene ontology analysis at months post-tbi have shown similar changes, with upregulation of inflammatory and immune-related genes ( ) . importantly, late downregulation of ion channel expression in the peri-lesional cortex and thalamus suggests that this delayed examination of the transcriptome could be valuable for revealing mechanisms relevant to chronic tbi morbidities, including epileptogenesis and prolonged cognitive impairment ( ) . in addition, tissue-specific analysis of gene diffuse axonal injury reproduces human tbi needs standardization, e.g., location of animal within shock tube and heard immobilization ( ) expression across cell types in brain could provide useful insight into cell-specific pathways. for example, temporal trending of microglial expression profile indicates a biphasic inflammatory pattern that transitions from downregulation of homeostasis genes in the early stages to a mixed proinflammatory and anti-inflammatory states at subacute and chronic phases ( ) . the list of antiepileptic drugs has expanded significantly in the past decade, reflecting substantial investment in the search for new therapeutics with better efficacy and tolerability. however, the list of options with demonstrated efficacy in status epilepticus (se) has remained limited. the utility of benzodiazepines, often deployed in the field as a first-line agent, decreases with increasing duration of se. in addition, - % of patients with se develop refractory se when they fail to respond to first-and second-line therapy, posing a significant management and prognostic challenge ( ) . the development of aeds has relied substantially on preclinical animal models to establish efficacy and safety prior to proceeding to human trials. different epilepsy models exist that are each useful for different aspects of drug development and no model is suitable for all purposes. the majority of animal models induce epilepsy using electroshock or chemical seizure induction. nearly all recent aeds have been discovered by the same conventional models, and the reliance on these common screening models has been implicated as one of the reasons for the low yield of drugs with efficacy in refractory epilepsy ( ) . the pros and cons for each epilepsy model are discussed in detail in several excellent reviews ( , ) . some of the chemicals used include kainic acid, pilocarpine, lithium, organophosphates, and flurothyl ( ) . sustained electrical stimulation to specific sites, including the perforant path, the ventral hippocampus, the anterior piriform cortex can induce se ( ) . the latency, length, and mortality of convulsive se are more variable in chemoconvulsant as compared to electrical models, which are in turn determined by the drug and route of administration, species, sex, age, strain, and genetic background among other factors ( ) . it should also be noted that the presence of behavioral convulsion does not correlate fully with the electrographic data and vice versa. this can have c r i t i c a l i m p l i c a t i o n s w h e n s t u d y i n g d r u g s f o r pharmacoresistant se. therefore, it has been suggested that electroencephalographic quantification be used to measure the severity of se ( ) . furthermore, the genetic background and expressivity of animals can have a significant effect on seizure susceptibility, even between batches of inbred mice ( ) . proteomic and transcriptomic approaches have been utilized for assessment of alterations in expression profile following se, demonstrating that certain subsets of genes are upregulated at each timepoint following the onset of se. specifically, upregulation of genes regulating synaptic physiology and transcription, homeostasis and metabolism and, cell excitability and morphogenesis occur at immediate, early, and delayed timepoints. in addition, related studies have demonstrated changes in expression of micrornas related to epileptogenesis, including mirna- and mi-rna- following se ( , ) . selective rna editing post-transcription is yet another potential source of proteomic diversity in preclinical models of se, and merits further investigation as a modulator of protein levels that may be less closely tethered to gene expression ( ) . aneurysmal subarachnoid hemorrhage (sah) has an earlier age of onset and is associated with higher morbidity compared with other stroke subtypes. the pathophysiology of insult has traditionally been studied under two time-intervals, early brain injury (ebi) and, cerebral vasospasm (cv) and delayed cerebral ischemia (dci). the prime goal of translational research in this arena is to identify the mechanisms and targets related to the risk, severity, evolution and outcome. about % of patients die immediately following sah ( ) . thereafter, early brain injury within the first days, followed by dci are the most feared complications. cv is the phenomenon with strongest association with the development of dci, which - % of patients experience between day to ( ) . the underlying mechanisms leading to cv remain poorly understood and have therefore been a prime focus of preclinical studies. the majority have used rodent models, but primate, swine, and dog models have also been employed ( ) . cerebral aneurysms are difficult to model and hence two common approaches to modeling sah use alternative strategies. the first is direct injection of blood into the subarachnoid space, specifically into either the prechiasmatic cistern or cisterna magna, to generate sah predominantly in the anterior or posterior circulation territories, respectively ( ) . the second model, endovascular suture, passes a suture or filament through the internal carotid artery, creating a hole in one of the major branches resulting in egress of variable amount of blood into the subarachnoid space ( ) . variations in some parameters of the first method, including injected blood volume, csf removal prior to injection to prevent egress of blood into the spinal canal, and replenishing intravascular volume to keep cerebral perfusion pressure constant through maintenance of mean arterial pressure, as well as the rapidity of injection have raised questions about comparability and biofidelity of the results ( ) ( ) ( ) ( ) . the latter model appears to remove some of the mentioned confounding factors, as the hemorrhage occurs at physiologic mean arterial pressure (map) and intracranial pressure (icp), but is limited by variable puncture site and ultimate hemorrhage volume. another potential drawback is the period of ischemia caused by the intraluminal suture, although the occlusion period is typically not judged to be long enough to cause significant ischemia. the missing element in these models is the absence of aneurysm formation and rupture, and consequently the vascular processes intrinsic to the aneurysm itself that influence dci. as such, some studies have used combinations of interventions to generate aneurysms, including induced hypertension via unilateral nephrectomy and administration of angiotension ii or deoxycorticosterone acetate, as well as elastase injection. the downside of these models is that the timing of aneurysm rupture cannot be reliably predicted, which limits close monitoring and physiologic assessments in the early phase following sah, blurring the timing of dci ( ) ( ) ( ) . the immediate hemodynamic changes following the hemorrhage are monitored via a variety of methods. regardless of the method chosen, reports on the direction and range of values of cpp, cbf, and map can be quite variable, both within the same model and between different models. a common technique to measure blood flow is laser doppler flowmetry that provides a continuous measure of cortical perfusion. although it does not measure global cerebral blood flow and has spatial limitation, it appears to be relatively reliable and technically reproducible. other methods of flow measurement include radiolabeling methods and mri with the latter has the advantage of capturing the dynamic nature of the condition, as well as global and region-specific blood flows. as noted, cv and dci are responsible for delayed morbidity and mortality. given that these manifestations typically occur while patients are inpatient for care of their sah, therapeutic interventions are more feasible compared to the hyperacute phase when the processes leading to initial damage may have already occurred. however, monitoring for cv in animal models is not straightforward. one method of identifying cv is measuring the intraluminal diameter of vessels on histological samples. in addition to being an end-measure and therefore precluding measurements at different time points in the same animal, varying degrees of tissue desiccation among samples may yield numbers different from actual in vivo values. digital subtraction angiography and magnetic resonance angiography can provide a real-time evaluation, but the severity of cvand its timing, as well as neuronal cell death varies depending on the model and the affected vessels ( ) . the foundational molecular pathways that orchestrate cv are complex and remain incompletely elucidated. however, translational research using many of the above models has demonstrated that endothelin- , nitric oxide, and an inflammatory cascade ignited by breakdown of blood products play predominant roles. endothelin- is a potent vasoconstrictor produced by infiltrated leukocytes, and based on this notion, clazosentan was developed as an endothelin- receptor antagonist to combat cv. in human trials, clazosentan was found to significantly reduce the incidence of the dci without improving the functional outcome, and this or a related approach could ultimately prove beneficial if off-target drug effects, including pulmonary complications, hypotension, and anemia can be mitigated ( , ) . hemoglobin and its degradation products are also a strong stimulus for cv through direct oxidative stress on arterial smooth muscle, decreased nitric oxide production and, increasing endothelin and free radical production ( ) . this suggests that facilitating clearance of hemoglobin degradation products from the csf may be a potential therapeutic target. modulating the intense inflammatory response is also intuitive and while preclinical results support this notion in general, the evidence has thus far not been judged adequate to justify clinical trials. for example, il- receptor antagonist (il- ra) reduces blood-brain barrier (bbb) breakdown, a biomarker that is itself correlated with the severity of brain injury, and work continues to determine whether this or related pathways mediating bbb permeability might have therapeutic promise ( ) . given these numerous and likely interconnected mechanisms of delayed brain injury, further research is needed to understand their relative applicability to humans, and whether targeting a single pathway or a number of pathways simultaneously is likely to be the most adaptive strategy to reduce cv and dci in humans. the results of genome-wide rna sequencing analysis have supported the primary role of neuroinflammation in the pathogenesis of early brain injury. some studies have specifically found a key role for long non-coding rna (lncrna), a type of rna without protein-coding potential that are particularly abundant in the brain, in modulating the inflammatory behaviors of microglial cells ( ) . high-throughput mass spectrometry has also been utilized in demonstration of differential expression of proteins in the cerebral vessels after sah, as well as for monitoring the effect of experimental therapeutics ( ). we will not cover these proteomic studies in detail here, as they typically fall outside the rubric of what is classically considered "genomics", but their approach, which leverages global protein signatures rather than restricting observations to specific compounds, shares many similarities with genomics. as mentioned above, reverse genomic translation refers to an approach to the study of a disease by starting with humans using either cohort-based or case/control genomic studies. the observations made through the course of these studies then inform on the best approach for target validation and refinement to prioritize candidate mechanisms and related endophenotypes for therapeutic development. it has been shown that candidate compounds with independent confirmation of their therapeutic target via human genomics are more than twice as likely to prove effective in clinical trials ( ) . therefore, the reverse translation approach would seem an adaptive strategy to identify disease-associated mechanisms and therapeutic targets with the best chance of impacting clinical care in the near term. however, the approach to reverse translation requires large sample sizes with well-characterized patient data in order to achieve a statistically confident result. these large sample sizes raise the issue of variability in risk and treatment exposures between participants, which could impact patient outcomes independently of genomic effects and therefore erode power to detect genetic risk. the utility of reverse translation in target refinement and mechanism exploration in model systems can be highlighted using an example from the stroke community. recent gwas and subsequent meta-analyses of ischemic stroke and stroke subtypes in very large case/control datasets have validated the histone deacetylase (hdac ) region in chromosome p . as a major risk locus for stroke due to large artery atheroembolism (laa). this locus was also previously discovered in association with coronary artery disease (cad) ( , ) . based on these findings, azghandi et al. sought to investigate the role of the leading single nucleotide polymorphism (snp) in this genomic region (rs ) in increasing laa stroke risk ( ) . they found that rs , both in heterozygotes as well as in homozygote human carriers, is associated with increased expression of hdac in peripheral blood mononuclear cells on a dose-dependent manner, suggesting that the effect of this locus in stroke risk may be mediated by increased hdac expression. additionally, they demonstrated that hdac deficiency in mice is associated with smaller and less advanced atherosclerotic lesions in the aortic valves, curvature, and branching arteries, suggesting that hdac may increase atherogenesis and therefore represents a novel target for atherosclerosis and laa stroke prevention. notably, recent studies have suggested that both nonspecific (e.g. sodium valproate) as well as specific hdac inhibitors can have a positive impact on both stroke recurrence risk, as well as other phenotypes, including cancer. this highlights the central role that reverse translation can have in therapeutic target investigation and refinement, with potential beneficial off-target properties ( , ) . while acute stroke care is a vital component of neurocritical care at many institutions, reverse genomic translation successes in other relevant traits also merit mention. acute respiratory distress syndrome (ards) is a frequent complication of severe neurologic injury due to sah or neurotrauma. in a recent gwas by bime et al., variation in the selectin p ligand gene (selpg), encoding p-selectin glycoprotein ligand (psgl- ) was found to be associated with increased susceptibility to ards ( ) . the most significant snp in this locus, rs , which results in a missense mutation, has been successfully replicated in independent cohorts. further functional analyses have demonstrated that selpg expression was significantly increased in mice with ventilator (vili)-and lipoprotein (lps)-induced lung injury, and that psgl- inhibition with a neutralizing polyclonal antibody led to an attenuation of inflammatory response and lung injury. in selpg knockout mice, inflammatory response as well as lung injury scores were significantly reduced compared to wild-type mice ( ) . these results highlight the value of reverse genomic translation in first identifying human-relevant genetic risk factors for disease, and using model systems to understand the pathways impacted by their introduction to select rationally-informed modalities for potential treatment. intracranial aneurysms (ia) are commonly encountered in the neurocritical care setting, albeit most commonly after rupture. even so, inroads leading to a better understanding of aneurysm formation may ultimately reveal opportunities for treatments to prevent acute re-rupture or prevent future aneurysm formation after sah. the strongest associations with ia have been reported in the region near cdkn a/cdkn b in p . as well as in a nearby intragenic region known as cdkn bas or anril ( , ). anril is a long noncoding region responsible for the regulation of cdkn a and cdkn b and has also been implicated in the pathogenesis of cad and atherosclerosis, among other traits ( ) . overexpression of anril in mouse models of cad has been associated with negative atherosclerosis outcomes including increased atherosclerosis index, unfavorable lipid profiles, thrombus formation, endothelial cell injury, overexpression of inflammatory factors in vascular endothelial cells, increased apoptosis of endothelial cells, and upregulation of apoptosis-related genes. notably, reduced anril expression has been associated with reduced inflammatory, biochemical and molecular markers of atherosclerosis, indicating a potential target for atherosclerosis and ia prevention ( ) . when utilizing the reverse translation approach in genomic studies, the aforementioned examples highlight two distinct but equally important considerations for a successful implementation of such approaches. the first major consideration is that large populations of well-characterized individuals must be selected to ensure adequate statistical power to detect meaningful associations. thorough and standardized phenotyping of study subjects is one of the main predictors of the success of a gwas ( , ) . careful assignment of cases based on strict phenotypic criteria permits well-executed gwas even in diseases with heterogeneous presentations and multiple pathogenic features, such as multiple sclerosis (ms) and stroke ( ) . in neurocritical care populations where subtle characteristics of disease presentation and intermediate outcomes may represent important phenotypes for genomic investigation, such as sah, these traits should be closely defined and recorded to the greatest degree possible in all participants. this initial step is critically important in the greater scheme of reverse translational genomics, as these associations with subclasses and endophenotypes of disease often provide the biological insights needed to continue translational efforts using model systems tailored to refine observations. the second major consideration is that the execution of genomic studies needs to be comprehensive and thorough so as to permit association testing in a hypothesis-free environment. at the moment, gwas array-based studies seem to remain a favorable option of the genome, considering the lower cost associated with their utilization and proven track record in discovery, but over time, wes and wgs studies will become reachable even on more modest research budgets. for the transcriptome, rna sequencing and rna microarrays both offer unbiased surveys of global transcriptional variation, but because gene expression varies substantially by tissue it is critical that rational choices are made regarding the suitability of specific tissues for specific conditions. in uncommon conditions with necessarily small sample sizes, including neurocritical care-relevant diseases like se, sah, and ards, external validation studies can strengthen associations from an initial small discovery dataset, and in many cases these follow-up studies can make use of freely available resources. for example, in a recent expression-based gwas (egwas), microarray data for ards from the gene expression omnibus (geo) were collected and combined in an effort to identify novel genetic targets ( ) . the study not only validated previously known lung injury-and ardsrelated genes, but also discovered new candidate genes that may prove to be useful in future translational work. identifying loci, variants, expression patterns, and genenetworks with the use of human genomic studies is only the initial step in the reverse translation process. these discoveries must inform and guide the research to further understand and refine the phenotypic effects of these variants in model systems, including some of those described above. there are several techniques with which we can utilize the discoveries made from case/control genomic studies to build or modify model systems. one approach is transgenesis, in which a larger dna sequence including a human gene containing a mutation of interest, called a transgene, is injected into the pronucleus of a mouse fertilized egg. the fertilized egg is then inserted into the oviduct of a pseudopregnant female mouse, which is a female who has been mated with vasectomized male in order to achieve the hormonal profile of a pregnancy state. the offspring produced from this female can create an animal line that contains the human gene and allele of interest ( ) . however, because the transgene is inserted randomly at one or more genetic locations as either one or more copies, the level of expression and regulatory influences of the gene of interest may not initially be well-controlled across animals. as such, there are several intermediate steps that can allow more specific genetic alteration using transgenesis, involving embryonic stem cells (escs). the first step is the introduction of regulatory sequences (such as expression cassettes) into escs. then, by injecting the transgene first into these modified escs, gene expression can be more closely controlled. the escs with the transgene can then be inserted into blastocysts and give rise to new strains, using the same methods previously described ( ) . there are multiple variations on the transgenic approach which are uniquely suited to the model system being employed and can give rise to models that express transgenes in response to a particular stimulus, or in particular tissues of interest. a newer method utilizing programmable endonucleases has allowed researchers to bypass more traditional escbased methods for direct and precise gene editing. endonucleases are enzymes that cause double stranded dna (dsdna) breaks that can further be repaired either with non-homologous end-joining (nhej), an imprecise method for rejoining the dna breaks that involves various enzymes and may result in inactivating mutations, or with homologydirected repair (hdr), in which the dna breaks are repaired based on a co-injected template. four categories of programmable endonucleases have been used for direct and precise gene editing: homing endonucleases (he), zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens) and the clustered regularly interspaced short palindromic repeats/crispr-associated (crispr/cas ) system. the common characteristic of these enzymes is that they possess sequence-specific nuclease activity, allowing researchers to cleave dsdna at desired, pre-specified sites. the crispr/ cas system has proven to be the most successful so far, in terms of efficiency, cost, and simplicity of use. perhaps the most important advantage of this approach is that programmable endonucleases do not require the use of escs and can directly be inserted into one-or two-cell stage embryos, thus allowing more specific and direct gene-editing in a single step ( ) . drawbacks include enzymatic limitations as to where dna breaks can be reliably introduced, as well as off-target endonuclease activity at other sites across the genome which can disrupt gene activity in unintended ways. work is ongoing to refine these tools, improving the number of sites where gene editing can occur while also improving the specificity of the system ( ). one illustrative example of human genomic studies being used to refine models to understand disease processes is the case of human ich-associated mutations in col a and col a . col a and col a are the most abundant proteins in basement membranes. they form heterotrimers consisting of one col a and two col a peptides and are produced and modified in the endoplasmic reticulum (er). after their production, they are packaged into vesicles in the golgi apparatus and transferred to vascular endothelial basement membranes ( ) ( ) ( ) . the initial identification of mutations in this region in familial forms of cerebral small vessel disease, coupled with the subsequent detection of common col a /col a variants associated with sporadic deep ich led to the development of animal model systems to explore their effects ( ) ( ) ( ) ( ) . through mouse models, representative col a /col a mutations were found to recapitulate human disease phenotypes, with multifocal ich in subcortical regions of the forebrain and the cerebellum, as well as porencephaly, small vessel disease, recurrent intraparenchymal and intraventricular hemorrhages, agerelated ich, and macro-angiopathy ( , ) . using cellular assays and tissue derived from mouse models, mutations in col a /col a have been associated with decreased ratio of intracellular to extracellular col a , retention of abnormal collagen proteins in the er, er stress, and activation of the unfolded protein response ( ) ( ) ( ) , suggesting that the intracellular accumulation and er-stress could be an important molecular mechanism underlying ich related to col a and col a mutations. notably, treatment with the molecular chaperone sodium -phenylbutyrate resulted in decreased intracellular accumulation and significant decrease of ich severity in vivo, which could point the way towards eventual forms of treatment for both familial and sporadic col a and col a -associated ich ( ) . another recent example of model system refinement for neurocritical care-relevant disorders is status epilepticus (se). pyridoxal phosphate binding protein (plpbp) variants have been associated with a rare form of b -dependent epilepsy, which, if left untreated can lead to se. in a recent study, johnstone et al. utilized crispr/cas to create a zebrafish model lacking its encoded protein ( ) . they observed that plpbp-deficient zebrafish experienced significantly increased epileptic activity compared to their wild type counterparts, in terms of physical activity (high-speed movements), biochemistry (c-fos expression) and electrophysiologicallyrecorded neuronal activity. additionally, treatment of plpbp −/ − larvae with plp and pyridoxine led to an increase in their lifespan, and a decrease in their epileptic movements and neuronal activity. lastly, in these plpbp-deficient zebrafish, systemic concentrations of plp and pyridoxine were significantly reduced, as well as concentrations of plp-dependent neurotransmitters. collectively, these results provide insights for biomarker development and preclinical target refinement in b -dependent epilepsy. understanding how novel treatments might impact rare disease presentations could ultimately lead to new insights for common forms of disease as well, just as the discovery of rare pcsk variants in patients with very low cholesterol ultimately led to pcsk -inhibitors to treat more common forms of familial hypercholesterolemia. however, the use of animal models is not always the ideal approach to describing the effects of genetic variation, as the phenotypic alterations may be too subtle to observe or require impractical prolonged observation in late-life animals to ultimately exhibit relevant phenotypes. in these cases, tissuebased systems can provide a useful tool to study these effects. for example, ia formation, as previously described, has been associated with variants in anril. although the direct impact of these variants in human tissue or animal models is difficult to discern, work with mutations of anril in endothelial models have provided valuable insight. specifically, upregulation of anril has been associated with increased expression of inflammatory and oxidative markers in the vascular tissue such as il- , il- , nf-κb, tnf-a, inos, icam- , vcam- , and cox- ( , ) . these observations provide vital information about cellular mechanisms impacted by human disease-associated genetic risk factors without requiring the expense and time investment of creating, validating, and studying animal models. ultimately such models may still be required, but prior knowledge about cellular phenotypes associated with genetic variation may be highly valuable in choosing the right model system and selecting efficient approaches to validate these systems. the aforementioned examples highlight significant contributions of the field of translational genomics in identifying novel therapeutic targets, developing biomarkers of disease severity and elucidating disease-relevant pathophysiology. undoubtedly, these contributions are valuable in application to existing model systems of disease, or through refinement of models informed by the reverse translation process. given that many of our current models have proven to be ineffective in many cases, the reverse translation approach offers a significant advantage in that the translational discoveries arising in established or refined model systems have already been proven to be relevant to human disease. this advantage provides us with reasonable expectation that observed effects in model systems will also remain relevant to human disease, providing a substrate for therapeutic development. certainly, the ultimate goal of translational genomics is to be able to transfer the discoveries found from experimental models into clinically useful information in order to improve human health. this aim, with regard to the translational genomic approach, can be satisfied with two distinct approaches. one is concerned with improving our understanding of the mechanisms of disease, providing novel targets for therapeutic development. the other is concerned with leveraging the conclusions of translational genomics through more direct applications to clinical care. we will discuss these in order. once genomic discovery and translational exploration have confirmed the mechanism and relevance of a particular genomic association, translational genomics offers the opportunity to use these same translational approaches to derive highthroughput assays for screening of compound libraries, which are collections of small molecules useful for early-stage drug-discovery ( ) . the same in vitro assays used to identify cellular phenotypes associated with genetic risk factors can be tested for amelioration or "rescue" of wild-type features after exposure to library compounds. this is particularly advantageous for the reverse genomic translation approach, as these assays are often critical components of the overall discovery cycle, and with optimization to provide ideal readouts, screening can proceed quickly. an example of success here is the identification of molecular chaperones that can ameliorate the unfolded protein response detrimental to cell survival in col a -mediated cerebrovascular disease ( ) . identifying hits in these assays has the potential to accelerate drug-discovery, provided that the mechanism can be targeted by a small molecule and not a designed biologic entity such as a monoclonal antibody. while screening can be performed using novel compound libraries, it can also be accomplished using libraries containing already-approved drugs, providing an innovative way for compound repurposing based on genetic interactions. numerous tools already exist for in silico evaluation of existing compounds based on known mechanisms, so this step can begin even in the genomic discovery phase prior to translational validation ( ) . the second approach in which translational genomics has proven to be of great potential is the rapidly evolving and highly anticipated field of precision medicine. the observations arising from translational genomics, even when not informing us about the specific mechanisms associated with the phenotype in question, may be of predictive value. this finds application in two relevant translational genomics tools: polygenic risk scores (prs) and biomarker development based on rna expression profiles. while common genetic variation can provide valuable information about disease-relevant mechanisms and help refine disease models, they are relatively weak in explaining a significant proportion of the genetic basis of complex polygenic disorders, such as cad, diabetes, stroke, or sah ( ) . by summarizing the impact of many variants of small effect across the genome simultaneously, a polygenic risk score (prs) can be developed which explains far more of the genetic risk of a disease than any common variant can individually (fig. ) . application of these prs in independent clinical populations as a predictive tool represents a novel translational approach. in a recent study examining stroke, a prs combining snps associated with atrial fibrillation (af) was found to be significantly associated with cardioembolic (ce) stroke risk and no other stroke subtypes, paving the way for a potentially useful tool to discriminate ce stroke from other etiologies without reliance on expert adjudication or longitudinal monitoring ( ) . another recent study compiled a prs of cad, demonstrating that individuals in the highest quantiles of the prs exhibited cad risk on par with known mendelian cardiac diseases ( ) . these studies highlight the potential uses of prs as a genetic biomarker of disease, capturing orthogonal risk information compared to clinical risk factors alone. much work is still needed in this arena, ranging from derivation of readily accessible clinical genomic testing, dissemination of prs results in an interpretable format, disclosure of off-target results that may be clinically meaningful in their own right, and, critically, the validation of prs in ancestrally-diverse populations ( ) . despite these challenges, utilization of polygenic risk data to directly inform patient risk independent of our understanding of the underlying mechanisms is an exciting and rapidly evolving use-case for translational genomics. development of biomarkers is another approach in translational genomics that focuses more on predictive utilization than on elucidating mechanisms, and critical care has seen some early potential applications of this approach. in sepsis, where clinical prs percentile across the population distribution. plotting percentiles by disease risk, patients in higher prs percentiles (red dots) are at correspondingly highest risk for the disease outcomes are highly heterogenous, tools that might identify patients who are more likely to respond to certain treatments or identify individuals at highest risk for morbidity and mortality would be highly useful. in a recent study by scicluna et al., the authors categorized sepsis patients based on peripheral bloodderived genome-wide expression profiles and identified four distinct molecular endotypes (mars - ) ( ) . the mars expression profile was the only category that was significantly associated with -day and -year mortality. in addition, combination of the apache iv clinical score with this genetic scoring system resulted in significant improvement in -day mortality risk prediction, compared to apache iv alone. to further aid translation to clinical application, the authors used expression ratios of combinations of genes to stratify patients to the four molecular endotypes. bisphosphoglycerate mutase (bpgm): transporter , atp binding cassette subfamily b member (tap ) ratio reliably stratified patients to mars endotype; while other protein ratios were able to assign individuals to the other three mars categories. using this approach, not only could bpgm and tap transcripts potentially be used to identify patients with increased risk of mortality, but if these categorizations can be demonstrated to be causal, these molecular pathways could also be explored for therapeutic target identification and validation. further work is required to extend these findings across clinical populations, but this approach could ultimately yield new tools for prognostication in sepsis. in ischemic stroke, tissue plasminogen activator (t-pa) response and risk for hemorrhagic transformation (ht) are highly correlated with functional outcomes, and biomarkers to predict each of these would have obvious clinical utility. in a recent study, del rio-espinola et al. found that two genetic variations (rs and rs ) were associated with increased risk for ht and mortality after t-pa administration in stroke patients ( ) . specifically, rs in a m was associated with ht and rs was associated with in-hospital mortality. in a subsequent validation study, researchers created a genetic-clinical regression score that was successfully used to stratify stroke patients treated with t-pa based on risk for ht and parenchymal hemorrhage (ph) ( ) . while in the current clinical landscape the vast majority of patients do not have readily accessible genome-wide genotypes prior to events like acute stroke, increasing uptake of clinical genomics and genomically-enabled electronic health record systems could soon enable real-time risk prediction calculations incorporating both clinical and genetic information, providing more accurate tools for clinicians to incorporate into medical decision-making. a separate set of tools that could potentially become diagnostically useful in the clinical setting is the transcriptomic approaches to identify biomarkers, using array-based screening or rna sequencing. in a recent systematic review, a total of mirnas were reported to be differentially expressed in the blood cells of patients with acute ischemic stroke within h after stroke ( ) . some studies reported the area under the curve (auc) ranging from . to . , indicating a potential for clinical utility as early diagnostic markers when neuroimaging is not immediately available or is limited by feasibility. subsequent studies were able to partially replicate these findings, showing three mirnas (mir- a- p, mir- b- p, and mir- - p) that were upregulated in the acute poststroke period, an effect independent of stroke pathophysiology and infarct volume ( ) . these transcripts were associated with an auc of . in differentiating ischemic stroke and healthy controls, a metric that significantly outperformed computed tomography, as well as previously reported bloodbased biomarkers. in ich, a recent report identified up to and transcripts from whole blood that are differentially expressed between ich and controls and, ich and ischemic stroke, respectively ( ) . when comparing ich and ischemic stroke transcriptomes in the first h, and transcripts were differentially expressed compared to controls, respectively. ich transcriptome was over-represented by t cell receptor genes compared to none for ischemic stroke and underrepresented by non-coding and antisense transcripts. t cell receptor expression successfully differentiated between ich, ischemic stroke, and controls. similarly, rna-seq of whole blood rna successfully differentiated between not only ich, ischemic stroke and controls, but also between different stroke subtypes ( ). the list of genetic mutations that can cause se is extensive with most genes are associated with infantile-onset or childhood epilepsy syndromes. only a minority are seen in adultonset status epilepticus ( ) . the patients in the former group usually have accompanying intellectual disability related to their epilepsy syndromes. however, the evidence supporting a genetic etiology in the latter group may be absent, posing a diagnostic challenge. the available options include gene panel sequencing, whole exome sequencing, or whole genome sequencing. sequencing a pre-selected panel of genes is more common, but with decreasing cost, exome and genome sequencing are being used with increasing frequency. bioinformatic filtering and genotype-phenotype correlation are the main challenges, particularly with the large number of genetic variants identified during whole exome or genome sequencing. the yield of sequencing studies depends on pretest probability that is determined by early age of onset, consanguinity, or affected siblings. as such, to date, only a few genes associated with adult-onset se have been identified, posing a practical limitation that predominantly limits next-generation sequencing to pediatric patients at present ( ) . as clinical tools for determination of putative functional significance and deleteriousness of variants identified through sequencing are refined, it is hoped that sequencing approaches find a home in the armamentarium of the clinicians treating refractory or recurrent se in the neurocritical care unit. translational genomics undoubtedly represents an important component to overall efforts to improve our understanding of the diseases we treat, and in principle should improve our ability to identify therapeutic approaches to improve outcomes and, in some cases, prevent disease altogether. given the inherent complexity and inaccessibility of the human brain and its tissues, combined with the relative infrequency of the conditions we treat at the overall population level, progress has been modest when compared to conditions such as hyperlipidemia ( ) , coronary artery disease ( ) , or atrial fibrillation ( ) . nevertheless, the observation-based, hypothesisfree experimental process inherent to translational genomics lends itself well to conditions such as stroke and tbi in which the search for the "master regulator" that governs response to injury has remained elusive despite carefully designed and executed hypothesis-driven studies. an important component to future translational genomic studies in neurocritical care is the pressing need for collaboration across centers with access to large, well-characterized patient populations. the success of the international stroke genetics consortium, and the track-tbi and center-tbi consortia in amassing large human populations with stroke and traumatic brain injury, respectively, is a proven model to accelerate the human genomic studies that serve as the basis for reverse genomic translational research ( , ( ) ( ) ( ) . similar efforts through the critical care eeg monitoring research consortium and other partners could lead to biorepositories of specific conditions relevant to neurocritical care that could provide sample sizes sufficient to drive unbiased genomic discoveries ( ) . close alliances with model systems researchers are another critical component to accelerating translational genomics in neurocritical care. as characterization of human disease through multimodal and continuous physiological monitoring, electrophysiology, medical imaging, and biomarker sampling continues to evolve, it is imperative that this information is shared and explored with allied model systems researchers to ensure that models are re-evaluated for their correlation with these endophenotypes, and potentially for dedicated exploration of how these human-derived phenotypes inform on the utility of specific model systems to investigate disease. finally, building relationships with biotechnology and pharmaceutical industry partners will be essential to efforts to extend therapeutic targets arising from translational genomic discoveries towards drug development ( ) . while repurposing existing drug compounds for new indications is an important consideration, small molecule and biologic targets are likely to require extensive research and development in the preclinical and clinical space, and industry partners are often optimized for these phases of the therapeutic development process ( , ) . relatedly, development of polygenic risk scores for assessment of risk, prognosis, or treatment response will also require commercial investment and infrastructure, as few academic environments exist that can manage cliacertified genotyping, quality control, and result reporting and interpretation for on-target and clinically relevant secondary results ( , ) . particularly in rarer or particularly challenging disease indications like those commonly encountered in neurocritical care populations, academic-industry partnerships are important to raise awareness of and interest in important clinical indications where investment could yield a large impact on a relatively small population of patients. translational genomics, in which genomic associations with risk, outcome, or treatment response are systematically identified and explored for functional relevance in humans or model systems of disease, is a valuable tool for identification of mechanisms, risk factors, therapeutic targets, and risk estimates in multiple diseases that are highly relevant to clinicians and scientists operating in the neurocritical care space. while there are undoubtedly challenges to studying some of the most complex diseases that affect the most complex organ in the body, translational genomic approaches may be uniquely suited to this task. coordinated 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official journal of the american college of medical genetics drug repurposing from the perspective of pharmaceutical companies insights into computational drug repurposing for neurodegenerative disease clinical providers' experiences with returning results from genomic sequencing: an interview study recommendations for reporting of secondary findings in clinical exome and genome sequencing, update (acmg sf v . ): a policy statement of the american college of medical genetics and genomics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - ggaqf authors: nan title: abstracts of the papers presented in the xix national conference of indian virological society, “recent trends in viral disease problems and management”, on – march, , at s.v. university, tirupati, andhra pradesh date: - - journal: indian j virol doi: . /s - - - sha: doc_id: cord_uid: ggaqf nan patients showed rashes on face, hand and foot. ev detection carried out in vesicular fluid, stool, serum and throat swab specimens by rt-pcr of ncr gene. serotyping was carried out by using rt-pcr of viral protein of vp / a junction region followed by sequencing and phylogenetic analysis using neighbor-joining-algorithm and kimura- parameter model of mega- software. overall ev positivity detected in hfmd patients from kerala, tamil nadu, west bengal and orissa states was found to be . %, . %, . % and . % respectively. typing of vp gene sequences indicated presence of ca- , ev- , echo- strains in kerala and ca- in west bengal, orissa and tamil nadu. phylogenetic analysis indicated ca- , ev- , echo- strains showed . - . % and - . % homology with japanese, australian and french strains. however, ca- strains were closer to malaysian strains with . - . % nucleotide homology. the present study documents the association of multiple types of ev's i.e., ca- , ev- , echo- and ca- strains contributing as prime viral pathogens in hfmd epidemics in the reported regions with new emergence of ca- circulating strain in kerala, india. tasgaon september . sera were collected from suspected hepatitis cases and there contacts and tested for anti hev igm/igg antibodies (elisa) and liver enzymes like alanine aminotransferase (alt). anti hev igm antibodies were detected in . % ( / ) of the suspected cases. the overall attack rate was . %. male to female ratio was : . majority ( . %) of the cases were in the age group - years and recovered without any clinical complications. weekly distribution of cases showed that the majority ( . %, / ) cases occurred between nd and rd week of june. dark urine ( . %), jaundice ( . %), fatigue ( . %), abdominal pain ( . %), anorexia ( . %), vomiting ( . %), fever ( . %), giddiness ( . %), diarrhoea ( . %) and arthalgia ( . %) were the prominent symptoms. sera collected from antenatal cases (ancs) showed anti hev igm antibody in . affected pregnant women had a normal outcome. a death of year, male hepatitis e case was reported during the outbreak period that had cirrhosis of liver with oesophageal varices. sanitary survey revealed that water pipelines were laid down in close proximity of sewerage system, and water posts were without tap. these are the likely sources of faecal contamination of water supplies. among water samples collected from various places, were found to be unfit for drinking based on the routine bacteriological tests conducted at state public health laboratory, pune. no case occurred after the pipelines were repaired. this typical outbreak of hepatitis e re-emphasizes need for proper water supply/sewage disposal pipelines and adequate maintenance measures. jayanthi shastri, nilima vaidya, sandhya sawant, umesh aigal department of molecular biology, kasturba hospital for infectious diseases, mumbai, india dengue and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographic distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. the gobal prevalence of dengue has grown dramatically in recent decades. who estimates - million cases of dengue virus infections worldwide every year resulting in , to , cases of dhf and , deaths each year. public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. laboratory diagnosis of dengue virus infection can be made by the detection of specific virus, viral antigen, genomic sequence and/or antibodies. currently basic methods used by laboratories for diagnosis of dengue virus infection are virus isolation and characterisation, detection of genomic sequence by nucleic acid amplification technology assay and detection of dengue virus specific antibodies/antigen. molecular diagnosis based on reverse transcription (rt)-pcr s.a. one step or nested pcr, nucleic acid sequence based amplification (nasba), or real time rt-pcr, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. several pcr protocols for detection have been described that vary in the extraction method, genomic location of primers, specificity, sensitivity and the methods to determine the products and the serotype. pcr-based dengue tests, due to the specificity of amplification, enable a definitive diagnosis and serotyping of the virus. in addition dna sequencing of the amplification product enables the virus to be genotyped, providing important information on the sources of infection. more recently tests have incorporated flurogenic probe, so called taq man technology for the specific real time detection of dengue - amplicons. product is detected by a specific oligodeoxy nucleotide probe that is labelled with carboxy-fluorescein (fam). this technology offers the advantage of being both rapid and potentially quantitative. second, the detection of product by hybridisation of flurochrome labelled probes increases specificity. third, as the product is detected without the need to open the reaction tube, the risk of contamination by product carry over is minimised. the advantages of speed, contamination minimisation and reduced turn around time justify application of this assay over the currently used nested pcr assay. during the period january to october , molecular laboratory received samples from patients presenting with acute onset fever for dengue . %) samples were tested positive by this method. the disease peaks in the monsoon season with a percentage of . %. rapid tests, igm and igg capture elisa are popularly used tests for diagnosis of dengue infection. its utility is limited for diagnosing dengue in convalescecce ( - days) . specificity is also compromised due to infections with flaviviruses: japanese encephalitis and chikungunya. dengue ns ag elisa with its cost effectiveness, specificity and sensitivity should be considered as the test of choice for diagnosing dengue in the acute phase of illness in the developing countries. molecular diagnosis enables confirmatory diagnosis of dengue in the acute phase of the illness and is suitable for further typing methods. assistant general manager and r&d coordinator, division of quality control and r&d, bharat immunologicals and biologicals corporation ltd., village chola, bulandshahr, up vaccine development in india, though slow to start, has progressed by leaps and bounds in the past years. it was dependent on imported vaccines but now it is not only self-sufficient in the production of vaccines conforming to international standards with major supplier of the same to unicef. the role of drug authorities is to enhance the public health by assuring the availability of safe and effective a indian j. virol. (september ) (suppl. ):a -a vaccines, allergenic extracts, and other related products. vaccine development is tightly regulated by a hierarchy of regulatory bodies. guidelines provided by the indian council of medical research (icmr) set the rules of conduct for clinical trials from phase i to iv studies as well as studies on combination vaccines. these guidelines address ethical issues that arise during a vaccine study. a network of adverse drug reaction (adr) monitoring centers along with the adverse events following immunization (aefi) monitoring program provide the machinery for vaccine pharmacovigilance. genetic modifications have been developed to develop effective and cheaper vaccines by the use of recombinant technology. to ensure safety of consumers, producers, experimental animals and environment, governments all over the world are following regulatory mechanisms and guidelines for genetically modified products. as with other industrializing countries undergoing rapid shifts, india clearly recognizes the need to restructure its regulatory system so that its biopharmaceutical industry can compete in international markets. genetic engineering approval council (geac), recombinant dna advisory committee (rdac), review committee on genetic manipulation (rcgm), institutional biosafety committees (ibsc) are responsible for development, commitment for parameters and commercialization of recombinant vaccines. to centralize and coordinate the whole system, government has taken to form two agencies to regulate the regulation laws to develop recombinant pharmaceuticals products including vaccines. the first is the creation of the national biotechnology regulatory authority (nbra), under the department of biotechnology (dbt), as part of india's long-term biotech sector development strategy. the second major initiative will affect the entire indian pharmaceutical industry. this is the replacement of most state, district, and central drug regulatory agencies with a single, central, fda-style agency, the central drug authority (cda). the cda is expected to have separate, semi-autonomous departments for regulation, enforcement, legal, and consumer affairs; biotechnology products; pharmacovigilance and drugs safety; medical devices and diagnostics; imports; quality control; and traditional indian medicines. it will set up offices throughout india and will be paid for inspection, registration, and license fees. its enforcement powers will be strengthened by a new law increasing the criminal penalties for illegal clinical trials. in the manufacturing area, though, the country has been tightening the rules and enforcement. an amendment to the regulations, ''schedule m'' of the drug and cosmetics act, now specifies the good manufacturing practice (gmp) requirements for factory premises and materials. these requirements were modeled after us fda regulations, to improve regulatory coordination between indian and us regulators. india has realized the importance of regulations in pharmaceutical specially in vaccine field but it will take several years to implementation of these. india has coordinated some of its regulatory functions with western organizations. the us pharmacopoeia established an office in hyderabad in . a representative of the indian pharmaceutical lobby also recently has expressed openness to an expansion of the fda's oversight of indian manufacturing. as india expands its global drug and biologicals production, us and europe, as the world's largest drug importers, will likely expand their regulatory support in the development of the country's regulatory systems. rapid diagnosis of japanese encephalitis virus (jev) infections is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. however, the speed and accuracy of diagnosis must be balanced against test cost and availability, especially in developing countries. an antigen capture enzyme-linked immunosorbent assay (elisa) for detection of circulating jev specific nonstructural protein (ns ) was developed by using monoclonal antibodies (mabs) specific to recombinant (ns ). the applicability of this jev ns antigen capture elisa for early clinical diagnosis was evaluated with acute phase serum/ cerebrospinal fluid (csf) specimens collected from different epidemics during [ ] [ ] [ ] . jev ns antigen was detected in circulation from day to . the sensitivity and specificity of jev ns detection in serum/csf specimens with reference to reverse transcriptase pcr was %, and . % respectively. no crossreactions with any of the other closely related members of the genus flaviviruses (dengue, westnile, yellow fever and saint louis encephalitis (sle) viruses) were observed when tested with either clinical specimens or virus cultures. these findings suggested that the reported jev specific mab-based ns antigen capture elisa will be a rapid and reliable tool for early confirmatory diagnosis as well as surveillance of je infections in developing countries. manmohan parida the recent emergence of a novel human influenza a virus (h n ) poses a serious global health threat. the h n virus has caused a considerable number of deaths within a short duration since its emergence. a two-step single tube accelerated rapid real-time and quantitative swine flu virus specific h rtlamp assay is reported by targeting the h gene of the novel h n hybrid virus. the feasibility of swine flu h rtlamp for clinical diagnosis was validated with a panel of suspected throat wash samples comprising confirmed positive and confirmed negative cases of ongoing epidemic. the comparative evaluation of h specific rtlamp assay with real-time rt-pcr demonstrated exceptionally higher sensitivity by picking up all the h n positive and additional positive cases amongst the negatives that were sequence confirmed as h n . none of the real-time rtpcr positive samples were missed by rtlamp system. the comparative study revealed that rtlamp was -fold more sensitive than rtpcr with a detection limit of copy number. these findings suggested that rtlamp assay is a valuable tool for rapid, real-time detection as well as quantification of h n virus in acute phase throat swab samples without requiring any sophisticated equipments. because of its recurrent nature. despite considerable progress in understanding of the virus at cellular and molecular levels, the proper management of the disease in its different stages is still a dilemma particularly whether to use antiviral or steroids or both. the risk of using steroids with its attendant complications has to be weighed against the risk of progression of the disease if avoiding the use of steroids. this dilemma can be reduced to a considerable extent if basic principles of virology and pathogenesis are kept in mind. this article reviews current concepts of virological and clinical aspects of hsv keratitis to enable a broad understanding of the disease process. it is recognized several influential host factors including the fact that hsk is more common in men than women. it is observed that the ability of hsv to establish latent infection in sensory neurons and possibly cornea, but have as yet been unable to use this knowledge to prevent the disease limitations. acknowledging limitations may further stimulate application of laboratory knowledge in coping with hsk which constitutes to present major challenge in terms of management. mvo- study on effect of human bhsp in immunity of hcv core protein and hbv hbsag there are more than million individuals with hepatitis b and c in the world. in spite of vaccination in the different areas there are several reports about patients who got vaccine before. also there is not efficient vaccine against of hepatitis c and one of the important problems in vaccine project is development of effective and suitable adjuvant in human vaccines. at present research we applied human bhsp protein as adjuvant and chaperon. this protein injected to balbc mice as adjuvant together with recombinant proteins of hcv core and hbv hbsag. then humoral and cellular immune systems of the mice were studied. core and hbsag genes were cloned into petduet- vector and thermal vector of pgp - was used for human heat shock protein expressions. the different combination of these three proteins was injected to mice and we evaluated the total igg and igg a of mice serums after a week. two weeks after booster injection, we studied the proliferation and cytokine secretion of spleen, inguinal and popliteal lymph nodes lymphocytes in vitro and ex vivo conditions. so the core/hbsag + hsp and core + hbsag + hsp complexes induced total igg and igg a secretion. the spleen lymphocytes proliferation were increased equal to serum igg a level that was constant in second time bleeding with significant different to complexes with freund's adjuvant. at first il- and il- cytokines were increased and then decrease of il- meaned no hypersensitivity. the chaperon effect of hsp on structure of core and hbsag proteins was studied by cd and flourometer. it could fold the proteins after heating and unfolding. hepatitis b virus (hbv) infection is vaccine preventable global public health problem. all commercially available vaccines contain one or more of the recombinant hepatitis b envelope protein or surface antigen (hbsag). measurement of antigen responsible for immunogenicity of vaccine is central to quality assessment. the problems associated with the use of a polyclonal antibody in an assay with regard to its poorly defined nature and batch-to-batch variation has been mitigated by the use of mabs as described in this paper. the initial capture of hbsag by the mab could orientate it such that the same antibody could bind to it as a detection antibody after labeling with out steric hindrance. the development of an immuno-capture elisa (ic-elisa) to measure the hbsag content using a monoclonal antibody (mab) specific to determinant ''a'' of hbsag in the experimental vaccine formulations is being discussed. murine mabs developed against hbsag, subtype adw were found to cross-react with the other subtypes viz. ad and ay too. the mabs have been characterized following which, one mab hbs was chosen for developing ic-elisa format for the quantification of the hbsag in the final algel adsorbed vaccines. the unadsorbed hbsag was used to establish the standard curve of hbsag/a. the elisa had a sensitivity of ng/ml of hbsag. the recovery rate of hbsag/a was found to be around % in the vaccines treated to desorb the antigen from algel. twenty seven experimental batches of monovalent hepatitis b vaccines were analyzed for the hbsag content, both by ic-elisa and a commercial kit (axsym kit, abbott laboratories, usa). the statistical analysis of ic-elisa results indicated that an experimental equation f(x) = . (x) + . , could precisely estimate the amount of hbsag in the adsorbed vaccines. the amounts of hbsag recovered from the adsorbed vaccines as estimated by the ic-elisa format had a good correlation with the estimates derived from a commercial kit, which is being used by several vaccine manufacturers in india for the quality control of vaccine antigen. the varying amounts of vaccine antigens that could be recovered seemed to depend on the quality of the hbsag and the methods of hbsag adsorption to the alum gel during vaccine manufacture. epidemiology of the spread of h n virus. children of school going age have become victim of this deadly virus as evident from the reporting data generated in the past few weeks. the mortality rate has also been slightly increased. the disease spread in wave pattern and presently the world is passing through the second wave of pandemic with more severity in young and otherwise health people with a predilection for lungs leading to viral pneumonia and respiratory failure. now the pandemic gained hold in the developing world affecting more severely as millions of people live under deprived conditions having multiple health problems, with little access to basic health care. current data about the pandemic from developed counties need to be very closely watched in relation to shift in virus sub type, shift of the highest death rate to younger populations, successive pandemic waves, higher transmissibility than seasonal influenza, and demographic differences etc. presently the world appears to be better prepared. vaccine is available in market in many countries. even vaccine trials are actively going on in indian population. effective antivirals are available. although till now h n diagnostic centers worked with cdc/who recommended h n specific primer, probes with taqman chemistry by real time pcr, efforts on the development of indigenous diagnostics, vaccines and chemoprophylaxis is going on to have a better combat against this highly infectious virus. were positive for rotavirus infection by either page or elisa methods. the available data highlights the importance of rotavirus as a cause of diarrhea in children, which is severe enough to deserve specialized care. the observed proportion of . % of all diarrhea cases being associated with rotavirus falls within the range of values reported by other workers. the reported positivity varies from . to . %. in our study a complete concordance of elisa and page results were observed in ( %) of the tested specimens. this finding closely correlates with the findings of other authors who found a . - . % concordance results between elisa and page methods. some authors found rna-page method that is as sensitive and rapid as elisa for detecting rotavirus in stool samples of cases of diarrhea and some others proposed elisa is more sensitive than page method fond to be % specific. the remaining ( %) samples showed conflicting results. in a lone sample in which the od value of elisa test was . , this value was almost at the cutoff level, the possibility of this sample being positive by elisa test is doubtful. negative result of the same sample in page method is difficult to explain, the possibility of presence of lot of empty virus particles or due to low concentration of viral rna in the fecal specimen and insufficient extraction of viral rna could be possible. on the other hand, of the samples which gave positive results by page method were negative by elisa test. these samples had a typical - - - rna pattern. the reason for their being elisa negative thus remains unexplained, however blocking factors or the presence of inhibitory substance in stools might have been responsible. the samples containing predominantly complete particles can also give false negative results. since, the group antigen is not exposed. earlier studies have also reported page to be the most sensitive technique although some are of view that it is laborious procedure. how ever, the page system used in this study was very simple to perform and the results were available on the same day. the main requirement was of trained personnel and proper standardization of the technique. most reports states that the greatest advantage of page and silver stain method are its lack of ambiguity and the fact that it provides information about viral electropherotypes. the modified page system was thus found to be reliable, simple and rapid, no expensive reagents were required. locally available reagents from hi media were used. the cost of the chemical for page per specimen was rs. approximately as compared to rs. per test by confirmatory elisa. a locally produced slab gel electrophoresis system with power pack was the only equipment required. this method could be used for the routine diagnosis of rotavirus infection in the laboratory. vaccine, rapid diagnosis plays an important role in early management of patients. in this study a qc-rt-pcr assay was developed to quantify chikungunya virus rna by targeting the conserved region of e gene. a competitor molecule containing an internal insertion was generated, that provided a stringent control of the quantification process. the introduction of -fold serially diluted competitor in each reaction was further used to determine sensitivity. the applicability of this assay for quantification of chikungunya virus rna was evaluated with human clinical samples and the results were compared with real-time quantitative rt-pcr. the sensitivity of this assay was estimated to be rna copies per reaction with a dynamic detection range of to copies. specificity was confirmed using closely related alpha and flaviviruses. the comparison of qc-rt-pcr result with real-time rt-pcr revealed % concordance. these findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of chikungunya virus in acute-phase serum samples. in india, measles vaccine was introduced as part of expanded programme of immunization in . measles, mumps and rubella (mmr) vaccine is still not part of the national immunization schedule of india. the indian association of paediatrics (iap) recommends measles vaccine at months of age and mmr vaccine at - months. however, in a recent policy update, iap committee on immunisation opined that there is a need for a second dose of mmr vaccine for providing adequate immunity against mmr. the aim of the present study was to assess the extent of sero-protection against mmr at - years of age in children who have received one dose of mmr between and months of age. an attempt has also been made to assess the sero-response to the second dose of mmr vaccine in - years old children. a total of consecutive children between the ages of - years who had received mmr vaccine between and months of age and attending the immunization clinic of gtb hospital, delhi were enrolled. the vaccination status, anthropometry and physical examination findings were recorded. three ml of venous sample was again withdrawn for estimation of post vaccination antibody titre. it was observed that . %, . % and . % children were seroprotected for mmr respectively after . - . year of receiving first dose of mmr vaccine. seroprotection rose to . %, % and % for mmr respectively after - weeks of receiving second dose of mmr vaccine. geometric mean concentration of antibody also rose significantly in all three diseases. in view of low seroprevalence of mmr and hence high susceptibility to infection at - years of age, who have already received mmr vaccine, there is need to boost the immune responses against these three diseases by giving a second dose of mmr vaccine. baseline information on the epidemiology of viral agents causing stis and types of risk behaviour of affected persons are essential for any meaningful targeted intervention. the present study documents the pattern of viral stis in patients attending a tertiary care hospital, correlating the syndromic approach and the laboratory investigations to determine the aetiology. three hundred consecutive patients attending the sti clinic were diagnosed and categorized according to the syndromic approach of the who along with detailed history and demographic data. majority of the patients were men ( . %) with a mean age of years. men received education up to middle school. half of the female subjects were illiterate. sixty percent of the patients were married and among these, % were regular condom users. first sexual contact at or before years of age was more in men ( % vs. . % in women). promiscuity was more among male patients who had contact with csw. genital herpes was the commonest viral sti ( / ) followed by genital wart ( / ). concomitant infection with more than one virus was seen in % of patients. hiv was prevalent in . % of sti patients. hepatitis b, hepatitis c, herpes simplex type and molluscum contagiosum were the other viral agents seen in sti clinic attendees at our centre. this disease currently prevalent in more than countries world wide and annually - million people are infected with dengue virus among which . - lakhs cases were dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) which are serious forms of dengue virus infection and due to this condition , deaths might occur annually world wide and approximately million children were hospitalized for the fast decades. this disease is characterized by sudden onset of high fever with sever headache, pain in the back and limbs, lymphadenopathy macuolo-papulur rash over the skin and retro-bulbar pain. early diagnosis can be established with simple and rapid lgg/ gm antibodies detection in the blood samples of the patients based on the bi-directional immunoassay system for its management and control to reduce morbidity and mortality. details will be presented. myocarditis and dilated cardiomyopathy (dcm) are common causes of morbidity and mortality both in children and adults. the most common viruses involved in myocarditis are coxsackievirus b or adenovirus. recently, the coxsackievirus and adenovirus receptor (car), a common receptor for coxsackieviruses b , b and adenoviruses , has been identified. increased expression of car has been reported in patients with dcm suggesting utilization of car by these viruses for cell entry. the present study was designed to study the expression of car in myocardial tissue of patients with dcm. formalin fixed myocardial tissues were obtained from autopsy cases. a total of cases of dcm and cases of controls which included non-cardiac (group-a) and cardiac disease other than dcm (group-b) were included in the study. expression of car was studied by immunohistochemical staining of myocardial tissue using car specific rabbit polyclonal antibody and biotin conjugated secondary antibody. the tissue sections were considered positive when[ % of the cell showed brown color staining by immunohistochemistry (ihc). the car positivity in dcm cases was found to be % ( / ) as compared to % in control group a and % in control group b respectively. the car positivity was significantly higher in the test group as compared to both the control groups. further car positivity in all the cellular types (myocytes, endothelial cells and interstitial cells) was found significantly higher in test group as compared to both the control groups. the expression of car was significantly higher in myocytes as compared to both endothelial and interstitial cells in all the groups. however, no significant difference was observed in car positivity between endothelial and interstitial cells. the present study highlights the increased expression of car in dcm cases with further significance of car expression in myocytes and endothelial cells. this may help further in understanding the tropism of viruses or cellular susceptibility, which in turn will help in appropriate diagnostic and therapeutic approach in management of viral myocarditis and dcm cases. food security and safety vary widely around the world, and reaching these goals is one of the major challenges, raising public concern for the wellbeing of mankind, in particular. industrialized production and processing as well as improper environmental protection have clearly shown severe limitations such as worldwide contamination of the food chain and water. contaminated water and food during the processes of production, processing and handling are essentially responsible for food and water borne viral infections/diseases. the cases of viral food borne outbreaks are on the rise, creating a threat to human health. recent researches indicate that epidemiological studies are meager to focus the frequently contaminated foods and food borne viral diseases. current paper projects the etiology of select food borne viral diseases, probable reasons for non availability of appropriate methods to detect the viruses responsible for the diseases, routes of water and food borne transmission of enteric viral infections, currently available methods of detection of select viruses and bio safety measures to prevent food borne viral infections. dietary/nutritional management in food borne viral diseases is crucial to control weakness and gastro enteric intolerance due to disease condition and antibiotic therapy. it will principally improve food intake, resulting in better nutritional status leading to optimum immune response. food borne viruses are mainly belong to rotaviruses, enteropathogenic viruses, astroviruses, adenoviruses and caliciviruses, causes acute gastroenteritis (ag) which is an important health problem. the frequency of rotavirus as a cause of sporadic cases of ag ranges between . % and . %. astroviruses cause ag, with a frequency ranging between and %: outbreaks have been described in schools and kindergartens, but also in adults and the elderly. the frequency of identification of adenoviruses and as causes of sporadic ag in non-immuno suppressed children ranges between . % and . %, although there is probably underreporting because the sensitivity of conventional techniques is low. caliciviruses are separated phylogenetically into two genera: norovirus and sapovirus. norovirus is frequently associated with food-and water-borne outbreaks of ag. it is estimated that % of cases of ag due to norovirus are food borne. in sweden and some regions of the united states, norovirus is the first cause of outbreaks of food borne diseases. sapovirus outbreaks due to person-to-person and food borne transmission affecting both children and adults have recently been reported in countries such as canada and japan. it has been predicted that the importance of diarrhoeal disease, mainly due to contaminated food and water, as a cause of death will decline worldwide. evidence for such a downward trend is limited. this prediction presumes that improvements in the production and retail of microbiologically safe food will be sustained in the developed world and, moreover, will be rolled out to those countries of the developing world increasingly producing food for a global market. sustaining food safety standards will depend on constant vigilance maintained by monitoring and surveillance but, with the rising importance of other food-related issues, such as food security, obesity and climate change, competition for resources in the future to enable this may be fierce. in addition the pathogen populations relevant to food safety are not static. food is an excellent vehicle by which many pathogens (bacteria, viruses/prions and parasites) can reach an appropriate colonization site in a new host. although food production practices change, the well-recognized food-borne pathogens, such as salmonella spp. and escherichia coli, seem able to evolve to exploit novel opportunities, for example fresh produce and even generate new public health challenges, for example antimicrobial resistance. in addition, previously unknown food-borne pathogens, many of which are zoonotic, are constantly emerging. awareness and surveillance of viral food-borne pathogens is generally poor but emphasis is placed on norovirus, hepatitis a, rotaviruses and newly emerging viruses such as sars. it is clear that one overall challenge is the generation and maintenance of constructive dialogue and collaboration between public health, veterinary and food safety experts, bringing together multidisciplinary skills and multi-pathogen expertise. such collaboration is essential to monitor changing trends in the well-recognized diseases and detect emerging pathogens. it is also necessary to understand the multiple interactions between these pathogens and their environments during transmission along the food chain in order to develop effective prevention and control strategies. to analyse the effectiveness of these sirnas targeting rabies virus l gene, the bhk- cells expressing sirnas in shrna form were produced by transduction of cells with radv-l. the transduced bhk- cells expressing sirna were infected with rabies virus pv- strain. there was reduction in rabies virus multiplication as analysed by reduction in fluorescent foci forming unit (ffu) count by . % ( ffu in bhk- cells expressing sirna-l compared to ffu in bhk- cells expressing negative sirna). the expression of l gene mrna was reduced by . fold in rabies virus infected radv-l transduced cells compared to radv-neg transduced cells (negative control) as detected using real-time pcr. after analyzing the effectiveness of radv-l in vitro, its effectiveness was also evaluated in vivo in mice after virulent rabies challenge. the mice were inoculated with plaque forming units (pfu) of radv-l in masseter muscle (i/m route) and challenged with ld rabies virus challenge virus standard (cvs) strain. the results indicated % protection with improved median survival from to days compared with group of mice treated with radv-neg. the results of this study indicated that sirnas targeting rabies virus polymerase (l) gene delivered through adenoviral vector inhibited rabies virus multiplication in vitro and in vivo. and were successfully produced and purified from the infected spodoptera frugiperda (sf- ) cells using these recombinant baculovirus. the morphology of the vlps was validated by electron microscopy in comparison to the authentic bt virions. the vlps produced here were stable and were highly immunogenic with intact outer layer which is rapidly lost during normal infection of btv. these btv-vlps elicited long lasting protective immunity in vaccinated sheep against virulent virus challenge. with the use of btv-vlps it was also possible to differentiate the infected and vaccinated animals (diva). vlp-based btv vaccine has potential advantages with regard to controlling the spread of btv with multiple serotypes. it is possible to produce milligram quantities of correctly folded and processed protein complexes using this baculovirus expression system and hence it is a more promising system for producing new generation vaccines like vlp subunit vaccine against any viral diseases in large scale. peste des petits ruminants (ppr), goatpox and orf are oie notifiable diseases of small ruminants especially goat and sheep. these diseases are economically important, in enzootic countries like india and cause significant loss and are major constraints in the productivity. considering the geographical distribution of ppr, goat pox and orf infections and prevalence of mixed infection, in the present study, safety and potency of the experimental triple vaccine comprising attenuated strains of thermostable-ppr virus (pprv jhansi, p- ) grown at °c, high passaged goat poxvirus (gtpv uttarkashi, p ) and attenuated orf virus (orfv mukteswar, p ) was evaluated in sub-himalayan local hill goats. goats simultaneously immunized with ml of vaccine consisting of either tcid or tcid of each of pprv, gtpv and orfv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to pprv, gtpv and orfv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, snt, respectively following immunization. all the immunized animals resisted infections when challenged with virulent strains of either gtpv or pprv or orfv on day dpi, while in contact control animals developed characteristic signs of respective disease. further, ppr viral antigen could be detected by using ppr sandwich elisa kit in the excretions (nasal, ocular and oral swab materials) of unvaccinated control animals after challenge but not from any of the immunized goats. triple vaccine was found safe at dose as higher as tcid and induced protective immune response even at lower dose ( tcid ) in goats, which was evident from sero-conversion as well as challenge studies. the study indicated that these viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this triple vaccine in combating these infections simultaneously. toll like receptors (tlrs), primary sensors of microbial origin, plays a crucial role in the innate immunity. till now mammalian tlrs have been identified, while there is no information available on tlrs of yak. this study is part of world bank funded-icar project. yak, named bos grunniens for its distinctive vocalization and relationship with cattle, is natural habitant of extremely cold environment. when these animals comes to a lower altitude grazing land, adjacent to villages, become susceptible to the diseases of cattle, buffalo etc. thus, present study was undertaken to with genetic characterization and evolutionary lineage analysis of yak tlrs. we worked on tlr gene, which plays an important role in recognition of ssrna viruses. total rna was extracted from mitogen stimulated pbmcs of yak. the rt-pcr conditions were standardized for full length amplification of tlr gene using specific self designed primers. the expected amplicon of bps was obtained. it was cloned in pgemt-easy vector followed by transformation in e. coli top strain. the recombinant clones were screened, picked up for plasmid isolation and release of tlr was confirmed by restriction digestion. the cloned tlr product was sequenced and analyzed for the nucleotide and deduced amino acid sequences, and d structure analysis. the results revealed that yak shows more than % sequence homology with other bos indicus breeds and bos taurus breeds. however, identity was less than % with other animal species (equine, murine, feline, canine etc.). the evolutionary lineage findings cluster yak more closely with bovine species. point mutations revealed changes at nucleotide positions with corresponding amino acid change at positions. smart analysis of yak protein domain architecture revealed toll-interleukin i receptor (tir), leucine rich repeats (lrr) and signal peptide region. the variations in yak mainly lie in the lrr region. homology modeling revealed horse shoe shaped structure with alpha helix. the additional alpha helix present in bos indicus was not detected in yak. the present study shows existence of genetic variability in tlr gene of yak, in particular the lrr region, which plays an important role in the pathogen recognition and the evolutionary lineage analyses shows its closeness with other bovine species. a.p. aquaculture and fisheries, tirupati in this new millennium, aquatic animal health management strategies in asia expanded and adjusted to the current disease problems faced by the aquaculture sector. this presentation will briefly discuss some of the most serious trans-boundary pathogens affecting asian aquaculture including a newly emerging disease and highlight recent regional and national efforts on responsible health management for mitigating the risks associated with aquatic animal movement. a regional approach is fundamental since many countries share common social, economic, industrial, environmental, biological and geographical characteristics. capacity and awareness building on aquatic animal epidemiology, science-based risk analysis for aquatic animal transfers, surveillance and disease reporting, disease zoning and establishment of aquatic animal health information systems to support development of national disease control programs and emergency response to disease outbreaks are needed. molecular diagnostics with emphasis towards standardization and harmonization, inter-calibration exercises and quality assurance in laboratories, accreditation program and utilization of regional resource centres on aquatic animal health will also be needed. whilst most of these strategies are directed in support of government policies, implementation will require pro-active involvement, effective cooperation and strategic networking between governments, farmers, researchers, scientists, development and aid agencies, and relevant private sector stakeholders at all levels. their contributions are essential to the health management process. generally, aquaculture plays an important role in economy as harvests from natural waters have declined or, at best, remained static in most countries. fish and shrimp, the main aquaculture product sources, have gained the most attention. many factors can cause losses in yields of fish products and infectious disease in fish and shrimp is the biggest threat to the fishery industry. shrimp and fish aquaculture has grown rapidly over several decades to become a major global industry that serves the increasing consumer demand for seafood and has contributed significantly to socio-economic development in many poor coastal communities. however, the ecological disturbances and changes in patterns of trade associated with the development of shrimp and fish farming have presented many of the pre-conditions for the emergence and spread of disease. shrimp and fish are displaced from their natural environments, provided artificial or alternative feeds, stocked in high density, exposed to stress through changes in water quality and are transported nationally and internationally, either live or as frozen product. these practices have provided opportunities for increased pathogenicity of existing infections, exposure to new pathogens, and the rapid transmission and trans boundary spread of disease. not surprisingly, a succession of new viral diseases has devastated the production and livelihoods of farmers and their sustaining communities. this review examines the major viral pathogens of farmed shrimp and fish, the likely reasons for their emergence and spread, and the consequences for the structure and operation of the shrimp farming industry. in addition, this review discusses the health management strategies that have been introduced to combat the major pathogens and the reasons that disease continues to have an impact, particularly on poor, smallholder farmers in asia. btv isolates from the same geographic region have been termed as 'topotypes' and initial observation on segment nucleotide sequences identified a correlation between topotypes and genetic information. later topotyping was proposed based on segment , on the premise that the encoding protein ns , which is involved in virus egress from insect cells, would lead to evolutionary fitness in parallel with the geographic distribution of the different culicoides species. further studies attempted to extend this to nucleotide sequence homology in segments and , but failed to identify clear cut correlations or any evidence for positive selection. for example, south african isolates were found not to cluster into separate african lineage. in this study, we carried out a more extensive analysis of segment sequences. our analysis showed no segregation of isolates into topographically distinct groups. instead we observed topological clustering of the clades, and we attribute this to genetic bottleneck resulting in genetic drift and founder effect leading to homogenous gene pool in a geographical area. we hypothesize that when a new virus enters a geographical area where local btv strains are already circulating, the new genes/segments would enter into a bigger gene pool. consequently, the newer incursions into a heavily endemic area tend to get diluted and disappear from the population because the rate of drift is inversely proportional to the population size, unless they are positively selected. use of live attenuated vaccine in israel, europe, south africa and usa also led to more homogenous population similar to the vaccine strains due to continuous infusion of the vaccine type genes into the gene pool. we conclude that restriction of specific strains to certain geographical areas could generate uniquely imprinted genotypes which would not only indicate origin but also predict movement of viral strains to new areas. vvo- viral diseases of zoonotic importance: indian context k. prabhudas pd-admas, ivri, campus, hebbal, bangalore zoonoses are generally defined as animal diseases that are transmissible to humans. they continue to represent an important health hazard in most parts of the world, where they cause considerable expenditure and losses for the health and agricultural sectors. the emergence of these zoonotic diseases are very distinct, hence their prevention and control will require unique strategies, apart from traditional approaches. such strategies require rebuilding a cadre of trained professionals of several medical and biologic sciences. the article discusses virus infections that have significant zoonotic implications for india. buffalopox is a contagious viral disease affecting milch buffaloes and rarely, cows, with a morbidity rate up to % in the affected herd. although the disease is not responsible for high mortality, it adversely affects the productivity of the animals, resulting in large economic losses. furthermore, the disease has zoonotic implications, as outbreaks are frequently associated with human infections, particularly in the milkers. the causative agent, buffalopox virus (bpxv), is closely related to vaccinia virus. the outbreaks of febrile rash illness among humans and buffaloes were investigated in the villages of districts solapur and kolhapur of western maharashtra. clinico-epidemiological investigations of humans and buffaloes were carried out and representative clinical samples were collected respectively. the samples include vesicular fluid, scab, and blood. laboratory investigations for buffalo-pox virus (bpxv) was done by pcr on blood samples, scabs and vesicular fluid. in vitro virus isolation attempts were carried out by using vero e- cells. negative staining electron microscopy was also employed for detection of virus particles. a total of human cases with pox lesions on hand and other body parts from village kasegaon, district-solapur and cases from different villages of kolhapur district were reported. besides pox lesions patients were having fever, malaise, pain at site of lesion and axillary and inguinal lymphadenopathy. in kasegaon village, attack rate in human cases was . % and in buffaloes . % ( / ). whereas in kolhapur area attack rate in buffaloes was . % ( / ). bpxv was confirmed in blood, vesicular fluid and scab specimens from human cases and scab specimen from buffalo by polymerase chain reaction (pcr) method. the bpxv was also isolated from different clinical specimens and further identified by pcr and electron microscopy. clinical manifestation of the disease in buffaloes from solapur district was as reported earlier like common pox lesions on teats and udders whereas the buffaloes from kolhapur district had lesions on hairless parts of ears and on the eyelids with purulent discharge. bpxv from human and buffalo cases showed similarity. vaccines have been made against several diseases and used for controlling the afflictions. however a few of them were not effective for successfully controlling the disease. the reasons for the failure are many, the major being, either the pathogen is not completely cleared from the vaccinated animal or it reemerges after changing its antigenic structure, thus making the vaccination programme less effective. in addition to this, emergences of newer diseases such as hiv the development of suitable vaccines have become a challenging task. this is especially true in the case of viral diseases. these challenges have warned the researchers ''that protection by vaccination is not that simple and strait forward approach'', and lot need to be understood in terms of host virus interaction and role of environment in perpetuating the disease. so the immediate step that was considered was the environmental safety by way using non infectious materials as vaccines. with the understanding that has been developed in molecular immunology and molecular biology and with the availability of molecular tools that have been developed through recombinant dna technology the field of vaccinology has changed dramatically to emerge as modern vaccinology. this presentation deals with the modern approaches that are being used to produce effective vaccines in the case of foot and mouth disease of cloven footed animals. the similar approach may be worked out for other viral diseases also. despite the availability of an inactivated vaccine that is noted to provide solid immunity against the disease over a short period of time, the search for an ideal vaccine, the criteria for which are; safety of the vaccine for environment, easy in its preparation, does not require a cold chain for its storage, provides longer lasting immunity, economically viable and may be able to clear the virus in case of persistent infection is on. the advent of recombinant dna technology together with the information available on the molecular biology of viruses has enabled to design the development of newer vaccines that can induce strong cellular and humoral responses. the underlying principal in the present vaccine development strategy world over is the virus antigen gene has to be expressed in the tissue and the vaccine backbone has to trigger the immune system for eliciting desired immune response. bangalore campus of ivri has been vigorously pursuing research to develop ideal vaccines for foot and mouth disease keeping above principal in mind to achieve the previously mentioned criteria. the approaches selected are to see that the virus antigen/s replicate transiently in the host. the self replicating vaccines that have been developed are pox virus vectored vaccines, alpha virus replicase based vaccines and fmdv vectored vaccines. the approach and the result obtained so far will be discussed. silkworm, bombyx mori is affected with various diseases caused by viruses viz., nuclearpolyhedrosis (bmnpv), densosnucleosis (bmdnv) and infectious flacherie (bmifv). silkworm viral diseases form major constraints for the silk cocoon production in all the sericultural countries. the losses due to silkworm diseases is estimated about - % and among them viral diseases are most common. in sericulture, prophylactic measures play a vital role in the management of silkworm diseases. these include disinfection of silkworm rearing house and appliances, rearing area, rearing surroundings, silkworm egg and body, and rearing bed disinfection associated with maintenance of general hygiene and personnel hygiene. all these activities are generally carried out as rituals by using general disinfectants often with partial success. recent trends in complete management of silkworm diseases include development of silkworm hybrids evolved from disease resistant/tolerant breeds, effective eco-and user-friendly disinfectants, anti-microbial feed-supplements and use of transgenic silkworms. biotechnological breakthrough in this regard is through rna interference (rnai) approach involving dsrna mediated nuclear polyhedrosis management and this is presently pursued by apssrdi, hindupur in collaboration with centre for dna fingerprinting and diagnostics (cdfd), hyderabad. nadu and karnataka. the disease appears to be more severe in rural flocks than organized farms. our investigations revealed the morbidity, mortality and case fatality rates among rural and organised farms as . %, . %, . % and . %, . %, . % respectively. higher morbidity and mortality in rural areas may be due to stress factors like poor nutrition, parasitic burden, fatigue due to long walks and non availability of veterinary aid. kulkarni et al. also reported the severe bt outbreaks in rural areas of maharashtra with overall morbidity, mortality and case fatality of %, % and % respectively. all the south indian sheep breeds were found to be susceptible and clinical farm of the disease is evident in all of them though saravanabava ( ) reported variations in susceptibility among the indigenous sheep. trichy black and ramnad white sheep were found to be more susceptible than the vambur and mecheri sheep of tamil nadu. prevalence of bluetongue in sheep, goat and cattle appears to be high in the region. serological surveys conducted in andhra pradesh during revealed the prevalence of btv antibodies in sheep ( . %) goats ( . %) cattle ( %) and buffaloe ( %). similar high prevalence of btv antibodies in sheep and goats were also reported from the other states in the region. clinical disease has not been recorded in kerala though btv antibodies were recorded in sheep ( . %) and goats ( . %) (ravi sankar ) . culicoides are the known biological vectors of btv. all the culicoides species are not capable of transmitting the btv. the occurrence of the disease is related to the presence of the competent vectors in the area. jain et al. ( ) established the involvement of the culicoides in transmitting the btv by isolating the virus from culicoides at haryana, the north indian state. c. imicola and c. oxystoma were found to be prevalent in andhra pradesh and tamil nadu. narladakar et al.( ) reported the presence of c. schultzei, c. perigrinus and c. octoni in marathwada region of maharastra. culicoid vectors are significantly affected by the climate and annual variations in the climate reflects the outcome of the disease. the monsoon season (june to dec) with the temperature ranging from . to . °c appears to be favourable period for the multiplication of culicoides. the maximum no of outbreaks were recorded during the north east monsoon period (oct-dec) followed by south west monsoon period (june to sep) in the region. however, details on the distribution of the competent vectors, feeding habits and their dynamics in the region is lacking multiple btv serotypes were found to be circulating in the region. (kulkarni and kulkarni ; janakiraman etal. ; mehrotra et al. ) a total of serotypes viz. - , , , , , and were identified based on the virus isolations. sreenivasulu et al. isolated btv serotype from an outbreak of bt in native sheep of andhra pradesh. btv serotype , and were also isolated from the outbreaks occurred in andhra pradesh. some of the isolates need to be serotyped. deshmukh and gujar ( ) isolated btv type from maharashtra. following is the summary of the distribution of btv serotypes in this region. clinical picture of bt in native sheep appears to be slightly different, the major difference being that swelling of lips and face was less conspicuous. mucocutaneous borders appeared to be very sensitive to touch and bleed easily upon handling. the classical signs of cyanosis of tongue and reddening of coronary band are not the common features of the disease in native sheep. the disease was also confirmed by the virus isolation and identification. clinical disease has not been reported in cattle, buffaloes and goats in spite of high seroprevalence. in conclusion bt is established in native sheep and causes severe economic losses to the farmers. the disease is concentrated in the southern peninsula of the country. the disease is seasonal and is associated with the rain fall. multiple serotypes appear to be circulating in this region. the btv serotypes were of virulent in nature as evident by severe outbreaks. s. janardana reddy*, d. c. reddy department of fishery science and aquaculture, sri venkateswara university, tirupati in less than three decades, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of u.s. dollars in revenue, and augmentation of the world's food supply with a high value crop. concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. in india viral diseases have become an important limiting factor for growth of shrimp aquaculture industry. although more than different viral pathogens have been identified in different species of shrimp world wide, only a few viruses have identified which are causing disease problems in cultured tiger shrimps in india, east coast of andhra pradesh, in particular. diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, and transmission electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. while tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. the need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. the industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like infectious hypodermal and hematopoietic necrosis (ihhnv), hepatopancreatic virus (hpv), taura syndrome virus (tsv), white spot syndrome virus (wssv), monodon baculo virus (mbv), and bp. highly sensitive detection methods for some pathogens that employ dna amplification methods based on the polymerase chain reaction (pcr) now exist, and more pcr methods are being developed for additional agents. these advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. the establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. for this, immune effectors must be first identified and characterised. in the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. the development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. however, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment. bluetongue is an important viral disease of sheep causing severe economic losses to the farmers. lack of effective vaccine is the major impediments in controlling the disease. multiple serotypes were found to be circulating in the state. attempts are being made to develop the vaccine employing the available serotypes to control the disease. hence, it is essential to identify the antigenic relationship among the serotypes to identify the candidate vaccine strains to be incorporated in the preparation of vaccine. reciprocal cross neutralization test was employed to find out the r% values between btv- , - and - which indicated the extent of antigenic relationship between the serotypes. r% value between btv- and btv- was recorded as . r% value of . and . were observed between btv- and - and btv- and - respectively. the r% values recorded in the present study revealed a weak antigenic relationship between the btv serotypes. the extent of antigenic relationship between the btv serotypes was also determined by multiple sequence alignment of the nucleotide and amino acid sequences of the reference btv serotypes , and . the sequence analysis of the vp gene revealed a homology of - % and - % at the nucleotide and amino acid levels respectively. r% values obtained using reciprocal cross neutralization test with the btv- , and serotypes isolated in native sheep of andhra pradesh and the genomic analysis of the reference serotypes of btv- , and revealed very weak antigenic relationship and were highly divergent. diseases especially those by viral pathogens cause greater economic losses in most horticultural crop species throughout the world as compared to agricultural crops. non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. however, none of these measures is likely to provide an enduring solution against these diseases especially those caused by viruses due sometimes to the huge expenditure involved, but mostly to the questionable effectiveness and reliability of those methods. as key control pesticides are getting increasingly abandoned, development of alternative methods to control diseases has been a felt-need in the recent past. though breeding for disease resistance generally provides a reliable security in a long run, introgression of host plant resistance did not materialise in most important crops. non-availability of an appropriate source of resistance in inter-fertile relatives, linkage to undesirable traits, or often times polygenic nature of such sources of resistance are the stumbling blocks in breeding programs. the limitations of conventional breeding and routine cultural practices prompted the need for the development of other approaches of virus control that could be fully incorporated into traditional methods. in this perspective, the concept of pathogen-derived resistance offers an attractive strategy to evolve newer methods of virus management, by transforming crop plants with nucleotide sequences derived from the pathogen's genome. an increasing number of molecular characterisation of plant virus genomes and the stable transformation of a number of horticultural crop species have in fact opened an avenue for molecular breeding against virus pathogens. successful field-testing of genetically modified crop cultivars renders proof of their supremacy over existing cultivars. it also contributes to demonstrate their capability with regard to environmental safety with a view to winning over public concern and scepticism. in general, the eventual commercialisation transgenic lines expressing virus resistance will rely upon a host of factors including their field performance, genetic stability, public acceptance and the resolution of environmental concerns and patent related issues. as such, elaborate field trials and allied studies are now required to adapt genetically engineered horticultural crops expressing virus resistance for their implementation into practical agriculture. a few examples from current research at tnau, in india or elsewhere will be discussed in this presentation. virology unit, division of plant pathology, iari, new delhi in recent times there has been greater emphasis on vegetatively propagated crops in india to help diversify the indian agriculture. fruit, flower, spice and plantation crops are important vegetatively propagated horticultural crops, which have become a driving force for economic development in several parts of india. however, most of the vegetatively propagated crops are threatened by biotic stress caused by plant pathogens in general and plant viruses in particular. plant viruses produce specific and non specific symptoms and in some cases no symptoms are produced. correct identification and diagnosis of viral diseases is first step in the management of any disease including viral diseases. there have been two major breakthroughs in virus diagnostics during last four decades. the first one was serological assay using monoclonal or polyclonal antibodies in enzyme linked immunosorbent assay (elisa) and the other one was the use of in vitro amplification of dna in polymerase chain reaction (pcr). a significant development in serological assays has been its simplification in form of user's friendly quick strip/dip stick method. the one-step lateral-flow (lf) tests have been developed for the on-site detection and identification of several plant viruses. rapid advancement in virus genome characterization has led to the development of novel approaches of nucleic acid based diagnostics which include conventional pcr, real time pcr, multiplex pcr, micro/macro arrays and biochips. pcr protocols already exist for many plant viruses of citrus, banana, apple, papaya, vegetables, ornamental and spice crops. a further advancement has led to development of realtime pcr assay which is relatively easy but requires training for diagnosticians. in real-time pcr assays, results can be available within min. the nucleic acid template preparation in pcr has been simplified. membrane based dna template protocol and co-isolation of nucleic acid template preparation are novel approaches in pcr detection of virus and virus like pathogens. since many of the horticultural crops are often infected by more than one virus, their individual detection by pcr is not only expensive but also time consuming. therefore, multiplex pcr has been developed where in genome of more than one virus could be amplified and detected in the same reaction mixture. development of nucleic acid based chip is now one of the fastest and recent growing areas in the field of pathogen detection. these nucleic acid based chips have been named as dna/rna chips, biochips, genechips, biosensors or dna arrays. when it comes to applications of microarray technology for plant viruses, it is not too difficult to see the value of a method that could potentially detect a whole range of viruses using a single test. however, microarrays are unlikely to become the only method in use in a diagnostic laboratory. processing of germplasm including transgenic planting material imported for research purposes into the country. during the last two decades, a total of , samples of wheat including transgenics were imported from cimmyt (mexico), icarda (syria) and many other countries. these were grown in post-entry quarantine nursery each year at nbpgr, new delhi and the transgenic samples were grown in national containment facility of level- (cl- ) since its inception to ensure that no viable biological material/pollen/pathogen enters or leaves the facility during quarantine processing of transgenics. in addition, post-entry quarantine inspections of the transgenic wheat grown by indenters are also undertaken by nbpgr quarantine scientists. virus-induced gene silencing (vigs) is a technique in which viral genomes are used, usually after appropriate modifications, for transient gene silencing in plants. the mechanism behind vigs is the phenomenon called rna-interference (rnai), which is widespread in many organisms and is believed to be form of inherent defence system against intracellular pathogens, such as viruses and transposons. double-stranded rna or rna containing strong secondary structures, commonly produced during viral infections, are believed to cause triggering of rnai, which employs a battery of proteins and nucleoprotein complexes to identify and degrade specific viral transcripts. in vigs, viral genomes not causing severe symptoms, but which can accumulate and spread efficiently in the host plant are used as vectors in which a host gene is cloned and introduced into the plant. upon replication, the viral vector triggers rnai response in the host plant, which also targets the host gene, leading to its silencing and subsequently, the silenced phenotype revealing gene function in vivo. vigs has been used extensively to study gene functions in dicot plants, such as tobacco, tomato, pea, soybean, etc., using vectors derived from reference genes are commonly used as an/the endogenous normalisation measure for the relative quantification of target genes. the expression (characteristics) of seven potential reference genes was evaluated in tissues of healthy, physiologically stressed and barley yellow dwarf virus (bydv) infected cereal plants. these genes were tested by rt-qpcr and ranked according to the stability of their expression (characteristics) using three different methods (two-way anova, genorm and normfinder tools). in most cases, the expression (characteristics) of all genes did not depend on the abiotic stress conditions or on the virus infections. all the genes showed significant differences in expression (characteristics) among plant species. glyceraldehyde- -phosphate dehydrogenase (gapdh), beta-tubulin (tubb) and s ribosomal rna ( s rrna) always ranked as the three most stable genes. on the other hand, elongation factor- alpha (ef a), eukaryotic initiation factor a (eif a), and s ribosomal rna ( s rrna) for barley and oat samples; and beta-tubulin (tubb) for wheat samples were consistently ranked as the less reliable controls. the bydv titre was determined in two oat varieties by rt-qpcr by three different quantification approaches. statistically, there were no significant differences between the absolute and the relative quantification, or between quantification using gapdh + tubb + tuba + s rrna and ef a + eif a + s rrna. the geometric average of gapdh, s rrna, tuba and tubb is suitable for normalisation of bydv quantification in barley and oat tissues. for wheat samples, a combination of gapdh, s rrna, tubb, eif a and e fa is recommended. department of microbiology, yogi vemana university, vemanapuram, kadapa large scale production and import of propagative material poses potential risk of introducing several destructive pathogens particularly viruses and mycoplasma like organisms in our country. this demands adequate quarantine safe guards such as growing them under approved post entry quarantine facility for specific period so as to facilitate virus detection, thereby curtailing risk. when such facilities are coupled with propagation by tissue culture will ensure virus free propagative plant material. the requirement of nationwide network of post entry quarantine facility working in close collaboration with crop institutions are very much emphasized for considering import of high risk plant genera for agriculture development. present paper discusses about virus disease of quarantine importance affecting ornamental and fruit plants such as chrysanthimum, dahlia, dianthus, rosabengalensis, cattleya, cymbidium, dendrobium, lilium, citrus, vitis etc. the paper also discusses on immunodiagnostic methods of detection and methods of obtaining virus free propagative material. rice tungro occurs as epidemics in regular cycles and has been reported in the last years from all the major rice growing regions of india, especially prevalent in the southern and eastern states. development of the durable resistant varieties to tungro is crucial for the management of the disease. molecular breeding, involving the use of dna markers linked to the resistant gene(s) for selection, can overcome the difficulties encountered in conventional resistant breeding programs. for successful marker-assisted selection (mas), the identification of closely linked markers through the process of gene tagging and mapping is a prerequisite. attempts have been initiated for identification of tungro resistance genes through molecular mapping and their introgression into the target varieties using marker-assisted selection at drr, hyderabad. the inheritance of resistance to rice tungro virus disease was studied in seven resistant rice cultivars with field evaluation at hot spot locations. the microsatellite markers linked to rice tungro resistance in utri merah was studied and found that resistance genes were linked to rm on chromosome . through molecular mapping two qtl were identified controlling rtv resistance on chromosomes and in 'utri rajapan' explaining . % and . % of the phenotypic variance. in variety 'vikramarya', another two qtl for rtv resistance were detected on chromosomes and explaining . % and . % of the phenotypic variance. the closely linked markers identified in this study flanking the gene of interest through mapping will improve the efficiency and precision of introgression programs in marker assisted breeding for rtv resistance. functional characterization of these qtl for rtv resistance is under progress. there is only a limited pool of natural virus resistance in cassava against cassava mosaic geminiviruses and cassava brown streak ipomovirus hence the development of transgenic resistance in this significant crop might present an option. rna mediated resistance through the expression of inverted-repeat dsrna sequences derived from the virus genome and the modification of plant microrna to produce antiviral artificial microrna are strategies that have recently been proven very effective for induction of virus resistance (immunity) against a number of rna viruses. results from rna interference strategies against geminiviruses never resulted in immunity of transgenes. however, it suggest that viral mrna are targets of rna silencing and that the success of the strategy depends on the relevance of the target gene in the systemic spread of the virus. we have generated a number of rna silencing constructs to induce resistance against cbsv and the indian cassava mosaic viruses icmv and slcmv. due to the serious problems inherent with transformation of cassava and subsequent resistance screening, these constructs were tested for efficiency either by transient-or by transgenic expression in n. benthamiana. complete immunity was reached in transgenic n. benthamiana against cbsv using inverted repeat or amirna constructs. using different species of cbsv for resistance screening, immunity was broken, to show the minimum context for broad spectrum resistance. similarly, highly specific resistance was reached in expression of amirna. in contrast, virus resistance against icmv/ slcmv using single amirna constructs was not successful. results from the experiments to generate virus resistance against cbsv and icmv/slcmv will be shown; methods to evaluate efficiency of rnai gene constructs by transient gene expression in n. benthamiana and strategies to develop efficient resistance against rna and dna viruses in cassava will be discussed. bitter gourd (momordica charantia l.) which is also called bitter melon, balsam apple and balsam pear belongs to family cucurbitaceae. it is an important traditional vegetable of nutritive and medicinal value that is cultivated in tropical and sub-tropical asia, but is considered as a weed host reservoir for viruses in jamaica. viral disease-like symptoms were observed occurring naturally on the crops of bitter gourd grown in the fields of northern india during - . an incidence of . % of diseased plants was recorded which showed chlorotic spots and mosaic ranging from mild mottling to green blisters along with leaf smalling, leaf and fruit deformations, bud necrosis and stunted growth whereas . % plants exhibited leaf curling alone or in combination with mosaic-type disease. a reduction of . % in fruit yield was recorded in mosaic-like disease which could be attributed to lesser fruit setting due to bud necrosis, smaller fruit size and stunted plant growth. such plants produced deformed, notched, irregularly shaped fruits wherein pre-mature yellowing and necrosis on the anterior and posteriors ends made . % fruits unfit for marketability. the dwindling yield and production of unmarketable fruits posed a major constraint for profitable cultivation of this economically important crop, thus warranting for studies on etiology and management of these diseases. the mosaic-like disease was transmitted to healthy seedlings of bitter gourd at -leaves stage by sap inoculation as well as by aphid viz., myzus persicae sulz. and aphis gossypii glov. initially studies were carried out to optimize protocols for efficient plant regeneration and agrobacterium-mediated transformation for nagpur sweet orange, which is a popular and elite citrus cultivar in india. organogenesis was induced in etiolated epicotyl explants of one-month-old axenically raised polyembryonic seedlings by culturing them in mt medium supplemented with g/l sucrose with varying concentrations of plant hormones. it was found that bap at mg/l without auxin was best for efficient shoot regeneration in citrus using epicotyl explants. a % regeneration frequency was obtained and multiple shoot formation was obtained from both the cut ends of all the explants. an average of . well-differentiated shoots per explant were obtained, all of which rooted normally under the influence of mg/l iba. this improved regeneration protocol was utilized in standardizing agrobacterium-mediated transformation of citrus using a. tumefaciens strain eha , containing binary plasmid pcambia that harbors gus reporter gene and npt-ii plant selection marker gene. one-month-old epicotyl explants infected with over-night grown agrobacterium (a . - . ) for min and co-cultured for days were found to be optimum for transformation as assessed on the basis of pcr analysis and gus activity displayed by the stem and leaf sections of putative transgenics. overall transformation frequency ranged from to %. current study focuses on the generation of citrus transgenics for ctv resistance using a. tumefaciens strain eha containing binary plasmid pbinar harboring portion of coat protein gene of ctv and npt-ii gene employing the standardized protocols. several putative transgenic shoots were recovered on selection medium and they are being utilized for molecular analyses and resistance against ctv. work is also in progress on the generation of citrus transformants using rnai construct harboring ctv cp and p genes, singly and in conjunction. our lab was also involved in developing rice transgenics for resistance against rice tungro disease, which is one of the most important and widespread virus diseases of rice in south and southeast asia, causing an annual estimated loss in crop yield of economic losses worth millions of rupees are caused due to these diseases annually. virus diseases are frequently less conspicuous than those caused by other plant pathogens and last for much longer. this is especially true for perennial crops and those that are vegetatively propagated. one further problem with attending to assess losses due to various diseases on a global basis is that what most of the data are from small comparative trials rather than wide scale comprehensive surveys, even the small trials do not necessarily give data that can be used for more global estimates of losses. this is for several reasons, including: ( ) variation in losses by a particular crop from year to year; ( ) variation from region to region and climatic zone to climatic zone: ( ) differences in loss assessment methodologies; ( ) identification of the viral etiology of the disease; variation in the definition of the term 'losses' and ( ) chilli is the major vegetable and spice crop grown in thar desert areas of rajasthan. leaf curl disease (chlcd) is one of the major constrains in chilli cultivation faced by farmers and cause yield loss up to %. a survey was conducted in major chilli growing areas of thar desert; bikaner, nagur, jodhpur and jalore districts of rajasthan during november, to understand the present status of leaf curl disease in chilli. among the four district surveyed for chlcd, the disease incidence was recorded maximum (up to %) in jodhpur district followed by jolore district (up to %). no relation was found between the disease incidence and varieties. the major varieties grown in these area are; mehsana, rch (mandoria), haripur raipur, mathania and local cultivars. the number of whitefly was also counted in top, middle and bottom leaf of chilli grown in these areas. the average number of whitefly per plant ranged from . to . . more number of whitefly ( . ) was recorded in jodhpur district and lowest ( . ) in jalore district. total dna was extracted from three leaf curl infected samples from each district and tested for the presence of begomovirus using coat protein (cp) and dna-b specific primers. all the samples were positive for cp and dna-b amplifications by pcr. the cloning and sequencing of selected cp gene and dna-b fragments are in progress. the preliminary investigations shows that the leaf curl disease of chilli is widespread in the arid region of rajasthan and may be caused by begomovirus associated with satellite dna-b. bittergourd (momordica charantia) is an important vegetable crop of kerala. the crop is affected by several diseases of which mosaic is a prominent one. a field experiment was conducted to evaluate the efficacy of potentised resistance inducing substances (ris) viz., mosaic affected bittergourd plant tissue, ash of mosaic affected bittergourd plant tissue, plumbago and salicylic acid for control of bittergourd mosaic in march . ris were applied as drench and foliar spray at three potency levels twice, before flowering of the crop. the experimental crop was grown as per the package of practice recommendations in split plot design with five replications per treatment. the disease incidence, disease severity and yield of the crop were recorded. the result of the experiment shows that spraying was more effective than drenching of treatments for reducing mosaic incidence and severity. among treatments, infected plant extract at potency was the most effective one for reducing mosaic incidence and it showed the maximum incubation period and minimum disease severity. the spray application of treatments produced significantly higher yield than drenching. among the treatments, ash of infected plant at and potency and infected plant extract at potency were on par and produced comparatively higher yield. elephant foot yam (amoprhophallus paeoniifolius), colocasia (colocasia esculenta) and tannia (xanthosoma sagittifolium) are the major edible aroids cultivated in india. the elephant foot yam cultivation is gaining importance due to its high production potential, nutritional and medicinal values and good economic returns. all these aroids are vegetatively propagated and viral diseases are spreading through planting materials. ctcri has the mandate of producing healthy planting materials of these edible aroids. accurate diagnosis and identification of the virus is essential for production of healthy planting material and effective management of the disease. though occurrences of viral diseases on edible aroids in india were known in s, not much attention was given for detection and identification of the virus involved. in case of elephant foot yam - % mosaic incidence was observed with varying symptoms of mosaic, puckering, filiformy etc. in colocasia and tannia, - % incidence was noticed. rt-pcr amplification with potyvirus group specific primers and subsequent cloning and sequencing of the amplified product has confirmed the association of dasheen mosaic virus (dsmv) with all the three edible aroids cultivated in india. the complete full length coat protein gene of dsmv infecting elephant foot yam was cloned in pgem-t vector and sequenced. further sequence analysis revealed that the cp of dsmv consisted of nucleotides and the utr comprised of nucleotides. blast and phylogenetic analysis showed highest similarity of % with that of dsmv isolate af , reported from usa. the deduced amino acid sequence of cp had . - . % identity with other dsmv isolates. blast analysis of the partial cp gene sequences of colocasia and tannia also confirmed that the virus involved is dsmv. rt-pcr analysis of large number of samples from all the three crops confirmed that the potyvirus group specific primers (mj and mj ) are good for rapid detection of dsmv in these crops. dsmv specific biotinylated cdna and digoxigenin labelled crna probes were also prepared and dsmv in elephant foot yam was detected through nucleic acid spot hybridization. yellow leaf disease (yld) caused by sugarcane yellow leaf virus (scylv) is a recently recorded disease in india and is found wide spread throughout country. in popular varieties, the disease incidence varied from to . % and attained epidemic levels under field conditions. detailed studies on the impact of yld on sugarcane revealed that the virus infection significantly reduces various cane growth parameters, cane yield and juice quality. sequence comparisons of the coat protein (cp) and movement protein (mp) of scylv isolates from india and database sequences showed a significant variation between indian isolates and the database sequences both at nt and aa level in the cp/mp coding regions. the significant variation in our isolates with the database isolates, even in the least variable region of the scylv genome showed that the population existing in india is different from rest of the world. further, comparison of partial sequences encoding for orf and revealed that yld in sugarcane in india is caused at least by three genotypes viz., cub, ind and bra-per, of which a majority of the samples were found infected with cuban genotype (cub). the genotype ind was identified as a new genotype and this was found to have significant variation with the reported genotypes. we have identified specific primers from cp region of the virus and optimized rt-pcr conditions to diagnose the virus. this assay has been found efficient in detecting the virus in asymptomatic plants and tissue culture derived seedlings. elimination of the virus through meristem culture has been demonstrated to purify the virus from the infected planting materials and this technique needs to be adopted to supply disease-free planting materials for effective management of the disease. studies are also in progress to identify the yld-resistant sources in sugarcane germplasm to initiate breeding for yld-resistance in sugarcane. mycoviruses are viruses that infect fungi. they have been identified in all major fungal families. in the present scenario, mycoviruses are the important means of biocontrol of plant fungal pathogens. most identified fungal viruses have double stranded rna genomes, often with more than one dsrna present per virus particle, and have been spherical in shape. these viruses are mostly vesicle bound, as other viruses have protein coatings. to be a true mycovirus, they must demonstrate an ability to be transmitted-in other words be able to infect other healthy fungi through anastomosis and spores. mycoviruses lead 'secret lives', reduce the ability of their fungal hosts to cause disease in plants. this property, known as hypovirulence (hypovirulence is a term used to describe reduced virulence found in strains of pathogens), this phenomenon was first observed in cryphonectria (endothia) parasitica (chestnut blight fungus) on european castanea sativa in italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. these slower growing hypovirulent strains of c. parasitica contain a single cytoplasmic element of double-stranded rna (ds rna) similar to that found in mycoviruses that was transmitted by anastomosis in compatible strains through natural virulent populations of c. parasitica. hypovirulence has also been reported in many other fungal plant pathogens, including rhizoctonia solani, gaeumannomyces gramini var. tritici, ophiostoma ulmi, sclerotinia homoeocarpa, diaporthe ambigua alternaria alternata, and fusarium sp. etc. hypovirulence has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. it reduces the use of toxic fungicides which also affect the plant growth. the symptoms resulted by the mycoviruses are reduction in growth, reduction in pigmentation and sporulation, excessive sectoring and aerial mycelial collapse. these are the consequences of alteration in complex physiological and biochemical processes involving interaction between host and virus. cassava (manihot esculenta crantz.) is the major tuber crop in peninsular india, it is grown in an area of . lakh hectares with the annual production of . million tonnes both for direct consumption and the starch grain (sago) producing industries, mainly in the southern states of tamil nadu, kerala and andhra pradesh (fao ) . in tamil nadu, cassava primarily produced for sago producing industries where it is considered as an industrial crop rather than food crop, so the resource rich farmers are cultivating the cassava as irrigated crop in their fertile land and the poor farmers are raising the crop under rainfed conditions. in south india in addition to cassava there is a practice of intercropping important vegetable crops like, tomato, brinjal, legumes and gourds in cassava fields since all the above mentioned crops are short duration and are money spinners for the farmers. unfortunately, the major production constraint in these vegetable crops including cassava is the geminiviruses belonging to the family of in recent years there has been growing concern regarding the standard of scientific researches in india. the strengths, weaknesses, opportunities and threats (swot) analysis on indian scientific research reviewed the progress of science during the last six decades. although the 'strengths' were highlighted in good measure, it was the list of 'weaknesses' that called for attention to upgrade the standard of research and 'opportunities' that provide scope for overall scientific growth. a comparison between india and other countries in terms of research papers published revealed that india's contribution to science has come down enormously. what ails indian science? should we compare the growth of indian science with other developed countries? what criteria should be adopted to judge the quality and standard of scientific research? how to motivate the scientists to improve their scientific output? how do motivate the scientists to improve their scientific output? how do indian journals perform in maintaining quality? this paper analyses critically the scientific journals around the world, based on the scores allotted by the national academy of agriculture sciences (naas) in and for and journals respectively. in general, the indian journals performed poorly irrespective of the disciplines with only - % in the high standard. the paper dealt with the reasons for low impact factor, the anomalies in the allotment of scores to wide spectrum of the journals and the disadvantages the scientists face with the scoring system. a case study was presented of an institute with over scientists whose publications were analyzed to discuss the merits and demerits of the system. the performance of the journals published by prestigious academics, societies and councils was also projected. the paper concluded with the need for enhancing the image of the country through research publications in high standard journals and the role of various scientific bodies with shore and long term measures. poster session herpes simplex virus (hsv) keratitis is a leading cause of corneal blindness throughout the world. the infection can be diagnosed by clinical manifestations but in case of atypical ocular cases, laboratory diagnosis is more helpful in timely management of disease. collection of corneal scrapings in all cases of stromal and epithelial keratitis may not be possible, but collecting tear fluid is a convenient procedure causing less discomfort to the patients. therefore, the present study was intended to evaluate the suitability of tear specimens for detecting hsv by polymerase chain reaction (pcr) and immunofluorescence (ifa). tear fluid and corneal scrapings were collected from patients of suspected herpetic keratitis. hsv- antigen was detected by ifa using rabbit anti-hsv antibodies. pcr was performed to amplify bp region of thymidine kinase (tk) coding gene and bp region from dna polymerase coding gene of hsv. out of patients hsv antigen was detected in ( . %) of corneal scrapings and ( . %) of tear specimens and in ( . %) patients from both the specimens. hsv gene could be amplified in ( . %) of corneal scrapings and ( . %) of tear fluids and in ( . %) patients from both the specimens. although, corneal scraping seemed to be marginally superior material for detection of hsv, tear fluid may also serve as an appropriate alternative clinical specimen, due to ease of collection and least discomfort to the patients. in either cases pcr detected higher number of hsv cases than ifa. therefore if and when feasible, both ifa and pcr should be used simultaneously on each specimen to obtain best results. cytokines play a key role in the regulation of immune responses. in hepatitis c virus infection (hcv), the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect response to therapy. il- is produced by a variety of cells including t cells, phagocytes and fibroblast. cytokine genes are polymorphic at specific sites, and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines in patients with hcv infection. to correlate the serum levels and polymorphism of il- gene in chronic hepatitis c patients and healthy controls. forty patients positive for hcv rna attending the medicine out patient department and wards of lok nayak hospital, new delhi as well as forty healthy controls were enrolled for the study. the serum level of il- was detected by using elisa. genomic dna was extracted from whole blood of hcv infected patients and healthy controls by using accuprep genomic dna extraction kit according to manufacture's instruction. the genotyping of il- promoter (- variant) was carried out by pcr and direct sequencing using the method of patricia woo et al. . the serum level of il- was significantly down regulated in hcv infected chronic patients as compared to the healthy controls. genotyping of - promoter variant of il- was performed by pcr and direct sequencing. il- polymorphism in the g/g, g/c and c/c allele was non significant when compared to hcv patients and healthy controls. the il- serum levels were significant among hcv infected patients when compared to healthy controls. the polymorphism in the promoter region of il- (- ) was found nonsignificantly associated in hcv patients compared to healthy controls. in conclusion, the present study suggests that the host il- polymorphism alone may not play a significant role in the outcome of hcv infection. acute gastroenteritis (age) is a global health problem and has been associated with multiple etiological agents, which include bacteria, protozoa and viruses. viral gastroenteritis is considered as the second most common illness in children after upper respiratory tract infection. among enteric viruses, rota, noro, enteric adeno, astro and enterovirus are found to be associated with gastroenteritis. although, association of enteric viruses has been established in children hospitalized for age no such data is available from hospitalized children other than enteric infections. to determine the prevalence of enteric viruses circulating in hospitalized children. fecal samples, n = ( symptomatic and asymptomatic for age) were collected from children \ year of age from three different hospitals across the city of pune from june to feb. . detection of group a rotavirus was carried out by using antigen captured elisa. rt-pcr and pcr was carried out for the detection of norovirus, enterovirus, astrovirus and enteric adenovirus detection by using primers targeted to rdrp gene, ncr gene and consevered gene for serine protease and hexon gene respectively. out of fecal samples tested for enteric viruses in age cases, the prevalence of rota, entero, noro, enteric adeno and astrovirus were . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) respectively. however, the presence of these viruses in the asymptomatic cases (n = ) was detected at . % ( ), . % ( ), . % ( ), . % ( ) and . % ( ) levels respectively. mixed infections of enterovirus and rotavirus were found in both symptomatic . % ( ) and asymptomatic cases . % ( ). however, mixed infection of enterovirus with adenovirus were found only in asymptomatic cases . % ( ). no marked difference was observed in the seasonal pattern of all viruses in the patients with or without gastroenteritis. the findings of this study document highest circulation of rotaviruses in patients symptomatic and asymptomatic for age. the entero and noroviruses remain second most important enteric viruses in these patients. influenza in humans is a major public health concern and the understanding of its evolution in the light of its ''antigenic drift'' helps prediction of epidemics and update of yearly influenza vaccine. to antigenically characterize influenza a (h n ) isolates and study antigenic drift during to in pune city. patients with influenza like illness were identified using a strict case definition from dispensaries located in different areas in pune and clinical samples (ns/ts) were collected after obtaining informed consent. these clinical samples were processed in vivo (in fertile eggs) and in vitro ( overall, an additional ( . %) positive cases of dengue could be detected when ns antigen assay was also used in the study. highest ns antigen positivity was encountered among the samples collected on the rd day of fever whereas mac elisa for anti igm antibody was positive after th day and gradually there was an increase in the positivity towards the convalescent phase of the disease. the results of this study indicate that ns antigen based elisa test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of igm antibodies usually occur after fifth day of the infection. concurrent use of both diagnostic assays namely ns antigen as well as mac elisa will improve the overall detection of dengue infection. early detection of acute dengue virus infection is crucial to provide timely information for the management of patients. human parvovirus b , a member of the parvoviridae family, is a pathogen associated with a wide variety of diseases. most commonly, it causes childhood rash erythema infectiosum, but in some cases more serious symptoms such as persistent arthropathy, critical failures of red cell production causing transient aplastic crisis, this infection in pregnancy causes hydrops fetalis and myocarditis. traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. functional annotation would provide role of viral proteome in its survival and pathogenic mechanisms. svmprot functional family annotations of vp protein had deciphered its zincbinding, coat protein, outer membrane, chlorophyll biosynthesis, dna repair and calcium-binding nature. vp protein is having a key role in viral assembly of b virus and being non-homologous to human proteome, it was identified as an attractive molecular target for structure based drug discovery. the vp protein crystal structure was energy minimized using charmm. a structure based virtual screening method was applied using ligandfit to identify potential inhibitors of vp protein from chembank database and ten potential human parvovirus b vp inhibitors were proposed. prism genetic analyzer. the drafting of the sequences was performed using bioedit software and were submitted in genbank. for phylogenetic interpretation denv representing the full extent of genetic diversity in denv- , denv- and denv- were collected from genbank. neighbor joining algorithm was implemented with bootstrap value of , replicates for phylogenetic inference using mega . . . the genomic region to (c-prm gene junction) of denv were amplified directly from patient serum. twelve of samples were positive for dengue viral rna. of these were dengue type , was dengue type and were dengue type . for molecular epidemiological survey and genotyping of the sequences more than sequences from different geographical areas including sequences form previously reported north indian isolates were compared with our present data set. the critical analysis of the sequences revealed: dengue type sequences were clustered within sub-type of genotype iii and all the sequences of den- clustered along with genotype iii. thus, among the dengue types , and currently circulating in north india, dengue type , genotype iii, being the predominant one followed by, genotype iii of dengue type . although there is no specific treatment or vaccine available currently, the confirmative rapid diagnosis based on detection of viral nucleic acid or igm antibodies in serum, an indication of recent infection, helps in epidemiological monitoring, symptomatic treatment of patients and determining prognosis. serological detection of anti-cgv igm antibodies was performed using rapid immuno-chromatographic assay (rica) and igm-antibody capture enzyme linked immunosorbant assay (mac-elisa). eighty convalescent sera were tested by rica and of them were found positive for anti-cgv igm antibodies. twenty-five anti-cgv igm antibody rica positive sera were further assayed using mac-elisa. more sera from the patients are currently being tested to compare the sensitivity of these two serological assays in anti-cgv igm antibody based early serological diagnosis of cgv infection and the findings will be presented. thus the present study was designed to evaluate the utility of multiplex pcr (mpcr) for simultaneous and rapid detection of dengue and chikungunya viral infections. seventy-two acute phase blood samples from clinically suspected dengue cases were subjected for dengue and chikungunya uniplex pcr using dengue genus specific primers and e gene specific primers for chikungunya virus as well as multiplex pcr was developed for simultaneous detection of dengue and chikungunya infection. standard strains of dengue and chikungunya virus were used as controls. of the clinically suspected dengue samples were found to be positive for dengue viral rna by dengue uniplex pcr as well as dengue chikungunya mpcr whereas none of the samples were positive for chikungunya virus infection by both uniplex chikungunya pcr and dengue chikungunya mpcr. the result of dengue and chikungunya uniplex pcr was found to be % concordant with dengue chikungunya multiplex pcr. dengue chikungunya multiplex pcr was found to be a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples. sheetal malhotra, neelam marwaha, karan saluja, ratti ram sharma department of transfusion medicine, pgimer, chandigarh transmission through blood and blood products can be reduced to a great extent by efficient and reliable testing of the blood. the newer fourth generation elisa assays simultaneously detect antibodies against hiv- and and the presence of p antigen and thus shorten the window period to about days, as compared to days with third generation elisa. to compare the hiv seroprevalance among blood donors using fourth generation elisa (antigen-antibody) versus third generation elisa (antibody) assay. this was a prospective study involving blood donors of which were voluntary donors ( being students and being non students) and were replacement donors. sex workers are one of the core group for transmission of sti/hiv and as a ''bridge group'' to the general population. accordingly, highest priority is given to this group in targeted intervention for prevention of hiv/aids. here we are describing one such female sex worker who was harbouring concomitant sti including viral sti. a year old female sex worker was brought to the sti clinic of a tertiary care hospital by ngo with complaint of genital discharge for days. on per speculum examination, cervix was slightly erythematous, tender with mucopurulent discharge. there was no vaginal discharge or ulcer in anogenital area. however, there was a wart at lateral wall of vagina. as per naco syndromic management guideline, treatment was given for n. gonorrhoeae, c. trachomatis and hpv. cervical swab was taken and subjected to various microbiological investigation for the detection of sti viz n. gonorrhoeae, c. trachomatis, t. pallidum, candida spp., t.vaginalis, hsv- , hsv- , hiv, hbv, hcv, hpv and m. contagiosum. saline wet mount showed pus cells, but no yeast cells or trophozoite of trichomonas vaginalis. gram stained smear showed more than four polymorphonuclear leucocytes in the absence of gramnegative intracellular diplococci and a presumptive diagnosis of non gonococcal urethritis was made. no organism was isolated on any culture media after appropriate incubation. cervical swab was negative for antigen of c. trachomatis. serum was tested positive for hbv, hcv, hsv- and t. pallidum though it was seronegative for hiv. in the present case, the female sex worker was harbouring four viral sti viz hsv- , hbv, hcv and hpv alongwith t. pallidum. however clinically she was diagnosed and treated accurately only for genital wart while cervical discharge due to hsv- was misdiagnosed. it is necessary to try to test alternative approaches such as periodic presumptive therapy of viral sti, because this will not only boost up the efforts of sti control in the target group but also help in hiv control. alternatively, regular clinical and laboratory screening for viral sti may be tried. densonucleosis viruses (dnv) belong to parvoviridae family. they are the etiological agents of insect's disease known as densonucleosis, which leads to death or loss of vital functions of the infected insect. densonucleosis virus of mosquitoes has generated lot of scientific interests because of its tremendous potential in biological control and its application as a transducing vector. earlier, we have reported the isolation and characterization of a dnv from aedes aegypti mosquitoes and its prevalence among different ae. aegypti populations from india. there are reports suggesting that when aedes albopictus mosquitoes co-infected with dengue- and dnv, the multiplication of den- is suppressed. the present study focus on the effect of coinfection of ae. aegypti mosquitoes with dnv and chikungunya virus (chik). the first instar mosquito larvae were infected with dnv and the emerging dnv infected females were then infected with chikv by oral feeding. thus obtained chik infected female mosquitoes were analyzed by real time pcr for both dnv and chikv on alternate days post-infection, up to the th day. the data showed no significant difference in the multiplication of either of the viruses after co-infection. results suggest that chikv neither stimulates the replication of dnv nor is its own replication suppressed due to co-infection. this study forms an initial step in understanding the role played by such endogenous viruses on the vector dynamics. chandipura virus pathogenesis is manifested as encephalitis in young children with a very high mortality rate. this damage could be due to direct replication of the virus in brain parenchymal tissue or immune system mediated. this study aims at elucidating the role of brain infiltrating lymphocytes in pathogenesis using mice as the model system. mice were inoculated intracerebrally with the virus and the perfused brain tissue was used to isolate the lymphocytes. control mice were inoculated with an equal amount of media. in order to standardize the procedure for isolation of lymphocytes from brain tissue, splenocytes were processed to isolate the lymphocytes using histopaque density gradient method. methods to isolate lymphocytes from brain tissue as described by earlier workers were tested for the ease and efficiency of procedure using known suspension of lymphocytes from spleen. percoll density gradient method provided optimum yield of lymphocytes with an ease of handling. in this, brain cell suspension used to prepare % percoll is layered over % percoll prepared using media in : ratio. density gradient centrifugation is carried out at g for min at °c to obtain lymphocyte layer at the interface. leishman staining was performed to analyze the morphological characteristics of isolated lymphocytes. normal lymphocytes showed dark blue stained nucleus. some bigger sized cells with diffused nucleus characteristic of atypical lymphocytes were observed and some of the cells were surrounded by hair like structures. phenotypic characterization was carried out using flow cytometry. the presence of cd + , cd + and cd + cells was observed. the percentages of cd + , cd + and cd + cells was found to be . %, . % and . % respectively in the lymphocytes isolated from infected animal and . %, . % and . % respectively from control animal. hence, cd + cells showed maximum infiltration after infection. (santosh et al. ; pradeep et al. ). in the present study chikv suspected blood samples were collected and the acute phase samples were subjected to rt-pcr for the presence of virus specific rna by using the primer pair dvrchk-f/dvrchk-r as described by us earlier (naresh kumar et al. ). the convalescent phase samples were screened for chikv specific antibodies by using sd bioline chikungunya igm rapid test. six sets of primers were designed to amplify the complete nsp and complete structural genes of chikungunya virus. the products were further gel purified, cloned in ptz r/t vector and the recombinant clones were sequenced and submitted to the genbank. the complete ns gene and structural genes were compared with other available sequences in the genbank. sequence analysis results will be presented. the present study discusses these aspects in detail. . some of these phages (viz. v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. the four major routes of transmission are unsafe sex, contaminated needles, transmission from an infected mother to her baby at birth (vertical transmission) and breast milk. screening of blood products for hiv has largely eliminated transmission through blood transfusions or infected blood products in the developed world. in , globally, about million people died of aids, . million were living with hiv and . million people were newly infected with the virus. hiv infections and aids deaths are unevenly distributed geographically and the nature of the epidemics vary by region. more than % of people with hiv are living in the developing world. there is growing recognition that the virus does not discriminate by age, race, gender, ethnicity, socioeconomic status-everyone is susceptible. however, certain groups are at particular risk of hiv, including men who have sex with men (msm), injecting drug users (idus), and commercial sex workers (csws). the present study indicates the prevalence of hiv infection among the people residing in the northern region of india predominantly among the foothills of the himalayas. the study was carried out on the patients visiting herbertpur christian hospital (a unit of emmanuel hospital association) under the integrated counselling and testing centre scheme at the respective hospital during the - . the study indicates the screening of people groups residing in the respective area through community health schemes. the diagnosis of the hiv infection is done by three types of assays namely. the tridot method which is the rapid method of diagnosis followed by the. hiv coombs test which involves the dot immunoassay principle. the third assay is the enzyme linked immunosorbent assay (elisa). the number of patients screened during the period of september to march is which include patients coming from four different states namely haryana uttarakhand uttarpradesh and himachal pradesh. the number of people who were tested positive are and the number of people who were tested negative are . the people tested positive are sent to the higher centre for other confirmatory tests such as pcr and western blot analysis. these patients are sent for treatment and prophylaxis at a respective recognised centre in dehradun. the present study determines a consistent community hiv screening and treatment approach through diagnostics counselling and awareness programmes. classical swine fever (csf) also known as hog cholera is a highly contagious and fatal disease of swine. csf became rapidly a major issue of pig industries. it still causes important economical losses worldwide. it is considered as a major health problem of swines in india. during the month of august to october there was an outbreak of classical swine fever in bihar. from three districts darbhanga, patna and supol, total numbers of different infected tissue samples like kidney, spleen and lymphnode were collected from the dead morbid/pigs. total rna was isolated from % homogenate of infected tissues in sterile pbs by tri-reagent (sigma, usa) according to the manufacturer's instructions and cdna was prepared by using commercial available kit. the cdna was stored frozen at - °c until used. for the molecular detection of classical swine fever virus specific nested pcr amplification of e and ntr was done along with ns b and e rns amplification. primarily these samples were found positive with these primers. further confirmation by sequencing was done by cloning of these pcr products in pgem-t easy vector. e and ntr sequences were considered for phylogentic analysis along with complete available sequences of csfv. nucleotide sequence alignments were carried out using the clustalw program (dnastar) and phylogenetic tree analysis (dnastar) showed that ntr have close proximity with taiwan strain (accession no. ay ) and e shows close proximity with chinese isolate csfv- (accession no. af ). peste des petits ruminants (ppr) and sheeppox are oie notifiable diseases of small ruminants especially sheep and goat. both the diseases are economically important, in enzootic countries like india and are major constraints in the productivity of animals. considering the geographical distribution of both ppr and sheep pox infections and prevalence of mixed infection, in the present study, safety and potency of the experimental duel vaccine comprising attenuated strains of thermostable-ppr virus (pprv-revati, p- ) grown at °c and attenuated sheep poxvirus (sppv-srinagar, p ) was evaluated in local non-descript sheep. experimental animals were grouped into four groups and each group was comprising six animals, received doses ( tcid ), dose ( tcid ) and / th dose of vaccines and normal saline as control in ml volume subcutaneously, respectively. serum samples were collected on , , , and th day post vaccination. sheep simultaneously immunized with ml of vaccine consisting of either or doses of each of pprv and sppv were monitored for clinical and serological responses for a period of - weeks post-immunization (pi) and post challenge (pc). specific immune responses i.e., antibodies directed to both pprv and sppv could be demonstrated by ppr competitive elisa kit and capripox indirect elisa, respectively following immunization. all the immunized animals' resisted infection when challenged with virulent strain of sppv (srinagar isolate at p- ) on day dpi, while in contact control animals developed characteristic signs of sheeppox. the challenge of the sheep against ppr was not carried out, however, the antibody titre after immunization determined by snt and elisa, indicated that protective titre, as per earlier report on the goats. dual vaccine was found safe at higher dose and induced protective immune response even at lower dose ( tcid ) in sheep, which was evident from sero-conversion as well as challenge study with sppv. the study indicated that both the viruses are compatible and did not interfere with each other in eliciting immune response, paving the feasibility of use of this dual vaccine in combating both infections simultaneously. goatpox is one of the highly contagious, oie notifiable and economically important viral diseases of goats. the disease is caused by goatpox virus (gtpv) is classified of the genus capripoxvirus in the family poxviridae. the disease incurs severe economic losses in terms of high morbidity in adults and heavy mortality in young kids and is a major constraint in goat farming in india. considering the enzotic nature and economic impact of the disease, it is all important to control the infection by developing an effective vaccine. recently, vero cell based a live attenuated goat pox vaccine; using gtpv uttarkashi isolate (p ) has been developed in authors' laboratory and evaluated in goats. the vaccine was found safe, potent and immunogenic experimentally and even at field trials. the vaccine has been evaluated at large-scale at different regions of the country and found suitable for mass vaccination. however, the longevity of potency was not evaluated. therefore, a long term potency trials were studied for a period of years with annual challenge by using virulent goatpox virus and sero-monitoring. a sufficient number of hill goats has been vaccinated with dose of vaccine ( . tcid /ml) and monitored for clinical and serological response. every year, significant number of vaccinated (n = ) and control animals (n = ) were used for challenge with virulent strain ( . srd /ml, gtpv mukteswar). sera of pre-and post-challenged ( dpc) animals including controls have been collected and monitored for serological response in the form of specific antibody production by snt and indirect elisa. all the vaccinated animals were protected on challenge, whereas, all unvaccinated controls developed infections. the same has been reflected in sero monitoring of collected sera. so the developed live attenuated goat pox vaccine was found safe, immunogenic and potent for a period of years of immunization and suitable for mass scale vaccination in control and eradication of goat pox along with a/are suitable diagnostic tool/s in goatpox enzootic country like india. rotavirus infection in avian species varies from subclinical infections to outbreaks of diarrhea. the economic significance of rotaviral enteritis to the poultry industry has not yet been defined, but by analogy to the situation in mammals, it is likely to be significant. unlike the extensive studies performed on rotavirus infection in humans and animals, limited studies have been carried out to determine the extent of exposure of poultry birds to rotaviruses. to determine the prevalence of avian rotavirus antibodies in commercial broiler chickens. a total of chicken serum samples were collected from the lairage of a poultry slaughter house where birds from four different broiler farms in and around pune city were supplied to. the serum samples were tested by an igg antibody capture elisa wherein purified chicken rotavirus ch was used as coating antigen. sera from specific pathogen free (spf) chick (n = ) served as negative control in the test. cut off was calculated as mean negative control ? sd (standard deviation). s/co (mean sample od /cut off) values above ( . - . ) in % ( / ) serum samples were indicating positivity to rotavirus antibodies. the result of the study indicates exposure of the birds to avian rotavirus or similar agent that is circulating in pune city. bluetongue has become established in south india causing regular outbreaks in sheep. btv serotypes , , and were isolated from native sheep of andhra pradesh. the other serotypes circulating in the state need to be identified. however the major constraint is the serotype identification. to overcome the difficulties of traditional serotyping methods (neutralization tests), nucleic acid based tests are being tried. rt-pcr for serotyping was standardized using primers specific to vp gene of btv- , and serotypes. rt-pcr resulted in bp product of btv- , bp product of btv- which was defined by specific primers. however non specific amplification at two different sites i.e. bp and bp was noticed for btv- . specificity of rt-pcr was evaluated. btv- and btv- specific primers could amplify only btv- and btv- respectively where as btv- type specific primers amplified not only btv- but also btv- and btv- . nucleic acid sequence data obtained from btv- pcr product and btv- cloned products were specific to vp gene of btv- and btv- respectively. however, and bp products of btv- were identical to vp gene of btv- , , , , and and vp gene of btv- , and respectively, indicating the non specific amplification of btv- . foot and mouth disease is the most contagions and highly economically impotent disease of cloven footed animals. the disease is controlled by regular vaccination using the vaccine produced from the virus grown in the cell culture. the vaccine strain used for vaccine production is selected from the field isolates based on the adaptability and growth kinetics in bhk cells and antigen coverage. however the field viruses need to be passaged several times to adapt in tissue culture. passage of field viruses in tissue culture may results in development of mutants whose genetic makeup may differ from the field samples also some of the field strains may fail to adapt or may grow poorly in the tissue culture, thus the efficiency of the vaccine gets affected. structural proteins of fmdv carry the sequences which determine the serotype specificity and immunogenicity. thus one may replace the gene coding for structural proteins from the full length cdna copy of the vaccine strain that has been adapted to the tissue culture with the poly-structural protein gene (pi) so that the chimeric virus gets the serotype specificity of the field strain besides retaining the other characteristics that are needed for a vaccine virus. we have made replication competent fmdv asia i full length genome and cloned under t and cmv promoter separately in plasmid vectors. bam h sites were created for inserting pi- a gene of other field strains. the p - a of type 'o' vaccine strain was amplified directly from the cattle tongue material, cloned in plasmid vector and studied the specificity by sequence analysis and gene expression. we have introduced 'o' p - a gene into the full length construct devoid of asia structural protein gene, p - a. the in vitro transcribed rna in case of t promotered construct and plasmid dna in case of cmv promotered construct were transfected into the bhk cells. after the passaging the virus obtained was studied for the speciality. this approach may be used not only for rapid selection of vaccine strain and also as a repository of the cdna copy of the virus. the p is composed of a, b, c and d (vp , vp , vp , and vp ) respectively of which the vp is the most immunogenic and subunit vaccine produced with vp alone was able to induce high level of neutralising antibodies. thus to control the disease in india polyvalent vaccine consisting of the inactivated virus of all the three serotypes are in use. however the conventional vaccines have several drawbacks which include safety and temperature sensitivity. hence alternatively sub-unit vaccines consisting of vp protein has been tried. however this showed limited success due to the antigenic variations occurring in the field viruses thus escaping the neutralization from the antibodies generated from single cloned protein. hence the present study was undertaken with an objective to include all the neutralizing epitopes present in the three serotypes by linking vp ( d) genes and produce a poly valent protein for using as poly subunit vaccine. in this study we have constructed a cassette by linking the genes of three serotypes 'o' ( bp), 'a' ( bp) and 'asia ' ( bp). these genes were cloned individually in commercially pbsk vector and confirmed by sequence analysis before linking in pc dna vector. the linked gene construct was sub-cloned in pet expression vector. the expression of the protein gene from the pet vector was induced with iptg and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). a fusion protein of size kda was observed in page gels. since the protein contains his residues from the vector at the n-terminal end, affinity purification was carried out using nickel nitrilo-tri-acetic-acid (ni-nta) agarose matrix. the immunoreactivity of the purified protein was assayed by western blot with the anti fmdv type 'o' and 'asia ' specific sera. the may be used as a subunit vaccine. silkworm diseases caused by viruses, bacteria, fungi and protozoans form major constraints for the silk cocoon production in all the sericultural countries and among these silkworm viral diseases viz., nuclear polyhedrosis and infectious flacherie caused by bmnpv and bmifv cause severe crop loss. the traditional disease management strategies include prophylactic measures and use of disease free silkworm eggs. the prophylactic measures such as disinfection of silkworm rearing house and appliances, egg surface, silkworm bed disinfection and rearing surroundings. the disinfectants used presently in sericulture are either formaldehyde or chlorine based products, but these chemicals are neither eco-nor user-friendly. the awareness about health hazards caused by formaldehyde and environmental pollution caused by cl necessitated the development of eco-and user-friendly disinfectant products for use in sericulture. these include alternative disinfectant products developed using biodegradable chemicals and plant based ingredients by apssrdi, hindupur and central silk board for the management of silkworm diseases in india. the ideal disinfectant for sericulture would be the one which can inactivate silkworm pathogens of diverse origin and economical for sericulture. the paper discusses on the disadvantages of hcho and cl based disinfectants and advantages of eco-and user-friendly disinfectant for the management of silkworm diseases especially the ones caused by viruses. the baculovirus expression vector system (bevs) is widely used for the production of high levels of properly post-translationally modified, biologically active and functional recombinant proteins and has facilitated basic biomedical research on protein structure, function, drug discovery and the roles of various proteins in disease. bevs is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. the resulting recombinant baculovirus lacks one of nonessential gene (polh, v-cath, chia etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. insect cell-bev system is widely used to produce recombinant proteins. bevs also eliminates concerns regarding pathogens that could potentially be transmitted to humans as it is non-infectious to vertebral animals. these features make silkworm system an ideal expression and delivery package for producing proteins of medicinal importance. the efficiency, low cost and large-scale production of proteins using bevs represents breakthrough technology that is facilitating highthroughput proteomic studies. the bevs has become a core technology for cloning and expression of genes for study of protein structure, processing and function; production of biochemical reagents; study of regulation of gene expression; commercial exploration, development and production of vaccines, therapeutics and diagnostics; drug discovery research; exploration and development of safer, more selective and environmentally compatible biopesticides. utilization of silkworm larvae and pupae as bioreactor with recombinant bmnpv producing foreign proteins extends the usages of silkworms. due to its large-size and high protein synthesis ability as well as the expediency in mass culture, silkworm is considered as good candidate for producing recombinant proteins. wssbv is the causative agent of a disease, which has recently caused high shrimp mortalities and severe damage to shrimp culture. wssbv has been found across different penaeid shrimp species. in order to develop a effective diagnostic tool, a wssbv genomic library was constructed by cloning wssbv genomic dna extracted from purified virions. in the present study wssv disease free (confirmed by pcr analysis) were collected from hatcheries from different areas of guntur and prakasam districts and analysed to study the effect of various physical parameters like temperature, p h , salinity and turbidity on the prevalence of above disease. the studies on the surface water temperature revealed fluctuations in the ponds ranging between to . °c in diseased ponds and . to . °c in healthy ponds. these results show definite influence of temperature on the prevalence of wssv. present day strategy in vaccine development is to include marker facility that helps in distinguishing antibody response due to vaccination vis-à-vis infection in vaccinated animals. such information becomes relevant for effective disease control programmes especially when using inactivated virus vaccines like foot and mouth disease (fmd). the antibodies generated in the animals, only through vaccination, is the measure of vaccine efficacy and safety. presently inactivated fmd virus (fmdv) vaccines are used to control the disease in the endemic countries like india. the quality assurance of the vaccine depends on the efficacy of the vaccine in generating protective antibody without causing subclinical disease due to improper inactivation. since protective antibody response in vaccinated animals can not be distinguished from that of infected animals one needs to assay the antibody response against non structural proteins (nsps) and the vaccine must be free of contaminated nsps. production of vaccine free of nsps requires the cumbersome method of virus purification which adds to the cost of the vaccine. alternatively one may develop a positive marker vaccine by including a foreign protein or epitope which is not expected to be present in the vaccine and the antibodies generated against which helps in detecting the vaccine related response. here we report a molecular approach by which we introduced a immuno-dominant epitope of green fluorescent protein (gfp) into the structural protein gene of foot and mouth disease virus vaccine strain asia ( / ). our laboratory has produced a mini-genome of fmdv asia that lacks structural protein gene (p - a) coding for all the structural proteins (vp - ) of fmdv asia as a vector (pcfl dasia ). the p - a of the asia vaccine strain was cloned separately into a plasmid vector and by successive pcr mutagenesis and cloning we have introduced nucleotide sequence corresponding to amino acid epitope of gfp into p - a gene. gfp epitope was inserted by replacement at n-terminal region of vp- which is not immunogenic. the modified p - a was expressed in e. coli and studied. the modified p - a gene with gfp epitope was inserted into the pcfl dasia to get full length replication competent cdna cloned under cmv promoter in pcdna (pcflasiagfp). this can be used to produce synthetic virus with gfp epitope that can generate antibodies not only against neutralizing epitopes but also against gfp epitope. presence of antibody against gfp epitope in the vaccinated animal will reveal vaccine efficacy. elisa against gfp can be used as a companion test not only for safety evaluation but also for quick evaluation of efficacy. further absence of nsp antibodies in the serum may reveal the quality of the vaccine in respect of safety. self replicating dna vaccines are developed to achieve robust immune response through enhanced antigen production and gamma interferon expression in vaccinated animals. since self replicating dna vaccines induce gamma interferon expression which helps in viral clearance such vaccines are expected to be useful to cure even the carrier and persistently infected animals. understanding the events that help in the elicitation of both the arms of immune response in vaccinated animals is necessary to understand the effectiveness of the vaccine. the work presented here deals with the immunological evaluation of a sindbis virus replicase based dna vaccine carrying linked fmdv vp genes in vaccinated guinea pigs. we have constructed self replicating dna vaccine vector and to the down stream of a sub genomic promoter we have inserted secretory signal followed by linked-vp genes of fmdv serotypes (o-a-asia ) with glycine and proline bridge in between. guinea pigs were vaccinated with the construct and the sera at days post vaccination were evaluated both for cellular response by studying the cd levels and by mtt and cytokine profiles by real time assays. the humoral response was evaluated by studying cd levels in the whole blood by facs analysis and serum antibody levels by snt and elisa. the animals were challenged with gp infective dose of fmdv type 'o' virus lesions were scored. further the replicative efficiency of the challenge virus was studied by ab elisa. the results showed that all the assays except antibodies against ab protein have positive correlation with the protection. as expected the titre of the antibodies against ab protein was lower indicating that the challenge virus replication was inhibited in the vaccinated animals. the limited studies conducted by us showed that self replicating vaccine has a potentiality to emerge as potent vaccine for fmd. ganjam virus (ganjv) belongs to the genus nairoviruses (family bunyaviridae). these viruses cause diseases in livestock. it has been isolated from different animal hosts and tick vectors from india. genus nairoviruses includes a total of tick-borne viruses, classified into serogroups. the important serogroups are crimean congo hemorrhagic fever (cchf) and the nairobi sheep disease (nsd). the main members of the nsd group are nsd and dubge viruses. their genome consists of three segments of single stranded rna, viz. s, m and l that encodes viral nucleocapsid protein, viral glycoprotein g and g and the viral polymerase respectively. ganjv is very closely related to (nsdv). nsdv is found in east and central africa, causes very high morbidity and mortality in livestock. the present study involves phylogenetic comparison of ganjv isolates from india with other nairoviruses based on complete n gene. it will help to understand the kind of nucleotide (nt) and amino acid (aa) changes that have occurred in ganjv strains from different geographical areas. eight strains of ganjv isolated at niv during - from different parts of india were used in this study. virus stocks were prepared in vero e cell line these were used as the source of viral rna. the n gene was amplified either as a complete gene in one reaction or in fragments whenever necessary. thus obtained sequences were analyzed; annotated to get a consensus sequence, aligned against the sequence of prototype strain of ganjv and other representative nairoviruses. the nt sequences were converted to aa sequences and analysis was done at both nucleotide and amino acid levels. based on what nt or aa phylogenetic tree was constructed and compared with other nairoviruses (cchf, dugv, hazv, kupv and nsdv) where complete s segment sequences were available gen-bank database (ncbi). the phylogenetic data at both the nt and aa levels showed that all the strains of ganjv form monophyletic lineage with the nsdv. cchfv and hazv together formed another clade, whereas dugv and kupv made a separate branch in the tree. the different ganjv strains showed - % difference with nsdv at the nucleotide level and - % difference at the amino acid level. hazv showed - % difference at the nt level and % difference at the aa level with ganjv as well as nsdv. the present data obtained suggests that ganjv and nsdv are minor variants of the same virus. diarrhoeal syndrome is one of the major concerns of the livestock industry. most of the diarrheic cases in animals go unnoticed and limited attention is paid on viral etiology. presence of large amount of fecal matter in animal shed acts as a source of infection for calves via drinking water, feed, or contaminated soil. keeping this in view, investigation was planned to detect the association of rotaviruses with diarrhea in dairy calves and to observe the genomic diversity among the circulating viruses in tarai area of uttarakhand. a total of diarrheic fecal samples collected from instructional dairy farm, nagla, pantnagar, uttarakhand were screened during the study. samples were collected from both cow calves and buffalo calves in - months of age. for the diagnosis of rotavirus, all the fecal samples were subjected to rna-electrophoresis after nucleic acid extraction. viral genome segments were visualized by silver staining. out of the total samples tested, seven were found positive in rna-page showing typical genome segments migration pattern of bovine rotavirus. in the given samples prevalence of bovine rotavirus was . % and % in cow and buffalo calves, respectively. on the basis of migration patterns of rotavirus in rna-page, group a were identified with typical : : : pattern. variation within movement of various genome segments among isolates of bovine rotaviruses was observed during the study that may be indicative of emergence of mutants in the circulating isolates. the vp gene based group a specific rt-pcr was standardized and all the isolates in this area were confirmed to be of group a type. work is in progress to genotype the bovine rotaviruses of this region based on vp and vp genes. this study emphasizes the need to explore the prevalence of bovine group a rotaviruses in different places of uttarakhand and their genetic characterization which could help in selection of control strategies for rotavirus infections. foot-and-mouth disease (fmd) is endemic in india causing enormous economic loss to the animal keepers and trade embargo with fmd free countries in livestock and animal products. rapid diagnosis of fmd is of immense importance in prevention and control of the disease. fmd is initially diagnosed clinically and confirmed by laboratory tests. virus isolation in cell culture and sandwich elisa for antigen detection are commonly practiced in laboratories. the virus isolation though is very sensitive but it can be slow and analytical sensitivity of the elisa is lower and can not be used with certain sample types. the use of molecular techniques in the diagnostic laboratory has greatly increased the speed, specificity and sensitivity of fmd diagnostic tests. molecular techniques like rt-pcr, pcr-elsa and dot hybridization can be used with more success for detecting carrier animals and animals harboring sub-clinical infection and can be applied in a wide range of clinical sample types. these techniques can be used as genus and serotype specific test including detection of particular lineage/genotypes with in the serotype. multiplex pcr has been used to differentiate serotypes of fmdv and the technique is sensitive, experimentally simpler, cost effective and less time consuming. the assay can be used for serotyping on elisa negative samples. the molecular techniques not only help in diagnosis but also useful for epidemiological studies. lineage differentiating rt-pcr has been useful in identifying different lineages of serotype asia (lineage b, c and d) before proceeding with sequencing of d region. similarly genotype differentiating rt-pcr has been developed and used in differentiating two different genotypes of serotype a (genotype vi and vii). these assays have the potential to be applied on clinical samples directly, thereby saving much time needed for sample processing and nucleotide sequencing. recent development of real time rt-pcr methodology has allowed the diagnostic potential of molecular assays to be realised. advancement in real time pcr technology made it possible to combine several assays within a single tube which is in the progress in our laboratory. integration of these assays onto automated high throughput platforms provides diagnostic laboratories with the capability to test large numbers of samples. microarray technology was provided greater screening capabilities for pathogen detection. the microarray allows the addition of large number of oligonucleotide probes for identification of mutant pathogen and also for subtype determination. the combined properties of high sensitivity and specificity, low contamination risk, and speed has made realtime pcr and microarray technology a highly attractive alternative to conventional methods in increasing percentage of outbreaks confirmed and analyzed and for tracing the origin of fmd virus responsible for outbreaks. dna vaccines are expected to elicit both humoral and cellular responses, cellular response being long lasting. however the approach has several limitations like poor stability of dna, poor expression and risk of integration. poor expression becomes the major limitation in the case of fmd as fmdv proteins are poor immunogens. also dna vaccine vectors carrying only eukaryotic promoters elicit strong cmi response and weak humoral response. the methodology to achieve humoral response involves the expression and secretion of the expressed protein so that the antigen presenting cells will be able to process the antigen and produce humoral response. in case of fmd humoral response is as important as cellular response. the present project aims at addressing these issues; achieving higher expression and getting the protein secreted out by constructing self replicating gene vaccines for fmd and studying their efficacy. the vector for humoral immune response contains eef promoter, sindbis virus polymerase gene and secretory and anchoring signals. the integrity of the vectors was confirmed by sequence analysis. the linked polyvalent protein genes of fmdv serotype a, o and asia were cloned into the vectors and the presence of the insert was confirmed by restriction enzyme digestion. the functionality of the constructed dna vaccine vector (pvac self rep ) was assayed by transfecting the dna into bhk cell monolayer and studying the s labeled proteins in immuno-precipitation assays. the studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein. the secretion of the expressed protein was assayed by immuno-fluorescence assay and found to be positive. encouraged with these studies the preliminary studies were conducted on vaccine efficacy studies in guinea pig model. the immunized guinea pigs showed high antibody titres by snt and elisa, as compared to conventional dna vaccines (pup cd) even at / th of the dose. this approach of constructing self replicating dna vaccine for humoral response is the first report. genetically engineered microorganisms are important sources of industrial and medicinal proteins. over the past decade, plant host system has been investigated as potential host system for expressing proteins of therapeutic and diagnostic use. however concerns regarding the stability and environmental safety need to be addressed. chloroplast engineering is expected to resolve some of these issues since, plastids/chloroplasts are inherited maternally and are not disseminated through pollen. this makes plastid transformation a valuable tool for transgenic creation besides offering biological containment. since foot and mouth disease (fmd) of cloven footed animals is a major concern in the world over. foot and mouth disease (fmd) is the most feared, viral disease of the cloven footed animals causing heavy losses to the live stock industry. the disease is enzootic in many parts of the world including asia. the conventional vaccines for fmd have several limitations which include safety, temperature sensitivity and duration of immunity. attempts have been made to overcome these limitations using recombinant dna technology. amongst the newer vaccines, edible vaccines are cost effective and easy to administer. since the stability of the gene of interest is the major concern in the case of plant transgenics, marker genes are used for regular selection. the detection methods based on the available marker proteins like b glucoronidase (gus) protein/antibiotic selection are cumbersome and cost intensive. however selection based on herbicide resistance is much simpler and easy. hence in the present study, the -enolpyruvylshikimate- -phosphate synthase (epsp) gene was used as a marker along with the immunogen gene of fmdv. epsp is the key enzyme in the shikimate biosynthesis pathway necessary for the aromatic amino acids production. in order to investigate the mechanism of long term immunity and the effect of protective immunity induced by cationic plg micro particle coated dna vaccination. we constructed the expression plasmid containing a foot-and-mouth disease virus (fmdv) id gene sero type a. intramuscular vaccination of guinea pigs with the micro particles coated plasmid dna induced a strong antibody response and neutralization antibodies, cellular mediated immune response which lasted year. we further analyzed the persistence and expression of id gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction and quantitative pcr. the results showed that id gene was present and expressed in the muscle cells up to year after days post vaccination. furthermore, guinea pigs vaccinated with micro particles coated plasmid dna were protected against a challenge with fmdv virus. therefore the micro particles coated plasmid dna vaccination dose induce a protective immunity and long term humoral, cellular immuno responses against fmdv, which could be maintained by persistent expression of id gene in muscle cells. foot and mouth disease virus (fmdv) causes a highly contagious viral disease of cloven hoofed animals, which has a considerable socioeconomic impact on the countries affected. interleukin- (il- ) enhances the il- driven th immune response that is important in immunity against intracellular pathogens. the multiple roles of il- in many physiological and pathological processes have generated a great deal of interest in recent years. antiviral effects of il- have been reported. we evaluated the effects interleukin- (il- ) on the replication of fmdv in vitro in bhk- cells. bovine il- mature protein coding sequence was amplified from the bovine pbmc cells and cloned into prokaryotic expression vector pet a. protein expressed was purified and specificity was confirmed by immunoblotting. bhk- cells were treated with purified expressed il- protein with ( lg/ml) h prior to fmd infection. cell culture supernatants were collected at h post infection were subjected for elisa and virus titration assay. rna extracted from the cells was subjected to real time pcr for viral rna quantification. log titer reduction was observed in the fmd virus titer in il- treated cells compared to the untreated cells where as virus antigen quantified by elisa has shown a reduction of -folds. -fold reduction in the fmd viral rna copy number was observed in the il- treated cell compared to the untreated measured by qpcr. current study demonstrated the potent anti viral activity of il- on fmdv by inhibiting the viral replication. these results further suggests that il- has the potential role of il- as molecular adjuvant in fmd vaccine development and development of therapeutic for fmd. foot and mouth disease is the most contagious viral disease of farm animals. control of the disease in animals is by vaccination and slaughtering of infected animals. conventional oil adjuvant vaccine has its own limitation. alternate to this genetic vaccines where the dna encoding viral antigen may be a promising approach. naked dna vaccine has limitations like poor uptake of dna by cells and more importantly by nucleus. as a result delivery of naked dna through calcium phosphate nanoparticle was attempted. calcium phosphate nanoparticle is a potential delivery agent which proved to enhance the immune response. fmdv p - cd ''o'' vaccine gene constructs in pcdna . ? entrapped by the nanoparticles was prepared by using different molarity of calcium chloride and disodium hydrogen orthophosphate. the nanoparticles entrapping fmdv p - cd ''o'' and naked dna were presented to the guinea pigs through intramuscular injection to study the mrna expression of antigen by rt-pcr. animals were sacrificed at defined time to collect different organs and total rna was extracted. each time blood was collected to analyse the fmdv specific serum antibodies. dna vaccines presented through calcium phosphate produced transcripts in the injected muscle up to days whereas naked dna up to days. serum antibody levels of naked dna vaccine showed antibody titre till days. whereas nanoparticle injected animals showed serum antibody till days. serum neutralization titres of . were observed in calcium phosphate dna vaccines at about - days, where as naked dna sn titers were observed for short period of - days. the study clearly showed calcium phosphate nanoparticle entrapping fmdv vaccine dna may be a better delivery system for dna vaccines as it confirms availability of the antigen and persistence of antibody for longer duration than naked dna. capripox is highly infectious, contagious, and oie notifiable disease of small ruminants, caused by sheeppox and goatpox viruses which are members of capripoxvirus genus of the family poxviridae. in the present study, we analyzed the partial gene sequences of p protein, an immunogenic envelope protein of capripox viruses (capv) to assess the genetic relationship among different sheep pox and goat pox virus isolates from different geographical areas of the country. product of this gene has been shown to be important in attachment of capv to host cell surface receptors during viral entry and host immune response. the following virus isolates have been used in the analysis: gtpv-uttarkashi, p , vaccine virus; gtpv mukteswar, p , challenge virus; gtpv (akola), gtpv bareilly/ , gtpv ladakh/ and gtpv sambalpur/ , field isolates and sppv srinagar, p ; sppv ranipet, p ; sppv-rf, p , vaccine viruses and sppv makdhoom/ , sppv cirg/ , sppv pune/ , sppv bareilly, sppv / and sppv / , field isolates. in this study, all virus isolates were confirmed by pcr amplification and analysed in pcr-restriction fragment length polymorphism (pcr-rflp) using ecori enzyme to confirm their specificity. further, the amplicons were cloned and sequenced commercially. nucleotide and the deduced amino acid (aa) sequences were compared with published sequences of the members of the genus capripox virus. sequence analysis of partial bp sequence has shown high sequence identity among all indian sppv and gtpv isolates at both nt and aa levels. it revealed a . - % and . for gtpv field isolates where as, % for sppv field isolates at both the nt and aa levels. in general, capv isolates in this study shown . - . and . % homology between gtpv and sppv at nt and aa levels as reported earlier. further, it revealed a unique change of g a in all gtpv isolates resulting in formation of drai site in place of ecori and possible development of restriction enzyme specific pcr-rflp for differentiation of sppv and gtpv from field isolates. orf or contagious ecthyma is considered as non-contagious, proliferative disease and is caused by orf virus of the genus parapox virus of the family poxviridae. it is reported most commonly in sheep and goats and also a zoonotic agent. camels are also infected by orf virus and reported in camel rearing countries as a mixed infection with camel pox, the later is caused by an orthopox virus. in india, there are few reports of the orf virus infection in camels and identified by clinical signs and pcr. in this study, we identified the presence of orf virus from clinical samples of suspected case of sporadic infection in camels by serological and molecular techniques. viral dna isolated from processed scabs used initially in nested polymerase chain reaction as diagnostic pcr which successfully amplified bp fragments and also sequenced to check the fidelity of the product. after confirming the infection by pcr, some of the structural and non-structural genes were amplified for sequence analysis. out of the five genes characterized, the major important one selected for sequence and phylogenetic analysis is b l gene which is homologous to a major envelope protein p k of vaccinia virus. full open reading frame of bp from orf b l was amplified by pcr, cloned and sequenced commercially. nucleotide and deduced amino acid sequences of b l were compared with other published sequences of the members of the genus papapox virus. sequence analysis shows a maximum percent identity of . and (indian orf virus isolates); . and . (other orf isolates); . and . (orf-camel/jodhpur/ ); and . (bovine popular stomatitis virus) and finally . and . (pseudo cowpox virus) respectively at nt and aa levels. phylogenetic analysis of the isolate was also performed using the neighbour joining method in mega program to know the phylogeny relatedness of the virus, which revealed that the isolate is well grouped with the jodhpur isolate and closely related to pseudo cowpox virus. it warrants further analysis of other potential genes to confirm the causative agent of the contagious ecthyma in camels as pseudo cowpox virus. chikungunya an arboviral disease is transmitted through the bite of an infected aedes mosquito. it causes a self limited febrile illness along with arthralgia and myalgia. in some cases neurological and severe hemorrhagic manifestations has been observed. chikv epidemic has been reported in africa, india, south east asian countries and during the current out break imported cases of chikv has been encountered in most of the european countries. the causative agent belongs to the genus alphavirus family togaviridae. human beings serve as the chikungunya virus reservoir host during epidemic periods. outside these periods the main reservoirs are monkeys, rodents, birds, and other unidentified vertebrates. antibodies to chikv have been detected in domestic animals. in the present study we surveyed madanapalli, palamaner, b. kotta kota and tirupati and collected a total of rodent samples, bovine samples; sheep samples and canine samples. total rna was isolated from all these samples and subjected to rt-pcr using a primer pair dvrchk-f/dvrchk-r which could amplify a bp e gene product specific to chikungunya virus (naresh kumar et al. ). all the serum samples were further screened for chikv specific igm antibodies using commercially available ctk biotech strips. none of the samples were found positive either for chikv specific rna or chikv specific igm antibodies. more number of samples from domestic animals as well as rodents are being screened to study their possible role if any in the maintenance of chikv in nature and during the inter epidemic periods. the present study discusses these aspects in detail. petunia hybrida is widely used as experimental host plant for begomovirus identification and its characterization. hitherto, natural infection of begomovirus on petunia has not been reported in india. recently, petunia hybrida grown in and around ludhiana were found to be depicting typical symptoms caused by begomovirus. the symptoms include severe reduction in leaf size, downward curling and distorted leaves. severely infected plant became bushy, stunted and produces no flower. total genomic dna was extracted from the plants showing symptoms of begomovirus, by ctab method. the presence of virus was confirmed by using degenerated primers, designed to identify all the begomovirus prevailing in the world. to identify the strain associated with the disease, the positive samples along with healthy control were tested against different strain specific primers of tomato leaf curl virus, so far reported in india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus. among these, only tomato leaf curl new delhi virus specific primer was able to give the desired amplicon of * bp. hence, it is confirmed that the leaf curl disease of petunia hybrida is associated with tomato leaf curl new delhi virus. this disease of petunia can become a sever production constraint in coming years. from last years ( and ) it was observed that some varieties of brinjal grown in rainy season, showed typical leaf curl type of symptoms. the symptoms include upward curling of the leaves, cupping, vein thickening, reduction in leaf size and distortion of leaves. the severely infected plant remains stunted and bushy, became unproductive or produces only few fruits. the disease was experimentally transmitted from naturally infected brinjal to healthy seedlings by whiteflies (bemisia tabaci) and grafting, but not by mechanical or aphid transmission. to detect the begomovirus associated, total genomic dna was extracted from the plants showing disease symptoms. the presence of virus was confirmed by using pcr based begomovirus geneus-specific primers designed by deng et al., wyatt and brown and rojas et al. these degenerated primers give the expected product size of * , * and * bp, respectively. core coat protein (cp) gene and dna-b was also amplified in the samples using specific primers. to identify the strain associated with leaf curl virus, dna was subjected against primers of different indian tomato leaf curl virus strain i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl new delhi virus primer was able to show the desired product size of * bp. therefore, it was confirmed that leaf curl disease of brinjal is caused by tomato leaf curl new delhi virus in association with satellite b-dna. to identify the strain associated with the disease, all samples were further subjected to the specific primers, designed to amplify all the tomato leaf curl virus strains, so far reported from india i.e. tomato leaf curl new delhi virus, tomato leaf curl palampur virus, tomato leaf curl banglore virus, tomato leaf curl karnataka virus and tomato leaf curl gujarat virus, using pcr. among these, only tomato leaf curl palampur virus specific primer was able to give the expected product size of * bp. this shows the association of tomato leaf curl palampur virus with leaf curl disease of calendula and marigold. thus, calendula and marigold can act as a reservoir for the tomato leaf curl palampur virus and may cause severe constrain in the production of these important ornamental plants. groundnut bud necrosis virus (gbnv) belongs to serogroup iv of the genus tospovirus in bynayaviridae family and infects several economically important crops all over india. the nucleocapsid protein (np) encoded by the small rna of gbnv encapsidates the viral rnas. apart from this structural role, the np has also been implicated in the replication, transcription, maturation and cell to cell movement. with a view to study the structure and function, the np of gbnvtomato isolate from karnataka was over expressed in e. coli and purified by ni-nta chromatography. the purified np was present as ribonucleoprotein complex and as heterogeneous mixture containing monomers, tetramers and higher order multimers. in order to determine the regions involved in oligomerization and nucleic acid binding, mutational approach was taken. n-and c-terminal deletion clones were generated (n np, n np, c np and c np), over expressed in e. coli, and were purified by a procedure identical to that used for the wild type protein. initial studies on oligomeric status suggested that in addition to n-and c-terminal regions there may be additional regions or residues which contribute to multimerization of np. the amount of rna bound to the truncated proteins was reduced in case of n np, n np and c np. interestingly removal of amino acid residues (natively unfolded region) from the c terminus resulted in complete loss of nucleic acid binding suggesting that the rna binding domain was located in c-terminal region of np. further np was observed to get phosphorylated in in vitro kinase assays by a kinase present in the soluble fraction of tobacco plant sap. both atp and gtp were utilized as phosporyl donors and mn ? was the preferred metal ion which suggests that np might be phosphorylated by a ck -like protein kinase. phosphorylation studies with n-and c-terminal truncated proteins revealed that the site of phosphorylation lies within the amino acid residues - . by mass spectrometric analysis of the protein threonine- and serine- were identified as possible phosphorylation sites. a naturally occurring isolate of virus infecting gherkin (cucumis anguira l.) showing mosaic symptoms of mosaic, leaf distortion and dark green islands in the lamina was identified in the export cultivars of gherkin grown in commercial fields of kuppam rural, chittoor district, andhra pradesh. the virus infection was deadly prevalent among the field that caused a lot of economic damage to the crop that resulted in yield losses and reduced quality of fruits meant for export. symptoms of the infected fruit included blistering and malformation of the fruit. the virus infected leaf samples were collected and initial host range tests were conducted with different cucurbit species showed that the host range include propagation hosts like cucumis anguira (gherkin), cucumis sativus, cucurbita pepo, cucumis melo, langeneria vulgaris, momordica charantia and local assay host like chenopodium amaranticolor. the virus host range was only restricted to cucurbit species and chenopodium. the virus was maintained for further studies on cucurbita pepo by sap or mechanical inoculation. the virus induced mosaic, vein clearing symptoms on pumpkin. electron microscopy of the leaf dip preparations stained with % uranyl acetate from the pumpkin leaves showing symptoms revealed the presence of a long flexuous filamentous particle measuring nm. the virus positively reacted to the polyclonal antisera of papaya ringspot virus-w, potato virus y, tobacco etch virus and also strongly reacted with the polyclonal antiserum of zucchini yellow mosaic virus in direct antigen coated-enzyme linked immunosorbent assay (dac-elisa). because of very strong reaction to polyclonal antisera of zucchini yellow mosaic virus, we tried to amplify the partial nib and cp genes of the virus along with the utr by using two primers zy gctccatacatagctgag acagc and zy taggctttttgcaaacggagtcta at c . total rna from gherkin infected leaves was isolated using trizol ls reagent (sigma). rt-pcr was performed to obtain an amplicon of * . kbp, cloned into fermentas ptz r/t vector and sequenced at mwg biotech, bangalore. sequence analysis revealed that the virus was isolate of zucchini yellow mosaic virus and was showing % of homology to that of the zucchini yellow mosaic virus strain b genome ay and zucchini yellow mosaic virus nat genome ef which were strains reported from israel. the sequence of the present study was submitted to the genbank gq . the results state a suspicion that the virus could have been mobilized by some infected source brought by the commercial israeli based companies into india due to poor quarantine regulations as the gherkin cultivation in these regions is chiefly supported, purchased, exported and marketed by these private companies that are based from israel. this is the first report on molecular characterisation of zucchini yellow mosaic virus infecting cucumis anguira (gherkin) from india. they also exhibited synergism with other virus which was region specific. fifty percent of the total symptomatic plant population was found be positive only for carla while remaining showed mixed infection of carla with tospo in some regions while in others carla virus was found to be associated with cmv. presence of only carlavirus was up to - % incidence, without association of tospo, cmv, poty or tobamo viruses was also observed in some fields. avijit tarafdar, raju ghosh, k. k. biswas plant virology unit, division of plant pathology, indian agricultural research institute, new delhi citrus tristeza virus (ctv), a brown citrus aphid (toxoptera citricidus) transmitted closterovirus under family closteroviridae, is one of the major limiting factors in cultivation of citrus worldwide. ctv is a longest known plant virus having flexuous particle of nm in size. ctv genome is a positive sense ssrna of about kb nucleotide containing open reading frames (orfs) encoding proteins. several biological as well as genetic variants of ctv are reported in all the citrus growing countries in the world. ctv causes decline and death of millions of citrus trees in the world. in india, ctv is a century old problem, and has killed an estimated one million citrus trees till today. in molecular and genetic level, ctv isolates from india were not fully characterized. genetic diversity and sequence divergence in ctv isolates of india are not fully established. further, evidence of recombination and causes of evolution of ctv variants in india have not been studied till date. therefore, in the present study, effort has been made to characterize several indian ctv isolates in genetic level, examine their genetic diversity, identify recombination events and analyze evolution of divergent ctv. a total number of ctv isolates from different regions of india ( from darjeeling hills, five from bangalore, from delhi and from vidarbha) were under taken for genetic study. two genomic regions of ctv, i.e., entire cp gene (cpg) ( nt) and a gene fragment of orf a (orf a) ( nt) were amplified, cloned, sequenced and nucleotides were analyzed. based on cpg, indian isolates shared - % nucleotide identity, and based on orf a they shared - % identity, among them. incongruence of phylogenetic relationship was observed as on sequence analysis five phylogenetic clades based on cpg, and eight clades based on orf a, were generated suggesting the recombination events have been occurred between the sequences of indian ctv isolates. thus, to identify the potential recombination events, and determine the parental sequences in ctv isolates, six recombination detecting algorithms, namely, rdp, genconv, bootscan, maxchi, chimera and siscan were used. out of indian ctv, cpg of and orf a of isolates of ctv showed recombination events suggesting orf a was more prone and fragile to rna recombination as compared to cpg. this findings indicated that high degrees of genetic diversity and incongruent relationships of indian ctv isolates are due to genetic recombination occurred, which may be the important factors in driving evolution ctv variants in india, that was also supported by a splittree decomposition analysis. b. v. bhaskara reddy, y. sivaprasad, k. rekha rani, k. raja reddy department of plant pathology, regional agricultural research station, acharya n.g. ranga agricultural university, tirupati, andhra pradesh sunflower (helianthus annus l.) is one of the most important oil seed crops in the world which ranks third in area after soyabean and groundnut. the sunflower necrosis disease (snd) is characterized by necrosis of leaves, necrosis streaks on petioles, stem, floral parts and stunted growth. the causal agent of the disease has been identified as tobacco streak virus (tsv) which belongs to genus ilarvirus of the family bromoviridae. the suspected tsv infected sunflower samples collected from chittoor district in andhra pradesh were found positive for tsv-dac elisa. total rna was extracted from sunflower using rnaeasy isolation kit (qiagen). the tsv coat protein (cp) gene, movement protein (mp) gene and replicase (rep) gene were amplified by rt-pcr with specific primers, cloned in ptz r/t vector, sequenced and deposited in genbank (gu , gu and gu ). the size of cloned cp gene was bp and codes for amino acids. the cp gene sequence analysis revealed that the tsv-tpt infecting sunflower has - % homology at nucleotide level with soybean, tagietus-tpt and okra-tn isolates and - % homology at amino acid level. the movement protein gene was bp and codes for amino acids. the mp gene sequence analysis showed that it has - % homology at nucleotide level and - % at aminoacid level. chilli (capsicum annuum), the important commercial vegetable/spice of himachal pradesh, is affected by several viral diseases; of them cucumo, tospo, poty and gemini viruses are the most common genera. however, these viruses are not identified clearly and characterized fully, which are foremost needed to formulate the management strategy. therefore, in the present study, effort has been made to identify and characterize the important viruses causing diseases in chilli. in this study, several farms in major chilli growing areas of bilaspur and kangra districts in himachal pradesh were surveyed and infected plant samples were collected randomly. virus infection in these samples were detected by das-elisa using antisera to cucumber mosaic virus (cmv) and potyvirus (group specific) and through slot-blot hybridization (sbh) using cmv, iris severe mosaic poty virus (ismv), tomato spotted wilt tospo virus (tswv) and chilli leaf curl gemini virus (clcuv). based on das-elisa and sbh, the incidence of disease was estimated and ranged from . to . % by cmv and . to . % by potyvirus. to detect tospo and geminivirus in the infected chilli, sbh test was carried out. infected samples showed maximum virus titer in both das-elisa and sbh test were further confirmed by pcr using specific primers. desired sizes of amplicons; * bp, * bp, * bp and * bp of cmv, poty, gemini and tospo viruses, respectively, were obtained. as the present study clearly indicated that cmv appeared as a major one among the viruses infecting chilli in the hilly region of himachal pradesh, two isolates of cmv were characterized in genetic level. thus the amplified products (* bp) of cmv, palampur and palampur were cloned in pgemt cloning vector, sequenced and the sequences were submitted to ncbi database (palampur : acc-fm and palampur : acc-fm ). the sequences were then analyzed and compared with other sequences available in the data base. based on sequence analysis, it was found that present cmv isolates shared % nucleotide identity between them, are closely related with australian cmv isolate cmv-ly (acc-af ) by % nucleotide identity. in phylogenetic tree analysis, it was observed that indian cmv isolates formed same cluster along with cmv-ly. as it is known that cmv subgroup ii comprises cmv-ly, it is concluded that the cmvs of this hilly region of himachal pradesh belong to subgroup ii. chilli is essentially a crop of the tropics and grows better in hotter regions. chlii (capsicum annuum), a member of family solanaceae is an important vegetable and spice crop of immense commercial importance. the pungency in pepper is due to an alkaloid known as capsaicine and peppers are characterized as sweet, hot or mild depending on capsaicine content. the present investigation were conducted to find out the highly resistant cultivars of capsicum annuum against cmv and tylcv among ten cultivars of chilli in agroclimatic condition of aligarh. the highest ( and ) percentage of infection was observed in hc- and kalyanpur type- by showing the positive reaction to cmv by elisa test. no symptoms was recorded in case of bc- , lca- and jca- and showed negative reaction to cmv by elisa. bc- and lca- also showed negative reaction to tylcv by elisa and these were symptomless. maximum infection ( and ) was registered in hc- and c , cultivar. so, the bc- , lca- and jca- has proved highly resistant varieties against cmv and tylcv and these may be used in breeding programmes against viruses. cotton leaf curl virus belongs to the family geminiviridae, genus begomovirus. the members of this family contain circular single stranded dna molecules as their genomes. there are two kinds of begomoviruses-bipartite viruses with genomes consisting of two dna molecules designated dna-a and dna-b and the monopartite viruses which contain only dna-a but not dna-b. in monopartite viruses, the dna-a is accompanied by a small circular dna molecule called dna-b which is essential for the development of typical disease symptoms. cotton leaf curl virus is a monopartite virus and causes the cotton leaf curl disease which has emerged as a major disease of cotton in the indian subcontinent. the non-structural protein ac of cotton leaf curl kokhran virus-dabawali isolate (clcukv-dab) was cloned into pgex x vector and overexpressed in bl (de )plyss e. coli cells. the overexpressed gst-ac protein was purified by glutathione sepharose chromatography. the purified gst-ac protein was found to possess atpase activity. the optimum temperature and ph for the activity were °c and . respectively. the atpase activity was inhibited in presence of edta, showing that it is dependent on divalent metal ions. the activity was supported by magnesium, manganese and zinc ions but inhibited in presence of calcium ions. it was also inhibited by the non-hydrolyzable atp analogue adenosine-b, c-imido triphosphate and in the presence of other nucleotides like ctp and gtp. the k m and the v max of the reaction for atp as the substrate are . mm and . nmol/min/ mg of the protein respectively. the enzyme could also utilize gtp as the substrate. the fact that ac is specifically an ntpase and not a general phosphatase is revealed by the finding that it does not hydrolyze p-nitrophenyl phosphate to yield yellow colour while a similar reaction carried out in parallel with alkaline phosphatase readily yields the colour. it has been suggested earlier that ac may be involved in cell to cell movement of the virus (rojas et al. ) . it is possible that by its ability to hydrolyze atp, ac serves to power viral movement in the plant. thirteen sugarcane yellow leaf virus isolates causing yellow midrib and irregular yellow spot pattern from six states of india were characterized by rt-pcr assays. scylv- f and scylv- r primers were used as forward and reverse primer pairs and the amplified products were cloned and sequenced. comparative coat protein sequence analysis confirmed that all the scylv-indian isolates were clustered into two major groups confirming the existence of two strains of scylv affecting sugarcane crops of india. in a separate experiment, the member of both of the phylogenetic groups were found to be transmitted by the sugarcane aphid, melanaphis sacchari from infected to healthy sugarcane suggesting its secondary spread in nature. the symptoms produced by the virus causing cotton mosaic disease were little bit different in both sap inoculation and under natural field condition. in natural field condition it has shown clear chlorosis type of symptoms on major leaves of plants but in sap inoculated plants veinal chlorosis and mosaic type of symptoms are found to be common. in field conditions infected plants grows erect and have less boll formation. there is no effect found on seed shape or seed size. the initial symptoms produced on cotton leaves after inoculation were wonderful. local lesions observed in second week from inoculation and then they changes to chlorotic type of symptoms and some are necrotic symptoms also. the plants at early stage are found to be affected, has less lateral branch development and hence reduction in yield production. the naturally field infected plants showing good symptoms are also difficult to identify in lateral stage of plant. because they disappear with time. the virus is very easily sap transmissible. the virus is found to be transmitted by thrips palmi and thrips tobacci in persistent manner. no seed transmission is observed. virus showed same physical properties as it shows in stem necrosis of peanut or sunflower necrosis disease. the physical properties are found to be thermal inactivation point (tip) - °c, dilution end point (dep) - to - and longevity in vitro (liv) h, virus infecting nineteen different host plants are identified belonging to five different types of families viz. malvaceae, chenopodiaceae, compositae, leguminaceae and solanaceae. however they found to produce same types of symptoms as in most of the host that have been tested before. in elisa test report it is found that the virus showing positive test only with anti serum of tsv of a cowpea and cotton but negative reaction with pbnv of cowpea and cotton which clearly denied possibility of presence of pbnv in cotton producing these kinds of symptoms elisa report clearly shows that tsv antiserum of cowpea is showing positive results with clear chlorotic types of symptoms. a powerful approach to functional genomics, and an alternative to the massive generation of transgenic plants, is the use of the recently described virus induced gene silencing (vigs) process, which allows viral vectors to knock out the function of a gene-of-interest. vigs is based on a silencing mechanism that regulates gene expression by the specific degradation of rna. as a tool for reverse genetics, vigs has many advantages over other common ways to study gene function because of the ability of viruses to replicate and move systemically within a plant. vigs can generate a phenocopy of a mutant without all the troubles of traditional methods of mutagenesis. geminiviruses with their small dna genomes and ease of inoculation through agrobacterium, are excellent candidates for vigs vector development. as a first step, the geminivirus bhendi yellow vein mosaic virus, characterized in our lab (jose and usha, virology : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) has been chosen. the satellite b dna associated with this virus has a single open reading frame (bc ). bc is essential for symptom development but not for replication. therefore, bc has been replaced by a multiple cloning site harbouring sali, xbai, bamhi, bsrgi and xhoi, initially in a cloning vector and then in the binary vector containing the partial tandem repeat of the b dna. in the place of the bc orf, the plant phytoene desaturase gene has been cloned and the resulting construct was used for agroinfiltration along with the partial tandem repeat clone of the begomovirus (dna a component chilli (capsicum annuum l.) plants exhibiting prominent symptoms of begomovirus like: leaf curl, vein swelling, shortening of petioles, crowding of leaves and stunting of plants were collected from rorkee, uttarakhand and dhaulpur, rajasthan, india. total genomic dna was isolated from naturally infected chilli samples and pcr was carried out with coat protein (located in dna-a) gene specific primers. as expected to the primers, * bp dna fragments were amplified from the infected chilli samples. to know the bipartite nature of the virus isolates, nuclear shuttle protein (located in dna-b) gene specific primers were employed which also resulted in positive amplification of * bp dna bands with all the coat protein tested positive samples. to ascertain the association of dnab component with the virus isolates, a set of dna-b specific primers were used which resulted in positive amplification of full length (* . kb) dna bands in the chilli samples collected from rorkee, uttarakhand, however, multiple sizes bands were resulted with the samples collected from dhaulpur, rajasthan. these findings confirmed that both the virus isolates under study are bipartite begomovirus associated with dna-b satellite. the sequencing of the pcr products is under progress which analysis will be discussed. groundnut bud necrosis virus (gbnv) belonging to the genus tospovirus, which is a unique member of the family bunyaviridae, infects several economically important crops. the virus has three genomic ssrna segments namely s (ambisense), m (ambisense) and l (negative sense). the s rna codes for nucleoprotein (np) and non-structural protein (nss) from viral complimentary and viral strands respectively. many viral nonstructural proteins such as ns of hepatitis c virus, yellow fever virus, dengue virus, sv large t antigen and cytoplasmic inclusion protein of tamarillo mosaic potyvirus are known to exhibit rna/dna stimulated ntpase, dntpase and helicase activity. nss of gbnv does not have any sequence similarity with any of the above mentioned viral rna/dna helicases but has a ntp binding domain. however, it has been implicated as suppressor of gene silencing in vivo. with a view to elucidate the mechanism by which nss could act as a suppressor of gene silencing and examine the other potential roles of nss in the life cycle of the virus, the gbnv (to) nss was over-expressed in e. coli and purified by ni-nta chromatography. in vitro studies with the purified rnss suggest that it exhibits an rna stimulated ntpase activity. many of the proteins that possess the rna/ dna stimulated ntpase and datpase activity, are also shown to have atp dependent nucleic acid unwinding activity. it was therefore of interest to examine whether nss has the nucleic acid unwinding activity. the helicase assays revealed that nss has dna/rna helicase activity. helicase activity of nss was absolutely dependent on atp and mg ? ion. nss could unwind dsdna substrate with overhang, or overhang. mutation of the crucial lysine in walker motif a (k ) severely affected the unwinding activity where as mutation of aspartate residue in walker motif b (d ) resulted in only % loss of activity. in this regard, rnss is a unique enzyme which does not have the canonical helicase motifs but can catalyze dsdna/dsrna unwinding in an atp and mg ? dependent manner. the rnss might act as a suppressor of by unwinding the dsrna, the substrate for dicer. in addition to being a suppressor of ptgs, nss may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. this new research finding on nss might pave way for further studies on its role in viral replication and transcription. yellow vein mosaic disease of pumpkin (cucurbita moschata) poses a serious threat to the cultivation of this crop in india. the disease was found to be associated with whitefly-transmitted bipartite begomoviruses were detected in varanasi field using polymerase chain reaction (pcr) with primer design through coat protein conserved region of begomoviruses from ncbi database. all plant samples showing symptoms were infected with begomovirus. the virus species were provisionally identified by sequencing * bp of the viral coat protein gene (av ageratum conyzoides is commonly known as billygoat-weed, chick weed, goatweed and whiteweed. in india it is popularly known as bill goat weed. it is an annual herbaceous plant with a long history of traditional medicinal uses in several countries of the world and also reputed to possess varied medicinal properties including the treatment of wounds and burns. in cameroon and congo, it is used traditionally to treat fever, rheumatism, headache, and colic. during survey in and around gorakhpur in , ageratum plants were found affected with the symptoms of leaf curling, mosaic mottling and leaf yellows. the infected leaf samples were processed for virus identification and association with pcr assays. total dna was extracted and pcr were performed with begomovirus specific primers (tlcv-cp). a * bp band was consistently amplified on % agarose. the pcr products were directly sequenced and sequence was submitted in genbank with the accession no. gq . the blast search analysis showed highest similarity of % with the ageratum enation virus. vernonia cinerea leaves with yellow vein symptoms were collected around crop fields in madurai. a bp product amplified from total dna extracted from symptomatic leaves with degenerate primers designed to amplify a part of the av gene from begomoviral dna a component was cloned and sequenced. based on the above sequences, specific primers were designed and the full length dna a of nucleotides with typical genome organization of begomoviral dna a was obtained and was submitted to embl data base (acc no: am ). the sequence comparison with other begomoviruses revealed the closest identity ( %) with emilia yellow vein virus from china and less than % with all known begomoviruses. the international committee on taxonomy of viruses (ictv) has therefore recognized vernonia yellow vein virus (vyvv) as a distinct begomovirus species. conventional pcr could not amplify the dna b or dna b from the infected tissue. however, the b dna ( bp) associated with the disease was obtained (acc no: fn ) by the rolling circle amplification-restriction fragment length polymorphism method (rca-rflp) using phi dna polymerase. sequence analysis shows that dna b of vyvv has the highest identity ( %) with dna b of ageratum leaf curl disease and - % with the b dna associated with other begomoviruses. infectious clones of vyvv dna a and dna b as dimers were made using the products of rca-rflp. these infectious clones will be used for agroinfection of vernonia and the results will be discussed. this is the first report of the molecular characterization of vernonia yellow vein virus (vyvv) from vernonia cinerea in india. production of bulb and seed crop of onion (allium cepa l.) is hampered by onion yellow dwarf virus (oydv) and iris yellow spot virus (iysv) with an incidence of . % and . % in bulb crop and . % and . % in seed crop, respectively in the popularly grown cv. hisar- . four symptom-based variants of oydv designated as grade a, b, c and d produced varied types of symptoms in onion crop incurring heavy losses in bulb and seed production. iysv caused tiny hay coloured spots of different shapes and sizes on leaves and scapes which later coalesced and led to drying and lodging of scapes. the plant height, bulb weight and bulb size were . cm, . g and . cm in plants infected with oydv, . cm, . g and . cm in iysv infection, . cm, . g and . cm due to their combined infection, as compared to . cm, . g and . cm respectively, in healthy plants of bulb crop. in plants infected with oydv grade a the plant height was minimum ( . cm) whereas the number of umbels was maximum ( . umbels/pl.) but other yield parameters viz., weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded to be the lowest. the minimum reduction in plant height ( . cm), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were recorded in oydv grade d. the plant height was . cm with . umbels per plant, . g weight/umbel, seeds/umbel, . g seed weight/umbel and . g seed yield/plant in iysv infected plants. the plant height ( . cm), umbels/plant ( . ), weight/umbel ( . g), number of seeds/umbel ( ), seed weight/umbel ( . g) and seed yield/plant ( . g) were found to be the lowest in combined infection of oydv and iysv diseases in comparison to higher values in healthy controls ( . cm, . , . g, , . g, . g, respectively). a minimum reduction in the test weight, germination and seed vigour index were found ( . g, . % and ) due to oydv grade a infection, whereas these were . g, . % and in iysv disease infected plants and . g, . % and in combined infection of oydv and iysv diseases in comparison to . g, . % and in healthy plants. the maximum hampering of seed vigour parameters was recorded due to iysv infection. lodging of scapes caused by this disease was responsible for heavy losses in seed production and seed quality. cotton leaf curl disease is one of the major threats to cotton cultivation from northern india. survey conducted during , observed the disease incidence ranged from to % from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar. in order to study genetic variability in the virus, twelve clcuv isolates were partially characterized ( bp common region, full length av gene and partial sequences of ac and av gene). full length characterization of representative isolates from bhatinda, abohar, fazilka, sri ganganagar, hanumanghar is under progress. partial sequence analysis of clcuv isolates revealed that, the virus isolates collected during cropping season are closely related to cotton leaf curl burewala virus from pakistan and results were discussed. pratibha singh, h. s. savithri department of biochemistry, indian institute of science, bangalore tospoviruses, belonging to the family bunyavirideae, infect economically important plants such as groundnut, tomato, watermelon etc. they have a tripartite genome, with l, m and s segments of rna, in pseudo circular (panhandle) form. the viral genomes encode four structural proteins (l, n, g and g ) in the antisense orientation, and two non structural proteins nss and nsm in the sense orientation. the nsm is the only protein unique to tospoviruseses that infect plants in the bunyaviridae family and hence is proposed to be important for cell to cell movement. ground nut bud necrosis virus (gbnv), a member of the tospovirus genus, is the most prevalent virus infecting several species of leguminosae and solanaceae plants in india. total rna was isolated from gbnv infected tomato leaves and rt-pcr was performed using appropriate primers to amplify the nsm gene. the pcr product was cloned in pgex x vector. the recombinant nsm clone was transformed into bl (de ) e. coli cells and over-expressed by induction with . mm iptg. sds-page analysis of induced and uninduced fraction revealed the presence of overexpressed protein of expected size. the soluble gst-nsm was purified by gsh sepharose affinity chromatography. purified gst-nsm was shown to interact with in vitro transcribed rna transcript by electrophoretic mobility shift assay. further nsm was shown to interact with viral encoded proteins np and nss using elisa and yeast two hybrid system. nsm was also shown to be phosphorylated in vitro by pellet fraction of plant sap. thus the recombinant gbnv nsm possesses the characteristic features of a movement protein such as nucleic acid binding, interaction with nucleocapsid protein, and ability to undergo posttranslational modification. solanum melongena, commonly called as egg plant is one of the most important vegetable crop in the world. it is cultivated widely in the tropical and sub tropical regions. several viruses such as cucumber mosaic cucumo virus (cmv), potato virus-y (pvy), potato virus-x (pvx) and tobacco ring spot virus (trsv) infect egg plant under natural conditions. in india major crop losses due to cmv infection in brinjal is % (fao stat- ) . in the present study the infected leaf samples were collected from local fields of ramapuram, chandamama palli, chandragiri, madanapalli, yadhamari, durgasamudram villages in and around tirupati, were tested for cmv infection by dac-elisa with cmv antisera. the resulting positive samples were further inoculated to the raised brinjal seedlings of selected varieties through mechanical sap inoculation. different varieties of brinjal like mullabadhine, ankhur, ravya, mattigulla, casper and easter egg were used for monitoring the susceptibility to cmv infection. the mosaic symptoms were observed after weeks of inoculation in all varities of brinjal except mullabadhina. among all these susceptible varities ankhur variety is selected to study induced biochemical changes such as chlorophylls, carbohydrates, proteins, nucleic acids and polyphenol oxidases in cmv infected brinjal leaves. in the infected leaves considerable reduction in chlorophyll and starch and increase in total proteins, sugars, rna and polyphenol oxidases was observed when compared to healthy leaves. the amount of total starch, protein and dna decreased to about , and lg/g respectively in infected leaves, where as sugars ( lg/g), rna content ( lg/g) and polyphenol oxidase activity was increased as compared to healthy leaves. the above results suggests that there is an altered concentrations of chlorophyll, proteins, nucleic acids, carbohydrates and polyphenol oxidase activity in the brinjal leaves due to the effect of cucumber mosaic cucumo virus infection. leaf analysis was found to be used as widely accepted diagnostic tool to assess the nutritional status of the vegetables. the present study deals with these aspects in detail. the total rna and dna was isolated from infected leaf samples. rt-pcr assays were performed using sugarcane yellow leaf virus (scylv) specific primers (scylv- f and scylv- r). the infection of scylv was detected in all the collected samples, which showed the expected size (* bp) amplicon during rt-pcr. in another experiment with nested pcr analysis, a phytoplasma characteristic . kb rdna pcr product were amplified from dnas of all infected samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs p /p and fu /ru . dna extracts from plants with yellow mid rib and leaf yellows produced products of bp, which gave typical phytoplasma profiles when digested with hae iii and hha i. no pcr amplifications were produced using dna from symptomless plants. our results suggest that the yellow mid rib and leaf yellows symptoms on sugarcane varieties in uttar pradesh and uttarakhand states of india exhibiting midrib yellowing and leaf yellows symptoms is mainly caused by mixed infection of scylv and scylp. the affected clumps showed reduction in stalk height as compared to healthy fields. thirty-one sugarcane mosaic isolates belonged to sugarcane mosaic virus (scmv) and sugarcane streak mosaic virus (scsmv were collected from china and india), confirmed in indirect elisa and rt-pcr amplification with scmv and scsmv-specific primers. the amplicons ( . kb) from the coding region of coat protein (cp) were cloned, sequenced and compared to each other as well as to the sequences of scmv isolates from sugarcane (australia, usa, china, brazil, mexico and south africa), maize (australia, china, iranian) and one scsmv isolate from sugarcane (india) in genbank. maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the scmv subgroup that included isolates from china and only isolates from india, and the scsmv subgroup that contained all isolates from india. maize dwarf mosaic virus (mdmv) and johnsongrass mosaic virus (jgmv) were not detected in any of the samples tested. a strong correlation was observed between the sugarcane groups and the geographical origin of the scmv isolates. the millable sugarcane samples from china contained a virus tentatively described as sorghum mosaic virus (srmv). three isolates from nine chewing canes in fujian, yunnan and guizhou provinces of china also contained srmv, and the other samples including five isolates from india was found infected with scmv. no srmv infection has been detected in sugarcane mosaic samples from india. sequence comparisons and phylogenetic analysis indicated that srmv can be considered as the most common and prevalent potyvirus infecting sugarcane in china, however in india sugarcane streak mosaic virus is dominant in causing mosaic symptoms on sugarcane. dig-labeled dna probe complementary to coat protein (cp) region of tobacco streak virus (tsv) sunflower isolate was designed for the sensitive and broad-spectrum detection of tsv isolates, the most devastating virus in india. dot-blot and tissue print hybridizations with the digoxigenin labeled probe were performed for the tsv detection at field levels. here, dot-blot hybridization was used to check a wide number of tsv isolates with a single probe and sensitivity with different sample extraction methods. the probe with cp conserved region prepared from sunflower pcr amplicon was hybridized with the tsv field isolates of gherkin, pumpkin, sunflower, marigold and globe amaranth samples because of highly conserved with little variability in cp region. the sensitivity limits were decreased from total nucleic acid to partially purified and crude extract preparations. in particular, tissue blot hybridization offers a simple, reliable procedure as dot-blot, but requires no sample processing. because there is minimal sample preparation, tissue-print hybridization could be an important component of tsv management programs. thus, the above non-radioactive labeled probe techniques can facilitate in screening the samples during tsv outbreaks and in quarantine services. savita patil, rupali sawant*, k. banerjee virology group, agharkar research institute, macs, g.g. agarkar road, pune two mycobacterium smegmatis strains (ari lab nos. v and v ) were employed for the isolation of mycobacteriophages from soil and sewage samples. mycobacteriophages were isolated from soil samples collected from an area surrounding the tuberculosis (tb) ward, naidu hospital, pune, against m. smegmatis strain v . these were numbered as v , v and v and were isolated by using washed-cell preparation method. the bacteriophages against the other m. smegmatis strain, i.e. v , were isolated from soil samples (collected from around tb ward, sassoon hospital, pune). some of these phages (viz.v , v ) showed plaques at °c but not at °c. thus they seem to be lysogenic. for propagating and increasing the titre of all the above isolates, various previously described methods were attempted, but none of these methods were satisfactory. but when siliconized glassware and plastic-ware were used, propagation was successful. we showed that siliconization of glassware and plastic-ware was essential for the propagation of our mycobacteriophage isolates v , v , v , v and v . also, phage dilution medium (pdm) as described by chaterjee et al. ( ) was found to be effective for picking out of the plaques made by the phages. in this way, the phage isolates were propagated up to p . the various passages of the phage isolates v , v , v , v and v (i.e. original, p , p and p ) were stored at - °c. pvp- effect on pigments due to geminivirus infection on cowpea (vigna unguiculata) shail pande*, naveen pandey, k. shukla mahatma gandhi p. g. college gorakhpur, d.d.u. gorakhpur university, gorakhpur geminiviruses are one of the most important group of viruses causing economic losses in tropics. the symptom produced are yellowing of leaves which directly affect the pigments of diseased plants it in turn affects productivity and yield of diseased plant. cowpea vigna unguiculata is one of the important crop cultivated throughout india for its green pods which are used as vegetables and seeds are used as pulse. cowpea is affected by many viruses amongst them geminiviruses are one of the important virus on the cowpea plant. in the present study total chlorophyll content was studied in leaf of cowpea of diseased and healthy plants using arnon's method. carotenoids were also studied using ikan's method. it was found that chlorophyll content in diseased plants were lower compared to healthy plant similar results were found with carotenoids so the geminivirruses infection lowers the chlorophyll and carotenoid content in diseased plants which reduces yield of diseased cowpea plant. shweta sharma , amrita banerjee , j. tarafdar , r. rabindran , indranil dasgupta * department of plant molecular biology, university of delhi, south campus, new delhi; bidhan chandra krishi vishwavidayalaya, kalyani, nadia, west bengal ; tamil nadu agricultural university, coimbatore, tamil nadu rice tungro disease is an important disease of rice, caused by a joint infection by two viruses: rice tungro spherical virus (rtsv) and rice tungro bacilliform virus (rtbv) in south and southeast asia. the complex of rtbv and rtsv is transmitted by an insect vector green leaf hopper (glh). previously we reported complete genomic sequences of two geographically distinct isolates of rtbv; rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) collected from the field in mid- s. both the sequences showed high homology all along the genome but showed divergence from previously reported southeast asian isolate i.e. rtbv-phil (philippines). to check whether a time period of a decade has resulted into variability in the genomic sequence of different isolates of rtbv in india, we cloned and sequenced the complete genome of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari collected from the field in . the complete nucleotide sequence of the dna fragments covering the whole genome of rtbv was determined using universal primers m f and m r and by primer walking, without any ambiguities remaining. the nucleotide sequences of overlapping clones were assembled and analyzed using the dna analysis software generunner and blastn program of ncbi. homology search at the nucleotide and amino acid level were performed using the blastn and blastp (respectively) programs of ncbi. multiple sequence alignments were performed using clus-tal-w software. sequence analysis results thus obtained showed that both the recently obtained complete genomic sequences of rtbv from two geographically distinct regions of india i.e. west bengal and kanyakumari showed very high homology (both at the nucleotide and amino acid levels) with the two previously reported rtbv isolates from india i.e. rtbv-wb (west bengal) and rtbv-ap (andhra pradesh) all along the genome. as observed earlier both the sequences diverged significantly from the southeast asian isolates. this suggests that even after the spatial and temporal difference (a time gap of approx years) between the two previously reported rtbv isolates and the recently reported one, there is very little sequence variability between them. this further strengthens the earlier reports that the rtbv genomes in india are highly conserved. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. complete genomic rna sequences of two geographically distinct isolates of rice tungro spherical virus (rtsv), a member of the genus waikavirus, family sequiviridae, were determined from india. out of the two previously reported sequences, the indian isolates were closer to the resistance breaking strain rtsv-[vt ] than rtsv- [phila] . between them, the indian sequences showed nucleotide as well as amino acid identities of %. a moderate homology was observed between the leader peptide and a putative helper component protein involved in insect transmission of the maize chlorotic dwarf virus, a closely related waikavirus, indicating its possible transmission-related function. unlike rice tungro bacilliform virus, which causes rice tungro disease jointly with rtsv, and is significantly different between isolates from india and philippines, rtsv genomes were observed to be much more conserved between isolates from the two countries. rice tungro bacilliform virus (rtbv) are believed to be the joint causative agents for the devastating tungro disease of rice prevalent in south and southeast asia [ ] . rice tungro disease has become the major cause of production losses in rice during last three decades in several rice growing states of india. here, we report, for the first time the complete sequence analysis of two geographically distinct indian isolates of rtsv. we analyze the deduced protein sequences and their phylogenetic relationship with the two complete rtsv sequences from philippines as well as with other members of sequiviridae family. we provide molecular evidence that the indian isolates of rtsv are closely related to those from the philippines. we had earlier reported that rtbv isolates between india and philippines differ significantly from each other [ ] . this study was undertaken in order to see whether rtsv isolates from india also show similar difference from those reported from the philippines. frequent outbreaks of tungro were reported near kanyakumari in the last - years. the present work was undertaken to clone and sequence the full-length rtbv and rtsv genomes from the infected rice plants collected from above region and to analyze the similarity of its genetic material with the existing indian isolates of rtbv and rtsv. a . kb dna fragment encoding the reverse transcriptase gene of rtbv genome was amplified and cloned in t/a vector and was sequenced commercially. homology search at the nucleotide level using blastn program with the previously existing rtbv isolates revealed a very high percentage identity of % with the rtbv west bengal isolate and % with the rtbv andhra pradesh isolate. this further strengthens the earlier reports that there is not much genetic variability in the rtbv genomes in indian subcontinent. similarly, the cp region of rtsv was amplified by rt-pcr and was cloned in t/a vector. recently, rice tungro disease has been reported from kanyakumari district of tamil nadu. it is important to determine the genetic nature of this isolate in order to develop resistance strategies. it is thus necessary to clone and characterize the viruses from kanyakumari and to determine the mechanism of virus resistance in transgenic lines. rice tungro disease is an important viral disease of rice. rice tungro is caused by infection by two viruses: rice tungro bacilliform virus (rtbv) and rice tungro spherical virus (rtsv). rtsv is a plant picornavirus with a kb single stranded rna genome. it belongs to genus waikavirus in the family sequiviridae and is necessary for transmission of the two viruses by the leafhopper vector nephotellix virescens. rtsv rna is translated to form a large polyprotein, which is then self cleaved to form the viral proteins, including the three coat proteins, replicase, protease. studies have been conducted on rtsv from philippines. correct information of sequence variability of viral isolates to check whether different geographical conditions like those present in india select for genotypically variable strain and to design for transgenic resistance strategy, information on rtsv from india is absolutely essential. the objective of this study was to clone rtsv isolates from india and compare the genetic diversity of indian isolates from other southeast asian isolates and amongst each other. also develop strategy to impair the attack of virus-complex on rice. the achieve this, complete genomes of two isolates from india were cloned by amplifying different genes by rt-pcr and subsequently cloned in ta vectors, followed by sequencing. subsequently constructs containing cp - , antisense replicase, sense replicase and double stranded replicase were cloned in plant transformation vector. these constructs were used to transform aromatic rice variety from indian-pusa basmati (pb ). pcr analysis of the above plants was done to check the stable insertion of insert in the transgenics. jatropha (jatropha curcas) of the family euphorbiaceae is being grown in india as a major commercial fuel (bio-diesel) crop. jatropha is cultivated in districts of potential states of india. unfortunately, the cultivation of jatropha is limited by the severe mosaic disease. recently, a severe mosaic disease with significant disease incidence was observed in - on j. curcas grown in experimental plots of nbri and j. gossypifolia, a weed growing road side around lucknow and kathaupahadi, madhya pradesh. the disease consisted of the symptoms of severe mosaic, blistering, leaf distortion and stunting of whole plant and no fruit/seed production in severely affected plants. symptomatology and whitefly population observed on them suggested the occurrence of begomovirus infection. to detect the begomovirus infection, the total dna from leaf samples of infected jatropha plants was extracted and polymerase chain reaction (pcr) were performed using three sets of begomovirus genus specific (cpit-i/cpit-t, paliv /paric and paliv /palic ) primers and the expected size * bp, . kb and . kb amplicons were obtained which confirmed the begomovirus infection. further to identify the begomovirus/es and investigate the genetic diversity among them exists if any, the * . kb amplicons were cloned and sequenced. the sequence data were deposited in the genbank database under accession nos.: gq and fj (from j. curcas) and eu and fj (from j. gossypifolia). during blast analysis gq and fj shared highest % sequence identity with each other and - %% with sri lankan cassava mosaic virus (aj , aj , aj , aj and aj ) and indian cassava mosaic virus from india (ay ) therefore, designated as two strains of jatropha mosaic india virus-lucknow. blast analysis of eu showed maximum % similarities with croton yellow vein mosaic virus (aj ), % with tomato leaf curl new delhi virus (dq ) and - % with papaya leaf curl virus (aj and y ), therefore, identified as strain of croton yellow vein mosaic virus. blast analysis of the virus isolate (fj ) showed highest % identities with tomato leaf curl virus-bangalore ii (tolcv-b ii-u ) and - % with tomato leaf curl karnataka virus (tol-ckv, ay , fj ), therefore, considered as new begomovirus species ''jatropha yellow mosaic india virus''. the phylogenetic analysis of gq and fj (from j. curcas) and eu and fj (from j. gossypifolia) was performed along with some selected isolates of begomovirus which showed [ % sequence identities during blast analysis. the isolate eu showed closest relationship with croton yellow vein mosaic virus while fj showed separate clustering of all the four begomovirus from jatropha species. during phylogenetic analysis these isolates formed three separate clusters, therefore, they were considered as three distinct begomoviruses. the above data clearly show that some genetic diversity exists among the begomoviruses infecting jatropha species in india. bitter gourd (momordica charantia l.) of the family cucurbitaceae, also known as bitter melon is extensively cultivated in north eastern region of uttar pradesh, india. it is regarded as one of the world's major vegetable crops and has great economic importance. a severe yellow mosaic disease on bitter gourd (momordica charantia) with a significant disease incidence was observed during the survey of different locations of eastern up, india in the year . the whitefly (bemisia tabaci) population was also observed in the vicinity. the characteristic disease symptoms and whitefly population indicated the possibility of begomovirus infection. total dna were isolated from infected as well as healthy leaf samples. two primer pair (tlcv-cp and roja's primer) were used to study, which resulted * bp with tlcv-cp in / samples and * . kb amplicons with roja's primer in / samples. for further identification of the begomovirus, the pcr amplicons were cloned and sequenced (genbank accession no. eu and eu , respectively). the blastn search analysis of eu indicated - % identity with several isolates of tomato leaf curl new delhi virus (tolcndv). the phylogenetic analysis also showed closest relationships of the isolate (eu ) with tolcndv isolates. based on highest sequence identity and closed relationships with tolcndv the virus isolated from bitter gourd was considered as an isolate of tomato leaf curl new delhi virus. while, blastn search analysis of eu isolate, shared highest - % identites with pepper leaf curl bangladesh virus (peplcbv) isolates. the phylogenetic analysis of the virus isolate with selected begomovirus isolates revealed a closest relationship with peplcbv. these results confirmed the association of peplcbv on bitter gourd. study revealed the variability of viruses on bitter gourd in eastern up, india. tobacco streak virus groundnut isolate was characterized biologically by taking six cultivars (jl , tmv , k , k , k ) and one pre-release culture (k ) using seedlings of - days old under glasshouse conditions. there were clear differences were observed among cultivars tested regarding incubation period, percent seedling wilt and time taken to death of seedlings. k- was least susceptible among all the cultivars tested and it supported least virus titer (a nm: . - . ). both localized (necrotic lesions on leaf, veinal necrosis, leaf yellowing, wilting) and systemic (petiole necrosis, necrotic lesions on young leaves, death of top growing buds not only on main stem but also on all primaries (side shoots), followed by stem necrosis, stunted growth, axillary shoot proliferation with small leaves having general chlorosis, peg necrosis, pod necrosis, pod size reduction, wilt of plants) symptom were observed in all cultivars tested. biological differentiation of tsv and gbnv was made by sap inoculation of both viruses separately using susceptible groundnut cultivar jl under glasshouse conditions. there were certain similarities and differences were observed between these viruses infecting groundnut. seed infection of tsv ranged from . to . % in seeds collected from naturally infected and sap inoculated groundnut cultivars/pre-releases (jl , tmv , k- , k- , k- and k- ) belonging to spanish and virginia types. tsv was detected both in pod shell and seed testa from pod samples produced by sap inoculation under glasshouse conditions. however, seed transmission of tsv was not observed in groundnut. coat protein (cp) gene of three groundnut tsv isolates (gn-ap- - ; gn-ap - ; gn-ap - ) were sequenced and all the three isolates contained a single open reading frame (orf) of bp nucleotide and could potentially code for amino acids (aa). cp gene of tsv isolates originating from different hosts shared high degree of sequence identity both at nucleotide ( . - %) and amino acid ( . - %) levels respectively. tones grown in an area of . . ha (fao stat ). in india papaya is grown in nearly , ha with an annual production of , , tones (fao stat ) and occupies fourth place in the world. the crop is severely affected by a number of viruses. papaya ring spot virus (prsv-p) is the most important virus. the detection of virus infection in plants has traditionally involved either bioassay on indexing plants and or immunological methods (hill , torrence and jones ) . use of nucleic acid probes has improved the detection and sensitivity of viruses. the most common non-radioactive probes are biotynilated probes, which are very specific and sensitive. papaya ring spot virus (prsv-p) is a positive sense ssrna virus belonging to the genus potyvirus family potyviridae and transmitted by aphids. prsv-p coat protein gene region was used as template cdna for probe preparation. dot-blot hybridization with the biotin labeled probe were performed for prsv-p detection. the clarified sap of healthy and infected plants were serially diluted and spotted onto the nitrocellulose membrane, hybridized to biotin labeled probe. biotin labeled rna's are employed as probes, with a subsequent detection based on streptavidin-alkaline phosphatase conjugates. the sensitivity for viral detection of the biotin labeled probe was found to be sensitive than enzyme linked immunosorbent assay (elisa). in recent years tospovirus is causing devastating damage to the yield of vegetables in india. it infects economically important crops viz., tomato, chilli, peppers, groundnut, watermelon and various legumes. now it is emerging as severe disease in brinjal also. in order to monitor the natural occurrence and distribution of tospovirus in vegetable, surveys were conducted in the predominant brinjal growing areas of gujarat, karnataka, maharashtra and andhra pradesh during - incidence ranging from to %, to %, to %, and to . % respectively. samples collected from different places of india were found positive to pbnv in direct antigen coating-enzyme linked immunosorbent assay (dac-elisa). pbnv infected brinjal plants showed mosaic mottling of leaves with leaf distortion, longitudinal streaks on the stem and necrotic rings on leaves and fruits. early infection led to severe stunting and abnormal fruiting. biological and molecular characterization of pbnv-brinjal isolates were compared with other isolates and results are discussed. for identification of virus causing mosaic symptoms on soybean various host plants were tested. plants species belonging to the different families viz. caricaceae, graminae, leguminosae, malvaceae and solanaceae were tested. the virus produced symptoms on diagnostic plant species like chenopodium album, c. quinoa, helianthus anus, phaseolus vulgaris and vigna ungiculata. among tested families the leguminosae that were the host of virus included arachis hypogea, the virus causing mosaic symptoms in soybean is inactivated between and °c and between dilution of - to - . all the inoculated plants of assay host showed the symptoms at °c but not at °c. similarly local lesions produced at - but not at - . the virus in crude sap was infectious up to h but not at h at room temperature. however, the percentage infectivity decreased progressively as the aging of the sap was increased at room temperature. on the basis of reactions on diagnostic hosts pvp- identification and characterization of potyvirus infected chilli (capsicul annum l the virus under study caused mild mosaic and severe mottling symptom in leaves of infected plants. the dilution end point (dep) of the virus was found to be - to - , longevity in vitro (liv) - days at room temperature ( °c), thermal inactivation point (tip) - °c. electron microscopy of purified virus preparation revealed the presence of flexuous particle of size nm long and nm in width with characteristic cytoplasmic inclusions: pinwheels and scrolls. the virus was transmitted by sap and by aphid myzus persicae. the host range study revealed that the host species were restricted to family chenopodiaceae and solanaceae. on the basis of above characteristic, the virus under study was identified as potyvirus associated with mild mosaic and severe mottling symptom in capsicum. phytoplasma causing grassy shoot disease and sugarcane yellow leaf viruses are important pathogens of sugarcane. these pathogens are causing severe losses in sugarcane productivity. with a view to producing virus and phytoplasma free planting material of sugarcane, experiments were undertaken using infected varieties of sugarcane growing at the farms of sugarcane research institute. apical meristems measuring about mm in length, were dissected out, surface sterilized and cultured on agar gelled murashige and skoog's (ms) medium containing growth regulators for shoot induction. the established shoot cultures were multiplied through repeated subcultures on fresh media at - days interval. elimination of gsd and scylv was confirmed through molecular analysis of regenerated plants using specific primers of scylv and gsd. results revealed that apical meristem culture technique is effective in eliminating the pathogens like scylv and phytoplasma (gsd) from the infected clones. this is probably the first report on elimination of grassy shoot disease in sugarcane through meristem culture. papaya ringspot virus (prsv), which causes the most widespread and devastating disease in papaya, isolates originating from different geographical regions in south india were collected and maintained on natural host papaya. the entire coat protein (cp) gene of papaya ringspot virus-p biotype (prsv-p) was amplified by reverse transcription-polymerase chain reaction (rt-pcr). the amplicon was inserted into pgem-t vector by t-a cloning method, sequenced and sub cloned into a bacterial expression vector prset-a using directional cloning strategy. the prsv coat protein was over expressed as fusion protein in e. coli. sds-page gel revealed that cp expressed as a * kda protein. the recombinant coat protein (rcp) fused with his-tag was purified from e. coli using ni-nta resin. the antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified prsv. the purified rcp was used as an antigen to produce high titer prsv specific polyclonal antiserum. the resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (ic-rt-pcr) assay and compared its sensitivity levels with elisa based assays for detection of prsv isolates. ic-rt-pcr was shown to be the most sensitive test followed by dot-blot immunobinding assay (dbia) and plate trapped elisa. key: cord- -qp bdd authors: mayer, kenneth h.; pizer, h. f. title: ecological studies of diseases: promise and praxis date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: qp bdd nan in the late s, a number of prominent public health experts predicted the end of infectious diseases as a serious threat to mankind. their optimism was based on successes from recently developed antimicrobial drugs, vaccines, and with infection control measures like improved public sanitation and regulating the food supply. unfortunately, their hopeful forecast was short-lived. soon there were epidemics caused by newly identified human pathogens, like legionella and ebola virus, and resurgent old ones like tuberculosis and malaria, often in forms resistant to previously effective antimicrobial drugs. the most catastrophic pathogen has been hiv- , the major human immunodeficiency virus that causes aids. in approximately years, it has spread throughout the world to infect more than million people and cause approximately million deaths. aids is now the fourth leading cause of death worldwide and accounts for about % of recent deaths in africa (kanki and essex ) . we have been asked to review the ostfeld et al. ( ) text, infectious disease ecology: the effects of ecosystems on disease and of disease on ecosystems, through the lens of our recently published multi-authored text, the social ecology of infectious disease (mayer and pizer, ) . the primary unifying thesis of our book is that many, if not most, human epidemics are primarily potentiated by patterns and changes in human behavior, e.g., the sexual revolution facilitating the spread of hiv, or increased ease of international travel leading to major global influenza epidemics and the spread of sars. the ostfeld et al. ( ) text is grounded in basic biological ecology, focusing on physical environment-host-pathogen interactions, while the foundations of our perspectives are public health and sociobehavioral sciences, relying heavily on medical epi- ecohealth , - , doi: . book review Ó international association for ecology and health demiology as a core investigative tool. while a superficial read of the two texts might lead one to think they are completely different, they are in fact complementary. both texts are looking for the fundamental factors in infectious diseases that impact the health of populations, and both seek to describe and explain the complex and active interplay of pathogens and hosts that occur in an everchanging pattern of exposures to new hosts, pathogens, and vectors, and changing physical environments. infectious disease ecology: the effects of ecosystems on disease and of disease on ecosystems is the product of the th biennial cary conference that convened in may to discuss the effects of ecosystems on infectious disease dynamics. sixty-seven scientists from diverse backgrounds who are grounded in the modern discipline of ecology contributed to the book. it is divided into four parts that explore the effects of ecosystems on disease and disease on ecosystems, management and applications, and a final chapter by the editors that looks at the challenges that lay ahead. in contrast, our book (mayer and pizer, ) is organized along the lines of the specific varied human activities that provide the most distinctive niches for microbes to flourish and spread. one example from the two texts can help shed light on how these two books and their respective disciplines of ecology and public health inform each other. more than % of emerging human pathogens are zoonotic, i.e., transmitted to humans from other animals usually by vectors like mosquitoes and ticks. a myriad of human and animal activities (e.g., moving into new physical niches), plus factors like land and water use, and climate change affect the distribution and dispersal of vector populations which, in turn, influences pathogen prevalence and transmission, and ultimately infectious disease outbreaks in specific human populations. lyme disease provides a good example of how this works. the causative agent is borrelia burgdorferi, a spirochete that is typically transmitted to people by the bite of ixodes ticks. while reports of b. burgdorferi infection in new england date back to at least the s, it was not until the s that lyme disease became recognized as a significant public health problem. michael begon (ostfeld et al., ) uses lyme disease to explore the question of whether having multiple hosts for a given pathogen will amplify or inhibit the potential spread of an infectious disease to humans. a variety of mammals and birds that live in the northeastern united states are known to serve as reservoirs for spreading b. burgdorferi to humans. based on ecological studies of the incidence of lyme disease in recent years, including a series of manipulative experiments with mammals, it appears that the risk of dissemination of this zoonotic infection to humans has been greater when there is relatively less diversity among potential host reservoirs. the full picture of which factors are most responsible for periods of increased clinical cases is quite complex, and many other ecological issues impact the intensity of lyme outbreaks in humans. examples include boom years in acorn production which can increase the number of deer reservoirs and thereby the number of ticks, and human behaviors such as the frequency of using tick repellant when working or playing outdoors in endemic areas. in our book (mayer and pizer ) , infectious disease specialist, gary wormser, and vector researchers, richard falco and thomas daniels, discuss how suburbanizing the landscape worked to create a favorable environment for lyme disease. from the s onward, the suburbs grew % faster than the cities they surround. farmland was rapidly replaced by clustered housing separated by parks and wooded areas. while the net amount of forest did not decrease significantly after world war ii, the suburbanization created a new landscape of clustered homes separated by patches of wooded land. the ideal house in the suburbs has lawn in front and back for children and pets to play, and is situated at the end of a quiet cul-de-sac street that is surrounded by conservation land. while no doubt idyllic for young families, the suburban landscape design also put people, mammals, birds, and ticks in close proximity, thereby exposing them and their pets to the bite of ticks infected with b. burgdorferi. without an effective vaccine, prevention so far has depended largely on public health education around personal protection activities and behavior modification. this means using repellant when outside and promptly removing ticks from people and pets. despite public education efforts, the number of new cases of lyme has been increasing steadily. it just is not practical to keep children and pets indoors during the summer, or always send them outside well-covered from head to toe and sprayed with deet insecticide. meanwhile, most suburban communities have been against public prevention strategies that could be aimed at controlling vectors and reservoir hosts, like widespread pesticide spraying and culling deer populations. research shows that lyme disease is driven largely by the abundance of nymphal i. scapularis (ostfeld et al. ) . looking ahead, it would seem that strategies for controlling lyme outbreaks are likely to be informed by ecological studies such as those described by begon in infectious disease ecology. there are other examples in this text of how basic ecology helps inform the public health approach to infectious disease outbreaks in humans. in chapter , johnson and carpenter discuss consequences of eutrophication, which occurs when excessive nutrients, like nitrogen and phosphorous from sewage and fertilizer run off, produce plant overgrowth and decay. the algal blooms that result can adversely impact water quality and marine life, as well as create opportune environments for microbial pathogens to flourish that can cause human disease. eutrophication has been associated with outbreaks of a type of allergic dermatitis (''swimmer's itch'') that occurs when trematode cercariae invades the skin, as well as cholera and malaria. in addition to eutrophication, land clearing produces large numbers of temporary pools for mosquitoes to breed and spread malaria. on the other hand, if eutrophication is not abetted by other human influences, such as overwhelmed sewerage systems in developing world slums in mega-cities, or poor sanitation in public bathing areas, its sequelae may not result in appreciable clinical and public health problems. to the modern ecologist, infectious disease outbreaks are inherent events that inevitably will occur and be structured by specific ecosystem dynamics. basic research is needed to understand how and why pathogenic organisms, and the reservoirs and vectors associated with their spread to humans, are able to thrive in different environments. although ecology is a theoretical discipline that does not focus principally on predicting the next emerging or reemerging pathogen or how it can be controlled, it is increasingly being applied to these questions and, over time, it will likely play a more important role in infectious disease control and public health planning. kenneth h. mayer the aids pandemic: impact on science and society unaids. unaids/who aids epidemic update keesing f ( ) climate, deer, rodents, and acorns as determinants of variation in lyme-disease risk key: cord- -dgqevs r authors: piel, s.; kreuter, m.; herth, f.; kauczor, h.-u.; heußel, c.-p. title: pulmonale granulomatöse erkrankungen und pulmonale manifestationen systemischer granulomatosen: inklusive tuberkulose und nichttuberkulöse mykobakteriosen date: - - journal: radiologe doi: . /s - - -z sha: doc_id: cord_uid: dgqevs r clinical/methodical issue: granulomas as signs of specific inflammation of the lungs are found in various diseases with pulmonary manifestations and represent an important imaging finding. standard radiological methods: the standard imaging modality for the work-up of granulomatous diseases of the lungs is most often thin-slice computed tomography (ct). there are a few instances, e. g. tuberculosis, sarcoidosis and silicosis, where a chest radiograph still plays an important role. methodical innovations: further radiological modalities are usually not needed in the routine work-up of granulomatous diseases of the chest. in special cases magnetic resonance imaging (mri) and positron emission tomography (pet)-ct scans play an important role, e. g. detecting cardiac sarcoidosis by cardiac mri or choline c‑ pet-ct in diagnosing lung carcinoma in scar tissue after tuberculosis. performance: the accuracy of thin-slice ct is very high for granulomatous diseases. achievements: in cases of chronic disease and fibrotic interstitial lung disease it is important to perform thin-slice ct in order to diagnose a specific disease pattern. thin-slice ct is also highly sensitive in detecting disease complications and comorbidities, such as malignancies. given these indications thin-slice ct is generally accepted in the routine daily practice. practical recommendations: a thin-slice ct and an interdisciplinary discussion are recommended in many cases with a suspected diagnosis of pulmonary granulomatous disease due to clinical or radiographic findings. hintergrund granulome [ ] , mod. nach kirsten [ ] , reiser et al. [ ] und kaufmann et al. [ ] . nichttuberkulöse mykobakteriosen und pilzinfektionen. charakteristisch ist die spezifische, granulomatöse entzündung mit ausbildung von granulomen als mikroskopische ansammlung epitheloider histiozyten. granulome können je nach ihrer genese zentrale nekrosen aufweisen, mehrkernige riesenzellen bilden, aber auch eosinophile granulozyten enthalten. die häufigste erkrankung mit einer granulomatösen entzündung der lunge ist die sarkoidose [ , ] . im folgenden wird auf die häufigsten bzw. klinisch relevantesten krankheitsbilder eingegangen, insbesondere auf die typischen radiologischen befunde in der dünnschichtcomputertomographie der lunge und ihre klinische signifikanz. das radiologische bild in der ct ist ebenso heterogen und führt nur im zusammenhang mit anamnestischen, klinischen, serologischen und pathologischen be- [ ] . weitere wichtige extrapulmonale manifestationen der sarkoidose betreffen haut ( %), herz ( - %), augen %, zentrales nervensystem ( %) und selten nieren [ , ] . typisch für das radiologische stadium i und ii ist die bihiläre lymphadenopathie (. abb. ). die lymphknoten zeigen in der regel keine zeichen einer nekrose sowie keine relevanten raumfordernden effekte [ ] . kavernen sind selten (. abb. ). bei langem krankheitsverlauf können sie verkalkungen aufwei-sen [ ] ; in der mrt kann schon früh ein schießscheibenzeichen der lymphknoten erkennbar sein (. abb. ; [ ] die häufigste pulmonale manifestation der sarkoidose sind mikronoduli und noduli mit einem durchmesser von - mm und unscharfer begrenzung. diese haben eine "random", d. h. eine "zufällige" verteilung, also sowohl zentrilobulär als auch perilymphatisch, wobei letzteres richtungsweisend für eine sarkoidose spricht. typischerweise liegt eine betonung der mittel-und oberfelder vor. dichteanhebung und irregulärer verlauf der bronchovaskulären bündel werden häufig beobachtet. oft konfluieren mehrere mikronoduli zu einem größeren knoten > mm, umgeben von zahlreichen kleinen, satellitenartig angeordneten mikronoduli, von nakatsu [ ] mit dem begriff "sarcoid galaxy" bezeichnet (. abb. ). konsolidierungen mit oder ohne bronchopneumogramm treten bei - % der patienten auf [ ] . retikuläre zeichnungsvermehrung sowie milchglasinfiltrate kommen ebenso vor und sind ein hinweis auf den Übergang der erkrankung in eine fibrosierende form (. abb. ). interstitielle lungenerkrankung · dünnschichtcomputertomographie · interdisziplinäre diskussion · ct-muster clinical/methodical issue. granulomas as signs of specific inflammation of the lungs are found in various diseases with pulmonary manifestations and represent an important imaging finding. standard radiological methods. the standard imaging modality for the work-up of granulomatous diseases of the lungs is most often thin-slice computed tomography (ct). there are a few instances, e. g. tuberculosis, sarcoidosis and silicosis, where a chest radiograph still plays an important role. methodical innovations. further radiological modalities are usually not needed in the routine work-up of granulomatous diseases of the chest. in special cases magnetic resonance imaging (mri) and positron emission tomography (pet)-ct scans play an important role, e. g. detecting cardiac sarcoidosis by cardiac mri or choline c- pet-ct in diagnosing lung carcinoma in scar tissue after tuberculosis. performance. the accuracy of thin-slice ct is very high for granulomatous diseases. achievements. in cases of chronic disease and fibrotic interstitial lung disease it is important to perform thin-slice ct in order to diagnose a specific disease pattern. thin-slice ct is also highly sensitive in detecting disease complications and comorbidities, such as malignancies. given these indications thinslice ct is generally accepted in the routine daily practice. practical recommendations. a thin-slice ct and an interdisciplinary discussion are recommended in many cases with a suspected diagnosis of pulmonary granulomatous disease due to clinical or radiographic findings. interstitial lung disease · thin slice computed tomography · interdisciplinary discussion · ct pattern im radiologischen stadium iv stehen fibrotische veränderungen im vordergrund. es zeigen sich deformierungen und distorsionen der bronchien, konsolidierungen, rundherde, retikuläre zeichnungsvermehrung, seltener traktionsbronchiektasen ( %), honigwabenmuster ( %) v. a. in den oberlappen, emphysem ( %) und zysten ( %) [ ] . dabei kann sich ein gewöhnliche-interstitielle-pneumonie(uip)-muster ergeben, welches aufgrund seiner apikalen betonung von dem für eine idiopathische pulmonale fibrose (ipf) typischen uip-muster mit basaler betonung manchmal unterschieden werden kann (. abb. ) [ ] . die dünnschicht-ct ist damit der diagnostische standard bei verdacht auf eine eaa, gerade auch bei unauffälliger thoraxübersichtsaufnahme [ ] . im akuten und subakuten stadium ist ein diffuses milchglasmuster v. a. in mittel-und unterfeldern zu erwarten (. abb. ). bei der subakuten form zeigen sich zusätzlich auch unscharf begrenzte, "wattebauschartige" zentrilobuläre knötchen sowie ein mosaikmuster durch "airtrapping" bei bronchiolitis der terminalen bronchiolen. letzteres kann in der exspirations-ct deutlicher gesehen werden. [ ] . zusammenfassend kann eine abgrenzung zu den differenzialdiagnosen nur im interdisziplinären kontext erfolgen. die granulomatose mit polyangiitis (gpa, früher morbus wegener) ist zu-sammen mit der mikroskopischen polyangiitis (mpa) und der eosinophilen granulomatose mit polyangiitis (egpa, früher churg-strauss-syndrom) eine anca-assoziierte (antineutrophile zytoplasmatische antikörper) vaskulitis der kleinen gefäße [ ] . die diagnostischen kriterien der gpa (in anlehnung an die klassifikationskriterien des american college of rheumatology, acr) sind ulzerierend-hämorrhagische entzündungen der nasen-oder mundschleimhaut, pulmonale infiltrate, granulome oder kavernen, ein nephritisches urinsediment und granulomatöse entzündungen in der histologie. somit ist auch hier die entscheidende bedeutung der thorakalen bildgebung ersichtlich. das konventionelle thoraxröntgen ist unzureichend für den nachweis kleinerer veränderungen und die zuordnung zu einem bestimmter muster, daher ist eine ct-untersuchung bei verdacht auf eine gpa zielführend. die beurteilung der interstitiellen beteiligung gelingt in der dünnschicht-ct. in seltenen fällen kann auch eine kontrastmittel-ct hinzugezogen werden, z. b. bei hämoptysen zur blutungsquellensuche und ausschluss einer lungenarterienembolie. da eine nierenbeteiligung vorliegen kann, sollte davor eine gründliche nutzen-risiko-abwägung erfolgen. die typischen pulmonalen manifestationen betreffen sowohl das tracheobronchialsystem als auch das lungenparenchym. tracheobronchial kommt es in - % der fälle zu stenosierungen, oft an mehreren lokalisationen [ ] ; am häufigsten ist dabei die subglottische trachealstenose. ferner treten schleimhautnekrosen an den unteren atemwegen auf, aus denen oft stenosen, postobstruktive infektionen und inflammatorische pseudotumoren hervorgehen. darüber hinaus kommt es manchmal zur tracheobronchialen malazie, ösophagobronchialen fisteln und bronchiektasen. der trachealbefall kann uni-oder multifokal sein und ist im mittel - cm lang. charakteristisch ist der befall der gesamten trachea inklusive ihrer hinterwand (pars membranacea), die bei anderen erkrankungen oft ausgespart bleibt. die häufigste veränderung (prävalenz von %) in der ct ist der rundherd im lauf der erkrankung [ ] mit neigung zur zentralen nekrose (in % der fälle, v. a. bei knoten > cm) und kavernenbildung (. abb. ) . diese können sich rasch entwickeln und auf die thoraxwand übergreifen. ein halozeichen zeigt sich dabei in % der fälle, seltener ein reverse-halo-oder atollzeichen. die rundherde haben eine variable verteilung, häufig bestehen auch wechselnde infiltrate. oft beobachtet man ohne adäquate therapie eine kavernenbildung, unter behandlung dagegen können kavernen ausheilen und plattenartige vernarbungen ausbilden. knoten, die nicht mit klinischer oder serologischer aktivität der erkrankung korrelieren und größenprogredient sind, sollten aufgrund des -fach erhöhten malignitätsrisikos bei der gpa invasiv abgeklärt werden [ , ] . ebenso kann es im rahmen der vaskulitis zu einer kapillaritis im lungenparenchym kommen. eine alveoläre hämorrhagie tritt dann bei ca. % der patienten mit kapillaritis auf [ ] . bei einer akuten alveolären hämorrhagie zeigen sich milchglasinfiltrate. bei rezidivierenden blutungen können aber auch retikuläre zeichnungsvermehrungen, ein peripheres honigwabenmuster und traktionsbronchiektasen entstehen [ ] . sowohl bei bei gpa als auch viel häufiger bei mpa ist die entwicklung einer fibrose mit uip-muster, seltener mit nsip-muster, möglich und kann anderen klinischen manifestationen der vaskulitis vorausgehen [ , ] . nebenbefundlich werden pleuraergüsse in bis zu % der fälle beobachtet, entweder als zeichen des pleurabefalls oder als hydropische dekompensationszeichen bei nierenbefall. selten zeigen sich auch mediastinale lymphknotenvergrößerungen (. abb. ). die silikose (quarzstaublungenerkrankung) entsteht durch einwirkung alveolengängiger staubpartikel, die kristalli-nen quarz (siliziumdioxid, sio ), cristobalit oder tridymit enthalten [ ] . die silikose ist eine der ältesten bekannten berufskrankheiten (in deutschland gelistet unter bk ). sie ist auch die häufigste pneumokoniose (staublungenerkrankung) [ ] . zur exposition kommt es v. a. bei arbeiten in stollenminen mit quarzreichem gestein, bei der formsandverarbeitung, in der steinverarbeitung der glas-, porzellanund keramikindustrie und auch bei sandstrahlarbeiten. pathophysiologisch kommt es nach inhalation alveolengängiger staubpartikel zu einer phagozytose durch alveolarmakrophagen, danach zu deren zerfall, was eine entzündungsreaktion mit anschließender granulombildung und überschießendem bindegewebigem umbau im interstitium auslöst. bei lymphogener elimination der staubpartikel können ähnliche veränderungen in den hiluslymphknoten nachgewiesen werden. lymphknoten mit typischen verkalkungen bezeichnet man als sogenannte eierschalenlymphknoten [ ] . im verlauf der krankheit können granulome zu größeren schwielen konfluieren, die zentrale nekrosen ausbilden können [ ] . die erkrankung kann in verschiedenen formen auftreten: die akute und die klassische silikose. die wichtigste ursache für die akute silikose ist eine massive exposition gegenüber auslösenden noxen. es kommt zu einem frühen erkrankungsbeginn innerhalb von jahren. die aku- die radiologischen befunde der silikose werden nach der international labour organization (ilo) klassifiziert. dort werden "kleine schatten" auf dem lungenbild mit rundlicher, unregelmäßiger und gemischter form ebenso erwähnt wie "große schatten". die klassifikation erfolgt anhand von größe, ausdehnung und befallenen feldern. bezüglich der pleura gehen diffuse pleuraverdickungen, pleuraplaques und die adhärenz des kostophrenischen winkels in die bewertung ein [ ] . selten ist der einsatz einer dünnschicht-ct notwendig, empfohlen wird dies laut der aktuellen s -leitlinie bei fortwährender diagnostischer unsicherheit nach einer zweitbegutachtung der röntgenübersicht im rahmen der diagnostik der berufskrankheit. die ct soll in low-dose-technik und nach standardisierten vorgaben erfolgen [ ] . das risiko für das auftreten eines bronchialkarzinoms, einer tuberkulose (in diesem fall silikotuberkulose) und einer kollagenose ist bei silikosepatienten erhöht. ebenso besteht eine gesteigerte infektanfälligkeit, sodass opportunistische infektionen, u. a. invasive mykosen, ebenso gehäuft auftreten. daher kann insbesondere bei akut kranken patienten oder inkonklusiver röntgenübersicht die ct von hoher klinisch-diagnostischer bedeutung sein [ , ] . bei der langerhans-zell-histiozytose (lch) handelt es sich um eine seltene erkrankung, die sowohl als klonale, generalisierte form im kindes-und jugendalter als auch lokalisiert und im erwachsenenalter auftreten kann. im folgenden wird über die diffuse pulmonale verlaufsform des erwachsenen berichtet (plch). die plch tritt bei rauchern auf und ist geht mit einer proliferation der langerhans-zellen in den atemwegen einher. histologisch zeigen sich granulome mit langerhans-zellen und eosinophilen granulozyten. das konventionelle röntgenbild ist in der regel nicht diagnostisch. in der hochauflösenden ("high resolution", hr) ct sieht man oft das pathognomonische "schrotschussmuster": irregulär verteilte, die ganze lunge betreffende, unscharf begrenzte zentrilobuläre noduli ( - mm im durchmesser), selten größere noduli mit durchmesser > mm, die teils zentral einschmelzen können, sowie begleitende zystische läsionen (. abb. ). die zysten sind oft kleiner als cm, unregelmäßig, zu beginn dick-und im späteren stadium der erkrankung dünnwandig (wanddi-cke < mm). ober-und mittelfelder sind vermehrt betroffen, die herde sind wahllos vom zentrum nach außen verteilt. selten treten milchglasinfiltrate und eine paratracheale und mediastinale lymphadenopathie auf [ ] . das charakteristische ct-bild erlaubt bei einem raucher im jüngeren alter auch ohne eine bioptische sicherung bei fehlen sonstiger pathologien die diagnose einer langerhans-zell-histiozytose [ , ] . unter den infektiösen granulomatosen sind an erster stelle die tuberkulose und die nichttuberkulösen mykobakteriosen zu erwähnen [ ] . eine immer noch große rolle bei screening, erstdiagnose und verlaufs-kontrolle der tuberkulose spielt das konventionelle röntgenbild [ ] . die ct besitzt jedoch sowohl eine höhere sensitivität (bis zu %) als auch einen höheren negativen prädiktiven wert (bis zu % [ ] ). eine höhere sensitivität zeigt sich insbesondere bei kleinen befunden oder beim nachweis von komplikationen [ , ] . in den seltenen fällen einer chirurgischen therapienotwendigkeit ist eine ct in der planung unverzichtbar [ , , ] . bezüglich nomenklatur, einteilung, diagnostik und mögliche manifestationen der tuberkulose verweisen wir auf den gleichnamigen artikel von kienzl-palma in diesem heft. die nichttuberkulösen mykobakteriosen (ntm) umfassen eine gruppe von erkrankungen, die von mykobakterien verursacht werden, die nicht dem mycobacterium-tuberculosis-komplex und nicht m. leprae zugerechnet werden. die erreger sind in verschiedenen umweltbereichen weit verbreitet. oft sind die betroffenen hiv-kranke, immunsupprimierte oder solche mit einer pulmonalen grunderkrankung. die klinik ist unspezifisch und besteht v. a. in produktivem husten, hämoptysen, fieber und gewichtsverlust. die lunge ist das hauptmanifestationsorgan, aber auch diverse extrapulmonale manifesta- granulomatöse lungen-und systemerkrankungen world association of sarcoidosis and other granulomatous disorders (wasog) ( ) statement on sarcoidosis abdominal sarcoidosis: crosssectional imaging findings ats/ers/wasog statement on sarcoidosis the pattern and distribution of calcified mediastinal lymph nodes in sarcoidosis and tuberculosis: a ct study the dark lymph node sign on magnetic resonance imaging: a novel finding in patients with sarcoidosis a clinical interpretation of bilateral hilar adenopathy large coalescent parenchymal nodules in pulmonary sarcoidosis: "sarcoid galaxy" sign sarcoidosis mit pulmonary fibrosis: ct patterns and correlation with pulmonary function empfehlungen zu diagnostik der exogen allergischen alveolitis headcheese sign klassische muster der interstitiellen lungenerkrankungen chronic hypersensitivity pneumonitis: differentiation from idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia by using thin-section ct hypersensitivity pneumonitis common and uncommon manifestations of wegener granulomatosis at chest ct: radiologic-pathologic correlation management solider pulmonaler rundherde ungewöhnliches pulmonales befallsmuster bei m. wegener pulmonary manifestations of anti-neutrophil cytoplasmic antibody (ancapositive) vasculitis pulmonary fibrosis indicative of granulomatosis with polyangiitis initially believed to be sjögren's syndrome merkblatt zu berufskrankheit nr. der anlage zur berufskrankheiten-verordnung institute of pathology heidelberg richtlinien für die anwendung der internationalen klassifikation des iaa von pneumokoniosen-röntgenfilmen, überarbeitete auflage diagnostik und begutachtung der berufskrankheit nr. quarzstaublungenerkrankung (silikose) hämoptoe bei einem patienten mit schwerer copd und silikose lungenkrebs durch die einwirkung von kristallinem siliziumdioxid bei nachgewiesener quarzstaublungenerkrankung (silikose odeer silikotuberkulose) pulmonary langerhans'-cell histiocytosis klinische pneumologie die lungentuberkuloseaktuelle radiologische diagnostik einer alten krankheit utility of ct in the evaluation of pulmonary tuberculosis in patients without aids empfehlungen zur diagnostik und therapie nichttuberkulöser mykobakteriosen des deutschen zentralkomitees zur bekämpfung der tuberkulose (dzk) und der deutschen gesellschaft für pneumologie und beatmungsmedizin (dgp) comparative chest computed tomography findings of non-tuberculous mycobacterial lung diseases and pulmonary tuberculosis in patients with acid fast bacilli smear-positive sputum radiologie. urban & fischer, münchen . conant ef ( ) pulmonary sarcoidosis in the older patient: conventional radiographic features clinical and radiographic correlates ofprimaryandreactivationtuberculosis: amolecular epidemiology study chronic diffuse interstitial lung disease: diagnostic value of chest radiography and high-resolution ct pulmonarytuberculosis: uptodate imaging and management pulmonary tuberculosis: comparison of ct findings hiv-seropositive and hivseronegative patients the clinical assessment of roentgenographically atypical pulmonary sarcoidosis distribution of thoracic lymphadenopathy in sarcoidosis usind computed tomography unusual manifestations of thoracic sarcoidosis pulmonary findings in churg-strauss syndrome in chest x-rays and high resolution computed tomography at the time of initial diagnosis silikose. electronic learning institute of pathology heidelberg die aktuelle tuberkulosesituation in deutschland und die auswirkungen der globalen situation aneurysms complicating inflammatory diseases in immunocompromised, value of contrast enhanced ct glossar thoraxradiologischer begriffe entsprechend der terminologie der fleischner society chronic pulmonary berylliosis: imaging with high-resolution computerized tomography extrapulmonale tuberkulose: die radiologische bildgebung eines fast vergessenen verwandlungskünstlers prognosis of intrathoracic sarcoidosis in england inzwischen wurden multiple weitere erkrankungen beschrieben, die eine pulmonale granulomatöse entzündung verursachen können, u. a. eine interstitielle lungenerkrankung bei rheumatoider arthritis, die bronchozentrische granulomatose, die lymphomatoide granulomatose, berylliose, pilzinfektionen sowie medikamentös induzierte granulomatöse lungenerkrankungen. key: cord- -i on authors: nan title: abstracts dgrh-kongress date: - - journal: z rheumatol doi: . /s - - - sha: doc_id: cord_uid: i on nan im namen der dgrh, der dgorh und der gkjr begrüßen wir sie ganz herzlich zu unserem diesjährigen kongress visualisierung therapeutischer effekte von vasodilatantien beim sekundären raynaud-syndrom mittels fluoreszenzoptischer bildgebung di. stellenwert der gelenksonographie bezüglich diagnose, behandlung und therapiekontrolle der bursitis intermetatarsalis -einer häufig übersehenen differenzialdiagnose. fünf fallbeispiele wegen der deutlich eingeschränkten nierenfunktion konnten therapeutisch keine nsar angewandt werden. wir haben mit × , mg colchicin täglich behandelt. die anfänglich schwerkranke bettlägerige patientin konnte innerhalb von h mobilisiert werden. um eine abschließende sicherung der diagnose einer uratinduzierten sakroiliitis erreichen zu können ist die patientin mit einem dual-energy-ct (dect) untersucht worden. ergebnisse. mit dieser methode konnten gichttophi in beiden sakroiliakalgelenken dargestellt werden, ebenso an beiden mtp -gelenken. schlussfolgerung. aktuell liegen bisher noch keine weiteren berichte vor, dass diese methode auch für die diagnostik einer gicht im bereich der sakroiliakalregion zuverlässige ergebnisse liefern kann. zudem zeigt dieser krankheitsverlauf, dass sich die gicht durchaus primär im bereich des achsenskeletts manifestieren kann und nicht in erster linie an den peripheren gelenken zu entsprechenden beschwerden führen muss. a. glimm , s. werner , s. ohrndorf , c. schwenke , g. schmittat , g. burmester einleitung. typische pathologische veränderung bei der rheumatoiden arthritis (ra) ist die synovialitis. auch bei der osteoarthrose (oa) lassen sich entzündliche veränderungen der gelenke finden. diese können mittels fluoreszenzoptischer bildgebung (foi) und dem gelenkultraschall (us) sichtbar gemacht werden. ziel der studie: vergleich der foi mit dem us bei patienten mit ra und oa. methoden. es wurde bei patienten ( ra, oa) die foi beider hände sowie die us des handgelenks (hg) und der fingergelenke (mcp, pip, dip) der klinisch beschwerdeführenden hand von dorsal und palmar sowohl im b-bild (b-us) als auch mit power-doppler (pd-us) durchgeführt. synovialitis und tenosynovitis im us sowie die intensität des fluoreszenzsignals im bereich der gelenke in der foi wurden qualitativ als auch semiquantitativ nach standardisierten verfahren für den primavistamode (pvm) und drei verschiedene phasen (p - ; [ ] ) bewertet. in der statistischen analyse wurden anschließend sensitivitäten und spezifitäten für die foi bei der ra und oa getrennt für synovialitis und tenosynovitis, dorsal und palmar jeweils für b-us und pd-us als referenzmethode berechnet. ergebnisse. in abhängigkeit von der betrachteten phase zeigen sich für die ra und oa moderate sensitivitäten und spezifitäten. für die ra wurden in der phase des foi die höchsten sensitivitäten mit % für b-us und % für pd-us berechnet. auch bei der oa ergaben sich die höchsten sensitivitäten in der phase des foi mit % für b-us und % für pd-us als referenzmethode. die höchsten spezifitäten für beide diagnosen wurden in der foi in phase erreicht. hierbei lag die spezifität bei der ra für b-us bei % und für pd-us bei %. der höchste spezifitätswert bei der oa sowohl für b-us als pd-us war % (. tab. background. pet is a nuclear imaging technique that depicts functional processes within the body with high sensitivity by detecting annihilation radiation from radioactive decay of a positron-emitting radionuclide that was labeled to a biologically active molecule (tracer) and introduced into the body. f-fluoride ( f) can be used for pet as a bone-seeking agent reflecting bone perfusion and remodeling. we inaugurated a pilot study with simultaneous pet/mr to examine whether addition of pet provides different and additional information in comparison to mri in axspa patients. methods. eleven axspa patients, median age y, disease duration range . - y, mean basdai . , were examined by pet/ -tesla mri minutes after injection of a mean dose of mbq of f using a integrated whole-body pet/mr scanner (siemens biograph mmr®). t-mris were scored blinded to patient's clinical characteristics by two readers ( rheumatologist and radiologist/nuclear medicine specialist) using the berlin mri score and also by recording inflammatory lesions on a vertebral edge (ve) level. in a second step pet/mris were read blindly by the same readers also based on the ve involvement of individual vertebral bodies. results. the procedure was successful in all patients. the resulting mean effective radiation dose per patient was . msv. co-registration of pet/mri fusion images was highly accurate, allowing a precise comparison of mri and pet. in the direct comparison of the mri and pet signal the two readers saw consistent signals in almost % of the sites studied. however, there were areas where signals differed, e.g. within existing syndesmophytes where pet signal was increased but conventional mris showed no signal, or the sternum area and lateral or posterior spinal elements such as facets and spinous processes. conclusion. the new technique of integrated pet/mri provides similar imaging signals as conventional mri. however, we observed differences between the two modalities in areas with less inflammatory activity but where bone metabolism seemed to be active or in areas with blurred resolution on conventional mri. the possibility that pet detects osteoblastic activity in areas where no inflammatory signal is detected with mri seems to be of interest. einleitung. sensitiven bildgebenden verfahren wie der hochauflösenden arthrosonographie kommen bei der detektion initial entzündlicher veränderungen im rahmen der frühdiagnostik der psoriasisarthritis (psa) eine große bedeutung zu. die vorliegende prospektive studie untersucht die diagnostische und prognostische wertigkeit der sonographischen befunde im vergleich zur klinischen untersuchung auf ebene einzelner gelenke bei früher psoriasis-arthritis (psa). methoden. rekrutierung von patienten mit therapienaiver früher psa. sonographie von gelenken mit semiquantitativer graduierung (grad - ) von b-bild (gsus) und power-doppler-aktivität (pdus; baseline, monate). klinische parameter: anzahl druckschmerzhafter und geschwollener gelenke (tjc , sjc ), visuelle analogskala, das -crp, health assessment questionnaire haq. für jede followup-visite erfolgte eine kategorisierung des klinischen ansprechens nach eular-response-kriterien und der für die psa validierten minimal-disease-activity(mda)-kriterien (coates et al.). ergebnisse. baseline patienten, nach monate patienten, erkrankungsdauer ( ± , monate). patienten ohne therapie ( ), mit nsar ( ), steroid i.a. ( ) , dmards ( ), biologicals ( ). bei diagnosestellung zeigte sich eine signifikante korrelation zwischen dem us synovitis score und folgenden klinischen parametern: tjc (r= , ), scj (r= , ), das -crp (r= , ). nach monaten zeigte sich eine gute korrelation zwischen der relativen veränderung des us synovitis scores und der relativen veränderung folgender klinischer parameter: tjc (r= , ), haq (r= , ), pasi (r= , ), das -crp (r= , ). zu baseline waren von gelenken sonographisch auffällig, davon zeigten kein klinisches korrelat (subklinisch). nach monaten zeigten % der initial subklinischen gelenke einen unveränderten befund, % waren sonographisch nicht mehr auffällig und % wurden klinisch manifest. bei den klinischen respondern war der rückgang deutlicher ausgeprägt. schlussfolgerung. der ultraschall-synovitis-score korreliert mit klinischen aktivitätsparametern sowohl zum zeitpunkt der diagnosestellung als auch im krankheitsverlauf unter immunsuppressiver therapie. die subklinischen veränderungen bilden sich unter immunsuppressiver therapie zu einem großen teil zurück, deutlicher bei klinischen respondern. ein geringer anteil der initial subklinischen gelenke wird im verlauf klinisch manifest, in höherem maße bei klinischen non-respondern. t. diekhoff , k. hermann charité -universitätsmedizin berlin, radiologie, berlin einleitung. die gicht ist mit einer prävalenz von , - , % insbesondere in den industrieländern eine häufige erkrankungen, die mit gelenkschmerzen einhergeht. bei typischer symptomatik und laborkonstellation ist die diagnose der arthritis urica oft einfach zu stellen, ein atypisches beschwerdebild kann jedoch gelegentlich die abgrenzung zu anderen erkrankungen erschweren. besonders die kalziumpyrophosphat-kristallarthropathie (cppd oder pseudogicht), die selbst mit sehr variabler symptomatik auftreten kann, ist eine relevante differenzialdiagnose, besonders bei älteren patienten. methoden. mit der dual-energy-computertomographie (de-ct) steht ein modernes, innovatives verfahren zur verfügung, das eine detektion von harnsäurehaltigen weichteilverkalkungen ermöglicht und darüber hinaus eine sichere abgrenzung zu kalziumhaltigen verkalkungen gewährleisten kann. das prinzip der de-ct ist relativ simpel und seit längerem bekannt: die messung des untersuchungsvolumens mit zwei unterschiedlichen röhrenspannungen macht es möglich, einen schwächungskoeffizienten zu errechnen, der spezifisch für das untersuchte material ist. allerdings ermöglichten erst moderne cts mit zwei röntgenröhren die klinische anwendung. in jüngster zeit werden jedoch anstrengungen unternommen, die de-ct auch für ein-röhren-systeme verfügbar zu machen. ergebnisse. mit der de-ct können gichttophi sicher vom knochen aber auch von anderen verkalkungen getrennt und zum beispiel farblich kodiert dargestellt werden. im gegensatz zum konventionellen röntgenbild verspricht die de-ct jedoch nicht nur eine höhere sensitivität für tophöse veränderungen, sondern als schnittbildverfahren auch eine bessere abgrenzung und einordnung von anderen morphologischen veränderungen wie zum beispiel von erosionen. schlussfolgerung. dieser vortrag fasst die vor-und nachteile der de-ct in der detektion und abgrenzung von weichteilverkalkungen bei kristallarthropathien zusammen und gibt darüber hinaus einen ausblick auf zukünftige entwicklungen in diesem gebiet. background. anionic glycosaminoglycans interact with a variety of soluble and membrane bound molecules. chondroitin sulfate was shown to have anti-inflammatory properties but its role in arthritis is controversial. methods. we have analyzed the effect of chondroitin sulfate on collagen induced arthritis starting treatment before and after induction of arthritis and in mice with established arthritis. results. in all of these settings chondroitin sulfate significantly reduced the severity of arthritis. it prevented joint destruction, diminished the inflammatory infiltrate and reduced proinflammatory cytokines in joints and plasma. splenocytes restimulated with collagen produced less il- and more il- and il- . the beneficial effects of chondroitin sulfate were transient and closely correlated to the suppression of the collagen-specific humoral immune response. chondroitin sulfate, but not other glycosaminoglycans induced a direct btk and syk-dependent proliferation of b cells and markedly expanded the number of plasma cells in the spleen. in immunized mice chondroitin sulfate reduced the number of antigen specific plasma cells in the bone marrow and was able to suppress established humoral immune responses. conclusion. displacement of disease inducing plasma cells from the bone marrow might contribute to the beneficial effects of chondroitin sulfate and could be an attractive strategy to suppress antibody mediated autoimmunity. background. in rheumatoid arthritis a functional deterioration of the hpa-axis in form of inadequately low secretion of glucocorticoids in relation to severity of inflammation can be detected. the reasons for this phenomenon are not known. the purpose of this study was to find possible reasons responsible for adrenal insufficiency during arthritis. methods. da rats were immunized with type ii collagen in incomplete freund adjuvant to induce arthritis. plasma corticosterone was evaluated by ria and plasma acth by elisa. adrenal cholesterol was quan-titatively studied by sudan-iii staining and scavenger receptor class bi (sr-bi, the hdl receptor) by immunohistochemistry. fluorescent nbd-cholesterol uptake kinetics were analysed by flow cytometry. ultrastructural morphology of adrenocortical mitochondria and lipid droplets was studied by electron microscopy. results. initially increased corticosterone and acth levels were reduced to baseline levels in the later phase of the disease. serum levels of corticosterone relative to il- β were markedly lower in arthritic than control animals (inadequacy). cholesterol storage in adrenocortical cells and expression of sr-bi did not differ between immunized and control rats. however, number of impaired mitochondria largely increased during the course of arthritis (maximum on day ), and this was paralleled by reduced numbers of activated cholesterol droplets (inhomogenous droplets relevant for generation of glucocorticoids). in addition, number of normal mitochondria positively correlated with serum corticosterone levels. conclusion. this first study on adrenal reasons for inadequate glucocorticoid secretion in arthritis demonstrated impaired mitochondria and altered cholesterol breakdown paralleled by low corticosterone levels in relation to ongoing inflammation. justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main, universitätsklinikum gießen und marburg, orthopädische klinik, labor für experimentelle orthopädie, gießen, universitätsklinikum gießen und marburg, orthopädie und orthopädische chirurgie, gießen, universitätsklinikum erlangen, medizinische klinik , rheumatologie und immunologie, erlangen background. obesity is a risk factor in osteoarthritis (oa), but there is limited information about the interaction between bone formation and adipose tissue-derived factors, the so-called adipokines. adipokines such as adiponectin, resistin or visfatin are associated with the pathogenesis of rheumatoid arthritis (ra) and oa. adipokines are produced also by other cell types than adipocytes in ra and oa joints, for example osteoblasts, osteoclasts or chondrocytes. however, in contrast to their joint-destructive role in ra, their role in oa joint remodeling is unclear. therefore, adipokine expression in osteophyte development and bone forming cells as well as their effect on these cells was analyzed. methods. osteophytes and bone were obtained from oa patients during joint replacement surgery. serial sections of bone tissue were stained (masson trichrome, trap) and scored from grade one (no ossification, mainly connective tissue and cartilage) to five (ossified, mineralized osteophyte, < % connective tissue, ossified remodeling zones). immunohistochemistry against alkaline phosphatase, collagen-type ii, adiponectin, resistin, and visfatin was performed. oa osteoblasts were stimulated with adiponectin and measurements of il- , il- and mcp- were performed in cell culture supernatants. results. adiponectin, resistin and visfatin were detectable in osteoblasts and all osteophyte grades. in non-ossified osteophytes (grade ), especially adiponectin and to a lower extend resistin and visfatin were localized in connective tissue fibroblasts. in ossified osteophytes (grade - ), resistin, visfatin and to a lower extend adiponectin protein expression was co-localized with osteoblasts. resistin and visfatin were expressed by osteoclasts. visfatin was found in chondrocytes of all osteophyte grades ( % of chondrocytes) and adiponectin was detectable in blood vessels. osteoblast stimulation with adiponectin increased the release of the inflammatory mediators il- ( . -fold), il- ( . -fold), and mcp- ( . -fold). zeitschrift für rheumatologie suppl · | conclusion. the expression of adiponectin and visfatin expression in osteophyte connective tissue and cartilage suggests their involvement in early osteophyte formation. resistin and visfatin expression by osteoblasts and osteoclasts in ossified osteophytes indicates a role in bone remodeling of osteophytes at later stages. osteoblasts respond to adiponectin stimulation with the release of inflammatory mediators. therefore, adipokines are most likely involved in osteophyte formation at different stages affecting different cell types of bone remodeling. free fatty acids contribute to promotion of arthritis k. frommer , a. schäffler , s. rehart , a. sachs , u. müller-ladner , e. neumann justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, universitätsklinikum regensburg, klinik und poliklinik für innere medizin i, regensburg, agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main background. obesity is a known risk factor for several arthritic diseases and mechanical stress has been shown not to be the only factor. due to increased levels of free fatty acids (ffa) in obese compared to nonobese individuals and due to the involvement of ffa in inflammatory cardiovascular and metabolic diseases, we hypothesized that ffa play a role in the promotion of arthritic diseases. therefore, we therefore investigated the effect of ffa on various effector cells of arthritis. methods. rheumatoid (ra) synovial fibroblasts (sf), osteoarthritis (oa) sf, psoriatic arthritis (psa) sf, human primary chondrocytes (hch), human osteoblasts (ob), human macrovascular (huvec) and microvascular (hbdmec) endothelial cells were stimulated in vitro with different ffa within their physiological range of concentrations. immunoassays were used to quantify ffa-induced protein secretion. sulfosuccinimidyl oleate sodium (sso) was used to inhibit fatty acid translocase (fat). results. ffa dose-dependently increased the secretion of the proinflammatory factors (il- , il and mcp- ) as well as matrix-degrading enzymes (mmp- and mmp- ) in rasf (e.g. for lauric acid [ µm] with rasf/il- : . -fold increase; il : . fold increase; mcp- : . fold increase; pro-mmp : . -fold increase; mmp- : . fold increase). saturated and unsaturated ffa had similar effects on rasf. however, saturated ffa induced strong secretion of il- in chondrocytes, while unsaturated ffa only had a weaker effect on this cell type. at µm, both saturated and unsaturated ffa significantly increased il- secretion by osteoblasts to a similar degree as for sf. a high concentration of ffa ( µm) significantly induced il- secretion in huvec and hbdmec, whereas a low concentration of ffa ( µm) did not have a significant effect (p> . ) on human endothelial cells. blocking ffa transport into rasf by using sso almost completely abolished the effect of palmitic acid on il- secretion. conclusion. ffa are not only metabolic substrates but can also directly contribute to articular inflammation and degradation mediated by various effector cells of arthritis. our data also show that ffa transport into the cell is required for ffa-induced effects in sf. background. chronically inflamed tissues in ra are characterized by local hypoxia and enhanced angiogenesis. the hypoxia inducible factor (hif)- and (hif)- serve as key regulators of adaptation to hypoxia thereby promoting both angiogenesis and metabolic adaptation of endothelial cells. to investigate the impact of hif- /hif- on the angiogenic and metabolic transcriptome under hypoxia ( % o ) versus normoxia ( % o ) we performed a knockdown of either hif- α or hif- α in human microvascular endothelial cells (hmec). methods. specific knockdown of either hif- α or hif- α was achieved using shrna-technology. angiogenic and metabolic transcriptome of hmecs was studied by performing an agilent human whole genome microarray under normoxia vs hypoxia. significantly regulated genes were allocated to angiogenic and metabolic processes using panther database. results. in comparison to normoxia the incubation of untransduced hmecs under hypoxia resulted in regulated angiogenesis related genes and regulated cellular metabolism related genes. in both hif- α and hif- α knockdown cells, hypoxia was still capable of inducing a differential gene expression pattern, but with a much less pronounced effect compared to control cells. analysis of angiogenesis related processes (vegf-pathway, hif-activation, egfr-pathway) showed that % of the differentially expressed genes are controlled by both hif- and hif- . another % of the regulated genes are controlled by hif- . the remaining % of regulated genes are under control of hif- . the differentially regulated genes involved in the cellular metabolism (atpsynthesis, glycolysis, tca-cycle) were found to be to % controlled by both hif- and hif- . the remaining % are dependent on the presence of hif- . conclusion. hif- and hif- are both key regulators of the adaptation of endothelial cells towards hypoxia with overlapping functions. however, they do differ in their capacity to regulate cellular energy metabolism and angiogenesis. this leads us to conclude that hif- affects angiogenesis via indirect effects on cellular energy metabolism as indicated by the regulation of metabolic transcriptome to one fifth. hif- does more influence angiogenesis directly via regulating the synthesis of proangiogenic factors (as has been previously shown).these findings provide new insights into the divergent regulation of angiogenesis in inflamed (hypoxic) tissues by hif- and hif- and are, therefore, considered to be of clinical relevance in ra. background. membrane bound glucocorticoid receptors (mgr) play a pivotal role in pathogenesis of chronic inflammatory diseases as indicated by clinical observations. patients with sle show high frequencies of mgr positive monocytes, sometimes even higher than found in patients with active ra. with increasing glucocorticoid dosages expression of mgr on monocytes of sle-patients is downregulated, suggesting a negative feedback loop to control glucocorticoid action. these receptors represent an effective target for diagnosis and monitoring of different inflammatory diseases, but a feasible detection method is still necessary. objectives. we compare two methods of high-sensitive immunofluorescence staining -the well established liposome procedure with the commercialized faser-technique. methods. hek t cells were cultured for h with/without µg/ml brefeldin a in a humidified incubator at °c. human cd positive t cells and cd positive monocytes were isolated via magnetic-activated cell sorting and subsequently cultured in rpmi . monocytes were incubated for h with/without µg/ml lps. for liposome based highsensitivity immunofluorescence staining cells were incubated with the monoclonal (digoxigenin conjugated) anti-gr antibody, followed by incubation with anti-digoxigenin/anti-biotin matrix. subsequently biotinylated cy liposomes were added. faser technique was performed as described by the manufacturer (miltenyi biotec). dead cells were excluded by adding pi before cell acquisition, using a bd facs calibur flow cytometer. the acquired data were analyzed using flowjo . . software. results. the human mgr, which cannot be reliably detected with conventional staining methods, is detectable with the liposome procedure as well as with the commercialized faser-apc technique. furthermore, the faser-apc-procedure is more sensitive ( . % vs . %) and more specific ( . % vs. . %) compared to the liposome technique. additionally, minor changes of mgr expression can also be demonstrated with the faser technique. the faser procedure shows technical advantages: the commercially available faser-apc-kit is performed according to a standarized protocol and is less time consuming compared to the liposome procedure. conclusion. the human mgr is easily detectable with the commercialized faser kits, which represent an alternative due to a consistent quality and a standardized production. this method facilitates the analysis of the role that mgr play in the pathogenesis of chronic inflammatory diseases and perhaps provoke new insights in glucocorticoid therapy. background. in previous studies we detected th-positive, catecholamine-producing cells in inflamed hypoxic synovial tissue. therefore, the aim of our study was to investigate the influence of hypoxia induced catecholamines on inflammatory responses in arthritis. methods. synovial cells of rheumatoid arthritis (ra) and osteoarthritis (oa) patients were isolated and cultivated under normoxia or hypoxia with/without stimulating enzyme cofactors of th and inhibitors of th. expression of th and release of cytokines and catecholamines was analyzed. the effect of th+ cells was tested by adoptive transfer into dba/ mice with collagen type ii-induced arthritis (cia). th+ cells were generated from mesenchymal stem cells by defined dopaminergic factors. results. hypoxia increased th protein expression and catecholamine synthesis and decreased release of tnf in oa/ra synovial cells compared to normoxic conditions. this inhibitory effect on tnf was reversed by th inhibition with alpha-methyl-para-tyrosine (αmpt). incubation with specific th cofactors (tetrahydrobiopterin and fe +) increased hypoxia-induced inhibition of tnf, which was also reversed by αmpt. adoptive transfer of th+ cells reduced cia in mice, and hydroxydopamine, which depletes th+ cells, reversed this effect. conclusion. in summary, this study presents that th-dependent catecholamine synthesis exhibits anti-inflammatory effects in human ra synovial cells in vitro, which can be augmented under hypoxic condi-tions. in addition, the anti-inflammatory effect of th+ cells has been presented the first time in experimental arthritis in mice. background. previously, we demonstrated that long-lived plasma cells contribute to the pathogenesis of antibody-mediated diseases and should therefore be considered as a promising therapeutic target in systemic lupus erythematous (sle). in bone marrow stromal cells expressing the chemokine cxcl organize these niches that provide for the plasma cell survival. cxcl is the ligand of cxcr expressed on plasma cells. in this study we investigated the contribution of cxcl -cxcr interaction to the longevity of plasma cells in the murine model of lupus. methods. plasma cells purified from spleens of nzb/w mice were incubated with the cxcr blocker amd and then adoptively transferred to immunodeficient rag −/− mice. after days we analyzed the number of plasma cells in bone marrow. furthermore, ova immunized nzb/w mice were treated intraperitoneally with amd after boost; anti-ova secreting plasma cells in bone marrow were checked on day and after boost. the effect of plasma cell depletion was investigated in nzb/w mice using amd alone or combined with bortebomib for two weeks. results. two weeks after adoptive transfer the number of plasma cells treated with amd was lowered by % in bone. after secondary immunization with ova the amd treatment resulted in a significant reduction of anti-ova secreting plasma cells in bone marrow by % on day and by % on day . after days the number of mhc class ii negative anti-ova secreting plasma cells significantly decreased by % in bone marrow of treated mice. amd efficiently depleted plasma cells including long-lived. after two weeks treatment, total plasma cell number was decreased by % in spleen and % in bone marrow; long-lived plasma cells were reduced by % in spleen and % in bone marrow. the combination of bortezomib with amd in nzb/w significantly enhanced the depletion of long-lived plasma cells compared to monotherapy. conclusion. cxcr blockade with amd can reduce the homing of plasma cells to the bone marrow and the survival of long-lived plasma cells. the combination of bortezomib with amd shows synergistic effects on plasma cell depletion. the findings highlight the importance of the cxcr -cxcl axis for the plasma cell niche. zeitschrift für rheumatologie suppl · | er. tnfr expression defines synovial tissue infiltrating cd + t cells in patients with rheumatoid arthritis k background. one hallmark of rheumatoid arthritis (ra) is the infiltration of the synovial membrane by cd + t cells. it has previously been shown that infiltrating cd + t cells differ from non-infiltrating ones in their increased expression of tnfr . furthermore, tnfr is expressed on a fraction of circulating cd + t cells from ra patients, but not from healthy controls. aim of the study was the characterization of tnfr + cd + t cells in patients with rheumatoid arthritis. methods. peripheral tnfr + cd + t cells from ra patients were analyzed by flow cytometry. the expression of naive and memory t cell markers (cd ra and cd ro), markers for t cell activation (cd , cd and cd ) and of icam- as well as the frequencies of the positive cells were determined. to identify the t helper cell signature of tnfr + cd + t cells, intracellular staining of the th , th and th master transcription factors t-bet, gata- and ror-γt, respectively, was performed. results. peripheral tnfr + cd + t cells have neither a preferential naive nor a memory phenotype, but showed higher expression of the activation markers cd , cd and cd than tnfr -cd + t cells. tnfr + cd + t cells express higher frequencies of the t-bet and rorγt than tnfr -cd + t cells. there is no difference in gata- expression between tnfr positive and negative cd + t cells. functionally, it has been shown that the cytokine tnf acts as chemokine to attract cd + t cells to the rheumatoid joints. beside this direct effect of tnf, there are known indirect effects of tnf including the upregulation of cell adhesion molecules like icam- . therefore, icam- expression of migrating tnfr + t cells was investigated. the results show, that migrating tnfr + t cells recovered from synovial tissue are more frequently icam- positive than non-migrating ones. conclusion. tnfr + expression characterizes cd + t cells functionally capable of infiltrating the rheumatoid synovium in an icam- dependent manner. the results show, that tnfr expression defines a pathogenic subset of activated cd + t cells with th and/or th signature in patients with rheumatoid arthritis. hypoxia increased the production of interleukin- β in lps-primed human monocytes n background. monocytes are major players in the innate immune system and are recruited to sites of inflammation, where the environmental conditions vary extremely compared to the interstitium under physiological conditions. for example, in rheumatoid arthritis the inflamed joints are severely hypoxic. this decreased oxygen level could be a triggering factor for the activation and survival of monocytes. aim of the study was to analyze the influence of hypoxia on lipopolysaccharide (lps)-induced cytokine production in primary human monocytes methods. immunomagnetically separated monocytes from the blood of healthy donors were cultured for h under hypoxic conditions ( % oxygen). results. cytokine measurement in the supernatant with elisa showed increased concentrations of interleukin- β ( . ng/ml vs. . ng/ ml, p= . ) and interleukin- ( . ng/ml vs. . ng/ml, p= . ), but not of tnf, after hypoxia and lps-stimulation. cleavage of the il- β proform to its active form is dependent on the assembly of the inflammasome and the recruitment of caspase- followed by their activation. when inflammasome assembly was blocked with high extracellular k+-buffer or by inhibiting intracellular ca-signalling with the ca-chelator bapta-am, hypoxia induced il- β release was abrogated. similarly, il- β release after culture under hypoxia was also abolished in monocytic thp -cells, which are genetically made deficient for the inflammasome components nlrp and asc. one activating signal for the inflammasome was shown to be the release of reactive oxygen species (ros), since mitochondrial ros staining with mitosox revealed an increased mitochondrial ros release under hypoxic conditions. accordingly, the induction of mitochondrial ros through decoupling of the electron transport chain with rotenone also triggered an increase of il- β release under normoxic conditions. analysis of blood monocyts from ra patients showed no difference in lps and hypoxia induced il- β release compared to healthy controls ( . ng/ml vs. . ng/ml). conclusion. this study shows, that hypoxia leads to the activation of the inflammasome, the recruitment of caspase- and the subsequent cleavage and release of interleukin- β in human primary monocytes. intracellular calcium mobilization and mitochondrial ros production were shown to be essential mechanisms triggering inflammasome assembly. background. cell-derived membrane-coated microparticles have been identified as important mediators in intercellular communication. during the process of apoptosis, dying cells start to dynamically release microparticles. polymorphonuclear neutrophils are the most abundant type of leukocytes, representing - % of all white blood cells. due to their very short lifespan, they are the source of massive amounts of apoptotic cell-derived microparticles (admps). while the interaction between neutrophils and t lymphocytes has been focus of extensive research, the influence of neutrophil-derived microparticles on t cells has not been analysed yet. in this study, we investigated the effect of membrane-coated microparticles released by apoptotic neutrophils on different t helper cell subsets. methods. different cd + t cell subtypes were sorted according to the expression of cd , cd , cd ra and cd ro and co-cultured with admps or apoptotic cell remnants purified from uv-irradiated neutrophils isolated from the peripheral blood of healthy donors. t cells were stimulated by okt and anti-cd antibodies and cell proliferation was measured by h-thymidine incorporation or pkh -staining. secretion of cytokines was quantified by elisa. results. admps released by neutrophils selectively suppressed the proliferation of cd +cd -cd + tc in a dose-dependant manner and prevented the upregulation of cd on the t cell surface, while maintaining the expression of cd . the secretion of tumor necrosis factoralpha (tnfα) by t cells stimulated in the presence of admps was significantly reduced. interestingly, in contrast to admps, the apoptotic cell remnants of neutrophils exerted no effect on t cells. the suppressive effect of admps could be completely abrogated by the addition of interleukin(il)- or il- or by the presence of cd +cd +cd + t cells. conclusion. neutrophil admps suppress the proliferation of cd +cd -cd + t cells under conditions of limiting il- and il- concentrations. this could represent an important mechanism to prevent inappropriate activation and expansion of resting t helper cells in the absence of sufficient stimulation and cytokine production. t. alexander background. recent reports have shown dysregulated micrornas in murine lupus models, among them increased expression of mirna- , which has been demonstrated to target the transcription factor foxo in activated cd + t cells. the loss of foxo activity in t cells is associated with spontaneous t cell activation, clonal expansion and autoantibody production, all of which are present in systemic lupus erythematosus (sle). methods. expression levels of microrna- and foxo were analyzed with rt-pcr in magnetic purified peripheral blood cd + t cells from patients with sle and healthy controls (hc). multicolor flow cytometry was performed to analyze cd + t cell expression for ccr , cd ra, ki- , foxp , the interleukin- receptor-α and phosphorylated stat- a (pstat ). analysis of serum il- levels was performed with elisa in sle patients and hc (r&d systems). results. mirna- was significantly upregulated in cd + t cells from sle patients compared to hc (median expression . × e- vs. . × e- , p= . ) while foxo mrna levels were decreased, yet without reaching statistical significance. analysis of ki- expression revealed an increased percentage of proliferating cd + t cells in sle ( . % vs . %, p= . ). overall, cd + t cellular proliferation in sle was associated with increased frequencies of cd ra-ccr -effector memory t cells and enhanced basal pstat levels (median mfi . vs . , p= . ), suggesting a recent stimulation with common gamma chain(γc)-signaling cytokines. in this regard, tcons from sle samples displayed decreased expression levels for the foxo target gene cd (mfi vs. , p= . ) and serum il- levels were significantly higher in sle compared to hc ( . pg/ml vs. . pg/ml, p= . ). conclusion. mir- expression has been shown to be dependent on stat activation and to promote clonal expansion of activated cd + t cells. our data suggest that enhanced il r/stat signaling mediates induction of mir expression, which in turn promotes the proliferation of tcons in sle. the relative contribution of il r/mir- /foxo axis on the enhanced proliferative capacity of sle tcons remains elusive and merit further investigation. collectively, our data provide new insights in the pathophysiology of t cell hyperactivity in sle and identifies mir- as a candidate target for future therapeutic approaches. background. cell activation and apoptotic cell death leads to the formation of membrane-coated vesicles (mcvs). mcvs have previously been identified as mediators of cell-to-cell communication and carriers of microrna. an impaired clearance of apoptotic debris caused by an increased rate of apoptosis or a defect in phagocytic-cell clearance has been observed in sle patients. in this study, we analyzed the microrna content of activated and apoptotic lymphocytes and their corresponding mcvs from both normal healthy donors (nhds) and sle patients. further we investigated the immunomodulatory effect of mcv uptake by monocytes. methods. microrna content of activated and apoptotic lymphocytes and corresponding mcvs of nhds and sle patients were compared in an agilent microrna array and validated by qpcr. apoptosis was induced by uvb-irradiation. mir- expression in monocytes after uv-mcvs engulfment was determined by qpcr. expression of mir- target protein tab- was analyzed by western blot. results. mir- * levels were decreased after apoptosis induction in lymphocytes and apoptotic mcvs compared to their viable correlates. mir- , mir- a and mir- b were decreased in apoptotic lymphocytes compared to viable ones but increased or not significantly changed in apoptotic mcvs compared to viable mcvs, indicating a directional transport of microrna into mcvs. mir- a was expressed at higher levels in viable sle lymphocytes and mcvs compared to nhds. mir- b expression was decreased in uv-lymphocytes and uv-mcvs of sle patients. functional assays confirmed higher mir- levels and consecutively decreased target protein levels in monocytes after engulfment of uv-mcvs. conclusion. within this study we could show an unequal distribution of distinct microrna into mcvs released by activated or apoptotic lymphocytes. further the microrna content was regulated in whole apoptotic cells after uvb-irradiation. this suggests a directional transport rather than a random distribution. thus, cells regulate their microrna as well as the microrna content within released mcvs. we could show a microrna and protein expression change in phagocytes after mcv engulfment. hence, our results suggest mcvs could serve as a transport vehicle for microrna to mediate cell-to-cell communication and influence intracellular processes in phagocytes. disturbances of this system might contribute to the pathogenesis of sle. results. we found in the spleens of nzb mice -times higher numbers of long-lived plasma cells and megakaryocytes compared to wildtype, in nzw mice equal numbers and in nzb/w mice numbers between those for nzb and nzw or wildtype. moreover, in the spleen a fraction of plasma cells clustered around megakaryocytes. we also detected a missense mutation in the c-mpl gene of nzb mice leading to an amino acid replacement within the essential tpo-binding site. upon tpo stimulation of splenocyte and bone marrow cultures nzb cultures responded significantly stronger resulting in the double amount of megakaryocytes compared to nzw cultures. conclusion. in summary, our data indicate that augmented megakaryopoiesis enables the accumulation of a greater number of autoreactive plasma cells in lupus prone nzb/w mice. thus, we assume that enhanced megakaryopoiesis and higher megakaryocyte numbers are contributing to the development and/or pathogenesis of sle. background. baff is a cytokine important for the stimulation and survival of autoreactive b cells and therefore might play a role in several autoimmune diseases, e.g. autoimmune arthritis. in psoriasis arthritis, baff correlates with disease activity and testosterone, but only in male patients, suggesting a role for sex hormones in the regulation of baff. therefore, we wanted to determine if baff production in rheumatoid arthritis and osteoarthritis fibroblasts was regulated by neuroendocrine mediators. methods. fibroblasts were isolated from synovial tissue of ra (n= ) and oa (n= ) patients and cultured in vitro under different conditions. baff was determined by elisa. results. isolated fibroblasts were cultured in the presence or absence of interferon-gamma (ifn-γ), il- , lipopolysaccharide (lps), tumor necrosis factor (tnf), cpg, poly i:c, and cortisol in different combinations for and hours to determine the optimal stimulation strategy for induction of baff production (measured by elisa in supernatants) in fibroblasts. ifn-γ best induced baff in ra and oa fibroblasts. ifn-γ-induced baff production in fibroblasts was decreased by dihydrotestosterone in a concentration dependent manner. the effect was specifically inhibited by nilutamid, a testosterone receptor antagonist. furthermore, stimulation of beta-adrenoceptor increased, whereas stimulation of alpha-adrenoceptors did not change inf-γ-induced baff in synovial fibroblasts. in general the effects were more pronounced in ra as compared to oa fibroblasts. conclusion. taken together, inf-γ-induced baff production in synovial fibroblasts is decreased by testosterone and increased by betaadrenergic stimuli. therefore, neuroendocrine regulation of inflammation in the inflamed joint might be in part mediated by regulating baff production in synovial fibroblasts. a. grützkau , c. kyogoku , b. smiljanovic , j. grün , r. biesen , t. alexander , f. hiepe , a. radbruch , t. häupl deutsches rheuma-forschungszentrum (drfz), berlin, charité -universitätsmedizin berlin, medizinische klinik mit schwerpunkt rheumatologie und klinische immunologie, berlin background. gene expression profiling experiments using peripheral blood mononuclear cells (pbmcs) revealed a crucial role of type i interferon (ifn) in the pathogenesis of systemic lupus erythematosus (sle). however, it is almost unknown how particular leukocyte subsets contribute to the overall type i ifn signature described for pbmcs. furthermore, a detailed analysis of how ifn signatures differ in autoimmune disease from that observed after viral infection is missing so far. therefore, we compared expression levels of ifn signature genes in peripheral cd + t helper cells and monocyte (mo) subsets isolated from patients with sle, healthy donors (nd) and nd vaccinated against yellow fever by global gene expression profiling. methods. peripheral blood from patients with sle and nd were recruited. same nd were examined before and after immunization by yellow fever vaccine. after sorting cells, isolated rna were applied to affymetrix human genome u plus . array. data analysis was done using bioretis database, genesis software and ingenuity pathway analysis (ipa). results. comparing gene expression profiles of yellow fever immunized individuals and active sle patients it was possible to identify a "common" and an "autoimmune-specific" ifn signature. although major ifn signature genes were commonly expressed in cd + t cells and mo of patients with sle and immunized nd, expression magnitudes of them were higher in patients with sle compared to immunized nd. in sle, in addition to the typical "viral-induced" ifn signature, genes that are involved in apoptosis signaling, antiviral pkr signaling, fcγ receptor-mediated phagocytosis and il- -/il- -/il- -mediated jak/ stat signaling pathways were identified by ipa. conclusion. this study demonstrated that ifn signature in autoimmunity and that in viral infection are quite different in the number of ifn-related genes activated and their expression magnitudes. autoimmunity is characterized by a much stronger expression of ifn signature genes and is obviously modulated by a separate set of co-regulated genes defining the "autoimmune-specific" ifn signature. in summary, "common" and "autoimmune-specific" ifn signature genes are of potential interest as clinical biomarkers in sle diagnostics to differentiate between a disease flare and a viral infection. of peripheral blood lymphocytes (pbl). there is currently no data available about nk cells in gpa. the aim of this study was to evaluate the presence of nk cells in gpa granulomas and their proportions in pbl as a basis for a potential role in gpa. methods. paraffin sections of granulomas of gpa, sarcoidosis and tuberculosis patients were stained with a cd monoclonal antibody. nk cell (cd -cd +) proportions of pbl in gpa patients and healthy controls (hc) were analysed by facs analysis. clinical data was extracted from medical records. results. contrary to granulomas from tuberculosis and sarcoidosis which showed a considerable infiltration by cd positive cells, there was not a single cd positive cell in granulomas from gpa patients. therefore, the tissue destructive character of gpa granulomas is associated with a lack of nk cells. gpa patients with inactive disease [birmingham vasculitis activity score (bvas) = , n= ] possessed a significantly higher nk cell proportion in pbl (mean ± standard deviation: . ± . %) than both gpa patients with active disease (bvas> , n= , mean= . ± . %) (p= . ) and hc (n= , mean= . - . %, p= . ). thus, clinical remission is accompanied by an increase in the nk cell proportion in pbl. interestingly, patients with inactive disease that had "normal" nk cell proportions of less than % of pbl (n= ) showed a more severe disease course than those with more than % of pbl. conclusion. nk cells might, therefore, be helpful to limit granulomatous inflammation. whether nk cell proportion in pbl might be a useful biomarker in gpa, e.g. as predictor for relapses, will be further evaluated in our future studies. v. gerl background. plasmacytoid dendritic cells (pdcs) are considered a crucial element in sle pathogenesis due to their potency to produce high levels of ifn-α. this innate immunological function of pdcs is lost by terminal differentiation into a professional antigen-presenting cell (pdc-derived dc), thereby upregulating costimulatory molecules and downregulating innate characteristics, e.g. bdca- and ifn-α expression. pdc-derived dcs have not been described in vivo yet, probably due to the fact that they lose their specific markers during differentiation. furthermore, pdcs can differentiate into myeloid dcs by various stimuli. in sle, where low expression of bdca- is commonly seen, this differentiation could be relevant and point to such a lineage switch as well as to an activated state of pdcs. aim. to characterize pdc subsets of differentiation/activation in human peripheral blood and to study their impact on autoimmune inflammation in sle. methods. -color-flowcytometric analyses were performed on whole blood of healthy donors and sle patients. pdcs were identified by cd -/cd -/cd -/cd high//bdca- +/hla-dr+ expression and characterized for cd c, bdca- and the macrophage-associated siglec- , expressed on monocytes of active sle patients in an ifn-α dependent manner. cd and cd expression were measured in parallel. results. we found a small subpopulation of siglec- expressing pdcs in human peripheral blood. compared to siglec- negative pdcs, siglec- positive pdcs express significantly lower bdca- and cd , higher hla- dr background. agonistic autoantibodies against the angiotensin ii receptor type (at r) and the endothelin receptor type a (etar) have been identified in patients suffering from systemic sclerosis (ssc). here we examined the expression of at r and etar in human immune cells and pathological effects mediated through these receptors by corresponding autoantibodies (aabs). methods. at r and etar protein expression on peripheral blood mononuclear cells (pbmcs) from healthy individuals and ssc patients was analyzed using flow cytometry, mrna expression was examined by real-time pcr in pbmcs from healthy donors. in addition, pbmcs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin g (igg) fractions from ssc patients positive for at rand etar-aabs, and with igg from healthy donors serving as control. alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, elisa, and chemotaxis assays, respectively. results were correlated with characteristics/clinical findings of the igg donors. results. both at r and etar were expressed on human peripheral lymphocytes and monocytes. protein expression of both receptors was decreased in ssc patients when compared to healthy donors and correlated negatively with disease duration. in addition, igg fractions of ssc patients induced t cell migration in an anti-at r and anti-etar aab level-dependent manner. moreover, igg of ssc patients was capable of stimulating pbmcs to produce more il- and ccl than igg of healthy donors. all effects could be significantly abrogated by the application of selective at r and etar antagonists. statistical analysis revealed a negative correlation between ssc igg-induced il- concentrations and disease duration, between ssc igg-induced ccl concentrations and time since onset of lung fibrosis as well as an association of ccl concentrations with vascular complications of the corresponding ssc igg donors. conclusion. we demonstrated the expression of both, at r and etar, on human peripheral t cells, b cells and monocytes and found signs for a chronic receptor activation in ssc patients. the inflammatory and profibrotic effects upon aab stimulation in vitro, and their associations with clinical findings suggest a role for autoantibody-mediated activation of immune cells mediated through the at r and etar in the pathogenesis or even the onset of the disease. the bioenergetic role of hif- and hif- during angiogenesis of human microvascular endothelial cells background. hypoxia and angiogenesis are features of inflamed and injured tissues. the transcription factors hypoxia inducible factor (hif)- and (hif)- regulate the cellular and metabolic responses to reduced oxygen tensions thereby promoting angiogenesis with implications on the pathogenesis of ra. we investigated the effects of a knockdown of either hif- α or hif- α in human microvascular endothelial cells (hmec) on angiogenesis and bioenergetics under hypoxia ( % o ) versus normoxia ( % o ). methods. specific knockdown of either hif- α or hif- α was conducted by shrna-technology. to assess angiogenesis of hmecs both tubuli and node formation under hypoxia versus normoxia were investigated. expression of hypoxia driven genes involved in the metabolic response to hypoxia (gapdh/pgk/glut /ldha) was quantified by realtime rt-pcr. the bioenergetic status of the cells was quantified via atp/adp measurements. results. knockdown of hif- α/hif- α resulted in a loss of hypoxia induced angiogenesis. focusing on bioenergetic aspects, we found hypoxia to significantly induce pgk, ldha and gapdh in control cells. knockdown of hif- α and hif- α, respectively, did not affect the hypoxic induction of pgk and ldha. in hif- α and hif- α knockdown-cells, hypoxia was still capable of inducing gapdh, with a less pronounced effect in hif- α knockdown-cells. hypoxia did not significantly up-regulate glut , neither in control nor in hif- α or hif- α knockdown-cells. the knockdown of hif- α resulted in significantly decreased expression of glut under hypoxia. we also found the atp/ adp ratio to be similar in control, hif- α and hif- α knockdown-cells under normoxia. under hypoxic conditions hif- α knockdown-cells showed significantly reduced atp/adp ratios -indicating that less atp is available -compared to hif- α knockdown-cells. conclusion. hif- α and hif- α are both key regulators of angiogenesis. however, they do differ in their potency to regulate cellular energy metabolism. this leads us to conclude that hif- α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (as previously shown), whereas hif- α affects angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of gapdh and the diminished atp/adp ratio. these findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in ra. low baseline complement levels, autoantibody persistence and delayed thymic reactivation are risk factors for development of relapses after hematopoietic stem cell transplantation for refractory sle background. our previous research has provided the evidence that an autoreactive immune system can be "reset" into a healthy, tolerant state by immunoablative treatment to eradicate pathogenic effector cells, followed by transplantation of hematopoietic progenitor cells (hsct). nevertheless, disease flares may occur in a subset of these patients posttransplantation. here, we longitudinally analyzed the immune reconstitution of these patients to identify markers for favorable long-term responses. methods. since , patients with refractory sle received a cd +selected autologous stem cell transplantation after immunoablation with antithymocyte-globulin (atg) and cyclophosphamide as part of a monocentric phase i/ii clinical trial. autoantibody titers were evaluated with elisa, peripheral t-and b lymphocyte subsets immunophenotyped using multicolor flow cytometry. results. clinical remission (sledai ≤ ) could be achieved in all patients, despite immunosuppressive drug withdrawal, associated with disappearance of anti-dsdna antibodies and marked reduction of protective antibodies in serum. unfortunately, two patients died due to transplant-related infections. from the remaining eight patients, five patients are in long-term clinical remission for up to years after hsct, while three patients suffered a relapse of sle at , and months post-transplantation, respectively. patients with early relapses (≤ months) had decreased baseline complement levels, showed persistence of antinuclear antibodies (ana), less significant reduction in protective antibody levels and had slower repopulation of cd + cd ra+ thymic-derived cd + t cells after hsct (< /µl at months) when compared to long-term responders. in addition, flow cytometric analyses revealed an expansion of circulating plasmablasts and increased coexpression of siglec- on monocytes (as surrogate marker for type-i interferon signature), preceding the clinical flares by ~ months. conclusion. low baseline complement levels, persistence of ana and delayed thymic reactivity post-transplantation could be identified as risk factors for development of lupus flares after hsct. since atg-mediated cell lysis is complement-dependent, we conclude that low serum complement is directly associated with incomplete depletion of immunologic memory cells in these patients, which provides a rationale for complement substitution before immunoablation. moreover, lupus flares may be predicted individually by flow cytometry with plasmablast expansion and recurrence of type-i interferon signature. background. systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by the generation of pathogenic antibodies directed against a variety of autoantigens. we have previously shown that long-lived autoreactive plasma cells can contribute to chronicity and refractoriness of sle. our study is aimed to develop new methods for depletion of long-lived plasma cells in nzb/w mice, a model of sle. methods. we studied different treatment protocols on plasma cell survival: irradiation-based and more selective depletive treatments. - week-old nzb/w f mice were exposed to three different irradiation doses ( , , and gy in two splitted doses with a -h interval). the following protocols were also investigated: ) two bortezomib (bz) injections ( , mg/kg, i.v.) combined with anti-mouse cd ( mg/kg, i.v.), ) three bortezomib injections combined with anti-mouse cd , ) three bortezomib injections combined with anti-lfa- and anti-vla- antibodies (affecting directly the plasma cell niche; µg, i.p.) in a -d interval, plus anti-mouse cd and anti-b ( µg, i.v.). the plasma cells were analyzed in spleen and bone marrow by facs and elispot. results. the frequency of remaining plasma cells in bone marrow after , and gy irradiation were , and , % respectively, and in spleen were almost , and , %. short-term treatments with agents that affect plasma cells (bortezomib, anti-lfa plus anti-vla ) effectively deplete plasma cells including long-lived plasma cells in spleen and bone marrow of nzb/w mice. because of the b cell hyperactivity in nzb/w mice, we observe a rapid regeneration of autoreactive plasma cells in spleen and bone marrow. therefore, plasma cell depletion protocols were combined with b cell depletion. especially, the combination of plasma cell targeting with bortezomib, anti-lfa and anti-vla with b cell targeting (anti-cd plus anti-b ) interrupted the repopulation of autoreactive plasma cells in spleen and bone marrow. conclusion. very high doses of irradiation result in effective depletion of long-lived plasma cells but lower doses not. depletion of long-lived plasma cells can be achieved by the proteasome inhibitor bortezomib and by targeting both adhesion molecules lfa and vla . the combination with b cell depletion is needed to prevent regeneration of autoreactive plasma cells. varicella-zoster-virus(vzv)-specific lymphocytes and igg antibody avidity in patients with juvenile idiopathic arthritis or rheumatoid arthritis background. varicella zoster virus (vzv) is a herpes virus that establishes a life-long latent infection with risk of reactivation (shingles) particularly in immunosuppressed patients with autoimmune disorders. patients with rheumatoid (ra) or juvenile idiopathic arthritis (jia) have a high risk for disseminating varicella zoster virus (vzv) infection or herpes zoster. this study was aimed to investigate the humoral and cellular immune response to vzv including assessment of igg-anti-vzv avidity and vzv-specific reactivity of lymphocytes in ra (n= ) or jia patients (n= ) on different treatments, including biologic agents, such as anti-tumor-necrosis-factor(tnf)-alpha or anti-interleukin- (il- ) receptor inhibition (tocilizumab), compared to healthy adults (ha) and children (hc). methods. igg-anti-vzv concentrations and avidities were quantified by an adapted elisa. vzv-specific interferon-gamma-producing lymphocytes (spot forming units, sfu/ , , cells) were analyzed by elispot. results. no significant differences in the vzv-igg concentrations or avidities were found between the groups. however, lower igg-anti-vzv concentrations were found in tocilizumab-treated ra compared to ha and ra without biologic agents. ra showed lower median sfu ( / , , cells) than ha ( / , , cells), with lowest sfu in adalimumab-treated ra ( / , , cells). sfu were not altered in tocilizumab-treated ra and after incubation with anti-il- in vitro. no differences regarding igg-anti-vzv concentrations, rai and cellular reactivity were found between jia and hc. conclusion. our study demonstrated that ra and jia patients are still able to maintain humoral and cellular immune responses to vzv despite immunosuppressive therapy or biologic agents. in ra, the role of lower cellular reactivity for risk of herpes zoster has to be considered for recommendations on vaccination. cmv-specific cd + t cells from ra patients contribute to autoimmune disease zeitschrift für rheumatologie suppl · | . increased frequency of lir- (also called cd j or ilt ) on cd + t cells has been associated with autoimmune disease. furthermore, it has been shown that latent cytomegalovirus (cmv) infection contributes to the expansion of cd − t cells. hence we were interested in the influence of cmv infection on the lir expression on t cells in ra patients. methods. we were interested in the role of lir + t cells in ra patients, which potentially contribute to the autoreactive t cell pool, especially in cmv+ patients. therefore, we investigated the expression and function of lir- on cd + t cells in peripheral blood mononuclear cells (pbmc) from patients with rheumatoid arthritis by flow cytometry and cytotoxicity assay. results. flow cytometry analysis revealed higher frequencies of lir- + cd + t cells in cmv seropositive ra (n= , mean%: . ) compared to cmv+ hd (n= , mean%: . , p= . ). using hla-a* /cmvpp dextramers we analyzed cmv-specific cd + t cells. patients with ra had higher frequencies of cvm specific cd + t cells (n= ; mean%: . ) compared to healthy individuals (n= ; mean%: . , p= . ). phenotypically, cmv-specific cd + t cells are mainly cd negative and express lir- . analysis of the cytolytic potential by cd a expression revealed higher numbers of cd a+cd + t cells in ra patients (n= , mean%: , ) compared to healthy donors (n= , mean%: , ). importantly, we found a significant correlation (p= . ) of high numbers of cd +lir- + t cells with high disease activity score (das ) in ra patients without immunosuppressive treatment (n= , r= , ) . tab. . conclusion. this is the first demonstration of significantly increased frequencies of lir- +cd + t cells and of cmv-specific cd + t cells in patients with rheumatoid arthritis. these cells are characterized by a terminally differentiated phenotype. the higher cytolytic potential of cmv-specific t cells likely can be attributed to their function in containing latent cmv infection and to prevent cmv disease, but might potentially contribute to disease severity in ra patients. background. systemic lupus erythematosus (sle) is an autoimmune disease characterized by an acquired il- deficiency, which leads to a homeostatic imbalance between regulatory t cells (treg) and effector t cells (tcon; humrich et al. ). we recently demonstrated that treg homeostasis in lymphoid organs of diseased (nzbxnzw) f mice can be restored by treatment with recombinant il- (il- ) resulting in an amelioration of kidney disease. the aim of this study was to investigate the impact of il- therapy on intrarenal foxp + treg and kidney infiltrating conventional cd + t cells (tcon) in the (nzbxnzw) f mouse model of lupus nephritis. methods. (nzbxnzw) f mice with active nephritis were treated with recombinant il- either for a short period of days or for a longer period of days in total. absolute numbers, phenotype and proliferation of kidney infiltrating cd + t cell subsets were determined by flow cytometry at different time points. results. short-term il- treatment resulted in an enhanced proliferation and increased numbers and frequencies of intrarenal cd +foxp + treg compared to untreated control mice. on the other hand, long term il- treatment did not result in a persistent expansion of the intrarenal foxp + treg population. however, total numbers of kidney infiltrating cd + tcon with a memory/effector phenotpye were diminished and cd + tcon showed markedly reduced signs of cellular activation. conclusion. our data indicate that short term il- treatment is able to expand the size of the intrarenal treg pool. in contrast, long term il- treatment decreases the numbers of kidney infiltrating memory/ effector t cells and reduces cellular hyperactivity suggesting that treg suppress the activation and expansion of infiltrating tcon. these results may in part explain the amelioration of disease induced by treatment with il- and underline the important role of intrarenal treg for the suppression of kidney disease in lupus mice. these results also provide additional important rationales for an il- based immunotherapy of human disease. from transcriptome to protein biomarkers in ra: joint compartment and monocytes outperform serum and whole blood background. a main challenge in disease-management of ra is to establish objective criteria relevant for diagnosis and therapeutic stratification of patients. this study focused on global approaches in dissecting inflammation in ra including transcriptome analyses of synovial tissue and blood monocytes and proteome analyses of synovial fluid and serum. methods. gene-expression profiles from synovial tissues and blood monocytes of ra and osteoarthritis (oa) patients were generated by affymtetrix arrays. elisa and multiplex immunoassays were used for validation of candidate markers at the protein level in synovial fluid (sf) from ra and oa patients and in serum from the same group of patients and healthy donors. results. transcriptome analyses of synovial tissues from ra and oa revealed more than differentially expressed genes. to avoid difficulties in sampling synovial tissue and to avoid fluctuation in cellular composition of various cell types in blood, the transcriptome analyses from peripheral blood was focused on a specific cell population. monocytes were selected as the favourable cell type involved in the production of cytokines, which are often considered as therapeutic targets in ra. comparisons between ra and oa monocytes disclosed differential expression of more than genes. in total, genes that were up-regulated in synovial tissues and/or monocytes were used for validation at the protein level as potential biomarkers for ra. among these biomarkers, chemokines (cxcl , ccl , ip ), adhesion molecules (vcam , icam , e-and p-selectins), proteolytic enzymes (mmp , a at), and the shedding form of cell surface molecules (cd , cd ) background. idiopathic membranous nephropathy (imn) is a common cause of nephrotic syndrome in adults and has recently been identified as an autoimmune-mediated disease [ ] . autoantibodies directed towards the m-type phospholipase a receptor (pla r) are fairly specific for idiopathic mn and only found to a small percentage in sera from patients with secondary mn [ ] . the outcome of patients with imn is quite diverse: about one third of patients have spontaneous remission, another third progress to require dialysis and the last third continue to have proteinuria without progression to renal failure. we performed serological profiles of imn patients in order to compare antibody profiles to antibody frequencies found in the normal healthy population and to hopefully identify factors that help to predict disease course in imn. methods. serum samples of patients with imn were assayed for a variety of autoantibodies by elisa, addressable laser bead immunoassay (albia) and to dsdna by crithidia luciliae assay. results. the prevalence of autoantibodies found in our imn cohort is summarized in tab. . anti-pla r antibodies were found in about % of imn patients whereas the frequency of other antibodies was mostly below %. the one exception is anti-dfs that was found in . % of imn patients. conclusion. the prevalence of anti-pla r positive patients in our imn cohort matches what has been previously described [ ] . the frequency of the other antibodies that we determined is comparable to what has been reported in the normal healthy population. it is important to note that anti-dfs antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases (sard; [ ] ) whereas anti-ro reactivity is often regarded as a marker for sard. the absence of anti-ro and the high prevalence of anti-dfs confirms that imn is a rather organ specific autoimmune disease. background. activity and the quality of movement belong to the most fundamental diagnostic parameters for neurobehavioural analysis but in the past it has been difficult to include this information into pre-clinical murine disease models. here we tested the applicability of a radiofrequency identification (rfid) based automated tracking system in the experimental murine model of ovalbumin induced arthritis. methods. c bl/ mice were immunized twice with cationized ovalbumin in freund's complete adjuvant and onset of arthritis was induced two weeks after the last immunization by direct injection of cationized ovalbumin into the knee joint of the right hind leg. severity of arthritis was assessed through measurement of joint swelling and evaluation of histological changes. additionally mice were implanted with a rfid transponder and throughout the experiment their activity level was monitored by an id-grid sensor plate placed underneath the homecage. results. the joint inflammation in the ovalbumin induced arthritis model showed a quantifiable impact on the activity levels of the mice. our experiments could also show that movement activity correlates with disease severity as evaluated by clinical and immunological parameters. in the past employing behavioral methods was often limiting by group size, observation time and reproducibility and the stress of handling and new surroundings made results difficult to interpret. in our experiments a rfid-based automated tracking system allowed us to monitor individual activity long-term without removal of the mice from their homecage environment. this allowed for the correlation of clinical parameters to behavioral factors and adds another level of analysis to an established murine model. progranulin antibodies in a wide spectrum of autoimmune diseases results. autoantibodies against progranulin, a secreted and direct inhibitor of tnf-α receptors & were frequently identified in primary vasculitides. in detail, progranulin-antibodies were found during the course of disease in giant cell arteritis/polymyalgia rheumatica ( / ), takayasu's arteritis ( / ), classical panarteritis nodosa ( / ), behcet's disease ( / ), in granulomatosis with polyangiitis ( / ), churg-strauss syndrome ( / ) and in microscopic polyangiitis ( / ). in extended screenings progranulin-antibodies were also frequently detected in autoimmune connective tissue disorders, in rheumatoid and psoriatic arthritis and in inflammatory bowel disorders. in contrast progranulin-antibodies were only detected rarely in healthy controls ( / ), patients with obesity ( / ), residents of nursing homes ( / ), not in patients with cutaneously limited psoriasis ( / ), not in patients undergone sepsis ( / ), and not in patients with melanoma ( / ). a significant association of progranulin-antibodies with active disease states in granulomatosis with polyangiitis suggested a pro-inflammatory activity of progranulin-antibodies. this was supported by an observed neutralizing effect of progranulin-antibodies on the levels of circulating progranulin in elisa and western-blot. moreover, functional assays revealed, that progranulin-antibody containing sera render wehi-s cells far more sensitive to effects of administrated tnf-α, providing evidence for the suspected pro-inflammatory effect of progranulin-antibodies. conclusion. progranulin-antibodies occur in a widespread spectrum of autoimmune diseases and have a pro-inflammatory effect by neutralizing the physiologic tnf-blocker progranulin. background. flow cytometry (fcm) is widely used in research for molecular characterization at single cell level. conventional analysis is a semiautomated process of user defined gating and investigation in -d projections. for multiple parameter analysis with hundreds of marker combinations, this manual process is most limiting and impedes high throughput analysis. therefore, we developed a new algorithm for automated and standardized analysis of multiplex fcm data. methods. automation included asinh-transformation of data, cell grouping, population detection and population feature extraction. for grouping of cells, an unbiased unsupervised model based t-mixture approach with expectation maximization (em)-iteration was applied. populations were identified by meta-clustering of several experiments according to position and extension of cell-clusters in multi-dimensional space and by including a general procrustes analysis (gpa) step. for validation, peripheral leukocytes from healthy donors and patients with rheumatoid arthritis (ra) were prepared by hypoosmotic erythrocyte lysis and stained with different sets of lineage-specific antibodies. in parallel, different leukocyte samples were depleted of one of these populations by magnetic beads. qualitative and quantitative characteristics of major populations were compared with conventional manual analysis. results. whole blood leukocytes stained simultaneously with up to markers were correctly distinguished in all major populations including granulocytes, t cells and their subpopulations, monocytes, b cells, and nk-cells. the result was comparable to the "gold standard" of manual evaluation by an expert. the new technology is able to detect subclusters and to characterize so far neglected smaller populations based on the new parameters generated. automated clustering did not require fluorescence compensation of data. cell-grouping is applicable even for large fcm datasets of at least parameters and more than million events. comparing the cell-clusters between ra and healthy controls, differences were detectable in several cell (sub-)populations, stable enough to perform correct classification into controls and disease. conclusion. this new approach reveals promising results for automated and time-saving analysis of large datasets from multiplex fcm. the algorithm avoids operator-induced bias, is able to detect unexpected sub-clusters and to characterize so far neglected populations. this may reveal not only new markers for disease activity but also for therapeutic stratification. background. lasp- localizes at focal adhesions, along stress fibres and leading edges of migrating cells regulating metastatic dissemination of different tumors. since rasf have been implicated in the spreading of disease by leaving cartilage destruction sites, migrating via the bloodstream and re-initiating the destructive process at distant articular cartilage surfaces, the underlying mechanisms are of special interest. therefore, we investigated the role of lasp- in sf migration and its effects on ra. methods. to identify lasp- expression and its sub-cellular distribution in human sf as well as in hind paws of wt and htnftg mice, we performed western blots and immunofluorescence. the migration of sfs derived from wt, lasp- -/-, htnftg and lasp -/-/htnftg mice was studied in a modified scratch assay as well as in live cell imaging studies. furthermore, a transmigration assay using sf from all four genotypes and murine endothelioma cells (bend. ) as an endothelial barrier was carried out. sf transmigration under inflammatory conditions was also evaluated by tnf-alpha stimulation of the endothelial cells in vitro. results. lasp- expression was up regulated in rasf and in sf from htnftg mice compared to healthy controls and was found at structures of cell adhesion and invasion. the scratch assay as well as the live cell imaging studies showed a significantly reduced migration of lasp- ( ng/ml) was applied. in parallel, il- stimulation significantly amplified the expression of anti-apoptotic bcl- in sle treg but not tcon. conclusion. in analogy to our previous findings in lupus-prone mice, treg from sle patients show the classical hallmarks of il- deficiency with loss of cd expression and a homeostatic imbalance between treg and tcon. these findings could be associated with a reduced il- expression by cd + t cells in sle patients. on the other hand, low-dose il- stimulation in vitro could restore these defects, underlying the potential of il- as a novel therapeutic option in sle. the glucocorticoid-dependent modulation of immune-mediated inflammatory arthritis by osteoblasts in mice is t cell independent background. at present, the role of adiponectin in rheumatoid arthritis is still controversial. there is some evidence indicating anti-inflammatory effects, for example adiponectin reduces the tnf release by macrophages. in contrast to its anti-inflammatory role, adiponectin also exerts pro-inflammatory effects locally in joints, inducing for example pro-inflammatory factors and matrix-degrading enzymes in ra synovial fibroblasts. moreover, our immunohistochemical analysis of ra bone tissue showed a co-localization of adiponectin with key cells of bone remodelling (osteoblasts, osteoclasts). however, the role of adiponectin in bone remodelling of ra still needs to be defined. in this study, we therefore focussed on adiponectin and its immunomodulatory properties on ra osteoblasts and osteoclasts. methods. human osteoblasts and osteoclasts were isolated from bone tissue and blood samples of ra patients. immunocytochemistry and rt-pcr were used to analyze the expression of adiponectin and its receptors in osteoblasts and osteoclasts. osteoblasts and osteoclasts were treated with adiponectin ( µg/ml). adiponectin-mediated effects on the cytokine expression in osteoblasts and osteoclasts were analyzed using elisa. results. the expression of adiponectin and its receptors (adipor , adipor , and paqr ) by cultured ra osteoblasts and osteoclasts could be confirmed on translational and transcriptional level. stimulation of primary ra osteoblasts and osteoclasts with adiponectin resulted in an alteration of cytokine release. osteoblasts showed a time-and dose-dependent increase in il- production. furthermore, adiponectin induced the secretion of il- and gro-alpha and significantly increased the il- and mcp- production (il- : -fold, p= . ; mcp- : -fold, p= . ). stimulation with adiponectin resulted in an increase in il- production in pre-osteoclasts ( -fold) but not in osteoclasts. the secretion of il- was increased in pre-osteoclasts ( -fold) and osteoclasts ( -fold). the results of the present study confirm the pro-inflammatory potential of adiponectin in ra. the cytokines released after adiponectin treatment by osteoblasts and osteoclasts promote osteoclastogenesis or the migratory potential of osteoclasts and monocytes. together with the finding that adiponectin is present in the bone compartment of ra suggest an involvement of adiponectin in articular destruction. acknowledgement: funded by the german research society (spp , immu-nobone, ne / - ). novel mechanisms of glucocorticoid therapy in arthritis anti-inflammatory acting glucocorticoids (gcs) are an important component of rheumatoid arthritis (ra) therapy. but their beneficial usefulness, especially in ra therapy, is hampered by severe side effects like glucocorticoid-induced osteoporosis (gio). until now the molecular mechanisms underlying the beneficial and side effects of gc therapy are poorly understood. gcs exert their actions via the glucocorticoid receptor (gr) that alters gene expression by either binding as a dimer to gc response elements in the promoter region of target genes or by interacting with and thus interfering with other transcription factors. for a long time gr dimerization was considered as the molecular base of side effects. interference of pro-inflammatory transcription factors, such as ap- and nf-κb by the gr monomer was believed to contribute to the therapeutic effects of gcs. in a model of gio we previously showed that unexpectedly interaction of the gr monomer with ap- , but not nf-kb in osteoblasts is decisive for bone loss (cell metabolism : ). in contrast, in antigen-induced arthritis (aia), we could demonstrate that gcs act in the acute inflammation of ra via the dimerized gr. particularly, gc therapy suppressed th and th cell derived pro-inflammatory cytokines in a dimerization dependent manner. furthermore th , rather than th cells seem to be the most crucial targets for an efficient gc therapy since il- -/-mice were resistant to gc therapy whereas ifnγ-/-mice responded as efficient as wild type mice to steroid treatment (pnas : ). in a more chronic arthritis model, the k/bxn serum transfer induced arthritis, we demonstrate now that, unexpectedly, dimerization of the gr in non-hematopoietic cells also contributes to the anti-inflammatory effect of gcs. thus, for immunosuppression of arthritis the gr is required in distinct cell types. taken together, for anti-inflammatory actions the gr dimerization dependent gene regulation is decisive in ra, whereas gio depends on the suppression of ap- dependent gene expression. intriguingly for anti-inflammatory activities of gcs immune and non-immune cells are involved. our approaches give new insights into gc action on arthritis and bone that can be translated into new concepts for anti-inflammatory therapies preventing gio. background. new bone formation and ankylosis are a hallmark of ankylosing spondylitis (as). the impact of cytokines and different mechanisms of new bone formation (endochondral vs. membranous) to syndesmophyte formation and joint ankylosis in as are still poorly understood. in order to analyze cartilage hypertrophy -as a potentially important element of endochondral bone formation -and to assess the possible influence of cytokines, we performed an immunohistochemi-cal study of the hyaline articular cartilage of facet joints of as patients in comparison to autopsy controls and patients with osteoarthritis (oa). methods. the cytokines interleukin(il)- , il- and il- , as well as the marker of cartilage hypertrophy runt-related transcription factor (runx ), matrix metalloproteinase (mmp ) and collagen type (col ) were determined in facet joints from patients with as (undergone correction surgery of rigid hyperkyphosis), oa patients (undergone surgery of the lumbar spine, because of neurological deficits) and controls (autopsies without spinal diseases). immunohistochemistry was performed and the entire cartilage area was analyzed for the frequency of positively stained chondrocytes. background. immunization with glucose- -phosphate isomerase (g pi) induces arthritis in susceptible strains of mice. depletion of regulatory t cells (tregs) prior to immunization switches the usually acute, self-limiting course to a non-remitting, destructive arthritis. this provides a possibility to study molecular switches for the transition from acute, self-limiting to chronic, destructive arthritis within one mouse model. to examine the role of fibroblast-like synoviocytes (fls), which are known to modulate immune responses via the production of pro-and anti-inflammatory mediators, the phenotype and function of fls from mice with either acute, self-limiting or non-remitting, destructive arthritis was studied. methods. fls from dba/ mice that developed either the acute or the chronic form of arthritis were isolated from joints over a time course of days. to investigate the phenotype of fls elisa studies as well as zymography have been performed. for the functional clarification of those cells the matrix-associated transepithelial resistance invasion (matrin) assay and a cartilage attachment assay have been used. furthermore, fls have been transferred in vivo into the knee joints of immunodeficient mice and the joints have been scored histologically. results. fls from treg-depleted mice produced significantly more cytokines (e.g. interleukin (il- )) upon stimulation with other cytokines, growth factors and tlr ligands. this increased susceptibility to cytokine stimulation in chronic animals compared to acute ones is observable throughout the disease course ( days). furthermore, the secretion and activity of matrix metalloproteases (mmps) was enhanced in the fls from chronic mice compared to samples from acute ones. additional functional differences include the collagen-destructive potential and the potential to attach and eventually invade wild type cartilage. here, fls from treg-depleted chronic arthritic mice showed a higher invasive and destructive potential. ultimately, fls from treg-depleted mice were able to destroy cartilage in immunodeficient mice. conclusion. our results are compatible with the hypothesis that uninhibited inflammation in the early phase of treg-depleted mice causes the acquisition of an autonomously aggressive phenotype of synoviocytes which contribute to the switch from acute to chronic arthritis even in the absence of late support from t and b lymphocytes. collagen-induced arthritis modulates reactivity to sympathetic neurotransmitter stimuli during osteoclastogenesis of bone marrow-derived macrophages from da rats background. osteoclast(oc)-mediated bone destruction contributes to increased disease burden in rheumatoid arthritis. simultaneously, changes in synovial tissue innervation occur, leading to a reduction in catecholaminergic nerve fibres. studies on sweat gland innervation revealed that catecholaminergic fibres are capable of phenotypic transition to cholinergic nerves. the sympathetic neurotransmitters norepinephrine (ne) and acetylcholine (ach) affect osteoclastogenesis oppositely prompting us to study osteoclastogenesis at different phases of collagen-induced arthritis (cia) in an altered neurotransmitter microenvironment. methods. for induction of experimental arthritis, da rats were immunized with bovine collagen type ii while controls received isotonic nacl solution. to generate oc, bone marrow-derived macrophages (bmm) were isolated and differentiated with recombinant m-csf and rank ligand. the influence of ne and ach stimulation on osteoclast differentiation and activity was compared between arthritic and control animals at the acute ( days post immunization, pi) and the chronic ( days pi) disease state. as the nicotinic α ach receptor subunit is involved in the cholinergic anti-inflammatory reflex, we also applied a specific agonist, arr- . additionally, the gene expression profile for ne and ach neurotransmitter receptors was analyzed. results. ach stimulation generated significantly more osteoclasts in controls ( days pi). arr- mediated effects were similar to ach. ne decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. cells from arthritic animals were less affected by ne and ach stimulation.oc from arthritic animals showed tendentially decreased activity in an enzymatic cathepsin k activity assay. ach and arr- stimulation decreased cathepsin k activity days pi, but the effect disappeared days pi, representing the chronic arthritis state. ne stimulation significantly inhibited enzyme activity days pi, but has little effect under chronic conditions. the receptor gene expression profile changed in the time course of arthritis. days past immunization muscarinic ach receptors m and m were significantly upregulated whereas after days adrenoceptors α d and α b were significantly downregulated. conclusion. we conclude that cia differentially modulates neurotransmitter influence during oc differentiation and activation but the underlying processes remain still unknown. the observed time pointdependent changes in neurotransmitter receptor gene expression may constitute a regulatory mechanism to counteract alterations in the local neurotransmitter composition. background. the generation of memory t lymphocytes allows effective and fast immune responses during antigen re-challenge and represents a hallmark of adaptive immunity. previous work from our group has demonstrated that murine memory cd + t cells reside in specific bone marrow niches and are characterized by the high expression of cd and ly- c. these cells were designated as resting in the context of gene expression and proliferation. here, we aimed to phenotypically and functionally characterize human memory t lymphocytes in peripheral blood and bone marrow of healthy individuals. methods. mononuclear cells were isolated from paired blood and bm samples from individuals undergoing hip replacement surgery. phenotypic analysis and cytokine profile of distinct memory t cell subsets were assessed by flow cytometry. proliferation and cell cycle status were analyzed using ki- and propidium iodide (pi) staining, respectively. results. distinct populations of cd -expressing cd +cd ra-and cd +cd ra-t cells were detected in bone marrow but not in the periphery. ccr and cd l expression was reduced on bone marrow cd +cd +cd ra-and cd +cd +cd ra-t cells compared to their cd -counterparts in bone marrow and peripheral blood. cell cycle analysis and ki- expression levels demonstrated the nonproliferative state of bone marrow memory t cells. furthermore, bone marrow resident memory t cells showed reactivity against various pathogenic agents, such as tetanus, measles and cmv. conclusion. we have identified a population of cd -expressing cd +cd ra-and cd +cd ra-t cells in the bone marrow. despite cd expression, which is generally regarded as early activation marker, the cells were resting in terms of proliferation. bone marrow cd + memory t cells have downregulated ccr and cd l indicating reduced homing capacity to secondary lymphoid organs. our data underline the role of the bone marrow as a major reservoir for resting memory t lymphocytes. therapeutic methods exerted an influence on satisfaction and future expectations in patients with rheumatoid arthritis (ra). methods. when visiting their rheumatologist, patients with ra were asked to complete a questionnaire at home after the consultation and then return it to an independent opinion research centre, where the data was collected and analysed. the form comprised various areas, namely demography, aspects of the diagnosis, medical care, therapeutic measures, and the illness in a personal context. results. patients ( females/ males) from the whole of austria with a particular emphasis on lower austria, upper austria and tyrol completed the questionnaire (of distributed), resulting in a response rate of %. % of the patients lived in settlements of under , inhabitants; a further % in settlements of under , inhabitants.. the rheumatologist attended could be reached within one hour for % of the patients and within minutes for %. in slightly fewer than % of the respondents the diagnosis was made within three months, in % within six months. in %, the diagnosis was made by a rheumatologist. after experiencing the first symptoms, % contacted their general practitioner. a high degree of satisfaction appears to originate from the information supplied by the rheumatologist attended. most patients felt they were involved in decisions regarding their therapy. % of the respondents were employed prior to their illness; as a consequence of the disease % had to leave their jobs. conclusion. the majority of the respondents came from rural areas. the correct diagnosis was made within six months for almost half of the patients questioned. most patients felt well informed by their rheumatologists and involved in therapeutic decision-making. , combinational therapy of synthetic dmards ( . ; . - . ), and biological-monotherapy ( . ; - ). all of these differed significantly on later observation periods. comparing the prescriptions separately by sequential treatments there were no differences in retention rates for the individual dmard classes. regarding retention, in the first treatment synthetic dmards showed the longest retention, but from the second on this was the case for tnfi-combinational treatment and non-tnfi-biologicals (. abb. ) conclusion. traditional dmards are the starting point of therapy and mtx is the anchor drug for the first and all subsequent courses. already at the second sequential course, the combination therapies of mtx+tnfi become numerically more relevant, and their retention is better than mtx. therapie der rheumatoiden arthritis im letzten jahrzehnt -was hat sich verändert? abb. | ev. anzahl eingeschlossener patienten nach einschlussjahren und therapie sowie zeitlichen entwicklungen im das und der eular-response cal features of ra (erosive arthritis with classical radiological features) plus specific laboratory markers (ccp-antibodies). the third patient, a -year-old female presented initially with features of ra and sle simultaneously (alopecia, subcutaneous nodules, leucopenia, positive ccp-antibodies, high titres of ana and dna-antibodies). the fourth patient, a -year-old patient presented with severe polyarthritis in the upper and lower limbs, subcutaneous nodules, fever and cervical lymphadenopathy, she had high titres of ccp-antibodies ( u/ml by a normal range of less than iu/ml), ana of (normal less than ), and dna of iu/ml (normal less than iu/ml). conclusion. the take home massage of this presentation is to be aware of rhupus if the sle patient develops erosive arthritis or subcutaneous nodules, or if the ra patient develops features of sle like leucopenia, active urine sediment, or clinically significant serositis. rhupus seems to be a distinctive entity and should be kept in mind while dealing with patients having ra or sle as it can affect the treatment and outcome. vorgeschichte. bei der abklärung eines akuten thoraxschmerzes sind auch seltene ursachen, so zum beispiel der einbezug thorakaler organe in eine entzündliche systemerkrankung, zu bedenken. anhand eines besonderen falles möchten wir den weg zur diagnose bei einem schwer kranken notfallpatienten zeigen. leitsymptome bei krankheitsmanifestation. ein -jähriger mann wird bereits zum dritten mal mit akuten thoraxschmerzen in die notaufnahme aufgenommen, jeweils war eine kardiale ischämie ausgeschlossen und ambulante diagnostik empfohlen worden. nach der schmerzcharakteristik, der vorgeschichte von rezidivierenden thoraxschmerzen und entsprechenden risikofaktoren wird bei massiver symptomatik jetzt von einem akuten koronarsyndrom ausgegangen und die invasive diagnostik durchgeführt. eine akute koronare ischämie kann jedoch ausgeschlossen werden. fieber, hohe entzündungszeichen, hinfälligkeit und gewichtsverlust von mehr als kg in den letzten wochen lassen dann eine infektbedingte oder tumorbedingte ursache vermuten, abdomensonographie und röntgen-thorax sowie ausführliches labor samt immunologie führen nicht weiter. wegweisende weitere organbefunde finden sich nicht. die behandlung mit antibiotika hat keinerlei effekt auf klinik oder entzündungsparameter. nach tagen wird der krankheitsverlauf kritisch evaluiert, eine nichtinfektiöse ursache der beschwerden wird in betracht gezogen, eine transösophageale echokardiographie wird zum ausschluss einer endokarditis und zur beurteilung der aorta (leitsymptome thoraxschmerzen und fieber!) durchgeführt. dabei zeigen sich die klappen sämtlich unverdächtig, die aorta ascendens weist eine massive echoarme wandverdickung auf. zum sicheren ausschluss eines intramuralen hämatoms wird sofort eine ct der thorakalen aorta durchgeführt, die eine ausgeprägte zirkuläre wandverbreiterung ohne hinweise auf dissektion zeigt. diagnose. riesenzellarteriitis mit aortitis. therapie. steroide, einleitung einer remissionsinduzierenden therapie mit cyclophosphamid boli. weiterer verlauf. innerhalb von tagen ist der patient beschwerdefrei, die entzündungsparameter sind halbiert, der bettlägerige patient lässt sich mobilisieren und verlässt nach tagen die klinik. bei der diffe-renzialdiagnose des akuten thoraxschmerzes sollten eine unklare entzündungsserologie und eine b-symptomatik frühzeitig an eine aortitis denken lassen. in diesem fall fand sich bereits in tee und ct ein ausgeprägter befund, der sich am ehesten durch den langen verlauf vor diagnosestellung erklären lässt. einleitung. die progressive familiär intrahepatische cholestase (pfic) gehört zu einer heterogenen gruppe seltener, autosomal rezessiv vererbter erkrankungen der gallensäurenexkretion mit intrahepatischer cholestase, hohem risiko der leberzirrhose bereits im kindesalter und hepatozellulärem karzinom. das auftreten eines sle bei pfic ist bisher in der literatur nicht beschrieben. methoden. wir berichten über eine jetzt -jährige patientin mit genetisch gesicherter pfic- (defekt der atp abhängigen gallensalz-exportpumpe bsep), biliostomaanlage im kindesalter, therapie mit udca und kontinuierlicher hepatologischer betreuung. / manifestierte sich ein sle mit az-minderung, florider polyarthritis, polyserositis, splenomegalie, anämie und leukozytopenie. die ana waren mit > : homogen erhöht, die dns-ak im elisa mit u/ml, ena und antiphospholipid antikörper waren negativ. eine steroidtherapie wurde mit prednisolon mg/tag begonnen. bei guter verträglichkeit und stabilen cholestaseparametern wurde die therapie um chloroquin mit mg an tagen der woche ergänzt. die betreuung wurde interdisziplinär fortgesetzt. ergebnisse. unter der steroidtherapie waren gelenkbeschwerden und serositis gebessert, das crp, die leukozytopenie und anämie normalisiert. die dns-antikörper fielen auf u/ml, c und c stiegen um %. es persistierten lediglich physiotherapeutisch behandelte muskuläre schmerzen und schonatmung nach pleuritis. bei steroidreduktion unter mg traten die gelenkbeschwerden wieder auf, die dns-antikörper stiegen auf u/ml und c /c fielen um %, das crp blieb normal. die steroiddosis wurde auf mg/tag und die chloroquindosis auf × mg/woche erhöht. neue organkomplikationen im rahmen des sle sind nicht aufgetreten. die gallensäuren waren mit µmol/l (norm < ) im jahr (letzte messung vor manifestation des sle) deutlich erhöht, bei manifestation des sle mit µmol/l bereits deutlich gebessert und unter hoch dosierter steroidtherapie mit , µmol/l nach woche sowie , µmol/l nach monat normalisiert. unter prednisolon mg/tag stiegen sie bei inaktiviertem sle auf µmol/l nach monaten an, bei steroidreduktion unter mg auf µmol/l. nach erneuter steroiddosiserhöhung auf mg prednisolon/tag fielen sie auf µmol/l ab. schlussfolgerung. die pfic- schützt nicht vor der manifestation eines sle. eine behandlung mit steroiden und chloroquin ist auch bei pfic sicher und wirksam. der floride sle und die höher dosierte steroidtherapie senken trotz bestehenden genetischen defekts unabhängig voneinander und synergistisch die gallensäurenspiegel im serum bei genetischer störung der atp-abhängigen sekretionspumpe bsep. augenentzündungen. eine ausgeprägte b-symptomatik mit fieber, nachtschweiß und abgeschlagenheit seit ca. zwei jahren hatte sich jeweils unter der therapie der hörstürze verflüchtigt. die mr-angiographie der aortenwand zeigte den typischen befund eines ausgeprägten wandenhancements der thorakalen aortenwand sowie der supraaortalen gefäße, duplexsonographisch fand sich eine zirkuläre wandverdickung der proximalen und mittleren acc beidseits ohne relevante stenosen. bei fehlendem nachweis zerebraler vaskulitischer oder embolischer veränderungen im kraniellen mrt und bei normalem eeg wurde der cerebrale krampfanfall als gelegenheitsanfall dd im rahmen der hochaktiven grunderkrankung interpretiert. diagnose. takayasu-arteriitis mit assoziiertem cogan-syndrom und rezidivierender polychondritis. therapie. steroidstoß, darunter rasche reduktion der entzündungszeichen und promptes ansprechen der b-symptomatik, beginn einer steroidsparenden therapie mit mtx. bei anhaltender krankheitsaktivität und hohem steroidbedarf wird der patient aktuell mit tocilizumab behandelt. schlussfolgerung. der präsentierte fall zeigt, dass mittels neuer bildgebender verfahren gelegentlich eine großgefäßvaskulitis detektiert werden kann, obwohl das klinische bild (hier: kopfklinik) eine kleingefäßvaskulitis vermuten lässt. das cogan-syndrom ist häufig mit einer großgefäßvaskulitis assoziiert, in diesem fall auch mit einer polychondritis. umgekehrt erweitert sich unser wissen um das befallsmuster der großgefäßvaskulitiden, die zwar überwiegend große, aber auch mittelgroße und kleine gefäße einbeziehen können. in unserem zentrum ist dies der zweite fall einer takayasu-arteriitis mit assoziiertem cogan-syndrom in einem kollektiv von fällen. diagnostik. im rahmen einer vorstellung in unserer rheumatologischen ambulanz zur abklärung eines möglichen sjögren-syndroms, konnte in der lungenfunktionsdiagnostik eine leichte restriktion und Überblähung festgestellt werden. in einem auswärtig durchgeführten mrt der halsweichteile zeigten sich die glandula parotis und submandibularis beidseits inhomogen und kräftig kontrastiert, ebenfalls mehrere grenzwertig große lymphknoten. aufgrund der erneut pathologischen lungenfunktion und dem verdacht auf eine lungenbeteiligung bei sjögren-syndrom erfolgte ein ct-thorax. hier zeigte sich eine zysti-sche rarefizierung vor allem der zentralen lungenanteile, differentialdiagnostisch mit einer langerhans-zell-histiozytose vereinbar. auch erschien der diabetes insipidus passend zur histiozytose. die immunologischen untersuchungen waren unauffällig, insbesondere konnten keine ssa-/ssb-antikörper oder eine hypergammaglobulinämie nachgewiesen werden. somit erschien ein sjögren-syndrom unwahrscheinlich. zur weiteren abklärung erfolgte eine bronchoskopie, in den biopsaten konnte immunhistochemisch eine langerhans-zell-histiozytose nachgewiesen werden. in einer pathologischen nachuntersuchung auswärtiger parotis-biopsate bestätigte sich diese, zudem konnte eine mutation des braf-proto-onkogens nachgewiesen werden. im abschließend durchgeführten pet-ct konnte eine vermehrte kontrastmittelaufnahme des hypophysenstiels, eine vermehrte stoffwechselaktivität der parotis beidseits sowie kutan axillär links diagnostiziert werden. abschließend lag somit eine langerhans-zell-histiozytose mit befall von hypophyse, lunge, parotis, haut, lymphknoten sowie einem fraglichen befall des darms vor. therapie. in absprache mit der adulten langerhans-zell-histiozytose studiengruppe erfolgt nun eine therapie mit cytarabin. weiterer verlauf. der weitere verlauf nach geplantem therapiebeginn bleibt abzuwarten. einleitung. die anti-gbm-erkrankung (goodpasture-syndrom) ist eine prototypische autoimmunerkrankung mit ernster prognose, wenn sie als "pulmorenales syndrom" mit der trias rapid-progressive glomerulonephritis, alveoläre hämorrhagie und nachweis von autoantikörpern gegen glomeruläre basalmembranen (anti-gbm-ak) auftritt. Über weniger aggressive verläufe ist wenig bekannt. in der literatur wird die bedeutung der frühen diagnosestellung für die prognose der patienten betont. wir berichten über eine -jährige patientin, die sich mit abgeschlagenheit, müdigkeit und blutbeimengungen im sputum vorstellt. sie betreibt einen nikotinabusus. methoden. wir sehen eine blasse patientin (hb , mmol/l, normochrom, normozytär), die keine weiteren auffälligen befunde in der körperlichen untersuchung zeigt. die apparative diagnostik mittels endoskopie und sonographie ergibt keine befunde, die die anämie erklären können; in der bronchoskpie zeigt sich das bild einer alveolären hämorrhagie ohne aktive blutungszeichen. neben der ausgeprägten anämie bestehen eine milde proteinurie mit mg/d sowie eine geringe erythrozyturie. die immunologische diagnostik ergibt gering erhöhte anti-gbm-ak, negative ana und anca. die nierenbiopsie zeigt eine rapid-progressive glomerulonephritis mit halbmondbildung in einem glomerulum, zusätzlich können lineare igg-ablagerungen in der immunfluorenz dargestellt werden. es ist ein glomerulum in der biopsie betroffen, die anderen glomeruli sind unauffällig. es bestehen einzelnen erythrozytenzylinder, diffuse entzündungszeichen lassen sich nicht nachweisen. die nachträglich durchgeführte immunfluoreszenz in der lungenbiopsie bestätigt den befund linearer igg-ablagerungen. ergebnisse. in unserem fall sehen wir eine junge patientin in einem relativ guten allgemeinzustand mit gering erhöhten anti-gbm-antikörpern und einer gering ausgeprägten nierenschädigung mit einem befallenen glomerulum in der biopsie. es wird angesichts des jungen alters der patientin und der geringen ausprägung der erkrankung eine therapie mit cyclophosphamid nach dem "euro-lupus-protokoll" von f. hossiau eingeleitet. im verlauf steigt der hb auf , mmol/l, die alveoläre hämorrhagie sistiert und die proteinurie ist rückläufig. schlussfolgerung. das goodpasture-syndrom mit einer inzidenz von , - , / mio. einwohner und jahr ist eine sehr seltene autoimmunerkrankung mit ernster prognose. möglicherweise spielen umweltfaktoren (hier: nikotinabusus) eine rolle für die manifestation der erkrankung. interessant ist, dass die erstbeschreibung im rahmen einer influenza-epidemie -wie auch in diesem jahr bei unserer patientinerfolgte. Über die therapie gibt es wenige informationen. die meisten empfehlungen gibt es zum pulmorenalen-syndrom, über weniger aggressiv verlaufende manifestationen gibt es nur wenige informationen. einleitung. wir berichten über einen -jährigen raucher mit akut aufgetretener polyarthritis und neu aufgetretenem raynaud-phänomen. auffallend war die diskrepanz zwischen nur geringer systemischer entzündungskonstellation und einer hochaktiven polyarthritis mit schwerem raynaud-phänomen. im verlauf kam es zu einer spontanen thrombophlebitis der v. cephalica. methoden. pathologische werte: crp maximal , mg/dl (norm < , ), zellzahl im gelenkpunktat . leukozyten/µl. im normbereich lagen: bsg mm/ h, ana, ena, ds-dns-ak, rheumafaktoren, anca, kälteagglutinine, kryoglobuline, tumorsuche initial ohne befund (ct-thorax, bronchoskopie, ct abdomen und becken, coloskopie, gastroskopie, skelettszintigraphie), fdg pet-ct: zwei suspekte lymphknoten rechts cervical sowie suspekte rechte tonsille. ergebnisse. selbst unter mg prednisolon weiterhin hochaktive polyarthritis. nach unauffälliger initialer tumorsuche veranlassten wir ein fdg-pet ct mit nachweis zweier suspekter lymphknoten rechts cervical sowie einer suspekten rechten tonsille. die biopsie der klinisch sich nur in der palpation diskret induriert darstellenden region ergab ein gering differenziertes, gering verhornendes plattenepithel mit %iger sequenzhomologie mit hpv typ . nach resektion des tumors und radiatio sistierten sowohl die polyarthritis als auch das raynaud-phänomen ohne weiteres rezidiv auch nach absetzen der glukokortikoide. schlussfolgerung. eine akut auftretende hochaktive polyarthritis mit hohem glukokortikoidbedarf in kombination mit einem raynaud-syndrom, auffallend niedriger systemischer entzündungsaktivität und fehlendem autoantikörpernachweis sollte insbesondere bei einem langjährigen raucher anlass zu einer intensiven tumorsuche geben. ein fdg-pet-ct kann bei okkulten tumoren zielführend sein. bemerkenswert ist hier der nachweis von hpv- im tumor als weiterer risikofaktor neben dem zigarettenrauch. methoden. bei der klinischen untersuchung fielen ein beidseits positives menell-zeichen und deutlicher klopfschmerz über dem lumbosakralen Übergang auf. labordiagnostisch konnte ein erhöhtes c-reaktives protein und ein positiver hla-b nachgewiesen werden. der bath ankylosing disease activity (basdai) index betrug , . die beckenübersichtsaufnahme zeigte eine definitive bilaterale sakroiliitis grad gemäß den modifizierten new-york-kriterien. der befund der kontrastmittelunterstützten mrt der iliosakralgelenke bewies das vorliegen einer bilateralen floriden sakroiliitis. bei gesicherter hla-b positiver ankylosierender spondylitis wurde die indikation zur einleitung einer biologikatherapie mit einem tnfα-inhibitor gestellt. während der abklärung von kontraindikationen wurde in der konventionellen röntgen-thorax-aufnahme eine rundliche, glatt begrenzte zystische läsion mit flüssigkeitsspiegeln im rechten mittellappen entdeckt. die weitere abklärung mittels nativer thorax-ct, bronchoskopie und biopsieentnahme bestätigte den verdacht auf eine bronchogene zyste. die erregerdiagnostik in der bronchoalveolären lavage zeigte lediglich eine kontamination mit der residenten standortflora. ergebnisse. in anbetracht der geplanten immunsuppressiven therapie, die mit einem erhöhten infektionsrisiko einhergeht, wurde die bronchogene zyste im september operativ entfernt. zur linderung der beschwerden erhielt die patientin eine schmerztherapie mit nsar. als sich die patientin sechs monate später erneut zur einleitung der biologikatherapie vorstellte, berichtete sie, dass die schmerzen etwa einen monat nach der operativen resektion praktisch verschwunden seien. die klinische untersuchung war unauffällig, der basdai-index lag bei , . auch das zur verlaufskontrolle angefertigte mrt der isg zeigte im vergleich zur voruntersuchung einen deutlichen rückgang der floridität. schlussfolgerung. es handelt sich um den ersten fall einer kompletten klinischen und radiologischen remission einer hla-b -positiven ankylosierenden spondylitis nach operativer entfernung einer bronchogenen zyste als potenziellen entzündlichen fokus. bei der systemischen verlaufsform der erkrankung treten neben kutanen erscheinungen zusätzlich muskuloskeletale, hämatopoetische ( - %), renale (ca. %), kardiale, cerebrale und pulmonale ( - %) manifestationen auf. eine polyserositis ( - %) ist häufig. mit - % werden im sle-schub abdominelle schmerzen beschrieben. nur in seltenen fällen (amerika , %, asien , - , % aller sle-patienten) kommt es zum bild einer lupusassoziierten mesenterialen vaskulitis (lmv). die Ätiologie der lmv ist weitestgehend unklar, eine genetische präsidsposition sowie auslösende faktoren (bakterielle darminfektionen, medikamente wie nsar, phosphodiesterasehemmer) werden diskutiert. pathogenetisch wird eine mesenteriale ischämie durch eine mikroangiopathie (arteriolen, venolen) bei inflammatorischer immunkomplexpräzipitation sowie thrombembolischen ereignissen angenommen. radiologisch/sonographisch zeigt sich ein segmentales darmwandödem mit darmdilatation. endoskopisch dominieren oberflächliche ulzerationen, perifokale hämorrhagien bis hin zur gangrän. eine erhöhte perforationsneigung wird beschrieben. mikroskopische befunde zeigen eine fibrinoide nekrose subseröser gefäße mit leukozytoklasie der gefäßwand bzw. ein submuköses Ödem mit nur diskreter invasion mononukleärer zellen. die prognose der lmv scheint abhängig von genetischer prädisposition, raschem beginn einer immunsuppression sowie restriktivem einsatz operativer interventionen und wird je nach literaturquelle mit einer letalität bis zu % angegeben. background. we report a patient with tma in the context of sle treated successfully with the c inhibitor eculizumab. the patient had sle with lupus nephritis (ln). before she developed tma with renal failure and neurologic manifestations, she was treated with various immunosuppressive regimens for mucocutaneous and musculoskeletal manifestations and later for ln. the diagnosis of atypical hemolytic uremic syndrome (ahus) was made based on the presence of coombs-negative hemolytic anemia, thrombocytopenia, renal failure, seizures due to cerebral ischemia and signs of tma in the renal biopsy. plasma exchange and hemodialysis were started immediately and could stabilize her condition. six weeks after the beginning of plasmapheresis but still severely compromised renal function and thrombocytopenia, complement inhibition with eculizumab became a therapeutic option. after the first infusions, renal function, anemia and thrombocyte counts markedly improved. dialysis could be stopped. extensive genetic testing of mutations associated with the overactivation of the alternative complement pathway was negative. after months, when the patient was still in remission, eculizumab infusion intervals were widened and it was finally stopped after months of treatment. since then, renal function remained stable with nearly normal glomerular filtration rates. background. il- signaling plays an important role in inflammation but is restricted by different regulatory mechanisms. these mechanisms include the decreased availability of gp , the signal transducing chain of the il receptor, on the cell surface. the aim of this study was to determine whether the inflammatory environment in the arthritic joint has an impact on monocytic gp surface expression and the extent to which regulatory processes in the synovial fluid (sf) can be transferred to an in vitro model. flow cytometry and live cell imaging were used to measure the cell surface expression and internalization of gp . stat phosphorylation was monitored by flow cytometry and western blotting. results. the level of cell surface gp expression on sf monocytes was reduced compared to peripheral blood (pb) monocytes from patients with juvenile idiopathic arthritis (jia). this reduction could be reproduced by stimulating pb monocytes from healthy donors with sf and was dependent on p mapk. the induction of p by il- β in pb monocytes interfered with il- signaling due to the reduced cell surface expression of gp . the results suggest that p -mediated pro-inflammatory stimuli induce the downregulation of gp on monocytes and thus restrict gp mediated signal transduction. this regulatory mechanism could be relevant in the inflamed joints of patients with jia. kr. sjia patient characteristics of those who successfully discontinued corticosteroids during canakinumab treatment: secondary analysis from a pivotal phase trial background. interleukin- β (il- β) is a key driver in the pathogenesis of systemic juvenile idiopathic arthritis (sjia). canakinumab (can), a selective fully human anti-il- β monoclonal antibody, has been shown to be efficacious in the treatment of sjia [ ] . corticosteroids (cs) are a mainstay of therapy for sjia, however due to the well-known long-term side effects, reduction of cs dosage is desirable. objectives. to assess patient features associated with cs discontinuation during can therapy. methods. patients ( - years of age) with active sjia received s.c. can ( mg/kg to mg max) every four weeks during the maximum week cs-tapering phase [ ] . cs tapering was to be initiated when at least an adapted acr was achieved and no fever. a -year-old boy presented with nocturnal tingling paresthesia affecting his feet and his calves. no excessive leg movements were noted at night. within a few months, his symptoms worsened. the paresthesia occurred both during the day and at night. moreover, the paresthesia came to be triggered by merely standing up. affecting a sharply demarcated area not corresponding to dermatomes, symptoms resolved promptly with movement. the paresthesia was associated with local skin erubescence in spots that slowly began spreading all over the affected area. symptoms did not occur while the patient was seated. mild painless swelling around both of the ankles was noticed in the evenings. approximately one and a half years after the initial manifestation, painful triphasic color changes of all fingers and toes triggered by cold or stress occurred. the family history was positive only for psoriasis. extensive laboratory studies excluded inflammatory and hematological conditions as well as occlusive arterial diseases known to be associated with secondary raynaud's phenomenon. polyneuropathy and other neurological disorders were excluded as well. inflammatory joint disease suspected from the initial imaging with magnetic resonance of the feet and ankles was not confirmed by repeated investigations and scintigraphy. the only consistent abnormality was a reduced pulse amplitude corresponding to vasospasm, which was revealed by photoplethysmography of toe vessels. additionally, paradoxical amplitude reduction after application of nitroglycerine was seen in finger vessels. placing his hands or feet in cold water did not trigger raynaud's phenomenon. initial treatment with non-steroid anti-inflammatory drugs, topical isosorbiddinitrate and local steroid instillation (suspicion of inflammation of tibialis posterior tendon) was ineffective. systemic therapy with the calcium channel blocker amlodipine was initiated. the initial dosing of mg ( . mg/kg/day) was slowly increased to mg ( . mg/kg/day) which lead to complete resolution of the patient's ailments. after three years of pharmacotherapy and . years in remission, a weaning off the treatment is planned. based on the patient's positive response to calcium channel blocker, we conclude that the lower-leg paresthesia was of vascular origin and can be considered an atypical presentation of raynaud's phenomenon. background. the initial treatments of choice for jdm are high-dose corticosteroids and methotrexate. however, no consensus exists about second line therapeutic options in refractory or recurrent cases. results. we present a -year-old boy who was diagnosed with jdm due to severe proximal muscle weakness, dysphagia, a heliotrope rash, gottron's sign, nail teleangiectasia and a characteristic muscle biopsy. creatine kinase levels were within normal range and no antinuclear antibodies were present. over a period of seven years, the patient was treated with high-dose corticosteroids, methotrexate, intravenous immunoglobulins, oral steroids, mycophenolate mofetil, rituximab and infliximab. despite all treatment efforts, skin and muscle inflammation persisted and the boy developed severe subcutaneous calcifications, rendering him wheelchair-bound. as il- production correlates with disease activity in adult and juvenile dm, treatment with tocilizumab ( mg/kg every weeks) was initiated, leading to a complete resolution of skin inflammation within months. within months of treatment, the disease activity score (das) decreased from to (out of ), the childhood myositis assessment scale (cmas) increased from to (out of ) and the kendall manual muscle test (mmt) increased from to (out of ). in daily life the wheelchair was no longer necessary. treatment was well tolerated but accompanied by a moderate increase in liver transaminase activities. interestingly, therapy with rituximab was associated with a decline in igm levels only, whereas igg and iga stayed markedly elevated. in contrast, following initiation of tocilizumab treatment, igg levels rapidly declined to normal range, emphasizing the role of the humoral immune system in the pathogenesis of dm. conclusion. taken together, treatment of a severely affected jdm patient with tocilizumab was safe, well tolerated and led to a significant improvement in disease activity. further investigations of il- -blocking agents as a treatment option in otherwise therapy-resistant jdm patients are warranted. functional capacity of jia patients with an initial adjustment to an anti-tnf-alpha therapy background. thirty three percent of patients with polyarticular jia are treated with biologics [ ] . despite substantial improvement achieved by anti-tnf-α treatment according to disease activity [ ] patients have joint-specific impairments. this factor should be considered when analyzing the functional effects on joint limitations while performing daily activities. methods. in a prospective study on polyarticular jia patients treated with anti-tnf-α therapies plus functional therapies we study the longitudinal effects on joint function. the measurements include -d gait analysis (eight vicon f cameras, omg, london, balance control (s -check, tst, großhoeflein), pedobarography (emed plate ( sensors/ cm², novel, munich), daily activity assessment (step watch, orthocare innovations, ok usa), acr pedi and joint mobility testing. we here present the cross-sectional data of the first patients ( . ± . yrs, . ± . cm, . ± . kg, pain-level: . ± . / vas, active joints: . ± . , chaq: . ± . ). results. preliminary results demonstrate that the ability to walk is slightly limited. patients have a reduced push off power generation within the ankle joint (patients: . ± . w/kg; healthy controls: . ± . w/ kg). further on they show limited sensory motor control and stability in comparison to patients with an inactive disease status while performing balance tests (patients: sensory index: . ± . , stability-index: . ± . , patients with inactive status: sensory-index: . ± . , stability index: . ± . (p< . ). note: lower indices values are better results. conclusion. it is one of the first studies which show functional joint-specific deficits during every day activities in patients who receive an initial anti-tnfα-therapy. the limited stability and motor control might be due to limited joint integrity in the ankle joint. this is supported by the impaired push off function while walking. the next study step will show possible effects of the anti-tnfα-therapy. background. the role of sport as a therapeutic tool in treating patients with jia is becoming more important recently [ ] . effects of exercise therapy are reviewed beneath others by takken et al. [ ] . they state that short-term effects look promising but the effects of long-term studies remain unknown. methods. the preventive mobility workout (pmw) is a whole body home-exercise-therapy ( min each day) for patients with an inactive diseases status. it counteracts the deficits which were observed during functional studies in the past [ ] . it consists of exercises for muscular strengthening (squads: hamstring to quadriceps ratio), hamstring flexibility (lift and raise), core stability (prone bridge -time-to-failure), shoulder griddle mobility (horizontal extension) and ankle joint integrity (mechanical power while walking . for statistical analysis an anova was calculated and the level of statistical significance was set to p< . . results. preliminary results show a group effect of the pmw for the hamstring to quadriceps ratio (h-q-r) for the right side (p< . ) and a tendency for the left side (p= . ). the h-q-r for the right side has changed in the tg and cg from . ± . to . ± . and from . ± . to . ± . , respectively. it has changed for the left side in the tg and the cg from . ± . to . ± . and from . ± . to . ± . , respectively. all other parameters regarding flexibility or joint integrity show low or no effects. we have re-tested n= out of so far and the pmw training show little training-effects. the preliminary results might be a reasonable proof for long-term effects. a possible reason for the little effects might be that patients are supposed to train every day but the training diaries show that they exercise approximately three times a week. the authors would like to thank the "deutsche kinder-rheumastiftung" for supporting the study. conclusion. we will validate these proposed definitions prospectively in a jia associated uveitis cohort. based on the results, we will weight these measures to develop an overall scoring system. background. minute walk is a primary outcome measure in studies in pulmonary hypertension. currently we have a two of sets of data [ , ] regarding test results in the minute walk test ( mwt) in healthy children with a large span in the norm values in the different age groups. aim of the study was to establish norm values for healthy german children for the minute walk test. methods. the team of an occupational therapist and a study nurse were visiting schools. permission from the parents was give before the test. always just probands from one class were invited to participate. the test were performed according the international guidelines [ ] . the demographic data of the probands were collected and the parents filled out a short survey regarding the physical activity and the health condition. children with chronic diseases, which decrease the stamina were excluded. up till now probands participated from the age to years. of them were female. the mean minute walk continuously increased with age (. tab background. juvenile idiopathic arthritis (jia) is the most common chronic disease in pediatric rheumatology which often results in foot impairments [ ] . patients with jia are reported to have smaller pressure loads underneath the foot while walking [ ] . the aim of the study was to analyze the peak plantar pressure distribution of a well described cohort of jia patients with an active symmetrical ankle joint arthritis and no history of foot involvement. [ ] ) wurden in bezug auf therapie und outcome anhand der wallace-kriterien beurteilt: "active disease" (ad), "inactive disease" (id), "clinical remission under medication" (crm), "clinical remission off medication" (crom; [ ] background. familial mediterranean fever (fmf) is one of the most common autoinflammatory diseases (aid). pathogenomic relevant mutations in the mefv gene show autosomal recessive inheritance, but co-dominant mutations have been described. we aimed to evaluate correlations between ethnic origin, phenotype and genotype for fmf patients in the german aid-net-registry. methods. we used two common scoring systems modified for children (mor et al., pras et al.) to assess disease severity in fmf patients of the aid-net-registry. for the five most frequent mutations, we tested for a correlation of the genotype with the phenotype, mean crp and ethnic origin, respectively. furthermore, we evaluated the applicability of the two severity scores for children. results. among the patients, we detected a total of pyrin mutations and different sequence variants, including one new mutation (p.gly asp). the five most frequent alterations were p.met val ( %, n= ), p.met lle ( %, n= ), p.val ala ( %, n= ), p.glu gln ( %, n= ) and p.met ile ( . %, n= ). ethnic origin could be determined in cases; the prevailing ancestry was turkish ( %, n= ), % (n= ) were lebanese. p.met val in homozygous form ( %, n= ) was correlated with a more severe disease activity, based on the score by mor, as well as with a higher mean crp ( mg/l, n= , mg/l, n= ) compared to patients without this mutation (p= . and p< . , respectively). the score suggested by pras did not yield a significant genotype-phenotype correlation; indeed, the two scoring systems were inconsistent with each other (κ< . ). although a typical distribution of mutations in different ethnic populations was obvious, this trend was not statistically significant, probably due to the divergent number of cases. conclusion. the homozygous p.met val substitution was associated with a more severe disease activity. there was no origin-genotype correlation in this fmf population. the well-known severity scores for children (mor, pras) are inconsistent. the aid-net is working on a new scoring system. . all patients with rp should be investigated by capillaroscopy. capillaroscopy will be classified into "normal", "aspecific changes" or "scleroderma pattern". . all patients who have additional symptoms pointing to a definite connective tissue disease should be evaluated according to disease specific guidelines. . ana-negative and capillaroscopy-negative patients should be followed-up at least every months. . ana positive patients without disease-specific antibodies and with negative capillaroscopy findings should be followed-up at least every months. . ana and disease-specific antibody positive patients should have organ specific evaluation according to symptoms, examination and relevant to that particular disease e.g. patients who are ana and scl- positive may need organ specific evaluation for jssc as per the juvenile systemic sclerosis inception cohort protocol (www.juvenilescleroderma.com). . ana-positive patients, who have no disease specific antibody but have positive capillaroscopy results, should be followed-up at least every months. . ana-negative patients with positive capillaroscopy result should be followed-up at least every months. . the group could not reach an agreement regarding treatment, due to a lack of data for the paediatric age group. the group agreed that implementation of adult recommendations conclusion. the group made a suggestion for a standard of good clinical practice for rp in children. our aim is that this will facilitate a large multicentre prospective follow-up study of children with rp. background. chronic non-bacterial osteomyelitis (cno) is an inflammatory disorder of the skeletal system of unknown etiology. long-term follow-up and response to treatment data have rarely been reported. the aim of the study was to characterize the clinical, radiological, histological and laboratory data at juvenile cno onset, and to analyze the long term treatment response. methods. the course of disease of juvenile patients with non-bacterial inflammatory bone lesions was evaluated retrospectively. clinical, radiological, histological and laboratory data were assessed at disease onset and for a median time of disease of months. results. the mean age at disease onset was . years, the mean time between the first symptoms and the diagnosis of cno was months. % of the patients had multifocal bone lesions. biopsy was performed in patients. only when bone biopsy was taken within months of symptom onset, cellular infiltrates could be observed. at later time points, fibrosis, hyperostosis and bone edema predominated. the initial treatment consisted of non-steroidal anti-inflammatory drugs (nsaids). % of the patients required second line therapy consisting of sulfasalazine and short term oral corticosteroids, % of the patients required bisphosphonates or tnf-blocking agents. the number of clinical lesions decreased to % within . months and reached . % after months of treatment. the number of radiological lesions, however, declined to only . % after months of treatment. in detail analysis of the tre-atment response revealed that initiation of sulfasalazine treatment in nsaid non-responders led to a significant and sustained decline of the clinical, as well as the radiological number of lesions. conclusion. the rapid clinical improvement in cno, following initiation of therapy with nsaids, is not accompanied by a likewise decrease of the number of radiological lesions. treatment with sulfasalazine is effective in childhood cno. background. exercise has a wide variety of beneficial health effects. it stimulates bone formation and maintains bone strength as well as decreases the risk of falls. moreover, exercise at regular intervals is also assumed to positively affect immune functions. conversely, in more than % of the astronauts during/after space flight and under simulated weightlessness immune functions are suppressed. to assess the effects of simulated weightlessness during the nd berlin bedrest study (bbr- ) on immunological parameters. furthermore, to compare the effects of two different exercise performances (resistive vibration exercise and resistance exercise without vibration). methods. physically and mentally healthy male volunteers ( - y) experienced days of six degree head down tilt bed rest. they were randomized to groups: resistive vibration exercise (n= ), resistance exercise without vibration (n= ), inactive controls (n= ). blood samples were taken days before bed rest, on day and after beginning of bed rest. composition of immune cells was analyzed by flow cytometry. cytokines and neuroendocrinologic parameters were analyzed by a multiplex suspension array/ elisa in plasma. general changes over time were identified by paired t-test, exercise-dependent effects by -group repeated measurements anova. results. for all cases pooled, the number of granulocytes (p< . ), nkt cells (p< . ) and hematopoietic stem cells (p< . ) increased during the study; the concentrations of dhea (p< . ) and eotaxin (p< . ) decreased. different impacts of the specific types of exercise on the change over time were shown for lymphocytes, nk cells, nkt cells, tcell subpopulations and the concentrations of ip- and rantes. conclusion. we found immobilization/simulated weightlessness to significantly impact immune cell populations, and cytokine and neuroendocrine factor concentrations. exercise was able to specifically influence immunologic parameters. interestingly, these changes resemble those found during the aging process. background. novel therapies have made remission and low disease activity (lda) achievable goals in ra. we assessed the impact of treatment with subcutaneous abatacept or adalimumab on these goals and on functional and radiographic outcomes in ample (abatacept versus adalimumab comparison in biologic-naïve ra subjects with background methotrexate), the first head-to-head trial of biologics in ra patients with inadequate response to mtx (mtx-ir). methods. ample is a -year, phase iiib, randomized, investigator-blinded study. biologic-naïve ra patients with mtx-ir were randomized to receive mg abatacept weekly or mg adalimumab biweekly, combined with a stable dose of mtx [ ] and radiographic non-progression (defined as change in modified total sharp score ≤ . ) were analysed in patients achieving or not achieving remission or lda at days or . results. baseline clinical characteristics of abatacept and adalimumab treatment groups were balanced, as was clinical, functional and radiographic efficacy and safety at day [ ] . the proportions of patients meeting each of the remission criteria or lda at day were similar for both groups, but significantly more patients achieved das (crp) remission compared to cdai, sdai or rapid remission, and the smallest proportion achieved boolean remission. compared to remission, a higher proportion of patients achieved lda. across all definitions of remission or lda, > % of the patients achieving remission at days and were haq responders at day . more than % of patients achieving remission or lda at days and were radiographic nonprogressors at day . improvement in physical function and radiographic outcomes were consistent between the two treatment groups in both remission and lda populations (. tab. ). conclusion. through year, patients treated with subcutaneous abatacept or adalimumab in ample achieved comparable rates of remission and lda. similar improvements in physical function and radiographic outcomes were observed. these data help to illustrate the relationship between remission, lda and functional and radiographic outcomes independent of treatment with subcutaneous abatacept or adalimumab. background. previous small studies suggest that responses to some immunizations may be attenuated by intravenous abatacept but remain clinically meaningful [ , ] . we investigated the magnitude of response to pneumococcal and influenza vaccination in a larger number of patients receiving subcutaneous (sc) abatacept therapy. the objective of the study was to evaluate the antibody response to the standard -valent pneumococcal polysaccharide vaccine and the - seasonal influenza trivalent vaccine in adult patients with ra on sc abatacept and background dmards. these multicentre, open-label sub-studies of the -valent pneumococcal polysaccharide vaccine and seasonal influenza vaccine enrolled patients in the acquire (pneumococcal and influenza) or attune (pneumococcal) studies. patients were enrolled at any point during their sc abatacept treatment cycle after completion of ≥ months' abatacept treatment. all patients received fixed-dose sc abatacept mg/week with background dmards. a pre-vaccination blood sample was collected and vaccines administered, while continuing background sc abatacept and dmards. after ± days, a post-vacci- background. in real life, dosage increases are common with biologic agents [ ] . intravenous abatacept is administered by patient body weight ( mg/kg) and weeks after the first infusion and every weeks the-reafter [ ] totalling infusions over the first months. no adjustments to this schedule are recommended. abatacept retention rates, efficacy and safety over months in action (abatacept in routine clinical practice) have been reported previously [ , ] this study was designed to assess adherence to recommended dosing of abatacept over the first months in action. methods. action is an ongoing, -year, international, non-interventional, prospective cohort of ra patients treated with intravenous abatacept. all patients on abatacept treatment for ≥ months, and with infusion data available at initiation and at months, were considered in this analysis. good adherence was defined as correct dose by patient body weight and number of actual-to-recommended infusions within the range - % (i.e. - infusions). results. in total, / ( . %) patients received abatacept ≥ months and had the infusion data available. most had established ra and failed ≥ anti-tnf agent ( . %). of patients with body weight data available at initiation, . % received the recommended initial dose, . % a lower dose and . % a higher dose than recommended. good adherence to the abatacept treatment schedule was found in / ( . %) patients. over months, . % of patients received infusions, . % received infusions and . % had infusions. change in dosage over time was assessed in / patients with data available at both time points. the majority of patients ( . %) maintained the recommended dosage. / ( . %) patients received abatacept at the recommended dose for body weight and at the recommended treatment schedule over months. conclusion. in the real-world action study, adherence to the recommended abatacept treatment regimen over months was good. few patients received changes in dose and/or frequency of administration over this time period. background. in rheumatoid arthritis (ra), synovial fibroblasts (sf) secrete large amounts of il- , il- and matrix metalloproteinases (mmps) which are crucial for cartilage destruction. rasfs are sensitive to the action of cannabinoids and they express cannabinoid receptors type i and ii (cb and cb ), the vanilloid receptor (trpv ) as well as endocannabinoid degrading enzymes. cannabinoids are regarded as antiinflammatory and since anandamide (aea) is found in ra synovial fluid we investigated how this endocannabinoid affects adhesion, proliferation, and production of inflammatory mediators of rasf. methods. adhesion was assessed by the xcelligence system. proliferation was quantified by the amount of incorporated fluorescent dye into cellular dna. mmp- and cytokines were detected by elisa. in oasf, aea dose-dependently decreased the il- β induced production of mmp- (by %) in a trpv -mediated manner. il- and il- levels were only weakly modulated. in rasf however, aea decreased il- β induced production of il- ( %), il- ( %) and mmp- ( %). the effects of aea were not inhibited by cb , cb or gpr antagonists but were blocked by the trpv antagonist capsazepine. the inhibitory capacity of aea was enhanced by cyclooxygenase- inhibition in rasfs and oasfs, but was unaltered or even slightly reduced by faah inhibition. aea was even more potent in reducing above mentioned mediators when rasfs but not oasfs were incubated under hypoxic conditions and treated with tnf. furthermore aea increased adhesion of oasfs and rasfs to fibronectin. adhesion was modulated by cb , gpr , and trpv antagonists. combined faah and cyclooxygenase- blocked the stimulatory effect of aea on adhesion. proliferation was decreased by aea in rasfs and oasfs via a cyclooxygenase- but not via cb , cb or trpv dependent mechanism. conclusion. in conclusion, aea promotes an antiinflammatory phenotype of rasfs and oasfs by activating trpv . cb , trpv , and gpr act in concert to modulate adhesion of sfs and this is highly dependent on the intracellular concentration of aea. additionally, cyclooxygenase- metabolites of aea exert their anti-proliferative effects independent of cb and cb . fc. it has been reported that lower levels of czp, compared to ada or ifx, are transferred from treated mothers to the neonate [ ] . this discrepancy may be due to active transport of antibodies across the placenta thought to be mediated by the neonatal fc receptor (fcrn). however, anti-tnf binding to fcrn, and fcrn-mediated transcytosis have not been studied. the objective of this study is to quantify binding of czp, ifx, ada and eta to fcrn and to measure fcrn-mediated transcytosis. a biacore™ assay was used to determine binding of czp, ada and ifx to human fcrn. anti-tnfs were passed over an fcrn-coated chip .mdck-ii cells transfected with human fcrn were used to measure fcrn mediated transcytosis. the anti-tnfs and the control antibody (p ), which possessed a fc modified to prevent binding to fcrn, were biotinylated to allow visualization. the amount of each anti-tnf transcytosed across the cell layer over hours was measured by msd assay. results. ifx ( nm) and ada ( nm) had high binding affinity to fcrn while the binding affinity of eta to fcrn was - -fold lower ( nm). in contrast, czp did not bind to the fcrn with any measurable affinity. the levels of transcytosis seen with ifx and ada were . ng/ml and . ng/ml, respectively (mean of experiments). transcytosis of eta ( . ng/ml) was lower than that of ada and ifx. in contrast, the level of czp transcytosis was significantly lower, at . ng/ml, than that observed with the other anti-tnfs and comparable to the control p ( . ng/ml). conclusion. czp didn't bind to fcrn and thus no fcrn-mediated czp transcytosis was detected. in contrast, ada and ifx had a relatively high binding affinity to fcrn and were actively transcytosed. eta showed lower binding affinity and transcytosis, but fcrn-mediated transport could still be measured. these results explain the previously observed active transport of anti-tnfs across the placenta seen in patients treated with ifx and ada, whereas only low levels were observed with czp [ ]. background. anti-cyclic citrullinated peptide (ccp) status was reported previously as predictive of abatacept response [ ] . predictors of retention with abatacept have not been published previously. this study was designed to identify predictors of abatacept retention after failing ≥ biologic agent. in routine clinical practice) is an ongoing, -year, international, non-interventional, prospective cohort including patients with ra treated with intravenous abatacept [ , ] . patients from canada, germany, greece and italy, where patient numbers were sufficient to explore between-country effects, were included. at data cut-off (february ), all patients had -year follow-up (interim analysis). abatacept discontinuations were reported by the investigator at any time point during follow-up. socio-demographics, disease characteristics and medical history at abatacept initiation, and previous and concomitant treatments were deemed potential predictive variables. clinically relevant variables and those with p≤ . (univariate analysis) were entered into a multivariate cox proportional-hazards regression model, adjusted for clustered data from one investigator. using backwards selection, variables with p≤ . were retained in the final model. . there were no interactions or effects of c-reactive protein level, rheumatoid factor status, type of previous anti-tnf failure, infection at initiation and abatacept monotherapy. sensitivity analysis, including all variables significant in univariate analysis, was consistent. conclusion. in this first report of real-world predictors of abatacept patient retention, anti-ccp positivity and failing < prior anti-tnf agents were associated with higher retention. differences in retention between some countries may reflect specificities in healthcare systems and populations. abatacept, a biologic agent with no contraindications or special warnings for cardiac comorbidity, seems to be a good option for these patients. weekly subcutaneous abatacept confers comparable onset of treatment response and magnitude of efficacy improvement over months when administered with or without an intravenous abatacept loading dose zeitschrift für rheumatologie suppl · | methods. patients from the intent-to-treat populations of the acqui-re [ ] and ample [ ] studies randomized to subcutaneous abatacept plus mtx were included. all patients received fixed-dose subcutaneous abatacept mg/week; in acquire but not ample, patients also received an intravenous loading dose (~ mg/kg based on weight range) on day . for this post-hoc analysis, assessments included acr and haq-di response (improvement ≥ . ) over months, with patients who discontinued considered non-responders. mean changes from baseline over months in das (crp) were assessed in patients with das > . at baseline (last observation carried forward) to account for differences in baseline disease activity between the two studies. results. all patients were biologic naïve at baseline, with mean disease duration of . and . years, das (crp) . and . , and haq-di . and . in acquire and ample, respectively. efficacy was compared throughout the study. for patients treated with subcutaneous abatacept with and without an intravenous loading dose, acr response rates were similar (. tab. ). haq-di response rates were also similar with and without the intravenous loading dose (. tab. ). for the overall populations, mean (standard deviation [sd]) changes from baseline to day in das were − . ( . ) and − . ( . ) in acquire and ample, respectively. for patients with baseline das > . , mean (sd) changes in das from baseline to day were − . ( . ) and − . ( . ) in acquire and ample, respectively. conclusion. time to onset and magnitude of acr and haq-di responses and das improvements were generally similar with subcutaneous abatacept with or without intravenous loading in patients with ra and an inadequate response to mtx. the findings from this posthoc analysis suggest that subcutaneous abatacept can be given effectively without an intravenous abatacept loading dose. background. ra is associated with pain and impairment of physical function, significantly impacting a patient's health-related quality of life (hrqol) and ability to perform daily activities. patient-reported outcomes (pros) related to hrqol and daily activity have become an essential part of assessment in ra. we continue to report here comparative findings from pros assessed with subcutaneous abatacept or adalimumab on background mtx in the first head-to-head study, ample. we compared changes in pros at year in patients with ra treated with abatacept or adalimumab, both on background mtx. methods. ample is a phase iiib, randomized, investigator-blinded study of months' duration. biologic-naïve patients with active ra and inadequate response to mtx were randomized to either mg abatacept weekly or mg adalimumab biweekly in combination with mtx. pros evaluated through day included: hrqol, assessed using short form- (sf- ; including physical and mental component summary subscores [pcs and mcs]); activity limitation over the previous days, using the activity limitation questionnaire (alq; [ ] ); productivity, using the work productivity and activity impairment questionnaire for ra [ ] ; physical and psychosocial independence, captured using items from haq, sf- score; and alq [ ] . other pros previously reported from ample include: patient pain, patient global assessment, fatigue, and physical function [ ] . all efficacy analyses were done using the intent-to-treat population, which included all patients who were randomized and received at least one dose of study drug. baseline characteristics were analysed descriptively and changes in pros from baseline were assessed using ancova. results. baseline demographic and clinical characteristics of the abatacept and adalimumab treatment arms were similar. improvements in all domains of the sf- , including pcs and mcs observed at day parameter baseline woche woche itt-gesamt das , mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) vas da pat., mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) sjc , mw ± sd , ± , (n= ) , ± , (n= ) , ± , (n= ) vas schmerz, mw ± sd zielsetzung der ole-studie beinhaltete die beurteilung der verträglichkeit und der wirksamkeit von czp. die retentionsraten sowie die wirksamkeit wurden bis woche und die verträglichkeitsdaten bis woche beobachtet. in die verträglichkeitsanalyse wurden alle pat einbezogen, die in die ole-studie eintraten und czp erhielten (n= ; n= kombitherapie; n= monotherapie), einschließlich der plazebo/czp-patienten, die die ausgangsstudien erfolgreich abgeschlossen/abgebrochen haben. bezüglich der wirksamkeit wurden folgende analysen vorgenommen: ) czp pat, die die ausgangsstudien erfolgreich beendet haben und zu irgendeinem zeitpunkt während der ausgangsstudien oder ole-studie andere dmards eingenommen haben (n= ; kombitherapie completer); ) czp pat, die die fast ward studie erfolgreich beendet haben und zu keinem zeitpunkt andere dmards eingenommen haben (n= ; monotherapie completer). ergebnisse. verteilung und häufigkeit der unerwünschten ereignisse (ue), einschließlich der reaktionen an der injektionsstelle (ereignisse/ patientenjahre: monotherapie , , kombitherapie , ) und der schwerwiegenden unerwünschten ereignisse (sue) waren mit dem vergleichbar, was bisher für czp berichtet wurde (. tab. ) . das auftreten von schwerwiegenden infektionen (si) und malignitätsraten war niedrig. es wurden todesfälle berichtet: kardiovaskuläre ereignis-se, infektionen, unfall und tumorerkrankung. die retentionsraten der pat, die die ausgangsstudien erfolgreich beendet haben, waren zur w in der czp monotherapie-( / , %) und der czp kombitherapie-gruppe ( / ; %) vergleichbar. der durchschnittliche das - (crp)-wert und dessen abweichung vom baseline-wert der ausgangsstudien zum zeitpunkt des eintrittes in die ole-studie und nach w der monotherapie-completer und der kombitherapie-completer, sowie die zugehörigen haq-werte sind in . tab. dargestellt. schlussfolgerung. vorliegende ole-studie konnte das günstige risiko-nutzen-profil der czp-monotherapie bestätigen. die langzeitwirksamkeitsdaten zeigten keine unterschiede zwischen pat, die czp als monotherapie erhielten und pat, die czp in kombination mit anderen dmards erhielten. background. rheumatoid arthritis (ra) is the most common disease of joints that non-or deficiently treated leads to functional loss and premature cardiovascular death within years. but nearly % of the ra patients fail to treatment with tnfα-inhibitor (tnfi) indicating a switch to rituximab (rtx). the urgency of personalized promising treatment in time presupposes predictive parameter. rheumatoid factor (rf) and anti-citrullinated protein antibodies (acpas; especially accp) are shown to be better diagnostic than less theranostic biomarkers. in that context we investigated the role of antibody subtypes against mutated citrullinated vimentin (amcv) that determine response outcome in rtx-treatment. methods. a cohort of only amcv igg positive ra patients was tested for amcv subtype igm and iga (additionally for rf igg, igm, iga and accp igg) by elisa at baseline (after failure to first approach with tnfi) and at week (after first rtx cycle). responders were characterized by a difference in their das of ≥ . (eular good-response) between baseline and week . the cohort comprises responders (rr) and non-responders to rtx (nrr). results. amcv igg, igm and iga showed higher treatment related decreases compared to rf and accp ig subtypes and additionally even diverged in both groups depending on response outcome: especially amcv iga exhibited a higher mean titer decline of rr by % at lo- wer baseline titers ( . to . u/ml) and a mean titer increase of nrr by nearly % at higher baseline titers ( . to . u/ml). at baseline rr displayed relatively more negative iga titers ( %; n= / ) than nrr, who in return showed more iga positive titers ( %; n= / ). amcv iga positive patients were more likely to show positively for rf iga ( %) and igm ( %), what could be inversely detected for iga negative patients with seronegativity of rf iga ( %) and igm ( %). conclusion. amcv immunoglobulin subtypes showed treatment dependent changes contrary to already known antibodies (accp). especially amcv iga reflects response outcome: amcv iga negativity at baseline and decreasing titers during treatment are predictive for good eular-response to rtx. einleitung. im rahmen der abklärung eines unter tocilizumab-therapie aufgetretenen arzneimittelexanthems erfolgte die bestimmung von c c und c -beide parameter waren erniedrigt. bei recht geringer und nur kurz andauernder ausprägung des exanthems wurde die therapie komplikationslos fortgeführt. die komplementfaktoren wurden im verlauf bestimmt und blieben erniedrigt. im weiteren verlauf erfolgte die konsekutive messung bei weiteren patienten. methoden. nephelometrische bestimmung von c c und c im serum vor und während der therapie mit tocilizumab (jeweils vor der nach wochen anstehenden infusion) bei patienten mit gesicherter rheumatoider arthritis (rf+, ccp+). ergebnisse. c c-und c -komplement wurden bei konsekutiven patienten mit rheumatoider arthritis vor und unter tocilizumab-therapie bestimmt. bei allen patienten fielen sowohl c c, als auch c unter der therapie mit tocilizumab ( mg/kg kg) ab. / patienten hatten eine c c-erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). / patienten hatten eine c -erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). drei patientinnen entwickelten unter der therapie ein exanthem, davon hatten eine komplementerniedrigung. keine "offensichtlich" erhöhte infektneigung in abhängigkeit von komplementspiegeln. bei verlängerten infusionsintervallen aufgrund von infekten zeigte sich, dass der effekt von tocilizumab auf die komplementspiegel reversibel ist. durch blockade des il- -rezeptors tocilizumab kann ein erworbener komplementmangel induziert werden. Ähnliche daten wurden im rahmen einer pilotstudie an sle-patienten erhoben, die mit tocilizumab behandelt wurden. der effekt ist bei der rheumatoiden arthritis nicht vorbeschrieben. der genaue umfang des komplementmangels ist bisher nicht untersucht (andere bestandteile der kaskade?) wurde in der sle-studie ausführlicher untersucht. da die verschiedenen komplementbestandteile erniedrigt waren wurde auf eine synthesestörung und nicht auf einen gesteigerten verbrauch geschlossen, was auch in dieser kohorte der fall zu sein scheint. der erworbene komplementmangel könnte einen teil der infektiösen komplikationen unter der therapie erklären. eine korrelation ist aber aufgrund der geringen fallzahl nicht möglich. schlussfolgerung. anhand dieser studie konnte eine exzellente korrelation zwischen den parametern der dxr und des bx verifiziert werden. mittels der neu entwickelten voll digitalisierten bx-technik ist somit eine quantifizierung der periartikulären demineralisation möglich und als surrogatparameter der radiologischen progression bei einer ra eingesetzt werden. jahre, die ra bestand im median seit , jahren. , % der patienten waren mit tnf-alpha-blockern vortherapiert, , % ausschließlich mit dmards. der mittlere das lag zur baseline bei , . zur woche zeigten , % der patienten eine das remission (< , ) und , % bzw. , % der patienten ein gutes bzw. moderates ansprechen gemäß eular-kriterien. Über den beobachtungszeitraum stieg der anteil der tcz-monotherapiepatienten von , % auf , %. die mtx-komedikation sank im gleichen zeitraum um , %. , % der patienten, die tcz zunächst zusammen mit einem dmard erhalten haben, konnten dieses absetzen. tcz zeigte in der mono-und kombinationstherapie eine vergleichbare wirksamkeit: , % bzw. , % der patienten erreichten eine cdai remission (≤ , ). der anteil von patienten ohne glucocorticoid(gc)-begleittherapie stieg über den beobachtungszeitraum um , % auf , % an, der anteil mit einer tagesdosis ≤ mg auf , %. bei , % war eine reduktion der gc-dosis möglich, nur bei , % war eine erhöhung notwendig. bei , % der patienten, die zur bl mit gc behandelt wurden, konnten diese komplett abgesetzt werden. die mittlere gc-tagesdosis verringerte sich kontinuierlich von , (bl) auf , mg/d (w ). schlussfolgerung. diese interimsanalyse der nichtinterventionellen studie ichiban zeigt bei den ersten patienten mit mittelschwerer bis schwerer ra über die bisherige beobachtungsdauer von wochen deutliche verbesserungen der aktivitätsparameter, sowie eine reduktionen der begleitenden dmard-therapien und des bedarfs von glucocorticoiden unter behandlung mit tcz. vergleichbar mit den kontrollierten studien ist die tcz-monotherapie auch unter praxisbedingungen der kombination mit dmards ebenbürtig. diese anhaltende wirksamkeit wird erstmals in rheumatologischen praxisdaten für den langzeitverlauf von , jahren gezeigt. zeitschrift für rheumatologie suppl · | einleitung. die arteriosklerose (as) steht als häufigste todesursache im besonderen fokus der medizinischen forschung. neuere erkenntnisse weisen auf einen starken zusammenhang zwischen parametern der systemischen entzündung und der pathogenese der as hin. patienten mit rheumatoider arthritis (ra) haben daher ein stark erhöhtes kardiovaskuläres risiko. ziel: untersuchung des zusammenhangs zwischen verschiedenen ra-krankheitsspezifischen risikofaktoren und dem auftreten einer arteriosklerose bei ra-patienten. methoden. ra-patienten, davon % weiblich, ± , jahre alt, wurden hinsichtlich der krankheitsaktivität (krankheitsdauer , ± , ; das , ± , ; serum-crp , ± , mg/dl,; anti-ccp-antikörper , ± , u/ml, radiologisches stadium , ± , ; davon , % mit erosionen), sowie klassischer kardiovaskulärer risikofaktoren der as erfasst, welche durch den score-wert (systematic coronary risk evaluation) zusammengefasst wurden. zur as darstellung wurde eine carotis-duplexsonographie mittels eines mhz-schallkopfes (ge vivid pro) durchgeführt. die mittlere intima-media-dicke (imd) der a. carotis communis wurde durch ein softwaregestütztes messverfahren ermittelt. ergebnisse. plaques waren bei patienten ( %) nachweisbar. diese korrelierten mit einer erosiven form der ra (p= , ), einer längeren krankheitsdauer (p= , ) und höheren anti-ccp-antikörpern (p= , ). die mittlere imd betrug , ± , mm. je ausgeprägter die radiologischen veränderungen sind, umso höher war die wahrscheinlichkeit der plagues (p= , ). mittels altersadjustierter partieller korrelationsanalyse wurde der das als altersunabhängiger einflussfaktor auf die imd ermittelt (p= , ). mittel-und hochgradige stenosen zeigten sich bei fünf ra-patienten ( , %), welche ausnahmslos eine erosive verlaufsform aufwiesen. normalbefunde stehen in zusammenhang mit einem crp-wert unter mg/dl (p= , ). auch die traditionellen kardiovaskulären risikofaktoren haben signifikanten einfluss auf as. der score-wert erwies sich als äußerst verlässlicher prädiktor für plaques (p< , ), imd-verdickung (p= , ) und stenosen (p< , ). durch elimination der traditionellen risikofaktoren mittels partieller score-adjustierter korrelationsanalyse bestätigte sich erneut die assoziation von pathologischen ultraschallbefunden mit dem das (p= , ). schlussfolgerung. die erhebung klassischer risikofaktoren bei ra-patienten ist unerlässlich. die nutzung des score-werts als screening-parameter ist besonders effektiv. zusätzlich sollten parameter der krankheitsaktivität von ra zum management von arteriosklerose herangezogen werden. besonders aussagekräftig hierfür sind der das , ein erosiver krankheitsverlauf, die crp-werte und die erkrankungsdauer background. apremilast, an oral small molecule specific inhibitor of phosphodiesterase- , works intracellularly to modulate inflammatory mediators. the palace - trials compared the efficacy and safety of apremilast vs placebo in patients with active psa despite prior dmards and/or biologics. the overall safety and tolerability of apremilast was assessed in a pooled analysis of the palace , and placebo-controlled phases. methods. safety data was pooled from phase , randomized, placebo-controlled studies; patients with active psa despite prior dmards and/or biologics were randomized : : to placebo, apremilast mg bid (apr ), or apremilast mg bid (apr ) stratified by baseline dmard use. at week , patients with < % reduction in swollen and tender joint counts were required to be re-randomized (early escape) to apr or apr (placebo group) or remained on initial apremilast dose through week . stable concurrent dmard therapy was allowed (mtx, sulfasalazine, leflunomide, or combination). the analysis comprises data from the placebo-controlled periods (weeks - ). results. patients were randomized to placebo (n= ), apr (n= ), or apr (n= ) and included in the safety population. baseline demographic and disease characteristics and prior/concurrent therapy were comparable across treatment groups; . % had prior biologic exposure. adverse events (aes) occurred in . % (placebo), . % (apr ), and . % (apr ) of patients. aes occurring in ≥ % of any treatment group were diarrhea, nausea, headache, and urti (. tab. ); most occurred within the first weeks of treatment and nearly half resolved within weeks. of patients with these aes, most ( - %) were mild or moderate. rates of discontinuation due to aes were low: . % (placebo), . % (apr ), and . % (apr ). serious aes occurred in . % (placebo), . % (apr ), and . % (apr ) of patients. one death occurred (apr ) due to multiorgan failure not suspected to be treatment-related. no cases of serious systemic opportunistic infections, lymphoma, vasculitis, or reactivation/de novo tb were reported. there were no clinically meaningful differences between apremilast and placebo in terms of major cardiovascular aes, changes in blood pressure, malignancies, or effects on laboratory measurements. conclusion. apremilast was generally well-tolerated with no new safety concerns identified compared with the known profile. the aim of the current study was to investigate the relationship between worsening of functional status, clinical disease parameters and radiographic spinal progression over two years in patients with early axial spondyloarthritis (axspa). methods. in total, patients with early axspa ( with as and symptom duration ≤ years, and with non-radiographic axspa (nr-ax-spa) and symptom duration ≤ years) from the german spondyloarthritis inception cohort (gespic) were included in the current analysis based on the availability of radiographic data and data on the functional status at baseline and after years of follow-up. spinal radiographs were scored according to the modified stoke ankylosing spondylitis spinal score (msasss). functional status was assessed by the bath ankylosing spondylitis functional index (basfi), and clinical disease activity by the bath ankylosing spondylitis disease activity index (basdai). results. basfi worsening in ≥ point after years (n= , . %) was significantly associated only with higher basdai worsening over years in comparison to those without functional worsening: . ± . vs - . ± . , p< . . basfi worsening by ≥ points (n= , %) was, however, associated not only with basdai change ( . ± . vs - . ± . , p< . ), but also with a higher rate of radiographic spinal progression measured by the proportion of patients with msasss worsening by ≥ units ( . % vs. . % in patients without basfi worsening, p= . ), or with new syndesmophyte formation ( . % vs. . %, p= . ). importantly, in the multivariate analysis both basdai increase and progression of structural damage in the spine remained statistically significantly associated with basfi worsening. no other disease-related parameters (e.g. sex, hla-b positivity, symptom duration etc) were found to be significantly associated with basfi worsening over two years. conclusion. in this prospective study we could demonstrate that only factors were significantly associated with worse functional outcome over two years in patients with early axspa: ) increase of disease activity and ) progression of structural damage. elevated serum vascular endothelial growth factor is highly predictive for radiographic spinal progression in patients with axial spondyloarthritis who are at high risk for progression background. vascular endothelial growth factor (vegf) is an essential mediator of the endochondral ossification and, therefore, might play a pathogenetic role in the process of syndesmophyte formation in axial spondyloarthritis (axspa). the aim of the study was to investigate the role of serum vegf as a predictor of radiographic spinal progression in patients with axspa. methods. altogether patients with definite axspa [ with ankylosing spondylitis (as) and with non-radiographic axspa] from the german spondyloarthritis inception cohort (gespic) were included in the current study. radiographic spinal progression was defined as ) worsening of the modified stoke ankylosing spondylitis spine score (msasss) by ≥ units after years, and ) development of a new syndesmophyte or progression of existing syndesmophytes after years. serum vegf levels were detected at baseline. results. mean baseline vegf values were significantly higher in patients with msasss worsening by ≥ units after years (n= ) as compared to those without progression ( ± vs. ± pg/ml, respectively, p= . ) and in patients with syndesmophyte formation/ progression (n= ) as compared again to those without progression ( ± vs. ± pg/ml, respectively, p= . ). area under the curve (auc) was . , p= . for the msasss worsening ≥ units and . , p= . for syndesmophyte formation/progression. importantly, the performance of vegf as a predictor of radiographic spinal progression was clearly in patients who were already at high risk for such a progression due to the presence of syndesmophytes at baseline (n= ): auc was . , p= . , and . , p= . , respectively. vegf serum level of > pg/ml in high-risk patients had a sensitivity of %, a specificity of %, and an odds ratio (or)= . ( %ci . - . ) as a predictor of msasss worsening by ≥ units over years. the same serum level of results. immediately after the second session of plasmapheresis, therapy with infliximab mg/kg resumed. after weeks of hospitalization with repeated administration of infliximab had good dynamics (bas-dai . , asdas (crp) = . , basfi . ), significantly reduced pain in the joints and spine, stiffness, increased mobility in the joints and spine. the treatment continued: holding plasmapheresis followed by infliximab. after infusions patient experienced a good effect -basdai . , asdas (crp)= . , basfi . . conclusion. plasmapheresis in some patients could be effective by reducing activity and dealing with secondary tnf inhibitors failure, since this procedure deletes the macromolecular blood proteins, including tnf-, igg antibodies, and circulating immune complexes results. there were still signs of osteitis in sacroiliac joints in patients at week , in patient the mri-determined sacroiliitis has resolved completely. the patient has improved clinically and fulfilled asas improvement criteria. there was a minor decrease in sparcc sacroiliitis score (from to ) in patients at week , indicating reduction of inflammation in sacroiliac joints. sparcc sacroiliitis score stayed the same in the remained patient. conclusion. rituximab may be of some benefit in decreasing mri-evident sacroiliitis in patients with highly active as, even in patients in whom tnf-α inhibitors have failed. background. despite the differences in the pathogenesis of ra and as, neck pain is a frequent clinical symptom in both diseases. we evaluated the correlation between subjective reports of neck pain and objective signs of inflammation as assessed by f bone marrow edema (bme) on mri in ra and as patients. methods. stir-mri of the cervical spine of patients ( ra, as) were included. mris were scored by two blinded readers using a recently published mri scoring system, with quantification of the extension of bme in the atlantoaxial region, corpus, facet joints and processus spinosus of all cervical vertebrae, ranging from - points. the presence or absence of degenerative changes was also recorded. conclusion. the majority of patients with ra and as had objective signs of bme but also degenerative changes on mri at different cervical locations. assessment of bme in the atlantoaxial region is important in clinical practice, in addition to degenerative changes, since its presence seems to influence the intensity of neck pain reported by these patients. x. baraliakos , having mri data at w . of these , ten were treated with secukinumab and with placebo in the core study. mris were rescored for this study. asspimri-a scores and the occurrence of vertebral edges (ve) inflammatory and fatty lesions were evaluated by an independent blinded reader. results. all pts completed this exploratory mri substudy. in pts receiving × mg/kg secukinumab followed by × mg/kg (n= ) secukinumab, spinal inflammation was reduced compared to bl at w -similar to the results of the core study -and this reduction was sustained up to w (abb. ). also in pts who had initially received placebo switching to secukinumab at w , mri inflammation at w was reduced. of the ves evaluated, the proportion of ves with inflammatory lesions was reduced from . % (n= ) at bl to . % (n= ) at w and . % (n= ) at w . in contrast, the proportion of fatty lesions at bl ( . %, n= ) remained largely unchanged at w ( . %, n= ) and w ( . %, n= ). secukinumab reduced mri inflammation at w and w . conclusion. mri analysis suggests that the il- a inhibitor secukinumab can reduce spinal inflammation and this effect may be sustained for up to years. unlike reports with tnf blockers, secukinumab appeared to leave the proportion of fatty lesions unchanged. the potential impact of these preliminary findings on radiographic progression under secukinumab therapy will be studied in larger trials. schlussfolgerung. es zeigen sich keine signifikanten unterschiede in der krankheitsaktivität der beiden gruppen (vor einleitung der ada-therapie nach dmard-und nach anti-tnf-versagen). auch bei der patientengruppe mit mehreren vortherapien mit tnf-inhibitoren können keine signifikanten unterschiede in der ausprägung der erkrankung nachgewiesen werden. im trend wurde ein früheres einsetzen der haut-und gelenkmanifestation sowie eine stärkere systemische entzündungsreaktion in den patienten mit vorheriger tnf-therapie festgestellt werden, während die dauer der erkrankung und der bmi mit den charakteristika der patienten mit ausschließlicher dmard-vortherapie vergleichbar sind. long-term ( -week) results of a phase , randomized, controlled trial of apremilast, an oral phosphodiesterase inhibitor, in patients with psoriatic arthritis (palace ) background. apremilast, an oral phosphodiesterase inhibitor, works intracellulary to modulate a network of pro-and anti-inflammatory mediators. the palace study assessed the efficacy and safety of apremilast in patients with active psoriatic arthritis (psa) despite prior dmards and/or biologics. methods. patients were randomized : : to placebo, apremilast mg bid (apr ), or apremilast mg bid (apr ). at week , patients with < % reduction from baseline in swollen/tender joint counts were required to be re-randomized (early escape) to apr or apr (placebo group), or remained on their initial apremilast dose. at week , all remaining placebo patients were re-randomized to apr or apr through week . results. patients were randomized. at week , significantly more apr ( . %; p= . ) and apr patients ( . %; p< . ) achieved an acr vs placebo ( . %). at week , all patients had a minimum weeks of apremilast exposure. response to apremilast was generally maintained over the treatment period. at week , acr was achieved by . % (apr ) and . % (apr ) of patients (table) . exposure-adjusted incidence rates for adverse events (aes), severe aes, and serious aes were comparable between - and - weeks. the proportion of patients remaining on apremilast to week who first reported the most common gi disturbances (e.g., diarrhea, nausea, and vomiting) after week was low (ranging from . - % for apr and - . % for apr ). there were no clinically meaningful laboratory findings with exposure up to weeks. no deaths beyond the previously reported in the - week period were observed in the - week period. no safety signals with respect to major cardiac events, malignancies, and opportunistic infections were observed, consistent with the - week period. no cases of lymphoma, tuberculosis, or tuberculosis reactivations were reported for the -week period (. tab. ). conclusion. apremilast administered to patients with psa beyond weeks continued to demonstrate meaningful clinical response. for patients who completed weeks of the study, acr response rates up to % were observed. apremilast continued to be well tolerated with an acceptable longer-term safety profile. methods. to identify how conventional cd + and cd + t cells and regulatory t cells are recruited into the inflamed kidneys in ln, serum and urine samples of sle patients were analyzed for chemokines using multiplex assays. based on the assay's results a group of corresponding chemokine receptors (ccr - , cxcr and cxcr ) was chosen, whose frequencies on urinary t cells were subsequently determined in patients with acute ln by flowcytometry. results. chemokines (ccl , ccl , ccl , ccl , ccl , ccl , ccl , ccl , cxcl , cxcl , cxcl and cx cl ) were significantly elevated in the urine of patients with active ln when compared to the control group. the other chemokines (ccl , ccl , ccl , cxcl , cxcl ) and cxcl showed no significant differences between the groups. ccr and cxcr were the most prominent receptors on both urinary cd + and cd + t cells, although cd + t cells also expressed high amounts of ccr and ccr . however, when compared to t cells in the blood, urinary cd + t cells showed significantly higher expression of all examined chemokine receptors but ccr while urinary cd + t cells only had higher expression of ccr and ccr . the chemokine receptor expression on cd +foxp +cd -regulatory t cells (treg) differed from conventional cd + t cells as well. treg expressed significantly more ccr and significantly less cxcr . conclusion. ccr and cxcr are the primary receptors in the mechanism of recruiting t cells into the inflamed kidney. key chemokines are ccl , ccl , ccl and ccl as well as cxcl and cxcl . however, at least for cd + t cells, there are secondary pathways of recruitment involving ccr /ccl and ccr /ccl . also, treg recruitment seems to rely more on ccr than that of conventional cd + t cells. methods. observe is a multicenter, retrospective medical chart review study. rheumatologists from german academic and non academic centers who treat > sle patients annually and have > years of practice experience were randomly recruited. physicians identified consecutively all their adult sle patients who had received belimumab as part of usual-care. index date was the first belimumab infusion date. the primary outcome was the change in overall sle disease manifestations months after index date based on physician judgment. the overall response rates as well as reasons for early treatment discontinuation within months were assessed. changes in formal disease area indices, e.g. selena-sledai if available and changes in oral steroid dose are also reported. results. previous analyses from us patients treated with belimumab have described significant clinical improvement across relevant organ systems based on clinical judgment and formal disease activity indices and marked reductions in corticosteroid use in patients that received at least infusions of belimumab. the current study is the first description of patient characteristics and outcomes after months of therapy with belimumab outside of the us. it is also the first time overall responder rates and reasons for discontinuation with belimumab have been described in a real world setting. the study provides insights into the effectiveness and safety of belimumab in an ex-us clinical setting. larger, prospective observational studies are needed to confirm the results. commercial support grant disclosure: research funded glaxosmithkline. background. toll-like receptor (tlr- ) signaling is considered to play an important role in b cell hyperreactivity in sle. b cells from slepatients express significantly more tlr- than those from healthy donors (hd), especially if patients have positive dsdna-antibodies and high disease activity. tlr- stimulation of b cells is tightly linked to their differentiation into plasma blasts and memory cells. the objective of this study was to analyze in a comprehensive manner the effect of tlr- signaling on cytokine production by b cells from sle-patients, in comparison to b cells from hd, and in relation with disease activity. methods. b cells from sle-patients and hd were stimulated in vitro using cpg for hours, and culture supernatants were then tested for cytokines and chemokines (bio-plex). the cytokine responses were compared between both groups. in addition, within sle patients, the patterns of cytokines produced by b cells were compared with indices of disease activity. results. cpg-stimulation significantly increased cytokine production ( out of parameters; p< . ) compared to baseline. striking increases were found for il- ra ( ± pg/ml), il- ( ± pg/ml), il ( ± pg/ml) and ip- ( ± pg/ml; p< . ). there was no significant difference between both groups. remarkably, production of il- , il- , il- , il- p , il , il- , il- a, eotaxin, basic fgf, g-csf, gm-csf, ifn-γ, ip- , mip- α, and vegf correlated inversely with the sledai (p< . ) and even more (additionally il- β, il- ra, mip- β and tnf-α) with anti-dsdna antibody titers. the frequency of cd + memory b cells showed a positive correlation between the production of ip- and tnf-α in sle, whereas the levels of il- β, il- , mip- α, and mip- β showed a positive correlation with cd + b cells in hd. conclusion. the current data indicate hitherto unknown perturbations of cytokine/chemokine production by b cells in active sle. the inverse correlation of cytokines/chemokines produced by b cells from sle patients with sledai and anti-dsdna titer suggests that the known enhanced b cell proliferation and differentiation upon tlr -stimulation possibly diminishes cytokine production. background. several cytokines, including ifn-γ, il- , il- , and il- have been implicated in the pathophysiology of autoimmune disease. il- , a potent inducer of ifn-γ, enhances th responses that are thought to be synergistic and dependent on il- . we tested the hypothesis that intra-renal il- mediates kidney and systemic disease in mrl-faslpr mice. methods. by constructing il- p /il- -/-mrl-faslpr mice and using an ex-vivo gene transfer to deliver il- intra-renally, we determined that il- , independent of il- and/or il- , incites kidney disease in mrl-faslpr mice. moreover, we provide the novel finding that local intra-renal il- mediates systemic disease (lung pathology, systemic auto-abs). results. thus, our data indicate that il- is a potential therapeutic target for immune mediated kidney and systemic disease in mrl-faslpr mice. using a caspase- inhibitor, that inhibits the release of active il- and il- β, we successfully treated kidney (improved renal function, pathology) and systemic disease (skin lesions, lymphadenopathy, and splenomegaly) in mrl-fas lpr mice, while administration of an il- receptor antagonist did not influence disease progression. probing further we found that inhibition of il- activation results in an amelioration of lupusnephritis by a reduction of intra-renal infiltrating leukocytes (macrophages and t cells) and reduced activation of these leukocyte populations. moreover, caspase- inhibition resulted in decreased inf-y and il- production, indicating an altered balance of th and th cell responses in this model. conclusion. taken together, our findings indicate that il- , independent of il- β, il- and/or il- , is the major mediator of kidney and systemic disease mrl-faslpr mice. therefore, caspase- inhibition is a potential therapeutic target for autoimmune disease in the mrl-faslpr mice. background. in the treatment of giant cell arteritis (gca) glucocorticoid-related adverse effects occur frequently, particularly in patients with relapsing disease. a -year-old woman presented with a month history of fever, chills, arthralgias and cephalgias and markedly elevated serum inflammatory markers. whereas further evaluation including ultrasound of the temporal arteries was unremarkable, a positron emission tomography-computed tomography (pet-ct) demonstrated an intense fluorodeoxyglucose uptake of the aorta, the subclavian, carotid and femoral arteries. gca was diagnosed and treatment with high dose prednisone was begun. results. because of disease flares at prednisone dosages below mg/ day and the occurrence of vertebral fractures, cyclophosphamide and methotrexate (mtx) were added as glucocorticoid-sparing agents. as these treatments had to be stopped because of intolerance and mtxpneumonitis, respectively, we started tcz infusions ( mg/kg body weight). the clinical status rapidly improved. after infusions of tcz follow-up pet scan showed resolution of the previously seen uptake and we were able to taper the daily dose of prednisone to mg. treatment was well tolerated. however, the patient developed mild hyperthyroidism with a rapid rise of the initially normal levels of anti-thyroid peroxidase and anti-thyroid antibodies, anti-tsh receptor antibodies remained normal. thyroid function normalized and the antibody-levels fell without further treatment in the following months. in conclusion, this case demonstrates the successful treatment of a patient with relapsing giant cell arteritis with tcz. for the first time, we report the occurrence of a transient autoimmune thyreoiditis possibly induced by tcz. klinik für pädiatrie mit schwerpunkt pneumologie und immunologie, sektion rheumatologie, berlin, vestische kinder-und jugendklinik der universität witten/herdecke deutsches zentrum für kinder-und jugendrheumatologie organización médica de investigación arthr care res ar&t in press sp division of rheumatology diagnosesicherung: a + b) mr-morphologisch myositistypische veränderungen (os) histologie + c) generalisierte myalgien und laborchemisch dtl. elevierter ck, sowie positivem nachweis von ana und jo- -ak, pulmonales ct mit diffusen milchglasinfiltraten, in bronchoalveolärer lavage neutrophile alveolitis. ergebnisse. vormedikationen: a) glukocorticoidmonotherapie, mtx-monotherapie, mtx in kombination mit etanercept, cyclophosphamidboli, und zuletzt intravenöse immunglobuline (ivig) in kombination mit mycophenolatmofetil . b) mtx-monotherapie, mtx in kombination mit glukokortikoiden, cyclophosphamidboli, intermittierend intravenöse immunglobuline, cyclophosphamid per os (fau-ci). c) cyclophosphamidboli jeweils gutes ansprechen des ck-wertes auf jeweilige rituximabgaben mit ebenfalls ansprechen des klinischen bildes mit guter regredienz des aus myalgien resultierenden schmerzniveaus. im fall a keine beatmung mehr notwendig. im fall von c) auch gute regredienz subjektiver dyspnoesymptomatik und besserung wichtiger lungenfunktionsparameter, regredienz ctmorphologischer milchglasinfiltrate, im verlauf fehlender nachweis neutrophilie in bal. weitere rituximabgaben bei a, b und c im verlauf zum remissionserhalt nach jeweiligem klinischem befund production of cytokines by b cells in response to tlr stimulation inversely correlates with disease activity in sle-patients berlin zeitschrift für rheumatologie suppl · | das muskuloskeletale system, eines der am häufigsten betroffenen organsysteme bei sle (bei - % der sle-patienten). das ziel dieser analyse war es um diejenigen parameter zu identifizieren, die zu diesem effekt beigetragen hatten, wurde jeder der einzel-parameter zur untersuchung und symptom-erfassung innerhalb des muskuloskeletalen bilag-organsystems analysiert. die post-hoc-analyse umfasste nur patienten, bei denen ein parameter zu studienbeginn als vorhanden gewertet wurde, und jeder parameter erforderte ≥ patienten-beobachtungen pro kohorte um einen vergleich zu erstellen dadurch wurde die zahl der patienten mit einer initialen beteiligung des muskuloskeletalen systems aufgedeckt, die eine in woche auflösung der manifestation aufwiesen auch im selena-sledai-score war die rate der verbesserung bei dem arthritis-parameter in der belimumab-gruppe mit mg/ kg ( , %; n= ) und mg/kg ( , %; n= ) signifikant höher als die daten weisen darauf hin, dass mg/kg belimumab effektiv auf muskuloskeletale organmanifestationen sind akzeptiert als posterbeitrag auf dem eular klinische forschergruppe für rheumatologie (kfr), freiburg i. br., universitätsklinikum ulm, klinik für dermatologie und allergologie die physikalische therapie (pt) ist ein wesentlicher bestandteil der medizinischen versorgung von ssc-patienten patientenregister des dnss erfasst prospektiv, jährlich klinische verlaufsdaten zur organbeteiligung und therapie von patienten mit systemischer sklerodermie. die mittels freitext erfassten angaben zur verordneten pt wurden ausgewertet hivamat n= ( , %) und hylase n= ( , %) anwendung. die anzahl der verfahren, die die patienten zeitgleich erhielten, variierte zwischen mind. und max. . Über % der patienten erhielten anwendungen gleichzeitig. insgesamt wurden therapiearten genannt. , % der patienten mit gelenkkontrakturen zeigten nach einem jahr physikalischer therapie eine signifikante verbesserung der symptomatik (p= , ) gegenüber den patienten die keine physikalische therapie erhielten. nach drei jahren waren es , % der patienten (p= , ). bei den patienten mit muskelschwäche zeigten % der patienten eine signifikante symptomverbesserung (p= , ) dieser studie kann erstmals gezeigt werden, dass pt-symptome wie gelenkkontrakturen und muskelschwäche bei ssc-patienten signifikant verbessern kann. dennoch erhält weniger als die hälfte der ssc-patienten eine physikalische therapie punkten zur kontrolle einer mmf-therapie in der klinischen praxis zu untersuchen bei patienten ( -mal sle, je -mal systemische sklerose, sharp-syndrom und primäres sjögren-syndrom) die mmf erhielten, wurde , und min nach einnahme von mmf die mpa-konzentrationen im serum per hplc bestimmt. die mpa-auc wurde durch die mathematische methode der bayes %) und in der standarddosis von g/tag bei von patienten ( %) eine mpa-auc von > µg.h/ml. bei zwei patienten wurde nach der messung die dosis adjustiert: eine patientin mit einem sle mit diffus-proliferativer lupusnephritis hatte trotz einer mmf-dosis von g/tag nur eine mpa-auc von , µg.h/ ml. die dosis wurde daraufhin auf g/tag erhöht. der mpa-auc stieg danach auf max. , µg.h/ml und die krankheitsaktivität nahm ab (sledai von auf , proteinurie von auf mg/ h und prednisondosis von auf mg/tag) pharmakokinetic study of mycophenolate mofetil in patients with systemic lupus erythematosus and design of bayesian estimator using limited sympling strategies mycophenolic acid area under the curve correlates with disease activity in pupus patients treated with mycophenolate mofetil colony stimulating factor- (csf- ) -neuer aktivitätsmarker der lupusnephritis? brigham and wome's hospital, boston, renal division the authors would like to thank pfizer for supporting the study. furthermore the authors would like to thank the "deutsche kinder-rheumastiftung". einleitung. bei patienten mit früher axialer spondyloarthritis (spa) mit einer krankheitsdauer von< jahren und nachweis von akut-entzündlichen veränderungen in der ganzkörper-magnetresonanztomographie (mrt) in der wirbelsäule und/oder den sakroiliakalgelenken (sig) zu baseline [ ] untersuchten wir die langzeit-effektivität über vier jahre. methoden. in der esther-studie wurden patienten mit etanercept (eta, n= ) vs. sulfasalazin (n= ) behandelt [ ] . ab dem zweiten studienjahr wurden alle patienten mit eta behandelt (einige patienten unterbrachen zwischenzeitlich die therapie (n= ) zur untersuchung der biologika-freien remission und wurden dann (erneut) mit eta behandelt) [ ] . klinische, laborchemische und mrt-daten der patienten, die zu den jeweiligen studienzeitpunkten vorhanden waren, wurden im vierten studienjahr analysiert (as-observed-analyse). ergebnisse. von patienten, die zu baseline eingeschlossen wurden, erreichten , % das ende von jahr (n= ). in der gesamtgruppe zeigte sich ein gutes bis sehr gutes ansprechen, wobei etwa % eine asas partielle remission und etwa - % eine asdas inaktive erkrankung erreichten (. tab. ). der anteil der patienten mit normalem crp ("crp-remission") stieg von , % zu screening auf , % zu woche , während der anteil der patienten mit negativem mrt ("mrt-remission" definiert als fehlen akut-entzündlicher veränderungen in den sig und der wirbelsäule gemäß beider scorer) auf von % auf , % anstieg. , % der patienten zu woche waren sowohl in asas-remission, im status einer asdas inaktiven erkrankung als auch in mrt-remission. das ansprechen nach vier jahren war sehr ähnlich in den gruppen unabhängig davon, ob im ersten jahr sulfasalazin gegen wurde oder die therapie im jahr unterbrochen worden war (ergebnisse werden nicht gezeigt).schlussfolgerung. es zeigte sich ein konstantes und anhaltendes ansprechen bei patienten mit früher axialer spa, die mit etanercept behandelt wurden. das ansprechen scheint besser zu als bei patienten mit etablierter ankylosierender spondylitsi mit einer langen krankheitsdauer (> jahren; [ ] ). einleitung. in einer -wöchigen placebokontrollierten studie mit -wöchiger offener verlängerung bei patienten mit aktiver nichtröntgenologischer axialer spondyloarthritis (nr-axspa) wies adalimumab eine gute effektivität auf [ ] . bei patienten, bei denen es zum wiederauftreten der krankheitsaktivität nach absetzen des medikaments in woche kam, wurde die therapie wiederbegonnen ziel der studie war es, die langzeiteffektivität nach wiederaufnahme der therapie von adalimumab nach stopp über jahre zu evaluieren. methoden. bei ursprünglich in die studie eingeschlossenen patienten wurde die therapie nach wochen beendet und patienten ( % männlich, mittleres alter jahre, range - , mittlere krankheitsdauer vor therapiebeginn jahre, range - , % positiv für hla-b ) hatten, definiert durch erreichen eines % ansprechens gemäß der assessments in spondyloarthritis society-kriterien (asas ), gut auf die therapie angesprochen. bei wiederauftreten von krankheitsaktivität (definiert durch nicht mehr erreichen von asas ) wurde adalimumab mg alle wochen über jahre (woche r ) weitergeführt. die asas kriterien und der bath ankylosing spondylitis disease activity index (basdai) wurden in form einer completer-analyse berechnet. ergebnisse. der patienten mussten wiederbehandelt werden: / ( %) erreichten jahr , / ( %) erreichten jahr und / ( %) der patienten erreichten jahr der wiederbehandlung. nach jahren wiederbehandlung mit adalimumab erreichten / ( %) wieder asas und / ( %) erreichten partielle remission gemäß der asas-kriterien. nach jahren erreichten / ( %) und nach jahren / ( %) asas . asas partielle remission wurde nach jahren von / ( %) und nach jahren von / ( %) patienten erreicht. in der completer analyse fiel der mittlere basdai von , ± , zum zeitpunkt der wiederbehandlung auf , ± , im jahr (p< , ), , ± , (p= , ) im jahr und auf , ± , (p= , ) im jahr der wiederbehandlung ab. schlussfolgerung. in dieser gruppe von patienten mit aktiver nr-axspa, die ein gutes therapieansprechen über wochen mit adalimumab erreicht hatten und die bei wiederauftreten von krankheitsaktivität nach stopp der therapie in woche weiterbehandelt werden mussten, sprach die mehrheit der patienten, die in der studie verblieben, gut und anhaltend auf das fortsetzen der therapie an. tab. | sp- langzeit-effektivität über jahre etanercept-therapie bei patienten mit früher axialer spondyloarthritis. daten zu baseline (bl), jahr (w ), jahr (w ), jahr (w ) und jahr (w ). daten background. secukinumab (ain ) is a new fully human monoclonal antibody (mab) targeting il- a for the treatment of inflammatory diseases. administration of mabs can be associated with immunogenicity via the induction of anti-drug antibodies (adas). adas can lead to unwanted clinical consequences, such as loss of exposure, loss of efficacy due to altered pharmacokinetics and/or functional neutralization and, in the worst case, anaphylactic reaction and immune complex diseases. the assessment of ada formation is therefore a critical component in the assessment of biotherapeutic safety. methods. the immunogenicity assessment strategy for secukinumab follows a three-tiered approach. first, samples are analyzed for presence of ada in a screening assay which takes a % false-positive rate into account. in a second step, screening assay positive samples are tested in a confirmatory assay that identifies true positive responses. finally, true immunogenicity-positive samples are quasi-quantified via titration. a biacore-based assay was used during the early stages of the secukinumab program, and an msd-based bridging assay was applied during the later stages of the program. in addition, pharmacokinetics and clinical efficacy as well as safety data are also evaluated. samples to assess immunogenicity were obtained from individual subjects encompassing clinical studies in different indications during treatment and during follow-up. dosing regimens included single doses such as mg subcutaneously in psoriasis patients as well as multiple × mg/kg doses intravenously in ms patients over a six-month period.results. none of the subjects tested for immunogenicity developed sustained adas. in total, subjects met the definition of treatment-related, transient positive immunogenicity showing low ada titers. none of these subjects had evidence of loss of efficacy, deviating pk behavior or reported anaphylactic reaction or immune complex disease.conclusion. based on the available data, secukinumab appears to carry a low risk of immunogenicity. in the very few transient immunogenicitypositive patients identified so far, there has been no indication of altered pharmacokinetics or loss of efficacy, and no adverse event that could be linked to immunogenicity has been detected. more data from the ongoing phase studies are required to strengthen this encouraging finding in a larger patient population. risikofaktoren für eine aa-amyloidose bei entzündlich-rheumatischen erkrankungen und bei der idiopathischen aa-amyloidose methods. we report a case of an -year-old woman suffering from ulceration and signs of infection of the ulnar aspect of the right forearm due to subcutaneous calcification in association with crest syndrome.results. this case presents an unusual case of extensive subcutaneous calcification in crest syndrome requiring surgical excision due to secondary ulceration, inflammation and infection. while a surgical approach has already been described for calcification in different connective tissue diseases, only scant data of massive subcutaneous calcification related to a forearm in crest syndrome followed by surgical excision exist. conclusion. in crest syndrome, extensive subcutaneous calcification related to the forearm can occur. surgical excision followed by primary wound closure can lead to an excellent postoperative result. background. the whole blood interferon signature (wbifns) is measured in several clinical trials studying inhibitors of interferon alpha (ifn-α) in sle, but failed repeatedly -in contrast to the less sensitive ifnα -to reflect longitudinal changes in lupus activity and to guide dosage finding of rontalizumab. therefore, better ifn biomarkers reflecting disease activity over time and individual response to the inhibition of ifnα are needed to optimize the risk-benefit ratio of ifn-inhibitors. here, we show that the highly sensitive monocyte restricted ifnα response protein siglec- , also known as sialoadhesin or cd , is a useful biomarker to monitor longitudinal changes in disease activity of sle patients. methods. ifn-α and siglec- were measured by delfia and flow cytometry, respectively, in accurately characterized lupus patients over a period of up to months (overall visits). changes of biomarker and changes of disease activity (bilag ) were correlated using spearman rank test (srt). disease courses of selected sle patients were plotted to demonstrate in detail the relations of ifn-biomarkers with disease activity, sle medication and clinical manifestations. background. a -year-old woman was admitted because of sudden attack of convulsion and somnolence situation with positive canca and myeloperoxidase antibodies. cerebral magnetic resonance imaging (mri) showed thickening and marked progression of the dura-meningeal enhancement and edematous changes at pre and post central gyrus left side. based on these findings, it was diagnosed as hypertrophic cranial pachymeningitis related to anca-associated vascultis as unusual presentation. there was only temporarily und partial responce to a -month therapy with cyclophosphamide mg i.v and oral glucocorticosteroids . taking into consideration the severe, life-threatening course of the disease in the case of our patient, the decision was made to use rituximab, a chimeric, monoclonal igg antibody directed against cd , leads to destruction of b cells via complement mediated lysis and antibody dependent cellular cytotoxicity. the first administration of the medication was performed according to the pattern for rheumatoid arthritis patients treated with rituximab, i.e. infusions for mg in -day intervals in combined therapy with glucocorticosteroids. a follow-up mri at months after start with rituximab showed significant regression of the meningeal pathology at temporo-occipatel aspects (pachymeningitis) and completely resolution of edematous changes at pre and post central gyres. the complete clinical remission was achieved by introducing rituximab. conclusion. rituximab seems to be successful therapie for the induction and maintenance of remission in patients with anca-associated vasculitis (aav) with cns involvement (hypertrophic cranial pachymeningitis ) , who had previously failed to respond to standard treatment with cyclophosphamide and steroids and a range of alternative treatments [ , ] . antikörperdiagnostik. mit prednisolon-therapie ( mg/kgkg, mg/ tag) und zusätzlich methotrexat mg wöchentlich war keine anhaltende normalisierung der entzündungsserologie zu erzielen. infliximab ( mg/kg) -wöchentlich i.v. erbrachte nur kurzzeitig eine normalisierung der entzündungswerte, dann trotz weiterer infusionen einen erneuten anstieg der bsg bis auf mm, crp mg/l. nach umstieg auf tocilizumab ( mg / mg/kgkg) alle wochen konnte nach wochen eine bislang anhaltende normalisierung der entzündungsserologie erzielt werden (crp , mg/l, bsg mm . std.). der allgemeinzustand der patientin besserte sich deutlich, der hb-wert normalisierte sich auf , mmol/l. in der kontrastverstärkten sonographie fand sich ein abfall in der kontrastmittelaufnahme der a. carotis communis. die maximale intima-media-dicke reduzierte sich bislang auf , mm. schlussfolgerung. die bisherige standardtherapie der takayasu-arteriitis mit prednisolon und mtx führte auch im vorliegenden fall nicht zur remission. für infliximab fanden wir ein frühzeitiges therapieversagen des sonst erfolgreich beschriebenen ansatzes einer tnf-α-blockade bei riesenzellarteriitis. dennoch gelang mit tocilizumab eine bislang über monate andauernde klinische, sonomorphologische und serologische remissionsinduktion bei monatlicher fortführung der il- -blockierenden therapie. background. cd is the prototypic nk receptor that is also expressed on a unique population of effector cd + cells. these cd -expressing t cells are expanded in rheumatoid arthritis patients and had features of senescent cells. nkg d is another nk receptor over expressed on effector cd + cells in aav patients. cd + as well as nkg d + t cells seem to be involved in tissue injury as they are capable of mediating tcr-independent immune activation. it is hypothesized that il- is able to up regulate the expression of nk cell receptors. interleukin- (il- ) is a proinflammatory cytokine that is over expressed in aav and is linked to the expansion of cd + effector memory t cells (tem). in aav in remission a persistent expansion of these cd + effector memory t cells has been observed. in the present study we assessed the expression cd on cd + t cells of aav and if expression of these molecules was influenced by il- . methods. the distribution of cd + tem and the proportion of cd +cd + t cells and nkg d+ cd + t cells were analysed in aav-patients and hcs by facs. in vitro effects of il- on the expansion of cd + tem and up regulation of cytotoxic markers were assessed in the same way. in addition il- serum levels were measured in patients and hc by elisa. results. we observed an increased proportion of circulating cd +cd + t cells in aav as well as nkg d+ cd + t cells in patients in remission compared to hc ( . vs . p< . and vs . p< . ). % to % of these cells were cd + effector memory t cells. the percentages of the cd +cd + t cells and nkg d+ cd + t cells were constant over time. we also observed elevated il- serum levels in patients in remission compared to hc (p= . ). in vitro stimulation of pbmcs with il- increased not only the proportion of cd + memory cells (cd ro+) but also the expression of cd and nkg d on these cells. conclusion. the driving force behind the persistent expansion of a cytotoxic subset of cd + effector memory t cells expressing cd and nkg d+ and being tcr -independent is likely the increased il- expression in aav patients . ergebnisse. unabhängig von regime der remissionsinduktion und der primären erhaltungstherapie lag am ende der nachbeobachtungsperiode bei % der patienten eine renale remission vor ( %; % pr). % hatten eine persistierende proteinurie von > , g/tag bei stabiler nierenfunktion, % eine persistierende niereninsuffizienz mit erhöhtem kreatinin bei inaktivem sle, bei % wurden eine persistierende aktive ln und/oder renale rezidive beobachtet. vier patienten verstarben. patienten mit langzeit-cr waren gekennzeichnet durch einen niedrigeren tubulointerstitiellen chronizitätsindex in der initialen nierenbiopsie ( , ± , vs. , ± , ; p= , ), eine hochsignifikant geringere proteinurie nach cyc-pulsen ( , ± , vs. , ± , g/tag; p= , ) und niedrigere dsdna-ak ( ± vs. ± u/ml; p< , ) zum zeitpunkt des beginns der erhaltungstherapie. eine proteinurie von < , g/tag nach pulsen cyc zeigte eine sensitivität von % und eine spezifität von % für eine langzeit-cr. schlussfolgerung. eine proteinurie von < . g/tag nach remissionsinduktion mit pulsen cyc sowie ein geringer tubulointerstitieller chronizitätsindex in der nierenbiopsie sind prädiktoren einer anhaltenden kompletten renalen remission bei ln. background. to evaluate and compare clinical efficacy of three biomarkers for interferon activity (measured directly and indirectly) and six traditional biomarkers to indicate current disease activity in sle. methods. ifn-α (delfia), ip- (elisa) and siglec- (flow cytometry) was measured in accurately characterized lupus patients and compared to serum titres of anti-dsdna (elisa and ria), anti-dsdna-ncx elisa, anti-nuc elisa, c and c . disease activity was evaluated using bilag- and a modified sledai- (msle-dai- k). additionally, clinically quiescent patients were monitored for flares over the course of days. results. increased levels of ifn-α, ip- and siglec- were found in %, % and % of active sle patients. ifnα (r= . ; p< . ) and siglec- (r= . ; p< . ) correlated better with bilag- than ip- (r= . ; p= . ), farr assay (r= . ; p= . ), anti-dsdna-ncx elisa (r= . ; p= . ), anti-dsdna elisa (r= . ; p= . ), anti-nuc elisa (r= . ; p= . ), c (r=- . ; p< . ) and c (r=− . ; p= . ). predictors of sle flares were disease duration ≤ months, mild clinical activity (in contrast to no activity), complement c ≤ mg/dl and ifn-α ≥ pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. conclusion. ifn-α, ip- and siglec- emerged as beneficial biomarkers for disease activity in lupus patients. therefore, implementation of ifn biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-ifn-directed therapies. . ) and carried significantly more often other antibodies ( . %; p< . ), which were separated into u rnp-( . %), ro-( . %), pmscl-( . %) antibodies, followed by . % with rheumatoid factors, . % with la-, . % with dsdna-and . % with jo- -and . % with ku-antibodies. the kaplan-meier analysis of the onset of organ involvement revealed a clear inclined position of overlap patients between patients suffering from lcssc and dcssc, especially regarding lung fibrosis and heart involvement. patients suffering from pah, oesophagus involvement and kidney involvement, overlap and lcssc patients showed nearly similar curve progression (log rank < . ). furthermore musculoskeletal involvement was significantly more frequent and more progressive in patients with overlap disease, followed by patients with dcssc and lcssc (log rank < . ). conclusion. these data support the current concept, that ssc-overlap syndromes should be regarded as a separate ssc subset, distinct from lcssc and dcssc, due to a different course of the disease, different proportional distribution of specific autoantibodies and skin/organ involvement. methoden. patienten mit gpa ( mit aktiver und mit in remission befindlicher gpa) wurden durchflusszytometrisch analysiert und mit gesunden verglichen. eine färbung für cd , cd , cd , igd, iga, cd , mhcii, wurde mittels flowjo-software analysiert. die statistische auswertung erfolgte mit "graph pad prism" und p-werte< , wurden als signifikant angesehen. die studie wurde von der ethik-kommission der charité genehmigt. ergebnisse. deutliche unterschiede (p= , ) wurden sowohl für die absolute zahl als auch die frequenz der plasmazellen im peripheren blut der patienten mit gpa mit krankheitsaktivität ( , ± , /µl) im vergleich zu denen mit einem bvas von ( , ± , /µl) oder gesunden ( , ± , /µl) gefunden, ähnlich wie bei sle. bei patienten mit gpa ist außerdem eine signifikante erhöhte anzahl der plasmazellen igapositiv (p= , ). die anzahl der plasmazellen sowie die frequenz der plasmazellen an den b-zellen im blut korrelieren mit dem bvas (r= , ; p< , ). interessanterweise zeigte sich keine expansion der doppelt negativen memory-zellen, die zum beispiel beim sle beschrieben ist. für die naiven b-zellen fand sich ebenfalls ein signifikanter unterschied zwischen patienten mit aktiver erkrankung im vergleich zu gesunden. bei den t-zellen fanden sich nur diskrete veränderungen. schlussfolgerung. die anzahl der plasmazellen ist bei patienten mit aktiver gpa deutlich erhöht, was eine rolle von plasmazell-vermittelten mechanismen in der pathogenese nahelegt. ein großteil dieser plasmazellen ist iga-positiv, diese könnten eine rolle bei der hno-beteiligung spielen. key: cord- -qom rokn authors: chen, long; li, jie; wang, nan; shi, yuan title: post-newborn: a new concept of period in early life date: - - journal: frontier and future development of information technology in medicine and education doi: . / - - - - _ sha: doc_id: cord_uid: qom rokn post-newborn infants refer to infants from > days to < days after birth. during this period, infants are still completely dependent on breast milk or/and formula milk for feeding. up to now, the concept of post-newborn has not been mentioned in classic textbooks. with the development of perinatal medicine, mortality rate of diseases in neonates such as premature infants, asphyxia, infectious diseases have decreased significantly, and consequently, issues of the quality of life for these survivors have aroused widespread concerns. the post-newborn infants have some important characteristics differing from both newborn infants and infants after the period: ( ) different fatal diseases and mortality rate; ( ) the diseases inherited from newborn period requiring early and prompt treatments; ( ) some peculiar diseases during this period requiring much attention; ( ) either similar or different immune function; ( ) rapid growth and uneven development of organ systems. establishment of the new concept of post-newborn will further reveal the nature of life, reduce the mortality rate of infants, and improve the quality of life. unit (nicu) could greatly reduce the mortality rate of newborn infant, but not improve the quality of life synchronously in the early s, the world's first nicu was established at new haven hospital of yale university, which became a milestone in the developmental history of modern neonatal medicine. after that, nicu has been established in the world in succession and developed rapidly, and thus, neonatal medicine has entered a booming era. with the establishment of transfer and treatment network for the critically ill neonatal infant in the developed areas, the constant in-depth development of the relevant basic and clinical research, improvement of the treatment techniques, and especially the use of pulmonary surfactant, the mortality rate for the seriously ill newborn infants such as low birth weight newborns, extreme low birth weight newborns, severe respiratory distress syndrome have been decreased significantly. the mortality rate of the newborn in united states in has been dropped by nearly half as compared with that in (from . to . %) [ ] , where as the neonatal mortality rate in china in has been decreased by % as compared with that of (from . to . %) [ ] . however, a few surviving infants suffered from different kinds complications in the post-newborn, such as recurrent respiratory infections, physical retardation, cerebral palsy, bronchopulmonary dysplasia, retinopathy, and congenital heart disease requiring early surgical treatment [ ] [ ] [ ] [ ] [ ] . moreover, the complication and sequelae could change the way of life and learning of the children to some extent, affect their formation of good personality and mentality, lead to various kinds of personal and social problems in adults, add a great deal of disease burden to the countries, families and individuals [ , ] . although most of these children in the postnewborn have obtained some treatment and their prognosis has been improved, the post-newborn is still a very fragile, important and special stage in early life, which lacks a specific concept to define the short but crucial time. the post-newborn is a continuation of neonatal period. in addition to the abovementioned sequelae and complications during the neonatal period, there is no clear distinction between the physiological and pathological condition of early infants. because the symptoms and signs of diseases are more atypical in early infants, pediatricians should hold very cautious attitude. nevertheless, it's lack of a specific term to cluster the infants during this period. so far most of literatures have described the early infant as ''baby'', ''early baby'', or ''small baby''. a clear and specific concept has not been mentioned in the previous classic textbooks. with the development of perinatal medicine, the neonatal mortality rate has been gradually decreased. chinese ministry of health recently announced the infant mortality rate was run down from . % in to . % in , achieving the un millennium development goals [ ] [ ] [ ] ahead of schedule. consequently the issues of quality of life for these infants surviving in the neonatal period have caused widespread concerns. the task of pediatrics is not only to focus on reducing morbidity and mortality rate, but also to guarantee children's health, improve the quality of life. the disease sequelae of the post-newborn might affect the health and well-beings of future life. within this period, the infant have a very strong repair and remodeling capacity. when given appropriate rehabilitation, the infant could obtain unimaginable therapeutic efficacy. at the same time, early prevention of some adult diseases such as hypertension and diabetes should be paid much attention. studies have suggested that these main diseases in adulthood have a close relationship with early infancy, especially nutritional status of the post-newborn [ ] . how to further promote the growth and development and correct the complications and sequelae at the post-neonatal period, make the ill infants reach the level of normal infant development as early as possible, and reduce the incidence of adult-related diseases has become an important issue. it is necessary to carry out professional studies. for this purpose, we give the definition for this period which refers to post-newborn from [ days to \ days. in this period, the infant still completely depend on breast milk and/or formula milk for feeding. up to now, the concept of post-newborn has not been mentioned in classic textbooks. the concept of post-newborn is mainly based on the following reasons: ( ) the supplementary food is usually introduced from the th month after birth. after this period, the gastrointestinal function of infant begins to adapt to the supplementary food and has significant changes. ( ) after days, most of the common neonatal diseases and complications have been cured. ( ) for most infant medications at this period, no reference for dosage has been made yet, which is not consistent with evidence-based medicine: whether the application of various drugs has an effect or not? how the level of such drug's effect is produced? is there any possible side effects or delayed benefits? the emphasis on this period is helpful for promoting the drug research and development in this specific period. after this period, most of the medications have some reference usage, which has greatly enhanced the safety. ( ) in this period, the antibody levels of infants from breast milk are high, while its own immune function is low, and there are few autoimmune diseases. investigation of the immunity and its tolerance in this period is expected to provide new treatment methods for immunological disease/rheumatic diseases in the future. however, due to different kinds of reasons, there is no infant mortality reported within months alone. it is reported that in some asian developing countries, neonatal disease is still the first cause of death, and infectious disease is the major killer after the neonatal period. in western developed countries, the first cause of death is the congenital malformation, and the death age of those infants is not at the neonatal period [ , ] . because of high infant mortality rate and various kinds of complicated diseases, this indicator can not fully reflect the health level of infants. clinical practice experience suggests that most of the deaths occurred in younger infants after the neonatal period, especially within days. in the united states, sudden infant death syndrome is one of the main causes of infant mortality [ ] , the peak age of death appeared in - months after birth, and the infant mortality rate decreased after months [ , ] . . . . the disease inherited from neonatal period requires early and prompt treatment. there might be a distinct defect in terms of the time concept for neonatal period. although during neonatal period, a few of the mother-born diseases have been corrected, such as hemolytic disease of newborns, premature rupture of membranes, gestational diabetes etc. [ ] , it is impossible for the sequelae and complications of neonatal diseases to be fully corrected in this period. how to better diagnose and treat these diseases in post-neonatal period is the issue which the pediatricians have to pay much attention to. these diseases often require the full co-operation of many specialties including screening and correcting for hearing anomalies, treatment of persistent pathological jaundice, rehabilitation for hypoxic ischemic encephalopathy, screening and treatment for retinopathy, therapy for repeated infection of bronchopulmonary dysplasia, congenital heart disease requiring early surgical treatment due to repeated or fatal respiratory infections affecting infant growth and development. . . . in this period, some special diseases need attention, such as late-onset vitamin k deficiency, which is frequently present in infant with exclusive breastfeeding, chronic diarrhea, and malnutrition. it is prone to have fatal intracranial hemorrhage, and the survivors often leave behind the neurological sequelae, seriously harming the health of babies. during this period, the nerve myelin development is obvious. early diagnosis and treatment of cerebral palsy can help reduce neurological sequelae. . . . the immune function has similarities and differences. the common point of post-neonatal infant and other infancy stages is the low immune function. during this period, the non-specific immunity, humoral and cellular immune function are very immature, and levels of siga and igg are low, with low resistance to infection, prone to have various bacterial and viral infections. clinical manifestations of pneumonia in the post-newborn is more insidious than those of children's pneumonia, the post-newborn infants suffering from pneumonia have usually very light and a typical symptoms such as refusing milk or milk feeding decreasing, no weight increasing, weak cry, and some even lack any clinical manifestation but with rapid disease progress. the difference between neonatal and post-neonatal infants is that the neonatal babies have significantly increased resistance because of colostrum feeding. as the time going on, the maternal antibodies decrease obviously, leading to a variety of infectious diseases such as respiratory syncytial virus, eb virus and ev virus. . . . there is a conflict between the rapid development and growth and uneven development of organ systems during the post-newborn period. this period is the stage for extremely strong growth. the weight of infant has been more than doubled that at the birth. the nutritional requirements are relatively high. at the same time, the uneven development of organ systems, especially the digestive system is often difficult to adapt to a large number of food digestion and absorption. it has been suggested that both nutritional deficiencies and overnutrition are able to significantly affect adult metabolic diseases [ , , ] . the fine mapping of the human genome indicated that diseases were genetically related. not only genetic (congenital) disease but also risk of acquired and some so called adulthood-starting diseases (such as diabetes, hypertension) might start to be increased before birth or at the infancy and childhood. moreover, it has made people realize that gene could not be the only factor to decide the human disease. through epigenetic modifications of gene, early nutrition and its regulation could alter genetic pathway, and then prevent or inhibit the occurrence of disease in adulthood. classical genetics indicated that individuals of the same gene should have exactly the same phenotype, but the truth is not the case. one pair of identical twins often has the differences in terms of appearance, personality of many aspects, which might be the result of epigenetic modifications. the epigenetics has made the research that under the situation gene dna sequence has not changed, the gene expression has the inheritable changes. the epigenetics has three important features: ( ) not involving dna changes; ( ) gene functions have changed; ( ) the change of gene function has heritability and reversibility. food, as a source of methyl donor may change the gene expression and thus affect the development of various organ systems, possibly through epigenetic methylation modifications. further reveal the nature of life and improve life quality of children pediatrics is the only vertical division of subject in clinical medicine in accordance with the process of human life (age group). the division of each age group has accordingly promoted the research progress of the stage. especially the establishment of neonatology and its specialties has made remarkable achievements. the infant stage, especially post-newborn is one of the critical periods for individual physique and neurogenesis, with time and spatial differences. not only the normal anatomy and physiology have the characteristics, but also diseases in different system in terms of etiology, clinical phenotype, assessment method, diagnosis and treatment are quite different from those newborns, children and adults. the research on the developmental law of post-neonatal infants will help to find new diagnosis, treatment and prevention methods for the diseases of children and adults. the task of the post-newborn research remains how to better promote physical and neurological growth and development of the infants, which are the most basic features and the most common issues in children's life course. the physical development integrates child nutrition, endocrine and metabolic diseases, genetics and environmental medicine. nutrition is the material basis for physical growth and development, whereas environmental factors are the important aspect affecting development. researches have revealed that developmental abnormalities are related to human abuse of the environmental substances such as melamine and phthalate esters, whose mechanism may be through endocrine and epigenetic modification. it's important to maintain the balance of nutrients, trace elements, minerals and vitamins. the neurological development is the essence of life quality, which includes the prevention and treatment of children's neurological diseases and mental illness, and promotion of neural development level. although neurological rehabilitation medicine has been significantly improved, the number of children with neurological and intellectual disabilities has been not significantly decreased because the mortality for neonatal disease, especially preterm infants, severe asphyxia, severe respiratory distress syndrome has been dropped significantly and many survivors suffer from neurological complications. it is a big challenge for pediatrician to carry out the early identification and intervention for the infants with the intellectual disability and high risk factors, and finally reduce the incidence of children with intellectual disabilities. prevention of infectious disease and immunization is important for infants during post-newborn. infectious diseases once led to the death of large number of infants. along with development of antibiotics and vaccine work, infectious diseases have been obviously under control. however, a few of infectious diseases such as tuberculosis and measles have flared up in recent years. more seriously, at the same time, new resistant strains such as the production ndm- ''super bacteria'' have been emerged constantly. in addition, a number of newly-emerging viruses including h n avian flu virus, ev virus, sars virus have brought forward new challenges. the strengthening of the development for anti-infective drugs, vaccines and vaccination during this period will help decrease the illness of infant, reduce costs for health care, lower the social and family burden, and thus abate infant mortality. the strengthening of the post-newborn research, especially the research on translational medicine [ ] , may help to find new ways and methods for the treatment of diseases in infants, children, and adult caused by nutritional abnormalities. the nutritional abnormalities could cause disease by means of epigenetic modification. dna methylation depends on the dietary intake of methionine and folate which are subject to individual nutrient levels. low dietary intake of methionine in rats could lead to the occurrence of dna demethylation, more prone to liver cancer [ ] . it has been suggested that mammals should have a critical developmental period before and after birth, and nutrition as well as other environmental stimuli have an impact on developmental processes and cause permanent change in terms of metabolism and susceptibility to chronic disease [ ] [ ] [ ] [ ] . a few of population epidemiological and animal model experimental data support this view, but the complex biological mechanism is still unclear. future research in this area is to select possible target goal to improve the nutritional regulation of intestinal development, namely the detailed understanding of the relationships among nutrients, epithelial cells, intestinal flora, enteric nerves and endocrine. it's of great value to find what nutrients and metabolic pathways get involved in regulation of early life and adult dietary regulation of epigenetic mechanisms. more importantly, epigenetic modifications might occur in the critical window period of early life, especially in the post-newborn. the premise of research is to enhance the degree of concerns on post-newborn. first of all, it is to strengthen the promotion of concept of post-newborn. in the s, the textbook set the ''newborn of diabetic mothers'' as the independent chapter for etiological diagnosis, which caused a high degree of perinatal medical attention, hence greatly contributed to the research on this disease. for this reason, it's strongly recommend that post-neonatal should be defined as an independent age group and be regulated. the post-newborn should be write into pediatric articles and textbooks. in addition, the relevant theoretical system should be built in time. at the same time, specialized training courses and academic conferences should be held to study on the basis of post-newborn so that the concept of postnewborn could go deep into the field. deaths: final data for causes of deaths in children younger than years in china research on prevention of bilirubin-induced brain injury and kernicterus: national institute of child health and human development conference executive summary the impact of pediatric vision disorders in adulthood retinopathy of prematurity-epidemics, incidence, prevalence, blindness delayed diagnosis of congenital heart disease worsens preoperative condition and outcome of surgery in neonates quality of life in children with heart disease as perceived by children and parents respiratory symptoms in preterm infants: burden of disease in the first year of life countdown to for maternal, newborn, and child survival: the report on tracking coverage of interventions ministry of health of the people's republic of china ( ) china health statistics yearbook . ministry of health of the people's republic of china the fetal origin of adult diseases evolution of cause of infant and mortality in some countries and regions in the world sudden infant death syndrome sudden infant death syndrome: seasonality and a biphasic model of pathogenesis the changing concept of sudden infant death syndrome: diagnostic coding shifts, controversies regarding the sleeping environment, and new variables to consider in reducing risk attention to mother-born diseases of newborn infant mortality, childhood nutrition, and ischaemic heart disease in england and wales fetal and infant growth and impaired glucose tolerance at age hypomethylation of hepatic nuclear dna in rates fed with a carcinogenic methyldeficient diet programming by early nutrition in man potential mechanisms of metabolic imprinting that lead to chronic disease developmental origins of disease paradigm: a mechanistic and evolutionary perspective developmental origins of the metabolic syndrome prediction, plasticity, and programming key: cord- - lkkez n authors: nan title: invited speakers date: - - journal: respirology doi: . /j. - . . .x sha: doc_id: cord_uid: lkkez n nan the physiology of respiration reaches its extreme limits when man is exposed to the effects of high altitude, which is basically a decrease in ambient pressure and temperature. the process of acclimatization of humans to high altitude occurs within minutes of ascent, starting with increases in cardiac output, and ventilation. these result in acute changes in pao and paco . slower changes of acclimatization include increase in hemoglobin concentration (polycythemia), and muscle capillarity, but decrease in mitochondrial volume density and cellular aerobic capacity. through acclimatization, animals and human inhabitants of high altitude areas have developed adaptations that allow them to function as near normal as those living at sea level. humans who undergo acute ascent to high altitude could acclimatize, but some fail to do so. if there is failure to acclimatize, oxygen diffusion impairment results, due to decreased partial pressure, and lower affi nity of hb for oxygen. in addition, there is signifi cant increase in ventilation/perfusion heterogeneity. the resulting hypoxia leads to hypoxic pulmonary vasoconstiction (hpv) which results from multiple components: changes in epithelial cell wall that lead to intracellular calcium increase and/or calcium sensitization. subsequently, pulmonary hypertension develops, with actual breaks in the capillary endothelium leading to an infl ammatory process (seen more during exercise), and decrease in alveolar fl uid clearance. these mechanisms contribute to the development of alveolar edema, high altitude pulnary edema (hape), which is one of the two major diseases due to acute mountain sickness (ams). both hape and the other ams, high altitude cerebral edema (hace) could be potentially fatal, and must be recognized and treated early. knowledge of the pathophysiology of the ams would allow more rational approaches to their prevention and treatment. high altitude illnesses can develop among healthy individuals who sojourn for recreation and work: cerebral form called acute mountain sickness (ams) and potentially fatal pulmonary form called high altitude pulmonary edema (hape). ams is generally self-limited. high-altitude cerebral edema (hace) is likely a continuum of ams and the end-stage of ams. ams is not a precondition for the development of hape. hape develops in non-acclimatized mountaineers after rapid ascent to altitudes above m. hape may develop even in the absence of ams. severe ams may be a risk factor for hape. the altitude, the rate of ascent to new altitude, (> m/day to an altitude above m), and individual susceptibility are major determinants of ams and hape. on ascent to high altitudes all people have swelling of the brain. patient with ams often experience "hangover headache." other symptoms occur within the fi rst to h. these include malaise, anorexia, nausea and vomiting, and insomnia. ams patient must be evaluated for signs of global encephalopathy rather than focal fi ndings, although retinal hemorrhage is commonly seen. when these are present, the subject has hace until proven otherwise. patient may die if not treated promptly. brain herniation is the usual cause of death. the hallmark of hape is an excessively elevated pap which precedes the development of pulmonary edema. symptoms are incapacitating fatigue, chest tightness, dyspnea with effort, orthopnea, cough, and pink frothy sputum in advanced stage of disease. prevention of all altitude complications requires ascending at an increment rate to allow acclimatization. at > m, one should not spend subsequent nights m higher than the previous night. trekker must take a rest day every to days. anyone with ams should not ascend until symptoms are resolved. acetazolamide and dexamethazone are effective in prevention and treatment of ams from proceeding to hape or hace. at the fi rst sign of hace, patients should descend to a lower altitude while supplementary oxygen is given. increasing oxygenation is the highest priority in the treatment of and prevention of hape. if supplemental oxygen is unavailable, then descent, use portable hyperbaric chamber, or both become lifesaving. nifedipine is necessary only when supplemental oxygen is unavailable or descent is impossible. because of its pulmonary vasodilatory effects, phosphodiesterase inhibitors can be used for prevention and treatment of hape. rudolf virchow in described that the determinant risk factors for venous blood clot formation are stasis, endothelial injury and hypercoagulable state. dr simpson et al , a british surgeon observed that during the london blitz, the world war ii, britons who were forced to remain on sitting in cramped position and deck chairs for hours during the air raids developed fatal pulmonary embolism. it was suggested in by homans that "prolonged dependency stasis" or immobilization is one factor that predisposes patients to develop thrombosis in the deep veins of the legs. several risk factors have been identifi ed for developing venous thromboembolism (vte). travel is one of the transient risk factors. it is not confi ned to one mode of travel such as air travel. it is also incriminated to other modes of land travel such as car, bus and train. the term "economy class syndrome" was proposed by symington and stock ( ) and by the group of cruickshank ( ) for venous thromboembolism occurring in patients during air fl ight travel. it is usually seen in patients sitting in limited or cramped circumstances in the economy coach or tourist class seats, however it is also found out that patients who were also seated in the business class also develop this syndrome. factors implicated were the long duration of travel, immobilization or inactivity in sitting position, and the low cabin pressure, low humidity and dehydration during air fl ights. in several studies performed on large airports in europe, the presence of genetic factors such as factor v leiden and environmental factors such as the use of oral contraceptives predispose patients several fold to develop venous thromboembolism. signs and symptoms pertaining to vte develop not only during and after the fl ight but also several weeks after the travel. nowadays, airlines as well as bus companies have advisories and measures impose to prevent development of vte and deaths due to vte during the travel period. with the steadily increasing use of air travel, more and more patients with pulmonary disease are fl ying long distances, at high altitude in partially pressurised aircraft. this is associated with long periods of reduced mobility and exposure to reduced inspired pressures of oxygen and reduced barometric pressure. some individuals therefore may be at risk of barotrauma, hypoxia or venous thrombo-embolism (vte). therefore it is important to identify these individuals and adequately assess the real risk entailed by fl ying. the effect of reduced atmospheric pressure is a potential risk for patients with recent or pre-existing pneumothorax but otherwise is unlikely to be associated with risk other than that due to the associated reduced inspired oxygen fraction (fio ), typically . ( %) in a commercial aircraft at cruising altitude. the reduced (fio ) may be problematic for patients with hypoxic lung disease or in patients with other co-morbidities that may exacerbated by hypoxia. medical history, lung function and resting oxygen saturation will help identify patients at risk although it is diffi cult to predict the clinical effects of altitude from tests (even hypoxic challenge tests) conducted at sea level. there is currently a lack of good data defi ning the clinical outcomes due to hypoxia during fl ight in patients with lung or other diseases. the risk of vte increases with duration of fl ight above four hours, presumably related to the duration of immobility, although the role of prolonged hypoxia remains to be determined. preventive measures are now currently invoked on most airlines and guidelines for the use of antithrombotic agents are available, stratifi ed by risk. it is anticipated that guidelines will continue to be updated as new data are made available. cardio-pulmonary exercise testing is now well accepted as an appropriate test for the investigation of shortness of breath on exertion. in addition the test has been found to be useful for the assessment of pulmonary vascular dysfunction and the assessment of fi tness for major thoracic surgery. even though there are well described and internationally accepted protocols to perform the test, the interpretation of a cardio-pulmonary exercise test often leaves the interpreting physician confused. importantly with the multiple facets of the test (respiratory, cardiac, peripheral vascular) that need to be interpreted it is easy for the interpreting physician to look at a certain aspect of the test relating to their specialty and to give the other facets relatively little attention. in this presentation we review the process of interpreting cardio-pulmonary exercise tests. in addition we will interpret a number of tests based on our previous discussion on how to interpret these tests. finally we will review the literature regarding new interpretive strategies for exercise induced pulmonary vascular disease. bronchoscopy as an image-guided intervention has benefi tted from advances in optical and non-optical imaging technologies. some current bronchoscopy advances incorporate higher resolution ccd (charged-couple device) digital-"chip" technology and magnifi cation lenses to enhance the image resolution. the hope is that improved visualization combined with analysis of concomitant tissue biopsies may realize so-called "in-vivo endoscopic diagnosis" without the need for tissue biopsies, however studies of highmagnifi cation endo-cytoscopy, co-focal micro-endoscopy and optical coherence tomography (oct) remain investigational. further limiting these near-histologic resolution imaging modalities is the need for an initial screening of "highly suspicious" mucosa to focus attention upon. to facilitate identifi cation of abnormal airway mucosa, there are advances in the bronchoscopic detection of dysplastic and malignant mucosa. newer generations of autofl uorescent (af) bronchoscopes combine video ccd technology with af signaling to enhance the visual resolution of the images. non-af technologies being evaluated for the same purpose include fi ltered-light narrow band imaging (nbi) and post image-capture processing by a number of other spectral estimation technologies (set). bronchoscopic image guided interventions (bigi) also benefi tted from advances in non-traditional bronchoscopy technologies. foremost has been endobronchial ultrasound (ebus), initially designed as radial probes modeled after intravascular us and modifi ed for the airways. while useful in advancing our understanding of endobronchial mucosal structure, predicting tumor invasion depth and responsiveness to endobronchial interventions, radial ebus did not permit real-time guidance. dedicated linear-array ebus bronchoscope has changed this dramatically as the . mhz needle-puncture ebus bronchoscope has increased diagnostic accuracy of peri-bronchial lymph nodes/masses from a previous average of < % to > % for even small targets (< cm) in experienced hands. simultaneously miniaturization of radial ebus probes (thin . mm) and incorporation of guide-sheaths have increased the utility of ebus in evaluating parenchymal lung pathology. concomitant work in image processing of radiology imaging data (dicom data of chest ct images) has made available a number of "virtual bronchoscopic navigation" programs to assist the bronchoscopists in navigating towards smaller peripheral focal targets, and to improve the historic diagnostic yield of smaller (< cm) peripheral nodules from - % up to - %. these systems include passive endobronchial "road-maps" view (similar to "mapquest"/"google earth") and more technology enhanced electromagnetic navigation bronchoscopy (enb) (similar to gps guidance). all these ancillary technologies have spurred improvements in the basic bronchoscope, as thinner bronchoscopes capable of reaching peripheral segments ( . mm and . mm with . mm working channel; . mm with . mm working channel) are coupled with new biopsy instruments. the eventual development of steerable single fi ber scanning endoscopes with multi-wavelength imaging may change our current concepts of the bronchoscopes and how we can use them. journal compilation © asian pacifi c society of respirology pg - diagnostic tests pleural effusions are common and often present diagnostic challenges. the new british thoracic society guidelines on investigation of pleural effusions detailed some of the new approaches to undiagnosed pleural effusions. traditional teaching recommends measurement of blood and pleural fl uid protein and ldh levels as the fi rst step of investigation to categorize the effusion into a 'transudate' and 'exudate' using light's criteria. the need to apply this to all effusions is questionable in . current efforts focus on the development of disease-specifi c diagnostic tools incorporating clinical, radiologic and biochemical parameters. • elevated ntpro-bnp levels in pleural fl uids are useful in confi rming cardiac failure as the etiology of a pleural effusion, especially in patients whose fl uid may be falsely elevated into the 'exudative' range by concurrent diuretic therapy. • pleural ntpro-bnp levels are elevated in cardiac failure effusions, but not in other transudative effusions (eg hepatic hydrothorax). • pleural fl uid ntpro-bnp appears a better marker than pleural fl uid bnp. variations in accuracy may also in part depend on the commercial kits used. adenosine deaminase: • ada measurements in pleural fl uids are useful in the diagnosis of tb pleural effusions with a sensitivity and specifi city of and % respectively. limiting the test to lymphocytic pleural effusions will further improve the diagnostic accuracy. • ada is cheap and fast to perform and is now widely used in endemic countries. false positives can occur with bacterial infections, rheumatologic effusions, and occasionally malignant effusions. false negatives are uncommon, and therefore present a valuable 'rule-out' test in regions of low tb rates. • ada is at least as diagnostically useful as pleural fl uid total interferon-gamma levels. • igras have been tested in pleural fl uid and blood of patients with tb pleural effusions in several studies. the diagnostic sensitivity and specifi city are poor and igras are not recommended for the investigation of tb pleuritis. • serum mesothelin is a fda-approved test for the diagnosis and monitoring of mesothelioma. • pleural fl uid mesothelin adds information to pleural fl uid cytology in the diagnosis of mesothelioma, providing a diagnostic sensitivity of % (specifi city %). elevated pleural fl uid mesothelin levels suggest epithelioid or biphasic mesothelioma or occasionally metastatic carcinomas. procalcitonin: • early evidence suggest that serum level of procalcitonin may aid differentiation of pleural infection from pleural effusions of non-infective etiologies. the value of pleural fl uid procalcitonin level is limited. management strategies imaging guidance for pleural procedures: • pleural procedural complications are often under-estimated and underreported. studies have now shown that mandatory imaging guidance (especially bedside pleural ultrasound), and restricting procedural privilege to certifi ed trained clinicians can signifi cantly reduce complication rates from pleural procedures. this practice is now incorporated into many national and professional society guidelines. intrapleural therapy for pleural infection/empyema: • recent clinical trials on intrapleural delivery of fi brinolytics alone have failed to improve important clinical outcomes of pleural infection. however, the combination of tissue plasminogen activator and dnase has shown promising results. • recent studies have revealed increasing concerns of complications of talc pleurodesis, and randomized studies have shown a much lower success rate than previous non-randomized literature, even in selected patients. the concept of drainage without needing to create pleurodesis has growing appeal and the use of indwelling pleural catheters is now regarded as fi rst-line therapy in increasing number of centers. chronic respiratory disease (crd) is non-communicable respiratory disease including asthma, chronic obstructive pulmonary disease (copd), allergic rhinitis, idiopathic pulmonary hypertension, hypersensitivity pneumonitis, occupational respiratory disease. among these crd asthma and copd are important for regional health. facts of asthma million people suffer from asthma. , people died of asthma in . prevalence of asthma has increased or is increasing. asthma is the most common disease among children over % of asthma death occurs in low and lower-middle income countries. asthma is underdiagnosed and under-treated (who, ). facts of copd copd is a life-threatening lung disease that interferes with normal breathing. it is more than a "smoker's cough". an estimated million people have copd worldwide. more than million people died of copd in , which is equal to % of all deaths globally that year. almost % of copd deaths occur in low-and middle-income countries. the primary cause of copd is tobacco smoke (through tobacco use or second-hand smoke). the disease now affects men and women almost equally, due in part to increased tobacco use among women in high-income countries. copd is not curable, but treatment can slow the progress of the disease. total deaths from copd are projected to increase by more than % in the next years without interventions to cut risks, particularly tobacco smoking (who, ). prevention and control of crd in asia pacifi c were held by dokkyo medical university group, later designated as who collaborating centre for prevention and control of crd (du-wcc). seven countries and a district in asia pacifi c joined the meeting. prevalence of asthma in adults was reported from . to . % with a median of . % based on reports. prevalence of childhood asthma ( - y/o) was from . to . % with median of . % based on reports. prevalence of copd was . to . % with a median of . % based on nation-wide surveys. in spirometry-based survey reported, prevalence of copd was . % in adults years and over in japan, % in adults years and over, and . % in adults years and over in china. management in most of the countries gina and gold were adopted for their national guidelines. major risk factors for crd, especially for copd were smoking and indoor air pollution for cooking/heating. pharmacological early interventions have been reported to improve the prognosis of asthma and copd. occupational respiratory diseases are disorders which are induced by occupational and industrial conditions. providing information of the risks of industrial activities would reduce this disorder. strategic direction for the prevention and control of crd most of crd are treatable and at least partially preventable. development of user-friendly guides for prevention and control of crd for offi cials in health care, fi rst-line health-care givers and patients and their family and its implementation would decrease the burden of these crd. the scientifi c foundation of asthma diagnosis and management has grown in leaps and bounds. evidence-based strategies to control asthma and treat its exacerbation are published yearly in the gina guidelines. the -year finland study showed that these strategies work. while cases treated did increase (through better detection), the hospital days and cost per case markedly decreased. the study also showed that widespread adaption and effective implementation of these strategies is best done through a national program. cmes for medical practitioners are important but are of limited reach. all stakeholders must be enlisted to buy-in. for asthma, the target stakeholders are the health care personnel, nurses and village health volunteers included; the patients and their families; the government and its public health offi cials; the asthma advocacy groups; the community-at-large; and the pharmaceutical industry. the idea is to present the problem to them, include their inputs in the formulation of the plan, collegially decide on target indicators of success and engage them to work for the implementation of the program in the context of what each one can do best. duplicating the finnish experience is a big challenge in the asia pacifi c region. while most countries have their own adaptation of the gina guidelines, few have working national asthma programs. in developing economies, the health infrastructure is not that well developed yet to absorb all guideline recommendations. spirometry may not be widely available nor affordable. government spending for health is commonly below the % of gdp level that who recommends. in the philippines, signifi cant out-of-pocket health expense is borne by the patient. furthermore, programs like tb control, dengue treatment and malaria eradication, which are no longer concerns in developed countries, compete for the meager public health funds. for low income countries, the international recommendations may have to be rewritten to emphasize on simple algorithm for separating non-infectious from infectious respiratory illnesses; practical objective measurements for diagnosis and management such as peak fl ow; available, affordable, and low-risk medications recommended for asthma control; and a simple regimen for recognizing severe asthma (gina). to be viable, the national asthma program will have to piggy back to the existing national health delivery infrastructure which must ensure, among others, access to free or cheap medication. lung cancer and copd commonly coexist in smokers, and the presence of copd increases the risk of developing lung cancer. in addition to smoking cessation and preventing smoking initiation, understanding shared mechanisms in these smoking-related lung diseases is critical, to develop new methods of prevention, diagnosis and treatment of lung cancer and copd. common mechanisms may involve infl ammation, abnormal repair, oxidative stress, epithelial-mesenchymal transition, altered nicotine receptor biology and epigenetic alterations. strategies to study genomics and epigenomics, in addition to gene-environment interaction, will yield greater insight into the shared pathogenesis of lung cancer and copd. copd clinical guidelines are important to guide diagnosis and management of people with copd. the australian 'copdx' guidelines are evidence-based guidelines that are prepared by the australian lung foundation and thoracic society of australia and new zealand. relevant literature is searched regularly and evaluated by a clinical committee. updates are then produced regularly during the year. challenges regarding critical appraisal, resources and dissemination to clinicians will be discussed. national copd guidelines in this region are different from country to country, but basically are adapted from gold. copd prevalence in asia pacifi c countries and region estimated by regional copd working group was . %. vietnam has the highest prevalence: . %. the copd management and guideline implementation problems in the asia pacifi c region are: smoking, biomass using are common; continuous medical education (cme) for health workers are not compulsory; lack of device and personnel for performing proper spirometric tests; over burden for health workers; low access to medical care and low affordability for copd medications. all of these problems result in that copd diagnosis are mostly in late stage, high rate of emergency room visit, icu admission and hospitalization. the consensus is expected to cover following resolutions: reducing the smoking and biomass smoke exposure, screening for copd in large scale using questionnaires and confi rming by spirometry, advocacy for compulsory cme on copd, establishing asthma and copd outpatient care unit (acocu) in different levels of health care settles and introducing copd medications into insurance medication list. infections caused by environmental mycobacteria are more common than tuberculosis in many parts of the world. the more than species of mycobacteria have similarities, but generally the diseases and hosts fi t in specifi c patterns. disease due to environmental mycobacteria can be diffi cult to diagnose and treat and can confuse workup for tuberculosis. mycobacteria have low virulence and even lower invasiveness. they form biofi lms that protect them and allow long term persistence. the treatment is often frustrating for the patients and physicians. learning their metabolic mechanisms and attacking them should be the strategy for combating the disease caused by these organisms. lung cancer and copd commonly coexist in smokers, and the presence of copd increases the risk of developing lung cancer. in addition to smoking cessation and preventing smoking initiation, understanding shared mechanisms in these smoking-related lung diseases is critical, to develop new methods of prevention, diagnosis and treatment of lung cancer and copd. common mechanisms may involve infl ammation, abnormal repair, oxidative stress, epithelial-mesenchymal transition, altered nicotine receptor biology and epigenetic alterations. strategies to study genomics and epigenomics, in addition to gene-environment interaction, will yield greater insight into the shared pathogenesis of lung cancer and copd. airway epithelial cells, which are the fi rst line of cells to contact with inhaled substances such as microorganisms, play an important role in the host defense by two major mechanisms. first, they actively contribute to the innate immune system by recognition of the pathogen and production of antimicrobial substances and cytokines. second, they provide a passive barrier function that prevents invading microorganisms, air pollutants and airborne allergens into the internal milieu. on the other hands, airway epithelial cells are involved in the production of airway infl ammation in asthma and copd by excessively and un-regulatory expressing pro-infl ammatory and pro-allergic cytokines, executing apoptosis and losing barrier function. there is very close relationship between epithelial barrier function and innate immune response of epithelial cells. for instance, losing barrier function results in not only allowing foreign substance and pathogens to invade into the internal milieu but also enhancing innate immune responses. asthma and copd are different diseases, but they may have the same mechanism in the pathogenesis of exacerbation of these diseases in term of losing epithelial barrier functions. in this symposium, we will present the latest information on and the regulatory mechanism of airway barrier function and discuss in the context with asthma and copd pathogenesis and exacerbations. key words; airway epithelial cells, barrier function, asthma, copd patients with severe and diffi cult-to-treat asthma ("refractory asthma", approximately % of total asthma) have impaired health status refl ected by persistent symptoms, severe airfl ow limitation and frequent asthma exacerbations despite taking maximally recommended doses of inhaled corticosteroids and long-acting β -agonists. a better understanding is thus needed regarding factors associated with such troublesome condition and, in our cross-sectional observational study, clinical and demographic characteristics of patients fulfi lling the american thoracic society workshop criteria for refractory asthma (ajrccm, , group a) were compared with those of patients with severe persistent asthma defi ned on the basis of the gina guideline (group b). there were no signifi cant differences between the two groups with respect to age, gender, smoking status, disease duration, pulmonary function (fev , pef, dlco), or markers of airway infl ammation in the induced sputum (eosinophils, neutrophils, ecp, tryptase). however, in contrast to group b, all patients in group a were adult-onset, and % of the patients already had severe symptoms at the time of disease onset. prevalence of atopy, postbronchodilator fev /fvc ratio and fev reversibility were signifi cantly less in group a than in group b. patients in group a complained of copious amounts of phlegm associated with chronic sinusitis and/or chronic bronchitis, and showed high concentrations of mucin (muc ac + muc b) in the sputum. in addition, nasal clearance time assessed by saccharine test was signifi cantly longer in the group a than in the group b patients, indicating impairment of airway mucociliary clearance. these fi ndings and other pathophysiological and clinical data suggest that "refractory asthma" may be a different form of asthma (phenotype) rather than a progression of asthma severity during follow-up of natural history of the disease. furthermore, it is likely that irreversible airway narrowing possibly due to airway remodeling and airway mucus hypersecretion are important factors contributing to the pathogenesis of severe and diffi cult-to-treat (refractory) asthma. the results prompt for further longitudinal studies and interventions to defi ne the mechanisms of this unique phenotype of asthma. journal compilation © asian pacifi c society of respirology drug development is a long and expensive process. on average it takes at least years and more than a billion dollars to develop a compound from basic science discovery through clinical trials and fi nal approval by regulatory authorities of a new therapeutic. one of the main obstacles to development of new compounds is the diffi culty in obtaining good pre-clinical proof of effi cacy for a new drug. most of this is currently obtained from experiments using animal models of disease or cell lines, neither of which refl ect well human disease nor predict whether responsiveness in these models predicts responsiveness in human disease. recent studies have focused on developing methods that employ human cells or tissues taken from the relevant organ and from relevant patient populations. of these models, the explant model, which uses whole tissue samples, is the closest to the in vivo situation because it maintains the complex cell-to-cell interactions. in asthma, studies have shown that this model can sometime be even better than in vivo study. thus, for example, the explant model vivo offers several advantages over in vivo allergen challenge of asthmatic volunteers. first, repeat bronchoscopy to sample the airways after initial allergen challenge is not required. second, tissue responses of more severe asthmatics, who for safety reasons cannot be challenged with allergen in vivo, can be studied. third, problems of dilution of secreted mediators during bal are avoided and released mediators are not consumed by in-coming infl ammatory cells, thus increasing the sensitivity of the model. finally, and most importantly when seeking pre-clinical proof of concept of drug effi cacy, the model allows testing of novel compounds at an early stage before its full safety profi le is established, a process that is both expensive and time-consuming. we have previously shown that the asthmatic airways generate increased t cell chemotactic activity compared to healthy controls. using a highly selective ccr antagonist we have recently shown that the ccr -chemokine axis plays a key role at least in the traffi cking of t cells into the asthmatic airways. having established this, we then showed that predominantly the ccr + t cells are recruited in response to allergen stimulation. we have further shown that the selective removal of these ccr + t cell from blood signifi cantly reduced allergic infl ammation as shown by a marked reduction in the production of the th cytokines il- , il- and il- but with no consequences for th responses. taken together, our studies have strongly suggested that inhibiting the migration of t cell to the asthmatic airway by targeting ccr is likely to abrogate the allergic infl ammation in the airways without affecting immune responses that serve to protect against infection. these studies have also shown the value of using such ex vivo models of asthma to provide proof of concept for new drugs, giving the pharmaceutical industry the necessary pre-clinical proof to proceed with confi dence into further clinical development. sleep-disordered breathing (sdb) or obstructive sleep apnea (osa) is a prevalent but largely undiagnosed sleep disorder. apnea-hypopnea index (ahi: the number of apneas and hypopneas per hour of sleep) is used to classify sdb severity. in icsd- (international classifi cation of sleep disorders ver. ), "osa syndrome" was defi ned as ahi ≥ with hypersomnolence/ daytime symptoms or as ahi ≥ regardless of the symptoms. epidemiological studies clarifi ed that sdb is associated with increased likelihood of hypertension, cardiovascular disease, stroke, motor vehicle accidents, depression, diminished quality of life, and even mortality. clinical guidelines for hypertension put weights on sdb as a cause of hypertension. international diabetes federation (idf) made a consensus statement on sleep apnea and type diabetes. "overlap syndrome" (coexist of copd and sdb) was reported to have much higher mortality than sdb alone. sleepiness was thought to be a major symptom for osa syndrome. it is true that there is a signifi cant trend that the severe the sdb is the more the subjects had sleepiness. however, the majority of sdb subjects (even the majority of subjects with ahi ≥ ) do not have sleepiness (ess: epworth sleepiness scale > ). the berlin sleep questionnaire was used to screen high or low risk subjects for sdb. four-item screening tool was also developed (gender, bmi, blood pressure, snoring frequency). these tools may be useful, when certifi ed with sleep monitoring in each population. prevalence of ahi ≥ , estimated from two-stage sampling, was - % in male and - % in female. two-stage sampling is oversampling the subjects with sleepiness or snoring to perform sleep monitoring, and weighting of results to the survey sample. when all the participants underwent sleep monitoring, the prevalence of ahi ≥ was - % in male and - % in female. there is a strong need for better recognition, screening and treatment of sdb. more studies are needed, especially for long-term outcomes of asymptomatic sdb. genome-wide association studies may be useful to elucidate causes or underlying mechanisms of sdb. continuous positive airway pressure (cpap) is a standard treatment for patients with obstructive sleep apnea (osa), especially for moderate to severe osa. the mechanism of action is to provide a pneumatic splint to preserve upper airway. the pressure level required to maintain airway patency is determined by manual pressure titration by a sleep technologist during attended laboratory polysomnography (psg) to eliminate obstructive respiratory-related events (e.g., apneas, hypopneas, respiratory effort-related arousals [rera], and snoring). despite wide acceptance as a standard therapy for treatment of osa patients, very few pap titration protocols have been published so far, and there are inconsistency and variations in cpap titration protocol among clinical sleep laboratories. for this reason, the pap titration task force developed evidence-and consensus-based standardized pap titration protocol and published its guideline entitled "clinical guidelines for the manual titration of positive airway pressure in patients with obstructive sleep apnea" in journal of clinical sleep medicine . in this lecture, i will explain about manual cpap titration guidelines as below based on publications which are recommended by positive airway pressure titration task force of the american academy of sleep medicine (aasm): ( ) important considerations prior to cpap titrations. ( ) criteria for cpap pressure to be increased. ( ) minimum and maximum starting cpap pressure. ( ) an interval and minimum pressure to eliminate obstructive respiratory events. ( ) different algorithms for cpap pressure to be increased to eliminate obstructive respiratory events observed for patients ≥ years and < years. interventional bronchoscopy has typically been associated with obstructive tumor removal to regain central airways patency; such interventions, whether with rigid or fl exible instruments were also limited to the navigable fourth or fi fth generation airways. tissue destructive techniques included ablative heat techniques (laser, electrocautery and argon plasma coagulation), cold techniques (cryotherapy) and mechanical coring with the rigid bronchoscope. a current new crop of tissue debridement devices include modifi cations of established technologies: fl exible co laser fi ber usable beyond the trachea, cryotherapy using non-contact surface cryospray, rotational microdebrider devices adopted from otolaryngology, pulsating balloon resectors. the latter three devices do not involve heat that may cause post-treatment infl ammation and cartilage destruction and subsequent airway fi brosis or malacia. previous direct intra-lesional injection, with mitomycin or steroid was directed towards non-malignant fi brotic lesions, conversely on-going studies with cytotoxic agents ( fu) and compounds thought to have immune adjuvant effects (pts) are demonstrating potential utility in endobronchial tumors. photo-dynamic therapy (pdt) compounds with shorter half-lives require fewer bronchoscopies and have shortened photo-toxicity side effects. therapeutic bronchoscopic image guided interventions (bigi) have benefi tted from advances in "virtual bronchoscopic navigation" software available to improve reaching small peripheral lesions. for radiation therapy for focal lung lesions not resectable because of patient co-morbidities or preference, the accurate placement of fi ducial markers (gold) are used to direct high-dose rate external-beam intentiy modulated radiation therapy (imrt) including cyberknife machine. trials also demonstrate feasibility and effi cacy of treating peripheral lesions by high-dose rate (hdr) brachytherapy through catheters placed with image guidance. one area of new focus is bronchoscopy in the management of chronic obstructive lung diseases (old) including emphysema and severe asthma. based on lung volume reduction surgery for severe emphysema with heterogeneous distribution and air-trapping, non-surgical bronchoscopic lung volume reduction (blvr) has taken on a number of innovative approaches including exclusion by spigots (watanabe), valves (emphysys, spiration), metallic coil retraction (pneumrx), airway bypass to relieve trapped gas (broncus), atelectasis by bio-glue (aeris) or by heat steaming (uptake). although none of the clinical devices in trials have shown unqualifi ed success, some devices are now being marketed (europe), or are available on a compassionate basis for management of broncho-pleural fistulas (bpf). airway radio-frequency ablation (rfa) of airway smooth muscle is usa-fda approved for management of severe asthma. future innovations in interventional bronchoscopy will likely incorporate advances in diagnostic bronchoscopy such as video-autofl uorescence and "in-vivo biopsy" techniques to guide local endobronchial therapies for in-situ cancers; image processing software to design custom stents for compromised airways; and drug-eluting stents to maintain airway integrity in a variety of malignant and benign airway diseases. pleuroscopy describes a minimally invasive procedure that provides the physician a window into the pleural space. it refers to a procedure that is performed in an endoscopy suite or operating room with the patient under conscious sedation and local anesthesia. increasingly these procedures are being performed by nonsurgeon pulmonologists to diagnose pleural pathology such as pleural effusions or pleural carcinomatosis; talc pleurodesis and chest tube placement under direct visual guidance. pleuroscopy was fi rst conceived in a report dated , which documented the fi rst endoscopic examination of the pleural space by richard cruise in a year old girl with empyema. it did not gain widespread application until when hans christian jacobaeus published his technique also known as the jacobaeus operation. in this procedure he created a pneumothorax by severing adhesions using galvanocautery that collapsed the underlying lung, and allowed safe entry as well as unobstructed examination of the pleural space. since then, pleuroscopy has been applied both as a diagnostic and therapeutic tool. for a hundred years, rigid endoscopic instruments such as stainless steel trocars and telescopes have been pivotal in the technique. smaller telescopes and instruments have been applied with excellent views of the pleural space and comparable diagnostic yield. a signifi cant advance is the creation of fl exrigid pleuroscope that is fashioned like the fl exible bronchoscope. the fl ex-rigid pleuroscope consists of a handle, and a shaft that measures mm in outer diameter, -cm proximal rigid portion and -cm fl exible distal end. the fl exible tip is movable by a lever on the handle, which allows -way angulation degrees up and degrees down. it has a . mm-working channel that accommodates biopsy forceps, needles and other accessories, and is compatible with various electrosurgical and laser procedures. the fl ex-rigid pleuroscope allows autoclaving. a notable advantage is its easy interface with existing processors and light sources made by the manufacturer for fl exible bronchoscopy or gi endoscopy at no additional costs. although certain endoscopic characteristics such as nodules, polypoid masses and "candle wax drops" are suggestive of malignancy, early stage mesothelioma can resemble pleural infl ammation. autofl uorescence and narrow band imaging have been incorporated to white light pleuroscopy to enhance diagnostic accuracy. both modes of imaging discriminate early malignant lesions from non-specifi c infl ammation, aid in selecting appropriate sites for biopsy and better delineate tumor margins for more precise staging, but are of little value at present in clinical practice since most patients with malignant pleural effusions have extensive pleural involvement that is easy to diagnose with white light pleuroscopy for pleuroscopic guided pleural biopsies, specimens obtained with the rigid forceps are larger than those with the fl ex-rigid pleuroscope since they are limited by size of the fl exible forceps, which in turn depends on the diameter of the working channel. the fl exible forceps also lacks mechanical strength in obtaining pleural specimens of suffi cient depth, which can be overcome by the use of insulated tip (it) diathermic knife. full thickness parietal pleural biopsies are obtained with it knife, and the electrocautery knife is particularly useful when smooth thickened lesions are encountered, of which nearly half are due to mesothelioma. to improve analgesia before talc poudrage, lidocaine can be administered to the parietal pleura via spray catheter inserted through the working channel of the pleuroscope. similarly talc poudrage can be administered under visualization using the spray catheter. with the introduction of the fl ex-rigid pleuroscope, similar in design and handling to the fl exible bronchoscope, and compatible with standard light source and video processor available in most bronchoscopy suites, pleuroscopy will enjoy an expanded interest as more practitioners acquire the skill. the fl exrigid pleuroscope is a signifi cant invention in the history of minimally invasive pleural procedures and will revolutionize the practice of pulmonary medicine by replacing conventional biopsy methods in future. the common known causes of interstitial lung disease (ild) are drug toxicities, environmental exposures and collagen vascular disease (cvd). among these causes, drug or environmental exposures can be excluded by medical history. however, cvd-related ild may often be confused with idiopathic interstitial pneumonia (iip) because the radiological and histological characteristics of cvd-related ild are often indistinguishable from those of their idiopathic counterparts and occasionally, systemic manifestations of the underlying cvd develop several months or years after the diagnosis of ild. early diagnosis of occult cvd is very important in patients presenting with ild, because there are signifi cant differences in prognosis between the iip and cvd-ild groups. patients with cvd-ild survive longer than those with iip. additionally, different treatment regimens and evaluation for additional systemic involvement or malignancy may be needed in patients with cvd-ild. although, cvd-ild and iip is often considered indistinguishable, there are some clues that can help clinicians detect occult cvd in patients presenting with ild. first, a thorough medical history and physical examination can detect occult cvd. its importance cannot be overemphasized. the cvds frequently associated with ild are scleroderma, rheumatoid arthritis (ra), polymyositis/dermatomyositis (pm/dm), sjögren's syndrome, mixed connective tissue disease (mctd), undifferentiated connective tissue disease (uctd) and systemic lupus erythematosus (sle). therefore, symptoms and signs that occur frequently in these cvds should be searched for. these symptoms and signs include raynaud's phenomenon, gastro-esophageal refl ux disease, telangiectasis, dry eyes, dry mouth, arthritis, the characteristic skin lesions of dm (heliotrope rash, gottron's papule, mechanic's hand) and various serositis etc. second, certain fi ndings on hrct can help in the diagnosis of cvd. although the parenchymal abnormalities are similar to their idiopathic counterparts, the presence of airway-related abnormalities -mosaic attenuation, bronchial wall thickening, and nodules -are more common in cvd-ild. the presence of extrapulmonary abnormalities may also provide important clues to the underlying diagnosis. patients with cvd more frequently have pleural and pericardial effusions, pericardial thickening, enlarged pulmonary artery and esophageal dilatation. hrct can also show joint abnormalities or soft tissue calcifi cations. third, there are some serologic tests that can help in the diagnosis of cvd even in patients with obscure symptoms. high titers of antinuclear antibody and rheumatoid factor are often found in patients with cvd. other more disease specifi c tests currently available are anti-ssa/ssb antibody for primary sjögren's syndrome, anti-scl- antibody for systemic sclerosis, anti-jo- antibody for pm/dm, anti-u ribonucleoprotein (rnp) antibody for mctd, antibody to cyclic citrullinated peptides (ccp) for rheumatoid arthritis and so on. fourth, the frequent pathologic patterns of ild associated with cvd are nsip, uip, op, lip and dad. among them, nsip is the most frequent pathologic pattern in cvd-ild. therefore, pathologic pattern consistent with nsip should raise suspicions about the possibility of cvd. other pathologic fi ndings that may be suggestive of an underlying cvd include follicular bronchiolitis and lymphoid follicles. however, it is still impossible to diagnosis all occult cvds at the outset of ild because the initial clinical presentations can be essentially indistinguishable from those of iip. therefore, close follow up for a developing cvd is very important especially in patients with nsip. the role of pathological diagnosis for non-neoplastic lung disease is important and critical. however, agreement of pathological diagnosis in iips may not be that high. despite the expectations after publication of ats/ers classifi cation of idiopathic interstitial pneumonias (iips), interobserver variability in the pathological diagnosis of iips is still problematic. there are several major reasons for the poor agreement in pathological diagnosis of iips in which the biggest reason is a lack of specifi c and diagnostic fi nding to any type of iips. in the session, i would fi rst share the virtual steps of making diagnosis on surgical lung biopsy with audience, indicate recent data of inter-observer agreement in iips cases, and then, introduce factors behind the poor agreements followed by several possible solutions to this important issue. children's interstitial lung disease (child) differs from adult interstitial lung disease in that certain classic idiopathic pneumonias described in adults are not seen in children and unique forms of interstitial lung disease are found in infants and young children but not in adults. the most common form of idiopathic interstitial pneumonia in adults is idiopathic pulmonary fi brosis (ipf), also known as cryptogenic fi brosing alveolitis (cfa), a progressive and fatal disorder, defi ned pathologically as usual interstitial pneumonia (uip). uip is characterized by a heterogeneous mixture of normal lung, mild infl ammation, and fi brosis and the presence of fi broblastic foci, felt to be the leading edge of fi brosis. previously, although many infants and children were given the diagnosis of ipf, cfa, or uip, they did not have the characteristic fi broblastic foci. thus although the uip pattern is occasionally seen in the context of another primary disorder, such as abca mutations, true ipf/uip does not exist in children. the tendency to use the term ipf in children merely serves to obscure the real diagnosis and creates anxiety in families whose affected children may not actually have a fatal disorder. unique conditions have been described mainly in infants and young children that do not occur in adults. these include growth abnormalities, inborn errors of surfactant metabolism, neuroendocrine cell hyperplasia of infancy (nehi), and pulmonary interstitial glycogenosis (pig). growth abnormalities occur as a consequence of an early insult to the lung that results in retarded or arrested lung development and alveolar simplifi cation. risk factors associated with growth abnormalities include prematurity, congenital heart disease, and chromosomal defects, most commonly down syndrome. the major advance in child has been the discovery of genetic mutations that lead to surfactant dysfunction. these include mutations in the sp-b, sp-c, abca , ttf- , and gm-csfra genes. clinical presentation can vary from severe respiratory failure at birth leading to death (sp-b, abca mutations) to more insidious onset with chronic lung disease (sp-c, abca , ttf- , gm-csfra mutations). nehi is a chronic benign form of child presenting in the fi rst year of life with tachypnea, crackles, hypoxemia, characteristic features of symmetric ground glass densities in the right middle lobe and lingula and central lung regions on hrct, and a mixed restrictive/obstructive pattern on infant lung function testing. lung biopsy shows increased numbers of neuroendocrine cells and neuroepithelial bodies in the distal airways with otherwise normal lung architecture. pig is another benign form of child seen in infants and characterized by interstitial widening with glycogen-rich interstitial cells. pig is seen as a primary disorder ("pure" pig) and as a patchy disorder seen in the background of some other primary disorder, such as a growth abnormality ("patchy" pig). in conclusion, it is important to recognize the differences between pediatric and adult interstitial lung disease so that the proper diagnosis and prognosis can be given and the appropriate treatment applied. journal compilation © asian pacifi c society of respirology not to treat acute bronchitis with initial antibiotics, with the following exceptions. those at high risk of serious complications because of preexisting co-morbidity, patients over years of age with acute cough and two or more of the following, or patients over years of age with one or more of the following; ( ) admission to hospital in the previous year ( ) type or type diabetes ( ) history of congestive heart failure ( ) current use of oral glucocorticoids. clinicians need to address patients' concerns, perspectives, and expectations about the treatment and explain to patients that antibiotics are not necessary for a self-limiting respiratory tract infection. physicians should tell patients that antibiotic use increases the risk of an antibiotic resistant infection. and physicians also need to spend time answering questions and offer a contingency plan if symptoms worsen, and advise patients to return for a consultation if symptoms are not starting to settle in accordance with the expected course of the illness or if symptoms worsen signifi cantly. some physicians are certain that patients will benefi t from antibiotics and prescribe for expectation of fast relief. they are mostly comfortable with their prescribing decisions by their clinical experiences. taiwan's study demonstrates substantial variations among physician groups in the practice of prescribing antibiotics for viral respiratory infections. older physicians and those practicing in clinics rather than medical centers were signifi cantly more likely to prescribe antibiotics, and dispensing doctors in contrast to those without dispensing privileges or on-site pharmacists were signifi cantly highly prescribing antibiotics. statistical data from nhic in korea showed that general physicians in clinics prescribe antibiotics in % of acute bronchitis patients, while doctors at tertiary hospitals showed less but still fairly high rate of %. efforts and interventions to reduce the potentially inappropriate prescription of antibiotics should target modifi able factors. quality improvement (qi) strategies like using active clinician education, delayed prescriptions and targeting management, may yield reductions in antibiotic use. anti-tussives are occasionally useful and can be offered for short-term symptomatic relief of coughing. a meta-analysis and systematic review found that beta- -agonists were not effective for the treatment of acute bronchitis or cough of < weeks duration in children or in adults unless airfl ow obstruction was present. summary acute bronchitis is one of most commonly diagnosed and treated diseases in daily clinical practice. however, since it is mostly a self limiting disease, the standardization of diagnosis and treatment has long been neglected leaving various controversies in the management, particularly the use of antibiotics. the inappropriate prescription of antibiotics for acute bronchitis will surely lead to the emergence of resistant organisms in the community let alone the increase of socio-economic burden. further attention and research is needed for the reasonable approach to the treatment of acute bronchitis in order to prevent overuse of antibiotics and improve health-economy. prevalence acute bronchitis is one of the most common conditions encountered in clinical practice, accounted for approximately million visits to korean physicians in , and consistently ranks among the top reasons for ambulatory visits in the united states. defi nition acute bronchitis refers to a clinical syndrome distinguished by a relatively brief, self-limited infl ammatory process of large and midsized airways that is manifested predominantly by cough with or without phlegm production which lasts for up to weeks and absence of fi ndings suggestive of pneumonia. acute bronchitis should be distinguished from acute exacerbations of chronic bronchitis and acute infl ammation of the small airways -asthma or bronchiolitis. those with underlying lung disease, congestive heart failure, or a compromised immune system are considered to be at high risk for complications of acute bronchitis. etiology acute bronchitis is one of the most common causes of antibiotic abuse. in healthy communities, there is little evidence of bacterial infection in people with bronchitis, but there are few practical studies to distinguish between bacterial and viral bronchitis. within this context, the use of antibiotics to treat acute bronchitis is controversial but common in real practice. viruses are usually considered the most common cause of acute bronchitis but have been isolated in a minority of patients. those isolated in acute bronchitis include, in order of frequency of occurrence, are infl uenza, parainfl uenza, respiratory syncitial virus (rsv), coronavirus, adenovirus, and rhinovirus. the yield of specifi c pathogens varies according to several factors, including the presence or absence of an epidemic, the season of the year, and the infl uenza vaccination status of the population. bacterial pathogens are thought to play a very minimal role in acute bronchitis. the bacteria that have been causally linked to acute bronchitis in otherwise healthy individuals include only mycoplasma pneumoniae, chlamydophila pneumoniae and bordetella pertussis. antibiotic treatment of patients with pertussis is indicated to limit transmission, but there are no compelling data to support the prospect that cough will be less severe or less prolonged with antibiotic therapy. clinical manifestations acute bronchitis cannot be distinguished from upper respiratory infections in the fi rst few days. acute bronchitis is suggested by the persistence of cough for more than fi ve days, and most often lasts from to days. approximately % of patients with acute bronchitis report the production of purulent sputum. it usually represents sloughing of cells from the tracheobronchial epithelium, along with infl ammatory cells, and does not signify bacterial infection. pulmonary function test fi ndings consistent with bronchial hyperresponsiveness are common. fev less than % at the initial visit was present in % of adults from the mid western united states with no history of underlying lung disease. pft abnormalities are usually transient, typically resolving after to weeks, although they may last as long as months. fever is a relatively unusual sign in acute bronchitis and, when accompanying cough, suggests either infl uenza or pneumonia. diagnosis acute bronchitis is established in a patient who has the sudden onset of cough, with or without sputum expectoration, and without evidence of pneumonia, the common cold, acute asthma, or an acute exacerbation of chronic bronchitis. the absence of the following fi ndings reduces the likelihood of pneumonia suffi ciently to eliminate the need for a chest radiograph: ( ) heart rate > beats/min; ( ) respiratory rate > breaths/min; ( ) oral body temperature of > °c; and ( ) chest examination fi ndings of focal consolidation, egophony, or fremitus. chest radiography should be reserved for use in patients with any of these fi ndings or cough lasting > weeks. an exception, however, is a cough in elderly patients; pneumonia in elderly patients is often characterized by an absence of distinctive signs and symptoms. rapid diagnostic tests exist for several pathogens currently linked to acute bronchitis. patients with severe paroxysmal cough, with or without post-tussive vomiting should be evaluated for pertussis regardless of the immunization history. rapid tests should be used primarily when the suspected organism is treatable, the infection is known to be circulating in the community, and the patient has suggestive symptoms or signs. treatment statistical data from korea national health insurance corporation (nhic) shows that approximately - % of patients with acute bronchitis receive antibiotics despite the evidence that, with few exceptions, they are ineffective. meta-analyses of randomized, controlled trials all concluded that routine antibiotic treatment is not justifi ed. the decision not to use an antibiotic should be addressed individually and explanations should be offered because many patients expect to receive an antibiotic based on previous experiences and public expectations. main challenges for appropriate antibiotic use in acute bronchitis are the diagnosis is based on clinical fi ndings, without standardized diagnostic methods and sensitive or specifi c confi rmatory laboratory tests. how to identify accurately the few patients who are seriously ill or whose symptoms could be meaningfully ameliorated by prompt antibiotic treatment is not standardized. recent studies have suggested that the annual decline in fev is greater in gold stage ii than in later stages of the disease. ( ) if the decline in pulmonary function predominantly occurs early in the course of the disease, then it is logical that diagnosis and intervention aimed at reducing the progression of the disease should mainly occur in the early stages of the disease. severity of copd at diagnosis differs enormously on where it is done: population based studies, screening or hospital patients. epidemiologic studies: prevalence, underdiagnosis and severity there are increasingly more data on the prevalence and distribution of copd from around the world. the prevalence of chronic obstructive pulmonary disease (copd) varies from country to country, mainly due to the effects of cumulative exposure to smoking and the increased life span of the population. ( ) ( ) ( ) ( ) systematic review of epidemiological studies concluded that the prevalence of copd, in adults aged years and more, worldwide ranges around - %. ( ) these differences may be related, at least in part, to differences in genetic background, smoking habits and exposure to other environmental risk factors, and are accompanied by differences in diagnostic rates and in management of the disease around the world. there is a large underdiagnosis of copd with about only one out of three or four of all subjects fulfi lling diagnostic criteria of copd identifi ed by the health care system. ( ) ( ) ( ) copd in general population is diagnosed at earlier stages. data from latinamerica (platino), similar from those from spain, showed severity was distributed as follows: stage i, % and stage ii, . %. screening for copd screening, combined with smoking cessation advice, help motivated smokers to attempt quitting smoking. ( ) in general practice, when individuals were preselected on the basis of smoking age and respiratory symptoms chronic cough was a better predictor of airfl ow obstruction than other symptoms, such as wheeze and dyspnoea. age was also a good predictor of obstruction; smokers over with cough had a % chance of having an obstruction. ( ) diagnosis at the hospital the decrease in lung function is gradual. the disease is usually diagnosed late because patients may adapt to the condition or doctors may not notice the symptoms. by then, the patient is diagnosed at the hospital, when lung function is often poor, sometimes less than % of normal. the relationship between lung disease and increased body weight can take two forms: the effects of increased body weight on the normal respiratory system and the association of increased body weight with diseases of the respiratory system. obesity (body mass index, bmi, greater than kg/m ) can reduce normal static lung volumes (principally functional residual capacity, but also total lung capacity and residual volume at very high bmi), ventilation (particularly during sleep and exercise) and gas exchange (increased gas transfer). the epidemic of overweight and obesity has been associated with the increased prevalence of asthma through proposed mechanisms such as dietary effects, reduced lung volumes and lung recoil affecting airway responses to breathing, a general infl ammatory effect and through associated sedentary lifestyle. however more recent epidemiological data suggests the association between asthma and obesity is not as close as once thought. overweight and obesity clearly are associated with the high prevalence of obstructive sleep apnoea with increased body weight increasing the likelihood of upper airway collapse on a background of reduced upper airway dimensions and snoring and reducing minute ventilation in patients with obstructive sleep apnoea, predisposing to hypoventilation and chronic hypercapnoeic respiratory failure. the increased load to breathing also affects breathing, especially during sleep, in patients with respiratory muscle weakness or abnormal chest wall mechanics due to kyphoscoliosis or previous chest wall surgery such as thoracoplasty. increased body weight will also exacerbate the effects on symptoms of existing chronic respiratory diseases including asthma, chronic obstructive pulmonary disease and interstitial lung diseases. avoidance of weight gain and continued efforts at weight loss remain an important goal in patients with respiratory disease. this presentation will be a review of recent fi ndings and implications from imaging studies in asthma. the use of -dimensional imaging in asthma has provided useful insights into the understanding of pathophysiology of disease, which may have implications on how asthma is treated. small airways disease plays a major role in asthma and has traditionally been diffi cult to probe. however, the combined use of new imaging methods combined with complex lung function has provided useful insights into pathophysiology. findings and implications from hrct, pet, spect and mri will be discussed. the assessment of pulmonary function has changed very little over the past to years. all techniques performed in the laboratory still view the lung as a very simple single compartment unit. measures of volume and fl ow only assess disease in the medium to large airways whilst the small airways receive relatively little attention. however there are a number of emerging techniques on the horizon that have the potential to give great insight in to the periphery of the lung where most respiratory disease emanates. the measurement of mechanics using the forced oscillation technique and gas mixing using the multiple breath nitrogen washout test are now emerging as very powerful tools for assessment of peripheral lung function. in this presentation we will be discussing the state of the art in terms of assessing pulmonary function and what may we expect to see in the future. copd is a major public health problem in asia. copd prevalence was . % ( . % in males and . % in females) in china (> yrs), . % in japan (> yrs). in most of the developing countries in asia, copd is always underdiagnosed by physicians owning to lack of routine spirometry test and only based on symptomatic diagnosis. smoking is the most important risk factor contributing to development of copd. however, copd prevalence was . % in chinese non-smokers (> yrs) and . % in korea (> yrs), similar to those in mexico city ( . %) and caracas ( . %). in china, non-smokers accounted for . % of copd patients compared with . % in usa and . % in the uk. exposure to environmental tobacco smoke (passive smoking) and indoor air pollution (particularly the coal and biomass combination) may contribute to the higher prevalence of copd in developing countries. to reduce the risk factors (smoking, coal or biomass fuel for cooking, indoor and outdoor air pollution) are the priority for reducing the prevalence in the asian developing countries. government in some countries had made some effect in tobacco control and reducing air pollution. unlike hypertension or diabetes, the management of copd is only based on symptomatic treatment, owing to lack of specifi c biomarkers. annual lung function test with spirometer is the only parameter in detecting early stage of copd. data showed that more reversibility of fev was demonstrated in stages i-ii copd patients with ics/laba or tiotropine administration, as compared with those in stages iii-iv. an intervention study at the community level has shown that early intervention (improvement of indoor ventilation, bronchodilators) was able to reverse rapid annual fev, decline in copd patients. more affordable medication should be developed in the low income countries. data showed that oral administration of carbocysteine (thio compounds) reduced exacerbation rate by . %, which was consistent with inhaled administration of fluticasone/formoterol (seretide) or tiotropine. in addition, orally administered low dose teophylline ( mg, bid) improved pre-bronchodilator fev, and reduced exacerbation rate. there was a synergistic effi cacy in fev , with the combination of teophylline and inhaled corticosteroids. chronic obstructive pulmonary disease (copd) is characterized by the presence of airfl ow limitation due to loss of lung elasticity and/or airway narrowing. the pathological hallmark of loss of lung elasticity is emphysema and airway wall remodeling contributes to the airway narrowing. using computed tomography (ct) these lesions can be assessed by measuring low attenuation areas (laa) and airway wall thickness/luminal area, respectively. recently, copd has been widely recognized as a systemic infl ammatory disease, and body weight loss is one of its clinical features. traditionally, the patients who had copd with predominant emphysema and a low body mass index (bmi) were called "pink-puffers". however, the relationship between body weight loss and emphysema had not been assessed. based on these back ground, we have evaluated the body composition, emphysema and airway dimensions in copd patients using ct images. bmi was signifi cantly lower in the emphysema dominant phenotype compared to the airway dominant phenotype. furthermore, bmi correlated with laa% (ρ = − . , p < . ) but not with wa%. chest subcutaneous fat mass was also correlated with laa% (ρ = − . , p < . ). these data indicated that the "pink-puffer hypothesis" is correct in some aspects. next, we postulated that reduced leptin and leptin receptor signaling could contribute the development of emphysema. serum leptin was correlated with bmi which was correlated with dl co /v a . the expression of leptin and leptin receptor was evaluated pcr in human lung tissues. both genes were detected in the lung tissues, but the expression of leptin gene was low. the leptin receptor gene expression was signifi cantly lower in copd patients and it was signifi cantly correlated with dl co /v a . the leptin receptor gene expression in the lung did not correlated with body weight. these data suggested that the patients' physique can be associated with the relative contribution of emphysematous lesions in copd and leptin and leptin receptor system might affect the mechanism of developing emphysema. nutritional support has been one of possible clinical approaches as a copd therapy these days. however, its effect is still controversial. to clarify the relationship between low bmi and emphysema and to classify the phenotypes of copd based on the patients' physique may help to fi ne the defi nite targets for nutritional support even in the early stage of copd. journal compilation © asian pacifi c society of respirology sy - at present copd is often only treated in gold stage iii or iv . it is without question possible to diagnose copd earlier. if spirometry would be more readily performed in general practice, copd could be diagnosed in gold stage i and ii, as is evident from the practice of "spirometry days" organized by the belgian thoracic society in which the majority of the patients were diagnosed in gold stage ii . whether gold stage i and ii truly represent the "early" stages of the disease may be debated , but this defi nition of early copd could certainly be used as an operational defi nition. it is clear now that spirometry is required for early diagnosis of copd . although at present there is no irrefutable evidence that early treatment of copd is warranted, there is accumulating suggestive evidence that early treatment of copd may result in better outcomes. this evidence is primarily related to secondary analyses of the torch and uplift , studies. first, it was demonstrated in a secondary analysis of the torch study, that inhaled corticosteroids, long-acting betaagonists and their fi xed combination reduced the rate of decline of fev by , and ml, respectively . if treatment indeed reduces the progression of the disease, then an easy case for early treatment is made. in addition, a subgroup analysis demonstrated that except for the effect on the sgrq (st. george's respiratory questionnaire) all other treatment effects were numerically larger in gold stage ii than in gold stage iii and iv . a subgroup analysis of the uplift study demonstrated numerically greater treatment effects in gold stage ii as well. in addition, tiotropium reduced the rate of decline of fev in these patients (albeit only by ml/year), which was not the case in the later gold stages . finally, in patients not taking any medication at the onset of the study (maintenance naïve patients) tiotropium substantially reduced the rate of decline of fev and the rate of deterioration of the sgrqscore . taken together these data demonstrated that: ) large treatment effects were obtained in early disease; ) indications of disease modifi cation were present in early disease. hence, they support early treatment of copd. pulmonary rehabilitation is now the standard of care for patients with chronic obstructive pulmonary disease (copd) who remain symptomatic despite bronchodilator therapies. combining the best of interprofessional, personalized and evidence-informed care, pulmonary rehabilitation allows clinicians and their patients to realize signifi cant benefi ts in a variety of important patientcentered copd outcomes. the fundamental elements required for an effective pulmonary rehabilitation program will be discussed, and the scientifi c evidence supporting their effectiveness will be summarized. issues relating to optimal site of delivery, components of effective rehabilitation programming, duration of rehabilitation, timing of rehabilitation and target populations will be reviewed. recent developments in this rapidly expanding area will also be highlighted. lastly, methods to establish or enhance an existing pulmonary rehabilitation program will be discussed, with the goal of fully realizing the many substantive benefi ts of pulmonary rehabilitation in copd. however, only very low gene transfer seen after a second dose with either day and day spacing. we attribute this to rapid upregulation of neutralizing antibodies against adenovirus. anti-tumor humoral immune responses were seen almost all patients with reactions seen against known meso tumor antigens (sv large t antigen and mesothelin) and against unknown proteins in cell lysates. given the caveats of phase trials (small numbers, different doses, heavily pretreated patients), we still saw clinical responses (defi ned as prolonged stable disease, prolonged survival, partial or complete responses by modifi ed resist criteria, decreased metabolic tumor activity by pet scanning, or "mixed" responses) in about / of the patients. we are currently administering two doses spaced only three days apart. this appears to be well tolerated. based on strong preclinical data supporting the combination of gene therapy and chemotherapy, we have started a trial using ad.inf instillation into the fi rst treatment cycle of fi rst line (cisplatin/pemetrexed) or second line chemotherapy (gemcitabine). our groups is also generating "designer chimeric t cells" in which a single chain antibody fragment is linked to the transmembrane and cytoplasmic regions of the t-cell receptor. this artifi cial t-cell receptor is then transduced into t-cells that are then reinfused. the t-cells are then activated by cells expressing mesothelin. preclinical data show striking activity against mesothelin-expressing tumors in mice. mesotheliomas make large amounts of the immunoinhibitory cytokine, transforming growth factor-beta (tgf-β). preclinical studies using tgf-β blockers have shown activity in mouse models of mesothelioma and a clinical trial using an anti-tgf-β antibody is now underway at the university of pennsylvania. in summary, gene-based and immunotherapies are being actively studied in mesothelioma and have shown some promising results. future trials are focusing on combining these approaches with chemotherapy and surgery. given the relatively mild and non-overlapping toxicities, we believe these, or other gene therapy and/or immunologic approaches will soon become part of the standard therapeutic armamentarium. the burden of asbestos-related diseases (ards), in particular mesothelioma, has been shouldered mostly by the developed countries of the west. this is a consequence of the heavy dependence on asbestos use up to around the s by those countries. in contrast, the majority of asian countries started to depend on asbestos since then and has not yet reached the suffi cient latency time for the related diseases to manifest. japan is an exception, because it heavily used asbestos during the period to catch-up with the west. japan has now become one of the global epicenters of ards. it is a tragic consequence of experiencing the burden of ards that many developed countries decided to move towards banning asbestos or a de facto non-dependence. in the asia-pacifi c, this group comprises australia, new zealand, japan, korea and singapore (the "forerunner" group in terms of ards). in contrast, the many other countries in asia still use asbestos at substantial levels, turning asia into the world's center of asbestos consumption today. however, as these countries start to use up the latency time, and manage to improve medical recognition, reporting and recording, ards will soon emerge as a major public health issue in the region. early indications of this forecast do exist. not only should lessons be learnt from the experiences incurred by the "forerunner" group of countries, but more importantly, they should crystallize in international collaboration involving national administrators, academia, ngos and international organizations, for the effective recognition and countermeasures of ards. i will also refer to the progress made and hurdles encountered by the asian asbestos initiative and the who global plan of action on the elimination of asbestos-related diseases. at the international level, ards present a domino-effect that needs to be coped with. sy - interstitial pneumonia (ip) can be classifi ed into two groups in terms of known causes. pneumoconiosis, drug-induced pneumonitis, radiation-induced pneumonitis, and hypersensitivity pneunonitis (hp) are categorized as ip with known causes. on the other hand, idiopathic interstitial pneumonias (iips), which include idiopathic pulmonary fi brosis (ipf), nonspecifi c interstitial pneumonia (nsip), cryptogenic organizing pneumonia (cop), and desquamative interstitial pneumonia (dip), have no known causes. most physicians tend to diagnose of ip patients as iips without intensive examinations. however, some environmental and occupational lung diseases, especially asbestosis and chronic hp, should carefully be differentiated from iips. asbestosis is one type of pneumoconiosis, which is induced by asbestos exposure with a latent period of usually more than years. bilateral fi ne crackles can be frequently auscultated and pulmonary function test shows restrictive and diffusing impairments. chest hrct shows subpleural dot, subpleural curvilinear shadow, ground glass opacity, interlobular septal thickening, traction bronchiectasis, and honeycombing. in our case series of asbestosis (n = ) in yokosuka, a town of shipyard for more than years, honeycombing was seen in cases ( %). chronic hp is an allergic disease induced by long-term exposure to antigens. major causative antigens are avian dropping and feather, mold, and bacteria. chest hrct tends to show traction bronchiectasis and honeycombing in advanced stages, which are similar to ipf. in surgical lung biopsy, most cases are classifi ed as nsip-like and uip-like patterns. to clarify the importance of unrecognized exposure to avian antigen, we precisely reviewed patients with bird-related chronic hp. in the patients, patients were bird-breeders with direct exposure to avian antigen by contacting their own birds, whereas patients seemed to be exposed to wild birds, neighbor's birds, feather duvets, stuffed bird, and fertilizer with chicken droppings without recognition of avian contact. number of patients is very limited both in asbestosis and chronic hp, which suggests that there is a small group of subjects who are susceptible to develop pulmonary fi brosis after exposure to asbestos or antigens. however, genetic background of susceptibility to pulmonary fi brosis has not elucidated. in our case series of chronic hp (n = ), cases ( %) had the fi rst-degree family members with ip, who might have common environmental and/or genetic factors. further studies are needed to determine host susceptibility to pulmonary fi brosis, which might contribute to clarify the pathogenesis of ip. long acting beta- agonists has been in the doctors' armory of asthma medications for about years (available in in uk and in usa). there is little doubt that laba when combined with inhaled corticosteroids can improve asthma symptoms for a subsection of people with asthma, and is generally superior to add-on leukotriene receptor antagonist. indeed addition of laba to inhaled corticosteroids is in all major asthma guidelines as a step-up therapy. however, after the salmeterol multicentre asthma research trial was prematurely halted, a focus on effi cacy and safety of the wide use of laba severe pulmonary arterial hypertension (pah) is a fatal condition associated with complex pathobiology. vasoconstriction, thrombosis, and remodeling of the pulmonary vessel wall contribute to increased pulmonary vascular resistance in pah. the pathology of pulmonary hypertension can be classifi ed into endothelial, smooth muscle, and/or adventitial lesions, although not all compartments of the pulmonary artery wall are involved in each case of severe pah. the classic lesion of severe pah is the plexiform lesion, an abnormal proliferation of predominantly endothelial cells. smooth muscle thickening can be seen in all forms of the disease but is not a constant feature in the idiopathic pulmonary arterial hypertension. the adventitia is often markedly remodeled in patients with certain forms of collagen vascular diseases associated with severe pah, most notably scleroderma. the obligatory lining of pulmonary arteries with a monolayer of endothelial cells is disrupted in severe pah. the three-dimensional vascular pattern is rather suggestive of an intravascular tumor-like proliferation (tumorlet), instead of a retracted scar if this lesion would represent an abnormal healing to an injury to the vascular wall. the evidence of a tumorlike endothelial cell proliferation was provided by the demonstration that plexiform lesions in patients with idiopathic pah were preferentially monoclonal, whereas similar lesions in lung of patients with secondary pah due to congenital heart malformations were polyclonal. monoclonality was also observed in plexiform lesions of patients from anorexigen-induded pah. vasoconstriction has been related to abnormal potassium channels and to endothelial dysfunction. endothelial dysfunction leads to impaired production of vasodilators such as nitric oxide and prostacyclin along with overexpression of vasoconstrictors such as endothelin (et)- . many of these abnormalities not only elevate vascular tone and promote vascular remodeling but also represent logical pharmacological targets. recent genetic and pathophysiologic studies have emphasized the relevance of several mediators in this condition, including nitric oxide, prostacyclin, et- , serotonin, angiopoietin- , and members of the transforming-growth-factor-beta superfamily. disordered proteolysis of the extracellular matrix is also evident in pah. the unraveling of the pathobiology of severe pah may lead us to novel therapies and approaches to better treat the disease. unfractionated heparin (ufh) and low-molecular-weight-heparin (lmwh), acted by enhancing the ability of antithrombin (at) to inhibit coagulation proteases, have been used as initial anticoagulant therapy for vte. they are delivered intravenously or by subcutaneous injection. subcutaneous fondaparinux, a synthetic pentasaccharide with specifi c anti-factor xa activity, is also recommended as the initial treatment for vte according to recent guideline. oral vitamin k antagonists, acting by reducing the activity of several coagulation proteases, are used for long-term anticoagulation. the major disadvantage of vitamin k antagonists is the need for frequent coagulation monitoring to maintain a therapeutic level. new anticoagulants, including oral direct thrombin inhibitors, such as dabigatran, and oral anti-factor xa inhibitors, such as rivaroxaban and apixaban, are emerging in recent clinical trials. these new drugs may replace heparins and vitamin k antagonists, which are expected to have a huge impact on the treatment of vte in the near future. thrombolytic therapy should immediately be used in patients with massive (high risk) pte. the effect of thrombolysis in patients with submissive (intermediate risk) pte remains controversial. further stratifi cation of these patients is necessary. patients with multiple poor prognostic indicators such as right heart dysfunction, thrombolytic therapy should be considered. several countries have started to support activities raising public awareness of vte, with the goal to decrease mortality and morbidity. in us, national institutes of health (nih) and centres for disease control (cdc) have fostered thrombosis activities to improve the prevention of vte and its long-term complications. in the united kingdom, a thrombosis group has been formed to promote awareness among parliamentarians about the risk and management of vte; to increase knowledge of its causes, effects, and treatments; and to monitor the implementation of government initiatives and other researches being and this program has corrected the wrong perception that pte is a rare disease in china pulmonary hypertension (ph) is a common complication of chronic respiratory diseases, such as chronic obstructive pulmonary disease (copd) or interstitial lung diseases (ild). ph associated with respiratory diseases is classifi ed as diagnostic group iii according to the current clinical classifi cation of dana point . it is suggested that the pulmonary vascular abnormalities originate at an early stage of the diseases. the functional (hypoxic vasoconstriction) and morphological factors (vascular remodeling, destruction of the pulmonary parenchyma) explain the elevation of pulmonary vascular resistance that leads to ph. the ph is mild to moderate in copd with mean pulmonary artery pressure (mpap) usually ranging between and mmhg, however, worsening during exercise and exacerbations. a small proportion of copd patients may exhibit severe ph defi ned by a resting mpap > to mmhg, whose prognosis is particularly poor. ph is relatively frequent in advanced ild, particularly in idiopathic pulmonary fi brosis, which predicts a poor prognosis. the diagnosis of ph is suggested by doppler echocardiography, but the confi rmation still requires right heart catheterization. the potential treatments of ph associated with respiratory diseases are as follows: ) treat underlying lung disease; ) provide supplemental oxygen therapy when appropriate; ) rehabilitation; ) treat right heart failure; ) consider vasomodulator therapy; and ) consider lung transplantation when indicated. journal compilation © asian pacifi c society of respirology sy - tobacco use is the most common preventable cause of death. about half of the people who don't quit smoking will die of smoking-related problems. the epidemic varies among countries and is increasingly affecting developing countries, where most of the world's smokers ( %) live. close to half of all men in low income countries smoke daily and this has been increasing. legislation the framework convention on tobacco control (fctc) was adopted by who member countries in may to commit all countries to protect nonsmokers from tobacco smoke in public places, to eliminate all tobacco advertising, promotion and sponsorship, to require warning labels of cigarette packs and to prohibit misleading tobacco product descriptors such as "light" and "mild". even though many countries have passed legislation mandating smoke-free environments and the total global population covered by comprehensive smoke-free laws increased from . % to . % in one year, the overwhelming majority of countries still have no smoke-free laws, very limited laws, or ineffective enforcement. compliance with smoke-free laws is low. treatment to aid smoking cessation support for smoking cessation or "treatment of tobacco dependence" refers to a range of techniques including motivation, advice and guidance, counselling and appropriate pharmaceutical aids, all of which aim to encourage and help tobacco users to stop using tobacco and to avoid subsequent relapse. the success of these interventions depends on their synergistic use in the context of a comprehensive country tobacco-control strategy. in many countries, provision for treatment, training of health-care providers, education and information on the wide use of cessation is scarce. therapies, as well as fi nancial resources are limited and rarely incorporated into standard health care. also, smoking cessation is not seen as a public health priority and is not necessarily approached as a key tobacco-control strategy. smoking cessation services are most effective when they are part of a coordinated tobacco control programme. brief cessation counselling is relatively inexpensive when integrated into existing primary health-care services, is usually well received by patients, and is most effective when it includes clear, strong and personalized advice to quit. communication technologies -such as telephone quitlines, text messaging, interactive telephony, and online counselling -as well as psychological and behavioural modifi cation therapies, offer important support. cessation prescription medicines, available in many countries like nicotine replacement therapies, bupropion and varenicline can double the likelihood that someone will successfully quit. bronchiectasis is an old disease that we all treat, but surprisingly little about this disease has been well studied. the defi nition, diagnosis, natural history, pathogenesis and treatment are all uncertain. this talk examines these points in the light of new information about biofi lms and "normal" bacteria. it asks more questions than it answers, but the questions raise thoughts on whether our usual approaches are the best. the mediastinum is generally split into three compartments strictly for the purpose of classifi cation of the most likely abnormality in the individual compartment. there are no anatomical boundaries or fascial planes that divide one compartment from the other. classically, the compartments have been classifi ed as anterior, middle, and posterior. a recent change in classifi cation has used the categories of anterior, middle-posterior, and paravertebral compartments. approximately % of mediastinal tumors are located in the anterior compartment, % in the middle compartment, and % in the posterior compartment. asymptomatic masses are more likely to be benign than malignant, and symptomatic masses are more likely to be malignant than benign; however, there is a large variation the most common tumors in the anterior mediastinum consist of thymoma, lymphoma, germ cell tumors, and thyroid tumors. thymoma is by far the most common anterior mediastinal tumor and approximately / are encapsulated and non-invasive while / are invasive. the most common paraneoplastic syndromes associated with thymoma include myasthenia gravis, hypogammaglobulinemia, and pure red blood cell aplasia. benign teratomas occur in both male and females, while malignant germ cell tumors of the mediastinum are almost exclusively in males. lymphoma can occur most commonly in the anterior or middle mediastinum and may be associated with systemic symptoms and occasionally superior vena cava syndrome. the most common abnormalities in the middle mediastinum include lymphoma, granulomatous disease, and developmental cysts. bronchogenic cysts are almost always benign, although they can cause symptoms such as obstructive pneumonia and are generally treated with surgical resection. recent mediastinal compartment classifi cation has switched from posterior mediastinum to naming this the paravertebral compartment. it is located posterior to an imaginary line drawn to connect the anterior aspects of the vertebral bodies on the lateral chest radiograph. the most common tumors in this location are neurogenic tumors, meningoceles, or thoracic spine lesions. the most common neurogenic tumors in adults are neurilemomas, and they are almost always benign. neurofi bromas also occur in this compartment. they are frequently benign, but may be malignant, especially in patients with neurofi bromatosis. in these individuals, malignant tumors of the nerve sheath origin are more common. ganglioneuroma, ganglioneuroblastoma, and neuroblastoma are more common neurogenic tumors in children or adolescents.. cough is an important lung defense. in a refl ex manner, noxious agents are expelled from the airways as these are sensed by the receptors in the airway epithelium. in addition to appreciating its cleansing function, understanding cough refl ex is important in the diagnosis and treatment of common clinical conditions. most diseases associated with cough are transient. a problem arises when cough persists and becomes chronic. an in-depth search for the underlying pathology is warranted since treatment directed to the cause of the cough is curative in over % of cases. an anatomic-diagnostic protocol ensures a systematic search for cough etiology but unfortunately, this approach can be lengthy and in many settings, impractical. thus an empiric syndromic approach is now recommended. this paradigm shift is borne by the following premises: ( ) the current awareness of the relative frequency of the disorders (alone or in combination) that can cause cough; ( ) the sensitivity and specifi city of many (but not all) diagnostic tests in predicting the cause of cough has been established; ( ) a sequential evaluation and treatment for the common causes of cough using a combination of selected diagnostic tests and empiric therapy has been proven effective; and ( ) a sequential and additive therapy is often crucial because more than one cause of cough is frequently present. a recent study by dr. aileen wang on "the management of chronic cough in a tertiary center: an asian perspective" showed that even in a filipino immunocompetent population, the most common causes of chronic cough is asthma, post-nasal drip syndrome (pnds) and gastroesophageal refl ux disease (gerd). the study concluded that: ( ) the accp recommendations are generally applicable to an asian setting. research of viruses, their structure, pathogenicity and host-interactions has burgeoned. we now understand how viruses infect cells, how they replicate, how they interfere with host defences and how they interact with other tissue events and pathologies. rhinovirus infection causing the common cold is the most frequent and 'common' infection in humans. it is also implicated in most exacerbations of asthma and copd. the patho-biology of rhinovirus will be used to illustrate virus pathogenicity, virus-host-interactions and to highlight potential future therapeutic options. sy - much hope has been placed in the discovery of biomarkers to help understand the mechanisms of diseases such as copd, help stratify the disease better and guide treatment. it is also hoped that some of these could speed up drug discovery by serving as surrogate markers that respond to novel drugs within a shorter timescale than is the case with current drug trials in which the main outcome is the spirometric measurement of forced expiratory volume in one second (fev ). although studies in patients with copd have identifi ed several biomarkers, most of these need to be validated and their prognostic value is unclear. many of the markers have been identifi ed in blood and although it is recognised that there are non-pulmonary consequences of copd, some of which can be viewed as systemic biomarkers measured and/or generated in the lungs are likely to be most informative. there are several methods to identify and quantify biomarkers of copd and different biological samples (blood, bal, sputum and lung tissue) can be used to provide material for such analyses. whilst most studies to date used commercial immunoassays (elisa), there has been a keen interest in applying unbiased approaches such as proteomics. we have recently completed a large programme of work which applied -dimenshional electrophoresis and mass spectrometric analysis to identify biomarkers of copd. induced sputum was obtained from copd patients with a spectrum of disease severity and control subjects. two-dimensional gel electrophoresis and mass spectrometric identifi cation of differentially expressed proteins was fi rst applied to induced sputum from gold stage copd patients and healthy smoker control subjects. initial results thus obtained were validated by a combination of immunoassays (western blotting and elisa) applied to a large subject cohort. the biomarkers were localised to bronchial mucosa by immunohistochemistry. of individual protein spots identifi ed, were quantitatively and qualitatively different between the two groups. protein spots were subjected to tandem mass spectrometry, which identifi ed separate protein species. seven of these were further quantifi ed in induced sputum from individuals. using this sequential approach, two of these potential biomarkers (apolipoprotein a and lipocalin- ) were found to be signifi cantly reduced in copd patients when compared to healthy smokers. their levels correlated with fev /fvc, indicating their relationship to disease severity. in summary, a potential role for apolipoprotein a and lipocalin- in innate defence has been postulated previously; our discovery of their reduction in copd indicates a defi cient innate defence system in airways disease that could explain increased susceptibility to infectious exacerbations. persistent chronic infl ammation, repetitive tissue injury, and dysregulated epithelial repair leading to tissue remodeling and fi brosis are the hallmarks of chronic lung diseases, such as chronic obstructive pulmonary disease (copd), chronic asthma, pneumoconiosis, pulmonary fi brosis and sarcoidosis. the innate immunity system with its pattern recognition receptors are recently identifi ed to involve in the pathogenesis of these chronic lung diseases. the neutrophilic infi ltration of the airway mediated through t h and il- family may play an important role in steroid-resistant asthma and status asthmaticus. in addition, the epigenetic modifi cation of gene expressions and cellular senescence may modulate the progression of chronic asthma and copd. histone deacetylase (hdac ), which can be inactivated by oxidative stress or pi k-akt pathway, may regulate corticosteroid-related anti-infl ammatory response. manipulation of hdac activity is a new treatment direction in steroid-resistant asthma and copd. sirt , a nad + -dependent deacetylase, is an important signaling pathway related to cell survival, dna repair, and apoptosis. the sirt is decreased in alveolar macrophages of smokers and copd patients, and associated with pro-infl ammatory response through the activation of nf-κb. understanding the complexity of infl ammatory and epigenetic regulations in chronic lung diseases may potentiate the development of novel therapies. fibrosis is a common fi nding in chronic lung diseases, with tgf-β-related pathways play important roles. myofi broblasts are the principal effective cells in the fi brogenic process. they can be evolved from the activation of resident pulmonary fi broblasts, marrow-derived fi brocytes, or the trans-differentiation of pulmonary epithelial cells through epithelial mesenchymal transition (emt). further understandings on the emt process in lung tissue repair may help to elucidate the mechanism of lung remodeling and fi brosis. in addition, lung stem/progenitor cell study appears to be a new and potential fi eld in lung injury and repair. it is anticipated that more clear mechanisms behind lung remodeling and fi brosis can be identifi ed and new treatment modalities be developed accordingly. sy - cancers are genetic diseases with constitutional genomic variations that are present in normal cells contributing to an individual's risk of developing cancer such as lung cancer. furthermore, cancer cells acquire genetic mutations and genome wide changes in their dna as well as their epigenome compared to their normal cellular counterparts. some of these mutations have turned out to be important driver mutations and are associated with exquisite sensitivity to targetted cancer therapies. some of these genetic changes include egfr and eml -alk fusion gene mutations. epigenetic changes include methylation abnormalities as well as histone modifi cations, and possess the characteristic of potential reversibility which is attractive for the development of new therapies. the human genome project and rapid technological advances including deep dna sequencing have contributed signifi cantly to the ability to detect cancer specifi c genetic, genomic and epigenomic aberrations. these genetic and genomic abnormalities have promise across the translational spectrum from identifying individual susceptibility to cancers, early diagnosis markers, molecular pathology and tailoring of treatment (predictive biomarkers) as well as informing on outcome (prognostic biomarkers). much work remains to be done to validate clinical utility and translate these potential useful biomarkers into useful clinical tools. this session will review some of the developments in the genomics and epigenomics of lung cancer and mesothelioma. asthma is a disease that is diagnosed largely on a history of variable symptoms and the demonstration of variable airway narrowing. it is associated with airway infl ammation (cd t-lymphocytes, b-cell lymphoid aggregates, macrophages, eosinophils and, in adults, neutrophils) and airway wall remodelling (airway wall thickening, principally increased airway smooth muscle and deposition of collagen below the basement membrane, with minor encroachment on the airway lumen). it is not associated with a prominent effect on the lung parenchyma. the cause(s) of asthma remains unknown and the severity of disease remains largely constant. copd is diagnosed with a spirometer and is defi ned as a fi xed reduction in fev , relative to fvc. when caused by cigarette smoke it is associated with airway infl ammation (cd t-lymphocytes, b-cell lymphoid aggregates, macrophages and neutrophils) and airway remodelling (mild increase in airway wall thickness including airway smooth muscle with encroachment on the airway lumen) and tissue destruction (emphysema and loss of small conducting airways). the severity of copd increases with age and continued exposure to irritants. copd has many causes including smoking cigarettes, burning of biomass fuels, asthma and early life respiratory illness and exposures (viral infections, bronchopulmonary dysplasia). as a group, patients with asthma have reduced lung function, in relation to disease severity, and may have a slightly increased rate of decline in lung function. patients with asthma who smoke have reduced lung function and an accelerated decline in lung function. apart from stopping smoking there is no current treatment that can improve the rate of decline in lung function in patients with asthma or copd. therefore prevention, early intervention and uncovering the unknown environmental and genetic contributions to airway diseases remain critical. sy - situation the isaac and ariap studies have showed the different prevalence but heavy burden caused by asthma in asia pacifi c countries is common. goals with inhaled corticosteroids and other medications, the goals in managing asthma in developing countries should attain those stated in gina: control of symptoms, maintaining normal activity levels, normal pulmonary functions, preventing asthma exacerbations, avoiding adverse affects and preventing asthma mortality. in developing countries, avoiding the abuse of short-acting beta agonists, systemic corticosteroids and antibiotics is also a major problem. accessing medical care and medications is crucial. steps a core group, preferably at an university medical center, is the basic step. the asthma and copd outpatient care unit (acocu) run by this core group will help in getting experience and capacity building. the following steps are gina dissemination and implementation, patient club formation, increasing awareness, training doctors and nurses, advocacy, researches, efforts for the availability of affordable asthma drugs, and multiplying acocu in the whole country. the sustainability of acocu is assured by successful implementation of gina. a network of acocu will encourage and improve the activities of acocus. successful provincial acocu will encourage the building of district and even commune acocus. diffi culties the continuous medical education for all doctors on gina, the medication affordability, spirometers and mechanism to maintain an acocu. despite the advances in asthma diagnosis and treatment, slta cases continue to present in the er, sometimes leading to unnecessary mortalities. these are highly preventable situations with inhaled steroid based chronic therapy. with expert care, most patients get through er/icu urgent phase with good outcomes. these strategies are published and updated regularly in the international asthma guidelines. the finnish experience confi rms that a national program that pushes for implementation of guideline recommendations is able to reduce asthma hospitalizations and cost of care. not all countries have such a program but in most developed economies, the cost of asthma care including medications is covered provided the guidelines are followed. unfortunately, even in these affl uent countries, a subset on patients still lands in the er with sltas. while there may have been a failure of health care delivery, the cross-country existence of this problem raises the prospect that some patients are prone to life-threatening exacerbation. sltas may have its own specifi c risk factors that are distinct from the risk factors for simple hospital admission for acute severe asthma. by defi ning risk factors for slta within the population of those admitted to hospital with acute asthma, we may be able to develop specifi c interventional strategies to reduce its occurrence. reported slta risk factors include advancing age, chronic severe asthma, increased infl ammation markers, asthma exacerbated by pneumonia and low nutritional status. other risk factors include recent hospital admission, prior intubation, steroid dependence, non-adherence to inhaled corticosteroids, psychological or psychosocial problems, lack of access to medical care, lower fev , and current cigarette-smoking. a specifi c phenotype of severe brittle asthma has been reported. the backbone of er management for slta includes quick assessment of severity, oxygen therapy, early use of systemic steroids and repetitive saba bronchodilator administrations. enhancements include the use of saba with high intrinsic effi cacy, the addition of ipratropium in refractory cases and the concurrent administration of inhaled corticosteroid for its non-genomic, airway edema-reducing effect. mgso can also be considered for refractory cases. when ventilator support is needed, niv may be attempted in some patient. for intubated cases, the ventilatory strategy includes low tv and rr, high i : e ratio, and monitoring for the development of dynamic hyperinfl ation. the burden of the slta problem can be mitigated by identifying its phenotype a priori, providing preventive therapy before actual exacerbation occurs and putting in the er/icu the appropriate treatment protocols. journal compilation © asian pacifi c society of respirology available treatment of asthma using inhaled corticosteroids and long-acting inhaled β -agonists (labas) is highly effective and safe. importantly, it is also relatively inexpensive. however, many patients remain poorly controlled despite the use of optimal treatment. most advances in asthma therapy have been achieved by improving these drugs and more recently several promising once-a-day labas have been developed. new corticosteroids are also being developed with differential effect on trans-activation and trans-repression of pro-infl ammatory transcription factors, thus giving them a better therapeutic index. the big challenge in asthma is posed by corticosteroid unresponsiveness which is relative and therefore requires high doses to achieve symptom control which inevitably leads to side-effects. one option being pursued is to develop activators of the nuclear enzyme histone-deactylase (hdac) which is recruited to the gene initiation site of pro-infl ammatory mediators. there is an increasing appreciation that asthma is not a single disease and is increasingly seen as a syndrome consisting of several phenotypes. so far, two relatively clear subsets have been identifi ed: eosinophilic and neutrophilic forms of asthma. with this notion in mind, attempts are being made to develop more-specifi c inhibitors for a range of mediators with the hope that sub-phenotypes of asthma will be identifi ed that respond well to either single mediator inhibitors or a combination of these. a number of cytokine modulators have been tested in clinical trials, the most notable example being anti-tnf inhibitors which is felt to be more relevant to neutrophilic asthma. unfortunately, large clinical trials with tnf inhibitors have not found them to be very effective. treatment with blocking antibody for the eosinophils growth factor, il- , has been slightly more effective, with early clinical trials showing that the treatment reduces the frequency of exacerbations in patients who have eosinophilia. whilst the exact mechanisms leading to the development of these two subphenotypes is not fully understood, it is thought that eosinophilia represents a risk factor for exacerbations which has led to eosinophils counts in sputum being used as a guide to treatment; this has been benefi cial in reducing exacerbations. neutrophilic forms of asthma represent a special challenge because patients with neutrophilia tend not to respond well to corticosteroids, making them reliant on bronchodilators. such patients' asthma may be driven by mechanisms that involve il- which induces the production of neutrophil chemoattractants by the epithelium, which makes il- and its chemo-attractant axis a target for novel therapies. the major unmet need in asthma is the treatment of infections. there are early indicators of antibiotic treatment (macrolides) being effective in the treatment of severe asthma. but the real hope comes from novel strategies aimed at the effects of viruses which are the cause of most acute exacerbations, both in milder and more severe forms of disease. recent studies have identifi ed a defi ciency in type i interferons (ifn), the production of which by the bronchial epithelium -the prime target of virus infection -has been shown to be reduced when epithelial cells from asthmatic are grown in culture and infected ex vivo. in the acute care setting, niv must usually start without delay to avoid further deterioration and an increased likelihood of failure. thus, the decision to start must be made quickly based on a bedside assessment. i recommend a simple two step process, the fi rst of which is to assess the patient's need for ventilatory assistance. if the patient has increased dyspnea (moderate to severe) and evidence of increased work of breathing including tachypnea (> /min in obstructive diseases and > /min in hypoxemic respiratory failure), increased accessory muscle use or abdominal paradox, the patient needs ventilatory assistance. arterial blood gases are helpful in making this assessment, but i discourage awaiting blood gas results before starting if the need is obvious, because the window of opportunity may close if initiation is too delayed. i do recommend obtaining baseline blood gases, however, and using them for comparison with later measurements to make certain that the patient is responding. the second simple step is to make sure patients have no contraindications to niv. these include patients with a need for immediate intubation by virtue of a respiratory arrest, hypotensive shock, or uncontrolled arrhythmias or upper gastrointestinal bleeding. the inability to fi t a mask because of a facial deformity, recent facial surgery or burns is also a contraindication. relative contraindications include agitation that prevents the patient from tolerating the mask, increased secretions or diminished ability to protect the airway. patients with these contraindications are at increased risk of failure if placed on niv and should be promptly intubated. patients with multiple risk factors for niv failure should be started only by experienced personnel under very close monitoring. in patients with hypercapneic respiratory failure, these include higher acute physiology scores, marked tachypnea, greater acidemia at baseline and a worse neurological score. in hypoxemic respiratory failure, risk factors for niv failure include the diagnosis of ards or pneumonia, greater age, hypotension and the failure to improve oxygenation substantially within the fi rst hour. although patients at high risk of niv failure can still be given a trial if the clinicians judge it to be indicated, but they must be watched very closely in an icu, with plans to intubate if there is no improvement within the fi rst hour or two. just as the decision to endotracheally intubate a patient in respiratory failure is a clinical judgment that requires the consideration of multiple factors, so is the decision to implement noninvasive ventilation. in the largest rct to date, cpap and nppv performed similarly, both improving dyspnea scores and ph more rapidly than with oxygen alone, but neither lowered intubation nor mortality rate (the major outcome variable) compared to oxygen-treated controls. however, this study enrolled patients whose intubation rate was slightly below % in all of groups, including controls, suggesting that they were too mildly ill to manifest a signifi cant mortality benefi t. meta-analyses of the rcts on cpap or nppv compared with o therapy alone have confi rmed the benefi ts described above, even showing a signifi cant mortality benefi t with cpap. meta-analyses comparing the modalities show equivalency of nppv and cpap with regard to reduction of intubation, lengths of stay and mortality, and with similar myocardial infarction rates. therefore, by virtue of its greater simplicity and potentially lower cost, cpap alone is generally regarded as the initial noninvasive modality of choice for cardiogenic edema patients. but because some studies have found that nppv reduces dyspnea and improves gas exchange more rapidly than cpap alone, nppv is preferred by some initially and can be substituted for cpap if patients treated initially with cpap remain dyspneic or hypercapnic. the success of noninvasive positive pressure to treat cardiogenic pulmonary edema has encouraged its extension into the pre-hospital setting. an emerging trend is to provide cpap devices on ambulances for initial therapy of cardiogenic pulmonary edema, a practice that has been associated with decreased need for intubations in the fi eld. patients with advanced chronic obstructive pulmonary disease (copd) experience poor quality of life and very high levels of symptom burden, including intractable shortness of breath, activity limitation, fatigue, social isolation, anxiety and depression. many of the these burdens are shared with caregivers, and resources in the community to support individuals and their families with chronic illness in the community are often lacking. with the recognition that patients with advanced copd and their caregivers have so many unmet needs, there is a growing acceptance for the need to improve the care and quality of life for patients with advanced copd. while signifi cant gaps in our knowledge and understanding of this area remain, factors contributing to these adverse experiences will be discussed. the importance of prevention, relief, reduction, and soothing of symptoms, without affecting a cure, will be emphasized as an integral component of the care provided for these patients. techniques and tools to optimize the care of patients with advanced copd, including optimizing pharmacologic therapies, inter-professional team care, anticipating and appropriately initiating end-of-life planning, patient and caregiver advocacy, as well as timely and effective communication will be reviewed. withdrawal or withholding life support in medically futile cases has been recognized as an ethical and a legal procedure. it is based on the inherent right of a person to autonomy in making health care decisions. the western model however may not apply to the asian setting being widely varied in terms of cultures, religions and economic progress. more than an individual decision, it may actually be a communal decision with a heavy reliance on input of relations, especially the elders. life support withdrawal often entails complete discontinuation of all measures. efforts to avoid feelings of guilt or abandonment may make families opt for partial withdrawals even when they are not shown to be any more benefi cial. studies have shown that distrust with the medical system does play a major role. active discussions may be diffi cult with reticent cultures or when there are gender differences between patients or their families and the physicians. in this era of globalization and migrations, an understanding of these differences may minimize potential confl icts that arise out of these discussions. awareness that the western approach may not fi t the asian medical model allows the health care providers to be sensitive to the needs and wants of their patients and their families. it is hoped that the data reviewed spurs the development of asia pacifi c guidelines that try to fi nd some uniformity in the diversity of the region. screening for lung cancer is not currently recommended by any major medical organization. multiple phase ii non-randomized trials of computed tomography (ct) screening have yielded enticing results. they have demonstrated that ct screening detects smaller size lung cancer of - mm in diameter. it has been documented that the chest radiographs miss - % of the cancers detected by screening ct. in prevalence studies, - % of detected cancers are stage i. when ct screening results were compared to a validated control group, ct has been shown to detect times more lung cancer than would be expected and results in ten times more thoracic operation than would be expected. additionally, no decrease in advanced stage cancers or decrease in lung cancer deaths were observed. to date, multiple small randomized controlled screening trials (rct) have been reported, but they have been too small to assess if ct screening reduces mortality. a meta-analysis of baseline fi ndings from six small randomized controlled trials observed more stage i and more total lung cancers in the ct screened group. for every individuals screened with low dose ct, stage i nsclc and false positive nodules were detected and thoracic operations were performed for benign nodules. the two large rct of ct screening that may defi natively answer the question of ct screening and its ability to decrease lung cancer mortality are the national cancer screening trial (nlst) and the nederlands-leuvens longkanker screenings onderzoek (nelson) trial. mortality results from those two trials are anticipated in and respectively. a recent report from the nelson trial validated the use of ct volumetric assessment of nodules to assess malignancy and determine which nodules should be treated surgically. currently, there is considerable effort to identify susceptibility genes for lung cancer with particular interest in q - which is strongly associated. this region contains several genes of interest, including three genes that encode nicotinic acetylcholine receptor subunits. however, these genes may just be associated with nicotine dependence. a recent report utilizing gwas (genome wide association scan) methodology identifi ed snps at q . associated with lung cancer susceptibility in never smokers. an enormous research effort is underway related to biomarkers in airway epithelial cells, blood, sputum, breath, and urine for early diagnosis or prediction of high risk. intense efforts are devoted to develop models of risk for determining which individuals should be offered screening. journal compilation © asian pacifi c society of respirology sy - lung cancer is the leading death-related cancer worldwide. molecular targeted therapy appears to be an alternative approach for patients with non-small cell lung cancer (nsclc). the epidermal growth factor receptor (egfr) is one of these targets, responsible for the cell growth, proliferation, apoptosis and metastasis of the tumors. egfr-tyrocine kinase inhibitor (tki) has been applied to target egfr and suppress the development of tumors. some egfr-tkis, including gefi tinib and erlotinib, have been approved, while the others are still under development or in clinical trials. several studies demonstrated that egfr somatic mutations might predict the high response rate and greater survival benefi t of egfr-tki. in addition, egfr amplication, k-ras mutation, met amplication or the egfr t m mutation might predict the clinical effect of these drugs. both erlotinib and gefi tinib have been undergoing several clinical trials for nsclc treatment as a single drug or in combination with chemotherapy. br. trial showed that erlotinib improved survival with previously treated nsclc patients in a randomized multicenter during phase iii study. thus, erlotinib was approved to be the second or third-line treatment of advanced nsclc patients. however, isel failed to demonstrate a statistically important benefi t of gefi tinib in overall survival as compared with placebo. different study population, dosing and drugs of br. and isel might explain the different results. in ipass trial, clinical selected nsclc patients with asian origin and characterised by adenocarcinoma histotype were treated with gefi tinib or paclitaxel/carboplatin doublets as the fi rst line therapy. the results showed that gefi tinib had the superiority in terms of progression free survival (pfs) in patients with egfr mutation. in eortc and perol trials, gefi tinib and erlotinib maintenance therapy showed the trend of improved pfs, but not overall survival in advanced nsclc patients. the toxicity of gefi tinib and erlotinib includes diarrhea, rash, etc., which can be well-tolerated. novel egfr-tkis include vandetanib, sorafenib, sunitinib, and cediranib, of which some are under evaluation in phase iii trials as monotherapy or in combination with standard chemotherapy. vandetanib targets both egfr and vegfr and was tested in the second phase trial, suggesting the addition of vandetanib to the single chemotherapy might improve response rates and survival. sorafenib has been applied to different carcinoma histology and in combination with different chemotherapy. when combined with paclitaxel and carboplatin to treat patients with squamous cell cancer, no survival benefi t was seen. however, another clinical trial was launched to investigate the effect of sorafenib, in which squamous cell cancer were not eligible. novel egfr-tkis are under development with hope of overcoming resistance to egfr-tki gefi tinib and erlotinib. there is a great need of further clinical trials. egfr-tki is one of the important alternatives in treatment of nsclc and has shown promising potential in the future. more promising results may come out if the combination and sequence of egfr-tki with traditional therapies, like chemotherapy, radiotherapy and surgery, can be optimized. there is also a need of disease-specifi c biomarkers to predict the effect of the drugs and identify the patients most likely to benefi t from the drugs. lung cancer is the number one cause of cancer death. most cases are found after distant metastasis, and outcome of drug therapy for these patients used to be disappointing. however, we have faced a new paradigm shift, i.e., the molecular targeted therapy and the individualized therapy. many promising data has reported from not only western countries but also asian countries such as the effi cacy of egfr tki to tumors with mutated egfr, that of alk inhibitor to tumor with eml -alk fusion protein, and that of pemetrexed to non-small non-squamous cell lung cancers. new questions have emerged from these new evidences derived from some important clinical trials. among them, questions regarding with ethnic difference would be one of the most important issues. is survival data same between asian and caucasian? (data from japan lung cancer registry study as well as some global trials have shown survival of asian patients with lung cancer appears to be obviously better than that of caucasian.) why egfr mutation is frequent in asian patients? is only egfr gene status related with prognosis of lung cancer? what would be the cause of alk abnormality? is the criteria of pathological diagnosis for lung cancer same between asian countries and western countries? here, newest evidences for treatment of lung cancer will be presented, and importance of ethnic difference and asian trials will be discussed. the burden of chronic obstructive pulmonary disease (copd) is growing. despite these growing numbers, many patients with patients with copd remain undiagnosed, the greatest number being those with milder disease. delays in the diagnosis of copd are common. evidence suggests that patients with mild copd experience increased symptoms, reduced activity levels and exercise capacity, and impaired health-related quality of life. this growing body of evidence has made it clear that mild copd is not 'normal'. with recognition of this reality and efforts to appropriately recognize copd at an earlier stage, clinicians must be aware of the various therapeutic options for their patients. the defi nition of mild copd will be discussed, as well as effective strategies for the targeted early diagnosis of copd. the numerous and varied disease manifestations and consequences for patients with milder copd will be reviewed. in addition, practical and effective management options available to clinicians caring for patients with mild copd will also be examined. clinicians have been long aware that neither the traditional distinctions of "emphysema" versus "chronic bronchitis" nor the traditional clinical phenotypes of "blue bloater" and "pink puffer" are suffi cient to categorize patents that suffer from chronic obstructive pulmonary disease (copd). recently, the global initiative for chronic obstructive lung disease (gold) workshop has used quantitative measures (fev and fev /fvc ratio) to defi ne copd, but this defi nition fails to take into account the full heterogeneity of copd. with an increased understanding of pathophysiologic variation, copd now clearly represents a spectrum of overlapping diseases with important extrapulmonary consequences. a "phenotype" describes the outward physical manifestations of a particular disease, and comprises anything that is part of the observable structure, function or behavior of an individual. such phenotypic distinctions in copd include: frequent exacerbator, pulmonary cachectic, rapid decliner, airways hyperresponsiveness, impaired exercise tolerance, and emphysema versus airways disease. these variable manifestations, each with unique prognostic, clinical and physiologic implications, represent distinct phenotypes within copd. while all of these phenotypes have smoking as a common risk factor, the other risk factors that determine these phenotypes remain poorly understood. an individual smoker has variable expression of each phenotype and there is mounting evidence that copd phenotypes have different clinical outcomes. these phenotypes can be broadly classifi ed into one of three groups: clinical, physiologic and radiographic. thus, the paradigm that copd is one disease may be incorrect, and suggests that copd should be considered as a spectrum of smoking-related diseases. failure to consider copd phenotypes is likely to limit the power of therapeutic trials since not all copd patients are likely to benefi t from each therapy. the challenge to future copd researchers is to better characterize these phenotypes and identify their risk factors. measurement of fractional exhaled nitric oxide (feno) is an attractive biomarker of diseases where airway eosinophilia dominates. indeed even before any randomized controlled trials were published some were advocating treatment tailored in accordance to feno data. commercially available bench-top and portable feno analyzers are now readily available and in some countries, feno measurements attract a payment. however, despite the ease of measuring feno, it has its drawbacks in biological and measurement issues. biologically feno is signifi cantly infl uenced by atopy, intake of caffeine, exercise, ethnicity, etc on the measurement front, variabilites include: feno measured by different analyzers may provide different values and on-line vs off-line measurements. the cut-off for determining 'abnormally high results' is yet unknown. not surprisingly, there is discordance on the effi cacy of tailoring asthma medications in accordance to sputum eosinophils [ ] and feno [ ] in people with asthma, although both are eosinophilia infl ammatory markers. tailoring of medications in accordance to sputum eosinophilia (compared to standard practice) significantly reduced exacerbations in adults with asthma (odds ratio . , %ci . , . ). in contrast, the benefi t of tailoring of medications in accordance to feno was, at best, modest. the utility and limitations of using feno levels in the clinical setting will be discussed in this talk. shortness of breath and activity limitation are cardinal symptoms experienced by patients suffering from respiratory illness or disease. cardiopulmonary exercise testing (cpet) allows for the objective evaluation of these symptoms, recognizing that exercise involves the effective integration of respiratory, cardiovascular, neuromuscular and metabolic functions. the organs involved in these varied and important roles have a sizeable reserve, with the consequence that clinical manifestations of a disease state or abnormality may not become readily apparent until the functional capacity of the organ(s) is markedly impaired. objective assessment of various parameters during exercise, which places an increased physiologic demand on the functional reserve capacity of these organs, can provide a sensitive method for the early detection of abnormal function and responses(s). the results from exercise testing also parallel functional capacity and quality of life more closely than measurements obtained only at rest, and have been shown to accurately predict important outcomes, such as mortality, in a variety of patients and clinical circumstances. a brief overview of normal exercise physiology and characteristic responses demonstrated by patients with various disorders frequently assessed by the pulmonologist will be offered. in addition, a summary of the indications, conduct, and practical interpretation of cpet will be presented in this session. effect of tiotropium on outcomes in patients with moderate chronic obstructive pulmonary disease (uplift): a prespecifi ed subgroup analysis of a randomised controlled trial obstructive lung disease and low lung function in adults in the united states: data from the national health and nutrition examination survey international variation in the prevalence of copd (the bold study): a population-based prevalence study chronic obstructive pulmonary disease in fi ve latin american cities (the platino study): a prevalence study prevalence of copd in spain: impact of undiagnosed copd on quality of life and daily life activities global burden of copd: systematic review and metaanalysis prevalence of chronic obstructive pulmonary disease in china. a large, population-based survey diagnostic labeling of chronic obstructive pulmonary disease in fi ve latin american cities copd prevalence in a random population survey: a matter of defi nition treatment of copd: the sooner the better clinical copd phenotypes: a novel approach using principal component and cluster analyses offi ce spirometry signifi cantly improves early detection of copd in general practice: the didasco study salmeterol and fl uticasone propionate and survival in chronic obstructive pulmonary disease clinical trial design considerations in assessing long-term functional impacts of tiotropium in copd: the uplift trial a -year trial of tiotropium in chronic obstructive pulmonary disease mortality in the -year trial of tiotropium (uplift) in patients with chronic obstructive pulmonary disease effi cacy of salmeterol/fl uticasone propionate by gold stage of chronic obstructive pulmonary disease: analysis from the randomised, placebo-controlled torch study effects of tiotropium on outcomes in patients with moderate chronic obstructive pulmonary disease (uplift): a prespecifi ed subgroup analysis of a randomised controlled trial tiotropium as a first maintenance drug in copd: secondary analysis of the uplift trial treating tobacco use and dependence: update. clinical practice guideline tobacco atlas the mpower package. geneva, world health organization implementing smoke-free environments. geneva, world health organization curbing the epidemic: governments and the economics of tobacco control clinical and public health signifi cance of treatments to aid smoking cessation we report that pulmonary emphysematous lesions appear to be a dynamic phenomenon that involves not only the gradual loss of alveolar structure, but apoptosis, cellular proliferation, and cellular senescence as well. cellular proliferation compensates for increased alveolar cell apoptosis in chronic obstructive pulmonary disease (copd) patients. however, smoking, age, and the increased cell cycle turnover that compensates for apoptosis accelerate alveolar cell senescence, thereby halting cellular proliferation and tipping the balance toward apoptosis, which, in turn, promotes the formation of emphysematous lesions. as a result, alveolar cells disappear and the emphysematous lesions progress. at the same time, cellular senescence is thought to induce infl ammation. more specifi cally, senescent alveolar cells induce infl ammation by producing various infl ammatory cytokines in tissue. lymphocytes and clara cells may also age more rapidly in the lungs of copd patients. lymphocyte senescence may induce an autoimmune reaction and increase susceptibility to infection, and clara cell senescence may impair airway regeneration as well as sustain airway infl ammation. thus, cellular senescence may be involved in arrested tissue repair, chronic infl ammation, and increased susceptibility to infection, which are the typical features of copd. there is increasing recognition that copd is an increasing global burden. new drug treatments continue to emerge suggesting that copd is more responsive to treatments than previously thought. however, there is still much that is unknown about copd that will contribute to further advances in treatment and management. pulmonary imaging can contribute by providing information on how structure and function relate to relevant clinical parameters, such as disease progression, treatment responses and exacerbations. in other words, imaging can help characterise copd in terms of clinical outcomes or phenotypes. there have been many advances in imaging methodology, including ct, mri, spect and pet. recent fi ndings from research studies using innovative methods of studying structure and particularly function, in copd will be reviewed. their clinical implications will be discussed. the spectrum of children's interstitial lung disease (child) encompasses a large, heterogeneous group of pediatric diffuse lung disorders that are diffi cult to diagnose and treat. as the differential diagnosis is large, a systematic approach is needed for accurate diagnosis. the classic fi rst step of obtaining a detailed history and performing a careful physical examination remains essential for providing diagnostic clues as well as assessing severity of illness. as examples, a history of hemoptysis and fatigue would suggest an alveolar hemorrhage syndrome; exposure to avian antigens, hypersensitivity pneumonitis; and a history of adenovirus pneumonia, bronchiolitis obliterans. the presence of growth failure, crackles, loud p , and clubbing on physical examination would point to a severe and progressive lung process with cor pulmonale. recent advances in diagnostic modalities have greatly improved the ability of clinicians to identify these disorders. in infants and children with diffuse lung disease, genetic testing can be diagnostic for surfactant dysfunction mutations (sp-b, sp-c, abca , ttf- , gm-csfra receptor). infant lung function testing has proven useful for assisting in the diagnosis of certain disorders, such as neuroendocrine cell hyperplasia of infancy (nehi) and distinguishing nehi from surfactant mutations. hrct may detect extent and severity of disease, but can also be useful in diagnosing specifi c disorders, such as nehi (symmetric ground glass densities in the right middle lobe and lingula and the central lung regions), bronchiolitis obliterans (mosaic perfusion, vascular attenuation, and central bronchiectasis), hypersensitivity pneumonitis (ill-defi ned centrolobular micronodules), and pulmonary alveolar proteinosis (crazy-paving). bronchoalveolar lavage can aid in the diagnosis of specifi c conditions, such as alveolar hemorrhage syndromes (hemosiderin-laden macrophages), aspiration (lipid-laden macrophages), hypersensitivity pneumonitis and sarcoidosis (lymphocytosis), eosinophilic pneumonias (eosinophilia), and histiocytosis (cd a + cells). lung biopsy performed by video-assisted thoracoscopic surgery (vats) has largely supplanted conventional open lung biopsy as the procedure of choice as it is equally accurate, but associated with less morbidity. although lung biopsy remains the gold standard for diagnosis of child, it must be interpreted in the context of the clinical and radiologic fi ndings. it should be emphasized that although lung biopsy can be diagnostic in some disorders, such as bronchiolitis obliterans, it may not be necessary because less invasive studies such as hrct may be suffi cient for diagnosis. finally, some pulmonary vascular disorders, such as pulmonary vein stenosis or atresia, may mimic child. for these disorders, echocardiography, mra, or cardiac catheterization may be required for diagnosis. with a systematic approach and improved diagnostic capabilities, it is reasonable to expect that a specifi c diagnosis can now be made in the vast majority of child cases. key: cord- -bs qk authors: venkatachalam, sangeeta; mikler, armin r. title: an infectious disease outbreak simulator based on the cellular automata paradigm date: journal: innovative internet community systems doi: . / _ sha: doc_id: cord_uid: bs qk in this paper, we propose the use of cellular automata paradigm to simulate an infectious disease outbreak. the simulator facilitates the study of dynamics of epidemics of different infectious diseases, and has been applied to study the effects of spread vaccination and ring vaccination strategies. fundamentally the simulator loosely simulates sir (susceptible infected removed) and seir (susceptible exposed infected removed). the geo-spatial model with global interaction and our approach of global stochastic cellular automata are also discussed. the global stochastic cellular automata takes into account the demography, culture of a region. the simulator can be used to study the dynamics of disease epidemics over large geographic regions. we analyze the effects of distances and interaction on the spread of various diseases. nowadays, the problem of emergent diseases and re-emergent diseases like influenza and sars, have caused increased attention towards public health in general and epidemiology specifically. with the ever-increasing population and ability to travel longer distances in short time, the spread of communicable diseases in a society has been accelerated [ , ] . growing diversity of the population, and globalization are leading towards increasing interaction among individuals. constant exposure to public health threats is raising people's concern and necessitates pro-active action towards preventing disease outbreaks. further, greater emphasis on infections and epidemics is rooted in the imminent threat arising from bioterrorism. as a result, public health professionals have been focusing on identifying the factors in the social, physical and epidemiological environment which aid to faster spread of diseases. as the significance of public health is being recognized, the role of epidemiologists has become more prominent. epidemiology deals with the study of cause, spread, and control of diseases. the goal of epidemiologists is to implement mechanisms for surveillance, monitoring, prevention and control of different diseases. to accomplish the above mentioned, epidemiologists need to deal with large data sets of disease outbreaks. these data sets are often spatially and/or temporally distributed. it is in fact ironic that, for epidemiologists to study the dynamics of different diseases, it is vital for an outbreak to occur. epidemiologists have been studying and analyzing the data sets using primarily statistical tools. in the vast variety of infectious diseases, expertise is needed in terms of epidemiologists for every disease. statistical tools, prove to be inadequate and fragmentary, when focusing on large spatial domains. these tools have been deemed limited, particularly in view of an emerging global computational infrastructure that facilitates high performance computing. hence, it is imperative to develop new tools that take advantage of today's computational power, and help epidemiologists to analyze and understand the spatial spread of diseases. the computational tools also enhance the quality of information, accelerate the generation of answers to specific questions and facilitate in prediction. such tools will take on an important role in surveillance, monitoring, prevention and control of different diseases. in the domain of computational tools, the cellular automata paradigm has been in use for several decades [ ] . nevertheless, in the field of modeling epidemics, this paradigm has rarely been utilized to its full potential [ , , , ] . a cellular automata as defined by lyman hurd is a discrete dynamical system, where space, time, and the states of the system are distinct [ ] . ca has been exemplified as an array of similar processing units called cells. the cells arranged in a regular manner constitute a regular spatial lattice. figure shows a regular lattice of cells. the fundamental property of each cell is a state, where the states of cells change based on a update rule, either local or global. the update rule is applied synchronously throughout the lattice and the state transitions of the cells are based on few of the close by cells, known as the neighborhood. for a two-dimensional lattice the most common neighborhoods defined are von neumann and moore neighborhood as shown in figure [ ] . in the von neumann neighborhood, the state of cell c i ,j depends on the states of the four neighborhood cells namely c i+ ,j , c i− ,j , c i,j+ , c i,j− . in the moore neighborhood, the state of cell c i,j depends on the states of the eight neighborhood cells namely as mentioned before, the ca's evolution is based on a global update rule applied uniformly to all the cells. the signature of this rule can be thought of as a state transition from time t- to t. as shown in the figure the state of the center cell changes to a state, which is in majority among the cells in the neighborhood. the update rule determines the deterministic or stochastic behavior of a ca. stochastic behavior is seen by probabilistic update rules in non-deterministic state transitions. our efforts to design and implement a cellular automata based simulator has been necessitated by the need to study the dynamic of spread of a vast number of infectious diseases. towards this goal, this paper proposes the use of ca paradigm to simulate an infectious disease outbreak. specifically, this paper focuses on the design and evaluation of epi-sim, a global disease outbreak sim- ulator. the following section summarizes some of the research effort in modeling disease epidemic and highlights principle approaches. the design of epi-sim is discussed in section . section presents the experimental analysis and results of the simulator. section discusses the geo-spatial model and the approach towards the global model to account for different demographics. section concludes the paper with a summary and direction for future work in the area of modeling infectious diseases outbreaks. most of the work in modeling infectious disease epidemics is mathematically inspired and based on differential equations and sir/seir model [ ] . differential equation, sir modeling rely on the assumption of constant population and neglect the spatial effects [ , ] . they often fail to consider individual contact/interaction process and assume populations are homogeneously mixed and do not include variable susceptibility. considerable research has been conducted in sir(susceptible, infectious, recovered) modeling of infectious diseases using a set of differential equations. both partial and ordinary differential equation models are so deterministic in nature that they neglect the stochastic or probabilistic behavior [ ] . nevertheless, these approaches/models have been shown to be effective in regions of small population [ ] . other approaches for modeling disease epidemics have been using mean field type approximations [ ] . even though the mft models are similar to the differential equations, they add a probabilistic nature by adding different probabilities for the mixing among individuals. although, according to boccara [ ] mean field approximations tend to neglect spatial dependencies and correlations and assume that the probability of the state of cell being susceptible or infective is proportional to the density of the corresponding population. this approach relies on the quantitative measures to predict local interaction. boccara and cheong [ ] study the sis model of spread of infectious disease in a population of moving individuals, thereby introducing non-uniform population density. in every update the cells take up a state of being either susceptible or infectious and randomly choose a cell location to move to. ahmed et al [ ] model variations in population density by allowing cyclic host movement. other approaches in modeling variable susceptibility of the population, have been done by inducing immunity in the population. ahmed et al [ ] introduce incubation and latency time, and suggest that the parameters have an accelerating impact on the spread of a disease epidemic. nevertheless, the underlying assumption is spontaneous infection of individuals. boccara and cheong [ ] concentrate on sir epidemic models and take into consideration the fluctuation in the population by births and deaths, exhibiting a cyclic behavior with primary emphasis on moving individuals. di stefano et al [ ] have developed a lattice gas cellular automata model to analyze the spread of epidemics of infectious diseases. the model is based on individuals, where individuals can change their state independent of others and can move from one cell to other. however, this approach does not consider the infection time-line of latency, incubation period, and recovery which have been shown to be important to model a disease epidemic. in our model the basic unit of cellular automata is a cell, which may represent an individual or a small sub-population. for each cell we use the moore ( ) neighborhood definition. each cell can be characterized with its own probability for risk of exposure, probability of contracting the disease and state. unlike the sir model, every cell comes in contact with the cells in its defined neighborhood. the time-line for infection that we consider is shown in figure . however, the moore neighborhood is restricted in modeling population demographics and travel patterns. the limitation is eliminated in the next version of the simulator with a global neighborhood which will be proposed in future publications. the following sections discuss the definitions, features and rules of the model and simulator. in order to understand the functioning of the simulator, we define definite number of states a can exists in, and define the infectious time-line. the following section describes the different states and definitions considered in the model. state 's' for susceptible is defined as the state where, the cell is capable of contracting a disease from its neighbors. in the infectious state, 'i' the cell is capable of passing on the infection to its neighbors. in the recovery state, 'r' the cell is neither capable of passing on the infection, nor is capable of contracting the infection. infectivity ψ, at any given time is defined as the probability of an susceptible individual to become infectious, if it has an infectious cell as a neighbor. latency λ, is defined as the time period between, the cell becoming infected and it becoming infectious. infectious period θ , is the period of time, when the infected cell is capable of spreading the disease to other cells. recovery period ρ is defined as the time period, the cell takes to recover, wherein it is neither capable of passing on the infection, nor is capable of catching the infection. the following rules are applied to the ca for simulating the spread of the disease. the rules describe the state transitions of individual cells. with an infected cell in its defined neighborhood. the cell acquires the disease from the infected neighbor based on the probability of given by the parameter of infectivity ψ. the cell remains in the latent state for the number of time steps (updates) as defined by the parameter latency λ. . the state of the cell changes from latent l to infectious i after being in state l for the given λ. in this model we assume for simplicity, that every cell exposed to the pathogen, will become infectious. in the state i, the cells are capable of passing on the infection to neighborhood cells. for example if for a disease d, λ= units, then after two time steps the cell will enter the infectious state i. . after a time period, defined by the infectious period θ, the state of the cell changes from infectious i to recovered or removed r. once the cells enter the state r, the cell is no more capable of passing on the infection. . from the state r, the cell's state changes back to either susceptible s or it remain in state r, signifying complete immunity. the 'healing mode' turned on determines the transition from state r to state s and vice versa. while modeling a disease epidemic, few parameters that are considered important are neighborhood radius, contact between individuals, infection probability (variable susceptibility), immunity, latency, infectious period and recovery period. the simulator is highly parameterized to let the user change and modify the above parameters. the neighborhood of every cell can be changed from a neighborhood to neighborhood depending on the region being simulated and the contacts among the individuals of the region. as mentioned, the infection probability represented as infectivity ψ is a significant parameter for the spread of a disease. in the case of our model, ψ is based on the virulence of the disease and contact rate among individuals. for some diseases individuals attain lifetime immunity, after being infected, while for disease like common cold, individuals attain temporary immunity. thus, to take this fact into consideration, the simulator has a feature of healing mode. with the healing mode enabled the simulation is executed in a mode that forces cells to turn into susceptible after the recovery state and with healing turned off, the cell attains complete lifetime immunity. as mentioned above, the infection time-line is also an important factor in modeling a disease epidemic. thus the time periods of latency λ, infectious θ, and recovery ρ are all expressed as time units, for example, latency of two days, can be represented as λ= units. the simulator allows the user step through the simulation at each time step, or execute it continuously. we will see in the next section, how changing these parameters, can change the dynamics of spread of diseases. an epidemic is a severe outbreak of an infectious disease which spreads rapidly to many people. for example, the occurrence of influenza in a region is considered as an epidemic. when a disease spreads to larger geographic regions or throughout the world it is known as pandemic. moving along the same direction an endemic is defined as a disease that is always present in certain group of the population. using our model we show both an epidemic and endemic. an epidemic is characterized by an exponential growth of the infected individuals in a population. in the case of an endemic the number of infected individuals fluctuates around a mean, there is no exponential growth. experiments were conducted on a by grid cellular automata with different values of ψ, λ and θ. the results in this section represent the mean over multiple random experiments and different random graphs of the same type. the analysis of results in this section have been conducted with reference to the above definitions. as mentioned earlier, ψ is an important factor in the analysis of spread of a disease. figure vaccination has contributed significantly towards the eradication and reduction of effect of many infectious diseases [ ] . the following experiments were con- on the simulator by vaccinating about % of the population at random and infecting few cells. figure (b) , shows the growth of infected individuals in a vaccinated and non vaccinated population. figure (b) depicts that the growth of infected individuals in a population with only % of the population vaccinated, is considerably less as compared to the growth in a non-vaccinated population. we study the effects of spatial distribution of population, by vaccinating a part of the population using the random vaccination and ring vaccination. every time a new vaccine is discovered, the question arises as to how should the vaccine be distributed to minimize the spread of a disease and maximize the effect of vaccination. thus, in this experiment we compare the random vaccination, which is also known as uniform strategy [ ] , and ring vaccination. the doses of vaccine available at our disposal is often limited, thus for the purpose of experiment we consider n doses of vaccine to be available to vaccinate the population, where n is about % of the population. in random vaccination, the n vaccines, are randomly distributed to individuals in a population, independent of the other. in the ring vaccination, individuals are vaccinated in a ring surrounding an area. the thickness and circumference of the ring depends on n. as figure shows, using random vaccination many more individuals are infected as compared to the ring vaccination. this experiment validates the result shown by fukś and lawniczak in [ ] . the previous model described poses a limitation of neighborhood. the model considers a neighborhood of cells, because of which after a time period the number of susceptibles reduce and saturate the neighborhood . in such a situation the variance of infectivity parameter plays no role and has the same effect on the spread of the disease. also, the need to simulate a disease, where an infective can spread the disease to twelve other individuals in one time step, will not be possible to simulate. another important issue to note is the movement of people, migration, or travel is not considered. some models, we saw in the previous section deal with movement of individuals from one cell to another in the defined neighborhood, where again the neighborhood is restricted. the saturation of neighborhood occurs due to overlapping of neighborhood, when more than one cell is infected in a neighborhood. cells in a neighborhood may get infected more than once in one time unit. the geo-spatial model is designed for simulating global outbreak of a disease in a environment with global interaction. even for this model the basic unit of ca "saturation.txt" using "saturation.txt" using "saturation.txt" using "saturation.txt" using "saturation.txt" using fig. . represents saturation of neighborhood ψ is a cell, which represents an individual. the neighborhood as defined for this model is global, where in a region of n cells every cell has n- neighbors. for the functioning of this model, the definitions for the states of cell, parameters for simulation are same as the ones for sir model discussed earlier. this model has an additional parameter of contact rate and the definition is as follows. contact rate parameter defines the number of contacts made by an individual per time unit. instead of having the same contact rate parameter for every cell in the lattice, for simulation purposes this parameter has a poisson distribution over the cells. the simulation of spread of disease is discussed further. where k is the contact rate defined for that cell. thus the cell has now established contacts with k cells. . once a contact has been established between cell 'a' and cell 'b', depending on the virulence of the disease defined by the infectivity parameter, cell 'a' can pass the infection to cell 'b' if cell 'b' is in a susceptible state s. if cell 'a' is not infected currently and cell 'b' is infected then cell 'a' can acquire the infection from 'b'. thus the infection can pass on in both directions. the geo-spatial model is different from the sir type model in terms of the neighborhood. the neighborhood saturation problem posed by sir type model is overcome by this model. however, this model is restricted in modeling population demographics and travel patterns. the choice of cells for contact, is random and is not based on distance from the cell or any other parameter. to study the effects of position of index case on spread of a disease, the simulation was run with different initial positions. after a certain time unit it is seen that locations of new infected cases are not very different for the two to analyse the contact rate the experiment was done with three different contact rates for cells. the result shows that as average contact rate increases, the number of infected individuals also grows. for this model the contact rate is directly proportional to number of infected individuals. it is important to note that the contacts made by cells are random. figure (a) shows the comparison. as seen before in the other model, as infectivity parameter ψ increases the number of infected individuals increases. the average contact rate was fixed for this experiment. figure (b) shows the comparison. the models described above may be used for simulating diseases over small regions with local interaction and global interaction respectively. as mentioned before, these models do not take into account the demographics of the region and may not be accurate for simulating disease spread over large geographic regions because of the neighborhood constriction posed by them. thus the global stochastic cellular automata with demographics will facilitate to understand the effects of different demographics, the population density, socio-economics of a region and culture. it can also be used effectively for investigating different vaccination strategies and understanding the effects of travel. in the following section we discuss the design of a global outbreak simulator with a global interaction and demography. even for this model the basic unit of ca is a cell, which represents an individual or a small sub-population. the neighborhood as defined for this model is global, where in a region of n cells every cell has n- neighbors. the neighborhood for a global sca is defined using a fuzzy set neighborhood. the definition of fuzzy set neighborhood is as follows. the set f ⊂ s where s is a set of all the cells f : { s, p |s ∈ s, ≤ p ≤ } s, : total/complete membership s, : no membership the variable p maintains the state of infection, if infected else . state of infection δ is defined as any number between and , indicating the level of infection present in the cell. indicates not infected, indicates fully infected. interaction coefficient i for a particular cell is defined as the interaction between that cell and every other cell in the lattice space. it is calculated as the reciprocal of the euclidean distance between the cells. euclidean distance as derived from the gis gravity model. global interaction coefficient Γ of cell ci,j is the summation of all the individual (n- ) interaction coefficients of the cell. every cell has one global interaction coefficient and n- interaction coefficients. the infection factor i is calculated as a fraction of the interaction coefficient to the global interaction coefficient Γ , for every cell to cell interaction. it is also based on the virulence of the disease and the state of infection of the infecting agent. i ci,j = ∀c k,l =ci,j ic i,j ,c k,l Γ c i,j ×δ c k,l ×ψ the global interaction coefficient and the interaction coefficients are calculated based on the distance. as the distance in between the cells reduce, the interaction coefficients increase which indicates more chances of interaction between them. Γ ci,j = ∀c k,l =ci,j √ i−k + j−l the global interaction coefficient and the interaction coefficients are calculated based on the distance and population. the distance between the cells and the populations of the cells are considered. for better understanding, the cells are considered to be small regions having certain populations. the product of the populations of the two cells, acts as a factor for the interaction coefficients. the population factor is directly proportional to the interaction coefficient and the distance between them is inversely proportional to the interaction coefficient.thus two cells with high populations are assumed to interact more than two cells with low populations, when the distance between them is same. Γ ci,j = ∀c k,l =ci,j √ i−k + j−l × p ci,j × p ci,j this paper describes a disease outbreak simulator using the cellular automata paradigm. the results show the variation in the spread of the disease for different parameters of infectivity ψ. the simulator has also facilitated the study of different vaccination strategies. geo-spatial model helps us in simulating disease spread in an environment with global interaction including travel and migration. in the same direction the global model can be used to simulate disease spread over large geographic regions. it deals with global interaction and the demographics of the region. while still working on the development of computational tools to facilitate surveillance, monitoring, prevention and control of dynamics of different diseases, the current simulators prove as valuable tools to study the dynamics of different diseases. global stochastic versions of the ca are currently being developed. on modeling epidemics. including latency, incubation and variable susceptibilityphysica a on some applications of cellular automataphysica a a comparison of simulation models applied to epidemics multiagent coordination by stochastic cellular automata presented at the international joint conference on artificial intelligence critical behavior of a probabilistic automata network sis model for the spread of an infectious disease in a population of moving individuals a probabilistic automata network epidemic model with births and deaths exhibiting cyclic behavior object-oriented implementation of ca/lgca modelling applied to the spread of epidemics individual-based lattice model for spatial spread of epidemics discrete dynamics in epidemiology through cellular automata case of study: avian influenza indonesia working paper wpf epidemiologic methods for the study of infectious diseases mean-field-type equations for spread of epidemics: the 'small world' model deterministic site exchange cellular automata models for the spread of diseases in human settlements epidemic modelling using cellular automata complex transmission dynamics of clonally related virulent mycobacterium tuberculosis associated with barhopping by predominantly human immunodeficiency virus-positive gay men educational uses of virtual reality technology key: cord- -l kjxhd authors: nan title: irish society of gastroenterology: proceedings for summer meeting – th/ th may in galway date: journal: ir j med sci doi: . /bf sha: doc_id: cord_uid: l kjxhd nan inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc) are associated with a diverse group of extra gastrointestinal features. however, unlike patients with irritable bowel syndrome (ibs), there have been few reports of gynaecological abnormalies in ibd. the aim of our study was to observe the effect of ibd and ibs on the age of menarche and menopause in female's. subjects (mean age yr, range - yr), resident in dublin, participated in the study. these included uc (n= ), cd (n= ), ibs (n= ) patients and healthy female controls. the different subgroups were matched for age and marital status. information from subjects was obtained by a written questionnaire. menarche was at a significantly later date (student t test, p value < . ) in patients with cd ( . + . yr) and uc ( . + . yr) than in normal controls ( . + . yr). no significant differences in age of menarche were observed between the ibs ( . + . yrs) and control groups or between the cd and uc groups (p value > . ). the date of menopause was also significantly early (mann-whitney u-test) in patients with cd ( . _+ . yr) and uc ( . + . yr) than in normal controls ( . + . yr). the differences between the ibs group ( . + . yr) and the controls or the cd and uc patients were not statistically significant. conclusion: the fertile period of females with ibd is significantly reduced compared to healthy controls. this difference is not observed in ibs patients. the underlying mechanism is not known but may involve the abnormal immune responses observed in these patients. chloride (ci) secretion by the human colon epithelium provides the osmotic driving force for fluid secretion into the colon lumen. under 'normal' conditions this fluid is readily reabsorbed. in secretory states the volume of fluid secreted secondary to ci secretion may exceed the absorptive capacity of the epithelium and diarrhoea results (secretory diarrhoea). increase in secretion by the colon epithelium is also thought to play a role in the diarrhoeas which occur in inflammatory disease of the colon. in this study we examined tile pathways for cisecretion in human colon and some aspects of their cellular control. normal human colon epithelium was dissected from colectomy specimens, mounted in ussing chambers and voltageclamped to milli-volts. when necessary a single membrane of the epithelial cell was permeabilised with nystatin in order to study the 'opposite' membrane in isolation. entry of ci across the basolateral membrane of the epithelial cells was inhibited by basolateral addition of a combination of bumetanide and dids. entry of ci at the basolateral membrane was found to be rate limiting for transceuular ci secretion. this suggests that basolateral ci entry to the cell ocurs via na*/k § c cotransport and/or ci/hco~-exchange. ci ~ diffuses across the cell and is extruded at the apical membrane via ci channels which were inhibited by a combination of anthracene- carboxylic acid (a- -c) and -nitro- -( -phenylpropylamino)benzoic acid nppb. the nppb inhibited channel was stimulated by experimental manoeuvres designed to increase intracellular i.j.m.s. ~vc,~.~ wvastroen.ero. ~ july, august. september, ca .. the a- -c inhibited channel was stimulated by an increase in cellular camp (addition of forskolin). this demonstrates that ci secretion across the apical membrane occurs via amp and also by ca * activated pathways. vectorial trans-cellular c i transfer occurs in a basolateral to apical direction. ci" uptake across the basolateral membrane is the rate limiting step in this process and occurs via na § cotransport and/or ci/hco " exchange. these mechanisms of ci" uptake are inhibited by bumetanide and dids respectively. ci secretion across the apical membrane occurs by two separate types of ci-channel (which may be active simultaneously), a camp stimulated ci" channel which is inhibited by a- -c, and a ca § stimulated c i channel which is inhibited by nppb. the understanding of the transcellular mechanism of ci" secretion provided here provides a basic model for ci (and fluid) secretion in human colon epithelium and allows for study of how these processes are affected by neuro-humoral and pharmacologioal agents. gastric carcinoma is the most common malignancy in china, the fourth most common in western.europe and a major cause of cancer morbidity and mortality worldwide. it is well known that metastases, especially distant organ metastases through the blood vascular system, have been the most important cause of patients' death. so far, markers valuable in predicting the metastatic potential of gastric carcinoma have not been identified and the mechanism of metastasis has been unclear. the aim of this study was to determine whether dna ploidy analysis had any objective value in predicting the metastatic potential of gastric carcinoma to distant organs. dna ploidy of gastric carcinomas with metastases ( liver, adrenal, ovary and lymph node) was measured by flow cytometry. dna aneuploidy was significantly related to liver metastases: out of gastric carcinomas with liver metastases were aneuploid ( %) compared with out of ( . %) cases without liver metastases (p< . ); the one gastric carcinoma with adrenal metastasis was also aneuploid. dna ploidy was not related to ovarian or lymph node metastases. the results suggest that an aneuploid dna pattern is a predictor of high risk potential for metastases to the liver and may be a useful tool in the "followup" of patients with gastric carcinoma in detecting those at high risk of developing metastases following surgical resection. liver transplantation (olt) is complicated by acute cellular rejection (acr) in up to % of patients with a peak incidence in the first two weeks following surgery. a diagnosis of acr is suspected on the basis of an acute deterioration in biochemical liver function, confirmed histologically with liver biopsy and associated with recipient t cell activation. to examine a component of the immunological events associated with acr we have assessed the changes in t cell surface molecules and t cell activation status following olt. using flow cytometry a total of twenty four peripheral blood t lymphocyte profiles were analysed in six patients over a four week period following liver transplantation, during which four episodes of histologically confirmed acr developed in three patients. blood was stained with monoclonal antibodies against cd , cd and cd molecules and with dual labelling of the same markers in combination with hla-dr to define the activation status of the t lymphocyte subsets. results: . consistent changes were not observed in the hla-dr negative cd + and cd + populations or the ratio of cd +:cd + cells during acr, a finding which has been equivocal in other studies. . all four episodes of acr were associated with an increase in the activation status (hla-dr antigen expression) of the cd +, cd + and cds+ populations for each patient as compared to baseline: mean % cd +hla-dr+, cd +hla-dr+ and cd +hla-dr+ at baseline and during acr was . vs . %, . vs . % and . vs . % respectively. . successful treatment of episodes of rejection was accompanied by a decrease in the activation status of the three populations: cd +, cd + and cds+. mean % cd +hla-dr+, cd +hla-dr+ and cd +hla-dr+ during acr and following treatment was . % vs . %, . % vs . % and . % vs . % respectively. in two patients no episodes of acr developed following transplantation. in neither of the subjects were there consistent changes in the activation status of the t cell subsets. in conclusion, we have identified consistent patterns of change in t lymphocyte markers following liver transplantation related to significant clinical events. serial measurements of these markers may be of use in evaluating patients following transplantation and may predict those at risk of acr. we have studied haemophiliacs. none presented with liver related disease and all were contacted by postal questionnaire. had haemophilia a, haemophilia b, von willebrand disease (vwd) and one was a carrier. there were males and females, ages ranged from to yr. all had elisa and recombinant immunoblot (riba) testing. tested positive by riba and of these have circulating virus, demonstrated by polymerase chain reaction (pcr). tested negative for hcv. mean alt in the positive groups was . ukat/l, range . to . , in those negative mean was . ukat/l, and range . to . . the positive group were older, (mean age yr, range to yr), than those negative (mean age yr range to yr). conclusions: hcv has been demonstrated in % of this group, which is at the lower end of the reported range. liver function tests reflected the presence of virus. hcv was commoner in older patients, possibly reflecting increased exposure to factor viii. although hcv is a slowly progressive diseases because of the improved life span in haemophiliacs, it is likely to be clinically significant in such patients. whether duodenitis is a distinct clinical entity on its own or a stage in the evolution or resolution of duodenal ulcer disease is still controversial. the aim of our study was to observe the histology of differnt endoscopic patterns of these lesions. the case records of consecutive patients (m:f ratio : , mean age yr, range - yrs) with endoscopic evidence of duodenitis were stud.ied. patients on anti ulcer medication before endoscopy were excluded from the study. all the endoscopies were performed in a single gi unit. biopsies for histology were taken from the endoscopically inflamed areas of the bulb. the presence of helicobacter pylori (hp) infection was checked with the deltawest test. different.endoseopic patterns of duodenitis were observed: patchy (n= , %), diffuse (n= , %), erosive (n=ll, %), and nodular (n= , %). ( %) had normal histology ( / of patchy, / of diffuse, / of erosive and / of nodular duodenitis). in the remaining patients, histology showed acute inflammation in ( %), chronic inflammation in ( %) and gastic metaplasia in ( %). acute inflammation was most frequently seen in erosive duedenitis ( %), and gastric metaplasia in nodfflar duedenitis ( %). of the patients with abnormal histology, deltawest test was positive in cases ( %), most frequently in erosive duodenitis ( % of the cases). conclusion: the endoscopic diagnosis of 'patchy' duodenitis is unreliable and therefore if suspected, should be confirmed on histology. erosive duodenitis is the type most accurately diagnosed endoscopically, most frequently associated with underlying acute inflammation and the presence of hp infection. nodular duodenitis is most commonly associated with underlying gastric metaplasia. non-invasive tests to detect h.pylori are necessary for epidemiological studies, investigation of dyspepsia and for follow-up after eradication treatment. the carbon urea breath test (ubt) is highly sensitive and specific. however, the current recommended protocol for the ubt, including an overnight fast, the use of large reservoir bags, multiple positional changes and sample collections render the test unsuitable for widespread use. the aim of this study was to assess the sensitivity and specificity of a modified, simple ubt. subjects undergoing endoscopy for investigation of dyspepsia or after a course of eradication therapy were recruited. nonfasting patients were administered a standard motility-inhibiting liquid meal ( mls calogen + mls ensure). immediately after, breath samples were collected in duplicate in ml vaeutainers via straws. subjects then received mg of carbon urea dissolved in ml of water. after sitting for min, repeat breath samples were collected in vacutainers. an excess of ~ co excretion of per ml was taken as a positive result. the ubt results were compared with antral and corpus histology (x ), antral and corpus culture (x ) and antral clo test. the "gold standard" was defined by the results of any two of the biopsy based methods. subjects were recruited ( male), mean age yr (range - ). subjects had a positive ubt; true positives and false positives. subjects had a negative ubt; true negatives and false negatives. therefore the sensitivity was . % and the specificity was . %. conclusion: this modified ubt is user-friendly, ideal for use in a primary care setting and is as reliable as other methods for the non-invasive detection of h.pylori. u.c.c. high oestrogen states (pregnancy, the use of the oral contraceptive pill, phases of the menstrual cycle) are associated with hypertension and oedema secondary to salt and water retention. the natriferic steroid aldosterone promotes salt and water retention by activation of the atp regulated potassium channel (karp) via a membrane-bound, protein kinase c dependant mechanism and a slower genomic pathway. we investigated whether oestrogens have similar effects. normal human colonic epithelium was mouhted in' ussing chambers and the transmembrane potential was clamped to omv by application of a short circuit current (s.c.c.) which is then a measure of electrogenic ion transport. the apical membrane was rendered electrically silent by treatment of the mucosal surface with the ionophore nystatin and a mucosa to serosal potassium gradient was established. under these conditions the s.c.c. is a measure of potassium ion transport across the basolateral kaa. p channel. subequent addition of the oestrogen b-oestradiol produced a . + . % increase in s.c.c. (n= ) within min. -a-oestradiol was more effective, it produced a . + . % increase within min (n= ). addition of chloride ions to the apical membrane of the nystatin treated colon causes cell swelling, this activates a calcium regulated potassium channel (kc,) and inhibits the kar r channel. under these conditions addition of -a-oestradiol caused a . + % decrease in s.c.c. after min (n= ). these effects on the ka~ v and the kca channels were abolished by treating the basolateral membrane with the diuretic amiloride (p< . ) or with sodium free krebs (p< . ), this suggests that these effects occur due to alkanization of the cell by stimulation of the na+/h § exchanger. upregulation of the kat p channel promotes salt retention by generating a favourable electrochemical gradient for sodium absorbtion by surface cells. inhibition of the ken channels prevents chloride secretion by colonic crypt cells. chloride secretion causes loss of salt and water from the colon. oestradiol simultaneously activates sodium absorbtion and inhibits chloride secretion thus causing net salt and water retention in human colon. this provides a model for oestrogen effects on salt absorbing epithelia and has important implications for the management of fluid balance disturbances associated with high oestrogen states. the aim of the study was to estimate the incidence of symptomatic ulcerative colitis (uc) and crohn's disease (cd) prospectively from st october to th september in the eastern health board region. cases were defined using the lennard-jones criteria and were identified primarily by systematic examination of hospital medical records. risk factor data was obtained via telephone interviews with the incident cases. data on each patient were collected using a standardised questionnaire by one researcher. a total of uc cases and cd cases were identified during the month study period. using population figures from the census, crude incidence rates for the diseases were . / , in uc and . / , in cd. mean age at diagnosis was . yr in uc (range - yr) and . yr in cd (range - yr). males accounted for . % of uc and . % of cd cases. the presence of blood or mucous in stools was the most frequent presenting symptom in all cases: . % in uc and . % in cd. risk factor data demonstrated that . % of uc and . % of cd cases were smokers (males and females) and that . % of uc and % of cd cases had ever used the oral contraceptive pill (females only). a positive family history of inflammatory bowel disease (in first degree relatives) was found in . % of uc and . % of cd cases. conclusions: this is the first study to estimate the incidence of uc and cd in the irish republic, from a study population of over million people. demographic and risk factor data among irish cases are consistent with previously published studies. these incidence rates may be used as a baseline to detect any change in the risk of inflammatory bowel disease in future studies. apoptosis (a form of programmed cell death) of lymphocytes is an important mode of immune regulation in the mucosal immune system. apopt sis also has a critical role in pathologic states including tumour-derived immune suppression, inflammation and acquired immune deficiency syndrome. the only physiological, receptor-mediated mechanism for lymphocyte apoptosis that has been characterized is the apo-l/ fas (fas) receptor-ligand pathway. we have used a partially purified oesophageal tumour-derived factor to demonstrate the presence of a hitherto unrecognised, non-fas mechanism of apoptosis in mucosal and systemic lymphocytes. the factor is selective, non-toxic, and is inhibitory to activated, proliferative but not resting lymphocytes. the fas antigen-deficient mrlllpr mice and their normal congenic control strain were obtained and bred under i.j.m.s. july, august, september, conventional conditions. the mrliipr mouse has a specific genetic mutation leading to defective apoptosis through the fas pathway and develops lymphoproliferative and autoimmune disorders. a partially purified preparation of oesophageal tumour-derived inimune suppressor factor that has been shown to be free of all known cytokines was tested in dose-ranging studies on cell proliferation and apoptosis using lymphocytes from the mutant and control mice. proliferation was shown using thymidine incorporation and apoptosis demonstrated by flow cytometry (propidium iodide/hoechst reagent). the lpr mice were confirmed to have deficient fas signalling as shown by defective labeling with fitc-tagged monoclonal anti-fas and by defective inhibition of proliferation in functional assays with fas antibody. lymphocytes from the congenic control strain expressed the fas receptor appropriately and had no functional deficit. when the responsiveness of lymphocytes from the two strains of mice to the tumour-derived immunesuppressor were compared, it was found that suppression leading to apoptosis occurred equally in a concentrationdependent manner in both the fas-mutant (lpr) and the faspositive congenic control. conclusion: there exists a non-fas-mediated pathway for triggering apoptosis in lymphocytes as shown by the ability of a human tumour-derived immune suppressor to mediate apoptosis equally in fas-negative (mutant) and fas-positive murine lymphocytes. this system can be now used to probe the non-fas pathway and associated secondary signalling events. the malignancy of colorectal cancer (crc) is related to its ability to disseminate as evidenced by the observation that the majority of crc deaths are due to the development of metastases. urokinase-type plasminogen activator (u-pa) is a serine protease implicated in tumour invasion. we previously employed semi-quantitative immunohistochemistry to demonstrate that u-pa antigen level is of prognostic importance in duke's b colorectal cancer (crc) . the aim was to quantitate u-pa levels in colorectal tissue and to determine the relationship between u-pa level, clinical features, tumour stage, tumour grade and patient survival following crc resection. there were crc cases ( males, females mean age yr, range - ). tissue level of u-pa antigen was measured on detergent extracts of crcs and corresponding colorectal mucosa remote from the cancer using an emsa assay (american diagnostica). there was a wide variation in the range of u-pa levels in normal mucosa and a significant difference between normal colonicand normal rectal levels (p-- . ). results are expressed as ratio of cancer u-pa to normal mucosal u-pa to correct for this variation, u-pa ratio was independent of age (p= . ), gender (p= . ) tumour site (p= . ), tumour size (p= . ) or tumour stage (p= . ). u-pa cancer/normal ratios were greater in poorly differentiated tumours (p= . ). patients with a high cancer/normal u-pa ratio had a significantly shorter survival than those with lower ratio (logrank test p= . ). no. cox regression analysis identified tumour stage (relative risk [rr] . , p< . ) and cancer/normal u-pa ratio (rr . , p= . ) as independent prognostic features. conclusion: u-pa antigen measurement is an independent variable which is predictive of survival in colorectal cancer. in recent years, the efficacy of treatments aimed at the inexpensive, one week bologna regime (omeprazole mg daily + clarithromycin mg b.i.d. + metronidazole mg b.i.d.) and the two week bordeaux regime (omeprazole mg b.i.d. + amoxicillin g~am b.i.d + clarithromyein mg b.i.d.) achieve eradication in - % of cases. there is as yet, however, no standardised approach to eradication therapy. a first line treatment should be reliable, well-tolerated, inexpensive and efficacious. the aim was ) to assess the suitability of the bologna regime as a first line eradication treatment. ) to determine the factors that lead to treatment failure. ) to evaluate the bordeaux regime as a second line eradication treatment. subjects with h.pylori-associated duodenal ulceration (du) or non-ulcer dyspepsia (nud) were recruited at endoscopy. h.pylori status was assessed before and weeks after treatment by histology (antral + corpus x ), culture (antral + corpus) and clo-test (antral); subjects were positive if or more tests were +ve and negative if all tests were -ve. all subjects were treated with the bologna regime. subjects were enrolled ( male), nud and du, mean age yr (range - ). patients completed the followup. h.pylori was eradicated in / ( . %). pre-treatment sensitivities were available in of the patients in whom treatment failed. / ( %) had primary metronidazole resistance, / had metronidazole and clarithromycin resistance and the remaining patient was sensitive to both antibiotics. of the subjects were subsequently treated with the bordeaux regime. h. pylori was eradicated in / ( . %). conclusion. the inexpensive, bologna regime eradicated h. pylori in . % of patients. primary metronidazole resistance is an important factor in treatment failure. the more expensive bordeaux regime is a highly effective second-line treatment. a prospective comparison of laparoscopy and combined imaging (computed tomography and ultrasound) was made in the pre-operative staging of distal oesophageal and gastric cancer in patients who were selected for surgery. patients with clinically overt metastases or a contraindication to major surgery were excluded from the study. all patients underwent chest radiography, computed tomography of mediastinum and abdomen, ultrasonography of abdomen and laparoscopy. of patients assessed consecutively, ( . %) had metastatic disease to the liver or peritoneal cavity, of whom were detected pre-operatively. metastases were detected by laparoscopy in patients, and by combined imaging in (ultrasound , contrast enhanced ct scan ). the distribution of metastases detected pre-operatively were peritoneal cavity in (laparoscopy versus combined imaging ) and hepatic (laparoscopy , combined imaging ). laparoscopy was significantly more sensitive than combined imaging in detecting metastases in patients with adenocarcinoma (lapar scopy , combined imaging ) than in those with squamous cell carcinoma (laparoscopy , combined imaging ). conclusion. patients with cancer of the oesophago-gastric region who are selected for surgery, have metastases to the peritoneal cavity more frequently than to the liver. these metastases are more frequently detected by laparoscopy than by imaging methods. the addition of laparoscopy to the staging protocol prevented unbeneficial thoraco-abdominal exploration in patients. laparoscopy should be used in the assessment of these patients immediately prior to performing excisional surgery. laparoscopic surgery has appeal for the performance of intraabdominal procedures with minimal invasion, however the range of its applicability requires further definition. the utilisation of laparoscopy in colorectal surgery has been limited by concern regarding the adequacy of 'clearance' of malignant disease with this technique. a series of patients in whom laparoscopic mobilisation of the colon was performed to assist colectomy, facilitating a less invasive abdominal incision, is reported. from july , patients have undergone a laparoscopically-assisted colectomy (lac). ( males, females; mean age: . yr, range: - yr). the indication for surgery and the procedure performed are listed in the august, september, with the patient in the lloyd-davis position, pneumoperitoneum was induced in the standard fashion. laparoscopic access was via ports ( ram x ; mm x ). depending on the procedure, the splenic flexure alone, or with the transverse colon and hepatic flexure, was mobilised laparoscopically. thereafter, access was via a transverse lower abdominal incision for completion of the procedure. in all patients, colonic mobilisation was successfully achieved at laparoscopy. there were no complications related to this procedure. in patient, a major pelvic bleed occurred during the open dissection. mean operating time (total) was . min (range: - ). colonic mobilisation was completed in less than hr in all cases. patients developed specific postoperative complications (anastomotic dehiscence: , pouch sepsis / excision: , pelvic abscess: ). initial results indicate reduced post-operative respiratory complications, analgesia requirements and hospital stay in patients undergoing laparoscopically assisted colectomy. colonic fluid hypersecretion is an important and life threatening consequence of diseases such as inflammatory bowel disease and infectious diarrhoeas.-secretion of chloride ion occurs in the crypts while sodium and water follow passively. the chloride ions leave the cell through an ion channel, this involves a net loss of positive ion from the apical membrane which must be balanced by an exit of positive across the basolateral membrane. in this studywe investigated the role of basolateral potassium channnels in regulating charge balance thus maintaining secretion of chloride and ultimately salt and water. samples of colon were obtained from humans, rabbits and mice. sheets of isolated epithelium were mounted in ussing chambers. the short circuit current was measured under voltage clamped conditions. bathing solutions were adapted to study chloride secretion and potassium recycling in isolation. addition of forskolin (a secretagogue which increases intracellular camp) produced an immediate and sustained increase in chloride ion secretion in human (a s.c.c. = + ua/cm n= ) rabbit (a s.c.c. = + uajcm n= ) and mouse (a s.c.c. = • ua/cm ? n-- ). treatment of the basolateral membrane with umol tetra-pentyl-ammonium (t.p.a. an inhibitor of the kca channel) produced a complete inhibition of the response in all three species, wheras tolbutamide (an inhibitor of the kar p channel) produced less than % inhibition in all colons. addition of carbachol (a secretagogue which increases intracellular calcium) produced an immediate but transient increase in chloride secretion which was completely inhihited by pre-treatment with t.p.a. chloride secretion in human and other mammalian colons is dependent upon potassium exit through the kca channel on thc basolateral membrane, this is essential to maintain charge balance within thc cell. fluid secretion into the lumen of the colon may be prevented by inhibition of these potassium channels. the basolateral kc~ channel may be a useful target for the pharmacological control of fluid secretion in the colon. anal canal pressures may be raised in patients with acute anal fissure. a prospective study of the effect of parturition on the pelvic floor has allowed study of the hypothesis that anal sphincter hypertonia predisposes to postpartum anal fissure. primigravid women were studied. anal manometry was performed six weeks before and after delivery in women. postpartum studies only were performed on a further women. a bowel symptom questionnaire and detailed obstetrical record were kept. anal fissure was diagnosed on history and examination. ( . %) women developed postpartum anal fissure. antenatal anal canal pressures were identical in those who did and did not develop fissure. postpartum resting and squeeze anal pressures were decreased equally (table). postnatal constipation was more common in those with fissure ( %) than those without ( %, chi . , p< . ). caesarean section ( %), operative delivery ( %), perineal tear ( %) or epidural anaesthetic ( %) did not effect the incidence of fissure. conclusion. anal canal pressures are normal prior to development of postpartum anal fissure, and decrease postpartum. gastrointestinal (gi) infection is a common cause of morbidity in young animals. evidence is emerging that passive oral immunisation of the g.i. tract with specific immunoglobulin (ig) provides protection against intestinal pathogens. hens immunised against such organisms lay eggs containing large amounts of specific ig. the aim of this study was to test the ability of specific hen egg ig to protect against salmonella infection, in the young calf. twelve isa brown hens were immunised with heat inactivated salmonella. an elisa was developed to measure specific ig levels, which reached a peak days post immunisation. specific ig rich eggs were collected, the yolks harvested and pooled. an animal model of salmonella induced gastroenteritis was developed using young calves. forty two animals were divided into three groups. two of these groups acted as controls, one receiving no egg yolk supplement, while the other received normal egg yolk from non-immunised hens as a supplement. the third group received a supplement of specific-ig rich egg yolk from immunised hens. after five days each animal was orally challenged with x live salmonella. in the groups of control animals, / animals given no egg supplement, and no. / animals fed normal egg, succumbed to infection within three days of challenge. however, in the group fed ig rich eggs, only / animals succumbed to infection after challenge. these preliminary results suggest that specific hen egg yolk ig has a potential use in passive oral immunisation against gi infections. an inverse relationship between appendicectomy and ulcerative colitis (u.c.) and smoking and u.c. has been proposed. our study examines the frequency of common surgical interventions and of smoking in a group of ibd patients attending a twice weekly g.i. clinic. patients were interviewed in a prospective, questionnaire based case control study. the subjects comprised patients with u.c., patients with crohn's and controls derived from aia orthopaedic traumatology clinic matched for age, sex and socio-economic group. subjects were questioned on all previous surgery and on smoking history. the appendicectomy rate amongst controls was . % ( / ) which was significantly greater than that of u.c. patients . % ( / p < . ) of the u.c. patients had appendicectomy prior to disease onset. the appendicectomies were evenly distributed through the categories of disease extent (proctitis, left sided and extensive colitis). there was no significant difference in appendicectomy rate between crohn's patients and controls. the groups had comparable rates of tonsillectomy and cholecystectomy. . % of the u.c. patients were non-smokers at the time of diagnosis in contrast to . % amongst the crohn's patients (p < . ). conclusions: u.c. is associated with significantly lower appendicectomy rate than in controls. this is not true for other surgical procedures unrelated to the treatment of u.c. the role of the appendix in the evolution of u.c. requires definition and clarification with large multicentre studies. the immune response to gliadin has been proposed to play a central role in th e pathogenesis of coeliac disease. in this study, sensitivity to gliadin was investigated in coeliac and normal individuals. pbmcs were cultured with soluble gliadin and latex bead coated gliadin (latex-gliadin) at varying concentrations. ppd was included as a control antigen. culture supernatants were removed after three days and cytokines (il ,il ,il .ifn)') measured using capture elisas. proliferation was assessed on day by measuring thymidine incorporation. gliadin induced proliferation in cell cultures from of twenty coeliac and of fourteen normal individuals. il production was seen in of thirteen coeliacs and of nine normal controls and these responses were independent of proliferation. gliadin failed to induce il or il production. whereas ppd induced ifn'r production in association with proliferation in all individuals, gliadin induced ifn), production was less frequently observed. of cd patients, four (all with active disease) demonstrated gliadin induced ifn t production (with both soluble and latex-gliadin). in the normal controls an ifn~ response was only observed with latex-gliadin in two of individuals tested. latex-gliadin also seemed more potent at inducing proliferation with subjects ( normals, coeliac) responding to this preparation alone. in preliminary experiments, cells cultured with gliadin for ten days when restimulated with gliadin demonstrated a suppressed response: in this cell proliferation, il and il production was inhibited but ifn~/ was increased. these results indicate that gliadin specific t cells are present in the peripheral blood of both coeliac and normal individuals. it is postulated that cells may be of the th phenotype in normal subjects and coeliac patients with inactive disease (due to the observed increase in il in the absence of ifn~t production) whereas in patients with active disease thl responses may predominate. adult coeliac disease in an irish provincial hospital b. twohig, k. gaynor, r e o'regan. st. joseph's hospital, clonmel. previous reports on adult coeliac disease in ireland came from teaching hospitals. these often serve as tertiary referral centres with a possible resulting different patient type from hospitals serving a district general type function. we report on all patients diagnosed over a year period in a county hospital serving a mixed urban/rural population of about , . a retrospective study of all patients newly diagnosed with coeliac disease between / / and / / who'were over the age of years was carried out. the sex, age, delay from onset of symptoms to diagnosis and mode of presentation were established and lab. parameters included haematology screen, serum calcium, serum albumin and specific antibodies were also recorded. there were patients diagnosed over the years at a mean age of (range - yr). the duration of symptoms prior to diagnosis ranged from three weeks to yr (average months). those with mild non specific symptoms and in particular females, were most likely to have long delayed diagnosis. the principal modes of presentation were diarrhoea ( %), weight loss ( %), anaemia ( %) of which % was iron deficiency, hypocalcaemia ( %), steatorrhoea ( %). in addition to haematological abnormalities, other abnormal lab. tests included low serum calcium in of tested, low serum albumin in of tested. alpha gliaden antibodies were raised in and normal in patients with definite coeliac disease before starting on a gluten free diet. endomysial antibodies in our population also seemed to be a poor marker for the condition, being raised in only of patients with definite coeliac disease before starting on a gluten free diet. conclusions: in adults the symptoms of coeliac disease may be non specific but certain symptoms such as diarrhoea with or without weight loss, inadequately explained anaemia (particularly iron deficiency) and hypocalcaemia should suggest early small bowel biopsy to rule out coeliac disease. our study demonstrates a very long delay in diagnosis in some patients and particularly in women of child bearing years with anaemia. _oc.e.j _j_astroen_ero_ooj july, august, september, despite early optimism alpha gliaden antibodies have poor specificity and sensitivity in adult patients with coeliac disease in our area and it appears that endomysial antibody may not be any great improvement. activated mos produce -and tnf, known tumouricidal mediators. tims have been reported to demonstrate impaired production of these mediators suggesting that these effector cells are supressed. we postulated that tim cytotoxicity is not dependent on the release of these cytotoxic agents but is mediated via the release of the enzyme-arginase. currie et al have demonstrated that arginase is cytotoxic to tumour cells by depletion of the essential amino acid l-arginine, therefore the aim of this study was to determine the role of this enzyme in colorectal tumour-derived mos. human peripheral blood monocytes (pbm) were isolated from aged-matched controls (con) and from blood pre-operatively obtained from patients undergoing surgery for colorectal cancer. colorectal tumour specimens ( - gm) were freshly procured and digested. the percentage of tims was then assesse d flow cytometrically, and both pbms and tims were seeded at a concentration of x / well. cytotoxicity was assessed using a standard cr release assay against the sw colorectal cell line. tnf, -generation and cytosolic arginase were also assessed. these results indicate that both pbm and tims have impaired production of tnf and o -, however, cytotoxicity is enhanced above con and is associated ~,th a paralleled increase in arginase activity. the arginase-dependent nature of this cytotoxicity was confirmed by addition of the inhibitor l-lysine which significantly abrogated cytotoxicity in all groups. these results suggest caution in the use of the arginase substrate larginine in the management of colorectal cancers. m~=macrophage, o -=superoxide anion, tnf=tumour necrosis factor, tims=tumour infiltrating macrophages. grosvenor road, belfast bti bj. the antitumour effects of l-arginine has been demonstrated in a number of experimental models including mammary and transplantable solid tumours. this study was to investigate the effect of dietary arginine in procarcinogen , dimethylhydrazine (dmh) induced colorectal cancer model. colorectal tumours were produced in male wistar rats by weekly subcutaneous injections of dmh at a dosage of mg/ kg body weight. l-arginine was given in a % solution of drinking water. animals were sacrificed weeks after the last dmh injection. animals were divided into groups. group i was given dmh injections, group ii was given dmh and % arginine for weeks, group iii dmh and arginine for the first weeks only, group iv dmh and arginine for the last weeks. fourteen out of animals developed tumours in group iii compared to out of in group i (p= . , yates-corrected chi test), out of in group ii and out of in group iv. decreased incidence concomitant with reduced tumour area and volume were found only when dietary arginine was given in the first weeks of colorectal carcinogenesis i.e. during the initiation stages. arginine supplementation during the promotion phases appeared to significantly enhance the tumour burden. conclusion. these results suggest that dietary arginine may be used for tumour prevention only in the early stages of colorectal carcinogenesis before tumour formation but that its effects may be harmful in the later stages of tumour development. early experience with interferon therapy in haemophiliacs chronically infected with hepatitis c suggested that they respond as well as non-haemophiliacs to interferon alpha. the aims of this study were to examine the effects of interferon therapy in hepatitis c in an irish haemophiliac population. we prospectively studied haemophiliac patients who received courses of interferon alpha b (ifn), using hcv-rna pcr to guide dosage and duration of.therapy as well as to determine virological response. all patients were asymptomatic, hcv riba and pcr positive, and had abnormal liver enzymes. seven had concomitant asymptomatic hiv infection; none had hepatitis b. genotyping in patients revealed type in , type in and type in . treatment was initiated with ifn - mu s.c. alt die and increased to a max of mu if lft's remained raised or pcr +ve after months. blood was drawn monthly for liver enzymes and pcr. responders were encouraged to continue treatment for months. three patients had sustained biochemical normalisation and negative pcr on treatment of which remains in remission year off treatment. fourteen became virologically negative by pcr on treatment but relapsed within months of stopping interferon. three were retreated with interferon and again responded while on treatment. twelve had no evidence of biochemical or virological response. we conclude that response to interferon is common in an irish haemophiliac population. however, sustaine d virological and biochemical response rates appear <t % using interferon monotherapy in this cohort. these results suggest that either combination therapy or maintenance therapy might be worthwhile treatment options. arginine is used therapeutically in humans to correct hypertensive states, but has been shown in high doses to induce pancredtitis in rats. as arginine is a producer of nitric oxide (no) this has been thought to be the cause of the pancreatitis. this study was designed to test this hypothesis. mg per g body weight of arginine was injected intraperitoneally either alone or with nitric oxide synthesis blockers ( -name and -nna) or a breakdown inhibitor (superoxide dismutase-sod). sprague-dawley rats were studied for or hr, before estimation of serum amylase, wet:dry lung weight ratio and histology of lung and pancreas. results are as shown; the relevant inter-group comparisons will be discussed. the neutrophil (pmn) has been implicated in sepsis-induced tissue injury. this may result from pmns dying by cell lysis rather than apoptosis (apop) with extrusion of deleterious cellular contents. the principle role of the pmn is to ingest and kill bacteria and we have previously shown that the ingestion of e.coli results in the induction of pmn apop. during systemic inflammatory response syndrome (sirs), there are elevations in both the acute phase and heat shock proteins, however the effects of these on e.coli induced pmn cell death is unknown. so the aims of this study were to determine the effects of heat shock and acute phase responses on e.coli induced pmn apop. pmns were isolated from healthy volunteers. x pmn/ ml were divided into three groups, control, lps ( ng/ml for one hour) or heat ( ~ for min). pmn activation was assessed after lps co-culture by ingestion of e.coli and cd lb expression. heat shock protein was assessed in the heated group by western blotting. lps resulted in a significant increase in both ingestion ( recent reports suggested that appendicectomy may protect against the development of ulcerative colitis. depletion of mucosal helper t ceils is the suggested mechanism. since mucosal t cell activity has an even more critical role in the pathogenesis of coeliac disease, we investigated whether the development or mode of presentation of this disorder is influenced by appendicectomy and/or tonsillectomy. one hundred and fifty consecutive adult coeliac patients diagnosed at three hospitals by the accepted international criteria were studied [ females and males; mean age at diagnosis (median , range - ) yr]. the control group consisted of consecutive patients examined at orthopaedic clinics in the two geographic areas [mean age yr (median , range - )]. lj.m.s. july, august, september, contrary to the findings in ulcerative colitis, where appendicectomy was rare before the onset of the condition, of our coeliac patients had undergone appendicectomy before the diagnosis of their disease, in comparison with of the controls (p=ns). similarly, there was no significant difference in pre-diagnosis tonsillectomy rate ( of patients vs of controls). in the coeliac patients, the age at presentation did not differ between those who had previous appendicectomy [mean (median ) yr] and those who did not have previous appendicectomy [mean (median ) yr]. presenting features, predefined in the study protocol, were compared in the appendicectomy and non-appendicectomy coeliac patients and did not differ significantly [diarrhoea/ steatorrhoea ( % vs %), weight loss ( % vs %), anaemia ( % vs %), dermatitis herpetiformis ( % vs %), other ( % vs %)]. conclusion: resection of mucosa-associated lymphoid tissue (malt) does not influence the occurrence or the mode of presentation of adult coeliac disease. therefore, in this classical example of a t cell-mediated disorder, the hypothesis that surgical depletion of mucosal t cells .has a protective effect or influences the clinical manifestations cannot be supported. abnormal gut permeability to enteric flora or their toxins is considered to be an aggravating factor in a variety of illnesses. this study investigates the effect of isolated lower limb ischaemia-reperfusion injury on gutpermeability. male wistar rats (n= ) were randomised into three groups: control, hr of bilateral hind limb ischaemia alone and hours of bilateral hind limb ischaemia followed by hr of reperfusion. at the end of the experiment blood samples were taken for measurement of systemic endotoxin concentration. four sacs from the terminal ileum ( cm each ) were removed, evaginated over a glass rod, tied at both ends and filled with a stock solution of polyethylene glycol (m wt. ) in bicarbonate buffer. the sacs were then placed in a beaker filled with ~ c-labelled polyethylene glycol in buffer solution and incubated for one hr in a shaking water bath at ~ and continuous oxygenation. at the end of the incubation period /a samples were taken from the each sac and its radioactivity counted. the count was then calculated as a percentage of the radioactivity in the incubation flask and taken as a measurement of.sac permeability to polyethylene glycol. .l the development of tolerance following orthotopic liver transplantation [olt] has always been of interest and mechanisms postulated to explain its development include clonal deletion and peripheral t lymphocyte anergy. although donor chimerism is traditionally associated with successful outcome following allogeneic bone marrow transplant, a partial or mixed chimeric state may exist following solid organ transplantation, and patients who exhibit stable mixed haemopoietic chimerism may not experience acute cellular rejection following oui'. haemopoietic chimerism has been suggested as a principal component in the development of tolerance after olt, i.e. two way cellular trafficking from the donor organ to recipient and vice versa. the aim of this project was to examine the incidence of chimerism after olt using a polymerase chain reaction [pcr] based method for microsatellite analysis of highly polymorphic simple tandem repeat [str] sequences. the number of repeats varies between individuals and can be used to identify haemopoietic cells from different individuals. patients were studied prospectively, and retrospectively to months after olt. dna was extracted from the donor organ and explanted organ at the time of transplantation. dna was extracted from peripheral blood at serial points post olt. donor and recipient ceils were examined for chimerism by pcr-based amplification of strs using a panel of microsatellites which included cyp- , int- and vwf. a radioactive label, (~ p was incorporated into the pcr reaction. following amplification, gel electrophoresis was performed and subsequent autoradiography identified the amplified strs. one patient, who required retransplantation following hepatic artery thrombosis, has demonstrated complete donor chimerism at and months post olt, which may reflect the presence of haemopoietic progenitor cells in the donor liver. this patient did not experience cellular rejection after olt. a second patient exhibited transient chimerism weeks post olt. three patients studied retrospectively at , , and months post olt did not demonstrate peripheral blood chimerism. conclusions: preliminary results indicate that chimerism occurs following olt, and that pcr based microsatellite analysis is a sensitive method of detecting chimerism. donor derived lymphocytes may exert beneficial immunomodulatory effects in olt recipients. the highest reported prevalence of coeliac disease is found in the west of ireland. in this population, an extended major histocompatibility complex relating to coeliac disease has been determined, which includes human leucocyte antigens hla-a and b , determined by a single gene on chromosome . secretion of blood group antigens including abo and lewis blood groups is determined by a single gene on chromosome . a possible increased prevalence of non-secretors amongst the coeliac population has recently been described. using lewis (le) antigen,'phenotype, secretor status can be inferred. of coeliacs who had either le(a) or le(b) antigens, ( %) were non-secretors le(a+,b-), compared with % of disease free controls (p= . , odds ratio = . . % ci . - . ). % of coeliacs had the recessive lewis phenotype le(a-,b-) versus % of controls. hla-a , b and non-secretion of lewis antigens were unrelated. an increased prevalence of complications and associated autoimmune disease was found in the non-secreting and recessive coeliac groups. the relationship between the non-secretor state and coeliac disease is not confirmed in this study, nor has an increased prevalence of class hla markers been demonstrated in non-secretors. the finding of an increased prevalence of complications and auto-immune conditions in the recessive and non-secretor groups may prove useful as a predictor of disease severity in coeliac disease. endoscopy findings which will be submitted separately for reporting in detail at this meeting showed no significant difference between the open access and the comparator group. conclusion: uncensored open access o.g.d. considerably increased the endoscopy burden, it provided a disappointing reduction in delay time from referral to o.g.d., it significantly reduced out patient visits and for the majority allowed the g.p. to control management. because we underestimated the human and technical resources required to provide this service, the increased workload without adequate resources, damaged staff morale and cooperation and resulted in a general reduction in the level of service offered by the unit. patients referred for uge are usually assessed first by the medical team in the outpatients. apart from extra cost, this sometimes involves unnecessary delays in diagnosis and treatment. an open access to the endoscopy unit can only solve this problem if this facility is used appropriately by the gp's. we compared direct referrals by gp's with outpatients referral by the medical team. the case records of consecutive patients (m:f ratio ll : , mean age yr) were analysed. all were referred for endoscopy to a single unit, either directly by the g.p's -group (n= , m:f ratio : , mean age yr) or by the medical team from the outpatients -group (n= , m:f ratio : mean age yr). patients referred for repeat endoscopy, inpatient referrals and casualty referrals were excluded from the study,-the findings on endoscopy and the duration of symptoms and drug treatment prior to endoscopy were compared between the two groups. in group l, patients had peptic ulcer disease (duodenal ulcer n= , gastric ulcer n= ), had either hiatus hernia or inflammatory lesions (oesophagitis/gastritis/duodenitis, alone or in combination), and had normal findings. the mean duration of symptoms before endoscopy was months. / ( %) patients received some sort of acid blockade treatment prior to endoscopy. in group , patients had peptic ulcer disease (duodenal ulcer n= , gastric ulcer n= ), had either a hiatus hernia or inflammatory lesions and had normal endoscopy. the mean duratioti of symptoms before endoscopy was weeks. / patients ( %) recieved acid blockade prior to endoscopy. no significant differences between the two groups were observed. conclusion: an open access endoscopy unit can help cut down extra cost and unnecessary delays in diagnosis and management. helicobacter pylori (h. pylori) leads to gastric cancer through im is subdivided into three subtypes, type i, type ii and type iii. type i may progress to type iii, the latter being closely associated with adenocarcinoma. the aim of this study was to determine the relationship between the subtypes of im and h. pylori in the gastric antral mucosa. patients undergoing endoscopy at the meath/adelaide hospitals were enrolled. the aldehyde fuchsin -alcian blue technique was used to identify the subtypes of im no significant difference in the prevalence of type i was found between h. pylori positive patients n= ) and h. pylori negative patients (n= ). however, the prevalence of type ii and type iii was significantly lower in the h im subtype - . pylori +ve h. pyori -ve conclusions: with the progression of im, the prevalence of h. pylori decreases. this may be due to the spontaneous clearance of the bacteria in the premalignant stomach. this study suggests that im, once present, will progress independent of h common bile duct stones: size and shape: a radiological review departments of gastroenterology and radiology ~ st. james's hospital however, since small (< mm) faceted common bile duct stones (cbds) are amenable to endoscopic sphincteroplasty, a potentially safer means of duct clearance than sphincterotomy, we decided to perform a radiological review of our common bile duct stones. hence the size (maximum diameter largest stone), number and shape of common bile duct stones at e.r.c.p. in consecutive patients ( females: males; mean age . yr; range - yr.) were reviewed independently by two radiologists. correcting for an x-ray magnification factor of . x, the mean stone size was . ram key: cord- -hg xsyr authors: buttery, philip c.; barker, roger a. title: gene and cell-based therapies for parkinson’s disease: where are we? date: - - journal: neurotherapeutics doi: . /s - - - sha: doc_id: cord_uid: hg xsyr parkinson’s disease (pd) is a neurodegenerative disorder that carries large health and socioeconomic burdens. current therapies for pd are ultimately inadequate, both in terms of symptom control and in modification of disease progression. deep brain stimulation and infusion therapies are the current mainstay for treatment of motor complications of advanced disease, but these have very significant drawbacks and offer no element of disease modification. in fact, there are currently no agents that are established to modify the course of the disease in clinical use for pd. gene and cell therapies for pd are now being trialled in the clinic. these treatments are diverse and may have a range of niches in the management of pd. they hold great promise for improved treatment of symptoms as well as possibly slowing progression of the disease in the right patient group. here, we review the current state of the art for these therapies and look to future strategies in this fast-moving field. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. the long-held promise of gene and cell therapies (gcts) for parkinson's disease (pd) is slowly starting to emerge as a realistic prospect. in the last decade, the first in vivo gene therapies (gts) for non-neurological conditions have met regulatory approval initially in the form of glybera in for hereditary lipoprotein lipase deficiency. this was followed not long after by luxterna in for the eye disorder leber's congenital amaurosis and zolgensma in for spinal muscular atrophy [ ] [ ] [ ] . the conditions that these therapies are aimed at are however all rare (or "ultra-rare"), and for this reason, such gts are unlikely to be transformative to substantial healthcare sectors, and their commercial viability has been questioned. in contrast, pd is a relatively common neurodegenerative disease that, owing to its debilitating long-term effects, carries a large socioeconomic impact. as such, gcts for pd could be transformative not only for the disease itself but also for the pharmaceutical economy. primarily a central nervous system (cns) disorder, the dominant effects of pd are on movement and cognition, and in early disease motor features correlate tightly with degeneration of dopaminergic neuronal projections from the substantia nigra pars compacta (snc) to the striatum. therapeutic responses to pharmacological agents, such as oral dopamine (da) precursor medications (levodopa), are often satisfactory in the early years after diagnosis, but become steadily less efficacious as the disease progresses. within years of disease onset, the progression is such that responsiveness to levodopa and other oral dopaminergics is unreliable, and additional da unresponsive features may begin to dominate the clinical picture. these features can also be aggravated by side effects from the dopaminergic therapies the patient is taking, for example postural hypotension and neuropsychiatric problems. the problems faced in treating advanced disease, coupled with the predictable progression and anatomically defined nature of the underlying da deficit, has led to the promotion of gcts as an attractive prospect for improved symptomatic treatment of pd, and potentially also for disease modification. one of the main burdens of living with pd are the fluctuations in motor performance that emerge and then worsen from years after diagnosis, such that after years of the disease, % of patients have motor fluctuations, and % have levodopa-induced dyskinesias [ ] . loss of striatal dopaminergic innervation is considered a major factor in the development of these clinical features, and da replacement strategies that more successfully achieve continuous exposure of striatal neurons to da (such as infusion therapies (it)), rather than the inconsistent levels provided by oral therapies, have demonstrated a clear ability to smooth out these fluctuations. the currently widely used its (reviewed in [ ] ) include the following: & levodopa-carbidopa intestinal gel (lcig [ ] [ ] [ ] ), which is administered by a pump and catheter enterically (intrajejunally), so bypassing gastric emptying. & apomorphine, an agonist for da receptors, which can be administered via subcutaneous infusion, so bypassing the enteric system entirely [ ] . over the last years, however, deep brain stimulation (dbs) has risen rapidly to become the gold standard for treating worsening motor fluctuations in patient who remain levodopa responsive but free of the more severe gait and cognitive issues of advanced pd [ ] . by providing round the clock electrical stimulation to the subthalamic nucleus (stn), this technology acts to revert neuronal activity within the movement circuitry from the abnormal pattern that arises with da depletion, to a pattern more typical of the da replete ("normal") state [ ] . using different strategies, therefore, both its and dbs achieve a smoother da response than oral therapies, with consequent beneficial effects on quality of life. however, each of these options carries its own problems. apomorphine is frequently poorly tolerated due to side effects, cognitive issues, or local injection site problems. lcig, often used in patients too elderly or frail to consider dbs, is prone to problems of delivery, for example relating to tube dislodgement or blockage. dbs carries not only immediate surgical risks, but also risks of stimulation-related speech and swallowing difficulties and of device infection. moreover, each technology has very significant recurring expenses either on a daily basis or in the form of infrequent but expensive hardware maintenance and replacement for dbs. although dbs technologies are still evolving, with promised improvements in targeting, tuning of stimulus effect, and battery life [ ] , there is nonetheless a "gap in the market" that gene and cell therapies (gcts) are well placed to fill. an attractive aspect of gcts is their promise of a one-shot solution, without the down sides and recurring costs of infusion therapies and dbs. additionally, by being targeted to the predominant site of pd pathology, they may be less prone to the off-target side effects seen with its and to a lesser extent with dbs. an ultimately higher goal is the potential for gcts to slow disease progression, or even to repair aspects of the neurodegenerative changes that occur in pd brain-for example, by using neuronal cell therapies that can release da in a normal synaptic fashion where it is needed. from the perspective of gct technology development, pd has both attractions and hurdles. on the plus side, it is a common condition that is hugely costly to treat by conventional means, and that is also increasingly well understood at a pathological and anatomical level. on the other hand, both its and dbs are now well-established and effective treatments, meaning that the bar to improvement is high. as such, the financial risks for companies trying to dethrone these proven technologies are not trivial. of the various viral vectors that have been investigated over the years, two have emerged as worthy of translation into the clinic for pd. these are adeno-associated viruses (aavs) and lentiviruses (lvs), and they will each be discussed separately. adeno-associated viruses have probably received the lion's share of attention for use as vectors for gt in pd. they are small ( . kb), non-enveloped, single-stranded dna viruses, of the parvovirus family, and have been considered as potential vectors for gene therapy for over two decades [ ] [ ] [ ] . as a family, they are able to infect a wide range of tissues, including both neural and non-dividing cells [ , ] , with long-term transcription of cargo sequences obtained episomally, without integration into host genome [ , ] . multiple aav serotypes exist and have varied tropism for different species and cell populations, meaning different serotypes have been used to target different tissues [ ] , with the aav serotype in particular showing robust and long-term neuronal expression in both animals and humans ( [ -- ] , reviewed in [ , , ] ). the main drawbacks of aavs are the limited size of transgene they can carry, the ability to upscale their manufacture, and also the theoretical issue of a pre-existing immune response to them in some patients. however, cargo size is only a challenge for certain gt strategies (as will be discussed below), and new methods have now largely resolved previous technical difficulties related to manufacturing large quantities of high quality virus [ , ] . the immunogenicity of aavs however remains debated. although this has not so far emerged as a consistent problem in the immune privileged cns, it is recognized that aavs can provoke immune responses that vary with serotype and that may limit the effectiveness of systemic treatments, or prevent repeated dosing [ ] [ ] [ ] . where more serious immune responses have been reported in the brain in animal models, they have been attributed to the non-native transgene in some cases (e.g., intracerebroventricular injection aav with green fluorescent protein [ ] ). however, it is also possible that the aav serotype is particularly susceptible to detrimental side effects under certain delivery conditions, possibly because of its ability to transfect a wide variety of cells types. thus, intravenous and intracisternal delivery in animal models has been reported to cause a sensory neuronopathy of variable severity [ , , ] . interestingly, perez et al. report that use of species-specific transgene may prevent this outcome, implying that an anti-transgene immune response may contribute in some cases, but autoimmune and toxic causes have also been speculated [ , ] . the reports have been concerning enough to put a halt to further use of the intrathecal route for delivery of zolgensma (aav ) for spinal muscular atrophy-a treatment that received licensing for human use (intravenously) in [ , ] . the use of aav in pd has to date not found similar issues, and this serotype has also been employed to carry the rpe gene in luxterna for eye disease (leber's congenital amaurosis) [ ] [ ] [ ] . it has also been used experimentally in children, in treatment trials for the rare conditions aadc deficiency [ ] and canavan's leukodystrophy [ ] . meanwhile, aav has been used in another approved gt, glybera, for hereditary lipoprotein lipase deficiency hlld (aav ) [ ] . the second vector type in current use are lentiviruses. these are markedly different to aavs, being enveloped rna retroviruses that obtain long-term expression through genomic integration. retroviruses have long been used in the biological sciences for non-clinical work, owing to their ease of manipulation and also their stable, regulatable expression. the main advantage of lvs over other retroviruses is their ability to infect postmitotic cells, including neurons, which facilitates their use as vectors for neurons in vitro. this has made them attractive candidates for use in cns diseases. the best known lentivirus is the human immunodeficiency virus (hiv), but the first to be taken forward towards human gt use was a non-human lv, the equine infectious anemia virus (eiav). this was engineered to include minimal viral sequences, with proteins required for making up the viral particle (gag) and replicatory enzymes (pol) being supplied "in trans" in the packaging process, so not incorporated in the genome of the resultant particles [ ] . these engineered viruses are thus replication defective and contain less than % (or < kb) of the original viral genome; the only protein expressed by the integrated dna within the target cell is the cargo protein of choice-from the transgene cassette. the tissue tropism of lvs tends to be quite broad, and they have proven to be less flexible than aavs, but the engineered eiav in current use is fully neurotropic, being pseudo-typed with the envelope protein of vesicular stomatitis virus (vsv-g), which facilitates a broad tissue (and species) tropism. other pseudo-typing has been used with different tissue tropisms [ , ] , but for fuller tissue specificity, lvs have the cargo capacity to include tissue-specific enhancers [ ] , something not possible in aavs. a theoretical concern with lvs is that the genomic integration that is integral to their function and also provides a potential for oncogenicity. such oncogenicity has been seen with some gts, but only for retrovirus-mediated gene delivery to bone marrow populations for immunodeficiency disorders [ ] [ ] [ ] [ ] . the key difference here from the eiav technology is that the viruses employed were not lvs, but γ-retroviruses, which have a distinct mode of integration into the host dna that makes them more prone to inducing oncogenicity [ ] . in addition, the transduced cells were actively proliferating, rather than postmitotic, and so are probably given a selective growth advantage, promoting the development of additional mutations and oncogenicity. such considerations make it unlikely that lv transduction of cns neurons will provoke oncogenesis, although some uncertainty around this issue must remain. the target of the current gts for pd is the motor circuitry that is deprived of dopaminergic innervation as a result of the degeneration of the nigrostriatal (ns) projection. there are however several potential approaches by which gt can be used to manipulate this pathological circuitry, so improving patient symptoms. the three main strategies are described below (see also fig. ): . gene therapy to modify motor circuitry: circuit modification by gt uses gene delivery to a critical node within the motor circuitry of the basal ganglia to provide a biological effect similar to dbs. . gene therapy to synthesize da: da-synthetic gt aims to restore the da synthesis capacity in the tissue where it is needed, in this case the putamen-the major target of the degenerating ns projection. . gene therapy to modify disease: growth factor (gf) gt uses gfs to boost survival and function of residual ns neurons, so slowing disease progression in this pathway and supporting residual da production. each of the approaches has potential benefits and drawbacks, and all have now been trialled in patients with pd. for each, aav and lv vectors might be considered as closely equivalent options, given that both have demonstrated longterm expression in humans over periods of years-up to years for lvs in ocular gene transfer of endostatin/ angiostatin for macular degeneration [ ] , and over years according to postmortem data on aavs in pd [ ] . aavs are though the more established technology, having been used in the majority of trials to date. lvs have so far been used exclusively for one of two da-synthetic approaches, but if regulatable expression becomes useful or necessary in due course, then they may well find roles in other areas [ ] . the da depletion that characterizes pd results in profound changes in the motor circuit of the basal ganglia and related structures. these changes derive from da's role as a modulator of neuronal activity (neuromodulator), which affects different pathway activities in distinct ways. for example, the direct and indirect pathways are respectively activated and inhibited by da, resulting in loss of the normal balance of activity between these two pathways as da levels decline. allied to this, the subthalamic nucleus (stn), a key node in pd normal the circuit, shows heightened synchronized activity in the absence of da, with a pattern that is dominated by low frequency neuronal spiking [ , ] . this synchronized hyperactivity, so-called β band activity, is felt to be specifically detrimental to motor performance, being tightly correlated with some clinical features of pd, including slowness (bradykinesia), stiffness (rigidity), and tremor. the ability of dbs to interfere with β band activity may be integral to how it achieves its beneficial effects [ ] (fig. c) . the use of aav to deliver glutamic acid decarboxylase (gad) to the stn is a logical extension of these insights. the stn has a physiological output that is glutamatergic and therefore excitatory to its downstream target nuclei. by transfecting stn neurons with a gene for gad, which converts the excitatory neurotransmitter glutamate to the inhibitory transmitter gamma-aminobutyric acid (gaba), the output of the stn can in principle be converted from excitation to inhibition. this is predicted both to dampen activation of stn target nuclei-the globus pallidus interna (gpi) and substantia nigra pars reticulata (snr)-and also, through effects on reciprocal interactions within the stn, to suppress synchronized activity of the nucleus itself. in essence, the strategy promises to achieve through gt a similar benefit to dbs, but without the need for long-term implantation of batteries and wires. the aim of this form of therapy is illustrated in fig. d(a) , and the relevant clinical trials are summarized in table . this approach was pioneered by a collaboration between investigators at weill college of medicine at cornell and a biotech company neurologix, and this group were in fact the first to use gt to treat adult neurological disease. early preclinical studies in rodents [ , ] , and later in primates [ ] , were encouraging, showing correction of clinical deficits and even a degree of neuroprotection. this data facilitated subsequent clinical studies, and a phase safety study of unilateral stn treatment using viruses carrying a mix of genes for two gad isoforms (gad and gad ) was published in (nct ) [ ] . three dose cohorts were used in patients with pd with an average disease duration of around years. aav -gad was infused into the stn unilaterally in an open-label study design. the results showed the procedure to be well tolerated, with no adverse events (aes) related to the intervention. there were also statistically significant changes in updrs scores and fdg-pet signal on the treated side. with this encouraging data, neurologix and the study team went forward to a phase double-blinded study of bilateral stn injections, recruiting patients split ( / ) between sham and a treatment arms (nct ) [ ] . again the procedure was well tolerated, with no aes related to treatment. however, this time, in the blinded format, efficacy results were disappointing. improvement in the defined off updrs part motor scores at months were significant, but only modest at % compared with % in the sham arm. for comparison, the first randomized controlled trial using dbs showed a % improvement in off updrs part score at months [ ] . for this reason, the neurologix trial was not considered a resounding success, and neurologix later filed for bankruptcy in . this strategic failure underlines the high bar to success that exists in the form of stn-dbs, the current gold standard competitor in this therapeutic space. however, this may not be the end of the road for stn-gad therapy, as a subsequent analysis has emphasized a real and potentially useful effect, with clinical improvements persistent at months and matched by measurable and correlatable changes in network activity as seen with fdg-pet imaging [ , , ] . these later results have yet to salvage the approach, but the technology has now been obtained by another biotech company, meiragtx, and is still currently considered to be one of their pipeline products for the treatment of pd. its potential advantage over other gt strategies lies in its relative ease of [ ] administration: the stn is a small and well demarcated target that functional neurosurgeons target frequently for dbs; crucially, it could be infused with the relevant vector more quickly and completely than the larger putamen (see below). however, for the same reason, off-target effects may be more difficult to control. within the basal ganglia, da is delivered to the striatum by axonal projections from the substantia nigra (snc), which thus regulate striatal function and motor and cognitive performance. in the striatum, the main population of striatal output neurons-medium spiny neurons (msns)-are exposed to da in both "tonic" and "phasic" modes, with phasic release being tightly correlated with circuit activity and behavior, and important for habit learning [ ] . phasic da responses cannot be reconstructed by gene delivery to the striatum, but tonic da exposure seems sufficient to ameliorate many of the features of pd in both patients and animal models. replenishment of da supply through gt is therefore an obvious approach. to date, two vector/cargo combinations have been employed to achieve resupply of da to the dadenervated putamen. the aim of this therapy is illustrated in fig. d(b) , and the relevant clinical trials are summarized in table . the first of the two da synthetic strategies aims to restore sensitivity to exogenous levodopa treatment by augmenting striatal levels of the enzyme aromatic l-amino acid decarboxylase (aadc). aadc is the final step in conversion of levodopa to da, and its striatal expression within the dopaminergic nerve terminals is lost with disease progression [ , ] . thus, although not a dose-limiting step physiologically, its levels become dose-limiting with disease progression, and aav-mediated transfection of aadc into striatal msns should theoretically correct this deficit. restoring aadc activity then allows striatal da to be synthesized in response to exogenous levodopa, which must be supplied orally [ , ] . this strategy has the benefit of retaining some control over the amount of da synthesized, via alterations in oral levodopa dose. a single aadc gene is well within the cargo size of the aav vector, and preclinical studies in rodents and parkinsonian non-human primates (nhps) have paved the way for using aav to deliver its aadc cargo to patients in the clinic [ , ] . the first studies of aadc gene therapy in patients with pd were performed in california under the sponsorship of the biotech company genzyme (nct ) [ , ] ). using an aav vector, the group completed a twin-dose safety study in patients with moderately severe disease (average disease duration of . years). two cohorts of patients were split into low and high doses that equated to × viral genomes (vg) and × vg per patient, administered via two injection sites at a volume of μl per putamen. from a safety perspective, the treatment was well tolerated over the months of the study, although three of the patients had small intracerebral hemorrhages along the trajectory of the catheter at the time of surgery (two of which were asymptomatic). secondary outcomes included clinical assessments in the defined off medication state, and these showed significant (up to %) improvements in both cohorts, whereas diary scores improved in all patients, and levodopa daily dose was reduced in three of the patients in the low dose cohort, and all five in the higher dose cohort. there were also significant increases in fluoro-l-m-tyrosine (fmt)-pet signal in both cohorts, rising to % of baseline in the high dose subjects. these initial results were encouraging and were also corroborated independently by a group in jichi university, using an identical vector and methods [ ] . six subjects received the same dose as the high dose patients from california, and again the procedure was well tolerated, with no sustained aes, although one venous hemorrhage at the site of a cannula insertion produced minor transient motor deficits. again, there was an apparent benefit as assessed by a number of secondary outcomes at months, with a % improvement in defined off updrs part motor scores and a % improvement in fmt-pet signal. due to concerns about tissue (putaminal) coverage, partly sparked by data from the contemporaneous neurturin (nrtn) trials (see below), these phase trials were followed by a refined phase study, again with a primary focus on vector delivery, dose finding, and safety (nct ) [ ] . now under sponsorship of the biotech company voyager therapeutics, a novel delivery technique was introduced which aimed to optimize tissue coverage. this employed a new stepped tip cannula (smartflow, mri interventions, inc.) and a novel surgical targeting system (smartframe and clearpoint neuronavigational system -mri interventions, inc., irvine, ca), working in conjunction with the brainlab software (iplan flow). intraoperative mri (imri) and the contrast agent gadolinium (gd) facilitated visualization of putaminal coverage in the operating theatre. this in turn allowed the use of higher volumes of infusate and also flexibility in the number of cannulae employed (two or three per side), so optimizing coverage. using this approach, previous volumes of μl per putamen were shown to be inadequate, and were therefore increased up to as much as μl per putamen, with viral doses being increased incrementally in cohorts, up to . × vg per subject ( times the low dose of the original study). results again showed the procedure to be well tolerated [ ] . one subject suffered three serious aes (saes) consisting of a deep vein thrombosis and pulmonary embolus and subsequent atrial fibrillation, all of which resolved and were felt to be linked to the immobility needed for the infusion rather than the agent itself. in the higher dose cohort, putaminal coverage was up to % by imri, with a % increase in baseline f-dopa pet signal. secondary endpoints included updrs part motor on scores, updrs part defined motor off scores, hauser diary entries, levodopa equivalent dose (led), and pdq- . all of these measures showed improvements across the three dosing cohorts, with dose dependency present for all except diary scores. there were particularly impressive reductions in daily levodopa equivalent dose (led), which in the high dose cohort was similar (at − %) to that obtained by dbs. mild transient increases in dyskinesia were noted at month, but the therapy was broadly well tolerated, in particular with no behavioral aes. although encouraging both in terms of the degree of improvement and persistence over the time of the study (up to months), these results do not exclude a significant placebo effect. thus, the current status of aav -aadc technology is that voyager therapeutics is undertaking a multicenter phase study (restore- ), using a slightly modified virus at a single dose level, in a sham surgery-controlled trial. a heavy emphasis in this study is being placed on safety, including psychiatric parameters, but primary outcomes may now also include striatal coverage by mri, f-dopa pet imaging, and diary scores. all being well, the company hopes to push forward towards a phase study in the near future (restore- ) [ ] . the direct competitor to the aadc therapy is the more comprehensive "triple enzyme" approach [ , ] . again, the anatomical target is the striatum, but in this format, the viral cargo encodes all three enzymes required to synthesize da from the precursor amino acid tyrosine. these include tyrosine hydroxylase (th), gtp cyclohydrolase (gch , an enzyme required for synthesis of tetrahydrobiopterin, a cofactor for th), and also aadc. the inclusion of all three enzymes is aimed at facilitating a more definitive reduction in medication doses than the aadc approach. additionally, with no reliance on an adequate biodistribution of exogenous levodopa, the strategy could facilitate a more efficient elimination of motor fluctuations. coding for three enzymes is however beyond the carrying capacity of aav-based vectors, and so this strategy has utilized a lv vector initially developed by the biotech company oxford biomedica [ , ] . initial preclinical work demonstrated the practicality and safety of using an eiav lv for cns gene therapy in both rodent and nhp models of disease [ , [ ] [ ] [ ] [ ] [ ] . the first in human use of a lv gene therapy to the cns was then undertaken, initially in paris and then cambridge, uk, by oxford biomedica using their prosavin® vector [ ] . patients had advanced disease (average duration . years), and dosing was between . × and . × transducing units (tu) per patient. surgical infusion was performed via five tracts per putamen initially, but this was simplified to three tracts in the final surgical format. safety outcomes in the initial patients showed no aes related to the agent, whereas secondary measures of efficacy showed improvements relative to baseline at and months in the updrs part motor score ( % and %) and also in pdq- (− . points, − . %) at months. dose reductions in led were however modest, matching those changes seen on raclopride pet binding (− . %, − . %, and − . % in low medium, and high dose cohorts). this good safety record and hints at efficacy serve to reinforce the aav data and emphasizes the broad potential for surgically delivered gts in pd. the unblinded data from the lv/prosavin approach [ ] is perhaps slightly less convincing than for the voyager aav-aadc study published more recently [ ] , but like the original genzyme study, the prosavin study was a first attempt and has set the scene for a further dose-finding and safety trial with higher doses of vector. this new trial, sunrise-pd, is currently underway and may produce more convincing effects. sunrise-pd addresses concerns of suboptimal production of the three enzymes through a vector that has been reengineered to optimize da production [ ] . specifically, the th gene, already truncated to avoid auto-inhibition, is now expressed as a fusion protein with gch , so optimizing expression and allowing for a single internal translational initiation site (ires) upstream of the second cassette that contains the aadc gene. published data suggests that da production in transfected cells will now be - times higher than with the previous version [ ] . as with the original trial, delivery of the vector (oxb- ) is by infusion, but will now rely on injections of μl per putamen (total μl per side). sunrise-pd commenced at the end of and is now sponsored by axovant gene therapies. once dose optimization is complete, a move towards a planned sham surgery phase is likely to happen in . although aadc and triple enzyme gene therapies do not represent a cure for pd, or even a slowing of disease progression, they do represent a potentially viable alternative to dbs, the current gold standard for treating motor fluctuations. as described above, the target patients in trials to date have been those with moderately advanced pd and motor fluctuations, which is exactly the group in which dbs has found its major niche. whether there is room for both of these two new gene therapies in this space, alongside dbs and infusion therapies, is not clear at present. how things work out is likely to depend on just how successful each modality is in improving fluctuating motor features in this problematic patient group. the triple enzyme approach, which is not reliant on a supply of oral levodopa, has the potential to achieve a greater reduction in oral medication than aav -aadc and a better smoothing out of motor fluctuations. in practice, how much this distinction really matters is yet to be understood, and the aadc approach may yield a lower risk of uncontrolled motor or neuropsychiatric side effects, because the da stimulus can be regulated not only by viral dose and tissue distribution, but also by oral levodopa dose after surgery. moreover, in the higher dose cohorts in the voyager/aadc trial, subjects not only benefited from reduced fluctuations but also managed to reduce their oral levodopa intake by very significant amount and still achieve improved off medication scores on formal motor testing [ ] . this suggests that increasing aadc enzyme levels in the striatum may improve some symptoms even in the absence of a recent oral levodopa dose (perhaps by improving utilization of residual endogenous levodopa). at present, the other distinction between the two approaches is turning out to be the reliance of the aav -aadc approach on imri to monitor the vector distribution obtained by their convection enhanced delivery (ced) technology. the voyager team has focused heavily on this aspect, using gd-imri monitoring as a means of optimizing vector delivery at the time of surgery. the technique of ced was originally described in the s [ , ] and relies on generation of a pressure gradient at the needle tip that enhances tissue spread of vector by bulk convective flow rather than by diffusion. it has been pioneered by the bankiewicz team and is now in use in both aav -aadc and aav -gdnf trials (see below). using a : mix of gadolinium contrast agent (gd) and viral sample, the distribution of viral infusate can be accurately monitored in real time using imri. nhp studies have confirmed a tight match between mri visualization and pathological distribution of vector expression [ ] , and needle design has incorporated a shoulder into the device, a short distance back from the exit point of the needle, to reduce backflow and optimize the pressure gradient [ ] . this strategy promises optimization of effect in individual patients, but more importantly it may also facilitate refinement of surgical technique over time. thus its benefits are already apparent in enabling safe administration of higher volumes of infusate than are planned for the axovant/lv study. indeed, such high volumes may well partly underlie the promising outcome data in the latest unblinded voyager study [ ] . however, visualization of tissue coverage also promises a greater understanding (and avoidance) of off-target effects, which could turn out to be extremely important, particularly as continuous dopaminergic stimulation of ventral striatum carries the risk of unwanted neuropsychiatric side effects. although the lv strategy has not yet used imri to monitor tissue distribution, it is unclear whether or not this is a true drawback. there are differences in how the two vectors move through brain tissue that may make imri less useful for lvs (aav diffuses poorly, so must be convected [ ] ), and lack of a need for imri technology could boost uptake of the eiav-triple therapy technology, in the context that many dbs centers outside north america do not currently employ imri in their dbs practice. this might give the lv vector a competitive advantage, at least in the short term. one current limitation to comparing these two approaches is the lack of any postmortem data on eiav vector distribution in humans, and so the field relies heavily on nhp data. ultimately, whether the lv strategy adopts some form of imri monitoring in due course remains to be seen. the extensive demonstrations of safety and efficacy of gt in late pd, by the twin strategies of da replacement and circuit modification, is very important for the future of the technology in both pd and other cns diseases. however, in reality, these approaches only offer a better means of managing motor features in a disease that is already advanced and irreversible. given the wealth of historical data examining da neuronal survival, a more ambitious strategy would look to use gt strategies to slow or halt the da neuronal loss that underlies disease progression. growth factors (gfs) have a long history of exploration in pd (reviewed in [ ] ), having been thoroughly examined in both cell culture and pd animal models over several decades. particularly, promising beneficial effects have been noted for two related gfs: glial cell line derived neurotrophic factor (gdnf) and neurturin (nrtn) [ , ] . both of these are able to enhance survival and neurite outgrowth of da neurons in vitro and in vivo [ ] [ ] [ ] [ ] , and both can rescue da neurons in preclinical models of pd [ , [ ] [ ] [ ] . such work has raised hopes for using these gfs as therapeutic agents in pd, both to slow da neuronal loss and to enhance function in the surviving da neurons and their axon terminals [ , ] . the aim of this form of therapy is illustrated in fig. d(c) , and the relevant clinical trials are summarized in table . the first in human use of gfs to treat pd was not through gt, but via surgical infusion of the recombinant gf protein (rgdnf) itself, which is relevant to briefly review. a sequence of trials were made, starting with the unsuccessful use of intraventricular rgdnf delivery [ ] , before moving on to procedures that targeted the putamen directly. these initial open-label intraparenchymal infusion studies showed some promising outcomes [ ] [ ] [ ] . however, a randomized study, using infusion pump driven delivery of rgdnf to the putamen, failed to meet its primary trial outcomes at - months [ ] . based on methodological concerns, additional evidence of benefit from postmortem studies, and anecdotal longer term follow-up data on some patients from the initial open-label trials [ , ] , a revised double blind placebo-controlled trial was undertaken in bristol, uk, and the results from this have been published recently [ , ] . this trial abandoned pumps, using instead intermittent (monthly) infusions of rgdnf, for up to months. a stronger emphasis was also put on tissue coverage, and a mri-monitored ced infusion approach was employed. although the trial did not meet its primary end point, improvements in f-dopa pet were documented, and % of the patients receiving rgdnf for the full weeks gained a meaningful clinical improvement in one or more of the core outcome measures. whether this constitutes a useful outcome is unclear, but the surgical delivery system employed in this study was complex and not without complications, as has also been reported in the new ongoing trial of recombinant cerebral dopamine neurotrophic factor (rcdnf) protein being undertaken in scandinavia [ ] . as such, these recombinant gf protein therapies are probably best considered as preparing the ground both for the more tractable genetic approach of gf gts. despite earlier work using lv-mediated gdnf delivery in animal models, the first gf to come to the clinic in the format of a gt was actually nrtn rather than gdnf, and used an aav vector, rather than lvs. aav -nrtn was manufactured by the californian biotech company ceregene, with their product cere- providing neuronal expression of nrtn as a pure protein without viral coding sequences [ ] . to enhance secretion, the preprodomain of human nerve growth factor was used in place of the nrtn sequence, and preclinical testing showed cere- to be well tolerated, with persistent gene expression for periods of over months and minimal biodistribution beyond the target area [ ] [ ] [ ] (reviewed in [ ] ). therapeutically, it showed promising effects on the survival of snc da neurons in rodent and nhp pd models. initial clinical studies followed, and a phase study of patients, with an average disease duration of years, found cere- to be well tolerated (nct ) [ ] . this study was not powered to determine whether the agent had trial category: phase , safety patients: subjects, cohorts disease stage: earlier (< years) and later (> years, moderate-severe disease) primary: safety tolerability over years secondary: updrs, nonmotor scores, and dat scan changes over months either a symptomatic benefit or a later disease-modifying effect, but even so f-levodopa pet findings were a little disappointing, with no clear change in striatal signal. the real stumbling block was the subsequent blinded study (nct ), which recruited patients to the active arm and patients to sham surgery [ ] . notably, the average disease duration in the recruited participants was . and years in the surgical and sham arms respectively, and by this measure, subjects were slightly more advanced than those in the gdnf protein infusion studies [ , ] . a viral dose of . × vg per brain was comparable to other putaminally targeted aav procedures, but this dose was delivered without mri guidance, and as eight separate deposits, each of μl, split between four needle tracts per hemisphere (total μl). the trial failed to meet its primary end point of a change in the updrs part motor score in the defined off medication state at months with the intervention. on the positive side though, the procedure was again well tolerated-saes occurred in both arms, but none were attributable to the novel therapy. however, a failure to meet the primary endpoint meant that the therapy as delivered could not be competitive with the established technology of dbs. things could have stopped there, but as with the bristol gdnf protein study [ ] , there were clues in the original data that a subset of patients might be getting significant benefit, with patients assessed at months demonstrating a significant benefit over placebo. this was not the primary endpoint, and the benefit was only seen in a small subset of the total patients, but this and some additional trends to improvement with surgery in some of the secondary endpoints suggested that modifications to the delivery might yet improve outcomes over a longer time course (see below). an important element in this decision to continue trialling this agent was postmortem data on four patients, who died of unrelated causes subsequent to receiving the gene therapy, which lent more insight into the disappointing outcome. two of these individuals died within a few months of treatment and two of them about years later, so giving a time course of transgene effects. there were three main findings from these pathological studies [ , ] , the first being the technical issue of poor tissue coverage by the gt within the putamen. thus, although putaminal nrtn expression evoked by the treatment was good and persistent, the volume coverage per infusion was less than % of total putaminal volume, which was probably insufficient to provide good clinical benefit. the second observation was that dopaminergic denervation of the striatum was surprisingly advanced even at years into the disease. probably at least partly as a result of this, the enhanced tyrosine hydroxylase (th) expression evoked by the nrtn was rather modest and only covered~ % of striatal area in the short-term cases and . % in the longer term ( years) cases. this suggested a resistance to trophic effect in these patients, albeit one that was partly overcome with time. thirdly, the striatal nrtn expression was not matched by similar nrtn expression in the snc as would be expected by the retrograde transport of gf observed in preclinical models. this was improved in the two longer term patients but was still poor even compared with aged animal models and was matched by deficient transport to other putaminal targets such as gpi. this suggests a retrograde axonal transport deficit in patients with pd, which may be specific for certain projections, as the transport of nrtn from putamen to cortex (via axon terminals within the putamen) seemed to be largely intact. together, these findings imply that the degenerating human ns system offers a significantly different environment for disease-modifying strategies than is apparent in either preclinical rodent or nhp pd models, and that both better tissue distribution of aav -nrtn and more protracted time points might be needed. more fundamentally, it is difficult to envisage a gf working on ns projections that no longer exist, and data from other groups also suggests that the α-synuclein pathology of pd may itself impair both axonal transport and gdnf/nrtn receptor (ret) expression and signalling in the surviving ns projections [ ] [ ] [ ] . this has been debated [ ] , as has the extent to which there may be unwanted detrimental effects of these gfs [ ] . the logical outcome from this pathological data was that nrtn delivery to relevant tissues still needed to be optimized. this would require several improvements on existing methodology, including larger volumes of infusate, delivery of vector to both putamen and snc, and inclusion of earlier stage patients. moreover, assessment would need to be done over longer time points than the months of the original studies. revised studies were therefore embarked up on which partly explored these issues, specifically by reinforcing putaminal expression of nrtn with co-expression of the same vector in snc. this line of investigation required further preclinical studies, which ultimately concluded in a revised blinded sham control study that reported in (nct [ ] ). patients in this study now received bilateral doses aav -nrtn to both snc and putamen, with putaminal doses being four times the previous dose, and subjects being followed for between and months. however, again the study failed to hit its primary endpoint of change in defined off updrs part motor scores. notably, given the pathological data, disease durations for participants in this study were slightly shorter than before, at . years for the treated and . years for the sham cohort. as fluctuations are a marker of ns degeneration, this suggests that denervation of the striatum in these subjects was probably still profound. one of these subjects has come to postmortem subsequently ( years after surgery), and this examination confirms that vector-mediated nrtn expression remains robust even at this time point, with correlated th expression, but that the coverage of expressing tissue was again very limited in extent [ ] . despite its overt failure, post hoc analysis of this trial again suggested that certain subgroups, in particular those with shorter disease durations, might show some benefit, backing up the pathological conclusions. this latter point is a crucial insight and one that is being tackled in the inclusion criteria of an upcoming aav -gdnf trial being run through the universities of ohio and california. on this background, aav -gdnf is the last therapy still standing in the arena of gf gt for pd at present, and recent reports suggest some hope. preclinical work suggests that gdnf may have more powerful neurotrophic effects than neurturin [ ] [ ] , and successful transduction of putaminal medium spiny neurons has been shown in both rodent, primate, and aged primate models with rescue of dopaminergic projections [ ] [ ] [ ] . following on from these preclinical studies, a phase trial of patients with moderately severe pd was undertaken sponsored by the national institute of neurological disorders and stroke (ninds) (nct ) [ , ] . aav -gdnf was delivered by imri-ced in dose levels between × and × vg per patient, and the procedure was well tolerated. gd-imri tracking of the infusions however showed that putaminal coverage (using μl infusate per putamen) was again limited at only %, suggesting that, despite demonstrated improvements in f-dopa pet signal, further optimization of delivery might be required in future studies. this conclusion was also reinforced by the variability in improvement in pet signal; although improvement was seen in of patients, the range of change in those that improved was wide (between and % above baseline, median %), and the other patient experienced unilateral deterioration. despite these limitations, the ninds study has provided proof of principle evidence, and a second study (nct ) is now being planned. this new study is penned to start imminently, based out of the universities of california and ohio, and sponsored by the biotech company brain neurotherapy bio, inc. its twin aims are to assess the utility of gf gt in a cohort of patients with earlier disease (less than years) and also to optimize tissue coverage using imri and a novel neurosurgical approach. this uses a posterior stereotactic trajectory and a single infusion cannula that should facilitate better coverage of the irregular volume of the putamen-allowing a "shape-conforming" infusion [ ] . the primary endpoint is safety over years, with secondary endpoints of changes in motor and nonmotor scores, alongside dat scan changes ( months). overall, these gf studies have been very valuable, in particular with respect to our improved understanding of the pathological human striatal environment in pd. the good safety and tolerability profiles of the aav vectors that has emerged is also important, but the overarching conclusion is that using gf-mediated gt to rescue the degenerating human ns system in pd is unlikely to be a panacea for all patients. even so, the potential prize remains substantial, and if a clear "disease severity threshold" could be drawn, before which gt could be given with a good prospect of benefit, then this would still be an important achievement. pd is relatively easy to diagnose, and such a therapy given early (e.g., before years) would have the potential to slow disease progression in this pathway across large numbers of patients, so ameliorating a large burden of disease that currently carries very significant impacts on patients, carers, and society. unfortunately such a threshold-a measure of responsiveness to gf therapy-is likely to vary considerably from patient to patient (perhaps up to years in some patients) and may be difficult to assess accurately a priori. however, there is currently a good prospect that the da replacement strategies (aav -aadc and lv-triple enzyme) will become important backup options in later stage patients, alongside the current mainstay of dbs. by comparison, the circuit modification approach seems to have faded as an option at present, but its ease of surgical administration to a small volume target means that it is still an attractive alternate approach if significant benefit can be demonstrated. the prospect of treating pd patients early with gf gt is not only enticing but also carries risks. short-term side effects of the gf treatments thus far have been minimal but the longer term risks are very unclear. nhp data has not so far produced clear evidence of long-term problems, but potential side effects, including aberrant sprouting of sn da neurons, weight loss, and hyponatremia, have been highlighted in some preclinical work, as well as one of the early trials that delivered rgdnf into the cerebral ventricles [ , , ] . for these reasons, some attention has switched to looking at regulatable vectors that could allow the gf stimulus to be switched off flexibly after transduction-perhaps even allowing an intermittent gf dosing regimen to be used over months and years. this sort of regulation would require additional gene regulatory sequences and might be difficult to achieve within the restrictive payload of aavs. this issue might drive a move to use lvs for gf therapy in due course, as these vectors can facilitate regulatable expression in different cells systems including the delivery of gfs to the striatum [ ] . however, there remains a good deal of focus on developing aavs in novel ways [ ] , so regulatable aav technology may yet emerge. gt therapy is still in its early days and is an emerging technology. several new avenues suggest that the studies described above may only be the forerunners of a wider array of gt options for pd in the future. although combining gf therapy with da synthetic gt has been postulated, it is unlikely to be a useful strategy given that different disease time points require different therapeutic strategies. by the stage of disease at which da synthetic treatment needs consideration, gf gt would be unlikely to work given the extent of ns degeneration; conversely, early treatment with gf gt ought to obviate the need for later gt to manage motor fluctuations. a more likely scenario is that gf gt will be combined with, or replaced by, other gt approaches that either promote responsiveness to gfs or otherwise provide a beneficial disease-modifying effect. although systemic (non-gt) treatments along these lines may well be feasible and easier, the gt approach may still retain some advantages. by "vectorizing" a treatment to certain neuronal populations (e.g., ns projection neurons or striatal msns), off-target effects might be minimized and a more potent diseasemodifying effect obtained. novel approaches along these lines might include manipulation of downstream pathways from nrtn/gdnf (e.g., overexpression of the gf receptor nurr ), or direct targeting of culprit disease pathways, including α-synuclein toxicity [ , ] , and lrrk kinase overactivation [ ] . virally mediated gene inhibition (rnai) or editing (crispr-cas ) are two options here [ ] , but where pd is seen in the context of gba mutations, expression of the normally functioning wild-type gene may prove beneficial [ ] . a new york-based biotech company is already pursuing this latter goal using intracisternal injection of an aav -based vector carrying gba , with phase results likely to be published in the near future (nct and [ ] ). also on the brink of clinical testing is vectorized antibody therapy against α-synuclein, a technology that has been previously been developed for treating hiv infection [ , ] and which is being pushed in the pd field by a collaboration between pharma companies abbvie and voyager. again, an aav vector is the proposed technology [ ] . with the aav and eiav studies being generally well tolerated, there will be a good deal of temptation to stick with vectors known to be safe. introduction of a novel vector would entail starting from scratch with safety studies for any evolving strategies, but such approaches may offer new potential advantages. in particular, aav is known to bind to heparan sulfate proteoglycans (hspgs), moieties that are abundant on the neuronal surface and contribute to vector uptake, but which also limit vector diffusion. although this has allowed tight control over vector targeting, arguably this control has been too tight, contributing also to the limited putaminal distribution of vector that has led to failed trials. other aav serotypes, such as aav , can diffuse more broadly within neuronal tissue [ ] , while maintaining neuronal infectivity, and may well have achieved better putaminal distribution of vector payload had they been employed. such vectors, and modifications of them, may yet come into their own for pd, in particular for strategies that aim to curtail the spreading pathology that characterizes the pd brain. some of these vectors may also allow peripheral (or intrathecal) delivery (see below). although the strategy of asking putaminal cells to start making da in situ is easily understood, another option being explored is a more fundamental reprogramming of endogenous putaminal cells, converting them directly into th expressing neurons. these brand new da neurons (perhaps converted from endogenous astrocytes) might be able not only to start producing da, but also to sprout an axonal arbor and provide more efficient da delivery to striatal cells via synapses. the technology for this, although more distant than standard gene therapy, is nevertheless on the horizon, with encouraging data emerging in animal models [ ] . future nonsurgically delivered therapies may also yet displace these options, particularly if delivery methods are eased while target specificity is maintained, and any immunological issues are side-stepped. one option proposed is the use of peripherally delivered vector (e.g., via intravenous infusion), in combination with targeting to specific brain regions by local disruption of the blood-brain barrier (bbb) with focused ultrasound [ ] . this could be an extremely attractive option for either advanced or early disease. indeed, more anatomically diffuse disruption of bbb can be obtained even more easily pharmacologically [ ] and might enable much more widespread cns uptake of vector and, with this, more extensive disease modification. we are currently at an exciting time in the arena of gt for the pd field. several therapeutic options are emerging in parallel as potential treatments for this condition, but with the safety profile of the technology so far holding, this bodes well for an even wider array of gt options in the future. time will tell which if any of the current and future candidate gt treatments emerges as useful and practical therapy and so ends up dominating the therapeutic arena for pd. it is also possible that gts could be used synergistically with cell therapies [ ] . the main rationale for the use of cell therapies in pd has been to replace the lost a da neurons of the snc-the ones that are principally affected by the disease process in this condition. other approaches have been tried which exploit less defined modes of action-e.g., spheramine® was trialled as a cell therapy that was thought to work more through a trophic and paracrine action [ ] , in much the same way as the parthenogenetic stem cell therapy being trialled at the present time by isco [ ] . nevertheless, the vast majority of approaches have sought to replace the few hundred thousand da neurons that are lost in pd, engrafting new cells in the striatum where da normally works. these therapies therefore are not being used to rebuild the circuitry as originally lost (since the grafts are placed heterotopically), and nor are they being used to cure pd-as the underlying disease process carries on in the pd patient brain and may even involve the grafted cells themselves (see below). rather, da cell therapies are being used as a symptomatic therapy to better treat the nigrostriatal dopaminergic aspects of pd, albeit that a core component of the pd pathology is being repaired in the process. although this approach is restricted in its ambition, it does have the potential (if it works) to make redundant almost everything we currently do for pd patients in the clinic, with drugs and surgery. this is because all current drug and surgical therapies essentially work on the dopaminergic aspects of pd, and the complications that these drugs produce arise through abnormal activation of this pathway (see above). there are a number of cell types that have been considered to be suitable for grafting as da neuronal replacements (including for example, peripheral sympathetic neurons, adrenal medullary cells, and carotid body cells), only some of which actually form neurons of the type lost in pd. this is important as preclinically it has been shown that the a neurons are the optimal da cells for effecting repair at this site [ ] , and other da cell types work less well, if at all. many of the other sources of cells that have been trialled either do not form neurons (e.g., adrenal medullary cells [ ] ), or do not form the da neurons of the midbrain (e.g., parthenogenetic stem cell-derived neurons [ ] ). in this review, we will concentrate on the work that has been done with fetal ventral mesencephalic (vm) tissue (that contains the developing da neurons of the midbrain) and stem cell (both es and ipsc)-derived da neurons. in terms of these latter cells, some comment needs to be made at this stage about their relative advantages and disadvantages. human fetal vm (hfvm) tissue is derived from elective termination of pregnancies and as such brings with it major ethical issues around abortion. thus the use of such tissue is strictly regulated in all countries, but in some it is not allowed to be used at all for clinical purposes and/or researchalthough this can also change over time (e.g., the usa). in part, this is because of concerns that the adoption of such an approach may encourage women to have terminations, especially if they have family members affected by conditions that may use such tissue for therapeutic gain. in addition, religious objections are also key arguments raised around not allowing human fetal tissue from termination of pregnancies to be used, centering on questions of the potentiality of embryos and the sanctity of life. however, in the uk for example, there is no evidence that using fetal tissue in transplant programs changes practice in terms of women deciding to have a termination of pregnancy or not. furthermore, there are strict guidelines stipulating that those donating fetal tissue cannot say which programs of work they want that tissue to be used for, either clinically or for research. in addition to these ethical problems are the logistical ones that in order to have enough surviving da neurons after grafting, tissue needs to collected from at least fetuses per side of the brain grafted. this puts major constraints on having sufficient tissue to graft patients, given that hfvm tissue cannot be cryopreserved successfully and can only be stored in hibernation medium for short periods of time (a few days only) [ ] . thus, in our recent trial using this tissue in patients with pd (transeuro), planned transplant operations were cancelled because of insufficient tissue being available for grafting [ ] . a final major problem with using this tissue is that the final grafted cell product cannot be standardized as each patient will receive their own unique tissue transplant, which also contains many cells of the type not needed, given only the minority of cells in the developing vm are of midbrain dopaminergic origin. however, there are advantages with using this tissue, including that the use of primary, non-manipulated tissue brings many regulatory and safety advantages, as well as greater confidence that one is replacing like with like-namely the da cells of the midbrain that are lost in pd are being replaced with genuine human midbrain a neurons. the use of human pluripotent stem cells gets around many of the above problems, especially logistical ones given these cells can be easily expanded and differentiated in culture and then cryopreserved [ , ] . however, such stem cell therapies are still not without problems. thus with human embryonic stem (es) cells, ethical concerns remain around the fact that the cell line so derived can only be achieved through destruction of that embryo. some have tried to circumvent this problem using single blastomeres, so in theory side-stepping the need for embryo destruction [ ] . this problem is avoided to some extent with human ipsc lines, although not completely, as questions of potentiality still exist with any human stem cell (hps) [ ] . in addition, the country of origin of the human stem cell line also has implications, as lines derived from countries that have had cases of new variant cjd cannot currently be used easily in the usa. given the size of the us market, this has major consequences on the commercial development of any product derived from such cellsthe investment in and thus development of that product may be significantly limited. finally, there are issues that have yet to be resolved about the genetic testing and variability seen in stem cell lines, and what this actually means for their safety clinically. although many different types of genetic variability have been shown in a range of cell lines (e.g., hps cells recurrently acquire and expand dominant negative p mutations) [ , ] , this also varies as a function of time in culture, and the significance of the variability from a safety perspective is still unknown. the position of the regulatory agencies on this aspect also remains to be defined. all of these issues, coupled with the commercialization of any future stem cell-based therapies for pd, has meant that many teams are now seeking to derive and use their own hps cell line, as this gives them greater control over what that line can be best employed to treat. this though can make it hard to extract exact information about the cell being developed for clinical trialling. however, reassuringly, most modern differentiation protocols mean that the major worry with these cells of teratoma development is a problem that is more theoretical than real. in addition, the regulatory agencies now all have clearer guidelines on what is needed to show that these cells are acceptable from a safety, biodistribution, and tumorigenicity perspective. the use of cell-based therapies for treating pd brings with it issues of immunogenicity, as would be the case for any allotransplant. one difference for cns disease is that the brain has a degree of immunological privilege, and the cells are also not especially immunogenic given their fetal origin or derivation. nevertheless, rejection issues are still important and need to be considered, as they may have contributed to the lack of efficacy in the double blind placebo-controlled fetal transplant trials done in the last part of the twentieth century (reviewed in [ ] ). the brain has long been known to have relative immunological privilege by virtue of having no professional antigen presenting cells within it, a limited lymphatic drainage system, and a blood-brain barrier (bbb). however, of late, it has been shown that the cns is actively patrolled by the immune system and that a relatively well-developed g lymphatic system is also present, which means that antigens within the cns can be delivered to the peripheral immune system and an immune response generated [ ] . as such, cells placed into the cns will likely evoke an immune reaction, especially as the grafted procedure itself will temporarily disrupt the bbb. thus, unless the cells are autologous, some sort of immunosuppressive treatment will be needed at least for a time after implantation. the extent of this immunosuppression will depend in part on the following factors: & when the bbb seals up (which is thought to be within a few weeks for cell suspension grafts [ ] ). & when dying cells and fragments from the transplant stop entering the peripheral immune system and thus being exposed to the peripheral immune system (which is probably in the first few weeks only after grafting [ ] ). & the immunogenicity of the cells being grafted, both at the time of grafting and as they mature relative to the host. for hvm tissue, this last issue has been looked at both in vitro [ ] and in vivo [ , ] , with such work showing that the tissue can both express major histocompatibility (mhc) antigens and invoke an immune response. this has also been seen with hpsc-derived allografted tissue in vitro, as well as in nhp studies using matched and mismatched stem cell grafts [ ] . in this latter study, it also shown that the rejection response could be abrogated with tacrolimus monotherapy [ ] . all of this has led some groups to consider less immunogenic cells-either using an autologous approach [ ] , or using a universal cell line where the major mhc antigens have been engineered out of the cells [ ] . whether either of these approaches really offers advantages for treating pd remains unproven, as is the case that autologous ipscs are really immunologically tolerated when grafted back into the donor host [ ] . there are though two other issues that need to be considered with the use of autologous approaches: the cost of making individualized cell therapies (which currently in the uk would be in the region of several million pounds per patient) the risk of promoting any pd pathology in the grafted cells given they are patient derived (and thus harbor the same genetic risks for pd) and αsynuclein pathology has consistently been found in unrelated fetal da cells years after grafting [ ] in spite of these possible risks, the first report has been published of a single patient receiving an autologous ipsc derived da cell transplant for their pd [ ] . in this case, the patient had bilateral transplants of cells without immunosuppression and responded well symptomatically, although not on other more objective measures. this case, while showing that this approach was safe, used protocols for making the da neurons that might not have been optimal and in addition there were other controversial aspects to how this transplant was done [ ] . at the present time though, most groups have elected to use allogeneic cells and immunotherapy after grafting. this latter regime is typically of the type normally used in other organ transplants and which to date has been used with fetal tissue trials in patients with pd without much in the way of complications [ ] . as to exactly what this regime will look like in future stem cell trials for pd, and for how long it will be given after grafting, is still to be finalized. the initial trials in the early to late s predominantly used adrenal medullary tissue for which the preclinical evidence was limited (reviewed in barker and dunnett ). the conclusion of the clinical trial work using this tissue, which was undertaken extensively across many centers especially in the usa, was that these transplants offered no real benefit and were associated with side effects and poor survival [ , ] . therefore they were abandoned, especially as more encouraging data was beginning to emerge with the use of hfvm allografts. the move to trial hfvm in the clinic came on the back of robust reproducible preclinical findings in neurotoxic animal models of pd that had been carried out in many different laboratories around the world. these studies showed that allografts of the developing vm into rats lesioned unilaterally with -hydroxy-dopamine ( -ohda) could survive, make and receive connections from the host brain, release da, and restore behaviors to normal in these animals (reviewed in [ ] ). this was also shown in nhp, most notably marmosets [ ] , and it was on this background that patients were first grafted in the late s and thereafter till the end of the century. although a number of groups took on this early work, the best studied cohort was a series of pd patients recruited to an iterative open-label study in lund sweden, led by olle lindvall and anders björklund, which also included two mptp patients from california [ ] . all this work involved grafting patients with relatively advanced pd with hfvm tissue, and following them over extended periods of time both clinically and with pet imaging of the da system. this study, along with other open-label studies showed that transplants of hfvm could: & survive long term in the human pd brain (e.g., [ ] ). & have long-term clinical benefits (e.g., [ ] ). & release da in a physiological way when the graft was pharmacologically stimulated (e.g., [ ] ) with reactivation of motor cortical areas after grafting [ ] . & improve quality of life measures not just motor features (e.g., [ ] ). & work as well when placed striatally as when placed in the striatum as well as the nigra [ ] . & not be guaranteed to work in every patient (e.g., [ ] ). & acquire the pathology of pd after several years of engraftment (e.g., [ ] ). these early studies led to more formal studies being undertaken in the usa in the early s once federal funding became available under the clinton administration for work using human fetal tissue. these two nih funded double blind placebo-controlled studies reported in and and failed to show clinical benefits that satisfied the trials primary end points [ , ] . in addition, both trials reported for the first time severe and disabling graft-induced dyskinesias (gids) some of which required further neurosurgical intervention [ ] , and more recently, one patient was shown to have a transplant that did not work clinically despite recovery of striatal da innervation at postmortem and on da imaging [ ] . as a result, the adoption of this approach for treating patients with pd fell out of favor, especially in light of the side effects and lack of efficacy compared with the results that were appearing at this time with dbs [ ] . this in effect led to a moratorium on this therapeutic strategy and debates around whether cell therapies had any future in pd. as part of these discussions, a review of the data from all these trials was undertaken [ , ] , and a number of conclusions were drawn, suggesting that this approach may still have merit, especially given that protocols for making human stem cell-derived da cells of an authentic midbrain type were starting to emerge. these conclusions were that: & those patients that seem to have the highest chance of benefitting from hfvm grafts tended to be younger with less advanced disease. & patients with extensive striatal da loss that extended into the ventral stratum did less well [ ] . & patients who developed gids had reported significant levodopa-induced dyskinesias (lids) pre-grafting, but also that the development of gids may relate either to a poor distribution of da cells across the striatum [ ] , or to the grafting of large numbers of ht neurons relative to the da cells in the transplant [ ] -all of which could be avoided using better preparation and delivery approaches. & the use of no immunosuppression in the first nih study was associated with low numbers of surviving da cells at postmortem, although there may be other reasons for this relating to the amount of hfvm tissue grafted. in addition, in the second nih-funded study, there was a distinct change in the direction of the clinical response of grafted patients when their ciclosporin a was stopped months after surgery. although the reasons for this remain unknown, it could relate to a partial graft rejection on stopping the ciclosporin a. & clinical improvements could take many years to appear and thus using primary end points relatively soon after grafting may miss the maximal benefit of the grafts. as a result of this, a new trial funded by the eu (transeuro) was set up that tried to address many of these factors in its design. this trial, which grafted its first patient in and its last in , will report next year at the earliest, as the primary end point is the change in the updrs part score in the defined off stage, years after the second transplant. the lessons learnt from this trial have recently been published with the hope that this will help in the design of the next round of stem cell-derived da cell transplant trials for pd [ ] . in addition, this article also highlighted why this therapeutic approach, using this cell source, has no future from a simple logistical perspective, but may be adapted if a stem cell source of da cells can be identified. in this last respect, in and , two seminal papers from the groups of studer and parmar published protocols showing that hesc could be converted into authentic da neurons of the midbrain type. theoretically, these could therefore be used for treating patients with pd in much the same way as hfvm tissue [ , ] . subsequently, it has been shown that this approach offers major potential therapeutic promise in treating pd, by virtue of the fact that these da neurons can: & be produced reproducibly, with a midbrain da phenotype, using different starting cell lines [ ] . & be manufactured in the numbers needed for clinical translation in a gmp compatible fashion [ ] . & be shown to have equivalence to human fvm grafts in terms of their functional effects in animal models of pd [ ] . & connect and establish connections appropriately with the host brain [ , ] . in addition, grafts of these cells do not form tumors or contain large numbers of ht neurons [ ] . as a result, several academic groups, often in conjunction with pharma or small biotech companies, around the world are now moving this technology towards clinical trials including in europe, the usa, and japan, with the first of these having started with an ipsc approach in kyoto in japan. this work has been led by jun takahashi, based on his preclinical work in parkinsonian nhps, where he showed long-term survival and efficacy of human ipsc-derived da cells [ ] . all these groups have over the last - years worked together as a consortium (gforce-pd [ ] ) to try and ensure that their respective clinical trials can be better coordinated, and also to facilitate the exchange of data around preclinical developments of these stem cell-derived da cell products [ ] an approach which we encourage others working on similar experimental therapies to adopt. these trials, which were due to start in the usa in and in europe in prior to the corona virus pandemic, have attracted commercial interest from major companies as the potential for this therapy to transform the natural history of treated pd at an affordable cost has now been perceived as realistic by many working in this field. what are the challenges moving forward with this approach and where will it end? the field of stem cell-based therapies for pd sits at an important stage in its development, since it is about to enter clinical trials where the effectiveness and utility of this therapy will be revealed, along with its competitiveness-both therapeutically and commercially. if these early trials prove successful, then one can imagine that this therapy will be moved to be trialled in earlier stage patients as there is a logic in doing this especially in younger onset cases of pd. ultimately this therapy has the capacity to transform how we treat pd by obviating the need for many if not all of the therapies currently given in the clinic to patients with pd for their motor problems. although this remains a long way off, the commercial investment in this area does mean that this field is likely to move faster through phase / trials than we might imagine. as to where next, there is great interest in using the technologies we have described in this review to directly reprogram cells in situ without the need to graft cells into the brain (see above). in other words, can we convert resident glia into da neurons and by so doing avoid the practical, ethical, and immunological issues that play a role in any cell transplant grafted into the brain? this is now being explored in animal models, although to date the results have been modest at best with little evidence that sufficient da cells can be generated of the type needed to reverse the deficits seen in pd [ , ] . nevertheless, this whole area is one that is changing every year as our ability to reprogram cells becomes ever better and with this the prospect of repairing the pd brain from within. required author forms disclosure forms provided by the authors are available with the online version of this article. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's 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human retinal pigment epithelial cells versus sham surgery in patients with advanced parkinson's disease: a double-blind, randomised, controlled trial. the lancet neurology the a dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of parkinson's disease the biology and behaviour of intracerebral adrenal transplants in animals and man human clinical-grade parthenogenetic esc-derived dopaminergic neurons recover locomotive defects of nonhuman primate models of parkinson's disease effects of cool storage on survival and function of intrastriatal ventral mesencephalic grafts designing stem-cellbased dopamine cell replacement trials for parkinson's disease chapter -strategies for bringing stem cell-derived dopamine neurons to the clinic: a european approach (stem-pd) cryopreservation maintains functionality of human ipsc dopamine neurons and rescues parkinsonian phenotypes in vivo human embryonic stem cell lines derived from single blastomeres scientific and ethical issues related to stem cell research and interventions in neurodegenerative disorders of the brain human pluripotent stem cells recurrently acquire and expand dominant negative p mutations detecting genetic mosaicism in cultures of human pluripotent stem cells fetal dopaminergic transplantation trials and the future of neural grafting in parkinson's disease. the lancet neurology chapter -the cerebrospinal fluid and barriers -anatomic and physiologic considerations cerebrospinal fluid in neurologic disorders blood-brain and blood-cerebrospinal fluid alterations following neural transplantation the time course of loss of dopaminergic neurons and the gliotic reaction surrounding grafts of embryonic mesencephalon to the striatum human fetal neural precursor cells can up-regulate mhc class i and class ii expression and elicit cd and cd t cell proliferation a double-blind controlled trial of bilateral fetal nigral transplantation in parkinson's disease neuropathology of fetal nigra transplants for parkinson's disease mhc matching improves engraftment of ipsc-derived neurons in non-human primates autologous induced pluripotent stem cell-derived neurons to treat parkinson's disease hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients immunogenicity of induced pluripotent stem cells characterization of lewy body pathology in -and -year-old intrastriatal mesencephalic grafts surviving in a patient with parkinson's disease personalized ipsc-derived dopamine progenitor cells for parkinson's disease stem cells: scientific and ethical quandaries of a personalized approach to parkinson's disease neurotransplantation registry on adrenal medullary transplants: presurgical, and -and -year follow-up neuropathological study years after autologous adrenal medullary transplantation in a parkinson's disease patient mechanisms and use of neural transplants for brain repair behavioral assessment of the effects of embryonic nigral grafts in marmosets with unilateral -ohda lesions of the nigrostriatal pathway fifteen months' follow-up on bilateral embryonic mesencephalic grafts in two cases of severe mptp-induced parkinsonism extensive graft-derived dopaminergic innervation is maintained years after transplantation in the degenerating parkinsonian brain long-term clinical outcome of fetal cell transplantation for parkinson disease: two case reports dopamine release from nigral transplants visualized in vivo in a parkinson's patient delayed recovery of movement-related cortical function in parkinson's disease after striatal dopaminergic grafts health-related quality of life following bilateral intrastriatal transplantation in parkinson's disease cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with parkinson's disease neural transplantation in parkinson's disease: the swedish experience lewy bodies in grafted neurons in subjects with parkinson's disease suggest host-to-graft disease propagation transplantation of embryonic dopamine neurons for severe parkinson's disease. the new england journal of medicine dyskinesias following neural transplantation in parkinson's disease robust graft survival and normalized dopaminergic innervation do not obligate recovery in a parkinson disease patient dopaminergic transplantation for parkinson's disease: current status and future prospects cell-based therapies for parkinson disease-past insights and future potential factors affecting the clinical outcome after neural transplantation in parkinson's disease. brain dyskinesia after fetal cell transplantation for parkinsonism: a pet study graft-induced dyskinesias in parkinson's disease: high striatal serotonin/dopamine transporter ratio. movement disorders : official journal of the movement disorder society human trials of stem cell-derived dopamine neurons for parkinson's disease: dawn of a new era dopamine neurons derived from human es cells efficiently engraft in animal models of parkinson's disease generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions generation of highpurity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation strategies for bringing stem cell-derived dopamine neurons to the clinic: a european approach (stem-pd) human esc-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of parkinson's disease target-specific forebrain projections and appropriate synaptic inputs of hesc-derived dopamine neurons grafted to the midbrain of parkinsonian rats hesc-derived dopaminergic transplants integrate into basal ganglia circuitry in a preclinical model of parkinson's disease predictive markers guide differentiation to improve graft outcome in clinical translation of hesc-based therapy for parkinson's disease human ips cell-derived dopaminergic neurons function in a primate parkinson's disease model fetal-cell revival for parkinson's. nature generation of induced neurons via direct conversion in vivo publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments rab has been supported by an nihr biomedical research award to addenbrooke's hospital/university of cambridge, as well as grants from parkinson's uk, cure parkinson's trust, rosetrees trust; chdi, eu-fp programme, michael j fox foundation, evelyn trust, nihr i i programme, mrc, and the wellcome trust.pcb is supported by an mrc-nihr clinical academic research partnership award. key: cord- -o cw ufy authors: horby, peter w.; hoa, ngo thi; pfeiffer, dirk u.; wertheim, heiman f. l. title: drivers of emerging zoonotic infectious diseases date: - - journal: confronting emerging zoonoses doi: . / - - - - _ sha: doc_id: cord_uid: o cw ufy this chapter discusses drivers of emerging infectious diseases (eid) of humans that have an origin in other vertebrate animals (zoonoses). this is a broad topic, worthy of a book in its own right. this chapter will therefore provide only an overview of key concepts of drivers of the emergence of zoonotic diseases, and particularly infectious diseases with a major disease burden in humans. as the authors mainly work in asia, the focus of this chapter is asia, but many of the lessons learned in this region are likely to apply elsewhere. more than % of the world population live in asia, a region with some of the fastest developing economies in the world. yet, despite tremendous advances, infectious diseases still remain a major burden for the human population in asia. of the estimated . million deaths in children aged less than years in southeast asia in , % are attributable to infectious causes (liu et al., lancet : – , ). as such, asia is both vulnerable to imported eids and a global focus of major social and environmental change that may facilitate the emergence and dissemination of new pathogens. however, it would be too simplistic to present the extensive changes in asia as inevitably increasing the risk of eids. some aspects of socio-economic change might serve to reduce the overall risk of infectious disease emergence, but all ecosystem changes have the potential to provide new opportunities for microorganisms to spill-over into human populations. the conditions or events (the 'drivers') that result in the successful cross-over of an animal microbe into humans are not well characterised, but emergence is often precipitated by changes to ecological or biological systems (wilcox and colwell ) . such changes include altered patterns of contact between wild and domestic animals (e.g. nipah virus), of direct human and wild animal contact (e.g. hiv, ebola), and changes in species abundance or diversity (e.g. hantavirus; lyme disease). species diversity, including the diversity of insect vectors and pathogenic microorganisms, increases towards the equator (guernier et al. ) , and jones et al. found a correlation between the emergence of zoonotic pathogens and the diversity of mammalian wildlife species (jones et al. ) . whilst high animal host and pathogen species diversity may be associated with a high burden of infectious diseases and an increased risk of disease emergence, biodiversity loss may, perhaps counter-intuitively, be associated with increased disease transmission. biodiversity loss directly disrupts the functioning and stability of ecosystems, producing effects that can extend well beyond the particular lost species (hooper et al. ) . changes in species abundance and diversity may favour pathogen amplification and 'spill-over' through a variety of mechanisms, including reduced predation and competition resulting in increased abundance of competent hosts, and the loss of 'buffering species' leading to increased contact between amplifying host species and compatible pathogens (keesing et al. ) . tropical regions with a rich pool of existing and potential pathogens that are increasingly connected, but also experiencing high rates of ecosystem disruption and biodiversity loss, may therefore be at a particularly high risk of disease emergence. human pathogens occasionally re-emerge as a result of dynamics that are beyond the control of humans. for example the hantavirus outbreaks in the southwestern u. s. in - and - have been attributed to changes in the abundance of infected rodents following periods of heavy snow and rainfall and vegetation growth leading to abundant production of rodent food. however, many ecological disturbances resulting in an eid seem to originate in the direct actions of humans. a wide range of anthropogenic factors have been linked to infectious disease emergence, including changes in land-use, travel, trade, and demographics. notably, most of these associations are speculative and supported (wolfe et al. ). stage : the pathogen is still exclusively infecting animals. stage : the pathogen has been transmitted from animals to humans under natural conditions but not yet from human to human. stage : there is limited transmission from animals to humans and between humans. these are often severe and lethal diseases due to for instance filoviruses (e.g. ebola). stage : animal disease that have sustained transmission between humans (e.g. influenza). stage : a microbe that exclusively infects humans (e.g. measles, syphilis) by little hard data because ecological and biological systems are highly complex and multi-layered (woolhouse ) . demonstrating or predicting the impact of particular conditions or events on the functioning of a system is difficult, with further inference of the impact of any changes on the risk of pathogen emergence posing a formidable challenge. socioeconomic development is associated with large increases in demand for natural resources. the demand for water, wood, pulp, agricultural land, living space, roads, minerals and power has had an enormous impact on the landscapes of asia. deforestation occurred throughout the s and the area of primary forest in asia has continued to decline (fao ) . deforestation, forest fragmentation, and afforestation are all alterations in habitat, which change species composition and the interaction between wild animals, domestic animals, insect vectors and humans, providing new opportunities for microbial transmission and potential emergence. there are well-documented examples of deforestation and forest encroachment resulting in increases in infectious diseases, such as yellow fever, mayaro, and chagas disease in the americas (saker et al. ). the clearing of forest and planting of large cacao plantations was linked to the emergence of oropouche virus in brazil. in asia there are already very high pressures on productive land, and the peak in land-use change in asia has probably passed. many areas are now in an era of increasing intensification of land productivity. this intensification is driven largely by demographic pressures, which are predicted to result in a % increase in food production by , with decreased consumption of grains and increased demand for meats, fruits and vegetables (fao a, b) . the increased demand for food, and meat in particular, when combined with demands for natural resources from industry and domestic consumers, and river damming for hydroelectric power, is resulting in a large increase in stress on water resources (fao b). the consequences of intensified agricultural production include the depletion and degradation of river and groundwater, reduced soil quality, and biodiversity loss. a direct and predictable effect of reduced access to clean water for low-income families is an increase in the risk of water-washed diseases (diseases that increase when the availability of water for personal hygiene is limited e.g. diarrhoeal and respiratory infections, trachoma), and water-borne diseases such as typhoid and hepatitis e. however, unquantified risks arising from the intensification of agriculture are pollution of freshwater with pesticides and fertilizers, loss of biodiversity, and land abandonment by small-scale farmers. the potential consequences of these changes on the risk of emergence of zoonotic infections have not been assessed. wild animals are an important source of food (bush meat) in some developing countries and bush meat has been implicated in the emergence of hiv, and the spill over of monkeypox, nipah and ebola virus (brashares et al. ) . whilst the reservoir of the sars coronavirus is thought to be bats, wild civet cats traded for food are thought to have acted as an intermediate host, transmitting the sars virus to humans through live animal markets (li et al. ) . wild animal products are popular in asia as traditional medicines, tonics, delicacies, or as symbols of wealth. although all ten countries in the association of southeast asian nations (asean) are signatories to the convention on international trade in endangered species of wild fauna and flora (cites), asia continues to host the largest illegal wildlife trade in the world (rosen and smith ) . the smuggling of h n -infected birds of prey into europe, the frequent smuggling of bush meat from africa into the u.s., and the importation into the u.s. of pet rodents infected with monkeypox show that both the legal and illegal trade in wild animals and wild animal products is a potential conduit for the international spread of zoonotic pathogens (bair-brake et al. ; van borm et al. ) between and the world population is expected to increase by . billion (a % increase), and the increase will be concentrated in urban areas of developing countries (un-desa ). whilst mega-cities (cities with a population of at least ten million) receive a lot of attention, most urban dwellers live in small cities, with half of the global urban population in living in cities of less than , people (un-desa ). this can be perceived positively as cities generally offer better economic opportunities, better educational opportunities, better living conditions, better nutrition, better sanitation, and therefore better health than underdeveloped rural areas. at the same time it is likely to mean that in many low to middle income countries, health and veterinary infrastructure in rural areas will not improve, or may even deteriorate, adversely affecting the likelihood of the early detection of eid. the demand for food in urban centres will increase and result in livestock and their products being transported over large distances from a wider catchment area, and thereby increase risk of spread and amplification of eid. urbanisation is one facet of changing human sociocultural systems, which also includes changing consumer demands and dietary habits (janes et al. ). the consequence is a spatial concentration of people and animals; not necessarily co-located, but connected through increasingly complex networks of rural and peri-urban farms and markets, distributors, agricultural workers, and consumers. due to the increase in global human population and economic development, demand for livestock products has risen dramatically over the last years, with the per capita consumption of meat in developing countries more than tripling since the early s and egg consumption increasing fivefold (fao a, b) . the increased demand for meat has been met by more intensive and geographically concentrated production of livestock, especially pigs and poultry (steinfeld et al. ). much of this has been through expansion of both the number of small-scale production units and large commercial farms. high-density monoculture of domestic animals is a form of low biodiversity that poses a particular threat for the spread of infectious diseases from farmed animals to humans. where domesticated animals are a conduit of spread from wild animals to humans, high density livestock production may promote spread of zoonotic diseases. genetic diversity within an individual host species is important since genetic diversity limits the potential for devastating epidemics (king and lively ) . the nipah virus outbreak in malaysia and singapore in - is a good example. once nipah virus crossed from wild bats to domestic swine, an explosive outbreak in high-density swine farms resulted in widespread exposure of humans and over human cases of encephalitis (pulliam et al. ) . other examples where intensified livestock production practices may have led to emergence of a zoonosis include: • streptococcus suis causes severe sepsis and meningitis in humans and is associated with areas of intensive pig production (see fig. . ). risk factors for human infection include swine slaughtering and the eating of undercooked pig products (wertheim et al. ). outbreaks in swine herds of porcine reproductive and respiratory syndrome virus also potentially increases the rate of invasive s. suis infection in swine, which in turn leads to an increased risk of s. suis infection in humans (hoa et al. ). • highly pathogenic avian influenza a subtype h n crossed-over from wild aquatic birds (the natural reservoir of influenza a viruses) to humans via massive amplification in domestic poultry. • the human q-fever epidemic in the netherlands during - caused by the bacterium coxiella burnetii is thought to have arisen when economic drivers led to an increased density of dairy goat farming, which resulted in amplification of coxiella burnetii prevalence and consequent increased spill-over to humans (roest et al. ; georgiev et al. ). the classical foodborne diseases such as e. coli, campylobacteriosis and salmonellosis associated with livestock products have been a significant problem in highincome countries for some time (cdc ; painter et al. ). one of the key factors, in particular in the case of campylobacter, has been the integration of highly intensive poultry production with poultry processing systems facilitating cross-contamination of meat (moore et al. ; nyachuba ) . it is notable that some foodborne pathogens cause subclinical infections in their animal hosts (e.g., campylobacter in poultry); therefore there is no direct incentive for farmers to control them. in addition, subsequent attribution of the source of foodborne disease in an affected human is notoriously difficult. it is likely that the global incidence of foodborne illness will rise as a result of increased industrial production of poultry in the emerging economies. the situation will be exacerbated by the emergence of antimicrobial resistant pathogens of foodborne pathogens (sahin et al. ; luangtongkum et al. ; altekruse and tollefson ) . despite the examples above, the intensification of livestock farming often entails more effective separation of domestic and wild animals, improved standards of animal health and welfare, reduced movement and species mixing: all of which reduce the risk of eids. reducing contact between domestic and wild animals, whether the wild animals are in their natural habitat or captive, is a key strategy of the food and agriculture organization (fao) to reduce risk to human health, and is part of the wider fao strategy of enhancing 'biosecurity'. improving biosecurity in farms is a major challenge since a large proportion of farming in asia is based on small-scale backyard production, and there is often a mix of commercial and backyard farming in any one location. achieving improvements in biosecurity without adversely affecting the livelihoods of small-scale farmers requires an approach to risk management that is adapted to their socio-economic context. the longer-term vision is to restructure the livestock production sector towards a more commercialised and controlled system, where controls benefit animal health, human health, and commercial profitability. but it has also been recognised that small-scale producers will continue to have a key role in providing food security in developing economies, and in fact their importance may increase due to their more efficient use of natural resources compared with large-scale industrial production (sjauw-koen-fa ; quan ). food production has become a complex national, regional and global network of food value chains (ercsey-ravasz et al. ; dickson-hoyle and reenberg ). many countries have begun to more tightly regulate live animal trade in the wake of bovine spongiform encephalopathy (bse), sars and avian influenza a/h n , but complex and poorly regulated food manufacturing and distribution chains still offer ample opportunities for disease outbreaks. in endemic regions, live bird markets are frequently contaminated with highly pathogenic avian influenza a subtype h n (davis et al. ; samaan et al. ; wan et al. ) . contaminated markets can become reservoirs and the source of infection for poultry and humans. measures to reduce the risk in live animal markets include the separation of species, not allowing animals to remain in the market more than h, not allowing poultry to exit the market alive, improved cleaning and disinfection, and weekly rest-days, when all animals are removed and the market is thoroughly cleaned. the re-emergence of brucellosis, one of the world's most common zoonoses, is thought to be the result of higher risk of transmission through increased within-and between country trade of susceptible livestock and their products (seleem et al. ) . the development of denser regional road transport networks may be especially important since, compared to air travel, roads offer a more egalitarian form of connectivity that includes animals and goods as well as humans. this provides opportunities for pathogens to disperse beyond their traditional niche by increasing opportunities for informal trade in live animals and their products (eisenberg et al. ) . veterinary medical authorities often struggle to enforce compliance with regulations, and it is common practice for farmers and traders to adapt their production and trading behaviors to avoid adverse economic consequences of regulations aimed at controlling zoonotic diseases. as a result, informal trade networks may intensify and re-structure in unpredictable ways during disease outbreaks. increasing intensification of animal husbandry in asia may be a trade-off between a lower risk of emergence events, as animals will be 'healthier' and better isolated, but should an eid event occur, an increased risk of massive amplification in large, naïve monocultures. perhaps the greatest risk arises when a limited number of intensive livestock production units are surrounded by substantial backyard farming with little or no biosecurity, thereby linking extensive and weakly regulated value chains with global food systems. livestock production practices can have major unintended and unanticipated impacts on the risk of zoonotic infections. perhaps the most notable example is bse. the feeding of ruminant-derived meat and bone meal to cattle, and possibly changes in practices for rendering animal tissue, led to an epidemic of bse in cattle, that was followed by an epidemic of a fatal neurological disease (variant creutzfeldt-jacob disease) in humans (taylor and woodgate ) . in contemporary livestock production, the use of antibiotics is a significant concern. almost % ( / ) of eids identified in asia since represent the emergence of a new pattern of antimicrobial resistance (jones et al. ) . bacteria in some areas show alarmingly high rates of resistance to anti-bacterial agents, and the recent emergence of the new-delhi metallo-beta-lactamase- (ndm- ) resistance gene, conferring resistance to a last resort antibiotic, and its rapid dissemination to other regions highlights the serious threat to human health of antimicrobial resistance (khan and nordmann ) . antibiotics are used extensively in the livestock and aquaculture sector to treat or prevent infections, or as growth promoters. non-therapeutic use of antibiotics as growth promoters involves the prolonged administration of sub-therapeutic doses. this practise has a demonstrable effect on the emergence and prevalence of resistant microorganisms in food animals and their environment, as well as resulting in the excretion of antibiotics into the environment, where environmental bacteria may be subject to antibiotic selection pressures (marshall and levy ) . synthetic antibiotics such as quinolones are quite stable in the environment over long periods of time. heavy metal contamination of animal food may also play a role in selection of antimicrobial resistance. whilst there remains some debate about the overall impact of these findings on human health, it is clear that the continued use of non-therapeutic antibiotics in an agriculture industry that is rapidly increasing in scale and intensity, has potential for becoming a very real threat through the inability to prevent/cure disease in production animals and the consequences for human food security as well as the transmission, for example, of resistant food-borne bacterial pathogens to humans. some recent examples concern transmission of methicillin resistant staphylococcus aureus (mrsa) from pigs to humans and esbl (extended spectrum betalactamase, an important resistance mechanism against most beta-lactam antibiotics) positive e. coli on chicken meat for human consumption (verkade et al. ; kluytmans et al. ) . swine-associated mrsa is now contributing significantly to invasive disease in patients in the netherlands (verkade et al. ) . antibiotic production and consumption continues to increase, often in an uncontrolled way, accelerating the evolution of antibiotic resistance, which then spreads rapidly across the globe. since the s few new antibiotics have been developed and we are on the cusp of returning to an era of untreatable infections. the consequences of antibiotic use in animals therefore require better surveillance, research and regulation. infectious diseases continue to be an important cause of human and animal morbidity and mortality worldwide. whilst important health advances (e.g. hygiene and vaccination) have been made, infectious diseases are dynamic and resilient, and continue to challenge local, national and global public health systems (fauci and morens ). the recognition of the linkage between anthropogenic changes, animals, and disease emergence has resulted in repeated calls for a more holistic, interdisciplinary, and integrated approach to the study of infectious diseases. the one health approach is one initiative aimed at realizing this aspiration. the task ahead is to understand how social, economic, and environmental changes are altering the landscape of infectious disease risks for both animals and humans, and how future emerging risks may be mitigated. a more analytical approach to these emergence events requires characterisation of the attributes of natural and artificial ecological systems before and after disturbance, and the functional relationship between changes in system attributes and pathogen emergence (woolhouse ) . human campylobacteriosis: a challenge for the veterinary profession is that a rodent in your luggage? a mixed method approach to describe bushmeat importation into the united states economic and geographic drivers of wildlife consumption in rural africa vital signs: incidence and trends of infection with pathogens transmitted commonly through food-foodborne diseases active surveillance network detection and characterization of clade high pathogenicity avian influenza h n viruses in chickens seized at ports of entry and live poultry markets in vietnam the shrinking globe: globalisation of food systems and the changing geographies of livestock production environmental change and infectious disease: how 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biocomplexity as an interdisciplinary paradigm origins of major human infectious diseases how to make predictions about future infectious disease risks key: cord- -gza hsv authors: tiew, pei yee; mac aogain, micheál; ali, nur a’tikah binte mohamed; thng, kai xian; goh, karlyn; lau, kenny j. x.; chotirmall, sanjay h. title: the mycobiome in health and disease: emerging concepts, methodologies and challenges date: - - journal: mycopathologia doi: . /s - - -z sha: doc_id: cord_uid: gza hsv fungal disease is an increasingly recognised global clinical challenge associated with high mortality. early diagnosis of fungal infection remains problematic due to the poor sensitivity and specificity of current diagnostic modalities. advances in sequencing technologies hold promise in addressing these shortcomings and for improved fungal detection and identification. to translate such emerging approaches into mainstream clinical care will require refinement of current sequencing and analytical platforms, ensuring standardisation and consistency through robust clinical benchmarking and its validation across a range of patient populations. in this state-of-the-art review, we discuss current diagnostic and therapeutic challenges associated with fungal disease and provide key examples where the application of sequencing technologies has potential diagnostic application in assessing the human ‘mycobiome’. we assess how ready access to fungal sequencing may be exploited in broadening our insight into host–fungal interaction, providing scope for clinical diagnostics and the translation of emerging mycobiome research into clinical practice. fungal disease affects over million people worldwide causing over . million deaths annually [ ] . despite their natural environmental abundance, few fungi are human pathogens, and while fulminant fungal infection is uncommon in the healthy individuals, invasive fungal disease is a concern in the immuno-compromised host with significant associated morbidity and mortality [ ] . increasing numbers of patients are at risk of invasive fungal disease including those with human immunodeficiency virus (hiv), malignancy and transplant recipients on immunosuppressive or immunomodulatory therapies, each contributing to the rising global trend of fungal infections among susceptible populations. global warming and climate change also have significant impacts on the pathogenicity and survival of fungi [ ] . acclimatisation to higher temperatures increases their ability to replicate in the human body with high basal temperature, resulting in increased pathogenic potential even for species previously reported as nonpathogenic [ ] . this impacts fungal distribution where increases in heat-resistant species facilitate human interaction, infection and transmission through skin contact, inhalation and/or ingestion [ ] . fungal infection therefore can manifest as superficial infections of the skin, nail, hair and mucous membranes, by dermatophytes and candida spp. or as invasive disease caused by opportunistic and endemic mycoses [ ] . superficial disease is generally mild and easily diagnosed with readily available treatment. however, in the immuno-compromised individual, such infections may progress to invasive disease. opportunistic fungal disease is primarily seen in the immunocompromised with aspergillosis, candidiasis and cryptococcosis most commonly reported. the annual incidence ranges from , to million, with high and variable mortality rates ranging from to % [ ] . table and fig. summarise the most common invasive fungal diseases and the anatomical sites involved. endemic fungal diseases occur in distinct geographic regions, apparently driven by environment and climate. irrespective of underlying host immunity, individuals residing in endemic regions are at higher risk of life-threatening disease. blastomycosis and histoplasmosis are endemic in the midwestern united states (us), coccidioidomycosis in the southwestern us, paracoccidioidomycosis in brazil and talaromycosis in southeast asia [ ] . worryingly, endemic fungal disease continues to propagate even expanding beyond traditional accepted boundaries, again driven at least in part by climate change, urbanisation, land development and ease of travel [ ] . a key example is the outbreak of cryptococcus gattii at vancouver island and the pacific northwest of the usa [ ] . this poses significant and emerging challenges for diagnosis, which in turn delays treatment initiation with adverse clinical consequence. the early diagnosis and initiation of therapy for fungal infection is critical, influencing outcome and mortality, particularly in the immuno-compromised [ ] [ ] [ ] . current available diagnostic modalities lack sensitivity and specificity, and therefore the diagnosis of invasive fungal disease relies on a combined clinical, microbiological and radiological approach with empirical treatment often initiated based on clinical judgment alone pending diagnostic confirmation [ ] . patient characteristics including the presence or absence of neutropenia or use of antifungal prophylaxis impact the diagnostic accuracy of tests, adding additional uncertainty [ ] [ ] [ ] . the ability to distinguish infection from colonisation poses an additional and important clinical challenge. conventional culture methods provide species-level identification and information on antifungal susceptibility. however, culture-based methods are poorly sensitive and delay time to diagnosis [ ] [ ] [ ] . their accuracy is contingent on sample source, type of fungus, as well as disease and host immune status. for instance, pneumocystis species are unable to grow on culture media [ , ] and isolation of aspergillus in blood is uncommon and ineffective for the diagnosis of invasive aspergillosis [ , ] . in addition, positive cultures obtained from non-sterile body sites may not be representative and should be interpreted with caution. direct microscopic examination is useful in providing rapid and more accurate diagnosis of fungal infection allowing differentiation by their morphology and staining [ ] . however, as with culture, the diagnostic yield is contingent on several factors and exhibits variable accuracy [ ] . non-culture-based techniques are increasingly employed to aid the diagnosis of invasive fungal disease. for example, detection of b-glucan, a component of the fungal cell wall, identifies patients with invasive disease with sensitivities ranging from - % and specificities of - % [ , , ] . the high false positive rate, however, limits the usefulness of this approach particularly in high-risk patient groups including patients with haematological malignancy, stem cell and lung transplant recipients, where co-existing bacterial infection is present and in those receiving haemodialysis [ ] [ ] [ ] . while b-glucan is a useful diagnostic target, it is not specific to any single fungal table review of major invasive fungal disease with organ involvement, disease manifestation, susceptibility, diagnosis and management [ ] species and may be detectable across a range of fungal infections including invasive aspergillosis, candidiasis, and cryptococcosis. galactomannan (gm) is another commonly used marker for the diagnosis of invasive aspergillosis in patients with haematological malignancy and/or neutropenia with sensitivities and specificities of - % and - %, respectively [ ] . however, sensitivity decreases with use of antifungal agents and among non-neutropenic patients [ , ] . serological testing is another option available for a range of fungal infections including candidiasis, cryptococcosis and endemic mycoses with diagnostic accuracies varying based on the type of fungal organism and sample source. while providing a good diagnostic yield for histoplasmosis and coccidioidomycosis, it is not particularly useful for the diagnosis of invasive aspergillosis [ , ] . polymerase chain reaction (pcr) provides an alternative, more rapid and accurate diagnostic tool for the detection of fungal infection [ ] . numerous pcr assays are described with variations in primer sequence, dna extraction method, positivity thresholds and assay performance, which coupled with limited multi-centre clinical validation, has curtailed widespread adoption [ ] [ ] [ ] [ ] . importantly, false positive results are common as fungi are ubiquitous and contamination possible at various sample processing stages necessitating rigorous and costly quality control processes [ ] . next-generation sequencing (ngs) is increasingly used in epidemiological studies and during infectious disease outbreaks for resolution of species, virulence and antibiotic resistance, to assist infection control and its management [ ] [ ] [ ] . in addition, ngs may be employed in the resolution of cryptic epidemiological cases where it has demonstrated superiority to traditional diagnostic methods in the detection of rare infections [ ] [ ] [ ] . however, demonstration of its utility within the medical diagnostic setting is currently limited. low abundance of fungi in relation to the overall microbial community (b . %) poses challenges to their detection via metagenomic sequencing in human samples, necessitating targeted amplicon sequencing approaches, which are still in their infancy [ , , ] . further refinement and standardisation of reproducible wet-lab and bioinformatic workflows coupled to automated and scalable user-friendly reporting systems remain key challenges for the field, which unless addressed, will continue to impede widespread adoption and translation into clinical practice [ ] [ ] [ ] . four distinct classes of antifungal drugs are currently prescribed: the polyenes, flucytosines, azoles and echinocandins. despite treatment, mortality rates for invasive fungal disease remain high with factors contributing to poor prognosis including delayed diagnosis and initiation of antifungal treatment, host factors, site of infection, emerging antifungal resistance and drug toxicity. clinical presentation of fungal infection is largely non-specific, and therefore a high index of suspicion is necessary for early diagnosis and initiation of treatment, particularly in the immunocompromised population. host factors of relevance include underlying comorbidities and long-term medications that interact with antifungal treatment. this is most marked in concomitant antifungal and antiviral treatments in hiv-related cryptococcus infection, which results in life-threatening inflammation secondary to immune reconstitution. the available tissue concentrations of antifungals also differ according to their chemical and pharmacokinetic properties leading to sub-optimal concentrations, therapeutic failure and the development of resistance in many settings, highlighting the importance of infection site to the appropriate selection of antifungal agent [ ] . drug toxicity is a further consideration as treatment is often prolonged and, in those with other comorbidities, increases the risk of drug-drug interactions [ ] . antifungal resistance is emerging rapidly, both intrinsic resistance and that driven by the widespread use of antifungal and environmental fungicides [ ] . resistance, however, varies based on geographic location, patient demographics and prior drug exposure [ , ] . azole resistance in candida and aspergillus spp. ranges from . - to . - %, respectively [ , [ ] [ ] [ ] [ ] . resistance to echinocandins-first-line antifungal agents for invasive candidiasis-is also described in invasive candida glabrata infections [ , , [ ] [ ] [ ] . the development of new antifungals with decreased toxicity and broad-spectrum coverage is urgently required to reduce mortality rates, and a better understanding of host-fungal interaction is necessary to facilitate future therapeutic development pipelines. furthermore, advances in ngs enable identification of antifungal resistance genes, informing our understanding of the resistome and guiding antifungal drug development [ ] [ ] [ ] . in the following sections, we aim to describe our current understanding of the mycobiome in the context of recent ngs advances and the methodological challenges and barriers that exist in terms of its clinical implementation. sample processing including storage conditions and various dna extraction protocols may inadvertently introduce confounders that affect experimental results (fig. ) . to date, studies comparing various methodologies of sample processing demonstrate conflicting results and varying efficacies, with implications for cross-comparisons of published mycobiome research. freezing samples within h to prevent overgrowth by fast-growing fungi and avoiding multiple freezethaw cycles has been advocated [ , ] . the addition of rnalater decreases the relative abundance of certain fungal taxa particularly in faecal samples suggesting possible introduction of bias in relative abundance assessments [ ] . nevertheless, taxonomic diversity generally remains assessable and largely unaffected by the freezing process [ ] [ ] [ ] . when extracting fungal dna for mycobiome sequencing, the highly rigid fungal cell wall, rich in glucans, chitin, mannans and glycoproteins, has necessitated additional steps that involve rigorous bead-beating or enzymatic lysis prior to dna extraction [ , , [ ] [ ] [ ] [ ] . notably, enzymatic lysis provides an alternative option for increasing dna yields [ ] [ ] [ ] . the harsher bead-beating method, however, is observed to have conflicting efficacies likely due to dna degradation [ ] . various dna extraction methods also significantly affect fungal dna yield and quality with phenol/chloroform extraction often outperforming the various available commercial kits ( fig. ) [ , , , , ] . this loss of dna using commercial kits is likely related to use of silica column purification [ ] . dna extraction kits illustrate inconsistent results when performed with different sample types and produce variable fungal dna yields [ , ] . while extraction methods affect dna yield and quality, their impact on mycobiome composition and diversity appears minor [ , , ] . however, recent work from our group, using spontaneous induced sputum, has demonstrated more consistent amplification of the its region from samples subjected to mechanical disruption as compared to enzymatic lysis [ ] . this was observed despite higher overall dna yields achieved by enzymatic lysis suggesting preferential liberation of fungal dna by mechanical lysis [ ] . extraction methods are critical and must be applied consistently and carefully both within and between mycobiome studies. ultimately, sample-specific mycobiome protocols are required and a need exists to incorporate specific methods adjusted to sample type or disease-specific application, importantly, with validation for routine use in future mycobiome research. in contrast to high-throughput sequencing (hts) of the bacterial microbiome, taxonomic classification and primer choice are not well established for the fungal mycobiome ( fig. ) [ , ] . as amplicon sequencing is well recognised to be subject to selective amplification bias, careful consideration of primer selection is crucial [ ] . key factors include taxonomic resolution, coverage, accuracy and amplicon length [ ] . the its region, located between genes encoding ribosomal subunits s and s, has been proposed as the universal genetic fungal barcode, primarily because these regions have high evolutionary rates and are flanked by highly conserved regions serving as suitable target sites for universal primers [ , , , ] . the its region typically spans - bps, and consists of two sub-regions, its and its , which are separated by the conserved . s region [ ] . although primers targeting its (i.e. its f and its ) have been used for decades in several large-scale microbiome projects, to date, it is still unclear which its fragment is optimal. recent findings demonstrate that these primers suffer from primer bias, amplification bias and fail to accurately profile mock communities analysed, leading to inconsistent representation of synthetic fungal communities of known taxonomic abundance [ , , [ ] [ ] [ ] [ ] [ ] . based on these observations, nilsson and colleagues provide a list of fungus specific, hts-oriented primers for amplification of its region, and recommend targeting the its sub-region by using degenerate forward primers gits ngs and the reverse primer its ng owing to their superior coverage of the fungal kingdom [ ] . advantages of the its sub-region include a more universal primer site and lower length variation, leading to less taxonomic bias compared to its [ , , ] . while this primer set (gits ngs and its ng) has a superior coverage of fungal kingdoms, its accurate taxonomic resolution has yet to be evaluated experimentally [ ] . further confirmatory studies are required before the field can advance and eventually reach consensus on methodology for targeted amplicon sequencing of the mycobiome, as has been the case for s rrna sequencing of bacterial microbiomes. in addition to primer choice, care must be taken to yield fewer compromised amplicon sequences. these include careful consideration of number of pcr cycles and dilution of dna samples where possible (fig. ) [ , ] . high fidelity proofreading pcr polymerase with low gc content bias is also necessary to reduce chimeras and random errors that accumulate in later cycles [ , ] . it is also essential to include appropriate negative controls and a mock community into the analysis. the former indicates sources of potential contamination, while the latter allows assessment of chimera formation, quantification of index switching and operational an alternative approach to targeted amplicon sequencing is the use of whole-genome shotgun (wgs) metagenomics to study the mycobiome. this entails the sequencing of all extracted dna in a given sample without the use of targeted pcr amplification of the its or other specific target sequences. here, all dna from resident microbes and host cells present in a sample is assessed. sequence reads are processed and classified relative to a reference database through either assembly-based or assembly-free methods [ ] . analysis will usually also include steps for removal of host dna sequences, which may contribute to a significant proportion of sequence data if removal of human cells is not attempted prior to dna extraction [ ] . while, in principle, metagenomic sequencing should provide a superior, unbiased assessment of the mycobiome, studies to date employing wgs metagenomics highlight the low abundance of fungi relative to bacterial dna across a variety of human samples (fig. ) [ ] . this represents a significant cost barrier given the sequencing depth therefore required to detect fungi in samples where bacteria predominate while lower sequencing coverage also hinders assembly-based analysis of fungi [ ] . currently, it appears that low fungal abundance in human samples hinders the widespread application of metagenomic wgs in human samples, a finding that appears unrelated to dna extraction methods and reflects a genuinely low overall in vivo fungal abundance [ ] . the development of tools for the analysis of itsderived amplicon sequences is still a nascent area of research, and many options are available for the analysis of metabarcoding amplicon data sets [ ] . while the field still has no consensus on standards and best practices, further studies are clearly required, and no optimal, broadly applicable protocol has been yet established. further, it is probable that amplicon sequencing and its accompanying analysis largely depend on the samples under investigation due to variable primer coverage, as demonstrated for comparable bacterial microbiome analysis targeting the s rrna gene [ ] . the increased variability of the fungal its region exacerbates this problem in the case of mycobiome research, and this requires careful consideration when interpreting its-derived mycobiome profiles. a further analytic hurdle of mycobiome metagenomic analyses remains the sparsely populated fungal databases on which read classifications are based (fig. ) . this leads to large numbers of unclassified reads or unidentified operational taxonomic units (otus), which may be addressed by generation of more high-quality metagenomic and whole-fungal genome assemblies [ , ] . major fungal its reference databases include the insdc, unite and warcup its [ ] . although accurate species-level classification is not possible using the its regions, there are currently no known alternate markers offering comparable lineage separation, while reported taxonomic thresholds for filamentous fungal identification at the genus, family, order and class levels are estimated at . %, . %, . % and . %, respectively [ , ] . the increased accessibility of third-generation long-read sequencing offers promise in this regard as the large stretches of repetitive and non-coding dna as well as introncontaining genes found in fungal genomes can be resolved by these technologies which may also be applied to targeted sequencing of the its [ , ] . although the literature on fungal metagenomics is lacking, two key studies (nash et al. and donovan et al.) represent the most in-depth assessment to date in terms of metagenomic identification of fungi in human samples [ , ] . nash et al. performed a large assessment of gut samples from the human microbiome project, performing both metagenomic analysis and comparative its sequencing. importantly, they showed that dna extraction methods did not have appreciable effects on the levels of fungi detected, reaffirming the apparent low abundance of fungi in these samples. secondly, they performed a comparative analysis of its and s targeted sequencing protocols demonstrating that its -targeting primers showed greater resolution of low-abundance taxa [ ] . the work of donovan and colleagues is notable for their bioinformatic dissection of the challenges inherent to analysis of the fungal metagenome. they present a solution in the form of the 'findfungi' pipeline, which highlights the challenges of fungal metagenomic analysis including false positive detection, for which they developed an important step to identify uneven read distribution across reference genomes indicating spurious fungal detection [ ] . finally, an additional challenge that remains is the implementation of these bioinformatic pipelines, which, necessitates execution on high-performance computing clusters due to the requirement to hold large genomic databases in memory (fig. ) . such computation requires domain expertise that may not be widely available to fungal research laboratories and remains an operational and logistical challenge. culture-based methods of detection require a priori selection of fungal growth conditions and are susceptible to bacterial overgrowth hindering the proliferation of selected fungi leading to bias. in addition, culture-based methods are prone to cross-contamination of spore-producing fungi, which can be minimised by the use of horizontal laminar flow clean bench [ , ] . while the addition of antibiotics to culture media is commonly employed to prevent bacterial contamination, its drawbacks include limited antibacterial spectrum, and its potential inhibitory effect on fungi [ ] [ ] [ ] . results from culture methods may be difficult to cross compare with the existing literature given heterogeneity of methodology and study designs as well as sample pre-processing protocols, thereby requiring a standardised protocol for detecting key fungal pathogens [ , ] . however, culture-based methods do allow for an assessment of antifungal susceptibilities and the isolates obtained by culture provides scope for further molecular characterisation. numerous culture-independent approaches have been developed to overcome the limitations of culture-based methodology but are not without their disadvantages. internal transcribed spacer-polymerase chain reaction (its-pcr) approaches could be used to detect mixed infections; however, careful target selection is necessary [ , [ ] [ ] [ ] . in some cases, its-pcr may appear even less reliable than culture for fungal detection [ ] . multiplex pcr assays allow for greater efficiency but require strict primer design [ ] . however, pcr-based approaches are susceptible to falsenegative results where sub-optimal sample storage or dna extraction methods are employed or where pcr inhibitors are present. the use of oligonucleotide array platforms represents a possible alternative but is expensive and subject to batch effects [ ] . arguably, sequencing of individual amplified its regions remains the gold standard for fungal identification, allowing for sensitive detection of unculturable or low-abundance fungi but may not be practical for all laboratories [ ] . the use of mycobiome profiling as an alternative to culture-based and molecular diagnostic detection methods represents potentially valuable applications for this technology, but one that requires further validation and research [ ] . the term mycobiome is used to describe the fungal component of the microbiome. it is yet unclear whether a core mycobiome, analogous to the bacteriome, exists in humans [ ] . relative to the bacteriome ([ % of the total microbiota), the human mycobiome is less diverse (generally \ otus per sample) and present at a lower abundance (b . % of the total microbiota) [ , ] . targeted sequencing approaches reveal that infants are colonised by fungi shortly after birth, predominantly by members of the genera candida, saccharomyces, cladosporium, cryptococcus and malassezia [ , ] . where correlated, stool mycobiomes from healthy adults reveal profiles predominated by saccharomyces, malassezia and candida suggesting that these genera are the key constituents of a healthy mycobiome throughout life. critically, however, and in contrast to human bacteriome studies, little correlation with host phenotype metadata is firmly established in healthy individuals and, as such, our knowledge of fungal mycobiome-host interaction remains limited to that in fungal colonisation and/or disease. it is clear from existing studies and their subsequent molecular analysis that intricate immunological systems have evolved to regulate fungal homoeostasis and, when perturbed, lead to infection or other pathologies [ ] . immunity to fungi is underpinned by innate signalling pathways recognising both spores and fungal cell wall components and include adaptive t cell responses that orchestrate tolerance and appropriate immune surveillance of ubiquitous fungi encountered daily [ , ] . immune responses to fungi are protective when functioning, however, where over-zealous, can manifest as allergy. consequently, the evolution of immuno-regulatory mechanisms in vivo is perturbed across a range of fungal-driven allergy. given the parallels with the human bacteriome, where host immunity acts as gatekeeper, it is arguable that similar ecological interactions occur with fungi [ ] . perhaps the strongest evidence for a conserved human mycobiome is evidenced by the restricted numbers of fungi that actually inhabit the human body, suggestive of niche specialisation [ ] . despite over described bacterial phyla, the human bacteriome is characterised by four: actinobacteria, bacteroidetes, firmicutes and proteobacteria. in corollary, fungal taxa exhibit lower diversity in vivo compared to that seen environmentally and associations between fungi and specific body sites indicate the likely existence of selection and adaptation [ , ] . given the limited available data, formal meta-analysis of clinical mycobiome studies, while important, is currently challenging to perform. however, emerging associations based on culture-independent mycobiome studies in humans are summarised in fig. . while fungal lung disease is now widespread and a growing global concern, the lack of dedicated mycobiome studies assessing the effects of fungi in chronic lung disease is hampering the field. in this particular organ system, the prior presupposition of sterility in a healthy lung puts back microbiome studies in this area for a number of years, and consequently, our understanding of the respiratory microbiome lags significantly behind that at other anatomical sites [ ] . the advent of culture-independent sequencing technologies has uncovered the diversity of organisms inhabiting the lung, including that in the healthy state [ , ] . through the use of internal transcribed spacer (its) sequencing, it was reported that ceriporia lacerata, saccaromyces cerevisiae and penicillium brevicompactum were possible pulmonary fungi, with the main pathogen being aspergillus fumigatus [ , ] . lung-dwelling microbiota include bacteria, fungi and viruses, which together maintain a crosskingdom ecological network [ ] . interactions between lung and gut microbiomes, through the lung-gut axis, have further immunological consequence, and changes in the gut mycobiome can influence allergic airway disease [ ] [ ] [ ] [ ] [ ] . the healthy lung mycobiome differs from that observed in patients suffering with chronic inflammatory respiratory disease such as asthma, chronic obstructive pulmonary disease (copd), cystic fibrosis (cf) and bronchiectasis [ , ] . the lungs in asthma, copd and cf all exhibit lower fungal diversity, which in turn is linked to poorer lung function postulated to be influenced by fungal overgrowth and/or loss of some fungal species [ ] . in asthma, higher loads with increase percentages of psathyrella candolleana, malassezia pachydermatis, termitomyces clypeatus and grifola sordulenta and a greater fungal diversity were observed compared to that of a healthy airway [ , ] . in children with severe asthma, a higher abundance of rhodosporidium, pneumocystis, leucosporidium and rhodotorula was detected when compared to those without asthma [ ] . bronchiectasis, a permanent and irreversible dilatation of airways, places patients at the higher risks of fungal acquisition and colonisation [ ] . this is reflected by airway mycobiomes demonstrating a higher abundance of potentially pathogenic taxa including aspergillus, penicillium and cryptococcus and the detection, in many patients, of an unfavourable allergic sensitisation and immune response profile associated with aspergillus [ , ] . in view of the dysfunctional mucociliary clearance inherent in bronchiectasis, the airway is susceptible to inhaled airborne fungi and therefore colonisation [ ] . in copd, aspergillus spp. has been isolated in % of patients and particular fungal taxa such as pneumocystis jirovecii associated with a copd airway have also been found in hiv and copd-associated hiv suggestive of immunological predisposition [ , ] . the overrepresentation of pneumocystis and other fungal species in hiv-related copd further illustrates the relationship between fungal communities in the lung and corresponding lung function and homoeostasis, warranting more detailed investigation by culture-independent sequencing [ ] . significant fungal variability is seen in cystic fibrosis (cf), a genetic disease caused by abnormal cystic fibrosis transmembrane conductance regulator (cftr) function, where fungal colonisation and infection for a. fumigatus range between and % [ ] . while dedicated cf mycobiome research is nascent, a number of groups have nevertheless started to characterise its main components. stable fungal communities are detected and exist in parallel to the more ephemeral fungal species that remain transient when assessed longitudinally [ , , , [ ] [ ] [ ] [ ] [ ] . targeted sequencing studies of the cf mycobiome reveal that in addition to the fungi detected by mycological culture alone, that (unculturable) fungal taxa are also present [ , , ] . overall, an increased abundance of aspergillus species (particularly a. fumigatus), c. albicans, c. parapsilosis and malassezia is identified [ ] . in both asthma and cf, an allergic response may be induced when aspergillus is encountered, with the key form being allergic bronchopulmonary aspergillosis (abpa) [ ] . the application of mycobiome sequencing approaches to the lung has started to gain significant traction and research interest with the transition from smaller pilotscale optimisation studies to a more detailed and comprehensive assessment in well-phenotyped cohorts of patients with respiratory disease [ , , , ] . efforts to examine gut mycobiomes have largely focused on gastrointestinal (gi)-associated disease, and key findings echo trends of mycobiome alteration in other non-gi diseased states. in inflammatory bowel disease (ibd), for instance, initial work using s rrna and rdna sequencing showed an increased fungal diversity in crohn's disease (cd) but not ulcerative colitis (uc) [ , ] . further differences were also identified between inflamed and noninflamed tissue [ ] . more recently, however, application of its sequencing has found distinct fungal microbial dysbiosis in ibd with increased basidiomycota/ascomycota ratios accompanied by higher proportions of c. albicans but reduced levels of s. cerevisiae [ ] . in cd, its pyrosequencing also confirms increases to overall fungal load during disease flares and a higher prevalence of the cystofilobasidiaceae family and c. glabrata species. in correlation, the application of its sequencing in another study revealed an increased candida tropicalis in cd as compared to non-cd relatives [ ] . interestingly, s. cerevisiae and filobasidium uniguttulatum are associated with non-inflamed tissue in cd [ ] . malassezia restricta was found to increase cd severity in a subset of patients with card polymorphisms, suggesting that the targeting of specific fungi in certain patient phenotypes may have value [ ] . it is further demonstrated that antifungal drugs lead to increases in colitis severity in animal models where its amplicon sequencing revealed gut fungal dysbiosis characterised by a reduction in candida spp. and concurrent increase in aspergillus, wallemia and epicoccum spp. [ ] . similarly, antibiotics influence gut inflammation through modification of the gut bacteriome with a consequential impact on fungal colonisation [ ] . here, alteration in enterobacteriaceae modulates fungal colonisation and colitis severity [ ] . although the mouse gut mycobiome does not fully represent that in humans, these findings lend support to the implication of fungal dysbiosis in ibd. paediatric ibd has also been assessed providing insight into potential age-associated effects, and s rdna pyrosequencing illustrates basidiomycota dominance [ ] . in other paediatric ibd works, its pyrosequencing reveals high candida burdens and a reduced fungal diversity [ ] . while dedicated study of the healthy gut mycobiome is limited and much required, nash et al. observed the healthy human gut mycobiome to be lacking in diversity and dominated by yeast genera such as saccharomyces, malassezia and candida, albeit with significant interand intra-individual variability [ ] . this suggests active and dynamic change to the gut mycobiome over time, further influenced by host immunity, even in the absence of disease. in irritable bowel syndrome (ibs), a functional gastrointestinal disease, its based metabarcoding of faecal samples reveals a gut mycobiome dysbiosis with a significant loss of diversity. saccharomyces and candida were dominant in ibs and healthy controls, but higher proportions were observed in the former [ ] . visceral hypersensitivity is linked to ibs with fungi implicated in animal models, suggesting that targeted manipulation of the mycobiome may provide scope for therapeutic intervention [ , ] . mycobiome signatures clearly differ between hypersensitive and normally sensitive ibs [ ] . while these findings are clearly of clinical interest, more compelling evidence and biological validation is required to further clarify mechanistic underpinnings of these early observations [ ] . interestingly, in the oncological setting, antifungal treatment is associated with decreased tumour progression in pancreatic ductal carcinoma, while posttherapeutic repopulation by malassezia accelerates tumour growth [ ] . in addition, an increased abundance of malassezia is described in colorectal cancer by faecal shotgun metagenomic sequencing, along-with moniliophthtora, rhodotorula, acremonium, thielaviopsis and pisolithus, while a higher abundance of basidiomycota is associated with more advance disease [ , ] . the gut mycobiome may additionally influence other organ systems such as the lung and central nervous system through their respective axes, but the precise mechanisms are poorly understood. in particular, the lung-gut axis, which includes the mycobiome, is highlighted as a key and emerging player in determining susceptibility to chronic lung disease, and the gut microbiome composition in itself has also been independently associated with several chronic inflammatory lung diseases [ ] . for instance, the presence of c. albicans in the gut influences th -related immune pathways, which in turn has implications for aspergillus-associated pathology in chronic airways disease supporting a role for lung-gut cross-talk in the pathogenesis of respiratory disease [ ] . gut mycobiome dysbiosis is reported in multiple neurological disorders, where a higher abundance of candida occurs in schizophrenia, autism and rett syndrome [ ] [ ] [ ] [ ] . this has prompted the investigation of gut fungi in the microbiome-gut-brain axis, an emerging biological concept already firmly established with respect to bacteria [ ] . while recent studies describe associations between fungal dysbiosis and ibs, the association with neurological symptoms such as anxiety and depression in ibs sufferers is intriguing, results that further support the role of gut fungi in the microbiome-gut-brain axis [ ] . while these findings remain speculative at present requiring more study to explain clearer biological mechanism, they nevertheless do illustrate the importance and emergence of the fungal mycobiome in human disease. the skin remains a key organ and provides barrier protection against pathogens but is also home to resident fungi [ , ] . different skin components demonstrate differing fungal patterns, and fungal species also vary by age and gender. for instance, children have greater numbers and variety of fungi on their skin compared to adults, who are dominated by lipophilic malassezia [ , ] . this is likely explained by the changes in sebum composition and sebaceous gland activation [ ] . most work reveals that healthy skin (across many body sites) is characterised by malassezia, which produces an aryl hydrocarbon receptor (ahr) ligand beneficial for epithelial cell health and uv protection [ , , ] . diverse domains of the skin mycobiome interact with one another, promoting connectivity and providing network stability. comparable to lung mycobiomes, the communities in skin mycobiomes help maintain ecological network structure [ ] . as with other organ systems and their microbiomes, the skin mycobiome associates with skin disease: malassezia, when in higher abundance or where an inappropriate immune response is observed is seen in association with seborrhoeic or atopic dermatitis [ , ] . conversely in psoriasis, which involves the multiplication of skin cells at much higher rates, patients had a lower abundance of malassezia when compared to healthy skin despite malassezia remaining the dominant phylum in both groups [ ] . relationships between the skin mycobiome and systemic diseases demonstrating dermatological manifestations are established: for example, the role of fungi in systemic sclerosis (ssc), an autoimmune disease characterised by skin thickening where rhodotorula glutinis is highly abundant. average r. glutinis abundance in normal subjects compared to ssc patients was . and . per million reads, respectively, supporting this association [ ] . tinea versicolor (pityriasis versicolor), a skin fungal infection, attributed to malassezia and a consequence of malassezia protein-induced melanocyte apoptosis and uv exposure has also been the subject of multiple investigations [ ] . affected lesions illustrate high burdens of m. globosa, m. sympodialis and m. furfur, and mycelial fungal forms predominate in contrast to yeast forms commonly seen in healthy skin [ ] . a role for the mycobiome has been established in seborrhoeic dermatitis, a condition where scaly patches, red skin and persistent dandruff ensue. while ascomycota and basidiomycota are prevalent in both healthy and dandruff-affected human scalps, the latter illustrates higher densities of acremonium, penicillium and malassezia [ , ] . malassezia restricta and malassezia globosa are identified in seborrhoeic dermatitis, and the presence of new colonising strains when compared to the healthy scalp is indicative of disease onset [ ] . malassezia may also more directly cause dandruff as antifungal agents are particularly effective as treatment [ ] . atopic dermatitis (ad) (eczema) is another important skin condition with established mycobiome associations: significant intra-and inter-individual variation is observed in ad where taxonomic diversity has been examined. however, consistently, as is the case in other skin conditions, m. sympodialis, m. sloofiae and m. dermatis all characterise disease and sometimes exacerbate it. ad patients demonstrate circulating ige to malassezia and on closer examination, secreted malassezia vesicles from ad patients differ in their rna content when compared to healthy individuals [ , , [ ] [ ] [ ] . in addition, malassezia may induce cutaneous inflammation via a th- response, with increased ccr ? th memory t cells (m. specific) observed in ad [ ] . while malassezia is a key ad correlate, it is important to recognise that non-malassezia fungi are also described [ ] . different methodologies have been employed to characterise and describe skin mycobiomes: ion-torrent sequencing has been used in ad, while other approaches include targeted amplicon sequencing of various fungal regions, such as the fungal s rrna large subunit d /d and the s rrna, internal transcribed spacer (its) regions its and its [ , , , , , , , [ ] [ ] [ ] . increasing evidence suggests the importance of the microbiome in neurological disease. changes to gut bacteria are linked to multiple sclerosis (ms), a neuroinflammatory disease characterised by demyelination and neurodegeneration, suggesting cross-talk via gutbrain microbiota in its pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] . higher abundance of candida, malassezia and trichosporon by targeted its sequencing is reported in ms compared to control brain tissue [ ] . similarly, metagenomic analyses of cerebrospinal fluid (csf) from ms patients reveal that the presence of malassezia, ascomycota, funneliformis, glomus, cladosporium, candida and alternaria, however as concluded by these works, likely reflects environmental contaminants [ ] . in contrast, jovel et al. using csf from ms patients did not report any detection of fungal reads [ ] . in alzheimer's disease, fungal cells are detected in neurons through immunohistochemistry and confocal microscopy within brain tissue and these fungal species are further identified by nested pcr, where candida, cladosporium, malassezia, neosartorya, phoma and sacharomyces are detected [ ] . using targeted amplicon sequencing, alternaria and malassezia are higher in alzheimer's compared to normal brain tissue [ ] . in other works, brain tissue from individuals with amyotrophic lateral sclerosis (als), a neurodegenerative disease with progressive motor neuron dysfunction, contains an increased abundance of candida, malassezia, fusarium, botrytis, trichoderma and cryptococcus [ , ] . the strong association between seborrhoeic dermatitis and parkinson disease (pd) also raises the suspicion of specific malassezia contribution to both disease states. importantly, genetic polymorphisms associated with pd and levodopa use are known to promote malassezia growth and invasiveness, and hence this fungal association is of clinical relevance [ ] . collectively, these studies implicate fungi in the pathogenesis of several neurological diseases underlining the importance of fungal biology and host-microbe interaction to overall human health and the inter-organ axis communication networks posited to promote disease onset. fungi are ubiquitous and present in indoor (built) and outdoor environments [ ] . while indoor and outdoor fungal communities are similar, indoor fungal particles are passively distributed and dominated by spores, hyphal fragments and other dormant fungi [ ] . fungi from indoor air may originate from outdoor air with air exchanges when windows are opened [ ] . outdoor airborne fungal composition is affected by location, temperature and humidity, with large numbers of fungi identified by metagenomic sequencing, with consequent and significant impact on indoor fungal communities [ ] . other sources of indoor fungi include humans, pets, plants, plumbing systems, heating ventilation and air conditioning systems (hvac), and also dust re-suspension [ , ] . exposure to indoor fungi and fungal particles is linked to health-related outcomes including allergies, fatigue and asthma [ , ] . in addition, damp dwellings pose an additional health risk due to fungal exposure [ ] . the built mycobiome is therefore of interest from microbiological, environmental and clinical perspectives. traditionally, morphology-based studies of fungal cultures and their spores, derived from indoor sampling, have recognised many common indoor fungal species; however, with the application of high-throughput sequencing in the built environment, the much larger scale and depth of the indoor fungal diversity have been demonstrated [ , ] [ , ] . in other works, fungal otus were detected in a homogeneous set of houses in a californian family housing complex [ ] . considering culture-dependent and culture-independent approaches (including next-generation sequencing; ngs), substantial fungal diversity is now associated with the built environment. these indoor fungi, whether persistent residents or transient visitors, likely hold clues to the increasing numbers of human allergies and other health problems observed globally in previously healthy individuals [ ] . one such example is 'sick building syndrome': associated with poor indoor air quality and fungal exposure and experienced by building occupants, with symptoms including headache, dizziness, nausea, eye, nose or throat irritation [ ] [ ] [ ] . as part of the effort in reducing harmful exposure of indoor fungi, sourcetracking of airborne fungi is necessary to locate and eliminate contaminated building surfaces that may serve as potential reservoirs, a major challenge given the highly dynamic movement of aerosols. fungal growth on building surfaces may also be invisible to the naked eye, and it is impractical to swab all surfaces for examination. therefore, future studies must instead attempt source-tracking by swabbing potential source surfaces in homes, classrooms and offices to compare them against air samples using probabilistic models [ , ] . some early works have reported mould and yeasts from the genera alternaria, aspergillus, cladosporium, penicillium, rhodotorula and wallemia, where rhodotorula mucilaginosa generally dominate moist spaces such as toilets, a. fumigatus in drier spaces such as the living rooms and cladosporium in almost every living space [ , ] . besides source-tracking, engineered building materials and surface coatings that prevent or minimise fungal colonisation are another area of active research [ , ] . these solutions, however, are expensive, and it is almost impossible to maintain sterility in a built environment unless medically warranted, for example, in the intensive care units of healthcare facilities, which in themselves poses a challenge [ ] . alternatively, buildings should be constructed using sustainable fungal-resistant materials complemented with architectural designs that consider operating factors such as heating, ventilation and air conditioning (hvac) systems for controlling indoor temperature and relative humidity, as well as plumbing designs with accessible sites for addressing water leaks, all of which can contribute to a safer indoor environment, minimal fungal contamination and potentially improved human health outcomes. the number of reported microbiome-related publications has increased exponentially in recent years and now includes numerous studies of the mycobiome [ , ] . in parallel, a better understanding of the role of microbes in health and disease has emerged with studies increasingly investigating the importance of the mycobiome and its contribution to disease [ , , , [ ] [ ] [ ] . it is likely that these organisms co-exist in the same environment and that interaction between bacterial, fungal and viral microbiomes achieves homoeostasis among healthy individuals, while dysbiosis leads to disease. although increasingly investigated in association with pathological states, it is still too early to make conclusions about the true clinical importance and relevance of the mycobiome. several challenges do remain including the validation and standardisation of robust sampling and analytic methodologies, which must be examined in larger-scale studies. currently, there is no standardised method in mycobiome sequencing with variation in dna extraction methods, primer selection, sequencing, reference databases and analytical pipelines (fig. ) , precluding robust meta-analysis of clinical studies performed to date, which continue to increase, but remain limited in number. nevertheless, the field of mycobiome research is evolving and the rapid adoption of sequencing and computational technologies may overcome current limitations and allow a better definition of the mycobiome from a clinical context [ ] . with an improved understanding of resident mycobiomes and their interaction with host immunity, as correlated with disease at difference stages, this potentially opens avenues for early diagnosis and more targeted therapeutic approaches. moreover, the use of ngs to characterise resistome pathways may have relevance for surveillance, antifungal stewardship and the development of new antifungal drug approaches. newer treatment approaches may emerge from better patient stratification based on mycobiome profiles, while manipulating or restoring a 'healthy' mycobiome also represents a potentially viable approach for precision medicine. these technological advancements combined with sequencing and analysis may be simplified and evolve into point of care diagnostics for fungal disease in the future. the number of microbiome studies focused on bacteria far exceeds that of the fungal mycobiome (and virome). this is largely attributed to pioneering efforts towards the standardisation of methodologies for bacterial s rrna gene sequencing and analysis, allowing scalability. a standardised and reliable method of mycobiome sequencing combined with more comprehensive fungal database coverage is crucial to achieve similar scalability for fungal microbiomes [ , ] . despite known interaction between various microbes, studies integrating bacterial microbiomes, fungal mycobiomes, viromes and parasites are limited. interaction between members of microbial kingdoms likely results in an alteration of function and behaviour of individual microbes, which in turn play important roles for disease pathogenesis. microbes housed at key body sites exhibit cross-talk and interaction with host immunity resulting in systemic manifestations of disease outside the initial site of dysbiosis. dysregulation of intestinal microbiome has been implicated in allergic airways disease, with increased intestinal clostridium spp. in asthmatics and an emerging role for the gut mycobiome in regulating fungal airway responses through the lunggut axis [ , , [ ] [ ] [ ] . such insight provides explanations for clinical phenomena including how disruption of the healthy intestinal mycobiome composition with oral antifungals can lead to exacerbations of allergy airway disease [ ] . changes in the composition of intestinal microbiota are also implicated in diseases of the central nervous system including autism spectrum disorder, parkinson disease and schizophrenia, further substantiating the key role of microbiome cross-talk, which is likely to include major fungal in addition to established bacterial players [ , , ] . to better 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respiratory initiative for pulmonary health (tariph) for collaboration support. conflict of interest the authors declare that they have no conflict of interest. key: cord- - u e q authors: nan title: selected abstracts from the th j project meeting, antalya, turkey, march - , date: - - journal: j clin immunol doi: . /s - - - sha: doc_id: cord_uid: u e q nan hans d. ochs the identification of single gene defects involving genes that play crucial roles in adaptive or innate immunity is not only important for confirming a pid diagnosis, but may contribute to optimal therapy, and contribute to genetic counseling, carrier identification and pre-natal diagnosis. to accomplish this, the diagnostician has to consider the inheritance of these disorders: x-linked, autosomal recessive, autosomal dominant and the type of mutation: loss of function, hypomorphic, dominant negative or gain of function. conventional techniques to screen for single gene mutations include flow cytometry to measure disease-specific expression of proteins (cell surface, cytoplasmic or nuclear) or to analyze relevant signaling pathways (e.g. stat b phosphorylation via the il- r; pstat via the il- receptor); and sanger sequencing of mrna or genomic dna using dye-terminator sequencing. next generation sequencing ("by synthesis") has been refined, and is being used increasingly to study families with multiple affected members with an atypical pid phenotype, or to explore consanguineous families with one member affected. whole exome sequencing requires less data analysis, compared with whole genome sequencing, but may miss intronic or regulatory elements. the challenge of whole exome/genome sequencing is to confirm that the multiple variants identified by these techniques are causative for the clinical phenotype of the study patient. however, with increasing experience, next generation sequencing will become a standard procedure for the identification of genetic defects responsible for inherited diseases, including pid. stephen jolles immunodeficiency centre for wales, university hospital of wales, cardiff, uk. immunoglobulin (ig)-replacement therapy represents the mainstay of treatment for patients with primary antibody deficiency and is administered either intravenously (ivig) or subcutaneously (scig). recent developments using a high-purity recombinant human hyaluronidase have allowed the longer term repeated use of this enzyme to facilitate the delivery of immunoglobulin and other molecules including antibiotics, local anesthetics, insulin, morphine and fluid replacement into the subcutaneous space. hyaluronidase facilitated scig (fscig) has helped overcome the limitations on the volume which can be delivered into the subcutaneous tissues by enabling dispersion of scig and its absorption into lymphatics. the rate of facilitated scig infusion is equivalent to that of ivig, and the volume administered at a single site can be greater than ml, an enormous increase over conventional scig, at - ml. the use of fscig avoids many of the systemic side effects of ivig, and has higher bioavailability than scig. over three years of safety data are now available for this approach though longer term safety data and information on anti-hylauronidase antibodies and their relevance will be required. fscig could aid several areas of patient management in both primary antibody deficiency and immunomodulatory indications. key factors influencing how it will be used in future are long-term safety data and cost-benefit analysis. date after the cd deficiency was firstly described in a turkish girl in . the patients with cd deficiency had normal b-cell differentiation in bone marrow, normal absolute number of b cells in peripheral blood and normal bcr repertoire. also, the patients had normal stimulation via the bcr, normal proliferation response upon antigen stimulation but reduced memory-b-cell compartment in peripheral blood. the cd deficiency leads to hypogammaglobulinemia and impaired antigen-specific humoral immune responses after vaccination. we had described thirty carriers in relatives of our two patients with cd deficieny and also showed that the mfi value of cd and cd expressions were lower in the carriers than in controls. during the last five years, it was also showed that the mutations of the other coreceptors of b cells such as cd and cd caused antibody deficiency. in conclusion, the description of cd deficiency reminds the importance of the molecules on b cells and contribute to identify new genetic defects (cd , cd ), and it was showed that coreceptors could affect the expressions and the functions of each other. ege university faculty of medicine, dept of pediatric immunology, izmir, turkey ig class switch recombination deficiencies are rare pids ( : , births) with normal or elevated serum igm and low igg, iga and ige levels, defective or normal somatic hypermutation, defective t/b cooperation ( %), intrinsic b cell defect ( %), susceptibility to bacterial infections begining from the first year of age (impaired b cell immunity) and lack of germinal centres in secondary lymphoid organs. we present a cd l defective case with clinical findings such as recurrent otitis media, recurrent upper and lower respiratory tract infections, sinusitis, arthritis, relapsing polychondritis , ebv-associated cervical lymphoproliferation, cmv infection, bronchiectasis, liver and spleen enlargement, multiple nodules in the liver, chronic diarrhea due to persistent cryptosporidium parvum, fungal pneumoniae, osteoporosis, and schwannoma. this case is remarkable with low igm levels and normal cd l ezpression on activated t cells although he had a novel mutation in cd l gene (a novel missense mutation in cd lg (c. c t), leading to an a. a. change from histidine to tyrosine at position (h y) at the start of the extracellular domain). in addition, we present two cases with cd deficiency with normal cd expression on b cells.both of these cases had homozygous-cd -mutation leading to a longer protein due to deletion of stop-codon. in conclusion; cd molecules although non-functional in b cells, may be normally expressed on cell surface. these cd molecules are unable to trigger signal, because cd l + il activation leads to complete lack of proliferation. evaluation of cd or cd l expression by flow cytometry may lead false results. study of cd l + cytokine (or cd + cytokine)induced b cell proliferation appears as a useful tool for these diagnosis. institute for immunology and physiology (ub ras). yekaterinburg, russia regional children clinical hospital № , yekaterinburg, russia the ural regional center of clinical immunology, which based on children clinical hospital number one (№ ) in yekaterinburg, observe patients from different territories of ural region and neighboring areas. it consists of laboratory department, consultative department, vaccination and treatment rooms, beds and boxes in special departments in the regional children hospital. the close collaboration with j-project started in . this is an example of such collaboration. in patient a. an international consilium was diagnosed a progressive neurodegenerative disease as a manifestation of primary immunodeficiency: x-linked agammaglobulinemia with b-cell deficiency. mri results: unspecified leukodystrophya rapidly progressive multifocal brain lesions with demyelinating, generalized cerebral atrophy, iii degree, signs of periventricular leukomalacia in the anterior horns of the lateral ventricles. the brain biopsy was recommended in order to clarify the nature of the defeat of the pathological process and define the role of the immune mechanisms of its development (held in the neurosurgical department with subsequent histological and immunohistochemical studies). histological and immunohistochemical study of the brain tissue of the right frontal lobe: a signs of productive meningoencephalitis in brain tissue with vasculitis, perivascular and focally moderate diffuse infiltration of mononuclear (accumulation of mononuclear cd rb+, vimentin+), most of which are cd + lymphocytes with granules of granzymeb. around -dystrophy and necrobiosis neurons, intracellular edema, small focuses of gliosis -there are isolated myeloid cells (myeloperoxidase +) and plasma cells with cytoplasmic expression of immunoglobulin light chains lambda and kappa; cells and the extracellular matrix of brain tissue expressing cd antigen and s protein. virological and bacteriological studies of brain tissue and liquor: connection of progressive degenerative changes and infectious process weren't obtained verified acknowledgments of an infectious or autoimmune process has not been received. search for a genesis of cytotoxic process in the brain continues. center for chronic immunodeficiency, university medical center freiburg and university of freiburg, germany the essential role for igg replacement therapy (iggrt) for common variable immunodeficiency (cvid) has been demonstrated in many studies and metaanalyses. while patients with "infection only" reach a nearly normal life expectancy -though still not quality -under iggrt, cvid patients with additional manifestations like inflammatory lung, bowel or liver disease, lymphoproliferative and/or autoimmune disease often require additional immunosuppressive treatment. there is little consensus on the form of immunosuppressive regimen, once steroids have failed, with possibly the one exception of rituximab treatment for autoimmune cytopenia. additional studies are essential to guide therapeutic algorithms. some of these patients suffer from late onset combined immunodeficiency (locid). as in classic forms of combined immunodeficiency, iggrt can be only a part of the treatment strategy, which needs to additionally address the cellular immunodeficiency of the patients. therefore a retrospective survey was performed on patients diagnosed with cvid who underwent hematopoietic stem cell transplantation. the results of this study are currently in revision. in summary, iggrt is the baseline therapy for cvid but does not address sufficiently the immune dysregulation in a subgroup of patients. better predictive markers have to be identified for the selection of patients for additional, potentially even definite forms of treatment in order to prevent the morbidity and mortality associated with these secondary manifestations of cvid. the first department of primary immunodeficiencies in russia was established on the basis of the institute of immunology in , when patients with pid were registered. currently, patients with pid are followed in the department of immunopathology in adults. % of pid adult patients have pid with immunoglobulin deficiency. analysis of this group of adult patients showed that the diagnosis of pid, on average has a delay of - years from the first symptoms. in % of cases, there is an infectious clinical phenotype, % -combined infectiouslymphoproliferative phenotype, % -infectious and enteropathy. the study of immunophenotyping of b-lymphocytes for the degree of maturation in this group of patients was begun. patients are currently included in this study. patients showed complete absence of b-lymphocytes, -the reduction of b-cells, patients of those have a normal amount switched memory b cells (mbc), people -a decrease amount of switched mbc. persons of the group with decreased amount of switched mbc had an expansion of transitional mbc. at present, a clear link of immunophenotypes with specific clinical phenotypes in not found, but this may be due to small sample of patients at the moment, the investigation continues. for the treatment of this category of patients only intravenous immunoglobulins are available in russia. we use drugs in various concentration of russian and foreign production. the availability of immunoglobulins for the adult patients unfortunately is not sufficient in russia, so the recommended pretransfusion level of igg is not achieved in about % of patients. our work presents the experiences of our center with the subcutaneous form of immunoglobulin therapy (scig). we have patients on such therapy. the youngest child is months old. the largest group consists of cvid patients, next-xla patients. we also substitute children diagnosed with the dgs and accompanying hypogammaglobulinemia and some children with subclasses deficiency as well as secondary hypogammaglobulinemia. in most cases we start therapy with intravenous preparates, but there have been some children to whom we proposed the subcutaneous form at the initial stage of the therapy. the main factors which made us change the mode of the drug application were adverse reactions to ivig, poor vein access and the parents`wish. the administration of scig is very rarely complicated by severe adverse reactions (the risk of their incidence amounts to about . %). even patients with serious side effects to previous immunoglobulin therapy and/or blood transfusion can be safely treated with scig. the most common side effects are local reactions but their incidence decreases during following substitutions. we can observe swelling, redness, induration, soreness. but we should remember that more severe side effects are also possible, for example: the first cvid patient presented with fever, weakness, difficulties in breathing during the following infusions. changing the brand of the drug turned out to be a sufficient method of getting rid of side effects. the second patient, also with cvid, suffered from nausea, headache, meningismus. we changed the drug brand, slowed down the infusion rate and introduced premedication with an antihistaminic drug. the third patienta girl with dgs and hypogammaglobulinemia, after having been operated on for hypoplastic left heart syndrome, responded to infusions with high fever, muscle and joint pain, skin changes (erythrodermia). we introduced premedication, changed the drug brand and slowed the infusion rate, yet without any positive effects. in two patients we observed adverse reactions after preparates at a concentration of twenty percent. there were: weakness, chills, fever, headache, and very intense pain in the place of injection. during the subcutaneous treatment of xla patients, we observe significant reduction in the number of infections and days of school absence. despite that, all our patients with xla suffer from chronic sinusitis. similar results occurred in cvid patients, but the severity of infections was the same. the use of scig results in more stable and higher igg through levels especially in xla patients. in our practice, we had only a few cases in which iv form appeared to be better than sc one. in the case of two boys with higm syndrome, we observed recurrent enthesitis of the first patient and progression of lung fibrosis of the other. ivig was better to control platelets levels in the girl with cvid and thrombocytopenia. it has also occurred that parents refuse to allow us to start subcutaneous therapy, giving two main reasons: they feel safer under frequent doctor`s control and they are afraid of making mistakes in procedures. as for the youngest children (below )their fear of needle is independent of its size, which is the third reason. in conclusion, we would like to emphasize that education programmes implemented by doctors and nurses are essential to make this form of therapy easier, safer and more satisfying for patients and their parents. despite intensive investigation into the nature of cvid, the exact molecular defect(s) and pathogenesis of disease remain unknown. our aim was to evaluate the role of t cells in the mechanisms of cvid development. additionally the impact of some innate and adaptive immunity related genes (hla, cytokine gene polymorphism, mbl genes) was investigated. based on previously observed by us constellation of shared immunogenetic profiles a comparison of t-cell phenotype of cvid patients, and elderly/young healthy individuals was performed. ten patients with cvid were enrolled ( male, female; average age - , years) presented mainly with pulmonary infections, followed by bronchoectasis and splenomegaly. our study demonstrated multiple t-and b-cell abnormalities in cvid patients such as: decreased cd +, increased cd + t cells and low cd /cd ratio, loss of naïve and early differentiated t cells, expansion of terminal effectors (cd + cd ra + cd l-) t cells, memory/effectors (cd + cd -cd -) and terminally differentiated (cd + cd +) t cells. excessive t-cell activation reflecting the prevalence of activated t cell phenotype was also detected, due perhaps to an antigen-driven process. the very low numbers of circulating mature (cd + cd +) and class-switched memory (igm-igd-cd +) b cells were pathognomic for our patients and could be used as an additional diagnostic criteria in the national guidelines. furthermore high level of nonclass switched (igm + igd + cd +) b memory cells and suppressed nk cell count was observed. decreased responsiveness to polyclonal stimuli via cd and cd pathway correlated with the loss of cd expression which was more pronounced in the treatmentnaïve cvid patients. these findings were further discussed in the context of the similarities that exist along with markers for immune senescence (lack of cd or expression of cd ). increased frequency of ifn-γ polymorphisms associated with low expression level found could indicate genetically predisposition to high activation of th lymphocytes in cvid and consequently support the concept of impaired th -type responses. in conclusion our study provided new insight into the pathogenesis of cvid. this work was partially granted by medical university sofia, grant# bcg vaccination at birth is the constant element of vaccination programmes in poland. high reactogenic bcg danish vaccine has been replaced in , by bcg moreau vaccine. frequency of disseminated bcg infection, in children with primary immunodeficiencies after bcg moreau vaccine manufactured by biomed, poland were estimated. one thousand five hundred sixty three cases of primary immunodeficiencies were diagnosed in the department of immunology, children's memorial health institute in warsaw between - . among patients with t cell predominant deficiency, group high risk of bcg infection, scid was recognized in children. mendelian susceptibility to mycobactarial diseases (msmd) was detected in four patients: ifgr deficiency and il deficiency -equally in two patients, and nemo -in one. in the group of primary immunodeficiencies regarded to be less prone to mycobacterium infections, cgd was diagnosed in , hies in patients, and xl-higm in patients. disseminated bcg infection was recognized in scid patients, of them died, because of bcg diseases. all patients with msmd developed bcg infection, one with il- deficiency died. during nearly -year-follow-up, no case of tuberculosis or disseminated bcg infection have been diagnosed among cgd , hies and xl-higm patients. early anti -tb drug prophylaxis and usage of wide range of antibiotics in therapy is crucial for cleaning of bcg infection. peter Čižnár ; julia horáková ; peter Švec ; ivana boďová ; sabina Šufliarska ; linda libai veghová ;, marieta hricová st pediatric department, comenius university medical faculty, children`s university hospital, bratislava, slovakia. transplantation unit, department of pediatric haematology and oncology, children`s university hospital, bratislava, slovakia. objectives: severe combined immunodeficiency (scid) is a group of disorders due to more than genetic defects, characterized by increased susceptibility to severe infections and early life death. the diagnosis is supported by the demonstration of low absolute t lymphocyte count variably associated with numerical defects of b and nk cells. patients are very heterogeneous regarding clinical course, immune parameters and clinical outcome. bcg (bacillus calmette-guerin) vaccine, a life attenuated vaccine was the part of slovak immunization program, administered at birth until . a comparison of clinical course of bcg exposed (bcg+) and non-exposed (bcg-) scid patients in slovakia in period of past years are given. results: incidence rate of diagnosed and treated scid in slovakia was calculated to : . , meaning , cases per year. in total cases represent ada patients, il rg deficiencies, case of complete del q and in cases genetic defect was not found by analysis of rag / , il rg, artemis, il ra, jak and ada genes. all patients were confirmed absent trec (t-cell receptor excision circles) copies in a retrospective neonatal guthrie card analysis. seven out of these patients underwent hsct, in the hsc source was a mud. favorite outcome was achieved in of them. half of our patients have been exposed to a live bcg vaccine during neonatal period. patients vaccinated with bcg faced severe complications and organ damage due to generalized skin and organ abscess formation, requiring prolonged (up to months) hospital care and complex antibiotic therapy with more than four types of anti-mycobacterium drugs, for more than years. average length of hospital care for bcg exposed patient was , months vs. , months in non-exposed group (p < , ). no statistical difference was found between the time of recognized first symptoms, and time of diagnosis in bcg + and bcg-group. the clinical presentation of non-bcg vaccinated patient differs in the initial symptoms when failure to thrive and pneumonia at months was the most common finding. post-transplantational recovery in bcg-group was less complicated. conclusions: two major improvements for the outcome for scid patients in slovakia have occurred in past years. early life vaccination for tuberculosis has been retreated and improvements in diagnostics for severe t cell defects have been made, including flow cytometry phenotyping and genetic testing within the middle european countries cooperation, the j-project. bcg and late diagnosis prolongs time for hospital care, immune reconstitution and carries severe complications, consequently it increase the costs of health care and decrease the quality of patients' life. the perspective of newborn screening for scid would be the next major step in improving the outcome of scid patients. ahmed aziz bousfiha ; leïla jeddane ; nahla erwa ; monika esser ; shereen m. reda in africa, primary immunodeficiencies are still largely undiagnosed, with no cases reported in of countries. though the african society for immunodeficiencies (asid) already organized international meetings and training schools, their impact outside the hosting country is still insufficient. at this time, only a few pid patients are reported in africa (less than patients), the majority of whom are in north africa and south africa. so, asid propose the a-project, a training program based on the j-project. some issues prevent effective training for pid in africa: diversity of languages, only a few are initiated to pid, lack of resources for travel expenses, difficulty to access to care and shading by the hiv pandemic. a-project is designed as a one-day training by an african pid expert in a small group of motivated caregivers. this project is adapted to the african context, as it only requests minimum funding and can reach more people. each a-project will be co-organized by asid and a local committee, and shall lead to some commitments to be realized by locals, in particular the establishment of a registry, a network between physicians and scientists and creation of a patient association. moreover, each a-project will be done in the medical language used in the country (english, french or portuguese). the first a-project was already done in benin, and five more are already planned for . our goal is to reach all countries where no patient were reported in years. this clinical program will raise pid awareness in africa and can potentially discover new aspects of the immunity. till very recently primary immunodeficiency diseases (pids) were not being frequently recognized in india. however, the scenario has changed over the last years or so. the indian society for primary immune deficiency (ispid) was founded in - . over the last years the ispid has organized international conferences (at new delhi and mumbai), national conferences (at chandigarh and varanasi) and continuing medical education programmes (at new delhi and lucknow). these meetings have served to act as catalysts for the cause of pids and have resulted in increasing the awareness about these conditions amongst paediatricians and physicians in our country. several centres now have the clinical skills and the technical wherewithal to perform laboratory investigations for these patients. the repertoire of tests includes nephelometery, elisa based tests and flow cytometry. facilities for molecular diagnosis of pids are also being developed at some of these centres. a lot more, however, needs to be done. the clinical phenotype of several pids in india is likely to be different from that in the west . further, the type of infections in these patients is also likely to be different. this is because of the differences in the micro-and macro-environment to which these patients exposed in developing countries. these differences have been well brought in the recent publication on chronic granulomatous disease from our centre . further, the genetic background of the indian population is diverse and several new mutations are likely to be identified amongst these patients. the indian council of medical research has taken up the lead in this regard and is proposing to set up centres for advanced research (cars) in pids - each at the post graduate institute of medical education and research, chandigarh and the institute of immunohematology, mumbai. the foundation for primary immunodeficiency (fpid), usa has also been closely involved in these efforts and has helped facilitate the development of these cars. the field of pid research in india is wide open and we are likely to witness new and exciting scientific developments in the coming years. references: . gupta s, madkaikar m, singh s, sehgal s. primary immunodeficiencies in india: a perspective. annals of the new york academy of sciences ; : - . the prevention of pid's complications, the improvement of the health care of patients with pid, the creation of the registry of the patients with pid, the implementation of the finance regulations for detection and treatment of patients with pid diseases in public health facilities, the training and professional development of medical professionals in this field. objective: to improve the detection and diagnosis of pid diseases and the life quality of patients with pid in the republic of kazakhstan. undertaken activities for realization of the project: . professor. l. marodi visited kazakhstan in october and the meeting was held in ministry of health of the republic of kazakhstan, presentations were given at the international conference held in national research center for maternal and child health, the negotiations with heads of the national medical holding' clinics and scientific center of pediatrics and pediatric surgery were held. international islamic university, kuantan pahang, malaysia. two hundred and sixty three ( ) suspected pid (primary immunodeficiencies) cases were referred to clinical immunologist led clinics in malaysia from - . patients referred were from all states of malaysia and seen at the institute of pediatric, hospital kuala lumpur and university associated hospitals in north and central peninsular malaysia the initial pid patients were seen by - pediatric immunologist between - followed by another , beginning in followed by the other in . there were ( . %) patients with at least abnormality on the immune prameter recorded and regarded as probable pid. however ( . %) were recorded as pid based on existing criteria. (who scientific committee , iuis scientific committee, primary immunodeficiency disease. ) our population were mainly children, % below years and % below year. only were above years. pid were classified as; predominant antibody deficiencies %, combined immunodeficiencies . %, other cellular immunodeficiencies . %, phagogocytic defect . %, immunodeficiency associated lymphoproliferative disorders %. our data differed from most classification where predominant antibody deficiency is most frequent as high as %, (steihm, ) . of the specific pid recorded, x linked a gammaglobulinemia (xla) , hyper igm syndrome (higm) , common variable immunodeficiencis (cvid ), selective iga deficiencies severe combined immunodeficiencies (scid) , di george syndrome (dgs) ,chronic granlomatous disease (cgd) , hyper immunoglobulin e syndrome (hige ) , primary cd deficiencies , ataxia telangiectassi % malaysia comprises of multi ethnic groups with a population of . million in . pid amongst them showed, malays at . %, chinese %, indians . % and others . % whilst the male predominate over female at a ratio of . : . family history of affected sibling or in first degree relative, or early death with suspected infant dying of infection was positive in . % which is higher than in most reports eg egypt . % (reeda ). this could be due to high consanguinity in the population. alternatively the symptomatic sibling of affected patients is more likely to be referred to the clinical immunologist. scid records the most varied organism from the positive microbiological isolate viz bacteria , fungus , virus , parasite . chromobacterium violaceum was seen in cgd patients in which deteriorated with eventual death. as in many national registries diagnostic delays remains prominent. in our series the mean diagnostic delay was o . ± . years. in comparison thailand stands at . ± . years, while france, a median of . years. malaysia remains committed to provide better diagnostic services and improved care of the pid patients through research collaboration with foreign partners with a drive for creating subspecialty training. patient groups aligned to ipopi is now closer to its formation with the creation of its protem committee ensuring that patients' interest will always be guarded aknowledgement. the authors thank all who had contributed directly or indirectly, especially medical officers, nurses, consultant pediatricians especially dr kamarul azhar, institut of pediatrics hkl, laboratory scientists especially dr shanaz murad of the imr kuala lumpur university of manchester, uk although the concept of grouping mendelian disorders associated with an upregulation of type i interferon (ifn) has not been previously recognised in the medical literature, our past and current work argues that this concept has scientific validity and clinical utility. i will discuss the possibility that such conditions can usefully be considered to represent a novel set of inborn errors of immunity, and that the recognition of diseases as type i interferonopathies will have significance for the development of targeted therapies, as well as informing our understanding of viral and retroelement biology, and the pathogenesis of some forms of autoimmunity. classic kaposi sarcoma (ks) is exceedingly rare in children from the mediterranean basin, despite the high prevalence of hhv- infection in this region. we hypothesized that rare single-gene inborn errors of immunity to hhv- might underlie classic ks in childhood. we report here autosomal recessive ox deficiency in an otherwise healthy adult with childhood-onset classic ks. ox is a costimulatory receptor expressed on activated t cells. its ligand is expressed on various cell types, including endothelial cells. the mutant ox protein was poorly expressed on the cell surface and failed to bind ox ligand, resulting in complete functional ox deficiency. the ox -deficient patient had a low proportion of effector memory cd + t cells in the peripheral blood, consistent with impaired cd + t-cell responses to recall antigens in vitro. the proportion of effector memory cd + t cells was less diminished. the proportion of circulating memory b cells was low, but the antibody response in vivo was intact, including to a vaccine boost. together, these findings suggest that human ox is important for cd + t-cell memory, but redundant for immunity to most common pathogens, with the notable and surprising exception of hhv- . the chronic mucocutaneous candidiasis disease (cmcd) is characterized by persistent or recurrent infection of skin, nails, oral, or genital mucosae with candida albicans. il- -mediated immunity has been concerned in host defense against candida on body surfaces. we have investigated nine patients with chronic mucocutaneous candidiasis disease (cmcd) and signal transducer and activators of transcription (stat ) mutations. the novel c. c > a (n k) and c. a > g (q r) mutations in the coiled-coil domain (ccd) and the c. c > t (t m) mutation in the dna-binding domain (dbd) of stat are gain-of-function (gof) for γ-activated factor (gaf)-dependent cellular responses to stat . low proportion of il- a-and il- -producing t cells, lower levels of intracellular il- a and il- by t cells and impaired candidainduced secretion of il- a and il- by leukocytes from cmc patients compared to that in healthy controls were found. the c. c > t (r w) mutation affecting the ccd and the c. c > t (t m) mutation affecting the dbd of stat and resulted in gain-ofphosphorylation and gof. these mutant alleles enhanced the cellular responses to cytokines via stat signalling pathway. these data provide further insight into the mechanism of host defense against candida. heterozygous gain-of-function stat mutation is known as a major etiology of chronic mucocutaneus candidiasis. gof mutation affecting the stat coiled-coil domen (d g) was initially discovered in -year boy with cmc. gof mutation of dna-binding domen (t m) was found in the second our patient with cmc. both patients have early manifestation of recurrent or persistent infections of the skin, mucous membranes, and nails with candida albicans. they also have skin infections with dermatophytes. patient presented from the first months of age with severe recurrent sinopulmonary infections. recurrent pneumonia and chronic bronchitis complicated by bronchiectasis, which resulted in cor pulmonale and congestive heart failure. patient suffered from recurrent hsv infection, recurrent aphthous stomatitis, has several episodes of bacterial skin infections. he also has chronic bronchitis and several episodes of pneumonia, but does not have bronchiectasis. both boys developed esophageal stricture, patient necessitating nissen fundoplication in the age of years. the patients have mild autoimmune features: uveitis (p ) and alopecia (p ). immunological investigation revealed different impairment of immune system: more severe, similar to combined immunodeficiency in p , which declines with age. the p does not have changes in lymphocyte number and immunoglobulin's, but impaired antibody production to pneumococcal antigens. western blotting performed with nuclear extracts of lymphocytes of both patients showed stronger stat phosphorylation after stimulation with cytokines ifnγ, ifnα, il- . mononuclear blood cells from both patients released much smaller amounts of il- a and il- than candida-exposed cells from healthy control. stat activation triggers transcription of interleukin (il)- which is crucial for mounting protective immune responses against fungi. several mutations affecting the stat /il- pathway have been reported, resulting in selective susceptibility to fungal (candida) infection, a hallmark of chronic mucocutaneous candidiasis (cmc). in patients with autosomal-dominant (ad)-cmc we previously reported defective th responses and identified an underlying gain-of-function (gof) stat mutation leading to hyperphosphorylation of stat . how this affects stat or leads to decreased il- remains to be determined. in patients with ad-cmc, we assessed how gof-stat mutations affect stat activation, dna-binding, gene expression, cytokine production and the effect of epigenetic modification. we show that stimulation of stat in the presence of gof-stat mutations leads to significantly reduced transcription of stat -inducible genes (rorc/il- / il- /il- /c-fos/socs /c-myc). this was not due to impaired stat phosphorylation, altered nuclear translocation nor sequestration of stat into stat /stat heterodimers. dna binding to a stat-consensus binding site construct (hsie) was intact but binding to an endogenous stat dna target was impaired. the reduced stat -dependent gene transcription could be normalized by inhibiting stat activation by fludarabine or enhancing acetylation with histone deacetylase (hdac) inhibitors trichostatin a or itf . silencing hdac , hda and hdac indicated an important role for hdac . impaired stat -dependent gene transcription likely underlies decreased th- cytokine production, susceptibility to fungal infections and other pathology seen in ad-cmc patients and could be a new target for defining novel therapeutic approaches for this potentially lethal disease. autoimmune features have been long thought as association with immunodeficiency disorders, but are now viewed as a crucial component of some diseases attributed to the breakdown of self tolerance or defects of immune regulators. it had been previously established that a single gene defect of the foxp gene (foxp in humans) caused widespread autoimmunity in both humans and mice. the clinical syndromes observed in both scurfy mice and humans suffering from ipex are similar to those observed in experimental models in which treg are selectively depleted. in , three groups demonstrated that these diseases were indeed the result of a regulatory cell deficiency. around one third of the patients with clinical manifestation closely resembling ipex syndrome, foxp is not mutated, these patients are referred to as ipex like. here we present a case; a female patient years with multiple autoimmune manifestations; dm, coeliac disease and ulcerative colitis with marked decrease in the percent of cd + cd + foxp + cells. as she has a siblings suffering from dm; the whole family was investigated. the father and the mother had % cd + cd + foxp + cells. background: hids is an autosomal recessive disease, first recognized as a separated entity at . in patients with hids, the activity of mevalonate kinase is reduced to - % of normal levels. hids is caused by mutations in the mevalonate kinase gene (mvk), located on the long arm of chromosome ( q ). it is manifested by cyclic attacks of fever initiated usually during first year of age. the frequency and severity of attacks tend to decrease later in life. materials and method: a retrospective analysis of medical history , clinical course and laboratory findings of two albanian children with periodic fever , diagnosed with hyper igd syndrome. results: case presentation : an -year-old boy admitted to the hospital because of periodic fever spikes, which occurred every - weeks and lasted - days, presented since the first year of life and coincided with the beginning of immunization. he had a tonsillectomy and adenoidectomy at the age of . the fever attacks were associated with chills, malaise, and abdominal pain without gastrointestinal signs. between attacks the patient was free of symptoms. from his family history, recurrent febrile episodes during childhood were reported to his father. physical examination showed normal findings, except for a cervical lymphadenopathy. laboratory: marked increase of erythrocyte sedimentation rate and crp. wbc-ranged from . to /mm . , high asto. serum igd was repeated several times and was always elevated (mean value: serum igd iu/ml). the mutation v i is found from the genetic examination done for gene mutations in chromosome ( q ). he repeated attacks after initial treatment with corticosteroid ,than is suggested. the second case was a four-year-old girl hospitalized five times because of prolonged fever, and diagnosed as pneumonia, tonsillitis, acute otitis media and sinusitis, treated by antibiotics. her laboratory findings were not remarkable except for increased acute inflammatory responses. serum amyloid a (saa) μg/l ( μg/l) and igd was extremely high . iu/ml. genetic examination for two mutations were negative, but reduced mevalonate kinase activity in white blood cells was demonstrated in more thorough investigations. treatment regime: colchicine conclusions: auto inflammatory syndromes always pose diagnostic and therapeutic challenges to the clinicians. the clinical description of the diversity of periodic fever syndromes is helpful in the assessment and management of these patients. although hids is predominantly identified in populations from northern european areas, it has to be considered in children with periodic fever. anastasiia bondarenko ; liudmyla chernyshova ; iryna sychova shupik national medical academy of postgraduate education, kiev, ukraine. dniepropetrovsk regional children's hospital, dniepropetrovsk, ukraine. background. aspergillus is an actual pathogen in chronic granulomatous disease responsible for about % of all infections. in - % lungs are involved and in % -cns. case. we report a case of combined loci in -years old female patient with ar cgd. the child was born from iii pregnancy, ii delivery on th week of gestation with body mass g. she received bcg vaccination at -th day of birth. at months the local inflammation in site of bcg with regional lymphadenitis developed which was treated with isoniazid for months. then bilateral purulent cervical lymphadenitis developed at , and months treated with wide spectrum antibiotics. culture from pus was negative. pcr for mycobacterium tuberculosis complex was negative. at months systemic infection without loci occurred with fever, lymphadenopathy, hepatosplenomegaly, loss of weight, progressive anemia, inflammatory changes in blood for almost months. bacteriological cultures were negative. treatment with wide spectrum antibiotics was insufficient for months. disseminated bcg infection was suspected and -compound amb treatment was started ex juvantibus with positive effect: the fever has stopped, the sizes of lymph nodes, liver and spleen have decreased, the weight of a body normalized. the child suffered from recurrent pyogenic infections, underwent disseminated salmonellosis. at the age of years blood samples were tested at the laboratory of human genetics of infectious diseases, inserm (paris, france). an absence of p phox protein expression detected by western blot conferring a complete defect in cyba due to compound of geterozigous mutations in q . at years primary tuberculosis complex of right upper lobe (mbt -) was diagnosed. at the age of during the unexplained fever multiple formations were identified in the liver, biopsy showed caseosis suspected the mycobacterial nature of lesions but mbt (-). at the age of years because of shade in the left upper lobe and ineffective standard antibiotic treatment the tuberculosis again was suspected. due to ineffective antimycobacterial treatment for months multi drug resistant tubercullosis was considered. anti-tb drugs ii line was appointed without clinical response. fever persisted. mri of brain revealed mass lesion in the left parietal lobe. because of suspected tumor the brain biopsy was done and the pus was obtained. microbiological studies revealed aspergillus fumigatus. at the same time subcutaneous tumor-like infiltrate х mm appeared on chest in a proection of lung lesions. the pus was obtained during thoracentesis. result of microbiological studies: aspergillus fumigatus. drainage of abscesses and intravenous voriconazolum led to dramatic clinical improvement and normalization of blood parameters. conclusion. features of our case is spread lesions of aspergillosis with relatively slow progression of infection. high incidence of tuberculosis in ukraine leads to a high suspicion regarding this infection. diagnosis of tb is mainly based on instrumental studies. radiological and histological differential diagnosis between tuberculosis and other infections with granulomas in cgd is difficult. high suspicion of tuberculosis led to late diagnosis of aspergillosis. great north children's hospital, newcastle upon tyne hospitals nhs foundation trust, and primary immunodeficiency group, institute of cellular medicine, newcastle university, newcastle upon tyne, uk even following the introduction of biologic disease modifying antirheumatic drugs (dmards), a small number of children suffering from severe, refractory autoimmune (ai), rheumatic and/or autoinflammatory disorders will not get into clinical remission (cr) and will potentially further suffer from multiple side-effects of combined and long-term immunosuppressive and anti-inflammatory therapies, in particular severe infections (marodi l, casanova jl. jaci ; abinun m. ped health ). whilst autologous t cell depleted hsct following the immunosuppressive conditioning regimen achieved complete clinical remission in majority of children with severe juvenile idiopathic arthritis (jia) (de kleer im et al. ann rheum dis ) , infection-related mortality remains significant (abinun m et al. mol immunol ) . therefore, following the success of allogeneic hsct in treating children with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome (nademi z et al. bmt ) , we treated further children with different severe ai (alps, autoimmune lymphoproliferative syndrome (n = ); complex ai disorder (n = )), rheumatological (jsle, juvenile systemic lupus erythematosus (n = ); jia (n = )) and autoinflammatory disorders (mkd, mevalonic kinase deficiency/ traps, tnf-receptor associated periodic fever syndrome (n = ); eoc, early onset colitis (n = )). overall, of the children are alive (follow up - years), in complete and (complex ai disorder) in partial cr, original disease (alps) relapsed in , and children died ( each with alps and eoc). children had significant, but transient acute (grade - ) and chronic (limited) graft vs. host disease (gvhd), experienced multiple virus reactivation(s), and remarkably we saw significant secondary ai diseases post-hsct (transient nephritic syndrome (n = ) and cytopaenias (n = ); psoriasis, n = ; and thyroid disorders (grave's thyrotoxicosis and hypothyroidism), n = ). our data add to the positive experience and evidence acquired over the last - years (daikeler t et al. bmt ; snowden ja et al. bjh ) to propose the allogeneic hsct as a viable treatment option for the small group of children suffering from severe autoimmune disorders. the wiskott-aldrich syndrome (was) is an x-linked primary immune deficiency disorder characterized by thrombocytopenia, microthrombocytopenia, recurrent, mostly respiratory tract infections, eczema and increased risk of autoimmune disorders and malignancies. was is caused by mutations in the wasp gene which encodes wasp, a -amino acid protein. wasp plays a critical role in actin cytoskeleton organization, signalling and different functions of immune cells. we present here the results of genetic analysis of patients with was from eleven eastern and central european (ece) countries, turkey, iran and azerbaijan. clinical and laboratory information of affected males and carrier females from was families were collected. the wasp gene was sequenced from genomic dna of patients with was, as well as their family members to identify carriers. in this large cohort, we identified unique mutations including novel sequence variants. the mutations were scattered throughout the wasp gene and included single base pair changes ( missense and nonsense mutations), small insertions, deletions, and splice site defects. this study was financially supported by the tÜbİtak project s and bap project tda- - . background: chronic granulomatous disease (cgd) is a rare primary immunodeficiency disorder of phagocytes. resulting in impaired killing of bacteria and fungi. a mutation in one of the four genes encoding the components p phox , p phox , p phox and p phox of the leukocyte nadph oxidase leads to autosomal recessive (ar)-cgd. a mutation in the cybb gene encoding gp phox leads to x-linked recessive cgd. methods: we report here the results of genetically and functionally characterized patients with cgd from turkish families in turkey. results: most of the families ( %) have an ar genotype (% p phox , % p phox and % p phox ) and % have an x-linked genotype. patients with a , a and x phenotypes with oxidase null activity (dhr stimulation index of ≤ . ) were found in patients. however, in p phox deficient cases and in other ar cases with high residual oxidase activity (dhr stimulation index ≥ ) were found in patients. conclusions: residual oxidase activity is similarly lack in the x , a and a phenotype except ar cases with missense mutation. in our cohort, the percentage of ar-cgd was different from european and usa registries (in comparison with % , % and % of p phox , p phox and p phox deficient ar-cgd cases, respectively) with the higher percentage of patients with p phox ( %) and p phox -deficent ( %) phenotypes, and the lower percentage of patients with p phox -deficient ( %) phenotype. the basic difference in our results from those reported is the higher percentage of patients with ar-cgd (% ), which was lower than in the european and usa registries, probably because of the higher prevalence of consanguineous marriage in turkey. introduction: schnitzler syndrome is an autoinflammatory disorder of unknown etiology. at least some of its clinical presentation is mediated through an activation of inflammasome and release of il- , as was repeatedly demonstrated by a prominent therapeutic effect of il- blockade. recent reports bring an evidence of an important role of mitochondria in inflammasome activation and in a pathogenesis of autoinflammatory diseases. we have therefore investigated mitochondrial function and structure in patients with schnitzler syndrome. materials and methods: activity and amount of oxidative phosphorylation complexes (oxphos) were analysed by spectrophotometry, histochemistry and imunoelectrophoretic methods in fibroblast cell lines derived from skin biopsies of three adult male patients with schnitzler syndrome. ultrastructure of mitochondria, mitochondrial network and reactive oxygen species (ros) were analysed by fluorescent and electron microscopy. results: the activities and amount of oxphos complexes i, iii and iv were decreased in patients with schnitzler syndrome. interindividual differences in the degree of impairment (from severe to moderate) in analyzed mitochondrial parameters were found. content of ros, previously suggested as main inducers of inflammasome, were not significantly increased in cells with schnitzler syndrome. we, however, did find consistent and prominent changes in mitochondrial structure of all three patients. disturbed mitochondrial network and mainly abnormal, partially swelling mitochondria with unusual and sparse cristae were characteristic for all patients. we did further notice marked accumulation of neutral lipids in all tested fibroblasts. conclusion: severe structural damage of mitochondria associated with milder functional changes represented a consistent feature found in all tested schnitzler syndrome patients. along with progress in basic and clinical immunology worldwide, the knowledge and activities in the field of primary immunodeficiencies (pids) have developed during last two decades. in , a group of junior doctors and students joined seniors in this filed to establish iranian primary immunodeficiency registry (ipidr). several national and international research projects have been done so far which led to lots of publications, while improving the diagnosis of patients with pids, construction of iranian primary immunodeficiency association (ipia), and establishment of research center for immunodeficiencies were other activities which lead to better management of the patients. organizing annual meeting on clinical immunology and immunodeficiencies, celebrating pi week annually and active participation in the international congresses were all helped in to increase knowledge of physicians in the country. the overall activities in the field of pids led to an increased trend in recognition of more patients in the recent years, which was associated with decreased delay in diagnosis. based on recent report of the registry, published recently in the j clin immunol, more than new patients with pid, in addition to previously reported patients, were presented. predominantly antibody deficiencies were the most common form of disease, followed by combined immunodeficiencies, congenital defects of phagocytes, and other well-defined syndromes with immunodeficiency. the rapid progress in identification and registration of the patients with pids is important not only as of epidemiological aspect, but also as of timely diagnosis and appropriate treatment of the patients. the j project physician education and clinical research collaboration program was launched in in eastern and central europe (ece). in less than years, it has achieved remarkable success. this project aims to increase knowledge in the field of primary immunodeficiency disorders (pid), and to improve the diagnosis and treatment of patients worldwide, particularly in countries with limited economic resources, which currently report fewer such patients than expected. in most ece countries, gene sequencing, which can provide a definitive diagnosis of pid, still remains unavailable. by contrast, such technology is used elsewhere to detect the more than pid-causing genes that have been discovered in the last three decades. thus, pid awareness programs like the j project remain critically important, to improve diagnostic facilities and treatment and to promote clinical research collaboration. this paper highlights the achievements of the j project and the spread of its concepts and spirit to the countries of western asia. primary immunodeficiencis (pid ) are rare genetically determined diseases , occurring with an incidence of per inhabitants. it is heterogeneous group of disorders, from quite commonly found and usually asymptomatic iga deficiency ( in ), to a very rare diseases such as chediak -higashi ( in inhabitants). in , within the framework of a government research project no. pbz-kbn- /p / -" development , improvement and implementation of highly specialized diagnostic procedures for immune-mediated diseases", a network of cooperating national centers for diagnosis and treatment of pid, named polish working group on primary immunodeficiencies (pgr pno) was founded. as a result of joint efforts of the group as well as an implementation of three eu grants [euro-pid -nas qlrt - - ( - , euro-policy -pid sp -ct- - , euro-gene -scan ( , )] the number of centers actively working in the diagnosis and treatment of primary immunodeficiencies increased. up todate pgr pno includes pediatric centers , and since - centers for adults. development and dissemination of new diagnostic and therapeutic standards contributed significantly to the increase in detection pid. with early diagnosis of the disease -the implementation of appropriate treatment, including gamma globulin replacement therapy together with quality of life has improved. in spite of all efforts recognition of pid in poland is very rare and currently is . to , what is almost times smaller than in europe (www.esid.org). at the moment, a nationwide registry of children and adults with pid consists of patients. in september pgr -pno summarized in the annual report the current status of the substitution therapy with intravenous and subcutaneous immunoglobulin therapy in children and adults with pid in poland. on the basis of available information, half of all patients ( children and adults) were diagnosed to have antibody deficiency. these data are similar to the register of a european database esid , where the percentage of patients with pid with a predominance of antibody deficiency is more than % (www.esid.org) development and dissemination of new diagnostic and therapeutic standards as well as a national cooperation contribute significantly to the increased detection of pid. early diagnosis of the disease is followed by earlier implementation of appropriate treatment, including gammaglobulin replacement therapy together with improvement of quality of life. mutations of was were identified in was boys and in heterozygote mothers. frequently genetic damages occur in , , , exones and , intrones: deletions ( ), splice-site mutation ( ), missens ( ), insertions ( ). deletions and insertions lead to stop-codon in cases. nbs patients were homozygous for del mutation and oneheterozygote for del had delt x-cgd: deletions ( ) and nonsens ( ) mutations in different exons of cybb were observed most often. in families was performed prenatal diagnosis: x-scid- , higm - , xla- , was- , nbs- , a-t- , cgd- , xlp- , dnalig - . healthy children - . recurrent severe complicated infections developed in % of pid patients. antibody deficiencies: bacterial infections - %, enteroviral - %, tuberculosis cases, atypical mycobacteriosis - case. combined pid: bacterial infections - %, fungal - %, viral - %, opportunistic (pneumocystis jiroveci) - %, mycobacterial - % ( -complication of bcg vaccination, -tuberculosis or atypical mycobacteriosis). cgd: bacterial - % (staph.aureus, e.coli, b.cepatia, salmonella spp., klebsiella spp.), mycobacterial - % ( % bcg origin, % -tuberculosis), aspergilosis - % immune disregulation syndromes (xlp [ ] and alps [ ]): bacterial infections - %, viral - % (ebv). autoimmune violations were observed in % of all pid cases: % of combined pid, % of antibody deficiencies, % of other well defined syndromes, % of autoimmune syndromes. cytopenias developed in %, vasculitisin %, ulcer colitisin %, arthritisin %. oncology diseases developed in % of patients: mainly in nbs, a-t, was and cvid: t-and b-leukemia's, lymphomas, solid cancer. the study of primary immunodeficiency is a unique model for studying the molecular basis of immunity. question about intravital pid verification is still relevant. a regional center of clinical immunology was established in at the regional children's clinical hospital № , yekaterinburg. the regional clinical hospital № joined the center in . institute of immunology and physiology, ural branch of the russian academy of sciences carries scientific management of the center. the centre works closely with foreign counterparts in the international project j project and it has been one of the centers of jmf since . over years we formed a regional register of patients with primary immunodeficiency, comprising patients: children and adults. analysis of the regional register allows to investigate the causes of deaths in patients with pid. infectious syndrome mortality -sepsis, generalized mycobacterial infection - patients, proliferative process - patients, dominate the mortality structure. creation of specialized immunological centers permit to raise the educational level of the medical staff, precisely identify nosological forms of pid among different groups of patients, to prevent the birth of children with pid, increase the length and quality of life such patients and take part in the collaboration with international experience in the pid. introduction: the aim of this retrospective study is to determine the frequency, and demographic, clinical and laboratory features of adult cvid patients referred to our clinic. materials and methods: we retrospectively evaluated adult patients ( female (% , ), male (% , ); aged to years: median years) who were diagnosed as cvid according to esid and pagid criteria during a year period (january -march ). results: the median current age of patients was , and the median cvid diagnosis age was , years. the diagnostic delay in patients with cvid was , years (median). cvid patients presented lower levels of igm ( patients, , %), iga ( patients, , %) and igg ( patients, , %). according to lymphocyte lmunophenotypes of cvid patients, cd ( patients, , %), cd ( patients, %) and cd /cd ( patients, %) values were observed the most lower ones. discussion: we found that both of the patients with bronchiectasis showed lower levels of immünoglobulins and lower imunophenotypes of b cell than the others that do not have bronchiectasis. in our patients cd , cd and cd /cd values have got enough priority to be mentioned about an immun deficiency. in conclusion, despite recent improvements in diagnostic tools, the diagnosis of mild or moderate cvid is often delayed. however, it seems that the diagnosis of cvid is delayed especially in adulthood on account of the fact that the lack of awareness of these illnesses among the medical professionals all over the world. primary immunodeficiency disease is important in turkey because of the high rate of consanguineous marriage. the lack of awereness about immunodeficiency can cause late-diagnosis and severe complications. the objective of this study was to assess pid awereness before and after clinical immunogy education among medical students. one hundred and thirty-two questionnaires with items ( ) were distributed to seventh somestre medical students and ( %) completed questionnaires were evaluated before (first) and after (second) their education about clinical immunology courses for hours. questionnaire scores (qs) were detected as total correct answers. the mean of the first qs was . ± . and second qs was ± . (p < . ). there was no statistically difference in gender ( m and f). of questions, there were related with pid directly. the correct responses rate less than % before education were of questions. all participants corrected their responses after education. the best improvement was detected in the responses of the clinical signs related with pid. it was remarkable that the participants have known the family history related with pid excellent before education. the majority of the participants ( %) believed that a lymphocyte count of /mm was related to immunodeficiency. nbt and ch test were not found to be related with pid before education. it is also important to increase the awareness of pid among the physicians during their education in medical school and more comprehensive education in pid appears to be useful for medical students. chronic granulomatous disease (cgd) is a rare primary immunodeficiency with mutations in nadph oxidase enzyme complex which causes failure of phagocytic cells to produce superoxide and subsequent intracellular killing of microorganisms. we retrospectively analysed medical records of patients diagnosed with cgd in the last years from immunological diagnostic centres from central and eastern european countries (estonia, poland, belarus, ukraine, czech republic, slovakia, hungary, serbia and slovenia) and russia. genetic sequencing from patients' dna was performed in genetic centres in ljubljana, belarus and netherlands for mutations in known genes involved in cgd pathogenesis: cybb, cyba, ncf , ncf . we included patients with cgd in our cohort, were female. the mean age at presentation of the disease was months and at diagnosis , years. lymphadenitis ( %), dermatitis ( %), enteritis ( %), pulmonary infections ( %), liver abscesses ( %) and septicaemia ( %) were the most common clinical presentation. complications of bcg vaccination ( %) were the most common presenting infection. in total , years of followup in our cohort, the patients suffered different severe infectious episodes ( . per year). respiratory ( %), lymph node ( %) and gastrointestinal tract ( %) infections represented the most prevalent severe infections. we identified different mutations out of genes tested. in patients we identified different mutations in cybb gene, unrelated patients had the same mutation in cyba gene and in patients had typical deletion in ncf gene. in our cohort we observed high incidence of bcg infections as a presenting symptom. apart from high bcg infections patients included in our study had similar frequencies of infections and infecting microorganisms as patients described in previous series. objectives: the aim of this study is to determine the carrier rate in healthy controls from central european and balkan region. methods: we screened more than healthy subjects from countries in the region. exon and was pcr amplified and subsequently sequenced with abi prism genetic analyzer. results: heterozygous mutations were found in % of apparently healthy hungarians, % of slovenians, % of bosnians, % of serbians and in % of apparently healthy macedonians. mutations found in hungarian population were as follows: v a ( ), k r ( ). mutations found in slovenian population were: v a ( ), k r ( ) and e q ( ). mutations found in bosnian population were: v a ( ), k r ( ) and f c ( ). mutations found in serbian population were: e q ( ), k r ( ). mutations found in macedonian population were as follows: e q ( ), k r ( ) and m v ( ). conclusion: we found higher than expected carrier rate in screened populations, from % to %. it is interesting to note that more than half ( %) of detected carriers in all analyzed populations has k r mutation. progress in the field of primary immunodeficiencies (pids) is reflected in national pid registries. data from slovenian pid registry were analyzed. patients' data were collected retrospectively before and prospectively afterward. patients were classified according to international classification and updated regularly. data of patients with different pids were analyzed. interestingly, complement deficiencies are the most common, accounting for % of all entries. second most common are antibody deficiencies with %, followed by well-defined syndromes ( %), immune dysregulation ( %), neutrophil defects ( %), combined deficiencies ( %), autoinflammatory disorders ( %) and defects of innate immunity ( %). prevalence of diagnosed pids in slovenia has changed in the last years; less complement deficiencies and more antibody deficiencies were diagnosed in comparison to previous decades. the number of new pid cases has been gradually increasing, a more prominent increase has been noted in the last years. the prevalence increased most for combined immunodeficiencies, cvid and autoinflammatory disorders. the spectrum of pid entities has also widened in the last decade. three patients with scid were diagnosed and successfully treated in the last three years (incidence - : . births). high prevalence of complement deficiencies reflects early implementation of good complement diagnostic facilities and awareness among infectologists. this group of patients was prospectively collected from . combined immunodeficiencies, cvid and autoinflammatory syndromes were all probably underdiagnosed before due to lack of awareness among physicians. distribution of pid groups is more consistent with esid registry in the last five years. identification and successful treatment of scid patients in the last years is an important quality marker. bulgarian association for clinical immunology was set up in aiming to get together all specialists working in the field of clinical immunology. one of the important objectives of the association was to raise the public awareness and attract attention of specialists, national health system, government and other related societies in order to improve the diagnosis and access to treatment for children and adults with pid. efforts of immunologists led to the following results: . consensus on the diagnosis and treatment of the basic pid groups was created by the pid national working group that was established in , and specific guidelines were disseminated as well. register for pid patients has been set up in bulgaria that allowed the collection of data on the incidence and prevalence of pid and the negative effect of these conditions on the population. . educational program to improve the qualification of the physicians and provide available resources to general practitioners and raise the public awareness were introduced. collaboration with patient's organizations was developed. . treatment of pid patients has been fully covered by the public health system since march . all these steps made it possible to advance the diagnosis and management of pid in our country. pediatric pid patients care -single center experience g. petrova; p. perenovska; s. mihailova; e. naumova umhat "alexandrovska", sofia, bulgaria j-project in bulgaria started in and up to now we have elaborated programme with diagnostic criteria, well equipped laboratory, established some mutual connections with foreign colleagues, held regional meetings, conferences; created clinical standards for treatment and ensured an immunoglobulin treatment and replacement therapy. here are examples of some of the problems we face: . seven-years old boy with hypogamaglobulinaemia (normal number b-ly with abnormal function). ivig had some initial effect, but lately we noted very fast deceleration in the overall health status with possible need of lung transplantation. the case is posing a question what more could we do, could we have prevented this rapid worsening. . ten-month old girl with scid with severe bcg infection after first vaccination, referred relatively late to our center, but successfully transplanted. the case is posing a question about timing of bcg vaccination and of referral to specialized center. . nine-years old girl with unidentified immune deficiency, normal immunological follow up but clinical course as an immune deficiency with very favorable effect of ivig according the parents. the case is posing the question should we stop or should we continue ivig, despite failing to find immunological defect, based on the good clinical response. unfortunately pids are not very well recognized and sometimes the patients are referred late. sometimes poverty and lack of knowledge of patients leads to miscalculation and neglecting of their conditions by themselves, or refusal for specific tests for clarifying the diagnosis. background: although rare, primary immune deficiencies (pid) are manifested with high rate infections as well as with autoimmune and malignant disorders that are treated hardly and inefficiently. pid are not only immunological problem; they require close collaboration between immunologists, pediatricians, ent, lung and gut specialists, dermatologists, hematologists, oncologists, patients and administration. the aim of this study was to summarize the activity on registration and replacement therapy of pid patients in plovdiv region at the university hospital "st. george"-plovdiv for year ( . - . ). methodology: children with pid of humoral immunity hospitalized at the clinic of pediatrics, and adults with hereditary angioedema (hae) were included in the study using immunological and other lab tests, clinical follow up and treatment: iv and sc ig for children with pid and c esterase inhibitor (ruconest, berinert) for hae patients. results: three national workshops and a national conference on pid were hosted and organized in plovdiv since . well established university hospital immunological laboratory, detecting serum immunogloblins, blood lymphocyte populations and subpopulations and complement proteins; clinic of pediatric and genetic diseases and an information center for rare diseases and orphan drugs function in plovdiv. an expert center for diagnosis and treatment of pid was created at the university hospital. it is a team of two competent pediatricians, an immunologist and an allergist. the targets are hospitalized pid children, outpatient pid children and hae adults. the center introduced regular replacement therapy with iv and sc iv and c inhibitor, reimbursed by the national insurance. together with icrdod the experts provide education for patients and parents how to perform sc ig application as well as consultations of patients and relatives about pid.since march indicated reimbursed replacement therapy with iv ig -octagam, started regularly in hospitalized pid children with bruton hypogammaglobulinemia, cvid, omenn syndrome or igg id. these patients, aged form to years, had to hospitalizations for one year. four outpatient children with omenn syndrome or igg subclass deficiency were subjected to sc ig -gammanorm, and hae type outpatients had good response for replacement c inhibitor therapy as follows: conestat alfa (ruconest)in hae adults ( iu/ml weekly), and berinert ( u/kg b.w. weekley)in hae adult patients. conclusions: the expert pid center in plovdiv university hospital provides competent diagnosis, therapy, education and consultations for pediatric and adult pid patients from plovdiv region. the recent introduction of reimbursed replacement therapy for pid patients (hospitalized or outpatients) allows regular immunological and clinical follow up of the diseases. background: intravenous immunoglobulins (ivigs) are scarce biological products used in a broad variety of disorders. tolerance to infusions is usually good but adverse events, including some serious ones, have been reported. methodology: a cohort study aimed for detection of adverse events that occur during and following intravenous immunoglobulin (ivig) infusions at cairo university children hospitals [patients were recruited at neonatal intensive care units (nicu), pediatric intensive care units (picu), general and specialized inpatient wards ] over a time period of six months, from april through september, . the study included transfusions for different disease conditions in patients.three maltose-stabilized intravenous immunoglobulin products were administered to patients. assessments were done before, during and after the infusions. results: there were symptoms and laboratory changes of adverse events during ivig transfusions, with some patients experiencing more than one adverse reaction. adverse events were noted to occur most frequently within to h from onset of ivig infusion (n = , . %). first hour after infusion onset was the most common timing for symptoms of adverse reactions (n = , . %). patient characteristics of those with adverse reactions: adverse reactions occurred in . % of the infusions (n = ) with the majority belonging to the - years old age group (n = , . %), with variable diagnostic categories. ten patients observed during infusions ( . %) had one or more risk factors for complications, while patients observed during infusions ( . %) had no risk factors. the commonest risk factor was administration of nephrotoxic drugs (n = , . %), followed by presence of a suspected autoimmune disorder (n = , . %) and preexisting renal insufficiency (n = , . %). using regression analysis, the predicting variables for each complication were noted .for example ,fever and chills were related to infusion rate and dose whereas the predicting variables for pallor were infusion rate and presence of existing risk factors. conclusions: clinicians should be aware of the high need for special monitoring while infusing ivig to patients with primary immunodeficiency disorders, autoimmune hematological disorders and sepsis. certain diagnosis of a primary immunodeficiency disorders (pid) is most confirmedly performed by investigation of a gene defect, allowing genetic counseling and screening. molecular diagnosis helps both parents and index pid patient by carrier detection and pre-implantation testing for selecting appropriate reproductive decisions. furthermore, for confirmation of diagnosis and establishment of the inheritance pattern genetic analysis is necessary. this survey in all pid cohorts should be considered for long-term planning such as bone marrow transplantation of a pid infant at birth. the result of this testing also is important for screening of newborns and for those in specific family or ethnic groups. the prevalence of pids has been estimated to be more than / worldwide. based on the total population of iran reported in ( , , ), the expected prevalence of pids in iran would be more than , individuals. however, because of the high rate of consanguineous marriages in iran and an increased risk for development of disorders with an autosomal recessive pattern of inheritance, this prediction is likely to be an underestimation. to date, clinically diagnosed patients of pids have been reported in iran, and a definite diagnosis, defined by mutation analysis, was made in individuals. as a result, . % of the expected pid patients have been identified, and among these, . % have been diagnosed at a molecular level. the proportion of genetically definite diagnosis varied between . and % in the different disease categories. this wide spectrum might be due to unknown underlying genetic defects or modifying genes, especially in patients with predominantly antibody deficiency. on the other hand, the latter patients also had the lowest percentage of clinically diagnosed cases. croatia is a small country with inhabitants . according to expected prevalence of pids we expected approximately four hundred patients with pid. for the past few years university hospital center zagreb is the reporting center for esid registry. it is also the national center for diagnosis and treatment of pids, including haematopoietic stem cell transplantation. current pid database is running manually, with the exception of patients reported to esid registry. most of them are of pediatric age, reffered to the hospital from all over the country. the pids patients are classified according to international classification ( iuis-international union of immunological societies ). there are pids patients included in the database at the moment. the majority of them have antibody deficiency. combined immunodeficiency and well defined syndromes appear in equal distribution. other types of pids are reported in small numbers. although no consanguinity was reported, we noticed the geografic distribution of severe combined immunodeficiency patients (scid) mostly in regions, istra and podravina. it can be explained with genetic isolation. the establishment of national online pid registry is in process. the aim is to improve the diagnosis od pid specially in adult patients, who are not included in present database, with exception of the patients diagnosed in childhood. primary immunodeficiency disorders are underdiagnosed in croatia, specially in adults. establishing national pid registry will improve the physicians awareness of pids, which is particularly important for adult patients. we expected the number of diagnosed pid patients will rise. this will give the opportunity to make progress in diagnosis and treatment, and the opportunity for further epidemiological and clinical studies. another patients have well defined immunodeficiency syndromes from which: are with di george anomaly; with wiskott-aldrich syndrome; with ataxia-teleangiectasia; with hyper ige syndrome; with schimke syndrome and are with nijmegen breakage syndrome. in early nineties, two ( ) of our patients had chronic mucocutaneus candidiasis. patients are registered as x-linked scid and with xlinked lymphoproliferative syndrome (xlp). patient has chronic granulomatous disease; have severe congenital neutropenia and have hereditary angioedema. patients are with whim syndrome. patients are with anti-inflammatory disorders: has hyper ig d syndrome (hids) and have familial mediterranean fever. the j-project realization in the republic of kazakhstan allows to update the pid problem, to raise the availability of early diagnosis of pid, to improve the life quality of patients with pid by providing substitution therapy and as a result, the infant mortality and disability rate has been reduced. common variable immunodeficiency (cvid) is the most common primary immunodeficiency (pid) characterised by impaired immunglobulin production and immune dysregulation. chronic and recurrent infections and its results are typical manifestation of this disease. in addition there is a higher risk of autoimmune disorders, lymphoproliferative or granulomatous diseases and malignancies. successful management of cvid patients is based on prevention and consistent therapy of infections with sufficient immunoglobulin replacement and/or antibiotics, prevention and active screening of cvid related complications. in our study we analysed data of patients gained from medical records. these data were also input into czech pid registry and pid registry organized by esid (european organisation for immunodeficiency). we aimed at the period before diagnosis-onset of the symptoms and their characters and the course of the disease-effect of therapy, occurence of the related complications. finally, we compared our data with similar performed studies. chronic and recurrent upper and lower respiratory infections were the most frequent first manifestation of our cvid patients, but developed chronic lung disease or autoimmune disorder as well. in all patients the intravenous or subcutaneous imunoglobulin replacement therapy, eventually combined therapy with antibiotic prophylaxis, was initiated. beside chronic lung disease the most common complications were autoimmunity disorders, especially autoimmune thyroiditis, evans syndrome, trombocytopenia (itp), autoimmune hemolytic anemia (aiha). on the contrary we revealed patients with insuline dependent (type ) diabetes mellitus and cvid. only few case reports have been published with such association. successful management of cvid patients is based on a prevention and a consistent treatment of infections with sufficient immunoglobulin replcement and/or antibiotic therapy, a prevention and an active screening of cvid related complications. such approach can significantly improve the prognosis of cvid patients and the quality of their life. laura zilinskaite ; ieva bajoriuniene , ; raimundas sakalauskas ; brigita sitkauskiene , department of pulmonology and immunology, lithuanian university of health sciences, kaunas, lithuania. background. primary immunodeficiency (pid) is considered to be a rare disease. despite that it is thought that six million people may be living with a pid worldwide. in the hospital of lithuanian university of health sciences (hluhs) patients with suspected immune disorders have been diagnosed and treated since . we aimed to review the structure of pid diagnosed and treated in hluhs during the last five years. methods. data about patients with pid consulted in the hluhs was collected from the department of medical statistics. case histories of these patients were revised and patients' data was collected: onset of symptoms, type and duration of disorder, type of treatment. all patients with pid were divided into several groups according to the classification of international union of immunological societies primary immunodeficiency diseases classification committee ( ). results. there were patients with pid diagnosed in the centre. antibody deficiency was diagnosed for patients: -bruton's disease, common variable immunodeficiency (cvid) and selective immunoglobulin (ig) a deficiency. complement deficiencies were diagnosed for patients: -c esterase inhibitor deficiency and -c deficiency. another well-defined syndrome with immunodeficiency was found in six patients. the most prevalent symptom in patients with predominant igg deficiency was recurrent pneumonias which occurred at the age . ± . yrs. the mean time between the onset of symptoms and confirmation of the diagnosis was . ± . yrs. thirteen patients are on the replacement therapy with intravenous immunoglobulin. these patients had - infections/year before the treatment initiation and only - infections/year during the treatment. •pid center has successful sideproject as regional charitable public organization of invalids "society of patients with primary immunodeficiency diseases in st. petersburg" solovushka (nightingale) "". web site for patients (www.opidspb.ru) was opened due to mutual efforts of pid center staff and patients. pid center laboratory is based on spb pasteur institute central clinical diagnostics laboratory and laboratory of molecular immunology and seroepidemiology. main groups of tests perform for pid patients are: general clinical assays; flow cytometry ( -color assay on facs canto ii); humoral factors assays (ig levels, igg subclasses, post-vaccination igg levels, etc.); burst-test on flow cytometer; genetic analysis of btk, rag , rag ,was, cybb genes. despite of relatively short story of spb pid center it has a variety of completely diagnosted and successfully cured cases of pid including agammaglobulinemia with b-cell in identical siblings, wiskott-aldrich syndrome, chronic granulomatous disease (cgd), etc. introduction: the primary immunodeficiency diseases are a group of disorders caused by basic defects in immune function that are intrinsic to, or inherent in, the cells and proteins of the immune system. there are more than primary immunodeficiency diseases.. laboratory studies are necessary to determine the presence of a primary immunodeficiency diseases. the standard screening tests for antibody deficiency starts with measurement of immunoglobulin levels in the blood serum. these consist of igg, iga and igm levels. the results must be compared to agematched controls. additional studies used to evaluate patients with antibody deficiencies include measuring the different types of lymphocytes in the blood by marking those cells with molecules that can identify the different types. a commonly used test is called flow cytometry that can identify b-cells and t cells present in the circulation. methods: in in our laboratory of immunology is created the sector of examination for the pid and we measure the immunoglobulin levels with a beckman coulter immage immunochemistry system fully automated rate turbidometry and rate nephelometry method. we have determine the aged-matched levels of immunoglobulins in our laboratory. we have install also a new flow cytometer of beckman coulter company and we can determine the b cells by cd marker and t cell with cd , cd and cd marker. this examinations has been of great help in diagnosis of primary immunodeficiencies. results: in years - from the examination of children aged from - years old reported in our laboratory for immunological examination from the pediatric department of uhc mother teresa in tirana we have selected cases with disorders regarding the primary immune deficiencies. we have cases ( cases reported in - and new cases ) with nul b cell in cytoflorometry (b cd cell = %). the level of immunoglobulins was indetectable for igg , iga and igm in the moment of diagnosis. they were boys and the age at diagnosis was and years and actually they are and years treated with ivig. we have classified them as bruton (xla). we have case with low cd b-cell in circulation but only in of them we have done the immunoglobulin level. the low level of cd b cell is accompanied with different kind of hypoglobulinemies: common variable immunodeficiency cvid, with isolated iga deficiency, with igg deficiency and with both igg + igm deficiency. the immunoglobulins disorders : we have classified them according the antibody deficiencies (tab ) and we noted that the most frequent one is the deficiency of iga ( / or . % -from which iga isolated, accompanied with low igm and with low igg ) with average age at diagnosis . years old. in children in the first years of live aged from . to years old we found case with transient hypogammaglobulinemia of infancy. according this survey we can report our cases of primary immunodefiecies in uhc mother teresa of tirana with different classification conclusion: the finding are to be completed with other cases in albania and it is necessary to do the national register for pid in order to estimate exactly the prevalence of this disorders in our country. nyíregyháza-debrecen, hungary the stat mutation was confirmed in as the cause of autosomal dominant hyper-ige syndrome (ad-hies). the disease, which also mentioned in the literature as job's syndrome, is a rare primary immunodeficiency. this disease can be characterized by the following classic triad: recurrent purulent skin infections, cold abscessus which are formed in the ground of chronic ekcematoid dermatitis, pneumatocele, formation causing pneumonia and extremely high ige level. dental training interdependency as well as bone and connective tissue disorders frequently occur in the nonimmunological symptoms as multi-systemic disease. the stat protein has an important role in the area of wound healing, immunity, tumor and neovascularization. we would like to show this in case of -years old kitti whom the perinatal medical history was eventless. there is a frequent hospitalization in kitti's case history since her infancy. due to the age of three months because of serious exsiccotoxicosis, months old bilateral bronchopneumonia and pleurisy required icu care. bronchoscopy was made because of recurrent pneumonia, which excluded the bronchial malformation. lately rather otitis, mastoiditis and airway obstructive symptoms dominated the clinical picture. from serum immunoglobulins-igg is very low, whereas high levels of igewas indicated. on this basis, the diagnosis of job's syndrome was up, which is a negative stat mutations as the molecular genetic test performed demonstrated. the disease has a great clinical importance, since the risk of emergence of serious and often life-threatining complications are high. because of the prevention of disseminated infections, the early detection and the appropriate treatment are essential. in default of causal therapy, the treatment primarily concern to prevention of infections, or their aggressive antibiotic therapy. calcium and vitamin d and as well as histamine on the case of itchy skin symptoms are reccurrended to use. laboratory of molecular immunogenetics, human genetics institute, cnrs and university montpellier , montpellier, france. we performed clinical, immunological and genetic studies of hyper-ige syndrome (hies) patients from hungarian, lebanese, one russian, one polish, and one swedish families with autosomal dominant (ad) or sporadic forms of the disease to reveal cross-ethnicity of recurrent and novel mutations in the signal transducer and activator of transcription- gene (stat ). four patients from hungarian families, and one russian, and one swedish patient carried the heterozygous r w germline mutation at the dna-binding site of stat . the recurrent v m mutation affecting the src homology (sh ) domain was detected in one lebanese and one polish family, and the v del deletion located in the dna-binding domain was unveiled in another lebanese family. a novel h y mutation affecting the dna-binding site of stat in three hungarian patients from a gypsy family was also found. the segregation of this mutation with hies, restriction fragment length polymorphism analysis of stat from patients and controls and the negligible production upon il- stimulation of monocyte chemotactic protein- by the patient's blood mononuclear cells suggested that the h y mutation was disease-causing. these data suggest, that dominant negative mutations of the dna-binding and sh domains of stat cause ad and sporadic cases of hies in different ethnic groups with r w as the predominant mutation found in of the families. functional and genetic data support that the novel h y mutation may result in the loss of function of stat and leads to the hies phenotype. published in molecular immunology. : - . males with an expressed mutation in the sap (signaling lymphocyte activating molecule [slam]-associed protein) gene have an x-linked syndrome characterized by an increased vulnerability to infection with epstein-barr virus (ebv). we evaluated two related male patients with fatal infectious mononucleosis (fim) and mutation in the sap gene. sequence analysis revealed hemizygous g to a transition at nucleotide position in exon in one of the patients, and heterozygosity for this mutation in the genomic dna from his mother and maternal grandmother. this mutation resulted in asparagine instead of glycine in the sequence of the sap protein at amino acid position . to analyse the effect of this missense mutation on protein function cdna was generated by site-directed mutagenesis and cloned in pcmv-flag vector. we found that the mutant sap (sap/g d) protein was defective in protein folding as manifested by the reduced half-life compared to that of wild type sap. furthermore, the sap/g d protein was defective in binding to its philological ligands slam and b . these results suggest that defects in protein folding and ligand binding collectively contribute to the loss of function of the sap protein in patients carrying g d mutation. dedicator of cytokinesis (dock ) deficiency is an innate error of adaptive immunity characterized by recurrent viral, bacterial and fungal infections, very high serum ige concentration and a progressive deterioration of t-and b cell-mediated immunity. traditional sanger sequencing may fail to identify mutations in dock , due to overlapping large deletions in heterozygous patients. we studied the genetic and immunological features of two sisters ( and years of age) born to healthy hungarian parents. mutational analysis of genomic dna and cdna from the patients and parents by a combination of pcr and bidirectional targeted sequencing failed to identify the mutation. however, a multiple ligation-dependent probe amplification (mlpa) assay revealed two previously unknown large deletions, del - exons and del - exons, of dock in both patients. the children's mother was heterozygous for the del - exons mutation, whereas the father carried the del - exons deletion. immunoblot analysis showed an absence of dock protein from the peripheral blood lymphocytes of both patients. these data suggest that the new compound heterozygous del - exons and del - exons mutations result in a loss of dock protein function and a typical dock deficiency phenotype. our findings suggest that traditional sequencing technology may give misleading results in such cases and that mlpa may be indispensible for the definition of the large deletions frequently observed in patients with dock deficiency. we describe here a patient with invasive cryptococcus laurentii infection and the x-linked form of hyper-igm syndrome (x-higm). c.laurentii is an extremely rare human pathogen. this fungus was previously considered saprophytic and non-pathogenic to humans, but it has been isolated as the etiologic agent of skin infection, keratitis, endophthalmitis, lung abscess, peritonitis, meningitis, and fungemia. most affected individuals had a compromised immune system because of leukemia, cancer, diabetes mellitus, aids, or prematurity. repeated isolation of c.laurentii from the oropharynx of an immunocompromised patient has also been docu- the wiskott-aldrich syndrome (was) is an x-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. was is caused by mutations in the wasp gene which encodes wasp, a -amino acid protein. wasp plays a critical role in actin cytoskeleton organization and signalling, and functions of immune cells. we present here the results of genetic analysis of patients with was from eleven eastern and central european (ece) countries and turkey. clinical and haematological information of affected males and carrier females from was families were collected. the wasp gene was sequenced from genomic dna of patients with was, as well as their family members to identify carriers. in this large cohort, we identified unique mutations including novel sequence variants. the mutations were scattered throughout the wasp gene and included single base pair changes ( missense and nonsense mutations), small insertions, deletions, and splice site defects. genetic counselling and prenatal diagnosis were applied in four affected families. introduction: mhc-class ii deficiency is an autosomal recessively inherited combined immunodeficiency disorder characterized by less than % expression of hla-dr on b cells or monocytes. it is caused by mutation of genes (ciita, rfxank, rfx , rfxap) regulating transcription factors which controls expression of mhc-class-ii molecules on cell surface. mhc-class ii molecules are expressed on thymic epithelial cells, antigen presenting cells (b lymphocytes, dendritic cells, monocytes and macrophages) and activated t cells. these molecules are of critical importance to immunity, cd + t cell development, antibody production, tolerance induction and inlammatory response. consequently, patients present with clinical findings related to combined immunodeficiency during infancy. material and methods: medical records of thirteen patients with mhc-class ii deficiency, followed-up in ankara university, medical school, division of pediatric immunology/allergy from to , were evaluated retrospectively. findings: during study period, male and female patients were diagnosed with mhc-class ii deficiency. age of diagnosis were between months and years of age. consanguinity were present in eleven out of thirteen patients. most frequent clinical findings during initial diagnosis were failure to thrive, pneumonia and oral moniliasis. lymphopenia was absent in all of the patients, however, low serum igg level was present in all of them. except for the yo female patient with a positive family history and whose hla-dr expression was , %, hla-dr expression was % in the rest of the patients. eight patients underwent hematopoietic stem cell transplantation (hsct). two patients were lost soon after hsct due to complications and three patients died of opportunistic infections. four patients died of severe opportunistic infections without underwent hsct. results: mhc-class ii deficiency is a combined immunodeficiency and not considered a rare disease in our country. to date, only known treatment is hsct. since it has poor prognosis, hsct should be performed before development of chronic viral infections and sequelae related to infections in patients who have hla-matched sibling. fatih celmeli ; giancarlo la marca ; ines santisteban ; michael s. hershfield purine nucleoside phosphorylase deficiency (pnp deficiency) is a rare autosomal recessively inherited type of immunodeficiency. pnp deficiency constitutes about to percent of all combined immunodeficiencies. it is characterized by progressive combined immunodeficiency and neurologic findings which includes ataxia, developmental delay, and spasticity. the immunodeficiency is progressive, with normal immune functions at birth, but severe t cell deficiency with variable b cell functions presented by the age of years . the only curative treatment is the hematopoietic stem cell transplantation (hsct). here, we present a year-old girl with recurrent respiratory tract infections, short stature and spastic paraplegia. immunological, biochemically and genetics investigation revealed pnp deficiency with a t mutation in pnp gene. case report: a years-old girl was referred to our pediatric immunology clinic for recurrent sinopulmonary infections since year of age. she was full term neonate and her parents were not consanguineous. she had a history of prolonged and resistant bronchopneumonia, and an attack of generalized chickenpox complicated with pneumonia. she had also severe zona infection resolved with ulceration in cornea, one year ago. she has been suffering from frequent infections like chronic sinusitis, oral moniliasis, recurrent pneumonia and sclerosing cholangitis. she has also nonprogressive cerebral palsy, spastic paraplegia, behavioral problems and limited motor and mental retardation. there was no family history of recurrent infections or immunological disorders. on her physical examination, there was failure to thrive, (her weight and height < rd p) oral moniliasis, bilateral crepitus ralls, and splenomegaly. she has also marked spasticity with brisk reflexes in lower extremities. laboratory tests revealed lymphopenia: hemoglobin g/dl and platelets /mm wbc /mm , absolute lymphocyte /mm . the laboratory results are shown in the table. lymphocyte proliferation is lower than normal limits in response to pha stimulation (wst assay). ppd response and hiv was negative. antihbs, and antihav were negative despite vaccination. uric acid level , mg/dl. direct coombs was negative, thyroid autoantibodies were within normal limits. genetic analysis revealed homozygous missense mutation (c. g > a), which causes the a t amino acid substitution in pnp gene, in exon , which has previously been reported. additionally, a homozygous g/a polymorphic site in ivs has been detected (c. + g (ivs + g)). discussion: pnp deficiency is caused by mutations in the pnp gene at q . . this gene encodes the protein purine nucleoside phosphorylase, one of the enzymes involved in the purine salvage pathway. adenosine deaminase (ada) deaminates adenosine to yield inosine, which is then converted to hypoxanthine by pnp. pnp also converts guanosine to guanine. a number of metabolites are elevated in the plasma and urine in pnp deficiency, including deoxyguanosine and deoxyinosine. there is an intracellular accumulation of their deoxy triphosphate compounds, particularly deoxyguanosine triphosphate (dgtp). the latter is toxic to t cells, a property similar to deoxyadenosine triphosphate in adenosine deaminase deficiency. in this report, we demonstrate the clinical characteristic of the patient with late diagnosis of pnp deficiency. pnp mutations likely lead to an intense alteration of the enzyme activity which in turn, cause severe and early onset of the clinical findings. however, in our case, the clinical onset of the disease is quite late (after years) which can be explained by the residual activity of the pnp. in conclusion patients with pnp deficiency can be late onset. additionally, late diagnosis of this patient can cause severe comorbidity which limits the chance of bone marrow transplantation. di george syndrome, a disorder caused by a defect in chromosome ( q . deletion), results in the poor development of several body systems. clinical features include congenital heart defects, hypoparathyroidism and thymic hypoplasia or aplasia leading to t-cell immunodeficiency. the aim of our study is to screen and determine the incidence of di george syndrome within only one tube of blood in children with congenital heart anomalies in our population. children who were found to have a cardiac defect during routine visits in pediatric cardiology and neonatalogy departments were included into the study. cases with known genetic syndromes and newborns younger than gestational weeks of age and small for gestational age (birth weight < gr) were excluded. a total of patients were included. there were ( %) males and ( %) females. age ranged between - months ( . ± . months). parental consanguinity was % (n = ) in the study group. the majority of patients diagnosed after murmur was heard during the routine physical examination (n = , %). five patients ( %) diagnosed antenatally. remaining clinical signs on admission were as follows; respiratory distress (n = , %), tachycardia (n = , %) and central cyanosis (n = , %). echocardiagorafic examinations revealed ventricular septal defect (vsd) (n = ), tetralogy of fallot (tof) (n = ), vsd-asd (n = ), aortic coarctation (n = ), double outlet right venticle (dorv) (n = ), transposition of the great arteries (n = ), truncus arteriosus (n = ) and pulmonary atresia (n = ). pulmonary stenosis, endocardial cushion defects, total pulmonary venous return anomaly and hypoplastic left heart were the other defects. q . deletion was ascertained in ( . %) patients; these patients were diagnosed to have tof ( . %), truncus arteriosus ( . %), dorv ( . %), vsd ( . %) and vsd-asd ( . %). preliminary results of the study showed that the frequency of q . deletion is % in patients with known cardiac defects. single tube of blood is enough for flow cytometric and genetic analyses. further studies involving higher number of patients is mandatory to give sufficient information about the exact incidence of the disease. di george syndrome -where do we stand now? małgorzata pac; małgorzata skomska; ewa bernatowska department of immunology, the children's memorial health institute, warsaw, poland di george syndrome (dgs) classically comprises t-cell deficiency (due to thymic hypoplasia), hypoparathyroidism, cardiac malformations,and facial abnormalities. deletions of the long arm of chromosome at position q. are most commonly associated with dgs. syndrome is also found associated with other genetic abnormalities ( p deletions, char ge), certain teratogenic influences (retinoid acid, foetal alcoholic syndrome, maternal diabetes). the dgs phenotype is very heterogenous with variable expression of the different features including the immunodeficiency. the initial treatment emphasis is to control the hypoparathyroidism. correction of congenital heart defects (if present) is usually needed. the best treatment of the immune defects of dgs is still controversial. both hsct and transplant with fetal thymus are the option for complete dgs (cdgs). long term survival after hsct has been reported, though at a lower rate ( - %) compared to survival after hsct for scid. survival in the subgroup receiving matched sibling donor transplants was better at over %. the use of post natal human thymus was pioneered by markert at duke university and has become established as the treatment of choice for cdgs, with the result of out of treated patients survived ( %). more recently this approach has also been used in london, at gosh. . under care of cmhi there are patients fulfilling esid criteria, girls ( . %) and boys ( . %), age / - y.o. in % of them q deletion in locus d s was found. the vast majority children were diagnosed as partial dgs. none of them had significant hypogammaglobulinemia and no regular ivig therapy or antibiotic prophylaxis were required.. the mean number and percentage of cd , cd and cd lymphocytes as well as lymphoproliferative answer to pha and cd in dgs patients were slightly diminished. in many improvement of cellular immunity was observed with age. about % presented with congenital heart disease, requiring surgery, while almost % had the symptoms of hypocalcemia and hypoparathyroidism, next % -speech and learning difficulties. one child was diagnosed as cdgs. scid is a rare, inherited condition, is caused by numerous molecular defects that lead to severe compromise in the number and function of t cells, b cells, and occasionally natural killer cells. seventeen patents with scid were registered during the period from till in the children's clinical university hospital. medical charts of these patients have been reviewed. there were boys and girls. positive family history was in families. mean age at the onset of symptoms and scid diagnosis was . ± . and . ± . months, respectively. pneumonia ( %), candidacies ( %), bcg infection ( %), diarrhea ( %) were the most important infections. anemia and relative lymphopenia were in % cases , growth retardation, hypotrophy had % children . pathogens such as candida albicans ( ), mycobacterium tuberculosis complex ( ), cmv ( ) and others have been identified. totally patients died.two girls are alive ( and months post-transplant). autopsy was done in patients. we saw different changes in thymus and lymphatic nodes. artemis deficiency (n = ), t-b-scid (n = ), t-b + scid (n = ), γc deficiency (n = mutation r w in γ-chain of receptor for il- ), unspecified scid (n = ) were detected. conclusion: generalized bcg infection had % of our scid patients. (incidence of tb is still high . / population in latvia and newborns obligatory are vaccinated on the second to fifth day of life). due to possibility of absence for pid routine genetic identification in only few scid forms were identified precisely. children's hospital, university of freiburg, freiburg, germany. purpose: ipex (immunodysregulation, polyendocrinopathy, enteropathy, x-linked) is a rare x-linked recessive life-threatening disorder characterized by autoimmunity and early death. pulmonary complication related with ipex has not elucidated exactly. here, we report i.e. patients, of which died from severe pulmonary disease. methods: clinical data and laboratory findings included autoantibodies, immunoglobulin levels as well as number of t, b and nk cells were evaluated. foxp expression was performed by flow cytometry. genomic dna was isolated and all exons and exon-intron boundaries of the foxp gene were sequenced by sanger sequencing. results: patient i (pi) presented with nephrotic syndrome at years of age and then developed autoimmune hepatitis without eczema, enteropathy or high ige and died at years of age due to acute respiratory distress syndrome (ards). two cousins of pi had the same hypomorphic splice site mutation leading to normal foxp protein expression and suppressive capacity. however, they exhibited typical symptoms such as eczema, diabetes and enteropathy with eosinophilia at early age (pii, piii) and were transplanted in infancy. one of them had severe respiratory distress right after birth (piii). patient iv from another family presented with chronic diarrhea without autoimmune manifestations and died due to ards. conclusion: lung disease related to ipex syndrome has not been reported before and this entity could be a critical factor in disease outcome. severe combined immunodeficiencies (scids) are a group of primary immunodeficiencies that comprise a number of monogenic disorders characterized by a block in t cell differentiation with or without impairment of b cell and natural killer (nk) cells. without early diagnosis and treatment most children die in the first year of life. a lack or very low number of t-cell receptor excision circle (trec) detected by realtime quantitative polymerase chain reaction assay (qpcr) is consistent with t-cell lymphopenia and has repeatedly demonstrated clinical validity in population based newborn screening for scid. however, the impact of population screening will be less in communities with high consanguinity and family history of scid, in which targeted screening may be more appropriate. here, we screened high risk neonates and infants (with one or more of the following: clinical presentation and/or family history suggestive of pid, failure to thrive otherwise unexplained, lymphopenia) at cairo university hospitals. their full history and clinical examination were recorded. immunoglobulin profile and immunophenotyping of peripheral lymphocytes were performed as confirmatory tests. sixteen classical scid cases were detected, as well as another scid variants (omenn syndrome and major histocompatibility complex class ii deficiency) (totally . % of all subjects screened). the rate of consanguinity in this group was . %. secondary causes of low trecs, other than scid, in our series included: bacterial septicemia ( preterm, full-term), prematurity ( cases), one preterm with omphalocele and facial dysmorphism, one preterm with congenital adrenal hyperplasia, one full term with microvillus inclusion disease, and one full term with idiopathic tcell lymphopenia. this demonstrates that in populations with high consanguinity rates, as in egypt, targeted (non-population based) trecs assay may provide a more efficient screening strategy. case : a is a years old female, st kid of non consanguineous marriage presented with fever for months. one week after the onset of the fever red patches appeared on the face, hands, abdomen, ll. mother sought medical advice and received antibiotics, antipyretics and oral steroids for about one month with no signs of improvements regarding fever. one week later the mother noticed pallor and sought medical advice and the baby was admitted to local hospital and received one bag of blood. and then she was referred to our hospital (zagazig university hospital) where she developed acute pallor again which needed recurrent blood transfusions. dark colored urine occurred in frequent attacks with abdominal enlargement and pain, and interestingly upto this time, no improvement regarding fever, bone pain or pallor. on examination she was underbuilt (all parameters are under rd centile), pallor, tinge of jaundice, generalized skin pigmentation, generalized lymphadenopathy, nd degree clubbing and hsm investigations: cbc (pancytopenia with reticulocytopenia) -lft (increased ast with indirect hyperbilirubinemia)-kft (normal)-ldh (highly elevated u/l)-esr ( ) -fibrinogen ( . gm/ l)serum triglycerides ( . mg/dl)-ebv-vcm igm positive and igg negative -c ( . normal) -rf and ana(-ve) -serum ferritin ( ng/ml) -cd is low -bone marrow biobsy revealed dysplastic changes -l.n. biopsy revealed non specific inflammatory changes. treatment: hlh protocol with no sct prognosis: patient passed initial phase with complete resolution and waiting for bmt case : m is months old boy, nd kid of non consanguineous marriage (the st is healthy years old female) presented with fever and difficult breathing since the age of months. the fever was of gradual onset stationary course and not responding to treatment with antipyretics and antibiotics. one week later the mother noticed abdominal enlargement with red rash over the abdomen that was associated with pallor but there was no evidence of bleeding from any site, no change in the color of urine, no jaundice, no ecchymosis, no joint swelling then he is referred to our hospital (zagazig university hospital). this boy is delivered by nvd at term with no evidence of any problem either during pregnancy or delivery, he is exclusively breast fed and vaccinated as scheduled. examination: his weight was kg, length cm, and head circumference cm all are average for age, he was pale with no jaundice or cyanosis he had hsm and other systemic examination was quite well. children with pid usually are admitting to the hospitals and intensive care units of infants' pathology regional children clinical hospital № , but at a later date, in serious condition, after the development of clinical manifestations in the form of severe generalized infectious disease, or various complications including hematological. the screening technology approves children to be diagnosed in newborn period and to be observing by immunologist and hematologist for the next preventative therapy and bone marrow transplantation or hematopoietic stem cell transplantation. costs for the differential diagnosis and verification of the diagnosis before the manifestation and complications development of pid - usd. costs for the differential diagnosis and verification of diagnosis after manifestation and complications development of pid would be usd in months. economic loss prevention in pid - usd. economic loss prevention in t -lymphopenia - usd. we also should include into account the contribution to the state's economy, which will later be obtained, due to the presence of a healthy member of society. at is an autosomal recessive multisystem disorder characterized by progressive cerebellar ataxia, telangiectasia and increased susceptibility to infections and malignancies, particularly lymphomas and leukemia. laboratory immune investigations typically show decreased peripheral tcells, particularly of naïve t cells, with abnormal in vitro response to mitogens. most at patients have decreased serum iga and igg subclass concentrations. while about % of patients with at show raised serum igm concentrations during the course of the disease, it is unusual to find a high level of igm at onset. as cerebellar ataxia and oculocutaneous telangiectasia are not present at very young age, these patients are often erroneously diagnosed as hyper igm syndrome (higm). to prevent mistaking a-t patients for higm it is proposed to add dna repair disorders as a possible cause of higm. . diagnosis of at, suggested by elevated alfa-feto-protein and increased sensitivity of patients' cells to irradiation, can be confirmed by identifying a mutation in the atm gene.we report female case of at that with diagnosis of hyper igm received ivig but later they had manifestation of ataxia in the course of their disease and then had telangiectasia of conjunctiva. first case was a yrs girl that was suffered from itp and granulomatosis lesion of skin associated with hyper igm before at diagnosis. second case was a years old girl with microcephaly, sever ftt , neurodevelopmental delay , abnormal faces and hypo and hyperpigmentation lesions and anemia. with respect to these manifestation and increased afp ,nijmegan breakage syndrome was suggested. third case was suffered from hyperigm before a t diagnosis for years. we present a patient with a dock deficiency. mutations in the dedicator of cytokinesis gene (dock ) cause a combined primary immunodeficiency syndrome that is characterized by elevated serum ige levels, depressed igm levels, eosinophilia, sinopulmonary infections, cutaneous viral infections, and lymphopenia. onset of the disease was observed at -month of age with severe eczema and recurrent respiratory infections (pneumonia, bronchitis, otitis). at years of age neuroblastoma was diagnosed. from the age of years he started with severe skin infection , subsequently recurrent mucocutaneous aspergillosis was established based on skin biopsy and bacteriological studies. immunological investigations revealed persistent leukocytosis, hypereosinophilia, low level of igm and increased ige up to iu/ml. so hyper ige syndrome was diagnosed and genetically confirmed when a large deletion of the dock -gene was identified. stem cell transplantation was performed in when the patient was y.o. one year later progressive multifocal leukoencephalopathy secondary to infection by polyomavirus jc was diagnosed. but after immunosuppressive therapy with cyclosporine awas suspended our patient's condition improved: the load of polyomavirus jc on plasma showed a decrease; mri brain was essentially stable ; immunological tests showed an initial improvement of subpopulations and proliferative response to mitogens; donor chimerism % stable. Özdemir Öner ; bozdoğan sıla department of pediatrics, division of allergy/immunology, sakarya university, medical faculty, adapazarı, türkiye. background: bronchiolitis and infantile asthma are the most frequent causes for typical wheezing signs in infants. however, when a physician comes across patients with recurrent wheezing are resistant to β -agonist and anti-cholinergic therapy, known as atypical wheezing cases; he should investigate for hypogammaglobulinemia in these patients. aim: here, three cases are reported to make pediatricians aware of hypogammaglobulinemia, which is one of the reasons causing recurrent and persistent wheezing attacks during infancy and beyond. case presentations: case : month-old girl presented to us with complaining of coughing and persistent wheezing. she has been having wheezing and breathing difficulty for the last months after she got upper respiratory tract infection. her symptoms persisted even though she was using religiously nebulized salbutamol + budesonid therapy. before this episode, she had had other wheezing attacks in her past medical history beginning from months of age. in her family history, her father has asthma. physical examination revealed her breathing difficulty. ronchi as well as rales were heard on the auscultation of her lungs. at the fourth day of admission, she was given ivig mg/kg/dose. later, her symptoms did improve and not recur for the last months. laboratory findings showed normal routine biochemistry, complete blood count and sedimentation rate. chest x-ray showed normal findings. echography was normal. ph-metry for reflux investigation was normal. sweat test was normal. in the serological evaluation: low igg level for his age ( mg/dl) was detected at two different times. igg subgroups, igm ( mg/ dl), iga ( mg/dl) and ige ( ) levels were within normal. case : -month-old girl came to our outpatient clinic with complaints of coughing and wheezing. at months of age, she had urinary and upper respiratory tract infections. despite antibiotic therapy, wheezing persisted for months and wheezing severity increased and it did not respond to β -agonist therapy. thereafter, she was admitted to the hospital for days and symptoms resolved. however, she came back to hospital due to recurrence of her symptoms in days. in her family history, grandmother and her cousins have asthma. physical examination showed breathing difficulty. ronchi as well as rales were heard on her lungs. although salbutamol, ipratropium, antibiotherapy (clarithromycin) and anti-reflux therapies were given, her symptoms did not improve for weeks. at the th day of admission, she was given ivig mg/kg/dose. later, her respiratory system symptoms did not recur for the last several months. once she was evaluated for persistent wheezing attacks during admission, biochemistry, cbc, esr were normal. chest x-ray and echography were normal. in serological evaluation: low igg level for his age ( mg/dl) was detected at two different times. igg subgroups, igm ( mg/dl), iga ( mg/dl) and ige (< ) levels were normal. case : month-old boy was brought to us complaints of having frequent lower respiratory tract infections (bronchiolitis). he was experiencing recurrent wheezing attacks almost every other week for the last months. in past medical history, he was diagnosed with trisomy and hypothyroidism at the months of age. he went thru an operation for atrio-venticular septal defect. physical exam revealed dyspnea, tachypnea and wheezing. crackles were heard on the chest auscultation. abdominal, cardio-vascular and the rest of the examination were normal. when he was evaluated for frequent wheezing attacks in our outpatient clinic, routine biochemistry, cbc and esr were normal. chest x-ray showed normal findings. in serological evaluation: low igg level for his age ( mg/dl) was detected twice. igg subgroups, igm, iga and ige levels were within normal. he was given ivig mg/kg/dose. for the last three months, he did not have any lower respiratory tract infection. conclusion: the awareness of immunodeficiency among pediatricians has been greatly improved. recurrent respiratory tract infections are major infections in these patients. thi is a relatively common condition associated with infant hypogammaglobulinemia. in patients with recurrent and/or persistent wheezing symptoms during infancy and beyond, especially resistant to therapy, hypogammaglobulinemia should be excluded from possible diagnoses. background: the acquisition of new food allergy after transplantation or transplant-acquired food allergy (tafa) is usually reported in adults and rarely in children. tafa is described mainly after liver, but also after small bowel/intestinal, lung and heart transplantations. in different studies, the male/female ratio is equal. literature data suggest that children with tafa typically present within the first year after surgery and they are typically allergic to multiple foods. aim: tafa is generally characterized with allergy to multiple foods and increased level of total and/or spesific ige. here, a patient although who had normal total. ige and specific ige test results, he developed reaction to skin prick test for cow's milk is presented and his clinical presentation will be discussed. case presentation: month-old-boy came to our allergy clinic with complaints of vomiting after drinking cow's milk and skin rush on the area where contact ed with chocolate. in his past medical histroy, left lateral segment of liver (donor was his mother) was transplanted to him when he was at months. the liver donor was not recorded as having a history of allergic disease. methylprednisolone and tacrolimus immunosuppression were used after the transplantation, and tacrolimus therapy was continued for prophylaxis of chronic rejection. when he was at months, family fed the patient with cow's milk but hours later he began to vomit. he vomited five times in two hours. then, he developed constipation. rectal irrigation was used. then oral intake stopped for two days. he was thought to be having food protein induced enterocolitis. his vomiting complaints repeated after intake of formula and baby food which include grain. so he fed with special formula including short-chain peptides and free aminoacids and his symptoms improved. past medical history: extrahepatic biliary atresia was diagnosed at weeks age with conjugated hyperbilirubinemia (according to scintigraphy and biopsy results). family history: his father has penicillin allergy and his aunt has asthma. at our outpatient clinic: height and weight were within normal percentiles. physical examination reevaled normal examination findings. laboratory findings: wbc was . /mm , with % neutrophils, % eosinophils and % lymphocytes. his hemoglobin was . g/ dl , platelet count was . /mm . total ige : < and immunocap specific ige against milk, grain and other classsic foods was < . . skin prick test results: saline: x mm, histamine x mm, fresh cow's milk: x mm, other food allergens (peanut, egg, fish, soybean, wheat): x mm. conclusion: our patient seemed to have cow's milk allergy related to liver transplantation. laboratory investigations and clinical presentation of the patient did not look like typical ige-mediated food allergy, which is expected in tafa. patient history: years old male patient, the first pneumonia at the age of followed by times pneumonia attacks/year, otitis media and sinusitis. hospitalization due to respiratory insufficiency caused by bronchiolitis obliterans and diagnosed with hypogammaglobulinemia at the age of . no consanguinity. patient findings at diagnosis ( / ) had hepatospenomegaly, bilaterally cervical, supraclavicular axillar lap, osteoporosis, bronchiolitis obliterans organizing pneumonia, hyper igm, lower igg, iga, ige, low b-cell. no response to tetanus toxoid. İsohemagglutinin antib was ¼. cd ,cd l, aid, taci, baffr, icos gene mutation were negative. protein electrophoresis revealed polyclonal igm increase, immunofixation was no clonality. lymph node biopsy result was available paracortical expantion . cd (+) t and cd were positive. b lymphocyte distribution were normal. malignancy ruled out. no giant germinal centers. evaluation of bone marrow aspiration/ biopsy were abnormal localization of megakaryocytes, dismegakaryopoiesis, blasts in normal range. lymphocyte ratio % mostly consisting of cd + cells, cd + cells were rare, plasma cell ratio was %, amyloid negative. progression of patient, igm level and spnenomegaly were increased and patient had respiratory failure. splenectomy could not be done due to respiratory failure. than patient was treated with rituximab ( mg/kg/week). after rituximab therapy, lymph nodes and splenomegaly were ( cm), regressed and igm level decreased (from mg/dl to mg/dl), increased effort capacity. after month after rituximab therapy splenomegaly and igm level were progressed. splenoctomy was performed. pathological evaluation of the spleen malignancy ruled out. current igm level is mg/dl. conclusion: the most convinient scenerio for this patient would be a csr defect of unknown etiology presented as cvid. the recent litarature revealed genetic defects of some molecules operating in dna repair pathways such as msh , msh , msh , msh , mlh , rad , rad , nbs leading to csr abnormality and impaired antibody maturation. cvid is characterized by hypogammaglobulinemia, recurrent respiratory and gastrointestinal bacterial infections. good's syndrome(gs) is a thymoma-related immunodeficiency and characterized by hypogammaglobulinemia, decreased b cell and variable deficiencies in cell-mediated immunity. case: a -year old male patient presented with a palpable anterior chest wall mass. in , he was diagnosed with rheumatoid arthritis. laboratory showed hypogammaglobulinemia and all autoantibodies were negative. cd + , cd + , cd + cells were < %. bone marrow(bm) examination demonstrated low cd + b-lymphocyte and increase in cd + t cells. tuberculin skin test was positive. t-cell proliferative response was normal. immunizations with h.influenzae type-b and tetanus toxoid revealed no response. anti-b titer was low. taci, btk and icos mutattions were negative. ultrasonography showed hepatosplenomegaly. mild edema, mononuclear cell infiltration (suggested the early stages of extrahepatic biliary obstruction) were detected on liver biopsy. in the medical history, he had reported chronic sinusitis, otitis and bronchitis dating back to rd decade of life. at age , he underwent surgery for thymoma. in follow-up, the computed tomography showed soft tissue mass on the anterior chest wall and pathology was thymoma. discussion: frequent respiratory infections with encapsulated pathogens beginning at the age of , lack of opportunistic pathogen infections, presence of hepatosplenomegaly and rheumatoid arthritis, bm examination findings and successful management of infections with ivig therapy all indicated a diagnosis of cvid. the coexistence of cvid and thymoma has been reported in the literature. introduction: common variable immunodeficiency (cvid) is a primary immunodeficiency disorder characterized by impaired b cell differentiation with defective immunoglobulin production. it has heterogeneous clinical manifestations including recurrent infections, chronic lung disease, autoimmune disorders, gastrointestinal disease, and susceptibility to lymphoma. patients with this disorder have evidence of immune dysregulation leading to autoimmunity. autoimmune cytopenias are a more common presenting disorder in children and may be the initial manifestation of the disease. we want to present a patient presenting with autoimmunue hemolytic anemia and finally diagnosed as cvid. case: a years old, previously healthy female patient applied to emergency clinic with complaint of paleness, light headedness and yellow discoloration of her scleras. her history was not compatible with blood loss. she denied having melena, hemotochezia or hematuria. her mensturation history was also normal. her hemoglobin level was . gr/dl, reticulocyte count was % . , complete blood count was otherwise normal. direct coombs was (+++). for the differential diagnosis of immune hemolytic anemia, viral serology, ana and anti-dsdna were studied, but the results were normal. igg, igm and iga levels were lower than normal normal for her age. other causes of hypogammaglobulinemia were excluded. blood group was arh (+), blood isohemagglutinin were anti a(-) and anti b (-/ +). lymphocyte subsets were also studided. as the patient has reduced immunoglobulin levels with normal lymphocyte subset analysis, she presented after puberty and other defined immunodeficiency states were excluded, she was diagnosed as cvid and monthly immunoglobulin replacement therapy was planned. she had aseptic meningitis after her first ivig transfusion. the ivig preparation was changed with another trade and she did not have any problem during the following treatments. it has been one year since she was diagnosed and she did not have any other medical problems. conclusion: the diagnosis of cvid requires decreased igg, igm and iga levels are also reduced but are less valuable for diagnosis. ige level is checked to exclude other disorders. igg subclass determinations are indicated if antibody titers are decreased but immunoglobulin levels are near normal. hypogammaglobulinemia secondary to other disorders should be excluded. autoimmune conditions can be the presenting signs/symptoms in cvid. autoimmune hematologic disorders may precede, present at the time of diagnosis or develope during the course of cvid in approximately one-half of the patients with autoimmune problems. selective immunoglobuline deficiency is an uncommon dysgamaglobulinemia, in which immunoglobuline levels except igm level are normal. it can be primary or secondary to cancer, autoimmune diseases, gastrointestinal system diseases and immunosuppressive therapy. patients can be asymptomatic or have recurrent infections, asthma, angioedema, autoimmune diseases, celiac disease and bronchiectasis. allergic diatheses are the second commonest presentation of selective igm deficiency. in this presentation, we report a case with asthma and angioedema who has selective immunoglobuline m deficiency. a year old male patient who has been diagnosed with asthma for years with a well controlled asthma for years presented with labial angioedema. he had labial angioedema daily without antihistamines. he did not have any suspected food or drug allergy. he did not have family history of angioedema. physical examination was normal under antihistamine therapy. laboratory evaluation revealed a . % percentage of eosinophils. absolute lymphocyte count was , absolute neutrophil count was cells/mm . immunoglobuline e value was ng/ml, levels of immunoglobuline g and a were within normal limits for age. immunoglobulin m value was . mg/dl ( - ). anti a was / , anti b was ½ positive, antihbs was above mlu/ml. lymphocyte subsets were normal. because of the continuous usage of antihistamines, prick tests could not be done. levels of d was . , d was . ku a /l. thyroid functions, antitpo, c , c and c esterase inhibitor values were normal. antinuclear antibodies and antitransglutaminase lga was negative. immunoglobuline m value of his father was normal, immunoglobulin m value of his brother was . mg/dl ( - ). selective immunoglobuline deficiency is a rarely seen dysgamaglobulinemia. it was reported in children with asthma, but it was not reported in children with angioedema. it can have value in the clinical evaluation of patients with angioedema. cukurova university, department of pediatric allergy and immunology, adana, turkey a patient who is at age of years at present and who has been followed up at the our clinic with xla diagnosis presented with pain in his ankles and wrists and swelling of his right knee. he also was suffering from skin tightening of the lower extremities. his physical examination revealed arthitis of the right knee and sclerotic changes were detected in the skin. skin biopsy was performed and it revealed morphea (localized scleroderma). after the diagnosis of morphea and arthritis, ivig gm/kg and nsaid were applied. following the treatment skin findings and arthritis resolved, however approximately two months later liver enzymes were detected to be high in his routine control. liver biopsy performed to clarify the aetiology of elevated liver enzymes was reported as autoimmune hepatitis. in addition to ivig gm/kg, budenofalk mg/day was started. after months of treatment, his liver enzymes normalized. currently he is being treated with ivig monthly and ursofalk daily. patients with xla typically present with recurrent bacterial infections and it might be associated with some autoimmune diseases. there are not any reports indicating an associaton of autoimmune hepatitis and scleroderma with xla. bruton agammaglobulinemia is an inherited immunodeficiency disease caused by mutations in the gene coding for bruton tyrosine kinase (btk). the median age at the diagnosis of the antibody deficiency is about . years in turkey. here, we report a case of bruton disease presenting with recurrent cervical abscess at two months old infant. a -months-old boy was firstly hospitalized for the treatment of the right cervical abscess at days old. after the recurrence of swelling on the cervical area, the patient was referred to the admitted to hospital secondly. there was no consanguinity between parents and, his family history was unremarkable except four of the mother's cousins, died because of unknown etiology in infancy. on his physical examination, his weight, height andhead circumference were normal range by age. there were no visible tonsils. there was a palpable, mobile x cm mass on right upper cervical area. the molecular analysis of the causal gene for bruton's tyrosine kinase (btk gene) revealed the mutation in exon . this mutation g. delg (c. + del) leads to the changes of amino acid order in the protein with the subsequent changes in activity of btk (at the level of dna: substitution of glutamic acid (p.glu *) causes non-sense mutation leading to the formation of stop codon with premature end of dna transcription to cause stop codon. after initiating the intravenous immunoglobulin with antibiotics, the cervical mass was getting smaller in a short period, and had not observed again. neutropenia was improved within the months. this case is an important example to diagnose bruton disease in early life. it demonstrates that maintaining a high level of clinical suspicion is essential for the diagnosis of bruton disease in a child with recurrent cervical masses. elif azarsiz; neslihan edeer karaca; guzide aksu; necil kutukculer ege university faculty of medicine, department of pediatric immunology, izmir, turkey transient hypogammaglobulinemia of infancy (thi) is characterized by recurrent infections and reduced serum immunoglobulin levels. typically, thi patients recover spontaneously, mostly within - months of age, but sometimes recovery may be delayed until - years. the use of intravenous immunoglobulin (ivig) as an alternative to antibiotic prophylaxis remains contraversial also in symptomatic patients. some authors believe that ivig therapy may cause a delay in the maturation of the humoral immune system because of the interference from passively transfered antibodies. the aim of this study was to investigate the effect of ivig replacement on recovery from immunodeficiency in these patients. patients ( %) received ivig therapy while patients ( . %) showed spontaneous normalization without ivig. the percentages of patients who had more than six times the number of febrile infections in a year decreased from % to % in the group receiving ivig treatment. at admission, before being recruited to ivig therapy, serum immunoglobulin g (igg) levels and anti-hemophilus b (hib) antibody titers were found to be significantly low in cases who were selected for replacement. the percentages of patients who did not have protective levels of anti-hib, anti-rubella or anti-rubeola-igg were also significantly high in ivig cases. there was no statistically significant difference in the age at which igg levels normalized between both groups. patients in the ivig group and non-ivig group reached normal igg levels at the age of . ± . and . ± . months, respectively. in conclusion, ivig infusions do not cause a delay in the maturation of the immune system in thi patients. the very low and non-protective specific antibody responses against previously applied vaccines are important factors to consider when selecting patients for ivig therapy. zoltán ellenes-jakabffy ; ibolya kovács ; mihaela bătăneanţ ; maria cucuruz ; margit Şerban ; lászló maródi department of pediatrics, clinical city hospital oradea, romania. objective: to study the amplitude of the chronic inflammatory phenomena: atopic and autoimmune diseases, as well as their associations in pediatric sigad patients and to study the sigad patients' family history ( st degree relatives) for pids (primary immunodeficiencies). methods: retrospective analysis of the clinical and laboratory records of pediatric sigad patients diagnosed between and at the departments for pediatric immunology of the medical universities of debrecen (hungary), oradea and timişoara (romania). results: out of patients, we found out ( %) with atopic diseases, mostly with respiratory localizations (asthma and allergic rhinitis), ( , %) patients with autoimmune diseases (jia, psoriasis, celiac disease, thyroiditis etc.) and other patients without clinical symptoms of autoimmunity but constantly elevated autoantibody levels. there were patients with coexistent atopic and autoimmune diseases. regarding the family history, we identified families with multiple cases of pids : with multiple sigad, with sigad and cvid, with sigad and higms (hyper igm syndrome). conclusions: the chronic inflammatory phenomena are present in the majority of the studied sigad patients: symptomatic atopic diseases in %, symptomatic autoimmune diseases in , %, that means a cumulative %. there are comorbid associations within the atopic and autoimmune disease groups and also between the two groups. sigad is the most common primary immunodeficiency. it's prevalence is / - / . aim was to assess the prevalence of co-morbidity in patients with sigad in latvian pediatric population and the analysis of some immunological abnormalities in these patients. the study included patients - years old. medical charts have been reviewed. into account were taken the data, which were made at time of diagnosis. patients were divided into groups: st -patients with allergic disease, nd -patients with autoimmune diseases, rd -patients with infectious diseases, th -asymptomatic patients or patients with sigad unrelated diseases. patients with multiple co-morbidities of various disease groups were not placed in any of these groups. each patient group was divided by age: - years, - years - years. results. . % were boys and . % girls. sigad in . % of the cases were diagnosed before years of age (inclusive). % of the patients had co-morbidities: allergic ( %), autoimmune ( . %) or infectious diseases ( . %). patients - years old: children with infectious and autoimmune diseases have . times greater igg than healthy children or children with allergic diseases (p < , ); children with autoimmune diseases has . times more cd cells than children with allergic diseases (p < , ).; children with infectious diseases have . times lower absolute number of cd cells than children with autoimmune diseases (p < , ); patients - years old: children with infectious diseases have . times more absolute number cd cells than children with allergic diseases (p < , ). patients - years old: children with autoimmune disease have . times higher cd /cd index than children with infectious diseases (p < , ) karakina m. l. , , , tuzankina i. a. , , vlasova e. v. introduction: antiepileptic drugs are known to cause immunosupression in some cases. levetiracetam is an anticonvulsant medication used to treat epilepsy in the posttraumatic seizures. we report a rare case of hypogammaglobulinemia and b cell aplasia associated with levetiracetam treatment. case report: a -year-old female was operated for pituitary tumor with transnasal surgery and required second operation for postoperative rhinorrhea. after operation, menengitis developed and antibiotic treatment was administrated. however, there was a poor response to this treatment after one month and craniotomy was performed due to the diagnosis of "shimic menengitis". her seizures occurred as a postoperative complication and levetiracetam was initiated. after the -month follow-up, the findings of menengitis could not be controlled with antibiotherapy. she was referred to our immunology department for chronic menengitis with fever, headache and high cerebrospinal white blood cell count. results: in her clinical evaluation, it was learned that she had previously healthy. laboratory examination showed that decreased levels of igg mg/dl (normal: - ) and iga mg/dl (n: - ). peripheral blood flow cytometric analysis revealed the absence of b cells (cd + b cells; < %). t cell subsets and natural killer cell numbers were normal. neutrophil function, chemotaxis, phagocytosis and oxidative burst activity were found to be normal. isohemaglutinin titer, levels of pneumococal and tetanus specific igg antibodies were also normal. antiepileptic drug was discontinued after epileptic seizure was controlled. b cells gradually increased three weeks later and returned to normal within two months (cd + b cells: . %). conclusion: patients requiring levetiracetam should have serum immunoglobulins measured and lymphocyte subsets analysis performed if they experience recurrent or persistent infections. mustafa gulec ; fevzi demirel ; ugur musabak ; ozgur kartal ; sait yesillik ; abdullah baysan ; ergun ucar ; osman sener gulhane medical school, division of immunology allergic diseases, ankara, turkey. gulhane medical school, department of chest diseases, ankara, turkey. introduction: common variable immune deficiency (cvid) may present with several clinical manifestations involved in different organs and tissues in adults. we present a case with a history of chronic cough for more than twenty years and further diagnosed as cvid. case: a -year-old male who works in a chemistry lab admitted to our clinic with a history of frequent upper respiratory tract infections for more than years. he also had intermittent diarrhea symptoms and his respiratory symptoms have been worsened since . he had been hospitalized due to pneumonia and empyema several times. he had undergone left lower lobectomy due to bronchiectasis in . he had been admitted to intensive care unit due to worsening of his medical condition. he was further diagnosed with cvid and ivig treatment was initiated. he is currently under remission with monthly ivig treatment and without any respiratory or gastrointestinal symptoms. discussion and conclusion: cvid is a clinical disorder in which the humoral part of the immune system is affected. most frequent presenting symptoms belong to respiratory, gastrointestinal systems and skin. however, due to the organ specific physical examination and lack of awareness, the diagnosis is frequently overlooked. in our case, frequent upper respiratory infection, loss of weight, diarrhea, bronchiectasis and empyema with unknown etiology are the most informative clinical signs. medical history is the most important part of patient evaluation. immunoglobulin replacement therapy is a basic treatment in primary immunoglobulin deficiency disorders. immunoglobulin substitution can be given intravenously (ivig) or subcutaneously (scig) for patients with antibody deficiency. both of these treatments are effective in prevention and cure of infections, although differences in advers events profile and patients' quality of life can be seen. the authors describe here their experiences in switching patients from ivig treatment to scig and a few years observation of scig therapy of patients with antibody deficiencies. sirje velbri , mirja varik tallinn children's hospital, tallinn, estonia. north-estonian regional hospital, tallinn, estonia. j clin immunol ( ) : - antibody deficiencies are the most common group of primary immunodeficiencies. the main hallmark of antibody deficiencies are recurrent infections but the patients have also higher risk of autoimmune and allergic diseases. we analysed retrospectively the frequency and character of autoimmune diseases in patients ( children and adult patients) with primary antibody deficiencies. there were ana-lysed patients with xla, patients with cvid, patients with selective iga deficiency, pa-tients with iga/igg subclass deficiency and patients with isolated igg subclass deficiency. autoimmune diseases were found in patients ( , %) besides in children ( %) and in adult patients ( %). in two boys with xla there was not found autoimmune diseases but in patients with other forms of antibody deficiencies in - % of cases. autoiimmune diseases were found more often in iga/igg subclass defi-ciency ( %) than in other forms of antibody deficiencies ( - %). the spectrum of auto-immune diseases differed in adults and child-ren and in different forms of antibody de-ficiencies. immune thrombocytopenia was found in adult patients with cvid or igg subclass deficien-cy, autoimmune connective tissue disorders in iga and iga/igg subclass deficiency. in children there was found mainly thyroiditis, diabetes i type and juvenile arthritis. rostov state medical university. research institute of clinical immunology, rostov-on-don, russia. primary immunodeficiency (pi) is currently one of most important genetically determined immunological clinical pathology which is hard to manage . among different types of pis almost % of cases occurs due to a deficiency in antibodies production. injections of immunoglobulin are current standard in the management of pi. however this treatment is expensive, often is hard for patients, and frequently has limited effectiveness. substitution of immune proteins frequently is inefficient for treatment of severe infectious conditions in pi patients. we investigated maturation, activation and differentiation of immune t-cells in the dynamics of ivig replacement therapy. we observed patients with with cvid ( ) and xla ( people) over a year of regular replacement therapy. we have found that recovery of the humoral component does not affect the maturation and the differentiation of t-cells, but can reestablish activation and regulatory properties. these changes are more evident among patients with cvid, which immuneregulatory and functional potential reestablish faster. obviously, the effects tlymphocytes increase the effectiveness of replacement therapy in patients with xla, cvid common variable immunodeficiency (cvid) is one of the most frequent symptomatic primary immunodeficiencies. the diagnosis is based on significantly decreased levels of immunoglobulins, with poor or absent response to vaccines and by excluding other defined causes of hypogammaglobulinaemia. as suboptimal antibody production is mainly due to b cell defects, therefore, we aimed to study lymphocyte subgroups of cvid patients and to compare the patterns with the clinical presentation in these patients. six adult patients with cvid diagnosis were studied. lymphocyte subpopulations were determined by flow cytometry. for b-cells subgroups cd , cd , igm and igd reagents w e r e u s e d . a l l l y m p h o c y t e s w e r e g a t e d f o r f i n d i n g cd + cd + memory b cells and from this population switched (cd + cd + igd -igm -) and non-switched (cd + cd + igd + igm + ) memory b cells were counted. all patients had normal levels of total lymphocyte count and absolute counts of cd + , cd + and cd + /cd + cells were also normal in all patients. only one patient showed low levels of cd + cells levels. according to paris classification scheme the patients could divided into two subgroups: mb and mb . although almost all our cvid patients had normal number of total b cells, most of them showed reduced number of memory b cells and/or switched memory b cells. all of our patients had very low or absent level of class-switched memory b cells, therefore can be possible associated with a higher risk of granulomatous disease and splenomegaly. detailed investigation of b-cell phenotypes can better characterise cvid patients and can provide more information about possible clinical outcome. background: common variable immunodeficiency (cvid) is one of the most frequent symptomatic primary immunodeficiencies, often related to spectrum of infectious and autoimmune diseases. in cvid patients wide spectrum of gastrointestinal disorders, including infections, are frequently seen. inflammatory bowel disease, helicobacter pylori infection, giardia lamblia infection, campylobacter or salmonella infection have been reported. however, abnormal liver function test and liver disease are found in approximately % of cvid patients. case history: a -year old female patient was admitted to infectious disease department due to recurrent pneumonia and purulent rhinosinusitis. blood analysis confirmed panhypogammaglobulinaemia with impaired responses to vaccinations, elevated liver function tests and anti-hcv positivity, interpreted as old and passed infection. due to cvid intravenous immunoglobulin substitution was started. however, liver function tests remained elevated and with hcv-rna analysis high hepatitis c viral load was detected. chronic hepatitis c virus infection was diagnosed and treatment with peginterferon α- a and ribavirin was started. conclusion: our case emphasizes the need for hcv-rna and hbv-dna analysis in patients with hypogammaglobulinaemia, as the serological detection is impaired and prognosis for chronic hepatitis in immunodeficiency patients is poor. outpaitent clinic of clinical immunology and allergology, east tallinn central hospital, tallinn, estonia background: primary antibody deficiencies are the most frequent primary immunodeficiencies. recurrent respiratory tract infections may result in permanent lung damage in - % of patients, most commonly presenting with the development of bronchiectasis. we aimed to evaluate the lung function and radiographic pulmonary changes in our patients with primary antibody deficiency. material and methods: we reviewed the records of adult patients with a confirmed diagnosis of primary antibody deficiency at our clinic. patients were included in this analysis in whom ct scan was performed during the last months, comprising patients with cvid and two with igg subclass deficiency (median age years; range - years; % females). all patients were on regular immunoglobulin replacement and one of the patients was on prophylactic antibiotic at the time of analysis. mean trough levels of igg were calculated based on the results measured during the last months prior to ct scan. the spirometry was performed according to published protocols. results: all patients demonstrated normal spirometry data based on fev and fvc. two patients had slightly lower mmef rates, however, the changes were not associated with higher rate of infection nor changes in ct scan. none of our patients had bronchiectasis or atelectasis. among parenchymal changes fibrotic lines were most frequently detected. in two patients ground glass due to fibrosis was noted. mean immunoglobulin trough levels in our patients were between . - . g/l, with the median of mean trough levels of all our patients . g/l. when comparing the trough levels to lung function and ct scan results, no significant associations were seen. conclusion: no remarkable changes in lung function or chest ct scan in our patients with primary antibody deficiency were noted. as regular immunoglobulin replacement therapy could have prevented the development of permanent lung damage. rationale: patients with primary immunodeficiencies (pi) (n = ) were treated subcutaneously (sc) with immunoglobulin g (ig) preceded by recombinant human hyaluronidase (ighy) at or week intervals based on their previous intravenous ig (igiv) dose. we report data for a subset of patients aged ≥ years from the final efficacy, safety and tolerability data of a pivotal phase trial of ighy. methods: patients received igiv for months at prestudy doses and frequencies. subsequently, ig % was administered sc, at % of the weekly equivalent of the iv dose, following rhuph infused through the same sc needle at a dose of u/g igg. after a ramp up from a -to a -or -week dose interval, patients received ighy every weeks for months. the primary efficacy endpoint was the mean rate of validated acute serious bacterial infections (sbis) per patient-year during the efficacy period. results: fifty-nine patients received ighy infusions; . % were completed without administration changes due to tolerability concerns or adverse events (aes). median infusion sites/month was . . the temporally associated systemic ae rate was . /infusion (ighy) vs. . /infusion (igiv). the local adverse drug reaction rate was . /infusion. the annual sbi rate was . and . /patientyear for all infections. conclusion: in adults with pi, ighy was effective in preventing infections. the majority of patients received full -to -weekly doses of ig using a single sc site with good local and systemic tolerability. rationale: in a pivotal phase trial of facilitated-subcutaneous (sc) infusion of human immunoglobulin g (igg), %, and recombinant human hyaluronidase (rhuph ) (ighy) in patients with pi, rhuph permitted most patients to have a single-site infusion (every - -week igg dosing) with bioavailability and infusion rates comparable to intravenously administered igg (igiv). we report the final analysis of the long-term extension of the initial phase study, with a duration of up to years of treatment with ighy plus additional follow-up. methods: sixty-six patients who completed the initial phase study enrolled in the extension study. patients continued their pre-study ighy dose/frequency every - weeks. after months, some patients switched to -week dosing to evaluate effects of shorter ighy interval on trough igg levels. from the final analysis, tolerability and safety after up to years of treatment were evaluated. the ighy part of the extension study was followed by a - week observation period during which patients received igg % administered iv, or sc weekly without rhuph . results: in the extension study, patients were treated with ighy and discontinued prior to safety follow-up. following discontinuation of rhuph , patients switched to follow-up. no patients withdrew due to ighy-related reactions (adrs). no serious adrs related to ighy were reported. the maximum ighy exposure for the initial and extension studies combined was years (total exposure = . patient-years; n = ighy infusions); during this time, there were no clinically observable long-term changes in the skin or sc tissue. the rate of temporally related systemic adverse events (aes), excluding infections, was . / infusion. the rate of all local aes was . /infusion. of the ighy infusions administered in the extension study, . % had no administration changes (rate reduction, interruption or discontinuation) due to tolerability concerns or adverse events. the annual rate of all infections under ighy treatment was . /patient-year. reducing the dosing interval from to weeks (same monthly dose) resulted in a % increase in trough igg levels. thirteen patients had at least non-neutralizing anti-rhuph antibody titer of ≥ : with no associated aes; no patients had neutralizing anti-rhuph antibodies. conclusions: in the extension study, ighy was well tolerated and effective, with no serious adrs for treatment periods up to years. over a maximum -year ighy exposure (for an individual patient) in the initial phase and extension studies combined, no long-term changes in skin or sc tissue were observed. the rates of infections and adverse reactions were stable or decreased over the course of the two studies, suggesting no increased risk with continued exposure to ighy. rationale: we report interim analysis of safety, tolerability and pharmacokinetics (pk) of igsc % in patients with primary immunodeficiencies (pi) aged ≥ years in europe. methods: epoch : igsc % or intravenous ig % (igiv) administered at pre-study doses every months. epoch : igsc % administered time per week for months at epoch doses. serum igg trough levels are maintained at > g/l. the primary endpoint is validated acute serious bacterial infection (sbi) rate. results: at the interim analysis in october , patients started the study. during igsc % treatment (n = ), acute sbi episode (pneumonia, moderate in severity) was reported. the infection rate per patientyear was . (igsc %). there were no serious adverse events considered related to any treatment. the rate of local adverse drug reactions (adrs) was . /infusion and all were mild in severity; no severe systemic adrs were reported with igsc %. of igsc % infusions, only . % required slowing or interrupting the administration rate. mean serum igg trough levels were . g/l (igsc %, n = ), . g/l (igiv -week interval, n = ) and . g/l (ivig -week interval, n = ). conclusion: igsc % provided an effective and well-tolerated therapy, with no dose adjustments needed from pre-study ig dose. this study is ongoing to confirm results over months. it is well known, that intravenous immunoglobulin (ivig) is the main therapeutic modality in b-cell primary immunodeficiencies (pid), it decreases mortality and morbidity in these patients dramatically. yet, it is also well known that all ivig products are very expensive, especially considering life-time use and almost normal life expectancy in these patients, if treated correctly. irregular treatment and problems with insurance/state coverage of ivig in some countries stems from this. goal: the goal of our study was to compare medical and other costs, related to the disease in patients with humoral pids with or without ivig treatment. patients: patient with b-cell deficiencies ( % x-linked agammaglobulinemia, all genetically confirmed, % common variable immunodeficiency). the age of patients varied from to years. methods: we analyzed medical and other disease-related costs during years preceding the diagnosis (without ivig therapy) retrospectively and during years on regular ivig therapy. the costs included those incurred by the state (hospitalization, home visits, emergency calls) and by the parents (costs of drugs, private consults, etc). the state costs caused by parents missing work were also considered. for standardization purpose for calculation we used the prices for the end of . results: the patients analyzed fell into different categories: . the st group -( % of patients studied) -patients with several severe infections before therapy, some chronic conditions as a result of those. age of diagnosis varied from years to years (with average time to diagnosis years). in this group costs of ivig treatment were , times higher than before the diagnosis (fig. ). nd group( % of all). patients with late diagnosis (average time to diagnosis years), who had multiple severe infections before the ivig treatment and acquired serious chronic lung complications due to it. in this group the costs before the treatment were higher, than on ivig treatment. . very early diagnosis -within the first year of life (mostly because of preceding family history) ( %). the comparative analysis was not possible, but it was noted that these patients had no history of serious infections before of while on ivig therapy. the only additional costs, besides ivig, were related to bad venous access, requiring occasionally day in-patient hospitalization for ivig infusion. as expected, in all groups, the number of infectious episodes, the number of hospitalizations (fig. ) and doctor visits (fig. ) after beginning of regular ivig treatment dropped dramatically. we also followed patients with xla, who did not receive ivig therapy because of social aspects. both patients died from severe infections. we evaluated this fact in economical prospective: in russia one worker, who works continuously and retires at years of age brings about mln roubles into the state budget (when costs for schooling and routine medical care are subtracted). if one supposes that an xla patient have been diagnosed very early in life, did not form complications prior to therapy and was on regular ivig therapy for life, this sum equals to years on ivig. conclusions: regular ivig therapy not only leads to reduction of infectious episodes, hospitalizations, and as a result improved quality of life. in some cases it even brings down the disease-related costs, incurred by the medical system and the family, and is economically advantageous for the state. introduction: replacement of immunoglobulins is a standard therapy for patients with primary immunodeficiency disease (pidd) characterized by primary antibody deficiency (pad). this poster represents our clinical experiences of initiation of home-based treatment with subcutaneous immunoglobulin (scig) with the patients diagnosed with primary variable immunodeficiency (cvid). case report: the patient (age ) has been treated at our immuno-allergy outpatient clinic since with the diagnosis of hypogammaglobulinemia (igg, iga, igm) with normal b cell count, withsusp. cvid. with the repeated administration of intramuscular and intravenous immunoglobulins (ivig, imig) repeatedly occurred serious adverse reactions, which resulted in discontinuation of the replacement therapy. in february the health condition of the patient worsened due to recurrent bacterial respiratory infections. there was a progressive decrease of serum concentrations of immunoglobulins (igg , g/l, igm , g/l, iga , g/l). the patient was admitted to the intensive care unit of the st internal department, university hospital bratislava, for a subcutaneous immunoglobulin replacement trial. despite serious adverse reactions with previous administration of several types of immunoglobulins, there have not occurred any clinically relevant side effects. conclusion: compared with im or iv formulations and administration, for selected patients, scig is better tolerated, clinically efficacious, safe, and appreciated by the patients. background: common variable immunodeficiency (cvid) is primary immunodeficiency (pid) classically viewed as antibody deficit. although, cvid is considerd to be a humoral immunodeficiency, approximately % of cvid patients have low t-cell counts or abnormal tcell function. despite adequate immunoglobulin replacement patient morbidity and mortality is variable and a number of complications are not those typically seen in pure antibody pid e.g. xla. so, t-cell rather than b-cell phenotype could determine outcome in patients with cvid. many patients with cvid have clinical history suggestive of allergic respiratory disease, but prevalence of asthma and role of atopy have not been well established. apart from recurrent infections and their sequelae, cvid patients suffer from other disease-related complications in up to % of the cases. about % have onset before the age of years. aims: ) to present one more case of tadolescent with cvid and allergic asthma, ) emphasise ultimate need of collaborative network of primary immunodeficiency centers. case report: parents of , y boy were sure that "something was wrong" with their son and were seaking for problem solution for many years. since age , child had frequent respiratory infections. adenoidectomy and tonsillectomy were performed at age . sinuitis was diagnosed several times. boy was complaining of fatique for a long time. last several years, his main problems were fever (max c) usually lasting days till weeks, accompanying running nose, coughing, conjunctival problems; intermittently headache (lasting for a few hours till all day). oral aphtae were present almost every two weeks. he was incompletely vaccinated (bcg, and once diteper). morbilli and varicellae infections passed without complications. in jan he was diagnosed as allergic asthma in sarajevo (allergy to pollen, soya, nuts and antibiotics ("ceclor" and "pancef"). in february was admitted at children's hospital sarajevo for suspected primary mmunodeficiency. he had slightly lower levels of igg and iga (twice measured), normal ige and decreased number of t helper ly. due to suspected pid, boy was checked up in two nearest regional pid centers : hypogammaglobulinemia was confirmed (igg , , iga , , igm , , as well as deficiency of igg , igg and igg ). flow cytometry showed slightly raised concentration of lymphocites b (cd ), slightly raised number of nondifferentiated b cells. cvid was suspected but not proved. in july child visited center for primary immunodeficiencies in munchen, germany, were he was diagnosed ad probable cvid on the basis of hypogammaglobulinemia, lower levels od switch memory b cells, normal number of t-cells, positive antibodies to vaccinations and overcome infection (morbilli, varicellae). allergic asthma was additionaly confirmed in specialised pediatric pulmology hospital in germany (abnormal spirometry, normal ige, positive skin prick test, abnormal fractional exaled nitric oxid test, incipient brochiectasies due to asthma confirmed by high -resolution lung ct scan). low human immunoglobulin replacement was started ( mg/kgbw) as well as antiasthmatic therapy (inhalatory steroids, antihistaminics). excellent therapeutical response were achieved : after one , y follow up, we can confirm patient has excellent general condition, no subjective symptoms, no tiredness, no severe infections. conclusion: diagnosing cvid is challenging task and quite often could be "per aspera ad astra". there is ultimate need of collaborative work of primary immunodeficiency network aimed of diagnosing patients on time. cvid patients with history suggestive of allergic asthma, are negativne on traditional tests, additional test designed to identify allergic asthma might be conducted. common variable immune deficiency (cvid) is a heterogeneous syndrome characterized by hypogammaglobulinemia, recurrent infections, immune disregularity (autoimmunity, autoinflamation) and propensity to malignancies. in the us report, . % of cvid patients had a lymphoid malignancy, and cancers of other sorts developed in % of patients. it is not clear why cvid patients have higher risk of malignancy but chronic antigenic stimulation, chronic inflammation and increased chromosomal radiosensitivity may be the cause. cvid patients with higher igm level, reduced or absent b cell numbers, cd t cells lower than and pli phenotype have higher prevalence of malignancy. allergy and clinical immunology department of rasool e akram hospital has registered cvid patients. mean age of the onset of cvid symptoms was . years. mean diagnosis age was . years with mean diagnostic delay of years. mean follow up time was years (minimum . -maximum y). malignancy occurred in the follow up of patients ( %). one patient had two different malignancies (breast cancer and gi adenocarcinoma). malignancy risk per case was %. hodgkin's lymphoma was the most common type ( % of cancers common variable immune deficiency is a heterogeneous syndrome characterized by hypogammaglobulinemia , recurrent infections , auto immunity and auto-inflammation . more than % of cvid patients have auto immune complications and among them, auto immune cytopenia is the most common. cvid patients with higher igm levelhigher low b cellslower t reg levelslower cd /cd ratio and lower class switched memory b cells have higher prevalence of autoimmunity. allergy and clinical immunology department of this hospital has registered cvid patients. mean age of onset of cvid symptoms was . years. mean diagnosis age was . years with mean diagnostic delay of years. mean follow up time was years (minimum . maximum y). autoimmunity was detected in cases ( %) and cases ( %) had more than one autoimmunity. autoimmunity was the first symptom of cvid in percent of cases. autoimmune disorders should be considered in the follow up of cvid patients. igg is major immunoglobulin and classified subgroups as igg , igg , igg and igg . igg and igg subclasses are rich in antibodies aganist proteins such as the toxins produced by the diphtheria and tetanus, as well as antibodies aganist viral proteins. recurrent ear infections, sinusitis, bronchitis and pneumonia are common in ig g subclass deficiency. ig g is the major subclass of ig g. igg subclass deficiency is very rare. chronic eosinophilic pneumonia is one of the eosinophilic lung disease and is seen rarely. in the presence of peripheral eosinophilia and radiological pulmonary infiltrates diseases suspected. when increase in the number of eosinophils in bronchoalveolar lavage fluids and/or presence of eosinophils in lung tissue diagnosis is confirmed. according to different recording systems chronic eosinophilic pneumonia is % - of the interstitial lung disase. there is not any criteria for diagnosis but also diagnoses is confirmed with suspected findings. symptoms inludes that: )in the presence of respiratory symptoms for two weeks long )eosinophilia at alveolar lavage and\or peripheral blood ( bal fluid cytological examination > % , blood > /mm ) )radiological imaging of the lung peripheral infiltration )exclusion of the other causes of eosinophlic lung disease there is eosinophilia over /mm nearly all patients. one of third or half of patients have diagnosed as asthma. disease begin with systemic symptoms such as night sweats, weight loss, anorexia and pulmoner symptoms such as cough, shortness of breath, wheezing. patients have restrictive or obstructive findings in pft. one of third patients, especially with history of asthma, have obstruction in pft. İn the pathologic biopsy findings include; thickening of alveolar walls and accumolation of eosinophils and lymphocytes. long time used corticosteroids treatment is recomended. relaps is common when treatment is discontinued. we present the patient who has ig g deficiency, chronic eosinophlia and % eosinophils in bronchoalveolar lavage fluid. the patient improved long time used oral steroid then inhaled steroids. this was presented in terms of clinical association. case: a years old female patient who were followed due to asthma in the other center for two years, although use of combination inhaled fluticasone and salmeterol, patient was admitted with cough and sputum production. in thorax ct there were, bronchiectasis at right lower lobe, pneumonic consolidation in the right lower lobe and ground glass opacities. we detected as igg mg/dl ( - mg/dl), iga mg/dl ( - mg/dl), igm mg/dl ( - mg/dl), ige . mg/dl, igg mg/dl ( - mg/dl), igg mg/dl ( - mg/dl), igg mg/dl( - mg/dl), igg mg/dl ( - mg/dl). because eosinophilia ( cells) and symptoms continued, bronchoscopy was performed. left main bronchus was normal, right bronchus were seen dilated. purulent secretion was aspirated on rigt bronchus with flexible bronchoscopy ..in cytological examination % eosinophils was detected in bal. bronchoalveolar lavage cultures was negative. the patient was diagnosed chronic eosinophilic pneumonia and mg/ kg oral steroid was began. ivig was given up to patient; because of frequently recurrent sinopulmonary infection and patient had igg subclass deficiency. in the third month of oral steroid therapy physical examination findings and pft were improved and started inhaled steroid. conclusion: immunodeficiencies often can be seen alone. although, the pathogenesis of immundeficiencies with lung disease is not well understood and associated with interstitial lung disease. therefore, investigations must be include bronchoscopy and bronchoalveolar lavage. scid is a congenital heterogeneous group of diseases characterized by severe impairment of t,b, nk cell development and function. the hematopoietic stem cell transplanted patients with scid in the time period from to were evaluated. male/female ratio is / . median follow-up time is years ( months- years). parental consanguinity ratio was , %. mean age of onset of the symptoms was ± , months. most common clinical findings on admission were; pneumonia ( , %), moniliasis ( , %), diarrhea ( , %), dermatitis ( . %). classification according to t, b, and nk cell counts; (figure ). molecular genetic defects were determined in patients (figure ) are given. there is no difference between age groups according to occurrence of acute and chronic gvhd. death ratio increases with the increasing age. acute and chronic gvhd and number of deaths were significantly higher in peripheral stem cell transplanted patients. there is no statistically significant difference in occurrence of acute/chronic gvhd between b-and b + scid, who had been hla idantically transplanted from family donor. both groups have similar ratios of ivig treatment need after hsct. there is no statistically significant difference in occurrence of acute/ chronic gvhd between nk-and nk + scid, who had been hla idantically transplanted from family donor. both groups have similar ratios of ivig treatment need after hsct. death ratio was similar in groups. bcg dissemination in bcg vaccinated patients is significantly higher in the nk + group. the rate of complications due to severe infections is high and increases with age in patients with scid. hsct is curative, should be considered as early as possible. trec analysis for neonatal screening will give chance for early diagnosis and treatment of the patients. the hyper-ige syndromes are rare pids and are characterized by atopic dermatitis, skin abscesses recurrent pneumonias, elevated serum ige levels and sometimes mucocutaneous candidiasis. we have been evaluating and following up a group of patients with features suggestive for autosomal recessive hyper ige syndrome (ar-hies). homozygous dock mutations were identified in several of these patients. however, two siblings from a consanguineous family in this group of patients showed homozygous block in chromosome p . in homozygosity mapping which includes stk gene. sanger sequencing was performed for stk deficiency and showed a novel c. - delgata mutation in stk gene causing a premature stop codon. clinical manifestations of stk deficiency, also known as a macrophage stimulating (mst ) deficiency, stk deficiency comprise recurrent and severe viral skin infections including molluscum contagiosum and warts, fungal and bacterial infections and autoimmunity which are also the features of ar-hies. our patients's main features include autoimmune cytopenias (aiha and itp), cutaneous viral (molluscum contagiosum and mild perioral herpetic lesion), mild atopic dermatitis, seborrheic dermatitis, lymphopenia (particularly cd lymphopenia), and intermittent mild neutropenia. serum ige level was mildly high in these patients. our results indicates that patients that show clinical phenotype of ar-hies needs to be also evaluated for stk deficiency. determination of the underlying defect and reporting the patients are required for the description of the phenotypic spectrum of pids and the frequency of these diseases as well as for genetic counselling. mendelian susceptibility to mycobacterial disease (msmd) is a rare entity. patients with msmd have susceptibility to salmonella and some other intracellular microorganisms in addition to weakly pathogenic mycobacterial species. il- rβ deficiency, most common form of msmd, is caused by mutations in the il rb gene. patients who have symptoms suggestive for msmd or history of sibling death due to bcgosis were evaluated. the expression of the il- rb was detected in patients and family members by monoclonal antibodies on the lymphocyte surface by flow cytometry after the lymphocytes were stimulated in vitro with pha. mutation analyses was done by sanger sequencing. all index cases were presented either with bcg or salmonella infection. two patients, though they were bcg vaccinated had no clinical symptom, six presented with the symptoms of salmonella infections, two developed leukocytoclastic vasculitis, candidiasis was the accompanying feature in seven. recurrent leishmaniasis that necessitated subcutaneous interferon-γ and prophylactic amphotericine b therapy was present in a patient. in all patients the percentage of lymphocytes with il rβ expression was found to be less than % . prophylactic antimycrobial treatment and in severe and resistant infectious episodes if-γ therapy, should be given. many patients have associated mucocutaneous candidiasis. the prognosis is good unless the patient admits at the later stages of bcg infection. the results of our patients showed that the analysis of the surface expression of il rβ on activated lymphocytes is an effective diagnostic method which can also be used in screening of the patients with probable msmd. ataxia-telangiectasia (a-t) results from the loss of ataxia-telangiectasia mutated gene (atm) function and is a heterogeneous, multisystemic disease and characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, immunodeficiency, premature ageing and increased neoplasia incidence. even in classic a-t with ataxia and telangiectasia, the onset of clinical symptoms and the rate of progression are variable. here, we report two siblings was diagnosed as a-t with severe and early hypogamaglobulinemia, decreased cd , increased cd ro, no ataxia and telangiectasia, purulent otitis and hemophagocytosis that harbor a rare frameshift mutation of atm gene. Özdemir Öner ; bozdoğan sıla department of pediatrics, division of allergy/immunology, sakarya university, medical faculty, adapazarı, türkiye. department of pediatrics, sakarya university, medical faculty, adapazarı, türkiye. background: the acquisition of new food allergy after transplantation or transplant-acquired food allergy (tafa) is usually reported in adults and rarely in children. tafa is described mainly after liver, but also after small bowel/intestinal, lung and heart transplantations. in different studies, the male/female ratio is equal. literature data suggest that children with tafa typically present within the first year after surgery and they are typically allergic to multiple foods. aim: tafa is generally characterized with allergy to multiple foods and increased level of total and/or spesific ige. here, a patient although who had normal total. ige and specific ige test results, he developed reaction to skin prick test for cow's milk is presented and his clinical presentation will be discussed. case presentation: month-old-boy came to our allergy clinic with complaints of vomiting after drinking cow's milk and skin rush on the area where contact ed with chocolate. in his past medical histroy, left lateral segment of liver (donor was his mother) was transplanted to him when he was at months. the liver donor was not recorded as having a history of allergic disease. methylprednisolone and tacrolimus immunosuppression were used after the transplantation, and tacrolimus therapy was continued for prophylaxis of chronic rejection. when he was at months, family fed the patient with cow's milk but hours later he began to vomit. he vomited five times in two hours. then, he developed constipation. rectal irrigation was used. then oral intake stopped for two days. he was thought to be having food protein induced enterocolitis. his vomiting complaints repeated after intake of formula and baby food which include grain. so he fed with special formula including short-chain peptides and free aminoacids and his symptoms improved. past medical history: extrahepatic biliary atresia was diagnosed at weeks age with conjugated hyperbilirubinemia (according to scintigraphy and biopsy results). family history: his father has penicillin allergy and his aunt has asthma. at our outpatient clinic: height and weight were within normal percentiles. physical examination reevaled normal examination findings. laboratory findings: wbc was . /mm , with % neutrophils, % eosinophils and % lymphocytes. his hemoglobin was . g/ dl, platelet count was . /mm . background: except for antibody deficiency and complement defects, hematopoietic stem cell transplantation (hsct) is the single best curative treatment defined for primary immunodeficiency (pid) so far. in the current study, we aimed to assess the role of pid type, donor type and clinical status on hsct success rates. materials and methods: we retrospectively reviewed the records of a total of hscts procedures performed in patients diagnosed with pid between and , in ankara university pediatric allergy and immunology department. results: of the patients, had severe combined immunodeficiency (scid) and had non-scid. the survival rates following hsct, in both scid and non-scid patients were %. when classified according to the source of donor, patients who had a hla-matched sibling donor (msd) in the scid group had . % survival rate post transplantation, those who had a matched related donor (mrd) had . % and those who received a haploidentical donor had . % survival rates. in the non-scid group there were patients with haploidentical transplants ( omenn syndrome and mhc class ii deficiency) and all patients died. we assessed several potential risk factors associated with survival in scid patients. patients diagnosed over months of age with a pre-existing pulmonary infection, requiring intensive care and/or mechanical ventilation had significantly lower survival rates. conclusion: hsct is the best curative treatment for pid. our results demonstrated that hsct performed from matched family donors or even haploidentical parents is a lifesaving treatment in various types of pid's, especially in scid. introduction: in case of donor availability allogeneic hematopoietic stem cell transplantation (hsct) can be regarded as a definitive therapy for a variety of primary immunodeficiency syndromes (pids), including severe combined immunodeficiency (scid) and non-scid pids. study period: we retrospectively reviewed the hospital records of consecutive children with pid, who had allogeneic hsct in the last years, between january and january . our median follow-up time is , years ( months - , years) patients and methods: the median age of children at hsct was , months ( , months- years). boys/ girls diagnoses were based on anamnestic data, clinical findings, and immunological and genetic analysis. conditioning regimens included busulphan + cyclophosphamide, busulphan + cyclophosphamide + atg, fludarabine + melphalan or fludarabine + anti-cd in b-, t-, nk-scid cases conditioning was not used. indications for transplantation: patients' diagnoses were severe combined immunodeficiency (n = ), wiskott-aldrich syndrome (was, n = ), chronic granulomatous disease (cgd, n = ), xlinked lymphoproliferative disease (xlp, n = ), whim-syndrome (n = ), hyper igm syndrome (cd ligand deficiency, n = ), leukocyte adhesion deficiency (lad, n = ), dock mutation (n = ) transplantations: hscts for children were performed. patients were retransplanted ( pt once, pt twice, pt times), because of rejections. at the first hscts in / cases sibling bone marrow, / sibling peripheral blood, / sibling cord blood, / unrelated cord blood, / unrelated bone marrow, / unrelated peripheral blood, / haploidentical donors were used median cd count was , x /kg ( , x - , x /kg) patients engrafted on median ± day (anc > , g/l) acute gvhd occured in / cases ( pathomorphological findes: distortion of the structure of lymph nodes due to enlargement of paracortical zones and follicules was found in all patients. presumed atypical cells (makrolymphoblastes and berezovsky-sternberg-reed cells), were detected in patients overwise during follow-up for - years in these patients revealed no specific infectious and malignansy. accumulation of proliferating dn cells (a lot of mitoses) was characteristic fiture in lymph nodes and spleen of alps patients. setting of plasma cells in follicular zones in the lymph nodes was reveald in cases, and eosinophilesin , pronounced immunoblast proliferation in , sinus histiocytosisin . multiple hyperplastic follicles, extended sinuses with many phagocytizing macrophages, lymphocytic infiltration was revealed by histological examination of the spleen in all cases. immunohistochemistry findes: t-and b-cells proliferation (ki- expression), atypical location of lymphocyte populations, settings of plasma cells in all lymph node zones. in paracortical zones was found cd +, cd +, cd + cd -, cd + cd -cells in patients, cd +, cd + cells in patient; and in restricted follicular zones -cd +, cd +, hla-dr + cells in patients, cd +, cd + and cd + in patients. the presentation of a case of kimura disease in a patient with was. the was was diagnosed in year months, based on infectious syndrome, atopic dermatitis, hematological syndrome -skin hemorrhages and thrombocytopenia - * /l. after he performed often sars. regularly received ivig at mg/kg, antibiotic therapy with a positive effect -infectious syndrome stopped, controlled hemorrhagic syndrome. since there was a herpes virus infection with frequent exacerbations on the face. patient received acyclovir, valacyclovir, famvir with a temporary effect. in . on the left side of the face -periorbital region, eyelids, malar region appeared the site of soft tissue hyperproliferation - cm in diameter with a thickness of - cm, with moderate moist, painful on palpation, the left eye was closed, the lid margin ciliated dramatically thickened, deformed. in october-november was admitted in diagnostic department, massive antibiotic, antimycotic, antiviral therapy, ivig therapy. the histological study showed the angiolimphoid hyperplasia with eosinophilia without immunomorphological signs of malignant tumor growth. based on histological, immunohistochemical studies of the skin, these clinical and laboratory findings, diagnosis: mass lesion of the left face -kimura disease. patient helded courses of chemotherapy with positive dynamics: mass lesion decreased, erosive surfaces disappeared and now receives romiplostimum, acyclovir, biseptolum, ivig . g/kg time per month. under the observation there is a family k., which is unique in the deletion variants of the same region q. . and the presence of an adult patient with the di george syndrome. clinical manifestations of the syndrome in family members is differ. methods: clinical, laboratory, instrumental and genetic ( dna was isolated using a kit «qiaamp dna mini kit» and dna from dried blood spots was isolated using a commercial kit "dna-sorb-b». mlpaanalysis set salsa mlpa probemix p -b digeorge, genetic analyzer applied biosystems ). mother ( years), had few incidents of pneumonia before years. she was diagnosed ullrich-turner syndrome at age years. all childbirths by caesarean section. she has not any laboratory and instrumental findings of immunodeficiency, endocrinopathy, cardiac and thyroid abnormalities. she has deletion in the starting area of the di george region (lcr -a), including genes cltcl , hira, cdc , cldn , gp bb, tbx , txnrd , dgcr . father ( years), healthy, current smoker. the dna microstructural violations in di george region haven't been identified. son was from i pregnancy, heart defect was set prenatally, marked growth retardation. childbirth at weeks. he had unstable reduced cell parameters and hypogammaglobulinemia, the size reduction of the thymus and congenital heart disease: truncus arteriosus, dc b stage by lang. after days of life operated for congenital heart disease. during the surgical intervention the thymus wasn't found in a typical place. the postoperative period was complicated by sepsis, heart and respiratory failure. received ivig, antibiotic and antifungal therapy. he suffered from sars, severe course, the degree of respiratory failure ( months) -death. in mlpa-dna defined microdeletion disorders starting region di george (lcr -a) -from the -year-old dry blood sample (postmortem study a year old boy with bleeding, eczema and recurrent pyogenic infection was admitted to our department. the child who received treatment in hematology accidentally a few times, but has not had the effect of treatment . laboratory parameters -cd -cells- % ( ), cd -cells - % ( ), cd -cells - % ( ), cd -cells - % ( ), cd / -cells - % ( ), cd / cd - . , hla-dr- , nbt- . igg- . q /l, iga- . q / l, igm - . q / l, ige- u / ml. hb- g / l, erythrocytes- . x / l, leukosytes - . x / l, neutrophils- %, lymphocytes - % ( ), eosinophils- %, monocytes- %, metamielosytes:- %, plateletsrare was observed.normal bone marrow cells, the separation of platelets from meqakariosyt has become weak. the advantage of erythroid unordered have been observed. bone marrow puncture the mielokariosytes - . x / l, meqakariosytes - . x / l, blasts- . %, myelosytes - . %, metamyelosytes - . %, seqments - . % neutrophils-% . , eosinophils- . %, lymphocytes- . %, monocytes- . % erythroblasts- . %, pronormoblasts- . %, normoblasts- . % meqakariosytes - . %, plasmatic cells- . %.his younger brother with eczema, pyogenic infections (furunculosis) was admitted to our department. cd - % ( ) ataxia telangiectasia (a-t), is a genetic disorder caused by the homozygous mutation of the atm gene and, frequently associates with variable degrees of cellular and humoral immunodeficiency. however, the immune defects in patients with a-t are not well characterized. to our knowledge, there is no work on major lymphocyte subpopulations and recent thymic emigrants of a-t patients comparing to age-matched healthy controls. according to esid criteria, patients diagnosed as a-t and agematched healthy children were assigned to the study. both patients and healthy controls were grouped as - , - , - years and older than years. using flow cytometer, major lymphocyte subpopulations and cd + cd ra + cd + recent thymic emigrants (rte) were determined as per cent and absolute cell numbers and compared. no significant differences regarding all lymphocyte subpopulations were observed between age groups of a-t. comparing to the healthy controls, there were a decrease (in t cells, effector memory t cells, b cells, naïve b cells, switched b cells and rte) and there were an increase (in active t cells, naïve t cells and nonswitched b cells) in the absolute number and percent of some cell populations in the a-t group. findings of this study showed effector functions in some cell lymphocyte populations were decreased and could be thought that bone marrow of patients should be tried to increase the cells numbers. however, the study has an important limitation about patient and healthy population. ataxia-telangiectasia (a-t) results from the loss of ataxia-telangiectasia mutated gene (atm) function and is a heterogeneous, multisystemic disease and characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, immunodeficiency, premature ageing and increased neoplasia incidence. even in classic a-t with ataxia and telangiectasia, the onset of clinical symptoms and the rate of progression are variable. here, we report two siblings was diagnosed as a-t with severe and early hypogamaglobulinemia, decreased cd , increased cd ro, no ataxia and telangiectasia, purulent otitis and hemophagocytosis that harbor a rare frameshift mutation of atm gene. nijmegen breakage syndrome(nbs) is a rare autosomal recessive disease usually presenting at birth with microcephaly. here we present a case of nbs diagnosed at the age of twenty seven who admitted to our outpatient clinic with malaise, loss of appetite, weight loss and dyspnea on effort . she was hospitalized in another centre years before because of pneumonia, pancytopenia, generalized lymphadenopathy, hepatosplenomegaly and hypogammaglobulinemia (very low igg and iga) where she couldn't have any definite diagnosis. her past medical history was remarkable for primary amenorrhea and basal cell carcinoma excision from preaericular region when she was . she had no severe infection history before except frequent upper respiratory tract infections. her parents were consanginous and she had a brother died at months of age. microcephaly together with short stature, multiple palpable lymphadenopathies , splenomegaly and absent secondary sexual characteristics were prominent features in physical examination. direct fluorescence sequencing of the nbn gene showed a homozygous mutation in exon (c. _ delacaaa) which confirmed the diagnosis of nbs in our patient. nbs is mostly diagnosed in childhood. the delay in diagnosis was partly due to the lack of severe infections in her past medical history until she was . another factor that lead to the delay is that it is an unknown disease among physicians in turkey. the longest known survival is years in a patient who had no clinical features of nbs other than primary amenorrhea. as a result, our patient is one of the oldest patients reported in the literature presenting nearly all of the classical features of the disease and carrying the most common pathologic mutation. wiscott-aldrich syndrome (was) is a rare x-linked recessive immunodeficiency disorder characterized by thrombocytopenia, small platelets, eczama, recurrent infections and an increased risk of autoimunity and malignancy. the gene responsible for was is located in chromosome xp . -p - , which consists of exons, and encodes a -amino acid protein. in this study, we aimed to screen was gene mutations and analyze the effects of determined mutations in boys with non-classical was phenotype. ivs + g > a gene alteration in intron of was gene was identified in case , previously. so, rna isolation and then cdna synthesized was carried out. in case , after amplification of exons of was gene by pcr, the amplicons were sequenced. in this patient, g > c alteration was detected in the first base of intron . afterwards, cdna synthesized for detecting the splicing effect. based on the gel image results, cdna found to be base pairs smaller than the normal in case . in case , g > c alteration was detected in the first base of intron and then we determined multiple splicing products. two different splicing mutations (ivs + g > a and ivs + g > c) were detected in two cases with non-classical phenotype. ivs + g > a splicing mutation was stated in the literature, previously, but ivs + g > c mutation was first time identified in this study. whim is rare (< / ), heterozygous, autosomal dominantly inherited pid, caused by mutations in the gene encoding for the chemokine receptor cxcr , mapped on q locus. the altered cxcr / cxcl interaction impairs cellular homeostasis and trafficking, resulting in immunological dysfunctions with abnormal retention of mature neutrophils in the bone marrow (myelokathexis) and consecutive severe neutropenia, variable degree of lymphopenia and hypogammaglobulinemia. whim patients suffer from recurrent bacterial infections since early childhood and later on manifest a specific susceptibility to hpv infections with developing widespread warts. because of rarity of the disease, heterogeneity in clinical presentation and usually incomplete phenotype, the diagnosis is often delayed and whim syndrome is not suspected. a year old boy who is suffering from recurrent bacterial infections, often complicated with bronchopneumonia, with severe neutropenia, but also, with lower level of lymphocytes and still, normal serum immunoglobulin level is presented. he has no developed warts; neither his parents nor relatives have warts. at the age of years was unveiled the diseasecausing mutation in the cxcr gene (c. c > t; p.r x; heterozygote). severe congenital neutropenia accompanied with lymphopenia and findings of mature neutrophils in bone marrow, might be an easy approach for getting closer to the clinical diagnosis of whim syndrome. early identification is important for clinical and therapeutic management, allowing a more comprehensive follow-up and administration of appropriate therapy. we report two cases of congenital heart disease (tetralogia fallot and interruption aortic arch) with confirmed microdeletion chromosome q . by karyotype and fluorescence in situ hybridization analysis (fish). children underwent surgical correction of congenital heart defects with good postoperative outcome, although were complex. the phenotype of these patients can be extremely variable, frequently leading to clinical confusion, diagnostic delay, excess morbidity, early mortality. identification of these patients is essential for their adequate management and genetic counseling. a multidisciplinary approach is fundamental to ensure that the patient will be able to attain his or her maximal potential. the underlying cause of the juvenile periodentitis is not well understood but is now thought to be related to an abnormal immune system and to invading bacteria in the cementum of the teeth. instead painful fissures and recurrent pyogenic infections of the skin seem to be the most common medical complications. however, a number of pls patients with abscesses or pseudotumors of the liver have been described. there have been reports of pls patients with other stigmata such as growth retardation, non-symptomatic intracranial calcifications and mental retardation furthermore, coinheritance of pls and albinism type has been reported. case presentation: a yr old girl admitted to our hospital with chief compliant of skin lesions since early months of birth. in the past medical history; she had skin abscess and failure to thrive. on admission she had erythematous, shiny skin with generalized dry scaly predominantly palmoplantar hyperkeratosis and loss of teeth except four or five molar teeth. she informed that she had malformed teeth since childhood which fell off one by one. she had also poor oral hygiene non-pitting edema on lower extremities. no hepatosplenomegaly detected. family history was negative. she investigated for probable immune-deficiency with regard to skin lesions, history of skin abscess and ftt the lab finding are as following: cbc diff = normal ,crp = neg, esr = , ast = , alt = , alph = , alb = . , total pr = . , urea = , cr = . igg = , igm = , iga = , ige = . , cd = %, cd = %, cd = %, cd = %, cd = %, cd = % conclusion: according to nearly normal lab values and presenting signs such as: generalized pyogenic periodentitis, palmoplantar hyper keratosis and negative family history were attributed to a very rare autosomal recessive disorder with ectodermal dysplasia known as papillon-lefevresyndrome manifesting with palmoplantar hyperkeratosis and severe early onset of destructive periodontal leading to pre-mature loss of both primary and permanent dentitions. nijmegen breakage syndrome (nbs) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. the disease is caused by mutations in the nbs gene, which encodes nibrin, a component of the hmre -rad -p complex involved in cellular response to dna doublestrand breaks. the aim of the present case report was to discuss two siblings with immunologically and clinically different phenotypes of disease presentation and to make an attempt to explain possible genotype-phenotype relation. the two patients and their non-consanguineous parents were clinically, laboratory and genetically investigated. for mother and father we observed no clinical presentation and no immunological abnormalities. sibling no. ( years old boy) had no history of recurrent infections and no deviation in immunological tests. sibling no. . ( month old girl) had recurrent infections since birth and iga, igg and igg deficiency as well as t-and b-cells deficiency. cytogenetic analysis revealed variable percent of spontaneous chromosomal instability which was more severe (in % of chromosomes analyzed) in sibling no. . additionally we sequenced bi-directionally ( amplicons) the dna samples from all family members to survey the germline genetic variation in the nbs gene. the del (exon ) was detected in both siblings in homozygous and in both parents in heterozygous feature. in order to explain different clinical and immunological presentation of two siblings the rest of the nbs exones were analyzed for genetic heterogeneity. no additional changes were observed. in conclusion patients with the same nbs genotype may show different phenotypes. other gene/epigenetic factors seem to play a role in phenotype modulation. omenn syndrome [mendelian inheritance (omim )] is an autosomal recessive form characterized by the presence of fatal generalized severe erythroderma, lymphoadenopathy, eosinophilia and profound immunodeficiency. objective: we studied clinical and immunological presentation of the disease manifestation and frequency c. - delaa (p.k vfs ) in rag gene among eastern slavs population. results: we collected clinical and immunological data of patients ( from belarus, -ukraine, -russia) females, males. age of omenn syndrome manifestation varied from st day of life to yr month. age of diagnosis - days to year months. in patients had classical immunological phenotype t(+/-)b-nk+, pt had tlowb + nk + with cd + tcrgd + expansion. in pts had mutation in rag gene, in had c. - delaa (p.k vfs ) in one or two alleles. at present moment in pts are alive, were transplanted, pt is prepared to bmt. conclusion: this study demonstrates that the most popular genetic abnormalities in eastern slavs children with omenn syndrome is c. - delaa (p.k vfs ) in rag gene. this information may be useful for rapid diagnostic of omenn syndrome in laboratories used sscp (single strand conformation polymorphism) before sequencing. under examination the patient particularly bright phenotype attracted attention: microcephaly, "birdlike" facial features (sloping forehead, nape, hypoplasia of brow ridges, broad nasal bridge and protruding midface, hypoplasia of the mandible). in addition, besides specific anomaly of the facial bones we noted: big ears, sparse hair and clinodactyly of the fifth fingers. clinical and immunological characteristics: the feature of the case is pancytopenia syndrome we have diagnosed at the early stages of observation and which is continued throughout the period of observation. erc - . - . x /l hb - - g/l leuk - . - . x /l neu - - % ( - cells/mcl) plt - - x /l data of immunological examination: iga - , g / l igm - , g / l igg - , g / l (other results of immunological examination are without features) the deep insufficiency of antibody production in our patient was the cause of serious, recurrent, and subsequently chronic bacterial sinopulmonary infections after years old. the results of clinical laboratory and immunological examination without significant features: erc - , - , x /l hb - - g/l leuk - , - , x /l neu - - % ( - cells/ mcl) plt - x /l iga - , g/l - . g / l igm - , g/l - . g / l igg - , g/l - . g / l (other results of immunological examination are without features) the x-linked chronic granulomatous disease (cgd) is a primary phagocytic cell deficiency characterized by severe bacterial and fungal infections of various organs. we report of a years of a male patient with xlinked cgd who presented with recurrent hepatic abscesses as the sole manifestation of the disease. phagocytic and bactericidal activities of granulocytes were studied by using microbiological assays. generation of superoxide anion by blood granulocytes was measured by the ferricytochrome c reduction test. cgd is an immunodeficiency caused by mutations in genes encoding subunits of the nadph oxidase complex. normally, assembly of the nadph oxidase complex in phagosomes of phagocytic cells leads to a "respiratory burst" essential for the clearance of microorganisms. cgd patients lack this mechanism, which results in life-threatening infections and granuloma formations. the leading cause of death are pneumonia and pulmonary abscess, septicemia and brain abscess. in neurogical manifestations various pathogens have been involved including aspergillus spp., s. prolificans, a. infectoria, salmonella and staphylococcus spp. there are only some several reports on fungal brain and spinal cord infection, aspergillus abscess resembling brain tumor, meningitis due to streptococcus and candida spp. in the past years we treated children with cgd. we present the infectological challenge of an x-linked cgd patient with brain abscess. in spite of our effort we were unable to identify its causative pathogen. empiric therapy sometimes resembles polypragmasia in cgd. to decrease mortality and morbidity from fungal infections in cgd the prophylactic use of itraconazole or voriconazole is widely recommended. a relatively new azole, posaconazole is active in pulmonary and cerebral fungal manifestations , indeed may be effective against fungi with inherent resistance to ampb or voriconazole. there was no etiological diagnosis in our case that did not respond to conventional antifungal and antibacterial treatment. based upon the findings and literature data we presume the causative agent might be some kind of moulds. we suppose the use of echinocandin and posaconazole as salvage ("prophylactic") therapy has resulted significant regression of the brain abscess. the diagnosis of chronic granulomatous disease (cgd) was verified in children during the past few years in the department of clinical immunology of regional children's hospital of chelyabinsk (russia). in our opinionin casesthe diagnosis was establishedearly enough. michael w. transferred to the neonatal pathology unit of our clinic at the age of days with vesiculopustules. take into consideration our own experience of observing children with this form of primary immunodeficiency in previous years, the child was conducted immunological examination, in particular, the test of restoration nitroblue tetrazolium by superoxide anionformed when oxygen explodes in leukocyte (nitro blau tetrasolium -nbt-test). this decision is caused by the fact that previously observed children with chronic granulomatous disease, had vesiculopustules in % at birth. the survey has revealed a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test, which allowed us to suggest the diagnosis of cgd. the baby was banned vaccination against tuberculosis, and after reliever vesiculopustules the boy was discharged home. however, at the age of month the baby suffered from glandular abscess and right-segmental pneumonia. in cbc there is expressed anemia (hb g/l, erythrocytes - . x /l), leukocytosis ( x /l), accelerated esr ( - mm/h). at the age of months in the university of debrecen, medical and health science center debrecen, hungary, molecular genetic studywas conducted by prof. dr. laszlo marodi. it was revealed a mutation in c. g > a in exon of gene cybb (encoding subunit gp -phox), after that the diagnosis of cgd was finally verified. it was assigned a basic preventive antimicrobial therapy by trimethoprimsulfamethoxazole, and itraconazoleto prevent fungal infections. despite this since months the child has repeatedly and consistently suffered from bilateral groin lymphadenitis during the year, acute hematogenous osteomyelitis of the left ulna, pneumonias, purulent mesadenitis, endoperitonitis. the fact that the child aged months was diagnosed sepsisis evidence of the severity of infectious complications. clinical and biochemical blood tests were distinguished by consistently high levels of esr and the presence of leukocytosis, and also severe anemia. now the child is years months, he is undergoing treatment at the department of clinical immunology, russian children's clinical hospital (moscow) after bone marrow transplantation. case history № , christina n. from the early history we know that the girl's newborn period was uneventful. she was vaccinated against tuberculosis in the nursing home (no reaction). it was noted the formation of abscesses after vaccination against whooping cough, diphtheria and tetanus in and . months. in years months she suffered from mezootit. for the first time the girl came under our observation in the children's hospital in chelyabinsk (russia) at the age of years with right segmental pneumonia, complicated by the destruction. after further examination it was diagnosed tuberculosis of intrathoracic lymph nodes to the right with upper lobe bronchopulmonary defeat. to take into consideration given above, it was conducted immunological test that revealed a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test. on the basis of the history, clinical manifestations and results of immunological examination chronic granulomatous disease was diagnosed. after fourmonth period treatment a recovery came from tuberculosis and child was transferred to outpatient monitoring. in years in this family a second daughter was born -arina n. taking into account revealed immunodeficiency of her sister, the child was not vaccinated against tuberculosis on our recommendations. a six-month-old child was conducted immunological examination, which, like her older sister, also shows a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test. during the molecular genetic studies of both sisters it was identified identical mutation c. - del gt gene ncf (encoding subunit p phox). now the older girl is and her sister is years old. observing them in the dynamics neither of them do not show any life-threatening infections. thus, the recovery test of nitrobluetetrazolium by superoxide anion formed when oxygen explodes in leukocyte (nitro blau tetrasolium -nbt-test) is a fairly reliable method of early diagnosis of chronic granulomatous disease. chronic granolomatous disease (cgd) is due to defective phagocyte superoxide production leading to impaired microbial killing. it is comprised of a group of five genotypes with a common phenotype, chracterized by recurrent severe bacterial and fungal infections and tissue granuloma formation. patients with cgd often present with pneumonia, liver abscess, skin infections, lymphadenitis, osteomyelitis. a five month old boy was referred to pediatric infection unit by lymphadenitis. on the medical history, he had taken antibiotic therapy for lymphadenopathy when he was months old. the abnormal eye movement was noticed by the family and on the eye examination peripheric chorioretinal hypopigmented lesions were determined when he was months old. İn his labratory examination, viral serology were negative. his parents were not consanguineous. his mother's brother had died at years old. he had history of skin infections and osteomyelitis. his grandmother had been diagnosed as having tuberculosis lymphadenitis one year ago. on his physical examination, his growth was normal, patological findings were horizontal nistagmus, / degree systolic murmur, fistulized lymph tissue on the left submandibuler region. on the laboratory findings; he had anemia and neutropenia. immunglobulin levels and lymphocyte subtypes were normal. his respiratory burst activity was very low. chronic granulamatous disease was thougth in the patient by the clinical and laboratory findings. İt was detected gp phox mutation in the genetical analysis. he was diagnosed as having x-linked cgd. antibiotic prophylaxis (tmpsmx, flucanazol) and interferon gamma were started. he is months old now and he is on the list of match unrelated donor screening. patiens who has history of lympadenitis, skin infections and chorioretinal findings should be evaulated for the x-cgd. leucocyte adhesion deficiencies (lads) are rare autosomal recessive inherited disorders. three different forms of lads have been described so far. in lad-i, the most common leucocyte adhesion deficiency, the function of β integrin cd is lost. . while one of the first signs of the disease consist in delayed separation of the umblical cord, severe infections already start early during infancy. another feature of lad-i includes impaired wound healing. therefore, mortality during infancy is high. a fifty day old boy was referred to the hospital due to diarrhea and leukocytosis. the patient was delivered following an uncomplicated full term pregnancy. the parents were first degree cousins. father's brother and mother's uncle had died during infancy period. his umblical cord had separated on the th day. patient had applied because of diarrhea by starting in the first days of life and leukocytosis was detected ( . / mm ). on the physical examination, his body weight was gr ( th to th percentile) and his height was cm ( th percentile). there was a granuloma on the umbliculus. other systems were normal. in the laboratory examination, leukocyte count was high, immunoglobulins, respiratory burst activity, gaita analysing and culture were normal. in the lymphocyte subtype analysing, cd + b cells ratio was mildly low. cd , cd a, cd b, cd c levels were found to be very low. in the genetic analysing, it was detected two deleterious mutations in the itgb gene. patient had been diagnosed as lad- . he treated by antibiotics and then started prophylactic antibiotherapy. family screened for tissue match. his older sister was found full matched. he was referred to transplantation unit. it was applied bone marrow transplantation when he was months old. presence of delayed umblical cord separation and leucocytosis should be considered in the diagnosis of lad but these patients might have different symptoms such as diarrea. mendelian susceptibility to mycobacterial disease (msmd) is a rare entity. patients with msmd have susceptibility to salmonella and some other intracellular microorganisms in addition to weakly pathogenic mycobacterial species. il- rβ deficiency, most common form of msmd, is caused by mutations in the il rb gene. patients who have symptoms suggestive for msmd or history of sibling death due to bcgosis were evaluated. the expression of the il- rb was detected in patients and family members by monoclonal antibodies on the lymphocyte surface by flow cytometry after the lymphocytes were stimulated in vitro with pha. mutation analyses was done by sanger sequencing. all index cases were presented either with bcg or salmonella infection. two patients, though they were bcg vaccinated had no clinical symptom, six presented with the symptoms of salmonella infections, two developed leukocytoclastic vasculitis, candidiasis was the accompanying feature in seven. recurrent leishmaniasis that necessitated subcutaneous interferon-γ and prophylactic amphotericine b therapy was present in a patient. in all patients the percentage of lymphocytes with il rβ expression was found to be less than % . prophylactic antimycrobial treatment and in severe and resistant infectious episodes if-γ therapy, should be given. many patients have associated mucocutaneous candidiasis. the prognosis is good unless the patient admits at the later stages of bcg infection. the results of our patients showed that the analysis of the surface expression of il rβ on activated lymphocytes is an effective diagnostic method which can also be used in screening of the patients with probable msmd. introduction: mendelian susceptibility to mycobacterial disease (msmd, online mendelian inheritance inman ) is a rare immunodeficiency characterized by predisposition to infections caused by weakly virulent mycobacteria, such as mycobacterium bovis bacille calmette-gue´rin (bcg), environmental nontuberculous mycobacteria (ntm), and salmonella strains in otherwise healthy individuals. il- rb deficiency is the most common form of msmd and is characterized by childhoodonset mycobacteriosis with frequent recurrence. it has been found that patients with il- rb deficiencies are also prone to developing infections with nontyphoidal salmonella species with bacteremia and lymphadenopathy. here we present a girl with recurrent cutaneous leukocytoclastic vasculitis (clv) with salmonella enteritidis due to il- rb deficiency. case report: a four year old girl that had been diagnosed serologically with recurrent salmonella infections, associated with lymphadenopathy and skin eruption was admitted as having henoch-schönlein purpura. she had been vaccinated with bcg and developed left axillary lymphadenitis which spontaneously drained and had recurrent oral monilia plaque. edema and purpuric eruptions were present on the upper and lower extremities and the abdomen.multiple mobile, painful,enlarged submandibular lymph nodes of about x cm in diameter were palpable. skin biopsy showed a dense inflammatory site with eosinophils, neutrophils and fibrin in the upper dermis and dermal vessel wall,compatible with leukocytoclastic vasculitis. serological studies to assess diagnostic markers for vasculitis and infectious agents were all negative. immune work-up were unremarkable other than hypergammaglobulinemia. salmonella enteritidis was identified in blood culture. she responded dramatically to ceftriaxone treatment within a few days and lesions cleared completely. extended immunological and molecular genetic examination of the patient was carried out for il- /ifn-γ pathway defects. on the facs analysis of t cells for cell surface expression of the cytokine receptor chains, she did not express any il- rβ . discussion: in thepresent report, we describe a child with clv with salmonella enteritidis due to il- rb defi-ciency.in a large cohort of patients with il- rb deficiency, ntm, m. tuberculosis, disseminated bcg infection after inoculation with the vaccine, and salmonella infection have been described. sporadic cases with other infectious agents have also been reported. salmonella infections reported in these patients were due to extraintestinal, or septicemic, recurring infections caused by nontyphoidal salmonella species. only two il- rb -deficient patients have been identified with vasculitis due to salmonella strains; both came from turkey, where consanguineous marriages are common. kutukculer and colleagues reported the first case of s. enteritidis-associated clv. sanal and colleagues reported a clv case associated with group d salmonella infection. leukocytoclastic vasculitis is an immune complex mediated disease predominantly involving small vessels of the skin and can be associated with drugs or can be found as a component of other disease, such as infections, connective tissue disorders, and malignancies. infectious agents can cause vasculitis directly or clinically mimic primary vasculitis .multiple infectious agents have been suspected as triggering or contributory factors in the vasculitic process . several factors contribute to the primary vasculitis related to infections: a type or immune-complex reaction, cell-mediated hypersensitivity, abnormal immune regulation, and direct endothelial cell invasion by infectious agents. in our case, extensive evaluation was performed to determine the underlying vasculitis process. clinical and laboratory examinations revealed no association between vasculitis and other infections or an underlying connective tissue disease or medication. she responded well to ceftriaxone treatment, and clinical manifestations gradually resolved within a few days, providing strong evidence that improvement of the vasculitic lesions was due to elimination of the salmonella with antibiotics conclusion: our patient has one of the exceptional forms of il- rb deficiency, with recurrent clv due to salmonella enteritidis. although common presentations for salmonella infection in individuals with il- rb deficiency are lymphadenopathy and bacteremia, it can be present clinically as clv. some infections such as salmonella may be responsible for different types of vasculitis even though they are not common . in this respect, clinicians should be aware of possible infectious causes of vasculitis, and children presenting with unusual recurrent infections caused by non typhoidal salmonella, bcg, or ntm should be investigated for ifn-γ ⁄ il- pathway defects. gülez n.; genel f. dr. behcet uz children hospital allergy-immunology department, izmir, turkiye the complement system is an important part of the innate immune defense and also plays a major role in shaping the adaptive immune response. these functions are required for a good defense against infections, especially bacteria. the c deficiency is a rare disease that is associated with recurrent neisserial infections, especially meningits caused by n. meningitidis. the patient, a seven years old girl was admitted to hospital with high fever and diffuse, purple-coloured skin lesions. her symptoms gave the diagnosise meningococcal meningitis. she had also earlier been diagnosed with the same disease when she was years old. a sister to the patient had died from meningitis at years of age. she has also one older and one younger sister. there is no consanguinity between her parents. the laboratory analyses of the classical pathway measured as complement hemolytic activity (ch ) and c concentration revealed no activity and absence of c , respectively. analysis of serum from her younger sister showed the same results, while her older sister's ch and c levels were found normal. thus, our patient and her younger sister were diagnosed with hereditary c deficiency. the genetic analyses have not been completed yet. we here report the third and fourth cases of c deficiency in turkish patients. interferon-g receptor- (ifngr ) deficiency is caused by mutations in ifn-γ receptor- gene and is characterized mainly by susceptibility to mycobacterial disease. we report a boy with complete recessive ifngr deficiency, afflicted by recurrent mycobacterial diseases with m. bovis, m tuberculosis, m. avium intracellulare and m.fortuitum. genetic analysis showed a homozygous mutation ( inst) in ifngr gene leading to complete ifngr deficiency. in addition, he had atypical mycobacterial skin lesions caused by m.avium intracellulare and he developed scrotal and lower limb lymphedema secondary to compression of large and fixed inguinal lymphadenopathies. to our knowledge, the patient is the first case with interleukin- /interferon − γ pathway defect and severe lymphedema. defects in the il- / ifn − γ pathway must be considered in patients with disseminated or recurrent mycobacterial infections and in patients with severe viral infections, especially in countries where bcg vaccination is part of the national health programme. it must be kept in mind that these patients may develop granuloma-like skin lesions and severe lymphedema. hsct must be applied at the earliest time before developing organ damages. t lymphocyte/nk cells. restricted defective molecules in the circuit and recently discovered cybb responsible for autophagocytic vacuole and proteolysis have been identified in around % of patients with the mendelian susceptibility to the mycobacterial disease (msmd) phenotype. primary defects in oxidase activity in chronic granulomatous disease (cgd) lead to severe, life-threatening infections. the role of phagocytic respiratory burst in host defense against mycobacterium tuberculosis was controversial. previous studied showed that the critical role at reactive oxidants is to serve as intracellular signals for activation of microbicidal enzymes, rather than excretions a microbicidal effect perse.the role of phagocytic respiratory burst in host defense against m. tb is further supported by recent studies discovered immunological defects secondarily affecting phagocyte respiratory burst function and resulting in primary immunodeficiencies with varied phenotypes, including susceptibilities to pyogenic or mycobacterial infections. the patients with severe pid's like scid have broader diverse infections susceptibility and mycobacterial infections as well, however, common variable immunodeficiency (cvid) mostly characterized by a deficiency of immunoglobulins and recurrent sinopulmonary infections. method: we overview the clinical rate of mycobacterial disease in our pid cases and evaluate the complex cases. results: two hundred pid cases were evaluate between - in our clinic, among % of them which diagnosed as msmd nearly all presented with mycobacterial infection. % diagnosed as cgd and interestingly % of them have been experienced mycobacterial disease sometimes in their life, as disseminated bcg or late onset complications of bcg including osteomyelitis or mtb once or more than one episode through their life. also we have presented a cvid patient with disseminated tb and granulomatouse hepatitis, tb arthritis , peritonitis and a patient with lad and nontubercolouse mycobacterial infectiouse abcesses of her skin. conclusion: pid cases like cgd, msmd or cvid which are living in area's with high prevalence of mycobacterial infection could have quiet different presentations and the study of these complex cases has provided essential insights into the functioning of the immune system. despite the conventional view we have confirmed that the generation of rois by phagocytic respiratory burst may play a role in the defense of the host against m. tuberculosis by clinical evidence. goran ristic, srdjan pasic, bojana slavkovic division of clinical immunology, mother and child health care institute of serbia, belgrade chronic granulomatous disease (cgd) is a rare disease caused by mutation in any of the five components of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase in phagocytes, resulting in recurrent, life-threatening bacterial and fungal infections of the affected individuals. our proband male patient presented at age of years with bilateral pneumonia and positive serology for aspergillus sp. the phorbol myristate acetate (pma) stimulated nitroblue tetrazolium (nbt) test showed no reduction ( %) in our patient and partial reduction ( %) in his mother. analysis of the cybb gene showed a deletion of nucleotide g (c. delg) exon causing frame shift mutation and early termination of translation (p.val serfs). this mutation was not previously described. at the moment of diagnosis, his mother was already pregnant, th week of gestation. fetal ultrasound showed that she was carrying a male fetus. the fetal blood sample, obtained by the percutaneous umbilical cord blood sampling, showed male karyotype and the pma stimulated nbt test showed % reduction. there were no complication during pregnancy or delivery and a healthy boy was born. the proband patient underwent allogenic hsct, his sister was the identical sibling donor. in the cases where the family-specific mutations are unknown, partial or complete gene deletions can be recognized by multiplex ligase-dependent probe amplification (mlpa) or array comparative genomic hybridization ( features of the patients with ar-hies dock- deficient and non-dock- deficient group are given in the table . all patients with dock- deficiency presented with cutaneous viral infections or early onset and severe atopic dermatitis. many have also food allergy and/or asthma. neurological complications and malignancy were seen in % and % respectively. sixty seven percent of patients had low t; %, low cd levels; %, high ige. the latter features were shared between patients with or without dock- deficiency, except atopic dermatitis which was mild when present in patients without dock- deficiency and ige levels were only mildly high or normal. we identified stk and coronin a deficiency in two siblings each among the patients who showed overlapping features with ar-hies and do not have dock- deficiency. our results showed that patients with dock deficiency have early onset and more remarkable eczema, food allergy, asthma, more marked eosinophilia, higher ige, low igm levels and development of malignancy. these features may be helpful in differentiation dock patients from patients without dock- deficiency. it seems routine lymphocyte subset study results are not helpful for this differentiation. alişan yıldıran , stephan borte murat elli , tunç fışgın ondokuz mayıs university, school of medicine, samsun-turkiye immunodeficiency center leipzig (idcl) at klinikum st. georg ggmbh, leipzig-germany hsct might be curative for some pids. our immunology and transplantation center was newly established. we retrospectively reviewed all children with pid who diagnosed and received hsct at ondokuz mayıs university or somewhere between june and december . twenty-two patients were identified. four of them were referred to us for hsct from other centers. the median age was months ( month- yr) at hsct. patients' diagnoses were scid (n = ), chs (n = ), leukocyte adhesion deficiency (n= ), mhc class ii deficiency (n= ), chronic granulomatous syndrome (n = ), hlh (n = ), was (n = ) and omenn's syndrome (n = ). seven patients received hla-matched related hsct; twelve haploidentical hsct and two matched unrelated hsct. one scid patient died just after her diagnosis. two patients developed bcgosis secondary to reactivation of pretransplant vaccination. one of them died due to hemophagocytic bone marrow aplasia, and the other has recovered. five patients had graft failure; two of them received no conditioning regimens because of general health status and the other because of cmv infection. at a median follow up of months (range - ), patients are alive, with overall survival of %. we conclude that; our clinic undertakes an important duty in our region for pid patients. also, different pid's could be seen in our region. nerological complication patient (epilepsy, brain infarct) outcome died (one with post -bmt comp., with malignancy all alive department of pediatrics and adolescent medicine department of allergology, rheumatology and clinical immunology, university children's hospital, university medical centre, ljubljana, slovenia. developed.there is no severe life-threatening complications development. we describe here a patient with invasive cryptococcus laurentii infection and the x-linked form of hyper-igm syndrome (x-higm). c.laurentii is an extremely rare human pathogen. this fungus was previously considered saprophytic and non-pathogenic to humans, but it has been isolated as the etiologic agent of skin infection, keratitis, endophthalmitis, lung abscess, peritonitis, meningitis, and fungemia. most affected individuals had a compromised immune system because of leukemia, cancer, diabetes mellitus, aids, or prematurity. repeated isolation of c.laurentii from the oropharynx of an immunocompromised patient has also been documented. invasive c.laurentii infection has not been reported in patients with any form of primary immunodeficiency disorder emphasizing the true rarity of disease due to this fungus. two groups have recently reported that dectin- deficiency due to the mutation of clec a or premature termination of the dectin- signal transduction molecule card may predispose patients to chronic mucocutaneous candidiasis (cmc). we studied the frequency of clec a mutation in healthy individuals, patients with the hyper-ige syndrome (hies), and patients with autoimmune polyendocrine syndrome type (aps- ), all aged between to years. genomic dna was isolated from peripheral blood. monocytes and monocyte-derived dendritic cells (mddcs) were used to study the phenotypic expression of dectin- . clec a gene was sequenced with the big dye terminator cycle sequencing kit. mononuclear cells (mcs) were isolated from heparin-treated venous blood. mdccs were obtained by culturing monocytes isolated by immunomagnetic cell separation assay. receptor expression was assessed by flow cytometry. secretion of il- a by mcs stimulated with killed c. albicans blastoconidia was assessed by elisa. we report here on healthy individuals with homozygous (one year-old man) or heterozygous ( men and women) tyr x mutation in the dectin- gene but no signs of cmc. dectin- levels on monocytes and mddcs were negligible in the homozygous man and the heterozygous individuals displayed intermediate levels of dectin- , between those of the homozygous man and the wild-type controls. markedly lower levels of il- a production were observed in the cells of the man with the homozygous mutation than in the control cells. levels of production of this cytokine were intermediate in heterozygotes. the frequency of tyr x heterozygous individuals was % among healthy donors and % in patients with hies and % in patients with aps . importantly, the year-old hies girl with heterozygous tyr x mutations has never had mucocutaneous candidiasis. we suggest that dectin- receptor-mediated immunity is redundant for host defense against cmc, possibly due to the involvement of multiple lectin receptors in the recognition and uptake of candida. chronic mucocutaneous candidiasis (cmc) is a heterogenous group with recurrent chronic candida infections spesifically involving nails, skin and oropharynx. several immunodeficiencies as dock- deficiency, severe combined immune deficiency, autoimmune polyglandular syndrome type (apeced), il- rβ and il- p deficiencies, card , stat- and stat- can cause cmc. we report here a years old boy, born to consaginous parents with onicomycosis, moniliasis, recurrent pneumonia, recurrent herpetic lesions, autoimmune thyroiditis and trombocytopenia. last admission was due to generalized tonic-clonic convulsion and mycotic aneurism on middle cerebral artery was detected. flow cytometry revealed cd lymphopenia, immunoglobulin values were in normal range. polymorphism on exon for aire gene (t/c heterozygot g g) and heterozygot stat mutation (c. > a; q h) were detected. various stat gof mutations (affecting the coiled-coin domain or the dna-binding domain) have been systematically associated with susceptibility to cmc. autoimmune manifestations associated with stat- mutations have been attributed to increased type interferon. the aneurism formation is not elucidated whether it's due to candida infection or vascular damage directly affected by stat- mutation. purpose: chronic granulomatous disease (cgd) is a rare genetic disease of phagocytic system. affected patients commonly present with bacterial infections associated with pneumonia, abscesses and lymphadenitis. in this study, we investigated the clinical and laboratory findings of our cgd patients. materials and methods: the demographic data (age at diagnosis, initial presenting symptoms, family history, follow-up period), mutation analysis, therapy options, complications, radiological findings and prognosis were evaluated retrospectively. results: among cgd patients, autosomal recessive form was detected in of them. the age at onset was statistically lower in x' linked cgd patients than ar form ( . ± . mo vs . ± . mo; p = . ).respiratory tract infections (sinusitis, otitis, pneumonia) and recurrent abscesses were more commonly seen at onset. microbiological culture revealed a. fumigates from lung biopsy in one patient and s.marcescens from blood specimen in other ones. bcgitis was observed in one patient and five patients received anti-tb therapy. non-infectious complications were granulomatous uveitis, recurrent pericardial effusion, skin granuloma, noduler formation in lung and brain area. conclusion: due to high rate of consanguinity, autosomal recessive inheritance was observed highly in our patient cohort. since, patients with cgd are susceptible to tuberculosis and bcg complications; initiation of tuberculosis prophylaxis is advisable in countries where bcg is still administrated at birth. key words: chronic granulomatous disease, consanguinity, bcg. acquired immunodeficiency research center, isfahan university of medical sciences, isfahan, iranbackground: defects of the immune system in primary immunodeficient diseases (pids) predispose individuals to recurrent infections. complex genetic components for susceptibility to mycobacterial disease have been suggested. natural human immunity to the mycobacteria group, including mycobacterium tuberculosis(mtb), bacille calmette-guérin (bcg) or nontuberculous mycobacteria (ntm) relies on the functional il- / -ifn-γ integrity of macrophages (monocyte/dendritic cell) connecting to key: cord- -qau gvw authors: willermain, françois; van laethem, yves; caspers, laure title: global variations and changes in patterns of infectious uveitis date: - - journal: emerging infectious uveitis doi: . / - - - - _ sha: doc_id: cord_uid: qau gvw before , most uveitis cases were supposed to be due to infectious agents, mainly syphilis or tuberculosis [ ]. progress in the understanding of intraocular inflammation led to the discovery that uveitis can be of infectious and noninfectious origin and that many pathogens can cause infectious uveitis. theoretically, koch postulates must be fulfilled, in order to formerly demonstrate that a disease is due to an infectious agent. however, in infectious uveitis, most often, serological evidence, molecular or histological demonstration, and treatment response are usually the only available elements to suggest the infectious origin of the uveitis. using those evidences, a large number of infectious organisms have been demonstrated to cause infectious uveitis. some have a global importance around the world, while others have more limited niches. many of them have been considered as emerging pathogens. before , most uveitis cases were supposed to be due to infectious agents, mainly syphilis or tuberculosis [ ] . progress in the understanding of intraocular inflammation led to the discovery that uveitis can be of infectious and noninfectious origin and that many pathogens can cause infectious uveitis. theoretically, koch postulates must be fulfilled, in order to formerly demonstrate that a disease is due to an infectious agent. however, in infectious uveitis, most often, serological evidence, molecular or histological demonstration, and treatment response are usually the only available elements to suggest the infectious origin of the uveitis. using these evidence a large number of infectious organisms have been demonstrated to cause infectious uveitis. some have a global importance around the world, while others have more limited niches. many of them have been considered as emerging pathogens. according to woolhouse, an emerging pathogen can be defined as an infectious agent whose incidence is increasing following its introduction into a new host population. a pathogen will be reemerging when its incidence increases in an existing host population [ ] . however, in practice, both expressions are likely used by many authors regardless of those biological and epidemiological criteria. the term emerging disease will thus also be used in situation of increase awareness or discovery of pathogen in previously supposed non-infectious diseases [ ] . as far as we know, most emerging infectious uveitis agents fall into the two last categories. emerging disease by definition, the question of emerging infectious disease addresses how a microbe becomes a pathogen in the human species. emerging infectious disease thus resumes the origin of human infectious disease. most of the emerging infections discovered during the last decades are zoonosis. major modifications in human behavior have facilitated their jump from animals to humans. the global population has increased from million humans around to . to . billion in , billion in , and more than billion in . more than % of those people reside in urban areas (from % in africa to % in the americas), in contrast with only % in . environmental modifications were mandatory to build those cities, with deforestation or other forms of land use than in the past. therefore, and also linked to widespread international trade of goods and animals, microbes have received a new and rapid access to an ever larger human population. in addition, the population is globally more mobile also; international travel rose above one billion people in , further increasing pathogen dissemination around the globe. in the past decades, most new pathogens were acute respiratory viruses. for instance, human metapneumovirus (first reported in in the netherlands but demonstrated thereafter in specimens stored since the s at least) is a paramyxovirus leading to very frequent and sometimes very severe respiratory tract infections in small children [ ] . a new betacoronavirus is the agent of sars (severe acute respiratory syndrome), discovered in after a physician infected a dozen patients in hong kong, with subsequent more than , cases in countries and deaths [ ] . another new coronavirus (mers-cov, middle east respiratory syndrome coronavirus) has been demonstrated as the causative agent of a deadly respiratory tract infection in the middle east since , with more than , cases and deaths [ ] . but of all these emerging pathogens, the flu viruses, are the most widespread and deadly. influenza a is a zoonose, with viruses mutating and mixing in swine and birds. if the spanish flu (new h n influenza a virus) killed more than million people in - , several pandemics (h n in , h n in , and new h n in ) appeared in the more recent decades, with a lower but consequent mortality. in more restricted areas, non-pandemic strains of influenza a as h n (asia and egypt) or h n (china) are still potential deadly sword of damocles [ ] . one of the most severe emerging infections of the last centuries is obviously aids (acquired immu-nodeficiency syndrome) linked to the hiv (and rarely hiv ) [ ] . if it was first recognized in in the united states, its emergence in the human world is older and faraway from our western world. indeed, the oldest known case (a posteriori) is a man in the present democratic republic of congo, in . analysis of the virus in his frozen serum as well as others suggests that hiv and hiv were separately acquired from monkeys in the s, in the western part of central africa. in more than siv (simian immunodeficiency viruses) presently known, only hiv and hiv were able to infect humans and to establish persistent human to human transmission. here again, urbanization and increasing local/regional trade, with all the sociologic modifications that it implicated, have largely contributed to the dissemination of the disease in sub-saharan africa. in a second time, international travel, sexual behaviors, and iv drug abuse have been keystone factors in the worldwide propagation of aids [ ] . if emerging disease brings us to the origin of infectious disease, re-emerging disease deals with the evolution of infectious disease and the impact of the human society on it. most of those reemerging infections are also zoonoses, often linked to a vector. climatic changes (global warming) have allowed ticks to reach higher latitudes as well as altitudes, with transmission of lyme disease or tick-borne encephalitis more in the north of scandinavia or at higher altitude in central europe [ ] [ ] [ ] . similarly, mosquitos as aedes albopictus are now found in the south of europe (as france and italy), with several local transmissions during the summer of dengue or chikungunya from imported cases. chikungunya virus was discovered years ago in tanzania and since that time has spread to several parts of africa and in all indian ocean/western pacific countries [ , ] . due to the introduction by an international traveler of an asian strain in the caribbean in , chikungunya is now an important public health problem not only in those islands but already also in central and south america [ ] . west nile virus infection was unknown in the americas until . due to migration from the old world of birds wearing infected ticks, the infection was introduced in the northeast of the united states, and in a few years, it spread to all the states [ ] . few studies, and mainly from the occidental world, address the incidence and prevalence of uveitis [ ] . it is therefore difficult to raise conclusions on global variations or evolution of this epidemiological aspect of intraocular inflammation. this contrasts with the important literature describing the causes of uveitis in different center and location in world which clearly shows important variations of the distribution of different etiologies around the globe. those differences are mainly due to genetic and environmental factors and often grouped between the so-called developed and developing worlds. accordingly, the distribution of both specific infectious and noninfectious causes varies greatly around the world. in the context of infectious uveitis, some uveitis type is logically limited to endemic regions. onchocerciasis, for example, has a limited distribution in africa, south america, and yemen [ ] . lyme disease is almost exclusively found in the northern hemisphere. leptospirosis occurs most frequently in tropical and subtropical area. brucellosis remains prevalent in the developing world, mostly in the mediterranean basin, the arabic gulf countries, india, and central america. htlv- infection is endemic in the caribbean, central and south america, south and intertropical africa, and japan. similarly other infectious agents such as dengue, west nile virus, rift valley fever, or chikungunya virus, as well as rickettsia only infect patients in limited endemic regions. there are thus only reported as causes of uveitis in studies from those regions. in the series of rathinam sr and namperumalsamy from india, leptospirosis was the most frequent cause of infectious uveitis but remains very rare in the united states and europe [ , ] . however, due to evolution in our societies, such as globalization, those causes of infectious uveitis begin to emerge in non-endemic regions in patients having traveled in endemic regions (see sect. . . . ) [ ] . in contrast some organisms have spread worldwide, some with a relative stable incidence and others with period of increase and/or decrease incidence. for example, across the world, toxoplasmosis and herpesvirus remain major causes of posterior and anterior uveitis, respectively [ ] [ ] [ ] [ ] [ ] [ ] . tuberculosis and syphilis are discussed in the next paragraph as classic examples of worldwide cause of uveitis with period of burden and decrease. an important example of decreased incidence of a ubiquitous infectious uveitis is cmv retinitis which made a steep decline in incidence following the introduction of haart [ ] . in europe, it is believed that syphilis has emerged around . interestingly, it has been reported that in its early years, the disease was much more severe that nowadays, suggesting the selection of a milder strain of treponema pallidum occurred. since that time, syphilis has continued to spread around the world and became one of the major health problems, illustrated by the fact that, in the nineteenth century, an entire medical subspecialty, syphilology, was devoted to its study [ , ] . the discovery of penicillin has been associated with a significant decrease of syphilis rate to the point that some authors have postulated that the disease might disappear. unfortunately, this was not the case and the incidence of syphilis has been the subject of important variation with frequent outbreak [ , ] . for example, in the united kingdom, there has been a % increase in the incidence of syphilis between and . this exponential increase has been attributed to unsafe sexual practices mainly among men who have sex with men (msms) [ ] . the same trend was found in other countries. as a consequence, many reports have warned the uveitis community of what was called by narsing rao and colleague "the reemergence of an old adversary" [ , ] . meanwhile, a tremendous number of studies have been published improving our knowledge on the epidemiology and clinical presentation of ocular syphilis. acute syphilitic posterior placoid chorioretinitis was in this context rediscovered with an exponential rate of publication from , the year of its publication by gass, to [ ] . tuberculosis is another old infectious disease which had a major impact on global human health. overall, the worldwide burden of tuberculosis is still growing, as control of the disease in many regions of the world is offset by the increase incidence in another part, mainly sub-saharan [ ] . tuberculosis remains one of the most important infectious causes of morbidity and mortality worldwide. in contrast with syphilitic uveitis, where the diagnosis can be easily made based on serological evidences, there is a great confusion regarding the diagnosis and treatment of ocular tuberculosis. progress in systemic and ophthalmological investigation together with a more accurate description of clinical signs has permitted to better define guidelines for the diagnosis and treatment of intraocular tuberculosis [ , ] . nowadays, tuberculosis is a leading cause of uveitis in endemic countries, but tuberculosis uveitis can also be found in non-endemic countries with a probable recent increased frequency [ , [ ] [ ] [ ] . this recent increment of tuberculosis uveitis in non-endemic countries is mainly attributed to the development of immigration and postulated by llorenc and coworkers to be one of the challenges of globalization [ , ] . there is thus an increased awareness of ocular tuberculosis among uveitis specialists all around the globe. one of the major recent breakthroughs in the uveitis field was the discovery that two entities, namely, posner-schlossman syndrome (pss) and [ ] . a possible role played by cmv infection in the development of pss was suggested by early work of bloch-michel in the eighties [ ] . since that time, several works have confirmed this hypothesis [ , , ] . in addition to pss, it was found in those studies that cmv-positive anterior uveitis can also present the clinical characteristics of fhi or chronic granulomatous uveitis. altogether, those data indicate that several previously thought idiopathic uveitis (pss, fhi, and some chronic granulomatous anterior uveitis) have indeed a viral origin. this evidence has not only important implications for the epidemiology but, of course, also for the management of uveitis. we have seen earlier that the success of humanity in terms of demographic expansion has create favorable conditions to increase the speed for the emergence and spreading of infectious diseases. in other terms, diseases might quickly jump between very distant part of the world and confront clinicians with diseases unusual in their region. fortunately, the dissemination of information has also been progressively accelerated allowing a quick exchange of information between specialist from endemic regions and recently affected countries. in this context, a series of infectious uveitis, mainly rickettsioses, west nile virus, dengue, or chikungunya, has been the subject of an increase awareness and careful descriptions from both endemic and non-endemic regions [ , , ] . the recent outbreaks of ebola and zika virus have been similarly associated with uveitis cases and those pathogens should be now included in the list of emerging infectious uveitis agents [ , ] . the epidemiology of infectious uveitis is a dynamic process and the consequence of the complex relationship between microbes and human. on one hand, some pathogens such as toxoplasmosis or herpesvirus remain major causes of uveitis, while others, such as tuberculosis, seem to progress despite our efforts to eradicate them. on the other hand, infectious uveitis previously limited to particular geographical niches can now be found almost all around the globe. this is clearly due to evolution of our lifestyle which has also important impact on the emergence of new infectious diseases which might become someday new uveitis causes. the decrease of cmv retinitis among aids patients following haart highlights that, in addition to this negative aspect, our civilization also has a positive impact on infectious uveitis epidemiology and is able to reduce the incidence of some devastating infectious uveitis causes. indeed, we should not forget that the development of our human society has also created better ways to diagnose, control, and eventually eradicate infectious diseases. diagnosis and treatment of uveitis. wb sauders company population biology of emerging and re-emerging pathogens? human metapneumovirus: a ubiquitous and long standing respiratory pathogen consensus document on the epidemiology of sars severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) th update ecdc. communicable disease threats report isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) reflections on years of aids joint factsheet on lyme borreliosis global climate change and infectious diseases chikungunya in europe: what's next? chikungunya: a re-emerging virus chikungunya outbreak in caribbean region global trends in emerging infectious diseases epidémiologie. in: brezin ap (ed) les uvéites. société française d'ophtalmologie elimination of onchocerciasis from africa: possible? global variation and pattern changes in epidemiology of uveitis leptospiral uveitis in the developing world emergent infectious uveitis epidemiology of ocular toxoplasmosis causes of uveitis at the eye center in saudi arabia: a retrospective review pattern of uveitis in a referral centre in tunisia etiology and clinical features of ocular inflammatory diseases in a tertiary center in lebanon uveitis subtypes in a german interdisciplinary uveitis centeranalysis of patients pattern of uveitis in a university-based referral center in southern thailand declining incidence of aids-defining opportunistic illnesses: results from years of populationbased aids surveillance syphilis in renaissance europe: rapid evolution of an introduced sexually transmitted disease? syphilis in the united states syphillis: a reemerging infection british ocular syphilis study (boss): -year national surveillance study of intraocular inflammation secondary to syphilis syphilis: reemergence of an old adversary syphilitic uveitis acute syphilitic posterior placoid chorioretinitis intraocular tuberculosis -an update classification of intraocular tuberculosis patterns of uveitis in patients admitted to a university hospital in riyadh, saudi arabia the manchester uveitis clinic: the first patients-epidemiology and casemix epidemiology of uveitis in a western urban multiethnic population. the challenge of globalization fuchs heterochromic cyclitis: rubella virus antibodies and genome in aqueous humor rubella virus-associated uveitis: clinical manifestations and visual prognosis epidemiologic relationship between fuchs heterochromic iridocyclitis and the united states rubella vaccination program clinical features of cytomegalovirus anterior uveitis in immunocompetent patients associations of fuchs heterochromic iridocyclitis in a south indian patient population possible role of cytomegalovirus infection in the etiology of the posner-schlossman syndrome long-term oral therapy with valganciclovir in patients with posner-schlossman syndrome cytomegalovirus anterior uveitis: long-term follow-up of immunocompetent patients west nile virus-associated optic neuritis and chorioretinitis dengue eye disease ebola virus disease and the eye uveitis associated with zika virus infection the cause of infectious uveitis varies greatly around the world. some widespread microbes continue to threaten vision in almost every part of the globe. some infectious uveitis previously limited to particular geographical niches can now be found almost all around the globe. this evolution is the consequence of changing in our lifestyle which has also important impact on the emergence of new infectious diseases as well as their diagnosis and management. key: cord- -ju bq authors: last, john title: a brief history of advances toward health date: journal: understanding the global dimensions of health doi: . / - - - _ sha: doc_id: cord_uid: ju bq three major discoveries determined the health and history of the human species. the first occurred almost a million years ago, when our hominid precursors discovered how to use fire to cook the meat they had hunted. they found that cooked meat tasted better, it didn’t go bad so quickly, and eating it was less likely to make them ill. our understanding of nutrition, a basic tenet of public health science, and the art of cooking have been improving ever since. as humans grew fruitful and multiplied, so did the variety and number of their diseases. permanent human settlements transformed ecosystems, and abiding by epidemic theory, the probability of respiratory and fecal-oral transmission of infection rose as population density increased. ecological and evolutionary changes in micro-organisms account for the origins of diarrhea , measles, malaria, smallpox, plague, and many other diseases. micro-organisms evolve rapidly because of their brief generation time and prolific reproduction rates. many that previously had lived in symbiosis with animals began to invade humans, where they became pathogenic. some evolved complex life cycles involving several host species, such as humans and other mammals, humans and arthropods, and humans and freshwater snails. these evolutionary changes in host-parasite relationships occurred at least several millennia before we had created written histories. our oldest written records that have a bearing on health date back about , years. the code of hammurabi (c. bce) contains ideas indicative of insight into the effects on health of diet and behaviour. it also suggests rewards and punishments for physicians who did their jobs well or poorly. information about the impact of diseases, especially of epidemic diseases, from those ancient times has come down to us in myths and biblical accounts of pestilences and plagues, although we cannot reliably identify the nature of the epidemics that afflicted ancient populations. the greek historian thucydides provided a meticulously careful description of an epidemic that struck the athenians in the second year of the peloponnesian war in bce, from which the forces of athens perhaps never fully recovered. modern infectious disease specialists have puzzled over this epidemic. was it typhus, a virulent form of epidemic streptococcal infection, that is, a variant form of scarlet fever, or something completely different? similar questions have been raised about other ancient epidemics, for instance the sweating sickness that recurred many times in mediaeval europe then vanished, never to be seen again, almost , years ago. there has been debate too about the exact nature of the black death, the terrible pandemic that devastated asia minor and the whole of europe in - . this is usually attributed to the plague bacillus, yersinia pestis, but revisionist historians and epidemiologists have raised the possibility that other pathogens, for instance, the anthrax bacillus, might have been responsible. here, as with the plague of the athenians, the plague of justinian, the medieval sweating sickness, the accounts of apparent fulminating epidemic syphilis (that may really have been another sexually transmitted disease or may have been caused by a highly virulent variant of the causative organism of syphilis, treponema pallidum, which has slowly lost its extreme virulence and infectivity), and, indeed, as with all other great epidemics of historical times before the rise of modern microbiology, we can only speculate about the exact aetiology and pathogenesis. this is a rather sterile, albeit fascinating, quest. it is more productive and useful to focus on what we know with reasonable certainty, and it is simplest to describe this knowledge in relation to some of the heroic figures who have contributed to advances in our understanding of epidemics and other diseases that have helped to shape history. this account therefore concentrates on a handful of the heroes of public health through the course of written history. hippocrates , the father of medicine, was also the father of public health. he practised and taught in a school of medicine at the temple of asklepios, near epidaurus in greece, and alone or with members of his school, laid the foundations of rational clinical medicine with careful descriptions of diseases and common sense ideas about ways to manage them. the hippocratic writings contain rich medical wisdom based on careful observation of sick and healthy people and their habits and habitats. epidemics is a series of case records of incidents of diseases, many of which we now know to be caused by infectious agents. the accounts of tetanus, rabies, and mumps, for instance, could have been written by a modern clinician. airs, waters, places outlines environmental health as it was understood two-and-a-half thousand years ago. the relationships of environment, social conditions, and behaviours to health and sickness is made explicit in the timeless advice of the opening paragraph: whoever would study medicine must learn of the following. first, consider the effect of each of the seasons . . . and the differences between them. . . . study the warm and cold winds . . . and the effect of water on health . . . when a physician comes to a district previously unknown to him he should consider its situation and its aspect to the winds . . . and the nature of its water supply . . . whether the land be bare and waterless or thickly covered with vegetation and well-watered, whether in a hollow and stifling, or exposed and cold. lastly, consider the life of the inhabitants-are they heavy drinkers and eaters and consequently unable to stand fatigue, or being fond of work and exercise, eat wisely but drink sparely. in short, study environment and life style, which are very modern concepts. for well over a thousand years after hippocrates' lifetime, human communities were afflicted with ever-present respiratory and gastrointestinal infections that cut deeply into the lives of everyone, and most deeply, as a rule, into the lives of young children who all too often died before they reached adolescence, carried off by measles, scarlet fever, diphtheria, bronchitis, croup, pneumonia, gastroenteritis, or typhoid. from time to time, this steady drain on long life and good health was punctuated by great and terrifying epidemics-smallpox, typhus, influenza, and, most terrible of all, the plague, or the "black death." the causes of these periodic devastations, the contributing reasons to why they happened, were a mystery. many at the time believed they were god' s punishment for sin, or the work of evil spirits. ideas about contagion were rudimentary, even though it had been dimly understood since antiquity that leprosy, perhaps the least contagious of all the infectious diseases, was associated with propinquity and uncleanliness. the th century italian monk, fracastorius, recognized some ways infection can spread. his conclusion, that disease could pass by intimate direct contact from one person to others, was easy to draw because he saw the dramatic epidemic of syphilis that was so obviously spread by sexual intercourse. he described this in a mock heroic poem, syphilis, sive morbis gallicus ( ) about the swineherd syphilis, and how he got and passed on to others the "french disease" then raging in europe. his anti-hero, of course, gave us the name of the disease. fracastorius' s other concepts, contamination by droplet spread and by way of shared contaminated articles, such as clothing and kitchen utensils, were published in de contagione in . fracastorius is important because he made a conceptual breakthrough-he brought about what thomas kuhn calls a paradigm shift in understanding of infection and some ways to control it. after fracastorius, the pathfinders on the road to health became numerous, but mention here will be made of only a handful of public health heroes: paracelsus, john graunt, antoni van leeuwenhoek, bernardino ramazzini, james lind, edward jenner, johann peter frank, john snow, ignaz semmelweiss, and louis pasteur. the swiss alchemist theophrastus bombastus von hohenheim, known as paracelsus ( - ), occupies the junction of medieval alchemy with scientific chemistry, pharmacy, medicine, and environmental health. he was a colourful character, a foul-mouthed drunkard who insulted and sometimes fought those who disagreed with him, whom he considered superstitious nincompoops. he recognized the relationship of goitre to cretinism, the fact that inhaled dusts caused lung disease, and that some common mental disorders were diseases, not caused by witchcraft or 'possession' by evil spirits. he experimented with chemical remedies containing compounds of mercury, lead, and other galenicals, observed their effects, and, thus, could be considered also a founding figure of pharmacology. john graunt ( graunt ( - , a london merchant haberdasher, was an amateur scientist and an early fellow of the royal society. he was interested in the impact of epidemics, especially the plague, and how plague outbreaks caused the numbers of deaths, and the age at death, to vary from one year to another. for over years before his time, parishes had kept records of baptisms and deaths, and what was then understood about causes of death was inscribed in the bills of mortality. graunt collected and analyzed these bills of mortality. he demonstrated statistical differences between males and females, between london and rural areas, and the ebb and flow of epidemics of plague. he published his work in natural and political observations ... upon the bills of mortality (london, ). this work was the foundation for the science of vital statistics. john graunt demonstrated the importance of gathering facts in a systematic manner, to identify, characterize and classify health conditions of public health importance. the diagnostic categories in the bills of mortality tell us what was understood years ago about the variety of human ailments and their causes. the nature of diseases caused by things not visible to the naked eye was long a mystery that began to unravel when antoni van leeuwenhoek ( - ), a dutch linen draper and amateur lens-grinder in delft, perfected the first functioning microscopes, with which he viewed drops of water, vaginal secretions, feces, his own semen, and the detailed structures of plants and insects. he lacked any formal scholarly training but in a series of letters to the royal society of london, he described accurately and in detail all that he saw. he did not suggest that the tiny creatures he was the first to see with his microscope were capable of causing diseases, but he is nonetheless regarded as the first of the 'microbe hunters' who sought and identified the pathogenic micro-organisms responsible for many diseases. bernardino ramazzini ( - ) was an italian physician who observed and classified workers in many occupations, and reported his observations and conclusions about the diseases to which workers in each of these were vulnerable in de morbis artificum diatribe (on the diseases of workers, ). it is a tour de force, a masterly account in the form of sweeping generalizations, and although the evidence supporting these generalizations was often flimsy, ramazzini introduced a new way of thinking about ways in which work conditions can affect health. james lind ( lind ( - was born and educated in edinburgh. he was apprenticed to a surgeon when he was , and spent nine years as a naval surgeon, during which time he saw many cases of scurvy, a disease that disabled and often killed sailors on long ocean voyages. lind thought this disease might be caused by a diet lacking fresh fruit and vegetables. he conducted an experiment, giving different diets to each of several pairs of sailors. this was the first clinical trial ever conducted-although the sample sizes were very small, there was no random allocation, and no informed consent was obtained from the sailors. the two sailors who received fresh oranges and lemons recovered rapidly from the scurvy, the others did not, or got worse. lind also initiated the first effective measures aimed at enhancing hygiene in the british navy, but he is best known for his work on scurvy, reported in a treatise of the scurvy ( ). not only was this the first reported clinical trial, it also was proof that a dietary deficiency can cause disease, that a well-balanced diet is essential for good health. thus lind, like fracastorius, was responsible for an important paradigm shift in the understanding of causes and control of disease. johann peter frank ( - ) studied medicine in heidelberg and strasburg, was a professor of medicine at göttingen and pavia, and taught in many other centres of learning including st petersburg, before he ended his career in vienna where he was professor of medicine at the allegemeines krankenhaus. early in his career he began writing system einer vollständigen medicinischen polizey, his great work on ways to improve population health. this appeared in a series of nine volumes from to . it was, as the title indicates, a system dealing with every then-known way to protect and preserve good health, including community hygiene, personal health protection by cleanliness, and a suggested set of laws and regulations to govern the control of conditions in lodging houses and inns, medical inspection of prostitutes, and so on. edward jenner ( jenner ( - was an english family doctor who practised throughout his life in the village of berkeley, gloucestershire. in his days, smallpox was a ubiquitous threat to life and health. in severe epidemics, it killed up to a quarter of all it attacked. when it did not kill, it often left disfiguring facial pockmarks and if it infected the eyes it caused blindness. the practice of variolation, inoculation into the skin of dried secretions from a smallpox bleb, was invented in china about years ago and spread along the silk route, reaching asia minor in the th century. lady mary wortley montague, wife of the british ambassador to constantinople, described the practice in a letter dated april , , and imported the idea to england when she came home. by the time jenner was a child, variolation had become popular among educated english families as a way to provide some protection against smallpox. jenner knew the popular belief in gloucestershire that people who had been infected with cowpox, a mild disease acquired from cattle, did not get smallpox. he reasoned that since smallpox in mild form was transmitted by variolation, it might be possible similarly to transmit cowpox. a smallpox outbreak in gave him an opportunity to confirm this notion. in he began a courageous and unprecedented experiment-one that would now be unethical, but that has had incalculable benefit for humankind. he inoculated a boy, james phipps, with secretions from a cowpox lesion. in succeeding months, until the summer of , he inoculated others, most of them children, to a total of . all survived unharmed, and none got smallpox. jenner published an inquiry into the causes and effects of the variolae vaccinae in -perhaps the most influential public health treatise of all time. the importance of jenner' s work was immediately recognized and although there were sceptics and hostile opponents, vaccination programs began at once. the frequency and ferocity of smallpox epidemics began to decline early in the th century, but the disease remained a menace until the mid- th century. in , the american epidemiologist donald soper worked out the strategy of containment, in other words, vaccinating all known contacts of every diagnosed case. in , who embarked on a global campaign to eradicate smallpox. the last naturally occurring case was a girl in somalia in . in , the world health assembly proclaimed that smallpox, one of the most deadly scourges of mankind, had been eradicated. at the beginning of the new millennium, samples of smallpox virus are preserved in secure biological laboratories in several countries, but, thanks to edward jenner, this terrible disease need never again take a human life-unless it is used illegally in biological warfare. john snow ( - ) was a london physician, and a founding father of modern epidemiology. (he was also a pioneer anesthetist who invented a new kind of mask to administer chloroform, which he gave to queen victoria to assist at the births of her two youngest children.) snow' s work on cholera demonstrated fundamental intellectual steps that must be part of every epidemiological investigation. he began with a logical analysis of the available facts, which proved that cholera could not be caused by a 'miasma' (emanations from rotting organic matter) as proposed in a theory popular at that time, but must be caused by a transmissible agent, most probably in drinking water. he confirmed the proof with two epidemiological investigations into the great cholera epidemic of . he studied a severe localized epidemic in soho, using analysis of descriptive epidemiological data and spot maps to demonstrate that the cause was polluted water from a pump in broad street. his investigation of a more widespread epidemic in south london involved an inquiry into the source of drinking water used in over households. he compared the water source in houses where cholera had occurred with that in others where it had not. his analysis of the information about cases and their sources of drinking water showed beyond doubt that the cause of the cholera outbreak was water that was being supplied to houses by the southwark and vauxhall water company, which drew its water from the thames downriver, where many effluent discharges polluted the water. very few cases occurred in households supplied with water by the lambeth company, which collected water upstream from london, where there was little or no pollution. john snow reasoned correctly that the cholera must be caused by some sort of agent in the contaminated water supply. this was a remarkable feat, completed years before robert koch identified the cholera bacillus. snow published his work in a monograph, on the mode of communication of cholera ( ). this book has been reprinted in modern editions and is still used as a teaching text. the hungarian physician ignaz semmelweiss ( - ) was a great but tragic figure. working in the obstetric wards of the allgemeines krankenhaus in vienna, he tried to transform traditional but ineffective treatment methods by using logic and statistical analysis to demonstrate the efficacy, or lack of it, when he compared treatment regimens. he believed in the germ theory of disease and was convinced that the terrible death rates from puerperal sepsis must be caused by germs introduced into the raw uterine tissues by birth attendants who did not disinfect their hands. he carried out a meticulous comparative mortality study in his own wards, where he insisted that all birth attendants must cleanse their hands in a disinfectant solution of bleach, and other wards run by senior obstetricians where hand-washing was not routine. his senior colleagues regarded his findings as a gross insult to their professional competence. semmelweiss' s rather abrasive nature and his jewish origins in the anti-semitic atmosphere of th century vienna made matters worse for him. he was hounded out of his hospital post, and ended his life in a mental hospital. his belatedly published comparative statistical analyses of the death rates from puerperal sepsis in his own and other wards of the allgemeines krankenhaus are a model of how to conduct such studies, but, unfortunately, no one in vienna heeded him and young women continued to die of childbed fever for another generation. medical science advanced rapidly in the second half of the th century, applying the exciting discoveries of a new science, bacteriology, which transformed public health. the great bacteriologists of the late th century identified many pathogenic bacteria, classified them, developed ways to cultivate them, and, most important, worked out ways to control their harmful effects, using sera, vaccines, and "magic bullets" such as the arsenical preparations that ehrlich developed to treat syphilis. it would be useful to discuss each of them, but i will focus on just one, louis pasteur ( - ). this french chemist evolved into a bacteriologist, and was a towering figure of th century bacteriology and preventive medicine. in , he had recently been appointed professor of chemistry in lille, and was invited to solve the problem of aberrant fermentation of beer that caused it to taste bad and made it undrinkable. he showed that the problem was caused by bacteria that were killed by heat. in this way he invented the process for heat treatment to kill harmful bacteria, first applied to fermentation of beer, then to milk-the process known ever since as pasteurization that has saved innumerable children from an untimely death. he went on to study and solve many other bacteriological problems in industry and animal husbandry. he developed attenuated vaccines, first to prevent chicken cholera, then, in , to control anthrax, which was a serious threat to livestock and, as well, occasionally to humans. before this, in , he began experiments on rabies, seeking a vaccine to control this disease, which without treatment is invariably fatal. as a result of the success of the anthrax vaccine, he believed that an attenuated rabies vaccine could be made. this, of course, was many decades before the virus was visualized. he prepared and successfully tested his rabies vaccine in on a boy, joseph meister, who had been bitten by a rabid dog. pasteur became not just a national but an international celebrity. born in the same year as louis pasteur, the austro-hungarian monk gregor mendel ( - ) was another amateur scientist, a botanist. experimenting with varieties of garden peas, he cross-pollinated them and observed and recorded the results. unfortunately, he published his findings in an obscure journal where they remained un-noticed for many years, but when they were unearthed about years after his death, gregor mendel was retroactively honoured as the father of a new science, genetics, which soon found many applications in clinical medicine, with the recognition of the fact that many inherited diseases were caused by genetic disorders. almost years after mendel' s death, other discoveries with great public health relevance include development of genetically modified sterile insect vectors of disease, genetically resistant strains of rice, wheat, and so on, and applications of genetic engineering to limit and even control and prevent some recessive inherited disorders. pasteur, henle, koch, virchow, and, soon after, battalions of bacteriologists and pathologists firmly established the fact that micro-organisms caused many diseases-the germ theory was a proven fact, not theory. however, many germ diseases require much more than germs before they can cause their worst damage. tuberculosis is caused by the tubercle bacillus acting in conjunction with poverty, ignorance, overcrowding, poor nutrition, adverse social and economic circumstances, and other enabling and predisposing factors. the diarrheal diseases, including cholera, are caused by various microorganisms, but these get into the gut when ingested with contaminated water or food, that is, they are really caused by poor sanitary and hygienic practices. by late in the th century, many of these factors had been clarified. the stage was set for the health reforms that included the sanitary revolution, the beginnings of a social safety net, provision of immunizations, nutritional supplements for school children, prenatal care for pregnant women, and other essential public health functions we take for granted years later. it required a dedicated army of public health workers to achieve all this. i have singled out and mentioned a mere handful of the public health pathfinders on the road to good health. many others could be added, but that would turn this brief chapter into a weighty monograph. often the physician−pathfinders used their own patients as experimental subjects for their path-finding discoveries. lind' s sailors, jenner' s young friends starting with james phipps, pasteur' s patient joseph meister, and all others known and unknown by name who provided the material for the great discoveries of robert koch and other members of the austrian and german schools of bacteriology, should be remembered and honored too. many others belong in their company: the great german pathologist rudolph virchow recognized that political action as well as rational science are necessary to initiate effective action to control public health problems; edwin chadwick and lemuel shattuck reported on the appalling sanitary conditions associated with the unacceptably high infant and child death rates that prevailed in th century industrial towns; william farr established vital statistics in england as a model for other nations to follow. and so the list grows from a handful of public health pathfinders to whole armies. more was needed than scientific discoveries. such discoveries had to be applied, and this often required drastic changes in the established social and economic order. so, other pathfinders appear on the road to health. they include politicians, administrators, journalists, creative writers, performing artists, and cartoonists. the journalists, creative writers, and artists who transmit the scientific concepts of public health to the general public and to the politicians are indispensable partners in the team that makes it possible for us to advance up the road to better health. the process continues in modern times with investigative journalism and tv documentaries. i have identified five essential ingredients of the processes that brought about the public health reforms called the sanitary revolution of the late th and early th century, and have shown that these five features are essential for the control of all public health problems. . awareness that the problem exists. john graunt began this process with natural and political observations. others consolidated his conceptual breakthrough, and it was applied to great effect after the establishment of formal national vital statistics in england and wales under the inspired leadership of william farr. by farr' s time, widespread literacy, the proliferation of daily newspapers, and word of mouth helped to enhance awareness among thoughtful people everywhere that there were massive public health problems in society at that time. modern computer-based record-keeping and effective health information systems with instantaneous worldwide notification of contagious disease outbreaks with public health significance continue to enhance the process. . understanding the causes. in the second half of the th century, understanding rapidly increased, as epidemiology and bacteriology, and nutritional and environmental sciences explored previously unknown landscapes of aetiology and pathogenesis. the new mass media-daily newspapers-propagated this understanding among literate people throughout the country. from the middle of the th century, news magazines and tv have ensured that knowledge of causal connections-smoking to cancer, diet and lack of exercise to coronary heart disease, alcohol-impaired driving to traffic fatalities, and many more-are very widely disseminated. this, however, has not necessarily led to effective control measures. . capability to control the causes. with astonishing speed, once the initial breakthroughs had occurred, sera and vaccines were developed to control many of the lethal microbial diseases that had plagued earlier generations. improved dietary practices, pasteurization of milk, improved personal hygiene and, above all, environmental sanitation to rid drinking water of polluting pathogens, all advanced rapidly in the final quarter of the th century and the first few decades of the th century. thus many ancient infectious disease scourges have been controlled, most dramatically being, perhaps , the eradication of smallpox. unfortunately, new infectious pathogens including the human immunodeficiency virus, viral tropical haemorrhagic fevers, the coronavirus of severe acute respiratory syndrome, and a score or more of others, have emerged to take their place . . . this is an essential prerequisite to the determination to act upon the problem. it is the most fascinating and challenging aspect of the essential features. this belief is the moral imperative that drives public health reforms. geoffrey vickers described the history of public health as a process of redefining the unacceptable-an endless process of identifying conditions, behaviors, and circumstances that individuals, communities, and cultures must no longer tolerate. throwing the contents of the chamber pot into the street, clearing one' s nostrils on the tablecloth, coughing and spitting on the living-room floor, all became unacceptable in the late th century. many people outside the boundaries of traditional medical science and public health practice played a role in this process. in the era of the great reforms of the th century, they included social reformers like edwin chadwick, journalists like henry mayhew and charles kingsley, novelists like charles dickens, cartoonists in punch and other periodicals-all of whom were aided by the rise of literacy in that period. collectively, they inspired a mood of public outrage that became an irresistible force for reform. in the second half of the th century, this sense of moral outrage found new targets-lighting a cigarette without permission in someone else' s home, carrying infant and child passengers in a car without safety equipment, dumping toxic industrial waste where it harms others, and more. yet, much else remains to be done. . political will. there is always resistance to change, there are always interest groups-often rich and powerful withal-who will do whatever it takes to obstruct necessary improvement. in the era of the sanitary revolution, it was the owners of water companies, factories, and tenement housing who resisted most vigorously. since the s it has been tobacco companies and a host of manufacturers of toxic petrochemical and other dangerous compounds released into the air and water. legislation and regulation are almost always necessary, and inevitably generate opposition. nevertheless, when the other four features-awareness, understanding, capability, and values-are in place, the political will to bring about reforms gathers momentum and usually succeeds eventually. these five essential ingredients required for public health reforms apply to several public health problems that have waxed and waned over time: tobacco addiction, impaired driving, domestic violence, child abuse, irresponsible domestic and industrial waste disposal, and so on. lately, mountainous barriers-of our own making-to maintaining our public health have appeared. the most formidable is a cluster of human-induced changes to global ecosystems and the global commons-the atmosphere, the oceans, wilderness regions, and stocks of biodiversity-that threaten all life and health on earth, not just the life and health of humans. another barrier is perhaps an inherent flaw in the human character that leads many individuals and national leaders to believe that disputes can be settled by violent means. currently, we have so many terrible weapons that violence done by them can and does cause immense suffering, innumerable deaths ( % or more of these deaths, as well as a similar proportion of permanent maiming and disability, are among non-combatants), and appalling damage to ecosystems, the environment, and the fabric of society. sadly, this is rarely recognized as a public health problem. the very first essential ingredient, awareness of the problem, is lacking. both these massive public health problems, in my view, are linked to the insatiable human craving for petroleum fuels, an addiction far more pervasive and dangerous to mankind and the earth than addiction to tobacco. so far in our only partially sentient and insightful civilization, this particular addiction is not even recognized as a public health problem. one public health problem that has been recognized is a worldwide pandemic of tobacco addiction and its many adverse effects on health and long life. recognition of this problem led the delegates to the world health assembly of to approve the framework convention on tobacco control. another universally recognized public health problem is the global pandemic of hiv/aids. tobacco addiction and the hiv/aids pandemic are both associated with the values of modern life and social behavior, including the marketing practices of transnational corporations. surmounting these barriers to health will require social, cultural, and behavioral changes and political action. i am an optimist. i believe that the pace of scientific advances will be maintained in the future, and that values will continue to shift in favor of essential changes towards global ecosystem sustainability. i do not know whether those who follow us will ever reach the ultimate summit or idealized who vision of halfdan mahler' s "health for all," but i am confident that they will continue to climb towards it. de contagione et contagiosis morbis et eorum curatione, libri iii natural and political observations mentioned in a following index and made upon the bills of mortality with reference to the government, religion, trade, growth, air, diseases and the several changes in the said city an inquiry into the causes and effects of the variolae vaccinae a treatise of the scurvy hippocratic writings. harmondsworth: penguin a green history of the world; the environment and the collapse of great civilizations the greatest benefit to mankind; a medical history of humanity from antiquity to the present a history of medicine, . primitive and archaic medicine on the mode of communication of cholera key: cord- -e bn ui authors: nan title: ecr book of abstracts - a - postgraduate educational programme date: - - journal: insights imaging doi: . /s - - - sha: doc_id: cord_uid: e bn ui nan for pet-ct, the ct exam is generally performed as standard whole-body exam from the base of the skull to the upper thighs. standard parameters are non-ionic iodinated intravenous contrast material at . g/kg bodyweight with a venous delay of seconds after injection and - mm thick continuous axial reconstructions with medium soft convolution kernels. ct images frequently contain important diagnostic information beyond mere 'anatomic landmarking', so generally a diagnostic, contrast enhanced normal-dose ct should be performed except if a recent diagnostic ct scan is available for fusion. then, a low-dose ct is sufficient for attenuation correction. a difference from routine ct protocols is the expiratory position to match the respiratory position of pet acquisition. also, negative oral contrast material should be applied and the whole body should be included in the field of view to allow effective attenuation correction of pet images. diagnostic reading has to include lung, soft tissue and bone windows. optimally, the assessment should be performed by one reader evaluating ct and pet images simultaneously on multiplanar reformats. learning objectives: . to get acquainted with standard ct examination parameters for oncological imaging including the requirements for an effective attenuation correction. . to see the diagnostic value of ct beyond 'anatomical landmarking'. . to learn effective ways to interpret pet-ct examinations. nuclear medicine perspective t. beyer; zurich/ch combined pet/ct images were first proposed as early . since pet/ ct became available for broader clinical testing. since its commercial introduction in more than ' pet/ct systems were installed worldwide. pet/ct is a logical and technical consequence of early, manual or semi-automatic efforts to align functional and anatomical images for easier and improved diagnosis. pet, or positron emission tomography, is an emission tomographic imaging method based on the application of radioactively labelled biomolecules in order to measure and quantitate signalling or metabolic pathways. ct, computed tomography, on the other hand, uses an external ionising radiation transmission source to generate projection data of the transmitted radiation, thus helping to generate high spatial resolution images of the anatomy of the subject. both sets of information can be combined easily in a pet/ct, whereby both the pet and ct components can be operated in close spatial proximity within a single gantry without cross-talk effects. through the combination of ct and pet overall examination times of oncology pet studies are reduced by %. in addition, pet instrumentation has been advanced to include time-of-flight measurements for improved signal-to-noise ratio, extended axial field-of-view-coverage for higher sensitivity and novel image reconstruction for improved contrast. today, high-quality one-stop shop staging with pet/ct is possible in min, or less. learning objectives: . to illustrate the origin of combined pet/ct imaging. . to motivate the strength of pet: high spatial sensitivity, quantification and functional information. . to appreciate the difference between "contrast" and "tracer" imaging. . to highlight novel developments in pet imaging: time of flight (tof), extended axial field-of-view. computed tomography is the main contribution to diagnostic medial radiation exposure to the public. in the year , ct accounted for only % of all radiationassociated examinations; however, at the same time it accounted for % of the total radiation exposure. since the beginning of this century numerous international surveys had been performed with the aim to define diagnostic reference levels in european countries. radiation exposure by ct has increased particularly by cardiac ct examination, what has caused awareness to utilise all strategies for radiation protection in ct. modern ct scanners are equipped with automated anatomical or organ sparing exposure control. it is the ct investigators responsibility to check the clinical indication, limit the scan range and decide for the appropriate scan protocol with the least radiation exposure. newer technical developments in ct scanner hard and software will enable to further reduce the dose from ct. all these strategies are essentially necessary since the number of ct investigations and the scope of clinical indications are expanding with advancing medical progress. learning objectives: . to understand the meaning of diagnostic reference values. . to become aware of dose intense ct protocols. . to learn about strategies for radiation protection in ct. nuclear medicine perspective s.p. mueller; essen/de different radiopharmaceuticals labelled with positron emitting radioisotopes are used to study a multitude of molecular processes using a positron-emissiontomography (pet) scanner which nowadays is typically integrated with a ct scanner (pet/ct). this lecture will enable the attendee to comprehend that the radiation exposure from pet depends on the biodistribution and kinetics of the radiopharmaceutical, the physical half-life of the positron emitting radioisotope used for labelling, and the injected activity, leading to an understanding that there is no generic "radiation exposure from pet or pet/ct". learning objectives: . to learn that the comparison of doses for different radiopharmaceuticals is based on the concept of the effective dose which expresses the total stochastic risk from the non-uniform radiation exposure to individual radiosensitive organs in terms of a uniform whole body radiation dose. . to understand that the effective dose from certain radiopharmaceuticals may be reduced by simple means, e.g. if it is renally eliminated by frequently voiding the bladder, and that the effective doses for the most prevalent radiopharmaceuticals lie within the typical range of other diagnostic nuclear medicine tests and compare favourably to the radiation exposure from the ct portion of a whole-body pet/ct scan. pet and pet/ct are non-invasive, -dimensional imaging modalities which have become standard of care in patients with malignant lymphomas. these modalities have been extensively studied for staging, restaging, monitoring response to therapy, surveillance after definite treatment, and assessment of transformation. more recently, pet tracers have been suggested as surrogate markers for cancer drug development. learning objectives: . to understand in which clinical scenarios pet and pet/ct imaging are superior to standard imaging modalities or other diagnostic tests. . to learn the diagnostic accuracy and predictive potential of pet and pet/ct for staging/restaging hodgkin's disease and non-hodgkin's lymphoma. of the examination. therefore, for oncologic applications, pet-ct has already gained widespread acceptance for the initial staging of cancer, the management of recurrent cancer, and for monitoring the response to therapy. the development of a large variety of radiotracers is an evolving procedure. the most frequent used radiotracer in clinical practice, f fdg, is based on the identification of the fundamental aspects of tumour glucose metabolism. new radiotracers, with promising potential for pet-ct, are also currently available to visualise specific cellular and molecular tumour pathway and more being developed. learning objectives: . to appreciate the advantage of a combined pet-ct technique. . to consolidate our knowledge of optimal examination protocols and to be aware of the pitfalls that may be encountered using this technique. . to understand the indications for pet-ct in the diagnosis, staging, and therapy monitoring of a large variety of gi tumours. . to become familiar with the different radiotracers to obtain a tailored and personalized diagnosis for the large variety of gi tumours. a- integrated positron emission tomography (pet)/ct provides combined metabolic and anatomic information of malignancies. the addition of ct to pet for urogenital purposes is very useful. ct should be performed with oral and intravenous contrast agent administration as a full diagnostic technique. if performed under these conditions, this technique can help to avoid pet pitfalls including focal retained activity in ureters and urinary bladder, and increased uptake in physiologic and benign pelvic processes such as endometrial uptake in the menstrual phase, leiomyomatosis, endometriosis or infection. we will describe the use of pet/ct in the characterisation, staging and surveillance of urogenital malignancies including kidney, prostate, bladder, uterine cervix, endometrium and ovaries. pet/ct is internationally accepted as the most useful surveillance imaging tool in patients with ovarian cancer, and its use as a problem-solving modality has also rapidly grown in the rest of urogenital malignancies. learning objectives: . to learn the appropriate protocols and settings of a diagnostic ct in pet/ct for urogenital purposes. computed tomography remains the workhorse of clinical cross sectional imaging due to its good availability, enormous speed, high spatial resolution and sufficient tissue contrast to evaluate most diseases based on morphology. compared to pet and spect, room for improvement remains in sensitivity and specificity for certain diseases and, compared to ultrasound and mri, in dynamic imaging. however, already today there are new ct techniques which can provide diagnostically equivalent information as spect or pet but with higher spatial resolution, in shorter acquisition time and without radioactive tracers. one of these techniques is ct perfusion imaging with repeated low-dose acquisitions of the same organ. with this method, a detailed evaluation of brain perfusion is feasible e.g, for stroke assessment, or of tumour perfusion, e.g. to assess early therapy response. another option is dual energy ct which does not require additional dose but can provide additional important diagnostic information similar to pet or spect. examples are the evaluation of lung ventilation und perfusion with xenon gas and iodine contrast. for oncological imaging, the evaluation of tumour perfusion based on spectral identification of iodine is an attractive option to increase specificity without additional dose or radioactivity. similarly, it is feasible to assess myocardial perfusion along with coronary ct angiography. for some diseases, even the molecular substrate can be identified with this technique, e.g. uric acid in gout patients. these new techniques provide significant advantages in oncological imaging and may further add to disease characterisation if combined in pet-ct. learning objectives: . to understand the strengths of current ct technology in oncological imaging based on morphology. . to learn about the diagnostic value of ct beyond anatomical referencing. energy ct as options to improve disease characterisation. tracers beyond fdg in daily routine m. beheshti; linz/at the ability of positron emission tomography (pet) to study different biological processes opens up new windows for both researches and daily clinical use. addition of computed tomography (ct) to pet improves detection efficiency and results in better localization of the lesions. the aim of this review is to consolidate knowledge of oncological applications of pet tracers other than [( )f]fluoro- -deoxy-d -glucose] (fdg) in the daily clinical practice. fdg, as a non -specific tracer, has limited value in the assessment of different cancers such as prostate cancer, neuroendocrine tumours (net), brain tumours, hepatocellular carcinoma (hcc), and some types of breast cancers. hence, due to high sensitivity of pet in performing non-invasive functional studies, further investigations and developments are warranted for defining specific pet radiotracers and theirs clinical applications regarding different tumour entities. oncological non-fdg pet tracers can be generally categorized into groups: those labeled with f- , c- and other non-fdg tracers. fluorine- and c- are labeled with different amino acids, substrates involved in fatty acid synthesis, protein synthesis, amino acid transport substrate and tracers linked to nucleic acid synthesis. these tracers are also labeled with specific ligands for receptor imaging. the other non-fdg a- with the increasing use of abdominal cross-sectional imaging, incidental adrenal masses are being detected more often. the important clinical question is whether these lesions are benign adenomas or malignant primary or secondary masses. benign adrenal masses such as lipid-rich adenomas, phaeochromocytomas, myelolipomas, adrenal cysts and adrenal haemorrhage have pathognomonic cross-sectional imaging appearances. however, there remains a significant overlap between imaging features of some lipid-poor adenomas and malignant lesions. the nature of incidentally detected adrenal masses can be determined with a high degree of accuracy using computed tomography and magnetic resonance imaging alone. positron emission tomography is also increasingly used in clinical practice in characterising incidentally detected lesions in patients with cancer. the performance of the established and new techniques in ct, mri and to a lesser extent pet, that can be used to distinguish benign adenomas and malignant lesions of the adrenal gland will be reviewed. with the increasing use of imaging, incidentally detected renal masses are very common. while masses detected by ct or mr usually can be properly classified, renal masses detected by ultrasound frequently require further workup. the following considerations determine the diagnostic workup: simple cysts are very common but may present atypically. renal cell carcinomas may be cystic but usually display at least a small solid component. renal cell carcinomas have a bad prognosis when metastasized but metastases hardly every develop before the tumour has reached cm in diameter. differentiation between solid tumours by imaging alone is exceedingly difficult, save for the identification of angiomyolipomas in adults. this course will discuss suitable diagnostic algorithms based on the initial presentation of the mass. typical imaging findings of various benign and malignant renal masses will be presented. the role of the bosniak classification will be illustrated. newer developments such as a waitand-see approach or primary biopsy for small solid renal masses will be discussed. learning objectives: . to learn how to detect and characterise a renal mass. . to understand how to apply adequate protocols according to the clinical situation. the wrist and hand are characterised by variability of bones, fibrocartilage, ligaments, muscles and neurovascular structures. coalitions (most commonly lunotriquetral, prevalence of . %), ulnar impaction positive variance of the ulna, carpe bossu and an accessory medial lunate facet associated with osteoarthritis are typical variants of bone. the triangular fibrocartilage complex (tfcc) and the interosseous ligaments often present with small defects. radial tfcc defects are present in % of symptomatic but also in % asymptomatic wrists. in addition, their prevalence increases with age, apparently without increasing symptoms. accessory muscles are common and may clinically mimick a neoplasm. disease may also be mimicked by imaging artifacts. magic angle effects cause increased tendon signal and may thus lead to the incorrect diagnosis of tendinopathy. the lunate appears to be more dorsally tilted on sagittal mr images than on standard radiographs (radiolunate angle ~ ° larger on mr images). this value increases to ~ ° if the wrist is positioned in ulnar deviated as is commonly the case when the wrist is examined in the "superman position", with the arm above the head. magic angle artefacts are commonly encountered in the wrist. the extensor and flexor pollicis longus are especially prone to such artefacts, due to their oblique course approaching the critical ° with regard to the b field. in conclusion, variability is rather the rule than the exception in the hand and wrist. only part of the findings have clinical meaning. technical aspects add another dimension of variability. learning objectives: . to become familiar with the normal anatomy. . to be able to identify normal variants. . to appreciate the range of pitfalls that may simulate pathology. the imaging of the trauma stratifies the severity and the treatment strategy. the leading modalities in low-energy trauma are x-ray and ultrasound. the different approach has to be used in high-energy trauma -the silent life-threatening injury must be actively searched. although the first examination on the site of the accident could estimate whether the severe trauma is present, the imaging must confirm or exclude it. besides fast transport to the trauma centre has the extraordinary impact on survival the diagnostic algorithm. the installation of ct, ultrasound and x-ray directly within the emergency department is extremely important for the trauma management. if the focused assessment sonography for trauma (fast) is replaced by whole body ct, the life-threatening injury is detected at fastest. protocol includes imaging of non-enhanced ct head and cervical spine followed by the contrast-enhanced ct of the entire thorax, abdomen and pelvis, in cases of lower extremities trauma covering whole body. ct could be performed also under resuscitation, because whole imaging takes about three to five minutes. following findings listed recently according to their impact on survival must be confirmed or excluded: intracranial injury, cervical spine trauma, aortic injury, overpressure pneumothorax, severe bleeding in the abdominal cavity, organs injury, peripheral vascular trauma, bone trauma; the trauma team including anaesthesiologist, surgeon and radiologist discusses the findings and plans of the treatment. over the last two decades, spiral-ct has become a highly reliable imaging modality to diagnose haemorrhage in trauma, while the role of catheter angiography has changed from a diagnostic to a therapeutic modality. traumatic injuries of the heart and aorta lead to sudden death occurring at the accident site, whereas uncontrollable haemorrhage from larger arteries and parenchymal organs is the most frequent cause of mortality during the first h following severe trauma. in patients with ruptured aorta or major arteries, stenting and temporary balloon occlusion may contribute to saving lives, while transcatheter embolisation may prevent exsanguination in haemorrhage from visceral organs, arteriovenous fistulas, and secondary onset haemorrhage. however, a haemodynamically stable patient is a prerequisite for all angiographic interventions. definite haemostasis using the above-mentioned techniques can be obtained in - % (major and periphery arteries) and - % (visceral organs), respectively. possible complications following angiographic haemostatic interventions depend very much on the treated vessel bed but are, in general, as low as %. learning objectives: . to understand potential treatment options and when to treat and when not to treat. a s c b d e f g h to current best practice, restriction of opinions to one's own expertise, expression of opinions that take into consideration all of the material facts, and the readiness to change that opinion if additional information becomes available. any unusual, contradictory or inconsistent features of the case should be highlighted. the expert should highlight whether a proposition is a hypothesis (in particular a controversial hypothesis) or an opinion deduced in accordance with peer-reviewed technique, research and experience accepted as a consensus in the scientific community. he/she should indicate whether the opinion is provisional (or qualified), stating the qualification and the reason for it, and identify what further information is required to give an opinion without qualification. when there is a range of opinion on any question to be answered by the expert, (a) the range of opinion should be summarised, (b) highlight whether that range of opinion includes an 'unknown cause' (because of limited facts of the case or limited research/peer-reviewed publications), and (c) give reasons for the opinion expressed. reprints of scientific publications will assist the court, but will also enable the attorneys to undertake a more robust cross examination! learning objectives: . to learn about imaging findings in relation to whether or not abuse has occurred. . to understand in which cases the diagnosis of child abuse should be raised in the radiology report. . to become familiar with the terms that should be used when highly specific imaging indicators are identified in an otherwise normal infant. the radiologist at the eye of the storm : imaging plays a central role in the diagnosis of child abuse. is there any risk of the radiologist having a detrimental impact on either the welfare of the patient or the judicial process by either under-diagnosing or over-diagnosing child abuse? ovarian cancer continues to be a challenge to radiologists and clinicians, as it is one of the most lethal female tumours. this is mainly due to its diagnosis in an advanced stage in the majority of patients. however, new developments can be observed: new insights in tumour biology, advances in imaging and new concepts of ovarian cancer treatment and surveillance. furthermore, a multidisciplinary expert team approach has also substantially changed the management of patients with suspected ovarian cancer. the findings of radiology are becoming pivotal in a more individualised patient care. the role of radiology includes (a) characterisation of sonographically indeterminate adnexal masses, (b) staging as guidance for surgery and treatment planning (including identification of sites of non optimal resectabilty) in suspected ovarian cancer, (c) assessment of recurrent disease, and (d) in selected cases image-guided biopsy. in this session we provide an update on the aetiology and current concepts of treatment of ovarian cancer and on the contribution of radiology in characterisation and staging in patients with the working diagnosis of ovarian cancer. the panel discussion will focus on the role of radiology in multidisciplinary conferences in suspected ovarian cancer. session objectives: . to learn about new concepts in etiology and treatment of ovarian cancer. . to become familiar with optimised imaging protocols to diagnose ovarian cancer. . to learn about the value of ct and pet/ct as a basis for treatment planning in ovarian cancer. . to appreciate the role of the radiologist in multidisciplinary consensus conferences. session objectives: . to learn about current imaging practices in the evaluation of suspected child abuse. . to learn about imaging changes which permit a firm diagnosis of child abuse. . to become familiar how to construct a comprehensive report providing evidence of child abuse. how to image and detect patterns of skeletal injury indicating child abuse p.k. kleinman; boston, ma/us (paul. kleinman@childrens.harvard.edu) in infants, skeletal injury may form the basis for the diagnosis of abuse. the first step is the acquisition of a skeletal survey utilising meticulous technique adhering to a rigorous imaging protocol. a proper interpretation is ensured when the radiologist is familiar with the various patterns of skeletal injury, their specificity for abuse and those entities which may simulate inflicted injury. appreciation of the lesion morphology, the fundamental pathologic alterations and mechanism of injury are essential in assessing the significance of the findings and placing them in the clinical context. dating fractures poses a significant challenge, but in most cases, injuries can be placed in a certain time frame, especially if a follow-up skeletal survey is obtained. although radiography forms the basis of skeletal imaging, ultrasound, ct, scintigraphy and mri may clarify findings and optimise diagnosis and management. the radiology report must be constructed with care and the language should be crafted with the expectation that the radiologist may be called to testify in court, a daunting challenge in an often highly adversarial environment. learning objectives: . to learn about the appropriate imaging protocols and quality for skeletal survey in suspected child abuse. . to learn about patterns of skeletal injury typical of child abuse. . to become familiar with findings that point to alternative diagnoses. imaging strategies to fully determine intracranial injury resulting from child abuse c. adamsbaum, c. rey-salmon; paris/fr (c.adamsbaum@svp.ap-hop-paris.fr) radiologists play a key role in the early diagnosis of abusive head trauma (aht). symptoms are various and may range from coma to asymptomatic children. changing elements of the history provided previous injuries, bruising in non-ambulatory children and delay in seeking care raise a high index of suspicion. the most common finding in aht is of multifocal subdural haematomas over the cerebral hemispheres, the convexity, the posterior interhemispheric fissure and the posterior fossa. the haematomas are often associated with hypoxic-ischaemic injury and retinal haemorrhages. ct is the first diagnostic examination to be used for patients with acute injury. it can reveal intracranial haemorrhage, fracture and soft tissue swelling with a high degree of sensitivity. ct should be repeated after a time interval if the findings are doubtful or if there is a discrepancy with the neurological picture. mri (including t , t , t *, diffusion sequences and cervical spine examination) is required to fully determine intracranial injury as it can exquisitely demonstrate hypoxic-ischaemic injuries by showing areas of cytotoxic oedema. it is impossible to date precisely a haematoma whose pattern is influenced by numerous factors. however, the main point is to determine the presence of 'age-different' lesions. this not only provides a strong argument for the diagnosis but also suggest repetitive violence and thereby, a high risk for further injury unless protective action is undertaken. brain imaging must be performed in all siblings younger than years, living in the same conditions as the index case. learning objectives: . to learn about imaging protocols designed to: a) detect acute treatable conditions b) depict fully and determine the timing of all injuries. . to become familiar with imaging findings that are highly suggestive of child abuse. . to learn about head trauma that may simulate child abuse. what is the information required by any court and how the radiological reports should be phrased s. chapman; birmingham/uk (stevechapman@doctors.org.uk) this presentation will describe the role of a paediatric radiologist as an expert witness (as opposed to a witness to fact). the expert has an overriding duty to the court that takes precedence over any obligation from whom the expert has received instructions or by whom the expert is paid. particular duties include advice that conforms a- : ovarian cancer: update and role of radiology j.a. spencer; leeds/uk (johnaspencer @hotmail.com) ovarian cancer is known as the 'silent killer'. it is usually diagnosed late and most women have disease disseminated to the peritoneum (and/or pleura) at presentation. standard treatment comprises cytoreductive surgery followed by platinumbased chemotherapy. an alternative is neoadjuvant chemotherapy followed by interval debulking surgery (ids) then completion chemotherapy. data from the eortc trial show this to be as effective as the standard of care. there are strong genetic predispositions to ovarian, fallopian tube and primary peritoneal cancers. there is linkage with breast cancer in women with brca gene mutations and with colon cancer with the hnpcc gene. ongoing trials are screening women in the general population and at high risk of the disease. these use the serum tumour marker ca followed by us as screening tools. mr imaging is an effective tool to assess sonographically indeterminate adnexal masses and offers an earlier diagnosis of cancer than interval reassessment with us. ct is the most commonly used modality for assessment of disease extent prior to surgery. image-guided biopsy is necessary prior to starting neoadjuvant chemotherapy. the eortc and mrc (uk) ov trials of women with treated ovarian cancer have shown no outcome advantage for women treated early at 'ca relapse' of disease versus later with clinical or ct evidence of relapse. this questions the role of ca in follow-up and argues against investigation of the 'ca +ve, ct -ve' woman with more expensive and scarce imaging resources such as ct-pet and dcemr. learning objectives: . to become familiar with clinical features of ovarian cancer. . to learn about the etiology of ovarian cancer, including genetic predisposition and the current research into screening. . to become familiar with new concepts of treatment ovarian cancer and surveillance of the treated patient and their impact for the use and choice of imaging modalities. imaging of adnexal masses: is it feasible to diagnose ovarian cancer? i. thomassin-naggara; paris/fr (isabelle.thomassin@tnn.aphp.fr) adnexal masses can be depicted by many imaging modalities (us, ct, mr and pet-fdg). however, the characterisation of adnexal tumours is mainly based on two techniques: us and mri. for complex adnexal masses, mr imaging add to conventional criteria of malignancy common to all imaging modalities (bilaterality, tumour diameter larger than cm, predominantly solid mass, cystic tumour with vegetations, and secondary malignant features, such as ascites, peritoneal involvement, and enlarged lymph nodes) specific features based on the characterisation of the solid tissue (including vegetation, thickened irregular septa, and solid portion) of the adnexal tumour. signal intensity of solid tissue on t sequence (fibrous tissue), perfusion (neoangiogenesis), and diffusion (cellularity) are combined to make a decision tree analysis. low t signal, absence of high b signal, and time intensity curve type are predictive of benignity whereas high t signal and time intensity curve type are predictive of malignancy. combining common classical features and specific mr features for predicting malignancy, mr imaging has a high degree of accuracy ( %) for characterising complex adnexal tumours. this high diagnostic confidence rate may help young women wishing to preserve childbearing potential to opt for conservative surgery and avoid the systematic removal of benign complex adnexal masses in menopausal women. learning objectives: . to understand the role of ultrasonography in assessing and managing complex adnexal lesions. . to learn how to optimise the mri protocol and how to improve the characterisation of indeterminate complex adnexal masses. . to understand the added value of functional sequences (dce mri and dwi) in diagnosing adnexal masses. the european and american guidelines include breast ultrasound in a restricted diagnostic scenario for breast cancer diagnosis. the fourth edition of the european guidelines ( ) reviewed in refers to us in only twice. acr in its acr practice guideline reduces breast ultrasound examination to seven situations. evaluation of the axilla and ultrasound as a screening tool on high risk women is considered an area of research. the progression of us makes guidelines become obsolete very fast. its application still remains pending on the results of clinical trials. image smoothing on sono-ct or multiple frequency transducers will produce images, eventually with more diagnostic information. second tissue harmonics will produce sharper and more clear images. cad systems, doppler and contrast doppler will help in diagnosis. d with the new software and automatic probes constitute a very promising work in progress. they will make a dramatic change in our workload. the radiologist will be released of performing the exam, to review the images in the workstation. sonoelastography has evolved from the manual to the actual automatic shearwave. this system measures the transversal transmission of sound in biologic tissues. it is a new and promising technology, probably more objective and non-operator dependant, that is able to differentiate benign from malignant conditions. all of these systems will be reviewed and evaluated for its actual situation. the problem now is how to introduce new terms, new descriptors, and new technologies in the bi-rads system, once clinical evidence is demonstrated. diffusion-weighted mri (dwi) is a promising technique in oncology. it can be used for in vivo quantification of the combined effects of capillary perfusion and diffusion. using echoplanar imaging (epi), dwi is possible with fast imaging times minimising the effect of gross physiologic motion from respiration and cardiac movement. in this lecture, we will discuss the acquisition, post-processing and quantification methods and results of dwi in abdominal and pelvis tumours. we will also review the mechanisms associated with diffusion changes in tumours. mri-pathologic correlation will be shown. finally, limitations and future directions of the technique will be reviewed. diagnostic potentials -as well as limitations -associated with morphological cross-sectional imaging on the one hand and functional imaging on the other are increasingly well understood. it has become obvious that in many cases both kinds of imaging complement one another. hence, hybrid pet/ct imaging must be considered one of the most promising new developments in medical imaging. however, some questions have to be raised and challenges have to be met to avoid overrating pet/ct in oncology. pet/ct with [ f]- -fluoro- -deoxy-d-glucose (fdg) as a radioactive tracer has been reported to be more accurate than either imaging modality alone and sometimes even more accurate than ct and pet read side by side. however, the clinically important question should rather be the following: does this higher accuracy have an impact on patient management? furthermore, some tumours do not have an increased glucose metabolism making them fdg-pet negative. the most recently launched line of pet/ct scanners combines high-definition pet with high-end multislice ct. these imaging systems not only provide a higher diagnostic accuracy based on detection of smaller lesions with ct and pet, but also offer integration of complex ct protocols into the pet/ct scan. these protocols include ct perfusion, three-dimensional ct image reconstruction or virtual fly-through. the aim of this talk is to give an overview concerning pet/ct in oncology. the mechanism of radionuclide uptake, different tracers, the indications of pet/ct in oncology and its accuracy will be addressed. learning objectives: . to understand why uptake of the tracer provides information on the viability of solid tumours. . to learn whether pet/ct is able to give reliable quantitative information. . to know whether pet/ct is a valuable tool for tumour response to treatment. to gain additional functional information. functional imaging techniques such as diffusion tensor imaging and tractography as well as fmri are increasingly used and relied on in clinical practice. with diffusion tensor imaging and tractography the location, course and integrity of the major white matter tracts can be depicted, while with fmri the brain's cortical function is visualised. despite major technical advances, these techniques are still time consuming, labour intensive and have several limitations. they therefore need to be used and interpreted with care. the purpose of this lecture is to review the functional anatomy of the brain in a clinically relevant context and to illustrate when additional functional imaging techniques may be indicated. the most important eloquent brain areas are addressed and include the motor, visual and language systems. routine clinical cardiac mri requires speed and efficiency as a result of physical motion. consequently, the challenges and benefits of rapid mri are nowhere more apparent than in the field of cardiovascular mr imaging. to meet these challenges, one must balance the competing constraints of signal-to-noise ratio (snr), contrastto-noise ratio (cnr), spatial resolution, temporal resolution, scan time, and image quality. one of the main determinants of snr is the static magnetic field strength. hence, cardiovascular mri at . t or more holds the promise to overcome some of the snr limitations and to extend the capabilities of cardiac mri. all studies in cardiac mri at higher field strength have proven the feasibility of cardiac mri for the comprehensive assessment of cardiac morphology and function. the studies demonstrated a significant snr increase, but also outlined image-quality problems associated with b -field inhomogeneities and specific absorption rate (sar) constraints. with regard to acquisition speed, parallel imaging mri capabilities form an important enabling factor, especially if enough snr is available. therefore, the combination of higher field strength and parallel imaging strategies may help to overcome several of the present limitations in cardiac mri like cardiac perfusion and coronary artery imaging. the present indications, advantages and limitations of cardiac mri at . t will be discussed. the risks associated with the exposure of ionising radiation has raised increasing concerns in the radiological community. the recent years have shown an overall increase in the use of ct for the imaging of the heart and the coronary arteries. the downside of this increased use of cardiac ct is the increase in the collective radiation dose with cardiac ct which have been reported to be associated with an effective radiation dose of msv or more. increased awareness of the radiation dose with cardiac ct led to the development of several effective radiation dose reduction strategies including prospective ecg gating technique, anatomy and ecg-based tube current modulation, high pitch acquisition, and adaptation of the ct scanning parameters to the body habitus. however, the dose reduction strategies should be selected carefully on an individual patient basis in order to avoid serious image quality impairment by noise and artefacts. the lecture outlines the different radiation dose saving techniques currently used in clinical practice, the anatomy of the brain is often perceived as being complicated. especially the cortex is seen as an irregular arrangement of variable structures, which are difficult to differentiate and to identify. we will review the overall subdivision of the brain into lobes and describe their boundaries and their major gyri and sulci. we will then describe the location of specific functions. . primary sensorimotor cortex: motor is located in the precentral gyrus, sensory in the postcentral gyrus around the central sulcus (cs), hence the importance of always correctly identifying the cs. we will present interlocked methods to identify the cs in the axial plane (a) knob, (b) lateral axial, (c) medial axial, (d) gyral/cortical thickness and to identify it in the sagittal plane (a) lateral sagittal, (b) hook, (c) medial sagittal. . primary auditory cortex (a ): centred at the postero-medial part of heschl's gyrus (hg), we will present simple landmarks in each of the planes: (a) axial: adhaesio interthalamica, (b) sagittal: omega/heart shape of hg, (c) coronal: omega shape of hg. . primary visual cortex (v ): centred on the calcarine sulcus, we will discuss the characteristic shape that allows the identification of this structure in all planes. at the end of this lecture, you will know the subdivision of the cortex; the methods and landmarks necessary to identify the primary sensorimotor, speech, auditory, and visual areas. magnetic resonance imaging (mri) has undergone a rapid development in the last decade with numerous new techniques. nevertheless, pattern recognition of brain lesions based on signal intensities on conventional mr sequences (t -and t -weighted, flair) is the first step in diagnostic "work up". good examples for pattern recognition are lesions with t -shortening ("bright" on t wi) such as: fat containing lesions (lipoma), melanoma, lesions with colloid content, calcifications, and haemorrhagic metastatic lesions. t -shortening ("dark" on t wi) in enhancing lesions is suggestive of lymphoma and certain infections (tuberculosis, fungal abscesses). flow void (absence of signal) reflects high velocity flowing blood or csf, and will help in diagnosing vessel abnormalities and related pathologies. the purpose of this lecture is to refresh knowledge on patterns and normal variants useful for clinical practice. a simplified "pipeline" consisting of easy consecutive steps will be introduced. learning objectives: . to learn important normal variants of different structures and lesions in the brain. . to learn about recognition patterns that might be helpful in suggesting the most likely etiology of common brain lesions. . to consolidate the key imaging findings in different types of lesions and normal variants. a- : c. clinical symptoms correlated to brain anatomy m. smits; rotterdam/nl (marion.smits@erasmusmc.nl) diagnostic neuro-imaging heavily depends on a thorough understanding of brain anatomy in relation to the brain's function. clinical neurological symptoms and deficits not only give us an indication of the brain area(s) involved but may also direct us towards the use of specific imaging techniques, such as diffusion tensor imaging and tractography, as well as functional magnetic resonance imaging (fmri). specific imaging findings, on the other hand, may direct clinical management in a neurologically intact patient, such as the decision to resect a brain tumour in or near an eloquent brain area. again, specific imaging techniques may be used evaluation of response to treatment allows an early assessment of tumour response typically after - cycles of chemotherapy. this evaluation is most commonly performed with pet/ct during therapy for high grade non-hodgkin's lymphoma. this review presentation looks at the potential roles that ct, regional mri and whole body mri do and could play in response evaluation from a radiology perspective. the presenter is a radiologist who is clinical director of a medium volume pet/ ct centre. the talk focuses on discrepancies between ct and pet findings in tumour response and on tumours or situations where ct is the primary modality to determine tumour response. potential roles for whole body mri in response evaluation and assessment of solid tumours by mri are examined. at a general level, the role of the radiologist as part of the multidisciplinary oncology meeting (mdm), including when and where to use image-guided biopsy in response evaluation and the use of recist versus precist criteria is discussed. the aim of the presentation is to outline the role of ct in tumour response evaluation in the era of pet/ct and to explore potential roles particularly for whole body mri in tumour assessment. the presenter hopes to encourage radiologists to get fully involved in mdm discussions regarding ct, mri and pet/ct evaluation. learning objectives: . to learn the standard method (tumour measurements) for the evaluation of tumour response to treatment. . to understand the limits of international standard. . to become familiar with methods that provide functional or structural information, like perfusion ctmr or dw-mri. evaluation of tumour response to therapy: the role of nuclear medicine a. chiti; milan/it (arturo.chiti@humanitas.it) the high costs and possible side effects of chemotherapy and radiation therapy treatments favour the use of effective ways to monitor the treatment efficacy in oncology. molecular imaging demonstrated to be effective in evaluating the response after and during the course of therapy, in order to assess chemo-sensitivity and chemo-resistance of a particular neoplasm. the use of pet-ct in this setting can vary from very sophisticated and complex quantitative evaluation to simple qualitative analyses. in malignant lymphoma, international criteria for monitoring response to therapy have recently been revised, and fdg now plays a central role in defining tumour response. in a variety of solid tumours, studies have indicated that fdg pet-ct may provide early and accurate assessment of tumour response, suggesting that it could play a significant role in personalising the treatment of malignant tumours. performed during and after therapy for hl and aggressive nhl, fdg pet results have a high prognostic value and correlate with survival. fdg pet has been incorporated into revised response criteria for aggressive lymphomas, and several ongoing trials are under way to investigate the value of treatment adaptation based on early fdg pet results for hl and aggressive nhl. many technical aspects must be taken in consideration to avoid critical errors in evaluating response. from patient preparation to image acquisition a series of possible pitfalls must be avoided. in the clinical practice, the most widely used parameter is the suv, which can improve the accuracy of qualitative image assessment in many clinical settings. learning objectives: . to understand why pet-ct can be used to assess tumour viability. . to become familiar with the principles of signal quantification and to discuss its advantages and limits. . to learn about the incoming tracers that might enhance the role of pet-ct in the evaluation of tumour response. alzheimer's disease: the role of radiology j. alvarez-linera; madrid/es (jalinera@ruberinternacional.es) neurobiological changes in alzheimer's disease (ad) occur in an stereotypical pattern that begins in the medial temporal lobe (mtl) years before the clinical manifestation (brain reserve). brain atrophy is a marker of neurodegeneration that reflects the neurobiological disorder and is correlated with the neuropsychological changes at all stages of the disease. other imaging markers may reflect changes in microstructural (diffusion), functional (perfusion) or metabolic (mrs) domains that would provide additional information but are awaiting wider validation. in the early stages of ad, the most effective mri markers are those that reveal atrophy in mtl, particularly the measures of the hippocampus. the mtl atrophy mea-the required conditions in whom they may be successfully used, and how these techniques could be implemented in the daily clinical practice. proper linking of the data acquisition to the patients ecg is among the crucial prerequisites for successful cardiac imaging. while cardiac ct data acquisition itself does not impose any effect on the patients' ecg, inherent physical effects in the mr environment does show impact on the ecg trace and may hamper proper r-peak delineation. in addition, patient-related factors such as arrhythmia might affect image quality (iq). dose saving strategies in cardiac ct limit the possibilities of retrospective iq optimisation and as such more emphasis is recommended prior to data acquisition which is mainly related to patient selection/heart rate control. in cardiac mr a high amplitude ecg without influence of magnetic or scanning effects is of outmost importance. arrhythmia also remains a challenge that might be overcome by arrhythmia-rejection algorithms or real-time imaging. suboptimal contrast enhancement ought to be avoided by proper planning and timing as the majority of post-processing algorithms is based on signal behaviour. in the unfortunate situation of suboptimal contrast enhancement often only the use of standard post-processing tools is possible while semi-automated tools for cardiac post-processing may fail or necessitate substantial user interaction. the use of straight forward visualisation techniques is recommended for diagnosis while complex visualisation tools may add on confidentiality but are mainly suited for case presentations. coronary evaluation is typically performed using centerline tools allowing for the easy assessment of cad. while these tools potentially allow for estimation of the degree of diameter and area stenosis, accuracy though may still be limited. this is the second session between esr and eanm at each society's annual congress. few would argue but that patient's interests are best served by crossfertilisation and open communication between specialties. this is particularly true for radiology and nuclear medicine. this session elaborates on clinical scenarios where cross-fertilisation between both specialties is particularly important. the session will elaborate the radiologist and nuclear medicine perspectives on two common clinical scenarios, i.e. tumour response to therapy and evaluation of alzheimers disease. session objectives: . to appreciate how nuclear medicine and radiology provide complementary information. . to learn how each method can enhance the mutual performance of the radiologist and nuclear medicine physician. . to learn about recent advances in the field of tumour evaluation and early detection of alzheimer's disease. shown on mdct-images in the axial, coronal, and sagittal plane. variants of the normal anatomy, which are important to describe and to know are also shown. some of these variants are also delineated on mr images. in the second part of the lecture, the different expressions of congenital malformations of the external and middle ear will be explained. the different findings of the severity of the malformations will be shown and their importance for further clinical-therapeutical procedures will be explained. imaging investigation of cholesteatoma is required before surgery. if no surgery has been performed previously, ct will provide information about the location of the lesion (epi, pro, meso, retro, hypotympanum), the partial or total destruction of the ossicles, and possible extension to the inner ear. if there is no doubt about any of these factors ct is sufficient. in doubtful cases an mri examination is performed to confirm or refute the presence of cholesteatoma using t sequences without iv contrast medium, and diffusion weighted imaging with or without high resolution t , depending on the age of the patient. in postoperative recurrent cholesteatoma, mri is becoming the modality of first choice for detecting cholesteatomas, appearing : low in signal on t sequences, high in signal on diffusion weighted imaging. however, care is required since performing diffusion weighted imaging without t may lead to false positives. a granuloma with a slightly or markedly increased t signal is often associated with a high signal on diffusion. measurement of adc is useful for detecting cholesteatomas, infected cholesteatomas or abscess. finally whilst mri is the first examination in the follow-up of postoperative patients, the use of contrast medium is not necessary in most of the cases. a- : c. implants and postoperative findings in the middle ear b. verbist; leiden and nijmegen/nl many conditions which affect the function of the middle ear may require surgical intervention. postoperative imaging will be requested either to evaluate complete removal of diseases (eg in cholesteatoma) or because of new, persisting or recurrent complaints of the patient (e.g. vertigo after stapes replacement). in this presentation, different surgical procedures will be reviewed including the indications for a certain surgical approach as well as the different types of prosthesis. the normal postoperative imaging appearance of the most common surgical techniques will be shown. it will be discussed whether ct or mri should be performed to answer the clinical questions. an overview of possible failures and complications will be given. the management of patients with vascular malformations is often suboptimal. the reasons for this are many but include confusion regarding classification, uncertainty about the most appropriate imaging of the various forms of malformation and a sures are helping to propose new diagnostic criteria for ad, allowing a diagnosis of probable ad in predementia stages, when memory loss criteria are attached to imaging criteria (mri or pet) or measures of amyloid/tau in csf. the use of atrophy markers (global or mtl) increases the effectiveness in clinical trials (both by reducing the size of the sample and increasing the statistical power) and is therefore contributing significantly to the development of new treatments. the association of multiple markers of structural and functional imaging (mri and pet) and the use of advanced computational analysis techniques will allow better management of ad but it needs a broader validation and know the most efficient combination of biomarkers at each stage of the disease, including the preclinical period. scientists, researchers and clinicians all benefit from molecular imaging in dementia providing exciting new insights into their basic biology and pathophysiology. targeting specific aspects of neurotransmission, metabolism, inflammation or plaque formation -just to mention some of the current molecular approaches -increasingly gains impact on establishing the correct diagnosis, following the course of dementia or developing cns drugs. this talk will highlight the molecular targets and major pet and spect tracers for application in dementia, and will update on the results of the clinical imaging studies published in recent years. typical imaging patterns of alzheimer's disease (ad) will be discussed, including also the diagnostic discrimination from other types of neurodegenerative dementias such as frontotemporal dementias, lewy body dementia, and others. assessment of mci patients and the probability of transition in manifest dementia (predominantly ad) will be addressed together with its prognostic relevance. furthermore, recent advances in analysing tools which further improved the high diagnostic accuracy already reached by visual assessments will be presented. even though in competition with other modalities, ‚standard fdg' pet has shown to be a robust and both, sensitive and specific marker in the diagnostic work-up of dementia. fdg information will be markedly extended in clinical practice by specific amyloid imaging in the near future when these tracers are approved and thus generally available. learning objectives: . to become familiar with the nuclear medicine method that enables detection and evaluation of alzheimer's disease. . to learn about the potential development of functional studies using nuclear medicine. . to understand how nuclear medicine and radiology can provide complementary information. symptoms may require explanation and reassurance only. lesions with a mainly cutaneous element may be treated with laser. deeper lesions are usually treated with several episodes of sclerotherapy. agents such as alcohol, polidocanol and std will be discussed and the relative advantages and issues explained. informed consent is vital, and the approach to this will be outlined. high flow lesions are frequently associated with severe cosmetic changes, invasion of surrounding tissues, haemorrhage, and high output cardiac failure. lesions may be life threatening. in the extremities peripheral ischaemia and ulceration due to steal of blood by the arteriovenous malformation is common. informed consent will again be discussed, as potential for major complications is high. key to endovascular management of these lesions is an understanding of the nidus, arterial inflow and venous outflow. the role of liquid and solid embolic agents and specifics of transarterial, venous and direct approaches will be discussed. avms pose a major problem regarding classification and treatment. a case report discussed by the panellists demonstrates how these patients should be approached. the importance of establishing an interdisciplinary outpatient clinic is also presented. the most important organisational steps for providing an efficient clinical service are given. in addition, the most common pitfalls and complications of treatment are illustrated. computer-aided detection/diagnosis (cad) is recognised as a workstation or a system developed in order to assist the radiologists (clinicians) in performing their daily diagnostic tasks. clinically implemented cads are available at workstations and (if dicom compliant) may serve as a plug-in to pacs. the three-layer cad system includes: ( ) image analysis procedures whose development requires a medical and technical knowledge, ( ) a database module that is managed by experienced radiologists and it professionals, and ( ) graphical user interface (gui) that enables a user-friendly access to the data, the processing tools, and the results. a modern cad development involves a multidisciplinary team whose members are experts in medical and technical fields. a close collaboration of all experts is required at all stages of system life-cycle. at each stage the physicians knowledge and experience are indispensable. it includes medical analysis of the diagnostic problem, data collection, image analysis evaluation, and clinical verification. design, testing, and evaluation have to be successful in order to ensure cad implementation in a daily clinical routine. in this session three experts will share their experiences in the area of the overall cad architecture, its evaluation, validation and acceptance by clinicians, advantages and restrictions of solutions and clinical implementation in lung, breast and colon cancer. reading paradigm (primary, concurrent, second) in oncology as well as results of cad clinical implementation will be presented. perspectives in clinical cad implementation in diagnosis and treatment will be discussed. patients presenting with vascular malformations mostly are nomadic and hopeless individuals looking for help. finally having reached a "multidisciplinary specialistgroup" after a sometimes long and misleading trip throughout the ocean of "singleplayers" of different specialities these patients do not ask for any more diagnostics -they strongly claim for therapy. vascular malformations are congenital lesions, although merely seen at birth they become evident throughout the individuals growth. these developmental errors can affect all components of the vascular tree in any area of the body. the therapeutic goal must be defined rather as "control" than "cure" of this disease. to make this point understandable for both patients and collegues a fundamental understanding of the pathogenesis and natural course must be created. in special cases of complex vascular malformations the precise diagnosis and the information about all potential side-effects as well as risk-factors of progression enables these patients to manage their daily life. therefore, indications for treatment vary depending on the specific type of slow flow or high flow lesion, location, pain, functional and cosmetic impairments and general side-effects of each particular lesion, since no single specialist has enough knowledge to diagnose or treat vascular anomalies beyond the border of his distinct speciality multidisciplinary working-groups emerged at these interdisciplinary interfaces. their common language in classifying and their overall understanding of pathogenesis, prognosis offer these mostly hopeless patients a custom-fit treatment addressing their symptoms. imaging of vascular malformations should be directed by clinical assessment of the type of malformation to be expected, clinical symptoms and need for treatment. in order to make the proper decision of the required imaging modality or treatment, it is essential that the (interventional) radiologist is a member of a dedicated vascular malformation team. imaging needs to be tailored to the individual patient although general rules can be applied. duplex ultrasound together with a clinical assessment is often sufficient to make a proper diagnosis. this is especially true for the paediatric population. if more information about the extent of the lesion is needed, mr is often used in case of low flow lesions (venous/lymphatic), and mra or cta in case of high flow lesions. angiography is mandatory if an avm is diagnosed and treatment is planned. high frame rate imaging and selective injections are the only options for a proper evaluation of the nidus architecture of the avm. there are exceptions that warrant deviating from the above general rules. in this lecture, both the general rules and the exceptions will be discussed. at the onset of the st century humankind is focusing its attention on a very small molecule, as controlling co in the atmosphere is becoming a major goal, economically, socially and politically. yet, there is another small molecule which is going to play a more prominent role in the near future. h o, especially in its liquid form, the 'blue gold', is just indispensable to our lives. water makes to % of the human body weight and is crucial to the working of the biological machinery. still, how such a tiny molecule with its ° 'magic' angle could have been at the origin of life remains largely a mystery. different organisms have adopted different strategies in the way they get the most out of water, depending on their environment, and water contributes to the biodiversity. faulty mechanisms in the use of water by tissues may lead to severe diseases or death. clearly, water deserves to be seen as the prime 'biological molecule', and radiologists have long recognised its importance from the days of 'dry' (bone and air) radiology to the advent of ct which allowed contrast from 'wet' tissues to be explored. with mri one went one step further, as magnetisation of water is the sole source of contrast. life has led to intelligence, and recent mri studies have suggested that water may also actively contribute to the mechanisms underlying brain function. could the 'molecule of life' also be the 'molecule of the mind'? clearly, water must be radiologists' best friend. learning objectives: . to comprehend how the structure of the water molecule makes it important to life. . to understand the importance of water in biological and cellular processes. . to become familiar with the different ways water is responsible for image contrast in radiology. a. the role of cad in modern-day imaging a. todd-pokropek; london/uk (a.todd@ucl.ac.uk) the use of cad in medicine is an important and growing area of research. firstly good data must be acquired including not just images but associated information. the first step in that of preprocessing, notably (but not only) noise reduction. the data are then passed onto the segmentation step. often this step is semi-automatic requiring some manual intervention. conventional edge detection methods are not often of value, but active shape and appearance models, the use of markov random fields etc are commonly used. the next step is that of feature extraction both of shape and texture. these data are then submitted to one of several classifiers such as artificial neural networks (including mtanns) support vector machines (svm) and data reduction using principle and independent component analysis, and multiple voting techniques such as adaboost are also of value. the output may simply be returned to the observer (clinician) or as further input for a decision support system. examples considered will be in breast imaging (mammography), lung nodule detection, virtual colonoscopy and lumber spine. the use of the cad system as a simultaneous assistant or as a second reader is important. the use of cad in therapy is of increasing important. the assessment of such cad system (evaluation and validation) is still controversial. the difficulty of bringing systems both instrumentation and software for use in clinical practice in often underestimated and there have been some notable failures. some example of 'failures' will be given. ct colonography has evolved rapidly and disseminated widely over the last decade. the ability to provide an accurate whole colon examination with near perfect completion rates, use of reduced laxative bowel preparations and extra-colonic organ review has attracted very considerable interest amongst the wider radiological and gastroenterological community. inclusion of ct colonography in several core radiological training programmes confirms its evolution from super-specialist technique (performed in only a few centres) to mainstream. however, evidence supports highly variable performance, which is perhaps unsurprising given the complexity of both technique and interpretation methods -both which require specific training. notably, attendance at a training workshop generally represents the beginning of a radiologist's experience of ct colonography. indeed, most delegates rapidly progress from unconscious to conscious incompetence, acknowledging the need for a planned implementation strategy prior to offering ct colonography in their routine clinical practice. thankfully a decade of intensive research and large volume clinical experience has equipped the radiological community with knowledge and experience to inform successful ct colonography implementation strategies. by combining this experience with training and accreditation practices developed for colonoscopy, the prospect of a robust quality assurance framework is realistic and necessary to reassure both service commissioners and the general public alike. step one: the publication of international ct colonography standards, co-authored by eminent radiologists from across europe and beyond has been achieved. this talk will review the likely next steps. computer-assisted detection (cad) for ct colonography is now widely available in europe from a number of different vendors. this presentation will detail factors that potential users will need to know in order to properly evaluate cad systems, use them in clinical practice, and evaluate their likely impact. the different ways in which the performance of cad systems can be assessed will be discussed and the difference between weak methodologies (e.g. internal validation) and more valid assessments (e.g. external validation) will be explained. now that the diagnostic performance of ct colonography has been well-established, this state-of-the-art symposium will deal with issues related to the implementation ct colonography in day to day clinical practice. the lecturers will deal with the diagnostic performance of ct colonography, the technical requirements necessary to obtain high quality diagnostic data, the factors that underpin a high-quality service (including reader training), how implementation differs across different countries, and the possible impact of new developments, including computer-assisted detection (cad). two decades ago spiral ct technology initiated a new era in diagnostic imaging with virtual colonoscopy or ct colonography (ctc) as a major innovation. introduced by david vining in , ctc was rapidly endorsed as a potential tool for colorectal cancer screening. after an initial pioneering period defining the basic ctc principles, the advent of multi-slice ct significantly improved spatial and temporal resolution, allowing for isotropic image reconstruction with detailed d rendering of the colonic wall and very short acquisition times, reducing motion artefacts. furthermore, application and refinement of (ultra-) low dose technique almost completely tackled the issue of radiation dose. these improvements have resulted in the perfect optical colonoscopy imitator with reliable fly through of the colon in a timely manner. consecutively, the primary d reading paradigm with d problem-solving is getting more and more adepts, although in experienced hands primary d-read with d-problem solving is a solid contender. to improve depiction of the colonic wall new d visualisation methods were developed. these technical improvements with the application of state-of-the-art ctc technique have resulted in a very good performance of polyp detection. in three multi-centre studies, totalising patients, sensitivity ranged between - % and - % and specificity between - % and - % for lesions > mm and > mm, respectively. finally, it may be expected that further refinements of cad, laxative-free ctc with electronic cleansing and dual energy ct will bring ctc to the next level and will enhance it as the reliable and cost-effective tool for colorectal cancer screening. learning objectives: . to review the evolution of ct colonography since its introduction, with a focus on data acquisition and methods of data visualisation and interpretation. . to review the currently achievable test characteristics of ctc (sensitivity, specificity, accuracy) via reference to current trial data. . to become familiar with imminent developments that may further enhance ctc test characteristics. astinitis and extension of infection from adjacent spaces (neck, pharynx, pleura or retroperitoneum). typical cases including the role of radiologic findings with respect to sensitivity and specificity, and important differential diagnosis will be discussed. the distinction between a parapneumonic pleural effusion and an empyema based on radiologic findings is often impossible. features suggesting a "complicated" course requiring interventional or even surgical treatment at some point will be discussed. features of empyema and lung abscess at ct will be illustrated as well as ct indices of severity of empyema and its effect on the underlying lung that allow some prediction of functional outcome after surgical decortication. an empyema necessitatis describes a chronic empyema that attempts to decompress through the chest wall. infectious agents include tuberculosis, actinomyces, staphylococcus and various types of fungi. it has to be differentiated from other mostly neoplastic diseases that cross fascial planes such as lymphoma or pancoast tumour. most lymphomas arise in lymph nodes or other lymphatic tissues. extranodal lymphomas arise in tissues normally devoid of lymphoid tissue. involvement of so-called extranodal organs is a common finding after staging investigation, however, and a substantial part of nhl even arises in these sites. the latter form is often referred to as primary extranodal nhl. splenic lymphoma is common in both hodgkin disease and non-hodgkin lymphomas but it may be difficult to detect by imaging techniques because lymphoma nodules in the spleen are often smaller than cm. splenic enlargement alone is not a good indicator of lymphomatous involvement. primary hepatic lymphoma is rare compared with disseminated diseases at both nodal and extranodal sites. several forms of hepatic involvement can be seen including mass lesions/nodules, diffuse infiltrative form and extrahepatic involvement of the hepatic ligament. lymphomatous involvement of liver hilum nodes often infiltrates along the hepatic artery and portal vein toward the head of the pancreas and produces an infiltrative bulky mass that involves the liver, pancreas and duodenum. primary pancreatic lymphoma is very rare and can be difficult to differentiate from pancreatic adenocarcinoma. definitive pathological diagnosis of lymphomas is often obtained using image-guided biopsy. this noninvasive procedure is important as the prognosis and management of lymphomas differ greatly from that of adenocarcinoma or metastatic diseases. staging (ann arbor classification or modifications) and response to therapy is primarily by ct. community acquired pneumonia (cap) is a major health care problem because of their high morbidity and mortality rates. patients exposed to non-hospital risks who develop pneumonia have been traditionally categorised as having communityacquired pneumonia (cap). healthcare-associated pneumonia (hcap) is a new designation for pneumonias affecting individuals residing in non-hospital health care facilities, patients undergoing outpatient procedures or therapies, and patients who have been recently discharged from the hospital setting. when the diagnosis of cap or hcap is suspected, imaging studies are mandatory for the evaluation of affected patients. a nosocomial pneumonia is defined as one not acquired in a hospital or a long-term care facility. it occurs most commonly among icu patients, predominately in individuals requiring mechanical ventilation. pulmonary infection is a major cause of morbidity and mortality in patients with impaired immune function. increasing numbers of patients are becoming immunosuppressed, because of solid organ and hematopoietic stem cell transplantation, the use of immunosuppressive agents for treating a host of inflammatory diseases, or congenital and acquired diseases such as acquired immune deficiency syndrome (aids). mildy impaired host immunity as it occurs in chronic debilitating illness, diabetes mellitus, malnutrition, alcoholism, advanced age, prolonged corticosteroid administration and chronic obstructive lung disease have also been regarded as predisposing factors of pulmonary infections. the rapid diagnosis and treatment of pulmonary infections are essential. combination of pattern recognition with knowledge of the clinical setting is the best approach to pulmonary infectious processes. acute mediastinitis is a potentially life threatening but fortunately rare condition that requires prompt diagnosis and treatment. spontaneous or iatrogenic oesophageal rupture is the by far most common cause. other causes include post-surgical medi-ask the patient for those information. at the time of mr imaging plain films and/ or results of an ultrasound exam of the shoulder, if possible, should be available. however, only such prerequisites allow to run a tailored examination protocol and to support the orthopaedic colleague with the needed answers for further adequate therapy. the patient has to be placed in the magnet in a pleasant situation to avoid artefacts due to patients movement, which usually cause motion artefacts. it is very important to explain to patients to keep quiet during the whole examination, because artefacts may need repetitions of sequences, and lead so to prolongation of total examination time. furthermore, they make diagnosis more difficult and eventually more or less inaccurate. lastly, sequence repetitions decrease patient throughput and therefore cost effectiveness. imaging in three orientations (axial, paracoronalparallel to the supraspinatus tendon, and parasagittal -parallel to the glenoid cavity) using t w (pdw) and/or t w sequences have to be performed. in case of specific questions regarding lesions of the labrum intraarticular administration of contrast agent (mr arthrography) should be considered, which is sometimes (different from country to country) possible after discussion with referring orthopaedic surgeon only. mr imaging (mri) and mr arthrography are the primary diagnostic imaging modalities applied to patients with degenerative, traumatic and sports-related shoulder lesions. in this categorical course the characteristic mr imaging and mr arthrographic features of articular pathologies of the shoulder, in particular, instability-associated injuries, rotator cuff tears and biceps tendon lesions are discussed. beside lesion detection the radiological analysis comprises the understanding of the underlying pathomechanism and recognition of possible interrelations between different type of lesions (for example, secondary impingement and associated lesions of the posterosuperior labrum). the use of classification systems for specific lesions is introduced in order to improve the radiological report. a reasonable structure for written reports is discussed comprising a brief and clear description of pathological findings with subsequent interpretation and categorisation of findings with a view of therapeutic decision making. tissue changes in the treated neck by surgery and/or radiotherapy (rt) make the detection of residual or recurrent tumour more difficult. clinical evaluation of the neck is also hampered by these changes. therefore, any (non-invasive) method helping in the detection of recurrence is welcome. in order to evaluate the treated neck, radiologists should be familiar with expected post-rt findings. histological changes post-rt will be discussed in combination with imaging examples. this knowledge will enable radiologists to recognise non-expected findings post-rt. non-expected post-rt findings can be caused either by tumour recurrence, or by complications of treatment (e.g. chondro-radionecrosis). imaging examples will be shown. especially after surgery, including various types of neck dissection, lymphatic drainage patterns of the head and neck are altered. it is important that radiologists are aware of these changes. imaging examples will be shown. ct and/or mr-findings in the treated neck may be inconclusive. in these cases, there may be an additional role for metabolic (pet) imaging. at present, the position of metabolic imaging in the imaging protocol for the treated neck is unclear. information from the recent literature will be discussed. also, examples from daily practice will be shown, with emphasis on the importance of base-line imaging after treatment and the timing of such base-line scans. this knowledge will help in understanding the current indications and limitations of post-treatment metabolic (pet) imaging of the head and neck. an increasing number of functional and metabolic imaging options reflecting relevant aspects of tumour biology have rapidly been incorporated into clinical trials and, progressively, into clinical practice. while tumour staging according to the tnm system still rules most decisions regarding treatment choice in head and neck cancer, biological information from the tumour and its microenvironment has proven to have important predictive and prognostic value heading for a tailored and individualised patient's management. ct, mr and pet are the mainstay imaging modalities to access tumour extent, both of the primary tumour, lymphatic and distant metastases and for early depiction of recurrence during patient's follow-up. hypoxia and angiogenesis, the major driving forces for tumour aggressiveness, long linked to chemo and irradiation treatment failure, can now be imaged both by pet ( f-miso or cu atsm) or mri (bold and pwi, respectively). this information is being used to define biological tumour volumes, to tailor conformational and intensity modulated radiation treatments and to select patients for specific treatments such as radiosensitizers, hypoxia selective cytotoxic and antiangiogenic drugs. overexpression/amplification of egfr (epidermal growth factor receptor) are common genetic abnormalities in scc linked to increased cell proliferation and worse prognosis. imaging techniques reflecting cell proliferation/ density ( f-flt -desoxyfluorothymidine-and diffusion-weighted mri) can be used to select patients for treatment with egfr inhibitors. this special focus session will review the use of these imaging modalities both prior, during and after treatment of head and neck cancer focusing on their specific advantages and accuracies in these different settings. contrast-enhanced ct and mri are routinely used in order to determine the precise localisation, size and anatomic extent of the primary lesion. on the other hand, positron emission tomography (pet) is the most sensitive and specific technique for in vivo imaging of metabolic pathways and receptor-ligand interactions in the tissues. a common question is which of these techniques should be used in a particular patient. the most widely used technique is ct, as it has a number of important advantages over mri, like wide availability, relative low cost and short examination time. however, ct also has a number of disadvantages compared to mri: relative low soft tissue contrast resolution, severe image quality degradation by dental fillings or other metallic foreign objects, and radiation exposure. pet in combination with ct and/or mri has a good sensitivity and specificity for the detection of primary tumour and for nodal staging as well as for detection in a single examination distant metastases, occult tumours, second synchronous tumours, and for radiotherapy planning. by combining pet with ct and mri studies, either sequentially or synchronous performed, the diagnostic accuracy is significantly higher. in those situations, it has been proven that magnetic resonance (mr) imaging is a valuable adjunct to delineate the pathology. in non-pregnant woman the radiological evaluation is less difficult. still, a multimodality approach may be needed in certain cases. in this lecture, radiological features of non-gynaecologic emergencies such as acute appendicitis, diverticulitis, renal calculi and pyelonephritis will be discussed. the purpose is to discuss the role of imaging and the potential of different methods applicable in childhood uti -with a focus on us. the imaging task in uti has changed, and new questions arouse for radiology. in addition, growing economical demands pressurise radiology to restrict imaging to those conditions where an evident benefit has been demonstrated. this efficacy-oriented approach is difficult in children due to the lack of evidence-based data. thus, controversies exist on if, when and how to investigate childhood uti, trying to minimise procedures and reduce burden on children and health budgets without missing important conditions that may pose a threat to the kidney. furthermore, modern imaging with new methods, applications and potential (e.g. contrast-enhanced voiding urosonography = ce-vus) may influence the imaging algorithm. the in utero and post-natal follow-up of foetuses with urinary tract dilatation has provided lots of information about the proper management of urinary tract malformations. nowadays, affected neonates are evaluated following standardised charts panel discussion: the three musketeers were actually four : ct, mr and pet: how to choose between modalities in head and neck cancer patients. an increasing number of imaging modalities reflecting functional and metabolic aspects of tumours have rapidly been incorporated into clinical practice. these modalities provide additional information on tumour vascularisation/ angiogenesis (ct and mr perfusion); on tumour metabolism, hypoxia and proliferation (pet using different radioactively labelled substances) and on tumour architecture and cellular density (dwi). hybrid imaging, pet-ct and, in the near future pet-mr, can provide morphologic, metabolic and functional information in a one-stop-shop examination. the choice of the best modality (ies) to answer specific questions in the diagnosis, follow-up and in the prediction of response to treatment and prognosis will be addressed. acute abdominal and pelvic pain in pregnant women may be the manifestation of various gynaecological and non-gynaecological conditions. the correct diagnosis of the causes of acute pain during pregnancy is critical to minimise maternal-foetal morbidity and mortality. although ultrasound (us) is the primary imaging investigation in the diagnostic evaluation of the pregnant patient, the role of magnetic resonance (mr) imaging in the evaluation of foetal and maternal diseases in pregnant patients continues to expand. mr imaging offers different potential advantages in comparison to us for evaluating acute abdominal and pelvic pain; these include multiplanar imaging capabilities, a higher soft tissue contrast and the ability to detect and distinguish blood from other fluid collections. when us is equivocal or nondiagnostic, mr imaging is a valuable complement to determine the exact aetiology of acute abdominal pain. the intrinsic safety and the accuracy of mri in diagnosing abdominal and pelvic disease make it an excellent choice for triage of pregnant patients with acute abdominal and pelvic pain. mr imaging provides important information that influences patient management, and it is important for the radiologist to recognise the mr imaging appearance of the common causes of acute abdominal and pelvic pain during pregnancy. this lecture will discuss the use of mr imaging for maternal diseases that cause acute abdominal and pelvic pain during pregnancy. moreover, this lecture will discuss the different mr imaging techniques to use, and will show how to detect and to differentiate the gynaecologic and non-gynaecologic causes of pain during pregnancy. in this lecture, the role of imaging in the evaluation of gynaecologic emergencies will be presented. a combined approach using both clinical findings and imaging features is necessary. accurate evaluation is important as failure to make a diagnosis may lead to serious consequences. presenting symptoms, such as pelvic pain or vaginal bleeding or discharge, may overlap with pregnancy-related emergencies and with non-gynaecologic abdominal emergencies. the range of conditions to be considered include ovarian cyst emergencies (cyst rupture, haemorrhage or torsion), infective conditions (bartholins' or vulval abscess, pelvic inflammatory disease or tubo-ovarian abcess) and acute bleeding (from inflammation, neoplasm, or trauma). pain may be related to the menstrual cycle, as in endometriosis or ruptured corpus luteum, or may be unrelated, such as in fibroid or ovarian torsion or pelvic inflammatory disease. the imaging features of these acute abnormalities will be reviewed and discussed in the context of the differential diagnoses. the major functional imaging tools for tumour management are obtained with dynamic contrast-enhanced imaging, diffusion-weighted mr imaging, and mr elastography. these acquisitions allow understanding of tumour angiogenesis and perfusion, and tumour architecture. dynamic contrast material-enhanced imaging allows assessment of perfusion parameters. diffusion-weighted mr imaging provides information that reflects tissue cellularity and the integrity of cellular membranes. mr elastography evaluates the mechanical properties of tissue such as stiffness and viscosity. to date, tumour detection is mainly based on morphologic features. however, changes in perfusion parameters have been shown as early parameters of liver metastases detection in patient with colorectal carcinoma or breast carcinoma. tumour characterisation is usually based on morphologic features. yet, perfusion parameters have been shown to correlate with microvascular density and tumour differentiation helping tumour characterisation. furthermore, apparent coefficient diffusion (diffusion-weighted mr imaging) and stiffness and viscosity (mr elastography) are significantly different in benign and malignant tumours. despite these differences, these last parameters usually do not allow definitive subtyping. functional imaging starts playing a major role in non-surgical treatment follow-up especially with targeted cancer therapy. changes in perfusion parameters, apparent coefficient diffusion, and stiffness and viscosity are observed in responders. interestingly, these changes appear promptly after treatment initiation. these functional variables are not included in recist . but might be part of response criteria in the next future. many examples of functional imaging for tumour management will be provided. learning objectives: . to understand the potential of functional imaging in tumour detection. . to understand the potential of functional imaging in tumour characterisation. . to learn about the future use of functional imaging in treatment planning and follow-up. (among others, thanks to the esur-espr working group). at birth, a confirmatory ultrasound is performed in order to evaluate the type and degree of the malformation. urinary tract dilatation are separated into mild, moderate and marked. mild and moderate dilatation will be followed by us. voiding cystogram and functional studies will be performed only if the dilatation is significant or persists. the prognosis is usually good. patients with marked dilatation would be managed more "aggressively". their work-up would be initiated as soon as diagnosed in order to diagnose cases that need therapeutic manoeuvre. for them voiding cystogram, anatomical and functional studies cannot be skipped and are important for the prognosis. long-term follow-up are needed to prevent further damage. learning objectives: . to learn which infants with antenatal diagnosis of urinary tract dilatation require imaging and when. . to become familiar with the most important differential diagnosis. . to learn about the imaging strategies in these infants. c. renal and pararenal masses: basic rules p. tomà; rome/it (paolo.toma@opbg.net) the differential diagnosis of renal and pararenal masses firstly depends on the age of the child. wilms tumour (nephroblastoma) is the most common abdominal tumour in - years old ( % of cases in children less than years old -peak age . years). renal non-wilms tumours represent a significant proportion of renal tumours in children, especially in children aged less than months or greater than years. neuroblastoma most commonly arises from the adrenal gland but can arise anywhere along sympathetic chain; it is the most common tumour in children under years of age ( % of cases in children less than years -mean age < years). adrenal adenomas and carcinomas also occur in childhood. us is the initial imaging modality to investigate an abdominal mass in children. ct or mri is used to confirm the us findings and not uncommonly add new, valuable information. concerning wilms tumour there is a very diverse approach to treatment according to geographical location. this variation in therapeutic attitude has consequences for the choice of imaging modality at diagnosis. neuroblastoma staging includes also i-mibg, and laboratory investigations (bilateral bone marrow aspirates with histochemical tests and urine catecholamine level measurements). we focus on the points under discussion: revision to the staging of neuroblastoma, the problems inherent in distinguishing nephrogenic rests from wilms tumour and the approach regarding small lung nodules in children with wilms tumour. the principle of bi-rads imaging finding should be that of "quasi-benign" type, i.e. with a very low associated risk of malignancy (< %), opening the possibility of a short-term imaging follow-up as an alternative to imaging-guided needle biopsy. however, a number of issues should be regarded as relevant to radiologists and patients. ) radiologists' differences in classification. . when needle biopsy is optioned, vacuum-assisted biopsy under stereotactical guidance for mammographic only findings (typically, microcalcifications) and core-biopsy for sonographic findings should be used; fine needle aspiration cytology should be avoided due to the high probability of benign lesions which need larger tissue samples. . when short-term follow-up (typically, months) is optioned, the same technique on which the bi-rads lesions was initially detected should be used. . in the screening setting, short-term follow-up option is commonly not considered and the reader is forced to give a dichotomic reading (recall/not recall). . the choice between the two options should be clearly discussed with the patient, including the psychological cost of waiting six months to get a conclusive report, and a really informed consent should be obtained. . till now, there is insufficient evidence for using tomosynthesis or mri for evaluating mammographic or sonographic bi-rads findings. the premise behind the bi-rads classification is to identify a group of low risk lesions that can be safely placed on follow-up rather than biopsy. in practice this can be very difficult and is anachronistic to the principles of the european population screening programmes where the radiology objective is to diagnose or discharge. the panel will discuss these dilemmas. starting in the late s an american college of radiologists task force developed the concept of guidelines to standardise mammography reporting: breast imaging reporting and data system (bi-rads). there is evidence that over time there have been improvements in consistency of use, sensitivity, specificity and positive predictive value. however, bi-rads 'probably benign; short interval follow-up recommended; less than % risk of malignancy' has always been the most difficult and controversial, with the highest levels of discordance between classification and recommendation, wide variation in both intra-and inter-observer agreement and a literature review demonstrating ppv for malignancy of between and %. management of breast disease has moved on a great deal since the s. in the main driven by increasing specialisation associated with population screening programmes. nowadays a specialist breast radiologist works with a wide variety of needle options at his hip to such an extent that the breast multi-disciplinary team increasingly feels that failure to obtain a non-operative diagnosis of discrete lesions is unacceptable. in this setting, and mindful of the medico legal consequences of 'delayed diagnosis', is there still room to allow a patient to leave the one stop clinic without a definitive diagnosis? as the leaders of the diagnostic team we also have to take account of the emotional and psycho-social consequences for our patients as well as the financial costs to patient and the healthcare economy. breast imaging reporting and data system (bi-rads) was developed for standardising reporting and include mammography, ultrasound, and mri. bi-rads category "probably benign finding -short-interval follow-up suggested" should have less than % risk of malignancy. category has been subgrouped into a, b, and c, and this influence the classification of bi-rads . mammographic bi-rads : three nonpalpable findings in this category include clusters of punctate microcalcifications, well-circumscribed and noncalcified solitary masses, and benign-appearing asymmetric density. digital mammography will increase detection of amorphous microcalcifications and the number of bi-rads (or a) lesions. intramammary lymph nodes and calcified fibroadenomas are bi-rads lesions. ultrasonographic bi-rads : a most important bi-rads lesion is a solid oval mass ("tumour") with gently lobulations, circumscribed margins, and parallel orientation. using strict criteria, the negative predictive value is approaching %. two other lesions are nonpalpable "complicated cyst" and clustered microcysts. the "complex" cyst, i.e., a mass with cystic and solid components, should be classified as bi-rads . mri bi-rads : there is lack of evidence which mri findings should be categorised bi-rads . nonmass-like enhancement (nmle) and enhancement kinetics are unique to mri. lesions less than mm ("foci") need no assessment. a challenge is mri-detected tumours < mm as morphologic analysis is of limited value. benign kinetic curves may justify short-term follow-up. in high-risk women, mri-guided vacuum-assisted biopsy should be considered. in conclusion, bi-rads is justified in diagnostic settings but should be avoided or kept at a minimum in screening. what will be the standard machine and field of the future? l. darrasse; orsay/fr (luc.darrasse@u-psud.fr) improving the sensitivity has still been an essential issue in mri, because the signal from nuclear spins relies on extremely weak magnetic interactions dominated by thermal fluctuation. to push the signal-to-noise ratio upward, the basic routes have been (i) to increase the field strength, (ii) to improve the signal detection with radiofrequency coils and (iii) to enhance the signal dynamically with contrast agents or alternative preparation techniques. the first route is the most obvious one, driven by the trends in analytical mr spectroscopy and small-animal mri. going up to or even above t represents a considerable challenge, both technically and in view to renew the mr equipment market. however, this way is limited by safety issues, radiofrequency penetration concerns and susceptibility artefacts. alternatively, improving the radiofrequency system relies on a complex electrodynamics background, where tissue conductivity and different sources of noise have to be considered. during the last half-period of mri development, radiofrequency coil arrays have appeared as a powerful mean to improve the signalto-noise and to accelerate the spatial encoding process, even able to overcome some pitfalls with high-field mri. finally, the topics of an optimal field strength has always been highly controversial because the contrast mechanisms, on which the diagnostic information is mainly founded, are essentially field-dependent. basically, the contrast mechanisms tend to be dominated, respectively, by macromolecular cross-relaxation at low field, and by susceptibility-weighted diffusion at high field. an emerging question is then to address different pathologies or organs with either general-purpose or dedicated mri systems. learning objectives: . to learn about the possibilities of gaining a jump in signal. . to understand the respective strategies of field increase and coil improvements. . to consider the probable system in . will new technologies allow a jump in sensitivity? j. hennig; freiburg/de (juergen.hennig@uniklinik-freiburg.de) imaging technologies have made breathtaking progress for several decades and in all aspects of medical imaging -mr, ct, us and pet. amongst the current developments in mri there are several areas which hold promise to redefine the boundaries of sensitivity. ultrahighfield mri with field strengths of t and more starts to reveal insights into tissue microstructure so far inaccessible to mr (or any other technique). this is still under intense technological development with some key issues to resolve notably safety issues related to the high radiofrequency power. the unique contrast and resolution does hold promise for highly relevant applications such as neurodegenerative disease, small vessel disease, ms and others -although definite clinical studies for a ‚killer application' are still lacking. c-hyperpolarisation of metabolites such as pyruvate, succinate, bicarbonate, etc. lead to an increase in detection sensitivity by factors of - , . finally, the development of targeted probes is progressing extremely rapidly and for all imaging modalities. most of this work is aimed at preclinical research, but the tremendous impact of the new insight in translational research promises to be of high impact for clinical application. will these technologies allow a jump in sensitivity? yes and no. image quality, contrast and sensitivity will grow significantly. the biggest impact will, however, lie in the combination of the new possibilities with other data -imaging as well as non-imaging. a true change in paradigm will only be achieved if the current parcellated knowledge about various aspects of the disease is unified into a comprehensive picture. learning objectives: . to review the recent developments in rf-coils and gradients. . to learn about the new measurements technologies. . to envision how these new tools will allow a huge gain in sensitivity. will new mr contrast probes compete with pet? s. aime; turin/it (silvio.aime@unito.it) purpose: molecular imaging is a new science that will have a tremendous impact in the development of innovative diagnostic tools. in the first stage of its enrolment, it has relied massively on pet/spect and optical imaging technologies because of the superior sensitivity of their probes. in the long-term, mri/mrs approaches may recover a central role, provided that further sensitivity improvements will be attained. high sensitivity mri probes have been designed and tested for molecular imaging applications. in the case of paramagnetic based systems they rely on improved design of the coordination cage as well as on the encapsulation/incorporation in proper nanocarriers. for multiple detection studies, cest as well hyperpolarized c- containing molecules have been considered. results: as molecular imaging is the evolution of biologists' in vitro work that has revolutionized the way living cells and intact tissues were investigated, mri multiplex-visualization of biological processes appears to be a key task for the forthcoming years for an efficient translation of such outstanding achievements. the search for frequency-encoding mri contrast agents represented by cestand hyperpolarized c- containing molecules have opened the interesting perspective of detecting more than one agent in the same anatomical region. this task is largely precluded to nuclear probes. moreover merging mri and nanotechnology allows the attainment of high sensitivity systems also with the classical relaxation agents. conclusion: recent achievements in amplification procedures allow to tackle the intrinsic insensitivity of mri probes to make them more competitive in the arena of molecular imaging applications. the use of imaging to monitor response to treatment has become central in the care of patients with cancer. it is crucial therefore that radiologists involved in the management of patients with cancer understand the place of imaging within the clinical context of the management of patients with cancer; that they are aware of the criteria of the accepted current criteria for assessing response and are familiar not only with new developments in imaging that act as a surrogate end-point for evaluating the success of treatment but are also aware of how imaging is used to predict the likely response early in the patient's pathway. this session will concentrate on all these aspects on the use of cancer imaging in monitoring response to treatment in patients with cancer. we are in the era of targeted cancer therapy, whether by small molecules derived from knowledge of the molecular pathogenesis of tumours, or from biological therapies emerging from our understanding of immunology and cell biology. these approaches convey new challenges for the monitoring of response. small molecule therapeutics often stabilise tumours for significant periods without producing clear reduction of masses, and for these the assessment of surrogate endpoints takes on increasing importance. the measurement of pharmacodynamics is central to early phase trials in which confirmation of on-target effects is required to determine the biologically effective dose, and in many cases this is best done by functional imaging. biological therapeutics such as monoclonal antibodies and cellular immunotherapy also need novel approaches for the determination of their actions in vivo, particularly as they are frequently tested in the setting of low level disease. randomised trials are underway to assess the contribution of functional imaging, in particular ct-pet as a means to guide therapy. the emerging data suggest that there are broad variations in accuracy, both according to the disease in question and the context in which studies are performed, even before factors such as imaging quality control and standardised reporting are included. for the future, standardisation of techniques and common quality control will play a vital part in advancing our understanding in this rapidly evolving field. tas. there is regression of portions of these arches, but several remnants normally persist. any failure in this process can result in congenital anomalies of the aorta or pulmonary vessels. these anomalies can be categorised into aortopulmonary anomalies, systemic arterial anomalies, and pulmonary artery anomalies. the aortopulmonary anomalies comprise truncus arteriosus, hemitruncus arteriosus, aorticopulmonary window, patent ductus arteriosus, and transposition of the great arteries. the systemic arterial anomalies include the anomalies of the aortic arch, of which the most common is a left aortic arch with aberrant right subclavian artery. double aortic arch is the most common cause of a vascular ring and is characterised by left and right aortic arches arising from the ascending aorta and encircling the trachea and oesophagus. a right aortic arch can have three "subtypes": aberrant left subclavian artery, mirror image branching, or isolated subclavian artery. the first type is the most common one and is the second most common cause of vascular ring. aortic coarctation, pseudocoarctation and interruption of the aortic arch are other systemic arterial anomalies. the most important pulmonary artery anomalies are idiopathic dilatation of the pulmonary trunk, absence or proximal interruption of a pulmonary artery, pulmonary arterial stenosis, and pulmonary sling. cardiovascular magnetic resonance (cmr) imaging has become integrated into the assessment pathways for congenital heart disease in both paediatric and adult patients. cmr provides a powerful tool, giving anatomical and haemodynamic information that echocardiography and catheterisation alone do not provide. extracardiac anatomy, including the great arteries, systemic and pulmonary veins, can be delineated with high spatial resolution. vascular and valvular flow can be assessed, shunts can be quantified, and myocardial function can be measured accurately and with high reproducibility, regardless of ventricular morphology. finally, cmr surpasses both catheterisation and echocardiography in providing high resolution, isotropic, three-dimensional ( d) datasets. this allows for reconstruction of data in any anatomical imaging plane, giving complete visualisation of complex congenital cardiac anomalies, without the use of ionising radiation. in the congenital heart disease, cmr can be justified for any patient in whom clinical or echocardiographic data are insufficient for monitoring, decision-making or surgical planning. due to the complexity of both the anatomy and physiology of congenital heart disease, it is essential to have a systematic approach for cmr when assessing these patients. with the development of novel ct scanners, especially the dual source ct, novel strategies of examining congenital heart disease became possible. especially newborns and infants younger years of age are difficult to examine, since compliance cannot be expected. also young children are especially susceptible to ionizing radiation and should be exposed as little as possible. in a first step the course will illustrate the underlying technical principle how to examine children in < sec with sub msv exposure. also the course will teach how to avoid sedation in children of any age. in a second step the course will show how to apply contrast media in children of all ages and what strategy to use. in a third step the course will show how to appropriately choose scanning parameters for the ct scan since the size and weight varies considerable in children as there are small to date newborns with < kg body weight up to adolescents with a body weight > kg. since little comprehensive data is available how to examine with an optimal compromise between sufficient image quality and unnecessary radiation overexposure the data of the erlangen study will be presented and discussed. in a forth part typical indications for the exam are shown and how to interpret the exam. also limits for the exams are shown. in a final step a comparison with mri is shown which method is used for which pathology. in this session contrast-induced nephropathy and nephrogenic systemic fibrosis will be discussed. measures to reduce the incidence these adverse effects will also be presented. the pathophysiology of cin is complex and not well understood. basically, a misbalance between vasodilatation and vasoconstriction takes place inside the kidney after intra-arterial or intravenous cm administration. furthermore, increased oxygen demand of tubular cells due to increased reabsorbtion of sodium and water is a second mechanism, leading to transient medullar ischaemia. identifying the patient at risk is the first step in prevention. knowledge of the patient's medical record and a recent basic kidney function are mandatory. high-risk patients should receive prevention. two major topics in cin prevention are the questions whether iso-osmolar cm cause significantly less cin than low-osmolar cm and whether hydration schedules with nahco give significantly less cin than hydration schedules with nacl . %. currently up to % of all mri examinations worldwide are performed using contrast agents, either an extracellular agent or an organ-specific agent. the extracellular mri contrast agents are chelates that contain the paramagnetic ion gadolinium which strongly affects the relaxation properties of water protons, leading to changes in tissue contrast. gd-dtpa was the first extracellular agent to be introduced in clinical practice. since the introduction of gd-dtpa in , various gadolinium chelates with different chemical properties became available for clinical use. for many years, it was believed that gadolinium-based contrast agents (gbca) a s c b d e f g principles in the use of conventional/anatomic imaging for response assessment l. schwartz; new york, ny/us (lschwartz@columbia.edu) the standard way to assess a patient's response to chemotherapy is to use computed tomography (ct) to measure tumour size using uni-dimensional (recist) or bi-dimensional (who) criteria. this methodology has changed little in the past years despite the emergence of new therapies and advances in imaging technology. measuring the changes in the size of tumours in one or two dimensions does not always capture the effects of novel therapies on primary tumours and metastases. radiographic changes in the size of tumours treated, for instance, with epidermal growth factor receptor tyrosine kinase inhibitors such as gefitinib or erlotinib or inhibitors of angiogenesis such as bevacizumab do not necessarily occur at the same magnitude or speed as observed in those individuals treated with standard cytotoxic therapies. with these newer agents, tumours respond by undergoing cystic change, central necrosis, and density changes that may not be captured by conventional measurements of the largest lesion diameter. learning objectives: . to understand and appreciate the use of conventional/anatomic imaging for response assessment in clinical practice as well as in clinical trials. . to understand general and disease-specific challenges associated with response assessment at imaging. . to recognise potential improvement in imaging and image post-processing for response assessment. pet in monitoring response w. weber; freiburg/de pet imaging and specifically pet/ct with the glucose analogue fluorodeoxyglucose (fdg) has been evaluated in a significant number of studies to monitor tumour response in patients undergoing chemotherapy, radiotherapy or targeted therapies. the clinical value of for differentiation of residual or recurrent viable tumour and therapy-induced fibrosis or scar tissue has been established for malignant lymphomas and various solid tumours. furthermore, there are now several reports suggesting that quantitative assessment of therapy-induced changes in tumour fdg-uptake may allow prediction of tumour response to chemotherapy and targeted drugs very early in the course of therapy. in non-responding patients treatment may be adjusted according to the individual phenotype of the tumour tissue. current studies investigate whether fdg-pet can be used to "personalise" treatment and to reduce the side effects and costs of ineffective therapy. in addition to fdg, several other imaging probes are in clinical studies to monitor tumour response to therapy. these include among others [ f]fluorothymidine (flt) for imaging of cellular proliferation, [ f]fluoromisonidazole for assessment of tumour hypoxia and radio-labelled rgd-peptides for angiogenesis imaging. preclinical and early clinical trials with these imaging probes are encouraging, but need to be confirmed in larger clinical trials. learning objectives: . to understand how image acquisition and reconstruction influence visual and quantitative analysis of pet studies. . to describe current criteria for assessing tumour response in lymphoma and solid tumours and recognise the limitations of these criteria. . to understand how differences between scanner models can influence response assessment by pet. panel discussion: why does the radiologist need to understand the importance of monitoring response and how it is done? : monitoring the response to treatment has become a critical part of the management of patients with cancer. the markedly improved diagnostic performance of crosssectional imaging in delineating the extent of malignancy has meant that imaging has become increasingly important as a surrogate end-point. these two factors have resulted in an increasing need for a standardisation of the criteria of response both in therapeutic trials and in clinical practice. it is vital that all radiologists imaging patients with cancer are familiar with the limitations, pitfalls and strengths of these changing criteria and also are aware of the possibility of using imaging to evaluate the changing molecular make-up of the cancer. stent-grafts is steadily increasing. therefore, knowledge of this treatment option is fundamental to provide basis for procedure planning and a meaningful radiological reporting. imaging plays a crucial role in the assessment of patients with aortic aneurysms. eligibility for endovascular treatment depends very much on detailed anatomical knowledge of the aorta, its branches and vascular access through the iliac arteries. we will review the role of imaging modalities: us, ct, mr for detection and evaluation of anatomy of thoracic and abdominal aneurysms. the strength and weakness of each modality will be reviewed and inclusion criteria for endovascular aortic aneurysm repair will be presented. the precise sizing of the stentgraft obtained from the images is certainly one of the most critical points of the endovascular procedure and a condition of its success. although it is a minimally invasive treatment it is associated with complications. there is a strong need for general radiologists to know about the most prevalent normal and abnormal findings of the post-treatment aorta. the session will end with discussion addressing the central role of imaging in pre-and post-treatment evaluation of the patients with aortic aneurysms. indications for and experience with placement of endovascular stent grafts in the thoracic aorta are still evolving. recent advances in imaging technologies have drastically boosted the role of pre-procedural imaging. the accepted diagnostic gold standard, digital subtraction angiography, is now being challenged by the state-ofthe-art computed tomography angiography (cta), magnetic resonance angiography (mra) and trans-oesophageal echocardiography (tee). among these, technological advancements of multidetector computed tomography (mdct) have propelled it to being the default modality used, optimising the balance between spatial and temporal resolutions and invasiveness. mdct angiography allows the comprehensive evaluation of thoracic lesions in terms of morphological features and extent, presence of thrombus, relationship with adjacent structures and branches as well as signs of impending or acute rupture, and is routinely used in these settings. in this presentation, we review the current state-of-the-art radiological imaging for thoracic endovascular aneurysm repair (tevar), especially focusing on the role of mdct angiography. after analysing the technical aspects for optimised imaging protocols for thoracic aortic diseases, we will discuss pre-procedural determinants of candidacy, and how to formulate interventional plans based on cross-sectional imaging. the purpose is to present the essential principles of endovascular repair of abdominal aortic aneurysms (evar). abdominal aortic aneurysms (aaa) larger than . cm should be treated. inclusion criteria for evar include satisfactory aneurysm neck morphology and suitable access vessels. preprocedural imaging involves cta from the diaphragm to the femoral arteries. several devices may be used to cover a wide range of anatomy. evar procedures are generally performed as combined procedures by teams of vasculr surgeons and interventional radiologists. patients are followed up indefinitely by cta (or ultrasound) and plain radiography. the periprocedural mortality is three times lower compared with surgery. late mortality rates are similar between surgery and evar. quality of life issues favour evar in the short and medium term. early published series reported high complication and reintervention rates for evar, although these have reduced with increased experience and improved device technology. gene-reporter imaging has been used to monitor cell-based therapies in neurology. one of the most promising applications of molecular imaging principles is the targeting of amyloid deposition in the patients with alzheimer's disease. it is hoped that early diagnosis of the neurodegeneration may lead to better therapies. the advent of hybrid-imaging will be associated with the need for multimodal contrast agents exploiting the imaging characteristics of the involved instrumentation for more differentiated visualisation of structure, physiology, and biology. learning objectives: . to understand the working mechanism and radiation of current clinical tracers. . to become familiar with the potential clinical indications and applications. . to learn about potential new tracers. panel discussion: what specific precautions are mandatory in order to guarantee contrast media safety to patients and healthcare professionals? : current contrast agents are safer than previous products. however there are still safety aspects to consider, including patient's kidney function, thyroid metabolism, allergy, radiation exposure, or specific medical history, and there is no such thing as total or complete safety. while these simple precautions may appear obsolete in the era of sophisticated technical approaches, these issues are still quite complex and crucial in maintaining a high safety level. the discussion will give guidelines and tips for achieving a high level of safety when using contrast agents. room q the abc of evar magnetic resonance imaging (mri) is the best tool to explore white matter disorders (wmd), including the most common demyelinating disease: multiple sclerosis (ms). the formation of demyelinating lesions is related to an "inflammatory attack". new techniques as diffusion tensor imaging (dti), perfusion weighted imaging and uspio enhancing can explore earlier and more specifically lesions formation. acute demyelinating lesions may resolve (remyelination) or persist as "chronic black holes". chronic demyelinating lesions lead to axonal injury and wallerian degeneration. a diffuse neurodegenerative process leading to cerebral atrophy is actually a major landmark in ms. conventional mri sequences allows visualisation of white matter lesions (seen as hyper t /flair lesions, low signal t lesions and possible gadolinium contrast enhancement), presently the basis of ms diagnosis. ms follow-up is usually clinic but imaging could provide prognosis and therapeutic parameters ("surrogate markers") even if disease progression and axonal loss are independent from "lesion load". mr spectroscopy, dti or magnetization transfer imaging (mti) provide measurement of diffuse tissue damage in clinical research, and may correlate to disease progression, as well as brain volume measurement. after exclusion of alternative diagnosis (mri "red flags" as other wmd), mri lesions dissemination in space and time criteria are the main points in ms diagnosis. the widely used revised mcdonald criteria may be replaced by more simple and efficient magnims criteria. such criteria could allow starting "modifying disease treatments" as soon as the first "clinical isolated syndrome". since blood oxygenation level-dependent (bold) signal changes have been observed using mri and modulated using neuronal stimuli, functional mri (fmri) has quickly become the most popular non-invasive functional neuroimaging technique in clinical practice and cognitive neuroscience. indeed, high-field mr scanners and bold-sensitive sequences are now widely accessible in both clinical and research settings. bold signal that relies on deoxyhaemoglin concentration is detectable without injection of external contrast media. the colourful activation maps combined with three-dimensional brain anatomy may have also made this imaging method as much attractive as controversial. numerous applications of fmri have been suggested in medicine. after a -year long history in clinical practice and thousands of scientific papers even in prestigious journals, the role of fmri remains mostly dedicated to map eloquent cortex before a neurosurgical procedure. in fact, bold fmri is challenging because the relationship between the neuronal response to a stimulus and the activation blobs relies on neurovascular coupling, haemodynamic response, mr signal detection, and complex time-series analyses. besides an obvious and partially elucidated complexity, and several concerns on the interpretation of experimental paradigms in cognitive neuroscience, fmri is based on a robust physiological and physical framework. bold signal is reproducible across subjects and mr scanners. fmri requires a rigorous methodology to acquire and analyse data, an advanced knowledge in sulcogyral and functional neuroanatomy to estimate spatial displacement and reorganisation in patient with focal lesion, and a solid experience in bold imaging to distinguish artefacts and potential confounds from appropriate results. learning objectives: . to know the key points of quality in fmri. . to know the main pitfalls in fmri interpretation. . to learn more about advances in fmri of brain perfusion. a- : the role of imaging in follow-up k.a. hausegger; klagenfurt/at (klaus.hausegger@lkh-klu.at) the goal of surveillance after evar is to prevent late rupture eurysm. high pressure els (type i and type iii el) are risk factors for late rupture therefore have to be treated. type-ii, iv and type v (endotension) els are low pressure els with a low rupture risk. thus, it is essential not only to detect els but also to classify els correctly. until recently stent graft surveillance has been performed with contrast enhanced multislice ct (msct), typically , , and months after evar and thereafter in yearly intervals. however, although msct proved to be a very effective in fu cumulative radiation exposure, repetitive contrast medium load and increasing work load have to be seen critically. therefore, alternative surveillance protocols especially including ultrasound have been evaluated. several studies have shown that a risk-adjusted follow-up regime might be most efficient. patients with low risk of aneurysm-related mortality after evar have a normal -and -month msct scan and sack shrinkage at months. in these patients regular contrast enhanced us in yearly intervals have been proven to be safe for further fu. a one yearly non-enhanced low-dose ct or a plain film may be recommended to detect distortion or migration of the stent-graft. patients with a persistent type ii el after one year need to be followed more closely and imaging modality is chosen depending on the character of the el. in most cases el classification can be made correctly by msct or us; however, sometimes dsa may be needed. panel discussion: the key role of imaging in endovascular aortic aneurysm repair : many patients with aortic aneurysms are currently treated endovascularly. therefore the number of patients imaged pre and post-procedurally is increasing. radiologists evaluating aortic aneurysms with different modalities should know the key imaging features to look for in pre-treatment as well as follow-up examinations. j.-p. pruvo; lille/fr (jppruvo@chru-lille.fr) neuroimaging is one of the fields of radiology with the most exciting recent advances. moreover, these advances show dramatically important clinical applications. some are useful for the specialised neuroradiologist, but most are critical for the general radiologist as well. in this session, we will try to address some of the most relevant issues: white matter disorders are common diseases. expertise of the radiologist is important in detecting the disease, and in the evaluation of the activity after treatment. with this respect, standardisation of criteria is desirable for appropriate medical decisions. functional imaging is a fascinating insight into the human brain. it opens new horizons and has found many applications in the evaluation of psychiatric diseases, and in the planning before treatment of brain lesions, among other potential applications. although it is obvious that we ignore much more than we know, this field is already one of the most exciting topics in neuroradiology. although these methods are still in the field of specialists, everybody should be aware of the possibilities of functional imaging of the brain and its recent developments. stroke is by excellence a clinically relevant problem. because it is an emergency situation, time really matters, and we have more than one imaging tool to explore these patients, it is mandatory to provide strong recommendations and guidelines for the radiologist, in accordance with the clinical situation and with the treatment options. at the end of this session, the attendees will be aware of important advances in the domain of neuroradiology, and will take home very important landmarks for their own clinical practice. jaundice is rare in children but imaging has a major role to establish the cause which differs according to age. in the neonatal period the leading cause is biliary atresia (ba) which consists of the obliteration of the extrahaepatic bile duct (bd) without dilatation of intrahaepatic bd and requires urgent surgical treatment to reduce the need for liver transplantation. ba must be considered when there are persistent white acholic stools and firm hepatomegaly. us shows the absence of dilated bd and in a few cases a cyst at the porta hepatis or findings of the splenic malformation syndrome, but often it is not conclusive. mrcp has not yet proved to be reliable for the diagnosis of ba. other neonatal cholestases include medical intrahepatic causes which can be identified by biological tests or histological findings and rare causes of extrahepatic obstruction with dilated bd such as lithiasis, choledochal cyst or spontaneous perforation of bd. in childhood, us easily identifies all causes of extrahepatic obstruction by showing dilated bd; the main causes include choledochal cyst (the most frequent congenital malformation, defined by an abnormally long common bilio-pancreatic channel), cholelithiasis, tumoural compression, sclerosing cholangitis, portal vein obstruction, postsurgical or posttraumatic stenosis… in all these cases, in our experience, mrcp has become the modality of choice and has replaced invasive procedures which are nowadays reserved for treatment. mdct may be useful in rare instances, for example, to differentiate calculi and aerobilia after surgery. imaging protocols and illustrative cases will be presented. neonatal bowel obstruction generally presents within hours of birth. the diagnosis of bowel obstruction is clinical but imaging is vital to determine the cause. the plain radiograph will distinguish upper from lower intestinal obstruction. the radiograph alone is diagnostic in duodenal atresia and jejunal atresia and the baby may be taken directly to surgery. if the radiograph shows multiple dilated loops of bowel, more than loops, then the obstruction is distal ileal or colonic. the radiograph may give some clues as to the possible cause, such as a large fluid level in the dilated loop proximal to an atresia. contrast enema, usually with low osmolar water soluble contrast, is used in most cases, however, to determine the cause of lower intestinal obstruction because the management of this is different for different causes of obstruction. meconium ileus and ileal atresia are the commonest ileal causes of obstruction and hirschprung's disease and meconium plug syndrome are the commonest colonic causes of obstruction. colonic atresia is rare and imperforate anus is a clinical diagnosis. radiologically guided reduction is used to treat meconium ileus. intestinal obstruction in the older neonate may be due to volvulous, pyloric stenosis, duodenal stenosis or omphalomesenteric band. in the baby born prematurely, obstruction may be caused by stricture secondary to necrotising enterocolitis. radiographs and contrast studies are also used to evaluate these conditions. learning objectives: . to learn about the most common causes of neonatal obstruction. . to understand the role of plain radiography in establishing the diagnosis. . to know when upper and lower gi contrast studies are indicated and the contrast medium used. stroke around the clock: will the challenger (ct perfusion) beat the champion (diffusion mri)? in patients admitted for a suspected stroke, time management is the most important criterion for therapeutic decisions. less than four hours and a half, all patients admitted in emergency for an acute neurological deficit without impairment of consciousness (nih < ) and presented with a brain ischaemia of less than one third of the territory of the middle cerebral artery should receive iv thrombolysis. brain imaging must exclude bleeding and quantify the extent of ischaemic damages. between . and hours after onset of symptoms, intravenous thrombolysis and/ or intraarterial mechanical or chemical thrombolysis may be indicated in selected cases. the assessment of ischaemic penumbra and vessel permeability is particularly important for the therapeutic decision. this may be evaluated either by mri or ct. mri is the technique of choice that should be used whenever possible because of the absence of irradiation and iodine-related side effects, the optimal anatomical coverage, the detection of lacunar infarct and its sensitivity for evaluating ischaemic brain damages of the posterior fossa. imaging protocol should include flair (fluid attenuated inversion recovery), t *, diffusion/perfusion and mr angiographic sequences. in case of unstable patient or if mri is not available, a non-contrast ct scan must be performed and completed by perfusion ct and ct angiography. the main advantages of this technique are the short time of data acquisition and the absolute quantification of perfusion due to the linear relation between attenuation and concentration. pneumonia is a common cause of attendance to hospital. chest infections in children are usually viral and self limiting, but sometimes chest infections can be prolonged or repeated. there are many underlying causes for this, for example, congenital anatomical causes, underlying patient susceptibility, inhaled foreign bodies and unusual organisms. one of the more common causes seen in hospital is when pneumonia has become complicated by empyema. very rarely other conditions such as kawasaki's disease or tumours can masquerade as pneumonia. this session will review these conditions together with the benefits and limitations of plain radiography. it will also consider times when other imaging modalities such as ct and ultrasound can help to guide diagnosis and treatment. in children, congenital heart disease (chd) is more frequent than acquired heart disease. diagnosis and subsequent management of patients with chd relies heavily on different and often multiple complementary imaging modalities. serial assessment of the morphology and function of the heart and thoracic vessels is needed at various stages of care. although chest radiography is frequently used for monitoring the cardiorespiratory status and complications that may arise during clinical care or intervention, it rarely provides a complete diagnosis. echocardiography, as first line imaging modality, often provides all information required for diagnosis and follow-up, especially in small children with good acoustic windows. contrastenhanced computed tomography (ct) and magnetic resonance (mr) imaging are valuable for detailed three-dimensional evaluation of the extracardiac vasculature and cardiac anatomy. in addition to being radiation free, mr has the advantage to provide both morphologic and functional information with the use of different techniques including ecg gated gradient-echo cine imaging, gadolinium-enhanced angiography and velocity-encoded phase-contrast imaging. a comprehensive mr evaluation including quantitative measurements of ventricular volumes and function as well as blood flow in vessels and across valves can give important information on long-term sequelae of the underlying cardiac defect, the significance of residual lesions, and potential complications of surgery. it is also valuable for planning and timing of future interventions. today, ct and mr have become the next line of investigation when echocardiography does not provide sufficient information, while catheter angiography is reserved for the assessment of coronary arteries, measurements of pulmonary vascular resistance and interventional procedures. over the past years, diagnostic imaging has witnessed a veritable explosion in the modalities available for studying patients. when applied to the study of the thorax, they contribute to earlier detection of abnormalities and greater diagnostic accuracy. yet, the conventional radiographic examination of the chest continues to be the most commonly performed imaging study and, when properly interpreted, continues to demonstrate a wealth of information. for a correct interpretation of the chest radiograph, several premises should be met by the radiologist: a proper knowledge of the anatomy and semiology of the thorax, stressing the need for a lateral projection, understand the importance of reviewing previous studies and try to avoid unnecessary cross-sectional studies. this lecture will address the radiological approach to imaging children of all age groups presenting with cough. this is a common symptom with very diverse causes, from acute viral infection to complex vascular and intrinsic congenital tracheal anomalies and can be the presenting symptom of acute (rapidly fatal if not managed adequately) and chronic effects of foreign body aspiration. the various causes of cough vary with age and whether cough is acute, sub-acute or chronic. this has effects on radiological investigations deployed, and thence on subsequent clinical management. we will present a pragmatic approach to radiological investigations in this diverse set of patients, using simple algorithms and illustrate the more important (and often rarer) causes of cough with discussion around deployment of examinations that are fit for purpose using alara principles. guidance on imaging algorithms and ct technique will be given to children with this important presenting symptom of myriad diverse pathological processes. the attendee will learn the value and optimal use of imaging with tips on optimising ct technique as fit for purpose. learning objectives: . to learn about the differential diagnoses in the very young child and in older children. . to learn which imaging modalities best help to determine the final diagnosis in the various age groups. . to understand radiation protection requirements in chest ct examinations. clinical examples of dual energy ct l.s. guimarães; viseu/pt (luis.s.guimaraes@gmail.com) dual energy (de) computed tomography (ct) allows the discrimination of different materials, which has several clinical applications. the ability to differentiate calcium from uric acid renal stones allows the identification of patients that will benefit from drugs that alkalinise urine. a musculoskeletal application of the same technique is in tophaceous gout, where the crystals can be differentiated from bone. postprocessing of ct angiography images can be facilitated using de techniques to identify and remove the calcium signal. such an approach removes both bones and calcified plaques. "virtual non-contrast" images can be obtained by identifying and removing the iodine-containing voxels of contrast-enhanced ct images, simulating non-contrast images and potentially eliminating the need for some non-contrast acquisitions, but it should be realised that small misclassifications could result in missing tiny caliceal tip stones, for example. dect also allows the utilisation of low energies without the prejudice of unacceptable noise. since iodine signal is significantly higher at low energies, disease conspicuity can be improved. this is particularly beneficial in the liver (for hepatocellular carcinoma identification), in the pancreas (for visualisation of hypo-and hypervascular lesions), and in enterography (to increase the identification of hyperenhancement). the approach for displaying the enhanced iodine signal may be one of blending information from the two energies (into a single grey-scale image), or via an iodine-only view. further validation of dual energy techniques and their limitations is needed to understand the patient populations in which such techniques can be utilised and where conclusions based on dual energy data can be trusted. ct perfusion imaging is a quantitative technique that employs rapid sequences of ct images after bolus administration of intravenous contrast material to measure a range of physiological processes related to the microvasculature of tissues. ct perfusion parameters can provide surrogates for tissue hypoxia as well as the physiological processes such as vasodilatation that represent vascular responses to hypoxia. although the basic techniques for dce-ct have been available for decades, more recently a range of technological advances have contributed to the greater applicability of perfusion ct in the clinical environment including wider ct detectors, shorter gantry rotation times, 'table-toggling', radiation dose reduction and software corrections for image mis-registration due to respiratory or other patient motion. consensus guidelines are now available for the acquisition and processing of ct perfusion studies for the brain and body. to date, the main applications of ct perfusion imaging in stroke have been the confirmation of stroke diagnosis and extent, identification of penumbra and selection of patients for thrombolysis. the main applications in oncology have been in lesion characterisation, risk-stratification and assessment of treatment response. computed tomography (ct) systems have provided three-dimensional (x, y, z) data since their clinical introduction in the s. this session will address the extension of ct imaging into the fourth and fifth dimensions. the rapid acquisition capabilities of modern ct scanners open the door to the fourth dimension -time -where a time-course ct scan can provide clinical information about blood flow, perfusion and other physiological measures regarding organ function. the door to the fifth dimension -energy -is opened by dual energy image acquisition techniques, which are made possible by dual source ct scanners or rapidly switching x-ray source ct scanners. dual energy ct images can be manipulated to provide quantitative information with regard to the elemental composition of tissues, which, in turn, can be used to differentiate between bone and iodine contrast, with many other applications possible as well. modern ct scanners now provide multidimensional data sets characterised as i (x, y, z, t, e), and the additional information provided by these five-dimensional data sets provide genuinely useful clinical information which add to the diagnostic potential of computed tomography. x-ray computed tomography (ct) usually measures the attenuation of the patient or object cross-section in question at a fixed chosen voltage value; the result is presented as the linear attenuation coefficient µ expressed in hounsfield units (hu). dual energy ct (dect) acquires data at two different mean energies and evaluates the differences in attenuation. dual energy ct imaging has been a topic since the s. the acquisition modes have changed over the years from two separate scans at different voltages to single scans with rapid kv-switching, and dual source ct operating with different voltages and pre-filtrations. these concepts will be explained in detail including future options of energy-discriminating detectors. the basic physics principles remain the same for all acquisition schemes. the two independent measurements allow separating two tissue characteristics contributing to attenuation. for example, low and high effective atomic number material densities can be determined in the so-called basis material decomposition. arbitrary combinations such as monoenergetic or electron density and effective atomic number images can then be provided. these will be illustrated and explained by examples such as differentiation between plaque and contrast agent or virtual unenhanced abdominal organ imaging. dect meanwhile offers quite a number of accepted clinical applications which will be covered in a separate talk. ous lesions (adenomas) and curable early-stage cancer, screening for crc has high potential for reducing incidence and mortality of the disease. nevertheless, the benefit of population-based crc screening needs to be weighed against potential risks, psychological distress and required resources since only a minority of the population develops the disease and may thus benefit from screening. several industrialised countries offer faecal occult blood testing as a primary screening tool, with positive test results being followed up by colonoscopy. some countries, such as the united states and germany, also offer colonoscopy as a primary screening tool. while the results of randomised controlled trials regarding screening colonoscopy are still outstanding, there is evidence from observational studies that support its effectiveness in population-based crc screening. recent evidence suggests that colonoscopy, when performed in the community setting, is more effective in protecting from neoplasms in the left colon and rectum than in the right colon. decision-analytic models support the (cost)-effectiveness of screening colonoscopy but results vary, among others, according to compliance, (country-specific) cost estimates and screening schedules. learning objectives: . to learn about the incidence and prevalence of colorectal cancer and its precursors in the target population of screening. . to understand the rationale for colorectal cancer screening. . to become familiar with estimated (cost) effectiveness of colorectal cancer screening when using optical colonoscopy as a primary screening tool. facts from the statistician (true for once?): how accurate is ct colonography the issue of diagnostic accuracy of ctc for crc and polyps has been debated for a long time, because of the conflicting results published in the literature. those results led researchers to design three important studies: two large, multicenter trials testing the performance of ctc in comparison with cc in asymptomatic individuals (acrin and impact) and one randomised, double-arm trial (siggar) conducted on symptomatic patients with the aim to detect crc. both the acrin and impact reported per-patient sensitivity of % for polyps > mm and - % for polyps larger than mm; per-patient specificity was extremely high, over %, independent of lesion size. the major drawback of the acrin was represented by the poor positive predictive value (ppv) ( % for polyps ≥ mm); a definitely better ppv was documented in the impact trial ( % for lesions larger than mm) as well as in studies obtained in high-experienced centers. however, the negative predictive value in both the acrin and the impact was rather high, approaching %; this is extremely important in order to reassure negative patients about the significance of the examination. excellent results were also obtained in the munich colorectal cancer prevention trial, a single-center study where around asymptomatic subjects underwent low-dose ctc in comparison with other screening tests (cc, sigmoidoscopy and fobt). despite the good results there are still some open issues: the significance of diminutive (< mm) polyps, the management of intermediate ( - mm) lesions and the detection rate for non-polypoid, flat lesions. colonoscopy is a very useful and important method to examine the colon. in recent years virtual colonoscopy has become a popular alternative to optical colonoscopy. disorder of the colon includes colorectal cancer, which is one of the most common cancers worldwide and presents a threat to life since the mortality is almost %. colorectal cancer screening has been shown to be an efficient method to find early forms of colorectal cancer and also to reduce mortality in this disease. in this session we will discuss the various methods used to examine the colon, how useful they are and the economy behind using them. facts from the epidemiologist: incidence, prevalence, rationale for screening, standard results of optical colonoscopy. colorectal cancer (crc) is the third most common cancer in the world, with about million new cases and more than . deaths per year. incidence of crc strongly increases by age and is higher in men than in women. the lifetime risk differs between countries and is about % in the united states. because most cases of crc develop from removable precancer-stabilisation complex system a dual echo t /dp sequence could be performed in a sagittal-oblique plane drawing an imaginary line which on an axial image used as reference plane, is done perpendicular to popliteous tendon. gradient echo (ge) sequences are useful in cartilage and traumatic pathology. t -weighted sequences understimate cartilage thickness since cortex and cartilage have the same signal. pd sequences may have the similar signal for cartilage and adjacent joint fluid, obscuring defects; fat suppression solves this. if available, acquisitions performed in orthostatism could give further information in the assessment of meniscal and patello-femural pathology. learning objectives: . to understand the influences of patient positioning, scan parameters and magnet/coil technology on image quality. . to learn how to optimise scan protocols to maximise patient throughput without compromising diagnostic quality. . to recognise how and when to modify scan protocols to answer specific clinical questions. this talk will review appearances of normal anatomic variants identified at mri of the knee including discoid meniscus, transverse meniscal ligaments meniscal flounce, ossicles, and pseudotears. subsequently the talk will focus on diagnostic pitfalls reflecting both imaging technique and variations in anatomy such as the effect of motion, imaging plane and anatomic variations in patella shape. mri of the knee is one of the most frequent performed investigations, thus indicating that common abnormalities need to be familiar to the general radiologist. a simple and straightforward method of enabling assessment of clinically relevant anatomical entities is based upon a (pre)structured radiology report. use of speech recognition facilitates implementation of pre-structured reporting in a kiss manner. since one now is enabled to structure the report, using digital tools, terminology used should be discussed between the reporting radiology department and the clinical partners. it is mandatory that the important message, the clinical relevance of the information that radiologists put in their report, is understood by the referring clinician in the same manner as it was meant to be. we should consider the background of our referring physician, and tailor our report, both in chosen terminology as well as detailed information. when working in a centre in which both specialised orthopaedic surgeons as well as general practitioners (gps) or sports physicians are referring for mri, each of the three needs a tailored report, especially considering advise for secondary referral: gps and sports physicians will need different approach, compared to medical specialists. also, the knowledge of terminology used should be considered. the presentation focuses on common encountered pathologies, considering common abnormalities. emphasis is given to common abnormalities, and pitfalls that should be dealt with. some sports specific entities are discussed. implication of clinical correlation is stressed. the presentation will include casebased clinical illustrations. learning objectives: . to review the imaging appearances of common abnormalities. . to understand the use of terminology to describe pathological findings. . to learn how to structure a radiological report to ensure clarity and brevity. of view and the "third eye" colonoscope are under investigation. at the moment, however, the combination of both, exact diagnostic and sufficient therapeutic action at the same time, is the reason why optical beats virtual in this particular indication. since its evolution ct colonography has been advocated as a safe well tolerated alternative to colonoscopy, particularly in the context of colorectal cancer screening. notably perforation rates are lower than those of colonoscopy and cardiovascular effects are less. however, patient preference studies have produced mixed results and it is not clear if standard ctc will increase compliance with screening. furthermore, issues over apparent inferior sensitivity for polyp detection compared to colonoscopy, need for prolonged radiologist education and radiation dose exposure have been cited as barriers to widespread implementation in a screening context. technical developments in recent years are beginning to address these issues. use of reduced laxative regimes supplemented with faecal tagging have shown good results with superior patient acceptability. software developments have also speeded up dataset analysis -notably panoramic or "virtual pathology" d views reducing or eliminating hidden areas within the colon. furthermore, computer-aided detection software is increasingly fit for purpose and good data show a positive effect on radiologist performance both in the research setting and day to day clinical practice. this presentation will review the data on patient preference and safety and highlight developments in those areas in which ctc holds advantages over conventional endoscopic techniques. discussion will focus not only on colon cancer screening but also the on data supporting ctc in older symptomatic patients who are at higher risk of adverse events during colonoscopy. learning objectives: . to review the patient safety of ct colonography and conventional colonoscopy. . to review advanced computer techniques for maximising visualisation of the colon during ct colongraphy including d visualisation methods and computer aided detection. . to review the data on ct performance with particular emphasis on patientfriendly reduced laxative regimens. . to consider patient preference data. osteoporosis, osteomalacia, hyperparathyroidism and paget's disease have conventionally been considered the disease entities that comprise metabolic bone disease. the lecture will follow this convention and discuss the imaging characteristics that suggest osteoporosis in the elderly or anorexia nervosa in younger patients by the distribution of fractures and marrow changes as well as the role of bone mineral density evaluations in osteoporosis. oncogenic osteomalacia will be discussed in the context of appropriate imaging for detecting the tumour and emphasise the basic biochemical abnormality that most radiologists should be familiar with that leads to the diagnosis. radiologists most frequently encounter hyperparathyroidism in renal osteodystrophy with over a million patients worldwide kept alive by dialysis. dialysis-induced imaging changes such as amyloid and the spectrum of findings from long-term dialysis will be shown and demonstrated. paget's disease is on the decline. its distinctive mri signal characteristics in uncomplicated cases, in contrary to most bone lesions encountered in clinical practice, will be explained. learning objectives: . to get an overview of the entities that are considered in the realm of metabolic bone disease. . to understand common, less common and rare findings. . to learn about some imaging findings that occur as a consequence of treatment. osteoporosis is of great socioeconomic impact, as approximately % of all postmenopausal women have osteoporosis in developed countries. ageing of populations worldwide will be responsible for a major increase of the incidence of osteoporosis in postmenopausal women. in , the who working group defined osteoporosis according to measurements of bone mineral density (bmd) using dual energy x-ray absorptiometry (dxa) as a bone density t score at or below . standard deviations (t score) below normal peak values for young adults. the relative risk of a fracture is between . and . for each unit decline in spine or total hip t score. due to limitations of the t-score concept a -year risk calculating tool has been developed by the who to determine intervention thresholds. fragility fractures are, however, not only related to bone mineral density (bmd). trabecular bone microarchitecture is a significant determinant of the bone's mechanical properties and is thus of major clinical relevance in predicting fracture risk. trabecular bone structure analysis can be based on images from multidetector computed tomography, high-resolution peripheral computed tomography, highresolution mri and projection radiography. advantages and disadvantages of the different methods depend on radiation, costs, availability and a reasonable time for in vivo scanning. the purpose of this refresher course is to demonstrate bone ablation. the main aim of thermal tumour ablation is to destroy the tumour using ionic movement to kill the malignant cells without damaging adjacent vital structures. multiple sources of energy have been used to induce cell death. rf energy is an electromagnetic one. radio waves emanate from the non-insulated distal portion of the electrode. heat is produced by resistive forces (i.e., ionic agitation) surrounding the electrode as the radio waves attempt to find their ground. other type of ablations (using different wave lengths) include microwave, electoporosis, laser and radiofrequency ablation is still seeking its place among bone interventional techniques. this presentation is an overview, in an area of non-vascular interventions in the spine and the appendicular skeleton. it will explain the patient selection, indication and possible approaches to a bone lesion. available material will be discussed, as well as possible combinations of them in order to yield maximum results, while reducing possible drawbacks. over the last two decades, open nephron sparing surgery has become the preferred surgical alternative to nephrectomy for treatment of patients with a single, small (< cm) localised renal mass and a normal contralateral kidney. because - % of newly diagnosed small renal masses are identified incidentally on crosssectional imaging, often in elderly patients, less invasive surgical nephron sparing alternatives have been advocated, including laproscopic partial nephrectomy and laproscopic cryoablation, for select tumours in an effort to reduce surgical mortality and morbidity while preserving renal function. percutaneous image-guided ablation offers potential advantages over surgical methods including the minimally invasive nature of the procedure, less mortality and morbidity than surgery, shorter hospital stay, and quicker recovery. local tumour control rates of up to % have been reported for small tumours. central tumours and tumours greater than cm in diameter are more difficult to successfully treat with local tumour progression occurring % of central tumours. tumours larger than cm in diameter typically require overlapping ablations and incomplete treatment can result from residual tumour at the ablation interfaces. while in some reports, local tumour progression occurred in up to % of tumours larger than cm, in contrast, others have shown that all exophytic tumours, despite their size, can successfully be treated using multiple overlapping ablations and complete treatment may require more than one ablation session. the most clinically relevant and potentially avoidable complication is ureteral injury with resultant obstruction of the intrarenal collecting system. percutaneous image-guided radiofrequency ablation (rfa) represents as a safe and effective minimally invasive procedure in selected patients with unresectable or medically inoperable lung malignancies. when adequately performed, in selected patients, the procedure is associated with over % immediate technical success rate and relatively low incidence of major ( - %) and minor complications ( - %). pneumothorax represents the most frequent complication (up to %) but presentation, the worse the outcome in adulthood. avn is a significant complication. ct/mr assists orthopaedic planning. imaging strategy: plain radiographs. small children localise pain poorly. whole limb radiographs may be needed. us detects hip effusions but cannot differentiate pus from effusion. nuclear medicine may need sedation for scanning phase. a full bladder may obscure the pelvis. mr is sensitive for soft tissue pathology and marrow disease included in the area imaged. radiographs are needed before mr, which is insensitive for bone detail. other causes: spinal problems -discitis, avulsions of muscle origins, trauma -toddler's fracture, diffuse bone disease, localised bone disease; blind areas: spine, pelvis/sacroiliac joints. learning objectives: . to learn about the imaging approach to the child with suspected developmental hip dysplasia. . to become familiar with the differential diagnosis in the older child with a limp. . to learn which imaging modalities best help to arrive at a diagnosis. skeletal injuries are the most common findings noted on imaging studies in cases of child abuse. in infants, certain lesions, such as the classic metaphyseal lesion and posteromedial rib fractures are sufficiently characteristic to point strongly to the diagnosis of inflicted trauma. other fractures are less specific for abuse, but when correlated with other imaging findings and clinical information, their presence may add strong support for the diagnosis. many of the most specific injuries produce subtle radiographic alterations, and meticulous technique, adhering to a strict imaging protocol, is vital in providing optimal detection of the fractures. in recent years, increasing attention has been given to those conditions that may simulate inflicted injury. a variety of normal variants, naturally occurring diseases and accidental injuries may be confused with the findings of child abuse. other conditions, real or hypothetical, may be suggested by consultants in medico-legal proceedings. to be credible, a witness must be prepared to counter reckless and irresponsible testimony given by uninformed or biased medical "experts". to ensure that investigators and finders of fact are provided with testimony that is clear, concise and accurate, the radiologist must be fully informed and prepared to address the radiologic alterations and their significance to a reasonable medical certainty. learning objectives: . to learn in detail the investigation of a child with suspected non-accidental injury. . to learn about the typical skeletal injuries seen on plain radiography and the differential diagnosis. . to understand when further imaging is required and which imaging modality is best. acute osteomyelitis and septic arthritis are diagnostic and therapeutic emergency in children. diagnosis remains challenging because of their variable expression (acute, subacute, chronic stages, causative agents and hosts). imaging strategy relies on a multimodality approach including radiographs and ultrasonography as first line examinations with a complementary role for bone scintigraphy and mri. mri is especially useful for misleading challenging presentations (such as axial skeleton, pelvis and calcaneus osteomyelitis) and for detection of reversible osteocartilaginous ischaemia requiring urgent surgical drainage. juvenile idiopathic arthritis (jia) is an heterogeneous group of chronic inflammatory disorders that are classified on their mode of onset (systematic, pauci and polyarticular diseases). it is a diagnosis of exclusion and imaging plays a major role in the differential diagnosis. radiographic scoring systems of jia are different from adult scoring systems with less emphasis on joint space narrowing. doppler-ultrasonography and mri play an increasing role in a the assessment of disease activity and response to treatment. learning objectives: . to learn about the imaging approach to a child with suspected osteomyelitis and septic arthritis and to outline the classical features. . to learn how best to investigate a child with suspected arthropathy and the specific findings. . to understand the differential diagnoses of bone and joint infections. semiquantitative scoring method. vertebral morphometry is based on radiographs or scans from dxa-machines calculating ratios of vertebral heights with normal values to identify vertebral fractures. great need for early, accurate and reliable imaging indicators of tumour response to anti-angiogenic drugs which is ultimately defined by overall survival rate, but is mostly based on changes in number and size of measurable tumour "targets", i.e. blood vessels. thanks to its characteristics of high temporal and spatial resolution in limited fields and lack of ionising radiation and side effects (i.e. easy repeatability), contrast-enhanced us (ceus) is currently the simplest and also most reliable imaging modality for detection, analysis and quantification of intratumoural macro-and microvascularity (up to μm in diameter). even minimal changes can be easily detected and quantified with ceus during anti-angiogenic treatments: early disappearance (or severe reduction) of tumoural vascularisation is highly predictive of complete (or significant) response even before tumour volume decreases. initial clinical studies were conducted on hypervascular hepatic metastases (e.g. metastases from gists), but recently also hypovascular hepatic metastases and primary cancers of different organs are being assessed. for the quantitative analysis of tumour vascularity changes several parameters (peak intensity, time-to-peak intensity, area under the curve, etc). are currently being investigated and specific perfusion software connected to working stations are being developed and clinically tested. the clinical application of mr-mammography (mrm) in the last years has shown that this imaging tool has had significant diagnostic advantages in the field of breast diagnosis, e.g. the proof of small breast cancers, the differentiation between scar and recurrent tumours, the detection of multifocality/multicentricity, the search for primary tumours, the delineation of implants, etc. the clinical use of mr-mammography is steadily increasing. however, many still describe a "high sensitivity but a low specificity", which is wrong. achieving a high quality is not an easy task; a lot of teachers have to be learned and considered. this refresher course is focussing on high quality concerning technical and diagnostic aspects, especially focussing the question of improving specificity. dynamic contrast enhanced computed tomography (dce-ct) is a noninvasive method showing haemodynamic changes in living tissue in various oncologic and non-oncologic applications. recently, it has gained an increased popularity for studying malignant tumour blood supply and formation of new vessels, also known as angiogenesis, that plays a critical role in the growth of cancer. technical remarks: repeated rapid ct scans are acquired at the same location to allow determination of time-attenuation curves (tac). several quantitative parameters are assessed: tumour blood flow (tbf) (ml/min/ g), tumour blood volume (tbv) (ml/ g), permeability surface product (psp) (ml/min/ g) and mean transit time (mtt) (s) are calculated using dedicated perfusion software. indication: perfusion ct may be used by the distinction of benign from malignant lesions when conventional methods are unreliable, may improve staging by demonstrating occult hepatic metastases, may guide biopsy to the tumour region most likely to be of highest grade. several tumours with higher perfusion are more sensitive to the chemoradiation than that with lower perfusion. findings at dce-ct images after chemoradiotherapy are a significant predictor of early tumour response and overall survival. its applications have been stimulated by the development of anti-angiogenesis therapy for monitoring the effects of therapy and additionally may be used as a noninvasive tool in detection of hepatic toxicity of chemotherapy. in the future, new prognostic information could impact clinical management. studying the response of cancers by measuring changes in their blood flow may provide useful information on oncologic patients for managing cancers in the future. advanced imaging is now widely utilised in the research and clinical settings. in the clinical setting, qualitative, semi-quantitative and quantitative approaches such as review of colour-coded maps to roi analysis and analysis of signal intensity curves are being applied in practice. we will also compare the relative advantages for t dce mri with t * dsc mri in the estimation of perfusion and permeability metrics in the clinic as well as some more automated histogram analysis methods. the role of advanced imaging in the characterisation of tumour biology and different pathologies will be reviewed. differentiating between recurrent tumour and therapeutic necrosis is often a challenge. we will review the role of advanced imaging and also the effects of anti-angiogenic therapies on tumour microvasculature/microenvironment resulting in changes in diffusion, perfusion and mrs. pseudophenomenon has made conventional imaging with gadolinium contrast agent almost obsolete necessitating mechanistic techniques to differentiate entities such as pseudoprogression which is seen more commonly as a result of therapy with temozolomide and radiation for high grade gliomas as well as pseudo-response and pseudo-pseudoprogression. lastly to combine and apply these different imaging techniques in a multi-parametric algorithmic fashion in the clinical setting can be shown to increase our diagnostic specificity and confidence. learning objectives: . to become familiar with different advanced mr techniques used in brain tumour imaging. . to learn the role of permeability, perfusion, diffusion imaging and mr spectroscopy in characterising brain tumour biology and differential diagnostics. . to become familiar with a complete imaging protocol, which can be implemented in a multi-parametric algorithm fashion in brain tumour diagnosis. monitoring and prediction of treatment response p.c. maly sundgren; lund/se (pia.sundgren@med.lu.se) high-grade gliomas have a poor survival rate despite improvements in surgery, radiation and chemotherapy. a contributing factor to the poor survival is the inability of currently available imaging techniques to accurately delineate the tumour which results that targeted focal treatment my not be effective. conventional imaging is not able to give an early assessment of the effectiveness of radiation and/or chemotherapy. early identification of patients with a poor treatment response or who suffer from tumour recurrence can be of great advantage: it provides the opportunity to adjust individual more rapidly, and sparing patients unnecessary morbidity, and breast mri has shown diagnostic sensitivities of - % for invasive breast cancer; however, specificities have been reported significantly lower with values between % and %. the specificity of breast mri is in a routine clinical setting based on the evaluation of morphologic features and relative "slow" dynamic characteristics of enhancing lesions. the only moderate specificity that is achieved using these characteristics can result in a significant number of false positive findings during, for instance, screening or pre-operative imaging. these findings will often require short-term follow-up, target ultrasound with biopsy or even mri-guided biopsy. fast dynamic imaging, spectroscopy and diffusion weighted imaging (dwi) have been described to have potential for improving the specificity of breast mri. the use of fast dynamic imaging sequences result in a more accurate evaluation of the initial enhancement of the lesion. in combination with pharmacokinetic modelling this can result in a more quantitative evaluation of enhancement. in proton-mr spectroscopy (mrs) the presence of a choline signal, a cell-membrane precursor, in breast lesions can be used to differentiate benign from malignant lesions. the specificity of mrs varies between and % in literature (average %). dwi has also shown potential in differentiating between benign an malignant lesions, but, like with other techniques, varying specificity values are reported ranging between % and %. in this presentation the value of these techniques for improving the performance of breast mri will be discussed. although bedside chest radiograph is one of the less elaborate imaging examinations in our diagnostic armamentarium, it remains the most frequent radiologic procedure performed in intensive care patients. despite its limitations chest imaging is an important tool in the management of the critically ill patient. though the advent of digital radiography has vastly contributed to improved image quality of the bedside radiographs, optimal positioning and technique remain a real challenge for the performing technologist. in addition, the interpretation of chest radiography in the critically ill patient poses a challenge for the radiologist, because findings are frequently unspecific and lung opacifications have similar appearances in a variety of different cardiopulmonary pathologies. clinical information and an interdisciplinary approach are therefore crucial for optimal interpretation of these chest radiographs. the american college of radiology has established expert recommendations for the use of bedside chest radiography. current recommendations suggest that routine daily chest radiographs should be reserved for patients with acute cardiopulmonary problems and in patients receiving mechanical ventilation. acquisition of a portable chest radiograph is recommended after insertion of endotracheal tubes, central venous catheters, pulmonary artery catheters, chest tubes, and nasogastric tubes. therefore, knowledge of correct positioning of catheters, tubes, and monitoring devices and of various malpositions and associated complications is essential for the interpreting radiologist. chest ultrasonography (chus) is a useful imaging tool in patients at intensive care units, because of its simplicity and reproducibility. however, there are some limitations such as low specificity of some crucial sonographic signs and limited searching area (soft tissues, pleural cavity and lung consolidations). the icu patient can be examined in supine or sometimes in lateral or partly lateral position using intercostal spaces as an acoustic window. supine analysis of the anterior chest wall rules out pneumothorax, while lateral approach detects clinically relevant pleural effusion and parenchymal consolidations. chus is a method of choice in detection as well as in characterisation and volume estimation of free and/or loculated pleural fluid. with chus we can explore and characterise lung consolidations from the moment they reach the visceral pleura. they can be in contact with pleural line or can be observed through an effusion. however, in case of subcutaneous emphysema and/or diffuse oedema of soft tissues the lung parenchyma can not be reliably assessed. also, in the question of pulmonary embolisms in critically ill patient, chus could be contributive. sometimes small pleural effusion can be visible with some peripheral lung tissue consolidations indicating minute pulmonary infarction. typical pulmonary infarction is triangular tissue consolidation with air bronchogram and absence of doppler blood flow signals within consolidated lungs but cect for confirmation is mandatory. chus exploration of the diaphragm can reliably evaluate respiratory movements since pleural effusion even substantial does not affect the amplitude of diaphragmatic excursion. learning objectives: . to understand the advantages and limitations of bedside thoracic ultrasonography. . to learn about the us findings of pleural and parenchymal diseases suitable for bedside ultrasonography. . to review current guidelines for estimation of pleural effusion volume. delay in initiation of other maybe more effective treatment. in recent years, different functional imaging approaches such as dynamic contrast-enhanced (dce) and dynamic susceptibility-weighted contrast (dsc) mri, diffusion-weighted imaging and spectroscopy have been complementary used for diagnosis and treatment response. in this lecture different advance mr and ct imaging methods as well as the traditional way of monitoring to assess treatment response will be discussed. in addition, a novel recently published promising technique will be described -the parametric response mapping (prm), a novel voxel-wise analytical method of monitoring physiological and environmental changes in a tumour volume during treatment will be presented and compared to the traditional methods used. finally, the aim of the lecture is to consolidate the present knowledge and novel ideas in brain tumour imaging for future monitoring of treatment response and the possibility and limitations for future individualisation of cancer therapy. learning objectives: . to gain an understanding of the present traditional model for the treatment cycle for brain tumours and how they are monitored. . to learn about different imaging biomarkers for early assessment of brain tumour treatment response that might result in individualisation of cancer therapy. . to consolidate present knowledge and ideas in brain tumour imaging for future brain tumour therapy and monitoring of response. radiation necrosis and pseudo-progression vs recurrent tumour pseudophenomenon has made conventional imaging with gadolinium contrast agent almost obsolete necessitating mechanistic techniques to differentiate entities such as pseudoprogression which is seen more commonly as a result of advanced multimodal therapeutic concepts. advanced, non-enhanced and contrast enhanced mr imaging techniques include mr-spectroscopy, perfusion mr imaging, dynamic contrast enhanced mri and diffusion tensor mr. in the presentation we will analyse the application of those techniques in brain tumour assessment with focus on the post-therapeutic brain to differentiate therapy induced from tumourinduced changes. the results of the available studies in literature, all with relatively limited patient numbers, indicate that the combination of functional mri proved to be useful in the post-therapeutic workup of gliomas, lymphomas and metastatic disesease. the typical patterns of tumour recurrence and the different therapyinduced effects will be presented. in perfusion (dsc-mri) and dynamic contrast enhanced magnetic resonance imaging (dce-mri) the signal intensity measurements of the tumour reflect a composite of tumour perfusion, vessel permeability, and the extravascular-extracellular space. in contrast to conventional enhanced mri, which simply presents a snapshot of enhancement at one time point, both techniques permits a fuller depiction of the wash-in and wash-out contrast kinetics within tumours, and this provides insight into the nature of the bulk tissue properties on its microvascular level. with the strong demand in drug development the identification of biomarkers that can assess tumour microvascular properties noninvasive dynamic mri is the method of choice to assess tumour response and to identify atypical tumour response findings. indications and spectrum of pathological findings functional abnormalities of the pelvic floor represent a significant health-care problem, as they affect approximately % of older multiparous women. moreover, nearly . surgeries in united states are annually performed to correct those disorders. the success of medical and surgical therapies relies on the correct classification of dysfunction and identification of the pelvic compartments involved. clinical classifications of pelvic floor abnormalities are primarily topographic, rather than functional. however, as the pelvic floor muscles tend to act as a unique functional entity, their dysfunction usually leads to dysfunction of more than one organ system (genitourinary or gastrointestinal), resulting in a wide spectrum of symptoms variably associated, including dysuria, urinary incontinence, uterine prolapse, anal or pelvic pain, obstructed defaecation, rectal prolapse or faecal incontinence. because of the variability of symptoms and complexity of physio-pathological mechanisms, diagnosis of pelvic floor disorders is usually achieved by combining different diagnostic tools. dynamic mri of the pelvic floor has emerged as an alternative and effective modality for assessing and understanding of these disorders. it currently offers a complete morphological and functional evaluation of all three compartments at the same time. constipation and pelvic organ prolapse are the most common indications for a dynamic mri. the physio-pathological features of the main pelvic floor disorders and their mri findings will be shown in detail. mr images and movies, with particular regard to posterior pelvic floor abnormalities, such as rectocele, rectal invagination and prolapse, enterocele, descending perineal syndrome and spastic pelvic floor syndrome, will be illustrated and discussed. the ability to perform and interpret dynamic pelvic floor imaging is essential for the modern gastrointestinal (and indeed urogynaecological) radiologist. the other speakers in this session will be reviewing indications, pathologic findings, clinical relevance, and mr technique. although i will detail the mr and fluoroscopic techniques used at my institution, i will focus on the specific technical and diagnostic advantages and disadvantages of both dynamic pelvic mr and conventional fluoroscopic evacuation proctography (defecography). many practitioners wising to initiate this type of imaging will wish to know what modality to chose and existing practitioners using fluoroscopy will possibly be interested in migrating their practice to mr. i will illustrate what imaging findings are best imaged by each technique, continually referencing this to whether the finding is ultimately important to the referring clinician or not. using examples from our own research programme and clinical practice, i will illustrate why we have migrated our practice exclusively to dynamic mr. although dynamic pelvic mr imaging is increasingly used for imaging pelvic floor disorders, the crucial question is whether the technique can completely replace conventional techniques. although mr imaging provides excellent soft-tissue contrast which is a particular advantage when imaging the pelvis, it has to be acknowledged, that the examination is performed in supine position and not in physiological sitting position. this presentation will review the ultrasound appearances of pleural disease in ward and itu-based patients, and will discuss the indications for pleural procedures and the complications associated with them. the use of colour doppler to aid the diagnosis of effusions will be discussed, as will the identification of septations and the need for intrapleural fibrinolytic therapy. the advantages of guided versus blind drain insertion will be presented, and the benefits of large versus small bore drains will also be discussed. pelvic floor function and structure is complex. imaging has a key role in guiding the clinician in managing patients with incontinence, constipation, difficult rectal evacuation and pelvic organ prolapse. dynamic imaging is of particular interest for assessment of the pelvic floor since this kind of imaging gives a near physiological data set of what is happening and gives us a better understanding of the multifactorial causes of pelvic floor dysfunction. whereas conventional evacuation proctography was over years standard of reference for dynamic imaging of the pelvic floor, dynamic mr imaging of the pelvic floor is gaining increasing acceptance among radiologists and clinicians. however, dynamic mr imaging of the pelvic floor is (due to the architecture of most the clinically mr magnets) usually performed in supine position which does exclude the axial load on the pelvic floor. the lack of a physiological patient positioning during dynamic pelvic floor mr imaging is still brought into discussion if dynamic mr imaging of the pelvic floor can replace conventional techniques or not. the pelvic floor is a complex anatomic and functional unit. in clinical routine a simple anatomic concept of the female pelvic floor has gained acceptance. especially for treatment planning, the female pelvic floor may be separated into three functional compartments: the anterior compartment (bladder and urethra), the middle compartment (vagina, cervix, uterus, and adnexa), and the posterior compartment (anus and rectum). intact structure of the pelvic floor is a basic prerequisite for a normal mechanism of defecation and continence. over the last years, mr imaging has gained increasing acceptance as imaging modality for evaluation of the pelvic floor, which enables a global and integrated approach to the pelvic floor. using static t weighted sequences the morphology of the pelvic floor can be visualised in great detail. a rapid half-fourier t -weighted, balanced steady state free precession (bssfp), or gradient-recalled echo (gre) sequence are used to obtain sagittal images while the patient is at rest, during pelvic squeeze, during pelvic strain and to document the evacuation process. on these images the radiologist identifies the pubococcygeal line (pcl) (which represents the level of the pelvic floor). in normal findings, the base of the anterior and the middle compartment are above the pcl at rest, and the pelvic floor elevates during contraction. during straining, although most clinical diagnostic imaging studies employ anatomic techniques such as computed tomography (ct) and magnetic resonance (mr) imaging, much of radiology research currently focuses on adapting these conventional methods to physiologic imaging as well as on introducing new techniques and agents for studying processes at the cellular and molecular levels in vivo, i.e. molecular imaging. molecular imaging promises to provide new methods for the detection of minimal changes in diseased tissue and support for personalised therapy. although molecular imaging has been practiced for over years in the context of nuclear medicine, other imaging modalities have only recently been applied to the noninvasive assessment of physiology and molecular events. nevertheless, there has been sufficient experience with specifically targeted contrast agents and high-resolution techniques for mr imaging and other modalities that we must begin moving these new technologies from the laboratory to the clinic. several projects relevant to oncology will be discussed with emphasis on how they were/will be moved from the bench to the clinic. aortic dissection is increasingly managed by endovascular means. it is important for all radiologists to understand the benefits and disadvantages of varying imaging modalities in the differential diagnosis and the fundamental anatomical requirements for assessing suitability for endovascular repair. the causes, detection, classification and complications of aortic dissection will be discussed followed by a panel discussion of imaging strategies which give the best information for diagnostic work-up, endograft planning and monitoring of patients during and after treatment. with the introduction of targeted therapeutics and personalised therapy regimen there is increasing need to improve diagnosis of diseases in a way that insight into pathophysiological and molecular regulation is provided. in this context, molecular imaging can be of tremendous help in basic research, drug development and the clinics. many interesting approaches of molecular imaging have been tried in small animal models. new methods and tools have been identified, which are easy enough to handle, aceptable in its costs and, most importantly, reliable enough to be translated to clinical practice. unfortunatenly, as soon as they have entered the clinics they are often not classified as molecular imaging anymore. among those are mr-spectroscopy, spio/uspio enhanced liver and res imaging but also contrast agents like gd-eob-dtpa and most applications for pet/spect. other interesting applications such as targeted ultrasound imaging are already established tools in preclinical research and very close to first clinical use. near infrared optical imaging is another example of a rapidly developing technology and first clinical devices and dyes (e.g. to detect arthritic lesions) are availibe now. it can be expected that with the availability of targeted and activatable probes the acceptance and the use of optical imaging methods will further rise. most proably, this will initially happen in context with intraoperative diagnosis and endoscopy. in summary, it is the aim of this talk to give a realistic overview on the available molecular imaging tools and on their potential for preclinical research and patient use. cardiovascular diseases remain the number one cause of morbidity and mortality, both in the developed and developing countries, and in men and women alike. it is expected that these numbers will continue to increase in the coming decades due to escalating proportions of obesity and the aging population. atherosclerosis is the major cause for cardiovascular disease. since in about % of cases a stroke or acute myocardial infarction is the first symptom of atherosclerosis, it is of paramount importance to identify patient at high risk. a first step in the identification is the use of clinical risk profiling, such as the framingham risk score, which has an area under the curve (auc) of about . using roc analysis. however, clinical risk profiling alone is not sufficient to identify individual patients at imminent risk to develop a cardiovascular event in the near future. a next step in a more precise identification of the patient at risk is the development of serum biomarkers for atherosclerotic disease. however, so far serum biomarkers have failed to contribute substantially to improve the auc in the prediction of cardiovascular events using roc analysis. a major step forward in risk stratification is provided by the rapid development of cardiac computed tomography, which offers a rapid visual access to the coronary tree, at low radiation dose and in a patient friendly manner. however, for the identification of the vulnerable plaque we need to go beyond anatomical imaging, and use molecular imaging tools. in the lecture i will discuss the different targets for molecular imaging within the vulnerable plaque, such as inflammation, apoptosis and angionenesis. imaging of these substrates of plaque vulnerability may offer opportunities for a precise identification of the patient at risk, at the individual level. the aim of the lecture is to familiarise the audience with the specific paediatric conditions in trauma radiology. it will be described the major pathophysiologic differences in childhood and the consecutive altered injury pattern. the standard radiological imaging protocol for various involved body regions and different trauma settings/varying queries will be described, with suggestion for standardised diagnostic algorithams in some typical settings. special regard will be given to radiation protection and the potential of imaging modalities such as ultrasound, multi-detector ct as well as mri in paediatric trauma patients. the roll of the lecture is to provide some basic guidelines for young radiologists and general radiologists who less often have to deal with paediatric patients. learning objectives: . to become familiar with the major pathophysiologic differences in childhood trauma . to standardised diagnostic algorithm in some typical settings in field of pediatric trauma. basic principles in the interpretation of signal intensities on t -and t weighted images g. wilms; leuven/be (guido.wilms@uz.kuleuven.ac.be) the signal intensities of normal structures and pathological findings on conventional t -and t -weighted mr images depend on many factors. the amount of water, proton density, chemical structure and/or binding, presence or absence of flow (of blood or csf), calcification, fat, blood degradation products, melanin, mucine and even air are all factors that influence the signal characteristics. t -and t -lenghtening is the rule in most tumoural, vascular and infectious lesions and therefore is rather unspecific. t -and t -shortening can be due to the presence of fat (lipoma), melanin (melanoma and metastasis of melanoma), mucine (metastasis of sigmoid carcinoma), colloid material (colloid cyst, rathke cleft cyst) and even calcification (falx!). t -shortening with t -lengthening can be due to cholesterine (cholesterol granuloma, craniopharyngioma), high protein content (tumoural cysts), and methaemoglobin (late haemorrhage, thrombus). t -lengthening and t -shortening is almost exclusively due to deoxyhaemoglobin. acute haemorrhage and meningioma can be iso-intense on t -weighted images, while some meningiomas and micrometastases can be iso-intense on t -weighted images. absence of signal can be due to high-velocity flowing blood (aneurysm, avm, hypervascular tumours), high velocity flowing csf (normal pressure hydrocephalus, cortical bone or extensive calcification, air and a large amount of iron (coils, clips). it is concluded that the simple use of a combination of t -and t -signal intensities on conventional mr images can be used to arrive at the diagnosis and differential diagnosis of brain lesions. complications occurring after acute aortic dissections should be separated into those related to the disease per se and those related to post-treatment conditions. life-threatening complications related to the disease should be familiar as immediate consequences for treatment may ensue. most of these are either located in the aortic root or related to mal-perfusion syndromes. the aortic root with very thin walled structures of the sinus of valsalva is located within the pericardial sac. the structure is prone to rupture and may produce life-threatening pericardial tamponade or present clinically as sudden onset of severe aortic regurgitation. all imaging modalities suitable for diagnostic workup in such conditions have to be rapidly accessible and performable. this precludes lengthy mr imaging procedures and renders ultrasound and ct as the mostly used modalities. the condition that has to be recognised as the most important inductor of malperfusion syndromes in the descending aorta is the progression of dissection into aortic branches and the true lumen collapse. both conditions are easily discernible with ct imaging. complications related to treatment should be sub-classified into those after pure surgical treatment and those related to endograft repair. the acute surgical complication with most deleterious effects is related to spinal cord ischaemia. the condition also applies to endograft repair. it is therefore beneficial for the radiologist involved either in pre-surgical imaging or interventional treatment to acquire thorough knowledge of the spinal cord blood supply. endograft complications of interest encompass endoleak formations, stent migrations and endoluminal stent collapse. learning objectives: . to learn the most common complications. . to learn the most appropriate imaging strategy for diagnosis. . to understand the clinical significance of the most common complications. which imaging modality is best for endovascular management? : the various imaging modalities for establishing the diagnosis of acute non-traumatic dissections will be discussed. the panellists will present recommendations for diagnostic work-up with special respect to differential diagnosis such as acute myocardial infarction and acute pulmonary embolism. also, the problem of sizing the endograft and planning the procedure will be addressed. the panellists will discuss strategies for detection of complications and for monitoring patients following treatment. the quality of radiographs is determined by many parameters from both technical and clinical origin. during the presentation, we will make an overview of the particular features of x-ray tubes and plain films that affect the quality of the radiological image. the training session will start from very simple x-ray tubes and film, and gradually introduce more sophisticated, state-of-the-art technology. we will briefly explain concepts such as focal spot, heel effect, beam quality, filters, grid, film sensitivity, automatic exposure control, etc. from a practical point of view. the european commission and selected research groups have developed criteria to judge the quality of the images and we will show how they can be used to improve the daily practice. a more technical evaluation of quality is possible with images of test objects, some of which are very straightforward and interesting if problems have to be retrieved. x-ray quality should be at a high level every day. therefore quality control procedures have a role. one example evaluates the stability of the imaging chain by means of acquisitions of homogeneous blocks of pmma. we will illustrate typical findings with repercussions on the radiological practice, and show that the fight against artefacts is a never ending process. both with film and digital detectors, radiologists should be aware that quality management and quality control procedures are needed. quality has to be organized, and quality control procedures have to be automated and included in the routine practice. there are diverse underlying causes for "diagnostic mistakes". missed lesions due to inadequate technique, "satisfaction of search" or lack of perception have to be differentiated from interpretative mistakes of lesions that have well been seen but erroneously interpreted due to the lack of experience, misconception or overlap of findings that complicates the differential diagnosis. each of these underlying reasons require a different approach to avoid them or reduce their risk of occurrence. the first type of mistake arises more often when interpreting chest radiographs and refers to the detection of mostly small focal parenchymal densities but also to the interpretation of mediastinal and hilar contours. by analysing typical cases, visual "tricks", helpful display techniques and strategies for systematic review and analysis will be outlined that are thought to be helpful in reducing the risk of "missing" a lesion. the second type of mistake mostly represents a problem of interpreting parenchymal abnormalities seen on ct. since the lung has limited means to "react" against an injury, many radiologic findings are aspecific. interpretation of pathology of parenchymal changes is based on pattern analysis, the knowledge of disease distribution and additional findings of pleura and mediastinum. in many cases, an interdisciplinary approach of radiologists and clinicians is essential for correct interpretation. cases will be analysed to illustrate difficult differential diagnosis occuring in daily routine (e.g., infectious pneumonia versus organising pneumonia) and key features helping in going into the right interpretative direction will be outlined. emphasis will lie on focal lung disease rather on the interpretation of diffuse interstitial lung diseases. learning objectives: . to learn about visual tricks and strategies to overcome typical perception errors in chest radiography. . to become familiar with overlaps of morphologic findings of focal parenchymal lesions frequently occuring in daily routine when interpreting thoracic ct. . to recognise those additional findings that represent the "helpful clue" for correctly narrowing the differential diagnosis. panel discussion: what have we learned from our mistakes? : interpreting the results of imaging studies is more and more challenging and time consuming due to the large volume of data to evaluate, compare and post-process. moreover, errors in the interpretation of imaging studies can have significant effects on patient care, particularly in acute medicine and oncology. so the question is how to be aware of the potential pitfalls that may be encountered in the realisation and the interpretation of imaging studies and how to avoid them or to learn from them. acute pancreatitis remains a potentially life threatening condition with an overall mortality rate of %. its outcome is strongly related to a precise and timely diagnosis, a correct estimate of severity and subsequent appropriate treatment. radiologic imaging, particularly ct, plays a key role in staging the severity and therefore helps guiding therapeutic decisions. management of interstitial or edematous pancreatitis is supportive while severe or necrotising pancreatitis requires intense monitoring and specific therapies. the latter has a higher mortality and guarded prognosis, since it may lead to organ failure, infection, pseudocyst formation and extrapancreatic parenchymal and vascular complications. a number of severity indices have been established to determine the prognosis of acute pancreatitis, based both on clinical and imaging criteria. a ct-based severity index has become the main prognostic method to predict outcomes. this course reviews relevant imaging findings of various stages of acute pancreatitis and its complications by ct, including special conditions such as groove pancreatitis and autoimmune pancreatitis. currently established and modified severity indices are reviewed to learn how to estimate prognosis and guide therapy. interpreting the results of imaging studies is more and more challenging and time consuming due to the large volume of data to evaluate, compare and post-process. radiology errors are inevitable, affect all radiologists and may be defined as a mistake that has management implications for the patient. errors can be broadly classified into technical errors, active errors (errors in perception, judgment or knowledge) and errors of communication. the majority of errors are false-negative interpretations and occur during interpretation of ct examinations. good communication between the referring physician and the radiologist is essential. unfortunately, only a small minority of radiologists keep a personal record of their errors. patient safety should benefit from the repeat organisation of "error meetings" through the act of collective learning. radiologists and radiology departments must continue to improve the process of recording and addressing errors. a- : radiologists do make mistakes, diagnostic errors can be the cause of severe consequences to patients or, luckily, in many cases, they can be corrected. learning from mistakes is quite important since understanding why a mistake has been made help to avoid it in the future. during this lecture a series of mistakes made in the field of gu will be presented and causes which have lead to each of them will be analysed. dyspnoea is a common symptom in patients presenting to the emergency room. in almost two-thirds of all cases, dyspnoea is caused by either a pulmonary or a cardiovascular disorder. imaging in patients with dyspnoea depends mainly on the clinical presentation and the medical history of the patient. chest radiographs are a cost-effective and rapid test for the evaluation of patients with dyspnoea, with a fair sensitivity and specificity. for this reason, chest radiographs are usually performed early in the diagnostic evaluation of patients with acute and chronic dyspnoea. chest radiographs enable the diagnosis of frequent causes of dyspnoea of pulmonary origin, such as pneumonia, pneumothorax, pleural effusions, interstitial lung diseases, and emphysema. the diagnosis of cardiovascular disorders, such as pulmonary venous hypertension, cardiomyopathy, as well as valvular diseases, is also facilitated by chest radiographs. if chest radiographs, clinical studies, and laboratory tests, however, are non-diagnostic or equivocal, ct is indicated. ct offers high sensitivity and specificity for the evaluation of pulmonary embolism and for diseases of the pulmonary parenchyma and the airways. high-resolution ct represents the method of choice for the evaluation of suspected diffuse lung disease. additional expiratory scans are useful for the evaluation of mosaic perfusion and air-trapping. chronic pancreatitis (cp) is an inflammatory disease of the pancreas, with irreversible morphologic changes and fibrotic replacement of the gland, which progressively result in loss of exocrine and endocrine function. cp is morphologically characterised by irregular sclerosis associated with destruction and permanent loss of the exocrine parenchyma which may be either focal, segmental or diffuse. the primary symptoms of cp are abdominal pain and maldigestion, which may be physically and socially debilitating, although it is acknowledged that chronic pancreatitis can occasionally be painless. a classification based on the causes of cp is useful in order to better define the therapeutic interventions. however, morphological changes of the pancreatic ducts are the main rule of thumb for the classification in order to better compare the results of the treatment. imaging techniques have a role both on the diagnosis -especially in the early phases of the disease -and on the classification of cp, either for the grading of the disease, to explain the aetiology -even for rare forms -and to differentiate the focal mass from ductal adenocarcinoma. ct, mri and ercp have a specific role in the assessment of cp, due to the different capacity of the techniques to explore the pancreatic gland. however, thanks to heavily t w sequences, mri has a competitive role with ercp, which actually has more an interventional role in case of obstructive cp. finally, mri thanks to secretin test and dwi sequences is able to give a non-invasive assessment of pancreatic exocrine function. complications of pancreatitis may include fluid collections and pseudocysts, vascular complications such as arterial pseudoaneurysm or thrombosis of the portal venous system, and stenosis of common bile duct and pancreatic duct. inflammatory fluid collections in the context of acute pancreatitis often resolve spontaneously. drainage may become necessary in the presence of clinical complications such as abdominal pain, compression of adjacent organs by large pseudocysts, or if superinfection of a pseudocyst occurs. depending on clinical, morphologic, or technical factors, drainage may be accomplished with image-guided external catheter placement, by endoscopic internal (transgastric) drainage or by internal surgical drainage. arterial pseudoaneurysm carries the risk of acute intra-or extraperitoneal bleeding, and transarterial embolisation is usually indicated when pseudoaneurym is detected. strictures of the common bile duct secondary to chronic pancreatitis may require percutaneous or endoscopic retrograde intervention. learning objectives: . to review interventional techniques that are used to manage fluid collections in the setting of pancreatitis. . to compare the results with those obtained by surgical and/or endoscopic approach. . to learn about algorithms used for clinical decision making, and for treatment evaluation and follow-up. . to understand major shortcomings and complications and how to avoid them. discussion : ants include the presence of accessory muscles, a low-lying peroneus brevis muscle belly, pseudosubluxation of the peroneus brevis tendon, and a bifurcated or mildly crescentic peroneus brevis tendon. accessory muscles in the ankle area include in the lateral aspect the peroneal tertius and peroneal quartus, in the medial aspect the flexor digitorum accessorius longus, and posteriorly the peroneocalcaneus internus, tibiocalcaneus internus and accessory soleus. as variations are commonly seen in asymptomatic ankles, matching with clinical symptoms is important. pitfalls include pseudoloose bodies in the ankle joint, pseudolipomas, and artefacts such as the "magic angle" effect, chemical shift, susceptibility, motion, "ghost" and partial volume averaging. knowledge of normal anatomy, pitfalls and variants, aids radiologists in making the precise diagnosis of various disorders. the aetiology of dyspnoea is varied. it may be due to pulmonary or pleural disease, cardiac pathology or extrathoracic causes. imaging is an useful adjunct in the diagnosis of majority of cases of dyspnoea. in this lecture we will present selected cases which will demonstrate the usefulness of different imaging modalities, emphasising when cross-sectional imaging is indicated. learning objectives: . to review typical cases illustrating the role of imaging modalities in the differential diagnosis of dyspnea cases. . to motivate the audience by the use of voting pads to be involved in the diagnostic process. . to highlight the conclusion that may be drawn on the basis of the discussed cases. the term bone marrow oedema was introduced to describe ill-defined bone marrow hypointensity on t wi and hyperintensity on t wi and water sensitive sequences. bone marrow edema can be found in many similar unrelated disorders, such as bone contusions, osteonecrosis, inflammatory or degenerative disease, being a non-specific mri abnormality representing a diagnostic challenge for radiologist. recently has been demonstrated that bone marrow oedema might be a prognosis marker for oa (osteoarthritis) and inflammatory disease, and could be used as a powerful predictive tool for treatment options. therefore our role as radiologist is to try to increase specificity to help patient management and decrease progression. subchondral bone marrow lesions (bmls) are a hallmark of osteoarthritis (oa) on mri. radiologically, bmls in oa are understood as non-cystic subchondral areas of ill-defined hyperintensity on t w images and of hypointensity on t w images, but only water-sensitive fat-suppressed sequences depict the lesions to their maximum extent. bmls are observed regularly in conjunction with adjacent cartilage alterations. higher grades of cartilage loss are associated with higher prevalence and greater volume of concomitant bmls. as the disease progresses, an increase in bml volume is seen in the same region subchondrally in many patients, which is positively correlated with an increase in cartilage loss and radiographic joint space narrowing. cysts are strongly associated with bmls in the same subregion and develop within non-cystic bmls. the published data on the natural history of bmls are ambiguous but recent reports showed that the majority of subchondral bmls may regress or resolve completely. note that progression and regression of bmls may be observed within the same knee simultaneously. histologic correlation studies showed that the lesions consisted of a mixture of different tissue patterns with only little oedema. specific changes in bone mineralisation and remodelling in areas of bmls have been shown, and they appear sclerotic compared to unaffected regions from the same individual. differential diagnoses of oa-related bmls include traumatic bone contusions and fractures with or without disruption of the articular surface. osteonecrosis, inflammation, idiopathic bmls, red marrow and post-surgical alterations should also be considered. learning objectives: . to learn about the basic physiopathology of oa and its relation with bme. . to analyse the distribution and natural history of bme in oa. . to understand the differential diagnosis and relevance of bme in staging oa and as a marker of prognosis. b. bme and early inflammatory disease a.j. grainger; leeds/uk (andrew.grainger@leedsth.nhs.uk) marrow oedema is identified as a feature of many forms of inflammatory and mechanical arthritis, but has been most studied in the inflammatory arthritides and particular in rheumatoid arthritis. it was first reported as a feature of ra as far back as . work has been undertaken both using human specimens and specimens from animal models which suggest that marrow oedema seen on mri in ra corresponds to areas of inflammation been associated with invading pannus, lymphocytic aggregates and hypervascularity. there is also evidence that the marrow lesions seen on mri in ankylosing spondylitis correspond to histopathological inflammatory change. marrow oedema has been shown to be an important predictor the recent technological advances of ctu and mru have had an exceptional impact on the assessment of chronic/intermittent obstruction. imaging studies should help answer the clinical questions raised concerning the presence, level, and cause of obstruction. in this session, the recommended techniques of ctu and mru will be described and their relative merits and limitations reviewed. d ct images should be used as an adjunct to the transverse images, instead of a replacement, because volume-rendered images best depict the lumen and not the wall of the urinary tract. thin-section reformatted ct images likely are as sensitive as transverse images in the detection of urinary tract abnormalities. mru is being increasingly used because it provides excellent anatomic and functional imaging in a single setting. it has proved particularly valuable in pregnant women and children. the relative advantages of static-fluid mru and excretory mru will be discussed. the main aetiologies of chronic/intermittent obstruction will be illustrated, including intraluminal diseases (stones, clots, etc.), wall abnormalities (transitional cell carcinomas, tuberculosis, etc.) and extraluminal diseases (pelvic and retroperitoneal tumours, retroperitoneal fibrosis, gi tract diseases, etc.) upj syndrome is the most common site of urinary tract obstruction in children. vessels crossing a ureteropelvic junction obstruction contribute to the degree of hydronephrosis in up to % of these patients. demonstration of these vessels and their location anterior or posterior to the obstruction facilitates surgical planning. at the end of the lecture, attendees will become familiar with moderate or severe urinary obstruction and their various features and causes. . technological advances in both computed tomography (ct) and magnetic resonance (mr) imaging have improved the diagnostic imaging of the urinary tract, surpassing ultrasound and the intravenous urogram. multidetector computed tomography urography (ctu) is defined as ct examination of the kidneys, ureters and bladder with at least one imaging series acquired during the excretory phase of contrast enhancement. mr urography (mru) can be performed using heavily t -weighted sequences without contrast material or t spoiled gradient -recalled echo sequences during the excretory phase after administration of gadoliniumbased contrast material. in adults, ctu or mru is now the preferred examination. technical aspects of image acquisition and processing will be explored and technical tips relating to protocol design given. . the typical and atypical appearances of upper urinary tract urothelial tumours and bladder cancers will be demonstrated. a method of fluoroscopic biopsy of upper tract tumours is described for validation of the imaging diagnosis. . early and accurate diagnosis of urinary tract tumours helps optimise prognosis but conventional investigative pathways are complicated and lengthy, utilising multiple imaging tests and many diagnostic algorithms exist without rigorous evaluation. ctu offers a single imaging test of high diagnostic accuracy with the potential to replace multiple alternative imaging tests in the diagnostic pathway, improve patient experience, improve diagnostic performance and accelerate diagnosis. mru is a promising technique that may be used for the initial evaluation of patients at high risk for developing upper-tract urothelial carcinoma when ctu or intravenous urography is contraindicated. rhage and periventricular echodensities. the posterior fontanelle approach improves the detection of grade ii haemorrhage in % more cases than the anterior fontanelle, and the mastoid fontanelle approach is essential for diagnosing cerebellar haemorrhage. ventricular dilatation is the main complication of intraventricular haemorrhage and resolves spontaneously in approximately % of cases. the main challenge with periventricular echodensities is to differentiate them from classical periventricular leukomalacia. when cysts appear during follow-up, the diagnosis is straightforward. mri is considered to be more sensitive than us for evaluating white-matter damage. congenital brain malformations including ventricular dilatation of diverse aetiologies, corpus callosus agenesis and posterior fossa malformations are usually diagnosed prenatally. mri complements us for this purpose. acquired abnormalities arise mainly from infections and hypoxic-ischaemic injury. although mr is considered the gold standard, us still plays an important role in the study of hypoxic-ischaemic lesions when used to its full capacity. depending on the duration and severity of the hypoxic insult, patterns different from those seen in premature infants may be observed. brain malformations are conditions where the brain has not formed properly during pregnancy. these problems in brain structure are almost often (with some exceptions) associated with neurological and developmental problems. often, brain malformations are part of syndromic complexes that require a multidisciplinary approach. malformations may be caused by inherited genetic defects, spontaneous mutations within the genes of the embryo, or effects on the embryo due to the mother's infection, trauma, or drug use. classification schemes are currently shifting from a morphological to a genetic approach. the most frequent congenital brain abnormalities may be categorised into anomalies of the corpus callosum and telencephalic commissures, holoprosencephalies and related entities, malformations of the cerebral cortex, and malformations of the cerebellum. these congenital brain defects are diagnosed either from direct physical examination or from imaging studies including ct and mri. prenatal mri offers a viable method to improve detection and characterisation of these entities in utero. learning objectives: . to learn about the common supra-and infratentorial congenital abnormalities. . to learn when mri is required and the appropriate imaging protocol. . to learn if and when ct is still useful in the investigation of congenital anomalies. in addition to predicting bone destruction for erosion, marrow oedema is independently predictive of joint space loss and therefore cartilage destruction. it also correlates well with other measures of disease activity. we have applied dynamic contrast enhancement techniques to show that treatment with anti-tnf therapy brings about a reduction in contrast uptake in areas of marrow oedema in patients with ra. in the seronegative arthritides marrow oedema in the spine in ankylosing spondylitis has been shown to be predictive of future changes and of response to treatment. diffusion weighted imaging of marrow lesions in ankylosing spondylitis can also be used to show a treatment response, seen as a change in the apparent diffusion coefficient. bone marrow oedema, also referred as bone contusion or bone bruise, is frequently identified at magnetic resonance imaging after an injury to the musculoskeletal system. it may result from a direct blow to the bone, compressive forces from adjacent bones impacting one another, or from traction forces that occur during an avulsion injury. its location reflects the mechanism of injury, which allows for a focused search for predictable patterns of associated internal derangements. it is seen in any joint but are particularly common in the knee reflecting mechanisms such as pivot shift, hyperextension, contrecoup or dashboard injuries, as well as lateral patellar dislocation. in a context of trauma, bone marrow oedema, identified at mr imaging as areas of poorly marginated signal intensity alteration (best seen on fat-suppressed sequences) in the cancellous bone and marrow, represents areas of oedema and haemorrhage secondary to trabecular injury. it can be seen as soon as one hour after trauma and usually resolves in the following six to eight months, except in case of subsequent chondral lesion. learning objectives: . to learn about bhe physiopathology in trauma scenario, direct and indirect mechanism. . to recognise bme as a footprint that allows other soft tissue injuries to be ruled out. . to analyse whether bme can be a value tool for follow-up. can we still use the term bme or should we be more specific? : the term bone marrow oedema was introduced to describe ill-defined bone marrow hyperintensity on t weighted images. since then many studies have demonstrated that it can be found in many similar unrelated disorders, such as bone contusions, osteonecrosis, inflammatory or degenerative disease, and that it is a non-specific mri abnormality. it has been demonstrated that bone marrow oedema might be a prognosis marker for oa (osteoarthritis) and inflammatory disease, and could be used as a powerful predictive tool for treatment options. therefore our role is to increase specificity to help patient management and decrease progression. room e sonography is an essential tool for studying the neonatal brain. brain scans are usually performed via the anterior fontanelle; however, a more complete assessment of the brain can be achieved using the posterior and mastoid fontanelles, high-resolution linear array transducers and colour or power doppler. the most common lesions in premature infants are intraventricular/periventricular haemor- at initiation, tumours in a pre-vascular phase are supplied by oxygen and nutrients that diffuse from pre-existing normal vessels. when the tumour reaches a critical size of approximately - mm diameter, the resultant ischaemia leads to secretion of angiogenic factors. these factors, such as vascular endothelial growth factor (vegf), recruit and maintain tumour vessels. "new" vessels (neovasculature) exhibit increased blood volume and permeability compared with normal vessels. various new specific therapies in oncology target tumour vasculature or tumour neoangiogenesis. it is not uncommon that these targeted therapies have pronounced cytostatic and not predominantly cytotoxic effects. this limits the usefulness of size-based morphological tumour response assessments. of newer magnetic resonance imaging (mri) modalities, perfusion mri has emerged as a valid marker of tumour-induced blood vessels and their function. mri perfusion measures the vascularity within a tumour, as well as its component heterogeneous parts. of parameters which can be measured to date, blood volume and permeability are commonly applied in patient studies. blood volume measures the aggregate size of the vascular space, while the permeability function informs about the integrity of vessels and their ‚leakiness' to contrast agents. we will describe the use of mr perfusion to monitor such new therapies and discuss its specific advantages and limitations in comparison to ct perfusion protocols. pet-based strategies for targeted treatment-monitoring in oncology will be briefly mentioned, with prospect on the significance of combined vascular and metabolic imaging for further optimising non-invasive response assessment in specific anticancer therapies. after a brief review of physical and technical principles of diffusion-weighted mr imaging and pet-ct, the lecture describes the ability of these techniques in evaluating functional parameters in tumour tissue. diffusion-weighted sequences have been used in an attempt to further increase the diagnostic capability of baseline and dynamic mr study by providing functional information. diffusion-weighted mr imaging is based on the random microscopic movement of molecules that can be quantified by means of apparent diffusion coefficient (adc). in the early post-treatment period after loco-regional therapies, tumours may not change in size. recent studies demonstrated that water diffusion can be used to differentiate viable and cellular regions from necrotic area in the tumour, regardless morphologic or dimensional changes. moreover, new classes of antitumour therapy have been developed that have an antiproliferative effect, inducing a delay in tumour shrinkage. diffusion mr imaging can be promising in this clinical setting as a biomarker to predict early response to systemic chemotherapy. on the other hand, pet/ct, combining the functional and the structural imaging approach, was shown to be superior regarding conventional imaging modalities in the identification of intrahepatic and extrahepatic metastases. less experience and less publications are available for pet-ct in monitoring tumour response after interventional therapies, but the ability of pet-ct to measure early metabolic changes could make this technique useful in the development of novel anticancer drugs. until now, in oncology, only the recist criteria based on anatomical measurement of the tumour size are used for drug trials or in clinical practice because this is a standardised way to assess the tumour response that allows the calculation of the progression-free survival (pfs) or the time to progression (ttp) that are usually accepted as surrogate end point for overall survival. however, tumour follow-up evaluation using only morphology is usually delayed and with the emergence of new numerous and very expensive targeted therapies there is now a need to move beyond morphology to find new ways to assess tumour responses or progression not only for clinical trials but also in clinical practice to maintain or to change quickly a treatment. this is the aim of the functional imaging using ultrasound, ct, mri or pet. the ultimate goal of these technique is to find biomarkers able to predict the likely course of disease, irrespective of treatment (prognostic biomarkers) or able to forecast the likely response to treatment (predictive biomarkers); before (baseline values of a parameter) or during the treatment (dynamic variation of the parameter during the follow-up). during this session the presenters will discuss the technical issues and the results obtained today using ultrasound with share-waves or microbubbles, functional ct, dynamic contrast enhanced mri, diffusion weighted mri and pet-ct. the candidate biomarkers will be presented as well as the limits and the problems that are still to be overcome. a. us and ceus m. claudon; vandoeuvre-les-nancy/fr (m.claudon@chu-nancy.fr) for tumour evaluation, the main advantages of ultrasound (us) associate a high frame rate and a large range of data and parameters potentially extracted from the signal backscattered from tissues. beyond morphology, elastography is a first modality, based on in vivo estimation of the mechanical properties of tissues. data on displacement or strain of tissues and lesions can be obtained by manual external compression, but shear wave generation techniques allow for a quantitative and more precise estimation of their visco-elastic properties. in oncology, clinical evaluation included first breast. contrast-enhanced us (ceus) is obtained after intravenous administration of microbubbles which are pure blood pool contrast agents. ceus is capable for detection, characterisation and follow-up of tumour lesions, based on enhancement profiles during bolus, destruction-replenishment, or contrast burst depletion imaging. quantification of perfusion in normal tissues and lesions may be obtained by extracting various blood flow and blood volumerelated parameters from time-intensity curves. protocols have entered validation processes to improve reproducibility. as a predictive technique, ceus is a promising tool for monitoring changes of haemodynamic parameters and evaluating the early response during chemotherapy or antiangiogenic treatment. it is helpful in the guidance and follow-up of lesions treated by radiofrequency or cryoablation. recent advances of us and ceus include d/ d real-time imaging with matrix technology, and the evaluation of targeted agents, to be released on site after bubble destruction by the us beam. interventional radiology (ir) is the part of clinical radiology based on the percutaneous or endoluminal treatment of widespread conditions. the procedures performed by the interventional radiologists require a deep level of knowledge of clinical imaging and specific training in patient management and care. technical skills are also needed because complex devices and materials are used in some procedures. therefore, specific training programmes are required to address the training needs for the interventional radiologist. a multidisciplinary approach is required (based on team work) with defined levels in patient care. ir procedures have become the treatment of choice for many conditions as an alternative for some surgical procedures. even for some conditions without a defined treatment are now being treated by ir. thus, it is a discipline with a great deal of interaction with other clinical specialities that requires a clear definition. ir specialists have to receive recognition in the patient care process and their activity has to be known by the medical community. the recognition of ir as a subspecialty of clinical radiology by the uems will contribute to the development of specific training programmes in the european community and will promote training centres with certified specialists. interventional radiology (ir) procedures are complex and require specific training to ensure good results. in the uk, a curriculum for subspecialty training was established in specifying the required knowledge, training and core procedures expected of trainees. this curriculum has undergone several reforms since that time. however, training is not uniform throughout europe and this stimulated the development of a europe wide ir training document to ensure similar training in all countries as a way of ensuring good medical practice. radiology training is based on years of common radiology training and years of subspecialty training with an option for further specialist training in the th year. regular appraisals and assessments of trainees' performance should guide progress at local training institutions with the aim that competence is assessed formally at the end of training by a european board examination. this qualification will be recognised thoughout europe and ensures that the required proficiency in ir procedures has been attained. competence in the core skills for ir occurs during the first years of training. in the next years, trainees undertake modular training depending on their areas of interest and ultimate goals. the majority of this training is practical, supervised training in interventional suites and theatres, with clinical exposure. simulators play a role alongside the more traditional training methods and allow early training in a more forgiving environment away from the patient. development and validation of such simulation models is progressing. discrimination of individual x-ray quanta, i.e. the detectors can 'see' the colour of the x-rays. different materials attenuate the energy spectrum in their own characteristic way. by comparing the measured spectra with the spectrum emitted from the x-ray tube the penetrated material can be characterised. colour x-ray imaging can be used in breast imaging to detect, for example the uptake of an iodinated contrast agent to show the vasculature of a tumour while reducing the impact of the structures from the surrounding tissue. it can also be used to estimate the breast density and as a material decomposition technique to separate the digital mammogram into compositional images, showing different material types separately. colour x-ray imaging can, for example be realised with pulse height discrimination in a photon counting detector, multiple exposures with different x-ray tube settings or filtering the x-ray beam before or after the object. the most simple form of colour x-ray is dual energy where two images are acquired at different x-ray energies. lung cancer staging is based on imaging techniques in combination with tissue diagnosis and surgical exploration. the tnm staging system describes the local tumour extent (t -t ), presence or absence of lymph node metastases (n -n ) and distant metastases (m /m ). different combinations of t-, n-and m-factors translate into tumour stages (stages ia-iv). therapeutic decisions and assessment of prognosis are based on these tumour stages. recently, the tnm system has been modified: tumour size is now used more precisely for t staging: tumours <= cm: t a, > - cm: t b, > - cm: t a, > - cm: t b, > cm: t . satellite tumour nodules in the same lobe are now classified as t (previously t ) and in a different lobe of the ipsilateral lung as t (previously m ). satellite nodules in the contralateral lung (previously m ) and pleural or pericardial metastases (previously t ) are now classified as m a, whereas distant metastases outside the chest are classified as m b (previously m ). also, the tnm staging system should now be applied not only to non-small cell lung cancer (nsclc) but also to small cell lung cancer (sclc). during this refresher course the different t-, n-and m-stages will be presented including the recent changes and examples will be presented and discussed with the audience. therapeutic strategies in different tumour stages will be described and key decisions highlighted. the accuracy of different imaging procedures and findings will be presented and the role of biopsy in specific clinical scenarios will be discussed. x-ray computed tomography (ct) has been proposed and evaluated recently as a potential alternative method for breast imaging. efforts so far showed success with respect to contrast-enhanced dynamic imaging, but suffered from limited spatial resolution. respective efforts and clinical results will be reviewed. the new concept presented here builds upon micro-ct scanning approaches and aims at providing both high spatial resolution at around µm for micro-calcification imaging and advanced dynamic scan capabilities with continuous acquisition and scan times of about seconds for differential diagnosis of lesions. to achieve this, spiral scan modes, slipring technology, high-resolution detectors and high-power micro-focus x-ray tubes are demanded. the concept has been evaluated and confirmed by simulations and basic experiments; feasibility studies are expected by the end of . colour x-ray imaging can best be described as the x-ray analogy to optical colour imaging. in optical imaging the wavelength -the energy -of the light gives the different colours that we see. emerging x-ray detector technologies enable energy maintenance of confidentiality of patient information. in contrast, more hierarchical cultures often defer to elders for decision-making whereas communal cultures may involve community leaders in a shared decision-making process. gender and religious issues can also affect the provision of high-quality procedures with same gender care being a requirement within some cultural groups and gowning procedures that maintain cultural values frequently being an expectation. in addition, in many countries in the developing world, radiation still has mystique and fear associated with it, affecting participation in screening programs and recruitment to medical radiation technology educational programs. this presentation will present findings from a variety of countries and cultures that will help to contextualise these issues through a cross-cultural imaging lens. first line image interpretation is now commonly used in the united kingdom. for many years radiographers have used a system commonly known as "red dot" in order to identify to the referring clinician that an abnormality has been recognised on a radiographic image. this has more recently evolved into radiographer comment where the radiographers' experience in recognising abnormalities can help referring clinicians. junior doctors are often inexperienced at image interpretation, thus this system can assist in ensuring that a higher percentage of fractures and injuries are observed and the appropriate treatment obtained. this presentation will demonstrate the fundamentals of basic image interpretation of the cervical, thoracic and lumbar spine in a trauma situation. it will include basic anatomy, mechanism of injury, common fractures and soft tissue signs. using these principles this will encourage radiographers to use the comment system, both developing the radiographer's role and helping to improve patient care. two fasciae cross the suprahyoid neck: the superficial cervical fascia (scf) and the deep cervical fascia (dcf). the latter can be divided into three parts and these layers define different fascial spaces or compartments. the descriptions of these compartiments in the literature vary almost as much as those of the fasciae themselves. in addition, the names of the formed compartiments vary within the literature. despite these controversies, the knowledge of these compartiments is inestimable for correct differential diagnosis of pathologies that arise in the suprahyoid neck. with the utilisation of cross-sectional imaging, it has been noticed that growth of some tumours appears restricted by fasciae and knowledge of the anatomy of these fasciae allows not only prediction of growth patterns. by allocation of a tumour to a certain compartiment the number of differential diagnosis drop dramatically due to the fact that in different compartiments different types of tissue occur. in addition, the exact localisation of infectious disease of the suprahyoid neck may predict further intracranial or mediastinal spread. even though the number management of cancer patients, imaging pitfalls must be recognised to avoid both false-positive and false-negative interpretation. the principles and good practices of pet/ct will be explained. normal distribution of fdg, pitfalls and normal variants will be presented. specific examples will be discussed to demonstrate how the combined information of images of human anatomy upon which biological information within body structures is added improves delineation of disease, can guide surgical and radiation planning and biopsy. advances in technology result in new training requirements for radiologists who should promote close collaboration with nuclear medicine specialists. the metabolic syndrome refers to the clustering of cardiovascular risk factors including diabetes, obesity, dyslipidaemia and hypertension. the association between metabolic syndrome and cardiovascular diseases raises important questions about the underlying pathological processes. insulin resistance and visceral obesity have been recognised as the most important pathogenic factors. metabolic syndrome generally precedes and is often associated with type diabetes. cardiovascular risk reduction in individuals with metabolic syndrome should include ( ) control of obesity, diet and physical activity and ( ) control of the individual components of metabolic syndrome, especially atherogenic dyslipidaemia, hypertension, hyperglycaemia and prothrombotic state. appropriate management of metabolic syndrome should be able to prevent the progression from impaired glucose tolerance to frank diabetes and thus to prevent the increasing prevalence of type diabetes and vascular diseases. each % increase in hba c is associated with a % increase in risk of incident pad. diabetes is also highly associated with progression of pad and especially with the development of critical limb ischemia. rigorous control of blood glucose prevents the microvascular complications of diabetes, although similar benefits on the macrocirculation have not been ascertained. patients with diabetes and pad should have an aggressive control of blood glucose levels with a hba c goal of < . % or as close to % as possible. in the new tasc paper this recommendation is graded as c, meaning that it is based on evidence obtained from expert committee reports or opinions and/or clinical experiences of respected authorities, while there are no applicable studies of good quality. peripheral arterial disease (pad) is a common cardiovascular complication in patients with diabetes. in contrast to non-diabetic pad, it is more prevalent and, because of the distal territory of vessel involvement and its association with peripheral neuropathy, it is more commonly asymptomatic. diabetic pad thus may present later with more severe disease and have a greater risk of amputation. the pervasive influence of diabetes on the atherothrombotic milieu of the peripheral vasculature is unique. the abnormal metabolic state accompanying diabetes results in changes in the arterial structure and function. the proatherogenic changes include increases in vascular inflammation and derangements in the vascular cellular components, alterations in blood cells and haemostatic factors. these changes are associated with an increased risk for accelerated atherogenesis as well as poor outcomes. in contrast to the focal and proximal atherosclerotic lesions of non-diabetic pad, in diabetic patients the lesions are more likely to be more heavily calcified, diffuse, and distal, sparing the proximal vessels and mainly affecting the more distal arteries in the calf and, in a later stage, the foot. by identifying a patient with subclinical disease and instituting preventative measures, it may be possible to avoid acute, limb-threatening ischaemia. the primary imaging modality to be used should be duplex ultrasound, due to its non-invasive nature, lower risks and costs. ct-angiography and mr-angiography are now replacing dsa as standard imaging methods, providing a non-invasive assessment of the localisation and extension of a vascular lesion and allowing an accurate planning of endovascular and/or surgical treatment. of spaces in the literature varies from seven to fourteen, the use of seven spaces or compartiments seems to be helpful for a sufficient diagnostic approach using cross-sectional imaging. anatomic landmarks and the radiologic appearance in a non-pathologic suprahyoid neck and a pathologic involvement of the suprahyoid compartiments will be reviewed extensively in a coherent manner. the anatomy of the suprahyoid neck is complex and the spectrum of diseases is wide. to improve studies of the neck, radiologists should become increasingly more familiar with the anatomy and expected pathology in the various spaces. this familiarity provides crucial information required for the selection of treatment options and therapeutic planning. in addition, radiologists should be aware of situations when diagnostic inaccuracies may lead to serious consequences and complications. for example, surgical approach to a deep lobe parotid tumour as though it is a parapharyngeal lesion will in all likelihood result in facial nerve injury. mri artefacts such as complex flow-induced increase in signals may be mistaken as a lesion prompting an unnecessary operation. an awareness of such pitfalls helps to improve the quality of imaging studies. this presentation highlights some pertinent anatomical knowledge that may help to improve the diagnostic accuracy of neck studies and at the same time explain the existence of pitfalls that may ruin imaging studies. three layers of the deep cervical fascia define the suprahyoid neck compartments, which include: prevertebral, retropharyngeal, carotid, masticator, parapharyngeal and pharyngeal mucosal space. knowledge of the structures inherent to these spaces will provide the radiologist with an accurate basis for differential diagnosis. also, expanding lesions will distort or displace adjacent structures and fascia in predictable fashion, which is crucial in defining the site of origin. both mri and ct are frequently used in the imaging of suprahyoid neck lesions. the introduction of functional imaging has also given some benefits. primary and secondary, benign and malignant processes occupying major suprahyoid neck spaces will be discussed, with regard to crucial findings necessary for appropriate treatment selection and treatment planning. since correct diagnosis requires close collaboration with ent surgeons, clinical findings will also be discussed, together with practical information needed for surgery. in vivo proton mr spectroscopy is a non-invasive mr technique that is routinely used to assess a number of paediatric neurologic conditions. it is based on the fact that protons belonging to different metabolites resonate at slightly different frequencies (chemical shift). using water suppression and volume localisation techniques we can obtain a spectrum (single voxel) or spectra (si) containing metabolite peaks corresponding to predetermined anatomical site(s). in paediatrics the majority of spectroscopy is performed in the brain and the metabolites detected usually are: n-acetyl aspartate, n-acetyl aspartyl glutamate, creatine and phosphocreatine, choline containing compounds (free choline, acetylcholine, phosphocholine, cytidine diphosphate choline and glycerophosphocholine), myoinositol, myoinositol monophosphate and glycine, aminoacids (glutamine, glutamate and gaba), lactate, lipids and macromolecules. these metabolites participate in fundamental metabolic pathways and their levels are being disturbed by various pathologies. thus, mr spectroscopy has a vast field of application including paediatric tumours, infarction, hypoxia, ischaemia, infection, inflammation, metabolic disorders neurological disorders and trauma. in many cases, it can redirect or narrow differential diagnoses; in selected instances, it may provide the key finding that points to a final diagnosis. however, mr spectroscopy alone is usually not specific, but can be very helpful especially in combination with other clinical, diagnostic and other mr methods. finally, particular caution is needed in data evaluation because spectral appearance and concentrations of the most prominent metabolites are affected by (a) experimental and preprocessing factors and (b) brain development. stroke in children is most often of ischaemic origin and thrombophlebitis is the second cause of stroke in children. stroke is considered as rare compared to the adult population. however, numerous aetiologies impose to look for a cause through series biological investigations. the goal of brain imaging is to confirm the diagnosis of stroke, to evaluate the extension of ischaemia and mostly to participate in the search of a cause for the stroke. prognostic is mostly related to the aetiology. a lot of cases are performed under general anaesthesia leading to limited indications of mra in the acute phase compared to adult. the role of cta at the acute phase is mostly to confirm thrombophlebitis and is the primary method to assess intracranial vessels at the acute phase of haemorrhage of arterial or venous origin. it is also the primary method to assess arterial vessels at the neck level (especially when dissection is suspected). cta permits to confirm or rule out arterial lesion that was suspected on mr: indeed, cases with pitfalls are numerous with mr angiography. the role of mra, also always performed in the acute phase or follow-up of vascular diseases, is not as clear as cta role because of artefacts especially in young children. the role of angiography (dsa) is finally limited to prove vasculitis when the other methods are not contributive and in cases of arterial or arterio-venous malformation to plan the treatment (endovascular procedure versus surgery or radiosurgery). diffusion tensor imaging (dti) studies demonstrate progressive apparent diffusion coefficient (adc) decrease within grey and white matter areas starting from foetal life as sign of brain maturation; meanwhile, fractional anisotropy (fa) increases in some white matter structures. changes in fa and adc, together with radial and axial diffusivity values, during cell proliferation-migration and during myelination, well correlate with modifications known from developmental histologic studies. acute adc decrease (i.e. ischaemia) can be detected already in foetal stroke and its measure is pivotal in monitoring neonatal hypoxic-ischaemic encephalopathy. adc calculation allows also to assess acute osmotic and metabolic anomalies in neonate (i.e. hypernatremia, hypoglycaemia, etc.). early fa decrease, with radial and axial diffusivity changes, is observed in white matter areas as sign or early wallerian degeneration after acute brain damage. chronic regional white matter fa reduction is detectable in sequelae of periventricular leukomalacia or adjacent to malformative cortex. also, in children with neurodevelopmental delay (autism, adhd, dyslexia, etc.). fa reduction and radial or axial diffusivity changes have been demonstrated, but in these conditions not on a single case basis rather in cohort quantitative studies. finally, building fiber tracking and colour coded (rgb) maps from dti data may help in better characterising suspected structural anomalies on a single case basis (i.e. corpus callosum, hippocampal fornix, optic radiation, cerebellar peduncles malformations, etc.). however, the potentials of fiber tracking applications still need to be fully exploited, especially in view of future higher spatial resolution image acquisitions. learning objectives: . to understand the relationship of adc-fa values with the structural characteristics of the normal developing brain. . to understand adc-fa value changes in paediatric cns diseases. . to learn about the potential impact of fibre tracking in clinical paediatric neuroradiological practice. congenital pulmonary malformations may involve the lung parenchyma, bronchi, arterial supply, and venous drainage. the pulmonary developmental abnormalities that most commonly result in clinically significant complications in neonates and children are hypogenetic lung syndrome, bronchopulmonary sequestration (bps), congenital lobar emphysema (cle), bronchial atresia, congenital cystic adenomatoid malformation (ccam), and bronchopulmonary foregut cysts. it should be noted that there is often considerable overlap between these conditions and that multiple lesions may be identified in the same patient either separately or as hybrid lesions. the aim of this presentation is to illustrate the characteristic imaging manifestations of the most important congenital lung diseases and their main differential diagnosis. emphasis will be given to pre-natal diagnosis using ultrasound or magnetic resonance imaging (mri) and post-natal diagnosis using mri or low dose ct in multidetector scanners. ultrasonography (us) is the primary screening modality in prenatal imaging. its diagnostic accuracy is usually very high so that the indications for foetal mri should be very rare. in some countries, because us is considered of secondary importance and is not exploited as much as it could be, there is a progressive shift from us to mri as a prenatal imaging modality. consequently, there are many discrepancies regarding the relative contributions of foetal us and mri in the prenatal literature. the main indications for foetal mri will be discussed, with emphasis on the central nervous system, which remains the main field of application. in daily practice, the technique of foetal mri has not changed during the last decade and most diagnoses can be assessed using t , t and t * sequences. the basic technique and patient preparation will be described. new techniques, based on a functional or a metabolic approach, have been developing during the past few years but their impact on daily practice is still very limited. many studies have underlined the high diagnostic contribution of foetal mri compared with us. however, the diagnostic accuracy of mri has not been studied in large series. comparison between pre-and postnatal data or between foetal mri and pathological findings make it possible to define the main limits of foetal mri. regarding the brain, migration or cortical development disorders are often overlooked or underdiagnosed. a tight collaboration between colleagues practicing prenatal us and mri should increase diagnostic accuracy, both modalities being complementary. founded on september , , the brazilian college of radiology comprises regional societies and ten thousand associates. the college is a branch of the brazilian medical association (amb) acting as its scientific department of radiology, imaging diagnosis and radiotherapy. it collects, under the same umbrella medical professionals and legal entities in the field of diagnosis and treatment using imaging methods and/or ionising radiation. session objectives: . to get an introduction to the practice of radiology in brazil. . to understand the importance of radiology as a method for diagnosing diseases in brazil. granulomatous interstitial lung disease: hrct path correlation c.i.s. silva; salvador/br (c.isabela.silva@gmail.com) granulomatous interstitial lung diseases are a group of lung diseases in which granulomas are an important component of the histologic findings. the most common conditions are hypersensitivity pneumonitis and sarcoidosis. hypersensitivity pneumonitis is very common in brazil because of the warm and humid climate in most regions and a large number of birds. the prevalence of sarcoidosis in brazil seems to be lower than that in north america and europe. other ubiquitous causes of granulomatous interstitial lung disease include intravenous talcosis, drug reactions, and some forms of vasculitis (e.g. churg-strauss syndrome). the aim of this presentation is to illustrate the characteristic high-resolution ct and histologic manifestations of the most common granulomatous interstitial lung diseases and to summarise the main differential diagnosis based on the pattern and distribution of abnormalities seen on high-resolution ct. learning objectives: . to become familiar with the characteristic high-resolution ct and histologic findings of the most common granulomatous interstitial lung diseases (hypersensitivity pneumonitis and sarcoidosis). . to learn about the most helpful features in distinguishing the granulomatous interstitial diseases from other parenchymal lung diseases. a- : the most common granulomatous lung diseases are fungal infections and tuberculosis. these infections are particularly prevalent in brazil, the most common fungal infection being paracoccidioidomycosis (south american blastomycosis). histoplasmosis and coccidioidomycosis may occur but they are uncommon. paracoccidioidomycosis is the most frequent endemic systemic mycosis in latin america, being seen particularly in brazil, argentina, colombia, and venezuela. the lungs are the main target organ of p. brasiliensis organisms, and infection of the lungs is the leading cause of morbidity and mortality in these patients. fungal infections need to be considered not only in patients living in endemic regions but also in patients who have travelled to these areas. although the prevalence of pulmonary tuberculosis has decreased in brazil in recent years it remains one of the most important causes of pulmonary morbidity and mortality. the aim of this presentation is to discuss the radiologic manifestations of the granulomatous pulmonary infections with emphasis in paracoccidioidomycosis and tuberculosis and to summarise the main differential diagnosis. magnetic resonance imaging (mri) is a highly sensitive technique that provides complementary information to conventional breast imaging (i.e. mammography and ultrasonography) for the detection of multinodular disease and for the assessment of primary tumour extent. the use of mr breast imaging has increased considerably in the past decade. although its use is well established for some indications, e.g. detection of unknown primary and assessment of uncertain disease extent, breast mri is still under investigation for other applications such as preoperative assessment in patients eligible for breast conserving therapy on the basis of conventional imaging and clinical examination. despite its superior ability to visualise disease spread, recent studies have shown increased rates of mastectomy without reduction in positive surgical margins or local recurrence rates. nonetheless, a shift in paradigm is occurring, focusing on the use of breast mri to reduce involvement of healthy tissue in breast-conserving therapy. this paradigm involves new methodology to optimise the transfer of information to the surgeons and radiation oncologists. new technological developments, such as diffusion-weighted imaging (dwi) and magnetic resonance spectroscopy (mrs) provide new insights to establish a link with underlying biology of the tumour. contrast-enhanced mri, dwi and mrs are examined for their ability to extract prognostically relevant information to individualise therapy to individual patients and tumours. this overview aims to summarise the current status of breast mr imaging and new developments geared towards providing therapists with patient-tailored information for treatment decision, treatment guidance and therapy response. this talk will review the current literature and clinical use of foetal mri in cases with ventriculomegaly. the initial part will discuss antenatal ultrasound in the detection, categorisation and diagnosis at the -week anomaly scan. the role of foetal mri will be discussed including which cases may benefit most from foetal mri when the resource is limited. the timing of the foetal mr with relation to gestational age and the value of further foetal mr scans at a later gestational age will also be discussed. this is still a controversial and debated area especially when the resource is limited or there are financial constraints. the current literature will be discussed on the issues around foetal mri in cases with ventriculomegaly both isolated ventriculomegaly and ventriculomegaly associated with other abnormalities. finally, this section will conclude by looking at the common associated abnormalities seen in cases referred with ventriculomegaly. the final section will discuss the role of foetal mr in imaging the posterior fossa, an area often difficult with ultrasound especially in the later stages of pregnancy. again the current literature will be discussed and the use of foetal mr in clinical practice. the concluding time will be used to summarise the referral pathway used by local centre in the uk. this will differ with other uk centres and centres worldwide. hopefully, time will allow an open discussion on this at the end of the session. learning objectives: . to learn about the differential diagnoses of ventriculomegaly identified on foetal ultrasonography. . to learn when mr should be performed and its value when compared to ultrasonography. . to understand the value of mri in assessing posterior fossa abnormalities. prenatal diagnosis involves obtaining genetic, anatomic, biochemical and physiological information about the foetus and analysing whether there are any alterations that might have repercussions during the foetal period or after birth. magnetic resonance imaging (mri) has been useful in the diagnosis of foetal anomalies for many years. thoracic abnormalities play an important role in infant morbidity and mortality. survival after birth depends largely on adequate lung development during gestation, and various pulmonary problems can affect foetuses. heart defects are present in of live births, and up to % of these are associated with other malformations. other thoracic anomalies (tracheoesophageal malformations, chest wall defects, and tumours, amongst others) can also cause morbimortality. congenital abdominal anomalies can affect many organs. it is essential to determine the location and morphological characteristics of the lesion to ensure an accurate diagnosis. mri's excellent contrast resolution between tissues is very useful in the diagnosis of these conditions. prenatal diagnosis also makes it possible to detect anomalies that can be difficult to recognise clinically in newborns but require early treatment to minimise possible complications.this presentation stresses the importance of diagnosing chest and abdominal problems before birth and analyses the usefulness of mri in this context. learning objectives: . to learn about the mr protocol when investigating the foetal chest. . to recognise the situations in which mri is helpful in assessing chest abnormalities. . to understand the value of mri when compared to ultrasonography in assessing congenital renal and gastrointestinal abnormalities. a s c b d e f g h structured reporting: improving the quality of radiology reports c.e. kahn; milwaukee, wi/us (kahn@mcw.edu) the radiology report communicates the results of an imaging procedure and provides the enduring legal record of the procedure. structured reporting uses standardised language and predefined formats to create reports. structured reports can integrate information collected during the imaging procedure, such as clinical data, technical parameters, measurements, annotations, and key images. in this presentation, well defined structured reporting describes its advantages and disadvantages, and identifies the motivations for its adoption. this session will discuss how structured reporting can make it easier to retrieve reported information, evaluate the appropriateness of exams, and aggregate data across health care enterprises. structured reporting can support radiology quality improvement, research, and education, and has the potential to improve the quality of communication between radiologists and their referring colleagues. beyond air-space disease is secondary to occupation of the acini and alveoli by oedema, exudates or malignant cells. it presents in the chest radiograph as ill-defined densities which tend to coalesce. the presence of an air bronchogram is pathognomonic. when the disease is widespread it shows the typical "butterfly" appearance. air- the pixel data set of modern imaging is transformed into a life altering insight for the individual patient through the radiologic report. a good report is patient and service centred, and forms the basis of the reciprocal relationship between radiologist and referring clinician. it is a creative process with serious scientific purpose and becomes part of the permanent record in a person's life. its primary role is communication of diagnostic and procedure-related imaging information but it has many vital adjunct roles in quality service delivery. in current practice of multidisciplinary care the report content must be of value to all the stakeholders beyond radiology. structured reports must have uniformity where possible to permit data-mining and audit whilst preserving the individual radiologist practice and style and the individual nature of each patient's health. report design has a particular role in modern alerting and safety-net systems when unexpected or critical imaging findings are discovered. the properly written report can be vital in medico-legal defence to reflect good practice. ultimately guidelines and standards on reporting must be meaningful to the users and appropriate to local resources, systems and needs. learning objectives: . to comprehend the role of the modern radiological report. . to understand the role of the report in communication and patient safety. . to become familiar with structured report formulations. structured reporting: european perspective r. silverio; grosseto/it (rsilverio@sirm.org) in the last decade, we observe an increasing interest in structured report (sr). this is a part of the dicom standard, where the technical and clinical information are organised in a standard format so that it can be retrieved and reused for clinical, statistical and research scopes. structured reports have three important features: a "structured" format, with fixed paragraphs, heading and subheading in which to describe technical data and clinical findings, impressions and conclusions; a consistent, "itemised" organisation, leading to a better way of reporting, even with automated speech recognition systems; and the possibility, or rather the need, of a common, shared language. when defined terms from a standard lexicon are associated with imaging reports, the information in the report becomes more accessible and reusable. moreover, sr allows the interoperability between the dicom world and the complex sphere of the e-health (electronic patient record, patient care, etc). it is possible to convert a radiological report created as sr in a cda (clinical document architecture) document. this clinical document will be used in every computerised healthcare application, as well in the several regional e-health projects (epsos, elga, calliope, renewing health, etc). promoted by european community. integrating the healthcare enterprise (ihe) initiative is as a key partner in fostering the adoption of structured report. in short, question of whether sr will be adopted by radiologists is now a question of "when", not "if." learning objectives: . to become familiar with structured report. . to keep up to date on the developments of e-health projects in europe. acute mesenteric insufficiency (ami) is due to arterial or venous occlusion. arterial ami is an emergency. the typical patient is elderly, may have atrial fibrillation and has recent onset intense visceral pain disproportionate to clinical signs. ct is the mainstay of diagnosis and may be supplemented by ct angiography (cta). waste no time if arterial ami is suspected. average mortality rates of % ( - %) have been reported and symptom duration before therapy is an independent predictor of mortality. age > years, metabolic acidosis and renal failure are other danger signs. interrupt routine ct lists; ensure that these patients are scanned as soon as possible -neutral oral contrast and high-dose iv contrast are essential. ct appearances should be correlated with serum lactate as acute arterial occlusion without reperfusion may produce falsely 'normal' bowel wall thickness. early diagnosis and aggressive surgical intervention combining bowel resection with revascularisation offer the best prognosis. venous ami may present with a more chronic, intermittent course of pain, fever, abdominal tenderness and ascites. therapy is bowel resection and anticoagulation. ischaemic colitis occurs in elderly patients with atherosclerosis spontaneously or after aortic aneurysm repair. this invited lecture explores the groundbreaking use of ct for studying antiquities and artworks from a wide range of cultures, irrespective of their age or material. dr. marc ghysels, a former interventional radiologist, comes from a family of artists and collectors. about ten years ago he set up a private radiology practice in brussels where he analyses antiquities and artworks. over the years he has built an international reputation among collectors and art dealers as well as museum curators and experts working in auction rooms. art specialists call on his radiological skills and knowledge to authenticate antiquities, and more specifically to show: what methods were used to make the works, what damage they have suffered over the years, how much restoration has been done, and to expose the many tricks used by forgers to deceive not only the discerning eye of the collector but also the methods of scientific analysis more commonly used than ct scans. his presentation will highlight the prominent role of ct as a nondestructive test to explore selected artworks made in wood, terracotta, stone, and ivory. acute abdominal organ ischaemia may be caused by hypovolaemic shock, spasm (ergotism), embolisation and aortic dissection. embolisation is the most common cause. it occurs typically in elderly patients with atrial fibrillation, after myocardial infarction or due to a thoracic aortic aneurysm (taa). the primary diagnosis is made by ct with contrast enhancement which can also demonstrate cardiac thrombi or a taa. interventional treatment can be performed with a thrombectomy device or fibrinolysis with recombinant tissue plasminogen activator (rt-pa; mg loading dose, mg/hr infusion dose). acute aortic dissection may cause dynamic compression of the true lumen with occlusion of the ostium of the visceral arteries (floating visceral sign). the primary diagnosis is made by ct. occlusion of the primary entry tear with a thoracic aortic stentgraft may decompress the false lumen followed by reexpansion of the true aortic lumen followed by reperfusion of the visceral arteries. chronic abdominal ischaemia in younger patients may be due to fibromuscular dysplasia, takayasu arteritis or neurofibromatosis. in the elderly it is usually caused by arteriosclerosis. the patient may have post-prandial abdominal pain (angina abdominalis), diarrhoea and/or weight loss. due to the collateral circulation, symptoms usually occur only if more than one of the major arteries (celiac trunk, superior and inferior mesenteric artery) is narrowed. the diagnosis can be made by color doppler ultrasound (cdus), ct and mr with contrast enhancement. endovascular treatment is done by pta and stent placement. to learn what to include in the report of cns infections for the treatment planning. . to learn how to report follow-ups and treatment monitoring of cns infections. finding of a palpable mass in the abdomen always raises the possibility of an important clinical problem. a potentially life-threatening process, especially malignancy is the major concern. the list of differential diagnosis of "abdominal mass" is a very long one. in the process of differential diagnosis the most common approach is to evaluate the patient according to the gender, age, patient history and co-existing clinical and laboratory findings. the accompanying symptoms and signs and the location of the abdominal mass are the key indicators in the clinician's way of thinking in the process of differential diagnosis. for instance, a palpable mass with acute abdomen or intestinal obstruction will be assessed differently than a mass found incidentally. imaging is commonly required to confirm or ascertain the diagnosis. cross-sectional imaging is required to accurately evaluate a palpable abdominal mass in most situations. ultrasound and computed tomography have each been used successfully in evaluating patients with palpable abdominal mass. although each modality is appropriate in most situations, the advantages and disadvantages of each modality in certain situations will be addressed and the appropriateness criteria will be reviewed in this lecture. knowledge of a detailed clinical history is as important to the radiologist as to the clinician. its impact on the diagnostic accuracy in the interpretation of the images will also be addressed. a palpable abdominal mass has a long list of benign and malignant differential diagnoses. these diagnoses may be as different as a hydatid cyst of the liver, a volvolus, an aneurysm, or a giant renal cell carcinoma. the diagnostic approach is based on two major steps: first, the affected organ must be indentified. second, the differential diagnosis must be established based on imaging characteristics. the choice of ultrasound, ct or mri should be based on location and size of the mass. despite the fact that ultrasound is frequently used as a first step, the overview and anatomic orientation in large masses may be hampered, making ultrasound a better technique for image-guided biopsy than for primary diagnosis. in the vast majority of cases, multidetector ct is the first technique of choice. a thin-section protocol should be used to allow for high-quality multiplanar imaging. a pre-contrast scan is usually not required but can be helpful in suspected hemorrhage. for most upper and middle abdominal masses, biphasic imaging in the arterial and portal phase is recommended in order to establish the relation of the mass to the vasculature and to assess vascularity. in the small pelvis, mr is the superior imaging technique. otherwise, mri is mainly used for problem-solving. this course will discuss how to use the various imaging tool efficiently to narrow the differential diagnosis, decide about the need for biopsy and establish a suitable therapy. the most common viral infection of the brain is herpes encephalitis (hsv ). it is a necrotising encephalitis with a a mortality of more than %. on imaging studies lesions will be seen in the "limbic system", i.e. the temporal lobes, hippocampi, insular cortex and cingulate gyrus. they appear as hypodensity on ct, t -and flair hyperintensity on mri, possibly with haemorrhagic transformation. diffusion is restricted in the early phase. enhancement occurs at a later stage. human immunodeficiency virus (hiv) infection is a multifocal giant-cell encephalitis eventually leading to a progressive leuco-encephalopathy. on mr atypical focal or diffuse symmetrical signal abnormalities are seen neither with mass-effect nor with enhancement and typically sparing the u-fibers. spectroscopy can show an increase of myo-inositol. progressive multifocal leuco-encephalopathy (pml) is due to reactivation of the jc polyomavirus in immunocompromised patients, % of which are hiv positive. on mri "scalloped" multifocal asymmetrical lesions are seen with minimal mass-effect and without enhancement. new treatments of hiv, especially highly active antiretroviral therapy (haart) can lead to a paradoxical worsening of patients due to the immune reconstitution inflammatory syndrome (iris). on mri mass lesions are seen with diffuse patchy enhancement. cerebral toxoplamosis appears as multiple enhancing lesions with marked perilesional oedema. calcification is possible. prion diseases are caused by a proteinaceous infectious particle leading to creutzfeld-jacob disease in humans. diffusion weighted mr-images show high signal intensities in the cortex and the basal ganglia. abnormalities on t -weighted images and flair occur at a later stage where atrophy is mostly prominent. to learn how to assist clinicians for the diagnosis and differential diagnosis of bacterial and parasitic cns infections. susceptibility effects, increased radiofrequency (rf) field inhomogeneity and more pronounced magnetic shielding effects. in the mean time, many investigators have proposed strategies to optimise imaging protocols and to decrease sar levels and to reduce artefacts including optimised coil and hardware design, in combination with parallel imaging and modulation of refocusing flip angles. many clinical applications in neurology and angiography for high field mri that were recently being investigated showed benefits over . t. however, as of to date, virtually no prospective comparative studies have been performed and published that could help to estimate whether or not there would be any clinical benefit of . t msk over . t. although many authors described the improved snr of msk imaging at . t allowing for higher spatial resolution this has not been prospectively investigated with comparison to . t. further studies have to show whether the improvement in speed and resolution will also translate into increased patient throughput and earlier depiction of disease in msk applications at . t. ultrasound is the best imaging modality for evaluation of acute scrotal conditions. mri is used only rarely, when us findings are equivocal and in cases of suspected infarction. ct is used exceptionally, for example, to detect air in the scrotal wall. high-frequency transducers with modern software like compound imaging and native harmonic, and very good colour sensitivity for low flow in small vessels provide excellent insight into testicular morphology and vascularisation. it is very important to distinguish testicular torsion from the acute inflammation, as it has important therapeutic and even medicolegal consequences. in testicular torsion rapid diagnosis is vital, and salvage rate is directly related to timely operation. torsion is more common in younger patients and orchiepididymitis is more common in adults. infection usually starts at the epididymis and then spreads to testis the patient presenting with an abdominal mass represents a common clinical problem. clearly, the differential diagnosis is large and obviously will depend on the age and sex of the patient as well as location of the mass. of course history and clinical examination are mandatory, but formal diagnosis will usually rest on radiological interpretation. contrast examinations have now been replaced by cross-sectional imaging and endoscopy and it behoves the clinical radiologist to be aware of the advantages and limitations of these methods in order to reach a diagnosis. particular difficulty may be encountered when the mass is so large that it is difficult to determine the organ of origin. the purpose of this interactive case discussion is to explore the relative merits of ultrasound, ct, mr and endoscopy in establishing a diagnosis in two different cases. the cases concerned are: . a -year-old man who presents with a right iliac fossa mass, but is otherwise asymptomatic. . a -year-old woman who presents with anaemia and a large upper abdominal mass. active audience participation will be encouraged by means of key pads in order to respond to issues raised during the debate. the most straightforward expectation from t mri scanners is a gain in snr that could be translated in improving spatial resolution and/or reducing imaging time at an at least constant cnr. together with field strength, susceptibility effects, chemical shift and t increase and t and t * decrease. also, relaxation properties of contrast agents are modulated. rf power deposition is higher at t and is often a limiting factor in sequence parameter optimisation. hence, the need to optimise mri sequence protocol parameters at t. for cartilage, muscle, synovial fluid and fat, t increases by between % and %, t decreases by between % and % and r of gd chelates decrease by % to % when b increases from . t to t. in most applications reduced t and increased t work synergistically towards a reduced snr. since the change in relaxation times is not linear for the different tissues, cnr varies with field strength, too. a relatively straight forward approach is to lengthen tr in order to compensate for longer t and to shorten te to compensate for shorter t (*). in non-fat-sat images bandwidth needs to be increased to control chemical shift effects. because of the better fat and water peak separation, t often helps improve frequency-based fat saturation but may also be greatly impaired by field inhomogeneities in the presence of metallic materials. field strength (b ) and appropriate dedicated multi-element coils are the hardware prerequisites to bring high resolution (hr) isotropic imaging into clinical routine. depending on the age of the child, hip symptoms may predict a variety of diseases. in the infant, developmental dysplasia and infection should be considered. after the age of , irritable hip most commonly due to transient synovitis is the commonest disease but infection is the more worrying condition. perthes disease affects some and this condition overlaps into those over who may have a slipped upper femoral epiphysis. fractures and rare cases of osteonecrosis and chondrolysis are a concern in the adolescent. in the young and indeed in most cases, ultrasound provides a useful first line test. aspiration of effusion may be both diagnostic and therapeutic. in the older child and, especially when sufe is a risk, conventional radiographs with an external rotated "frog leg" view is very important. despite the logistics problems in children, there is an important role for mri especially in cases where the initial imaging does not explain the symptoms. i will review the imaging pathways and provide examples of common diseases. i will also discuss how to manage the difficult case and show recent audit data on detection rates and clinical management. fai refers to a conflict between the proximal femur and the acetabulum. both an abnormal shape of the proximal femur (fai of the "cam-type": aspheric femoral head with a laterally increasing radius and/or a waist deficiency of the femoral neck) and the acetabulum (fai of the "pincer-type": acetabular retroversion or a deep acetabulum) or a combination of the two may be present. for a long period the fai may be asymptomatic and the only clinical finding will be an impaired internal rotation of the hip joint. later as the disease progresses, cartilage damage (outside-in abrasion of the acetabular cartilage/cartilage delamination) and labral tears occur. labral tears are most common in the anterosuperior aspect of the acetabulum. a typical labral tear is an incomplete detachment with a tear located at the base of the labrum. anatomic variants such as a sulcus may be present and should be distinguished from tears. a sulcus is common at the junction of the labrum with the transverse ligament and is generally located beyond the equator of the hip joint. insufficiency fractures about the hip commonly occur in the region of the femoral neck. another site vulnerable to overuse is the symphysis pubis. often, a bone marrow oedema pattern around the symphysis pubis is observed. a characteristic finding is the "secondary cleft sign", which reflects a partial tear of the adductor tendon aponeurosis at the symphysis pubis. understanding age-related changes is essential for interpretation of imaging studies. age is a risk factor strongly correlated with osteoarthritis (oa) which is the most common hip joint disease seen in adults. the diagnosis of oa is based on a combination of radiographic findings and characteristic subjective symptoms. the lack of a radiographic consensus definition has resulted in a variation of the that is painful and hypervascularised on colour doppler imaging. in cases of torsion b-mode findings are non-specific, while on colour doppler flow is absent in complete torsion, but may be present and diminished in incomplete torsion, or increased in intermittent torsion. in cases of trauma ultrasound is important to diagnose haematoma, haematocele to evaluate integrity of testis and assess whether the testicular fracture is present or if the surgery is needed in cases of testicular rupture. imaging is helpful in cases of rare causes of the acute scrotum, such as fournier gangrene, testicular appendage torsion, vasculitis, and also in evaluation non-scrotal causes of symptoms, like in cases of renal colic, abdominal inflammatory and traumatic conditions, etc. a scrotal mass is an important clinical problem and a source of anxiety to the patient. radiologists play an important role in the management of these cases, since imaging is required to provide information about precise anatomical location of the lesion, its size and extension and, possibly, to elucidate the nature of the disease. the us and mri findings of the many different scrotal tumours will be presented in this lecture. special attention will be given to two topics. the first are the possibilities offered by imaging to differentiate among the different pathologies, especially to differentiate between benign and malignant lesions. the second will be the problem of the small, non-palpable, indeterminate testicular mass. such lesions have been shown to be relatively frequent. prevalence of malignancy varies widely in the literature, and orchidectomy seems not justified in all cases. the use of contrast-enhanced mri and of advanced us techniques (contrast-enhanced us and elastography) may help to narrow the differential. furthermore, the use of intraoperative us to guide removal of non-palpable testicular nodules can help to perform conservative surgery in lesions with no malignant potential. learning objectives: . to understand the typical imaging appearance of benign and malignant scrotal tumours. . to review differential diagnosis of intra and extratesticular scrotal tumours and the impact of imaging. c. imaging of the penis m. bertolotto; trieste/it (bertolot@univ.trieste.it) ultrasonography (us) is the first-line imaging modality in patients with penile disease. using high-end equipment after pharmacologically induced erection penile anatomy is well defined and virtually all clinically significant penile vessels can be evaluated in normal and in impotent men. the superior soft-tissue contrast resolution afforded by mr imaging provides an opportunity to advance imaging evaluation of the penis in selected cases. in the clinical practice, erectile dysfunction is the most frequent penile abnormality which is investigated with doppler us. the clinical role of this evaluation, however, reduced after the introduction of oral medications for impotence. differentiation among different forms of erectile dysfunction is mainly based on evaluation of doppler waveform changes in the cavernosal arteries. peyronie's disease is the most frequent cause of penile induration. imaging is often required to evaluate the extension of the plaques, involvement of the penile septum, and relationship between the plaques and penile vasculature. in patients with penile traumas, imaging allows accurate evaluation of albugineal tears, extra-albugineal and cavernosal haematomas, vascular lesions producing high flow priapism and other pathological changes. compared with us, mr imaging has some advantages in identification of small albugineal tears, and is more accurate in identification of urethral or spongiosal involvement. other situations in which penile imaging can be required are circumscribed or diffuse cavernosal fibrosis, tumours, priapism, severe inflammation, and evaluation of postsurgical complications. most of these conditions are first investigated with us; mr imaging is optimal for tumour staging. endocrine tumours of the pancreas are derived form the apud cell system. they often show early clinical sign related to the hormones produced by these tumours. the usually very specific symptoms raise the suspicion of an endocrine tumour of the gastro-entero-pancreactic tract and initiate imaging studies for tumour detection. in this setting usually the pancreas is among the most frequent tumour sites. since endocrine active tumours of the pancreas usually show a strong vascularisation, they can be differentiated from other solid tumours of the pancreas -including adenocarcinoma of the pancreas. metastases in the pancreas are very rare -however, they represent a potential differential diagnosis either for non-functioning neuroendocrine tumours or for adenocarcinoma of the pancreas. since the evaluation of the whole pancreas is crucial, transabdominal ultrasound plays an only limited role for the detection of endocrine pancreatic tumours. usually mri and ct -alone or in combination with specific nuclear medicine tracers in pet -are needed for accurate diagnosis and staging of these tumours. published incidences and prevalence of oa. the progression of oa traditionally has been measured using radiographic joint space width (jsw). weight-bearing radiographs centered on the hip are the most reproducible and reliable ones. the sequence of degeneration includes the following radiographic findings: joint space narrowing, osteophyte formation, subchondral sclerosis, and cyst formation. current definitions of radiological oa based on reduced jsw and osteophytes display predictive validity for clinical hip oa. radiographs are also useful for assessing developmental dysplasia and other congenital disorders which may lead to early oa. there are cases though, that radiographs show minor changes and the clinical suspicion of early disease can be confirmed with more sophisticated imaging methods, such as ct and mri. ct is helpful for additional measurements such as femoral and acetabular abnormal version which might lead to oa. femoroacetabular impingement has been shown to cause labral and chondral lesions and leads to oa. prompt recognition of abnormal head-neck junction on radiographs enables proper conservative or surgical treatment planning. mr arthrography is the method of choice for assessing the labra whereas ct arthrography might be used as an alternative for articular cartilage assessment. cystic tumours of the pancreas include a variety of masses which can be classified into cystic tumours of the exocrine pancreas, cystic tumours of the endocrine pancreas, cystic tumour-like lesions of the exocrine pancreas. in order to simplify the approach only the relatively common cystic tumours -the intrapapillary mucinous tumours (ipmt), the serous cystoadenoma (sc) and the mucinous cystoadenoma (mc) -will be considered. ipmt are characterised by ductal dilatation. imaging characteristics of ipmt are best displayed at mr-cholangiopancreatography. on the basis of the extent, these tumours can be divided into ipmt of the main duct (or central), ipmt of the secondary ducts (or peripheral) and mixed ipmt. central and mixed ipmt have a malignant behaviour and therefore need to be treated surgically, while peripheral ipmt have a benign behaviour and, in general, need only follow-up. sc are characterised by a microcystic architecture in most cases. imaging features are typical in cases of microcystic appearance and consist of small amounts of fluids interspersed within multiple septae of a "sponge like" mass. the differential diagnosis with a mucinous cystoadenoma is difficult in cases with a oligo-macrocystic appearance. mc appears as a uni-oligolocular cystic mass, with a simil-ovaric stroma in the tail of the pancreas. imaging features include a cystic mass with a definite wall and inner septae. when calcifications and enhancing nodules are present, a mucinous cystoadenocarcinoma has to be suspected. therefore, a careful evaluation of imaging features of pancreatic cystic tumours is needed to differentiate benign form malignant masses. learning objectives: . to understand the classification and management of cystic pancreatic tumours using rad/path correlation. . to learn how to provide a differential diagnosis and how, when and why imaging techniques should be used. . to review pathological and imaging findings of intra-ductal cystic tumours. . to learn how to deal with the incidental pancreatic cyst. a s c b d e f g the evolving role of the radiologist p. brader; vienna/at (peter@brader.md) in the past two decades, enormous strides have been made in medical imaging and many new technologies and agents are now available for breast cancer research, clinical trials and patient care. advances in experimental and clinical breast imaging are likely to improve our knowledge of how breast cancer arises at the cellular level, which will help not only to identify and locate tumours but also to assess the activity of biological processes within these tumours. this recent and concomitant progress, in imaging in general, and in breast imaging in particular, has been facilitated by the convergence of molecular cell biology, pathology, chemistry, physics and engineering in a multimodality and multidisciplinary way. the challenge for radiology is to begin thinking at the molecular level. it is, therefore, important that the next generation of radiologists become more involved in multidisciplinary research and clinical work, and that younger radiologists receive special training in diagnostic imaging and nuclear medicine, as well as a solid understanding of physics, radiochemistry, pathology and biology. applying this approach to breast cancer patients should allow earlier detection, stratification of patients for treatment, and objective evaluation of new therapies. the outcome will be considerably better management and care of those with breast cancer. breast cancer is the leading cause of cancer death among women worldwide. imaging plays a key role in the early detection of breast cancer. mammography is an accepted screening modality with some limitations such as over-treatment. to overcome these limitations research is going on to characterise breast cancer more accurately. currently researchers are working on different strategies spanning the spectrum from tomography-based systems to mri and even molecular imaging. this panel discussion will focus on these different strategies. the auditorium will learn if in imaging still plays a major role in breast cancer detection or whether a simple blood test will eventually solve the problem. breast cancer is the leading cause of cancer death among women world wide. imaging plays a key role in the early detection of breast cancer. mammography is an accepted screening modality with some limitations such as over treatment. to overcome this limitations, research is going on to characterise breast cancer more accurately. currently researchers are working on different strategies spanning the spectrum from tomography-based systems to mri and even more molecular imaging. this panel discussion will focus on these different strategies. the auditorium will learn if in imaging still plays a major role in breast cancer detection or a simple blood test will eventually solve the problem. is mammography still an accepted modality for breast cancer imaging in ? m.j. yaffe; toronto, on/ca (martin.yaffe@sunnybrook.ca) x-ray mammography is currently the only imaging modality that when used in routine screening has been demonstrated to contribute to reduced mortality in women in the age range - . but, the accuracy of mammography is limited with respect to both sensitivity and specificity. digital mammography has provided increased sensitivity in women with dense breasts and three-dimensional methods such as digital breast tomosynthesis or dedicated breast ct promise to yield further improvement. however, these techniques are dependent on detecting rather gross physical changes as masses, microcalcifications or architectural distortions develop and, therefore, their ultimate potential is limited. it is likely that before , detection tools that are more specifically targeted to molecular characteristics or early functional changes associated with cancer will be clinically available. these may be either in the form of an imaging test, based on a specific biomarker for the cancer or possibly an innocuous test designed to sense a circulating biomarker in the blood or urine. in the latter case, a positive test would be followed by an imaging study to provide localisation for therapy. such tests could not only detect the presence of disease but would also provide prognostic information to guide the type and aggressiveness of therapy so that overtreatment could be avoided. promising imaging techniques based on targeted imaging with ultrasound, x-ray contrast agents, mr and radio-labelled agents are already under investigation. by exploiting new molecular or functional signals one of these is almost certain to replace mammography before . can we assess cell density of tumours with imaging techniques? d.-m. koh; sutton/uk imaging is increasingly used to define endpoints of clinical trials in oncology. tumour cellular density may be inferred by diffusion-weighted mr imaging (dw-mri) and measurement of the t relaxation time. dw-mri is unique as the mechanism of contrast is based on differences in the mobility of water between tissues, which can be quantified by the apparent diffusion co-efficient (adc). tumours are frequently more cellular compared with their tissue of origin which impedes water diffusion, resulting in increased conspicuity on dw-mri and lower adc values. studies have shown negative correlations between histopathologically determined cell density and adc in several tumour types, including prostate cancer, soft tissue sarcomas and cerebral tumours. dw-mri is sensitive for tumour detection, especially for disseminated peritoneal disease, liver and bone metastases. whole body dw-mri with background suppression (dwibs) is an emerging technique that has shown substantial promising for staging of lung cancer, prostate cancer, lymphoma and multiple myeloma. effective treatment results in reduction in cell density due to cell lysis, apoptosis and necrosis, leading to a rise in adc value. adc increase has been observed within days of anti-tumour treatment (including chemotherapy and radiotherapy); and as early as one to two weeks after treatment. quantitative adc measurements may also be prognostic as tumours with higher pre-treatment adc have been shown to respond poorer to chemotherapy and radiotherapy. nevertheless, a number of challenges still have to be overcome to qualify adc as a response and prognostic biomarkers in a multi-centre setting across different imaging platforms. angiogenesis is an essential process whereby tumours derive vascular supply from adjacent tissue, to sustain tumour growth and metastatic spread. newer targeted antiangiogenic therapies differ significantly from current cytotoxic therapies for cancer. the methods of dose selection are either invasive, such as biopsy and histology, or time consuming, such as tumour shrinkage and time to disease progression, both of which take months to assess and fraught with other limitations. moreover, cancer is a very variable disease, which means that some patients will respond to a particular therapy while others will not. there is, therefore, a great need to establish surrogate markers for drug response that are both rapid and reliable, not only for clinical trials of new drugs but also to aid in the selection of optimal treatment for individual patients. multiparametric imaging techniques provide a non-invasive insight into tumour cell density, vascularisation, and biochemistry. imaging data have the potential to provide information on disease profiling pertaining to diagnosis, prognosis, selection of therapy, monitoring of response to therapy, and pharmacokinetic information of drugs. undoubtedly, these methods hold great promise, but how can we standardise these techniques, in terms of acquisition parameters and image analysis (post-processing), and validate imaging parameters as biomarkers in multicenter international cancer trials. beginning with a review of the frequently used response evaluation criteria in solid tumours (recist), the development and optimisation of new imaging parameters as biomarkers of treatment response and optimised monitoring of tumour therapy in multicenter eortc clinical trials will be presented. dw-mri is sensitive for tumour detection; adc is affected by the different cellular density of a tumour. still, a number of challenges need to be overcome to qualify adc as a response and prognostic biomarker in a multicenter setting. the large variety in primary tumours and imaging methods, as well as the large variety and continual evolution of imaging equipment, point towards the need for a concerted design of imaging protocols in order to assure that these protocols are applicable to multicenter trials. furthermore, reliable evaluation of imaging data requires imagers experienced in the area of the definite cancer targeted in this trial and in the use of imaging tools for lesion quantification. from this session, a roadmap for future collaboration between eortc and esr combining different imaging modalities will emerge. session objectives: . to prove that advanced multiparametric imaging techniques play a crucial role in the diagnosis, staging, treatment monitoring and follow-up of oncological patients. . to explore avenues for future collaboration between eortc and ecr. . to suggest input of radiologists in the design and implementation of multicenter trials. the eortc imaging group: vision and strategy on cancer imaging s. stroobants; antwerp/be (sigrid.stroobants@ua.ac.be) response to cancer treatment is evaluated by subsequent assessments of target lesions and is defined as a significant decrease in measurable tumour dimensions (who, recist). the new targeted therapeutics which cause cytostasis rather than cytotoxicity have challenged volume-based response criteria and tumour regression is increasingly recognised as an unreliable end point. new imaging modalities looking at tumour biology, like positron emission tomography (pet) or diffusion weighted (dw) and dynamic contrast enhanced (dce) magnetic resonance imaging (mri) or increasingly used to identify subpopulation of patients most likely to respond. recently, new response criteria were introduced that incorporate fdg-pet (recist . for solid tumours and new cheson criteria for lymphomas). since imaging is becoming increasingly important in novel trial design, the eortc decided to invest in an imaging platform. eortc has established a medical imaging exchange infrastructure allowing to store imaging data linked to the clinical databases. a functional imaging expert group is set up to review protocols, organise central review and develop specific initiatives for imaging biomarker validation. eortc is part of the quicconcept consortium that within the eu innovative medicine initiative (imi) will try to validate the use of novel pet probes and dw-mri as biomarkers for apoptosis and proliferation. functional imaging techniques can only evolve successfully into biomarkers that are clinically valuable and important for drug development, when there is agreement on the standards for measurement and analysis and working groups are set up in collaboration with the different professional organisations (esr, eibir, eanm) to set up imaging guidelines. conditions such as ttts in twin pregnancies and foetal demise. the living foetus can also suffer from abnormal blood supply to the cns, and thus showing small or even large hemispheric infarcts. other diffusion technique, dti is also used to demonstrate the white matter formation of the foetal brain. mrs is also used in the evaluation of the foetal brain. the values of the naa, choline and the existence of lactate are different than the values in the paediatric and adult brain. learning objectives: . to explore the new imaging techniques for the study of the foetal brain. . to understand the use of diffusion-weighted imaging in the foetal brain. . to become familiar with the accurate use of t -weighted sequences in the foetal brain. . to understand the possibilities of tractography in the fetal brain. the paediatric central nervous system is a complex structure undergoing rapid development. as such, there is a rapid, continuous modification of what is "normal" in relation with age and the stage of development. knowledge of the normal patterns of brain development in the clinically relevant ages from to years is necessary to interpret neuroimaging findings correctly. knowledge of embryology and normal variants is also greatly helpful. mr imaging equipment and parameters need to be adjusted and optimisation for paediatric studies. pitfalls often occur from the misunderstanding of normal conditions that are perceived as abnormal based on a comparison with the appearance of the normal brain in adults. this includes, for instance, the evaluation of the brain in the first - years of life during the course of the process of myelination. a summary of the most frequent conditions that may lead to misinterpretation of findings will be provided here. learning objectives: . to understand a different approach to neuroimaging in the developing paediatric brain and spine. . to appreciate that, despite the wide and potentially complex spectrum of diseases seen in neuropaediatrics, most interpretations can be made through the thoughtful application of basic neuroradiological analytical techniques. . to be aware of certain areas where common pitfalls, myths and misunderstandings occur. the ability to develop more aggressive treatments of acute neurological disorders in children is nowadays improving; however, infants and children are often uncooperative, clinical signs are not always easily localised, and diagnosis and therapy may be both delayed. effective imaging of the central nervous system assumes an increasingly important role in the evaluation of these critically ill children. this lecture will review the main indications to perform a neuroimaging procedure in children with a neurological emergency. with didactic purposes, acute conditions will be classified in traumatic and non-traumatic (ischaemic and haemorrhagic stroke, infection and acute metabolic disorders). differential diagnosis will be discussed based on representative cases selected from the daily routine in a paediatric tertiary hospital. the varied neurological complications that can occur in the child admitted at the intensive care unit will also be presented in a didactic manner, including pres (posterior reversible encephalopathy syndrome), acute liver failure, osmotic demyelination syndrome, hypoglycaemic encephalopathy or induced neurotoxicity. eventually, some peculiarities in oncologic and immunosupressed children, particularly opportunistic infections, will be highlighted. practical algorithms with the preferential use of either ct or mri will be developed for each section. ct continues being the primary modality for trauma, although it should be better used in cases of non-available mri in the other acute conditions to save radiation in children. mr imaging is nowadays better for imaging these children owing to the new techniques that can be used, such as diffusion imaging (di), spectroscopy, arterial spin labelling (asl) or susceptibility-weighted imaging (swi). learning objectives: . to learn the characteristic neuroimaging findings that may be useful in establishing differential diagnoses. . to understand the various neurological complications that can occur in the intensive care unit and to become familiar with their most typical imaging patterns. . to consolidate knowledge of the best neuroimaging protocols for the acutely ill child and establish the main indications for the use of mr imaging, particularly diffusion and spectroscopy. challenges for morphologic imaging in oncology trials: reproducibility and reading f.e. lecouvet; brussels/be (frederic.lecouvet@uclouvain.be) beside clinical endpoints and biological or molecular parameters, beside emerging perfusion or diffusion imaging techniques, morphologic imaging remains the cornerstone of the evaluation of treatment response in the majority of primary or metastatic tumours. the large variety in cancers and imaging methods, as well as the large variety and permanent evolution in imaging equipments, raises the need for a concerted design of imaging protocols to guarantee transposability of these protocols to multiple centers. the reliable evaluation of imaging studies obtained in trials requires readers experienced in the area of the definite cancer targeted in this trial, and in the use of imaging tools for lesion quantification. the imaging group of the eortc targets this optimisation of imaging protocols. first, the different "organ groups" of the eortc will benefit from the availability of expert radiologists in the different fields of oncology, involved in the choice and tuning of adequate and generalisable imaging tools from the beginning of trials design. the design of ct or mri studies should target acquisition parameters transposable in a large number of centers; there is no need for "cutting edge" protocols for a reliable assessment of response in a majority of cancer patients. second, the central review of imaging studies will be possible based on the involvement of expert radiologists, and on a robust imaging platform that guarantees availability, quality control, and "side by side" evaluation of baseline and follow-up examinations. principles and limits of response evaluation by morphologic imaging in oncology will be illustrated. a practical approach to hrct of the chest for diagnosis of diffuse lung diseases includes: recognition of the abnormalities, definition of their distribution within the secondary lobule or the lung, identification of associated findings. when combined with the patient clinical history, these steps allow to shorten the list of differential diagnoses and may sometimes lead to a specific diagnosis. a reticular pattern consists of multiple lines as the result of interlobular septal thickening, intralobular lines or cystic walls of honeycombing. when present as a predominant abnormality, thickening of interlobular septa has a limited differential diagnosis which includes pulmonary oedema/haemorrhage, lymphangitic spread of cancer, sarcoidosis and alveolar proteinosis. honeycombing represents destroyed and fibrotic lung tissue containing numerous cystic airspaces with fibrous walls and is considered a ct feature of established pulmonary fibrosis. when honeycombing is present, uip is likely the histologic pattern and ipf is the most likely diagnosis, in the absence of a known disease. a nodular pattern consists of multiple rounded opacities - mm in size. the distribution of nodules is the most important factor in making an accurate diagnosis. a centrilobular predominance of nodules that typically spare pleural surfaces is a frequent sign of bronchiolitis and airway disease. a perilymphatic distribution is most frequently seen in patients with sarcoidosis, silicosis and lymphangitic spread of cancer. a random distribution with nodules diffusely and uniformly distributed can be the result of infection, haematogenous metastases and other rare diseases such as langerhans cell histiocytosis. the diffuse interstitial lung diseases (dilds) are a heterogeneous group of disorders which principally affect the lung parenchyma. basic hrct patterns are common to many disease processes and are usually non-specific. however, their distribution and their temporal evolution are often characteristic enough for diagnostic purposes. increased lung opacity: air-space consolidation, by definition, occurs when alveolar air is replaced by fluid, cells, or other material. on hrct, consolidation results in an increase in lung opacity associated with obscuration of underlying vessels. conversely, ground-glass opacity is defined as: "... hazy increased attenuation of lung with preservation of bronchial and vascular margins". the significance of ground-glass opacity depends on the clinical scenario. cysts and decreased lung opacity: a cyst appears as a round parenchymal lucency or low-attenuating area with a well-defined interface with normal lung. lung diseases characterised by cysts include langerhan´s cell histiocytosis, lymphangioleiomyomatosis, lip, postinfectious pneumatoceles, and amyloidosis. recently, lung cysts have been reported in association with extrinsic allergic alveolitis. honeycombing is a process characterised by the presence of cystic spaces.the determination of the presence or absence of honeycombing on hrct in patients with idiopathic interstitial pneumonia is of great importance. honeycombing may have an atypical distribution particularly in asbestosis, sarcoidosis, non-specific interstitial pneumonia (nsip), drug-related fibrosis and hypersensitivity pneumonitis. hrct is now an integral component of the clinical investigation of patients with suspected and established interstitial lung disease. a knowledge of the close relationship between histopathological changes and ct appearances. learning objectives: . to understand the different patterns on hrct scans of the chest. . to learn about a systematic approach to differential diagnosis of diffuse lung diseases. . to review key imaging findings. how do we report ct of the chest? : the radiology report is often the primary method of communicating imaging findings to the referring physician. practical guidelines on how to report ct scans of the chest for diffuse infiltrative lung diseases will be provided by the panel. emphasis will be placed on both report content and style in order to provide the clinician a tool for good patient care. with the advent of volumetric data acquisition and with increasing knowledge of patho-radiological correlations, hrct has further matured over the last decade and provides the radiologist with an excellent tool for accurately demonstrating gross lung anatomy and precisely analysing abnormal findings. the radiologic diagnosis of many pulmonary abnormalities is based on an assessment of their pattern and distribution. while the pathologist has the advantage of being able to evaluate specimens microscopically, the radiologist is confined to the assessment of more gross disease. however, the radiologist is able to examine the entire lung providing him with information about the distribution of disease and about additional diagnostic clues in mediastinum and pleura. both together, characterisation of the gross findings and knowledge of their distribution represent the key features for arriving at a confident diagnosis. session objectives: . to become familiar with the standardized and internationally accepted terminology for describing and analysing hrct findings. . to learn how a structured analysis of the predominant pattern and its distribution represent the key for making a specific diagnosis in the best case scenario or to understand how it helps to narrow the differential diagnosis. . to promote the understanding how the variability of manifestations of some diseases can cause an overlap of findings being responsible for difficulties the radiologist encounters when categorizing diffuse diseases of the lung parenchyma. a. most radiological reports consist of a ‚descriptive part' in which the radiologist describes the findings and a short ‚conclusion part' containing the interpretation of what is seen. both parts are filled with ‚jargon'. some terms are typical radiological ‚language' while others refer to terminology also used by clinicians and pathologists. some terms are purely descriptive but others may contain already some interpretation of what is seen and hence narrow the differential diagnosis. the meaning of a term may also change over time. it is very important that both radiologists and the readers of their reports speak the same language and know and understand the meaning and full content of each term. this is especially important when radiological findings in patients with diffuse lung diseases are described and interpreted. diagnosis of diffuse lung disease is indeed largely based on the recognition and description of the appearance pattern of the disease because this often allows developing an appropriate list of differential diagnosis. members of the fleischner society have introduced a glossary of terms for thoracic imaging. this presentation will emphasise on the importance of such a glossary for both describing and interpreting chest images. special attention will be given to the terms used to describe the different patterns in diffuse lung diseases: ( ) reticular and short linear opacities, ( ) nodular opacities, ( ) increased lung opacity and ( ) cysts and decreased lung opacity. a s c b d e f g h the purpose is to describe ethical dilemmas related to the use of radiation in finnish radiographer's work in diagnostic imaging. the data were collected during the spring and summer of , and it consisted of thematic interviews of diagnostic radiographers (n= ) whose working experience varied from to years. the data were analysed by the method of data-oriented qualitative inductive content analysis. ethical dilemmas related to the use of radiation were found to concern justification and optimisation principles, which were found to be inadequately implemented by radiographers. the background factors of these dilemmas were found to be both dependent on and independent of the employee, resulting in worsened well-being at work and in seeking for change. the current processing methods of dilemmas were found to be insufficient, and suggestions for better processing methods were made. the results suggest that there may be shortcomings in radiographers' knowledge of radiation usage. attitudinal problems within the work community seem to maintain ethical shortcomings. instead of ethically inadequate routines and passing the baton to other professionals, radiographers should be encouraged in committing themselves into responsibility and safety culture. respondents repeatedly described powerlessness and inability to intervene in ethical problems. the common cold is one of the most frequent illnesses in europe and the united states. although most cold are mild and resolve within a short time period, colds cost billions of dollars per year, mostly due to lost time at work and school. the common cold is a group of symptoms caused by one of a large number of viruses. rhinoviruses cause the greatest number of colds; there are more than different varieties of rhinovirus. the average adult experiences two to three colds per year, while children average - colds per year. in most cases, colds do not cause serious illness. most colds last for - days, although many people continue to have symptoms (coughing, sneezing and congestion) for up to weeks. some viruses that cause the common cold can also depress the immune system or cause swelling in the lining of the nose or airways; this can lead to bacterial infection. one of the more common complications is sinusitis, which is usually caused by viruses and rarely (about % of the time) by bacteria. however, it can be difficult to distinguish bacterial sinusitis from sinusitis caused by a cold because the signs and symptoms can be similar. however, due to the fact that a runny nose can also result from inflammation, trauma, foreign body and other abnormal processes, including tumours, an excellent diagnostic workup is necessary. the purpose is to examine the relationship between man and technology within radiography without considering man and technology as separate entities. the study is designed as an observational study which took place in a danish radiological department. it involved examinations followed up by three semi-structured interviews. through emergent narratives radiographers construct a practice in which the relationship between man and technology is marked by a struggle of domination of one over the other. the struggle expresses itself through two competing plots: a diagnostic plot with a prevailing, but not merely chronological structure mostly composed of events tied to pathology. the life story, in opposition, plots the examination as a significant experience in the patient's life, transforming it into events stretching towards a future yet unknown. most often the radiographers contributed to the domination of man over technology through active engagement of their narrative alertness. errors and failure to keep the time schedule can, however, lead the radiographers to a change of perspective that makes them displace man from the scene of radiography by playing on the premises of technology. in several stories technology can be labelled ‚setting' while the patient plays the main role. the radiographers act with the patient as point of origin, but they reason with technological arguments. hence, the human aspects of radiography constitutes an underground practice, which is not explicitly articulated valuable. this creates a field of tension between man and technology, with risk of technological dominance. the private life of everyone of us as well as the everyday practice of our profession is deeply embedded in ethical/deontological aspects which play a critical role into: our profession, the relationship with our patients, with our colleagues, with the health care team we work with, and with ourselves. the radiographer's everyday practice has to take this aspects into account. ethics finds its origins in the fusion of three different sources, as if it were a compromise between: the species we belong to, the society we live in, and ourselves. yet, we are not obliged to abide to these three elements. it is through our actions that we decide how to behave in certain situations. and this is the reason why every single action towards the others gathers importance, the simple rites when individuals meet play a pivotal role in the creation of a relationship with the others. shaking hands, greeting and using polite forms of speech, all these actions are ethically important, they allow us to catch the benevolence of our interlocutor and to discourage any possible hostility. what would happen if our professional identity were perceived and acknowledged through the way we can interact and relate to others? in this case, the work no longer ennobles the man, but it is the man, through his actions, who ennobles his profession. behaving ethically towards one's own profession, towards the others and towards ourselves, is it, or could this be the common ground of european professional ethics? whenever a diagnostic x-ray examination of a pregnant patient is considered to be necessary, conceptus dose estimation is an important step in assessing the risks to the unborn child. accurate estimation of conceptus dose is also needed after inadvertent irradiation of a pregnant patient from a diagnostic x-ray procedure. several methods have been developed to estimate conceptus dose from radiologic examinations. when the uterus is remote from the directly exposed tissues, the conceptus is exposed to scattered radiation and its dose is negligible (< mgy). examinations involving the abdomen-pelvis may deliver higher dose to the child. variations in maternal body size and uterus position should be taken into account to obtain accurate conceptus dose estimation. multidetector ct (mdct) scanners have replaced conventional ct technology. conceptus doses from abdominal mdct range from about to about mgy during the first post-conception weeks for a scan acquired at kvp, mas with a pitch of . , depending on maternal body size and uterine position. multi-phase abdominal ct examinations may deliver relatively high doses to the unborn child. doses to the conceptus below mgy should not be considered a reason for termination of pregnancy. the risk to the embryo/foetus for stochastic effects is assessed on the basis of dose using appropriate risk factors. although these risks from a single diagnostic procedure are low for the majority of diagnostic x-ray examinations, it is important to ensure that doses are kept as low as reasonably achievable. learning objectives: . to learn how to manage and counsel pregnant patients in case of (a) intentional and (b) accidental exposure. . to learn how to estimate conceptus radiation dose from diagnostic x-ray examinations. . to learn how to assess the radiogenic risks to the embryo/foetus from diagnostic x-ray examinations. epithelial cells. the mucociliary transport drains each sinus in a specific pattern through its ostium to the nasal cavity and ensures a constant flow of mucus containing bacteria and particulate matter. functional endoscopic sinus surgery (fess) aims to enlarge the preformed ostia to allow physiologic drainage. preoperatively, anatomic variants have to be ruled out or shown by ct to avoid injury. special emphasis lies on the ostiomeatal unit, a complex anatomic region at crossroads of mucociliary drainage from frontal, anterior ethmoid and maxillary sinuses (uncinate process, haller, agger and frontoethmoidal cells, inverse turbinate). description of the level of the cribriforme plate in relation to the roof of the ethmoid is another key element in a report to avoid skull base injury (keros i-iii). preoperative identification of variations in the location of the optic nerve and the cavernous portion of the internal carotid artery is also mandatory. furthermore, the close proximity of the sinuses to neighbouring structures and their thin bony walls predispose to certain pathways of spread. runny and stuffy noses may be due to a long list of pathologic conditions, which require distinct imaging strategies and treatment options. acute rhinosinusitis, for example, is normally managed by gps without any imaging study. occasionally, signs and symptoms suggest orbital or intracranial complications: in such cases msct allows a prompt diagnosis and is preferred to mri for wider availability and faster acquisition. persistence of signs/symptoms for longer than weeks classifies rhinosinusitis as chronic (crs); the condition may or may not be associated with the presence of nasal polyps. in patients affected by crs cross-sectional imaging aims at precisely depicting bone structures and air conduits. in detail, imaging shows the anatomic arrangement of sinus drainage pathways and their patency, maps anatomic variants that may facilitate crs or increase surgical risk, depicts bone changes induced by crs or by the mechanical pressure exerted by polyps. these tasks are better accomplished by ct, even more so after the advent of multislice technology allowed multiplanar reconstructions along oblique planes. after surgery, imaging is required when signs/symptoms recur -to asses presence and extension of crs or nasal polyps -or when late complications are suspected -such as mucosal synechiae impairing mucus drainage or mucocele. mri plays a limited role in sinusitis because it fails to demonstrate thin bone structures; nonetheless, its use is advocated in aggressive inflammatory diseases (such as invasive mycoses and wegener's granulomatosis) to demonstrate the involvement of deep spaces of the face and of the skull base. sinonasal tumours are rare and comprise only % of all malignancies. the clinical and radiological challenge is to differentiate malignancy from benign or inflammatory changes. most malignant tumours present at advanced stages. malignant tumours are most common in the th - th decades and have a male preponderance. squamous cell carcinomas are the most prevalent ( %) followed by adenocarcinomas. squamous cell carcinomas are most common in the maxillary sinus, next the nasal cavity and ethmoid sinuses, while adenocarcinomas are most often seen in the ethmoid sinuses, and commonly caused by occupational exposures, e.g. nickel and hardwood workers. inverting papilloma is one of the most common benign tumours and have been associated with human papillomavirus. ct should be the first modality in paranasal sinus imaging, while complementary mri before and after gadolinium i.v. is mandatory to differentiate tumour from inflammatory disease. a general rule is that tumours more often have unilateral extension and that malignant tumours usually erode the bone. however, malignancy may mimic benign tumours with thickening or remodelling of the adjacent bone. while ct is superior to demonstrate changes in the bone, mri can differentiate tumour from inflammatory changes and demonstrate tumour invasion and perineural spread. malignant tumours are: squamous cell carcinoma, adenocarcinoma, adenoid a s c b d e f g h b. x-ray imaging and pregnancy: justification and optimisation of exposure p. vock; berne/ch (peter.vock@insel.ch) as outside pregnancy, justification and optimisation are the main steps to be done when an imaging examination using ionising radiation is considered during pregnancy. however, the risk concerns the embryo/foetus in addition to the mother which means that justification has to be more critical whenever the uterine dose is not neglectable. the practical approach to an examination in any woman of child-bearing age starts by ruling out pregnancy, whether by taking history or by a laboratory test. when pregnancy cannot be ruled out, further steps will depend on the type of examination needed and the urgency of diagnostic clarification. ultrasound is the alternative to be preferred when it can answer the clinical question. but even among x-ray examinations, the uterine dose is varying widely which asks for a careful selection, optimisation and, maybe, for postponing the test. once pregnancy is confirmed, the major question is whether the specific type of diagnostic examination will include the uterus in the primary radiation field. examinations not involving the uterus by direct radiation -despite a potentially significant exposure by scattered radiation -can usually be performed without a relevant risk to the embryo/ foetus. the situation is more critical when the uterus is within the examination field and when therapeutic interventions are considered. the presentation will discuss the practical approach to these different situations, the influence of the stage of pregnancy, optimisation methods and the choice between alternative methods in some frequent clinical situations. this paper explores the risks to the foetus when magnetic resonance imaging (mri) is used. mri uses three main components to produce images from inside the body: a static magnetic field; a pulsed radio-frequency (rf) fields and time-varying gradient electromagnetic fields. the exact frequencies of these fields depend on the mri system purchased, for example; a . t scanner uses mhz rf, a . t system uses mhz and a t system uses mhz rf. there is also a wide range of options for gradient strengths and slew rates to be considered as well. the overall exposure for the foetus depends ultimately on the imaging sequence used and the area being scanned. this paper will discuss particular hazards that need to be addressed for pregnant women including biological effects of the static and time-varying magnetic fields, heating effects of the rf pulses and acoustic noise generated by the spatial encoding gradients. the circumstances for foetal exposure in mri will also be discussed including the following situations: the patient may not be aware that she is pregnant, likely to be in the first trimester; the mother is referred for direct foetal imaging after ultrasound (normally second or third trimester); the expectant mother may need diagnosis; research on pregnant volunteers. the exposure for pregnant staff working in mri is also an essential consideration. finally, how to minimise the exposure for the foetus during mr imaging will be discussed. a b c d e f g h in terms of cerebral perfusion (rcbf, rcbv, mtt and ttp) and cell metabolism (cytotoxic versus vasogenic oedema, diffusion restriction). the fundamental goals of stroke imaging in the acute phase are: . to rule out intracranial haemorrhage, and other non-stroke causes of the patient's symptoms; . to show occlusion of a major blood vessel, e.g. by ct or mr angiography; . to demonstrate ischaemic brain tissue (cytotoxic oedema), e.g. by diffusion-weighted mri; . to reveal tissue blood flow and to identify areas of salvageable brain tissue ("penumbra"), e.g. by perfusion ct or mri; . to select candidates for thrombolytic therapy within the critical to hour time window. the purpose of this presentation is to review how new developments in neuroimaging improve our understanding of the pathophysiology of acute stroke and to demonstrate that multiparametric techniques now play a crucial role in the rapid diagnosis, clinical management, therapy and outcome prediction of patients with an acute stroke. organisation is crucial to the practice of dethrombosis. the first step is to grasp the fact that ischaemic stroke is an emergency. stroke victims require an organisation similar to that provided for heart attack patients. patients must be taken to hospital in an ambulance manned by a specifically trained crew. ideally equipped with a mini ct scanner recently made available. this device provides an immediate morphological scan and ct angiogram to be sent by satellite to the reference hospital so that the problem will be clear and the medical team ready on the patient's arrival. clinical and neurological assessment of the patient must be undertaken immediately along the lines of uk practice implementing a simple, clear and rapid procedure. if the clinical, ct and ct angiography findings indicate intra-arterial treatment, responsibility shifts to the neuroradiology team, organised to work a hour shift, at the hospital or at least on call. dedicated imaging systems must be available: a multi-layer ct device (ct angiography and perfusion studies). dedicated angiography suite is essential. a flat panel system would be ideal. there is growing evidence supporting the availability of an mr system. this device would certainly enhance the possibilities of a stroke unit but it is not essential in the initial stages. a stroke unit should be based on a dedicated facility, with emergency access separate from the general emergency room, dedicated equipment, dedicated ambulances and appropriate links including a dedicated ward and a rehabilitation unit essential to the success of the project. learning objectives: . to learn about the benefits and risks in stroke intervention. . to consolidate knowledge of different methods of intervention in stroke therapy. . to learn about the different imaging methods used to decide on and perform stroke intervention. where do we stand in stroke therapy today? : stroke is a common health problem with high socio-economic costs. in recent years much effort has been focused on finding ways to ensure early intervention as well as new methods not only for early detection but for early treatment. the discussion will focus on where we stand today and whether we think stroke imaging and treatment will advance further. during the last decade imaging of stroke has developed from a minor field dealing with a barely treatable disease to one of the most dynamic parts of neuroradiology. the routinely treatment with intra-venous thrombolysis within the . hour time window and the continuously expanding treatment of severe strokes with intra-arterial thrombectomy have lead to increasing requests from clinicians and interventional neuroradiologists to provide imaging information for treatment guidance. all radiologists involved in the management of stroke are facing mainly three demands: first, to have good knowledge of the causes and the pathophysiology of ischaemic stroke and a proper understanding of the current models for infarct development, as a prerequisite for rational decision making and efficient communication with the clinicians. second, to be able to choose and to use appropriate imaging modalities for the acute work up of patients with stroke and to be aware of the benefits and drawbacks of ct and mri. finally, is good knowledge about the therapy options mandatory in order to be able to support the clinicians in the time critical treatment decision. this session will try to cover these three demands in order to give an overview on the current status and the opportunity to discus future perspectives. a. aetiology and pathophysiology of stroke r. von kummer; dresden/de (ruediger.vonkummer@uniklinikum-dresden.de) "stroke" is the commonly used diagnosis for disturbances of brain blood supply due to ischaemia, haemorrhage, or venous obstruction. it is evident that "disturbances of blood supply" in stroke patients do not represent a useful concept in order to develop effective treatment. it is the radiologist's task to describe the acute brain pathology, its conditions (pathophysiology), and its aetiology to give directions for specific treatment and prophylaxis. the approach is vascular and brain tissue imaging. brain ischaemia is the cause of stroke in about % of patients, intracranial haemorrhage in % of patients. arterial obstruction causing brain ischaemia is thrombotic or embolic. it can as well be caused by arterial wall dissection or inflammation. arterial wall diseases and heart diseases including a patent foramen ovale are sources for brain embolism. the obstruction of a brain supplying artery causes brain tissue ischaemia of various degrees depending on the capacity of collaterals supplying the same arterial territory. the chances for collateral compensation depend on the site of obstruction and the development of collateral vessels. neurons cannot tolerate low cerebral blood flow (cbf) below ml/ g x min for more than minutes and develop first cellular then irreversible ionic oedema. they survive, but do not function with low cbf between and ml/ g x min. enhancement of cbf in such regions, e.g. by arterial recanalisation can mean functional recovery. the imaging diagnosis of damaged brain is therefore the first aim in acute ischaemic stroke patients. focal liver lesions are part of our everyday practice because radiologists performing various types of examinations may encounter them without being aware of any clinical condition rising the suspicion of these changes. in a situation like this several important decisions are to be made by the radiologists, like: is the lesion clinically significant, is the lesion benign or malignant, are additional imaging examinations necessary, does it require medical, surgical or interventional radiological action, should it be, and if yes, how frequently followed up, etc. for providing a well-established answer to these questions the reporting radiologist has to be familiar with medical history and results of former examinations (physical, laboratory, imaging, etc.); consider the age, gender, physical condition, eating and drinking habits, potential drug abuse of the patient; bearing in mind the prevalence and statistical probabilities of different pathologic conditions. with all this information at hand the thorough analysis of images obtained by different modalities will allow a rather reliable differential diagnosis and a well-established planning of therapy of the incidentally detected lesions. learning objectives: to learn more about the clinical conditions that may result in the appearance of focal liver lesions. . to be informed about the clinician's way of thinking in the process of differential diagnosis. the spectrum of differential diagnoses is broad in the liver. therefore, incidentally discovered liver lesions represent a challenging clinical situation. fortunately, there are specific imaging features for the most common benign and malignant liver lesions (such as, e.g. hemangioma, fnh, cysts, vascular pseudolesions, hcc and metastases) so that a minimal-invasive diagnosis with a biopsy is not needed in a lot of cases. in ultrasound, the echogenity and recently also the contrast agent behaviour are used for liver lesions characterisation. in ct, attenuation and also contrast agent behaviour are used for characterisation. mr imaging offers several options including t -and t -weighted images, use of chemical shift imaging, gre sequences with long echos and diffusion weighted images, so that tissue components such as fat, water, glycogen, iron, etc. can be evaluated already in the pre-contrast examination. beside the evaluation of dynamic signal characteristics in the early dynamic phase after contrast agent application, mr can utilize also tissue-specific contrast agents dedicated to the res or to the hepatocytes. other modalities such as angiography, pet or other nuclear medicine methods usually only play a minor role nowadays in the evaluation of incidental liver lesions in nononcological patients. in oncological patients the clinical consequences and also the range of diagnoses and pre-test probabilities are different from the non-oncological group; therefore, the demands to imaging are even higher. in case of atypical presentation of otherwise benign liver lesions such as sclerosed hemangioma close follow-up or even biopsy can be necessary in such a setting. thyroid nodules are common. in - % of adults nodules can be palpated and at ultrasonographic examination nodules are seen in - %. most nodules are benign, the eventual diagnosis in patients with a palpable nodule is carcinoma in only % of cases. the incidence of thyroid carcinoma is low. papillary carcinoma is the most common type and is found in - % of thyroid cancer. lymph node metastases in thyroid carcinoma are primary to the paratracheal and prelaryngeal nodes (level vi), and the upper (level iii) and lower (level iv) jugular nodes. malignancy should be suspected when there is rapid growth, a firm or fixated mass, when enlarged lymph nodes are present or in case of vocal cord paralysis. in patients with multiple thyroid nodules the cancer risk per patient is not altered. ultrasound is the primary imaging technique in patients with a palpable thyroid nodule. it is best performed with the patient supine, the neck slightly extended and using a high frequency linear-array transducer. several ultrasonographic features are more often associated with benign or malignant nodules. however, while some ultrasonographic features may strongly suggest malignancy it may be very difficult to rule out malignancy by means of these features alone. ultrasound-guided fine needle biopsy can be performed in an attempt to differentiate between benign and malignant nodules. unfortunately, even with ultrasound guidance, an important number of cytological examinations will be non-diagnostic. in addition, in follicular lesions it is usually impossible to differentiate cytologically between benign and malignant follicular nodules. learning objectives: . to learn about the optimal settings for us examination of the thyroid. the neck is host to a large variety of benign and malignant diseases ranging from simple cysts to acute inflammation with complications to highly malignant sarcomas and imaging plays an important role to assess the nature and the extention of disease. patient history and clinical findings will decide which imaging technique is best suited to resolve the diagnostic dilemma. ultrasound is recommended to differentiate between a cystic or solid lesion, to guide fna, being quick and accurate. pathologic flow within or around a lesion can be assessed adding doppler technique. in acute illnesses with anticipated complications, such as a deep neck abscess, contrast-enhanced ct is required to evaluate local extention and demonstrate spread from the neck deep to the mediastinum. mri is indicated to assess non-inflammatory, subacute or chronic diseases, such as vascular malformations, branchial cleft cysts, benign and malignant soft tissue tumours, brachial plexus pathology and neurologic diseases. vascular malformations are easily depicted with mri using t sequences in different image planes. persistent fistulas of the branchial system can also be depicted by mri running from the anterior border of the sternocleidomastoid muscle to the lateral pharyngeal wall. assessing the origin of a soft tissue tumour has major implications for surgery. staging a malignant soft tissue tumour must comprise the skull base and upper mediastinum including local extention, pathologic neck nodes and perineural spread. follow-up during chemotherapy and postoperative monitoring of malignant disease are important indications for (mr) imaging, the more so in young patients, diminishing ionising radiation. learning objectives: . to identify different compartments in the neck. . to be able to choose the optimal imaging technique relating to request. . to become familiar with the most common pathology in adults and children. . to be able to address a short list of differential diagnoses. patients ( %) and by ultrasound in patients ( %). the activity of the disease was assessed correctly in % patients by ultrasound, compared to % patients by enteroclysis. there was no statistically significant difference. bowel us is a useful investigation for the diagnosis and follow-up of patients with crohn´s disease. learning objectives: . to learn about us techniques used in the assessment of patients with inflammatory bowel disease. . to learn tips for interpretation of us in the diagnosis and follow-up of patients with inflammatory bowel disease. . to understand the pros and cons of using us, including ceus, in the diagnosis and follow-up of patients with inflammatory bowel disease, including assessment of disease activity. . to appreciate the accuracy, reproducibility and usefulness of us compared with other imaging techniques in assessing patients with inflammatory bowel disease. a- : the use of computed tomography (ct) in the diagnostic imaging of patients affected by inflammatory bowel disease (ibd) has a long standing history. ct techniques such as ct-enteroclysis and ct-enterography have been reported as efficient tools for a radiological assessment of disease. the state-of-the art methods will be reported and their potentialities in diagnostic accuracy discussed in different clinical type of ibd. the acute setting of unknown ibd could be demanded as well to ct examination performed in emergency without endoluminal contrast medium administration. the ct appearance of the intestinal wall of the segments involved by an active disease could be not correctly interpreted if the knowledge of basic and classic typical findings of ibd is not combined to consider different pathologic entities that could affect the intestine, offering similar but not even equal signs. the entire abdominal background needs to be evaluated according to the clinical symptoms, signs, laboratory parameters and history of the patient. ct examination could be an efficient tool to determine the degree of disease activity in patient with known ibd as well as to assess the presence of an acute inflammatory disease of the intestine in patients with unknown ibd affection. the actual role of ct in the early diagnosis of ibd, in disease staging and detection of complications as well as monitoring therapy in follow-up will also be considered and discussed. learning objectives: . to learn about state-of-the-art ct techniques for the assessment of patients with inflammatory bowel disease. . to learn tips for interpretation in terms of diagnosis and differential diagnosis of inflammatory bowel disease. . to discuss the value of ct in determining disease activity and monitoring therapy. the aim of this lecture is to give an overview of the techniques and typical findings for imaging crohn's disease (cd) with either multidetector row computed tomography (mdct) or with magnetic resonance imaging (mri) and compare the two different modalities. optimal imaging of cd begins with the preparation phase. the small bowel has to be distended for a concise examination. this can be done either invasively, i.e. enteroclysis or orally, which is named enterography. there is much debate on which of these two preparation methods is better. for the administered intraluminal contrast, best is water with some additives, which are neutral in ct and biphasic in mr. imaging in mdct is done sec and sec after iv administration of iodinated contrast with a thin collimation. all image data are reconstructed in axial, coronal and sagittal planes. the aim of imaging in cd should be to establish the following: ( ) presence, severity, and extent of the disease; ( ) its activity; and ) extra-intestinal complications. both modalities have proven to be a good tool to evaluate the extent, the activity of the disease and the presence of extraluminal complications. both are also able to identify the type of the disease, being either the active/inflammatory, the fistulising/perforating, the fibrostenosing or the reparative/regenerative type. the challenge for mri is mostly the in-plane resolution of the image data whereas radiation dose is for ct. incidental lesions are frequently discovered during routine radiographic evaluations. correlation with clinical history and additional confirmatory imaging is essential to the development of an accurate, focused differential diagnosis and for appropriate management. the objective of this presentation is to describe the imaging findings associated with incidentally found liver lesions and to review those clinical and radiologic features, which should be considered in development of an ordered and accurate differential diagnosis. learning objectives: . to introduce typical cases illustrating the role of imaging modalities in the differential diagnosis of unexpected liver lesion cases. . to motivate the audience by the use of voting pads to be involved in the diagnostic process. . to highlight the conclusion that may be drawn on the basis of the discussed cases. the diagnosis of ibd is based on complex evaluation of clinical signs, endoscopic appearance, radiological imaging, laboratory tests and histology. besides crohn's disease (cd) and ulcerative colitis (uc) the modern classification advises to use the term ibdu (ibd unclassified) colitis to those % of cases, where the distinction between cd and uc is impossible based on the results of multiple exams. these cases are usually clinically more severe, with humbler therapeutic results. the new classification of cd types includes besides the phenotype (b : inflammatory non stenosing non penetrating, b : stenosing, b : penetrating) the age (a : ≤ y, a : - y, a : > y) the location (l : ileum, l : colon, l : ileocolic, l : upper gi tract) and p perianal involvement. in uc the most important clinical factors are extent, type and severity. the classification includes length and clinical types as acute, relapse-remission and chronic continuum. the radiological imaging in cd is needed for lesions not reachable by the endoscope, for categorising, for follow-up and detecting complications. different modalities fit best for these different tasks. the goal of imaging in uc is rather to help differential diagnosis and detection of complications. while in uc there are good correlation between laboratory and endoscopic signs of activity and severity so there is not much need for radiological activity indices, in cd the clinical activity does not correlate with the histological changes. although cdai is widely used for clinical studies, it includes several subjective factors, thus radiological activity indices may be of help. crohn's disease is more likely a systemic disease governed by a shift in the immune response, thus affecting the whole malt system. bowel ultrasound is a quick, simple and effective investigation. the method is simple and requires no bowel preparation. we use both -convex (c - -overview, anatomical orientation) as well as linear transducer (l - -preferred, incl. ceus, l - -children and very slim patients, l - -tdps, deep structures). we examine patients in supine position, first small bowel, than mesentery and large bowel. finally, we examine the terminal ileum and cecum or the areas of anastomoses. we assess folds, hastrum, wall thickness, vascularity, echogenity of the surrounding fat, the presence of nodes and peristalsis. in some cases we use intravenous contrast (ceus). at least four studies have prospectively compared the diagnostic accuracy of us with radiological studies, endoscopy or surgery in those with suspected crohn's disease. in these studies, the sensitivity of us ranged between % and % and its specificity reached % and %. in our prospective study we investigated patients by ultrasound and enteroclysis. we established the diagnosis by enteroclysis in the central venous vasculature and particularly the upper venous system may be obstructed by either benign or malignant lesions. malignant central venous obstructions are mainly due to bronchial carcinoma, mediastinal metastasis, mesothelioma or lymphoma. the most common cause for benign central venous obstruction is haemodialysis related; other benign reasons are rather uncommon but increasing due to the omnipresent use of permanent central venous access catheters as well as implantable cardiac rhythm management devices. the incidence of haemodialysis-related central venous obstruction resulting in angioplasty is described to be more than times higher compared to, for example, pace-makerinduced stenosis. in any case, early detection and treatment of complications are essential to provide adequate care. contrast venography for many years has been the standard of reference; yet this procedure has its shortcomings. sonography is not consistently reliable for detection of central pathologies, owing to difficult access to these vessels. today contrast enhanced ct is employed to define the site of the obstruction and the presence of possible thrombosis and reveal surrounding soft tissue alterations. mri is of comparative or even higher sensitivity and specificity in evaluating the patency of the central veins. the efficacy and safety of stent placement in central venous occlusion of benign and malignant origin lead to rapid relief. stenting provides fast symptomatic help. repetitive percutaneous intervention can prolong the cumulative patency. thrombolysis may be required to improve the final result, should, however, not be employed as a sole means for revascularisation. us and mr are excellent imaging techniques for studying tendons and muscles. during this integrated session we will review the advantages and disadvantages of us and mr for the diagnosis and follow-up of sports injuries. tricks of both techniques will be addressed, together with some useful guidelines for specific sports injuries. ultrasound is being used at the pitch side and in sports medicine practice as an adjunct to clinical practice. with this in mind there are a number of questions that will be answered in these talks. . when us and mri are the primary imaging and when they are complimentary. . what advances have there been in us and mri imaging to help advance our use of these techniques in tendon and muscle injury. . should we be aspirating haematomas and using autologous blood injections or prp to treat tendon or muscle disease. . can we predict the athletes return to sport? a- : muscle imaging is inherently complex and presents unique morphologic challenges and continuing integration of dynamic, physiologic and functional capabilities. in sports medicine, ultrasound (us) has proved to be an excellent tool to evaluate muscle strain and contusion injuries in athletes providing good correlation with clinical findings. in the acute phase, us has nearly equal sensitivity to mr imaging to diagnose muscle strains, except in the first few hours after the injury, when fresh haemorrhage and oedema have similar echogenicity to normal muscle and strains may go unnoticed. later in the process, us has been shown to be a useful tool in assessing the sequential stages of muscle repair, showing progressive resolution of blood fluid products, decrease in oedema and formation of scar tissue, thus aiding rehabilitation planning. dynamic us during muscle contraction can be valu- . to compare the diagnostic accuracy of ct versus mri in crohn's disease. . to learn about a strategy for the use of ct and mri in the radiological workup of patients with inflammatory bowel disease. five good reasons for the radiologist to be at the forefront : the chairman and speakers will debate the optimum imaging paradigms according to ( ) the clinical indication, ( ) assessment of those with suspected but as-yetundiagnosed disease, ( ) staging of the small bowel in those with newly diagnosed crohn disease, ( ) evaluation of response to therapy and ( ) assessment of those with long standing disease. the optimum approach to assessing and monitoring disease activity will be discussed. deep vein thrombosis (dvt) is a common condition which can lead to possible lifethreatening pulmonary embolism (pe). the primary imaging modality nowadays is duplex ultrasound. in cases where the pelvic veins and/or the vena cava is involved, a venous-phase ct is helpful to determine the exact extend of the clot burden. standard therapy for dvt is anticoagulation. however, if there is a contraindication or a complication (bleeding) to anticoagulation a vena cava filter is a good option to prevent pe. a filter should also be considered if a catheter directed thrombolysis is performed. with the new optional filters the filtration can be terminated either by removing the filter or by converting the filter into a stent. one problem which was observed with these optional filters is that (too) many of them are left in place. despite a low complication rate of filters, a filter may cause an increased risk of further dvt, or a caval occlusion and in rare cases even a migration or penetration is possible. therefore, patients with optional filters should be followed and filters should be retrieved when clinically no longer needed. varicose veins are an early manifestation of venous insufficiency of the lower limbs which can also lead to skin changes including extensive ulceration. it is a very common problem affecting up to % of adults in europe. patients presenting with venous insufficiency must first be assessed by a history and clinical examination to determine the presence and extent of the disease. a duplex ultrasound examination is essential to determine the cause from which an appropriate treatment plan can be decided. depending on the patients wishes and their funding options it is perfectly reasonable to treat all stages of the disease even if just cosmetic. treatment aims to eliminate the reflux in all contributing veins starting proximally and moving distally. surgery used to be the commonest treatment offered to patients but this has been largely superceded by endovenous methods which have significant advantages including: no general anaesthesia, outpatient based, no cuts, no scars, low recurrence, rapid convalesence. there are several endovenous methods using laser, rf, foam sclerotherapy and most recently steam heating and a combined mechanical/sclerosant system (clarivein (r)). laser and rf give almost identical results and % permanent truncal vein closure is to be expected. foam sclerotherapy is cheaper and quicker but long-term results are poorer and patients often have to return for repeat procedures. having ablated the main truncal veins using laser or rf at least % of patients require additional treatments for residual varicosities, e.g. microavulsions or foam sclerotherapy. adenocarcinoma is the most common pancreatic malignancy, affecting the head in - %. mdct is very effective in detection and staging of adenocarcinoma, with a sensitivity of up to % for detection and accuracy of - % for staging, but it has limitations (detection of small cancers, characterisation). mri is a problem-solving tool in equivocal ct to depict small cancers. mri with mrcp helps to differentiate between adenocarcinoma and focal pancreatitis. the "duct penetrating sign" at mrcp is indicative of an inflammatory mass. examination protocols for mdct include oral administration of - . l of water ("hydro-ct"), iv contrast administration of ml/kg b.w. (app. - ml), a flow rate of - ml/s, and a biphasic scan in parenchyma and venous phases. mri protocol should include non-fatsat and fat-sat t w gre, t w tse, dwi, mrcp, and contrast-enhanced sequences. gadoliniumenhanced t w d-gre are helpful for assessment of vessel infiltration and cystic tumours, whereas mangafodipir-enhanced sequences improve delineation of small tumours. in patients with locally advanced tumours, neoadjuvant chemoradiation may be used for tumour downsizing to make it amenable to radical surgery. however, pancreatic cancer often does not shrink after tumour response, which renders mdct or mri unreliable for tumour assessment in this setting. pet/ct may play a role in this indication. in conclusion, ce hydro-mdct is an excellent and robust tool for pancreatic cancer detection and staging. mdct in combination with mri or eus provides high accuracy for characterisation of tumours. further improvement is needed to assess tumour response after neoadjuvant chemoradiation. ct is the established imaging technique for evaluation of pancreatic adenocarcinoma. mri, however, can play a major role in this disease. technical advances of mri including parallel imaging techniques, multichannel receive coils of the abdomen, dynamic gadolinium-enhanced t -weighted fat sat d gre sequences, d mrcp sequences, mangafodipir-enhanced mri and diffusion weighted imaging (dwi) have greatly improved the results of mri in the assessment of pancreatic tumours. standard imaging sequences include t -weighted gre with fat saturation, in-phase and opposed phased t -weighted, axial single-shot turbo spin-echo (tse) and coronal/oblique d and/or d mr cholangio-pancreatography (mrcp) pulse sequences, post-gadolinium-enhanced d t -weighted fat sat sequence and dwi with multiple b values. mr imaging may be useful as subsequent examination for: ( ) detection of small non-contour-deforming tumours with isoattenuated appearance at ct, ( ) evaluation of local extension and vascular encasement, ( ) detection of the presence of lymph node and peritoneal metastases, and ( ) detection and characterisation of associated liver lesions and liver metastases. diffusion-weighted imaging may be helpful as a complementary imaging method in the differentiation between mass-forming focal pancreatitis and pancreatic adenocarcinoma. due to its superior soft-tissue contrast compared with ct, mri ap-able to monitor the healing process. local complications, such as vein thrombosis, irritation of adjacent neurovascular bundles, chronic haematoma and myositic ossificans can be demonstrated with this technique as well. however, us tends to underestimate the extent of injury and the abnormalities seen disappear more quickly when compared with mr imaging. at least in elite athletes, mr imaging seems, therefore, to play a more significant role in management of muscle injury, particularly when decisions regarding the time at which the patient can return to play are needed. by contrast, us is more accessible, and cheaper than mr imaging. in most clinical settings, us should be regarded as the first-line imaging choice for assessing skeletal muscle injury. learning objectives: . to understand the mechanism of injury of muscles in athletes. sports activity can affect tendons due to chronic overuse or acute injury. both can result in complete tendon rupture. us is helpful in precise assessment of rupture severity and extend, but enables alsofor assessment of tendon degeneration, where rupture of individual collagen fibres stimulates a chronic cycle of reparative response caused due to repetitive microtrauma. in chronic tendinopathy histopathological changes, such as hypoxic, mucoid, calcifying, or lipoid degenerations are present. us enables for differentiation of partial tears, tendinosis, tenosynovitis or paratendinosis, because of active and passive dynamic examination possibilities, and high-resolution capability when using high frequency probes. us developments as power doppler us, sonoelastography and contrast enhanced us allow further for new insights into tendinopathy. with the use of us, tendon changes can be diagnosed before they become symptomatic and a reduction of tendon load and initiation of treatment before the condition becomes chronic seem to gain important place in therapeutic regimes. furthermore, us-guided therapies are advisable over blinded-guided injections to minimise side effects and to allow an accurate targeted therapeutic approach. although diagnosis of acute muscle injuries in athletes is usually clinical, magnetic resonance imaging (mri) is a very helpful adjunct tool in this setting, showing the location, extent and severity of the injury and thereby streamlining the management of the patient. portability of ultrasonography (us) on the playfield in the acute setting is not matched by mri, which nevertheless has distinct patterns of grade - muscle strains; however, it is usually with nonacute and deeper injuries of the muscles that mri is especially helpful. presence of bone contusion, stress reaction, or stress fracture is readily displayed by mri and this is practically beyond the limits of us. evolving haematomas, fibrosis, scarring, and myositis ossificans are sequelae of direct or indirect muscle injury which are usually outlined in a single field of view by mri. delayed onset muscle soreness and chronic exertional compartment syndrome, as well as acute and chronic stages of muscle denervation changes, are readily diagnosed with mri. by providing an understanding of distinct muscular denervation changes, mri may actually noninvasively point to the entrapped or involved nerve and guide medical or surgical intervention. foci of mucoid degeneration within the muscle, myotendinous junction, or tendon itself are readily shown by mri, which thereby displays potential sites of failure during sport activities. diffusion tensor imaging may have a role in displaying the subtle architectural disruptions of directly or indirectly injured muscles. learning objectives: . to understand the specific role of mri in the evaluation of muscle and tendon injuries in athletes. . to recognise imaging patterns of tendon abnormalities in athletes: acute and over-use injuries. . to review different mechanisms of muscle injuries: direct and indirect. . to understand how mri might be used in the management of athletes. when a defect occurs in the bowel wall, air will appear within the peritoneal cavity, most frequently due to perforated peptic ulcer and perforated sigmoid diverticulitis. in most perforating gastrointestinal conditions however, the -imminent-perforation is walled-off by neighbouring bowel loops, mesentery and especially by the omentum, nick-named "policeman of the belly". if this walling-off process occurs timely and effectively, no or only minimal free air will appear. the most important causes of walled-off gastrointestinal perforation are appendicitis, peptic ulcer disease, sigmoid diverticulitis, bowel malignancy, crohn disease and -often underdiagnosed-accidently ingested sharp foreign bodies, as toothpicks, fish bones, chicken bones, etc. the extent to which the perforation is walled-off, determines the eventual course of the disease. the us hallmark of -imminent-perforation is inflamed fat around the involved bowel structure. inflamed fat on us corresponds to what is often called "dirty fat" on ct scan: hypodense fat is interspersed with hyperdense streaks. this represents oedema or cellular infiltration of the fatty mesentery and omentum, which have migrated towards the site of the imminent perforation in an attempt to seal it off. on us inflamed fat is recognised as hyperechoic, non-compressible fatty tissue often interspersed with hypoechoic streaks. if fluid collections occur within the inflamed fat, this implies abscess formation. inflamed fat is an important and valuable sign in perforating gastrointestinal conditions. if found in the absence of bowel pathology, the diagnosis is usually epiploic appendagitis or omental infarction. mdct is an extremely powerful tool when the search for a gi perforation is required. the high spatial and contrast resolutions make mdct the most accurate imaging method to identify even small amount of free intraperitoneal air. there is general consensus about the acquisition of a contrast-enhanced scan acquired during the portal venous phase of enhancement (delay of around - sec). controversies are still present in the literature about the utility of a pre-contrast scan as well as the need for a preliminary administration of an oral soluble iodinated cm or for an enema or gaseous distention of the distal gi tract. image reviewing needs the use of a workstation because multiplanar reformations have been demonstrated to improve the detection of small amount of free air. an appropriate window setting is mandatory and it makes mdct % accurate for identification of free air and almost % accurate in the identification of the precise site of perforation. the aim of the examination is not only to assess the presence of free air but also to detect the site of perforation: this is an extremely useful clinical information especially if surgeons decide to perform a laparoscopic repair. several ct signs have been described, able to guide the diagnosis and to differentiate between a perforation originating in the upper gi tract (stomach and duodenum), in the small bowel or in the colon. evidence-based radiology' (ebr) is based on best current evidence, traditionally acquired radiological expertise, the alara principle and the values of referring doctors and patients. any appropriately trained radiologist can formulate an answerable question, search the literature, appraise the retrieved evidence, apply their findings to local practice and evaluate the results. this presentation will describe and illustrate the 'ebr' process. patients with chronic pancreatitis (cp) may present with features resembling pancreatic carcinoma, for which cp is a risk factor with an incidence of . % after yrs. ebr methods were applied to the problem of differentiating mass-forming cp from pancreatic adenocarcinoma. a focused question and literature search found no secondary literature or imaging guidelines. primary literature searching found relevant papers, comprised current best evidence. for ct, time-attenuation curve characteristics had a sensitivity of % and a specificity of % for carcinoma. for mr, dynamic time intensity curve characteristics were only reported in a descriptive study but the duct penetrating sign (mrcp) had a sensitivity of % and a specificity of %. for pet/ct, f-fdg had a sensitivity for carcinoma between % and % with specificity of %- %. for eus/fna, the sensitivity for carcinoma in pre-existing cp was consistently low ( - %), but the specificity was %. simple bayesian analysis was used to establish the best order in which to apply these studies in practice. an algorithmic approach will be presented. the presentation will also consider other 'grey areas' in the field of pancreatic adenocarcinoma imaging using ebr methods. learning objectives: . to learn about evidence-based methods of literature searching and appraisal. . to understand how these methods can be used to produce diagnostic algorithms using the differentiation of pancreatic adenocarcinoma from mass-forming chronic pancreatitis as an example. . to learn about diagnostic algorithms based on different clinical scenarios (local and distant staging, advance versus early disease) involving multiple imaging techniques. : - : room f the hole in the guts in recent years, continuing trends in radiology have diminished the importance of plain films of the abdomen significantly. ultrasonography and mdct are applied with enormous success to the investigation of many abdominal conditions in the emergency setting. in the eyes of the radiologist, plain films, therefore, seem irrelevant in the presence of such powerful imaging procedures. surprisingly, referring physicians, mostly surgeons, gastroenterologists and urologists, still request plain films although the potential of mdct is obvious to them as well. in their perception, the plain film is either a definitive examination before initiation of treatment (e.g. stone at the ureteropelvic junction in us proven hydronephrosis) or a preliminary study prior to mdct or surgery (exclusion of pneumoperitoneum or ileus). in the present climate of cost and radiation consciousness this trend may continue. moreover, many surgeons, gastroenterologists, urologists, etc. have greater skills in reading plain films than in understanding mdct. therefore, the radiologist should poration. all these techniques remain investigational at this time for the treatment of breast cancer. the limitations of and challenges associated with each ablation technique and the issues raised by early pilot studies, which have so far prevented these techniques from replacing standard surgical techniques, will be discussed. in the elderly the co-existence of several diseases, the prevalence of involutional and degenerative aspects, together with physical and cognitive problems represent 'the norm'. it is therefore important to know how to distinguish the healthy elderly from those in need of treatment to avoid overdiagnosis and overtreatment. so the question is how to be aware of the potential and limits of diagnostic imaging and its applications in geriatric patients. brain development occurs rapidly during the last trimester of pregnancy and continues at a rapid pace in the first two years of life. more subtle maturation, for example in the white mater, occurs well into the third decade of life, as evidenced by diffusion tensor imaging (dti). evidence of brain degeneration is occurring soon afterwards and includes mild brain volume los, reductions in white mater integrity on dti, widening of virchow-robin spaces and accumulation of incidental white matter lesions (wml). severe wml and incidental cerebral microbleeds (mbs) are associated with know cardiovascular risk factors and while the concurrent impact may be subtle in terms of cognition, they carry a poor prognosis in the long run. the same is true for silent cerebral infarcts, which increase the risk of dementia. neurodegenerative (general brain volume loss, ventricular dilatation and hippocampal atrophy) changes also occur well before the onset of clinical signs of dementia. in genetically predisposed subjects (e.g. apoe carriers) abnormal (compensatory) brain activity on functional mri and increased (compensatory) cerebral metabolism on fdg-pet have been observed that predict subsequent cognitive decline. more recently, pet studies employing amyloid tracers have shown abnormal binding in a significant proportion of cognitively elderly, suggesting that these subjects are at risk to develop alzheimer's disease -the pace of which is currently undetermined. the objective of percutaneous needle biopsy of the breast is to obtain an accurate preoperative diagnosis with a low upgrade rate post-surgery. indications arise in both symptomatic and screen-detected lesions. a range of biopsy needle designs are available and their selection depends on being aware of the range of needle design types, their advantages and limitations. cutting needles, guns, vacuum systems and mr compatible devices will be discussed. guidance technique for ultrasound, x-ray stereotaxis and mr-guided procedures is extremely important, beginning with patient positioning and anaesthesia prior to commencing the procedure. accurate needle placement under direct vision using high frequency ultrasound probes, x-ray stereotaxis with digital systems and mr guided -with the option of using a cad system for distance calculation -will be presented. typical imaging signs and potential pitfalls of each technique, both anatomical and technical, will be highlighted. methods to increase accuracy including the use of meticulous technique, adequate sampling and correlation of specimen pathology with imaging findings at multidisciplinary meetings are essential. there is always the possibility of a false negative biopsy results in any type of percutaneous image-guided needle biopsy (fnac, core biopsy, vacuum-assisted biopsy). the risk depends upon the quality of the harvested cytologic or histologic material. the quality is closely related to the amount of material collected and the accurate targeting of a lesion. some lesions are at a higher risk of underestimation (sizes < mm, architectural distortions, microcalcifications, stellate lesions) than others (i.e. focal lesions). the false-negative rates for microcalfications using vacuum-assisted biopsy were reported to be . %, for mass lesions . %. using ultrasound-guided g-core needle biopsy the false-negative rates were reported to be in the range of . %. radiologic-histologic correlation plays a key role in the definite and correct judgement of the diagnostic result. in case of imaging-histologic discordance re-biopsy, possibly using larger needle diameters, or even open biopsy should be considered. the european guidelines for quality assurance of breast cancer screening and diagnosis define outcome parameter for breast biopsies. guidelines from the european society of breast imaging published in define the standards and skills necessary to perform these procedures. documentation standards should be used (b -b ) to allow continuous yearly evaluation of the individual institution results and quality improvement. the aim of any needle biopsy is to get as much, i.e. representative material as possible. nevertheless, a certain amount of underestimation, caused by lesion type (adh, dcis, lin, papilloma, for example) will remain and has to be reflected. the presentation will discuss the role of large vessel arteritis within the spectrum of thoracic vascular diseases. it will detail the pathological entities and their morphological, functional, and clinical characteristics. it will present typical ct and mri findings and discuss key elements to the differential diagnosis. it will finally discuss the clinical relevance of this diseases, with a special emphasis on overall evolving importance of thoracic vascular disorders. severe haemoptysis can occur in about % of patients. it is associated with high mortality due to asphyxiation, if not treated, and needs urgent and comprehensive evaluation of the lung parenchyma, airways, and thoracic vasculature. multidetector row ct angiography is a very useful noninvasive imaging modality for initial assessment of haemoptysis in stable patients. it can accurately identify the source and the most common predisposing causes of haemoptysis (bronchiectasis, chronic bronchitis, lung malignancy, tuberculosis and fungal infection) and the effects of haemorrhage on the lungs and airways. moreover, the combined use of thin-section axial and complex reformatted images allows clear depiction of the origins and trajectories of abnormally dilated systemic arteries that may be responsible for the bleeding in over % of cases requiring intervention with arterial embolisation or surgery. nonbronchial arteries may also represent an important cause of haemoptysis. the road maps of dilated bronchial and nonbronchial arteries provided by ct angiography represent a useful guide for endovascular treatment. ct angiography is a quick and noninvasive tool that is helpful in the diagnosis and management of haemoptysis. disorders that affect the elderly population. some misleading radiological presentations typically occurring in this population will be also presented. in the elderly the coexistence of several diseases, the prevalence of involutional and degenerative aspects, together with physical and cognitive problems represent 'the norm'. it is therefore important to know how to distinguish the healthy elderly from those in need of treatment to avoid overdiagnosis and overtreatment. so the question is how to be aware of the potential and limits of diagnostic imaging and its applications in geriatric patients. fast development in the area of imagining modalities demands a lot of work in dose and image quality optimisation and management. deterministic harms have been reported both in interventional and diagnostic radiology. there are also some special groups (e.g. children) which need a lot of attention and especially tight indications for x-ray examinations. radiation dose and image quality optimisation can be applied with small steps in everyday clinical work as a part of self assessment, if the safety culture is agreed by all professionals. the vendors put the settings high in order to reach the best image quality. after installation the settings must be re-evaluated before starting clinical use of the equipment. also, the sensitivity of aec (automatic exposure control) must be turned according the detector. using the air gap ( cm) instead of grid, e.g. in hip axio-lateral project or scoliosis, dose decrease can be even two thirds. the role of radiographer is expanding to new areas. quality assurance and dose and image optimisation could be parts of the expanded role. the new technical solutions in imaging offer a lot of possibilities for dose reduction, if we want, but there is also danger of dose creeping if the doses are not followed up frequently. the management and legislation offer the basics but the work must be done among those who are working in radiological departments. commitment to the safety culture on all levels in medical radiation offers better care and procedures with lower doses to the patients. radiological procedures are performed because of medical benefits to patients, but they also cause some harm because of the dose. if medical benefit overweighs detriment the procedure is justified. the purpose of the optimisation is then to adjust the procedure in a way to maximise the ratio of benefit over harm. question is who is responsible to do it and how it can be done. who should participate: we believe there is a major role of radiographer as she/he is present at each and every radiological procedure performed. but it is absolutely mandatory for them to understand benefits of the procedure and understand harm (dose). radiographer's position is the connection between radiologist, medical physicist and vendor's engineers. radiologists usually do not have in depth knowledge of technology and dose and physicists and engineers on the other hand do not understand in depth the medical aspects of procedure. radiographers must also understand operational possibilities of every x-ray machine. to get to know them they should be there at the acceptance of the x-ray machine and talk to service engineers and applicators. they should also talk to physicist when he/she is doing tests. and finally simulation of procedure(s) using different phantoms is the best way to understand how machine parameters are affecting image quality and dose. in presentation, some examples for different modalities will be given. learning objectives: to gain knowledge about how to start with optimisation of radiological procedures. . to understand the basics of quality control (qa) for radiographic modalities and how to use experience acquired from qa in daily work. . to become familiar with guidelines and applications for good radiological practice and how to implement them in the optimisation process. . to consolidate knowledge of technology of radiographic modalities and the use of materials in daily practice according to guidelines, and to be familiar with the radiographers responsibility in the field of radiation protection. the availability of magnetic resonance (mr) scanners operating at t and above has already proved beneficial for mr imaging and spectroscopy of the human brain and promises similar benefits in the human body. these advantages result from the increases with the magnetic field of the intrinsic signal-to-noise ratio, blood oxygenation level dependent (bold) contrast, which forms the basis of the vast majority of functional mr imaging (mri) experiments, and chemical shift dispersion. these gains can be exploited in improving the spatial and/or temporal resolution of anatomical and functional mri experiments and in increasing the spectral resolution in volume selective spectroscopy or chemical shift imaging. operation at the increased magnetic field also offers easier access to t * -contrast and improved implementation of susceptibility-weighted imaging (swi) in which the phase of gradient echo images provides information about local variation of magnetic susceptibility. in the brain, such variation appears to be dominated by differences in iron concentration and myelin content, so that high-field swi may provide useful information about the progression of neurodegenerative disease. the elevated t relaxation times at t also offer benefits for arterial spin labelling and time of flight angiography. current and potential future applications of high-field mri in clinical and pre-clinical studies in a number of areas and will be discussed in this presentation, along with the barriers to wider usage of t systems for clinical studies. the purpose is to present and discuss the role of imaging in non-traumatic acute abdomen, with particular focus on ultrasound (us). the neonatal gi tract emergencies, associated with specific disorders and imaging strategies, are excluded. acute abdomen in children refers to a wide variety of conditions ranging from benign disorders, such as gastroenteritis, to threatening disease, such as midgut volvulus or intussusception. the recognition of a surgical emergency requires usually, after a competent clinical evaluation, an imaging investigation in order to avoid negative or unnecessary surgery. abdominal plain film is known as poorly sensitive in most situations, except bowel occlusion. ultrasound requires experience and a thorough examination but has demonstrated a high sensitivity and specificity for diagnosing bowel obstruction, midgut volvulus, intussusception, acute appendicitis, over the recent years the clinical mri field strengths have gradually been increased to tesla; however, whole body mri systems with higher fields of up to . tesla have become available in experimental settings. compared to clinical field strengths, mri at very high magnetic fields has several advantages but also some unique challenges. with increasing field strength the signal-to-noise ratio increases, which can be used to either increase the spatial resolution in the images, or to acquire the images more rapidly. unfortunately, the energy deposited in the human body via the rf excitation scales quadratically with the field strength. thus, the specific absorption rate (sar) is a critical factor in all rapid imaging protocols, and requires the design of rf pulses with low sar, (e.g. verse pulses). inhomogeneities of the rf field which are induced by standing wave phenomena have to be compensated, and make the design of efficient spin echo pulse sequences very difficult. at higher field also the field inhomogeneities become larger and stronger imaging gradients are required to overcome the susceptibility-induced image distortion. stronger gradient systems are difficult to manufacture, and the usable gradient slew rate is limited by peripheral nerve stimulation thresholds. furthermore, stronger and faster gradients become very loud at high fields, and special measures for sound protection are required. despite these limitations, high-field mri offers image with very high resolution, it provides unique contrasts, a better spectral separation of the resonance lines and high signal for non-proton applications. clinical mri usually aims to depict anatomic regions of interest with uniform coverage and contrast behaviour. to this end it is essential to use suitably homogeneous radiofrequency (rf) magnetic fields for spin excitation, refocusing, and saturation. traditionally, such uniform transmit fields are generated by volume resonators based on quasi-stationary electrodynamics, which, however, gradually cease to apply as clinical mri explores high field strengths of t and beyond. the concomitant increase in operating frequency entails shorter rf wavelength and increasing tissue interactions, which render the tailoring of rf fields substantially more complex and patient-dependent. one promising response to this challenge is to depart from volume resonators and perform rf transmission by multiple, individually fed transmitter elements. with such transmit arrays the effective rf field can be tailored on a per-patient and per-scan basis by adjusting the relative magnitude and phase of driving the elements (rf shimming). in advanced imple- : - : room z neural stem cells are in the process of finding their translation into the clinic. however, it is well-known that the cells by themselves do not regrow lost tissue. to this end, neural stem cells need further support and guidance. tissue engineering is using biomaterials to provide a structural support for cells, but can also incorporate the release of factors that guide the fate of transplanted cells. ideally, an in vivo imaging approach would encompass all these processes. we here demonstratethe use of a f mri contrast agent to detect clinical-grade human neural stem cells non-invasively over days within a tissue cavity formed by stroke. to provide structural support for these transplanted cells within the tissue cavity, cells were mixed with an engineered de-cellularisedextracellular matrix (ecm). using diffusion mri, we were able to detect the presence of the ecm within the stroke cavity. this approach hence provides a novel approach as to how we can study transplanted cells and tissue regeneration in vivo by mri. our lab has been among the first to exploit dendritic cell (dc) therapy to treat melanoma patients. over the past years, immunological responses are increasingly reported and clinical responses have consistently been observed. moreover, dc therapy often has much milder side effects than standard chemotherapy. a key hurdle in the development of the dc therapy is accurate delivery of the cells to lymph nodes (lns), or their successful migration from the site of injection to lns. in particular, tools for measuring cell migration in vivo are necessary. ideally, we would be able to quantify the number of dcs at the relevant site, with high resolution anatomical context to allow differentiation of lns and the possibility of longitudinal data acquisition. furthermore, functional data on the ensuing immune response is also required. towards these ends, we have been working on developing imaging techniques to study dcs in vivo, for example with scintigraphy on in-labeled dcs, and magnetic resonance imaging (mri) on iron-labelled dcs. scintigraphy is quantitative, but it is restricted to the relatively short half-life of the radioisotope and is unable to resolve individual lns. mri allows high resolution anatomic localisation, but the use of contrast agents such as iron oxide is not quantitative. our recent work has focused on imaging the functionality of these dcs using positron emission tomography (pet) to study ln activation. finally, we have also developed in vitro assays that closely mimic in vivo dc migration in d scaffolds imaged using quantitative f mri, as a substitute for in vivo optimization. we plan on applying f mri to the tracking of dcs in vivo, as the technique allows both quantification and high-resolution anatomic detail. hypertrophic pyloric stenosis, etc. it enables some differential diagnosis such as infectious ileocolitis, ischaemic colitis, henoch schonlein purpura, complicated meckel diverticulum or duplication, etc. or even pyelonephritis. it can provide some findings suggesting mesenteric adenolymphitis, viral gastroenteritis, etc. the us findings will be described as well as the potentialities and limitations of us in each of these conditions. the accurate knowledge of the clinical findings together with the results of us will guide the need for another imaging modality: enhanced ct in case of suspected appendicitis with inconclusive us, upper gi series in an infant with bilious vomiting and impossibility to display the mesenteric vessels, etc. at last, some aspects of pancreatic and biliary tract emergencies will be shown. the purpose is to discuss the role of emergency imaging in acute paediatric gu conditions, with particular focus on the potential of ultrasound (us). besides gu tract trauma conditions such as urosepsis, renal failure, renal colics, ovarian or testicular torsion with all the respective relevant differential diagnoses have to be considered and urgently addressed adequately. particularly with respect to radiation protection (alara-principle) and due to the superb us potential in childhood, us is often used as the primary imaging tool. in many conditions us will reveal all treatment relevant information and no additional imaging is necessary in the acute setting. however, in other conditions such as severe (multiple) trauma ct remains the best primary imaging tool, however, only by using age-adapted ct protocols. the work-up and follow-up may also require imaging, usually achievable by us and partially by mri, scintigraphy and/or cystography. all these lead to imaging algorithms that differ from adults. the most important conditions, their imaging appearance, the potential pitfalls and some important methodical details will be presented, also discussing the recommended dedicated paediatric imaging algorithms. in most paediatric acute gu conditions us is adequate as the first and often sufficient imaging modality. rarely other imaging is necessary particularly in the acute setting -except for ct in severe (multiple) trauma. all imaging should be indicated with respect to therapeutic consequences and the alara principle, and proper paediatric protocols as well as methododical skills (e.g., dedicated paediatric us) should be provided hours throughout the year. learning objectives: . to learn about the diagnostic imaging approach in acute urinary tract disease. computed tomography (ct) is the imaging method of choice in the evaluation of abdominal injury after blunt trauma in haemodynamically stable children. an unstable patient needs to be stabilised prior to ct or to proceed directly to surgery. evaluation with ct allows for accurate detection and quantification of injury to solid and hollow viscera, and can also identifies an active haemorrhage. ct can help prioritise optimal management by diagnosing the major or most life-threatening site of haemorrhage or injury. ct can also demonstrate associated bony injury to ribs, spine, and pelvis. a normal ct examination may prevent other unnecessary explorations. the decision for operative intervention in the small percentage of children who require surgical haemostasis is primary made based on clinical criteria and not on ct findings. interventional techniques deeply changed approach to treatment of diseases. in each country, interventional radiology practice establishment varies according to local factors, but following a standard strategy seems better to set up this facility. according to above-mentioned points, i decided to establish this specialty in our hospital since as the pioneer center in interventional radiology iran. at first the procedures included percutaneous laser disc decompensation (pldd), and vertebroplasty followed by vascular procedures such as uae in later years. now, we are performing all interventional procedures in our country including aortic stent and all neurointervention procedures. the following items will be discussed: . prepare step by step strategies for establishing intervention. . solving issues regarding interventions procedures. . modified techniques for lowering costs to cover interventions in poor people. . setting of fellowship courses. . co-operation with dedicated and pioneer international centers such as using neocrylate for treatment of brain aneurysms in conjunction with ucsd for the first time on human. when presenting our experience we want to review how we should start interventional radiology in developing countries. fibroids are the most common benign tumours of the uterus during female reproductive age. uterine artery embolisation (uae) is a procedure in which using embolic particles (pva, gelfoam, etc.) we block blood supply to uterine fibroids. it has been reported as a relatively safe, effective, and durable nonsurgical alternative method diminishing fibroid-related symptoms. uae is typically performed in both uterine arteries by an experienced interventional radiologist. after uae, reduction in menorrhagia has been reported as - % and the mean decrease in fibroid size varies from % to % in the literature. complications including amenorrhoea are frequency ranging from % to % in the literature. uae may be followed by menopause in % of the cases. nevertheless, it is usually encountered in women in their late s. it seems that the future of uae depends on optimal selection of patients based on the prediction of volume-shrinkage, the outcome of fertility, and the long-term efficacy. although pregnancy is possible after embolisation, neither fertility preservation nor its improvement can be definitely guaranteed. women who desire to become pregnant should definitely be cautioned about potential complications during pregnancy. in this presentation we are going to review the position of uae in iran and to present our experience about the efficacy, and safety of uae. besides, we will discuss some methods of dose reduction during uae and the effects of uae on fertility and pregnancy outcome. purpose: labelling pancreatic islets (pi) with superparamagnetic markers enables their detection as hypointense spots on magnetic resonance (mr) images. we tracked pi transplanted into the liver and quantified their signal loss in liver tissue using mr imaging over a six-month period. methods and materials: pi were labelled with ferucarbotran μl/ml for - hours and transplanted into the portal vein in c-peptide negative recipients ( infusions). the liver was examined prior to transplantation and subsequently day and , , and weeks after transplantation using a t mr scanner. results: in all recipients significant c-peptide levels and near-normal hba c values were achieved with - % insulin dose reduction. no side-effects related to the labelling procedure were documented. a significant decrease in the number of islet spots was detected at week post-transplantation (on average %) with a subsequent only slight decrease for up to weeks. in two subjects with a labelling period of less than and hours respectively only a few islet spots were detected corresponding to poor islet visualization in phantoms labelled for the same period of time. conclusion: pi visualization was successful in all recipients but was less efficient if the labelling period was less than hours. a significant decrease in islet spots occurred at week post-transplantation suggesting early islet destruction or impaired engraftment. then, the decrease in islet spot numbers slowed and islets were still detected at weeks post-transplantation. data shows that mr detection of ferucarbotran-labelled islets correlates with sustained c-peptide production. areolar complex), the presence of other imaging findings (extension to the skin, inflammatory cancers) and the status of regional lymph nodes. this information influences the type of treatment given to the patient and it is very important to gather as much information as possible in order to obtain a one-step surgical excision with free margins at pathology. on the basis of this information and on discussions held in multidisciplinary meetings, markers will be placed wherever needed in order to locate the exact distribution of the tumour(s). the different parts of a structured report (clinical data relevant to the report, technical description of the diagnostic or the interventional modality, pitfalls during the exam, description of breast composition, description of lesions, categories and overall assessment) should always be included in order to guarantee that all the important information is there. breast imaging, like other subspecialties in radiology, is a multimodality area of knowledge where it is vital to integrate the information obtained through diagnostic and interventional procedures in order to supply the clinician with a body of data that is useful and straightforward. liver metastases are of the most difficult therapeutic challenges in oncologic managements. surgery is frequently impossible due to disease extent and systemic chemotherapy usually fails. in other body parts, the combination of radiotherapy and systemic chemotherapy is used for several types of malignancies. we successfully adopted a novel image-guided form of this combination for such masses referred to as radiochemoembolisation, which substantially intensifies the treatment locally. patients underwent transarterial radiochemoembolisation with chemotherapy protocol of mitomycin, doxorubicin, and cisplatin and embolisation with radioisotope particles of p. the effectiveness of method was determined by comparing contrast-enhanced ct images, pre-and post-intervention. treatment response was evaluated using recist criteria. we also used some arbitrary criteria such as tumoural mean density and enhancement pattern. for stable and partial-response cases, the procedure was repeated. we did not repeat the treatment for progressive diseases. complete-response cases were only followed. after completing treatment sessions, regardless of the tumour source, considerable amount of patients showed substantial response. in terms of recist criteria, a significant portion of patients gained at least a "partial response". but the decrease in tumour density and the appearance of non-enhancing tissues were more promising. radiochemoembolisation is an effective method for the treatment of patients with unresectable hepatic metastases. although this study lacks a control group, it shows the effectiveness of the treatment. literature review showed the response to treatment with regard to recist criteria had been better in our study comparing with studies only used chemoembolisation or radioembolisation. , which showed the superiority of digital mammography for younger and perimenopausal women, as well as those with dense breasts; the national ct colonography trial (nctct), demonstrating the equivalence of ctc with colonoscopy; and the national lung screening trial (nlst) which has preliminarily reported a % decrease in lung cancer-specific mortality associated with annual ct screening. acrin's current focuses include the assessment of emerging imaging biomarkers for the prediction of and monitoring of response to treatment and the extension of its activities to disease processes beyond cancer. according to the second approach, termed as ‚dark lumen mrc', dense barium or tapped water is used to distend the colon in conjunction with post-gadolinium d gradient echo with fat-saturation sequences. the colonic wall and the corresponding polyps exhibit high signal intensity as a result of gadolinium uptake while the colonic lumen presents with low signal intensity. mr imaging of the pancreas and bile ducts relies upon a combination of t -w and t -w strategies in order to safely acquire anatomic, cross-sectional and functional information. tissue-imaging strategies include free breathing t -w imaging (obtained with echo-train spin-echo sequences with or without fat saturation) and breath-hold d-gradient echo t -w imaging (obtained with fat saturation) without gadolinium and in the capillary phase and in the interstitial phase post-gadolinium. this combination is valuable in the assessment of the full spectrum of pancreatic diseases and malignant bile duct obstruction. hardware improvements (gradients) and the advent of more sophisticated phased-array coils and parallel imaging capabilities in modern mr scanners allowed to increase the spatial and temporal resolution of "conventional" t -w and t -w sequences. furthermore, it provided the possibility of adding high-b-value diffusion weighted imaging (dwi) to our routine protocol in order to increase the sensitivity of mri in detecting malignant and inflammatory diseases. to obtain conventional mrcp imaging thick-slab heavily t -w tse sequences or d heavily t -w tse sequences may be used. advantages and limitations of both sequences will be underlined. functional bile duct imaging is obtained imaging modality or technique for this problem. increasing technological capabilities and knowledge of how to optimise ct/mr contrast utilisation provides better ways to characterise tumours than ever before such that key clinical specialties outside of radiology now recommend nonbiopsy imaging diagnosis of hcc to triage patient treatment. radiologists must work extra diligently to learn the clinical issues, triage points and implications that are associated with specific clinical presentations and imaging findings. the perfect test that does not require cognitive interpretation of the constellation of imaging findings with integration of the patient clinical presentation to optimise patient care is not in our near future. this lecture emphasises historical advances in liver imaging and how data in the literature impacted patient care decisions in ways that may be different than expected from radiologists. an emphasis on pathologic-imaging correlation will be used to put apparent disparate published results in proper perspective to allow radiologists to meaningfully interact with clinical colleagues in determining optimal patient treatment. liver mri is undertaken to assess the liver parenchyma, vasculature, and biliary system. it is necessary to use a variety of unenhanced and contrast-enhanced mr pulse sequences to achieve lesion detection and characterisation. a set of t -, t -w and dwi sequences is now standard for lesion delineation and assessment of liver fat and iron content. in-phase and opposed-phase t -w gre images show focal or diffuse fatty infiltration and focal sparing. a fat-suppressed t -w tse sequence is robust and provides high tumour contrast, with . t units now allowing acquisition of isotropic d-tse sequences. the quite popular t -w half-fourier single-shot tse (e.g., haste) pulse sequences show anatomic detail (including bile ducts), but lack lesion contrast. in case of suspected iron overload (i.e., haemosiderosis and haemochromatosis) an additional t *-w gre is recommended. acquisition of dwi patient transport into the radiology department, contact to other potentially infectious persons, and things such as breath-holding are reasonable burdens and dangers to immunocompromised patients. when searching the focus of fever, imaging should help to identify an affected organ system in order to eventually guide invasive procedures to identify underlying micro-organism or non-infectious disease. equally relevant is the exclusion of its involvement with a reasonable specificity. depending on local epidemiology, organ system, and the clinical signs and symptoms, suspected differential diagnosis can be derived from image patterns. some of these diagnoses might be exclusion diagnosis, others might require invasive procedures including time consuming and costly analysis to be verified. invasive procedures, however, require adequate hemostasis, which is usually not available for a substantial duration due to pancytopenia in patients who underwent chemotherapy. if imaging fails to derive the underlying disease confident and conclusively in a fast way, clinicians might need to treat on an empirical basis. empirical treatment plays a major role in immunocompromised or severely ill patients at risk, because mortality rises within hours of untreated disease. on the other hand, empiric treatment causes relevant toxicity and substantial costs, while imaging might become cost-effective. using contrast agents that are taken up by hepatocytes and excreted through the biliary system in combination with volumetric t -w sequences. we use this technique in postoperative complications and to elucidate complex biliary anatomy. functional imaging of the pancreas is obtained by combining mrcp along with hormonal stimulation with secretin. in addition, secretin improves anatomic delineation and allows mri quantification of fluid production by the exocrine pancreas. despite the advance of cross-sectional techniques, the chest radiograph remains a basic tool for the initial approach to heart diseases. in this lecture we will present our current approach to evaluate cardiac pathology in conventional radiography illustrating it with selected cases. the basic approach consists of seven steps, evaluating the size and shape of the cardiac silhouette, cardiac calcifications, pulmonary circulation and lung changes, great vessels and implanted devices, all of them correlated with the clinical findings. looking at the heart on a chest film may be considered at a first glance as wasting time since, today, many other techniques are available today to offer an impressive luxury details of the morphological and functional evaluation of heart. nevertheless, the chest film remains often the first modality performed in many situations in which symptoms can be both from pulmonary or cardiac origin and then, careful analysis of heart may avoid rough mistakes in patient management. acquired and congenital pericardial or cardiac abnormalities may be responsible for a deformity of the cardiac silhouette and lead to explore the patient by a chest ct. ct with fast scanning capabilities can acquire images of the thorax with reduced cardiac motion artefacts, improving the evaluation of the heart in the course of a thoracic ct. unexpected findings of cardiac structures on both unenhanced and enhanced acquisition can dramatically influence the patient's clinical management. in many situations, a chest ct is performed without cardiac gating and it may be necessary to complement the initial examination by a gated acquisition to provide a more dedicated analysis. the normal appearance of cardiac structures and the most common cardiac abnormalities should be known by all the radiologists. various conditions such as idiopathic and acquired cardiomyopathy, ischaemic heart disease, valvular dysfunction can be identified even on non-gated scans. pulmonary diseases may also involve pericardium and cardiac structures and this should be clearly identified. learning objectives: . to learn how to detect and characterise cardiac abnormalities in the chest radiograph. . to learn how to detect and characterise cardiac abnormalities on chest ct. . to learn the limitations of the interpretation of cardiac abnormalities on ungated chest ct. . to learn when further evaluation is required. nal in discs and vertebrae to the adult shape and signal pattern will be reviewed. the normal pattern of unfused bony segments and apophyses in the child and fusion to the mature adult form will be reviewed. normal variants such as persistent segmentation/non-union leading to limbus vertebrae and dysraphism will be reviewed. intervertebral discs, vertebral body endplates, posterior elements and bone marrow show a wide degree of variation and the borderland of normal variant versus pathological abnormality is often difficult to navigate. common borderland findings such as schmorl's nodes versus scheuermann's disease will be discussed. transitional vertebrae as such are normal but incomplete transitional changes are associated with clinical symptoms. even without associated pathological changes the terminology of transitional vertebrae is a common pitfall. simple coping strategies will be reviewed. various imaging artefacts can have an influence on image interpretation though lumbar spine mr imaging is more resistant to these than imaging of the cervical and thoracic spine. this talk will review an approach to imaging complications of total hip replacements. the talk will briefly review surgical techniques and types of prostheses. the talk will subsequently review early and late complications of prostheses and focus on the integrated use of all modalities including radiographs, aspiration arthrography, scintigraphy and mri in achieving a diagnosis. there are many surgical techniques to repair meniscal tear, focal cartilage defect, cruciate ligament tear, malalignment, fracture, osteoarthrosis, etc. conventional radiography, ct-scan, ct-arthrography and mri play an important role in evaluation of the knee after surgery or arthroscopy. indications for postoperative imaging are infection, persistent pain and dysfunction. every radiologist should be familiar with "normal" imaging findings after arthroscopy, osteosynthesis, ligament reconstruction, osteotomy, knee prosthesis and meniscal or (osteo)chondral repair, but also recognise the main complications after knee surgery or arthroscopy. orthopaedic hardware is usually evaluated on plain radiography or ct, and only a relative contraindication for mri. microscopic metal artefacts and fibrotic scarring are frequently seen along the course of the instrumentation tract. after partial meniscectomy, an obtuse angle at the apex of the meniscus and increased signal intensity of the remnant part of the meniscus are normal findings, whereas fibrillation and recurrent tear may explain the complaints of the patient. various intra-and extra-articular reconstructive procedures exist for anterior and posterior cruciate ligament reconstruction. besides the neoligament, an osseous tunnel, screws and metal artefacts are also visible. postoperative findings of the extensor apparatus include a thickened patellar tendon, focal myxoid degeneration, fibrosis and focal defects, e.g. after harvesting tendon tissue for acl reconstruction or after release of the lateral patellar retinaculum for 'unstable' patella. accelerated osteoarthritis may be a late postoperative finding. mri very well depicts incorporation and alignment of osteochondral auto-or allografts, and the position, morphology and integrity of the meniscus after repair or transplantation. to evaluate postsurgical patients it is important to know the primary clinical diagnosis, the surgical treatment, the interval since surgery, and patients' current clinical symptoms. radiography is the most common imaging modality to evaluate the postoperative ankle, particularly in traumatic cases; after reduction and fixation of a fracture or dislocation it is generally carried out as routine. ultrasonography is highly sensitive and specific in postoperative tendon assessment, thanks to the superb resolution, and the opportunity for dynamic evaluation of tendon integrity. chest drains ( - fr) with underwater seals achieved decompression of pleural fluid collections. recent literature suggests small bore drains may be adequate, with image guidance playing an important role. pleural space anatomy, including fissural locations will be revised. aetiology of transudates, exudates and empyemas, along with typical clinical presenting features are outlined. various examples of plain film, ultrasound, multi-detector computed tomography and mri appearances of pleural collections and their aetiologies are presented. selection of patients, image guidance methods, catheter size, insertion technique, pitfalls and procedure complications are discussed. importance of physician-radiologist liaison with respect to catheter management and catheter dwell times is emphasised. pleural space fluid collections are common clinical entities that radiologists can accurately diagnose as well as successfully treat. overview of aetiology, radiological appearances and method of image-guided drainage of infected pleural fluid collections is provided. pelvic abscess drainage may employ more difficult access routes due to anatomic restrictions. the procedure may carry a higher risk of complications compared to simple abdominal drainage. to optimise patient preparation, correction of coagulation deficits, optimisation of antibiotic treatment, and sedation or anesthesia may be needed. the usual access routes for deep pelvic abscesses are the anterolateral and the posterior transgluteal approach. in selected cases, a transvaginal or transrectal approach may be the safer and more efficient option. depending on the viscosity of the drained fluid, the presence of necrotic solid tissue and gas, different sizes of drainage material may be needed. do not hesitate to employ large drainage catheters if indicated. an overview of the available material will be given. intestinal laceration is a major complication which can be managed percutaneously in selected circumstances. often, the weakest part of percutaneous drainage is the postinterventional management. it is advocated that the management is actively guided by the interventionalist who ensures that the drainage is properly handled, rinsed, and cleaned, that follow-up imaging is performed at the right intervals, and who is also responsible for indicating drain removal. chest ct and mri are commonly performed to evaluate patients with acute chest pain. a number of imaging techniques are available that may cover the entire chest, including the cardiovascular system. the radiologist will encounter more and more cardiovascular disease that was not visible with ungated ct and mri techniques. the differential diagnosis of acute chest pain may include over possible diagnoses, a substantial number related to cardiovascular disease. it is important to recognise chronic and acute coronary disease, myocardial infarction and its complications, left ventricular function, pericardial disease, atrial disease and large vessel disease. case material will be presented and discussed using both ct and mri techniques to illustrate the spectrum of usual and more unusual cardiovascular pathology that should be considered in the differential diagnosis of unexplained chest pain. pulmonary hypertension (pht) remains a disease difficult to diagnose because the clinical findings are nonspecific, often leading to a delayed diagnosis. once recognised, it is necessary to determine the underlying cause and to estimate the severity of pulmonary hypertension. among the noninvasive methods of assessment of pht, ct angiography (cta) now plays an important role in the diagnosis and post-therapeutic management of pht. numerous recent technological advances of multidetector-row ct technology (mdct) have reinforced the clinical impact of this technology by introducing new tools for the morphological evaluation of small-sized pulmonary arteries and pulmonary capillaries, the latter being accessible with dual energy ct and often referred to as pulmonary "perfusion". the role of ct now also includes the possibility to investigate the presence of pht on the basis of functional parameters, such as the distensibility of the pulmonary arterial wall, and to integrate cardiac functional information, with great interest towards right ventricular function. the major clinical impact of these new scanning modes is that morphology and function can be obtained from the same data set, with no restriction on the diagnostic performance of high-resolution ct angiographic images. the purpose of this lecture is to review these new trends in imaging of pht and estimating right ventricular function through practical clinical situations, including the most common causes of pht. ct scans of the chest are usually performed and reported as if the lungs were the only organs in the thoracic cage. however, the heart and pulmonary arteries can show significant abnormalities either as associated findings of the parenchymal disease or as incidental findings. so the recommendation is: do not forget the heart and pulmonary vessels when reporting a ct scan of the chest. mri has rapidly become important in post-operative assessment of the ankle, because it provides high soft-tissue contrast, multiplanar capability and osseous structures visualisation. it shows signal changes of ligaments and tendons, hypointense subchondral sclerosis, subchondral bone marrow oedema, joint effusion, capsular thickening, fibrosis, and synovitis. mri has also an important role in the evaluation of post-surgery ankle pain due to impingement syndrome and in the hindfoot chronic instability related to postoperated sinus tarsi syndrome; it demonstrates the anatomy of sinus tarsi, chronic synovitis and nonspecific inflammatory changes, synovial cysts, fibrosis and subtalar joint effusion. it is important also in the follow-up of tumours and tumour-like conditions of bone and soft tissues after surgery. computed tomography is the most valuable method to define the osseous anatomy of the postoperative ankle, so it is important in the follow-up of the operated osteochondral lesions of the talus. ct allows the evaluation of irregularities or degenerative changes, and progressive degenerative arthritis; however, ct usually fails to evaluate soft tissue's ankle lesion. the development of subsecond mdct scanners with high temporal and spatial resolution has significantly reduced scanning times and now it is possible to have very good quality images of the heart during routine chest ct examinations. therefore, the importance of looking at the heart and pulmonary vessels on a ct scan performed for non-cardiac reasons will be emphasised. anatomic cardiac details that every radiologist should know will be discussed in the first talk; the presentation is aimed mainly at the non-cardiac radiologist. it will review normal cardiac anatomic details, as routinely encountered on modern multi-detector ct studies. it is now possible to delineate much of the intracardiac anatomy on a contrast enhanced study and significant pathology may also be evident on non-enhanced ct examinations. important incidental findings and their clinical relevance will be discussed in the second talk. unexpectedly, cardiac abnormalities may be diagnosed when lung disease is clinically suspected and vice versa; the focus of this presentation will be the comprehensive evaluation of lung and heart/large vessels diseases.the strong correlation existing between pressure in the pulmonary artery system and dilatation of the pulmonary trunk and central branches will be discussed in the last talk. ct is considered more useful than echocardiography because it can depict the cardiac structures in all patients including those with extensive parenchymal abnormalities. at the end of the presentations we should try to answer the question about the routine reporting of cardiovascular findings on ct scan of the chest. a. anatomic cardiac details that every radiologist should know s.p.g. padley; london/uk (s.padley@ic.ac.uk) this presentation is aimed at the non-cardiac radiologist. it will review normal cardiac anatomic detail, as routinely encountered on modern multi-detector ct studies. it is now possible to delineate much of the intracardiac anatomy on a contrast enhanced study, and significant pathology may also be evident on non-enhanced ct examinations. this study will primarily review normal cardiac anatomy, including cardiac chambers, valves and coronary vessels. the typical radiological appearances of innocent incidental intra-pericardial abnormalities will then be reviewed. important incidental pathology will be discussed in the next talk. liver fat, inflammation, and fibrosis are important pathological features in patients with diffuse liver disease. the clinical gold standard for assessing these features is liver biopsy. due to its invasiveness and sampling variability, however, liver biopsy is suboptimal for screening, monitoring, and clinical research. there is, therefore, a need to develop biomarkers to assess liver fat, inflammation, and fibrosis non-invasively. in recent years, many quantitative imaging techniques have been developed, refined, tested, and made available. the question becomes: are these techniques ready for routine clinical use or are they most appropriate for research? in this special focus question, we will begin with a brief overview of diffuse liver disease and discuss basic concepts of biomarker validation and qualification. dr. reeder then will discuss conventional and state-of-the-art imaging-based biomarkers of liver fat. dr. cobbold will discuss the current status of non-invasive biomarkers for liver inflammation. dr. van beers will discuss ultrasound-and mr-based biomarkers of liver fibrosis, with emphasis on techniques such as transient elastography and mr elastography that measure visco-elastic properties of liver tissue. we will conclude with a panel discussion asking the question: are the repeatability, reproducibility, and robustness of the non-invasive biomarkers presented in this session adequate for routine clinical implementation? session objectives: . to review the clinical importance of diffuse liver disease. . to understand that key features of diffuse liver disease include fat accumulation, inflammation, and fibrosis. . to understand the need for non-invasive biomarkers to assess fat, inflammation, and fibrosis. . to review basic concepts in biomarker validation and qualification. quantification of liver fat s.b. reeder; madison, wi/us (sreeder@wisc.edu) intracellular deposition of fat within hepatocytes (steatosis) is a common condition of the liver. fat is the histological hallmark of non-alcoholic fatty liver disease (nafld) but also may occur with alcohol abuse, viral hepatitis, hiv and genetic lipodystrophies, and chemotherapy. nafld alone afflicts an estimated - million in the us and is a rapidly growing condition in other western societies, paralleling the expanding epidemics of obesity and diabetes type ii. this talk will review the clinical, pathogenic and histological features of liver fat, including an overview of fatty liver disease and diseases where fat is an important disease feature. next, it will review the current use and limitations of non-targeted biopsy in diffuse liver disease, and why quantitative non-invasive biomarkers of liver fat and iron would be beneficial. currently available conventional magnetic resonance imaging techniques that attempt to detect and quantify liver fat will then be discussed, as well as known confounding factors that corrupt the ability of conventional methods to quantify fat. this lecture will then describe emerging quantitative imaging methods for accurate and precise quantification of liver fat, and the advantages offered by these methods address in comparison with conventional methods. finally, remaining challenges and unsolved problems for quantifying liver fat will be discussed. conventional mri techniques, such as t w and gadolinium-enhanced t w sequences, which are highly sensitive for detecting demyelinating plaques, are recognised as the most important paraclinical tool for diagnosing multiple sclerosis (ms). however, the changes seen on mri in patients with ms are not diseasespecific, as focal white matter t hyperintense lesions (thl) are also commonly observed not only in the elderly but also in middle age and young patients, and in a large list of different disorders such as hypoxic-ischaemic vasculopathies (atherosclerotic and hypertensive small vessel disease, cadasil, fabry's disease and susac's syndrome), cns vasculitis, sarcoidosis, adult forms of leucoencephalopathies, trauma and radio chemotherapy, and acquired metabolic conditions (hepatic encephalopathy, alcoholism), among others. while it is recognised that a combination of findings from clinical history, physical examination and laboratory tests is commonly required to correctly establish a firm and clear aetiological diagnosis of thls, a detailed analysis of different mri features should also be considered essential, e.g. lesions shape, size, and distribution; contrast-uptake; and associated structural lesions (microbleeds, infarcts, spinal cord, brainstem and cerebellar involvement, …). in addition to these conventional mri-based features, non-conventional mr techniques (diffusion, mrs and perfusion) may also provide in some cases useful diagnostic information. knowledge of these features will assist the diagnostic work-up of patients presenting with thls, and should be considered a first step to take full advantage of the potential of mri, and in doing so should result in a reduced chance of misdiagnoses and facilitate the correct diagnosis of sometimes treatable disorders. learning objectives: . to be aware of the limited specificity of brain multifocal t abnormalities. . to learn about recognition patterns that might be helpful in suggesting the most likely etiology of brain multifocal t lesions. . to learn about the role of spinal cord imaging and advanced neuroimaging techniques for the differential diagnosis of brain t hyperintense lesions. . to be able to establish a neuroimaging diagnostic strategy in patients with multiple brain t lesions of unknown origin. breast imaging reporting and data system (bi-rads) was developed by american college of radiology in order to set standards for mammography reporting, common terminology and data collection. bi-rads is being used widely for over a decade and many studies have assessed the validity of the system. an important component of bi-rads is the lexicon which gives descriptors of specific imaging features that facilitate image interpretation and unify the reports. two main titles for these descriptors are about masses and calcifications. an asymmetry is called a mass when it is seen in two projections. a mass is defined with its shape, margin and density. definition of a mass with these three modifiers would help the radiologist to determine the type of the mass and probability of its malignancy. calcifications are divided into three categories by their shapes and another descriptor is defined for their distribution. the first category is for typically benign calcifications. the other two are for probably malign calcifications where biopsy should be suggested. the distribution modifiers for calcifications can also play an important role in assessing the probability of malignancy. bi-rads defines assessment categories from to for the final report that facilitate data management for yearly audits. category is the transition zone between malignant and benign lesions where the suspicion for malignancy should be less than % and requires short-term follow-up. bi-rads morphology and distribution descriptors are effective in assessing the risk of malignancy with a reasonable positive predictive value. the breast imaging reporting and data system (bi-rads ® ) for ultrasound (us) was developed by the american college of radiology (acr) and published in . although this lexicon was created to achieve a consensus among radiologists when describing breast abnormalities, clinical practice shows substantial intraobserver agreement but only moderate interobserver agreement. most problems are reported for descriptors related to shape (when trying to classify abnormalities containing lobulations and/or elliptical with not-parallel orientation), echo pattern and margin. especially mass margin is a critical feature for determining whether a lesion should be biopsied or not. other problems are related to the final assessment, including difficulties in applying the subcategories and the relatively high percentage of false negative cases for lesions interpreted as category . many computer-aided diagnosis software have shown the potential to improve performance amongst less experienced readers and decrease interobserver variability; still they do not solve all the problems. the bi-rads ® lexicon is actually under advanced revision by an international panel and ellen b. mendelson chairs the us subcommittee. revision is reviewing both descriptors and categories; it will include also new parameters linked to the evolution of us technology as colour flow mapping and elastography. troubles. characterisation of aml is possible, based on identification of its fatty content mainly using ct. when microscopic, this component may be missed and biopsy is required with immunostaining. mr imaging may be helpful using chemicalshift sequences but thresholds, to rule-out clear cell carcinoma, have to be better defined. presence of necrosis or calcifications rules-out diagnosis of aml. amls must be treated if haemorrhagic, painful or if diameter exceeds cm. embolisation is the main nonsurgical method, using microparticles, alcohol and/or coils but postembolisation syndrome must be prevented. volume regression may require all agents whereas aneurysm occlusion requires coiling. the effect on volume and on haemorrhagic risk is substantial making surgery as an alternative method for nephron sparing purposes. secondary surgery is required in less than % of embolisation. rf ablation is also possible but its efficacy is still under evaluation. the number of small renal tumours is increasing due to the large amount of imaging examinations of the kidneys performed with various modalities and the true increasing incidence of renal cancer. conservative therapy of small renal tumours is now widely recognised as the reference technique for the treatment of these small lesions. the increasing rate of chronic renal failure in the elderly and the efficacy of conservative therapy to treat cancer as demonstrated by the urologists performing partial nephrectomy and tumourectomy emphasise the role of percutanaeous minimally invasive ablative procedures, particularly in patients with surgical contraindications. radiofrequency ablation and cryotherapy are the two most developed techniques, but new technical approaches are under development such as microwave ablation, electroporation, etc. the evaluation of the success of the procedure relies on imaging techniques showing the lack of enhancement within the lesion and the size and shape of the necrotic covering the entire tumour area. the techniques, indications, results, and complications of both radiofrequency ablation and cryotherapy will be discussed. a variety of diseases including neoplasms, but also infectious, inflammatory, vascular and traumatic processes, may present as focal intracranial mass lesions. modern neuroimaging, primarily with mri, enables differentiation of these entities allowing for accurate diagnosis in almost all cases. the requirements are appropriate image acquisition and detailed analysis of imaging findings, while pertinent clinical information may be very helpful in certain cases. the distinction is frequently broad, between different disease processes, such as with tumefactive demyelination versus neoplasm, which is often sufficient for clinical decision making and patient management; at times this may be more specific, approaching histological diagnosis, such as with pilocytic astrocytoma versus medulloblastoma. this presentation will go briefly over the imaging techniques and various disease processes, while heavily concentrating on the differentiating features of intraaxial primarily non-haemorrhagic mass lesions. the emphasis will be on the key distinguishing imaging features, such as presence or absence of mass effect or vasogenic oedema, signal intensity characteristics, presence and pattern of contrast enhancement, as well as diffusion and perfusion features. decision making process will be discussed. a number of cases with intracranial masses will then be reviewed and analysed, using a step by step approach, accentuating the most reliable distinguishing findings. the role of mr imaging in adult gliomas consists of many steps. ( ) is there a lesion in the brain? ( ) is the lesion a tumour? ( ) is the tumour a glioma? ( ) is it a high-or low-grade glioma? ( ) if there is a suspicion and a biopsy is planned, which part of the tumour should be targeted at biopsy. ( ) how the surgery should be performed to avoid injury to functional areas of the brain (e.g. motor cortex). ( ) if there is need for adjunctive radiotherapy, how the extent of the tumour should be defined? ( ) after radiotherapy, if a new enhancing lesion occurs, is it a recurrent neoplasm or radiation necrosis? conventional anatomical mr imaging is not always powerful enough to answer these questions. however, with advanced mr imaging techniques such as diffusion mr (including tractography), perfusion mr, mr spectroscopy and functional mri (fmri) we can answer the above questions reliably in most cases. the purpose is to gain numerical information of accuracy in the treatment of localised prostate cancer and calculate the necessary size of the safety margin around the clinical target volume (ctv-ptv). in addition, the purpose was to evaluate the accuracy in treatment set up with patients with high bmi to find out whether patients with high bmi need a larger safety margin. portal imaging was used to measure off-line random set-up error from bony structure. the sample size was , i.e. all the patients who received radiation treatment for localised prostate cancer in landspítali -university hospital (lsh) . a total of portal images were acquired. in , the task was repeated for patients having gold fiducial markers in the prostate. the calculated -d imrt safety margins in lsh was to be . mm. correlation is with high bmi and set-up errors in medio lateralis (m-l) direction (r= . ). this indicates that a patient with an increased pelvic circumference has a tendency to have more set-up errors. reformed procedural workflow resulted in more acceptable calculated margins in . when corrected on line times per week the margin is to be . mm and . mm if corrected times per week. in conclusion, for delivery of - gy to the prostate with imrt and spare surrounding healthy tissue effort is needed to secure the accuracy in the overall procedure of treatment planning and delivery. learning objectives: . to gain knowledge on how to improve the target accuracy in treating prostate cancer with radiation therapy using electronic portal imaging. . to understand the importance of gaining numerical information of accuracy in the treatment of clinically localised prostate cancer and how to use the information to calculate the necessary size of the safety margin around the clinical target volume. . to become familiar with the preparation needed before starting treatment with imrt and be aware that there is a need to look at all the treatment elements including the technical side, quality, education, staff and patients. . to consolidate knowledge of radiation therapy for prostate cancer and the meaning of giving high dose to the target while minimising radiation dose to healthy tissue. b. image-guided radiation therapy: when imaging meets therapy a. sarchosoglou; athens/gr (anastasia @hotmail.com) the aim of this presentation is: to understand the necessity of imaging in the delivery of external radiotherapy; to be familiar with the technology of image-guided radiation therapy and to to gain knowledge about the implementation of d igrt. the aim of radiotherapy is to deliver high dose to target volume while minimising the dose to surrounding healthy tissues. however, during treatment delivery many uncertainties may arise that can result in higher toxicity and poor local control. thus, it is crucial to monitor and adjust if necessary, the actual treatment. image-guided radiation therapy is the process where images are taken immediately prior or during a course of radiation treatment, to assess and improve the accuracy of therapy. manufactures have developed a number of systems to perform igrt providing the options of ionising radiation images/non-ionising imaging technology and gantry mounted/room mounted equipment. d igrt can be performed by acquiring computed tomography images on the treatment unit. these images are matched with reference images from planning ct and potential discrepancies are recognised, calculated and corrected by couch shifts giving optimal results. such advanced technologies require quality assurance systems to be in place and high trained personnel. when properly implemented, igrt meets the aim of radiation therapy. imaging provides confidence to radiation therapy to be aggressive, to fight cancer. imaging promises radiation therapy a great future with improvements in clinical outcomes and patient survival! multicentre trials have shown that ct colonography is ready for widespread clinical use. however, these studies have also highlighted the inconsistent performances of ct colonography, with divergent results being recorded in different centres and between readers. inter-observer and inter-centre variability may be related to reader experience but it is also affected by how ct colonography is performed. while faecal tagging has been accepted universally as the proper way to prepare patients for ct colonography, there is no consensus on which tagging agent is better, on the timing of contrast agent administration and on whether laxatives should be administered and in what dosage. in a similar way it is not yet clear which is the best way to obtain colon distension, whether by administering spasmolytic agents routinely or by changing patient's decubitus. standardising bowel preparation and technique is one of the most important goals that need to be achieved for ct colonography to be universally accepted and this presentation will attempt to deal with the issue using an evidence-based approach. the evaluation of ct colonography (ctc) studies is based on detection, interpretation and reporting of colonic findings. it is performed on a computer workstation equipped with dedicated ctc software by a primary d or a primary d approach. in either case, the alternative viewing technique must be available for rapid correlation and characterisation of suspicious findings. primary d evaluation is based on "lumen tracking" by interactively scrolling through the axial slices and multiplanar reformatted images, focusing only on the air-distended colonic lumen from one end to the other one. primary d evaluation provides information about the attenuation of findings during the search process and is time-efficient. primary d evaluation is based on d virtual endoscopy in an antegrade and retrograde fashion and increases both, the conspicuity, especially of small and medium-sized polyps, and the duration of visualisation. the use of advanced d displays like virtual dissection or unfolding techniques may reduce the interpretation time for primary d evaluation. computer-aided detection (cad) algorithms used as a nd reader were shown to reduce the number of perceptual errors by pointing out possible abnormalities that might otherwise be missed. colonic findings are characterised by their morphology, their attenuation characteristics, and by their mobility. knowledge of ctc imaging features of common colonic lesions and artefacts is necessary for characterisation of findings and differentiation between definite colonic lesions and pseudo-lesions. standardisation of ctc reporting facilitates classification and communication of findings and the comparison with previous studies, thereby better assisting physicians in making appropriate management decisions. the revolutionary development in multidetector ct (mdct) technology during the past decade has contributed to a substantial increase in its diagnostic applications and accuracy in children. a major drawback of mdct is the use of ionising radiation with the risks of radiation-induced side effects, of which the induction of secondary cancer is the most important. therefore, justification and optimisation of paediatric mdct is of great importance in order to reduce these risks as much as possible (‚as low as reasonably achievable' principle). optimisation of paediatric mdct starts with a solid understanding of all technical aspects of ct, including the most relevant scan parameters, dose reduction techniques and technique of iv contrast material administration. furthermore, due to the smaller size and lack of visceral fat in young children the interaction and absorption of radiation will be different which will influence the choice of the various technique and scan parameters. although all these issues are pivotal for a successful ct examination, it may become worthless if the importance of pre-scan issues such as justification and patient preparation are ignored. after a short overview concerning the current knowledge on radiationrelated risks in children, this lecture will focus on several aspects relevant for mdct optimisation in children. issues such as justification, patient preparation, technique and scan parameters will be addressed. finally, some guidelines for radiation dose level-based ct protocols will be given. the purpose is to present an overview of the safety hazards and safety protocols related to infants undergoing mri examinations. mri infrastructure-dependent safety hazards originating from: (a) static and fringe magnetic fields, (b) gradient subsystems, (c) radiofrequency subsystems and (d) acoustic noise sound pressure will be reviewed and discussed. safety hazards related to upcoming technological issues and future trends concerning mri will be presented. the current status of the organisations responsible to the problems of mr safety will be reviewed. which authority is responsible and where the responsibility is addressed to (directives, legislation policies, etc). an optimised protocol related to a variety of clinical mr sequences in reference to temperature measurements, emf measurements, sar and acoustic noise figures using basic commercially available infrastructure will be presented. d/ d tse sequences with different etl's, d/ d gre, d/ d ssfp and se/gre epi sequences with multi-(b) diffusion gradients and d tof mr angiography sequences will be examined. in vitro measurements of snr, spatial resolution and scan times will be performed for each clinical mr sequence. a comprehensive mri equipment operational policy (optima: (etl, tr, te, b-value, epi factor etc).) for a safety protocol for infants undergoing mri is proposed. recommendations for safe infant mri examinations will be summarised and presented. conclusion: mri equipment can operate safely for infant imaging but require policies and procedures beyond those required for standard diagnostic mri examinations. learning objectives: . to get an overview of the three basic safety hazards related to infants undergoing mri examinations. . to become familiar with safety hazards related to upcoming technological issues and future trends. . to learn about a safety protocol that could be applied in mri examinations for infants. a s c b d e f g h perfusion imaging. new possibilities arise in mri almost every day. imaging algorithms are to provide the correct diagnosis in the shortest time with the lowest expenses and harm to the patient. mri is the first method of choice in neurological diseases with two exceptions: acute stroke and acute trauma, ct providing the necessary information, more available and with easier patient care. adequate imaging protocols optimally visualise the anatomical region of interest and pathological processes, optimise the comparison between serial examinations and provide the information in reasonable examination time. appropriate and well understood clinical information is indispensable (neuroradiology means good clinical knowledge combined with expertise in imaging methods and their evaluation). complicated protocols are superfluous without competent clinical background but adequate information must be provided to experienced clinicians. basic protocols help to gather important information but do not exclude additional necessary methods. examples to be demonstrated are mri protocol for the pituitary, epilepsy, multiple sclerosis and spine. the up-to-date concepts of image evaluation and interpretation will be demonstrated for brain ct, brain mri and spinal mri, including the importance of follow-up. the body and conclusion part of the structured neuroradiological report will be discussed. consultation with other neuroradiologists and with clinicians is the final tool to reach the goal: to help in recovering the patients' health. ten authentic and original typical clinical cases illustrating the importance of imaging modalities in the differential diagnosis of focal neurological symptoms from headache to epilepsy will be presented while respecting the privacy of the patient. the spectrum of pathologies includes vascular, inflammation, metabolic, degenerative diseases, congenital malformations and neoplasms of the central nervous system. each case story will be described shortly in a standard form followed by demonstration of typical ct and/or mri images. in some cases, conventional mri will be supplemented by multi-voxel mr spectroscopy, dti, mr tractography images, and morphological pictures. follow-up images will be presented where appropriate. several diagnostic options will be offered for attendants. the audience will be asked to participate in the diagnostic process by the use of voting pads. after highlighting of final diagnosis the basic differential diagnostic considerations will be briefly summarised and emphasised from the clinical and imaging point of view for each presented case. learning objectives: . to introduce typical cases illustrating the role of imaging modalities in the differential diagnosis of focal neurological symptoms. . to motivate the audience by the use of voting pads to be involved in the diagnostic process. . to highlight the conclusion that may be drawn on the basis of the discussed cases. diagnostic quality mri of soft tissue masses can be performed using a variety of magnetic equipments and a variety of field strengths. regardless of system design, efforts should be made to maximise signal-to-noise ratios (snr) using the most appropriate coil to include the lesion and associated oedema. fov should be tailored to the size of the patient and the size of the mass. it is important to obtain images in at least two planes through the lesion. slice thickness vary depending on the size of the lesion and interslice should be not more than one-half of the slice width. imaging matrix should be balanced to in-plane spatial resolution. parallel while ctc has achieved excellent results in average risk individuals with regard to detection of clinically relevant polyps, it has not yet been implemented on a large scale in colorectal cancer (crc) screening programmes. this is mainly due to the fact that it uses ionising radiation. there is uncertainty about adequate screening strategies and the risk of radiation-induced malignancy. this presentation will summarise recent results of ctc in a screening setting, will look at the dose associated with ctc, and summarise calculations of radiation-associated risk of malignancy. the key question is whether low doses from ctc will cause relevant negative effects in the screening population. the lecture will also feature a comparison between ct and mr colonography, an imaging test that might be able to provide high sensitivity crc screening without ionising radiation. from headache to epilepsy… or from "normal", physiological "headache" to life threatening pathological conditions. when do we need "neuroimaging" and what is the optimal diagnostic work-up? the radiologist must and should remain first of all a medical doctor: he/she should keep close contact with the clinical world and always keep an active dialogue with the referring clinician. indeed, medical imaging has become more and more sophisticated but also more expensive. imaging is not a "screening procedure" in brain pathology but should be performed in order to confirm a clinical, suspected diagnosis keeping still in mind that differential diagnosis must be considered as well. imaging plays also a unique role in lesion evaluation and treatment monitoring. the radiologist must answer precise questions about the patient's suspected pathological condition and if necessary, discuss the imaging findings with the clinician in order to narrow the differential diagnosis. each mri or ct must be performed with a clear knowledge of the clinical question and the suspected pathology. therefore, the radiologist will make the proper choices of imaging techniques especially with mri where imaging sequences have become numerous. headaches and seizures are symptoms only and may be present in multiple and very diverse pathological conditions as infectious, neoplastic, haemorrhagic, vascular diseases: by knowing the clinical history a good "clinically conscious" radiologist will undoubtedly be of greater "added value"! learning objectives: . to learn more about the clinical conditions causing focal neurological symptoms. . to be informed about the clinician's way of thinking in the process of differential diagnosis. neuroradiology has a continuously changing and developing array of modalities. conventional radiography has lost its importance. angiographic practice has shifted from diagnostics to therapy. ms-md ct scanners provide high-quality ct angiography and a s c b d e f g h t w-mri to localise pz-zone pca. in the t-zone, stromal nodules commonly have a low adc value simulating pca. significant differences in tumour adc values existed between patients with low-risk, and those with higher risk localised p-zone pca. with s-mri, the best accuracy for diagnosing pca is obtained by combining a positive t w-mri and a choline+creatine/citrate ratio > . . in the t-zone, s-mri has the same limitations than dce and dw-mri. multiparametric functional mri accuracy of mri to detect pca can be improved by combining different functional sequences. at the moment, it seems that the most widespread used protocol is a combination of dw and dce-mri for detection of p-zone tumours. performance of functional mri to detect t-zone pca is less well established. multiparametric mri can now be used to perform stereotaxic trus-guided biopsies after trus-mri image fusion. the role of imaging in patients with increased psa level after radical prostatectomy or radiation therapy is to aid in differentiating locally recurrent disease which can be managed with local therapy from distant metastatic disease requiring systemic therapy. although the majority of local recurrences in post-surgical patients can be detected by mri in the perianastomotic region which can also be evaluated with trus and trus-guided biopsy, some recurrences can occur at pelvic sites that are beyond the range of trus; mri has a role of labelling these sites for trusguided biopsy. the combination of an external phased-array coil and endorectal coil is recommended for detecting local recurrent cancer. current protocols involve t -weighted mri combined with functional techniques such as dynamic contrastenhanced mri (dce-mri), magnetic resonance spectroscopy and diffusionweighted mri. in the post-prostatectomy bed, recurrences present as lobulated masses having low to intermediate signal intensity on t -weighted images and showing early, nodular enhancement with early washout of gadolinium on dce-mri. the predominant finding after radiotherapy is a diffusely low signal intensity with an indistinct zonal anatomy where the contrast between a hypointense recurrence and benign irradiated tissue decreases. on dce-mri, peripheral zone enhancement is lower after radiotherapy and any focal enhancement should be regarded as suspicious. the overall diagnostic efficacy of dce-mri for detecting recurrent prostate cancer is better than t -weighted mri alone. on mri, bone metastases have low and high signal intensity on t -and t -weighted images, respectively, and enhance after intravenous gadolinium administration. learning objectives: . to understand the role of mri in the follow-up of the patients with prostate cancer after radical prostatectomy or radiotherapy. in this presentation new techniques with potential clinical value will be described with a focus on prostate cancer multi-modality mr imaging. techniques such as t w, dwi, dce and mrsi will be addressed, and their role in screening, determination of tumour aggression and localisation, mr-guided biopsy, mr-guided minimal invasive focal therapy (laser, cryo, hifu), and mr-guided radiotherapy will be discussed. examples will be shown. the major area of debate is how to approach patients with increased psa levels. the discussion has two parts: one is the patient who has not had a prior cancer diagnosis and the other one is the patient who has already been treated for prostate cancer. what is the role of mr (as well as mrs, dw mr and dynamic mr) in precluding the need for multiple biopsies? when should we stop in cases with high or increasing levels of psa despite consecutive negative biopsy outcomes? a s c b d e f g h and that radiation therapy and systemic treatments play a major role for curing minimal residual disease. for evaluating and predicting response to neo-adjuvant treatments, the major question is the debate between morphology (size and volume) and function (perfusion, spectroscopy and diffusion). for follow-up after treatment, considering the number of women concerned, cost benefit analyses are mandatory to offer the most efficient imaging strategies based not only on defined subgroups (risk factors of local relapse) but also over time. one of the first take home messages of this course is the multidisciplinary approach of this disease or in another way: we do not treat images but patients! a. evaluation of residual disease after excisional biopsy c. boetes; maastricht/nl (c.boetes@mumc.nl) mammography can have an additional value in evaluating if microcalcifications are left behind in the case of dcis and irradical operation. ultrasonography has proven to have no additional value in evaluating the postoperative breast, because of haematoma and scar tissue. mr imaging of the breast can of all imaging techniques predict residual disease in the most accurate way. however, false positive results can exist because of enhancing scar tissue. also, false-negative results exist, especially owing to residual low and intermediate grade dcis. it is recommended to perform postoperative breast mri in premenopausal women in the right time of the menstrual cycle, that means between the th and th day after the start of the menstrual cycle. unclear is how soon after the operation one should perform breast mri. if a large mass of residual disease is suspected, mri is an excellent tool to confirm this fact. but, if a mastectomy is considered, pathological confirmation before the re-operation is recommended. another imaging technique is tc- m-mibi scintimammography. this seems a promising technique with a high sensitivity and specificity. neoadjuvant chemotherapy is regularly used for downstaging of locally advanced breast cancer. while it is equivalent to adjuvant therapy regarding overall survival and occurrence of distant metastasis, more patients are eligible for surgical treatment by less aggressive breast conservative therapy. neoplastic growth depends on blood supply with nutrients and oxygen. hypoxia-induced angiogenesis is an early step in tumour progression. mri is the imaging modality providing highest sensitivity for detection of breast cancer, highlighting tumour vascularisation by injection of t -shortening contrast agents. characteristic differences between benign and malignant lesions are cause. ultrasound (us) and computed tomography (ct) are readily available and widely used imaging techniques for this work up. us comprises an examination of the abdomen with the graded compression technique. a transducer should be used optimised for the visualisation of the bowel. the ct protocol includes the use of intravenous contrast medium, while oral contrast medium will not be routine in many institutions. findings are bowel wall thickening (or enlarged appendiceal diameter), fat infiltration, free air and fluid collections. the present evidence on the role of imaging in appendicitis and diverticulitis is substantial and has been summarised in systematic reviews. although us is accurate in diagnosing appendicitis and diverticulitis, ct is more accurate than us. further advantages of ct are better identification of alternative diagnoses and better comparison between consecutive examinations. thereby, ct is more helpful in communicating the diagnosis to the referring physician. cost effectiveness of ct in acute appendicitis has been demonstrated as well as the positive impact on management. for colitis, the evidence is more limited and imaging has more limitations here. drawback of ct is ionising radiation exposure. an imaging strategy with initial us and ct only in inconclusive of negative us cases results in the highest sensitivity, reduces ionising radiation exposure and is cost effective. accurate and rapid diagnostic imaging is essential for the appropriate management of acute biliary tract disorders. ultrasonography (us) continues to be the first and often the only test needed to confirm suspected diagnoses affecting the ducts and gallbladder. however, there has been an increase in the overall use of computed tomography (ct) in the emergency room setting, mostly because for its widespread availability and the relative speed, ease and uniformity with which evaluations can be performed. thus, ct may be the first imaging examination performed on patients presenting with signs and symptoms that are less specific for biliary diseases. magnetic resonance (mr) imaging has similarly robust potential as ct, although its integration into the acute care setting requires greater technical and logistical effort. improved mr imaging sequences, advances in coil technology, streamlined imaging protocols, and increased technical and professional familiarity with the modality make it an increasingly attractive option when there is concern about patient radiation exposure or allergy to iodinated contrast material, as in pregnant patients with acute abdominal symptoms. mr can also be used as a problem-solving modality. in this presentation, the common and uncommon imaging appearances on us, ct and mr of acute diseases of the biliary tract and gallbladder will be reviewed. potential pitfalls to be avoided with the three modalities are also illustrated. learning objectives: . to learn the etiology and clinical presentation of acute inflammatory liver diseases. . to understand imaging strategies using us, ct and mri. . to know typical findings and the spectrum of differential diagnoses. pancreatitis is an abdominal condition potentially life threatening. imaging plays a key role not only in the diagnosis but also in the staging and patient management of acute and chronic pancreatitis. pancreatic inflammation and necrosis can be easily identified by imaging methods and has prognostic implications. the clinical prognostic criteria in acute pancreatitis are currently coupled with imaging criteria. ct plays a central role in the evaluation of patients with known or suspected pancreatitis. a ct-based severity index is the main prognostic method to predict outcomes. in addition, mri and mrcp play increasingly important roles in pancreatitis evaluation. integrity of the pancreatic duct can be easily evaluated by this last method, particularly if enhanced with the use of secretin. imaging appropriateness criteria determine the value of distinct imaging modalities with regard to the stage of disease. we review the modern imaging criteria for the diagnoses, staging and patient management in acute and chronic pancreatitis. we also discuss current severity indices and imaging appropriateness criteria. advancements in ct and mri technology have lead to an increasing use of these modalities in the non-invasive assessment of coronary arteries, myocardial perfusion, and cardiac function. while their role in detecting coronary artery disease and functional disorders has been widely accepted, it is still unclear whether they could be adopted in triaging patients for the best therapeutic approach. large studies have already suggested that indication for surgery and percutaneous interventions cannot be solely based on the demonstration of morphologic alterations and that such "cosmetic" interventions are not always leading to the expected outcomes. therefore, non-invasive imaging techniques have to offer more then just the detection of grades of coronary artery stenosis, of areas of infarcted myocardium, or of valvular alterations. adjustment of imaging protocols for additional evaluation of coronary flow reserve, of myocardial perfusion and contractility and of valve size, position and damage with subsequent quantification of degree of stenosis and/ or regurgitation are necessary in order to allow to choose the most appropriate therapeutic approach and thus become the "gold standard" for prognosis and pretherapeutic diagnosis of cardiac diseases. a. can ct predict the outcome of percutaneous intervention? c. loewe; vienna/at (christian.loewe@meduniwien.ac.at) the outcome of coronary revascularisation is not only defined by primary technical success but also by improvement of symptoms and quality of life. thus, despite the individual comorbidities, the outcome and thus the potential benefit of coronary revascularisation depends on many different factors, including morphology, distribution and severity of coronary lesions, myocardial viability, and ventricular function. consequently, the detection of coronary stenosis is not sufficient for planning an optimised treatment. it should be evaluated if the myocardial territory supported by the diseased artery is still vital. in addition, lesions at risk for plaque rupture (culprit lesions) should be identified and treated to avoid major coronary events. cardiac ct allows for the exact assessment of coronary morphology including length, calcification and severity of lesions. based on this morphological information, success of a percutaneous revascularisation procedure can be anticipated with high prognostic accuracy. in addition, the possibility of identification of culprit lesion by means of coronary ct has been described recently. by this, dedicated treatment of only the relevant stenosis should become possible avoiding multiple, potentially unneeded, stents. finally, even the assessment of myocardial viability by means of ct becomes possible. using all the possibilities of cardiac ct optimised treatment plan can be established, and outcome can be estimated. radiological interpretation always incurs some degree of error due to the nature of disease presentation coupled with the difficulty in diagnosis, especially where early signs of disease need to be identified such as in medical screening. early research studies of radiologists' performance concentrated upon the chest radiograph but more recent work has studied breast screening extensively, as well as mri and ct. as radiology is now almost fully digital then research has also examined observer behaviour with a range of digital images, viewing conditions, and image display presentations. it is possible that radiological interpretation will never be accomplished without some errors occurring; however, it is important that steps are taken to minimise any causes of errors as far as possible. the reported error rates found in numerous investigations across different radiological domains will be reviewed and the reasons for these will be elucidated. appropriate reporting conditions will be highlighted for different image viewing scenarios. a theoretical framework for understanding error causation, especially where abnormalities are missed will be detailed. furthermore, the underlying visual, perceptual and cognitive processes which lead to errors will be detailed and approaches to minimise error occurrence will then be proposed. the relationship between human perceptual and cognitive skills and computer imaging processing will be discussed and the usefulness of cad systems outlined as how they can best aid the radiologist from the human performance viewpoint. learning objectives: . to review the basic principles of perception, detection and detectability. . to learn about specifics of perception in medical imaging. . to learn how image processing can help us with the perception process. ing using graf's method led to higher treatment and follow-up rates than that based on nhi alone, i.e. - % vs. . - . % and - % vs. - %, respectively. however, improved examination techniques and a better understanding of the findings have led to a more tailored approach, and an extensive meta-analysis performed in , including papers, could not find any differences in treatment rates due to different ultrasound techniques. in this lecture i will present a crude status for us techniques used and also give recommendations for a worth while screening strategy based on present knowledge, and on work performed within the espr's ddh task force group. with technological advances in recent years, paediatric whole body imaging is now a clinically feasible and increasingly accessible technique. the two principle modalities available are pet, with or without co-registered ct images, and whole body mri (wb-mri), both of which can be utilised to evaluate widespread disease states efficiently. the main application of these techniques has hitherto been oncological; however, as these techniques become more accepted, their use is becoming more varied. pet provides functional assessment, utilising a radiotracer, most commonly fdg- , to assess metabolic activity within tissues, with areas of greater metabolic activity depicted as increased tracer uptake. the use of combined ct-pet enables accurate specific uptake values (suv) to be determined, via attenuation correction, and anatomical co-registration which reduces perceptual errors. wb-mri primarily provides an anatomical assessment, using tissue contrast to identify pathology. this typically uses water sensitive sequences to provide high sensitivity. "physiologic" mri sequences, such as diffusion weighted imaging (dwi) can also be employed to obtain functional mri data, either qualitatively or quantitatively. there are advantages and disadvantages to both techniques, including ionising radiation exposure, acquisition times, movement and other artefacts, and reproducibility, which all need to be considered when choosing a particular technique for whole body assessment in any given paediatric patient, with the disease process being investigated also influencing the technique used. the relative merits, clinical applications and evidence base for the use of pet/ct and wb-mri in paediatrics will be discussed. near future developments, such as pet-mri will be touched upon. the practice of medical image diagnosis is currently undergoing a fast transformation. vast amounts of data can be generated in standard examinations and focus is shifting from improving the collection of relevant data for diagnosis to development of effective methods to analyse, visualise, navigate and interact with medical information. it is now becoming generally accepted in the medical community that one of the most important keys to manage the increasing information work load is the use of d and d applications. this talk will take its starting point in state-ofthe-art medical visualisation and then discuss the need for a research agenda that focuses on the development of the next generation of medical visualisation tools, emphasising the fact that these tools must be based on medical user requirement and work flow studies as well as on new technical developments. childhood osteomyelitis is a relatively rare finding in childhood with an estimated annual incidence of per and is predominantly seen in young children (< years). it can be caused by via haematogenous spread, contiguously from local areas of infection or from penetrating trauma or surgery. usually it is caused by bacterial infections, but occasionally fungi, viruses or parasites are causative agents. clinical findings can range from mild to severe and depends on many factors such as age, site of infection, acute versus chronic osteomyelitis, and causative agent. the crp and sedimentation rate are usually elevated; however, this is not always the case. given the variability in clinical presentation radiology plays a crucial role in the detection of osteomyelitis. although conventional radiography (cr) has a limited sensitivity and specificity for the diagnosis of osteomyelitis, its wide availability makes it the first diagnostic technique of choice in children. for further analysis both mri and radionuclide bone scintigraphy (rbs) have shown to have a high sensitivity for the detection of osteomyelitis. the advantages and disadvantages of both the techniques will be addressed. ct mostly plays a role in the diagnosis of chronic osteomyelitis or in the pre-surgical work-up. the use of radiology also allows for a differential diagnosis of osteomyelitis, based on clinical cases, an overview of this differential diagnosis will be given. the aim of this lecture is to present an evidence-based diagnostic strategy for childhood osteomyelitis. developmental dysplasia of the hip (ddh) is the most common musculoskeletal disorder in childhood, with a reported prevalence of - % according to method of ascertainment and definitions used. ultrasound has enabled a detailed view of both neonatal hip stability (nhi) and morphology, and two different schools have developed; one arguing that nhi alone is the major pathology warranting splinting, the other including acetabular dysplasia as an important feature. both static (graf, morin) and dynamic (harcke) ultrasound techniques, as well as a combination of the two (modified graf (rosendahl)), have been described and are currently used. in europe, graf's ultrasound technique or a modification of this is commonly used within the german speaking countries and areas, in parts of scandinavia, the uk, italy, france, hungary and the netherlands. others use a modified morin's method while harcke's method is used only occasionally. initially, universal ultrasound screen-(helium or xenon), oxygen-enhancement or other technologies. all of these might also provide quantitative read-outs for disease and/or therapy monitoring. learning objectives: . to learn how ct and other imaging methods can be used to examine the structure-function relationship in sad. . to review the state-of-the-art imaging methods that can provide information about disease extent, disease activity and global and regional lung function in patients with sad. . to become familiar with the current use and the future developments of these techniques. panel discussion: signs of small airways disease can be seen on ct but when and why do they really matter? : signs of small airways disease are a frequent finding on ct especially when expiratory scans are performed in addition to inspiratory scans. when should we report these changes? are they always important, do they perhaps predict the development of more severe disease, or are there cases where they have little influence on diagnosis and therapy? when should an expiratory scan be performed? careful preoperative staging and preoperative using high-resolution mri together with preoperative multidisciplinary team discussion has been shown to reduce margin involvement by tumour from % to < % by identification of patients that require more radical preoperative therapy and surgery. the detailed demonstration of preoperative prognostic factors also recognises patients on imaging that are not at risk of local recurrence and therefore the avoidance of unnecessary preoperative therapy in a proportion of patients. the use of eus can help in the assessment of early stage flat lesions suitable for local excision and is a powerful complementary tool. pet-ct is crucial for the preoperative work up of patients undergoing radical procedures such as metastatectomy. follow-up for colorectal cancer patients at high risk for developing recurrent disease is now well established, and the emergence of specialised multidisciplinary teams, combined with a range of treatment options for recurrent disease has improved curative resection rates following metastatectomy and pelvic recurrence surgery. radiologists with a solid understanding of not only the pathological manifestations of primary and recurrent rectal cancer but also the treatment options available play a key role in enabling the appropriate selection of patients -increasing overall cure rates and reducing treatment-related morbidity. this refresher course aims to provide radiologists with an understanding of local staging of rectal cancer, the assessment of recurrent disease, and assessing response to treatment. the course will highlight how imaging underpins the key preoperative decisions for surgical and oncological treatment planning in rectal cancer. a. staging with us and ct a. maier; vienna/at (andrea.maier@meduniwien.ac.at) for rectal cancer surgery a variety of alternative operations are currently possible. furthermore, there is an increasing trend towards treating patients with radiotherapy before surgery. the choice of operation and the decision whether to employ radiotherapy is based on preoperative staging. in patients with primary rectal cancer accurate assessment of tumour extent and the presence or absence of lymph node invasion are factors for determining prognosis and risk of tumour recurrence. endorectal ultrasound (eus) is effective for t-staging. it has been recommended as the investigation of choice in the selection of potentially curative local excision. lymph node staging by this method is less precise than tumour staging. initial reports of the use of ct for tumour staging were encouraging. studies which compare ct with eus staging consistently show the latter to be more accurate for both tumour stage and lymph node stage. thus, the usefulness by the speakers at the end of the session. at the other end of the clinical and imaging spectrum to obliterative bronchiolitis is exudative small airways disease, typified by (japanese) diffuse panbronchiolitis. the exudative bronchiolitides are characterised by direct signs on hrct, notably a tree-in-bud pattern. while this is a readily appreciated and specific sign, other diseases that mimic this hrct finding will be discussed. bronchiectasis of variable severity is a usual accompaniment to both obliterative and exudative bronchiolitis and the relationship between large and small airways disease will be explored. the instances in which hrct findings of obliterative and exudative bronchiolitis co-exist are relatively few, and the differential diagnosis for this situation will be considered. in practice, hrct will often show signs of bronchiolitis (whether obliterative or exudative) limited to a segment or even subsegment, and the interpretation of the clinical significance of such a chance finding is sometimes a difficult judgement. learning objectives: . to become familiar with the direct and indirect signs of small airway involvement on ct. . to know how to correlate these signs with the pathological changes. . to understand the differential diagnosis with other diseases that can show similar ct findings. from pattern to diagnosis c. beigelman; paris/fr various schemes of classification of sad have been proposed according to clinical, pathologic or imaging criteria that are confusing. an optimal approach, mainly based on ct analysis of direct and indirect features, usually allows the recognition of the two main types of sad, namely inflammatory/exudative and fibrotic/constrictive/ obliterative bronchiolitis. a miscellaneous group that corresponds to bronchiolar involvement in diverse diffuse lung diseases may also be individualised. direct signs of sad that refer to direct visualisation of diseased bronchioles strongly suggest inflammatory bronchiolitis. they mainly consist in centrilobular nodules with tree in bud appearance. conversely, indirect signs mainly represented by mosaic attenuation, air trapping, bronchial wall thickening and dilation characterise fibrotic bronchiolitis. volumetric acquisition, performed a dose reaching that obtained with classical hrct protocols, may be of interest in several ways. particularly, maximum intensity projection tool may facilitate the recognition of the tree in bud pattern. the minimum intensity projection mode may optimise the recognition of the mosaic attenuation pattern requiring an appropriate contrast resolution, as well as the analysis of proximal airways. expiratory ct, optimally performed on a dynamic mode, may be useful in some conditions at a dose equivalent to around chest x-rays. a combination of post-processing tools from a volumetric acquisition performed with carefully chosen parameters might therefore appear useful in the evaluation of sad. furthermore, additional co-existing findings such as ground glass opacity or other features of interstitial pneumonias may be detected. their analysis may help to recognise the cause of sad in addition to clinical data. structural changes associated with sad are difficult to depict directly on ct. indirect signs of sad, such as mosaic attenuation on an inspiratory scan and/or air trapping on an expiratory scan, are common findings. they nicely illustrate the structure-function relationship between obstruction or expiratory collapse of the small airways and the subsequent effects on ventilation (local hyperinflation) and perfusion (hypoxic vasoconstriction). these "functional" signs are thought to be more sensitive than the direct visualisation of the underlying structural changes. careful interpretation and software tools help to generate data about disease extent as well as global and regional lung function. beyond paired inspiratory-expiratory ct scanning, dynamic expiratory cine as well as perfusion and ventilation imaging on the basis of both, ct and mri, can be applied to increase sensitivity, specificity and accuracy of the diagnosis of sad. some of them are ready for routine clinical use, such as dynamic expiratory cine ct, cine mri during continuous breathing as well as gadolinium-enhanced perfusion mri. future developments in the fields of ct and mri will provide novel technical approaches for functional imaging of sad. these will include novel applications of dual energy/spectral ct using iodine-enhancement for perfusion and xenon-enhancement for ventilation enhancement as well as ventilation mri using hyperpolarised gases to understand the advantages and limitations of bi-rads in clinical practice to appreciate the potential of an automated analysis of the descriptors to trace continuous modifications required by technological advancements. . to review bi-rads categorisation with case illustrations a. how i do it p. richards; stoke-on-trent/uk (paula.richards@uhns.nhs.uk) . always report in the same logical manner for each examination, which imprints normal pathology 'jumps out at one'. . evaluate all images before looking at the clinical information to prevent bias and satisfaction of search. . review every scout image. scoliosis transitional vertebrae and pseudoarthrosis become more obvious on coronals. single kidneys, hydronephrosis and renal tumours may explain 'back pain', especially if there are mets. . review any x-rays or old ivus, remembering that abdominal x-rays show the spine. . indications; summarise the reason for the scan. think of the differential diagnosis to exclude. . technique; allows one to check the levels scanned. be sure there has been no area missed between studies. . report vertebral alignment and disc height. . mri just like an x-ray, i.e. there is normal alignment from d to s . . start at the far sagittals and look at the nerve roots in the 'key hole', the pars and facets. . determine the worst abnormality on the axials and report the most significant findings first. check the facets at each level. . have a checklist of normals at the end; 'the bone marrow, cord and csf return normal signal. the conus ends normally with no pars or metastasis'. .opinion: explain what you think is causing the patient's symptoms. assume this is the only part read, so if there is only one kidney reiterate here. the lecture will illustrate additional information on scout images and benefits of coronal images. learning objectives: . to understand the influences of patient positioning, scan parameters and magnet/coil technology on image quality. . to learn how to optimise scan protocols to maximise patient throughput without compromising diagnostic quality. . to recognise how and when to modify scan protocols to answer specific clinical questions.a- the lumbar spine undergoes morphological changes with age. normal appearances and relevant normal variants will be discussed. in particular, the morphological change from an almost round intervertebral disc in newborns with high water sig-postgraduate.educational.programme s a b c d e f g h radiotherapy is an area with a rapidly improving development of new techniques and improved possibilities for accuracy. this lecture aims to illustrate and discuss points of contact and the need for collaboration between radiotherapy and radiography. european educational programmes and working fields for radiotherapy nurses and radiotherapy technicians are compared. radiotherapy nurses and radiotherapy technicians are responsible for the administration of radiotherapy to cancer patients and for the clinical care related to the treatment. apart from prevention and treatment of side effects and psychosocial support during the treatment, it encompasses preparations, delivery and verification of the radiation dose. educational programmes differ in terms of academical level and target groups, whether it is nurses or technicians in radiology or radiotherapy exclusively. areas in the radiotherapy process where competences from radiography are needed are discussed, considering both technical development and research progresses. learning objectives: . to gain knowledge about the differences and similarities between the areas of education and profession in radiography and radiotherapy from a historical perspective. the demand from the public for greater accountability and standards has increased the importance of audit and accreditation in modern healthcare delivery. specifically radiology departments participate in audit and accreditation by a number of entities. these include: . national bodies concerned with improving radiation safety particularly originating from eu / directives. . hospital wide accreditation surveys from state and private accrediting organisations. . auditing of standards from within professional bodies. . following from investigation of specific incidents such as misdiagnosis or radiation concerns. . modality-specific standards.although the specific requirements of the accrediting or auditing body may differ, the processes and practical aspects in demonstrating compliance with standards and quality improvement are generally similar. mechanisms such as outcomes, quality cycles, and performance indicators are critical in the success of any accreditation or audit program. in addition to fulfilling basic regulatory requirements, the medical physicist is playing an increasing role in developing performance indices particularly in radiation safety, clinical image quality and equipment management. the increasing role of the medical physicist in audit underpins the requirement for inclusion of audit and accreditation as part of their education. recent experience of setting up and participating in eu / directive-based clinical audit in ireland highlighted the critical need for clarity of roles and ownerships of processes to be communicated to those who are carrying out audit and those who are being audited in hospitals and dental practices. performing imaging studies in a child requires basic knowledge. the first point is to reassure and to avoid stressful/painful procedure. training of radiographer and radiologist is fundamental. explanations, presence of a parent (if not pregnant…), are preferable. specific devices may be useful for immobilisation and to avoid repeated exposures. antalgic drugs (traumatic circumstances) or sedative nitrous oxide inhalation (mcug) may be useful. x-rays exposition in relation with conventional radiology is lower than the one encountered with ct. but dose depends on type of examination and also on imaging equipment. new devices, such digital fluoroscopy with x-rays pulsed emission, flat-panel detector, slot-scanning x-ray imager need less ionising radiation than conventional screen-films devices or even photostimulable phosphor imaging plates. the scale between the higher and the lower dose for the same examination can be from ten to one. common principles of protection that apply to all x-ray imaging procedures, known as alara concept (as low as reasonably achievable), have to be used daily. act in accordance of the medical justification of the examination, without any non-ionising alternative method, remains the first step. the second one is the optimisation of each procedure, the limitation of expositions, with control of the x-ray beam adjusted to optimise the critical balance between image quality and exposure to the child. measure of the dose is mandatory to demonstrate appropriate levels on child exposure and to be sure in the future that very low radiation doses received during conventional imaging procedures will not produce adverse effects. the council directive / /euratom introduced the concept of clinical audit to medical radiological (diagnostic radiology, nuclear medicine and radiotherapy) procedures. clinical audit is a systematic review of the procedures in order to improve the quality and the outcome of patient care. according to the directive, clinical audits shall be implemented in accordance with national procedures. the review of its implementation in europe has revealed a high variation of approaches and many practical problems. therefore, the european commission has published further guidelines on clinical audits in its report radiation protection no. ( ). the purpose is to improve the implementation of clinical audits and to enable the member states to adopt the model of clinical audit with respect to their national legislation and administrative provisions. the guideline points out the importance of both internal and external assessments for clinical audit. it deals with all types and levels of clinical audit and gives practical guidance for application. it defines the list of topics which should be covered, while the actual criteria of good practice are discussed on generic levels only. the borderline between clinical audit and other quality assessments (accreditations, certifications, peer reviews) and regulatory inspections is also discussed. the guideline is addressed to all professional groups, hospital management, auditing organisations and regulatory bodies. it is important to recognise that the guideline is not a legal requirement. this refresher course lecture will give a summary of the ec guidelines and discuss briefly the implementation of clinical audits in europe.learning objectives: . to understand the purpose and essential contents of the european commission guideline on clinical audit. b. national perspective: clinical audit inspections s. ebdon-jackson; didcot/uk (steve.ebdon-jackson@hpa.org.uk) european council directive / /euratom requires under article ( ) that "clinical audits are carried out in accordance with national procedures". implementation of this requirement across europe has been varied. this paper will reflect on the experience in the uk and will consider examples of the various initiatives undertaken in the uk by professional bodies and organisations. this will be contrasted with the role of the regulator and the aims of the inspection process with regard to compliance with the specific regulation addressing clinical audit and the remaining regulations addressing justification, optimisation, etc. examples will be provided of the type of audits undertaken within uk hospitals. a uk perspective of ec and iaea initiatives in clinical audit will be provided. percutaneous radiofrequency ablation (rfa) is a safe and effective treatment for well selected patient with hepatic tumours such as hepatocellular carcinoma (hcc) and liver colorectal metastases (lcm). an appropriate selection is crucial. it is based on clinical and technical arguments. clinically, surgical resection of the haepatic tumours remains the gold standard. indeed, survival data following rfa are not as good as surgical resection. the only exception seems to be the rfa of the very early hcc (≤ . cm) in cirrhosis that is not candidate for liver transplantation. most often, the rfa offers an alternative for patients with medical comorbidities, poor liver function or prior hepatectomy. technically, there are three decisive points for complete ablation. the first point is the good visualisation of the tumour either under ultrasound or un-enhanced computed-tomography (ct) examination. metallic coil placement, lipiodol tattoo and virtual ct sonography with magnetic navigation are technical tricks that allow the rf ablation of "invisible" tumour. the second point is the tumour size: in most series, a diameter less than - mm is commonly admitted as a prognostic factor. this is probably because the maximal ablation diameter is slightly larger than mm with the electrode needles available now. micro-waves ablation might improve the local control by increasing the ablation diameter. the last point is the "heat-sink effect" that requires a temporary occlusion of a large vessel close to the tumour. the quality of follow-up imaging is a key factor for evaluation of tumour destruction. partial hepatectomy for liver metastases or primary liver tumours can only be performed when the future remnant liver volume (frlv) is considered large enough to avoid the risk of post-operative liver failure. in normal livers a frlv of - % is considered safe whereas in compromised livers a frlv of at least % is required. in patients whom the flrv is considered to small, pve may be performed pre-operatively to increase the frlv. pve involves percutaneous selective embolisation of the portal venous (pv) system, usually of the right liver lobe, which leads to atrophy of the embolised lobe. this, in turn, leads to hypertrophy of the frlv. because of the dual vascular supply to the liver (arterial and portal venous), necrosis of the embolised lobe does not occur. this compensatory hypertrophy of the left lobe facilitates resection in patients in whom the frlv would otherwise have been to small to allow resection. pve is most often performed percutaneously under conscious sedation and local anaesthesia through an ipsilateral approach, using a combination of particles, such as polyvinyl-alcohol (pva) and embolisation coils. potential complications of pve include thrombosis of the contralateral pv, liver abscess formation or cholangitis and are rare. most of the hypertrophy occurs in the first - weeks after pve and increase in volume of the frlv averages % after this time period. ct-volumetry should be performed both before pve and after - weeks to measure increase in frlv. accurate identification and reporting on soft tissue extremity masses is essential for correct diagnosis and optimal treatment planning. this lecture focuses on the mri signs to stage and grade-characterise soft tissue mass lesions. these two objectives are the major structural components of the mri report and fit the request of the referring clinician. this implies an accurate description of these signs with appropriate terminology in the report. local staging is essential for the preoperative work up. important staging parameters are size, compartmental location, skip lesions and relationship to neurovascular structures and joints, as well as distance to the nearest joint space. grading and characterisation is complex and multifactorial and classifies the lesions as "certainly benign" (no biopsy needed) and "possibly " or "certainly malignant" (biopsy needed). grading parameters are homogeneity, (changing) signal intensities in multiple acquisition techniques and static and dynamic gadolinium enhancement pattern. these are used to define the composition of the lesion, i.e. fat, met-hb, hemosiderin, myxoid tissue, collagen, viable, cystic or necrotic components. other important grading and parameters are lesion margin, shape, perilesional invasion or destruction, multiplicity, specific location and associated findings but also age and gender of the patient. image guidance and imaging fusion techniques represent an integral element in oncologic interventions and liver surgery. in addition, several liver planning techniques such as the virtual liver surgery planer enhance simulation of a proposed liver resection. using roboter-assistance or navigational guidance combined with thermal ablation techniques such as radiofrequency ablation, significantly enhances accuracy of ablation probe placement and efficacy of ablation necrosis. furthermore, the local recurrence rate can be considerably reduced, and the amount of complete tumour ablation is significantly more likely. integrating magnetic resonance imaging, and focused ultrasound to deliver and activate nano-capsules carrying anti-cancer drugs to effectively target tumours will be another task. this will involve drugs being injected into the body in the form of tiny capsules, which are harmless until they are activated by a concentrated focused ultrasound `blast'.the mri scanner will then be used to track the passage of the drugs, visualise the target and monitor the delivery of the drug treatment. the risks associated with radiation exposure in ct is of concern to radiologists, medical physicists, government regulators, and the media. thoracic ct is a technically robust, non-invasive imaging technique for the evaluation of several traumatic and non-traumatic thoracic emergencies. technical advances in the past years have resulted in improvements in image acquisition speed, spatial resolution, and the temporal resolution. consequently, thoracic ct can now be performed rapidly in emergency conditions without substantial delay in treatment. state-of-the-art ct systems are now capable of imaging the entire thorax within a few seconds. however, radiation exposure associated with thoracic ct have been increased with the advantages of modern ct systems particularly in the evaluation of chest pain syndrome. thoracic aortic ct angiography with cardiac gating may now be considered the preferred technique for the evaluation of chest pain syndrome in selected patients but is associated with a substantial higher radiation dose than routine non-gated thoracic ct. several effective strategies have been developed to limit the radiation exposure in cardiac gated thoracic ct including prospective ecg gating techniques, anatomy and ecg-based tube current modulation, high pitch acquisition, and adaptation of the ct scanning parameters to the body habitus. in addition, several techniques are available to reduce the radiation exposure in nongated thoracic ct depending on the patient's body habitus and the clinical indication. because of advancing technical developments and increasing diagnostic utility of mdct in emergency care, its use has expanded significantly and has changed patient care, especially in chest emergencies. however, mdct has important drawbacks in cost and radiation exposure. the first presentation will discuss mdct in acute chest pain (acp). scan techniques in different scanners will be addressed. the reasons why cta/ctca can be a viable application for acp and the current evidence for cta in acp will be discussed followed by an update on indications. the next presentation will address mdct in chest trauma. its significance, the utility of this imaging method and its advantage compared to other modalities will be discussed. the examination protocol and the role of post-processing methods will be addressed. the interpretation of mdct, findings, benefits, limitations and pitfalls will be shown. the utility of chest x-ray and ultrasound as primary examinations and the indication for mdct, routine or selective, will be discussed. our last speaker will familiarise us with procedures to measure radiation dose in thoracic mdct, followed by an update of radiation exposure associated with the protocols for the different clinical indications. finally, available techniques for radiation dose reduction in ecg-gated and non-gated mdct and their effectiveness in radiation dose reduction will be demonstrated. however, the best way to reduce radiation is to perform mdct only when there is an appropriate indication and this will be the focus of the concluding panel discussion of this session. chest trauma is, particularly in younger population, a significant cause of morbidity and mortality. it is directly responsible for - % of trauma-related deaths and in other % of deaths it is an important contributing factor. imaging methods play the key role in management of this group of patients. introduction of mdct in the last decade of the th century markedly changed the diagnostic approach to the thoracic trauma. fast data acquisition and increased resolution in the z-axis enabled reliable assessment of all chest anatomical components (often together with other parts of the body) in one examination. compared to x-ray mdct defines more accurately the extent and severity of traumatic changes and may detect serious in % of nontraumatic cases, subarachnoid haemorrhage (sah) is the result of aneurysmal rupture. other causes of sah include perimesencephalic sah ( %) and other disorders ( %) such as arteriovenous malformation (avm), vertebrobasilar artery dissection, dural av-fistula, cortical vein thrombosis, amyloid angiopathy, … sah represents an emergency situation and diagnosis should be established asap.plane computed tomography (ct) is the initial diagnostic test of choice (wide availability, easy accessibility, high sensitivity and specificity). if ct is negative, lumbar puncture and/or mri of the brain (including flair images) and spine should be performed. d tof mr angiography (mra) sequences have high sensitivity and specificity in detecting cerebral aneurysms; but because of their lower spatial resolution are insufficient to analyse in detail aneurysm morphology. this in contrast with cta. both cta and mra may be proposed as a first-choice, noninvasive examination, but the negative predictive value is poor, and therefore digital subtraction angiography (dsa) is mandatory for all sah cases with negative cta or mra. catheter angiography remains the golden standard in the detection and evaluation of cerebral aneurysms (size, relationship between neck and adjacent vessels, etc). increased attenuation (ct) and hyperintense signal (flair) within the basal cisterns and sulci are a characteristic finding of sah; however, it is aspecific. pattern and location of the blood may help to locate the ruptured aneurysm; sometimes helpful when multiple aneurysms are found. pseudo-sah may be a potential imaging pitfall because it may be observed in other acute neurological conditions (cerebral edema, bacterial meningitis, etc). recurrent disease following treatment for primary breast cancer can occur in the same breast following conservation surgery, ipsilateral breast tumour recurrence (ibtr) or in the contralateral breast, metachronous contralateral breast cancer (mcbc). estimations for the rate of recurrence are between and . % each year. the rate of recurrence is higher than breast cancer incidence in the general population. risk factors for recurrence are young age (< ), high grade disease, incomplete tumour excision and no radiotherapy. due to the large numbers of women who develop and survive breast cancer the cost and resource required to follow-up these women is considerable. there are variable guidelines in europe for the surveillance of women but most regimes include clinical follow-up and mammography. variations concern the frequency of mammography, the length of follow-up required, and whether clinical examination is required. the clinical effectiveness and cost-effectiveness of different surveillance mammography regimens after the treatment of primary breast cancer will be reviewed together with the results of systematic reviews. modelling of two data registries was undertaken to ascertain the survival benefit. alternative methods of surveillance will be considered such as mri with the diagnostic accuracy of the various imaging techniques considered. a summary of the economic evaluation will be presented to demonstrate the assumptions that require to be made in this complex area where there is a paucity of evidence.learning objectives: . to understand the risk of recurrent disease and second primary following the treatment of breast cancer. . to review literature on surveillance mammography and other imaging methods for detection. . to appreciate the range of recommendations for surveillance mammography and clinical follow-up with cost benefit analysis. the new challenge in breast cancer: evaluation of response : evaluation of the treated breast is one of the major challenges in breast imaging.there is especially much debate on the evaluation of response to therapy. the best imaging modality, the best imaging criteria, timing, accuracy and limitations of imaging are currently being investigated. whether it is time to reach conclusions regarding these aspects is the issue of this panel discussion. intracranial aneurysms are abnormal dilatations of the arteries, and will be found in % to % in general population. in % cerebral aneurysms are saccular and develop from the arteries of the circle of willis or its major branches. aneurysms typically become symptomatic between the age of and years, with subarachnoid haemorrhage (sah) or intracerebral haematomas. less common are giant aneurysms usually found in middle-aged women presenting with signs more indicative of a mass lesion. ct angiography (cta) has been proven to be an excellent tool to visualise intracranial arteries. the average sensitivity of cta for the detection of intracranial aneurysms reaches %. post-processing allows assessment of the aneurysm with maximum intensity projections (mip) and surface-rendered d projections in multiple plains. mr angiography (mra) is a practical and noninvasive tool for screening of high-risk individuals for aneurysms. dsa is diagnostic method of choice for assessment of intracranial aneurysms and was considered to be a 'gold standard' for evaluation of cerebral vessels. a clear shift from invasive to noninvasive imaging of cerebral vessels has been noticed in the last decade. this lecture will discuss advantages and disadvantages of cta, mra and dsa in detection, assessment, and post-treatment evaluation of intracranial aneurysms. intracranial aneurysms have a multifactorial origin. a heterogeneous and complex group of pathogenic mechanisms including luminal, mural and extramural factors interact for the development and growth of aneurysms. there are several types of intracranial aneurysms, being the saccular the most common type. the aneurismal treatment is dependent on its type and needs a multidisciplinary approach including endovascular and microsurgical teams. endovascular treatment is recognised as the first-line treatment for most of the ruptured saccular intracranial aneurysms. there has been an enormous improvement in the endovascular technology and techniques offering a wide spectrum of treatment possibilities that broadened the variety of aneurysms that can be treated. the treatment options include the use of coils with/without the help of balloon-remodelling and/or stenting. other options include the use of covered stents, of "flow-diverter" stents, of liquid embolics, or the parent vessel occlusion. the multislice computer tomography angiography (mscta) is a reliable method for pretreatment evaluation of intracranial aneurysms. for the posttreatment evaluation, magnetic resonance angiography (mra) can be used for the follow-up of embolised aneurysms; and mscta may be used for the follow-up of surgically treated aneurysms. there are several challenges remaining in aneurysm management. in the diagnostic field, improvement of the non-invasive techniques for the diagnosis, for the morphological and haemodynamic characterisation and for the posttreatment follow-up of aneurysms is expected. in the endovascular field, technological breakthroughs to improve the occlusion rate, to increase the treatment durability, and to promote the vessel wall healing are desired. learning objectives: . to learn about present treatment of brain aneurysms. . to understand the strategies for multimodal ct and mr aneurysm imaging pre and post-treatment. . to present the challenges for neuroimaging in the field in the near future. where do we stand in brain aneurysm treatment today? : there is a significant difference in the management of patients with ruptured versus un-ruptured brain aneurysms. this panel discussion will focus on the management of patients with brain aneurysms and will with simple case presentations show the management in ruptured and un-ruptured aneurysms and discuss the involvement of the neurosurgeon in more complex cases.cone beam ct (cbct) is a method for obtaining ct-like images using a c-arm system. our aim was to investigate the accuracy of these images compared with multi-detector ct (mdct) as a gold standard in radiological imaging of abdominal aortic aneurysms (aaa). patients with aaa referred for elective evar were included in a pre-therapy protocol ( patients) and in a post-therapy protocol ( patients). all were exposed to standard mdct and one additional cbct. image data were evaluated by two radiologists and statistically compared using a linear mixed model. first, predefined arterial measurements were performed, then predefined anatomical areas were assessed and scored for visibility on a scale from to . all measurements were chosen to be relevant for evaluating of aaa before and after evar. for the arterial measurements no significant differences were found between mdct and cbct. visibility for the anatomical areas was significantly better for mdct; however, most of the cbct readings were above lowest acceptable level. visualisation of the iliac arteries was suboptimal. we think that our results support the start of clinical trials that scientifically can test the outcome without the use of mdct immediately before and after evar. the problem with poor visualisation of the distal iliac arteries might be solved with newer technology. in conclusion, the result indicates that cbct in the operation room gives sufficient image-based information to support evar in a pre-and post-therapy setting. undertaking theatre radiography can appear a daunting task for the radiographers lacking in confidence in their ability but others (like all expert practitioners) make it look easy and uncomplicated. theatre radiography requires all the knowledge, skills and abilities of a competent radiographer together with assertiveness, reflection and the ability to modify your technique at a moment's notice. there are a multitude of 'traits' which distinguish the expert from the novice. these include production of optimum images, effective use of the equipment, radiation protection, infection control, effective communication and teamworking. there is also a clear depth of knowledge together with a multitude of skills and abilities. the key to a stress-free experience is preparation. it is essential that there is a mechanism for theatre staff and the radiology department to communicate effectively and give the radiographer prior notice of all cases requiring their attendance. it is also desirable to be in the theatre when the patient arrives for their examination to enable you to check the patient's identity, pregnancy status and ensure the patient is positioned correctly to facilitate screening. there are few excuses to give the surgeon if you cannot screen the area of interest because of poor positioning of the patient and equipment. this paper will discuss the knowledge, skills and abilities required to undertake theatre radiography in a safe and proficient manner without feeling threatened by the experience. learning objectives: . to understand the key skills required to undertake radiographic procedures in the operating theatre. . to gain an insight into the interpersonal skill required for effective radiographic practice in the operating theatre. prediction of coronary revascularisation outcome represents a major clinical question because a large number of medical and surgical options have become available for ischaemic cardiomyopathy with need to identify more rigorous criteria for patient's selection. the combination in a single examination of function, stress-perfusion and tissue characterisation with t -weighted 'oedema-sensitive' and late-gadolinium enhancement (lge) techniques supported the role of cardiac mr (cmr) as an important technique for the evaluation of patients candidates to revascularisation. besides more "traditional" indicators such as ejection fraction, end-diastolic wall thickness or endsystolic volumes, extent and distribution of myocardial scar depicted with lge has been identified as one of the most important predictors of post-revascularisation outcome with direct influence on functional recovery and on major adverse cardiovascular events (mace) due to the potential induction of arrhythmias from the scar.lge technique has been shown to be superior to nuclear medicine for the assessment of myocardial viability due to the higher spatial resolution (up to -fold greater than spect) and an intrinsic high contrast resolution. a further technique that could be adopted before revascularisation is stress imaging. myocardial ischaemia detected by either cmr adenosine first-pass perfusion or dobutamine-induced wall motion abnormalities has been shown to predict subsequent cardiac death whereas normal stress perfusion showed a high negative predictive value for mace. in conclusions, although as a relatively new diagnostic modality prognostic evidence is predominantly derived from singlecenter studies, cmr is increasingly becoming an important tool for risk stratification of patients before revascularisation, offering indications about outcome and mortality. cardiac valve diseases are an important public health problem, strongly linked to the general increasing age of the western population. the most frequent valve disease is aortic stenosis, for which percutaneous aortic valve replacement (pavr) is currently evolving to a feasible alternative therapy for the classical surgical approach in high-risk patients. nevertheless, careful evaluation of all aspects of this new approach is still required to avoid uncontrolled diffusion. imaging plays a key role in selecting patients who may be eligible for pavr, focusing on the evaluation of leaflet anatomy, severity of valve dysfunction, haemodynamic consequences and potential problems in the access route. while echocardiography is commonly used for both the anatomical and functional evaluation, multidetector ct (mdct) has important intrinsic advantages providing state-of-the-art d imaging with a high spatial resolution over a large anatomic coverage. during this course, we will discuss the advantages and disadvantages of mdct compared to other imaging modalities. the relevant anatomy of the aortic valve and annulus will be reviewed, with emphasis on correct alignment of the imaging planes, and its implications for correct reporting of the necessary measurements targeted at the clinicians need. furthermore, mdct scan protocol design will be reviewed, focusing not only on optimal implementation of common scan parameters but also on the need of ecg-triggering and its consequences. finally, we will present the current status of evidence on using mdct in pavr procedures, and discuss future challenges and perspectives. learning objectives: . to understand how to optimise the imaging protocol for aortic valve imaging. . to learn how to report the findings and what to include in the report. . to understand the impact of this approach on patient management. the need for quantitative image analysis in radiology is universal: computer-aided detection, segmentation for d volume visualisation, image enhancement, pattern recognition, etc. all need effective, robust and preferably generic (not 'ad hoc') algorithms for the computer. how to design such algorithms? a good inspiration source is the functionality of the visual system, the best investigated brain structure today. in this talk we will explain how we think the brain calculates features in images, why the retina measures at a wide range of resolutions and how we can exploit this. the visual system is strongly adaptive and self-learning. new optical recording techniques have given new insight in how the cells in the visual cortex are functioning. we will go through these functionalities step-by-step. what we discover is quite amazing. we recognise huge amounts of filter banks in the first stages of vision: many filters analyse each pixel of the incoming image at a range of scales, orientations, derivative order, for each colour, and also as a function of time. extensive feedback loops take care of optimal settings locally. we programmed these filters into the computer, and were able to build many interesting applications for computer-aided diagnosis: detection of catheters at seriously reduced levels of x-ray radiation dose, automatic polyp detection, quantitative analysis of ischaemic heart ventricle deformation, breast cancer cad, pulmonary emboli cad and analysis of in vivo microscopy images now so abundant in modern life-sciences research. postgraduate.educational.programmethe gi tract are leading causes but any tumour involving the abdominal cavity or haematological malignancy may be implicated. during treatment with chemotherapy or radiotherapy acute abdominal symptoms are common as a consequence of direct toxicity. imaging must be used as an adjunct to clinical assessment. patterns of abdominal involvement are often modified in the later phases of disease. gi tract obstruction may be due to progression or the mechanical consequences of prior surgery and is more commonly at multiple levels. perforation or fistulation may occur with either progressive disease or tumour lysis in response to therapy. cancer does not protect against the common inflammatory conditions but poor host response and the use of steroids may mask signs leading to extensive abnormality at diagnosis. bone marrow suppression increases susceptibility to infection including neutropenic enterocolitis and haemorrhage. bone marrow transplantation and consequent graft versus host disease is a potent cause of acute abdominal symptoms. ct is the major imaging technique although mr has an increasing role particularly in the assessment of the female pelvis. major determinants when considering the differential diagnosis include; the nature and initial stage of the primary, any prior anatomical modification (surgery), the nature and relationship to current treatment and the bone marrow status. this workshop will begin by describing the current limitations of mri in evaluating prostate cancer patients and will show how to overcome these with advanced imaging techniques including diffusion weighted mri (dw-mri), mr spectroscopic imaging ( h-mrsi) and dynamic contrast enhanced mri (dce-mri). the emphasis will be on the use of processing tools that are readily available for clinical practice. we will describe how to analyse images and provide a scheme for reporting findings back to surgeons/oncologists. we will show that using more than one mri tool improves imaging performance and that the relative importance of each technique remains unresolved. we will demonstrate new indications for advanced mri in prostate cancer patients. abdominal hernias are common in daily practice and can be divided into: external or abdominal wall hernias, internal hernias and diaphragmatic hernias. external hernias typically involve protrusion of abdominal contents through a defect in the abdominal wall. internal hernias involve protrusion of viscera through congenital or acquired defects in the mesentery or the peritoneum. diaphragmatic hernias involve protrusion of abdominal contents into the chest. among these conditions, the diagnosis of internal hernia is the most challenging. bariatric surgery and liver transplantation with biliary-enteric anastomosis, with the roux-en-y loop placed in a retrocolic position, are recognised predisposing factors for internal hernia development. symptoms of hernia are nonspecific and vague, and clinical and radiologic diagnosis can sometimes be challenging. immediate diagnosis is mandatory because misdiagnosis can be complicated by bowel obstruction, volvulus, strangulation, incarceration, or trauma. mdct with its multiplanar capabilities is widely believed to facilitate this diagnosis, because it is able to delineate hernia type, location, size, and shape and is particularly useful to diagnose unsuspected hernias. it also allows distinguishing hernias from masses of the abdominal wall, such as tumours, haematomas and abscesses. as a result, knowledge of the ct findings of abdominal hernias may allow early and more accurate diagnosis, with a resultant decrease in the mortality rate. the title of this talk limits the discussion of torsion to the mesentery and omentum. it has to be mentioned, however, that an abnormal twist (torsion) can involve any peritoneal reflection of those suspending and fixating hollow viscera, (e.g. stomach, small bowel, cecum, sigmoid and gallbladder), solid organs, (e.g. spleen, ovaries), or even extraperitonel (extraabdominal) organs such as the testicles, leading to the pathologic entity of volvulus or torsion of the corresponding viscous with its associated (different) imaging findings, prognosis and treatment. moreover, twisting of the mesentery and volvulus can occur secondary to a variety of conditions including congenital anomalies of intestinal rotation with variable prognosis. finally, torsion of the omentum can be either primary or secondary, also associated with different pathology, imaging findings and treatment. consequently, a broad spectrum of abnormalities ensues related to the pathophysiology of torsion which requires a detailed classification, an accurate description and the use of correct terminology making it impossible to be covered within the time limits of a presentation. therefore, focusing the discussion on to abnormal twist of small bowel mesentery and omentum will simplify the approach to avoid overlaps and confusion. this case-based lecture will present typical clinical cases of pelvic pain as well as some unusual but important causes. cases of acute and of chronic pelvic pain, and benign as well as malignant disease will be included. the audience will have the opportunity to participate in case discussion by the use of interactive voting pads. the selection of imaging modality for each clinical presentation and the importance of knowing the clinical findings at the time of the radiological interpretation will be discussed. for each case, the key radiological features will be illustrated. the essential elements of the radiology report and the key information required by the clinician will be discussed. in each case, the differential diagnosis and the need for follow-up imaging will be considered. the key teaching points for each diagnosis will be reviewed. this lecture provides a practical approach to the fundamentals of normal cross-sectional anatomy of the hip. basic knowledge necessary to identify the different portions and contents of the joint anatomy is provided. a special emphasis is on cartilaginous structures, the acetabular fossa, and capsular fibers and ligaments. functional anatomy of muscles and tendons about the hip is reviewed. the location of bursae and their association with adjacent structures is discussed. in the second part of the lecture attendees will learn how to avoid commonly seen pitfalls about the hip. a special focus on anatomic variants of the acetabular labrum simulating disease is given. osseous variants including acetabular ossicles and the superior acetabular notch will be explained and explored. debates about the role of herniation pits referred to as a normal variant are reviewed. the lecture offers an overview of muscular and tendinous variations around the hip joint. pelvic pain is an important part of clinical practice for any clinician who provides health care for women. pelvic pain may be acute, recurrent or chronic. acute pelvic pain (app) rarely lasts more than one month without crisis, resolution, or cure. pain of more than or or months of duration is considered as chronic pelvic pain (cpp) and in many settings may be considered and treated as an illness itself. women who present with app frequently exhibit nonspecific signs and symptoms. diagnostic considerations encompass multiple organ systems, including obstetric, gynaecologic, urologic, gastrointestinal, and vascular aetiologies. as the first priority, urgent life-threatening conditions (e.g. ectopic pregnancy, appendicitis and ruptured ovarian cyst) and fertility-threatening conditions (e.g. pelvic inflammatory disease and ovarian torsion) must be considered. adolescents and pregnant and postpartum women require unique considerations. ccp is a common and significant disorder of women, with a prevalence of . - %. many disorders of the reproductive tract, gastrointestinal system, urological organs, musculoskeletal system, and psychoneurological system may be associated with ccp, the most common being endometriosis, adhesions, irritable bowel syndrome and interstitial cystitis. ultrasonography should be the initial imaging test because of its sensitivities across most aetiologies and its lack of radiation exposure. computed tomography (ct) serves an important role in patients with nonlocalizing symptoms, an indeterminate us evaluation, or in patients who require a wider search beyond the field of view available with us. magnetic resonance imaging is an extremely useful second-line modality for problem solving after us or ct. fat-suppression sequences help to establish fat-containing lesions and increase the conspicuity of inflammatory lesions. different sequences will be discussed. mr is rated below us and ct for evaluation of acute pelvic conditions, but is excellent alternative when administration of iodinated contrast media or radiation exposure is undesirable, especially in young or pregnant patients. long imaging times, limited access and cost are major drawbacks of mri. imaging findings of various gynaecologic and nongynaecologic conditions causing female pelvic pain will be presented. an overview of the interventional radiology management of painful osseus metastases will be presented. consideration will be given to patient, lesion and treatment modality selection (including radiofrequency ablation, cryoablation, microwave ablation, laser ablation, ethanol ablation, mr focused ultrasound ablation, cement osteoplasty, transarterial embolisation and combinations therein, e.g. combined radiofrequency ablation and cement osteoplasty). the evidence supporting these techniques and patient outcomes will be reviewed. molecular imaging -defined as the non-invasive assessment of biological mechanisms at molecular and cellular level -will play a major role in future disease diagnosis and treatment planning. in this context, the aim of this session is to introduce in simple terms several major research areas in molecular imaging and to discuss their future potential for clinical radiology. the differential diagnosis of hip pain is broad and includes intra-articular pathology, extra-articular pathology including referred pain from the spine, and mimickers including groin pain and pain from the joints of the pelvic ring. the diagnosis of all causes of hip pain/pathology has improved with greater use of magnetic resonance imaging (mri) to complement traditional investigations. in this session pathology involving the hip and groin will be reviewed in a systematic way which will outline an approach to the hip that will allow the reader to maximise their diagnostic ability. the review will include evaluation of bone marrow disorders such as avascular necrosis and transient osteoporosis and intra-articular pathology including labral tears and femoroacetabular impingement (arthritis and traumatic fractures will not be reviewed). extra-articular diagnoses such as bursitis, groin pain (osteitis pubis and adductor/ gracilis dysfunction) and common muscle and tendon injuries will then be reviewed. today, many procedures of bone and joint are performed under imaging control. minimally invasive procedures require less resources, time, recovery, and cost, and often offer reduced morbidity and mortality, compared to other modalities. many percutaneous techniques are available. some aim to treat pain and consolidate bone (cementoplasty). others ablate or reduce the tumour (chemical and thermal ablation techniques). the interventional radiologist with an efficient imaging-guided technique (flat panel fluoroscopy, ct, and mri) can increase the precision of the above-described procedures allowing an improvement of the results and reduction of the complications. furthermore, the presented interventions are carried out either on an outpatient basis or with hours hospitalisation which contributes to the reduction of overall costs and presents a major advantage for patients of working age. some of these minimally invasive procedures can be considered as alternatives to surgery without excluding further surgical options if necessary. back pain is one of the most common complaints, it is estimated that almost % of working adults will experience it in any given year. diseases of peripheral skeleton may produce painful symptomatology as well. some of the algogenic structures of the spine and peripheral skeleton are lumbar intervertebral discs, facet joints and atlanto-axial/occipital joints, sacroiliac joints, nerve root dura, periosteum, ligaments, fascia. among several aetiologies which can involve these structures, spine degenerative pathology plays the leading role. nowadays, several different interventional techniques are available, each one with its specific target of action. with the right indication each of them has been proved to be effective as painkilling modality. herein, we present an up to date comprehensive overview on the interventional techniques available with their own indications. small airways and small airways diseases (sad) have attracted a lot of interest that has resulted in a large number of publications since the early s. despite the several histopathological and clinical subtypes that have been described diagnosis is not always obvious. the introduction of thin section ct and the fact that this ct technique is able to depict signs of sad has not only renewed interest but has also improved insights in these disorders. in this session the direct and indirect ct signs of sad will be presented and the ct techniques that can improve their detection will be explained. correlations between pathological findings and the presence of these ct signs will be made and diseases that can mimic these ct findings will be discussed. schemes of classification of sad based on clinical and pathological findings will be discussed briefly, but most attention will be given to the radiological classification, which is based on the recognition of the direct and indirect signs of sad. algorithms that help to identify the cause of sad will be presented. finally, the use of ct and also of mr as a tool to examine the structure-function relationship in sad will be discussed. it will be shown how careful interpretation and software tools can help to generate data about disease extent and global and regional lung function. the ability to recognise direct and indirect signs of small airways disease on hrct has led to renewed interest in these elusive disorders. the "purest" of these diseases is constrictive obliterative bronchiolitis which is manifested on hrct by the indirect sign of a mosaic attenuation pattern; the differential diagnosis for mosaicism and an algorithm that helps to identify the correct cause of small airways disease will be presented. the necessity for routine expiratory hrct to make the diagnosis of obliterative bronchiolitis is controversial and will be discussed jointly whole body fluorescent imaging and bioluminescent imaging are now widely applied in small animals to study all kinds of biological and molecular processes like i.e. gene expression, tumour progression and metastasis, apoptosis, inflammation, angiogenesis, proteolysis and to follow trafficking, differentiation and fate of cells (i.e. stem-, immune-and tumour cells). this has been done mainly by using gene reporters expressing fluorescent proteins or luciferases. recently new mutated red shifted fluorescent proteins (with better light penetration and less absorption and autofluorescence) and codon optimized and mutated luciferases have been developed making optical imaging more sensitive and offering the possibility to use dual gene reporters. apart from new "smart gene reporters" there has also been a great development in injectable near infrared fluorescent (nirf) probes, especially for tumour detection. these nirf probes can either be targeted or enzyme-cleavable. these new developments has opened up the possibility to apply nirf imaging in the clinic especially to image tumour tissue and to identify sentinel lymph nodes during operation. the assessment of the tumour-free margin during cancer surgery is critical to completely remove the tumour and improve the prognosis of the patient. by injecting a tumour specific nirf probe, tumour tissue and local metastases can be visualised in real-time during operation using a dedicated nirf camera system. we now already use nirf imaging in the clinic to detect the sentinel lymph node (sln) in several types of cancers. in the current presentation preclinical and clinical applications of nirf imaging in image-guided surgery will be discussed. targeted ultrasound contrast agents have opened up the door for molecular imaging with sonography. these contrast agents, which consist of encapsulated gas microbubbles, are coated with antibodies or specific ligands. injected into the circulation, microbubbles are retained in diseased tissue where they can be detected and quantified by different approaches such as "semiquantitative -d" or "quantitative -d" imaging techniques. due to their size, microbubbles behave similar to red blood cells and remain within the intravascular space. therefore, the disease process must be characterised by specific molecular changes on the surface of the endothelial cells to be assessable by ultrasound. several angiogenic markers such as vegfr , α v β -integrins, icam- and vcam are known to be overexpressed by the endothelium in neoplastic, inflammatory and vascular diseases. thus, molecular ultrasound imaging seems perfectly suited to detect these markers and monitor changes which might occur during treatment response or disease progression. today, targeted ultrasound contrast agents are becoming a routinely used preclinical tool and the first application of specific microbubbles in a clinical scenario is expected for the near future. this talk will introduce into the principles of molecular imaging with ultrasound. based on recent studies, basics of tumour biology, potential endovascular targets, synthesis of molecular probes and different imaging approaches for a preclinical and clinical application of molecular ultrasound will be discussed. in this rc, a general insight of imaging in epilepsy, including indications, protocols, and the most common neuroradiological conditions to be identified, will be covered. particular interest will be given to tumours as a cause of epilepsy, and to their more relevant neuroradiological aspects. finally, the contributions of different imaging techniques in the specific context of epilepsy surgery will be reviewed. brain tumours are a common cause of epilepsy more often in adults, less in children.tumours detected in patients with chronic epilepsy are predominantly located in the brain cortex area, affecting the temporal lobe most often. any benign or malignant brain tumour can be responsible for seizures, but some are more frequently associated with epileptic symptoms. low-grade astrocytomas, oligodendrogliomas, gangliomas, dysembryoplastic neuroepithelial tumours (dnets) and glioblastomas multiforme are the tumours significantly often presenting with seizures in adult population. brain tumour-related epileptogenesis in not fully understood yet, but one can list a number of factors playing an important role in this process, including: disruption of physiological neuronal structure, tumour affection on the release of neurotransmitters and abnormal electrical activity of the brain. different imaging techniques are widely used for evaluation for neoplasms in epileptic patients. ct is reserved for acute conditions, one must remember to exclude other possible aetiologies of seizures like haemorrhage, trauma or inherited malformations. mri remains the gold standard in imaging investigation of patients with epilepsy. conventional pre-and post-contrast se sequences are mandatory to perform in every subject. newer and more sophisticated techniques such as diffusion-weighted imaging (dwi), functional studies (fmri), spectroscopy (mrs) and pet are helpful in qualification for surgery and preoperative functional mapping. mri has become established as the modality of choice for preoperative local staging of rectal cancer. the most important general advantages of mri compared to other crosssectional imaging modalities are the soft tissue contrast resolution between the propria muscle layer of the rectum and the perirectal fat, the ability to visualise the different pelvic compartments including the visceral pelvic (mesorectal) fascia and the surrounding tissues in the pelvis. high resolution t -weighted sequences sagittal, transaxial and perpendicular to the tumour is the basic standard for morphological evaluation of the tumour, the distance of the tumour to the anal verge and for evaluation of extramural extension and the distance to the circumferential resection margin. presence of other adverse features such as extramural venous invasion and local lymph node metastases is also noted. the mr-images are ideally demonstrated by the radiologist in a local multidisciplinary conference to make sure that the information is used to select the best possible treatment for the patient. when neo-adjuvant treatment is administered, mri is usually performed both before and after treatment to assess treatment response. when planning surgery, both the pre-as well as the post-treatment images should be available for surgical planning. finally in this lecture, the potential benefits of t compared to . t for pre-operative imaging of rectal cancer as well as the present role of additional techniques, such as diffusion-weighted imaging (dwi) and specific contrast agents that have been evaluated for assessment of rectal cancer will be addressed. locally recurrent rectal cancer is the main concern after rectal cancer surgery and has long been regarded as a rarely curable disease. patients were treated palliatively, and subsequent median survival was months and the -year survival rate was %. however, during the past - years, more patients were considered candidates for curative treatment due to better treatment options. patients with distant recurrences from colorectal cancer, especially those in the liver or lung, have improved chances for cure with better imaging, better surgery and alternative minimal invasive treatment. in the follow-up after colorectal cancer surgery it is thus important to detect recurrences at an early stage. besides cea, imaging is often used as a surveillance tool. it is still unclear which is the most cost-efficient (imaging) tool for monitoring distant and local recurrences. this lecture aims at providing the evidence for surveillance by imaging and reviewing the guidelines for the detection of recurrences after colorectal cancer surgery. it will also discuss the role of mri for establishing resectability of locally recurrent rectal cancer and the imaging patterns and features of recurrent disease. its strength comprises a high sensitivity in detecting coronary stenosis > % and an excellent negative predictive value. the stringent limitation of cta lies in its confinement to anatomic grading of coronary stenosis and a lack of information regarding whether a stenosis causes reversible myocardial ischaemia indicating the need for coronary revascularisation. recently published data in animals and humans indicate that myocardial computed tomography perfusion (ctp) imaging is feasible, promising and accurate. the advantage of cta is the comprehensive evaluation of coronary arteries and myocardial perfusion defects from the same datasets, which permits both visualisation of coronary anatomy and physiology.further, ct provides information about regional and global myocardial function. the aim of this course is to understand basic principles of ct perfusion and functional imaging, to learn "how-to" perform ctp and comprehensive cta/ctp scans, to review current scientific evidence and to discuss potential clinical applications. nuclear medicine tests (spect and pet), mri and more recently mdct have been involved in myocardial perfusion imaging. in clinical practice, perfusion analysis is routinely performed with qualitative or semiquantitative assessment, both based on relative evaluation of uptake or enhancement of myocardium, considering a remote region as normal. however, the assumption of part of myocardium as normal can be wrong and cause false negatives. absolute quantification has been introduced mostly with pet perfusion imaging (water, ammonia and rubidium), but its incremental value for the clinical decision making has not been widely investigated. more recently, mr perfusion imaging has been used for quantitative analysis with different technical approach. there are some clinical scenarios where quantification can change the clinical interpretation: . multivessel coronary artery disease (cad), . balanced multivessel cad, . exclusion of cad in symptomatic patients, . microvascular disease, . revascularised patients. however, there are still some limitations in the use of absolute quantification: first, the setting of cutoff value for normal or abnormal perfusion; second, some clinical situations, such as heart failure patients, where perfusion is reduced and heterogeneous. finally, studies concerning the prognostic value and the cost-effectiveness are needed. epilepsy is a common disorder with a prevalence of up to % in the general population. epilepsies are broadly classified into generalized and focal. though most generalised seizures are controlled pharmaceutically, % of focal seizures are medically intractable. in this subset of patients, the overall sensitivity of mri in identifying responsible substrates is approximately %. the purpose of neuroimaging in epilepsy patients is to identify underlying structural abnormalities that require specific treatment (usually surgical) and to aid in formulating a syndromic or aetiologic diagnosis. in this presentation imaging findings of the most common non-neoplastic lesions responsible for focal epilepsy, namely: a. hippocampal sclerosis, b. malformations of cortical development, c. vascular abnormalities and e. gliosis, will be discussed along with their differential diagnosis and pertinent imaging pitfalls. since routine mr imaging is suboptimal in identifying epileptogenic substrates, imaging should be tailored accordingly. hippocampal sclerosis, the most common cause of mesial temporal lobe epilepsy, is best demonstrated when the temporal lobes are imaged with thin sections in coronal plane perpendicular to the longitudinal axis of the hippocampus. inversion recovery sequences best demonstrate morphology and volume loss in the hippocampus, mammilary body and fornix. t and flair images best demonstrate the increased signal due to gliosis. for malformations of cortical development flair is useful in assessing hyperintense signal. t gradient volume sequences can demonstrate subtle developmental malformations. finally, because many epileptogenic lesions are subtle and easily overlooked, a systematic diagnostic approach to mri interpretation in the clinical setting of epilepsy is helpful and will be discussed. epilepsy surgery is an effective and safe therapy for selected patients with intractable localisation-related epilepsy. when morphological mri fails to reveal focal, structural pathology (e.g. tumour, dysplasia, etc). as the putative aetiology for the seizures, other modalities may be taken into account. in the absence of structural pathology it is essential to identify the epileptogenic zone as exactly as possible in order to (a) increase the chance of a good outcome (reduction or termination of seizures) and (b) limit post-surgical sequelae. this lecture will cover some modalities that may add valuable information in this process. the need to individualise the pre-surgical evaluation and the concept of a multimodality epilepsy protocol will be discussed. the main focus will be on spect (single-photon-emission computed tomography), functional mri, pet (positron-emission tomography) and the added value of co-registration on morphological mri (e.g. siscom). briefly, ictal spect may help to identify focal areas of hyperperfusion (ictal zones). functional mri is used for several reasons. one is to determine language laterality (dominant hemisphere); another is to localise eloquent cortical structures to aid the planning of the surgical approach. pet may detect hypometabolic areas corresponding to areas involved in epilepsy. mrs (magnetic resonance spectroscopy), meg (magnetoencephalography) and intradural eeg-registration and stimulation will be mentioned. the term epilepsy covers a wide spectrum of symptoms and underlying etiologies. an essential part of the work-up of patients with epilepsy includes the radiological examination. in recent years more sophisticated methods radiological methods have emerged and the discussion will focus on how these new advanced techniques may help finding underlying causes and be of help in the pre-surgical work-up. excellence in teleradiology: key issues in workflow management j. schillebeeckx; bonheiden/be (jan.schillebeeckx@imelda.be)the healthcare market is undergoing significant change. the market is evolving from a provider centric to a patient centric model, requiring relevant data to converge at patient level in a timely and structured fashion. the push towards setting up collaborative networks for radiology is strong in most european countries. the most important challenge for teleradiology is to ensure that it develops in a manner that benefits patient care and ensures overall patient safety, and does not in any way reduce the quality of radiology services provided to the citizen. therefore, these collaborative networks require more than just it infrastructure with it support. but as important is case management, workflow management and the administrative and management support that provides all the stakeholders with operational, analytical and statistical qa reports. through a centrally operated hub, the radiology workflow is optimised to ensure throughput of cases, involving radiologists in the network, with the difference that only the services are provided by the hub to the network, not the medical acts, which remain in the hands of the radiologists. learning objectives: . to learn about the technical needs of a teleradiology infrastructure adapted to a distributed environment. . to understand that teleradiology involves much more than just technology.optimising the workflow and time management are also important. . to appreciate how creating collaborative networks can improve the efficiency of radiology procedures and bring improved work/life balance to radiologists. to make teleradiology an integrated part of clinical radiology, it must change from being a provider of radiology reports into a close collaboration with the client radiology department. there are many different aspects on workflow in a clinical setting and the teleradiology service should adapt to these specific needs in order to make a seamless collaboration. however, there are advantages with the global aspects of teleradiology that could improve diagnostic accuracy and efficiency in the clinical setting that should be woven into the collaboration. teleradiology services have developed substantially over the last few years from limited use between hospitals and tertiary care centres for second opinions and patient transfer to the international provision of reporting services. there is no doubt that teleradiology provides a valuable service in some circumstances, but it also has a number of inherent limitations regarding the proper provision of imaging services to the patient and therefore may increase risks for the patient. we will highlight the problems that have arisen and reiterate key parts of the guidelines which were developed for the benefit of patient care. this lecture will demonstrate a multimodality approach to the imaging of salivary colic. the relevant us and mri salivary anatomy will be highlighted and the ultrasound, computed tomography and magnetic resonance appearances of salivary grafts. respectively. graft sclerosis develops in % of nonoccluded venous bypass vessels after years and in % after years. this sclerosis causes more than % luminal narrowing in approximately half of the affected vessels. non-invasive imaging of coronary bypass grafts by md-ct require information about the operative procedure. with the increasing implementation of slice ct scanners and beyond, it is possible to scan the heart and the full anatomic extent of grafts with sub-millimeter slice-thickness within a single breath-hold. when analysing the grafts, three graft segments should be assessed: the origin or proximal anastomosis, the body of the graft and the cardiac anastomosis, either single or sequential. recent studies have shown that graft patency and the presence of significant graft stenosis can be assessed with an accuracy of % using most recent md-ct technology ( / slice ct or dual source ct). the assessment of native coronaries with respect to the progression of cad may still be problematic in cases with severe calcifications of the native coronary arteries. in these cases, mr perfusion imaging in combination with cine and late gadolinium enhancement (lge) imaging may be helpful in detecting newly developed, stress-induced myocardial ischaemia. teleradiology services have developed substantially over the last few years from limited use between hospitals and tertiary care centres for second opinions and patient transfer to the international provision of reporting services there is no doubt that teleradiology provides a valuable service in some circumstances, but it also has a number of inherent limitations regarding the proper provision of imaging services to the patient and therefore may increase risks for the patient. we will highlight the problems that have arisen and to reiterate key parts of the guidelines which were developed for the benefit of patient care. it is now well understood that teleradiology cannot be considered just as 'telereporting', i.e. the simple interpretations of images remotely acquired and sent as a message in the bottle. teleradiology procedures, being medical acts, must ensure the full involvement of the interpreting radiologist in all phases of the well established practice of diagnostic imaging: appropriateness check, personalised acquisition protocols, access to clinical history and prior imaging examinations, communication with referring physician and patient. any obstacle that teleradiology causes to these activities may put our patients at risk (and therefore may expose us at liability suits). the presentation will describe the organisational as well as technological remedies for reducing such risk. for several decades, monte carlo simulation has been recognised as a powerful technique to simulate the transport of radiation in media, as well as to provide solutions for calculations too complex for classical approaches. several general purpose codes and databases providing particle interaction cross-sections have been developed, and with the increase in computing power, simulation has become more and more popular within the field of medical imaging. using monte carlo simulation methods, the different steps involved in the formation of a medical image can be analysed in detail, and the whole system optimised. a topic that has deserved particular attention in the past is the simulation of x-ray tubes and x-ray spectra, which is the first step in the simulation of a medical imaging system. when simulating an x-ray tube, various complex phenomena need to be taken into account, such as the electron multiple scattering, bremsstrahlung interactions, characteristic x-rays emitted from the k-shell and auger electrons emitted during a photon interaction. together with the simulation of the target material and the filtration of the x-ray beam through permanent (e.g. a be window) and added filtration (e.g. al, mo, rh, ag, etc)., this technique can lead to a good estimation of the emitted x-ray spectrum, a task which is hard to achieve with experimental methods. this review will provide an overview of the basic knowledge necessary to start the simulation of an x-ray tube as well as of how to accelerate the calculations. emphasis will be given to mammographic x-ray tubes. breast cancer screening and diagnostic imaging, as all clinical imaging, are increasingly multimodality. a number of new imaging modalities have been developed, including digital breast tomosynthesis and dedicated breast ct. they have shown promise in early studies; however, their technological complexity present obstacle for optimisation. the ultimate technology tests are clinical trials, which are, however, challenging, particularly for breast cancer screening, as large studies are needed due to the small number of detected lesions. clinical trials are costly, long, and they involve repeated exposure of women to radiation. as an alternative, we have been developing virtual clinical trials, based upon our virtual breast phantoms and simulation of phantom images. this talk will describe the development of anthropomorphic computer breast phantoms, and illustrate their use in the analysis of phantom digital mammography and digital breast tomosynthesis images. different currently used phantom designs will be compared in terms of their flexibility and realism. specific phantom requirements related to different imaging modalities will be emphasised. the role of phantoms in tissue-specific analysis of the radiation dose during mammography will be discussed. in addition to the anthropomorphic phantoms for breast imaging, the use of virtual patients in various clinical imaging disciplines will also be illustrated. learning objectives: . to learn about requirements for anthropomorphic phantoms (virtual patients). . to understand the value of anthropomorphic phantoms for breast imaging. . to compare advantages and disadvantages of several types of anthropomorphic phantoms. . to learn how to estimate typical patient doses from simulations with virtual phantoms.colic will be shown and techniques for optimising the imaging of salivary colic given. the use of us, ct, mri, and both mri and conventional sialography in the imaging of salivary colic will be discussed. the role of interventional sialography and minimally invasive techniques in benign salivary gland obstruction will be demonstrated. trigeminal neuralgia is defined as recurrent episodes of lancinating pain most common in the second (v ) or third division (v ) of the trigeminal nerve. the pathogenesis is a neurovascular conflict by an artery or vein associated with focal demyelination of sensory fibers at the glia-schwann cell junction. apposition of demyelinated fibers induces abnormal generation and transmission of impulses. mr imaging is based on high-resolution d sequences: ciss/fiesta/ d t space with coronal and sagittal oblique mpr and a d tof intracranial sequence with axial and coronal thin mip reconstruction to delineate the course of vessels along the trigeminal nerve form the pons, and glia schwann cell junction to the gasserian ganglion. the brainstem and brain are assessed by t , flair and d isotropic gd-enhanced t sequences; the viscerocranium is examined by a noncontrast and coronal t gd fat suppressed sequence. analysis of images is focussed on recognition of displacement and distortion of the proximal trigeminal nerve by the sca, rarely by the aica, ba or petrosal, pontomesencephalic or peduncular vein. correlation of the circumferential site of distortion at the root entry zone with the somatopic representation of fibres increases the, in general, low specificity of the neurovascular contact. ruling our compressive tumours, neoplastic infiltration, inflammation or demyelination and delineating a neurovascular conflict stratifies patients for potential microvascular decompression in case of failure of medication. the purpose of this lecture is to provide an overview of the key imaging features of painful swallowing with or without associated mucosal pathology. in the presence of a mucosal lesion, painful swallowing is most often caused by infectious, neoplastic or traumatic lesions of the pharynx itself, whereas in the absence of mucosal alterations, painful swallowing is the result of functional disorders (dysfunction of the cricopharyngeus muscle), infectious, inflammatory or neoplastic diseases affecting adjacent neck spaces (retropharyngeal and paraphyrngeal space), neurologic impairment (glossopharyngeal neuralgia), carotidodynia and eagle's syndrome. the indications for ct, mri, us and videofluoroscopy will be reviewed and their respective role in the detection and precise description of the underlying cause. major emphasis will be put on how to report the findings in a comprehensive way. learning objectives: . to recognise the most common causes of painful swallowing in patients with a normal pharynx at clinical examination. . to review the role of different imaging techniques in the diagnosis and treatment of painful swallowing. . to review the key imaging techniques in the diagnosis and treatment of painful swallowing. . to review the key imaging features of the most common causes of painful swallowing as seen with the respective imaging techniques.the typical hrct features of interstitial lung disease are ground-glass opacity, consolidation, pulmonary nodules, tree-in but sign, bronchiolar wall thickening, mucoid impaction, air trapping, septal thickening, mosaic perfusion and honey combing. most frequent diseases in children to be dealt with are bronchiectasis, cystic fibrosis, asthma, constrictive bronchiolitis, bronchiolitis obliterans and extrinsic allergic alveolitis and they will be demonstrated with use of a systematic approach. during the lecture we will present and validate methods to simulate radiographic images with the monte carlo software mcnp/mcnpx in a time efficient way. we will start the lecture by introducing three image detector models that can be used in mcnp/mcnpx. the first detector model that will be presented is the standard semideterministic radiography tally, which has been used in previous image simulation studies. furthermore, we will present two alternative stochastic detector models: a perfect energy integrating detector and a detector based on the energy absorbed in the detector material. the image detector models will be validated by comparing calculated scatter-to-primary ratios (sprs) with published and experimentally acquired spr values. subsequently, we will introduce a method to modify the images, generated with the mcnp/mcnpx image detector models, for the physical characteristics of a computed radiography (cr) imaging systems. the method presented in this lecture takes into account the signal intensity variations due to the heel effect along the anode-cathode axis, the spatial resolution characteristics of the imaging system and the various sources of image noise. to demonstrate the accuracy of our model we will compare the threshold-contrast detectability in simulated and experimentally acquired images of a contrast-detail phantom. thoracic trauma in children is most commonly seen in a polytrauma context, and is associated with significant morbidity and mortality. blunt trauma accounts for the majority of cases, often resultant from motor vehicle accident and pedestrian crash. common thoracic injuries include pulmonary contusion, rib fractures, pneumothorax and haemothorax. diaphragmatic and mediastinal injuries, such as aortic rupture and tracheobrocheal tear, are rare but potentially life threatening. different patterns of injury are seen in children due to anatomical and physiological differences, and these should be recognised. chest radiography is the first and most important imaging modality. mdct allows accurate diagnosis for most traumatic injuries, and is usually performed for severe chest and/or polytrauma. adapted paediatric protocols are essential. foreign body inhalation is a common paediatric domestic accident, with potential serious or even fatal consequences. clinical history is the key for the diagnosis. with a definite history, bronchoscopy is the modality of choice for both diagnosis and treatment. however, in many cases the aspiration event is not witnessed and the diagnosis is often delayed or overlooked. the majority of aspirated foreign bodies are non-opaque and imaging findings largely result from complete or incomplete airway obstruction. chest radiography is the first imaging modality. expiratory films (or lateral decubitus or fluoroscopy) are very useful to demonstrate air-trapping. chest mdct offer excellent details of the tracheobronchial tree and pulmonary parenchyma, and is usually reserved for more complex cases and/or long-standing foreign bodies. high-resolution ct of the chest is the imaging technique of choice for the evaluation of most infiltrative diseases of the chest. in children dose-adapted protocols should be used and recommendations for suitable protocols will be given in the course. the mediastinum is a region of the thorax that separates both lungs and communicates with the neck and the abdomen. these two anatomic features are very important to understand the behaviour of some diseases and their radiological manifestations. most asymptomatic mediastinal masses are benign, while clinical symptoms might raise the possibility of a malignant lesion. imaging plays a very important role, especially ct and mri. in the presence of a mediastinal mass we must ask ourselves two questions: . where is the mass located? the classic divisions of the mediastinum in compartments remains very useful, because it narrows the differential diagnosis. . is the lesion cystic or solid? pure mediastinal cysts are benign and their characterisation depends on their location. thymic cyst (anterior mediastinum), bronchogenic and duplication cysts (middle mediastinum) and menyngoceles (posterior mediastinum). solid lesions may be benign or malignant while some lesions may have a cystic component. solid lesions of the anterior mediastinum are usually thymomas, germ cell tumours or lymphomas. in the middle mediastinum most masses are of lymphatic origin but we should also include aortic or oesophageal pathology. intrathoracic thyroid usually follows the trachea and thus is situated in the upper-middle mediastinum although posterior and anterior extensions may occur. in the posterior mediastinum most masses are of neural origin. there are some locations that will typically indicate specific diagnosis or a narrow differential. such is the case of the cardiophrenic angle masses, juxtadiaphragmatic lesions and thoracic inlet pathology. colorectal cancer is common. approximately , new cases occur each year in the million population of the states which comprise the eu. after lung, it is the second commonest cause of cancer death resulting in approximately , deaths per annum. these relatively high mortality figures are a reflection of the fact that the disease is often advanced at the time of presentation. efforts to reduce mortality, therefore centre on early detection as well as accurate staging. the latter is particularly important in rectal cancer-the commonest site for colonic cancer. detection depends on presentation, which is often protean or non-existent; hence, the introduction of screening programs. typical diagnostic tools include the barium oenema, optical colonoscopy and ct colonography. the limitations of the barium enema have been exposed by optical colonoscopy and it can no longer be advocated in this role. ct colonography, however, rivals optical colonoscopy in the detection of polyps and tumours, although of course has no therapeutic potential. major advances in the treatment of rectal cancer include total mesorectal excision (tme) of the rectum as well as neo-adjuvant therapies such as chemo-radiation. their use depends on highly accurate staging of the primary disease, which can only be achieved by mr examination, although of course ct or pet/ct is required for the assessment of more distant metastatic disease. finally, once treated, it is essential that these patients are followed-up; usually by a regime of colonoscopy and ct. these issues will be discussed in an interactive lecture. decreased opacity of the lung may be a bilateral or unilateral process. if unilateral, it may involve an entire lung, a lobe or a segment. faulty radiological technique must always be excluded. the most common cause of unilateral decreased opacity is a previous mastectomy. bilateral decreased opacity occurs in copd and asthma. it is also caused by decreased blood flow in the lung/s. expiratory films separate the true lung causes from all the others, by demonstrating air trapping. pulmonary nodules are spherical radiographic opacities (solid and subsolid) that measure up to mm in diameter. extremely common in clinical practice, pulmonary nodules, especially small ones under cm in diameter, are a challenge to manage. it is important to identify malignant nodules because they are potentially curable. the first step in assessing a pulmonary nodule on a chest radiograph is to determine that it is indeed a lung nodule rather than a pleural or chest wall abnormality. it is essential to review images from previous examinations, because a solid nodule that remains stable for at least years is probably benign. topics discussed in this talk include the importance of nodule size, growth rate, margin morphology, density (solid, ground-glass and part solid), calcifications or fatty components within the nodules, the significance of cavitations or bubble-like densities, enhancement patterns at dynamic contrast-enhanced ct, and findings on positron emission tomography (pet). the talk also covers the current guidelines for the management of incidentally detected nodules (solid and subsolid). this presentation has the purpose to familiarise radiologists with the spectrum of mesenteric and omental cystic masses, demonstrating the additional correlation with the underlying pathology. cystic masses of the mesentery or omentum are not common lesions; however, radiologists should be aware of these entities as well as with other cystic lesion of the abdomen. important tool for the diagnosis is to determine the organ from which the mass originates. common types of mesenteric and cystic masses include lymphangioma, enteric duplication cyst, enteric cyst, mesothelial cyst and non-pancreatic pseudocyst. other entities such as cystic mesothelioma, cystic spindle cell tumour and cystic teratoma could be located in the mesentery also. due to the overlap in the imaging features, not always the final diagnosis could be reached by imaging only and therefore histologic examination is necessary to establish the diagnosis. however, it is important for the radiologist to define the cystic nature of the mass and demonstrate the potential mesenteric or omental origin, targeting to the correct differential diagnosis of the cystic lesion. cancer cells from intraabdominal neoplasms, carried by peritoneal fluid throughout the abdominal cavity, result in widespread metastases in the form of implants, the socalled peritoneal carcinomatosis. the location of implants development is governed mostly by peritoneal fluid circulation and by specific anatomic pathways formed by peritoneal reflections. the most common sites where the peritoneal fluid may temporarily arrested facilitating implantation of cancer cells include cul-de-sac, distal small bowel mesentery, right paracolic gutter, posterior sub-hepatic space, greater omentum and sub-phrenic spaces. the role of imaging is to disclose the presence and extent of the disease -i.e. fundamental in candidates for cytoreductive surgery -to monitor response to treatment and to reveal recurrences. mdct with thin collimation and i.v. contrast material supplemented by multiplanar reconstructions is the primary imaging modality for the investigation of peritoneal carcinomatosis. ascitis, contrast enhanced smooth, nodular, or plaque-like peritoneal thickening, peritoneal nodules, plaques or masses, rounded, ill-defined soft-tissue or cystic mesenteric masses, mesenteric fixation with increased attenuation values and thickening, irregular soft-tissue permeation of omental fat or confluent solid omental masses are the most frequent ct findings of peritoneal carcinomatosis. ct has a sensitivity and specificity between and %, depending on the size/location of implants and examination protocol used. mr imaging employing a post-gadolinium-enhanced d flash sequence with fat saturation may alternatively be used and it is advantageous in cases of diffused layered type of peritoneal/mesenteric involvement. diffusion mri may be of value in post-treatment imaging evaluation. primary solid tumours of the peritoneum and mesentery occur much less frequently than metastatic disease in the same location. however, these rare primary neoplasms (peritoneal mesothelioma, primary peritoneal serous carcinoma, desmoplastic small round cell tumour, mesenchymal tumours, mesenteric fibromatosis or mesenteric desmoid tumour, mesenteric sarcoma, etc.) are often first detected at ct and should be considered in the absence of a known primary organ-based malignancy. ct appearance combined with patient's relevant clinical and demographic data can help narrow the differential diagnosis for a primary peritoneal or mesenteric tumour in many cases; diffuse sheetlike thickening of the peritoneum and stellate appearance of the mesentery at ct or mri are suggestive of primary malignant mesothelioma in older men with high level of asbestos exposure. absence of an ovarian mass is mandatory in suggesting the diagnosis of primary peritoneal serous carcinoma in a post-menopausal woman. desmoplastic small round cell tumour occurs in young men and often presents with a large primary peritoneal mass with calcification. a solid mesenteric mass at ct or mri, regardless of its pre-and post-contrast appearance, occurring in a patient with familial postgraduate.educational.programme embolisation of hcc with drug eluting beads k. malagari; athens/gr (kmalag@otenet.gr)drug eluting beads have proved predictable pharmacokinetics and achievement of higher doses of the chemotherapeutic, prolonged contact time with cancer cells.in addition, research data today have shown response, and tolerance benefit of drug eluting beads compared to conventional chemoembolisation for the more advanced subgroup of bclc -class b patients. for diameters larger than μm dc bead loaded with doxorubicin have proven to be more effective with respect to local response, recurrence rates and time to progression (ttp) compared to bland embolisation with similar diameters. in this session results of studies on dc bead loaded with doxorubicin for the treatment of hcc will be discussed, and guidelines for optimal clinical use will be presented. selective internal radiotherapy j.i. bilbao; pamplona/es (jibilbao@unav.es)selective internal radiotherapy, also called radioembolisation (re), consists in the delivery of beta-radiation to liver tumours using microspheres loaded with yttrium- (y ) that are injected into the hepatic artery or its branches. y is a pure beta-emitting radioisotope, with a limited tissue penetration (average: . mm and maximal: mm) and a half life of hours. y can be either incorporated or labelled into glass or resin microspheres ( µm). once the particles are infused into the hepatic artery, they travel to the distal tumoural arterioles, from where the beta-emissions from the isotope irradiate the tumour. with traditional external beam radiation, doses are limited to - gray (gy) due to the risk of radiation-induced liver disease that may occur with higher doses. with re, tumours can receive a higher dose of radiation due to their preferentially arterial vascularisation and a higher tolerance of the non-tumoural liver parenchyma to this form of radiation. re has shown an encouraging antitumoural activity with a good safety profile in patients with hepatocellular carcinoma, even in the presence of portal vein thrombosis or invasion. local tumour growth control is achieved in the majority of patients although response rates using volumetric criteria are achieved in only - % of patients. in liver-predominant unresectable metastases, there is promising evidence that re combined with systemic chemotherapy significantly extends the time to progression of liver metastases and increases objective response rates as well as enabling patients to receive systemic chemotherapy for a longer period of time. learning objectives: . to learn about the technique, legal and safety requirements in the cathlab. . to understand the diagnostic and interventional procedures before radioembolisation. combined therapies before and after ablation r. lencioni; pisa/it (lencioni@med.unipi.it) image-guided rfa is currently established as the standard of care for patients with early-stage hcc when transplantation or resection is precluded. however, histologic data from liver specimens of patients who underwent rfa as bridge treatment for transplantation showed that the rate of complete tumour eradication is highly dependent on the size and the presence of large abutting vessels. combined percutaneous-transcatheter approaches that aim at increasing the ablation volume by minimising heat loss due to perfusion-mediated tissue cooling have been developed, using either a balloon catheter occlusion of the tumour arterial supply at the time of the rfa or by performing a prior tace. experimental studies in animal tumour models have shown that lowering the temperature threshold at which cell death occurs by combining sublethal heating with cell exposure to trans-arterial chemoembolisation takes advantage of the largely portal vascularisation of liver tissue, while metastatic tissue is supplied almost exclusively by hepatic arteries. the benefit of intra-arterial application of chemotherapeutic drugs is proportional to the first pass extraction of the drug by the target tissue and inversely proportional to the body clearance of the drug. these figures vary greatly with different chemical properties of the drug. intratumoural drug concentration after transarterial application (compared to intravenous application) is approximately x for thp-adriamycine, x - x for fu, x - x for mitomycine, x for cisplatin or oxaliplatin, and x for doxorubicin. several trials support superiority of intraarterial fu over i.v. application in response rate, and partly with a moderate survival benefit. however, with the advent of novel chemotherapeutics (mainly oxalyplatin, irinotecan), response rates of i.v. chemotherapy approached the results after i.a. fu. more recently, i.a. oxaliplatin has shown a % response rate in a multicenter trial on non-responders to i.v. oxalyplatin. also, combinations of i.a. oxalyplatin and i.v. fu and cetuximab have achieved promising response rates as first line therapy. even though intraarterial chemoembolisation alone can achieve promising response rates, the actual survival benefits are limited to date. also, the beneficial effect of additional embolization (over conventional arterial injection) remains largely unproven for a large number of different embolisation agents in hepatic metastases. in an attempt to further increase tumour uptake, chemotherapeutic agents (anthracyclines and irinotecan) have been electrostatically coupled to microspheres. irinotecan-eluting microspheres have been untilized in the treatment of crc metastases in smaller case series. due to the high parenchymal drug uptake, appropriate medications to mitigate postembolization side effects need to be emphasised. while the initial response rates were over % (according to easl), tumour progression was observed within months in the majority of responders, suggesting potential stimulation of angiogenesis at the tumour boarders. potentially, adjuvant antiangiogenic treatment can provide an overadditive effect in these patients.primary bone tumours require both local staging and the identification of distant metastases to guide management. mr imaging is the modality of choice to determine local disease extent and allows excellent depiction of intra-and extraosseous disease. chest ct enables pulmonary metastases to be identified and bone scintigraphy allows evaluation of the presence of bone metastases. the roles of whole body mri and pet/ct in the staging of bone tumours will also be discussed. whilst imaging may allow a narrow differential diagnosis to be reached, histological confirmation of the nature of the lesion is required pre-operatively to plan appropriate treatment. image-guided biopsy may be performed using fluoroscopy, ct, mri and occasionally ultrasound guidance. the relative values of each of these techniques will be covered. percutaneous therapies are increasingly being utilised in the treatment of a number of primary bone tumours. radiofrequency ablation is the method of choice for osteoid osteoma and is now used in the treatment of chondroblastoma. alternatives include microwave therapy, cryotherapy and sclerotherapy. these percutaneous techniques may also be used for local disease control where disease recurrence is encountered. magnetic resonance imaging (mri) has evolved to become the most important diagnostic method for local staging of primary bone tumours and for detecting postoperative tumour relapse. it allows accurate preoperative staging of local tumour extent and helps to obtain adequate safety margins. mri is a noninvasive technique that can be used to obtain information regarding tumour vascularisation, metabolism, and pathophysiology, and allows early assessment of therapeutic effects of cancer drugs. one approach is dynamic contrast-enhanced (dce) mri, which measures tumour vascular characteristics after administration of a contrast medium. mri enhanced with small-molecular-weight contrast agents is extensively used in the clinic to differentiate benign from malignant lesions, as well as to monitor tumour microvascular characteristics during treatment. diffusion-weighted mri (dwi) is a more recent technique and it allows noninvasive characterisation of biologic tissues based on the random microscopic motion of water proton measurement. several studies have shown that dwi allows early detection of tumour response to chemotherapy. the use of water diffusion is a surrogate marker used to distinguish highly cellular regions of tumour from acellular and necrotic regions. whole body diffusion-weighted sequence (wb dwi) is a new promising technique feasible to evaluate multifocal disease. dwi has revealed great potential in the evaluation of patients with cancer or benign disease, as it supplies both quantitative and qualitative information of the whole body. this presentation will focus on the potential role of dwi in combination of dce mri in bone tumours as well as on the possibilities of wb dwi. in this integrated refresher course, the impact of basic and advanced imaging on the entire process, from diagnosis to treatment of bone tumours, will be addressed. diagnosis is based on understanding the imaging features from a histopathologic chemotherapeutic agents is an attractive alternate strategy to increase tumour necrosis. the efficacy of a combination therapy, including rfa plus the intraarterial administration of drug-eluting beads has been recently demonstrated, while the use of intravenously administered, thermally sensitive drug carriers is currently being explored. despite the advances in local treatment, the long-term outcome of treated patients remains unsatisfactory because new tumours emerge in about % of the cases within years. clinical trials evaluating the usefulness of adjuvant molecular targeted therapies with anti-angiogenic and anti-proliferative activity in preventing early recurrence after successful ablation are ongoing. in this integrated refresher course, the impact of basic and advanced imaging on the entire process from diagnosis to treatment of bone tumours will be addressed. diagnosis is based on understanding the imaging features from a histopathologic perspective. staging, biopsy and image-guided treatment require an integration of imaging findings with basic knowledge of surgical-oncological principles, as well as skills. can technically driven development of advanced mr techniques change how we diagnose, monitor therapy and determine prognosis. techniques and procedures that improve patient outcome in a cost-effective way will be identified based on presentations and a panel discussion. a. diagnosis: from radiographs to mri k. wörtler; munich/de (woertler@roe.med.tum.de)the diagnosis of a bone tumour is based on clinical findings, the age of the patient, the location of the lesion, its radiologic appearance, and, if imaging does not allow for a specific diagnosis, its histopathologic features. radiography remains the initial imaging modality for evaluation of the localisation of the lesion with respect to the longitudinal and axial planes of the involved bone, for the depiction of matrix mineralisations, and for estimation of biologic activity by analysing the patterns of bone destruction and periosteal response. ct can add "radiographic" information particularly in regions of complex skeletal anatomy such as the spine, pelvis and shoulder girdle. mr imaging has classically been used to determine the local extent of a bone tumour (local staging). in addition to radiography and/or ct, it can at times also be valuable in establishing the differential diagnosis, especially in cystic bone lesions and cartilaginous tumours. whole-body applications have recently gained importance in demonstrating the presence and extent of bone (marrow) involvement in benign and malignant systemic/polyostotic tumourous diseases. this course reviews the basic principles of diagnosing bone tumours in a multimodality approach (with an emphasis on conventional radiography). the different steps of morphologic analysis as well as the advantages and disadvantages of the individual imaging techniques are illustrated on the basis of pathologically confirmed cases. s a b c d e f g h review: ( ) the biological rationale for using perfusion imaging in brain tumours, ( ) methods available for the imaging of microvascular structure and function in brain tumours. we will discuss the relevant advantages and disadvantages of t versus t weighted acquisition strategies. early diagnosis and treatment of acute stroke is crucial for a favourable prognosis. while non-contrast mri is much more sensitive to ischaemia in comparison with non-contrast ct, perfusion studies make both techniques comparable. ultrafast ct scanners cover most of the brain with perfusion imaging; the scanning is faster and quality imaging results depend less on patient co-operation than in mri. however, mri enables diffusion imaging adding more specific information to the diagnostic process. because of the short time window to eventual vascular intervention, in many institutions ct is the technique of choice. besides depiction of hyperacute stroke by means of diffusion weighted imaging, mri has the advantage of performing a contrast agent-free perfusion study using a promising new technique called arterial spin labelling (asl). thus, an mri perfusion study can be performed even in patients with impaired renal functions where iodine and gadolinium-based contrast agents may be dangerous. perfusion imaging is an emerging non-invasive tool that enables evaluation of brain function via assessment of various hemodynamic measurements such as cerebral blood volume, cerebral blood flow, and mean transit time. these techniques have become important clinical tools in the diagnosis and treatment of patients with cns disorders via evaluation of brain tissue during cerebrovascular diseases, noninvasive histopathologic assessment of tumours, evaluation of neurodegenerative conditions and assessment of the effects of drugs. perfusion imaging is helpful to analyse and assist in judging the biological behaviour (especially haemodynamic features) of central nervous system diseases. qualitative and quantitative information can be obtained to evaluate pathoanatomical structures and pathophysiological changes of the lesions. cerebral blood flow (cbf), cerebral blood volume (cbv), mean transient time (mtt), permeability surface (ps), regional blood volume, microvascular permeability measurements and more information can be obtained for the diagnosis and differential diagnosis of the diseases. advantages and disadvantages of the ct perfusion imaging (ctp) and magnetic resonance perfusion imaging (mrp) in the different diseases will be evaluated in detail. hints and tips for the better applications and postprocessing will also be discussed. techniques for ct and mr, post-processing, radiation r.a. meuli; lausanne/ch (reto.meuli@chuv.ch)brain perfusion can be assessed by ct and mr. for ct, two major techniques are used. first, xenon ct is an equilibrium technique based on a freely diffusible tracer. first pass of iodinated contrast injected intravenously is a second method, more widely available. both methods are proven to be robust and quantitative, thanks to the linear relationship between contrast concentration and x-ray attenuation. for the ct methods, concern regarding x-ray doses delivered to the patients need to be addressed. mr is also able to assess brain perfusion using the first pass of gadolinium based contrast agent injected intravenously. this method has to be considered as a semi-quantitative because of the non linear relationship between contrast concentration and mr signal changes. arterial spin labeling is another mr method assessing brain perfusion without injection of contrast. in such case, the blood flow in the carotids is magnetically labelled by an external radiofrequency pulse and observed during its first pass through the brain. each of this various ct and mr techniques have advantages and limits that will be illustrated and summarised. learning objectives: . to understand and compare the different techniques for brain perfusion imaging. . to learn about the methods of acquisition and post-processing of brain perfusion by first pass of contrast agent for ct and mr. . to learn about non contrast mr methods (arterial spin labelling). brain tumours a. jackson; manchester/uk (alan.jackson@manchester.ac.uk) this presentation will discuss the current 'state of the art' in the use of perfusion imaging techniques in neuro-oncology. the term "perfusion imaging" is commonly used but is in fact a misnomer since perfusion and blood flow are not the only imaging biomarkers of microvascular structure and function in common use. indeed, in oncological applications measurements of proportional blood volume, endothelial capillary permeability or vessel size can be of equal or greater importance. we will reproduction is by spore formation which is prolific and therefore human respiratory tract exposure is almost constant. the spectrum of aspergillus infection includes aspergilloma (mycetoma), chronic pulmonary aspergillosis, chronic necrotising pulmonary aspergillosis (subacute invasive, chronic airway invasive), invasive pulmonary aspergillosis (angioinvasive, non-angioinvasive including acute tracheobronchitis, exudative bronchiolitis and bronchopneumonia), and allergic disease (allergic bronchopulmonary aspergillosis (abpa) and bronchocentric granulomatosis). immunocompromised individuals are particularly susceptible to invasive aspergillosis. the risk factors for invasive aspergillosis include: profound neutropenia, haematopoietic stem cell transplant recipients, solid organ transplantation, potent immunosupressive therapy, prolonged corticosteroid use and aids. invasive aspergillosis can be further subdivided into angioinvasive and non-angioinvasive forms. the imaging features of angioinvasive aspergillosis are characterised on ct by nodules with a 'halo' of surrounding ground glass opacity due to alveolar haemorrhage. in the context of neutrophil recovery, nodules may demonstrate cavitation. airway invasive aspergillosis may manifest as areas of consolidation (bronchopneumonia), nodular 'tree-in-bud' densities on ct (exudative bronchiolitis), or an acute tracheobronchitis with large airway thickening. falling rates are stabilising in europe. tuberculosis is developing new faces due to changes in host cellular immunity, such as hiv infection, immunosuppressive therapy, malignancy and due to multi-drug resistance. chest radiography plays a major role in screening, diagnosis, and response to treatment of patients with tb; however, radiographs may be normal or show only mild or nonspecific findings in active disease. ct and high-resolution ct (hrct) are more sensitive than chest radiography in the detection and characterisation of parenchymal disease, small foci of cavitation, mediastinal lymphadenopathy, and pleural complications. based on these findings, hrct is useful in determining disease activity and plays also an important role in the management of tb. radiological manifestations of primary tuberculosis are lymph node enlargement, airspace consolidation and pleural effusion. the most common findings of post-primary tuberculosis are centrilobular nodules, branching linear and nodular opacities, patchy or lobular areas of consolidation, and cavitation. miliary tuberculosis results from acute haematogenous dissemination of tb bacilli in lungs and other organs and ct-findings consist of innumerable small nodules randomly distributed throughout both lungs. airway tuberculosis is characterised by circumferential wall thickening and luminal narrowing, with involvement of a long segment of the bronchi.sensitive diagnostic tool. radiological semiotics are always useful and often very specific in addressing the diagnosis: some fundamental concepts include the peculiar pattern of vasogenic oedema, which, in opposition to what happens in the brain, tends to involve the central grey more than the peripheral white matter. distribution pattern of the lesion can also often address to aetiological diagnosis. in fact, lesions selectively involving posterior or lateral columns, asymmetrically, and sparing the central grey matter, are more typical expression of demyelinatinginflammatory diseases (multiple sclerosis, adem, devic's disease, les, behcet's disease), while selective and symmetrical involvement of both posterior columns, extended for more than myelomers, and possibly associated with involvement of lateral columns, is typical expression of combined sclerosis (b vitamin deficit). ischaemic lesions are also usually symmetrical, but they involve both grey and white matter, extending to the anterior two-third of the cord, or, sometimes, remain limited to the central grey matter. on the other hand, selective involvement of the anterior horns is typical of poliomyelitis. early stage head and neck cancer can be cured by surgery or radiotherapy. the choice depends on the functional and cosmetic result to be expected, tumour histology, patient's condition and preference, and institutional policy. in advanced lesions, nowadays concomitant chemoradiotherapy is offered, with surgery reserved for salvage. a relatively high locoregional control rate can be obtained, at the expense of acute and late toxic side effects, and a higher incidence of treatment complications. on post-therapeutic imaging studies, treatment-induced tissue changes are often visible; these changes should not be misinterpreted as evidence of persistent or recurrent tumour, or treatment complication. after radiotherapy, the visible changes depend on the radiation dose and rate, the irradiated tissue volume, and the time elapsed since the end of treatment. basically, thickening of the laryngeal and pharyngeal walls, increased attenuation of fat planes, postirradiation sialadenitis, lymphatic tissue atrophy, and retropharyngeal oedema will be seen. these irradiation-induced tissue changes usually appear symmetrical. the acute effects of radiotherapy occur during or immediately after treatment, and usually settle spontaneously. complications of radiotherapy are usually seen months to years after the end of treatment. there is no clear consensus regarding optimal time points for surveillance, but clinical assessments are more frequent in year and are performed over at least - years, during which time most locoregional failures and second primary tumours are detected. ideally for imaging surveillance at least one post-treatment baseline head and neck scan (mri/ct) should be performed at - months, and often closer surveillance is desirable. candidates for salvage surgery after (chemo) radiotherapy undergo a post-treatment scan at - weeks, followed by regular scans ( - months in year ; - months thereafter, the exact time period being tailored to the patient the most recent advances of radiotherapy techniques are characterised by the increased precision with which the radiation energy is released to the target, the reduced collateral damage to adjacent non-neoplastic tissues, and the synergic viral agents are part of the spectrum of organisms which cause community acquired pneumonias. furthermore, they are thought to function as a trigger for bacterial infections in the hospital or health care setting. in addition, viruses play a significant role as causative agents for infections in the immunocompromized host. within the last years, outbreaks of viral infections have challenged regional, national and even global health care systems, have effected thousands of individuals and have resulted in significant morbidity and mortality. most of the involved viral agents represented emerging organisms with an unpredictable impact on individual and society health. the radiologic community has learned several lessons from the documented outbreaks, and radiologists around the world have contributed to the early diagnosis of the disease, the monitoring of its course, and the documentation of complications as well as of response to therapy. thus, imaging plays an important role in the diagnosis and management of these patients. in this course, epidemiologic aspects, patho-physiology and clinical features of emerging viral infections will be presented. in addition, their radiologic features and the role of radiology in diagnosis and management will be discussed. attendees will learn how to understand, recognize, report and follow patients with emerging viral infections. major changes in the treatment of head and neck neoplasms encompass the advances of endoscopic-based surgical techniques, mainly for nasosinusal and laryngeal tumours, and the application of sophisticated radiation therapy techniques, combined with chemotherapy. as most tumours arise from the mucosa of the upper aero-digestive tract, clinical surveillance is necessary to detect superficial recurrences, while morphological and 'functional' imaging techniques are indispensable to detect subclinical extra-mucosal and nodal recurrences. how can imaging techniques discriminate recurrence, inflammation, necrosis or scar? key points include the knowledge of the normal appearance of tissues (morphology and signals) on ct, mri, and pet-ct after surgery and chemo-radiotherapy. specifically, when non-surgical treatment has been used, that means to become familiar with the expected changes both of tumour and adjacent tissues. morphology-based imaging techniques are often inadequate to discriminate small recurrences from vascularised scar tissue (enhancing). ct or mri do require to be integrated by information provided by functional-based imaging techniques, fdg-pet-ct being the most established. recently, a great interest among radiologists is focused on the application of dce-ct or dce-mri and dwi-mri in the follow-up of head and neck neoplasms. in fact, several studies have credited these techniques for providing functional information about tissues (perfusion, water exchange) that help to discriminate scar from recurrences. obviously, the horizon pursued is to combine morphology and functional data in a single examination. though clearly promising, these new techniques share significant limitations, like the reproducibility of ct and mr-based functional results, their introduction and feasibility in the day practice. today, ct angiography (cta) is considered as a safe, non-invasive and wellestablished procedure for vascular imaging. modern multi-slice ct technology allows for coverage of larger vascular territories -even the whole body -in just several seconds. further technical improvements such as wide detectors of up to cm width or moving table techniques are making new applications possible, such as perfusion imaging, or time-resolved ct angiography. especially with the moving table technique (the so-called "shuttle-mode"), vascular territories of up to cm can be covered in a dynamic fashion, which can be helpful in the diagnosis of aortic dissections or peripheral vascular occlusive disease. on the other side, radiation exposure has to be maintained within a reasonable range applying these repeated acquisition modes. another interesting field opening new options in cta is dual energy ct (dect). a number of technical setups may allow for spectral ct imaging, such as systems with two tubes (dual source ct), ct systems with switching kv modes or with special detectors. in vascular applications, dect is especially helpful for automated bone removal, plaque removal, and potentially for "perfusion" imaging (iodine mapping). in this course, the basics of modern ct angiography will be highlighted, with a special focus on new applications such as time-resolved cta and dual energy cta. angio- d with digital flat-panel detector has recently been adapted for use with c-arm systems and provides a higher detector quantum efficiency (dqe) than conventional detectors based on ii camera. this configuration represents the next generation of imaging technology available in the interventional radiology suite and is predicted to be the platform for many of the three-dimensional ( d) roadmapping and navigational tools that will emerge in parallel with its integration. it provides projection radiography, fluoroscopy, digital subtraction angiography, and volumetric computed tomography (ct) capabilities with the ability for immediate multiplanar post-treatment assessment in a single patient setup, within the interventional suite. such capabilities allow the interventionalist to perform intraprocedural volumetric imaging without the need for patient transportation. the clinical benefits of d angiography with these new systems have been assessed in the fields of cardiology and interventional radiology. these key features alone may translate to a reduction in the use of iodinated contrast media, a decrease in the radiation dose to the patient and operator, and an increase in the safety and performance of interventional procedures. proper use of this new technology requires an understanding of both its capabilities and limitations. this article provides an overview of the potential of this new technology. learning objectives: . to learn the basic principles of flat panel ct. . to review imaging protocols, results and radiation exposure aspects. . to become familiar with the most common applications.effect of chemo-radiotherapy. as the treatment planning becomes progressively 'tailored', strong predictive factors for the individual tumour arising in a specific patient have to be identified. these factors would ideally provide a quantitative assessment of the risks of both relapsing (in the primary, nodal or distant sites) and developing treatment-related (early or late) complications. predictors are related to tumour's characteristics (biology, metabolism, site, volume and spread) and to patient's overall clinical conditions. imaging-based predictive factors have been founded upon morphological findings ( d, volume) until the development of new techniques, which analyse 'functional' parameters like fdg-pet-ct, the most established, and perfusion-ct or dwi-mri. evidence of level a has been provided on the impact of negative predictive value of fdg-pet-ct in ruling out residual disease, whilst there is no clear data regarding the role of pre-treatment intensity of glucose metabolism (suv) in predicting the outcome after radiotherapy. this limitation can be addressed to the enrolment of tumours arising in various sites in the head and neck. a major disadvantage common not only in pet studies but also in most dce and dwi-mri studies. perfusion-ct and dwi-mr are promising techniques, as they provide information about neo-angiogenesis and water-flow in submicroscopic tissue compartments. however, these techniques still require randomised trials and confirmation studies about the reproducibility of their interesting results. in the follow-up after minimal invasive surgical techniques or chemo-radiation, a major limitation of standard morphological imaging is the differentiation of the highly vascularised scar tissue (enhancing, with mass effect) from persistent or recurrent neoplasm. is it time for integrating standard imaging with the functional information provided by dwi and/or ct or mr perfusion techniques? which evidence level are we at presently? is it now feasible in daily practice? three-tesla mri scanners offer an increased signal and contrast for mr-angiography (mra) compared to . -tesla machines. mra can be performed within a shorter time enabling the acquisition of temporally resolved three-dimensional datasets with high spatial resolution. due to high signal and contrast the dose of gadolinium may be reduced. furthermore, novel imaging protocols for mra at . -tesla with intravasal contrast agents and prolonged enhancement during high-spatial-resolution steady-state enable new possibilities for angiography of the upper and lower extremities including veins and vessels below the knee, spinal arteries, vascular malformations, and double-gated angiography of coronary arteries and bypass vessels to compensate for the complex cardiac motion pattern. a . -tesla mri system equipped with a matrix coil system allows for whole body mra with continuous table movement, which is an applicable technique for imaging peripheral vessels without the need for planning different steps and field of view positioning, thereby considerably reducing the examination time. phase contrast magnetic resonance angiography may develop into an important, noninvasive method for obtaining quantitative information on blood flow. in addition, non-enhanced three-dimensional mr angiography using turbo spin echo (tse) imaging with non-selective refocusing pulses may be a promising imaging technique for vascular imaging in patients with renal insufficiency. the advent of minimally invasive surgery has made even more important the place of preoperative imaging assessment of patients selected for this type of surgery. in fact, the loss of tactile feed-back and the bi-dimensional intraoperative vision have limited the capability of surgeons to assess extent and anatomic relationship of a given disease, particularly cancer. preoperative planning can be further enhanced by the use of d models of the target anatomy, derived from ct scan dataset. in addition, dedicated technology can be implemented to introduce mixed reality environments in the operative room. using d helmets with built-in microcameras, the surgeons' view of the operative field can be fused with the preoperative d anatomy of the patient. localiser should be used, either infrared based or electromagnetic. the last step would be intraoperative navigation. this offers special problems to be addressed, due to organ shifting and soft tissue in the setting of abdominal surgery. the appropriate use of information and communication technology (ict) and associated systems is considered by many experts as a significant contribution to improve workflow and quality of care in clinical settings. a conceptual design and prototypical implementation of such an infrastructure, i.e. a therapy imaging and model management system (timms) will be introduced as a solution to a patientspecific medicine. a timms is an information technology concept and framework for the collection, organisation, and utilisation of medical information from sources such as the electronic medical record, pacs, etc. timms was originally designed as a surgical assist system, but has many general medical uses as well, including all forms of model-guided medicine and may therefore be generalised to a medical information and model management system. the architectural framework and a number of individual ict components of a timms have been realised. these include standardised interfaces for communication of patient-specific and workflow models, thereby creating a unified environment for the input and output of data, including the representation and display of information and images, as well as the electromechanical control of interventional and navigational devices. in conclusion, the patient-specific model (psm) is the central construct for a patient within a personalised medicine environment in order to provide a clinician with a real-time representation of critical information about the patient. the required information concerning the patient for model-guided therapy is extracted by timms agents and assembled within the framework of an active psm and workflow management system. the introduction of picture archiving and communication systems (pacs), through a much more effective image sharing, has dramatically changed the role of radiology both within the hospital and on a geographic perspective. historically, the latter has become apparent first with the early implementation of teleradiology applications. physicians and the general public have understood that digital images can be read, processed, and stored independently on the site of production. therefore, teleconsultation and telereporting activities have been carried out among medical users, and generic users have learnt to include radiological images in their own on-line personal health records. only later the full potentialities of multidisciplinary image sharing within the hospital have been discovered, and now it is increasingly common to see advanced integration between radiologists and surgeons for planning and guiding surgical interventions. during this session, the lecturers -exceptionally expert in their respective fields -will give insights into image sharing: from the geographical applications (teleradiology) to the hospital-based applications, with specific reference to the support to surgeons (intraoperative guidance and model-guided surgery). medical imaging is part of a changing medical environment, a changing patient environment and consequently a new medical world. in the recent decennium one of the most important changes in radiology is the conversion from analogue to digital. in no time medical images have become interchangeable through the digital highway and could be post-processed in a different location. teleradiology has become a reality since then. we have seen the maturation of commercial international teleradiology companies offering a wide portfolio of services. another aspect is the availability of image data for all medical specialties beyond radiology and beyond the regular medical disciplines. an increasing number of surgical or oncological specialties and even pharmaceutical companies increasingly use image data to prepare a strategy for operative procedures, to choose the right therapy, to decide which prosthesis to the best to use, for follow-up or for post-processing purposes. they are supported by many new techniques and software. an increasing number of medical computer applications such as complex navigation and visualisation tools based upon digital images is already in clinical use or under development. another trend is the increasing interest in e-health and telemedicine in europe, also among european policy makers. now we see mobile health that brings care directly into the patient environment. the purpose of this presentation is to give a comprehensive overview of and insight into these new developments and to create awareness among radiologists of the increasing importance of integration of medical imaging in a multidisciplinary environment. key: cord- -qa bcsbu authors: starkel, julie l.; stapke, christina; stanley-o’malley, abigail; noland, diana title: respiratory date: - - journal: integrative and functional medical nutrition therapy doi: . / - - - - _ sha: doc_id: cord_uid: qa bcsbu lung disease rivals the position for the top cause of death worldwide. causes and pathology of the myriad lung diseases are varied, yet nutrition can either affect the outcome or support treatment in the majority of cases. this chapter explores the modifiable risk factors, from lifestyle changes to dietary intake to specific nutrients, anti-nutrients, and toxins helpful for the nutritionist or dietitian working with lung disease patients. general lung health is discussed, and three major disease states are explored in detail, including alpha- antitrypsin deficiency, asthma, and idiopathic pulmonary fibrosis. although all lung diseases have diverse causes, many integrative and functional medical nutrition therapies are available and are not being utilized in practice today. this chapter begins the path toward better nutrition education for the integrative and functional medicine professional. anti nutrients and inhibitors of lung physiology - lung disease is far more prevalent worldwide than commonly thought. in fact, death from chronic lung disease is increasing, and as of , chronic obstructive pulmonary disease (copd) has become the third leading cause of death in the united states in the past decade, disproportionately affecting the elderly [ ] . another lung disease, asthma, affects in , or about million americans, according to the centers for disease control and prevention and the national center for health statistics [ ] . this is . % of adults, more women than men, and . % of children. asthma is the leading chronic disease in children [ ] . this disease has been increasing since the early s in all age, sex, and racial groups. in europe, lung disease represents % of all deaths -the fourth leading cause. according to the world health organization (who), in , . million people died from acute or chronic lung disease, representing one sixth of the global total deaths [ ] . worldwide, four respiratory disease categories appear in the top ten leading causes of death in [ ] . specifically, copd was the third leading cause of death, followed by lower respiratory infections as the fourth, lung cancer as the fifth, and tuberculosis as the tenth [ ] . the major risk factor is smoking, leading to % of all lung disease-related deaths in europe, where smoking is more prevalent ( % prevalence) than in the united states ( % prevalence) by nearly twofold [ , ] . lung cancer, particularly non-small-cell lung cancer (nsclc) subtype, is the leading cause of cancer-related death worldwide [ ] . added together, lung disease rivals the position for the top cause of death. throughout the life cycle, diet and lifestyle are important modifiable risk factors in the development, progression, and management of obstructive lung diseases, such as asthma and copd [ ] , as well as restrictive lung diseases such as pulmonary fibrosis and sarcoidosis. inflammation, in particular, seems to be the leading contributor toward the progression of lung diseases. as with many diseases, maintaining a healthy lifestyle, including sufficient sleep, low stress, regular exercise, a whole foods diet rich in phytonutrients from plants (fruits and vegetables), and potential anti-inflammatory supplements, is beneficial in supporting the body during these difficult diseases. inflammation, in particular, seems to be the leading contributor toward the progression of lung diseases. high inflammatory foods should be avoided, such as fried foods and foods disproportionately high in carbohydrates, sugar, alcohol, and excessive protein. a healthier suggestion would be a diet with more than half of all food consumed as vegetables, about one third as protein, and the remainder (one sixth) as other foods, such as fruits, dairy, grains, or starches. some dietary supplements may also be recommended for their anti-inflammatory benefits, which will be discussed later in the chapter. as human life expectancy increases, we can expect to see more chronic disease. the world health organization estimates that by , chronic lung disease will account for % (one fifth) of all deaths [ ] , up from one sixth in . despite these growing numbers, relatively little human nutrition research exists for respiratory health, compared to other, less prevalent, diseases. investigators in the areas of aging and lung biology suggest some hope, using genetics and animal models, as well as epidemiological research, to further the general medical approach to lung disease. the pulmonary system is composed of the upper and lower respiratory tracts. air flows in through the nose or mouth, past the frontal and maxillary sinuses, down the pharynx (throat), past the larynx (voice box), and then down the trachea. this makes up the upper respiratory tract. once past the trachea, the air divides into the left and right bronchi, which supply the left and right lungs, each divided into five sections called lobes. the bronchi then divide into smaller bronchioles, at the end of which are air sacs called the alveoli. this makes up the lower respiratory tract [ ] (. fig. . ). the diaphragm is the central muscle that is used for breathing. the intercostal muscles, located between the ribs, and the abdominal muscles are helpful for breathing out when the breath becomes labored, such as during exercise. the neck muscles and the muscles in the collarbone area help with breathing when the other muscles are compromised or impaired. in some neurological diseases, such as amyotrophic lateral sclerosis (als) [see chap. , newton], nerve damage from the brain to breathing muscles can result in impaired movement of these muscles and thus impaired breathing. in certain cases, such as in lung cancer when a lobectomy, removal of part of the lung, is required, there is an expected decrease in short-and long-term pulmonary function and oxygenation. however, respiratory muscle strength may be preserved [ ] . in a pneumonectomy, removal of the entire lung, dramatic changes in thoracic anatomy take place, such as elevation of the hemidiaphragm, hyperinflation of the remaining lung, and influx of fluid into the postpneumonectomy space [ , ] . there are phagocytic macrophages on the cellular surface of the alveoli, type i epithelial cells and type ii epithelial cells. phagocytic macrophages destroy inhaled bacteria and serve an important role in suppressing or activating the immune response to antigens and pathogens, similar to dendritic cells discussed below. macrophage function has been shown to be inhibited by cigarette smoke [ ] . alveolar macrophages also secrete enzymes, arachidonic acid metabolites, growth factors, immune response components, cytokines, and lymphocytes [ ] . type i cells are responsible for maintaining the structure of the alveolar wall, whereas type ii cells and clara cells are responsible for the production of pulmonary surfactant (composed of - % lipid and - % protein as lecithin and myelin), which is essential for lung function. the surfactant reduces surface tension, facilitating easier stretching and collapsing of alveoli during respiration [ ] . diseases associated with inadequate surfactant production are acute/adult respiratory distress syndrome (ards) and infant respiratory distress syndrome (irds) [ ] . irds is seen in premature babies born prior to weeks of gestation due to immature development of pulmonary surfactant, which only begins to develop around the th week of gestation [ ] . dipalmitoylphosphatidylcholine, phosphatidylglycerol, and cholesterol compose the lipid portion of the surfactant, where apoproteins and proteins found in blood plasma compose the protein portion [ , ] . the importance of cholesterol is minimized in today's medical community. those with higher levels of cholesterol tend to have more in their fatty cell membranes which resist pathogenesis at a cellular level. low cholesterol predicts a greater risk of dying from gastrointestinal, neoplastic, or respiratory diseases. it occupies - % of our cell membranes, enhances the mechanical strength of the membrane, and reduces permeability [ ] . it suppresses main-phase transition of the lipid bilayer [ ] . collagen, a fibrous protein, along with elastin and proteoglycans, is a fundamental component of the connective tissue that composes the lungs, and collagen is present in the blood vessels, bronchi, and alveolar interstitium [ ] . connective tissue in the lung is key for the passive diffusion of oxygen and carbon dioxide that characterizes alveolar-capillary gas [ ] . collagen homeostasis is vital to maintaining respiratory function, where collagen production and degradation are balanced. dysregulated collagen homeostasis that favors collagen production over degradation can lead to pulmonary fibrosis and compromised lung function [ ] . some key nutrients to consider for collagen synthesis and crosslinking to maintain connective tissue integrity are vitamin c, vitamin b , iron, copper, zinc [ ] , riboflavin, thiamin, and pantothenic acid [ ] . the airways of the respiratory system (with the exception of parts of the nose and mouth) have cilia, special hairs coated with mucus that trap pathogens and other particles that enter with the air that is inhaled. cilia are responsible for triggering this mucus upward toward the pharynx where these particles or bacteria can be coughed out or swallowed. mucus present in the lungs can also trap inhaled particles such as viruses, bacteria, and smoke particulates [ , ] . along the lining of the respiratory tract, there are several types of cells that are involved in immune response, such as secretory cells (i.e., goblet cells and clara cells) and mast cells. ciliated epithelium and mucus secreted by glands present on airways, goblet cells, and the secretory products of clara cells serve an important mechanism for lung protection. however, excessive goblet cells or hypertrophy of mucous glands may result in increased viscosity of mucus seen in pathologies like bronchitis [ ] . ciliary function is also impaired by cigarette smoke [ ] . dendritic cells are also found in the airway lining from the trachea to the alveoli. immature dendritic cells phagocytize bacteria or other antigens, where they then mature and travel to lymphoid tissues to communicate with the immune system. this delivery of antigens can promote tolerance of the antigen by releasing anti-inflammatory cytokines. conversely, this delivery can also trigger the opposite response if the antigen is recognized as a pathogen, where t lymphocytes are activated and inflammatory cytokines are released [ ] . one potential cause of infections in the upper respiratory tract or bronchial tubes, such as bronchitis, or deep in the lungs, such as pneumonia, is when cilia become damaged and do not trap inhaled germs and particles as effectively. in diseases such as cystic fibrosis, thick mucus secretions can accumulate in the airways and lungs, making it hard to clear and thus increasing risk for infection. in asthma, specific inhaled particles can trigger a reaction causing the airways to narrow, restricting breathing [ ] . surface enzymes and factors can also be found in the lining of the airways that compose the majority of the innate immune system of the respiratory tract. these include: lysozymes: found in leukocytes with bactericidal properties lactoferrin: a bacteriostatic agent (inhibits bacterial reproduction) synthesized by lymphocytes and glandular mucosal cells alpha- antitrypsin: an antiprotease to protect lung tissue from excessive enzymatic activity interferon: an antiviral substance that may be produced by lymphocytes and macrophages complement: participates as a cofactor in antigenantibody reactions [ ] gas exchange takes place in the alveoli so oxygen can enter the body to support metabolic function and the carbon dioxide product from these functions can be removed. this is accomplished through millions of capillaries in the alveoli. these capillaries in the alveoli then connect to arteries and veins that move blood throughout the body. the pulmonary artery supplies carbon dioxide-rich blood to these capillaries within the alveoli to remove carbon dioxide, and the oxygen-rich blood then gets delivered to the heart through the pulmonary vein. the lungs also serve the vital function of maintaining acid-base balance through changes in minute ventilation. these changes affect the ph of the blood by either retaining or excreting carbon dioxide [ ] . poor physiologic management of co and bicarbonate can lead to the conditions of respiratory acidosis and respiratory alkalosis. respiratory acidosis is characterized by higher blood concentrations of co and h + , caused by hypoventilation or decreased rate of breathing. hypoventilation can have acute or chronic etiologies, resulting from copd, interstitial lung diseases, respiratory muscle fatigue (i.e., extended asthma attack), or mechanical abnormalities (i.e., deformities). respiratory alkalosis is characterized by lower blood concentrations of co and h + due to hyperventilation, or increased rate of breathing. possible causes of hyperventilation can also be chronic or acute, such as pneumonia and fever, increased stress and anxiety, liver disease, stroke or meningitis, pregnancy, overuse of aspirin and/or caffeine, excessive mechanical ventilation, or increases in altitude [ ] . a pulse oximeter tool can be used to measure the percentage of oxygenated hemoglobin in an individual's blood to determine their overall respiratory status. typically, oxygen saturations of % or less are indicative of central hypoxia [ ] . pulse oximetry is especially useful for assessing individuals with asthma and copd [ ] . oral health must also be considered as a contributing factor to respiratory health [ ] . for example, in patients affected with periodontal disease, mm of dental plaque could contain around of bacteria. one potential mechanism of this connection is aspiration of bacteria from the oropharynx into the upper or lower respiratory tracts, leading to their adherence to the alveolar and bronchial lining, potentially colonizing respiratory ducts and causing respiratory infections. in addition, cytokines and enzymes associated with inflammation of periodontal tissues can be transferred into the lungs, potentially triggering or exacerbating lung infections [ ] (. fig. . a systematic review done in examined oral health in the elderly and its association with risk of aspiration pneumonia. this review suggested that maintaining oral health, such as brushing after each meal, cleaning dentures once per day, and professional oral healthcare, potentially reduced the amount of potential respiratory pathogens that resulted in lower incidence of aspiration pneumonia [ ] . several other systematic reviews have found that adequate oral hygiene plays an important role in preventing pneumonia, particularly in clinical settings where there is increased risk for hospital-acquired pneumonia (hap) and ventilatorassociated pneumonia (vap), as well as in older populations [ ] . in addition, associations have been made between copd and the risk of periodontitis, although systematic reviews have established that these associations are preliminary and further studies are needed [ ] . another important consideration in respiratory health is orofacial development and structure. anatomical obstructions at the level of the nose and pharynx, such as those caused by allergic rhinitis and hypertrophy of the tonsils, pose an increased risk for obstructive sleep apnea syndrome and respiratory infections due to lack of airflow through the upper respiratory system [ ] . it has been established that the lung has a microbiome of its own that may have a large impact on health and disease [ ] . the fungal microbiome, or mycobiome, may also have a significant impact on respiratory health, although more research is needed to determine definitive associations [ ] . dysbiosis may occur in the lungs with a bacterial infection. a few specific bacterial strains have been studied, and one, in particular, pseudomonas aeruginosa, seems to grow in inflammatory conditions. it then seems to encode inflammatory components causing further inflammation. anti-inflammatory nutrients could help stop the cycle, and vitamin d use has some research supporting this. recurrent bacterial respiratory infections may damage lungs and lead to worse outcomes in future lung disease [ ] . an increased interest in research of the relationship of the airway and gut microbiome is indicating potentially positive results regarding the use of probiotics in pediatric populations that may aid in asthma prevention and intervention [ , ] . the gut-lung axis has also been established, where the microbiomes of the lung and gut have been immunologically linked and are thought to have an impact on respiratory disease [ , ] . the autophagy mechanism within our microenvironment provides a constant "cleanup" system to recycle cell debris from microscopic biowaste generated by dynamic cellular biochemistry [ ] . enzymes such as neutrophil elastase function like garbage disposals recycling waste molecules. alpha- antitrypsin is a thermostat-like control factor that signals the proteolytic enzymes to stop and protect healthy tissue from being affected. antiproteases in the lung, such as alpha- antitrypsin, are required to prevent the overactivity of neutrophil elastase to prevent the degradation of healthy lung tissue. those with the genetic mutations of a at deficiency are at disadvantage, and subsequent lung tissue damage can occur promoting lung diseases like copd, asthma, bronchitis, and emphysema. key components of lung structure are elastin and collagen, which provide support for the bronchioles and clusters of alveoli (acini). the key enzyme present in these cells is neutrophil elastase, which is responsible for the destruction of respiratory bacteria. protease and antiprotease imbalance in the lung resulting in emphysema can be caused by alpha- antitrypsin deficiency and nicotine in cigarette smoke or polluted inhalant exposure [ ] . ifmnt approaches to the a at-deficient patient assess for nutrient insufficiencies for some of the important connective tissue, collagen, and elastin system key nutrients: vitamin c, vitamin d, biotin, balanced fatty acids, and gut microbiome. when insufficiencies or deficiencies are identified, appropriate food and dietary supplementation interventions can be recommended. it should be noted that if an individual is identified with a at deficiency genotype, the status of liver health should also be assessed, as a at pathophysiology can express in liver cirrhosis. more recent studies of respiratory disease [ ] have revealed the relationship with bacterial or viral infections exacerbating the individual's genotype eliciting expression of the associated diseases. one of the most recognized inherited conditions of altered autophagy mechanisms is alpha- antitrypsin deficiency, with - genetic variants affecting severity of lung expression. low levels of circulating a at allow potentially harmful enzymes like neutrophil elastase to remain in the lungs unchecked. low levels of a at, and the consequent proliferation of neutrophil elastase, leave lung tissue vulnerable to destruction, resulting in a decline in lung function. there are several categories of lung disease and many diseases within those categories (. table . ). some micronutrients and phytonutrients have important antioxidant and methyl-donating properties important for the lungs and therefore have great role in a nutritional approach to lung health. iron's interaction with the lungs is essential. it carries oxygen from the lungs to the peripheral parts of the body, as well as carbon dioxide back to the lungs to be exhaled. however, too little or too much iron can pose a problem for the lungs. before iron administration, it is important to rule out hemochromatosis, or iron overload, for an individual. iron-deficiency anemia often presents in many chronic diseases including those of the lung, such as copd, lung cancer, and ipf [ ] . increased mortality, decreased quality of life, increased hospital admissions, and cost of treatment have been reported for those with chronic disease and low iron [ ] . anemia of chronic disease (acd) is usually at the root of this. acd is often the result of inflammation. inflammatory proteins, including il- , stimulate the production of hepcidin in the liver, which inhibits absorption and increases storage of iron resulting in a functional iron deficiency. typical iron markers, such as transferrin saturation, total iron binding capacity (tibc), and ferritin, are also affected by inflammation and are less useful markers in chronic disease. soluble transferrin receptor (stfr) seems to be a lesser known marker that is less affected by inflammation [ ] . because of the difficulty with iron absorption, intravenous iron is often used to replete deficiencies. as iron is a pro-oxidant, researchers studied any negative repercussions. there does not seem to be any increased oxidative stress with intravenous iron, but glutathione, the body's endogenous super antioxidant, does seem to decrease, likely in response to the pro-oxidative activity of iron. in a recent study, administration with vitamin e was seen to eliminate these negative effects [ ] . excessive iron can also be problematic for lung health for those with the genetic mutation for hemochromatosis (hfe). disorders of iron overload are increasingly being recognized as risk factors for most of the chronic diseases like cardiovascular, alzheimer's, and cancer [ ] . high iron can catalyze the formation of highly reactive hydroxyl radicals, oxidative stress, and programmed cell death. in the instance of lung cancer and other cancers affecting the lungs, tumors sequester iron for their own growth, usually leaving the patient with iron-deficiency anemia. in fact, % of cancer patients undergoing chemotherapy are iron deficient. inflammation also plays a role in iron homeostasis. the pro-inflammatory cytokines cascade down to affect the proteins that regulate . chronic obstructive pulmonary disease (copd) disease that restricts airflow through either inflammation of the lining of the bronchial tubes or destruction of alveoli increased risk of emphysema if genetic variant of alpha- antitrypsin deficiency and smoking or exposed to high levels of air pollution [ ] bronchiectasis a disorder of the airways that leads to airway dilation and destruction, chronic sputum production, and a tendency toward recurrent infection [ ] bronchiolitis airway injury that can be caused by infections, irritants, toxic fumes, drug exposures, pneumonitis (typically viral), organ transplants, connective tissue disorders, vasculitis, or other insults [ ] dyspnea shortness of breath or difficulty breathing [ ] emphysema thinning and destruction of the alveoli, resulting in decreased oxygen transfer into the bloodstream and shortness of breath. increased risk of emphysema if genetic variant of alpha- antitrypsin deficiency and smoking or exposed to high levels of air pollution [ ] alpha- antitrypsin deficiency a deficiency of a at, a protein produced in the liver that protects the lungs from excessive neutrophil elastase, an autophagic enzyme. a at may also accumulate in liver and cause liver disease [ ] obstructive asbestosis fibrotic lung disease resulting from extensive inhalation of asbestos fibers [ ] desquamative interstitial pneumonitis (dip) form of idiopathic interstitial pneumonia that is more common in cigarette smokers but may be seen in nonsmokers, in patients with underlying connective tissue diseases or those exposed to inorganic dust/particles [ ] sarcoidosis immune-mediated systemic disorder that is characterized by granuloma formation of the lung parenchyma and the skin [ ] restrictive pathophysiologyneuromuscular weakness amyotrophic lateral sclerosis (als) progressive neurological disease that affects the motor neurons of the nervous system [ ] guillain-barre syndrome progressive immune system attack on the peripheral nerves, usually following an infectious illness such as a respiratory infection. may eventually cause respiratory distress syndrome [ ] restrictive pathophysiologychest wall/pleural disease kyphoscoliosis kyphoscoliosis: a deformity of the thoracic cage that results in restriction of the lungs and impairs pulmonary function [ ] ankylosing spondylitis autoimmune inflammatory disorder characterized by inflammation of the axial skeleton and peripheral joints [ ] chronic pleural effusions chronic accumulation of fluid between the two outer membranes surrounding the lungs [ ] pulmonary vascular disease pulmonary embolism blood clot that typically originates from thrombi in the deep venous system of the legs and travels to the lungs pulmonary arterial hypertension (pah) progressive disorder of primary pulmonary arterial vasculopathy characterized by a mean pulmonary arterial pressure > mm hg at rest (> mmhg during exercise) [ ] iron homeostasis [ ] . iron can also impair cytokine secretion, which can leave those with an iron overload much more susceptible to infection, increasing the morbidity and mortality of infectious diseases, including those of the lung [ ] . oxidative stress may contribute to injury of lung tissue, causing further fibrosing in those lung diseases with that characteristic. allele variants in the genes associated with iron homeostasis (c y, s c, and h d hfe) are significantly more common in those with idiopathic pulmonary fibrosis (ipf) than those without ipf ( . % ipf patients vs . % non-ipf) and are associated with higher irondependent oxygen radical generation [ ] . iron is implicated in lung pathology. monitoring iron status and using supplements or diet to aid the body in increasing or decreasing the iron load are imperative for the nutritionist working with lung disease patients. choosing a good non-constipating form of iron is important, such as iron glycinate. the b vitamins are also important to monitor for lung health. vitamin b and its bioactive form, p- -p, are typically known to protect dna from mutation or damage [ ] . however, there is mixed evidence on its role for lung cancer. some research has shown that it is helpful for lung cancer patients as it is important for apoptosis when using chemotherapy, because it sensitizes cancer cells to apoptosis [ ] . however, research in showed that adult male smokers taking greater than mg vitamin b /day for long periods tended to have a greater risk for lung cancer. many variables, including genetic variants, form of b , and the status of other co-nutrients may be at play [ ] . other studies showed that men in the top quintile of vitamin b serum concentration had about one half the risk of lung cancer, and specifically, vitamin b and folate were inversely associated with risk of lung cancer [ ] . . squamous cell (epidermoid) carcinoma about - % of all lung cancers. these start in early versions of squamous cells, which are flat cells that line the inside of the airways in the lungs. often linked to a history of smoking and tend to be found in the central part of the lungs, near the bronchus [ ] large cell (undifferentiated) carcinoma about - % of lung cancers. it can appear in any part of the lung and tends to grow and spread quickly. a subtype of large cell carcinoma, known as large cell neuroendocrine carcinoma, is a fast-growing cancer that is very similar to small-cell lung cancer [ ] small-cell lung cancer (sclc) about - % of lung cancers are sclc. typically start in the cells lining the bronchi and parts of the lung such as the bronchioles or alveoli [ ] infectious diseases pneumonia inflammation of the lungs, usually caused by bacteria, viruses, or fungi [ ] bronchitis inflammation and eventual scarring of the lining of the bronchial tubes accompanied by restricted airflow, excessive mucus production, and persistent cough [ ] tracheitis bacterial infection that can develop in the trachea [ ] infant respiratory distress syndrome also known as hyaline membrane disease (hmd) or respiratory distress syndrome, this condition affects the alveolar ducts and terminal bronchioles in which the hyaline membrane is a fibrinous material composed of blood and cellular debris, caused by the absence of proper surfactant production due to an immature or poorly developed lung [ ] upper respiratory infection (uri) acute infections involving the nose, sinuses, pharynx, larynx, trachea, and bronchi, referred to as the common cold [ ] bronchopulmonary dysplasia (bpd) chronic lung disorder which may affect infants who have been exposed to high levels of oxygen therapy and ventilator support [ ] other cystic fibrosis disease characterized by abnormally thick mucus secretions from the epithelial surfaces of many organ systems, including the respiratory tract, the gastrointestinal tract, the liver, the genitourinary system, and the sweat glands [ ] acute lung injury clinical and radiographic changes in lung function associated with critical illness (acute respiratory distress syndrome is most severe form) [ ] respiratory because of disagreement in research, particularly with smokers or former smokers, using food first for b vitamins may be a prudent way forward. good sources of vitamin b are fish, chickpeas, chicken, potatoes, turkey, bananas, ground beef, and winter squash. pyridoxal kinase (pdxk) is the enzyme that converts pyridoxine and other vitamin b precursors to its bioactive form of p- -p. dysfunction of this enzyme is a good prognostic for lung cancer and other lung diseases. mthfr aa genotype is associated with a higher risk of lung cancer in women but not in men. the mthfr tt genotype was associated with a significantly decreased risk of lung cancer in women but not in men. in contrast, the mthfr c t and a c polymorphisms interacted with smoking status in men but not in women [ ] . methylation gene testing is imperative to understand the patient's status. some studies suggest that a higher intake of riboflavin (vitamin b ) may protect against lung cancer in smokers [ ] . folate deficiency was also associated with asthma and attacks of shortness of breath [ ] . correcting acidosis may preserve muscle mass in diseases where wasting is an issue, such as copd or ipf. for those receiving chemotherapy, a higher ph (more alkaline status) is helpful for muscle mass protection. high alkaline diets contain more fruits and vegetables, and those supply more magnesium, which is needed to activate vitamin d. as discussed below, vitamin d is extremely helpful for lung health. sleep quality involves maintaining adequate - hours with good sleep hygiene (see chap. ). good rem cycling, feeling refreshed upon awakening, and other characteristics of good sleep play significant roles in maintaining healthy acid-base balance. dietary intake of the minerals magnesium, potassium, sodium, chloride, and calcium promotes the balance of acidbase microenvironment. after exposure and tissue retention of toxic minerals and metals, these substances can contribute to perturbations in the acid-base metabolic milieu. some conditions reduce oxygen intake and should be addressed. one of the most common oxygen-impairing conditions is sleep apnea, altered sleep with random halting of breathing during sleep that is often accompanied by snoring. other limiting conditions are respiratory diseases like copd, a at deficiency, asthma, cystic fibrosis, etc. vitamin a is an important antioxidant and a general umbrella term for several fat-soluble retinoids, including retinol, retinal, and retinyl esters. there are also other substances that are provitamin a carotenoids or precursors to vitamin a. two forms are found in foods, the preformed forms of retinol or retinyl esters, which are found in dairy, fish, caviar, and meats (especially liver), and the provitamin a carotenoids, including the most important and common provitamin a carotenoid, beta-carotene, as well as others including alpha-carotenes and cryptoxanthin, which are found in plant-based foods. our bodies must convert these two forms within our cells to retinal and retinoic acid, the active forms of vitamin a in the body. new studies of the gene, β-carotene , ′-monooxygenase (bcmo ), which is responsible for the enzymatic conversion of β-carotene to vitamin a, are revealing that individuals with heterozygous or homozygous bcmo snps have - % less efficient conversion than those with normal gene function (see chap. ) [ ] . other carotenoids found in food, such as lycopene, lutein, and zeaxanthin, are not converted to vitamin a but have other antioxidant benefits in the body. most vitamin a is stored in the liver as retinyl esters, and deficiency is not visible until these stores are nearly depleted. vitamin a's role as an antioxidant helps the lungs in several ways, including maintaining alveolar epithelium cells and preventing development of respiratory tract infections. most of the developed world's population does not have a risk of deficiency due to sufficient vitamin a intake. however, most people with cystic fibrosis have pancreatic insufficiency, which reduces the ability to absorb fat and therefore the fat-soluble vitamins a, d, e, and k. according to a study in , between % and % of people with cystic fibrosis had a vitamin d deficiency, also a fat-soluble vitamin. with the addition of pancreatic replacement treatments, better nutrition, and vitamin a supplementation, deficiency has become rare. however, improved vitamin a status has not been thoroughly studied as of , and therefore it is largely unknown if an improved vitamin a status has any effect on cystic fibrosis [ ] . vitamin a deficiency has been shown to be associated with emphysema in rats. smoke exposure significantly decreases vitamin a concentration in lung tissue, significantly more in those with copd [ ] . retinoic acid seems to play a beneficial role in the treatment of ipf. a review showed that in all studies, retinoic acid decreased fibrosing, the formation of collagen, and reduced the expression of alpha-smooth muscle actin (alpha-sma), all hallmarks of ipf [ ] . it is important to not take large doses of vitamin a if one is in a malnourished state as it can cause toxicity and should be monitored with blood testing of vitamin a retinol. nourish the body with all foods and all nutrients slowly. the non-provitamin a carotenoids have also shown some benefit. lycopene, found in high amounts in guavas, watermelon, tomatoes, papaya, grapefruit, sweet red peppers, asparagus, purple cabbage, mangos, and carrots, slowed forced expiratory volume (fev) decline in former smokers [ ] . vitamin d's importance with lung health cannot be understated. vitamin d deficiency, or even insufficiency, is linked to accelerated decline in lung function, increased inflammation, and reduced immunity in chronic lung diseases. vitamin d has a role in the regulation of inflammation, immunity, cellular proliferation, senescence, differentiation, and apoptosis. sufficient vitamin d levels are correlated with better asthma control, better immune response related to respiratory infections, and reduced severity of exacerbations with copd and asthma when exposed to inflammation-causing pathogenic activity [ ] . vitamin d is obtained through sunlight on the skin (without sunscreen) and very few dietary sources. therefore, supplementation is generally recommended. higher vitamin d levels are shown to be protective in many lung disease states. sufficient levels improve treatment response with medications and reduce asthma severity [ ] . with infectious diseases of the lung, higher vitamin d concentrations are shown to have a protective action [ ] . vitamin d has a protective effect on lungs of smokers, and higher levels of vitamin d inhibit the pro-fibrotic phenotype of lung fibroblasts and epithelial cells. current data suggest an inverse association between serum vitamin d and lung cancer risk, and vitamin d deficiency at - weeks' gestation is associated with impaired lung function and asthma at years of age [ ] . lower levels of vitamin d are associated with an increased risk for respiratory infections, cystic fibrosis, chronic obstructive pulmonary disease, and interstitial lung disease [ ] . vitamin c is an important antioxidant that helps decrease oxidative damage in the body, including in lung tissue. it is also essential for lipid metabolism. it is present in the airway surface liquid and creates an interface between the epithelial cells and the external environment. vitamin c is a cofactor in collagen synthesis, which can aid in repair of bronchial and alveolar tissue when damaged. it also provides beneficial control of lipid peroxidation of cellular membranes, including those surrounding as well as those within intracellular organelles. vitamin c has some of the best lung protective capabilities, according to current research. vitamin c may also diminish oxidative attack on nonlipid nuclear material and is an antioxidant component of plasma and extracellular fluids surrounding the lungs. it is an antioxidant that not only fights oxidative stress but also reduces oxidized vitamin e and glutathione, allowing them to become active as antioxidants again. vitamin c is antiinflammatory and is helpful in all inflammatory states of the lung, even allergies. there are many ways in which vitamin c, along with its antioxidant partners, glutathione, vitamin e, vitamin a, and plant-based phytonutrients, affects lung health. it is well established that increased levels of vitamin c in the diet improve health outcomes for smokers and their offspring, as smoking depletes vitamin c [ , ] . vitamin c is also helpful in fighting infectious diseases such as respiratory infections and pneumonia, copd regardless of smoking status, asthma, and lung cancer [ ] . specifically, in certain lung cancers, vitamin c, along with other nutrients such as lysine, proline, epigallocatechin gallate, and zinc, can inhibit the proliferation of certain carcinoma lines and induce apoptosis, as well as inhibit lung cancer metastasis [ ] . even in lung transplants, vitamin c is helpful against oxidative stress by reducing glutathione and lowering lipid peroxidation, along with vitamins a and e [ , ] . the literature suggests these benefits can be achieved at - mg/day. check iron status before administering vitamin c supplementation as vitamin c doubles iron absorption from foods. vitamin e's primary role is as an antioxidant, breaking free radical chain damage and preventing peroxidation of lipid molecules. this vitamin also is promising with regard to beneficial effects on lung function preservation. oxidative stress and inflammation are key features in many lung diseases; therefore nutrients with antioxidant capacity can be useful. a few studies suggest that alpha-tocopherol found in sunflower and olive oils has a beneficial effect on fev (forced expiratory volume), whereas gamma-tocopherol found in canola, soybean, and corn oils has a negative effect on fev [ ] . however, from these authors' perspective, this is likely due to the source and type of the oils, which can be inflammatory, rather than the form of vitamin e. for example, a recent study showed that gamma-tocopherol was protective in allergic asthma [ ] . in addition, sufficient levels of vitamin e, in the alpha-tocopherol form, were found to reduce susceptibility of the elderly to acquiring pneumonia. some of the positive effects of vitamin e are synergistic with vitamin c [ ] . phytonutrients have been found to have two effects with respect to lung disease: one is a symptom-improving pattern, and the other is a rate-reducing pattern [ ] . idiopathic pulmonary fibrosis (ipf) is largely characterized by reduced antioxidant and increased inflammatory action. recent literature is showing the ability of certain flavonoids, in particular quercetin, to reduce inflammation and act as a strong antioxidant countering the pro-oxidant environment of ipf. quercetin is recognized as the most potent ros scavenger. taken together with glutathione, the impact is even greater, and it seems to help improve the antioxidant and inflammatory status more for those with ipf than non-diseased controls [ ] . curcumin has been shown to slow or limit fibrosing in murine studies related to lung, liver, or kidney fibrosing [ ] [ ] [ ] [ ] . it has also been shown to attenuate metastatic melanoma in the lungs when delivered in a nanoparticle [ ] . the potential for curcumin is interesting and hopeful. fisetin and fenugreek have also been studied as useful phytonutrients that help combat inflammation in lungs [ , ] . fisetin is found in apples, strawberries, persimmons, cucumbers, and onions, among many other fruits and vegetables. fenugreek is a plant used frequently in south and central asian cooking, where both the seeds and leaves are used. there are now supplements available for both of these phytonutrients. this is a reminder to eat a primarily plantbased diet when combating inflammation and to broaden our palates to include healthy foods and ingredients from other cultures than our own. lastly, the powerful antioxidant cannabidiol (cbd), from the cannabis and closely related hemp plants, is a powerful shield against oxidative stress, prevalent in lung disease [ ] . the research is not robust regarding lung function and minerals, and most has been done with regard to cystic fibrosis where bone density is associated with general nutritional status, including minerals. there have also been many studies trying to determine a correlation between mineral status and copd, where, again, the research shows that mineral status is not predictive but overall nutrient status may fall if not monitored. in contrast, one study in japan showed an inverse association between dietary calcium and the risk for copd [ ] . in an nih-aarp diet and health study, magnesium, iron, selenium, zinc, and copper intakes, both dietary and supplemental, were studied with respect to lung cancer. mineral supplementation did not affect lung cancer risk, yet dietary intake of calcium, along with vitamin d, and iron reduced the risk, and dietary intake of magnesium increased risk [ ] . boron has been shown to be protective against lung cancer, along with other nutrients, at levels of mg/day [ ] . there is some research showing that selenium is helpful, particularly for smokers, for improved fev. higher magnesium status is correlated to better fev but is not yet seen as an association. this may be due to magnesium's role as the vitamin d activator. there have been a few studies showing increased copper levels are related to decreased fev. some recent research has also shown that dietary zinc and iron are associated with reduced lung cancer, but the same was not seen with calcium, copper, magnesium, or selenium [ ] . low mineral bone density is prevalent at a higher rate among cystic fibrosis patients, and therefore supplementation with vitamin d, vitamin k , magnesium, calcium, and the trace minerals can be helpful [ ] . alpha-lipoic acid (ala) is a powerful antioxidant endogenously produced in the human body from foods such as yeast, organ meats, spinach, broccoli, and potatoes and is both water-and fat-soluble. ala, along with n-acetyl cysteine (nac), glycine, and vitamin c, is an important precursor to glutathione, which is a powerful endogenous antioxidant and the primary antioxidant in the lungs. ala has been shown to be anti-inflammatory in lung tissue in those with acute lung injury, and the proposed action is via inhibition of the nf-kappab signaling pathway [ ] . ala has also been shown to downregulate some cancerpromoting actions prevalent in lung cancer, likely by this same pathway [ ] . it also may alleviate nicotine-induced lung oxidative stress [ ] . n-acetyl cysteine (nac), another precursor to glutathione, is a powerful antioxidant on its own as well. in relation to the lungs, nac helps the clearance of mucus in the lungs by pulmonary cilia. this has been shown to be effective at - mg/day in divided doses [ ] . there is significant research on nac and lung health, showing improvement with nearly all lung issues, including nearly studies showing improvement for bronchitis [ ] , infectious diseases by reducing the bacterial count [ ] , smokers, and people with asthma and copd, through both its antioxidant effects and by reducing the viscosity of sputum and mucus. at an oral dose of mg/day, the mean glutathione concentration in lung tissue increased by % on one study [ ] . there are additional studies showing improvement for those with copd, asthma, cystic fibrosis, pulmonary fibrosis, and symptoms related to allergies or other infections. the dose that has been studied and has been shown to be most useful is mg twice daily and more effective if nebulized [ , ] . both ala and nac supplementation should be accompanied by vitamin b and the complex of b vitamins to prevent an elevation in liver enzymes (. fig. . ). there are several specialty labs that conduct micronutrient analysis and functional testing, such as genova diagnostics and spectracell. these tests can be useful for evaluating levels of individual nutrients as they function in the body, rather than just in serum, which is not an accurate indicator of tissue or functional status. patients suffering from copd, interstitial lung disease, and other diseases tend to have muscle and weight loss related to respiratory acidosis, and increasing weight and muscle mass helps with quality of life. respiratory acidosis occurs with co buildup where the lungs are no longer able to effectively exchange o and co . nutritional supplementation should attempt to reduce metabolic co production. fat metabolism produces less co than carbohydrate metabolism, so emphasizing a higher fat, lower carbohydrate diet can be helpful [ ] . in general, a high intake of omega- fatty acids is associated with poorer forced expiratory volume (fev) in patients with lung disease because of their pro-inflammatory nature. however, a complete fatty acid panel or a red blood cell membrane fatty acid test would reveal more details about the status of an individual's omega- pathway. certain omega- s and the work of their corresponding metabolizing enzymes such as elongase and delta- or delta- -desaturase may allow healthful omega- s (linoleic (la), gamma-linolenic (gla), lipoxins [ ] , prostaglandin series metabolites) to flow down an anti-inflammatory pathway instead. important cofactors for this pathway are vitamin b , vitamin b , vitamin b , vitamin b , biotin, vitamin c, zinc, and magnesium. lipid metabolism dysregulation is understood to be part of the pathogenesis of idiopathic pulmonary fibrosis. in ipf, free fatty acids play a role in the proliferation of fibroblasts. certain fats, in particular palmitic acid, oleic acid, and linoleic acid, are elevated in the lungs of those with ipf, whereas stearic acid is low. stearic acid is found in meat, poultry, fish, grain products, and milk and milk products. the palmitic, oleic, and linoleic acids enhance the tgf-ß -induced expression of α-smooth muscle actin (sma) and collagen type in mrc- cells, which can lead to fibrosis. stearic acid inhibits the levels of these fibrosing cells. stearic acid also improves the thrombogenic and atherogenic risk factor profiles [ ] . in one study on patients with copd, omega- fatty acids were found to reduce inflammation in bacterial infections of the lungs without suppressing the ability to clear the bacteria. those taking epa, dha, ala, and gla had improved exercise capacity and had lower risk of developing copd [ ] . although results have been mixed over the years possibly due to doses used in studies, a recent prospective study showed that pufas (omega- s) from fish help prevent lung cancer and can be part of treatment during lung cancer. in general, the strongest evidence for improved lung function and slowing decline is with the epa and dha forms of omega- fatty acids [ ] . because of toxicity issues in fish, increasing quality supplements vs fish intake may be more prudent. protein is essential for all lung conditions, and lack of it can result in poorer pulmonary function, decreased exercise capacity, and increased risk exacerbations. since many lung diseases have oxidative stress as a characteristic, it can cause protein carbonylation which may negatively affect dna expression and lipid membranes. nutritional supplementation with added protein and healthy carbohydrates can increase body weight and muscle strength and improve quality of life. those with copd, interstitial lung diseases, and others that affect oxygen absorption and co exhalation have greater levels of hypoxia and sometimes respiratory acidosis, which exacerbates the loss of muscle through oxidative stress and inflammation. supplementation of free essential amino acids versus complete proteins has been shown to help prevent muscle wasting among copd patients. muscle-building exercise is often prescribed for those with copd and interstitial lung diseases [ ] . supplemental l-carnitine at - g/day for - weeks increased the capacity of copd patients to rehabilitate and build muscle and helped inspiratory muscle strength. carbohydrates should be monitored for sufficient but not excessive levels. more co is produced with the utilization of carbs versus fats for energy. therefore, with gas exchange being an issue with most lung disorders, a slightly higher fat and lower carbohydrate diet may be indicated. it is worth mentioning fiber for a moment, as it is mostly delivered in carbohydrate-rich foods. there is evidence that consuming whole fruits and vegetables higher in dietary fiber is associated with reduced severity of asthma and copd [ ] . a diet that derives its carbohydrates from vegetables and fruits rather than from processed carbohydrates such as grains, breads, pasta, or added sugars will deliver fewer carbohydrate grams. glutathione (gsh), a tripeptide composed of cysteine, glutamine, and glycine and produced from methionine, is in every cell in the body. it is the most powerful and abundant endogenous antioxidant in the airway epithelial lining and is responsible for detoxification of electrophilic compounds, the scavenging of free radicals, and modulation of cellular processes such as dna synthesis and repair, differentiation, apoptosis, and immune function [ ] . it is also a heavy metal chelator. it is more effective than some other antioxidants because it is intracellular and extracellular. in isolated type ii alveolar epithelial cells, extracellular glutathione inhibits hyperoxia-induced injury, inhibits pro-inflammatory cytokine release, and promotes cell growth. it is obviously very important to maintaining lung function as this is the inflammatory process that begins lung cell or tissue damage, as mentioned above. the highest levels of glutathione concentrations in the body are in the lungs, liver, and brain. gsh depletion leads to activation of nf-kb (pro-inflammatory signaling) and increased pro-inflammatory gene transcription and cytokine release from histone deacetylase suppression in epithelial cells. total and reduced gsh concentrations are much lower in people with ards, pulmonary fibrosis, and hypersensitivity pneumonitis than observed in healthy adults. alterations in alveolar and lung gsh metabolism are widely recognized as a central feature of many inflammatory lung diseases such as idiopathic pulmonary fibrosis, acute respiratory distress syndrome, cystic fibrosis, and asthma [ ] . we make glutathione in the body with cysteine and methionine, and it is difficult to take exogenously because digestion can destroy it. the precursors of cysteine (essential), glutamine, and glycine and cofactors (vitamin c, vitamin e, vitamins b , b , b , and b , folate (b ), minerals selenium, magnesium, and zinc, and alpha-lipoic acid, see below) are therefore recommended so that the body can produce it on its own. the two enzymes necessary to produce it, gamma-glutamylcysteine synthetase and glutathione synthetase, must also be functioning well. we also recycle glutathione if the precursors and cofactors are available. cysteine is usually the most rate-limiting precursor, and many people supplement with n-acetylcysteine to provide the body with this nutrient. although glutathione is produced in every cell of the body, the greatest production is in the liver, so focusing on liver health is important to maintain good glutathione production. production declines with age and with lung disease, as well as other conditions. there are very few foods containing glutathione; they are raw or very rare meat, especially liver, unpasteurized milk and other unpasteurized dairy products, and freshly picked fruits and vegetables, such as avocado and asparagus. however, as mentioned earlier, it may be destroyed during digestion. glutathione contains sulfur molecules, which may be why foods high in sulfur help to boost its natural production in the body. these foods include: cruciferous vegetables, such as broccoli, cauliflower, brussels sprouts, and bok choy allium vegetables, such as garlic and onions eggs nuts legumes lean protein, such as fish and chicken other foods and herbs that help to naturally boost glutathione levels include: milk thistle (a liver-regenerating herb) flaxseed guso seaweed whey glutathione is also negatively affected by insomnia. getting enough rest on a regular basis can help increase levels. addressing a drop in glutathione for lung health involves maintaining good levels of the precursors and cofactors mentioned above. a good way to bring in the less abundant amino acid cysteine is to take n-acetylcysteine (nac). doses of - mg were more effective than placebo in reducing symptoms [ ] . supplemental selenium can also help with glutathione production. glutathione supplementation has also become more effective. there are several forms, from capsules to topical liposomal, which have shown good absorption. inhaled gsh has good research for use in cystic fibrosis (cf), chronic otitis media with effusion (ome), hiv seropositive individuals, idiopathic pulmonary fibrosis (ipf), and chronic rhinitis. it is not recommended for asthma due to significant side effects, and additional evidence is needed to determine if use with emphysema is recommended although theoretically it should be useful. it is also not recommended to use inhaled gsh during cancer chemotherapy treatment as it may interfere with the medication's actions. the mechanism of action of inhaled glutathione is limited to the upper airways and lungs and does not seem to affect serum levels. before considering inhaled gsh treatment, the patient should undergo urine sulfite sensitivity testing using a readily available special test strip called "em-quant sulfite test. " if positive, inhaled gsh should not be used as bronchoconstriction may occur. the recommended dose is - mg per day, depending on response, and whether inhaled gsh is considered safe. efficacy should be tested using a baseline pulmonary function test and a follow-up test after a prescribed time later [ ] (. fig. . , box . ). there are also serum tests for glutathione levels. these cofactors are vitamin c; vitamin e; vitamins b , b , b , and b ; folate (b ); minerals selenium, magnesium, and zinc; and alpha-lipoic acid. what do the glutathione cofactors do that makes them so important? direct cysteine toward glutathione production and increase cellular uptake of cysteine help form the glutathione molecule out of the three precursor amino acids help recycle glutathione from its oxidized gssg form back to its reduced (active) gsh form help maintain glutathione levels and keep the gssg-gsh ratio balanced recycle each other, improving overall antioxidant activity stimulate the activity of the whole glutathione enzymatic system co is a fat-soluble compound produced endogenously and also available through food and supplementation. it is required in the production of atp, is a powerful antioxidant, and therefore is helpful against oxidative stress, an important issue in lung disease. coq achieves its strong effects through a set of different mechanisms. it influences genes through its epigenetic effect to reduce inflammation, helps with the immune system, and even reduces aging by reducing systemic oxidative stress and mitochondrial aging [ ] . lungs are the most susceptible organ to oxidant damage because they interact directly with oxygen. therefore, it makes sense that antioxidants, and those that especially affect the lungs, are helpful in tissue and lung cell preservation [ ] . coq levels are significantly lower in those with copd and asthma with insignificant amounts of research on the levels of coq with other lung issues. it has been shown that supplementing patients with coq resulted in measurable benefits. in one study, patients with copd using steroids to reduce inflammation were able to reduce their steroid dosage when using coq [ ] . in another study, benefits were shown for copd patients during exercise, measuring performance, tissue oxygenation, and heart rate at a low dosage of mg/day [ ] . the levels of coq in the blood have been shown to indicate the degree of systemic oxidative stress, which implies it could be used as a marker to assess copd [ ] . several studies confirm the beneficial role of coq in decreasing oxidative stress, cardiovascular risk, and modulating inflammation during aging. dosage levels of mg/ day of coq have been shown to be therapeutic. however, in the reduced, more absorbable form, ubiquinol, mg/ day, was shown to be as effective. there is a wide range of toxins and anti-nutrients that can significantly impact the respiratory system. this can occur through acute or chronic exposure to these agents. the earth's air is the source of oxygen, and the lungs provide access to that oxygen to support life. the human need for oxygen is precarious because humans can only survive for about minutes without the precious gas. from about to sometime between and in europe and the united states, the ramp-up of new industrial revolution manufacturing processes opened a new era of increasing chemical and heavy metal atmospheric contamination. these pollutants can enter the body through breathing the polluted air. the more concentrated atmospheric pollutant densities cluster around areas of dense population. the dirty air provides a serious direct threat to those with respiratory diseases. an integrative and functional approach to assessing an individual with respiratory disease needs to include consideration of potential environmental contributors to the etiology of a condition. . table . lists environmental pollutants that are known to promote lung pathology. a study published in the canadian respiratory journal examined exhaled fractional nitric oxide (feno)an indicator of inflammation in the lungs -in school children at three different schools located three different distances from a large steel mill [ ] . steel processing is known to be a source of ambient iron, nickel, lead, copper, vanadium, and zinc. the study found statistically significant differences in feno between the two closer schools compared to the farthest school from the mill, indicating potential increased lung inflammation caused by heavy metals and/or air pollutants [ ] . although acute metal toxicity is possible, chronic, low-grade exposure is more common and may contribute to respiratory complications and disease. an individual's ability to vitamin c -as an antioxidant, it assists glutathione in this function and has been shown scientifically to raise glutathione levels short term; it is recycled by glutathione from its oxidized state back to its active state, thus strengthening antioxidant defenses; vitamin c also recycles vitamin e and alpha-lipoic acid vitamin e -as an antioxidant it also assists glutathione in eliminating free radicals much like vitamin c; it is also required for the proper functioning of glutathione enzymes; it recycles vitamin c and alpha-lipoic acid b vitamins -vitamins b and b maintain glutathione and its enzymes in their active forms; vitamin b participates in the formation of a glutathione molecule; vitamin b influences glutathione synthesis indirectly as it is important for the proper functioning of amino acids including gsh precursors; vitamin b increases the amount of magnesium (a vital cofactor) that can enter cells; folate (b ) pushes cysteine toward glutathione production rather than homocysteine production; folate and vitamin b work together in amino acid metabolism and protein synthesis. you can read more vitamin b deficiency and its effect on immune health at http://www. immunehealthscience. com/vitamin-b -deficiency. html selenium -part of the enzyme glutathione peroxidase (gpx). glutathione peroxidases, also known as selenoproteins, are a family of antioxidant enzymes that speed up the reaction between glutathione and free radicals magnesium -required for the proper functioning of the enzyme gamma-glutamyl transpeptidase (ggt) involved in the synthesis of glutathione zinc -zinc deficiency reduces glutathione levels, especially in red blood cells. however, zinc levels above normal have pro-oxidant properties and reduce glutathione too alpha-lipoic acid -an antioxidant produced by the body; it has been scientifically proven to enhance and maintain glutathione levels by stimulating enzymes involved in the synthesis of glutathione; it also helps increase the cellular uptake of cysteine, the crucial building block of glutathione; in addition, alpha-lipoic acid recycles vitamins c and e based on data from ref. [ ] eliminate these metals via detoxification in conjunction with gastrointestinal health and other factors can serve as important factors in whether or not these metals accumulate in the body. chronic arsenic exposure may be linked to respiratory complications [ ] . chronic arsenic ingestion via contaminated drinking water may be connected to respiratory symptoms such as chronic cough, shortness of breath, blood in sputum, and abnormal breath sounds [ ] . arsenic can also be ingested through foods such as rice and rice products, shellfish, and seaweeds, which have been shown to have high levels of inorganic arsenic (more toxic than organic arsenic found in fish) [ ] . however, ingested inorganic arsenic is typically biotransformed and excreted in the urine [ ] . that said, altered biotransformation has been observed depending on an individual's age, gender, nutritional status, and genetic polymorphisms responsible for the biotransformation of inorganic arsenic [ ] . chronic inhalation versus ingestion may result in irritation of the throat and respiratory tract [ ] . individuals most affected by arsenic exposure are children, nursing children, and infants of exposed pregnant mothers [ ] . acute inhalation of cadmium may lead to dyspnea and coughing [ ] . long-term exposure to cadmium has been reported to contribute to emphysema, dyspnea, and inflammation of the nose, pharynx, and larynx [ ] . individuals most affected by cadmium toxicity are those with occupations with cadmium exposure, such as those who work in certain types of factories, women, due to higher intestinal absorption because of low iron stores, and residents of asia due to high intake of rice grown in contaminated soil [ ] . the us national health and nutrition examination survey (nhanes) demonstrated an association between obstructive lung disease and serum lead and cadmium concentrations in the blood, where cadmium was shown to partially mediate the association between smoking and obstructive lung disease [ ] . in the korean nhanes, obstructive lung function was found to be associated with higher serum blood levels of cadmium and lead as well [ ] . the specific mechanism of heavy metal burden and its effects on respiratory health must be further investigated. although testing and treatment of heavy metal burden have its limitations, it is worth considering as heavy metal accumulation can wreak havoc on the body. an example of heavy metal testing that can be used in practice is urine provocation testing with a chelating agent, such as fda-approved dmsa. eliminating heavy metals from the body can be potentially harmful and requires careful monitoring and guidance by an experienced healthcare professional. air pollutants that are used as indicators of air quality are carbon monoxide, lead, nitrogen dioxide, ozone, particles, and sulfur dioxide [ ] . air pollution has been shown to have adverse effects on human health [ ] . a systematic review and meta-analysis done in china showed an association between respiratory disease and ambient nitrogen dioxide, which is increased through fuel combustion, industrial production, and fuel exhaust [ ] . diesel exhaust particles in particular have been associated with an increase in cytokines such as il- , il- , and ige in nasal mucosa [ ] . nitrogen dioxide in particular can potentially contribute to respiratory disease as it is a free radical that is highly reactive and poorly water-soluble and can be deposited in the lungs when inhaled [ ] . in another study performed in england, air concentration of nitrogen dioxide was significantly associated with respiratory hospital admissions [ ] . other pollutants, such as fine particulate matter and ozone, have been shown to significantly affect respiratory function in copd patients [ ] . increased ozone exposure has also been associated with increased airway inflammation and respiratory symptoms along with decreased respiratory function in children [ ] . optimization of nutrition and antioxidant status is essential to combating the potential health effects of air pollutants. . [ ] . it would be reasonable to assume having adequate stores and ability to utilize these nutrients may protect against other insults to the respiratory system as discussed in this section through their anti-inflammatory properties. acute and chronic exposure to certain chemicals can also pose a risk to respiratory health. obtaining a full occupational and social history when assessing individuals is important in order to identify any potential exposure to chemicals. one of the most well-known and common toxic chemical exposures that affects respiratory health is cigarette smoke. smoking cigarettes has been identified as a main cause of copd [ ] . increased oxidative stress from inhaling cigarette smoke appears to activate the nf-kb inflammatory pathway, increasing the production of pro-inflammatory cytokines such as interleukin (il)- , il- , and il- and tumor necrosis factor-ɑ (tnf-ɑ) [ ] . it also appears to reduce anti-inflammatory cytokines such as il- [ ] . electronic cigarettes, or e-cigs, have been increasing in popularity in recent years and are marketed as a better alternative to tobacco cigarettes. however, recent evidence suggests that the vapor and associated chemicals produced by e-cigs may be harmful to the respiratory system, although further research is needed to determine the mechanism [ , ] . exposure to metalworking fluid aerosols has been associated with asthma, hypersensitivity pneumonitis, impaired lung function, allergic alveolitis, and sinusitis [ ] . a review also identified an association between occupational exposure to pesticides and increased risk of asthma and chronic bronchitis [ ] . there are many chemicals that are toxic when inhaled. for example, inhalation of chlorine is toxic to the lungs, where low doses can cause airway injury and high doses can cause both airway and alveolar injury [ ] . these injuries can manifest as dyspnea, hypoxemia, pulmonary edema, and pneumonitis [ ] . high doses of carbon dioxide, such as that released from dry ice, can also induce respiratory failure. stress may also play a role in respiratory health and the body's ability to combat insults imposed on the respiratory system. from a physiological standpoint, it is worth noting that acute stress via activation of the sympathetic nervous system increases ventilation through the production of glucocorticoids [ ] . repeated acute stress may also affect growth and repair mechanisms [ ] . chronic biological stress in the form of infections can also be inflammatory and negatively affect the immune system and may affect an individual's susceptibility to respiratory complications. see the chronic infections and respiratory health section on page # below for further information on this association. however, appropriate amounts of physical stress, such as in the form of exercise, can be beneficial to respiratory health. some research has indicated a benefit of aerobic exercise to respiratory muscle strength in cystic fibrosis patients [ ] . chronic stress can be defined as recurrent acute stress or inability to moderate acute stress responses [ ] . this can be in the form of physical or emotional stress. chronic stress and negative emotions such as depression, anxiety, and anger may be linked to endocrine and immune processes [ ] . immunoglobulin e (ige) and cytokine production, as well as respiratory inflammation, are markers that characterize the asthma response and have been shown to respond to stress in some capacity [ ] . it has been hypothesized that increased stress may increase susceptibility to air pollution given its effects on the inflammatory response [ ] . another connection between emotions and respiratory health is acknowledged in east asian medicine, noting the association between the lungs and feelings of sadness, grief, and anxiety [ ] (. table . ). asthma is a chronic inflammatory lung disease, triggered by either an ige allergic reaction or nonallergic factors, and results in reversible airway obstruction and inflammation of the airway [ ] . it is characterized by recurrent episodes of wheezing, breathlessness, coughing, and chest tightness [ ] . severe asthma or asthma that is chronic or poorly controlled may lead to airway and lung remodeling that involves deposition of fibrotic tissue which leads to constriction of the bronchi [ ] . although the exact mechanisms have not yet been identified, compromised nutritional status, such as deficiencies in selenium, zinc, and vitamins a, c, d, and e, has been connected to asthma [ ] . the pathophysiology of asthma, nutrition considerations, genotypic characteristics, and lifestyle influences will be discussed in this section. there are numerous potential triggers to the development and/or exacerbation of asthma which can be summarized in box . . the various causes of asthma have led to the classification of several different subtypes and endotypes of asthma in hopes of choosing more targeted treatments. the pathophysiology of asthma is complex and not fully understood, due in part to its heterogeneous nature, which necessitates its organization into individual phenotypes and endotypes. this organization is important to be able to utilize targeted treatments by identifying the root causes of the symptoms. however, more research is needed to more clearly identify the specific pathological mechanisms of each phenotype and particular treatment responses [ ] . two of the most common asthma phenotypes are allergic and nonallergic asthma [ ] ; allergic is characterized by increased th immunity (th high) and nonallergic defined by varying mechanisms depending on the trigger (th low) [ ] (see also chap. ) . allergic asthma involves the ingestion of typically harmless environmental triggers (listed in . table . ) by antigenpresenting cells in the bronchi, which interact with immature helper t cells that, in turn, trigger an unwarranted allergic response [ ] . this reaction occurs from repeated exposure to a trigger and is referred to as the type hypersensitivity response [ ] . this increased th immunity upregulates eosinophilic inflammation, tissue damage, airway hyperresponsiveness, and bronchoconstriction [ ] . mast cell activation disorders, which is characterized by diseases and conditions related to mast cell mediators and the activation of mast cells, must also be considered when addressing allergic asthma [ ] . in contrast, nonallergic asthma can be caused by other factors such as anxiety, exercise, stress, dry air, cold air, viruses, hyperventilation, smoke, or other irritants [ ] . . inhaled cadmium (cd) is deposited in the alveoli where it is then absorbed into the bloodstream cd is transported to erythrocytes or bound to albumin, where it is then taken up by the liver to form a complex with metallothionein (mt) cd interferes with the absorption of zinc and competes for the same enzyme binding sites enzymatic activity of zinc-dependent enzymes reduces preferential binding of cd to mt can cause zinc deficiency altered biotransformation and excretion of ingested arsenic via contaminated water are linked to respiratory complications chronic inhalation of arsenic may result in irritation of respiratory tract diesel exhaust particles in particular have been associated with increase in cytokines such as il- , il- , and ige in nasal mucosa [ ] nitrogen dioxide is a free radical that is highly reactive and poorly water-soluble and can be deposited in the lungs when inhaled [ ] rising pollen and mold counts [ ] increasing ozone [ ] chemicals increased oxidative stress from inhaling cigarette smoke may activate the nf-kb inflammatory pathway, increasing the production of pro-inflammatory cytokines such as interleukin (il)- , il- , il- , tumor necrosis factor-ɑ(tnf-ɑ) cigarette smoke may reduce anti-inflammatory cytokines such as il- [ ] stress repeated acute stress may also affect growth and repair mechanisms [ ] ige and cytokine production, as well as respiratory inflammation, are markers that characterize the asthma response and have been shown to respond to stress in some capacity [ ] increased stress may increase susceptibility to air pollution given its effects on the inflammatory response [ ] ( individuals suffering from nonallergic asthma will tend to be less responsive to th -targeted treatments due to a differing immune response at play [ ] . some of the additional proposed phenotypes are eosinophilic, exacerbation-prone, exercise-induced, fixed obstruction/airflow limitation, poorly steroid-responsive, and adult-onset obesity-related [ ] . several of the proposed endotypes are summarized in . the american partnership for eosinophilic disorders defines eosinophilic asthma as a type of asthma characterized by especially high levels of eosinophils, more commonly developed later in adulthood, although may occur in some children [ ] . many with eosinophilic asthma do not have underlying allergies or history of allergic conditions such as eczema, food allergy, and hay fever, which are thought to be seen more in people with allergic asthma [ ] . in contrast to allergic asthma, the cause of eosinophilic asthma is still unknown. histamine intolerance must also be considered in assessing the root cause of asthma. ingesting histamine-rich foods and beverages such as bananas, grapes, strawberries, citrus fruits, tomatoes, nuts, chocolate, pineapples, fish, spinach, fermented foods, and beverages [ ] has been shown to provoke a histamine response that may result in asthma exacerbations, among many other potential signs and symptoms [ ] . disruptions in redox, or oxidation/reduction, reactions in addition to hindered antioxidant defense have been . usually not responsive to glucocorticoids [ ] based on data from ref. [ ] found to be a risk factor for asthma severity and development [ ] . the levels of glutathione, one of the lung's most predominant antioxidants in both reduced and unreduced forms, are thought to be important for lung homeostasis and tied to asthma [ ] . more research is needed to determine the exact differences in the pathophysiologies of the various subtypes of asthma in order to develop more targeted treatments. minerals such as zinc, selenium, copper, and manganese may serve as cofactors to major enzymes with antioxidant activity in the lung, such as superoxide dismutase, catalase, and glutathione peroxidase [ ] . asthma has been associated with decreased activity of these enzymes [ ] . low selenium intake has been associated with multiple chronic diseases including asthma [ ] . selenium serves as a cofactor to glutathione peroxidase, an enzyme with antioxidant activity in the lung that is responsible for maintaining gsh/gssg redox balance [ ] . imbalance between oxidants and antioxidants seems to serve an important role in asthma. levels of nonenzymatic antioxidants glutathione, ascorbic acid, alpha-tocopherol, lycopene, and beta-carotene, in addition to antioxidant enzymes superoxide dismutase (sod) and glutathione peroxidase, were significantly lower in asthmatic children compared to healthy controls [ ] . the amino acids glycine and glutamine, which are important in glutathione synthesis, were also found to be significantly lower in children with asthma [ ] . dha has also been found to be abundant in airway mucosa, where it is decreased in individuals with asthma and cystic fibrosis [ ] . magnesium is known to elicit the relaxation of bronchial smooth muscle, decrease responsiveness to histamine, have an anti-inflammatory effect, and decrease the susceptibility of animals to developing anaphylactic reactions [ ] . it is estimated that two-thirds of the population in the western world is not consuming the recommended daily allowance of magnesium [ ] . magnesium can be used intravenously as an effective treatment of acute asthma attacks. one double-blind controlled trial that used . g of magnesium sulfate when patients did not respond to treatment with beta-agonists found decreased likelihood of hospitalization and improved lung function [ ] . magnesium sulfate as an adjunct therapy with bronchodilators and steroids has also been shown to have a benefit in children with moderate to severe asthma [ ] . although the exact mechanism is not yet known, magnesium is thought to increase glutathione concentrations in the lung [ ] . more research is needed to determine additional associations between specific nutrients and asthma. however, optimization of the nutrients discussed in this section has the potential to reduce the severity and/or progression of asthma (. fig. . ) . asthma has a strong genetic component, with more than genes associated with it in varying degrees across many populations [ ] . more recent potential genetic associations include filaggrin, which encodes for the epithelial barrier; ormdl , which encodes transmembrane protein; beta- adrenergic receptor gene, expressed throughout smooth muscle and epithelial cells of the lung; and interleukin- receptor gene, which has a variant associated with elevated ige [ ] . a systematic review and meta-analysis showed that deficiencies in selenium, zinc, vitamins a, c, d, and e, and low fruit and vegetable intake could be associated with the development of asthma [ ] . although this data is tenuous due to lack of randomized controlled trials, it does give some indication of the relationship between nutrition status and dietary patterns with respect to asthma development. more research needs to be done to isolate the impact of these nutrients and dietary patterns on asthma prevention and development. a review conducted by berthon and wood noted the protective effects of the mediterranean diet for allergic respiratory diseases as evidenced by epidemiological studies. this diet emphasizes minimally processed plant foods in the form of fruit, vegetables, cereals, beans, breads, nuts, seeds, and olive oil and low to moderate intake of dairy, poultry, fish, and wine, as well as low intake of red meat [ ] . this association was the strongest in children, where the mediterranean diet had a protective effect on atopy, wheezing, and asthma symptoms [ ] . however, there is less data available to support this pattern in adults. the same review noted an association between the "western" diet, which emphasizes refined grains, red and cured meats, french fries, sweets and desserts, and highfat dairy products and increased risk of asthma in children [ ] . a meta-analysis and systematic review done in showed a reduction of risk in childhood wheezing with high fruit and vegetable intake and also showed negative association between fruit and vegetable intake and asthma risk in adults and children [ ] . in contrast, food allergy has been especially linked with allergic asthma in children [ ] . a study examining food allergy in asthmatic children identified higher serum levels of ige in asthmatic children compared to healthy controls, where all asthmatic children in the study were also identified as having a positive skin prick test (spt) to various food allergens [ ] . a study done on children under the age of diagnosed with asthma, with or without allergic rhinitis, was placed on a meat-based formula of carrots, beef, broccoli, and apricots for weeks. it was found that % had nearly complete resolution of symptoms [ ] . this same study also found that the most common food triggers were milk, egg, chocolate, soy, legumes, and grains [ ] . while food allergy as a cause of asthma is more common in children, hidden food allergy has been reported to be the root cause of asthma in around % of adults [ ] . improvement in respiratory symptoms was also seen in a small study of adults given an antigen-free elemental diet in a hospital setting [ ] . removal of food triggers has also been linked to improvement in exercise-induced asthma [ ] . identifying food allergies can be a complicated process because many of the testing methodologies such as skin prick tests (spts) and blood tests can yield false-positive results for up to - % of cases, according to the food allergy research & education organization [ ] . a food elimination diet and/or oral food challenge can be a powerful tool in determining food allergy specific to asthma symptoms, where a dietitian or nutritionist in conjunction with physician and/or allergist can serve an important role through this process to support the individual. oxidative stress may play a key role in the development of asthma, which can also be true for the development of chronic diseases such as cardiovascular disease, diabetes, and cancer [ ] . it has been shown that obesity may be a risk factor for people with and without allergy and may worsen pre-existing asthma [ ] . individuals with asthma are twice as likely to have gastroesophageal reflux disease (gerd) than people who do not have asthma, especially those resistant to treatment [ ] . celiac disease and asthma have also been linked. an italian cohort study was done that showed a significant association between treated asthma and celiac disease, where antibiotic exposure in the first year of life was controlled for and not found to contribute to this association [ ] . it has also been found that individuals with celiac disease following a glutenfree diet experienced improvement in asthma symptoms [ ] . it is well-known that toxic exposure to particulate matter, airborne pollutants, or cigarette smoke can trigger asthma symptoms [ ] . more specifically, a dose-dependent relationship between cigarette smoke exposure and rates of asthma has been shown [ ] . traffic density and asthma exacerbations have also been clearly demonstrated [ ] . certain medications may also serve as triggers to asthma. aspirin-exacerbated respiratory disease (aerd) is considered another asthma subtype caused by nonsteroidal anti-inflammatory drugs (nsaids) and is characterized by asthma, chronic rhinosinusitis, and acute respiratory reactions [ ] . in addition, overuse of antibiotics in childhood has been linked to asthma [ ] , indicating a connection between the microbiome and asthma development. allergic bronchopulmonary mycosis (abpm) noted in . table . is caused by a hypersensitivity reaction to fungal colonization of the airways [ ] . this is typically caused by the fungus aspergillus fumigatus. without treatment, this may lead to fixed airflow obstruction and bronchiectasis [ ] . the progression of asthma is complex and multifaceted, from preconception through childhood and adulthood. research suggests that early life events are largely predictive for regulatory mechanisms within the pulmonary immune system [ ] . for example, prenatal exposure to a farming environment, one rich in microbial compounds, is thought to influence innate immune patterning in the mother which may affect the development of the neonatal immune system [ ] . this influence in immune patterning can be seen through higher expression of toll-like receptors and and cd on peripheral blood cells, which implies possible desensitization to allergens in children [ ] . t regulatory cells, which serve an important role in immune regulation and are thought to play an important role in asthma by suppressing the th inflammatory response to harmless air particles, have been shown to be impaired in the cord blood of neonates at hereditary risk for allergy [ ] . in the study performed by singh et al. looking at serum ige and cutaneous sensitivity to food allergens in asthmatic children here was a negative correlation of total ige and duration of breastfeeding, indicating a connection between breastfeeding and the immune response [ ] . additionally, reduced maternal intake of vitamins d and e and zinc during pregnancy has been associated with increased asthma symptoms in children [ , ] . vitamin d has been associated with the maintenance and/or development of the t regulatory cells stated earlier in mice; however more research is needed to determine a definitive association in humans [ ] . a clinical trial performed on non-smoking asthmatic patients showed higher vitamin d levels were associated with greater lung function; furthermore, supplementation with vitamin d showed improved treatment response to glucocorticoids [ ] . vitamin d may also directly increase the antiinflammatory cytokine, interleukin (il)- and also enhance steroid-induced il- production (see . fig. . ) [ ] . more research is needed to determine the exact mechanism of vitamin d in asthma and respiratory disease. beta-agonists, combined with corticosteroids, serve as the primary conventional therapy [ ] . typically, a short-acting beta-agonist will first be prescribed to manage symptoms as needed, where low-dose inhaled corticosteroids may also be prescribed [ ] . if symptoms persist, it is recommended to evaluate problems such as adherence to use, inhaler technique, or persistent allergen exposure and comorbidities [ ] . once these are ruled out, the step-up treatment is a combination of an inhaled corticosteroid with a long-acting beta-agonist [ ] . a summary of other conventional treatments and their mechanisms can be found in . table . below. unfortunately, conventional methods for the treatment of asthma may have harmful side effects. for example, the use of . fig. . effect of asthma treatments on regulatory pathways. (reprinted from lloyd and hawrylowicz [ ] . with permission from elsevier) systemic glucocorticoids may lead to immunosuppression, cataracts, and osteoporosis, where long-acting beta-agonists have the potential of increasing asthma exacerbation risk and death [ ] . beta-agonist desensitization is thought to be one of the reasons for increasing asthma exacerbation risk and death [ ] . related to several subtypes of asthma and their differing pathophysiologies, it is important to first determine the subtype before deciding on treatment. for example, in an individual with allergic asthma, this could be a potentially simple fix once the allergen that exacerbates symptoms is identified. a more conventional approach may involve starting the individual on an inhaled corticosteroid or an ige antagonist (i.e., omalizumab) [ ] , rather than identifying the root cause of the patient's symptoms. while medications may be warranted until the trigger is identified, finding the underlying causes may not be common practice in many conventional settings. in contrast, the ifmnt assessment takes a much deeper dive into identifying triggers and any nutrient insufficiencies, inflammation or immune dysregulation, biochemical individuality, lifestyle, energy dysfunction, toxic load, sleep, and stress issues are taken into account. with this information, the practitioner can make more targeted dietary, lifestyle, and supplement recommendations to obtain sustained resolution of symptoms by treating the root cause (. table a -year-old female presented with a complaint of reactive airway disease, which was diagnosed as asthma and had been prescribed inhalers. she reported that she felt like she had difficulty breathing most of her life, especially when exercising. however, her condition was not severe enough to seek help until she was years of age. she reported a lot of stress during this time related to applying for a postgraduate training position. she also reported year prior to diagnosis developing new allergic symptoms. her past medical history was significant for conditions related to airways, including chronic sinus infections, strep throat, bronchitis, and recurrent pneumonia. she could not remember the last time she felt well but assumed it was sometime as a young child. her nutrition and health goals were to breathe better and to not have to rely on inhalers. the following data was collected on her initial visit. . methyl xanthine found in tea used less commonly due to side effects relaxes airways due to inhibition of phosphodiesterases; acts as a functional antagonist in airway smooth muscle [ ] based on data from ref. [ ] . table . summary of an integrative and functional medical nutrition therapy assessment adequacy of nutrient-dense foods to begin to assess nutritional status organic or nonorganic to assess toxic load and nutrient intake food preparation and processing to assess nutrient content and identify potential contaminants (e.g., plastic endocrine disruptors) assess food sensitivities or intolerances to identify potential triggers microbiome status: assess comprehensive digestive stool analysis for microbiology and fermented food intake; history of antibiotics or microbiota agonists (medications, toxins, stress, etc.) toxin intake via plastics or inhalation and skin absorption which may affect immune response assess flavonoids intake as they are antioxidant and anti-inflammatory compounds with mast cell inhibitory action; adequacy may reduce airway reactivity consider celiac disease and gluten intake as potential inflammatory antigens mineral assess and restore zinc, selenium, magnesium, manganese, iron, and iodine status to normal reference. caution to not supplement or intake of food sources higher than reference antioxidants assess and restore antioxidant balance; vitamins a, c, d, and e and glutathione assess quercetin intake (leafy vegetables, broccoli, red onions, peppers, apples, grapes, black and green tea, red wine) as it may act as mast-cell stabilizing agent inhibiting release of histamine, tnf-alpha release, formation of prostaglandin d , reducing interleukin production consider supplementation of quercetin if quercetin intake is low [ ] protein status assess and restore to support connective tissue and immune status ensure adequate glutamine and glycine intake oils/lipid/fatty acids assess fatty acid balance as dha important in lung tissue integrity assess adequate serum cholesterol and fat intake to support lipid bilayer important for cellular function in lung (epithelial cells, surfactant production, etc.) methylation assess methylation status and detoxification capacity of toxins related to asthma exacerbation; important assessment biomarkers suggested: mcv/mch, homocysteine, methylmalonic acid, rbc folate, genomic methylation snps inflammation/immune dysregulation assess asthma biomarkers to help identify root cause (see . fig. extreme exhaustion, depression, add, anxiety (accompanied by panic attacks), constipation, pain in legs, neuropathy in feet (numbness and tingling), rapid heartbeat, and a very severe rash on feet known as chilblains. utis -recurrent as a child. poor immune function (frequent infections). antibiotic use (very frequent from childhood into adulthood). sinus infections, strep throat, and bronchitis -she had recurrent sinus infections and strep throat about once a year every year and often this would lead to bronchitis, she could not remember if she had these issues before middle school. depression, anxiety, add. acne. peptic ulcers. yeast infections -multiple throughout college. eczema. two recent episodes of pneumonia the last episode resulted in her asthma diagnosis. asthma. evaluate exposure to fungus to identify allergic bronchopulmonary mycosis assess individual's medication history, considering short-and long-term use of conventional treatments evaluate exposure to particulate matter, airborne pollutants, cigarette smoke, or toxic metals such as cadmium and arsenic sleep and stress assess sleep adequacy ( - hours with -hour rem sleep) and quality (good sleep hygiene with little light/ sound/emf disturbance) to support detoxification of toxins that may worsen respiratory status and aid in repair of damaged lung tissue . periostin plays a role in the pathogenesis of allergic diseases, including asthma, as it is associated as a downstream molecule of the cytokine, il- . periostin is used as a biomarker for type immunity and can be used to determine the potential effectiveness of medications used to treat asthma, such as anti-ige antibodies and anti-il- antibodies. asthmatic patients with high serum periostin tend to be aspirin intolerant, eosinophilic, late asthma onset, and have a high nitric oxide fraction. high periostin can also indicate a reduced response to inhaled corticosteroids [ , ] . periostin in the right panel is stained brown and is localized in the thickened basement membrane in asthmatic patients. (reprinted from izuhara et al. [ ] . with permission from the korean academy of asthma, allergy and clinical immunology) for several weeks, led to being immobile for almost weeks th- th grade: was often sick (strep, sinus infections, bronchitis); described it as being constantly sick from fall through winter every year; also developed eating disorder during this time; had severe menstrual cramps (induced vomiting) accompanied by acne, which led to being put on birth control at age as a precursor to accutane (never prescribed); chronic constipation starting during this time university freshman -sophomore year: eating disorder was most severe during this time. first semester of freshman year: developed digestion issues, after eating certain foods (especially mexican or salsas), stomach would become distended, experienced pain, and often would result in vomiting. pain so severe during finals week she was admitted to er with no diagnosis. ct scan revealed possible peptic ulcers. junior-senior year: depression, anxiety, and inability to focus were most severe during this time which resulted in missing a lot of class and struggling as a student; suffered multiple panic attacks; gained a lot of weight (from to lb); end of senior year became engaged to be married -moved to dallas, tx. lived in dallas for months, continued to experience depression and anxiety and weight gain, and moved back to home state initially started running (~ miles a day) and experiencing inability to breathe, diagnosed with pneumonia, prescribed inhaler to help with running; other symptoms: eczema around the eyes and neck (after running outside), pain in calves, numbness and poor circulation in feet (pulse not detected by several health professionals), and development of chilblain rash (very painful, itching, lasts about - weeks from development to resolution); increased running -ran a half-marathon. visited pcp and several specialists for help with chilblain rash with no resolution or diagnosis; lost a lot of weight (from to lb). ongoing increased depression, anxiety, and inability to focus; pcp rx cymbalta (depression and anxiety); cymbalta discontinued after ~ months (did not tolerate side effects), continued psychological therapy for several months; chilblain rash continued. stopped running long distances. gained weight back (from to lb); subsequently saw blog for integrative rd and followed suggestion to eliminate gluten and focusing on whole foods diet. chilblains and eczema began to resolve while following integrative rd recommendations of gluten-free diet with some improvements. however, difficulty breathing got worse, and diagnosis of asthma was made with fast-acting inhaler used for exercise; as time progressed, breathing continued to worsen, led to daily inhaler use. weight at this time is still at around lb. high dairy diet (consumed dairy products at most meals and snacks), consumed three smaller meals with three snacks in between meals and snacks balanced with protein, fat, and carbs, with carbs coming from fruits and vegetables and fat mainly from full fat cheese, greek yogurt, and butter mostly nonorganic produce and commercially raised meats digestion, assimilation, and elimination hx of peptic ulcers and chronic constipation (bm ~ - times a month) bms currently at about × per week on encounter utilization, cellular, and molecular (mapdom) hx of likely gluten sensitivity. presented symptoms of possible dairy sensitivity (bloating, acne, asthma). evidence for compromised intestinal barrier. minerals: infrequent bms could indicate low fiber or low mineral status (mg); when bms do occur, they are hard and dry (low mg); severe menstrual cramps (low mg); labs showed low k and na, on yaz birth control (low zinc and low b vitamins). antioxidants: consumed adequate fruits and vegetables each day. protein: has some evidence of poor/slowed wound healing as evidenced by sore on leg that has not completely healed after a year; cuts that take months to heal. d and fat-soluble a, e, and k vitamins -hx of poor immune function (low d), vdr +/+ (low d and possibly a). oils/fatty acids: high omega- /omega- ratio, higher intake of damaged fats, very low intake of omega- . methylation: symptoms of depression, anxiety, add combined with mthfr c t snp and on yaz (low b and folate). eicosanoid fatty acids status -suspect issues with pge series pathway to control inflammation due to following signs and symptoms: allergies, autoimmune condition (asthma), peptic ulcers, eczema, and severe menstrual cramps immune function -suspect gut dysbiosis due to following s&s: poor immune function, yeast infections, hx frequent antibiotic use, cyst, and constipation body composition genetic makeup that indicated prone to gluten and dairy sensitivity, low vitamin d status, and impairment in methylation broad spectrum probiotic + fermented foods bioactive b complex (includes mg p p b and mcg thf) mg gla evening primrose oil and zinc . aim to eat three larger meals a day, allowing space in between of ~ hours; increase omega- intake by adding in small fatty fish, such as sardines or anchovies, once per week and taking fish oil; decrease omega- intake, switch from conventionally raised meats to organic, pasture-raised; and replace fat in diet from dairy with coconut sources, more nuts, and avocados. patient presented ~ months after the initial visit (september ). her breathing had improved immensely. she was able to stop taking her albuterol inhaler before exercise, recently stopped daily inhaler. after dairy-free diet for months, reintroduced dairy (cheese, butter, yogurt). asthmatic symptoms returned about - days after the addition of each. noticed the more dairy consumed, the worse her symptoms became. at time of appointment, diet whole foods, gluten-free, and dairy-free. weight loss lb within the first month of going dairy-free, continued to lose some weight. when reintroduced dairy symptoms of bloating and increase in weight, which resolved returning to dairyfree diet. bms are regular now at ~ × daily. this patient case followed some common patterns in the development of chronic disease and the comorbidities that are common, especially autoimmune conditions like asthma. the first is the genetic susceptibility of the individual; several snps are prone to dairy sensitivity. second, significant evidence for gut dysbiosis, promoted compromised gut barrier, can contribute to the development of dairy sensitivity. third is the exposure to dairy protein antigen. diet history evidenced trigger for asthmatic condition. additionally, inflammation, immune dysfunction, and methylation issues present. signs and symptoms significant for decrease in pge series anti-inflammatory pathways. low dietary omega- s potential contributor to asthma. immune dysfunction evidenced by extensive history of infection-antibiotic use. genomic snp mthfr c t gene, which indicated a greater need for folate. the use of yaz birth control and symptoms of depression, anxiety, and add known further to deplete b and folate. the diet and supplements recommended targeted control of inflammation, restore gut ecology, promote proper methylation, and replete nutrient insufficiencies. results from -month follow-up showed successful outcome in helping improve breathing and wean her off of inhalers. this case is an example of the ifmnt approach able to address the complexity of the whole patient story and bring the metabolic priorities into a manageable intervention program for the individual. one study found that the composition of the nasopharyngeal microbiota in children was linked to the frequency of upper respiratory tract infections and acute sinusitis [ ] . a study that intranasally inoculated mice with lactobacillus fermentum reduced the amount of s. pneumoniae in the respiratory tract and increased the number of macrophages in the lung and lymphocytes in the trachea [ ] . these findings may indicate a benefit of manipulating the upper respiratory tract microbiota with orally or nasally administered probiotics in the prevention and/or treatment of upper respiratory tract infections. allergic bronchopulmonary mycosis (abpm) is caused by a hypersensitivity reaction to fungal colonization of the airways. this is typically caused by the fungus aspergillus fumigatus. without treatment this may lead to fixed airflow obstruction and bronchiectasis [ ] . guillain-barre syndrome (gbs) is a rare neurological disorder in which the body's immune system attacks the peripheral nervous system, known as the network of nerves located outside of the brain and spinal cord [ ] . it is often preceded by a bacterial or viral infection. there are several potential mechanisms in which these infections trigger gbs. if an individual contracts a campylobacter jejuni bacterial infection, antibodies made to fight this infection can attack axons in motor nerves, which can potentially cause paralysis and respiratory failure [ ] . campylobacter can be ingested via contaminated food or other exposures [ ] . pérez-guzmán states that hypocholesterolemia is common among tuberculosis patients and suggests that cholesterol should be used as a complementary measure in antitubercular treatment [ ] . alpha- antitrypsin (a at) deficiency is an underrecognized disease in the united states, with around documented , people suffering from it, according to the alpha- foundation. this deficiency is inherited through autosomal codominant transmission, meaning affected individuals have inherited an abnormal aat gene from each parent [ ] . individuals with this deficient allele present with aat levels at less than % to low-end normal levels [ ] . however, it is also possible for individuals with a variant of this allele to be asymptomatic given different environmental conditions or lifestyle factors, such as refraining from smoking to reduce lung disease development risk [ ] ( box . ). a at deficiency most often manifests in the lungs as chronic obstructive pulmonary disease (copd) (i.e., emphysema or bronchiectasis or "genetic copd"). a at deficiency is often undiagnosed because people with genetic copd experience the same symptoms as people with copd, such as [ ] : shortness of breath wheezing the only way you will know for sure if you have genetic copd due to alpha- is to get tested. ________ a at deficiency can manifest in the liver as cirrhosis. symptoms related to the liver unexplained liver disease or elevated liver enzymes eyes and skin turning yellow (jaundice) swelling of the abdomen (ascites) or legs vomiting blood (from enlarged veins in the esophagus or stomach) a at expresses sometimes in the skin as panniculitis [ ] . panniculitis typically appears as raised red spots on the skin, which may break down and give off an oily discharge. while panniculitis spots (called nodules) may appear anywhere on the body, the most common places are the thighs, buttocks, and areas subject to injury or pressure. normal genotype m m most common abnormal genes are called s and z abnormal variant combinations: zz (highest risk) sz (lower risk increasing if smoker, inhalant pollutants) mz (lower risk of carrying an a at gene variant; considered "carriers") alpha- is the most commonly known genetic risk factor for emphysema up to % of all people diagnosed with copd may have undetected alpha- alpha- can also lead to liver disease. the most serious liver diseases are cirrhosis and liver cancer the world health organization (who), american thoracic society (ats), and the european respiratory society (ers) recommend that everyone with copd be tested for alpha- alpha- is a progressive disease that benefits from early detection. it can cause serious lung diseases, such as copd and emphysema when undiagnosed. in some cases, alpha- can also cause liver disease [ ] symptoms related to the lung [ ] : shortness of breath wheezing chronic bronchitis, which is cough and sputum (phlegm) production that lasts for a long time recurring chest colds less exercise tolerance year-round allergies bronchiectasis the alpha- antitrypsin (a at) protein protects the body, especially fragile lung tissues, from the damaging effects of a powerful enzyme called neutrophil elastase that is released from white blood cells. in a at deficiency, a genetic mutation reduces levels of the protective protein in the bloodstream. a at deficiency can lead to chronic obstructive pulmonary disease (copd), specifically emphysema, and liver disease. smoking, which can inhibit what little a at protein an affected person does have, increases the risk of lung disease. alpha- antitrypsin deficiency is completely determined by mutations in a single gene. the severity of symptoms is mostly a function of which mutations a person has and how many copies. however, smoking can greatly increase the risk of lung disease due to aat mutations. andme reports data only for the pi * m, pi * s, and pi * z versions of the gene that encodes aat. keep in mind that it is possible to have another mutation that causes this condition that is not included in this report [ ] . a at deficiency is a genetic disorder that reduces circulating levels of a protein that protects the lungs by trapping a at in the liver, where the protein is produced, and prevents a at from entering circulation. a at deficiency can lead to chronic obstructive pulmonary disease (copd), specifically emphysema, and liver disease. when a disease-causing mutation is fairly common, as the pi * s and pi * z mutations are in europeans, it suggests that the mutation actually conferred an evolutionary advantage at one time. some researchers have suggested that several thousand years ago when the pi * z and pi * s mutations first arose, these versions of the gene for a at gave people a survival advantage by creating an environment in their lungs that helped fight off infections. the scientists theorize that the antimicrobial benefits of the aat mutations outweighed the cost of an increased risk of copd and liver disease in the era before antibiotics were available [ ] . in contrast to lung disease, manifestation of liver disease related to a at can be referred to as a "toxic gain of function, " due to accumulation of mutant a at protein rather than protease deficiency within the liver [ ] . when taken together, fibrotic lung diseases are the leading cause of mortality worldwide. under the umbrella of interstitial lung disease (ild), pulmonary fibrosis (pf) is the most common. any ild that involves scarring of the lungs falls in the pulmonary fibrosis category. pulmonary fibrosis is the scarring of lungs, which destroys tissue over time, making it impossible to transfer oxygen from inhaled air into the bloodstream. there are more than different diseases under the pulmonary fibrosis umbrella. because pf is often misdiagnosed or goes undiagnosed, there is not an accurate count of those with these diseases. however, it is estimated that as many as in adults over , or , people in the united states, are affected [ ] . there are more than , deaths from ipf every year in the united states. more people die each year from idiopathic pulmonary fibrosis than from breast cancer [ ] . there are other forms of interstitial lung disease including the newly identified pleuroparenchymal fibroelastosis, cryptogenic organizing pneumonia (cop), desquamative interstitial pneumonitis, nonspecific interstitial pneumonitis, hypersensitivity pneumonitis, acute interstitial pneumonitis, interstitial pneumonia, sarcoidosis, and asbestosis [ ] . symptoms include cough and dyspnea, restrictive pulmonary function tests with impaired gas exchange, and progressive lung scarring. the disease progresses with an initiation of inflammation. fibrosing starts with the action of transforming growth factor-β (tgf-β)-dependent differentiation of fibroblasts to myofibroblasts, which then express α-sma (smooth muscle actin) [ ] . after the tgf-β-dependent differentiation of fibroblasts to myofibroblasts, which express α-sma, there is sustained, excessive deposition of collagen by the myofibroblasts in the lung interstitium leading to the progressive lung damage in patients with pf [ ] . research published in supported the idea that dysfunctional type ii aecs (alveolar epithelial cells) facilitate lung fibrosis through increased susceptibility to injury, leading to excessive and dysregulated remodeling [ ] . the disease seems to progress in steps, and inflammation is not typically present continuously, except during certain periodic episodes of deterioration (. fig. . ). there are five main categories of pf causes: drug-induced, radiation-induced, environmental, autoimmune, and occupational. of these five, four have identifiable causes. some of the autoimmune diseases that can lead to pf are rheumatoid arthritis, scleroderma, sjogren's syndrome, polymyositis, dermatomyositis, and antisynthetase syndrome. idiopathic pulmonary fibrosis (ilp) is defined as pf with an unknown cause, including a genetic cause for some families [see . fig. . ]. the symptoms of ilp are a dry, hacking cough, shortness of breath, fatigue, chest discomfort, loss of appetite, and unexplained weight loss, all caused by the fibrosing of the lungs. diagnosis can be difficult, and pf is often misdiagnosed as copd or other more common lung diseases. in addition, in the recent past, path to a true diagnosis was invasive. since damage to the lungs, even through a diagnostic biopsy, can trigger further lung damage or a period of fibrosis, many physicians or patients are cautious with a biopsy approach to diagnosis. since the current treatments are limited, one must evaluate whether defining the exact form of pf is necessary for treatment and follow-up. difficulty breathing, crackling sounds while breathing, and low oxygen levels are the first indicators. clubbed fingernails may also be a symptom. high-resolution ct scans are performed, which can show scarring. the pulmonologist will ask many questions and order more blood tests to try to distinguish between the forms of pf. the future is pointing to molecular endotyping as a more accurate way to diagnose. molecular endotyping includes genetic, metabolic, transcriptional, and environmental factors to help determine the pathophysiology [ ] . genetic research has been progressing for a couple decades with illuminating results. there are more than a dozen genetic variants that have been associated with this family of diseases. researchers now believe at least % of idiopathic pulmonary fibrosis (ipf) patients with multiple family members suffering from ipf have some common familial genetic variants, which may allow researchers to eventually drop the term idiopathic and further define various forms or categories, with differing progression or outcome. the name given to this version of interstitial pneumonias is familial interstitial pneumonia (fip) [ ] [see . currently two categories of genetic focus have been defined: those genes related to telomere biology (shorter telomeres) and those related to surfactant protein processing. the genes related to shorter telomeres are tert, terc, htr, dkc , and rtel . more mutations have been found in the tert gene, which encodes the protein component of telomerase, than any other gene. further research may allow targeted therapies to affect the genetic expression associated with the development of ipf [ , ] . a common variant within the promoter of the muc b gene is the most replicated single-nucleotide polymorphism related to familial and sporadic forms of ipf as well as early radiographic findings of ipf [ ] (. figs. . and . ). wound contraction and re-epithelialization . fig. . the cellular and molecular mechanisms of fibrosis in multiple organs. the cellular and molecular mechanisms of fibrosis in multiple organs. once an injury occurs in an organ, epithelial and/or endothelial cells are impaired, which results in the release of chemokines and growth factors, including il- and tgf-b . macrophages and monocytes are recruited and activated, both of which further release cytokines and chemokines and further induce fibroblast activation. activated fibroblasts transform into a-sma-expressing myofibroblasts and migrate into the wound along the fibrin lattice. ecm is excessively accumulated, and some parenchymal cells (hepatic stellate cells in the liver, tubular epithelial cells in the kidney, alveolar epithelial cells in the lung, or cardiomyocytes in the heart) are further differentiated into myofibroblasts or fibroblasts by the stimulation of cytokines and chemokines, especially for tgf-b . after the inflammatory phase, two events occur. one is the regeneration of injured tissues followed by wound contraction and reepithelialization. in contrast, once chronic injury, inflammation, and necrosis occur, myofibroblasts are perpetually activated, and excessive ecm is deposited, finally resulting in fibrosis formation. ctgf, connective tissue growth factor; ecm, extracellular matrix; egf, epidermal growth factor; emt, epithelial-mesenchymal transition; hsc, hepatic stellate cell; il, interleukin; mmp, matrix metalloproteinase; tgf, transforming growth factor; timp, tissue inhibitors of metalloproteinase. (reprinted from chen et al. [ ] . with permission from elsevier) conventional treatment is typically palliative. the american thoracic society recognizes that supplemental oxygen and transplantation are the only suggested treatments for ipf. supplemental oxygen is prescribed, and the need for oxygen increases over the progression of the disease. keeping the oxygen saturation level over % (normal is in the upper s) is ideal and is how healthcare providers determine the level of supplemental oxygen to be used. cardiovascular exercise, in this case called pulmonary rehabilitation, is recommended to maintain as much use of the lungs as possible. infrequently, nutrition and counseling are recommended and are placed into the category of symptom management. nutrition can have a significant role in the management of this disease, but little implementation exists in some of the proposed protocols. there are currently two medications available in the united states with minor impact on the disease progression: nintedanib (commonly called ofev) and pirfenidone (esbriet). histopathological quantification showed similar amounts of dense collagen fibrosis, fibroblast foci, and alveolar macrophages in untreated or pirfenidone-or nintedanibtreated ipf patients [ ] . both have significant side effects, including fatigue and gi issues, and patients may have to evaluate their quality of life versus length of life. other antiinflammatories or immune-suppressing medications used are corticosteroids, mycophenolate mofetil/mycophenolic acid (cellcept®), or azathioprine (imuran®). immunesuppressing drugs may be harmful for those with short telomeres, and researchers are exploring this potentially contradictory recommendation [ ] . lung transplantation is a final effort. about is half of all transplants. with the prevalence of this disease closer to , , this is a small fraction of those with the disease. some of those with the transplant go on to live productive lives, while others develop pf again, in the transplanted lungs. overall, there is a shorter life expectancy in those with pf, because of telomere shortening. bone marrow or immune response abnormalities have been found in some ipf cases before and after lung transplantation, which increases the associated morbidity. as stated above, inflammation occurs at the beginning and throughout the progression of all fibrosing diseases, including those of the lungs. therefore, reducing inflammation is one wise strategy to slow fibrosing. there are several nutrients that can help slow or reverse the inflammation involved in the fibrosing process. the following two-part diagram shows where in the fibrosing pathogenesis each phytonutrient acts [ ] (. fig. . ). a few of those compounds are discussed in more detail here. curcumin, the active constituent in the common spice turmeric, has been shown to reduce fibrotic activity in several studies. in mice, curcumin inhibited collagen secretion of ipf fibroblasts. it affects the signaling of tgf-β, in a dosespecific manner, resulting in reduced expression of α-sma, which is responsible for inappropriate fibrosing. this was shown in vitro and in vivo in mice, with intraperitoneal, but not oral, administration. at the time of the study, oral ingestion of curcumin was not adequately absorbed into plasma, and there was greater than ten times plasma concentration of curcumin following an intraperitoneal injection [ ] . however, some new oral products on the market are showing greater absorption. the results of this study suggest more research into curcumin, including improved delivery into patients. for example, some delivery options may include nebulized curcumin directly into the lungs, binding it to highly absorbable agents for oral use or liposome-encapsulated curcumin suitable for intravenous use (already shown to be effective in an animal model). according to manufacturers of curcumin products, some are more readily absorbed than others. one study on fibrosing suggested that a dose of around mg curcumin split into three doses taken with meals including pepper (bioperine) achieved doses that were sufficient to exert the desired therapeutic effect. research into using quercetin also has some promising results in slowing the progression of ipf. quercetin reversed lung fibrosing in mice and reversed the disease progression normally caused by typical pulmonary senescence markers [ ] . it is worth mentioning that n-acetylcysteine (nac), a long-used therapeutic agent for breaking down mucus in the lungs, has not been found to be effective in those with ipf. in fact, due to its acidic nature, it has even been shown to be harmful when used in the inhaled form [ ] . several of the drugs being developed have a natural product as a model or foundation. until a drug or gene therapy is developed that stops or reverses this disease, it may make sense for the patient to focus on anti-inflammation and reducing myofibroblast activation, the extracellular matrix (ecm) accumulation, and the epithelial-mesenchymal transition (emt) process. the phytochemicals listed in . fig. . would be good ones to investigate. with the recent identification of genes associated with ild, a call for gene-related therapies both related to telomere lengthening and connective tissue disease has been initiated, and this type of therapy, as with any disease, could be personalized [ ] . one recent study looked at various biomarker values as a more precise way of diagnosing. the biomarker molecules were classified according to their involvement into alveolar epithelial cell injury, fibroproliferation, and matrix remodeling as well as immune regulation. furthermore, genetic variants of tollip, muc- b, and other genes associated with a differential response to treatment and with the development and/or the prognosis of ipf were identified. research into personalized medicine for treatment is starting [ ] . although controversial, because of the lack of research on interpretation of the results, telomere length testing is available directly to consumers and through healthcare . fig. . antifibrosis therapy. the molecular mechanisms and therapeutic targets of natural products against fibrosis. a tgf-b exerts a profibrotic effect through smad-dependent [target ( )] and smadindependent pathways [target ( ) ]. in the smad-dependent pathway, tgf-b directly phosphorylates and activates the downstream mediator smad and smad through tgf-b receptor i, and then smad and smad bind smad , which forms a complex that moves into the nucleus and initiates gene transcription. smad , transcribed by smad , is a negative regulator of tgf-b/smad signaling, and the imbalance between smad and smad contributes to fibrosis. pi k, erk, and p mapk are downstream mediators of the smad-independent tgf-b pathway. pparg [target ( )] could inhibit tgf-b to reduce fibrosis, while ctgf [target ( )], a matricellular protein, contributes to wound healing and virtually all fibrotic pathology. additionally, gas contributes to fibrosis through the tam receptor, which further activates the pi k/akt pathway. similarly, lpa triggers fibrosis through the lpa receptor [target ( ) ] that stimulates b-catenin to induce fibrogenesis. the activation of the hedgehog pathway [target ( ) ] induces the transcriptional activity of gli to express target genes, which have an important role in interstitial fibrosis, undergoing myofibroblast transformation and proliferation. il pathway [target ( ) ] stimulates nf-kb [target ( ) ] to activate tgf-b to induce fibrogenesis, while nrf [target ( ) ] antagonizes nf-kb activity to protect against fibrosis. b the chemical structures of isolated compounds and their therapeutic targets are presented. ctgf, connective tissue growth factor; il, interleukin; lrp, low-density lipoprotein receptor-related protein; ri, transforming growth factor-b receptor i; rii, transforming growth factor-b receptor ii; sara, smad anchor for receptor activation; stat, signal transducer and activator of transcription; tcf, t-cell factor; tgf, transforming growth factor. (reprinted from chen et al. [ ] practitioners. there are a few different methods: quantitative polymerase chain reaction, or qpcr, which has a % variability rate, and flow cytometry and fluorescent in situ hybridization, or flow-fish, which has a % variability rate. most research labs use flow-fish for research. telomere length is a hot topic in research, the antiaging industry, and with popular health blogs. shorter-thanaverage telomeres have also been linked to heart disease and heart failure [ , , ] , cancer [ ] , diabetes [ ] , and osteoporosis [ ] . research has shown ways to slow telomere shortening. some include reducing stress, meditation, practicing loving kindness (a technique encouraging compassion) [ ] , reducing exposure to air pollution and toxins [ ] , cardiovascular exercise [ ] , and a healthy fat and high vegetable diet [ , ] . one study showed that minutes of cardiovascular exercise three times per week resulted in longer telomeres representing years of biological age, similar to those of marathon runners, compared to those who didn't exercise much or at all [ ] . intermittent fasting, which reduces oxidative stress and keeps weight in check, has exploded in the scientific literature as a way to increase longevity and slow telomere shortening [ , ] . nicotinamide adenine dinucleotide (nad+) supplements may also help maintain telomere length by activating sirtuins, the antiaging enzymes; parps, which are involved in dna repair; and cd , which plays a role in insulin production. another supplement, cycloastragenol, derived from the herb astragalus, has also been shown to activate telomerase in mice. an ingredient called ta- has been derived and is used in supplements [ ] . overall, a healthy lifestyle and diet seem to delay the shortening of telomeres. with relation to pf, the gene mutations involved in telomere shortening may or may not be influenced by the above interventions. more research is needed for this. pulmonary fibrosis is a devastating disease with no management or a known cure. the integrative and functional medicine nutritionist can help her/his patient by managing weight, encouraging a healthy diet full of anti-inflammatory foods and encouraging a healthy lifestyle with exercise and stress reduction. there is some promising research into natural supplement use to target the different areas of progression within the disease process and some ongoing drug and gene therapy development to follow. the prevalence of lung disease in the united states and worldwide is growing and will continue to grow rapidly with the deterioration of earth's atmosphere, which is caused by pollutants such as industrial and construction toxins and volcanic and wildfire particulates. poor maternal, childhood, and adult nutrition from micronutrient-poor diets resulting in nutrient insufficiencies, not necessarily nutrient deficiencies, is also contributing to increased lung disease diagnoses or poorer results during treatment [ , ] . lifestyle choices and habits also play a role in the development of many of the lung diseases in today's world, such as smoking or vaping, which uses chemicals that are poorly studied to date. other lung diseases have their roots in genetics. some key processes drive many lung diseases, with the inflammatory process being the most important, according to current literature. nutrition can be of great help with inflammation, using a diet rich in whole foods providing micronutrients and phytonutrients. understanding genetics is also key to unraveling the causes and potential future treatments for many lung diseases. those patients with both genetic and environmental determinants, such as in those who smoke and have genes associated with copd, are at the greatest risk [ ] . despite the prevalence of lung disease, there is a general lack of nutrition knowledge among practitioners, including familiarity with the research about the use of nutrition for prevention, slowing disease progression, or as a treatment of lung disease. historically, nutrition has been used in a supportive role, primarily monitoring macronutrients to prevent weight loss, muscle atrophy, and acid/alkaline balance. although this is extremely important, more attention needs to be directed toward emphasizing micronutrients and phytonutrients. research is strong regarding the benefits of vitamins, minerals, and pre-and probiotics, and indeed, some integrative and functional practitioners are using vitamin and mineral nutritional therapy in oral, intramuscular, and intravenous applications, when allowed, in practice. a newer area of research is around nutraceuticals, including targeted vitamins, minerals, and plantderived constituents concentrated to therapeutic doses. some exciting research around the use of curcumin and quercetin, for example, has been shown to dampen inflammation to the point of disrupting the disease process (see above). the expanding knowledge of the microbiome is identifying the importance of the lung 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is shorter in women with osteoporosis loving-kindness meditation practice associated with longer telomeres in women association between leukocyte telomere shortening and exposure to traffic pollution: a cross-sectional study on traffic officers and indoor office workers physical exercise prevents cellular senescence in circulating leukocytes and in the vessel wall association of marine omega- fatty acid levels with telomeric aging in patients with coronary heart disease telomere length, oxidative damage, antioxidants and breast cancer risk physical activity and telomere length: impact of aging and potential mechanisms of action protein restriction, epigenetic diet, intermittent fasting as new approaches for preventing age-associated diseases fasting mimicking diet is very relevant for health and longevity cycloastragenol is a potent telomerase activator in neuronal cells: implications for depression management periostin: an emerging biomarker for allergic diseases zemaira® | alpha- antitrypsin deficiency diagnosis assessment of quality of life in children suffered from asthma identifying maternal conditions affecting altered embryologic development. neonatal advanced practice nursing: a case-based learning approach susceptibility for cigarette smoke-induced damp release and damp-induced inflammation in copd key: cord- -yhi hgq authors: kovács, katalin title: social disparities in the evolution of an epidemiological profile: transition processes in mortality between and in an industrialized middle income country: the case of hungary date: - - journal: mortality in an international perspective doi: . / - - - - _ sha: doc_id: cord_uid: yhi hgq the present paper seeks to understand the transformation of mortality patterns in hungary, by which mortality inequalities by education began to appear in the early s, continued to grow in the following years, and now seem to be stabilising. the first part of this paper overviews the theoretical innovations of the last decades regarding the interpretation of cause-specific mortality dynamics, often referred to as epidemiological transition theories, and their relevance for the analysis of mortality inequalities. the paper then analyses the cause-specific trends of mortality for two educational classes between and . the trends were corrected for changes in the coding system and divided into linear (stagnating, increasing or decreasing) periods. causes of death were grouped according to the relationship between the sequences of these periods for the two educational classes. the causes of death were finally clustered into six groups. one group, which is dominated by nutrition-related and cardiovascular diseases, is largely responsible for the onset of mortality inequalities in . the results imply that the quality of nutrition has diverged for the educational classes since , and this fact has left its footprint on the pattern of mortality. the history of food production and availability seems to be in line with nutrition-related mortality, and it is argued that nutrition transition theory provides a very plausible explanatory framework for the growth of mortality inequalities. countries in central and eastern europe (cee) experienced rapid industrialisation under equalising state-controlled regimes, and entered into the globalising international economy two decades ago. their transformation into service economies is still an on-going process. over the past years social inequalities have increased sharply, reaching medium-level income inequalities in an eu context, which is considered high by the citizens of these countries. in the present paper i look at the implications these changes have had on the level and distribution of mortality in hungary, as an example of this group of countries. all-cause mortality is considerably higher in cee countries than in the rest of the european union, but it was recently shown to correspond to the income level of these countries (spijker and von wissen ) . on the other hand, inequalities in mortality by education have been found to be extremely high in all of these countries (mackenbach et al. ) . so far the explanation for these developments has only been provided within a larger context that applies to the whole of the eastern european region, including not only cee and baltic countries but also countries like russia, belarus and ukraine. one of main conclusions has been that they have not so far undergone the healthier life style changes that have occurred in western europe, and this has resulted in a "reversed epidemiological transition", in which an elevated burden of cardiovascular diseases dominates the pattern of mortality (vallin and meslé ) . is this framework applicable to central and eastern europe and does it explain the evolution of their cause of death pattern and high level of inequalities? if so, what role did income play in these processes and what are the specific social processes that triggered these developments? in hungary, inequalities in all-cause mortality were negligible during the 's and widened during the 's. the next one and a half decades brought a further, dramatic, increase in inequalities, which appear to have stabilised at this very high level for the past half a decade ( fig. . ) . as regards broad groups of causes of death, the data suggest that the apparent similarities in all-cause mortality during the s might be attributable to causes other than the lack of inequalities in living conditions between people with different education. this period was characterized by the overmortality of the less educated from cardiovascular diseases and the over-mortality of the more educated from malignant tumours (fig. . and . ). an explanation is called for which will look at the historical development of cause-specific mortality within the framework of the epidemiological transition. in this paper i shall review recent developments in epidemiological transition theory, and test the applicability of some of these theories to the evolution of cause-and education-specific mortality inequalities in hungary between and . smr, / smr, / long-term mortality trends are commonly interpreted within the framework of epidemiological transition theory, outlined years ago by omran ( ) . the original statements of the theory on mortality, fertility and population growth have already been tested, analysed, criticised and modified. by now, epidemiological transition theory and demographic transition theory have split: the first one has gradually shifted towards a focus on cause-and age-specific mortality patterns, while the second is now far more concerned with patterns of fertility and family formation. the original postulates of omran are, without doubt, of a heuristic nature: based on limited empirical basis (in its original form it was based on the long-term causespecific mortality trends of just six countries), it provided a comprehensive picture of the evolution of cause-of-death patterns throughout the history of mankind. in a rather vague division of human history, three transitional phases were distinguished: the 'age of pestilence and famine', the 'age of receding pandemics', and the age of 'degenerative and man-made diseases' (omran ) . stages were differentiated on the basis of average life expectancy, and age-and cause-specific mortality. during the first phase, which encompassed most of human history (the "pre-industrial period", omran ), mortality due to chronic malnutrition, endemic infectious diseases, and high prenatal and maternal mortality shaped the overall high level of mortality, which was further increased by epidemics, famine and wars in the "peak years". in the second stage, which started in the eighteenth or nineteenth century in western societies, mortality declined considerably, mainly due to factors other than medical interventions: improved nutrition, improvement of personal cleanliness, ecological recession of certain diseases, better housing conditions and the start of using contraceptive methods. the cause-of-death pattern was less and less characterized by diseases caused by pandemics but communicable diseases-tuberculosis in particular-were still dominant. the third stage is characterised by the dominance of non-communicable diseases, such as diseases of the circulatory system and different types of cancer. from the perspective of the future development of the theory, the additional characteristics of the stages are less important, though omran's approach, which has been modified several times by himself and others over the past years, remained complex and aimed at explaining the whole of population dynamics. the evidence which accumulated subsequent to omran's original article, coming mainly from countries of the americas, shows little correspondence with this original framework and offers an amazing variety of cause-specific mortality patterns and their changes over time (albala and vio ; castillo-salgado et al. ; costello and osrin ; hill et al. ; huicho et al. ; marshall ; vigneron ; vigneron ) . this evidence was incorporated into the original model as three models, the classic, the delayed and the accelerated models (omran ) . later on this was expanded to six models, the classical western model, the semi western model and four non-western models: the rapid, the upper intermediate, the lower intermediate and the slow (omran ) . other authors suggested a different classification of countries (frenk et al. ) in order to incorporate new evidence that did not fit into the original sorting in the first form of the epidemiological theory. from the perspective of countries in latin america, the concept of an epidemiological transition was in sharp contrast to the mortality experience of many countries of the region, which were characterized by a sharp divide between the mortality patterns of different population groups within one country. this experience questioned the choice of countries as the units of analysis, and even raised doubts about the usefulness of the whole concept of an epidemiological transition. apart from total refutation, the experience of latin american countries are best summarized as a "patchwork pattern" in which different social groups are often segregated geographically, and display diverse mortality patterns corresponding to different stages of the epidemiological transition. in other words, "different epidemiological worlds" live next to each other. for industrialised countries, on the other hand, a large collaborative study of who did confirm the previously proposed trend of age-specific death rates for two broad groups of causes of diseases in the last half of the twentieth century (salomon and murray ) . the more and more sophisticated classification, however, did not help to overcome one of the major theoretical drawbacks of the original theory. despite the very complex, and somewhat apocalyptic, view of the future presented by omran in his last article ( ), epidemiological transition theory presents a linear view of changes in mortality patterns, according to which more developed stages follow less developed ones, alongside with the course of 'modernisation'. this process may take place slowly or quickly, and with some variations, but it also follows a linear route. in this respect, epidemiological transition theory does not differ from theories of modernisation propounded in the 's (carolina and gustavo ) and is very similar to the dominant view of demographic transition theories (melegh andŐri ) . the theory of an epidemiological transition was attractive not only for public health researchers, it can also be viewed as a major contribution to the on-going debate of historians and historical demographers centred around the nature of mortality changes in the last centuries. in countries with a long history of collecting detailed mortality data, the distinctive phases of receding epidemics and the death toll of infectious diseases in general could be identified. due to the great variability within the regions of one single country (for sweden: rogers and nelson ) , this further classification, unlike the contribution coming from the discipline of gerontology, has not become a commonly accepted modification of the original epidemiological transition theory. olshansky and ault ( ) carried out a detailed examination of the age-specific death rates of the us and pointed to the onset of an epidemiological phase that differs from the one specified as the third stage of the epidemiological transition in the original form of the theory. this fourth stage, they suggested, is characterized by the dominance of the same major causes of death as the third stage but with a continuing delay in mortality from some of these causes, leading to a further significant improvement in life expectancy. the new stage, called the 'age of delayed degenerative and man-made diseases' has become a standard part of the most commonly accepted form of the epidemiological transition theory. anthropology or 'evolutionary biology' has also made its contribution to refining epidemiological transition theory by adding a new transition stage, thus refining how the original theory divided up the other end of the historical time-scale (armelagos et al. ). the addition of the "baseline" mortality pattern, called the 'palaeolithic stage', however, is less relevant from the perspective of the current research. the approach of evolutionary medicine, which emphasises the links between the specific nature of human production, diet and other aspects of living conditions, and causespecific mortality, can, however, be beneficial in interpreting contemporary mortality trends as well. another major discipline contributing to the further refinement of epidemiological transition theory was epidemiology itself. recent trends in epidemiological research clearly exhibit some fragmentation. epidemiology was first concerned with certain diseases in detail but recently more comprehensive approaches have emerged. alongside the continuing research of the risk factors associated with specific diseases, trends in mortality due to major groups of diseases have also been studied and the results and hypotheses presented in the framework of "sub-transition models" such as cancer transition and cardiovascular transition (also known as the cardiovascular revolution). the cancer transition is an extension of the classic transition theory that takes into account new discoveries on the role of infections in the development of certain types of cancers. the discovery of the presence of bacteria in the majority of stomach cancer cases promoted the recognition of how important infections are in cancer in general, though the infectious origin of certain other cancers (such as cancer of the cervix, testicular cancer and certain lymphomas) was already well-known. new discoveries triggered the formulation of "cancer transition theory" (gerstein and wilmoth ) , according to which there is a definitive restructuring process in operation within cancer mortality: those with an infectious origin lose their importance and other non-infection-related cancers emerge. most cancers, however, are known to be influenced by some major risk factors such as non-appropriate diet, smoking and excessive alcohol consumption. these well-known risk factors are now more and more closely linked to societal transformation processes, mostly of a global nature. from among these theoretical frameworks we note in particular the theory of a nutritional transition (popkin , popkin and mendez ) . in this framework, major features of food production, distribution and several other characteristics of living conditions are connected to mortality patterns. nutritional transition theory, just like epidemiological transition theory, divides human history into five distinctive patterns, out of which the fourth corresponds to the living conditions of contemporary cee countries. the fifth profile describes the living conditions and dietary habits of the most health-conscious members of the most affluent countries. labelling the phases not as stages but as profiles obscures the fact that these patterns are arranged in historical order so that they also represent some "developmental route". the patterns, however, are connected with a large number of dimensions of actual living conditions. as regards the transition to the fourth ("obesity characterized") profile, several processes, such as "supermarketisation", are connected to several social processes like the demand for safer food, the changing opportunity cost of females' time, technological changes, and changes in logistics and production systems. altogether this transition is technically characterized by the growing importance of edible oil and animal products in human diet. additionally sugar consumption is on the rise, often in the form of consuming sweetened beverages. the shift from high fibre intake to refined grains and additionally declining fruit and vegetable intake is also documented in many countries (popkin ) . transition theories regarding other risk factors are less developed at the moment, but the term "smoking epidemic" is also in use and the influence of strong economic forces has already been recognised (yach et al. ) . regarding the other dominant groups of diseases, cardiovascular mortality has always been regarded as being strongly related to the epidemiological transition. ischemic heart disease in particular often serves as a "marker disease" that indicates a country's position in the phases of the epidemiological transition (heuveline et al. ) . based on the observations of the contemporary occurrence and frequency of different cardiovascular diseases in different regions of the world, a complete framework for "cardiovascular transition" has gained popularity in the past few years. this framework provides a correspondence between particular cardiovascular diseases and stages of the epidemiological transition (califf et al. ) . the linearity of the occurrence of the stages is not stated but it is inherent in the logic of this scheme. the 'pestilence and famine' stage, with life expectancy around years, is characterized by a modest share of cvds in total mortality ( - %) and the dominant forms of cardiovascular mortality are rheumatic heart disease and other infection-related diseases of the circulatory system, cardiomyopathy in particular. the latter disease may also be connected to malnutrition. in the second stage the proportion of deaths caused by cvd grows to - % and cardiovascular mortality is dominated by rheumatic valve disease, ischemic heart disease and haemorrhagic stroke. in this stage life expectancy reaches about years. in the third stage, in which life expectancy reaches years, the proportion of deaths due to cardiovascular diseases is greater than %. the dominant causes of death within cvds are ischemic heart disease, and ischemic and haemorrhagic stroke. in the stage of 'delayed degenerative diseases' the proportion of cvds among all deaths falls below % and life expectancy exceeds years. major cardiovascular causes of death are the same as in the previous stage, with the addition of congestive heart failure. another important observation not exactly linked to stages is a major shift between stroke types: haemorrhagic stroke declines while ischemic stroke emerges (lawlor et al. ) . a fourth stage also appears in some variations of the "cardiovascular transition" schemes. in some cases (yusuf et al. a ) a stage of 'health regression and social upheaval' is visualised, characterised by the re-emergence of rheumatic heart disease and a new increase in ischemic heart disease due to increasing alcoholism. in the increasingly unregulated social environment, violence also becomes more common and hypertensive disease-which is otherwise characteristic of stage according to these authors-also re-emerges. this visualisation, of course, relies heavily on recent russian mortality trends. other authors have predicted the emergence of heart failure as the main characteristic of a future scenario for cvd mortality (bonnux et al. ; gaziano et al. ) . risk factors for cardiovascular diseases were found similar to the ones identified for cancers but the linkage between the single diseases and the exact role of single risk factors is less clear, with some exceptions. for two major different stroke types, for instance, different set of risk factors had already been identified (o'donnall et al. ) , though inappropriate diet, smoking and excessive intake of alcohol play an important role in the development of all cardiovascular diseases. predictions on the future trends of mortality and cause-specific mortality are not restricted to the field of cardiovascular diseases. in his last publication omran ( ) also outlined a fifth stage, the 'age of aspired quality of life with paradoxical longevity and persistent inequalities'. in this he expressed his hope for a future decrease of inequalities in survival, together with an expectation that there was a high probability of the re-emergence of infectious diseases. nevertheless, the 'invisible perils' in the future of mankind are considered by omran as well, such as the possibility of the evolution of an (airborne) virus with abilities similar to those of hiv; the potential misuse of atomic bombs; and high, uncontrolled population growth. omran's view on the unpredictable role of infectious diseases is not unique. several other authors foresaw the future emergence of new diseases and the re-emergence of "old" infectious diseases that were previously believed to have been controlled by medical interventions. notable examples are the emergence of multi-drug resistant tuberculosis and avian flu. following the emergence of the hiv/aids pandemic, the fear of new infectious diseases is spreading. scientific examination reveals, however, that the majority of the emerging and newly recognised diseases are in fact not new but were known only in some peripheral regions of the world and have reached the consciousness of the wealthy only recently (farmer the circumstances of the onset of "newly identified" diseases during the s pointed out that human activity played a triggering role in the majority of the cases. in the integrated view of evolutionary medicine that divides human history only into three epidemiological transitions, the "third transition" is the new era of emerging and re-emerging infectious diseases (harper and armelagos ) . the "end of the antibiotic era", as this approach calls it, results mostly from the intensification of the globalisation process, especially that of the transportation system, which serves as a 'virtual superhighway' for pathogens. figure . outlines the theories providing a comprehensive explanation for changes in patterns of mortality and their phasing. while many epidemiological transition theories cover the whole of the history of mankind, others refer only to developments in the latest centuries, or even just decades. most of them inherently treat the process of change in mortality patterns as "development", i.e. as a linear, and in some respect hierarchical process. possible reverses and uncertainties mostly appear regarding the latest stages-which is probably due to empirical observations being more numerous and diverse regarding the near past. the issue of social disparities is present in nearly all approaches to the epidemiological transition. in most cases social inequalities in mortality or diverse mortality patterns that are characteristic of social classes, strata or groups are discussed in relation to major drivers (or causes) of the epidemiological transition. in some cases drivers or causes are stated only in general, like modernisation, industrialisation and urbanisation. in other cases propositions are well-formulated and corroborated by some empirical evidence. omran, starting from his very first publication, continuously mentioned social disparities in mortality as well as the driving forces listed above but he did not provide a theoretical framework for the application of these in connection with particular mortality or disease patterns specific for single countries or population sub-groups. mckeown ( a mckeown ( , b, mckeown and record ) studied the disappearance of infectious diseases in industrialising england and formulated his famous nutrition hypothesis. detecting a time-lag between the almost complete disappearance of numerous infectious diseases, and a very notable drop in tuberculosis mortality, before the discovery of the appropriate treatment methods (mostly antibiotic drugs), he concluded that the major cause of decreasing mortality was the improvement in the living conditions and, in particular, the nutritional status of the population during the nineteenth century. the nutritional thesis provides an obvious explanation for social disparities in mortality, whose modified versions later appear in recently developed explanatory approaches. evolutionary medicine, with its anthropological orientation, considers the "palaeolithic" baseline to have been free from social hierarchies in early human communities (harper and armelagos ) . notably they also focus on dietary habits. they suggested that there was a low mortality period before human communities settled down, as a result of their varied diet as well as small population size. mortality started to grow when diet became heavily reliant on crops, which were unequally distributed across population strata. in parallel, the growth of average community size led to new, infectious, diseases becoming the leading causes of death. based on this approach one can conclude that unequal access to food results in unequal resistance to diseases, thus inevitably leading to inequalities in mortality. historians and historical demographers, however, present a rather different picture of those centuries of human history which can be characterised by the dominance of infectious diseases. they suggest that some of the infectious diseases exhibit a "discriminative" nature: there is a long incubation period between the moment of infection and the development of the disease and the resistance of the host matters during the process of battling with these diseases. other infections, by contrast, are "quick" enough not to allow time for the host (the human body) to develop resistance and they kill in a short time; consequently, they can be considered "nondiscriminative". several infectious diseases, such as smallpox and mumps, have been observed to change over the centuries, as their originally "non-discriminative" nature turned into "discriminative". it is still unclear if the changing nature of some formerly fatal diseases is due to increased community-level resistance to those diseases or merely to the changing nature of the disease-scape. however, the disappearance of some infectious diseases, notably the plague, is still explained in several alternative ways (slack ) . during early modern times, when infectious diseases dominated mortality, the excess mortality of those in disadvantaged social positions was likely to be more pronounced in those causes of death that were connected with epidemics and pandemics. according to historical demographers, excess deaths were indeed connected to the availability of food, though this relationship was largely influenced by the effectiveness of supportive networks (bengtsson et al. ) , which helped to mitigate the effect of economic hardships (e.g. famine). regarding the plague outbreaks in london, it was observed that the locus of epidemics moved from the central, relatively wealthy parishes to the poorer suburban ones during the seventeenth century. overall, it is likely that social disparities existed in the era of infectious diseases, though their importance might have changed over time, partly due to factors that operated independently of social organisation and human activity and partly due to greater awareness and ability to cope among the wealthy (slack ; hall ) . the early industrial era inevitably brought large mortality inequalities, which are well documented for some countries. time series of mortality data by social groups, however, are not available for many countries. studies using time series on income inequality for industrialised countries have suggested that mortality inequalities were narrowing from the first years of the twentieth century until about , and widening afterwards. detailed british data (pamuk ) has reinforced this view. this process probably took place in varying ways in different regions: in sweden no sign of the emergence of social inequalities in mortality was found till the s (bengtsson and dribe ) . theoretical explanations for modern inequalities have emerged in the fields of both epidemiology and sociology. in the epidemiology of cardiovascular diseases a particular "disease mobility" was observed first: in the beginning of the twentieth century myocardial infarction used to be the disease of the affluent in developed north american and european countries, but in the s and the s mortality rates due to infarction started to decline earlier and more rapidly among the better educated and the better off (marmot et al. ; kaplan and keil ) . these experiences led to the formulation of the social "following pattern" of diseases. based on the concept of the diffusion of innovation, pearson ( ) suggested an "adoption theory": population groups with higher education and/or better income adopt new ideas, products and behavioural patterns more readily. once a risk factor is recognised, it first becomes public knowledge among those with higher social status, mediated by health education or mass media. the messages reach the poorer and/or less educated groups of societies later. the life course perspective for understanding the occurrence of chronic diseases also originates from cardiovascular epidemiology (forsdahl ; kuh and ben-shlomo ) . its scope, however, is much wider, identifying risk factors that act during the in utero period and early childhood, risk factors which are associated with the social position of the parents (davey-smith and hart ). from the point of view of social sciences, these findings call for integrating intergenerational mobility into epidemiological transition models that are used to understand the particular mortality patterns of single countries. in the social sciences the "fundamental causes" concept was introduced in order to understand the relationship between socio-economic status and disease. these fundamental causes do not refer to causes of death but to dimensions of social position which are causally linked to resources that can be used to avoid risk or to minimize the consequences of diseases once they occur. resources include money, knowledge, power, prestige and interpersonal relationships. fundamental causes act, according to the proponents of this idea, when new diseases, new risk factors or new knowledge on risk factors emerge or new medical technologies are introduced (link and phelan ) . in these cases living conditions and access to resources act directly to grant or restrict different groups' access to, and application of, the new technology. social position, therefore, is the fundamental cause of a disease (or death) and not a "proxy", as it was previously treated in epidemiological research. the concept of fundamental causes has only recently been applied to the analysis of cause-of-death patterns (miech et al. ) . the examination of education-specific mortality inequalities and their dynamics over the last decades of us history aimed at testing the fundamental cause hypothesis. a large number of causes of death ( ) were included in this examination. in accordance with the concept of fundamental causes, the analysis found increasing inequalities for most "emerging" causes, e.g. those whose overall rate was in an increase. omran's classic paper on the epidemiological transition ( ) positioned hungary together with the rest of "eastern europe", in the same model as japan. mortality developments have diverged significantly since then. the latest additions to the concept of epidemiological transition provide no direct guidance for understanding overall mortality trends and educational inequalities in mortality. detailed knowledge has accumulated on the changes in mortality profiles in developed high-income countries. mortality trends, especially the burden of infectious and non-communicable diseases, are widely discussed with regard to low income countries. industrialized middle-income countries seem to be neglected in the discussion of the epidemiological transition. in order to fill this gap, first we examine the applicability of one of the previously outlined theories that focus primarily on other regions of the world: the plausible "following" hypothesis. the higher overall level of mortality as well as the cause of death patterns in hungary (and other cee counties), often referred to as "lagging behind" those of western europe, might be interpreted as the mortality pattern of a society in which large population segments who are "lagging behind" produce an overall "delayed" cause-of-death pattern and large mortality inequalities at the same time. if this proposal were true and meaningful, one would observe the same mortality dynamics for the more and the less advantaged segments of the population but with some time lag regarding the latter group. existing data allow us to examine these processes by education only: i shall therefore compare the mortality development of the less and the more educated hungarian adults. as a contrast i also examine the applicability of another popular branch of theories known as risk behavioural factor approaches, in particular, the possible role of nutrition in shaping cause-of-death pattern differences. mortality data for hungary, by education, are available from . for the period between and we calculated age-standardized cause-specific mortality rates by education for the population aged and above. cause-specific death rates were also calculated for the whole population and by education groups. data on the number of deaths by education is provided by the mortality register of the hungarian central statistical office. corresponding population estimates and forecasts were prepared by lászló hablicsek, based on census data from , and (hablicsek and kovács . underlying causes of death were included in the analysis. education level was dichotomized: high (completed years and passed the matura exam) and low. these two groups will be referred to as the less and the more educated. selecting the relevant causes of death was a multi-stage process. first we selected causes cited in discussions of the epidemiological transition theory that linked their theoretical considerations to empirical analysis. the starting point, however, was the broad categorization into the two distinctive groups of causes of death which came out of the who global burden of diseases study (salomon and murray ) . group included the infectious diseases; diseases of the pulmonary system and several diseases connected to malnutrition and maternal mortality. group encompassed all other diseases, except the external causes: injuries, homicide and suicide. looking at a large number of countries over shorter or longer observational periods (from to ) and taking into account total mortality and wealth (as measured by gdp), salomon and murray ( ) found no consistent relationship between external causes and total mortality or wealth, and we decided therefore to leave them out of the present analysis. the next step in selecting the causes of death was based on those considerations which have been summarized in the introduction. additional results from studies that analysed time trends for a number of diseases in specific countries with regard to the epidemiological transition were also included, particularly studies on the epidemiological transition in the netherlands (wolleswinkel-van den bosch ; wolleswinkel-van den bosch et al. ) and in canada (lussier et al. ) . for group causes, the identification of nutrition-related, pulmonary or maternal causes of death is not problematic. the large group of 'infectious and parasitic diseases', as the international classification of diseases calls it, was much more difficult to break down into smaller and meaningful causes of death, because if anything is clear from the literature, it is that infectious diseases are generally declining but they still vary significantly by country. therefore we decided to select all those causes for which more than cases were found for each year during the period between and . this procedure resulted in a list of one disease: tuberculosis. we also added the "new diseases" such as hiv/aids and newly recognised and antibiotic-resistant infectious diseases. these categories turned out to be almost empty. in practice, the study also includes a number of infectious diseases which are traditionally classified under pulmonary diseases (such as influenza, pneumonia) or other major disease groups (peptic ulcer, appendicitis), or whose coding in some periods overlaps other broad cause-of-death groups (meningitis, enteritis). group included different types of cancers and cardiovascular diseases, divided up according to those "sub-theories" of the epidemiological transition which we briefly introduced earlier. for cardiovascular diseases, the categorisation was based on the list of diseases that appear in different versions of the "cardiovascular transition". apart from these, some other distinctions were also made according to major coding categories such as chronic and acute ischemic heart diseases. among cancers, we distinguished in particular all those cancer types with are connected with infections. a further distinction was made by major risk factors, including not only smoking, excessive drinking and obesity, but also environmental and occupational exposures (for a short summary see table . ). this categorisation, however, does not lead to easy interpretation, due to the pervasive and complex nature of the everyday operation of risk factors. some other diseases, specifically discussed by certain authors with respect to the epidemiological transition, such as alzheimer and parkinson's disease, were also added. the list of the causes of death that we selected for analysis is included in the appendix, together with the coding used. age-specific death rates by the selected causes of death (where possible) were used to create standardized mortality rates using the european standard population. mortality trends, resulting from the standardisation process, did not form continuous time series in most cases, as illustrated in fig. . . there were three different icd coding versions in operation during the observed period, and in addition, "automatic coding" was introduced in , which again affected the structure of the (underlying) causes of death, as if another new icd version had been introduced. icd- was introduced in and icd- in . first we fitted the different versions of icd codes, often with the help of literature, in order to achieve the same content for each disease over time. when code-fitting was not obvious, we relied on code-fitting used by others wolleswinkel-van den bosch et al. ; hashibe et al. ; lawlor et al. ) . the resulting time series called "original values" still did not construct continuous curves in this study. there are three known methods to deal with the changes of icd coding system. the first one, the "double or bridge coding" would require coding death in a certain period according to both the outgoing and the new coding systems. this task was carried out only in for the hungarian mortality data. the second method follows the exact matching of the disease categories by four-digit coding (meslé and vallin ) . this method was partly used in this study but only for some specific causes of death. after establishing the coding we followed a third method of fitting the curves (janssen and kunst ) but applying a simpler method than they did. fitting coefficients were calculated by causes of death, but always for the entire population. the same coefficients were used to fit curves for those with lower and higher educational background. the values of the coefficients, listed in the appendix, provide an overview about the reliability of the estimated time series : the closer the coefficients are to , the higher the reliability. no fitting was applied in the case of those causes which were too small to calculate standardized rates or for those which showed outliers "too often", such as influenza. overall mortality trends were similarly not fitted ( fig. . ) . we now turn to consider the relation between the two estimated mortality time series for groups with lower and higher education. for certain causes of death, almost exclusively in those years when the annual number of deaths is very low, it was not possible to determine definitive relations since the low number of deaths did not allow for standardisation, so fitting was also not applied. therefore the general trends of overall mortality due to these causes are difficult to establish. this fact is well illustrated in the case of obesity. from this cause less than deaths were reported annually between and , but about in the following years. as for inequalities, a clearer picture emerges from the distribution of the number of deaths: most of them appeared among the less educated. deaths due to nutritional anaemia, malnutrition and obesity, as well as maternal death almost exclusively happened among those with lower education. for other rare causes of death such as hiv/aids and "newly emerging infectious diseases", however, no such pattern evolves. hiv/aids mortality was the highest in , when deaths were attributed to this disease. the number of cases declined afterwards and people with lower and higher educational attainment seem to be equally affected. among the newly emerging diseases only deaths were reported from , again distributed proportionally between the educational classes. after disregarding the above-mentioned causes of death, we categorized the remaining causes by the relationship between the two mortality time series displayed by the groups with lower and higher education. the classification of the relations rested on a simplified view of the time series. given that we worked with estimated values in the classification, the dynamics represented by the time series were the focus. the time series were broken down to linear (growing, stagnating or declining) phases and the classification was based on the relationship between the sequences of these phases by causes of death, presented by the two mortality time series. time series were broken down into phases using join-point regression analysis, with software provided by the national cancer institute of the united states. this regression is for analysing trends and the software fits data in the simplest possible sequence of linear trends which are connected by the join-points. first a linear trend for the overall period is fitted, then trends with a growing number of joint-points are also fitted and their significances are tested against the null-hypothesis (e.g. having join-points). the tests of significance are based on a monte carlo permutation test. the breakdown of the time series was successful in most of the cases, though the method applied involves some uncertainties. the location of the join-points is provided together with confidence intervals, which were often very wide, covering even - years. in the following classification only those periodicities were considered when confidence intervals for the joint points were shorter than years. uncertainties were taken into account in all those cases when confidence intervals were wider than - years. the sequences of linear trends and the corresponding set of join-points by cause of death are not given here but are available from the author. to examine the "follow-up" hypothesis, first one has to give a clear definition of a follow-up pattern of two curves. the method chosen for this analysis was not to construct a general definition but first to regard the estimated mortality time series for the two educational groups, then to classify them by their type of relation and then to examine the possible interpretations of their being "follow-up" by type. the application of this method resulted in six different groups of diseases, according to the relationship between the mortality trends estimated for the more and the less educated. this classification allows us to investigate the possibility of providing a proper definition of follow-up. in the case of diseases with strongly declining mortality (type i) the definition of follow-up is not obvious at all. the dynamics of decline did not provide any meaningful definition of follow-up, since for the major diseases of this category (pulmonary tuberculosis, haemorrhagic stroke and cancer of the stomach) the timing of strongly declining and the less strongly declining periods, represented by the mortality of the less and more educated, mostly coincide ( fig. . ) . the existence of sequences of declines with a different pace also means that a definition based simply on when mortality of the less educated reached the mortality level of the better educated also gives no clear-cut answer: for instance, the value of tuberculosis mortality of the more educated in was reached years later by the less educated, but the values for the more educated in or in were reached, by the less educated, only or years later, respectively. in the case of influenza, the level of fluctuation highly exceeds the level of inequalities. for rarer diseases that also belong to this class of causes of death, temporal but irregular high peaks of mortality among the better educated would make it difficult to define a follow-up pattern (table . ). in the case of some other diseases, mortality of the less and the more educated also shows similar sequences of periods of linear trends, but the overall trends are not declining (type ii, fig. . ). inequalities change little or not at all over time and the mortality of the more educated never (in "regular" cases such as the hypertensive diseases of the circulatory system or cervical cancer) or just in exceptional years (in the case of mesothelioma and epilepsy) reaches the level of the less educated. providing any follow-up definition seems meaningless in these cases (table . ). in a number of diseases, however, the sequences of the linear periods of different types are also similar for the less and the more educated, but the overall dynamics of the curves turn to be very different. for these causes of death mortality levels are quite similar at the beginning of the period considered here, but at a certain point of time mortality of the two groups starts to diverge quite distinctly (type iii, fig. . ) . regarding most diseases in the class of type iii mortality, negligible differences in mortality characterise the beginning of the observed period and then the same types of linear trends apply to both educational groups, but the levels of mortality iv. the two educational groups start with the same type (inclining, declining or stagnating) of trend and mortality from the disease is higher for the better educated. mortality of the better educated changes its trend in a certain year for the better and finally huge over-mortality of the less educated is present only mortality of the better educated shows major trend change during the period resulting in higher mortality among the less educated by the end of the period , similar to the one we give for type i diseases. in other cases, however, when the mortality of both educational classes increases, there is no sign that mortality of the less educated would follow that of the more educated by any means. it is more plausible that "the same story is played out" for both of the educational groups concerning risk factors or general conditions of life but with very different risk levels. for type iv causes of death ( fig. . ) , the less educated population is characterised by growing mortality, while the mortality of the better educated changed from a growing to a declining trend. similar trend changes can be expected in the future for the mortality of the less educated, but this change will appear later than the end of our observation period. approximate minimum time-lags for the onset of this change are given in table . . in practice these time lags can also be a bit longer, since we cannot be sure if the last couple of years of the observation period represented the beginning of a new type of trend or not. altogether, a clear follow-up pattern was detected only for three-though very important-causes of death (fig. . ) . as regards acute ischemic heart disease, ischemic stroke and breast cancer, the sequences of the rising and declining periods are similar for the less and the more educated with a time-lag, so the mortality of the less educated seems to follow the mortality of the more educated. though it is impressive that the estimated follow-up time is the same for ischemic heart disease and ischemic stroke, it is important to mention that these "scenarios" are also "played out" at different mortality levels. at their maximum values, breast cancer and ischemic stroke mortality of the better educated is % higher and that of acute ischemic heart disease is % higher than those of the less educated, suggesting that follow-up type explanations need to be supplemented for a full understanding. type v causes of death are characterized by different trends for the two educational groups for the whole of the period (see fig. . ). for some, the mortality of the less educated increases and that of the more educated decreases (aneurysm); for others, both are in increasing but with different intensity (cancer of the oral cavity, non-hodgkin disease) or the mortality of the better educated is declining while the mortality of the less educated is stagnating (cancer of thyroid, prostate cancer, peptic ulcer). the possibility that these diseases start to decline or strongly decline among the less educated can certainly be hoped for, but the follow-up time would be longer in these cases than our observation period. there is thus no point in laying down a definition for the purpose of this study. some diseases, typically rare causes of death, could not be classified into the previous types and they are placed into type vi, represented by fig. . . though a clear follow-up pattern was identified for only three causes of death, there is evidence of some kind of follow-up for a large number of diseases but it is not easily identified. time lags are usually long, exceeding more than one or two decades, so that while follow-up may provide a vague and partial explanation for mortality developments and the development of inequalities for the chosen relatively short time period, it certainly does not a provide a full picture. taking a closer look at the onsets of trend changes, it is quite obvious that they cluster in time. most of the changes occurred in the very first years of the s and around . both these periods were important turning points, and the two clusters can thus be interpreted as indicators of two diverging trends in living conditions, in the widest possible sense of the term. in the first cluster we find, surprisingly, a number of nutrition-related causes of death: diabetes, other endocrine diseases, and two strongly nutrition-related cancers (cancer of the uterus and gallbladder cancer). trends of mortality by education diverge from about the same point of time for a number of causes of death related to the circulatory system: chronic ischemic heart disease, arrhythmias, heart failure, atherosclerosis, other diseases of the veins and arteries and pulmonary heart disease. causes of death which are possibly nutrition-related, such as cancer of the rectum, acute ischemic heart disease and ischemic stroke, also show signs of changing mortality relations by education between and . some other causes of death, which are clearly not nutrition-related, such as meningitis, cancer of the liver, cancer of the brain, melanoma, other skin cancer and leukaemia, join this cluster. the most likely interpretation of the existence of this cluster is that these changes reflect the widening inequalities in the quality of diet for the two social groups distinguished by educational level. alternatively, within the risk-factor oriented explanatory framework, one can argue that all these changes are attributable to diverging trends of excessive alcohol consumption, noting that cirrhosis of the liver, the only cause of death which is clearly related to alcohol consumption, started to emerge a couple of years earlier. alcohol-related changes are known to have an immediate mortality impact but some possible effects of the divergence in alcohol consumption cannot be ruled out. altogether, divergence in nutrition seems to provide a more suitable explanatory framework. the second cluster includes causes of death with important trend changes between and . smoking-related causes, such as cancer of the larynx and cancer of the trachea, bronchus and lung, clearly dominate this cluster, joined by some other diseases such as colon, pancreas, kidney and bladder cancer and valve diseases with other than rheumatic origin. attributing the evolution of this cluster to the appearance of the divergence in smoking habits in the two educational classes, it is to be noted that this divergence point seems to be more diffused in time than the one related to the divergence in nutrition: trends of important smoking-related causes of death (cancer of the oesophagus) started to diverge a year earlier than , though this cancer type is also influenced by nutrition. to explain the rise of mortality inequalities between the less and the more educated from the very beginning of the s in hungary, one might turn towards basic sociological approaches which would focus on the changing relations of education and income, assuming that the relationship between the two was non-existent in the s and became gradually stronger over the period between and . from a simplified point of view on the former state socialist states that assumes that these countries had no income inequalities at all, the onset of mortality inequalities during the s must be a mystery. in fact, income inequalities were already present and connected to educational levels during the s in hungary. even after taking compensation in-kind into account-since a large share of incomes was undoubtedly distributed in this formthe income of those with higher education can be estimated as being twice as high as that of people without this qualification (pető and szakács ) . during the s the maturation of the "second economy" partly confused this relationship. in this period the state made some form of economic activities free from its direct control; therefore, in this sector (especially in agriculture) a limited market economy developed. social status was distributed along two axes: in the formal economy, in which income and education were correlated, creating very mild income inequalities, and in the informal economy, in which education and income did not correlate strongly (kolosi ) . since the emergence of the free market economy following , the correlation of income and education has become stronger and stronger, just as in most european countries (tóth ) . this, coupled with the lack of a significant improvement in gdp, led to widening social inequalities and the extension of poverty. the changing relation between income and education therefore plays a certain role in explaining widening mortality inequalities, but it cannot explain the negligible mortality inequalities which existed during the s nor their revival during the s. we should look, therefore, at nutrition-related risk factors. the food supply in hungary was mostly based on domestic production during the s and s. limited exchange with other state socialist countries existed but imports were mainly limited to a small amount of tropical fruits. domestic products, however, were satisfactory for domestic demand. agriculture had developed into one of the leading ones in europe and from the 's there was no food shortage in hungary. the distribution of food was rather even and quality differences by education hardly existed. during the s, with the growth of the "second economy", food provision varied and prices were already partly market-driven. the better-off could use their resources to purchase better quality food and these provisions were available to a large share of the population, but obviously not for everyone. low food prices, together with energy prices which were still subsidized, made it possible for a larger proportion of the population to buy food of satisfactory quality. in the countryside, "around-the-house" agricultural activity was widespread, producing mostly for the household (occasionally producing for the market, too). during the 's the proportion of food grown "around-the-house" was estimated at % of the overall food consumed (ksh ) . from the s the food supply and the price system of the country were placed into a global context. open trade relations provided a great variety of available food, while domestic production, including around-the house output, started to decline. food prices relative to income represented a greater and greater share of household expenditure and competed with rising energy costs. around-the-house agricultural production, which had been characteristic for many households for decades, halved in less than a decade: its share in overall food consumption of % in had shrunk to % by (ksh ). as a result of these processes, the availability of quality food has been shrinking for an ever growing proportion of the population. domestic agricultural production, however, started to recover in the last years of our observation period, as the states that joined the european union in came to benefit from the unified european agricultural policy. the history of food production and food availability seems to run in parallel to the inequalities in nutrition-related mortality, so this narrative provides a very plausible explanatory framework for our findings. if this framework is supported by similar findings from other countries, then we can conclude that the mortality of the middle income industrialised countries, with moderate income inequalities, is still strongly determined by nutritional differences and by the lack of availability of quality food for large proportions of their populations. social differences in food intake have been described both in wealthy and poorer countries and are usually discussed in connection with obesity. major changes in human nutrition have also been described, characterized by a growth in sugar and animal source food intake (popkin ) . in the context of wealthier countries, the poorer nutritional habits of the less educated is usually understood in the context of lack of knowledge, forced habits by tradition or lack of awareness due to putative or real economic interests. in the case of poor countries the phenomena is understood in the context of absolute deprivation and poverty. several facts indicate that none of these scenarios are appropriate for middle income countries. hungarian household surveys, for instance, indicate that the amount of sugar and sweetened beverages consumed is much lower in low income households than in households with higher income. the difference in this respect between the lowest and the highest income quintile households was fivefold in . some features of the differences in food consumption, however, run parallel with the pattern of the western countries, such as the similar levels of pork consumption of households with different income and the large gaps in poultry, fruit and vegetable consumption. relatively high pork intake is the only fact which would suggest that tradition also plays some role in forming nutrition patterns. differences in fruit and vegetable consumption fluctuate and depend on yearly prices (polgár ; ksh ) so there is good reason to attribute these differences to the decline of around the house production and the lack of financial resources. food intake differences by education can largely be explained by rising poverty among the less educated and the changes in the system of food production and pricing. as a generalisation of our findings, we note that the nutritional elements of living conditions are rarely measured in europe and they are usually restricted to the poorest countries. in the first relevant eurobarometer survey, however, less than % of the west european population answered "yes" to the question if paying for food causes any (some or serious) problem, and the corresponding proportion was between and % for central, eastern and baltic countries (not including the czech republic and slovenia). these data refer to the years around . publicly available raw data of the second european quality of life survey ( ) indicate that the question of food quality is still relevant in cee and baltic countries. for the only directly foodrelated question ("can you afford a meal with meat, chicken or fish every second day if you want it?") no more than % of the population gave a negative answer in west european countries, whereas this proportion was around % in most cee and baltic countries and in greece, and even higher in some countries such as slovakia, bulgaria and hungary ( , and %, respectively) . the same question was included in the same year in the european statistics on income and living condition survey and released results (ward et al. ) suggest that that survey yielded a similar picture: no more than % of the population in western europe was affected and %- % in central and eastern europe and in the baltic countries (except for estonia, romania and bulgaria). data indicate that even if starvation-related mortality is negligible in lower-middle income european countries, there are good reasons to assume that the quality of nutrition is still not satisfactory for large proportions of the populations in these countries, and leaves its footprint on their mortality pattern. as far as the history of the hungarian food provision regime is concerned, some of its elements can be regarded as similar to other countries of the region, while some other elements are certainly different. the above-mentioned developments in income inequalities and food provision in the s are probably similar in all cee countries, while the introduction of the second economy was unique to hungary. the development of free market conditions from the s and the degree of exposure to the global competition varied over time and between the countries, as did the role of around-the-house agricultural production. rising income inequalities and the application of a global pricing system, however, seem to lead to similar levels of mortality inequalities in these countries, though the composition of over-mortality by cause differs (leinsalu et al. ). cee and baltic countries, therefore, probably share more common features than differences in this respect. the generalization of the findings for the whole region of "eastern europe", however, seems less fruitful, allowing for the fact that the cee and baltic countries have had consistently lower income inequalities than countries of the former soviet union other than the baltic countries. several other aspects of household economy, such as the overwhelming role of energy expenditure in cee countries, are not present in the same way. the analysis of cause-specific mortality is a challenging task. these studies typically go beyond the time periods of consistent registration systems of causes of death and creating credible time series is demanding. the solution chosen in this paper can be criticized and other alternatives of code bridging should be considered in further research. the classification of causes of death by their relation to mortality developments between the more and the less educated can also be questioned and other alternatives should also be regarded. the method followed by this paper was to decompose the overall time series to sequences of linear trends and there is no doubt that other than linear approximate trends could also have been considered. moreover, the linear approximation itself was carried out with a high level of uncertainty: the exact point of time when trends changed was hard to establish, which introduces some uncertainties about the findings. the changing composition of the population over time is an inherent problem of studies examining long term developments. in our case the share of the population aged years or more with less than secondary school graduation was % in and % in . a more detailed educational classification of the population would have been desirable but was impossible to carry out with consistency due to major changes in the schooling system during the observed period. in our discussion we deliberately avoided some important issues which might naturally be regarded as good candidates for explaining mortality inequalities, such as health care provision and differences in health care utilization. the reason for this neglect was the lack of space to cover all elements of cause-specific mortality inequalities in one paper. instead, we aimed at identifying some general driving forces contributing to widening inequalities. setting up an accurate statistical record of the different health services, which would have been necessary to evaluate their role, was beyond the possibilities of this study. similarly, we had to disregard other, similarly important elements of welfare policy, except for some aspects of income distribution. our discussion addresses only some of all the arguments raised in different theoretical approaches to the epidemiological transitions theory. we limited the scope of the paper to looking at the role of nutrition in the long term development of mortality and mortality inequalities. the intention of providing an explanation for the observed mortality trends in connection with the social processes of hungary in the last four decades has left little space for discussing the applicability of other, similarly attractive, explanatory frameworks that undoubtedly 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cardiovascular diseases: understanding the links the uneven tides of the health transition epidemiologic transition interrupted: a reassessment of mortality trends in thailand mortality profiles in a country facing epidemiological transition: an analysis of registered data icd coding changes and discontinuities in trends in causespecific mortality in six european countries socioeconomic factors and cardiovascular disease: a review of the literature zöldség és gyümölcsfogyasztás, statisztikai tükör, ii. évfolyam . szám a life course approach to chronic disease epidemiology secular trends in mortality by stroke subtype in the th century: a retrospective analysis educational inequalities in mortality in four eastern european countries: divergence in trends during the post-communist transition from social conditions as fundamental causes of disease does the recent evolution of canadian mortality agree with the epidemiologic transition theory? demographic research the second fatal impact: 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absolute and relative income hypotheses revisited income composition and inequalities in hungary convergences and divergences in mortality. a new approach to health transition the epidemiological transition in an overseas territory: disease mapping in french polynesia epidemiological transition and geographical discontinuities: the case of cardiovascular mortality in french polynesia european inequalities. social inclusion and income distribution in the european union the epidemiological transition in the netherlands reclassifying causes of death to study the epidemiological transition in the netherlands mortality decline in the netherlands in the period - , a turning point analysis globalization and tobacco global burden of cardiovascular diseases part i: general considerations, the epidemiologic transition, risk factors, and impact of urbanization global burden of cardiovascular diseases part ii: variations in cardiovascular disease by specific ethnic groups and geographic regions and prevention strategies key: cord- - wfyaxcb authors: ubokudom, sunday e. title: physical, social and cultural, and global influences date: - - journal: united states health care policymaking doi: . / - - - - _ sha: doc_id: cord_uid: wfyaxcb in chap. , we examined the technological environment of the health care policy-making system. specifically, we examined the classification, evolution, and diffusion of medical technology; the effects of medical technology on medical training and the practice of medicine; effects on medical costs, quality of care, and quality of life; effects on access to care; the ethical concerns raised by medical technology; and the practice of technology assessment. we concluded the chapter by observing that the growth of technology, as well as other human endeavors, affects other important aspects of our lives, most notably, the air we breathe, the food we eat, the generation of radioactive by-products and toxic chemicals, the manufacture of illicit drugs, and the generation of natural and man-made hazards. in other words, in addition to their effects on the health care system, technology and other human activities affect many other aspects of our lives that are associated with health. the who's defi nition of health as "a complete state of physical, mental, and social well-being, and not merely the absence of disease or infi rmity" (who ) , is primarily based on the wellness model. in this defi nition, emphasis is put on the fact that health is not merely the absence of disease, but also involves a social dimension. therefore, it also emphasizes the social and fi nancial support systems identifi ed in table . of chap. . this defi nition of health, as involving the combination of physical, mental, and social well-being led to the concept of the "health triangle." the health triangle left out the spiritual dimension of health, which has recently gained signifi cant attention in the literature due to a growing interest in the notion of holistic health. holistic health stresses the importance of all the things that make a person whole and complete. in addition to the three dimensions of the health triangle, of his analysis (szreter , p. ) . subsequent studies revealed that the cessation of the large-scale redistribution of income and wealth from the very rich to the poorest in society had adverse effects on the health of the population. for example, when unhealthy behaviors and lifestyles were held as constant as possible, studies showed that people of lower socioeconomic status were more likely to die prematurely than were people of higher socioeconomic status (isaacs and schroeder , p. ; smith et al. , p. ; davey smith et al. , p. ) . the relationship between physical, social and cultural, and global environmental factors and health status is very well documented. in a letter to the editor of the jama , winkelstein ( winkelstein ( , p. argues that curative medical care, or those practices that are used for the care and rehabilitation of the sick, which involve most of the physical and designed social technologies listed in table . of the previous chapter, is not the same as health care. medical care, as he defi nes it, makes only modest contributions to the health status of the population. on the contrary, the health status of the population is largely determined by a different set of factors that involve important physical, social, and economic components. these include preventive medicine, genetic predisposition, social and economic circumstances, environmental conditions, lifestyles and behaviors, and medical care (mckeown ; kannel et al. ; belloc and breslow , p. ; bunker et al. ; bunker et al. , p. ; marmot et al. marmot et al. , p. bell and standish , p. ; mcginnis et al. , p. ; wilkinson wilkinson , p. . we briefl y examine each of the identifi ed determinants of health below. preventive medicine seeks to minimize the occurrence of illness and disease. unlike the medical model that is reactive and seeks to contain disease and ill-health after they have occurred, preventive medicine is proactive and seeks to minimize the likelihood of the occurrence of disease and ill-health. generally, there are three areas or types of preventive measures, namely: primary prevention, secondary prevention, and tertiary prevention. primary prevention seeks to stop or minimize the development of disease or ill-health before it occurs. primary prevention may involve counseling against smoking, in order to prevent the development of chronic emphysema or chronic obstructive pulmonary disease (copd) and lung cancer. other primary interventions may include the promotion of an active lifestyle or exercise program, in order to minimize the likelihood of excess body fat and heart disease; driver education and mandatory seatbelt and motorcycle helmet laws, in order to reduce motor vehicle accidents and accidental head injuries; vaccinations for various forms of diseases and illnesses, such as measles and rubella, which can minimize the occurrence of early childhood diseases and mortality; and water purifi cation and sewage treatment programs that can minimize the occurrence of typhoid, cholera, and other waterborne diseases. secondary prevention involves the early detection and treatment of disease. health screenings and periodic and regular health examinations, such as hypertension screenings, mammograms, and pap smears, serve as examples of secondary prevention measures. these examples fall under the broad category of health promotion discussed in chap. . the benefi ciaries of these programs are currently healthy people who are targeted to improve their health-related behaviors in order to minimize their chances of developing catastrophic and expensive illnesses. as was discussed in chap. , secondary prevention measures are some of the most cost-effective steps employers take to lower their health benefi t costs ( coffi eld et al. , p. ) . tertiary prevention measures involve steps taken to reduce the complications of diseases or illnesses, or to prevent further illnesses. they involve rehabilitative practices and the monitoring of the process of health care delivery. the infection control practices in hospitals and other improvements in the methods of health care delivery discussed in chap. , under the postindustrial period of the evolution of the health care system, which are intended to reduce the occurrences of nosocomial infections and iatrogenic illnesses, are practical examples of tertiary prevention measures. other examples include patient education, nutrition counseling, and behavior modifi cation programs that seek to prevent the recurrence of disease and illness (timmreck , p. ) . since the mid- s in the united states, there have been signifi cant reductions in heart disease, stroke, personal injury, and non-tobacco-related death rates foege , p. ; banta and jonas , p. ) . similarly, the data presented in table . of chap. show signifi cant declines in death rates related to heart disease, cancer, stroke, infl uenza and pneumonia, chronic liver disease or cirrhosis, human immunodefi ciency virus (hiv) disease, suicide and homicides, from to . these particular declines appear to be the result of preventive health measures, such as early screening, detection and treatment of hypertension, the provision and utilization of pneumonia and infl uenza vaccinations, moderate alcohol intake or abstinence, safe sex practices, suicide prevention and anger management programs, increased use of seatbelts and reductions in driving-underthe-infl uence episodes, smoking cessation, and the lowering of dietary fat and cholesterol. if, at least, some of the declines in mortality discussed above are due to preventive measures, the preventive strategy has yielded signifi cant gains in health. perhaps, it is this recognition of the importance of preventive services that led to the establishment of the us preventive services task force (uspstf) in . most likely, it was the recognition of the crucial role that preventive medicine plays in enhancing population health that led to the convening of the uspstf in by the us public health service. the task force is a leading independent panel of nationally recognized nonfederal experts in prevention and evidencebased medicine. programmatic responsibility for the task force was transferred to the agency for health care research and quality (ahrq) in (uspstf procedure manual ). the uspstf is assigned the responsibility of making evidence-based recommendations that address primary and secondary preventive services targeting conditions that represent a substantial burden in the country, and that are provided in primary care delivery settings or made available through primary care referrals. the task force's recommendations are intended to improve clinical practice and promote the public health. tertiary prevention measures are outside the scope of the uspstf. even though the main audience for task force recommendations is the primary care provider, the recommendations are also used to guide programmatic, funding, and reimbursement decisions by policy-makers, managed care organizations, public and private payers, quality improvement organizations, research institutions, and consumers. beginning at the end of may , the uspstf changed the grades it assigns to its recommendations. it assigns one of fi ve possible letter grades, a, b, c, d, or i, to each of its recommendations, including "suggestions for practice" associated with each grade. the agency also defi nes the levels of certainty regarding the net benefi t of each of its recommendations. the task force's reduction of the grade given for evidence quality from "b" to "c" for routine mammograms in women under the age of years generated signifi cant controversy among health professionals and politicians (kinsman ) . in addition to the mammography recommendations stated above, the uspstf has recently recommended against screening for testicular cancer in adolescent or adult males (grade d recommendation) (uspstf , p. ) . it has also concluded that there was insuffi cient evidence to assess the balance of benefi ts and harms of screening for bladder cancer in asymptomatic adults (moyer , p. ) , and that prostate-specifi c antigen (psa) screening was associated with psychological harms, while its potential benefi ts remained uncertain (lin et al. , p. ) . table . shows the approach adopted by the agency in june , to rank its recommendations. health is dependent upon biological factors. our predispositions to health or disease begin to take shape at the moment of conception. these predispositions are embedded in our genetic code. the genetic code guides the development of the proteins that determine our phenotypes (sizes, shapes, personalities, hair color, etc.) and genotypes or those aspects of our genetic codes that we cannot see, such as the biologic limit of our life expectancies (mcginnis et al. , p. ; khoury et al. ; bell and standish , p. ; starfi eld , p. ; blum ; centers for disease control and prevention (cdc) ) . genetic factors predispose individuals to certain diseases. but although an individual may have a strong likelihood of developing a particular disease, this propensity to develop the disease is signifi cantly enhanced by environmental factors. for example, some studies demonstrate that there is a genetic basis for alcoholism (reich ) . but a person who has never taken a drink will not become an alcoholic. some triggers, in this case, the availability and consumption of alcohol, are necessary for the individual to progress from being genetically predisposed to alcoholism to actually (berkman and breslow ; burnett ; banta and jonas , p. ; davis and webster , p. ) . these examples suggest that the interaction between genetic factors and the environment in producing a particular disease is complex. while people have little or no control over their genetic makeups, the lifestyles and behaviors they freely choose and the surroundings where they live can have signifi cant infl uences on the likelihood of developing a particular disease to which they are genetically predisposed. to further the discussion of the infl uence of genetics on health, mcginnis et al. ( , p. ) cite studies which show that although only about % of deaths in the united states may be attributed to purely genetic diseases, about % of late-onset disorders, such as diabetes, cardiovascular disease, and cancer, have some genetic component. for example, the brca gene accounts for only between and % of breast cancers in the united states; only about % of colon cancers may be explained by genes, and only about % of elevated serum cholesterol levels may be explained by familial hyperlipidemia. similarly, studies of identical twins focusing on the occurrence of schizophrenia, and other twin studies examining the occurrence of dementia in older people, have found that about half of each might be explained by genetic factors. further, while about two-thirds of the risk of obesity might be genetic, the risk is expressed only with exposure to controllable lifestyle factors (baird , p. ; muller , p. ; panjukanta et al. , p. ; kendler kendler , p. rowe and kahn ) . the institute of medicine (iom) ( , p. ) reported that americans in , compared with those who lived in , were healthier, lived longer, and enjoyed lives that were less likely to be marked by injuries, ill health, or premature death. but the gains in health reported by the iom were not shared equally among the population of the united states. at the moment, as was also the case in , gains in health status are not shared fairly or equally by all americans. americans with a good education, those who hold high-paying jobs, and those who live in serene and comfortable neighborhoods live longer and healthier lives than those with lower levels of education and income, and those who live in crime infested, overcrowded, and less comfortable and cohesive urban areas (isaacs and schroeder , p. ; bell and standish , p. ; lantz et al. lantz et al. , p. navarro , p. ; satcher , p. ; williams , p. ; metzler , p. ; kilbourne et al. kilbourne et al. , p. berkman and lochner , p. ) . there are several pathways through which social and economic circumstances affect health. those with good educational achievements are more likely to attain higher socioeconomic status than the poorly educated (angel et al. ; barr ; bartley ; mirowsky and ross , p. ) . people of lower socioeconomic status die earlier and are more susceptible to undesirable life events than people on higher socioeconomic levels, a pattern that holds true in a progressive fashion from the poorest to the richest (mcleod and kessler , p. ; adler et al. , p. ; adler and newman , p. ; guralnik et al. , p. ; mcdonough et al. mcdonough et al. , p. . this trend also holds whether one looks at education or occupation (national center for health statistics , p. ; kaplan and keil , p. ). these differences are said to be due to the fact that people of higher socioeconomic status have healthier behaviors and lifestyles than those of lower socioeconomic status. people of higher socioeconomic status are less likely to smoke, and are far more likely to eat healthier foods and to engage in leisure-time physical exercise (national center for health statistics , p. ; pratt et al. , p. s ; giles-corti and donovan , p. ). according to isaacs and schroeder ( , p. ) , as a result of "a sedentary lifestyle and unhealthy eating habits, obesity and the diseases it fosters now characterize lower-class life." poor eating habits and a sedentary lifestyle alone do not explain the differences in health between high and low socioeconomic people. rather, another explanation for the differentials lies in the distribution of income or the income gradient between the low and high socioeconomic groups. in a study of white americans using census data, undertaken by smith et al. ( , p. ) , men earning less than $ , per year were . times as likely to die prematurely as were those earning $ , or more. a similar study of british civil servants conducted about years before the american study showed that when smoking and other risk factors were controlled for, those who were in the lowest employment category were more than twice as likely to die prematurely of cardiovascular disease as were those in the highest employment category (davey smith et al. , p. ) . the fi ndings of these studies have led to the theory that inequitable distribution of income and wealth, or the socalled income and wealth gradient, causes poor health (sen (sen , p. , daniels et al. ; deaton , p. ). as noted above, the relationship between health and income is referred to as a gradient. this terminology emphasizes the gradual relationship between the two variables. health improvements are directly related to improvements in income throughout the income distribution, and poverty has more than a "threshold" effect on health (deaton , p. ) . the us national longitudinal mortality study (nlms) published by the national institutes of health (nih) ( ) showed that the proportional relationship between income and mortality was the same at all income levels, implying that the absolute reduction in mortality for each dollar of income was much larger at the bottom of the income distribution than at the top. apart from income, mortality is also known to decline with wealth, rank, and with social status (marmot et al. (marmot et al. , p. (marmot et al. , (marmot et al. , p. . similarly, studies also show marked differences in life expectancy by race and by geography or people's places of residence. for example, there is a -year gap in life expectancy between white men who live in the healthiest counties or localities and black men who live in the unhealthiest counties (murray et al. , p. ; gittelsohn , p. ; marmot marmot , p. kawachi and berkman ) . the brief discussion in this section points to the effects of numerous, and possibly interrelated, social and economic factors on health. income might affect health just as health might affect income; the distribution of income and wealth might affect health. similarly, education, race, minority status, geography, employment, housing, discrimination and social isolation, nutrition, lifestyle, stress, health practices, and coping skills might affect health. it does not appear to matter very much which of the above factors is stressed, especially since they are more likely to be interdependent than independent. disease risks exist, most often, along a continuum (rose ) . risks are rarely dichotomous. according to lochner ( , p. ) , there is no clear division between risk and no risk with regard to, for example, levels of blood pressure, cholesterol, alcohol or tobacco use, physical activity, diet and weight, etc. this gradient of risk also exists for many social and environmental conditions, such as socioeconomic status, social isolation, occupational and environmental exposure, and air quality. put differently, the numerous studies on the determinants of health that we are unable to fully summarize individually here for lack of space, point to the fact that even though the human and material resources at our disposal, the foods we eat, our levels of education, the houses we live in, the quality of the environments where we live and work, to name but a few, affect every person's health, the effects may vary in direction and scope from person to person, depending on the differences in their unique circumstances. improvement in environmental conditions is an important goal of the us government, as can be inferred from the emphasis on environmental quality outlined in healthy people . that document clearly states that factors in the physical and social environment play major roles in the health of individuals and communities. the physical environment is operationalized to include the air, water, and soil through which exposure to chemical, biological, and physical agents may occur. the physical environment can harm individual and community health, especially when individuals and communities are exposed to toxic substances, irritants, infectious agents, and physical hazards in homes, schools, and work sites. the physical environment can also promote good health, for example, by providing clean and safe places for people to work, exercise, and play ( healthy people , p. ). therefore, the physical environment is perhaps one of the most important factors that should be considered when classifying the health status of an individual (wikipedia ) . environmental factors, such as air and water quality, exposure to pesticides and toxic waste, and housing conditions, have major effects on health and human development. for example, substandard air and water quality have been directly associated with diseases such as cancer, asthma, certain birth defects, and some neurological disorders (grant makers in health , p. ) . similarly, many forms of cancer are associated with dioxin, polychlorinated biphenyls (pcbs), and mercury (friis ) . also, airborne particulate matter, tobacco smoke, and ground-level ozone, have been known to cause asthma attacks in children. exposure to lead, which can be found in peeling paint or in the soil and air in many poor communities, has been associated with impaired cognitive and behavioral development and low birth weight among children born to exposed mothers, and is also known to cause kidney damage (friis ) . in recognition of the danger of environmental contamination, bell and standish ( , p. ) urge communities to act on their behalf to make changes in the policies that affect their physical, social, and economic environments. they state, plausibly, that "policy, place, and community" matter. combined, policy and community can alter or ameliorate the underlying forces that lie at the heart of the determinants of health. for example, they argue that policy determines the behaviors or things that are allowed, encouraged, discouraged, and prohibited. policy also determines whether industrial facilities will be sited near residential neighborhoods, how industrial facilities treat their neighbors; how dense neighborhoods will be; what materials can be used to build houses; who will live in a neighborhood; whether businesses can locate in a neighborhood; and whether there are tax or other incentives available for locating in a neighborhood (bell and standish , p. ). in the developed communities or countries, environmental epidemiologists are concerned about such things as gene-environment interactions, environment-environment interactions, particulate air pollution, nitrogen dioxide, ground-level ozone, environmental tobacco smoke, radiation, lead, video display terminals, cellular telephones, and persistent organic pollutants (pops) that act as endocrine disruptors. exposure to these downstream or proximate environmental vectors (exposures that are closely related in time and space to the ill-effects they cause) affect both health and well-being (encyclopedia of public health ) . in the developing communities, the primary environmental determinants of health are said to involve biological agents in the air, water, and soil that account for most deaths. for example, diarrheal diseases acquired from contaminated food or water, malaria, intestinal parasitic infections, respiratory diseases caused by biological and chemical agents in both indoor and outdoor air, wreak havoc in the developing countries. these environmental hazards take a far greater toll on human life and suffering in absolute terms compared to those environmental vectors of concern in the developed countries (encyclopedia of public health ) . the above environmental vectors that cause havoc in the developing countries also abound in the poor localities of the united states and other developed countries. wealthy people are more likely to live in better homes and locations where they are less exposed to environmental risks than poor people (friis ; mcleod and kessler , p. ; giles-corti and donovan , p. ; shi and singh , p. ; grant makers in health , p. ) . for example, although the rates of asthma have been rising in the country, the disease affects low-income people disproportionately. whereas the national prevalence rate of childhood and adult asthma is put at about %, some african-american communities report about % of children suffering from asthma. also, puerto rican children are reported to have the highest prevalence of active asthma of any us ethnic or racial group. in california, latino children are reported to be hospitalized for asthma at a rate that is % greater than that of white children. obviously, environmental hazards are some of the reasons for these disparities ( healthy people ; joint center for political and economic studies and policylink , p. ; flores et al. , p. ) . despite the gains in environmental quality since the advent of the environmental movement in the s, mainstream environmental policies neglected the problems identifi ed in low-income communities because the inhabitants of those areas lacked the political and economic resources to press for environmental justice. however, since its start around , the environmental justice movement has resulted in the cleanup of hazardous waste sites, the redevelopment of brown-fi elds, the shutdown of incinerators, and the establishment of parks and conservation areas in low-income communities. additionally, in low-income communities, local pollution problems are being addressed, cleaner and more accessible means of public transportation are made available, and wild lands and unique habitats are being protected (faber and mccarthy ) . these changes are due to interest group pressure, the recognition of the externalities associated with environmental degradation, and the value of a clean environment to the health and well-being of all persons, rich and poor. mcginnis et al. ( , p. ) contend that behavior choices constitute the single most important domain of infl uence over health prospects in the united states. lifestyle and behaviors involve many dimensions, including dietary choices, engagement in physical activity, sexual behavior and recreation, including the choice to smoke and to ingest alcohol, the wearing of motor vehicle seatbelts and motorcycle helmets, and other responsible behavior when operating motor vehicles. because lifestyle and behavioral factors are under the control of individuals, the public is very likely to defi ne lifestyle and behavioral health problems as being self-induced. the choices we make with regard to the many dimensions of lifestyle and behavior enumerated above have signifi cant impacts on personal and population health. for example, dietary factors have been associated with coronary heart disease and stroke; colon, breast, and prostate cancers; and diabetes (us department of health and human services ) . similarly, a sedentary lifestyle has been associated with increased risk for heart disease, osteoporosis, dementia, diabetes, and colon cancer (us department of health and human services ) . furthermore, research shows that diets rich in fruits and vegetables, low-fat dairy foods with reduced saturated and total fat, and low sodium diets can lower blood pressure (appel et al. (appel et al. , p. svetkey et al. , p. ; sacks et al. , p. ) . the primary differences between how we perceive behavioral change now from much earlier perceptions is the great awareness that individual behavior occurs in a social context (berkman and lochner , p. ) , be it the place of work or abode, the family, the place of worship, the peer group, the school system, the stage of development, etc. for example, the results from the national youth risk behavior survey (yrbs) demonstrated that numerous high school students engaged in behaviors that increased their chances of dying from motor vehicle crashes, other unintentional injuries, homicide, and suicide. specifi cally, the survey results showed that . % of those surveyed had rarely or never worn a seatbelt during the days preceding the survey; . % had ridden with a driver who had been drinking alcohol; . % had carried a weapon during the days preceding the survey; . % had drunk alcohol during the days preceding the survey; . % had used marijuana during the days preceding the survey; and . % had attempted suicide during the months preceding the survey (grunbaum et al. , p. ) . the authors of the yrbs concluded that "priority health-risk behaviors, which contribute to the leading causes of mortality and morbidity among youths and adults, are often established during youth, extend into adulthood, are interrelated, and are preventable." the examination of the main causes of death in the united states, which we shall shortly discuss in the next section of this chapter, will shed further light on behavioral risk factors. meanwhile, suffi ce it to say that lifestyle and behavioral factors constitute some of the important determinants of health that health policy must seek to address. even though it is agreed that the contribution of medical care to improved health is not as pronounced as the other factors just examined, curative medical care-those practices, technologies, and organizations that society and the medical profession use to cure and rehabilitate the sick-is nonetheless a key determinant of health (blum ; cdc ) . the centers for disease control and prevention (cdc) estimate that only about % of premature deaths in the united states can be attributed to inadequate access to medical care, while the remaining % can be accounted for by individual lifestyle and behaviors ( %), genetic profi les ( %), and social and environmental conditions ( %) (cdc ) . the reason why medical care is the least important determinant of health is because it is reactive, not proactive-it waits for disease and illness to occur before intervening, so to speak. in other words, while individual and population health are somehow associated with having access to curative care, access to preventive services is of greater signifi cance. therefore, health can improve signifi cantly, and the prevalence of disease can decline dramatically, without effective medical care, due to the other determinants of health (sigerist , p. ; mckeown , p. ; banta and jonas , p. ). this knowledge is very likely the reason why williams and jackson ( , p. ) and isaacs and schroeder ( , p. ) advocate the broadening of the concept of health policy to include the other determinants of health that were not usually seriously considered when discussing health policy. this knowledge, too, is the primary reason for this chapter of the book. we can elaborate further on the importance and relevance of the determinants of health by linking them to the ten leading causes of death in the united states. where possible, the analysis will link the incidences of mortality reported in the country that are associated with each, some, or combinations of the determinants of health. table . shows the ten leading causes of death in the united states for and . we present, below, the ten leading causes of death in the country for and in order to attempt to link some of them to treatable or preventable behaviors and exposures. in other words, we shall attempt to show that most of the deaths can be associated with factors that mainly fall under the social, economic, environmental, and lifestyle and behavioral determinants of health that we have just discussed. most of the ten leading causes of death presented above are nongenetic and can be prevented or treated. diseases of the heart, cancers, cerebrovascular diseases or strokes, chronic lower respiratory diseases, unintentional injuries, diabetes, infl uenza and pneumonia, and infection-and high blood pressure-induced nephritis can be curtailed, prevented, or treated. for example, cigarette smoking is linked with an increased risk of heart disease, chronic lower respiratory disease, and cancer; obesity is a major health risk for diabetes, hypertension, coronary heart disease, and some forms of cancer; alcohol causes a wide variety of accidents and injuries, increases the risks for high blood pressure, irregularities of the heart, and stroke; fl u vaccines can minimize infl uenza deaths; and seeking treatment for infections can prevent septicemia. additionally, although there is a genetic basis for nephrosis and nephrotic syndrome, the conditions can occur as a result of infection (such as strep throat, hepatitis, or mononucleosis), use of certain drugs, and diabetes. furthermore, although age and family history are important risk factors for alzheimer's disease, longstanding high blood pressure and a history of head trauma are suspected risk factors for the disease as well mcginnis and foege ( , p. ) identifi ed and quantifi ed the major external or nongenetic factors that contributed to deaths in the united states in . deaths associated with socioeconomic factors and access to medical care, although important contributors to the total deaths recorded in the country, were not included in the study because of the diffi culty quantifying them independent of the other factors reported in the study. about years after the mcginnis and foege study, mokdad et al. ( mokdad et al. ( , p. ) used a similar methodology to quantify the nongenetic factors that contributed to deaths in . the results of the two studies cited above showed that about half of all deaths that occurred in the united states in both and could also be attributed to a small number of largely controllable behaviors and exposures, including tobacco, diet and activity patterns, alcohol, microbial and toxic agents, fi rearms, sexual behavior, motor vehicle accidents, and illicit drug use. the results of the causes of death studies reported by mcginnis and foege and mokdad and his colleagues are consistent with the fi ndings of the national yrbs cited earlier in this chapter. the survey results showed that in the united states, . % of all deaths among youth and young adults aged - years were due only to four causes: motor vehicle crashes, other unintentional injuries, homicide, and suicide. the deaths attributable to these causes among the identifi ed population group were . , , . , and . %, respectively (grunbaum et al. , p. ) . furthermore, substantial morbidity and social problems were said to result from the approximately , pregnancies that occurred each year among women - years (ventura et al. , p. ) , and from the estimated million cases of sexually transmitted diseases (stds) that occurred each year among persons - years (institute of medicine ; eng and butler ) . similar to the studies on the actual causes of death in the united states in and , the yrbs also found that the leading causes of mortality and morbidity among all age groups in the country were related to behaviors that contributed to unintentional injuries and violence, tobacco use, alcohol and other drug use, sexual behaviors that contributed to unintended pregnancies and stds, including hiv infection, unhealthy dietary behaviors, and sedentary lifestyles. in , almost years after the yrbs discussed above, the cdc quantifi ed the death rates among teenagers aged - years between and . not surprisingly, the ten leading causes of death for the teenage population remained constant throughout the period. they were as follows: accidents or unintentional injuries, % of deaths; homicides, % of teenage deaths; suicide, %; cancer, %; and heart disease, %. further analysis showed that motor vehicle accidents accounted for almost three quarters ( %) of all deaths from unintentional injury; and that non-hispanic black males had the highest death rate among all teenagers, with homicide being the leading cause of death for them (minino ) . the determinants of health that have occupied our attention up to this point are not only affected by the broad national and personal factors we have identifi ed but are also affected by broad global or international factors (shi and singh , p. ) . therefore, the rest of this chapter is devoted to examining the infl uences of global factors on the health care system and the health policymaking process. foreign policies involve the political relationships between countries and the outside world. foreign policy development generally concerns the protection of a country's national interests, usually defi ned in terms of security, economic prosperity, and ideological goals (lee et al. , p. ) . increased globalization has led to the broadening of foreign policy concerns to include health. conversely, it is now recognized that international trade and fi nance, migration and population mobility, environmental change or global warming, the emerging and reemerging infectious disease paradigms, natural disasters, and global insecurity or terrorism have clear and observable consequences for human health (kassalow ; mcinnes and lee , p. ; lee et al. , p. ; katz and singer , p. ; campbell-lendrum et al. , p. ; fidler , p. ; macpherson et al. , p. ; labonte et al. ) . we shall briefl y examine how these components of globalizationinternational trade, population mobility, infectious diseases, global warming or climate change, and natural disasters and terrorism-affect countries' health care and policymaking systems generally, and the united states' health care and policymaking systems in particular. we begin with international trade. the principal agents of global international trade and fi nance include such international agencies as the world bank, the international monetary fund (imf), and the world trade organization (wto). it has been reported that the market-biased or effi ciency-oriented austerity policies these organizations promote or sponsor have resulted in reduced expenditures for social programs in developing countries, thereby impairing population health and slowing the advances in literacy, fertility reduction, and improved reproductive health of the women of the developing countries (kinnon , p. ; gray ; watts ) . some specifi c examples of international trade and fi nance policies include the following: trade liberalization or the lowering of tariffs and other barriers to imports that has led to the doubling of the value of world trade from % of world gdp in to % in (world bank ; the reorganization of production and service provision across multiple national borders by multinational or transnational corporations, such as outsourcing or the pursuit of integration into global value chains, resulting in a global labor market (world bank , p. woodall ) ; the conditions attached to world bank and imf loans, and to the rescheduling of loan payments, including structural adjustment programs (saps); fi nancial liberalization, which exposes national economies to the uncertainties created by large and volatile short-term capital fl ows; the signifi cant growth in the world's urban population caused by transnational economic integration; the promotion of export-oriented agricultural development that does not consider the social and environmental consequences of such actions, which result from the pressures on governments around the world to increase export earnings (stonich and bailey , p. ) ; and the promotion and reinforcement of a market-oriented concept of health sector reform that strongly favors private provision and fi nancing (petchesky ; koivusalo and mackintosh , p. ). critics of the above international trade and fi nance policies argue that it is not at all clear that globalization leads to substantial poverty reduction. they point to the large-scale and extreme unequal distribution of wealth and income in the countries that have been identifi ed as "globalizers" witnessing rapidly growing economies. it is argued that even a little redistribution of income through progressive taxation and targeted social programs would go farther in terms of poverty reduction than many years of solid economic growth (jubany and meltzer ; paes de barros et al. ; de ferranti et al. ) . further, it is argued that as countries compete for foreign direct investment and outsourced production, the need to appear business-friendly may limit their ability to adopt and implement labor standards, occupational safety and health regulations, and other redistributive programs (cornia ) ; global integration of production may cause a sharp decline in the wages of, and demand for, low-skilled workers; large amounts of debt limit the ability of many developing and developed countries to meet other human needs related to health, education, water, public safety, sanitation, nutrition, etc.; globalization may lead to an intensifi cation of worldwide social relations which link distant localities in such a way that local happenings are shaped by events occurring many miles away, and vice versa (giddens , p. ) ; much of the urbanization caused by international fi nance and trade policies occurs in countries that have limited resources to provide urban infrastructures; and the emphasis on private fi nancing and provision of health care leads to large-scale underinsurance and uninsurance in both the developed and developing countries (labonte and schrecker , p. ) . globalization and the quest for exports are also blamed for increased smoking and tobacco-related mortality in the developing countries (murray and lopez , p. ) . also noteworthy is the escalation in the sale of weapons, much of it facilitated by western governments. the wars that have raged on and off in sub-saharan africa, latin america, and asia are tragic examples of the ill effects of aggressive weapon sales to these places (mcmichael and beaglehole , p. ) . although the adverse effects of globalization discussed above tend to affect developing countries more than the united states, there are signifi cant adverse consequences of globalization for the united states as well. some of these include the perpetuation and exacerbation of the gap between the rich and the poor, a large public debt profi le that puts signifi cant pressure on social and other safety net policies and programs, the prevalence of uninsurance and underinsurance, job insecurity and reduced wages, the collapse of large manufacturing businesses, increased availability and demand for illicit drugs, and the emergence of new infectious diseases that spread more easily due to increased migration and population mobility (ubokudom and khubchandani , p. ) . for example, american labor unions complain that the north american free trade agreement (nafta) with canada, mexico, and the united states, which came into force on january , , has led to the loss of american jobs. job loss causes stress, loss of income and the fi nancial means to pay for medical care. from the onset, health issues were not at the heart or margins of foreign policy theory or practice for two reasons. first, the protection and promotion of population health did not factor into world leaders' calculations of what "competition in anarchy" (the condition from which foreign policy dynamics fl ow) required of their countries, nor was health for all seriously (as opposed to rhetorically) considered a pathway to a better world. second, those who were engaged in public health did not participate signifi cantly in discussions of foreign policy (fidler , p. ) . therefore, there were only small and nonsubstantial linkages between health and foreign policy (harris , p. ) . actions linking health issues or problems with foreign policy have been strongest when the potential impact on economic prosperity, national security, the environment, and development is severe. this has resulted in attention to health threats that are acute and severe, those that are projected to result in mass casualties, and those that are believed to be geographically widespread. in contrast, long-term health risks, or health risks that cause minor health problems, affect a limited number of people, or are not geographically widespread, attract little attention in relation to foreign policy. in other words, acute epidemic infections and major public health emergencies, such as natural or human-induced disasters, bioterrorism, and chemical and radiation accidents, have received signifi cant attention (fidler , p. ; lee et al. , p. ; katz and singer , p. ) . a few specifi c examples of "attention-receiving" public health problems include the previously unknown human immunodefi ciency virus/acquired immunodeficiency syndrome (hiv/aids) which appeared in the united states in the early s; the hantavirus, believed to have originated in korea; eastern equine encephalitis, which is found in the eastern and north-central united states, canada, parts of central and south america, and the caribbean islands; western equine encephalitis, which occurs primarily in the western and central united states, canada, and parts of south america; the polio virus that is believed to have originated in india in ; the spread of severe acute respiratory syndrome (sars) from china in ; and the outbreak of the deadly h n -swine flu-infl uenza believed to have originated in mexico (cdc ; shi and singh , p. ; friis , p. ) . in summary, many health problems, particularly infectious diseases, are widely recognized as global concerns that cross national and international boundaries. consequently, countries frequently include in their foreign policies strategies on these diseases that have the potential to threaten their domestic interests. this is likely to lead to higher prioritization, more attention, greater political support, and more funding. for example, in the united states, projections of the impact of hiv/ aids on the workforces of many countries, and the prevalence of hiv among military personnel in several regions of the world, contributed to the determination that hiv/aids was a security issue. similarly, awareness of the havocs caused by previous infl uenza pandemics and the economic impact of the small and short outbreak of sars led to serious preparations by the who and its member states for the next infl uenza pandemic (katz and singer , p. ) . this understanding has led to many international agreements covering health and the environment, including the agreement on sanitary and phytosanitary measures, the international standards organization's classifi cation system for food labeling, the un framework convention on climate change, and the kyoto protocol, to name a few. data from the national aeronautics space administration (nasa) show that the earth's surface has warmed by about . °c between january and november . that period was reported to be the warmest january-november in the nasa goddard institute for space studies (giss) analysis, which covers years. the period was only a few hundredths of a degree warmer than , so it is possible that the fi nal giss results for the full year, , would be warmer or in the same range as . further, the available data also show that the earth's surface has warmed by more than . °c over the past century and by about . °c in the past decades (nasa ) . therefore, contrary to frequent assertions that global warming has slowed in the past decade, global warming has proceeded in the decade that ended in just as fast as it did in the prior decades (nasa ) . the health hazards posed by climate change and global warming are inequitable, diverse, global, and probably irreversible over human time scales (patz et al. , p. ; campbell-lendrum et al. , p. ) . they include increased risks of extreme weather, such as fl oods and storms, fatal heat waves, long-term drought conditions in many areas of the world, surface water pollution and groundwater contamination, the melting of glaciers that supply freshwater to large population centers, salination of sources of agricultural and drinking water, increased rates of water extraction that may precipitate declines in supply, and creating a conducive environment for the global killers that are very sensitive to climatic conditions, such as malaria, diarrhea, and protein-energy malnutrition (campbell-lendrum et al. , p. ; friis , p. ) . as we noted under the actual causes of death, these three global killers cause many deaths in the united states; they are also said to account for about three million deaths worldwide each year (who ) . the relationship between migration, population mobility, and health is receiving renewed attention due to the emerging and reemerging infectious diseases that were discussed previously in this section. the health of both legal and illegal migrants to any country are affected by the determinants of health discussed earlier in this chapter, as well as by the risks that are present in their country of origin or that arise from the migration process itself (macpherson and gushulak , p. ) . this is very true of the united states where a signifi cant portion of the annual population growth is due to migration. the effects of population mobility and migration on the country's health care system and the provision of health services are reported daily in the pages of newspapers. first, there is likely to be increased demand for services due to population growth, whether that growth is due to increased fertility rates or migration. for example, the exponential growth in medicaid expenditures in states that border mexico are said to be due to the increased demand for medical services by illegal immigrants as well as by the medical needs of an aging population. second, offi cials of the states that share boundaries with mexico complain about increased violent crimes committed by illegal immigrants, crimes that take a heavy toll on population health and health care expenditures. third, increased migration compels more health services planning, infrastructure maintenance, development and training of a diverse medical workforce to cater for the increasingly diverse population, and the establishment of public health programs for health promotion, health protection, and disease prevention (macpherson et al. , p. ; cohen et al. , p. ) . and, fourth, the opinion pages of newspapers carry citizens' letters that attribute the success of previous terrorist campaigns to the nearly open border policy the united states maintained prior to september , ( / ). since the / attacks, border security and entry visa requirements have been tightened. border control measures are now centered on inspecting and excluding goods, vessels, and people that pose serious health or terrorist threats to the united states. other countries have similar measures. the world has changed. indeed, the world has changed signifi cantly. while most people are actively planning on how to make their lives better, a few others are actively planning on how to destroy lives and settle political and ideological differences through acts of violence. no place and people are immune from the threats of violence, terrorism, and natural disasters. in the past or years, the united states has experienced disasters that have led to a rethinking of how to keep the population safe. the terrorist attacks in the united states on september , , an unsuccessful attempt to initiate an anthrax epidemic in october , and the devastation caused by hurricane katrina of the atlantic hurricane season led to signifi cant loss of lives and property and revealed defi ciencies in the public health and emergency response systems in the country. because of both underfunding and understaffi ng, and perhaps because the changes that have taken place in the world were not anticipated, the public health system was unable to develop or implement a comprehensive program of preparedness, prevention, response, and recovery (us general accounting offi ce ) . following the disasters, state, local, and federal public health agencies began to identify weaknesses in the nation's public health infrastructure and to reevaluate existing disaster response plans (baker and koplan , p. ). the shortcomings revealed in the nation's disaster response plans elevated public health to an important national instrument for anticipating and dealing with terrorism, infectious disease outbreaks, and natural disasters. the guidance on responses to chemical, biological, radiological, nuclear, and explosive threats provided by the cdc, and by other national organizations and universities, helped individual state governments to develop statewide policies that took their unique concerns into account (ziskin and harris , p. ; shah shah , p. gebbie and turnock , p. ) . public health plans to deal with terrorist threats, infectious diseases, and natural disasters now involve public health agencies at the federal, state, and local levels of government; other government and private agencies, such as the departments of justice and defense; the food and drug administration; private, public, and nonprofi t hospitals, clinics, and nursing homes; private and public practitioners, such as nurses and physicians; blood supply organizations, such as the american red cross; police and fi re departments; and individuals and groups throughout the country. as would be expected, expenditures for government public health activities, while still low relative to expenditures for medical care, rose from $ billion in to about $ . billion in , an increase of . % from (centers for medicare and medicaid services (cms) ) . it remains to be seen if this enthusiasm for public health, demonstrated by increased funding since , can be sustained. the law that is used as the basis for most of the new emergency preparedness measures is the homeland security act of . in addition to the strengthening of the public health infrastructure, the law also called for improved inspections of food products entering the united states. it calls for better measures to contain attacks on food and water supplies, to protect vital infrastructures, such as nuclear facilities, and to track biological materials anywhere in the country. further, the provisions of the law have been used to justify tough and controversial interrogation techniques, such as waterboarding. similarly, presidential executive order , signed by george w. bush on april , , authorizes the apprehension, detention, or conditional release of individuals with suspected communicable diseases, such as sars, cholera, diphtheria, infectious tuberculosis, plague, smallpox, yellow fever, and viral hemorrhagic fevers such as ebola (the free dictionary ) . in summation, international trade and fi nance, infectious disease epidemics, global warming and climate change, population mobility, and natural disasters and terrorism signifi cantly affect the united states health care delivery and policymaking systems. in addition, medical technology and us health care professionals and consumers are also affected by global factors. for example, because the united states is widely believed to be the world leader in the development and utilization of high-technology medical protocols, foreign dignitaries come here for specialty care. also, nurses and foreign medical school graduates (fmgs) move to the united states to acquire licenses to practice in the country. this so-called brain drain causes shortages of medical practitioners in the developing countries and alleviates some of the shortages in the health professional shortage areas of the united states. furthermore, telemedicine allows us physicians to transmit radiological images to other countries where they are analyzed at lower costs. on the other hand, us consulting pathologists and radiologists provide their services to other parts of the world. also, advanced medical equipment and supplies that are abandoned here a few years after deployment are shipped to the developing and less technology-intensive developed countries at low costs. the high costs paid by us consumers are used to subsidize the low costs paid by the developing countries (ubokudom and khubchandani , p. ) . this chapter has identifi ed the impacts of physical, social, cultural, and global factors on health and health policymaking. health can be defi ned under the medical or wellness models. the health status of the us population, or the population of any other country for that matter, is largely determined by factors that have important physical, social, and economic dimensions. these include preventive medicine, genetic disposition, social and economic circumstances, environmental conditions, lifestyles and behaviors, and medical care. these determinants of health are associated, in various degrees, with the real or actual causes of death in the country. research demonstrates that most of the deaths in the country are attributable to a small number of largely controllable behaviors and exposures, or due to factors that fall under the preventive, social, economic, environmental, and lifestyle and behavioral determinants of health. these determinants of health are not only affected by the broad national and personal factors identifi ed in the chapter, they are also affected by global or international factors, including trade and fi nance, outbreaks of infectious diseases, climate change, natural disasters, and the threats of terrorism and population mobility. but even though most of the deaths in the country are the result of social, cultural, economic, environmental, and global factors, medical care is also an important determinant of health that cannot be ignored. an insurance card is one of the important factors that infl uence access to medical services. consequently, the next chapter examines demographic factors, most especially americans' ability to access medical services, and the disparities in health among segments of the population. socioeconomic inequalities in health: no easy solution socioeconomic disparities in health: pathways and policies poor families in america's health care crisis a clinical trial of the effects of dietary patterns on blood pressure in why are some people healthy and others not? strengthening the nation's public health infrastructure: historic challenge, unprecedented opportunity in jonas's health care delivery in the united states health 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between human and ecosystem health date: - - journal: biol conserv doi: . /j.biocon. . . sha: doc_id: cord_uid: ls qus y nan intellectual ghettos are passé . there was a time when the control of wildlife diseases was the domain of veterinarians while conservation was that of biologists. that false dichotomy has long since passed as infectious disease has become a central issue in biological conservation, which itself has become enmeshed in an inter-disciplinary web that embraces the health of ecosystems and people (e.g. riordan et al., ) . indeed, these issues are set to become more entwined, as globalisation, climate change, human population expansion and the natural and unnatural movements of species interact to catapult emergent and mobile diseases to prominence beyond either the public health or conservation agendas, to land firmly on the global political table. infectious diseases have passed in both directions between people and wildlife since time immemorial, but today the enormous rise in human populations, their penetration to every corner of wilderness with concomitant land-use changes, and their transportation of organisms around the world creates an explosive mix of risks. is infectious disease generally a matter for concern by conservation biologists? one general answer might be that infectious diseases are hazards to ecosystems when they affect keystone species such as top predators, or when they undermine ecosystem support systems (foresight, ) . however, infectious disease is a natural phenomenon, and a general tenet of biological conservation might be not to meddle where natural processes operate naturally. compassion might prompt the rescue, or even euthanasia, of a sick animal, but such an intervention could be said to have little relevance to conservation, much of which is focused on the viability of populations and ecological communities. of course, even before since it was formalised in the models of anderson and may ( ) , ecologists have realised not only that parasites (in the widest sense of pathogens) were not merely a source of morbidity and mortality in nature, but could also limit, even regulate, some populations. in that sense, pathogens are clearly relevant to conservation biologists, as part of natural processes, but that certainly does not qualify them as a problem, nor does it constitute a justification for meddling in population processes insofar as these processes are natural. this line of thought and it has merit, leads to the conclusion that infectious diseases may often not be of concern to conservation. it also leads sometimes to conservationists being disquieted by a too ready eagerness to intervene when disease afflicts wildlife. on the other hand, there are clear and pressing cases where infectious disease in wildlife conspicuously affects, or is affected or caused by, humans, and human involvement is an operational definition of topics within the ambit of conservation . so, as is characteristic of conservation issues, the decision of when an infectious disease justifies intervention is not always straightforward, and indeed the position of infectious disease within conservation is both technically and philosophically challenging. it was discussions of such topics that prompted us to convene a conference to explore the diverse ways in which infectious disease threaded through issues in mammalian conservation. to say that this special issue of biological conservation is merely the proceedings of a conference would not be correct. nonetheless, it was catalysed by a two-day event held on st- nd may : the mammals trust uk's international conference on wild mammals and disease, and the ensuing think tank hosted at tubney house by oxford university's wildlife conservation research unit. from these events grew the papers that comprise this volume. our starting point was that infectious diseases can have profoundly damaging consequences for mammal populations, particularly those that are already small or isolated. some of the diseases affecting wildlife also pose potential threats to human health. of known pathogens that have infected humans historically, % had zoonotic origin . furthermore, emergence of human pathogens is associated with the ability to infect wildlife for bacteria, fungi and viruses (cleaveland et al., ) . it is estimated that % of all diseases emerging over the last two decades have been zoonoses; these include sars, avian influenza, ebola, monkey pox, and the west nile virus (brown, ; hart et al., ) . a brief scan of the major issues in conservation biology worldwide reveals infectious disease as a recurrent linking thread between them. for example, alien species bring with them a host of other, less obvious, creatures that can cause disease: ticks and fleas, intestinal worms and protozoans, viruses and bacteria. in britain, an obvious case is the grey b i o l o g i c a l c o n s e r v a t i o n ( ) - a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c o n squirrel, sciurus carolinensis; more than half a century of research and puzzlement passed before the breakthrough that their impact on native red squirrels, sciurus vulgaris, was mediated by a virus. habitat loss and modification, the most important issue for biodiversity conservation, which is also due to the expansion of human populations and activities, has widespread ramifications for infectious disease transmission to and from wildlife. worldwide, the expansion, both in range and density, of domestic dogs and cat populations with their human masters has led to an apparent increase in incidence of outbreaks in wild carnivores, particularly canids . for example, rabies spread by dogs threatens canids around the world, including highly endangered ethiopian wolves, canis simensis, and african wild dogs, lycaon pictus (laurenson et al., ) . the few recent isolated cases of rabies transmitted to humans by daubenton's bats, m. daubentonii, have given a new impetus to studies of the prevalence of this group of viruses in bat populations in the uk and its implications for human health (harris et al., ) . these were some of the issues we sought to explore as we began planning the weekend of brain-storming that gathered together experts from the cutting-edge of wildlife disease studies from around the world. and from their energetic deliberations we identified a series of seven themes which we propose as the key topics in this field of conservation biology, and around which this special issue is constructed. while biological conservation is not solely about rarity, and seeks to solve problems involving species that span the pestilential to the imperilled, extinction risk is the ultimate indicator. our first theme, therefore, was to explore the circumstances in which infectious diseases can threaten extinction. several papers, including those on rabies in ethiopian wolves, canis simensis (randall et al., ) , and african wild dogs, lycaon pictus (vial et al., ) , disease in island foxes, urocyon littoralis (clifford et al., ) , squirrel parapox virus (sqpv) in red squirrels, sciurus vulgaris (gurnell et al., ) , and devil facial tumour disease (dftd) in tasmanian devils, sarcophilus harrisii (hawkins et al., ) examine this theme. wild canids are particularly susceptible to generalist pathogens transmitted from domestic dog reservoirs, such as rabies and canine distemper virus . the three papers on canids, case reports and population viability analyses, illustrate how dramatic die-offs of around % can threaten the persistence of such rare populations, particularly when combined with other causes of population decline, as in the case of island foxes. as canids generally exhibit rapid population growth rates, only population viability analyses can estimate population persistence and determine the effectiveness of disease control strategies. pathogen-mediated competition, which can lead to unviable populations, is starkly illustrated by analyses of the dynamics of sqpv in red and grey squirrels. gurnell et al. ( ) describe how sqpv causes disease with high mortality in red squirrels but appears non-pathogenic in grey squirrels. however, not all populations of introduced grey squirrels carry the virus -those in scotland and italy do not -but the rate of red squirrel replacement by grey squirrels is some twenty times faster in those areas where grey squirrels carry the virus. the conservation of red squirrels will depend on minimising contact between these species. these examples illustrate that diseases has the potential drive extinction when a pathogen infects a variety of host species and is maintained, often with low pathogenicity, in a more numerous reservoir host, spilling over into a less numerous and sometimes isolated threatened host (lyles and dobson, ). yet diseases can also decimate large populations of endemic species. hawkins et al. ( ) record the extraordinary case of devil facial tumour disease (dftd) that now constitutes a serious threat to the tasmanian devil sarcophilus harrisii. the tasmanian devil is the world's largest extant marsupial carnivore. dftd is a cancerous disease found exclusively in wild devil populations, and hawkins et al. report that it is consistently fatal to afflicted individuals. the tumours were first reported in and have subsequently been histologically confirmed in individuals from separate sites, covering % of tasmania (with a maximum of % of individuals infected). on the basis of the threat posed by dftd, the devil has been listed as a threatened species in tasmania. a recurring issue in infectious disease epidemiology, from public health, conservation and livestock management perspectives, is the difficulty in identifying the reservoir of infection for multi-host pathogens (e.g. courtenay et al., ; haydon et al., ; mathews et al., ) . reservoir identification can aid disease management, ensuring that the correct host is targeted with appropriate and effective disease control tools. when disease control measures in wild species of conservation concern are difficult to implement, due to complicated logistics or limited finances, identification of a reservoir domestic host may offer potential control measures that might be both effective and feasible. the importance of reservoir identification is classically illustrated by a range of papers in this special issue, for example the ongoing dilemma facing bovine tuberculosis control , the diseases emerging from bats (breed et al., ) , phocine distemper virus (pdv) in northern seal population (hall et al., ) and the canid pathogens threatening island foxes (clifford et al., ) . in the southwest of england bovine tuberculosis has proved increasingly recalcitrant and is currently increasing in cattle at an annual rate of %. macdonald et al. report on studies of the possible role of various wild mammals and conclude that if any wildlife species plays a significant role in the epidemiology of btb in cattle, it is likely to be badgers, meles meles, although the role of deer, particularly fallow deer, dama dama, in a local context is still unclear. other species simply do not occur in sufficiently large numbers or transmission does not appear sufficiently frequent to allow m. tuberculosis persistence and thus constitute potential reservoirs. identifying the reservoirs of the emergent henipaviruses, which caused mortality in humans and domestic animals (hendra virus in horses, nipah virus in pigs) in the s has been challenging, but these viruses have now both been identified in fruit bat species (breed et al., ) . this paper highlights the role of bats as reservoirs of these and other emerging diseases. behavioural and physiological characteristics of species such as the fruit bats, including the ability to cover long distances between islands and continents, close association at roosts and their mammalian physiology, ensure a large effective population size for pathogen persistence and adaptation. identification of fruit bats as reservoirs for henipaviruses means that control can be directed at minimising their contact with domestic species. ecological communities are complex and elucidating the reservoir host, when situations cannot be experimentally manipulated is challenging. hall et al. ( ) illustrate this with the case histories of two outbreaks of phocine distemper that have severely affected harbour seal (phoca vitulina) populations in european and uk waters. both outbreaks were detected on the danish island of anholt, the first in and the second in . harbour seals were highly susceptible to infection while sympatric grey seals are more resistant. in this phocid community the most likely reservoir of the virus are arctic species of seals, but grey seals, halichoerus grypus, could be important asymptomatic carriers that link these reservoir hosts to the harbour seal populations further south. hall et al. emphasise that understanding of the determinants of the host range remains poor, and that development of more realistic epidemiological models should be combined with studies into the factors controlling species and individual susceptibility. only then will further understanding of reservoirs hosts and transmission routes be built up. clifford et al. ( ) paper on island foxes also illustrates the approach that must be taken when reservoirs cannot be identified. serological studies suggest that feral cats are not putative reservoirs for the canid pathogens under study, but have not revealed whether the pathogens can be maintained in the small island fox populations or are introduced from domestic dogs. in this situation, the practical solution seems to be targeted vaccination programs against the most virulent pathogens and continued intensive disease surveillance. using models to improve disease management for conservation it is clear from this special issue that modelling is a powerful tool for understanding and planning the management of infectious disease. recent stochastic, mixed models offer novel predictions about the role of culling, fertility control, and oral rabies vaccination in rabies control and are reviewed by sterner and smith ( ) . furthermore, both vial et al. ( ) and randall et al. ( ) illustrate how alternative management strategies might affect disease persistence and spread in african wild dogs and ethiopian wolves, respectively. targeting only a viable minimal 'core' of the population, through oral or even parenteral vaccination, is likely to be effective as well as more affordable and logistically less demanding. even if only % of animals were protected with a vaccine lasting only two years, this would ensure persistence of even small populations through suppression of the largest outbreaks of disease, that reduce populations to below minimum viable population sizes. similarly, modelling of the spread of both grey squirrels and their sqpv, by gurnell et al. ( ) has identified four main corridors whereby grey squirrels will reach kielder forest (one of the red squirrel's last strongholds in england), initially within two years and in large numbers within years. assuming that greys will not settle within kielder because of the unfavourable nature of the spruce habitat, the authors predict that sqpv disease will burn out at the edges of the forest, although many red squirrels will die. this burn-out is unlikely to be the scenario in other refuge areas where the habitat is more favourable to greys. gurnell et al. conclude that the conservation of red squirrels will therefore depend on minimising contact between red and grey squirrel populations. this will necessitate monitoring grey squirrels as they approach refuge areas, and removing them. morgan et al. ( ) illustrate both the power and the limitations of a modelling approach, through their study of the pathogens of the saiga antelope (saiga tatarica) and domestic ruminants in central asia. for both foot and mouth disease and gastrointestinal nematodes, they reveal that the main risk is associated with infection of saigas from livestock, the putative reservoir host and subsequent geographical dissemination of infection through saiga migration. their discussion on the trade-off between adding biological reality to models, and thereby increasing mathematical complexity and intractability, highlights that the main contribution of modelling is probably to force researchers to formalise understanding in a logical way, highlighting the areas in which too little is known. these areas of uncertainty can then be made the focus of further research, and sound a warning not to accept too readily the predictions of simpler models. sterner and smith ( ) go on to adopt a further multidisciplinary approach and integrate models of disease spread with economic analyses of medical, public health, and veterinary costs. this reveals how post-exposure prophylaxis and increased pet vaccinations have been major costs during and after epizootics in north america. they recommend that this approach should be expanded when considering disease control options in biodiversity conservation. disease surveillance as a cornerstone in disease and conservation management disease surveillance and monitoring self-evidently provide crucial evidence to underpin management decisions. the advances in recent years in molecular techniques have been rapid and have provided powerful tools for the investigation of wildlife disease. this is forcefully illustrated by leendertz et al. ( ) who describe approaches and techniques that can be used in the field when investigating baseline health status and disease outbreaks in great apes, focusing in particular on non-invasive sample collection and mortality investigations. this provides the very basis for diagnosis and surveillance and can be extrapolated to other species. this paper also demonstrates how synergies can also be obtained through close collaboration between the fields of public health and conservation (see below). harris et al. ( ) , also report on exemplary programmes of both passive and active surveillance in the united kingdom to investigate the prevalence of european bat lyssaviruses (eblv) a rabies-related virus, in bats. since , cases of eblv have been recorded in bat species in europe. some bats have been tested for lyssaviruses for surveillance, and an antibody prevalence level of - % of eblv- has been found in daubenton's bat. however no cases of live lyssavirus infection or lyssavirus viral rna have been detected through active surveillance. the authors emphasise how research and monitoring regarding prevalence, transmission, pathogenesis and immunity is required to ensure that integrated bat conservation continues throughout europe, whilst enabling informed policy decision regarding both human and wildlife health issues. disease surveillance is also a cornerstone of diagnosis and control when considering the effect of reintroductions and translocations. however, as mathews et al. ( b) argue, despite guidelines recommending health-screening and surveillance of translocated individuals and in re-established populations, few reports are available and best practice is rarely observed. these authors present two case studies of health surveillance in wildlife reintroduction programmes -in water voles (arvicola terrestris) in the uk, and in marsupial dibblers (parantechinus apicalis) in australia. these illustrate the potential importance of even basic screening strategies in helping to avoid disease transfer and identifying predictors of survival. building on improved techniques for surveillance, one would expect strong parallels between disease in humans and wildlife, and hence that advances in new research fields such as comparative genomics and molecular genetics would hold lessons for conservation biology. o'brien et al., argue exactly this case, using examples drawn from wild species of the cat family, felidae. they conclude that resolving the interaction of host and pathogen genomes can shed new light on the process of disease outbreak in wildlife and in people, by reviewing a highly virulent feline coronavirus epidemic in african cheetahs and a disease model for human sars, which illustrate the critical role of ancestral population genetic variation. furthermore, widespread prevalence of species specific feline immunodeficiency virus (fiv), a relative of hiv-aids, occurs with little pathogenesis in felid species, except in domestic cats, suggesting immunological adaptation in species where fiv is endemic. o'brien et al. conclude that conservation management may benefit greatly from advances in molecular genetic tools developed for human biomedical research to assay the biodiversity of both species host and emerging pathogens. leendertz et al. ( ) provide a further illustration of how these synergies can be built up, by demonstrating how collaboration between human laboratories, veterinarians and field biologists can successfully tackle emerging diseases in great ape and human populations. successful pathogen detection in wild great apes has been achieved several times thanks to approaches and techniques that have been developed for human pathogen detection, focusing in particular on investigation of deaths and non-invasive sample collection. most of the host-pathogen systems reported in this special issue concern situations where pathogens have been defined as 'emerging', that is they have recently increased in incidence, impact or geographic range; have recently moved into new populations; or are caused by recently-evolved pathogens (morse, ; dazak et al., ; lederberg et al., ) . a variety of reviews have highlighted the situations and risk factors for emergence in human and domestic animals populations (morse, ; taylor et al., ; cleaveland et al., ) . the expansion of human populations is paramount, influencing agricultural development, urbanisation, deforestation and habitat fragmentation. this in turn influences disease emergence by changing the densities and ecology of disease hosts vectors and pathogens, and altering human interaction with them (mcmichael, ) . for example, the penetration of remote forest areas through logging or bushmeat hunting is rapidly escalating the contacts between people and primates (zommers and macdonald, ) . not only does this have important implications for direct persecution through the harvest of bushmeat, but it also raises sinister opportunities for disease transmission as described by leendertz et al. ( ) . they describe recent outbreaks of disease in great apes, including ebola, and indications of cross-transmission of ebola and other viruses between primates and humans. there is no doubt that research that integrates infectious disease with primate ecology provides insights to emerging diseases in humans and the role of disease in primate evolution. international travel and global trade hugely increase the capacity for disease spread and are thus other factors that determine disease emergence. a prime example was apparently rabbit haemorrhagic disease virus (rhdv) which was first identified after thousands of domestic rabbits died suddenly in china in . similar epidemics subsequently occurred in other regions of asia, the middle east, europe and north america, suggesting that the virus had dispersed widely following its emergence in china. however, forrester et al. ( ) report that rhdv had circulated apparently harmlessly for many years before the first recognised epidemic in china. they have therefore studied the evolution, emergence and dispersal of this virus in relation to its impact on conservation of wildlife species. using phylogenetic analysis they show that the chinese epidemic virus represents a relatively recent lineage derived from more divergent european viruses that circulated for many years prior to . they show that the genetic lineages of the pathogenic viruses that emerged in the uk in the early s, are distinct from and pre-date those of the chinese virus. in short, several other divergent pathogenic european strains of rhdv emerged from apparently harmless strains to cause epidemic outbreaks, independently of the chinese epidemic virus. forrester et al. also illustrate the complexity of conservation issues surrounding the rabbit in the uk -originally an invasive species, now an agricultural pest, yet a source of food for rare native predators and an important tool for habitat conservation. contact with wildlife has also been identified as a risk factor for emerging human pathogens . this is illustrated in this special issue by papers describing high profile emerging viruses that have caused significant disease in domestic animals and humans from wild fruit bats in asia and australia (breed et al.,) and european bats (harris et al.) hendra virus has caused disease in horses and/or humans in australia every five years since it first emerged in . nipah virus has caused a major outbreak of disease in pigs and humans in malaysia the late s and has also caused human mortalities in bangladesh annually since . emergence may have been precipitated by unsustainable hunting of fruit bats and deforestation which have changed the distribution and thus contact rates between fruit bats and humans or their domestic animals, combined with an increase in domestic pig production in asian countries. animal behaviour, as the central factor, more often than not, in determining transmission of infectious disease between infected and susceptible individuals, is unquestionably important in wildlife epidemiology and conservation. this point was originally made in the context of failing attempts to control rabies in european red foxes, vulpes vulpes, which largely ignored their behavioural ecology (macdonald, ) , and in this volume it is made stridently by randall et al. and vial et al., who illustrate how knowledge of, respectively, ethiopian wolf and african wild dog social systems is important to understanding and managing the extinction threat they both face from rabies. further emphasise this in the context of attempted control of bovine tuberculosis (btb) in cattle. the worsening situation of btb in cattle has occurred despite a succession of government schemes involving killing badgers with the intention of reducing transmission of btb to cattle. macdonald et al. discuss the perturbation hypothesis which postulates that killing individuals may affect the survivors in ways (behavioural, physiological, immunological) that cause a disproportionate, and perhaps counterproductive, effect. they conclude that the perturbation hypothesis is supported by the data and does provide one plausible mechanism to explain why culling badgers has not generally achieved control of btb in cattle. macdonald et al. draw on various studies to argue that to have any prospect of contributing significantly to controlling btb in cattle, a badger cull would have to be undertaken over a very large area. considering the likely very important role of cattle-to-cattle transmission, and the opportunities for solutions in terms of farm management and surveillance, they judge it would be inappropriate (and probably impractical) to undertake such a cull now (especially in the context of revised agricultural payments which increasingly put a premium on custody of the countryside and its biodiversity (mathews et al., a) ). remarkable satellite tracking studies have revealed long distance migrations by the fruit bats (breed et al., ) . this behaviour clearly has far-reaching implications for disease transmission within and across continents, particularly for migratory species. saiga movements, that determine the timing and scale of contacts with domestic animals, also crucially affect disease transmission and persistence. morgan et al. ( ) explain how for both foot and mouth disease and gastrointestinal nematodes, the main risk is associated with infection of saigas from livestock, and subsequent geographical dissemination of infection through saiga migration. the chance of this occurring for foot and mouth disease is predicted to be highly dependent on saiga population size and on the time of viral introduction. for nematodes, the level of risk and predicted direction of transmission are affected by key parasite life history traits, such that prolonged off-host survival of marshallagia in autumn enables infection of saigas and transfer northwards in spring. these seven themes have been in the news regularly fzs since our weekend workshop, as the world anxiously watches the progress of h n avian flu, west nile virus and sars on the front-pages. sars, appearing in southern china in late , illustrates that issues at the heart of biodiversity conservation and ecosystem health are fundamental to human wellbeing. sars may have emerged in humans from sars-like coronaviruses (cov) in himalayan palm civets (paguna larvata) and other small carnivores in the wet markets of asia. specimens collected from animals found in live wild-game markets in guangdong china have yielded a sars cov-like virus and several of the early sars patients in guangdong province worked in the sale or preparation of wildlife for food (bell et al., ; peiris et al., ) and half of civet dealers at the market were found to have antibodies that cross reacted to sars (bell et al., ) . recent research suggests that the disease may have jumped to civets from rhinolophid bats in the marketplace, since sars-like coronavirus has been detected in three species from china, australia and the usa lau et al., ) . greater genetic variation between these bat strains (as revealed by nucleocapsid protein sequences) than seen in the human or civet sars indicates that the viruses and bats have had time to co-adapt, and hence that bats are probably the origin of sars. bats find themselves along with civets and people in wildlife markets, to which live mammals are brought from increasingly remote areas into contact with an increasingly large human population with global links. bats are also important pollinators and dispersal agents, and many are endangered species and thus their conservation is a priority for the continuing function of many ecosystems. this blend of factors links the concerns of public health, agriculture, biodiversity conservation, animal welfare, third world development and global economics. a salient reality shrieks from the sars story and is touched on elsewhere in this volume: the inter-connectedness of global populations of humans and wildlife. hunters, farmers, market vendors and consumers experience direct risk of zoonotic disease transmission from bushmeat and animals (karesh et al., ) . however, human to human transmission then spreads the risk to other individuals around the world and can turn a local outbreak into a global crisis. the local sars outbreak in hong kong and southern china, quickly spread to countries across five continents, through human air travel (peiris et al., ) . the fact that over million people travel by air annually (karesh et al., ) means that the risk of global epidemics has never been higher and is the factor that could accelerate the spread of a global flu pandemic, ahead of vaccine production, should the mutations for human-human transmission occur. all these studies bring repeatedly to the fore the inextricable linkages that ensure that no wildlife disease issue is the preserve of any one discipline. ecologists to economists, virologists to veterinarians, philosophers to politicians, are all enmeshed in understanding the linkages, and in working together to find solutions. the need for an interdisciplinary approach has been reiterated widely in recent years and is highlighted again in the recently published uk office of science and innovation foresight report ''infectious disease: preparing for the future''. this report brought together diverse experts to consider the threats from disease for wildlife and for humans. their conclusions emphasise the pressing nature of the issue, not least because of advancing climate change which may affect host and vector abundance and distribution and thus contact networks and transmission rates, but also because of the huge costs of disease outbreaks that affect human or livestock health. for example, bse in the united kingdom in - is estimated to have cost £ . billion whereas avian influenza in vietnam in / is costed at £ . billion (foresight, ) . against this background, progress forward must be made. but which way is forward? whilst evidence-based policy is surely essential, and thus science must underpin disease management, we live in a rapidly changing, unpredictable world where a complex web of risk factors can give rise to new disease-related problems at any turn. science thus may not always have complete answers prepared in anticipation but, with investment, it can prudently look ahead. one delphic circle of the wise recently identified the following priority areas (foresight, ) for investment (i) novel information technology for capture, analysis and modelling of data for the early detection of infectious diseases, (ii) early detection and characterisation of new or newly resistant/virulent pathogens using genomics, (iii) improving technology for the rapid identification and characterisation of infectious diseases in the field and (iv) high-throughput screening for infectious diseases of people, animals and plants using surrogate, noninvasive markers in airports, sea/road containers and livestock markets. others could add to this list. for example, this, and all other branches of conservation biology, must urgently develop inter-disciplinary syntheses, must achieve alignment with other major guests around the table of environment, sustainability and development, and must not forget the underpinning importance of a deep understanding of natural history . some of the roads leading to these new initiatives are charted in the papers gathered in this special issue. biological conservation stands to benefit from the anticipated advances in new diagnostic, monitoring and control tools. given that the vast majority of endangered species occur in the developing world, these tools and technologies must also be cheap and practical to use in areas where infrastructure is poor. moreover, although we are easily seduced by the new technology, we must keep an eye on the fact that old, lo-tech methods might frequently be best in the developing world and we must keep at eye on the fact that new technology is seductive and that in many instances, old methods might still be best. several spectres loom as infectious diseases, the exploding human population and the biodiversity and extinction crises travel together into the century. some wild populations that were once sufficiently abundant to withstand epizootic disease are now so reduced that an outbreak could tip the balance towards extinction. many of these find themselves encircled and infiltrated by burgeoning hoards of infectious domestic species. some wildlife diseases that once smouldered in isolated wilderness now challenge people that have penetrated their isolation and, through remarkably few links, populate a transmission chain that spans the globe. on the bright side, however, the papers in this volume reveal how the problems are starting to be understood, in some cases sufficiently to achieve solutions. furthermore, biodiversity conservation has taken its place, along with others concerned with infectious diseases, at the table where environmental futures will be decided. one conservation truth is particularly, and perilously, clear when it comes to infectious disease: biodiversity and humanity are in it together. through the kindness of professor john beddington and jill nelson, the mammals trust uk sponsored the weekend of conference and think-tank that spawned this volume, and professor rob mars agreed to publish this special issue of biological conservation. dr. andrew pullin, as the editor guiding us, has been both wise and helpful, and at elsevier julie millard has been unwaveringly supportive and unflustered. all the papers in this volume have been reviewed by at least three reviewers, and by both of us; the result is that all the authors have had to forebear writing at least two, and sometimes more, revisions. we thank both the reviewers and authors for their hard work, and tolerance of our pernicketiness. this paper, which serves as a preface, benefited from the comments of angela mclean, gus mills and zinta zommers. emerging henipaviruses and flying foxes -conservation and management perspectives. biological conservation, this volume emerging disease of animals diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the role of pathogens in biological conservation pathogen exposure in endangered island fox (urocyon littoralis) populations: implications for conservation management epidemiology of canine leishmaniasis: a comparative serological study of dogs and foxes in amazon brazil emerging infectious diseases of wildlife-threats to biodiversity and human health unravelling the paradox of the emergence of rabbit haemorrhagic disease virus using phylogenetic analysis squirrel poxvirus; landscape scale strategies for managing disease threat phocine distemper virus in the north and european seas -data and models, nature and nurture. biological conservation, this volume european bat lyssaviruses: distribution, prevalence and implications for conservation emerging disease and population decline of an island endemic, the tasmanian devil sarcophilus harrisii. biological conservation, this volume identifying reservoirs of infection: a conceptual and practical challenge wildlife trade and global disease emergence severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats assessing and managing infectious disease threats to canids emerging infections: microbial threats to health in the united states pathogens as rivers of population declines: the importance of systematic monitoring in great apes and o other threatened mammals. biological conservation, this volume bats are natural reservoirs of sars-like coronaviruses infectious disease and intensive management: population dynamics, threatened hosts and their parasites rabies and wildlife: a biologist's perspective principles, practice and priorities: the quest for 'alignment biological hurdles to the control of tb in cattle: a test of two hypotheses to explain the failure of control. biological conservation, this volume bovine tuberculosis (mycobacterium bovis) in british farmland wildlife: the importance to agriculture health surveillance in wildlife reintroductions. biological conservation, this volume keeping fit on the ark: assessing the suitability of captive-bred animals for release environmental and social influences on emerging infectious disease: past, present and future assessing risks of disease transmission between wildlife and livestock: the saiga antelope as a case study factors in the emergence of infectious diseases factors and determinants of disease emergence plagues and adaptation: lessons from the felidae models for severe acute respiratory syndrome integrated disease management strategy for the control of rabies in ethiopian wolves modelling wildlife rabies: transmission, economics, and conservation. biological conservation, this volume risk factors for human disease emergence development of vaccination strategies for the management of rabies in african wild dogs. biological conservation, this volume infectious disease in the management and conservation of wild canids the wildlife trade and global disease emergence. foresight. infectious diseases: preparing for the future laurenson frankfurt zoological society, p.o. box , arusha, tanzania and royal (dick) school of veterinary studies all rights reserved key: cord- -due tloa authors: nan title: ecr , part a date: - - journal: insights imaging doi: . /s - - - sha: doc_id: cord_uid: due tloa nan percutaneous needle biopsy has been a mainstay of oncologic diagnosis for almost three decades, since the advent of ultrasound and ct. the basic principles of fine-needle aspiration and core needle biopsy can be applied to almost any site in the body, with subtle differences in technique depending on the organ being investigated and the imaging modality utilised. while excisional biopsy is still appropriate in certain cases, percutaneous needle biopsy has become the standard of care in the diagnosis of most tumors throughout the body and is also used to diagnose noncancerous conditions, such as infection. percutaneous biopsy is also beneficial in the staging of patients with cancer, particularly when another treatment method may be more appropriate than surgical resection. the advantages of percutaneous biopsy over surgical excisional biopsy include time and cost savings and reduction in morbidity. the aim of this course is to discuss the practical aspects of biopsy, needle selection, and guidance techniques and to show how to approach difficult lesions and avoid complications. imaging after treatment of breast cancer is for confirmation of lesion removal, identification of postprocedural fluid collections, detection of residual or recurrent cancer and screening for metachronous cancers. posttherapy changes -which include fluid collections, edema, skin thickening, architectural distortion, scarring and calcifications -are mainly due to surgery, axillary dissection and radiotherapy. the greatest treatment-related changes occur - months after therapy, and mammographic stability is achieved after two to three years. for mammography, pre-and all posttherapeutic images have to be compared. ultrasound is the method of choice for evaluation of fluid collections. mri is for problem solving (i.e. differentiation between scar and relapse) and should not be performed prior to months after therapy to avoid false-positive diagnoses. dystrophic calcifications may develop in areas of fat necrosis mimicking malignancy. fat necrosis predominantly occurs at the treated site; however, it can develop anywhere in the ipsilateral breast. its appearance may be indistinguishable from cancer at all imaging modalities. to differentiate between fat necrosis and other common post-treatment changes from relapse, it is important to know the timeline when these changes occur and schedule follow up imaging accordingly. mammography serves as the basis for postoperative surveillance. ultrasound is helpful in the early postoperative phase, whereas mri is the method of choice, especially for differentiation of scar and relapse in the later postoperative phases. breast radiologists need to be familiar with post-treatment imaging findings in patients with breast cancer, but often patients are also imaged after a diagnosis of benign entities which are treated surgically (fibroadenomas, radial scars, papillary lesions) or after a percutaneous diagnosis of a high-risk lesion which has undergone a surgical biopsy to avoid underestimation (atypical ductal hyperplasia, lobular neoplasia or flat epithelial atypia). these procedures will leave an imprint on the breast which can be a cause of concern due to the surgical scar. furthermore, imaging findings after plastic surgery for mastopexy, reduction mastoplasties, implants or auxiliary techniques (lipofilling, hyaluronic acid) are becoming frequent in our daily practice and have their peculiarities that can overshadow breast cancers. interventional percutaneous procedures can also be a cause of tissue distortion. there are advanced systems for biopsy (bles) and for percutaneous removal of benign lesions (fibroadenomas, papillomas), which use large-gauge needles and have to be accounted for due to their trace in the breast tissue. endoscopy is currently considered the reference standard for the evaluation of colonic disease activity in patients with inflammatory bowel disease (ibd). however, it only allows evaluation of the mucosal surface and is not always complete. it cannot, therefore, help to estimate the depth of involvement of transmural inflammation and extraluminal complications, both characteristics of ibd. an evolving role for cross-sectional imaging in the evaluation of patients with ibd is increasingly recognised, especially in the setting of crohn's disease (cd) since the cross-sectional imaging has been demonstrated to have a high diagnostic accuracy not only for assessing the presence and extension of luminal disease, but also for evaluating the cd-related acute or chronic complications. available evidence suggests that ultrasound, computed tomography and magnetic resonance have similarly high diagnostic accuracy in the detection of disease activity, location, severity and complications: in particular, the penetrating and stricturing lesions which are characteristic of cd. thus, the choice of the technique for assessing cd may be influenced by local availability or expertise. in the case of ulcerative colitis, cross-sectional imaging, although less evaluated, may also be helpful in certain circumstances. there is evidence indicating that cross-sectional imaging is an alternative problem-solving tool to endoscopy whenever tissue sampling is not required and can provide valuable guidance for performing medical and surgical treatment with maximised efficacy and safety. overall, findings from crosssectional imaging accurately reflect disease activity and provide reliable information for decision-making and patient care optimisation. learning objectives: . to learn about optimised examination protocols for ulcerative colitis and colonic crohn's disease in the acute, subacute and chronic disease setting. . to become familiar with the criteria for the assessment of disease activity through ct, mri and ultrasound. . to learn about an integrated approach to the use of cross-sectional imaging in colonic inflammatory bowel disease. liver transplantation is the accepted treatment of patients with irreversible liver cell failure and some metabolic disorders and in a selected group of patients with hepatocellular carcinomas. over the last decade, major transplant centres have reported improving survival rates, though during this period they have developed more complex surgical techniques, including split-liver, auxiliary and live-related transplantation, and have treated more marginal higher risk patients. this successful outcome has been dependent on appropriate recipient selection, robust surgical technique, improvements in immunosuppression and intensive care management and the prompt recognition and treatment of complications. diagnostic and interventional radiology have been core specialties in achieving the goals of improved graft and patient survival. improvement in surgical techniques has decreased the more common vascular and biliary complications, but the newer techniques present differing diagnostic and interventional challenges, particularly in paediatric recipients. developments in mr and mdct allow many of these vascular, biliary and infective complications to be diagnosed non-invasively. vascular techniques of angioplasty and stent placement may reverse the sequel of graft ischaemia or portal hypertension. mrc allows the diagnosis of biliary strictures that may be treated by dilatation or stent placement. imaging is also important in the diagnosis of recurrent disease and the acquired diseases of prolonged immunosuppression including atypical infections and the post-transplant lymphoproliferative disorders (ptld). this interactive session will present these appearances by case example and provide guidance of the appropriate diagnostic and treatment paradigm. learning objectives: . to understand the common imaging findings after liver transplantation. . to recognise significant complications following liver transplantation. a- : b. imaging of treated liver tumours i. bargellini; pisa/it (irenebargellini@hotmail.com) imaging findings after systemic and loco-regional treatments vary greatly depending on baseline features of the treated tumour and treatment modality. conventional uni-dimensional and bi-dimensional criteria (such as recist . and who criteria) have been extensively validated in metastatic lesions treated with conventional chemotherapy. however, their prognostic value is limited in patients treated with new molecular targeted therapies and after locoregional treatments. in the setting of hepatocellular carcinoma (hcc), specific response criteria (such as easl and mrecist) have been proposed that take into account variation in the size of the viable tumour. these criteria have been extensively validated, although their interpretation could be troublesome after some specific treatments, such as molecular targeted agents and y radioembolisation. on the other hand, there is no consensus regarding tumour response criteria for metastatic lesions after loco-regional treatments. the role of morphological criteria and several imaging biomarkers (such as those provided by diffusion and perfusion imaging, fdg activity, dual source ct) is under investigation, being able to provide additional information on tumour activity and biology. while new drugs with different mechanisms of action and new treatments are becoming available, the work of radiologists is changing and there is increasing evidence that tailored radiological response criteria are required for these new targeted and tailored treatments. learning objectives: . to understand the common imaging findings after chemotherapy for liver tumours. . to recognise common imaging findings after radiofrequency ablation of liver tumours. . to be aware of the common imaging findings following transarterial treatment of liver tumours. vessel size is practical, since it is related to the pathological changes as well as to the clinical and radiological presentation. the two main large vessel vasculitides are giant cell arteritis and takayasu arteritis. behcet disease may combine large and small vessel vasculitis. imaging plays an important role in primary vasculitis. chest radiographs are not especially useful in large vessel vasculitis. contrast ct and mri further detect and especially help in the characterisation of large vessel vasculitis. vessel wall changes are well detected with both techniques. contrast enhancement, distribution of vessel involvement and morphological vascular changes should be considered. today, pet/ct is the recommended imaging technique in the assessment of vessel wall inflammatory changes and in the evaluation of treatment response. although the clinical scenario differs usually between takayasu and giant cell arteritis, the radiologist should combine the imaging findings with clinical and laboratory data to suspect a specific vasculitis. therefore, this presentation will concentrate on the basic signs and associated findings in large vessel vasculitis, pathologic correlation, imaging protocols and the differential diagnosis. the term vasculitis refers to a variety of clinico-pathological entities. the most widely used chapel-hill classification divides the vasculitis syndromes into three groups based on the size of vessels primarily involved. small vessel vasculitis such as anca-associated granulomatous vasculitis (wegener's disease), churg-strauss granulomatosis and microscopic polyangitis are most often associated with pulmonary abnormalities. the spectrum of hrct findings is quite variable and differs by entity. the hrct findings will be discussed together with clinical and laboratory findings to be integrated into a multidisciplinary diagnostic approach. the typical findings in wegener's granulomatosis include solitary or multiple, often cavitary nodules or masses, or focal or diffuse consolidations, churg strauss is dominated by interlobular thickening or transient multifocal and nonsegmental consolidations frequently in subpleural distribution. all types of pulmonary vasculitis may present with focal or diffuse pulmonary haemorrhage that produces pulmonary ground glass or consolidations in various distributions. a number of collagen vascular diseases (e.g., lupus erythematosus) or other granulomatous diseases (e.g. sarcoidosis) may affect the small pulmonary vessels causing haemorrhage or pulmonary hypertension. per disease entity, the course will review typical and more rare hrct features and key features that allow for diagnosis and classification, discuss side-to-side overlapping of morphologic features important for differential diagnosis and illustrate the findings of other diseases that represent the most challenging differential diagnosis, e.g. oedema, infection or malignant diseases. learning objectives: . to learn when hrct is of value in investigating pulmonary vasculitis. . to appreciate the different appearances of pulmonary vasculitis on hrct. a- : d. inflammation and remodeling a.a. bankier; boston, ma/us (abankier@bidmc.harvard.edu) the presentation will lay out the pathological and pathophysiological basis for the complex processes of inflammation and remodelling. the implications of these processes for imaging will be discussed and illustrated by selected pathologies. finally, the presentation will indicate how the imaging reflections of these processes could be used in the future as imaging biomarkers for the diagnosis, follow-up, and outcome evaluation of disease. at (marcus.hacker@meduniwien.ac.at) appropriate diagnosis and therapy of coronary artery disease (cad) frequently require information about both the morphological and functional status of the coronary artery tree. thus, combined imaging consisting of invasive coronary angiography (ica) plus spect myocardial perfusion imaging (mpi) is practiced in clinical routine diagnostic of patients with stable angina since many years and can therefore be accepted as the reference standard in the diagnosis of hemodynamically relevant coronary artery stenoses. both morphological and functional information are mandatory for the decision of performing an interventional therapy or initiating/maintaining medical treatment in numerous symptomatic patients. the hemodynamically relevance of coronary artery lesions is a major condition to decide whether an interventional therapy should be performed or not. a non-invasive concept providing both information could provide accurate allocation of perfusion defects to their determining coronary lesion and specific morphological and functional classification of patients with coronary artery disease. in symptomatic patients, a normal stress mpi confers a very low short-term risk for cardiac death and/or acute myocardial infarction. however, a normal mpi does not exclude the presence of underlying coronary atherosclerosis, which may be extensive although not yet flow-limiting. in this regard, ct will unmask a sizeable subgroup of patients with coronary atherosclerosis who should receive more intensive antiatherosclerotic intervention than would have been indicated by mpi results alone. knowledge regarding the presence and extent of subclinical coronary atherosclerosis in patients who do not have ischemia by mpi can be of importance in patient management. learning objectives: . to appreciate the scope of information a spect/ct cardiac study can deliver. . to become familiar with protocols of spect/ct studies. . to learn a structured approach to performing and reporting a spect/ct study. a- : c. mr/pet: do we really need it? h.h. quick; erlangen/ de (harald.quick@imp.uni-erlangen.de) following pet/ct and spect/ct, mr/pet hybrid imaging is the most recent addition to the palette of hybrid imaging modalities. mr/pet synergistically combines the excellent soft tissue contrast and detailed image resolution of mr with metabolic information provided by pet. integrated mr/pet systems furthermore offer the ability to acquire hybrid imaging data simultaneously. this can be applied to mr-based motion correction of pet data. these features open up several cardiac applications, e.g. evaluation of cardiac function and viability, diagnosis of cardiac inflammatory diseases and tumorous diseases. to fully assess the diagnostic potential of mr/pet, however, several technical challenges have to be considered: attenuation correction (ac) of the patient tissues in mr/pet has to be based on mr-images and is currently hampered by a limited number of tissue classes and undercorrection of bone tissue. cardiac radiofrequency coils and ecg gating equipment are currently not considered in ac. consequently, quantification of pet data therefore might be biased. the clinical workflow is rather complex and needs to be tailored to cardiac examinations. few research groups currently explore this new hybrid imaging modality in selected cardiac applications. cardiac mr/pet: do we really need it? considering the sparse clinical experience that is available today, it is quite early to answer this question yet. however, once the remaining technical hurdles are overcome and the diagnostic potential can be fully exploited clinically, the answer is most likely positive. the majority of malignant bone tumours can be detected on plain radiography. the age of the patient, location of the tumour in a bone and history of a preexisting bone abnormality should be included in determining the likely diagnosis. careful analysis of the pattern of bone destruction, periosteal reaction and matrix mineralisation allow for characterisation of most cases of osteosarcoma, ewing's sarcoma, chondrosarcoma and adamantinoma. mri is the best imaging technique for staging by displaying . the extent of bone marrow involvement, including epiphyseal infiltration and skip lesions, . the presence and extent of extraosseous soft tissue mass, . involvement of the neurovascular bundle, muscle compartments and adjacent joint. this allows to find the best biopsy approach and establish the feasibility of limb salvage as opposed to amputation. longitudinal non-contrast t sequences are the most accurate for determining intraosseous extent. axial fat-suppressed pd or t sequences optimally demonstrate soft tissue extension, and dynamic contrastenhanced mri is useful for differentiation of extraosseous tumour from edema and for assessment of tumour response to chemotherapy. chest ct is the most sensitive modality for detection of pulmonary metastases, and technetium skeletal scintigraphy is still frequently used for detection of osseous metastases. the most valuable, readily available and easy-to-use techniques to assess response to radiation therapy and chemotherapy are dynamic contrast-enhanced mr imaging, diffusion mri and colour-doppler ultrasound. to evaluate local control of disease and for detection of local recurrence, mri is usually the best imaging technique. plain radiography may detect complications of prosthesis. learning objectives: . to consolidate knowledge on malignant primary bone tumors on plain radiography, ct and mri. . to understand the principles of tumour staging and monitoring chemotherapy. . to become familiar with imaging findings following chemotherapy and surgery. oncologic approach to malignant primary bone tumours c. dhooge; ghent/be (catharina.dhooge@ugent.be) the most common bone sarcomas are osteosarcoma (os) and ewing sarcoma. os occurs primarily in long bones, ewing sarcoma occurs also in the pelvis, spine or chest wall. localised disease confers a % cure rate, and initially metastatic disease %. a multidisciplinary approach which includes neo-adjuvant systemic chemotherapy and local control measures for primary and metastatic sites followed by adjuvant chemotherapy has become the standard of care. complete surgical excision is mandatory in os; in ewing, radiation can also be used. standard chemotherapy for os is based on a combination of cisplatin, doxorubicin and high-dose methotrexate. ifosfamide and etoposide will be further considered (euramos). systemic therapy for ewing includes vincristine, doxoribicin, cyclofosfamide, ifosfamide and etoposide. high-dose chemotherapy with autologous stem cell reinfusion is evaluated in intermediate prognosis and metastatic disease (euro-ewing). among the different prognostic factors, response to neo-adjuvant chemotherapy by measuring chemotherapy-induced necrosis is a powerful indicator of outcome in bone sarcomas. patients who achieve a good histological response to pre-operative chemotherapy, defined as < % viable tumour at the time of tumour resection, have a better survival than those who show poor response (>= % viable tumour). five-year survival for good responders is o- % compared to - % for poor responders. however, the outcome for malignant bone tumours has improved little in the last years. large international studies exploring new drugs such as biologic agents (interferon) or mechanisms conferring drug resistance (topoisomerase inhibitors) in randomised controlled trials will hopefully lead to therapeutic innovation. learning objectives: . to become familiar with the principles of chemotherapy in malignant primary bone tumours. . to understand what the oncologist expects from the radiologist. . to recognise the value and limits of chemotherapy in malignant primary bone tumours. thursday a- : surgical approach of malignant primary bone tumours g.m.l. sys; ghent/be (gwen.sys@ugent.be) in primary malignant bone tumours, three surgical procedures are necessary: biopsy, resection and reconstruction. whether a biopsy is performed in an open or a percutaneous way is a matter of preference, but in each case it should provide sufficient and representative material for pathological investigation without compromising the following treatment. the area of interest and the trajectory should be determined preoperatively in a multidisciplinary meeting. as the biopsy tract has to be removed during the resection surgery, it should be close to the planned approach for the subsequent resection. if a biopsy is performed improperly, the diagnosis may be wrong or the following resection may be impaired because of extensive contamination of compartments, resulting in a severe functional deficit. surgery planning is based on clear imaging and a multidisciplinary discussion of tumour extension, aiming for a wide resection of the tumour with clear margins. correct perioperative measurements are based on predefined fixed bony landmarks that should preferably be visible on the same image as the tumour. an amputation is performed if a surgical limb-salvage procedure is expected to result in a non-functional limb. nowadays, several reconstruction techniques for bones (bone grafts or prostheses), tendons (artificial or human ligaments) and skin (skin flaps or grafts) are available. invasion of the soft tissues such as the neurovascular structures, muscles and skin, will determine which reconstruction technique (s) are necessary to restore the patient's function. each reconstruction technique bears inherent complications requiring a regular follow-up. we outline the current opportunities and threats in diagnostic radiology: traditional diagnostic radiology has been rapidly replaced by clinical radiology and the role of the radiologist is changing from image interpreter to clinical manager of imaging data. to care for the patient`s medical problem and not only his images is important. this contains great opportunities for radiology to develop and for the radiologist to become a central player in patient management. however, this requires not only clinical knowledge and involvement, but also visibility and sometimes even / availability. the need for " only" image reporting is declining as clinical subspecialisation easily brings image interpretation into the domain of non-radiologists. to understand the importance of identity, visibility is very important. visibility can be achieved by being part of mdt s and also in many other ways. clinical radiology is an idea which, after all these years, has not been very well embedded in current radiology and radiology training. to understand the importance of certified training, maybe the most important action we have to undertake now is to capitalise on our radiological expertise. image interpretation in a clinical context can only be done or franchised (to non-radiologist) under conditions of certification and accreditation by radiology. we should never let the primate of education and certification slip to non-radiologists. radiology certification should be a quality standard, recognised by international and national medical bodies, such as medical chambers and specialist societies. the latter should have high priority. session objectives: . to learn about the current opportunities and threats in diagnostic radiology. . to understand the importance of identity. . to acknowledge the importance of certified training. where are the turf battles in diagnostic imaging? g.m. bongartz; basle/ch (georg.bongartz@usb.ch) turf battles in radiology are a foreseeable development for most imaging techniques which evolve push-button methods and readily interpretable imaging results. we as radiologists must learn to accept competition. turfs should rather be seen as challenge than as threat. predominantly, nonirradiating techniques like ultrasound and mri have become progressively easier to apply (us) or to understand (mri), where the final image in some standardised areas (eg joints) can readily be interpreted by medical specialists in this field. but also x-ray diagnostics in dental medicine or orthopedic imaging is largely applied outside the radiologic department. ct today seems still relatively excluded because of its complexity. but with increasing resolution, optimal d reconstruction and fast accessibility, it is only a question of time when the added value of the radiologist will be under question. radiology has changed. we must become experts with respect to patho-anatomy and radiological differential diagnosis. this requires profound education, both initial training and ongoing education. radiology must be advertised internally and externally. in radiological publications, outcome studies are still underrepresented. demonstration of our dedicated skills, fostering our special expertise and offering this as support to our medical colleagues will preserve radiology for the future. cooperation with partners seems the only way out of a turf battle -we need to search for alliances and integrate our partners to create a win-win situation. a- : beating threats in europe with radiological training a.k. dixon; cambridge/uk (akd @radiol.cam.ac.uk) by introducing radiology to medical students, it is hoped that they will begin to appreciate how difficult it is! likewise, when teaching residents, they must come to understand the basic anatomy and principles better than their clinical colleagues; this is very much the tenet of subspecialty training. they should also appreciate that patient care is optimal when there is close collaboration between clinicians and radiologists. passing of an examination (national or edir) offers some proof that a radiologist has attained a certain standard, but the principle of testing oneself formally or informally at regular intervals again provides a measure of continuing competence. such certification can be of value in the case of litigation. radiology is one of the fastest growing specialties and the techniques that we learn during training are quickly outdated; hence, the need for life-long learning and effective continuing professional development. learning objectives: . to understand how to teach radiology to undergraduate medical students. . to become familiar with the principles of self-assessment. . to appreciate the necessity for life-long learning. history of ultrasound in radiology: lessons learned l.e. derchi; genoa/ it (derchi@unige.it) the relationship between radiology and us has never been an easy love story, and still isn't. in the early s this new imaging field was not readily covered by radiologists and many other colleagues established us sections within their departments. then, important fields of us (cardiology, obstetrics, gynecology) became almost exclusive domain of non-radiologists. in the s, the increasing success of us initiated turf battles with other clinicians. nowadays, clinical specialists perform > % of us examinations wordwide. in europe, most in-hospital us examinations is still performed in radiology, but this is not the case in all countries. a strong practice in us is quite important since this is often the first approach to patients. if initial us referrals are kept, radiologists will continue to guide decisions in subsequent imaging workup. furthermore, us keeps close to patients; in europe the examination is usually done directly by radiologists or, if by sonographers, the radiologist usually checks the case with limited directed us imaging. this clearly shows our role as physicians. to maintain a key role in us radiologists need to: ) have the best specialists in us; attention must be given to us in residency programs. ) invest in us technology (as examples: ceus, elastography, d/ d). ) invest in us research. ) have high visibility in the us community, keeping close contact with all clinical colleagues. ) present higher visibility of us within radiology, with leaders supporting us within the radiological community and each radiologist promoting us in his/her environment. learning objectives: . to understand why ultrasound has moved out of the hands of radiology in some subspecialties. . to learn what this means for daily radiology practice and the quality of work. . to know about the threats and how we should deal with them. thursday a- : interdisciplinary cooperation without losing identity m.f. reiser; munich/de recently, major shifts in the paradigms of patient care took place: interdisciplinary counseling and including the patient in the decision making process for diagnostic and therapeutic measures. the new concept is that the patient no longer comes to the specialist, but the specialists come jointly to the patient. this is reinforced by the competition among health care providers, political decision makers and the public. in many hospitals this general trend has resulted in the establishment of various centers and boards such as the breast cancer centers, vascular centers and tumor boards for several cancer entities to name only few. at the university hospital of munich centers and boards have been implemented up to now. in almost all of them the participation of radiologists is required. this results in a major challenge in terms of personal resources. together with the regular clinical-radiological rounds the time necessary for these activities equals full-time radiologist posts. the participation of radiologists has many beneficial aspects: acknowledgement of radiology as an important clinical discipline and of the radiologist as a clinical consultant; participation in and influence on the creations of sops and (internal) guidelines; opportunity to advocate appropriate radiological methods for diagnosis and interventions; increase of knowledge in clinical medicine and new concepts of therapy; close personal links with our clinical partners. in order to prevent radiologists from losing their identity as radiologists it is mandatory that they remain firmly integrated in the radiology department and to strengthen the position of radiology as the central institution for providing cost-effective imaging and interventional services. moreover it must be taken care to offer attractive career options within the field of radiology. at (michael.fuchsjaeger@medunigraz.at) ultrasound (us) is the adjunct method of choice to mammography in breast imaging. over the past two decades, us technology has undergone profound advancements and refinements. us has, therefore, become an assessment tool with a defined field of indications as well as a unique set of diagnostic descriptors for breast lesion differentiation. the acrin study has demonstrated the special benefit of us in patients at risk with dense breast tissue. the bi-rads lexicon for ultrasound, in its second edition, expanded the role of breast us. further enhancement of image quality as well as the recent advent of automated breast us has even fueled scientific discussions on the potential role of us for breast cancer screening. automated breast us, which is based on computed generation of a d imaging data set obtained from many parallel d images, offers a different approach with a variety of benefits. images are obtained by the sonographer in standardised fashion, whole breast data sets can be reviewed at any time after the examination, reducing operator dependence, and image fusion with, i.e. mri is easily possible. the basic physical background, the significance as well as important aspects of practical use of handheld and automated breast us will be explained and illustrated by the respective imaging examples. emphasis will be laid on strengths and potential weaknesses of both us technologies with regard to breast imaging. the terms "complicated cyst" and "complex-cystic lesion" are based on breast ultrasound (us) terminology. the simple cyst is a fluid-filled, clearly defined, anechoic lesion without any suspicion of a solid intracystic mass. if a simple cyst is complicated by echogenic fluid, it is called complicated cyst. this echogenic fluid can be caused, e. g., by cell debris. thin echogenic septa are common in complicated cysts. vascularisation can never be found in these echogenic components. a complicated cyst has no solid component and no thickened wall or thickened septa. the expected probability of malignancy is %. the us criteria of a complex-cystic lesion following berg et al. categorise it into types: type , thick outer wall and thick internal septa (alone or in combination of both); type , one or more intracystic masses; type , mass of mixed cystic and solid components (at least % cystic); type , predominantly solid with eccentric cystic foci (at least % solid). complex means there is a suspicion of a tumour inside a cyst or a solid tumour with cystic components. a complicated cyst will usually be followed by us after months. a complexcystic lesion needs a correlation with mammography. us-guided large core needle biopsy in combination with a us and mammography visible marker placement is useful in type and in type complex-cystic lesions. in type and type complex-cystic lesions, a preoperative hook wire placement and excision surgery is the typical procedure. learning objectives: . to learn about the us appearance of complicated cysts and complex-cystic lesions. . to consolidate knowledge on differential diagnosis for these respective lesions. . to understand the diagnostic algorithm for a work-up of these lesions. renal colic is the most frequent non-obstetric cause for abdominal pain and subsequent hospitalisation during pregnancy. intervention is necessary in patients who do not respond to conservative treatment. ultrasound (us) is widely used as the first-line diagnostic test in pregnant women with nephrolithiasis, despite that it is highly nonspecific and may be unable to differentiate between ureteral obstruction secondary to calculi and physiologic hydronephrosis. magnetic resonance imaging (mri) should be considered as a second-line test, when us fails to establish a diagnosis and there are continued symptoms despite conservative management. moreover, mri is able to differentiate physiologic from pathologic dilatation. in fact in the cases of obstruction secondary to calculi, there is renal enlargement and perinephric oedema, not seen with physiological dilatation. in the latter, there is smooth tapering of the middle third of the ureter because of the mass effect between the uterus and adjacent retroperitoneal musculature. when the stone is lodged in the lower ureter, a standing column of dilated ureter is seen below this physiological constriction. mri is also helpful in demonstrating complications such as pyelonephritis. in the unresolved cases, computed tomography remains a reliable technique for depicting obstructing urinary tract calculi in pregnant women, but it involves ionising radiation. nephrolithiasis during pregnancy requires a collaboration between urologists, obstetricians, and radiologists. learning objectives: . to list the us, mr and ct imaging findings of urolithiasis and urinary tract infections. . to discuss the role and the appropriate uses of us, mr and ct for imaging these suspected conditions in pregnancy. . to discuss how imaging can influence the management of nephrolithiasis during pregnancy. oncology t.f. hany; zurich/ch (thomas.hany@gmail.com) in malignant tumours, pet/ct imaging using -fluoro-deoxyglucose (fdg) is widely used nowadays. fdg-uptake is an unspecific process and results in physiological as well as pathological malignant and non-malignant tissue uptake. to properly understand and interpret pet/ct imaging, knowledge of the mechanism and pathways of fdg in different tissue types like brain and the gastrointestinal tract is key. intrinsic as well as extrinsic factors influence uptake pattern not related to oncological disease like uptake in brown fat in patients exposed to cold ambient temperature and psychological stress or increased muscular uptake in nonfasting patient. typical treatment-related changes occur during and after treatment and must be recognised correctly so as not to overstage patient disease. a systematic analysis and knowledge of all these factors facilitate and improve the reading of oncological clinical cases. learning objectives: . to understand pattern of physiological fdg-uptake. . to learn about the pattern of non-pathological uptake in several tissue types. . to understand the influence of chemotherapy and other agents on fdguptake in the body. radiologists mostly prefer to use the comparative technique while evaluating radiological images, especially when the anatomy is identical for both sides. comparative analysis mostly works if the anatomic details are not complex or the right resembles the left. however, the complex anatomy and relatively high incidence of individual variations in head and neck may hinder this way of interpretation. variable pneumatisation of paranasal sinuses and temporal bone may cause difficulty. vascular system, especially venous structures may be sources for pseudolesions. for example, asymmetrically enlarged jugular vein or venous plexuses at any location may be misinterpreted as mass of any origin. veins may also become problematic on mr imaging due to entry slice phenomenon, in-plane flow, and flow turbulence effects and can have variable enhancement. normal anatomical structures such as facial nerve may enhance and can be mistaken to be pathologic on mri. technical issues can be considered as a group that may cause pseudolesions. images of the improperly positioned patient may become problematic while evaluating tiny structures. pet and pet-ct carry their own risks for pitfalls. normal structures may confound interpretation and result in false-positive findings. the situation may become much more complicated when there is asymmetry. similarly, atrophy and unilateral absence of a structure may be misread as a mass on the contralateral side. because of the complex anatomy and relatively high incidence of individual variations, head and neck imaging demands much more attention and careful analysis. the appropriate imaging technique and detailed knowledge of anatomy are essential to recognise pseudolesions. as a general rule, detailed knowledge of the patient's clinical history is essential before any imaging study is performed, to evaluate the appropriateness of the indication, tailor the acquisition protocol and correctly interpret the study. in head and neck imaging, this rule particularly fits in the emergency setting. in certain circumstances, the swelling and enhancement of soft tissues produced by infectious lesions may mimic a neoplasm. even more so, in patients already treated with surgery or chemoradiation. in these patients, the challenge is double: to identify any abnormality in the new anatomy produced by treatment and to discriminate between inflammation, complication and relapse. these can be very difficult without knowledge, for example, of the reconstructive procedure after surgery. previous images are extremely useful, not only because they help detect abnormalities, but also because through indirect information on the growth rate of the lesion they are crucial for correct interpretation of findings. tablets in radiology represent a novelty. since the introduction of the tablets on the market, the radiological field has been probably the first medical discipline to discover the many advantages of these devices. in fact, many applications for image management have been made available on the app stores (apple and android), and allow radiologists and non-radiologists to handle dicom images on the tablet, as part of the patient's record. however, the emerging applications are driving the process from the simple dicom image viewing to the full integration of the tablet with the pacs, allowing the handling of a patient's full record and presumably the possibility to report. in view of this rapid technological development, again radiology falls in the middle of a storm and is asked to find a solution to problem: are tablets suitable to read and report dicom images? and if so, which kinds of images (ct, mri, x-ray, etc) .? how can we manage the portability of patients' data (security issues, data loss, etc).? what will be the impact on teleradiology? these issues will be addressed by the panel of experts who will speak in the refresher course. session objectives: . to appreciate the current state of tablet technology and its practical use in radiology. . to understand the pros and cons of the use of tablets. . to learn about specific critical areas of utilisation (dicom images reading and teleradiology). a. tablet-computers: a technical overview j. fernandez-bayo; sabadell/es (jfernandezb@cspt.es) since their introduction in , tablet pcs have evolved extensively. they have become very popular, filling the gap between laptop computers and smart mobile phones. we will review technical aspects like the processors, storage space and memory, size and weight, connectivity and networking, software, autonomy, and battery life in different devices. special focus will be on the displays and different possible uses in radiology. for diagnostic purposes, different technical aspects must be taken into consideration, like the display size, resolution, pixel size (pixel pitch or pixels per inch), luminance, and contrast. displays in radiology normally have sizes between " and " and resolutions up to mpx, with luminance that ranges from cd/m to cd/m . by contrast, tablets have screen sizes between " and " and resolutions up to mpx, with luminance that ranges from cd/m to cd/m . due to their screen size, tablets score better in pixel pitch, with around ppi versus ppi in medical displays. the two systems have similar contrast ratios. other technical aspects of display that we should take into consideration are the number of distinguishable grays that can be represented, which in tablets correlates with the number of colours that can be displayed (color gamut) and colour accuracy (delta-e). additionally, we should take into consideration that displays should be calibrated for medical diagnostic purposes and tools to calibrate tablets have recently become available. learning objectives: . to learn about the pc evolution: from desktops, to laptops and tablets. . to appreciate the versatility features of a tablet. . to understand the hardware features with a specific focus on display. . to understand the hardware features with a specific focus on networking. a- : b. reading dicom images on the tablet o. ratib; geneva/ch touch screen tablets are becoming widely available, providing convenient mobile solutions for physicians and health-care providers. this is particularly attractive in medicine to "nomad" physicians, who need to be able to access relevant patient data and images anywhere-anytime in their daily practice where they are rarely at a single location. while they may not always be adequate for routine diagnostic tasks, they provide a convenient mobile solution for on-call and remote consultations. there are different types of software architecture that can be implemented for such tasks. two different major designs are: ( ) online web-based applications where the device serves as a "thin-client" to display images rendered and manipulated on a remote computer and ( ) local applications that reside on the mobile device and can run independently after images have been downloaded on the device. the first solution requires the user to be constantly connected to the network, while the second solution can continue to function after being disconnected from the network. most pacs vendors are starting to provide web access to their imaging solutions that can be accessed from mobile devices. web access can however be slow and dependent on reliable access to wireless network. we chose to develop a stand-alone companion application to our open source imaging platform osirix. with the increasing capacity and computing power of mobile devices, users will soon be able to perform most of the processing and image manipulation functions that are today only feasible on desktop or laptop computers. learning objectives: . to learn which dicom readers are available for tablets. . to appreciate the different approaches to dicom reading (local vs remote) and the pacs/tablets integration. . to understand the pros and cons of dicom image reading with the tablet in regards to image quality and displays. the optimisation of clinical breast cancer care comprises the prevention of over-diagnosis, reduction of over-treatment, and avoiding unsuccessful treatments. these goals are targeted in the vph-prism project by seven european and two us partner institutions. from the prospectively collected data, we will derive optimised imaging protocols that comprehensively take a woman's history and risk factors into account. the tight integration of radiological and histopathological images enables joint assessment of quantitative tissue parameters from microscopic and macroscopic imaging. more informed therapy decisions can be taken by such enhanced multidisciplinary data, backed by a powerful case database. the reduction of overtreatment, and highly individualised diagnosis and therapy decisions are closely related. we ultimately aim at deriving predictive parameters from the multi-modal and multi-disciplinary database to select the treatment option with the best chance of enduring recurrence-free survival. prospectively collected data, personal risk factors, and corresponding imaging data fed into the database from three large cohort studies are expected to provide insights into the individual preconditions and factors affecting disease progression. to optimise therapy, the decision between therapy options is key. this will be addressed by a decision support system using the project database. for surgical excision planning, vph-prism will aim at providing better estimates of tumour size, and with visual planning aids supporting radiologists and surgeons. for chemotherapy, quantitative lesion parameters, tracked over time during therapy, may help to detect success or failure of the treatment early on, such that a chemotherapy regime can for a non-responder be switched to a different regime in due time. quantitative imaging biomarkers in dementia; the fp vph-dare@it project w.j. niessen; rotterdam/nl (w.niessen@erasmusmc.nl) the number of individuals suffering from dementia today is roughly million. due to ageing societies, this number is predicted to increase to million by . worldwide annual costs are estimated to be € billion. in , the who declared dementia a global health priority, highlighting the urgent need for improvements in this area. vph-dare@it's aim is to enable more objective, earlier, predictive and individualised diagnoses and prognoses of dementias to cope with the challenge of an ageing european society. the expected impact of vph-dare@it will influence the scientific, clinical and industrial communities across europe and internationally to improve health care of dementia patients. in this presentation we will introduce the vph-dare@it project. we will then focus on the development, validation and implementation of quantitative imaging biomarkers for the early detection and differential diagnosis, which will take place as part of the project. author disclosure: w.j. niessen: advisory board; part-time detached to quantib bv as scientific director. shareholder; co-founder and shareholder of quantib bv. horizon : improving diagnosis and medical interventions and support to medical imaging j.-l. sanne; brussels/be (jean-luc.sanne@ec.europa.eu) research and innovation contribute to increasing europe's competitiveness. at the same time, research and innovation help make people's lives better by improving things like healthcare. in january the european union launches horizon , the biggest eu research and innovation programme ever, with a budget of € billion (current prices) over seven years ( ) ( ) ( ) ( ) ( ) ( ) ( ) . eu support for research and innovation helps drive international projects across the european union and beyond, and promotes the progress of knowledge and technology. one of the three key pillars of horizon will be tackling societal challenges (priority ) such "health, demographic change and well-being". one feature is the development of new and more effective diagnostics. relevant tools and technology innovations will be supported in view to improve disease outcomes through earlier, more accurate diagnosis and by allowing for more patient-adapted treatment. support will be provided through the work programme - of societal challenge "health, demographic change and wellbeing". additional opportunities are offered in the work programme - of priority (industrial leadership) "leit -information and communication technologies". practically, all imaging modalities have been used in space research since a long time ago. first of all, radiology is targeted in research activities, because by definition all cosmonauts and astronauts are healthy persons. the biggest volume of examinations had been performed on crew members for studies of organs and functions of the human body before and after long-term flights. health effects of zero gravity were of special interest. for example, multiple studies with involvement of both russian and international specialists were done on bone mineralisation (bone densitometry), muscle volume (mri) and metabolism (mr-spectroscopy) both in astronauts and healthy control subjects. first, ultrasound (us) examinations were conducted in space onboard russian orbital stations in . complex us studies were done by the author himself during his flight as a crew member of "salut- " orbital station in . the author had found some complex changes in blood and fluid dynamics under weightlessness. teleradiology was used for data transfer to medical specialists on the earth. today, we are continuing to use radiology for research on astronauts. for example, applications of sophisticated brain mri and fmri methods are of special interest for studies of subtle brain changes in space travellers. among our targets are flights to the moon, mars and large asteroids. to be better prepared for that task, we need better diagnostic tools. new versions of mobile us systems, miniature x-ray and ct machines suitable for use inside spaceships are under development. there are several mri pitfalls that should be recognised when imaging the female pelvis. mri appearances of uterus and ovaries are dependent on the phase of menstrual cycle/use of exogenous hormone therapy. normal postsurgical and post-radiation appearances of the pelvis can sometimes mimic tumour recurrence. it is important to become familiar with these appearances to avoid potential pitfalls. one very common pitfall is differentiation of transient myometrial contraction from adenomyosis. interrogation of all imaging planes over the duration of the entire mri examination can be useful to distinguish between the two, although myometrial contractions can last up to min. the choice of the correct imaging plane is crucial for the precise classification of uterine anomalies (coronal oblique) and accurate evaluation of parametrial invasion (axial oblique) in patients with cervical cancer. both dynamic contrast-enhanced mri and diffusion-weighted mri improve the accuracy of mri in the evaluation of malignant pelvic conditions. however, certain pitfalls related to each technique should be recognised to avoid misinterpretation. it is crucial to be familiar with the anatomy of the uterovesical (uv) ligament, as it is often the site of pelvic lymphoma (such as bladder or cervix lymphoma). however, some benign conditions such as endometriosis can involve the uv fold and invade both bladder and uterine wall. certain mri features can be helpful in making the correct diagnosis. learning objectives: . to become familiar with normal variations in mri appearances of female pelvis resulting from physiological conditions (e.g. different phases of menstrual cycle) and treatments (including exogenous hormone therapy, surgery and radiation) potentially mimicking disease. . to consolidate knowledge on the role of the correct mr imaging plane in avoiding potential mis-classification of uterine anomalies and parametrial invasion in patients with cervical cancer. . to learn about certain pitfalls related to dynamic contrast-enhanced mri and diffusion-weighted mri. a- : b. pitfalls in ultrasound k. kinkel; chêne-bougeries/ch (karen.kinkel-trugli@wanadoo.fr) pitfalls of sonographic findings in the pelvis can be related to technical issues, interpretation errors or to the patient's specific condition or pathology. common problems consist of insufficient bladder filling, misinterpretation of posterior enhancement or shadowing according to the anatomical structure and pathology of a size that goes beyond the field of view of the probe. organspecific problems will be illustrated in interactive questions, particularly for the uterus and the ovaries. identification of diseases can be difficult in many areas of abdominal imaging and therefore misses are easily made, also by the more experienced radiologist. it is difficult in the mesentery and peritoneum, covering a large area but being a very thin structure where abnormalities are easily missed. therefore, knowledge of the peritoneal anatomy and pathophysiology of peritoneal diseases is not widespread and differential diagnosis can be difficult. hence, special attention should be paid to prevent misses. another difficult area is diagnosing occult gastrointestinal bleeding. here, the choice of the proper imaging method is crucial as well as understanding the pros and cons of the method. the technique used should be optimised, as otherwise sometimes subtle signs of bleeding are missed. the presence of bowel dilatation itself is often correctly diagnosed, but the extent of the obstruction, the cause of the obstruction and especially complications (ischaemia) might be misdiagnosed. ct is the preferred technique for evaluation of bowel dilatation. care should be taken to scrutinise the examination for principal findings in patients with bowel obstruction. a. mesentery and peritoneum d. akata; ankara/tr (dakata@hacettepe.edu.tr) imaging findings of neoplastic or inflammatory diseases within the peritoneal cavity and the mesentery sometimes overlap and cause difficulties in interpretation. even disease processes in the peritoneum, mesentery or omentum may not be recognised on radiological examination, causing major difficulties in the management. peritoneal anatomy and physiopathology of peritoneal diseases must be well understood for better evaluation. ct is the best modality to assess the whole cavity. mr is equally sensitive with better contrast resolution; however, both modalities have advantages and limitations. ultrasound has a complementary role in evaluating the peritoneal fluid content. the presence of lacelike mobile thin septa is highly significant for tuberculosis. for better diagnosing the pathology, systematic approach is needed such as assessing the presence or absence of fluid in the peritoneal cavity, its character and location, accompanying soft tissue densities and their location. some inflammatory or infectious causes, such as acute pancreatitis and tuberculosis, involve typically parietal peritoneum and certain peritoneal reflections. peritoneal carcinomatosis involves typically both visceral and parietal peritoneum as well as subdiaphragmatic space. contrast enhancement patterns of the peritoneal membranes and the mesentery also have a complementary role in differentiating a variety of diseases. learning objectives: . to learn about the imaging characteristics of peritoneal and mesenteric masses and their differentials. . to appreciate the potential and limitations of imaging techniques in the detection of such lesions. . to understand the common pitfalls in diagnosis. a- : a. filippone; chieti/ it (a.filippone@rad.unich.it) patients with persistent, recurrent, or intermittent bleeding from the gastrointestinal tract for which no definite cause has been identified by initial oesophagogastroduodenoscopy, colonoscopy, or conventional radiologic evaluation are considered to have an occult gastrointestinal bleeding (ogib). the management of ogib is clinically challenging, since the causes of such a bleeding frequently arise in the small bowel (sb), until now considered as the ''dark continent''. conventional barium contrast studies allow only a limited evaluation of sb, whereas angiographic diagnosis is strictly related to active bleeding. the introduction of capsule endoscopy (ce) as well as of crosssectional imaging dedicated to the sb visualisation, such as multidetector-row computed tomography enteroclysis (cte) and magnetic resonance enteroclysis (mre), represents significant technological advances that have overcome the limitations of older diagnostic tests. although ce is recommended as a first-line investigation in ogib patients, cte or mre are alternative diagnostic tools when ce is contraindicated due to suspected/known obstruction or stricture. moreover, in patients in whom a tumour is suspected, cte or mre may be the preferred initial test. therefore, radiologists have to be familiar with cte and mre techniques, in terms of bowel distension, scanning parameter selection, contrast administration as well as with specific imaging findings. similarly, they have to be aware of the potential pitfalls such as suboptimal bowel distension, artefacts due to peristalsis or breathing, intraluminal food debris and previous surgery. learning objectives: . to understand the causes of gi bleeding and underlying pathophysiology. . to appreciate the strengths and limitations of the imaging techniques used in diagnosis. . to learn about common pitfalls in diagnosis. dilatation. presently, ct takes a major place in this setting with a reduced role for plain films. sonography is an alternative method when ct is not recommended. the questions of mondor in were pointed before the advent of cross-sectional imaging; these are still valid and have to be answered: ) is this a true occlusion (how to differentiate it from adynamic ileus)? ) does the obstruction concern the small bowel or the colon (impacting on the decision to operate or not)? ) what is the cause of the obstruction? ) are there signs of bowel ischaemia? the key points to avoid missing imaging diagnoses in bowel dilatation are to make the distinction between true mechanical obstruction and adynamic ileus (an early sign of mesenteric infarct) and to detect signs of strangulation leading to ischaemia (such imaging findings are present despite normal blood tests). in this setting, ct is the preferred technique; if it is not available, plain films and sonography can help in an optimal medical decision. the national lung screening trial (nlst) found a % reduction in lung cancer mortality with three annual screens using helical ct relative to chest xray. the us preventive services task force has issued draft recommendations to provide annual ct screening to high risk individuals defined by age and smoking criteria. questions remain regarding the implementation of ct screening: the determination of risk to identify those who should be screened, the frequency and duration of screening, definitions of screen positivity based on detected nodule characteristics, diagnostic algorithms for the follow-up of positive screens, overall societal costs of screening, and methods to adequately diffuse this technology across all socioeconomic groups at risk. answers to these questions are being addressed by several groups through ongoing research and secondary analyses of trial data. several models exist to identify individuals at highest risk of lung cancer (and lung cancer mortality), which may be enhanced if validated molecular markers are included. ongoing analyses of nodule features and likelihood of lung cancer will better inform screening interpretation guidelines and diagnostic algorithms. estimates from the nlst suggest that screening as performed in the nlst will be cost effective: the base case estimates $ , per quality adjusted life year gained (qaly). subset analyses suggest that screening is much more cost-effective in women than men, in higher versus lower risk groups, and in current versus prior smokers. while several variables influence cost-effectiveness, major drivers of cost are the cost of ct screening exams as well as the number of follow-up ct scans per positive screen ( . in nlst). while the national lung screening trial (nlst) had shown a significantly reduced lung cancer-specific mortality and all-cause mortality, the current results from the various european trials do not show such positive results. the dutch-belgian nelson trial, the german lusi trial and the british ukls have not yet published their results. so far, more cancers were detected in the screening arm of published danish and italian trials, but the positive effect on cancer mortality could not yet be shown. the italian mild trial even showed higher mortality in the yearly screening arm, and no difference between yearly screening and the control arm. this presentation discusses the implications of these findings and the consequences on implementation on screening in clinical practice. while limited in number of participants, european trials used advanced approaches that are most likely to be used if screening will ever be implemented. this presentation will discuss issues such as growth assessment and volumetry as critical factors for nodule assessment, handling of sub-solid lesions, computer aided detection and evaluation, and use of nonradiologists for reading and standardised follow-up. the goal is to provide an insight into how the european trials have affected our idea on practical implementation of lung cancer screening. the discussion will address the following issues: . are there sufficient data to support the implementation of individual lung cancer screening in clinical practice? . if yes, how to move from efficacy to effectiveness: what are the most optimised risk profiles for screening? what is the minimum level of infrastructure support and organisation required? how should subjects be informed about screening? what are the optimal follow-up duration and screening interval? what are the technical and positive screen management requirements? . what about the alternatives of waiting for additional data from ongoing trials or the validation of new biomarkers of lung cancer, permitting better selection of screened population before implementing individual lung cancer screening? attendees' opinions will be collected interactively. the speakers will be asked to comment and debate. treatment of prostate cancer depends strongly on the stage of the disease at the time of detection. however, treatment is controversial, even in prostate cancers found through early detection. this presentation will be based on a careful recent literature review. early detection of prostate cancer by use of psa testing leads to a significant reduction of at least % of disease-related mortality in screened men. this is however at the price of - % of overdiagnosis, usually resulting in overtreatment. diagnostic developments which reduce overdiagnosis and the proper identification of cases which may not require treatment are central issues of current research and will be addressed by referring to available evidence. active observation of potentially nonaggressive cancers is an option in the management of this disease until, preferably, the diagnosis of such cases can be avoided all together. radical prostatectomy and radiotherapy are the options for treating those cancers which are judged to be aggressive. treatment recommendations for prostate cancer continue to evolve and are affected by technological advances in surgery, new discoveries in tumour biology, and the development of predictive and prognostic biomarkers. with the increased complexity of treatment decision-making, the role of mri is also evolving. the more individualised and targeted the treatment approach, the greater is the role of imaging in treatment selection, planning and follow-up. mri is especially crucial for planning technologically sophisticated treatment approaches such as robotic surgery, igrt, or focal therapy and in assessing patients' eligibility for active surveillance. though needs for uniform interpretation and standardised reporting remain unresolved, the value of mri in pretreatment staging of prostate cancer, particularly for evaluating extracapsular extension and seminal vesicle invasion, has been documented. not only the tumour stage, but also its size, volume and grade (aggressiveness) are important factors that influence treatment selection. therefore, the introduction of functional mr is essential. when added to t weighted mri, diffusion-weighted mri, dynamic-contrast-enhanced mri, and mr spectroscopy, in addition to improving tumour detection, can provide an indication of tumour aggressiveness. mri performance on all sequences is dependent on lesion size/volume and grade, and this should be considered when applying mri results to treatment selection and planning. while it has been shown that multiparametric mri is generally more accurate and informative than anatomic mri alone, evidence-based guidelines specifying which combinations of sequences are essential for specific indications in patients with prostate cancer are yet to be developed and validated in welldesigned studies with robust outcome measures. in major trauma, it is essential to immediately recognise life-threatening conditions and to initiate early treatment. the majority of these patients succumb at the site of the injury to severe injuries of the central nervous system, heart and great vessels. however, there is another peak of early deaths within the first four hours after the injury. in the majority, these patients are at risk due to haemorrhage which is basically controllable by early minimally invasive treatment. consequently, this means that the vast majority of internal injuries can be survived as long as bleeding control is achieved within the first four hours after an injury. minimally invasive interventional techniques are already in use at a very early stage of the clinical decisionmaking process. particularly, we have witnessed a major paradigm shift in the treatment of blunt aortic injuries. endovascular repair has replaced open repair in many trauma centres. in appropriately selected patients, this adoption of endovascular stent grafts has resulted in a reduction in perioperative mortality, stroke and paraplegia, as compared to open repair. this entire session will provide knowledge about indications, requirements, standard procedures, and outcome of vascular emergencies, pardon me, urgencies. a. aortic emergencies m. cejna; feldkirch/ at (manfred.cejna@lkhf.at) aortic emergencies can be classified into several categories. "acute aortic syndrome": acute aortic syndrome is an entity of changes of the aortic wall. these include aortic dissection, intramural thrombus, and penetrating atherosclerotic aortic ulcer. besides coronary malperfusion and aortic valve insufficiency (type a dissection), emergency presentation is often due to visceral/limb ischaemia. sequelae of aortic syndromes are pseudoaneurysm formation and potentially aortic rupture. endovascular treatment has been established as the treatment option in at least type b dissections and penetrating aortic ulcers. aortic aneurysm rupture: with increasing diameter, aneurysms in the thoracic, thoraco-abdominal and abdominal aorta are prone to rupture. signs of impending aortic rupture are pain and imaging signs are sometimes subtle just like blurred contours of the aorta and (blister like) vascular wall deformation. endovascular treatment is an alternative to vascular surgery. aortic trauma: in severe thoracic trauma (mostly deceleration trauma), pseudoaneurysm formation and aortic rupture occur at the level of the aortic isthmus. endovascular treatment is considered the treatment of choice. inflammation, mycotic aneurysms and aorto-enteric fistulae: with acute and chronic inflammation, aneurysm and pseudoaneurysm formation and fistulae to enteric structures can occur. endovascular treatment is more often considered as bridging to vascular surgery instead of being a definitive cure. iatrogenic aortic injuries: besides accidental aortic injury during operations or interventional procedures, transaortic valve replacement and placement of aortic balloon pumps can result in iatrogenic trauma. endovascular treatment may be a minimally invasive approach. the focus will be on systematic presentation of aortic emergencies and the role of endovascular treatment. there are a number of vascular visceral emergencies that may be traumatic or nontraumatic in origin. the degree of urgency or potential for urgency varies with each condition and the subsequent management and intervention techniques will vary accordingly. an understanding of the natural history, pathophysiology, diagnostic tests and interventional techniques for these conditions will aid the diagnostic and interventional radiologist in identifying the pathology and knowing when to refer or initiate definitive treatment. i talk about embryonal development and anatomical differentiation of cardiovascular system, with special attention to the peripheral vascular system. the goal of this part of presentation would be to understand the anatomical structure and architecture of arteries and capillaries. we will discuss the casual classification of different kinds of peripheral arterial emegencies; e.g. congenital, posttrauma, arteriosclerotic and iatrogenic. the audience will learn about different therapy approaches, interventional and non-interventional, with accent on pad (classification, pathophysiology, risk factors, statistical facts and therapy). at the end, we will review different studies about combination therapy (conventional and interventional) with different possibilities and outcomes. modern cancer research and increasing therapy are mechanism based with the development of disease-modifying therapies that target the hallmarks of cancer. modern imaging tools enable the visualisation and quantitative assessment of the expression of molecular targets, of their interaction with potential ligands, as well as of the functional consequences of interactions at a molecular, cellular, metabolic, physiological, and morphological levels in a temporo-spatially resolved manner. the ability to gather such information from the intact organism renders imaging highly attractive for biomedical research and drug development. the determination of cell proliferation with radiolabelled thymidine is a wellestablished method in various life science branches where [ h]thymidine has been used for more than years and still represents the gold standard for the assessment of tumour cell proliferation. since non-invasive determination of this parameter is required in clinical studies, considerable efforts have been made in radiopharmaceutical research. the synthesis of a large series of proliferation markers resulted therefrom mostly focussing on pyrimidine nucleosides. the labels used were gamma as well as positron emitting radionuclides. here, [ f]flt ( '-deoxy- '-fluorothymidine) is examined and discussed with respect to its preclinical and clinical application. since apoptosis is an important mechanism of cell death in tumours responding to treatment, the non-invasive assessment of apoptosis with tracers for the detection of phosphytidyl-serine presentation and/or caspase activation could be used as surrogate marker for therapeutic efficacy. several approaches have been followed during the last years; their potential and limitations will be presented. angiogenesis is one of the hallmarks of cancer that is considerably easy to characterise with many different imaging modalities and methods. it is not only crucial for invasive and metastatic tumour growth, but is also a prerequisite for the accumulation of anticancer drugs and highly impacts the success of radiotherapy. this talk summarises our experiences in microstructural, functional, and molecular imaging of tumour angiogenesis. the microarchitecture of vessels can longitudinally be studied using high-resolution ( )ct. dce mri and hf-us are introduced as favourable tools to characterise perfusion, vessel permeability and vessel maturation during antiangiogenic treatments. using targeted probes, it is shown that "fluorescence molecular tomography (fmt)", mri and us are capable of estimating the expression of angiogenic marker molecules on tumour vessels and of matrix-associated enzymes in the interstitial space during vascular remodelling. additionally, examples are also given for epr-based nano-sized theranostics and it is shown how us can aid in better accumulating them at the target site by inducing vascular permeation. many of these novel imaging concepts and tools can relatively easily be translated into clinics. thus, one can expect them to play a major role in the clinical management of tumour treatments soon. mammography technique from the point of view of radiographic compression and positioning is no different from film screening and radiologists ignore this at our peril. we will discuss the danger areas at the edges and the back of the breast and help you to find lesions only seen on one view. with digital capture the technologist can view images immediately, but the temptation is for them to repeat exposures to obtain radiographic perfection on otherwise clinically acceptable images. however, the task of assessing quality cannot be left to them, as viewing conditions on the 'lower resolution' acquisition monitor in a bright x-ray room is not enough. there is now evidence from population screening programmes that poor image quality can reduce cancer detection from between % in france to % in ontario. 'quality' can be lost at keys points in the imaging chain: the x-ray beam and dose, the detector and finally display. in flanders, poor detector performance was compensated for by increasing the x-ray dose by % and by meticulous quality control. postprocessing is less well understood, but reader studies now show different algorithms will affect performance particularly for the detection and classification of micro-calcification. finally, it does not matter how good all the equipment is if we try and read the images with the lights on full. mammography is the most important breast imaging technique allowing the visualisation of masses and micro-calcifications. however, in conventional mammography the three-dimensional breast tissue is reduced to a twodimensional image. therefore, small lesions may be undetectable due to superimposed glandular tissue. digital breast tomosynthesis (dbt) has emerged as a new imaging modality to overcome this limitation. several lowdose mammographic projections are performed over a limited angle of up to degree using a standard mammography system. the average glandular dose of a tomosynthesis scan is somewhat higher compared to one-view mammography, but lower compared to a standard mammography in two projections. based on the low-dose projections, a stack of cross-sectional images covering the entire breast is reconstructed with an interslice distance of about mm. several studies have shown that tomosynthesis improves both tumour detection and the characterisation of focal masses. due to the crosssectional nature of dbt, the techniques allow on the one hand the reliable differentiation of true focal masses from summation artefacts. on the other hand, tomosynthesis improves the detailed analysis of the lesions' border to differentiate benign lesions and carcinomas. additionally, contrast-enhanced spectral tomosynthesis may allow analysing signal-intensity time curves. in conclusion, dbt adds important information to standard two-projection mammography and can replace spot compression and rolled views as problem solver for difficult cases. however, the impact of tomosynthesis on breast cancer screening has to be investigated in larger clinical trials. reading screening mammograms is not the same as reading diagnostic mammograms. the vast majority of women attending a screening programme are usually asymptomatic and do not have abnormalities associated with a malignancy. the task of a mammogram reader is to scan a mammogram and perceive any possible abnormalities that could be associated with a malignancy. in other words, we are talking about perception and not diagnosis of a finding. examples will be shown about this crucial step. in modern population-based screening programmes, we use computerised reading and recording systems. since the work flow is rather quick, we have to ensure basic steps such as identity control and see that both ris and pacs are synchronised so that decisions of screening readings are recorded in both the systems of one and the same patient. in computerised screening programmes, we use standard reports informing a woman that no signs of malignancy have been detected on her screening mammograms. in case of findings that need further assessment, there are several ways to deal with this situation. the most common way is via a phone call, but this can also be done with a letter. a screening report should be short and concise and should only inform whether we see signs of a possible abnormality that can be associated with a malignancy. extensive reports describing the contents of a breast or findings of no clinical relevance should not be part of a screening report. emergency radiology services are very much on the rise in the last decade. medical emergencies and trauma are of enormous importance and a leading cause of death in all age groups. the use of radiological imaging in emergency departments showed an exponential increase since the s. the annual growth rate in ct is - % per year depending on the institution. even for advanced level ° medical centres running an own emergency radiology unit, it is a challenge to integrate advanced radiology services in an interdisciplinary team treating patients with acute traumatic and non-traumatic emergencies. general principles: radiology and radiological imaging procedures should be integrated in an interdisciplinary team. imaging should be guided by interdisciplinary clinical algorithms or guidelines (e.g. nexus or canadian cspine rule). many clinical diagnoses relay today on a early and thorough initial radiological diagnostic workup -mostly based on mdct. emergency radiology (er) is still a relatively young subspecialty in european radiology -it deserves specific training. the lecture will cover: the development of emergency radiology and its future perspectives; the use of conventional radiography, ultrasound and mdct; logistics and management of the patient; clinical guidelines and mdct in the primary patient survey (e.g. using atls for trauma) and advanced scanning protocols for mdct. infants and children with abdominal emergencies present special diagnostic challenges because of similarities in clinical findings between various paediatric conditions. imaging is frequently a critical part of the diagnostic process, but the choice of the best imaging modality can be controversial. patient age and safety issues such as radiation exposure must be taken into consideration when planning imaging. because certain pathologies are more likely to occur in specific age groups, patient age is a primary determinant of the imaging approach. this presentation will illustrate the differences in pathology between several paediatric age groups and discuss effective imaging algorithms for congenital and developmental, inflammatory, and obstructive conditions. the optimal use of radiographs, ultrasound, ct, and mri will be discussed. pitfalls that should be avoided in ultrasound and ct will be emphasised. in diagnostic imaging, we tend to focus on radiation protection, justification and optimisation, as the primary opportunity for radiographers and radiologists to ensure the safety of patients, both adults and children; however, this ignores some other important issues. this session aims to raise awareness amongst medical imaging professionals of some key considerations in relation to a number of child protection issues. when imaging a child in case of suspected non-accidental injury, every detail of that examination from conversations with the child and their parents or guardians, technical aspects of the examination, observations during the examination and the language used following the examination need to be carefully considered. another area of medical imaging requiring attention is how we communicate any radiation risks associated with examinations or procedures with children and their parents. there is evidence that while consent processes for invasive procedures are usually in place, these often lack focus on the risks associated with a high exposure to ionising radiation. these two medico-legal areas, suspected non-accidental injury and risk communication, are commonly encountered by radiographers and radiologists and as such it is important to make sure that we operate at the highest possible professional standards and in keeping with both regulations and best practice including the production of witness statements/contemporaneous notes. on rare occasions, we may find ourselves involved in legal proceedings as a witness or expert witness and it is thus important that we are all aware of the key considerations in this regard. session objectives: . to appreciate the need for radiographers to focus on more than just the imaging procedure in paediatric examinations. . to understand the extremely vulnerable nature of paediatric patients and the need for all health professionals to pay particular attention to this. a. professional responsibilities: an international perspective m. davis; dublin/ie (michaela.davis@ucd.ie) radiographers play a vital role in imaging children with suspected nonaccidental injuries. this session will also explore the wider role that radiographers have in child protection. current legislation and its implications will be explored from a variety of countries including those outside europe. in addition, case studies will be presented focussing upon child protection issues that have arisen in the x-ray department during imaging procedures. several of the barriers to radiographers becoming involved in child protection will be explored. radiographers and their contribution to child protection will be discussed and practical examples given. learning objectives: . to become familiar with current legislation and guidelines which have particular relevance to radiographers from an international perspective. . to appreciate best practice in child protection and the need for all radiographers to be aware of this. . to learn about child protection case reports involving imaging, radiologists and radiographers. a- : b. risk communication in paediatric examinations j.l. portelli; msida/ mt (jonathan.portelli@um.edu.mt) medical imaging plays a vital role in the diagnosis and treatment of numerous medical conditions for millions of patients worldwide. however since most medical imaging examinations utilise ionising radiation, the associated risks of radiation exposure also need to be acknowledged, especially since high doses of radiation are known to cause adverse biological effects. medical imaging professionals should have a good understanding of the benefits and risks associated with the imaging examinations they perform, so as to ensure a high benefit-risk ratio for all patients undergoing diagnostic imaging procedures, and to be able to appropriately communicate such information to patients, their families and other healthcare professionals. this may be particularly more important when imaging paediatric patients, who are relatively more susceptible to the effects of radiation and receive a higher effective dose per unit of radiation when compared with adults undergoing the same medical imaging examination. indeed, following worldwide media attention about radiation incidents and their adverse effects, some parents/legal guardians may be concerned and reluctant to pursue important medical imaging examinations requested for their child, as they may have a misconception of the risks involved. in this regard, this lecture will seek to enhance awareness about the radiation doses associated with common paediatric imaging examinations; highlight the importance of medical imaging professionals having an appropriate understanding of benefits and risks associated with these examinations; and encourage such professionals to improve patient care by making use of this knowledge to have better discussions with referring clinicians, patients and their families. learning objectives: . to appreciate the frequency and radiation dose associated with certain paediatric imaging examinations. . to become familiar with the current status quo in the communication of radiation dose-related risks. . to understand best practice and potential considerations in providing accurate information to referring clinicians, patients and their families. c. what to do if you find yourself being called to give evidence m.d. viner; london/uk radiology is a powerful tool in the investigation of cases of suspected nonaccidental injury and suspicious death in children. the evidence obtained can be pivotal in bringing successful prosecutions against suspects and protecting the child and others from further harm. it is essential that evidence is obtained, recorded and presented in accordance with applicable rules of evidence in order to be admissible in court. it is thus important that those undertaking such examinations are appropriately trained, conversant with standards of evidence and maintain continuity of evidence throughout the investigation. this presentation will review rules of evidence for cases of suspected child abuse and their application to imaging examinations. it will examine the role of the radiographer and the importance of training and familiarity with legislation and guidelines that underpin forensic practice, highlighting the pitfalls that lawyers may exploit if procedures have not been followed. drawing on examples, it will discuss best practice from the radiographer's perspective, detailing the importance of continuity of evidence and maintenance of contemporaneous notes and witness statements. it will address key considerations for radiographers and others who provide evidence or act as an expert witness. imaging professionals play a pivotal role in the investigation of suspected child abuse. it is essential that this evidence is obtained, recorded and presented with regard to the applicable standards of evidence. for those called to give evidence, the importance of appropriate imaging protocols and procedures, detailed documentation and thorough preparation cannot be understated. learning objectives: . to understand the importance of contemporaneous notes and witness statements if involved in any forensic or suspected non-accidental injury examination. . to become familiar with the importance of continuity of evidence in forensic examinations. . to appreciate the key considerations if you are asked to provide evidence or act as an expert witness in court. brain tumours are the second most common neoplasms in children after leukaemia. as "paediatric brain is not just a small brain", paediatric brain tumours are not the same as tumours in adults. mri is a method of choice in their diagnosis in accordance with the principle alara in the paediatric population. by the age of approximately years, supratentorial tumours dominate; later ( - years of age), the majority of children show infratentorial neoplasms which are pilocytic astrocytomas, most often found in the hemispheres, and medulloblastomas or ependymomas -in the fourth ventricle. after the age of , both groups are diagnosed with equal frequency. cerebral hemispheric tumours include astrocytoma which is the most frequent, teratoma in neonates, and in older children atypical teratoid/rhabdoid tumour, ependymoma, pnet, ganglioglioma and dnet. in the midline, we deal with chiasmatic or hypothalamic glioma, craniopharyngioma and germ cell tumours of the pineal region. extraparenchymal tumours are uncommon in the paediatric population. apart from the location, imaging pattern in various sequences, and contrast enhancement of the tumour, advanced mri techniques are helpful in establishing the diagnosis. dwi helps to differentiate between tumours of high and low cellularity showing low adc values in case of medulloblastoma and high in astrocytoma. mrs, pwi and dti also add important information to the diagnosis. mri of the spinal canal is obligatory in case of these primary brain neoplasms that have a high propensity to metastasise to other parts of the central nervous system such as medulloblastomas, ependymomas, high-grade gliomas, cerebral neuroblastomas, and pineal region neoplasms. learning objectives: . to learn about the difference between paediatric and adult brain tumours. . to understand the imaging strategy for the paediatric population. . to become familiar with the most common paediatric brain tumours. pancreatic endocrine tumours (p-nets) include both pancreatic neuroendocrine tumours associated with a functional syndrome (functional p-nets) and those associated with no distinct clinical syndrome (non-functional p-nets). non-functional p-nets may show immunohistochemical positivity for hormones which may be produced, but not secreted, which are clinically inert and whose serum concentrations are insufficient to induce symptoms. among functional p-net, the most common are gastrinomas and insulinomas, whereas those secreting vasoactive intestinal peptide, glucagon, somatostatin and other hormones are considered together as a group called rare functional p-nets. non-functional p-nets are classified, according to the who classification, in well-differentiated neuroendocrine tumours and poorly differentiated neuroendocrine carcinomas (necs) of small or large cell type. well-differentiated neuroendocrine tumours are then divided according to a grading scheme based on mitotic count or ki index in nets-g (with a mitotic count < per high-power fields (hpf) and/or % ki index), and nets-g (with a mitotic count - per hpf and/or - % ki index). all poorly differentiated neuroendocrine carcinomas are graded g (with a mitotic count > per hpf and/or > % ki index). most pancreatic nonfunctional p-nets are well-differentiated tumours, whereas poorly differentiated neuroendocrine carcinomas are uncommon. pancreatic endocrine tumours (p-net) are divided into functional p-nets and non-functional p-nets. diagnostic imaging of these tumours includes detection, characterisation and staging. there are significant differences when dealing with functional p-nets and non-functional p-nets. in fact, in the first case the main aim of diagnostic imaging modalities is the detection of the tumour, while for non-functional p-nets the main aim is the characterisation and the differential diagnosis with other tumours of the pancreas, mainly with pancreatic adenocarcinoma. staging is also important for both tumours, but mainly for non-functional p-nets. a large number of imaging modalities are available, both "morphological" and "functional". we can arbitrarily divide the imaging modalities for the diagnosis of p-nets into three levels: the first level includes us (with contrast-enhanced us), mdct, mri and nuclear medicine techniques; the second, endoscopic us, angiography and venous sampling; the third, intra-operative us. functional p-nets are represented mainly by insulinomas and gastrinomas which are small in size in most cases. their detection is still difficult, but significant improvements have been made with us, mdct and mri, so that these tumours are detected in most cases if appropriate imaging techniques are performed. however, still, no single modality is % effective. non-functional p-nets can be differentiated from pancreatic adenocarcinoma, since they are hypervascular and usually large in size. since they are potentially malignant, they require accurate staging by imaging modalities, both morphological (mdct, mri) and functional (pet/ct) to plan surgery and chemotherapy. there is worldwide consensus about surgical treatment of resectable p-nets in the following clinical situations: proven malignancy, whenever it is possible to remove the primary tumour and at least % of liver metastases (debulking); p-nets associated with a clinical syndrome caused by hormonal hypersecretion (insulinomas and gastrinomas mainly); non-functional p-nets with a size larger than cm. vice versa, there are different expert opinions about the surgical treatment of functional and non-functional p-nets in men syndromes and in sporadic non-functional p-nets less than cm in size. in men all insulinomas should be resected, while for gastrinomas the extent of resection is patient tailored, deserving standard whipple for selected cases. in the most common sporadic small incidentalomas, where long-term prognosis is favourable, surgical treatment has to be weighted with co-morbidities and morbidities of the patient and the procedure planned. these tumours have a risk of node metastases at the time of diagnosis in less than % of patients. whenever possible, mini-invasive approach with pancreas-sparing operations should be recommended. in elderly patients, a first period of follow-up should be preferable and surgery should be offered only if the size of the tumour demonstrates increasing trends. the "acute aortic syndrome" includes three closely related emergency entities of the thoracic aorta: aortic dissection, intramural hematoma and penetrating atherosclerotic ulcer. as these conditions present with similar symptoms, cross-sectional aortic imaging is essential for diagnosis, triage and treatment planning. initial imaging features of these pathologies including typical early complications as well as their differential diagnosis will be reviewed. as nowadays several of these patients will undergo early endovascular thoracic aortic repair, an update on its indication, the different technical options and their clinical results will follow. finally, the focus will be on repair-related late complications and progression of aortic disease. imaging follow-up protocols, features of chronic complications and their interventional repair will be discussed. a. pre-therapeutic radiological evaluation j. ferda; plzen/cz (ferda@fnplzen.cz) the acute thoracic aorta syndrome covers patients suffering from acute chest pain and those injured during high-energy trauma. despite the similar symptoms, the aortic pathologies are heterogeneous, including acute aortic injury, intramural haematoma, aortic dissection, and ruptured thoracic aortic aneurysm and mycotic aneurysms. the diagnostic approaches could include the whole pallete of the imaging modalities: chest x-ray, transesopheageal echocardiography (tee), multidetector ct angiography (mdcta), mri and pet/ct. because of some disadvantages of tee, like problems with intubation in injured patients or problems of mri with seriously ill patient monitoring, mdcta has become the most used modality due to its ability of discrimination between aortic and non-aortic pathologies, and also due to its high sensitivity, specificity, availability and especially superior geometrical resolution and potential of d imaging. the proper pre-therapeutic mdcta protocol is using sub-millimeter isotropic resolution covering the entire aorta with pelvic arteries. when the pathology of thoracic aorta is found during the triple rule-out triage protocol, additional mdcta of the rest of the aorta and pelvic arteries has to be completed. ecg triggering could be an important advantage in the reduction of pulsation-derived artefact. the volume of applied contrast material used should be as small as possible to plan applications during endovascular treatment. d reconstruction of the thoracic aorta with its side branches and advanced vessel analysis have a crucial role in the measurement of the aortic diameter, diameter profile and lengths in the selection of proper stent graft type and size. they can be accommodated even in patients with small vessels and tortuous anatomy. calcified arteries can be no longer considered absolute exclusion criteria. some devices are designed to reduce the incidence of endoleaks, improving sealing capabilities by the use of a polymer to better adjust to the variable morphology of the proximal neck or to fill the aneurysmatic sac completely. more importantly, the introduction of fenestrated devices has resolved the problem of facing difficult anatomies when the aneurismatic pathology involves renal or visceral arteries. obviously, the use of these new devices requires a learning curve. beyond an increase of cost, they permit treating those patients who in the past were considered unfit candidates for evar. the evar new frontier requires performing the procedure in an outpatient fashion. a multicentre study was conducted on patients who were discharged / hours after uncomplicated stent-graft implantation. cases ( %) with access vessel complications required additional procedures and patients were hospitalised overnight. the -day readmission rate was % for access vessel stenosis or false aneurysm. from the time of the first evar procedure, significant improvements have been achieved but there is still room for further progress. the standard imaging modality after endovascular treatment for thoracic aortic pathology (tevr) is ct angiography (cta). cta enables the radiologist to assess for the patency of the endografts, the presence of any complications after tevr, and the development of any new pathology related not only to the aorta, but elsewhere above and below the diaphragm. the role of mr is reduced compared with cta, particularly in the presence of stainless steel endografts. however, good images can be obtained by mri and mra if nitinol endografts have been inserted. mr is a useful modality for follow-up in young patients who require lifelong follow-up and, therefore, limited exposure to ionising radiation. ultrasound has no practical role in the follow-up after tevr, and although plain thoracic radiographs are routinely performed by some centres post-tevr, in practice almost equivalent information can be obtained by ct. the main complications after tevr are endoleaks, type a dissection, paraplegia and stroke. the classification of endoleaks is similar to the classification for evar. type and type endoleaks require treatment by additional endograft coverage with or without supraaortic or visceral artery bypass. other treatment options include the use of branched, fenestrated endografts or the use of chimneys and snorkels. type endoleaks are less common than post-evar. similar to the evar population, intervention is mandated for an enlarging aortic diameter and involves embolisation in most cases. interventional oncology is a discipline that uses imaging-guided procedures in the treatment of cancer patients. when the malignant disease in not curable, interventional techniques such as biliary and oesophageal stenting can provide useful palliation and improve the patient's quality of life. in patients with potentially curable tumours, thermal ablation, cryotherapy and radioembolisation provide useful alternatives to radiotherapy and surgery in carefully selected patients. interventional radiologists who treat patients with cancer should practise as clinicians, participating in multidisciplinary meetings where treatment choices can be discussed with specialists from other oncological disciplines. they should involve themselves in all stages of clinical practice, including pre-treatment consultations, ward rounds and patient follow-up. radiation oncology and interventional oncology have much in common, including the potential for curing cancer using minimally invasive techniques. interventional oncologists can usefully adopt many of the principles and methods of practice in radiation oncology, including quality assurance and the collection of robust outcome measures. an alliance between these disciplines would be beneficial to clinical practice and research in both disciplines. session objectives: . to understand the contribution of interventional radiology to oncology. . to appreciate the areas of overlap between radiation oncology and interventional oncology. . to understand the need for a clinical pattern of practice in interventional radiology in oncology. the current scope and future challenges j.i. bilbao; pamplona/es (jibilbao@unav.es) together with chemotherapy, surgery and radiotherapy, interventional radiology (ir) is the fourth pillar in oncology. transcutaneous and endovascular procedures, guided by imaging, constitute a fundamental knowledge essential for the multidisciplinary management of oncologic patients. the range is wide and covers most of the needs in patient´s care. in short: obtaining samples for making an accurate diagnosis and also to know more about its specific biology (biomarkers); ir covers any possibility needed for vascular access; percutaneous needle ablation is the best alternative in selected patients; endovascular embolisation with "vehiculisation" of therapies is an outstanding method for selective treatment, and sometimes precise ablation, of different tumors; ir is a unique way to offer palliation in a wide range of tumoral complications, such as embolisation for bleeding, stenting for vein obstructions or drainage of fluid collections. there is an obvious need of continuous technical refinement. image-guided therapy is a continuous process that implies many disciplines and that includes every step, from staging through guidance of the procedure to the evaluation of the outcomes, both immediately, during and in the follow-up. future developments in ir may be focused towards the personalisation of therapies to each patient and to a better understanding of the biological mechanisms of tumour response or progression and their image expression. finally, radiologists must be actively involved in the whole process of clinical trials, from the creation of new lines to the performance of therapy until the evaluation and diffusion of the data. radiation treatment has a fundamental role in the multidisciplinary management of people with cancer. the evidence base for radiation therapy is robust and approximately % of people diagnosed with cancer will benefit from radiation treatment. most of this benefit comes from an increase in cure. the technical quality of delivery of radiation treatment is of great importance in avoiding a geographic miss and also in reducing the amount of normal tissue being irradiated unnecessarily. as well as the excellent technical delivery of any treatment offered, of paramount importance is the intelligent integration of care with attention to evidence base choices for both local and systemic treatment. multidisciplinary teams are in the best position to integrate surgery, radiation treatment, interventional oncology and systemic therapy in a planned and deliberate fashion. best practice: how to organise an interventional oncology unit a. gangi, j. garnon, g. tsoumakidou; strasbourg/fr (gangi@unistra.fr) interventional oncology is a fast growing field. interventional oncology is a clinical and technical speciality. solid clinical knowledge associated with the technical skills is mandatory for a successful practice. the technical skills should be associated to the clinical part. the knowledge of all treatment modalities and the participation of a multidisciplinary team is vital for the best practice. every single patient should be seen by the interventional oncologist and followed-up. the interventional oncologist is part of a multidisciplinary team, taking care of the patient. the team includes oncologists, radiotherapists, pain specialists, psychologists, surgeons, and anesthesiologists. the interventional oncology unit should include specialised nurses, technician, anesthesiologist, anesthesiologist nurses, and the interventional radiologist. the team should be able to take care of the patient's follow-up and eventual complications. to summarise, high-quality interventional oncology services are essential for safe and effective patient care. the technical skills of the interventional oncologist should be associated with an excellent clinical knowledge of the disease. a multidisciplinary team work is mandatory for a successful interventional team. harmonisation of training and appropriate credentialing is required for clinical services of the best quality. interventional radiologists continuously improving their skills and knowledge, patient access to safe and effective minimally invasive treatment options is increasing. whereas cardiac ct and mri are routine examinations for specialised radiologists, it has been necessary to wait for the introduction of fast scanning techniques to modify the behavior of nonspecialised radiologists regarding the interpretation of cardiac cavities. the objective of this session is twofold. first, cardiac radiologists will underline the role of these imaging modalities in the assessment of congenital heart disease but also in ischaemic and nonischaemic cardiomyopathies. in the second part of this session, chest radiologists will focus on the diagnostic information accessible at the level of the right ventricle, known to be the target of numerous respiratory disorders. two practical situations will illustrate this approach: pulmonary thromboembolic and chronic respiratory diseases. accurate assessment of the right ventricle (rv) is crucial for the management of congenital heart disease. echocardiography can provide some information, but as the rv sits directly behind the sternum and as many patients have undergone multiple operations, visualisation can be difficult. furthermore, for long-term follow-up and treatment planning, accurate measurement of ventricular size and mass, and valvular flow (tricuspid and pulmonary), and assessment of anatomy in d and d are vital. this presentation will outline the role of cross-sectional mri and ct for assessing the rv, and describing the indications, imaging protocols, technical pitfalls and relevant information for treatment planning. right heart failure in ischaemic and non-ischaemic cardiomyopathies m. grothoff; leipzig/de (matthias. grothoff@herzzentrum-leipzig.de) the role of the right ventricle (rv) has been underestimated for many years. today, we know that the rv is of importance for keeping up a sufficient circulation and that right heart failure in both ischemic and non-ischemic cardiomyopathies can result in hemodynamic compromise. rv ischemia has a higher in-hospital morbidity and mortality but post-infarction chronic right heart failure is a rare finding. as the rv has a different physiology from the left ventricle (lv), also the pathophysiology of ischemia is different here. using our standard cmr tools t weighted imaging for detection of edema and late gadolinium enhancement imaging for detection of necrosis/scar we can find that the rate of myocardial salvage is much higher in the rv than in the lv and that rv failure in the acute phase is basically caused by reversible ischemic effects. in non-ischemic cardiomyopathies like hcm and dcm changes in the rv myocardium mostly resemble changes of the lv. in arrhythmogenic right ventricular cardiomyopathy/dysplasia (arvc/d) however, there are specific findings in the rv and the role of imaging in diagnosing arvc has been strengthened by the revised task force criteria published in . cine imaging should be performed carefully for detection of rv akinesia, dyskinesia or small aneurysms as well as the planimetry of the rv. findings like the detection of fibrosis in lge imaging and the detection of fat in t imaging can provide additional information, but are not diagnostic for arvc. learning objectives: . to learn about the different ct and mri techniques for evaluating right heart morphology and function. . to understand the prognostic impact of right heart involvement in ischaemic heart disease. . to learn about the basic morphological and functional features of different non-ischaemic cardiomyopathies. right heart and chronic respiratory diseases: can ct be used as a onestop-shop? m. remy-jardin; lille/fr (martine.remy@chru-lille.fr) over the last decade, ct technology has evolved towards fast scanning capabilities and high temporal resolution, enabling improved evaluation of the heart and surrounding structures in the course of routine thoracic ct imaging. this has progressively modified the radiologist's implication in the interpretation of chest ct examinations. firstly, it is more and more common to integrate the physiological interactions between heart and lung when analysing the ct features depicted on a given examination. secondly, it is now possible to integrate cardiac functional information into a diagnostic ct examination of the chest, providing prognostic information in the management of patients with a wide variety of chronic respiratory disorders. the purpose of this presentation is to provide non-specialised radiologists with some practical guidelines in the choice of the scanning protocols and range of functional information that can be deducted from chest ct examinations. right ventricular dysfunction and levels of pressure within the pulmonary circulation are the key points of such approaches. learning objectives: . to appreciate the clinical impact of a cardiothoracic evaluation from the same ct examination. . to learn about the scanning protocols enabling such a combined analysis. . to understand its compatibility with other non-invasive modalities. rv and prognosis in pulmonary thromboembolic disease n.j. screaton; cambridge/uk (nicholas.screaton@papworth.nhs.uk) right ventricular failure is the most common cause of early death following acute pulmonary embolism and in patients with chronic thromboembolic pulmonary hypertension. echocardiography is sensitive in diagnosing and quantifying rv dysfunction. ct is the reference standard for the diagnosis of acute pe diagnosis and has the potential to provide comprehensive evaluation of the pulmonary arteries, heart and lung parenchyma. ct signs of right ventricular dysfunction which have shown predictive value for adverse outcome include: flattening of the inter-ventricular septum or bowing towards the left ventricle, reflux of contrast medium into the ivc, relative rv/lv diameter ratios on transverse and -chamber reconstructions, and ventricular volumetry as assessed both on routine non-gated ctpa and ecg-gated ct. in patients with cteph, mri permits imaging of the pulmonary vasculature and functional evaluation of the right ventricle and pulmonary circulation, enabling both morphological assessment and prognostic evaluation. learning objectives: . to learn about clinical and imaging factors associated with prognosis of thromboembolic disease. . to understand the benefits and weaknesses of different imaging modalities in predicting prognosis in thromboembolism. . to discuss the merits of comprehensive imaging evaluation in the routine diagnostic workup of suspected thromboembolic disease. multimodality imaging including ultrasound has within recent years been introduced in several anatomical regions. pet, ct and mri datasets can after an initial co-registration be fused and shown either side by side or using overlay on screen while doing real-time ultrasound; thus, the pet, ct or mri dataset is reformatted in a projection to fit the real-time ultrasound images. several methods of aligning the images are available and have been shown to have a high accuracy. image fusion involving ultrasound has been tested in phantoms, animals and in patient studies, mainly in abdominal imaging. by combining several imaging modalities, it is possible to benefit from the strengths of each modality. it is useful for characterisation of and intervention on liver lesions, abscesses containing air and lesions in areas with poor overview by ultrasound. also, the method is useful for characterisation and biopsy of pet-positive lesions, especially in patients with a history of cancer. the background of the method and clinical examples, mainly from abdominal applications, will be covered. also, perspectives and future research topics will be highlighted. contrast-enhanced ultrasound (ceus) has proved to improve the detection and characterisation of pathologies compared to conventional ultrasound, ct and mri in a number of indications. ultrasound contrast agents (uca), which are purely intravascular, do not show any interstitial diffusion or glomerular filtration like iodinated complexes or gadolinium chelates. dynamic contrastenhanced ultrasound (dce-us) allows to display the enhancement of a lesion with a high frame rate after bolus or infusion administration of uca, and to compare enhancement profiles between normal and abnormal tissue. quantification of dce-us is useful to quantify tumour enhancement and to limit intra-and interobserver variability. mathematical models with several perfusion parameters can be used. dce-us has shown to be of interest after antiangiogenic therapies as it allows an earlier evaluation of tumour response than usually done with ct and mri, which remain mainly based on the recist criteria. the common perception of d ultrasound (us) is of surface-rendered images, especially of the foetus. the same approach, enhanced by new transducer technology and software, can be used to take ultrasonography to another level in general body imaging, providing different image presentations and working practices similar to those of ct and mr. the technology involved in a us volume acquisition was represented initially by mechanical d transducers, and more recently by purely electronic, matrix probes. this latest technology allows bi-plane real-time acquisitions, fast switch to volume acquisition up to degrees, and d acquisitions with acceptable frame rates. protocols have been described for major abdominal and retroperitoneal organs, with fast optimisation of acquisition settings. the technology supports contrastenhanced ultrasound (ceus). after volume acquisition, interpretation for volume us datasets requires a post-treatment phase, including multi-planar reconstruction, multislice imaging, and volumetric analysis. virtual cystoscopy and volume ceus quantification are new options. the implementation of this new, efficient us modality in a radiology department supposes deep changes in the daily practice with delayed post-treatment reporting times. it should represent a more effective way for comparison between different examinations and a better communication tool with clinicians. the proven and potential benefits, in terms of clinical accuracy, training, workflow and overall efficiency, will be discussed. the purpose of this lecture is to emphasise some pitfalls in liver imaging. morphologic changes in the liver are usually attributed to chronic liver disease where liver cirrhosis represents the most important cause. however, noncirrhotic diseases may also induce atrophy-hypertrophic changes of the liver. the most common mechanisms are related to venous obstruction (either portal or hepatic venous) and biliary obstruction. multidetector ct and mr imaging are essential to highlight these abnormalities. when dealing with liver tumours, the most important question that has to be solved is tumour characterisation. yet, it is often difficult to assess whether a large tumour is intra-or extrahepatic. imaging findings that might be helpful will be shown. last, some liver lesions can mimic liver tumors. vascular disorders and focal fatty changes or focal fatty sparing are the most common causes. some other conditions can be also mimickers and such cases will be shown. the technique and the anatomy of the bile duct and pancreatic duct will be described. the anatomical variants of the biliary duct and the pancreatic duct system will be analysed and their possible role in generating diagnostic imaging pitfalls described. strategies to avoid pitfalls in diagnostic imaging of the bile duct and the pancreatic duct system will be illustrated, considering the possible source of pitfalls. diagnostic imaging findings of different diseases involving the biliary ducts and pancreatic duct system will be illustrated, as well as the diagnostic imaging criteria useful for the differential diagnosis. over the last two years, the number of ct and mri units has increased by %, and modern pet centers and leading centers for radiotherapy have been built. we can identify an approximately % increase in the number of radiological research projects--mainly ultrasound, ct, and mri investigations. during the last ten years the total number of vascular interventions and mr studies has increased by ten times, four times for ct, and the annual growth of us and x-ray investigations is about %. unfortunately, these technical innovations are not always accompanied with pacs and ris installations; this is a reason why all advantages of digital equipment are not achieved. we see rapid development of private medicine; new private hospitals and ambulances have grown by more than % and investment companies are looking for new projects in this field. however, the professional education of radiologists is not enough to work with new diagnostic equipment. this is a reason why in we will start a pilot project to create a new system of continuing medical education in clinical medicine, including diagnostic radiology. we hope, this pilot project will increase the efficiency of clinical practise. differential diagnosis (dd) including tumour and non-tumour brain pathologies is an integral part of neuroradiological diagnostics and in some cases is a challenge to experienced neuroradiologists. more than patients with different cns pathologies have been investigated in the department of neuroradiology, burdenko neurosurgical institute since . in the vast majority of the cases, there were tumours. we used deconvolution method for bv, bf, mtt and ps maps reconstruction on commercially available software (perfusion . . , . protocols, adw, ge). ct perfusion has demonstrated high level of information in determining the degree of malignancy in glial brain tumours. a strong correlation between bv and ps and tumour degree has been demonstrated. the use of ct-pwi makes it possible to successfully carry out dd between radiation necrosis and tumour progression. some histological types of brain lesions are characterised by unique haemodynamic properties which enable using this method as non-invasive "biopsy". ct-pwi has been especially useful in dd of the skull base region, bone and extracranial pathologies. the absence of artefacts created by the skull base bones makes this method especially valuable as compared to the mri t *-and asl-perfusions. ct-pwi makes it possible to successfully differentiate tumours and a number of non-tumour cns lesions including demyelinating diseases, infectious and non-infectious granulomas, etc. ct-pwi is a powerful method for differential diagnosis of brain pathology. t-pwi can provide unique functional information regarding tumour pathophysiology and haemodynamics, which are not available with routine ct and mri. learning objectives: . to become familiar with the technique of brain perfusion ct. . to understand the added diagnostic value of perfusion ct. . to learn about input of ct perfusion for assessment of treatment. interlude: development and use of web-based teleradiology in russia o.s. pianykh; newton highlands, ma/us (opiany@gmail.com) the largest territory in the world, russia, presents a perfect teleradiology use case of extremely unevenly distributed health care, concentrated in moscow and spread thin outside over an enormous area and sparse population. this unevenness deeply affects the quality and availability of radiological services, and cries out for efficient and practical teleradiology solutions. however, the same unevenness in medical expertise, financing and it infrastructure frequently acts against the advancement of teleradiology projects, blocking their natural progress. as a result, technical, financial, educational and clinical matters, interleaved into the nascent russian teleradiology ventures, require serious and predictable solutions, based on analysis and adaptation of already developed, internationally proven strategies. currently, russian teleradiology simply takes advantage of whatever is available to make its progress better, but needs to develop itself up to the tasks it faces. tuberculosis in russia: a challenge for a national radiological service i.e. tyurin; moscow/ ru (igortyurin@gmail.com) approximately, . billion people worldwide are infected with mycobacterium tuberculosis, of which million are active cases. the reasons for resurgence of tb infection include the hiv epidemic, a rise in reactivation disease in the elderly, a growing migrant population and spread of drug-resistant strains. the incidence of atypical tb infection is also on the rise, of which mycobacterium avium intracellulare is the most important human pathogen. the pathologic form of the pulmonary infection depends on the sensitivity of the infected host and is classified as primary or postprimary. primary tb pattern represents infection resulting from recent contact with the pathogen. postprimary tb pattern results from reactivation of a dormant focus within the lungs. thoracic tuberculosis produces a broad spectrum of radiographic abnormalities. the radiological patterns had been described as parenchymal, airway, vascular, mediastinal, pleural, and chest wall lesions. common causes of a missed diagnosis of thoracic tuberculosis are failure to recognise hilar and mediastinal lymphadenopathy as a manifestation of primary disease in adults, overlooking of minimal productive lesions or reporting them as inactive, and failure to recognise that an upper lobe or superior segment of lower lobe mass might be tuberculosis. in aids patients, the imaging features depend on the degree of immune suppression. a pattern of postprimary tb is also usually seen among patients with decreased immunity due to alcoholism, renal failure, diabetes mellitus, ageing, malignancy, and renal and cardiac transplantation. learning objectives: . to understand the scope of the problem with tuberculosis screening in the russian federation. . to learn about the changing imaging patterns of thoracic tuberculosis. . to appreciate the current role of imaging in the differential diagnosis and follow-up of this pathology. interlude: mystery of denisov's cave and paleoradiology m. mednikova; moscow/ ru (medma_pa@mail.ru) this study focuses on the anatomical identification of fossil bones from altai highland in southern siberia, their radiological description, and parallels among other hominin fossils. methods describe the small and isolated tubular bones. we used microct versaxrm- by xradia inc. and digital microfocus x-ray. denisova cave is the best studied paleolithic cave in north asia. archaeological data suggest that about thousand years before the present, a group of humans migrated to southern siberia from the west. their descendants had lived in the altai mountains in relative isolation for thousand years. the taxonomic status of the early inhabitants of southern siberia was clarified thanks to palaeogenetic studies. it was concluded that the altai hominin was genetically twice further from modern humans than were neanderthals. phylogenetic analysis suggests that this clade diverged from that ancestral to humans and neanderthals very early -about billion years ago. descendants of this clade apparently survived in the altai refugium until the period - thousand years ago. due to structural patterns of preserved pedal and manual phalanxes, humans from denisova cave could be expected to be the bearer of "archaic" traits. the extraordinarily thick walls of tubular bones in adults reflect a high level of occupational stress. radiological volumetric microscopy of the denisovan girl phalanx gave evidence for the histological estimation of the developmental age. radiological methods seem to be the perspective approach in the study of rare and fragmentary fossil human remains, differentiating between archaic and modern humans. advances in imaging of pancreatic masses g.g. karmazanovsky; moscow/ru (karmazanovsky@ixv.ru) modern diagnostic radiology allows not only to detect pancreatic tumours, but also to carry out their differential diagnosis with high accuracy. this is especially important because many benign tumours of the pancreas do not require immediate surgery. at the same time, many benign tumours may become malignant. the differential diagnosis of the pancreatic cystic lesions using endoscopic ultrasound and magnetic resonance pancreaticocholangiography has now become particularly accurate. contrast enhancement allows detecting signs of tumor invasion into the main blood vessels and adjacent organs. modern developments of radiology help to use extended operations for malignant tumours and sparing surgery with minimal removal of intact functioning pancreatic tissue in benign tumours. learning objectives: . to become familiar with major types of pancreatic masses and their radiological manifestations. . to understand approaches to the selection of diagnostic pathways in pancreatic tumours. . to become acquainted with new developments in diagnostic imaging of pancreatic tumours. interlude: an artistic view of ct a.l. yudin; moscow/ ru (prof_yudin@mail.ru) radiology is a specialty based largely on figurative perception of diagnostic imaging. the radiological picture of many symptoms reminds one of objects and environmental phenomena (for example, vanishing lung, draped aorta, geographic liver, porcelain gallbladder, dirty fat and others). training of creative mentation promotes lasting assimilation of educational material at the associative level. metaphorical thinking skills can also be improved by searching for amusing fragments in radiological images similar to the objects in everyday life; animals, cartoon characters, and others. the essential help in the study of the physical foundations of radiology and functional capabilities of workstations provide the execution and study of x-ray films, ct or mri of fruits and vegetables, flowers, various artifacts and other objects in the environment. eventually, it is just beautiful. ultrasound of the breast is an established imaging technique with several advantages. it has been used to differentiate solid from cystic breast lesions for a long time. it has also been increasingly used to visualise small lesions in mammographically dense breasts and is the initial imaging method of the breast in young women. the features of the lesions that need to be analysed are: shape, relation to anteroposterior to laterolateral diameter, margins, internal structure and absorption of sound (distal acoustic phenomena). color doppler enables the thorough assessment of flow within the breast lesions. elastography is established as a useful method to differentiate breast lesions on the basis of their stiffness, which can be quantified. the accurate staging of axillary disease is an important aspect in the management of patients with breast cancer. it is recognised, however, that underlying malignancy can be found in lymph nodes that appear morphologically normal. a variety of morphological features that may be seen in pathological nodes have been described (cortical thickness of > . mm and absence of a fatty hilum). the automated breast ultrasound technique has been known for a long time, but with recent improvements now may play an important role. the automated breast volume scanner (avbs) employs frequencies of - mhz. avbs has various major advantages, such as being independent of the experience of the operator as well as supplying consistent and reproducible results. additionally, for the first time it has become possible to obtain coronal images of the complete breast, which greatly assists in the planning of surgical interventions. to study the technical basics in breast ultrasound (us), a breast scanning phantom helps to understand different us settings of greyscale imaging. typically, breast us work has a linear array transducer with a minimum field of view of . cm and a high frequency of minimum mhz. a broadband transducer (e.g. - mhz) with different bands is preferred. in the central axillary area, a lower frequency band offers a deeper penetration depth. high frequencies improve the tissue contrast and optimise the axial and lateral resolution. the transmitter focus is adapted to the region of interest. highly sensitive color doppler detects slow flow rates ( cm/s and less). for frequencies and power settings used in diagnostic greyscale and color doppler studies, no safety limitations have to be considered. breast us is mainly used as a second-line imaging tool to mammography (mg). a correlation between both imaging modalities is crucial. skin markers and additional mgs may help to solve the correlation problem. in bi-rads or us lesions with unclear correlation in mg, a marker placement after us-guided large-core needle biopsy helps to clear the problem. the pathomorphological growth pattern of a lesion is the basis to understand different imaging aspects with different modalities. second-look us after mri is an important correlation of lesions, which were first visible with mri. understanding the complementary imaging criteria will help to reduce the number of false-positive results. a practical imaging approach to brain and neurovascular trauma will be reviewed, with an emphasis on understanding the correlation between pathophysiology and imaging signs. guidelines on when to order acute neurovascular studies will be presented. special technical considerations to help optimise ct and mr imaging protocols for suspected brain injury, cns haemorrhage, and arterial dissection will be discussed. neurological emergencies are often associated with high morbidity and mortality, and thus require prompt diagnostic and therapeutic action. nontraumatic emergencies may however have a subacute onset, and radiological signs may be subtle, which can lead to delay in diagnosis and treatment. since clinical features are often nonspecific, the radiologist may be the first to point the clinician in the direction of the correct diagnosis. it is therefore of great importance that the radiologist is aware of and familiar with the various imaging findings, on both computed tomography (ct) and magnetic resonance imaging (mri), of non-traumatic neurological emergencies. these include vascular, infectious and inflammatory diseases. commonly encountered emergencies are ischaemic and haemorrhage stroke, venous thrombosis, arterial dissection, abscess, acute disseminated encephalomyelitis (adem), and encephalitis. radiological findings in rarer diseases may mimic those in the more commonly occurring diseases, but need to be correctly interpreted as therapeutic strategies and prognosis may be entirely different. such entities include for instance posterior reversible encephalopathy syndrome (pres), reversible cerebral vasoconstriction syndrome, susac's syndrome, and status epilepticus. furthermore, initial findings of (impending) complications of brain disease, such as hydrocephalus and herniation of brain structures, may be subtle, while early recognition allows for prompt and adequate intervention. finally, diagnostic and therapeutic interventions performed in an emergency setting may interfere with the diagnosis and interpretation of clinical and imaging findings. associated limitations and pitfalls therefore need to be recognised to avoid false-negative or false-positive diagnosis, respectively. learning objectives: . to learn about the modalities (ct/mri) and protocols for non-traumatic neurological emergencies. . to learn how to diagnose the main non-traumatic neurological vascular and non-vascular emergencies. . to become aware of the pitfalls and limitations of clinical presentation and imaging findings in non-traumatic neurological emergencies. the current century has brought with it new challenges in radiation protection, many of which follow the development of mdct which greatly enhanced diagnostic capabilities and substantially reduced scanning time to just a single breath-hold for chest ct, making ct scanning both patient-friendly and the physician's preferred tool. ct has also been added to nuclear imaging systems to create hybrid scanners (pet/ct; spect/ct image gently years later: lessons learned in radiation protection for children. the alliance for radiation safety in pediatric imaging was founded in to promote radiation protection for children worldwide through awareness, education and advocacy. comprising over medical professional societies and agencies, the alliance seeks to change practice locally for improved paediatric patient safety in medical imaging. since its inception, the alliance has had campaigns in ct, diagnostic and interventional radiology, nuclear medicine, digital radiography and an outreach campaign to parents and educational summits. yet, there is still work to be done. there is a need for parents and patients to receive information about their imaging test prior to the performance of imaging procedures, particularly those involving ionising radiation. the paediatric medical imaging community should work tirelessly with the manufacturers of imaging equipment to tailor equipment for children to optimise the study and ensure that the users of the equipment are knowledgeable in its safe use in children. the routine display of size-specific dose estimate on ct scanners will better estimate patient dose for quality improvement within facilities and in dose registries. finally, there is a need for diagnostic reference levels in paediatric radiology. this presentation will update the audience in progress to date regarding these challenges. the image wisely programme continues to move forward, guided largely by its steering committee, composed of two representatives each from acr and rsna and one representative each from aapm and asrt. the image wisely website (www.imagewisely.org) has educational content for imaging professionals (radiologists, medical physicists, and technologists) and referring physicians. a patient tab features links to radiologyinfo.org and the image gently website. a tab for "my equipment" links visitors to radiation dose-related 'microsites' developed by ge, hitachi, philips, siemens and toshiba. as of august , , , individuals, facilities and associations have taken the image wisely pledge. to attract traffic to its website, image wisely has recently instituted 'news' and 'top reads' sections on the splash page highlighting recent articles and announcement, highlighting breaking news coverage and journal publications on relevant topics. in , content on nuclear medicine procedures was finalised, completing two of the three targeted foci of the initial action plan_ct and nuclear medicine. in october , a launch meeting will initiate the process of developing content for the third target area, fluoroscopy. in and , image wisely has concentrated on efforts to engage referring physician communities, with particular interest in paediatricians and emergency medicine physicians. progress has been slow, but has recently gained traction with emergency medicine in the form of radiologist participation in a symposium, diagnostic imaging in the emergency department: a research agenda to optimise utilisation, organised by the society of academic emergency medicine. learning objectives: . to learn about the image wisely campaign for radiation protection of adults. . to understand the lessons learnt while communicating messages. lessons from a national approach to patient safety in radiation protection p. cavanagh; taunton/uk (petecavanagh@gmail.com) in considering a european campaign on radiation awareness, it is useful to gain information from similar campaigns in health care. there have been a number of national patient safety campaigns based on the institute for healthcare improvement (ihi) , lives campaign initiated in the usa in . following the success of this, a similar campaign was launched in england. part of this process included an analysis of the success of the programme and the key interventions and drivers that were considered essential at both the national and local level. these included strong leadership, . bone (pseudo)-tumours: the majority of bone tumours can be detected on plain radiography. the age of the patient, location of the tumour in a bone and history of pre-existing bone abnormality should be included in determining the likely diagnosis. careful analysis of pattern of bone destruction, periosteal reaction and matrix mineralisation allow for characterisation of most bone tumours and differentiation from pseudotumours. ct may be useful for osteoid osteoma and mri is the best imaging technique for further diagnosis and staging by displaying tumour composition and extent of bone marrow involvement, including skip lesions, presence and extent of extraosseous soft tissue mass, and involvement of neurovascular bundle, muscle compartments and adjacent joint. this allows to find the best biopsy and surgical approach. to evaluate local control of disease and for detection of local recurrence, mri is usually the best imaging technique. . soft tissue (pseudo)-tumours: the majority of soft tissue tumours can be detected on ultrasound. except for cysts and subcutaneous lipoma, mr will be the next imaging technique for diagnosis, local staging, and eventually to find the best biopsy and surgical approach. the age of the patient, location of the tumour and careful analysis of the signal characteristics on mri allow for characterisation of many soft tissue tumours and differentiation from pseudotumours. plain radiography and ct may be useful for detection of calcifications and myositis ossificans. to evaluate local control of disease and for detection of local recurrence, ultrasound and mri are the best imaging techniques. in this honorary lecture, i will review a number of personal impressions that may contribute to the successful performance of research projects in cardiovascular radiology. also, issues and obstacles in performing successful research in the field of radiology will be considered. key factors for success include guidance by inspiring mentor, genuine interest and motivation, some creativity and serendipity. radiology has developed tremendously with many innovative modalities over the last years, providing ample opportunity to participate in clinical research. potential issues that should be addressed are subspecialisation, turf battles, competition from other clinicians/ imaging specialists and adequate training in radiology research. the field of imaging research includes traditionally more clinically oriented research by focussing on developing and testing of new modalities and applications, now becoming more basic by including molecular imaging, population imaging, outcome studies and alike. silicone-filled implants (single or double lumen) require a dedicated sequence where silicone exhibits a high signal intensity, whereas signals from fatty and fibro-glandular tissues are cancelled. diagnosis of intracapsular rupture relies on the detection of a linguine sign (direct sign) or multiple "key-hole" and/or " teardrop" images (indirect signs). extracapsular rupture is defined as the presence of silicone outside the capsule (breast, soft tissues, lymph nodes). breast reconstruction after mastectomy may involve autologous tissue flaps (skin, fatty tissue, muscle and fascia). the transferred muscle is inserted in front of the major pectoralis muscle and enhances after contrast medium injection. the most common benign findings seen in such reconstructed breasts and also after a breast-conserving treatment are oedema, seroma, haematoma, fat necrosis and fibrosis. diffuse thickening of the skin and trabeculae is usually observed the first year after completion of radiation therapy. small focal areas (less than mm) of non-mass-like enhancement and thin linear or rim enhancement at the lumpectomy site can all be expected findings. these benign findings decrease progressively, but residual enhancement may be observed in % of women at or more years. in patients who were switched from tamoxifen to aromatase inhibitors, a stronger or re-appearance of a background enhancement may be observed. before classifying such findings as benign or probably benign, clinical examination and standard imaging data must be taken into account. highly suspicious lesions in such patients are similar to bi-rads lesions in the nontreated breast. prevention strategies for women at high lifetime risk of breast cancer are usually adapted to the individual woman's risk of breast cancer. women at high risk (brca mutation carriers or women with a % likelihood of brca-heterzygosity) are offered primary prevention, which includes chemoprevention by tamoxifen or surgery (risk-reducing mastectomy and/or salpingooopherectomy, where the latter also helps reduce the risk of subsequent breast cancer by %). if women opt for secondary prevention (active surveillance/screening), this has to start at at the latest. mri is considered a compulsory component of all programs worldwide. due to the very limited added cancer yield of mammography in this subset of women, and because of the still unsettled issue of possibly increased radiosensitivity of brca-mutation carriers, mammographic screening is not recommended until the age of . there is broad agreement that (at least) an annual screening is important; especially for brca mutation carriers, screening every months may be more appropriate. we recommend interleaved screening, with annual mri and annual ultrasound (± mammography) phase-shifted by months. there are important differences between brca -and brca -associated cancers in terms of age distribution and incidence rates, histologic features, but also specific imaging features which must be known by radiologists. mri screening protocols must be adjusted to the specific needs of this (usually very young) screening population. this presentation will review the current evidence with respect to screening of women at high familial risk of breast cancer and will provide information on all aspects mentioned above. chest trauma is directly responsible for % of all trauma deaths and is a major contributor in another % of all trauma mortality. blunt trauma, accounting for % of chest injuries, is the third most common site of injury in polytrauma patients. plain radiographs still have a role in recognition of some acute thoracic pathology that requires immediate further management, either diagnostically and/or therapeutically, such as tension pneumothorax, major transdiaphragmatic herniation, large hemothorax or obvious mediastinal hematoma. mdct of the chest is now typically included in a whole body scan with iv contrast to facilitate rapid diagnosis on polytrauma cases using less radiation than selected segmental scans. mdct is the well-proven diagnostic gold standard for chest injury evaluation. the major advantages of mdct over other modalities include identification of active bleeding, direct signs of trachea or esophageal injury, direct evidence of major arterial vascular injury, such as pseudoanurysms, pneumo and hemopericardium, location and extent of lung contusion and laceration, and assessment for thoracic spine, shoulder girdle and rib fractures. diaphragm injuries are well depicted by mdct, especially on the left by identifying both the torn diaphragm edges, herniation and constriction of abdominal contents at the level of the torn diaphragm (collar sign), and direct contact of herniated structures with the posterior chest wall (dependent viscera). tracheal injuries are suggested by diffuse and progressive pneumomediastinum, dilated tracheostomy cuff, ectopic endotracheal tube, and direct connection of mediastinal air with the trachea lumen. ct-angiography eliminates the majority of indications for diagnostic catheter angiography. pulmonary symptoms such as chest pain, shortness of breath or wheezing are common non-traumatic symptoms prompting er visits. because clinical symptoms are very non-specific, imaging plays a major role in differentiating life-threatening from less severe diseases and forming a diagnosis. the chest radiograph remains the first imaging despite its limited sensitivity for certain diseases and being prone to inter-observer variability. comprehensive cardiothoracic ct examinations using most modern ct equipment are well evaluated in their diagnostic accuracy to determine the presence of vascular life-threatening events such as aortic dissection, acute coronary disease or pulmonary embolism. protocols and literature evidence will be discussed. the main focus of the course, however, will lie on the analysis of pulmonary ct findings and its contribution to the differential diagnosis of pulmonary emergencies (e.g. pneumonia, oedema, pneumothorax, exacerbation of fibrotic or obstructive lung diseases) and how to further integrate imaging findings, laboratory findings, patient history and clinical information to tackle the differential diagnosis. imaging findings will be discussed by clinical case studies, key findings and also overlapping morphological features of other differential diagnosis will be discussed side by side, illustrating the options and also limitations of imaging findings. mentor was first introduced in greek mythology, and in its modern usage first recorded by a french writer, françois fénelon, in . since then, this concept has been widely used in the development of mentoring, mentorship and more recently of mentees, with an overarching principle based on accompanying, sowing, catalysing, showing and harvesting. this concept remains today as a fundamental one in all teaching activities and implies generally a close contact between the mentor and the mentee. however, the development of internet with concomitant e-learning capabilities, which are hugely increasing, raises the very challenging necessity to re-think this concept and to adapt it to this new era. the esr is very concerned by this endeavour in the context of the striking development of esor, on one hand, and with the launching of its e-library, on the other. n. gourtsoyiannis; athens/gr (gournick@gmail.com) the presentation "esor in action" gives an overview on all esor activities in and . a detailed report on the esor courses and teaching programmes in is presented, containing statistics about the number of course participants and of scholarship and fellowship grants. additionally, all educational activities in , including dates, venues, topics and local organisers, are announced. evidence-based radiology: the basics evidence-based medicine (ebm) was derived from clinical epidemiology during the last century. it "is the conscientious, explicit, and judicious use of current best evidence in making decision about the care of individual patients" (dave l. sackett, ) . this means integrating ( ) individual clinical expertise with ( ) the best available external evidence from systematic research and ( ) patient's values and preferences. when considering evidence-based radiology (ebr), the needed expertise is not only clinical, but also technical, while ( ) radioprotection issues assume a relevant value according to the "as low as reasonably achievable" (alara) principle. ebm (and ebr) can be practised according to the "top-down" model, when using guidelines issued by governmental, professional, or academic bodies, or the "bottom-up" model, when local physicians ( ) formulate an answerable question, ( ) search for the best evidence; ( ) appraise critically; ( ) apply findings to practice; and ( ) evaluate the performance. a hierarchy of radiological studies has been established according to the investigated matter: . technical performance; . diagnostic performance; . diagnostic impact; . therapeutic impact; . patient outcome; . societal impact. different degrees of recommendations are based on different levels of evidence, with experts' opinion as the lowest level and meta-analyses of high-quality homogeneous studies and multicenter studies being the best level of evidence. to practise ebr, radiologists should be familiar at least with basic statistics and epidemiology as well as of methods for study design, thus recognising the principal types of bias which can limit the value of published studies. learning objectives: . to understand the historical development and general principles of evidence-based medicine (ebm) with its top-down and bottom-up approaches. . to learn how to apply ebm principles to radiology (evidence-based radiology, ebr) and to illustrate the main reasons for the relative delay in developing ebr, including the challenges of fast technological innovation in medical imaging. . to learn about the relationship between levels of evidence and the strength of recommendations for diagnostic imaging and interventional radiology. . to learn how radiologists could play a stronger role in building the evidence in favour of diagnostic imaging and interventional radiology, taking the patient's interest as the primary aim of clinical practice and science. education in research: action plan j. hodler; zurich/ch a survey regarding education in research has been commissioned by the esr and published in insights imaging ( ) : - . the results indicate that education in research, associated with career models, needs promotion. the research committee of the esr is working on possible future steps to promote research through education. several problems will have to be addressed, most notably inhomogeneity within europe, motivation issues, illdefined career paths as well as funding issues. several stakeholders will play an important role, including scientific societies, although this is the core business of academic radiology chairmen. most young professionals starting a career in academic institutions are eager to expand their professional and personal aptitudes. an effective way to support young professionals in this regard is by mentoring. mentorship is a relationship where the mentor supports a junior faculty member (mentee) in personal and professional development. the mentee benefits from early determination and initiation of career-relevant steps, greater productivity in research and publishing activity and developing techniques in networking and collaboration. moreover, the mentee gains perspectives on how his/her discipline operates academically, socially and politically, improves self-efficacy and deals more confidently with challenging intellectual work. the mentor, a knowledgeable, experienced and regarded person, benefits from fresh ideas, energy and curiosity of mentees, as well as through acceptance as a mentor an enhanced status in the department. it is a trusted relationship of dialogue, learning and challenges, based on mutual interests. however, the mentor is not automatically the best friend or the sole exclusive advisor of the mentee. more and more academic medical institutions offer facilitated mentoring programmes, which help the mentoring pairs to create an effective mentoring relationship to accomplish the desired skills. evaluation of faculty mentoring programmes in academic medical institutions demonstrates the importance of mentoring in research and academic development as well as in patient care. especially in radiology where over many years there have been challenges in recruiting and retaining talented young professionals, mentoring is considered as an essential way for preparing the next generation of scientific and intellectual leaders. pitfall: "a hidden or unexpected danger or difficulty". imaging methods can provide an extraordinary amount of useful data to specialists treating head and neck (cancer) patients. it is crucial that these data are used to full advantage of individual patients. the most important factor in this process is mutual cooperation between the physicians in charge of patient care and the diagnostic imaging specialist. pitfalls in the head and neck may present in various ways: normal variants may look like disease, incidental findings are frequently encountered and suboptimal technique may obscure important findings. moreover, many pitfalls are directly related to technical errors. the presentation aims to familiarise general radiologists, who have an interest in head and neck imaging, with common pitfalls encountered on ct and mr studies focussing on the neck. in an interactive setting, examples from daily practice will be discussed. imaging of the skull base and maxillofacial skeleton requires a meticulous imaging technique and a good knowledge of normal anatomy and possible anatomical variants. asymmetry in the pneumatisation of the paranasal sinuses, skull base or temporal bone is a common reason for misinterpretation. for example, hypoplasia of the maxillary sinus may be misinterpreted on conventional radiography as maxillary sinusitis or orbital blowout fracture, depending on the context; asymmetric pneumatisation of the petrous apex or mastoid bone may mimic, respectively, a cholesterol granuloma and fluid effusion in the non-pneumatised side on mri. vascular variants may also cause interpretation problems. for example, turbulent flow in a large jugular bulb may mimic a jugular foramen tumour on mri. variants in the vascular plexus surrounding the trigeminal or facial nerve may occur, and cause asymmetric findings on mri, possibly mimicking neuritis or perineural tumour spread. incomplete maturation or arrested development of skull base structures may also cause confusion. examples are the cochlear cleft, not to be confused with otosclerosis, or arrested pneumatisation of the sphenoid sinus, possibly mimicking a tumoural lesion in the central skull base. to avoid problems, one should keep in mind the existence of such variants, while correlating the imaging findings with the clinical problem; in some cases, an additional imaging study may be needed to exclude a pathological process more confidently. this short -min introduction by the session chairman will give an overview of the current state of cardiac computed tomography (ct) for diagnosis and prognosis. in addition, the introduction will provide the basis for the subsequent dedicated lectures covering low radiation dose, diagnostic accuracy, and incidental findings in cardiac ct. what are the protocols with the lowest radiation dose in clinical practice? j.-f. paul; le plessis robinson/fr (pauljf@ccml.fr) the radiation dose delivered for coronary ct angiography using retrospective gating is high, because only a small part of the total radiation delivered is used for the reconstruction of the image in the retrospective mode. on average, % of the radiation burden is used to reconstruct one phase of the cardiac cycle. ecg-triggered tube current modulation allows reducing the nominal dose by up to % in the systolic phase. another approach for reducing radiation exposure is to use prospective, sequential acquisition, with this approach being associated with % dose reduction compared to the spiral retrospective acquisition. exposure time may be also shortened by an increase of pitch, in particular on the dual-source ct. however, applicability to each dose-sparing technique depends on the heart rate and its regularity. dose-sparing strategies should be modified in case of high or irregular heart rate and radiation dose reduction may be less effective. for coronary ct angiography, individually weight-adapted protocols have been successfully applied, by adjusting mas to patient's weight. more recently, settings of kv and even kv have been successfully used for cardiac studies, especially in slim patients or children. reduction of exposure time and individual adaptation have an additive effect on dose reduction: it has thus been possible to scan down under msv in selected, slim patients, using this combined approach. in many cases, using the latest technology, radiation dose may be lower than the mean radiation dose associated with conventional angiography. learning objectives: . to understand various protocols to lower radiation dose in cardiac ct with regard to the clinical situation and the risk of image quality impairment. . to learn how to tailor the radiation dose level to each patient in terms of morphology. . to become familiar with modulating exposure windows in regards to a patient's ecg. how accurate and prognostically valid is coronary ct angiography? f. cademartiri, e. maffei; monastier di treviso/it (filippocademartiri@gmail.com) the role of cardiac computed tomography in cardiovascular diseases is becoming increasingly important. cct is a robust and reliable investigation for the detection and exclusion of significant coronary artery disease. in addition, cct provides reliable noninvasive information concerning coronary plaque burden (severity, distribution, type), morphology and function of left and right ventricle, valves, aorta, and so forth. in the past years, the prognostic role of cct has been established. the prognostic information that can be extrapolated from cct is very diverse and most of it is still under investigation. there are converging evidences that cct can stratify cardiovascular risk better than conventional clinical methods, especially in symptomatic patients. for asymptomatic individuals, the role is still controversial since coronary calcium score alone has a very important role already. several aspects of cct information seem to have importance for prognostic stratification: plaque presence, obstruction, plaque type and distribution. more recent studies have started showing the relationship between cct and pharmacological treatment. this is a very new and appealing topic, since the evidence is towards the fact that the most effective pharmacological treatment (e.g. statin) should be restricted to patients with at least some nonobstructive cad as detected by cct. this might become a revolutionary concept in cardiovascular medicine with several implications. learning objectives: . to learn about and integrate the prognostic information from coronary ct angiography into conventional methods. . to learn about coronary ct angiography and conventional methods. . to appreciate the prognostic impact of coronary ct angiography and conventional methods. incidental findings in cardiac ct: how to report and proceed s. leschka; st. gallen/ch (sebastian.leschka@kssg.ch) rapid advances in computed tomographic (ct) technology have facilitated the widespread use of ct for cardiac imaging worldwide, and can be considered nowadays an essential part of the clinical workup of patients with suspected coronary artery disease. albeit that the performed ct study is focussed on the cardiac structures and the coronary arteries, adjacent noncardiac structures are in the field of view whenever a patient undergoes cardiac ct to quantify coronary calcium or to perform a noninvasive coronary angiogram. in addition, the location of the heart near other anatomic regions, including the lungs, mediastinum, upper abdomen, and bones, necessitate that these structures are included when acquiring coronary ct angiographic images. several publications have demonstrated a high frequency of incidental findings on coronary ct angiography. the availability of this information is part of an ongoing clinical debate. while some radiologists believe that every image should be interpreted completely, others believe that examining noncardiac structures reveal too many incidental findings of uncertain clinical significance, which may harm because additional testing increases risk, cost, and patient anxiety. the aim of this lecture is to separate solid facts from opinions and beliefs. the diagnosis of pulmonary embolism (pe) is usually established by a combination of clinical assessment, d-dimer testing, and imaging with either pulmonary ventilation-perfusion (v/q) scintigraphy or pulmonary multidetector ct angiography (ctpa). v/q scintigraphy is a functional imaging method using isotopes for the visualisation of ventilation and perfusion distribution in the lungs. in recent years, the imaging techniques for diagnosing pe have improved. many nuclear medicine centres have adopted the single photon emission computed tomography (spect) technique as opposed to the planar technique when diagnosing pe. the introduction of -dimensional v/q spect technology instead of -dimensional planar v/q scintigraphy has resulted in fewer indeterminate results and a higher diagnostic value. the latest improvement is the addition and combination of a low-dose ct without contrast to the v/p spect (spect/ct) technique. the spect/ct in combination has been compared to ctpa, planar scintigraphy and spect alone, and one study has shown that spect/ct in combination had the highest diagnostic accuracy for pe diagnosis. spect in combination with low-dose ct without contrast enhancement is superior, especially for small subsegmental emboli; however, consensus is lacking regarding the clinical impact and treatment. spect and spect/ct may provide alternative diagnosis if pe is refuted and are feasible in almost all patients, because there are no definitive contraindications. in the present lecture, spect and spect in combination with low-dose ct are discussed in the context of diagnosing pe. over the last few years, emergency radiology (er) has been a subspeciality with growing interest. because of rapid developments in technology, ct has become the most useful tool in evaluating trauma and other emergency patients. in this lecture, the basics of planning and organising an er department are presented and discussed. besides optimising technical equipment and protocols for imaging, different logistic concepts have to be considered in planning and organising er departments. first of all, logistic concepts have to be considered for obtaining an optimal workflow: the radiology department has to be in close proximity to the emergency department and the admitting area, in particular. the whole workflow must be optimised for speed and accuracy. this also mandates having dedicated and standardised examination and viewing protocols for ct. in contrast to the usa where dedicated emergency radiology departments are well established, nonspecialised radiologists still frequently do the reading of emergency radiology cases in european countries. the radiologic staff involved has to be trained for interpretation of trauma and other emergent cases. this does not only account for residents, but also for consultants and attending radiologists. since a large number of cases will arrive during after hours and on weekends, staffing has to be adjusted to this fact, which includes attending radiologists to be available during these hours on call or, preferably, on-site. this lecture will give an overview of logistic concepts and organisation of an emergency department and will also discuss critical issues in polytrauma imaging. at least during the radiological training in hospitals, every radiologist will personally be confronted with the treatment of polytraumatised patients. it is a fact that standardised whole body mdct (wb-ct) as an independent predictor is able to save around % of patient lives. thus, wb-ct is the major diagnostic tool and should be performed as fast as possible. some institutions bypass the emergency room and directly start patient stabilisation in the ct cabinet. however, radiographs and focused ultrasound remain important in general settings as long as they are performed in a way which does not delay ct diagnostics. after scanning, specially trained radiologists support the whole interdisciplinary team with correct, appropriate and prioritised diagnoses. besides optimisation of parameters like logistics, patient positioning preferably 'feet first', reading and communication, 'choosing the right protocol' is a crucial factor for ideal radiological patient care. four types of protocols should be differentiated: there is a wide agreement that for patients with polytrauma, integrating whole body computed tomography (ct) scan into early trauma care significantly increases the probability of survival. therefore, ct is today considered the most important imaging technique in the diagnostic workup of polytrauma patients. the downside of whole body ct represents the relatively high radiation exposure. to overcome this handicap, the following measures can be taken: . positioning of the arms during whole body ct above the head results in reduction of radiation dose; . adaption of scan parameters, especially reduction of tube voltage to kv reduces radiation dose; . installation of recent technical improvements like iterative reconstruction algorithm reduces noise in the image, allowing a downward adjustment in radiation dose to obtain standard diagnostic quality images; . installation of dose control software enables optimisation and improved dose management over time. another disadvantage of high-resolution whole body ct is the amount of acquired data. to optimise the workflow in the evaluation of a couple of thousand submillimetre images it is necessary to connect the mdct scanner with a dedicated workstation for volume image reading, which offers the possibility of quick multiplanar online evaluation. in this introduction i will define the connectome (a comprehensive map of neural connections in the brain) and related concepts such as scaling, structural and functional connectivity as well as the integration-segregation paradigm. the development of mri-based techniques such as white matter tractography and segmentation of white and gray matter has played a crucial role in the emergence of connectomics by providing tools to map in vivo the entire human structural connectivity at a macroscopic scale. i will explain these methods and how from mr imaging a human connectome can be mapped and represented as a network (set of nodes and edges). finally, i will also review the validation work related to those techniques and mention the foreseen technical advancements in the field. session objectives: . to learn what the human connectome is and how it is affected by brain disease. . to understand how the human connectome can be imaged and characterised with mri. . to become familiar with the network formalism and its interpretation. . to understand the relationship between brain function and underlying structural connectivity. . to understand the conceptual ideas behind connectomics. . to learn about the general workflow, from diffusion imaging to mapping a structural brain network. . to become familiar with the robustness of the technique by reviewing validation studies and getting a glimpse of the technical challenges. the economics of brain networks e. bullmore; cambridge/ uk (etb @cam.ac.uk) we review the methods and recent results of network analysis of human neuroimaging data. human functional mri and structural mri data can be analysed using mathematical tools drawn from graph theory to quantify the complex (non-random) topological properties of brain networks. these results can be substantiated by meta-analysis of prior neuroimaging results and analysis of non-human nervous systems. brain networks consistently express complex topological features, such as small-worldness, hubs, modules and rich clubs. some of these features entail disproportionate biological cost, but may be "worth it" by supporting integrative information processing and adaptive behaviours. highly connected hub nodes are high cost/high value network components that likely also represent special points of vulnerability for diverse brain disorders. these economical principles of brain network organisation are expressed at the microscopic scale, e.g., in the nervous system of the nematode worm c. elegans, and may therefore provide a new axis for translation between macro and micro systems neuroscience. neuroimaging can be used to elucidate the economical principles of human brain network organisation in health and disease. connectomics in brain pathology m.p. van den heuvel; utrecht/nl (m.p.vandenheuvel@umcutrecht.nl) healthy brain function depends on efficient functional communication within a complex network of structural neural connections, a network known as the connectome. conversely, damage to the brain's network, disrupting local neuronal processes and/or global communication between remote functional systems may lead to brain dysfunction. in the last few years, emerging evidence from a wide variety of studies suggests that connectome abnormalities may indeed play an important role in the aetiology of several brain disorders. in my talk, i will discuss the results of recent studies suggesting an important role for affected connectome organisation in a number of neurological and psychiatric disorders. in particular, i will highlight the findings of affected functional and structural brain network in neurodegenerative disorders such as alzheimer's and als, as well as discuss how the application of network science and connectomics may aid our understanding of the biological basis of psychiatric disorders such as autism and schizophrenia. learning objectives: . to understand the role of connectome architecture in (cognitive) brain function and dysfunction using diffusion mri/functional mri. . to become familiar with connectomics as a tool for examining disease pathology in a wide range of neurological and psychiatric brain disorders. linking structure and function: the role of modeling in understanding the pathophysiology the interplay of the brain's intrinsic activity and the external world has seen a revival in the last decade, especially in neuroimaging. a long-held assumption in many of these studies has been that ongoing brain activity is sufficiently random that it averages out in statistical analysis. hence, imaging studies are termed 'activation' paradigms, where experimental manipulation results in the activation of cerebral circuits that are necessary for performing the task. nevertheless, a large amount of recent literature reports a body of observations that there are consistently distributed patterns of activity during rest. this fact has led to the suggestion that it might be possible to characterise network dynamics without needing an explicit task to drive brain activity. indeed, numerous neuroimaging experiments have evidenced the solid existence of spontaneous long-range correlations, i.e. functional connectivity (fc), by fmri, meg, and eeg techniques. the functional connectivity is defined as the statistical dependence between remote neurophysiological dynamics. the emergence of resting functional connectivity is intrinsically linked with the underlying anatomical connections between those areas, i.e. the structural connectivity. whole brain modeling can indeed establish a direct link between structure and function. furthermore, the modeling can shed light on the origin of pathological functional disfunction by making the link with the underlying anatomy explicit. we will show how, structural dti tractography, functional imaging and modelling studies can be combined for reaching this goal. traumas to the paediatric pelvis and hip include traumatic dislocations of the hip, fractures of the femoral neck, fractures of the pelvic ring, acetabular fractures and apophyseal avulsion fractures. traumatic dislocations of the hip, fractures of the femoral neck, fractures of the pelvic ring and acetabular fractures are rare in children (less than % of paediatric fractures), as compared to adults. these fractures are commonly the result of high-energy trauma. imaging is based on plain radiographs, but ct and mri are very useful to precisely assess bone (ct) and cartilage and soft tissue (mri) lesions. imaging enables accurate diagnosis, appropriate treatment and detection of potential complications (femoral head osteonecrosis, premature physeal closure …). in contrast with the previous injuries, apophyseal avulsion fractures of the hip and pelvis are common in children and adolescents, usually associated with athletic activities. in most cases, these fractures are of good prognosis and can be treated conservatively when minimally displaced. plain radiographs confirm avulsion injuries to ossified apophyses, but mri and ultrasound are the modalities of choice to demonstrate injuries to nonossified apophyses and to assess apophyseal displacement. the elbow is a very common site for fractures in a child. the challenge for the radiologist is to differentiate normal variants of growth from possible injury and this is usually achieved by having good-quality radiographs and an understanding of normal growth. it is important to recognise those fractures which require surgical intra-operative treatment. in a small number of cases when assessing for vascular integrity, intra-articular extent and injury to cartilaginous structures, ultrasound and mr imaging have a valuable role. this lecture will give an overview of the radiographic appearance of fractures, highlight the features which require orthopaedic intervention and illustrate the use of additional imaging modalities. learning objectives: . to become familiar with the types of injuries seen in the paediatric elbow. . to understand the strengths and weaknesses of different imaging modalities. a- : the diagnosis of c-spine injury is more complex in children than in adults. early diagnosis is crucial since delayed diagnosis results in high morbidity and mortality. leading mechanisms of pediatric c-spine trauma are motor vehicle accidents, sports and pedestrian injuries. due to their anatomy, children are prone to different types and locations of injuries. children < yeas of age are more likely to injure the spinal cord itself and are prone to dislocations and high (c -c ) bony injuries. children > years of age more often sustain c-spine fractures. in children under the age of years, radiographs are rarely helpful. ap and lateral radiographs are helpful in children aged between and years. in children aged > years, additional lateral and odontoid views are obtained. anatomical variants such as pseudosubluxation of c -c , widening of the atlantodental interval and ossification centres may appear to be of concern on imaging, but are normal. abnormal radiographic findings require additional imaging to differentiate them further with ct on the area of concern. mri is mandatory if signs of atlantorotary subluxation and spinal cord injury without radiologic abnormality (sciwora) are present. mri identifies injuries to the spinal cord that are not apparent with other modalities, and should be used when a child presents with a neurologic deficit but normal radiographs on ct scan. nowadays, ultrasound (us) has become the first choice for performing most breast biopsies. the main advantages of this technique are non-ionising radiation, full control of the needle position in real time and widespread use of us equipments at all centres. us allows access to difficult places (such as the axilla or near the nipple), multiple lesions can be safely biopsied in one single session, the breast is not compressed, there is excellent comfort for patients and radiologists, local anaesthesia and haematoma do not hide the lesion and it is a cost-effective technique. however, the main limitation is that the lesion must be visible on us. us-guided fine-needle cytology was widely used in the past, but today its use has decreased. us-guided core-needle biopsy has proven to be a reliable technique to perform a biopsy for breast lesions, showing a sensitivity value of about %. furthermore, it can be safely used for performing biopsies of axillary lymph nodes. vacuum-assisted devices can be used not only for diagnostic purposes, but also for therapeutic ones, because small palpable benign lesions (such as fibroadenomas), papillomas and radial scars can be completely removed. stereotactic breast biopsy is the performance of tissue sampling under mammographic guidance. as with any imaging technique, some lesions are only seen with mammography. when these lesions are suspicious (e.g. birads or higher), there are no imaging techniques that can downgrade this suspiciousness directly, although for birads lesions follow-up might be a viable alternative. in mammography, most lesions that are only visible on the mammogram consist of clusters of microcalcifications. these harbour a likelihood of around % being malignant and should thus be classified as birads . this implies that tissue sampling is mandatory. different from ultrasound-guided biopsy, it is not possible to perform a real-time biopsy under mammographic guidance. rather, stereotactic biopsy uses triangulation to assess the depth of a lesion within the breast, while the lesion location in the xand y-plane is assessed on a scout view. the needle is positioned using the coordinates thus obtained. since there is no real-time feedback of the accuracy of needle positioning, it is necessary to obtain substantially more tissue than under ultrasound guidance. therefore, vacuum-assisted systems are essential. in case of calcified lesions, the biopsy result is controlled by x-ray. calcifications should be present in the sample. biopsy complications that occur under stereotactic guidance are usually limited. most common is the formation of large haematomas. scarring may also occur. breast magnetic resonance (mr) imaging is the most sensitive modality available to evaluate the breast for cancer. it can detect lesions that are occult at mammography and ultrasound. it has a limited sensitivity and the positive predictive value ranges between and %. histopathological assessment of mr-detected lesions is therefore mandatory. some of these lesions will be visible at second-look ultrasound and can be biopsied sonographically. a significant number however will only be discernable on mr, and mr-guided biopsy is therefore the sampling technique of choice. mr biopsy has become an essential component of any breast imaging practice. several mr-biopsy techniques are available including the grid-localising, pillar and post and the freehand techniques. the technique for preparing and performing a mr-guided vacuum-assisted biopsy using the grid-localising technique is reviewed. potential complications, limitations of mr breast biopsy and actions to prevent failure are discussed. imaging-histologic correlation is essential to ensure accurate sampling. clip placement and follow-up imaging should be performed to ensure sampling, including ultrasound correlates. finally, new developments in mr-guided therapeutic interventions are discussed. abdominal injuries require a timely and reliable diagnosis to prevent potentially lethal outcomes. the armoury of clinical tools (physical examination, lab tests) does not fulfil these criteria, since they are either not fast or not reliable. imaging diagnostic modalities help the clinician to acquire the necessary amount of information to initiate focused and effective treatment. however, the selection of the appropriate imaging algorithm, modality and technique, as well as the precise detection and interpretation of essential imaging findings are frequently challenging, especially because the circumstances, under which these examinations are performed (open wounds, bandages, non-removable life-supporting equipment, lack of patient cooperation, etc)., are frequently less than optimal. knowledge of critical imaging signs, symptoms and the role they play in the evaluation of the patient's condition, as well as fast decision-making and ability to closely cooperate with the clinicians are skills of key importance for radiologist members of the trauma team. this presentation will describe the technique and important findings for ct stone studies as well as the accuracy of this study. an explanation of when iv contrast should be given to augment a ct stone study and findings of gu and non-gu diseases that can mimic symptoms of stone disease will be provided. examples and descriptions of acute mesenteric vascular abnormalities, bowel obstruction and infectious conditions will be included. ways to differentiate and categorise emphysematous infections for proper treatment will be described. lower tract emergencies will also be illustrated and described. a systematic approach to evaluating abdominal cts will be described to avoid mistakes. using cases and an audience response system, this segment of the course will go over the optimal imaging approach for patients presenting with acute abdominal pain and abdominal injuries. ct findings will be emphasised. key imaging findings of traumatic and nontraumatic causes of acute abdominal pain including gastrointestinal tract and urinary tract pathology will be explained. a systematic approach for the imaging evaluation of patients with abdominal emergencies will be illustrated and explained including proper scan protocols and analysis of imaging findings. imaging diagnosis of blunt and penetrating abdominal injuries, urinary tract obstruction, infection, bowel obstruction, and ischemia will be emphasised. transcatheter renal denervation represents a novel therapy for treating patients with treatment-resistant hypertension, leading to higher risk of major cardiovascular events. an overview of sympathetic nervous system anatomy, physiology and physiopathology will be followed by a description of the technical aspects of renal denervation. first, results in patients with therapyresistant hypertension look very promising. however, these data need to be confirmed. importantly, long-term efficacy and safety need to be assessed. as a consequence, the treatment cannot be considered, at the moment, standard therapy. future studies need to address other disease conditions characterised by sympathetic hyperactivity, including heart failure, chronic kidney failure and others. it is a general opinion that in the near future the treatment will also be applied to lesser severe hypertensive patients. future studies should also include formal cost-effectiveness analyses. renal denervation can be achieved by a number of different techniques that include radiofrequency ablation, intraluminal and high-intensity focussed ultrasound ablation, chemical ablation and radiation ablation. radiofrequency and ultrasound ablation are currently the most commonly used techniques. most of the evidence to date is derived from studies and trials based on devices that use radiofrequency ablation technology. there are a number of currently ce-marked devices available and each device differs in the catheter design, generator design and energy delivery. these device types include single-point ablation, multi-point ablation, balloon-mounted systems and irrigated technology. the indications for renal artery denervation have been formulated by various international and european societies based on the various clinical trials and studies. these indications include patients with true resistant hypertension, age - years, egfr > ml/min/ . m (mdrd formula), single renal arteries with diameter > - mm with a landing zone of - mm depending on the device, no significant renal artery stenosis > %, no renal artery aneurysm, no previous renal artery angioplasty or stenting and the absence of significant valvular heart disease where lowering blood pressure will be dangerous. renal denervation of the sympatic nerves around the renal arteries is a new catheter technique to treat patients with resistant hypertension. resistant hypertension is seen in about %- % of all patients with hypertension. the mechanism of sympatic nerve driven resistant hypertension will be explained. the available techniques for rdn will be discussed, focusing on the pros and cons of each technique. currently, rf ablation, hifu and cryo-ablation are the techniques under investigation. the technique and physics of renal denervation with radiofrequency ablation will be explained in more detail, focusing on available devices in the market. patient selection and nonresponders will be discussed. the current evidence from the available trials will be reviewed. the future of rdn and the role of radiology and the radiologist will be illuminated. the adrenal glands are composite endocrine organs consisting of the steroid hormone-producing cortex and the catecholamine-synthesising medulla. the increased use of imaging modalities has demonstrated the presence of varying sized mass lesions in up to % of individuals subjected to ct studies for reasons unrelated to adrenal dysfunction. most of these incidentally discovered lesions are non-functioning benign lesions of cortical origin. however, incidentalomas may also represent functioning lesions and malignant masses. clinical diagnostic and biochemical evaluation is used to further subdivide functional and non-functional adrenal lesions. f-dopa has been found to be of high sensitivity and specificity in pet imaging of pheochromocytoma. ct and mr imaging are first choice in characterisation of adrenal lesions. techniques of dual energy ct and histogram analysis may offer additional information. pet-ct has been shown to contribute to the diagnostic power, especially in oncologic patients. knowledge of the physiologic appearance of adrenal glands in -fdg pet is necessary to correctly identify pathologic processes. fdg-pet also has the ability to detect metastatic lesions in non-enlarged adrenal glands. in addition, fdg-pet has the advantage of simultaneously detecting metastases at other sites. the role of mr-dwi and mr-spectroscopy in characterising adrenal masses has to be defined by further studies. differentiating benign from malignant adrenal masses using non-invasive imaging methods can reduce the need for percutaneous adrenal biopsy in patients with underlying malignant disease and the follow-up imaging of incidentally detected adrenal adenomas. renal cell carcinoma (rcc) is the most common malignant tumour of the kidney. as response rates to radiation and nonantiangiogenic chemotherapy are low, surgical excision, i.e. radical nephrectomy has been the treatment of choice. however novel treatment options have emerged, so that imaging of rcc is of increasing interest. nephron sparing partial nephrectomy has become an accepted operative option and antiangiogenic agents such as inhibitors of tyrosine kinase (tk) and mammalian target of rapamycin (mtor) have been approved for treatment of advanced rcc. thus, it has become important to perform correct local staging and identify patients suitable for partial nephrectomy using standardised scoring systems such as renal or padua. furthermore, optimal imaging strategies for monitoring of advanced and metastatic rcc are discussed, as current antiangiogenic therapy evaluation in clinical routine is based only on morphological imaging information, but changes in tumour size may lag behind functional changes. finally, common side effects, i.e. pneumonitis, of vascular disruptive agents are addressed. characterisation of renal masses can be performed using three categories with respect to the lesion size and gross architecture: the indeterminate very small masses, the cystic, and solid renal masses. lesions with diameters below mm are usually difficult to classify due to partial volume effect that prevents accurate ct attenuation measurement. in the general population, these lesions are likely to be microcysts and do not require further workup. better characterisation remains needed in selected patient populations such as patients with hereditary renal tumour disease and previous history of renal carcinoma. in this case, mri combining t w, diffusion-weighted imaging and dynamic contrast enhanced sequences or contrast-enhanced us may help differentiate very small cysts from solid neoplasms. cystic renal mass characterisation still relies on bosniak's classification with categories: benign (i) and minimally complicated (ii) cysts, indeterminate cystic lesions (iif and iii) and malignant cystic masses (iv). some cystic masses remain unclassified at ct because of atypical attenuation characteristics or enhancement properties. us, contrast-enhanced us, and mri are now playing a key role by providing additional diagnostic information that help distinguish between atypical fluid filled masses and atypical solid neoplasms, especially poorly enhancing solid papillary rcc. the characterisation of small solid renal tumours starts at ct with the identification of macroscopic fat, a typical feature of angiomyolipoma. in the case of non-fatty indeterminate renal neoploasms, percutaneous-guided biopsy can be performed when accurate characterisation is needed before surgery or when renal metastases or lymphoma are suspected. drug development being a complex and costly process, there is an increasing need for imaging biomarkers to take go/no go decisions in the early clinical phases. the recist criteria based on tumour size measurements at ct are currently used for this purpose. however, additional functional and molecular biomarkers have been developed to assess the early biological effect of drugs on tumours. development of imaging biomarkers is a structured process in which new biomarkers are discovered, validated and qualified against biological processes and clinical end points. the validation not only concerns the determination of the sensitivity and specificity, but also the measurement of reproducibility. reproducibility assessments, standardisation of the acquisition and data analysis methods and quality control are crucial when imaging biomarkers are used in multi-centre trials. functional and molecular parameters obtained at perfusion imaging, diffusion-weighted mr imaging and pet are being developed and validated. the perspectives (earlier assessment of response to treatment) and limitations (limited validation and standardisation) of these imaging biomarkers in cancer drug development will be presented. more recently, pharmacodynamic imaging biomarkers such as fdg-suv and k trans have been introduced. drug developers are reluctant to use more exploratory unvalidated imaging biomarkers (i.e. cannot distinguish a true negative from a false negative). the extensive literature on biomarker validation mostly refers to biochemical biomarkers extracted as analytes from biospecimens and is unhelpful to radiologists. unlike biospecimen biomarkers, the quality and validity of imaging measurements as biomarkers depend crucially on the use of a diagnostic imaging device, in the presence of the patient, in a manner for which the device (a) was not designed, (b) has not received regulatory approval and (c) may be unfamiliar to the user in the trial site. technical validation and biological validation are orthogonal activities. ''technical validity'' is confidence that the imaging biomarker can be measured reliably anywhere in the world. ''biological validity'' is confidence that the biomarker correctly reports some underlying biology that is important to the patient's future clinical outcome. since the physician always has access to the patient's clinical status and history, the biomarker is only useful if it provides a better forecast than clinical data alone. (if the forecast is near perfect, the biomarker might be a surrogate end point). technical and biological validations are massive undertakings best achieved by consortia, and in particular publicprivate partnerships, of which the innovative medicines initiative in europe and the biomarkers the standard imaging assessment of tumour response relies on size measurements, which, with predominantly cytostatic targeted agents, may not reflect the drug effect. functional imaging biomarkers have the potential to quantify the biological characteristics of tumours and measure on-target and off-target effects that indicate early likelihood of response to a specific therapy, which can then be used to guide the optimal biological dose and drug schedule. serial, non-invasive assessments of whole tumour are possible. this is particularly important in the context of inter and intra-patient tumour heterogeneity, as different parts of the tumour and primary vs metastatic lesions may be biologically different and these characteristics may change with treatment. however, functional imaging end points suffer from variability, which can be very significant in a multicentre setting. strict quality assurance and quality control measures need to be implemented at the start of a trial and the variability across centres documented. data acquisition protocols need to take account of equipment variations. data analysis methodology needs standardisation of software, central review and preferably double reading of scans. automation may not always prove the most robust and reliable option. this presentation will focus on the factors that are crucial in determining the compatibility of data in multicentre trials with functional imaging end points. learning objectives: . to learn about the potential role of quantitative imaging in processes related to tumour growth such as cell metabolism, cell death, and vascular function in the assessment of tumour response. . to become familiar with the issues of accuracy, reproducibility and standardisation for using functional imaging biomarkers in drug development. quantitative nuclear medicine in drug development w. weber; new york, ny/us nuclear medicine techniques can detect and quantify very low concentrations of radiolabelled pharmaceuticals in the human body. this allows investigators to use nuclear imaging for various purposes during drug development. nuclear imaging can visualise drug targets that are only present in nanomolar concentrations and can thus identify patients most likely for therapies directed against these targets. a recent example is folate receptor spect imaging for the selection of patients for treatment with a folate-targeted drug conjugate. in addition, nuclear imaging and specifically pet can measure the concentration of pharmaceuticals within tumour and normal organs over time. nuclear imaging can also be used to monitor target inhibition, for example the blocking of oestrogen receptors by anti-oestrogens. finally, nuclear imaging can assess tumour response to therapy by measuring changes in tumour metabolism or proliferation, e.g. with fdg-and flt pet/ct. this allows an earlier and more sensitive detection of tumour response than morphologic techniques. since pet is a whole body imaging technology, pet imaging can also be used to study the heterogeneity of target expression, tissue pharmacokinetics, target inhibition and response. pet imaging is now widely available in many countries and has become internationally standardised. it is therefore a robust clinical technique that will increasingly be used during drug development. there are about . patients per year in europe who lose their leg. about % are due to diabetic ischaemic and ulcerative leg problems. in diabetic patients the prevalence of foot ulcerations is about % and the risk for developing foot ulcerations is increased four times. diabetic foot problems may be due to neuropathy and malperfusion or a combination of both. the neuropathic ulcer and the neuroischaemic ulcer are usually at the plantar pedis, have a punched-out appearance and are painless. bone deformities (charcot foot) may be associated. the arterial ulcer is usually at the toe, forefoot and ankle, with pale, cold skin and it may be painful. patients need a multimodality approach involving a diabetologist, vascular specialist (angiologist, vascular surgeon, interventional radiologist) and podiatrist. pain control, antibiotic and antithrombotic treatment and the treatment of cardiovascular risk factors and other co-morbid disease have to be done first. in case of ischaemic pain and ulceration imaging such as mra and cta is the next step. patients should be referred to a team of vascular specialist early in the course of their disease to plan for revascularisation options. revascularisation is the optimal treatment for patients with an ischaemic and neuroischaemic diabetic foot. a. pathophysiology of the diabetic foot v. bérczi; budapest/hu (berczi@hotmail.com) the incidence of diabetes mellitus, associated with both predisposing genetic and environmental factors, is increasing globally. several major clinical trials have proved that complications may occur many years following proper glycaemic control. besides peripheral arterial disease, sensory and motor neuropathy along with an altered response to infection is of crucial importance. recent studies have showed that microvascular occlusive arterial disease is not a major factor affecting the diabetic foot; infrapopliteal macrovascular disease and microvascular dysfunction (e.g. arteriovenous shunting, precapillary sphincter malfunction, capillary leakage, venous pooling) are major components of impaired perfusion of diabetic foot. there are no randomised controlled trials analysing the major outcome following endovacular or open bypass surgery. the major outcomes, however, were similar in the case series: -year limb salvage showed a median of % (interquartile range - %) and % (interquartile range . - . %) following open surgery and endovascular treatment, respectively. limb salvage rate, however, was considerably higher with either type of revascularisation compared to medical therapy. negative pressure wound therapy, hyperbaric oxygen therapy, effective off-loading or nonweight-bearing therapies (total contact casts rather than removable devices) have also shown promising results in recent publications. endovascular arterial revascularisation is today a solid option in the management of cli with low complication rates and limb salvage rates comparable with surgery. the restoration of adequate blood flow to the foot is crucial to facilitate wound healing, provide pain relief, and avoid whatever amputations. the angiosome concept was first introduced in by taylor and further developed by attinger for planning treatment of ischaemic lesions of the foot. they divided the foot into six distinct angiosomes, arising from tibial and peroneal arteries. planning the procedure on the basis of this concept will yield the best local results of wound healing, compared with the indirect intervention. a proper pre-procedure assessment through colour doppler us and dsa of lower limbs is mandatory for guiding the procedure through the vessels of the foot. several studies have evaluated the efficacy of pta in the btk and the reliability of the angiosome model, approaching % with a limb salvage rate of up to % at months. on the basis of these data, we can conclude that pta in diabetic patients with btk disease is a safe and effective technique. the first endovascular treatment option is related to the angiosome model, but when not feasible the indirect technique is also a valid and similarly effective procedure. to face technical failures, up to % in crural chronic total occlusion a decade ago, different approaches and dedicated devices and technologies have been developed in the last few years. furthermore, percutaneous revascularisations are gaining more interest, particularly in patients with critical limb ischaemia not only as first-line treatment, but also as the only possible treatment for complex lesions and high-risk patients. special techniques for crural endovascular revascularisations are by design techniques rarely used in routine practice, but could offer wide possibilities for the interventional radiologist to solve challenging situations and manage complex lesions. these techniques include options for arterial access (trans-popliteal, trans-tibial, trans-pedal), approaches for crossing chronic total occlusion (assisted endoluminal, lambda technique, re-entry technologies) and uncommon routes for angioplasty/recanalisation (trans-metatarsal loop technique, trans-collateral techniques). combined antegrade and retrograde approaches have also been developed as well as related methods for successful re-entry. deciding when the patient/lesion is a potential candidate for special techniques is also a crucial issue. the interventional radiologist should be able to convert at any time if necessary the strategy of treatment to another one. he should also be able to select the most appropriate technique for the patient in planning revascularisation. continuous medical education and training is however mandatory and for some techniques the learning curve is relatively long. pathology of the eye and orbit is rare in the radiologist's practice. however, differential diagnosis is not too difficult if the compartment model is applied. in the orbit, different anatomical structures like the optic nerve as part of the cns, muscles for moving the globe, vascular structures and glandular tissue are present in a very small space. each anatomical structure is found in a special compartment; each compartment may give rise to a different group of pathologies, and only to these pathologies: glioma in the optic nerve, rhabdomyosarcoma in the eye muscles, varix in the venous vessels (intraconal compartment) and pleomorphic adenoma in the lachrymal gland. understanding the compartments in the orbit is therefore the key to differentiating different pathological entities. this presentation explains the orbital anatomy, how the compartments are differentiated, and what changes in anatomy treatment may induce. orbital congenital lesions are uncommon. they can be diagnosed prenatally, at birth or later during childhood. several orbital components can be involved. we will focus on congenital globe lesions (such as staphyloma, coloboma, persistent hyperplastic primary vitreous, coats disease), the developmental cysts such as epidermoids and dermoids, and vascular malformations such as lymphangioma and vascular tumours, e.g. capillary haemangioma. orbital inflammatory and infectious lesions are on the other hand common. orbital pseudotumour can involve any area of orbit, being one of the great mimickers in the orbit. if located in the orbital apex and/or cavernous sinus, it will be called tolosa-hunt syndrome. other inflammatory processes in the orbit are sarcoidosis and wegener and sjogren syndrome. among infections the most common is the orbital cellulitis, mostly secondary to a sinusitis and frequent in the urgency setting. the role of the radiologist is to assess whether it is preseptal or already postseptal or complicated by a subperiosteal phlegmon or abscess. also of importance is to know and evaluate the possible intracranial complications. finally, we will focus on inflammatory and infectious lesions of some specific regions such as the globe and lachrymal gland and give some clues for their differentiation. as more than pathologies can be seen in the orbit, a systematic approach is very important to come to the right diagnosis. the main and most helpful criteria of differential diagnosis of any orbital pathology is the definition of the affected orbital compartment, as some tumours may only or preferentially involve specific orbital structures. the criteria of the most frequent masses of the globe, malignant melanoma and retinoblastoma are presented as well as those of cavernoma and lymphoma, the main representatives of intraconal tumours. there are numerous extraconal neoplasms, only few arising from the nasal sinuses, and only a little number of tumours of the optic nerve. the presentation will include the most frequent as well as rare, but important tumours. the routine practice of oncologic imaging requires standardisation, which means that we need to harmonise technical protocols and agree on the meaning of selected words for the radiological report. the words "response, "progression" and "stable disease" are precisely defined according to internationally accepted thresholds and criteria. although the rules are quite simple and rather easy to apply, they are very efficient in the classification of the response to treatment, and therefore for the medical decisions. however, the role of the radiologist is not limited to measurements and calculation. the detection of new lesions may be challenging and requires experience. the differential between cancer progression and complications of the treatment might be very difficult and requires an adequate communication with the referring clinician. overall, most of the decisions taken by the clinician will be related to imaging results, stressing the importance of adequate protocols and reports. in solid as well as non-solid tumours, pet/ct imaging using -fluorodeoxyglucose (fdg) has demonstrated the ability to a) correctly stage disease, b) demonstrate therapy response and c) predict therapy outcome. fdg uptake can be measured objectively; however several factors in the standardisation processes of tracer application, image acquisition and post-processing are needed for reproducibility. the term standard uptake value (suv) measurement is used for compensating the influence of injected dose, decay time and body mass and represents fdg uptake in any selected pixel of the image. for therapy assessment, drop in fdg uptake represents tumour cell kill, notably a negative pet scan does not exclude viable tumour cells but overall has a better outcome. pet response criteria in solid tumours (percist . ) have been introduced to refine previously established pet response criteria by eortc. major changes concern the use of lean body mass-based suv (sul), sulpeak measurement in a fixed roi, use of only a single target lesion and normalization to liver uptake. metric measurements in ct component of the pet/ct as an intrinsic asset like in recist . have not yet been introduced, but might be crucial in the future. the proposed percist . criteria are not yet standard, since several limitations hamper its general use but may improve metabolic tumour response assessment. malignant gliomas (who grade iii and iv) are the most common primary tumours of the brain. according to consensus guidelines, the standard of care of these tumours includes maximal safe surgical resection followed by combined treatment with chemoradiotherapy. radiological assessment is critical in the follow-up and should be performed at four different times: . within hours after surgery, an early post-operative mri must be done to evaluate residual tumour and to be used as baseline for follow-up. . two to six weeks after completing radiotherapy, a new mri examination is recommended to evaluate the response to treatment. four possibilities are envisaged according to the rano criteria: complete response, partial response, stable disease or progressive disease. the evaluation of this first mri examination after rt is challenging and the pseudoprogression phenomenon can appear. advanced mr techniques may be of help in this respect. . additional follow-up should then be performed by mri every to months to rule out clinically silent progression. . in any case, an mri examination must be completed when there is a clinical suspicion of progressive disease. several options for treatment can be offered when progressive disease is detected, including antiangiogenic drugs. evaluation of response to these drugs is challenging too, and the pseudoresponse phenomenon can then appear. due its complexity, it is recommended that the management of malignant gliomas be performed in the context of multidisciplinary teams and that the radiologists are strongly involved in these teams. we are facing complex times with no parallel in human history. the worldwide economic crisis, in combination with a non-regulated process of globalisation, is pressuring countries to change their social and political model. these changes are affecting academic institutions and the health-care sector. higher education was always more internationally open than most sectors because of its immersion in knowledge, which was never worried about country juridical boundaries. it is well recognised that it was the knowledge created by universities research that contributed to the development of societies. we, as european citizens, must be able to transmit the message that having more knowledge and being capable of understanding better the world we live in will make us stronger, more successful and more competitive. european academic institutions must be prepared for this shift of paradigm and understand that they are no longer educating only for their country but for the globalised world. it is also important to bear in mind that developed health-care systems are suffering several pressures and a call for a new inter-professional collaboration concept is needed, based on each one's roles and responsibilities, towards a better and more efficient health-care delivery for the patients. in this session, the invited speakers will give us an overview about the challenges that academic institutions will face in the near future and the way they must adapt to become more attractive and at the same time guarantee that radiographers and radiologists are educated and trained according to the highest professional standards. session objectives: . to appreciate the challenges that academic institutions must face in order to educate graduates with the ability to work internationally and to actively contribute to the healthcare sector of the future. . to explore innovative approaches in education and assessment. . to highlight the importance of inter-professional collaboration in order to ensure that graduates are ready to meet the demands of the modern healthcare sector. challenges in undergraduate education l.j.o.c. lanca; lisbon/pt (luis.lanca@estesl.ipl.pt) radiographers operate a wide range of sophisticated equipment and perform a range of techniques in several radiological procedures. they are responsible for the radiation delivered to the patient while assuring safety and image quality at an acceptable level for an accurate diagnosis. education and training in radiography, in line with the constant technological developments, is a requirement to be fulfilled by higher education institutions (hei). this is of major importance in undergraduate education where radiographers learn to play an effective role as healthcare providers in planning, preparing and performing accurate and safe radiological procedures within the diagnostic or therapeutic field. the european qualifications framework (eqf) for radiography, in terms of the development of knowledge, skills and competences, provides a european standard for the development of radiography learning outcomes at eqf level . this constitutes an opportunity to develop, tune, advance and promote standards of radiographic practice, education and research throughout europe. the eqf provides guidance to increase the educational and professional recognition of radiography in europe. at a european level, the cooperation between hei in undergraduate radiography education would be an opportunity to provide discussions of mutual concern, explore new perspectives and views of different cultures, and also share learning and teaching methods. international cooperation is an opportunity to improve and provide the harmonisation of education regarding a professional group that plays an effective role in healthcare in their field of competence and expertise. there is a vast amount of evidence available from the published literature that higher education institutions continue to explore innovation in both teaching and assessment practices. this is especially true of health professional programmes as evidenced by the abundance of literature relating to medicine, nursing, radiography and other allied health professions. such higher education, health professional programmes would appear to be more innovative than many other disciplines often as a direct attempt to mirror the dynamic clinical environment in which most graduates will work. there are, however, challenges associated with any such educational innovations and novel approaches to delivery and assessment. these specific challenges must be carefully considered in parallel with the larger challenges facing higher education institutions, both nationally and internationally, along with the professions which they contribute to. through a case study approach, supported by published peer-reviewed literature, institutional reports and research, this presentation will consider international best practice in education along with the value of collaborative, inter-professional approaches to teaching and assessment. haemodialysis accesses have become the most commonly performed type of vascular surgical procedure. however, only % of all haemodialysis accesses remain patent at years. autogenous fistulas have a higher rate of primary failure compared to prosthetic grafts ( % vs. %), but the long-term patency of fistulas is superior to that of grafts. autogenous fistulas fail after a median of to years, whereas prosthetic arteriovenous grafts fail after a median lifetime of only to months. the pathogenic stenoses causing access failure occur in various locations, but the most common site in prosthetic arteriovenous grafts is at the anastomosis between the graft and outflow vein, as identified in % ( , ) to % of cases, or in the outflow vein itself in autogenous arteriovenous fistulas. clinical assessment alone will detect a large number of failing fistulas. by assessment of the thrill, pulse character, palpation of the fistula itself, excessive bleeding and difficulty needling a fistula may all indicate a failing fistula. if the venous pressures during haemodialysis exceed mm hg, fistula flow falls to< ml/min, graft flow decreases to< ml/min, or access blood flow falls by more than %. a fistulogram is recommended if monitoring or surveillance suggests that thrombosis is either imminent or has already occurred. duplex can be used to assess the fistula, but cannot visualise the central veins. mr and ct venography has also been used in many patients successfully. treatment of symptomatic stenosis of vascular access is essential as it can lead to thrombosis and loss of vascular access. but treatment can lead to restenosis. the treatment of stenosis remains a challenge which requires a good knowledge of different materials (guidewire, catheters, balloons and stents). though the pathophysiological mechanism of stenosis is different for native fistulas, grafts or restenosis, the treatment technique remains the same. the basic principle is the use of high inflation pressure balloon angioplasty. whatever the material used, the key is to have no residual stenosis greater than %. so far, no study has demonstrated the superiority of the "new" tools (cutting balloon, drug-eluting balloon, cryotherapy) regarding restenosis. except perhaps for anastomotic grafts stenosis, stents should be reserved to treat complications or failure of balloon angioplasty. the stents indications are residual elastic stenosis, wall obstructive damage, acute rupture during angioplasty, aneurysm or restenosis. complications of stenosis treatment are rare, but can lead to the loss of vascular access. the main complications are thrombosis and acute rupture. as with any treatment, there have been contraindications: infection, distal ischaemia, high flow, newly created or surgically revised access (< weeks). when dealing with a vascular stenosis access, never compromise on the future, but think about the surgical alternative. so, the management of stenosis vascular access must also be multidisciplinary. percutaneous treatment of a thrombosed dialysis access can be extremely challenging. the major concern does not stem from the fact that the procedure can be complexly long, but arises rather from the management of its complications, which can be serious particularly when the access is branched onto the brachial artery. access infection is an absolute contraindication. temporary contraindications include fluid overload and severe hyperkalaemia. percutaneous thrombectomy of grafts is very well standardised and predictable: size of mm, well palpable wall which is easy to needle, small average clot burden, and an underlining stenosis almost always found at the venous anastomosis. thrombectomy of avfs is subject to variations in approach and technical difficulties depending on their anatomical particularities. all techniques employed follow rules: first, the removal of thrombi and, second, dilation of the stenosis responsible for the thrombosis. heparin and antibiotics must be injected. the basic principle is to place in the arterialized vein or graft introducer sheaths in opposite directions to work on both the venous outflow and arterial inflow. thrombus lysis or removal can be achieved by mechanical, pharmacological and pharmaco-mechanical methods. all techniques may work in grafts given that the modest amount of thrombus ( . ml on average) can be simply pushed into the lungs. prosthetic grafts are thus easier to declot than native fistulas, but they are much more prone to early rethrombosis. thrombi located at the arteriovenous anastomosis form a firm and rubbery plug, frequently and notoriously resistant to thrombolysis. drug-induced lung disease is an increasingly common cause of morbidity and mortality. the diagnosis is based on clinical history and consistent radiologic findings. lung biopsy is performed in a small percentage of cases. highresolution ct may demonstrate different parenchymal patterns including diffuse alveolar damage, acute or chronic alveolar haemorrhage, nonspecific interstitial pneumonia (nsip), hypersensitivity pneumonitis, organising pneumonia, and eosinophilic pneumonia. the imaging and histologic manifestations are often nonspecific. a systematic approach to the radiological evaluation of drug-related lung diseases is essential and includes not only chest imaging pattern recognition, but also integration of available clinical information. in this interactive session, we will present and discuss several cases illustrating thoracic changes after instrumental procedures in the thorax such as surgery, radiofrequency ablation, interventional endoscopy, and intensive care. radiologists have an important role in assessing the results of these procedures as well as in depicting the complications. however, knowledge of the normal appearance after those procedures has to be presented. the initial imaging workup of polytrauma patients remains challenging. besides hardware requirements, workflow issues continue to evolve, with the current focus on the introduction of whole body ct into the early resuscitation phase of severely injured patients as a standard and basic diagnostic imaging method. this session is aimed at a thorough discussion on the requirements for advanced imaging in the early clinical situation in emergency radiology. focus is directed on the issues of radiation dose as well as on ct and contrast media protocols. a. chest and abdomen m. scaglione; castel volturno/it (mscaglione@tiscali.it) thoraco-abdominal injuries are a significant cause of death in the polytraumatised patients. early recognition and communication of lifethreatening thoraco-abdominal injuries is the major task of the radiologists involved in the emergency room. although most of these patients reach the hospital prior to dying, lethality continues to remain high. heart, thoracic great vessels, trachea, bronchus, pleura, lung, diaphragm, abdominal/retroperitoneal, vascular and solid organ injuries are potential cause of death. any appropriate surgical/interventional management approach must be carried out "around the clock", before thoraco-abdominal injuries reach the level of clinical evidence. on the other hand, non-operative management has actually become the standard of care for the most serious thoraco-abdominal injuries. these goals become feasible if a correct contrast-enhanced mdct diagnosis, in a dedicated facility in which the trauma team works effectively h a day, days a week, is performed. thus, in this lecture, the most serious thoraco-abdominal injuries will be illustrated, with special emphasis on vascular/injuries as well as the value of post-processing techniques, protocols, pitfalls, tips and tricks. furthermore, the importance of a rational and integrated imaging approach will be pointed out and, finally, the role of the radiologist in the emergency room will be emphasised. spinal and musculoskeletal trauma account for significant morbidity in severely traumatised patients. traumatic injuries to the spine encompass a variety of frequently occurring disorders and primarily result in stable injury. although rare, unstable disorders comprise injuries to the bone, the discs, and the ligaments. in the majority of cases, spinal cord injuries result in devastating medical and social consequences. severe musculoskeletal disorders usually are the result of high-impact accidents, such as motor vehicle accidents and falls from a height. for instance, the injured pelvic ring causes one of the most life-threatening conditions that the trauma team must handle. pelvic injuries are often associated with severe arterial, venous, and/or osseous bleeding. radiography is no longer recommended as the primary screening modality in spinal and pelvic trauma for adults. despite the radiation dose burden of ct, patients with a high risk of spine injury receive mdct imaging, as it is the method of choice. mr imaging is indicated primarily when patients present with myelopathy, and to search for spinal cord pathology. interventional radiology plays a major role in the therapy of complex traumatic pelvic disorders. extremity injuries in patients after polytrauma can be complex and are initially often difficult to be fully diagnosed. emergency radiology diagnosis is today mostly based on a standardised whole body ct (wbct), which can be extended with cta and adapted to cover extremity injuries. extremity injuries comprise: fractures of ( ) long bones, ( ) articular joints, ( ) complex fractures of hands and feet, ( ) vascular, ( ) soft tissue, ( ) nerve and plexus injuries and ( ) amputations. imaging protocol: *mdct is indicated in all major and complex bony fractures and is carried out early or integrated with wbct. cta using mips and mprs enables a thorough workup. * the role of us and cr is limited. * the role of mr and mra (in stable patients only) is to evaluate unstable articular injuries, injuries of tendons or major ligaments and nerve and plexus injuries). * the role of dsa is mostly for intervention. clinical findings and findings from wbct determine how to proceed, "first things are done first" in a priority-oriented clinical algorithm. treatment of extremity injuries must therefore be priority oriented and carefully planned in the context of possible concurrent injuries and a possible risk of multi-organ failure (mof). systemic drugs can be used for the treatment of cns and non-cns diseases. both classes can affect the brain inadvertently. common general drugs that affect the brain in a bystander manner are steroids (~ % brain volume reduction), recreational drugs (alcohol, cocaine, heroin, xtc), metronidazole and anti-epileptic drugs (corpus callosum demyelination) and immunosuppressants like cyclosporine/tacrolimus (pres/rpls) and methotrexate. mechanisms of action include neurovascular compromise, fluid/metabolite shifts and toxic effects to myelin of other tissue components. among cns-targeted drugs, especially immunomodulating agents may cause specific side effects. cytokine-release syndromes may occur with broad-acting agents such as general t cell antibodies. specific side effects may occur in multiple sclerosis, where treatment with natalizmumab may cause reactivation of jc virus leading to progressive multifocal leukencephalopathy (pml). upon withdrawal of therapy, this may then evolve into an immune reconstitution inflammatory syndrome (iris). in alzheimer disease, novel antibodies or vaccinations against amyloid may cause amyloid related imaging abnormalities (aria), which may present with microhaemorrhage on t * images, or with edema and effusion on flair. the objective of this session will be to review the diagnostic value of conventional sequences as well as the use of contrast in the monitoring of brain tumours, with special attention to gliomas. also, we will address the advantages and limitations of advanced techniques: perfusion, diffusion, and spectroscopy. immediately after surgery, the main objective of neuroimaging is the detection of the remaining tumour. it may also be necessary to rule out complications such as haemorrhage, ischaemia or infection. the fundamental technique is mri with contrast in the first hours and also diffusion sequences. in late follow-up, the goal is to differentiate the changes secondary to treatment of those related to tumour progression or recurrence. in these cases, conventional sequences present important constraints and are useful studies of diffusion, perfusion and spectroscopy. during follow-up after chemoradiotherapy, the objective is to assess tumour response. the response according to the new rano criteria will be revised. the combination of chemotherapy and radiotherapy, as well as the use of anti-angiogenic drugs causes changes that complicate the assessment of the response to treatment, with cases of pseudoprogression and pseudoresponse. in these cases, perfusion techniques, diffusion and spectroscopy can provide relevant information, although it is necessary to standardise the quantification to make its wide use possible. the speakers in this course will update the audience on contrast media safety such as steps to be taken before contrast administration and present the newest safety guidelines. the first speaker will cover new concepts of non-renal reactions to contrast media explaining which hypersensitivity reactions are allergic and non-allergic. the audience may learn assessing symptoms according to the ring and messmer classification and understand the importance of tryptase sampling and skin testing in the follow-up. the second speaker will address nsf. the presentation will review the pathophysiology, risk factors, recent recommendations and prevention of nsf. patients with gfr less than ml/min/ . m have increased risk of developing nsf. lowstability gadolinium contrast media show the strongest association with nsf. following existing guidelines on the use of gadolinium contrast agents minimises the risk of nsf. potential long-term harm from gadolinium accumulation in the body and legal issues are discussed. the last speakers will cover contrast medium-induced nephropathy with more recently published guidelines related to that issue. the presentation will include the definition of cin and the choice of contrast medium and prophylactic measures. recent changes in esur guidelines will be explained. the risk of cin is considered significantly lower following iv. cm administration and patients referred for enhanced ct are genuinely at risk if they have an egfr < ml/min/ . m . volume expansion with isotonic saline or sodium bicarbonate may be used for preventing cin in at-risk patients. acute immediate hypersensitivity reactions occur within the hour following the administration of contrast media. they can be seen with iodinated and gadolinium-based contrast agents. over the last ten years, new concepts have emerged in the way of understanding, managing and exploring hypersensitivity reactions. the clinical appearance is best classified by the ring and messmer scoring, from grade (cutaneous and subcutaneous signs) to grade (cardiovascular arrest). the mechanisms involve either true ige-mediated hypersensitivity or non-allergic hypersensitivity. the differential diagnosis in favour of allergy is made on a triad: clinical signs (the more severe, the more are the chances to be allergic), elevated tryptase levels in the plasma (indicating mastocyte triggering) and positive skin tests performed one month after the reaction. these new concepts induce important consequences in managing hypersensitivity reactions: be prepared to treat the patient adequately, be prepared to draw blood after the reaction to dose tryptase levels, send the patient to a dedicated allergologist, and forget about the preventive role of premedication against severe reactions. the authorities have introduced several restrictions on the use of high-risk agents, which will be followed by every physician in the eu. the agents are contraindicated in ) patients with severely reduced renal function including dialysis, ) acute renal insufficiency, ) neonates and ) pregnant women. they may only be used with caution in patients with moderately reduced renal function and children less than year old. there must be at least days between injections in those patients. renal function must always be determined by laboratory methods before use of high-risk agents. women should stop lactation for hours. these agents should never be given at doses higher than . mmol/kg per examination in any patient. for the intermediate and low-risk agents, the restrictions are significantly less; they should only be used with caution in patients with severely reduced renal function including dialysis. if a physician does not follow these rules, he or she will have legal problems as they have been introduced into the spc by the authorities. the contrast media safety committee (cmsc) of the esur has updated its guidelines on contrast medium-induced nephropathy (cin). the acr recently updated its guidelines as well. new guidelines were produced by nephrological societies. the topics reviewed include the definition of cin, the choice of contrast medium, and the prophylactic measures used to reduce the incidence of cin. the cmsc considered it appropriate to keep the definition of cin that was agreed in . however, nephrologists have recently agreed on a new definition. in the previous guideline, a number of risk factors were listed (raised s-creatinine levels, particularly secondary to diabetic nephropathy, dehydration, congestive heart failure, age over years, concurrent administration of nephrotoxic drugs). the significance of these risk factors has been confirmed and new risk factors were added. the cmsc agreed that the risk of cin was significantly lower following intravenous cm administration and concluded that patients referred for enhanced ct were genuinely at risk if they had an egfr < ml/min/ . m . the previous cmsc guideline suggested the use of low or iso-osmolar cm in patients with risk factors for cin and the cmsc considered that this previous guideline should not be changed. the cmsc considered that there was enough evidence to recommend that either volume expansion with isotonic saline or sodium bicarbonate may be used for preventing cin in at-risk patients, while the efficacy of nac and other drugs in reducing the incidence of cin remained unproven. guidelines produced by other societies provide very similar suggestions, thus further validating these recommendations. jia is the most common rheumatic entity in childhood and includes a subset of childhood arthritis, all of which are characterised by chronic synovitis with a potential risk of progressive joint destruction. radiological investigations in jia should ideally be able to determine the presence and degree of active inflammation, precursors of bony destructions and established erosions. however, there are many pitfalls in the interpretation of joint pathology in children. ultrasonography is often the initial tool in the assessment of arthritis and can depict joint fluid and synovitis. erosions and cartilage destruction of small joints may also be seen. the major problems are standardising the imaging technique and the lack of normal standards of anatomy in us in children. radiographs can show bone erosions and may depict cartilage loss indirectly through joint space narrowing, but are insensitive to inflammation and early joint destruction. mri is the only imaging modality that can assess all relevant anatomical structures in joint inflammation and is sensitive to early inflammation and destruction. however, large variations in the amount of joint fluid, bone marrow oedema-like lesions and changes resembling erosions are seen in children and also in healthy individuals. the differentiation between true pathology and normal findings on mri in children remains a challenge, particularly in early disease. in this lecture, the role of radiographs, ultrasound and mri and the typical radiological findings in joint pathology in jia will be presented. current knowledge on validity and reliability of the different imaging techniques in jia will be discussed. brain mri plays an important role in those criteria, as it can demonstrate the classical dissemination in time and space and helps earlier diagnosis, which is of major importance since the present recommendation in children is to start immunomodulating treatments as soon as diagnosis is established. among paediatric ms, % begin before the age of years and % before the age of years, frequently with an adem presentation in young children as initial manifestation of ms. however, only % of the adem indicate ms onset and the most predictive factors should be known, i.e. periventricular, deep white matter, corpus callosum high t signal lesions and black holes on t sequences. mri evaluation is also instrumental in differential diagnoses such as nonrelapsing adem, vasculitis, immunogenetic diseases and occasionally leukodystrophies. finally, brain mri is useful to evaluate the risk of more severe ms. the obesity epidemic represents one of the most significant european and public health challenges in the st century with prevalence of the disease having tripled in many countries during the past years. this is resulting in an ever increasing cost to healthcare systems including hospital and in particular radiology services which face unique challenges when imaging this group of patients. obesity significantly increases the patient's risk of various comorbid diseases including the incidence of cancer, diabetes, and cardiovascular and liver pathology with a multidisciplinary team approach mandatory for optimal patient care. this multidisciplinary symposium will review the implications of the epidemic with cutting edge, in-depth lectures presented by european experts addressing the epidemiology, role of imaging in the bariatric surgical patient as well as the importance of abnormal fat deposition in the liver. session objectives: . to learn about the impact of the obesity epidemic on european healthcare. . to appreciate the value of imaging techniques in the management of the post-operative bariatric patient. . to understand the role of radiology in fatty liver disease and the importance of imaging during subsequent patient surveillance. obesity: causes and consequences to the patient r. batterham; london/uk (r.batterham@ucl.ac.uk) obesity is one of the greatest st century public health challenges. its prevalence has tripled in many european union (eu) countries since the s. currently, % of eu adult population is overweight and % obese, and the numbers of those affected continue to rise. overweight and obesity are risk factors for numerous health problems, including hypertension, diabetes, cardiovascular diseases, respiratory problems, musculoskeletal diseases and some forms of cancer. mortality also increases sharply once the overweight threshold is crossed. because obesity is associated with higher risks of chronic illnesses, it is linked to significant additional health care costs and is already responsible for - % of health costs. changes in our environment are the main driver for this increase in overweight/obese. however, a person's genetic make-up can either increase or decrease their chances of becoming overweight. the gastrointestinal tract is the body's largest endocrine organ producing hormones that regulate bodyweight. dietary modifications, such as caloric restriction, are the first-line obesity treatments. however, dieting produces only moderate weight-loss with poor weight-loss maintenance. compensatory gut hormone changes induced by dieting are thought to contribute to the failure of dieting. in contrast, bariatric surgery is an efficacious treatment modality for obesity, producing durable weight-loss, amelioration of obesity-associated co-morbidities and reduced mortality. consequently, the number of bariatric procedures undertaken within europe has doubled in the last years with , procedures undertaken in . there is increasing evidence that surgically-induced alterations in circulating gut hormones mediate the weight-loss and metabolic beneficial effects of bariatric surgery. learning objectives: . to understand the epidemiology of obesity and its impact on european healthcare provision. . to appreciate the aetiology of obesity and the scientific rational for surgical treatment. . to learn about the effects of obesity on health. imaging of modern surgical procedures and their complications m. rengo; latina/ it (marco.rengo@gmail.com) we will illustrate the common bariatric procedures, in particular their normal appearance on different diagnostic technique as well as their early and late complications. we will illustrate the correlation between conventional barium studies and advanced imaging with mdct and mr. we will explain how to optimise mdct and mr acquisition protocol according to the clinical indication. we will explain what to evaluate before redo surgery, in particular quantitative and functional analysis. we will illustrate the role of interventional radiology in the management of early postoperative complications, in particular in the management of patients subjected to gastric banding. non-alcoholic fatty liver disease comprises a variety of pathological disorders ranging from simple steatosis to steatohepatitis. this condition is common in the western population and is typically associated with obesity and the metabolic syndrome. its incidence increases dramatically. diagnosis of fatty liver and distinction between simple steatosis and steatohepatitis are keys because the latter can lead to extensive fibrosis and cirrhosis with an increased risk of hepatocellular carcinoma (hcc). imaging plays a crucial role in diagnosing fatty liver. the two most important imaging modalities are ultrasound and mr imaging. ultrasound can exclude major steatosis but lacks accuracy to precisely quantify fat, while mr is the most accurate for quantification. the technique of reference is mr spectroscopy, but sophisticated sequences based on chemical shift principle have been shown as accurate as mr spectroscopy. unfortunately, imaging has still limitations to assess the presence of fibrosis and inflammation which are associated with steatohepatitis and functional tools could be of interest. today, imaging is combined with clinical and biological biomarkers to evaluate the risk of steatohepatitis. due to the increased risk of hcc and cirrhosis-related complications in patients with steatohepatitis, patients at risk should be enrolled in the surveillance programme. learning objectives: . to understand the pathophysiology of fatty liver disease and its link to cirrhosis. . to become familiar with the role of imaging in the detection and quantification of fat in the liver. . to learn about the importance of imaging in the surveillance of patients with fatty liver disease. the concept of breast cancer units originates from the need for making available to all women in europe high-quality breast services where breast disease could be looked after by specialists working as teams. such teams have to provide all the services related to breast cancer, including genetics and prevention, treatment of the primary tumour, care of advanced disease, palliation and follow-up of previously treated women. the breast unit is made up of a group of dedicated breast cancer specialists including a radiologist, radiographer, surgeon, reconstructive surgeon, pathologist, medical oncologist, radiation oncologist and breast care nurse. the specialists involved in breast cancer units have access to all the facilities required for high-quality care and spend most of their working time dealing with breast cancer. in our hospital, we routinely work as a team and to make it possible we organise weekly meetings involving specialists from different disciplines to evaluate and plan patient care at any step of the diagnostic and therapeutic process. each year, we take care of about . newly diagnosed patients with primary breast cancer and of . patients in follow-up. about are patients who undergo rt. after a short introduction from our fellows from pathology, surgery, oncology and radiation therapy, who will discuss their role in the breast unit, we will try to give a practical demonstration of the everyday work of breast units with particular emphasis on the role of the radiologist. the european society of urogenital radiology (esur) published, in , the new clinical guidelines for evaluation of the multi-parametric mri of the prostate. this structured reporting/scoring system (pi-rads) is based on literary evidence and consensus of experts' opinions. the pi-rads scoring system is similar to that already employed by breast imaging (bi-rads) and reflects the probability of a prostatic lesion to be significant. each lesion can be scored in all sequences used in the multi-parametric prostate mri protocols (t -and diffusion weighted imaging, dynamic contrast enhanced imaging and mr spectroscopy) based on defined mri criteria, which are specific for each sequence. based on the scoring in the particular sequences a final pi-rads score for each lesion can be assessed. for each lesion a five-grade scoring systems was created where, for example, the score means that clinically significant disease is highly unlikely to be present and score clinically significant disease is highly likely to be present. this lecture elucidates the principles of this scoring system and its impact on the target definition for the invasive diagnostics and therapy. prostate cancer screening with psa and extended systematic biopsy protocols have led to the over-detection and over-treatment of small and well differentiated cancers, considered clinically insignificant. these cancers cover up to % of the overall prevalence and may not be of any threat. multiparametric (mp)-mri has shown recently its value in the detection, localisation and characterisation of prostatic tumour foci larger than . cm, and may be of value to address the issue of over-detection and over-treatment. an mri-targeted biopsy strategy alone, without any additional systematic biopsies, has been suggested to decrease the detection rate of insignificant tumours while increasing that of potentially aggressive tumours. in patients with no evidence of lesion on mp-mri, the biopsy may probably be deferred. in patients with localised prostate cancer, mp-mri may also become a cornerstone in the selection, guidance and surveillance of patients managed with focal therapy. the rational for this new modality of treatment is to decrease over-treatment by destroying exclusively the index lesion detected on mp-mri. the precision of such treatments may be increased with mritransrectal ultrasound image fusion, allowing for real time navigation during the procedure. the accuracy of multiparametric mri has greatly improved the ability of localising tumour foci of prostate cancer. this property can be used to perform a trus-mr image registration, a new technological advance, which allows for an overlay of an mri onto a trus image to target a prostate biopsy towards a suspicious area. three types of registrations have been developed: cognitivebased, sensor-based and organ-based registration. cognitive registration consists of aiming at a suspicious area during biopsy with the knowledge of the lesion location identified on multiparametric mri. sensor-based registration consists of tracking in real time the trus probe with a magnetic device, achieving a global positioning system which overlays in real time prostate image on both modalities. its main limitation is that it does not take into account prostate and patient motion during biopsy two systems (artemis and uronav) have been developed to partially circumvent this drawback. organbased registration (koelis) does not aim at tracking the trus probe, but the prostate itself to compute in a d acquisition the trus prostate shape, allowing for a registration with the corresponding d mri shape. this system is not limited by prostate/patient motion and allows for a deformation of the organ during registration. the pros and cons of each technique and the rationale for a targeted-biopsy only policy are discussed. radiotherapy of the prostate typically takes the form of either external beam radiotherapy or alternatively, brachytherapy using radioactive seed implants. in both cases, the use of ultrasound has been proven to be highly useful and this presentation will give an current state of the art overview of ultrasound in external beam and radioactive seed radiotherapy. in the case of external beam radiotherapy, accurate localisation of the prostate is essential to ensure adequate target coverage with minimal damage to normal tissue. dedicated ultrasound scanners are able to provide sub millimeter localisation of the prostate superior to the imaging traditionally obtained using ct scanners. this positional information can be obtained daily. in addition, ultrasound allows the imaging of patients with metal hip implants which are normally difficult to image using ct. apart from the benefits of the technique, some pitfalls will also be highlighted. in the case of prostate brachytherapy, the use of ultrasound in the -d volume imaging of the prostate both prior and during treatment will be discussed. in both cases, some time will be spend discussing the quality assurance requirements for ultrasound imaging systems. image-guided radiation therapy (igrt) with onboard kilovoltage cone-beam ct (cbct) allows image guidance during radiotherapy treatments for patient setup and dose replanning. all these items will be discussed in connection with other topics: organ dose and image quality. it is compulsory to convert cbctimage's pixels from arbitrary grey scale to hounsfield unit (hu). this conversion was obtained using a catphan phantom. the same phantom is used for image quality evaluation; standard ctdi head and body phantoms and a farmer chamber were used to measure the cbdi to estimate organ and effective doses by monte carlo software of different protocols of acquisition. ctdosimetry software (ver. . . -impact) and pcxmc . rotation software were used. to verify the dose replanning techniques by cbct, horizontal and vertical dose profiles were compared with the same obtained from ct. patient replanning was verified using cbct vs ct in terms of conformity index. image quality parameters of cbct (in comparison with ct images) are fine for spatial resolution, but are less useful for low contrast. cbdi of . mgy/ mas was measured; msv effective dose, mgy prostate dose and mgy bladder dose were evaluated and discussed during the presentation. replanning on cylindrical phantom shows a mean percentage difference for each profile % and a variation on d mean in all inserts < %. the mean percentage difference between parameters characterising ct and cbct-based plan values is less than %. the replanning showed a substantial agreement with doses evaluated on the reference ct-image; patient dose must be evaluated for all radiation sources. cranial nerves i-vi have an anteroposterior course and are best examined in the coronal plane. cranial nerves vii-xii run in an anterolateral direction and are therefore best examined in the axial plane. these two planes also allow left-right comparison which makes lesion detection easier. lesions involving the cranial nerve nuclei can be detected on axial proton-density/t /multi-echo-ge images and diffusion-weighted images can exclude acute infarction. the cisternal segment of the cranial nerves is best detected on heavily t -weighted images (drive/fiesta/ d-tse/ciss). at -tesla, d-sequences (e.g. b-ffe) can be used and it is often possible to cover the cisternal segments of all cranial nerves. in the cavernous sinus, the cranial nerves can only be evaluated on coronal gadolinium-enhanced high-resolution t -weighted images and similar axial images are needed to evaluate the nerves in the jugular foramen and hypoglossal canal. axial and coronal gd-enhanced highresolution t -weighted with or without fat suppression are used to image the extracranial course of the cranial nerves. today, the tse-dixon sequences provide non-fatsat and fatsat images simultaneously, making cranial nerve imaging faster and easier. time-of-flight images can be used to study neurovascular conflicts, although these can also be detected on submillimetric gd-enhanced t images. of course, it is important to know the major anatomy of the cranial nerves and the most frequently occurring lesions. the abovementioned imaging techniques, the most important anatomy and the most frequently occurring lesions will be demonstrated and discussed in this lecture. mri study of the lower neck space includes the study of anatomy and pathologies of the thoracic outlet, or cervicothoracobrachial junction, extending from the cervical spine and the mediastinum to the lower border of the pectoralis minor muscle, the brachial plexus and the supraaortic vessels (carotid, subclavian arteries and veins). for this, use of a dedicated head and neck coil is fundamental to avoid frequent artefacts arising from air and the passage between different surfaces. only in case of thoracic outlet evaluation, a dynamic angio-mri study should be performed and this means the use of a surface coil. mri protocol of the lower neck should include t -and t weighted sequences. both fat-saturated proton density and t with stir sequences can be used to overcome the inhomogeneity of the magnetic field, especially with large fov. sand bags can be placed on either side of the neck and suprascapular region of the patient to improve image quality. also, flow saturation bands can be utilised to limit blood flow artefacts. also, volumetric sequences are in common use to obtain a quick examination. breath or cardiac gating can be helpful. abnormal findings of brachial plexus consist of nerve signal abnormalities with mild or marked hyperintensity on t -weighted, being aware of magic angle effects and swelling. in case of thoracic outlet syndrome, mri protocol should define the compression of brachial plexus components arteries and or veins, both in indifferent and dynamic mri acquisition. learning objectives: . to learn how to overcome difficulties in performing a lower neck study. . to understand how to avoid the most common pitfalls. . to become familiar with differential diagnosis. c. ct and mri of temporal bone: user's guide f. veillon; strasbourg/fr ct and mr imaging of the temporal bone must be performed with a precise technique. in ct it is important to locate the box of the study above and not in the orbit to avoid the lenses. irradiation is divided by compared with a study through the orbit. the axial sections must be parallel to the lateral semicircular canal the coronal, sagittal sections are completed by oblique coronal views through the long process of the incus and the head of the malleus to get the ossicular v. double oblique sections through the componants of the v permit a view of the stapes and also the malleus and incus in d (mip : mm). mri must be performed parallel to the roof of the orbit permiting very good axial sections in the plane of the lateral semicircular canal (t and t ). the internal auditory meatus and the inner ear must be analysed with mm axial sections (se) after intravenous gadolinium injection completed by a high resolution t (gradient echo or se) . - . mm, depending on the machine ( t, , t). the middle ear in chronic otitis media must be studied with t , diffusion and high resolution t . there is no need of contrast medium injection apart from the complication: fistula of the lateral semicircular canal, thrombosis of the sigmoid sinus. the different pathologies are then discussed: external auditory meatus and middle ear pathologies: ct first. mri is useful for postoperative cholesteatomas. inner ear malformations, otosclerosis, trauma: ct. labyrinthitis, inner ear hemorragia, schwannomas, internal auditory canal content: mri. revascularisation time windows for patients with acute ischemic stroke are generally restricted up to . hours in the anterior and up to hours is the posterior perfusion area. later treatment attempts require more accurate prediction of risk and benefit, as safety and efficacy at these time strata are less well. thus, rapid and effective imaging is important for decision-making concerning intrarterial catheter based recanalisation and/or thrombolytic therapy. advanced imaging techniques identify irreversible infarction as well as tissue at risk. diffusion-weighted mri detects ischemia within minutes of onset, whereas perfusion-weighted mri and ct perfusion studies disclose the ischemic penumbra. combined, they provide information on mismatched tissue, i.e. potentially salvageable brain. in addition, non-catheter angiographic techniques like ct or mr angiography are a useful adjunct to localise arterial occlusion. as an attempt to a reliable emergency examination, the following protocol has been proven to be robust: for the anterior perfusion area, a non contrast ct may exclude cerebral bleeding and is followed by ct angiography (including supraaortic and intracranial vasculature). if technical available, ct perfusion should be performed in addition. in ischaemic strokes of the vertrebrobasilar region, cta is essential to exclude basilary stenoses or thrombotic occlusion. if the stroke onset remains unclear or might extend the above mentioned time window, mr stroke imaging (i.e. diffusion, flair and perfusion sequences) is suggested as mr offers a higher sensitivity. anyway, the best method for each emergency stroke imaging center is depending on clinical availability h/ days, technical equipment and -finally -individual experience of the emergency team. some recent publications have questioned interventional treatment of stroke as an alternative to iv thrombolysis only. these papers -albeit published in highranking sources -usually do not describe the modern concepts of interventional stroke treatment. interventional radiologists, therefore, are convinced by their practical experience that the modern concept of combining iv thrombolysis with mechanical thrombectomy by stent retrievers offers a benefit to a subgroup of patients with severe stroke. this includes anatomical level of occlusion -basilar artery or single vertebral artery, proximal carotid occlusion with distal tandem occlusion, carotid t obstruction and m occlusion. m occlusions are debatable. besides location, the clinical status of the patient before stroke and time of onset, absence of early ct signs of stroke or bleeding and clinical contraindications to iv thrombolysis such as recent surgery influence the decision-making. more difficult than the description of technical success is prediction of clinical success. there are a couple of scores such as thrive available that may be used for outcome forecasting. the amount of collateral flow is frequently used as a decision tool, but is not always easy to quantify. mismatch scores and penumbra have been questioned recently. in conclusion, anatomical factors are pretty easy to identify to indicate treatment, but the clinical and functional setting still lacks quick and reliable parameters that allow a clear decision-making, particulary in borderline cases. the refresher course addresses the current state-of the-art use of different mechanical revascularisation strategies, devices and potential complications. in addition, multimodal imaging applications with a focus on patient selection for endovascular recanalisation therapies, as well as new techniques to guide endovascular therapy within the angio suite are presented. finally, some organisational aspects important for providing an interdisciplinary interventional stroke service are discussed. the ultimate goal of an acute endovascular stroke intervention is neurological recovery or improvement. recanalisation of an arterial occlusion is key in achieving this goal. clinical data suggest that endovascular stroke treatment results in higher recanalisation rates and may provide superior clinical outcomes when compared with intravenous thrombolytic therapy only. however, these higher recanalisation rates are far away in being paralleled by equally higher rates of favourable outcomes in recanalised patients. thus, patient selection remains crucial. besides the careful neurological assessment, brain imaging is here of major importance. the case presentations illustrate that imaging may help in patient selection for subsequent thrombolytic/endovascular therapies by differentiation of patients who may profit from intravenous or interventional therapy in an even extended time window from those who do not. there are a number of key areas supported by evidence-based medicine necessary for a high-level interventional stroke service. as a precondition, a neurointerventional stroke service has to be organised within a multidisciplinary acute stroke team. inside the hospital, it is all about streamlined pathways. any possible delays should be minimised at every step. sacroiliac joint pain may arise from a number of conditions including inflammatory arthritis, degeneration, fractures and tumours. studies suggest a prevalence of % to % pain arising from the joint in patients with positive clinical signs. temporary effect is provided by a mixture of local anaesthetics with steroids with a response varying between % and % in reported series. dual blocks using agents of differing duration are considered more precise, but are less often used in practice. imaging including mr and scintigraphy are of limited predictive value. injections may be into the synovial joint, around the joint or adjacent to the nerve innervation of the joint. there is evidence that para-articular sources of pain are common and injection outside the joint may be more effective. sl-joint injections are performed through a dorsal approach guided by ultrasound, fluoroscopy or low-dose ct. shortacting agents may have lasting benefit, but radiofrequency ablation has been employed in an attempt to obtain long-term response. the evidence for lasting therapeutic response to intra-articular or periarticular injection of steroids and conventional radiofrequency neurotomy is weak. there is fair evidence of longterm response to cooled radiofrequency neurotomy. facet joints account for - % of all low back pain. they are affected by osteoarthritis, joint space narrowing, intra-articular vacuum phenomenon/fluid, osteophytes, synovial cyst and ligament hypertrophy. conservative therapy is initially proposed. percutaneous facet joint steroid infiltrations are minimally invasive procedures involving injection of corticosteroid with or without local anaesthetic inside the joint. they also can provide diagnostic verification of a certain facet joint acting as the pain source. the injectate usually contains a long-acting corticosteroid mixed with a local anaesthetic. sodium hyaluronate solutions or ozone were tested; however more and extensive studies are necessary. other options are either percutaneous ablation or surgical arthrodesis. fluoroscopy, computed tomography or magnetic resonance can be used for guidance. fluoroscopy has the advantage of real-time imaging. cone-beam ct can also be used. computed tomography provides better anatomy information, but has increased radiation dose for the patient. magnetic resonance has higher cost and longer duration. a recent study concluded that it is twice the cost of ct-guided infiltrations. mri can be used in combination with focal ultrasound for ablation, a technique which is still under investigation. success depends upon patient selection ( - % immediate and - % long-term relief). the level of evidence is moderate for lumbar spine concerning short-and long-term improvement. however, the most recent guidelines released (american society of pain physicians) state that it is the oldest and most commonly used technique. the success rates with its safety profile and least invasiveness seem to make it an attractive therapy. although promising for early assessment of response to treatment, these newer functional biomarkers need extensive validation and standardisation for their wide clinical use. validation includes the assessment of reproducibility and accuracy, whereas standardisation concerns image acquisition and postprocessing. the added value of the more complex functional biomarkers relative to the viability parameters should also be shown. viability and functional imaging biomarkers are evolving and emerging parameters for the early assessment of response to treatment. mri biomarkers must be able to show how tumours respond to specific treatment. they need to allow assessment of the effectiveness of new treatment more rapidly than classical clinical end points. these biomarkers must be easy to obtain to facilitate a large spread of the technique. they have to be reproducible. the longest diameter of the tumour remains the easiest biomarker that can be obtained from any kind of morphologic acquisition with no need of post-processing. additional information about the tissular organisation and cellularity can be now easily obtained using modern scanners through diffusion-weighted sequences. the ease with which those sequences are obtained for a while masked the necessity to perform a more complex postprocessing than the one initially done to get reliable biomarkers. there are numerous mri biomarkers of microcirculation, reflecting o consumption, blood volume, blood flow, vessel permeability and extravascular volume. to get them, we need more sophisticated acquisitions and image processing that take into account the t of the tissue, arterial input function, respiratory motion, etc. most of these new mri biomarkers are now used in research and in phase i studies, but have not been validated in more advanced clinical trials or in clinical practice. to use them widely and reliably, we need to perfectly understand the consequences of the choices we make during the acquisition and post-processing of these biomarkers. new targeted treatments in cancer can be effective without significantly reducing tumour size. there are already a large number of targeted treatments that are licensed to treat a range of cancers. for some of these cancers, there is currently no reliable method to tell whether the drug is effective and new response, predictive and prognostic biomarkers are required. functional imaging techniques such as diffusion-weighted mri, dynamic contrastenhanced mri and fdg-pet imaging are being developed or applied as response biomarkers. however, for these to be useful in a wider multi-centre setting, the measurements need to be precise, repeatable and reproducible. we discuss these properties in the context of emerging imaging response biomarkers. learning objectives: . to understand imaging biomarker precision (repeatability and reproducibility) and accuracy and how it is evaluated. . to learn how to interpret biomarker precision and accuracy in the context of the biomarker's intended use. coronary artery disease (cad) and its related cardiac disorders are still the number one cause of death in the usa and the western world. up to date, single photon computed tomography (spect) using traditional radiotracers like thallium- or tc- m sestamibi is the most utilised imaging technique for the assessment of myocardial perfusion. however, over the past decade, there has been a growing interest in cardiac imaging with positron emission tomography (pet) and, indeed, a paradigm shift has been witnessed in the use of myocardial perfusion imaging (mpi) with pet taking advantage of the superior imaging properties of pet over spect. therefore, pet mpi is now being increasingly used for routine clinical evaluation of patients with known or suspected cad. furthermore, it is being used not only at large academic institutions, but also at community hospitals and even in private practice. several factors contribute to this shift in the use of pet mpi, including the growing availability of combined pet and computed tomography (ct) systems, mainly driven by oncological applications, radiotracer, like rubidium- or fflurpiridaz, which can be used in clinical routine, changes in reimbursement, and the increasing clinical evidence supporting the value of pet/ct mpi. the lecture "pet for evaluation of perfusion, absolute myocardial blood flow and coronary flow reserve" will cover several aspects of the growing field of pet mpi. besides the visualisation of coronary morphology, computed tomography (ct) has shown feasibility to also assess myocardial perfusion. currently, there are two different approaches to ct-based myocardial perfusion imaging: singleshot and dynamic, sequential acquisitions over a predefined scan time. the presentation will cover basic concepts of both approaches and highlight protocol details and findings in these stress acquisitions. also, emerging scientific results with respect to diagnostic accuracy, the detection of hemodynamically relevant coronary stenosis and prognostic implications will be discussed. over the past few years, cardiovascular magnetic resonance imaging (cmr) has been increasingly established as an important method in the diagnosis of cardiovascular disease. many studies have shown the equality or even superiority of cmr compared to other imaging modalities (e.g. nuclear medicine and echocardiography). cmr offers important advantages like the absence of ionising radiation, high spatial resolution, and the combination of perfusion imaging with tissue characterisation. the main clinical applications in the assessment of coronary artery disease (cad) include ventricular function, myocardial viability and perfusion. in clinical routine, myocardial perfusion is determined by contrast-enhanced first-pass perfusion techniques during pharmacological stress using coronary vasodilators (e.g. adenosine) or ßadrenergic agents (e.g. dobutamine). non-invasive characterisation of myocardial microcirculation is thought to reflect myocardial tissue supply much better than mere luminographic detection and quantification of epicardial coronary stenosis, and has been shown to be useful for planning of revascularisation procedures and cardiac risk stratification. in several studies on the prognostic value of cmr in cad assessment, normal stress perfusion cmr was highly predictive for a good prognosis, thus able to identify patients in whom invasive angiography can be deferred safely. the purpose of this lecture is to demonstrate how it is possible to sensitise the mr signal to water molecules diffusion in tissue and how to use calculated indices to reflect structural integrity. the concept of diffusion-weighted imaging will be introduced with particular emphasis on the pulsed gradient spin echo (pgse) sequence. methods to calculate the apparent diffusion coefficient (adc) will be described and the concept of the diffusion tensor (dt) will be explained. image processing of diffusion data includes steps like eddy current distortion correction, model fitting and potentially registration of maps to a reference space. indices that are reproducible and rotationally invariant will be described, such as fractional anisotropy (fa), mean diffusivity (md), axial diffusivity (ad) and radial diffusivity (rd). examples of when to use the adc maps or the dt indices will be given in relation to pathologies such as stroke, multiple sclerosis and neurodegenerative diseases. strategies for result presentation such as region of interest approach, histogram analysis and tractbased methods will be shown. limitations and advantages of diffusion imaging methods such as dt do not preclude the use of this technique in research and clinical radiology for investigating structural changes in disease. diffusion imaging methods have a lot of potential to be used in clinical practice. however, although diffusion-weighted imaging has been increasingly applied, clinical use of diffusion tensor imaging is limited to date. this is in part due to the lack of standardisation and the need for more complex analysis tools to evaluate the data. during this lecture, an overview will be provided of some analysis methods for diffusion-weighted and tensor imaging. i will discuss the different parameters that can be obtained as well as some advantages and limitations of the different analysis methods. it is highly recommended to include diffusion-weighted imaging (dwi) in the routine protocols for mri of the brain. dwi as well as automatically calculated apparent diffusion coefficient (adc) map is used for evaluation. the adc values of gray and white matter are identical in the adult brain. restricted diffusion (high signal on dwi and low signal on adc) is seen not only in acute stroke, but can also be present in some brain tumours, the necrotic centre of abscesses, some acute mr plaques, some contusions, encephalitis, creutzfeldt-jakob disease, metabolic diseases, etc. this finding is thus not specific, but is very helpful combined with clinical information and the signal pattern on other sequences. acute stroke has restricted diffusion during the first - days and the diffusion gradually increases to become very high in a chronic infarct. the diffusion changes in ischemic brain and spinal cord tissue are usually irreversible, but may be reversible. brain tumours with high cellularity, such as lymphoma, glioblastoma multiforme, medulloblastoma and metastases from small cell lung carcinoma, usually have restricted diffusion. the surrounding vasogenic oedema has increased diffusion. in the central necrosis of a pyogenic abscess the diffusion is restricted, while it is increased in the necrotic centre of a malignant brain tumour. reversible diffusion restriction in the cortex, the hippocampi and thalami can be seen in patients with status epilepticus. diffusion tensor imaging has so far had limited clinical use, but may be useful, e.g. for the preoperative evaluation of brain tumours. learning objectives: . to understand the differential diagnostic possibilities of high signal intensity lesions on diffusion weighted images (dwi) of the brain and spinal cord. . to become familiar with the appearance of acute, subacute and chronic stroke on dwi. . to learn about the appearance of cerebral tumours, infection/inflammation, neurodegenerative diseases and traumatic lesions on dwi. . to understand the present use of diffusion tensor imaging (dti) and diffusion tensor tractography (dtt) in clinical neuroradiology. the signal intensity in diffusion-weighted imaging (dwi) reflects the cell density in the tissue. dwi, including apparent diffusion coefficient (adc) maps, can therefore be used to differentiate highly cellular from acellular regions of tumours, distinguish cystic from solid lesions, and monitor change in tumour cellularity over time, reflecting response to therapy. in general, tumours have high signal intensities in dwi, but low corresponding adc values compared to normal/benign/reactive tissues. dwi has a wide range of clinical applications, which includes cancer imaging, imaging of infections and inflammations, evaluation of trauma and visualisation of peripheral nerves. of these, the most promising application appears to be in oncological imaging. there is, e.g. now evidence to recommend inclusion of dwi in mri protocols for lesion detection and characterisation in the liver. the advent of whole body (wb) mri including wb-dwi (from root of the neck to groin) has introduced tumour imaging with a systemic approach compared to established multi-modal diagnostic algorithms. it has been found valuable for staging, therapy evaluation and surveillance of tumours, especially in children. however, dwi is generally recommended to be incorporated into oncologic mri protocols of both wb-mri and of selected organs, because it provides additional valuable information to the conventional mr sequences. dwi of the body is frequently prone to imaging artefacts, which can obscure or mimic lesions. to minimise misinterpretations, analysis of the raw b-value images directly in conjunction with adc maps and conventional coregistered sequences is recommended. spinal surgery is most frequently performed to decompress (disc herniation, stenosis, malignant infiltration), fuse and stabilise (particularly following trauma or infiltrative destructive processes) and correct deformity. often, there may be a combination of these procedures at one operation. surgical instrumentation or bone graft is sometimes employed. patients may present themselves with symptoms early or late following the procedure. this interactive session seeks to address the variety of surgical procedures undertaken and subsequently imaged post-operatively because of symptoms. the session aims to help one to understand and become familiar with the expected post-operative imaging appearances related to the surgical procedure, learn about abnormal pathological features as a cause of symptoms in the acute and more chronic situation and explore the diagnosis and differential diagnosis. this may include post-operative fibrosis versus recurrent disc herniation versus post-operative infection. failure of fusion due to failure of instrumentation or inadequate take of bone graft can give rise to pseudoarthrosis. recurrent stenotic symptoms may relate to an inadequate decompression, recurrent disc herniation, postoperative haematoma, extension of a malignant process or ischaemic damage. joint replacement surgery for the treatment of arthritis most often offers the patient excellent results. however, there are potential complications that a radiologist should know about. it is essential to understand the importance of pre-and postoperative imaging for evaluating patients. most commonly, standard radiographs are used to assess the patients after joint replacement. however, ct, nuclear medicine methods as well as mr imaging play an increasing role in such patients. the radiologist should be aware of the most common type of prosthesis and the most common complications after joint replacement. these complications include postoperative prosthesis loosening, prosthesis fractures, periprosthetic fractures, postoperative infection, rotation failure of the prosthesis, and soft tissue abnormalities such as surrounding tendon tears. this interactive session seeks to address the variety of joint replacements which are undertaken and subsequently imaged postoperatively as a result of symptoms. the session aims to help one to understand and become familiar with the expected postoperative imaging appearances related to joint replacement, learn about abnormal pathological features as a cause of symptoms and explore the diagnosis and differential diagnosis. learning objectives: . to learn about changes related to surgery. . to understand changes related to non-surgical treatments. serbia is a beautiful country where east and west merged and often collided for centuries, and where nowadays eastern cultural heritage meets and mixes with the western heritage in a lovely and unprecedented way, with a lot of charm and with a variety of extraordinarily natural beauty and plenty of both traditional and gastronomical wonders to explore. it extends from the edges of the pannonian flatland in the north, over the danube and sava rivers to the gradually growing wonderful mountains in the south. already in , the first x-ray unit was installed, and only twenty years later x-ray units are present in most state-owned and private hospitals in serbia. nowadays, slightly more than half of the thousand radiologists in serbia perform a broad variety of diagnostic imaging and interventional techniques in different radiological units, including numerous ct, mri, and few pet/ct units distributed in five university centers, a number of public clinical centers and hospitals, and also in the private sector. several centers are fully equipped with pacs and ris systems, and are capable of performing teleradiology services. there are still significant challenges in serbian radiology in the field of education and research, planning and implementation of national radiology networks, improvement of standard clinical practice and financing the new equipment. yet since radiologists' enthusiasm overwhelms most of the difficulties, radiology in serbia provides nowadays a broad variety of modern and competent diagnostic and interventional procedures, improving the management and treatment of our patients. radiology in serbia, since its beginning, has been inextricably linked to european radiology. there are written documents testifying to the fact that the first x-ray appliances in serbia had emerged and had been used for medical purposes only two years after wilhelm conrad röntgen discovered x-rays in . the real beginnings of diagnostic radiology in serbia can be linked to the establishment of the general state hospital in , where the first x-ray cabinet was installed. later discoveries in the field of radiological techniques, materials and contrast agents were also immediately accepted by the serbian radiology community. the first cerebral angiography was performed in , which may be considered as the beginning of neuroradiology in serbia. the second half of the twentieth century is characterised by the introduction and wide application of ultrasound, ct and mr diagnostics and outbreak of vascular and nonvascular interventional radiology procedures in the areas of neuroradiology, uroradiology, and cardiovascular and gastrointestinal radiology. for years, serbian interventional radiology has successfully kept pace with achievements of the most relevant centres worldwide. nowadays, serbian radiologists monitor and enforce the most complex modern procedures in different areas of radiology. the rapid development of science and information technology has enabled the daily monitoring of all modern developments, methods and approaches and their increasingly faster introduction into practice. non-ischaemic cardiomyopathies (nicm) refer to myocardial diseases caused by mechanical and/or electrical disorder in the absence of significant coronary artery disease, valvular heart disease, hypertension, or congenital heart disease. nicm often present with genetic mutations, but no clinically apparent disease. in some cases, the presence of fibrotic tissue could cause adverse events and, therefore, the use of late enhancement (le) post-gadolinium technique is necessary in evaluating these patients. le suggestive of fibrotic tissue could be found in up to % of patients with nicm. in the majority of cases (up to %), midwall/subepicardial enhancement could be found; patchy enhancement, often at septal junctions, has been registered in % of patients, while the least common pattern of le was subendocardial ( %). in ischaemic cardiomyopathy (icm), subendocardial or transmural is a typical pattern of hyperenhancement and is related to coronary artery distribution. furthermore, le has the potential to provide important information for risk stratification in clinical practice. in nicm, the extent of fibrosis has been shown to be independently associated with increased rates of future adverse events including all-cause mortality, while in icm myocardium with le in more than % of wall thickness is unlikely to recover contractile function following coronary revascularisation. as many nicm disorders are associated with the presence of scar tissue, le in nicm is nonspecific and should be correlated with clinical presentation and history, which is critical in the evaluation of these patients. interlude: hop-on, hop-off sightseeing tour of serbia s. stojanovic; novi sad/ rs (tupsons@gmail.com) in the interlude between scientific lectures, you are most welcome to hop on a sightseeing tour around serbia during which we shall stop at a few stations where the natural and cultural heritage of our country will be shown. nota bene: some of the sights will be visible to everyone and some only to radiologists. ten thousand years of human effort and five billion years of european soil refinement will blink in front of your eyes through the window we have made with red churches' walls, azure skies and a silver sparkle of our spirit. all the time, a bit of grayscale shadow will supplement the picture as we in radiology are so used to. fragments of past and present will make a mosaic, the glance of which will hopefully light up your day. when you hop off our tour, we believe, some pictures will become part of your emotional memory and maybe will make you wish to see them in vivo. the role of prenatal mri in foetal central nervous system abnormalities: a case-based pictorial review k. koprivsek; sremska kamenica/rs (katarina.koprivsek@gmail.com) mri provides a unique opportunity for studying in vivo central nervous system development. its higher contrast resolution compared with prenatal sonography allows better visualisation of sonographically occult normal cns structures as well as structural abnormalities. in clinical praxis, prenatal mri has become a mandatory tool in evaluating: a) foetuses that have abnormalities suspected on the basis of foetal sonography; b) foetuses with increased risk for brain abnormalities even in the setting of a normal prenatal sonogram (family history of cns anomalies or a genetic/metabolic disorder); c) foetuses potentially at risk due to maternal illness; d) foetuses with congenital malformation, which are candidates for prenatal or neonatal surgical treatment; e) foetuses that could not be evaluated by sonography due to oligohydroamnion, maternal obesity, difficult position of the foetal head, or us reverberation on foetal calvaria in advanced gestational age. we will present a selection of foetal cns abnormalities detected in our institution during the last years, including ventriculomegaly, abnormalities of the posterior fossa, agenesis of the corpus callosum, cerebral cortex developmental abnormalities, spine developmental abnormalities and a variety of encephaloclastic lesions. in all previously listed cases, prenatal mri could provide either crucial or additional information, which can lead to the accurate diagnosis of different cns developmental abnormalities (cortical, commissural and posterior fossa malformation), specific disorder or syndrome, further enabling adequate pregnancy management and parents' counselling. this lecture will cover the standard mammographic views required, the reporting systems for mammograms together with birads and other european systems, breast density, feature analysis, information the clinician requires in a cancer case and how to approach a b lesion. acr practice guidelines ( ) recommend obtaining previous mammograms as you can dismiss an abnormality that is unchanged for years or that has been previously worked up and are able to detect subtle new lesions more easily, although this has not been confirmed in the screening situation. in a structured report the following information is recommended: indication, breast density, description of lesion, size, location, comparison to previous findings, overall assessment, management recommendations and a score to indicate the level of suspicion. examples of different common conditions seen on mammography will be shown including soft tissue masses, microcalcification, architectural distortion and asymmetry. breast ultrasound is one of the main imaging modalities in breast radiology, it allows us to characterise lesions and also guide interventional procedures. the bi-rads categorisation of ultrasonographic findings facilitates the diagnostic approach and also helps the radiologist to use a common language, understood not only by the rest of the radiological community, but also by other breast cancer professionals. ultrasound can be a diagnostic procedure on its own, but is mainly a modality that characterises the findings of other modalities (mammography and mri) and, as such, correlation between all these techniques is the mainstay of everyday clinical practice. this act of correlating and integrating the information of all modalities is what makes a breast radiologist a key actor in the diagnosis, staging and follow-up of breast cancer and other benign or high-risk entities. the final product of this integration will be the radiological report, the means by which we convey all the information we have gathered through all the procedures to our clinical colleagues. this report shall also follow some composition rules to be clear and concise. as with any other modalities, breast magnetic resonance imaging (mri) examinations should be reported in a structured way following the guidelines of the respective national, continental or international societies. adequate nonclinical information (i.e. patient name, date and type of examination, etc). is indispensable. any breast imaging report has to follow a stringent structure including indication, clinical history, clinical findings, brief description of technology used, assessment of parenchymal density, detailed description of significant findings, comparison with previous imaging studies and final assessment according to bi-rads. indications and contraindications of breast mri will be discussed. significant findings at breast mri include foci (small contrast-enhancing spot, nos, < mm), mass lesions, "non-mass-like enhancement" (no mass lesion, partly diffuse regional contrast enhancement of various size) and associated findings. furthermore, as breast mri is a functional study, the different appearance patterns of kinetic contrast enhancement will be presented. the current status and the appropriate use of the bi-rads mri lexicon will be discussed. any breast mri report should not only follow the guidelines, but also follow a red thread, be consistent, express confidence and be comprehensible to clinicians. the overall final bi-rads assessment is based on the most worrisome finding, taking into account both breasts and all imaging methods (mammography, ultrasound, mri) evaluated. furthermore, adequate communication of the result, as well as do's and don'ts of the report wording will be discussed. the prostate is a small glandular organ, whose main function is to secrete seminal fluid. in young men it comprises of mainly the peripheral zone ( %) and a smaller central gland. however, the central gland enlarges with age to form the larger part. the main diseases of the prostate are inflammation, hyperplasia, and cancer (the most important). approximately % of cancers arise in the peripheral zone. the main applications of mri are for cancer detection, staging, and recurrence. mri is increasingly used for radiotherapy planning owing to its good soft-tissue contrast when compared with ct scanning. endo-rectal rf receiver coils, usually inflated with air or perfluorocarbon but sometimes of a rigid design, improve signal detection of the prostate. phased-array surface coils may also be used or (preferably) in combination. t -weighted imaging helps to distinguish haemorrhage caused by biopsy. on t -weighted images the peripheral zone appears brighter, owing to a larger proportion of prostatic ducts, while tumours generally appear darker. more sophisticated mr methods are increasingly used to help diagnosis. in diffusion-weighted images (dwi) tumours appear relatively bright, but dark in the corresponding calculated map of apparent diffusion coefficient (adc), owing to the more cellularly-dense nature of tumours, and hence, reduced diffusion. in dynamic contrast-enhanced mri (dce-mri) tumours appear bright owing to increased vasculature. magnetic resonance spectroscopic imaging (mrsi) of normal prostate is dominated by high levels of citrate from the prostatic ducts; in cancer this is reduced while choline is increased. prostatectomy samples allow one to verify image contrast against histology. recent studies on prostate cancer have provided a number of relevant targets for imaging and treatment purposes. today, imaging plays an important role in different aspects of the disease, but its role should grow in answering clinical questions at various phases of the disease. relevant targets for imaging include metabolites (including glucose, acetate, choline, and amino acids), antigens (including prostate-specific membrane antigen), receptors (grp receptor, cxcr , androgen receptor), proliferation, angiogenesis, and hypoxia. in this presentation, a range of radiotracers for potential use in the imaging of prostate cancer will be discussed, as well as options to tailor nuclear imaging tracers to the various phases of the heterogeneous disease. the fusion of morphologic (ct and mri) and functional (pet, spect) imaging modalities (pet, spect) has become widely available and part of the standard diagnostic workup in cancer patients. for many years, neurosurgeons have already relied on multimodal imaging data during brain tumour surgery to identify hyper-or hypometabolic tissue. the availability of pet and spect data during percutaneous ablation procedures may also be helpful to facilitate treatment planning, probe placement, probe re-positioning, and early detection of residual disease following percutaneous image-guided ablation of cancer. however, up to now conventional us-and ct-guided thermal ablation procedures rely on morphologic information only. intraoperative implementation of multimodal data requires the application of frame-based or frameless stereotactic techniques, both relying on the establishment of an accurate relationship between the patient's preoperative image space and the intraoperative physical space. in image-guided neurosurgery, ct/mrt/spect/pet data are registered to the patient using rigid body transformation. due to respiratory motion and different patient positioning during image acquisition and the actual intervention, soft tissue deformations in the liver occur. thus, the images and the surgical presentation do not match between preoperative imaging and intervention, leading to an extensive degradation of spatial guidance. this presentation illustrates solutions for these fundamental problems that occur during d-navigated liver interventions, including patient immobilisation and repositioning, respiratory triggering and intraoperative imaging. r. bale: equipment support recipient; isys has supported the section of microinvasive therapy with testing equipment. patent holder; rb is a (co-) inventor of the atlas aiming device and the bodyfix immobilisation device and a (co-)shareholder in its financial returns. the goal of ultrasound-triggered, image-guided drug delivery is to increase the therapeutic index and decrease adverse effects of drugs. the bio-effects of focused ultrasound can lead to local tissue heating, cavitation, and radiation force, which can be used for ) local drug release from nanocarriers circulating in the blood, ) increased extravasation and cellular uptake of drugs and/or carriers, and ) enhanced diffusivity of drugs. thermo-sensitive liposomes have been suggested for local drug release in combination with local hyperthermia more than years ago. microbubbles may be designed to enhance cavitation effects. real-time imaging methods, such as magnetic resonance, as well as optical and ultrasound imaging have led to novel insights and methods for ultrasound-triggered drug delivery. image guidance of ultrasound can be used for: ) target identification and characterisation; ) spatio-temporal guidance of actions to release or activate the drugs and/or permeabilise membranes; ) evaluation of biodistribution, pkpd; ) physiological read-outs to evaluate the therapeutic efficacy. liposomes may carry both hydrophilic and hydrophobic drugs in their aqueous interior and lipid bilayer membrane, respectively. the circulation half-life may be increased by incorporating polyethylene glycol (peg)-lipids in the bilayer. recent publications have shown that ultrasound-triggered delivery is feasible. realtime imaging methods, such as magnetic resonance, optical and ultrasound imaging may lead to novel insights and methods for ultrasound-triggered drug delivery. up to now, the success of surgical tumour resection has always been limited by the surgeon´s vision. the human eye is not an accurate detector of small cancer cell clusters and it cannot accurately differentiate cancerous from healthy tissues due to lack of "visible" contrast. by combining a fluorescent probe targeting the folate receptor and a multi-spectral real-time fluorescence camera to observe the operating field, we found that, using fluorescence molecular imaging, -fold more lesions can be identified compared to conventional surgery. these first in-human results point to molecular-based clinical decision-making in surgical and endoscopic procedures as a paradigm shift over decision-making based on human vision. the talk describes current progress with instruments, methods and applications in the field of intraoperative imaging. pre-clinical and clinical results are presented and the advantages and limitations of the method as well as future directions will be discussed. the emerging field of multispectral optoacoustic tomography (msot) is also outlined. mysteries of the human brain unveiled: imaging of white matter microstructure and neuroplasticity p.m. parizel; antwerp/be (paul.parizel@uantwerpen.be) the human brain remains one of nature's great mysteries, and is considered science's final frontier. this greyish lump of tissue with a weight of . kg contains some billion neurons, each of which are connected to thousands of other nerve cells in an intricate network of white matter fibers. in the previous century, the brain was perceived as a fixed three-dimensional landscape, with brain functions confined to certain locations (e.g. motor and sensory cortex, visual cortex, auditory cortex). this concept of neurolocalisationism was in line with the philosophy that there should be "a place for everything, and everything should be in its place". advanced quantitative mri techniques for acquisition and data analyis (fmri, dwi, dti, vbm), help to unravel white matter connectivity of the human brain, and are able to show that the brain can modify its structure and function in response to changing circumstances (such as learning, memory, hormones). this process, which is known as neuroplasticity, occurs at different levels and different time scales. for example, in response to injury, changes may occur at cellular level as well as on a larger scale with cortical remapping. some processes may take months or years (e.g. physical therapy and training) whereas some forms of neuroplasticity happen within hours or days (e.g. changes in brain volume and connectivity during the female menstrual cycle, which have been linked to behavioral changes). in summary, quantitative mri techniques help to unveil the mysteries of the human brain and have opened exciting new fields of active research such as white matter connectivity and neuroplasticity. there are a host of inflammatory and infective insults that can manifest focally or diffusely within the musculoskeletal system. the appearances of the underlying pathological processes in both the soft tissues and skeleton cover a very wide imaging spectrum. the appearances vary, depending on the timing and degree of inflammatory insult and the host response in the involved tissues. the approach of this lecture will cover the imaging manifestations using all modalities covering radiography, ultrasound, ct, scintigraphy and magnetic resonance imaging. the basic knowledge that is required will be displayed in major musculoskeletal categories covering disorders involving the soft tissues, joints, bones and entheses. the imaging manifestations will also be linked with the evolution of the pathological processes covering acute, sub-acute and chronic stages of the inflammatory/infective disorders. by the end of the session the audience should have a clear understanding in making best use of the imaging modalities in the correct diagnosis of a wide variety of inflammatory and infective conditions that can affect the musculoskeletal system. arterial pta and stenting is firmly established as primary treatment in the management of athero-occlusive disease, particularly of peripheral limb arterial disease (pad). patients are selected on the basis of symptoms such as short distance claudication or critical limb ischaemia for lower limb disease or appropriate ischaemic symptoms in other territories in the renal and mesenteric territories and head and neck vessels. commonly used grading systems such as the fontaine or rutherford scores can be useful in patient selection. where treatment is required, noninvasive imaging is utilised to look for the site, severity and extent of arterial disease. most centers use duplex imaging as the primary imaging modality; mra or cta are often used with a higher sensitivity and specificity; and invasive angiography is reserved for problem solving or immediately prior to intervention. the tascii classification has been devised to select patients for either endovascular or surgical intervention. however, most centres will adopt an endovascular approach first. all patients should be on good medical therapy, i.e. aspirin and statins, with good blood pressure and diabetic control where appropriate. when carrying out interventions, an access sheath is used, peri-procedural heparin administered and lesions crossed using selective catheters and commonly hydrophilic guidewires. subintimal crossing of occlusions can be much simpler than trying to cross lesions luminally. balloon angioplasty and or stenting may be applied differently depending on the site and extent of the lesions. the evidence for drug-eluting technologies has also been growing with improved patency rates compared to standard balloon angioplasty. during the efrs meets russia session, radiographers have the opportunity to get information about the role and education of radiographers in the different european countries. the radiographer is a health-care team member who interacts with other professionals in the primary and secondary health-care environment to provide an optimum diagnostic or therapeutic outcome. radiographer education therefore requires that the curriculum covers a wide range of scientific, medical, pathological, sociological, ethical and technical subjects together with the development of appropriate clinical skills. the curriculum should also include the development of research and audit skills to ensure the constant improvement of service quality for the benefit of service users. the session will include a panel discussion about the role of the radiographer in image acquisition and processing. the tasks of the assistant to the radiologist are: filling of the protocol of research, survey of the patient, instructing the patient according to safety measures, positioning the patient, placement of coils of strengthening, beginning of magnetic and resonant research -the research is approved by the doctor, introduction of contrast substance (in the presence of the certificate of the procedural sister) and supervision over the patient during mr research. furthermore, the other tasks are briefing on patient safety, excluding introduction of the patient to metal objects on mr tomography, explaining the procedure of the study, informing about the duration of the study, wearing noise-cancelling headphones, warning about possible vibration and loud sounds, and providing a globular button to call the nurse. today coronary ct-angiography has become a well-established and proven diagnostic modality. quality of coronary and cardiac cta datasets has tremendous significance for correct diagnosis. a radiographer has a pivotal role in performance of coronary cta. coronary cta is one of the most challenging examinations for radiographers because even a small mistake at any stage of examination may ruin the whole study. before the scanning procedure, the radiographer performs a check for possible contraindications to contrast media injection (history of adverse reactions to contrast media and impairment of renal function are the most important ones). the radiographer checks the patient's heart rhythm and takes part in administration of betablockers for control of the heart rate. a detailed knowledge of different scanning modalities of ct-machine is a must. radiation exposure to the patient from coronary cta examinations has been one of the major public concerns. this is why the radiographer should be aware of different approaches to the performance of low-dose cta (prospective gating, tube current modulation, high-pitch scanning) according to patient body composition. the radiographer should have knowledge of when and how to use radiation dose control in an appropriate way. another big issue is injection protocols. selection of optimal bolus timing and iodine load results in highquality cta images. the radiographer has to know the basics of ct image reconstruction and interpretation in order to perform different types of reconstructions from the raw data according to the radiologist's requests and be able to recognise image artifacts. clinical examples of the radiographer's role for performance of coronary cta will be given. every country is famous for its architecture, history, literature, music, museums, etc. however, there are some distinctive features for russia. "russia is a riddle wrapped in a mystery inside an enigma." these words by the famous british statesman winston churchill aptly throws light on the pulsating art and culture of russia. the different aspects of russian art and culture find its best expression in the rich heritage. just feeling the nature and boundless expanses of the country can understand the mysterious russian soul, works of art and real masterpieces. cardiac mdct in children with congenital heart diseases v. bereznitskiy, k. serkova; moscow/ ru (vsber@mail.ru) in ct studies of children with congenital heart diseases, it is mandatory to keep the x-ray dose, amount of contrast agent and study time as low and as short as possible and simultaneously get the best diagnostic image quality. how can we reduce the x-ray dose? by minimising the volume coverage, reducing the voltage, minimising the effect of "overranging", use of x-ray protective equipment, use of tube current modulation technique, use of iterative reconstruction technique. how can we reduce the amount of contrast agent by volume? by covering of only necessary anatomic volume, minimising scanning time, use of adequate speed injection in ml/sec, which depends on catheter position and size, blood flow and anatomic structures. how can we reduce the amount of iodine concentration in the contrast agent? by lowering the voltage we may reduce the iodine concentration and get higher absorption ct values (hu). how can we reduce the time of cardiac ct studies? by preparing the study protocol ahead, preparing all necessary equipment in the ct room, warming up of the contrast agent before the study. what is the radiographer's role in cardiac ct studies? to take care along with the responsible radiologist the above-mentioned needs, to take care of adequate room and instrument temperature, to take care that only absolutely necessary people with adequate x-ray protection are in the study room, for performing the necessary image post-processing, archiving and distribution of studies. the purpose is to evaluate the quality of imaging in forced position of patients with anesthetic support. the role of laboratory technician in ct post processing. we analysed images of unconscious patients on artificial lung ventilation on -and -slice computer homographs ge and siemens and monitored them through the infusion. during investigation the patients were in forced positions, as a result they got artifacts from upper extremities on the abdominal cavity. we analysed patients -hands under the butt, patients -hands on the chest (abdomen), patients -arms along the body. according to the images the smallest number of artifacts was identified when the patient was on the table with hands under the butt. different variations of the patient's hands position and the problem of choice of this position will be offered for researching. the reconstruction the laboratory assistant can execute and also the results of tests will be presented. by order of the doctor the laboratory technician builds d and d reconstructions of fractured ribs, pelvis, facial skeleton, sagittal and coronary reconstructions of spine in case of its fracture. gastrointestinal stromal tumours (gist) are a rare disease that metastasises in up to % of patients with subsequent median progression-free survival (pfs) of around months. tumours are characterised by activating mutations in the kit or the pdgfra gene and treatment is mainly based on tyrosine kinase inhibitors designed to block mutated receptors. however, drug resistance is often based on mutations changing the conformity of the receptor, leaving little effective therapeutic options. to date, second line chemotherapy offers a median pfs of - months and external beam radiotherapy is limited by organs at risk close to the tumour. alternative approaches like endoradiotherapy or minimally-invasive ablation techniques are effective for local control but are inconsistently used and are not tailored to the individual patient's type of disease. to address these issues, the mitigate project proposes a closed-loop personalised treatment concept combining endoscopic-assisted tissue sampling, inline biotechnology and targeted molecular pet imaging probe development combined with minimally-invasive treatment monitored by new mr imaging techniques. a consistent value chain across european research institutes and smes will be established for mass spectrometry of tumours, linkage of radiochemical molecular imaging probes, design of new animal models and targeted therapeutic radiopharmaceuticals. this closed-loop platform will minimise fragmentation of treatment approaches by a coherent molecular-based multimodality concept, thus providing new treatment options. the mitigate platform can be expanded to further patient cohorts with oligometastatic diseases such as other sarcomas or renal cell carcinoma. gastrointestinal stromal tumours (gist) are characterised by highly specific genetic mutations. several specific targets are overexpressed in a majority of the tumours. on the other hand, secondary resistancies limit the usability of highly specific chemotherapeutics such as imatinib. instead of applying cascades of alternative tki inhibitors in order to find an efficient one, quantitative molecular imaging and targeted endoradiotherapy could be considered. for the detection of gist by imaging, ffluorodeoxyglucose (fdg)-pet is widely accepted to visualise the tumour burden. however, as fdg gives no molecular information of potential targets for treatment, new radiopharmaceuticals have to be developed to visualise and quantify other target-structures expressed by gist that would be able to be addressed. this molecular information can be used both for designing a conventional treatment or an advanced strategy: i.e. targeted endoradiopharmaceuticals. compared to conventional chemotherapy this method has the great advantage that not only tumour cells expressing the targeting vector are affected but also tumour cells within the range of the radiation of the decaying radiopharmaceutical. this method is used with great success in the case of neuroendocrine tumours using labeled octreotate derivatives (a small cyclic peptide binding to the somatostatin-receptor) and non-hodgkin's lymphoma applying y- labeled zevalin (a monoclonal antibody binding to the cd antigen). the success of these methods relies on the crossfire-effect, meaning that a certain margin of tissue surrounding the cell targeted by the specifically accumulating endoradiopharmaceuticals is destroyed as well, depending on the penetration range of the emitted radiation. thus, the development of new radiopharmaceuticals for the specific diagnosis and treatment of gist would strongly improve the outcome of the tumour patient. stereotactic radiofrequency ablation of liver tumours: does improved targeting increase tumour response? r. bale, g. widmann, p. schullian, m. haidu, w.r. jaschke; innsbruck/at (werner.jaschke@i-med.ac.at) the purpose is to describe stereotactic radiofrequency ablation (srfa) and to review its inital results in the treatment of patients with primary and secondary liver tumours. one retrospective review includes consecutive patients with srfa sessions for the treatment of hepatocellular carcinomas (hcc) and metastases ( ). in two other studies the outcome after srfa of intrahepatic cholangiocellular carcinomas (icc) ( ) and colorectal liver metastases (crlm) ( ) was evaluated. the overall technique effectiveness was . % with a local recurrence at year of . %. technique effectiveness was not significantly different for lesions< cm ( . %) and - cm ( %). after treatment of inoperable consecutive patients with iccs a median overall survival of months was achieved. a median os of months and os rates of %, % and % at , and years in patients with resectable crlm were achieved. tumour size did not affect os and dfs. rfa probes can be precisely planned, positioned and distributed by means of stereotaxy. especially in large liver tumours the local recurrence rate can be greatly reduced by precise stereotactic placement of multiple radiofrequency probes. these improved local control rates result in better long-term survival rates. author disclosure: r. bale: shareholder; coshareholder atlas aiming device. other; coinventor atlas aiming device. functional and molecular imaging in tumour therapy response assessment s. aime; turin/ it (silvio.aime@unito.it) in the mitigate project further development of currently used mri and ct techniques will be tackled in order to obtain functional and metabolic information which are mandatory to detect early response to the targeted radiopharmaceutical approach. new gd-based agents are under scrutiny in order to improve dce-mri procedures and attain more precise information on the tumour microvascularity level as well on the vessel permeability and the assessment of extracellular ph and mmp activity. the x-nuclei mri ( na sodium) is a non-invasive imaging technique, which enables the measurement of the tissue sodium concentration (tsc) in humans. the direct link of the tsc with the tissue integrity and viability provides a promising approach for monitoring tumour tissue, and could also help to assess cytoxicity and cell death by means of breakdown of the sodium-potassium-pump. however, na mri is challenging because of the low in vivo abundance, the fast transversal relaxation rate and the low gyromagnetic ratio of the na ions in human tissue. new mr measuring sequences for x-nuclei mri will be developed to establish a standardised tsc measurement method within the eu for better comparability. furthermore, the x-nuclei mr data will be reconstructed, scaled and fused with further imaging modalities. lung cancer is the leading cause of death related to cancer. most patients are inoperable as they present with advanced stage disease or even a localised tumour associated with poor general condition, limited cardiopulmonary function or a too high surgical risk. according to the stage of the disease, chemotherapy, radiotherapy and percutaneous ablation therapies are the current therapeutic options for inoperable patients. it is important that radiologists are familiar with the various response and complication imaging patterns related to each of those treatments. the timeline modifications after radiation therapy and percutaneous ablation and the diagnostic management of drug-induced lung disease will be reviewed. this presentation will summarise the current evidence and how to detect early recurrences after those treatments. learning objectives: . to learn about changes after radiotherapy. . to learn about sequela after percutaneous ablation of lung tumours. . to understand changes after chemotherapy of lung cancer. cardiovascular medicine owes much of its spectacular development to the parallel evolution of imaging techniques in the last thirty years. the complex contemporary techniques in interventional cardiology, electrophysiology, and cardiac surgery require advanced imaging modalities. in current times, a wide spectrum of imaging techniques -such as fluoroscopy, mri, non-contact mapping, echo techniques -is performed in organised cardiovascular labs while computed tomography and nuclear techniques are provided by the collaborative departments of radiology and nuclear medicine. it is indeed the collaborative spirit among specialists that bring about the results patients desire. cardiac magnetic resonance (cmr) is a very versatile imaging technique for the assessment of patients with left ventricular dysfunction and has become a central method for characterising the etiology of the dysfunction. indeed, wall thinning less than mm and aneurysmal motion are morphological signs of transmural necrosis. furthermore, a low dosage of dobutamine ( - microg/min) is used to detect functional reserve in the case of viable tissue. a further option offered by cmr is late contrast enhancement (le) imaging, which allows identifying the presence of fibrotic tissue as well as necrotic infarcted myocardium. the sensitivity and specificity of late hyperintensity is, respectively, . and . . similar results can be obtained with mtctetrofosmina g_spect. there is a relation between transmural extension of hyperenhancement and the recovery of contractile function after coronary revascularisation. the chance of recovery decreases progressively with the transmural extension of late hyperintensity, since the average value of the latter is ± % in the segments that recover and ± % in the segments that do not recover contractile function. cmr was also shown to be able to predict response to cardiac resynchronisation therapy differentiating between nonischemic and ischaemic cardiomyopathy le-cmr may allow to precisely delineate the presence and the location of fibrosis, avoiding the placement left ventricular leads in necrotic areas and in particular when the scar is located in the posterolateral area. at the beginning of the presentation, a short overview will be given on the relevance and prevalence of valvular heart diseases with special focus on europe and the data collected in the euroheart survey. also, the aetiologies of the various valve diseases will be briefly mentioned. in the second part, special attention will be given to aortic stenosis (as) and mitral regurgitation (mr), as these two entities are responsible for up to % of all valvular heart diseases. echocardiography is well accepted as the backbone examination of any valvular heart disease evaluation. echocardiographic criteria for severe as and mr are reviewed as well as their importance for the indication of valve repair or valve replacement. in the third part, we will focus on newer "cross-sectional" imaging technologies such as cardiac mr and ct. in particular, the strengths of cardiac mr in the assessment of regurgitant lesions will be demonstrated. in addition, the value of cardiac mr for the pre-interventional evaluation of congenital heart diseases will be discussed. similarly, the crucial role of cardiac ct in the workup for transaortic valve implantation (tavi) will be illustrated. finally, the yield of these novel techniques in the context of combined valvular and ischaemic heart disease will shortly be reviewed. learning objectives: . to better understand the role of cardiac mri in valvular heart disease. . to appreciate the added value of mri vs other imaging modailities. cardiac resynchronisation therapy (crt) and transcatheter valve heart (thv) interventions have been important therapeutic breakthroughs for heart failure patients and patients with valvular heart disease and contraindications for surgery. patient selection is crucial to optimise procedural outcomes and therapeutic efficacy. multimodality imaging plays a central role in patient selection and procedural strategy planning. assessment of left ventricular dyssynchrony, cardiac venous anatomy and extent and location of myocardial scar are key to identify heart failure patients who will respond to crt. stepwise use of non-invasive imaging modalities, including echocardiography, nuclear imaging, magnetic resonance imaging and multidetector row computed tomography (mdct), has been common practice to select patients for crt. recent technological advances have allowed integration or fusion of imaging modalities to create -dimensional models that provide relevant information. multimodality imaging is also crucial to optimise the results of thv interventions. detailed anatomical and geometrical evaluation of the mitral valve with current -dimensional imaging techniques has permitted better selection of patient candidates for transcatheter edge-to-edge mitral valve repair and accurate procedural guidance. for transcatheter aortic valve implantation, accurate measurement of the aortic annulus with -dimensional echocardiography or mdct allows us accurate selection of the prosthesis size to minimise the risk of complications, and evaluation of the procedural access (transarterial or transapical) with mdct permits accurate procedural strategy planning. the learning objectives of this session include: providing an overview of current imaging modalities to select patients for crt and thv interventions and how to use them in clinical practice. the inner layer of the anal canal consists of squamous and columnar epithelium with the transitional zone at the dentate line. the muscular component of the anal sphincter consists of an inner layer of circular smooth muscle (internal sphincter), extending downwards from the rectum, and an outer striated muscular layer extending downwards from the levator ani muscle, comprising the puborectalis muscle and the external sphincter. between these layers is the fat-containing intersphincteric space, including the continuation of smooth-muscle fibers of the longitudinal muscle of the rectum. outside the anal sphincter is the fat-containing ischioanal space. magnetic resonance imaging (mri) and endoscopic ultrasonography have become the mainstay for preoperative imaging of perianal fistulas. for complex tracts, mri seems preferable. mri can be performed using an endoluminal coil or a phased-array surface coil. a state-of-the-art imaging protocol should include t tse sequences in three orthogonal planes, with the axial and coronal sequences angulated at the anal canal. addition of a fat-saturated t tse sequence is recommended for optimal conspicuity of inflammatory changes and post-contrast t -weighted imaging can also be helpful. intersphincteric infection is the principal feature of perianal fistulas; this is generally not found in other conditions. veins can be mistaken for fistulas, but in contrast to fistulas, veins usually are thin-walled, tortuous, symmetric structures. a pilonidal sinus may resemble a fistula, but absence of extension to the intersphincteric space helps one to discriminate between them. haemorrhoids and anal tags may resemble small submucosal fluid collections, but are easily diagnosed at clinical examination. this lecture will describe the pathogenesis of fistula-in-ano, with a focus on cryptoglandular disease, so that the radiologist can understand how the various classifications of fistulas arise. the park's classification for fistula-inano will be described with reference to cryptoglandular disease and other aetiologies. the surgical questions that need to be answered by imaging will be presented, followed by a description of what the radiologists need to include in their report for it to be clinically useful. the role of anal ultrasound and mri for preoperative fistula imaging will be described, with explanation as to why the latter is fundamental to modern management of the disease. perianal fistulising disease develops in approximately half of the adult and paediatric patients with crohn's disease (cd), with a relevant impact on their quality of life. in patients with cd, particularly those with severe fistulising ileal or colo-rectal disease, complex ramified anal fistulas associated with abscesses (parks' classification type - or s.james hospital classification type - ) are more frequently observed than in other patients. perianal disease thus represents a severe complication of cd, which may change disease management, by requiring specific pharmacological and/or surgical treatments. a non-responsive disease eventually may lead to total proctectomy with ileostomy. mri represents the gold standard diagnostic modality, because it provides a comprehensive staging of both enteric and perianal disease, with high accuracy and without invasiveness. moreover, mri is the ideal tool for monitoring disease response to therapy, being able to detect the inflammatory activity of perianal fistulas with high accuracy. so far, several clinical indexes have been proposed to measure fistula's activity, including the perianal disease activity index (pdai). similarly, several mri classifications of fistula's activity, based either on t -weighted or t -weighted contrast-enhanced imaging, have been proposed, although a widely accepted imaging severity score is not available yet. several cases of complex perianal fistulising cd of increasing severity will be shown, with anatomical and clinical correlations, staging and activity description, particularly in patient follow-up and treatment monitoring. finally, differential diagnoses with other benign conditions which may affect the anal canal, including inflammatory infective diseases, hydradenitis and pilonidal disease will be discussed. ground glass opacity (ggo) is characterised on hrct by the presence of a hazy increase in lung opacity that does not cause obscuration of underlying bronchial and vascular margins. although a very common finding, it also constitutes a very nonspecific term since it can be seen in a variety of different intraalveolar and interstitial processes with different histology including inflammatory, infectious and neoplastic diseases that have a common physiologic mechanism: partial displacement of air. ground glass opacity may even be seen in normal processes such as poor ventilation in dependant lung areas and in expiration. moreover, ggo can represent either an ongoing, active and potentially treatable disease or an irreversible process. to interpret correctly this highly nonspecific but very significant finding, it is crucial to attempt to further classify the different large main entities in which this radiological finding appears. are there specific radiological and hrct findings that can help us differentiate ggo in autoimmune inflammatory conditions from infectious and neoplastic processes? are there associated findings other than ggo, such as nodules, reticulation or focal disease, and distribution of findings that can narrow the differential diagnosis? systematic evaluation of ggo and associated findings as well as integration with clinical information (acute, subacute or chronic symptoms) is essential in defining ggo subtypes to improve the radiological diagnosis. radiologists who regularly review high resolution ct (hrct) should be aware of the range of patterns and, more importantly, their potential pathological meaning. a pattern of ground-glass opacification is one of the more common hrct findings but, to the unwary, its interpretation can be problematic. an important underlying principle is that a ground-glass pattern may be caused by any process -physiological or pathological -which partially displaces air. physiological (i.e. non-disease-related) ground-glass opacification is perhaps most commonly seen in subjects who, for whatever reason (e.g. breathlessness, obesity), are unable to maintain or achieve a satisfactory inspiratory effort during scanning. a generally increased lung density (in contrast to adults) is also a feature in infants and young children simply because there are fewer alveoli in the developing lung. finally, it is worth noting that intravenous contrast administration (presumably because of a relative but temporary increase in capillary blood volume causing partial displacement of air) can unpredictably increase lung density. disease processes which lead to partial filling of the air spaces, thickening of the interstitium, partial collapsing of alveoli, and/or increased capillary blood volume will also manifest as a pattern of ground-glass opacification. in clinical practice, the recognised causes of ground-glass opacities on hrct include pulmonary oedema (cardiogenic or otherwise), infections (e.g. pneumocystis jiroveci pneumonia) and some of the idiopathic interstitial pneumonias (e.g. non-specific interstitial pneumonia and respiratory bronchiolitis associated interstitial lung disease). the presentation will review and revise the causes of physiological and disease-related ground-glass opacification on hrct. ground-glass opacity (ggo) is defined as increased attenuation of the lung parenchyma without obscuration of the pulmonary vascular markings on ct images. ggo may be the result of a variety of interstitial and alveolar infectious and noninfectious inflammatory diseases. as an imaging finding alone, ggo does not usually allow a specific diagnosis. ggo in inflammatory disorders is often present in the company of other interstitial or alveolar findings. however, the number of diseases that cause diffuse isolated ggo or ggo as the predominant finding is relatively small and can be prioritised with clinical information. the most common cause of diffuse isolated ggo in immunocompromised hosts are a variety of diffuse, opportunistic pneumonias, e.g. pneumocystis jiroveci pneumonia (pcp), cytomegalovirus pneumonia (cmv) or herpes simplex pneumonia (hsv), which constitute the first differential. chronic onset disorders in immunocompetent patients include cellular nonspecific interstitial pneumonia (nsip), subacute hypersensitivity pneumonitis (hp), organising pneumonia, air-space sarcoid, and drug-induced lung disease. in these disorders, ancillary findings such as an associated reticular pattern with traction bronchiectasis/bronchiolectasis (nsip), mediastinal lymphadenopathy (sarcoidosis), superimposed nodularity or cysts or may help to refine the diagnosis. in patients with collagen vascular disorders, e.g. scleroderma, ggo secondary to pulmonary involvement needs to be differentiated from drug-induced lung disease. this refresher course will put ggo in the context of outpatients versus inpatients, the acuity of clinical symptoms, e.g. fever, cough and dyspnoea, signs of massive systemic inflammation, and the clinical situation such as inhalational history, pneumotoxic drug administration, immunocompromise, or bone marrow suppression. in this course, we will review the dysplastic and neoplastic conditions that are associated with persistent ggo in the lung parenchyma. we will separate these conditions into those that are responsible for localised diseases such as single nodular ggo and those responsible for more extended diseases. nodular ggo can be separated into pure or mixed or part-solid nodules. pathology-radiology correlations show that nodular ggo are related to atypical adenomatous hyperplasia (aah), adenocarcinoma (adc) in situ (ais), minimally invasive adc (mia), and invasive adc according to the new iaslc/ats/ers lung adenocarcinoma classification. differential diagnosis includes exceptional metastases of angiosarcoma and melanoma as well as infection, inflammation and localised fibrosis. diffuse ggo related to neoplastic conditions is rare and may be due to lepidic adc (former advanced adc with bac component), diffuse large b-cell non-hodgkin's lymphoma, intravascular lymphomatosis (ivl) or mucosa-associated lymphoid tissue (malt) lymphoma. these neoplastic diseases should be differentiated from infectious and inflammatory causes of diffuse ggo. we will review the value of different morphological ct criteria to differentiate benign from malignant localised ggo, such as the size, the morphology (round, oval, flat), presence of mixed vs. pure ggo, and the multiplicity of nodular shadows. discussion will also include the changes that may occur within the nodule as well as the mean doubling time. the role of pet ct and transthoracic biopsy will be discussed. finally, we will present the current recommendations regarding the management of nodular ggo. it is the purpose of this lecture to briefly discuss ankle distortion-related lesions with emphasis on failed radiographic diagnosis, discuss their significance and management implications and define the diagnostic pathways in the acute and subacute stage. significant lesions are those with no return to sports activities after months; these have our major interest. in the acute setting of ankle distortion, the ottawa clinical decision rules are accepted to decide whether or not radiographic evaluation is needed; about % of radiographic examinations can be avoided. additional evaluation is restricted to patients with residual pain during reevaluation one week after trauma. assessment of fractures with the lauge hansen classification has a major advantage, as it defines the stability at the talocrural and distal tibiofibular joint. only in restricted cases, additional examinations may be needed to rule out instability. although radiographs easily detect displaced fractures, occult fractures may occur; also, grade ii-iii ligament lesions and peroneal tendon dislocation may go undetected. additional imaging evaluation should focus on the detection of significant lesions. significant lesions are talar fractures and complete calcaneofibular ligament tears. the latter is explained by the instability at the level of the subtalar joint with associated sinus tarsus syndrome. in case of residual local tenderness posterior and inferior to the lateral malleolus, the fibulocalcanear ligament and retinaculum peroneorum lesions are best evaluated with (dynamic) ultrasound. mri or ct is used to detect occult fractures in case of residual talocrural joint effusion with anterior talar tenderness during endorotation and plantar flexion. the ankle and foot can be affected by inflammation from a number of diseases. the main focus of this lecture will be inflammation secondary to infection and systemic inflammatory disorders. due to its function in locomotion and weightbearing, the foot is specifically susceptible to infections secondary to penetrative trauma including foreign bodies or to abnormal repetitive pressure. in addition, both seropositive and seronegative arthropathies can affect the foot. other unusual causes of inflammation include chronic recurrent multifocal osteomyelitis, osteoarthritis, mechanical disorders and sensory loss. this lecture will deal with the various patterns of inflammation in the context of pathogenesis and discuss the imaging features that aid in diagnosis. the importance of the clinical context in diagnosis will also be discussed. characterising tumours before deciding on the most appropriate treatment is a general rule in oncology. cystic tumours are now adequately graded based on bosniak's classification. however, this pre-therapeutic characterisation is not currently systematic in case of solid renal tumours, as it remains difficult in clinical practice due to the high prevalence of renal cell carcinomas (rcc) and the lack of reliable imaging criteria for recognition of benign versus malignant tumours. as a result, approximately % to % of surgically excised renal masses are actually benign. to avoid these unnecessary ablations, pretherapeutic image-guided biopsy has been proposed, but its use is still debated as it is invasive and time consuming. new validated imaging criteria, able to accurately differentiate the most common tumour types but also indolent vs more aggressive malignant lesions, would be useful to reduce the number of unnecessary biopsies or to consider the most appropriate treatment for a tumour or active surveillance. among these, mr imaging plays a major role based on chemical shift gradient echo (gre) sequences, signal intensity on t -weighted images, dynamic contrast-enhanced sequences, diffusionweighted sequences and late contrast-enhanced images. multiparametric mr imaging is now performed in clinical practice in most primary care centres. therefore, using different combinations of two or several parameters, it is now possible to distinguish certain renal tumours. a larger validation of all these combinations is still necessary to define those having a clinical significance for routine practice. targeted therapies such as vegf mab bevacizumab, vegf tyrosine kinase inhibitors (sunitinib, pazopanib, axitinib and sorafenib) and mtor inhibitors (temsirolimus and everolimus) have been approved and included in european guidelines. pre-therapeutic imaging prerequisites are mainly based on the natural history of mrcc. this justifies imaging through a thoracoabdominal ct scan. outside clinical symptoms, there is no need for bone imaging or brain ct scan; the latter nevertheless could be done because of influencing the therapeutic strategy. pet scan should not be done due to low sensitivity. the evolution of metastatic sites by comparison from previous imaging is useful to evaluate the natural history. outside clinical trials or research imput the strategy is to use the drug in an optimal way to increase pfs. so far, monitoring has to inform about change in recist criteria for cr, pr, sd or pd, and if pd is a real pd such as recist pd or a slowing pd. in conclusion, standard ct scan and recist are adapted to real life. nevertheless, more information on the mode of action of targeted drugs should be interesting for knowledge and research purpose, without any impact on the management of patients. additional imaging could be done with additional criteria than recist on ct scan, by vascular functional imaging and pet scan. breast mri is recognised as a useful tool for a number of clinical indications, but remains relatively challenging. recognising the importance of an evidencebased approach to the development of protocols will influence the further integration of this technique into everyday clinical practice in the management of breast cancer patients. there remain concerns regarding the potential of over-diagnosis from breast mri. the aim of this integrated rc is to inform the clinical practice with regard to the establishment of breast mri in specific clinical and imaging scenarios, namely, using mri for surveillance in high-risk patients, monitoring response to neo-adjuvant chemotherapy and developing protocols for the indeterminate imaging scenario of non-mass-like enhancement. the establishment of protocols to maximise the specificity and sensitivity of the technique of breast mri in such indeterminate clinical and imaging scenarios will be discussed. non-mass like enhancement is a frequent finding in breast mri. it relates to the fact that enhancement occurs in the normal-appearing fibroglandular tissue that surpasses that of the other parts of the parenchyma. there is no associated space-occupying lesion. in pre-contrast non-fat-suppressed or fatsuppressed t -and t -weighted images, there is usually no correlate observable. the differential diagnosis of non-mass-like enhancement is between benign nonspecific fibrocystic disease/adenosis, hormonal stimulation, and subclinical mastitis on one hand, vs dcis or (less likely) diffusely infiltrating (usually lobular) cancer on the other. key components of differential diagnosis is configuration of the enhancement (does it follow the orientation of the milk duct or not?) and symmetry (symmetric or asymmetric). less important criteria are internal enhancement (internal architecture) and enhancement kinetics. it is important to realise that enhancement kinetics can only be used to corroborate the suspicion of dcis -but they cannot be used to alleviate the indication to biopsy a finding which, based on configuration and asymmetry, is suspicious. management depends on the different constellation of clinical, mammographic, and mri findings. it usually includes short-term follow-up ( months) and, if stable/persistent, mr guided vacuum biopsy. assessment of the change in tumour burden is an important feature of the clinical evaluation of cancer therapeutics. tumour shrinkage and time to the development of disease progression are important end points in cancer clinical trials. however, these end points are useful only if based on widely accepted and readily applied standard criteria. criteria, known as recist, were published and are updated on a regular base. the revised recist includes a new imaging appendix and underlines the importance of moving from anatomic unidimensional assessment to either volumetric anatomical assessment or functional assessment with pet or mri. the goal of the lecture is to discuss the value of the different mr imaging techniques (e.g. mr spectroscopy, diffusion-weighted imaging, angiogenesis mapping, pet-mri), which evaluate response to treatment in breast cancer. participants will learn about particular imaging challenges of assessing response to neo-adjuvant chemotherapy. in addition, they will learn to understand clinical aspects of neo-adjuvant chemotherapy based on recist. currently, the esr has been using social media in many successful ways to improve the contacts among members and to boost our fast development as one of the most advanced scientific communities in the world. our session "the role of social media in radiology" presents the view of experts, acknowledged leaders of international it projects. it seems to be important to discuss the major implications of social media for radiology, the leading clinical discipline in information technology. davide caramella will clarify the relationship of social media to professional societies and especially to the esr, including their basic features. the "social media generation" has developed from schoolkids to students and, recently, to teachers as well. alexander sachs represents the young professional expert of elearning in radiology and will reflect the current user behaviour. osman ratib will share his view on how social networks enhance the communication between radiologists, nonradiologists and patients. critical aspects of the pros and cons of large communication scenarios are followed by some ideas of future developments. jürgen brandstätter will indicate the patient´s view. most patients see social network communications with their radiologist as useful for, e.g. appointment management or other non-sensitive communication. besides communicating personal health data, a wide area of potential useful other possibilities are at hand, like anonymised health data for reviewing purposes, second opinion or education of patients. the fast development of social media may induce hopes and fears; the four speakers of this session will present them in a panel discussion. the widespread use of social networks in all professional activities has attracted a great interest in the recent years. in medical applications, the main concerns include the possible consequences in terms of patients' confidentiality protection, perceived role of the physician, patient-physician relationship, and caregivers' distraction during professional activities. there are no doubts, however, that social networks are beneficial in enhancing the information flow between scientific societies and their professional members, without any interference at the point of care. social networks have proved to be extremely effective in updating members about relevant news concerning their areas of interests and in allowing them to be informed about the educational activities promoted by their scientific societies. this presentation focuses on the social network activities promoted by the esr via facebook, youtube, twitter and other blogs. impact of social media on the training of radiologists a. sachs; vienna/ at (alexander.sachs@meduniwien.ac.at) in modern times, social media have gained more and more important value in exchanging first experiences in radiology from resident to resident. social networks such as facebook, twitter, google plus and others are used to start discussion groups about cases and share the growing knowledge with different educational concepts in each hospital. growing e-learning modalities as moocs, applications for smartphones or tabs and web-based case series or learning platforms support the training of radiologist in social media. modern elearning platforms use social media as their first choice of sharing knowledge around the whole world about certain illnesses, cases or different decisions of therapy. though sharing information seems to be easier and shows several advantages than in former times, social media can also highlight new problems. this lecture focuses on user behaviour to improve learning, the pedagogic evidence for large group learning with networks and the importance of using networks to manage time. social media networks in communication between radiologists, nonradiologists and patients o. ratib; geneva/ch while social networks have gained unprecedented popularity in our daily environment, they have only limited utilisation in medical applications. the paradigm of communication within a given community for exchange and sharing of imaging data would certainly have its place in a medical environment where there is an increasing need for multidisciplinary collaborative work. the required security and confidentiality in management of sensitive patient data have often precluded the usage of existing public social networks. professional proprietary systems have emerged in a more restricted way with much less flexibility leading to limited use. the exchange of medical images adds a level of complexity due both to the size of the data and the need for appropriate browsing and visualisation tools. traditional pacs and teleradiology system offer such features, but in a very limited way, far from the desired convenience of social networking between groups of users. a prototype of such a system aimed essentially at the community of users that need to exchange anonymised medical images and data for academic and research purposes will be presented and discussed. the project nicknamed dicom sandbox is being tested and evaluated by some users in europe under the coordination of the esr subcommittee of ehealth and informatics. developed with open source software components, it allows using existing cloud-based storage services with additional layers of secure dicom file management and visualisation. it allows the community of users to share dicom data with all social networking and notification features beyond simple peer-to-peer exchange. interactions between patients and radiologists through social media j. brandstätter; wiener neudorf/ at (j.brandstaetter@codewerk.at) social networks have become integral parts of most citizen's use of the internet. especially, the younger generation uses social networks extensively; however the way those tools are used should be chosen carefully. it is one thing to use a social network for private interests, but a different one to use it as transport media for sensible data like personal health-care data. the sensitiveness of citizens regarding privacy has been increased recently by different events of misuse or fraud, especially accelerated by the recent privacy affairs caused by governmental agencies. though most patients see social network communications with their radiologist useful for, e.g. appointment management or other non-sensitive communication, personal medical reports or images shall not be shared across this media. besides communicating personal health data, a wide area of potential useful other possibilities are at hand (e.g. anonymised health data for reviewing purposes, second opinion, education of patients, …). future developments shall make use of social media where useful, but always with deep respect to the patient's privacy rights. apart from the historical, but ever present clinical expectation from the neuroradiologist is the answer to the most important question if the patient has a brain tumour or not. nowadays, due to the outstanding technological advances in imaging techniques during the last decades, the stakes are growing much higher and it is the neuroradiologist on whom lies the initial responsibility of answering a large number of questions that are of utmost importance for further brain tumour patient care and treatment. having the opportunity to provide the usual and generally basic information regarding tumour morphology and exact location and to narrow the differential diagnostic possibilities, neuroradiologic expertise now opens a wide spectrum of techniques, especially those derived from mri, such as mr spectroscopy, mr perfusion, dwi, dti, swi, etc. these provide us the unique possibility of not only obtaining and comparing images, but also of exploring multiparametric brain maps, allowing us to gain insight into the internal architecture of the tumour and the functional and molecular features of the tumorous tissue. this consequently defines the actual stand of neuroradiology, indicating the uprising clinical urge not only to provide the answers regarding the tumour type, grade and possibilities of the most appropriate and patient-individualised therapy modality, whether surgical or nonsurgical, but also to accurately assess the post-treatment tumour behaviour and status, by differentiating the true tumour progression from pseudoprogression, represented by inflammatory responses to the radio-and/or chemotherapeutical treatment, and to monitor other neurooncological response assessment criteria. a. differential diagnosis and pseudo-tumoural lesions h.r. jäger; london/uk (r.jager@ucl.ac.uk) conventional contrast-enhanced mr imaging gives an indication about the likely tumour type and histological grade, but advanced mr imaging methods, including perfusion-weighted (pwi) and diffusion-weighted (dwi) imaging, mr spectroscopy (mrs) and pet imaging provide additional information which helps to refine the diagnosis of intrinsic brain tumours. contrast enhancement can be a feature of low-grade (pilocytic astrocytoma and ganglioneuromas) as well as high-grade gliomas (who grade iii anaplastic gliomas and who grade iv glioblastomas). infiltrative low-grade (who grade ii) astrocytomas do not usually enhance, whereas who grade ii oligodendrogliomas can enhance. higher-grade glial tumours (who grade iii and iv) tend to have an elevated relative cerebral blood volume (rcbv) on pwi, a decreased adc on dwi and increased choline turnover on mrs and pet imaging. a raised rcbv can, however, also be seen in low-grade oligodendrogliomas, particularly in those with a p/ q chromosomal translocation, which is associated with a better response to chemotherapy. pwi and dwi are useful in the differentiation of glial neoplasms from cerebral lymphoma, which has an increasing incidence in the immuno-competent population. dwi and pwi are also very useful in the differentiation of enhancing pseudo-tumoural lesion such as cerebral abscesses, tumefactive demyelination, pseudoprogression and radiation necrosis. perfusion imaging plays an important role in the characterisation and management of brain tumours. low-grade brain tumours may transform into high-grade gliomas at some point, which is a transformation that is highly variable and difficult to predict in an individual patient. conventional contrastenhanced computed tomography (ct) and magnetic resonance (mr) imaging are found to be insufficient to depict this transformation and angiogenesis and therefore to detect malignancy. however, perfusion imaging provides additional information over conventional imaging in terms of tumour physiology and haemodynamics, providing important biomarkers for malignancy and prognosis. perfusion ct and mr perfusion techniques such as arteriel spin labelling and dynamic susceptibility-weighted perfusion imaging are more practical and widely used compared to nuclear medicine methods. these perfusion techniques should be considered and used in routine clinical workup of brain tumours to asses grading and prognosis. maximal safe resection, radiation therapy and temozolomide chemotherapy are the current standard of care for newly diagnosed high-grade gliomas. still, hgg have a poor survival rate. a contributing factor to the poor survival is the inability of currently available imaging techniques to accurately delineate the tumour, with the result that targeted focal treatment my not be effective. conventional imaging is not able to give an early assessment of the effectiveness of radiation and/or chemotherapy. in addition, conventional imaging has difficulties in differentiating pseudo-progression, which is a common phenomenon in conventional chemo-radiation therapy, from true progression. early identification of patients who suffer from tumour recurrence can be of great advantage: it provides the opportunity to adjust individuals more rapidly, sparing patients unnecessary morbidity and delay in the initiation of other, maybe more effective, treatment. in recent years, different functional imaging approaches such as perfusion mri, diffusion-weighted imaging and spectroscopy, have been complementarily used for imaging evaluation of treatment response. in this lecture, different advanced mr and ct imaging methods used to support differentiation between pseudo-progression and true tumour progression to assess treatment response will be discussed. in addition, a novel recently published promising technique, the parametric response mapping (prm), a novel voxel-wise analytical method of monitoring physiological and environmental changes in a tumour volume during treatment will be presented and compared to the traditional methods used. finally, the aim of the lecture is to consolidate the present knowledge and novel ideas in brain tumour imaging for assessment of pseudo-progression versus true tumour progression. learning objectives: . to understand the challenges and limitations of routine mri in monitoring brain tumour treatment. . to become familiar with the role of advanced imaging biomarkers for early assessment of treatment response. . to learn how to integrate routine and advance mri into clinical practice after tumour therapy. the purpose of this lecture is to describe the role of functional radiological imaging modality-ct and mr perfusion in the evaluation of head and neck tumors. conventional contrast-enhanced ct and/or mr imaging are the current standard techniques for the diagnosis and treatment evaluation of the head and neck tumors. however, this method is limited in its ability to depict the angiogenesis which is a hall-mark of tumor growth. perfusion imaging provides a rapid evaluation of tissue perfusion and can be easily implemented in every head and neck ct or mr protocol. the determination of tissue perfusion is based on examining the relationships between the arterial, tissue and potentially the venous enhancement after the introduction of a bolus of contrast material. the quantification of the perfusion values helps to outline the malignant tissue as well as to differentiate recurrent disease from nonspecific post-therapeutic changes and can be used as a therapeutic monitoring tool during and after tumor therapy. in recent years diffusion-weighted mri (dwi) gained increasing importance and also acceptance in head and neck radiology. although this functional technique is challenging due to air tissue interfaces, its ability to improve the diagnostic accuracy is widely accepted. dwi can be applied to detect lesions not only in adults, but also in children. it is helpful in lesion characterisation and has a great potential to differentiate recurrence from posttherapeutic changes. initial promising results to improve lymph node staging however are difficult to reproduce. image interpretation is performed qualitatively based on visual assessment of the high b-value images (b= - sec/mm ) and the corresponding apparent diffusion coefficient (adc) map. quantitative image analysis is based on the measurement of the underlying adc value. solid tumors including recurrences are bright on the high b-value images and dark on the corresponding adc map, whereas posttherapeutic changes are bight on both the high b-value images as well as the adc map. cystic and necrotic lesions are typically dark on the high b-value image and bright on the adc map. furthermore, malignant lesions tend to have a lower adc compared to benign and cystic lesions provided that no necrosis is present. therefore, image interpretation always has to be performed together with morphological images to avoid misinterpretation of functional images. pet/ct has established itself as a robust, rapid and reliable technique in head and neck oncology. it is mainly used to stage nodal disease, to detect distant metastases and synchronous tumours, to identify unknown primary tumours in patients with metastatic neck nodes, to assess treatment response and prognosis after chemoradiotherapy and for radiotherapy planning. mri with diffusion-weighted imaging (dwi) is routinely used for the assessment of submucosal tumour spread, to stage nodal disease, to monitor treatment response and to detect recurrent disease. the information provided by pet/ct and mri is often complementary and the recent implementation of hybrid mr/pet systems in clinical settings holds promise because it can combine morphologic, functional and molecular information. this lecture focuses on clinical applications of pet/ct and mr/pet in the head and neck. current evidence about the combined use of pet/ct and mri with dwi is discussed. the principles of mr/pet data fusion are summarised as well as current knowledge regarding the diagnostic performance of mr/pet in the head and neck. typical radiologic findings of tumour manifestations are reviewed with particular emphasis on the early detection of lesions, their appearance on different imaging modalities and the added value of hybrid imaging techniques. the variable appearance of functional phenomena mimicking disease as well as the potential pitfalls of image interpretation and how to avoid them are equally addressed. major emphasis will be put on how to report the findings in a comprehensive way. multi-energy and functional imaging ct has reached a robustness allowing the use of this new technology in clinical routine for a variety of different clinical questions. this presentation will discuss the basic principles and the strengths and limitations of the techniques. implementations of multi-energy methods for material characterisation and of ct methods for functional imaging will be discussed. contrary to normal single-energy ct systems, multi-energy ct scanners allow simultaneous scanning at two peak x-ray energies. when the attenuation is measured at two energies, their values are not exactly proportional to each other, which open new diagnostic possibilities. measurements at two spectra can be achieved using multiple kvp and/or filtration or with detectors with energy discrimination. these methods have different pros and cons such as sensitivity to subject motion and dose efficiency. to be able to acquire functional data such as perfusion, images are acquired dynamically following the injection of a contrast agent and physiological models are used to convert the measured contrast agent concentration to perfusion estimates. methods that acquire multiple images have the potential to increase the radiation dose to the patient, so ct protocols need to be optimised. in computed tomography, d images are reconstructed from a number of x-ray radiographs that were acquired at different angles. tomographic reconstruction relates to the generation of a d image based on these acquired radiographs. most tomographic reconstruction methods are based on a technique called filtered back projection (fbp) in which a sharpened version of the radiographs are back projected to generate the d image. while it has been the standard reconstruction method, mostly due to its speed and good quality if many radiographs are available, iterative reconstruction methods are emerging. the reason for that is that fbp suffers from important disadvantages. in this talk, the basic concepts of iterative reconstruction are explained and its virtues are detailed. the flexibility of iterative reconstruction methods is demonstrated for the field of x-ray computed tomography. finally an outlook is given with respect to future research on tomographic reconstruction. interventional radiology (ir) has been increasingly applied in the management of obstetric and gynaecological haemorrhage. national reviews of maternal deaths from post-partum haemorrhage have recognised that maternal deaths may be prevented by ir and that all obstetric units should have protocols and arrangements in place to ensure appropriate and timely referral to ir. the important role that irs play in the management of fibroids has also highlighted how these techniques can be applied to other gynaecological conditions which may result in haemorrhage. this session will start by describing which gynaecological disorders may result in haemorrhage and the technical aspects of treatment followed by the published evidence for embolisation. the session will then focus on obstetric haemorrhage, with an emphasis on prophylaxis and how women deemed at high risk for haemorrhage might be managed safely. the techniques used for prophylaxis of haemorrhage will be described in detail and the published data presented. the final presentation will concentrate on the technical aspects of embolisation when haemorrhage has occurred to ensure the best results by knowing the relevant anatomy and appropriate technique of embolisation. the published evidence of the results of embolisation in pph will be presented. these presentations will be followed by a panel discussion on how irs can reduce the radiation dose whilst simultaneously ensuring successful outcomes for their therapies, an important issue in a young and fertile group of women. gynaecologic massive bleeding related to benign and malignant causes is under the scope of all radiologists because of being diagnostic and also because of its implication in interventional radiology procedures. the causes are uterine fibroids (uf) and post-partum hemorrhage (pph). however, oncologic disease, arterio-venous abnormalities, or iatrogenic injuries are less commonly known to be eligible for embolisation procedures. usually, surgical homeostasis attempts or radical hysterectomy is preferred to embolisation procedures. despite the good clinical outcome for hysterectomy and the hazardous success rate of conservative treatment, both are unsatisfactory to patients. pelvic artery embolisation (pae) could dramatically change the patients' clinical issue. the purposes of this lecture are to: revise the pelvic arterial anatomy (possible arterial anastomosis or variation); outline the aetiology of non-obstetric massive menometrorrhagia; expose the typical clinical presenting features; describe the imaging appearances of the etiologies; and discuss patient section, procedure technique, pitfalls and complications. obstetric haemorrhage occurring mainly in the postpartum period is a significant cause of maternal morbidity and death. blood loss of over and ml after vaginal delivery or caesarean section, respectively, complicates % of deliveries. major blood loss of more than ml occurs following less than % of all deliveries. the causes include uterine atony, retained placenta products, placental abnormalities, uterine rupture, lower genital tract laceration, cervical ectopic pregnancy and coagulopathies. generally, treatment is based on administration of uterotonic drugs, vaginal packing, surgical ligation of uterine arteries and even hysterectomy is sometimes needed. transcatheter embolisation of uterine, pudendal, vaginal arteries and ovaries is considered to be superior and should be the first-line treatment for intractable obstetric haemorrhage where interventional radiology is available. angiography reveals extravasation of contrast agent in more than % of these patients. the most frequent extravasation is from vaginal artery, and then from pudendal and uterine arteries. depending on the extravasation location and haemodynamic stability of a patient, f catheters or coaxial microcatheter technique is utilised for catheterisation. gelatin foam, microparticles acrylic glue and microcoils are the most frequent embolic agents. the embolisation procedure usually does not require general anaesthesia and can be repeated if bleeding continues. primary success rate in bleeding control is reported from to %. the most commonly reported long-term side effects after embolisation were transient buttock numbness and urinary frequency. the transcatheter pelvic artery embolisation for obstetric intractable bleeding is a fertility-preserving alternative to hysterectomy. medical imaging has nowadays integrated the diagnostic armamentarium of anosmic patients regarding not only qualitative assessment of the olfactory tract, but also quantitative evaluation of olfactory bulb volumes which are known to closely correlate to the olfactory function. many clinical studies on various pathological conditions have evidenced the value of such measurements in the workup of olfactory dysfunction for both aetiologic and prognostic purposes. imaging workup also plays a role in the medico-legal evaluation of post-traumatic anosmia together with electrophysiological and clinical olfactory tests. technical improvements in fibre tracking (ft) using diffusion-tensor imaging (dti) and appropriate designs of olfactory stimulation at bold-based functional mri (fmri) are expected to allow insights into the neurophysiological processes and circuitry of olfaction in the very near future. imaging workup of the anosmic patients will be the cornerstone of this lecture. the relevance of the different imaging techniques will be detailed. beyond the workup of anosmia, a comprehensive overview of the most common lesions of the olfactory tract seen in clinical practice will be given. additional review of lesions observed at the anterior cranial fossa in the near vicinity of the olfactory tract will be done, which comprehensively includes all commonly observed developmental, traumatic, inflammatory and neoplastic conditions not arising from the olfactory tract. their potential impact on the olfactory function or on other sensory/neurological functions will be underlined. clues to clinical/radiological differential diagnoses of the most common conditions and radiological features of the anterior cranial fossa will be highlighted. the central skull base, the floor of the middle cranial fossa, has a complex anatomy and is pierced by a variety of foramina and canals providing crossroads for spread of disease between the extra-cranial head and neck and the intracranial compartment. disease spread into the intracranial compartment indicates a dismal prognosis, as often it becomes inoperable due to involvement of vital structures including cranial nerves, carotid artery and/or brain parenchyma. imaging plays a pivotal role in the assessment of these structures which are not amenable to clinical inspection and can dramatically change the patient's management. in this complex anatomical area, ct and mr have a complementary role in providing a comprehensive roadmap for treatment planning. of major importance is the pterygopalatine fossa, the cavernous sinuses and orbital apex. the former, lying between the pterygoid plates and the maxillary sinus, is a major crossroad between the intra-and extracranial compartments and should be carefully inspected. pathologies affecting the central skull base are varied and may originate from the skull base proper, from the middle cranial fossa or from the extracranial head and neck. by looking at the centre of the lesion, its tissue characteristics and pattern of growth, it is often possible to provide a useful differential diagnosis and, most importantly, to map the lesion's extent. here, we present a radiologist-friendly approach to central skull base pathology highlighting the most important features in the differential diagnosis and treatment planning. . the jugular foramen: the jugular foramen is an opening in the skull base. the radiologic evaluation requires high-quality imaging with ct and mr. angiography is reserved for preoperative embolisation. it is important to recognise the "pseudo lesions". the most common tumour of the jugular foramen is the paraganglioma. the second is the schwannoma of the lower cranial nerve, and the jugular foramen meningioma is the third most common. the differential diagnosis shall be discussed. . the cerebellopontine angle (cpa) and the internal auditory canal (iac): the iac is a bony conduit for several nerves and a vessel, the neurovascular bundle. the cpa is a cistern of the peripheral cerebral spinal fluid, and several anatomical structures pass through this. it is also the place of some frequent disease processes. most of the lesions in the cpa are benign tumours with order of frequency: vestibular schwannoma and meningioma. the third most common lesion is a benign cystic lesion: the epidermoid cyst. in the iac, you can encounter the same benign tumoural lesion as in the cpa, but also inflammatory lesions, viral lesions and malignant lesions. why is it important in your imaging protocol of the iac and cpa region to use gadolinium? the differential diagnosis of different lesions shall be discussed. learning objectives: . to learn about the imaging techniques and anatomy in the posterior cranial fossa. . to become familiar with the imaging findings of common posterior cranial fossa pathologies. . to learn how to differentiate between the lesions in the posterior cranial fossa. health care is characterised by several specific features making it different from traditional markets. two of the key features are: the impossibility of patients to make choices, due to asymmetry of knowledge between them and their health-care providers; the fact that patients create an agency relationship with health-care providers, delegating to them the decision about the care delivered. due to these characteristics and to the fact that patients are fragile when they need care, there is a natural tendency for patients to rely on healthcare professionals. it is in this complex socio-technical environment that radiographers must be aware of their roles and responsibilities and ensure professional autonomy and accountability; demonstrate an ethical and knowledgeable understanding of the profession; apply professional practice in securing, maintaining or improving health and well-being; develop knowledge, skills and competences that underpin their education and training, contributing to the wellbeing of the patient; understand that professional advancement arises out of evidence-based practice and is acquired through focused research. this demands for a focus on patient care and safety, based on high professional standards. ebp is the best way to achieve this desideratum, as it combines the best research evidence with clinical knowledge and expertise, addressing the patient in a holistic way. since the radiographer is the ultimate interface between patient and technology, it is crucial to create a real teamwork concept, using guidelines for the roles and responsibilities of each team member as the pathway to minimise practice error, maximising patient and staff safety. the use of ionising radiation in medical exposures is well documented across countries. recent studies indicate that a substantial fraction ( to %) of these procedures is unnecessary when taking into consideration patient clinical indication. defensive medicine, economic interest, poor patient clinical information, patient pressure and the lack of knowledge about other alternative procedures, taking into account the benefits and risks, are the principal causes of unnecessary radiological examinations. patients must be referred to a medical imaging department for an examination that should be justified according to clinically appropriate image criteria and informed of the potential benefits and risks. following justification, the optimisation of practice is essential to ensure the minimal exposure of radiation dose to the patient whilst diagnostic efficacy is maintained. the principal aim of optimisation is getting a narrower dose distribution, with lower mean and th percentile values of the diagnostic reference level. optimisation processes need to consider several factors including the type of examinations, body region, clinical information, available equipment technology and image processing tools. to implement this process, a multidisciplinary teamwork is essential where radiographers, being the pivot between patient and technology, have an essential role. to understand and apply optimisation procedures, radiographers should be aware of the international guidelines, standards and directives. all medical imaging procedures must be audited to analyse if the workflow processes (referral, justification, optimisation and radiation protection principles) are according to the recommendations to create a radiation protection safety culture. although we are approaching the year , a lot of clinical radiography is not based on best evidence. lot of clinical decision-making is based on, for example, traditional practices that we cannot argue as being the best possible ones. in this presentation, barriers to using research and other sources of evidence and facilitators of evidence-based (eb) radiography are described. the role radiographers have in developing eb guidelines and procedures may vary according to educational level and work description. developing eb guidelines is not the work of every radiographer, but obeying them is. the speaker gives some suggestions on what to do in a situation if there are no eb guidelines and procedures in ones' country or institution and no group that develops them. with the framework of "the new work order" and increasing implementation of new work processes, professionals are faced with new modes of collaboration requiring communicative skills as well as multiprofessional competencies between all the stakeholders across the hospital. some evidence about the consequences of ineffective communication in the field of radiography is presented. in addition to benefits associated with the procedure of clinical audit, uniform good quality protocols for examinations and uniform national/international criteria help to perform comparisons between radiology units. clinical audit also works as an educational tool and as a feedback tool for the staff on their performance. it also points out the need for corrective actions. initiatives from professional organisations are probably the most powerful engines for the construction of europe, and in many cases open the road for legal and political advances. many medical specialties felt that there was a need for the harmonisation and standardisation of medical training over europe. the esr has shared this opinion and has elaborated a unanimously appreciated training curriculum, which is constantly updated. establishing the ebr (european board of radiology), with the mission to organise a european diploma was the logical next step. today, examinations have already been organised and more that candidates from european countries and abroad have applied. the trainee who passes the examination proves his professional skills and has reached the objectives assigned in the curriculum. in this session, we will explain how esr organises the diploma. we will review, capitalising on our current experience, the main lessons learnt from the past editions, the keys for success and reasons for failure, as seen by the examiners. we will also learn about the "diploma adventure", from the point of view of a successful candidate. national radiology training programmes in europe differ to a large extent, regarding length of training, content, and the presence or absence of a national board examination for certification. thus, for cross-border radiologic activities, the level of training and expertise applying to radiologists is difficult to assess under these circumstances. there is an increasing number of medical subspecialties, which organise european diplomas. more than subspecialties (e.g. anaesthesiology, ophthalmology, urology, etc). organise european examinations, which are well received among the respective communities. growing numbers of residents in training successfully stand this test of knowledge and practical skills. recognition of european diplomas by national authorities is an issue, which will increase the level of acceptance. the present status of the recognition process in european countries will be summarised. case material for the diploma will test knowledge in general radiology at a standard to be expected at the end of training as outlined in the esr curriculum. all ten esr-recognised subspecialty areas of the curriculum will be tested. the written exam comprises two computer-based sections, a multiple response section, and a short case section. there will be multiple response questions. these questions comprise both multiple options format, a single stem with answer options, and short imaging studies where candidates are asked to either annotate the image from a drop-down menu, or select the radiological features displayed or differential diagnosis from the options given. a special scoring system has been developed to prevent guessing. the short case section comprises or case-based questions. you may practice using the self-assessment tests on the esr website which follow the same format as the written examination. for the oral cases, you must have a systematic method and perform under pressure, which takes practice. you may rehearse with experienced radiologists who feedback on your performance. you can break a radiology report into areas, observations, interpretation and conclusion. conclusions should take into account the clinical information. candidates often forget this under pressure, so ask for it to be repeated. your differential diagnosis should be logical and comprehensive. the commonest causes of an abnormality is a good topic to revise. suggest if further imaging tests are indicated and give an indication of urgency. each case is marked separately. as a well-known statement in the academic world of education, "assessment drives learning". with the help of carefully prepared and performed examinations, it is possible to emphasise and instill the training curriculum. due to different reasons, training of radiology in european countries is highly varied. like in our experience in turkey, a central examination system, accompanied by a neatly prepared training charter and curriculum, is a good starting point to solve the problem. we believe that a common european assessment system like edir will contribute a great deal to the harmonisation of radiological training and establishment of quality standard in every country. the need for harmonising the training is not unique to radiology. this is why in more than medical disciplines, european diploma examinations are now being organised. furthermore, in some disciplines like urology, annual and online "in-service assessment" of residents and practising urologists allows the participants to evaluate their current knowledge base against the current european standards, and also provides the trainers and directors knowledge about the teaching and learning process in education centres. although there are huge diversities among different european centres, the urgent need of harmonising radiology training and setting a standard of quality are felt by all radiologists in europe. establishing and institutionalising a common assessment system in european radiology appears to be the shortest way to reach this goal. this is why we should support "european diploma in radiology" by any means and work hard to promote its recognition throughout the continent. the majority of emergency ct studies reported by junior radiologists or general radiologists out-of-hours comprise brain scans. brain ct studies are often performed to detect acute life-threatening abnormalities, such as stroke, intraparenchymal or subarachnoid haemorrhage, cerebral edema, etc. misrecognition of these often subtle, but life-threatening abnormalities can lead to inappropriate patient management and worsen patient outcome. errors in interpretation can generally be categorised as either perceptual or cognitive in nature. perceptual errors are those in which the radiologist does not see the abnormality, resulting in a false-negative interpretation (e.g. basilar artery thrombosis, deep cerebral venous thrombosis, pres,…). cognitive errors, on the other hand, are those in which an abnormality is identified but the meaning or significance of the abnormality is not recognised. cognitive errors can result in false-positive interpretation if, for example, a normal anatomic variant is mistaken for a pathologic condition. while the more common normal variants and artefacts often do not present a problem to experienced clinicians and neuroradiologists, less experienced individuals should beware of these diagnostic hazards. to reduce false-negative and false-positive reporting, a checklist of the review areas (blind spots) to be verified on any brain ct scan will be offered and the most frequent normal variants will be discussed. pitfalls in brain imaging comprise perceptual or cognitive errors of interpretation of imaging findings. errors of perception refer to a missing of a pathology that is present on an image, while cognitive errors consist of a wrong interpretation of a seen structure or pathology. interpretation may be false negative or false positive. in addition, a wrong conclusion may be drawn resulting from missing, incomplete or false clinical information, or as a consequence of a lack of knowledge of differential diagnoses. perception errors are frequently the consequence of incomplete examination protocols where lesions are missed (for instance, acute ischaemic changes without the use of diffusion-weighted imaging) or signals, for instance, flow-related phenomena are misinterpreted. asymmetric positioning of the patient`s head may also lead to misinterpretation of side differences, especially in the temporal lobe. another source of perception errors might be reduced attention (especially at night shifts) and have been proved to occur in regions that are "potential blind spots", as the sulci, cavernous sinus, meckel cave, dural sinuses, brainstem, and skull base foramina. perceptual errors may be avoided by following a checklist when reading images. all sequences should be used for comparison of signal intensities to distinguish artefacts from pathology. another very important fact to avoid misinterpretation lies in a profound knowledge of morphologic and metabolic age-related changes of the brain and of anatomical normal variants (such as, for instance, accentuated virchow robin spaces in certain locations). cystic liver lesions can be classified based on their nature as benign and malignant. in the benign category are, among others, developmental and infectious/inflammatory cysts, while neoplastic cyst can be subdivided into primary and secondary. developmental cysts originate from abnormal ductal plate malformation and consist of hepatic (bile duct) cyst, bile duct hamartomas, caroli's disease and polycystic liver disease. infectious/inflammatory cysts include, among others, abscesses (pyogenic and amoebic) and hydatid cysts. primitive neoplastic cystic lesions are cystoadenoma and cystoadenocarcinoma. secondary lesions can originate mostly from mucinous tumours such as colon and ovary. the role of crosssectional imaging in the detection and characterisation of these entities will be discussed, with an emphasis on the differential diagnosis with ct and mr imaging. benign hepatocellular neoplasms are being recognised with increased frequency using cross-sectional imaging. one of the main goals is to be able to make a clear-cut differential diagnosis between focal nodular hyperplasia (fnh) and hepatocellular adenoma (hca) since patient management is substantially different. despite the sound knowledge acquired in the last few years about the morphological features of fnh using various cross-sectional imaging techniques, new players in the field have arisen such as diffusionweighted mr imaging (dwi) and use of hepatobiliary contrast agents. these new biomarkers offer a different view over fnh and allow a more accurate characterisation even in more atypical cases. concerning hca, current knowledge implies that the sub-types of this neoplasm should be known since, again, patient management differs and these patients are no longer seen as compulsory surgical candidates. these sub-types will be further discussed and the role of imaging for risk stratification (haemorrhage and malignant transformation) will be addressed. further, illustrative cases of combined fnh/hca cases will be demonstrated along with the main imaging clues for a successful differential diagnosis. imaging in the early postoperative period is complex. it is essential for radiologists to know the new anatomic arrangement after surgery and to understand the range of normal postoperative appearances. this latter aspect can be particularly difficult, because the radiological findings are affected by the type of operation performed (open vs. laparoscopic) and the complexity of the surgery (such as the presence of adhesions or haemorrhage) as well as the underlying comorbidity of the patient. patients may be considered in broad categories: haemodynamically unstable patients who may be bleeding; patients with distension or unopened bowels who may have obstruction or ileus; patients with sepsis in whom to search for an intraabdominal collection or leak; and patients who do not follow an expected recovery for which the cause is unclear. ct is the primary imaging technique, with ultrasound reserved predominantly for liver and renal transplants or assessment of perihepatic collections, and fluoroscopy for routine postoperative assessment of upper and lower gi anastomoses. ct protocols should be optimised to detect particular complications with positive luminal contrast for assessment of anastomotic leaks ( %), triple phase imaging for bleeding (without positive oral contrast) and delayed phase imaging to detect ureteric injury. since each operation has a specific range of expected complications, these should be considered when deciding on the protocol and in light of the patient's clinical status. excellent communication with the surgical team is required to make an accurate diagnosis and in particular where haemostatic compound or mesh for hernia repair was used. learning objectives: . to understand the best imaging options for investigating suspected postoperative complications. . to appreciate the normal appearances of the abdomen and pelvis after surgery, in particular with regard to free fluid, haematoma and free gas, and when to suspect complications. radiologists should be aware of the newer surgical techniques and expected post-operative alterations, to yield a correct interpretation of a post-surgery imaging examination, thus being able to differentiate a normal post-operative finding from a potential complication. in this setting, it is crucial to perform a technically adequate imaging examination, so that post-operative anatomical and functional findings may be evaluated. therefore, the aim of this lecture is to give an overview of the contribution of various imaging modalities in the diagnosis of late post-operative complications following gi tract surgery. it will include a mention of the technical issues that need to be considered to achieve better diagnostic accuracy, as well as a description and illustration of the main imaging findings of late post-operative complications. those include diseaserelated (recurrence for malignancy or inflammatory disease) and procedurerelated (anastomotic strictures, internal herniation, adhesions and intussusception, among others) complications. focus will be placed on crosssectional imaging techniques, which at present constitute the workhorse for detecting and characterising late post-operative complications after gi tract surgery. in this way, imaging has an obvious and pivotal role in planning therapy, since some late complications are usually treated in a conservative way while others require a surgical approach. lung biopsy of a suspected lung cancer remains a well-established technique for histopathologic examination in patients with suspected lung cancer. complications can nevertheless occur and therefore indication should be given after carefully balancing the benefits and drawbacks for a particular patient. the method should be clearly considered complementary to alternate methods of tissue sampling, fiberoptic endoscopic, mediastinoscopy or us-guided endoscopy, surgical biopsies and resection during a multidisciplinary discussion. several steps are needed in the preparation of the biopsy procedure, including informed consent, management of anticoagulation and anti-platelet medications, imaging assessment of the lesion and its environmental lung, and planing of the needle trajectory procedure. the choice of the guiding method and the most appropriate biopsy needle is still largely dependent on local skills and habits. ct-guided percutaneous biopsies using coaxial automated core biopsy needles offer many advantages. needle manipulation can help to reach difficult targets. participation of the patient is needed in terms of respiratory manoeuvres and breathhold. complications may occur such as pneumothoraces haemorrhage and air embolism. knowledge of the respective risk factors and the potential method of prevention or treatment by operators are essential. the therapeutic response of lung cancer can be objectively evaluated on the basis of changes in the tumoural size or depiction of metastases, such proposed in the recist criteria (response evaluation criteria in solid tumours). recent advances in cancer biology have triggered development of novel targeted therapies designed to disrupt specific biologic pathways. among them, antiangiogenic drugs represent a promising strategy for non-small cell lung carcinomas (nsclc). these agents are more cytostatic rather than cytotoxic, explaining the limitations of tumour response assessment based on morphological criteria. dynamic contrast-enhanced imaging techniques have the potential to quantify tumoural angiogenesis. in particular, dynamic contrastenhanced multidetector-row ct (dce-ct) represents a promising tool to provide non-invasive and repeatable assessment of the angiogenic process within non-small cell lung cancers (nsclc), offering the possibility to generate morphological and functional information from the same examination. dce-ct allows the calculation of regional tumour blood flow, blood volume, flowextraction product, and permeability-surface area product, over the entire tumoural volume, using mathematical models and dedicated softwares. dce-ct can depict early changes in lung cancer vascularity, before tumour shrinkage, that could help predict response to antiangiogenic drugs. becoming familiar with this technique enables the radiologist to integrate it into clinical practice and to assist the thoracic oncologist in the pre-therapeutic and follow-up evaluation of nsclc patients treated by antiangiogenic drugs. this new approach should improve patient's selection for targeted therapies. a- : it is now accepted that pet-ct is of value in the staging of lung cancer and reduces the number of unnecessary thoracotomies. its role has been further clarified by recent publications on its value in assessing mediastinal nodal involvement, tumour aggressiveness using either standardised uptake value or total glycolytic volume, and response to neoadjuvant chemotherapy. more recently, mri has been investigated as a tool to detect distant disease using diffusion-weighted imaging, and there are a number of trials that have compared both pet-ct and dwi mri retrospectively and prospectively. this presentation will summarise the current available data and the likely benefits of either imaging modality neurological deficits in children are an urgent condition that depends significantly on imaging for a prompt and accurate diagnosis because of the significant overlap present in clinical history, presentation and neurological examination. different imaging modalities, such as ultrasound, computed tomography (ct) or magnetic resonance imaging (mri), are utilised dependent on the age of the patient and neurological symptoms. the purpose of the present lecture is to discuss the causes and imaging appearance of acute neurological conditions in childhood, broadly classified into stroke, infection, inflammatory demyelination, metabolic disorder, cerebral neoplasm or neurotoxicity. imaging also plays an outstanding role in the management of paediatric oncology patients presenting with acute neurological symptoms related to disorders of blood cell counts, immunosuppression, neurotoxicity of therapy, or progression of the primary malignancy. a review of the main indications to perform a neuroimaging procedure in these children will be undertaken as well as a differential diagnosis based on representative cases selected from the daily routine in a paediatric tertiary hospital. practical algorithms with the preferential use of either ct or mri will be developed for each section. ct continues to be the first imaging modality in these patients in many centres, despite the accompanying radiation, because of the easy availability and no requirement of sedation. however, mr imaging is nowadays better for imaging these children owing to no radiation, the more completely provided information and the useful advanced techniques that can be used, such as diffusion imaging (di), spectroscopy, arterial spin labelling (asl), and susceptibility weighted imaging (swi). learning objectives: . to learn about the currently limited role of ct in the non-traumatic acute setting. . to become familiar with radioprotection strategies and protocols adapted to children. . to consolidate knowledge on the role of mri as the modality of choice for acute non-traumatic neurologically ill children, with an emphasis on newer techniques. . to become familiar with imaging findings and the main differential diagnosis of acute neurological conditions in children. b. imaging of acute chest pain and distress in children c.e. de lange; oslo/no (clange@ous-hf.no) acute chest pain in children is a common complaint in the emergency department, but patients rarely present with significant distress or lifethreatening symptoms requiring immediate care or resuscitation. the most frequently reported cause is benign musculoskeletal pain followed by respiratory and gastrointestinal causes, while cardiac causes are less frequent. a thorough clinical history and careful physical examination will determine, in most cases, the patients in need of further investigation to establish a diagnosis. in this regard, radiology plays an important role, especially in the emergency setting in patients with more serious associated symptoms like acute breathing difficulties, swallowing problems, fever or sepsis. when choosing the appropriate technique for investigation, the consequences of radiation exposure in children must be considered. plain radiography and fluoroscopy still remain the most important and frequently used tools to gain information on various acute chest/pulmonary problems. ultrasonography is the first choice for diagnosis/treatment of pleural effusions. multidetector computed tomography and magnetic resonance imaging are mainly used for investigating pulmonary/mediastinal masses and congenital abnormalities of the great vessels and the lungs. this lecture will discuss the choice of imaging technique and the urgency of radiological management depending on the symptoms and age of the patient. the imaging characteristics of the different causes of acute chest pain and/or distress in children will be reviewed, represented by the more common conditions involving the chest wall, respiratory tract, oesophagus and the heart, as well as less frequent causes such as tumours, manifestations of congenital malformations and nonaccidental trauma. outcome data from trauma series have demonstrated improved patient survival by the prompt diagnosis of a range of injuries and the recognition of life-threatening sequels, principally that of active arterial haemorrhage. widely used trauma scoring systems are applicable in the paediatric population, although the mechanisms and combination of injuries may differ from those encountered in adults. a ct diagnosis of active haemorrhage demands early operative or non-operative intervention with embolisation or use of alternative endovascular techniques including deployment of vascular stents. this principle is applicable to blunt or penetrating liver, splenic, renal or pelvic trauma. it is also applicable in some cases to severe mediastinal and limb vascular injury. important caveats are firstly the use of ct in an overall trauma service and avoidance of any inappropriate irradiation. secondly, in paediatric interventional practice, due regard needs to be given to the delivery of implantable devices that may have adverse sequels in later life. nevertheless, the demand for immediacy in intervention may outweigh any such consideration. this presentation will summarise the importance of imaging and intervention in both the early and delayed complications of trauma with an emphasis on abdominal and thoracic trauma. brachial plexus originates from the ventral branches of the cervical nerve roots from c to t . it is composed of roots, trunks, divisions, cords, and branches, topographically divided into supraclavicular (roots and trunks), retroclavicular (divisions), and infraclavicular sections (cords and branches). mri has the advantages of multiplanar imaging, high tissue contrast, and relative paucity of artefacts. the t -weighted sequences display topographic anatomy including muscles, blood vessels, and nerves stressed by tissue fat planes. the nerves appear as elongated fibres that are isointense to the scalene muscle, posteriorly and superiorly to the curvilinear flow void of the subclavian artery. the fat-suppressed pd and t -weighted and stir sequences detect signal alterations of the bp. general abnormal findings include: loss of fat planes around part or all bp components, nerve signal abnormalities with mild or marked hyperintensity on t -weighted images that can be associated with swelling, focal or extensive enhancement of the nerves after gad injection on t fat sat images. differences in signal intensity should be judged by visual inspection, which is susceptible to a subjective point of view; the comparison of both sides of bp has a relevant role in judging the presence of signal alterations, especially when they are smooth and/or focal. the pathology of brachial plexus can be divided into nontraumatic and traumatic brachial plexopathies. among nontraumatic brachial plexopathies, we find radiation fibrosis, metastatic breast cancer and primary or metastatic lung cancer. other pathologies are tumours and inflammatory disease or thoracic outlet disease. the peripheral nerves of the upper limb are affected by a number of entrapment and compression neuropathies. these syndromes involve the brachial plexus as well the musculocutaneous, axillary, subscapular, ulnar, radial and median nerves. clinical examination and electrophysiological studies are traditionally the mainstay of diagnostic workup. however, ultrasonography and magnetic resonance imaging (mri) may provide key information about the exact anatomic location of the lesion or may help to narrow the differential diagnosis. in certain patients with the diagnosis of a peripheral neuropathy, imaging using either ultrasonography or mri may help establish the cause of the condition and provide information crucial for conservative management or surgical planning. in addition, imaging is particularly valuable in complex cases with discrepant nerve function test results. a variety of peripheral neuropathies can be encountered in the lower limb. most are entrapment syndromes affecting many nerves, such as the sciatic, gluteal, femoral, lateral femoral cutaneous, obturator and pudendal around the hip, the peroneal and its branches and the saphenous at the knee, the superficial peroneal at the lateral leg, the tibial with its plantar and calcaneal branches at the ankle, the deep peroneal and the interdigital nerves in the foot. although clinical examination and nerve conduction studies are the mainstay of the diagnostic workup of peripheral neuropathies, ultrasound (us) and magnetic resonance (mr) imaging may provide key information about the exact anatomic location of a lesion and the nature of the constricting finding or may help narrow the differential diagnosis. in patients with peripheral neuropathies of the lower extremity, us and mr imaging may provide critical information for planning an adequate treatment strategy. although us and mr imaging have followed parallel paths for nerve imaging with little comparison of the two modalities, us seems to have some advantages over mr imaging, including higher spatial resolution, time effectiveness, the ability to explore long nerve segments in a single study and to examine tissues in both static and dynamic states. advances in diagnosis of acute stroke have been achieved by brain imaging. ct or mri distinguish ischaemia from haemorrhage and may identify acute clot (dense artery sign) or vessel wall pathology by "black blood mri". dwi provides evidence of early infarction. ct and mra enable identification of large vessel pathology as potential source of embolic or haemodynamic ischaemia. supplementary techniques are ct or mr perfusion. on mri perfusion, a mismatch between the area of restricted diffusion and perfusion (pwi) is a signature of the penumbra. the tissue of pwi restriction outside of the dwi abnormality represents "tissue at risk". similarly on ct perfusion, the region of reduced cerebral blood volume presumably represents irreversible infarction and the area of reduced cbf and extended mtt visible beyond the cerebral blood volume abnormality represents potentially reversible ischaemia. morphologic imaging and advanced standardised perfusion/penumbral stroke protocols aim at identifying lesions that are amenable to acute stroke therapy and exclude pathologies that mimic stroke but do not represent vascular disease. since iv tpa has become an approved therapy from , the number of acute stroke patients entitled for treatment has remained limited (~ ) despite extension of the time frame from to . h. dwi-flair mismatch and an individualised assessment of the ischaemic penumbra may serve in the identification of patients within the appropriate time frame. by selection of patients with advanced brain imaging and initiation of appropriate therapy, a further increase in good outcomes and reduction of the incidence of symptomatic haemorrhage may be achieved. in recent years, the role of imaging in the patient admitted with an acute neurologic deficit has changed significantly, due to the arrival of new treatments for acute stroke, aimed at re-establishing blood flow, reducing infarct size and protecting the brain at risk. the first goal is to differentiate haemorrhagic from ischaemic stroke and to rule out other stroke mimics. this can be achieved by performing nonenhanced ct scan of the brain. the next strategic imaging objectives include: demonstration of major blood vessel occlusion (e.g. by ct angiography) and identification of potentially salvageable brain tissue, the so-called "penumbra" (e.g. by ct perfusion). ct angiography is performed during rapid intravenous bolus injection of a high-concentration iodinated contrast agent. the fov should cover not only the intracranial arterial circulation, but also the arteries of the neck, to exclude dissection. ct perfusion is also achieved by bolus injection of contrast, followed by a series of fast images or volume acquisition. ct densities change over time and reflect the iodine concentration. perfusion images are processed to generate parametric maps, which reflect: regional cerebral blood flow (rcbf), blood volume (rcbv), mean transit time (mtt), and time to peak (ttp).the combination of ct angiography and ct perfusion provides a unique insight into the pathophysiology of the cerebral circulation. these techniques are now essential tools in the management of acute stroke and in selecting those patients who are potential candidates for advanced therapies such as thrombolysis or thrombectomy. learning objectives: . to become familiar with a comprehensive imaging protocol in patients with suspected stroke. . to understand the advantages and limitations of cta and perfusion in the initial work-up of stroke patients. . to recognise the different imaging patterns in stroke and their prognostic value. a- : b. stroke mimickers and pitfalls p. vilela; lisbon/pt (ferrovilela@sapo.pt) "stroke mimic" is classically used to describe nonvascular diseases that present with acute focal neurologic deficit corresponding to a consistent vascular distribution, which may resemble or may even be indistinguishable from stoke. it is estimated that up to % of stroke-like presentations are due to mimickers. there are several neurological and psychiatric disorders that can have such a clinical presentation, such as seizures and/or postictal status, the most frequent one. the other classical clinical stroke mimickers include: multiple sclerosis, migraine (specially hemiplegic migraine), metabolic disturbances (more commonly hypoglycaemia/hyperglycaemia), intracranial tumours or infections and the conversion reactions. brain imaging plays a key role in the diagnosis and management of acute stroke by differentiating ischaemic from haemorrhagic lesions, identifying the vessel occluded and estimating the viable brain tissue that is at risk for stroke. imaging is also important to exclude some of the aforementioned stroke mimics. the more comprehensive stroke imaging protocols, with ct perfusion/angiography and/or with mri, have reduced significantly the misdiagnosis of stroke. mri-dwi is the most sensitive and specific imaging modality for depicting ischaemic stroke. however, it is also essential to be aware that there are some pathological processes that can mimic stroke in imaging studies. these include some disorders that may present themselves with lesions associated with restricted diffusion, like transitory postictal brain abnormalities, migraine and encephalitis. the author reviews the most common clinical and imaging stroke mimics and highlights the importance of brain imaging in depicting these mimickers and avoiding the potential adverse effects of stroke therapy in these patients. learning objectives: . to learn the differential diagnosis of stroke. . to understand the role of ct and mri in the work-up of stroke and related disorders. . to recognise imaging patterns that may mimic stroke clinically and radiologically. imaging plays a central role in the management of stroke, which is an important health issue according to the number of patients involved and the severity of the disease. it helps to select patients who will benefit from a revascularisation therapy (iv thrombolysis or endovascular treatment) at the acute phase of stroke. if the value of perfusion/diffusion mri mismatch in the evaluation of penumbra is still a matter of debate, other imaging factors are associated with the clinical outcome including length of clot, flair positivity, and collateral circulation. if iv thrombolysis using rtpa is still the reference treatment, endovascular treatment is emerging since several years as a feasible and efficacious alternative singularly since the appearance of stent retrievers. recent neutral randomised trials comparing iv thrombolysis and endovascular treatment have shown the critical role of appropriate imaging in the selection of patients who can benefit from acute revascularisation as well as the importance of using the most efficacious endovascular devices. prevention of stroke is partially based on the treatment of cervical and intracranial stenosis. several randomised trials comparing carotid angioplasty and carotid endarterectomy were initially negative, but a recent meta-analysis showed that below the age of years both techniques were equivalent. the place and value of endovascular treatment of intracranial arterial stenosis is still controversial after the publication of the sammpris results. the management of thyroid nodules has been continuously evolving. i hope that during this session you will understand the important differences in the upto-date national and international guidelines on thyroid nodule management and understand the role of the radiologist in the multidisciplinary thyroid meetings. the features that are suggestive of benign, indeterminate and malignant nodules will be demonstrated. advances in both radiological and cytological techniques such as us elastography and braf mutation analysis that may help triage patients with thyroid nodules will be covered. the differing biopsy techniques (fine needle aspiration, non-aspiration (fna and fnnac) and trucut biopsy) and their roles will be highlighted. there will be time for an open question and answer discussion with all the speakers before the end of the session. the management of thyroid nodules and the guidelines of the various interested parties, surgeons, endocrinologists, nuclear medicine physicians, radiologists and ultrasound specialists, vary across the world, indeed between different countries within the eu, and are constantly evolving. i will discuss how the thyroid multidisciplinary meeting (mdt) works at our institution and the role of the radiologist in it. the current differing international guidelines on the management of thyroid nodules will be mentioned, highlighting their salient points and differences. the role of the differing imaging modalities used in the management of thyroid nodules will be discussed based on differing clinical scenarios. finally, the management of the common clinical scenario of the incidental thyroid nodule will also be discussed. learning objectives: . to understand the role of the radiologist as part of a multidisciplinary team. . to learn about current guidelines on the management of the thyroid nodule. . to understand the role of various imaging modalities in the patient with thyroid nodule. thyroid disease is ubiquitous and usually benign; therefore accurate imaging of the thyroid is a major challenge for radiology. significant advances in conventional ultrasonography allow superb visualisation and anatomic characterisation of thyroid pathology and the impact of broader bandwidths, higher frequencies, spatial compound imaging and novel signal processing techniques will be discussed in this session. the key b mode features of thyroid nodules are discussed with reference to relevant clinicoradiological guidelines, specific pitfalls and limitations. a significant number of nodules remain indeterminate after conventional assessment, and imaging advances that enable more accurate, non-invasive thyroid characterisation are highly attractive, potentially reducing unnecessary biopsy and surgery for a large number of patients. ultrasound elastography offers a potentially useful adjunct to conventional sonography in thyroid disease with a growing evidence base indicating that thyroid malignancies are typically stiffer than benign lesions and that a 'soft' cutoff value with a high negative predictive value is feasible. however, elastography studies have included a variety of different techniques, patient/nodule selection and assessment methods; therefore the current role of elastography will be discussed. fine needle biopsy of the thyroid nodule f. campoy-balbontín, m.c. jurado-gomez; sevilla/es (fcampoy @gmail.com) thyroid nodules are very common. the clinical importance of thyroid nodules rests with the need to exclude thyroid cancer, which occurs in - %. ultrasound (us) has been widely attempted to differentiate benign from malignant nodules and to guide fine needle biopsy (fnb). however, considerable overlap between benign and malignant characteristics has been found. nowadays, there are a number of different guidelines for the management of thyroid nodules. at our hospital, we have adopted the guideline proposed by the society of radiologist in ultrasound ( ). the different elements necessary to perform the fnb procedure are demonstrated; the transducer probe, the needles, the slides, the syringe. the way of managing these elements is shown by figures and videos, with different skills and performances to optimise the procedure and get the best sample. uscontrol fnb can be in parallel or perpendicular, both methods are demonstrated. thyroid fnb is usually non-aspirated, but sometimes it has to be undertaken with aspiration; when and how to change from one method to another is explained. with good technique, the insufficient cytological rate can be significantly reduced, with which the need for core biopsy diminishes. although the complication rate for core biopsy is low, fnb is safer and if performed correctly has a high diagnostic yield. core biopsy should be limited to those nodules with a second insufficient fnb cytological result, with us findings suspicious for malignancy. multimodality breast imaging is emerging as radiologists have access to new technologies coinciding with the refinement of operative techniques. one of the challenges for radiologist remains the correct preoperative staging, especially the metastatic involvement of axillary lymph nodes. multimodality imaging has new accurate solutions. the imaging follow-up of patients with sophisticated oncoplastic techniques is another challenge where multimodality breast imaging is the solution. coincident with this increasing contribution to the diagnostic process is the emerging role of the breast imager as a therapeutic clinician, making use of innovative image-guided procedures. in this integrated rc, experts will present a clinical update on these newer techniques and there will be an opportunity to discuss how the evolution of such techniques is changing the nature of the modern multidisciplinary team meeting. a. conventional, functional and interventional lymph node assessment r.m. pijnappel; utrecht/nl (r.m.pijnappel@umcutrecht.nl) metastatic involvement of axillary lymph nodes has been known to be one of the most important prognostic factors for women with breast cancer. the traditional approach to staging the axilla is either lymph node sampling procedures or sentinel node biopsy. the main diagnostic features of potentially malignant involvement of the axilla are abnormal lymph node morphology (shape and cortical thickness). there has been a concerted effort in recent years to find imaging techniques that might facilitate accurate axillary staging. so far, no imaging technique alone (ct, mri, scintigraphy and ultrasound) has been capable of differentiating between positive and negative lymph nodes of the axilla in breast cancer. recent techniques like gadofosveset-enhanced magnetic resonance imaging and high spatial resolution -t mri imaging appear promising; however ultrasound-guided sampling of suspicious lymph nodes by core or fna remains the standard of care in assessing the axilla prior to treatment planning. ultrasound-guided axillary lymph node sampling is an effective method of assessing the axilla. a recent meta-analysis shows that ultrasound can be expected to achieve sensitivity approaching % for detecting abnormal nodes in those that eventually prove to be nodal metastatic disease. the false-negative rate where biopsy is performed is around %. it is impossible to detect the sentinel node by ultrasound alone. therefore a new promising technique was introduced, injecting an ultrasound-contrast agent around the nipple. using this method, it is possible to detect the sentinel node in % and therefore lower the false-negative rate of us-guided biopsy with another %. oncoplastic breast procedures were introduced to fill the gap between comprehensive oncologic surgical treatment of breast cancer and the achievement of cosmetic results that fulfill patient expectations in both, body imaging and psychological well-being. due to its complexity and relevance for the patient, the decision of performing these procedures must be made as part of the multidisciplinary approach of breast cancer treatment. as a consequence, the role of breast radiologists has expanded beyond the anatomic region of the breast and the usual imaging techniques. basic knowledge of the different oncoplastic techniques is mandatory to understand the spectrum of findings from a multi-modality approach. implants and/or autologous reconstruction techniques (pedicle, free or perforator flaps, as well as lipofilling techniques) are widely applied. the role of the radiologist in the multidisciplinary team is twofold: assessment during the planning stage, and imaging evaluation at follow-up. the assessment during the planning stage includes the determination of the local extent of the disease that makes the choice of the appropriate surgical technique possible, and the imaging study of the donor site in those cases where autologous reconstruction is elected. imaging evaluation at follow-up comprises the recognition of changes and potential pitfalls after reconstruction, the identification of short/mid/long-term reconstruction complications, and the detection of recurrent/second carcinomas. controversial aspects will be reviewed, such as the probability of recurrence after oncoplastic surgery, the need to establish multimodality follow-up protocols and the interrelations between the autologous tissues and the mastectomy bed or remaining breast. the use of image guidance for surgical planning has only recently been accepted. the use of intraoperative ultrasound in women undergoing lumpectomy is associated with a higher rate of negative margins. the combination of image guidance and radiological intervention techniques also harbours the possibility of tumour treatment without surgery. most techniques make use of the placement of needles within the tumour. treatment is performed by heating, freezing or extracting the tumour tissue. currently, radiofrequency ablation under ultrasound guidance is being furthest evaluated. however, in rfa even after optical complete tumour ablation, residual vital tumour tissue is still found in many cases. this is mainly dependent on tumour size with poorer results ( - % success rates) in tumour over cm, and better results (~ % success rate) in smaller tumours. adequate tumour size estimation is thus of vital importance. currently, since remaining vital cells cannot be excluded, the technique is limited to use in patients who cannot undergo surgery due to poor health state. larger series in which rfa is combined with radiotherapy have not been reported. it is therefore not clear whether it is important to actually excise residual vital tumour. similar restrictions currently hold for cryotherapy, laser ablation and hifu. large vacuum-assisted needles and the breast lesion excision system can be used to remove the tumour through a minimal incision in the skin. however, margin evaluation is difficult. nevertheless, these techniques are already optional for the removal of high-risk lesions and can potentially be combined with (focused) radiotherapy. the müllerian ducts are paired embryologic structures that undergo fusion and resorption between the th - th weeks of gestation to give rise to the uterus, fallopian tubes, cervix, and upper two-thirds of the vagina. non-development, defective vertical or lateral fusion, or resorption failure of the müllerian ducts can result in formation of müllerian duct anomalies (mdas), whose reported prevalence ranges between . % and %- %. the classification revised by the american society for reproductive medicine in is the most widely accepted. obstructive mda can occur in infants as palpable pelvic mass, at menarche with cyclic pelvic pain, or pelvic mass, and primary amenorrhea. complications include hematocolpos, hematometra, hematosalpinx, endometriosis, pelvic adhesions, and obstruction of urinary tract. mdas are commonly associated with renal and other anomalies; thus, identification of both kidneys is important. accurate diagnosis of an mda is essential because of the high associated risk of infertility, endometriosis and miscarriage, and since the management approach varies depending on the type of malformation. hysterosalpingography (hsg) is routinely used in evaluation of infertility. because a key component of mdas characterisation is the external uterine fundal contour, hsg is limited for this purpose. ultrasonography (us) is initially performed to evaluate uterine anatomy and to assess for associated renal abnormalities. mri is reserved for complex or indeterminate cases to characterise the uterine anomaly and evaluate the possibility of a vaginal abnormality, or after a seemingly normal us when clinical opinion ensures its use, or to make decisions on management strategies and preoperative planning. this refresher course will focus on the radiologist's approach to using pet-ct within the context of the gynaecologic oncology multidisciplinary meeting. the current indications for using fdg-pet/ct will be discussed for cervix, endometrial and ovarian cancer as well as the potential use of fdg-pet/ct in rare gynaecologic cancers. the value of adding fdg-pet/ct to the patient management pathway will be presented alongside the potential pitfalls in interpretation as well as controversies. future developments, including potential new tracers, will be mentioned. cystic lesions account for the vast majority of ovarian masses and are detected incidentally in - %. their spectrum ranges from non-neoplastic cysts to benign tumours, e.g. cystadenomas and dermoids to the rare ovarian cancer. diagnostic criteria in us, ct and mri are based on the clinical background (age, menopausal status, medical history, and tumour markers) and the morphology of the ovarian mass. typical of a benign cystic lesion is its pure cystic structure or thin septations, fatty or haemorrhagic contents, and mild enhancement of solid components in a complex solid and cystic ovarian mass. thus, patients can be categorised into different risk groups. sonography has been established as the first-line imaging modality to assess the ovaries. recently, management guidelines have been adopted for cystic ovarian lesions. in these guidelines, also cystic adnexal incidentalomas are included. mri is most useful as a complementary technique in sonographically indeterminate masses. an algorithmic approach will render a specific diagnosis in the vast majority of cases. in complex cystic lesions, integration of dwi and dynamic contrast-mri allows differentiation of rare benign complex tumours, e.g. cystadenofibroma from ovarian cancer. pitfalls of cystic ovarian tumours include cystic fibroids, peritoneal cysts, and extraperitoneal cystic tumours. compared to us, both ct and mri are superior in assessing large cystic pelvic masses. criteria to differentiate between intra-and extraperioneal origin include displacement patterns and identification of the vascular pedicle. careful analysis of imaging and clinical findings usually allows differentiation of inflammatory tumours from ovarian cancer. learning objectives: . to learn about specific imaging algorithms of ovarian cystic tumours. . to understand certain imaging features that can differentiate ovarian from non-ovarian cystic tumours in the pelvis. . to become familiar with the pitfalls in imaging of ovarian cystic tumours and the lessons to be learned from them. the diagnostic methods available in patients with conductive hearing loss are: personal and family history, otoscopy, audiology, imaging, surgical inspection and counseling. the most important reasons for imaging are: atypical history, suspicion of congenital hearing loss, suspected otoscopic image, asymmetric bone conduction thresholds, profound mixed hearing loss and suspected tympanometry. ct can be used to detect otosclerosis, tympanosclerosis, posttraumatic ossicular lesions, superior semicircular canal dehiscence and cholesteatoma. mri can be used to exclude schwannoma, congenital cholesteatoma and residual and recurrent cholesteatoma after prior surgery. this lecture will illustrate the value of ct and mri in the evaluation of cholesteatoma patients as well as prior to first stage surgery and prior to second look surgery. different subtypes of surgery will be highlighted and illustrated. the growing importance of mr imaging and diffusion weighted (dw) imaging in particular will be illustrated in the evaluation of cholesteatoma patients. the role of echo-planar and non-echo-planar dw imaging will be discussed as well as the eventual use of delayed gadolinium-enhanced t weighted imaging. mr is the modality of choice in patients with congenital or acquired sensorineural hearing loss (snhl) and the complete auditory pathway from the cochlea to the auditory cortex must be studied in these patients. the labyrinth, internal auditory canal (iac) and cerebellopontine angle (cpa) are best studied using submillimetric heavily t w images, showing the intralabyrinthine fluid and nerves. but submillimetric gd-enhanced t w images or d-flair images remain more sensitive. the auditory pathway in the brainstem is best studied with a multi-echo sequence (m-ffe/medic/merge) or t w tse sequence and the auditory cortex is best studied on t w tse images. the clinical information provided by the otologist will direct the radiologist to a certain part of the auditory pathway or will let him use certain imaging techniques. for instance, congenital malformations in the labyrinth and iac must be excluded in case of congenital snhl, and in case of mixed hearing one will start with ct to exclude otosclerosis. the most frequent pathology in the labyrinth (labyrinthitis, intralabyrinthine schwannoma, congenital malformation, post-traumatic lesions), the iac and cpa (cochleovestibular schwannomas, meningiomas, epidermoid tumours and facial nerve neuritis) and central auditory pathway (ms, infarction, tumor and trauma) will be illustrated using clinical examples and the important "otologist-radiologist" team work will be stressed. for tumours that nearly always arise from the investing mucosa, like in the larynx and hypopharynx, it is endoscopy that directly maps the superficial extent and grades the functional impairment (arytenoid and vocal cord movement, pyriform sinus distensibility). in addition to white light, the recent use of narrow band imaging endoscopic technology, enhancing fine capillaries in the mucosal surface, enables the detection of neoangiogenesis caused by early squamous cell carcinoma. conversely, submucosal spread is the domain of cross-sectional imaging techniques. therefore, it is the integration of the two classes of data, along with the information about patient conditions, which permits the choice of the most proper treatment strategy. in early and advanced glottic cancer, treatment planning is modulated by information about deep neoplastic invasion into the paraglottic space, the cartilage or the suprasubglottis subsites. this information can be derived by discriminating the tumour tissue from intra-laryngeal muscles, fat spaces and the cortical rim of ossified cartilages. though mr shows a greater contrast resolution than ct, it is hampered by a significantly longer acquisition time. this means more artefacts caused by breathing and movement. nevertheless, mr has the potential to unravel the signals from submucosal tissues, separating tumour from oedema, muscles, fat and ossified cartilages. advanced laryngeal and hypopharyngeal cancers need to preliminary assess local (nodes) and distant (metastases) spread. ct-pet is the more accurate tool. the multidisciplinary approach to the processing of the whole frame of data is essential to offer the patient the best care. traditionally, x-ray ct scanners were equipped with a single or a few detector rows only, image reconstruction was assuming parallel slices, which was a good approximation for these fan-beam systems. about a decade ago the number of slices increased to or more and the parallel slice approximation was no longer valid. the cone-beam nature of these multi-slice ct (msct) scanners had to be taken into account by specific cone-beam reconstruction algorithms. while these msct systems -today they are simultaneously acquiring up to slices -are cone-beam ct systems in a general sense, the notion of cone-beam ct (cbct) is also associated with a specific kind of conebeam ct, namely those equipped with flat detectors, i.e. the non-diagnostic or non-clinical ct systems. msct, in contrast, is a cone-beam ct system for diagnostic use, i.e. a clinical ct system. this distinction between msct and cbct is useful, as there are decisive differences in application, performance, and in image reconstruction. these differences will be reviewed in the presentation, with a particular focus on why image quality in msct is so much better than in cbct. cone-beam (cb) imaging with flat detectors is performed on a variety of different imaging systems. the most important area is cb imaging using interventional c-arm systems. the minimally invasive procedures carried out on these systems benefit from the availability of high-resolution d images for intervention planning, guidance and outcome control. interventional cb imaging was first applied in neuroradiology using rotational angiography acquisitions. today, it is used for a variety of procedures in interventional radiology, cardiology and oncology based on angiographic and soft tissue protocols. one of the inherent advantages of this approach is the direct registration of the volume images into the interventional procedure. other application areas of cb imaging using different scanning systems are onboard imaging in radiation therapy or the integration of flat detector tomography and spect. this part of the course teaches the generation of d volume information from flat detector systems and its utilisation in different clinical applications. image acquisition protocols, scan modes, and system design parameters are explained. application-specific calibration and processing steps are introduced to show how cb imaging is tailored for specific clinical applications. examples are presented for angiographic and soft tissue volumetric imaging. medical applications utilising the tomographic images for diagnosis, intervention planning, guidance, or outcome control are discussed. in the last decade, three-dimensional dentomaxillofacial imaging through conebeam ct (cbct) technology has become widely available. dental cbct (digital volumetric tomography; dvt) equipment is compact and often affordable to dentists. the driver for dental cbct was implant dentistry, but its use has spread to other areas, including paediatric applications. radiation doses are variable. some systems offer a fixed, large, field of view and others fixed exposure factors, obstructing attempts at optimisation. typically, doses are at least an order of magnitude greater than for "conventional" imaging. image quality is also variable, which means that certain equipment may be unsuitable for some clinical applications. there is often scope for lower exposure factors to be used than those recommended by manufacturers. a key aspect of using cbct relates to justification. imaging in three dimensions may be perceived by dentists as inevitably superior, a view which is often implicitly encouraged by those selling equipment. the research on diagnostic efficacy is, however, limited. in , the european commission published "radiation protection : evidence-based guidelines on cone beam ct for dental and maxillofacial radiology". this provides a comprehensive set of recommendations, including referral criteria, a quality assurance programme and optimisation strategies. priorities for future research relate to exposures to the necessary image quality requirements for specific clinical applications, along with research on the impact on clinical outcomes of cbct-based treatments. it is clear that much work remains before the place of cbct in dentistry can be established. the role of nonvascular interventional radiology of thorax for both diagnosis and treatment has significantly increased in the last years. although these procedures are technically easy with highly successful results, thoracic nonvascular interventional procedures are not unfortunately routinely performed in all of the interventional units. the most commonly performed procedures generally under ct guidance are transthoracic biopsies of the lung, mediastinal lesions and pleura. the others include percutaneous drainage of thoracic collections such as pleural effusions, empyema and mediastinal collections. image-guided tumour ablation performed by either radiofrequency ablation (rfa) or microwave (mw) as a nonvascular thoracic intervention has become a serious treatment alternative in patients with lung tumours (either non-small cell lung cancer or lung metastasis) since the year . interventional radiologists are expected to understand the clinical indications of these procedures at the first step. in addition, the techniques of the procedures and possible risk of complications in their management methods should be learnt by interventional radiologists as well. this refresher course will help us to discuss the main indications, techniques and complication management of these procedures. a. lung biopsy t. sabharwal; london/uk (tarun. sabharwal@gstt.sthames.nhs.uk) lung biopsy is most commonly now performed under ct guidance. it has a high technical success rate. common complications include pneumothorax, bleeding, sepsis, pain and failure. air embolism is a rare complication. pet scan is a useful tool to guide for appropriate target. ultrasound is useful for biopsying pleural-based lesions. drainage of thoracic fluid collections is a relative emergency. we will review the different thoracic fluid collections that can be drained by radiologists: pleural (parapneumonic, empyema, and malignant effusions), mediastinal, chest wall, pulmonary (lung abscesses and infected tumours) and pericardial effusions. we will answer the following questions about the main thoracic fluid collections: how to diagnose, when and how to drain, which guidance (ultrasound or ct-fluoroscopy), and procedures to perform before and after the drainage. the choice of imaging to detect thoracic fluid collections and guide drainage depends on the availability of the modalities, the imaging characteristics, location of the collection, and the comfort and expertise of the operator with the specific modalities. we will also review the main complications of percutaneous drainage of thoracic fluid collections (pneumothorax, pain, drain dislodgement and drain blockage) and compare these complications with those related to large-bore chest catheters inserted by thoracic surgeons. the incidence of lung cancers continues to increase and primary lung cancer remains the leading cause of cancer-related deaths in both women and men. therapy of lung tumours includes resection, radiation therapy, chemotherapy, thermal ablation or a combination of these treatment modalities. image-guided percutaneous thermal ablation therapies are minimally invasive techniques established in the local treatment of hepatic, renal, or osseous tumours. among these techniques, radiofrequency ablation (rfa) has now attained consideration for therapy of small pulmonal tumours. other ablation techniques that have been used for treatment of lung tumours include cryoablation, laser (litt) and more recently microwave ablation (mwa) and irreversible electroporation (ire). ablative techniques may produce a complete and irreversible tumour tissue destruction through application of either hot or cold thermal energy, or through permeabilisation of the cell membrane while using ire. ct is currently the imaging modality that is most widely used for planning, monitoring the ablation's course and for assessment of treatment response. only patients with stage i and stage ii lung cancer and those with small metastases are potential candidates for ablation. the medical history and physical examination of the patient as well as recent imaging with ct should be evaluated to determine the indication for thermal ablation. percutaneous lung rfa is considered as a safe procedure with an overall morbidity rate from . % to . % and mortality rate from % to . %; it requires less costs and offers faster recovery, and reduced morbidity and mortality. sorafenib, a tyrosine kinase inhibitor, has shown clinical efficacy in patients with hepatocellular carcinoma (hcc) and is the standard of care for patients with advanced-stage hcc. nowadays, many targeted therapies are evaluated in hcc either as sole treatment or in combination with other treatments such as tumour ablation, chemo-embolisation, and surgical resection. therefore, there is a need to assess the efficacy of targeted therapy in hcc. recist is the reference method to evaluate treatment efficacy in solid tumours, but does not seem appropriate in evaluating targeted therapy as objective responses are seen in very few cases in patients treated with sorafenib or sunitinib. new criteria have been proposed to evaluate the treatment efficacy of nonsurgical treatments in patients with hcc. the most common ones are the choi criteria, the easl criteria, and the modified recist criteria. all these criteria mainly focus on internal tumour changes such as appearance of necrosis or disappearance ot tumour hypervascularity. many examples will be shown during the lecture. another approach is based on functional imaging and especially perfusion-related imaging. contrast-enhanced ultrasound, ct perfusion and dynamic contrast-enhanced mr imaging have the capability to assess perfusion changes in patients under treatment. the advantages and disadvantages of these modalities will be discussed. lastly, other functional tools that are not routinely used will be presented. the evaluation of treatment efficacy is a key issue with prognostic and patient survival implications. it is crucial to have objective and reproducible criteria for specific groups of patients. the goal of ablative therapies of hcc is to induce tumoural tissue destruction. complete response (cr) after initial chemical and thermal percutaneous ablation, defined as the absence of contrast enhancement of the treated tumour at ceus, dynamic ct or dynamic mr, has been reported to correlate to long-term survival. nevertheless, the clinical effectiveness of imaging techniques to assess initial treatment success differs according to tumour size. the success rate of rf has been demonstrated to be superior to pei in hccs > cm, and depends on the ability to ablate all viable tumour tissue including an adequate tumour-free margin all around the lesion of . to cm. thus, the effectiveness of rf directly depends on the tumour location and size. rf is considered an effective treatment in lesions cm and its effectiveness is progressively reduced along with tumour size and it is not effective in lesions > cm. ceus beyond month may confirm or detect residual tumour, deserving a final ablation procedure. ct and mr are more effective in the follow-up to confirm cr and detect local recurrence, or additional hcc lesions in the liver parenchyma. the presence of transient hyperaemic inflammatory changes in the periphery of the treated area is a common finding that should be considered to avoid overestimation of the recurrence rate. one of the major characteristics of medical imaging in the twenty-first century is the dramatic influx of novel technology. this impact of new technology and techniques is experienced in all imaging modalities. the continuous development and implementation of highly sophisticated medical products and devices is key to the evolution of medical imaging leading to the improvement of patient care in terms of quality and positive outcomes. this session will analyse state-of-the-art displays (monitors, smartphones, tablets) and their applications in modern imaging. the session will provide a brief overview of displays in the current imaging chain. it will then evaluate the various specifications and parameters associated with displays. the session will evaluate current qa standards of modern displays as well as the challenges surrounding such novel technologies. image quality can be described in terms of resolution and contrast. an image displayed on a monitor consists of differences in brightness. the display monitor is at the end of the image chain in radiology, which starts at the x-ray tube. there are several factors in this chain that have influence on the contrast displayed on the monitor. diagnostic image quality can be described as observed contrast, which is not only dependent on the displayed image, but is also influenced by ambient conditions. for the primary class displays, typical characteristics of display monitors; such as resolution, homogeneity, luminance, contrast, monochrome or color, must be appropriate for the radiologist to execute a reliable diagnosis. calibration of display monitors is also important. which parameters are significant and what is the effect of variation of these parameters. there are standards like dicom gsdf and technical standard from acr-aapm-siim. both within and outside the clinical environment, the use of smartphones and tablet computers is increasing. reported applications range from teaching and education to navigation in surgical procedures. interest in the use of smartphones and tablets for viewing radiological images has been growing. however, these portable displays can differ significantly from conventional radiological displays. in order to determine where and how smartphones and tablets can be used appropriately, it is important to understand these displays and the challenges associated with them. this presentation will outline the current and potential uses of smartphones and tablet computers in radiology and compare their performance with other radiological displays and standards. the possible limitations/benefits of smartphones and tablets as display devices for radiological images will be discussed. recent research in the field will be reviewed, with emphasis on studies of diagnostic efficacy. in modern clinical environment, diagnostic monitors have replaced glowboxes and films and have become an important part of the imaging chain. the required specifications and the quality of monitors largely depend on their purpose, beginning from quite simple monitors used for the preview of radiographic images placed on modality or in control room to monitors used by high-end diagnostic workstations. decision about the kind of monitor for certain workplace is also very important from the economic standpoint as there are large differences in their prices. besides monitor specifications, also ambient conditions and ergonometry in the reading rooms are also very important issues which need to be considered to ensure optimal environment for clinical image reading. some of good and bad examples will be given in the presentation. as with other radiological equipment, also diagnostic monitors need to be controlled to ensure their optimum performance. quality control begins with monitor acceptance testing and setting up measurement parameters which are to be followed. practically all monitor vendors include some qc software within their workstations which can, together with some independent measurements, form an efficient quality control programme. in the last steps, how to deliver all information regarding image quality and possible presets on the diagnostic monitor and how not to confuse the user are important. sitting an examination is always a source of anxiety. the edir exam is no exception. this workshop is dedicated to those who want to take the edir exam, or become examiners, and would like to learn more about its format and discover some tips and tricks. the edir organisers and examiners have decided to build an interactive, friendly and enjoyable session in order to teach the essentials. some will play the role of the examiners, some the role of the candidate. in order to simulate the candidate's experience, this session will have no safety net. some senior members will expose themselves to real difficult questions in general radiology. fortunately, we hope that the attendees will be able to help the "candidates" to succeed. hopefully, you will enjoy this session and get some very useful information for your edir exam. abdominal malignancies are an important health problem in mexico. like in many other middle-income countries, there has been an epidemiological transition with an overall increase in oncologic diseases. colorectal, gastric and liver carcinomas are some of the frequent abdominal malignancies. colorectal cancer is a disease that is curable if detected early and even preventable if precursor polypoid lesions are removed. imaging plays a critical role in staging at diagnosis. additionally, virtual colonoscopy is an accepted modality in cancer screening. high-resolution magnetic resonance imaging (mri) has become a pivotal modality in the pretreatment assessment of rectal carcinoma. computed tomography (ct) is an excellent modality in the preoperative staging of gastric cancer and follow-up after treatment. positron emission tomography (pet) combined with ct (pet/ct) is particularly helpful for gastric and colonic carcinoma staging. hepatocellular carcinoma (hcc) is also common. cirrhosis related to alcohol and hepatitis c infection are by far the commonest aetiologies. ultrasound (us) in conjunction with alpha-protein is the most widely used modality for screening purposes. ct and mr are confirmatory modalities. the advent of hepatospecific gadolinium contrast agents has been very helpful in the characterisation of focal lesions in cirrhotic patients. the use of multidetector ct (mdct) in pancreatic carcinoma for the detection of vascular and adjacent organ invasion is crucial in treatment planning. over the last few decades, there has been an important improvement in imaging techniques resulting in better quality images. imaging is critical not only for the diagnosis, but also for treatment planning and follow-up. learning objectives: . to learn about the imaging of common oncologic gastrointestinal diseases. . to understand the importance of imaging in liver and colon cancer. . to learn how imaging supports oncologic institutions. activity, ultrasound is readily available and gives detailed local information but is limited by the restricted field of view, communication of results to clinicians and comparison of examinations on time. computed tomography (ct) is fast, readily available and gives a detailed, reproducible overview, but radiation exposure and contrast resolution are limitations. magnetic resonance imaging combines a good, reproducible overview with high contrast resolution, dynamic information and no radiation exposure and is therefore preferable in many situations, but the longer examination times than for ct, availability and costs are limiting factors. for the diagnosis of stenoses, fistulas and abscesses, either technique can be used, although the unrestricted view of ct and mri favour these techniques in many situations. in acute situations, us and ct are more accessible than mri. in that setting us can be considered, but when the examination is inconclusive or the patient has clear inflammatory signs, ct is preferable. endoscopy is currently considered the reference standard for the evaluation of colonic pathologies, including colitis. it allows direct visualisation of the mucosa and in obtaining tissue sample. however, it has major limitations including the invasiveness of the technique, incomplete endoscopy and risk of perforation. endoscopy cannot, therefore, help to estimate the depth of involvement of transmural inflammation and extraluminal complications. by contrast, an evolving role for cross-sectional imaging in the evaluation of patients with colitis has been increasingly recognised, especially in the setting of crohn's disease (cd), since cross-sectional imaging has been demonstrated to have a high diagnostic accuracy not only for assessing the presence and extension of luminal disease, but also for evaluating cd-related acute or chronic complications. establishing the ultimate cause of colitis may sometimes be challenging and histology cannot be conclusive. however, cross-sectional imaging may provide additional information that is useful in the workup of colitis. the use of cross-sectional imaging has been increasing in the evaluation of inflammatory bowel disease. following their presentations on imaging protocols and features of small bowel disease and colitis, each speaker will present a case to illustrate the learning points covered in their lectures. the session will be moderated by the chairman and the audience will have full opportunity to ask questions of all the panel. multimodality imaging is essential in a wide variety of oncology situations. anatomic imaging, whether using us, ct or mri, is mandatory for tumour localisation. moreover, the evaluation of treatment response mostly relies on size assessment, whether uni-or bi-dimensional. with the advent of new targeted bio-therapies, functional imaging has progressively been integrated in the imaging strategies whether for better tumour characterisation or for optimal evaluation of treatment response. hence, assessment of molecular targets by pet is supplemented by the recent developments of diffusion mri, reflecting tissue architecture and cellularity, tissue perfusion, reflecting angiogenesis, and magnetisation properties of tissues. hcc is a primary liver tumour where the use of multimodal anatomic, functional and metabolic imaging appears of particular interest. according to easl and aasld recommendations, noninvasive diagnosis of hcc can be performed using dynamic contrast enhanced cross sectional imaging. liver mri provides optimal sensitivity and specificity for the detection of as small as cm large hcc. recent reports have suggested that the prognosis of hcc lesions could be anticipated based on the combined analysis of metabolic f -fdg pet ct, as well as with diffusion weighted imaging. the objective of this lecture will be to highlight the potential of combining both metabolic and multimodal cross sectional imaging in order to improve the management of patients with hcc. the (extra)ordinary night shift at the er k. petrovic; novi sad/rs (smakap@sbb.rs) besides the accuracy, working at the er requires maximal shortening of the diagnostics time, which has a significant influence on the patient's outcome. thus, it is the radiologist's duty to be familiar with the pathophysiological mechanism of the disorders, specific protocols, possibilities and limits of certain examinations, and also to be aware of all possible pearls and pitfalls, to make the correct diagnosis. pathology encountered at the er has an extremely wide range and requires expertise in all imaging modalities from head to toe. the more the radiologist is aware of all possible situations, the more is the diagnosis accurate, and information given to the clinician is more valuable. the aim of this presentation is to point out the possibility of encountering different pathological conditions in different body regions. moreover, it is important to be reminded that in radiology there are different modalities, each of which has its own indications and limits. this lecture reviews the selected series of cases from the er department of clinical center of vojvodina, pointing out the most common possible differential diagnosis and possible diagnostic mistakes, which often occur due to the lack of experience. learning objectives: . to point out the in-depth knowledge and experience in all imaging modalities and all body regions required for a radiologist to perform a routine work at the er. . to present a series of cases that are most and less often encountered at the er, discuss the possible differential diagnosis and mistakes which could potentially be made. diagnosis and management of pancreatic cystic lesions r.j. méndez; madrid/ es (ramiro.mendez@salud.madrid.org) pancreatic cystic lesions are frequently diagnosed in patients with clinically suspected pancreatic disease, but they are also increasingly detected on imaging studies performed due to other abdominal processes. the prevalence of pancreatic cysts increases with patient's age. most symptomatic cysts will be surgically resected, whereas asymptomatic lesions should be managed depending on the likelihood of causing harm to the patient. cystic lesions can be inflammatory or neoplastic. the incidence of pseudocysts is low without a history of pancreatitis. some pancreatic cystic neoplasms are malignant, but even benign lesions can become symptomatic depending on its size and location in the pancreas. the radiologist detects most of the pancreatic cystic lesions and should also play an important role in the clinical decision-making process. cyst size, morphology, calcifications, contrast enhancement, location in the pancreas, relation with pancreatic ducts, patient's age and gender are important data to classify the lesion. a specific radiological diagnosis is not possible in every patient, but in many cases this information is enough to decide on a conservative approach with imaging follow-up. if a more aggressive lesion is a concern, then endoscopic ultrasound and fluid analysis can help to better characterise some pancreatic cystic lesions. cyst puncture is only recommended if fluid analysis results will modify the patient's management. patient's age, status and preferences should also be taken into account. when follow-up is recommended, the preferred imaging technique and interval should be indicated. the chair will set the scene for the need for effective radiation protection in clinical radiology and highlight the role of clinical audit as a tool in achieving this. clinical audit is defined as a professionally led method of improving patient care through the systematic examination of systems, processes and outcomes against chosen and agreed standards. it can readily be carried out by individuals, groups or whole departments. if properly conducted, clinical audit can be an effective way of examining what we do to provide reassurance on performance, and to improve patient care. the clinical radiology audit committee (crac) of the royal college of radiologists (rcr) co-ordinates national radiology audit activity. it promotes and facilitates audit through nominated audit leads in each hospital, who act as a link between the rcr and their department. at least one national audit is carried out annually, with data collected via electronic submission and the anonymised results presented at an annual audit forum. individual departmental results are analysed using statistical process control (spc) methodology. this enables identification of departments underperforming against the national mean, and recommending corrective action, by redesigning the process being audited, or by identifying and eliminating specific root causes locally. the committee has developed a web-based tool for facilitating local audit, "auditlive", a fully searchable collection of templates which can be downloaded and adapted. fellows are also able to submit their own templates for publication hence sharing best practice. trainees are encouraged to participate in audit through audit poster competitions at national radiology scientific meetings. our experience leads us to believe that audit succeeds when relevant, locally owned and properly structured, and multi-professional, and the rcr model encourages this. models of external audit in the netherlands s. geers-van gemeren; utrecht/nl in the netherlands clinical audit is legally obligatory for healthcare professions in order to be able to practise since . clinical audit is a tool designed to improve the quality of patient care, experience and outcome through formal review of systems, pathways and outcome of care against defined standards, and the implementation of change based on the results. the quality of the provision of care by professionals is assessed by peers. in the fields of radiology, nuclear medicine and radiotherapy, different models of clinical audit are used. for radiotherapy a multidisciplinary audit has been used since . for nuclear medicine a multidisciplinary audit has been implemented since . for radiology the clinical audit for radiologists and for radiographers are separate. implementation of the multidisciplinary audits needs requirements and adjustments of the audit system. this process is complex and needs approval of the members of all involved societies. to support the clinical audit a web based tool adas (general digital audit system) is used. the development of professional standards is a prerequisite to start clinical audit. the use of adas in multidisciplinary audits is a requirement to be able to audit different professions and focus on the content and the quality of their contribution to patient care. clinical audit is a good tool to improve the quality of patient care. important are the professional standards, the culture of learning and willing to improve by the professionals. "every defect is a treasure." learning objectives: . to learn about the different dutch models of external clinical audit. . to comprehend the importance of professional standards, the culture of learning and willingness to improve. . to become familiar with the use of a digital audit system to support multidisciplinary clinical audit. external regulatory audit in finland r. seuri; helsinki/fi (raija.seuri@hus.fi) directive / /euratom states that the clinical audit of medical imaging should be carried out in accordance with national procedures. the finnish solution has been regulatory external audits every five years by radiological professionals with special training for audits. all radiological units have so far been audited at least twice, and the third time is performed on site in the unit. although the purpose is to audit the process of radiological practises compared to "good practise", special focuses are recommended by the finnish advisory committee for clinical audit, set by the national institute for health and welfare. at the beginning focus was on the organisation and documentation of the structure and processes of imaging practise like justification, optimisation and quality control, but also encouragement and guidance to self-assessment. later special focus has been on paediatric imaging and ct, and the third cycle will target deeper to ct practises. the auditing group includes at least a radiologist and a radiographer to audit a small unit, and a medical physicist if the unit has high-dose modalities like ct. during the audit the auditors work for an independent organisation. multi-professionality gives both insight and the possibility to contact all professionals in imaging practise. the focus of clinical audit is on quality improvement; it is a way to both give and get feed-back and education. it is not control or inspection, but encouragement and guidance to self evaluation and implementation of good practises. we often think of the scientific researcher as detached, objective, and dispassionate, nobly labouring without any expectation of reward. nothing could be further from the truth. scientists are as subject to ambition, competitiveness, envy and even guile as any person. lifting the veil on scientific discovery reveals to us the human dramas that underlie not only the coveting of recognition, but also great conflicts over priority and credit. nowhere are the stakes higher than for that pinnacle of scientific honour: the nobel prize. mri has had a notably tempestuous genesis -a cogent episode that has much to teach us. it involved an explosive clash of personalities; deceit and rampant self-interest; challenges to the basic concept of scientific behavior; legal suits and validation by the united states supreme court; and declamations of denunciation in the international press. it persisted as a year battle -a prize fight, a blood feud -between two protagonists: one whose seminal contribution established the biologic basis of mri, and the other whose flash of insight served as a cornerstone of diagnostic imaging. . metabolic disorders of the skeleton involve the mineralised components of the skeleton. they affect all bone components histologically, but involvement patterns may vary depending on the age of the patient (growing versus adult skeleton) as well as the type of bone (cortical versus trabecular bone). they can be depicted on radiographs and ct images, but remain occult on mr images because the bone marrow is spared in the vast majority of these disorders. mr imaging can help in the assessment of local complication such as fractures. . metabolic disorders of the bone marrow can affect either red (anemia…) or yellow marrow as well as the balance between these marrow types in the body. medical imaging plays a limited role in the assessment of these disorders, but marrow changes associated with these metabolic conditions must be recognised to avoid confusion with neoplastic conditions. in primary and secondary malignant tumours of solid organs, in most of the cases surgical resection is considered the curative treatment. however, this is possible only in about % of the cases with tumour-dependent recurrence rates of % and even more. the main components for adjuvant, neo-adjuvant and finally palliative therapy are the permanently growing number of chemoand antibody therapies. nevertheless, in many cases these therapies provide no definitive or long-lasting success, necessitating multimodality treatment concepts. in the meantime, local ablative techniques represent the main components of these concepts. three main minimally invasive tumour-ablative techniques can be differentiated: chemo-ablation (percutaneous alcohol injection, transarterial chemo-embolisation, chemotherapy, chemo-perfusion), thermo-ablation (radiofrequency-, microwave-, laser-ablation, high-intensified focused ultrasound, cryo-ablation) and radio-ablation (radio-embolisation, interstitial brachytherapy, percutaneous stereotactic radiation). these ablation techniques differ significantly among each other with respect to their mode of action and indications; nevertheless, their benefit is high tolerance and at the same time high therapeutic efficacy, which can easily be combined with other treatment modalities. the different minimally invasive techniques will be discussed together with specific short-and long-term results and complications. missed lung lesions are one of the most frequent causes of malpractice issues. chest radiography plays an important role in the detection and management of patients with lung cancer, chronic airways disease, pneumonia and interstitial lung disease. among all diagnostic tests, chest radiography is essential for confirming or excluding the diagnosis of most chest diseases. however, numerous lesions of a wide variety of disease processes affecting the thorax may be missed on a chest radiograph. the chest radiograph will also help narrow a differential diagnosis, help to direct additional diagnostic measures, and serve during follow-up. the diagnostic usefulness of the radiograph will be maximised by the integration of the radiological findings with the clinical features of the individual patient. ct has a tremendous spatial resolution that helps detect lesions in the chest and has proven to be more sensitive and specific than chest radiographs. however, missing lesions or misinterpreting lesions in ct of the chest is not uncommon. in this session, we will provide interactive cases of chest examinations (radiographs and ct) in which lesions have been missed and or misinterpreted, with a special focus on how correlation with mdct of missed lung lesions can help improve interpretation of plain chest radiographs. this session will explore the value of imaging for facilitating precision medicine, in which molecular data (including genomics, proteomics and metabolomics) is used to classify patients into subpopulations and tailor treatments to the specific molecular characteristics of their diseases. imaging already plays a limited role in precision medicine, primarily through the use of molecular imaging techniques (e.g. pet/ct, mri/pet and hyperpolarised mri) and theranostics (the combination of an imaging agent with a therapeutic entity). in the near future, the role of imaging in precision medicine will increase at a gradually accelerating pace through the development of new molecular imaging probes and theranostic agents and through progress in radiogenomics (the correlation of radiologic findings with genomic features). this session will discuss recent findings and methodologies in radiogenomics as well as the use of nanotechnology for designing novel molecular imaging probes and theranostics. in addition, as theranostics is often considered the essence of precision medicine, the session will provide an in-depth look at current and emerging theranostic strategies and their special benefits for treatment selection, assessment of dose distribution of targeted therapies, and treatment follow-up. radiogenomics and personalised (precision) medicine g.p. krestin; rotterdam/nl (g.p.krestin@erasmusmc.nl) "precision medicine" as well as related notions, such as personalised medicine, or stratification medicine, all revolve around the idea that the consideration of individual characteristics -molecular and otherwise -can improve medical research and practice. precision medicine is a multi-faceted approach to medicine that integrates molecular and clinical research with patient data and outcomes. individual assessment of the location and extent of an alteration is and always has been the basis of medical imaging, whether the 'alteration' is a disease, a malformation, or an injury. as such, medical imaging intrinsically enables "precision medicine". the addition of genomic data in the last twenty years allows new correlations to be made between cellular genomics and tissue-scale imaging. structural and functional imaging and the automated analysis of large amounts of image data have only recently reached a stage where they can be used on a large scale and in a population setting. identifying common genetic variants that contribute to explain variance in imaging phenotypes by a systematic analysis of the genome is based on genome-wide association studies (gwas). the power of gwa analyses has been recently demonstrated with the identification of susceptibility genes involved in a range of imaging phenotypes like coronary artery calcifications, intracranial volume, or size of the hippocampus. these developments highlight new etiological pathways and are expected to improve the understanding of the molecular basis of some diseases. correlation between genotype and imaging phenotypes may be relevant for further characterising the development of disease states. use of nanotechnology, imaging and therapy t. lammers; aachen/ de (tlammers@ukaachen.de) advances in nanotechnology and chemical engineering have led to the development of a significant number of novel materials for diagnosis and therapy. many different diagnostic and therapeutic nano-and micro-materials have been designed and evaluated over the years, including, e.g. gadoliniumcontaining dendrimers, uspio nanoparticles and microbubbles for functional and molecular imaging, and drug-loaded liposomes, polymers and micelles for temporally and spatially controlled drug delivery to tumors and to sites of inflammation. in the literature, such advanced nano-and micromaterials are generally claimed to be highly useful and broadly applicable. a critical reflection on their specific capabilities, however, as well as on their pharmacokinetic properties, biodegradability and toxicity is often lacking. in the present lecture, i will briefly introduce the rationale for using diagnostic and therapeutic nanomaterials. i will highlight several clinically relevant examples in which nano-and micromaterials hold potential for improving disease diagnosis and detail how combining diagnostic and therapeutic properties within a single 'nanomedicine' formulation can be used to individualise and improve (chemo-) therapeutic treatments. learning objectives: . to learn about the design and construction of nanoparticles for use in imaging and therapy. . to appreciate both the advantages and limitations of using nanoparticles as agents for both imaging and therapy. . to learn how nanotechnology enables multimodality imaging and therapeutic agents. theranostics w. weber; new york, ny/us precision medicine requires diagnostic tests that predict the effectiveness of specific therapeutic interventions for individual patients. this close interaction between therapeutics and diagnostics is expressed in the term "theranostics". a frequently used example for a theranostic is the staining of tumours for her expression prior to therapy with her antibodies, such as trastuzumab or pertuzumab. theranostic has also been practised since many years in nuclear medicine by using radioiodine scans to select patients for treatment with iodine- . a more recent example is the selection of patient for peptide receptor-targeted radiotherapy (prrt) by somatostatin receptor imaging. imaging is highly attractive for theranostics, because it allows investigators to study the spatial and temporal heterogeneity of target expression, whereas in vitro assays generally analyse a small part of the tumour at one point in time. several molecular imaging probes, such zr-trastuzumab or folate receptor targeting ligands, are in clinical development as in vivo companion diagnostics. imaging companion diagnostics can also be used to assess tissue pharmacokinetics and thereby help to determine the optimal dose and dose schedule of therapeutic agents. they can also identify unexpected interactions between two therapeutic agents. for example, studies with c-docetaxel have shown that bevacizumab can markedly decrease the intratumoral delivery of docetaxel. finally, imaging-based companion diagnostics can assess target inhibition, as demonstrated by the inhibition of uptake of the androgen receptor ligand f-fdht in metastatic prostate cancer treated with anti-androgens. imaging-based companion diagnostics are therefore expected to play an important role in establishing precision medicine. erus technique as well as the state-of-the-art mr imaging protocols tailored according to the presenting rectal tumor site, allowing trained radiologists to obtain all necessary information for appropriate treatment decision-making, will be described. normal cross-sectional appearances as well as morphological and signal changes encountered in pelvic structures/tissues that may be involved in primary rectal cancer will be illustrated. the influence of imaging findings on initial therapeutic approach and potential limitations of different imaging techniques will be discussed. the staging and categorisation of malignant lymph nodes in patients with rectal cancer is a topical issue and has resulted in some degree of confusion. much enthusiasm has been expended in determining whether mesorectal nodes are, or are not, involved in the primary disease process, as there is a perception that nodal disease is an important determinant of local recurrence. this incorrectly perceived association of nodal status and a high risk of pelvic recurrence have propagated the concept that this should be the primary indication for neoadjuvant therapy regardless of whether tme surgery is to be performed. furthermore, misinterpretation of involved local nodes results in over-treatment of patients, if firstly involved nodes alone are considered as a poor prognostic factor, and secondly the optimal treatment of involved nodes is pelvic radiotherapy plus a non-systemically acting but radiosensitising dose of chemotherapy within chemoradiotherapy (crt) schedules. in this lecture, the evidence for risk factors associated with nodal disease and validated risk factors for local recurrence will be reviewed. the evidence for both the optimal techniques and objective criteria for assessing lymph nodes with magnetic resonance imaging (mri) will be presented. the standard treatment for advanced rectal cancer is preoperative chemoradiotherapy (crt) followed by standard resection of the rectum and mesorectum. neoadjuvant crt allows downsizing and downstaging of the tumour, leading to improved resectability and local control. while the role of mri in rectal cancer treatment is recognised and mri is recommended as part of the standard staging workup, its role for restaging after preoperative treatment is more controversial. this lecture will provide an understanding of whether and how mri can assess treatment response in rectal cancer and how it may impact treatment decision. the attendees will learn about the difficulties in image interpretation and learn about potential new imaging techniques to improve its performance. primary tb typically presents with consolidation in the middle and lower lobes, and necrotic lymphadenopathy. fibrosis, tuberculomas and calcified nodes may result as sequelae. cavitation and tree-in-bud appearance involving the apical and posterior segments of the upper lobes and the superior segments of the lower lobes are the hallmarks of reactivation tb is typically seen in immunocompetent patients. in immunocompromised patients, primary tb findings are more commonly observed, as miliary or disseminated disease or atypical manifestations in case of severe immunosuppression. pleural effusion and tracheobronchial involvement may be observed. the "classical" appearance of non-tb mycobacteria (n tm ), less common and more indolent than tb, typically affects males more than years old with pre-existing pulmonary disease or underlying immunologic disorder. despite a great overlap in the radiologic appearances of both infections, the presence of cavities on sites other than the upper lobes should suggest an n tm infection. the "nonclassical" form related to mac infection that predominantly affects elderly women with no pre-existing pulmonary disease mainly consists in mild bronchiectasis and centrilobular nodules predominantly located in the lingula and middle lobe. a high rate of lymphadenopathy and disseminated disease are seen in immunocompromised patients. the radiologist has to ensure the diagnosis of active tb and mention scarring before starting specific treatments; to suggest n tm infections, immune reconstitution inflammatory syndrome with paradoxical worsening in haart treated hiv-infected patients as multi-drug resistant tb; to appreciate the extent of disease and follow-up; to diagnose the complications; to propose mri or pet scans in some situations. learning objectives: . to appreciate the imaging features of primary and post-primary infections in tb. . to learn about the similarities and differences between tb and non-tb mycobacterial infections. . to understand the radiologist's role in diagnosis. the appropriate investigational technique, frequently targeted differential diagnosis, and the special needs of immunocompromised patients need to be understood by the referring physician as well as by the radiologist. thus, an intensive interdisciplinary co-operation on a patient basis, as well as on a department basis is essential. early detection of a focus is the major goal in febrile neutropaenic patients. as pneumonia is the most common focus, chest imaging is a special radiological task. the sensitivity of chest x-ray, especially in the supine position, is known to be low. therefore, the very sensitive thinsection multislice-ct became the gold standard in neutropenic hosts and might be cost-effective in comparison to antibiotic treatment. the infiltrate needs to be localised, so that this information can be used as guidance for invasive procedures for further microbiological workup. furthermore, the radiological characterisation of infiltrates gives a first and rapid hint to differentiate between different sorts of infectious (e.g. typical bacterial, atypical bacterial, fungal) and non-infectious aetiologies. follow-up investigations need careful interpretation according to disease, recovery, and concomitant treatment. due to a high incidence of fungal infiltrates, interpretation of the follow-up of an infiltrate must use further parameters besides the lesion size. besides the lungs, also other organs systems such as brain, liver and paranasal sinuses need attention and are to be imaged with the appropriate technique. despite advances in diagnosis and treatment, new pulmonary infections have been diagnosed. streptococcus pneumoniae remains the main aetiological agent in outpatients with community-acquired pneumonia (cap). elderly patients or those with toxic habits and various comorbidities favour the development of severe cap. in addition, the development of nucleic acid amplification techniques has emphasised the role of concomitant bacterial and viral pneumonia in the outcome of cap in elderly patients. healthcareassociated pneumonia has been recently defined as a different infectious condition by the american thoracic society/infectious diseases society of america (ats/idsa). the main concern of this new disease is the risk of having an infection due to multidrug-resistant pathogens. with the advent of haart and increased long-term survival of hiv-positive patients, the range of pulmonary manifestations has also evolved. in patients with haematological malignancies or after hsc transplant, aspergillus is a common infection. actually, aspergillus spp. isolation from lrt samples in copd may indicate an increased diagnosis possibility of ipa. new emerging viruses such as human metapneumovirus (hmpv), sars-associated coronavirus, and avian influenza caused by the h n virus have been diagnosed. in , an outbreak of a novel swine-origin influenza a (h n ) virus was reported. the clinical diagnosis of new pulmonary infections as well as the presence of concomitant bacterial and viral infections has been significantly enhanced by improved laboratory methods. a systematic approach to the radiological evaluation of lung infections is essential and includes not only chest imaging pattern recognition, but also integration of available demographic, clinical and laboratory information. the paediatric central nervous system is a complex structure undergoing rapid development. as such, there is a rapid, continuous modification of what is "normal" in relation to age and the stage of development. knowledge of the normal patterns of brain development in the clinically relevant ages from to years is necessary to interpret neuroimaging findings correctly. knowledge of embryology and normal variants is also greatly helpful. mr imaging equipment and parameters need to be adjusted and optimised for paediatric studies. pitfalls often occur from the misunderstanding of normal conditions that are perceived as abnormal based on a comparison with the appearance of the normal brain in adults. this includes, for instance, the evaluation of the brain in the first - years of life during the course of the process of myelination. a summary of the most frequent conditions that may lead to misinterpretation of findings will be provided. a wide spectrum of anomalies of thoracic and abdominal organs may be encountered on radiologic evaluation. these anatomic variants and developmental anomalies can all pose a diagnostic challenge to the radiologist. familiarity with these anomalies, the imaging techniques available for their study, and their variable imaging manifestations is necessary for differentiating them from pathology. a basic understanding of the embryologic development and normal anatomy of thoracic and abdominal organs and vessels is also essential for identifying these anomalies. this review explores the wide variability in appearance of the normal thoracic and abdominal organs during imaging, stressing a thorough understanding of normal anatomy to recognise normal variants. the skeleton of a child is a developing system with a variety of changing normal appearances. imaging studies, especially plain films, are requested for many clinical reasons, and the radiologist is in the position to determine if an image is a normal finding or we are dealing with a lesion. the way the physis and epiphysis grow, ossify, and fuse constitutes a great source of physiologically bizarre appearances, which the radiologist must be familiar with. this talk will concentrate on the plain film diagnosis of some of the most common musculoskeletal variants. other imaging modalities will also be shown when appropriate for the case. irregularities, asymmetries, partial fusions, hypo-or hyper-dense bone areas, accessory bones, prominent normal structures, external artifacts, and potential fracture lines are the most often encountered pseudolesions. a defective radiological technique may also be potentially misleading. patient age, location of the supposed "abnormality" and lack of significant local symptoms are key factors. usually plain films, correlated with regional clinical findings, are the only imaging method that is required. however, in certain doubtful situations, ultrasound, ct, mri, bone scan, or even biopsy may be needed to reach the right diagnosis. unnecessary overuse of these imaging modalities, and the subsequent family anxiety that ensues from this overuse, should be avoided with careful analysis of the x-ray and clinical findings. the purpose of this lecture is to cover the clinical presentation, underlying pathological processes and essential mri features of relatively common conditions affecting the hip. as the hip is afflicted by different conditions according to age, this is how the various pathological entities will be presented. a simple imaging algorithm is presented showing the role of mri. the role of mr arthrography in the assessment of the dysplastic hip and femoroacetabular impingement is covered. a structured approach to mri reporting is outlined. (claudia.schueller-weidekamm@meduniwien.ac.at) mri of the hand requires high spatial resolution, perfect immobility, and homogeneous fat suppression. the hand can either be placed in the superman position, which often is uncomfortable for the patients, or both hands can be placed above the abdomen next to each other while the patient is in the supine position. standard sequences are axial pd, followed by coronal t tse, and t tse fs in the coronal and sagittal planes. additional d gradient echo sequences with fat saturation are recommended to assess ligaments and cartilage, as well as the triangular fibrocartilage complex (tfcc). in certain cases, mr arthrography with thin-slice t fs is helpful for further evaluation of the wrist ligaments. in inflammatory diseases, such as rheumatoid arthritis, static or dynamic t fs sequences are useful to assess tenosynovitis and synovitis. the most common injuries and inflammation of the hand and wrist are discussed with special emphasis on key findings for an accurate diagnosis. the awareness of advantages and drawbacks of other imaging modalities, such as conventional radiographs and ct, should be strengthened. the clinical impact of the radiological report is discussed to strengthen the importance of proper terminology for the description of pathological findings. the structure of the report should be clear and concise, and should allow an interaction and broad communication with clinicians. gastro-enteropancreatic neuroendocrine tumours (gep-net) are a heterogeneous group of cancers that differ in their biology and clinical presentation. diagnosis of these tumours has been improved by advances in pathology and classification and by the combined use of structural imaging and functional imaging modalities. multimodal imaging is increasingly recognised both in detecting and staging disease and also in characterising biological patterns of lesions that may be relevant to the selection and delivery of therapy. in this course, the complex nature of gep-net and the intrinsic uses and limitations of each diagnostic imaging modality will be underlined. insights to hybrid structural and molecular imaging techniques will be provided and discussed. a. tumour biology, pathogenesis and classification b. wiedenmann; berlin/ de (bertram.wiedenmann@charite.de) neuroendocrine tumour cells are characterised by the coexpression of neuronal and epithelial proteins and cellular organelles such as synaptic vesicles containing synaptophysin of neurons and intermediate filaments/cytokeratins of the epithelial cells. based on the presence of secretory vesicles and the continous, uncontrolled vesicular release of biogenic amines, neuropeptides and hormones, patients suffer in half of the cases of so called functional symptoms and syndromes. examples are the carcinoid syndrome (excessive release of serotonine) or the zollinger-ellison syndrome (excessive release of gastrin). activation or inhibition of certain g-protein coupled receptors (e.g. somatostatin receptors) or channel proteins (r-type calcium channels) can lead to the control of the hypersecretion or so called functionality of the affected patients. medical interference with signal transduction pathways involving tyrosine kinase receptors as such mtor and channel proteins can lead to an inhibition of cellular and tumour growth. these observations have led to the establishment of new therapies, especially for pancreatic nets using especially mtor and tyrosine kinase inhibitors. whereas the tumorigenesis is unknown in the case of sporadic nets, hereditary nets appear to develop via a menin mutation through the intermediate stage of hyplasia before they develop the full metastatic potential. based on the above given tumour biological and histopathological findings, a rather robust classification for nets has been developed by the european neuroendocrine tumor society (enets) using a tnm-classification together with a gradingsystem. furthermore, the formerly used terms such as apudoma, neurocrestoma, carcinoid, etc. have been largely replaced by the meanwhile generally accepted term neuroendocrine tumour. this presentation will focus on the pivotal role of nuclear medicine in the diagnosis and treatment of neuroendocrine tumours (nets). the metabolic and molecular imaging capabilities of both positron emission tomography (pet) and single photon emission tomography (spect) have made a great impact on the clinical management of patients with these tumours. a poster child of molecular imaging in oncology is scintigraphy of the somatostatin receptor (ssr). in-dtpa-octreotide has a substantial value in detection, diagnosis and staging of net and more particularly in gastroenteropancreatic nets. newer ssr-binding radiopharmaceuticals have been developed for pet, mainly with gallium- as radiolabel, with higher affinity and more advantageous pharmacokinetics. when used with high-resolution and rapidthroughput multimodal whole body imaging of modern pet/ct cameras, these tracers provide the current state-of-the art ssr imaging. we will also discuss the clinical value of metabolic tracers, such as [ ] -hydroxytryptophan) for amino acid metabolism imaging. the clinical merits and indications of these tracers will be explained. the continuously evolving quest to develop tracer for other receptor systems expressed on nets will be illustrated, e.g. bombesin, vip, cck and glucagon-like peptide receptor ligands. finally, the role of imaging as selection for metabolic and peptide receptor radionuclide therapy will be discussed. gastroenteropancreatic neuroendocrine tumours (gep-nets) are a heterogeneous group of neoplasms that arise from cells of the diffuse neuroendocrine system and may present with a wide spectrum of clinical presentations. their prognosis is mainly related to their biology, proliferation and differentiation. the main goals of imaging are the diagnosis and the staging of these tumours. the diagnostic challenge is very different in functional tumours where clinical presentation and laboratory parameters are of utmost importance and in nonfunctional tumours where imaging may show characteristic features such as hypervascularisation and calcifications. staging is also essential as locoregional involvement and distant metastases (such as liver metastases) may change the therapeutic approach and are major prognostic factors. multimodal workup included morphological imaging modalities with ct and mr imaging, and endoscopic ultrasound was the most useful and functional imaging. the latter includes somatostatin receptor scintigraphy, fdg pet and more recent functional tools such as pet using ga and f-dopa. imaging may also play a role in assessing prognosis in combination with tumour differentiation and tumour proliferation, obtained from pathologic examination. last, imaging is useful in evaluating tumour response after treatment. although surgery remains the only potentially curative therapy for patients with primary gep-nets, other available treatments include chemotherapy, interferon, somatostatin analogues, and targeted therapies. imaging criteria rely not only on changes in tumour size, but also on internal tumour changes. the purpose of this session is to bring the audience up to date with a novel mri method capable of measuring brain perfusion. this method is called arterial spin labelling (asl) and provides a complete non-invasive means to quantitatively assess cerebral blood flow (cbf). as with any mri technique, asl relies on proper setting of many technical parameters to provide an adequate image quality, with minimal influence from potential artefacts. a recent position paper, co-signed by members of the perfusion study group from the international society for magnetic resonance in medicine and the european cost-funded action on 'asl in dementia', has established a series of simple guidelines to help promote this technology in clinical practice. these guidelines will be provided within this session. the created cbf maps will then be analysed, and a simple description of the main features and clinical characteristics of these physiological images will be given to the audience. in particular, a proper difference in the meaning of cbf in several diseases will be highlighted, as it can be either causative, such as e.g. in stroke and cerebrovascular diseases, where a reduced cbf leads to a breakdown in tissue homeostasis, or consequential, as in e.g. dementia, in which a reduction of cbf is the mere reflection of impaired metabolism, combined or not with an underlying brain atrophy. following this session, the radiologist or clinician should be able to better appreciate asl-based sbf maps in several neurological conditions. perfusion is an important parameter to assess the status and liability of organs and tissue. typically, exogenous contrast agents are administered to measure this quantity. arterial spin labelling (asl) is capable of estimating perfusion without the use of exogenous contrast media by labelling inflowing blood magnetically. this labelling process can happen either in a short time over an extended region or over a longer time period, but in a localised area. these techniques are called pulsed asl (pasl) or continuous asl (casl), respectively. for quantification, a critical parameter is the time the labelled blood takes to flow from the region of labelling to the imaging region. this time is typically called bolus arrival time (bat) or arterial transit time (att). it is important to either reduce the influence of this transit time on measured signal intensity or directly estimate it. applying proper bio-physical models, the perfusion-weighted data can then be converted in absolute measures of perfusion. more sophisticated methods allow measuring of the main feeding artery of each imaging voxel (vascular territory mapping) or assessing other parameters beyond perfusion, allowing more detailed assessment of tissue status and function." the use of asl in cerebrovascular disease j. hendrikse; utrecht/nl (j.hendrikse@umcutrecht.nl) obvious applications of arterial spin labelling in clinical mr protocols are cerebral blood flow measurements in patients with acute or chronic cerebrovascular disease. in patients with acute stroke, the cerebral blood flow measurements may indicate the infarct core, with severely decreased perfusion and the infarct penumbra, with decreased perfusion but still viable brain tissue. in chronic cerebrovascular disease, arterial spin labelling cbf measurements show the regionally impaired haemodynamics distal to a carotid obstruction. with adequate collateral blood flow, these areas may be relatively small and with a failure of compensatory mechanism the cerebral blood flow may fall below a critical level. in acute stroke patients, asl mri may show compensatory hyperperfusion in stroke regions after the recanalisation of an occluded artery. other applications of arterial spin labelling are clinical mr protocols in children. in addition to cbf-weighted images, asl mri has also the ability to measure timing parameters: typically, the time it takes for the magnetically labelled arterial blood to flow from the arteries in the neck to the brain tissue, which may be delayed in patients with cerebrovascular disease. furthermore, asl mri has the ability to visualise the (collateral) perfusion territories of the brain feeding arteries in many clinical applications. in patients with cerebrovascular disease, a pitfall may be the absence of label in brain regions due to delayed (collateral) flow, which also may result in high asl signals in (collateral) arteries. learning objectives: . to appreciate the different roles of asl in cerebrovascular diseases. . to become familiar with the different types of calculated images obtained from multi-time points and selective pulses asl. . to become familiar with the limitations and pitfalls of asl. the use of asl in non-vascular brain disease m. smits; rotterdam/nl (marion.smits@erasmusmc.nl) arterial spin labelling (asl) is a non-invasive mri technique with which cerebral blood flow (cbf) can be measured quantitatively. although the first publications of asl mri date over years back, it is only now that asl is commercially available on mri systems from all major vendors and is making its way into clinical practice. the main areas of interest for current and future nonvascular clinical application of asl mri of the brain are dementia and neuro-oncology. asl is proposed as a diagnostic alternative to fluorodeoxy-dglucose (fdg)-pet in the workup of dementia patients. asl has several advantages over fdg-pet. crucially, it can easily be added to the routinely performed structural mri examination. feasibility studies show that asl provides reliable cbf maps in dementia. in patients with established alzheimer's disease and frontotemporal dementia, hypoperfusion patterns are seen that are similar to hypometabolism patterns with fdg-pet. current and future studies in this field will need to demonstrate the validity of asl in the diagnostic workup of the individual patient, early in the disease process. studies on asl in brain tumour imaging indicate a high correlation between areas of increased cbf as measured with asl, and increased cerebral blood volume as measured with dynamic susceptibility contrast-enhanced perfusion imaging. the major advantages of asl for brain tumour imaging are the fact that cbf measurements are not influenced by breakdown of the blood brain barrier, as well as its quantitative nature, facilitating multicentre and longitudinal studies. the analysis of a sinonasal tumour requires mri for differentiation between tumour and secretions or polyps, to assess the local extension and spread into adjacent compartments (skull base, intracranial, orbit, deep facial compartments) and to depict perineural spread. t , t and high-resolution postcontrast series preferably in three, but at least in two different planes, are needed. ct provides additional information of bony structures and serves as a road map for surgical navigation. a fascinating benign sinonasal tumour is inverted papilloma, which has a high recurrence rate if not removed in toto and may harbour carcinoma in < % of patients. ct may show the attachment of the tumour as an area of increased sclerosis, which directs the surgeon to plan the operation. in patients with recurrent inverted papilloma, providing information to the radiologist about its presumed localisation is important, the more so in patients suffering from polyposis. malignant tumours are uncommon and of a large histologic variety. about % arise in the maxillary sinus; % are squamous cell carcinomas. they are often large at diagnosis, because clinical symptoms of nasal obstruction or headache are non-specific. diagnosis is by biopsy. in view of the rapid development of advanced endoscopic techniques, preoperative imaging is extremely important to assess accurately tumour extension. the skull base and dura as well as the lamina papyracea should be scrutinised for tumour locations to foresee an additional cranioendoscopic approach to a standard endonasal endoscopic approach. finally, imaging may play a role in differentiating non-nasal disease, e.g. primary bone tumours, from mucosal disease. to discuss risk/benefit considerations in medical imaging from the perspective of patients and highlight some of the pre-requisites to ensure trust and confidence. this presentation will draw on the work of the european patients' forum on patient safety, quality of care, health literacy, and patient empowerment, to explore what are the key factors to consider in radiation risk analysis, from a patient's perspective, recognising that 'one size does not fit all'. it will examine what constitutes quality information in this environment, effective dialogue between the patient and the radiologist, and the interdisciplinary team, informed consent and transparency of data. it will also highlight some of the particular challenges regarding vulnerable patients, and those requiring on-going treatment due to chronic disease (s). the justification for medical procedures is based on the assumption that the benefit outweighs the risk. this presentation looks at the radiation risk factors from recent high-profile papers (pearce et al., ) and publications from international bodies such as beir and icrp. at lower radiation doses (< msv), quantifying, understanding and communicating radiation risk to staff, patients and referring clinicians presents a range of challenges. advances in health information systems will facilitate more precise dose risk relationships. there are a number of approaches to stochastic radiation risk assessment such as organ-/age-based assessment and effective dose equivalent assessment. at-risk groups, such as children and pregnant women, need special focus. eu directives demand special attention for high-dose interventional procedures and ct scans. these techniques, particularly if repeated, require further risk assessment regarding potential deterministic effects such as erythema, hair loss, and radiation-induced cataractogenesis. this paper looks at strategies in consenting, monitoring, and follow-up of such high-dose effects. the practical implications of the new occupational icrp eye dose limits for interventional practice are also examined. with the advent of higher field scanners in clinical practice and the construction of 'mri compatible' implanted devices, the list of the do's and don'ts while performing an mri examination changes constantly. in this presentation basic safety guidelines and rules will be explained regarding static magnetic field effects, time varying magnetic field effects, radiofrequency field effects and acoustic noise effects both with regard to the patient as well as the personnel using the equipment. due to the advances in medical technology the list of possible 'safe' and 'unsafe' items changes almost daily. therefore it is very important to have all the information about the patient's condition and implants prior to the mri procedure in order to asses possible contraindications in advance. while until a couple of years ago cardiac pacemakers and neurostimulators were contraindicated in the mri environment, the advent of 'mri compatible' pacemakers and other implanted devices introduces challenges in patient safety. in fact these devices are only safe in certain configurations and also in a lot of cases specific mri scan sequences and rf antennas are only allowed. following the european emf directive, where the mri part is derogated, the protection of staff working with em fields also became a topic of debate. what are the possible risks for staff working with mri magnets and how can one implement practical rules for the safe use of the mri equipment. the field of image-guided ablation has expended recently with new ablation techniques like micro-wave, irreversible electroporation, cryoablation. nevertheless, after each treatment, whatever the technique used, we will leave in place in the treated organ, a scar instead of the tumour. follow-up of these treated areas are done by radiologists using ct of mr or contrast enhanced ultrasound. standardisation of terms has been done in by an international committee presided by dr. goldberg. this allows reporting criteria identical from one center and from one country to another. furthermore, it allows a more homogeneous literature and evaluation of success. nevertheless, techniques are not all the same and the cellular and tissue damage in the tumour and around it in the healthy parenchyma are not identical and does not have the same evolution over time. thermal ablation is frequently used to treat liver, lung and bone tumoural lesions because of its safety, efficacy and tolerability. one issue is the lack of reliable imaging modality to assess treatment efficacy and to detect early relapse. several papers have demonstrated that fdg pet/ct is a useful tool to follow-up ablated liver tumour, detecting residual disease easily and earlier than conventional imaging. few reports evaluating the usefulness of fdg-pet/ct in the follow-up of ablated lung lesions are available, but prospective studies show promising results, with a high negative predictive value of this technique. very few data are available on bone lesions and further studies are needed to assess the role of fdg-pet/ct in the follow-up of these lesions. in general, the in vivo typical aspect of completely ablated lesion on fdg-pet/ct is a ring shape, diffuse, peripheral, homogeneous fdg uptake at the treated lesion; on the other hand, the presence of heterogeneous and focal uptake is more frequently related to disease relapse. suvmax (standardised uptake value) is not a reliable indicator. it is higher in case of disease relapse than in completely ablated lesions, but also in case of important inflammatory reaction. finally, the best time point to evaluate ablation efficacy still remains to be defined, but fdg-pet/ct should be performed at least months after treatment to avoid dubious or inconclusive findings due to inflammatory reaction. the aim of thermal ablation treatment is to generate an area of thermocoagulation whose diameter is larger or at least equivalent to that of the tumour. this necrotic scar usually shrinks with time, but most often very slowly. therefore, the criteria of response based on size measurement cannot be applied. the pattern of thermal ablation is similar whatever the thermo-ablation technique used. on ct imaging, thermal ablation areas are well circumscribed and oval shaped. the criterion commonly used to assess the efficacy is the absence of enhancement in the thermal ablation necrosis, which corresponds to tissue devoid of viable tumour. on mr imaging, the thermo-ablation areas are typically hyperintense on unenhanced t due to the presence of proteinaceous material, and hypointense on t , explained by the dehydrating effect of thermal damage. the residual tumour is typically round in shape and located at the periphery of the necrotic area or in contact with large vessels. mr imaging allows earlier detection of residual liver tumour than ct imaging. neurodegenerative diseases, cancer and heart disease perhaps summarise the major challenges to medicine in this century, a challenge in accurate diagnosis and also in treatment. recent technological advances in medical imaging technology mean that these challenges can be dealt with more efficiently. anatomy can be studied faster, safer and with higher spatial resolution and precision. in addition to anatomy, the study of function has become feasible; more sophisticated hardware and software are being used to get the required data to map out the functionality and understand how this relates to anatomy to offer a more comprehensive view of normal development and explain pathological conditions. at the same time, researchers and clinicians are urged to create a platform for reciprocal communication to facilitate translation of the research findings to a graspable clinical benefit for the patients. this translational platform will allow flagging the major clinical questions, inform current and future research experiments and at the same time help utilise recent research findings in the clinical setting. this session will review state-of-the-art mri techniques and document different approaches to translational imaging. it will also allow understanding the obstacles and remedies in implementing translational medicine. many neurological diseases are associated with progressively reduced cognitive function. recent evidence suggests that proficient cognitive function depends on an appropriate interaction between large-scale cognitive control networks in the brain. it is hypothesised that damage to white matter microstructure, as found in many neurological diseases, disrupts the integrated operation of these networks and results in impaired cognitive function. diffusion tensor imaging (dti) has been used to investigate alteration to white matter micro-structure and resting state fmri (rsfmri) has proven to be a useful technique to assess brain function in multiple cortical systems. however, the combined use of these techniques in the clinical setting has not yet taken hold. in this presentation, i will demonstrate the flexibility of these mri techniques in assessing brain structure and function and address their feasibility of use in a clinical setting. imaging in oncology is a growing field within radiology. different tomographic techniques are used either isolated or as multimodality-multivariable imaging, as there is an increasing need to combine morphological and functional information. different cutting-edge modalities, such as pet-ct, dw-mr, ce-mr and mr-spectroscopy, are standard in most clinical scenarios. new images have advantages providing excellent soft-tissue contrast and multidimensional functional, structural and morphological information. the development of new diagnostic imaging research areas, mainly in the field of oncology, cardiology and neuropsychiatry, will impact the way medicine is performed today. both clinical and experimental multimodality studies, in humans and animals, will have to demonstrate an efficient use of the imaging information provided by the modalities to affect the future of medical imaging. we will comment on the impact of continued technological developments in medical imaging on patients with cancer (diagnosis, staging and follow-up) and the challenges of imaging technologies from research into clinical reality. the recent advances and developments in measurements and biomarkers which have led to a greater understanding of cancer will be commented, together with the morphologic, metabolic and functional oncological imaging research and clinical practice. finally, the role of the radiographer in advanced oncological imaging techniques will be evaluated. when imaging the head and neck region with ct or mri, teeth are always present. we frequently encounter radiolucent or radiopaque lesions of the jaw on scans performed for other indications. familiarity with typical dental conditions is necessary to subject the patients to the right therapy. to correctly evaluate and describe those lesions, this special focus session on dental imaging gives the radiologist a tool to report dental lesions. the anatomy of panoramic and ct images will be reviewed in the first talk. technical details as well as pitfalls will be presented. in the second talk, the focus is on pre-and post-surgical imaging with ct where typical lesions of the jaw will be presented. an insight into imaging the teeth with mri will be given by the third speaker. the discussion on the impact of radiologists on dental images will complement this special focus session's theme. panoramic radiography produces a single image of the maxilla, mandible, teeth, temporomandibular joints and maxillary sinuses. during the exposure, the x-ray source and the detector rotate synchronously around the patient producing a curved surface tomography. this horseshoe-shaped zone of sharp image is called the focal trough/image layer. advantages of this technique are a good overview of lower facial hard tissues, the convenience of an extraoral examination, a low patient radiation dose, and low costs. disadvantages are limited width of the sharply imaged layer, variable magnification, and oblique projection, especially in the upper premolar region. patient positioning is most important for obtaining a detailed panoramic radiograph. in addition to patient position errors, patient movement during exposure and metal objects can cause artefacts. also, ghost images caused by structures located between the x-ray source and the rotation centre can hamper diagnostics. cbct is a radiographic imaging method that allows accurate d imaging of hard tissues. during a cbct scan, the scanner rotates around the patient's head ( °- °) obtaining multiple sequential planar projection images by a d detector. the scanning software collects the data which is processed to create a volumetric data set with isotopic voxels with varying fov. disadvantages of cbct imaging are poor soft tissue contrast and artefacts. in addition to normal panoramic and cbct anatomy, after this presentation the audience will be familiar with the panoramic and cbct techniques as well as common errors and pitfalls of these techniques. pre-surgical and post-surgical imaging with mdct and cone-beam ct a. gahleitner; vienna/at (andre.gahleitner@meduniwien.ac.at) multislice-ct and more recently cone-beam-ct have become an established method for anatomic imaging of the jaws prior to dental implant placement. commonly referred to as "dental-ct", these high-resolution imaging techniques have gained importance in diagnosing dental-associated diseases of the mandible and maxilla. especially in patients with inflammations, cysts, oro-antral fistulas, odontogenous sinusitis, impacted teeth and dental fractures, new indications have emerged. unfortunately, most radiologists have had little experience in this area and many of the dental-ct findings remain undescribed. hence, we will determine the ct appearance of frequent dentalrelated diseases of the jaws and demonstrate typical pre-and postoperative findings. this presentation reflects our experiences from years of performing dental-ct with . investigations, closely working together with dentists and oral surgeons. learning objectives: . to learn about the typical findings from pathological conditions of the jaw, which confront dentists and oral surgeons. . to understand these findings in cross-sectional imaging like multislice and cone-beam ct. . to learn about the usual treatments for these conditions in order to know how they are used in dental radiology. dental mri s. rohde; dortmund/ de (stefan.rohde@klinikumdo.de) this lecture will discuss the potential of dental mri in the field of inflammatory and neoplastic disease of the periodontal space. experimental and preliminary clinical data from high-field dental mri will be presented with a special focus on the visibility of critical anatomical structures on high-resolution sequences. the results will be compared to high-resolution cone-beam ct. based on representative cases, we will review the main causes of thoracic non-traumatic vascular emergencies (acute aortic syndrome, pulmonary thromboembolism and haemoptysis); mdct angiography has become the firstline imaging test for the diagnosis of these entities. as acute aortic disease is the most common fatal condition in patients with chest pain and prompt recognition and treatment are of paramount importance, we will review the spectrum of acute aortic pathology focusing on the distinctive findings of each entity (classic dissection, intramural haematoma and penetrating aortic ulcer) and upgrading the clues for their diagnosis. acute pulmonary embolism (pe) remains a common clinical challenge. mdct pulmonary angiography has become the first-line imaging study in the diagnosis of pe because of its speed, accuracy, low interobserver variability and ability to provide alternative diagnoses. we will review the role of mdct in the evaluation of acute thrombotic pe: findings of acute pe (including how to evaluate the severity of an episode of pe at ct pulmonary angiography) and some potential pitfalls. massive haemoptysis is a life-threatening condition that is associated with a high mortality rate. haemoptysis usually involves bleeding from the bronchial arteries or, less frequently, from nonbronchial systemic arteries. haemoptysis of pulmonary arterial origin is rare, estimated at less than % of haemoptysis cases. mdct angiography permits noninvasive, rapid, and accurate assessment of the cause and consequences of haemorrhage into the airways and helps guide subsequent management. acute respiratory failure can have multiple underlying causes including infection, fluid overload, immunological diseases or exacerbation of preexisting lung disease. since the clinical symptoms are nonspecific, imaging plays an important role. the first imaging method is mostly the chest radiograph, easy to access and to obtain, but non-diagnostic in many cases. (hr)ct offers more possibilities to define the differential diagnosis. the option of this interactive workshop will be to get familiar with the spectrum of diseases that can cause acute respiratory failure and learn about key findings in radiography as well as ct to reduce the differential diagnosis. the interaction between preexisting lung disease, clinical information (e.g. chemotherapy, rheumatoid arthritis, copd) and imaging findings will be discussed using clinical case studies. options and also limitations of imaging findings will be illustrated. the following scenarios will be taken into account: acute cardiac failure and various appearances of oedema; acute immunological-toxic disorders including druginduced lung disease and inhalational injuries; exacerbations of preexisting lung disease including fibrotic and obstructive lung disorders; severe infections causing respiratory failure and their complications. contrast-enhanced ultrasoud (ceus) and elastography are evolving us techniques that have already found their ways into clinical practice (ceus) or will do so in the next few years (elastography). ceus is performed with nd generation us contrast media, but these contrast media are not available worldwide. in many european countries, sonovue® (bracco, it) is the only available contrast, consisting of micro-bubbles less than the size of red blood cells. in contrast to ct and mr contrast media, these bubbles are strictly intravascular. for us imaging, low mechanical index us techniques are used to see the resonance of sound by these bubbles. ceus is excellent for the differential diagnosis of focal liver lesions based on vascularisation and specific contrast uptake (imaging the wash-in and wash-out of us contrast over time with excellent temporal resolution) and increases us sensitivity to liver metastases in colorectal cancer patients. however, ceus is only useful when there are excellent us conditions. in pancreatic us, ceus allows to differentiate adenocarcinoma from neuroendocrine tumours based on vascularisation (us contrast uptake). whereas elastography (strain elastography or shear wave elastography) is established for the diagnosis of liver fibrosis, there are no general recommendations for using these various techniques to measure the "hardness of a focal lesion" in focal liver and pancreas lesions. diffusion-weighted mr imaging (dw-mri) provides information on tissue cellularity, extracellular space tortuosity and integrity of cell membranes by measuring the motion of water molecules in tissues modified by flows (blood vessels, glandular ducts,.). and interaction with cellular components. integrated in conventional-mr acquisition protocol, dw-mri increases sensitivity for lesion detection. the apparent diffusion coefficient (adc) can be calculated using a mono-exponential relationship between signal attenuation and b-value. this fitting is influenced by microcapillary perfusion. a more sophisticated approach (intravoxel incoherent motion, ivim) would enable estimation of parameters that separately reflect tissue diffusivity and microcapillary perfusion. using ivim-analysis, the derived quantitative parameters describe tissue diffusivity, perfusion and perfusion fraction. by providing qualitative and quantitative information, dw-mri is applied in oncology to characterise malignancy, including lesion aggressiveness, and to monitor treatment response. perfusion imaging is a useful tool to achieve information concerning tissue vasculature, microvascular permeability and interstitial space characteristics. dynamic contrast-enhanced techniques (dce-ct, dce-mr) are based on the analysis of the contrast agent (tracer) biodistribution in tissues. quantitative parameters are obtained using pharmacokinetic models, as transfer constant ktrans, extracellular-extravascular space fraction ve, vascular tissue fraction vp and the rate constant kep. quantitative dw-mr and dce-mr parameters are increasingly used as imaging biomarkers to predict tumour response and/or to monitor the effects of therapy, especially when anticancer agents with novel models of action are used (anti-angiogenic, tyrosine kinase inhibitors and several others). therefore, these new imaging biomarkers may have a pivotal role in correctly evaluating tumour response and stratify and managing cancer patients. elderly and the young can all present special problems that require a greater depth of understanding to obtain a diagnostic study. this session will help you in your quest for reliable high-quality investigations for all. a. coronary cta in patients with severe arrhythmias and high heart rate c. loewe; vienna/ at (christian.loewe@meduniwien.ac.at) besides all advances in scanner technology, heart rate remains a critical issue for coronary cta, and motion artefacts due to cardiac function represents still the most frequent reason for limited diagnostic image quality in cardiac cta. thus, the target heart rate is defined depending on the scanner system used and ranged between below bpm and below bpm. however, in a number of patients heart rate control is not possible or not successful (i.e. children, emergencies, heart transplant recipients), whereas a relevant proportion of ctas are performed in patients with heart rates higher than the target frequency. during this presentation, the possibilities for heart rate control by beta blocker injection and beyond will be discussed and possible workflows will be presented. furthermore, examination strategies for patients with high heart rates will be presented including reverse dose modulation, high-pitch scanning, systolic scanning and more. finally, tools for reconstruction and assessment of patients scanned at higher heart rate will be introduced. different to high heart rates and even more critical with regard to image quality is arrhythmia. due to the complexity of cardiac synchronisation, a ct suite is not the place for cardiac resynchronisation, whereas the indication to cta has to be reevaluated depending on referring diagnosis and severity of arrhythmia. since there are patients undergoing cta because of arrhythmia including patients prior to ablation treatment, strategies for examination and image reconstruction in case of arrhythmic patients have to be established and will be presented. cardiac computed tomography (ct) has become a widely available diagnostic tool used in a range of heart conditions. the commonest application of this technique is in the evaluation for coronary artery patency (coronary ct angiography) in patients with chest pain. when coronary arteries are heavily calcified, or post-coronary angioplasty with stent implantation, diagnostic problems can occur. in these circumstances, the evaluation of the coronary arteries on ct is hampered by the occurrence of high-density artefacts caused by calcifications and stent struts. these artefacts may preclude the appropriate assessment of the coronary lumen. the presence of motion artefacts in the dataset or image noise in very large patients may exacerbate the problem. in this scenario, accurate patient selection and preparation remain key to ensure that the diagnostic yield of the cardiac ct study is good. optimisation of scan parameters (kv), contrast injection protocol and use of appropriate postprocessing techniques (e.g. dedicated convolution filters) play an important role in daily clinical practice. recent technical developments include dualenergy scan techniques and gemstone spectral detector systems that acquire simultaneously high and low kilovoltage datasets. this is done to achieve tissue differentiation. in principle, by using monochromatic image reconstruction, the effect of high-density artefacts may be decreased. using a similar principle, high-density structures can be subtracted from the image. the introduction of iterative reconstruction algorithms may play a role in that these algorithms are theoretically more accurate in the modeling of physical noise and tissue geometries. lack of movement artefacts is one of the major prerequisites for good image quality in cardiac mr. it is crucial to have the minimum possible image acquisition time. it is important to make both te and tr as short as possible. data acquisition should be synchronised with patient's ecg or pulse. special attention should be paid to good quality of ecg recordings (good contact of electrodes with the skin, electrodes positioning, using of dielectric pad). in patients with arrhythmia, prospective ecg synchronisation should be used instead or a retrospective one, or special protocols may be applied for arrhythmia correction. breathing artefacts are usually not a problem for cardiac mri, because most sequences are acquired during a single breath-hold. using ssfp and parallel imaging allows obtaining a complete set of cine mr images through the whole heart in - short breath-hold periods. the technique of realtime cardiac mr is a good way to perform successful examinations even in difficult patients, especially the ones with heart failure. other systemic mrartefacts (aliasing, chemical shift, magnetic susceptibility, off-resonance) should be recognised and diminished or eliminated by the operator. these artefacts are more prominent in case of t systems. late-enhancement studies with gd are very dependent on the correct selection of the ti time. phase-contrast mri requires venc calibration in cases of valve stenoses. to meet the challenges and the benefits of cardiac mri, one must balance the constraints of signal-and contrast-to-noise ratios, spatial and temporal resolution, scan time and image quality. scoliosis, a common spinal deformity in teenagers, especially girls, progresses during their growth until the end of puberty. therapeutic choices, which are either medical -with corset -or surgical, depend on this progression. follow-up is mainly clinical. however, radiographs are often indicated to give precise information: ap and lateral views of the spine in standing position remain the basis of this follow-up. obtaining a good image quality at all levels of the spine has been a technical challenge for a long time, and the question of radiation dose is also a main concern, as these radiographs require a high radiation dose. fortunately, radiographic techniques have greatly improved over the past few years: from the conventional or cm film to digital screens and now flat screens, we now obtain better quality images with less radiation. today, eos system using xenon chamber is the best to give good ap and lateral views. it allows d reconstructions with minimal radiation dose. on these radiographs, measures can be performed, essentially cobb's angle. it helps in evaluating progression of the curves and making surgical decisions. other imaging techniques are requested only in particular situations as in the presence of neurological symptoms or general disease such as neurofibromatosis, or in young children with malformative scoliosis and sometimes preoperatively. ct is best to study the vertebrae; it objectivates vertebral malformations (agenesis, hypoplasia, blocks). mr will be requested to study the cord, nervous roots, craniospinal junction and the soft tissues. collaboration between the radiologist and spinal surgeon is essential. the key elements in the diagnostic process, as for most bone lesions, are: the age of the patient, the type of bone and bone segment involved, the location within the bone, the presence of signs of aggressiveness (type of osteolysis, limits, presence and type of periosteal reaction). conventional x-rays should never be absent from the initial workup of a focal bone lesion in a child, as they provide essential features for differential diagnosis and, in most cases, eliminate the need for other more costly techniques. the need and priority for other imaging techniques, such as ct, mri and bone scintigraphy, are decided on a case-by-case basis and sometimes a combination of these techniques, which complement each other, is needed. it is the radiologist's responsibility to recommend histological analysis of a lesion via biopsy whenever the imaging aspect is not characteristic or indicates signs of possible malignancy. learning objectives: . to recognise the most common benign bone tumours and pseudo-tumours. . to understand the differences between benign bone tumours, pseudotumours and malignancies in children. . to understand imaging modalities that could help in the differential diagnosis of benign bone tumours and pseudo-tumours in children. a- : a.c. offiah; sheffield/uk (amaka.offiah@nhs.net) the nosology and classification of genetic skeletal disorders subdivides conditions into groups defined by molecular, biochemical and/or radiographic criteria. conditions are associated with mutations in at least one of different genes. some of these conditions are rare or even extremely rare (occurring in fewer than in , of the population). clearly, the general paediatric radiologist cannot be expected to correctly recognise and diagnose all of these conditions. the approach is to perform a set of radiographic images dependent on the patient's age/size and to develop a standard system for reviewing these images. the standard set of images for a patient over to years of age consists of ap and lateral skull, ap chest, ap pelvis, lateral thoracolumbar spine, ap one upper limb, ap one lower limb and dp left hand. variations on this routine dysplasia skeletal survey will be discussed and a standard system for interpreting the images will be presented using radiographs of the commoner skeletal dysplasias as examples. in the last few years, interest in body composition (bc) analysis, which is the quantification and characterisation of relative amounts of muscle, fat, bone, and other vital parts composing the human body, has grown rapidly for clinical, research and epidemiological purposes. studying body composition has gained great importance for the comprehension and decoding of a multitude of patho-physiological processes (e.g. obesity, diabetes and endocrine diseases and also gastrointestinal, renal, nervous, infectious diseases, etc). and physiological and para-physiological conditions as in athletes or growth and ageing processes. although the main imaging techniques, which include dualenergy x-ray absorptiometry (dexa), computed tomography (ct), and magnetic resonance imaging (mri), offer a differentiated and attractive analysis of bc, they still need to find a definite position in clinical practice. through 'programming' low birth weight is associated with increased rates of coronary heart disease, stroke, hypertension & non-insulin dependent diabetes. prediction of morbidity and mortality from body composition, particularly fat, stimulates the importance and application of accurate and precise methods for quantitative assessment of body composition; the gold standard for which is cadaver analysis, but other methods have to be implemented in vivo. body composition alters in many chronic diseases, starvation cases, metabolic syndromes, and senescence stages, and is a key component of health. several techniques are available which vary in simplicity and complexity of use. each make assumptions and may not measure body composition directly, but make predictions from other measurements made. skinfold thickness measurements assess regional fat and are quick and simple to perform at all ages. reproducibility is good, but less so in obesity. there is some limitation of reference data to express results as standard deviation scores (sds). body mass index (bmi; weight/height ) is widely used as an index of relative weight expressed as sds for gender, age and ethnicity. waist circumference (wc) is a measure of central fat, and can also be expressed as a ratio to hip circumference. bioelectric impedance analysis (bia) measures body impedance to a small electric current, which estimates total body water (tbw) from which can be derived fat free mass (ffm). tbw can also be measured by neutron activation. air displacement plethysmography (bod pod) is a new method for body composition assessment. learning objectives: . to understand normal body composition in children and adults. . to learn how body composition can be altered by disease. . to appreciate the clinical relevance of assessing body composition. . to understand the non-imaging methods available for measuring body composition, with their advantages and limitations. computed tomography: what does it measure and how? j. damilakis; iraklion/ gr (damilaki@med.uoc.gr) ct allows measurement of total body fat and enables differentiation of subcutaneous from visceral adipose tissue. body fat can be estimated using the conventional technique of manual planimetry. using planimetry, the user delineates manually the boundaries of subcutaneous and visceral fat in each ct image. however, this method is labour intensive, time-consuming, and, therefore, not easily applicable in routine clinical practice. another ct method used for the estimation of abdominal fat is the semiautomatic measurement of adipose tissue area. abdominal fat is assessed in a ct slice by using a fixed range of hounsfield units to define the area of adipose tissue. a limitation of this method is that the attenuation range of fat may vary among individuals. the stereological volume estimation method is based on the cavalieri's principle. according to this principle, the volume of an object can be measured by cutting it into equally spaced slices and measuring the area of the object on each slice. to measure body fat using stereology, a square grid of test points is randomly superimposed on each ct section. all points lying inside the fat tissue region are selected by the user and the software automatically calculates the total number of points hitting the fat. stereology provides the possibility for efficient fat volume assessment. however, research studies are needed to optimise the stereological estimation of fat and compare stereological measurements with those from other adipose tissue measurement ct techniques. learning objectives: . to appreciate the role of ct imaging in body composition analysis. . to learn how to measure visceral and subcutaneous adipose tissue using ct. . to understand the advantages and limitations of ct techniques (planimetry, thresholding, stereology) in evaluating adipose tissue. mri: current and future applications d.c. karampinos; munich/ de (dimitrios.karampinos@tum.de) mri has been emerging as an ionising radiation-free imaging modality to measure fat distribution in the human body and fat content in different organs. the present talk will first introduce mri methods to measure fat distribution, including the well-established t -weighted imaging and the emerging dixon imaging approaches. the challenges related to the data acquisition and image post-processing of the two imaging approaches in the study of fat distribution will be reviewed. examples will be shown from the current use of mri in investigating adipose tissue distribution alterations or differences in patients with metabolic disorders, including obesity and diabetes. quantitative mri methods enabling the measurement of fat content with high spatial resolution will be then presented, with a focus on water-fat separation methods. the technical challenges associated with the establishment of mri-based fat content, as an accurate and reproducible imaging biomarker will be discussed. the selection of pulse sequence parameters and image reconstruction algorithm in a state-of-the-art water-fat separation experiment will be explained. applications will be shown from the growing literature of mri-based fat quantification in abdominal organs (e.g. liver) and in skeletal muscles, aiming to show the great premise of quantitative water-fat mri in quantifying fat content with high spatial resolution in different body parts. at the end of the talk, the potential of quantitative water-fat mri will be discussed in the emerging application of measuring bone marrow fat content and its association with bone health and metabolic disorders. the key role of dual-energy x-ray absorptiometry (dxa) in the management of metabolic bone diseases is well known. the role of dxa in the study of body composition and in the clinical evaluation of disorders which directly or indirectly involve the whole metabolism as they may induce changes in body mass and fat percentage is less known or less understood. dxa has a range of clinical applications in this field, from assessing associations between adipose or lean mass and the risk of disease to understanding and measuring the effects of pathophysiological processes or therapeutic interventions, in both adult and paediatric human populations as well as in pre-clinical settings. dxa analyses body composition at the molecular level that is basically translated into a clinical model made up of fat mass, non-bone lean mass, and bone mineral content. dxa allows total and regional assessment of the three abovementioned compartments, usually by a whole body scan. since body composition is a hot topic today, manufacturers have steered the development of dxa technology and methodology towards this. new dxa machines have been designed to accommodate heavier and larger patients and to scan wider areas. new strategies, such as half-body assessment, permit accurate body scan and analysis of individuals exceeding scan field limits. although dxa is a projective imaging technique, new solutions have recently allowed the differential estimate of subcutaneous and intra-abdominal visceral fat. the transition to narrow fan-beam densitometers has led to faster scan times and better resolution; however, inter-or intra-device variation exists depending on several factors. in cancer patients, disease-free survival is a good indicator for tumour response, but for many common cancers, treatment of disseminated disease is often noncurative. the increased duration of survival is related to changes in tumour size after treatment. however, the anatomical determination of tumour response has some limitations, especially when non-cytotoxic targeted therapies are used. with these new treatments options, the lack of progression may be associated with a good improvement in outcome, even in the absence of major shrinkage of tumours. new imaging biomarkers are therefore needed to assess therapeutic response. molecular imaging is now playing a prominent role in the monitoring of cytostatic targeted therapies. pet-ct, dynamic contrast enhancement studies or diffusion-weighted imaging are the most promising ones. the aim of this session is to present the state of the art in tumour response assessment with regard to new therapeutic regimens. in early oncologic times, treatments of different malignancies were reported in different ways. c. gordon zubrod and others first articulated the model of multicentre clinical trials and argued for standards to be agreed for included and excluded subjects, the method of assigning treatment and in measuring response. nowadays, we have better knowledge of tumour biology; however, we continue using the classical response criteria (who and recist) and overall survival as a primary goal for effective treatment in most of the malignancies. in the last few years, different criteria for responses have been reported to evaluate the disease and a lot of discussions between authors have been reported. now, we can use the recist criteria v . , implement the percist criteria and also forget the immunotherapy criteria. furthermore in the last few years, different treatments such as vaccines, antiangiogenics and targeted therapies have increased our arsenal in the treatment of different malignancies such as kidney cancer, prostate cancer, breast cancer and so on. another problem for our patients is that those diseases have a long evolution after the first-line treatment. we have to be able to define in each disease the treatment line and type of treatment to determine which evaluation criteria are necessary and which is the objective of treatment, pfs or os. we have to be aware that if we are not able to define such topics, we may be losing active treatments. recist . are the criteria most often used in studies to evaluate response to treatment in solid tumors. on each follow-up examination, response is defined by a combination of unidimensional measurement of 'target lesions', qualitative evaluation of 'non-target' lesions, and presence/absence of 'new lesions'. ct and mri are the preferred modalities for recist evaluation, and evaluations must be performed using the same modality, in the same plane. we will review anatomic locations which should be avoided, and how to deal with intercurrent events resulting in the impossibility of measurement. cases when recist seems inadequate will be discussed, including evaluation of bone metastases, and focal therapy. finally, though these criteria were developed for drug trials, they may provide a frame for reading examinations and writing reports in routine practice. reproducible, objective and quantitative criteria help to define or in the obese patient. ct is especially helpful in those with suspected retroperitoneal pathology, in the investigation of immunocompromised patients and those with complex post-operative problems. ct guidance may be required to facilitate percutaneous abscess drainage, particularly for deep pelvic collections. in addition, ct plays a vital role as a 'problem-solver' when ultrasound has failed to fully answer the clinical question posed. this short presentation will utilise clinical studies to illustrate the benefits of ct in the investigation of the acute abdomen, both as the initial imaging modality and as a second line tool following ultrasound. learning objectives: . to understand the advantages of ct as a primary imaging modality for children with acute abdomen. . to learn about clinical scenarios in which ct is relevant. . to appreciate the use of ct as an adjunct to ultrasound. abdominal trauma: us is better v. miele; rome/it (vmiele@sirm.org) ultrasonography (us) is a reliable technique whose advantages are rapidity, portability and accuracy in depicting intraperitoneal fluid without interrupting resuscitation and without radiation exposure. in the haemodynamically unstable paediatric patients, it represents the first line together with the x-ray evaluation. us for trauma has become more standardised and is worldwide known with the acronym of fast (focused abdominal sonography for trauma) or e-fast (extended-fast) used in depicting also pleural and pericardial effusions and pneumothorax. nevertheless, many parenchymal injuries are not correctly visualised at baseline us and some traumatic solid organ lesions can occur without hemoperitoneum. in case of haemodynamically stable patients, who have suffered a low-energy trauma, the greater time available allows the use of specific us contrast agents, enabling a better identification of traumatic organ injuries. contrast-enhanced ultrasonography (ceus) has a greater sensibility and specificity in the identification of parenchymal traumatic lesions, both in the first evaluation and follow-up, and could avoid unnecessary radiation and iodinated contrast medium exposure. in case of haemodynamically stable paediatric patients, who have suffered a high-energy trauma, us is not recommended as first-line investigation, because ce-mdct should be performed first. only in the follow-up, ceus can be considered an alternative to ct. in conclusion, e-fast and ceus should be considered as a useful tool in the assessment and monitoring of paediatric trauma. this examination can be performed at the patient's bedside, representing a useful alternative to ct in the paediatric traumatised patients and in the follow-up of a known abdominal injury. learning objectives: . to understand the differences in the diagnostic paths of paediatric patients. . to learn about the diagnostic efficacy and limitations of ultrasonography. . to become familiar with the use of the contrast-enhanced ultrasonography (ceus). abdominal trauma: ct is better m. raissaki; iraklion/gr (mraissaki@yahoo.gr) ct has been considered a sensitive, specific, and accurate test in the identification and grading of injuries, especially in the severely injured child. it should be overemphasised that traumatised children differ from adults: haemodynamically stable children may actually be actively bleeding and rapidly deteriorate. conversely, children have smaller calibre vessels, stronger vasoconstriction, and stronger solid organ capsules; bleeding may stop spontaneously, organ rupture is more difficult and delayed rupture is rare. this is why few children will undergo laparotomy or trans-arterial embolisation. the goal of imaging is to clear the abdomen in multi-traumatised children, identify those that may rapidly deteriorate because of clinically silent active bleeding, increase the surgeon's confidence level and define short-and long-term medical management. due to children's increased radiosensitivity, all ct scans should be indicated based on appropriate early clinical evaluation, assessment of risk factors for abdominal injuries and evaluation of the closely monitored child. alara includes the availability of outside scans upon admission, avoiding non-contrast scans and multiple phases and applying age-and weight-dependent exposure parameters. ct should not be performed as follow-up unless there is clinical deterioration. ct has the advantage of rapidly identifying and delineating with high resolution solid organ, vascular, mesenteric, bowel injuries and pending oligaemic collapse (hypoperfusion complex or shock bowel). important fractures and thoracic injuries may be simultaneously revealed with ct. the objective documentation of intraabdominal injuries is extremely important in suspected child abuse. evaluation of mr imaging in a patient with an intramedullary lesion should focus on key features: a) the location of the lesion on the cross-sectional area of the cord, best evaluated on axial images, b) the length of the lesion evaluated on sagittal images, c) the presence of cavitation and cysts, d) signal intensity on t -wi, e) the presence of enhancement and enhancement type, and f) associated leptomeningeal enhancement. the knowledge of the presence or absence of the brain lesions is important information for narrowing the differential diagnosis. the clinical picture and the onset of symptoms will help the differentiation between the neoplastic and inflammatory and vascular lesions. in this lecture, the imaging characteristics and typical patterns of intramedullary lesions will be presented. a diagnostic algorithm, which includes imaging, clinics and csf analysis, will be discussed. a common clinico-radiological situation is the differentiation between spinal infection, degenerative changes and osteopaenia, and spinal tumour. in most cases, conventional ct and/or mri will allow a certain differentiation, and additional biopsies are mainly used to obtain tissue for bacteriological or histopathological classification. advanced mr techniques such as dwi may sometimes help in increasing diagnostic certainty, but are seldomly used in day-to-day practice. spinal infections typically present around the intervertebral disc, but may also present as isoloated spondylitis without disc involvement, and sometimes the infection quickly spreads to the epidural space. degenerative changes my sometimes mimic infection due to strong gadolinium enhancement. the main differential diagnosis of spinal tumorous processes is osteopaenic changes (oedema), and ct may be helpful in such cases, for example to demonstrate degenerative gas in the vertebral body. rare causes of spinal abnormalities such as atypical pathogens and rare tumours may cause diagnostic problems; often the fastest road to a diagnosis is biopsy in those cases. the correct clinical diagnostic approach to spine disease is mandatory in the selection of patients to be treated. the anatomy and the relationship between different structures must be known in detail to understand the source of the pain and so to treat it percutaneously. mr, ct, dynamic x-ray and nm bone scan in selected cases can be used to understand the reason for the pain. mr with t stir sequence or t fat supp technique is mandatory to show bone marrow oedema and to decide on which metamer to perform the treatment. ct is often necessary after mr in primary and secondary spine tumours. disk disease can be seen either on ct or mr. two major treatments must be considered: discogenic pain and vertebrogenic pain, and for this reason disk treatment and vertebral treatment. disk treatment includes many mini-invasive systems to use; however, no treatment has been shown to be superior to the others, with good clinical results in - % of the cases, even at long-term follow-up. vp and assisted techniques are available for the treatment of not within a few minutes following ablation, ct of the ablation zone will demonstrate an extensive area of ground glass opacity (ggo). a ggo of at least . mm peripheral to the tumour is predictive of complete ablation. as early as hours post-treatment, the entire ablated region usually appeared as a well-demarcated homogeneous dense opacity on ct that corresponded to necrotic tissue and its surrounding rim of granulation tissue. this zone of ablation is the "baseline post-ablation imaging" for follow-up. then, a relatively slow involution of the ablation zone will occur with various patterns, including nodular, fibrosis, disappearance, cavitation and atelectasis. morphologic features of local tumor progression are an increase in the overall size or a change in the shape of the ablation zone (even without enhancement). it is generally considered that an ablation volume that does not increase in size on subsequent imaging after the baseline post-ablation imaging is a complete ablation. the relatively slow decrease in size of the ablation zone renders ct morphologic evaluation responsible for late discovery of local tumour progression. pet/ct is able to discover incomplete ablation earlier than ct at a stage the disease remains small. patients who have very early evaluation with pet/ct are at risk of either false-positive result due to early inflammation process, or false-negative result due to early inflammation masking active tumor foci. or months after ablation can be a reasonable time for pet/ct. learning objectives: . to consolidate knowledge on the imaging aspects of successful ablation. . to become familiar with the main pitfalls of post-ablation imaging. . to learn about the imaging aspects of most common complications. a- : c. follow-up imaging of thermal ablative therapies for kidney tumours d.j. breen; southampton/uk (david.breen@uhs.nhs.uk) image-guided ablation of kidney tumours has been increasingly set to become the standard of care treatment for smaller (< cm) renal tumours, yet ablation remains a non-extirpative technique and is therefore paramount, in this curative setting, that imaging follow-up should reliably confirm complete tumour eradication. early on in radiofrequency ablation (rfa), control remained problematic and inadequate treatment was evidenced by residual marginal crescents of viable, enhancing disease. in the current era of more definitive ablation with multipolar techniques, cryo-and microwave ablation (mwa), this pattern of treatment failure should rarely, if ever, be encountered. typically following adequate ablation imaging should confirm a completely nonenhancing tumour and a related cortical ablation zone. whilst nonenhancement is an accepted surrogate of tumour non-viability, most practitioners look for additional collateral features such as the 'halo' sign, appearing as a soft tissue ring in the adjacent peri-renal fat, usually a sound marker of complete tumour eradication. follow-up data have shown that cryoablation (cra) can yield robust outcomes, but often incurs notable haemorrhagic change and a 'rind-like' feature around the ablation zone. cra also appears to induce faster involution of the treated tumour, whereas rfa and mwa can induce a persistent granulomatous mass which only very slowly involutes over a number of years. late local recurrence is increasingly rare at around - %, but can occur as nodules of enhancing disease as late as - years after the initial treatment. subtraction mr can be useful, but to date pet and perfusion techniques still lack resolution and specificity. multiple percutaneous image-guided therapies are currently available for thermal ablation of bone tumours. thermal sources for these treatment modalities include high-intensity ultrasound, laser, microwave, radiofrequency, and cryotherapy. the predictability of thermal ablation is adequate to limit collateral damage and complications, however, is limited by biologic and anatomic variability of tissue. clinical evaluation is essential in symptomatic tumours. close imaging follow-up with ct, mri, bone scan, and pet plays a vital role in the management of the post-thermal ablation patient and detection of complications. recurrences or regrowth can be considered for repeated thermal ablation if the lesion is discovered early, before the tumour geometry, location, or distribution become unfavourable. the imaging features could vary with the different ablation method. the radiologist reporting the follow-up imaging should be familiar with different ablation methods. mri with dynamic contrast enhanced imaging and subtraction allows to detect recurrences in hypervascular tumour. contrast enhancement on t -weighted mri imaging seems to be predictive of clinically unsuccessful ablation. on ct scan, bone reconstruction can be visualised but is a slow process, particularly in adults. pet scan is an excellent morphologic and metabolic image to follow-up the tumours. however, the inflammation produce after ablation can be misleading, particularly during the first three months. imaging follow-up plays an essential role in the management of the post-thermal ablation patient and detection of complications. interventional radiology (ir) is an evolution of radiology that treats many diseases, originally treated by traditional surgery. the clinical importance of ir has been demonstrated over the years by performing procedures that offer the benefit of therapeutic treatments, competitive from the point of view of a shorter hospitalisation time. indeed, thanks to ir, patients are treated in a less invasive manner, hospitalisation is limited and thus promotes the containment of social costs. to maintain standards clinical oversight needs to focus upon correct organisational, functional and technological appropriateness of practice. to achieve correct ir performance, it is necessary to guarantee that all the instrumental guides (fluoroscopy, ultrasound, computed tomography equipment) function optimally, the procedural equipment including guides/catheters/stents, etc. are chosen according to their technical features and their characteristics are appropriate for use, incorporating cost considerations. as the complexity and variety of interventional procedures grow, there is an increasing need to ensure the appropriate training of specialised ir staff. consideration of dedicated education and continued professional development options for radiographers working in ir is of priority. the italian association of interventional radiographers (aitri) is very important in sharing guidelines in a multidisciplinary environment to standardise and harmonise the knowledge and skills of the team in hybrid theatre and interventional suites. interventional radiology technology is rapidly expanding and, to maintain safe and efficient practice, careful consideration of current and potential future organisational needs and the training of staff within an ir suite require detailed consideration. the lecture will discuss where new and current knowledge about radiation risk and effects are coming from. a brief overview of the different sources of knowledge, including hiroshima and nagasaki life span study, will be given. the most important stakeholders involved in research and formulating guidelines for radiation protection, with special emphasis on the new main publication from the international committee on radiological protection (icrp), and the impact on radiographic practice will be discussed. new tissue weighting factors for breast glandule gives a significant higher risk for females today, especially in the pubertal age. on the other hand, they have the risk for hereditary effects significantly decreased. estimation of risk, both in general and in the individual, will be discussed. regarding staff protection, there is a new recommendation for annual dose limit for the eye lens. the previous annual dose limit was msv, and this is now recommended to be decreased to msv averaged over five years with no single year exceeding msv. the new recommendation can be a limiting factor in interventional radiology and cardiology, if sufficient protection is not used. learning objectives: . to understand the importance of radiation protection in interventional radiology for patients and staff. . to appreciate the latest recommendations for staff radiation protection in interventional radiology. . to appreciate the important role of the interventional radiographer in radiation protection. a- : the improvement of patient care and aims for the highest possible levels of service are to the forefront of the modern "interventional suite". role development experiences will be explored, considering need, effect and management of development. service demands, the need to extend those provided, the limited number of radiologists, financial constraints and the requirement for continuous professional development at state level provide much stimulus for professional radiographic role development. increased job satisfaction, reduced waiting lists and improved patient experience all provide largely positive results it appears. resistance from radiologists, multidisciplinary team members and radiographers themselves however, can provide unnecessary barriers to change. once abridged the blurring of professional lines and insecurity within multidisciplinary team members may also occur. various experiences would suggest that only by allowing ownership of change by all the clinical stakeholders involved, thus empowering them, leads to successful development. on examination clear guidelines and distinct protocols must be established, taking account of current workload, to create the ideal scenario for good multidisciplinary team ethos, where members have overlapping yet clearly defined roles. professional accountability, responsibility and an understanding of those role's are all key, but without correct training, including support and resources, no regime for role development can be achieved successfully. clinical audit not only ensures the delivery of high quality and effective care in line with best practice, whilst ensuring cost effectiveness, but also provides the confidence for all involved, especially the interventional patient. learning objectives: . to understand the factors influencing change in professional roles within interventional radiology. . to learn about the potential impact of enhanced professional roles and a multidisciplinary team approach on service delivery and patient outcomes. . to understand the need for clear protocols and guidelines along with appropriate training and audit of practice when implementing such changes. there have been many attempts to develop gene reporters for mri, however these give relatively modest image contrast that can be difficult to detect. i will describe in this talk a reporter that gives intense and positive contrast in mr images (up to ~ x increase in signal), which can also be used with radionuclide imaging, thus combining the sensitivity of radionuclide imaging with the spatial resolution of mri. the contrast obtained is directly related to the degree of gene expression and is readily reversible, thus allowing longitudinal studies of changes in expression. the transplantation of pancreatic islet into the liver is an excellent example of successful cell therapy. transplanted islet visualisation in vivo using a noninvasive imaging method, for example mri, is necessary to prove technical success. monitoring of transplanted islets in vivo and long-term tracking their fate using mri requires their labelling by a suitable contrast agent in vitro prior to transplantation. under encite project we successfully performed animal experiments, which proved therapy potential of labelled pancreatic islets. labelled islets implanted into the rat liver were viable and induced long-term normoglycemia in diabetic rats. mri proved their viability and even distribution in the host tissue. these results allowed performing a clinical study on a group of patients, which were transplanted by iron labelled pancreatic islets from cadaver donors. the successful pilot experiment enabled introducing of transplantation of native islets into routine clinical practice. search for better alternative sites, improved immunosuppression and alternative insulinproducing cells require multimodal and multifunctional molecular probes. currently we test polymer meshes as artificial transplant sites, effect of mesenchymal stem cells as a supportive net for beta cells, and novel cellular probes combing h, f labels and fluorescent ones. optical imaging in the clinic j. dijkstra; leiden/nl (j.dijkstra@lumc.nl) recently a lot of developments have been done in the field of optical imaging. new sensitive devices made it possible to use both visible and near infrared light as diagnostic tool and guidance tool. the advantage of near infrared is that the penetration depth in tissue is much better than for visible light, allowing to see deeper. by using techniques like augmented reality, the data from infrared, both anatomical and functional, can be presented to the operator in real-time. optical imaging is used e.g. as an evaluation tools for mammography where the optical spectral properties of the tumor cells is used to monitor the effectiveness of chemo treatment. the spectral properties of the breast are measured using a laser at different wavelengths and a camera to create tomographically a d volume. dedicated near infrared probes which bind to certain tissue types make it possible to look for e.g. remaining tumor tissue in resection margins during surgery. this techniques also allow to visualise structures which should be avoided during procedures like nerves. by adding multispectral imaging, the endogenous contrast can be used. different tissues have their own absorption spectrum which can be shown as additional information, for instance the ration hb/hbo . optical imaging also allows for acquiring images at near microscopic resolution real time in-vivo by using optical coherence tomography. this modality is being tested to provide information about the presence of certain cell types very fast where otherwise histology is needed. mesenchymal stem cells constructs for image-guided cell therapy in myocardial ischemia and digestive fistulas o. clément, e. blondiaux, g. rahmi, l. pidial, a. silva, f. gazeau, c. wilhelm, g. autret; paris/fr (olivier.clement@inserm.fr) regenerative medicine has recently emerged as a potential therapeutic tool. a number of preclinical and clinical trials have been conducted in many diseases ranging from diabetes to myocardial infarction and neuro-regenerative diseases that assess the feasibility and benefits of injecting stem cells. however, outcomes relating to graft survival remain generally unsatisfactory, whether the process of injection is direct, intravenous or catheter-guided. to overcome such issues, tissue engineering has potential to improve cell engraftment and therapeutic response including functional parameters. this work aimed to evaluate mesenchymal stem cell constructs for image-guided cell therapy in myocardial ischemia and digestive fistulas. we tried to options: . fibrin patches based on fibrinogen monomers polymerised with thrombin and seeded with cells. . constructs based on d multilayers of confluent cells sheets. tissue constructs were labelled with iron oxide particles and evaluated in a model a myocardial infarction for the fibrin patches or digestive fistula for the d constructs. mri at . was performed at various time points after treatments using a high resolution coil. fibrin patches could induce a therapeutic effect by increasing the left ventricular ejection fraction compared to sham. d constructs induced an increased number of fistula healing and enhanced micro-vasculature density compared to controls. mri of labelled stem cell constructs allowed a good evaluation of the models and showed increased therapeutic efficacy. the proposed paracrine mechanisms will be discussed. is cell imaging relevant for the clinic? lessons to be learned from preclinical research u. himmelreich; leuven/be (uwe.himmelreich@med.kuleuven.be) non-invasive imaging of therapeutic cells has become a popular field of research over the last decade. this interest was mainly based on the hope that the location, migration but also function of immune, stem and other cells can be visualised over time in individuals. the development of novel contrast agents and mechanisms for mri but also other imaging methods has resulted in exciting basic research findings. in particular, the application of relatively biotolerant iron oxide based nanoparticles has fostered the hope for direct translation into clinical research and general practice. however, pre-clinical research has also highlighted several limitations of nanoparticle based cell imaging by using mri including the generation of unspecific contrast, difficulties to quantitatively image engrafted cells, unambiguous contrast, adverse effects on cell biology, limitations for longitudinal follow-up or the lack of functional information. such shortcomings are traditionally overcome in preclinical research by combining mri with other imaging modalities like bioluminescence imaging or positron emission tomography. our research focuses on the optimisation of cell labeling strategies for robust, sensitive and potentially quantitative visualisation of stem and progenitor cells in therapy models in vivo to assess cell behavior after engraftment. the sensitivity, stability, toxicity and adverse effects on the cell biology by the labeling procedure were studied for iron oxide based particles. the potential of gdchelates and f labeled compounds for cell labeling has been assessed in vitro and in vivo. based on our preclinical research finding the potential of future applications in patients will be explored. cardiac ct is becoming the imaging modality of choice for an increasing number of clinical indications, not only to rule out coronary artery disease but also to evaluate cardiac morphology and function, and to determine patient outcome after coronary artery revascularisation. however, as with any other imaging tools, appropriate interpretation of cardiac ct examinations is required to assess the clinical value of this newly established diagnostic imaging modality. this process requires performance of thorough cardiac ct acquisition protocols, detailed knowledge of standard cardiac anatomic and physiologic terminology, as well as appropriate postprocessing, reading and reporting. in particular, radiologists need to recognise and be aware of the imaging findings that may confound and lead to interpretation errors. this lecture will summarise the practical aspects of postprocessing, reading and reporting of non-invasive cardiac ct examinations. the value and limitations of every available ct postprocessing technique including two-dimensional multiplanar reformations, curved multiplanar reformats, maximum intensity projection (mip) and volume rendered images will be explained. moreover, hints for improving reading results by recognising technical causes for various artefacts in cardiac ct will be elucidated and reading approaches to diminish false positives, false negatives and inaccuracies when assessing coronary artery stenosis will be suggested. cardiac magnetic resonance (cmr) is a complex imaging technique due to the intrinsic anatomical and technical peculiarities of the exam. these include the non-orthogonal cardiac orientation within the chest cavity requiring dedicated acquisition planes and the complex respiratory and cardiac motion to which the heart is subject and requiring a combination of ecg-gated and breath-hold sequences. potential additional anatomical pitfalls also include normal structures and variants like the moderator band, papillary muscles, and the presence of prominent crista terminalis or myocardial trabeculations, whose recognition is mandatory and may mimic in some cases a pathological condition. technical issues of cmr concern the continuous intracavitary inflow of protons and the associated "slow-flow" artifacts (limiting visualisation of endomyocardial border in some cases) pitfalls related to ecg gating, like inadequate synchronisation or the t-wave swell phenomenon; and finally a series of specific artifacts intrinsically related to the use of different pulse sequences that may interfere with image quality. an additional, more complex issue to consider is also the widespread diffusion of high-field magnets which have further enhanced those aspects. knowledge of the spectrum of those cmr peculiarities is mandatory to approaching and providing a correct diagnosis according to the main clinical request. the present lecture will review the most important anatomical and technical pitfalls of cmr examination and offer, when possible, practical solutions to overcome those limitations. shoulder imaging and intervention are becoming increasingly important in clinical practice. this session considers the indications, techniques, imaging findings and relative merits of diagnostic ultrasound and mri of the shoulder. the indications, techniques and results of us-guided interventional procedures are also considered. a panel discussion will deal with controversies in shoulder imaging such as the accuracy of us and mri in assessing rotator cuff tears, tendinosis, impingement and muscle atrophy, and the efficacy of us-guided interventions. audience participation in the discussion will be welcome. the shoulder is an anatomic area that is very commonly evaluated with musculoskeletal ultrasound. ultrasonography is widely recognised as a reliable means of assessing rotator cuff disease with accuracies reaching % for fullthickness tears and - % for partial-thickness tears. diagnostic accuracy depends mostly on the skills and experience of the examiner. a comprehensive ultrasound (us) examination requires, first of all, sound knowledge of the anatomy. a specific scanning protocol must be adopted in every us examination in patients with shoulder disease, because focal symptoms do not correlate with the location of the disease. the greatest importance of ultrasonography in rotator cuff assessment lies in its dynamic character. several dynamic manoeuvres can reveal pathologies such as subacromial impingement. last, but not least there are several scanning pitfalls such as anisotropy. anatomy key structures as well as the us technique and scanning protocol will be presented in this lecture. dynamic manoeuvres will be described and demonstrated with videos. scanning pitfalls will be emphasised. advantages and disadvantages of the us examination compared to other imaging modalities will be discussed. guidelines concerning the mr examination technique of the rotator cuff will be presented in this session. on intermediate-weighted mr images, tendinopathy and partial and complete tears of the rotator can be differentiated with high diagnostic accuracy. indications for mr arthrography, especially for the detection of small articular sided partial tears in athletes, will be presented as well. established classification systems for the description of rotator cuff tears will be discussed. besides characterisation of rotator cuff lesions, especially in view of therapeutic decision-making, recognition of the underlying pathomechanism is necessary. therefore, the role of imaging is to detect different structural findings that are suggestive of a possibly underlying impingement syndrome. in primary impingement syndromes imaging abnormalities of the rotator cuff, the overlying bursa and the coracohumeral arch represent the centre of imaging findings. primary extrinsic impingement is caused by structural abnormalities of the coracoacromial arch, whereas secondary extrinsic impingement is related to glenohumeral instability. types of internal impingement (posterosuperior and anterosuperior impingement) are secondary to rotator cuff and/or capsular dysfunction. posterosuperior impingement can be diagnosed on mr arthrograms by identification of the socalled "kissing lesion" pattern, with corresponding lesions of the undersurface of the rotator cuff, posterosuperior labrum, greater tuberosity and superior glenoid. imaging abnormalities of this condition will be discussed. the shoulder is one of the joints in the human body that is most subject to a number of pathologic conditions, both in young and in elderly subjects, such as subacromial-subdeltoid bursitis, calcific tendinopathy, and degenerative conditions. being inexpensive, readily available, and radiation-free, ultrasound is the imaging modality of choice to guide interventional procedures around the shoulder. thanks to its high resolution and multiplanar capabilities, ultrasound can be used to guide needles precisely in the tendons of the rotator cuff or within the joint space, both gleno-humeral and acromio-clavicular. this approach can be used to perform a number of different procedures. when dealing with bursitis, a needle can be guided within the subacromial bursa to aspirate fluid and to inject anti-inflammatory drugs. in case of calcific tendinopathy, one or two needles can be used to dissolve calcium deposit and drain it, providing patients prompt relief. in selected patients with overuse tendinopathy, ultrasound can be used to guide intratendinous injection of platelet-rich plasma that has been reported to be helpful in stimulating tendon healing. although minimally invasive, these procedures should be performed in an ultrasound ward with a high degree of sterility, as risk of infection can be concrete. multi-detector computed tomography (ct) offers new opportunities in the imaging of the gastrointestinal tract. its ability to cover a large volume in a very short scan time, and in a single breath-hold with thin collimation and isotropic voxels, allows the imaging of the entire oesophagus, stomach, and the whole chest and abdomen with high-quality multiplanar reformation and threedimensional reconstruction. preparation of the patients by fasting from solid food approximately hours prior the examination is important. proper distention of the oesophagus and stomach by oral administration of effervescent granules and water, and optimally timed administration of intravenous contrast material are required to detect and characterise the disease. preoperative staging of oesophageal and gastric carcinoma appears to be the main indication for mdct and may replace endoluminal ultrasound (eus) in the staging of advanced cancers. the use of various reconstruction techniques, including virtual gastroscopy (vg) using a volume-rendering (vr) technique, is promising for the detection of early gastric cancer. the application of the texture analysis technique to distinguish between the different types of gastric and esophageal tumors is still evolving. finally, the introduction of fdg pet, in combination with mdct, has resulted in further optimisation of the diagnostic workup of oesophageal cancer, as well as specific types of cases of gastric cancer. by providing morphologic and functional information in the same setting, this technique has come to be the modality of choice, when available. the diagnosis of oesophageal and gastric cancer is usually based on endoscopic findings accompanied by biopsy. however, staging is a matter for diagnostic imaging and is the major determinant of disease management. this should be discussed within a multidisciplinary forum (multidisciplinary team [mdt] meeting) in which the radiologist plays a crucial part. accurate stagingusually based on the tnm staging criteria -is essential and the radiologist's report should reflect this pivotal role. the tnm staging of oesophageal cancer and gastric cancer will be discussed in detail. the phases in staging is essentially a filtering process which seeks to initially exclude distant metastasis and/or advanced local disease, initially by optimally protocolled ct scanning, if ct shows advanced disease, treatment is palliative, but even under these circumstances imaging will help determine the method of palliation. conversely, if ct demonstrates localised disease, f fdg-pet scanning for oesophageal cancer and for selected cases of gastric cancer is indicated. if this, too, shows no nodal or distant metastasis, accurate t staging with eus will help determine whether the patient proceeds directly to surgery or undergoes neo-adjuvant chemo/radiotherapy prior to surgery, or in the case of oesophageal cancer may be suitable for emr. the role of staging laparoscopy in gastric cancer will also be discussed. in summary, the radiologist and nuclear medicine physician are crucial in determining treatment. their reports are lynch pins in the mdt discussion of patient management. it is therefore essential that the imaging report should optimally inform this discussion. learning objectives: . to learn about the latest tmn staging in oesophageal and gastric cancer. . to appreciate the imaging criteria for local, nodal and metastatic disease, and understand the accuracy of imaging staging. . to become familiar with the structure of a perfect imaging report. a- : c. assessment after treatment a.m. riddell; london/uk (angela. riddell@rmh.nhs.uk) it is now established that for the majority of patients with oesophageal and advanced gastric cancer, there is survival benefit from the use of neoadjuvant therapy. therefore, there is a requirement for imaging to accurately restage the tumour and to assess the response to neoadjuvant therapy, to provide prognostic information and to direct future management. restaging following therapy is challenging, as differentiating treatment-related fibrosis/oedema from viable tumour is problematic with both ct and endoscopic ultrasound. the t and n staging accuracy for both modalities falls following neoadjuvant therapy. inconsistencies in measurements due to alterations in the degree of gastric/oesophageal distension can also limit the accuracy of recist criteria to determine the response. functional imaging techniques such as pet-ct offer an improved method for assessing response. alterations in the standardised uptake value (suv) occur much earlier than changes in size; therefore a metabolic response can be detected sooner, allowing for more rapid alterations in treatment strategies. acute complications following oesophagogastrectomy generally occur within the thorax and are either related to a leak at the anastomosis/mediastinitis or respiratory complications such as pneumonia or a pleural effusion. intra-abdominal collections may develop following oesophagogastrectomy and gastrectomy. late complications following both procedures are often due to tumour recurrence: locoregional such as lymph node recurrence or at the anastomosis; metastatic spread such as haematogenous spread or via the peritoneum or pleura. currently, there is no consensus on the most appropriate timing or frequency of postoperative imaging. a. an overview of pulmonary artery hypertension n.j. screaton; cambridge/uk (nicholas.screaton@papworth.nhs.uk) pulmonary hypertension is defined by increased mean pulmonary arterial pressure > mmhg at rest or > mmhg during exercise. ph causes significant mortality and morbidity, but commonly presents with non-specific clinical signs and symptoms resulting in significant delay in accurate diagnosis and specific treatment. untreated ph is progressive with increased pulmonary vascular resistance leading to right ventricular failure and ultimately death. the current dana point classification of pulmonary hypertension is clinically based. it groups diseases with similar pathophysiological mechanisms and therapeutic approaches. groupings include conditions characterised by diffuse small vessel narrowing (group and group '), ph secondary to left sided cardiac disease (group ), chronic hypoxic pleuro-parenchymal disease (group ), chronic thrombo-embolic pulmonary hypertension cteph (group ), and a miscellaneous group of diseases with either unclear or multi-factorial aetiologies (group ). in the dana point classification, small vessel diseases are subdivided into group which primarily affect the pulmonary arterioles and group ' affecting the capillary/venous pulmonary circulation (pulmonary capillary haemangiomatosis and pulmonary veno-occlusive disease). the differentiation of group from group ' diseases is important since in group ' arteriolar dilatation treatments can cause life-threatening pulmonary oedema. group is synonymous with cteph with other causes of large vessel obstruction (vasculitis and pulmonary artery tumour) being considered as group disorders. recent advances include an increased understanding of molecular mechanisms underpinning pah, facilitating targeted therapy development, a rapidly expanding role of surgical pulmonary endarterectomy in proximal cteph, and recognition of imaging as a potential therapeutic end point. ct allows depicting pulmonary hypertension (ph) and helps identifying its cause, therefore playing a crucial role in the diagnostic workup. ct features of pulmonary arterial hypertension include dilatation of the pulmonary artery trunk, with a diameter greater than or equal to mm, a ratio to the aortic diameter greater than : and a segmental artery-to-bronchus ratio greater than : in at least three pulmonary lobes. on ecg-gated ct, right pulmonary artery distensibility shows the best diagnostic value with % sensitivity and % specificity for a cutoff value of . %. among the various causes of secondary ph, ct is especially useful for detecting signs of chronic thromboembolic pulmonary hypertension, including wall-adherent thrombi, bands, webs or chronic arterial occlusion, mosaic lung attenuation and systemic collateral supply. ct shows signs of pulmonary edema, such as thickening of the interlobular septa, centrilobular ground glass opacities, mediastinal lymph node enlargement and pleural effusion in ph caused by pulmonary veno-occlusive disease, left heart diseases or mediastinal fibrosis. signs of lung parenchyma diseases may be indentified on ct; ph is a late complication in patients with pulmonary fibrosis, sarcoidosis or chronic obstructive lung disease, but may affect systemic sclerosis patients with limited lung parenchyma involvement. congenital cardiac abnormalities with untreated right-to-left shunting resulting in eisenmenger syndrome, such as ventricular or atrial septal defect and patent ductus arteriosus are easily recognised on ct. conversely, signs of peripheral pulmonary arteriovenous shunting in portopulmonary hypertension and ph caused by hepatopulmonary syndrome are more difficult to assess. learning objectives: . to learn about the ct diagnosis of pulmonary artery hypertension. . to become familiar with the causes of pulmonary artery hypertension on ct. a- : c. mri in pulmonary artery hypertension j. biederer; heidelberg/ de (juergen.biederer@med.uni-heidelberg.de) for the assessment of pulmonary arterial hypertension (pah), the dedicated min mri protocol would comprise a free breathing and noncontrastenhanced examination, short t -w sequences, dynamic contrast-enhanced perfusion imaging, a high-resolution angiogram, a d breath-hold acquisition, dynamic steady-state free precession or gradient echo sequences of the heart and a study of myocardial late enhancement. the morphologic sequences show typical features of pah: right atrial/ventricular dilatation, enlargement of the pulmonary trunk/main pulmonary arteries and peripherally attenuated pulmonary vessels. incidental infiltrates, nodules or masses of the lung, mediastinum and chest wall would be covered. the first pass contrastenhanced perfusion imaging demonstrates an increased mean transit time/decreased pulmonary blood flow, but a relatively homogeneous lung perfusion (important to differentiate from cteph, where multiple segmental perfusion defects would be expected). the cardiac part shows right ventricular mass, wall thickness and functional changes correlating with elevation of pulmonary arterial pressure: distortion of the interventricular septum, area change of the pulmonary trunk, right ventricular volume/stroke volume as well as pathologic right/left ventricular end-diastolic volume indexes. late enhancement of the right ventricular wall would correlate with myocardial fibrosis. furthermore, optional experimental velocity-encoded sequences (ideally for multidirectional flow visualisation, " d flow") show a decreased pulmonary artery blood flow velocity, increased retrograde flow and inhomogeneous velocity profiles. in conclusion for the near future, given the availability of scanner time and appropriate experience of the team, thoracic mri is probably the most comprehensive and effective single examination for the diagnosis and follow-up of pah. in the era of organ-based radiology, the group of diseases known as multisystemic malignancies represents an obvious challenge to both radiologists and oncologists. the necessity to match the growing possibilities of different imaging modalities with widespread multisystemic pathology and clinical sufficiency resulted in upgrading well-known diagnostic algorithms. the precise knowledge of clinical staging systems and classification, and the pathologic and physiologic mechanisms of the disease pathways are important for planning imaging modalities and specific protocols. perfect imaging of multisystemic malignancies now includes not only traditional anatomic-based modalities, but also much more often different types of whole body scanning such as ct, mri, pet and their combination in spect/ct, pet/ct and now even pet/mri. new imaging modalities and growing possibilities of traditional imaging techniques obviously influence the current clinical guidelines for this disclosure: c. heussel: consultant boehringer ingelheim, grifols, novartis. a common language between radiologists and clinicians, so that the latter can make an informed treatment decision based on sound conclusions to become familiar with the conditions necessary to implement them to understand the limits of their application to learn useful lessons from these criteria for routine clinical practice chairman's introduction a. palko this requires a thorough knowledge of the relevant brain anatomy, choice of appropriate structural and molecular imaging modalities and interpretation of mri and pet/spect in the most prevalent disorders in a structured fashion the presentation has the objective of giving the audience a synthesised panorama of our country situated in north america, with more than million inhabitants and a large prehispanic history based on the aztec, toltec and maya cultures, and also of interesting facts related to the three centuries of colonial existence under the spain influence and dominium until the war of independence. after , mexico has never had an international war; the last was against the northern border country, the united states. also, we will give important data on how in the mexicans rebelled against a -year dictator, general porfirio diaz. the mexican revolution was a long and cruel war, but later opened the doors to democracy and a complex developmental era began that is still in process. some demographic and contributions to health, science and culture issues will be mentioned and also the works of mexican scientists, writers and philosophers who have been presented awards including the nobel prize. in relation to well-known mexican artists such as diego rivera and frida khalo, some facts of their works will be presented. interventional radiology in oncologic patients g. elizondo-riojas; monterrey/ mx (elizondoguillermo@hotmail.com) interventional radiology (ir) is becoming an increasingly prominent subspecialty in the care of oncologic patients. its role extends from initial diagnosis to minimally invasive treatment of the malignancy and its complications. image-guided biopsies are increasingly performed using minimally invasive techniques. also, an integral part of care of these patients is vascular access as a means of medication, chemotherapy or parenteral nutrition, and interventional radiologists can place required the devices with well-established safety and efficacy. ir also plays a substantial role in the therapy of oncologic patients, through local tumor treatments such as transarterial chemo-embolisation and locoregional control with radiofrequency/cryo ablation, as well as management of complications of malignancy such as pain, obstruction (biliary, ureteral, etc) ., venous thrombosis and drainage of thoracic and abdominal collections. in mexico, ir is a growing subspecialty, and more medical students nowadays want to be radiologists and eventually become interventional radiologists. this is a paradigm shift. more and more radiologist wants to be involved with patient management and to be more than just "observers" in the process of patient care. we have to be prepared to offer this opportunity to our residents; otherwise other specialties will "have to fill the empty space" that we have left. interventional oncology has all the advantages to fulfil this opportunity. it is our chance to contribute to the advancement of medical care. learning objectives: . to appreciate the role of interventional radiology in the management of oncologic patients. . to learn how interventional radiology changes the quality of life for patients with cancer. . to understand the impact of interventional radiology procedures in the outcome of some neoplastic diseases. interlude: origins and development of radiology in mexico m.e. stoopen-rometti; mexico/mx (mstoopen@clinicalomas.com.mx) mexican radiology started in , just a few months after the discovery of xrays. during the last few decades, as well as in other countries, there has been a great development both in public and private sectors, which will be described in this interlude. learning objectives: . to learn about the history of radiology in mexico. . to learn about the development of radiology in mexico. . to learn about the present and future of radiology in mexico. modern issues in oncologic ultrasound j. mexico/mx (jtanus @hotmail.com) advances in ultrasound (us) technology allow confident characterisation of masses. these include harmonic imaging, compound imaging, power doppler, faster frame rates, higher resolution transducers, three-dimensional ( d) us, us contrast agents and, more recently, elastography and fusion imaging. highfrequency transducers provide superb spatial and soft-tissue resolution, permitting substantially improved differentiation of subtle lesion, margin resolution, and lesion conspicuity in the background of normal tissue. elastography features such as size ratios, shape, homogeneity, and maximum lesion stiffness complement conventional us in the analysis of lesions. ultrasound contrast agents have overcome some of the limitations of doppler ultrasound techniques with demonstration of irregular branching central or penetrating vascularity within a solid mass raising suspicion of malignant neovascularity (neoangiogenesis). ultrasound contrast agents can provide important information in the assessment of lesions to be treated by locoregional therapies, which include ablation (feeding vessels), trans-arterial chemo/radio-embolisation, detecting viable tumour persistence following this treatment; facilitation of needle positioning in cases of poor lesion delineation, and assessment of local tumour progression. it facilitates needle positioning in cases of incomplete or poor lesion delineation on unenhanced ultrasound. us contrast agents are an important key during the evaluation of the immediate treatment effect of ablation and guidance for immediate re-treatment of residual tumour. learning objectives: . to understand the role of ultrasound in the management of oncology patients. . to learn how ultrasound is used in large oncology centers. . to learn about the modern concepts of ultrasound in oncology patients. the aim of this lecture is to give an overview of the techniques for imaging inflammatory bowel disease (ibd) of the small bowel and the colon with either ultrasound (us), multidetector row computed tomography (mdct) or with magnetic resonance imaging (mri) and compare the different modalities for its strength and weakness. optimal imaging of the bowel begins with the preparation phase. the small bowel has to be distended for a concise examination. this is mainly done orally, which is named enterography. a solution of . % mannitol seems to be the one preparation technique mostly used for small bowel distension. another technique is the enteroclysis, application of contrast after intubation of the small bowel. the comparative advatages and disatvantages of the two preparation methods will be discussed. this intraluminal contrast gives a neutral contrast in ct and a biphasic signal in mr. the colon can be prepared in a fashion similar to colonoscopy meaning total cleansing. another possibility is the so called fecal tagging whereas the stool will be contrasted with an additive to standardised food. therefore no cleansing is needed for preparation. imaging parameters will be discussed for mr and ct. the aim of imaging for the bowel should be to establish the following: ) presence, severity, and extent of disease; ) activity of the disease and ) extra-intestinal complications. us, mr and mdct have proven to be a good tool to evaluate the extent, the activity of the disease and the presence of extraluminal complications. pros and cons will be discussed when to use which technique. learning objectives: . to understand state-of-the-art mri, ct and us protocols for imaging ibd. . to appreciate the comparative advantages and disadvantages of enterography and enteroclysis protocols. . to learn about protocol modifications when evaluating the colon.a- : b. small bowel disease j. stoker; amsterdam/nl (j.stoker@amc.uva.nl) classification of small bowel crohn's disease is helpful for assessing disease activity and treatment monitoring. similar to clinically based classification, also imaging-based classification systems have been developed, of which some have been externally validated. important imaging features for determining disease activity include bowel wall thickness and vascularity/enhancement; at mri, also wall oedema plays a role. stenoses, fistulas and abscesses are important sequels. for assessment and monitoring of small bowel disease the breast imaging reporting and data system (bi-rads®) for mammography of the american college of radiology (acr®) consists of several components, a standardised lexicon of terms to be used during reporting, a -step coding system for the mammographic density as a surrogate parameter for the mammographic sensitivity, and a group of assessment categories ranging from to for structured communication regarding the recommended further management. the goal of bi-rads® is to improve the quality of breast imaging reporting and communication. in addition, by providing structured reports it facilitates regular quality assurance measures. the bi-rads® atlas for mammography is currently in its fourth edition and was released in . the upcoming th edition is expected soon and will be incorporated into the course as it becomes available. the breast imaging reporting and data system (bi-rads) was first developed by the american college of radiology for standardising the reporting of mammography. since its first publication in , new editions have also addressed breast ultrasound (us) and mr imaging examination. the new (fifth) edition will be published soon. on the form, this new version was designed to include a web-based format. substance includes updates in lexicon descriptors, e.g. masses, calcifications, associated features (now comprising surrounding tissue with stiffness assessment), and special cases. us descriptors will be reviewed in the lecture. of note, guidance on how to link bi-rads descriptors with management recommendations has been added in the report section. a new approach to outcome assessment (audit section) is being proposed for screening us. the appropriate use of descriptors is expected to increase the accuracy of imaging interpretation. for relevant patient management, us analysis is to be integrated with other available imaging, as well as with clinical data. learning objectives: . to learn about the bi-rads lexicon. . to understand the usefulness of bi-rads system. a- : c. mri k. pinker-domenig, p.a.t. baltzer; vienna/at (katja.pinker@meduniwien.ac.at) dynamic contrast-enhanced magnetic resonance imaging (dce-mri) of the breast is a well-established non-invasive imaging technique. it has clinical application in the screening of high-risk patients, diagnosis and staging of breast cancer, monitoring neoadjuvant chemotherapy and post-treatment follow-up. to standardise the reporting of dce-mri of the breast and minimise false-positive results without compromising sensitivity, the american college of radiology (acr) introduced the breast imaging-reporting and data system (bi-rads®) mri lexicon in . bi-rads relies on the combined analysis of morphological appearance and lesion enhancement kinetics. it is widely used for reporting dce-mri of the breast and is applicable at any given field strength. it aims to provide an up-date report on the bi-rads® lexicon and instill confidence in using bi-rads® descriptors. the bi-rads® system may be placed in a broader clinical context to highlight its value for standardised reporting of dce-mri of the breast. mr-guided focused ultrasound is a new therapeutic modality which can allow selective destruction and or heating of tissues in deep body areas under close image guidance control. these talks will introduce the field and allow you to understand the areas of current work and areas of future development in this rapidly expanding field. session objectives: . to become familiar with mr-guided focused ultrasound. . to understand the advantages of focused ultrasound. . to learn in which areas focused ultrasound is evolving successfully. description of technique c. moonen; utrecht/nl (c.moonen@umcutrecht.nl)high-intensity focused ultrasound (hifu) is the only clinically viable technology that can be used to achieve a local temperature increase deep inside the human body in a non-invasive way. mri guidance of the procedure allows in situ target definition and identification of nearby healthy tissue to be spared. in addition, mri can be used to provide continuous temperature mapping during hifu for spatial and temporal control of the heating procedure and prediction of the final lesion based on the received thermal dose. the primary purpose of the development of mr-guided hifu was to achieve safe non-invasive tissue ablation. the technique has been tested extensively and is now accepted in the clinic for ablation of uterine fibroids. mr-guided hifu for ablation shows conceptual similarities with radiation therapy. however, thermal damage generally shows threshold-like behaviour with necrosis above the critical thermal dose and full recovery below. mr-guided hifu is being clinically evaluated in the cancer field. this presentation will cover the basic technologies for treatment of stationary tissues, and some advances towards treatment of mobile abdominal organs. for state-of-the-art mr liver and pancreatic imaging, a field strength of at least . t is required. all non-blood pool gadolinium chelate-based contrast agents are suitable for dynamic liver and pancreatic mri. all gadolinium chelates should be routinely administered at a rate of - ml/s followed by a ml saline flush at - ml/s using a power injector. to obtain hepatobiliary phase imaging in addition to dynamic phase imaging, the use of liver-specific contrast agents is required. gd-eob provides the highest hepatocyte enhancement, but an overlap between delayed phase and hepatocyte phase has to be considered during dynamic evaluation. the hepatocyte phase can be considered adequate when contrast is detected in the intrahepatic bile ducts. hepatobiliary phase imaging benefits from a gradient echo high flip angle, depending on magnet field strength. in the absence of liver function impairment and biliary obstruction, contrast-enhanced mr cholangiography can be obtained with gd-eob at - minutes, and with gd-bopta at - minutes. when the differential diagnosis is primarily between solid benign lesion vs. metastasis, the use of a liver-specific ca is recommended, due to the ability to diagnose fnh confidently. the combined interpretation of dynamic and hepatobiliary phase improves diagnostic accuracy of mr imaging for the detection of hcc.hemangiomas and intrahepatic ccc result in relative hypointensity in the late vascular phase after gd-eob administration. when combined with t weighted mrcp, contrast-enhanced mrc allows morphologic and functional assessment of the biliary system. gastrointestinal stromal tumours (gist) are rare malignant tumours arising within the wall of the gut from the interstitial cells of cajal, which act as pacemaker cells controlling peristalsis. the crude annual incidence of clinically detected gists is approximately cases per million in europe. the median age at diagnosis is approximately years, and % of patients are older than years. however, a small number of cases do occur in younger people and children, and these are usually syndromic gists. gists can occur at any site of the gut from the oesophagus to rectum, although they can also arise in the extra-gastrointestinal abdominal and pelvic locations, so-called e-gists. the commonest location is the stomach ( %), followed by the small intestine ( %) and rectum ( %). diagnosis is by biopsy, with spindle cell or epithelioid morphology, and immunohistochemical staining for cd (the protein product of the kit gene) and/or dog- receptors. approximately, % of gists have mutations in the kit gene, % have mutations of the pdgfra gene, and the remaining % have no mutation (wild-type) or rare gene mutations. early stage disease is managed by surgery, followed by years of adjuvant imatinib (a tyrosine kinase inhibitor with activity against kit and pdgfra receptors) for cases at high risk of relapse. advanced metastatic disease is treated with imatinib, with a median duration of response of approximately years. at disease progression, second-line treatment is with sunitinib, with a median duration of response of approximately months. imaging plays a major role in the detection, characterisation and staging of gastrointestinal tumours (gists). imaging of gists depends on the mode of presentation and the local availability. endoscopic ultrasound and computed tomography (ct) are the most widely used imaging methods. gists have a spectrum of radiological appearances depending on tumour size and site of origin, but often show certain key radiological features. multiphase multidetector row ct is necessary to detect and stage gists and multiplanar imaging is important so as to define the likely organ of origin of the mass, which may be a challenging task. the clinical diagnosis of a gist is based mainly on imaging, as biopsy runs the risk of tumour rupture or seeding of the biopsy tract. in patients with an unresectable primary tumour or metastatic disease at presentation, biopsy confirmation is important before starting medical treatment. ct or/and mri are used to assess primary tumour extension, local invasion and the presence of metastases, with main sites of metastasis being the liver and the omentum. gastrointestinal stromal tumours (gists) are treated with targeted therapy regimes. these treatment strategies are based on the suppression of tumour vasculature using the tyrosine kinase inhibition pathway. drugs like imatinib and sunitinib exhibit specific changes in tumours and metastases that can be detected on cross-sectional imaging. these changes differ significantly from standard treatment effects of cytotoxic chemotherapy. while the latter leads to destruction of tumour cells and thereby to a reduction of tumour size, the former will cause a decrease in vascularity and thereby in attenuation and enhancement of lesions while the size may remain unchanged. radiologists have to be aware of these specific patterns of response to treatment. this presentation will include a review of the choi criteria which have been developed for the assessment of gist lesions under treatment. also, current imaging strategies focusing on time-resolved ct imaging (ct perfusion imaging) will be presented. the aim of this lecture is to provide a practical approach to response imaging in gist patients integrating both existing and novel strategies. radiological response evaluation using anatomical imaging was developed in the s in early phase ii clinical trials. potential new anti-cancer agents were assessed by measuring tumour size before and during therapy and a predetermined reduction in size constituted response. radiological response represents an objective surrogate for patient benefit and was the primary end point in early phase ii studies. if a pre-defined percentage of patients achieved the required response, then the agent proceeded to large phase ii and iii studies where clinical time to progression or progression-free survival comprised the primary end point. between and , there were extraordinary advances in cross-sectional imaging techniques which became widely available. different imaging techniques were introduced piecemeal with different co-operative groups adding different requirements, so meaningful comparisons from one trial to another could not be made. in , the recist criteria were introduced requiring specific imaging stipulations and a minimum baseline tumour size to unify the different criteria and enable meaningful comparisons from one study to another. further advances continue to be made regarding anatomical and functional imaging. not all anti-cancer agents are cytocidal and many studies now use time to progression or progression-free survival defined by radiological imaging as the primary end point. certain tumour types respond in a unique manner requiring the employment of specific response criteria in these tumour types to adequately demonstrate response. some of these tumour types and their specific response criteria will be discussed. learning objectives: . to learn about the role of radiological response evaluation in clinical trials. . to understand how radiological response evaluation has evolved and continues to evolve. . to understand which response criteria are utilised in specific tumour types. emerging biomarkers for response assessment: pros and cons m.c. roethke; heidelberg/ de (m.roethke@dkfz.de) non-invasive response assessment of new specific anti-cancer therapies is an emerging field of oncologic imaging. in the past few years, large efforts were undertaken to develop new functional biomarkers to enable an earlier diagnosis, improved risk stratification and treatment monitoring of oncological diseases. imaging biomarkers reflect changes in tumour biology, which can be differentiated into certain categories (e.g. cell density, tumour heterogeneity, iron concentration, elastic properties, or cellular receptors). in a first step, alternative qualitative and quantitative imaging biomarkers for the different modalities (mri, ct, pet) are elucidated. for magnetic resonance imaging, new techniques with potential for treatment response monitoring such as diffusion-weighted imaging, susceptibility weighted imaging, and elastography will be assessed. then, quantification of iodine uptake of contrast-enhanced ct as an imaging biomarker will be addressed. novel pet imaging strategies for therapy monitoring will be mentioned with focus on receptor targeting tracers (e.g. psma, dotatoc). furthermore, the role of pet-ct/mri is mentioned to facilitate functional techniques in oncological imaging with imaging biomarkers. the potential clinical use of prior introduced biomarkers is demonstrated for several cancer entities (e.g. hcc, prostate cancer, malign melanoma, multiple myeloma, glioblastoma). advantages and disadvantages of the illustrated imaging biomarkers are discussed during this part of the lecture. the goal is to image the right patient, at the right time, using the right test and with the right interpretation so that patients will be advised on the right therapy. ultimately, the aim is to maximise patient outcomes at an affordable cost. this session will cover the state of the art in ebr, cer, and hta and demonstrate how the results are used in imaging decisions. the application of evidence-based medicine to medical imaging was relatively delayed in comparison to other specialties. radiologists should be aware of the necessity to justify radiological examinations and imaging-based interventional procedures on the basis of the best available evidence. diagnostic tests can be evaluated with studies exploring their value in terms of: . technical performance; . diagnostic performance; . diagnostic impact; . therapeutic impact; . patient outcome; . societal impact. notably, this is a one-way logic chain. while improvement at the upper levels implies improvement at some of the lower levels, the vice versa is not always true (e.g., a better diagnostic performance does not always imply a therapeutic impact or a better patient outcome). moreover, different degrees of recommendations are based on different levels of evidence, with experts' opinion as the lowest level of evidence and meta-analyses of high-quality homogeneous studies and multicentre studies being the best level of evidence. the quality of studies needs to be evaluated in terms of internal and external validity, the former regarding study implementation (protocol violations, reference standard, measurements, and readers' independence), the latter regarding study planning (study design, subjects selection, methods, and statistical analysis). biases influencing the internal validity limit the intrinsic value of study results, while those influencing external validity limit the generalisability of study results to clinical practice. finally, high-quality research must be planned to build the evidence in favour of radiological procedures, especially for new technologies which tend to enter the market without any preliminary demonstration of efficacy. state-of-the-art methods in ebr, cer, and hta relevant to imaging u. siebert; hall i. tirol/at more and more, clinical guideline developers and reimbursement decision makers need to base their work and decisions on solid evidence. this presentation will introduce the key concepts and principles of evidence-based medicine (ebm), comparative effectiveness research (cer), and health technology assessment (hta) and their application to the field of radiology. in particular, it will be discussed which role observational studies, clinical trials, and decision-analytic modeling play in ebr, cer, and hta and when each of these study types is needed to assess imaging technologies. specific topics include the assessment of bias, optimising cutoffs and multiple test strategies, making causal inferences, explicitly weighing benefits, risks and costs, and considering ethical, legal and social implications (elsi). the excitement generated by the discovery of x-rays led to the early adoption of this technology in many hospitals around europe. over the last three decades, the explosion of new medical imaging technologies together with the recognition by clinicians of the value of these for their patients has meant that imaging budgets have increased exponentially. evidence based radiology (ebr) is a relatively new approach designed to inform clinicians of the most appropriate technique to use in a given clinical scenario. the comparative effectiveness research (cer) is used by healthcare systems to develop a strategic approach to rationalise the availability of imaging investigations. the health technology assessment (hta) funding approach is a mechanism to assess new and emerging imaging technologies in a systematic timely manner. the uk has a nationally funded healthcare system which is designed to deliver equitable care for the population free at the point of delivery. the challenge for the uk has been to ensure the highest quality service by delivering the most appropriate technology and care for patients in a timely manner. the national institute for clinical excellence (nice) is an independent body which gives guidance on new drugs and medical devices. this body requires robust scientific evidence on which to base their decisions. to create the evidence base, the imaging studies need to be of a certain standard to be included (consort, stard, etc). an example from oncologic imaging will be used to show how ebr influences daily practice. acute abdomen and abdominal trauma are both emergencies with potential fatal sequela when under/ misdiagnosed. imaging plays a crucial role in the diagnosis and management. us is the imaging technique of choice in most cases, as it is rapid, portable, lacks radiation and there is no need for sedation in young infants. us accuracy can be enhanced by iv contrast agents administration. however, limitations may occur in obese children, in deep structures, or because of gas filled bowel loops. on the other hand, ct provides excellent visualisation without limitations by obesity, gas, or deep lesions. however, there is always the radiation exposure risk of the radiosensitive paediatric patient, while sedation is required in non co-operative young children. the aim of this session is to understand "which" is the modality of choice and "why", for the most common paediatric abdominal emergencies. the acute abdomen: ct is the answer a. paterson; belfast/uk (anniezunz@gmail.com)abdominal pain is a common symptom in children, and whilst the majority will have a self-limiting condition, in some the pain may indicate an acute medical or surgical condition that requires prompt investigation and treatment. in the paediatric setting, the primary imaging modality for those with an acute abdomen is ultrasound. however, there are certain patient groups -often older children or adolescents -in whom ct plays an important role. it is well acknowledged that ct is non-operator dependent: an important factor for children presenting to emergency departments outside children's hospitals. the speed of a ct exam is valuable in the acute setting, as is the capability to obtain images without having to touch the tender abdomen of a distressed child. ct offers a global perspective of the abdomen, and image quality is not hindered by the presence of excessive bowel gas, an abnormal body habitus only porotic fragility fractures, but also of primary and secondary spine tumours such as traumatic fractures. a wide variety of lesions in and around the orbita can impair eye movement. ct and mr imaging is frequently used to confirm or exclude lesions in and around the orbit in patients with impaired eye movement. first of all, it is very important to know the exact clinical history of the patients. ct is excellent for confirming a mass; however mri is more sensitive and arrives often at a single most likely diagnosis. characteristic imaging features may help distinguish among lesions that have overlapping clinical presentations. this review focuses on some of the common orbital masses. in this lecture common benign and malignant lesions will be discussed. vascular lesions include capillary (infantile) haemangioma, cavernous haemangioma, and lymphangioma. benign tumours include optic nerve sheath meningioma, schwannoma, and neurofibroma. malignancies that are reviewed include: lymphoma, metastasis, rhabdomyosarcoma, and optic glioma. in addition, benign and malignant lesions affecting the eye movement from outside of the orbit (e.g. from the paranasal sinuses, nasopharynx and skull base) will be discussed. trigeminal neuralgia is a unique form of facial pain, defined by the international association for the study of pain as a sudden, unilateral, brief, stabbing, recurrent pain in the distribution of one or more trigeminal nerve branches, triggered by a specific event. additional diagnostic criteria of the international headache society include paroxysmal, stereotypic pain attacks and absence of associated neurological deficits. it is due to trigeminal nerve compression at the root entry zone, a transitional zone between central and peripheral myelination, where the myelin sheath is thinner and more prone to compression and electrical stimulation. the most common cause is vascular compression, by aberrant loops of the sca, aica, pica or vertebro-basilar dolichoectasia, leading to a neurovascular conflict. small size of the prepontine and cpa cisterns has been identified as a predisposing factor. occasionally, expansile lesions of the skull base or cns compress the root entry zone and lead to atypical trigeminal neuralgia. mr imaging is the modality of choice to evaluate these patients and requires specific tailored protocols to depict its causes, including d heavily t w images reconstructed in the three planes and an angiographic sequence for the depiction of neurovascular conflicts. as neurovascular contacts are quite common in the general population, strict imaging criteria for the diagnosis of nvc have emerged. the offending vessel needs to cross the nerve perpendicularly and deviate or indent its course at the rez. a pictorial review of the most common causes of trigeminal neuralgia and trigeminal neuropathy will be presented. learning objectives: . to understand the causes of trigeminal neuralgia and its clinical symptoms. . to learn about the relevant anatomy and appropriate imaging methods. . to become familiar with the relevant radiological images, differential diagnosis and limitations of the method. c. facing problems of the face: facial pain, tics and palsy b. verbist; leiden/nl (b.m.verbist@lumc.nl) facial impairments may be due to neuropathies of the trigeminal and facial nerve. this lecture will focus on the seventh cranial nerve. this nerve is a complex, mixed nerve with motor fibres, parasympathetic fibres, sensory fibres to the external auditory canal and special sensory (taste) fibres to the tongue. the most common presentation of facial nerve dysfunction is facial palsy. the paresis or paralysis of facial muscles may be caused by several conditions such as developmental abnormalities, infectious or inflammatory disease or tumoural lesions. other presentations of facial nerve dysfunction are facial tics or hemifacial spasm and pain. the indications for imaging, choice of imaging modality and possible imaging findings in case of facial palsy, facial tics and facial nerve-related pain will be discussed. in this presentation, the procedures used to create the standard will be reviewed and some of the most recent changes discussed. as a collaboration of professional societies and companies, integrating the healthcare enterprise (ihe, www.ihe.net) seeks to establish methods wherein health computer systems can communicate to achieve specific functional objectives. these involve development of methodologies to implement standards such as dicom, hl , etc. in this presentation, the procedures used to create the ihe profiles will be reviewed. patient dose tracking is rapidly growing in the usa as a large number of commercial products are available in response to user demand. these products leverage the work that has gone into the development of the dicom dose structured report and the proliferation of digital imaging systems. medical physicists are crucial team members as they are most capable of performing patient dose measurements and calculations. noteworthy is that the 'toolbox' of the medical physicist is expanding. informatics with patient dose tracking tools invariably includes familiarity at some detail with dicom structure and tags, dose structured reports (sr), sr readers, ris content and orders, modality work list broker content, modality (imaging) specific content as well as pacs, archive rules for storage/retrieval and emr. patient-specific information and the highly detailed specifics of the individual dose prescription are necessary for advanced estimates of information. a dose tracking system (a qa tool outside of the normal imaging device that uses content provided by dose sr and other information systems) usually strives to elevate the quality of the dose estimate, using informatics tools such as patient and organ models, dosimetry engines, and predictive statistics. general use cases for dose tracking will be presented including advanced dose estimations for individual patient exams and qa review for both ct and fluoroscopy. examples of tracking of prescriptions by patient, protocol/procedure and operator for determination of patient dose history, 'outliers', continuous improvement (using dmaic tools) or for meeting regulatory or accrediting bodies will be included. with the introduction of digital radiology, it is possible to have automatic systems to collect and archive patient dose data individually, in addition to demographic, geometric, and other procedural parameters, as part of the dicom header or through other dicom services. these automatic systems mean significant benefits for patient dosimetry and quality control. different approaches were used, depending on the availability and level of implementation of the dicom standard, including extracting the technical information from the headers, using the radiation dose structured reports (which contain accumulated dose over several irradiation events), analysing the mpps messages sent by the modalities to the radiology information system, and implementing optical character recognition techniques on saved screen images. before issuing a formal patient dose report, the medical physicists should verify and correct all patient dose data. all these approaches allow managing more information and provide better capacity to audit the full imaging procedure and to help with the optimisation. the current level of technology allows doing so at a reasonable cost and with a great benefit for the clinical practice. automatic detection of abnormal patient doses or mistakes in the technical parameters used and their prompt correction is possible. diagnostic reference levels will be effortlessly reviewed with such systems. some examples of pitfalls and possible optimisation actions will be presented. pathology. finally, the perfect assessment of clinical course of the disease and possible outcomes, the understanding of tumor response criteria and therapyinduced changes are significant for image interpretation in patients with multisystemic malignancies. multiple myeloma is a haematologic disorder characterised by the infiltration and proliferation of monoclonal plasma cells mainly in the bone marrow. the main symptoms are hypercalcaemia, renal impairment, cytopaenia/anaemia and bone disease -summarised as crab-criteria. symptomatic multiple myeloma is consistently preceded by asymptomatic premalignant stages called monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. these stages also present with detectable monoclonal protein and/or monoclonal plasma cells in bone marrow, but do not show any end organ impairment. staging of multiple myeloma is based on the measurement of the monoclonal protein in serum and urine as well as the assessment of impairment of haematopoiesis, renal function and mineralised bone. in the last decade, the development of novel therapeutic agents has led to an increase in response rates and survival time of patients with multiple myeloma, which further stresses the value of response assessment by imaging. cross-sectional imaging like mri, ct and pet-ct is currently replacing conventional radiological surveys in the initial workup and follow-up of patients with monoclonal plasma cell diseases. recent studies demonstrate the added value of mri in improving initial staging by unravelling a diffuse infiltration of bone marrow by plasma cells, a focal pattern or a combination of both. also, pet-ct can detect response to therapy earlier than conventional response criteria. furthermore, recent studies revealed that a complete remission of myeloma confirmed by mri or pet-ct goes along with a better prognosis compared to a complete response based only on serological parameters. learning objectives: . to learn about the role of clinical staging systems and imaging in work-up and classification of plasma cell disorders with a focus on multiple myeloma and to learn about parameters of disease activity. . to understand the pathophysiologic mechanisms of multiple myeloma and to learn about the effects of abnormal plasma cells on bone remodeling cells. . to become familiar with response criteria and therapy-induced changes and to compare the significance of imaging and serological markers for response evaluation in multiple myeloma. . to learn about recent studies on imaging based response assessment and prognostic significance.a- : over the last years, there have been numerous efforts to classify lymphoid malignancies, culminating in the who classification introduced in . so it is clear that malignant lymphomas require a sophisticated diagnostic approach based on clinical features, morphology, immunophenotyping and genetic analysis. it is essential that such an approach underpins the clinical management of these diseases, many of which are amenable to cure. the same situation is present in radiology staging procedures -it has a multidisciplinary approach. a wide range of staging procedures can include all radiology methods from cxr and chest and abdominopelvic computed tomograph to whole body mri, scintigraphy and pet-ct scans. lesions of the regional lymph node system. distant spread of mm is thought to transpire intra-and extravascularly (along the external vessel lattices) supported by specific mechanisms of extravasation and protection of mm cells against the local immune system. to date, many imaging methods, such as ultrasound, computed tomography (ct), magnetic resonance imaging, and positron emission tomography/ct, are used in the diagnosis of mm and its metastases, whereas lymphoscintigraphy has become an important tool for workup of the lymph node drainage patterns. radiological challenges include improved staging and re-staging of multi-systemic mm. accordingly, current imaging guidelines and the role and applications of the different imaging modalities in advanced mm (stage iii / iv) are reviewed with regard to their usefulness in patient management. however, standardised imaging protocols and integration of functional information to morphological imaging are needed in the future to allow for improved detection and guidance of treatment in patients with mm. the original atlanta classification of acute pancreatitis established in is outdated. in , an international working group has modified the atlanta classification for acute pancreatitis to update the terminology and provide new morphologic classifications.the revised classification of acute pancreatitis identified two phases of the disease: early (first week) and late (after the first week). acute pancreatitis is subdivided into two types: interstitial oedematous pancreatitis and necrotising pancreatitis. if the diagnosis of acute pancreatitis is established by abdominal pain and by increases in the serum pancreatic enzyme activities, a contrast-enhanced ct is not usually required for diagnosis in the early phase. this revised classification introduces new terminology for pancreatic fluid collections. depending on the presence or absence of necrosis, acute collections in the first weeks are called acute necrotic collections or acute peripancreatic fluid collections. once an enhancing capsule develops, persistent acute peripancreatic fluid collections are referred to as pseudocysts and acute necrotic collections, as walled-off necroses. all can be sterile or infected. this classification of acute pancreatitis allows a consistent, worldwide classification and should avoid confusion in the terminology of pancreatic fluid collections. the role of diagnostic imaging in chronic and inflammatory pancreatitis and ipmns is to detect structural changes of the ducts and of pancreatic parenchyma, assess the functional integrity of the gland, detect associated complications, and assist in management. these goals are generally fully achieved using mdct and mr imaging. in this lecture, the advantages and limitations of each technique will be discussed and illustrated. key features allowing differential diagnosis of both entities will be underlined. autoimmune pancreatitis (aip) was first described in and represents a rare form of immune mediated chronic pancreatitis which is characterised by a marked infiltration of lymphocytes and plasma cells into pancreatic tissue. whilst the majority of cases present with diffuse gland involvement, approximately % of patients demonstrate either segmental or focal involvement of the pancreas. clinical presentation is very variable with patients describing a range of symptoms; severe pain however is uncommon. imaging plays a central role in the diagnosis and management of aip and knowledge of the radiological appearances, which can vary significantly due to the various degrees of fibrosis and inflammatory infiltrate, is critical. cardinal features include focal or diffuse pancreatic enlargement with the loss of normal lobular architecture. in addition, pancreatic duct involvement as demonstrated by single or multiple focal strictures with limited more proximal dilatation is common. whilst these appearances may suggest a diagnosis of aip correlation with clinical history, serology and histopathology is mandatory to accurately diagnose atypical cases. in contrast, paraduodenal pancreatitis is a specific and rare form of segmental chronic inflammation characterised by fibrous tissue formation in the "groove" area between the duodenum, head of pancreas and lower common bile duct. the pathology was first described in and has since been subdivided into pure, segmental and non-segmental forms. whilst the radiological diagnosis of both aip and paraduodenal pancreatitis remains challenging, the presentation will emphasise relevant imaging strategies as well as provide tips and tricks for accurate diagnosis. bone scintigraphy is often used in combination with ct and clinical information for staging and therapy response evaluation of bone metastases. magnetic resonance (mr) imaging is a technique that is known to be valuable both for detection of bone metastases and for evaluation of their response to treatment. integrated positron emission tomography with computed tomography (pet/ct) combines the functional data from pet with anatomic information of ct in a single examination. f-fluoride is a non-specific pet tracer that has recently shown very high sensitivity for bone metastases. for accurate response evaluation, it is important to recognise not only changes in bone metastases, but also the effects of treatment on unaffected, normal parts of the bone marrow. these latter therapy-related benign changes could otherwise be misinterpreted as disseminated disease or vice versa. despite the fact that the methods mentioned above, alone or in combination, have improved the assessment of bone metastases, they often remain non-specific. a specific diagnosis can in many cases only be achieved with a bone biopsy. this refresher course will give you an overview of the present imaging methods for detection and characterisation of bone lesions. indications for, and how to perform, ablation and cementoplasty of bone metastases will be presented. optimal algorithms for treatment follow-up of bone metastases and advantages and disadvantages with different imaging methods will be reviewed. also, future development of diagnostic and therapeutic methods will be included in the presentations, e.g. potential role of emerging pet imaging probes for assessing bone malignancies. author disclosure: h. ahlström: research/grant support; astrazeneca. a. diagnostic approach: the role of hybrid imaging k. herrmann; würzburg/ this presentation will include a short review of the epidemiology of primary and metastatic bone disease, its classification and staging definitions. then we discuss the role of radionuclide imaging in initial and subsequent treatment strategy considerations. another aim is to provide guidelines for the appropriate use and applications of diagnostic radionuclide approaches for the assessment of bone diseases. the focus of this presentation will be on the role of radionuclide imaging in metastatic prostate, breast and lung cancer in the context of other available diagnostic imaging approaches. the ability of hybrid imaging technologies to achieve improved diagnostic accuracy will be emphasised and when appropriate the potential role of emerging pet imaging probes for assessing bone malignancies discussed. if feasible, the availability and effectiveness of radionuclide-based therapeutic approaches for malignant bone diseases will be reviewed. skeletal metastases are associated with significant morbidity and have high impact on health costs. effective palliative treatments are available, but less than % of patients respond in terms of tumour markers, pain or quality of life and all treatments are associated with some toxicity. it is generally accepted that the current imaging techniques are inadequate for measuring treatment response in a clinically relevant time frame such that skeletal metastases are usually considered as non-measurable disease in clinical routine and trials. there is therefore a need for non-invasive, objective methods to evaluate treatment response of skeletal metastases at earlier time points to guide clinical care. in addition to imaging structural changes in bone (e.g. x-ray, ct), it is now possible to image a number of biological characteristics of bone metastases including tumour metabolism, tumour cellularity, and osteoblast and osteoclast activity. methods currently available, or being actively investigated, include m tc-mdp spect/ct, f-fluoride pet/ct, f-fdg pet/ct, c/ f-choline pet/ct, rgd-spect/pet and dw-mri. some of the tracer methodologies will potentially be applicable for and complementary to pet/mri. the timing of follow-up imaging after commencing treatment is still under investigation. it is likely that tumour-specific methodologies may reflect response/non-response earlier than bone-specific methods due to a prolonged flare in reactive bone in responding metastatic sites. learning objectives: . to understand the techniques and quantitative parameters used for treatment response evaluation. . to learn the optimal timing for follow-up imaging after treatment. . to become familiar with common pitfalls in the interpretation of posttreatment mri and pet. panel discussion: how to differentiate between bone metastases and chronic inflammatory or treatment-induced changes : structured reporting is an opportunity and a challenge at the same time. expert speakers of this session will present and discuss their views on the subject, contributing to the clear vision of the members of audience on this complicated subject, convincing us to take this technique as an opportunity to improve and standardise the quality of our reporting activities. even though structured reporting (sr) has been appreciated as one of the effective methods of electronic reporting, there are not that many real applications to facilitate utilisation of this technique in practice. why it is so?the radiologists want a flexible tool to create clear, concise reports in a reasonably short time. like all template-based reporting systems, the application must be tailored to preferences of multiple users in a radiology department. the report templates should also be flexible in terms of length to avoid inappropriately detailed reports. the final appearance of the report is critical for acceptance by clinicians. accordingly, the technical challenges of development of sr systems may be grouped as: "user interface", "database structure", and "output file/document" problems. flexibility to comply with different user preferences will lead to both user interface design and database structure challenges. one of the major technical challenges is the transformation of structured data to a final text to be acceptable and applicable for the referring physician. also, exporting of reports to dicom format using dicom-sr concepts is still a challenge. multi-media formats are becoming more and more popular specially in sr reports. effective communication of radiology results is critical to high-quality health care. to that end, the radiological society of north america (rsna) has developed a library with more than best-practice radiology reporting templates. the templates are freely available online (www.radreport.org) and have been accessed more than , times. the new ihe "management of radiology reporting templates" (mrrt) profile builds upon the web's hypertext markup language version (html ) to provide an international standard for describing and exchanging reporting templates. this presentation will describe the rsna report template library, introduce the international mrrt standard for report templates, and describe opportunities for esr members and affiliated societies to participate. the broad goals of this initiative are to improve the quality of radiology reports, increase the productivity of radiologists, promote the use of practice guidelines, and advance data-driven health care.learning objectives: . to explore a library of 'best practices' radiology reporting templates. . to become familiar with international standards for radiology reporting templates. . to understand how structured reporting will promote the use of practice guidelines and will advance data-driven health-care. structured reporting in europe: the esr initiative o. ratib; geneva/chthe rsna has initiated a new initiative called radreport with the aim of helping promote standard reporting and improve reporting practices by creating a library of clear and consistent report templates (http://reportingwiki.rsna.org/). this project also supported in part by the national institute of biomedical imaging and bioengineering (nibib) has already gained the participation of numerous countries around the world, contributing with reports in different languages. these report templates are "structured" in the sense that they incorporate reusable knowledge, or meaning, to the clinical reporting process. structured interpretation and reporting for detecting significant prostate cancer is crucial, as it allows comparing inter-observer interpretation variability, reduces this variability by stimulating discussion of the individual scores, enhances communication with the clinicians in a uniform way, facilitates quality assurance plus research, and in this way improves patient outcome. in this presentation, the pi-rads scoring system will be shown and discussed, and fast effective structured reporting using computer software will be presented. in a rapidly ageing society, dementia prevalences are sharply increasing. furthermore, knowledge about disease mechanisms is constantly evolving. a more nosologic approach in the workup of dementia is required to improve prognostication, patient management as well as select appropriate treatment. a. anatomy of the limbic system t.a. yousry; london/uk (t.yousry@ucl.ac.uk)the term "limbic" has been used in many different connotations. to understand the anatomy, we have to define the term first. the clearest definition stems from broca: "le grand lobe limbic". defined as such, the limbic lobe is composed of main structures: the limbic and the intralimbic gyri. . the limbic gyrus is composed of the subcallosoal gyrus anteriorly, followed in an arc by the cingulate gyrus, isthmus and parahippocampal gyrus. the latter is composed of parts: i) a posterior narrow segment, the superior surface of which is called subiculum, and ii) a more voluminous anterior segment, also known as the piriform lobe. the latter consists of the anterior part of the uncus and the entorhinal area. . the intralimbic gyrus arches within the limbic gyrus. it is divided into parts: i) anterior (prehippocampal rudiment); ii) superior (indusium griseum); and iii) inferior (hippocampus). the latter consists of lamina rolled inside each other: the cornu ammonis and the dentate gyrus, with the cornu ammonis consisting of neuronal fields (ca - ). to understand the function, we have to understand the connections that define this function. the interplay with the insula is of particular importance. in summary, we will review the mri characteristics of the components of the limbic lobe, their connections, and their function, using . t, t and . t high field imaging. at the end of this lecture, you will know definition (s) of the limbic lobe, its major subdivisions, connections and functions. structural neuroimaging is increasingly used in the diagnosis of neurodegenerative diseases. in a memory clinic setting, computed tomography (ct) and magnetic resonance imaging (mri) have become the most important paraclinical diagnostic tools. recent guidelines on the diagnosis and management of disorders associated with dementia state that neuroimaging should be performed at least once during the diagnostic workup. in the past, neuroimaging had been performed to exclude potentially treatable causes of dementia. however, this exclusionary approach of neuroimaging in the diagnosis of dementia has been replaced by an inclusionary approach driven by conclusive evidence that ct and mri can substantially support the clinical diagnosis by the demonstration of a distinct atrophy pattern and the assessment of vascular (co)morbidity. for these purposes, the use of visual rating scales allows a fast and reproducible assessment of global/regional cortical atrophy and vascular white matter pathology. more recently, new imaging markers such as cerebral microbleeds and superficial siderosis haven been identified and linked to alzheimer pathology. in addition, next to structural mri, advanced and quantitative mri methods have been introduced in the clinical setting. mri perfusion techniques such as arterial spin labelling (asl) demonstrated a diagnostic value in memory clinic patients, particularly in those showing no or inconclusive pathology on structural mri. de (horst.urbach@uniklinik-freiburg.de) cognitive decline is a clinical symptom in many neurodegenerative disorders. mri is typically performed in the initial diagnostic workup of these patients. as a structural imaging modality, mri is often unrevealing while several molecular imaging modalities show disease-associated alterations: f-fdg-pet shows reduced glucose metabolism in neocortical association cortices, mainly in the posterior cingulate, precuneus and temporoparietal regions. since glucose metabolism is normally higher in the posterior cingulate and precuneus, reduced glucose metabolism must be specifically sought for or highlighted by voxel-based analysis. dynamic imaging of swallowing from the mouth to the duodenum is the only clinical test providing a fast and accurate diagnostic overview of the upper gi function. swallowing disorders are divided into those with dysphagia that means problems during eating or drinking, with suspicion of aspiration as an important subgroup and those with sensations like the globus syndrome or noncardiac retrosternal pain. oropharyngeal dysphagia in patients with neuromuscular diseases or postoperative conditions can be evaluated precisely and within the same session as oesophageal motility disorders, stenoses or postoperative problems are detected and in a complementary fashion to endoscopy, ph monitoring and manometry. the exclusive ability of dynamic imaging of swallowing is to diagnose combined disorders of the pharynx and the oesophagus, to detect and localise subtle and multiple benign stenoses of the upper gi tract and to combine visualisation of pathophysiology and bolus transport as well. radiologic evaluation concentrates on functional units, which represent the visible "moving areas" of deglutition, where evident pathologic conditions can be found: ) oral cavity and tongue, ) soft palate, ) epiglottis, ) hyoid and larynx, ) pharyngeal constrictors, ) pharyngoesophageal segment, ) oesophagus and stomach. this lecture intends to explain the common causes of dysphagia, the approach to a tailored examination and the use of an advanced imaging technique. further, the basics of taking the swallowing history and the structure of reporting swallowing disorders along the seven functional units will be provided. hoarseness is defined as a rough or noisy quality of voice. the possible causes of hoarseness are manifold, ranging from benign diseases such as the common cold to malignant tumours. therefore, hoarseness as a symptom should always be taken seriously. evaluation of a patient with hoarseness by a head and neck specialist will always starts with a history and a physical examination including laryngoscopy. after these examinations, a diagnosis can be established in most patients without additional imaging. the aetiologies of hoarseness are: ) inflammation/infection, ) trauma, ) neoplasms, ) others (including medical conditions, e.g. hypothyroidism) and ) vocal cord paralysis. this presentation will focus on imaging of vocal cord paralysis. vocal cord paralysis should not be considered a diagnosis, but as a symptom of (possible) underlying disease. when vocal cord palsy is discovered, this is frequently a reason to perform cross-sectional imaging. the radiological workup needs to include the full course of the vagus nerve. moreover, reporting radiologists need to be able to recognise the radiological signs of vocal cord palsy in the absence of hoarseness, since many patients with cord palsy are asymptomatic (up to %). the anatomy of the vagus nerve and its laryngeal branches that innervate the intrinsic muscles of the larynx will be discussed. then the (expected) course of the vagus nerve and recurrent laryngeal nerve will be shown. finally, radiological signs of vocal cord palsy will be discussed using examples from daily practice. in contemporary medicine, endovascular techniques often replace conventional methods of treatment, including surgery, in the management of an increasing range of diseases. this requires development of specific methods assessing the effectiveness of treatment and able to detect complications that may be much different from those occurring in conventional therapy. this is first of all seen in the endovascular treatment of patients suffering from vascular lesions and neoplastic diseases. commonly, there is a high initial technical success of endovascular treatment, but the durability is not always satisfactory and needs to be further examined. endovascular treatment is bound to the risk of specific complications, often without clinical symptoms. therefore, there is a need to use imaging follow-up of these patients.interventional radiologists are aware of the importance of those issues and should play an important role in developing and deciding about the follow-up scheme and choice of the best imaging methods for these patients. besides indepth knowledge of the treatment, radiologists are also familiar with the possible complications and can offer an optimal, minimally invasive and cost effective diagnosis and treatment. the potential imaging methods for follow-up after angioplasty (pta) and/or stenting include doppler ultrasound, cta and mra. although excellent images can be obtained by cta and mra, the drawbacks of these studies for routine follow-up are obvious. (access to ct and mr equipment, cost, contrast medium, ionising radiation, etc). in the uk, the most common imaging method is duplex ultrasound. there is no established role for the use of ultrasound contrast agents. ultrasound is freely available in the majority of centres, is inexpensive and complication free. follow-up by ultrasound may be undertaken by radiologists, although more usually by technologists in vascular laboratories. in practice, few patients are followed up by any imaging study at all unless they develop symptoms. the main category of patients who undergo some form of imaging follow-up after pta/stenting are patients with surgical bypass grafts, who are usually routinely followed up whether they have undergone angioplasty/stenting or not. in view of the fact that this is a lecture on follow-up after intervention, procedural complications are excluded from the discussion. the complications of angioplasty and stenting are similar. the main early complications are restenosis or reocclusion. other early complications are related to the arterial puncture site and include haematoma, dissection, occlusion and pseudoaneurysms. the main late complications are restenosis and reocclusion. treatment of restenosis and reocclusion usually involves additional angioplasty or stents using conventional or drug-eluting balloons or stents. cutting balloons, atherectomy and cryotherapy have a controversial role based on limited evidence. imaging follow-up of oncologic patients after embolisation procedures j.i. bilbao; pamplona/es (jibilbao@unav.es)the term "embolisation" groups different procedures in which imaging guidance is fundamental for both performing the procedure and for the early detection of complications. it has been demonstrated by several studies that "tumoural response" is tightly associated with survival, thus it is of major interest to measure accurately how the tumour is modified by the treatment. the "world health organisation" -who -first published the objective criteria for the evaluation of response. these criteria were slightly modified in and named as "response evaluation criteria for solid tumors"-recist. accordingly, "complete response" (cr) corresponds to the disappearance of the tumour, "partial response" (pr) is defined when there is a decrease in the tumour size equal to or higher than % and "progressive disease" (pd) is applied when there is an increase equal to or higher than %. "stabilisation of the disease" (sd) corresponds to measurements in between > % (pr) and < % (pd). tumours (until five nodules in the same viscera), no matter if necrotic or viable, were evaluated with one measure (its largest diameter). a new version (" . recist) has introduced modifications, among which is the number of nodules ( instead of ) that needs to be evaluated. in some circumstances, when morphologic criteria (recist and who) fail to predict the outcome, then new functional criteria have been recently established. "mrecist" (which only measures the tumour that uptakes contrast) is an accurate classification for hcc. new methods "beyond recist" have been established for some specific treatments such as targeted therapies. in this session, the audience will have a comprehensive overview of the elastography, its principles and current state-of-the-art clinical applications. the session will deliver an overview of currently available methods of both ultrasound elastography (use) and magnetic resonance elastography (mre), their physical principles, technical and equipment requirements as well as the influence of various factors for obtained results. following this session, the audience will have insight into clinical applications of elastography in liver, prostate and brain diagnostics. the strengths and weaknesses of elastographic modalities will be compared and discussed under consideration of more established imaging modalities. the session will present potential developments of use and mre and possible future clinical applications. panel discussion will aim at defining the place of elastography in current and future diagnostic algorithms. the well-known sensitivity of the palpating hand for detecting lesions is related to the incredibly wide range of values of the shear modulus in the body, spanning over eight orders of magnitude. elastography was invented to exploit this high variability of constants by introducing the shear modulus into the image contrast of medical ultrasound or magnetic resonance imaging. today, both ultrasound elastography (use) and magnetic resonance elastography (mre) are in clinical use, predominantly for staging liver fibrosis or discriminating tumours in the breast, liver, and prostate. the talk reviews the physical principles, technical requirements and current methods of both use and mre. furthermore, the strengths and weaknesses of elastographic modalities are compared and discussed under consideration of more established imaging modalities. an outlook of elastography is given, highlighting the real-time feasibility of use and the sensitivity of mre to tissue pressure. suspicion of prostate cancer (pca) exists in the case of elevated psa serum values and/or suspicious findings on digital rectal examination (dre). systematic biopsy (sb) will be done to confirm or rule out pca. unfortunately, an elevation of psa serum values often is caused by benign changes, e.g. prostatitis or adenomyomatosis and not every cancer is palpable on dre. furthermore, sb may miss clinically significant disease in up to %. therefore, a more targeted approach would be desirable and imaging of pca is currently under strong effort. one possibility for visualisation of pca is the representation of tissue elasticity. usually, cancers have higher cell and vessel density than the normal surrounding tissue and therefore are associated with increased stiffness. in contrast to dre, where only the posterior parts of the prostate can be reached, rte does not have this problem, since all anatomical regions can be evaluated. furthermore, this noninvasive technique is time-and cost-effective and targeted biopsy or focal therapy of the prostate can be done under real-time conditions. another important issue is that rte can be performed by both urologists and radiologists. nevertheless, rte is of limited value in the detection of small cancer lesions and there may be problems in visualising pca with predominantly gleason pattern . the lack of data about inter-and intraobserver variability and of multicentre studies (now in process) are probably the reasons why rte has not been included in the guidelines of urological societies so far. cerebral tissue structure is altered in many neurodegenerative diseases, but also during physiological processes like maturation or ageing. structural changes directly affect the mechanical tissue properties. magnetic resonance elastography (mre) is an imaging technique capable of assessing biomechanical brain parenchymal properties non-invasively. viscoelasticity can be quantified by analysing the propagation of mechanically elicited shear waves in the investigated tissue. thus, mre could be a helpful tool to detect physiological or pathological processes influencing the cerebral tissue integrity. learning objectives: . to appreciate the advantages and limitations of a virtual palpation of the brain. . to become familiar with mechanical wave induction and detection in cerebral measurements. . to learn about current and potential clinical applications. . to learn about the relationship between microscopic and macroscopic, as well as focal and globally diffuse biomechanical brain parenchymal alterations. are based on the generation of shear waves inside the liver tissue with specific mr liver drivers and ultrasound probes. subsequently, phase images revealing the displacement of the waves will be investigated for healthy and pathological (alcoholic fibrosis) livers. moreover, the development of phantoms mimicking the stiffness of biological soft tissues (liver, muscle, …) will be also presented. the viscoelastic (elasticity and viscosity) properties of healthy and fibrotic livers were measured, and the relevance of these parameters, used as a liver diagnostic marker, was analysed. in parallel, an increase of the wavelength as a function of the stiffness was obtained for the developed set of phantoms. the characterisation of the elastic properties is an excellent marker to differentiate minor and major fibrosis. a summary of the advantages and disadvantages of us and mr elastography techniques will be presented. a- : the breast lesion excision system (bles) has evolved as a breast radiological technology over the last decade and is now in widespread use in europe and across the world. it was designed as a large biopsy device, but more recently due to its unique capability to obtain a single large breast tissue specimen in only a few seconds by utilising a radiofrequency cutting and cauterising wave, it has increasingly been explored in the therapeutic setting. it is easy to use under ultrasound or mammographic guidance with procedures taking a similar length of time to that of a vacuum biopsy, and with patient preparation and anaesthetic essentially identical. the technical aspects of performing these procedures will be detailed as well as its use specifically to perform excisional biopsies. this is limited by patient and lesional factors, all of which will be discussed in more detail. following a bles, the post-procedure appearances need to be considered so that follow-up imaging can be interpreted accurately. the risks and complications of this procedure are outlined as well as a discussion made of the latest papers in this field that may suggest future applications and developments. learning objectives: . to understand the mechanism of the bles technique. . to become familiar with the post-bles aspects of various types of imaging. during the past few years, it has been shown that there is not a single disease entity called "breast cancer". there are different subtypes that entail diverse recurrence risks. this is the first issue to take into account, and patients will be stratified accordingly before any follow-up is planned. imaging findings in a patient treated for breast cancer will depend on the type of treatment: breast conserving therapy (bct), mastectomy (and all the reconstructive techniques), radiotherapy and minimally invasive techniques. to differentiate between fat necrosis and other common post-treatment changes from relapse, it is important to know the timeline when all these changes take place and also schedule the follow-up imaging procedures accordingly. other important issues to take into account are the limitations and indications of the different modalities (mammography, ultrasound and mri). although ultrasound and mammography have traditionally been used in the follow-up of these patients, mri is being used more and more often due to its superior multiplanar capabilities and the functional information not supplied by the other techniques. blood clot formation in the veins is referred to as venous thrombosis (vt). vt is most common in the deep veins of the legs and pelvis. if the thrombus dislocates to the pulmonary arteries this is referred to as pulmonary embolism (pe). the combination of deep vein thrombosis (dvt) and pe is referred to as venous thromboembolism (vte). the diagnosis of dvt and vte is essential, as vte is potentially fatal. furthermore, undiagnosed, non-fatal dvt may result in negative long-term pathologies (e.g. post-thrombotic syndrome). major risk factors for the development of vte are thrombophilia, history of dvt, age > , surgery, obesity, long travel, cancer, immobility and pregnancy. in addition, underlying congenital abnormalities or compression syndromes (may-thurner syndrome) are also important to diagnose. diagnosis of vte is not always easy. primary diagnosis includes clinical (wells score) and lab testing (d-dimer). first line imaging testing is done by leg ultrasound (us). if pe is suspected ct is indicated immediately. ct of the pulmonary arteries (ctpa) may be combined with indirect ct venography (ctv). the question of when to combine ctpa and ctv still remains. a favourable approach is to add ctv in a negative ctpa. recently mr venography (mrv) is emerging as comprehensive imaging tool especially in patients with chronic dvt to assess the extent and underlying causes. in this respect imaging without radiation dose with high spatial delineation of vascular abnormalities facilitates intervention for thrombolysis or interventional therapy including recanalisation and stenting. alternatively, intravenous unfractionated heparin is used in situations when a quick reversal is necessary. b) non-medication: early mobilisation has been widely adopted to activate the muscle pump. if early mobilisation is not possible, compression stockings and/or pneumatic compression boots are applied to better empty the veins. . treatment: a) anticoagulation: the main goal of anticoagulation is to prevent progression and recurrence of dvt. typically, anticoagulation is started with lmh, followed by vitamin k antagonists (coumadin). the first dvt is generally treated for - months; recurrent dvt for months. b) compression therapy: compression stockings ameliorate pain and leg swelling of acute dvt. in addition, the development of a post-thrombotic syndrome (pts) should be reduced. c) thrombolysis: the main goal of thrombus removal is prevention of pts. in addition, thrombolysis is used for severe ilio-femoral dvt, especially with phlegmasia coerulea dolens. there are different ways of thrombolysis: systemic thrombolysis was abandoned because of high bleeding complications. catheter-based thrombolysis has shown to reduce the bleeding risk, but it is quite time and resource consuming. the latest development is pharmaco-mechanical catheter thrombus removal which combines thrombolytic drugs with a mechanical maceration. recent studies (cavent,torpedo) have shown a reduction of pts compared to anticoagulation. the use of inferior vena cava filters (ivcfs) is a controversial method for the prevention of pulmonary emboli. there are large variations in their usage; often with ten to one hundredfold differences in placement between different countries. ideally, ivcf usage is based on sensible protocols derived from clinical experience and trials. in practice, this is less frequently the case. indications, contraindications and questionable indications (mainly during venous thrombolysis) will be discussed. familiarity with ultrasound guidance will be stressed. the key technical steps in the insertion and removal of ivcfs will be discussed. the question that this refresher course is aimed to answer is whether novel it tools may actually help in improving quality and workflow efficiency in daily radiological practice. in fact, since the early installations of pacs, it tools have often been considered as mere productivity tools rather than enabling technologies for fostering quality in medical care. the three distinguished lecturers in this course will address the following topics: improving quality and efficiency of computerised order entry through decision support, improving quality and efficiency of reporting through structure and templates, and improving quality and efficiency of dose management through exchange between modalities and registries. they will cover all aspects of the radiological workflow: from the selection of the most appropriate diagnostic procedure, to the efficient reporting of diagnostic and non-diagnostic data (such as radiation dose information and contrast media information). they will demonstrate how newly adapted it tools may provide assistance throughout the radiological workflow, with potentially enormous gains in terms of patients' safety and total quality management. session objectives: . to appreciate the need for it tools to ensure quality control. . to understand how to collect data concerning radiation dose. a. improving the quality and efficiency of computerised order entry through decision support p. mildenberger; mainz/ de (peter.mildenberger@unimedizin-mainz.de) electronic communication of requests for radiological procedures is a logical and relevant extension to integrated information systems in health care. this communication of orders and the entry (oe) into ris improve the workflows. but, quality improvements require additional efforts to link order-entry-solutions with health knowledge for ordering the appropriate imaging procedure. these clinical decision support (cds) tools should be based on generally accepted and implemented criteria, e.g. evidence-based medicine. it is known that the acceptance of such systems, if successful and efficient implementations are given, is very good. it concepts for cds are well known and standards for classifications are available, but semantic interoperability is still a developing area. actually, clinical information systems provide different levels of integration of oe and cds. further developments could be an implementation of interoperability profiles and the representation of cds knowledge in webbased services of non-commercial organisations. b. improving quality and efficiency of reporting through structure and templates e. neri; pisa/it (emanueleneri @gmail.com)the report represents the final stage of the diagnostic imaging process, for which the radiologist is responsible. this process is driven by an accurate selection of the diagnostic tests, based on the clinical question. therefore, the report should be an expression of this path, depending on the question asked by the clinician. the structure of a radiology report for the same clinical problem and the same method of investigation varies from country to country and between different radiologists. this diversity can be a problem in the global world where a patient can have imaging tests from different places with different reporting languages, or when patient data is transmitted and shared between different centres. there is, therefore, the need to standardise the report as much as possible; finalising its structure and contents to a specific clinical problem, and possibly using a standard terminology. an attempt to solve this problem is in progress with the joint initiative on structured report esr-rsna (http://www.rsna.org/reporting_initiative.aspx), aimed at improving reporting practises by creating a library of clear and consistent report templates. the main expected benefits of structured reporting templates are report uniformity and improvement of communication between referring physicians. literature evidences that structured reporting can also quicken report turnaround, and reduce costs and lexical errors; thereby improving the quality and safety of radiological practise. the european directive on medical exposures requires the assessment and evaluation of patient doses, especially in procedures involving high doses to the patient. in the current draft of the new directive on basic safety standards, some requirements on patient dosimetry in diagnostic and interventional radiology have been reinforced: x-ray systems should provide dosimetric information with the capability of being transferred to the examination report (for all ct and interventional systems). diagnostic reference levels (drls) shall be reviewed regularly. these requirements will push the industry and the users to develop better strategies to evaluate patient doses, transfer these values to patient reports (contributing to the patient exposure tracking system) and also use available software to process these dosimetric data and perform some automatic analysis. this analysis should: a) include periodic calibration factors for patient dose quantities, b) include automatic detection of high dose values (especially relevant for interventional procedures), c) include statistical analysis to update drls and compare with the existing ones and d) suggest corrective actions to fulfil the quality assurance programmes and the clinical audit requirements. dicom radiation dose structured reports represent a significant advantage, but more efforts will be necessary for the automatic process of the relevant data contained in the report to verify that the radiological risk is acceptable and to suggest, if appropriate, corrective actions to improve clinical practice. without these last steps, patient dosimetry efforts and european regulations for radiation safety could only have a moderate impact. gastrointestinal tumors include a variety of lesions, with the most frequent being adenocarcinoma of the small and large intestine, small bowel lymphoma, neuroendocrine tumours (nets) and gastrointestinal stromal tumours (gists). according to histology, location and initial imaging staging, those lesions may require completely different therapies: surgery alone, adjuvant chemotherapy (cht), neoadjuvant cht followed by surgery or a combination of neoadjuvant chemoradiotherapy (crt) followed by surgery. after treatment, imaging follow-up is mandatory. the most common post-operative findings after small bowel or colonic resections will be presented, together with clues for early detection of recurrence. in those cases which underwent adjuvant cht, imaging findings and diagnostic criteria related to the use of either cytotoxic or cytostatic drugs will be shown, to facilitate the interpretation of radiologists in assessing response to therapy. finally, tissue changes following neoadjuvant crt will be discussed, particularly in rectal cancer. the possibilities of different imaging modalities in assessing either complete or partial response to therapy will be presented, with a critical analysis of different imaging findings. unlike infectious enteritis, in patients with crohn's disease (cd) the changes in clinical activity have poor correlation with changes in inflammatory lesions after medical treatment. mucosal healing (mh) and improvement in patient's symptomatology are usually considered the main objectives of medical treatment. however, improvement in clinical symptoms is not always associated with mucosal healing and the persistence of severe inflammatory lesions augurs a more aggressive disease course in terms of flare-ups, hospitalisation requirements and the need for surgery. endoscopy is the gold standard for the assessment of luminal lesions in cd, but given the good correlation shown between endoscopy and cross-sectional imaging in assessing the activity and severity, imaging is increasingly introduced as a tool to monitor medical treatment. there is some evidence indicating that crosssectional imaging can be considered a responsive and reliable tool as it detects meaningful changes in patients' status over time after therapeutic interventions. besides this, cross-sectional imaging is capable of monitoring the colon and the small bowel and penetrating lesions that cannot always be assessed by endoscopy. becoming familiar with the radiological changes during and after treatment will be key in the decision-making process. degeneration is routinely defined as a morbid change of cells, tissues and even organs. in the msk system degeneration occurs in bones, joints (synarthrosis and diarthrosis), muscles and tendons. primary degeneration implies ageing as well as overuse, based on mircrotrauma. the latter may be related to constitutional, professional or sports-related factors. a distinction between ageing and overuse is not possible with the use of histology and imaging. only the intensity of the changes, which may be inadequate in relation to the age of the patient, may give a hint. on the contrary, the term "secondary degeneration" should be used for clearly defined events or diseases, which finally will result in the degeneration of tissues. the causes are inflammation (ra, infection), metabolic disease (e.g. cppd) or trauma. other ways to classify degeneration of the msk system are related to the anatomy. accordingly, degeneration of the particular parts of the peripheral or axial skeleton, muscles and tendons has to be discussed. one has to have in mind that bones, cartilage, muscles and tendons form a complex unit. the single elements of these complex units jointly undergo degenerative deterioration, e.g. bone, cartilage, fibrocartilage and capsule are jointly involved in case of oa of the knee joint. the same holds true for the synchondroses of the spine. at the rotator cuff not only the tendons, but also the underlying bone and the muscle tissue are changed. (o.m.vandelden@amc.uva.nl) there are many different types of embolisation, but all share many basic principles. the specific approach, technique, materials and equipment used may differ depending upon many variables such as the type of vascular territory involved (venous, arterial, small vessels, large vessels), clinical setting (elective or emergent procedure), and type of underlying disease to be treated (e.g. tumor embolisation, acute haemorrhage, vascular malformation, arteriovenous fistula, etc). pre-procedural imaging and planning are essential for most embolisation procedures and intra-procedural navigation can be done with fluoroscopy, dsa, and new d-techniques. with proper imaging workup prior to embolisation procedure time, radiation dose and contrast load can be significantly reduced. embolisation materials can be roughly divided into liquid agents (alcohol, glue, polymers), particulate agents (pva, calibrate microspheres, drug-eluting beads) and coils (detachable, non-detachable) and plugs (amplatzer®-plugs). plugs and coils are deployed at the exact site of destination and usually easy and safe to use. particles and liquid agents reach their site of destination by flow guidance and can be more difficult and unpredictable in their use. complications include puncture-site complications (thrombosis, dissection, haematoma), systemic complications (contrastinduced nephropathy, allergic reactions) and specific embolisation-related complications (non-target organ embolisation, end-organ ischaemia, postembolisation syndrome). when used with expertise and proper experience, most types of embolisation have good results with acceptable complication rates. although there has been advancement in imaging techniques, several pitfalls in the assessment of diseases of the pancreas, small bowel and rectum remain. the differentiation and the correct characterisation of tumours and inflammatory disease in some cases may be difficult. also, atypical presentation of common tumours or uncommon tumours persists and is a challenge. problematic situations are constantly represented as intestinal bleeding and unexpected findings. in this session, we point out the procedure of the best imaging modality and protocols of challenging cases. a. pancreas c. triantopoulou; athens/gr (ctriantopoulou@gmail.com)pancreatic imaging and interpretation of focal lesions remains a challenging issue. despite the advances in imaging techniques and the dedicated protocols that are now in use, a possibility for pitfalls remains. these pitfalls are related either to the inability of the early recognition of a lesion, or to the wrong characterisation of a "mass". both pitfalls are of great importance, taking into account the lethal pancreatic cancer and the possible complications that may follow an unnecessary pancreatic surgery. there are many variants of the pancreatic parenchyma, the ducts or even vessels, and deep knowledge of the pancreatic anatomy and embryology is needed, to be able to recognise these variations in imaging. a pancreatic variant may not only be misinterpreted as a severe pancreatic disease (e.g. ectopic spleen vs neuroendocrine tumour), but may also be the underlying cause of pancreatic inflammatory lesions (e.g. ectopic pancreas on the duodenal wall causing groove pancreatitis). other pitfalls are related to the atypical presentation of a common lesion (e.g. isoattenuating pancreatic adenocarcinoma), the presence of uncommon tumours, diffuse or multifocal diseases, the co-existence of two different entities and the heterogeneous or overlapping appearance of cystic lesions. in any challenging case, a focused methodology should be applied and the diagnostic procedure based on predefined imaging parameters, taking also into consideration the clinical and laboratory findings. excluding pancreatic cancer should be the first goal and every attempt should be made to differentiate between surgical and non-surgical cases. endoscopy is the gold standard in small bowel diseases, but it is a challenging technique: the pillcam is a commonly used tool and multidetector ct is a diffuse investigation technique. ct enteroclysis/enterography is a worldwide tool in intestinal exploration. throughout the investigation of the small bowel, morphologic anomalies or embryonal development defects may be present (meckel's diverticulum, duplications of intestinal tracts). artefacts may be determined and there may be misunderstanding. mdct is the first tool in emergency diagnosis; imaging pitfalls are possible. unsuspected diseases may be revealed: we will see a case of gist in a patient affected by intestinal bleeding of an obscure origin. ct enteroclysis may find unknown or unsuspected diseases: correct technical parameters have to be respected, as artefacts may be derived from an improper acquisition technique, such as a limited distension or poor intravenous enhancement. in follow-up, mr is preferred to investigate the intestine, mainly in the young population. unexpected findings may be due to rare pathology: we met a case of endometriosis, with ambiguous presentations; patients affected by autoimmune pathologies (pyoderma gangrenosum or lupus erythematosus), with unknown inflammatory intestinal chronic diseases. in oncology, pet-ct imaging is the main diagnostic tool, but there are abdominal pitfalls that limit its effectiveness (intestinal hot spots, artefacts from peristalsis): the small bowel may frequently obstruct an easy diagnosis. in case of emergency, if the radiological diagnosis is not clear, the surgeon determines the management of the patient. in case of a defective study technique, a correct examination may be repeated. endoscopy or pillcam may help to detect mucosal lesions invisible at ct. mri plays a key role in the assessment of disease of the rectum and in particular for the staging (and restaging) of rectal cancer. in this session, the relevant mr anatomy of the rectum will be discussed. furthermore, the strengths and weaknesses of mri in the assessment of rectal cancer during primary staging and restaging after chemoradiotherapy will be highlighted using various rectal cancer cases. finally, we will discuss some rare tumours of the rectum (including gist and carcinoid). optimisation of ct and mr techniques has provided new challenges for imaging in the assessment of airway and lung diseases. phenotypic abnormalities which can be recognised on visual and quantitative evaluation of ct images in copd patients may improve the diagnostic accuracy, help optimise treatment and provide a framework for clinical trials. although expiratory ct helps assess air trapping due to small airway obstruction and excessive dynamic collapse of large airways, potential tricks in technique and interpretation of ct images must be known. owing to multiple and successive improvements over years, functional and morphologic evaluation of the lung using mri has become reality for clinical practice. already regarded as a favoured cross-sectional imaging in paediatric chest radiology, mri of the lung is gaining new indications also in lung diseases occurring in adults. the potential for high-resolution computed tomography (hrct) to uncover several morphological subtypes that come under the umbrella term chronic obstructive pulmonary disease (copd) is now more familiar to both radiologists and specialist respiratory physicians. a complete approach to the classification of copd would ideally assimilate several parameters through a combined visual-quantitative hrct analysis. the insight that some subjects given the label of copd have "pure" airways diseases or emphysema can be readily provided by a simple visual evaluation. the subtypes -centrilobular, panlobular, and paraseptal emphysema -can be reliably distinguished on hrct images. the visual assessment of bronchial abnormalities and accompanying smoking-related interstitial lung disease may also complete the phenotypic classification of copd. furthermore, it is now possible to objectively quantify the global extent of emphysema, gas-trapping, and bronchial metrics by two types of softwares which are now increasingly available on latest ct workstations. learning objectives: . to learn about the classification of copd. . to appreciate the role of hrct in the classification of copd. a- : this presentation will examine the role of expiratory ct in the diagnosis of small and large airways diseases. small airways disease or air trapping may be a feature of asthma, copd and hypersensitivity pneumonitis, and is typically characterised by a mosaic attenuation pattern on ct. tracheobronchomalacia and excessive dynamic airway collapse (edac) are large airways conditions that may be recognised by tracheal narrowing on inspiratory and/or expiratory ct. the typical clinical and ct manifestations of these diseases will be reviewed. additionally, a number of important caveats with regard to ct in small and large airways disease will be discussed: ) the comparative strengths and limitations of ct, bronchoscopy and lung function testing in making the diagnosis; ) the overlap in appearances that exists between diseased and healthy individuals; ) the variability in definitions of conditions such as tracheobronchomalacia. the optimal ct technique for imaging of airways disease, including the role of dynamic expiratory ct, will also be reviewed. for routine clinical applications, a standardised mri protocol has been widely implemented. it addresses the major challenges of mri of the lung, i.e. low proton density, susceptibility artefacts as well as respiratory and cardiac motion. beyond visualisation of lung morphology, which is done in an inspiratory breath-hold, functional imaging has become an integral part of the routine protocols: perfusion, blood flow, ventilation, respiratory motion, diffusion. for perfusion imaging, inspiratory or expiratory scans can be used, blood flow measurement should be performed during shallow breathing, and ventilation and diffusion are mainly assessed in inspiration. respiratory motion is best assessed by dynamic image acquisitions. continuous breathing can also be combined with triggering or navigators to improve image quality or derive functional maps. mri is recommended as the first-line cross-sectional imaging modality in paediatric chest radiology, including cystic fibrosis, complicated pneumonia, any pulmonary or cardiovascular anomaly, as well as the assessment of mediastinal masses in children and adults. mri is also challenging pet/ct in the staging of lung cancer (tnm) as well as ct and echocardiography in the assessment of pulmonary arterial hypertension. mrieven without contrast -is an alternative in the diagnosis of acute pulmonary embolism if iodinated contrast medium is contraindicated or in pregnant women. mri should also be used in complicated pneumonia, e.g. in immunocompromised patients as well as for the functional assessment of chronic obstructive pulmonary disease (copd) or severe asthma. the role of mri in interstitial lung disease stills needs to be determined. unlike the simple forms of cranisynostosis whose modalities and timing of surgical correction are well established, the management of faciocraniosynostoses requires a multispecialistic and repeated evaluation of the single patients. their evolution, in fact, depends on the specific genetic anomalies but also on the phenotype, which can vary in time, of their clinical expression which may lead to different degrees in severity of the associated functional disturbances (csf dynamics impairment, visual deficits, respiratory anomalies, etc). consequently these conditions may require several surgical steps the timing of which are dictated by the exact clinical diagnosis and the evaluation of the functional status at different ages. current management of faciocraniostenoses is thus based not only on the radiological studies but also on the functional assessment of the brain function (e.g. mri imaging studies, metabolic and cerebral blood circulation investigations, respiratory pathways volume evaluation, sleep recording). today's surgical multidisciplinary management may benefit a large variety of techniques which extend from free bone cranioplasty with intraoperative active fixed expansion to more dynamic and less invasive methods which exploit the physiological brain expansion in infants or the gradual cranial vault expansion mechanically stimulated by springs or distractors. in the present report, we will review the decision making process and the rationale on which, at the necker-enfants malades, we base the use of the currently available techniques for skull expansion and correction of cranial malformations according to the accurate dynamic radiological and functional evaluation at different stages of the disease. learning objectives: . to learn about the imaging patterns of faciocraniosynostoses. . to learn how and when to image. . to become familiar with associated brain anomalies. b. all about the paediatric pituitary gland m.i. argyropoulou; ioannina/ gr (margyrop@cc.uoi.gr) mr is the imaging modality of choice for the assessment of the pituitary gland (pg) and the hypothalamus. the normal adenohypophysis is bright during the first two months of life and appears isosignal to the brain parenchyma afterwards. the neurohypophysis is bright, provided that the child is well hydrated. the pg height decreases during the first year of life and then increases until puberty. adenohypophyseal deficiency has been associated with a small pituitary size, an ectopic neurohypophysis, a hypoplastic or absent pituitary stalk, hypothalamic gliomas, craniopharyngiomas and iron overload states. precocious puberty may be idiopathic, but it has also has been associated with hypothalamic hamartomas, craniopharyngiomas and hydrocephalus. diabetes insipidus may be secondary to histiocytosis x, germinomas and trauma causing pituitary stalk interruption. the hypothalamopituitary axis is evaluated by using sagittal and coronal t -weighted images without and with contrast administration. dynamic contrast enhancement may be useful in the evaluation of disorders responsible of diabetes insipidus. magnetisation transfer imaging may be useful in the evaluation of pituitary deficiencies or precocious puberty. familiarity with orbital and ocular anatomy is crucial to the understanding of disease processes of the orbit. lesions of the orbit may be divided into those which are intraocular and those which are extraocular. the underlying aetiology and geneses of many different lesions that can occur in the orbits vary depending on the location of the lesions and sometimes on additional nonorbital conditions. ct and mri play crucial roles in the evaluation of orbital pathology where often mri still is a complement to ct examination in the evaluation of orbital lesions familiarity with the radiologic appearance of common orbital lesions is important, as many of these lesions will not be seen on physical examination. after a very brief anatomic overview, most of this lecture will focus on the more common benign and malignant lesions as well as differential diagnosis involving the orbit and lachrymal gland in both the paediatric and adult population. the lecture will also present suggested imaging protocol and standard of care with respect to imaging. learning objectives: . to understand the embryology and imaging findings of the most common malformations. . to learn about space-occupying lesions and the differential diagnosis of tumours and inflammatory conditions. . to be aware of the role of conventional and advanced mr sequences in the diagnostic approach to lesions in the orbit. room e the hand and wrist injury to the wrist and hand is an important clinical problem. first, such injury is relatively common. second, the spectrum of abnormalities is complicated. the purpose of this presentation will be to learn more about the imaging appearances of soft tissue and osteoarticular injury of the wrist and hand using multiple imaging techniques. emphasis will be placed upon pathomechanics and characterisation of greater and lesser arc injury patterns of the wrist. the various posttraumatic instability patterns of the wrist will be also discussed. rheumatoid arthritis (ra), psoriatic arthritis (psa) and other inflammatory disease can be diagnosed and sometimes differentiated in the early stages of the disease on the basis of mri and/or pdus features of the hand and wrist. rheumatoid arthritis (ra) activity is closely correlated with inflammation. the synovial membrane is the principal site of inflammation in which the inflammatory process enhances capillary perfusion and permeability. doppler ultrasonography (dus), using the amount of colour pixels in the region of interest, and dynamic magnetic resonance imaging (de-mri) are both able to detect this inflammation in the wrist and hand. although these techniques are both capable of monitoring synovium inflammation modifications after ra treatment, pdus has become an essential tool for ra joint monitoring in routine practice in view of its sensitivity in the detection of synovitis, feasibility in outpatient clinics, and low cost. (ellopis@hospital-ribera.com) radiological study of the wrist and hand is challenging due to its complex anatomy with many small structures and the number of normal bone and soft tissue variants that might mimic injuries. moreover, many findings can be asymptomatic. their knowledge is important to avoid misdiagnosis. during this lecture we will also review the role of the different imaging modalities, such as plain films for wrist alignment and bone structures as well as the important role of us and mr in differentiating tumour from tumour-like conditions. we will become familiar with some specific radiological findings that allow us to make accurate diagnoses of soft tissue and bone lesions. learning objectives: . to learn more about the spectrum of intra and para-articular soft tissue tumours, and soft tissue tumour-like lesions. . to become familiar with us and mri findings of specific soft tissue lesions. room e oncologic imaging chairman's introduction c. pfannenberg; tübingen/ de (christina.pfannenberg@med.uni-tuebingen.de) with recent advances in cross-sectional imaging, the frequency of detecting "incidental findings" has markedly increased. incidental findings (=incidentaloma) are defined as unexpected, asymptomatic abnormalities detected by imaging performed for an unrelated reason. these incidentalomas have created a management dilemma for both radiologists and clinicians, particularly in the cancer patient in whom any mass warrants further evaluation. discovery of incidental findings often leads to a cascade of additional tests that is costly, provokes anxiety and exposes patients to the risk of unnecessary radiation and intervention. the workup of incidentalomas varies widely by physicians, and strategies for optimising patient management are only beginning to emerge. in this course, guidelines concerning a rational approach to some of the more common incidental abnormalities in cancer patients are presented. basic principles and tools for interpretation of incidental findings, common pitfalls and protocol issues regarding the differentiation of benign and malignant lesions as well as treatment-induced abnormalities will be addressed with a focus on the liver, lung and skeletal system. at the end of the session, the speakers will discuss the role of functional imaging techniques for characterisation of incidental lesions. session objectives: . to become familiar with basic principles and common pitfalls in interpretation of incidental findings in the lung, abdomen and skeletal system. . to learn how functional imaging (dwi, pet/ct, bs) can help in lesion characterisation. . to provide attendees with clear and practical messages for the management of the most common incidental findings in cancer patients. a. abdomen: common pitfalls and protocol issues a. ba-ssalamah; vienna/ at (ahmed.ba-ssalamah@meduniwien.ac.at) an incidentaloma is quite a frequent diagnosis in medicine, in general, and in radiology, in particular. by definition, an incidentaloma is an abnormality or a tumour found by coincidence without clinical symptoms or suspicion. these incidental findings have long posed challenges to physicians, and particularly to radiologists as well as health-care providers due to the resulting high costs. the scope and scale of these challenges have increased with the introduction of new technologies, in particular cross-sectional imaging, such as mdct and mri using sub-millimetre thin slices. incidentalomas still cause a management dilemma for clinicians, radiologists and even for the patients themselves. this dilemma is particularly pertinent to oncology patients. therefore, a precise knowledge of the broad spectrum of incidental findings is crucial. based on this knowledge, we can choose the appropriate radiologic examination that will provide a confident diagnosis. this way, needless uncertainty, for both the patient and the physician in charge, is avoided. furthermore, invasive procedures, such as biopsies, with the potential for complications, as well as cost-intensive follow-up examinations, can be reduced. furthermore, mri is usually used to detect the side effects of chemotherapy of the liver. simple steatosis is usually reversible, unless the liver receives a 'second hit' of damage from other causes leading to other manifestations, including chemotherapy-associated steatosis, sinusoidal obstructive syndrome, nodular regenerative hyperplasia, veno-occlusive disease, peliosis, pseudo-cirrhosis, and sclerosing cholangitis largely due to microvascular injury. finally, the effects of chemotherapy on peritoneal and mesenteric structures after performing intraperitoneal chemotherapy will be discussed. this presentation will discuss the complex topic of incidental findings in thoracic imaging of oncological patients. incidental findings will be presented with respect to their respective organs and anatomical regions. the presentation will emphasise the clinical relevancy of the findings as well as the specific of risk estimation in oncological patients. finally, the presentation will discuss the usefulness (or lack thereof) of current management guidelines for incidental findings, as designed for the general population. illustrative clinical scenarios will be discussed. radiographs and ct often detect incidental observations in bones, corresponding either to normal variants or benign conditions, which require additional imaging to rule out malignant conditions. mri most often provides nonambiguous explanation for these observations. beside this, mri, targeting either the whole body or only the axial skeleton, often including diffusionweighted (dwi) sequences, is increasingly used for bone tumour detection in many "osteophilic" cancers and haematologic malignancies, without need for contrast material injection and without irradiation, with unparallelled sensitivity to bone lesions. an important advantage and also a challenge for musculoskeletal radiologists are the all organ screening capabilities of dwi, demanding careful study of the huge information provided and knowledge extending far beyond bones in terms of organs and metastatic spread of different cancers. a second major advantage is its ability to detect lesion changes under therapy. however, benign lesions and non-neoplastic conditions, as well as artefacts may also lead to confusing observations on mri, and particularly on dwi. anatomical mr sequences as well as other imaging modalities are of utmost help to recognise these pitfalls. whole body mri including dwi sequences has to position itself among other diagnostic tools, bone scan, spect, and especially pet with its variety of tracers, in the currently evolving strategy of bone screening techniques, which will most likely vary according to the primary cancer. learning objectives: . to present most frequent x-ray and ct pseudo lesions and benign conditions and show how mri often enables straightforward interpretation of these abormalities. . to highlight the strengths of mri with diffusion weighted imaging (dwi) for malignant lesion detection and characterisation within bones, but also beyond bones. . to highlight the typical appearance of malignant lesions on projectional and cross-sectional imaging. . to learn to differentiate these lesions from normal marrow variations, stress lesions, bony pseudo-lesions and other benign conditions, and to become familiar with treatment induced changes within lesions and their environment. . to situate mri amongst other functional imaging techniques (pet, spect, ). colorectal cancer is the rd most commonly diagnosed cancer in the world with % of cases diagnosed in the developed world. with an estimated . million new cases clinically diagnosed and over , people killed worldwide by this disease on an annual basis, colorectal cancer is a true public health concern. survival of colorectal cancer is directly related to the extent of disease and specifically presence of liver metastases. imaging plays a key role in the initial staging of colorectal cancer and is the gold standard in evaluating extra colonic disease, primarily liver metastases. imaging is also widely used for therapy monitoring and staging. ct, mri, pet/ct and pet/mri are the key imaging modalities. in addition, tumour response therapy assessed with morphological and functional biomarkers is increasingly used by advanced gastrointestinal oncologic programs. furthermore, image-guided therapy is widely used for patients with unresectable lesions. liver colorectal metastases were the first liver metastases to be treated with image-guided ablation techniques. a gamut of ablative techniques exists ranging from transarterial embolisation, radiofrequency ablation, highly intensified focused ultrasound, brachitherapy etc. in short, diagnosing, characterising and finally assessing tumour response after neoadjuvant and image-guided therapy are all functions where imaging plays a pivotal role. this course is divided into three logical segments dealing with current treatment options, morphological and finally functional biomarkers and it is followed by a panel discussion which includes audience participation. a. current treatment options t.k. helmberger; munich/de (thomas.helmberger@klinikum-muenchen.de) hepatic metastases in colorectal cancer may occur in % to % of the cases. considering the general oncological (isolated hepatic tumour load; prognostic benefit), and technical (size, number, location of hepatic metastases; expected hepatic functional reserve) framework, surgical resection is still considered to be the method of choice -even if this statement never had been verified by rtcs. nevertheless, in clinical reality only - % of patients with liver metastases may qualify for resection. in consequence, the majority of patients need other or at least modified therapeutical pathways including adjuvant or neoadjuvant chemotherapy and more and more image-guided local ablative therapies. the latter encompass chemo-(transarterial chemoperfusion/-embolisation), thermo-(radiofrequency-, laser-, microwave ablation, high intensified focused ultrasound), and radio-ablative (radio embolisation, interstitial brachytherapy, etc). techniques. particularly, thermalablative techniques gained wide acceptance over the last years, since ample evidence could be presented showing that this method can be applied as primary and also complementary therapies in resectable and non-resectable metastatic disease. furthermore, recent data confirm that in multimodality therapy concepts, progression free survival and overall survival in patients with primarily unresectable and with unfavourable prognosis is comparable to surgery with -year survival rates more than %. minimally invasive, imageguided therapies will not replace surgical resection; however, these therapy modalities are eligible in a large number of cases and should be implemented consequently in multimodality treatment regimens according to the interdisciplinary consensus of oncologists, interventional radiologists, and surgeons. colorectal liver metastases are typically identified as low attenuation lesions on portal venous phase ct. planning of optimal therapy depends on accurate localisation and characterisation of all focal liver lesions and this is best achieved with mri, which should include diffusion-weighted imaging and use of hepatocyte-specific contrast medium. the typical mri signature of colorectal metastases includes low signal on t w, moderately increased signal on t w, irregular peripheral arterial phase enhancement with low signal on portal venous phase, restricted diffusion and absent hepatocytes. morphological tumour response can be assessed with either modality. size-based systems for assessing tumour response are widely applied, with recist . the most widely used at present. recist . includes several modifications that make it more user friendly than recist . . more advanced morphological criteria have been described for new targeted and molecular therapies, including overall attenuation, the tumour-liver interface and the appearance of the peripheral rim of enhancement. as hepatic surgeons become more aggressive in their approach to resection in patients with liver metastases, it is important to understand that disappearance or calcification of liver metastases after treatment does not necessarily equate to a complete pathological response. the timing of imaging is therefore critical in assisting the surgeon to remove all previously affected hepatic segments after chemotherapy. learning objectives: . to learn about the algorithm for detecting and characterising liver metastases. . to understand conventional imaging criteria for assessing tumour response. . to learn about the rationale for monitoring patients after radical and palliative treatments. conventional size measurement criteria remain the most widely used method to determine the response of colorectal liver metastases to treatment. however, tumour size reduction is assessed relatively late (e.g. weeks after treatment) and new targeted treatment may be effective without reducing tumour size. new functional imaging techniques can be applied to quantify different aspects of tumour biology and to develop response, and predictive and prognostic biomarkers. we discuss the use of diffusion-weighted mr imaging, dynamic contrast-enhanced mr imaging and fdg-pet imaging in the evaluation of treatment response in patients with colorectal liver metastases. digital breast tomosynthesis (dbt) is a promising technique for breast imaging based on a full-field digital mammography (ffdm) platform. the x-ray tube moves through a proscribed arc, and several low-dose projection images are acquired. images are reconstructed into a stack of mm slices. the radiation dose is about the same or slightly higher than for ffdm. the most important advantage of dbt is the elimination of superimposed tissue, which improves detection of lesions otherwise hidden by dense breast parenchyma and reduces the interpretation problems caused by overlapping tissue. dbt is suggested to improve sensitivity as well as specificity in women with dense breast. dbt is superior to ffdm in tumour size assessment and for mass visibility and cancer conspicuity. dbt is comparable to ffdm for evaluation of microcalcifications. dbt is superior for the detection of spiculated masses and architectural distortion. the potential to improve sensitivity and specificity is of interest for screening. an open question is whether dbt should be applied in one or two views. two-view dbt would mean a doubling of radiation dose, but a solution is the implementation of synthetic d images reconstructed from the d dataset of dbt. the synthesised images are created by summing and filtering the stack of reconstructed dbt slices. using synthetic d allows "combo mode" to be implemented in screening with the same radiation dose as for conventional d. results using dbt in breast cancer screening are very promising. different study design of published studies may explain the variations of results. magnetic resonance imaging of the breast provides a multitude of techniques for detection of breast cancer and differentiation of benign from malignant lesions. in the last few years, significant improvements of scanner hardware and equipment in terms of coils as well as sequence software have been achieved. to date, multiparametric quantitative measurement of perfusion, diffusion and tissue chemistry is possible at field strengths up to tesla. although higher field strengths and improved sequence protocols provide higher signal and allow faster imaging, specific technical issues have to be considered to avoid artefacts and pitfalls. this talk aims to demonstrate the possibilities and limitations of multiparametric and (ultra-)/high-field mri. furthermore, these new techniques are put into the broader clinical context to determine the potential value for breast lesion detection and differentiation. positron emission mammography is a recently developed imaging device using positron emission technology. after injection of a positron-emitting radiotracer, the radiation is detected by a dedicated high-resolution camera, providing high spatial resolution. commercially available systems include cameras where the breast is compressed and which display a d image similar in its layout to a mammogram and hanging breast devices where a d image similar in its layout to mri is produced. these devices have been compared to pet/ct and have shown higher spatial resolution, accuracy and sensitivity in detecting malignant breast lesions. the most widely studied tracer in pet/ct and in pem is fluorodeoxyglucose (fdg). fdg is a glucose analogue and has had a major impact on oncology imaging with pet/ct. breast carcinomas demonstrate increased glucose consumption and the intensity of the uptake has been correlated with prognosis, hormonal receptor and her status, ki and nodal involvement. current potential indications for pem are detection and characterisation of breast lesions, local staging in patients with known breast cancer and baseline assessment prior to neoadjuvant chemotherapy. future developments of pem will involve new tracers and fusion with other breast imaging modalities. fluorothymidine is a proliferation tracer that seems promising in early assessment of response to chemotherapy. fluoroestradiol has the potential to image oestrogen receptor status in vivo. the possibility of pem-guided biopsy is a very exciting development and has the potential to select lesions with the highest intensity of uptake. fusion with mri, us and mammography will increase the diagnostic accuracy. the presentation will focus on two frequent findings on mri with increasing incidence during ageing, notably "unspecific" hyperintense lesions on t /flair and cerebral mircobleeds (cmbs) on t * or swi images. clinicoradiologic studies suggest that these t /flair lesions are associated with increased risk of cognitive decline, stroke and death. the clinical relevance of these lesions probably depends on periventricular versus deep white matter location. radiologic-histopathologic studies correspondingly indicate that the degree of histopathologically confirmed demyelination also depends on the location of these t /flair lesions. cerebral mircobleeds (cmbs) are punctate hypointense lesions on t * or swi images. while multiple cmbs are associated with diseases such as cerebral amyloid angiopathy (caa), clinicoradiologic studies demonstrate increased incidence of sporadic cmbs in mild cognitive impairment (mci) and alzheimer dementia, indicating a degrading effect of cmbs on cognition. on the other hand, radiologic-histopathologic studies show that not all black spots on t * or swi correspond to cmbs. cmb mimics include micro-calcifications. the presentation will demonstrate typical manifestations of these "unspecific" t /flair lesions and cmbs during ageing and provide tips for the interpretation of these lesions during daily clinical routine. with the increasing use of mri, it has become more common to discover incidental brain findings. these findings may be non-specific or have some morphological characteristics and geographical distribution highly suggestive of a demyelinating disease. the term "radiologically isolated syndrome" (ris) was introduced by okuda in to describe those asymptomatic patients with radiographic abnormalities highly suggestive of multiple sclerosis (ms). sometimes, these lesions may even meet the mri criteria for dissemination in space, which are currently used to predict future development or conversion to ms in patients presenting a clinically isolated syndrome (cis). however, we must not forget that these radiological criteria should be applied only when the patient had experienced any clinical manifestations suggestive of a demyelinating disease. therefore, what happens in those patients in whom we found highly suggestive lesions but have never had symptoms? due to lack of knowledge about the natural history or evolution of this new syndrome, clinical and therapeutic management of these patients is not well established, nor is the risk of conversion to ms. recent data, however, indicate that the presence of gadolinium-enhancing cervical cord and cortical lesions significantly increases the risk of conversion to cis or ms. all these data have improved the characterisation of ris subjects and in our understanding of risk factors for initial symptom development. incidental findings on brain imaging are defined as previously undetected abnormalities of potential clinical relevance that are unexpectedly discovered and unrelated to the purpose of the imaging. incidental findings are increasingly detected in clinical practice, with screening, and in the research setting. data on the prevalence of these abnormalities are scarce, the clinical course of the findings is often unknown, and the management of such lesions is not clear. the prevalence of incidental findings can be expected to vary depending on the purpose of the imaging exams. with improvements in imaging technology (higher field scanners, new pulse sequences), the number of detected incidental findings will increase dramatically. another important point is the advent of imaging biomarkers. advanced techniques for postprocessing and analysis, such as automated segmentation of brain structures or voxel based morphometry, will lead to the discovery of imaging biomarkers. once the predictive value of these markers has been established, most mri studies of the brain, both in the clinical and research setting, will reveal information that might be relevant for the well-being of patients or participants. although still incidental, these findings can unfortunately no longer be considered unexpected. we will soon face large medical, ethical, and practical problems as a result of technical improvements. in this lecture, the most important incidental findings and their prevalence will be reported. the management of incidental findings in the clinical and research setting will be discussed. finally, recommendations for follow-up will be provided. the primary assessment of laryngeal cancer is provided by endoscopy, which is accurate in delineating both the superficial extent and functional impairment. imaging techniques are recommended to grade submucosal invasion, local and distant spread. in the last few decades, open and endolaryngeal surgical procedures as well highly sophisticated rt techniques have been developed to treat the tumour while preserving critical laryngeal functions. for example, early glottic tumours (tis-t ) can be treated by endoscopic laser excision or rt. the integration of information acquired by endoscopy and imaging is essential for proper treatment planning. the radiologist has to know that the site of origin (supraglottis vs glottis or subglottis) and local extent (superficial and submucosal) are the key tumour-related factors in planning the type of treatment. submucosal invasion has a variable impact depending on the location of the primary tumour. the key issues in glottic cancers include the invasion of: the posterior paraglottic space, crico-arytenoid joint, and anterior commissure and contralateral true vocal cord, and vertical spread towards supra and/or subglottis, thyroid or cricoid cartilage. the key issues in supraglottic cancers encompass the invasion of: base of the tongue, preepiglottic space, piriform sinus, vertical spread into glottis and subsequent cartilage invasion (observed exclusively in transglottic cancers). functionsparing surgical techniques -as supracricoid laryngectomies -can be applied in advanced cancers when sparing of the posterior commissure, the ipsilateral crico-arytenoid joint, the mucosa investing the upper cricoid and the outer perichondrium of the thyroid cartilage is demonstrated by endoscopic and imaging mapping. the detection of recurrent laryngeal carcinoma is often challenging. to better appraise the expected findings after initial surgery of laryngeal cancer, the classical partial laryngectomy and laser laryngeal surgery are briefly presented.(chemo)radiotherapy has become an important treatment modality for laryngeal carcinoma. the recurrence rate in t -t laryngeal carcinoma is reported to be between % and %. the difficulty in differentiation between radiation reaction (such as oedema, fibrosis and soft tissue and cartilage necrosis) and recurrent disease is discussed. a histologic study on wholeorgan sections of recurrent glottic carcinomas is also presented: recurrent disease presents with different tumour spread than does a primary carcinoma, resulting in difficulties in estimating the extent of tumour recurrence. currently, selected recurrences of early glottic carcinoma are, whenever possible, treated by larynx-preserving salvage surgery. in those cases, a precise evaluation of the recurrent tumour extent is mandatory for planning larynx-preserving salvage surgery. this is especially true in critical regions such as the preepiglottic space, infiltration into cartilage, contralateral tumour spread and subglottic tumour extension. the accuracy of crt-classification is relatively low: many patients with signs and symptoms suggesting tumour recurrence present with post-radiotherapy changes; small tumor foci, often localised in the subglottic region, are undetectable by imaging studies. tissue changes in the neck treated by surgery and/or radiotherapy (rt) make the detection of residual or recurrent tumour more difficult. clinical evaluation of the neck is also hampered by these changes. rt-induced fibrosis and postsurgical scar tissue make palpation of the neck very difficult. endoscopy is hampered by endolaryngeal oedema which can persist for many months post-rt. therefore, any (non-invasive) method helping in the detection of recurrence is welcome. patients at risk for local failure after rt can be successfully identified by a post-rt ct (or mr) study between to months after rt. the optimal time point to perform such a 'baseline study' seems to be about to months post-rt. patients with indeterminate findings are candidates for 'imaging surveillance'; i.e. follow-up imaging every to months up to a period of years after rt. however, ongoing studies suggest that metabolic imaging (fdg-pet) may detect local recurrences with a higher accuracy than 'conventional' anatomically based imaging techniques, such as ct and mr. ct and/or mr-findings in the treated neck are frequently inconclusive. at present, new techniques are available to detect recurrent cancer. pet-ct is widely applied in the post-treatment setting. also, advanced mr-techniques such as diffusion weighted imaging (dwi) and dynamic contrast-enhanced mr (dce-mr) can be applied. focusing on the larynx, the advantages of these techniques as well as the limitations will be shown using imaging examples from daily practice. during the last years, several hundred new radiotracers for pet imaging of cancer have been developed and tested preclinically. these tracers are based on proteins (e.g. antibodies and antibody fragments), peptides (e.g. g proteincoupled receptor ligands) or small molecules (e.g. radiolabelled protein kinase inhibitors or metabolic substrates). using these radiotracers, the expression and function of a variety of proteins can be studied in patients, including for example glutamine/glutamate metabolism, neutral amino acid transport, psma, grpr, sstr, cxcr , folate and alpha-v beta- integrin expression. some of these radiopharmaceuticals are potential companion diagnostics and may allow selection of patients for treatment with radiopharmaceuticals or targeted drug conjugates. preclinical and initial clinical data are promising for several new radiotracers, but the key challenge is the clinical validation and dissemination of these compounds. validation and dissemination have been hampered so far by regulatory hurdles as well as by the lack of standardised trial designs for the validation of imaging agents and the lack of accepted and appropriate end points to prove clinical utility. addressing these issues will be critical for the future clinical use of pet and other molecular imaging technologies. imaging targets in cancer range from simple size measurements to more specific biomarkers on functional, cellular, metabolic and molecular levels. as our understanding of basic tumour biology has advanced, techniques have been developed to exploit this information to produce increasingly specific molecular imaging tools. the biodistribution of these molecular imaging probes should be more specific in diagnosing and assessing cancer than the morphological information acquired using anatomical imaging alone. this lecture will discuss current and emerging functional and molecular imaging techniques using mri and their applications in oncology. functional measures of tumour blood flow and vascular permeability can be made using dynamic contrast-enhanced mri. diffusion-weighted imaging is a surrogate for the cellular content of the tumour and emerging methods can be used to probe features of the extracellular space such as tumour ph and stromal content. on the molecular level, cell surface expression of specific proteins and enzyme activity within the cell can be imaged; labelled probes have been developed which bind to these proteins and a new mr technique is being developed for assessing tumour glucose in a similar way to pet. hyperpolarisation methods are emerging to overcome the major limitation of mr: low sensitivity. one such approach is dynamic nuclear polarisation, which can probe carbon metabolism non-invasively in patients with cancer. functional and molecular imaging techniques with mri will increasingly be used in radiology in conjunction with anatomical imaging methods to improve diagnosis and prognosis, target biopsies, as well as predict and detect response to treatment. with mr-spectroscopy, diffusion-weighted imaging, pet-and spect-ct, as well as pet-mri, molecular imaging has already become part of clinical routine diagnosis. also, some contrast agents such as spio for mps-labelling and scavenger-receptor binding gadolinium chelates are clinically applied for liver and lymph node imaging. however, there are still a number of highly promising novel tools that are expected to emerge clinically in the near future. in this context, the first part of the talk will give an overview on how optical imaging basically works and which future clinical applications can be expected. in this context, raman spectroscopy, fluorescence optical reflectance imaging, fluorescence mediated tomography, photoacoustic imaging and cerenkov luminescence imaging will be addressed. the second part of the talk introduces molecular ultrasound imaging as a safe and preclinically wellevaluated method. besides the diagnostic use of molecular ultrasound contrast agents, which have been already evaluated in clinical trials, the potential of this method also for theranostic purposes will be highlighted. the assessment of the amount of arterial calcification with computed tomography is a standard method in the risk stratification of coronary heart disease. coronary calcium detection by ct has been shown to identify atherosclerotic plaque and to quantitatively assess coronary calcium. many studies have demonstrated the association between the degree of coronary calcium, the burden of atherosclerosis, and the risk for cardiovascular events associated with coronary calcium. the cac scoring can provide individual risk assessment and reclassify the low and particularly intermediate framingham risk cohort into lower-and higher-risk strata. the absence of cac is associated with a very low risk of future cardiovascular events, with modest incremental value of other diagnostic tests in this very low-risk group. the role of cac in the workup of symptomatic patients is under discussion and there is evidence that the absence of cac has a high negative predictive value for ruling out acute coronary syndrome. although cac measurement is highly standardised, some technical aspects have to be considered and all possibilities of dose reductions utilised. this presentation will show the technical and epidemiological fundamentals of cac scoring and discuss the clinical applications of the method. coronary cta has gained an important role in the evaluation of patients with chest pain, suspected of coronary artery disease (cad). in comparison to invasive coronary angiography, coronary cta with -multidetector ct has an excellent sensitivity for coronary stenosis. specificity for stenosis is less excellent, but has improved with newer ct generations. the strongest impact of coronary cta has been on the exclusion of stenosis in symptomatic patients at low-intermediate pre-test probability. in contrast to invasive angiography, coronary cta yields more information than just the severity of stenosis. coronary cta also allows determination of plaque types (calcified, non-calcified, partly calcified) and quantification of the atherosclerotic plaque burden. also, new developments point to the possibility to assess the haemodynamic significance of stenosis based on ct density measurements in the coronary arteries, or evaluation of myocardial perfusion. thus, coronary cta yields quantitative imaging biomarkers that could be used for risk stratification in the future. recent studies have shown that whether or not a patient has stenosis and plaque, as well as the type and burden of plaque, affects the risk of myocardial infarction and mortality. therefore, the impact of coronary cta imaging biomarkers reaches beyond the mere assessment of stenosis for symptomatic treatment and could potentially alter medical management of patients for improvement of long-term prognosis. a- : c. added value of carotid and peripheral artery imaging for risk assessment l. natale; rome/ it (lnatale@rm.unicatt.it) technical protocols optimisation of mra and cta of carotid and peripheral arteries is crucial for image quality and to complete the analysis of both lumen and vessel wall. furthermore, mra protocol needs to be integrated with dedicated pulse sequences in order to obtain vessel wall imaging that is already included in cta datasets. new ct and mr scanners allow this optimisation in terms of radiation exposure, contrast media dose reduction and spatial resolution. all these parameters will be discussed for both techniques. according to international guidelines, non-invasive imaging indications will be discussed, with particular attention to high risk population (e.g.: diabetic patients). some possible imaging biomarkers of plaque composition will be discussed. learning objectives: . to become familiar with optimal technical considerations for performing cta and mra of the carotid and peripheral arteries. . to understand clinical indications for carotid and peripheral cta and mra. . to discuss the role of cta and mra parameters including plaque imaging as biomarkers of cardiovascular disease. key: cord- - l mua authors: menotti-raymond, marilyn; o’brien, stephen j. title: the domestic cat, felis catus, as a model of hereditary and infectious disease date: journal: sourcebook of models for biomedical research doi: . / - - - - _ sha: doc_id: cord_uid: l mua the domestic cat, currently the most frequent of companion animals, has enjoyed a medical surveillance, as a nonprimate species, second only to the dog. with over hereditary disease pathologies reported in the cat, the clinical and physiological study of these feline hereditary diseases provides a strong comparative medicine opportunity for prevention, diagnostics, and treatment studies in a laboratory setting. causal mutations have been characterized in felid genes, with the largest representation from lysosomal storage enzyme disorders. corrective therapeutic strategies for several disorders have been proposed and examined in the cat, including enzyme replacement, heterologous bone marrow transplantation, and substrate reduction therapy. genomics tools developed in the cat, including the recent completion of the -fold whole genome sequence of the cat and genome browser, radiation hybrid map of integrated coding and microsatellite loci, a -cm genetic linkage map, arrayed bac libraries, and flow sorted chromosomes, are providing resources that are being utilized in mapping and characterization of genes of interest. a recent report of the mapping and characterization of a novel causative gene for feline spinal muscular atrophy marked the first identification of a disease gene purely from positional reasoning. with the development of genomic resources in the cat and the application of complementary comparative tools developed in other species, the domestic cat is emerging as a promising resource of phenotypically defined genetic variation of biomedical significance. additionally, the cat has provided several useful models for infectious disease. these include feline leukemia and feline sarcoma virus, feline coronavirus, and type c retroviruses that interact with cellular oncogenes to induce leukemia, lymphoma, and sarcoma. mankind has held a centuries-long fascination with the cat. the earliest arch eological records that have been linked to the domestication of felis catus date to approximately years ago from cyprus, with recent molecular genetic analyses in our laboratory suggesting a middle eastern origin for domestication (c. driscoll et al., unpublished observations) . currently the most numerous of companion animals, numbering close to million in households across the united states (http://www.appma.org/ press_industrytrends.asp), the cat enjoys a medical surveillance second only to the dog and humankind. in this chapter we review the promise of the cat as an important model for the advancement of human hereditary and infectious disease and the genomic tools that have been developed for the identification, and characterization of genes of interest. for many years we have sought to characterize genetic organization in the domestic cat and to develop genomic resources that establish f. catus as a useful animal model for human hereditary disease analogues, neoplasia, genetic factors associated with host response to infectious disease, and mammalian genome evolution. , to identify genes associated with inherited pathologies that mirror inherited human conditions and interesting pheno-types in the domestic cat, we have produced genetic maps of sufficient density to allow linkage or association-based mapping exercises. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the first genetic map of the cat, a physical map generated from a somatic-cell hybrid panel, demonstrated the cat's high level of conserved synteny with the human genome, which offered much promise for the future application of comparative genomic inference in felid mapping and association exercises. several radiation hybrid (rh) and genetic linkage (gl) maps have since been published. [ ] [ ] [ ] [ ] [ ] [ ] , , although previous versions of the cat gene map, based on somatic cell hybrid and zoo fish analysis, , revealed considerable conservation of synteny with the human genome, these maps provided no knowledge of gene order or intrachromosomal genome rearrangement between the two species, information that is critical to applying comparative map inference to gene dis covery in gene-poor model systems. radiation hybrid (rh) mapping has emerged as a powerful tool for constructing moderate-to high-density gene maps in vertebrates by obviating the need to identify interspecific polymorphisms critical for the generation of genetic linkage maps. the most recent rh map of the cat includes markers: coding loci, selected markers derived from the cat x whole genome sequence targeted at breakpoints in conserved synteny between human and cat, and short tandem repeat (str) loci. the strategy used in developing the current rh map was to target gaps in the feline-human comparative map, and to provide more definition in breakpoints in regions of conserved synteny between cat and human. the markers cover the length of the feline autosomes and the x chromosome at an average spacing of one marker every . mb (megabase), with fairly uniform marker density. an enhanced comparative map demonstrates that the current map provides % and % comparative coverage of the human and canine genomes, respectively. ninety-six percent of the cat markers have identifi able orthologues in the canine and human genome sequences, providing a rich comparative tool, which is critical in linkage mapping exercises for the identification of genes controlling feline phenotypes. figure - presents a graphic display of each cat chromosome and blocks of conserved syntenic order with the human and canine genomes. one hundred and fifty-two cat-human and cat-dog homologous synteny blocks were identified. alignment of cat, dog, and human chromosomes demonstrated different patterns of chromosomal rearrangement with a marked increase in interchromosomal rearrangements relative to human in the canid lineage ( % of all rearrangements), as opposed to the more frequent intrachromosomal rearrangements in the felid lineage ( % of all rearrangements) since divergence from a common carnivore ancestor my ago. with an average spacing of marker every . mb in the feline euchromatic sequence, the map provided a solid framework for the chromosomal assignment of feline contigs and scaffolds during assembly of the cat genome assembly, and served as a comparative tool to aid in the identification of genes controlling feline phenotypes. as a complement to the rh map of the cat, a third generation linkage map of strs is currently nearing completion. the map has been generated in a large multigeneration domestic cat pedigree (n = informative meioses). previous first-and second-generation linkage maps of the cat were generated in a multigeneration interspecies pedigree generated between the domestic cat and the asian leopard cat, prionailurus bengalensis, to facilitate the mapping and integration of type i (coding) and type ii (polymorphic str) loci. the current map, which spans all autosomes with single linkage groups, has twice the str density of previous maps, providing a -cm resolution. there is also greatly expanded coverage of the x chromosome, with some str loci. marker order between the current generation rh and gl maps is highly concordant. approximately % of the strs are mapped in the most current rh map of the cat, which provides reference and integration with type i loci. whereas the third-generation linkage map is composed entirely of str loci, the sequence homology of extended genomic regions adjacent to the str loci in the cat x whole genome sequence, to the dog's homologous region, has enabled us to obtain identifiable orthologues in the canine and human genome sequences for over % of the strs. thus, practically every str acts as a "virtual" type locus, with both comparative anchoring and linkage map utility. combined with the cat rh map, these genomic tools provide us with the comparative reference to other mammalian genomes critical for linkage and association mapping. the domestic cat is one of mammalian species endorsed by the national human genome research institute (nhgri) human genome annotation committee for a "light" -fold whole genome sequence, largely to capture the pattern of genome variation and divergence that characterizes the mammalian radiations (http:// www.hgsc.bcm.tmc.edu/projects/bovine/, http://www.broad.mit. edu/mammals/). although light genome coverage provides limited sequence representation, (∼ %), one of the rationales for these light genome sequences included "enhancing opportunities for research on species providing human medical models." the -fold assembly of the domestic cat genome has recently been completed for a female abyssinian cat, "cinnamon," and a x whole genome sequencing effort is planned in the near future. a total of , , reads were assembled to , contigs, covering . gb with an n (i.e., half of the sequenced base pairs are in contigs <n ) contig length of bp. assembled supercontigs (n = , ) had an n length of kb (http:// hosted. abcc.ncifcrf.gov/cgi-gin/gbrowse/cat/). the estimated size of the genome was . gb and the genome coverage was approximately -fold, predicting an average inclusion of - % of the eukaryotic genome sequence. feline coding genes were identified using a comparative approach based upon sequence homology and syntenic orthology of neighboring gene homologues in the genomes of six index mammal species (human, chimp, mouse, rat, cow, and dog). the results revealed nearly , feline genes plus , conserved sequence blocks (csbs) used to build the gene map, depending upon the framework rh map of ordered type markers. the x feline genome sequence detects % of human genes, % of chimp or cattle genes, and % of dog genes based upon sequence identity to approximately bp of reciprocal base match between the cat sequence and the genome sequence of the six index mammals. a genome browser has been developed from the cat assembly, named genome annotation region field (garfield), which provides a physical map of the autosomes and the x chromosome, which can be inspected for sequence representation, including genes and the proportion of that gene available in the x cover, single nucleotide polymorphisms (snps), and strs, which can be used in linkage and association mapping, and other genome features (http://ccr.cancer.gov/ labs/lab). figure a total of , snp variants were identified in cinnamon's sequence, representing an incidence of / bp. approximately % of cinnamon's genome was heterozygous and % homozygous, which was not unexpected in a breed cat that is also the member of a highly inbred pedigree for retinal atrophy. , long stretches of alternating homozygous and heterozygous segments were observed ( figure - ), which represent the consequences of close inbreeding during the domestication process, and the more recent generation of fancy breeds and inbred disease pedigree. similar patches of homozygous/heterozygous segments were observed in the recently released whole genome sequence of the dog. the length of the segments is influenced by breedspecifi c history including effective population sizes, use of popular sires, and population bottlenecks. , linkage disequilibrium (ld) mapping has recently emerged as a powerful approach in humans for association mapping. long stretches of linkage disequilibrium in the target population greatly facilitate the success of the strategy and decrease the number of markers required for analysis. the extended lds observed in dog breeds, up to one hundred times the length observed in human populations, is proving to be a powerful mapping strategy for identification of genes associated with breedspecifi c phenotypes, including hereditary pathologies. , the potential of this mapping approach in cat breeds was evaluated by examining breed-specific patterns of common segment homozygosity in cat fancier association (cfa) (http://www.cfainc.org) breeds. the level of homozygosity refl ected in a group of snps was roughly half that seen in dogs, likely reflecting a more extensive recent inbreeding within heterozygosity across cinnamon's chromosomes is represented in nonoverlapping windows of kb. black represents regions with more than two snps per kb while gray represents the homozygous region (less than two snp, / kb). white represents gaps in the chromosome assembly. dog versus cat breeds. this level of homozygosity was used to estimate that some , equivalently spaced snp variants would be required for a linkage disequilibrium/haplotype-based association genome search of a complex heritable disease within cat breeds. recently, tyrosinase-related protein (tyrp ), one of the key enzymes in the melanogenic pathway, was linked to two coat color variants in the cat by association mapping in cat breeds due to extensive ld. two dna polymorphisms in tyrp , an a g substitution in the signal peptide and an in-frame insertion tyrp - ins / , were associated with the chocolate (b) allele. a premature uag stop codon at position of tyrp was associated with a second allele of the b locus, cinnamon (bl). snp discovery is planned in the x whole genome sequencing of the cat through a resequencing strategy of selected genomic regions in several cat breeds as was recently performed in the x whole genome sequencing of the dog. the majority of hereditary pathologies in the domestic cat for which the gene defect has been elucidated have resulted from the analysis of candidate genes (table - ) . however, with the availability of a detailed comparative map, and integration with developing gl and rh maps, and the cat x whole genome sequence, linkage and association-based mapping techniques have recently identified causative mutations for hereditary disease genes, , as well as several feline phenotypes (table - ) . , , once a genomic region is implicated from association-based or linkage mapping exercises, fine mapping has been accomplished by development of new strs or snps in the targeted region using the cat x whole genome sequence data accessed sequence tagged sites (sts), utilizing sequence information from the cat x whole genome sequencing effort, which ultimately identifi ed an ∼ kb deletion and a novel gene candidate, lix ( figure - ) . though the function of lix is unknown, the predicted secondary structure is compatible with a role in rna metabolism. an exon sequence screen of human sma cases, not otherwise explicable by mutations at the smn locus, failed to identify comparable lix mutations. the smn gene product, smn, is a ubiquitously expressed protein member of multiple ribonucleoprotein complexes with diverse roles in rna metabolism, splicing, and transport in all cells. , a central focus of sma research remains to discern the disease mechanism(s) and to understand why the primary disease pathology is localized to spinal lower motor neurons when all cells require smn function. lix expression is largely restricted to the central nervous system (cns), primarily in spinal motor neurons, thus offering an explanation of the tissue restriction of pathology in feline sma. determination of lix function may well provide fresh insight into the mechanisms of human sma pathology, impetus for more targeted therapeutics, and answers to fundamental questions of motor neuron development, maintenance, and/or function. through the cat genome browser, garfield. a recent report of the mapping and characterization of a novel gene causative of feline spinal muscular atrophy marked the first identification of a disease gene purely from positional reasoning. human spinal muscular atrophies (smas) are a genetically heterogeneous group of neuropathies that varies in clinical severity, from lethal in infancy to onset of mild weakness in adulthood, but all are characterized by neurogenic muscle atrophy due to degeneration of lower motor neurons of the spinal cord. for approximately % of people affected with sma, disease pathology is attributable to a mutation in the smn gene, on human chromosome q , which is subject to a high frequency of deletions and gene conversion events with the divergent and only partially functional centromeric copy/copies of the duplicated smn locus. , a domestic cat model of sma has been described that is a model of autosomal recessive juvenile-onset sma. with the feline smn gene excluded as the disease locus, a full genome linkage scan was conducted in a pedigree segregating for sma. the disease phenotype was linked to chromosome a q, in a region of conserved synteny to human chromosome q . fine mapping was accomplished with development of new strs and the world's veterinary schools produce thousands of practitioners each year, most of whom carefully document genetic and chronic diseases of our pets. the result is a comprehensive veterinary literature that has described over feline hereditary pathologies (http://www.angis.org.au/). the clinical and physiological study of these feline hereditary diseases provides a strong comparative medicine opportunity for prevention, diagnostics, and treatment studies in a laboratory setting. additionally, large animal homologues are similar to humans in natural genetic diversity and offer the possibility of evaluating long-term effects of treatment. to date, causal mutations have been characterized in felid genes that cause hereditary disease (table - ). the largest representation comes from lysosomal storage enzyme disorders that arise from defects in genes playing a role in degradation of macromolecules targeted to the lysosomes. many of the genes that cause these pathologies have been mapped in the cat. corrective therapeutic strategies have been proposed and examined in the cat, including enzyme replacement, heterologous bone marrow transplantation, and substrate reduction therapy. , limitations to these treatment strategies include high morbidity and mortality, limited positive outcomes, incomplete response to therapy, cost, and in some cases requirements for continuous lifelong therapy. gene therapy poses the most recent of intervention strategies. feline models have been important in elucidating molecular pathogenesis and are now playing a critical role in evaluating and optimizing the range of therapeutic strategies prior to clinical trials in humans. the mucopolysaccharidoses are disorders that result from the defi ciency of lysosomal enzymes involved in the degradation of mucopolysaccharides. the cat offers homologous models for mucopolysaccharidosis types i, vi, and vii. mucopolysaccharidosis type i (mps i), which results from a genetic deficient activity of the enzyme α-l-iduronidase (idua), can lead to mental retardation, growth abnormalities, and shortened life span in humans. naturally occurring models have been characterized in the cat , and dog. immune responses can nullify the effect of gene corrective therapy. it has been demonstrated that cats, but not dogs, mount a potent ctl response to canine idua after neonatal gene therapy, which can be prevented with transient ctla -ig. the efficacy of neonatal retroviral therapy has also been explored in the cat. the cat model, additionally, provides an ideal system to study mechanisms of brain neurodegeneration and neural-directed strategies, especially given a large body of preexisting literature on cat neurology. mps vi or marteaux-lamy disease, deficient activity of arylsulfatase b (arsb), is characterized in humans and cats with growth retardation, coarse facial features, corneal opacity, and skeletal deformities. [ ] [ ] [ ] [ ] the feline model also exhibits abnormal lysosomal storage in occasional neurons and glia distributed throughout the cerebral cortex. fibroblast-mediated in vitro gene therapy has been examined in this cat model. recently, an adeno-associated vector containing feline arsb has demonstrated gene therapy-based correction of corneal clouding in the mps vi cat. mps vii results from deficiency of β-glucuronidase (gusb), which in humans manifests as cartilaginous and bony malformations, growth and mental retardation, abdominal organ enlargement, and corneal clouding. naturally occurring animal models have been described in mice, dogs, and the cat. enzymatic activity has been restored in fibroblasts and restored by retroviral gene transfer of rat gusb cdna. as gusb is an essential housekeeping enzyme, this feline model is important for examination of exogenous genes and gene product delivery to a variety of tissue types, and could prove especially valuable due to extensive research conducted on the anatomy and physiology of the cat central nervous and visual systems. three different serotypes of adeno-associated viral constructs of gusb demonstrated the efficacy of this vector to achieve gene transfer in the normal cat brain, as a model for the efficacy of this construct in a large mammalian brain. defi ciency of lysosomal α-mannosidase leads to an accumulation of mannose-rich oligosaccharides, which leads to mental retardation, recurrent infections, skeletal changes and hearing impairment. this feline model was initially important in achieving bone marrow transplantation as corrective strategy for neuronal storage diseases of the cns. , recently, adeno-associated viral (aav) constructs of feline α-mannosidase were used to demonstrate the efficacy of cns gene therapy. treated cats exhibited widespread improvement of neuropathology, showing the effi cacy of this treatment in a large mammalian brain for cns correction of human lysosomal enzyme defi ciencies. lipoprotein lipase (lpl) is a crucial enzyme involved in the regulation of lipoprotein and lipid metabolism ability to thrive. cats with lpl deficiency display a remarkably similar phenotype to humans, including severe pancreatitis, chylomicronemia, and failure to thrive. there is currently no adequate treatment for this pathology in humans. cats could prove to be the most valuable animal model of lpl deficiency, as of numerous animal model systems examined including the mouse, the cat most closely resembles the lipoprotein pattern and lipid transport system of humans. recently, aav-mediated transfer of a human lpl s x variant into feline muscle cells demonstrated correction of the hypertriglyceridemia associated with feline pathology; this offers much promise for treatment of human lpl defi ciency. a separate class of lysosomal storage disorder characterized in the cat is the gangliosidoses, g m and g m , which are heritable neurodegenerative diseases. a deficiency of lysosomal β-galactosidase results in the neuronal accumulation of the g m ganglioside, while the degradation of the g m ganglioside is initiated by coordinated action of at least three gene products, the α and β submits of β-n-acetylhexosaminidase and the g m activator (gm a) protein. , mutations in any of these enzymes result in an accumulation of gangliosides g m and g m in the lysosomes of affected neurons, resulting in progressive deterioration of the cns. feline models have been especially important in characterizing the pathobiology and molecular biology of these diseases. g m -gangliosidosis has been characterized in cat models defi cient in the g m activator protein and hexb, , , , exhibiting remarkably similar pathology to human sandhoff's disease. limited reduction in g m neuronal storage has been reported following bone marrow therapy. feline models will be important in the development of therapeutic strategies for these disorders. mucolipidosis ii (i-cell disease) is caused by deficient activity of the enzyme n-acetylglucosamine- -phosphotransferase, which leads to a failure to internalize enzymes into lysosomes. the cat is the only known animal model for this pathology. congenital diseases of feline muscle and neuromuscular junction have been reviewed by gashen et al. some pathologies have been observed in isolated breeds, including hypokalemic myopathy of burmese cats, glycogen storage disease type iv in norwegian forest cats, and myopathy observed in the devon rex. the cat is the only reported animal model for type iv glycogen storage disease. myotonia congenital, , muscular dystrophy (dystrophin defi cient), and laminin α defi ciency , have also been reported in the cat. x-linked muscular dystrophy in humans is characterized by progressive degeneration of skeletal and cardiac muscle. mutations in humans lead to either an absence of or abnormality in the protein product dystrophin. , a deletion in the dystrophin muscle promoter characterized in the cat eliminates expression of muscle and purkinje neuronal dystrophin isoforms. marked clinical heterogeneity is observed in these models, from severe disability exhibited in human and dog, to minor muscle fi brosis and an actual regenerative process leading to muscle hypertrophy in mouse and cat. [ ] [ ] [ ] these different sequellae could be important in characterizing immediate and secondary consequences of the lack of dystrophin and points out the importance of multiple animal models. hypertrophic cardiomyopathy is a clinically heterogeneous myocardial disease contributing to one of the most common causes of sudden cardiac death in young adults. the cat represent the first spontaneous large animal model for this familial disease and will prove to be valuable for examining pathophysiological processes and therapeutic interventions. there is a continued demand for alternative mammalian models for studying human diseases. compared to traditional murine models, the cat's more similar physiology, increased size, and longevity have made it ideal for testing the safety and efficacy of some therapeutic modalities in naturally occurring feline disease models. [ ] [ ] [ ] [ ] additionally, cats have a genetically heterogeneous background, similar to humans. a good example of a human disease in need of a better mammalian model is cystic fibrosis, as mice targeted with the most common human mutation (∆f ) in the cystic fibrosis transmembrane receptor (cftr) fail to spontaneously develop the same opportunistic lung infections that plague human patients with cystic fi brosis. , one group has been working to produce a ferret cystic fibrosis model through gene targeting of somatic cells for nuclear transfer. , while reproductive cloning has a consistently low success rate ( - %) across mammalian species, targeting genetic loci through homologous recombination in somatic cells is currently the only viable method for producing knockout models in all mammalian species other than the mouse, for which targeted embryonic stem cells are routinely used. nuclear transfer of targeted fibroblasts has been successfully used to produce viable α- , -galactosyltransferase knockout pig and cow models for xenotransplantation studies. , with the successful reproductive cloning of cats by several groups, - the development of gene-targeted cat models through nuclear transfer is now feasible. the imminent release of the annotated feline genome project, integration of recombination and radiation hybrid maps, [ ] [ ] [ ] [ ] [ ] [ ] and availability of multiple pac, bac, flow-sorted autosome, and y-chromosome libraries , (j. pecon-slattery et al., unpublished observations) at the laboratory of genomic diversity will facilitate efforts by researchers to develop new cat models of specific human genetic diseases. the coat color loci influence the development, maturation, and migration of melanocytes as well as the synthesis of melanin and the formation, transport, and transfer of melanosomes. genes involved in these processes often have pleiotropic effects, which impact other important biochemical pathways. coat color loci in the mouse have been known to be part of diverse cellular, developmental, and physiological processes and in some cases to be implicated in pathologies such as anemia, sterility, and neurological disorders. [ ] [ ] [ ] the cat is an excellent model system with which to study coat color phenotypes. at least nine different coat color loci have been identified in the cat, and several are now characterized on a molecular genetic level including, a (nonagouti) responsible for melanism, b (brown), which changes black pigmentation to brown or variants of brown, c (color), causing the darker pigmentation at extremities (i.e., ears, tail), observed in siamese and burmese cats , and albinism, and d (dilute), which causes dilution of expected color (i.e., black pigmentation appears gray). the cat is also unique in mammalian species in exhibiting a variation in coat pattern, demonstrating agouti (a) (nonpatterned coat) and variants of the t (tabby) locus, which affect striping and spotting patterns. the cat has provided several useful models for infectious disease. these include feline leukemia and feline sarcoma virus, feline coronavirus, and type c retroviruses that interact with cellular oncogenes to induce leukemia, lymphoma, and sarcoma. [ ] [ ] [ ] historically, many of the human oncogenes that defi ne signal transduction pathways were originally discovered in the context of feline leukemia virus interaction in cat models. the cat provides the only naturally occurring model for human aids pathogenesis, in its endemic fatal transmissible feline immunodefi ciency virus (fiv). , similar to its close phylogenetic relative hiv, fiv induces cd -t lymphocyte depletion in affected cats, an immune system collapse, and susceptibility to adventitious microbial agents as a prelude to wasting disease and death. , interestingly, over wildcat species (including lions, leopards, cheetahs, ocelots, pumas and other big cats) are endemic with their own species-specific strain of fiv [ ] [ ] [ ] [ ] [ ] [ ] ; however, unlike strains in domestic cats, the wildcat fiv strains do not appear to cause acute immunodeficiency in the wildcat species, perhaps a consequence of historic natural selection of host genetic resistance to the fatal virus. , lion and puma-specific fiv strains have recently been demonstrated to utilize novel, more promiscuous mechanisms for cell entry than fiv, suggesting a divergent tropism and biological properties of these viruses. the world health organization reported a new human respiratory illness outbreak (severe acute respiratory syndrome, sars) that emerged in guangdong province, china in . , sequence analysis demonstrated that the infectious agent was a previously unrecognized coronavirus. , an animal model demonstrating clinical symptoms and pathology of sars-infected patients has not been reported. of interest, the recent report of a highly virulent feline coronavirus epidemic in captive african cheetahs, a disease model for human sars, illustrates the critical role of ancestral population genetic variation. in addition, cats injected with the sars virus developed clinical symptoms, an important insight in implicating the virus in the sars epidemic. [ ] [ ] [ ] [ ] the feline panleukopenia (feline distemper) virus has revealed a natural history parable in its abrupt transformation of the cat virus to an epidemic, fatal canine parvovirus, that emerged in the world's puppy population in . in contrast, the canine distemper virus, which is normally restricted to canid species, precipitously adapted to and decimated east african lions in , killing one-third of the lions in the serengeti ecosystem within a -month outbreak. a clear involvement of host defense mecha- nisms in these and other infectious disease episodes renders the cats and their pathogens an excellent candidate species for characterizing the interaction of microbial adaptation and host disease gene defenses. given the critical importance of infectious disease in scores of chronic and acute human disease, there are powerful research opportunities in the cat family. , finally, recent concern over the emergence of avian flu h n has shown a strong susceptibility of cats, both domestic and large cats, again raising possibilities for pathogenesis and therapy development. , with the development of genomic resources in the cat (table - ) and the application of complementary comparative tools developed in other species, the domestic cat is emerging as a promising resource of phenotypically defined genetic variation of biomedical significance. exploration of similar resources in other species, particularly the dog and mouse, has provided important insight into otherwise unexplained biomedical disorders. early taming of the cat in cyprus the feline genome project integration of the feline radiation hybrid and linkage maps radiation hybrid mapping of novel microsatellites in the domestic cat genome a genetic linkage map of microsatellites in the domestic cat (felis catus) a . megabase resolution radiation hybrid map of the cat genome and comparative analysis with the canine and human genomes second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (felis catus) a third-generation rh map of the domestic cat extensive conservation of sex chromosome organization between cat and human revealed by parallel radiation hybrid mapping genetic mapping in mammals: chromosome map of domestic cat a radiation hybrid map of the cat genome: implications for comparative mapping novel gene acquisition on carnivore y chromosomes comparative genomics: lessons from cats comparative gene mapping in the domestic cat (felis catus) the domestic cat genome sequence annotation and comparative inferences genome sequence, comparative analysis and haplotype structure of the domestic dog identifi cation and characterization of multi-species conserved sequences an initial strategy for the systematic identification of functional elements in the human genome by low-redundancy comparative sequencing hereditary progressive retinal atrophy in the abyssinian cat genetic structure of the purebred domestic dog dog star rising: the canine genetic system linkage disequilibrium maps and association mapping extensive and breedspecifi c linkage disequilibrium in canis familiaris the canine genome understanding the genetics of autoimmune disease: two loci that regulate late onset addison's disease in portuguese water dogs canine rpgrip mutation establishes cone-rod dystrophy in miniature longhaired dachshunds as a homologue of human leber congenital amaurosis cat domestication age an str forensic typing system for genetic individualization of domestic cat (felis catus) samples tyrosinase and tyrosinase related protein alleles specify domestic cat coat color phenotypes of the albino and brown loci feline polycystic kidney disease mutation identified in pkd an ∼ -kb deletion associated with feline spinal muscular atrophy implies an essential lix function for motor neuron survival a homozygous single-base deletion in mlph causes the dilute coat color phenotype in the domestic cat albinism in the domestic cat (felis catus) is associated with a 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causes mucolipidosis ii in domestic shorthair cats two mutations within a feline mucopolysaccharidosis type vi colony cause three different clinical phenotypes molecular basis of feline beta-glucuronidase deficiency: an animal model of mucopolysaccharidosis vii deletion of the dystrophin muscle promoter in feline muscular dystrophy mutation analysis of feline niemann-pick c disease a tyrosinase gene missense mutation in temperature-sensitive type i oculocutaneous albinism. a human homologue to the siamese cat and the himalayan mouse molecular basis of erythrocyte pyruvate kinase (rpk) deficiency in cats cats lack a sweet taste receptor nomenclature for the description of human sequence variations spinal muscular atrophy identifi cation and characterization of a spinal muscular atrophy-determining gene quantitative analysis of survival motor neuron copies: identification of subtle smn mutations in patients with spinal muscular atrophy, genotype-phenotype correlation, and implications 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against sars virology: sars virus infection of cats and ferrets the emergence and evolution of canine parvovirus-an example of recent host range mutation a canine distemper virus epidemic in serengeti lions (panthera leo) plague time: how stealth infections cause cancer, heart disease, and other deadly ailments germany says people in areas with bird flu should keep cats indoors h n virus attachment to lower respiratory tract infl uenza a virus (h n ) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts avian h n infl uenza in cats feline friend or potential foe? comparative gene mapping in the domestic cat (felis catus) zoo-fish analysis: cat and human karyotypes closely resemble the putative ancestral mammalian karyotype patterns of y and x chromosome dna sequence divergence during the felidae radiation phylogenetic reconstruction of the felidae using s rrna and nadh- mitochondrial genes complete nucleotide sequence of the domestic cat (felis catus) mitochondrial genome and a transposed mtdna tandem repeat (numt) in the nuclear genome comparative genome organization of human, murine, and feline mhc class ii region key: cord- - r rsio authors: jani, meghna; dixon, william g.; matteson, eric l. title: management of the rheumatoid arthritis patient with interstitial lung disease date: - - journal: lung disease in rheumatoid arthritis doi: . / - - - - _ sha: doc_id: cord_uid: r rsio the treatment of rheumatoid arthritis (ra) has undergone considerable changes over the last – years. with an expansion in the armamentarium of therapies available for ra comes a wider choice in selecting the best treatment in terms of comparative safety in the presence of comorbidities. clinicians frequently encounter patients with ra-associated interstitial lung disease with uncontrolled joint disease and have to make decisions about the safest treatments in this context with the eventual goal of joint remission. in this chapter, available evidence is reviewed on the comparative pulmonary safety of non-biologic disease-modifying antirheumatic drugs (nbdmards), biologic dmards, biosimilars and targeted synthetic dmards in ra-ild. in addition, the potential role for additional immunosuppression in ra-ild is reviewed as well as overarching recommendations proposed for patient assessment to guide treatment decisions and management. the management of rheumatoid arthritis (ra) has changed dramatically over the past - years. new classification criteria for ra have been introduced [ ] that allow the study of patients earlier in their disease course, and recommendations have been developed to treat patients with ra using a strategic approach by targeting an optimal outcome, with the primary goal of joint disease remission [ , ] . the armamentarium of treatments available for the treatment of ra has also expanded. however, with the introduction of novel therapies comes a wider choice in selecting the best treatment for the individual patient not only in terms of comparative efficacy but also safety in the presence of comorbidities. interstitial lung disease (ild) is one of the most common respiratory manifestations in patients with ra and is a major cause of morbidity and mortality. ra-ild encompasses several histopathologic patterns; the most frequent considered here are usual interstitial pneumonia (uip), nonspecific interstitial pneumonia (nsip), and obstructive pneumonia (op). the development of respiratory complications of treatment is thus particularly problematic in patients with such coexistent lung disease, and there have been many reports of respiratory complications of both non-biologic disease-modifying antirheumatic drugs (nbdmards) and biologic dmard (bdmard) therapy. clinicians are frequently encountered with decisions about balancing risk and benefit of treatments in patients with ra-ild who have active articular disease. serious respiratory adverse events (sraes) in the patient with ra on treatment for their joint disease may be due to induction of 'pneumonitis' or idiosyncratic adverse drug reactions (adrs), acceleration of pre-existing ild or increased predisposition to infection in a susceptible host. several nbdmards and bdmards have been implicated in the development of ild. conversely, treatment of the underlying disease process may be beneficial in halting the progression of the lung disease. this chapter first considers methods for assessing drug safety and then reviews the available evidence for respiratory outcomes of nbdmards and bdmards in ra-ild and the role of additional immunosuppression and overarching recommendations for patient assessment and management of ild. whilst randomised controlled trials (rcts) are considered the gold standard in assessing efficacy of treatments, they have limited utility in terms of assessing safety. although safety has been evaluated as part of a number of rcts, uncommon events such as new-onset ild may not be captured. rcts often have restrictive eligibility criteria to ensure a homogenous population (leading to exclusion of patients with known ra-ild), small numbers of patients and a short follow-up period. this also limits their external validity, as real-life patients may be older and have multiple comorbidities compared to trial patients. open-label extension (ole) studies are often an adjunct to double-blind rcts, and although they do allow surveillance of participants for a longer duration, patients who are intolerant to the drug during the previous study will not be able to participate in the extension period, therefore potentially leading to underreporting of adverse events (aes). ole studies also lack a comparator group and are prone to loss to follow-up, making it difficult to interpret the rate of aes. post-marketing spontaneous pharmacovigilance involves the reporting of suspected adverse drug reactions by healthcare professionals or patients. this is done either in the form of case reports/series to medical journals or to national or international monitoring centres, such as the uk medicines and healthcare products regulatory agency (mhra) 'yellow card scheme', european medicines agency eudravigilance database and the us food and drug administration (fda) medwatch programme. whilst these are useful for drug safety signal detection, there is no available denominator to calculate rates of ae or to determine whether these reported cases represent an increased incidence above the background population rate. observational cohorts have the advantage of being able to examine drug safety in a 'real-world' setting and can follow large numbers of patients for long periods of time. they can therefore study the medium-to long-term side effects of a drug that might otherwise be missed in clinical trials or spontaneous pharmacovigilance. however these come with the challenge of interpreting results in the context of clinical decisions, with their consequent biases and confounding. each of these methods can employ a wide array of terminology to describe various forms of respiratory aes. for example, parenchymal lung disorders are called many things from 'pulmonary fibrosis' to 'allergic pneumonitis'. there are also challenges in differentiating between respiratory conditions with different aetiologies but similar clinical presentations. rcts, for example, can report on culturenegative pneumonias or 'community-acquired pneumonitis', which may indeed be idiosyncratic drug reactions or pneumonias secondary to a resistant microorganism: often patients are administered a combination of glucocorticoids and antibiotics due to initial uncertainty of the diagnosis. the data available for this chapter come from all of the above study designs. having been in use for ra for the longest duration, nbdmards and tumour necrosis factor inhibitors (tnfis) have the most observational evidence, but not in all cases. newer tnfis, nbdmard and emerging novel therapies recently licensed for ra have not had the opportunity for longer follow-up; therefore, experience about pulmonary safety may be restricted to clinical trials and spontaneous pharmacovigilance. methotrexate (mtx) has been described as the anchor drug in ra treatment, as it is often used first line at diagnosis, in combination with other nbdmards and concomitantly with biologics, following failure of traditional nbdmards for controlling articular disease. it inhibits folic acid and purine metabolism along with t-cell activation. mtx-induced pulmonary injury was initially reported in children with leukaemia in the s [ ] , followed by a case series in treated patients with ra in [ ] . following a review of cases of methotrexate pneumonitis in the year , % of cases arose in patients with ra (dose range . - mg/week), % occurred during intensification/consolidation treatment for leukaemia (dose range - mg/week), and % were in patients treated for other malignancies (dose range - mg/week) [ ] . whilst mortality rates have been reported up to %, hypersensitivity pneumonitis is reported to be a rare ae in ra patients. in a systematic literature review of patients with ra on mtx, patients ( %) had some type of lung toxicity, but only patients were felt to be definitive cases of pneumonitis attributable to methotrexate ( . %) [ ] . estimates of reported incidence vary between . and % of treated patients (in up to -year follow-up) [ , ] . the clinical presentation of acute mtx pneumonitis is generally nonspecific, with symptoms (fever, rigors, malaise, nonproductive cough, dyspnoea, chest pain) that can be progressive over several days. criteria proposed by searles and mckendry [ ] and carson et al. [ ] are generally accepted for defining mtx pneumonitis and can sometimes help in differentiating the disease from ra-ild and respiratory infections, although it is possible to fulfil the criteria with conditions other than pneumonitis, for example, infection or a progression of pre-existing ra-ild (table . ). searles and mckendry criteria have since been adapted by kremer et al. [ ] categorising them into major and minor. all rely on a combination of clinical features, radiological, histology and exclusion of infection. studies have explored other factors that might differentiate these clinically similar respiratory diseases. histological findings in mtx pneumonitis such as cellular interstitial infiltrates, diffuse alveolar damage, tissue eosinophils and granuloma formation are nonspecific and have all been seen in ra lung disease [ ] . highresolution computer tomography (hrct) studies in mtx pneumonitis typically show ground-glass changes, centrilobular nodules +/− diffuse parenchymal opacification [ ] . bronchoalveolar lavage (bal) cell profiles in mtx pneumonitis show a lymphocyte alveolitis with a preferential increase in cd + cells compared to normal ra controls [ , ] , though comparisons have not been made between bal in mtx pneumonitis and ra-ild. a challenge with all these studies is the problem of measuring the accuracy of a test against no accepted gold standard. it has been argued on several counts that mtx hypersensitivity pneumonitis is a unique hypersensitivity reaction rather than the development or progression of ra-ild. firstly, mtx-associated lung injury tends to occur early in the [ ] , the close temporal relationship arguing in favour of causality. secondly, acute lung injury has resolved after discontinuation of the drug and treatment with high-dose glucocorticoids [ ] . lastly, case reports of ild have been reported in other conditions including psoriatic patients treated with mtx [ , ] as well as in other conditions not typically associated with ild. however, there are issues with each of these arguments. clinicians are more likely to diagnose and report mtx pneumonitis if there is a close temporal relationship. if cases in question are identified by asking rheumatologists to identify them [ ] , it is inevitable that a strong temporal relationship will be found. the second argument of resolution on drug withdrawal would be more convincing if there were no concurrent glucocorticoid treatment. acute exacerbations of ild, which may resemble drug pneumonitis, tend to be responsive to glucocorticoids. that said, there are cases of improvement on mtx withdrawal with no increase in pre-existing glucocorticoid dose [ ] . third, dose and duration of mtx treatment does not appear to be associated with pulmonary toxicity in the reported literature [ ] . furthermore a recent systematic review and meta-analysis evaluating the risk of pulmonary disease amongst mtx-treated patients with psoriasis, psoriatic arthritis and inflammatory bowel disease in rcts concluded no increased risk [ ] . it is not clear whether pre-existing ra-ild increases the risk of mtx pneumonitis with studies both supporting and opposing this view. the largest study was a multicentre case-control study from the usa [ , ] , which identified cases and controls matched for mtx use. the strongest predictors of lung injury were diabetes, hypoalbuminaemia, previous use of nbdmards, rheumatoid pleuropulmonary involvement (or . ( % ci . - . )) and older age (> ). a history of copd was modestly associated with mtx-induced lung injury. since all the cases were identified following clinical presentation, the observed association between pre-existing lung disease and mtx pneumonitis may represent an increased likelihood of presentation (because of a reduced physiological lung reserve) or a surveillance bias rather than an increased predisposition to pneumonitis. meta-analysis of six studies that suggested an association between pre-existing lung disease (of varying definitions) and an increased risk of mtx pneumonitis found a pooled odds ratio of . ( % ci . , . ) [ ] . however, two studies that suggested no such association [ , ] were not included in the meta-analysis for reasons that are not clear. howes et al. [ ] followed patients who received mtx for ra for a median treatment duration of months, all of whom had baseline pfts. three patients developed pulmonary toxicity according the established criteria [ ] , all of whom had abnormal baseline pfts. of the patients studied, / patients had a transfer factor of < % at baseline, one of whom developed pulmonary toxicity. the authors report a relative risk for pneumonitis of , though this estimate is not robust given the small numbers. there is currently no conclusive evidence that lung function is likely to decline faster in patients with ra-ild on mtx who do not develop pneumonitis. in a retrospective cohort analysis, following weeks of high-dose glucocorticoid treatment, one study reported treatment with mtx vs. leflunomide or azathioprine was associated with an improvement in fvc at months in patients with less fibrosis at baseline, although there were no another differences in other major outcomes such as mortality [ ] . another study by dawson et al. [ ] followed patients with ra for years, % of whom were on low-dose mtx (mean . mg/week), with hrct in all patients at baseline and pulmonary function tests (pfts) at baseline and at -month follow-up intervals. there was no significant difference in the change in pfts over the follow-up period either between the mtx and non-mtx patients or between mtx and non-mtx patients with proven ild on hrct at baseline. the authors concluded that they found no association between mtx therapy and progression of chronic pulmonary fibrosis. current guidelines on the use of mtx recommend that all patients should have a baseline chest radiograph with or without pfts. there appears to be little evidence to support this; however, pfts may be useful in patients with ra deemed to be at high risk of ild or known to have ra-ild to assess for progression (discussed further in section "patient management and treatment"). in summary, diagnostic uncertainty and the lack of any gold standard test for mtx pneumonitis leaves ambiguity about the true pattern of disease. however mtx has not been shown to consistently accelerate the progression of underlying ra-ild. whilst few studies have been conducted in pre-existing ra-ild stratified by severity of lung disease, the risk of pneumonitis means that it may not always be the safest first-line nbdmard in patients with ra who have severe pre-existing lung disease. leflunomide is an isoxazole derivative, which inhibits de novo pyrimidine synthesis, resulting in several downstream anti-inflammatory effects such as suppression of tnf-induced cellular responses and inhibition of matrix metalloproteinases and osteoclasts. leflunomide-induced pneumonitis is rare but well reported. a signal of concern was raised in after an investigation by the japanese ministry of health following post-marketing surveillance in ( . %) of the first japanese patients treated with leflunomide, resulting in five fatalities [ , ] and the committee on the safety of medicines also reporting cases in the uk of which five were fatal. further case reports have subsequently been reported especially in the japanese and korean populations [ ] [ ] [ ] . however leflunomide is often used second line after mtx exposure or in combination with mtx in patients with active articular disease, which make studies evaluating leflunomide-induced pneumonitis difficult to interpret. in a report of such cases from australia and new zealand, were co-prescribed mtx [ ] . chikura et al. [ ] reported on pneumonitis cases following leflunomide exposure, classified using searles and mckendry criteria (table . ), and found % had a history of mtx exposure, whilst % were on concomitant mtx at the onset of ild. the majority presented within weeks of initiation. clinical features of those who died were pre-existing ild and diffuse alveolar damage on histology (n = / ). pre-existing ild was also shown to be a risk factor for leflunomide-induced pneumonitis in a large case-control study using a canadian claims database [ ] . the risk of ild was increased with the use of leflunomide (adjusted rr . [ % ci . - . ]); however, in patients without previous mtx exposure or ild history, the risk associated with leflunomide treatment was not elevated (rr . [ % ci . - . ]). however, it was acknowledged given the probable association between prior ild and mtx pneumonitis; clinicians may be more likely to prescribe leflunomide than mtx to patients with prevalent ild, leading to channelling bias explaining the increased risk observed. leflunomide-induced pneumonitis appears to occur more frequently in japanese and korean population (reported rate ~ %) [ , ] , whilst in the western caucasian population, a rate of < . % is reported [ ] . genetic susceptibility in japanese patients has been described in a study which investigated human leukocyte antigen (hla) class i associations with mtx pneumonitis in japanese patients with ra and found hla-a × : as a possible predictor. the prevalence of this allele is proportionally higher in the japanese population ( . %) than in the caucasian population ( . %) [ ] . therefore such genetic differences and undiscovered genetic predictors may explain some of the differences in frequency observed in such populations. several risk factors of leflunomide-induced pneumonitis have been reported in small numbers of patients in case series and retrospective studies including preexisting lung disease [ , , ] , a prescribed loading dose, smoking, low body weight [ ] and increased c-reactive protein, hypoalbuminaemia, hypoxia and lymphopaenia [ ] . treatment includes cessation of the drug, treatment with glucocorticoids with some benefit reported with activated charcoal and cholestyramine as washout treatments. whilst conclusions of use in ra-ild are limited from studies due to channelling bias, leflunomide should be avoided in patients with previous mtx pneumonitis and should be used with caution in patients with pre-existing ild. sulphasalazine is a -aminosalicyclic acid ( -asa) derivative metabolised to sulphapyridine, which is the active moiety in ra. pulmonary hypersensitivity reactions such as eosinophilic pneumonias [ , ] , fibrosing alveolitis and bronchiolitis obliterans have been well described, with over case reports in the literature [ , ] . drug reaction with eosinophilia and systemic symptoms (dress) is also reported [ , ] . typical presentation of sulphasalazineinduced lung disease reported is with new-onset dyspnoea and infiltrates on chest radiograph (with or without peripheral eosinophilia with eosinophilic pneumonitis). cough and fever are the most common symptoms with sputum production, whilst allergy history, rash, chest pain and weight loss were inconsistent findings [ ] . histology is often variable; the most frequent appears to eosinophilic pneumonia with interstitial inflammation with or without fibrosis. drug cessation often results in resolution of symptoms in patients who develop eosinophilic pneumonia [ ] . the role of systemic glucocorticoids is not wellstudied, as most patients improve with withdrawal of sulphasalazine. there are no studies that have systematically evaluated the safety of sulphasalazine in preexisting ra-ild; however, it is worth observing that the rates of sraes with sulphasalazine appear much lower than those seen with methotrexate and leflunomide in the literature. hydroxychloroquine is an antimalarial drug and is a -aminoquinoline derivative, often used in combination with other nbdmards. it is usually well tolerated and serious aes are rare. few cases of drug-induced pneumonitis exist [ ] , with some reports in association with drug reaction with eosinophilia and systemic symptoms (dress) syndrome [ , ] . hydroxychloroquine has been tried in childhood ild (diffuse parenchymal lung diseases) in case reports given the lack of therapeutic options. in a systematic review from to [ ] , case reports were identified: a favourable response to hydroxychloroquine was reported in cases ( %), with an unclear effect in the rest. whilst idiosyncratic reactions leading to lung toxicity have been reported, no studies have demonstrated a benefit or harm of this drug in ra-ild. in summary, case report evidence for drug-induced pneumonitis exists for all nbdmards commonly used to treat joint disease; therefore, incident ild may be a theoretical risk in most treated patients. with treat-to-target guidelines [ ] and the eventual aim of abrogation of joint inflammation as soon as possible, most patients with ra are likely to be treated with a nbdmard early in the course of their disease. whether the risk of progression of ra-ild or sraes increases with nbdmards is not clear and may vary according to the individual patient profile including their comorbidities, concomitant treatments, severity of ild at the outset of treatment and their genotype. evidence to date suggests caution should be exercised whilst treating patients with pre-existing ra-ild with all nbdmards, particularly mtx and leflunomide, with careful monitoring of disease progression and vigilance for (sometimes rare) sraes associated with such treatments (summarised in table . ). five tnfi agents have been approved for treating patients with ra: three monoclonal antibodies (infliximab, adalimumab and golimumab), the recombinant soluble tnf receptor etanercept and pegylated certolizumab. as biologic therapies such as tnfis target key cytokines and cells in the inflammatory cascade with pleiotropic effects on the immune system, there have been particular concerns regarding their long-term safety profile. although these drugs are highly effective in controlling joint disease, and often are used as first-line biologics in combination with mtx, they are reported to be associated with various autoimmune aes [ ] . whilst the most common of these appear to be lupus-and vasculitis-like events, tnfiassociated lung injury has been reported [ ] . the safety of these drugs in patients with established ra-ild continues to be scrutinised. conversely elevated levels of tnf-α have been detected in the lungs of both experimental animal models [ ] and patients with idiopathic pulmonary fibrosis (ipf) [ ] . interestingly serum levels of tnf-α have also been found to be elevated in patients with rituximab-induced ild [ ] . etanercept has been used in a randomised double-blind placebo-controlled exploratory trial in clinically progressive ipf subjects, however failed to show a difference in primary endpoint of improvement in fvc over weeks. the study was underpowered; however, a nonsignificant reduction in disease progression was seen in several physiologic (including transfer factor), functional and quality-of-life endpoints amongst participants receiving tnfi. importantly there was no decline in lung function or higher incidence of sraes in the etanercept group compared to placebo. therefore whilst legitimate concerns about pulmonary toxicity have been highlighted, tnfi treatments may have plausible bidirectional effects. the next section will review the levels of evidence for both incident ild and safety in pre-existing ra-ild. clinical trials of tnfi therapy have been significantly underpowered to detect even a large increase in the incidence of serious ra-ild. the original rcts leading to eventual approval of tnfi provided no ra-ild safety signals. in the first reported phase iii trial of infliximab and mtx versus placebo and mtx over weeks, there was a death in both the placebo arm due to ild and the infliximab arm due to cardiopulmonary failure as a result of suspected pulmonary embolism or ild [ ] . in a two-year extension of this trial, there was a single death attributed to ild in the infliximab arm; however, it was not clear whether this is the same patient reported in the previous paper. a clinical trial of etanercept versus mtx in early ra reported three cases of pneumonitis in the mtx group at year follow-up [ ] and four cases at year follow-up [ ] with no reported cases in the etanercept arm despite a ratio of etanercept/mtx of : . there was one case of death from respiratory failure in the -year ole of this study in the etanercept arm [ ] . no cases of ild were reported in the -year ole of etanercept [ ] . ild was not reported in any of the initial adalimumab clinical trials, in either the adalimumab or placebo arms [ ] . however, within an adalimumab ole study [ ] , saes coded as 'parenchymal lung disorders' occurred at a rate of . - . / patient-years, accounting for % of all sae's. in this trial, however, there was no placebo-controlled or nbdmardexposed arm, making the interpretation of these figures challenging. similarly no signal of concern for incident ild was observed in the initial golimumab-ra trials [ , ] in the two-year [ ] or five-year [ ] extension studies. in the certolizumab rapid and rapid trials [ , ] , and subsequent oles [ ] , no cases of druginduced pneumonitis were reported in any arms. therefore original rcts of tnfi agents did not raise concern over the potential development of ild. these trials typically had short duration, included between and patients in the tnfi arm each and excluded patients with ra-ild at baseline. concerns regarding the respiratory safety of tnfi agents arose initially following three case reports of rapid fatal exacerbations of ra-associated fibrosing alveolitis following commencement of infliximab in [ ] , which was extended in a later publication to five cases [ ] . of these cases, three patients were taking concomitant azathioprine, and one taking leflunomide developed rapid progression of ra-ild (known uip preceding treatment). the fifth patient did not have a history of lung disease pretreatment and, following infliximab, developed nonfatal cryptogenic organising pneumonia (cop or bronchiolitis obliterans organising pneumonia [boop]) [ ] . in a case series of autoimmune diseases associated with tnfi, / (~ %) were reported to develop ild (interstitial pneumonitis in patients, sarcoidosis in three, pulmonary haemorrhage in two and boop in one patient) [ ] . exacerbation of previous ild was reported in four patients. perez-alvarez et al. described a further series of patient case reports from the literature ( % with ra), with incident ild or exacerbation of pre-existing ild ( patients, %) [ ] . the outcome was available in of the described cases: ( %) had complete resolution, improvement or partial resolution in ( %) and no resolution reported in ( %). case reports of incident ild have been reported with newer tnfis such as certolizumab [ ] [ ] [ ] [ ] and golimumab [ ] , including possible deterioration of pre-existing ild in a patient with ra treated with certolizumab and leflunomide after months of therapy [ ] . whilst the temporal association with onset of ild and repeated reporting of such events provides a signal of concern, the interpretation of such data for extrapolation into clinical practice is limited by a likely reporting bias, a possible 'bandwagon' effect (a phenomenon where following the publication of an index case, further cases are consequently more likely to be reported), and lack of an adequate denominator. furthermore, reports of stabilisation of ra-ild in patients exposed to tnfi therapy have also been published [ ] [ ] [ ] , suggesting a further bidirectional influence of tnfi on ild. to accurately evaluate the risk of incident ild or exacerbation in patients with ra-ild on tnfi in patients who are most likely to receive them in the real world, observational studies offer the best design to examine rare outcomes. however all studies evaluating such events have faced methodological issues, namely, with channelling bias and challenges with classification. using the national databank for rheumatic diseases, wolfe et al. used a combination of hospital records, patient descriptive reports and physician and mortality records to classify patients who had severe ra-ild (requiring hospitalisation or who died) [ ] . they reported a significant association with previous use of infliximab (hr . , % ci . - . ) and etanercept (hr . , % ci . - . ). however the study was confounded by the fact that physicians were, at the time, prescribing tnfis for treatment of ra-ild, possibly accounting for such an association. the incidence of hospitalisation for ild requiring hospitalisation was reported per , patient-years with a % mortality, likely reflecting the types of patients included in this analysis. whilst the authors concluded that there was no evidence of an association between tnfi and hospitalisation for ild as the observed effect with tnfi was likely to be due to confounding by indication, the specific effect of tnfi on patients with ra-ild at baseline was not assessed [ ] . two claims database studies have evaluated ild incidence in patients treated with biologics including tnfis. to assess the risk of incident ild in patients with autoimmune diseases who were members of kaiser permanente, northern california, herrington and colleagues compared new users of tnfi and nbd-mards [ ] . ild cases were identified using icd codes, with a pilot of the first cases verified using ct reports. the study demonstrated no increased risk in the tnfi-exposed group of new ild; however, patients with known ild at baseline were systematically excluded. using data from the commercial claims and benefit (medicare) databases, curtis et al. assessed the ild incidence and exacerbation amongst users of rituximab, abatacept and tocilizumab to tnfi agents [ ] . two definitions of ild were used (one more sensitive, the other more specific in the absence of ct results and lung histology). there were no significant differences in the risk of ild and its related complications between patients with ra receiving tnfis and those on non-tnfis. of the patients' studies, patients had a history of ild, of which were put on tnfi agents [ ] . however there were clear baseline differences between the groups receiving the different drugs, which were not adjusted in the analysis. therefore the true risk in the tnfi group may be confounded if patients with higher levels of ra joint disease or ild disease severity were channelled to specific therapies, which may have led to an overall underrepresentation of risk in this group. in a recent single-centre retrospective evaluation from japan, nakashita et al. described up to one-year outcomes of ra patients with (n = ) or without (n = ) established ild on a biologic drug [ ] . in patients with established ra-ild, ( %) had an exacerbation of ild, which the authors concluded was greater in patients exposed to tnfi agents using descriptive statistics. none of the patients on tocilizumab (n = ) or abatacept (n = ) were reported to have an exacerbation; however, conclusions were limited due to low numbers. similar to curtis et al.'s study [ ] , it did not adjust for severity of ra or any confounders potentially introducing channelling bias. in this case, it is likely that patients on tnfi (approved earlier than the non-tnfi drugs in this study) had more severe disease at the outset, in turn is known to be associated with the outcome. data from the british society for rheumatology biologics register for ra (bsrbr-ra) evaluated the effect of tnfi in patients with ra who have established ild [ ] and reported no difference in all-cause mortality between patients on tnfis and nbdmards. however ra-ild as an underlying cause of death was reported in a higher proportion of tnfi-treated patients than nbdmard-treated patients ( vs. %), suggesting a signal for concern. methodological challenges included potential for misclassification, as prevalent ild was identified from clinician questionnaires since attempts to retrospectively verify such cases were unsuccessful. whilst it could be that tnfi truly increased the proportion of deaths attributable to ra-ild, given the well-publicised respiratory concerns with tnfi, it may be that physicians at that time were more inclined to report ra-ild on death certificates of patients exposed to tnfis leading to the observed relationship. pfts, imaging or nonfatal exacerbation of ild details were not available to examine the effect of treatment on such outcomes. challenges in interpreting the data common to all studies is delineating the effect of tnfi on ra-ild in the presence of nbdmards, the majority of which are linked to reports of pneumonitis or exacerbation of ra-ild. furthermore, it can be difficult to ascertain if severity of ra associated with both development of ild and also being the indication for tnfi could explain the association between tnfi and ra-ild exacerbation. however, no studies have completely excluded a clinically meaningful risk, and the multiple case reports, observational evidence and possible increase in ild-specific mortality in patients with known ra-ild indicate that careful monitoring and caution is required when considering use in this setting. with several biologics approaching patent expiration, substantial interest has been in the development of biosimilar products that are not bioidentical but highly similar to already approved reference products listed above [ ] . to date, biosimilar infliximab ct-p (marketed as remsima and inflectra) and biosimilar etanercept sb (marketed as benepali) have been approved for use by the fda and ema. there were no cases of pneumonitis or ild in the biosimilar infliximab ct-p trials in ra [ , ] or ankylosing spondylitis [ ] , although there was a case of noninfective dyspnoea reported within the ct-p arm of the latter trial felt to be related to the drug by the investigators. open-label extensions of these trials have not suggested a signal for concern [ , ] . similarly no cases of new ild were reported with biosimilar etanercept sb , albeit treatment-emergent aes in ≥ % of patients were reported only [ ] . one patient in the sb arm died due to cardiorespiratory failure (felt to be unrelated to the drug by the investigators), and further details of the event were not provided [ ] . whilst this provides some initial reassurance, biosimilars should be considered as having similar risks to the originator reference products and therefore be used with vigilance in the context of established ra-ild. rituximab is a monoclonal chimeric anti-cd antibody licensed for the treatment of non-hodgkin lymphoma and ra in tnfi nonresponders. rituximab-induced ild has been a well-described ae in haematology patients often presenting as acute/subacute hypoxaemic organising pneumonia, nsip or hypersensitivity pneumonitis [ ] [ ] [ ] . whilst its pathogenesis is unknown, it may involve induction and release of cytokines, and it has been reported to occur in patients receiving rituximab for several months [ ] . in ra, a number of rituximab trials have reported incident ild [ ] [ ] [ ] , including subsequent deaths from acute respiratory distress syndrome (ards) [ ] and culture-negative (non-infective) bronchopneumonia [ ] . hadjinicolaou et al. identified > cases of rituximab-associated ild up to june in a systematic review of published studies and reports to the fda and ema [ ] . in addition, uk spontaneous pharmacovigilance reporting systems (via its yellow card system) have recorded hundreds of rituximab-associated cases of non-infectious respiratory disorders till march (table . ). of the patients with possible rituximab-associated ild/pneumonitis, deaths have been reported (table . ). further observational evidence has been discussed in section "other agents with limited agents in ra-ild"). abatacept is a selective t-cell costimulation modulator. it is a fully human recombinant protein that comprises of the extracellular domain of ctla and the fc portion of an igg molecule that has been modified to prevent complement activation. pooled safety data from eight trials of abatacept involving patients with ra reported incident ild in patients ( . cases per person-years), with no events reported in the placebo control groups [ ] . patients with pre-existing ra-ild were excluded from all of the initial abatacept trials [ ] . therefore limited conclusions can be drawn from existing trial data, which may suggest a small increase in incident ild in patients with ra. despite its fda licence back in , few case reports exist describing abataceptinduced lung injury. one case reported drug-induced respiratory failure, weeks after the second abatacept dose [ ] , however was unable to distinguish if infection was the reason for deterioration by the time of death. relatively few respiratory adrs have been reported to the regulatory agencies after treatment with abatacept compared with other biologic agents (table . ). indeed, only one case of pneumonitis and cases of non-infectious respiratory events had been reported through the uk medicines and healthcare products regulatory agency (mhra) 'yellow card' system by march . however it is worth noting at exposure to abatacept may be limited in the uk due to lack of its initial approval by the national institute of health and care excellence (nice) until as a second-line biologic (following tnfi failure and if rituximab was contraindicated) and first-line biologic approved in . whilst experience of using abatacept in the context of pre-existing ild is limited, a case report of rapid-onset interstitial pneumonia days post initiation of administration, mhra medicines and healthcare products regulatory agency, na data not available, ra rheumatoid arthritis a includes cases reported as idiopathic pulmonary fibrosis, pulmonary fibrosis and pulmonary toxicity treatment has been described in a japanese patient [ ] . conversely, there are reports of stabilisation of ra-ild on abatacept [ ] and improvement in some patients [ ] . in a case series of patients with ra-ild (with varying grades of severity at baseline), all patients completed weeks of abatacept treatment, and no one was reported to have an exacerbation of their pulmonary disease [ ] . in the three patients with ra-ild on abatacept, as part of nakashita and colleagues' retrospective study [ ] , none had ild-related complications reported by one-year follow-up. however using claims data, in the curtis et al. study [ ] , of the patients with ra-ild on abatacept, there was no significant decreased risk of ild events compared to tnfis. in comparison with other biologic agents introduced to the market at around the same time, the spontaneous pharmacovigilance and case report figures are cautiously encouraging. the lack of a clear denominator or the number of patients treated with each drug in these settings makes accurate interpretation challenging. whilst experience in baseline ra-ild is limited, emerging observational data thus far appears tentatively reassuring. additionally patients on abatacept may have a more favourable infection profile in comparison to other biologics in those who have experienced a hospitalised infection previously [ ] . this study also forms the basis of its use as a conditional recommendation in the context of prior serious infection in the acr guidelines for ra [ ] . therefore given the limitations of spontaneous pharmacovigilance and inconsistent findings from observational data, more observational data is required before robust conclusions can be formed regarding safety of abatacept in ra-ild. however, there may be a role for abatacept particularly in patients in whom serious infection is a concern. tocilizumab tocilizumab is a humanised monoclonal antibody that inhibits il- receptor signalling through its membrane-bound and soluble forms. in the treatment of active joint disease, where mtx is contradicted or not tolerated, tocilizumab monotherapy has been shown to be as effective as combination treatment with mtx [ , ] . given the potential concerns regarding the respiratory safety profile of mtx, there may be a preference for using this drug over other biologics in ra-ild by some physicians. experimental work has demonstrated profibrotic effects of il- on lung fibroblasts, which may have the potential to be antagonised by blocking the il- / il- receptor pathway, suggesting a potential benefit of tocilizumab in ra-ild [ ] . however, clinical data on the use of tocilizumab in pre-existing ra-ild is sparse and inconclusive. unlike other biologics, early rcts of this biologic agent have raised some concerns regarding an association with development of ild. a single case of 'allergic pneumonitis' after tocilizumab monotherapy exposure in patients with ra was reported in by nishimoto et al. [ ] . in , two of patients treated with tocilizumab and mtx in the option study [ ] developed ild at weeks and , with a further two cases of 'culture-negative pneumonia'. a similar case was also reported in the satori [ ] trial, which included patients on tocilizumab monotherapy. in one of the few head-to-head biologics studies, the adacta trial compared tocilizumab monotherapy with adalimumab monotherapy and reported two deaths in the tocilizumab arm, one of which occurred suddenly in a -year-old man with multiple comorbidities, including pre-existing ild [ ] . further deaths due to interstitial pneumonitis have been reported in tocilizumabexposed arms in recent trials such as the surprise (tocilizumab + mtx arm) [ ] , as well as the summacta trial, the latter reporting one death due to ild and ards each [ ] . of note other anti-il- drugs being evaluated for ra, such as sarilumab (a fully human anti-il- rα mab that binds membrane-bound and soluble human il- rα), have also reported sraes within trials. of the two deaths reported in the saril-ra-mobility trial (sarilumab +mtx), one was due to ards (investigator felt was drug-related) [ ] . within the phase iii study published to date, four deaths in the sarilumab arms were described [ ] , one due to unspecified respiratory complications post surgery. whilst the consequences of such reports are not clear at present for the patient with pre-existing ild, such observations suggest the need for alertness especially since most trials exclude patients with multimorbidity. case reports have suggested an association between incident ild and tocilizumab exposure in patients on a concurrent nbdmards both in ra [ ] [ ] [ ] and adult-onset still's disease [ ] . spontaneous pharmacovigilance figures from the uk have reported five deaths due to sraes in tocilizumab-exposed patients to date, four due to ild and one secondary to ards (table . ). whilst the total number of patients exposed to treatment (denominator) is not available, cautious comparison of these figures with abatacept, which was approved earlier than tocilizumab by fda and ema, is of interest. furthermore, the reaction study, a retrospective study of tocilizumabexposed patients with ra in japan, reported interstitial pneumonia in two of the patients followed up over months and an additional six discontinuations for pneumonia (presumed infective), raising further concerns of pulmonary toxicity in this population. ra-ild was found to have improved in one patient with pre-existing ra-ild within weeks after administration of tocilizumab [ ] , whereas another patient with biopsy-proven uip and emphysema developed a fatal exacerbation of ild after treatment with tocilizumab [ ] . of the ild events reported in patients who received tocilizumab every weeks over weeks ( . per patient-years) in a post-marketing surveillance programme in japan, the presence of a known ild was a risk factor for acute presentation with ild exacerbation and serious infections [ ] . recent small retrospective observational studies demonstrate limited reassurance. in the nine patients with ra-ild selected to receive tocilizumab treatment over year in the nakashita et al. study [ ] , no patients were reported to have an ild exacerbation. however patients who did not complete at least year of follow-up lacked imaging data or discontinued treatment because infections were excluded from the study leading to a likely selection bias of reported outcomes. in a retrospective case-control study in ra, patients were stratified according to the presence of baseline ra-ild (n = ) or without (n = ) [ ] . during observation period of . patient-years and . patient-years for the ra-ild and non-ild groups, respectively, six patients developed an acute exacerbation in the ra-ild group (none in the non-ild group). interesting all the patients who developed an exacerbation of ild were on tocilizumab monotherapy, and there was a suggestion that all patients who developed an exacerbation had more uncontrolled articular disease that may in turn be associated with the outcome [ ] . the administrative data study by curtis et al. concluded no significant differences between tocilizumab and other biologic drugs in the risk of ild or its exacerbation [ ] . however exacerbation of ild can be difficult to define in such databases as recognised by the authors. hence surrogate measures such as hospitalisations for ild, pneumonia or lung transplant were used, which as expected were observed infrequently and capture a heterogeneous overall outcome that includes infection. therefore, whilst current evidence does not allow robust conclusions about the pulmonary safety of tocilizumab in ra-ild, ongoing vigilance is recommended if used in this context in the absence of clear predictors of ild exacerbation. anakinra is a recombinant il- antagonist, which binds competitively to the type i il- receptor and therefore acts as a competitive antagonist to il- . initial trials that supported the licensing in ra did not demonstrate a signal of concern in relation to incident ild either as monotherapy [ ] [ ] [ ] or in combination with mtx [ ] . whilst such data supported the initial licensing of anakinra in ra, in clinical practice, it is not widely used in ra for active joint disease as it appears less efficacious than tnfi agents and of no additional value in early ra [ ] . moreover, combination of anakinra and etanercept provided no added benefit compared to etanercept monotherapy, and indeed increased serious safety concerns were reported including two patients with new ild and pneumonitis in the combination biologic arms [ ] . there are sparse reports of non-infective sraes on anakinra from spontaneous pharmacovigilance (table . ), possibly reflecting fewer exposed patients with ra. therefore apart from one case of improvement in tocilizumab-induced pneumonitis following anakinra in a patient with adult-onset still's disease [ ] , there is no further literature indicative of non-infective sraes with its use [ ] but also minimal evidence to suggest a potential beneficial role in ra-ild. janus kinase (jak) inhibitors, such as tofacitinib and baricitinib, are synthetic orally administered compounds that block jak, a protein that mediates signal transduction of multiple cytokines. although these are not biologic drugs, though they interact with biological pathways, they have been classed as targeted synthetic dmards (tsdmards) for use in moderate to severe ra, who are nonresponders or intolerant to mtx [ , ] . tofacitinib has been approved in several countries, such as in the usa, latin america and asia although not in the european union or uk. baricitinib has completed phase iii trials, and early results from head-to-head trials even suggest that it may be more efficacious than a tnf inhibitor [ ] . rct data thus far with regard to respiratory safety of jak inhibitors are inconclusive. phase iii trials of tofacitinib with concomitant mtx in patients with ra have reported cases of new-onset ild and pulmonary sarcoidosis [ ] . other sraes reported in tofacitinib-treated arms in rcts include bronchopneumonia [ ] and a death due to ards [ ] . a combination of pulmonary fibrosis and chronic obstructive pulmonary disease has been observed in such trials using tofacitinib monotherapy [ ] . however data from two long-term extension studies from japan following up tofacitinib-treated patients over patient-years reported no additional ild events [ ] , nor did subsequent meta-analysis of tofacitinib trials [ ] . in addition phase iib studies and phase iii studies of baricitinib also have not reported incident ild cases [ , ] and is currently under regulatory review. in , the fda released a risk evaluation and mitigation strategy (rems) document highlighting the known concerns with tofacitinib, instructing the pharmaceutical company to send information out to healthcare professionals regarding the risk of serious infections, malignancies, decreases in peripheral lymphocyte counts, neutrophil counts and haemoglobin and derangement in lipid profiles [ ] ; lung toxicity was not mentioned. no case reports or regulatory reports of ild have been published to date, although tofacitinib was licensed for the treatment of ra a few years ago in november in the usa and remains unlicensed in most of europe, limiting the numbers of exposed patients. conclusions drawn regarding the association between jak inhibitors and ild in patients with ra are therefore restricted, given limited data and further observational evidence are required to assess its effect on pre-existing ra-ild. although ra-ild is a fairly heterogeneous extra-articular manifestation of ra, the majority of cases mimic the two idiopathic interstitial pneumonia patterns of nsip and uip. whilst nsip and organising pneumonia appear to be more responsive to glucocorticoid treatment, the presence of the uip pattern on hrct in patients with ra may be associated with a significantly shorter survival compared to other forms of interstitial disease [ ] . as a general clinical guidance, lung disease features that may be predictive of treatment benefit include histopathologic patterns other than uip (especially nsip and op, younger age of the patient and worsening of symptoms, pfts or finding on hrct over the preceding - months [ ] [ ] [ ] . a dlco of less than % is associated with progression and poor prognosis and may identify patients who could be considered for treatment [ ] . numerous medications have been described for potential therapies for ra-ild, but currently there are no large rcts to help guide physicians specifically in the management of the pulmonary manifestations of ra-ild. in addition to immunosuppressive treatments, general considerations for management of ra-ild include smoking cessation and age-appropriate vaccinations for pneumonia and influenza, as well as prophylaxis for pneumocystis jirovecii pneumonia in patients who are profoundly immunosuppressed [ ] . typically glucocorticoids were used as first-line therapies in an attempt to stabilise and improve the disease course of ra-ild based on limited evidence from ipf. because ra-ild is a heterogeneous spectrum of histopathologic patterns beyond uip (ipf), experience extrapolated from management of idiopathic ild suggests that some forms, including nsip and op, may respond to glucocorticoid therapy [ ] . given the paucity of evidence in ra-ild, it was felt that the addition of azathioprine maybe beneficial in glucocorticoid-responsive patients and could result in improved survival compared to glucocorticoids alone, again based on historic data from ipf studies [ ] . however, the multicentre prednisone, azathioprine, and (n) acetylcysteine [nac]: a study that evaluates response in ipf (panther-ipf) trial had to be prematurely stopped as it is found that combination of prednisone, azathioprine and nac was associated with greater mortality (eight vs. one death), more hospitalisations ( vs. ) and more serious aes ( vs. ) compared to placebo in ipf patients with mild to moderate disease [ ] . a large proportion of deaths were due to pulmonary infection, highlighting the need for adequate precautions to be taken against respiratory infections in patients with ra-ild who are susceptible to serious infections [ ] . indeed in elderly patients with ra, prednisolone has been shown to have a dose-dependent risk of infection, with current/recent glucocorticoid therapy demonstrating the greatest impact on infection risk [ ] . therefore use of glucocorticoids specifically for ild in ra is best performed after liaison with respiratory colleagues, with use of the lowest possible dose prescribed for the shortest duration. pirfenidone is an anti-fibrotic drug that inhibits transforming growth factor beta (tgf-β)-stimulated collagen synthesis, decreases the extracellular matrix and blocks fibroblast proliferation. it has demonstrated efficacy in ipf [ ] [ ] [ ] and has been approved by the national institute of health and care excellence in the uk and the fda in the usa for use in patients with mild or moderate ipf. in ctd-ild, specifically secondary to systemic sclerosis, case reports and small retrospective studies have suggested modest benefit [ ] [ ] [ ] [ ] , whilst a recent open-label study in systemic sclerosis demonstrated acceptable tolerability of pirfenidone, especially important since . % of patients were on concomitant mycophenolate mofetil [ ] . currently there is no evidence for its use in ra-ild. theoretically, however, given its action on tgf-β and fibroblast proliferation, there may be justification of its use in the fibrotic nsip pattern and fibrotic stages of other ra-ild subtypes [ , ] . currently there is a phase ii trial underway to assess the safety and tolerability of pirfenidone mg/day for the treatment of ra-ild [ ] , with a need for more rcts the effect on respiratory function in this setting. other anti-fibrotic agents such as nintedanib, a tyrosine kinase inhibitor that targets multiple tyrosine kinases, including vascular endothelial growth factor, fibroblast growth factor and platelet-derived growth factor receptors, have been recommended by the international thoracic guidelines for its use in ipf [ ] . it was recently found in various murine systemic sclerosis models to effectively inhibit the endogenous as well as cytokine-induced activation of fibroblasts and exert potent anti-fibrotic effects [ ] . whilst there is currently no clinical evidence to date in ctd/ra-ild, there may be potential for future clinical trials with this drug. cyclophosphamide has been commonly used to treat ild unresponsive to glucocorticoids. however evidence of its use in ipf is lacking [ ] . conflicting data exist regarding its use in scleroderma-related ild [ ] , with additional concerns regarding its toxicity profile. rcts using cyclophosphamide suggest moderate benefit in scleroderma-ild patients with early disease [ ] [ ] [ ] , although a previous metaanalysis concluded no improvement in pulmonary function following months of treatment [ ] . no rcts have been performed assessing the use of cyclophosphamide in ra-ild. limited evidence suggests that there may be some role in rapidly progressing patients with restricted therapeutic options in the acute or subacute setting [ ] or in refractory drug-induced pneumonitis unresponsive to glucocorticoids [ ] . the use of this agent is not recommended for mild/moderate stable ra-ild disease. experience of using cyclosporine in the treatment of ra-ild is limited and not recommended currently due to its poor safety profile and absence of proven benefit on pulmonary or joint disease. few publications in ipf have been less than encouraging [ , ] ; however, it appears to yield some benefits according to anecdotal reports in myositis-related ild particularly anti-synthetase syndrome [ ] [ ] [ ] . mycophenolate mofetil often used in the treatment of scleroderma-ild is an inhibitor of lymphocyte proliferation and additionally targets nonimmune cells such as fibroblasts and smooth muscle cells. the majority of the evidence for use has been derived from small prospective case series and retrospective reviews and has been shown to stabilise scleroderma-ild [ ] [ ] [ ] and ctd-ild [ ] . in the latter series, patients with ra-ild were included; mycophenolate was associated with modest improvements in forced vital capacity (fvc), diffusing capacity and reductions in the prednisone dose [ ] . a head-to-head rct assessing the use of a twoyear course of mycophenolate compared to oral cyclophosphamide for months in ssc-ild has recently been published, demonstrating a more favourable safety profile with mycophenolate; however, both treatment arms demonstrate similar efficacy on lung function [ ] . in the treatment of ra-ild, mycophenolate has been prescribed at doses of - g per day in patients with ra who have limited pulmonary disease with some benefit [ , ] . however it is not effective in the treatment of active articular disease on ra, requiring the use of concomitant nbdmards, which may have additional consequences on tolerability. whilst studies supporting the use of mycophenolate in ra-ild represent a relatively small number of patients, further work on tolerability and safety in the context of active ra is required, and if promising well-designed trials may be helpful before advocating its use in this area. of the biologics having potential utility for treatment of ra and other connective tissue disease-related ild, perhaps rituximab has had the most interest and shown some promising results in published case reports and case series, not inclusive of ra [ ] [ ] [ ] [ ] . a recent retrospective review of ctd-ild cases treated with rituximab, which included anti-synthetase syndrome (n = ), dermatomyositis (n = ), systemic sclerosis (n = ), systemic lupus erythematosus (n = ) and unclassifiable ctd-ild (n = ), suggested stabilisation of lung disease in patients and worsening in nine patients. four patients with myositis had a reported clinically significant improvement with an fvc of > % post treatment. whilst encouraging in a ctd-ild setting, most published work is in the form of case reports, subject to reporting bias, or retrospective case series. evidence of rituximab use for the treatment of ra-ild has not been encouraging and studies to date have been small or inconclusive. in an open-label pilot study of rituximab in ra-ild [ ] of seven patients who completed a -week follow-up, one showed improvement in respiratory function, five were stable and one deteriorated. the study initially recruited ten patients, and of the three who did not complete the study, one patient died of ards/possible pneumonia, weeks post treatment (no infective source identified). four small retrospective observational studies assessing the use of rituximab in ra-ild have been inconclusive (all still in abstract form, full papers not published). dass and colleagues in [ ] presented data from patients with baseline ra-ild; three deaths were reported in patients with ra-ild following rituximab treatment, one due to pneumonia and possible acute progression of ild, weeks after the first cycle of rituximab therapy. furthermore, five patients had a decline in transfer factor of > % over an unspecified time in the presented results of this study. the same group reported their rituximab experience in ra-ild over years (january -july ) in [ ] . of the patients with ra-ild on rituximab with a total of patient-years, the authors concluded there was no significant improvement or deterioration in the majority (as measured using fvc/ dlco; actual data not available in abstract). however there were deaths reported, nine of which were attributed to progressive ild (median dlco of % [range - %] pre-rituximab). other reasons for death were lung cancer, colon cancer and infection post surgery (n = each). becerra et al. [ ] reported on a single-centre retrospective review of patients with ra and known lung involvement who were undergoing therapy with rituximab, of whom had established ra-ild. progression of ild over years was described in one patient with severe uip at baseline. improvement in lung function was observed in none of the patients, % (n = ) reporting respiratory infections, two of which required hospitalisation. recent work from the bsrbr-ra compared mortality of patients with physician-reported ra-ild, of whom were treated with tnfi and on rituximab [ ] . the differences in mortality between the two groups were not statistically significant, and adjustment for baseline confounders made little difference to the estimates (hr adj . , % ci . - . ). however, methodological issues included inherent differences in the two treated groups (the tnfi group being a more historic cohort, therefore likely to have more severe disease), low numbers of deaths and no information on baseline ra-ild severity. the results of all four of these studies are difficult to interpret often due to a lack of well-matched comparator groups and uncertainty about the natural history of ra-ild. therefore, currently, few data exists that suggest that rituximab can markedly improve ra-ild. in comparison to other biologics, there did not seem to be a significantly lower risk of complications related to ild in the curtis et al. study [ ] , although channelling bias may exist. furthermore rituximab is known to reduce igg levels, which in turn may be associated with an increased infection risk, which bears consideration whilst making such treatment decisions [ ] . in the context of ra overlap with another ctd, however, rtx may have a role in stabilising ild, especially with other extra-pulmonary manifestations of ctd which exist that may benefit from b-cell depletion. a clinical trial is currently underway comparing rituximab versus cyclophosphamide in patients with systemic sclerosis, myositis and mixed connective tissue disease (recital, clinicaltrials.gov identifier: nct ). currently, therefore, the evidence does not support preferentially switching to rituximab in all patients with ra-ild who have active joint disease, regardless of the severity of lung disease. in end-stage refractory ra-ild, single lung transplantation may be considered. however there are limited data on long-term outcomes for lung transplant in ra-ild. amongst ten patients with ra-ild who underwent transplantation, survival at year was similar to ipf lung transplant recipients ( and %, respectively), although higher for scleroderma-ild at % [ ] . eligibility for lung transplantation may be restricted due to age (typically < years), contraindicated with certain comorbidities common in ra including osteoporosis, whilst other extra-articular manifestations of ra may in turn complicate transplantation. nonetheless, comparable survival outcomes of ra-ild with ipf are somewhat reassuring. in summary at present, there are no current treatments available that have appeared to consistently stabilise ra-ild as well as adequately treat joint symptoms. the risks and limited benefits of such medications bear consideration, especially since most evidence for efficacy has been demonstrated in ipf and ctd-ild. glucocorticoids may be used in ra-ild, however, with the lowest effective dose for the shortest possible duration to minimise risk of infection amongst others. robust rcts are required to support the use of novel antfibrotic agents or immunosuppressive therapies directed towards treatment of ra-ild. however, at present there is a paucity of evidence to support the routine use of these strategiest. evidence to guide management and treatment of ra-ild is of low quality or absent, hence extrapolated from systemic sclerosis-ild or ipf. however, despite the lack of robust data, clinicians are required to advise patients on the comparative safety of treatment and strategies to minimise complications of ra-ild such as infection and reduce the overall burden of morbidity to prolong survival. in addition ra management has become increasingly complex over the last decade. several effective dmards are available, and treat-to-target strategies support the use of early, rapidly escalating treatment often in combination with each other. as discussed earlier, there are no treatments that conclusively improve both joint and pulmonary disease in all patients; therefore, careful baseline assessment, adequate risk assessment for adverse outcomes and regular monitoring and vigilance whilst using the majority of dmard strategies are essential. to enable guidance of management strategies, a recent approach in idiopathic interstitial pneumonias (iip) has been to classify pulmonary disease as self-limiting, reversible, stable, progressive or irreversible [ , , ] . a thorough assessment of baseline ra-ild severity should consider clinical features, pfts and imaging pattern/extent on hrct, low dlco and a high radiological fibrosis score on hrct being predictors of poor survival [ ] [ ] [ ] . such information in combination with monitoring disease progression over time allows mapping of the disease trajectory and may better inform management decisions in the face of poor evidence. clinical assessment including quantification of exercise tolerance using instruments such as the five-point medical research council (mrc) breathlessness scale [ ] can help aid serial assessment of the symptoms of ra-ild (table . ). bibasilar crepitations are an associated feature that may help trigger detection of asymptomatic ild, but have limited value in assessing severity or monitoring progression. the -min walk test is a commonly used instrument for assessing ipf and can be performed easily. reduced walk distance and oxygen desaturation below % are poor prognostic factors in ipf [ , ] ; however, practically this may be challenging to perform in ra patients with poor mobility. pfts provide objective measures of lung function and should be performed in all patients with respiratory clinical features, or confirmed ra-ild, prior to decisions about therapy. ilds share a restrictive pattern, with reductions in lung volumes and a reduced dlco [ ] . low baseline fvc and dlco (fvc < % and dlco < % of predicted values) are independent predictors of early death in patients with ipf [ , ] . importantly, a - -month decline in fvc of ≥ %, or a decline in dlco of ≥ %, is associated with increased mortality in ipf [ ] ; this association might also exist in patients with idiopathic fibrotic nsip. bdmards biologic disease-modifying antirheumatic drugs, dlco diffusing capacity of the lungs for carbon monoxide, fvc forced vital capacity, ild interstitial lung disease, hrct highresolution computed tomography, nbdmards non-biologic disease-modifying antirheumatic drugs, pfts pulmonary function tests, tb tuberculosis, uip usual interstitial pneumonia hrct imaging is indicated in patients with ra-ild clinical features or in asymptomatic patients with a dlco of < % of predicted [ ] . individuals with hrct findings consistent with uip, such as basally dominant honeycomb cysts with little or no ground-glass change, have poorer prognosis than those with hrct-detected features indicative of other types of iip [ , ] . in ssc-ild, an hrct-based prognostic staging system has been developed, categorising patients as having limited (< % lung involvement) or extensive (> % lung involvement) ssc-ild, with extensive disease found to be a strong predictor of mortality [ ] . similarly quantifying the extent of fibrosis and disease on hrct has been shown to predict disease progression in ipf [ ] . no comparable studies have assessed the use of similar hrct-based scoring in ra-ild, although a small study suggested that the degree of interstitial changes detected using hrct imaging was predictive of prognosis [ ] . on the basis of these factors, the authors have previously proposed a framework for assessment of ra-ild, risk assessment before initiation of biologic therapy and post-treatment monitoring [ ] (fig. . ). the proposed approach is designed to help predict short-term progression of ild irrespective of biologic therapy. in patients at higher risk of progression, even in patients who appear to have stable lung disease over a one-or two-year period, treatment decisions should involve a multidisciplinary approach including a respiratory physician as well as a careful discussion with the patient regarding the benefits and risks of treatment options available. in all patients, including those with self-limiting and stable ra-ild on treatment for their joint disease, rheumatologists should prompt questioning of new or evolving respiratory symptoms at follow-up, as patients may not spontaneously disclose such information. all patients who smoke should be educated about the important associated risks. whilst being implicated in the pathogenesis of ra-ild, it may also lead to deterioration of lung disease and is associated with higher joint disease activity and reduction in the effectiveness of medications such as mtx and tnfi drugs [ ] . smoking cessation advice and further support such as nicotine replacement therapy should be made available for all patients with ra-ild [ ] . following an adequate period of monitored observation, classification of the trajectory of ra-ild may be possible in the individual patient. in patients in whom a - -month period of stability is observed, pft follow-up intervals can be extended to monthly. routine monitoring of pfts is suggested every - months in individuals at high risk of progression of ra-ild or on any biologic drug [ ] . an observed decline following serial pfts could be due to progression of ra-ild, either its natural [ ] history or accelerated by therapy or attributable to other causes that may or may not have an association with the dmard prescribed. 'reversible' decline has been categorised into potentially reversible with risk of irreversible disease (e.g. cases of drug-induced lung disease) or reversible disease with risk of progression (e.g. ra-cellular nsip, some ra-fibrotic nsip, ra-organising pneumonia) [ ] . management strategy for the former would involve cessation of the putative drug with or without further treatment with glucocorticoids if required, whilst the latter may require more prolonged therapy and monitoring. in practice, the two are difficult to delineate given their similar presentations, and therefore rheumatologists faced with such patients demonstrating worsening respiratory symptoms or lung function should consult with respiratory colleagues early. if the deterioration in clinical picture is found to be due to progression of ra-ild, the decision to continue any dmard, but especially bdmards or tsdmards, necessitates clinical decisions on a case-to-case basis. the choice of whether to continue with effective dmard therapy for joint disease or the risk of potential pulmonary toxicity may be based on the perceived likelihood that the dmard in question is driving deterioration (e.g. temporal relationship of starting the drug with the clinical scenario). currently there is no observational evidence that preferentially switching between biologics, for instance, helps prolong survival or stabilises the disease; however, this may be tried in individuals that require management of their articular disease following careful discussion. in patients deemed to have progressive, irreversible disease, management of patients with ra-ild in a joint pulmonary and rheumatology clinic should be especially considered where possible, with attempts made at stabilisation. in all patients, and especially in those with multimorbidity, conservative and nonpharmacological options that may be worth exploring include education, psychological support, pulmonary rehabilitation and supplemental oxygen if appropriate [ , , ] . sraes following treatments for ra may be due to incidence/exacerbation of ra-ild or due to other reported pulmonary complications summarised in table . . careful consideration of the underlying aetiology of sraes is essential, as deterioration in chest symptoms may not be due to pneumonitis or progression of ild, which has been the focus of review in this chapter. a thorough investigation of common and opportunistic infections is imperative in patients with ra, especially on high glucocorticoid doses, certain nbdmards, bdmards and tsdmards. a full review of infection risk and biologics is beyond the scope of this chapter, as has been extensively reviewed recently [ , ] . rare reports of pulmonary manifestations of certain drugs have also been reported in the literature and are summarised in table . . for instance, lymphoproliferative disease (including non-hodgkin lymphoma) have been described during treatment with mtx and may regress following cessation [ , ] . tnfis have been associated with paradoxical aes (associated with the management of the rheumatoid arthritis patient with interstitial lung disease treatment and possible induction of the same event) such as sarcoidosis and vasculitis [ ] . the authors recommend a low threshold for withholding treatment with such cases, close liaison with respiratory and infectious disease physicians and imaging using hrct, which in turn may help determine the cause of the deterioration. the respiratory safety of ra therapy is an important consideration whilst deciding on the best treatment for patients with ra with active joint disease and coexisting ild. this chapter summarises the current evidence available of the use of dmards in the context of pulmonary toxicity. however several limitations of the existing literature exist. whilst ra-ild is increasingly recognised, its natural history is still poorly understood. with newer treatments, case reports are helpful to identify a signal for concern, although such case reports are insufficient alone to provide a clear picture of drug toxicity. where observational evidence is available, confounding by indication and channelling to certain treatments may limit robust conclusions. the lack of adequate comparator groups in several retrospective studies may no conclusive data on pneumonitis risk bdmards biologic disease-modifying antirheumatic drugs, dress drug reaction with eosinophilia and systemic symptoms, ild interstitial lung disease, nbdmards non-biologic biologic disease-modifying antirheumatic drugs, tsdmards targeted synthetic disease-modifying antirheumatic drugs further limit inferences drawn from the evidence. it appears that legitimate concerns are associated with several therapies; therefore, involving patients and multidisciplinary teams in such decisions is important. careful discussion of the benefits and harms of treatments is encouraged, although this is made more challenging by the uncertainty of the current safety profile. systematic pre-and post-treatment assessment can help identify the trajectory of patients with ra-ild and enable more effective risk stratification, enabling clinicians and patients to make better-informed decisions. unfortunately at present, there are limited therapeutic options for additional treatment for specifically stabilising or reversing established fibrosis; however, emerging treatments such as novel anti-fibrotics may hold promise. given the current evidence base, the decision to start an dmard in patients with ra who have ild should be based on its potential for improving joint disease, individual patient characteristics, patient education about the known risks and benefits of treatment and multidisciplinary team input. rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative treating rheumatoid arthritis to target: update of the recommendations of an international task force eular 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ankylosing spondylitis: the planetas study efficacy and safety of ct-p (biosimilar infliximab) in patients with rheumatoid arthritis: comparison between switching from reference infliximab to ct-p and continuing ct-p in the planetra extension study efficacy and safety of switching from reference infliximab to ct-p compared with maintenance of ct-p in ankylosing spondylitis: -week data from the planetas extension study a phase iii randomised, double-blind, parallelgroup study comparing sb with etanercept reference product in patients with active rheumatoid arthritis despite methotrexate therapy rituximab-induced lung disease: a systematic literature review rituximab-induced interstitial lung disease rituximab-induced interstitial lung disease: five case reports lung involvement and drug-induced lung disease in patients with rheumatoid arthritis safety and effectiveness of rituximab in patients with rheumatoid arthritis following an inadequate response to prior tumor necrosis factor 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rheumatoid arthritis patient after the administration of abatacept a case of rheumatoid arthritis complicated with deteriorated interstitial pneumonia after the administration of abatacept refractory rheumatoid arthritis with interstitial lung disease: could abatacept be the answer? abatacept can be used safely for ra patients with interstitial lung disease risk of hospitalised infection in rheumatoid arthritis patients receiving biologics following a previous infection while on treatment with anti-tnf therapy american college of rheumatology guideline for the treatment of rheumatoid arthritis adding tocilizumab or switching to tocilizumab monotherapy in methotrexate inadequate responders: -week symptomatic and structural results of a -year randomised controlled strategy trial comparison of adding tocilizumab to methotrexate with switching to tocilizumab in patients with rheumatoid arthritis with inadequate response to methotrexate: -week results from a prospective, randomised, controlled study (surprise study) inverse effects of interleukin- on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs treatment of rheumatoid arthritis with humanized anti-interleukin- receptor antibody: a multicenter, double-blind, placebo-controlled trial effect of interleukin- receptor inhibition with tocilizumab in patients with rheumatoid arthritis (option study): a double-blind, placebocontrolled, randomised trial study of active controlled tocilizumab monotherapy for rheumatoid arthritis patients with an inadequate response to methotrexate (satori): significant reduction in disease activity and serum vascular endothelial growth factor by il- receptor inhibition tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind, controlled phase trial efficacy and safety of subcutaneous tocilizumab versus intravenous tocilizumab in combination with traditional dmards in patients with ra at week (summacta) sarilumab, a fully human monoclonal antibody against il- rα in patients with rheumatoid arthritis and an inadequate response to methotrexate: efficacy and safety results from the randomised saril-ra-mobility part a trial sarilumab plus methotrexate in patients with active rheumatoid arthritis and inadequate response to methotrexate: results of a phase iii study a case of organizing pneumonia induced by tocilizumab a fatal case of acute exacerbation of interstitial lung disease in a patient with rheumatoid arthritis during treatment with tocilizumab exacerbation of combined pulmonary fibrosis and emphysema syndrome during tocilizumab therapy for rheumatoid arthritis acute pneumonitis in a patient with adult-onset disease after toclizumab treatment with good response to anakinra interstitial lung disease in rheumatoid arthritis: response to il- r blockade postmarketing surveillance of tocilizumab for rheumatoid arthritis in japan: interim analysis of patients association of disease activity with acute exacerbation of interstitial lung disease during tocilizumab treatment in patients with rheumatoid arthritis: a retrospective, case-control study recombinant human interleukin- receptor type i in the treatment of patients with active rheumatoid arthritis dose-range and dose-frequency study of recombinant human interleukin- receptor antagonist in patients with rheumatoid arthritis. the il- ra arthritis study group treatment of rheumatoid arthritis with recombinant human interleukin- receptor antagonist treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin- receptor antagonist, in combination with methotrexate: results of a twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial a randomised trial evaluating anakinra in early active rheumatoid arthritis combination therapy with etanercept and anakinra in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully with 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meta-analysis safety and efficacy of baricitinib at weeks in patients with rheumatoid arthritis who have had an inadequate response to methotrexate baricitinib in patients with refractory rheumatoid arthritis risk evaluation and mitigation strategy (rems) nda usual interstitial pneumonia in rheumatoid arthritisassociated interstitial lung disease rheumatoid arthritis and lung disease: from mechanisms to a practical approach an official ats/ers/jrs/alat clinical practice guideline: treatment of idiopathic pulmonary fibrosis: an update of the clinical practice guideline the lung disease of rheumatoid arthritis azathioprine combined with prednisone in the treatment of idiopathic pulmonary fibrosis: a prospective double-blind, randomized, placebo-controlled clinical trial prednisone, azathioprine, and n-acetylcysteine for pulmonary fibrosis risk of serious infection in patients with rheumatoid arthritis-associated interstitial lung disease immediate and delayed impact of oral glucocorticoid therapy on risk of serious infection in older patients with rheumatoid arthritis: a nested case-control analysis pirfenidone in patients with idiopathic pulmonary fibrosis (capacity): two randomised trials pirfenidone in idiopathic pulmonary fibrosis pirfenidone for idiopathic pulmonary fibrosis: analysis of pooled data from three multinational phase trials open-label compassionate use one year-treatment with pirfenidone to patients with chronic pulmonary fibrosis clinical experience with pirfenidone in five patients with scleroderma-related interstitial lung disease improved pulmonary function following pirfenidone treatment in a patient with progressive interstitial lung disease associated with systemic sclerosis a case report: the efficacy of pirfenidone in a chinese patient with progressive systemic sclerosis-associated interstitial lung disease: a care-compliant article an open-label, phase ii study of the safety and tolerability of pirfenidone in patients with scleroderma-associated interstitial lung disease: the lotuss trial is pirfenidone ready for use in non-idiopathic pulmonary fibrosis interstitial lung diseases? treatment of rheumatoid arthritis-associated interstitial lung disease: a perspective review phase ll study of pirfenidone in patients with ra-ild. clinicaltrials.gov identifier: nct nintedanib inhibits fibroblast activation and ameliorates fibrosis in preclinical models of systemic sclerosis combined corticosteroid and cyclophosphamide therapy does not alter survival in idiopathic pulmonary fibrosis cyclophosphamide for scleroderma lung disease cyclophosphamide versus placebo in scleroderma lung disease a multicenter, prospective, randomized, doubleblind, placebo-controlled trial of corticosteroids and intravenous cyclophosphamide followed by oral azathioprine for the treatment of pulmonary fibrosis in scleroderma predicting treatment outcomes and responder subsets in scleroderma-related interstitial lung disease effects of cyclophosphamide on pulmonary function in patients with scleroderma and interstitial lung disease: a systematic review and meta-analysis of randomized controlled trials and observational prospective cohort studies efficacy of intensive immunosuppression in exacerbated rheumatoid arthritis-associated interstitial lung disease rheumatoid arthritis associated with methotrexateinduced pneumonitis: improvement with i.v. cyclophosphamide therapy cyclosporin as a treatment for interstitial lung disease of unknown aetiology progression of fibrosis in usual interstitial pneumonia: serial evaluation of the native lung after single lung transplantation cyclosporine in anti-jo -positive patients with corticosteroid-refractory interstitial lung disease the efficacy of calcineurin inhibitors for the treatment of interstitial lung disease associated with polymyositis/dermatomyositis survival benefit associated with early cyclosporine treatment for dermatomyositis-associated interstitial lung disease experience of mycophenolate mofetil in patients with autoimmune-related interstitial lung disease demonstrates promising effects a prospective observational study of mycophenolate mofetil treatment in progressive diffuse cutaneous systemic sclerosis of recent onset cyclophosphamide versus mycophenolate mofetil in scleroderma interstitial lung disease (ssc-ild) as induction therapy: a singlecentre, retrospective analysis mycophenolate mofetil improves lung function in connective tissue disease-associated interstitial lung disease mycophenolate mofetil versus oral cyclophosphamide in scleroderma-related interstitial lung disease (sls ii): a randomised controlled, double-blind, parallel group trial mycophenolate mofetil is safe, well tolerated, and preserves lung function in patients with connective tissue disease-related interstitial lung disease b-cell depletion salvage therapy in rapidly progressive dermatomyositis related interstitial lung disease successful experience of rituximab 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with rheumatoid arthritis survival and quality of life in rheumatoid arthritisassociated interstitial lung disease after lung transplantation pragmatic prognostic approach of rheumatoid arthritis-associated interstitial lung disease an official american thoracic society/european respiratory society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias prevalence and prognosis of unclassifiable interstitial lung disease lung function estimates in idiopathic pulmonary fibrosis: the potential for a simple classification a multidimensional index and staging system for idiopathic pulmonary fibrosis the mrc breathlessness scale an official ats/ers/jrs/alat statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management a clinician's guide to the diagnosis and treatment of interstitial lung diseases fibrotic idiopathic interstitial pneumonia: the prognostic value of longitudinal functional trends rheumatoid arthritis-associated interstitial lung disease: diagnostic dilemma radiological versus histological diagnosis in uip and nsip: survival implications interstitial lung disease in systemic sclerosis: a simple staging system update in diffuse parenchymal lung disease patients with limited rheumatoid arthritis-related interstitial lung disease have a better prognosis than those with extensive disease the safety of biologic therapies in ra-associated interstitial lung disease patients with early rheumatoid arthritis who smoke are less likely to respond to treatment with methotrexate and tumor necrosis factor inhibitors: observations from the epidemiological investigation of rheumatoid arthritis and the swedish rheumatology register cohorts rheumatoid arthritis (ra) associated interstitial lung disease (ild) opportunistic infections and biologic therapies in immune-mediated inflammatory diseases: consensus recommendations for infection reporting during clinical trials and postmarketing surveillance risk of infection with biologic antirheumatic therapies in patients with rheumatoid arthritis lymphoproliferative disorders in rheumatoid arthritis: clinicopathological analysis of cases in relation to methotrexate medication spontaneous remission of 'methotrexate-associated lymphoproliferative disorders' after discontinuation of immunosuppressive treatment for autoimmune disease. review of the literature key: cord- -n mlxe p authors: nan title: cis annual meeting: immune deficiency & dysregulation north american conference date: - - journal: j clin immunol doi: . /s - - - sha: doc_id: cord_uid: n mlxe p nan a y.o. female was referred to our clinic with a history of multilineage cytopenias/evans syndrome, a history of idiopathic thrombocytopenic purpura, hemolytic anemia, chronic neutropenia, lymphopenia, and hypogammaglobulinemia treated with ivig. our patient was healthy until she was years old; at that time, she developed joint pain, rash, and bruising. she was found to have evans syndrome with idiopathic thrombocytopenic purpura (itp), neutropenia, and lymphopenia. she was initially diagnosed with lupus and was given steroids. her bone marrow biopsy did not conclude myelokathesis. when she was years old, she remained thrombopenic and was started on high dose of immunoglobulin replacement therapy. in ( years old), she developed polyarthritis in her upper and lower extremities. in ( years old), she had a severe nosebleed, for which she was admitted and treated with amicar twice; her platelets were found to be , k/ul. she received rituximab weekly for weeks resulting in an increase of platelet count to - k/ul. she recently (march ) had a splenectomy to remove her large spleen, and since then, her platelets have rebounded to - k/ul. in , she was placed on long-term immunoglobulin replacement therapy after being hospitalized for bilateral pneumonia for nights requiring iv antibiotics for treatment. in , she developed and was treated for another pneumonia. her family history is characterized by multiple members with autoimmune multilineage cytopenia as well as autoimmune diseases such as multiple sclerosis (mother), thyroiditis and enteropathy. on physical examination, she did not present with any warts and the remainder of her physical examination being unremarkable, except for her scar from her splenectomy and a cervical lymphadenopathy. immunologic evaluations showed igg mg/dl, iga < mg/dl, and igm mg/dl. cbc with differential and lymphocyte screen were as follows (cell/mm ): wbc . x , hemoglobin . g/dl, platelets x ; % neutrophils (anc: ), % lymphocytes, % monocytes, % eosinophils; absolute total t-cell number was ( - cells/mcl), cd + t-cells ( - cells/mcl), cd + t-cells ( - cells/mcl), natural killer cells ( - cells/ mcl), and absolute number of b cells was ( - cells/ mcl). she came to our clinic with her sister, who also had multilineage cytopenia and hypogammaglobulinemia, treated with monthly ivig; and her nephew whom had neutropenia. based on this family presentation all three underwent whole exome sequencing (wes). the patient, the patients sister and the patients nephew were all found to have a variant on cxcr (frameshift mutation on chromosome , p.val fs; refnt: tca; altnt: t). as an important note, the patient had a bone marrow biopsy, which did not conclude myelokathesis. in summary, our patient with trilineage cytopenia and hypogammaglobulinemia, without any warts or myelokathexis, had whim syndrome (warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis), which was discovered by studying her wes. with the identification of her specific diagnosis, this allowed us to discuss the potential future indication of plerifaxor (antagonist of the alpha chemokine receptor cxcr ). and equally important, we discussed family planning and future pregnancies given that the mutation is autosomal-dominant. ( ) submission id# taha al-shaikhly, mbchb , kathleen mohan, arnp , matthew basiaga, do, msce introduction: complement component- (c ) is shared by the classical, lectin and alternative complement activation pathways. c , a major opsonin, facilitates phagocytosis of encapsulated microorganisms. inherited c deficiency is rare and is associated with increased risk of bacterial infections. subjects with connective tissue diseases (ctd) and c nephritic factors can have low and occasionally undetectable c levels, yet they are at an underappreciated infectious risk. we hypothesize that excessive c consumption in secondary complement deficiency disorders (scd) is associated with higher risk of bacterial infections similar to primary complement deficiency disorders (pcd). objectives: to compare the rate of bacterial infections between pcd and scd patients and evaluate the association between c level and bacterial infection risk. methods: we performed a retrospective cohort study. subjects with an undetectable complement activity (ch ) or any of the complement components measured at seattle childrens hospital from - were included in our study. we recorded the number of infections, observation periods, diagnosis (pcd, scd and its underlying etiology), lowest complement component levels, and the immunosuppressive agents used. the date of birth, and date of lowest c level were considered as start points to calculate the observation periods for pcd and scd subjects respectively. infections requiring hospitalization or parenteral antibiotics were categorized as serious bacterial infections (sbis). descriptive analyses were performed to determine medians and ranges for continuous variables. differences in rates of bacterial infection were assessed using the chi-square and kruskal-wallis tests when appropriate. among subjects with ctds, we treated every c measurement as a single observation (n= , ) and studied the association between c concentration and the -day odds of having a sbi. multivariable logistic regression was performed to determine infection risk based on c level while controlling for contributing factors. results: we identified subjects with pcd, and subjects with scd. scd consisted of three subgroups (ctd-related (n= ), nephritic factor-related (n= ), and infection-related (n= )). collectively, ctd subjects had a lower median rate of sbi compared to pcd subjects (p = . ). subjects with ctd and c level < have higher rate of bacterial infection (of any severity) (p = . ) and of sbi (p = . ) when compared to ctd subjects with c >= at the beginning of observation period ( figure ). while controlling for immunosuppression level pediatric resident, baystate medical center faculty advisor, baystate medical center introduction: zap codes for a -amino acid enzyme, zap , a member of the syk-protein tyrosine kinase family that plays an important role in t cell development and activation. zap is phosphorylated at tyrosine kinase residues upon t cell receptor (tcr) stimulation resulting in tcr-mediated signal transduction with src family kinases. zap deficiency results in a rare t+b+ nk+ severe combined immunodeficiency (scid). we report a novel compound heterozygous mutation in zap leading to presumed absent zap function in an infant with a normal trec newborn screen and scid. case description: the patient is a term, fully immunized female, born to non-consanguineous parents who was hospitalized for rsv bronchiolitis at mo. at mo she developed an erythematous, papular rash on her face and extremities, nonresponsive to topical antifungal therapy. at mo she was re-hospitalized with rsv bronchiolitis and subsequently treated with multiple courses of antibiotics for presumed bacterial pneumonia followed by albuterol and oral steroids for possible reactive airways disease. during this course of treatment, her rash resolved. at mo she presented with failure to thrive (wt < . % for age), multifocal pneumonia and respiratory failure requiring intubation. bronchial alveolar lavage confirmed pneumocystis jiroveci pneumonia prompting an immune evaluation. total immunoglobulins were normal for age, however antibody titers to tetanus, diphtheria and streptococcus pneumoniae were absent. lymphocyte enumeration revealed elevated cd t cells and markedly diminished cd t cells, normal b and nk cells. t cell proliferation to mitogens (pha, pwm) and antigens (candida, tetanus) was absent, however t cells proliferated normally to stimulation with pma and ionomycin. trec number was normal by newborn screening, but was std deviations below the mean and would have resulted in a positive screen upon repeat. invitae gene scid panel revealed two variants of unknown significance, c. c>g (p.arg gly) leading to substitution of arg with gly and c. _ dupgcat (p.ile metfs* ) resulting in a premature translational stop signal expected to disrupt the last amino acids of zap protein. parental sequencing revealed these variants to be on opposite chromosomes. the patient was successfully treated for pjp pneumonia and has since successfully engrafted a / matched unrelated donor stem cell transplant. discussion: we report a novel compound heterozygous mutation in zap which we presume led to t+ b+ nk+ scid. our patients clinical presentation of failure to thrive, recurrent lower respiratory tract infections, dermatologic findings and pjp pneumonia are consistent with previously reported cases of zap scid. her paucity of cd t cells, abundance of cd t cells and absent proliferation to mitogens are also consistent with previously described cases of zap . normal proliferation of t cells when bypassing the tcr by stimulating cells with ionomycin and pma confirms a defect in the tcr. we believe this is the second documented case of missed scid by newborn screen in ma since the implementation of trec screening in . pediatric resident (pgy iii), goryeb children's hospital attending physician, pediatric and adult asthma, allergy and immunology, llc introduction: acute otitis media (aom) is one of the most common reasons for antibiotic use in early childhood. we explored the challenges when aom fails traditional therapies and immunologic evaluation does not identify a commonly described immunodeficiency. case description: an eighteen-month-old male presented with episodes of aom and recurrent purulent otorrhea requiring intravenous antibiotics. laboratory evaluation revealed a normal cbc, normal immunoglobulins (igg , iga , igm , ige ) and igg subclasses. lymphocyte subset panel was normal. initial responses to dtap and prevnar boosters were normal, however, there was rapid decline to tetanus and pneumococcal antibody titers. a sub optimal response to haemophilus influenza type b vaccine was noted. although vaccinated twice for mmr, he never mounted mumps specific igg. mitogen response to pha was normal with decreased responses to cona and pokeweed and no detectable tetanus nor candida responses. further investigation revealed decreased non-class and class switched memory b-cells. the patient was recently vaccinated to pcv and at the present time has protective titers. discussion: it has been previously suggested that decreased memory b cells may contribute to decreased antibody responses to select vaccine antigens resulting in recurrent aom in children. our case supports the need to investigate beyond typical immunologic screening for immunodeficiencies. introduction: dna mismatch repair (mmr) system corrects replication errors in newly synthesized dna, and prevent recombination between dna sequences when they were not identical ( ) . msh is a part of mmr genes, ( ) ( ) ( ) . case: a ten-year-old girl presented with fever, brown spots on her skin, hair loss, recurrent pulmonary infections, arthritis on the left hand and right ankle. she has also been followed up with nf ( figure ). there was a first-degree cousin marriage between her parents. physical examination revealed findings of pneumonia and nf. anti-nuclear antibody, anti-ndna, anti-dsdna, anti-histone, anti ro and anti-nucleosome antibodies were positive. in her immunologic assessment showed low igg and iga levels associated with high igm level ( table ). the coexistence of nf, hyper igm syndrome, sle, were considered in the patient. intravenous ig ( mg/kg, every weeks) treatment was started due to hypogammaglobinemia. the frame shift mutation in exon of the msh gene was detected in the boztug's laboratory. in the follow up period, she admitted at years old with back pain. a mass in the left paravertebral area, related to the spinal canal and neural foramina, was detected at the l -l levels in spinal mri. the lymphadenopathy around the liver and hilum and the left parietal bone lesions were developed within two months despite surgical excision of primary mass ( figure ). as a result of pet examination; suvmax was found to be around . in the mass lesion in the paravertebral region and suvmax values did not exceed . in other lymphadenopathy and masses. atypical cellular infiltration suggesting neoplastic events, which were including small-medium size atypical pleomorphic mononuclear cells and t cells. since all these formations did not indicate definite cancer, chemotherapy was not started. interestingly, although chemotherapy was not given, progression stopped, and partial spontaneous regression was observed. discussion: the effect of msh mutations on patients may significantly vary with the inheritance pattern ( ) . leukemias or lymphomas are not common in heterozygote mmr gene defects ( , ) . however, homozygote mutations in mmr genes show a different pattern. wimmer and etzler proposed the new term constitutional mismatch repair-deficiency syndrome (cmmr-d) for patients who have a homozygous mutation in mmr ( ) . cmmr-d characterized by development of childhood cancers, mainly hematological malignancies and/or brain tumors, as well as early-onset colorectal cancers, and neurofibromatosis type ( ) . bi-allelic germline mutations in any of the mmr genes in which msh is involved increases hematological malignancies by % ( , ) . msh mutation has been associated with many cancers since its identification. leukemia, lymphoma, colorectal cancer, endometrial cancer, brain tumors are some of these cancer types ( ) ( ) ( ) ) . msh deficiency is an important disease that can affect different systems at the same time. there is a high risk of malignancy in the cases and therefore they must be closely monitored. this case has also shown that atypical lymphoproliferation may occur in msh homozygous mutant cases. (normal rage: - ) background: advances in inborn errors of human immunity have supported the discovery of new syndromes that are marked by striking features of autoimmunity and immune dysregulation often associated with cytopenias, lymphoproliferation, and a predisposition to reticuloendothelial malignancies leading to evaluation with hematologists/oncologists. moreover, hematologists/oncologists have also seen an increasing use of effector cell-based therapies, checkpoint inhibitors, immunomodulatory and targeted therapies resulting in autoimmunity and hyperinflammatory complications. a working knowledge of clinical immunology could help practicing hematologists/oncologists in the identification and management of these conditions. objectives: to support the advancement of aspho members and the field by facilitating education regarding the best practices in diagnosis and management of immunological disorders. to create a platform for the development of collaborative clinical research in patients with hematological/oncological manifestations of immunological disorders or those requiring hematopoietic stem cell transplantation for a underlying immunological disorder. design/methods the aspho clinical immunology sig was initiated based on collaboration with the clinical immunology society (cis). aspho members who are pediatric hematology/ oncology clinicians, clinical researchers, and trainees are eligible to participate. we have established a steering committee with representatives from across the united states and canada with diverse clinical and research expertise. through regular teleconferences and annual in-person meetings, we have developed a platform to provide our members with a network of immunology resources to ensure a strong foundation of knowledge and tools to conduct clinical care and research pertaining to the diagnosis, evaluation, and treatment of patients with immunological disorders. results we currently support over members within our online community. several educational initiatives have been successfully launched. we have submitted an invited review to pediatric blood and cancer which provides a case-based review of primary immune regulatory disorders. we hosted the first immunology for hematology oncology practice (i-hop) cased-based webinar series. this series features case-based discussions of patients with primary immunodeficiency disorders presented by fellow trainees and mentored by senior clinicians. we will also be hosting an aspho webinar focusing on the laboratory evaluation of primary immunodeficiencies and immune dysregulation syndromes. we have also begun the process of laying the groundwork for clinical research initiatives. conclusion: the aspho clinical immunology sig seeks to serve as a collaborative resource for pediatric hematology/oncology clinicians and researchers. through the development of educational and research initiatives, we envision improving the care of patients with immunological disorders that are often managed by pediatric hematologists/oncologists. moreover, we hope to broaden our understanding and application of clinical immunology within pediatric hematology/oncology. we hope that this successful initiative will serve as a blueprint for the development of future collaborations with other specialty societies and patient groups. autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a rare autosomal recessive disease caused by aire gene mutations. clinical diagnosis is established by the presence of at least two components of the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and addisons disease. in europe, the classic presentation is widely recognized and nonendocrine autoimmune manifestations are rarely reported. a recent study of american apeced patients demonstrated a more heterologous presentation, with many nonendocrine manifestations including urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis and sjogrens-like syndrome, all uncommon in european reports. within the american cohort, % of patients developed a mean of three non-triad manifestations before reaching the classic triad. finding of aire mutations and high-titer antiifn-autoantibodies is seen in both european and american cohorts. we present the case of two siblings, who demonstrate an apeced-like phenotype with both classical and atypical features. they share the same heterozygous c + _ + delinsct aire mutation. the older, an eight-year-old boy, with history of prematurity, bronchopulmonary dysplasia and onychomadesis in infancy, came to medical attention at months of age due to failure to thrive (ftt), in addition to fevers and urticarial rash lasting months after his mmr vaccine. the fevers resolved with anakinra, which was discontinued two years later due to pneumonia. from age - he developed an alps negative lymphadenopathy which self-resolved. lung issues include chronic cough, initially treated as asthma but with poor bronchodilator response, and frequent lung infections, including - pneumonias per year. at age five evaluation for ftt revealed growth hormone deficiency. two years later he was diagnosed with primary addisons disease. chronic abdominal discomfort, bloating, cyclical constipation/diarrhea, recurrent rashes, dystrophic nails, and sicca symptoms are also present. his sister, age five, shows ftt, but no growth hormone deficiency. at age one, she too developed a fever and rash syndrome lasting months. severe gerd and constipation started in infancy and are ongoing. at age three she developed a transaminitis, initially diagnosed as ebv, but later thought to be autoimmune hepatitis. she has frequent viral respiratory infections, and pneumonia at age two. she has had a chronic cough, with poor bronchodilator response, for most of her life. evaluation of seizure at age three showed normal brain activity. brain mri revealed partial agenesis of the corpus callosum and microgyria. her brother has similar mri findings. both children have had developmental motor delay and poor tone. brain dysgenesis and neurodevelopmental delay has not previously been described in apeced. although there were both typical and atypical symptoms, the history in combination with genetic findings led to further investigation of an apeced-like syndrome. autoantibody testing confirmed high-titer antiifn-autoantibody typical of apeced in both children and hightiter bpifb autoantibodies found almost exclusively in apeced pneumonitis in the brother. whole exome sequencing and copy number variation analyses are underway to further evaluate the patients condition. this case demonstrates the importance of clinical presentation in the evaluation of genetic results and in the guidance of therapeutic management. ( ) submission id# rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties are referred to our center for further evaluation. this study elucidates the demographic and laboratory characteristics of referred infants with transient or persistent idiopathic t cell lymphopenia (tcl) without clearly identified genetic or acquired etiology. methods: a retrospective analysis was performed from september (when trec screening started) through the end of december . descriptive statistics were calculated for demographic and laboratory characteristics. t-test or mann-whitney tests were used to compare laboratory variables. pearson or spearman tests were used to determine correlation between initial trec levels and t cell counts. by definition, the cd +, cd +, and cd + populations of transient tcl patients normalize by age year. results: eighteen infants with transient and with persistent tcl were identified. males comprised . % of the transient and . % of the persistent tcl cohorts. whites comprised . % of the transient and . % of the persistent tcl cohorts. the mean initial trec levels did not differ between the transient and persistent cohorts ( . vs. . trecs/l of blood, p = . ). mean initial absolute counts of cd + ( vs. cells/l, p < . ), cd + ( vs. cells/l, p < . ), and median initial absolute counts of cd + ( vs. cells/l, p = . ), were higher for transient vs persistent cohorts. initial trec level did not correlate with initial cd +, cd +, or cd + absolute counts. the median age of resolution for the transient cohort was . days (range - ). the absolute cd +, cd +, or cd + counts rarely exceeded the reported median values for age, and remained closer or below the th percentile for age up to days of life. the majority of both transient and persistent tcl patients demonstrated unremarkable lymphocyte proliferation to mitogens. conclusion: our centers transient tcl cohort appears to be predominantly male and non-white, whereas the persistent tcl cohort is more evenly distributed by sex but still predominantly non-white. the transient cohort had lower initial trec levels, but higher initial t cell counts. both cohorts appear to have relatively intact in vitro function. introduction: primary immune deficiency disease (pidd) is typically considered a pediatric illness, although advances in treatment and diagnosis are changing this paradigm. currently, data on pidd in older patients are very limited. objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd- codes d , d. . , d . , d . , d . , d . , d . , d . , d . , and d . . a total of records from geographically-diverse clinics were identified and characterized by age, gender, and pidd diagnosis. results: of the pidd patients in the ciic registry, ( %) were between - years of age (see figure) . within this age group, most patients were female (n= , %). the most common diagnoses among patients > years of age included common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d . ; n= , %) and antibody deficiency with near normal immunoglobulins (d . ; n= , %) . in comparison, the registry included ( %) patients aged - years; this age group was predominantly male (n= ; %). the most common icd- codes within the younger cohort were relatively evenly distributed between hereditary hypogammaglobulinemia (d . ), antibody deficiency with near normal immunoglobulins (d . ) , and common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d . ). conclusions: our data suggest that pidd in patients over age may be more prevalent than previously reported. additional research is needed to corroborate these findings, further characterize the nature of pidd in this population, and determine whether there are unique diagnostic and treatment considerations within this demographic. introduction/background: increased susceptibility to invasive infections with neisseria has been well documented in patients with deficiency of terminal complement proteins. the molecular attack complex is constructed with complement components c to c . a deficiency in complement c has been described previously in both african american and south african populations. complement c deficiency is inherited in a co-dominant pattern, with multiple known mutations. we present a case of a -year-old, previously healthy male, who presented with invasive n. meningitides infection. he was found to have a novel mutation noted on genetic sequencing of the complement c gene. objective: we present the case of a -year-old, previously healthy male, who presented with invasive n. meningitides infection. on genetic sequencing, he was found to have three mutations of the complement c gene. two of which have been described previously, and a third novel mutation. methods: a -year-old male with no known history presented to us with a -hour history of emesis. he was found to be febrile, and quickly decompensated, developing septic shock. blood cultures were drawn, and within hours grew n. meningitides. he was treated with broad spectrum antibiotics upon arrival, and subsequently narrowed to ceftriaxone. his hospital course was complicated by disseminated intravascular coagulation, as well as acute tubular necrosis, leading to endstage renal disease for which he is listed for kidney transplant. results: on immunodeficiency evaluation, he was noted to have an undetectable ch (< , reference range - ). complement levels returned with c of . (reference range - ) and c r of . % (reference range - %). complement c function screen returned at % (reference range . - %). all other complement levels were within normal limits. genetic sequencing showed the patient to be compound heterozygous for two of known four variants which have been reported to recur in african patients with complement c deficiency. this included c. del and c. del, which are predicted to result in frameshift and premature protein termination. he was also found to be heterozygous for sequence c g>a, which results in amino acid substitution p.arg lys. this variant is rare, with one large database reporting it in of alleles, and not in a homozygous state. it has not been reported in a case of c complement deficiency previously. conclusions: we present the case of a previously healthy -year-old male with invasive meningococcal disease. he is compound heterozygous for two mutations that have been associated with total complement c deficiency; however, he was found to have subtotal c deficiency. furthermore, he has a third novel mutation of the complement c gene. further investigation is warranted on the significance of this finding and impact on relevance to possible kidney transplant. background: measuring the function of the classical pathway of complement activation is useful in several disease states, including complement deficiency, autoimmune conditions such as systemic lupus erythematosus and certain forms of nephritis. the original method for assessing classical pathway activity was the haemolytic ch method, but this assay can be time consuming and has reagent stability issues due to the use of sheep red blood cells. there can also be high lab-to-lab variability due to differences in the protocols used. here we report the assay characteristics of an automated, commercial, liposome-based assay to measure ch activity. we also compare the results obtained using the traditional haemolytic method with the automated, liposome-based method used on the spaplus turbidimetric analyser. methods: a linearity study was performed based on clsi guideline ep -a. the linear range of the spaplus ch liposome assay was established by analysis of a series of sample dilutions and evaluation of results against pre-defined goals for recovery and %cv. precision was assessed based on clsi guideline ep -a over days. samples with different ch activities ( . - . u/ml) were run in duplicate, with two runs per day using reagent lots and different analysers. interference analysis was performed by spiking haemoglobin, bilirubin, chyle, ascorbic acid or saline (as a control) into samples before measuring the ch activity. for the assay comparison study, sera from routine patient samples were used. samples were collected from chulalongkorn hospital, faculty of medicine, chulalongkorn university, thailand. ch classical pathway activity was assessed using a haemolytic method and also using the liposome based ch assay for use on the spaplus turbidimetric analyser (the binding site ltd., birmingham, uk). c protein concentrations were also available for of these samples. results: the liposome ch assay gives a linear response over the range . - . u/ml, covering the measuring range of the assay ( . - . u/ml) at the standard analyser dilution (neat). the within run, between run and between day %cvs were all . %. the total %cv was . % in all samples. minimal interference was observed with the four common interferents tested. a significant correlation was observed between the two ch methods (p< . , r= . , y= . x± . ), with . % agreement between the methods in determining whether patients were above or below the lower limit of the assay normal range. the individuals in disagreement had normal ch results using the haemolytic method, and low ch values in the liposome assay. of these, c values were available for / , and had c concentrations below the lower limit of the assay normal range. conclusion: the liposome ch assay for use on the spaplus analyser has passed assay development guidelines based on those set out by the clsi for linearity, precision and interference, and there is a strong correlation between this automated assay and the haemolytic ch method used here. five additional patients with low c concentrations were defined as having a low ch using the spaplus liposome method compared to the haemolytic method. ( ) submission id# background/aims: rotavirus vaccine is a live viral vaccine that is part of the routine u.s. childhood immunization schedule. live viral vaccines administered to infants of mothers who received biologic medications during pregnancy can potentially cause vaccine-associated disease. infant death from disseminated mycobacterial infection after vaccination with bacille calmette-guerin (bcg) in infants whose mothers received infliximab during pregnancy has been reported. it is currently recommended that infants born to women who received biologic therapy during pregnancy not receive live viral vaccines, however there is a paucity of information regarding adverse events from live viral vaccines. we report two infants, born to mothers receiving infliximab during pregnancy, who tolerated the complete series of rotavirus vaccine. methods: two infants who received rotavirus vaccine and whose mothers received infliximab (monoclonal antibody against tumor necrosis factor alpha which blocks the inflammatory response) during pregnancy were identified and their charts were reviewed. each mothers chart was assessed for timing of the biologic doses during pregnancy and concurrent immunosuppressant therapy. results: the mother of the first infant had crohn's disease and received infliximab every weeks throughout her pregnancy (final infusion at approximately weeks estimated gestational age [ega] ). she did not take additional immunosuppressive drugs throughout her pregnancy. the infant was born at weeks ega. the infant received rotavirus vaccine at , , and months of age. the infant did not have coexisting medical conditions or recorded hospitalizations during the first year of life. there were no side effects from rotavirus vaccine documented during well child examinations. the childs growth was normal during the first year of life. the mother of the second infant also had crohn's disease and received infliximab infusions every six weeks during pregnancy until weeks ega. additionally, she took mesalamine (anti-inflammatory) daily. the infant was born at weeks ega. the baby had a brief and uncomplicated neonatal intensive care unit stay. she did not have medical conditions diagnosed at the time of birth, or in the first year of life. the child received rotavirus vaccination at , , and months of chronological age, and the infant did not experience documented adverse reactions. the child presented to the emergency department twice in the first year of life: once for thrush at months of age and once for viral gastroenteritis at months of age. the childs growth curve was unremarkable. conclusions: we report two infants, whose mothers received infliximab during pregnancy, who safely tolerated the -dose series of rotavirus vaccination. neither infant in this case series suffered from minor or severe adverse events as a direct consequence of receiving rotavirus vaccine. this suggests that administration of rotavirus vaccine may be safe in infants whose mothers received biologic therapy. introduction: combined immunodeficiencies (cids) can arise from partial loss of function variants in recognized scid genes, which can lead to relative lymphopenia with poorly functioning and oligoclonal t cells. cids have been most commonly associated with variants of the rag genes, but other genes are also implicated. clinical symptoms may be less severe, and the onset generally is delayed, compared to typical scid presentations. case report: a -year-old female presented with a history of recurrent and progressively worsening infections involving multiple microorganisms and organs, starting in infancy and requiring frequent hospitalizations. bacterial or viral infections included rhinosinusitis, otitis media, herpetic stomatitis, dental abscesses, pneumonias, pulmonary mycobacterial abscesses, cmv hepatitis, urinary tract infections, dermal abscesses, and groin hidradenitis. fungal and yeast infections included cryptococcal meningitis, oral thrush, dermatophytosis of the face, osteomyelitis of a finger, and onychomycosis. laboratory tests in showed: mildly low t cell counts ( /ul) with a reversed ratio of cd /cd t cells ( . ); almost absent b cells ( /ul) ; and low nk cell counts ( /ul). cd + t cells were mostly of the memory phenotype ( %). t cell development showed low counts of th cells. t-cell stimulation tests demonstrated poor proliferation responses (< %) to concanavalin a, tetanus toxoid, and candida albicans, with near-normal responses to pokeweed (> %) and pha (> %). she had low ig levels (iga , igm , ige < ), except for igg ( mg /ml; due to replacement since early childhood). limited genetic evaluation at age showed a heterozygous variant in the rag gene (g. t>c, c. t>c, p.met thr; nm_ . ). discussion: loss of function variants in rag or rag genes are known to cause a t-b-nk+ type scid. more than missense variants have been reported for rag , with disease-associated variants predominantly in zinc binding regions. the rag missense variant in our patient also lies within the zinc binding region (amino acids - ). the variant is rare (mean allele frequency . in gnomad) and has been identified in at least one other individual with scid (t-, b cell-, nk+). although classified as a variant of unknown significance, occurrence in at least two individuals with deficiencies of t and b cells-within a functionally important rag domainsupports an interpretation that the variant may be pathogenic. most patients with cid with rag variants are either homozygous for a poorly functional allele or have one nonunfucitonal and a second, poorly functional allele. we detected only a single potentially pathogenic allele. our patient has decreased nk cells in addition to t and b cell defects. further genetic studies including whole exome sequencing, are planned to identify further variants in rag or other relevant genes. rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties in new york were referred to northwell health for further evaluation after abnormal newborn screens. the demographic and immune parameters of infants with transient t cell lymphopenia (ttcl) without clearly identified genetic or acquired etiology are described. tcl is considered transient if the lymphopenia resolves by months of age. similar data from the following infants with low lymphocytes (fill) program of the united states immunodeficiency network (usidnet) are presented. methods: a retrospective analysis of two separate patient cohorts with ttcl are described. cohorts include patients referred to a single center, northwell health, in ny from september to december and at usidnet using data tracked by fill from june to july . results: out of , referrals at northwell, infants with ttcl were identified. infants were predominantly male ( . %) and non-caucasian ( . %). out of fill participants, infants with ttcl were identified. infants were predominantly male ( . %) and non-caucasian ( . %). initial laboratory parameters for the northwell versus fill cohorts are summarized: a) median trec levels: . vs. . trec/l of blood; b) median absolute cd + count: vs. cells/l; c) median cd + count: vs. . cells/l; d) median absolute cd + count: . vs. . cells/l. initial naïve cd + t cell information was available for northwell and fill infants (median %). mitogen proliferation studies were performed in ( . %) northwell and ( . %) fill infants with % of these northwell and % of these fill infants demonstrating normal proliferation. genetic testing, such as targeted genetic panels or chromosomal microarrays (cma), was performed in northwell and fill infants. no genetic or chromosomal aberrations were identified. whole exome sequencing (wes) was not performed in either cohort. of ( . %) northwell and of ( . %) fill infants did not receive the initial rotavirus vaccine. no fill infants were vaccinated but no adverse effects were reported in of ( . %) northwell infants who received the first rotavirus dose. of these, of ( . %) had normal mitogen proliferation while ( . %) had decreased proliferation to phytohemagglutinin. conclusions: identifying biomarkers for ttcl and developing evidencebased guidelines for the diagnosis and management of ttcl are important knowledge gaps. this descriptive study is limited by small sample size and the constraints of registry-based research. although there appear to be differences between these cohorts, our findings suggest that ttcl may disproportionately affect different segments of the population. ttcl infants with normal mitogen proliferation may be able to tolerate rotavirus vaccination. thus, routinely checking proliferation studies in all ttcl infants may help risk stratify these patients and minimize vaccinerelated adverse events. currently, there is insufficient evidence to recommend more extensive genetic testing such as genetic panels, cma, or wes. systematically collecting information about patient characteristics and outcomes, as well as encouraging increased participation in registries such as fill, may help address these shortcomings. background: systemic lupus erythematosus (sle) is a chronic, inflammatory disease that affects multiple organs. the measurement of anti-dsdna antibodies (abs) is a gold standard serological test used in the diagnosis and monitoring of sle, with higher serum levels associated with worse prognosis. however, not all anti-dsdna abs are pathogenic, and some patients have consistently high levels with low disease activity. one mechanism suggested for the pathogenicity of these antibodies is complement activation. here we describe an assay to measure the c q binding activities of anti-dsdna abs in sle patients. materials & methods: the concentration of anti-dsdna abs was determined using the quantalite dsdna elisa kit (inova) as per the manufacturers instructions. in order to determine the c q binding capacity of bound abs, samples were added to the pre-coated plate and incubated. bound anti-dsdna ab/c q complexes were then detected using a biotinylated anti-c q antibody ( ng/ml) and streptavidin peroxidase ( mg/ml). normal reference ranges were developed in serum samples from healthy controls, and upper limits of these normal ranges were used as cut-offs. the dsdna abs and c q binding capacity of bound abs was then assessed in sle patients, and compared to other markers and the sle disease activity index (sledai) score. results are displayed as absorbance at nm (au). results and conclusions: the th percentile ranges for anti-dsdna abs ( . - . au) and c q binding activities ( . - . au) were developed from the measurements generated in healthy serum samples. sle patients with an increased anti dsdna ab concentration (> . au) were then separated into those with low (< . au) and high (> . au) c q binding activities. patients whose dsdna abs had high c q binding activity were found to have significantly higher sledai scores (mean . vs . ) . serum c q concentration, serum dsdna abs (measured by another method) and serum c and c concentrations were not significantly different between the two groups. this assay suggests that dsdna abs from sle patients differ in their ability to bind complement, and that high complement binding activity of these antibodies may be linked to a more active form of disease. x-linked lymphoproliferative (xlp) is a primary immunodeficiency, caused by signaling lymphocyte activation molecule (slam)-associated protein (sap) deficiency. patients with xlp have severe immune dysregulation, usually triggered by ebv infection, leading to fulminant infectious mononucleosis, dysgammaglobulinemia and lymphoproliferation. without hematopoietic stem cell transplant (hsct) fatality is reportedly % by age . we report the natural history of xlp in a patient, and describe the lessons learned. our patient was healthy and developed normally until -years of age, when he developed progressive respiratory symptoms. lung biopsy revealed mature lymphoplasmacytic infiltrate in the alveolar septa, consistent with lymphoid interstitial pneumonia (lip). he received corticosteroids and cyclophosphamide with significant improvement. at age , he developed severe infectious mononucleosis (fever, hepatosplenomegaly, lymphadenopathy, lymphocytosis). he had a protracted clinical course, but eventually recovered and seroconverted to a typical convalescent pattern. he subsequently developed hypogammaglobulinemia, and was started on intravenous immunoglobulin (ivig). during the same year, his -year-old brother developed lip, and subsequently hemophagocytic lymphohistocytosis (hlh) and died within months from overwhelming candidiasis. unfortunately, his youngest brother (age ) then developed lip and died months later from a massive gastrointestinal bleed. both siblings were treated with corticosteroids and cyclophosphamide; they did not have detectable ebv infection. at age years, our patient experienced recurrent strokes and was found to have biopsy-proven cns vasculitis. he was treated with interferon-and recovered with residual left sided weakness, but was lost to follow-up. he continued on ivig, with no other immunomodulatory agents for several decades. he had progressive lung disease and recurrent seizures controlled with anti-epileptics. at age , he developed sudden vision change, headache and right-sided weakness, followed by a seizure. mri of the brain revealed small bilateral areas of acute infarction suggestive of a central embolic event, however, no primary thrombus was identified. he did not receive any immunosuppression but was anti-coagulated. eventually he was discharged home with resolution of weakness to his baseline. the patient was referred to our clinic after discharge and we re-evaluated him after years. immune profiles at the time showed therapeutic igg troughs, low/undetectable igm/a/e, normal t/b/nk-cell counts, normal spontaneous, but decreased antibody-dependent nk cytotoxicity, % sap protein expression (on cd +cd +, cd -cd + and cd + cd + cells), and deletion on the x chromosome encompassing the sh d a gene which encodes sap. his mother was a carrier of the same deletion. his functional status excluded the option of hsct. a year later, he had rapid deterioration with recurrent lung infections, liver failure, and thrombocytopenia. bone marrow biopsy revealed hodgkins lymphoma. he declined chemotherapy and died few days after diagnosis. our case represents a rare patient with xlp surviving to the fifth decade without hsct, particularly having experienced mononucleosis and non-ebv related cns vasculitis. our patient survived decades longer than his brothers (who most likely shared the same genetic defect) without evidence of somatic reversion ( % sap expression in cd +cd +) to explain his milder clinical phenotype. this case may help in understanding the natural history of xlp, and confirms that prognosis remains poor without hsct. haematology and oncology, chu de québec ctla- is a major negative regulator of immune responses, and ctla- haploinsufficiency has been identified as a monogenic cause of primary immunodeficiency in patients presenting with a common variable immunodeficiency (cvid) phenotype with autoimmunity. here we present the case of pb, a -year-old man who had been followed by the immunology service of our center for years. a diagnosis of cvid had first been made when the patient presented with atypical transverse myelitis, low immunoglobulin levels, and lymphopenia. over the years, his clinical picture was dominated by various forms of autoimmunity, namely inflammatory demyelinating disorder of the central nervous system, autoimmune haemolytic anemia, immune thrombocytopenia, cryptogenic organizing pneumonia, rheumatoidlike polyarthritis, chronic liver transaminitis with biopsy-proven moderate fibrosis, and lymphocytic colitis with malabsorption. immunoglobulin replacement therapy was started at diagnosis, and autoimmunity was sequentially treated with methotrexate, interferon beta -a, cyclophosphamide, mycophenolate mofetil, rituximab, and finally a combination of low-dose prednisone and sirolimus, with stabilization of his neurological condition, the most debilitating complication of his immune dysregulation syndrome. bone marrow transplant had been offered, but declined by the patient due to perceived good quality of life compared to transplant-associated risks. the patient was later referred to our hematology ward in july of for septic shock complicating febrile neutropenia, which was part of a twomonth, gradual-onset pancytopenia. the diagnosis of immune-mediated aplastic anemia soon became apparent, as demonstrated by a bone marrow biopsy performed in a peripheral center two days prior to admission. the underlying pneumonia and thereafter biopsy-induced staphylococcus aureus iliac osteomyelitis and soft-tissue abscess were treated with broad-spectrum antibiotics as well as multiple surgical interventions. the patient was started on eltrombopag, high-dose corticosteroids and cyclosporin a, the latter promptly switched to tacrolimus due to liver enzymes disturbances, all of which resulted in no significant hematologic response despite over seven weeks of treatment (with concurrent treatment of complicating infection, upper gastrointestinal bleeding, and intensive-care-unite myopathy). during that time, genetic confirmation of ctla- haploinsufficiency was received, and the patient was thereafter started on abatacept on day of current hospitalization. administration of equine anti-thymocyte was initially foregone because of perceived infectious risk in the setting of poor iliac wound healing and superimposed adenovirus viremia; however, given the lack of response, it was given on days through of hospitalization. haematologic response began on day of hospitalization with a steady rise in alllineage myelopoiesis up to a complete neutrophil response, platelet near-complete response as well as resolution of transfusion needs by day . while waiting for a well-matched bone marrow donor, isolated platelet decrease was observed and attributed to multiple factors, including low-grade thrombotic microangiopathy, inflammatory consumption and drug-related thrombocytopenia, but the patient remained well. to our knowledge, our patients presentation is one of the most severe manifestation of ctla- haploinsufficiency to have responded to targeted therapy with abatacept, as a bridge to hematopoietic stem cell transplantation, with resolution of both immune and infectious complications, showing that genetic diagnosis is helpful in optimizing the management of presumed cvid patients. hospital de octubre health research institute (i+ ), madrid, spain, dept. of immunology, university hospital octubre. madrid. spain background: xlf/cernnunos deficiency is a rare primary immunodeficiency classified within the dna repair defects. these patients present severe growth retardation, microcephaly, lymphopenia and increased cellular sensitivity to ionizing radiation. here, we describe two unrelated cases with the same nonsense mutation in the nhej gene showing significant differences in clinical presentation and immunological profile but a similar dna repair defect. methods: missense nhej mutation was identified by targeted next-generation sequencing with an in-house designed panel of genes. for foci experiments, primary skin fibroblasts were irradiated with ionizing irradiation ( cs) or treated with mm etoposide for hour. after irradiation, the cells were seeded at a density of x cells/ml in t flasks in triplicate. to evaluate cell sensitivity to gamma-ir ( and gy),adherent cells were trypsinized and counted days later. pbmcs from patient and healthy controls were irradiated with gy, fixed and stained for cd , cd and phospho-histone h ax. mean fluorescence intensities (mfi) of gamma-h ax were evaluated on gated cd + lymphocytes. results:we report two patients harboring the same homozygous mutation in cernunnos/xlf/nhej gene. strikingly, their clinical phenotype ranges from severe combined immunodeficiency to isolated thrombocytopenia followed until escolar age (table ) . they harbour the same c. c>t mutation in nhej gene but different immunologic features (table ) . p presented with mild t lymphopenia, hypersensitivity and nhej repair defect, typical for patients with xlf/nhej defects. on the other hand, p presented a more severe phenotype (t-b-) , however hypersensitivity and nhej repair defect was similar to p .of note, p has survived into the first decade of live. both patients are alive and well after hsct. discussion: usually the repair defect in these disorders is assessed by immunofluorescence assays of irradiation-induced gamma-h ax foci using skin fibroblasts. a high throughput, sensitive and reliable assay to quantify gamma-h ax foci in pbmcs isolated from blood samples would be a valuable tool to diagnose these patients and perform hsct early. flow cytometry (fc) can be applied as a rapid diagnostic tool for dna repair disorders. patients with the same homozygous mutation (p.r x) in nhej gene have been previously reported. two patients died at . and years while another of the patients is already years old and is alive (without hsct). however,none of these patients presented severe t lymphopenia as it has been observed in our first patient. conclusions: the assignment of a timely and accurate diagnosis is of paramount importance in the management of patients with defects in dna repair. in the era of nbs an abnormal trec assay should be followed by ngs approach as cernunnos deficiency may present early in life as scid,as other rs-scid defects. since genetic diagnosis takes time,functional radiosensitivity assays in peripheral blood may lead to the correct diagnosis and avoid exposure to alkylating agents during the conditioning regimen prior to genetic diagnosis. it would also be helpful in cancer patients to individualize and to guide the dosing of ionizing radiation (ir) and/or genotoxic agents to avoid accumulation of cells with genomic instability that could accelerate cancer development. figure ). her skin lesions also significantly improved after starting the medication ( figure ). her hospitalizations were complicated by fluid overload and hypertension. both fluid overload and hypertension resolved prior to discharge. she remains on mg prednisone daily, cetirizine, ranitidine, cromolyn and benadryl and hydroxyzine prn. to our knowledge, this is the youngest patient successfully treated with midostaurin and she is doing very well on therapy with no apparent side effects. she has had resolution of many of her systemic mastocytosis symptoms including skin lesions, axillary mass and improvement in her diarrhea and growth as well as objective improvements in her tryptase levels. case report: a two-year-old male presented to the hospital with a painful, non-pruritic facial and groin rash. the rash started one week prior to presentation. he had no associated fevers. his history was remarkable for failure to thrive (ftt) and chronic bilateral leg pain with antalgic gait. over the preceding months, he had been diagnosed with hand-foot-mouth disease and varicella. he had also had recurrent cervical lymphadenopathy (lad) for greater than one year requiring incision and drainage. gram stain and gomori methenamine-silver nitrate stain (gms) were negative and pathology showed only acute and chronic inflammation with areas of necrosis. his family history was negative for autoimmune disease or immunodeficiency. infectious exposure history was significant for an incarcerated father with unknown tuberculosis status and history of living in a shelter. on physical examination, the patient was well appearing with multiple erythematous papules, with superficial erosions and scabbing on the face (figure ), lower abdomen, genital area, buttocks and proximal lower extremities. he had large, firm, non-tender submandibular lymph nodes. he also had small palpable axillary and inguinal lymph nodes bilaterally. his laboratory workup revealed normal white blood cell and platelet counts, but microcytic anemia, an erythrocyte sedimentation rate of mm/hr, and c-reactive protein of . mg/dl. full body magnetic resonance imaging (mri) revealed bilateral cervical, supraclavicular, right hilar and inguinal lymphadenopathy and a patchy right upper lobe consolidation with at least one small area of cavitation ( figure ) and an adjacent smaller area of ring enhancement. it also revealed three small nonspecific hypodense foci within the right lobe of the liver and borderline splenomegaly. given these findings, there was concern for granulomatous diseases. the patient underwent a liver biopsy ( figure ) which showed non-specific evidence of necrotizing granulomatous disease. microbiological cultures and stains for bacteria, acid-fast bacilli and fungi were negative. his infectious work-up was negative for hsv, tuberculosis, hiv, syphilis, histoplasmosis, and toxoplasmosis. superficial bacterial cultures from the face and groin grew mixed gram positive and negative organisms, including methicillin-susceptible staphylococcus aureus (mssa). his immunologic workup revealed borderline elevated iga and igg with normal igm, normal t,b, nk-cell counts and pneumococcal and tetanus titers. a dihydrorhodamine (dhr) flow cytometric test was positive, consistent with a diagnosis of chronic granulomatous disease (cgd). genetic testing confirmed x-linked disease. he was treated with acyclovir and ceftriaxone with resolution of his rash. conclusion: we present a case of a two-year-old male with newly diagnosed x-linked cgd. though he had been seen by multiple healthcare providers for recurrent lymphadenopathy over the preceding year, he had no other history of recurrent viral or bacterial infections or significant family history that might implicate a primary immunodeficiency. at time of presentation, he had diffuse rash which could have caused his palpable lymphadenopathy on exam. a high index of suspicion for cgd in the setting of recurrent lad and ftt prompted sending the dhr, which led to the diagnosis. chronic granulomatous disease (cgd) is an inherited primary immunodeficiency (pid) which results in both inflammatory response dysregulation and an increase in susceptibility to certain bacterial and fungal infections. without curative treatment such as a bone marrow transplant, it remains a chronic disease with daily medication management, intermittent treatment and life-long surveillance. in general, chronic disease involves physical, psychological and social effects which can affect the patients quality of life. although some research has been done on how pid affects quality of life, there is little research in the united states about how cgd affects patients quality of life. to examine the effect of cgd on patients quality of life, as a part of a voluntary research protocol examining the natural history of immune deficiencies, we administered the who qol-bref instrument to adult cgd patients enrolled on a nih irb approved protocol and seen in the infectious disease clinic at the national institutes of health (nih) over a five-month period. the who qol-bref is comprised of items, which measure the following broad domains: physical health, psychological health, social relationships and environment. each item is rated on point likert scale. it has been validated cross culturally and has been widely field tested. the survey was interview administered to patients ( males, females) with genetically confirmed cgd. the age range was - years old (mean age . years) with a distribution of % x-linked cgd and % autosomal recessive cgd. results have been obtained and will be presented. rationale: common variable immunodeficiency (cvid) is the most common primary immunodeficiency with an estimated prevalence of : , . we aimed to analyze the clinical presentations and their associated comorbidities amongst cvid patients in usa. methods: data on , cvid patients reported in the united states immunodeficiency network (usidnet) from to were analyzed based on clinical, immunological and genetic factors. univariate analysis with spearman rank coefficients was done to analyze correlations between disease outcomes. observed survival was estimated using the kaplan-meier method. results: among the patients, ( . %) were female and ( . %) were male. median age at diagnosis was years [mean (sd), . ( . ); range, - ; iqr, - ] with median age of onset of years (mean (sd), . ( . ) ; range, - ; iqr, . females showed a longer delay in diagnosis ( . vs. . years, p= . ). higher body mass index (bmi) linearly correlated with the age of diagnosis (r= . ). in survival analysis, a -year delay in age at diagnosis increased the risk of death by . % (hr: . , % ci: . - . , p= . ). conclusions: our study suggests a longer delay in diagnosis in female subjects and a strong association with diagnosis of cvid in patients with higher bmi. females may have a longer period without symptoms leading to a diagnostic delay. gender-based and disparities-based inquiry into these trends may need additional study. the physical well-being of those with primary immunodeficiency (pi) and the physical maladies of those with pi are well-documented. since the s, advances in identification and treatment of pi has for many led to lives where the physical infections of these groups of diseases are manageable. however, not as well understood are the emotional and mental health aspects of living with pi. as part of a larger survey project the idf national patient survey, this study aims to quantify any potential mental health issues or challenges faced by adults with pi. our hypothesis-those with pi, suffer from statistically higher rates of depression when compared to the u.s. general population. the idf national patient survey was a nationally distributed, unincentivized, mail-based survey of , persons in the idf patient database identified as being either adults with pi or the parent/caretaker of a child with pi. the questionnaire comprised approximately main questions about pi as well as the validated sf- v , brief fatigue inventory and the patient health questionnaire- (phq- ) instruments. additional questions asked about current use of prescription medications for anxiety, depression, stress and pain. for the purpose of this study, only adult respondents with pi are included as the basis for analysis. the two-item patient health questionnaire (phq- ) meets the criteria for general screening of depression suggested by the u.s. preventive services task force. scored on a scale of - , a score of three or higher is suggested as the cut-point for depressive screening. according to a ahrq study that utilized meps data, , of the , ( %) respondents scored three or greater. in our survey of the ( %) adults scored three or greater ( <. .) overall, those in our survey scored lower on the sf- v mcs scale when compared to the u.s. population ( . v. . , p<. ) . further, adults with pi who scored three or higher on the phq- had an average mcs of . . those who met the phq threshold in our survey were also more likely to report moderate to severe limitations in normal activities as a result of emotional problems than those that fell below the threshold ( % versus %, p <. ). not surprisingly, those that met the phq threshold reported much higher use of prescription medications for anxiety, depression, stress ( % versus % below threshold, p <. ) as well as a higher reported use of prescription pain medications ( % versus % below threshold, p <. ). though moderate to severe fatigue was reported by % of those below threshold, % of those with phq scores at threshold reported experiencing moderate to severe fatigue (p <. ). health care providers should consider including the phq- in the overall health assessments of their patients with pi. those scoring three or higher should be referred to the appropriate professional for further evaluation. (lek et al., ) . the w l is a semi-conservative amino acid substitution, which may impact secondary protein structure. in-silico analyses supported a deleterious effect, located within the sh domain, which is a critical functional domain (chandesris et al., ; koskela et al., ) . it was thus determined that this variant is likely pathogenic. the patients prophylactic treatment was optimized with tmp-smx ( mg- mg) twice daily for prevention of infections. she was also started on hibiclens (chlorhexidine) baths once per week. she was referred to pulmonology for optimization of pulmonary health in the setting of bronchiectasis and mild decline in dlco. she was advised to followup on a yearly basis to the primary immunodeficiency clinic to assess for recurrent infections and for changes in pulmonary health. finally, targeted testing and clinical evaluation of both of the patients parents was recommended to determine if w l was inherited or arose de novo. the pathogenic role of the w l missense change would be further supported if it had occurred de novo or if it segregates with the disease in the family. uploaded file(s) uploads pulmonary function testing results.pdf j clin immunol ( ) (suppl ):s -s s introduction: lipopolysaccharide-responsive and beige-like anchor protein (lrba) deficiency is a rare autosomal recessive disease of the immune systems characterized by hypogammaglobulinemia and decreased ctla expression on t regulatory cell (t regs) due to defective intracellular trafficking of ctla . previous in vitro study has shown a significant increase of ctla expression on lrba deficient t cells after overnight culture with chloroquine, an older anti-malarial agent. this effect is likely due to increasing lysosomal ph. however, there is no evidence of such effect in human subjects after administration of weight appropriate doses anti-malarial agents. we are presenting a set of siblings with lrba deficiency who had ctla expression measured before and four weeks after starting hydroxychloroquine. case reports: case is a -year-old east-indian boy with autoimmune thyroiditis, type diabetes mellitus (dm), short stature, autoimmune cytopenias, and lymphadenopathy. he was referred to immunology clinic at years of age for suspicion of autoimmune lymphoproliferative disorder. primary immunodeficiency genetic panel was sent which revealed a homozygous mutation in lrba gene (c. _ del). this novel variant resulted in a frameshift and created a premature stop codon amino acids downstream from this location which may lead to absent or abnormal protein. lung ct scan showed interstitial lung disease. lung biopsy showed interstitial nodular and diffuse lymphoid proliferation. this diagnosis led to the testing of his sister (case ) given her history of autoimmune illnesses and the family history of consanguinity. case is a now -year-old girl with type dm, autoimmune thyroiditis, lymphadenopathy, psoriatic arthritis, and seizures. her lung imaging showed pulmonary nodules without interstitial lung disease. both cases received hydroxychloroquine while waiting for insurance approval of abatacept. ctla expression on tregs was measured prior to and four weeks after starting hydroxychloroquine treatment. at baseline, . % of case s cd cells were treg (foxp +ve, cd hi) and . % of them expressed ctla- (in contrast to . % tregs in the healthy control) with mean fluorescence intensity (mfi) of . this ratio and mfi did not change after weeks of hydroxychloroquine treatment ( mg/kg/day). soluble interleukin- receptor levels were measured: case had a baseline level of pg/ml, which decreased to pg/ml after weeks of hydroxychloroquine treatment. for case : . % of her cd + t cells were found to be foxp +cd hi and . % of these tregs expressed ctla- . this ratio increased by % after one month of hydroxychloroquine. increase in mfi was also noted from to . case had a drop in soluble interleukin- receptor level from pg/ml to pg/ml after treatment. conclusion: in contrast to the previous in vitro assays, we did not find a significant increase in ctla expression on t regulatory cells in vivo after weeks of mg/kg/day hydroxychloroquine. interestingly, soluble il- receptor levels improved dramatically with hydroxychloroquine. ( ) submission id# human nf-kappab defect results in defective intrinsic b-cell differentiation, function and class switching introduction/background: autosomal dominant heterozygous mutations in nfkb (encoding for the protein nf-kb ) have been identified in the etiology of a form of primary immunodeficiency disorder that presents with hypogammaglobulinemia, defects in b-cell maturation, endocrinopathy, and autoimmune manifestations. in humans, the effects of altered nf-kb and mechanisms of immune system impairment have not been fully delineated. objectives: to understand the mechanism of the antibody deficiency in patients with hypomorphic mutations in nfkb (c. dela; p.lys serfs* ) by evaluating b-lymphocyte proliferation, differentiation, function, and gene expression. methods: immunophenotyping of primary b-cells from subjects with mutant nfkb was completed by flow cytometry. proliferation of b-cells was assessed by cfse stimulation of primary cd + b-cells from healthy and nfkb mutant subjects. differentiation of healthy and affected naïve b-cells (cd -cd -) into plasmablasts (cd +cd +) following stimulation was assessed by flow cytometry. the supernatant from these cells were assayed for iga, igg and igm production by elisa. to study the defect in class-switch recombination, naïve b-cells and ebvtransformed b-cells from affected and healthy individuals were stimulated and expression of the aicda gene was quantified by qpcr. in parallel experiments, ebv b-cells from wildtype and nfnb mutant individuals were stimulated and aid (activationinduced cytidine deaminase) protein levels were determined by western blot. results: patients with hypomorphic mutations in nfkb (c. dela) had low memory b-cell (cd + cd + igd-igm+) and class-switched memory b-cell (cd + cd + igd-igm-) numbers. in vitro, primary bcells from these patients demonstrated a % reduction in proliferation and cell division in response to cd l and il- (p = . ). compared to healthy naïve b-cells, mutant naïve b-cells had a significant reduction in plasmablast differentiation (p = . ) and secreted significantly lower levels of immunoglobulins in response to cd l and il- stimulation. mutant naïve b-cells and mutant ebv b-cells failed to increase aicda expression and aid protein levels in response to cd l and il- stimulation. conclusions: our studies demonstrate that a hypomorphic nfkb mutation in humans affects intrinsic b-cell proliferation and differentiation. the mutation impairs transcription of the aicda gene that encodes aid, a key protein involved in b-cell class-switch recombination. the nfkb gene defect also impairs immunoglobulin production, as seen in common variable immunodeficiency-like cases. these studies provide unique translational insights into physiological activities of nf-kb in downstream immunologic outputs in humans, expanding those suggested by experimental observations in mice. background: few studies have evaluated the quality of life (qol) and patient reported outcomes of primary immunodeficiency disease (pidd) patients, and no studies have assessed medical provider perceptions of their pidd patients qol, neurocognition, physical well-being and psychosocial health. understanding provider beliefs regarding patient reported outcomes is essential to improving clinical management of pidds. here we report our pidd medical provider survey results. methods: providers were contacted via email with the assistance of the clinical immunology society. participants completed adult and/or pediatric-based likert scale survey questions via a secure online survey service. in addition to demographic information, survey questions assessed provider perceptions of patients overall qol and their impression of the impact of disease or its associated treatment on mental health, physical well-being, neurocognition, social relationships and school/work performance. clinicians were expected to make their assessments based on their pidd patient cohort as a whole rather than on specific diagnoses or patients. given the small sample size, a p-value < . was considered statistically significant; repeated measures anova and paired t-test analyses were used. results: study participants (n= ) were primarily from the united states ( %), born between - ( %) , and trained in allergy/ immunology ( %). % of survey takers practiced within an academic center, % were female and % cared for children with % of providers concurrently caring for adults. there was a statistically significant difference (p= . ) in the perceived overall qol of pediatric versus adult pidd patients with % of providers feeling as though their pediatric patients had a good qol while only % believed their adult patients had a good qol. clinicians believed adult pidd individuals had more difficulties related to associated co-morbidities rather than their actual pidd compared to pediatric pidd patients (p= . ). providers felt that the neurocognition and school performance of children were more often negatively affected by a pidd than the neurocognition and work performance of immunodeficient adults (p= . ). clinicians believe children with pidd more frequently had difficulties related to their concentration than memory (p< . ). % of those who care for pidd adults believe their patients work performance or daily mental functioning is at times negatively impacted. anxiety symptoms and social relationships were viewed as being more negatively impacted by a pidd diagnosis or treatment than anger or depressive symptoms in both children and adults (p< . ). % of pediatric clinicians feel their pidd patients experience anxiety symptoms often or almost always. of physical health parameters, energy, rather than mobility or pain, was deemed to be more deleteriously influenced by an immunodeficiency in adult and pediatric patients (p< . ). conclusions: our results show that medical providers perceive the overall qol of pediatric pidd patients to be superior to that of adults with pidd, but most clinicians feel a diagnosis or associated treatment regimen for pidd can negatively impact the physical well-being, psychosocial health, school/work performance and neurocognition of both children and adults. [cbm] complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed cbmopathies) result in broad clinical manifestations ranging from severe combined immunodeficiency (scid) to lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card mutation (c. c>t; p.r *). results: we have identified an -month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a -year-old boy with a history of severe pulmonary infections (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. both patients possessed absent tregs, absent memory b cells, and hypogammaglobulinemia. however, only the -month-old had poor t cell proliferation to pha, cona, and cd . both patients were found to be homozygous for the same novel variant of card (c. c>t; p.r *). the mutation rendered card protein expression unstable and it was undetectable by immunoblot. to confirm card deficiency, we stimulated patient b cells with phorbol -myristate acetate (pma) and ionomycin across a time-course and immunoblotted for various signalling proteins in both the nf-b (ikk/, ib, p ) and mapk (mek / , mkk , jnk / , erk / ) pathways as well as various cleavage substrates of the malt paracaspase (relb, cyld, bcl , hoil ). nf-b and jnk activation were completely absent and malt paracapase activity was lost, but surprisingly, mkk (which acts upstream of jnk) was intact. furthermore, co-immunoprecipitation experiments revealed that card was required for optimal malt association with bcl in response to stimulation. conclusions: these two cases highlight the crucial role of card in regulating lymphocyte development, function, and humoral responses. in addition, we have identified the oldest known living individual with card deficiency and he presented uniquely with inflammatory gastrointestinal disease in addition to scid, further adding to the spectrum of phenotypes associated with card -related primary immunodeficiencies. abstract: the usidnet registry began in with an niaid contract with the immune deficiency foundation, which continues today. it aims to provide a resource for clinical and lab research through enrollment of known immunodeficiency patients into a national registry, the usidnet. nih is a major national and international referral center for clinical trials on inborn errors of immunity, or primary immunodeficiency diseases. it is a mechanism for depositing nih data into usidnet. a registry of patient information may help us understand how many people have each disease. the information may improve how we diagnose and treat these conditions. the patient registry is designed to obtain longitudinal data on a large number of patients with primary immunodeficiency diseases who come to nih to participate in research. the data is collected from the nih electronic medical record system, cris and is deposited into a secure registry with restricted and monitored access. all medical information is anonymized for patient privacy. department of biochemistry, emory university, atlanta, ga oas is an intracellular sensor for dsrna that generates the second messenger '- '-oligoadenylate to activate rnase-l as a means of antiviral defense. we describe four patients with a complex early-onset autoinflammatory and immunodeficiency disease caused by heterozygous de novo oas mutations. patients presented early in life with lung inflammation including pulmonary alveolar proteinosis and interstitial lung disease. they had febrile flares with dermatitis specifically with macular, pustule and bullous features often progressing to ulceration. infants had episodes of bloody diarrhea in patients (assoc. with villous blunting and cryptitis in two patients and oesophagitis in one patient). immunoglobulin igm, igg, and iga levels were low while t cell, b cell, and nk cell numbers were generally in the normal range. exome sequencing identified de novo heterozygous oas missense mutations in all patients. one patient had a heterozygous de novo oas mutation p.ala val, with mutant oas protein being expressed in ex vivo generated t cell blasts. in sorted primary patient monocytes and b cells, oas p.ala val was associated with spontaneous rna degradation and apoptosis as determined by rna chip technology and flow cytometry, respectively, while t cells were not affected. monocytes displayed disturbed terminal differentiation and functioning as indicated by reduced gm-csf-r expression and signaling. b-cells display reduced class-switch-recombination. proliferation of allogeneic t-cells was reduced in response to sorted oas mutated monocytes and b-cells. activation of interferon response genes in pbmcs was detected. two further unrelated patients had a heterozygous de novo oas mutation p.cys tyr, which appeared to compromise protein stability in transformed patient fibroblasts and when transfected. cells transfected with this mutant protein had reduced - oligoadenylate synthesis compared to wild type transfected cells. immortalized fibroblast lines demonstrated higher levels of inflammatory cytokines and spontaneous cleavage of rnas. a th patient with the clinical phenotype had a heterozygous de novo oas variant p.val gly, but has yet to have formal validation of the variant. three patients underwent hematopoietic stem cell transplants in an effort to control their diarrhea and skin inflammation. one patient died with ongoing chronic graft versus host disease, while the two others (p.ala val, cys tyr) are alive and reasonably well with a followup of . - years. the untransplanted patient died as a result of respiratory failure. in summary, patients with de novo heterozygous oas mutations have chronic ongoing inflammation of multiple organs. this is at least in part due to spontaneous rna cleavage, apoptosis and production of inflammatory cytokines and type i interferons. this defines a new category of autoinflammatory disorder. introduction: increased susceptibility to infections is the most common complication of chronic granulomatous disease (cgd). hemophagocytic lymphohistiocytosis (hlh) is a severe disorder resulting from hyperinflammation and hypercytokinemia that can lead to multi-organ system dysfunction ( ) characterized by certain criteria: fever, splenomegaly, cytopenias, hypofibrinogenemia or hypertriglyceridemia, hyperferritinemia, increased soluble cd /il- ra, evidence of hemophagocytosis, or decreased/absent nk cell cytotoxicity ( ) . secondary hlh occurs infrequently but often is preceded by smoldering infection in cgd ( , , ) . we present a case of hlh in a -day old male, the youngest reported case with cgd. case: a -day old male with previously diagnosed x-linked cgd, due to known family history, presented with fevers. initial evaluation was unrevealing including chest x-ray, urinalysis, and blood and csf cultures. he was admitted and treated empirically with cefepime. ct demonstrated multiple multifocal nodules of the lungs and spleen. after lung nodule biopsy was performed, antimicrobial therapy was broadened to iv meropenem, voriconazole, and micafungin. despite this, he continued to have fever and developed new onset tachycardia, respiratory distress, and lactic acidosis. further decompensation with vasoactive refractory shock was treated with vasopressors and stress dose hydrocortisone. additional laboratory evaluation revealed rising liver enzymes (ast u/l, alt u/l), cytopenias (hemoglobin g/dl, anc /ul, platelets , /ul), and coagulopathy (fibrinogen - mg/dl). splenomegaly was present on abdominal ultrasound. a diagnosis of evolving hlh was considered and dexamethasone was administered. within hours of clinical decompensation, the patient died of multiorgan failure. subsequent blood cultures returned with gram-negative rods (and ultimately burkholderia cepacia). autopsy confirmed hemophagocytosis within the bone marrow. no mutations were found in genes associated with primary hlh. discussion: patients with cgd are susceptible to infectious complications and auto-inflammation most commonly involving the lungs, gi, and gu systems ( , ) . patients with cgd can be at increased risk of hyperinflammatory syndromes secondary to infections and chronic inflammation. as shown in the included case, hlh can present in infancy and can be deadly. early consideration and directed treatment of hlh is imperative, even in the setting of sepsis malignant proliferation of gamma-delta t cells include hepatosplenic t-cell lymphoma (hstl), primary cutaneous t-cell lymphoma and t-cell large granular lymphocytic leukemia (t-lgl). the former two have often been associated with splenomegaly and cytopenias. however, reactive proliferation of gamma-delta t cells in spleen mimicking malignancy has only been reported once and has a significant risk of misdiagnosis. a -year-old female presented with two years of unintentional weight loss, persistent leukopenia and thrombocytopenia, with leucocytes around - x ^ /l and platelets around x ^ / l. she also had associated macrocytic anemia (hemoglobin= - g/dl) with laboratory evidence of dat (direct anti-globin test) negative hemolysis. physical examination and computed tomography (ct) imaging showed splenomegaly. there was no hepatomegaly or lymphadenopathy. serum liver function test, auto-immune studies, hemolysis and hereditary diseases workup, viral and bacterial serologies were all normal or negative, except for mild hyperbilirubinemia and ldh elevation. bone marrow examination performed four months prior to the splenectomy revealed mildly hypocellular marrow ( %) with trilineage hematopoiesis. flow cytometric analysis and cytogenetics of the bone marrow aspirate and peripheral blood were normal except for small population of large granular lymphocyte and mild low absolute b cell counts in peripheral blood. a laparoscopic splenectomy was performed for diagnostic and therapeutic purposes due to patients worsening luq pain. there was no other treatment given prior to surgery. hours postsplenectomy her leucocytes increased to . and platelets to . her three-month post-splenectomy wbc count and platelet count was . and , respectively. hemoglobin also improved to . . pathology showed red pulp expansion by small lymphocytes (fig. ) and subsequent ihc (immunohistochemistry) was positive for cd ( fig. ) , cd , cd , tia- and negative for cd , cd and cd . cd was difficult to interpret. eber was negative. flow cytometry ( fig. ) showed increased gamma-delta t-cell population ( %) with positive cd , cd and cd and negative cd , cd and cd . molecular studies by pcr didnt reveal any t-cell receptor gamma or beta gene rearrangement. cytogenetics was negative for isochromosome q or any other abnormalities. she was symptom free at months from her splenectomy. the morphology and immuno-phenotype of these gamma-delta t cells show significant overlap with the malignant cells seen in hstl and t-lgl, such as loss or downregulation of cd , cd and cd . awareness of this reactive condition is necessary to prevent making a wrong diagnosis of a malignant disease with a potentially benign, spontaneously resolving disease. additional studies of similar cases is needed in order to establish more definitive criterion to separate benign from malignant processes and delineate the role of gamma-delta t cells. uploaded file(s) uploads fig . flow cytomtery.pptx background: sex steroids in the human thymic environment influence aire expression as well as interactions with its partners, i.e. genes coding for aire interactors. here we investigated the effects of sex steroids on these interactions during minipuberty the surge of sex hormones that occur along the first six months of life -and up to months of life. we employed a network-based approach for investigating aire-interactors gene-gene relationships and how abundantly co-expressed thymic mirnas covariate with those genes. aire-interactors networks allowed the measuring of gender-related differences in gene-gene expression correlation disclosing relevant differences between minipuberty groups. methods: total rna was extracted from thymic surgical explants obtained from male (m) and female (f) infants -aged - months (groups mm and mf, for minipuberty) and - months (group nm and nf, for nonpuberty) and used in dna microarray assays. gene coexpression network (gcn) analyses were performed for aire and its interactors and for mirna-gene coexpression analysis. the set of genes coding for the aire-targeted proteins was previously identified in tecs by abramson et al. (cell : - , ) . aire-interactors networks were obtained for all groups (link strength cut-off for gene-gene > | . | and for mirnagene < - . ). aire expression in mtecs was quantified by immunohistochemistry. these methodologies are described in moreira-filho et al. (sci rep : , ) . results: the mm x mf networks comparison showed that abundantly expressed mirnas are interacting with the different aire interactor genes in both networks. it is interesting to note that network topology were more similar between nm and nf groups, although aire interacts with only one distinct mirna in each network (mir- - p in the nm group or mir- in the nf group). conversely, in the non-puberty networks the sets of mirnas and their interacting genes are distinct for each network. immunohistochemistry analysis revealed a higher percentage of mtec aire positive cells in the minipuberty groups: i.e. there is a significant difference between mm x nm (p = . ) and between mf x nf (p = . ). conclusions minipuberty and genomic mechanisms shape thymic sexual dimorphism along the first months of life. this process does not involve changes in aire expression between genders, but differences in the interactions of aire with its partners that persist throughout the non-puberty period, probably regulated by mirnas and also by genetic and epigenetic factors. introduction: neutrophils are presumed to defend against aspergillus species by releasing reactive oxygen species (ros) and neutrophil extracellular traps (nets) to degrade fungal hyphae. triazole antifungals synergistically enhance neutrophil mediated hyphal degradation. patients with cgd are particularly susceptible to aspergillus species likely due to their inability to create ros and nets, and in severe cases may not be amenable to antifungal therapy alone. objective: we present a case of severe disseminated aspergillosis in a patient with cgd in whom gt served as an important adjunct to antifungal therapy and bridge to transplant. results: a -year-old boy with known cgd, lost to follow up and nonadherent to prophylaxis, presented acutely with right-sided hemiparesis. neuroimaging revealed an embolic left middle cerebral artery infarction and cardiac magnetic resonance imaging showed extensive vegetations involving both right and left ventricles and atria, with an ejection fraction of %. the patient was admitted to intensive care, started on liposomal amphotericin b, meropenem and vancomycin, and underwent debulking of the intracardiac masses on post admission day (pad) . operative findings showed severe constrictive pericarditis with multiple abscesses and intracardiac vegetations. thorough debridement of the vegetations was undertaken, however some deep seated abscesses in the myocardium were not amenable. operative cultures were positive for aspergillus fumigatus. clinical status remained precarious, with ongoing requirement for inotropic and ventilator support. antimicrobial therapy was refined to voriconazole, with amphotericin b remaining on board until therapeutic levels of voriconazole were achieved. as effective neutrophils are integral in the immune response against aspergillus, the decision was made to start granulocyte transfusions to aid in clinical stabilization prior to hsct. interferon gamma infusions were not administered because of the risks of adverse effects and potentially increasing transplant rejection. gts were started on pad , at a dose of approximately x ^ granulocytes, three times a week. the patient tolerated the infusions well, with no allergic or inflammatory response. neutrophil oxidative burst measured one hour post infusion showed . % mean fluorescent intensity, compared to a baseline of % ( figure ). clinical improvement was seen, with inotrope cessation on pad and extubation to bipap on pad . human leukocyte antigen (hla) allosensitizaton was tested on pad , days after the first gt, with no evidence of hla antibodies. a total of gts were given over months, prior to proceeding to a / hla matched related donor transplant (pad ), with two transfusions given before neutrophil engraftment (anc ) on day + . the patient is now stable months post transplant, with no evidence of graft rejection. he remains on chronic suppressive antifungal therapy, to continue until full lymphoid reconstitution. conclusion: gt may be a useful adjunct to antifungal therapy in patients with impaired neutrophil function with severe invasive aspergillosis, and potentially provide a life sustaining bridge to hsct. methods: subjects were enrolled in irb protocol for rvt- . rvt- was implanted into the quadriceps with immunosuppression. results: subject was normal at q . but had hypocalcemia, an asd, pda, and abnormal ears. the subject received a cord blood transplant mismatched at hla-b and hla-c alleles at age months. subsequently mild graft-versus-host disease (gvhd) developed and was treated with antithymocyte globulin, steroids and cyclosporine. donor t cells developed in low numbers. twelve years later, the subject developed epstein barr virus lymphoma and suffered two relapses. while in remission, subject received unmatched rvt- . two weeks after rvt- implantation, the subject developed an adenovirus infection resulting in skin and gut gvhd, presumably from activation of the cord blood t cells. subject was treated with corticosteroids, cyclosporine, cidofovir and infliximab. four years post rvt- , subject is healthy with genetically recipient t cells/mm and % naïve cd t cells. subject was normal at q . but had an asd, pda, hypoparathyroidism, and no t cells at birth. his genetic defect is unknown. subject was treated with a ric myeloablative, allogenic, unrelated, / cord blood transplant, and a subsequent myeloablative, unrelated / cord blood transplant. hematopoietic chimerism was established without t cell development. rvt- expressed the one allele in the recipient that was not expressed by the second cord donor. the post-thymic transplant course included immune thrombocytopenia requiring rituximab and splenectomy and generalized adenopathy for years but no gvhd. he failed weaning of immunoglobulin replacement. three years post rvt- , he has cd , cd , and cd t cells/mm . he is active in school. subject had absent trecs on newborn screening with cd + t cells/ mm. a single mutation in foxn was identified; she has sparse scalp hair. subject received a / matched unrelated umbilical cord transplant. the post-transplant course was complicated by significant morbidity, and no naïve t cell development. rvt- expressed the one allele in the recipient that was not in the cord blood donor. the subject did not develop gvhd, is healthy and at months has naïve cd + t cells. she had resolution of longstanding norovirus and sapovirus gastroenteritis. conclusion: rvt- can improve t cell immunity after poor or failed correction with allogeneic hematopoietic transplants. in subject , gvhd post rvt- was related to an acute viral infection; cord t cells attacked hla mismatches in the recipient. subjects and were given rvt- matched to recipient alleles that were not expressed in the hematopoietic donor. we hypothesize that thymocytes developing in rvt- , if strongly reactive to the recipient-mismatched allele, are deleted by the bonemarrow-donor dendritic cells (that acquire recipient mhc from the recipient-allele-matched thymic epithelial cells) thereby preventing gvhd. rationale: ctla haploinsufficiency is an autosomal dominant immune dysregulation syndrome characterized by variable phenotypes. here we present a young woman diagnosed with evans syndrome and lymphoproliferation as a child, found to have a novel ctla variant as a young adult, and who developed hypogammaglobulinemia and a bacterial endocarditis while stabilized on ctla- replacement therapy. methods: sequencing of genes, including ctla , in primary immunodeficiency panel. results: our patient was diagnosed with evans syndrome at age with manifestations of anemia and thrombocytopenia recalcitrant to treatment over many years with steroids, cyclosporine, and vincristine. bone marrow biopsy reportedly showed normal trilineage maturation and her symptoms responded for a short time to splenectomy at age . symptoms recurred at age when she was also found to have pulmonary reticular opacities, prominent lymph nodes, and elevated b cells. repeat bone marrow and lymph node biopsies at that time were unrevealing. minor responses to treatment with ivig, rituximab, mycophenolate mofetil and gcsf were noted. at age , she developed varicella-related encephalitis shortly after vaccination. with a strong suspicion of an immune dysregulation syndrome, immune evaluation revealed normal immunoglobulins with good vaccine responses, elevated b cell numbers, normal t cell numbers, and normal mitogen proliferation. ctla sequencing revealed a mutation in exon [c. c>a, p.tyr *] causing a premature translational stop signal, which was consistent with previously reported cases of ctla haploinsufficiency. she was started on rapamycin initially for her cytopenias but was then transitioned successfully to abatacept with almost complete resolution of her anemia, neutropenia, and pulmonary opacities. after months of stable control, she developed a precipitous drop in her platelets and was eventually diagnosed with streptococcus viridans endocarditis of her native mitral valve. this responded to antimicrobial therapy, but eventually needed surgical intervention due to ongoing insufficiency. around this time, she was also found to be newly hypogammaglobulinemic, necessitating ongoing igg supplementation therapy. during successful replacement of her mitral valve with a biosynthetic prosthesis, it was noted that her aortic valve also had evidence of previous disease, implicating a prior endocarditis as part of her clinical syndrome as well. conclusions: in this patient, the presentation of recalcitrant cytopenias, lymphadenopathy, elevated b cells, vaccine-induced viral infections and lung findings precipitated concern for immune dysregulation syndromes and allowed for identification of a novel deleterious ctla mutation. in addition to previously reported clinical findings, our patient presents with the first reported case of repeated endocarditis in the setting of ctla insufficiency disease. given the finding in this patient of prior (unrecognized) disease, regularly screening patients with ctla insufficiency for evidence of cardiac affectation may be prudent. clinical research nurse, johns hopkins university background: the relationship between elevated serum alpha fetoprotein (afp) concentration and age, mortality, genotype and neurologic outcome in ataxia telangiectasia (a-t) patients has remained inconclusive over the past decades, leaving afp as a useful marker for disease diagnosis without further clinical significance. objective: to examine the relationship between afp levels and age, mortality, genotype and neurologic outcome using a data set larger than any prior study. methods: we retrospectively collected data on a-t patients at johns hopkins medical center ( - years of age) with both classical (predicted protein null) and variant a-t. this included serum afp measurements ( serial levels in a-t patients, max observations per patient). mixed model compound symmetry covariance was used for statistical analysis to examine the effect of age at visit on afp levels. subgroup analysis by mutation type, mortality, feeding/swallowing scores as a surrogate for neurologic function, x-ray induced in vitro chromosomal breakage and serum transaminase levels were similarly analyzed. results: significant association between age and afp level was found such that for every year increase in age, afp level increases ng/ml (p< . ). subgroup analysis by mutation type found that the patients with missense mutations showed a negative linear relationship be-tween log afp levels and age (r= - . , p= . ). we found greater afp levels in patients who subsequently died, after controlling for age (least square mean afp level in log scale . greater in deceased patients versus living patients, p= . ). we found a significant decline in feeding score by . units (score range - ) per ng/ml afp increase (p= . ) after adjusting for age. there was no significant relationship between afp levels and serum transaminase levels. conclusion: afp increases with age in a-t patients, though this may not apply to patients with missense mutations. there is a statistically significant increase in mortality and worsened swallowing scores with increasing afp levels, but this remains to be proven clinically significant. here we present a pediatric hae patient who had recurrent abdominal attacks in which constipation, secondary to the adhd medication dexmethylphenidate (focalin), appears to be a trigger. of importance, this is the first pediatric patient with hae to be described as having safely undergone a capsule endoscopy for direct visualization of the gastrointestinal tract. this was done to decrease the risks associated with the more invasive procedure of traditional endoscopy and colonoscopy. case presentation: the patient was an -year-old male with hereditary angioedema who presented with day history of diffuse abdominal pain and nausea. in the ed, patient was in no acute distress. abdominal ultrasound showed severe circumferential thickening of the wall of multiple bowel loops and a large amount of simple ascites. x-ray revealed stool in the colon. he was admitted for pain control and hydration. in the next year, he visited the ed five more times for exacerbations of angioedema of his hand, penis, and bowel. each time, he presented he had underlying abdominal pain and constipation. he was seen by gastroenterology and had a workup that was negative for helicobacter pylori, parasites, and other gastrointestinal infections. to further evaluate his abdominal pain, capsule endoscopy was performed and well tolerated. during an admission in january he received a full inpatient bowel cleanout, after which, his angioedema finally improved. of note, he was diagnosed with adhd and started on dexmethylphenidate (focalin) just prior to this period of recurrent angioedema attacks, and he did not have attacks during the summer months when he was off the medication. discussion: abdominal pain is a common complaint in pediatric hospitals, and further workup consists of endoscopy and colonoscopy. this may be easily accomplished in the general population, however, in patients with hae, these procedures carry greater risk and may be avoided, leading to delayed diagnosis and treatment ( , ) . a newer and less commonly used alternative for direct visualization of the gastrointestinal tract is capsule endoscopy. some benefits are that it does not require sedation, is less invasive, and is less likely to be irritating to the mucosa ( ). additionally, since psychological stress may be a trigger for angioedema attacks, the decreased stress associated with a noninvasive procedure such as capsule endoscopy, makes it safer to use ( ) . limitations of capsule endoscopy include dependence on battery life and its inability to biopsy or administer therapy if needed ( ) . hereditary angioedema treatment consists primarily of avoiding triggers and managing acute episodes. in this first case of hae in a pediatric patient where capsule endoscopy was used, the procedure was well tolerated without any complications. recognizing constipation as a trigger and capsule endoscopy as a safe method of direct visualization of the gastrointestinal tract will help others to control and decrease the severity of their hae attacks as well. a year old male with past medical history of common variable immune deficiency (cvid) and related autoimmune complications, including granulomatous-lymphocytic interstitial lung disease (glild), hepatosplenomegaly, leukopenia, and thrombocytopenia tolerated monthly subcutaneous immunoglobulin replacement as outpatient for several years with infrequent infectious complications. four months ago, he was found to have elevated liver enzymes on routine chemistry. a liver biopsy two months later showed pathology consistent with nodular regenerative hyperplasia (nrh) without overt cirrhosis. a hepatic venous pressure gradient (hvpg) of mmhg was found, consistent with portal hypertension. his hepatitis viral markers were negative, he did not drink, and portal venogram was negative for thrombosis. in early october, the patient was admitted to the hospital with anasarca and tense ascites. he underwent a diagnostic and therapeutic large volume paracentesis and was also found to have spontaneous bacterial peritonitis (sbp) and bacteremia with group b streptococcus. the patients course was complicated by polymicrobial peritonitis, vre bacteremia, fungemia, variceal hemorrhage, hepatic encephalopathy, and hepatorenal syndrome. his hepatic complications from portal hypertension were out of proportion to his liver parenchymal disease. transjugular intrahepatic portosystemic shunt (tips) was considered to alleviate portal hypertension but was not feasible due to his degree of encephalopathy. immunosuppressants such as high dose steroids were given while in the hospital with plans to start rituximab to treat patients glild after he had recovered from the acute infections. unfortunately, after two months in the hospital, the patient succumbed to sepsis and progressive liver failure. this case emphasizes the importance of systematic screening and continued vigilance for hepatic complications in patients of cvid as studies have shown that nrh of the liver is present in more than % of cvid patients who undergo a liver biopsy (pmid: ). a cross-sectional study of patients with primary hypogammaglobulinemia and hepatic dysfunction found that histological findings of nrh were present in % of cvid patients and was associated with portal hypertension in % of cases (pmid: ). another study estimated the minimal prevalence of nrh in cvid patients as % (pmid: ), stating that this was likely a gross underestimate as nrh may also be present in patients with normal liver function tests that are not routinely biopsied. therefore, liver enzyme levels may not anticipate the severity of liver involvement. there is currently no treatment for cvid-related liver disease. other causes of non-cirrhotic portal hypertension, including hepatic veno-occlusive disease and budd-chiari syndrome should be ruled out or treated in cvid patients presenting with hepatic disease. in the case of hepatic nrh in cvid patients, early detection could lead to earlier interventions (such as tips prior to hepatic encephalopathy), to mitigate complications. we describe the application of epigenetic quantification of t regulatory (treg) cells in addition to cd +, cd +, cd + t cells, b cells, nk cells, monocytes and neutrophils from as little as μl of fresh, frozen or dried blood. the method yields identical results to flow cytometry from fresh blood samples of a healthy donor cohort, with the advantage of being more sensitive and precise with limited amount of blood and minimal sample preparation (sci transl med ). we have used this method ) to immunophenotype patients with early onset immune regulatory disorders (pird) and primary immune deficiency (pid), and ) to evaluate cell subsets reconstitution early after hematopoietic stem cell transplantation (hsct). patients with immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) and ipex-like pird were evaluated by analyzing the treg-specific demethylated region (tsdr) of the foxp locus in the total of cd + t-cells. despite the dysfunctional foxp mutated protein, ipex patients exhibited elevated treg/cd + cell ratios which seemed to correlate with disease severity. in contrast, most of the patients with ipex-like symptoms without foxp mutations exhibited decreased treg/cd + cell ratios -in line with the possible central pathogenic role of treg function and number in pird. using epigenetic quantification of cd +/b-and nk cells, out of confirmed scid and xla cases were correctly identified within a cohort of newborn dried blood spot (dbs) samples ( % sensitivity, % specificity). the method identified one delayed onset scid as well as a xla case that were missed by combined trec/krec testing. epigenetic immune cell quantification missed one scid case with maternal engraftment that was identified by combined trec/krec testing. abnormally elevated treg/cd + ratio was also detected in a dbs from a newborn who was subsequently confirmed to be affected with ipex syndrome. when applied to serial blood samples during engraftment and reconstitution post-hsct, the epigenetic method allowed identification of the different blood cell subsets, including treg cells, at earlier time points than flow cytometry according to current clinical practice. this opens the way to a better understanding of the correlation between early immune reconstitution events and graft vs. host disease or viral reactivation, earlier than with the current methods, in different types of hsct. these studies underscore the suitability of epigenetic immune cell quantification for accurately measuring multiple immune cell types from limited blood sample sources. we propose this method as uniquely suitable for novel molecular diagnostic applications in settings with limited fresh blood sample or limited cell number, at the point of care as well as for newborn screening. we evaluated a -year-old male with hyperpyrexia, hypertrichosis, conical hypodontia, and a history of illnesses concerning for nemodeficiency syndrome. starting at six months of age, he suffered recurrent episodes of acute otitis media (non-typeable hib and actinobacter iwolffli), pneumonia, and rsv bronchiolitis. whole exome sequencing demonstrated a de novo heterozygous c. g>a (p.r q) mutation in the eda-receptor (edar) gene not present in the parental dna. his physical exam findings and mutation were consistent with hypohidrotic ectodermal dysplasia (hed), a rare genetic condition characterized by abnormal development of skin, teeth, hair, and sweat glands. hed is caused by defects in the ectodysplasin-a (eda)-nfkb signaling pathway but is not typically associated with immune deficiency. consistent with this, immunophenotyping showed normal sub-populations of t-, b-, and nk-cells. immunoglobulin and complement levels were quantitatively appropriate. he had normal mitogen-induced lymphocyte proliferation and normal antibody response to pneumococcal vaccination. nk-cell studies demonstrated robust cytotoxicity. however, nasal mucosa biopsy showed diffuse squamous metaplasia and the absence of ciliated epithelial cells. we hypothesize that recurrent infections in our patient arose from impaired mucociliary clearance due to a ciliary defect. this case raises the possible association between edar variants and ciliary dysfunction. it also underscores the importance of evaluating the immune status of hed patients with recurrent infections which could mimic nemo-deficiency and have broad implications about clinical management. the rapid pace of new gene discovery and phenotype expansion for primary immunodeficiency diseases (pidds) creates challenges for genetic testing and variant interpretation. whereas well-described clinical case reports in published literature have traditionally served as the source of phenotypic data used for variant interpretation, for pidds the causal variants are often private to the patients family and thus the sole source of phenotypic information for a novel genetic variant is frequently the history provided by the clinician on the test requisition form. taking into account such heterogeneous information during variant interpretation requires establishing objective criteria for its inclusion as part of the variant interpretation process. to this end, we adapted our laboratorys preexisting, evidence-based variant classification framework, called sherloc, by developing point-based criteria for the inclusion of clinical information such as a patients phenotype, familial segregation patterns, and whether the variant is inherited or de novo in the patient. as part of this process, we defined clinical criteria for pidd genes. here, we illustrate the application of this method and the importance of integrating clinical information into variant interpretation. between april and october , our commercial diagnostic laboratory performed immunological genetic tests, and information about the patients clinical history was provided in ( %) of these orders. restricting our analysis to just the genes for which case report information is currently used in variant interpretation, these tests revealed variants, ( %) of which were classified as pathogenic or likely pathogenic (p/lp). information from case report descriptions, segregation patterns, and de novo status were applied for %, % and % of p/lp variants, respectively. in ( %) cases, the clinical information provided by the clinician on the test requisition form was used as evidence in the classification of the patients variant as p/lp. ten variants were initially classified as being of uncertain significance and reclassified following receipt of further clinical information or testing of additional relatives. in addition, suspicious variants of uncertain significance were identified in which one or two additional patient case reports would allow for reclassification from uncertain significance to p/lp. these data illustrate the importance of providing good quality clinical information to the genetic testing laboratory both at the time of sample submission and following the receipt of genetic test results. background: cartilage-hair hypoplasia (chh) is a skeletal dysplasia with combined immunodeficiency, variable clinical course and increased risk of malignancy, mostly non-hodgkin lymphoma and basal cell carcinoma. there is a paucity of long-term follow-up data, as well as knowledge on prognostic factors in chh. objective: we conducted a prospective cohort study in finnish patients with chh to describe clinical course and analyze risk factors for adverse outcomes. methods: we recruited finnish patients with chh in - and performed clinical follow-up in - . we obtained health information from finnish national medical databases (covering time period of - ), the finnish cancer registry and the cause-of-death registry of the statistics finland and analyzed all patients' health records. standardized mortality ratios (smrs) were calculated based on the population data. primary outcomes included immunodeficiencyrelated death (from infections, respiratory diseases or malignancies), the development of lymphoma and the development of skin cancer. results: the study cohort included males and females. median age at recruitment was . yrs (range weeks - . yrs) and median duration of follow-up for the surviving patients was . yrs (range . - . yrs). half of the patients ( / , %) had no symptoms of immunodeficiency, while ( %) and ( %) patients manifested symptoms of humoral or combined immunodeficiency respectively, including six cases of late-onset immunodeficiency. in a significant proportion of patients ( / , %), clinical features of immunodeficiency progressed over time. of the patients with non-skin cancer, eight had no preceding symptoms of immunodeficiency. altogether patients had deceased (smr= . , % confidence interval (ci)= . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] including deaths due to pneumonia (n= ), malignancy (n= , smr= , %ci= . - ) and lung disease (n= , smr= , %ci= . . malignancy was diagnosed in / ( %) patients, mostly lymphoma (n= ) and skin cancer (n= ). severe short stature at birth (compared to normal, smr/smr ratio= . , , symptoms of combined immunodeficiency (compared to asymptomatic, smr= ( %ci= . - ) vs smr= . ( %ci= . - . ), hirschsprung disease (odds ratio (or) . , %ci= . - ), pneumonia in the first year of life or recurrently in adulthood (or= . / , , and autoimmunity (or= , %ci= . - ) in adulthood associated with early mortality. in addition, recurrent pneumonia in childhood was associated with the development of lymphoma, while warts and actinic keratosis were associated with the development of skin cancer. birth length standard deviation score correlated significantly with the age at the diagnosis of first malignancy (p= . ), lymphoma (p= . ) and skin cancer (p= . ), demonstrating that patients with shorter birth length developed malignancies at an earlier age. conclusions: patients with chh have high mortality due to infections and malignancies, but also from lung disease. some subjects present with late-onset immunodeficiency or malignancy without preceding symptoms of immune defect, warranting careful follow-up and screening for cancer even in asymptomatic patients. we provide clinicians with the risk factors for adverse outcomes to assist in management decisions. autoimmune lymphoproliferative syndromes (alps and related disorders) are characterized by insufficient apoptosis due to defects in the fas apoptosis pathway. fadd deficiency (omim ) is an autosomal recessive disorder resulting from a mutation in fas-associated protein with death domain (fadd), the adaptor protein involved in fas signaling to caspases and . we present a case of fadd deficiency identified by whole exome sequencing with a novel genetic mutation we describe two brothers with recurrent febrile episodes accompanied by seizures and respiratory compromise. the older sibling initially presented with status epilepticus following the measles mumps rubella vaccination later experiencing similar episodes until his demise at months of age. the younger sibling, who is unvaccinated, presented at months with fever, rash, vomiting, and diarrhea. he developed status epilepticus with respiratory depression that required intubation. he also had enlarged cervical lymph nodes that regressed with antibiotics and steroids. he recovered from that episode but subsequently had a series of similar illnesses with fevers, altered mental status and seizures. with the exception of elevated hhv igg, extensive infectious workup up in all instances was negative. previously described fadd deficiency patients demonstrate an alps like phenotype with increased circulating double negative t cells, lymphocyte apoptosis defects, elevated fas ligand and il , encephalopathy, functional asplenism but no splenomegaly or lymphadenopathy. our patients clinical and laboratory findings were similar. he had normal igg and iga, decreased igm, and lack of isohemagglutinins. absolute cd + count is elevated, with elevated percent of cd + tcr+ cd -cd -. normal mitogen and antigen t lymphocyte stimulation, but with defect in pokeweed induced b cell proliferation. fas ligand and il level are increased (see table ). no hepatosplenomegaly, but howell jolly bodies were detected in peripheral blood indicating functional hyposplenism. whole-exome sequencing revealed two different genetic alterations in the fadd gene: a maternally inherited nonsense mutation predicted to severely truncate the protein and a paternally inherited missense mutation in codon . although this paternal mutation has not been described as pathogenic, a different variant in same nucleotide of fadd has been associated with fadd deficiency (reference ). there are very few cases in the literature of fadd deficiency patients and the overall prognosis is poor compared to classical alps patients, as these patients are at significant risk of deadly sepsis from encapsulated organisms or death from neurologic complications. of the fadd deficiency patients described in the literature, several died prior to years old. while pneumococcal prophylaxis may reduce the risk of sepsis, hematopoietic stem cell transplant has been reported for patients with fadd deficiency (reference ), and is being considered for our patient. rationale: hcuvp is a patient product-introduction program that provides cuvitru® (immune globulin subcutaneous [human], % solution [ig gly]) free of charge for the first infusions to eligible patients with primary immunodeficiency disease (pid). using patient data from this ongoing program, our analysis described the clinical characteristics and infusion parameters of pediatric and adolescent patients who were initiated on ig gly through hcuvp. methods: hcuvp eligibility criteria were: patients aged years old, with a primary icd- -cm code verifying diagnosis of pid, and no current or prior use of ig gly at program initiation. data from patients who received the first ig gly infusion between january , , and september , were included. data from patients receiving infusions after october , were censored. descriptive statistics were calculated for patients demographic and clinical characteristics and prescribed and actual infusion characteristics by age group (< years and years). results: in total, patients who completed all infusions were included in the analysis, of whom were aged < years. among those who previously received immunoglobulin (ig) therapy, a greater percentage of patients aged < years were treated with intravenous ig therapy (n= ; %) compared with adult patients (n= ; %) before initiating ig gly. nine patients aged < years were treatment naïve. the mean infusion volume per site was lower among patients aged < years ( years: . ml; years: . ml; and years: . ml) than among patients aged years ( years: . ml and years: . ml). however, the mean infusion rate per site was similar between patients aged < years ( xmen disease (x-linked immunodeficency with magnesium defect, epstein-barr virus infection and neoplasia) is a primary immune deficiency caused by mutations in magt and characterized by chronic infection with epstein-barr virus (ebv), ebv-driven lymphoma, cd t-cell lymphopenia, and dysgammaglobulinemia. magt gene codifies to magt protein, a mg +-selective transporter, expressed in the human immune system, specifically in the spleen and the thymus. functional studies have established the key role of magt in t cells and natural killer (nk) cell activation. upon cd + t-cell receptor stimulation, magt mediates a transient mg + influx that is necessary for phospholipase c gamma (plcy ) activation, which drives ca + rise and downstream signaling. this mg + influx also regulates cytotoxic functions of nk and cd t cells through nkgd , reason why these patients have impaired cytolytic responses against ebv. eleven male xmen patients have been described. we present the case of a -year old hispanic infant with a pathogenic variant in magt gene that clinically manifested with early pneumocystis jirovecii and cytomegalovirus (cmv) interstitial pneumonia, and ebv chronic infection with good response to intravenous immunoglobulins supplementation without hematopoietic stem cell transplantation or gene therapy. laboratory study highlights low levels of nkg d ligands. the objective of this case report is to broaden the spectrum of clinical presentation of xmen disease, that manifests initially as a combined immune deficiency (cid) and evolved with a favorable course of the disease with intravenous immunoglobulins supplementation therapy and chemoprophylaxis with trimethoprim-sulfamethoxazole. introduction: lysinuric protein intolerance (lpi) is a recessively inherited disorder of the cationic amino acids transporter subunit y+lat caused by variants in the slc a gene. the disease is characterized by protein-rich food intolerance has a heterogeneous presentation. the clinical findings are a result of depletion of lysine, ornithine, and arginine. symptoms can include hyperammonemia, failure to thrive, protein aversion, neurologic disease, and lung disease. there is also evidence that inflammatory manifestations are mediated through upregulation of nfb, il , and tnf that occur independent of intracellular arginine levels and can lead to lifethreatening episodes of hemophagocytic lymphohistiocytosis (hlh). case presentation: a -year-old male presented with history of anxiety, depression, eating disorder, delayed puberty and complex partial seizures. due to poor nutrition and failure to thrive, a gastrostomy tube was placed. following commencement of enteral feeds, he presented with altered mental status, bilateral mydriasis, hyperreflexia, and agitation which lead to a picu admission. ammonia peaked as high as μmol/l and episodes ceased with cessation of enteral feedings. prior to enteral feeds, he had been self-restricting protein in his diet. biochemical testing was consistent with lpi and illumina next-generation sequencing revealed compound heterozygous variants in slc a (p.s lfs* and p.e dfs* ). hyperammonemia resolved quickly with cessation of protein intake and high rate dextrose infusion without the need for ammonia scavenging agents. he was subsequently started on proteinrestricted enteral feeds. at diagnosis he did not have any respiratory symptoms, ct scan of chest showed patchy areas of groundglass opacification that was suggestive of early pulmonary alveolar proteinosis (pap). bronchoalveolar lavage demonstrated foamy, cloudy pink fluid and elevated bronchioalveolar macrophages on cell differential. his clinical course and slc a genotype led to suspicion for smoldering hlh. the findings of elevated ferritin, hypertriglyceridemia, decreased fibrinogen, splenomegaly, elevated il- receptor, decreased nk cell function, along with hemophagocytosis on bone marrow biopsy confirmed the diagnosis. because of his pap and hlh, in addition to dietary modifications, a trial of il- beta inhibition (anakinra) at mg/kg/day was initiated. follow up ct scan of chest months after initiation of anakinra showed complete resolution of pulmonary groundglass opacifications and pap. bone marrow evaluation showed continued hemophagocytosis in spite of the normalization in ferritin, soluble il- receptor, nk function, and triglycerides levels. overall, he is significantly improved on daily anakinra and no longer meets criteria for hlh or pap. discussion: recent data has shown in y+lat models that thp- macrophages and a airway epithelial cells upregulate il and tnf regardless of intracellular arginine content. this suggests that inflammatory manifestations may continue independent of dietary modifications. we present a year old patient with newly diagnosed lpi who was treated dietary modification and anti-il therapy resulting in resolution of hlh and pap. more research is needed to see if long-term il blockade that can consistently control both the immunologic and pulmonary manifestations of lpi and positively impact morbidity and mortality. learning objective: recognize that symptoms of bartonella endocarditis and associated complications can share features of certain immunocompromising conditions. case description: an -year-old caucasian boy with history of repaired pulmonary atresia and aortic root dilation was diagnosed with pancytopenia and splenomegaly during a brief hospitalization for atypical pneumonia. pancytopenia persisted, splenomegaly worsened, and five months after presentation, he developed hypertension and renal insufficiency. he was diagnosed with hypocomplementemic, diffuse sclerosing and crescentic glomerulonephritis and was started on mycophenolate mofetil with improvement in kidney function and stabilization of cytopenias. as part of a comprehensive immune work-up, alps (autoimmune lymphoproliferative syndrome) panel was sent and demonstrated elevated double-negative t (dnt) cells with out of positive immunologic criteria for alps. neither targeted sequencing for alps and alpslike disorders nor whole exome sequencing revealed pathogenic mutations. by age , the patient remained on mycophenolate, but developed failure to thrive, with weight dropping from th percentile to less than rd percentile. he was hospitalized again for low-grade fever, increased work of breathing, left shoulder pain and fatigue and was found to have right lower lobe pneumonia. pancytopenia worsened, and he was started on cefepime and azithromycin without improvement in symptoms. echocardiogram revealed vegetations in his pulmonary conduit and bilateral branch pulmonary arteries, but multiple blood cultures were negative. upon further history, the patient reported contact with kittens. bartonella henselae titers and polymerase chain reaction (pcr) from blood were sent and were both positive. he completed a -week course of gentamicin, -month course of ceftriaxone, and was transitioned to doxycycline and rifabutin. after initiating antimicrobial therapy, his weight and energy significantly improved, his blood bartonella pcr became negative, and his splenomegaly resolved. approximately one year later, the patient underwent pulmonary artery conduit replacement and bartonella pcr testing of the tissue specimen was positive. he has had sustained weight increase, resolution of hypocomplementemia and splenomegaly, decrease in dnt cell frequency from > % to . %, and improvement though not resolution of cytopenias. he currently remains on doxycycline and rifabutin and continues treatment with mycophenolate. discussion: alps is characterized by defective lymphocyte apoptosis and clinical features such as lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and glomerulonephritis. the hallmark laboratory finding is expansion of dnts. our patient met criteria for a probable alps diagnosis based on the presence of both required criteria (chronic splenomegaly and elevated dnt cells) and secondary additional criteria (typical immunologic findings noted on alps panel). pediatric cases of bartonella henselae endocarditis have been associated with splenomegaly, cytopenias, and glomerulonephritis which mimic many features of monogenic immune dysregulatory disorders. the diagnosis of bartonella endocarditis in our patient therefore raises the question of whether his immunosuppression predisposed him to infection or if his entire clinical presentation can be explained by bartonella endocarditis. physicians taking care of patients with immune dysregulatory disorders should consider bartonella endocarditis in the differential diagnosis of onset or exacerbations of immune dysregulation. rationale: while fever is considered a sign of infection, many individuals with primary immunodeficiency (pi) anecdotally report a lower than normal average body temperature. on immune deficiency foundation (idf) friends and idf pi connect research forum online, pi patients report a diminished fever response even when other signs of infection are present. there is limited knowledge about the average body temperature in persons with pi. however, the implications of missing an infection in those with pi is well established. methods: study investigators partnered with patient investigators to design a prospective cohort study to determine whether body temperature differed between persons living with and without pi. three hundred fifty adults with pi were recruited from idf and one adult household member without pi was also recruited. mckesson digital oral thermometers (model - bgm) were provided and used to record temperatures in all participants three times a day for five consecutive days. descriptive statistics were calculated. median body temperatures were compared between the two cohorts at each time point using mann-whitney test. results: data from households were used for analysis ( . % participation rate). the pi population was largely female ( . %) with a median age of years and largely caucasian population ( . %). the non-pi population was largely male ( . %) with a median age of years and largely caucasian population ( . %). pi diagnoses included cvid ( . %), hypogammaglobulinemia ( . %), igg subclass deficiency ( . %), selective iga deficiency ( . %), specific antibody deficiency ( . %), agammaglobulinemia ( . %), chronic granulomatous disease ( . %), combined immunodeficiency ( . %), and complement deficiency ( . %). a total of individuals with pi ( . %) reported a lower than normal non-sick body temperature, while individuals with pi ( . %) reported a normal (between °f - °f) non-sick body temperature. a total of individuals with pi ( . %) reported absence of fever with infection, while individuals ( . %) reported a normal fever response with infection. the median body temperature was significantly higher for the pi patients in the morning, but not evening or bedtime, reading in of the days (monday: pi = . °f vs. non-pi = . °f, p = . ; tuesday: pi = . °f vs. non-pi = . °f, p = . ; wednesday: pi = . °f vs. non-pi = . °f, p = . ; thursday: pi = . °f vs. non-pi = . °f, p = . ; friday: pi = . °f vs. non-pi = . °f, p= . ). conclusions: despite the limitations of this non-clinical study, individuals with pi are knowledgeable about their conditions and can offer unique insights and direction to researchers. this study demonstrates that collaboration with patient advocacy groups may facilitate patient-centered and patient-driven research with high participation among the target population. introduction: familial mediterranean fever (fmf) is a hereditary condition characterized by recurrent episodes of painful inflammation caused by mutations in the pyrin (mefv) gene. alterations in the mefv gene affect pyrin production leading to recurrent fevers and painful inflammation in the peritoneum, synovium, and pleura. amyloidosis may also develop as a complication. arabic, turkish, armenian, and sephardic jewish populations are most commonly affected. homozygosity for mefv mutations are associated with a more severe course. there is a paucity of information regarding pediatric fmf in the literature. case: we present a case of a -year-old male with minor speech delay diagnosed with compound heterozygous fmf. patient was initially referred due to recurrent fevers and infections. at months of age, he was hospitalized with septic shock requiring intubation secondary to adenovirus. at months of age, the patient began to have recurrent fevers every to weeks, leading to multiple blood draws and courses of antibiotics prior to referral. at , , and months of age, he developed three separate episodes of febrile seizures. a total of - lifetime episodes of acute otitis media occurred prior to bilateral myringotomy tube placement. four episodes of streptococcus pyogenes pharyngitis confirmed by throat culture preceded tonsillectomy. no oral ulcers, joint pain, or abdominal pain were reported. no other infections such as pneumonia, sinusitis, uti, non-viral gastroenteritis, fungal infections, or skin infections were reported. both parents are ashkenazi jewish and a maternal history of early miscarriage was noted. family history was negative for immunodeficiency, malignancy, and autoimmunity. the patients vital signs and physical exam were unremarkable. serology indicated leukocytosis of . k/l with elevated monocytes of cells/l, elevated eosinophils of cells/l, and slightly elevated cd t cell count of cells/l. neutrophil, cd t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus, diphtheria, rubella, streptococcus pneumoniae, and haemophilus influenzae b titers were protective. genetic analysis identified that the patient was compound heterozygous for the e q and v mutations in the mefv gene. family was instructed to keep a fever diary. colchicine . mg once a day was given initially, then increased to . mg once a day for inadequate response. loose stools were observed while patient was maintaining a lactose free diet so he was switched to colchicine . mg bid with resolution of loose stools. apart from two occasions when his colchicine dose was missed, the patient remained afebrile at his follow up visits. conclusion: we present a pediatric case of compound heterozygous fmf (e q and v mefv mutations) in an otherwise healthy -year-old male of ashkenazi jewish background, initially symptomatic at months of age. individuals who are compound heterozygous for the e q and a second mevf mutation are generally symptomatic, although severity cannot be predicted. additional pediatric research on symptomatic heterozygous and compound heterozygous fmf is recommended. natural killer (nk) cells are innate lymphocytes that play a key role in defense against virally-infected cells and in tumor surveillance. nk cells can be divided in two subsets. the majority of nk cells in peripheral blood expressed intermediate levels of cd and are referred to as cd (dim). these nk cells are responsible for nk cell cytotoxicity. a minor population of nk cells express very high expression of cd and are referred to as cd (bright). these nk cells are responsible for cytokine production and are precursors to cd (dim) nk cells. a few immunodeficiencies have been described in which there are abnormal nk cell subsets, such as autosomal dominant gata deficiency where cd (bright) nk cells are absent and irf where there is a paucity of cd (dim) nk cells and relative expansion of cd (bright) nk cells. here we present a patient with an absence in cd (bright) nk cells secondary to cd deficiency. our patient is a -year-old african american female born to non-consanguineous parents. the patients past medical history is significant for chronic lung disease secondary to prematurity, recurrent acute otitis media, failure to thrive and congenital hypothyroidism. family history is significant for an older sister that presented at age with ebv-associated hodgkin lymphoma whose treatment was complicated by chronic activated ebv infection and who ultimately underwent hematopoietic stem cell transplantation (hsct). our patient presented with pancytopenia, fever, lymphadenopathy and splenomegaly. she was found to have ebv viremia with greater than , copies in whole blood by pcr. she was treated with two doses of rituximab followed by etoposide and dexamethasone as a bridge to hsct. whole exome sequencing demonstrated a homozygous mutation in cd . cd is a member of the tumor necrosis factor receptor family and influences the function of t cells, b cells and nk cells. in nk cells, cd is primarily expressed in cd (bright) nk cells. cd deficiency is an autosomal recessive disorder associated with persistent symptomatic ebv viremia, including ebv-driven hemophagocytosis and lymphoma, hypogammaglobulonemia and specific antibody deficiency. our patients immune evaluation prior to initiation of chemotherapy and immunosuppression was notable for very elevated igg, iga and igm. despite hypergammaglobulonemia patient had only out of protective titers against streptococcus pneumoniae. the patient had pan-lymphopenia with appropriate percentages of lymphocyte subsets. assessment of her b cell subsets showed a slight increase in the percentage of transitional b cells/plasmablast and a nearly complete absence of cd -expressing b cells. her nk cell phenotyping demonstrated a complete loss of cd (bright) nk cells with reduced nk cell cytotoxicity, comparable to what has been previously reported in patients with gata deficiency. previous reports of patients with cd deficiency denote normal nk cell numbers with normal to moderately reduced nk cell cytotoxicity, however, cd deficiency causing a specific loss of the cd (bright) nk cell subset has not been previously reported. cd deficiency should be consider in patients with ebv driven disease and abnormal nk cell studies. introduction/background: the transcription factor ikaros is encoded by the ikzf gene and plays a crucial role in lymphopoiesis. somatic, and more recently also germline mutations of ikzf are associated with a hematologic malignancies, most notably b-cell precursor acute lymphoblastic leukemia. germline mutation in ikzf was first reported as a monogenic cause of human disease characterized by marrow failure and immune deficiency in a single neonate in . subsequently, mutations leading to haploinsufficiency were discovered to underlie a proportion of patients with cvid and low b cell numbers, and dominant-negative mutations have been observed to cause more severe combined immune deficiency phenotypes. at this time, there is very little known regarding allogeneic hematopoietic cell transplantation (hct) outcomes for patients with severe dominant-negative ikzf mutations. concerningly, ikaros deficiency has been observed to have a negative impact on graft versus host disease in mouse models. objective: to describe allogeneic stem cell transplant outcomes in patients with the dominant-negative ikaros mutation. methods: we collected transplant data from patients who underwent allogeneic hct at transplant centers around the world. results: patients underwent allogeneic hct using a variety of conditioning regimens. patients received bone marrow (n= ) or cord blood (n= ) grafts from an hla-matched sibling donor (n= ) or single allele hlamismatched unrelated donor (n= ). neutrophil engraftment occurred between day + and + post-transplant. platelet engraftment occurred between day + and + except in one patient who did not have return of normal platelet counts due to underlying liver dysfunction. all patients were documented to have greater than % whole blood donor chimerism at a median of days (range - days) following transplant and maintained > % donor chimerism until last follow-up. only one patient developed grade ii acute gvhd. no patients developed chronic gvhd. one patient died approximately year post transplant related to cryptosporidium cholangitis which existed prior to hct. at the most recent follow up of the surviving patients (range: . - . y), ivig had been discontinued, antimicrobial prophylaxis had been stopped, and patients had received routine vaccinations. they all had excellent performance status. conclusions: allogeneic hct may be a safe option to consider for patients with dominant-negative ikaros mutation as there does not appear to be an increased risk of death or gvhd. moreover, -out-of- of the transplanted patients are alive and well and show no features of the disease. however, because of the limited number of patients evaluated and the retrospective nature of this analysis, our data do not allow firm conclusions to be made, and further studies will be needed to evaluate outcomes in larger cohorts. introduction: when evaluating patients with t-cell lymphopenia, we often are concerned about defects in lymphocyte production and function, especially in the setting of frequent infections. here we outline a case demonstrating t-cell lymphopenia due to increased loss, which should be considered in the differential diagnosis. case report: we report a -year-old male who initially presented with recurrent, right-sided pneumonias requiring frequent hospital admissions including severe episodes necessitating intensive care unit admission. his work up for the pneumonias included a bronchoscopy revealing normal anatomy with minimal inflammation, and a chest ct with mild peribronchial wall thickening. as his pulmonary disease progressed, he developed a persistent, productive cough with expectorated mucous plugs that were plastic-like in appearance. while his pulmonary symptoms responded to steroids, his mucous plug production persisted. sputum cultures were intermittently positive, isolating cryptococcus neoformans and aspergillus niger. he underwent vats and wedge biopsy, concerning for recurrent aspiration. an immunologic evaluation initially demonstrated normal t-and b-cell counts, but serial evaluation of his lymphocyte population demonstrated low cd + cells (ranging - cells/cumm), and low normal cd cells (ranging - cells/cumm) with normal b-and nk-cell numbers. further t-cell evaluation revealed normal ratios of naive and memory p o p u l a t i o n s ( c d c d r a + % , c d c d r o + % , cd cd ra+ %, cd cd ro %), normal trec ( copies per ^ cd cells) and normal thymic emigrants (cd cd cd ra+ : , normal - ), indicative of sufficient thymopoiesis. mitogen and antigen stimulation assays demonstrated normal responses to phytohemagglutin, concanavalin a, and pokeweed mitogen, with a low lymphocyte response to candida. he had normal quantitative immunologlobulins, normal diphtheria, tetanus and streptococcus pneumonia titers. his dihydrorhodamine flow cytometry and fish for chromosome q . deletion were negative. given normal function and thymic output, his immunologic profile was concerning for t-cell loss. our patient was registered with the undiagnosed disease network, and had a second review of his lung biopsy, concerning for plastic bronchitis. subsequent lymphatic imaging demonstrated abnormal lymphatics within the bilateral clavicular space, right greater than left, with questionable partial thoracic duct, explaining his unilateral symptoms. he was diagnosed with plastic bronchitis secondary to abnormal lymphatic drainage, with lymphatic fluid filling his airways and secondary t-cell loss. discussion: plastic bronchitis is a rare and potentially fatal disorder, seen commonly after the fontan procedure for congenital heart disease. this process has resulted in t-cell loss into the airway and subsequent t-cell lymphopenia. in patients with fontan-related protein losing enteropathy, multiple immune abnormalities have been described including reduced immunoglobulins, lymphopenia, and selective cd lymphocyte deficiency. similar findings have been reported in patients with lymphatic malformations. although the impact of t-cell loss on adaptive immunity is not entirely known, there is no indication of increased risk for atypical infections. given his normal mitogen assay, our patient did not start prophylactic antibiotics. he continues to have symptomatic episodes with lymphopenia, but has had no opportunistic infections, and remains stable with an aggressive pulmonary regimen. we conclude by reiterating the importance of considering t-cell loss in patients presenting with lymphopenia, particularly with evidence of normal thymopoeisis and t-cell function. introduction: granulomatous disease (gd) has been described with a variable incidence ( . - . %) in patients with common variable immunodeficiency (cvid). an increase in malignancies has been reported in cvid patient cohorts, particularly for lymphoma, reported in . - . % of the cvid patients depending on the cohorts. prior analysis of a cohort of cvid patients included patients with gd (gd+). in these, there was a suggestion of more cases of lymphoma ( . %) when compared to cases without (gd-) ( . %) although the difference was not statistically significant (p=. ). objectives: compare the frequency of lymphoma in gd+ and gd-patients in the cvid patient cohort from the usidnet registry. methods: we submitted a query to the usidnet registry requesting deidentified data for patients with the diagnosis of cvid, through august . statistical analysis was performed on spss, with comparisons done with pearson chi-square or fisher's exact test, depending on the sample sizes, using an alpha level of . . results: a cohort of cvid patients from the usidnet registry was analyzed. ninety-one patients ( . %) were gd+. overall, patients ( . %) had a malignancy diagnosis, of these ( . %) with lymphoma. lymphoma was present in / gd+ patients ( . %) versus / gdpatients ( . %) (p=. ). overall malignancy was present in / gd+ ( . %) versus / ( . %) (p=. ). discussion: in the cohort of cvid patients from the usidnet registry, we found a frequency of lymphoma of . %, which is in the range of previously described cohorts. the frequency of lymphoma was . % in patients with gd, higher than the . % frequency for gd-patients, but these differences were not statistically significant. our identified frequency of lymphoma in gd+ patients was lower than the one previously identified in the cvid patient cohort, but with similar proportional differences between gd+ and gd-patients. despite no statistical significance, the frequency of lymphoma, as shown here and elsewhere, was higher in cvid patients gd+ than gd-in both studies, with no full understanding of this increased risk of lymphoma. expanding this analysis to larger groups of cvid patients may help to confirm, or deny a more robust association, which may have a meaningful impact in the outcomes of this particular population. introduction: patients with refractory pericarditis have been treated with intravenous immunoglobulin (ivig) or interleukin receptor antagonist (anakinra) with limited and transient benefit. separate or combined therapy with subcutaneous immunoglobulin (scig) and interleukin (il) inhibitor (rilonacept) for refractory pericarditis in a cohort of patients has not been previously described. case descriptions: patients were referred for recurrent pericarditis refractory to traditional therapies at ages ranging from to years. they all had multiple serious sequelae of their pericarditis and abnormal immune parameters including hypogammaglobulinemia, poor responses to vaccines, poor mitogen induced lymphocyte proliferation, and/or b cell lymphopenia. the patients had varied past medical histories and associated conditions. patients were started on ig, with some initiated on ivig, though all were transitioned to hyaluronidase-facilitated scig (hyqvia). patients were then started on either anakinra or rilonacept with patients continuing on rilonacept and remaining on anakinra. all patients had complete or near complete resolution of their pericarditis on dual therapy for greater than year. the markedly elevated il prior to therapy seen in all of the patients normalized post-therapy. some patients had elevated il prior to therapy that also improved post-therapy. patient who has also been diagnosed with familial mediterranean fever (fmf) has stopped both therapies for greater than year with no further episodes of her pericarditis. discussion: patients with recurrent refractory pericarditis and signs of immunodeficiency and autoinflammatory disease on laboratory testing responded to dual therapy with hyqvia and rilonacept or anakinra resulting in resolution of pericarditis. inflammasome and immune abnormalities may be implicated or associated with recurrent pericarditis and may respond to targeted therapies. chief, laboratory of clinical immunology and microbiology, idgs, dir, niaid, nih, bethesda, md, usa hypomorphic recombination activating gene (rag ) mutations result in residual t-and b-cell development in both humans and mice and have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (cid-g/ai). recent studies have shed light on how hypomorphic rag mutations alter the primary repertoire of t and b cells, but less is known about their effect on immune dysregulation in targeted organs. in order to investigate the role of these mutations in determining intestinal disease, we set out to evaluate gut immunity and microbiota interplay in rag mutant hypomorphic mice. we evaluated two mouse models carrying homozygous rag mutations (r q and r w), corresponding to human mutations (r q and r w, respectively) described in patients with cid-g/ai. both mutations fall in the coding flanksensitive region of the rag c-terminal domain. on the basis of aminoacid properties and in vitro studies, the r q mutation has demonstrated a moderate effect on rag protein stability while the r w mutation resulted highly disruptive. analysis of intestinal pathology in rag mutant mice (niaid animal protocol lcim e) revealed different degrees of spontaneous colitis, with the most severe inflammatory infiltrate observed in mice carrying the most disruptive mutation, r w. colonic inflammation was characterized by crypt elongation, epithelial hyperplasia, and an abundant inflammatory infiltrate extending to the colonic lamina propria, with occasional crypt abscesses. a significant increase in activated cd hicd lcd + t cells expressing the gut homing receptor was observed in mesenteric lymph nodes (mlns) of both mutant strains, and was especially prominent in r w mutant mice. additionally, the proportion of mln cd + t regulatory (treg) cells was increased in both mouse models. finally, mln of mutant mice contained a high number of myeloid cells (cd b+ ) along with a decreased number of b + b cells, and these abnormalities were also more prominent in r w than in r q mice. in summary, we have shown that rag mutant hypomorphic mice present with different degrees of inflammatory bowel disease, with the mouse model carrying the most disruptive mutation presenting with the most severe phenotype. we are currently performing studies to evaluate the impact of rag mutations on microbiome composition and diversity in these mouse models of cid-g/ai. background: hypogammaglobulinemia or low serum immunoglobulin g (igg) levels either inherited (primary) or acquired (secondary) is associa t e d w i t h i n c r e a s e d i n f e c t i o n r a t e s . p r i m a r y ( °) hypogammaglobluinemia can be caused by many primary immune deficiencies (pid) including combined variable immune deficiency (cvid), while secondary ( °) hypogammaglobluinemia can be caused by many acquired conditions such as lymphomas, leukemias, or chemotherapies and other immunosuppressive drugs. immunoglobulin replacement therapy (irt) has been the mainstay of treatment in patients with hypogammaglobulinemia by reducing infection through replenishing the quantitative igg. there are other applications of ig therapy such as in autoimmune diseases, where the mechanism of action is thought to be ig mediated immunomodulation. innate immune cells have shown to be involved in such mechanism, but whether irt modulates adaptive immune cells in patients with hypogammaglobulinemia is not well known. hypothesis: irt has an immunomodulatory effect on t-cell function and proliferation in patients with hypogammaglobulinemia. methods: blood from thirty patients with °(n= ) or °(n= ) hypogammaglobulinemia recruited from the immunodeficiency clinic at the ottawa hospital was drawn for peripheral blood mononuclear cell (pbmc) isolation, before starting irt and minimum weeks after starting irt. data regarding igg level, number and type of infections after receiving irt was collected. pbmcs were analyzed using flow cytometry for quantitation of t-cell subset. cultured and anti-cd /cd stimulated pbmc were also analyzed for extracellular and intracellular cytokine production, measured by e l i s a a n d f l o w c y t o m e t r y, r e s p e c t i v e l y. c o m b i n e d cytomegalovirus, epstein-barr virus and influenza virus (cef) peptides were used to study specific t-cell responses. anti-cd / cd stimulated pbmc were used for celltrace t-cell proliferation a s s a y s . d a t a w a s g r o u p e d b a s e d o n n a t u r e o f hypogammaglobulinemia i.e. °or °. results were compared between before and after irt using wilcoxon matched-pairs signed rank test. results: irt was not found to significantly alter proportion of treg, cd +, or cd + t-cell populations or activation state as measured by cd ra/r expression. however, irt was found to significantly increase expression of intracellular ifn-y in cd + and cd + t-cells post-cd /cd stimulation in °(p = . ), but not in °h ypogammaglobulinemia patients. there was no change in extracellular il- and il- cytokine production in both groups. in contrast, cd + tcells in °hypogammaglobulinemia patients showed significantly higher expression of intracellular ifn-y and tnf-a post-cef viral peptide stimulation (p = . ). cd + and cd + t-cell proliferation after cd /cd stimulation was found to be decreased after irt for both groups (p = . & p = . ). conclusions: our results suggest that irt can alter cd + and cd + t-cell function with differential effect in patients with °o r °hypogammaglobulinemia in addition to replenishing serum igg level. more experiments assessing cytotoxicity of t-cells will be conducted to further study t-cell subset function as well as bcell function. these laboratory results will be analyzed for association with clinical outcomes. uploaded file(s) uploads background: severe congenital neutropenia (scn) is a rare immunodeficiency disorder characterised by the extremely low absolute neutrophils count (anc) less than . x /l. the clinical feature of scn is recurrent bacterial infections and the patients the risk of leukemia development. the incidence of scn is estimated to be in individuals. mutations in more than genes have been described causing scn and it is either recessive, dominant or x-linked inheritance. case presentation: we described an years old malaysian girl who presented with recurrent abscesses over the whole part of the body, recurrent oral candidiasis, growth failure and recurrent pneumonias since months old. she also had history of a few episodes of acute tonsillitis, chronic suppurative otitis media and herpes zoster infections. throughout her age, she had persistent neutropenia less than . x /l but in few occasions, her anc elevated up to more than . x /l . she was treated as autoimmune neutropenia, respectively due to few positive results of autoimmunity workout such as antinuclear antibodies (ana) and double stranded dna (dsdna) but eventually later to be negative. later at the age years old, whole exome sequencing was performed and confirmed by sanger s e q u e n c i n g , f o u n d a h e t e r o z y g o u s v a r i a n t i n e l a n e gene(c. g>t; p.gly ter), an autosomal dominant which was described to cause scn. both parents do not carry this mutation, hence, it is a de novo mutation. currently, she had few on and off recurrent infections. despite that, she is relatively well and on prophylaxis antibiotic. conclusion: to our knowledge, we report for the first time a malaysian girl with scn, with confirmed mutational analysis of the elane gene. the delayed diagnosis might be due to the insufficient awareness of the phenotypic presentation of this rare disease. moreover, the genetic analysis is not available in malaysia and need to be done outside of the country. this case demonstrates the importance of the genetic analysis which may help in improving the diagnosis and management of the patient. ( ) submission id# professor of paediatrics and immunology, university college london; great ormond street hospital nhs trust; orchard therapeutics, london, uk background: ada-scid is a rare genetic disorder which causes severe combined immunodeficiency. historically, ada-scid has been treated using enzyme replacement therapy (ert) followed by allogeneic hematopoietic stem cell (hsc) transplant (hsct) from a matched related donor (mrd) or, if none is identified, a non-mrd (matched/mismatched unrelated or mismatched related donor). we developed a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by the short form of the elongation factor- alpha (efs) promoter (efs-ada lv). the drug product (otl- ), composed of autologous hscs transduced ex vivo with the efs-ada lv, was evaluated in a prospective, historically-controlled phase i/ii clinical trial in ada-scid pediatric subjects. we report safety and efficacy at months in ada-scid subjects treated with lentiviral gene therapy (gt) compared to a historical cohort of ada-scid patients treated with hsct. methods: twenty subjects ( male, female; mo . yrs) were treated with gt. autologous cd + hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. busulfan was administered at a single dose ( mg/kg) prior to infusion of otl- . the control group included patients ( . mo . yrs) treated with allogeneic hsct (mrds n= , non-mrds n= ) at great ormond street hospital, uk (n= ) or duke university childrens hospital, usa (n= ) between . results: at months, overall survival (os) and event-free survival (evfs), defined as survival in the absence of ert reinstitution or rescue allogeneic hsct) were statistically significantly higher in the gt group compared with the hsct group (table) . successful engraftment of genetically modified hsc was observed in all gt subjects at months, which persisted over months, based on vector gene marking in granulocytes (median . copies/cell [range . - . ] at months) and peripheral blood mononuclear cells (median . copies/cell [range . - . ] at months), and was associated with increased red blood cell ada enzyme activity and metabolic detoxification from deoxyadenosine nucleotides. over months, none of the gt subjects required peg-ada ert reinstitution and % were able to stop receiving immunoglobin replacement therapy (igrt), whereas % hsct patients required rescue hsct or reinstitution of peg-ada ert, and % were able to stop receiving igrt (table) . nine subjects in the gt group experienced a serious adverse event (sae), most frequently infections and gastrointestinal events; only one was considered treatment-related. in the gt group, there were no events of autoimmunity during the study. due to the autologous nature of the product, there was no incidence of graft vs host disease (gvhd) in the gt group; whereas patients in the hsct group experienced acute gvhd and experienced chronic gvhd events, one of whom died. conclusions: treatment with lentiviral gt for ada-scid is well tolerated and has a favorable benefit-risk profile at months based on sustained gene correction and restoration of immune function, as well as improved os and evfs compared with hsct (mrd or non-mrd) at months. background: ada-scid is a rare genetic disorder that causes severe combined immunodeficiency, with minimal or absent b cell function. prior to, and often after, treatment with allogeneic hematopoietic stem cell (hsc) transplant (hsct) or autologous ex vivo hsc gene therapy (gt), patients are managed with enzyme replacement therapy (ert) and immunoglobulin (ig) replacement therapy (igrt). we evaluated a gt treatment with autologous hscs transduced ex vivo with a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by an internal short form of the elongation factor- alpha (efs) promoter ("efs-ada lv"). at months follow-up, pediatric ada-scid subjects treated with gt were compared to a historical cohort of ada-scid patients treated with hsct. here, we report on b cell reconstitution in these cohorts. methods: twenty subjects ( male, female) aged mo - . yrs received gt. autologous cd + hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. genetically modified cells were administered after conditioning with single dose busulfan ( mg/ kg). the control group included patients aged . mo to . yrs treated with hsct at great ormond street hospital (uk) (n= ) or duke university children's hospital (us) (n= ) between - . the hsct patients received an allogeneic transplant from matched related donors (mrds) (n= ) or non-mrds (n= ). subjects continued to receive igrt post-gt until a clinical decision was made to stop, factoring in b cell reconstitution, general medical condition and seasonal infections. results: by month , in the gt group, % had stopped treatment with igrt compared to % in the hsct group overall. by months and , higher proportions of gt-treated subjects had stopped igrt ( % and %, respectively) compared with mrd hsct patients ( % and %, respectively) and non-mrd hsct patients ( % at both timepoints) (table) . in the gt group, vector gene marking was detectable in peripheral blood mononuclear cells within months and persisted at months post-infusion (median . copies/cell [range . - . ]), suggesting successful gene modification. as evidence of b cell reconstitution, iga and igm levels in peripheral blood sera more than doubled by months, from . mg/dl (range to ) to . mg/dl (range to ) and . mg/dl (range to ) to . mg/dl (range to ), respectively. additionally, antibody response following tetanus vaccination, was evaluated in subjects. all subjects mounted a protective response to the vaccine (median antibody response . iu/ml [range . to . ]), based on a normal threshold of . iu/ml (hammarlund clin infect dis ) and a laboratory reference range ( . to . iu/ml). conclusions: gt with autologous hscs transduced ex vivo with efs-ada lv resulted in b cell reconstitution, as evidenced by doubled iga and igm production at months, cessation of igrt in % of patients by months, and protective specific antibody responses to tetanus vaccine in patients that were evaluated. background: x-linked chronic granulomatous disease (xcgd) results from mutations in cybb encoding the gp phox subunit of phagocyte nadph-oxidase. attempts to treat xcgd with gene therapy (gt) using transduced autologous hematopoietic stem cells (hsc) transduced ex vivo with a gammaretroviral vector have met with limited efficacy due to transient engraftment of gene corrected hscs, gene silencing, and vector insertion-mediated activation of oncogenes leading to myelodysplasia. we developed a novel self-inactivating (sin) lentiviral vector (g xcgd lv) with a chimeric cathepsin g/cfes myeloid-specific promoter driving gp phox expression from a codon optimized cdna. following transplant of g xcgd lv ex vivo transduced autologous hscs into busulfanconditioned xcgd patients, there was long-term restoration of oxidase activity in peripheral blood polymorphonuclear neutrophils (pmn) at months in of severely affected xcgd patients without evidence of genotoxicity. here we present data about the multiple assays used to assess quality and quantity of restoration of pmn oxidase activity. methods: similar trials of gt with g xcgd lv were initiated in the uk (n= , plus compassionate use patient) and usa (n= ). all patients had histories of inflammatory disease and severe, persistent infections (some non-responsive to conventional therapy at time of gt). g-csf plus plerixafor-mobilized cd + hscs were transduced with ex vivo g xcgdlv. subjects received myeloablative conditioning with singleagent busulfan, targeted to net area-under-the-curve of , ng/ml*hr. freshly prepared or cryopreserved quality-tested genetically-modified hsc, manufactured on-site, were administered intravenously. pmn oxidase activity post-gt was assessed by p-nitroblue tetrazolium (nbt) reduction, dihydrorhodamine (dhr) flow cytometry assay, and quantitative ferricytochrome c assay (ferric) measurement of superoxide generation. results: we report results for patients (aged - years) with - . years of follow-up; two additional patients were treated but died within three months of gt from complications deemed related to pre-existing diseaserelated co-morbidities (severe pulmonary disease and anti-platelet antibodies). within month post-gt, oxidase (+) pmn were present in peripheral blood based on nbt testing and dhr flow cytometry. expression of the corrective transgene was confirmed by flow cytometry using antibody detection of gp phox. quantitative biochemical measurements of oxidase activity were also confirmed in some samples using the ferric assay, demonstrating quantitative levels of superoxide production per corrected cell that were within the normal range. functional testing of oxidase burst activity using dhr fluorescent assays was applied serially to follow levels of corrected pmn where oxidase activity per corrected cell also were in the normal range. all patients had > % pmn dhr+ within one month, which remained stable for most patients over the follow-up period ( figure) . follow-up demonstrated sustained stable persistence of - % oxidase burst positive neutrophils in of surviving subjects at months, with restoration to clinically beneficial levels (defined as % of pmn being dhr+) in these patients as of december . conclusion: these results demonstrate corrected pmn function within month in x-cgd patients treated with autologous gt. pmn oxidase activity was sustained at levels which restore biochemical function and provide clinically beneficial levels of immunity for months in / patients. the formulation for igsc % was developed based on the knowledge acquired from the formulation of grifols currently licensed % immune globulin (human), gamunex®-c; however, the protein concentration was increased from % to % to facilitate efficient subcutaneous administration. gamunex-c has an extensive record of safety and tolerability when administered intravenously and subcutaneously for greater than years in diverse patient populations. the igsc % manufacturing process employs the same purification steps as gamunex-c and was demonstrated to be robust and to provide an igg product with the required potency, purity, and quality. the formulation excipient characteristics and compatibility with the drug product have been well established. glycine has been an excipient of intramuscular immune globulin (human) for fifty years and intravenous immune globulin (igiv) for over twenty years. the igsc % formulation has low buffering capacity, and a low ph was selected to achieve a product with low aggregates, low fragments and viscosity suitable for subcutaneous administration. to improve visual clarity, the igsc % formulation contains a small amount of polysorbate (ps ), which is widely used in biopharmaceutical products. subcutaneous administration of the igsc % formulation has been well tolerated in clinical studies. objectives: the goal was to provide the pid population with a new % immunoglobulin liquid product for subcutaneous administration (igsc %). methods: igsc % is manufactured using the current manufacturing process for gamunex-c, followed by an additional concentration step so that the product can be formulated at a higher protein concentration. igsc % and gamunex-c batches were produced at full industrial scale and then subjected to a series of analytical testing including assessment of purity, composition and neutralizing activity. results: the igsc % and gamunex-c manufacturing processes and formulations have preserved the igg integrity, molecular characteristics and potency. the manufacturing processes have eliminated lipids, alcohols, and acetate and coagulation factor impurities, including fxia, which were undetectable by either specific or global methods. the igsc % and gamunex-c batches were % gamma globulin by agarose membrane electrophoresis, and have a subclass distribution similar to normal plasma and acceptable specific antibody content. igsc % was shown to be primarily monomer plus dimer igg ( ± %) with minimal aggregate or fragment, which confirms that appropriately gentle processing conditions were used during the concentration of % igg solutions to % igg. conclusions: igsc % is a highly concentrated igg solution with characteristics comparable to gamunex-c, but with twice the igg concentration in order to facilitate subcutaneous administration with reduced volumes and shorter infusion times. analytical testing demonstrates suitable potency, purity, and neutralizing activity for a number of specific antigens. funding: this study was funded and conducted by grifols, a manufacturer of % immunoglobulin for subcutaneous administration. disclosure: all authors are employees of grifols. frequent respiratory tract infections and seizures cause recurrent hospitalizations in these children and are typically considered a result of neurological impairment and poor airway clearance. evaluation of these patients for immunodeficiency is not a common clinical practice. here we report combined immune deficiency in patients with mds and recurrent respiratory tract infections. case presentation case : a boy with mds was initially referred at age months for an abnormal newborn screen with low t cell receptor excision circles (trec) for severe combined immunodeficiency (scid). initial evaluation revealed moderate cd + and cd + t cell lymphopenia (figure ). initial immunoglobulins levels were normal. he was placed on antiseizure medications. he later developed recurrent and severe respiratory tract infections starting in infancy. at months of age, he developed hypogammaglobulinemia ( figure ). in addition, t cell counts progressively decreased and stayed around cells/ul. immunoglobulin replacement therapy started at months of age. hospitalizations due to respiratory tract infections significantly decreased. case : a -year-old boy with mds had recurrent bacterial and viral respiratory infections which required numerous hospitalizations including intensive care unit stays. newborn screening for scid was negative. he had been on anti-seizure medications. immunologic evaluation at years of age revealed low total cd + cells and cd + t cells (cd +: cells/ul[normal range - cells/ul], cd +: cells/ ul[normal range - cells/ul]), hypogammaglobinemia (igg: mg/dl[normal range - mg/dl]), and non-protective igg levels to tetanus, varicella and pneumococcus serotypes. immunoglobulin replacement therapy started at years of age which resulted in reduced frequency and severity of respiratory infections, and improved quality of life. discussions: t cell lymphopenia and hypogammaglobulinemia were seen in both our cases of miller-dieker syndrome. to our knowledge, immune deficiency has never been reported in mds. one of our cases suggests that low t cell counts may start as early as at birth and may be detected by newborn screening. hypogammaglobulinemia may be primary or secondary due to antiepileptics. both children had reduced frequency and severity of respiratory infections and improved quality of life after immunoglobulin replacement highlighting the importance of screening and early management of immunodeficiency. conclusion: miller-dieker syndrome is likely another syndromic primary immune deficiency disorder. a high index of suspicion with early screening and management of immunodeficiency may be beneficial for children with miller-dieker syndrome. uploaded file(s) uploads this prospective, multi-center, open-label study assessed the pharmacokinetic (pk), safety, and tolerability of immune globulin subcutaneous (human), % caprylate/chromatography purified (igsc %) in subjects with primary immunodeficiency (pi). the objectives were to determine a weekly subcutaneous (sc) dose of igsc % that is noninferior to the intravenous (iv) dose of immune globulin injection (human), % caprylate/chromatography purified (igiv-c %) and to determine the steady state trough igg levels after igsc % and igiv-c % infusions. there were possible phases. if not on a qualifying igg regimen at enrollment, subjects (n= ) were required to enter the run-in phase, receiving igiv-c % to achieve steady-state before entering the iv phase to determine steady-state area-under-the-curve (auc) of iv infusions. subjects with a qualifying igiv-c % regimen ( - mg/kg) (n= ) directly entered the iv phase for steady-state iv pk assessments. upon completion of the iv pk assessments subjects entered the sc phase, receiving weekly doses of igsc % for up to weeks, with steady-state auc determined at the th dose. igsc % was not associated with any reports of serious local infusion site reactions (isrs). the majority of local isrs were mild-to-moderate. igsc % (at a dose conversion factor of . ) provided equivalent exposure to igiv-c % as assessed by steady-state auc - days, with % higher mean igg trough values, lower fluctuations in igg concentrations and the flexibility of at home administration. igsc % was well tolerated with a safety profile comparable to igiv-c %. clinicaltrials.gov identifier: nct disclosure: kecia courtney, elsa mondou, and jiang lin are employees of grifols, a manufacturer of igsc %. grifols is the sponsor of this study. background: in two reports described the deficiency of adenosine deaminase (dada ) as early-onset lacunar strokes, intermittent fevers, livedoid rash, and early onset polyarteritis nodosa (pan). since these first reports, the clinical spectrum has dramatically expanded to include antibody deficiency, liver disease, vasculopathy, pure red cell aplasia, cytopenias, and lymphoproliferative disease. methods: forty-two patients were enrolled in an irb approved study at the nih. sequencing of ada , the gene encoding adenosine deaminase (ada ), was performed in all patients. information was obtained by chart review of all clinical, serologic, and radiographic testing. results: all patients had germline biallelic loss of function mutations in ada , leading to absent or significantly decreased protein expression and function of ada . the cohort comprises females ( %) and males ( %). there were sibling pairs and families with affected individuals. twenty-seven patients had a history of at least one ischemic stroke and experienced a hemorrhagic stroke. the average age at the time of first stroke is . years (range months - years), and the average number of strokes is (range - ). no new strokes have occurred in patients on anti-tnf therapy. skin manifestations occurred in % of patients and include livedo ( %), cutaneous vasculitis resembling pan ( %), and raynauds ( %). hepatomegaly ( %) and splenomegaly ( %) were also notable. portal hypertension was observed in ( %) patients, with patient requiring a spleno-renal shunt for a massive variceal bleed. abdominal mra revealed arteritis and aneurysm in / patients evaluated; patients developed bowel necrosis. peripheral vasculopathy was seen in patients, with one requiring amputation of gangrenous digits. the most common immune abnormality seen in this cohort is hypogammaglobulinemia ( %); patients have low igg, patients have low igm, and patients have low iga. ten of these patients are on immunoglobulin replacement. specific antibody responses to vaccines were inadequate in / patients challenged. lymphocyte phenotyping revealed decreased class-switched memory b cells in / patients ( %) tested. however, there was no relationship between absolute number of class switched memory b cells and hypogammaglobulinemia or infection frequency. hematologic abnormalities include transfusion depended anemia ( %), neutropenia ( %), lymphopenia ( %), and thrombocytopenia ( %). seven patients developed pancytopenia, presented with pure red cell aplasia, and developed aplastic anemia. three patients have undergone bone marrow transplant, with two of those patients requiring a second transplant for graft failure. conclusions: the spectrum of dada has expanded from strokes, intermittent fever, and cutaneous manifestations to include portal and systemic hypertension, immune deficiency, cytopenias, vascular abnormalities, and bone marrow failure. while initiation of anti-tnf therapy improves inflammatory markers, and no new strokes have occurred while on therapy, cytopenias do not seem to improve. bone marrow transplantation should be considered in patients with findings of bone marrow failure, although transplant of our patients has been complicated by immune mediated neutropenia. disease manifestations are heterogenous, making a comprehensive evaluation critical to our understanding of this disease. given the increase in neonatal diagnosis of athymia, clinical care is provided by the referring medical centers prior to rvt- implantation and patients return to the referring centers earlier after rvt- . this creates the need for clear, concise guidelines for the care of these patients. primary goals of pre-transplantation clinical care are ( ) management of pre-existing medical needs such as feeding difficulties, airway obstruction, congenital cardiac defects and developmental disabilities; ( ) management of symptoms related to oligoclonal recipient t cell expansion (autologous gvhd/atypical complete digeorge anomaly) and ( ) prevention of infections. most deaths in the pre and early post-transplantation period are secondary to pre-existing infections. necessary surgical and medical procedures (ie cardiac surgery, hearing aids) should not be delayed. for the first to months after rvt , patients have profoundly low naïve t cell numbers and may require immunosuppression to prevent rejection of rvt- by oligoclonal recipient t cells. immunosuppression needs to be closely monitored and titrated for desired effect while minimizing side effects such as renal toxicity, electrolyte abnormalities and hypertension. t cell counts should be performed every months and are used to guide weaning of immunosuppression. most patients with successful transplants develop greater than /mm naïve t cells by months post rvt- . infection prevention, clinical stability and optimal nutrition are critical for lasting engraftment. clinical guidelines have been developed to address immunosuppression, management of autologous gvhd symptoms (gut, skin and liver), preservation of renal function, and developmental considerations. after the development of naïve t cells, patients should continue to be monitored regularly by an immunologist. patients may develop autoimmune complications such as thyroid disease and transient cytopenias. while risk of complications related to viral infections is greatly decreased after development of naïve t cells, patients with comorbidities (central venous access device dependence, tracheostomy, chronic lung disease) continue to require complex care from multidisciplinary teams. medical conditions associated with athymia but not alleviated by thymus transplantation, such as hypoparathyroidism or cardiac defects, may require lifelong medical care. lastly, patients must be evaluated for readiness for killed and live vaccines. transplant outcomes are influenced by the clinical condition at the time of rvt- implantation and optimization of immunosuppression, nutrition and clinical stability in the first months following rvt- . clinical care that maintains a well-nourished, clinically stable, infection free patient yields the best chance for successful t cell development. guidance documents supporting these goals ensure patients are best prepared to receive rvt- and develop long lasting thymic function. hemophagocytic lymphohistiocytosis (hlh) is a life-threatening disease of immune dysregulation characterized by unchecked inflammatory responses leading to end-organ dysfunction. primary hlh results from inherited mutations that impair capacity for immune regulation whereas secondary hlh arises from inappropriate response to an immune stimulus such as infection, malignancy or autoimmunity. we report a -monthold male who presented with symptoms of hlh as an initial manifestation of congenital disorder of glycosylation (cdg) due to mutations in the gene component of oligomeric golgi complex (cog ) resulting in cog -cdg (cdg-iij). a -month-old male with history of mild motor delay presented with days of fever, vomiting, and diarrhea. initial evaluation identified highly elevated ferritin and triglycerides, transaminitis, coagulopathy, and hyperammonemia. he subsequently developed generalized seizures. liver and bone marrow biopsies demonstrated erythrophagocytosis consistent with hlh. immunologic evaluation was notable for mild hypogammaglobulinemia, neutropenia, thrombocytopenia, and anemia. serum cd levels and nk functional studies were later found to be normal. the patient was initially treated with ammonia-scavenger therapy and fresh frozen plasma (ffp) for coagulopathy with subsequent intravenous immunoglobulin and dexamethasone several days later. within hours after starting ffp, the patients ferritin level declined sharply. hyperammonemia and transaminitis also resolved, and his fever curve improved. additional immunosuppression was considered, but not initiated due to the patients ongoing clinical improvement. over the next months, the patient experienced two further acute episodes of fever, liver dysfunction, coagulopathy, and sepsis physiology. the second episode was successfully treated with ffp, though no clear infectious trigger was identified. the third episode occurred days after routine vaccinations. the patient had prolonged hypotension requiring ionotropic support that resolved after receiving daily ffp, and hypoxia with pleural effusions that resolved after a single treatment with protein c concentrate. as the patient had met / clinical diagnostic criteria for hlh, but also had a history of hyperammonemia, he underwent concurrent biochemical and genetic evaluation for both primary hlh and inborn errors of metabolism. whole exome sequencing identified compound heterozygous mutations in cog , part of an oligomeric protein complex involved in golgi apparatus structure and function. cog mutations have previously been reported in two patients with autosomal recessive cog -cdg (cdg-iij), who were described to have similar clinical symptoms of hypotonia, seizures, coagulopathy, and liver dysfunction, as well as recurrent infections. subsequent immune phenotyping while the patient was healthy was notable for slightly low numbers of nk cells, but normal cd a mobilization and perforin/granzyme b expression in vitro. our patient represents a novel presentation of cdg due to cog defect with associated immune dysfunction manifesting as recurrent episodes of inflammatory crisis with features of hlh. cdg and inborn errors of metabolism should be considered during diagnostic evaluation for patients with hlh symptoms, as cdg patients may develop acute episodes of severe inflammation, in the absence of cellular regulatory defects, for which ffp and protein c concentrate may have therapeutic value. of the deaths with identifiable causes, ( %) were related to infections. the rate of death per person-year was . . the most common autoimmunity-related complication was sweets syndrome, seen in patients ( %) with anti-ifn-g autoantibodies. sixteen of those patients ( %) had recurring sweets syndrome. additionally, patients ( %) developed lymphatic obstruction, which continued to recur in patients ( %). seven patients ( %) in this study did not have anti-ifn-g autoantibodies. the median [iqr] age of autoantibody-negative patients was [ , ] years and patients ( %) were female. none of the autoantibody-negative patients developed new infections during follow-up. at the end of the follow-up period, none of the patients had active/progressive disease and patients ( %) had died. conclusions: ninety-one percent of hiv uninfected thai patients with disseminated ntm infection with or without other opportunistic infections had detectable anti-ifn-g autoantibodies. about one third of patients with autoantibodies to ifn-g had recurrent infections during follow-up. after approximately years of follow-up, % of patients with anti-ifn-g autoantibodies had inactive disease following multi-drug antibiotic therapy while % had active/progressive disease and % had died. patients with anti-ifn-g autoantibodies are at risk for recurrent infections and autoimmunity-related complications. therefore, longterm follow-up is recommended. life-long secondary antibiotic prophylaxis may be required to prevent recurrence of infection in the setting of persistent anti-ifn-g autoantibodies. the study of early t cell development in patients with severe t cell immunodeficiencies is challenging because of the rarity of these diseases, the difficulty to obtain hematopoietic stem cells (hscs), and limitations in the assays to assess in vitro differentiation of hscs to mature t cells. we recently developed a serum-free system that allows faithful analysis of sequential steps of t cell differentiation. in this system, artificial thymic organoids (atos) are generated, based on the d aggregation and culture of a delta-like canonical notch ligand (dll )-expressing stromal cell line (ms -dll ) with cd + cells isolated from bone marrow (bm) samples of normal donors (nd). in this project, we set out to evaluate the possibility of using the ato system to study t cell differentiation in patients carrying t cell defects, in order to define the exact steps of t cell development affected by different genetic defects. using the ato system, we studied in vitro t cell differentiation from cd + cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag , rag , ak , il rg) or that affect thymus development (digeorge syndrome, dgs). the ak -deficient patient showed a markedly decreased viability in cd + cells and a very early defect in t cell development, already at the pro-t cell stage. this defect was very similar to that observed in a patient carrying a null il rg mutation who was reported to show autologous reconstitution after unconditioned haploidentical hsc transplantation. in contrast, cd + cells from a patient carrying a missense il rg mutation and with a leaky scid phenotype were capable of differentiating into mature t cells in vitro, although with -fold decreased efficiency as compared to normal donors (nd). interestingly, in the patient carrying the null il rg mutation, we noticed very few cells that could reach full maturation, with an absolute number of cd + tcrab+ cells around -times less than in nd. at variance with pro-t cells (that failed to express the gc protein), these mature t cells did express normal levels of gc, suggesting that they may have derived from residual cd + cells from the bm donor. in addition, cd + cells from the patients carrying rag and rag hypomorphic mutations were able to differentiate to cd +cd + double positive cells, but not to cd +tcrab+ cells. finally, the dgs patient showed a completely normal in vitro t cell differentiation, confirming that t cell deficiency reflected thymic abnormalities. in summary, our data show that the ato system could be extremely useful in determining whether the lack of t cells in patients with unknown gene defects reflect hematopoietic or thymic intrinsic problems, and may therefore provide critical evidence in deciding whether hsc or thymus transplantation is warranted, even without knowing the actual gene defect. introduction: ataxia-telangiectasia (at) is an autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene, which aids in detection and repair of dna damage. at is characterized by progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, variable degrees of t-cell lymphopenia (tcl) and immune compromise. patients are at an increased risk for malignancy, particularly leukemia and lymphoma, and are unusually sensitive to ionizing radiation. with the advent of trecbased newborn screening (nbs) for scid, at patients are being recognized with asymptomatic tcl in early infancy. objectives: we present an older child with at and chronic granulomatous lesions and discuss how this may be avoided in individuals with at diagnosed following abnormal nbs. case report: a y/o male was born at term following an uncomplicated twin pregnancy and delivery, prior to institution of scid nbs. he demonstrated mild gross motor and speech delay as an infant and was diagnosed with at at age . he had received all routine immunizations, including live vaccinations. he developed granulomatous skin lesions at age , initially small papules on his cheeks and ears, which subsequently formed large disfiguring plaques on sun-exposed areascheeks, arms and hands (fig ) . following an extensive workup, his lesions were found to be secondary to a mutated vaccine-strain rubella (ra / ) based on bp genotyping, previously described in other immunocompromised individuals [perelygina/sullivan et al. jaci ] . his lesions have been refractory to multiple treatments including nitazoxanide. he is currently on daily oral and topical steroids, tmp/smx and ivig. retrieval of his nbs for trec determination revealed that he would have screened positive [mallot/puck et al. j clin immunol ] . when first measured at age , cd t-cells were low, /ul, with cd /ul and cd /ul. b and nk cell numbers were normal. since april , cases of at were seen at ucsf in infants with non-scid tcl on nbs. these males and female were all born at term and discharged from well-infant nurseries. at was diagnosed at - months of age. their initial trecs ranged from - /ul (normal with perkinelmer enlite kit > ), and all had low t-cells on initial flow cytometry ( - cd /ul, ref range> ) with decreased cd ( - /ul) and cd ( - /ul) t-cells; however naïve t-cells were present, ruling out typical scid and raising concern for non-scid tcl. three infants also demonstrated low b-cells (< - /ul), while nk cells were normal in all. two are currently receiving ivig, one of whom is also on tmp/smx. all have avoided not only rotavirus but also mmr and varicella live vaccinations. conclusions: at is now often diagnosed in infants with low trecs on scid nbs, prior to neurologic manifestations. benefits of early diagnosis include avoidance of live vaccines, including mmr, which led to the debilitating granulomas in our older patient. additionally, patients receive prompt immunologic monitoring and treatment, avoidance of unnecessary radiation, specialty referrals and family genetic counseling. while there is no cure for at, ongoing research may bring neuroprotective treatments in the future. introduction: subcutaneous immune globulin %, ig gly, was well tolerated in the phase / north american study in patients with primary immunodeficiency diseases (pidd). here we assess comorbidities, use of concomitant medications, infusion parameters, and tolerability in advanced age patients ( y) treated with ig gly in the north american study. methods: patients aged years with pidd received weekly ig gly infusions at volumes ml/site and rates ml/h/site for~ . years in the north american study (nct ). the medical history at baseline, medical conditions that were ongoing (defined as comorbid events), use of concomitant medications, adverse events (aes), tolerability, and infusion parameters were assessed by age: in advanced age patients ( y; n= ), adult ( < y; n= ), and pediatric/adolescent patients (< y; n= ). results: the mean number of medical history events at baseline was higher in advanced age patients ( . events/patient; events in patients) versus adult ( . events/patient; events in patients), and pediatric/adolescent patients ( . events/patient; events in patients). of these, the medical conditions that were ongoing at baseline (comorbid events) were also higher in the advanced age patients ( . events/patient; events in patients) versus adult ( . events/ patient; events in patients), and pediatric/adolescent patients ( . events/patient; events in patients). in the advanced age patients, neurological comorbidities ( events) were the most common, followed by those related to eyes, ears, nose, and throat ( events), gastrointestinal ( events), and musculoskeletal comorbidities ( events). concomitant medications were given to treat a preexisting condition in all patients in the advanced age group ( medications in patients). despite the higher mean number of comorbid conditions, infusion parameters in the advanced age patients were comparable to those in the adult age group. median maximum infusion rates and infusion volumes/site were comparable in the advanced age patients ( ml/h/site; . ml/site) and adults ( ml/h/site; ml/site); lower infusion rates and volumes/site were reported in the pediatric/adolescent patients ( . larger infusion volumes and faster infusion rates were not associated with increases in causally related local aes in the advanced age group, consistent with the trends seen in the pediatric/ adolescent and adult patients. conclusions: despite the higher mean number of comorbidities in advanced age patients with pidd, ig gly was infused at relatively high rates and volumes and was well tolerated. introduction: hyqvia (ighy; immunoglobulin infusion % with recombinant human hyaluronidase [rhuph ]) is an immunoglobulin (ig) replacement therapy approved for patients with primary immunodeficiency diseases (pidd) that allows larger infusion volumes, up to ml/site, and has improved ig bioavailability compared with conventional subcutaneous ig products. a post-authorization safety study is being conducted in the united states to acquire long-term safety data on ighy and to assess prescribed administration regimens in routine clinical practice. infusion characteristics and treatment-related adverse events from an interim analysis are reported here. methods: patients aged years with pidd receiving ighy were included in this ongoing, prospective, non-interventional, open-label, uncontrolled, multicenter study. as a part of routine clinical practice, patients are treated with ighy according to standard medical care and their treatment regimen is at the discretion of the treating physician. adverse events (aes) are collected from enrollment to study completion/discontinuation using a subject diary and assessed at every study visit (every months or standard practice). aes are assessed based on seriousness, severity, and causal relatedness to ighy. the presence of anti-rhuph antibody is evaluated on a voluntary basis. treatment preferences for various attributes of ig therapy were assessed annually using a treatment preference questionnaire. results: a total of patients were enrolled at us study sites (data cutoff date: august , ). infusions were self-administered at home ( %) or at the clinical site ( %) most commonly using -week infusion intervals ( . %). the mean maximum ig infusion rate was . ml/h and the mean ig dose was mg/kg bodyweight/ weeks. the mean number of infusion sites used for administration was . and mean infusion duration was . hours. most infusions ( . %) were administered without a rate reduction, interruption, or discontinuation due to aes. there were no serious aes (saes) related to ighy. sixteen patients experienced a causally related non-serious local ae ( . %; . events/patient-year, . events per infusion) and patients experienced a causally related non-serious systemic ae ( . %, . events/patient year, . events per infusion). seven of patients who were tested for anti-rhuph antibody had positive binding antibody test to rhuph (titer : ; maximum titer : at enrollment, : during the study); no neutralizing rhuph antibodies were detected. of the patients who responded to the treatment preference questionnaire at the end of year , the majority ( / [ . %]) preferred to receive their ig therapy at home; . % ( / ) preferred the doctors office; patients preferred treatment at the hospital, had no preference, or indicated other. almost all patients ( / [ . %]) indicated a preference to continue treatment with ighy. conclusion: this interim analysis of patients with pidd treated with ighy in routine clinical practice supports previous observations that ighy is a well-tolerated and preferred therapy with no reports of treatment-related saes or neutralizing anti-rhuph antibodies. background: cartilage hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with variable immunodeficiency. pathogenic defects in rmrp, encoding the untranslated rna subunit of ribonucleoprotein endoribonuclease complex (rmrp), result in reduced mrna and rrna cleavage. rmrp c. a>g is the most common variant, increased in finnish and amish populations. while cellular immunodeficiency is associated with increased morbidity and mortality, there is no established correlation between clinical and immunological phenotype. lymphocyte radiosensitivity has not been described. case: a full-term amish female infant had low trec copies on newborn scid screen. flow cytometry at months-old demonstrated severe t and b cell lymphopenia (cd +t-cells cells/mcl, range: , - , cells/mcl; cd +b-cells cells/mcl, range: - , cells/mcl) with normal nk quantitation (cd / + cells/mcl, range: - , cells/mcl) and cd + memory t-cell expansion ( . %) relative to the naïve subset ( . %). t-cell functional mitogen responses were normal. she was diagnosed with chh with homozygous rmrp c. a>g mutation. lymphocyte subset (t, b and nk cells) radiosensitivity was evaluated by flow cytometric analysis of phosphorylated (p) atm, smc and gamma-h ax after low-dose ( gy) irradiation. an increase in gamma-h ax level was observed in a subset of non-irradiated t cells ( . % v. . % gamma-h ax+) and nk cells ( . % v. . % gamma-h ax+) in the patient, suggestive of a constitutive defect in dna repair. the relative distribution of t, b and nk cells expressing patm, psmc and gamma-h ax at hour postirradiation (ir) was not significantly different from the experimental healthy control (ehc) or pediatric reference range (prr). however, the kinetics of dephosphorylation at hours post-ir was altered with residual gamma-h ax expression in a subset of the patients t cells (delta . %, mode ratio mean fluorescence intensity (mfi)= . ; ehc: delta . %, mode ratio mfi= . ; prr: delta . %, mode ratio mfi= . ). a similar finding was observed in a subset of patient b-cells for gamma-h ax (delta . %, mode ratio mfi= . ; ehc: delta . %, mode ratio mfi= . ; prr: delta . %, mode ratio mfi= . ). the frequency of the patient's lymphocytes with residual gamma-h ax persistence at h post-ir was prominent, with . % t-cells demonstrating persistence of gamma-h ax (compared to . % in the ehc, and . % in the prr), and . % b-cells gamma-h ax+ (compared to . % in the ehc, and . % in the prr). there has been lack of follow-up, but verbal report suggests no significant immunological or infectious concerns at year of age. discussion: lymphocyte radiosensitivity is a novel finding in chh with t and b cell lymphopenia. the ability of rmrp to associate with telomerase reverse transcriptase (tert) and function as an rna-dependent rna polymerase, yielding distinct silencing rna sequences, may underlie radiosensitivity in rmrp mutants. systematic characterization of lymphocyte radiosensitivity and immunological phenotype could provide useful information on whether this could serve as a biomarker for the magnitude or complexity of immunodeficiency. assessment of radiosensitivity has implications in conditioning regimen selection for patients requiring allogeneic hematopoietic cell transplantation. we recommend lymphocyte radiosensitivity assessment in chh infants identified by nbs scid and chh patients with significant immunodeficiency and/or malignancy. novel primary immunodeficiency with lymphoproliferative disease due to biallelic defects in nckap l background: three children from non-consanguineous families and different ethnic backgrounds developed lymphoproliferative disease by years of age. they also had recurrent infections, including pneumonia and bronchiectasis, otitis media, and skin pustules. immune phenotyping revealed low cd + t cell percentages, an accumulation of memory-like cd + t cells, impaired t cell proliferation, and low total nk cell numbers. methods: the affected individuals, unaffected parents, and other unaffected family members underwent exome sequencing. results: all affected cases had rare and bioinformatically damaging biallelic variants, with appropriate familial segregation, in nckap l, which encodes hem . hem is an essential component of the wave regulatory complex (wrc). immunoblotting confirmed destabilization of the wrc in all patients. immunofluorescence microscopy demonstrated defective f-actin and wave localization to immune synapses in nk cells. significant abnormalities were identified in patient lymphocyte and neutrophil migration and morphology, consistent with altered wrc-mediated cytoskeletal dynamics. all patients exhibited impaired inside-out integrin activation. knockdown of hem produced deficient proliferative responses and mtorc -mediated akt activation in control t cells. conclusions: the immunologic and clinical phenotype in the affected individuals recapitulates the phenotype observed in hem -deficient mice. biallelic defects in nckap l therefore result in a novel human primary immunodeficiency disease characterized by lymphoproliferation and susceptibility to infections. background: concurrent existence/significance of immunodeficiency with new onset lymphoproliferative disease remains understudied. just two studies to date have evaluated the prevalence of hypogammaglobulinemia in chronic lymphocytic leukemia (cll) and neither studied prevalence and impact of ige deficiency on outcomes in cll [ , ] . therefore, the objective of this study was to examine the prevalence of hypogammaglobulinemia, examining all isotypes, in newly diagnosed cll patients and to test the hypothesis that patients with hypogammaglobulinemia have a distinct clinical profile and outcome. methods: using the banked sera of newly diagnosed, treatmentnaïve, cll adult patients from the lymphoma molecular epidemiology resource (l-mer), ig (igg, iga, igm and ige) levels were measured. the l-mer was initiated as an observational cohort study of prospectively enrolled newly diagnosed lymphoma patients evaluated at the mayo clinic (rochester, mn) and the university of iowa (iowa city, ia) [ ] . igg/a/m levels were measured using immunoturbidimetric assay whereas the ige level was determined using electrochemiluminescence immunoassay. the associations between ig deficiencies and clinical factors were evaluated with wilcoxon rank sum and chi-squared (fishers exact, where appropriate) tests. cox regression models were used to assess the effects of clinical variables on overall survival (os). time was calculated from biopsy to death due to any cause; patients still alive were censored at last contact. all tests were two-sided and assessed for significance at the % level using sas v . (sas institute, cary, nc). results: the mean age (sd) of the selected cll cohort was . ( . ) years with a male predominance ( . %). . % of the patients were white. with a median follow-up of five years, there were deaths. hypogammaglobulinemia in newly diagnosed, treatmentnaïve cll was common in our cohort with ( . %) patients having a measurable isotype deficiency. the most common ig deficiency was igm ( . %, % ci . - . %), followed by igg ( . %, % ci . - . %), ige ( . %, % ci . - . %) and iga ( . %, % ci . - . %). multiple deficiencies in the same patient were common ( figure ). iga and ige deficiency were associated with higher rai stages (grading system for cll) at presentation (p< . and . respectively) as well as with higher white blood cell counts at presentation (p= . and . respectively). a higher proportion of iga deficient patients needed second treatment during follow-up ( % compared to %, p= . ). when comparing predictors of overall survival, higher rai stage [ - vs , hazard ratio (hr) . , % ci . - . , p= . ] and age (hr . , % ci . - . , p< . ) correlated with worse overall survival. individual immunoglobulin deficiencies did not correlate with overall survival. conclusions: a significant proportion of treatment-naïve patients with cll have underlying ig deficiencies-both in isolation and a combination of different isotypes. a deficiency of iga or ige was associated with severe disease at presentation. the underlying relationship between these two immunologic disorders deserves further study. background: patients with primary immunodeficiency (pid) have an increased risk of developing autoimmune diseases, including rheumatoid arthritis (ra). management of these patients is challenging as immunomodulators can further increase their risk for infections. additionally, patients with ra that undergo therapy with drug modifying antirheumatic drugs (dmards) may develop a secondary immunodeficiency. there are few studies reviewing the characteristics of patients with a pid who later develop ra, and no studies have been reported comparing these patients to those who develop an immunodeficiency after starting dmard therapy for ra. methods: patients were identified as having inflammatory arthritis and a concomitant immunodeficiency (id) at our institution between / / - / / using icd- and codes. manual chart review was performed to confirm and identify the timing of diagnosis of these disorders. patients were excluded if either there was no definitive diagnosis of id or ra (clinically diagnosed by a practicing allergist/immunologist and meeting acr criteria for ra with a score of or higher, respectively), or rituximab was administered prior to diagnosis of id . clinical symptoms, treatment, and laboratory data were extracted. fishers exact test was used to compare the categorical variables between the groups; ttest was used to compare the continuous variables. results: patients met the inclusion criteria. patients were diagnosed with an id and developed ra later in life (group ), and patients were diagnosed with ra and subsequently developed a clinically significant id (group ). the mean ages of diagnosis of id and ra in group patients were . years (sd ± . ) and . years (sd ± . ), respectively. in group , the mean age of diagnosis of ra was . (sd ± . ), compared to . years (sd ± . ) for the diagnosis of id. most patients in both groups were female ( % in group and % in group ). all patients in both groups had a humoral id, including common variable immunodeficiency (cvid) ( % of group patients), specific antibody deficiency (sad) ( % of group and % of group patients), and hypogammaglobulinemia ( % of group and % of group patients). all patients in group were seropositive for rheumatoid factor (rf) or anti-cyclic citrullinated peptide (anti-ccp), whereas only % of patients in group were positive for rf or anti-ccp (table ). most patients in both groups were treated with immunoglobulin replacement therapy. treatment of ra in both groups was similar, but combination dmard therapy was not used in group patients in contrast to group patients. conclusions: our study indicates that even though clinical characteristics and management are similar in patients with coexisting id and ra, rf and anti-ccp are usually negative in patients who develop ra after id, possibly due to impaired antibody production in immunodeficient patients. assistant professor of allergy and immunology, arkansas children's hospital, university of arkansas medical sciences introduction/background: complement deficiencies are relatively rare, comprising less than % of primary immunodeficiencies. they are associated with increased risk for infections with encapsulated organisms and autoimmunity. of all complement deficiencies, the rarest are defects in the alternative complement pathway. properdin deficiency is the most commonly described alternative pathway deficiency, with factor b and factor d deficiency more rarely described. fewer than patients with factor d deficiency have been reported with all reported cases being children of consanguineous parents who succumbed to meningococcal sepsis. objectives: to describe a case of factor d deficiency associated with recurrent respiratory infections with streptococcus pneumoniae pneumonia with associated lung abscess and empyema. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, complement assays and functional evaluation, and genetic evaluation by next generation sequencing. results: a year old marshallese male was transferred from an outside hospital to our facility for further evaluation of worsening pneumonia and was found to have right-sided pleural effusion and pulmonary abscess in the right lower lobe. the abscess was drained and was found to be positive for streptococcus pneumoniae via polymerase chain reaction. he improved after chest tube placement and treatment with intravenous antibiotics. his medical history was significant for recurrent acute otitis media and prior hospitalization out-of-state for pneumonia with empyema secondary to streptococcus pneumoniae, which required chest tube placement and admission to the pediatric intensive care unit at months of age. immunologic work up revealed age-appropriate lymphocyte subpopulations, lymphocyte proliferative responses to mitogens, quantitative immunoglobulin levels, pneumococcal/tetanus/diphtheria titers, and ch complement assay. ah complement assay was decreased to units/ml. complement testing was repeated -with normal ch and ah of units/ml. further evaluation revealed normal levels of factors b, h, i and properdin. factor d level was . mcg/ml, and factor d function was decreased to units/ml, indicating a diagnosis of factor d deficiency. sequencing of the cfd gene revealed a previously undescribed homozygous deletion (c. _ del and p.lys del). the parents were not agreeable to personally undergoing genetic evaluation to determine if this was a de novo mutation. the patient was managed with pneumococcal and meningococcal immunizations, prophylactic amoxicillin and intravenous gamma globulin (ivig) without any further infections. unfortunately, after two ivig infusions, he was lost to follow up. conclusion: factor d deficiency is an extremely rare alternative complement pathway deficiency, described in less than patients. all infections described thus far have been secondary to neisseria meningitidis. this case represents not only a novel mutation in the cfd gene leading to factor d deficiency, but also the first description of a patient with factor d deficiency developing invasive infection secondary to streptococcus pneumoniae. background: viral infections are a significant cause of morbidity and mortality in patients with primary immunodeficiency disorders and following hematopoietic stem cell transplantation. adoptive immunotherapy using virus specific t-cells (vsts) has been shown to prevent and treat viral infections in immunocompromised hosts. human parainfluenza virus- (hpiv ) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies and has not previously been used as a target for t cell therapeutics. introduction: we previously reported that fatigue is increased in common variable immunodeficiency (cvid). however, in previous studies, fatigue was not defined using validated tools. our aim from this study is to identify the prevalence of patient-reported fatigue, using validated questionnaires, and determine the factors predisposing to fatigue in cvid methods: data from cvid who responded to the idf patient national survey a were analyzed. fatigue was measured using the brief fatigue inventory (bfi) questionnaire, which includes seven items to identify fatigue, and measure fatigue severity. a total of patients with cvid and responses to bfi were enrolled. demographics, co-morbidities, immunoglobulin replacement therapy (iggrt) route and dose, co-morbidities, infections, depression, quality of life (qol) (using the sf- v ) and disability were compared between fatigued and non-fatigued. logistic regression was used to identify the significant variables. ebv reactivation without ptld, treated with rituximab. alive and well. j clin immunol ( ) (suppl ):s -s s granulomas are the most significant day-to-day problem for cvid patient management. currently, there are limited options for their treatment and the optimal therapy is unknown. in case reports and small series, infliximab has been reported effective while others found it useless. we here describe a yo white male referred for monthly ivig in august . at age , he developed large areas of erythematous polymorphic plaques in his cheeks, arms and legs. a skin biopsy showed tuberculoid granulomas negative for bacteria, baar and fungi, with infiltrating cd + lymphocytes. a prolonged course of steroids did not improve his skin. he also had multiple pneumonias and bronchiectasis, and oral candidiasis. he received all vaccines, including bcg with no complications. with low immunoglobulins and a poor response to pneumococcal polysaccharides and tetanus toxoid he was diagnosed as cvid and placed on ivig at yo with excellent infectious control since then. at age , his skin lesions persisted and deepened to the bone on his left leg. broad spectrum antibiotics for months were unsuccessful. at yo to yo, skin grafts were performed on his arms, legs and both cheeks. two ulcers persisted on his left leg until august that increased in size, deepened and became erythematous and extremely painful (fig. ) . in september, two new ulcers appeared on his right cheek and right gluteus, respectively. one week later a third ulcer was found on his left calf. on september th, infliximab mg/kg ( mg) was administered. on the second infliximab dose, october th, the pain was completely gone and all ulcers were shrinking, and those ones in the cheek, gluteus and calf almost completely resolved. by the third dose, on november rd the ulcers in his right leg were almost closed (fig. ) . infliximab mg treatment continues every weeks. lab test remained unchanged from till , when his wounds got worsened. (table ) granulomatous disease in cvid is a challenge. both b and t cell directed therapies are encouraged. we add a new case of an infliximab responsive patient to others already reported. over genes have been reported to cause monogenic cvid. a year old girl presented with recurrent pneumonias and a diagnosis of cvid. the parents sought a second opinion. born at weeks gestational age, she was "always smaller and sicker than her friends," and in the prior months she had episodes of pneumonia with fever to f requiring emergency department treatment. two of these were associated with rsv and metapneumovirus, respectively. laboratory evaluation confirmed low levels of igg ( mg/dl) iga ( ) and igm ( ) congenital tuberculosis (ctb) is a rare disease most often associated with maternal genitourinary (gu) tuberculosis (tb) or disseminated tb. due to infertility caused by gu tb, ctb is rarely reported even in endemic countries. infants can acquire tb hematogenously via the placenta or umbilical vein or by fetal aspiration of infected amniotic fluid. presenting symptoms include respiratory distress, fever, hepatosplenomegaly, poor feeding, lethargy, and low birth weight. we report a premature female infant conceived via in vitro fertilization (ivf), who was born to indian immigrant parents at weeks of gestation due to preterm premature rupture of membranes. maternal history was significant for pulmonary tb at years of age. she denied abdominal or gu symptoms. infants nicu course was complicated by opacifications in the right lung and leukocytosis with neutrophil predominance, identified during evaluation of frequent apnea and bradycardia episodes at month of age. clinical improvement was noted after treatment with vancomycin, amikacin and piperacillin-tazobactam; however, leukocytosis of unknown etiology persisted. at . months of age she was discharged to inpatient rehabilitation. at months of age, she was readmitted for fever and respiratory distress. during this admission, an immune evaluation was undertaken due to persistence of symptoms along with unresolved leukocytosis with a peak of , cells/l with neutrophilia to , cells/l, and chest ct evidence of progressive multifocal lung disease worse in the right upper lobe despite empiric treatment with broadspectrum antibiotics. infectious work-up was negative, including acid-fast bacilli testing from bronchoalveolar lavage. due to the pronounced and persistent leukocytosis and neutrophilia, a primary immune defect was suspected. immune evaluation included: normal immunoglobulins (ig) g, a, and e, elevated igm, vaccine-specific antibody titers protective to diphtheria and of streptococcus pneumonia strains, mildly elevated t and b cells, a normal flow cytometry for dihydrorhodamine, myeloperoxidase stain and glucose- -phosphate dehydrogenase level, as well as a peripheral smear with no giant azurophilic granules. her primary immunodeficiency genetic panel was unrevealing. she underwent lung biopsy via video-assisted thoracoscopic surgery, which showed noncaseating granulomas and eventual growth of multi-drug-resistant mycobacterium tuberculosis (mtb). upon treatment with an appropriately adjusted anti-tuberculosis regimen, she showed rapid clinical and laboratory improvement. endometrial samples obtained from mother showed gu tb, confirming the diagnosis of ctb. the slow-growing nature of mtb that resulted in delayed diagnosis, along with the presence of non-caseating granulomas and persistent neutrophilia, prompted an immune work up that was completely normal. this case demonstrates the importance of considering ctb in the differential diagnosis of an infant presenting with severe lung infection, persistent neutrophilia, suboptimal response to broad-spectrum antibiotics and relevant epidemiologic risk factors. furthermore, in the setting of appropriate parental exposures and infertility prompting the use of ivf, maintaining a high level of suspicion of ctb can aid in earlier diagnosis of affected neonates. -year-old caucasian male who initially presented with recurrent otitis media, persistent hsm, lad, and hypogammaglobinemia (igg < mg/dl) at years of age. he was diagnosed with common variable immunodeficiency (cvid) and chronic arthritis when he was and years of age, respectively. subsequently, he developed hepatitis and recurrent pneumonia with mycobacterium avium complex (mac). his arthritis partially responded to anti-tumor necrosis factor (tnf) agents and tofacitinib, but did not respond to anti-interleukin- treatment. a combination of anti-tnf inhibitor, tofacitinib, and low dose prednisone was required to control his arthritis. hypogammaglobulinemia (igg < mg/dl), recurrent otitis media, pneumonia, crohn's disease, celiac disease, lad and failure to thrive at years of age with more recent development of hsm. he required only immunoglobulin replacement therapy. case# is a -year-old caucasian male, the half-brother of case# , who initially presented with recurrent pleural effusion and bilateral pulmonary infiltrates, hsm, lad, abdominal distension and ascites at years of age. a transbronchial lung biopsy revealed chronic eosinophilic pneumonitis. liver biopsy showed increased eosinophils in the sinusoids with diffuse enlargement of hepatocytes, but without hepatitis. colon biopsy revealed minimal colonic eo-sinophilia. his pulmonary infiltrates and pleural effusion responded to prednisone, and he has not required additional treatment for past . years. conclusions: the clinical manifestations of the same genetic variant may be variable and unpredictable even in the same family. stat gof syndrome should be considered in children with multisystem autoimmune diseases, lad, hsm and low switched memory b cells regardless of presence of hypogammaglobulinemia or history of recurrent infections. background: patients with primary immune deficiencies characterized by severe t lymphopenia and/or poor t cell function and patients posthematopoietic cell transplantation are at high risk of severe viral infections. antiviral medications are expensive, not always effective and associated with significant toxicity and/or long-term side effects. as such, there has been increasing interest in the use of donor-derived or thirdparty virus-specific t cells (vsts), and several studies have demonstrated efficacy of vsts generated using various manufacture strategies. however, in depth immunologic and metabolic characterization of vsts has not been reported, limiting correlative investigations into efficacy. methods: ebv-vsts were generated from apheresis t cells collected from healthy donors using three methods: ( ) stimulation and expansion with hla-matched ebv-lymphoblastoid cell lines (lcls) purchased from astarte biologics or sigma-aldrich over a period of weeks, ( ) stimulation with ebv peptivator from miltenyi followed by expansion over - days with different cytokines, and ( ) stimulation with ebv peptivator followed by isolation of activated cells using the ifn-gamma capture system from miltenyi. immunophenotyping by flow cytometry was performed using the miltneyi macsquant analyzer. the nanostring ncounter system was used to measure gene expression for metabolic pathway analysis, and the agilent seahorse xf cell mito stress test system was used to measure mitochondrial respiration. results: ebv-vsts generated using lcls or peptivator plus il- both resulted in a high percentage of cd t cells skewed to the effector memory and terminal effector memory phenotype with high expression of the exhaustion markers pd- , tim- , and lag- . conversely, ebv-vsts generated using peptivator plus il- and il- and the ifn-gamma capture system resulted in a mixed cd and cd t cell population with a high number of central memory t cells and lower percentage of cells positive for pd- , tim- , and lag- . stimulation with peptivator followed by expansion with il- resulted in an intermediate immunophenotype. nanostring results demonstrated upregulation of the glycolytic pathway in ebv-vsts stimulated with peptivator followed by expansion with il- or il- compared to ebv-vsts generated using the other manufacture approaches. the seahorse mito stress test demonstrated that the peptivator plus il- ebv-vsts had a significantly lower spare respiratory capacity than other ebv-vsts and a low extracellular acidification rate despite upregulation of the glycolytic pathway. the peptivator plus il- and il- ebv-vsts had the highest basal oxygen consumption rate, atp-linked respiration, and extracellular acidification rate. conclusions: manufacture of ebv-vsts using the various approaches currently employed clinically results in t cell pools with different immunophenotypes and different metabolic profiles. ebv-vsts stimulated with peptivator followed by expansion in il- and il- and ebv-vsts isolated using the ifn-gamma capture system have immunophenotypes and metabolic phenotypes suggestive of potential greater in vivo persistence, whereas ebv-vsts expanded in il- and il- have characteristics correlated with increased effector function. however, these vsts are more likely to be short-lived and to have impaired metabolic fitness. these phenotypes will enable better correlation with clinical results and suggest combinatorial approaches depending on clinical indication. introduction: majority of patients with primary immunodeficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors for patients. a new . % ig preparation octanorm (octapharma, lachen; tradename cutaquig® in north america) has been developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was assessment of the efficacy of octanorm in preventing serious bacterial infections. main secondary endpoints included (among others) evaluation of tolerability and safety of octanorm, the number and rate of other infections, number of days missed at work, and use of antibiotics. methods: a prospective, open-label, non-controlled, single-arm phase study involving adult patients with pid was conducted at russian centers. patients treated with at least infusions of ivig prior to enrollment and with igg trough levels . g/l underwent an -week wash-in/wash-out period followed by a week efficacy period. during the study, patients received weekly administrations of octanorm at the same monthly dose as during previous ivig treatment (monthly ivig dose divided by for weekly dose). in total, each patient received scig infusions. results: twenty-four patients completed the study. one patient terminated early (after infusion , during wash-in/wash-out phase; personal reasons). mean age was . years (range - years). fifteen patients ( %) were female and patients ( %) male. no serious bacterial infections were recorded. during the efficacy period a total of non-serious infections was observed in patients. seventeen infections in patients were of mild and infections in patients of moderate intensity. the infection rate per person-year was . . in total patients received infusions of study drug. the average dose of cutaquig® was . g/kg/week. during the entire study, systemic adverse events were reported (including infections). three of these systemic adverse events were rated as related to study drug, all were non-serious. there was no serious or significant adverse event nor was there an adverse event leading to withdrawal. infusion site reactions were reported for % of infusions. serum igg trough levels were nearly constant during the efficacy period. median igg trough levels were . g/l at screening, . g/l at the end of wash-in/wash-out period and . g/l at the termination visit. one patient had a trough level g/l at visits during the efficacy period and the dosing was subsequently adjusted for this patient. during the primary treatment period patients ( . %) used antibiotics in treatment episodes (total of treatment days; range - days) and patients had absences from work or school due to infections (total of days of absence). conclusion: this study demonstrated that the new subcutaneous human normal immunoglobulin . % is well tolerated, safe and effective in adult patients with pid. background: children with chronic granulomatous disease (cgd) are at high risk for fungal infections (especially with aspergillus species) and these infections usually have contiguous site involvement. most patients have pulmonary presentation. infective endocarditis and fungal osteomyelitis of skull are distinctly unusual. we report one such case. case: a -year-old boy, born out of a non-consanguineous marriage, presented with soft tissue swellings of skull for months. his past history was significant with an episode of pneumonia at year and recurrent soft tissue swellings all over the body since ½ years of age. on examination he was wasted, had signs of micronutrient deficiency, rickets, pallor, cervical lymphadenopathy and two abscesses, x cm on right temporo-parietal region and x cm over left frontal region. he was also found to have hyperdynamic precordium with an ejection systolic murmur. investigations revealed hemoglobin g/l; platelet count . x /l; total leukocyte count x /l(n /l /m /e ); elevated c-reactive protein( mg/l) and a raised erythrocyte sedimentation rate( mm sthr). chest x ray revealed cardiomegaly (cardiothoracic ratio %) and d echocardiography showed vegetation of x mm over the anterior mitral leaflet suggestive of infective endocarditis. blood and urine cultures were sterile. culture from pus over the temporo-parietal abscess showed growth of aspergillus fumigatus. human immunodeficiency virus serology was non-reactive. immunoglobulin profile revealed elevated igg . g/l ( . - . g/l) and iga . g/l( . - . g/l); igm was . g/l( . - . g/l). in view of strong suspicion of cgd, nitroblue tetrazolium dye reduction test (nbt) was carried out-it revealed no reduction and dihydrorhodamine (dhr) assay showed a low stimulation index ( . ). flow cytometry for gp phox and gp phox was normal and dhr of mother did not reveal x linked carrier state. contrast enhanced computerized tomography (cect) of head showed osteomyelitis of the calvarial bones. contrast enhanced magnetic resonance imaging (cemri) brain showed heterogeneously enchancing soft tissue lesion in the scalp at right fronto-parietal region and left frontal region with underlying bony destruction suggestive of osteomyelitis. he was given intravenous antimicrobials (ceftriaxone, gentamycin, cloxacillin, voriconazole). after weeks of therapy, he showed resolution of findings on mri brain and a repeat d echocardiography showed significant decrease in size of mitral leaflet vegetation. conclusion: this case highlights a rare presentation of cgd with infective endocarditis and skull osteomyelitis due to aspergillus fumigatus. to the best of our knowledge, this has not been reported previously. background: genetic defect in il r affect cellular immunity, underlie mendelian susceptibility to mycobacterial disease (msmd) and inflammatory bowel disease (ibd) through different pathways. we present for the first time a patient with il- r deficiency from a consanguine family with two different phenotypes. initially diagnosed as crohn's disease prior to the msmd diagnosis. method and material:patient was referred to the clinical immunology and allergy clinic at the at alzahra university hospital for immunological and genetic evaluation . blood samples from patient, his family and healthy donor controls were collected upon informed consent. in this study, we investigated effect of il r mutation in il- /ifnaxis by evaluation of patients whole blood cell response to il- and ifn-, il- r expression in pbmcs and t cell blasts. also wholeexome sequencing has been performed. result and discussion: a years old male from consanguine family , with history of right sub-axillary bcg lymphadenitis, recurrent mouth ulcers , chronic diarrhea in childhood and appendectomy at age of was investigated. based on his clinical presentation abdominal pain, significant weight loss, chronic and bloody diarrhea , endoscopic and pathological findings treatment for crohn's disease (cd) was started at the age of seven . unfortunately, protracted patient's symptoms ends up to resection of his colon and colostomy two years later. he was presented with multi focal osteomyelitis at the age of . although no bacteria was detected in pcr and tissue culture of the bone biopsy and the patient was not responded to antibacterials , he had a dramatic response to empirical anti mycobacterial treatment and his severe bone pain and lesions were healed. even though the bone manifestations were completely controlled, he continuously was under treatment for his gastrointestinal symptoms. genetic analysis was confirmed segregation of homozygous mutation in splice site of exon in il- r . expression of gene was completely abolished in pbmcs of patient and the surface expression of il rb was not detectable in t cell derived pbmcs of the patient compared to healthy control. furthermore, did not response to il stimulation since we could not detect increase of inf-after stimulation with il and bcg. our patient received bcg vaccination at birth and had bcg lymphadenitis as an infant, cd and mycobacterial multifocal osteomyelitis as a child. furthermore there are some evidences which indicate the role of atypical mycobacterial infections as a trigger for cd. conclusion: we reported for the first time contemporary msmd and ibd in years old patient, who had impaired il- signaling and abolished il r expression in pbmcs and t cell blast. however, mycobacterial osteomyelitis is a typical phenotype of msmd patients with deficiency in ifn-r or stat, there were no mycobacterial osteomyelitis reported in il- r deficient patients. background: advanced genetic studies help explain the occurrence of many undiagnosed, rare conditions. recently, nbas variants were identified as a causative basis of recurrent liver failure in infants (infantile liver failure syndrome , ilfs ). the nbas (neuroblastoma amplified sequence) gene encodes a protein involved in golgi to er retrograde transport. nbas functions seem to be broad and loss of function variants in nbas have been associated with multisystem manifestations. case report: a y m old chilean male presented to the er with a three day history of vomiting, diarrhea and one day of fever ( . °f). on examination he was pale, lethargic, and tachycardic. a chemistry profile revealed markedly elevated liver enzymes, increased bilirubin, and coagulopathy, consistent with the acute hepatic failure (alt , ast > , total bilirubin . ( . db), ggt , and inr of . ). he was hospitalized, given vitamin k, and kept on intravenous fluids, ursodiol, and antipyretics. his liver function improved significantly within days of admission (alt was down to , ast , total bilirubin . ). work-up of possible etiologies including autoimmunity and infectious hepatitis was negative. liver sonogram was normal, but liver biopsy was consistent with acute hepatitis with some necrosis. urine organic acid and plasma amino acid screens were not consistent with any inherited metabolic disorders. his parents recalled two previous episodes of liver failure at ages and years. both were preceded with a mild febrile illness and non-specific symptoms including fever, coughing, vomiting, diarrhea, lethargy, and decreased po intake. these subsequently were followed by jaundice and marked elevation of liver enzymes. flu a and adenovirus were identified as causes of febrile illnesses of the two previous episodes. for this admission, adenovirus was found in the respiratory secretions and a mild ebv viremia was also detected. genetic evaluation in chile was reportedly normal. after a literature review we obtained sequencing of nbas which revealed two variants: c. g>t,p.glu * and nbas c. t>g, p.iie ser. both variants have been previously reported in patients with an infantile onset, recurrent liver failure syndrome. his other clinical features include developmental and speech delays, failure to thrive, and facial dysmorphism. he also has a history of recurrent ear infections and has had sets of tympanostomy tubes. further testing was limited due to the lack of insurance coverage. conclusion: nbas deficiency is a newly described syndrome of recurrent acute liver failure that occurs early in life. once individuals have survived to adulthood they do not seem to develop liver failure with illness. typically, liver crisis is triggered by a common childhood febrile illness. the mechanism of disease is thought to be thermal instability of hepatocytes which improves over time in most cases. however, although spontaneous recovery can occur following the crises, each episode can be fatal or result in permanent liver damage required liver transplantation. increased awareness of this disease will lead to the early establishment of the diagnosis. appropriate and timely management of fever at the onset of illness can significantly improve outcome in this potentially fatal disease. associate prof., infectious diseases and tropical medicine research center, isfahan university of medical sciences, isfahan, iran background: pre-eclampsia, a pregnancy-specific complication, has been shown to be associated with cytomegalovirus (cmv) infection. cmv specific t-cell response plays the major role in cmv infection or disease .we explored whether a change in cmv-specific cell-mediated immunity (cmi) is related to the development of preeclampsia. method: cmv-specific cmi was assessed using cmv-quantiferon (qf-cmv) assay in serum from women with pre-eclampsia as well as normal pregnancy controls retrospectively. participants were matched for gestational age individually. proportion of reactive results, mean value of interferon-level produced in mitogen and antigen tubes were compared between the cases and controls via chi-square, wilcoxon rank-sum tests, respectively. odds ratio (or) and confidence interval (ci) were calculated as well. result: no significant differences observed between demographic characteristics of the case and control groups. the qf-cmv assay turned reactive (qf-cmv [+]) in of of patients ( %) vs. of controls ( . %) (p = . ). women with pre-eclampsia had lower mean ifn-levels in antigen tube ( . ± . ) compared with normal pregnancy controls ( . ± . ) (p = . ). there was no statistically significant differences in this value of mitogen tube between cases ( . ± . ) and controls ( . ± . ) (p = . ). women with suppressed cmv-cmi were . times more likely to manifest pre-eclampsia (or= . , % ci: . - . ). this result even strengthened after adjustment for age, gestational age and gravidity (or = . , % ci: . - . ). conclusion: our finding support an association between suppressed cmv specific cmi and pre-eclampsia. introduction: the triad of susceptibility to infections, auto-inflammation, and cancer in a patients personal and family history are always suggestive of an underlying primary immunodeficiency; however, in some cases the diagnosis might be delayed for years. furthermore, the results of immunological and inflammatory evaluation can also be affected by ongoing immunomodulatory therapy initiated by different specialists upon clinical diagnosis. objective: to describe a unique presentation of auto-inflammatory disease with combined immunodeficiency in an adult patient. case presentation: we report here the case of a year old male, who had a long history of infections including recurrent sino-pulmonary bacterial infections starting during childhood, osteomyelitis at years of age, recurrent tonsillitis requiring tonsillectomy at years of age, recurrent cellulitis, an episode of prostatitis with septicaemia, as well as recurrent varicella zoster and warts. the patient was also diagnosed with sclerosing mesentheritis, and reynauds phenomenon, recurrent oral ulcers, arthritis, uveitis, autoimmune thyroiditis, lung fibrosis and suffered repeated episodes of abdominal pain. furthermore, there is a family history of early childhood death, multiple soft tissue cancers, crohns disease, and autoimmune thyroiditis. upon physical examination, the patient had multiple telangiectasia, baseline erythroderma, and flushing. immunological evaluation showed lymphopenia with significant reduction in both circulating b and t cells, however, assessment of humoral immunity revealed low igg and decreased igm with normal iga levels. at the time of the evaluation he had been on low dose daily prednisone ( . mg), colchicine, and methotrexate as immuno-modifying therapy. genetic evaluation revealed a heterozygous mutation in nod as well as compound heterozygous mutations in the mefv gene. discussion: mutations in nod have been described in association with blau syndrome a multisystem auto-inflammatory syndrome which may explain many of the features experienced by our patient. to our surprise next generation sequencing revealed a second aberration in the mefv gene which causes familiar mediterranean fever, another multisystem auto-inflammatory disease, which might lead to the phenotype observed in the patient. conclusion: this is the first report of genetic lesions in two different genes leading to a severe course of auto inflammation. monogenic autoinflammatory syndromes (mais) are a diverse group of disorders characterized by primary over-activation of the innate immune system. induction of the inflammasome complex by innate immune sensors and increased production of il- b are implicated in the pathogenesis of mais. macrophage activation syndrome (mas) is a life-threatening illness defined by acute hyper-inflammation and unopposed cytokine release. it is considered an acquired condition secondary to infection, rheumatoid disease or malignancy. the early therapeutic use of il- b inhibition has profoundly improved the prognosis mas. it has recently been shown that increased free il- levels in the blood are causatively linked to the development of mas. significant overlap in clinical presentation and laboratory markers between patients with mais and mas led us to explore the role of free il- and therapeutic use of il- b inhibition in a patient with cdc mutation. here, we report the case of an months-old female who presented with hydrops fetalis in utero, and later developed failure-to-thrive, splenomegaly, anemia, thrombocytopenia, arthralgias, rashes, frequent febrile episodes and mild facial dysmorphism along with massive increase in crp, esr and ferritin. whole exome sequencing (wes) identified a heterogenous likely pathogenic de novo variant in cell division control protein homolog (cdc ) c. g>a (p.c y). cdc encodes a small rho family gtpase that regulates multiple signaling pathways controlling cell polarity, migration, endocytosis and cell cycle progression. single allele mutations in the cdc gene were recently reported to cause takenouchi-kosaki syndrome manifesting with growth retardation, developmental delay, facial dysmorphism, and thrombocytopenia however systemic autoinflammation has not been described. cdc closely interacts with the wiskott-aldrich syndrome protein but little is known about the mechanism underlying immune abnormalities associated with cdc mutations. our patient had an inflammamosopathy-like syndrome. because of significant clinical overlap to mas, we measured il- , il- , free il- and il- binding protein, all of which were significantly increased. this increase in free il- heightened her risk of developing mas. her il b level was normal, but an increase in il- b is hardly ever detectable in the serum despite playing a critical role in this type of inflammation. indeed, chronic il- b excess in the tissues promotes systemic inflammation and is associated with chronically elevated crp and esr. with this rationale we started the il- receptor antagonist anakinra. within hours from starting anakinra, the parents observed an increase in appetite, resolution of arthralgias and improved mobility. over the course of the following weeks, fever, anemia, thrombocytopenia and rash disappeared, the spleen massively decreased in size and the patient started to meet developmental milestones. crp, esr eventually normalized while ferritin and free il- are still trending down. conclusions: significant increase in free il- and extremely encouraging clinical response to therapy with anakinra in a patient with novel cdc mutation suggests a link between mas and defects in cdc . elucidating the mechanism of inflammasome activation and the drivers of il- increase in mas and mais more broadly may shed light on novel therapeutic targets like the use of human recombinant il- binding protein. j clin immunol ( ) (suppl ):s -s s maintenance; smarcal is enriched in cells that maintain telomeres via the alternative lengthening of telomeres pathway and smarcal decifient cells demonstrate telomere instability with replication fork collapse and increased telomere-associated dna damage. [ , ] telomere analysis of siod patients, including one patient who received a hematopoietic stem cell transplant (hsct) years prior, as well as heterozygous family members revealed significantly shorter telomeres in siod patients compared to heterozygous family members and compared to agematched, healthy controls. methods: peripheral blood mononuclear cells were isolated using a ficoll-hypaque density gradient, cryopreserved, then sent to repeat diagnostics in north vancouver, bc. telomere length measurements were performed at a single-cell level using flow-fluorescence in situ hybridization as previously described. [ ] telomere length was measured in total lymphocytes, naive and memory enriched t cells, b cells, and nk cells and compared to reference samples from age-matched, healthy individuals. results: compared to age-matched healthy controls, three siod individuals had mean telomere lengths (mtls) less than the st percentile for age across all lymphocyte subsets (total lymphocytes, b cells, nk cells, naïve and memory t cells). in comparison, three unaffected family members had normal mtls ( th percentile< x < th percentile) across all subsets, and two unaffected family members had low mtls ( st< x < th percentile) in some subsets. the siod individual who received a matched-sibling hsct years prior, had normal mtl in nk cells ( th < x < th percentile) but low mtls ( st< x < th percentile) for all other subsets. conclusions: these data show that siod patients have significantly impaired telomere lengths across multiple lymphocyte lineages and support a limiting role for smarcal deficiency in telomere maintenance. in comparison, unaffected family members, heterozygous for smarcal mutations, have mean telomere lengths that are normal or slightly low for age. this suggests that abnormally short telomeres are seen in individuals with homozygous but not heterozygous smarcal mutations. for the individual who received a hsct, we do not have pre and post-hsct telomere data, but these results support obtaining pre and post-hsct telomere length analysis in future cases. abnormally short telomeres have been linked to widespread perturbation of gene expression. [ ] we hypothesize that smarcal deficiency, by the effect of stalled forks and shortened telomeres, leads to perturbation in the transcriptome of affected tissues. shortened telomeres may explain the reduced hematopoietic bone marrow production in siod, as bone marrow failure is a cardinal feature of dyskeratosis congenita, a disorder of impaired telomere maintenance. future studies to investigate the role of telomere maintenance in siod include measurement of telomerase activity in polyclonally activated t cells and transcriptome analysis using rna-seq background: yellow fever is a potentially fatal disease for which only supportive treatment is available. vaccination is the primary strategy for prevention of this disease and the vaccine is extremely effective, but there are a few specific populations where it is contraindicated. regarding iga deficiency (the most frequent primary immunodeficiency), current recommendations in the literature are controversial. there are no specific studies in this disease, so case series addressing the safety or possible adverse events after vaccination are essential for decisionmaking during epidemic scenarios, as experienced in brazil in the last years. in this context, this study aimed to describe adverse events after the use of the yellow fever vaccine in iga deficient patients. method: a retrospective cross-sectional study was conducted including iga deficient patients followed at a specialized pediatric outpatient clinic between and . all patients had at least one year of follow-up. immunoglobulin levels, antibody response to vaccines and lymphocyte subset count were evaluated to exclude other immunodeficiencies or the presence of abnormalities that could contraindicate vaccination. demographic data, the presence of infections and comorbidities, use of immunosuppressive medication and adverse events after vaccine administration of the vaccine were described. results: thirty-eight patients with iga deficiency were included in the study and received the vaccine. vaccinated patients had a mean age at the time of the study of . years (sd ± . y). six out of the presented comorbidities: thyroiditis (n= ), type diabetes mellitus (n= ), celiac disease (n= ) and juvenile rheumatoid arthritis (n= ). all patients were atopic and only one had recurrent infections in the last year despite the use of antibiotic prophylaxis. all patients had normal igg and igm levels for their age, positive vaccine responses for measles, rubella and mumps, and age-appropriate lymphocyte subset count. after months of observation, no immediate or late adverse events were reported. among the non-vaccinated patients, only one had a formal contraindication (systemic erythematosus lupus using immunosuppressive therapy). five out of the non-vaccinated patients reported being afraid of receiving the vaccine, still intended to receive it and for other patients data regarding vaccination was unavailable. conclusion: despite the small number of patients, the absence of adverse events in this case series suggests that immunization with yellow fever vaccine may be safe in iga deficient patients, excluded other contraindications. more studies are essential to confirm the safety and help the decision-making process regarding the vaccine administration for iga deficient patients, especially in this yellow fever outbreak scenario. introduction/backround: immunodeficiency, centromeric instability, and facial anomalies syndrome (icf) is a rare group of autosomal recessive disorders involving the triad of hypogammaglobulinemia, centromeric instability, and facial anomalies. the majority of patients have hypo-or agammaglobulinemia, but t cell defects have also been reported. we present the case of a child with icf- who presented with nk deficiency and ultimately developed an ebv-driven malignancy and was successfully treated with bone marrow transplant. methods: whole exome sequencing and nk cell function via -cr cytotoxicity assay and phenotyping via flow cytometry were performed at baylor college of medicine and texas childrens hospital. centromeric banding studies were performed at university of pittsburgh medical center. results: the female patient presented at months of age with cmv pneumonitis and persistent cmv viremia requiring treatment followed by prophylaxis with valgancyclovir. she initially had hypogammaglobulinemia and low t, b, and nk cells; she had normal trecs, lymphocyte mitogen proliferation responses and zap , mhci and mhcii expression. the hypogammaglobulinemia and t-and b-cell lymphopenia resolved within months after initial presentation as she clinically improved from her cmv infection. she was found to have nk cell deficiency on three separate commercially tested samples. whole exome sequencing revealed a homozygous variant in zbtb indicative of icf- syndrome that was confirmed with sanger sequencing (c. _ del, p.q vfs). repeat nk cell studies confirmed impaired function, and phenotyping showed an increase in cd -bright and a decrease in cd -positive cells, suggesting either impaired transition from immature to mature nk cells or impaired survival of mature cells. her karyotype and centromeric banding studies were normal, as were centromeric instability studies. she later developed a memory b-cell defect and presented at months of age with persistent fever, respiratory distress, loss of vaccine titers, hypogammaglobulinemia and low b and t cells. she was found to have ebv viremia and an eber-positive diffuse large b-cell lymphoma in her right lung. due to tenuous clinical status, she received rituximab for treatment of ebv prior to definitive lymphoma diagnosis. she was treated with chemotherapy per protocol anhl , group b (pre-phase with cop, courses and with copadm, and courses and with cym) and her course was complicated by seizures attributed to methotrexate toxicity. she ultimately underwent reduced intensity conditioning with hydroxyurea, alemtuzumab, fludarabine, mephalan, and thiotepa followed by a cd- selected, hla-matched, unrelated donor peripheral blood stem cell transplant. her early post-transplant course was complicated by adeno-, ebv, and cmv viremia, all successfully treated with antivirals and a donor lymphocyte infusion. she is now greater than months posttransplant, off immunosuppression with % donor engraftment, no evidence of organ toxicity or gvhd, and with excellent immune reconstitution. conclusions: this is the first reported case of impaired nk cell function and phenotype and ebv-driven malignancy in a patient with icf- . this case expands the phenotype of icf- and suggests that early bone marrow transplant should be considered in these children. it also demonstrates a novel requirement for zbtb in human nk cell maturation and function. rationale: common variable immunodeficiency (cvid) is a disorder that affects the production of immunoglobulins and is associated with development of autoimmunity. multiple mutations have been described that are associated with cvid, but plcg mutations have only been described in patients with phospholipase c gamma (plc ) associated antibody deficiency and immune dysregulation (plaid) and autoinflammatory plc associated antibody deficiency and immune dysregulation (aplaid). we present a case of a y/o male cvid patient with recurrent upper respiratory tract infections, steroid-dependent autoimmune thrombocytopenia, low b cell count, hepatosplenomegaly, and restrictive lung disease. he was found with a variant of unknown significance at the plcg gene. in contrast to plaid our patient does not exhibit cold urticaria. method: case presentation of a cvid patient followed in our clinics. patients chart and previous laboratories were reviewed. sequence analysis and deletion/duplication cvid panel testing was performed using invitae© discussion: genetic testing has revolutionized the diagnosis of immune deficiencies, but variants of unknown significance are being increasingly reported. in this case, a variant of uncertain significance was identified which replaces threonine for alanine at codon of the plcg protein. this codon is located at the sh domain, which is part of a region that provides auto-inhibitory enzymatic functions. plaid mutations have been identified in sh domain, but it has been known that both sh and sh domains facilitate plcg association with other proteins. studies with deletion of plcg gene have shown functional abnormalities in b cells, natural killer cells and mast cells. to our knowledge, there has not been any previous report of a cvid patient with a variant mutation at the sh domain of the plcg gene without being diagnosed as plaid or aplaid. our patient has immunodeficiency, recurrent upper respiratory tract infections, steroid-dependent recurrent autoimmune thrombocytopenia, rheumatoid arthritis, hepatosplenomegaly, early-osteoporosis and restrictive lung disease. he does not have cold urticaria as seen in plaid, but exhibits autoimmunity not observed in aplaid. conclusion: conclusion: plcg is an important protein in the pathway of b cell development. a novel mutation in the sh domain of the plcg gene may be associated with the cvid phenotype of low b cells and autoimmunity. this could lead to a gain-of-function mutation as seen in plaid but without early-onset cold urticaria. functional studies are required to confirm the significance of this mutation. primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory disease affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, and is characterized by extreme inflammation and massive tissue infiltration by activated t cells and macrophages. to this day, hematopoietic stem cell transplantation is the only available curative treatment with a transplantrelated mortality of %. thus, the development of new, more efficient anti-inflammatory treatments would be a significant advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting both jak-dependent and independent cytokines will be more effective than either one alone to reduce the lifethreatening symptoms induced by this pathology. using a perforin-deficient mouse model of hlh, we first compared the effect of targeting individual cytokines with blocking antibodies on the progression of the disease. we show that blocking ifng and il- , but not il- , significantly reduces the severity of hlh. targeting the jak-stat signalling pathway with ruxolitinib, a specific inhibitor of jak and jak , downstream of ifng and il- , but not il- , is similarly beneficial. more importantly, combination therapies using ruxolitinib and blocking antibodies to either ifng or il- show synergistic effects, further mitigating the progression of the disease. these results suggest that jak-dependent and independent cytokines drive the pathogenicity of hlh in perforin-deficient mice. it further supports that ruxolitinib, although effective in reducing the symptoms of hlh, should be used in combination with anti-ifng and/or anti-il- antibodies to prevent hlh progression. this is particular relevant since the former were recently approved for the treatment of hlh while the latter (il- binding proteins) are in clinical trials for il- -dependent macrophage activation syndromes. despite the increased risk of opportunistic lung infection in patients with severe t cell dysfunction (e.g. cd l deficiency) and/or severe cd t cell lymphopenia, we are not aware of any reports of disseminated pneumocystis jiroveci infection in non-human immunodeficiency virus (hiv) patients with primary immunodeficiency (pid). we report the first case, to our knowledge, of disseminated pjp in a patient with cvid like/ctla haploinsufficiency. he had been diagnosed with common variable immunodeficiency (cvid) in , approximately eight years prior to being referred to us, and was on intravenous immunoglobulin (ivig). he was also diagnosed with multilineage evans syndrome in . his medical history was also significant for potential granulomatous lymphocytic interstitial lung disease (glild) (lung biopsy in the remote past with interstitial disease), significant splenomegaly ( . cm), severe portal hypertension, nodular liver disease (likely nodular regenerative hyperplasia) complicated by anasarca, history of chronic diarrhea (potential enteropathy), lymphadenopathy s/p biopsy with nodular lymphoid hyperplasia, and a history of multiple pneumonias. in , he had developed disseminated pjp with lung, liver, and bone involvement. the t vertebra pjp invasion was confirmed with a bone biopsy; gomori methenamine silver staining and pcr were performed and concluded pjp. he was treated with trimethoprim sulfamethoxazole (tmp-smx) and steroids, then was continued on tmp-smx prophylaxis. due to his liver damage and his chronic neutropenia, tmp-smx was replaced by atovaquone as a secondary prophylaxis for pjp. his laboratory studies were significant for an absolute neutrophil count of . k/ul, absolute lymphocyte count of . k/ul, hemoglobin of . g/dl, platelets of k/ul, total bilirubin of . t-cell receptor beta chain repertoire analysis showed an oligoclonal distribution. severe combined immunodeficiency panel through ambry genetic testing was negative as was genetic testing for cd l deficiency. given his complex clinical history, whole exome sequencing was obtained and detected an autosomal dominant heterozygous missense mutation (c. g>a) implicated in ctla- haploinsufficiency and previously reported by schwab et al. our patient is currently undergoing therapy with abatacept (ctla- fusion protein), which has been reported to improve glild, splenomegaly and enteropathy in patients with ctla- haploinsufficiency. he is improving on this regimen. he has met with the stem cell transplant team, but at this point of time, due to his abnormal lung function, his liver damage and his significant splenomegaly, he is not a good candidate. defects in the nf-b signaling pathway are implicated in the pathogenesis of several primary immune deficiencies in humans. the clinical features of these conditions vary significantly, reflecting the complexity of the pathway, and its broad role in innate and adaptive immune responses, and the development and differentiation of lymphoid organs. here we report the first case of a human pid caused by a homozygous mutation in nfkbid in a year-old male. he was the second child of consanguineous parents, and was diagnosed with possible cvid at the age of , after recurrent episodes of pneumococcal pneumonia. however the clinical features have evolved over time; he developed severe ebv infection at age , causing hepatitis and pancreatitis. at the age of , he presented with an anca-negative systemic vasculitis, manifesting as pulmonary haemorrhage, and acute necrotizing pauci-immune glomerulonephritis. pulsed methylprednisolone and cyclophosphamide induced an initial remission, however, relapse a year later led to end-stage renal failure. he is now dialysis-dependent, and due to the underlying pid, and chronic cmv viraemia, is not a candidate for renal transplantation. genomic dna was subjected to whole-exome sequencing. variants were filtered using a model of autosomal-recessive inheritance and functional analysis of primary cells was performed. we identified a novel, homozygous, single-base deletion resulting in a frame-shift, and premature stop in nfkbid. nfkbid encodes ibns, a non-classical inhibitor of nf-b signaling. at diagnosis the patient had reduced levels of igg , iga and igm, elevated ige, with absent humoral immune responses to pneumococcal polysaccharide vaccine, and an intact response to tetanus. lymphocyte numbers were initially within normal reference ranges, albeit with an increased proportion of cd +:cd + t cells. however, over time there has been a significant reduction in b cells and cd + t cells. cd + t cells demonstrated a skewing towards a central memory phenotype (cd ro+/ccr +), and cd t cell proliferative responses to pha were comparable to a healthy control. functional analysis of primary cells from the proband revealed a complete absence of bns protein expression, dysregulated nf-b signaling, and elevated pro-inflammatory cytokine production. the patient is currently receiving a trial of targeted therapy to modulate the aberrant immune responses. this novel pid highlights the importance of regulation of nf-b signalling, in orchestrating an appropriate immune response, maintenance of self-tolerance, and protection against viral pathogens. primary immunodeficiency diseases (pid) are a heterogeneous group of conditions with variable clinical features that are frequently associated with significant diagnostic delay. accurate diagnosis has significant therapeutic benefit and may lead to personalized therapies. we established the immunology flagship of melbourne genomics health alliance in australia to determine the clinical utility of genomic sequencing for diagnosis and management of individuals with suspected and confirmed cases of pid. adults and children with suspected or confirmed pid (n= ), autoinflammatory disease (n= ) and hereditary angioedema (hae, n= ) were recruited to the melbourne genomics immunology flagship. whole-exome sequencing (wes) was performed, with targeted gene analysis. variant curation and reporting was performed according to the american council of medical genetics guidelines. overall, wes was diagnostic in % ( / ), confirming a preexisting diagnosis in % ( / ), and offering a new or more specific diagnosis in % ( / ). variants of uncertain significance were identified in a further patients ( %) in genes known to be associated with their clinical diagnosis, that warrant further functional validation. in the hae group, diagnosis was confirmed in only patients ( %), suggesting that wes may not be the appropriate technique for genetic diagnosis in this condition. a higher diagnostic rate was observed for autoinflammatory disorders ( %; / ) compared to pid ( %; / ). of those who received a diagnosis, immediate changes to patient management and treatment occurred for / patients ( %), including hsct for and specific targeted therapy for ( %) individuals. we have demonstrated the utility of wes for accurate diagnosis of complex immune diseases, with the potential to change diagnoses, guide therapeutic intervention and provide opportunities for genetic counseling. further longitudinal analysis will determine clinical outcomes and health economic implications of genomic sequencing for diagnosis and management of immunological conditions in australia. at birth he had neonatal asphyxia and cerebral palsy. at years old he had presented involuntary movements, left paresis, bilateral horizontal nystagmus. at years of age, he had a right nasal obstruction. it was resected by otorhinolist and informed by biopsy: inflammatory polyp and chronic sinusitis. he has had pneumonias, sinusitis and diarrhea. at the age of years, the ataxia telangiectasia was confirmed by sequencing with pcr ( exons, bp) of the atm gene: transition g> a, nucleotide position , codon , affecting splicing. alpha fetoprotein - . u/ml. brain mri, say cerebellar atrophy. he had igg mg / dl - mg / dl, iga . mg / dl, < mg / dl, igm mg / dl - mg / dl, ige . -< iu / ml. subclasses of igg: igg : . g / dl, igg : . gr/dl, low. igg anti hepatitis b , . no seroconversion. hiv negative tcd + lymphocytes: , %, = cells / mm , ltcd +: , % = , cel / mm , ltcd +: , % = , cells / mm , cd / cd : . . for all of the above, common variable immunodeficiency was diagnosed. he receives human immunoglobulin. at , i arrived at this hospital due to fever, respiratory distress and lymphadenopathy in the neck. ct showed ganglionic conglomerate on right side neck. lymph node biopsy: strong tumors with cd and bcl , weak and moderate diffuse pax- ; negativity with cd , cd and cd , and a cell proliferation index with ki of %, diagnosis: diffuse large b cell lymphoma. treated with rituximab and chemotherapy. lymphoma completely remitted. conclusion: the association ataxia telangiectasia and lymphoma is frequent. by contrast, cvid and ataxia telangiectasia are extraordinarily rare. introduction: chronic granulomatous disease (cgd) is a primary immunodeficiency wherein affected patients are susceptible recurrent infections caused by specific bacteria and fungi as a result of defective nadph activity. additionally, inflammatory complications involving the bowel and lungs can cause significant morbidity. currently the only proven permanent cure to cgd remains hematopoietic stem cell transplant. case: a -year-old patient was diagnosed in infancy with x-linked cgd. at age yrs he received a nonmyeloablative peripheral blood stem cell transplant from his / non-carrier sister as previously reported (nejm : , ) . conditioning was cyclophosphamide ( mg/kg) on d- and d- ; daily fludarabine ( mg/m ) on d- through d- ; antithymocyte globulin at mg/kg on d- through d- . posttransplant immunosuppression consisted of cyclosporine on d- through d+ . he received . x cd + peripheral blood stem cells which were t-cell depleted with x add back of cd + cells on day . after days of neutropenia (anc < ) there were signs of engraftment. per protocol, he received donor peripheral-blood lymphocytes containing . x cd + cells/kg on d+ after transplantation. since donor t cells constituted less than percent of his circulating cd + t cells and he had no graft versus-host disease, he received . ¬ cd + cells/kg on d+ . after the discontinuation of cyclosporine, he received a total of three donor-lymphocyte infusions ( . ¬ cd + cells/kg) at -day intervals achieving % t cell and myeloid engraftment at months post-transplant with no acute nor chronic gvhd. at last follow-up years post-transplant ( ) he had % and % lymphoid and myeloid peripheral chimerisms, respectively. the patient and family declined further periodic followup. then, in october he presented with malaise, cough and fevers. he eventually was found to have a large consolidation and a bal grew burkholderia cepacia. his dhr showed % activity and peripheral blood myeloid and lymphoid chimerisms were % and %, respectively. discussion: this late graft failure following peripheral blood transplant occurred following a conditioning regimen which is not the current standard for transplant in cgd. in the case series in which this patients transplant is reported (nejm ), another patients myeloid chimerism fell to % by years post-transplant, remaining stable at that level of chimerism without any serious infections over regular periodic follow up to the present time. current regimens typically include busulfan to enhance engraftment and prevent graft failure. this case reinforces the need for prolonged monitoring of primary immune deficiency patients after transplantation. introduction: with the introduction of severe combined immunodeficiency (scid) newborn screen (nbs) in the state of kansas in , a case of complete digeorge syndrome (dgs) was discovered in an infant born to a diabetic mother with atypical features. this is the first dgs case diagnosed after adding the scid nbs, which emphasizes the need to establish scid nbs in all states. case presentation: the female infant was born via spontaneous vaginal delivery at / weeks to a year old g now p mother. maternal history was significant for chronic hypertension, obesity, insulin dependent type diabetes, anxiety, depression, and scoliosis. the infant was noted to have a left sided abdominal wall defect and hernia, imaging identifying left renal agenesis, and was initially suspicious for vater syndrome. fortunately, the infant's scid nbs revealed low t cell receptor excision circles (trecs). her initial white blood cell count was . with an absolute lymphocyte count of . k/ul. ebv pcr, cmv pcr, and hiv studies were negative. chest imaging discovered absent thymus, abnormal vertebrae with only ribs on the right and ribs on the left, and abnormally formed thoracic vertebrae (t ). echocardiogram detected an atrial septal defect measuring . cm, possible pfo versus secundum asd. endocrinology was consulted for management of labile calcium and phosphorus levels. fish was negative for q . deletion. microarray r evealed a variant of unknown signif icance arr[grch ] p . ( _ )x . sequence analysis of combined and severe immune deficiency genes showed a variant of uncertain significance c. c>a (p.leu met). management and outcome: additional evaluation included: cd ul ( - ul), cd ul ( - ul), cd ul ( - ul), cd ra cells/ul ( - cells/ul), normal cd , and cd / , normal immunoglobulin g level, and normal dihydrorhodamine assay. skeletal survey, ct abdomen and chest, and hla typing were performed in preparation for thymic transplant. discussion: patients with complete dgs, a form of scid found in less than percent of patients with qds, have absent thymus and a t cell count < standard deviations below normal for age (typically < naïve cd + t cells/mm ). in a large series of patients with complete dgs, only percent had an identifiable q . deletion [ ] . infants of a diabetic mother have various genetic and syndromic associations including diabetic embryopathy. [ ] despite the importance of immunological aspects in pregnancy, few studies have reported on the cellular immune modifications of diabetic embryopathy. diabetes during pregnancy may affect the development of the thymus and thus maturation of the immune system in the offspring. [ ] the recent addition of a trec assay to newborn screening can identify such a subset of infants with atypical presentations. scid nbs uses an assay for trecs, a biomarker of t cell development. [ ] [ ] [ ] this initial presentation now places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients, who may present with atypical features. newer genetic and molecular techniques now allow for earlier identification of immune defects in such disorders with life-long clinical concerns. [ ] references: introduction/background: goods syndrome is a rare cause of combined b-and t-cell immunodeficiency occurring in association with a thymoma. affected patents are susceptible to bacterial, fungal, viral, and opportunistic infections. an association with autoimmunity has also been reported. current knowledge of goods syndrome is primarily limited to case reports and small series. objectives: to examine the spectrum of clinical and laboratory features of a major cohort of goods syndrome patients in the us. methods: we conducted a retrospective analysis of patients with goods syndrome in the usidnet registry and the mount sinai hospital (msh) cohort. r e s u l t s : we i d e n t i f i e d p a t i e n t s w i t h t h y m o m a a n d hypogammaglobulinemia (usidnet, n= ; msh, n= ; median age: years; female: %), representing data from patient years. the median age at diagnosis of thymoma and hypogammaglobulinemia were years (range - ), and . years (range - ), respectively. two patients were deceased (at age and years, cause unspecified). all patients had low igg (median mg/dl, range - ). iga and igm were reduced in % and % of patients, respectively. low cd + b cells (median . /mm^ , range - ) were reported in all available records. the absence of cd + b cells was observed up to years postthymectomy. a wide range of additional laboratory abnormalities were identified: low cd + t cells (n= ), low cd + t cells (n= ), low cd / cd ratio (n= ), low nk cells (n= ), and absent peripheral eosinophils (n= ). the most common sites of infections were lower respiratory ( %), upper respiratory ( %), and gastrointestinal ( %). in addition, sepsis ( %), meningoencephalitis ( %), osteomyelitis ( %), and urinary tract infection ( %) were also observed. identifiable infectious agents included: bacteria ( %), virus ( %), fungus ( %), parasites ( %), and protozoa ( %), with opportunistic infections recorded in % of patients. opportunistic infections were significantly associated with absolute cd lymphopenia (p= . , fishers exact test). enterovirus was identified as a previously unreported cause of meningoencephalitis in this population. autoimmune manifestations were reported in % of patients, with a higher prevalence of inflammatory colitis ( %) than previously reported. hashimoto thyroiditis, fibromyositis, and bronchiolitis obliterans organizing pneumonia (n= each) were identified as previously unreported autoimmune/inflammatory conditions in this population. a case of alopecia areata was also observed. additionally, bronchiectasis was recorded in % of patients. all patients were initiated on immunoglobulin replacement, with antibiotics prophylaxis in %, and immunosuppressive medications employed in % of patients post diagnosis of immunodeficiency. conclusion: goods syndrome is a combined immunodeficiency, with a wide range of autoimmunity in a subset of patients. we expanded upon the spectrum of associated infectious and inflammatory complications through a major us cohort. persistent immune dysregulation was observed up to decades post-thymectomy. introduction: primary immunodeficiencies (pids) constitute a large group of rare disorders that affect the immune systems function. some pid patients develop autoimmunity in addition to having increased susceptibility to infections due to their impaired immunity [ ] . ( ) case presentation/ management: a year old caucasian female with history of bipolar disorder, factor v leiden deficiency, anti thrombin deficiency, pulmonary embolism, endometriosis, and seasonal allergies was evaluated for chronic granulomatous disease (cgd) in . the main symptoms were inflammatory breast lesions necessitating surgeries on the right breast, and back, facial, genital, ocular, mouth, and scalp sores. biopsy with cultures of the wounds was positive for corynebacterium, coagulase-negative staphylococcus, enterococcus, bacteroides species, and provatella. neutrophil oxidative burst was ordered by the infectious disease specialist and showed normal and abnormal neutrophil populations, a finding consistent with cgd carrier. patient was started on interferon gamma- b after failing multiple courses of antibiotics. her symptoms were well controlled on interferon gamma- b mcg/ . ml sq every other day, trimethoprim mg tab ( tabs in am and tab in pm), cefixime mg once daily, and topical mupirocin as needed except for her recurrent genital ulcers. cgd can be rarely associated with oral ulcers however there is a limited literature describing associated genital ulcers. according to the international study group diagnostic criteria published in ( ), the patient was diagnosed by a rheumatologist as having behcets disease (bd). there are no pathognomonic laboratory tests in bd; as a result, the diagnosis is made clinically. patient failed a trial of colchicine and was later started on cyclosporine, which resulted in decrease of her mouth and genital ulcers. discussion: bd is a rare disease mostly seen along the silk road. the prevalence has been reported as . (usa) to (in a single village, northern turkey) for inhabitants. ( ) cgd is a primary immunodeficiency caused by defects in any of the five subunits of the nadph oxidase complex responsible for the respiratory burst in phagocytic leukocytes. patients with cgd are at increased risk of life-threatening infections with catalase-positive bacteria and fungi, and inflammatory complications such as cgd colitis. ( ) reports of cgd female carriers with discoid lupus erythematosus, photosensitivity rashes, and other autoimmune phenomena have been published [ , ] ( ) . to the best of our knowledge, this is the first case to report bd in an affected cgd carrier. the treatment of inflammatory disease in patients with cgd poses a difficult balance between therapeutic immunosuppression and the increased risk of severe infection. ( ) . high dose intravenous immunoglobulin, and targeted therapies such as ctla -ig for t cell mediated pathologies, rituximab for b-cell mediated pathologies, and anti-tnf for ibd, may be preferable over the broad immunosuppressive activity of glucocorticoids. in addition, emerging evidence suggests that hematopoietic stem cell transplantation has indication for cases that have been difficult to control using immunosuppression. ( ) given all that, our case emphasizes the need to maintain suspicion for autoimmune disorders / immune dysregulation in patients with pid. introduction: cd -ligand deficiency is an x-linked combined immunodeficiency, characterized by susceptibility to infection, often with associated neutropenia, malignancy, and autoimmunity. central nervous system (cns) manifestations are less commonly reported than respiratory or gastrointestinal complications, but are most often attributed to infection. herein we describe a challenging case of gradual onset episodic memory loss, confusion, and unilateral hemiplegia in a young male with cd ligand deficiency. case presentation: the patient is a -year-old male with cd -ligand deficiency on immunoglobulin replacement therapy presenting with recurrent, episodic altered mental status (ams) and gradual neurocognitive decline. initial neurologic symptoms began at age years, and included fever, nausea, and eyelid fluttering. initial comprehensive infectious workup at this time, including blood and urine cultures, lyme antibody, serum pcr for hsv, cmv, ebv, respiratory viral pcr including atypical viruses, csf studies including culture, lyme eia, pcrs for enterov i r u s , v z v, e b v, c m v, h s v / w e r e u n r e v e a l i n g . electroencephalogram (eeg) and mri displayed generalized slowing and global atrophy, respectively. definitive diagnosis was not made. the patient continued to decline with worsening developmental delay and memory loss. one year later, at age years, he had a recurrent episode of ams with repeat negative infectious workup including blood and urine cultures, respiratory virus pcr including atypical viruses, csf culture including acid fast bacillus and fungi, cryptococcal antigen, viral encephalitis panel by pcr, and serum pcr for ebv and hhv- . eeg at this time showed left hemispheric epileptogenic potential, consistent with seizure activity. his presentation, at age years, was notable for right-sided hemiplegia with facial numbness, dysarthria, nausea, and fever. he was found to have anello virus on pcr of csf, abnormal left temporal region on eeg, and global atrophy with stable, diffuse generalized volume loss on mri. he was diagnosed with occult anello virus-induced encephalitis with hemiplegic migraine and discharged on valproate. discussion: here we present the first reported case of anello virus detected by pcr in a cd -ligand deficient male with neurocognitive manifestations, attributed primarily to hemiplegic migraine. given the anello virus prevalence and relatively avirulent character, it is presumed to be unlikely culprit for encephalitis; however, the significance of this finding is as yet unknown. this case highlights diagnostic challenges in immunodeficiency: infection may go undetected by standard diagnostic techniques; however, the significance of infections identified with advanced techniques may not yet be understood. background: henoch-shönlein purpura (hsp) is an iga-mediated small vessel vasculitis that presents with a tetrad of abdominal pain, arthritis, glomerulonephritis, and purpura. hsp is typically a selflimiting disease of childhood following a viral illness. there is no universal treatment for patients with chronic or recurrent hsp. we report a chronic refractory case of hsp that was successfully treated with a tumor necrosis factor inhibitor (tnfi), etanercept. etanercept functions as recombinant protein that consists of a tnf-alpha receptor ligand-binding region that links to the fc portion of human igg. it is currently approved for use in diseases: juvenile rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis. tnfi are categorized into two broad categories, recombinant receptors (etanercept) and neutralizing antibodies (ex. infliximab and adalimumab). there have been prior case reports of hsp associated with tnfi agents during the treatment of other autoimmune conditions in the adult population. to our knowledge, there have been prior etanercept related hsp reports, one report associated with adalimumab, and one with infliximab. however, there has been no prior report of etanercept use successfully treating chronic refractory hsp. case presentation: a -year-old native american male with year history of chronic hsp, hla-b positive, and enthesitis related arthritis who was initially treated with steroids, sulfasalazine and methotrexate for symptoms of joint pain and purpura. his iga level was mg/dl prior to therapy. despite treatment for one month of steroids, eight months of sulfasalazine exclusively and eight months of methotrexate and sulfasalazine, he continued to have persistent purpura on bilateral extremities without improvement. he was subsequently initiated on etanercept mg weekly and methotrexate was discontinued. approximately one month later, his rash significantly improved. his rash and joint pain recurs when he misses a dose of etanercept. punch biopsies were taken months after initiation of etanercept. the biopsies of a lesion from his left arm showed early leukocytoclastic vasculitis and from his left leg showed weak granular deposition of iga, igm and c within vessel walls. there is controversy whether this is a true iga vasculitis. however, we believe that his clinical presentation and the deposition of iga and c within blood vessel walls seen on biopsy correlates with chronic henoch-shönlein purpura. conclusion: there is no standard treatment of chronic hsp, but there are reports of benefit with nsaid and corticosteroids. per our literature review, there are no prior reports of etanercept use in the treatment of chronic hsp. tnf inhibitor, etanercept should be considered as a treatment for chronic refractory hsp in the pediatric population as it has showed rapid resolution of purpura in this case report. further studies of etanercept in the treatment of chronic hsp should be conducted given the controversial literature of anti-tnf ab induced hsp during the treatment of other autoimmune diseases. although clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers of hfa mutation, we present cases that appear to suggest an increased risk non allergic rhino-sinusitis. case report: we present a year old gentleman with perennial colored rhinorrhea, with facial pressure and tenderness, constant post nasal drip, dry cough and bilateral congestion that had been going on for the past several years. he also had a frequent urge to clear his throat and had frequent episodes of sore throat despite having no history of gerd or lpr. he reported to have multiple sinus infections every year that would progress to pneumonia and eventually require long courses of oral antibiotics. all started in his s intensified in the recent past. he had other siblings; one died in his s due to liver complications of hh and had a carrier sister and brother with a hx of sino nasal problems exactly similar to the patients. his exam was remarkable for bilateral narrowed nasal passages and moderate edema of the mucosa. his rhinolaryngoscopy showed significant edema and purulent drainage, most notably from bilateral middle meati. his skin test was negative. his cbc showed a wbc count of . /ml with % eosinophils and his immunoglobulin panel showed an iga of mg/dl, igg of mg/dl and ige of mg/dl. patient was placed on alkalol sinus rinses and azelastine nasal spray, which he reported to work pretty well. he left for costa rica and is expected to return back with his siblings to a&i clinic in the coming months. discussion: hh is one of the most common inherited disorders in people of northern european descent with an incidence of : and carrier rate of : .. most affected hh patients are homozygous for the mutation designated c y at the hfe gene located at the th chromosome. unlike hereditary hemochromatosis, clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers. hh patients are at risk for a number of infections with bacteria whose virulence is increased in the presence of excess tissue iron. hh is also a risk factor for acute fulminant frs . here the mechanism is postulated to be due to quantitative or qualitative neutrophil defects as this condition is mostly seen in patients with dm, aplastic anemia, and can happen in patients undergoing antineoplastic chemotherapy. no known increased susceptibility for infections through either mechanism is postulated for patients with the heterozygous carrier state. here we present hh carrier patients who present with recurrent rhinosinusitis with no allergen sensitizations and normal ige levels. since most fungal immunity is at the tissue level and is cytokine driven, it can be speculated that increased tissue levels of iron might interfere with mechanisms of innate immunity. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md background: dedicator of cytokinesis (dock ) mutations are associated with a combined immunodeficiency disorder marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. almost all cases can be diagnosed by documentation of the loss of dock protein expression. methods: we describe a -year-old male with a diagnosis of pre-b cell acute lymphoblastic leukemia (all) followed by epstein-barr virus (ebv) associated diffuse large b cell lymphoma (dlbcl). compound heterozygous mutations in dock were documented following the completion of whole exome sequencing (wes). the pathogenicity of the variants was assessed. flow cytometric quantification of intracellular dock protein was completed. dock protein function was assessed by evaluating the morphology of patient lymphocytes when migrating in a d collagen matrix. results: a concern for a primary immunodeficiency was raised due to a history of recurrent otitis media which began at months of age. by years of age, numerous warts were noted on his fingers; however, they were transient for a duration of only years. no atopic features were appreciated. at years of age, a diagnosis of pre-b cell all was made. during all therapy, infectious complications were severe including an intestinal perforation, osteomyelitis, and sepsis. at years of age, still in an ongoing remission from his all, an incidental finding of a lung nodule led to a diagnosis of ebv-associated dlbcl. during therapy, however, infectious complications were again severe including a soft tissue infection and sepsis. wes was performed and compound heterozygous mutations in dock (c. _ del and c. - g>c) were documented. flow cytometric quantification of intracellular dock protein was normal when compared to a normal control. nevertheless, additional functional assessment of dock protein was completed. when migrating through a d collagen matrix, % of the patient lymphocytes studied demonstrated abnormal elongation (stretch ratio > defined by length/width) compared with % of lymphocytes from a normal control. he is being evaluated for hematopoietic stem cell transplant. conclusion: autosomal recessive mutations in dock are a rare cause of a combined immunodeficiency marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. here, pre-b cell all followed by the development of a subsequent malignant neoplasm (ebv-associated dlbcl) led to the discovery of dock deficiency. hence, as our case underscores, for rare instances of high clinical suspicion despite normal dock protein expression, additional functional testing is crucial to make a definitive diagnosis and plan treatment. understanding the spectrum of dock mutants and their phenotypes will improve our understanding of dock deficiency. background: autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) is a rare primary immunodeficiency caused by heterozygous loss-of-function mutations in the signal transducer and activator of transcription (stat ) gene. ad-hies classically characterized by recurrent cold staphylococcal abscesses, pneumonia, eczema, and an elevation of ige level. other additional clinical manifestations of hies have been recognized including skeletal dysplasia (scoliosis, pathologic fractures, delayed dental deciduation), pneumatoceles, coronary-artery aneurysms, brain lesions, and chiari malformations. objective: to describe a unique case of abdominal abscesses in a patient with ad-hies. method: a -year-old female with known ad-hies (c. c>t (p.arg trp)) and a complicated history of early pneumococcal pneumonia and meningococcemia resulting in bilateral amputation below the knees along with loss of several digits, presented for evaluation of skin infection. she had a history of recurrent staphylococcal skin abscesses and presented with inability to use her prostheses due to pain from inflammation around her amputation sites. she underwent imaging and was found to have bilateral extremity abscesses with an associated osteomyelitis of her l tibia (which was found to be mrsa after incision and drainage). while receiving intravenous antibiotics for her osteomyelitis, she developed intractable abdominal pain. imaging showed a thick-walled, multi-septated, paranephric abscess as well as several smaller abscesses scattered throughout her abdomen. she underwent multiple drain placements and drainage of retroperitoneal fluid collections via interventional radiology (ir). purulent fluid from the abdominal abscess drainage grew mrsa. the patient continued to have re-accumulation of abscesses despite multiple drainages. repeat imaging noted increased paranephric abscesses which were not communicating with drains. given lack of response to several ir-placed abdominal drains and to weeks of intravenous antibiotics, she had an open surgical washout with minimal improvement. hospital course was further complicated by development of a left lower lung lobe consolidation and sub-segmental pulmonary embolism necessitating treatment with heparin. finally, after several weeks of escalating antimicrobial therapy and with additional drain placements, the retroperitoneal abscesses started to recede. repeat abdominal imaging several months later while asymptomatic revealed slow but continuing resolution of the abscesses. conclusion: the present case raises awareness of an unusual location for infection in a patient with ad-hies. although the majority of complications of ad-hies are sinopulmonary and skin infections, recalcitrant intra-abdominal abscesses should be considered in the differential of infections in hies. introduction/background: the recent epidemiologic studies have revealed that primary immunodeficiencies (pids) are more common than previously thought. however, there are very few data on epidemiology of pids in korea. objectives: we attempted to estimate the pid epidemiology and disease burden in korea and provide the background information for pid registry for future. methods: to review the previously reported scientific studies, pubmed, koreanmed, google scholar were searched. any studies on pids reported in scientific journal (korean or international) from january to november were searched. both korean and english reports were searched. diagnosis for pid was categorized from group i to group xi according to iuis phenotypic classification. study period was divided into two periods: period from to and period from to , because there was a multicenter study to estimate pid epidemiology from to . in addition, the number of pid patients and the cost for care were estimated among patients who requested reimbursement to health insurance review and assessment service (hira) korea for one year in . results: a total of pid patients were identified in reports. one hundred and ninety-nine patients ( reports) and patients ( reports) were found in period and period , respectively. the pids were reported in patients for immunodeficiencies affecting cellular and humoral immunity, patients for combined immunodeficiency with associated or syndromic features, patients for predominantly antibody deficiencies, patients for diseases of immune dysregulation, patients for congenital defects of phagocyte, patient for defects in intrinsic and innate immunity, patients for auto-inflammatory disorders, patients for complement deficiencies, and none for phenocopies of pid. from hira reimbursement data, the number of pid patients were for combined immunodeficiency, for predominantly antibody deficiency, for common variable immunodeficiency, for functional defect of neutrophils, for immunodeficiency associated with other major defects, for other immunodeficiencies. a total of , pid patients were treated for , days and $ , , was reimbursed in . conclusions: we performed a systematic review on published studies for pid in medical journals and national open data system of hira to estimate the pid disease burden for the first time in korea. to obtain more information on true pid epidemiology and disease burden in korea, a national multicenter study for pid registry is required in the future. micro-thrombocytopenia is one of the most serious challenges for wiskott-aldrich syndrome (was) and x-linked thrombocytopenia (xlt) patients. thrombocytopenia leads to severe, potentially life-threatening, bleeding episodes, which require frequent transfusions and account for % of deaths in patients experiencing was mutations. the gold standard treatment for was patients is hematopoietic stem cell transplantation (hsct) from an hla-identical donor but more recently a number of gene therapy (gt) trials in europe and usa showed promising results. in particular, it has been shown that was patients receiving lentiviral mediated gt, consisting of autologous cd + cells transduced with lentiviral vector encoding the human was gene under the control of the endogenous promoter, in combination with a reduced intensity conditioning regimen, have a significant increase in platelet (plt) counts. even though plt counts do not reach normal levels, treated patients decreased the severity and frequency of bleedings. here, in a cohort of xlt and was patients, fifteen treated with gt, the plt phenotype and function were analyzed by electron microscopy, flow cytometry and proteomic profile. the aim of the project is to assess the presence of plt defects in was untreated patients and the impact of gt treatment on the correction of plt behavior. we demonstrate that plts of untreated was patients have reduced size and abnormal ultrastructure along with hyperactivated phenotype at steady state, showing increased expression of cd p, activated iib integrin and cd l; conversely, activation response to agonist and aggregation capacity are both decreased. analyzing plt samples isolated from treated patients, we found that gt restores plt size and ultrastructure very early after treatment and reduces the hyperactivated phenotype proportionally to was protein (wasp) expression and follow-up length. plts isolated from gt treated patients showed a normal activation response to agonists and restored aggregation capacity in out of analysed patients. by proteomics, various protein pathways were found downregulated in untreated plt samples, mainly involving cytoskeletal-rearrangement proteins, integrins, signal transduction molecules, vesicles-transport proteins; additionally, decreased metabolic capacity were observed. these results are in line with the functional defects observed in plts in terms of activation and aggregation. conversely, the expression of protein-pathways found downregulated in untreated patients is comparable to healthy controls in gt-treated plt samples, reflecting the amelioration of plt phenotype and function. overall, our study highlights the coexistence of multiple defects in the activation and aggregation responses occurring in was patient plts in absence of wasp. gt was able to normalize the plt proteomic profile followed by consequent restoration of plt ultrastructure and phenotype, suggesting gt is responsible for the observed reduction of bleeding episodes in treated patients. introduction: pik cd is an autosomal dominant genetic disorder of the immune system that results in persistent activation of pi k. signaling through pi k is essential for immune cell regulation of metabolism, migration, proliferation and differentiation, leading patient to present with lymphadenopathy, immunodeficiency and senescent t cells. the mutated protein causes t cells to over activate and mature too quickly leading to their death, this over activation also blocks the maturation of b cells. case presentation: a -year-old female with a childhood history of failure to thrive, asthma, chronic rhinitis and common variable immunodeficiency on intravenous immunoglobulin replacement, was seen in immunology clinic to establish care. she reported frequent episodes of pneumonia and bronchitis in her childhood. her family history was significant for family members with leukopenia, but no diagnosed immunodeficiency. patient had son who did not report symptoms concerning for immunodeficiency. physical exam was within normal limits with no lymphadenopathy. laboratory examinations exhibited normal iga ( mg/dl), igg ( mg/dl), and igm ( mg/dl). while flow cytometry showed normal absolute cd ( - cells/ul), cd ( cells/ul), nk cells ( cells/ul), cd ( cells/ul), cd ra ( cells/ul), cd ro ( cells/ul), cd ( cells/ul), and hla-dr ( cells/ul), nonswitched memory cells ( cell/ul) and class-switched memory cells: ( cells/ul). ( - cells/ul). vaccine response was not pursued as patient had been on ivig. genetic testing was pursued, and revealed a mutation in pik cd gene, specifically a mutation in the c. g>a; p.val met variant (rs ). this mutation though seen in databases, is not currently reported in medical literature as associated with this condition. based on these, ct chest was ordered to screen for bronchiectasis, adenopathy and lymphoma. ct showed no cardiopulmonary disease or adenopathy, but did show an incidental adrenal mass which is now being worked up. while the pattern of inheritance of this mutation is autosomal dominant, her son is asymptomatic and testing of her son has not been pursued, though it was advised for her cousins given history of leukopenia. patient has continued on igg replacement therapy. conclusion: recent publication by the clinical immunology society suggests consideration for next generation sequencing when it can affect future family planning or it has treatment and prognostic implications. this case highlights all aspects of the importance of genetic testing as part of the diagnosis of cvid, since it can affect progeny, it offers the possibility of treatment with immune modulating agents and has implications on screening, since patients are at increased risk for malignancies. background: abnormal v(d) j recombination activity in patients with mutations in the recombination-activating genes and (rag / ) results in markedly reduced usage of distal vand j genes at the t cell receptor alpha (tra) locus. mucosa-associated invariant t (mait) cells express a semi-invariant t cell receptor containing the distal trav - gene. mait cells can be identified by flow cytometry using a mab directed against valpha . , which recognizes the product of the trav - gene. by performing high throughput sequencing (hts) of tra rearrangements and flow cytometry, we have confirmed lack of t cells using distal valpha genes in patients with known rag mutations. we now report that flow cytometry with mab against valpha . successfully identified rag deficiency in two patients with an atypical presentation. methods: tra rearrangements were analyzed by hts using gdna from sorted t cell subsets from rag-mutated patients and healthy donors. distal valpha usage was measured in whole blood by flow cytometric analysis with an anti-valpha . antibody. rag mutations were detected by sanger sequencing. patients were enrolled in niaid protocol -i- . results: hts of tra rearrangements revealed lack of distal trav and traj gene usage in patients with rag / mutations. the presence of circulating mait cells in controls and patients with known rag / mutations and various clinical phenotypes was analyzed by flow cytometry using mab against valpha . . we found a virtual lack of valpha . expression in rag mutated patients (< . %) compared to controls ( - %) . we used the valpha . assay to test two patients with unknown immunodeficiency manifesting as skin granulomas and autoimmune cytopenia, and found nearly absent expression ( . % and . %). targeted sequencing of rag / revealed that both patients were compound heterozygous for rag mutations: p.r h/p.c y and p.r w/p.r q, respectively. conclusions: patients with mutations in rag / demonstrate a skewing of their tcralpha repertoire. the reduction in recombinase activity in these patients does not allow for rearrangements of the most distal valpha segments. rapid identification of patients lacking valpha . + t cells by flow cytometry may prompt sanger sequencing and identification of rag / mutations in a matter of days. this assay represents a simple but powerful tool to reduce the cost and time associated with other analysis methods. acknowledgements: supported by dir/niaid/nih. director, centro de inmunología clínica dra.bezrodnik y equipo introduction: the fate of effector t cells is strongly dependent on the expression of bcl- or blimp- , which are inhibited reciprocally through a complex signaling pathway. several studies have shown that bcl- is a key transcription factor for differentiation towards the follicular helper t cells (tfh) lineage able to collaborate with b lymphocytes (bl). on the contrary, the transcription factor blimp- is highly expressed in t lymphocytes th , th and treg, thus regulating the differentiation towards tfh. materials and methods: whole fresh blood and peripheral mononuclear cells from a patient with homozygous mutation in stat b were analysed by flow cytometry. analysis of ctfh (cd +cd ra-cxcr +), ctfh (cxcr +), ctfh (ccr +), ctfh (cxcr -ccr -), naïve bl (lb igm+igd+cd -), memory (mbl) (lb igm+ igd-cd +), switched (mbl-sw) (igd-igm-) and plasmablast (pbc) (cd +cd ++) cells was performed. immunoglobulins were measured in serum. results: the patient with stat b deficiency showed increased values of ctfh ( %) (healthy donors p -p : , - , %) that presented an activated phenotype (icos+ and pd- +) with a skewed to a th profile (ccr +), consistent with her hipergammaglobulinemia and the marked and sustained increase in the switched mbl and pbc subpopulations in peripheral blood over the years. discusion: this immunological phenotype described in the patient with stat b deficiency could explain in part the pathophysiology of the autoimmune disorders. this patient (as well as the other two patients with mutations in stat b previously described by our group), have had chronic hypergammaglobulinemia, autoantibodies and consequently autoimmune processes (psoriasis, hypothyroidism, eczema, alopecia and celiac disease, among others). we believe that the link between this clinical symptomatology and the molecular defect relies in the fact that the absence of stat b promotes a greater expression of bcl- , which generates a bias towards the production of ctfh cells, that give rise to a greater activation of lb, generation of lbm and plasma cells (dysregulation in the cg), events that manifest as hypergammaglobulinemia and autoimmunity. in summary, we provide promising evidence of the mechanisms that lead to autoimmunity in this type of patients that could also be a consequence of the defect in the regulation of gc, highlighting the crucial role of stat b in the humoral immune response and maintenance of the tolerance of the immune system. background/introduction: the term primary immunodeficiencies (pid) encompasses a phenotypically and genetically diverse group of conditions. genetic testing for these conditions can guide treatment, reduce morbidity and mortality, allow for genetic counseling, and identification of additional at-risk family members. however, this testing can be complicated by a number of factors, including pseudogenes, high homology, methodology limitations, and the heterogeneous nature of pids. methods: mayo clinic laboratories launched their first set of nine pid next generation sequencing (ngs) tests approximately one year ago. these tests include one single gene assay for gata deficiency and eight targeted next generation sequencing panels for: atypical hemolytic uremic syndrome (ahus), autoinflammatory disorders, b-cell disorders, monogenic irritable bowel disease (ibd), phagocytic defects, severe combined immunodeficiencies (scid), and severe or cyclic neutropenia. herein we summarize our first year of experience with these ngs tests, with a focus on the eight targeted panel tests. results: from march through november we performed testing for cases. our highest volume of tests was for the ahus panel ( / cases, %). a variant was reported in / cases ( . %). these variants included variants of uncertain significance, likely pathogenic variants and pathogenic variants. the indication with the highest percentage of cases where a variant was reported was scid ( / cases, . %). the number of cases that were considered solved, where the genotype likely explains the patients phenotype, varied widely by indication. twenty cases were found to have a pathogenic or likely pathogenic variant or variants; however / cases were heterozygotes for an autosomal recessive condition and were not considered solved cases. the panel with the highest percentage of solved cases is our scid panel ( / cases, . %). conversely, we have yet to solve an autoinflammatory, irritable bowel disease, or telomere defects case; however % of cases in each of those three panels have had a variant of uncertain significance reported. we hypothesize that one of the reasons for the low detection rate for these three panels is inappropriate test orders. we are also actively looking for ways to update all panels to increase detection rates and clinical utility, for example expanding the gene list of our ibd panel, including large deletion/duplication detection, and including ncf , a difficult gene to capture by ngs, on the phagocytic panel. finally, we present the molecular findings from a number of interesting cases that were solved using our targeted ngs panels. conclusions: the launch of our pid ngs tests in march of has allowed us to aid patients by confirming diagnoses and providing molecular diagnoses that will enable more accurate genetic counseling and risk assessment. we have also uncovered areas for improvement, both on the clinical side: provider education is important to enable better identification of patients who can benefit from molecular genetic testing for pids, and on the laboratory side: introduction of more expanded panels and additional methodologies. the progressive decrease of red blood cells, platelets or neutrophils via a self-directed immune process is jointly termed as autoimmune cytopenias. while autoimmune cytopenias, including autoimmune hemolytic anemia (aiha), immune thrombocytopenic purpura (itp), and autoimmune neutropenia (an), are a common presentation of autoimmunity in the general population, they are particularly frequent and can appear as the first sign in patients with primary immunodeficiencies (pids). possible causes of cytopenia in pids comprise mainly immune dysregulation, bone marrow failure (bmf) and myelodysplasia. our goal is to investigate possible immune mediated mechanisms underlying chronic cytopenia in children in order to achieve an early diagnosis and consequently offer timely and appropriate therapy. we selected patients affected by chronic cytopenia, evaluated with immunophenotyping by flow-cytometry; data were subjected to multivariate analysis by principal component analysis (pca). next generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. among the patients, were affected by bone marrow failure, of which were diagnosed with fanconi anemia and severe congenital neutropenia; were affected by immune-mediated cytopenia and by idiopathic cytopenia. the immunephenotyping showed a typical pattern of cd t cell subpopulations expression in patients compared with healthy donors with an increase of naïve t cells and a reduction of central memory (cm) and effector memory (em) t cells levels. we observed a decrease in total b cells, b switched and b memory cells and an increase in cd low cells. pca showed an overlap between groups, however it revealed a peculiar trend of some single patient, suggesting the pathway involved in immune defect. preliminary results from ngs studies revealed genetic variations in genes previously associated with pids in out of patients investigated. in particular we identify one patient with a mutation in fas, one with a mutation in aire and one with a mutation in ikaros. concerning the remaining patients further studies are ongoing to validate the pathogenicity of the genetic variations. pca is a very effective tool to analyze several parameters at the same time, highlighting patients whose phenotype shows the main peculiarities. the presence of specific lymphocyte subpopulation patterns can be important indicators of immune-mediated cytopenias and helpful signs of specific pids that should promptly be investigated with genetic analysis. the rapid of discovery of novel, monogenic primary immunodeficiencies has been made possible by the broad availability of clinical whole exome sequencing (wes). however, clinical wes has major shortcomings that should be understood by practicing immunologists. focusing on the iuis list of~ monogenic primary immunodeficiency genes, we show here limitations in coverage that could significantly impact clinical interpretation. on the agilent whole exome capture kit, the most common wes platform, there are a number of genes with exons that are poorly covered. specifically, there are at least genes with less than % exonic coverage, with less than % coverage and with less than % coverage (e.g. ikbkb, ncf , taci, unc b and tbx ). beyond this challenging technical issue, there are more subtle issues as well. these include the presence of pseudogenes in at least of our genes (e.g. ak , c qbp, cd , cftr, cr , msn, ncf , ncstn, ikbkg, nhp , pms , pten, rnaseh c, rps, sbds and was), which can make accurate sequencing very challenging. finally, there are many known causative intronic (e.g. btk, ctla- , wasp) and copy number variant mutations (e.g. rag and xiap) as well as large deletions (e.g. dock ) that we cannot expect to be optimally covered using wes. this list of genes requires consideration even with a negative exome and may require additional approaches including whole genome sequencing, sanger sequencing, cnv arrays and/or long-read ngs sequencing. wes is a powerful genomic diagnostic tool, but to avoid missing key diagnostic insights using these alternative approaches may be critical when certain genes are in the differential diagnosis. going forward, as pid phenotypes continue to broaden, these issues remain fundamentally important even if these genes are not obviously implicated in a given clinical phenotype. more physicians are utilizing targeted genetic panels to reach a definitive diagnosis for their patients with immunodeficiency. however, this increase in testing also has led to the discovery of many more variants of uncertain significance (vus) in the genes tested. these findings can often leave the patient and the physician with more questions than answers. we present a patient with recurrent infections found to have multiple variants of uncertain significance in several genes associated with primary immunodeficiency. a -year-old female who was diagnosed with crohns disease at age after intestinal perforation and jejunal resection experienced two discrete episodes of epstein barr virus (ebv) meningoencephalitis and septic shock. the first episode was diagnosed when patient had fever and altered mental status and occurred prior to her crohns disease diagnosis and the second episode was complicated with altered mental status, disseminated intravascular coagulation (dic) and hypotension requiring picu admission. aside from these two major infections, the family denied any other infections requiring antibiotics in the last years and reported a remote history of repeated streptococcal pharyngitis that have not recurred. immunology was consulted at the time of the second episode of meningoencephalitis and work up was mainly unremarkable with normal immunoglobulins, adequate vaccine response to hib, tetanus, diphtheria, rubella, measles and pneumococcus ( out of protective titers). she had normal t cell numbers with slightly decreased natural killer numbers for age. neutrophil studies showed normal dihydrorhodamine (dhr) analysis, glucose- -phosphate dehydrogenase levels and myeloperoxidase (mpo) stain. commercial testing of her toll like receptors ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) showed normal function. invitae primary immunodeficiency panel demonstrated a heterozygous variant in nod (c . c>t; p.arg trp) as well as heterozygous variants of uncertain significance in il r (c. g>t; p.ser ile) and tlr (c. c>g; p.leu val). the patients nod variant is known to be associated with an increased risk for crohns disease. even with our patients presentation with recurrent severe viral infections and ibd, it is not immediately clear how these genetic results explain the pathology. innate immune defects probably contribute to her presentation and it is currently unclear if and how the combination of multiple genetic variants has left her immunologically vulnerable. we use this case to demonstrate that even when genetic testing does not elucidate a clearcut diagnosis of primary immunodeficiency, it can still provide helpful insight into a patients underlying immune phenotype. introduction: xiap deficiency is a rare primary immune deficiency characterized by hemophagocytic lymphohistiocytosis, recurrent fever and inflammatory syndromes, inflammatory bowel disease, hypogammaglobulinemia, recurrent infections, and other manifestations. loss of xiap results in abnormal tnf receptor signaling and nlrp inflammasome actvity which leads to dysregulated production of il- beta and il- . we hypothesized that suppressing the nlrp inflammasome with either targeted deletion or pharmacologic inhibition would suppress abnormal production and secretion of inflammatory il- beta and il- . methods: bone marrow derived macrophages (bmdms) from control, xiap-deficient, and xiap and nlrp double knock-out mice were derived with week of culture in l -cell conditioned media. bmdms were stimulated with a variety of tlr agonists or tnf-alpha, with or without a variety of inhibitors including the nlrp inhibitor mcc , the cathepsin b inhibitor ca- , and quercetin, which is a natural flavonoid (antioxidant) found in many fruits and vegetables, and available as a nutritional supplement. il- beta, il- , and tnf-alpha were measured in supernatants by elisa, and cell death was evaluated by flow cytometry using pi exclusion. results: as expected, bmdms from xiap deficient mice had markedly increased tlr-agonist-or tnf-alpha-induced il- beta production compared to normal bmdms. genetic deletion of nlrp and the pretreatment of cells with the nlrp inhibitor mcc greatly reduced abnormal il- beta production; residual production of il- beta could be inhibited by caspase- inhibition. pre-treatment of cells with the cathepsin b inhibitor ca- also decreased cytokine production but was toxic at higher concentrations. quercetin reliably abrogated il- beta, and also il- . quercetin was found to inhibit priming of the nlrp inflammasome (decreased upregulation of pro-il beta and nlrp ) and also decreased tnf-alpha secretion following tlr agonist stimulation. conclusion: quercetin suppresses the nlrp inflammasome and may be a promising therapeutic option for patients with xiap deficiency. it prevents il- beta and il- secretion. it is a particularly appealing option given that it is a naturally occurring antioxidant, has a great safety profile, and is readily available as a nutritional supplement. human studies are needed. recently, single cell rna sequencing (scrnaseq) analysis in mice has disclosed an unexpected complexity of thymic stromal cells, and medullary thymic epithelial cells (mtecs) in particular. however, the developmental origin, hierarchy, and function of these subpopulations remain ill-defined. moreover, although cortical tecs (ctecs) are thought to represent a more homogeneous population, their characterization has been largely restricted to the adult thymus. we have previously shown that impaired lymphostromal cross-talk in the thymus of patients with combined immunodeficiency (and of corresponding mouse models) is associated with abnormalities of thymic architecture and tec maturation. here, we sought to compare tec distribution and gene expression in wild-type (wt) and in mice carrying rag hypomorphic mutations observed in patients with combined immune deficiency and immune dysregulation. methods: multi-color flow cytometry and scrnaseq were used to analyze composition and distribution of ctec and mtec subpopulations in wt and rag mutant mice at various weeks of age (niaid animal protocol: lcim- e). results: we observed that rag mutant mice have an excess of ctecs, and that their mtec compartment is predominantly represented by cells with high levels of mhc class ii (mhc-ii) expression, recapitulating the phenotype of neonatal wt thymi. while mhc-iihi mtecs are thought to represent a minor fraction of mtecs in adult wt mice and include mature aire+ cells, a relative abundance of mhc-iihi mtecs is observed also at neonatal age, where they are thought to represent immature mtecs. to define more precisely tec maturation, we performed scrnaseq on sorted cd -epcam+ cells, and identified and distinct clusters of tecs in wt and rag mutant mice, respectively. a large proportion of cells in rag mutant mice could be ascribed to the ctec compartment, confirming our previous flow cytometry and histopathology results. furthermore, scrnaseq analysis also disclosed a different distribution of mtec subsets in wt and rag mutant mice. to address the hypothesis that this difference in ctec and mtec abundance and subset distribution may reflect different maturation stages in tec development in wt and rag mutant mice, we will perform lineage tracing and transplantation experiments, and we will also extend tec scrnaseq analysis to wt and mutant mice of embryonic and neonatal age. in parallel, to evaluate the contribution of thymocyte maturation in shaping the stromal populations, scrnaseq will be performed on thymocytes. conclusions: we have further refined the complexity of tecs, and shown that impaired development of t cells in combined immune deficiency (as exemplified by rag mutant mice) has profound effects on the composition and maturation of tecs and may thus contribute to abnormalities of immune tolerance that are often associated with these conditions. the advent of next-generation sequencing (ngs), with the development of whole-exome sequencing (wes) in particular, has allowed the identification of unknown genetic lesions for many diseases and the implementation of specific therapeutic strategies. primary immunodeficiencies (pids) are a group of rare diseases which have benefited from ngs, with the discovery and molecular characterization of previously genetically undefined diseases and the identification of novel molecules involved in the regulation of the immune system. pids are often associated with autoimmune disease due to the dysregulation of the immune system as a whole. the clinical phenotypes are heterogeneous and often overlapping. while a monogenic cause of disease has been identified in a most subsets of patients, the recent application of whole-genome sequencing has found that a polygenic cause is likely. our aim is to investigate the genetic background of patients with immunedysregulations and autoimmunity and to evaluate the possible pathogenicity of the identified gene variants through extensive functional studies. we select patients with sign of immunedysregulation and autoimmunity, extended immunophenotyping and next-generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. in six of them we identify a single gene as responsible of the clinical feature. in particular, we identify two patients with gain of function mutation in stat , one patient with a mutation in ctla , one patient with an activating pik cd mutation, one with a rag mutation and one with a fas mutation. in most of them variants in multiple genes have been detected. interestingly, we find that some genes are recurrently mutated in more then one patient such as was, dock , casp , casp , nfatc and fcgr a. further studies are ongoing to validate the effect of the variations identified. our results strongly suggest that the old hypothesis, based on a single gene mutation as a cause of illness, should be revised in favor of the concept that "is the sum that causes the effect" and that a different point of view on pids now seems inevitable. physician, omni allergy, immunology, and asthma introduction/background: immunoglobulin replacement therapy (igrt) may be optimized to reduce the severity and incidence of infections and potentially delay or abrogate the development of pulmonary complications of primary immune deficiencies. pulmonary complications including bronchiectasis are common in common variable immune deficiency (cvid) and contribute significantly to morbidity and mortality in these patients. it remains unclear whether continued obstructive bronchial changes are a result of repeated respiratory infections, associated inflammation and immune dysregulation, or simply lung-damage that is irreversible by the time therapy is initiated. it has also been suggested that under-treatment in addition to the diagnostic delay may contribute to the development of bronchiectasis in patients with pid. lower serum igg levels with any given dose of immunoglobulin replacement therapy have been demonstrated in patients with bronchiectasis compared to those pid patients without this complication. in addition, earlier studies have shown that greater doses of ig ( mg/kg/ month) may reduce the frequency and duration of infections and help prevent or slow progression of chronic lung disease. objective: to evaluate the prevalence of bronchiectasis in a cohort of patients with a diagnosis of cvid and identify associated ig dosing patterns and clinical outcomes. methods: data were analyzed from the ideal (immunoglobulin, diagnosis, evaluation, and key learnings) patient registry. this is a prospective, longitudinal registry study of patients receiving ig replacement therapy in the home or ambulatory infusion suite with one national home infusion provider. nursing and pharmacy standard of care forms were collected, and dose, infection rate, and prevalence of bronchiectasis were evaluated in patients with a diagnosis of cvid (icd- codes: d . , d . ) results: there were patients in the registry with cvid, ( . %) of which bronchiectasis was also observed. seventy-nine percent (n= ) of the study population was female, and % (n= ) of the cases of bronchiectasis were observed in females. the mean age of the patients with concurrent bronchiectasis was ± . at start of care compared to ± . in those without this observed bronchial obstruction. most bronchiectasis patients (n= ) received igrt subcutaneously every week with a mean dose of . ± . mg/kg/wk. the mean dose of ig in the remaining patients receiving ig intravenously was . ± . mg/kg/month. the average annual rate of infection in ivig and scig patients with bronchiectasis was . ± . and . ± . , respectively, however many were serious bacterial infections. at time of analysis, of the bronchiectasis patients remained active in the registry and had withdrawn. reasons for withdrawal included stopping igrt due to the following: patient decision (n= ), physician decision (n= ) insurance change (n= ), and patient expired (n= ). conclusions: there were documented cases of bronchiectasis in our cohort of cvid registry patients, and dosing patterns aligned with standard doses despite the presence of bronchial obstruction. further studies are necessary to assess evolution of lung damage with respect to ig dosing in patients with cvid. background: activated phosphoinositide -kinase syndrome type (apds ) is a combined immunodeficiency resulting from gain-offunction (gof) mutations in pik cd, the gene encoding the catalytic subunit of phosphoinositide -kinase (pi k). this form of pid is characterized by recurrent respiratory tract infections, susceptibility to herpes virus infections, impaired antibody responses, lymphoproliferation and autoimmunity. previous studies showed that patients with apds have b cell defects that contribute to the clinical phenotype. furthermore, these patients display t cell abnormalities, including increased numbers of memory t cells and t follicular helper cells (tfh), reduction of naïve t cells and impaired t regulatory cell (treg) function. whether these t cell abnormalities are also associated with perturbations of t cell repertoire in unknown. objective: we aimed to investigate the effects of increased pi k signaling on the t-cell repertoire of patients with apds. methods: high throughput sequencing was used to study composition and diversity of t-cell receptor (tra) and t-cell receptor (trb) repertoire in sorted treg, tfh, conventional cd + (tconv), and cd + t cells from patients with pik cd gof mutations and healthy controls. results: treg cells of patients with apds show restriction of tra and trb repertoire diversity, and increased clonality. no repertoire restriction was detected in tfh, tconv, and cd + t cells from the same patients. however, the trb repertoire of treg and cd + cells was enriched for the presence of hydrophobic amino acids in position and of the cdr , a biomarker of self-reactivity. conclusion: these data demonstrate that the t-cell repertoire of patients with apds is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. furthermore, our result support the notion that the pi k pathway is a key regulator of treg cell development and homeostasis in humans. j clin immunol ( ) (suppl ):s -s s ( ), iii. predominantly antibody deficiencies ( ), i. immunodeficiencies affecting cellular and humoral immunity ( ), vii. auto-inflammatory disorders ( ), ix. phenocopies of pid ( ) . two non related cases of ataxia-telangiectasia and one case of schimke syndrome (smarcal compound heterozygous mutation) were diagnosed in the last year. we observed a wide range of age (we evaluate adult and pediatric population) with a male:female ratio close to : immunodeficiency, immune dysregulation, and systemic autoimmunity. clinical diagnosis of these disorders is complicated by overlapping phenotypes. in april , a -gene next generation sequencing (ngs) panel inclusive of copy number variation analysis was launched by a commercial laboratory to facilitate clinical diagnosis of primary immunodeficiency (pid), monogenic autoimmunity and autoinflammatory disorders. we assessed the outcomes of genetic testing utilizing this panel on a cohort of pediatric patients with immunohematologic phenotypes evaluated at our tertiary care center during an -month period ( / / - / / ). eligible subjects were evaluated by at least two of three providers from a multidisciplinary pediatric hematology-immunology team, including a hematology physician, immunology physician and a geneticist or genetic counselor. twenty-three patients met inclusion criteria; ( %) were caucasian, ( %) were male with an average age of . years. the two most common phenotypic diagnoses included cytopenias, single-or multilineage (leukopenia, neutropenia, anemia, thrombocytopenia) primarily attributed to autoimmune causes or hypogammaglobulinemia. five ( %) were given a definitive genetic diagnosis as a result of panel testing, though in two of these cases, the causative mutations were listed as variants of uncertain significance (vus). diagnoses included common variable immunodeficiency due to a pathogenic variant in nfkb , stat multiorgan autoimmunity due to gain-of-function mutation, and familial cold autoinflammatory syndrome due to a pathogenic mutation in nlrp . biallelic dnmt b vus were found in a patient whose phenotype and further laboratory studies (including karyotype) were consistent with immunodeficiency-centromeric instability, facial anomalies syndrome. further, a stat vus was identified in a patient with multiorgan autoimmunity and his father with hypothyroidism; studies from an outside research laboratory were consistent with gain-of-function with this variant (private communication). an additional three patients had vus identified that were suspected to be related to their phenotype, prompting eligibility for research studies. four ( %) patients had increased risk alleles in nod , conferring an increased risk of crohns disease. three ( %) patients had pathogenic or likely pathogenic carrier findings warranting genetic counseling. in addition, vus (an average of per patient) thought to be unrelated to phenotype were identified, necessitating further investigation and counseling. the use of an ngs panel in a cohort of pediatric patients with immunohematologic disorders led to a definitive diagnosis in % of previously undiagnosed patients and prompted further research investigation in several more. genetic testing also led to the identification of clinically significant carrier findings, risk alleles and vus unrelated to phenotype, necessitating genetic counseling. our experience illustrates the value of genetic testing for diagnosis of immunohematologic disorders, and the importance of multidisciplinary care, including genetic counseling, for the proper evaluation and management of these patients. background: allogeneic hematopoietic cell transplantation (allohct) is curative for primary immune deficiencies (pid). however, many patients lack a fully-matched unaffected sibling, or may have an unknown underlying genetic defect, rendering it undesirable to use related donors. many pid patients have significant comorbidities at the time they are referred to allohct, precluding the use of myeloablative conditioning. the use of alternative donors with reduced-intensity conditioning (ric) has historically led to increased rates of graft failure, graft-versus-host disease (gvhd), and transplant-related mortality (trm). posttransplantation cyclophosphamide (ptcy) as gvhd prophylaxis immunomodulates the graft through the preferential sparing of regulatory t cells and hematopoietic stem cells from its cytotoxic effects, thus allowing for robust donor engraftment that overcomes the hla barrier while effectively preventing severe acute and chronic gvhd. we report the outcomes of two institutions using a ric allohct regimen with alternative donors and ptcy in patients with pid. design: we transplanted pid patients (table ) using alternative donors and ric, either serotherapy-free (n= ) or alemtuzumab-based (n= ). all patients received ptcy for gvhd prophylaxis on days + and + , either alone (n= ), or combined with mycophenolate mofetil and either sirolimus (n= ) or tacrolimus (n= ). donors included haploidentical family members (n= ), matched unrelated (n= ), and mismatched unrelated (n= ). stem cell source was t cell-replete bone marrow (n= ) or peripheral blood stem cells (n= ). results: the median follow-up is months (range . - years). at months, overall survival is %, and event-free survival (defined as alive without graft failure) is %. the median days of neutrophil and platelet engraftment are (range - ) and (range - ), respectively. there were patients who developed acute gvhd, grade (n= ) or grade (n= ), and there were no cases of grade or agvhd. seven of eight patients treated with systemic corticosteroids responded, and one was corticosteroid-dependent, then responded to second-line therapy. one patient developed skin-only chronic gvhd, which responded to corticosteroids and puva light therapy. five patients developed graft failure, either primary (n= ) or secondary (n= ), and four were successfully re-transplanted and remain engrafted. one patient with secondary graft failure had autologous recovery and has not required a second allohct given some durable infection control gained during initial engraftment. there were three deaths prior to day due to infection, and one death at . years secondary to presumed overdose. in ongoing follow-up of engrafted survivors (n= ), evidence of phenotype reversal has been demonstrated in all patients, with complete or ongoing resolution of some or all of their underlying disease manifestations, including infection, transfusion-dependence, autoimmunity, malignancy, and/or immune dysregulation. discussion: we have observed high rates of engraftment, low rates and severity of acute and chronic gvhd, and low trm in patients with pid transplanted using alternative donors, ric, and ptcy-based gvhd prophylaxis. ric allohct with ptcy shows promise for curing pid, and its use minimizes toxicity and widely expands the donor pool, thus allowing us to offer this curative therapy to many more patients with pid. chronic granulomatous disease (cgd) is a primary immune disorder that involves mutations in the nicotinamide adenine dinucleotides (nadph) oxidase complex (deffert, cachat, & krause, ) . two-third of cgd cases are caused by loss-of-function mutations in the cybb gene that encodes the gp pox subunit of the nadph. the increased in patients' life expectancy thanks to progress in diagnosis and management has underlined the burden of inflammatory manifestations occurring independently of infectious agents (dunogue et al., ; marciano et al., ) . cgd patients develop inflammatory granulomatous disorders, notably colitis, as a consequence of a dysregulated inflammasome activation. the treatment of inflammatory manifestations remains challenging, as it can be associated with an increased risk of infections. thus, understanding the pathophysiological mechanism of auto-inflammation in cgd could help improve the therapeutic arsenal for the management of these manifestations. to reveal the precise pathophysiological mechanism of auto-inflammation in cgd, we have developed a cellular model that reproduces the cgd phenotype in phagocytic cell. through crispr-cas gene-editing we generated a thp- c e l l l i n e h a r b o r i n g t h e p r e v i o u s l y d e s c r i b e d mu t a t i o n c. _ delccginsggt (p.tyr ter) in the cybb gene responsible for gp phox knock-out by early termination of translation. this cell line recapitulates the phenotype of cgd phagocytes: (i) decreased h o production (ii) and enhanced inflammatory responses after pma stimulation as evidenced by increased il- , il- and tnfa secretion levels (kuijpers & lutter, ) . these features were rescued by complementation through lentiviral transduction of a wild type cybb gene. this new model will help us to investigate the auto-inflammation reported in cgd patients and also to propose new therapeutic targets of inflammatory manifestations in this disorder. interleukin- (il- ) driven responses. children with irak- deficiency are predisposed to recurrent and invasive infections secondary to streptococcus pneumoniae, staphylococcus aureus and other pyogenic bacteria with high mortality rates in early childhood. the frequency and severity of infections is thought to decrease with age due to the acquisition of humoral immunity and immunologic memory, however due to the rarity of the disease, the natural history of this condition beyond early childhood is not well described. objectives: we present three unrelated irak- deficient patients with persistent chronic rhinosinusitis with nasal polyposis that developed in childhood. cases: patient is a y/o male with compound heterozygous mutations in irak (p.g afs* /c. - g>t) with a history of recurrent s. pneumoniae osteomyelitis (left hip at age and left knee at age ) and c. septicum sepsis at age following acute bowel perforation. additionally, he experienced recurrent aom during infancy and recurrent uti since age . despite prophylactic antibiotics and ivig, he has had recurrent polymicrobial (mrsa, s. pneumoniae, h. influenzae, p. aeruginosa, a. fumigatus) rhinosinusitis with nasal polyposis since age refractory to medical management requiring surgical intervention and prolonged courses of iv antibiotics. patient is an y/o female with homozygous deletions (exons - ) in irak with a history of ruptured appendicitis complicated by pseudomonas abscess and bacteremia at age , culturenegative sepsis with septic arthritis and osteomyelitis of the right leg at age , and septic shock secondary to mssa bacteremia complicated by rhabdomyolysis and dic at age . she has a history of chronic rhinosinusitis, and despite ivig and prophylactic antibiotics, she developed polymicrobial (h. influenzae, b. fragilis) rhinosinusitis with associated nasal polyposis pending surgical management. patient is a y/o female with homozygous mutations in irak (q x/q x on exon ) with a history of s. pneumoniae meningitis at months, m. catarrhalis epiglottitis and neck cellulitis at months, rsv bronchiolitis at months, enterococcus bacteremia at months, s. pneumoniae sepsis at age and streptococcus lymphadenitis at age . despite ivig and prophylactic antibiotics, she developed recurrent polymicrobial (h. influenzae, b. fragilis, mssa, v. cholera, p. aeruginosa, a. fumigatus) rhinosinusitis refractory to medical management requiring surgical intervention and iv antibiotics. conclusions: in our centers experience, irak- deficient patients continue to suffer from infectious complications, most prominently recurrent polymicrobial sinus infections beyond early childhood. the consistent presence of sinonasal polyps in these children is unusual, as it is not typically found in uncomplicated pediatric chronic rhinosinusitis. these infections have occurred despite antimicrobial prophylaxis and ivig, highlighting the role of irak- in sinopulmonary epithelium. additionally, the infectious organisms identified in our patient cohort are not commonly associated with irak- deficiency. further study of chronic rhinosinusitis and nasal polyposis in a larger cohort of irak- deficient patients and other innate immunodeficiencies may help identify pathways for targeted treatment of these patients. introduction: chronic granulomatous disease (cgd) is an inherited phagocytic defect associated with inability to clear catalase positive organisms. infections in patients with cgd are severe and recalcitrant. commonest infections are pulmonary followed by soft tissue infections and suppurative lymphadenitis. osteomyelitis is an uncommon infection in patients with cgd. it poses several diagnostic and therapeutic challenge. we herein report our experience of osteomyelitis in cgd over the last years. material and methods: review of records was carried out to describe the profile of osteomyelitis in cohort of patients with cgd at pediatric immunodeficiency clinic, advanced pediatrics centre, postgraduate institute of medical education and research, chandigarh, india. the diagnosis of cgd was based on nitroblue tetrazolium dye reduction test (nbt) and dihydrorhodamine reduction (dhr) assay. results: of the patients with cgd, ( . %) had osteomyelitis ( males and females; age range - years). most patients had their first episode of serious infection in early childhood (mean age: . years). stimulation index (si) of dhr assay ranged from to . . mutational analysis was done in / patients ( x-linked; autosomal recessive). site of involvement was variable ribs- ; vertebrae- ; radius- ; skull- ; tibia- . aspergillus fumigatus was the most common isolate ( %; / ); others had aspergillus flavus, aspergillus terreus and serratia marcescens each. all patients with rib osteomyelitis had concurrent pneumonia, and fungus was isolated in all of them (aspergillus fumigatus- , aspergillus flavus- , zygomyces spp.- ). antifungals (intravenous amphotericin b) were given for a duration of - weeks and were followed by oral voriconazole in therapeutic doses for to months in majority of them. debridement and resection of ribs was required in one patient, while other patients were managed conservatively. out of patients, ( %) succumbed to pneumonia and respiratory failure. conclusion: osteomyelitis in the context of cgd is usually caused by aspergillus spp. involvement of ribs and vertebra usually occurs with the contiguous spread of infection from the lungs. therapy often requires prolonged duration of anti-microbials, and may require surgical debridement in addition to it. a -year-old woman with history of hypogammaglobulinemia and acute liver failure a -year-old woman with a -month history of nausea, vomiting, and abdominal pain was admitted to an outside hospital with new onset of jaundice and anasarca. liver biopsy was thought most consistent with alcoholic steatohepatitis, and she was discharged with counseling on alcohol cessation and medical management of liver disease. she presented to our facility for a second opinion. over the following days, she developed further rise in direct hyperbilirubinemia up to . mg/dl, new coagulopathy with an inr . and hypoalbuminemia to . mg/ dl in the absence of ongoing alcohol consumption. liver sonography revealed course echotexture and patent vessels. pcrs directed against multiple hepatotropic viruses were negative and copper studies were normal. due to a history of moderate alcohol consumption, she was started on high-dose corticosteroids due to a presumptive diagnosis of alcoholic hepatitis. additional history raised concern for a possible primary immunodeficiency, including idiopathic thrombocytopenic purpura at years of age, multiple episodes of sinusitis treated with antibiotics and sinus surgery, one episode of suspected bacterial pneumonia, and one hospitalization for influenza a during which she developed neutropenia. in her s, she developed refractory genital warts, prompting infectious diseases evaluation. initial immune evaluation had revealed low immunoglobulins (iga < mg/dl, igg mg/dl, igm mg/dl) with very low responses to tetanus and diphtheria, despite a recent booster dose, and b and t cell lymphopenia (cd + cells/μl, cd + cells/μl, cd + v, cd + cells/μl, cd / + cells/μl); antigen and mitogen proliferation were not assessed. intravenous immunoglobulin replacement was initiated but discontinued by the patient due to infusion-related adverse effects, and she was lost to follow up until she presented with liver failure. both parents were deceased from cardiovascular disease in their s and she had no siblings. she had limited knowledge of family history but no known immune diseases. due to suspicion for genetic etiology of immune disorder and liver disease, we performed next-generation sequencing of a panel of over genes implicated in primary immune deficiencies. patient was heterozygous for a nucleotide substation (c. + g>a) within a splice site at the exon /intron boundary of the nfkb gene. during the hospitalization, immunoglobulin replacement and trimethoprim-sulfamethoxazole prophylaxis were initiated. an attempt was made to refer the patient for additional immunological evaluation and transplantation evaluation but unfortunately, she developed worsening liver failure and multiple complications, including extended-spectrum beta-lactamase (esbl)-producing e. coli bacteremia, hypotension requiring vasopressors and extensive bowel ischemia, and died in the hospital. in summary, this case highlights both the risk of diagnostic delay in adult patients presenting with a primary immune deficiency and potential for genetic testing to clarify the diagnosis. while the particular genetic change has not been described, other splice site and predicted loss-offunction mutations have been reported as pathogenic in this gene, which have been implicated in autosomal dominant common variable immunodeficiency. this case further expands on the genetic causes and spectrum of disease associated with changes in the nfkb gene. introduction: malnutrition and micronutrient deficiency are underrecognized causes of acquired immunodeficiency in adults, and may occur even in patients with high body mass index (bmi). methods: a -year-old woman with a medical history significant for one remote urinary tract infection presented to the emergency department after sudden onset of severe right flank pain. the pain was accompanied by urinary frequency and not relieved by ibuprofen; she denied fevers or chills. she was diagnosed with pyelonephritis and discharged on ciprofloxacin, which was later changed to trimethoprim-sulfamethoxazole after her culture grew resistant e. coli. her pain continued despite treatment, prompting her to return to the hospital three days later. upon presentation, she was afebrile with blood pressure of / mmhg and heart rate of bpm. her body mass index was . kg/m^ . her physical exam was otherwise notable for right costovertebral angle tenderness. laboratory studies revealed a leukocyte count of , /ul with % neutrophils; alkaline phosphatase of units/l and albumin of . g/dl, but otherwise normal liver function tests; normal lactic acid; and urinalysis with , wbc/hpf, rbc/hpf, moderate bacteria, and the presence of wbc clumps. ct scan of the abdomen and pelvis demonstrated an obstructing mm right renal stone with hydronephrosis and a right renal abscess contiguous with a right-sided hepatic abscess measuring . x . x . cm. she was treated with ceftriaxone and metronidazole, and underwent imaging-guided drainage of the abscesses. abscess cultures again grew resistant e. coli. she was discharged from the hospital with drains in place and a plan to continue trimethoprim-sulfamethoxazole until definitive management of her nephrolithiasis with ureteroscopy and lithotripsy. discussion: there remained the question of how an ostensibly immunocompetent patient had developed such severe intraabdominal infection with little systemic inflammatory response (e.g. no fever and only mild leukocytosis). a hiv antibody screen was negative. on further interview, she described a lb intentional weight loss over the preceding years, accomplished by dietary restriction to less than calories per day. nutritional assays revealed prealbumin, vitamin c, and vitamin b levels below the threshold of detection. she had low-normal b and b . out of concern for an acquired immunodeficiency resulting from malnutrition with micronutrient deficiency, balanced nutrition was discussed with the patient who agreed to liberalize her diet. background: the past decade has brought dozens of new mendelian disorders of immunity. yet, the genetic contribution(s) to diverse disorders of the immune system remain largely unelucidated. the majority of research participants referred to the national institute of allergy and infectious diseases (niaid) for what may be a mendelian disorder evade molecular diagnosis. making progress in this area requires a coordinated, systematic, and transparent approach to clinical genomics research which leverages the unique environment at the national institutes of health clinical center (nih cc). methods/design: this study is designed to systematically apply exome sequencing and related technologies with clinical grade interpretation and reporting to niaid research participants at the nih cc under a single protocol in order to facilitate research and clinical genetics care across niaid. we are recruiting approximately participants per year from approximately intramural clinical investigators. we generate genomic data, collect standardized phenotyping and report clinical interpretation in the medical record, all while providing linked genetic counseling. results: to date, we consented participants, we sent out samples for exome sequencing and samples underwent copy number variant analysis. we have completed analysis for families ( individuals) and finalized and resulted cases. here we present a case series illustrating some of our findings. case : a year-old female was referred to niaid for neonatal onset multisystem inflammatory disease (nomid). developmental delay and mild intellectual disability were appreciated on clinical evaluation. exome sequencing detected a mosaic novel likely pathogenic variant in nlrp . chromosomal microarray analysis (cma) showed ã mb interstitial deletion of chromosome previously associated with developmental delay and intellectual disability. case : a year-old ukrainian male was referred to niaid for the clinical diagnosis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced). exome sequencing and cma did not detect pathogenic variants in aire, but did find a de novo variant in fam b. defects in fam b are associated with poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis (poiktmp). the clinical features of the patient were consistent with poikmp. case : a -year-old man had a history of brain, liver and kidney nocardiosis, disseminated mac infection, prostate cancer and lymphoma. family history was significant for prostate cancer. exome sequencing showed a heterozygous pathogenic variant in brca , associated with susceptibility to breast-ovarian, male breast, pancreatic and prostate cancer. conclusion: this case series illustrates that multiple diagnoses, unexpected diagnoses, secondary genomic findings, and data sharing helped identify variants in candidate genes. process standardization supports data integrity and efficiency while accommodating the need for investigator flexibility and providing tailored patient care. rationale: activated pi kinase delta syndrome (apds) is a primary immunodeficiency caused by dominant mutations that increase activity of phosphoinositide- -kinase (pi k). the catalytic subunit p is mainly expressed in cells of the hematopoietic system, primarily lymphocytes and myeloid cells, and mutations affect both b-and t-cells. we sought to further evaluate the role of the t-cell receptor (tcr) repertoire in immune dysregulation and the pathogenesis of autoimmunity and lymphoproliferation in patients with apds. methods: we evaluated the tcr repertoire in the peripheral blood in patients with pik cd mutations and compared these to the peripheral tcr repertoire in patients with common variable immunodeficiency (cvid) and healthy controls to investigate the role of the tcr in disease. the tcr repertoire in affected tissue of patients with pik cd mutations was also evaluated (tissue included lymph nodes for both patients, in addition to gastrointestinal tract and lung tissue in one patient). a fixed number of tcrs were subsampled ( , for blood and , for tissue) and diversity was calculated using the gini and shannon indexes. results: using the shannon and gini diversity indexes, the tcr repertoire in patients with pik cd mutations had less diversity/ increased clonality as compared to healthy controls and those with cvid ( figure ). for the two apds patients with biopsy tissue available for analysis, the diversity of the tcrs in tissue was increased as compared to the peripheral blood tcr repertoire ( figure ). conclusions: pi k plays an important role in the development and function of both b-and t-cells. patients with apds were found to have decreased tcr repertoire diversity in the circulating t-cell compartment compared to healthy controls and other cvid patients. the increased tcr diversity in the affected tissues compared to peripheral blood implicates the pi k/akt signaling pathway with t-cell trafficking and tissue immune homeostasis, and suggests this pathway may play a role in the development of inflammatory and lymphoproliferative complications in these patients. gain-of-function mutations in pi kd result in a human primary immunodeficiency, named apds (activated pi k-delta syndrome), characterized by lymphopenia, lymphoproliferation, respiratory infections and inefficient responses to vaccination. however, what promotes these immune disturbances at the cellular and molecular level remains unknown. we have recently published a mouse model that recapitulates major features of this disease and used this model and patient samples to probe how hyperactive pi kd fosters aberrant humoral immunity. we found that mutant pi kd alters the intrinsic function of t and b cells, leading to icos-independent increases in t follicular helper (tfh) and germinal center (gc) b cells, disorganized gcs, and poor class-switched antigen-specific responses to immunization. these phenotypes were associated with increased phosphorylation of akt and s in t and b cells, and lower threshold of activation, with altered regulation of foxo and bcl family members. moreover, b cells showed enhanced responsiveness and proliferation to both antigens and innate stimuli, accompanied by reduced cell death. strikingly, aberrant responses were accompanied by increased reactivity to gut bacteria, and a broad increase in autoantibodies that were dependent on commensal microbial stimulation, as demonstrated by striking reduction of self-reactivity upon antibiotic treatment in mutant mice. we now have further examined b cell function in these mice and demonstrate that altered foxo plays a major role in disruption of both b and t cell function. we further provide evidence for altered activation of metabolic pathways in b cells, compared to wt cells, that may contribute to the dysregulated b cell reactivity. our findings suggest that proper pi kd regulation is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome. this research was supported in part by the intramural research program of the nih, nhgri and niaid. autoimmune cytopenias are seen in a significant proportion of patients with immunodeficiencies affecting antibody production. previous b-cell maturation studies using fluorescence-activated cell sorting (facs) have associated various phenotypes of primary immunodeficiency diseases affecting antibody production with differing levels of b-cell differentiation. in this study we analyzed the peripheral b-cell compartment of patients with a hypogammaglobulinemia and > % b-cells with and without a history of autoimmune cytopenias. b-cells were isolated from peripheral blood using monoclonal anti-cd and these cells were gated to identify the proportion of memory b cell (cd +cd + ), igm+ memory b (cd +igm+), marginal zone b-cells (igm+ igd+), isotype-switched memory b-cells (cd +igm-igd-) and transitional cells (igmhicd hi). pid patients with a history of aic had decreased proportions of total cd + b-cell ( . % vs . %; p= . ) and igm memory b cells ( . % vs . %; p = . ). conversely, the proportion of marginal zone b-cells was increased in this group ( . % vs . %; p = . ). consistent with previous reporting, the proportion of isotype-switched memory b-cells was significantly lower in the aic group ( . % vs . %; p = . ). statistically significant inter-group difference was not seen within the transitional b-cell subset. our data suggest that maturation arrest of marginal zone (cd +igm+ igd+) b-cells may be implicated in the development of autoimmune cytopenias in humoral immunodeficiency. ( ) submission id# taissa de matos. kasahara , sudhir gupta, md phd student, state university of rio de janeiro and university of californis irvine professor, university of california at irvine, irvine, ca, usa introduction/background: common variable immunodeficiency (cvid) is the most frequent form of primary hypogammaglobulinemia with decreased serum igg and iga levels and variable levels of igm in adults. in addition to decreased serum immunoglobulins, - % of cvid patients present autoimmune manifestations. the mechanisms that lead to a breakdown of selftolerance in cvid are not completely understood. however some differences in b and t cells subsets and autoreactive b and t cells can be detected. elevated expression of surface igd and downregulation of igm receptor are hallmarks of anergic naïve b cells that contain autoreactive receptors in human peripheral blood. moreover, memory b cells that have class switched to igd and present an igd+igm-phenotype are also highly reactive to self-antigens in healthy individuals. the role of these autoreactive naïve and memory b cells in the immunopathogenesis of cvid has not been evaluated. here we investigated the frequency of cd -and cd + b cells expressing igd and igm in peripheral blood of cvid patients. methods: peripheral blood mononuclear cells (pbmc) from cvid patients (n= ) and health subjects (n= ) were separated by ficollhypaque and incubated with anti-human cd -percp, cd -fitc, igd-bv and igm-apc to identify different subsets of b cells by flow cytometry. cd +cd -igd+igm-and cd +cd -igd+ igm+ b cells were sorted, loaded with cfse and cultured with cpg and ant-cd for days to evaluate the proliferation. results: among the compartment of cd -b cells, cvid patients showed an increased frequency of igd+igm+ cells and a lower frequency of igd-igm-cells as compared to control group. no differences were observed in the frequency of igd+igm-cells in cd -b cells between cvid patients and controls. in contrast, in the compartment of cd + b cells, cvid patients showed an increased frequency of igd+igm-, igd+ igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. when the patients were divided in two groups based on autoimmune manifestations, the group with autoimmune disease showed an increased frequency of igd+igm+ and igd-igm+ cells in cd -b cells when compared to the control groups. both patient groups showed an increased frequency of igd+igm-, igd+igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. regarding the proliferation, naïve b cells from cvid patients showed a reduced proliferative capacity in response to in vitro stimulation as compared with naïve b cells from health subjects. conclusion: our results suggest that the increase of cd +igd+igm-b cells can be related to the susceptibility of autoimmunity in cvid patients. introduction: immunoglobulin g -related disease (igg -rd) is a group of immune-mediated conditions where tissues are affected with dense lymphoplasmacytic infiltrations with a predominance of igg -positive plasma cells and storiform fibrosis, usually in the setting of elevated serum concentrations of igg . common presentations include autoimmune pancreatitis, sclerosing cholangitis, retroperitoneal fibrosis, salivary gland disease, and orbital disease, among others. symptoms of asthma or allergy are present in approximately percent of patients and they typically exhibit a good initial therapeutic response to glucocorticoids. case presentation: a -year-old female with a history of gastroparesis, cutaneous lupus erythematosus and suspected autoimmune pancreatitis was referred to allergy/immunology clinic for evaluation of elevated igg . she reported a -year history of recurrent abdominal pain attributed to recurrent pancreatitis based on previous mild lipase elevations. prior endoscopic ultrasound (eus) of the pancreas revealed edema. there was concern for gallstone pancreatitis but ercp followed by cholecystectomy, biliary and pancreatic sphincterotomy had no change in her symptoms. in , she was noted to have a positive ana and high serum igg , per patient (values from osh records could not be obtained). symptoms improved with a course of steroids, hence suspicion for autoimmune pancreatitis. in she developed a rash on her arms and face. biopsies of the affected areas revealed cutaneous lupus erythematosus on the arms and a basal cell carcinoma on the face, which was excised. ana was only : at that time. at the visit, she complained of severe allergic rhinitis, joint pains, as well as a malar rash, which responded to intermittent courses of prednisone by prior providers. laboratories obtained at initial visit were significant for thrombocytopenia ( thou/cu mm), positive lupus anticoagulant ( sec) and elevated igg ( mg/dl; normal range - mg/dl). c , c , c q, ana, anti-double stranded dna, anti-smith antibodies, antiphospholipid panel, upep and spep were all unremarkable. ct chest and abdomen were also normal. given the patient's history of cutaneous lupus erythematosus, plaquenil was started as a steroid sparing agent. eus of the pancreas with possible biopsy was ordered in an attempt to obtain a histopathologic diagnosis of igg -rd. conclusion: this case exhibits the association between elevated igg , pancreatitis of unknown origin, allergic rhinitis, and cutaneous lupus erythematosus, highlighting the value of identifying a pathologic connection between seemingly unrelated disorders in patients with elevated igg , as they may be manifestations of igg -rd. in order to make the diagnosis, histopathologic findings showcasing lymphoplasmacytic tissue infiltration consisting mainly of igg -positive plasma cells and small lymphocytes is essential. the majority of patients respond to glucocorticoids, and while the duration of response is variable, most patients flare during or after glucocorticoids are tapered, as noted in this patient. rituximab has been shown to be effective in some patients and will be considered in this patient if symptoms persist. ( ) submission id# rationale: pnp deficiency is an autosomal recessive disorder due to defective purine metabolism leading to severe combined immunodeficiency (scid) and neurological deterioration. newborn screening utilizing t-cell receptor excision circle (trec) assay can detect affected patients before complications arise. herein, we describe an infant initially identified by newborn screening with pnp deficiency and congenital cmv, a previously unreported presentation. methods: cmv quantitative pcr (qpcr) was performed by nebraska medicine, pnp enzyme activity by duke and genetic sequencing by invitae. results: a small for gestational age (sga) male infant was reported to have an abnormal trec assay on day of life (dol) . he was hospitalized for further evaluation. initial studies revealed profound lymphopenia, normal lymphocyte proliferation to mitogens and no evidence of maternal engraftment. additionally on dol , he had cmv viremia and viruria; thus with sga, failed unilateral hearing screen and head ultrasound with bilateral parenchymal calcifications, congenital cmv was suspected. pnp enzyme activity was abnormal. cmv treatment was initiated with ganciclovir on dol . foscarnet was added on dol . cmv qpcr levels decreased below the limit of detection by dol . genetic testing found a pathogenic homozygous mutation in pnp (c. - g>a). the infant has a / hla-matched, unaffected, cmv positive sibling and will proceed to hematopoietic stem cell transplantation. conclusions: to our knowledge, this is the first reported case of pnp deficiency identified through newborn screening. this novel case of congenital cmv and pnp deficiency highlights the importance of cmv screening and need for treatment strategies for congenital cmv in scid. despite a dramatic increase in the use of next generation sequencing over the last decade, the majority of the more than million identified human genomic variants do not have well-established clinical implications. progress is being made on this complex challenge through multiple approaches, including data sharing. to maximize our understanding of genomic data, platforms that enable effective and responsible data-sharing are essential. this means that genotypic and phenotypic data must be findable, accessible, interoperable, and reusable under conditions that are ethical and transparent. to highlight innovations in data-sharing and their potential to advance discovery, we present three data-sharing mechanisms. for each platform, we will present a case highlighting its key functionality and discuss opportunities and challenges that may arise as each platform is scaled up. ( .) genomic research integration system (gris) is a collaborationengendering web application that facilitates the identification of genetic variants associated with rare immunological disorders. users can access integrated and standardized phenotypic and genomic data that is analyzable within the platform. gris enables systematic and automated capturing, and links patient data from disconnected systems and paperbased records. standardized annotations allow for the comparison of data from different clinical studies. the main goal of this tool is discoverability of other affected individuals enrolled in separate protocols within the niaid intramural research program. this internal database was used to find a second family with a rare variant in a candidate gene. ( .) the genomic ascertainment cohort (tgac) is a resource that aims to improve our understanding of the phenotypic consequence of genetic variation by providing access to aggregate, de-identified genomic data from large nih intramural and related cohorts. participants have provided informed consent to be re-contacted for additional phenotyping in the future. the main goal of this tool is to enable further study of the clinical consequence of variants in a large, unbiased cohort of patients ascertained for many indications. this database was used to investigate findings in participants with previously published pathogenic variants in genes associated with primary immune deficiency based on medical record review. ( .) clingen is dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for precision medicine and research. through the sharing of genetic and health data, clingen seeks to answer whether a given gene is associated with a disease (clinical validity)?; whether a given variant is causative (pathogenicity)?; and whether the information is actionable (clinical utility)? this resource is meant to convene disease-and gene-specific expert groups to curate the medical literature on mendelian disease to better define gene-disease and variant-disease relationships using many lines of evidence. this resource was used to clarify clinical validity of disease-gene assertions. together these efforts help create a clinical research ecosystem that maximizes the value of clinical research data and ultimately improves patient care. this research was supported by the intramural research program of the nih, niaid. introduction: according to the population reference bureau, the number of elderly americans, defined as age and older, is projected to more than double from million to million by , rising from % to % of the total population. the impact of immunodeficiency in this important segment of the population remains understudied. methods: the usidnet registry was queried to obtain demographic, clinical data of elderly patients defined as age and older. descriptive analyses were performed on the data. results: participants ( . %) were eligible out of total registry participants. the median age of the cohort was years and predominantly female ( . %) and white ( . %) with a median bmi of . ± . .the majority ( . %) of subjects were living. humoral deficiencies comprised the majority of diagnoses ( . %), with common variable immune deficiency being the most frequent ( . %). of the remaining non-humoral diagnoses, immune dysregulation ( . %) and immunodeficiency with myelodysplasia ( . %) were the most frequent. the majority ( . %) of subjects reported having received immunoglobulin replacement therapy (igrt) at some point, with . % reporting via iv route. of the infections that occurred in this cohort, sinopulmonary infections were the most commonly reported, specifically sinusitis ( . %), pneumonia ( . %), upper respiratory infection ( . %), and otitis media ( . %). in this cohort, autoimmune, cardiovascular, and granulomatous complications were reported . the number of patients with malignancy was , with some patients diagnosed with multiple malignant disorders. of the reported malignancies, the majority ( . %) were solid tumors. conclusions: compared to the age-matched non-immunodeficiency united states population, this cohort had more females . % (usidnet) versus . % (us population) and fewer whites . % (usidnet) vs . % (us population. humoral immunodeficiencies, specifically cvid, were most common diagnoses, similar to other age groups of immunodeficiency patients. majority of these patients have received igrt, with approximately half via iv route. this cohort reported living with a variety of non-infectious complications, including autoimmunity and malignancies. more research which specifically focuses on elderly patients with immunodeficiency is needed. clinical microbiologist and infectious disease physician, university of calgary x-linked agammaglobulinemia (xla) is a primary immunodeficiency caused by mutations in the bruton tyrosine kinase gene which leads to b cell maturation failure and defective antibody production. this puts patients at risk of recurrent sinopulmonary infections, gastrointestinal infections, and recurrent skin infections including infections caused by helicobacter sp. helicobacter sp are gram negative bacilli commonly found in the gastrointestinal tract of various animals. helicobacter sp. have been linked with gastritis most notably helicobacter pylori causing gastric ulcers in humans. helicobacter sp. has been found in rare cases to cause disseminated infections including pyodermic gangrenosum and cellulitis notably in patients with agammaglobulinemia. infections caused by helicobacter bilis are challenging to diagnosis due to difficulties with culturing the pathogen as well as poor guidelines for antimicrobial management. case report: the patient was diagnosed with x-linked agammaglobulinemia at the age of months with a history of recurrent sinusitis and was started on ivig q weeks. despite regular ivig, he developed bronchiectasis. at years of age in , he developed a chronic rash around his left knee resembling erythema nodosum. by , he had developed a left knee effusion associated with left sided calf pain. his knee pain was found to improve during courses of ciprofloxacin to treat recurrent lung infections. given case report data of h. pylori causing erythema nodosum in patients with agammaglobulinemia, he was treated empirically for an h. pylori infections with no improvement. in he was found to have progressive cellulitis with pyomyositis of the left leg. a skin biopsy of a calf nodule was found to be culture negative but s pcr was positive for h. bilis. he was started on treatment with ertapenem and levofloxacin with subsequent resolution of his rash. his left ankle pain progressed and by late and was found to have possible osteomyelitis of the left ankle on mri. in he was found to be bacteremic with h bilis. due to progressive symptoms with significant impact on function and rising inflammatory markers despite months of antimicrobial treatment, doxycycline and flagyl were added leading to clinical improvement and normalization of his inflammatory markers. he was continued on oral doxycycline and flagyl for months for a chronic osteomyelitis. discussion: h. bilis is a slow growing pathogen which is challenging to culture in the laboratory often requiring special agar plates and prolonged incubation. in patients with agammaglobulinemia and associated chronic skin infections or erythema nodosuma, h bilis should be suspected as a possible pathogen. due to challenges with culturing, s pcr or amplification of the s ribosomal subunit should be considered to try to identify the pathogen. there are poorly delineated clinical antimicrobial breakpoints to help guide therapy with minimal evidence. case reports suggest prolonged therapy with aminoglycosides and penicillin. other studies have successfully treated patients with a carbapenem, azithromycin and levofloxacin. in the absence of sensitivity data, prolonged treatment ( months) should be considered with a combination of antimicrobials. patients should be followed closely as recurrent infections are not uncommon. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md introduction: dock deficiency is a combined immunodeficiency characterized by eczema, recurrent sinopulmonary infections, viral skin infections, malignancy and early mortality. in recent years, liver disease and vasculopathy have been increasingly recognized as a complication of dock deficiency. we clinically characterized our cohort of dock deficient patients, with a specific focus on these newly identified areas of disease involvement. methods: chart reviews were performed on patients seen at nih with genetic and clinical diagnosis of dock deficiency. patients were all enrolled on irb approved niaid protocols. results: we identified patients from families with dock deficiency in our nih cohort, ranging in age from - years. of the families, had homozygous mutations. of the patients, food allergy was diagnosed in ( %), eczema in ( %), and asthma in ( %). chronic or recurrent viral skin infections were seen in / ( %). chronic ebv viremia by pcr positivity was seen in / patients ( %); only patients were known to be ebv immune without viremia. cmv viremia was infrequent. sinopulmonary infections were common, with bronchiectasis occurring in / ( %) with available imaging. liver disease was diagnosed in ( %), with having biliary tract abnormalities on imaging and stool positive for cryptosporidia; most patients with cryptosporidia were without diarrhea. the incidence of cryptosporidia is likely under-represented due to more recent availability of sensitive assays for cryptosporidia detection. other liver abnormalities included fatty liver, metastatic disease from malignancy and medication related hepatitis. vasculopathy, predominantly of the aorta and cerebral arteries, was diagnosed in , with patients in the last years being prospectively imaged. autoimmunity was rare ( %) including autoimmune cytopenias and hypothyroidism. of with follow-up are alive ( %) with age range - years. of the living patients, ( %) have had a hsct. causes of deaths include malignancy ( ), infection ( ) , and hsct complications ( ) . long-term follow-up of patients with hsct (up to years) has revealed resolution of the infection susceptibility and eczema, no new cancers, and stabilization of vasculopathy. conclusions: in addition to the well described manifestations of dock deficiency including eczema, allergy, recurrent sinopulmonary infections, skin viral infections and malignancy, our cohort revealed a relatively high incidence of liver disease, frequently associated with stool positivity for cryptosporidia, as well as vasculopathy. both of these clinical manifestations should be considered during preparation for hsct as they may affect management through transplant. autoimmunity has likely been over-estimated in prior descriptions of dock deficiency. long-term follow-up after hsct is needed to determine the prognosis from the vasculopathy, liver disease, and malignancy risk. ( ) submission id# yasuhiro yamazaki , stefano volpi , luigi d. notarangelo introduction/background: extl (exostosin like glycosyltransferase ) is an exostosin family member which initiates heparan sulfate (hs) chain biosynthesis and elongation. we have reported homozygous extl hypomorphic mutation (r w) as a cause of immunoosseous-dysplasia syndrome. fourteen patients who have extl homozygous mutation were reported so far. eight of them manifested t cell lymphopenia, and presented with severe combined immunodeficiency (scid) or omenn syndrome. using patient-derived induced pluripotent stem cells (ipscs) as a model, we have previously reported that extl mutations affect differentiation to thymic epithelial progenitor cells as well as expansion of hematopoietic progenitor cells. consistent with the latter, previous studies have suggested that mutations in other genes involved in hs biosynthesis affect hematopoietic stem cell (hsc) differentiation. however, the exact mechanisms by which extl mutations affect hematopoiesis are not known. objectives: we tried to clarify gene expression difference in hscs derived from wild-type, extl hypomorphic and extl knock-out (ko) human ipscs. methods: the control bj ipsc line was engineered by crispr/cas gene targeting. extl ko ipscs were obtained which carried compound heterozygous extl mutations (c. _ inst; c. _ insgatattt). hsc differentiation was induced using the stemdiff hematopoietic kit (stemcell technologies). bulk rna from each ips cells and each differentiated cd +cd +cd + was analyzed by rna sequencing. results: as compared to control ipscs, patient-derived cells showed slightly lower capacity to generate cd +cd +cd + cells. on the other hand, extl ko cells showed no differentiation into cd + cd +cd + cells. gene set enrichment analysis showed enriched expression of genes involved in hematopoietic progenitor cell differentiation, regulation of hemopoiesis, and positive regulation of hemopoiesis in both control and patient-derived cd +cd +cd + cells compared to parental ipscs. moreover, these gene sets were more abundantly enriched in control than in patient-derived cd +cd +cd + cells. the gene set of response to type i interferon was significantly enriched in control versus patient-derived cd +cd +cd + cells. conclusions: these results confirm that extl plays an important role for hsc homeostasis in human cells. because type interferons play a role in hsc proliferation, the decreased type i interferon signature may account for the reduced number of hscs that we have previously reported upon in vitro differentiation of extl -mutated versus control-derived ipscs. this study was supported by the division of intramural research, niaid, nih, under protocol -i-n . a case of autoinflammatory syndrome with osteoporosis and specific antibody deficiency autoinflammatory syndromes are inherited disorders with an exaggerated inflammatory response with no specific trigger. the clinical phenotypes of variants of autoinflammatory syndromes may overlap. we report a case of a year old male with prior diagnosis of specific antibody deficiency, periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (pfapa) syndrome, arthralgia and moderate atopic dermatitis. he was diagnosed at years of age with specific antibody deficiency based on persistently low pneumococcal titers against repeat immunizations. due to recurrent infections, he was placed on immunoglobulin replacement therapy (igrt) at years of age. igrt was discontinued at years of age due to full resolution in infections and patient demonstrated robust response to immunizations. patient had lifelong history of recurrent fevers (every weeks) associated with pharyngitis and aphthous ulcers consistent with diagnosis of pfapa. as he became older these episodes became less frequent. last episode of fever was over a year ago. the father had similar symptoms of recurrent fevers and oral ulcers as a child but currently remains asymptomatic. paternal grandfather died of kidney disease. patient has been generally in good health until recent year with intermittent abdominal pain, arthralgia and several long bone fractures with no history of prior trauma. a bone density scan revealed osteopenia and osteoporosis with a z score of - . of lumbar spine, - . of left femoral neck, - . of left hip. given history of familial autoinflammatory disease, and antibody deficiency genetic testing was obtained which identified a pathogenic heterozygous variant of taci and mefv c. g>a (p.met lle). taci mutation has been linked to antibody deficiency syndromes. genetic study for family members is pending. the mefv gene is associated with autosomal recessive familial mediterranean fever (fmf) and has been reported in autosomal dominant fmf as well. fmf is characterized by recurrent episodes of fever associated with serositis, arthralgia, and arthritis. patients with fmf have elevation in acute phase reactants during attacks with most returning to normal levels during the episode-free periods. multiple studies have shown that patient with fmf have lower bone mineral density and zscores than the general population. inflammation in fmf is thought to be mediated by several different cytokines (il- , il- , il- , il- , il- , il- , il- and tnf-). these same cytokines play a role in osteoclast activity and bone resorption. it has been suggested chronic inflammation during acute attacks and subclinical inflammation during the disease-free period lead to bone loss and osteoporosis. regular use of colchicine, the main treatment for fmf, may slow down osteoporosis. beside careful monitoring of clinical and laboratory phenotype, genetic evaluation is an important step in distinguishing between overlapping entities and can prevent complication and promote targeted intervention. a year old previously healthy boy was referred for periodic fever/ pfapa and mosquito bite hypersensitivity. eight weeks earlier he developed fever to f, mouth sores and exudative tonsillitis; a rapid strep screen was negative. one week later he developed moderate cervical lymphadenopathy and had a positive ebv early antigen antibody.. one month later he had several severe local reactions to mosquito bites. each manifested - cm of erythema and induration with a + cm bullae which left an ulcer after rupture and healed with a hypopigmented scar. the bites were accompanied by fever to f for days. one febrile episode was treated with low dose prednisolone for presumed pfapa, and the fever resolved within hours. his past history was positive for nasal allergy and mild asthma. his parents are not related: mom is of european-indonesian and dad european-african (creole ancestry. testing prior to this visit showed normal igg, iga and igm, elevated ige ( , u/l) and normal cbc. lymphocyte subsets revealed cd + % ( /mcl), cd + % ( /mcl), cd + % ( / mcl), cd + % ( /mcl), nk cells % ( /mcl). on examination he appeared well with height at th%ile and weight at th%ile. there was no lymphadenopathy, hepatosplenomegaly or inflammed skin lesions; there was a cm round scar on the right plantar surface at the site of a prior mosquito bite. laboratory studies confirmed nk lymphocytosis % ( /mcl) and elevated ige ( , u/l). lymphoproliferation to mitogens, cd /cd , cmvand hsv were normal, but absent to tetanus and candida antigens. ebv antibodies reflected past infection (vca-igg+, vca-igm-, ebna+); quantitative ebv pcr was > , , copies/ml whole blood. nk cytotoxicity and cd a expression were decreased. bone marrow nk analysis suggested conality. the patient was diagnosed with "hypersensitivity to mosquito bites with ebv-associated t-/ nk lymphoproliferation." this disorder represents a subset of chronic active ebv (caebv) that is rarely seen outside of east asia. the lack of organomegaly or lymphadenopathy with hyper-ige and nk lymphocytosis and decreased nk function support the likelihood that nk cells are the target of ebv infection in this patient. this diagnosis may be a precursor to hemophagocytosis, liver necrosis or lymphoma/leukemia, and the only curative treatment is bone marrow transplantation. the patient's sister is a / hla match. she is seropositive for past ebv infection, and she has no history of extreme reactions to mosquito bites. genetic mutations that cause familial hemophagocytic lymphohistiocytosis have not been reported in caebv, and to the best of our knowledge familial cases of this disorder have not been identified. the response to bmt in this patient is pending. introduction/background: a number of case reports have described symptomatic hypogammaglobulinemia following administration of anti-epileptic drugs (aeds), specifically lamotrigine, carbamazepine, and levetiracetam. the mechanism by which symptomatic hypogammaglobulinemia develops is unclear. we evaluated the prevalence and the clinical significance of hypogammaglobulinemia associated with use of these aeds. objectives: our aim was to characterize the prevalence of aed-induced hypogammaglobulinemia, identify specific aeds associated with hypogammaglobulinemia, and characterize the timeline to development of hypogammaglobulinemia after initiation of therapy. methods: a retrospective, multicenter, electronic medical record review spanning years identified patients with hypogammaglobulinemia who were on aed therapy (lamotrigine, carbamazepine, or levetiracetam). patients were excluded if they had a pre-existing primary immunodeficiency (pid), malignancy, protein-losing enteropathy, or significant proteinuria. patients on chronic immunosuppressive therapy, those without laboratory criteria for hypogammaglobulinemia, or those on one of the aeds for less than one month were also excluded. results: of the cases reviewed, patients met our inclusion criteria. the median age was ; % were adults, % were female, and % were white. lamotrigine was implicated in / of the cases, carbamazepine in / , and levetiracetam in / . tetanus and pneumococcal titers were available for / patients. of those patients, / had protective titers to both per report with responses to > % of the serotypes. only one patient reported severe, recurrent infections while the remaining four had little to no symptoms. interestingly, the patient with severe infections did have protective titers. of the five laboratory proven hypogammaglobulinemia patients, one died of an infection, two have continued on the medication due to refractory seizures responsive only to these medications, and two are currently being tapered off of their aed. conclusion: while it appears that aed-induced hypogammaglobulinemia is quite rare, it should be considered in a patient without other secondary causes of hypogammaglobulinemia on aed therapy. many antiepileptics downregulate nfkb signaling suggestive that patients who develop symptomatic hypogammaglobulinemia may have hypomorphic mutations in the nfkb signaling pathway. ( ) submission id# autoimmune lymphoproliferative syndrome (alps) results from defective apoptosis of lymphocytes mediated through the fas/fas ligand (fasl) pathway. the hallmark lab finding is an expansion of t cells that express the alpha/beta t cell receptor, but lack both cd and cd (double negative t cells) in the setting of normal or elevated lymphocyte counts. patients present with chronic, nonmalignant, noninfectious lymphadenopathy or splenomegaly. for definitive diagnosis, patients need to have ( ) a pathogenic mutation in fas, fas ligand or caspase or ( ) a defective fas-induced lymphocyte apoptosis. we describe a probable case of alps with heterozygous mutation in fas c. a>g(p.his arg), a variant that has not been previously reported (his lymphocyte apoptosis assay is pending). unique to this case is the patients castleman disease-like features on pathology. a year-old male referred from hematology clinic presented with an year history of chronic lymphadenopathy, splenomegaly, anemia, and no underlying diagnosis. malignancy had previously been excluded by bone marrow aspirate and biopsy years prior. however, he had a right sided lymph node that had increased in size for the past months. he was otherwise asymptomatic. a lymph node biopsy years prior was reportedly normal. his exam demonstrated significant bilateral lymphadenopathy, greater on right, with an approximately x cm mobile right neck mass. he had splenomegaly palpated cm down and across to midline. he was therefore admitted for excisional lymph node biopsy to evaluate for possible malignancy and labs were sent to evaluate for alps. labs were supportive of alps. he had elevated t cell receptor alpha beta double negative t cells (tcr a/b dntcs) in blood ( . %). b level was elevated (> pg/ml). plasma soluble fasl level was elevated ( pg/ml). interleukin- (il- ) and il- levels were elevated ( and pg/ml respectively). he had multilineage cytopenias: anemia with hgb of . g/dl and neutropenia (absolute neutrophil count of k/ul). he had hypergammaglobulinemia with an igg level of mg/dl. broad infectious work-up was negative, including hiv, quantiferon, cocci, bartonella, toxoplasma, coxiella burnetii, ebv pcr and, cmv igm. lymph node biopsy showed no evidence of malignancy. immunostains and flow cytometry showed the presence of expanded tcr a/b dntcs in the lymph node, consistent with alps. interestingly, lymph node histology showed morphologic features typical of plasma cell variant castleman disease. numerous castlemanlike follicles showed typical regressive changes with onion-skinning morphology. paracortical hyperplasia with sheets of plasma cells was noted. there was negative staining for hhv (a well-known cause of plasma cell variant castleman disease). the diagnosis of idiopathic multicentric hhv -negative castleman disease was excluded by definition in the setting of alps, per evidence-based consensus criteria published in . in addition, our patient did not show any symptoms typically associated with it, such as fever, night sweats, weight loss, weakness or fatigue. should his fas-induced lymphocyte apoptosis be defective (in separate assays), this would confirm his alps-fas diagnosis and we would start the patient on sirolimus. head of immunology unit, children' s hospital ricardo gutierrez introduction: slc a gene encodes the proto-couple folate transporter (pcft), which supports intestinal folate uptake, and participates in folate transport into the central nervous system. slc a mutations cause pcft defects, resulting in low folate levels in serum and cerebrospinal fluid. hereditary folate malabsorption (hfm) is a rare, autosomal recessive disorder with pcft deficiency resulting in cerebral folate deficiency. most of the patients present megaloblastic anaemia, moderate pancytopenia in the first few months of life, failure to thrive, diarrhoea and/or later onset neurological symptoms including seizures and developmental delay. i m m u n o d e f i c i e n c y i n h f m c a n m a n i f e s t i t s e l f w i t h hypogammaglobulinemia with normal t-cell function. b-cell precursor compartment seems to be particularly vulnerable to folate deficiency in some hfm patients. this immunodeficiency can be restored with specific treatment with folic acid. aim: to describe a female patient with a homozygous pathological variation in the slc a gene. results: a months old girl, born of non-consanguineous parents. she started at months old with diarrhoea due to rotavirus, low weight and bicytopenia with normal bone marrow aspiration. she presented low levels of folic acid . ng/ml (nv . - . ng/ml) at first thought due to secondary to malnutrition. treatment with folic acid supplementation was administrated, improving platelets counts. at months old she presented steatorrhea with severe perianal panniculitis which required surgical treatment. no germs were rescued after a skin biopsy. moreover, she suffered from a respiratory infection due to picornavirus with two episodes of pneumothorax which required intensive care. at that moment ivig treatment was administered due to hypogammaglobulinemia and clinical severity. chronic diarrhoea worsened with bloody depositions. three rectal ulcers were found in the gut biopsy. bowel inflammatory disease was suspected and mesalazine administration was started with weight improvement. furthermore, at months old she presented status epilepticus, with pathological eeg and normal mri; one of them related to a cmv infection, successfully treated. in the immunological evaluation igg and iga were low with normal igm and igd. the protein-antibody response was not evaluated. she presented normal lymphocyte and t cells extended populations, t cells proliferation assay, dhr, treg cells, complement, cd a expression, alpha-fetoprotein, without autoantibodies a molecular panel testing was done by ngs and a homozygous variant in slc a gene was found, causing impaired intestinal folate absorption. conclusion: hfm should be considered in the diagnosis of patients with cytopenias and hypogammaglobulinemia in order to provide specific treatment. hfm has wide clinical manifestations, not only with megaloblastic anaemia and neurological impairment but also with gastrointestinal and skin manifestations. with folate treatment, clinical and immunological defects can be normalized. introduction: multifocal epithelial hyperplasia (meh), or hecks disease, is a rare, benign infection of the mucosa caused by human papilloma virus (hpv). clinically, meh manifests as numerous painless, soft, sessile papules or plaques, and typically occurs in the labial, lingual, and buccal mucosa. meh lesions are usually associated with hpv types and , and seen more commonly in patients of caribbean or central/south american descent. prior studies in adults have shown that tumor necrosis factor alpha (tnf) promotes hpv, and may influence duration of hpv infection. case: we present a five-year-old full term male of haitian descent referred for assessment of multiple flesh colored, papular lesions on the buccal and labial mucosa that had persisted and quantitatively increased over one year, although some lesions regressed. he had no pain or difficulty eating. medical history significant for one seizure; negative for infection. no family history of infection, immunodeficiency, consanguinity, or miscarriage. head and neck examination failed to reveal cervical lymphadenopathy, masses, or hypertrophy in the salivary glands. intraoral examination revealed multiple papular nodules, mostly flat although some were corrugated. the greatest concentration was noted on the lower left labial surface extending to the mucosal vermillion interface, not involving the vermillion or commissure region. lesions extended into the mandibular vestibule and the left buccal mucosa. no other lesions were noted on extremities, genitalia, or any other visualized mucosal surface. based on history and exam, he was diagnosed with meh. white blood cell count, neutrophils, lymphocytes, cd and cd t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus and streptococcus pneumoniae titers were protective. cytomegalovirus igg and igm were negative. epstein-barr virus igg was positive, igm and early antigen ab negative. serology was significant for elevated tnf ( pg/ml; reference range < pg/ml) while interferon gamma and interleukins , , , , , , , , , and were normal, as was il- receptor cd . one month after the initial visit, lesions were stable and unchanged. nine-valent hpv vaccination was considered, but not administered. conclusions: meh is a rare but benign disease caused by hpv. awareness of the disease and its course is important to prevent unnecessary expanded immunodeficiency work-up and possible procedures to eliminate lesions. although mucosal immunity can be site specific, especially with hpv, our understanding of t-cell cytokine and chemokine responses to hpv in cervical and laryngeal lesions may be instructive. the mechanism which allows hpv persistence in meh is not characterized, but it likely is due to increased viral persistence and an inability for the host immune response to successfully induce viral latency and successful containment. elevated tnf levels, with normal levels of il- , il- , il- , il- , may correlate with decreased clearance of hpv and prolonged duration of meh. it remains unclear if viral persistence is the cause of, or the sequela of, increased tnf. longitudinal monitoring of cytokine (tnf, il- , il- , il- , il- ) and chemokine (ccl , ccl , ccl , ccl , ccl , and ccl ) serum concentrations may be useful biomarkers for disease resolution. introduction: autosomal dominant hyper ige (jobs) syndrome is a rare primary immunodeficiency characterized by eczema and sinopulmonary infections as well as musculoskeletal and vascular complications. as in all chronic illnesses, patient education is an ongoing need. in the rare disease population, patient education is especially important as patients must be able to explain their unique healthcare concerns in a variety of medical settings. we focused on ad-hies, due to our relatively large cohort of patients, the frequent lack of classic signs of illness often impairing diagnosis of severe infection, and the diverse nonimmunologic clinical features of this disease. objectives: we aimed to increase understanding of the clinical manifestations of ad-hies to promote earlier recognition of symptoms and to increase self-efficacy for symptom management in the adult hies population. methods: adult patients were asked to participate in a patient education project. demographic information was collected from participants. they also completed a -item multiple choice test about symptom recognition in ad-hies and promis self-efficacy for managing symptoms, an item validated survey. then, patient education handouts that focused on pulmonary symptoms, eczema, bone health, and cardiovascular complications were reviewed with the participant. six weeks later, participants were asked to repeat the -item test and the self-efficacy survey. the demographic information, test, and self-efficacy were collected anonymously. results: participants provided demographic information, completed the test and the self-efficacy survey. of the participants, were male and were female. participants ranged in age from to years. / ( %) reported looking for information about ad-hies using search engines and most patients ( %) report that they have been given information about ad-hies from a doctor. / ( %) participants identified pulmonary symptoms as the symptom that concerns them most and / ( %) participants identified more than one symptom of concern. participants returned the second test and second survey. the mean test score increased from . to . with / participants achieving a score of / or higher. the self-efficacy scores were unchanged with a mean score of . before reviewing the patient education handouts and . after. conclusions: participant feedback to this project was generally positive. ad-hies patients are seeking information and an educational intervention can improve their understanding of disease. self-efficacy results were mixed and unchanged overall, but suggest that ad-hies patients manage symptoms as well as other patients with chronic illnesses. patient education should continue at each encounter. this project can be expanded to include more topics, pediatric patients, and other rare disease populations. funded by the nci contract no. introduction: bcl b plays an important role in the development and maintenance of the immune system and the central nervous system. expression of bcl b represses nk and myeloid factors while inducing t cell lineage genes in thymocytes at the dn stage. conditional loss of bcl b expression in murine thymocytes leads to t cell deficiency while complete knockout of bcl b was fatal within a few days of birth. recently, specific heterozygous bcl b mutations have been reported in individuals with global development delay. however, only of these cases, both carrying heterozygous missense variants, had low trec values with other cases having frequent infections. little is known regarding the impact of bcl b on human nk and t cell function. methods: we identified a novel heterozygous truncating mutation in bcl b in an infant who was first detected by trec newborn screening. she subsequently developed severe autoimmune hemolytic anemia at the age of months. we used standard immunoblotting and flow cytometry methods to assess protein expression and the impact of this bcl b mutant on t cell and nk cell development and function. results: the patient has a novel single base-pair deletion in the bcl b gene, which is predicted to produce a truncated protein with the loss of of zinc finger domains in bcl b. immunoblotting of t cell blast lysates revealed a reduced bcl b expression in the patient consistent with the heterozygous defect in bcl b but also generated a novel band with a smaller molecular weight that we postulate represents the truncated protein product. while mitogen responses to cona and pha were normal, both cd + and cd + t cell counts were decreased, especially cd + naïve and cd +cd + naïve t cells, suggesting reduced thymic output. the function of th cells was skewed with reduced il- production but increased ifn levels after pma and ionomycin stimulation. moreover, t regulatory cell counts were below normal range. nk cell counts were normal but these were mostly cd bright nk cells. of the few cd dim nk cells that presented, approximately half did not express cd , the fc receptor for adcc. perforin was only present in cd expressing nk cells. as such, anti-cd stimulation understandably led to low but not defective nk cell degranulation. function after stimulation with k cells was normal when controlled for nk cell counts. conclusion: we report a novel bcl b truncating mutation with a leaky scid phenotype that manifested with t-cell lymphopenia and autoimmunity. lowered thymic-derived naïve t and regulatory t cells, skewed th cytokine response, and incomplete nk cell development suggests that bcl b is important for the development and differentiation of multiple lymphocyte lineages. introduction: chronic diarrhea is one of the most common gastrointestinal complaints in patients with common variable immune deficiency (cvid) and can lead to life-threatening complications such as malabsorption and malnutrition. chronic diarrhea in cvid could be caused by infections, an inflammatory bowel disease-like picture, as well as malignancy. giardia lamblia is one of the most common parasites causing diarrhea in cvid (up to %), and can be refractory in these patients, leading to villous atrophy, weight loss, and failure to thrive. case report: a -year-old female with a history of cvid presents with chronic diarrhea and significant weight loss. her cvid was diagnosed by hypogammaglobulinemia (low levels of igg, igm, and iga), inadequate responses to protein and polysaccharide-based vaccines, decreased memory b cells (cd +cd + . %), and recurrent sinopulmonary infections. she was started on immune globulin replacement therapy and had significant improvement in her rate of infections. four years before her presentation to our center, she developed chronic, severe diarrhea. work up revealed giardia lamblia infection on endoscopy and colonoscopy. biopsy showed intraepithelial lymphocytes, villous blunting, and atrophic gastritis with rare plasma cells concerning for non-infectious enteropathy related to her cvid, in addition to the high burden of giardia organisms. she was initially treated with metronidazole for several weeks. however, her diarrhea did not improve, and she developed significant peripheral neuropathy leading to lower extremity weakness and limited mobility. her diarrhea persisted and was associated with approximately a -pound weight loss. repeat endoscopy and colonoscopy two years later showed persistent high burden giardiasis of the small intestine, as well as reactive lymphocytic infiltrates and atrophic gastritis. she was treated with nitazoxanide but continued to have diarrhea, and her stool continued to show trophozoites. given the significant inflammation and the lack of response to multiple antiparasitic agents, she was referred to our center for further evaluation. she was started on oral budesonide ( mg daily) and oral immune globulin ( grams weekly for weeks). with this regimen, she had significant improvement in her diarrhea with a -pound weight gain. repeat colonoscopy showed considerable improvement in inflammation and resolution of her giardia infection, though her stool antigen continues to be positive. conclusions: persistent diarrhea in our patient is most likely due to a combination of cvid enteropathy and giardiasis. a prolonged course of metronidazole and later nitazoxanide did not control her diarrhea and led to significant side effects. switching to an immunomodulatory approach significantly decreased the inflammation in her bowel and may even have helped to reduce the burden of giardia in the gut. targeting both underlying bowel inflammation as well as active infection in cvid patients with chronic diarrhea might be needed to control symptoms. introduction: sphingosine- -phosphate (s p) is a lipid chemoattractant that is critical for lymphocyte egress from lymphoid organs. following a s p concentration gradient maintained by s p lyase ubiquitously expressed in tissues, lymphocytes within lymphoid organs are drawn to efferent lymph and blood unless their s p receptor is internalized or downregulated. owing to diminished degradation of not only s p, but also other sphingoid bases, deleterious mutations in sgpl (encoding s p lyase) perturb sphingolipid catabolism in numerous tissues. correspondingly, human s p lyase deficiency results in multiorgan dysfunction including kidney, skin, endocrine gland, and neurologic impairment alongside expected lymphopenia. although severe t cell lymphopenia (< cells/microliter) rivaling that of severe combined immunodeficiency (scid) can be seen in patients with s p lyase deficiency, no such patients have been identified by newborn screening of t cell receptor excision circle (trec) counts, which are a surrogate measure of effective t cell production. herein, we describe an infant boy with an undetectable trec count at birth who was found to have two novel, biallelic sgpl mutations resulting in s p lyase deficiency. case description: a -day-old boy with a preceding history of fetal hydrops is born at a gestational age of weeks and presents with renal failure, anasarca, and respiratory failure. trec analysis of a dried blood spot obtained at hours of life reveals zero copies/microliter. subsequent peripheral blood studies show profound lymphopenia, with diminished cd + t ( /microliter; cd +, cd +), cd + b ( /microliter), and cd / + natural killer ( /microliter) cell counts. recent thymic emigrants are reduced ( . % of cd + t cells are cd ra+cd +), as is the ratio of naïve-to-memory cd + t cells ( % cd ra+, % cd ro+). expedited whole genome sequencing identifies two novel variants in sgpl a paternally inherited splice site variant (c. + t>c) predicted to impact a canonical splice donor site, and a maternally inherited missense change (c. g>a; p.cys tyr) located in a well-established functional domain of s p. in addition to nephrotic syndrome and lymphopenia, the patient displays evidence of adrenal insufficiency and has increased plasma levels of sphingoid bases and ceramides. before further analyses could be pursued, the infant dies at days of age due to ongoing complications of renal failure and eventual cardiorespiratory failure. summary: we report the first case of s p lyase deficiency identified by newborn trec screening for scid. as sgpl is not included in most commercially-available, scid-tailored gene panels, s p lyase deficiency would be missed by conventional genetic testing. therefore, analysis for variants in sgpl should be considered in neonates with low-to-undetectable trec counts, nephrotic syndrome, and other suggestive sequelae. w a r t s , hypogammaglobulinemia, recurrent infections, and myelokathexis) is a rare autosomal dominant primary immunodeficiency. it is caused by a defect in the gene encoding the chemokine receptor cxcr . this receptor, along with the associated ligand cxcl , regulates leukocyte migration. we present the case of a -year-old female, who presented after she self-identified the signature signs of whim syndrome in herself and multiple family members. objectives: we present the case of a -year-old female who presented with a history of recurrent warts, leukopenia of unknown cause, and recurrent infections as a child. as a child, she experienced multiple ear and sinus infections, along recurrent warts on her upper and lower extremities that have persisted to this day. furthermore, during a routine examination when she was -years-old, she had a complete blood count drawn significant for leukopenia. no further workup was undertaken at that time. when continued leukopenia was noted at the age of , referral to a hematologist and a bone marrow biopsy was completed. bone marrow was significant for myelokathexis with borderline hypercellular marrow for patient age ( % cellularity), and normal cell line quantity. a trial of neupoegen was undertaken, without significant improvement. her family history is significant for father and brother with both leukopenia and recurrent warts. results: genetic analysis showed a heterozygous pathogenic variant in the cxcr gene, c. _ dup (p.ser phe fs* ). recent complete blood count was significant for a total wbc count of . k/ul, with a differential consisting of % neutrophils and % lymphocytes. lymphocyte subsets were significant for quantitatively low cd +, cd + and cd + subsets, with normal numbers of cd + and nk cells. immunoglobulin levels revealed an igg of mg/dl, iga of mg/ dl, and igm of mg/dl; igg anti-diphtheria and tetanus titers were protective, however, none of the s. pneumoniae serotype titers were > . ug/ml. mitogen (pha, cona and pwm) and antigen (candida and tetanus) stimulation of lymphocytes were normal for all stimuli. conclusions: we present the case of a -year-old female with a history of recurrent infections, warts, and myelokathexis. on genetic analysis, she is noted to have a pathogenic mutation of the cxcr gene. the substitution of a phenylalanine for a serine decreases one of the seven serine phosphorylation sites in the carboxy tail of the molecule that occurs upon binding to its ligand, cxcl (sdf ). additionally, the variation generates a premature stop condon terminating the remainder of the carboxy terminal amino acids including ser - , known to have a role in carboxy terminial beta-arrestin binding. failure to generate adequate beta-arrestin binding sites leads to prolonged cxcr cxcl interaction resulting in myelokathexis. background: lacking protective antibodies, patients with primary antibody deficiencies (pad) suffer from frequent respiratory infections leading to chronic pulmonary damage. macrolides prophylaxis has been proven effective to successfully manage chronic lung diseases as cystic fibrosis, bronchiectasis, copd. we conducted a trial to evaluate the efficacy and safety of orally low-dose azithromycin prophylaxis when added to the usual care in pad patients. methods: a -year, phase ii, prospective, multicenter, randomized, double-blind, placebo-controlled trial on pad patients (age - years) with chronic infection-related pulmonary disease. patients received azithromycin mg or placebo once daily three-times a week for months. the primary outcome was the decrease of annual episodes of respiratory exacerbations. secondary endpoints included: time to the first exacerbation, number of hospitalizations, additional doses of antibiotics, health related quality of life measures, and safety. results: forty-four patients received azithromycin and patients received placebo. the mean number of exacerbations was · per patientyear ( %ci · - · ) in the azithromycin arm, and · ( %ci · - · ) in the placebo arm (p= · ). in the azithromycin group the hr for having an acute exacerbation was · ( %ci , - · , p= , ) and the hr for hospitalization was . ( %ci , - · ) (p= · ). the rate of additional antibiotic treatment per patient-year was · ( %ci · - · ) in the intervention and · ( %ci · - · ) in placebo groups (p= · ). improvement in hrqofl was observed in intervention group. azithromycins safety prole was comparable with placebo. conclusion: in pad with respiratory exacerbation, azithromycin prophylaxis led to reduction of exacerbation episodes, of additional courses of antibiotics, and of risk of hospitalization. given the deleterious effects of respiratory diseases adding azithromycin to pad treatment should be considered as a valuable option. background: the autosomal-dominant hyper-ige syndrome (hies), is a primary immunodeficiency caused by mutations in signal transducer and activator of transcription (stat ) that leads to defective th immunity. adverse reactions following -valent pneumococcal polysaccharide vaccine (ppsv ) have been reported in % of stat -hies patients, including severe local reactions that appear to be specific to this vaccine. case report: we present the case of a six-year-old girl, second child of nonconsanguineous healthy parents, that developed an extensive inflammatory skin reaction at the vaccination site following a single dose of ppsv . the vaccine was prescribed due to history of recurrent respiratory tract infections and an incomplete vaccine calendar with no previously administered pneumococcal vaccines. the reaction began after hours with local erythema and edema at vaccination site, expanding in hours to a phlyctenular lesion with no well-defined borders. within the first weeks, it progressively evolved to a deep necrotic lesion that required surgical debridement. the subsequent skin defect required surgical repair with a split-thickness skin graft from her right thigh as the donor site. the complete wound healing process took about months, leaving a large scar ( figure) . the patient had a longstanding history of recurrent infections with multiple hospitalizations including severe neonatal pneumonia that required respiratory support, a colon perforation with secondary peritonitis and septic shock that required a hemicolectomy at months of age, recurrent oral candidiasis, recurrent pneumonias of different lobes, recurrent acute otitis media, a cervical phlegmon, three episodes of dental abscess and multiple kidney abscesses due to gram-negative bacteria treated with intravenous antibiotics and surgical drainage. family history is notable for an older sibling that died due to sudden infant death syndrome. the patients mother has large and wide nose suggestive of stat -hies phenotype, but no history of infections. immunological work up showed mild eosinophilia ( cells/ mm ), elevated ige ( mg/dl), normal igg, iga, igm and lymphocyte subsets (cd , cd , cd , cd , cd ). peripheral th cells were markedly decreased ( . % vs. . % of normal control). specific pneumococcal antibodies evaluated month after psv revealed / serotypes in protective levels. high resolution thorax ct showed multilobar bronchiectasis. echocardiogram and total spine x-rays were normal. stat -hies was suspected with a national institutes of health score of . a novel heterozygous missense variant in stat affecting the src homology (sh ) domain (p.lys glu) was found by next-generation panel sequencing. a variant in the same position (p.lys met) has been previously reported in a hies patient (clinvar). currently, she is on monthly ivig and prophylactic antibiotics (cotrimoxazole, azithromycin and fluconazole). conclusions: the case presented raises awareness on the risk of severe local adverse reactions to ppsv in stat -hies patients. the etiology of such reactions is unclear and warrants further study. the benefits and risks of immunizing stat -hies patients with ppsv should be weighed carefully by medical providers. abstract (max words) introduction: dock deficiency is a rare primary immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective t-cell activation and th differentiation, impaired eosinophil homeostasis and dysregulation of ige. to date, there are no reported cases from malaysia. objective: we aimed to describe the clinical, immunological profile and mutational analysis of three siblings of consanguineous parents, presented with hyper-ige and lymphopenia between the years and , which were solved by mutational analysis of the second and third siblings. methods: clinical data and investigation results were collated from the medical record. scoring of the symptoms and physical examination findings using nih score was performed. t, b, nk lymphocyte subsets and serum igg, iga, igm, total ige quantification, lymphocyte proliferation test and pneumococcal specific antibody response were performed. mutational analyses were performed in freiburg, germany. result: three siblings presented at different time points over a -year span with raised ige levels, recurrent infections, eczema, hypereosinophilia and bronchiectasis. the nih scores for hyper-ige syndrome (hies) ranged from . we also documented two serious infections in the siblings, which were disseminated cryptococcus neoformans and salmonella sp. immunological results showed t-cell lymphopenia, defective t-cell proliferation, decreased igm, raised ige, hyper-eosinophilia and defective pneumococcal antibody responses present but not in all siblings. we identified a large deletion in dock starting from exon - in of the siblings from mutational analysis performed. we will proceed with next generation sequencing and dock protein assay in malaysia to further characterize the defect. conclusion: our on-going study is the first description of dock in a family from malaysia. the diagnosis of dock should be suspected in cases with raised ige levels, recurrent infections and lymphopenia, despite no warts infection in the history. this study emphasized the importance of international research collaboration and networking in solving complicated cases. the index patient presented at the age of years with increased susceptibility to lower airway and gastrointestinal infections (hospital admissions x/year until puberty). she suffered from mumps and varicella disease despite immunization, as well as from recurrent local, partially destructive hsv infections. she was diagnosed with common variable immunodeficiency (cvid) at age and started on immunoglobulin replacement therapy. following a hypoglycemic seizure at age , the patient was diagnosed with isolated acth insufficiency with secondary adrenal insufficiency requiring hormone substitution. during and following her first pregnancy at age , she suffered from recurrent bronchopneumonias including pneumocystis jirovecii infection, resulting in bronchiectases documented on chest ct at age . currently, chronic lung disease is severely limiting her quality of life (table ) . her daughter was noticed to be hypogammaglobulinemic soon after birth and failed to develop antibody responses to inactivated vaccines. she was started on immunoglobulin replacement therapy. she has not suffered from severe lower airway infections, but developed alopecia totalis at age and nail dystrophy. w h o l e e x o m e s e q u e n c i n g r e v e a l e d a h e t e r o z y g o u s c. _ insacccgag (p.lys profster , nm_ ) mutation in exon of nfkb in both mother and daughter. this monoallelic loss-of function frameshift mutation was not found in gnomad, gvs washington or clinvar databases. as previously published, a monoallelic mutation in this c-terminal domain leads to impaired phosphorylation and subsequent reduced nuclear translocation of the nfkb /p active form. pediatricians and internal specialists need to be aware of the combination of hypogammaglobulinemia, acth deficiency, immune dysregulation and ectodermal dysplasia which is unusual for cvid -possibly indicating nfkb deficiency. this clinical syndrome may overlap with symptoms and signs found in both apeced/ aire (ar) and eda-id/nfkbia (ad) deficiencies. besides ig and hormone replacement therapy, curative treatment with hematopoietic stem cell transplantation is a therapeutic option for patients with nfkb deficiency, although the experience is limited. table introduction: the modes of immunoglobulin (ig) administration for primary immunodeficiency diseases (pidd) differ in pharmacokinetics, infusion parameters, and tolerability. during consecutive clinical studies, a cohort of patients with pidd experienced all modes of administration with the same ig % product in sequence from intravenous (iv) to subcutaneous (sc), then to hyaluronidase-facilitated sc (ighy), providing a unique opportunity to assess each administration modality within the same patient cohort treated and observed at the same sites. here we report the rates of infections stratified by igg trough levels, and the rates of adverse events (aes) with the modes of ig administration (ivig, scig, ighy) within this patient cohort. design and methods: this analysis included patients with pidd aged years who participated in clinical studies: in study (nct ) patients received ivig % every weeks followed by weekly scig %; in study (nct ), patients were treated with ighy every weeks; in study (nct ; extension of study ), patients continued with the same ighy dose. to assess a potential association between the administration route at comparable igg trough levels and the infection rate, igg trough levels were categorized as < mg/ dl, < mg/dl, < mg/dl, < mg/dl, < mg/dl and mg/dl. periods where patients had trough levels within these strata were assessed, and the infection frequency was calculated. the time periods for this analysis were months for ivig and months each for ighy and scig % ( . years) treatments. in order to account for differences in the frequency of administration, rates of systemic and local aes were assessed as aes/patient-year for each mode of therapy. results: for igg trough levels of < mg/dl, the associated annual infection rates were lower or similar for ighy than scig ( the treatment involves the control of infections and immune dysregulation with chemotherapeutic regimens followed by definitive treatment with hematopoietic stem cell transplant (hsct). aim: to describe a female patient with a pathogenic variation in stx with normal cd a expression. results: she was a years old female, the th daughter of nonconsanguineous parents, without relevant personal or family records. she was admitted due to a prolonged febrile syndrome, lymphoproliferation, pancytopenia and hepatitis, with hhv rescued in bone marrow and blood. gancyclovir treatment started with good response. she was admitted one month later with similar clinical symptoms with relapsed hhv infection. furthermore, hemophagocytosis was found in the bone marrow and evaluation of nk cell cytotoxicity demonstrated slightly reduced cytotoxic activity. functional studies for primary fhl were performed: perforin expression and cd a surface expression were normal. she fulfilled criteria of fhl, and treatment with gancyclovir and steroids was administered. despite this treatment, she persisted with activated macrophagic parameters, and started with hlh treatment protocol. she improved the clinical symptoms and laboratory parameters, but persisted with hhv low viremia. three months later, when immunosupression was decreased, she was readmitted with similar clinical manifestations and added neurological symptoms (facial paralysis, abnormal movements and sleep tendency). cerebral spinal fluid was pathological with hhv positive rescue. immunosupresive treatment was adjusted, but hhv copies in blood increased markedly. foscarnet treatment was administered and immunosupression was suspended for days in order to control viral infection. unfortunately the patient died days later. although specific functional tests were normal, sequencing of stx gene by ngs revealed a homozygous variation in c. _ deltgcc, which is a previously reported mutation responsible for fhl. conclusion: despite the fact that cd a was normal, the strong clinical and laboratory results must keep the fhl diagnosis in mind and intensive treatment should be early administered; in order to give the patient the opportunity to achieve the curative treatment. objectives: to report and characterize the clinical course of a patient with apeced and specific antibody deficiency. methods: retrospective chart review was performed. the patient was enrolled in niaid irb-approved protocol -i- . results: the patient is a year-old-girl with apeced caused by homozygous aire c. _ del , who manifested cmc, hypoparathyroidism, adrenal insufficiency, sjogrens-like syndrome, autoimmune hepatitis, intestinal dysfunction and autoimmune pneumonitis. she suffered from recurrent sinusitis and severe pneumonias requiring hospitalization and administration of intravenous antibiotics several times per year. at age , she presented to our institution with fever and cough, a computed tomography (ct) of the chest revealed bilateral pulmonary infiltrates and bronchiectasis. bronchoscopy showed mucopurulent secretions in the bilateral lower lobes with culture of the bronchoalveolar lavage fluid growing streptococcus pneumoniae. further evaluation for an underlying disorder such as primary ciliary dyskinesia and cystic fibrosis including exome sequencing and sweat chloride testing was unrevealing. quantitative immunoglobulins were normal. despite prior vaccination, specific antibody testing showed negative rubeola igg and protective levels (> . mcg/ml) to only of pneumococcal serotypes. lymphocyte enumeration showed normal b cell subsets. as approximately % of apeced patients may experience asplenia, splenic ultrasound was performed confirming the presence of a cm spleen and peripheral blood smear did not reveal howell-jolly bodies. serotyping of the s. pneumoniae isolate confirmed serotype f, which is part of the -valent vaccine. follow up vaccine challenge with the valent pneumococcal polysaccharide vaccine showed an inadequate response. hence, she was started on monthly immunoglobulin replacement and over the following years she has experienced a single methicillin sensitive staphylococcus aureus pneumonia. she has missed very few school days and other parameters including linear growth have improved, she is now along the fifth percentile for height and along the tenth percentile for weight. although she continues to experience intermittent cough she remains active participating in sports without limitation. conclusions: we report the evaluation, treatment and outcome of a patient with apeced complicated by autoimmune pneumonitis and specific antibody deficiency. as infectious susceptibility of apeced classically pertains to the signature infectious disease, cmc, patients with invasive or recurrent infections should be evaluated for underlying immune deficiency. investigation should include assessment for asplenia, quantitative immunoglobulins and specific antibodies with response to antigens. in patients with predominate respiratory symptoms, autoimmune pneumonitis should be evaluated given the near % prevalence of pneumonitis observed in american apeced patients. acknowledgements: supported by dir/niaid/nih introduction: autoinflammatory diseases are genetically heterogeneous disorders of innate immunity characterized by recurrent fever, rash, and/ or serositis, which generally are considered distinct from autoimmune diseases. we report a case of a patient with lupus-like disease and a mutation of nucleotide-binding oligomerization domain-containing protein (nod r w, yao syndrome) suggestive of an overlap between autoinflammatory and autoimmunity processes. case presentation: a -year-old man was evaluated for recurrent pleural effusions, morning stiffness, erythematous rashes, and fever up to °c. history was notable for hashimotos thyroiditis and multiple admissions for presumed pneumonia with recurrent bilateral lung infiltrates and pleural effusions. transbronchial biopsy showed nonspecific pneumonitis and organizing pneumonia. antinuclear and anti-dsdna antibodies were positive. he received prednisone for presumed lupus pneumonitis leading to improvement. prednisone was tapered and hydroxychloroquine was started, but his fevers, pleuritic pain and pleural effusion reoccurred. genetic testing revealed a nod sequent variant (r w) associated with autoinflammatory disease. hydroxychloroquine was stopped and colchicine was added to his regimen, allowing prednisone to be tapered without recurrence of symptoms. further immunological testing revealed increased signaling through the type i interferon receptor (interferon signature). conclusion: although this patient had several clinical (serositis, arthralgia) and immunological (antinuclear and anti-dsdna antibodies, interferon signature) manifestations of lupus, his clinical presentation also was consistent with yao syndrome. in retrospect, he had been having recurrent inflammatory symptoms for many years. recent studies in both mice and humans suggest that inflammasome activation and il- production are involved in the pathogenesis of lupus. this case provides further support for the idea that lupus and hashimotos thyroiditis, prototypical autoimmune diseases, may have overlapping autoinflammatory features. background: the implementation of severe combined immunodeficiency (scid) newborn screening by trec assay has played a pivotal role in identifying these patients early in life. the screen has also led to the identification of infants with other immunologic abnormalities, of which the clinical implications have been unclear and there are limited data on their outcomes. objective: to review immunologic and genetic outcomes of infants referred to an immunology service of a tertiary care center with abnormal newborn scid screens. methods: we retrospectively reviewed charts of infants with positive scid screen from july to november . we excluded patients who had positive screen at < weeks corrected gestational age. we classified outcomes into groups including scid, non-scid t-cell lymphopenia (nscid-tcl) and normal t-cell count. idiopathic t-cell lymphopenia was defined as nscid-tcl (cd + < , cells/mcl) with negative chromosome microarray and negative whole exome sequencing/or genetic panel (either genedx® scid panel or invitae® primary immunodeficiency panel). results: of infants, % were male, % were caucasian, and % were african-american. fifty-four % and % of infants were identified by illinois and missouri screens, respectively. the mean age at initial evaluation was days ( - days). % of infants had a normal tcell count (n= ) or normal repeat newborn screen (n= ), % had nscid-tcl, including mild (cd + , - , cells/mcl, n= ) and moderate (cd + - , cells/mcl, n= ) tcl, and % had scid (n= ), leaky scid (n= ) or complete digeorge (n= ). genetic etiologies of nscid-tcl included q deletion (n= ), trisomy (n= ), and mutations of tbx (n= ), foxn (n= ), and cd e (n= ). three of these infants had novel variants at the time of diagnosis. secondary causes of tcl were identified in infant (thoracic infantile fibrosarcoma). one infant had idiopathic tcl. eighteen infants with nscid-tcl were followed clinically without complete genetic testing performed. for scid, mutations were found in jak (n= ), ada (n= ), il rg (n= ), and rag (n= ). the patient with leaky scid had negative whole exome sequencing. all patients with scid and leaky scid underwent hematopoietic stem cell transplantation at a median age of weeks ( weeks - months), with successful engraftment in all but patient. of idiopathic and nscid-tcl cases followed clinically, had at least one follow-up visit at median age months ( . months . years) and the majority had improved or stable lymphocyte count without serious infections requiring intravenous antibiotics, though had a hospitalization for rsv infection. the mysm patient died after cord blood transplant from unclear etiology. our study had limitations. half of infants with nscid-tcl did not have a complete genetic workup, and only a fifth of patients with nscid-tcl were inpatients, potentially explaining the relatively low number of infants with secondary lymphopenia. conclusions: in our cohort, one-fourth of infants with abnormal scid screen had nscid-tcl. although the majority of nscid-tcl did well, approximately one-third of them had underlying genetic abnormalities associated with their t-cell lymphopenia. ( ) submission id# introduction: accumulation of intracellular adenosine and deoxyadenosine nucleotides (daxp) due to adenosine deaminase deficiency results in profound lymphopenia and severe combined immunodeficiency. left untreated this form of scid is uniformly fatal. while allogeneic hematopoietic cell transplant (hct) and autologous gene corrected stem cell therapy (gt) are potential cures for ada-scid , initiating enzyme replacement therapy (ert) immediately upon diagnosis regardless of definitive treatment is standard of care. hct and gt are not therapeutic options for all ada-scid patients and ert offers immediate therapeutic intervention for these patients leading to partial immune reconstitution, and durable survival in most patients treated. adagen (pegademase), approved by the fda in in the usa, is a pegylated bovine ada (nada) with the enzyme harvested from bovine intestines. this unsustainable production process led to the development of a recombinant enzyme source based on the bovine protein sequence and an improved pegylated linker by using succinimidyl carbamate (revcovitm-(elapegademase-lvlr). methods: a phase ii/iii clinical trial was performed at us sites under institutional irb approval. eligible ada-scid subjects were stable on adagen and without complicating underlying conditions. demographics, medical history, lymphocyte counts, immunoglobulin levels, trough plasma ada activity and rbc daxp measurements were collected. patients were treated with adagen as a single, weekly im dose adjusted to achieve a trough plasma ada activity of > mmol/hr/l and rbc daxp < . mmol/l (protocol target levels). once patients had achieved this level ( - weeks), a seven-day pk on adagen was done and the patients were transitioned to revcovi based on the formula for enzyme equivalent activity of mg revcovi = units adagen. after weeks on revcovi, trough ada and daxp were assessed and a seven-day pharmacokinetic study was conducted at week . patients were assessed periodically for clinical and laboratory values and evaluation of the study endpoints was done at week . subjects subsequently continued on revcovi and were assessed periodically. results: six patients, ages - entered the trial with initial adagen dosing at . - . u/kg/wk (see table ). adagen dosing was adjusted to target endpoints of ada trough activity (> mmol/hr/l) and rbc daxp (< . mml/l). patients transitioned to weekly revcovi using the aforementioned conversion formula at doses of . - . mg/kg/wk. the spectrum of clinical manifestations range from infections to autoimmunity and inflammation among patients with hypomorphic recombination gene and (rag / ) pathogenic variants. auto-antibodies targeting cytokines ifn-alpha, ifn-omega and il- were reported in a large proportion of these patients and their occurrence often coincides with viral infections. we report the time of emergence and relative frequency of anti-cytokine antibodies in children and adults, and their persistence among patients with hypomorphic rag deficiency. antibodies were measured from plasma samples of patients by enzyme linked immunoassay (elisa). our rag cohort includes patients with rag (n= , %) and rag deficiency (n= , %). antibodies targeting ifn-alpha ( %) were most common followed by il- and ifn-omega ( % each). two asymptomatic patients who were detected by newborn screening for scid and received hematopoietic stem cell transplantation had no detectable anti-cytokine antibodies. in the cohort of young children (ages mo- years, n= ), all patients had detectable antibodies to ifn-alpha, prior history of severe viral infection and subsequently developed autoimmune cytopenias. other anti-cytokine antibodies were less common (ifn-omega %, il- %). similarly, children between - yo age (n= ) also had high fraction of anti-ifn-alpha antibodies ( %) with prior history of infections ( %) and continued to have other anticytokine antibodies less commonly (ifn-omega %, il- %). in the adult cohort (n= , ages - years) the frequency of anti-ifnalpha anti-cytokine antibodies were lower ( %,) and il- and ifnomega ( % each) continued to persist. three adult patients had anticytokine (ifn-alpha, ifn-omega and il- ) antibodies tested at multiple timepoints and elevated titers persisted up to years. our data demonstrates that anti-cytokine antibodies, especially those targeting ifn are frequent and emerge early in life in association with viral infections in patients with rag deficiency. a lower fraction of adult patients have detectable anti-cytokine antibodies, and maintain these over several years. anti-ifn-alpha may serve as a useful biomarker for identifying partial rag deficiency among young and adult patients with history of viral infections and autoimmune cytopenias. the role of these antibodies to cytokines is yet to be determined but a specific signature of these antibodies may help to identify an underlying immunodeficiency and initiate early definitive treatment with bone marrow transplantation. anti-cytokine antibodies appear to be a novel tool in evaluation of autoimmune diseases including rag deficiency. introduction: norovirus is one of the most common pathogens causing gastroenteritis in immunocompromised patients, often leading to chronic infection, causing villous atrophy, malabsorption, weight loss, organ failure, need for parenteral nutrition, and death. norovirus treatment in immunocompromised patients is challenging. oral immunoglobulin (poig) has been used to treat norovirus gastroenteritis with variable success. our aim in this study was to determine the outcomes of treating norovirus gastroenteritis in immunocompromised patients methods: electronic medical records were reviewed for patients with norovirus infection confirmed by rt-pcr since january . our initial cohort was focused on patients with primary immunodeficiency (pid), lung, and liver transplant. data on demographics, immunological phenotype, treatment with poig, the number of bowel movements (bm), and virus clearance were collected. descriptive statistical methods were used to describe treatment outcomes. further analysis of patients immunophenotype, immunosuppression medications, and co-morbid illnesses is underway. results: twenty-six immunocompromised patients ( norovirus infection episodes, as one patient had reinfection) were analyzed twelve females, age range months- years. twelve patients had pid diagnosis ( common variable immunodeficiency, severe combined immunodeficiency, x-linked agammaglobulinemia, wiskott-aldrich syndrome, digeorge syndrome, hyper-igm, stat gain-of-function, nemo and lymphopenia in a patient with trisomy ), patients were status-post liver transplant, and two patients were status-post lung transplant. of patients were on ig replacement therapy at the time of the norovirus infection. the average number of bm/day in all patients was . (range - ) . eight patients received poig ( - mg/kg) weekly for a duration from - weeks. three of those received additional nitazoxanide and received ribavirin. / patients in the poig group were receiving total parenteral nutrition (tpn), and / on no treatment group received tpn. the average number of bm/day in poig before treatment was . (range - ), and . (range - ) in those who did not receive any treatment. of ( %) on poig vs. of ( %) in the no treatment group cleared the virus. the average number of weeks to return to baseline bm was . (range - ) in the poig group vs. . (range days- weeks) in the no treatment group. of on poig continued to have chronic diarrhea that is still ongoing. conclusion: despite anecdotal reports suggesting successful use of poig in immunocompromised patients, our data did not show a significant decrease in stool output in patients treated with poig, compared to no treatment. however, poig led to a higher rate of virus clearance. a study with larger sample size might be warranted to identify the patients who benefit from poig in the context of norovirus infection and ensure the appropriate use of ig products, especially given the concerns for the national shortage of ig products. chief medical officer, novimmune sa primary hemophagocytic lymphohistiocytosis (phlh) is a life-threatening, immune regulatory disorder characterized by immune hyperactivation that is driven by high production of interferon (ifn)-. patients with hlh typically develop fever, splenomegaly, cytopenias and coagulopathy. until recently, there have been no fda approved treatments for hlh, and standard dexamethasone/etoposide-based treatment has not evolved significantly in + years. emapalumab-lzsg (ni- ) is a fully human, anti-ifn-monoclonal antibody that neutralizes ifn-and which was recently approved (november ) by the fda for the treatment of adult and pediatric (newborn and older) patients with phlh with refractory, recurrent, or progressive disease or intolerance with conventional hlh therapy. results of the pivotal trial supporting this approval are presented herein. methods: this open-label pivotal study (nct ) includes patients years with a diagnosis of phlh and active disease. data presented were from patients, of whom had failed conventional hlh therapy prior to study entry. the initial emapalumab-lzsg dose was mg/kg given intravenously every - days. subsequent doses could be increased up to mg/kg based on the evolution of response parameters. dexamethasone was administered concomitantly at to mg/m /day and could be tapered during the study. treatment duration was weeks, with possible shortening to a minimum of weeks, or extension up to the time of allogeneic hematopoietic stem cell transplantation (hsct). the primary efficacy endpoint was the overall response rate (orr) at end of treatment, assessed by pre-defined objective parameters, including normalization or at least % improvement from baseline of fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen, d-dimer, central nervous system (cns) abnormalities, and with no sustained worsening of scd serum levels. the primary analysis used an exact binomial test to evaluate the null hypothesis that orr be % at a one-sided . significance level. patients were eligible to enter an extension phase for follow-up after completing the main study (nct ). the data cut-off applied is july . results: patient characteristics are summarized in table and efficacy is summarized in table . disease at study entry was consistent with the broad spectrum of phlh abnormalities. over % of patients had signs and/or symptoms of cns disease. orr was significantly higher than the pre-specified null hypothesis of %, meeting the primary endpoint. the response rate based on investigators clinical judgement was . %. emapalumab-lzsg infusions were in general well tolerated, with mild to moderate infusion-related reactions reported in % of patients. the observed safety events (pre-hsct conditioning) mostly included hlh manifestations, infections or toxicities due to other administered drugs. infections caused by pathogens potentially favored by ifn-neutralization occurred in patient during emapalumab-lzsg treatment (disseminated histoplasmosis), and resolved with appropriate treatment. no off-target effects were observed. conclusions: treatment with emapalumab-lzsg was able to control hlh activity with a favorable safety and tolerability profile in a very fragile population. the majority of patients proceeded to hsct with favorable outcomes. our results indicate that emapalumab-lzsg should be considered as a new therapeutic option in phlh thanks to its targeted mode of action. results: a total of genes were differentially expressed between t cells of qds patients (n= ) and healthy controls (n= ) (log fold change range (- . , . )).when these genes were tested for pathway enrichment, the top pathways in t lymphocytes based on their p value included communication between innate and adaptive immune cells, cross talk between dendritic cells and natural killer cells, allograft rejection signaling, dendritic cell maturation, and b cell receptor signaling. the top biological processes with differential expression included immune response, inflammatory response, apoptotic process, interferon gamma mediated signaling pathway, nucleosome assembly, defense response to virus, lipopolysaccharide mediated signaling pathway, positive regulation of nf-kappa b import into nucleus, type i interferon signaling pathway, and neutrophil chemotaxis genes. we compared gene expression between qds participants with low t cell counts (n= ) and qds participants with normal t cell counts (n= ) and found genes that were differentially expressed (q< . ) (log fold change range (- . , . ) patient began experiencing recurrent high fevers and developed splenomegaly. elevated transaminases and concern for lymphoproliferative disease prompted a splenectomy and liver biopsy. both the spleen and liver biopsy were positive for ebv but were negative for malignancy. bone marrow biopsy was unrevealing. genetic testing identified a pathogenic variant in xiap/ birc ( c>t), and the patient was treated with high dose oral steroids resulting in an improvement in symptoms. subsequently, therapy with anakinra was started and steroids were tapered. during the steroid taper, he noticed a change in the vision of his left eye consistent with naion, as well as worsening of his colitis. there was loss of the inferior visual field and fundoscopic exam was significant for left optic disc swelling. oct noted superior retinal nerve fiber layer thinning. oral steroids were restarted with improvement in optic disc swelling, but without improvement or change in vision. as of his most recent exam, the patient has persistent bilateral inferior visual field defects with segmental optic nerve atrophy typical of naion. he has continued therapy with anakinra, and subsequently tapered off of prednisone; though he remains on a physiologic dose of hydrocortisone. conclusions: this case demonstrates an unreported ocular manifestation in a patient with xiap deficiency, which clinically appeared sensitive to immunomodulation. our patient is an unusual candidate for naion due to his young age, the average age of onset being the mid to late s, and lack of vascular risk factors. we hypothesize that his hyper-inflammatory condition contributed to irreversible vascular damage in the optic nerve head, resulting in naion. therefore, it may be useful to consider the involvement of systemic inflammatory and immune dysregulatory conditions when treating patients with atypical naion. additionally, naion should be considered in patients with xiap deficiency and sudden unilateral vision loss. the importance of de novo mutations in causing severe sporadic immune disease is well described, yet significance of such a variation in less severe and later onset of immune disease is poorly investigated. whole exome sequencing has been a powerful tool to resolve and explain the genetic basis of novel syndromes in immune related diseases. however, proving causation can be complicated due to low number of the affected individuals. we performed whole exome sequencing in a cohort of patients with noncongenital immune defects, along with detailed cellular biochemical phenotyping. we report and describe a novel non-congenital combined immune deficiency arising from a de novo gain-offunction mutation in ikbkb(c. g>a). this gene encodes ikk , and activates canonical nfkb signalling. cellular and biochemical studies of the proband revealed that ikk v i results in enhanced nf-kb signaling, as well as t and b cell functional defects. ikk v is a highly-conserved residue, and to prove causation, we generated a crispr/cas mouse model that carry the precise orthologous missense mutation. we show that mice and humans carrying this missense mutation exhibits remarkably similar cellular and biochemical phenotypes. dysregulation in patients. total rna isolated from cryopreserved peripheral blood mononuclear cells was reverse transcribed to generate cdna. we selected four known gata transcriptional targets, gata , gata , tal and zfpm (encoding fog ) and used droplet digital pcr to quantify transcript levels normalized to the low-expressing gene tbp . we used samples from individuals with wild-type gata (wt), known gata mutation patients (mut) and two individuals suspected of gata deficiency but without identified mutation or allelic imbalance (unk , unk ). transcript analysis revealed significantly decreased transcript levels of gata , gata and tal in mut pbmcs compared to wt. most wt samples had higher zfpm transcripts than gata mutated patients however it did not reach statistical significance. strikingly, we were able to use this analysis for two individuals suspected of gata deficiency. in the first case (unk ) a yr old female with primary lymphedema, hypogammaglobulinemia, recurrent infections and possible family history of leukemia was referred for gata testing. no mutation was identified however it was noted that she was homozygous across the gene preventing allelic evaluation. the second patient (unk ), a yr old female, had erethemya nodosa on legs, mycobacteria kansasii and cytopenias. in each of the targets analyzed, transcript levels from unk were lower than the wt samples and in a similar range as the gata mutation samples while unk had a profile consistent with the wt samples. we propose the use of gata targets as surrogate markers in cases where a mutation is not identified and allelic expression analysis is uninformative. are often under-reported and under-recognized. we sought to further understand and evaluate the prevalence, type, and association with serum immunoglobulin e (ige) for cvid patients with atopic manifestations. methods: we performed a retrospective analysis of cvid patients with atopic manifestations in the partners healthcare cvid cohort. we evaluated baseline patient characteristics, atopic diagnoses, and serum ige levels. results: in the partners cvid cohort, the average age was years old (± ) and % female. / ( . %) of patients had a diagnosis of asthma, with the majority of these diagnosed by an allergist ( %) or pulmonologist ( %). eczema/atopic dermatitis was diagnosed in / patients ( %), by either an allergist ( %) or a dermatologist ( %). allergic rhinitis was diagnosed in / ( . %) with positive skin prick testing in % of these patients. food allergy was diagnosed in patients ( . %). the median cohort serum ige was . iu/ml. the median serum ige was higher in patients with or more atopic complications compared to those with one or less atopic condition ( vs. iu/ml), which was statistically significant (p= . ). conclusions: we report higher rates of atopy than previously described in other cvid cohorts. consistent with previous reports, we find a low median cohort serum ige level in cvid patients compared to the general population. however, we identify a subset of patients with a predisposition towards atopy and higher ige levels within the broader characterization of cvid, and these patients may have a more specific molecular diagnosis that leads to elevated ige and atopic conditions. whole exome sequencing is underway to further evaluate this hypothesis. whim (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency with autosomal dominant inheritance. in most patients, the genetic cause of the disease is a gain-offunction variant in c-x-c chemokine receptor type (cxcr ) that results in arrest of neutrophil migration from the bone marrow. most patients develop hypogammaglobulinemia and early waning of antibody response with vaccination. however, the exact origin of aberrant humoral immunity in whim syndrome patients is yet to be clarified. here we describe a -year-old iraqi female with a heterozygous cxcr p.ser ter variant, which is presented with haemophilus influenzae meningitis, history of tetralogy of fallot, early onset intermittent neutropenia, lymphopenia, recurrent bacterial and viral infections. immunologic evaluation revealed hypogammaglobulinemia, elevated igm level and a lack of protective vaccine titers after tetanus and prevnar vaccinations. a bone marrow biopsy was consistent with myelokathexis. immune phenotyping, functional studies and apoptosis assays were performed on peripheral blood cells by flow cytometry in our whim patient and controls. although we found that all lymphocyte compartments were reduced, naïve cd t helper cells and switched memory b cells were predominantly affected. spontaneous apoptosis was most pronounced in b rather than t cell compartments in whim patients. in addition, naïve b cells easily activated and died upon activation in vitro. cxcl , a ligand of cxcr , induced elevated t helper cell migration and increased actin polymerization in p.ser ter mutant cells. we conclude that intrinsic b cell abnormalities, such as increased rate of apoptosis and altered activation, might be responsible for defective antibody response in whim patients. although most individuals effectively control herpesvirus infections, some suffer from unusually severe and/or recurrent infections requiring anti-viral prophylaxis. a subset of these patients possesses defects in nk cells, innate lymphocytes which recognize and lyse herpesvirus-infected cells; however, the exact genetic etiologies are rarely diagnosed. plcg encodes a signaling protein in nk cell and b cell receptor-mediated signaling. dominant-negative or gain-of-function mutations in plcg cause cold urticaria, antibody deficiency, or autoinflammation. however, loss-of-function mutations and plcg haploinsufficiency have never been reported in human disease. we examined families with autosomal dominant nk cell immunodeficiency with mass cytometry and whole-exome sequencing to identify the cause of disease. we identified two novel heterozygous loss-of-function mutations inplcg that impaired nk cell function, including calcium flux, granule movement, and target killing. although expression of mutant plcg protein in vitro was normal, phosphorylation of both mutants was diminished. in contrast to plaid and aplaid, b cell function remained intact. plcg +/-mice, as well as targeted crispr knock-in mice, also displayed impaired nk cell function with preserved b cell function, phenocopying human plcg haploinsufficiency. we report the first known cases of plcg haploinsufficiency, a clinically and mechanistically distinct syndrome from previously reported mutations. therefore, these families represent a novel disease, highlighting a role for plcg haploinsufficiency in herpesvirus-susceptible patients and expanding the spectrum of plcg -related disease. we pursued genetic diagnosis, which identified bi-allelic frameshift mutations in the rag gene which had not been previously described: c. delg (p.v sfsx ) and c. _ del insaaaagagtg (p.v kfsx ). taken together, his presentation suggested significant immune dysfunction had evolved since transplant leading to extensive pulmonary nontuberculous mycobacterial infection and possible bronchiolitis obliterans. he therefore will undergo a subsequent unconditioned cd + stem cell boost from his sister, the original donor, once he completes mycobacterium abscessus treatment. this case highlights the potential long-term immune dysfunction which may evolve after unconditioned allogeneic stem cell transplant for scid, in which full engraftment in all myeloid and lymphoid compartments is not expected. it also highlights the importance of guideline-driven follow-up of these patients to monitor for said dysfunction, to prevent serious infection and long-term sequelae. somatic hypermutation (shm) in the b cell receptor (bcr) heavy (igh) and light chain genes promotes affinity maturation and also mutation away from self-reactivity, therefore serves as an important peripheral tolerance checkpoint. as an example, unmutated bcr ighv - genes give rise to antibodies that bind to i/i antigen on red blood cells (rbc) and may elicit cold agglutinin disease (cad), a variant of autoimmune hemolytic anemia (aiha). in case of healthy individuals, frequent shms in the i/i binding site of bcr ighv - genes decrease rbc reactivity and cad. patients with primary immunodeficiencies (pid) paradoxically develop autoimmune diseases, including autoimmune cytopenias, especially aiha. it is unclear if impaired shm of bcr, in particular mutation away from i/i binding, is relevant in the development of rbc reactivity and consequently aiha in a pid background. our studies focus on pid patients with hypomorphic recombination activating gene (rag and ), combined immunodeficiency phenotype and history of autoimmunity, in particular aiha (rag cid/ai). we detected increased frequency of unmutated ighv - bcr in memory b cell repertoires of rag-cid/ai patients as well as elevated titer of unmutated ighv - antibodies in the patients' plasma. lower level of shm likely reflect abnormal germinal center (gc) reaction. as rag and heterotetramer primarily shapes the pre-immune t and b cell repertoire, we studied the interaction of follicular helper t cells (tfh) and naive b cells via in vitro co-culture experiment. interestingly, tfh cells from rag cid/ai patients exhibited highly activated phenotype with increased expression of cd l and il- compared to healthy controls and were able to initiate exaggerated response (class switching and shm) of healthy donor naive b cells. on the contrary, in vitro activated naive b cells from rag cid/ai patients showed impaired proliferation, class switching and decreased level of shm with diminished induction of genes involved t cell co-stimulation (cd , il- r) and shm (aicda, repair enzymes) compared to healthy donor naive b cells indicating intrinsic defect in patient b cells. furthermore, b cells from rag cid/ai patients also showed increased apoptosis and accumulation of gamma-h ax foci at steady state indicating reduced cellular fitness. these findings suggest that the development of aiha is a multifactorial process in partial rag deficiency. our studies highlight that impaired germinal center reaction is an important tolerance checkpoint with the inability of patient's b cells to respond to hyperactive tfh cells and introduce proper level of shm. hence, we propose that b cell fitness is compromised which impairs proper gc interaction, shm, including mutation away from self and sustains rbc reactivity in hypomorphic rag deficiency. introduction/background: the forkhead box n (foxn ) transcription factor is an essential regulator of t cell development, affecting the differentiation and expansion of thymic epithelial cells (tecs). autosomal recessive mutations in foxn cause a t-b+nk+ lymphocyte phenotype due to a thymic aplasia in conjunction with alopecia universalis and nail plate dystrophy resulting from keratinocyte dysregulation. this is a classic nude/scid (omim # ) phenotype. we report on the identification of two independent patients, identified through newborn screening with absent trecs and with a t-nk+b+ scid phenotype who presented with a t cell lymphopenia who had compound heterozygous mutations in foxn . notably, these individuals had normal hair and nail beds. objectives: to determine whether distinct compound heterozygous mutations in foxn cause a novel t-nk+b+ phenotype in the absence of a classic nude presentation. neutralizing autoantibodies (autoabs) against cytokines increase the susceptibility for selected infections (e.g. anti-ifn-autoabs for nontuberculous mycobacteria and non-typhoid salmonella, anti-il- -autoabs for mucocutaneous candidiasis and anti-gm-csf-auotabs for infections by cryptococcus, nocardiae and aspergillus spp). however, the role of anti-il- -autoabs is less clear. il- is a key mediator of the acute-phase response and released early in bacterial infections. patients with impaired signaling or affected production of il- are at increased risk for severe bacterial infections. only three patients with high-titer and neutralizing anti-il- -autoabs who suffered from severe infections caused by s. aureus, s. intermedius and e. coli have been described so far. to investigate the prevalence of anti-il- -autoabs in patients with bacterial infections, we investigated a cohort of patients and identified three further patients, all previously healthy, with neutralizing auotabs against il- who hardly developed an acute-phase response. the first patient suffered from life-threatening pneumonia caused by s. pneumonia, the second patient developed a submandibular abscess and septic arthritis caused by s. pyogenes and the third patient suffered from life-threatening pneumonia caused by s. aureus. we also discovered neutralizing anti-il- -autoabs in two adults among a cohort of patients with autoimmune diseases (n = ), in one adolescent among a cohort of obese individuals (n = ) as well as in three mothers of neonates with impaired il- signaling. so far none of the later individuals developed a severe bacterial infection. this suggests that naturally occurring and neutralizing anti-il- -autoabs are a risk factor for severe bacterial infections yet with incomplete penetrance. ( ) submission id# persistent transaminitis in copa syndrome researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city social service intern, immunodeficiencies research unit, national institute of pediatrics pediatrics resident, pediatrics hospital, st century national medical center, mexican institute of social security researcher, data science department, mexican autonomous institute of technology researcher, department of research methodology, national institute of pediatrics background: inborn errors of immunity constitute a heterogeneous group of over individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build hundreds or thousands of decision trees randomly (bootstrapping), to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of inborn errors of immunity (iei). methods: with a local database of patients with suspected iei, we built a decision tree using c . dtc, and a random forest on python (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term immunodeficiency in their electronic medical records, and then hand-picking cases in which an iei had been confirmed or ruled out. it consisted of patients, of which had been diagnosed with iei. we first split the dataset randomly into training ( %) and testing ( %) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set. the random forest classified all cases by majority vote into nine groups ( to ), according to the iuis pid group. next, we repeated the process on a larger scale with a dataset of , patients from usidnet. accuracy was assessed by out-of-bag (oob) error estimates. results: accuracy was greater than % for the local dataset (pid/ not, groups), and for the usidnet dataset ( groups). we provide a list of decision nodes and a diagnostic route with those questions that achieved a greater information gain and less entropy. this might help clinicians direct their interrogation and diagnostic approach of suspected iei patients. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical red flags. researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city dna repair defects are inborn errors of immunity that result in increased apoptosis and oncogenesis. dna ligase -deficient patients suffer from a wide range of clinical manifestations since early in life, including: microcephaly, dysmorphic facial features, growth failure, developmental delay, mental retardation; hip dysplasia, and other skeletal malformations; as well as a severe combined immunodeficiency, radiosensitivity and progressive bone marrow failure; or, they may present later in life with hematological neoplasias that respond catastrophically to chemo-and radiotherapy; or, they could be asymptomatic. we describe the clinical, laboratory and genetic features of five mexican patients with lig deficiency, together with a review of other patients available in pubmed medline. four out of five of our patients are dead from lymphoma or bone marrow failure, with severe infection and massive bleeding; the fifth patient is asymptomatic despite a persistent cd + lymphopenia. most patients reported in the literature are microcephalic females with growth failure, sinopulmonary infections, hypogammaglobulinemia, very low b-cells, and radiosensitivity; while bone marrow failure and malignancy may develop at a later age. dysmorphic facial features, congenital hip dysplasia, chronic liver disease, gradual pancytopenia, lymphoma or leukemia, thrombocytopenia and gastrointestinal bleeding have been reported as well. most mutations are compound heterozygous, and all of them are hypomorphic, with two common truncating mutations accounting for the majority of patients. stem-cell transplantation after reduced intensity conditioning regimes may be curative. department of laboratory medicine, clinical centre immunology, allergy and rheumatology division, department of pediatrics, baylor college of medicine, texas children's hospital, houston,texas, usa laboratory of clinical immunology and microbiology, fungal pathogenesis section, national institute of allergy and infectious diseases, department of intramural research, national institute of allergy and infectious diseases (niaid), national institute of health, bethesda maryland, usa card deficiency is an autosomal recessive primary immunodeficiency known to underlay increased fungal infection susceptibility mostly presenting as invasive cns candida infections (in infancy or adulthood) and dermatophyte infections. more recently, a rare card variant (c. + g>c, leading to exon skipping, card del ) showed a significant protective association towards inflammatory bowel disease (ibd) when present in heterozygosity. at the nih we studied an -year-old male patient (p ) born to a non-consanguineous marriage who presented as an infant with recurrent/severe thrush, candida esophagitis, and an episode of tinea pedis; p also has mild hypogammaglobinemia (igg mg/dl at age y). p s gdna was tested by whole exome sequencing and showed a card c. + g>c mutation in homozygous state. segregation analysis and sanger confirmation determined that both parents and p s elder brother carried the same variant in heterozygosity, while his asymptomatic younger brother (p ) was also homozygous. as previously described, this variant caused card exon deletion as determined in p and p s pbmcs by cdna sequencing and by a lower molecular weight card protein by immunoblot evaluation. p and p s pbmcs, as well as the heterozygous parents cells, showed a defective cytokine generation (tnf-, il- , il- and gm-csf) in response to heat killed candida (hkc), but not to lps. while patients pbmcs failed to induce phospho-erk and phospho-p- upon hkc-stimulation but presented an intact response to pma+ionomycin; the parents cells responded normally to both stimuli. moreover, t-cell activation and proliferation was affected in response to hkc but not to pha in both patients, whereas the parents exhibited normal results under the same conditions. when hek cells were transiently transfected with wt or card del vectors together with a trim plasmid (e -ubiquitin ligase, naturally associated to card ), we confirmed that card del failed to bind trim by immunoprecipitation. furthermore, malt , bcl and trim were only co-precipitated by wt card , but no by card del , strongly suggesting trim is an integral part of the card /bcl /malt -cbm-complex. in summary, herein we demonstrate that the card del allele fails to bind trim , and in turn is unable to conform a complete/functional cbm complex. our data also show that card del acts in a dominant negative fashion in terms of cytokine generation (previously reported), but one wt allele seems sufficient to generate normal levels of hkcinduced p-erk and p-p- , as well as t-cell proliferation. while decreased cytokine generation associated with card del in heterozygosity has been described to be sufficient to protect towards ibd, other defective pathways are affected in homozygosity and likely necessary to confer increased susceptibility to fungal infections. altogether these results suggest that card del acts through a gene dosage mechanism that can dissect pathways that associate ibd protection and fungal infection susceptibility. further work is warranted to explore card del role, if any, in b-cell and t-cell biology. professor, endocrinology, university of michigan medical school background: acquired generalized lipodystrophy (agl) syndromes are a heterogeneous group of diseases characterized by selective dysfunction and loss of adipose tissue after birth. this causes ectopic lipid deposition and deficiency of the adipokine leptin, which promotes metabolic dysfunction through impaired glucose handling resulting in insulin-resistant diabetes mellitus, dyslipidemia and steatohepatitis. while the metabolic effects of altered adipokine secretion are known, the molecular mechanism is less clear. many agl cases are suspected to have an autoimmune etiology. effector and regulatory t cells, dendritic cells and macrophages reside in normal adipose tissue. t cells within adipose tissue highly express pd- and regulatory t cells express ctla , which limits immune activation in the adipose tissue under normal circumstances. thus, inhibition of these immune checkpoints may hypothetically cause immune activation, leading to adipocyte dysfunction and autoimmune destruction. we have encountered two cases that raise clinical concern for this process. patient cases: patient is a -year-old female who presented with failure to thrive at months. she was diagnosed with insulin-resistant type diabetes and hypertriglyceridemia at ages and years with progressive subcutaneous fat loss and low leptin levels culminating in a diagnosis of agl. her childhood clinical course was complicated by hypertrophic cardiomyopathy, hepatomegaly, autoimmune hemolytic anemia with massive splenomegaly and severe chronic diarrhea secondary to autoimmune enteropathy. she presented at years with acute liver failure, thrombotic microangiopathy, nephrotic syndrome and progressive kidney insufficiency. evaluation for her multi-faceted autoimmune presentation identified a familial heterozygous pathogenic variant in the ctla gene (c. _ insgttgg,p.ala glyfster ). despite aggressive immune therapies, including ctla -ig (abatacept), her kidney disease and enteropathy have progressed. patient is a -year-old male diagnosed with localized malignant melanoma of the right neck in july . he underwent excisional biopsy and regional lymph node dissection with negative margins. he relapsed in november and underwent a modified radical neck dissection with lymph node positive for disease and received external beam radiation from january-february . additionally, he was started on anti-pd- therapy with the humanized antibody drug pembrolizumab in april but discontinued the drug in february in the setting of toxicities including hypothyroidism. subsequently, he developed up to . % weight loss with progressive loss of subcutaneous fat first in his face, then generalized to the rest of his body. in the ensuing months, imaging with pet-ct demonstrated loss of subcutaneous fat concurrent with elevations in alt and triglyceride levels plus a low leptin level consistent with agl. conclusion: these cases raise concern that inhibition of the immune checkpoints ctla and pd- may facilitate the development of agl. we hypothesize that these defects significantly increase t cell autoimmune activity in the adipose tissue and/or alter t cell metabolism resulting in agl. disorders of immune dysregulation should be considered in the etiology of agl. similarly, patients with either genetic or pharmacologic inhibition of immune checkpoints should be monitored for the development of agl with careful physical exam and periodic monitoring of glucose and triglyceride levels. background: rosai-dorfman disease (rdd; also known as sinus histiocytosis with massive lymphadenopathy) is a rare non-langerhans cell histiocytosis. it is characterized by proliferation and accumulation of activated histiocytes in affected tissues. classically, rdd presents with bilateral, non-tender, and often markedly enlarged cervical lymphadenopathy. case presentation: a -year-old female presented with a -week history of asymptomatic, persistent and bilaterally enlarged cervical lymph nodes. she was otherwise healthy with no significant past medical history. operative excision biopsy of the largest lymph node confirmed the diagnosis of rdd. three months following diagnosis, routine bloodwork revealed that she had developed lymphopenia (lymphocyte count . x /l). between -year and -and-a-half-years post-diagnosis, the patient was hospitalized and treated with intravenous antibiotics for presumed episodes of osteomyelitis and presumed episodes of lymphadenitis. given the recurrent presumed infections and persistent lymphopenia, the patient was referred to immunology for evaluation. she received a full immunologic work-up. lymphocyte immunophenotyping revealed low cd ( cells/mm ) and low cd ( cells/mm ) counts. the rest of her immunologic work-up was within normal limits. approximately -and-a-half-years post-diagnosis, the decision was made to initiate treatment for rdd. she was started on a -week tapering course of prednisone therapy. within -weeks of starting corticosteroid therapy, the lymphadenopathy had diminished, and by -weeks, the lymphopenia completely resolved. at her most recent clinic visit, she had been free of serious infections for more than -years, and her lymphocyte counts had remained stable and within normal limits for over one year. discussion: in the literature, immune system dysfunction has been reported in rdd, with both auto-antibodies and cellular immunodeficiency implicated. in this patient, the persistent lymphopenia and recurrent episodes of presumed infections appeared consistent with an immunodeficiency. given the known association of rdd with immunologic dysfunction, this was certainly a reasonable assumption; however, when these issues resolved following corticosteroid therapy, we questioned whether her clinical presentation could instead represent a manifestation of her underlying rdd. this case highlights the diagnostic challenge of differentiating between an infection and an rdd exacerbation. the episodes of presumed infections were considered probable but not confirmed with microbiologic or histopathologic specimens. the mechanism underlying lymphopenia in rdd is not clear but may involve decreased production, increased destruction, or sequestration of lymphocytes. to our knowledge, this has not been specifically studied in rdd in the past, however lymphopenia has been linked to lymphocyte maldistribution in other diseases. for example, studies have shown that experimentally altering either the surface of the lymphocyte or the environment through which the lymphocyte travels through can cause sequestration of lymphocytes in various lymphoid organs including lymph nodes. conclusion: we describe the case patient with rdd that developed persistent lymphopenia, and multiple episodes of presumed infections resulting in hospitalization and intravenous antibiotic therapy. her lymphopenia resolved and she had sustained remission of rdd following treatment with corticosteroids. we hypothesize that lymphocyte sequestration in enlarged lymph nodes may have resulted in lymphopenia. this, combined with recurrent rdd exacerbations that clinically resemble infections created a presentation that mimicked an immunodeficiency. background: there is an expanding spectrum of immunodeficiency phenotypes linked to dna repair defects, and some patients may not be diagnosed until adulthood. the most well recognized genetic defect linked to dna repair is in the gene, ataxia telangiectasia mutated (atm), which causes ataxia telangiectasia, characterized by combined immunodeficiency, neurodegeneration, radiation sensitivity, and ocular telangiectasias. however, there are several other dna repair defects associated with immunodeficiency, including some syndromic and severe combined immunodeficiency (scid) disorders. objective: we present the case of an adult patient with prolonged history of recurrent infections, facial abnormalities, and autoimmunity who was found to have radiosensitivity suggestive of a dna repair defect. methods: retrospective chart review, immunodeficiency evaluation, flow-based radiosensitivity assay, gene sequencing. results: a -year-old female was referred to our clinic due to a complex history of recurrent infections and immune dysregulation. the patient had a lifelong history of sinopulmonary infections and panhypogammaglobulinemia with low vaccine responses, leading to a diagnosis of common variable immunodeficiency (cvid), necessitating treatment with immunoglobulin replacement. clinical features were also notable for congenital dysmorphia (strabismus, thin and angular face, high arched palate, nasal septal defect, small mouth, missing dentition, clinodactyly, severe equinovarus, and scoliosis). she was subsequently diagnosed with autoimmune features of vasculitides requiring trial of cyclophosphamide, azathioprine, rituximab and belimumab, which was later discontinued due to neutropenia and worsening sinopulmonary and skin infections despite immunoglobulin replacement. in the course of our evaluation she was revealed to have severe b cell lymphopenia ( %), cd naïve t cell lymphopenia, persistent iga and igm deficiency one-year post rituximab therapy, and elevated alpha fetoprotein (afp). radiosensitivity assay revealed decreased atm phosphorylation and elevated levels of h ax -hours after low-dose ( gy) radiation in her lymphocyte subsets (t, b and nk cells) . due to the evidence of radiosensitivity and elevated afp levels, there was concern for an atm or other genetic defects in a dna repair pathway. therefore, a targeted (primary immunodeficiency genes) panel was pursued for genetic testing ( genes, invitae, san francisco). the evaluation did not identify a variant in the atm gene but rather a variant of uncertain significance was identified in the chd gene, in exon , c. g>a (p.gly arg), which may be mosaic. this variant has not been reported in population databases. chd is typically associated with charge syndrome, and while this patient has some dysmorphic features, she is not typical for charge syndrome. currently, studies on copy number variation (cnv) and deep intronic variants in atm are pending. conclusion: dna repair defects may occur in adult patients with a primary diagnosis of cvid. our patient exhibits some phenotypic features of both a chd variant, and atm leading to possible abnormal dna damage responses (ddr). the exact cause of the immune deficiency in our case remains presently unsolved. this case highlights the relevance of both functional studies and genetic evaluation of complex cases of immune dysregulation, for improving our understanding of the phenotypic variability in these immunological disorders. background: womens health issues in patients with immunodeficiency are largely underrepresented in the literature. there are no studies assessing for fertility issues in patients with antibody deficiencies, and there are few sizable studies examining pregnancy and outcomes on progeny in the same cohort. the two largest studies of pregnancy in antibody deficiency, an idf survey and a study of the czech population, provide conflicting data about the safety of pregnancy for these patients. immunoglobulin replacement has been shown to be safe and beneficial in pregnancy for patients with cvid, however, dosing strategies are unguided. we sought to further understand these and other issues associated with fertility and pregnancy in a large cohort of patients with antibody deficiencies. methods: we performed a retrospective chart review of over patients with icd and/or icd codes of cvid or another antibody deficiency from january to december . inclusion criteria also comprised of having reached at least years of age, the beginning of child bearing years. data collected included disease characteristics, comorbidities, laboratory values, and outcomes. this was followed by a phone survey to elucidate data regarding fertility, pregnancy, delivery complications, and outcomes of children. this study was irb approved. results: the current age of women included ranged from to years of age, currently being in childbearing years to being post-menopausal. forty percent of the women had been pregnant, delivering an average of babies per woman who had been pregnant. fertility issues were not a prominent factor for women who never became pregnant. a majority of women who had babies ( %) did not receive a diagnosis of antibody deficiency until after their child bearing years. recurrent upper respiratory tract infections, bacterial sinusitis, and urinary tract infections during pregnancy were common even in those not yet diagnosed with antibody deficiency. immunoglobulin levels and dosing of intravenous and/or subcutaneous replacement were recorded for a subset of patients with recent pregnancies. the data re-enforced that increases in dosing are needed in the third trimester. cord blood igg levels were also recorded for baby and were the same or higher than the mothers most recent igg prior to delivery. it was rare for children of our patients to be diagnosed with antibody deficiency or a related condition, although cvid, hypogammaglobulinemia, combined immunodeficiency, lymphoma, rheumatoid arthritis, and other diagnoses were found. conclusion: this is the largest report of outcomes before, during, and after pregnancy for patients with antibody deficiencies in the united states. this report highlights the importance of closely monitoring women during pregnancy for recurrent infections regardless of whether a diagnosis of antibody deficiency is present. it also highlights that close monitoring of igg levels during pregnancy is necessary for women with antibody deficiencies. backgrounds: autoinflammatory diseases (aids) are a group of disorders with an inborn error of innate immunity, characterized by recurrent episodes of fever and inflammatory attacks. the spectrum of aids is expanding, but no data on clinical presentation and symptom variability exist for the iranian population for timely precise diagnosis. this study aims at establishing the first autoinflammatory registry of an iranian population focusing on the clinical and laboratory features that may help clinicians toward a better understanding and diagnosis of these disorders. methods: clinical and laboratory characteristics of patients who clinically and or genetically diagnosed with aids collected. we used the updated version of classification criteria from the eurofever registry for the clinical diagnosis. results: in our retrospective study, clinical and laboratory characteristics of the participants collected. mean age of disease onset, disease course manifestation, the mean duration of episodes, atypical symptoms, laboratory and imaging studies as well as complications, and response to treatment also reviewed. data resulted in patients of whom were male. their age ranged from to years. out of were genetically diagnosed. familial mediterranean fever (fmf) was the most common clinically and genetically approved diagnosis. there were also patients suspected of nlrp and nod mutations. age at disease onset differed variably and ranged from the neonatal period to adulthood. fever was present in all the participants and the duration of episodes was - days. the frequency of attacks was between to more than per year. some of the common clinical manifestations were as follows: myalgia or fatigue ( %), arthralgia and arthritis ( %), abdominal pain ( %), aphthous stomatitis ( %), chest pain ( %), chronic gastrointestinal symptoms ( %), skin lesion ranging from urticarial rash and severe nodular acne to pyoderma gangrenosum ( %), exudative and or erythematous pharyngitis ( %), consanguineous parents ( %), symptoms of a type of allergy ( %), lymphadenopathy ( %), splenomegaly ( %), increased acute phase reactant ( %), elevated liver function test ( %) . out of of the individuals reported positive family history and in one of the cases, a patient carrying the homozygous mutation in the mefv gene has shown no clinical manifestation. conclusion: this study highlights the most common manifestations of aids in the population of iranian origin and can be used as evidencebased clinical criteria for their diagnosis. background: the term benign ethnic neutropenia (ben) is used to describe patients of african/arabic descent with absolute neutrophil counts (ancs) less than cells/ul in the absence of other causes. historically, race has been used to support the diagnosis of ben, but self-reported race is notoriously imprecise. the duffy null phenotype (fya -/fyb-) is a known molecular cause of ben and may be a more reliable marker of ben than self-reported race. in addition, although the anc is known to be lower in patients with ben, the lower limit of ancs is poorly described. it is important to differentiate patients with ben from primary immunodeficiency diseases (pidd) and to recognize their expected anc values. methods: eligible subjects included patients less than years seen at the university of michigan between january -july . duffy null (fya -/fyb-) patients were identified using electronic medical record search engine (emerse) software and search terms duffy and fyab. potential subjects were identified; patients met inclusion criteria including duffy null status and the absence of other conditions or medications, potentially impacting ancs. unique healthy anc values were recorded from the duffy null patients. age and sex matched controls were identified using emerse software with search terms tonsillectomy, department of anesthesiology and absolute neutrophil count. subjects with conditions or medications that might impact the anc or of african/arabic descent were excluded from the control group. asian and caucasian patients included as controls were presumed to be duffy null given that < % of these populations are expected to be duffy null. control subjects were identified; met inclusion and exclusion criteria. statistical analysis was performed using two-sided two-sample t-test, anova and onesample t-test. results: the median age of the duffy null cases was . years (iqr: . - . ) with . % (n= ) male and all of african or arabic descent. mean anc for duffy null patients was cells/ul (n= , sd= ) while mean anc for controls was cells/ul (n= ; sd= ) with a mean difference between controls and duffy null cases of cells/ul ( % ci: - ; p= . ). the anc levels between duffy null individuals and controls were evaluated by five age categories (p= . for all age categories). however, there was no difference in anc levels between duffy null cases at different age categories (anova, p= . ). ( . %) duffy null cbcs had anc levels in the nonneutropenic range (> cells/ul), ( . %) cbcs had mild neutropenia ( - cells/ul), ( . %) cbcs had moderate neutropenia ( - cells/ul), and ( . %) cbcs had severe neutropenia (< cells/ul). conclusions: although neutropenia can be associated with pidds and is often a sign of a compromised immune system, duffy null patients have a wide range of values that are often much lower than previously appreciated. the degree of neutropenia related to duffy null phenotype appears to persists throughout childhood and young adulthood. in the context of patients of african/arabic descent presenting with asymptomatic neutropenia, duffy null status should be assessed, and ben should be considered in the differential. complications, hypogammaglobulinemia and a unique characteristic of decreased susceptibility to enveloped viral infections. objective: to investigate the role of impaired host n-linked glycosylation on viral susceptibility to ebola virus. methods: to mimic the condition observed on cdg-iib patients, we tested in vitro three proprietary iminosugars (emergentbiosolutions©), uv b, uv , and uv , which act as competitive inhibitors of -glucosidase i and ii. their ability to inhibit the trimming of n-glycans was compared to known n-glycans modifiers as castanospermine, tunicamycin, as well as the bacterial enzyme peptide-nglycosidase f (pngase-f). ebola virus envelope protein gp was chosen as a prototype glycoprotein, as it is heavily glycosylated with nglycosylation sites. hek t cells were seeded at x ^ cells/well in well plate. after h, cells were transfected with pflag-ebolavirus gp by coupling with effectene®. after h, cells were treated with the inhibitors and harvested h after treatment. trimming of n-glycans was evaluated via molecular weight assessment by western-blot. results: all three inhibitors had comparable effectiveness in inhibiting trimming of nglycans from ebola gp glycoprotein compared to castanospermine. a greater molecular weight shift was seen with tunicamycin and pngase f as expected. conclusions: chemical inhibition of the n-linked glycosylation pathway was successfully achieved using three new mogs inhibitors. this approach merits further investigation on potential applications on antiviral therapies. investigator, laboratory of human genetics of infectious diseases, necker branch, inserm u , necker enfants malades hospital, paris, france head, immunodeficiencies research unit, national institute of pediatrics stat gof mutations are associated with infections, autoimmunity and inflammatory manifestations; the rosacea is one of the manifestations described in this disease, however, the etiology rosacea is not clearly established. the characteristics of rosacea are not described in stat gof in the different clinical series. we describe the different characteristics of rosacea in a family with affected members with stat gof. a family with eight members with stat gof mutation were diagnosed through a first affected member affected with tuberculosis and onychomycosis. seven members more had a clinical history of mycobacterial, viral and fungus infections and autoimmunity disease, in all the seven, was documented the same mutation stat gof. in six of these adults patients, we documented rosacea, it started after adolescence, it was localized in the face and/or eyes, was progressive and not ameliorated with medical treatment and caused nose deformity. rosacea has been described previously as a unique manifestation, and the etiology is not clear, an autoimmune hypothesis has been proposed. the fact that is present in patients with stat gof could suggest that have effectively an autoimmune component. physicians face the patients with rosacea must look for other manifestation presents in stat gof mutations. genetic studies in rosacea patients could evidence an new gene defect. introduction: homozygous mutations causing loss of function of the transcription factor forkhead-box n (foxn ) underlie autosomal recessive severe combined immunodeficiency with congenital alopecia and nail dystrophy (nude scid). affected humans, like the scid mouse, have small or absent thymus, absent or severely diminished t cells, alopecia, and nail dystrophy. infants with nude scid have had neonatal lymphopenia and severe, life-threatening infections. studies of heterozygous carriers of foxn mutations are limited, some having been reported with no phenotype or mild disease manifestations, such as nail dystrophy without lymphopenia or recurrent infections. objective: we describe six infants, including two brothers, with t-cell lymphopenia (tcl) following abnormal california newborn screens (nbs) for scid. each had a single heterozygous variant in foxn . case reports: six infants ( female, male) were referred for evaluation after abnormal california nbs for scid (table ) , with t-cell receptor excision circle (trec) counts from undetectable to (normal > ). all infants were well at the time of initial evaluation. five infants with absolute cd t cell counts > cells/ul and cd t cell counts > cells/ul began evaluation as outpatients on home isolation. patient , with undetectable trecs, cd t cell count , and cd t cell count was urgently admitted for inpatient evaluation and management and immediately started on antimicrobial prophylaxis. patient further evaluation was significant for lymphocyte proliferation to mitogens that was initially normal but waned with time, prompting treatment with a paternal haploidentical hematopoietic cell transplant at months of age. patients and developed neutropenia within weeks of birth treated with granulocyte colony stimulating factor (gcsf). patient remains well on gcsf but has had persistent growth failure under continued evaluation. patients , , and remain stable off antimicrobial prophylaxis, but with persistent moderate tcl. as part of an immune evaluation, patients and - had gene panel testing revealing heterozygous variants in foxn . only the variant of patient (presumed shared by patient , his brother) was predicted to be pathogenic; patient had dystrophic nails and sparse hair most evident after years of age, features shared by his mother and his brother, patient . the other patients lack the clinical features of the previously described phenotype of nude scid. their heterozygous foxn variants are of unknown significance; the functional role of these variants in the patients clinical phenotype is unknown. conclusion: six infants with abnormal nbs for scid had lymphopenia and heterozygous variants in foxn . for these infants, variation exists in level of tcl and presence of hair and nail findings. heterozygous variants of unknown significance in foxn have been uncovered in others, including infants with abnormal nbs for scid, highlighting the need for functional studies to address the possible role of each heterozygous foxn variant in congenital lymphopenia and neutropenia. more work is needed before attributing tcl to a novel foxn variant of unknown significance in the absence of family history, abnormal hair or nails, or functional evidence. remains poorly understood. we characterized the intestinal microbiome and metabolome in patients with cgd to determine if intestinal microbiome and metabolomic signatures could distinguish subpopulations of patients with cgd while using the metabolome to add a functional dimension to observed microbiome signatures. methods: clinical metadata and fecal samples were collected crosssectionally from healthy volunteers (hv; n= ) and patients with cgd (n= ). metabolomic profiling and s rrna (v ) sequencing was performed on fecal samples (total samples: ; reads/sample: , to , ; median: , ) . results: samples from patients with cgd had distinct intestinal microbiome signatures and metabolomic profiles depending on genotype, presence of cgd-ibd and specific interventions (e.g. treatment with an elemental diet). notably, samples from patients with active cgd-ibd (compared to samples from patients without a history of cgd-ibd) had significantly different alpha-and betadiversities, and were enriched for enterococcus spp. signal transducer and activator of transcription gain of function (stat -gof) is a primary immunodeysregulatory disease in which a subset of patients have features of autoimmunity and autoinflammation. enteropathy with growth failure and nutrient wasting is a more common feature of immunodysregulation. ruxolitinib is a janus kinase-stat inhibitor that has been shown effective for the treatment of immunodysregulatory features in stat -gof. our patient is a year old male with stat -gof (c. a>g p.h r) with severe total parenteral dependent enteropathy that led to growth failure (weight . kg). treatment with ruxolitinib led to resolution of diarrhea, return of normal diet, and catch up growth. a dose of . mg twice daily was initially started but was decreased to . mg every morning and mg every evening due to elevated transaminases and thrombocytopenia. over the following year the patient thrived gaining . kg with normal every other day stools. despite weight gain, he remained stable on the same dose of ruxolitinib. as he outgrew his dose, he developed an increased frequency of upper respiratory infections (parainfluenza, coronavirus, rhinovirus). one year after initiation of ruxolitinib, he again developed profuse watery diarrhea that was norovirus positive (weight kg, bsa . ). he was placed on bowel rest and ruxolitinib was dose escalated with a goal of mg/m /day. when he reached mg twice daily, enteropathy completely resolved but liver function tests began to rise. he gained weight and began thriving after weeks of therapy. six months later, enteropathy is controlled, and transaminases have remained elevated (alt iu/l, ast iu/ml) but stable. the appropriate dose and pharmacokinetics for ruxolitinib for the treatment of immunodysregulatory symptoms in pediatric patients has not been thoroughly studied. the dose used was extrapolated from data on the use of ruxolitinib in pediatric myelofibrosis. a dose of mg/m /day appears to provide the most benefit with tolerable adverse effects. this dose should be maintained in order to prevent recurrence of disease related manifestations. abstract clathrin-mediated endocytosis (cme) is the major endocytic pathway by which eukaryotic cells internalize cell-surface cargo proteins and extracellular molecules, thereby allowing for a broad range of biological processes, including cell signaling, nutrient and growth factor uptake, and cell fate and differentiation . the fbar domain only proteins and (fcho /fcho ) are involved in the initiation of clathrin coat pit formation. whether fcho and fcho are functionally redundant or have distinct functions is unclear. we report here the first cases of a severe immunodeficiency due to a genetic defect affecting cme. by using whole exome sequencing and genomic analysis of a targeted pid gene panel, we have identified biallelic loss-of-function fcho mutations in five patients from unrelated families of italian (p ), turkish (p , p , and p ) and algerian (p ) origin with severe t cell lymphopenia manifesting as recurrent and severe infections of bacterial, mycobacterial, viral and fungal origin. p developed ebv-associated diffuse large b cell lymphoma. three patients (p -p ) died in childhood, whereas p and p are alive with full donor chimerism at and . years after allogeneic hematopoietic stem cell transplantation, respectively and have cleared pre-transplant infections. patients p , p , and p carried homozygous frameshift mutations predicted to cause premature termination. western-blotting analysis of ha-or flag-tagged fcho constructs showed expression of truncated products in p and p , whereas no protein was detected in p , presumably due to mrna decay. p and p carried homozygous splice-site mutations at the invariant - and + positions, respectively, leading to skipping of exon in p 's fcho cdna. qpcr analysis demonstrated differential expression of the fcho and fcho genes, with the former being predominantly expressed in lymphoid cells, whereas fcho was more abundantly expressed in fibroblasts and k cells. analysis of t cell activation in p (the only patient for whom pre-transplant pbmc were available) revealed reduced t cell proliferation. while tcr internalization in response to cd cross-linking was normal (consistent with recent evidence that tcr internalization occurs through a clathrin-independent pathway), chase experiments demonstrated that transferrin internalization was abolished in activated t cells from p . we had previously reported that a missense mutation in tfrc, encoding transferrin receptor , impairs transferrin internalization and intracellular iron delivery, causing a combined immunodeficiency with defective t cell proliferation. our data identify the first form of severe immunodeficiency due to defects of clathrin-mediated endocytosis, and provide additional evidence in support of the critical role played by iron cellular metabolism in t cell function and homeostasis. natural history of anti-interferon-gamma autoantibody-associated immunodeficiency syndrome in thailand submission id# centralized sequencing initiative at niaid: year therefore, we set out to investigate the pneumococcal-specific responses of igg, igg , iga and igm to prevnar ® in igg subclass deficient (iggscd) patients in this study. pneumococcal responses were measured using the vacczyme pneumococcal capsular polysaccharide igg, igg , iga and igm elisas (the binding site group, birmingham, uk) in control (n= , median age years, range - ) and iggscd patients (n= , median age years, range - ) recruited from the immunodeficiency unit at the karolinska university hospital iga and igm antibodies in response to pcv vaccination was observed weeks post vaccination in iggscd patients (median, . th and . th percentile these median concentrations were lower than those observed in control patients (median, . th and pcv igg mg/l, - however, percentage changes between pre to post vaccination concentrations of igg, igg and iga in response to pcv in iggscd patients were not significantly different to the control patients u/ml vs . u/ml, respectively) iga u/ml and pcv igm u/ml) responders and non-responders of pcv igg iga and igm in response to pcv in iggscd patients were generally lower compared to the control population. these results support the fact that in addition to igg and igg , measurement of iga and igm could also provide useful information for the clinician gain-of-function ikbkb mutation causes human combined immune deficiency submission id# neutralizing anti-il- -autoantibodies are a risk factor for pyogenic bacterial infections national institutes of health, national institutes of allergy and infectious diseases service of immunology and rheumatology, garrahan national pediatric hospital copa mutations impair er-golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis copa syndrome: a novel autosomal dominant immune dysregulatory disease analysis of pulmonary features and treatment approaches in the copa syndrome expanding the phenotype of copa syndrome: a kindred with typical and atypical features the forest and the trees: machine learning to classify cases of suspected inborn errors of immunity using decision tree and random forest algorithms submission id# card Δ gene dosage: from mono-allelic protection to ibd, to bi-allelic increased fungal infection susceptibility yamanaka d , walkiewicz m , lionakis m and rosenzweig s stim mutation associated with a syndrome of immunodeficiency and autoimmunity a novel hypomorphic mutation in stim results in a late-onset immunodeficiency clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in stim gain-of-function mutation in stim (p.r w) is associated with stormorken syndrome gain-of-function mutations in stim and orai causing tubular aggregate myopathy and stormorken syndrome stormorken syndrome caused by a p.r w stim mutation: the first italian patient and a review of the literature by studying ecs-pre and ecs-post patients we were able to describe the bona-fide effect of gcs on the immune system in general, and t lymphocytes in particular. decreased lymphocyte/thymic output, as well as increased apoptotic tcell death underlies lymphopenia in ecs/chronic gcs-exposed patients. under such conditions, il- was significantly decreased in plasma and our in-vitro studies showed that il- replenishment was able to increase bcl (anti-apoptotic molecule) and bcl expression, and efficiently counteract the apoptotic effects of gcs. recombinant il- has been explored as a co-adjuvant treatment for multiple human cancers and may offer a treatment option for lymphopenia and its genetic counselor, co-director of personalized medicine, division of hematology/oncology/bmt and the institute for genomic medicine, nationwide childrens hospital genetic counselor, division of hematology/oncology/bmt, nationwide children's hospital acknowledgments. genetic sequencing was kindly provided by drs. raif geha and janet chou at the division of immunology, allergy, rheumatology and dermatology, boston children's hospital, harvard medical school. the following grants are acknowledged: . rui . /cippt/ (usm) . bmbf eo (freiburg) the authors would like to thank the director general of health of malaysia for permission to publish this scientific presentation. while severe viral infections may also be an initial presentation of primary immunodeficiency, an immune evaluation is not always obtained in this scenario. patients with xla have an increased susceptibility to severe enterovirus infections, manifesting as chronic meningoencephalitis, which can be fatal. the following case describes a patient with newly diagnosed xla presenting as suspected coxsackievirus and confirmed hhv- meningitis, pseudomonas meningitis and bacteremia. this may be the first reported new diagnosis of xla presenting with both severe bacterial and viral coinfection. case description: a year old, partially vaccinated, hispanic male with a history of febrile seizures presented to the emergency room with fever, oliguria, watery diarrhea, lethargy, meningismus, ecthyma gangrenosum and lower abdominal pain. eight days prior to presentation, he was seen by his pediatrician for facial rash and low grade temperature, and was diagnosed with hand-foot-and mouth disease. he worsened on empiric antibiotics. he had no history of sinopulmonary infections. he did not attend daycare. his vaccines were delayed due to parental choice, and he had not received live vaccines (rotavirus, mmr or vzv). full sepsis evaluation was performed. csf demonstrated pleocytosis, and he was started on empiric antibiotics and transferred to picu. due to worsening abdominal pain, ct of the abdomen was performed, which was consistent with ruptured appendicitis and septic emboli at the lung bases. csf pcr panel was positive for hhv- and he was started on gancyclovir. csf and blood cultures subsequently grew pseudomonas aeruginosa. immune evaluation was performed. serum immunoglobulins were undetectable. in addition to iv antibiotics, he received mg/kg ivig and lymphocyte subsets revealed profound b cell lymphopenia ( . %, cells/ul). btk protein analysis revealed hemizygous btk pathogenic variant confirming the diagnosis of x-linked agammaglobulinemia. the hospital course was further complicated by brain abscesses and pyoventriculitis. he was treated with additional doses of mg/kg ivig and iv antibiotics. repeat mri of the brain nearly weeks after admission demonstrated significant improvement. there was significant clinical recovery. he was discharged home at baseline neurological status. his igg level upon discharge home was mg/dl with the plan to increase dose to mg/kg per month with close monitoring. conclusion: both severe opportunistic bacterial infections and severe viral infections as the initial presentation of xla have been well reported in the literature. this case describes the first reported severe pseudomonas aeruginosa and hhv- co-infection in a newly diagnosed xla patient. this case further highlights the necessity for an increased index of suspicion of primary immunodeficiency in a patient who presents with a severe first infection, despite lack of recurrent infections. we present two patients with dock deficiency due to compound heterozygous variants including a copy number loss at chromosome band p . spanning approximately . mb with partial deletion of the dock gene and a novel c. c>t (p.ser leu) missense variant [chr : (grch ) nm_ ] in dock . functional data is presented to support the pathogenicity of the missense change, along with a review of the literature on dock variants. the proband is a -year-old female with elevated serum ige, severe atopic dermatitis, mild persistent asthma, food allergies, and seasonal allergic rhinitis. she is currently healthy following haploidentical bone marrow transplant in june . she has a -year-old brother with dock deficiency with the same compound heterozygous variants. the brother had later onset of symptoms and a milder presentation of intermittent asthma and seasonal allergic rhinitis. each of the parents is heterozygous for one of the two variants. we evaluated the pathogenicity of the c. c>t missense variant with western blots of dock protein expression, intracellular flow cytometry, and dock stretch assays. flow cytometry showed decreased dock protein expression and stretch assays revealed t cells that were stretched in collagen gels. notably, dock is a large gene containing exons spanning kb and it is relatively common to be a carrier of a rare missense change. in fact, gnomad has approximately individuals with rare (< . frequency) missense alleles in dock . therefore, it is important to demonstrate the potential pathogenicity of any given rare missense change, since few pathogenic missense variants in dock have been reported. of the published dock variants listed in the human gene mutation database (hgmd) only are missense. the majority are gross deletions, of which were reported in hgmd. the remaining reported dock variants include nonsense, splicing, small deletions (all frameshifting), small insertions (all frameshifting), small indels, and gross insertions/duplications. this case demonstrates the relatively infrequent but important contribution of missense changes to pathogenic dock alleles. functional validation of missense alleles is critical in the complex evaluation of dock deficiency. background: hsct is the only known curative option currently for cd l deficiency, an x-linked disorder. in cd l deficiency and other x-linked immune deficiencies, there is an ongoing debate regarding the use of a carrier female sibling or mother as hsct donor. skewed lyonization despite complete donor chimerism has raised concerns for incomplete disease control post-hsct. no data exist regarding the efficacy of related female carrier as hsct donor for cd l deficiency. we herein report outcomes of three patients with cd l deficiency who underwent hsct using a related female carrier donor. method: retrospective review of patients who received hsct from carrier female related donor at three separate institutions. results: three patients with cd l deficiency underwent hsct between - . patient had recurrent episodes of pneumocystis jiroveci pneumonia (pjp) despite being on bactrim and immunoglobulin replacement. patient presented with pjp and severe neutropenia. patient presented with acute respiratory failure from severe respiratory viral infections, cmvand had severe neutropenia requiring g-csf treatment. age at the time of hsct ranged from . - yrs. all three underwent reduced toxicity hsct with busulfan and fludarabine-based preparatory regimens. two of them received matched sibling bone marrow hsct and one received tcr and cd depleted mobilized maternal pbsc haploidentical hsct. donor cd l expression varied from % - % on activated cd cells. immunoglobulin profile and lymphocyte subset were done in two of donors, they were within normal range for age, and none had significant infection history. no history of intermittent neutropenia or oral ulcers noted in donor and the absolute neutrophil count of the donor varied between /l. donor age ranged from . yrs years. cd dose ranged from . x - . x cells/kg and cd dose ranged from x . x cd + cells/kg. gvhd prophylaxis consisted of csa/mmf (n= ) and tcr-a/b depletion and no csa (n= ). neutrophil engraftment ranged from - days and platelet engraftment ranged from days. none of the patients developed acute or chronic gvhd. all three patients maintain full donor myeloid chimerism at the latest testing ( months months); t cell chimerism was % in one and mixed in two patients ( % at nine months, % at months). all three patients had excellent t cell immune reconstitution; two patients came off immunoglobulin replacement - months post hsct, whereas the rd patient is ivig dependent, though iga level was mg/dl at nine months post-transplantation. latest evaluation, months post-hsct, revealed % - % cd l expressing activated cd t cells, which correlated with donor cd l expression and t-cell chimerism. conclusion: our data suggest that hsct utilizing x-linked carrier appears to be safe and results in durable engraftment with excellent humoral and cellular immune reconstitution in patients with cd l deficiency. longer follow-up and data from a larger cohort is needed to make a definitive determination of safety and efficacy of utilizing female carrier as hsct donors in this disease. chief, immunology service, department of laboratory medicine, nih clinical center, bethesda, md, usa background: ikaros belongs to a hematopoietic-specific zinc-finger (zf) family of transcription factors. after dimerizing and dna binding to pericentric-heterochromatin (pc-hc) regions, ikaros is described as a central regulator of lymphocyte differentiation. somatic mutations/ deletions affecting ikaros n-terminal zf have been identified in b-acute lymphoblastic leukemia (all) patients, and germline n-terminal mutations were reported in cvid patients with progressive lack of b cells, hypogammaglobinemia, autoimmune diseases and b-all. methods: we performed targeted sequencing panel for known inborn errors of immunity disease-causing genes in a previously healthy male pediatric patient with burkitt lymphoma, followed by benign lymphoproliferation, thrombocytopenia and neutropenia. b-cells and immunoglobulin levels were normal. ikaros dna-binding, nuclear localization and protein binding were evaluated by emsa, fluorescence microscopy and immunoprecipitation. protein modeling was also performed. results: a novel heterozygous germline mutation in ikaros c-terminal zf dimerization domain (p.r l) was detected in this patient. this mutant showed normal pc-hc localization but dna-binding was markedly reduced in terms of ikaros dimerization and multimerization. moreover, reduced wt-mutant binding was also detected. mutant/wt cotransfection experiments suggest a haploinsufficient defect. geometry based docking of wildtype ikaros predicted that r is within the homodimer interface and may abolish cation-pi interactions and destabilize the ikaros-zf dimerization domain. conclusion: a novel germline ikaros c-terminal mutation affecting homodimerization/multimerization and resulting in reduced dna binding to its dna consensus site was detected in a patient with burkitt lymphoma, benign lymphoproliferation and cytopenias. further studies are warranted to formally establish the casual connection between this genotype and phenotype.( ) submission id# patricia pichilingue-reto, md , prithvi raj, phd , igor dozmorov, phd , quan-zhen li, md, phd , edward wakeland, phd , nancy kelly, md , maria teresa de la morena, md , nicolai s. van oers, phd methods: mice were generated by crispr/cas technology to genocopy the foxn compound heterozygous mutations identified in one of the human patients. thymopoiesis and hair follicle extrusion was analyzed in the various heterozygous and homozygous mutant mice. gene expression analyses of the hypoplastic and normal-sized thymii and the developing skin were performed. in addition, a structure-function analysis was performed with luciferase reporter assays using distinct and previously unreported foxn mutations uncovered in patients who presented with low trecs. results: mice harboring compound heterozygous mutations in foxn that match the human patient phenocopy the t-b+nk+ scid phenotype with normal hair and nails. a functional characterization of the diverse foxn mutations suggests that the severity of the block in thymopoiesis depends on whether the mutations affect the dna binding or transactivation domains of foxn . a -amino acid segment at the end of the dna binding domain appears to be essential for tec development. however, this segment is not required for normal keratinocyte functions in the skin and nail plate. gene expression comparisons are revealing key targets of foxn that suggest a dichotomy in its function in the thymus versus the skin. conclusions: novel compound heterozygous mutations in foxn are causal to a t-nk+b+ phenotype with normal hair shaft extrusion and nail plate extension. this differs from the classic nude/scid (omim # ) reported for individuals with autosomal recessive mutations in foxn . assistant professor of medicine and pediatrics, department of allergy and immunology, uva introduction: copa syndrome is a recently described monogenic immunodysregulatory syndrome. the cop protein, encoded for by the copa gene, is expressed in all cell types and is involved in trafficking from the golgi complex to the endoplasmic reticulum ( ) . the most common clinical features of copa syndrome are interstitial lung disease, pulmonary cysts or follicular bronchiolitis, pulmonary hemorrhage, arthritis, glomerular disease, and autoantibody development ( , ) . atypical features of copa syndrome identified thus far include: extrapulmonary cysts in the liver and kidney, renal and neuroendocrine malignancies, autoimmune neurological disorders such as neuromyelitis optica, and infections, such as meningitis ( ) . clinical case: we present a case of a year-old male with copa syndrome (de novo heterozygous mutation in exon , c. g>c; p.ala pro) manifesting as lymphocytic interstitial pneumonitis, peripheral blood b-cell lymphocytosis, mediastinal lymphadenopathy and persistent transaminitis (alt and ast - u/l, nl ast< u/l, alt < u/l) with normal bilirubin, alkaline phosphatase and pt/inr. the transaminitis was noted prior to diagnosis of copa syndrome, and has persisted despite seven months of therapy with pulse dose steroids, two cycles of rituximab and maintenance therapy with hydroxychloroquine and prednisone. he has had a normal ck and aldolase excluding muscle injury as a source of his transaminitis. a congenital cholestasis panel was normal. markers of autoimmune liver disease including ana, anti-liver kidney microsomal antibody and anti-smooth muscle were negative. serum ceruloplasmin and alpha- -antitrypsin level were normal and celiac serologies, were negative. liver ultrasound was normal. a liver biopsy did not demonstrate inflammatory changes, hepatocyte necrosis, mononuclear cell infiltrates or fibrosis. nonspecific biopsy findings included occasional intraparenchymal neutrophils. it is unclear if these scattered neutrophils and the transaminitis are due to an early as yet unidentified autoimmune process, perhaps in response to hepatocellular stress exacerbated by the copa mutation. discussion: liver involvement has not been reported in copa syndrome. we describe a child with copa syndrome who has had chronic transaminitis with no clear alternative cause. if the phenotypic spectrum of copa syndrome involves the liver, it may limit immunomodulatory options for the treatment of this disease. background: in humans, biallelic stat lost-of-function (lof) mutations lead to a very low or complete absence of the wild-type (wt) protein. whereas, heterozygous mutations can lead to partial loss of function. these patients are susceptible to mycobacteria and herpes virus infections. on other hand, heterozygous gain-of-function (gof) mutations in the stat gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections, viral infections and autoimmune disease. methods: in this study, we evaluated novel stat mutations, three gof and one lof. in vitro, pbmcs from these patients were stimulated with ifn-and ifn-for , , and minutes and levels of phospho-stat were measured by flow cytometry. the stat phosphorylation and activity (firefly and renilla luciferase activities) were evaluated in u a-stat deficient cells transfected with a reporter plasmid (for luciferase), wt or mutant-stat plasmids. results: we observed higher levels of stat phosphorylation after two hours of stimulation from three gof mutations compared to wt. however, a lof mutation showed absent stat activation at baseline and in response to ifn-and ifn-. luciferase reporter assay confirmed gain of function and loss of function stat activity observed by flow cytometry. conclusions: using flow cytometry followed by a luciferase assay, we confirmed four novel stat mutations. measuring phosphorylation of stat by flow cytometry is sufficient to determine whether the stat mutation is disease causing. this assay can be translated to a clinically accessible test for stat related disease. background: variants in recombination-activating genes (rag) are common genetic causes of autosomal recessive forms combined immunodeficiencies (cid) ranging from severe combined immunodeficiency (scid), omenn syndrome (os), atypical scid (as) and cid with granulomas and/or autoimmunity (cid-g/ai). the clinical and immunological presentation is broad, ranging from severe infections secondary to near absence of t and b lymphocytes and hypogammaglobulinemia to the occurrence of autoimmunity with late manifestations with partly preserved immune subsets and near normal immunoglobulin levels and broad spectrum of autoantibodies. objective: we aim to estimate the incidence, clinical presentation, genetic variability and treatment outcome with geographic distribution of patients with the rag defects in populations inhabiting south, west and east slavic countries. due to shared ancestry, we also investigated our cohort for founder variants in rag and rag genes. methods: demographic, clinical and laboratory data were collected from rag deficient patients of slavic origin via chart review, retrospectively. results. based on the clinical and immunologic phenotype, our cohort of patients from families represented a wide spectrum of rag deficiencies, including scid (n= ), os (n= ), as (n= ) and cid-g/ai (n= ). sixty-six ( . %) patients carried rag and patients ( . %) carried rag biallelic variants. we estimate that the minimal annual incidence of rag deficiency in slavic countries varies between in , , live birth and it may vary secondary to health care disparities in these regions. in our cohort, % of the patients carried rag p.k vfs* (c. _ delaa), either in homozygous (n= , %) or compound heterozygous (n= , %) form. the majority ( %) of patients with homozygous rag p.k vfs* originated from vistula watershed area in central and eastern poland, and compound heterozygote cases distributed among all slavic countries except bulgaria. clinical and immunological presentation of homozygous rag p.k vfs* cases was highly diverse suggestive of strong influence of other genetic and/or epigenetic factors in shaping the final phenotype. survival of rag deficient patients without hematopoietic stem cell transplant (hsct) (n= , . %) is poor and dramatically improved in the last decade with access to hsct and tailored conditioning regimens. conclusion: we propose that rag p.k vfs* is a founder variant originating from the vistula watershed region in poland, which may explain a high proportion of homozygous cases from central and eastern poland and the presence of the variant in all slavs. our studies in cases with rag founder variants confirm that clinical and immunological phenotype only partially depend on the underlying genetic defect. hsct is becoming available for rag deficient patients in eastern europe with improving outcome. clinical immunologist, centre hospitalier universitaire de montréal (chum) background: acute gvhd following solid organ transplantation is a rare complication. intestinal and liver transplantation have the greatest risk of gvhd among solid organs due to high number of donor lymphocytes in these organs. prevalence of acute gvhd after liver transplantation is estimated to be around , - % and has a poor prognosis ( ) . chronic neurological gvhd is a rare form of gvhd with three subtypes described: cerebral vasculitis, demyelinating disease and immune mediated encephalitis. acute neurological gvhd has no clear definition and is still considered a controversial entity. case presentation: a year-old male underwent cadaveric liver transplantation for alcoholic cirrhosis and hepatocellular carcinoma. the donor was a year-old man who died from anoxic brain injury. the receiver was induced with basiliximab and then put on prednisone, azathioprine and tacrolimus. he was readmitted weeks later for myalgia, headache, fever and neutropenia. clinical state initially improved with empiric antibiotics. he then developed a skin eruption, colitis and dic. the latter was thought to be tacrolimus-induced. he was switched to cyclosporine. skin and rectosigmoid biopsies were compatible with acute gvhd. he received basiliximab and ivig and developed a refractory convulsive state. csf analysis showed elevated proteins and slight pleocytosis. cerebral mri showed non-specific white matter lesions and conventional angiography was normal. chimerism on peripheral blood was % but was % donor on csf. with the presence of chimerism on csf, evidence of cutaneous and digestive gvhd and no infectious cause, neurological gvhd was considered the most likely diagnosis. brain biopsy showed non specific change including neuropil spongiosis, microglial activation and reactive gliosis; but no signs of vasculitis or demyelinating disease. he was treated with atg, highdose systemic corticosteroids, cyclosporine, ivig and intrathecal methotrexate and corticosteroids. csf pleocytosis, proteins and chimerism improved with treatment ( % to % donor). no improvement was noted regarding his neurological state and he developed pancytopenia. he was then transfer to palliative care and died shortly after ( month and a half after liver transplant). discussion: to our knowledge, there is only one prior case published of neurological gvhd following liver transplantation ( ) . both patients were old, had hepatocellular carcinoma and had at least one hla match. age > year, hepatocellular carcinoma and shared hla antigen are known risk factors for gvhd following liver transplantation ( ). our patient had only one hla match with the donor. this case is intriguing as there was a great discrepancy between blood and csf chimerism. acute neurological gvhd following transplantation is a real complication. it must be taken into consideration in patients with neurological involvement after transplant, even solid organ transplantations. introduction: hyper-igm syndrome are rare. although no data are available on the frequency of activation-induced cytidine deaminase (aid) deficiency, this disorder is estimated to affect less than : , , individuals. by the year , cases worldwide ( ) with such mutation have been described. we describe a patient with hyper igm by mutation in the aicda gene. case report: mvv, -year-old boy, born to consanguineous parents, was referred with recurrent pneumonia, which started shortly after discontinuation of breastfeeding at months old. repetitive otitis evolved with bilateral tympanic and partial hearing loss. he was submitted to adenoidectomy without improvement. immunological evaluation showed normal numbers of b and t cells with cd + ( /mm , %), cd + ( /mm , %), and cd + ( /mm , %). immunoglobulin concentrations were: igg = mg/dl (p ). treatment with intravenous immunoglobulin and prophylactic antibiotic was initiated and he had no infections during the follow up except for one episode of sinusitis. at years of age, molecular evaluation was performed and a mutation in homozygosity in the aicda gene (omim * ) at position chr : . . was found, confirming the clinical suspicion. conclusion: the role of aid in the immunoglobulin class-switch recombination (csr) and somatic hypermutation (shm) have not been fully elucidated. summarizing within the shm and csr processes, aicda mutation can induce dna lesions in directed sequences in the s and v regions required for dna cleavage. recurrent infections and consanguinity raised the suspicion of inborn errors of immunity in this patient. the literature described late diagnosis as in the second or even the third decade of life. it was suggested that high levels of igm antibodies may provide effective defense, at least, against some infectious agents. it is important to emphasize that the impossibility to obtain genetic diagnosis did not prevent to introduce therapy. * aicda: activation induced cytidine deaminase gene patients with chronic granulomatous disease (cgd) are at risk for recurring infections and non-infectious inflammation, reduced quality of life and life expectancy. conventional treatment with life-long anti-bacterial and antifungal prophylaxis prolongs lifespan but does not eliminate the lifelong risk of infection and inflammation. allogenic stem cell transplantation is currently the only curative option for this disease. although sct with reduced intensity conditioning has improved treatment-related mortality and efficacy, it remains a matter of debate whether all patients with cgd benefit from sct, whether pre-existing infections and non-infectious inflammation are risk factors and at what age sct should be performed. we compared patients with cgd on conventional treatment with those after stem cell transplantation for their prognosis and evaluated potential risk factors for stem cell transplantation outcome followed up in six european centers. frequency of infections, inflammatory complications, hospitalizations, operations and immunomodulative/immunosuppressive therapy, height and weight were compared in patients on conventional treatment /before stem cell transplantation versus patients after sct. correlation between transplantation outcome and patient characteristics or medical history was tested. patients were recruited, on ct, after stem cell transplantation. before/without transplantation % of patients suffered from at least one infection, , % from inflammatory complications. patients on conventional treatment developed infection/inflammation/ hospitalization/surgery at a median of , (range [ , - , ] , iqr , ) per year, versus (range , iqr , ) in the first year after stem cell transplantation but (range [ - ], iqr , ) after the first year post stem cell transplantation. there was a significant decrease of all complications after stem cell transplantation (p < . ). growth improved significantly after stem cell transplantation (z-score weight - , versus - , (p. ), z-score height - , versus - , (p. )). nevertheless, complications post stem cell transplantation are frequent: % of patients had at least one infection, % had severe acute gvhd, % chronic gvhd, % had graft rejection, % died. preexisting active mold infection increased the risk for complications after stem cell transplantation. in summary infections and non-infectious inflammation are common in patients with cgd on conventional treatment, their growth is significantly impaired. stem cell transplantation, if successful, significantly reduces the risk for infections and non-infectious inflammation. however, treatment related mortality of stem cell transplantation in patients with cgd remains considerable. introduction: development of a diverse t cell repertoire is essential for full immune recovery following definitive treatment for severe combined immunodeficiency (scid), whether by allogeneic hematopoietic cell transplantation (hct); autologous gene therapy (gt); or, in the case of adenosine deaminase deficiency, enzyme replacement therapy (ert). however, the time course and depth of diversity of t cell receptor rearrangements have been difficult to measure directly, necessitating estimates from total and naïve t cell counts and from spectratyping, in which t cell receptor (tcr) beta chain diversity is estimated by the length distributions of cdna amplicons between a series of tcr beta chain variable (v-beta) segments that have productively recombined with the tcr beta-chain constant region. analysis of the actual sequences of rearranged tcrs could indicate more precisely the status of the t cell compartment of these patients, and might reveal oligoclonal expansion of dysregulated t cells, t cell insufficiency, or t cell exhaustion. objectives: we wished to ascertain whether deep sequencing of individual tcr v-beta rearrangements in peripheral blood could be performed sequentially following diagnosis and treatment of scid to differentiate satisfactory immune reconstitution from incomplete or skewed repertoire development that might require further cellular therapies. methods: equal amounts of total rna were obtained from peripheral blood of controls and scid patients pre-hct and at d, and mo, and yearly post-treatment(s). cdna was used as template to semi-quantitatively amplify rearrangements at the tcr-beta locus (trb). raw sequences were filtered to remove pcr errors, and resulting fastq files were converted into fasta format (seqtk software, github, inc), filtered for productive rearrangement, and analyzed for v, d, and j gene composition and length (imgt highv-quest software). the vdj statistics file (past program) was used to calculate a shannon entropy (h) index to measure repertoire diversity, taking into account both abundance and richness of the overall repertoire; and a gini-simpson index of unevenness, measuring inequality in the relative representation of species in a given sample. graphical representations of repertoire diversity were generated by hierarchical tree maps of the trb repertoires (irepertoire software): each dot represents a unique sequence and the dot size corresponds to frequency of that sequence in the total sample. results: tcr v-beta sequence analysis of scid patients (image) showed (top) baseline poor diversity due to pre-treatment ada deficiency followed by improvement to normal complexity (shannon h > . ) after receiving peg-ada and autologous lentivirus gene therapy at age m; (middle) increasing diversity in xscid after maternal t-depleted unconditioned hct, although b cells did not recover; and (bottom) failure of initial unconditioned maternal t-depleted hct in another xscid patient at m, followed by autologous lentivirus gene therapy with subsequent improvement (shannon h increasing from . to ) months later. conclusions: tcr v-beta diversity sequence analysis provided a detailed assessment of repertoire diversity in response to cellular therapies for scid. this method could become a useful predictive tool to measure successful t cell immune reconstitution, both as early as d and in the years following treatment. background: the stim (stromal interaction molecule ) protein, encoded by the stim gene, is involved in calcium regulation in the endoplasmic and sarcoplasmic reticulum. pathogenic variants in this gene are associated with three different disorders. homozygous loss-of-function (lof) pathogenic variants in stim have been reported to cause autoimmune cytopenias, lymphoproliferation, enamel defects, anhydrosis, and iris hypoplasia. the first described cases had frequent mortality in early childhood due to recurrent life-threatening infections and development of kaposi sarcoma ( ), while recently discovered cases have had more prolonged survival, though still with recurrent serious infections ( ) . heterozygous gain-of-function (gof) pathogenic variants in stim have been associated with both tubular aggregate myopathy (tam) and stormorken syndrome. tam is a clinically heterogeneous progressive muscle disorder with a variable age of onset. muscle biopsy characteristically demonstrates tubular aggregates, with type ii muscle fiber atrophy ( ) . stormorken syndrome has a phenotype that includes miosis, thrombocytopenia, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis ( ) . the thrombocytopenia has not been reported to be immune-mediated; rather it is due to abnormal platelet calcium regulation ( ). we report a patient with stim pathogenic variant presenting with tam and immune-mediated thrombocytopenia, along with lymphoproliferative features, arthritis, and a mild immune deficiency. case: the patient is a -year-old with a history of congenital thrombocytopenia (platelets ranging , - , ) who presented with acute arthritis of bilateral hand joints after exposure to cold temperatures, which resolved with naproxen. he had back pain without muscle weakness, and preceding sore throat and general fatigue. labs were significant for leukocytosis and elevations in his inflammatory markers and creatine kinase. mri of his lower extremities was negative for inflammatory myositis, but did demonstrate bilateral hip and knee effusions, and significant inguinal lymphadenopathy and hyperintense linear signal changes in the mid-and distal femurs with patchy red marrow signal. abdominal ultrasound could not identify a definite spleen. bone marrow biopsy was negative for malignancy but significant for toxic granulation of neutrophils, evident of inflammation. alpha-beta double negative t cells were not elevated. interferon-gamma was mildly elevated. flow cytometry demonstrated normal t, b, and nk cell absolute counts. circulating antibodies against platelets (both igg and iga) were detected. on lymphocyte antigen and mitogen proliferation testing, he did not exhibit any proliferation when stimulated with tetanus toxoid even though he had been fully vaccinated against tetanus. muscle biopsy demonstrated large vacuoles consistent with tam on both light and electron microscopies. invitaes primary immunodeficiency panel identified a pathogenic variant in stim (c. c>t; p.arg trp), consistent with a diagnosis of autosomal dominant stim -related conditions, including stormorken syndrome ( ) . conclusion: this patient expands the phenotypic spectrum of stim related disease. based on previous evidence, gof pathogenic variants in stim are associated with tam and stormorken syndrome, while lof pathogenic variants in stim are associated with immune deficiency. however, our patient with a stim gof pathogenic variant has features of lymphoproliferation and immune dysregulation in addition to tam. stim gof pathogenic variants should be considered in the differential of patients with immune thrombocytopenia and lymphoproliferation. references: introduction / background: card is critical for protein binding upstream of nf-kb (nuclear factor kappa b) and mtorc (mammalian target of rapamycin complex ) the signaling pathway involved in t-cell activation and inflammatory response. prior testing of card mutations demonstrated variable t-cell dysfunction. in vitro studies have demonstrated reduced interferon gamma cytokine production, interference of t-cell receptor (tcr) signaling, and th phenotype skew in t-cells with card defects. while homozygous mutation causes severe combined immunodeficiency deficiency, heterozygous card defect is associated with atopy by way of inappropriate th skewing. heterozygote atopy is characterized by eosinophilia, elevated ige, and severe dermatitis. despite multiple studies demonstrating in vivo consequences of card on t-cell function, little is known of the clinical significance. moreover, few studies have demonstrated the impact of card mutations on b-cell maturation and development, despite the recognized tcr and interleukin signaling deficits. objectives: this case demonstrates a card defect that evolved from atopy to combined immunodeficiency requiring intravenous immunoglobulin therapy. it highlights the poorly understood effect of card mutation on t-cell function, and the downstream impact on b-cell quality. methods: -year-old male, with past medical history of t-cell lymphoma and no evidence of disease status post autologous stem cell transplant, was found to have card e d missense mutation by genetic testing. consistent with previous literature regarding heterozygous card defects, the patient suffered from frequent asthma exacerbations, aeroallergen sensitivity, and eczema. lab work was consistently positive for elevated ige and eosinophilia. family history was positive for a son born with congenital molluscum, and multiple other children with recurrent infections. one child was also identified with card mutation. the patient had flow cytometry demonstrating % of circulating cells with atypical immunophenotyped cd + t-cells, and positive gene rearrangement studies. his qualitative immunoglobulin levels were significant for consistently low igm, but normal quantity igg. in the patients adulthood, he had recurrent bronchitis and pneumonia requiring hospitalization and intravenous antibiotics. given his recurrent infections, the patient underwent immunodeficiency evaluation. despite previous infection with herpes zoster, the patient did not have protective titers. additionally, the patient had received the pneumococcal conjugate vaccine once, and the pneumococcal polysaccharide vaccine four times. the most recent vaccination was one year prior to evaluation. despite repeated vaccinations, titers were unprotective. consequently, the patient was diagnosed with combined immunodeficiency, and initiated on intravenous immunoglobulin therapy. results: in summary, card defect is a cause of atopy, observed to become less severe with age. studies of card heterozygote mutations have demonstrated in vitro deficiencies in t-cell activation, likely secondary to skewed or decreased inflammatory cytokine production and tcr activation. our patient demonstrates that the variable t-cell dysfunction seen in vitro can have significant clinical implications evidenced by his inadequate vaccine response, and recurrent infections. his combined immunodeficiency poses a connection between card defects and, not only t-cell, but also b-cell function. conclusions: further studies are needed to determine deficits in t-cell and b-cell function in the setting of card defect, as this case suggests the clinical implications span further than atopy. genetic variants in the scaffold gene card cause disorders of the immune system. the clinical course and treatment depends on whether the card variant causes gain-or loss-of-function. however, lymphocyte immunophenotyping and proliferation assays in cells expressing card variants don't easily distinguish between gain-and loss-of-function. to address this challenge in variant interpretation, we used multiplexed genome editing in a lymphoma b cell line (tmd ) to generate cell populations expressing all possible singlenucleotide variants in the n-terminal amino acids of card . to assess function in each variant, we tracked its relative abundance over multiple conditions using dna sequencing. since card is required for survival of tmd lymphoma b-cells, cells expressing clinically identified gain-of-function variants grew faster relative to cells expressing other variants, even in the presence of upstream pathway inhibitors. upon evaluation of the relative abundance of each variant in genomic dna and mrna, we found that clinically identified loss-of-function variants were depleted in mrna, which could be attributed to alterations in splicing or to nonsensemediated decay. to address the impact of splicing, we modeled a newly-identified splice donor mutation (c. + g>a) found in two patients from one family diagnosed with combined immune deficiency, autoimmunity and atopy that was also observed in our screen. we show that the variant causes deletion of exon four and that card missing exon four exerts a dominant-negative effect leading to decreased nf-kb signaling and cell growth. these experiments demonstrate the utility of multiplexed functional assays for determining variant effect in clinically-relevant genes, which will improve diagnosis and treatment in patients. mutations in the rag and rag genes in humans cause a wide spectrum of phenotypes, ranging from severe combined immunodeficiency (scid) with lack of t and b cells to omenn syndrome (os), atypical scid (as) and combined immunodeficiency with granulomas and/or autoimmunity (cid-g/ai). here, we sought to investigate the molecular basis for phenotypic diversity presented in patients with various rag mutations. methods: we have recently described a novel flow-cytometrybased assay in which mouse rag -/-pro-b cells containing an inverted gfp cassette flanked by recombination signal sequences (rss) are transduced with a retroviral vector expressing either wild-type or mutant human rag (hrag ). the green fluorescent protein expression directly relates to the activity of rag proteins, representing a quick and powerful tool to correlate between defective activity of hrag mutant and severity of the clinical phenotype. the genetic variants of hrag analyzed in this study were affecting the various domains of the protein: ring, zinc finger ring type domain (amino acids - ); nbr (amino acids - ); hbr (amino acids - ) and the core domain (amino acids - ). using this sensitive assay, we tested the recombination activity of human rag variants that have been reported in patients. results: we have demonstrated correlation between the recombination activity of the mutants and the in vivo clinical phenotype of patients. in particular, similarly low levels of recombination activity were observed in patients with scid and os, whereas patients with as and especially those with cid-g/ai carried mutations that retained significant residual levels of activity. conclusions: these data provide a framework to better understand the phenotypic heterogeneity of rag deficiency. here we report a case of a child with b. cepacia lymphadenitis, ultimately diagnosed with takayasu arteritis. takayasu arteritis is a large vessel vasculitis which may have a nonspecific clinical presentation in childhood possibly leading to difficulty in diagnosis. case: a -month-old female presented with two weeks of fever, respiratory distress, and lymphadenopathy, and was treated with ivig for presumed atypical kawasaki disease. imaging studies performed due to worsening respiratory distress revealed retropharyngeal abscess with bilateral cervical lymphadenopathy, culture-positive for prevotella oralis and melaninogenica, with improvement following incision and drainage and antibiotic therapy. recurrence of fever and respiratory distress prompted ct imaging of her neck significant for worsening lymphadenopathy. cultures from lymph node biopsy grew b. cepacia. following treatment, she was readmitted with respiratory distress requiring chronic steroid treatment and found to have candida albicans on bronchoalveloar lavage and necrotizing granulomatous inflammation on lung biopsy. an immunologic evaluation was notable for two normal dhr assays. cgd genetic panel was negative for pathogenic variants in cybb (p ), ncf (p ), cyba (p ), ncf (p ). testing was also notably negative for hiv pcr, bartonella pcr, cryptococcal antigen, histoplasma antigen, bal afb stain and mycobacterial cultures, cmv pcr, ebv pcr, anca, serial blood cultures, and sweat test. lymphocyte subsets were normal for age. mitogen stimulation test, myeloperoxidase antibody igg, serine protease igg, c level, lad panel, and cytokine panel were normal. autoimmune lymphoproliferative disorders (alps) panel was negative. whole exome sequencing demonstrated heterozygous mutations in cfi and jak , not considered to be clinically relevant given the patients clinical picture and laboratory evaluation. the patient was then lost to follow-up for over a year. at the age of years, the patient presented with fever and back pain. imaging revealed severe large vessel vasculitis involving the aorta and subclavian, vertebral, mesenteric, and renal arteries. she also had evidence of cardio-embolic strokes on brain mri. she had had no significant interval infections, and her immunologic evaluation remained unrevealing. in the context of her new vasculitis, evaluation for deficiency of ada (dada ) was negative. she was ultimately diagnosed with takayasu arteritis and has begun therapy with systemic corticosteroids, aspirin, and etanercept. conclusions: we describe a case of b. cepacia infection in a child without identified immunodeficiency, ultimately diagnosed with a large vessel vasculitis. the presence of b. cepacia infection warrants a thorough investigation. burkholderia has been previously associated with giant cell arteritis, another type of large vessel vasculitis, though causation has not been established. to our knowledge b. cepacia infection has not been associated with takayasu arteritis. christopher santaralas, valentine jadoul, jacqueline squire, john cannon, jessica trotter, susan aja, neil goldenberg, david graham, jennifer leiding background: chronic granulomatous disease (cgd) is a primary phagocytic immunodeficiency secondary to mutations in any of the components of nadph oxidase. in addition to infection susceptibility, patients with cgd can develop auto-inflammatory disease that is difficult to manage. metabolomics is the systematic study of small molecule biomarkers of the clinical phenotype of disease. we sought to investigate plasma metabolic profiles in cgd as we hypothesized that unique signatures may differentiate patients with cgd. methods: plasma collected from subjects with cgd ( x-linked, p phox-deficient, p phox-deficient) and x-linked cgd carriers was analyzed using a targeted multiplex assay by liquid chromatography mass spectrometry (lc-ms) and simultaneously a profiling assay by lcms. sufficient signal was present for metabolites. x-linked cgd and p phox-deficient groups were sufficiently sized for multivariate and univariate analyses in metaboanalyst. twelve patients had a single time point of plasma metabolomics analysis and three had multiple time points, including one in whom both pre-and post-hematopoetic cell transplantation time points were assessed. post-hoc comparisons were also performed for those with, versus without, clinical comorbidities of autoinflammation. results: plasma from patients with x-linked and p phox deficient cgd had a differential metabolomic signature at baseline. many metabolites as measured by ion intensity were present at high levels, particularly homocysteine, kyneurine, tryptophan, citric acid, carnitine, methionine, and adenosine. increased values of metabolites reduced to that of normal (compared to post hct). homocysteine levels were elevated among patients with (mean . x ), versus without (mean . x ), clinical comorbidities of auto-inflammation (i.e., colitis, lupus). baseline samples showed elevated kynurenine among all cgd patients, relative to historical normal controls (unmatched, separate analysis). patients with colitis had elevated citric acid levels that were higher among patients with (mean . x ), versus without (mean . x ), colitis irrespective of genotype. conclusions: preliminary data with a small patient subset suggest that patients with cgd have metabolomic signature distinguishable by phenotype. citric acid cycle metabolites are elevated in crohns disease and ulcerative colitis. based on our data, citric acid may too act as a biomarker for inflammatory bowel disease in cgd. analyzing a larger number of samples, across time points, will likely describe a metabolomics profile for cgd and identify biomarkers for auto-inflammation in cgd. no significant medical history in mother; paternal history is unknown and unavailable.no significant medical history in mother or father. rationale: ataxia telangiectasia is a disorder with variable phenotypes characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition which may correspond to the degree of atm protein expression and/or radiosensitivity. we used in vitro cytometric assessment of atm, smc and h ax phosphorylation to assess dna damage in response to radiation and found that two siblings with the same copy number gain in atm have variable clinical neurologic and immunologic phenotypes. methods: chart review and radiosensitivity assays using cytometric assessment of patm, psmc , and h ax expression after irradiation with gy. results: patient a is a month old male identified after having low trecs on newborn screening, then found to have lymphopenia and elevated igm. he has diffuse café au lait macules and no neurologic symptoms. his year old sister, patient b, was being followed by neurology for several years for ataxia. she has selective iga deficiency, normal lymphocyte counts, lymphocyte proliferative responses, gammaglobulins, and vaccine specific antibodies. both patients have a copy number gains in atm (exons - ). mother and father both have copy number gains in atm and are healthy without neurologic symptoms or recurrent infections. both patient a and b have normal atm protein expression. phosphorylated atm, smc , and h ax was assessed in lymphocyte subsets (t, b, and nk cells) after low-dose irradiation to induce dna double-stranded breaks (dsbs). these parameters were assessed at hour post-irradiation when they are expected to be maximal and at hour post-irradiation, when under conditions of normal and effective dna repair, the phosphorylation state returns to baseline. patient a had abnormal patm and psmc but normal h ax expression hour and hours after irradiation of t, b, and nk cells. patient b had normal patm, psmc , and h ax expression in t cells but abnormal patm and psmc expression in b and nk cells hour after irradiation. patient b, however, had abnormal atm phosphorylation at hours after irradiation of t, b, and nk cells.conclusions: our results indicate that a unique copy number gain in atm within a family can correspond to different clinical and immunologic phenotypes as well as variable degree of radiosensitivity. the persistence of h ax at hours post-irradiation and impaired phosphorylation of atm and smc at hour post-irradiation demonstrates defects in dna dsb repair, and this is variably altered in different lymphocyte subsets. correlation between atm phosphorylation in lymphocytes with outcomes may be an area for future studies and particularly important in counseling patients regarding outcomes. antibodies have been implicated in both protection and pathology of dengue virus infections. however, much of this data is gathered from serum/plasma responses that is a cumulative of historical and ongoing infection. to precisely understand the role of antibodies with respect to the ongoing dengue virus infection, we employed the cutting edge approach of generating of human monoclonal antibodies from individual plasmablasts from peripheral blood of dengue patients that allows us to probe for answers at a single cell level. this method involves ex vivo single cell sorting of plasmablasts from peripheral blood of well-characterized dengue infected patient followed by single cell molecular cloning of immunoglobulin heavy-and light-variable regions into expression vectors containing the defined constant region followed by transient cotransfection of hek a cells with the heavy and light chain expression vectors made from genes arising from the same cell. thus far, using this powerful technology, for the first time in india, we have made number of human monoclonals, of which are specific to dengue and neutralize dengue virus at various concentrations. all the neutralizing antibodies are dengue-envelope specific and bind the highly conserved fusion loop of the dengue virus envelope. together, with the ongoing comprehensive analysis of the b cell repertoire and somatic hypermutations, these studies provide a detailed understanding of the dengue-specific plasmablast cell response at a single cell level and create a platform for testing these antibodies for basic research, diagnostic, prophylatic and as well as therapeutic applications. surviving. six of the ( . %) surviving patients remain dependent on ig replacement despite robust donor chimerism of - % and no active gvhd. all but two received rituximab pre-hsct. of the patients who are independent of ig replacement, only one ( . %) received rituximab post-hsct, whereas / of the ig dependent patients received rituximab post-hsct. t cell immune profiling revealed that the absolute numbers of lymphocyte subsets, cd + naïve t cells, and cd + recent thymic emigrants were not statistically different between ig independent and dependent patients ( figure ). however, there was a marked decrease in the number of total b cells, the percentage of memory b cells (cd + b cells), and classswitched memory b cells (cd + igd-igm-cells) in ig dependent patients ( figure ). t follicular helper (tfh) cell populations (cd +cd ra-cxcr +pd +) were evaluated in four patients and the frequency was similar to healthy controls ( . +/- . vs. . +/- . %). the ability of the patients naïve b cells to class-switch was assessed following exposure to il- , anti-cd antibody, and anti-human igm, and revealed normal b cell class-switching and differentiation to plasmablasts ( figure ) . additionally, t cell ability to provide b cell help was assessed by coincubating naïve b cells with activated cd + t cells. this revealed comparable b cell class switching to that of healthy controls. conclusion: the high incidence of poor long-term functional b cell reconstitution following allogeneic hsct for xlp- could be related to the use of rituximab in the post-hsct setting rather than pre-hsct. normal tfh numbers and function, and ability of b-cells to class-switch in-vitro suggest that persistent hypogammaglobulinemia is these patients is unlikely from a b or t-cell intrinsic defect. the possibility of rituximab induced acquired lymph nodal stromal defect in these patients is being explored. further studies are needed to understand the biology of persistent hypogammaglobulinemia in xlp- . additionally, due to the high incidence of persistent hypogammaglobulinemia, exposure of rituximab should be limited post-hsct. background: tandem mass spectrometry (ms/ms) has emerged as a primary platform for many clinical and newborn screening laboratories. the application of ms/ms mainly focuses on the quantification of accumulated small metabolites in plasma resulting from various metabolic defects. however, many disorders do not yield such metabolic markers and would benefit from the direct quantification of intracellular target proteins. unfortunately, the extremely low (e.g., pmol/l range) protein concentrations in blood cells limit their detection via ms/ms. in recent years, peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) has emerged as a promising technique for the quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). our lab has demonstrated that immuno-srm methods are able to reliably distinguish affected patients from the normal controls for wilson disease (wd), wiskott-aldrich syndrome (was), severe combined immunodeficiency (scid), and x-linked agammaglobulinemia (xla) (j. proteome res., and front. immunol., in press). these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed quantification of signature peptides in the low pmol/l range. methods: several candidate peptides for each protein were selected based on uniqueness using in silico blast tools and lc-ms/ms response. monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, wd, xla, scid, and was patients was spotted onto filter paper, dried, and stored at - °c until use. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed using srm mode with a waters xevo tq-xs. results/conclusions: to date, immuno-srm methods have been generated for wd, was, scid, xla, and cystinosis. preliminary data shows immuno-srm methods are able to reliably quantify target proteins using signature peptides and accurately distinguish affected patients from normal controls. analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (was and btk: p = . , scid: p = . ). intra and inter-assay precision ranged from - % and - %, respectively, and the multiplexed assay showed a broad linear range ( . fmol peptide) . in a blinded sample set of pidd patients and normal controls, immuno-srm-predicted diagnoses showed excellent agreement with clinical or genetic diagnoses. every molecularly-confirmed case of was and btk was also diagnosed by immuno-srm analysis. in addition, randomly selected samples provided by the nbs laboratory of washington state were tested and peptide concentrations were found to be within normal ranges. efforts are underway to validate and incorporate peptide biomarkers for adenosine deaminase deficiency, dock deficiency, and ataxia telangiectasia, as well as general markers for nk cells and platelets into a single multiplexed assay. in addition, scid, was and xla samples continue to be run while we focus on reducing assay costs, time, and necessary sample input. our data herein provides proof of concept for the immuno-srm workflow to be extended to various other genetic diseases as potential multiplexed newborn screening methods.( ) submission id# the background: the long-term effects of glucocorticoids (gcs) on the immune system have been extensively studied in patients with different underlying conditions (e.g, malignancies or autoimmune conditions), as well as in healthy volunteers receiving short-term courses of these drugs. although these approaches provided highly relevant data, neither of them answered the unbiased/bona-fide effect of long-term gcs use on the immune system. endogenous cushing syndrome (ecs) may be caused by pituitary or ectopic acth-producing adenomas, or by tumors or hyperplasia of the adrenal cortex. patients with ecs present with different gcsdependent manifestations, including those affecting the immune system as neutrophilia and lymphopenia. when tumors are removed, most of the effects of gcs tend to progressively regress. methods: paired samples from patients with ecs due to acth-producing adenomas (age range - y, females) were studied before (ecs-pre) and - months after tumor removal (ecs-post). extended lymphocyte phenotypes and apoptosis in different cell subsets were evaluated by flow cytometry. cytokine production (elisa) and responses, as well as their effects on cell proliferation and viability, were evaluated using cell trace violet and annexin-v staining. results: among multiple immunophenotypic changes, ecs-pre patients showed significantly reduced naïve t cells and recent thymic emigrants (rte) as well as increased apoptosis in t cells when compared to themselves (ecs-post) or age matched healthy controls. moreover, significantly increased exhausted cd t cells were observed in ecs-pre patients. interestingly, ecs-post patients showed full cellularity recovery of t cells and rte with increased proliferation and reduced apoptosis, in addition to correction of most of the other changes evidenced. significantly lower il- plasma levels were also detected in ecs-pre when compared to ecspost patients. to determine the role of il- in an ecs-resembling condition, healthy control pbmcs were treated with gcs in-vitro and the effect of il- and other cytokines was tested. a significant reduction in apoptosis was observed in the il- -treated cells that almost completely countered the pro-apoptotic effects of gcs; il- was also significantly more efficient than il- , il- , ifn-alpha and ifn-gamma in rescuing cells from apoptosis. il- -specific upregulation of bcl and bcl expression was evidenced in these cells.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - glgeft authors: possas, cristina; antunes, adelaide maria de souza; de magalhães, jorge lima; mendes, flavia maria lins; ramos, mateus pinheiro; de simone morais, juliana; homma, akira title: vaccines: biotechnology market, coverage, and regulatory challenges for achieving sustainable development goals date: - - journal: bioeconomy for sustainable development doi: . / - - - - _ sha: doc_id: cord_uid: glgeft this chapter provides an overview, from bioeconomic and global sustainability perspectives, of the main constraints to the current global vaccine innovation system for achieving sustainable development goals – sdgs. biotechnology market trends, gaps in vaccine coverage against emerging and neglected diseases, and patent protection and regulation are discussed. a structured long-term “public-return-driven” innovation model to overcome vaccine market failure is proposed. innovative preventive vaccines against emerging and neglected infectious diseases, such as zika, dengue, chikungunya, influenza, and hiv/aids, are examined here from bioeconomics and global sustainability perspectives, aiming to integrate public health and biotechnology market approaches. novel vaccines with reduced adverse effects can have an enormous impact on life expectancy and on the quality of life of the global population, significantly reducing government, individual, and business costs . nevertheless, there are significant production, technological development, market, coverage, regulatory, and governance constraints to achieving sustainable development goals (sdgs) . in this chapter we examine vaccine biotechnology market and the factors contributing to market failure, discussing policy strategies to optimize science, technology, and innovation (sti) and drastically reduce current constraints to vaccine development (singh et al. a (singh et al. , b, . for achieving sdg, it should be noted that only one of these goals, sdg , refers specifically to vaccines ( .b. ). however, in addition, we have also identified other sdg goals strongly related to vaccines and sdg goals related to vaccine, in a total of vaccine-related goals in sdgs. two of these goals are related to innovation and technological development of vaccines (sdg and sd ). we discuss the main vaccine development challenges for achieving sdg and current technological and regulatory obstacles particularly affecting developing countries. from this perspective, we propose sti governance strategies to overcome these gaps and increase global access to vaccines, focusing on institutional and regulatory perspectives, including intellectual property and ethics. policy recommendations for vaccine funding and incentives for innovation, development, and production are made. finally, we emphasize the enormous potential role that access to innovative vaccines can play on global sustainability (milstien et al. ; possas et al. ) , benefiting particularly the poorest countries in a global context permeated by sharp social inequalities. the global market for human vaccines is projected to reach usd . billion by from . billion in at a cgar of . % (markets and markets ) driven by the growing importance of vaccines in public health, reducing healthcare costs and contributing through prevention of diseases toward a more sustainable healthcare system. drastic changes in the dynamics of the global vaccine market occurred between and , with a sharp growth from usd billion in to usd billion in (access to vaccines index ) and to usd . billion in (markets and markets ) , with sales to high-income countries representing about % of the total value of this market (access to vaccines index ). in fig. . we indicate the evolution of the global human vaccine market from to and the forecast for . recently, other reports have been released anticipating an even more favorable scenario for the global human vaccines market. a recent study estimated that this market would grow from . billion in to . billion by (grand view research ) . these market forecasts also anticipate rising r&d investments in vaccine development projects by the main global players in the vaccine market. table . indicates the top pharmaceutical players according to global revenue share in . pfizer is expected to increase its participation in the market in the next decade due to the success of its pneumococcal vaccine prevnar and increasing investments in vaccine development. other important vaccine players include emergent biosolutions, csl, inovio pharmaceuticals, bavarian nordic, mitsubishi tanabe, serum institute of india pvt. ltd., alk-abelló a/s, altimmune, inc., bharat biotech international, and medimmune. the world vaccine market consists of four segments: gavi (the global alliance for vaccine and immunizations), unicef, paho revolving fund (rf), and rest of the world (row). about of the lowest-income countries in the world rely on gavi for funding of some key vaccines (who ) . unicef supply division (sd) is the procurement agent for most of these countries and for an additional approximately middle-income countries (mics) (totaling about countries). the paho rf provides financial and procurement support to about countries and territories in the americas. the row consists of self-funding and self-procuring countries spanning all income levels and receiving only marginal, mostly indirect, financial, procurement, market shaping, or other related support. these increasing investments in vaccine innovation, development, and production are guided by the growing global need for preventive vaccines and immunotherapy strategies against cancer, zika, hpv, hsv, hiv, and a broad range of infectious diseases that currently burden the healthcare system and societies worldwide (who (who , . this scenario of increasing global demand for vaccines in the next decade is supported by epidemiological indicators: annual burden of new hpv-related cancers worldwide to the tune of , ; rise of zika into a public health emergency with over countries reporting , cumulative confirmed cases of infection between and ; very high prevalence of hsv which infects approximately % of the world population under years of age; continued prevalence of tuberculosis which infects million and takes . million lives each year despite the progress made toward eliminating the disease; and rise in hiv infections worldwide over . million (who ; global industry analysts ). developments in reverse vaccinology and synthetic vaccinology are expected to help increase the rate of successful vaccine design and development (sette and rappuoli ) . emerging countries with mandatory immunization programs represent large markets with enormous potential for future growth and expansion. with large population base and relatively high proportion of young children and teen population, emerging markets including china and india represent the fastest growing markets in asia-pacific, with this region expected to grow at the fastest rate of . % in the next decade. the pharma industry is rapidly becoming a competitive player in the bioeconomy market, a new global paradigm that will introduce novel technologies such as genomics and proteomics across multiple economic sectors and industries. immunome, resulting from advances in sequencing technology and a bioinformatics resource, is also contributing to vaccine innovation and development. these advances in genomics, proteomics, immunome, bioinformatics and new information technologies and their increasing convergence are driving these new market trends. this accelerated innovation scenario is revolutionizing healthcare with new preventive and therapeutic technologies, expected to provide longer, healthier lives to the global population. physicians will eventually be able to predict a person's predisposition to a broad range of diseases and intervene appropriately, insert new genes to replace faulty ones, and tailor therapies to an individual's needs and profile. the immune system is a highly complex system, based on a coordinated expression of a wide array of genes and proteins. one of the major gaps in vaccine innovation, particularly affecting the development of new vaccines against emerging and neglected diseases, is related to the inability of scientists to explain the diversity of individual immune responses and clinical outcomes to the same vaccine and how this diversity relates to innate and acquired immunity. the human immunome, a specific set of genes and molecular structures underlying the response of the immune system to fight disease, is vast and estimated at billion times larger than the human genome project in terms of data output. because of this scale, scientists have never been able to characterize the core parts by which the immune system responds to pathogens and develops a disease. only recently, with the dramatic advances in sequencing technologies and bioinformatics, exponentially extending their informational scale, it became possible for the first time for scientists to uncover the complexity of the human immunome (soto et al. ; briney et al. ) . immunome, a bioinformatics resource, has been conceived for the characterization of the human immune system. it contains information about immunity-related proteins, their domain structure, and the related ontology terms and contains also information about the localization and mechanisms involved in the coding genes. determining the core parts of the immune system in the human immunome could drastically transform how we diagnose, prevent, and treat disease through the identification of new biomarkers while enabling highly targeted, computationally designed vaccines and therapies that reduce time and risk of product development. the immunome program of the human vaccines project is sequencing, in a global collaborative -year effort, receptors from a group of genetically diverse individuals in several continents and determines the structure and function of a key subset of receptors. through an open-source procedure, data will be made available to researchers across the world. in this program, laboratory analyses of biospecimens will be combined with an array of other genetic, lifestyle, and health information provided by volunteers to help researchers to identify individual genetic differences that contribute to diverse immune responses. the initial study will assess immune responses of ten healthy adults (ages - ) to a licensed hepatitis b vaccine (considered an ideal model to study human immunological protection), and it is expected that this study will expand to include several hundred people from neonates to the elderly in middleand low-income countries. the immunome program can thus bring crucial information to the development of more effective vaccines against emerging and neglected infectious diseases. vaccine manufacturers in developing countries, particularly affected by these diseases, should be actively involved in its international scientific and technological collaborations. in the near future, sophisticated technology will allow patients to search and manage their medical records, comparing them to current public health information based on individual genomic profiles. intelligent marketing agents will aggregate patient information from a variety of sources to provide timely and relevant responses. networks of distributed processing systems will offer new insights into data by mining all enterprise and public data sources. and supercomputing platforms and information management will enable the rigorous manipulation of genomic data. advances in remote sensing and artificial intelligence technologies have already created intelligent operating rooms with sensory control mechanisms and devices that transmit health information via telephones and personal digital assistants. the industry can now develop programmable microchips for the subcutaneous delivery of precisely timed doses of drugs and vaccines. interactive chips with built-in sensors may mimic the body's own regulatory ability. as the bioeconomy evolves, the industry faces an unprecedented era of opportunity and challenge. companies recognize that alliances are critical to their future and are now making them a major component of their strategy. virtual research organizations are now conceived searching to provide discovery technologies to scientists in pharmaceutical enterprises; providing them links to gene database, proteomics database, or high-throughput screening capabilities; and enabling fast and efficient access to vaccine and immunotherapy information. the world is facing multiple public health challenges, such as outbreaks of vaccinepreventable diseases, increasing reports of drug-resistant pathogens, climate change, and multiple humanitarian crises. the world health organization (who) included several emerging and neglected diseases among the ten global threats for (who ): influenza, dengue, hiv, and also high-threat pathogens, such as ebola, several other hemorrhagic fevers, zika, nipah, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome (sars) and disease x, which represents the need to prepare for an unknown pathogen that could cause a serious epidemic. to address these and other threats, started its new -year strategic plan: the th general programme of work. this plan focuses on a triple billion target: ensuring billion more people benefit from access to universal health coverage, billion more people protected from health emergencies, and billion more people in better health and well-being. reaching this goal will require addressing these threats to health from a variety of angles. the world will face another influenza pandemic; the only thing we don't know is when it will hit and how severe it will be. global defenses are only as effective as the weakest link in any country's health emergency preparedness and response system. who is constantly monitoring the circulation of influenza viruses to detect potential pandemic strains: institutions in countries are involved in global surveillance and response. every year, who recommends which strains should be included in the flu vaccine to protect people from seasonal flu. in the event that a new flu strain develops pandemic potential, who has set up a unique partnership with all the major players to ensure effective and equitable access to diagnostics, vaccines, and antivirals (treatments), especially in developing countries, in order to make possible the supply of required vaccines as soon as possible. dengue, a mosquito-borne disease that causes flu-like symptoms and can be lethal and kill up to % of those with severe dengue, has been a growing threat for decades. a high number of cases occur in the rainy seasons of countries such as bangladesh and india. now, its season in these countries is lengthening significantly (in , bangladesh saw the highest number of deaths in almost two decades), and the disease is spreading to less tropical and more temperate countries such as nepal that have not traditionally seen the disease. an estimated % of the world is at risk of dengue fever, and there are around million infections a year. who's dengue control strategy aims to reduce deaths from the disease by % by . hiv infections in sub-saharan africa despite being only % of the population. this year, who will work with countries to support the introduction of self-testing so that more people living with hiv know their status and can receive treatment (or preventive measures in the case of a negative test result). who has included in these ten global threats for diseases and pathogens that have potential to cause a public health emergency but lack effective treatments and vaccines. this list for priority research and development includes ebola, several other hemorrhagic fevers, zika, nipah, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome (sars) and disease x, which represents the need to prepare for an unknown pathogen that could cause a serious epidemic. international recognition of vaccines' impact and increased global demand for vaccines have stressed the need for global strategies to assure timely provision of lowprice vaccines (meissner ) through policies supporting free and universal access. in this scenario, the decade of vaccines (dov) initiative was launched at the world economic forum in davos in , signed by international agencies, such as the world health organization (who), unicef, the us national institute of allergy and infectious diseases (niaid), and the bill & melinda gates foundation, with the mission: "to extend, by and beyond, the full benefits of immunization to all people, regardless of where they are born, who they are, or where they live." this declaration was supported by a commitment by the bill & melinda gates foundation to donate usd billion to research and development and to delivering vaccines for the poorest countries. the dov initiative gained significant international support and visibility. two years later, after consultations with dov stakeholders, including industry groups, a global vaccine action plan (gvap) was launched by the member states of the th world health assembly in may , aiming to deliver universal access to immunization by . following the collaborative dov strategies, the gvap brought together multiple stakeholders to achieve the ambitious goals of the plan: the leadership of the bill & melinda gates foundation, gavi alliance, unicef, us national institute of allergies and infectious diseases (niaid), and who, mobilizing many partners (governments, health professionals, academia, manufacturers, funding agencies, development partners, civil society, media, and the private sector). if the gvap is translated into action and resources are mobilized, it is expected that between . and . million deaths could be averted by the end of the decade, with gains in billions of dollars in productivity. nevertheless, it is important to note that actions and resources will not be sufficient for the success of gvap if the plan does not conceive a global strategy to support manufacturers in the developing world to overcome the main ipr and regulatory barriers that delay and hinder vaccine development and production. the millennium development goals (mdgs) for - were incorporated by governments worldwide and had a strong global mobilization power on promoting development and social initiatives, engaging national leaders in elaborating and monitoring these goals (un ) . this mobilization was facilitated since the targets were quantifiable and could potentially be attained. although the two healthrelated goals, mdg (reduce under- mortality from to by two-thirds) and mdg (reduce maternal mortality from to by three-quarters), had not been met by and it is estimated by who that . million infants worldwide are still missing out on basic vaccines, significant progress has been made, with child and maternal mortality approximately halved, with significant global progress. in sequence to mdgs, the united nations promoted an in-depth revision of this strategy (un (un , and formulated a new global strategy, sustainable development goals (sdgs) for - with goals, with one of them (sdg ) directly related to health (un ). the target of sdg is to "ensure healthy lives and promote well-being for all at all ages." its sub-targets include ones that could be met: two-thirds less maternal mortality and a third less noncommunicable disease (ncd) mortality. they also include ending preventable newborn and under- deaths and ending hiv/aids, tuberculosis, malaria, and neglected tropical diseases, besides other non-vaccine related sub-targets. in this chapter, we argue that a major component of sdgs is crucial for attaining sdg goals and should not be minimized: innovation and technological development of vaccines. we discuss how this component should be incorporated into monitoring the sub-targets of this goal, and we emphasize the need for a new vaccine innovation model based on an expanded role of the state and incentive mechanisms to pharmaceutical companies and public manufactures to correct the current scenario of "market failure" constraining access to vaccines. it is certainly unacceptable, from ethical and sustainable development perspectives, to simply recognize this "market failure" as a detrimental and inevitable consequence of the rationale of a global market economy. on the contrary, it should be seen as a massive public health failure and a global failure to direct economic development for the benefit of societies (trouiller et al. ). the "valley of death" although vaccine candidates are in the development pipeline for neglected and emerging infectious diseases mainly affecting the poorest countries such as malaria, dengue, hiv, tuberculosis, and pneumonia, only of them have made it through the pipeline recently and are widely used in these countries: a conjugate vaccine for meningitis serogroup a diseases and a vaccine against japanese encephalitis virus (kaslo et al. ; who ) . it has been estimated by these authors that unfortunately much of this promising pipeline could go to waste and fall into the so-called valley of death, failing to move from proof-of-concept to second-phase trial due to lack of market interest in vaccines against these emerging and neglected diseases affecting only the poorest populations in developing countries. no single organization or group is interested in supporting the costly and more complex late-stage clinical trials for neglected diseases that mainly affect the poor nations. this scenario raises great concern for two reasons. first, around % of these vaccine candidates in the development pipeline target the mentioned neglected and emerging infectious diseases, a much higher problem in lower-and middle-income countries (kaslow et al. ). second, this means a significant waste of global resources in a crucial area for sustainable development, considering that these vaccine candidates received billions of dollars for the first phase of vaccine development from prestigious donors, such as the us national institutes of health (nih), the european union, the welcome trust, and the bill and melinda gates foundation. taking a vaccine candidate from a discovery at the laboratory bench to widespread deployment is a complex, lengthy, and expensive endeavor, with many financial, licensing, and regulatory barriers. no organization or group plans to support the emerging and neglected diseases vaccines from the beginning to end. therefore, it could take many decades to incorporate these vaccines into the national immunization programs in these poorest countries (kaslow et al. ) . in table . we provided a selection of promising projects for vaccines for emerging and neglected infectious diseases affecting the poorest developing countries that could significantly impact on achieving sdg targets. science and technology have made enormous progress and are now prepared to provide the innovative-intensive vaccines that the poorest populations in the world urgently need. but innovation and discovery are not the major bottleneck, which reside in technological development, production, and timely provision of vaccines to people (homma et al. ). r&d-based pharmaceutical major industry players are reluctant, due to freemarket rationale, to invest in the development of vaccines to treat the major neglected and emerging diseases affecting mainly the poorest nations, since return on their investments cannot be guaranteed. national and international policies currently support a free-market-based global order, with economic opportunities, rather than global public health needs guiding the direction and rationale of vaccines development. it is certainly unacceptable, from ethical and sustainable development perspectives, to simply recognize this "market failure" as a detrimental and inevitable consequence of the rationale of a global market economy. on the contrary, it should be seen as a massive public health failure and a global failure to direct economic development for the benefit of societies (trouiller et al. ). an urgent redefinition of priorities in vaccine development is needed. this strategy cannot rely only on fragmented contributions of researchers, funding agencies, and the pharmaceutical industry. effective national and international policies need to be urgently conceived to redirect the global economy to address the true public health needs of society (homma et al. ; røttingen et al. ) . "political will," identified as the need for a strong commitment to prioritize health considerations over economic interests, has been frequently emphasized by policy-makers as a major issue to ensure access to vaccines but is not sufficient. it is necessary to go beyond "political will," with a clear goal in mind and a realistic plan to achieve it. from this perspective, it will be necessary to promote effective global implementation of strategies to accelerate innovation, technological development, and production of new vaccines and to ensure timely global access to them. moreover, a global vaccine policy strategy should be conceived to promote the necessary enforcement of regulations and other mechanisms to stimulate vaccine development, production, and global access to these products. novel, creative, and effective strategies involving both the public and the private sector are needed to ensure low-price vaccines, accelerating innovation and technological of vaccines against emerging and neglected diseases. priority action areas should include: . advocating a preventive vaccines r&d agenda . conceiving capacity-building programs adequate to the conditions of developing countries' manufacturers . promoting technology transfer to public and private manufacturers in emerging countries . elaborating an adapted legal and regulatory framework to increase flexibility and "fast-track" procedures . prioritizing funding for vaccine development . securing availability, accessibility, and distribution of these vaccines consensus is building among the main stakeholders in the global vaccine community that the spiraling costs of risks associated with vaccine r&d are detrimental to global access to these products, particularly in the poorest developing countries. most of them agree that these vaccine r&d costs should be instead rewarded by means other than financial returns in the market from charging high product prices. novel mechanisms such as incentives, prizes, and "patent pools" for drugs and vaccine innovation and development have been proposed in the last two decades. there is now vast literature on the subject, claiming for alternative models that should be urgently implemented to meet the increasing global demand for vaccines, particularly in the poorest developing countries. the main question is: how to conceive a feasible long-term mechanism to minimize these risks faced by pharma companies? which global organizations should be responsible for this alternative model? we recommend this new vaccine incentive model should be coordinated by three international organizations: who, gavi, and unicef. these organizations would, in collaboration with the main stakeholders, identify from the list of candidates the priority vaccine candidates, identify the funding mechanisms necessary to these candidates to enter the second-phase clinical trials, and specify which organization, or alliance, would be responsible for these selected vaccine candidates from beginning to end. in this innovative global collaboration strategy, these three leading international organizations should bring together the main players and stakeholders in the vaccine market, with funding agencies such as the bill & melinda gates foundation, nih, welcome trust, and other organizations as path, iavi, and the international vaccine institute in seoul and vaccine manufacturers, in collaboration with other nongovernmental organizations in order to conceive and implement this alternative long-term model for sustainable development and provision of vaccines which are uncertain business products or require a great amount of public funding to go beyond the initial proof-of-concept phase. a novel global priority-setting strategy, driving adequate implementation, will be necessary to assess the vaccine candidates in the pipeline, trying to identify the most favorable candidates which are uncertain business cases that will require significant public funding to move into second-phase clinical trials. funding mechanisms supported by subsidies from governments, such as those of the g countries, and philanthropic organizations, such as the bill & melinda gates foundation, could remedy the market failure threatening vaccine development for lmics. gavi already provides one form of subsidy (gavi a, b). support to develop vaccines or to make them available during epidemics is also provided by public organizations, such as the coalition for epidemic preparedness innovations in oslo and the biomedical advanced research and development authority, part of the us department of health and human services. such schemes need to be expanded and rethought to give vaccine developers more certainty and upfront financial backing (kaddar et al. ) . for instance, gavi could commit to purchasing a vaccine before it has been developed, on the condition that the developers meet certain regulatory milestones. at present, the alliance buys vaccines to distribute to lmics after they have been licensed or recommended by the who for general use (gavi (gavi , b . only with this kind of leadership will the global community secure vaccines for some of the world's most debilitating diseases. there is an urgent need for a paradigm shift in global governance of health innovation systems to achieve sustainable development goals (buse and hawkes ; possas et al. ; seib et al. ; mazzucato ) . "mission-oriented" approaches have been proposed to overcome current constraints in innovation systems (mazzucato and penna ) . recently, in a new report, "the people's prescription: re-imagining health innovation to deliver public value" (mazzucato ) , the authors call for restructuring research and development innovation systems in order to create, rather than extract, value. it also calls for long-term "missionoriented" public investment and a public return on this investment. in this report the authors argue that health innovation is about making new treatments and cures available to the people that need them. profits might be earned but not at the cost of doing what the health system is meant to do: heal. this report is the outcome of result of collaboration between the ucl institute for innovation and public purpose, stopaids, and global justice now and just treatment. the report identifies gaps of the current health innovation system and sets principles for a new model. it proposes concrete policy actions that can be taken in the long term to actively shape and co-create a health system that delivers real public value. the report is structured into two sections. the first is "diagnosis" with chapters on "problems with the current health innovation system" and "principles for a health innovation model that delivers public value." the second section, "remedies," includes chapters on "immediate policy actions: getting better prices today" and "transformative proposals: re-imagining our health innovation system to deliver public value." the report focuses on the unethical and unacceptable current global scenario for health innovation, highly inefficient, with a pharmaceutical industry that makes billions in profits without providing the affordable products that people need. the report examines all those problems, and then it sets out some key principles of how a "healthy" innovation model for health would work, based on an analysis of case studies from different countries and different contexts, looking at where innovation has been done well. in vaccine development, as in drugs development, there is a tremendous waste of resources because public health is not driving the r&d agenda. we have all the money going into proof-of-concept studies instead of developing public accountability. track" the need to provide more flexible and expedite new vaccine products and processes resulting from biotechnology is challenging both developed and developing countries to accelerate the implementation of adequate regulations and intellectual property rights (crager ; possas et al. ) . ipr are granted by the state to individuals, enterprises, or organizations under temporary monopolistic conditions (patents) in order to compensate them for the investments made in their creations/innovations. in industry, a patent is clearly an instrument to guarantee the returns of the investments on r&d through the commercialization of the patented products and through the payment of property rights. patents are viewed as a crucial incentive to innovation. nevertheless, arrow ( ) recognized in his pioneer theory that in spite of its advantages, the patent system creates a suboptimal situation in economic terms: patents create a monopoly that restricts the diffusion and dissemination of innovation. the argument is that this restriction is temporary (after years the patent protection "falls" to public domain) and is compensated by the fact that the knowledge related to the patent is necessarily published in the moment that the patent is granted. nevertheless, several authors have noted the detrimental impacts of the monopoly created by the patent system on health products' innovation, particularly on the development and accessibility to new drugs for neglected and emerging diseases and proposed incentive mechanisms, such as prizes, "patent pools," and awards to compensate this "market failure." although in the vaccine sector many intellectual property and market issues affecting price remain unclear, in the current regulatory scenario, the access to new technologies in multipatented vaccines, such as adjuvants for vaccine compositions, remains a main challenge (possas et al. ) . for vaccine manufacturers in emerging countries, access to patent information on vaccine adjuvants is a crucial issue, detrimental to vaccine development. the incorporation of new adjuvants for vaccines which boost the immune response has become crucial to the development of innovative vaccines, as new antigens, with purer and smaller molecules, may have less then optimal immune responses, necessary to vaccine protection for a lengthy period of time. the malaria vaccine candidate rts provides a good example of the crucial role new adjuvants can play: this vaccine, based on the plasmodium falciparum sporozoite antigen circumsporozoite protein (csp), was successful in providing protection against clinical malaria only when combined with a powerful adjuvant (as or as ). another example are the tests using hybrid flagelins also in malaria vaccines. adjuvants have emerged thus as an alternative route for vaccine development with enormous potential in the global market (mbow et al. ) . the development of new, powerful, and safe adjuvants is therefore a key component of vaccine research. we present in table . some of licensed vaccine adjuvants, with company and class. figure . indicates the countries concentrating patent deposits for adjuvants to vaccine compositions and the diseases related to them (zika, dengue, hiv/aids, influenza), china ( %), the usa ( %), south korea ( %), and the uk ( %), with these three countries accounting for nearly % of all patent deposits. it is also observed that chikungunya had no deposit in the period from to , as indicated the espacenet base. it should be stressed that very few deposits are related to zika and dengue vaccine, only in china and the usa. it also indicates that most of these adjuvant deposits are concentrated in vaccine compositions related to just two diseases, influenza ( %) and hiv/aids ( . %), while zika, followed by dengue, is more neglected. the increasing risk of a pandemic of influenza, rapidly affecting all continents, might explain the concentration of r&d efforts on vaccine compositions and adjuvants for this disease. table . provides an overview of the patent holders of deposits for vaccine adjuvants against zika, dengue, and hiv in the - period, listed by country, number of deposits, and deposits with partnerships and partners. this table indicates that the number of partnerships is very low and should be stimulated. figure . shows the temporal evolution of patent deposits in the period - of the three largest patent depositors of adjuvants and formulation of vaccines with adjuvants for the diseases studied, showing that only in the last decade there has been a greater r&d effort. this figure also indicates the leadership of china and the increasing role played by this country in the development of adjuvants for vaccine compositions. finally, it should be noted that in addition to these intellectual property barriers to access to vaccine formulations and vaccine adjuvants, such as confidentiality and constraints to patent information sharing (possas ; possas et al. ) , other regulatory obstacles remain also a challenge to vaccine development and access to timely immunization: virtual inexistence in many countries, particularly the developing ones, of expedite and "fast-track" review processes (fda ; u.s. dept. of hhs et al. ) for evaluating priority and emergency projects; lack of flexible regulatory procedures for sharing biospecimens and samples; legal constraints in access to biorepositories and to biobank information; and the difficulties in defining the standard of care to be provided during clinical trials. we examined here, from bioeconomics and global sustainability perspectives, the current innovation system for vaccine development, providing considerations on how this system should be better designed and implemented to address sdgs' vaccine coverage targets. public investment plays a crucial role in biomedical r&d worldwide, but research and development activities supported by governments with public resources are in most cases not directed into targets that have the most public health value, such as vaccines. for this reason, there is increasing awareness and concern in the global vaccine community on the fact that public-supported vaccine products should be shared by the public and not just privately appropriated by pharmaceutical companies. this scenario evidences a need for conceiving a new innovation governance paradigm for vaccine development compatible with the market rationale, aiming profitable products with social return: an expanded role of the state, with clear procedures for planning, development, regulation, and forecast; a redefinition of the patterns of relationship between private and public players; and finally, a political agreement between the main players and stakeholders on the more adequate mechanisms to balance the risks and the rewards between those players. moreover, there is a need to conceive a more flexible intellectual property regime for emerging and neglected diseases mainly affecting the poorest populations in developing countries. the monopoly created by patent protection must be compensated by new incentive mechanisms such as awards, prizes, and "patent pools" to accelerate global access to vaccines. it is also crucial to strengthen the local capacity of vaccine r&d institutes and manufacturers in emerging developing countries in order to accelerate the incorporation of new technologies for production of innovative vaccine products. the multinational companies have the intellectual property of these new technologies, such as adjuvants for vaccine compositions, but they do not have sufficient production capacity to meet the global demand for these products, a gap that should be overcome with expanded global collaboration with developing countries' manufacturers. in addition, these players should identify gaps and priorities in infrastructure and capacity building in developing countries' manufacturers in order to facilitate technology transfer agreements with leading pharmaceutical companies and ensure a sustainable long-term supply of vaccines to the poorest populations. finally, clear expedite and "fast-track" regulatory procedures and pathways should be conceived and implemented. in other words, it will be necessary for international organizations and pharmaceutical companies to move from a short-term "shareholder-driven innovation model" to a long-term "public-return-driven innovation model", aiming global sustainability and social welfare for meeting the needs of low-income populations while searching for innovative and profitable vaccine products. access to vaccines index ( ) how vaccines companies are responding to calls for greater immunization coverage economic welfare and the allocation of resources for invention the broad socioeconomic benefits of vaccination health in the sustainable development goals: ready for a paradigm shift improving global access to new vaccines: intellectual property, technology transfer, and regulatory pathways commonality despite exceptional diversity in the baseline human antibody repertoire espacenet patent search. epo, brussels, belgium. accessed fast track, breakthrough therapy, accelerated approval and priority review for patients and patient advocates global alliance for vaccines and immunizations ( a) gavi sustainable development sustainable development goals monitoring framework human vaccines: market analysis, trends and forecasts market report on pharmaceuticals vaccine research, development, and innovation in brazil: a translational science perspective global support for new vaccine implementation in middle-income countries vaccine candidates for poor nations are going to waste vaccine markets by diseases and technologies, report the people's prescription: re-imagining health innovation to deliver public value mission-oriented finance for innovation: new ideas for investmentled growth new adjuvants for human vaccines immunization policy and the importance of sustainable vaccine pricing access to vaccine technologies in developing countries: brazil and india propriété intellectuelle et le sida dans les pays en dévéloppement: innovation et accès aux produits pharmaceutiques access to new technologies in multi-patented vaccines: challenges for brazil innovation and intellectual property issues in the "decade of vaccines new vaccines against epidemic infectious diseases policy making for vaccine use as a driver of vaccine innovation and development in the developed world reverse vaccinology: developing vaccines in the era of genomics a) intellectual property issues in biotechnology. cabi, wallingford. pages biotechnology in agriculture, medicine and industry: an overview intellectual property issues in microbiology high frequency of shared clonotypes in human b cell receptor repertoires drugs for neglected diseases: a failure of the market and a public health failure guidance for industry: expedited programs for serious conditions -drugs and biologics secretary general. the road to dignity by : ending poverty, transforming all lives and protecting the planet. synthesis report of secretary-general on the post- sustainable development agenda the millennium development goals report united nations development programme ( ) sustainable development goals who's vision and mission in immunization and vaccines - . who, geneva world health organization ( ) fact sheet v p pricing report world health organization ( ) ten threats to global health in key: cord- -dltsdqcm authors: siegel, frederic r. title: lessening the impacts from non-tectonic (natural) hazards and triggered events date: - - journal: mitigation of dangers from natural and anthropogenic hazards doi: . / - - - - _ sha: doc_id: cord_uid: dltsdqcm floods are a global problem. they are predictable to some degree by weather forecasting but to a greater degree and with more accuracy when drainage basin monitoring equipment is in place. this includes stream gauges that telemeter the elevation of stream/river surface in a channel and the rate of water flow to a central computing station. the computed data from the telemetered sites plus the input of stream/river channel cross-sections data allow prediction of where flooding will be a problem, when the flooding will reach an area, and to what level out of a channel (magnitude) the flood is estimated to reach. this gives the populations at risk of the flooding early warnings (hours, days) and time to prepare for the floodwaters or to gather important documents and evacuate to safe higher ground. will be issued for residences or other structure for areas proximate to rivers, whether fl ood insurance for structures will be written for a given zone, and if written, what the cost of fl ood insurance will be. floods can cause landslides and potential health problems for populations when there is torrential rain that is long lasting. first, depending on the location of a fl ooding river channel, rushing water can undermine bank material or erode base of valley walls causing landslides that could affect people living in the threatened areas. second, torrential rains can overload sewer systems so that sewerage carrying pathogens are discharged into waters that may affect water users downstream. populations warned of these potential fl ood-triggered hazards can follow advisories to evacuate if necessary and drink bottled water if their water supply is tainted. preparedness against fl oods includes warning/alert systems sent by weather bureaus and fl ood management agencies and, of course, evacuation plans showing routes to safe sites with staffs and supplies in place to help displaced citizens. an intergovernmental panel on climate change report suggests that fl ooding has been more frequent and more severe in some regions and that there is less fl ooding in other regions [ ] . the report projects that extreme precipitation events will occur over most of the mid-latitude land masses and that those over wet tropical regions will become more intense and frequent. these extreme precipitation events often cause fl ooding that endangers people and property especially if early warning systems are not in place. natural changes in river fl ow other than those that result in fl ooding can be categorized as disasters for people and for a country. for example, the semliki river marks the border between uganda and the democratic republic of congo (drc) . in recent years, and perhaps as a result of global warming/climate change, the volume of water discharged into the river from melting snow and ice from mountain peaks has been reduced as the ice has receded and less snow has fallen. in addition, there have been periods of erratic rainfall and the wet season is wetter. the result has been a meandering of the river that has fl owed through uganda fi elds and left them as part of the drc . ugandan farmers and herders have to pay drc owners of the "new land" a tribute to be able to cross m of river water to continue to work the fi elds and care for their herds on the other side of the river that was ugandan territory before the border shifted as the river meandered. there is international intervention that is working to set a fi xed border so that land that was lost can be assigned correct ownership and ugandan territory can stop shrinking [ ] . landslides also called landslips are the best known of the disaster class called mass movements. mass movements are a world wide problem that cause deaths and hundreds of millions of dollars damage each year to homes, infrastructure, and utilities. other movements in this grouping include: ( ) subsidence or a lowering of an area of the earth's surface; ( ) collapse of soil/rock into a void in the subsurface; ( ) rockfalls; and ( ) mudfl ows (debris fl ows). the latter is activated when torrential or sustained rain over a period of time loads water weight into slopes and seeps into earth materials there, lubricating them and also pushing grains apart because of increased water pressure. the heavy, lubricated, destabilized matter fi nally breaks loose and speeds downslope without warning at as much as a mile a minute ( km/ ph). the destabilization can be further abetted when some of the earth materials comprising a slope is composed of the clay mineral class called smectite (montmorillonite/bentonite), a mineral that expands when it is wetted and shrinks when it dries. a combination of the expansion and lubrication from added water and its weight can bring down a hillside. populations at the base of slopes and beyond are at risk of sudden burial with major loss of life. it should be noted that away from hillsides, soils that expand when wet and shrink when dry cause cracks in basements and foundations of buildings that rest on them, a property damage problem but a problem that is not a threat to the well being of people. avoidance of possible building sites underlain by expansive/shrinkage soils or extraction of the soils prior to construction can mitigate the potential basement/foundation problem. keeping the soils wet with an irrigation system that activates during dry weather has been used to stabilize the reactive soils. landslides occur in hilly topography (elevated terrain, > ° slopes) where the soils are well developed, where poorly consolidated sedimentary rocks comprise the hills, where the structure of the rocks dip (angle) downward and outward from the at risk slopes, and where there is considerable rainfall. these are predictive observations that suggest the possibility of a landslide prone region. landslides are a threat when water seeps into the hills adding water weight that increases the pull of gravity on the earth materials, increases groundwater (pore) pressure that pushes grains apart, and acts as a lubricant for subsurface earth materials. other observations that alert citizens that there is a downslope movement of soil are cracks in the ground and trees that tilt back into a slope. a geologist's observations of scarps in a hilly area attests to it being landslide prone. scarps are mini-cliffs often with exposed soil caused by a landslide but that may be hidden from the untrained eye by overgrowth. tilt-meters (inclinometers) are electrical driven pieces of equipment that can be installed in slopes to telemeter movement or deformation data that suggests the possible onset of a landslide. a recent approach to identify the strong probability of a landslide and thus could serve as an early warning system that allows people to evacuate to safety uses fi ber-optic strain sensors complemented by rainfall data [ ] . these sensors render continuous monitoring in time and space. fiber-optic sensors that can detect displacement, groundwater pore pressure, displacement (slow slope slippage), ground vibrations, and temperature, are glued equally spaced to the surface of a pvc (fl exible) pipe and embedded in shallow trenches in a soil. the pipe . mass movements can bend or twist with pre-slope failure tensile strain (e.g., elastic, plastic, shear, viscous volumetric) registered as d deformation. the various sensors will register the location of the deformation on monitors at an off slope location. physically, a landslide appears as a block of earth materials that breaks from a slope along a scarp and slides down along a concave surface pushing lobes of slide materials out at the base of the block. the areas affected by landslides are generally limited and distances landslide move are generally small although in areas of very steep topography the momentum of a landslide can carry the detached mass a signifi cant distance and across adjacent terrain. as noted previously, landslide moving into a river valley can act as a dam, cause fl ooding initially upstream and subsequently downstream when the temporary dam is breached. in addition to landslides starting as a response to an earthquake's shaking, jarring, rolling motions and excessive rainfall, human activity can lead to landslides as well. this can happen when toes of slopes that give stability to hills in landslide prone regions are cut away to establish a road. this disturbs a slope's equilibrium and is an example of bad land use planning that creates conditions that can lead to landslides. similarly, landslides may be caused if the head of a slope at equilibrium between gravity stress trying to pull soils and rocks down and the strength of the mass resisting slope is weakened by overloading the head with housing and welltravelled roads. the added weight (stress) and vibrations from vehicular traffi c can result in a slope failure. also, sliding can be abetted by loss of anchoring vegetation especially where logging has been active. in landslide prone areas as evaluated by geologists and geological engineers, citizens can be protected to a good degree from injury, loss of property, and even death in two ways. one is zoning that prevents habitation where conditions are conducive for landslips if a slope were to be loaded with destabilizing weight such as the earlier cited homes and well traffi cked roads. second, for existing habitation or planned habitation, geological engineering can diminish the threat of landslides. this is done by ( ) redirecting rainwater or snowmelt so that it does note invade slopes at risk; ( ) by installing retaining walls with perforations that allow in seeping water to exit a slope; and ( ) by installing concrete caissons, a few feet in diameter down to base rock along the front of a vulnerable slope. these efforts are costly so that economic constraints prevent their general use. in areas of japan where topography is hilly and where landslides threaten infrastructure including critical transportation systems, the japanese government has invested in landslide prevention . they sealed hills with shotcrete so that rainwater or snowmelt can not seep into the hills and destabilize them with added weight and lubrication at earth material slide planes. preparedness would require that earth moving equipment and search teams be available to rescue people who survived but are trapped by displaced earth materials. recurrence of landslides is associated with the amounts of rainfall an area receives over a period of time. landslide activity will increase in some global areas and in some regions as a result of increased rainfall because of climate change. as mentioned in the previous section on fl ooding, extreme precipitation events, or simply increases in annual mean precipitation is likely for the high latitudes, the equatorial pacifi c region, and mid-latitude wet regions and can provide the stimulus for landslide activity. central america and south america are especially ar risk [ ] . an avalanche is a large mass of snow that suddenly slides downslope. there are two general types of avalanches: ( ) sloughs that are small fl ows of powdery snow that are unlikely to kill people or destroy structures; ( ) full depth avalanches that are massive slabs of snow that break loose from a mountain and cause death and destruction. the latter may carry ice, soil, and rock debris. avalanches generally occur without warning on slopes > ° and < °at alpine areas worldwide (e.g., swiss mountains, western canada, new zealand, alaska, the himalayas). one or a combination of factors can contribute to an avalanche event. these include storminess, slope shape, orientation of steep slopes with respect to the sun, the rough or smooth character of the ground beneath a snowpack, vegetation, the nature of the layers of a snowpack, and vibration. thus, if there is a cm ( in.) or more snowfall in a h periods, there is likely to be an overloading and an avalanche depending on how the layers in a snowpack are bound together. most avalanches occur during snow storms and blizzards. clearly, a steeper convex slope is more conducive to avalanche activity. a rapid temperature increase can cause melting of a layer in the snowpack that results in an avalanche. the more vegetation there is deters the down fl ow of an avalanche as would a rough rocky surface beneath a snowpack. vibrations from activity on a slope (skiers, snowboarders, snowmobiles), thunder storms, low fl ying jet planes, and explosions can set off an avalanche. during wwi, thousands of soldiers in alps regions were killed by avalanches triggered by artillery fi re. avalanches can fl ow downslope at speeds of km/h ( mph) or more. depending on the mass being moved, an avalanche can kill people, and damage or destroy structures and infrastructure (homes, recreational areas, bridges, tunnels [block road and/or railway movement]), pipes and utility lines (water, natural gas, electricity), and put workmen maintaining an infrastructure at risk. hundreds of people are killed by avalanches each year. during and , avalanches in nepal , triggered by an earthquake and by unseasonal severe rain and snow blizzards, roared downslope from mount everest and other peaks in the region. many trekkers were killed, as were sherpa guides and climbers preparing for the climb to the summit of mount everest. people can be protected by zoning that evaluates the history of avalanches, the paths they follow, and their frequency and reach in an alpine area to prepare risk maps to prevent use or allow limited or full use of the zoned terrain. in areas where use of terrain is allowed, warning systems are in place so that when sounded, citizens will not enter a threatened area, or if there, evacuate it immediately. avalanches can not be prevented but defensive structures can be used in established populated areas to try to divert them such as snow fences or snow walls. avalanche sheds can be used to protect structures and transportation routes by making the fl ow of snow ride over them. dangerous buildups of snow on slopes that are known for avalanches can be set off as snow slides or snow slips using vibrations caused by controlled explosions with explosives implanted in the snowpack, dropped by helicopter, or delivered by artillery shells. excellent sources of information on avalanches can be found at www.ussartf.org/avalanches.htm and at www.conserve-energy-future. com/types-causes-effects-of-avalanches . a rockfall happens when a mass of rock from a very steep to vertical cliff detaches from the face of a cliff and free falls down. the rocks bounce off underlying rocks, often detaching them as well. the falling rocks crash onto the base of a cliff sometimes running out damaging structures and/or infrastructure, blocking roads and putting vehicles at risk. in addition to steep topography, the geological character of the rocks comprising a cliff, the climate, and sometimes vegetation are factors that infl uence the risk of a rockfall but do not predict when one may occur. fractures and fi ssures in rocks can fi ll with water during a winter day and freeze at night causing ice wedging that weakens rocks against the pull of gravity. trees that root in cracks and crevices in a rock cause root wedging that does the same. stress when an earthquakes strikes an area can loosen rocks to the point that they fall from a cliff face. a geological evaluation of an area can reveal areas that have suffered rockfalls and areas likely to have rockfalls but geologists can not reliably predict when a fall will occur. to protect an area and its inhabitants, infrastructure, and businesses from rockfalls, municipalities have options. they can install catchment fences at the base of cliffs to prevent run outs, require that rock bolts be inserted to stabilize an at risk rock face or that chain-link fencing be fastened to the rock face. subsidence of an area of the earth's surface is the result of the continuous extraction of large volumes of groundwater or petroleum from underlying sedimentary rocks without recharge or replenishment of fl uids. fluids in subsurface rocks provide buoyancy pressure that strengthens the resistance of the rocks to compaction and hence subsidence of the earth's surface . if volume loss does take place in subsurface rocks, subsidence may or may not occur depending on the strength and thickness of overlying rocks. overlying rocks may be inherently strong enough so that they do not subside. conversely, they can respond to the compaction of underlying rock by subsiding. subsidence can be arrested or even reversed somewhat in some geological settings if a degree of buoyancy pressure is reestablished by re-injecting fl uid (e.g., brine) into the rock. for example, in oil fi elds, eight barrels of brine are extracted with one barrel of crude oil. the brine can be recycled into the oil reservoir under pressure. similarly, if extraction of groundwater from aquifer rock is replenished by groundwater recharge, any subsidence that has taken place should stop. oil production at the wilmington field in southern california, usa, began in and caused a subsidence that by reached . m ( ft) at long beach harbor and extended to parts of los angeles harbor. the subsidence damaged oil wells, pipelines harbor infrastructure, railroad tracks, streets, and bridges and reversed the fl ow of sewers and storm drains. repair cost more than u$s million at that time. brine reinjection was used to arrest the subsidence and there was stabilization and a rebound of cm ( in.) [ ] . mitigation of a possible subsidence problem can be determined by geologists and geological engineers who study samples of the sequence of rocks from the surface to the oil or water reservoir. they can predict whether or not subsidence will take place and if it is realistic to plan to recharge the extraction reservoir with volumes of fl uid that equal the volumes withdrawn, thus maintaining buoyancy pressure in the reservoir/aquifer. this can prevent or at least minimize subsidence if it begins. governments can be stressed two ways economically because of subsidence. first is the cost to repair the damage done to infrastructure and structures in the areas affected by subsidence. second are the economic losses that can be incurred if a productive sector is slowed down by not being able to extract critical fl uids from the subsurface without having to invest more funding to improve extraction by drilling wells deeper. this would likely mean an increase in the price of a commodity used to make or grow a product and hence be infl ationary for the public. a case in point is the more than four year severe drought being suffered in in the central valley (especially the san joaquin valley) of california, usa where subsidence has been a recurring problem during past droughts as groundwater was extracted, but not excessively, from aquifers to make up for the shortage of surface water. however, excessive groundwater pumping during the existing extended drought, mainly by the agricultural sector, has lowered groundwater tables to ft (~ m) lower than recorded in the past. as a result some areas experienced a subsidence of up to two inches ( cm) a month as fi negrained layers in the aquifer were depleted of their buoyancy pressure and compacted. if the compaction is tight enough, part of the storage capacity (porosity) and permeability (ability to transmit fl uids) of an aquifer could be lost. the subsidence varies with location in the valley. one area subsided at / in. a month. the maximum subsidence was about a foot ( in. or cm) a month [ ] . another result of the over pumping is a reduction of fresh water pressure in the aquifer that could result in salt water intrusion where the aquifer extends to the marine continental shelf. the differential subsidence in the san joaquin valley has caused damage to infrastructure, the most important of which may be the california aqueduct comprised of canals, pipelines, tunnels and pumping stations. the aqueduct carries water from northern california rivers that receive melt from the sierra nevada snowpack and from rainwater some miles (~ km) to southern california. change in the fall (inclination) of sections of the aqueduct from subsidence and low spots on the system prevent the water from fl owing as well as it did pre-subsidence and thus needs reworking. changes in the fall of sewer lines have to be repaired to prevent sewage backup and its consequences from a reversal of slope. similarly, water pipelines have to be reset to allow effi cient fl ow of water. some bridges have subsided to the degree that they are no longer above the water surface. roads are ruptured and have to be repaired. levees in place for fl ood control when the rains come have sunk and have to be raised. building foundations sink as well and need correction. very important in this agriculturally dependent valley and its towns has been the destruction of thousands of public and private well casings. california and the agriculture industry have the economic wherewithal to make the necessary repairs once the drought breaks. the central valley grows about % of the vegetables and fruits sold in the united states. as previously indicated, if crops fail or yields drop because of the lack of water for irrigation, the cost of the produce and fruit will increase. the state is investing large sums of money to develop a capital improvement program. collapse of a small areas of the earth's surface into voids in the subsurface causes sinkholes. this is an action that takes place most often where there is a relatively high water table in terrain underlain by limestone, a rock type that houses many famous cave systems worldwide. in this scenario, groundwater moving slowly through limestone continually during geologic time (millions of years) dissolves the limestone leaving voids in the subsurface. when these are large enough and the roof rock strength cannot resist the pull of gravity, the roof rock collapses into the subsurface cavity. the areas affected are generally small, rounded or oval shaped, and tens of meters or less across. sinkholes have caused structural damage to buildings, swallowed homes and car dealerships, ruptured roads (pavements) including highways, and in rare cases have caused injury and death. sinkholes have developed as well in terrain underlain by salt-rich (evaporite) strata that are dissolved by irregular groundwater fl ow. in some cases, water pipes underlying roadways have broken and the released water has washed out the earth materials around a ruptured waterlines creating open spaces in the subsurface into which earth/road materials have collapsed. underground coal fi res can create cavities into which the overlying rock and soil can subside and ultimately collapse. to lessen the risk of suffering loss from collapse from a future sinkhole when buying a home or business or land to develop and build on, one should commission an evaluation of the terrain by experts that will predict its vulnerability for collapse. for example, a geologist will fi rst assess the geology of the area under study (especially when underlain by limestone or dolostone), the topography, and determine the position of the water table that could cause dissolution of underlying rock. he/ she will review the history of insurance claims against collapse situations in the area. in a surface analysis, the geologist will look for signs of potential surface movement that could portent sinkhole activity by examining building foundations in the neighborhood for visible cracks, especially arcuate ones, cracks in roads or sidewalk pavement, and depressions or low spots in the terrain as well as the presence of small ponds that could represent former sinkholes. this study will suggest the level of risk in an area but only an investigation of the subsurface conditions can give more accurate vulnerability information. this can be done using ground penetrating radar (gpr) that takes a short time using modern equipment that digitizes data for ready presentation and interpretation [ ] . in one florida study, the gpr data for nine m lines were generated in h. another option is to do a seismic study that will yield information as to the solid rock nature or void presence in the subsurface. studies such as these, where collapse is an endemic problem, but on a larger, perhaps county scale, can result in sinkhole vulnerability maps that can guide land use planning for small scale evaluations of potential sites for buildings or infrastructure. where there is a void in the subsurface but there is a need to use the terrain, it may be possible to fi ll the void with grout to stabilize the surface but this can be very costly. a question exists as to whether scientists can spot an incipient sinkhole as it develops so as to be able to warn inhabitants of at-risk homes to evacuate. the possibility exists using the nasa satellite system insar (interferometric synthetic aperture radar) that detects small movements on the ground. a study of an area in louisiana, usa and noted that the ground shifted horizontally in. ( cm) in a section of the survey. a sinkhole opened up there a month later and the horizontal displacement observed was towards the center of the sinkhole [ ] . scientists believe that there is the potential to use this deformation as an early warning system in other sinkhole prone areas so that people can remediate the condition or evacuate buildings that might be at risk of a calamitous collapse. this is a step towards preparedness. however, they noted that not all sinkhole sites have surface shifting before a collapse, making necessary the previously cited methods to detect subsurface cavities that could become sinkholes. mitigation of an economic loss of home or business to a collapse into sinkhole event can be achieved by having collapse/sinkhole insurance as part of a home owners policy or an insurance policy that covers a business structure and inventory. the policies should cover collapse whether the event is from natural processes (e.g., subsurface dissolution of limestone) or from the failure of a water or sewer pipe and subsequent washout of supporting earth materials that leads to a collapse of an overlying home, business structure, road, highway, or bridge. because many home buyers and home owners are unaware of their vulnerability (risk) from a collapse/sinkhole event, the state of florida, usa mandates that home owners carry catastrophic ground collapse coverage. in the united states, alabama, kentucky, missouri, pennsylvania, texas, and tennessee are states with collapse into sinkhole problems and their own insurance requirements. infectious/communicable diseases are natural hazards abetted in some instances by human actions or inactions. some of these diseases have been eradicated (smallpox) or nearly eliminated (polio, measles, guinea worm disease [dracunculiasis]). others are treatable (malaria) or can be stabilized (hiv/aids), and still others are not preventable (dengue fever) but the exposure to which can be alleviated to some degree. still others for which prevention and/or curing/stabilization medications are not available are being researched intensely such as an ebola vaccine because of the - west african outbreak of the disease. in the most recent clinical trial of an ebola vaccine, the success rate was %, encouraging but requiring more testing before it is approved for universal application. the threat or onset of infectious disease can be mitigated in several ways. first is prevention. there are vaccines available that can protect citizens against contracting specifi c infectious diseases. in the case of measles and polio, the actions of religious zealots (taliban sect) who have burned down health clinics and injured or killed health workers have prevented vaccinations for all so as to achieve global eradication of poliomyelitis in two countries where it still occurs (pakistan and afghanistan) and measles (globally), especially in asia and africa. however, this not withstanding, vaccination programs for measles are making good progress. from to the number of deaths from measles has dropped %, from , to , . mumps and measles (mmr vaccine) and whooping cough (dtap vaccine) are two other infectious/communicable diseases that can be eliminated if vaccinations are given. in the case of children, a second application of the mmr vaccine is necessary to give them lasting immunity to mumps and measles. it should be noted that vaccinations may not be close to % effective. scientists have modeled the probable effectiveness of vaccinations for endemic infections [ ] . they conclude that vaccines denominated as "leaky" provide the same degree of resistance to a disease to all who have been vaccinated and gives a partial immunity after being vaccinated. a vaccine called " all-or-nothing (aon)" is best applied when the probability of re-infection is high, transmission is likely, or when a vaccine has low power to reduce the risk of infection. in contrast to a "leaky" vaccine, an aon vaccine completely protects a major number of vaccinated persons but others in the population receive no direct benefi t from it. some infectious/communicable diseases such as tuberculosis can be cured with medicines taken over a prescribed period of time. chagas is a disease that is endemic in latin american nations. it can be cured if medication is taken early enough after the onset of the disease. if this does not happen the persons with chagas disease can look forward to a middle age with cardiac and gastrointestinal problems with the healthcare burden this implies. cholera is an infectious disease that can be controlled with access to adequate sanitation or cured by rehydrating victims with oral rehydration salts. if a cholera victim's dehydration is severe, iv plus antibiotics are used to cure the patient. a short term lasting cholera vaccine is available and used by health workers where there is a cholera epidemic. aids/ hiv is an infectious/communicable disease that is not curable and that has killed million people. the disease can be stabilized if an infected individual has access to (or the funds to pay for) a cocktail of antiretroviral medications taken during a lifetime. this allows an hiv/aids carrier to live an otherwise healthy life and be productive in his/her community. about . million of the . million people with the disease are now taking the retroviral medications. seventy percent of the global total of two million new cases are in sub-saharan africa [ ] . unless the . million infected individuals not on antiretroviral medications get access to them and follow prescription protocols, the disease will continue spreading through populations. some infectious diseases can be controlled but not eliminated. infl uenza is controlled by a seasonal vaccination that is prepared according to what scientists consider will be the dominant strains of the infl uenza virus during a coming infl uenza season. a strain was missed during the - season and more people suffered sickness as a result. although some people do contract the disease, the vast majority of the vaccinated population is protected. if some do contract infl uenza from the unaccounted for strain, its effects are generally less than they would be without the vaccination. the earlier cited ebola epidemic that raged in liberia, sierra leone, and guinea, western africa controlled and the region declared free of the disease after more than , deaths of more than , infected. the ebola epidemic shows how vulnerable many countries/regions are because of an inadequate health infrastructure that is not prepared to cope with a disease once identifi ed, its spread, and the care and treatment of large numbers of infected people. ebola is transmissible by body fl uid contact and this had to be learned by family members and others caring for infected individuals as a fi rst phase in controlling and ultimately leaving a country ebola free. the spread of this disease to other countries, regions, or around the world was limited by the general immobility of people from affl icted countries and controls at transportation centers and by immigration controls at adjacent countries for those from the disease ridden countries who were mobile and presented a transmission risk. as the disease progressed in the affl icted nations, help arrived rapidly from developed nations that had experience in disease control that experts applied on site and taught to health givers in spite of the fact that who delayed before putting out a public health emergency alert. laboratories that had been researching vaccines against ebola increased their efforts and laboratory testing on animals. test results of the promising vaccines on human subjects have been encouraging for two of the vaccines developed. although the vaccines may prove to be effective against ebola after future positive test results, they were not available at the time the epidemic was identifi ed in west africa . preparedness is the key to dealing with the outbreak of an infectious disease that could develop into an epidemic/pandemic. as noted above, initial preparedness such as protocols in the physical contact with affl icted persons that caused transmission of the disease, their treatment, and burial practices of their kin were lacking in the west african ebola outbreak. a november, report by a panel from the harvard global health institute and the london school of hygiene and tropical medicine critiqued the who for not declaring a public emergency until months after being informed of the ebola outbreak by guinea and sierra leone [ ] . the panel recommended several ways in which the who could improve its role in preparing for and dealing with an infectious disease crisis. among the recommendations were the following: ( ) the need to invest in developing a nation's core capacity, that is, its ability to detect, report, and respond rapidly to outbreaks; ( ) the strengthening of incentives for early reporting of outbreaks and science-based justifi cations for trade and travel restrictions to prevent transmission of a disease; ( ) the creation of a who center with adequate capacity to respond quickly to a disease outbreak; ( ) an assurance of access to the benefi ts of research that yield improved diagnostics, and the most effective medicines and vaccines; ( ) information on best protocols to follow in treating an infectious disease and on cultural awareness and traditions to account for when dealing with families of the affl icted or dead; and ( ) availability of funding to put these and other recommendations into practice [ ] . with respect to improved diagnostics ( above), nature published a december supplement in which contributors modeled the impact of new diagnostic and prognostic technologies for lessening the global burden of infectious diseases [ ] . they are of the opinion that new diagnostics can more rapidly direct patient treatment and limit disease transmission to the general population thus effectively reducing the spread of epidemics. the effectiveness lies in the education of on-site health providers in the new diagnostics and protocols as they become operational. the contributors also believe that research can come up with new and rapid diagnostic protocols for multiple diseases that affect populations worldwide, that are effective and reduce costs that existing protocols incur, especially in less developed and developing countries. the ebola outbreak and spread by direct or indirect contact of an infectious disease raises the question of whether the world is prepared to combat an infectious disease, natural or from bioterrorism, that is transmitted through the air we breathe. given the ease of transmission, the mobility of disease carriers within a country or internationally, the populated venues where transmission through respiration can take place (e.g., airplanes, cruise ships, theaters, arenas, subways, malls), the answer is no for many less developed and developing countries that do not have a well functioning health infrastructure … not enough doctors, nurses, well equipped clinics, hospitals, laboratories, medicines, or vaccines. thus, a virulent disease transmitted through the air could infect and kill millions of people before medical care to treat it or vaccines and medicines to cure it are found and tested so as to control and/ or eliminate a killer virus. it is possible to minimize the spread of an easily transmittable infectious disease but this requires a major investment. a proactive action would be for the health capable and economically advantaged nations to work with less prepared ones to create a good healthcare infrastructure, a mission of the who that has been neglected [ ] . this would include training personnel as deemed necessary, building clinics where hospital care may be lacking, setting up laboratories that can identify diseases at their onset rather than waiting for results from a central laboratory, and by educational programs for citizenry. response team training in all countries is essential to initially deal with a disease. obviously, there has to be open and clear lines of instant communication between health organizations locally, regionally, nationally, and internationally when disease surveillance detects an outbreak node of a known or unidentifi ed infectious disease. this allows a global response to begin on how to cope with the outbreak of a deadly viral disease in order to minimize its spread and develop a treatment protocol. diarrheal disease that affect s of millions of people each year can be controlled in two ways. first, they can be countered and their impact on society lessened by the use of antibiotics assuming medications are available and economically accessible. second, is the identifi cation and elimination of the sources of diarrheal diseases. basically this means improving sanitation conditions and food storage, preparation, and handling operations. investment by governments to provide access to advanced sanitation systems and for education with respect to food cleanliness can lessen the onset and spread of diarrheal infections and limit employee sick days, thus helping a country's productivity and economic development. there are several infectious/communicable diseases that do not fall into the categories described in previous paragraphs. vaccines and medications are being researched with some in trial stages. these diseases include malaria, dengue fever, yellow fever, west nile virus, plague, and most recently ebola because of the west african epidemic. as already noted, in the case of the ebola epidemic, preliminary tests of new vaccines being researched have had good results on human volunteers. however, it is uncertain whether the positive results are because of the vaccines or the care factor given to those with the disease. a zika vaccine is scheduled for phase clinical trials during the latter period of . lastly it should be noted that science magazine commissioned a survey to prioritize ten potential vaccines that would merit increased government and industry funding for their research and development. these vaccines do not show clear scientifi c or safety obstacles and would benefi t societal health conditions mainly in less developed and developing countries. the surveys were sent to vaccine experts globally who were asked to prioritize the listed vaccines based on scientifi c feasibility, morbidity and mortality, and societal/economic impact. fifty experts fi lled the survey. the overall priority ranking results in were ( ) ebola sudan, ( ) chikungunya, ( ) mers, ( ) lassa fever, ( ) marburg, ( ) paratyphoid fever, ( ) schistosomiasis, ( ) rift valley fever, ( ) sars, and ( ) hookworm [ ] . vaccines for some of these ( , , , , , and ) could prevent or reduce the chances of serious outbreaks in the near future. zika would be a addition. the psychological impact of living through a natural or anthropogenic hazard that injures or kills family and friends, and damages and destroys homes, business, and places of employment is a shock to citizens that puts them in a state of disbelief and distress. for that reason, the preparedness sections on hazards recommends that mental health professionals be available to the suffering populations. mental health is considered here as a health hazard that in this text is triggered by a primary event but may result from a secondary event (e.g., earthquake aftershock) or a triggered hazard. the stresses caused can affect populations for reasons other than suffering physical, biological, and chemical dangers environments can present [ ] . citizens living through a natural or anthropogenic hazard and secondary hazards that can occur suffer stress from several results of the event. the immediate stress factor is a concern for families in the degree of destruction citizens see wreaked on their environment. stress levels heighten from the loss of or injury to family and friends and loss of home and possessions. the greater the loss, the higher the level of stress. other factors that stress populations suffering through the immediate . mental stress aftermath of a major hazard event include displacement, sense of vulnerability and insecurity, apparent slowness of assistance midst the chaos a hazard can cause, fear of what might come next, and physical exhaustion. there are ongoing discussions on coping with the stress of disaster [ , ] . post hazard stress arises from loss of employment and income, apparent slowness in recovery leading to societal normalcy, and reconstruction. in a latter section of this book, different classes of insurances are discussed that can, if purchased, mitigate economic stress from hazards losses. the number of people susceptible to hazard-caused stress and their resulting mental health problems will increase in the future because of population growth and increased population density in urban centers that have experienced recurring hazards. this is especially true for major cities (perhaps greater than one million inhabitants) and for mega-cities with more than ten million people. easing or mitigation of stress levels involves having access to mental health professionals who can help relieve people's lasting anxieties, receiving social support from family and friends, focusing on the present and future rather than reliving the past, and taking care of oneself physically. climate change the shifting river that is making uganda smaller brillouin optical time-domain analysis for geotechnical monitoring subsidence, rebound and surface strain associated with oil producing operations progress report: subsidence in the central valley sinkhole detection in florida using gpr and cpt ( pp.) sinkhole: precursory deformation measured by radar interferometry vaccination programs for endemic infections: modeling real versus apparent impacts of vaccines and infection characteristics hiv/aids fact sheet n . geneva: world health organization will ebola change the game? ten essential reforms before the next pandemic ( pp.). the report of the harvard global health institute and the london school of hygiene and expanding the role of diagnostic and prognostic tools for infectious diseases in resource-poor settings vaccine priority survey the role of public health within the united nations post- framework for disaster reduction impact of natural disasters on mental health coping with the stress of natural hazards key: cord- - ut bu authors: lane, j. michael; summer, lila title: smallpox as a weapon for bioterrorism date: journal: bioterrorism and infectious agents: a new dilemma for the st century doi: . / - - - - _ sha: doc_id: cord_uid: ut bu smallpox, the only disease ever eradicated, is one of the six pathogens considered a serious threat for biological terrorism (henderson et al., ; mahy, ; whitley, ). smallpox has several attributes that make it a potential threat. it can be grown in large amounts. it spreads via the respiratory route. it has a % mortality rate. the potential for an attack using smallpox motivated president bush to call for phased vaccination of a substantial number of american health care and public health workers (grabenstein and winkenwerder, ; stevenson and stolberg, ). following september , , the united states rebuilt its supplies of vaccine and vaccinia immune globulin (vig), expanded the network of laboratories capable of testing for variola virus, and engaged in a broad education campaign to help health care workers and the general public understand the disease (centers for disease control and prevention, a). this chapter summarizes the scientific and theoretical bases for use of smallpox as a bioweapon and options for preparation for defense against it. smallpox, the only disease ever eradicated, is one of the six pathogens considered a serious threat for biological terrorism (henderson et al., ; mahy, ; whitley, ) . smallpox has several attributes that make it a potential threat. it can be grown in large amounts. it spreads via the respiratory route. it has a % mortality rate. the potential for an attack using smallpox motivated president bush to call for phased vaccination of a substantial number of american health care and public health workers (grabenstein and winkenwerder, ; stevenson and stolberg, ) . following september , , the united states rebuilt its supplies of vaccine and vaccinia immune globulin (vig), expanded the network of laboratories capable of testing for variola virus, and engaged in a broad education campaign to help health care workers and the general public understand the disease (centers for disease control and prevention, a) . this chapter summarizes the scientific and theoretical bases for use of smallpox as a bioweapon and options for preparation for defense against it. variola major, the virus that causes smallpox, is an orthopox. variola minor, its less pathogenic cousin, has little theoretical expectation for use as a bioweapon. v. major is a large dna virus ( ϫ nm), with one of the most complex genomes of human viruses ( kbp double-stranded dna). the genome of several strains of v. major has been completely sequenced, but the functions of the genes have not all been elucidated (moss, ) . like most orthopox viruses, variola is host specific. humans are the only natural hosts. experimental infection of small numbers of monkeys with large intravenous doses of virus has been accomplished (leduc and . the virus grows well on many tissue cultures and on the chorioallantoic membrane of embryonated chicken eggs. vaccinia virus, a close cousin of variola, is routinely grown in many laboratories, and can be lyophilized to ensure stability to heat. the same techniques could be used with variola. the genetics of vaccinia are well known, in part because vaccinia strains have been proposed as carrier viruses for genes from other agents, including hiv (moss, ; smith et al., ) . this work shows that the genes of orthopox viruses are amenable to deletions and additions. researchers working with ectromelia (mousepox), another close cousin of variola, have created a very virulent strain, able to escape the protective effect of prior immunization with vaccinia (born et al., ; jackson et al., ) . recent research suggests that smallpox virus could be recreated by synthesizing long strands of dna, thus enhancing its availability for bioterrorism. the viral genome has been deposited in public databases, making such work possible although time-consuming and highly technical (wade, ) . variola virus is fairly hardy in the environment if protected from heat and ultraviolet light. however, it is easy to kill with standard hospital disinfectants or ultraviolet light (fenner et al., ) . there is little information about its ability to survive when aerosolized; by analogy to vaccinia virus, it probably could survive for an hour or more if not in direct sunlight (harper, ; thomas, ) . in summary, variola has several virologic attributes that make it attractive as a terrorist weapon. it is easy to grow. it can be lyophilized to protect it from heat. it can be aerosolized. its genome is large and theoretically amendable to modification. the pathogenesis of smallpox is believed to resemble that of mousepox (ectromelia), which was elucidated by fenner and colleagues during the s and s. subsequent work with rabbitpox and monkeypox, orthopoxes that also cause species-specific systemic disease, have refined our understanding of pathogenesis (fenner et al., ) . infection is via the respiratory route. during the first or days after infection, the virus multiplies in the epithelium of the upper respiratory tract. it is then released into the bloodstream in a primary (asymptomatic) viremia. this viremia is cleared by the reticuloendothelial system, where the virus again multiplies. about or days after the initial infection, the reticuloendothelial cells release a secondary viremia, which is probably cell-associated. this is a massive viremia that causes an intense and prostrating prodrome, with fever, myalgias, and other symptoms of a vigorous viremia. the secondary viremia is cleared toward the end of the prodrome, when the leukocyte-associated virus becomes localized in small blood vessels in the dermis and upper respiratory epithelium. the skin lesions evolve in a stately and characteristic way (see section on "clinical disease"). the histopathology is characteristic. the lesions are full of virus. the fluid from vesicles and pustules and scabs is infectious, and virus can be isolated from scabs (breman and henderson, ; fenner et al., ) . the main source of natural infection is the secretions from the upper respiratory tract, where lesions quickly break down and excrete virus because the epithelium in the nose and throat lacks the firm keratinized layer that seals in the virus in the lesions on the skin. patients are therefore infectious from about - hours before the initial faint macular rash appears on the skin, and remain infectious throughout the course of the rash (breman and henderson, ; dixon, ; fenner et al., ) . the clinical illness and fatality rate roughly parallel the density of the skin lesions. when lesions are sparse, cases are unlikely to die and probably are not efficient transmitters. however, their mobility may allow them to have enough social interaction to result in transmission. vaccine-modified smallpox can be very mild and nonfatal, although if vaccination was more than years prior to exposure, the fatality rate is not trivial and patients can still transmit the disease. as lesions become denser and confluent, the fatality rate increases, the amount of virus in the respiratory secretions increases, and patients are more infectious (fenner et al., ; mack, ; rao, ) . hemorrhagic smallpox has a fatality rate of nearly %, and patients are highly infectious. about - % of unvaccinated patients with v. major get hemorrhagic smallpox, probably with disseminated intravascular coagulation (bray and buller, ; mckenzie et al., ; rao, ) . they are usually very sick, usually unable to get out of bed and thus may not transmit efficiently. the clinical presentation (from mild to discrete to confluent to hemorrhagic) is a function of the host response, not the virus. the clinical types do not breed true, in that transmission from any patient can give rise to any of the clinical presentations, and the virus is the same. the immune response in smallpox includes both cell-mediated immunity and production of neutralizing antibodies. both probably appear about days after the onset of the rash (fenner et al., ) . immunity is essentially life-long in recovered patients, although very rare cases of second infections have been reported several decades after initial infections. the immune response in hemorrhagic smallpox is probably poor, contributing to the very bad outcome. in summary, smallpox causes an acute illness with a devastating prodrome, with virus primarily transmitted via respiratory secretions. the fatality rate roughly parallels the density of the skin lesions and hence the intensity of the preceding viremia. the clinical spectrum of variola major has been well described (breman and henderson, ; dixon, ; fenner et al., ; rao, ) . the illness starts with a dramatic prodrome, with high fever and signs and symptoms indicative of massive viremia. the patient usually improves somewhat when the viremia is cleared, although the fever does not return to normal. as the fever decreases (about - days after the onset of the prodrome), the characteristic rash becomes evident. during the first day or two of the rash, it may be impossible to distinguish from measles or many other viral exanthms. on dark skin, the rash may not be apparent on the first day or two, being simply faint macules. if the mouth is carefully examined, an enanthem can be detected. the lesions of smallpox have a predilection for the cooler parts of the body and are most dense on the face and peripheral extremities. lesions are present on the palms and/or soles in the majority of cases. the individual lesions undergo a slow and predictable evolution. excellent photographs can be found at the centers for disease control and prevention (cdc) website (centers for disease control and prevention, a; world health organization, ) . by about the rd day, the macules become papular, and the papules progress to fluid-filled vesicles by about the th day. these vesicles become large, hard, tense pustules by about the seventh or eighth day. figure . is a typical patient with semiconfluent smallpox on about the th day of rash. the pustules are "in" the skin, not just "on" the skin. they are deep-seated and feel like dry garbanzo beans, usually nearly cm in diameter. about the th or th day, the lesions begin to dry up and umbilicate. by about weeks after the onset of the rash, lesions are scabbing. about weeks after onset, the scabs begin to separate, leaving pitted and depigmented scars. the causes of death from smallpox are not well elucidated. massive viral toxemia probably causes a sepsis cascade. cardiovascular shock may be part of the agonal syndrome. in hemorrhagic cases, disseminated intravascular coagulation probably occurs. antibacterial agents are not helpful. loss of fluid and proteins from the exudative rash probably contribute to death. modern medical care might reduce the fatality rate, but there is no way to prove that contention (bray and buller, ; breman and henderson, ; fenner et al., ; koplan and foster, ; rao, ) . in summary, smallpox produces a serious and prostrating clinical disease. the characteristic pustular rash is easy to diagnose if smallpox is known to be circulating. there is no proven therapy. no data exist to show whether modern supportive care could reduce the death rate. when smallpox is known to be circulating, the clinical presentation and characteristic rash make diagnosis fairly easy. diagnosis can be difficult when smallpox is not high on the index of suspicion. initial cases after a covert bioterrorist attack will probably be missed, at least until the th or th day of the rash. transmission may have already taken place by this time. for that reason, but also as good clinical and public health practice, patients with undiagnosed rashes accompanied by fever should always be isolated until the diagnosis is established. the cdc has produced a diagnostic algorithm based on experience with the differential diagnosis of suspect smallpox cases. this algorithm is figure . and can also be found at the cdc website (centers for disease control and prevention, b; seward et al., ) . most cases initially considered suspect smallpox turn out to be chickenpox, disseminated herpes simplex, secondary syphilis, or drug eruptions. if this algorithm indicates that a patient is high risk to be smallpox, local and national public health authorities should be immediately notified by telephone, and laboratory specimens taken for polymerase chain reaction (pcr), electron photomicroscopy (em), and viral culture. a network of laboratories around the united states can do real-time pcr on very short notice (centers for disease control and prevention, b) . state health department laboratories are or know of the nearest one of these laboratory resource network laboratories. instructions for obtaining, handling, and shipping specimens can be found at the cdc website (centers for disease control and prevention, b) . rapid diagnosis requires a sophisticated viral diagnostic laboratory. rapid testing is best done using em to identify actual virions and real-time pcr assays to detect viral dna. these tests are highly sensitive if adequate specimens are provided to the laboratory ropp et al., ; sofi et al., ) . pcr is very specific, whereas most mammalian orthopoxviruses, including vaccinia and monkeypox, have the same brick-like viral structure and cannot be reliably differentiated from variola with em. public health action should be initiated by either a positive pcr or em test, but confirmation by viral culture should also be attempted. laboratory tests require adequate specimens. copious specimens must be provided if smallpox is seriously suspected (i.e., if the clinical picture fits the "high-risk" category in the algorithm, or if intelligence or communications from terrorists suggests an attack). at least six lesions should be unroofed. the roof tissue and the pus should be placed in separate sterile vials. em grids should be touched to the base of the opened lesions. pus can be dried directly onto plastic slides. punch biopsies of several lesions should be taken, with half put into formalin fixative and half sent unfixed. shipping and handling of specimens are important; guidance should be sought from cdc (centers for disease control and prevention, b). efforts are currently underway to detect smallpox virus in the environment, including in air distribution systems in large buildings. these involve filtration of large volumes of air and testing the material from the filters with pcr (nbc news, ). these techniques are conceptually encouraging, but have unknown sensitivity and specificity. in summary, clinical diagnosis is easy when smallpox is suspected and the rash is fully developed. sophisticated laboratory tests are available to diagnose suspect cases j. michael lane rapidly and accurately. laboratory specimens must be adequate in volume, and carefully handled. suspect smallpox is a public health emergency and proper public health authorities must be immediately informed. there is no nonhuman host for smallpox, and there are no subclinical carriers. once patients have recovered, they are immune and cannot transmit the infection (fenner et al., ) . since there have been no cases since , any new cases must be the result of bioterrorism, or a highly unlikely escape from one of the two official laboratories. the epidemiology of naturally occurring smallpox has been well studied. the work of downie et al. ( ) , rao et al. ( ) , heiner et al. ( ) , mack ( ) , mukherjee et al. ( ) , sommer and foster ( ) , and the recent demographic analysis by gani and leach ( ) provides a good picture of the occurrence and spread of the disease. smallpox does not ordinarily spread rapidly. transmission requires prolonged face-to-face contact, such as that which occurs among family members or caregivers. transmission is most efficient when the index patient is less than feet from the recipient, so that the large-droplet respiratory secretions can be inhaled (downie et al., ; mack, ; sarkar et al., ) . since virus is not secreted from the respiratory tract until the end of the prodrome, patients are usually bedridden when they become infectious and usually do not transmit the disease widely. smallpox has been documented to spread from bedding containing pus and scabs, and from dead bodies (dixon, ; fenner et al., ; hopkins et al., ) . modern hospital systems for handling infectious wastes and cremation of bodies should eliminate such transmission (centers for disease control and prevention, c) . under natural conditions, smallpox is a highly seasonal disease. transmission is most efficient in cool dry seasons, possibly because respiratory droplets evaporate quickly in dry conditions -creating small droplets that remain suspended in the air. although the vast majority of smallpox is acquired by prolonged face-to-face contact, a well-documented outbreak proves that true aerosolization does occur. a patient whose smallpox had not been diagnosed was hospitalized in meschede, germany, in . he was coughing vigorously, which probably contributed to creating an aerosol. patients and visitors on a floor above his room became infected with smallpox (wehrle et al., ) . modern hospital infection control practices should keep such transmission from occurring in hospitals, but the outbreak proves that aerosols can be dangerous. these epidemiological observations led the world health organization in to switch tactics from mass vaccination to the "surveillance and containment" method. this method is also sometimes called "ring vaccination". c.w. dixon describes, in his once-definitive textbook on smallpox ( ), how he used "expanding ring" vaccination to control a smallpox epidemic in north africa after world war ii. vaccine supplies and workers were in short supply, so mass vaccination was impractical. the identification of patients and vaccination of their contacts was his first priority. the next was vaccinating people living in tents surrounding the infected family. finally, an entire infected village was vaccinated if time and vaccine supply permitted (dixon, ) . this concept formed the basis of the surveillance and containment method, after foege and his colleagues made similar observations about the ease of controlling smallpox by vaccinating close contacts in west africa in - (foege et al., foege et al., ) . surveillance and containment methods work well, even in large and geographically extensive outbreaks, such as that which occurred among several tens of millions of people in the ganges floodplain in (fenner et al., ) . it is the method that eradicated smallpox when years of mass vaccination failed. surveillance and containment consists of five steps. the first step is to identify and report cases. the diagnostic algorithm developed by cdc ( figure . ), and the widespread availability of pcr lab tests expedite this process. the second step is to isolate the patient(s). the profound illness and fear of transmission make isolation readily acceptable to the patients and the public. legal authority exists for isolation if necessary (centers for disease control and prevention, c) . the third step is to identify contacts, the persons who might have had prolonged face-to-face interactions with the patients during the time they were clearly ill. often patients themselves are dead or moribund and cannot be interviewed. their family members should be considered contacts, and one or more of them can usually provide information about other possible contacts. contacts are usually easy to find; they want to be vaccinated and will seek health officials once the patient has been diagnosed. the fourth step is to vaccinate the contacts. vaccination prevents smallpox from developing if performed within or days of exposure (massoudi et al., , kennedy et al., . the copious supply of bifurcated needles and lyophilized vaccine now available means that vaccination can be rapid and efficient. the rate-limiting factor will be the paperwork demanded by modern medicolegal systems. the contacts are placed under fever surveillance, with temperatures taken twice a day. if they become febrile, and therefore possibly prodromal, they are immediately isolated before they become capable of transmitting the virus. the fifth step is to vaccinate people who have been or might be associates of one of the first-ring contacts, particularly if the process is not initiated until several days after the index patient(s) became infectious. there is ample time to vaccinate these "second-ring" contacts, because they have not yet been exposed to actual illness. most new cases detected after initiation of containment activities will be known contacts vaccinated in the incubation period and can be promptly isolated. in the unlikely event that a case develops in a missed contact, the containment process is promptly restarted. in summary, under natural conditions, smallpox does not spread rapidly. transmission is by prolonged face-to-face contact. true aerosol spread can occur, but is rare. surveillance and containment tactics should quickly control outbreaks, even large ones resulting from widespread bioterrorist activity. patients suspected of having smallpox must be immediately isolated under full contact and airborne precautions (centers for disease control and prevention, d) . suspect smallpox is a public health emergency, and public health authorities should be immediately notified. smallpox outbreaks in europe and the united states had a high degree of nosocomial transmission (mack, ; mack, ) . if the diagnosis is suspected, modern infection control procedures and effective isolation should virtually eliminate nosocomial transmission. early in a bioterrorist attack, if smallpox is not high on most clinicians' index of suspicion, cases may be missed and transmit the disease before being effectively isolated. most hospitals have procedures to isolate patients with fever and an undiagnosed rash to reduce nosocomial transmission of measles and chickenpox, but these procedures may be difficult to implement in a large, busy city emergency ward. only recently vaccinated personnel should be allowed to attend patients. if the health facility does not have such personnel prevaccinated and designated as clinical team members, anyone entering the room must be vaccinated with fresh vaccine and vigorous technique, and should also wear a properly fitting n- respirator. personnel should be vaccinated even if they claim to have had a recent successful vaccination; if they are immune, such vaccination carries no risk, and it will eliminate the possibility of an error in the vaccination history. supportive care is the basis of the clinical management of smallpox (breman and henderson, ; fenner et al., ) . adequate food and fluids must be provided in a clean environment. if patients are obtunded, intravenous hydration with monitoring of electrolytes is important, although intravenous access may be difficult through the edematous and pock-laden skin. smallpox is disfiguring. older texts suggested removing mirrors from patients' rooms (dixon, ) . there is controversy about the optimal locus for medical care for smallpox patients. there is an ethical imperative to provide the best care possible, but modern hospitals have many immunosuppressed patients (hiv, cancer therapy, transplants, etc.) who would do poorly if they contracted smallpox or had to be vaccinated. facilities with good isolation possibilities -such as motels, older tuberculosis, mental, or veterans administration hospitals, or mobile hospitals, such as those available from the federal emergency management authority or the military -might make adequate smallpox hospitals. medical care can be brought to such facilities, and isolation of the entire facility may be possible. (centers for disease control and prevention, d) dead bodies, bedding, and wastes are potentially infectious (fenner et al., ; hopkins et al., ) . bodies of patients dying from smallpox should be cremated. modern handling of infectious bedding and wastes will kill the variola virus. rooms where patients have been cared for should be disinfected with any standard hospital disinfectant. personnel handling bodies, linen, or wastes must be vaccinated. in summary, careful consideration should be given to the locus of medical care during an outbreak because smallpox is transmissible nosocomially. scrupulous adherence to infection control guidelines must be maintained, including judicious disposal of medical wastes and dead bodies. only recently vaccinated personnel should provide care to patients. no historical evidence exists that smallpox was an effective bioweapon. over several centuries of colonial settlement in north and south america, anecdotes, diaries, and public letters expressed intent to use smallpox against indigenous people. much like the current discussions about the potential use of smallpox as a weapon, what has been written into historical texts and some medical journals may have been fueled more by fear than plausibility. the correspondence between the british general jeffery amherst and his colonels about infecting hostile indians with smallpox is prolifically referenced in recent journals. the general made this suggestion in july , as strategy against tribes near fort pitt, pennsylvania, involved in pontiac's rebellion: "could it not be contrived to send a small pox among the disaffected tribes of indians?" a week later, his colonel replied in the only mention of it ever made again: "i will try to inoculate the ___ with some blankets that may fall in their hands, and take care not to get the disease myself" (knollenberg, ) . several weeks before that communication william trent, an indian trader at fort pitt, suspicious of two delaware indian visitors, wrote in his personal journal, "we gave them two blankets and a handkerchief out of the small pox hospital. i hope it will have the desired effect." intent is clear, but the epidemiological record shows that smallpox was raging among the tribes the previous spring, weeks before these documents were written (knollenberg, ) . more reasonably, the seasonal nature of smallpox caused subsequent outbreaks rather than blankets. the disease, particularly devastating to american-indians, had been endemic among them for over a century. during the american revolution, george washington may have believed that british soldiers infected fleeing citizens by using variolation, despite the fact that variolation was a procedure normally used to prevent smallpox. in an record, a council of indian chiefs faced a pacific fur company trader, who called himself "the smallpox chief," and threatened to uncork a bottle of the virus if the council decided to attack; fear of smallpox may have averted war. oral history relates the use of scabs in blankets, linen, clothing, and virus-contaminated tobacco, spanning activities throughout north america and brazil (knollenberg, ; wheelis, ) . smallpox virus currently exists legally in only two laboratories: the cdc in atlanta and at the state research center for virology and biotechnology in the novosibirsk region of russia. possession of smallpox virus in any place other than these two laboratories is illegal by international convention. a former deputy director of the soviet union's bioweapons program has written that, during the cold war, their laboratories produced smallpox in large amounts, and made efforts to adapt it for loading into intercontinental missiles (alibek, ) . scientists defecting from the former soviet union, or leaving russia seeking work in other nations, may have illegally carried stocks of the virus to "rogue" nations (alibek, ; gellman, ; mangold et al., ; warrick, ) . there is no publicly accessible proof that such defectors actually transported smallpox out of russia, but no way of disproving that they did. allegations have been made that iraq, iran, north korea, and france may have stocks of the virus; these allegations have neither been proved nor disproved (gellman, ; johnson et al., ) . during the middle of , a team of top american scientists found no physical or anecdotal evidence to suggest that iraq was producing smallpox or had stocks in its possession. (linzer, ) . smallpox can be grown in large quantities and lyophilized for stability. large amounts can be stored in relatively small containers. the virus would be relatively stable in an aerosol if protected from heat and ultraviolet light (harper, ) . the infectious dose may be fairly small (fenner et al., ). an outbreak of cases occurred in aralsk, kaza-khstan, in and was kept secret by the soviets. the index case was allegedly on a boat on the aral sea when she was apparently infected (enserink, ; zelicoff, ) a recent claim suggests that an aerosol released from a bioweapons installation on an island in the aral sea infected her while the boat passed close to the island. the fact that only one of several workers on the boat was infected makes this distant mode of infection improbable. the subsequent additional cases, experiencing close face-to-face contact, were consistent with natural spread of the disease (henderson, ) . "dark winter," a widely publicized political exercise intended to educate public health and government leaders about biological terrorism, used smallpox in its script (o'toole and inglesby, ) . the narrative exaggerated transmission rates, and many leaders and media took the fictitious results literally. bbc television followed with a docudrama, "smallpox ," in which a crazed terrorist infected himself and spread smallpox through casual and biologically impossible contact (bbc england, ) . the epidemiology of smallpox renders these fictitious accounts highly improbable. such zeal to alert us to general biosecurity issues obscures many specific aspects of an attack using smallpox. while no official statements identify specific modes of spread that terrorists might choose, unofficial speculations abound. a fear-inducing hoax, or virus sprayed into a building's air circulation system, or the use of nebulizers to infect thousands at a large airport, are all realistic scenarios (bozzette et al., ) . the concept of a volunteer suicidal terrorist who walks around a busy mall, or a big city subway, is unrealistic because smallpox renders people so sick that they are rarely mobile, and they are so visibly sick that they would be avoided by the general public (piller, ) . given the respiratory portal of entry of the virus, a spray or aerosol may be the likely method of introduction. a large aerosol spray from a light airplane, such as a crop duster outfitted to release lyophilized smallpox virus over a public event such as a political rally or sports competition, is technically feasible. the federal aviation administration, or any developed country's aviation security system, would quickly impound such an unauthorized airplane. if smallpox virus is found, the containment efforts, beginning with a public announcement that attendees of the relevant event should get vaccinated, would abort the attack. if smallpox virus is introduced into the air circulation of a large building, large numbers of respiratory infections could take place. several large cities are currently using air filtration systems and doing daily laboratory tests for smallpox, anthrax, and other biohazards. the u.s. government's "biowatch" program deploys devices to sample air throughout cities and operates in open air, but not closed arenas (nbc news, ) . the sensitivity and specificity of these tests in realistic situations have not been determined, but they are theoretically very sensitive because they use real-time pcr. some terrorist groups are associated with the illegal drug trade (hastert, ) . cocaine is sniffed into the upper respiratory tract, so contamination of cocaine with smallpox virus could seed the infection in a widespread population. many drug users do not readily access the health care system when they are ill and might not be quickly diagnosed, and thus transmit the virus. terrorists might spray virus in an airport bus outside the target nation, and infect passengers bound for widespread destinations within that nation. multiple simultaneous cases would thus occur in many cities, with resultant panic. large numbers of smallpox cases are not necessary for an effective bioterrorist attack. a small number of cases could generate public panic. terrorists could put a solution of smallpox virus into hand-held atomizers, and station volunteers outside of such places as entertainment theme parks, military installations, and critical industries. the virus could be sprayed directly into the faces of persons leaving such facilities, under the guise of marketing a new perfume, etc. half a dozen cases, with obvious connections to well-known institutions, could invoke panic and social disruption, such as that which followed the anthrax cases in the united states in . terrorists with access to a modern virus laboratory might genetically modify smallpox in ways similar to the published manipulations of ectromelia (jackson et al., ; roos, ) . such a strain could not be tested for pathogenicity because there is no animal model for human infection. genetically altered strains might pose problems of transmission; alteration of pathogenicity might have unknown effects on the transmissibility of the virus. experienced intelligence observers feel that terrorists would avoid creating a strain with enhanced virulence. such strains could devastate developing countries with poor public health systems, and a widespread outbreak would quickly spread to such countries (johnson et al., ) . natural smallpox could similarly boomerang. terrorists with the ability to manufacture it would realize that an effective attack might cause widespread disease in nations harboring their colleagues. many such nations have poor public health systems and little vaccine, and would be more devastated than the nation initially attacked (johnson et al., ; oxford, ) . in summary, smallpox virus may exist in "rogue" nations. scenarios involving aerosol spread of smallpox are technically feasible, but have limitations. even small numbers of cases might suffice to create considerable panic. the most important aspect of prevention of a bioterrorist attack using smallpox is reliable intelligence. the location of the virus and the abilities and intent of its possessors drive preventive efforts. allegations that iran has weaponized smallpox must be viewed through our knowledge that these allegations have been made by an iranian opposition group (warwick, ) . the claims that france, iraq, north korea, and yemen have the virus await firm data (gellman, ) . unproven statements in an internationally politicized climate must be weighed with evidence-based intelligence. reliable intelligence, the key factor, is an unknown. public health efforts, and the extensive publicity about them, have direct preventive value for averting an attack or a resulting major epidemic of smallpox, and can be emulated in many nations. the announced british strategy is to stockpile vaccine and train a cohort of health workers who can identify and isolate smallpox cases and start surveillance and containment activities (oxford, ) . in the months after september , , the united states increased its vaccine supply from million doses to about million doses. the supply of vig increased more than -fold. the number of laboratories capable of doing real-time pcr for orthopoxviruses increased from two to at least . courses for clinicians, public health officials, and others have been widely held. several new websites have been created to assist professionals with the diagnosis and management of smallpox, and acquaint them with all aspects of vaccination. posters and training materials have been widely distributed. nearly , front-line medical personnel have been vaccinated (centers for disease control and prevention, f) . in the absence of solid information about the risk of a bioterrorist attack using smallpox, nations face a policy dilemma. if vaccination carried no risk, there would be no dilemma; widespread vaccination would be reinstated. however, vaccinia is a pathogenic live virus. vaccination carries well-known risks, particularly in populations that are largely naive to the virus, and in which large numbers of persons are immunosuppressed by hiv, posttransplant therapy, cancer chemotherapy, and steroid medication (lane and goldstein, ) . the potentially fatal complications of vaccination have recently been reviewed (centers for disease control and prevention, e; lane and goldstein, ) . postvaccinial encephalitis occurs about four or five times per million vaccinees, but carries a % fatality rate. progressive vaccinia is rare, but in our highly immunosuppressed population could be more common than in the past. it may be fatal in % or more of cases. eczema vaccinatum is more common, and the background prevalence of eczema (atopic dermatitis) is about three times higher today than in the s when studies of the frequency of eczema vaccinatum were performed. it has a fatality rate of about %. figure . is the face of an eczematous woman who acquired vaccinia from her child. recently, myocarditis has been found to occur about once in every in , vaccinees (arness et al., ; eckart et al., ; halsell et al., ) . postvaccinial myocarditis has an unknown fatality rate, but deaths have been reported in the past (dalgaard, ; ferry, ; finlay-jones, ) . in addition to these serious complications, a number of vaccinees suffer from "robust" major reactions, with fever, pain, and inflammation at the site; about % develop inconsequential, but sometimes, unsightly rashes (frey et al., ; grabenstein and winkenwerder, ) . vaccinia is transmissible by direct contact. transmission is rare and generally only to very close contacts such as family members sharing a bed (grabenstein and winkenwerder, ; neff et al., ) . the presence of immunosuppressed patients on the wards of major hospitals has kept many medical personnel from accepting vaccination. careful management of the vaccination site and scrupulous hand hygiene can minimize transmission . vaccination is not the only means of preventing smallpox. rigorous isolation of patients coupled with quarantine of contacts until their incubation period is over will prevent transmission (bozzette et al., ; eichner, ; eubank et al., ; mack, ; meltzer et al., ) . the prolonged incubation period ( days) provides time to allow public health action and education of the population once initial cases are recognized. in the absence of circulating smallpox, but the presence of a theoretical threat of a bioterrorist attack, there are several options for the use of smallpox vaccine. in , the united states government's advisory committee on immunization practices (acip) recommended that vaccinia be reserved for laboratory personnel who work with orthopoxviruses capable of infecting humans (e.g., variola, vaccinia, and monkeypox) (centers for disease control and prevention, ) . these guidelines were expanded to include animal handlers working with animals infected with orthopoxviruses. after september , , when smallpox vaccine in the united states was limited to million doses, the acip formed a smallpox working group, which held public hearings and studied scientific data and political opinions. many public health and academic officials suggested that vaccine be made available to limited numbers of workers who would form response teams in the event of a terrorist attack. in june , the acip recommended that vaccine be made available to selected state and local officials with responsibility for protection of public health (centers for disease control and prevention d). this was consistent with cdc's plan for containment of a smallpox attack and coincided with proof that the million doses of vaccine could be diluted : (centers for disease control and prevention, d; frey et al., ) , that additional vaccine was on hand, and that production of new vaccine was well under way. vaccine supplies are now more than adequate to immunize the entire u.s. population, and suggestions have been made to resume vaccination more widely (bicknell, ; derugy and pena, ) . public announcements suggested vaccinating considerable numbers of health care workers, particularly in big city emergency wards and other areas where initially undiagnosed smallpox cases might seek medical care. many states sought to revaccinate predominantly older workers who had a past history of vaccination, because the residual immunity in such revaccinees is not trivial, and they are less likely to experience adverse events after vaccination (hammarlund et al., ) . the number of health care workers in big city hospitals is large, and they turn over rapidly. many health care workers vaccinated under such a policy are in frequent contact with patients with hiv, status postorgan transplants, atopic dermatitis, etc. given the recent data (frey et al., ) that adults given primary vaccination frequently need to take one or more days off work because of fever and malaise, many hospitals expressed concern about the effects of widespread vaccination (connolly, ; gettleman, ) . other "first responders" -including policemen, firemen, ambulance workers, and other emergency medical personnel -should be considered for preevent vaccination (bush, ; centers for disease control and prevention, g; stevenson and altman, ) . in part reflecting these concerns, in august the institute of medicine recommended shifting emphasis away from vaccinations to focus on measures to improve coordination and quicken response time to any public health threat (olson, ) . several writers suggest that individuals have the right to decide for themselves about the risks and benefits of vaccination, particularly given the current copious vaccine supply (bicknell, ; derugy and pena, ) . the morbidity and mortality from vaccinia, the problems of screening potential vaccinees and their household members for contraindications, and the efficacy of surveillance and containment methods for controlling smallpox outbreaks, argue against such a policy (bozzette et al., ; eichner, ; eubank et al., ; lane and goldstein, ; meltzer et al., ) . the united states stopped routine vaccination in . it could be resumed if the threat of smallpox becomes considerable. only in a scenario where smallpox becomes widespread would it be wise to resume mass vaccination. such a scenario would inevitably include inadvertent exportation of the disease to other nations, including those with poor fiscal and public health resources and no vaccine. such resumption of endemicity of smallpox would be a human tragedy of untold proportions. thus, developed nations must stockpile vaccine and maintain preventive public health efforts for rapid containment of terrorist-induced outbreaks. in summary, intelligence about the existence of smallpox in the hands of terrorist groups, and their intent for it use, is the best means of preventing an attack. readiness to control an outbreak resulting from an attack entails a high index of suspicion among clinicians, a good network of diagnostic laboratory capabilities, and a plan for use of surveillance and isolation techniques to quickly contain outbreaks. vaccination policies need periodic review, because the risks and benefits of vaccination of various medical and law enforcement groups are controversial. resumption of widespread vaccination is dangerous and unnecessary. the united states has rebuilt its vaccine supply, trained diagnostic laboratories, enhanced the availability of clinical and epidemiologic information, and completed a detailed plan for handling a smallpox attack. what should be done to reduce the threat of smallpox further? there are scientific and political efforts currently underway to prepare for an outbreak, to reduce its likelihood, and to enhance the safety of vaccines. the intelligence community and the new department of homeland security have increased the antiterrorism budget and the amount of attention paid to possible bioterrorist threats. presumably much of this work is classified, but rumors of nations that may still have (illegal) stocks of variola virus are under investigation. rapid detection of variola virus in the environment would be useful. air filtration systems are being improved so that huge volumes of air inside large buildings, such as commercial or government offices, can be routinely and rapidly filtered. the filters are then tested by pcr for the presence of variola virus dna (fackelmann, ; nbc news, ) . the sensitivity and specificity of such systems cannot be calculated, because no real smallpox has been found or can be injected into these systems. given the high sensitivity of pcr, they should be good at detecting variola. a test that could find virus in the throats of patients early in the prodrome could help identify patients who need immediate isolation in the event of an outbreak. such tests have been developed, again using pcr techniques, and are being explored using material taken from experimentally infected monkeys . antiviral compounds, particularly ones derived from cidofovir and its analogues, are being investigated. a nontoxic antiviral with good activity against variola, or indeed against the serious complications of vaccinia, would change the risk-benefit calculations for both pre-and postevent vaccination programs. compounds that are more soluble and thus more bioavailable than cidofovir are currently being screened for activity against variola (bray and roy, ; langbein, ; nyets and declercq, ; smee and sidwell, ) . a vaccine that provides protection against variola but is less pathogenic than vaccinia would be useful. several candidate strains derived from vaccinia are being actively investigated. modified vaccinia ankara, nyvac, and lc m are prominent among these attenuated strains (bonnilla-guerrero and poland, ; drexler et al., ; earl et al., ; henderson, borio, and lane, ; lane and goldstein, ; mccurdy et al., ) . ultimately, the united states, russia, and the world health organization will have to decide whether retaining the existing legal stocks of variola virus is worthwhile. as long as the virus exists in any laboratory, terrorist groups may claim that one motivation for retaining the virus is its potential as a bioweapon. if the stocks are destroyed, this argument no longer exists. international cooperation under the aegis of the world health organization is needed to affirm the illegality of biowarfare using variola, and maximize efforts to find and destroy illegal stockpiles (breman et al., ) . biohazard myopericarditis following smallpox vaccination produced by simon chinn, in association with the learning channel and granada international the case for voluntary smallpox vaccination smallpox vaccines: current and future a poxvirus protein that binds to and inactivates il- and inhibits nk cell response a model for a smallpox-vaccination policy looking back at smallpox antiviral prophylaxis of smallpox preventing the return of smallpox diagnosis and management of smallpox u.s. to vaccinate , workers against smallpox. the new york times protecting americans: smallpox vaccination program smallpox vaccination and myopericarditis: a clinical review smallpox vaccine: recommendations of the immunization practices advisory committee (acip) smallpox images diagnostic algorithm for rash illnesses smallpox response plan and guidelines. guide c. legal and quarantine authority smallpox response plan and guidelines (version . ) guide d-specimen collection and transport smallpox response plan and guidelines (version . ) environmental control of smallpox virus annex . medical management of smallpox patients and vaccination complications smallpox vaccination and adverse events. guidelines for clinicians smallpox vaccination program status by state recommendations for using smallpox vaccine in a pre-event vaccination program: supplemental recommendations of the advisory committee on immunization practices (acip) and the healthcare infection control practices advisory committee (hicpac) medical professionals cite possible side effects, uncertainty of threat. the washington post lack of vaccinia viremia after smallpox vaccination fatal myocarditis following smallpox vaccination orthopoxviruses responding to the threat of smallpox bioterrorism smallpox in tripolitania, ; an epidemiological and clinical study of cases, including trials of penicillin treatment smallpox. little brown and company the recovery of smallpox virus from patients and their environment in a smallpox hospital identification of vaccinia virus epitope-specific hla-a* -restricted t cells and comparative analysis of smallpox vaccines immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox department of defense smallpox vaccination clinical evaluation team. ( ) incidence and follow-up of inflammatory cardiac complications after smallpox vaccination case isolation and contact tracing can prevent the spread of smallpox did bioweapons test cause a deadly smallpox outbreak? modelling disease outbreaks in realistic urban social networks handheld bioterror detectors in works smallpox and its eradication. who adverse reactions after smallpox vaccination fatal myocarditis after vaccination against smallpox: report of a case selective epidemiologic control in smallpox eradication smallpox eradication in west and central africa clinical responses to undiluted and diluted smallpox vaccine smallpox vaccination (vaccinia): a review. part i. background, vaccination technique, normal vaccination and revaccination and expected normal reactions smallpox vaccination (vaccinia): a review. part ii. adverse events transmission potential of smallpox in contemporary populations tertiary contact vaccinia in a breastfeeding infant four nations thought to possess smallpox two hospitals refuse to join bush's plan for smallpox. the newyork times us military smallpox vaccination program experience myopericarditis following smallpox vaccination among vaccinia-naïve us military personnel duration of antiviral immunity after smallpox vaccination airborne micro-organisms: survival test with four viruses house speaker hastert introduces the speaker's task force for a drug free america a study of intrafamilial transmission of smallpox commentary on dr alan zelikoff's epidemiological analysis of the aralsk outbreak (n ) smallpox and vaccinia smallpox as a biological weapon: medical and public health management frequency of vaccinia virus isolation on semipermeable versus nonocclusive dressings covering smallpox vaccination sites in hospital personnel two funeral-associated smallpox outbreaks in sierra leone expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox terrorists' most likely weapon here? induction of human t cell-mediated immune responses after primary and secondary smallpox vaccination general amherst and germ warfare. mississippi valley smallpox: clinical types, causes of death, and treatment smallpox and pan-orthopox virus detection by real-time '-minor groove binder taqman assays on the roche lightcycler and the cepheid smart cycler platforms evaluation of ist century risks of smallpox vaccination and policy options transmission of vaccinia virus, and rationale for measures for prevention new compounds show promise as smallpox drugs. reuters strenthening national preparedness for smallpox; an update modelling responses to a smallpox epidemic taking into account uncertainty u.s.team in iraq finds no smallpox smallpox in europe, - a different view of smallpox and vaccination epidemiology of smallpox in west pakistan i: acquired immunity and the distribution of disease an overview on the use of a viral pathogen as a bioterrorism agent: why smallpox? plague war, pbs videotape modified vaccinia ankara: potential as an alternative smallpox vaccine haemorrhagic smallpox. . specific bleeding and coagulation studies modeling potential responses to smallpox as a bioterrorist weapon poxviridae: the viruses and their replication pattern of intrafamilial transmission of smallpox in calcutta government monitoring philadelphia air for biotoxins. sensors check for anthrax, smallpox, in cities contact vaccinia-transmission of vaccinia from smallpox vaccination therapy and short-term prophylaxis of poxvirus infections: historical background and perspective panel urges shift of focus in preparing for smallpox. the new york times shining light on dark winter smallpox bioterrorism unlikely: populations are easily protected. basic notes smallpox strike called unlikely. the los angeles times epidemiological studies of smallpox. a study of intrafamilial transmission in a series of infected families. ind smallpox. the kothari book depot scientists research antidotes to super mousepox virus pcr strategy for identification and differentiation of smallpox and other orthopoxviruses virus excretion in smallpox. . excretion in the throats of household contacts mouse models for studying orthopoxvirus respiratory infection development and experience with an algorithm to evaluate suspected smallpox cases in the united states a review of compounds exhibiting anti-orthopoxvirus activity in animal models the formation and function of extracellular enveloped vaccinia virus real-time pcr assay to detect smallpox virus the smallpox outbreak in khulna municipality, bangladesh. . methodology and epidemiologic findings bush lays out plan on smallpox shots; military is first. the new york times smallpox shots will start soon under bush plan. the new york times vaccination success rate and reaction profile with diluted and undiluted smallpox vaccine: a randomized controlled trial air sampling of smallpox virus mass smallpox vaccination and cardiac deaths us paper to face russian lawsuit. gazeta, ru an airborne outbreak of smallpox in a german hospital and its significance with respect to other outbreaks in biological warfare before an epidemiological analysis of the smallpox outbreak in aralsk key: cord- -qnp m o authors: taylor, robert b. title: medical words linked to places date: - - journal: the amazing language of medicine doi: . / - - - - _ sha: doc_id: cord_uid: qnp m o many medical terms come from places: towns, rivers, islands, forests, mountains, valleys, countries, and continents. these toponymous diseases, syndromes, descriptors, and other entities bring us colorful names that help us recall some of their history. today we have zika virus, its name coming from the zika forest in ghana. caucasian comes from the caucasus mountains, lesbian from the island of lesbos, and epsom salts from a mineral spring in epsom, surrey, england. chapter . / - - - - _ tells the stories behind these place-named diseases and how many of them affect us today. future disease nomenclature based on places will be one more loss of the richness of our amazing medical language. in this chapter, i will begin with the current major concerns-the viral diseases causing today's outbreaks, epidemics, and pandemics. i will then present disease names linked to a variety of places, ending with those in europe and america. in , indian automobile manufacturer tata motors decided not to call its new hatchback car by its planned name zica, derived from "zippy car." this is just one more reaction to the epidemic of zika virus infections that the world health organization has declared a global emergency (fig. . ). in addition to causing fever and malaise, when the patient is pregnant, the zika virus may also cause birth defects, notably microcephaly (from greek words meaning "small" and "head"). some adult zika virus patients go on to develop the guillain-barré syndrome. the zika virus has been a dark cloud over the fragile economy of brazil in many ways, including its adverse impact on attendance at the rio de janeiro summer olympic games. the zika virus is, for most of the world, a newcomer. on new year's day , for example, hardly anyone had heard of the infection. now the disease is well known, but where did it arise and how did it get its unusual name? in the s, researchers identified a transmissible agent in the blood removed earlier from a rhesus macaque laboratory monkey sick with a fever. the monkey had come from a mosquito-infested jungle in uganda called the zika forest, the name coming from the word for "overgrown" in the luganda language of uganda. from here, and only in the past few years, the virus has migrated across asia and the pacific to central and south america, reaching pandemic proportions in some areas. in addition to mosquito-borne infection, we now have discovered sexually transmitted zika virus disease and continue to learn more each year. one might assume that the west nile virus came from egypt, but in fact, the organism was first discovered in the west nile district of uganda in . this mosquitotransmitted arbovirus was considered a problem only for birds and horses until the s, when human infections began to be reported. most west nile viral infections are subclinical, but a few are complicated by meningoencephalitis and a flaccid paralysis reminiscent of polio. the west nile virus is a member of the family flaviviridae, from the latin flavus, meaning "yellow." the family was named for the yellow fever virus, which tends to cause liver damage, giving its victims a yellow jaundiced appearance ( fig. . ). viruses of the flaviviridae family have turned up in various places and have often acquired the names of those locations. japanese encephalitis is a disease of domestic pigs and birds, notably herons, that can be spread to humans by mosquitoes. it is the chief cause of viral encephalitis in asia. another member of the flaviviridae family is mansonia pseudotitillans, the cause of saint louis encephalitis. the mosquito-borne disease harkens to in st. louis, missouri, when more than a thousand cases were reported. la crosse encephalitis is caused by a virus from the family bunyaviridae, the same family that causes crimean-congo hemorrhagic fever. discovered in the early s in la crosse, wisconsin, the mosquito-transmitted disease is found chiefly in the midwestern and appalachian regions of the united states. several families of rna virus can cause hemorrhagic fevers. the hantavirus takes its name from the place where the virus was first isolated in the late s: the hantaan river valley in south korea. rodents, such as the cotton rat, spread the hantavirus, and humans acquire the virus through contact with rodent feces, urine, or saliva. the hantavirus belongs to the family bunyaviridae. lassa virus, a member of the arenaviridae family, causes lassa hemorrhagic fever. the disease was initially described in in lassa, a town in borno state, nigeria. a zoonotic disease, the reservoir is rodents, notably multimammate mice (mastomys natalensis), whose excreta-feces and urine-can be aerosolized, and the disease can be spread by inhalation of these tiny particles. two members of the family filoviridae are the ebola virus and the marburg virus. ebola hemorrhagic fever, spread among humans by direct contact with body fluids, such as blood, semen, or breast milk, of an infected individual, causes death by internal and external bleeding in approximately half of infected persons. the name ebola comes from the ebola river, which flows near the village of yambuku in the democratic republic of the congo (previously zaire), where the disease was identified in ( fig. . ). the name marburg virus did not originate in africa, but in germany. in the s, several monkeys were sent from uganda to europe for use in laboratory experiments. unknown to the scientists involved, these monkeys carried a filoviridae virus, resulting in infections in several dozen researchers in three cities, including the university town of marburg, giving the organism the name it carries today. one of the disease names specifically criticized by the who is the middle east respiratory syndrome (mers). the term "middle east" may not have the exotic flavor of a river in south korea or a forest in uganda, but it does give a good indication of where the disease began, and a hint as to why it was first noted in this part of the world. mers, first reported in saudi arabia in , is a viral disease of the respiratory tract causing cough, fever, dyspnea, and sometimes death. only a few cases have been reported in the united states, and, so far, these have been contracted during travel in the middle east and imported by returning travelers. other cases have been reported in more than two dozen countries including great britain, germany, greece, korea, china, malaysia, and the philippines. the intriguing aspect of mers is the likely connection to camels. the causative virus (mers-cov) has been found in camels, and some patients with the disease have told of contact with camels. the who warns against drinking raw camel milk or camel urine or eating undercooked camel meat. so perhaps the "middle east" part of the disease name is on target. where else but the middle east do humans live in close communion with camels? and drinking camel urine? yes, some in the region believe that drinking camel urine has medicinal value. what could be the connection between the vitamins essential to our health, a polish biochemist, and camel dung in the middle east? the story goes back more than three millennia. as early as the eighteenth dynasty (c. - bce and the time of king tutankhamun), the early egyptians worshiped the god amun, also called amun-ra or ammon, as the leading god of the empire, later to be identified with the greek god zeus (fig. . ). in what is now libya, they built a temple to ammon where egyptians came to worship their god; while they did so, their camels fertilized the nearby sand with urine and feces. it was from this sand that sal ammoniac, the "salt of ammon," was first derived. in fact, the ancient greek word for "sand" is ammos, probably related to the name of the god (shipley, p. ). from sal ammoniac comes ammonia (nh ), a pungent-smelling, colorless gas, so named in by swedish chemist torbern bergman ( - ), and from ammonia comes our word amine. amines are derived from ammonia by chemical substitution of one or more of the hydrogen atoms with other radicals. the next etymologic landmark in the story of the word vitamin came in when polish biochemist casimir funk ( - ) introduced the concept of amines as being vital to life. he postulated that there were at least four necessary amines and that without them patients would develop beriberi, scurvy, rickets, and/ or pellagra. to form his word, funk combined -amine with the latin word for life, vita (recall the fellini movie, la dolce vita-the "sweet life"). funk's original word vitamine was later shortened to vitamin when it was learned that not all vitamins contained amines [ ] . casimir funk was nominated for a nobel prize in , , , and . he never received the award. today we have vitamins a, b (a number of these), c, d, e, and k, the latter so designated because of its action in coagulation (originally named koagulationsvitamin in german). there are also vitamin wannabes, such as the bogus cancer drug amygdalin (laetrile), a cyanogenic substance found in apricot kernels and dubbed vitamin b , perhaps alluding to the world war ii flying fortress. we have come a long way from camel urine to drugstore shelves full of colorful vitamin bottles and a few misbegotten imitators. in chap. , i told of prometheus chained by zeus for giving humans the gift of fire; the location where he was restrained was the caucasus mountains near what are now iran and turkey ( fig. . ). it was in these mountains that german scientist johann friedrich blumenbach ( - ) discovered a skull. blumenbach, the skull from the mountains considered the father of physical anthropology, suggested that the various human subspecies ("races") could be classified by the study of their skulls. based on his anthropometric comparisons, in , he proposed five families of humans: yellow (mongolians), black (ethiopians), red (american indians), brown (malaysians), and white. for the name of this last category, he used the location of the finest skull of all, the caucasus mountains, and from that moment on, white persons became caucasian (gershen, p. ). it started with the greek lyric poet sappho (ca. - bce). she was born on the island of lesbos, located in the aegean sea off the west coast of turkey and a center of civilization even before the golden age of greece in the fifth century bce. she was the most renowned poetess of her day, and her lines had a distinctive "sapphic meter." sappho shared her poetry with a cohort of young women, and probably because of the erotic passion of some of her poems, the tale has evolved regarding homosexual relationships between the poetess and her students. thus female homosexuality came to be named for the island, lesbos, and a female homosexual, wherever she lived, became a lesbian. a word less often used to describe women who love other women is sapphism, a synonym for lesbianism. in fact, according to hendrickson (p. ), sappho was probably married and had a son, although many will argue that this would not be solid evidence of her sexual preferences. the crimean war was fought in the mid- s between russia and an alliance of the united kingdom, france, and turkey. it was the war that gave us florence nightingale and the charge of the light brigade. during this war british medical officers on the island of malta noticed a disease characterized by sweating, joint and muscle pains, and a fluctuating fever. a logical name for the mysterious malady was malta fever. in , british medical officer jeffrey allen marston ( - ) described his personal experience with the disease, and in , scottish microbiologist david bruce ( - ) linked the disease to an organism that came to be called brucella abortus. the "bruce" in the name of the genus honored bruce; the species designation abortus reflects the tendency of the disease to cause abortions in cattle. the disease was briefly called bang's disease, after danish veterinarian bernhard bang identified b. abortus as the agent causing cattle to abort. because of the wavelike nature of the fever, the disease acquired the name undulant fever. other names enjoyed popularity in various settings: scottish delight, milk sickness, goat fever, cyprus fever, gibraltar fever, and mountain fever. in the end, the medical and scientific communities have come to favor the name brucellosis, honoring the man who discerned the cause of the disease. this story begins in the town of magnesia, named for an ancient greek tribe, the magnetes. the town lies in the thessaly district of greece, an area conquered by the romans following their victory in the battle of magnesia in bce, ending the roman-seleucid war. in this conquered area, the romans discovered a white mineral that, applying the name of the town, they called magnesia. when a different substance with a dark color was found, the white substance became magnesia alba, from the latin word for "white," and the darker substance became magnesia nigra, latin for "black." in , french chemist antoine bussy ( - ) found that the magnesia alba yielded an element, which he named magnesium. magnesia nigra came to be called manganese. there is another twist to the magnesium story. in the early seventeenth century, in the town of epsom in surrey, england, a farmer offered his cows water from a nearby well. but the cows refused to drink. the farmer learned why when he tasted the bitter water. he also observed, however, the water seemed to heal skin abrasions and sores. the bitter-tasting substance in water came to be identified as hydrated magnesium sulfate, better known as epsom salt. epsom wells became a destination spa, visited by diarist and member of parliament samuel pepys; nell gwyn, mistress of king charles ii of england; and other seventeenth-century notables (evans, p. ). in the united states, there is a magnesia temple at the town of sharon springs in schoharie county, new york (fig. . ). in addition to epsom salt, magnesium has other medical uses: a water solution of magnesium oxide is milk of magnesia, popular as a laxative. magnesium has had a role in treating ventricular arrhythmias of the heart and preeclampsia/eclampsia. it is sometimes prescribed for management of migraine or of the restless leg syndrome. and the white powder that gymnasts and weight lifters use to improve their grips is magnesium carbonate. returning to the town of magnesia, in this region was found an iron oxide stone that had the apparently magical quality of attracting iron to itself. it was called the "magnesian stone" and served as the source of our word magnet (haubrich, p. ). the guinea worm is a parasitic nematode that is spread when a person drinks water containing the guinea worm larvae. the cause of the disease in humans is dracunculus medinensis, and the disease is properly called dracunculiasis. approximately a year following ingestion of the larvae, the female guinea worm finds its way to the skin, where it forms a blister. a little later the blister breaks and the worm begins to emerge. an adult female guinea worm can be two to three feet long and as thick as a strand of spaghetti. affected persons sometimes facilitate extraction of the worm by winding it around a small stick. the name guinea worm arose when european explorers first encountered the disease on the guinea coast of west africa in the seventeenth century. in latin dracunculus means "little dragon; dracunculus medinensis means the "little dragon from medina," so named because the disease was once rampant in the muslim holy city of medina in saudi arabia. dracunculiasis is no longer endemic in either location. the prevention of dracunculiasis requires nothing more than drinking filtered water, and the disease is on the threshold of being exterminated. the source of the drug colchicine is the autumn crocus, colchicum autumnale, so named because it was first discovered in the colchicum region of the republic of georgia on the black sea. the plant's nickname "naked lady" refers to the appearance of the flower without surrounding leaves. the extract of the plant can be highly toxic, and severe accidental poisonings have occurred, several involving cases in which the autumn crocus was mistaken for wild garlic (fig. . ) . use of the drug to treat joint pains can be traced to early egyptian writings, circa bce, found in the ebers papyrus. therapeutic use of colchicum extract is mentioned in the writings of persian physician avicenna ( - ) and french surgeon ambroise paré ( - ). american statesman benjamin franklin ( - ) brought the colchicum root to america from france and used it to treat his own attacks of gout. in addition to its use in the treatment of gout, colchicine is sometimes used to treat behçet disease, pericarditis, and familial mediterranean fever; the latter is a hereditary disorder also known as armenian disease. the word "plaster" in plaster of paris came from greek emplastron, to latin emplastrum, to french plastre. but what about paris? throughout history, various products had been used to bind wounds. french surgeon guy de chauliac ( - ), author of the seven-volume chirurgia magna in , introduced the use of egg white to stiffen bandages [ ] . however, the "paris connection" came in when dutch army surgeon antonius mathijsen ( - ) began incorporating gypsum into dressings used to immobilize fractures ( fig. . ) . mathijsen was not french, but the gypsum was quarried in the montmartre section of paris, hence the name plaster of paris. although the word is probably never seen today in medical records, clap remains a vulgar term for gonorrhea. the common old french term for brothel was clapier. the term technically translated to "rabbit burrow," and perhaps this image had something to do with the "red light" section paris, where a number of brothels were located, being called, in the middle ages, le clapier. as language moved across the english channel in the sixteenth century, clapier in french became clapper in english. eventually the word was shortened to clap, generally expressed with an article as "the clap" [ ] . the french had another word for this distressingly common malady, chaudepisse, describing a frequent manifestation of gonorrhea. chaude means "hot" in french. and pisse? is translation really needed? it was first called bramble disease by a norwegian physician who reported a cluster of cases of "acute muscular rheumatism" occurring in the village of bramble in norway. but alas, the original report and a few others that followed using the term "bramble disease" were published only in norwegian, and the name failed to catch on. then in , doctoral candidate ejnar sylvest ( - ) published his thesis describing a disease outbreak on the picturesque danish island of bornholm, in the baltic sea, that he termed "bornholm disease-myalgia epidemica." today we know this as bornholm disease, as well as epidemic pleurodynia, epidemic myalgia, devil's grip, and the grasp of the phantom. the disease causes the usual viral symptoms of fever and headache. an additional and distinguishing feature is severe pain in the lower chest, giving rise to the more colorful names of the disease. fortunately for those with the devil's grip, the disease is self-limited and rarely fatal. it was an american, not a german, who gave rubella its everyday name: german measles. the disease has been well known to health-care professionals and parents since first described by german physician friedrich hoffmann ( - ) in . it was given the name rubella, from the latin word meaning "little red," by english military surgeon henry veale in his description of an outbreak in india. american physician j. louis smith coined the term "german measles" in . working at new york's bellevue hospital, smith described an outbreak of the disease and, reading of similar outbreaks in germany, he named the disease "german measles" (bordley and harvey, p. ) (fig. . ) . then, amid the anti-german fervor of world war i, rubella was briefly renamed liberty measles (fortuine, p. ). this chapter is about diseases named for places. is a trench a "place"? it certainly was to the world war i soldiers who spent weeks and months slogging shoulder to shoulder in deep ditches filled with icy cold water and filth. from these barely habitable ditches came the "trench" in the names of three different diseases. the first is trench fever, with an estimated one million cases occurring in western europe during the first world war. it was first noted in an infantry private in , and by , the allied general headquarters stated: "trench fever is a matter of national importance… and it merits the attention of every physician and pathologist who has the opportunity of working among the troops" [ ] . trench fever, caused by bartonella quintana and spread by the human body louse, causes fever, prostration, a macular rash, and bone pain. despite these manifestations, some doughboys welcomed the infection because a stay in the medical facility offered respite from the trenches. trench fever has also been called five-day fever, shin bone fever, and meuse fever; the latter is a reference to the wwi meuse-argonne battle of . today, we no longer have trench warfare, but we do have urban trench fever occurring in demented, homeless, and alcoholic persons. from the muddy ditches of wwi, we also get the disease name trench foot, describing damage to the tissues that occurs with prolonged exposure of the extremity to moist cold (fig. . ). dominique jean larrey ( - ), a surgeon in napoleon's grande armée, noted the prevalence of what we now call trench foot in the french troops during their ill-fated invasion of russia in . trench foot was seen not only in wwi but also in world war ii and in the vietnam war; it was called immersion foot or paddy foot in the latter jungle-based campaign. then there is trench mouth, also known as acute necrotizing ulcerative gingivitis, caused by a mixed bacterial infection. recognized in greek soldiers in the fourth century bce, the disease was described by scottish surgeon john hunter ( - ) in . at the pasteur institute in paris in , french physician jean hyacinthe vincent ( - ) identified the fusospirochetal cause; one of the other names for acute necrotizing ulcerative gingivitis is vincent angina [ ] . in this term, angina reflects its true latin meaning, "infection of the throat." the disease did not acquire the name trench mouth until world war i, when it occurred in men spending weeks and months in trenches under physical and psychological stress, receiving a poor diet, and with scant options for oral hygiene. nantucket is a small island off cape cod, massachusetts, usa. in april of , the cape cod times, in a story titled "cape cod a hot spot for babesiosis from ticks," reported health officials calling for "hospitals to screen blood transfusion products for babesiosis or nantucket fever." in the article, dr. al demaria, state epidemiologist with the massachusetts department of public health, declared nantucket fever [ ] . what is this toponymous disease that many health professionals have never heard of? nantucket fever, caused by a tick-borne protozoan parasite called babesia microti, causes manifestations not unlike malaria, including fever, arthralgia, lymphadenopathy, and hemolytic anemia. the formal name for the febrile illness is babesiosis, first described by romanian scientist victor babeş ( - ) as a disease of cattle and sheep. the first human case of babesiosis was reported in . in cattle, babesiosis is sometimes called texas cattle fever, tick fever, or redwater fever, the latter referring to the appearance of blood in the urine. in the united states, human babesiosis has been reported chiefly in northeastern and midwestern states and occurs most often during warm weather. if a young physician today were asked to identify a disease named for a place, the answer might well be lyme disease, also called lyme borreliosis. although the disease had been recognized in europe since the eighteenth century and documented the dangers of the deer tick in wisconsin in , the full spectrum of the disease was not recognized until , in connection with a series of cases in southeastern connecticut, including the small towns of lyme and old lyme. the disease is caused by borrelia bacteria, notably borrelia burgdorferi, and is spread by the same vector as nantucket fever/babesiosis: the ixodes tick, also called the deer tick. a curious, perhaps pathognomonic, feature of the disease is the early appearance of a "bulls-eye" rash, called erythema migrans, although this helpful diagnostic clue is not seen in all patients (fig. . ). later manifestations may include fatigue, neurocognitive manifestations, disorders of heart rhythm, polyneuropathy, and arthritis. lyme disease is the most commonly occurring tick-borne disease in europe and north america. in the united states, the areas of greatest prevalence are the northeast and middle atlantic states and western wisconsin. in these locations, woodsmen and woodswomen, beware of the deer tick. another malady, along with lyme disease, well known for its geographic label is rocky mountain spotted fever. also sometimes called tick typhus or blue disease, rocky mountain spotted fever was first recognized in in the snake river valley in the rocky mountains of the western united states. the disease is spread by ticks carrying the causative organism, rickettsia rickettsii, a name that seems unnecessarily repetitive, like the name of the common black rat, rattus rattus; the latter is an animal reservoir for fleas carrying the bacteria causing bubonic plague. the name of the organism redundantly honors american pathologist howard taylor ricketts ( - , who first isolated the pathogen that causes the disease. a potentially fatal disease, rocky mountain spotted fever is the most commonly reported rickettsial disease in the united states. curiously, because of the epidemiologic distribution of the disease in america, you and i are at more risk of contracting rocky mountain spotted fever in north carolina than we are in colorado. first found in in a -year-old boy living on tangier island in virginia's chesapeake bay, tangier disease is also known as familial alpha-lipoprotein deficiency. it is a congenital disorder causing a severe deficiency of high-density lipoprotein (hdl) in the blood. in addition to abnormal serum lipids found on laboratory analysis, patients with tangier disease may have splenomegaly, hepatomegaly, neuropathy, atherosclerosis, and cloudiness of the corneas of the eyes (fig. . ) . there is a long list of other medical entities that came from cities, countries, and even continents. in , scientists working in the city of philadelphia, pennsylvania, discovered the philadelphia chromosome, an abnormality of chromosome associated with chronic myelogenous leukemia. milltown, new jersey, originally named for a local gristmill, was the manufacturing site of the anxiolytic drug meprobamate (miltown). in the s, wallace laboratories, in an effort to maintain secrecy about their new drug, applied the code name miltown, after the new jersey borough. when the drug went to market in , the company decided to keep the town name as the trade name. named originally for the peoples of the nation of mongolia, the mongolian spot is a melanocytic birthmark that may appear blue, or some shade of gray, black, or brown. the term was coined in by german anthropologist erwin bälz ( - ), who was actually working in japan and not in mongolia. in mexico, the birthmark is called rabo verde, "green butt," and in spanish the term sometimes used is mancha ("stain") de baelz (bälz), eponymously honoring the man who first described it. mongolian spots generally disappear during later childhood. the influenza pandemic of - is sometimes called the spanish flu. caused by the h n virus, the disease affected million people worldwide and resulted in more than million deaths (fig. . ). it seems ironic that spain has had its name attached to a flu that has been traced to a single index case in a wholly different country: a us army cook at fort riley, kansas. situated on the hepatitis b virus (hbv) is the surface antigen, a.k.a. the australia antigen (hbsag). its presence upon laboratory testing indicates current infection with the hbv. the name australia antigen was suggested when american physician baruch blumberg ( - ) discovered its presence in the blood of a member of the australian aboriginal population. who issues best practices for naming new human infectious diseases casimir funk: his discovery of the vitamins and their deficiency disorders catechism in medical history medical history in medical terminology trench fever: the british medical response in the great war the relation of peri-dental gingivitis to vincent's angina cape cod a hot spot for babesiosis from ticks. cape cod times key: cord- -u mb h authors: lu, pu-xuan; zhou, bo-ping; gui-lin, yang; qi, an title: introduction of emerging infectious diseases date: - - journal: diagnostic imaging of emerging infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: u mb h emerging infectious diseases (eids) refer to contagious diseases newly appeared, or with drug resistance, whose incidences have been rapidly increasing and are likely to further rise in the future. eids are usually discovered in three ways. firstly, some existing diseases are ascertained as eids due to the recent discovery of pathogens. secondly, previously considered noninfectious diseases are identified contagious as a result of new etiological findings. thirdly, new infectious diseases are incurred by various complicated reasons such as evolution of pathogens. due to their uncertainty and unpredictability, eids could result in high mortality and great impacts on social stability and economic development as people are unable to react immediately and take specific preventive or control measures. therefore, eids have become a major public health problem worldwide. cases in point are the epidemics of sars in and h n avian influenza around , which have eloquently demonstrated their great threats to human health, society, and economy. in the coming twenty-first century, contagious diseases are expected to remain as a crucial public health concern for countries around the world. pu-xuan lu , bo-ping zhou , yang gui-lin , and an qi emerging infectious diseases (eids) refer to contagious diseases newly appeared, or with drug resistance, whose incidences have been rapidly increasing and are likely to further rise in the future. eids are usually discovered in three ways. firstly, some existing diseases are ascertained as eids due to the recent discovery of pathogens. secondly, previously considered noninfectious diseases are identifi ed contagious as a result of new etiological fi ndings. thirdly, new infectious diseases are incurred by various complicated reasons such as evolution of pathogens. due to their uncertainty and unpredictability, eids could result in high mortality and great impacts on social stability and economic development as people are unable to react immediately and take specifi c preventive or control measures. therefore, eids have become a major public health problem worldwide. cases in point are the epidemics of sars in and h n avian infl uenza around , which have eloquently demonstrated their great threats to human health, society, and economy. in the coming twentyfi rst century, contagious diseases are expected to remain as a crucial public health concern for countries around the world. at present, more than kinds of eids have been found worldwide, and over kinds have been reported successively in china. eids can be classifi ed into the following fi ve categories. such contagious diseases did not exist in the past and newly emerge due to new pathogens such as aids, severe acute respiratory syndrome (sars), human infection with highly pathogenic avian infl uenza h n , infl uenza a (hln ), and human infection with avian infl uenza h n . some existing diseases are not recognized as eids until recently, such as hepatitis d and e, legionnaire's disease, and lyme disease. some existing diseases considered noninfectious are found contagious in recent years, such as peptic ulcer caused by h. pylori and t-cell leukemia. contagious diseases endemic in some places become prevalent in new places and then are generally considered as eids, such as west nile virus (wnv) that was once popular in middle east and europe and broke out in the usa in . pandemic and subject to multiple infl uential factors. for example, diseases like lyme disease, legionnaires' disease, and peptic ulcer disease are globally distributed, sars appears in countries and regions, and bse rages over european countries. highly contagious and complex in dissemination routes. eids like ebola hemorrhagic fever, sars, west nile encephalitis, and mad cow disease are all highly contagious through various channels. fast transmission with severe damages. aids, described as super cancer and the top killer in twentieth century, has been spreading across the world in an astonishing speed since diagnosed in . in , both the outbreak of sars in spring and the highly pathogenic avian infl uenza h n after mid-december caused casualties and huge economic losses. sars, human avian infl uenza, and infl uenza hln have all become rampant worldwide in a short time. besides, convenient transportation and close international contacts also facilitate their spread. zoonotic. more than three quarters of eids are zoonotic and closely related to animals. researches by jones et al. revealed that . % eids were zoonotic, with . % caused by wild animals, such as human avian infl uenza and ebola virus. diffi cult to prevent, diagnose, and treat due to the varied pathogens. pathogens of eids mainly encompass viruses, bacteria, rickettsia, and chlamydia, predominantly viruses which are highly elusive and infectious. free of natural immunity in human populations. with strong variability, the viruses of eids are able to escape the immune barrier and develop resistance to drugs in new hosts as the environment changes. therefore, people have no immunity to eids. biological factors. pathogenic microorganisms may mutate to adapt new environment. therefore, nonpathogenic strains may become pathogenic, and attenuated strains become virulent, or evolve into new pathogens, thus giving rise to eids. pathogens may generate numerous mutant strains by acquisition, recombination, or transfer of genes in a short time, part of which may develop into new pathogens of contagious diseases. natural factors. global warming has changed the geographical distribution of vector insects and increased their reproduction speed and invasiveness. as a result, breeding time of pathogens outside human bodies is curtailed, making insect-borne contagious diseases more frequent. social factors. deforestation, construction of dams, and other human activities can change the ecological environment. additionally, population movement, sexual promiscuity, drug abuse, and other bad behaviors may cause and spread contagious diseases. clinical and imaging characteristics of eids similar to other contagious diseases, eids progress through four periods: incubation period, prodromal period, period of apparent manifestation, and convalescent period. this period commences from the invasion of pathogens into human body and ends upon the appearance of clinical symptoms. this period spans from onsets to manifestation of evident symptoms. clinical manifestations are usually nonspecifi c and shared by all eids, such as headache, fever, fatigue, and muscle soreness. it commonly lasts - days. after prodromal period, some patients with acute contagious diseases enter the period of apparent manifestation. during this period, all the signs and symptoms of the infectious diseases usually fully manifest. when human immunity grows to a certain extent, the pathophysiological changes would come to an end, and patients' symptoms and signs basically disappear, which is clinically called convalescent period. some diseases can reoccur, and some may cause sequelae. clinical imaging diagnosis is of great signifi cance for the diagnosis, differential diagnosis, and effi cacy evaluation of eids. in light of the strong infectiveness and variability of eids, imaging data are needed for their clinical diagnosis, differential diagnosis, treatment evaluation, and prognosis. mostly caused by viruses, eids have mainly such pulmonary imaging changes as rapid occurrence of ground-glass opacity and (or) pulmonary consolidation. cavity and cystic changes can also be seen in some cases. for some patients, imaging presentations and clinical symptoms and signs are not consistent. for example, the imaging fi ndings may be serious, while clinical symptoms are mild. for example, the pulmonary interstitial fi brosis lesions of patients with human avian infl uenza h n may procrastinate for several years. during convalescent period, absorption of lesions is slow and imaging changes such as interlobular septal thickening and interstitial hyperplasia may be observed. for example, pulmonary fi brosis may be left in some sars patients with severe pulmonary injuries after recovery as the absorption of pulmonary lesions take a long time. moreover, patients with sars or avian infl uenza h n may be complicated with avascular necrosis of femoral head. efforts shall be made to strengthen the surveillance of eids from organization system, personnel, equipment, and tech-nological competence. by monitoring and investigation, we could timely detect new sources of infection or new pathogens and infl uential factors and take prompt and effective measures to rein in their spread and contagion. a global coordinated information platform for eids prevention and control shall be established and improved where countries shall timely communicate epidemic situation of eids and share experience for eids prevention and control so as to block large-scale spreading. public health infrastructure is fundamental to support public health preventive measures and to assess public health status. the high quality training of public health practitioners plays an vital role for the control of infectious diseases. faced with the threats of emerging infectious, public health professionals should strengthen their training, and establish team culture to evaluate epidemiology of infectious diseases, thereby in the face of emerging infectious diseases outbreaks reasonable measures can be taken in an orderly manner to control the situation. . conduct epidemiological studies to clarify the epidemic stages, characteristics, and infl uential factors of eids, so as to provide scientifi c evidences for formulation of prevention countermeasures . accelerate the research and development of vaccines . speed up the research of diagnostic reagents to form experimental methods for rapid diagnosis of eids . carry out studies on pathogenic mechanism and early warning techniques for eids . accelerate the research and development of new drugs, especially antiviral drugs . build a strain resource bank of pathogenic microorganisms overview of eids researches isolation and characterization of viruses related to the sars coronavirus from animals in southern china research contents and methods of eids chest x-ray manifestations of sars clinical characteristics and chest x-ray manifestations of severe sars study about indicators for beside radiogram and protection principles of infl uenza a (h n ) role of imaging in diagnosis of human avian infl uenza people's medical publishing house, beijing human avian infl uenza epidemiological characteristics and pulmonary imaging fi ndings of human avian infl uenza chest clinical imaging diagnosis of aids viral hepatitis. people's medical publishing house ev- hand-foot-mouth disease. people's medical publishing house the epidemic situation and t prevention and treatment strategies for eids the epidemic characteristics and countermeasures of eids cd( ) (+) t lymphocyte count, and plasma hiv viral load: a study in consecutive aids patients relationship between respiratory viral load and lung lesion severity: a study in cases of pandemic h n infl uenza a pneumonia radiological features of lung changes caused by avian infl uenza subtype a h n virus: report of two severe adult cases with regular follow-up preservation of ecological balance and natural environment is the fundamental way to control and reduce zoonotic contagious diseases, therefore precluding eids from the source. key: cord- -w hed w authors: blatt, amy j. title: geographic medicine date: - - journal: health, science, and place doi: . / - - - - _ sha: doc_id: cord_uid: w hed w this chapter uses a sub-discipline of medicine, known as geographic medicine, to describe how human movements contribute to the transmission of parasites on spatial scales that exceed the limits of its natural habitat. traditionally, public health programs have focused on the health of populations, whereas the practice of medicine has focused on the health of individuals. it should be noted, however, that the population health management owes much to the effective delivery of clinical care. this chapter demonstrates how public health is intimately linked to patient care through human movement. nearly a century ago, people typically did not develop a disease where it is contracted or even close to that place. today, daily travel is a common way of life in modern metropolitan areas. large, localized mosquito populations in areas that people visit regularly may be both reservoirs and hubs of infection, even if people only pass through those locations briefly. by examining of the role of human movement across different scales, this chapter examines how public health communities can use information on pathogen transmission to increase the effectiveness of disease prevention programs and clinical care. in , the united states' institute of medicine convened an expert committee on the u.s. commitment to global health and reaffi rmed the notion that local health and local health care are linked to sources of disease and disability occurring elsewhere in the world [ ] : the last three chapters of this book will illustrate the importance of geography in the practice of medicine, both on a local and global scale. the current chapter will highlight the role of geographic forces in the fi eld of travel medicine (also known as geographic medicine) and emphasize the role of geographic knowledge and thought in a study of disease that views humans has vectors and hosts. chapter will introduce a relatively new discipline, called geospatial medicine, that utilizes the advances in geospatial data and technology to uncover the genetic, social, and environmental effects of disease as it occurs throughout human development. this is an exciting new fi eld in which there is a tremendous potential for geographers to take center stage in answering some of the hard research questions that scientists have been grappling with for a long time. lastly, chap. puts the fi nishing touches on the new portrait of medical geography that we have been painting, and frames it in a more centralized role on the national stage of the u.s. health care reform. a main objective of this book is to present a new model of patient care that emphasizes the patient's geographic and medical history, against the backdrop of contemporary globalization, while taking advantage of the current advances in geospatial data and technologies. the current chapter will describe the main principles of travel medicine, and emphasize how human movements contribute to the transmission of parasites on spatial scales that exceed the limits of its natural habitat. studies have shown that the ability to identify the sources (origins) and sinks (destinations) of imported infections due to human travel and locating the high-risk sites of parasite importation could greatly improve the control and prevention programs [ , ] . for instance, a group of researchers have combined mobile phone data with a high-resolution malaria prevalence map to analyze the regional travel patterns of nearly million individuals over the course of a year in kenya, in the context of parasitic dispersals. in this way, they were able to identify and map the sources and sinks of human and parasite travel in this country ( fig. . ) [ ] . to describe the challenges of travel medicine on human terms -in , an estimated . billion travelers crossed international borders, including an estimated . million u.s. travelers [ , ] . an increase in the number of international travelers and local commuters has caused a surge of global and regional epidemics in the past several decades [ ] . travel-related risks include infections from food, vectors, and bodily fl uids. most travelers ( - %) report health problems while traveling, but many do not seek pre-travel advice -such as vaccinations, prophylactics, and therapeutic medications [ , ] . moreover, the risk of travel-related diseases is . times higher in those with an underlying medical conditions than in health individuals [ ] . given the unique health situations of travelers, there is a need for better characterization of the geographical and environmental risk factors underlying travel-related illnesses. to address these concerns, this chapter describes how human travelers who are infected can serve as an important route for the transmission of the virus from their place of origin to their place of destination. by understanding the effects of human movement on disease transmission, researchers can appropriately identify high-risk areas for effective intervention and control. historically, epidemiologists have viewed human movement from two main perspective. the fi rst perspective is from that of the populations of susceptible hosts moving into high-risk areas. the second perspective is from that of populations of infected hosts moving into susceptible populations. movements of infected hosts across different spatial scales affect pathogen transmission in a variety of ways. noted historian and geographer r. mansell prothero published one of the fi rst studies describing the role of human movements in epidemiology based on his experience in africa, in . drawing on the geographic literature concerning diffusion and migration processes, he discussed the relevance of these movement patterns to public health in his seminal paper published in the international journal of epidemiology . he carefully outlined the differences between circulatory and migratory movements and categorized these movements by their spatial scale (i.e., a rural-urban gradient) and temporal scale (i.e., the time and timing of displacements). circulatory movements are those in which the individuals return home after some period, and migratory movements are those which usually result in permanent changes of residence. prothero's argument was that knowledge of the nature of these movements would help inform the public's understanding of the incidence and prevalence of disease on a population level and provide informed options for control [ ] . for instance, seasonal migratory movements from one rural area to another for agriculture could potentially expose individuals to different areas where the risk of african trypanosomiasis, or malaria, is high [ ] . at broad spatial scales (e.g., national, international), individual movements can drive pathogen introduction and reintroduction. for example, the global spread of dengue virus via shipping routes was characterized by periodic, large, spatial displacements. globalization and mass air transportation have changed the transmission of pathogens by dramatically shortening the time required to travel around the earth. at fi ner scales (e.g., regional, urban-rural, intra-urban), movement associated with work, recreation, and transient migration into high-risk areas not only lead to individual infection, but also contribute to local transmission when infected hosts return home and infect other individuals ( fig. . ) [ ] . indeed, vector-borne diseases place an enormous burden on public health and require effi cient control strategies that are developed through an understanding of the origin (or sources) of infections and the relative importance of human movement at different scales. a number of social and environmental factors -such as human population density, regional settlement patterns, population movements, precipitation, and other weather-related factors -contribute to local and regional transmission dynamics [ ] . human movement -which determines exposure to vectors -is a key aspect of vector ecology that is poorly understood. this is due to the variations in exposure based on individual host movement that can strongly infl uence the pathogen's transmission dynamics [ ] . the types of movement most relevant for exposure will depend on site-specifi c differences, the ecology of the arthropod vector, human behavior, and the relative scale of host and vector movement. for instance -although fi ne-scale host movements are not important to pathogens transmitted by vectors that are able to move long distances in search of a host -these fi ne-scale host movements are very important for pathogens transmitted by sessile vectors. aedes aegypti is the principal vector of dengue virus. it bites during the day, disperses only short distances, and is heterogeneously distributed within urban areas. humans, on the other hand, move frequently and allocate different amounts of time to multiple locations on a regular basis. not only does this infl uence the individual risk of infection with dengue virus but it also infl uences overall patterns of transmission [ ] . in addition, commuting and non-commuting patients have different diffusion patterns and determinants in a dengue epidemic. non-commuters (e.g., elderly adults and housewives) may initiate a local epidemic, whereas commuters carrying the virus to geographically distant areas can cause a large-scale epidemic [ ] . dengue is a global threat and is endemic or epidemic in almost every country located in the tropics (fig. . ). while new tools (such as vaccines, antiviral drugs and improved diagnostics) are being developed, better use should be made of the interventions that are currently available. along with comprehensive tracking of commuting cases, the concomitant rapid notifi cation and diagnosis of non-commuting cases can enable the appropriate interventions and faster response times, thus preventing subsequent large-scale epidemics. as illustrated in the previous sections of this chapter, human movement is a largely ignored variable in the study and diagnosis of pathogen exposures. there are few, if any, well-established clinical guidelines that utilize a person's place history in the diagnosis of infected individuals, despite the fact that disease management protocols can be made more effective and effi cient by targeting the sources or agents of transmission. the study of human movement is critical to identifying high-risk factors (e.g., hostpreferences) because these factors are always conditioned by exposure rates which are, in turn, closely related to variations in human movement and behavior. although geographical movement is becoming increasing easy to measure through advanced geospatial technologies, a patient's movements and place history have been largely ignored by the medical community. quantifying and describing human movements provides valuable information necessary to predict disease outbreaks and to evaluate control alternatives to halt epidemics [ - ] . in addition, the ability to apply this knowledge to a variety of diseases creates an opportunity to identify common areas where infection occurs across multiple diseases and to leverage public health programs to target the most important locations that serve as sources for more than one disease. by examining of the role of human movement across different scales, public health communities can use this valuable information on pathogen transmission to increase the effectiveness of disease prevention programs. as transmission rates are reduced through intervention efforts, scientists can expect the importance of heterogeneity in exposure to increase and play an even more important role in pathogen persistence. therefore, characterization of human and population movements can facilitate not only the elimination of disease, but also help to prevent its return [ ] . key considerations in the analysis of human movement patterns include, but are not limited to: spatial scale, the type and periodicity of movement, and the time period of observation. spatial scale refers to the geographical extent of the pathogen and the spatial interpretation of the data. the geographical extent of the pathogen and its transmission can be determined by the disease dynamic -i.e., the spread of a pathogen to new geographical areas versus a sustained transmission at a given location. if the transmission is local, then the relevant movements will be those placing the susceptible hosts in high risk locations at times when infection risk is high. assumptions regarding the importance of movements should be made with care because heterogeneity in exposure can have a dramatic effect on infection risk. the type of movement refers to what the researcher is aiming to measure. for instance, is the study interested in the sites where individuals spend their time on a . geographic considerations in human movement regular basis (high spatial and temporal resolution) or when they are traveling outside of their home city? what is the value of travel information (outside of an urban area) that specifi es exactly where people go? are specifi c routes important, or should only destinations be considered? these specifi c details will depend on the nature of the questions, systems, and resources and methods involved for measuring movements. finally, the time period of observation is concerned with how long to observe individual movements for. the correct answer will depend on the questions being asked and available resources. in the case of dengue, infection can occur up to weeks prior to the manifestation of symptoms. for a retrospective study, - days would be an appropriate observation period. conversely, in a prospective study, the length of the observation period will depend on the relative importance of rare movements. studies of human movements in developed societies reveal markedly regular patterns, especially during the work-week [ , , ] . additionally, there may be significant instability in movements on weekends or at other times (e.g., vacations). for regular movements during the work week, at least weeks of observation are desired. for more variable movements/times, longer observation periods will be necessary. over the last century, human civilizations and urbanization have witnessed a huge increase in mobility and population growth. the combined effect of these factors means that, despite great improvements in hygiene, sanitation and vector control, the containment of disease remains one of the biggest challenges of our modern contemporary society. the importance of increased human mobility in disease transmission cannot be under-stated. on national and global scales, the airline transport network has played a key role in the global dissemination of infl uenza and sars [ ] . in addition, migrants, tourists, and commercial travelers also have a signifi cant infl uence on the spread of hiv [ ] . on a local scale, human movements in metropolitan areas are frequent and extensive, but are often composed of highly structured commuting patterns between the home and places of employment, education, or business. the ability to quantify human movement and its effects are vital in the ongoing development of strategies to eradicate vector-borne diseases (such as dengue) from urban centers [ ] . using a meta-population analysis where mobile humans connect with static mosquito subpopulations in a very structured pattern, a group of researchers found that, due to frequency dependent biting, infection incidence in the human and mosquito populations is independent of the duration of contact [ ] . the researchers hypothesize that, since the biting rate is frequency dependent (and independent of the density of the human population), a mosquito will bite the same number of people per day. in addition, their modeled results indicate that people who travel regularly to areas with large mosquito populations form a high-risk group, and have a relatively high level of infection compared to people that travel regularly to patches with small mosquito populations. furthermore, extensive variation in human movement patterns causes the number of interactions between human and mosquito populations to increase. more variable human movements increases the likelihood that people will carry the infection from these highly infested areas to mosquito subpopulations where the pathogen has died out. therefore, a large mosquito population in a frequently visited area may be suffi cient to ensure infection is endemic, even if there are relatively few mosquitoes elsewhere. when people do not vary their travel patterns very much and there is no direct connectivity between mosquito populations, the transit corridor can signifi cantly enhance disease persistence by acting as a reservoir and hub. if people vary the areas they visit even occasionally, the effect of the transit corridor is overridden [ , ] . mosquito and human movements become even more important as remote rural villages are connected to each other by mass transportation networks. nearly a century ago, it was observed that people do not develop a disease where it is contracted or even close to that place [ ] . today, widespread mass transportation makes that observation even more relevant. the incidence and persistence of vector-borne diseases on relatively small spatial scales may be strongly infl uenced by infectious humans who remain mobile because the infection is mild or silent. increased human movement on a local scale may be a key factor behind increased incidence of vector-borne diseases. in modern metropolitan areas, daily travel is a common way of life. distant subpopulations of mosquitoes may be connected by this daily movement. large, localized mosquito populations in areas that people visit regularly may be both reservoirs and hubs of infection, even if people only pass through those locations briefl y. this implies that surveillance with the goal of controlling vector-borne disease may be a much greater challenge than originally anticipated. ultimately, successful public health intervention must focus on both hosts and vectors. large mosquito populations that are also visited by a large fraction of the human population need to be identifi ed. it is vital to employ surveillance strategies that reveal the variability in the distribution of mosquitoes and target areas where the mosquito population is signifi cant and human movement is extensive. further study of networks formed by human movement in urban areas are need, and cell phone records are one potential source of such detailed information [ ] . committee on the u.s. commitment to global health. the u.s. commitment to global health: recommendations for the public and private sectors a global view of health -an unfolding series stable and unstable malaria hotspots in longitudinal cohort studies in kenya international population movements and regional plasmodium falciparum malaria elimination strategies quantifying the impact of human mobility on malaria international tourism results and prospects for international trade administration ( ) u.s. travel to international destinations increased three percent in . department of commerce, international trade administration airports, localities and disease: representations of global travel during the h n pandemic advising the traveler health risks of travelers with medical conditions -a retrospective analysis disease and mobility: a neglected factor in epidemiology population mobility and trypanosomiasis in africa dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem climate change and risk projection: dynamic spatial models of tsetse and african trypanosomiasis in kenya the role of human movement in the transmission of vector-borne pathogens using geographic information systems to analyze the distribution and abundance of aedes aegypti in africa: the potential role of human travel in determining the intensity of mosquito infestation population movement and vector-borne disease transmission: differentiating spatial-temporal diffusion patterns of commuting and non-commuting dengue cases modelling disease outbreaks in realistic urban social networks understanding individual human mobility patterns large-scale spatial-transmission models of infectious disease what about people in spatial science habitual travel behaviour: evidence from a six-week travel diary the role of the airline transportation network in the prediction and predictability of global epidemics travel and the spread of hiv- genetic variants dengue and dengue hemorrhagic fever man bites mosquito: understanding the contribution of human movement to vector-borne disease dynamics household and community transmission parameters from fi nal distributions of infections in households stegomyia indices and their value in yellow fever control key: cord- - v brjf authors: nicholson, felicity title: infectious diseases: the role of the forensic physician date: journal: clinical forensic medicine doi: . / - - - : sha: doc_id: cord_uid: v brjf infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in . despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in . despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. for the forensic physician, dealing with infections presents two main problems. the first problem is managing detainees or police personnel who have contracted a disease and may be infectious or unwell. the second problem is handling assault victims, including police officers, who have potentially been exposed to an infectious disease. the latter can be distressing for those involved, compounded, in part, from an inconsistency of management guidelines, if indeed they exist. with the advent of human rights legislation, increasing pressure is being placed on doctors regarding consent and confidentiality of the detainee. therefore, it is prudent to preempt such situations before the consultation begins by obtaining either written or verbal consent from the detainee to allow certain pieces of information to be disclosed. if the detainee does not agree, then the doctor must decide whether withholding relevant details will endanger the lives or health of those working within custody or others with whom they may have had close contact (whether or not deliberate). consent and confidentiality issues are discussed in detail in chapter . adopting a universal approach with all detainees will decrease the risk to staff of acquiring such diseases and will help to stop unnecessary overreaction and unjustified disclosure of sensitive information. for violent or sexual assault victims, a more open-minded approach is needed (see also chapter ) . if the assailant is known, then it may be possible to make an informed assessment of the risk of certain diseases by ascertaining his or her lifestyle. however, if the assailant is unknown, then it is wise to assume the worst. this chapter highlights the most common infections encountered by the forensic physician. it dispels "urban myths" and provides a sensible approach for achieving effective management. the risk of exposure to infections, particularly blood-borne viruses (bbvs), can be minimized by adopting measures that are considered good practice in the united kingdom, the united states, and australia ( ) ( ) ( ) . forensic physicians or other health care professionals should wash their hands before and after contact with each detainee or victim. police officers should be encouraged to wash their hands after exposure to body fluids or excreta. all staff should wear gloves when exposure to body fluids, mucous membranes, or nonintact skin is likely. gloves should also be worn when cleaning up body fluids or handling clinical waste, including contaminated laundry. single-use gloves should only be used and must conform to the requirements of european standard or equivalent ( ) ( ) ( ) . a synthetic alternative conforming to the same standards should also be available for those who are allergic to latex. all staff should cover any fresh wounds (< hours old), open skin lesions, or breaks in exposed skin with a waterproof dressing. gloves cannot prevent percutaneous injury but may reduce the chance of acquiring a bloodborne viral infection by limiting the volume of blood inoculated. gloves should only be worn when taking blood, providing this does not reduce manual dexterity and therefore increase the risk of accidental percutaneous injury. ideally, a designated person should be allocated to ensure that the clinical room is kept clean and that sharps containers and clinical waste bags are removed regularly. clinical waste must be disposed of in hazard bags and should never be overfilled. after use, the clinical waste should be doublebagged and sealed with hazard tape. the bags should be placed in a designated waste disposal (preferably outside the building) and removed by a professional company. when cells are contaminated with body fluids, a professional cleaning company should be called to attend as soon as possible. until such time, the cell should be deemed "out of action." there is a legal requirement in the united kingdom under the environmental protection act ( ) and the control of substances hazardous to health regulations to dispose of sharps in an approved container. in the united states, the division of health care quality promotion on the centers for disease control and prevention (cdc) web site provides similar guidance. in custody, where sharps containers are transported off site, they must be of an approved type. in the united kingdom, such a requirement is contained within the carriage of dangerous goods (classification, packaging and labelling) and use of transportable pressure receptacles regulations . these measures help to minimize the risk of accidental injury. further precautions include wearing gloves when handling sharps and never bending, breaking, or resheathing needles before disposal. sharps bins should never be overfilled, left on the floor, or placed above the eye level of the smallest member of staff. any bedding that is visibly stained with body fluids should be handled with gloves. there are only three acceptable ways of dealing with contaminated bedding: the bbvs that present the most cross-infection hazard to staff or victims are those associated with persistent viral replication and viremia. these include hbv, hcv, hepatitis d virus (hdv), and hiv. in general, risks of transmission of bbvs arise from the possible exposure to blood or other body fluids. the degree of risk varies with the virus concerned and is discussed under the relevant sections. figure illustrates the immediate management after a percutaneous injury, mucocutaneous exposure, or exposure through contamination of fresh cuts or breaks in the skin. hbv is endemic throughout the world, with populations showing a varying degree of prevalence. approximately two thousand million people have been infected with hbv, with more than million having chronic infection. worldwide, hbv kills about million people each year. with the development of a safe and effective vaccine in , the world health organization (who) recommended that hbv vaccine should be incorporated into national immunization programs by in those countries with a chronic infection rate of % or higher, and into all countries by . although countries had achieved this goal by the end of , the poorest countries-often the ones with the highest prevalence-have been unable to afford it. in particular these include china, the indian subcontinent, and sub-saharan africa. people in the early stages of infection or with chronic carrier status (defined by persistence of hepatitis b surface antigen [hbsag] beyond mo) can transmit infection. in the united kindgom, the overall prevalence of chronic hbv is approx . - . % ( , ) . a detailed breakdown is shown in table . the incubation period is approx weeks to months. as the name suggests, the virus primarily affects the liver. typical symptoms include malaise, anorexia, nausea, mild fever, and abdominal discomfort and may last from days to weeks before the insidious onset of jaundice. joint pain and skin rashes may also occur as a result of immune complex formation. infections in the newborn are usually asymptomatic. * in the united kingdom, written consent from the contact must be sent with the sample, countersigned by the health care practitioner and, preferably, an independent police officer. the majority of patients with acute hbv make a full recovery and develop immunity. after acute infection, approx in patients develop liver failure, which may result in death. chronic infection develops in approx % of neonates, approx % of children, and between and % of adults. neonates and children are usually asymptomatic. adults may have only mild symptoms or may also be asymptomatic. approximately - % of chronically infected individuals (depending on age of acquisition) will develop cirrhosis over a number of years. this may also result in liver failure or other serious complications, including hepatocellular carcinoma, though the latter is rare. the overall mortality rate of hbv is estimated at less than %. a person is deemed infectious if hbsag is detected in the blood. in the acute phase of the illness, this can be as long as months. by definition, if hbsag persists after this time, then the person is deemed a carrier. carriers are usually infectious for life. the degree of infectivity depends on the stage of disease and the markers present table . the major routes include parenteral (e.g., needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized), mucous membrane exposure (including mouth, eyes, and genital mucous membranes), and contamination of broken skin (especially when < hours old). hbv is an occupational hazard for anyone who may come into contact with blood or bloodstained body fluids through the routes described. saliva alone may transmit hbv. the saliva of some people infected with hbv contains hbv-dna concentrations / - / , of that found in their serum ( ) . this is especially relevant for penetrating bite wounds. infection after exposure to other body fluids (e.g., bile, urine, feces, and cerebrospinal fluid) has never been demonstrated unless the fluids are contaminated with blood. intravenous drug users who share needles or other equipment are also at risk. hbv can also be transmitted through unprotected sexual contact, whether homosexual or heterosexual. the risk is increased if blood is involved. sexual assault victims should be included in this category. evidence has shown that the virus may also be spread among members of a family through close household contact, such as through kissing and sharing toothbrushes, razors, bath towels, etc. ( ) ( ) ( ) . this route of transmission probably applies to institutionalized patients, but there are no available data. studies of prisoners in western countries have shown a higher prevalence of antibodies to hbv and other bbvs than the general population ( ) ( ) ( ) ; the most commonly reported risk factor is intravenous drug use. however, the real frequency of transmission of bbvs in british prisons is unknown owing to the difficulty in compiling reliable data. hbv can be transmitted vertically from mother to baby during the perinatal period. approximately % of babies born to mothers who have either acute or chronic hbv become infected, and most will develop chronic hbv. this has been limited by the administration of hbv vaccine to the neonate. in industrialized countries, all prenatal mothers are screened for hbv. vaccine is given to the neonate ideally within the first hours of birth and at least two more doses are given at designated intervals. the who recommends this as a matter of course for all women in countries where prevalence is high. however, the practicalities of administering a vaccine that has to be stored at the correct temperature in places with limited access to medical care means that there is a significant failure of vaccine uptake and response. in industrialized countries, hbv vaccination is recommended for those who are deemed at risk of acquiring the disease. they include the following: . through occupational exposure. . homosexual/bisexual men. . intravenous drug users. . sexual partners of people with acute or chronic hbv. . family members of people with acute or chronic hbv. . newborn babies whose mothers are infected with hbv. if the mother is hbsag positive, then hepatitis b-specific immunoglobulin (hbig) should be given at the same time as the first dose of vaccine. . institutionalized patients and prisoners. ideally, hbv vaccine should be administered before exposure to the virus. the routine schedule consists of three doses of the vaccine given at , , and months. antibody levels should be checked - weeks after the last dose. if titers are greater than miu/ml, then an adequate response has been achieved. in the united kingdom, this is considered to provide protection for - years. in the united states, if an initial adequate response has been achieved, then no further doses of vaccine are considered necessary. vaccine administration after exposure varies according to the timing of the incident, the degree of risk involved, and whether the individual has already been partly or fully vaccinated. an accelerated schedule when the third dose is given months after the first dose with a booster year later is used to prevent postnatal transmission. where risks are greatest, it may be necessary to use a rapid schedule. the doses are given at , , and - days after presentation, again with a booster dose at - months. this schedule is currently only licensed with engerix b. hbig may also be used either alone or in conjunction with vaccine. the exact dose given is age dependent but must be administered by deep intramuscular injection in a different site from the vaccine. in an adult, this is usually into the gluteus muscle. hbig is given in conjunction with the first dose of vaccine to individuals who are deemed at high risk of acquiring disease and the incident occurred within hours of presentation. it is also used for neonates born to mothers who are hbeag-positive. between and % of adults fail to respond to the routine schedule of vaccine. a further full course of vaccine should be tried before deeming the patients as "nonresponders." such individuals involved in a high-risk exposure should be given two doses of hbig administered mo apart. ideally, the first dose should be given within hours after exposure and no later than weeks after exposure. other measures include minimizing the risk of exposure by adopting the safe working practices outlined in subheading . any potential exposures should be dealt with as soon as possible. in industrialized countries blood, blood products, and organs are routinely screened for hbv. intravenous drug users should be encouraged to be vaccinated and to avoid sharing needles or any other drug paraphernalia (see subheading . . .). for staff or victims in contact with disease, it is wise to have a procedure in place for immediate management and risk evaluation. an example is shown in fig. . although forensic physicians are not expected to administer treatment, it is often helpful to inform persons concerned what to expect. tables and outline treatment protocols as used in the united kingdom. detainees with disease can usually be managed in custody. if the detainee is bleeding, then the cell should be deemed out of action after the detainee has left until it can be professionally cleaned. contaminated bedding should be dealt with as described in subheading . . if the detainee has chronic hbv and is on an antiviral agent (e.g., lamivudine), then the treatment course should be continued, if possible. hcv is endemic in most parts of the world. approximately % ( million) of the world's population is infected with hcv ( ) . for many countries, no reliable prevalence data exist. seroprevalence studies conducted among blood donors have shown that the highest prevalence exists in egypt ( - %). this has been ascribed to contaminated needles used in the treatment of schistosomiasis conducted between the s and the s ( ) . intermediate prevalence ( - %) exists in eastern europe, the mediterranean, the middle east, the indian subcontinent, and parts of africa and asia. in western europe, most of central america, australia, and limited regions in africa, including south africa, the prevalence is low ( . - . %). previously, america was included in the low prevalence group, but a report published in ( ) indicated that almost million americans (i.e., . % of the population) have antibody to hcv, representing either ongoing or previous infection. it also states that hcv accounts for approx % of acute viral hepatitis in america. the lowest prevalence ( . - . %) has been found in the united kingdom and scandinavia. however, within any country, there are certain groups that have a higher chance of carrying hcv. these united kingdom figures are given in table . after an incubation period of - weeks, the acute phase of the disease lasts approx - years. unlike hepatitis a (hav) or hbv, the patient is usually asymptomatic; therefore, the disease is often missed unless the individual has reported a specific exposure and is being monitored. other cases are found by chance, when raised liver enzymes are found on a routine blood test. a "silent phase" follows the acute phase when the virus lies dormant and the liver enzymes are usually normal. this period lasts approx - years. reactivation may then occur. subsequent viral replication damages the hepatocytes, and liver enzymes rise to moderate or high levels. eighty percent of individuals who are hcv antibody-positive are infectious, regardless of the levels of their liver enzymes. approximately % of people develop chronic infection, one-fifth of whom progress to cirrhosis. there is a much stronger association with hepatocellular carcinoma than with hbv. an estimated . - . % of patients with hcv-related cirrhosis develop liver cancer ( ) . less than % of chronic cases resolve spontaneously. approximately % of cases are parenteral (e.g., needle-stick, etc.) ( ) . transmission through the sexual route is not common and only appears to be significant if there is repeated exposure with one or more people infected with hcv. mother-to-baby transmission is considered to be uncommon but has been reported ( ) . theoretically, household spread is also possible through sharing contaminated toothbrushes or razors. because the disease is often silent, there is a need to raise awareness among the general population on how to avoid infection and to encourage high-risk groups to be tested. health care professionals should also be educated to avoid occupationally acquired infection. an example of good practice blood or blood-stained body fluids need to be involved for a risk to occur. saliva alone is not deemed to be a risk. the risk from a single needlestick incident is . % (range - %). contact through a contaminated cut is estimated at %. for penetrating bite injuries, there are no data, but it is only considered a risk if blood is involved. blood or blood-stained body fluids have to be involved in transmission through mucous membrane exposure. this may account for the lower-than-expected prevalence among the gay population. follow the immediate management flow chart, making sure all available information is obtained. inform the designated hospital and/or specialist as soon as possible. if the contact is known and is believed to be immunocompromised and he or she has consented to provide a blood sample, it is important to tell the specialist, because the antibody tests may be spuriously negative. in this instance, a different test should be used (polymerase chain reaction [pcr] , which detects viral rna). the staff member/victim will be asked to provide a baseline sample of blood with further samples at - weeks and again at weeks. if tests are negative at weeks but the risk was deemed high, then follow-up may continue for up to weeks. if any of the follow-up samples is positive, then the original baseline sample will be tested to ascertain whether the infection was acquired through the particular exposure. it is important to emphasize the need for prompt initial attendance and continued monitoring, because treatment is now available. a combination of ribavirin (antiviral agent and interferon a- b) ( ) or the newer pegylated interferons ( ) may be used. this treatment is most effective when it is started early in the course of infection. unless they are severely ill, detainees can be managed in custody. special precautions are only required if they are bleeding. custody staff should wear gloves if contact with blood is likely. contaminated bedding should be handled appropriately, and the cell cleaned professionally after use. this defective transmissible virus was discovered in and requires hbv for its own replication. it has a worldwide distribution in association with hbv, with approx million people infected. the prevalence of hdv is higher in southern italy, the middle east, and parts of africa and south america, occurring in more than % of hbv carriers who are asymptomatic and more than % of those with chronic hbv-related liver disease. despite the high prevalence of hbv in china and south east asia, hdv in these countries is rare. hdv is associated with acute (coinfection) and chronic hepatitis (superinfection) and can exacerbate pre-existing liver damage caused by hbv. the routes of transmission and at-risk groups are the same as for hbv. staff/victims in contact with a putative exposure and detainees with disease should be managed as for hbv. interferon-α (e.g., roferon) can be used to treat patients with chronic hbv and hdv ( ) , although it would not be practical to continue this treatment in the custodial setting. hiv was first identified in , years after the first reports were made to the cdc in atlanta, ga, of an increased incidence of two unusual diseases (kaposi's sarcoma and pneumocystis carinii pneumonia) occurring among the gay population in san francisco. the scale of the virus gradually emerged over the years and by the end of , there were an estimated million people throughout the world living with hiv or acquired immunodeficiency syndrome (aids). more than % of the world's population lives in africa and india. a report by the joint united nations programme on hiv/aids and the who in stated that one in five adults in lesotho, malawi, mozambique, swaziland, zambia, and zimbabwe has hiv or aids. there is also expected to be a sharp rise in cases of hiv in china, papua new guinea, and other countries in asia and the pacific during the next few years. in the united kingdom, by the end of , the cumulative data reported that there were , individuals with hiv, aids (including deaths from aids) reported, though this is likely to be an underestimate ( ) . from these data, the group still considered at greatest risk of acquiring hiv in the united kingdom is homosexual/bisexual men, with , of the cumulative total falling into this category. among intravenous drug users, the overall estimated prevalence is %, but in london the figure is higher at . % ( , ) . in the s, up to % of users in edinburgh and dundee were reported to be hiv positive, but the majority have now died. individuals arriving from africa or the indian subcontinent must also be deemed a risk group because % of the world's total cases occur in these areas. the predominant mode of transmission is through unprotected heterosexual intercourse. the incidence of mother-to-baby transmission has been estimated at % in europe and approx % in africa. the transmission rates among african women are believed to be much higher owing to a combination of more women with end-stage disease with a higher viral load and concomitant placental infection, which renders it more permeable to the virus ( , ) . the use of antiretroviral therapy during pregnancy, together with the advice to avoid breastfeeding, has proven efficacious in reducing both vertical and horizontal transmission among hiv-positive women in the western world. for those in third-world countries, the reality is stark. access to treatment is limited, and there is no realistic substitute for breast milk, which provides a valuable source of antibodies to other life-threatening infections. patients receiving blood transfusions, organs, or blood products where screening is not routinely carried out must also be included. the incubation is estimated at weeks to months after exposure. this depends, to some extent, on the ability of current laboratory tests to detect hiv antibodies or viral antigen. the development of pcr for viral rna has improved sensitivity. during the acute phase of the infection, approx % experience a seroconversion "flu-like" illness. the individual is infectious at this time, because viral antigen (p ) is present in the blood. as antibodies start to form, the viral antigen disappears and the individual enters the latent phase. he or she is noninfectious and remains well for a variable period of time ( - years). development of aids marks the terminal phase of disease. viral antigen reemerges, and the individual is once again infectious. the onset of aids has been considerably delayed with the use of antiretroviral treatment. parenteral transmission included needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized. risk of transmission is increased with deep penetrating injuries with hollow bore needles that are visibly bloodstained, especially when the device has previously been in the source patient's (contact) artery or vein. other routes include mucous membrane exposure (eyes, mouth, and genital mucous membranes) and contamination of broken skin. the higher the viral load in the contact, the greater the risk of transmission. this is more likely at the terminal stage of infection. hiv is transmitted mainly through blood or other body fluids that are visibly blood stained, with the exception of semen, vaginal fluid, and breast milk. saliva alone is most unlikely to transmit infection. therefore, people who have sustained penetrating bite injuries can be reassured that they are not at risk, providing the contact was not bleeding from the mouth at the time. the risk from a single percutaneous exposure from a hollow bore needle is low, and a single mucocutaneous exposure is even less likely to result in infection. the risk from sexual exposure varies, although it appears that there is a greater risk with receptive anal intercourse compared with receptive vaginal intercourse ( ). high-risk fluids include blood, semen, vaginal fluid, and breast milk. there is little or no risk from saliva, urine, vomit, or feces unless they are visibly bloodstained. other fluids that constitute a theoretical risk include cerebrospinal, peritoneal, pleural, synovial, or pericardial fluid. management in custody of staff/victims in contact with disease includes following the immediate management flow chart (fig. ) and contacting the designated hospital/specialist with details of the exposure. where possible, obtain a blood sample from the contact. regarding hbv and hcv blood samples in the united kingdom, they can only be taken with informed consent. there is no need for the forensic physician to go into details about the meaning of the test, but the contact should be encouraged to attend the genitourinary department (or similar) of the designated hospital to discuss the test results. should the contact refuse to provide a blood sample, then any information about his or her lifestyle, ethnic origin, state of health, etc., may be useful for the specialist to decide whether postexposure prophylaxis (pep) should be given to the victim. where only saliva is involved in a penetrating bite injury, there is every justification to reassure the victim that he or she is not at risk. if in doubt, then always refer. in the united kingdom, the current recommended regime for pep is combivir ( mg of zidovudine twice daily plus mg of lamivudine twice daily) and a protease inhibitor ( mg of nelfanivir twice daily) given for weeks ( ) . it is only given after a significant exposure to a high-risk fluid or any that is visibly bloodstained and the contact is known or is highly likely to be hiv positive. ideally, treatment should be started within an hour after exposure, although it will be considered for up to weeks. it is usually given for weeks, unless the contact is subsequently identified as hiv negative or the "victim" develops tolerance or toxicity occurs. weekly examinations of the "victim" should occur during treatment to improve adherence, monitor drug toxicity, and deal with other concerns. other useful information that may influence the decision whether to treat with the standard regimen or use alternative drugs includes interaction with other medications that the "victim" may be taking (e.g., phenytoin or antibiotics) or if the contact has been on antiretroviral therapy or if the "victim" is pregnant. during the second or third trimester, only combivir would be used, because there is limited experience with protease inhibitors. no data exist regarding the efficacy of pep beyond occupational exposure ( ) . pep is not considered for exposure to low-or no-risk fluids through any route or where the source is unknown (e.g., a discarded needle). despite the appropriate use and timing of pep, there have been reports of failure ( , ) . unless they are severely ill, detainees can be kept in custody. every effort should be made to continue any treatment they may be receiving. apply universal precautions when dealing with the detainee, and ensure that contaminated cells and/or bedding are managed appropriately. cases of this highly infectious disease occur throughout the year but are more frequent in winter and early spring. this seasonal endemicity is blurring with global warming. in the united kingdom, the highest prevalence occurs in the -to -years age group. ninety percent of the population over the age of is immune ( ) . a similar prevalence has been reported in other parts of western europe and the united states. in south east asia, varicella is mainly a disease of adulthood ( ) . therefore, people born in these countries who have moved to the united kingdom are more likely to be susceptible to chicken pox. there is a strong correlation between a history of chicken pox and serological immunity ( - %). most adults born and living in industrialized countries with an uncertain or negative history of chicken pox are also seropositive ( - %). in march , a live-attenuated vaccine was licensed for use in the united states and a policy for vaccinating children and susceptible health care personnel was introduced. in summer , in the united kingdom, glaxosmithkline launched a live-attenuated vaccine called varilrix. in december , the uk department of health, following advice from the joint committee on vaccination and immunisation recommended that the vaccine be given for nonimmune health care workers who are likely to have direct contact with individuals with chicken pox. any health care worker with no previous history of chicken pox should be screened for immunity, and if no antibodies are found, then they should receive two doses of vaccine - weeks apart. the vaccine is not currently recommended for children and should not be given during pregnancy. following an incubation period of - days (this may be shorter in the immunocompromised), there is usually a prodromal "flu-like" illness before the onset of the rash. this coryzal phase is more likely in adults. the lesions typically appear in crops, rapidly progressing from red papules through vesicles to open sores that crust over and separate by days. the distribution of the rash is centripetal (i.e., more over the trunk and face than on the limbs). this is the converse of small pox. in adults, the disease is often more severe, with lesions involving the scalp and mucous membranes of the oropharynx. in children, the disease is often mild, unless they are immunocompromised, so they are unlikely to experience complications. in adults (defined as yr or older), the picture is rather different ( ) . secondary bacterial infection is common but rarely serious. there is an increased likelihood of permanent scarring. hemorrhagic chicken pox typically occurs on the second or third day of the rash. usually, this is limited to bleeding into the skin, but lifethreatening melena, epistaxis, or hematuria can occur. varicella pneumonia ranges from patchy lung consolidation to overt pneumonitis and occurs in in cases ( ) . it can occur in previously healthy individuals (particularly adults), but the risk is increased in those who smoke. immunocompromised people are at the greatest risk of developing this complication. it runs a fulminating course and is the most common cause of varicella-associated death. fibrosis and permanent respiratory impairment may occur in those who survive. any suspicion of lung involvement is an indication for immediate treatment, and any detainee or staff member should be sent to hospital. involvement of the central nervous system includes several conditions, including meningitis, guillain-barre, and encephalitis. the latter is more common in the immunocompromised and can be fatal. this is taken as days before the first lesions appear to the end of new vesicle formation and the last vesicle has crusted over. this typically is - days after onset but may last up to days. the primary route is through direct contact with open lesions of chicken pox. however, it is also spread through aerosol or droplets from the respiratory tract. chicken pox may also be acquired through contact with open lesions of shingles (varicella zoster), but this is less likely because shingles is less infectious than chicken pox. nonimmune individuals are at risk of acquiring disease. approximately % of the adult population born in the united kingdom and less than % of adults in the united states fall into this category. therefore, it is more likely that if chicken pox is encountered in the custodial setting, it will involve people born outside the united kingdom (particularly south east asia) or individuals who are immunocompromised and have lost immunity. nonimmune pregnant women are at risk of developing complications. pneumonia can occur in up to % of pregnant women with chicken pox, and the severity is increased in later gestation ( ) . they can also transmit infection to the unborn baby ( ) . if infection is acquired in the first weeks, there is a less than % chance of it leading to congenital varicella syndrome. infection in the last trimester can lead to neonatal varicella, unless more than days elapse between onset of maternal rash and delivery when antibodies have time to cross the placenta leading to either mild or inapparent infection in the newborn. in this situation, varicella immunoglobulin (vzig) should be administered to the baby as soon as possible after birth ( ). staff with chicken pox should stay off work until the end of the infective period (approx - days). those in contact with disease who are known to be nonimmune or who have no history of disease should contact the designated occupational health physician. detainees with the disease should not be kept in custody if at all possible (especially pregnant women). if this is unavoidable, then nonimmune or immunocompromised staff should avoid entering the cell or having close contact with the detainee. nonimmune, immunocompromised, or pregnant individuals exposed to chickenpox should seek expert medical advice regarding the administration of vzig. aciclovir (or similar antiviral agent) should be given as soon as possible to people who are immunocompromised with chicken pox. it should also be considered for anyone over years old because they are more likely to develop complications. anyone suspected of severe complications should be sent straight to the hospital. after chicken pox, the virus lies dormant in the dorsal root or cranial nerve ganglia but may re-emerge and typically involves one dermatome ( ) . the site of involvement depends on the sensory ganglion initially involved. shingles is more common in individuals over the age of years, except in the immunocompromised, when attacks can occur at an earlier age. the latter are also more susceptible to secondary attacks and involvement of more than one dermatome. bilateral zoster is even rarer but is not associated with a higher mortality. in the united kingdom, there is an estimated incidence of . - . per -person years ( ). there may be a prodromal period of paraesthesia and burning or shooting pains in the involved segment. this is usually followed by the appearance of a band of vesicles. rarely, the vesicles fail to appear and only pain is experienced. this is known as zoster sine herpete. in individuals who are immuno-compromised, disease may be prolonged and dissemination may occur but is rarely fatal. shingles in pregnancy is usually mild. the fetus is only affected if viremia occurs before maternal antibody has had time to cross the placenta. the most common complication of shingles is postherpetic neuralgia, occurring in approx % of cases. it is defined as pain lasting more than days from rash onset ( ) . it is more frequent in people over years and can lead to depression. it is rare in children, including those who are immunocompromised. infection of the brain includes encephalitis, involvement of motor neurones leading to ptosis, paralysis of the hand, facial palsy, or contralateral hemiparesis. involvement of the oculomotor division of the trigeminal ganglion can cause serious eye problems, including corneal scarring. shingles is far less infectious than chicken pox and is only considered to be infectious up to days after lesions appear. shingles is only infectious after prolonged contact with lesions. unlike chickenpox, airborne transmission is not a risk. individuals who are immunocompromised may reactivate the dormant virus and develop shingles. people who have not had primary varicella are at risk of developing chickenpox after prolonged direct contact with shingles. despite popular belief, it is untrue that people who are immunocompetent who have had chicken pox develop shingles when in contact with either chicken pox or shingles. such occurrences are merely coincidental, unless immunity is lowered. staff with shingles should stay off work until the lesions are healed, unless they can be covered. staff who have had chickenpox are immune (including pregnant women) and are therefore not at risk. if they are nonimmune (usually accepted as those without a history of chicken pox), they should avoid prolonged contact with detainees with shingles. pregnant nonimmune women should avoid contact altogether. detainees with the disease may be kept in custody, and any exposed lesions should be covered. it is well documented that prompt treatment attenuates the severity of the disease, reduces the duration of viral shedding, hastens lesion healing, and reduces the severity and duration of pain. it also reduces the likelihood of developing postherpetic neuralgia ( ) . prompt treatment with famciclovir (e.g., mg three times a day for days) should be initiated if the onset is d ays or less. it should also be considered after this time if the detainee is over age years. pregnant detainees with shingles can be reassured that there is minimal risk for both the mother and the unborn child. expert advice should be given before initiating treatment for the mother. this tiny parasitic mite (sarcoptes scabiei) has infested humans for more than years. experts estimate that in excess of million cases occur worldwide each year. the female mite burrows into the skin, especially around the hands, feet, and male genitalia, in approx . min. eggs are laid and hatch into larvae that travel to the skin surface as newly developed mites. the mite causes intense itching, which is often worse at night and is aggravated by heat and moisture. the irritation spreads outside the original point of infection resulting from an allergic reaction to mite feces. this irritation may persist for approx weeks after treatment but can be alleviated by antihistamines. crusted scabies is a far more severe form of the disease. large areas of the body may be involved. the crusts hide thousands of live mites and eggs, making them difficult to treat. this so-called norwegian scabies is more common in the elderly or the immunocompromised, especially those with hiv. after a primary exposure, it takes approx - weeks before the onset of itching. however, further exposures reduce the incubation time to approx - days. without treatment, the period of infectivity is assumed to be indefinite. with treatment, the person should be considered infectious until the mites and eggs are destroyed, usually - days. crusted scabies is highly infectious. because transmission is through direct skin-to-skin contact with an infected individual, gloves should be worn when dealing with individuals suspected of infestation. usually prolonged contact is needed, unless the person has crusted scabies, where transmission occurs more easily. the risk of transmission is much greater in households were repeated or prolonged contact is likely. because mites can survive in bedding or clothing for up to hour, gloves should also be worn when handling these items. bedding should be treated using one of the methods in subheading . . professional cleaning of the cell is only warranted in cases of crusted scabies. the preferred treatment for scabies is either permethrin cream ( %) or aqueous malathion ( . %) ( ) . either treatment has to be applied to the whole body and should be left on for at least hours in the case of permethrin and hours for malathion before washing off. lindane is no longer considered the treatment of choice, because there may be complications in pregnancy ( ) . treatment in custody may not be practical but should be considered when the detainee is believed to have norwegian scabies. like scabies, head lice occur worldwide and are found in the hair close to the scalp. the eggs, or nits, cling to the hair and are difficult to remove, but they are not harmful. if you see nits, then you can be sure that lice are also present. the latter are best seen when the hair is wet. the lice bite the scalp and suck blood, causing intense irritation and itching. head lice can only be passed from direct hair-to-hair contact. it is only necessary to wear gloves when examining the head for whatever reason. the cell does not need to be cleaned after use, because the lice live on or near skin. bedding may be contaminated with shed skin, so should be handled with gloves and laundered or incinerated. the presence of live lice is an indication for treatment by either physical removal with a comb or the application of an insecticide. the latter may be more practical in custody. treatment using . % aqueous malathion should be applied to dry hair and washed off after hours. the hair should then be shampooed as normal. crabs or body lice are more commonly found in the pubic, axillary, chest, and leg hair. however, eyelashes and eyebrows may also be involved. they are associated with people who do not bath or change clothes regularly. the person usually complains of intense itching or irritation. the main route is from person to person by direct contact, but eggs can stick to fibers, so clothing and bedding should be handled with care (see subheading . . .). staff should always wear gloves if they are likely to come into contact with any hirsute body part. clothing or bedding should be handled with gloves and either laundered or incinerated. treatment of a detainee in custody is good in theory but probably impractical because the whole body has to be treated. fleas lay eggs on floors, carpets, and bedding. in the united kingdom, most flea bites come from cats or dogs. the eggs and larvae fleas can survive for months and are reactivated in response to animal or human activity. because animal fleas jump off humans after biting, most detainees with flea bites will not have fleas, unless they are human fleas. treatment is only necessary if fleas are seen. after use, the cell should be vacuumed and cleaned with a proprietary insecticide. any bedding should be removed wearing gloves, bagged, and either laundered or incinerated. bedbugs live and lay eggs on walls, floors, furniture, and bedding. if you look carefully, fecal tracks may be seen on hard surfaces. if they are present for long enough, they emit a distinct odor. bedbugs are rarely found on the person but may be brought in on clothing or other personal effects. bedbugs bite at night and can cause sleep disturbance. the detainee does not need to be treated, but the cell should deemed out of use until it can be vacuumed and professionally cleaned with an insecticide solution. any bedding or clothing should be handled with gloves and disposed of as appropriate. staphylococcus aureus is commonly carried on the skin or in the nose of healthy people. approximately - % of the population is colonized with the bacteria but remain well ( ) . from time to time, the bacteria cause minor skin infections that usually do not require antibiotic treatment. however, more serious problems can occur (e.g., infection of surgical wounds, drug injection sites, osteomyelitis, pneumonia, or septicemia). during the last years, the bacteria have become increasingly resistant to penicillin-based antibiotics ( ) , and in the last years, they have become resistant to an increasing number of alternative antibiotics. these multiresistant bacteria are known as methicillinresistant s. aureus (mrsa). mrsa is prevalent worldwide. like nonresistant staphylococci, it may remain undetected as a reservoir in colonized individuals but can also produce clinical disease. it is more common in individuals who are elderly, debilitated, or immunocompromised or those with open wounds. clusters of skin infections with mrsa have been reported among injecting drug users (idus) since in america ( , ) , and more recently, similar strains have been found in the united kingdom in idus in the community ( ) . this may have particular relevance for the forensic physician when dealing with idus sores. people who are immunocompetent rarely get mrsa and should not be considered at risk. the bacteria are usually spread via the hands of staff after contact with colonized or infected detainees or devices, items (e.g., bedding, towels, and soiled dressings), or environmental surfaces that have been contaminated with mrsa-containing body fluids. with either known or suspected cases (consider all abscesses/ulcers of idus as infectious), standard precautions should be applied. staff should wear gloves when touching mucous membranes, nonintact skin, blood or other body fluids, or any items that could be contaminated. they should also be encouraged to their wash hands with an antimicrobial agent regardless of whether gloves have been worn. after use, gloves should be disposed of in a yellow hazard bag and not allowed to touch surfaces. masks and gowns should only be worn when conducting procedures that generate aerosols of blood or other body fluids. because this is an unlikely scenario in the custodial setting, masks and gowns should not be necessary. gloves should be worn when handling bedding or clothing, and all items should be disposed of appropriately. any open wounds should be covered as soon as possible. the cell should be cleaned professionally after use if there is any risk that it has been contaminated. during the last decade, there has been an increasing awareness of the bacterial flora colonizing injection sites that may potentially lead to life-threatening infection ( ) . in , a sudden increase in needle abscesses caused by a clonal strain of group a streptococcus was reported among hospitalized idus in berne, switzerland ( ) . a recent uk study showed that the predominant isolate is s. aureus, with streptococcus species forming just under one-fifth ( % β-hemolytic streptococci) ( ) . there have also been reports of both nonsporing and sporing anerobes (e.g., bacteroides and clostridia species, including clostridia botulinum) ( , ) . in particular, in , laboratories in glasgow were reporting isolates of clostridium novyi among idus with serious unexplained illness. by june , , a total of cases ( definite and probable) had been reported. a definite case was defined as an idu with both severe local and systemic inflammatory reactions. a probable case was defined as an idu who presented to the hospital with an abscess or other significant inflammation at an injecting site and had either a severe inflammatory process at or around an injection site or a severe systemic reaction with multiorgan failure and a high white cell count ( ) . in the united kingdom, the presence of c. botulinum in infected injection sites is a relatively new phenomenon. until the end of , there were no cases reported to the public health leadership society. since then, the number has increased, with a total of cases in the united kingdom and ireland being reported since the beginning of . it is believed that these cases are associated with contaminated batches of heroin. simultaneous injection of cocaine increases the risk by encouraging anerobic conditions. anerobic flora in wounds may have serious consequences for the detainee, but the risk of transmission to staff is virtually nonexistent. staff should be reminded to wear gloves when coming into contact with detainees with infected skin sites exuding pus or serum and that any old dressings found in the cell should be disposed of into the yellow bag marked "clinical waste" in the medical room. likewise, any bedding should be bagged and laundered or incinerated after use. the cell should be deemed out of use and professionally cleaned after the detainee has gone. the health care professional managing the detainee should clean and dress open wounds as soon as possible to prevent the spread of infection. it may also be appropriate to start a course of antibiotics if there is abscess formation or signs of cellulites and/or the detainee is systemically unwell. however, infections can often be low grade because the skin, venous, and lymphatic systems have been damaged by repeated penetration of the skin. in these cases, signs include lymphedema, swollen lymph glands, and darkly pigmented skin over the area. fever may or may not be present, but septicemia is uncommon unless the individual is immunocompromised (e.g., hiv positive). co-amoxiclav is the preferred treatment of choice because it covers the majority of staphylococci, streptococci, and anerobes (the dose depends on the degree of infection). necrotizing fasciitis and septic thrombophlebitis are rare but life-threatening complications of intravenous drug use. any detainee suspected of either of these needs hospital treatment. advice about harm reduction should also be given. this includes encouraging drug users to smoke rather than inject or at least to advise them to avoid injecting into muscle or skin. although most idus are aware of the risk of sharing needles, they may not realize that sharing any drug paraphernalia could be hazardous. advice should be given to use the minimum amount of citric acid to dissolve the heroin because the acid can damage the tissue under the skin, allowing bacteria to flourish. drugs should be injected at different sites using fresh works for each injection. this is particularly important when "speedballing" because crack cocaine creates an anerobic environment. medical help should be requested if any injection site become painful and swollen or shows signs of pus collecting under the skin. because intravenous drug users are at increased risk of acquiring hbv and hav, they should be informed that vaccination against both diseases is advisable. another serious but relatively rare problem is the risk from broken needles in veins. embolization can take anywhere from hours to days or even longer if it is not removed. complications may include endocarditis, pericarditis, or pulmonary abscesses ( , ) . idus should be advised to seek medical help as soon as possible, and should such a case present in custody, then send the detainee straight to the hospital. the forensic physician may encounter bites in the following four circumstances: a detailed forensic examination of bites is given in chapter . with any bite that has penetrated the skin, the goals of therapy are to minimize soft tissue deformity and to prevent or treat infection. in the united kingdom and the united states, dog bites represent approximately three-quarters of all bites presenting to accident and emergency departments ( ) . a single dog bite can produce up to psi of crush force in addition to the torsional forces as the dog shakes its head. this can result in massive tissue damage. human bites may cause classical bites or puncture wounds (e.g., impact of fists on teeth) resulting in crush injuries. an estimated - % of dog bites and - % of human bites lead to infection. compare this with an estimated - % of nonbite wounds managed in accident and emergency departments. the risk of infection is increased with puncture wounds, hand injuries, full-thickness wounds, wounds requiring debridement, and those involving joints, tendons, ligaments or fractures. comorbid medical conditions, such as diabetes, asplenia, chronic edema of the area, liver dysfunction, the presence of a prosthetic valve or joint, and an immunocompromised state may also increase the risk of infection. infection may spread beyond the initial site, leading to septic arthritis, osteomyelitis, endocarditis, peritonitis, septicemia, and meningitis. inflammation of the tendons or synovial lining of joints may also occur. if enough force is used, bones may be fractured or the wounds may be permanently disfiguring. assessment regarding whether hospital treatment is necessary should be made as soon as possible. always refer if the wound is bleeding heavily or fails to stop when pressure is applied. penetrating bites involving arteries, nerves, muscles, tendons, the hands, or feet, resulting in a moderate to serious facial wound, or crush injuries, also require immediate referral. if management within custody is appropriate, ask about current tetanus vaccine status, hbv vaccination status, and known allergies to antibiotics. wounds that have breached the skin should be irrigated with . % (isotonic) sodium chloride or ringer's lactate solution instead of antiseptics, because the latter may delay wound healing. a full forensic documentation of the bite should be made as detailed in chapter . note if there are clinical signs of infection, such as erythema, edema, cellulitis, purulent discharge, or regional lymphadenopathy. cover the wound with a sterile, nonadhesive dressing. wound closure is not generally recommended because data suggest that it may increase the risk of infection. this is particularly relevant for nonfacial wounds, deep puncture wounds, bites to the hand, clinically infected wounds, and wounds occurring more than - hours before presentation. head and neck wounds in cosmetically important areas may be closed if less than hours old and not obviously infected. • dog bites-pasteurella canis, pasteurella multocida, s. aureus, other staphylococci, streptococcus species, eikenella corrodens, corynebacterium species, and anerobes, including bacteroides fragilis and clostridium tetani • human bites-streptococcus species, s. aureus, e. corrodens, and anerobes, including bacteroides (often penicillin resistant), peptostreptococci species, and c. tetani. tuberculosis (tb) and syphilis may also be transmitted. • dog bites-outside of the united kingdom, australia, and new zealand, rabies should be considered. in the united states, domestic dogs are mostly vaccinated against rabies ( ) , and police dogs have to be vaccinated, so the most common source is from racoons, skunks, and bats. • human bites-hbv, hbc, hiv, and herpes simplex. antibiotics are not generally needed if the wound is more than days old and there is no sign of infection or in superficial noninfected wounds evaluated early that can be left open to heal by secondary intention in compliant people with no significant comorbidity ( ) . antibiotics should be considered with high-risk wounds that involve the hands, feet, face, tendons, ligaments, joints, or suspected fractures or for any penetrating bite injury in a person with diabetes, asplenia, or cirrhosis or who is immunosuppressed. coamoxiclav (amoxycillin and clavulanic acid) is the first-line treatment for mild-moderate dog or human bites resulting in infections managed in primary care. for adults, the recommended dose is / mg three times daily and for children the recommended does is mg/kg three times daily (based on amoxycillin component). treatment should be continued for - days. it is also the first-line drug for prophylaxis when the same dose regimen should be prescribed for - days. if the individual is known or suspected to be allergic to penicillin, a tetracycline (e.g., doxycycline mg twice daily) and metronidazole ( mg three times daily) or an aminoglycoside (e.g., erythromycin) and metronidazole can be used. in the united kingdom, doxycycline use is restricted to those older than years and in the united states to those older than years old. specialist advice should be sought for pregnant women. anyone with severe infection or who is clinically unwell should be referred to the hospital. tetanus vaccine should be given if the primary course or last booster was more than years ago. human tetanus immunoglobulin should be considered for tetanus-prone wounds (e.g., soil contamination, puncture wounds, or signs of devitalized tissue) or for wounds sustained more than hours old. if the person has never been immunized or is unsure of his or her tetanus status, a full three-dose course, spaced at least month apart, should be given. penetrating bite wounds that involve only saliva may present a risk of hbv if the perpetrator belongs to a high-risk group. for management, see subheadings . . . and . . . hcv and hiv are only a risk if blood is involved. the relevant management is dealt with in subheadings . . . and . . . respiratory tract infections are common, usually mild, and self-limiting, although they may require symptomatic treatment with paracetamol or a nonsteroidal antiinflammatory. these include the common cold ( % rhinoviruses and % coronaviruses), adenoviruses, influenza, parainfluenza, and, during the summer and early autumn, enteroviruses. special attention should be given to detainees with asthma or the who are immunocompromised, because infection in these people may be more serious particularly if the lower respiratory tract is involved. the following section includes respiratory pathogens of special note because they may pose a risk to both the detainee and/or staff who come into close contact. there are five serogroups of neisseria meningitidis: a, b, c, w , and y. the prevalence of the different types varies from country to country. there is currently no available vaccine against type b, but three other vaccines (a+c, c, and acwy) are available. overall, % of the uk population carry n. meningitidis ( % in the - age group) ( ) . in the united kingdom, most cases of meningitis are sporadic, with less than % occurring as clusters (outbreaks) amongst school children. between and , % of cases were group b, % were group c, and w and a accounted for %. there is a seasonal variation, with a high level of cases in winter and a low level in the summer. the greatest risk group are the under year olds, with a peak incidence under year old. a secondary peak occurs in the -to -year-old age group. in sub-saharan africa, the disease is more prevalent in the dry season, but in many countries, there is background endemicity year-round. the most prevalent serogroup is a. routine vaccination against group c was introduced in the united kingdom november for everybody up to the age of years old and to all firstyear university students. this has since been extended to include everyone under the age of years old. as a result of the introduction of the vaccination program, there has been a % reduction of group c cases in those younger than under years and an % reduction in those under year old ( , ) . an outbreak of serogroup w meningitis occurred among pilgrims on the hajj in . cases were reported from many countries, including the united kingdom. in the united kingdom, there is now an official requirement to be vaccinated with the quadrivalent vaccine (acwy vax) before going on a pilgrimage (hajj or umra), but illegal immigrants who have not been vaccinated may enter the country ( ). after an incubation period of - days ( , ) , disease onset may be either insidious with mild prodromal symptoms or florid. early symptoms and signs include malaise, fever, and vomiting. sever headache, neck stiffness, photophobia, drowsiness, and a rash may develop. the rash may be petechial or purpuric and characteristically does not blanche under pressure. meningitis in infants is more likely to be insidious in onset and lack the classical signs. in approx - % of cases, septicemia is the predominant feature. even with prompt antibiotic treatment, the case fatality rate is - % in meningitis and - % in those with septicemia. ( ). a person should be considered infectious until the bacteria are no longer present in nasal discharge. with treatment, this is usually approx hour. the disease is spread through infected droplets or direct contact from carriers or those who are clinically ill. it requires prolonged and close contact, so it is a greater risk for people who share accommodation and utensils and kiss. it must also be remembered that unprotected mouth-to-mouth resuscitation can also transmit disease. it is not possible to tell if a detainee is a carrier. nevertheless, the risk of acquiring infection even from an infected and sick individual is low, unless the individual has carried out mouth-to-mouth resuscitation. any staff member who believes he or she has been placed at risk should report to the occupational health department (or equivalent) or the nearest emergency department at the earliest opportunity for vaccination. if the detainee has performed mouth-to-mouth resuscitation, prophylactic antibiotics should be given before receiving vaccination. rifampicin, ciprofloxacin, and ceftriaxone can be used; however, ciprofloxacin has numerous advantages ( ) . only a single dose of mg (adults and children older than years) is needed and has fewer side effects and contraindications than rifampicin. ceftriaxone has to be given by injection and is therefore best avoided in the custodial setting. if the staff member is pregnant, advice should be sought from a consultant obstetrician, because ciprofloxacin is not recommended ( ) . for anyone dealing regularly with illegal immigrants (especially from the middle east or sub-saharan africa) (e.g., immigration services, custody staff at designated stations, medical personnel, and interpreters), should consider being vaccinated with acwy vax. a single injection provides protection for years. detainees suspected of disease should be sent directly to the hospital. human tb is caused by infection with mycobacterium tuberculosis, mycobacterium bovis, or mycobacterium africanum. it is a notifiable disease under legislation specific to individual countries; for example, in the united kingdom, this comes under the public health (control of disease) act of . in , the who declared tb to be a global emergency, with an estimated - million new cases and million deaths occurring each year, the majority of which were in asia and africa. however, these statistics are likely to be an underestimate because they depend on the accuracy of reporting, and in poorer countries, the surveillance systems are often inadequate because of lack of funds. even in the united kingdom, there has been an inconsistency of reporting particularly where an individual has concomitant infection with hiv. some physicians found themselves caught in a dilemma of confidentiality until , when the codes of practice were updated to encourage reporting with patient consent ( ) . with the advent of rapid identification tests and treatment and the use of bacillus calmette-guérin (bcg) vaccination for prevention, tb declined during the first half of the th century in the united kingdom. however, since the early s, numbers have slowly increased, with some cases reported in ( ) . in , % of reported cases were from people born outside the united kingdom and % were associated with hiv infection ( , ) . london has been identified as an area with a significant problem. this has been attributed to its highly mobile population, the variety of ethnic groups, a high prevalence of hiv, and the emergence of drug-resistant strains ( . % in ) (phls, unpublished data-mycobnet). a similar picture was initially found in the united states, when there was a reversal of a long-standing downward trend in . however, between and , the number of cases increased from , to , ( ) . there were also serious outbreaks of multidrug-resistant tb (mdr-tb) in hospitals in new york city and miami ( ) . factors pertinent to the overall upswing included the emergence of hiv, the increasing numbers of immigrants from countries with a high prevalence of tb, and perhaps more significantly, stopping categorical federal funding for control activities in . the latter led to a failure of the public health infrastructure for tb control. since , the trend has reversed as the cdc transferred most of its funds to tb surveillance and treatment program in states and large cities. from to , the annual decline averaged by . % ( ) , but the following year this was reduced to %, indicating that there was no room for complacency. the who has been proactive and is redirecting funding to those countries most in need. in october , a global partnership called stop tb was launched to coordinate every aspect of tb control, and by , the partnership had more than member states. a target was set to detect at least % of infectious cases by . the acquisition of tb infection is not necessarily followed by disease because the infection may heal spontaneously. it may take weeks or months before disease becomes apparent, or infection may remain dormant for years before reactivation in later life especially if the person becomes debilitated or immunocompromised. contrary to popular belief, the majority of cases of tb in people who are immunocompetent pass unnoticed. of the reported cases, % involve the lung, whereas nonrespiratory (e.g., bone, heart, kidney, and brain) or dissemination (miliary tb) are more common in immigrant ethnic groups and individuals who are immunocompromised ( ) . they are also more likely to develop resistant strains. in the general population, there is an estimated % lifetime risk of tb infection progressing to disease ( ) . there has been an increase in the number of cases of tb associated with hiv owing to either new infection or reactivation. tb infection is more likely to progress to active tb in hiv-positive individuals, with a greater than % lifetime risk ( ) . tb can also lead to a worsening of hiv with an increase in viral load ( ) . therefore, the need for early diagnosis is paramount, but it can be more difficult because pulmonary tb may present with nonspecific features (e.g., bilateral, unilateral, or lower lobe shadowing) ( ). after an incubation period of - weeks, symptoms may develop (see table ). the main route is airborne through infected droplets, but prolonged or close contact is needed. nonrespiratory disease is not considered a risk unless the mycobacterium is aerosolized under exceptional circumstances (e.g., during surgery) or there are open abscesses. a person is considered infectious as long as viable bacilli are found in induced sputum. untreated or incompletely treated people may be intermittently sputum positive for years. after weeks of appropriate treatment, the individual is usually considered as noninfectious. this period is often extended for treatment of mdr-tb or for those with concomitant hiv. patient compliance also plays an important factor. the risk of infection is directly proportional to the degree of exposure. more severe disease occurs in individuals who are malnourished, immunocompromised (e.g., hiv), and substance misusers. people who are immunocompromised are at special risk of mdr-tb or mycobacterium avium intracellulare (mai). staff with disease should stay off work until the treatment course is complete and serial sputum samples no longer contain bacilli. staff in contact with disease who have been vaccinated with bcg are at low risk of acquiring disease but should minimize their time spent in the cell. those who have not received bcg or who are immunocompromised should avoid contact with the detainee wherever possible. detainees with mai do not pose a risk to a staff member, unless the latter is immunocompromised. any staff member who is pregnant, regardless of bcg status or type of tb, should avoid contact. anyone performing mouth-to-mouth resuscitation with a person with untreated or suspected pulmonary tb should be regarded as a household contact and should report to occupational health or their physician if no other route exists. they should also be educated regarding the symptoms of tb. anyone who is likely to come into repeated contact with individuals at risk of tb should receive bcg (if he or she has not already done so), regardless of age, even though there is evidence to suggest that bcg administered in adult life is less effective. this does not apply to individuals who are immunocompromised or pregnant women. in the latter case, vaccination should preferably be deferred until after delivery. detainees with disease (whether suspected or diagnosed) who have not been treated or treatment is incomplete should be kept in custody for the minimum time possible. individuals with tb who are immunocompromised are usually too ill to be detained; if they are, they should be considered at greater risk of transmitting disease to staff. any detainee with disease should be encouraged to cover his or her mouth and nose when coughing and sneezing. staff should wear gloves when in contact with the detainee and when handling clothing and bedding. any bedding should be bagged after use and laundered or incinerated. the cell should be deemed out of action until it has been ventilated and professionally decontaminated, although there is no hard evidence to support that there is a risk of transmission from this route ( ). on march , , the who issued a global warning to health authorities about a new atypical pneumonia called sars. the earliest case was believed to have originated in the guandong province of china on november , . the causative agent was identified as a new corona virus-sars-cov ( , ) . by the end of june , cases had been reported from different countries, with a total of deaths. approximately % of cases occurred in china (including hong kong, taiwan, and macao). the case fatality rate varied from less than % in people younger than years, % in persons aged - years, % in those aged - years, and more than % in persons years or older. on july , , the who reported that the last human chain of transmission of sars had been broken and lifted the ban from all countries. however, it warned that everyone should remain vigilant, because a resurgence of sars is possible. their warning was well given because in december , a new case of sars was detected in china. at the time of this writing, three more cases have been identified. knowledge about the epidemiology and ecology of sars-cov and the disease remains limited; however, the experience gained from the previous outbreak enabled the disease to be contained rapidly, which is reflected in the few cases reported since december . there is still no specific treatment or preventative vaccine that has been developed. the incubation period is short, approx - days (maximum days), and, despite the media frenzy surrounding the initial outbreak, sars is less infectious than influenza. the following clinical case definition of sars has been developed for public health purposes ( ) . a person with a history of any combination of the following should be examined for sars: • fever (at least °c); and • one of more symptoms of lower respiratory tract illness (cough, difficulty in breathing, or dyspnea); and • radiographic evidence of lung infiltrates consistent with pneumonia or respiratory distress syndrome or postmortem findings of these with no identifiable cause; and • no alternative diagnosis can fully explain the illness. laboratory tests have been developed that include detection of viral rna by pcr from nasopharyngeal secretions or stool samples, detection of antibodies by enzyme-linked immunosorbent assay or immunofluorescent antibody in the blood, and viral culture from clinical specimens. available information suggests that close contact via aerosol or infected droplets from an infected individual provide the highest risk of acquiring the disease. most cases occurred in hospital workers caring for an index case or his or her close family members. despite the re-emergence of sars, it is highly unlikely that a case will be encountered in the custodial setting in the near future. however, forensic physicians must remain alert for the sars symptoms and keep up-to-date with recent outbreaks. information can be obtained from the who on a daily basis from its web site. if sars is suspected, medical staff should wear gloves and a surgical mask when examining a suspected case; however, masks are not usually available in custody. anyone suspected of sars must be sent immediately to the hospital, and staff who have had prolonged close contact should be alerted as to the potential symptoms. the most consistent feature of diseases transmitted through the fecaloral route is diarrhea (see table ). infective agents include bacteria, viruses, and protozoa. because the causes are numerous, it is beyond the remit of this chapter to cover them all. it is safest to treat all diarrhea as infectious, unless the detainee has a proven noninfectious cause (e.g., crohn's disease or ulcerative colitis). all staff should wear gloves when in contact with the detainee or when handling clothing and bedding, and contaminated articles should be laundered or incinerated. the cell should be professionally cleaned after use, paying particular attention to the toilet area. this viral hepatitis occurs worldwide, with variable prevalence. it is highest in countries where hygiene is poor and infection occurs year-round. in temperate climates, the peak incidence is in autumn and winter, but the trend is becoming less marked. all age groups are susceptible if they are nonimmune or have not been vaccinated. in developing countries, the disease occurs in early childhood, whereas the reverse is true in countries where the standard of living is higher. in the united kingdom, there has been a gradual decrease in the number of reported cases from to ( , ) . this results from, in part, improved standards of living and the introduction of an effective vaccine. the highest incidence occurs in the -to -year-old age group. approximately % of people older than years have natural immunity, leaving the remainder susceptible to infection ( ) . small clusters occur from time to time, associated with a breakdown in hygiene. there is also an increasing incidence of hav in gay or bisexual men and their partners ( ) . an unpublished study in london in showed a seroprevalence of % among gay men (young y et al., unpublished). the clinical picture ranges from asymptomatic infection through a spectrum to fulminant hepatitis. unlike hbv and hcv, hav does not persist or progress to chronic liver damage. infection in childhood is often mild or asymptomatic but in adults tends to be more severe. after an incubation period of - days (mean days) symptomatic infection starts with the abrupt onset of jaundice anything from days to weeks after the anicteric phase. it lasts for approximately the same length of time and is often accompanied by a sudden onset of fever. hav infection can lead to hospital admission in all age groups but is more likely with increasing age as is the duration of stay. the overall mortality is less than %, but % of people will have a prolonged or relapsing illness within - months (cdc fact sheet). fulminant hepatitis occurs in less than % of people but is more likely to occur in individuals older than years or in those with pre-existing liver disease. in patients who are hospitalized, case fatality ranges from % in - years olds to nearly % in those older than years ( ). the individual is most infectious in the weeks before the onset of jaundice, when he or she is asymptomatic. this can make control of infection difficult because the disease is not recognized. the main route is fecal-oral through the ingestion of contaminated water and food. it can also be transmitted by personal contact, including homosexuals practicing anal intercourse and fellatio. there is a slight risk from blood transfusions if the donor is in the acute phase of infection. it should not be considered a risk from needlestick injuries unless clinical suspicion of hav is high. risk groups include homeless individuals, homosexuals, idus, travellers abroad who have not been vaccinated, patients with chronic liver disease and chronic infection with hbv and hcv, employees and residents in daycare centers and hostels, sewage workers, laboratory technicians, and those handling nonhuman primates. several large outbreaks have occurred among idus, some with an epidemiological link to prisons ( , ) . transmission occurs during the viremic phase of the illness through sharing injecting equipment and via fecal-oral routes because of poor living conditions ( ) . there have also been reports of hav being transmitted through drugs that have been carried in the rectum. a study in vancouver showed that % of idus had past infection of hav, and they also showed an increased prevalence among homosexual/bisuexual men ( ). staff with disease should report to occupational health and stay off work until the end of the infective period. those in contact with disease (either through exposure at home or from an infected detainee) should receive prophylactic treatment as soon as possible (see subheading . . .). to minimize the risk of acquiring disease in custody, staff should wear gloves when dealing with the detainee and then wash their hands thoroughly. gloves should be disposed of only in the clinical waste bags. detainees with disease should be kept in custody for the minimum time possible. they should only be sent to the hospital if fulminant hepatitis is suspected. the cell should be quarantined after use and professionally cleaned. any bedding or clothing should be handled with gloves and laundered or incinerated according to local policy. detainees reporting contact with disease should be given prophylactic treatment as soon as possible (see subheading . . .). contacts of hav should receive hav vaccine (e.g., havrix monodose or avaxim) if they have not been previously immunized or had disease. human normal immunoglobulin (hnig), mg, deep intramuscular in gluteal muscle should be used in the following circumstances: • has the detainee traveled to africa, south east asia, the indian subcontinent, central/south america, or the far east in the last - months? • ascertain whether he or she received any vaccinations before travel and, if so, which ones. • ask if he or she took malaria prophylaxis, what type, and whether he or she completed the course. • ask if he or she swam in any stagnant lakes during the trip. • if the answer to any of the above is yes, ask if he or she has experienced any of the following symptoms: a fever/hot or cold flushes/shivering. diarrhea ± abdominal cramps ± blood or slime in the stool. a rash. persistent headaches ± light sensitivity. nausea or vomiting. aching muscles/joints. a persistent cough (dry or productive) lasting at least weeks. • take temperature. • check skin for signs of a rash and note nature and distribution. • check throat. • listen carefully to the lungs for signs of infection/consolidation. staff at higher risk of coming in to contact with hav should consider being vaccinated before exposure. two doses of vaccine given - months apart give at least years of protection. there is no specific treatment for hav, except supportive measures and symptomatic treatment. although the chance of encountering a tropical disease in custo dy is small, it is worth bearing in mind. it is not necessary for a forensic physician to be able to diagnose the specific disease but simply to recognize that the detainee/staff member is ill and whether he or she needs to be sent to the hospital (see tables - ) . this is best achieved by knowing the right questions to ask and carrying out the appropriate examination. tables - should be used as an aide to not missing some more unusual diseases. guidance for clinical health care workers: protection against infection with blood-borne viruses; recommendations of the expert advisory group on aids and the advisory group on hepatitis guidelines for hand hygiene in health care settings. recommendations of the healthcare infection control practices advisory committee and the hicpac/ shea/apic/idsa hand hygiene task force national model regulations for the control of workplace hazardous substances. commonwealth of australia, national occupational health and safety committee good practice guidelines for forensic medical examiners and the hospital infection control practices advisory committee. guideline for infection control in health care personnel report from the unlinked anonymous surveys steering group. department of health a strategy for infectious diseases-progress report. blood-borne and sexually transmitted viruses: hepatitis. department of health universal precautions for prevention of transmission of human immuno-deficiency virus, hepatitis b 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of acyclovir for varicella pneumonia during pregnancy outcome after maternal varicella infection in the first weeks of pregnancy outcome in newborn babies given anti-varicella zoster immunoglobulin after perinatal maternal infection with varicella zoster virus varicella-zoster virus dna in human sensory ganglia epidemiology and natural history of herpes zoster and post herpetic neuralgia clinical applications for changepoint analysis of herpes zoster pain treatment of scabies with permethrin versus lindane and benzoyl benzoate treatment of ectoparasitic infections; review of the english-language literature nasal carriage of staphylococcus aureus: epidemiology and control measures centers for disease control and prevention. community-acquired methicillinresistant staphylococcus aureus infections-michigan methicillinresistant staphylococcus aureus, epidmiologic observations during a community acquired outbreak emergence of pvl-producing strains of staphylococcus aureus bacteriology of 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severe acute respiratory syndrome epidemiological determinants of spread of causal agents of severe acute respiratory syndrome in hong kong alert, verification and public health management of sars in post-outbreak period age-specific antibody prevalence to hepatitis a in england: implications for disease control phls advisory committee on vaccination and immunisation. guidelines for the control of hepatitis a infection control of a community hepatitis a outbreak using hepatitis a vaccine seroprevalence of and risk factors for hepatitis a infection among young homosexual and bisexual men outbreaks of hepatitis a among illicit drug users identifying target groups for a potential vaccination program during a hepatitis a community outbreak multiple modes of hepatitis a transmission among metamphetamine users past infection with hepatitis a among vancouver street youth, injection drug users and men who have sex with men implications for vaccination programmes key: cord- -lvb oxo authors: efthimiou, petros; yadlapati, sujani title: adult-onset still’s disease date: - - journal: auto-inflammatory syndromes doi: . / - - - - _ sha: doc_id: cord_uid: lvb oxo adult-onset still’s disease (aosd) is a rare systemic, autoinflammatory disorder that often presents in adolescence and early adulthood with fever, rash, and polyarthritis. there are significant genetic and clinical similarities with systemic juvenile idiopathic arthritis (sjia) with a different chronological disease onset. the disease can have many protean characteristics leading to delays in diagnosis. treatment includes corticosteroids; traditional immunomodulators, such as methotrexate; and targeted biologic treatments that include il- and il- inhibitors. aosd remains a diagnosis of exclusion, since more common illnesses can present with very similar symptoms. definitive diagnosis should only be made after ruling out infectious, malignant, and other autoimmune and autoinflammatory diseases. yamaguchi criteria are often used to aid in diagnosis and, especially, classification [ , ] . patients frequently have a favorable prognosis with timely diagnosis, unless life-threatening complications such as macrophage activation syndrome (mas) ensue. prevalence of aosd is estimated to be less than one case per , people. aosd is rare, and hence there are currently no consensus on its incidence and prevalence in different populations. based on larger reviews from the s, it appears that aosd occurs worldwide and may affect slightly more women than men. this disease characteristically affects younger people between the age of and years of age [ , ] . a bimodal peak at ages - and - without a sex bias has been described in a retrospective study of patients from western france [ ] . however, an epidemiological survey from japan described that % of the cases presented after the age of , the majority ( - %) being women [ ] . aosd affects all ages, and stress has been suggested as an important risk factor for all ages [ ] . no familial trend has been reported in the recent literature; however, an association with certain hla alleles has been observed in certain populations. these hla subtypes include hla dr , b , b , b , dr , dr , and dq . pathophysiology of aosd was largely obscure until the recent past. a myriad of factors such as genetics, infectious (bacterial and viral) agents, and environmental factors have been thought to play a causative role. concurrent elevations of serologic markers of infectious agents have been noted in some patients with still's disease. these infectious triggers include ebv; parvovirus b ; cmv; hhv; hiv; coxsackievirus; mumps; rubella; echovirus; hepatitis a, b, and c viruses; campylobacter jejuni; chlamydia pneumoniae; adenovirus; influenza virus; parainfluenza virus; and mycoplasma [ , ] . however, to date definite insight in to precise role of infection in aosd is lacking. several associations with distinct hla alleles and aosd have been described thus far. pouchot et al. described a strong association between hla-b , b , b , and dr and aosd [ ] . in a small study of aosd patients, hla-bw was associated with disease susceptibility conferring a favorable prognosis [ ] . wouters et al. noted an increased frequency of the hla-dr allele in patients with aosd compared to normal controls, with the presence of hla-drw being linked to root joint involvement [ ] . an association between a chronic articular form of aosd and hla-drb * (dr ), drb * (dr ), and dqb * (dq ) was previously reported, while hla-dqb * (dq ) have been also associated with the systemic form of the disease in japanese population [ ] . statistics from a korean report supported an association between hla-drb * and drb * and aosd, while hla-drb * seemed to be protective. conversely, hla-drb * alleles were more frequently present in patients with the monocyclic systemic type of aosd [ ] . hallmark of aosd involves neutrophil and macrophage activation triggered by the pro-inflammatory cytokine il- . neutrophil (pmn) cd , a marker of neutrophil activation, has been found to be upregulated in patients with active aosd. calprotectin (calcium-binding protein) secreted by neutrophils and macrophages and macrophage migration inhibitory factor (mif) have been found to be useful markers of disease activity [ ] . intercellular adhesion molecule (icam- ) upregulated by il- has been implicated as a useful clinical marker whose expression typically reflects the level of disease activity. macrophage colony-stimulating factor, a cytokine which orchestrates proliferation and differentiation of macrophages, also appears to play a role in aosd. more recently, regulation of cytokine production has been noted in patients with aosd. a predominance of th subset of cytokines has been seen in peripheral blood and tissues of active untreated aosd patients. th immune cascade is characterized by elevated secretion of interferon γ (ifnγ), interleukin- (il- ), and tumor necrosis factor α (tnfα) cytokines that direct b cells toward igg a production, activate macrophages and natural killer (nk) cells, and promote cell-mediated immunity [ ] . when compared with controls, serum levels of il , tnfα, and ifnγ were significantly increased in patients with active aosd [ ] . il is a pro-inflammatory cytokine that is overproduced in the acute phase of aosd and is believed to be the cytokine initiating the inflammatory cascade that includes ifnγ, il , and tnfα [ ] . genetic polymorphisms of the human il gene have been described to confer disease susceptibility in a japanese study [ ] . conversely, in another japanese study, serum levels of soluble il receptors, il , and il correlated with chronic articular aosd activity, whereas ifnγ and il levels were found to be persistently raised, even in disease remission. understanding of the still's disease was also enhanced by the description of autoinflammatory syndromes. these disorders are associated with recurrent bouts of inflammation without an instigating antigenic stimulus. defective interleukin- processing, regulation of nuclear factor-b transcription factor, and possible uncharacteristic apoptosis are all anticipated mechanisms that may possibly play a role in the generation and perseverance of an inflammatory cascade. patients with autoinflammatory syndromes, in particular, the typical hereditary periodic fever syndromes, may share certain genetic traits; mefv gene mutation associated with familial mediterranean fever (fmf) and il- hypersecretion was seen with augmented frequency in turkish children with sjia. mutation of perforin and the munc - genes have been seen in patients with macrophage activation syndrome (mas), a known severe, life-threatening complication of aosd [ ] . mutations in genes encoding the tumor necrosis factor (tnf) receptor and pyrin superfamilies of molecules may result in the endurance of leukocytes that would customarily go through apoptosis [ ] . as a result, relatively minor pro-inflammatory triggers may lead to an exaggerated and potentially harmful, inflammatory response. il- b, the pivotal cytokine in aosd and other autoinflammatory syndromes, activates the thermoregulatory center, resulting in fever; may activate il- receptors on the endothelium, resulting in rash; and can also act on the bone marrow to increase mobilization of granulocyte progenitors and mature neutrophils, resulting in peripheral neutrophilia. il- also causes an increase in platelet production, which results in thrombocytosis, and decreases the response to erythropoietin, causing anemia. il- induces the production of il- . circulating il- stimulates the hepatocytes to synthesize several acute-phase proteins, such as crp, ferritin, and d-dimer. in general, three types of aosd have been described. the monocyclic pattern, which is the most benign form, is characterized by a single episode of aosd without recurrence. in the polycyclic pattern, the patient experiences recurrent attacks, although the subsequent aosd attacks often seem not as severe as the first one. in both the monocyclic and the polycyclic forms, systemic symptoms (rash, fever) are very prominent. the worst prognosis is carried by the chronic articular form, which is thought to be an unfortunate evolution of the polycyclic form. often, the systemic symptoms are absent or so remotely in the past that the patient may not even remember them and the main morbidity is from a chronic articular polyarthritis that mimics ra. common findings of aosd are fever, arthralgia, rash, and sore throat. other accompanying symptoms include, but are not limited to, myalgia, pharyngitis, lymphadenopathy, splenomegaly, and serositis. fever is usually quotidian and often precedes other manifestations. temperature spikes of > °c frequently occur and are associated with chills and rigors, joint pain, or rash. aosd is one of the main causes of pyrexia of unknown origin (puo). temperature fluctuations can be dramatic. fever may persist between spikes in approximately % of cases, and complete defervescence is not always a characteristic of the quotidian fevers [ ] . high-grade temperatures, more than . ° c, can be associated more strongly with monophasic pattern of aosd [ ] . skin rash associated with aosd is a salmon-pink colored, maculopapular eruption that tends to accompany or, more frequently, be exacerbated by fever. rash usually presents centrally at the trunk and the adjacent extremities (arms/thighs). histopathology of the rash often reveals non-specific findings including dermal edema and mild perivascular inflammation in the superficial dermis with lymphocytes and histiocytes. complement deposits (c ) have been described with immunofluorescence. arthralgias and myalgias are common manifestations of aosd. most commonly involved joints include the wrists, ankles, knees, elbows, proximal interphalangeal joints, and shoulders. these manifestations can evolve into more severe and potentially destructive polyarthritis that can mimic other systemic inflammatory arthritides, such as rheumatoid arthritis [ ] . myalgia can be debilitating and often associated with fever spikes. the muscle involvement, when severe, may be accompanied by an elevation of serum creatinine kinase and aldolase concentrations. however, muscle biopsy and electromyographic (emg) studies are typically normal. sterile pharyngitis manifesting as throat pain can occasionally precede the development of fever or rash by weeks, or even months, as a prodromal symptom and can often reoccur with disease relapses. hepatomegaly and modest elevation of serum hepatic aminotransferases and alkaline phosphatase are not uncommon in patients with aosd. several cases of fulminant liver failure in association with aosd have been described and may be associated with overexpression of il- [ ] . myocarditis, pericarditis, and pleural effusions have also been observed in aosd patients, and they seem to respond to anti-inflammatory treatment. uncommonly, some patients may develop severe interstitial lung disease and some progress to acute respiratory distress syndrome (ards). enlarged, symmetrical, cervical nodes are seen in about one half of patients with aosd. lymphadenopathy is often accompanied by fever, leukocytosis creating diagnostic confusion with lymphoma. lymph node biopsy typically shows intense, paracortical immunoblastic hyperplasia [ ] . splenomegaly is also seen in up to one third of patients. macrophage activation syndrome (mas) or reactive hemophagocytic syndrome (rhs) is a life-threatening complication of aosd. mortality rate ranges between % and % [ ] [ ] [ ] [ ] , and an incidence of - % has been noted in two recent series, a rate higher than other rheumatic diseases [ , ] . it is categorized by an uncontrollable activation of the reticuloendothelial system within the bone marrow, reticuloendothelial system, and central nervous system, with successive phagocytosis of hematopoietic cells by tissue macrophages (histiocytes) [ , ] . patients developing mas present with acute high fever, lymphadenopathy, and hepatosplenomegaly. laboratory findings include pancytopenia, elevated ferritin levels, triglycerides, and liver enzymes, often accompanied by normal erythrocyte sedimentation rate (esr). the most commonly implicated triggers include infections, medications, and disease flares [ ] [ ] [ ] . patients with mas have a decreased ability to eliminate antigen stimulation, thereby inducing t cell activation and proliferation resulting in cytokine secretion (interferon-gamma and granulocyte macrophage colony-stimulating factor) and macrophage hyperactivation. the end result is an uncontrollable increase in cytokines, specifically tnfα, interleukin- , and interleukin- production resulting in severe systemic inflammatory reaction, i.e., "cytokine storm" [ ] . there is also a suggestion that certain therapeutic agents, such as nonsteroidal anti-inflammatory drugs, methotrexate, sulfasalazine, penicillamine, and lately tnf-α, il- , and il- inhibitors may be capable of provoking mas, often complicating their therapeutic use [ ] . in theory, these therapies may create a state of immunodeficiency resulting in the reactivation of latent viruses (epstein-barr virus or cytomegalovirus) which in turn can stimulate mas. the counter-argument would be that anti-inflammatory medications may not be able to prevent the development of mas, at least in the dosages used in aosd or sjia. early suspicion of mas is most commonly raised by the detection of subtle laboratory changes, whereas clinical symptoms may be delayed. a recent international effort to identify candidate markers using an expert consensus process identified nine criteria that included a falling platelet count, hyperferritinemia, evidence of macrophage hemophagocytosis in the bone marrow, increased liver enzymes, falling leukocyte count, a persistent continuous fever ≥ °c, a falling erythrocyte sedimentation rate (esr), hypofibrinogenemia, and hypertriglyceridemia [ ] . hemophagocytosis, seen in bone marrow aspiration and biopsy, establishes the diagnosis, even though hemophagocytosis could be seen more frequently in biopsies from the liver, spleen, and/or lymph nodes. bone marrow aspiration is considered the gold standard and is usually required in atypical cases, causing a diagnostic dilemma. there is significant overlap between aosd and mas, and these two conditions are often thought to be anchoring the same disease spectrum, with aosd representing the milder form. aosd, unlike other systemic rheumatic diseases driven by adaptive immunity, is not typically associated with rheumatoid factor (rf) or antinuclear antibody (ana) positivity, although several cases have been published with low-level positivity of these autoantibodies. this has been considered in various sets of classification criteria. elevated esr is a common finding in most patients [ , ] . c-reactive protein (crp) may also be found to be raised. other laboratory abnormalities include leukocytosis, thrombocytosis, and anemia which often accompany increased disease activity. pancytopenia is often an alarming sign of coexisting or developing mas and necessitates prompt intervention. abnormal coagulation testing can rarely be seen and, in extreme cases, may develop into full-blown disseminated intravascular coagulation (dic) which can be fatal [ ] . abnormal liver and biliary function tests (increase in lactic dehydrogenase, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, and bilirubin) can also be seen in up to % of patients and often accompany fever and exacerbation of arthritis. mild periportal inflammation with monocyte infiltration may be seen on liver biopsy. serum ferritin has gained attention as both a diagnostic test for aosd and a marker of disease activity (biomarker). in aosd, ferritin is an acute phase reactant involved in inflammation, is linked to initiation of histiocyte-macrophage system and/or augmented release from damaged hepatocytes, and is not related to iron metabolism and storage. ferritin production is driven by cytokines such as il β, il , tnfα, and il [ ] . serum ferritin levels as high as ng/ml, five times the upper limits of normal, have been described in these patients. levels ranging from ng/ml to , ng/ml are not uncommon. fautrel et al. evaluated the validity of hyperferritinemia as a diagnostic tool in a retrospective analysis looking at patients with still's disease [ ] . a fivefold increase in serum ferritin had % sensitivity and % specificity. similar results were seen in a japanese study with % sensitivity and % specificity [ ] . serum ferritin levels are seen in other diseases such as hemochromatosis, gaucher's disease, infections (sepsis, hiv), and malignancies (leukemia, lymphomas). serum ferritin levels correlate with disease activity and often normalize when the disease flare subsides. however, the absence of elevated serum ferritin does not exclude active aosd. some patients with active aosd do not have elevated serum ferritin levels at all, or the rise of serum ferritin may lag behind the symptom presentation. glycosylated fraction is a more specific diagnostic marker than ferritin, albeit not commercially available in the usa. in healthy subjects, - % of ferritin is glycosylated. saturation of glycosylation mechanisms results in a drop in glycosylated fraction to drop to - %. this phenomenon is particularly prevalent in aosd. glycosylated ferritin often remains elevated for months after the disease goes into remission and hence cannot be used to monitor disease activity or response to treatment. improved diagnostic tests are clearly desirable, and new immunological tests, such as il , may prove useful in the near future for diagnosis as well as monitoring disease activity and response to treatment [ ] . currently available tests: complete blood count and differential, esr, crp, ana, and rf (both negative), liver function tests (lfts) and albumin, ferritin, and glycosylated ferritin (if available) are of practical use to most clinicians. radiographs are often of limited significance in the early phase of the disease. images are either normal or show soft tissue swelling, joint effusion, or mild periarticular demineralization. bone scan and gadolinium-enhanced magnetic resonance imaging were assessed in small series and may prove to be more sensitive imaging modalities for early diagnosis and successful treatment in follow-up. in one study, distinctive pattern of intercarpal and carpometacarpal joint space narrowing was seen in % of the subjects (bilateral in %) that led to pericapitate ankylosis in % of the cases [ ] . other investigators have also reported a tendency for distal interphalangeal, intertarsal, and cervical zygapophyseal ankylosis. patients who have the chronic articular disease pattern can present with joint erosions making the differential diagnosis from ra problematic, especially in the absence of systemic signs and symptoms. the differential diagnosis of aosd is extensive, especially at presentation, when the systemic symptoms predominate. conditions such as infections, neoplasms, and autoimmune disorders should be ruled out before the diagnosis of aosd can be confidently made. viral syndromes (e.g., rubella, cytomegalovirus, epstein-barr virus, mumps, coxsackievirus, adenovirus) can be excluded if the symptoms persist for more than months and serologies are negative. neoplastic disorders that can mimic aosd include leukemia and lymphoma, such as angioimmunoblastic lymphoma. however, the clinical presentation can differ substantially, with atypical rashes and/or isolated lymph node enlargement. at times bone marrow and/or lymph node biopsy may be needed to differentiate these entities. common mimickers of aosd include reactive arthritis and the other spondyloarthropathies, hemophagocytic syndrome, dermatomyositis, kikuchi's syndrome, sweet's syndrome, granulomatous disorders, and the vasculitides. other differentials of aosd are the periodic fever syndromes and, in particular, familial mediterranean fever, hyper igd syndrome (hids), and tnf receptor-associated periodic syndrome (traps). patients with familial mediterranean fever often present with acute, self-limited episodes of fever accompanied by signs of peritonitis, pleuritis, synovitis, and erysipelas-like erythema. the disease commonly starts in childhood or early adolescence. a significant family history, clinical presentation, and response to colchicine can aid in making the correct diagnosis. this can be verified, in many cases, with genetic analysis for the mefv gene. traps and hids commonly start in childhood and also have strong familial distributions. however, adult-onset cases of those rare syndromes do exist, and the genetic tests that are commercially available can be helpful in excluding them, especially when atypical presentations are present. several classification criteria have been developed from retrospective data available on aosd. one such study attempted to validate these classification criteria: yamaguchi's criteria were found to be the most sensitive ( . %), followed by cush's ( . %) and calabro's ( . %) criteria (table . ) [ , ] . of late, a french group has proposed a new set of criteria which takes into consideration the two new disease markers: serum ferritin and its glycosylated fraction (table . ). this set provided a sensitivity of . % and a specificity of . %, which remains to be authenticated in a different population before becoming widely accepted [ ] . fever of at least degrees c ( . f) last at least week arthralgias or arthritis lasting weeks or longer a non-pruritic macular or maculopapular skin rash that is salmon-colored in appearance and usually found over the trunk or extremities during febrile episodes leukocytosis ( , /microl or greater), with at least % granulocytes lymphadenopathy hepatomegaly or splenomegaly abnormal liver function studies, particularly elevations in aspartate and alanine aminotransferase and lactate dehydrogenase concentrations negative tests for antinuclear antibody (ana) and rheumatoid factor (rf) *several classification criteria have been developed from retrospective data available on aosd. one such study attempted to validate these classification criteria: yamaguchi's criteria were found to be the most sensitive ( . %) therapeutic goals include controlling physical signs and symptoms of inflammation, which is typically associated with the improvement in laboratory parameters. decisions about optimal therapy are influenced by the severity and chronicity of the disease and involve taking into consideration the adverse effect profile of the treatments, both long and short term, and the clinical response to initial therapies. the primary goal of therapy not only involves achieving control of acute symptoms but also preventing end-organ damage, including joint injury and major organ complications. the initial choice of therapy depends upon disease severity and extent of organ involvement. there has been a lack of head-to-head randomized controlled trials comparing different therapeutic modalities given the rarity of disease. treatment options constantly keep evolving as more insight is gained into the pathogenesis of aosd. support for the use of biologics as first-line therapy in severe disease is based upon the published experience in aosd and, especially, more extensive evidence supporting use of these agents in children with sjia, since recent evidence supports that sjia and aosd are different chronological disease onsets of the same "still's disease." patients with mild-to-moderate disease may present with fevers and rash, as well as with arthralgias or mild arthritis. while nsaids, such as naproxen or ibuprofen, were the first medications to be used in aosd, their role today is a very limited one. their use can be justified either as adjunct treatment or as monotherapy in mild, monocyclic cases for symptomatic relief. in addition to adverse effects that are commonly associated with nsaids, an association between the use of nsaids and macrophage activation syndrome (mas) has been described, as well [ ] . patients responsive to nsaids alone, who remain asymptomatic for at least a month, could have nsaid doses gradually reduced. glucocorticoids can be started immediately, as first-line treatment, or when nsaids are insufficient to control signs and symptoms of the disease. glucocorticoid dose can be gradually tapered once disease activity is controlled. oral prednisone is typically initiated at a dose of . - mg/kg per day, depending on the severity of disease. intravenous, high-dose steroids are reserved for those with refractory disease [ ] . approximately % of patients respond to glucocorticoids alone or to glucocorticoids used after a trial of nsaids [ ] . in addition to systemic steroids, those with one or two inflamed joints despite systemic therapy may benefit from intraarticular glucocorticoid injections. disease-modifying antirheumatic drugs (dmards) are typically used in the event of inadequate response to corticosteroids or as steroid-sparing medications. methotrexate remains the first-line steroid-sparing agent in aosd and is also a useful agent to treat still's arthritis. methotrexate can result in complete remission in up to % of patients and is effective in corticosteroid weaning [ ] . sulfasalazine is contraindicated because of lack of efficacy and association with mas development. internal organ damage and debilitating joint symptoms with radiographic are characteristic of moderate-to-severe disease. severe disease is defined as refractory disease, especially when terminal organ and/or life-threatening involvement exists, such as mas, severe hepatic injury, cardiac involvement with/or without tamponade, or disseminated intravascular coagulation (dic). biologic therapies such as il- inhibitors (anakinra, canakinumab) and il- receptor antagonists (tocilizumab) are used as first line or second line for sjia often without any prior use of corticosteroids or traditional dmards and that practice may be justified in moderate-tosevere aosd as well. small series have reported some efficacy of ivig when used early in the course of aosd [ , ] . target biologic agents have been historically reserved for refractory aosd (fig. . ) [ ] . these agents include a recombinant antagonist of the il- receptor (il- ra, anakinra), a human monoclonal antibody directed against il- β (canakinumab), and a soluble il- trap fusion protein (rilonacept). anakinra is particularly efficient in the rapid relief of the systemic symptoms. several retrospective case series and one open-labeled prospective randomized trial have evaluated the use of anakinra in aosd patients [ , ] . anakinra is approved as a daily subcutaneous injection ( mg) in ra. in aosd, higher dose may be necessary, since anakinra has a very short half-life ( - h). while anakinra does not work in all aosd cases, for the ones it does work, its onset of action is very rapid. in many cases, resolutions of systemic symptoms and normalization of inflammatory markers were reported within weeks of anakinra use, allowing for rapid tapering of corticosteroids, if they were ever used in the first place. however, relapses are not uncommon with the cessation of these agents. in certain patients, gradual reduction in dose has enabled the weaning of anakinra. daily anakinra injections are often complicated by frequent injection site reactions. if response to anakinra is incomplete or tolerability issues ensue, rilonacept and/or canakinumab can be considered. they have longer half-lives and can be administered at greater intervals, once or twice weekly for rilonacept and every - weeks for canakinumab, respectively. additional supplemental data is available in the setting of sjia. swart and associates reviewed data pertaining to children with sjia treated with anakinra [ ] . systemic symptoms resolved in % of the patients, and fatigue and well-being improved in % of cases. arthritis improved in % of the cases in time. absolute disease remission was mostly detected in patients with systemic symptoms, less arthritis, and a shorter duration of disease. similar findings were corroborated in a large, multicenter, randomized, placebo controlled trial by quartier et al. [ ] . sample population included patients with a systemic-onset jia for duration of more than months and steroid dependency. in month, anakinra was effective in out of patients (versus one in the placebo group) who reached the modified american college of rheumatology (acr) pediatric score. after months, majority of patients ( / ) who had been switched to anakinra were also responders. nigrovic and associates studied patients who received anakinra as first-line treatment. rapid resolution of systemic symptoms was observed in about % of cases, along with a supplementary preventive effect on refractory arthritis in almost % of the patients. based on these results, it was postulated that there could be a benefit for il- blockade therapy in initial phase of the disease (i.e., within months after onset) [ ] . these findings were also further reinforced by vaster et al. in [ ] . in a prospective series of patients who received anakinra as first-line therapy, % of the patients showed an american college of rheumatology pediatric score response or had inactive disease within months. overall, % of the patients treated with anakinra achieved remission. these results clearly indicate that il- blockade has an early place in the treatment strategy. tocilizumab, humanized monoclonal antibody directed against il- receptor, is used in refractory aosd. this agent has been studied with randomized placebocontrolled trials in sjia patients but not yet in aosd. effects of tocilizumab have been described to persist for ≥ months after its discontinuation. tocilizumab appears to have a marked corticosteroid-sparing effect and has a good safety and tolerance profile [ ] . most of the early data was with tocilizumab administered intravenously (iv) at a dosage of - mg/kg body weight every - weeks. nevertheless, larger randomized studies are still needed to further determine the optimal therapeutic scheme for tocilizumab, i.e., optimal dosing, interval, and duration of treatment for both the intravenous but also the subcutaneous route of administration. although those agents were the first biologics to be used in refractory aosd, these agents are no longer recommended as first-line treatment in aosd. anti-tnf-α agents, in particular infliximab, etanercept, and adalimumab, have been used to treat refractory aosd, but data on adalimumab are limited to a few cases [ ] . although complete resolution of symptoms has been observed, efficacy of the tnf-inhibitors has been mostly limited to still's arthritis. efficacy was better with the monoclonal antibodies, as compared to the soluble receptor, and switching from one agent to another had no additional effect [ ] . moreover, in one published series, two patients who were started on etanercept and adalimumab developed mas that could be allied with the initiation of therapy [ , ] . overall, tnf-α blockers should be considered for the treatment of chronic polyarticular disease, after the use of il- and/or il- inhibitors. non-refractory disease comprises of monocyclic and polycyclic aosd. nsaids can be used in monocyclic course of aosd without major systemic or articular involvement. preferred nsaid is high-dose indomethacin ( - mg/day). corticosteroids should be started promptly once the diagnosis is confirmed. usually, corticosteroid therapy starts at a dosage of . - mg/kg/day. pulse dose methylprednisolone is used if there is severe visceral involvement or there is suspicion of mas complicating the clinical presentation. higher dosages seem to be more efficient in controlling the disease and lessening the number of relapses. tapering of corticosteroids can start after - weeks, when symptoms have resolved, and biological parameters have returned to baseline. methotrexate (mtx) could be considered early for its steroid-sparing effect. typically, methotrexate ( . - mg/week) enables complete remission of the disease ( %) and limits frequent corticosteroid use. blood count and renal and hepatic function should be monitored before initiation of methotrexate and then at monthly intervals. alternative dmards may be use in the event of methotrexate failure; however, more recent literature suggests better results with targeted biologic treatment. an open-label, multicenter, phase ii study of subcutaneous tadekinig alfa (il- bp) in patients with aosd was recently published and showed promise. tadekinig alfa is the drug name for recombinant human interleukin- -binding protein (il- bp). this study was based on the principle that high levels of il- were noted during active flares of aosd. ten patients were assigned to receive mg tadekinig alfa, and patients received the mg dose. at week , of patients receiving mg and of patients receiving mg achieved the predefined response criteria. the agent was overall well tolerated with the exception of one case of optic neuropathy [ ] . it is becoming exceedingly evident that aosd patients fall into two distinct subsets, i.e., those presenting with systemic manifestations and those presenting with prominent articular manifestations. in addition, these findings are also reinforced by molecular evidence, cytokine profiles, clinical course, and response to therapy. predictors for a prominent articular pattern include female sex, proximal arthritis at disease onset, thrombocytosis, and corticosteroid dependency, whereas high fever, transaminitis, or elevated acute phase reactants are more likely to be connected with a systemic pattern of aosd [ , ] . alternative evidence to identify the systemic subtype of aosd are the following: thrombocytopenia, rhl, and hyperferritinemia. il- , interferon-γ, il- , and il- are typically associated with systemic aosd, whereas il- , il- , and il- are associated with arthritic aosd [ ] . this dichotomy aids in management as patients fall into one of the two categories and should benefit from different therapies. patients with systemic symptom predominant aosd often benefit from systemic corticosteroid therapy. from traditional dmards, methotrexate has been studied the most and may continue to have a role as steroid-sparing and in the treatment of chronic articular disease. il- antagonists should be considered first line in severe or refractory aosd, either alone or in initial combination with systemic corticosteroids when necessary. regularly reported side effects with anakinra include injection site reactions. longer acting il- inhibitors (rilonacept or canakinumab) may play a role in refractory disease or when tolerability issues exist with anakinra. tocilizumab, the il- receptor antagonist, has shown efficacy in both systemic and articular disease predominant aosd, even in cases where il- inhibition has been unsuccessful. in contrast to il- and il- inhibitors, anti-tnf-α agents typically have less sustained effect on systemic symptoms, but they may have a limited role for the chronic inflammatory polyarthritis. in an effort to standardize therapeutic management and evaluate 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adalimumab (anti-tnf-alpha) therapy to improve the clinical course of adult-onset still's disease: the first case report exacerbation of adult-onset still's disease, possibly related to elevation of serum tumor necrosis factor-alpha after etanercept administration a rare trigger for macrophage activation syndrome open-label, multicentre, dose-escalating phase ii clinical trial on the safety and efficacy of tadekinig alfa (il- bp) in adult-onset still's disease clinical manifestations of adult-onset still's disease presenting with erosive arthritis: association with low levels of ferritin and interleukin- distinct subsets of patients with systemic juvenile idiopathic arthritis based on their cytokine profiles consensus treatment plans for new-onset systemic juvenile idiopathic arthritis key: cord- -id fjgye authors: djikeng, appolinaire; nelson, barbara jones; nelson, karen e. title: implications of human microbiome research for the developing world date: - - journal: metagenomics of the human body doi: . / - - - - _ sha: doc_id: cord_uid: id fjgye the human microbiome refers to all of the species that inhabit the human body, residing both on and in it. over the past several years, there has been a significantly increased interest directed to the understanding of the microorganisms that reside on and in the human body. these studies of the human microbiome promise to reveal all these species and increase our understanding of the normal inhabitants, those that trigger disease and those that vary in response to disease conditions. it is anticipated that these directed research efforts, coupled with new technological advances, will ultimately allow one to gain a greater understanding of the relationships of these species with their human hosts. the various chapters in this book present a range of aspects of human microbiome research, explain the scientific and technological rationale, and highlight the significant potential that the results from these studies hold. in this chapter, we begin to address the potential and long-term implications of the knowledge gained from human microbiome research (which currently is centered in the developed world) for the developing world, which has often lagged behind in the benefits of these new technologies and their implications to new research areas. new high-throughput sequencing and data analysis approaches (costello et al., ; turnbaugh et al., ) , along with novel diversity screens and even more intrinsic single cell approaches to isolating new species (lasken, ) , have presented the sciences with a unique opportunity to investigate and interrogate the microorganisms that are associated with the human body, all at a greater depth than previously appreciated. from the earliest studies, the greater scientific community has recognized a high level of microbial diversity in nature beyond imaginations. this includes observations on the oceans, soils, and on animals. with respect to humans, it became increasingly apparent that the species on and in our bodies make significant contributions to our health and development. these species maintain normal cell function in the gastrointestinal tract (for example, see eckburg et al., ; bik et al., ; gill et al., ) . we are also dependent on these species for the efficient digestion of food components, such as plant material and xenobiotics , and to ward off certain diseases. in parallel, these microbes have been associated with, and can result in, many common diseases such as cavities, stomach ulcers, bacterial vaginosis (bv), and irritable bowel syndrome (foster et al., ; dorer et al., ) . most of the initial studies on the microbial diversity associated with the human body focused either on traditional culturing approaches or on sequencing and phylogenetic analysis of the s ribosomal (r)rna genes derived from microbial samples taken from the human body (eckburg et al., ; bik et al., ) . the limit to culturing or s rrna sequencing was primarily a reflection of the availability of molecular tools and approaches, and the cost associated with earlier versions of available sequencing technologies. the s rrna sequencing and analysis invariably revealed a higher level of microbial diversity than that seen with conventional culture techniques (gao et al., ; gao et al., ) . from the human stomach, for example, although the highly acidic environment was thought to only contain helicobacter types, bik et al. ( ) used , s rrna sequences obtained from gastric endoscopic biopsy samples to identify phylotypes of bacteria that potentially reside in the human stomach. the majority of these phylotypes was shown to belong to the proteobacteria, firmicutes, actinobacteria, bacteroidetes, and fusobacteria. this work also described that % of the clones represented organisms that were previously uncharacterized. subsequent ongoing studies continue to reveal high levels of diversity from the microbial species that inhabit the human body, with high levels of both intra-and interspecies diversity (costello et al., ; turnbaugh et al., ) . most of these studies that have investigated the diversity of the microbial species associated with the human body have however left important questions unanswered such as the identity of the nondominant community members and their biological roles, and which metabolic processes the populations that are present encode and carry out. in addition, the past years of genomic research have made it clear that closely related species, and even species that are identical at the s rrna level, can have wide variation in gene content (perna et al., ; kudva et al., ) . terms such as pan-genome and core-genome have been coined over the years to address the variations that are apparent in closely related species and have now been applied in a similar fashion to metagenomic populations (callister et al., ; bentley, ) . the initial publication of a shotgun sequencing of the human microbiome focused on the analysis of fecal samples from the human gastrointestinal tract . this study along with subsequent applications of shotgun techniques to the study of the human microbiome again highlighted the extent of microbial diversity associated with the human body (grice et al., ) . gordon and co-workers, for example, investigated the interplay between the gastrointestinal ecology and energy balance of animals on a western diet. here they found that obesity that was induced by the diet resulted in an increased proportion of one class of the firmicutes and that this same population could be reduced by manipulation of the diet. transplantation of the microbial populations from the obese mice to lean germ-free mice resulted in a higher level of the deposition of fat than when these microbial populations were taken from lean donors (turnbaugh et al., ) . more recently, gordon's group presented the results of a monozygotic and dizygotic twin pair study, where twins were concordant for leanness or obesity, and their mothers (turnbaugh et al., ) . the aim of this study was to address how host genotype, environmental exposure, and host adiposity influence the gut microbiome (turnbaugh et al., ) . the comparative analysis of fecal samples that were derived from individuals yielded , near full-length and , , partial bacterial s rrna sequences. in addition, . gb of metagenomic data was obtained from their microbiomes. the results from this analysis suggest that the gastrointestinal microbiome is shared among family members, but variations are present within each individual with respect to the lineages that can be observed. this variation was evident in both the monozygotic and dizygotic twin pairs. the results however suggest that there is a core functional microbiome that can vary depending on physiological states. the genomic era created the possibility of studying the detailed genetics of many microbial species. these include pathogens and nonpathogens and species that have both positive and negative impacts on the environment. the developments in the genomics arena have taken advantage of emerging and improving sequencing technologies, as well as improved assembly algorithms and approaches coupled with reduced costs for generating genomic data. the developments also placed the greater scientific community in positions to ask in-depth gene-based questions and get real answers. the advent of metagenomics was a natural progression of the genomics field, and in particular took advantage of the ability to sequence dna that was derived from a mixed community and assemble this genetic information to reconstitute metabolic and physiological information of any community of choice. metagenomic approaches have by now been successfully applied to environments as diverse as soils, the oceans rusch et al., ; yooseph et al., ; yutin et al., ) , and the human body costello et al., ; turnbaugh et al., ) in an attempt to describe and decipher the microbial species that are present in these niches. entire systems can now be studied with respect to viral, microbial, and fungal diversity, over varying time courses, and before and after perturbation (costello et al., ). on the human side, when coupled with s rrna analyses for detailed estimates of microbial diversity, metagenomics approaches present the perfect opportunity to address questions related to human health and associated problems. this is particularly relevant in the developing world, which presents its own series of challenges, some of which are presented below. one of the most valuable examples to date of the potential benefits from knowledge gained with human microbiome studies comes from a series of studies performed by dore and colleagues at inra. the significance of the studies conducted by this group relates to how the evolution of initial studies focusing on the microbiome can result in recommendations to improve human health conditions. their results evolved from initial metagenomic studies on human gastric samples using fosmid libraries from six healthy patients, and six patients with crohn's disease (cd). the group was able to identify nonredundant ribotypes mainly represented by the phyla bacteroidetes and firmicutes, of which distinct ribotypes were identified in the healthy microbiota, and only in cd. this metagenomic approach that was initially published gave the first insight into the reduced microbial diversity in patients with cd. their work continued to focus on microbiology of cd sokol et al., ; sokol et al., ; seksik et al., ) . they most recently compared fecal samples of patients active for cd (a-cd) patients, cd patients in remission (r-cd), active ulcerative colitis (a-uc) patients, four uc patients in remission (r-uc), eight infectious colitis (ic) patients, and by s pcr and found that members of firmicutes (clostridium leptum and c. coccoides groups in particular) were less represented in a-ibd patients compared to healthy subjects (hs) with faecalibacterium prausnitzii species (a major representative of the c. leptum group) in lower abundance in a-ibd and ic patients compared with hs. as a result of the initial work, the group proposed that f. prausnitzii was important for gut homeostasis. in members of the same group had published on the composition of the mucosa-associated microbiota of cd patients at the time of surgical resection and months following using fish analysis (sokol et al., ) , and again found reduced abundance of f. prausnitzii being correlated with an increased risk of postoperative recurrence of ileal cd. they further studied the anti-inflammatory effects of f. prausnitzii and showed that the bacterium exhibited anti-inflammatory effects on cellular and tnbs colitis models. the us national institutes of health (nih) initiated a roadmap program focused on the human microbiome (peterson et al., ) . the project has been described as a community resource, with overarching aims to help determine the core human microbiome, to understand the changes in the human microbiome that can be correlated with changes in human health, to develop new technological and bioinformatics tools to support these goals, and to address the ethical, legal, and social issues raised by human microbiome research (http://nihroadmap.nih.gov/hmp/). the project has a heavy sequencing and data analysis component that currently is underway at the four large nhgri/niaid-funded sequencing centers (j. craig venter institute (jcvi), baylor college of medicine, the broad institute and washington university). the current sequencing focus includes generating at least reference genomes at various levels of finishing (chain et al., ) , coupled with significant s rrna sequencing and metagenomic sequencing from to body sites on individuals some of which would be repeat sample donors (http://nihroadmap.nih.gov/hmp/). a number of "investigator"-driven demonstration projects have also been awarded. these demonstration projects aim to understand the implications of a number of diseases including cd, bv, psoriasis, and esophageal cancer to name a few (peterson et al., ) . it is anticipated that the results from these demonstration projects will give additional insights into the relationship between human health and disease and changes in the human microbiome. finally, the human microbiome project (hmp) roadmap initiative has awarded funds to investigate new technologies for improving knowledge of the human microbiome, as well as for the development of computational tools that will increase the value of metagenomic data (http://nihroadmap.nih.gov/hmp/fundedresearch.asp), and the ethical, legal, and social implications of this work. in summary, and as captured in the recent publication by peterson et al. ( ) , the goals of the hmp are to demonstrate the characterization of the human microbiome with population, genotype, disease, age, etc., and also catalog the influence of disease. the aim also is to present a standardized data resource and technological benefits. the project will go over years at a funding level of close to million us dollars. since the launch of this roadmap initiative in , a number of other hmps have been described. an overview of available projects as of mid- was presented in an editorial (mullard, ) . projects beyond the large nih usa based human microbiome efforts include work in europe, china, australia, and canada. in , the european commission committed close to million us dollars to a -year initiative called the metagenomics of the human intestinal tract (metahit) where the primary focus is the microorganisms that inhabit the gut, and how they contribute to obesity and inflammatory bowel disease (mullard, ) . a review of this effort is presented in another chapter written by ehrlich and colleagues. we are all cognizant of the fact that age, diet, and geographical location contribute to variations in the human microbiome. consequently, the more initiatives that we have in diverse parts of the world, the better positioned we will be to fully understand the key components of the microbiome and their interactions with the host under various environmental and physiological cues. because of a slow rate of progress in the areas of scientific research, along with low levels of available funding and investment in sciences in most developing countries, there has been very little scientific contribution toward solving major problems that hinder their global development. as coloma and harris ( ) nicely put it, "researchers in many developing countries will not be participating in genomics research, mainly because of their technological isolation and their limited resources and capacity for genomics research combined with the urgency of many other health priorities." areas such as public health, emerging infectious diseases, and agricultural development, which are key to long-lasting and sustainable national development, still lack the funding and innovation required to mitigate their inability to contribute to global development. the global health sector is of particular importance given the increasing number of diseases that plague the developing world (some of which are making a comeback after several years under effective control). examples of some of these are detailed below. consequently, in most developing countries throughout the world, and specifically in sub-saharan africa, south america, and asia, there is a serious need to improve public health. in these countries, communicable diseases caused by known and even unknown pathogens (see below) remain a leading cause of mortality. emerging infectious diseases are a major cause for alarm, and malnutrition and associated effects are also major issues that need to be effectively addressed. if one takes emerging infectious disease as an example, this captures many viral and bacterial agents. severe acute respiratory syndrome (sars) was the first infectious disease to emerge in the st century, and other emerging viral infectious diseases according to the world health organisation (who) include but are not limited to ebola and marburg hemorrhagic fevers. in addition, in an earlier publication by who ("new who office to help developing countries control emerging diseases"; j environ health , ) it was stated that in alone, communicable diseases caused the death of over million people worldwide, mainly among the poorest populations of developing countries. since then, more than new communicable diseases have been identified, and this list includes several diseases that were thought to be almost extinct that apparently have come back into the human population. certain food-borne diseases are also considered to be emerging as they now occur more often, and that list includes outbreaks of salmonellosis, which have increased significantly in the past years. listeria monocytogenes also falls into this category as its major role in food-borne diseases has become more recently appreciated, and some food-borne trematodes are also emerging as a serious public health concern. although food-borne infections with escherichia coli serotype o :h were first described in , it has rapidly emerged to be a leading cause of infections, which in turn result as a major cause of bloody diarrhea and acute renal failure, with an infection that is sometimes fatal. finally, while cholera devastated much of asia and africa for years, its introduction for the first time in almost a century on the south american continent in makes it another example of an emerging infectious disease. in addition, very little to none of the successful metagenomics stories in understanding the human microbiome and its role in aspects of human health have been conducted or duplicated in developing countries. notwithstanding ongoing efforts focusing on vaccines, better diagnostics, and improved treatment of many of these diseases, it is becoming increasingly essential to complement such approaches with an investigation of the role of the human microbiome on human health. several areas of anticipated important contribution of the human microbiome include zoonotic diseases and other emerging and re-emerging infectious diseases, sexually transmitted diseases, diarrheal diseases, respiratory diseases, eukaryotic diseases, malnutrition, and the integration of probiotics for improving human health. in addition to the translation of findings into practical approaches for improving human health, other opportunities offered by the human microbiome initiative are related to the transfer of technology to developing countries and the associated long-term benefits to training local populations in these developing sciences so that nations can retain the benefits. this will further strengthen capacity in genomics and bioinformatics and all the associated downstream applications that come with these areas of research. it is now appreciated that there is a resident (which constitutes the core microbiome) and a nonresident microbiota. the nonresident microbiota contains known and unknown microbes that cause a wide range of human diseases, most of which remain to be effectively controlled in both the developed and the developing world. human losses in the developing world in terms of mortality and contributions to economic development appear to be greater, however. currently, for example, communicable diseases caused by eukaryotic parasites such as plasmodium spp., leishmania spp., trypanosoma spp., and various viruses, among others, remain serious public health concerns in the developing world and affect more than . billion people (mahmoud and zerhouni, ) . in this context, scientific challenges that include genomics, metagenomics, proteomics, and metabolomics-related activities need to be expanded to ultimately encompass system and ecological understanding of communities of microbes and their interactions with humans. it has in fact been anticipated that the control of these diseases may be accelerated by the complete understanding of the genomes of these species, coupled with an understanding of the changes of the human microbiome that favor or reduce/eliminate their virulence and/or transmissibility. the adaptation processes, for example, by which zoonotic microorganisms that enter the human population adapt to become pathogens overtime can also be accelerated by longitudinal studies that focus on the populations on the bodies of both healthy and diseased individuals. as with most advanced technological and scientific approaches, and as is evident from the developing countries reports presented above, the development and applications of technological advances probably will take a significant amount of time to trickle to the developing world. in the realm of genomics and metagenomics as applied to human health, there is limited evidence that this will happen soon enough to allow developing countries to actively participate and shape research in these new fields. however, a recent award from the bill & melinda gates foundation (bmgf) to dr. jeffrey gordon at the washington state university in st. louis to study childhood malnutrition in developing countries (http://www.gatesfoundation.org/pressreleases/pages/child-malnutrition-microbial-cells-study- .aspx) suggests that there will be more movement in the direction of applying these technologies to problems in the developing world. for the above-mentioned study, gordon's group will investigate the microbes in severe malnutrition with a major focus on severely malnourished infants living in malawi and bangladesh, and whether their microbial flora varies from that found in healthy infants who live in the same environment. it is anticipated that as a result of these studies, we will be better positioned to develop effective treatment regimes for these disease conditions. this award is part of an initiative by the bmgf to fund research on malnutrition (http://mednews.wustl.edu/news/page/normal/ .html?emailid= ). in addition to that award and the anticipated outcome, there have been a significant number of plant and microbial genome projects initiated and conducted in the developing world. the range of microbial species that have been sequenced includes human, plant, and animal pathogens, as well as organisms that have potential benefit to the environment. some of these species include actinobacillus pleuropneumoniae serovar str. jl that causes fibrinous and necrotizing pleuropneumonia in swine, and that was sequenced by the huazhong agricultural university in china and haemophilus parasuis sh also sequenced by the huazhong agricultural university/institute of pathogen biology/chinese academy of medical sciences/peking union medical college. chromobacterium violaceum atcc was sequenced by the lncc (national laboratory of scientific computing in rio de janiero, brazil); this bacterium carries the bacteriocidal pigment violacein and can also cause diarrhea and septicemia in humans. ehrlichia ruminantium str. welgevonden was sequenced at the university of pretoria, south africa. leifsonia xyli subsp. xyli str. ctcb , the causative agent of ratoon stunting disease in sugar cane, was sequenced by the sao paulo state (brazil) consortium and leptospira interrogans serovar copenhageni str. fiocruz l - and xylella fastidiosa were also sequenced by the same group. leptospira interrogans serovar lai str. sequenced by the chinese national hgc, shanghai, and lysinibacillus sphaericus c - sequenced by the chinese academy of sciences/beijing genomics institute, chinese academy of sciences. the developing world has also been involved in the sequencing and analysis of some of the major eukaryotic parasites such as trypanosoma brucei, trypanosoma cruzi, leishmania major, and theileria parva (nene et al., ; berriman et al., ; bishop et al., ; el-sayed et al., ; gardner et al., ) . there have also been several initiatives that have looked at host genotyping in many developing countries. according to coloma and harris ( ) , thailand, south africa, indonesia, brazil, mexico, and india have all devoted resources to studies on human genetics and variation in human populations. as a result of many of these initiatives in developing countries, a limited capacity of tool development for genomics and bioinformatics approaches has occurred. however, much more remains to be achieved in technology and knowledge transfer, particularly in sub-saharan africa and latin america. the main focus should be on developing genomics platforms leveraging on the next-generation sequencing approaches that remain to be established in much of the developing world. events of emerging and reemerging infectious diseases in the human population remain constant occurrences in sub-saharan africa, southeast asia, and south america. emerging infectious disease events such as sars (field, ) and the most recent pandemic of h n illustrate and confirm the constant flow of pathogens from wild and domesticated animals, and other reservoirs into the human population. chikungunya fever, which is an arboviral infection, reemerged in asia in - after a long period of quiescence (bhatia and narain, ) . it is thought that factors including microbial, climatic, social, and economic aspects influenced the reemergence of the disease and the pace at which it spread, eventually resulting in high death rates (bhatia and narain, ) . indeed, there are many microorganisms (viral, bacterial, and eukaryotes) that have moved into the human population and remain part of the human microbiota, which, when able to effectively survive, can cause either new diseases or disease with a much higher severity. such cases of unknown and potentially pathogenic microorganisms in circulation in the human population are usually favored by factors related to ( ) the ability of microorganisms to adapt in new hosts, ( ) human actions (interactions with wild animals, hunting and effects on the environment leading to ecological disturbances), and ( ) human movements as a result of global world travel (field, ) . consequently, at any particular time, there could be a set of known and unknown microorganisms present in a given individual or a population as a result of their presence in and interaction with a specific environment or organisms therein such as animal reservoirs (i.e., bats, mice, and rats) of known and unknown microorganisms. the main challenge in the context of forecasting, and better yet preventing emerging and reemerging infectious diseases has been early detection and genetic identification of such known and unknown microorganisms. global human microbiome studies using metagenomics analysis of known and unknown microorganisms provide unique but powerful opportunities to uncover the near-complete composition of the microbial content of an individual or a population at any given time, thus setting the stage for a comprehensive inventory of the genetic characteristics of potential human pathogens. studies of the human microbiome in the developing world will likely contribute significantly to the discovery of emerging pathogens (viruses, bacteria, and others) in circulation in humans. in fact, in both developed and developing countries the issues of early identification of emerging pathogens have been an impediment for the prevention of emerging and reemerging infections. based on recent studies, human metagenomics coupled with the next-generation dna sequencing provides an opportunity for early detection of microbial organisms even when there is significantly low abundance (relman, ) . because of the extreme importance of monitoring zoonotic infections, metagenomics studies should in principle be extended from humans to domesticated and nondomesticated animals. in fact, based on the technologies already available for human metagenomics studies, there has been increasing interest in launching animal metagenomics initiatives. such initiatives will not only provide insights into the resident and transient microbial populations but also, in the case of natural reservoirs of given microorganisms, provide an opportunity to pinpoint the genetic changes that must occur for their adaptation to a new host -the human body. this is applicable in particular to invertebrate vectors and bats that are known to be host to a number of highly pathogenic viruses that pose significant public health problems to humans. developing countries are particularly affected by the impact of mycobacterium tuberculosis virulence and tb drug resistance. this has been primarily because of genome plasticity in the causative agent. unfortunately, available microarray-based platforms to identify strain diversity have not been fully implemented with the greatest tb incidence largely due the hiv/aids epidemic. the renewed interest and funding for top infectious diseases has recently revamped efforts to accelerate tb research, with a particular focus on the use of integrated approaches to find better control measures. in this context, it is proposed and highly anticipated that key aspects such as the integration of large-scale "omics," datasets focusing on parasite genetic determinants, host genetics, and host-parasite interactions will be crucial for this quest for better control measures. in addition, and given recent reports, the human microbiome would be a great addition to this integration of data in the context of systems biology. to this end, the evaluation of the human microbiome in cases of latent, nonlatent tb, and drug-resistant tb infections will provide insights into the role of the human (resident and nonresident) flora in various aspects of tb infections. such information would most likely contribute to improving diagnosis, control, and spread of tb infection. another example of the potential to come from using human metagenomic research and approaches in the developing world relates to another emerging infectious pathogen that causes leptospirosis. the leptospires cause an infection that is associated with very high levels of mortality annually, but have received relatively little attention, probably because the infection is concentrated in the tropical regions and in the developing world. more than half a million cases are reported annually, and majority of these cases are associated with human exposure to pathogenic leptospira species in the environment. mortality rates as high as % have been recorded. the genus leptospira is serologically divided into two species: l. interrogans, which is pathogenic to humans and animals, and l. biflexa, a free-living nonpathogenic species found in water and wet soil. more than pathogenic and saprophytic species have been recognized. many animals including rodents and dogs are known to be reservoirs of leptospira, and humans are considered to be the accidental hosts of this pathogen. transmission of the pathogen is primarily from soil and water to mammalian tissues (often noticed following on large-scale flooding), with the infection occurring via penetrating leptospires through mucosa or open skin. symptoms of leptospirosis include meningitis, pneumonitis, hepatitis, nephritis, pancreatitis, erythema nodosum, and death. no human vaccine against leptospirosis is available, and mild leptospirosis is treated with doxycycline, ampicillin, or amoxicillin. for severe leptospirosis, the primary therapy is penicillin g. the molecular diagnosis of leptospirosis has been with traditional approaches such as restriction enzyme analysis, nucleic acid probes and hybridization, pulse field gel electrophoresis (pfge), and varying ribotyping approaches. genome sequences from at least six leptospiras have become available in the past few years, and these genomes are providing insight on the diversity of these species. in addition, the availability of these genomes is allowing for the identification of novel virulence factors, and ultimately will facilitate vaccine development. recently, the genome sequence of the free-living l. biflexa was completed (picardeau et al., ) and shown to contain , protein-coding genes distributed across three circular replicons. in the current study, it has been estimated that , genes ( %) represent a progenitor genome that existed before divergence of pathogenic and saprophytic leptospira species. basically, nearly one-third of the l. biflexa genes are absent in pathogenic leptospira. in addition, , pathogen specific genes that are found in the pathogenic leptospires are not present in l. biflexa. of these, genes have no known function suggesting that there are mechanisms that are unique to leptospira and that the pathogenic specific genes need further study. the resulting genome studies suggest that there is still a significant amount of information that is not understood about the leptospiras, particularly as it relates to how the species adapts to new environments and how the genomes mutate. metagenomic studies of samples derived from infected populations will present an opportunity to study the pathogen without repeated passage where it has been shown to have genome rearrangements. in parallel, the pathogen can be studied directly in the environment when it is in transition from its natural host to humans (the accidental host). interestingly, there is a large niaid-funded project underway at the jcvi to sequence the genomes of an additional leptospira isolates (joe vinetz, personal communication; http://gsc.jcvi.org/). leptospirosis is another example of an emerging infectious disease that is prevalent in tropical environments and has not received as much attention as the major diseases in the developed world although the causative organisms result in a high mortality rate. genomics and metagenomics approaches have the potential to increase the understanding of these species and their impact on human health. diarrheal diseases remain one of the leading causes of deaths worldwide (culligan et al., ) . specifically, diarrheal diseases are the second most common cause of child deaths worldwide, and more than % of child deaths due to diarrhea occur in africa and south asia. worldwide, % of deaths from diarrhea are due to unsafe water and poor sanitation or hygiene. three-quarters of all deaths from diarrhea in children younger than years occur in countries. there are about . billion cases of diarrhea among children each year, in addition to those who die from the disease. the un reports that vaccines and better hygiene could decrease the number of deaths from diarrhea among children. since the s, oral rehydration therapy has been the cornerstone of treatment programs. this therapy prevents dehydration that is associated with diarrhea. giving zinc supplements with oral rehydration salts has also been shown to reduce the length of the illness and also the risk of more diarrhea episodes. sixty percent ( %) of children in developing countries do not get the recommended treatment for diarrhea, which is vaccination against rotavirus, the leading cause of the disease. in fact, rotavirus causes about % of hospital admissions of children below years suffering from diarrhea. current therapies are focused on rehydration therapies but the studies from a human microbiome approach, coupled with the development of novel antibiotics and/or probiotics holds significant potential (culligan et al., ) . many diarrhoeal diseases have been associated with viruses (ramani and kang, ) . recent results suggest that viruses are present in as much as % of diarrheal samples in the developing world (ramani and kang, ). there are however a significant number of cases of diarrhea without obvious causes, thus making it difficult to control them. in addition and specifically in the case of rotaviruses, because of their high genetic diversity, the emergence of new genotypes, and the reassortment between different genotypes (matthijnssens et al., ) , there is constant need for surveillance of circulating strains. human metagenomics studies hold the promise for increasing our understanding of the diversity of rotavirus and other etiological agents of diarrheal diseases. based on previous studies, gastrointestinal tract metagenomics studies in both healthy and diarrheal patients in developing countries may lead to the identification and association of additional microorganisms (bacteria, viruses, and eukaryotes) with various cases of diarrheal diseases . as an example, recent human microbiome studies have led to the discovery of a novel virus of the cosavirus genus and its association with acute diarrhea in a child in australia (holtz et al., ) . regular and comprehensive metagenomics analyses focusing on acute and difficult-to-cure cases of diarrhea and diarrhea cases with known and unknown causes primarily in developing countries may provide opportunities for ( ) a constant assessment of the diversity of known causative agents of diarrhea and ( ) identification of new microorganisms as they relate to cases of diarrheal diseases. sexually transmitted diseases (stds) are common infections throughout the developed and the developing world. stds can result in premature birth, stillbirth, and neonatal infections (de schryver and meheus, ) . many ongoing studies on bv aim to understanding the microbial populations that are present in the vaginal ecosystem and how they vary under health and disease conditions. recent studies that are focused on s rrna gene analysis have suggested that the extent of microbial diversity in the vaginal tract is not fully understood, which in turn has implications for current treatment regimes. this has potentially significant implications for asymptomatic disease conditions for example. additional results show a lack of homogeneity within the vaginal tract, highlighting a complex ecosystem (kim et al., ) . metagenomic approaches to studying this environment promise to give additional insights into the extent of diversity within this niche. ongoing studies in several parts in sub-saharan africa reveal that there is some relationship between the population of microbes that exists in the vaginal tract and stds. recently, van de wijgert et al. ( ) described a study in which they investigated the relationships among bv, vaginal yeast, and vaginal practices, mucosal inflammation, and hiv acquisition. from a cohort of , hiv-negative women, they observed that women who were positive for bv or vaginal yeast had a higher likelihood to acquire hiv, and they concluded that bv and yeast may contribute more to the hiv epidemic than previously appreciated (van de wijgert et al., ) . similar observations have been made in a review of all available literature on the extent to which bv may increase the risk of hiv acquisition (atashili et al., ) . earlier, in , van de wijgert et al. ( studied zimbabwean women to determine if intravaginal practices could be associated with changes in the vaginal flora and acquisition of stds. in this study, they found that some disturbances of the flora could be associated with increased likelihood of stds and hiv; the absence of lactobacilli from the vaginal flora was associated with being positive for hiv (van de wijgert et al., ) . martin et al. ( ) had similarly looked at a cohort of sex workers in kenya and demonstrated that although only % of these women were colonized with lactobacillus species at baseline, follow-up studies showed that the absence of culturable vaginal lactobacilli could be associated with the increased likelihood of acquiring hiv- . abnormal vaginal flora on grams-stain was associated with increased risk of both hiv- acquisitions. this group proposed that the treatment of bv and the use of lactobacilli to colonize the vaginal cavity should be evaluated for reduce risk of acquiring hiv- , gonorrhea, and trichomoniasis (martin et al., ) . how the microbial populations in the vaginal cavity can contribute to reduce chances of hiv infection is one of those major areas that need attention, and that will undoubtedly benefit from human microbiome research. according to the world health organisation (who, http://www.who.int/ mediacentre/factsheets/fs /en/), every seconds a child dies of malaria, a disease that can be prevented and cured. in there were million cases of malaria, and these resulted in nearly million deaths mostly among african children. in fact, % of all malaria deaths occur in sub-saharan africa. people who live in lower-income communities, i.e., approximately half of the world's population, are at risk of the disease. the who reports that in malaria was present in countries and territories. the disease, however, can be eradicated, says bill gates. in an interview with the bbc world services world today program in january , gates said "we have a vaccine that's in the last trial phase -called phase three." he added that "a partially effective vaccine could be available within years." a vaccine that is fully effective against malaria would take - years to develop. although most cases of malaria are found in sub-saharan africa, there are other countries, including in asia, latin america, the middle east, and parts of europe, that are also affected. key interventions include prompt and effective treatment with artemisin-based combination therapies; people at risk using insecticide nets; and indoor residual spraying with insecticide to control the vector mosquitoes. genomics approaches have already been used to elucidate the genomes of several of the plasmodium species (gardner et al., ; carlton et al., ; pain et al., ; mitsui et al., ), but new metagenomics approaches present opportunities to monitor the impact of the parasite of the microbial communities that reside on and in the human body, with a longer-term potential to develop novel probiotic approaches to supplement nutrition of infected individuals while the parasite runs its course. in countries that have a high rate of malaria, economic growth rates may be cut by as much as . %. in addition, genomic studies on the environments, in which the mosquitoes reside and breed, are being and will continue to allow for an increased understanding of the communities that they require for their survival (ponnusamy et al., a (ponnusamy et al., , b, ). this is particularly relevant since mosquitoes breed in areas where there are wet conditions, and the transmission of the disease can differ according to local factors such as rainfall, proximity of mosquito breeding sites to people, and the mosquito species in the area. a november report from susan anyangu in nairobi, kenya, carried by inter press service (ips) states that the rts.s vaccine being developed is to be used specifically in africa. it will be for infants and children aged less than years (the most vulnerable to malaria). the vaccine could be ready for use in years time. supplementing nutrition of people with malaria with probiotic solutions that have been derived from a metagenomic approach to understand the human microbiome holds significant promise. the fao/who defines probiotics as "live microorganisms which, when administered in adequate amounts confer a health benefit on the host." probiotics have become more and more valuable over the past few years and are available in a number of food sources, including yogurts and other milk products, fermented and unfermented milk, and some juices. these live microorganisms are in most cases bacteria that are similar to beneficial microorganisms found in the gastrointestinal tract. each species that is present in the gut environment would seem to hold some potential for use as a probiotic and therefore in human health. probiotics have been shown to be effective in treating irritable bowel syndrome (ibs), childhood and traveler's diarrhea, prevention and treatment of vaginal yeast infection and urinary tract infection, preventing and treating inflammation of the colon after surgery, reduction of the recurrence of bladder cancer, shortening the time of intestinal infections, and preventing eczema in children. although the benefits of probiotics are evident, they have yet to be adapted extensively in the developing world (reid and devillard, ) . other ideas on the use of probiotics for reducing the morbidity and mortality associated with hiv/aids have been explored and proposed (reid et al., ) where it has been proposed that lactic acid bacteria could play a role in maintaining the health of the human gut. we can only hope that as a result of the initiatives of the human microbiome project, new probiotics for a range of human health conditions may be developed based on baselines for people in different geographic locales. the efficient implementation of human microbiome research relies on the advanced instrumentation necessary for the processing of collected clinical samples, preparation and amplification of nucleic acid, and dna sequencing. in addition, dna sequence analysis also requires advanced bioinformatics resources. all genomicsrelated technologies developed over the past years remain very expensive to be acquired by developing countries. this is usually justified by low-use volume and high costs of equipment and maintenance (coloma and harris, ) . therefore, as suggested by these authors, involvement of laboratories and institutions in developing countries should take advantage of "north-south" and "south-south" collaborations. previous examples of successful "north-south" collaborations could be leveraged to initiate new ones in the context of human microbiome studies. for the past several years, there have been numerous initiatives in developing countries to reduce the technological divide and hence begin to actively contribute to genomics research. in this context, activities have included training and capacity building in genomics and bioinformatics. in addition, there has also been an emphasis on the development of "centers for excellence" to provide resources and a critical scientific mass at regional levels. four such regional "centers of excellence" are currently being established in eastern and central africa, southern africa, west africa, and north africa. one of the most advanced "centers for excellence," biosciences for eastern and central africa (beca), located at the international livestock research institute (ilri) in nairobi, kenya, has established facilities (with advanced genomics and bioinformatics resources) to support and accelerate research in a wide range of disciplines, including plant/crop sciences and animal sciences. such infrastructure would ideally be poised for use as a focal point for the implementation of a regional initiative on the human microbiome. the existence of such facilities would normally be used to engage various african institutions in south-south collaborations. the "south-south" collaborations indeed provide opportunities to strengthen the scientific capacity of institutions in developing countries, which would be translated into their effective participation in north-south initiatives. genomics and metagenomics initiatives are usually quite expensive, and obviously, most institutions in the developing world would not be able to fund such activities independently. however, given the existence of several human microbiome projects in the united states, canada, europe, china, japan, singapore, and australia, components in developing countries could easily be justified. for example, an african component of the human microbiome would provide elements to answering important outstanding microbiome questions, among which are included: ( ) is there a core human microbiome? ( ) does the composition of the human microbiome vary from one geographical region to another? given the anticipation of such interesting outcomes, existing initiatives could further provide seeds to launch other initiatives in the developing world. furthermore, in the context of the use of biosciences for africa's development, a strong case should be made to various stakeholders such as the african union and other regional organizations to fund the african component of the human microbiome. this next wave of genomics research will not be without its own set of challenges. recent studies, for example, show that many diseases present with similar observations, and as such initial surveys into the human microbiome under health and disease may give unexpected outcomes (yazdanbakhsh and kremsner, ) . bacterial vaginosis and hiv acquisition: a meta-analysis of published studies sequencing the species pan-genome the genome of the african trypanosome trypanosoma brucei re-emerging chikungunya fever: some lessons from asia molecular analysis of the bacterial microbiota in the human stomach analysis of the transcriptome of the protozoan theileria parva using mpss reveals that the majority of genes are transcriptionally active in the schizont stage comparative bacterial proteomics: analysis of the core genome concept comparative genomics of the neglected human malaria parasite plasmodium vivax molecular genomic approaches to infectious diseases in resourcelimited settings bacterial community variation in human body habitats across space and time probiotics and gastrointestinal disease: successes, problems and future prospects epidemiology of sexually transmitted diseases: the global picture helicobacter pylori's unconventional role in health and disease diversity of the human intestinal microbial flora comparative genomics of trypanosomatid parasitic protozoa bats and emerging zoonoses: henipaviruses and sars metagenomic analysis of human diarrhea: viral detection and discovery application of ecological network theory to the human microbiome molecular analysis of human forearm superficial skin bacterial biota substantial alterations of the cutaneous bacterial biota in psoriatic lesions genome sequence of theileria parva genome sequence of the human malaria parasite plasmodium falciparum from where did the 'swine-origin' influenza a virus (h n ) emerge? metagenomic analysis of the human distal gut microbiome topographical and temporal diversity of the human skin microbiome identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea heterogeneity of vaginal microbial communities within individuals strains of escherichia coli o :h differ primarily by insertions or deletions, not singlenucleotide polymorphisms genomic dna amplification by the multiple displacement amplification (mda) method neglected tropical diseases: moving beyond mass drug treatment to understanding the science reduced diversity of faecal microbiota in crohn's disease revealed by a metagenomic approach vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type and sexually transmitted disease acquisition rotavirus disease and vaccination: impact on genotype diversity phylogeny of asian primate malaria parasites inferred from apicoplast genome-encoded genes with special emphasis on the positions of plasmodium vivax and p. fragile microbiology: the inside story metagenomics and the global ocean survey: what's in it for us, and why should we care? theileria parva genomics reveals an atypical apicomplexan genome the genome of the simian and human malaria parasite plasmodium knowlesi genome sequence of enterohaemorrhagic escherichia coli o :h the nih human microbiome project genome sequence of the saprophyte leptospira biflexa provides insights into the evolution of leptospira and the pathogenesis of leptospirosis species composition of bacterial communities influences attraction of mosquitoes to experimental plant infusions identification of bacteria and bacteria-associated chemical cues that mediate oviposition site preferences by aedes aegypti diversity of bacterial communities in container habitats of mosquitoes viruses causing childhood diarrhoea in the developing world probiotics for mother and child probiotics for the developing world new technologies, human-microbe interactions, and the search for previously unrecognized pathogens the sorcerer ii global ocean sampling expedition: northwest atlantic through eastern tropical pacific the role of bacteria in onset and perpetuation of inflammatory bowel disease incidence of benign upper respiratory tract infections, hsv and hpv cutaneous infections in inflammatory bowel disease patients treated with azathioprine temperature gradient gel electrophoresis of fecal s rrna reveals active escherichia coli in the microbiota of patients with ulcerative colitis molecular comparison of dominant microbiota associated with injured versus healthy mucosa in ulcerative colitis faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of crohn disease patients diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome a core gut microbiome in obese and lean twins intravaginal practices, vaginal flora disturbances, and acquisition of sexually transmitted diseases in zimbabwean women bacterial vaginosis and vaginal yeast, but not vaginal cleansing, increase hiv- acquisition in african women influenza in africa the sorcerer ii global ocean sampling expedition: expanding the universe of protein families assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the atlantic and pacific oceans using the global ocean sampling expedition metagenomes the authors wish to acknowledge the invaluable information found on the world health organisation (who) website and on the mayo clinic website. key: cord- - xvzvdg authors: nan title: national scientific medical meeting abstracts date: journal: ir j med sci doi: . /bf sha: doc_id: cord_uid: xvzvdg nan zero months months months months group a . . * . ** . "* . ** zero months group b . . ** values are means; *p< . and **p< . l~ most other studies show neutral effects on insulin sensitivity, with minimal incidence of glucose intolerance. this may be partly explained by the diversity of age, diagnosis (whether insufficiency or deficiency state), other pituitary deficiencies and replacement therapy, and possibly by dosage ofgh utilised in these studies. clostridium difficile-associated disease (cdad) is primarily a nosocomial condition. community-acquired disease has been described but the incidence is low "). during a recent outbreak, we prospectively reviewed all new cases of cdad to determine what proportion of cytotoxin positive cases were community or hospital acquired. during a month study period, cases were identified. history of diarrhoeal episodes were recorded for each case. selected isolates were typed using pyrolysis mass spectrometry (pms). community-acquired cdad was defined as diarrhoea, on or within hours of admission, in association with a positive stool cytotoxin test for c.difficile and in the absence of hospitalisation within days. sixty-five cases ( %) were hospital acquired. fifteen patients ( . %) had cdad on admission; ( %) were community acquired, ( . %) had been recently hospitalised ( at st. james's hospital, at other hospitals). pms typing of faecal isolates revealed that predominant strains were responsible for the hospital outbreak; one of these strains was also isolated in community-acquired cases. this study suggests that the incidence of communityacquired cdad may be higher than previously reported. we suggest that all newly admitted or transferred patients with diarrhoea should be screened for this organism. several studies have confirmed that activated protein c resistance (apc-r) occurs in - % of thrombosis patients and is therefore more common than congenital deficiencies in the inhibitors of coagulation such as atii , proteins c and s. homozygosity for the factor v (fv) gene mutation is associated with a - fold increased risk of venous thrombosis while heterozygosity is associated with a - fold increased risk. the mutation, however, is highly prevalent in the general population, a prevalence of % has been reported in several european countries. its frequency in the population of northern ireland (ni) has not yet been reported. we screened a group of generally healthy elderly individuals (av. age . ; range - yr on several occasions for apc-r using an assay based on the prolongation of activated partial thromboplastin time by the addition of apc. a mean ratio of . (range . - . ) was measured. seven individuals ( . %) had ratios < . (av. . ; range . - . ). these subjects were then analysed for the fv mutation by pcr amplification and restriction analysis. the individuals with the lowest ratios (av. . ; range . - . ) were found to be heterozygous for the mutation. none of these individuals were deficient for protein c, protein s or atiii. the frequency of apc-r ( . %) within this ni elderly group is similar to that reported by others in the uk whose studies would have included a generally younger population. the successful ageing of these individuals perhaps underlines the low risk associated with heterozygosity. alternatively a higher prevalence of the mutation may exist in the general population of ni, where the incidence of heart disease is one of the highest worldwide. the insulin-like growth factor ii gene (igf ) is imprinted. thus, in contrast to most genes where both maternal and paternal copies (alleles) are transcribed into rna and expressed, gf is normally only expressed from the paternal copy (monoallelic expression). alterations causing biallelic expression of igf may lead to excess growth factor production, and thus, to tumourigenesis. this study evaluated igf expression in a series of childhood cancers. to date tumours have been evaluated using pcr based methodology ( wilm's tumours, rhabdomyosarcomas, miscellaneous). dna was extracted and a polymorphic site apal within the igf gene was amplified and digested. this identified samples as heterozygous for igf , meaning that separate maternal and paternal alleles were distinguishable. rna from these informative samples was extracted, pcr amplified and restriction digested, to identify monoallelic versus biallelic profiles at the expression level. samples with normal imprinting (monoallelic) displayed allele a ( bp) or allele bl/b ( / doublet). biallelic samples displayed both alleles. using this approach / informative wilm's tumours and / rhabdomyosarcomas demonstrated biallelic expression. in conclusion, biallelic expression of igf was detected in a significant number of wilm's tumours and rhabdomyosarcomas, and should be considered; with other genetic alterations, as a candidate mechanism in tumourigenesis. the proinflammatory cytokines, tnf~, il and il- , may mediate host metabolic and immune responses to cancer possibly leading to paraneoplastic phenomena such as cachexia. the cellular origin of these cytokines in the cancer patient, in many cases, remains unknown. we examined proinflammatory cytokine levels intracellularly, using flow cytometry, in pbmcs from oesophageal cancer patients (n= ) and age and sex matched controls (n= ). tnf~ and il- levels were significantly increased (p< . ) in pma stimulated t cells and monocytes from the cancer patients when compared to the healthy controls. these results were confirmed using standard elisa assays. following cotlagenase digestion, increased levels of tnfa and il- ,were detected in oesophageal tumour infiltrating t cells when compared to cells from normal mucosa. there was also increased production of tnf~ and il- , but not il-ib, in malignant epithelial cells when compared to normal and halothane and maintained by % nitrous oxide-oxygen, . - . % halothane, with spontaneous ventilation. tc rose marginally in group and fell in group (not significant, ns). tp in groups and at induction and and minutes were, respectively, (mean + sem, celsius) . + . vs . + . , ns; . + . vs . + . , ns and . + . vs . + . , (p< . ). overall incidence of shivering-was .%, but not significantly different between the groups. the data suggests that preemptive application of the space blanket increases tp in paediatric patients during general anaesthesia and tends to conserve tc. chronic actinic dermatitis (cad) an uncommon, eczematous, photosensitive eruption is diagnosed on history, clinical examination, skin biopsy and abnormal light tests. drug induced photosensitivity may look identical clinically, have a similar history and patients with cad may be treated with potentially photosensitising drugs. we therefore reviewed all patients with cad and compared their monochrumator light tests with patients who had drug induced photosensitivity. phototesting was performed on unaffected skin of the back with an irradiation monochromator; the minimal erythema dose (med) determined for a series of wavelengths between and nm, in patients with drug induced photosensitivity and patients with cad. of ten females, four males with drug induced photosensitivity, age range - (mean yrs), ten ( %), were photosensitive in the uva range ( - nm), the implicated drugs including, quinine, sparfloxacin, amiodarone, doxycycline, mefenamic acid, nalidixic acid, fenbrufen, diclofenac, enalpril and prochlorperazine maleate. three patients with rosacea were photosensitive to doxycycline. the re/nainder ( %), were tested after discontinuation of the drug and their light tests were normal. in the cad group, (four males and three females), age range - (mean . yrs), three patients ( %), were sensitive to uva, uvb and visible light, four ( %) to uva and uvb. in conclusion, uva dissociated from uvb photosensitivity seems a relative but not absolute sign of drug induced photosensitivity. this pattern of light tests should prompt a detailed drug history to elucidate the causative agent. phototesting should be performed while on the offending drug as testing days or weeks after discontinuation will give normal results. patients at risk of bone fractures by measuring bone mineral density (bmd) and markers of bone turnover and to assess the correlation of these with the severity of cld. twenty three patients with cld had bmd measured by dual-energy x-ray absorptiometry scanning of hip and lumbar spine. bone formation was assessed using serum levels of procollagen type peptide, osteocalcin and bone alkaline phosphatase, and bone resorption was assessed using hour urinary excretion of hydroxyproline, pyridinoline and deoxypyridinoline. % and % of patients had evidence of osteoporosis at the lumbar spine and femoral neck respectively. biochemical results showed that % of patients had an increase in all bone resorption markers and % had an increase in markers for bone formation. bmd at the lumbar spine was lower in patients with cholestatic liver disease compared to patients with other types of'liver disease (p= . ). no correlation was found between bmd and patient age, bilirubin, albumin, inr or duration of liver disease. conclusions: osteopenia occurs in up to % of patients with cld due to a high bone turnover state where bone resorption exceeds formation. osteoporosis is most severe in those patients with cholestasis. a detailed profile is presented of all leukaemia and multiple myeloma (icd-o code ) patients registered by the southern tumour registry during the -year period / "). annual age-adjusted rates of . and . per , were seen for males and females respectively. these levels indicate a lifetime (up to yr) risk of in for males and in for females. the main morphological sub-types registered were multiple myeloma ( %), cll ( %), aml ( %) cml ( %) and all ( %). one, two and five-year survival rates were examined; age at diagnosis and lesion type were extremely significant factors in relation to patient outcome. the overall incidence levels indicate that irish rates were relatively high by internatiomil standards r we assessed effects of reducing the volume of hyperbaric bupivacaine by giving half the volume as isobaric bupivacaine. when using . % hyperbaric bupivacaine for spinal blockade, the segmental spread and cardiovascular effects of the block have been shown to be dependent on the volume of local anaesthetic injected. patients undergoing elective surgery were studied in a prospective, randomised, double-blind trial: group ( patients) received their local anaesthetic as two equal aliquots of . % hyperbaric bupivacaine and . % isobaric bupivacaine respectively; group ( patients) received their local anaesthetic as two equal aliquots of . % hyperbaric bupivacaine. there was no significant difference found between the two groups with regard to maximal height of block (group , mean (range), t (t -ti ); group , t (ts-t )), rate of onset of blockade, or time to maximal blockade (group , mean (sem), . ( . ) rain; group : . ( . ) min). there was no difference found between each group in either cardiovascular stability or vasopregsor usage. the administration of a mixture of . % isobaric bupivacaine and . % hyperbaric bupiv/~caine confers no advantages over administration of the same volume of . % hyperbaric bupivacaine alone. propofolis used as a sedative during regional anaesthesia. providing titrateable sedation, it can compromise haemodynamic stability. a propofol ketamine combination provides stable haemodynamics during total intravenous anaesthesia, avoiding emergence phenomena m. we compared two sedative regimes in patients having spinal anaesthesia. following informed consent, patients, asa i-ii, undergoing spinal anaesthesia were randomized to one of two groups (n= ). group i (propofol-ketamine) received loading doses of . mg/kg propofol, . mg/kg ketamine followed by an infusion of . mg/kg/h and . mg/kg/h respectively. group ii (propofol) received bolus . mg/kg and infusion . mg/kg/h. subsequent infusion rates were titrated to effect using a sedation score. heart rate, blood pressure, oxygen saturation, end tidal c and oxygen requirements were recorded. observation continued for the recovery period and patients visited the following day. data were analysed using t-test, chi test and anova. groups were demographically comparable. sedative and respiratory indices were similar for both groups. there was no difference in total propofol requirements between the groups; group i - _+ mg, group ii - _+ : mg (mean _+ sd). there was a large difference in mean arterial pressure, being much lower in the propofol only group. both groups had an uneventful recovery without emergence phenomena. our results do not confirm the described additive effect of andketammc . ketamine.with propofol for sedation propofol ' =~ ) confers haemodynamic stability during spinal anaesthesia. we designed a controlled study to investigate whether there is a direct relationship between the degree of postoperative pain and the development of negative middle ear pressure in adults following tonsillectomy. middle ear pressure was measured by tympanometry. pressures were classified as type a (o to - mmh ), type b (flat) or type c (- to - mmh ) tympanograms. patients with type a tympanograms, undergoing tonsillectomy were enrolled in the study. patients had daily tympanometry whilst in hospital and then weekly until amrmalisatign. a questionnaire incorporating visual analogue pain scores was filled in at the same time. a control group of patients with type a tympanograms, undergoing appendicectomy and endotracheal intubation was used. follo~v up was available on patients. patients ( %) developed type c tympanograms, patients %) type b and ( %) patients remained unchanged. no member of the control group developed any change in middle ear pressure (chi squared = . , p < vol. , irish journal of supplement no. medical science . ). there was no relationship between pain scores for throat pain or otalgia and the development of negative middle ear pressure. patients recorded higher pain scores for throat pain at day then day , only of this group had negative middle ear pressure. middle ear pressure reverted to normal at day in / patients and in the remaining / it was normal at day . this study demonstrates the development of transient negative middle ear pressure following tonsillectomy in % of patients. this change is unrelated to the degree of postoperative pain nor is it associated with otalgia. postoperative ward analgesia remains suboptimal. this may be partially related to inadequate early use of opioids to attain minimum effective analgesic concentration (meac). we examined the incidence and predictors of severe postoperative pain on admission and discharge from our postoperative recovery room (rr). verbal pain scores were obtained in a pilot study of patients on rr admission and discharge. procedures were classed as open cavitary, laparoscopic, orthopaedic, ent or body surface surgery. intraoperative use and dosage of narcotics and nonsteroidal (nsaid) analgesics, anaesthetists' experience (prefellowship or post-fellowship nchd, consultant) were noted, and rr opioid usage recorded. pain scores and analgesic use were examined using mann-whitney, ~ analysis and logistic regression. moderate or severe pain was experienced by % of patients on either arrival or discharge. median intraoperative morphine dosage was mg. opioid use was slightly (median morphine dosage mg, p < . ) higher in patients undergoing cavitary surgery; these patients had the highest pain scores on rr arrival and departure. patients ( %) received > mg morphine intraoperatively. discharge scores of / or higher occurred in patients ( %). opioid usage and pain scores were unrelated to level of training. nsaid use/nonuse was unassociated with differences in opioid use or rr pain scores. no morbidity attributable to analgesic use (desaturation, slow respiratory rate) occurred. nonattainment of meac is frequent after open cavitary surgery. conservative opioid dosages continue to be employed despite inadequate early postoperative pain relief; this does not change with increasing experience. reporting such findings in departmental audit may help to alter perioperative management; such data may serve as a baseline for future interventional studies. diclofenac is frequently used for analgesia after tonsillectomy. recently concern has been expressed about the effect of diclofenac on prolonging bleeding time. one recent retrospective study found its use in tonsillectomy was associated with an increase in reactionary haemorrhage. we designed a randomised controlled study to compare the effects of rectal diclofenac and im pethidine given at induction with pethidine alone, in children undergoing tonsillectomy. fifty nine patients were entered into the study. there were males and females, mean age years, range ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . patients were randomised according to chart number. thirty five patients received rectal diclofenac after induction. twenty four patients acted as controls. there were no significant differences in operating time or operative blood loss between the two groups. in the recovery room the diclofenac group was significantly less restless than tl~e control group (p < . , chi squared test), with less crying, movement and agitation. there was no difference in postoperative recovery and no primary or reactionary haemorrhage. one patient in the diclofenac group developed a secondary haemorrhage. this study demonstrates a significant reduction in restlessness in the recovery room in children receiving rectal diclofenac. no increase in reactionary haemorrhage was demonstrated. diclofenac remains a safe and effective analgesic agent in children undergoing tonsillectomy. elderly patients have decreased dose requirements for many drugs compared to the young. few studies have examined dose requirements of opioids in the elderly when administered via patient controlled analgesia (pca) . we compared the pca morphine requirements between young and elderly patients. records were retrospectively analysed from , consecutive patients receiving pca for post-operative pain. inclusion criteria (i) age less than years or greater than years, (ii) upper abdominal surgery, and (iii) morphine pca usage. patients fulfilled the inclusion criteria. patients were young and were elderly. the mean age in the young was years and in the elderly was years. % were female in the younger group, % were female in the older group. pain scores at rest and on movement were similar in both groups . and . respectively in the young, . and . in the elderly. (p > . , students t-test). morphine usage over hours was + . mg in the young, and + . in the elderly. (mean + s.d.) (p.< . , students t-test). elderly patients required significantly less morphine via pca to achieve the same pain scores as the young. these findings are consistent with studies showing decreased requirements of other drugs in elderly. the erythrocyte sodium-lithium countertransport (slc) is abnormal in essential hypertension and some other forms of cardiovascular disease (cvd) but the considerable overlap in its activity in patients with these conditions and in normotensive healthy subjects remains a strong point against its possible utility as a marker for cvd. we sought to address this issue in greater detail. twenty-nine hypertensive patients ( with family history of cvd) aged . + . years (mean + se) and normotensive subjects ( with family history of cvd) aged . + . years, participated after informed consent. slc were determined in , , , and mm sodium chloride; and the vmax and km of the transporter determined. hypertensive and normotensive individuals with family history of cvd (n = ) had higher slc activity ( . + . vs . + . , p < . ), greater vmax ( . + . vs . + . mmol/lcell.h, p < . ) and lower km . mm (median) vs . (p < . ) than hypertensive and normotensive subjects without such a history (n = ). however, none of these parameters was sufficiently discriminatory as evidenced by the considerable overlap in the scattergrams for the two groups. on the other hand not only was the median quotient vmax/km significantly different . vs . (p < . ), but also the scattergram separated the two groups. this may reflect an effect of hereditary factors on the identified rate-limiting step in the transport system. capsaicinadministration results in depletion of substance-p sensitive nerves. this study was carried out to observe the impact on the morphology of mitral valve endothelium. the experimental group received capsaicin i.p. on day four of life; control animals received drug vehicle only. animals were anaesthetised by chloral hydrate and hearts were removed following perfusion of % glutaraldehyde, and were routinely processed for scanning electron microscopy. normal endothelial morphology showed an ordered and structured pattern, with large raised nuclei covered in discrete microappendages: no zoning was observed over the valve surface. following capsaicin administration, valves were seen to be torn and possessed a denuded endothelium. nuclear bulges changed in both apparent height and area, with the surface partially denuded of microappendages. one month following systemic administration of capsaicin to neonatal rats, a serious alteration of mitral valve endothelium morphology and integrity had occurred. depletion of substance-p may have resulted in mechanical insufficiency of the mitral valve. this study was funded by the health research board. with expanding applications and increasingly aggressive stress protocols, concerns about the safety of dobutamine stress echocardiography (dse) have arisen. the purpose of this study was to analyse prospectively the safety, adverse event profile, and complication rate of dse. prospective data was recorded in a consecutive series of patients undergoing dse for diagnostic evaluation of chest pain, for risk assessment following myocardial infarction or for detection of hibernating myocardium. the maximum dose of dobutamine used was mcg/kg/min in . % of patients and mcg/kg/min in . %. atropine was used in . % long-term outcome following coronary artery bypass grafting may be related to the prevalence of major risk factors and their treatment following surgery. we aimed to establish the prevalence and current management of coronary risk factors in a group of consecutive patients attending our hospital. this is a report of the first patients. data was collected by a structured patient interview, chart review, physical measurements and blood sampling. there were male and female patients, average age . years. the mean length of time since surgery was . years. thirty-nine patients had a recurrence of angina and this occurred on average ll months after surgery. as regards risk factors, were active smokers, were ex-smokers and only had never smoked. two thirds of the patients were taking regular exercise; only took no exercise at all. seventy-two percent of patients had a cholesterol greater than . mmol/l, yet only of the patients were on lipid lowering drug therapy and a further were on a lipid towering diet. twenty-nine patients had a systolic bp > or a diastolic bp > and of these were on antihypertensive therapy. seventy-seven patients were overweight but most of these had received specific advice regarding weight reduction in the preceding year. our results show a high prevalence of treatable risk factors in this high-risk group with inadequate treatment in many cases. new combined primary and hospital care strategies for cardiac rehabilitation and long-term secondary prevention of coronary heart disease are required. if the goal of modern therapy of acute myocardial infarction (ami) is preservation of myocardium, the occurrence of cardiac failure could be regarded as a treatment failure. in recent studies of iv thrombolysis and primary ptca the frequency of left ventricular failure (lvf) following ami has been as low as . %. this very low rate might be explained by selection bias in patients recruited to randomized trials. the purpose of this study was to examine the frequency of lvf in a consecutive alterations in nitric oxide (no) synthesis have been implicated in cardiomyopathy, ischaemic heart disease and septic shock. recent work has suggested a possible role for nitric oxide in cardiac arrhythmogenesis. the effects of inhibiting no synthesis with l-name (n c -nitro -l -arginine methyl ester) on cardiac electrophysiology have not been fully determined. the dominant frequency of electrically induced ventricular fibrillation was determined in l anaesthetised pigs ( - kg) using a fourier transform. the dominant frequency ( . _+ . hz in lead ii) was not altered by treatment ( . _+ . hz) with l-name in a group of pigs ( - kg) the effect of l-name ( mg/kg) was assessed in relation to energy required to defibrillate. there was no significant difference in the energies required to achieve successful defibrillation on % of attempts between the l-name group ( . _+ . j) and the control group ( . _ . j), and on % of occasions, l-name ( . + . j) and control ( . _+ . j). the results show that inhibition of no synthesis has no significant effect on the dominant frequency of ventricular fibrillation or on the efficacy of defibrillation in the pig heart. received either mg ( pts) or mg ( pts) oral flecainide followed by a maintenance dose of mg bd. all pts were euthyroid. none had significant hypertensive, valvular or ischaemic heart disease. pts had normal echocardiograms, had mildly dilated atria. of the pts ( %) converted to sinus rhythm, within hrs and at and days respectively. subsequently, he was found to have paralysis of all the muscle groups of his right upper limb apart from some flexor movements in his fingers with areflexia and sensory loss over the c /c dermatomes. the findings were thought to be in keeping with a brachial plexus.lesion. an mri scan showed a "haematoma" or "fibrosis" around the brachial plexus~ emg studies revealed a complete lesion from c /c with evidence of partial function at c and a good function at c . despite physiotherapy, at follow up months later there was no improvement in the emg findings. though brachia~ plexus injury has always been considered a complication of central venous line insertion <l), there have been no cases described, in the literature in the last years. central lines are mandatory irt major surgical cases but one must always be aware of the possible complications caused by them. body weight (bw) in icu patients fluctuates due to alterations in body mass ( ) and water ( ) and is usually estimated rather than measured. we hypothesised that (i) estimated weights are inaccurate and (ii) changes in true body mass (tbm) are masked by changes in water content. on admission, icu patients whose expected length of stay (los) was > days were placed on a bed incorporating load cells (accuracy of _ . kg). bw was estimated by icu staff. mbw was then recorded hourly under standard conditions. changes in mbw due to pack insertion/dressing changes were excluded. we calculated tbm as mbw minus cumulative fluid excess, corrected for insensible losses c ~. patients received standardized nutritional support from day and urinary nitrogen on day was calculated we studied patients whose mean (sd) age, mbw, los and apache ii score were . yr ( . ), . kg ( . ), . days"( . ) and ( . ) respectively. mean error in estimated weight on admission was . % (nurses) and . % (doctors). mean protein and calorie intake was g and kilocals/day. mean decrease in mbw during icu stay . kg/day (range . kg (gain) to . kg). mean reduction in tbm was . kg/day (range . - . kg/ day) and this correlated with urinary nitrogen loss (r= . ). in icu, (i) estimated weight is significantly inaccurate and should not be used in physiological calculations and (ii) rapid and significant decreases in body mass occur which may be underestimated due to fluid accumulation. dopamine appears to influence pituitary function and is associated with decreased circulating human growth hormone and insulin-like growth factor i m which could exacerbate catabolism, and therefore wasting, during critical illness. at hrly intervals from admission, patients whose expected length of stay exceeded days, were weighed (measured body weight, mbw) under standardized conditions,-using a bed incorporating electronic load cells (accuracy + o. kg). standard demographics, apache ii score and use of dot~amine by infusion was recorded. changes in weight due to removal./ insertion of prostheses, packs/dressings were excluded mbw was converted to true body mass (tbm) using measurements of cumulative fluid balance ( ml = kg) and insensible lossr patients received nutritional support, starting on day , based on their admission mbw. there were no significant differences in mean age, weight, length of icu stay, admission apache ii scores and mean daily protein/calorie intake between patients who had received dopamine by infusion (group d) and who had not (group nd). mean (sd) decreases in mbw during icu stay were . ( . ) kg/day (group d) and . ( . ) kg/day (group nd) (p< . i). mean decrease in tbm was . ( . ) kg/day and . ( . ) kg/day respectively (p< . ). thus group d were losing an additional . kg body mass per week relative to group nd. the use of dopamine by infusion is associated with an accelerated loss of lean body mass during critical illness. adhesion of polymorphonuclear leucocytes (pmns) to pulmonary endothelial cells is an initial step in the inflammatory process characterising the adult respiratory distress syndrome. previous studies using human umbilical vein endothelial cells (huvecs) have suggested that lipopolysaccharide (lps) is a potent stimulus for pmn adhesion to endothelial cells. the aim of this study was to investigate the effect of lps on pmn adhesion to human pulmonary artery endothelial cells (hpaecs). human pmns were coincubated with hpaecs _+ lps ( . - mg/ml) with % serum for hour. the effect of phorbol myristate acetate (pma) ( ng/ml) was also examined. percentage adhesion stimulated by pma was + sd. lps did not significantly increase adhesion at any of the concentrations used. to confirm the activity of lps pmns were incubated at ~ with . - mg/ml lps + % serum for hour and labelled with flourescent antibodies to the mac adhesion molecule complex (cd /cd lb). facs analysis indicated upregulation of both cd and cdllb. thus in the system used, lps did stimulate pmn adhesion molecule expression, indicating that the lack of adhesion reflects a difference in hpaec response to lps compared to that reported for huvecs. this work was supported by the health research board ireland. although inhaled corticosteroid therapy is of undoubted benefit in the management of asthma, dysphonia is a recognised sequela. this study was designed to examine longitudinally the effect of inhaled steroids on the voice and vocal cords of newly diagnosed and previously untreated asthmatics. twenty subjects were recruited and underwent voice and vocal cord assessment prior to and months after starting inhaled steroid treatment. the assessment consisted of ) rating dysphonia using a visual analogue scale, ) acoustical analysis of the voice and ) videostroboscopic examination of vocal cord activity. prior to commencing inhaled steroid therapy for their asthma subjects had normal voices, subjects were mildly hoarse and one was moderately hoarse. vocal cord pathology was noted in subjects, patients had vocal cord nodules and the remainder were noted to have mildly oedematous cords together with a glottic chink. at month follow up, improvement in voice was noted in subjects, one patient felt more dysphonic but there was no change in vocal cord appearance. one subject was noted to have developed a mid glottic chink with no associated change in voice. one subject had clearing of mild vocal cord oedema and improvement in voice. this study demonstrates that % of subjects commencing inhaled steroid therapy for asthma have mild vocal cord pathology. voice is more likely to be improved following use of inhaled steroids for months then made worse. although the relationship between elastin degradation and emphysema is well known, recent evidence suggests that a more complex process of pulmonary remodelling occurs within the emphysematous lung. the aim of this study was to assess the extent of extracellular matrix remodelling by ultrastructural examination of its two major components, elastin and collagen. emphysema was induced in rats by the intratracheal administration of porcine pancreatic elastase ( . u/g body weigh and human lungs were obtained at surgical resection for lung carcinoma. emphysema was confirmed histologically in both animal and human samples by measurement of the mean. linear intercept. matching sections were immersed in . m naoh and % formic acid to digest elastin and collagen respectively. scanning electron microscopy with stereo-pair imaging allowed -d visualisation of elastin and collagen frameworks. the distribution of emphysema was primarily panacinar in rat lungs a'nd centriacinar in human lungs. as expected in both types of emphysema, elastic lamellae were disrupted and perforated with multiple fenestrations. accompanying this disintegration was a marked increase in thickness of collagen fibrils which in some cases coalesced irish journal of medical science imparting a sheet-like appearance to the airspace walls. unique to human centriacinar emphysema, collagen formed helices which spiralled around alveolar septae to form bulky walls between adjacent airspaces. in conclusion, these findings lend support to the novel concept of aberrant collagen remodelling in the pathogenesis of emphysema. small cell lung carcinoma (sclc) is the most aggressive of the four common cell types of lung carcinoma. less than % of patients with sclc are alive two years after diagnosis. staging procedures, treatment regimens and survival results were reviewed in a small regional centre to make a comparison with larger treatment centres. thirty-one cases of sclc seen by one physician from to were reviewed. staging was clinical. treatment was undertaken in conjunction with the local oncology and radiotherapy services. % of patients had limited disease where as % had extensive disease at diagnosis. in patients with limited disease, % were alive at months and there was a % long term survival rate i.e. greater than three years. average length of survival in limited disease was days. survival results were comparable with those treated with chemotherapy alone and combination chemotherapy and radiotherapy. in patients with extensive disease the best results were from those treated with a combination of chemotherapy and radiotherapy with art average survival of days. these figures compare favourably with those from larger multidisciplinary centres. the factors contributing to our relative success may relate to continuity of care achieved in a smaller centre. nasal cpap is a very effective therapy for osa, but is cumbersome, and compliance varies. we prospectively evaluated consecutive osa patients treated with ncpap, who were asked to complete questionnaires before, and to months after starting therapy. this stttdy intended to examine both the patient's subjective response to ncpap and their bed-partner's impressions also. replies were received in ( %) patients. patients were divided i~to groups depending on whether they had a bed-partner, and according to their response to an initiat question assessing overall improvement in sleep quality and daytime al'ertness with ncpap, ranging from (minimal/none) to + (excellent). patients were called responders if they scored > . group a ( pts, %) were responders with bed-partners; group b ( pts, %) non-responders with bed-partners; group c ( pts, %), were responders ( pts, %) and nonresponders ( pts, %) without bed-partners; and group d ( pts, %) had stopped ncpap. nine questions were directed at the bed-partner, and assessed their perception of changes in both the patient's and their own sleep quality, daytime alertness, mood and quality of life, and also to changes in the relationship between patient and bed-partner following institution of ncpap. these questions scored from - (worse) to + (marked improvement). significant improvement in all parameters for the patient (mean + sd = . + . ) were noted in group a. in addition, group a bed-partners reported ~ubjective improvement in the same parameters ( . + . ). group b improvements were less, ( . + . in patients, and . + . in partners). overall, the data indicate a subjective success of therapy in % of patients, but the bed-partner's replies indicate this figure underestimates the true response rate. furthermore, the results show significant improvements in the bed-partner's sleep quality, daytime alertness, mood and quality of life, indicating that successful treatment of osa patients with ncpap also gives significant benefits to their bed-partners. vincent's hospital, dublin. neutrophil collagenase (mmp ) is a member of the matrixmetalioproteinases (mmps), a family of highly homologous zinc endopeptidases which play a crucial role in many physiological processes. the aim of this project was to develop a purification system for mmp from purulent sputum and raise polyclonal antibodies. after initial extraction, contaminating proteins were removed with a zinc chelate affinity column. mmp was then separated, from another closely related mmp, gelatinase b, on a q sepharose ion exchange column using a nacl gradient. the final purification step was carried out with an orange sepharose affinity column. sds-page analysis indicated the presence of purified protein with bands corresponding to latent neutrophil collagenase ( kd) and products of coll~igenase autodegradation at lower molecular weights ( kd & kd). a fold increase in specific activity was observed, with a % final yield, which provided mg quantities of pure enzyme. this work is funded by forbairt and the. irish american partnership. cd is a protein first described as a surface marker on hodgkin's lymphoma cells. recently cd has been demonstrated on th -type t lymphocytes (produce il- , , , l and ), which have a pro-inflammatory cytokine profile. but is not found on thl-type t lymphocytes (produce il- and ifn-gamma). its ligand, cd l, has also been described, cd -cd l interaction has been shown to aid the development oft lymphocyte clones into a th rather than a th phenotype. th -type cytokines are inextricably linked to the aetiology of inflammatory airway disease. firstly we investigated serum cd levels in various patient groups. we have demonstrated significant differences in serum cd levels in the following groups, atopic asthmatics (mean = iu/l, n = ), non-atopic asthmatics (mean = iu/l n = ) and atopic rhinitis/dermatitis (mean = iu/l n = ) and normal controls (mean = iu/l, n = ). secondly we cultured peripheral blood mononuclear cells from allergic individuals and normals. when these cultures were stimulated with house dust mite antigen (der p ) and il- or der p with both il- and i - , surface expression of cd on t lymphocytes could be demonstrated using fluorescent staining and flow cytometric analysis, after days culture in the allergic individuals but not in normals. the presence of i - in the culture increased the degree of surface cd expression. these results are important as they show that allergic individuals have an expandable population of memory t lymphocytes which respond to allergen by expressing cd and developing th- phenotype. most work on cd and th- cytokines has hitherto been carried out an t cell clones. we have developed a relatively simple in vitro system of looking at t lymphocyte response to allergen which will allow the testing of novel therapeutic interventions with a view to modulating the immune response in allergic disease. our work also suggests that even non-allergic patients with inflammatory airway disease may have increased th activity, which has not been shown previously. scimitar syndrome is a rare congenital disorder consisting of a spectrum of abnormalities including hypoplasia of the right pulmonary artery, dextroposition of the heart, anomalous pulmonary venous drainage of the right lung into the inferior vena cava and anomalities of the right diaphragm. bronchiectasis and respiratory tract infections on the right side are the usual clinical presenting features. a year old male patient was referred to the outpatient clinic with a history of recurrent chest infections which were slow to resolve following antibiotic therapy. physical examination revealed decreased air entry, coarse crepitations and a prolonged expiratory wheeze in the right lower lobe. the only abnormalities on routine biochemical and haematological screening were an elevated esr of and a slightly raised white cell count of . a chest x-ray revealed hypoplasia of the right lung when compared to the left. in addition to this there was a vascular shadow present in the right lower robe representing an anomalous pulmonary vein which appeared to drain to below the diaphragm on the night side. bronchoscopy showed a normal left bronchial tree. on the right, no apical segment was detected in the right lower lobe. otherwise no endobronchial lesion was seen. a dynamic computerised axial tomographic scan of the thorax was performed. this showed a dilated anomalous right lower pulmonary vein which was clearly seen to enter the inferior vena cava below the diaphragm. in addition the right lung was again noted to be hypoplastic when compared to the left. these findings were pathognomonic of the scimitar syndrome. "the patient was treated symptomatically and is presently stable. conclusion: scimitar syndrome, with its wide spectrum of abnormalities should be considered when reviewing plain chest x-ray in patients with recurrent right lower lobe respiratory tract infection. recent studies indicate that the ability of circulating neutrophils to regulate surface levels of adhesion molecules may be altered in disease situations. the aim of this study was to determine if changes in neutrophil responsiveness accompanies chronic inflammation in cf. neutrophils in blood samples from cf patients and age-matched control subjects were analysed by flow cytometry for expression of l-selectin and mac- (cd lb) following stimulation by interleukin- (il- ) and fmlp. as expected, both il- and fmlp provoked a decrease in surface levels of l-selectin and an increase in cdi lb levels. however, the magnitude of these changes was significantly lower in cf patients than in control subjects (table) . these results suggest that chronic exposure to inflammatory stimulii in vivo may alter neutrophil responsiveness in cf. given the emphasis on rational prescribing, we reviewed drug use in a bedded long-stay unit. prescribing patterns were analysed on an appointed day thereby obtaining a "snapshot" of prescribing practices. one hundred and ninety four long-stay residents, with a mean age of , were on drugs, the maximum number of drugs per patient was , the minimum and the average . . sixty percent of prescriptions fell within one of the following therapeutic categories:-central nervous system (cns) preparations ( prescriptions), analgesics ( ), gastrointestinal preparations ( ) and cardiovascular preparations (i ). there were ,prescriptions for respiratory drugs and only prescriptions were for antibiotics. the most commonly prescribed cns preparations were anti-psychotics ( ), benzodiazepincs ( t ), anti-depressants ( ). % of all analgesics prescribed ( ) were nsaids. the most commonly prescribed h blocker was cimetidine ( ). nuseals aspirin ( ), digoxin ( ) and captopril ( ) were the most commonly prescribed cardiovascular drugs. % of drugs were issued on an as required basis, i.e. "prn". the most commonly prescribed prn therapeutic classes were analgesics ( prescriptions) followed by gastrointestinal ( ) and cns preparations ( ). these results contrast with prescribing patterns in hospitals and general practice and may provide an insight into the challenges and realities of management in long-stay units. supported by the health research board. evaluation of physician requests to hospital based clinical pharmacist for ( ) drug information, ( ) possible adverse drug reaction (adr) was undertaken over a two year period from jan ' to dec ' (admissions - , , opd attendances - , ). overall requests were made. ( ) drug information: advice/information on new drugs, formulation, dosage, safety consideration prior to drug prescribing was given in cases. ( ) suspected adr: a total of suspected adverse drug reactions were investigated. in cases, no adr link was established, after extensive literature/data base search. adr's were confirmed in cases of which were reported to n.d.a.b. regular on-going interaction between physicians and clinical pharmacy allowed critical analysis of new drugs and heightened awareness of, potential adverse drug reactions in current clinical practice. we previously demonstrated that commonly prescribed medications are not easily identified by patients, doctors or nurses in the hospital setting o, ~. we then investigated the ability of hospital pharmacists, in all, to identify the same commonly prescribed branded and generic drugs. correct identification as follows:-bendrofluazide k ( / ), cimetidine ( / ), diazepam mg ( / ), diazepam mg generic ( / ), digoxin ( / ), ferrous sulphate ( / ), frusemide ( / ), mefenamic acid ( / ), paracetamol ( / ), prednisolone ( l/ ), temezepam ( / ), theoph /lline ( / ). pharmacists had % correct answers compared with % for nurses and % correct for doctors. the pharmacists had no difficulty recognising drugs with brand names written on them e.g. cimetidine, but like nurses and doctors had difficulty identifying the plain white tablets e.g. prednisolone. generic drugs were tess well recognised. a number stated that they were unwilling to definitively identify medication with no clear marking. pharmacists were also asked to list the top prescribed drugs, l got / correct and got / correct. in contrast out of doctors got / correct, got right and only got right. we conclude that hospital pharmacists are generally better than doctors or nurses at identifying commonly prescribed drugs but their knowledge of the top prescribed drugs is not as good at that of doctors. all professionals need assistant in this important task. suggesting reduced activity or more iranians with inherited variants of cholinesterase. one iranian subject with very low activity (dibucaine number below , atypical) had a history of apnea. these data indicate that the frequency of atypical and heterozygote genes for cholinesterase activity leading to prolonged apnea with succinylcholine (suxamethonium) is much higher in iranian than irish populations. this study emphasises the importance of ethnic pharmacology. it is has been advocated that funding for the prescibing of methadone in general practice should be provided separate from the indicative drugs budgeting scheme on the assumption that this may act as a disincentive to g.p.s to take on care of drug addicts. the objective was to analyse the current level and cost of methadone prescribing in general practices in the eastern health board over a six month period. there was a review of methadone prescriptions for gms patients from jan. to jun. . , persons received prescriptions. , scripts were issued. the age-specific prescribing rate for the total population was / , (males /i , , females / , ). males aged - years had the highest age specific rates ( / , ). the cost of methadone prescriptions amounted to s for the six months there was a trend towards an increase in the number of g.p.s who prescribed methadone over the period. only four of the g.p.s ( . %) who prescribed methadone issued in excess of scripts for the period studied. for a small number of g.p.s methadone prescribing is a significant cost item on their budget. in the light of this, government policy should be reviewed with a view to excluding methadone from the indicative drug budgeting scheme. sciences, mashhed, lran. there is increasing evidence that some of the wide variation in the response to medicines has a genetic origin which may be expressed on a racial basis. to further study inter-ethnic differences in pharmacology we compared the activity of an enzyme responsible for the breakdown of endogenous substances and drugs -serum cholinesterase (pseudocholinesterase), dibucaine and fluoride numbers -in irish and iranian healthy subjects. irish subjects had significantly higher serum cholinesterase activity ( . + . vs . + . u/ml, mean + sem, p < . drug prescribing data may reflect changes in therapy and disease pattern. we reviewed current drug use among patients (n = ) in a dublin teaching hospital in "snapshot" fashion on a designated day, and compared it with that obtained in . in , patients received an average of . different drugs each, with % on or more and % on none. by , the average was . (range i - ), with % on or more. the percentage of patients receiving heparin fell from % to %, due mainly to a reduction in use on the medical side. the proportion of patients prescribed hypnotics fell from % to %, while ssri's are now the most used anti-depressants. antibiotic choice changed from amp/amoxicillin to coamoxiclav and the cephalosporins. diuretics remained the most frequently used cardiovascular agents, accounting for % of all drugs used and prescribed for around one quarter of patients. digoxin use remained constant, and by , % of patients were on anti-platelet doses of aspirin. at least four different agents were in use in each of calcium antagonist, beta blocker and ace inhibitor classes. some of these changes in therapy reflect therapeutic advances, changes in disease management, greater choice of therapy and amendment of less than desirable therapeutic practices. on the other hand, some may reflect fashion or pharmaceutical promotion, rather than change as a consequence of evidence-based practice. acknowledgements: pharmacy staff, st. james's hospital and the health research board. studies of in vivo endothelial function in humans have usually involved intraarterial cannulation and the subsequent administration of substances that stimulate the endothelium to produce nitric oxide (no). such techniques are invasive and potentially hazardous. an alternative non-invasive method would be of benefit. animal studies have indicated that reactive dilation of vascular beds may be at least partially endothelium dependent. this study aimed to determine whether reactive hyperaemia in the human forearm was an endothelial dependent process with the potential to be used as a non-invasive method of stimulating the endothelium. ten volunteers underwent brachial artery cannulation and randomly received either placebo or n-monomethyl-l-arginine (l-nmma) ( ~mol/min), an no synthase inhibitor, for minutes. following this reactive hyperaemia was induced by the inflation of an arm cuff to mmhg for minutes and the response to this was measured by strain-gauge plethysmography. when flows had returned to baseline the process was repeated with the remaining substance. results were analysed by repeated me~isures anova. l-nmma resulted in significant reduction of basal forearm blood flow (p< . ). there was no significant difference in reactive hyperaemia with either l-nmma or placebo. in conclusion, no does not contribute to reactive hyperaemia in the human forearm. dublin . home-based infusion therapy has been widely recognised as the optimum for treatment of disease states that require daily intravenous therapy from a patient-care aspect. conditions necessitating intravenous therapies in hiv disease include: cmv retinitis, intractable cryptosporidial diarrhoea, azole-resistent candidosis, nutritional support with total parenteral nutrition, chemotherapy for aids-related malignancies and palliative care in the terminal phase of the disease. the need for such therapies is increasing as patient survival improves. in , the home-infusion service was set up in recognition of the need to treat patients, requiring intravenous therapy, in the home environment. this has been brought about by the development of small, light-weight pumps suitable for ambulatory use, the development of a service for aseptic compounding and the availability of permanent in-dwelling venous catheters. we describe the impact of this service on our patient cohort. to date, sixty-five hiv positive patients have received parenteral therapy at home. patients' age, sex, risk group, cdc stage, cd count, indication for therapy, complication rate and response to treatment are described. the provision of this service has reduced the number and length of patient admissions with associated improvement of quality of life. in addition, it recognises that patients prefer to be treated in the home environment aided by a co-ordinated multidisciplinary approach. since blood alcohol levels over mg/ ml are now illegal for vehicle drivers we have investigated if the commonly held "safe" limit of two drinks will bring the young adult over the legal limit and if this amount of alcohol will affect their psychomotor skills. following informed consent healthy volunteers, nonhabitual drinkers on no medication ( male, female), with a median age of (range - ) years participated and refrained from alcohol for at least days. each drank within minutes two standard drinks ( . ml each ) of . % vodka ( . units of alcohol) plus ml of orange juice at about minutes after a standard mid-day meal. their psychomotor performance was estimated by the number connecting technique at minutes after alcohol consumption and they were also asked to rate their feelings (which included alertness, clear-headedness, competence and attentiveness) using a visual analogue scale of to . blood samples at one and two hours later were collected from the antecubital vein and analysed on the same day for alcohol content using enzymatic methods. mean (+ sem) blood concentrations of alcohol at and hours respectively were . + . and . + . mg/ ml. in males and . + . and . + . mg/l- ml in females. values were significantly higher in females. blood concentrations in females were also higher (p < . ) than in males when expressed per kg body weight. while the blood alcohol in both the genders was considerably lower than the current legal limit in ireland their psychomotor skills as estimated from their task completion time and their answers to questionnaires were indicative of an impaired cns function. thus while drinks may keep many subjects below the legal limit, there is considerable inter-individual variation with females showing higher concentrations and both genders have evidence of impaired performance at these lower levels. a non-linear approach was used to develop an hrv parameter, robust to both data non'-st~itionary and missing data points. unlike the standard chaos approach, using higher dimensional embeddings and time-delays, we employed a onedimensional correlation integral plot. the parameter thus obtained, allows an estimate of the spatial spreading of the attractor (ssa) or spatial variation of rr intervals along a straight line. heart rate data from volunteers ( : to : hr), ~ifter oral placebo or propranolol mg, investigated the ability to detect drug effect. vitamin e (~-tocopherol) is the most important dietary antioxidant in lipid and cell membranes and its intake reversely relates to the incidence of coronary heart disease and certain cancers. estrogen regenerates oxidized tocopherol radical in vitro m but such interaction has not been investigated in postmenopausal women receiving estrogen containing hormone replacement therapy (hrt) although estrogen containing oral contraceptive may reduce plasma vitamin e levep. we studied healthy post-menopausal women (aged - ) ammenorrheic for at least one year. fifteen subjects took a combination of harmogen provera therapy and acted as a control group. blood samples were taken from all subject at baseline and after weeks. in the hrt group, serum fsh levels were greatly reduced ( . + . vs . + . iu/ , p < . , mean + sd, after hrt) with an increased serum oestradiol level (< - . vs . + . umol/ , p < . ). no change occurred in the control group. vitamin e status, measured either as plasma or red cell ~-tocopherol respectively showed no change in both groups (hrt group . + . vs . + . , . + . vs . + . gmol/ , p > . ). we conclude that in post-menopausal women, weeks estrogen containing hrt did not alter vftamin e concentrations in vivo. we assessed the clinical benefit of the newer markers of bone formation: osteocalcin (oc), procollagen carboxyterminal peptide (picp), bone alkaline phosphatase (balp), and bone resorption: carboxyterminal telopeptide of type collagen (ictp) and urinary deoxypyridinoline crosslinks (dpd) over traditional assays such as total alkaline phosphatase (talp) and urinary hydroxyproline (oh/pr) in patients with primary hyperparathyroidism (phpt). patients were sampled basally, then at , , , and hours post surgery and again at . , , and months post op. the mean basal p cp level was + ug/l (normal: - ) this increased to a peak at h ( +_ ug/l), then declined to normal at weeks ( + ug/l). mean basal urinary dpd levels were raised at . + . nm/mm cr. (normal . - , ), they had normalised by months to . -+ . nm/mm cr. mean balp levels were always normal, although normal the yearly mean oc level was significantly lower than the basal value. mean ctp, oh/pr and talp levels were always normal. therefore bone turnover in phpt is best assessed by the newer markers picp and dpd. we have previously described seasonal variation in fibrinogen with higher levels in winter. as fibrinogen is an acute phase reactant, the winter rise may be a response to seasonal infections. the present study investigates this hypothesis by examining seasonality infibrinogen and markers associated with infection: white cell count (wcc), interleukin- ( l- ), human herpes virus (hhv ) and herpes simplex virus (hsv) antibodies. monthly blood samples from healthy volunteers age and over were measured for fibrinogen, wcc, il- , hsv and hhv reactivation over a year time period. a rhythmometric method was used to examine the data for seasonality. statistical significance was measured using the fstatistic. a highly significant seasonal variation (sv), peaking in mid-february, was found for fibrinogen (n= ; sv= . g/ ; f= . ; p< . ). no significant seasonal variation was present for measures of wcc (n= ; sv= . e /l; f= . ; p> . ), hhv (n= ; sv= . au; f= . ; p> . ), hsv (n= ; sv= . au; f= . ; p> . ) or il- (n= ; sv= . pg/ml; f= . ; p> . ). the present investigation does not support the hypothesis that seasonal variation in fibrinogen is a direct effect of the acute phase response, initiated by a seasonal variation in level of infection. the explanation for the seasonal changes in fibrinogen remains unknown. increased plasma homocysteine and reduced plasma antioxidants are risk factors in the development of vascular disease. design: subjects drawn from elderly people living in the community (median age yr, range - yr; female). total plasma homocysteine, vitamin c, gamma tocopherol, retinol and beta carotene were measured by high pressure liquid chromatography. homocysteine levels in elderly males [median (range) = . um ( - . ), n= were significantly higher than in vol. , supplement no. irish journal of medical science elderly females [ . um ( . - . ), n= ]. these values were also higher than in a younger ( - years) male cohort [mean = . um, n= ]. no correlations to vitamin concentrations were found, nor was there a correlation to age within the elderly cohort. within the elderly females, a significant negative correlation with age was found in vitamin c, gamma tocopherol and beta carotene (p< . ). however a significant increase in retinol was noted. a very strong correlation between vitamin c and gamma tocopherol levels was noted in the elderly population sample (p< . after multiple regression). conclusion. homocysteine levels in the elderly are higher than in samples of a younger population. a gender difference is maintained in the elderly. the provision of extended care forms one part of a spectrum of health care for older people. in the eastern health board area all patients over the age of must be assessed by the multidisciplinary geriatric team prior to placement. we report on the experience of the total number of referrals for assessment for extended care to one department of geriatric medicine in a bed teaching hospital. ninety-eight patients listed for extended care in . the mean number of days between listing for long term care and placement was _+ days (range to ). almost one quarter of patients died while in hospital awaiting long term care: this underlines the frailty of patients who are admitted to hospital and request long term care. two patients were transferred to other institutions and patients were able to get home. of the remaining patients ( %) were placed in statutory or voluntary long term care accommodation and only % were eligible (usually financially but in some cases due to significant disability) for nursing home care using the terms of the nursing home act. patients who are listed for long term care through a general hospital are in general very frail, they tend to have a very extended length of stay and the provisions of the nursing home act only apply to a minority. these findings underline the need for provision of adequate statutory and voluntary extended-care places within the eastern health board area. there are over screening assessments for cognitive function and choosing the most appropriate may be difficult. increasingly the importance of behavioural dysfunction is recognised. can any of the cognitive assessments help to predict behavioural dysfunction.'? we compared and contrasted the folstein mini mental state examination (mmse) and the cognitive assessment schedule (cas) of the clifton assessment procedures for the elderly (cape) and compared them with the behavioural rating scale (brs) of the cape. the study was carried out on a total of referrals to the occupational therapy departments by geriatricians in the meath hospital and st. james's hospital. all subjects were over and medically stable. the time scale involved was may-july . the mmse and the cas were administered within the one sitting and each was timed. brs was rated the same day by either a staff or family, member. the average time to complete the mmse ( + s) was longer than the cas ( + s) but this was not statistically significant. the mmse and cas were significantly correlated (r = , p < . ). the cas was significantly correlated with the behaviour scale (r = , , p < . ) whereas the mmse was not. these results suggest that equivalent assessments of cognitive function may be made with the mmse or cas, but a low cas score will be a better prediction of behavioural dysfunction. a spectrum of neurological and myopathological changes are associated with patients in intensive care units. we observed several patients post discharge from icu who presented with unexplained dysphagia which we suspected may be associated with the neurological complications of sepsis. the particular complication of dysphagia as a neurological manifestation of sepsis has not been documented. our descriptive study presents a series of three patients with persistent dysphagia which may represent a similar phenomenon. we selected patients for the study ranging from - years of age and on the basis of medical history including icu stay, sepsis, and intubation. all patients presented with dysphagia as observed on videofluoroscopy. we studied the video findings in-depth in order to ascertain if similar swailow patterns were present in these patients and if this could be correlated with their medical history. each of the three patients presented with similar dysphagia signs. the oral phase of the swallow was moderately atypical but the pharyngeal phase was significantly atypical. it was felt that intubation alone was not the sole causative factor of this dysphagia. the polyneuropathy associated with sepsis in icu may explain the atypical swallow patterns observed in these patients. the severity of the persistent dysphagia can cause serious respiratory and medical consequences. there is a need for further investigation of this phenomenon to identify patients who are at risk. little attention has been paid to the prevaience and phenomenology of behavioural disturbances among medical patients despite awareness of the high prevalence of cognitive vol. , supplement no. impairment in this patient population. we screened consecutive admissions to a department of acute geriatric medicine. patients were evaluated over a week period using a modified version of the brief agitation rating scale. medication use, cognitive function and impact on nursing time were also measured. the prevalence of behavioural disturbance in this population was / ( %). the most frequent behavioural abnormalities were restlessness ( ), complaining ( ) and screaming ( ). the most common underlying disorders were dementia, stroke disease, personality disorder and paranoid psychosis. the behavioural disturbance was only documented in the medical notes in patients ( %) and in only cases was a psychiatric consultation sought. these findings demonstrate that behavioural disturbances are not only common but also under-documented in elderly medical patients and there is a need for training in the detection and management of behavioural symptoms in this patient group. in lower limb trauma where there is severe compound fracture, the successful treatment of this depends on adequate bone and soft tissue debridement. as a result, subsequent bone defects can lead to instability and often require large amounts of bone grafts, and major soft tissue reconstruction is reaquired to obtain skin cover. large soft defects can by reduced bv primary bone resection and shortening of the limb. this will improve the chance of bone healing if performed in the presence of an external fixator, then lengthening at a site away from the traumatised area can gradually restore limb length. two cases are presented to demonstrate .the effect of compression / distraction techniques on soft tissue and bone injuries in these difficult situations. wegener's granulomatosis -wg ( ), churg strauss syndrome -css ( ), polyarteritis nodosa -pan ( ) and unclassified ( ). using the chc definitions, the diagnoses were wg ( ), microscopic polyangiitis -mpa ( ), pan ( ) and undefined ( ). there was concordance in only patients (all wg). there is significant discordance between these two criteria sets. since the acr criteria does not recognise mpa, they tend to overdiagnose wg. in addition, the chc criteria cannot be applied without a biopsy and therefore surrogate features which predict the underlying histology are required to allow more practical application of the chc definitions. the objective was to determine the value of examination of dried freshly produced saliva, under light microscopy, in patients with xerostomia related to secondary sjogrens syndrome. ten patients with known connective tissue disease or rheumatoid arthritis attending rheumatology clinic were enrolled into the study, all with symptomatic xerostomia and dry eyes. all had an abnormal schirmer's test. five normal patients were enrolled, all of whom were without clinical evidence of rheumatological disease. control patients were enrolled who had no clinical evidence of rheumatological disease, a salivary sample was collected and examined by light microscopy. serum was also examined for the presence of anti-ro/la, rheumatoid factor, and anti-nuclear factor. all ten patients demonstrated 'reindeer horn' type ferning of saliva, a pattern of shorter thicker clubbed branches of crystallised mucus, in contrast to the normal ferning pattern of the healthy subjects. conclusion: we have shown in this preliminary report that light salivary microscopy is a simple test easily performed in an outpatient setting which could be a useful diagnostic procedure in sjogrens syndrome. recently, two sets of criteria have been proposed for the nomeclature of primary vasculitides, the american college of rheumatology (acr) classification criteria and the chapel hill consensus conference (chc) definitions. the aim of this study was to determine the concordance of these two systems in a cohort of patients with primary systemic vasculitis. patients with systemic vasculitis were recruited who had a biopsy proven diagnosis or, who had typical clinical features associated with a postive antineutrophil cytoplasmic antibody (anca). the case notes were reviewed and patients were classified according to both sets of criteria. twenty-six patients were recruited, of whom had a positive biopsy. applying the acr criteria, the diagnoses were, primary pulmonary hypertension (pph) typically affects young individuals, and has a high morbidity and mortality. secondary pulmonary hypertension complicating connective tissue diseases likewise carries a poor prognosis. we evaluated the acute and chronic effects of ketanserin, a selective serotonin type- receptor antagonist in patients with pulmonary hypertension in the acute study ketanserin was administered as a peripheral venous infusion during right heart catheterisation. following encouraging results during catheterisation oral administration of ketanserin mg daily in divided doses was instituted. in patient , a year old female with probable pph, serial cardiac catheterisations over a year period showed a significant, sustained reduction in both mean pulmonary artery pressure from mmhg at baseline to mmhg at year (normal - mmhg) and pulmonary vascular resistance units at baseline to units at year (normal < units). in patient , a year old female with limited scleroderma (crest) echocardiography after month's oral ketanserin showed a reduction in estimated peak right ventricular systolic pressure from mmhg at baseline to mmhg (normal range - mmhg). the acute and long term response to ketanserin with improyement in pulmonary haemodynamics in these patients suggests that if a beneficial effect is detected during catheterisation long term oral therapy may be worthwhile. levels were low (< . iu/ ); normal though above average (>- -< iu/l) and moderate high (> -< iu/l) respectively. gonadotrophins for ovarian stimulation were commencing initially at iu for group a & b and at iu for group c. ivf performance was poor in most aspects (total follicles, oocytes & embryos transferred) in group b comparing with group a or c; the cumulative ongoing pregnancy rate (pr) over ivf cycles in group b; was % comparing with . % in group a (p < . ) however there was no significant difference in pr in group c ( %) comparing with other two groups. cycle day fsh screening is predictive of follicular development in ivf. high initial dose of gonadotropins help to improve the pregnancy rate in the presence of moderate high level of fsh. the purpose of this study was to evaluate patient satisfaction with antenatal care provided in the perinatal day centre (pndc). a self administered questionnaire was administered to consecutive patients. the main indications for referral were suspected small-fordates ( %), non-proteinuric hypertension ( %), glucose tolerance testing ( %), reduced fetal movements ( %) and post-term evaluation ( . %); % were nulliparae. thirty-two percent of patients were reviewed in the pndc on the day of referral; the rest within days. twenty eight percent of patients lived more than miles from the hospital and % spent more than minutes in travelling there. eighty five percent of patients scored their level of satisfaction with the service provided in the pndc as > out of ; only . % would have preferred admission; % said that they would prefer to visit the pndc times per week to avoid admission. the main area of dissatisfaction related to the waiting time for review prior to discharge, with . % of patients waiting over hours. patients attending the pndc report a high level of satisfaction; changes to reduce the visit duration have been introduced. to examine the change of taking-up the essential preconceptual measurements; rubella immune status, cervical cytology and prophylactic folic acid intake; following specific advice and publicity through general public meetings with new patients prior to in vitro fertilization (ivf) programme. in we studied new couples for ivf for the presence of some specific pre-conceptual data (group a). in this study we follow-up the same intake in another new women interviewed to commence ivf programme from january till september (group b). in group (b) the taking-up measurements were dramatically improved. however, % and % stilldid not have rubella immunity test and cervical cytology performed; compared to % and % in group (ai respectively (p< . ). while folic acid intake was sustained at > % in both groups. following specific advice the rate of taking-up of preconceptual measurements prior commencing ivf programme was improved. there is a future need for continuous enhancement of the publicity and advice regarding the importance of preconceptual measurements. the aim of this study was to introduce icsi to ireland for treatment of specific cases of male factor infertility. following an introductory proving period using the bovine model, thirtyeight couples with infertility attributed to the male were selected for an icsi attempt. ovulation induction, oocyte retrieval and luteal management were as described for conventional ivf tm. the average age of patients selected for icsi were . + . years and . + . years for the female and male respectively, with an average duration of infertility of . + . years. a year old woman presented with a three day history of parasthesia in her lower limbs and difficulty walking. neurological examination revealed sensory loss in her limbs and truncal ataxia. rombergs sign was positive. pelvic examination revealed a large pelvic mass that was distinct from the uterus. routine blood investigations were normal. csf culture, ct brain and serum electrophoresis were negative. anti-purkinjie cell antibodies were not present. ca- levels were elevated at micrograms/litre. laparotomy revealed a cm left ovarian tumour. a total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy was performed. histology revealed a poorly differentiated clear cell adeno-carcinoma of the left ovary. the capsule was intact and peritoneal washings were negative. she made a good postoperative recovery. she received six courses of carboplatin without ill effect. her neurological symptoms resolved. subacute cerebellar degeneration can occur as a paraneoplastic disorder in ovarian carcinoma. the mechanism by which cancer can cause neurological disorders is not fully understood. paraneoplastic cerebellar degeneration occurs with or without the presence of purkinjie cell antibodies. the aim was to review all red cell transfusions in gynaecological surgery in . a retrospective review of blood bank records and individual charts was carried out. patients underwent gynaecological surgery; ( %) were cross matched and ( . %) were transfused. units were transfused. there were no single unit transfusions. the mean number of units transfused per patient was . this accounted for % of all units transfused this year. % of patients were undergoing elective surgery. the overall cross match/transfusion ratio was . intraoperative difficulty was recorded in % of cases. % of patients were transfused perioperatively and % postoperatively. the percentage of patients requiring blood transfusions in the the main individual operation categories was as follows: radical surgery: %; total abdominal hysterectomy and salpingo-oopherectomy, %; vaginal hysterectomy and repair, %; subtotal abdominal hysterectomy, %; vaginal hysterectomy alone . %; and total abdominal hysterectomy alone %. adverse reactions to transfusions were seen in % of patients. conclusion: the majority of patients transfused were undergoing elective surgery. vaginal hysterectomy was associated with greater blood loss than abdominal hysterectomy. only half of all units cross matched were transfused. dilatation and curettage (d+c) is the most common operation performed in the u.k. the liberal use of d+c has been criticised. the objective of this study was to evaluate the use of outpatient endometrial pipelle biopsy and determine its safety in terms of detecting abnormalities. complications and financial costs were also evaluated. data was reviewed from an active gynaecological unit from february to january , using theatre and outpatient records. a total of d+cs and endometrial pipelle biopsies were performed in this period. malignancies were detected by d+c and by pipelle biopsy. a total of and benign abnormalities were detected by each method respectively. there was a higher complication rate in the d+c group but the failure rate was higher in the endometrial pipelle biopsy group. the monetary savings over this period is estimated at s there were no missed malignancies to our knowledge over the year period since endometrial pipelle bioposy was introduced to this hospital. our study indicates that outpatient endometrial pipelle biopsy appears to be safe, efficacious and economical. while ultrasound findings may sometimes be in conflict with clinical examination, it is the case that there are instances when ultrasound findings have, following subsequent laparotomy, been found to be wholly incorrect. it is therefore not surprising that there remain some gynaecologists who view ultrasound with scepticism, preferring to rely solely of their clinical findings. there have been few studies that directly compare clinical, ultrasound and surgical findings in the detection of pelvic masses. the objective of the study was firstly to directly compare the reliability of clinical and ultrasound examination findings in the detection of pelvic masses proven by subsequent laparotomy and secondly to determine the accuracy of ultrasound in detecting malignancy. this was as a retrospective review of women who underwent a laparotomy because of a pelvic mass between january to february . information was obtained from theatre and patient records. real time abdominal ultrasound was used. findings at laparotomy were correlated with clinical and ultrasound findings. the sensitivity and specificity of ultrasound in detecting a uterine mass was % and % respectively. this contrasts sharply with clinical examination (sensitivity = % and specificity = %). similar findings were obtained when ultrasound was compared to clinical examination in detecting ovarian masses. ultrasouud is capable of predicting benign disease with reasonable confidence but the prediction of malignancy is less reliable. in conclusion, ultrasound is more sensitive and specific in detecting pelvic masses compared to clinical examination. vincent's hospital, dublin. osteoporosis occurring during pregnancy or lactation is a rare event despite the homeostatic demands of the foetus for calcium. we investigated the case of a year old woman who, immediately following vaginal delivery of her first child, developed severe back pain due to a vertebral compression deformity of the second lumbar vertebrae. bone mineral density (bmd) was measured by dual-energy x-ray absorptiometry. calcium metabolism and bone turnover were studied. there was a severe reduction in bmd in the spine (z-score = - . ) and f e m o r a l neck ( z -s c o r e = - . ); but, serial measurements showed no further reduction in bmd. indices of calcium metabolism and bone turnover were normal. pregnancy-induced osteoporosis is a severe but self-limited disorder in calcium homeostasis of unknown aetiology. women with low bmd prior to pregnancy may be at increased risk. in view of increased demand, supplemental calcium and vitamin d should be considered during pregnancy and lactation. crumlin, dublin . the aim of this study was to assess the clinical status on admission and the critical care m a n a g e m e n t of children p r e s e n t i n g with m e n i n g o c o c c a l i n f e c t i o n . t h i s was a retrospective study of the charts of consecutive admissions. mean age was . years (+ . ). the average duration of symptoms prior to admission was . hours (+ . ). on admission . % were hypotensive, . % had clinical signs of haemodynamic instability and . % of cases that had a wood gas analysis on admission had a metabolic acidosis (bases excess < - . ). the mortality rate was . %. % of deaths were hypotensive on admission and all had a metabolic acidosis. of the survivors . % were hypotensive on admission, % had clinical signs of cardiovascular compromise, % were admitted to the high dependency unit, % required invasive pressure monitoring and . % were ventilated and received inotropic support. in this study children presenting with m e n i n g o c o c c a l infection have a high incidence of cardiovascular instability. successful management is dependent on early presentation and initiation of therapy and on aggressive intensive care monitoring and support of the cardiovascular i and vital organ systems. the normal crying curve and incidence of colic for term infants are well known. we studied prospectively the crying pattern and the incidence of colic in preterm'infants to determine if prematurity influenced these behaviours. the subjects were consecutive preterm infants admitted to the cork neonatal units for two and a half months from july . a continuous hour diary was completed on each infant by the neonatal nurses, when the babies were on full oral feeding and no longer required intensive care. the parents completed the diaries 'after discharge. colic was defined according to wessel's rule of threes. two unwell babies were excluded. the duration of follow up was from to weeks. fifty infants were recruited and completed the study ( lost to follow up and one withdi'awn due to sepsis). their mean (range) gestational age was ( - ) weeks and birthweight was . ( . - . ) kg. the mean (range) age of crying onset was x(y-z) weeks; crying, peak 'was x(y-z) weeks and crying offset was x(y-z) weeks. one baby developed colic in the period of follow up. conclusions: the incidence of wessel's colic was less than expected in these preterm babies. the crying pattern according to chronological age was different from that clescribed in term babies. in general preterm babies had a delayed onset of crying, but the pattern became similar to term babies when allowance was made for gestational age. the findings'suggest that the crying patterns of early infancy have a developmental basis. we re-evaluated children who had had rs ( - ), with their closest-age siblings using the wechsler scales, coopersmith self esteem inventory and achenback child behaviour checklist (acbc) (duffy j. et al ). the rs patients' means were consistently lower than that of their sibs. however, comparison of mean raw data, using "t-tests", yielded significant differences only in the acbc scores, in that rs children exhibited significantly more problem behaviours than their sibs (p= . ). after categorisation of iq data, further comparisons between the groups (using x ), found rs patients were significantly more likely to score "below average" in tests of verbal iq compared to their sibs (p= . ). age of onset and clinical stage were also found to be more important predictors of outcome. children less than year of age at onset of rs had significantly lower iq scores on all measures of cognitive ability (p= . ) and more problem behaviours (p= . ) than children over year of age. no significant differences were found in comparison with sibs. clinical stage to which rs progressed affected only verbal iq scores. children in whom consciousness had been impaired had significantly lower iq scores than both their sibs and rs children in whom consciousness was less impaired (p= . ). in conclusion outcome remains cautiously positive, with / rs children attending mainstream schools or in employment without apparent difficulties. a national breastfeeding policy was introduced by the department of health in . factors identified for promotion of breastfeeding were based on the who/unicef "ten steps for successful breastfeeding". we present clinical cases which suggest that one step may need to be modified. the charts of breastfed babies admitted to the special care baby unit were reviewed for one year following the introduction of the national breastfeeding policy to this hospital. thirteen term breastfed babies were admitted because of fever and dehydration. none of the babies had water or bottle feed supplements. ten of the thirteen mothers were primigravida. eleven babies were admitted in the six months following the introduction of an exclusive breastfeeding policy. the nursing staff were then alerted to the risk of dehydration, but two further babies of mums committed to exclusive breastfeeding were admitted in the subsequent six months. routine biochemistry, haematology and a limited septic screen was performed in all babies. three of the thirteen babies had lumbar punctures. the mean (range) weight loss on admission was . ( . - )%. the mean (range) plasma sodium level was . ( - )meq/ and the mean (range) urea was . ( . - . )meq/t. there was no growth from the cultures of the blood, urine, csf and swabs. all the babies were given intravenous fluids and parenteral antibiotics for hours. the outcome was satisfactory in all babies and breastfeeding was reestablished in eleven of the thirteen babies. conclusions: the common factor to these babies was inadequate fluid intake prior to admission associated with stricl~ adherence to the policy, and avoidance of all supplements including water. we conclude that the who/unicef step "not to give food or drink other than breastmilk unless medically indicated" is too restrictive in the immediate postpartum period. . % of children reported headache in the previous months . % of girls and . % of boys reported headache (p < . ). . % of children reported daily headache . % of children reported weekly headache . % of children reported monthly headache . % of children reported headache less often than monthly the percentage of children with headache at each frequency, other than daily, increased with increasing age. in girls headache showed a marked rise at ages , and years. reported prevalence of headache in the past year in to year old aberdeen children was % and % was recorded for swedish children aged , , and years old. this study is the first community based prevalence study of headache in irish schoolchildren. our aim was to test the hypothesis that there is no correlation between the type of feeding & swallowing disorder the child has with the neurological diagnosis or the radiological findings. a further purpose was to develop a classification of the feeding & swallowing disorders which would guide us towards a management plan. a retrospective analysis of the data collected between the years - from the feeding & swallowing clinic at booth hall children's hospital was done. children were included in our study ages ranged from months to yrs. all the children were assessed by the members of the feeding & swallowing team and had videofluroscopic assessment by the same radiologist. neurological signs, speech therapy assessments & videofluroscopy findings were compared between children with spastic quadriparesis & those without. significant differences were noted. a clinical classification was devised using cluster analysis. we conclude that there is no causal relationship between the neurological diagnosis & the type of dysphagia. there are three distinct groups of children who require different strategies of clinical management. surveillance commenced in january to continuously monitor the incidence of surgical site infections (ssi). employing modern optical scanning technology (formic for windows version . , formic limited, london) a questionnaire was designed, which required minimal completion time. the questionnaire includes relevant data based on the american national nosocomial infections surveillance system for ssi including the ssi risk index. surveillance commences at the time of surgery and continues until the patient's discharge. optical scanning technology allows rapid reading of surveillance questionnaires thereby bypassing the bottleneck of manual data entry. by october , details of , procedures had been recorded. the crude ssi rate for these patients was . %. the patient risk index used demonstrated that there were increased chances of developing ssi in certain patient groups. seventy-nine per cent of ssi had presented by the th post-operative day. the length of stay increased by an average of days in patients developing ssi. regular feedback to individual surgeons, theatre and ward staff maintains awareness and highlights possible problems. we recommend optical scanning technology to all those engaged in surveillance work. this system would be especially useful were data collected is transported from outlying hospitals to a central receiving centre for collation and analysis. in francis crumpe published a paper ~ in which he described the therapeutic effect of poisonous mussels (psp) on a case of tetanus. he obtained the mussels from tralee ship canal on the occasion of its infrequent emptying, and entertained the idea of using them in tetanus after treating a young girl with psp who recovered after hours. prior to the use of psp he described it's paralytic effect in two cockrels who both recovered. in concluding his successful use of psp he speculated as to the clinical nature and role of the toxin tralee ship canal was opened in . the water was relatively stagnant and would have contained plenty of nutrients. as such it would have been an ideal habitat for toxic algae which may have been brought across the atlantic as spores in bilge water . the emptying of the canal may well have been done at times of algal blooming. this is the first irish report of psp and a most remarkable use of saxitoxin (?) in the treatment of tetanus, antedating the current management by seventy years. this study was carried out to quantify the published research on smoking in irish medical journals; to ascertain the type of research carried out; and to identify the authors of that research. during the years under study, papers explicitly dealing with smoking were published. only papers appeared in forum. there was a decline in published papers in the eighties with a resurgence in the early nineties. of the papers, a majority were observational and ten were editorials. only one paper dealt with smoking cessation, and one with preventive work. general practitioners were poorly represented as authors. one doctor (prof. r. mulcahy) published at least one paper on smoking in each quinquennium since . this study underlines the relative insignificance of smoking as a topic for research in ireland. a major sea change in attitude will be required if the government's targets for smoking cessation are to be realised, particularly if they are to be achieved by relying on the medical profession. radiology represents a major cost centre within a hospital. lack of awareness of cost amongst doctors may result in the inappropriate requesting of radiology services. this study assesses doctors knowledge of the cost to a tertiary referral hospital of commonly performed radioj'ogical procedures and investigations. doctors were asked to estimate the cost of items namely -chest,x-ray, arch aortogram, ultrasound abdo., lumbosacral spine, barium enema, ct brain, ct abdo., ultrasound abdomen, i.v.p. and percutaneous gastrostomy tube insertion under radiological screening. doctors in st. vincent's hospital were surveyed. doctors as a group overestimated the cost of all individual tests, by margins ranging from % (i.v.p. & gastrostomy insertion) to % (ct brain/ct abdo.) the total cost to the hospital of all items was s consultants'overestimated this total cost by %, followed by registrars, interns and s.h.o's who overestimated the total cost by margins of %, % and % respectively . conclusion: doctors tend to overestimate what radiological procedures and investigations cost a public hospital, often by quite wide margins. thus, any excessive requesting of radiology services by docto.rs is not due to a lack of awareness of their true cost to the hospital. (ded) in dublin. secondly, to identify the major cancers contributing to years of potential life lost (ypll) for the ehb, each cca and ded in males and females. in ireland little work has been done to date on disease specific premature mortality. crude death rates weight all deaths equally; in comparison, ypll emphasise deaths among younger persons and provide a measure of the burden of premature mortality. premature mortality for the deds in dublin for the years and was estimated using ypll, which was calculated by subtracting the date of death form . ypll due to each of the major disease groups were ranked for each ded. seventy-one thousand four hundred and sixty-eight & ) years of potential life were lost in dublin in the and . . % of ypll was due to injury and poisoning, . % to cancer, . % to circulatory disease. however, when the major cause of ypll was established for each ded, injury and poisoning was the number one cause of death in . % of the deds; cancers %, congenital and perinatal conditions . % and circulatory disease %. by emphasising deaths in younger individuals ypll is a valuable tool for planning and monitoring local health promotion initiatives. serum total homocysteine (thcy) levels are inversely associated with dietary intake of folic acid and b vitamins and raised levels have been linked with chd. we have examined the association between thcy concentration and the risk of chd in middle-aged men in british towns. we used a nested case control study design, within an ongoing prospective study, thcy concentration was measured in serum samples, stored at entry to the study, from incident cases of myocardial infarction and controls. cases and controls were frequency matched by town and age group. levels of homocysteine [geometric mean ( %c ) were significantly higher in cases than'controls: homocysteine . ( . - . )gmol/l vs . ( . - . )lamol/l; p = , . there was a graded increase in the relative risk (odds ratio; or) of chd in the nd, rd and th quartile of thcy (or . , . , . ; trend p = . ) relative to the first quartile. adjustment for age, town, social class, body mass index, smoking, physical activity, alcohol intake, hypertensive status, serum cholesterol and serum creatinine did not attenuate this association, (or . , . , . ; trend p = . ). the findings suggest that thcy is an independent risk factor for chd with no threshold level. in summer , a diagnosis of cryptosporidiosis was made in a child who had visited a pet farm. this child had participated in a summer project involving children and nine adults. reports of a similar illness among other project members, prompted an outbreak investigation. a cohort study consisting of two phases was initiated. % ( / ) of project participants responded to a self-administered questionnaire in the first phase. thirteen children met the case definition, of whom seven had cryptosporidium detected in their stools. illness was significantly associated with having visited a pet farm. (p< . ). % of those ill sought medical attention, of whom two were hospitalised the second phase of the cohort study was conducted among those who had visited the pet farm. % ( ) were interviewed. illness was significantly associated with play in sand, to which animals had access, at a stream's edge beside a picnic area (p< . ). contact with various animals was not statistically significantly associated with illness. however the small numbers involved may have obscured such an association. this outbreak highlights a potential hazard for children visiting pet farms and also that cryptosporidiosis is a significant but often overlooked cause of morbidity in healthy children. managers of pet farms need to be aware of the potential for transmission of disease to visiting children. strict implementation of hygiene measures is essential to minimise risk. the mrc vitamin trial highlighted the importance of folic acid in the prevention of neural tube defects(l). since , the department of health has recommended periconceptional folic acid supplements. the objective of this study was to document the knowledge and behaviour of women in child bearing years to periconceptional folic acid. a cross sectional community survey was conducted using an interviewer administered questionnaire in dublin. three hundred and thirty five women took part in the study. approximately two thirds / ( . %) had heard of folic acid. knowledge was significantly associated with higher social class and higher education (p< .o ). few / ( . %) had been advised to take folic acid before pregnancy. only / ( . %) of the women in the study were currently taking folic acid supplements. three quarters of the group ( . %) would be willing to take periconceptional folic acid supplements if they knew it would reduce the risk of malformations. the majority ( . %) would prefer to take folic acid in tablet form. this study clearly shows that few women in childbearing years have been advised on folic acid. however, if advised appropriately the majority would be willing to take periconceptional folic acid in tablet form. future publicity campaigns involving all health professionals should address these issues. unstable intra-articular fractures with or without dislocation of the phalangeal joints often lead to joint stiffness ana loss of function. nine patients with comminuted intra-articular phalangeal fractures were treated in our unit by dynamic external fixator using "pins and rubber bands traction system". the mean age was . years, and the follow-up average was . months. five patients had full and good range of motion in the involved joints. three patients had poor results, and one patient underwent open reduction one week following the original procedure. the technique and our results are discussed. this dynamic frame is compact, comfortable for the patient, easy to apply and allows early mobilisation. careful selection of patients and close follow-up in the first few weeks are needed. this study examines how gp's store and handle vaccines. all gp's in a health board region were invited to take part. gp's were interviewed in their practice premises about how they dealt with vaccines fridges were examined and temperature recorded post interview. oral polio was taken from randomly selected fridges for potency testing. cold chain monitors and freeze watch indicators were used to monitor batches of vaccine stored. of the gp's, ( . %) agreed to participate, used fridges to store vaccine, store vaccine at room temperature. of the fridges, ( . %) had the power supply safeguarded, ( . %) had thermometers, ( . %) had vaccine only stored therein. during defrosting, vaccine was inadequately protected in ( . %). of the gp's who use multi-dose vaccine vials, ( . %) keep them for further use at the end of a day/session, store them at room temperature. ( . %) fridges had temperatures outside the recommended range. ( . %) coldchain monitors indicated vaccine exposed to more than ~ ( %) of the oral polio samples showed a reduction in total titre of live virus, however, none were below the minimum acceptable. this study indicates that vaccine potency could be seriously compromised due to breaks in the cold-chain and suggests the need for guidelines to be drawn up, implemented and monitored to ensure the integrity of immunisation schemes. comparison was made with a study carried out in . in addition the range of antimicrobial agents tested included new oral cephalsporins and quinolones that were not then available. three hundred microorganisms isolated from mid stream urine (msu) samples were examined by standard microbiological techniques. antimicrobial susceptibility testing to antimicrobial agents was performed on significant pathogens (> organisms per ml) by disc diffusion test, and minimum inhibitory concentrations of antibiotics was carried out by e test on organisms found resistant by disc testing. by comparison with resistance amongst e.coli, the most commonly isolated pathogen, had increased for the following: ampicillin by % to %, co amoxyclav by . % from %, trimethoprim by % to % and nitrofuradantin by % from %. no increase in resistance occurred to cephradine ( %), or nalidixic acid ( . %). resistance to cefixime, ofloxacin and ciprofloxacin, was %, no resistance was encountered to cefotaxime. for proteus species resistance to ofloxacin, ciprofloxacin and cefotaxime was %, and for enterobacter sp %. enterococci were sensitive to ampicillin and augmentin but the numbers were small. pathogens isolated from patients domiciled in the inner city were significantly more resistant to nalidixic acid ( %), cefotaxime ( %), cefixime ( %), ofloxacin and ciprofloxacin ( %) than those isolated from patients in rural areas. the purpose of this study is to examine the relative importance of obstetric complications ( c%) in the aetiology of schizophrenia and mania. using the dublin psychiatric case register, birth records of patients with an icd- diagnosis of schizophreniaor mania were obtained. these records were evaluated, for obstetric complications using two scales, the lewis, owen and murray scale (lom) it~ and the parnas scale . the mothers of those going on to develop schizophrenia did not differ from those going on to mania as regards maternal age, parity, social class, or period of pregnancy. however, males who developed schizophrenia when compared to males developing mania, experienced significantly more oc's when rated by the lom scale (p= . ) and.more frequent oc's on the parnas scale (p< . ) of greater severity (p= . ). no significant differences were found between females with schizophrenia and those with mania. dublin. the aim was to evaluate the diagnosis, symptomatology and level of functioning of patients presenting with a first episode of psychosis to a catchment area service and a private psychiatric hospital. all patients presenting with a first episode of psychosis were assessed using the scid-p,,the positive and negative syndrome scale (panss) and the global assessment of functioning scale (gaf). fifty-eight patients ( male, female) ranging in age from to years (mean + sd = . + . ) were included in the study. the mean total panss score was . (sd + . ) and was strongly correlated with the gaf score (p < . ) but independent of age (p = . ). males had a significantly lower gaf score compared to females (p = . ) but there was no gender difference in the total panss score (p = . ), twenty-five patients ( %) had a lifetime prevalence of drug abuse or dependence but only patients ( %) had signs of drug abuse or dependence in the month prior to presentation. level of functioning was strongly influenced by the severity of psychopathology. substance abuse is common in individuals presenting with a first episode of psychosis. the aim was to evaluate the presence of involuntary movements in patients presenting with first episode psychosis to a catchment area service and a private psychiatric hospital. patients presenting with first episode schizophrenia and schizophreniform psychosis were assessed for involuntary movements using the involuntary movements scale (a.i.m.s.). patients ( m., llf.), age range - years (mean= . years.) were included in the study. one patient ( . %) satisfied the strict criteria of schooter and kane for spontaneous dyskinesia. patients ( m., if.), had minimal involuntary movements in at least body areas, predominantly orofacial. the total a.i.m.s. score was positively correlated with the number of days spent in hospital per year of follow up (p= . ). the group with involuntary movements were found to have spent more days in hospital per year of follow up, v. days (p= . ). for patients with first episode schizophrenia or schizophreniform disorder spontaneous dyskinesia is not common. however involuntary movements at presentation may be a predictor of poorer outcome. psychiatry has moved from custodialcare towards care in the community. adequate reprovision will have to be made in order to discharge the remaining continuously hospitalized patients. the objectives of this study were to describe a.n entire long-stay hospital population, to examine the differences between the old and new long stay groups within this populatian and to evaluate the needs for community residential and day care facilities in order for hospital closure to take place. the study group consisted of the total long-stay population of st. davnet's hospital, monaghan. the patients were assessed using the community placement questionnaire (cpq) one hundred and twenty four patients were included in the study. fifty-six were female and % were single. the mean age of the total group was . years. the majority suffered from schizophrenia. the assessment revealed a globally disabled group with multiple handicaps. the new long-stay group were disabled as the old long-stay group. the patients were characterised into four groups with regard to placement recommendations. these were a specialist unit for chronically disturbed geriatrics, a geriatric unit, a high support hostel and a medium support hostel. the remaining population of this hospital were highly dependent with multiple handicaps but would live in community with adequate support. there is little difference between the needs of the old long-stay and those of the new long-stay. failure of the immune system to identify self peptides is likely to lead to the development of an autoimmune reaction. susceptibility to autoimmunity is strongly influenced by genes clustered in the hla region (chromosome p) particularly class i (a, b and c) and class ii (d, q and p). it has been suggested that there is an autoimmune component in the aetiology of schizophrenia. of many conflicting reports from case/control studies using hla antigens the most consistent finding has been an increased frequency of hla-a (now split into a /a ). additionally, a susceptibility locus for schizophrenia has been reported near the hla locus. to attempt to confirm the hla a hypothesis, we have genotyped a preliminary sample of familial schizophrenic probands and unrelated controls at the hla-a region, using a pcr-ssop technique. the frequency of hla-a (the major component of a ) in patients and controls respectively was . % vs . %. these findings do not support the hypothesis. some of the discrepancy may be due to unspecific cross-reactions produced by commercial antisera used in the microlymphocytotoxicity method of previous studies. however it is also possible that the hla associatton with schizophrenia may reflect linkage disequilibrium with unidentified gene(s) within the hla region which is less strong in the irish population. schizophrenia is a common mental disorder affecting about % of the general population with a devastating disturbance of mind and personality. family, twin and adoption studies have demonstrated that the disease is largely genetic with a polygenic mode of: transmission. dopamine receptors have been implicated in the aetiology of the disease. as yet dopamine genes have been identified (d -d ). in particular the d receptor is expressed in the limbic regions of the brain, implicated in the control of emotions. association studies of a d polymorphism (glycine to serine substitution at position ,) with schizophrenia have produced conflicting findings, many of which, however, have demonstrated a significant excess of homozygosity, or excess of the -l genotype at this polymorphism. in this study, familial schizophrenics and irish unrelated controls were genotyped. the result show a small increase in the frequency of the - genotype which did not attain statistical significance (patients, . % vs. controls, . %). homozygosity (alleles - and - ) was also slightly increased in the patients (patients, . % vs. controls, . %). the small increase in the frequency of the - genotype and of homozygosity in the patients is in keeping with earlier findings but suggests that the effect, if any, of d sequence variation in the development of the disease is small. lack of information about general practitioners' (gp's) ability to prescribe psychotropic medication may affect patients' compliance. in this study, out of patients attending a psychiatry out-patient clinic completed a questionnaire which documented how many had run out of medication, the steps taken if they had and the role each patient thought their gp played in their treatment. % indicated that their gp knew what their current medication was but only % thought that their gp could provide them with a prescription if they did not have one from the clinic. this figure was similar in those who had ( %) and had not ( %) run out of medication in the past. on running out of medication, % of patients waited until their next appointment, % attended their gp and the remainder either contacted the department or went to a chemist. in conclusion, many patients do not appreciate the entitlement of their gp's to prescribe psychotropics for them. literature regarding whether or not the social class distribution of patients with psychiatric illness may differ from the general population remains controversial. we sought to clarify this by examining social class at the time of birth, to see whether patients with serious psychiatric illnesses (schizophrenia and mania) differ from the general population. paternal occupation of schizophrenic patients, and manic patients, from the dublin psychiatric case register, were obtained from birth registration details and categorised according to central statistics office criteria, the same-sex previous live birth was used as a matched control. there was no difference between the social class of patients with schizophrenia or mania (p= . ). neither patients with schizophrenia (p=b. ) nor mania (p= . ) differed from controls in social class distribution. paternal social class was found to be related to amount of time spenr in hospital (p< . ', mean= . ), and educational age (p< . , mean= . ) and "age at onset of the illness" (p= . , mean= . ). these results suggest that social class of origin may not be related to the development of either schizophrenia or mania. however, social class of origin may be relevant in terms of presentation of schizophrenia for treatment. cognitive function is widely recognised to be impaired in schizophrenia but there is an ongoing debate as to whether this impairment is generalised or localised, progressive or static, similar in both sexes, or related to symptoms. using the positive and negative syndrome scale (panss)'we measured psychopathology in chronic in-patients ( m, f; mean age . + . ) who satisfied feighner criteria for schizophrenia. subsequent to this, we assessed their global cognitive function using the mini-mental state examination (mmse) and their frontal cognitive function using a new instrument, the executive interview (exit). poor performance on the exit was associated strongly with increasing severity of negative (r=- . , p< . ) but not positive (r=-- . , ns) symptoms,'in both males (r=-- . , p< . ) and females (r=-- . , p< . ). overall exit performance declined modestly with increasing age (r=-- . , p< . ) but this phenomenon was co'nfined to females (r=-- . , p< . ; males: r=-- . , ns). mmse performance was also associated with negative symptoms (r=-- . , p< . ) but decreased mm:e prominently with age (r=-- . , p< . ) and showed no gender difference. frontal dysfunction in schizophrenia appears to be intimately related to negative symptoms over the course of severe chronic illness, and may reflect among males a more static' trait deficit than is accessed by the mmse. this study was supported by the stanley foundation. while determinants of the course of schizophrenia are unclear, emerging evidence suggests that the longer psychosis proceeds unchecked before initiation of anti-psychotic therapy, the poorer may be long-term outcome. we have reported that, among older in-patients, increasing duration of initially untreated psychosis in the pre-neuroleptic era was associated with a deterioration to a state of muteness (after controlling for intervening variables). the current survivors of this population have now been examined more extensively using the positive and negative syndrome scale (panss), the mini-mental state examination (mmse) and the executive interview (exit). among these patients (mean age . + . ), after controlling for age and for the duration and continuity of subsequent antipsychotic treatment, increasing duration of initially untreated psychosis was associated with greater severity of negative symptoms (p< . ) and with lower scores on the mmse (p< . ) but not with executive dysfunction on the exit (p= . ). increasing duration of initially untreated psychosis appears to be associated with the evolution of more prominent negative symptoms and cognitive impairment in a manner consistent with an active, morbid process in schizophrenia that can be ameliorated by anti-psychotic drugs. this study was supported by the stanley foundation and the health research board. patients who are selected and who agree to participate in the royal college examinations play an important role. as psychiatrists and exam organisers, we should be aware of the potentially stressful experience which this might present. the purpose of our study was to elicit attitudes to the exam, and also knowledge of the examination procedure. a questionnaire comprising questions was circulated to patients who had participated in the royal college examinations. responses were received from ( %) of the patients. there were males and females in the responding group. none of the patients had previously participated in the examinations. all of the respondents (n= ) felt that the candidate had been polite towards them during the interview. % (n= ) of the patients were nervous prior to the examination, and this group was predominantly female (n= ). % of the patients (n= ) did not know that they would receive payment for their participation. % (n=l ) did not know that they might be physically examined as part of the examination procedure. % (n= ) of the patients described experiences which had been upsetting for them the results of our study suggest that on the whole patients tolerate the exam procedure quite well. one of the central issues concerning physiotherapists in stroke rehabilitation is the emergence of abnormal tone. rehabilitation involves re-establishing a normal postural control mechanism (ncpm)"~. abnormal tone may develop in the presence of severe sensory and proprioceptive loss. the patients' attempts to move and find a stable base can lead to compensatory movement patterns and asymmetrical postures. positioning is used by physiotherapists to influence the distribution of muscle tone and facilitate symmetrical postures. it is essential that the patient is made aware of his 'position in space' as failure to do so presents no feedback regarding movement resulting in inertia ~ ~. standardised positioning charts have been used in hospitals. the physiotherapist liaises with nursing staff regarding correct use on a hour basis. this study looked at the role of a more individualised approach to positioning in the form of a photograph. patients were randomised to two positioning groups group a standard vs group b photograph, and their positioning was scored by a 'blinded' research physiotherapist over an eight week period. the results of a pilot study on a small number of patients revealed that nursing staff preferred a positioning chart individually tailored to the patient's problems. from a physiotherapy perspective, improved postural awareness correlated with better positioning scores in group b. the prevention of compensatory movements, posture is critical in stroke rehabilitation, the use of an individualised positioning chart requires further evaluation. we discuss the case of a fourteen year old boy who presented with bilateral ptosis present since birth, microcephaly and pigmentory retinopathy"!. he was found to have mild facial and proximal limb weakness. creatinine kinase and ldh were raised. muscle biopsy showed ragged red fibres consistent with a mitochondrial myopathy ~ ~. electron microscopy showed abnormal mitochondria. the term mitochondrial myopathy describes a diverse range of clinical disease ~ ~ and this is discussed. she developed a vasculitic skin rash with pruritis and oedema associated adenopathy, low grade fever and mouth ulcers. lab tests showed leucocytosis, eosinophilia and abnormal liver function. skin biopsy indicated an inflammatory picture without vasculitis. ct thorax confirmed axillary and para-aortic adenopathy. lymph node biopsy confirmed a reactive lymphadenopathy. the aim of. this study was to assess the characteristics of patients referred for pudendal nerve studies over a one year period. consecutive patients were asked a standard questionnaire and nerve studies were performed as described by kiff and swash m and swash and snook~ k of the patients, only were male. the age range was from to (mean ). presented with constipation and with faecal incontinence. two had both symptoms. bladder incontinence was presented in of patients. of these, faecal incontinence was the cardinal symptom in patients, constipation in . patients were nulliparous. of the remaining , had a history of complicated births involving forceps ( ), caesarean section ( ), post partum haemorrhage ( ), breech without forceps ( ) . patients had pelvic surgery and one had major trauma. of patients had bilaterally delayed pudental nerve terminal motor latency (pntml). of people had unilaterally delayed pntml, were right sided, were left sided. had normal studi~s. the range of measurements was . - . ms with a mean of . ms. in conclusionl delayed pntml was seen in of patients with constipation and of with faecal incontinence. pelvic surgery and a complicated obstetric history were significant. urological symptoms were also a common association. the p component of the middle latency-auditory evoked potential is attenuated in response to the second of paired clicks in a normal population. in schizophrenia, this attenuation is minimal. in alzheimer's disease (ad), the results have varied between centres depending on the frequency of the stimuli and the interclick interval. we studied ad patients, elderly controls (ec) and young controls (yc) using a paradigm of sets of paired clicks. in contrast to previous studies, our study demonstrates significantly larger absolute p generation and recovery amplitudes in ad patients compared to elderly controls and young controls. the purpose of study was to establish a simple screening vol. , irish journal of supplement no. medical science test to identify asymptomatic intracranial aneurysms (icas). an association between atherosclerosis and icas is recognised. elevated serum lipoprotein (a) [lp(a)] is an independent risk factor for atherogenesis. we aimed to assess the degree of correlation between serum lp(a) and the occurrence of sporadic ruptured aneurysms and familial asymptomatic aneurysms. lp(a) levels were measured in (a) patients with icas and normal controls, (b) first degree relatives of patients with familial subarachnoid haemorrhage (sah). icas were detected by cerebral angiography. patients with sporadic icas had significantly elevated lp(a) levels when compared with matched controls. mean level was . mg/dl in patients and . mg/dl in controls. in the familial studies, out of i subjects with asymptomatic icas had elevated lp(a) levels. one young female with elevated lp(a) had a pre-aneurysmal dilatation at operation. six out of subjects without icas had elevated lp(a) levels; four of these were in the second or third decade of life and may yet develop aneurysms. conclusions: lp(a) has potential as a biological marker for icas. follow-up studies are required on angiographically negative subjects. we have begun a genetic case-control study to establish if particular apoprotein (a) gene polymorphisms can be correlated with the occurrence of icas. post mastectomy breast reconstruction has undergone several changes in the recent years. attitudes have changed towards the problem from both the patient and the reconstructive surgeon, as aesthetic outcome receives a greater emphasis than previously. there is a shift towards using autologous tissue as a means of reconstruction; these new technically difficult procedures entail a longer learning. centralization of this type of reconstruction in highly specialized centres only will serve the patient better. we share our experience of post mastectomy breast reconstruction spread out over the past five years. seventy-eight consecutive cases of breast reconstruction are included in the study. different techniques of breast reconstruction were used with a recent switch to transverse rectus abdominis myocutaneous (tram) flap; we feet that tram flap is the gold standard of breast reconstruction as far as the ultimate cosmetic result is concerned. ours is only a moderate sized study compared to some published, yet it is representative of the experience of most of the plastic surgery units in the british isles. clinically significant paraneoplastic neurologicaldisorders are rare, most are associated with small cell lung, female genital tract and breast carcinoma. the malignancy is often silent and the neurological manifestations vary from encephalomyelitis, cerebellar degeneration, sensory neuropathy to neuromuscular block. prognosis is usually poor. pathogenesis is thought to be related to cross reaction with neurons of antibody produced to tumour antigens. detection of these antineuronal antibodies in serum has assisted diagnosis of paraneoplastic encephalomyelitis in which anti-hu antibodies are present and cerebellar degeneration, in which anti-yo are found. in our lab, we used avidin-biotin-complex immunocytochemistry to detect anti-hu (cortical neuron antibodies) and anti-yo (purkinje cell antibodies) in patients' sera. tests were performed on human frontal cortex and cerebellum, at in and in , dilutions, with positive and negative controls. of sera, were positive. two patients had repeat positives; in one, antibody titre rose in the second sample. subsequent patient review showed positives ( patients) had identifiable carcinoma with paraneoplastic cns signs, had no identifiable malignancy but had no other cause of their cns disorder and are being followed up; details of one patient were unavailable. these results are similar to other centres. the proliferation of tumour cells despite the presence of tumouricidal mediators could be due induction of a heat shock response, a universal cellular defence mechanism in host cells and possibly tumour cells. protection may be mediated either by increasing intracellular levels or surface expression of heat shock proteins (hsp). the aim was to assess the effect of heat shock induction on tumour cell protection against host effector cells. the heat shock response was induced in sw colorectal cells by either sodium arsenite ( - ~tm for hr) or by hyperthermia ( ~ for rain). monocyte (m )-mediated cytotoxicity or flow cytometry to evaluate surface expression of hsp and hsp were assessed. cytotoxicity showed a significant decrease in all treated groups (p< . ) when compared to the control value. there was also a significant decrease in all groups (p< . ) when compared to the ~ value. no significant alteration in surface expression of either hsp or hsp was seen. conclusion: heat shocking tumour cells significantly protects them from m -mediated tumour cell lysis. since the flow cytometric data indicate that there is no concomitant increase in surface expression of hsp and hsp on the tumour cell following heat shock, it can be inferred that induction of intracellular hsp levels are responsible for the protective effect on the tumour cells. a week qol study in consecutive advanced cancer patients was undertaken to compare the subjective question fact-g with simple subjective global tools (visual analog, categorical scales: vas, cas), objective tools (spitzer qli and ecog performance status) and verbatim patient description. we anticipated the high drop out rate enrolling to achieve complete study patients for statistical purposes. the study sample appeared representative of the advanced cancer population in the usa. generally qol was satisfactory despite the severity of illness. there were significant differences in all measures between those who described qol in verbatim responses as positive and negative, particularly cas, vas, and qli (p< . ). there were significant intercorrelations between qli and ps (observer rated), vas and cas (subject rated) respectively (p< . ). taking patient description as the gold standard, simple, global qol measures e.g. vas or cas are as effective as multidimensional ones (fact-g and qli). irish journal of medical science males had more dysphagia. survival from diagnosis was greater for females </= years (female median months, male median months, p< . ). anorexia was associated with reduced survival since diagnosis in the total population and in males. anorexia anddysphagia were associated with reduced survival from referral. advanced cancer patients were polysymptomatic. symptom prevalence differed with age, gender and cancer site. the pattern of g symptoms was related to gender and severe weight loss. specific symptoms were associated with reduced survival. there was a gender difference in survival favoring females. the administration of recombinant interleukin- (rll- ) is limited by the induction of increased microvascular permeability. the in vivo antineoplastic effects of taurine in combination with rll- were investigated and its impact on the associated vascular leak was examined. lung metastases were established in mice via tail vein injection. ten days after injection mice were randomized into treatment groups. on day the mice were sacrificed; lungs were removed, weighed and metastases counted. treatment of tumour-bearing mice with rll- alone resulted in a significant reduction in tumour nodule incidence compared to a control group, while the~group recei'~ing rll- + taurine showed an even fuither reduction in the incidence of lung nodules. animals receiving rll- showed a significant increase in mean wet lung weight compared to control lung weight, while mean wet lung weight of the rll- + taurine group was significantly less than that of the rll- group.and comparable to control values. animals receiving ril- + taurine in vivo demonstrated significantly enhanced splenocyte-mediated antimelanoma activity ex vivo compared to animals receiving rll- alone. thus taurine may have an important role in modulating both metastatic growth and the associated toxicity of rll- immunotherapy. a prospective analysis of symptoms in , patients on initial referral to the palliative care service of the cleveland clinic was made using the paradox relational database. the median number of symptoms per patient was (range - ). the most frequent symptoms were pain %, easy fatigue %, weakness %. anorexia %, > % weight loss %, lack of energy %, dry mouth %,'eonstipation %, dyspnea % and early satiety %. patients years and under had more pain, sleep problems, depression, anxiety, vomiting and headache (all p< . ).'the prevalence of early satiety, nausea, vomiting and anxiety were greater in females; dysphagia nd hoarseness in males. patients with >/= % weight loss had more gi symptoms; of these females had more nausea, early satiety; the progn.o'stic significance of abnormalities in the p tumour suppressor gene and in the expression of its protein in colorectal carcinoma may be influenced by the method of analysis used. we studied p abnormalities in patients with colorectal cancer followed for more than years. single-strand conformation polymorphism analysis (sscp) was used to detect alterations in exons - of the p gene. paraffin sections were examined immunohistochemically for p overe,xpression with the monoclonal antibody do- (dako) both with and without microwave antigen retrieval. abnormalities of the p gene were found in % of cases by sscp analysis but were unrelated to age, sex, tumour size or differentiation. outcome was unrelated to sscp abnormalities (p= . ). overexpression of p protein was seen in % of cases by immunohistochemistry without microwave antigen retrieval and in % of cases with microwaving. poor long-term survival was related to immunohistochemical expression of p protein either with (p= . ) or without (p= . ) microwave antigen retrieval. these results suggest that immunohistochemical detection of the p protein product may be more useful than sscp analysis of the encoding p gene in identifying those at high risk of colorectal cancer recurrence and death. dublin . the anti-tumour activity of tumour infiltrating lymphocytes (tils) is known to be poor and therapeutic manipulation of these cells has met with little success. suppressor macrophages (smo) influence t cell cytotoxicity and proliferation. we hypothesized that smo are a component of the lymphoreticular infiltrate and that these cells may be related to lymphocyte numbers within the tumour. tgf-b may influence macrophage phenotype. colorectal and breast tumours were obtained within an hour of resection. tumours were dissaggregated with collagenase and dnase for three hours. antibodies was used to identify smo (rfd and rfd ) and t cell subsets (cd and cd ) by flow cytometry on the resulting cell suspension. pre-op blood was collected from patients and tgf-b levelsdetermined by elisa. conclusions: we have shown for the first time that smo, defined by the antibodies rfd and rfd , are present within breast tumours. we have also shown that the balance of t cell subsets is different in these tumours and may be related to smo content. circulating tgf-b levels are increased in breast cancer and associated with greater smo numbers. this was not found to be the case in colorectal cancers. these results imply the existence of a fundamental difference in the make-up of the lymphoreticular infiltrate between these cancers. swelling of the upper limb is an uncommon but well irish journal of medical science recognised complication of breast cancer treatment. in severe cases, patients have limited arm function and feel disfigured. in a pilot study, the incidence of arm swelling following complete axillary clearance in the immediate post-operative period and at long term follow-up was investigated. arm volume measurements were performed using an opto-electronic volometer (bosl medizintechnick, hamburg). both ipsilateral and contralateral arm volumes were assessed. the expected volume of the ipsilateral arm volume was calculated using the formula vr = v = mls for right handed people and v = vr = mls for left handed people (vr and v = volume of the right and left arms respectively). the difference between the expected and actual volume of the ipsilateral arm was expressed as a percentage of the expected volume. twelve patients undergoing axillary clearance for breast cancer were prospectively evaluated pre-operatively, hours and days post-operatively. a second group of patients who had had axillary clearance at least months previously (range to months) were also evaluated. there was no significant change in arm volume in the immediate post operative period. clinically detectable arm welling was found on patients who had undergone axillary clearance at least months previously but none had any impairment of arm function. we conclude that axillary clearance can be performed safely and that arm swelling is an uncommon complication. a larger study is planned to investigate factors such as the influence of pectoralis minor division, duration of the operation and the number of axillary nodes retrieved on upper limb volume. epidermal growth factor (egf) is a potent mitogen and has been shown to accelerate healing of epithelial damage both in the skin an.d the gut. in the skin egf is not produced locally as the requisite mrna is not present but egf receptors are present on the surface of basal keratinocytes. egf is produced in various sites in the gi tract including the submandibular salivary glands. we have hypothesised that as there is upregulation of salivary egf production in some enteropathies a similar situation may occur in disorders of the skin with an associated enteropathy. using a sensitive radio-immunoassay, egf activity was estimated in stimulated saliva from patients with various skin disorders, patients with gastrointestinal disease, patients with mixed dermatological and gut disease and normal healthy volunteer controls. elevated egf activity was found in the following groups of patients : skin cancers, psoriasis, acne, oesophagitis and ulcerative colitis. the hypothesis of up-regulation of salivary egf production in skin associated enteropathy was rejected but the discovery of elevated egf activity in skin cancers and psoriasis may have aetiological and therapeutic implications. the malignant fibrous histiocytoma (mfh) is considered an uncommon malignancy. its potential for invasion, metastasis and death of patient has been reported in literature. it can be confused with other tumours including fibrosarcoma. salient histologic features include cells of both the fibrocytic and histiocytic series. mfh with its high recurrence rate and lethal potential merits an aggressive evaluation and treatment. we present unusual case of recurrent mfh treated in our unit with an open question as to what qualifies to be adequate primary surgical excision. the recommended management of localised merkel cell carcinoma has been wide surgical excision, combined with adjuvant radiotherapy in selected cases. the risk of recurrent regional disease is reported to be between % and %. a year old woman with merkel cell tumour on the cheek is presented; this patient was treated exclusively with radiotherapy to a total dose of gy over days. the tumour regressed rapidly during the treatment, and there were no signs of local or regional recurrence. the patient is still alive and free of disease for months. immune in origin with a heightened cutaneous immune response to ultraviolet light. the coexistence of cad and pbc is a new association which has not previously been documented and may not be fortuitous given the similar pathogenesis of both diseases. chronic actinic dermatitis (cad) is a rare photosensitive disorder which primarily affects elderly men resulting in an eczematous reaction to ultraviolet-radiation and sometimes visible light. the pathogenesis has been attributed to an autoimmune process, possibly in response to a photoallergen which has yet not been identified. we report a year old female patient who developed cad four years after being diagnosed with primary biliary cirrhosis (pbc). abnormal monochromator irradiation tests were detected with narrow band ubv, uva and in addition visible light wavelengths. phot provocation tests induced florid vesicular eczema and multiple patch and photo-patch tests were positive, findings typical of cad. immunoglobulin g was elevated at mg/dl and liver histology was typical of pbc with an elevated anti-mitochondrial antibody. routine biochemical and immunological tests were ~ormal and porphyrin screen was negative. azathioprine mg/day induced remission of cad. pbc is an auto-immune disorder where cell-mediated immunity is impaired, suggesting that sensitized t lymphocytes may cause damage to bile ducts. the pathogenesis of cad may be auto- we present two cases of cutaneous polyarteritis nodosa (pan) associated with seronegative arthritis: the first patient, a year old male presented in complaining of a year history of pain, stiffness and swelling affecting his right ankle. he also noted intermittent tender nodules on the dorsum of his foot and over his ankle over the preceding three years. the second patient, a year old male, presented .in complaining of a month history of tender nodules on his shins, and pain and swelling of his right ankle. skin biopsies of the nodules in both cases showed medium vessel vasculitis consistent with polyarteritis nodosa. neither patient had any symptoms or signs to suggest systemic involvement. the only abnormality. n laboratory investigations for vasculitis was elevated esr. x-rays showed periosteal elevation and new bone formation in case , and were normal in case. . bone scan demonstrated increased uptake at the talo-navicular joint in case and at the fight ankle in case . synovial biopsy and mri confirmed the presence of an inflammatory arthropathy in patient . joint involvement has been a prominent feature throughout the course in both cases requiring aggressive treatment with vol. , irish journal of supplement no. medical science cyclophosphamide and systemic corticosteroids in case . cutaneous pan is a localised cutaneous vascular disorder with a benign chronic relapsing course. in one reviewl, of patients had arthralgias but an association with arthritis has not been emphasized in the literature to date. we conclude that this condition may present as a seronegative arthropathy in which the joint symptoms may be the most prominent feature and aggressive immunosuppresive therapy may be required for control. cardiac transplantation patients have an increased risk of skin disease. in our centre, heart transplants were performed with a year survival of %. eighty three patients are now alive and have required dermatological assessment. the mean age of patient was . years, (range - years); males, females. skin infections were diagnosed in of patients. drug side effects, including sebaceous hyperplasia and steroid acne, were common. in the patients who developed skin cancer, mean time from transplant to development of lesions was . years. eleven of patients had non melanoma skin cancers (nmsc), squamous cell carcinomas (scc), basal cell carcinomas (bcc), giving scc/bcc ratio of : . three of patients had multiple skin cancers, one had tumours. nine of patients had actinic keratoses, two thirds of whom had sccs. nineteen of patients had viral warts, two of whom had sccs. viral warts, premalignant and malignant lesions were located on sun exposed sites. skin complications of cardiac transplantation though mild were very common. the observed incidence of nmsc in age matched cardiac transplant recipients, appears much higher than the expected incidence of . per , population (national tumour registry ). regular dermatological assessment of cardiac transplant patients is necessary to detect skin disease and early skin cancer. the increased incidence of warts and skin cancer in renal transplant recipients {rtr} is well known. the oncogenic potential of unusual human papilloma virus {hpv} types has been postulated from warts and in both premalignant and nonmelanoma skin cancer (nmsc). the possible etiological role of sun-exposure in facilitating the development of hpv associated skin disorders is also suggested. a clinical study to assess the risk factors for development of these lesions in rtr attending the dermatology servic& age and sex matched haemodialysis patients were similarly examined as controls. male and female patients with a mean duration of transplant of . years, range to years. a total of nmsc (range to ), scc and bcc, ratio . : , were excised from rtr of which over % had viral warts, mosle commonly occurring on sun exposed sites and always predated the development of neoplastic lesions. both were associated with mean duration from transplantation, years for warts and . for skin cancer and not the type of immunosuppressive treatment. none of the control patients had similar findings. conclusions: the close clinical association of viral wart lesions and development of skin cancer in these patients suggests a close relationship to immunosuppression, in addition to exposure to ultraviolet radiation. this study highlights the high rate of nmsc in rtr. these patients justify early and regular skin assessments soon after transplantation with advice on sun protection. sensitivity to ultraviolet (uv) light may be established by exposure to broad band uva and uvb radiation. the minimal erythema dose (med) can be determined at individual wavelengths using a monochromator. uv action spectra of photosensitive disorders may thus be constructed. we examine the value of this process in distinguishing two clinically similar photosensitive disorders. the radiation from a xenon arc is separated into component wavelengths using the monochromator. each wavelength is focused on unaffected skin, on the patient's back. the patient is exposed to a range of doses of w radiation. the med is determined for a series of wavelengths from to nm. chronic actinic dermatitis (cad) and drug induced photosensitivity are photosensitive disorders which may have similar clinical history and presentation. ten cad and drug induced photosensitivity patients were tested. uva photosensitivity was seen in % of the latter group. the remaining % had normal mlts as the implicated drug had been discontinued prior to testing. cad patients were sensitive to both wa and wb radiation. forty-three percent of these patients were also sensitive to visible light ( to nm). monochromator light test (mlt) results show that uva photosensitivity dissociated from wb photosensitivity is indicative of a drug induced light sensitive disorder. sensitivity to both wa and wb however indicates a diagnosis of cad. mlts can therefore distinguish between clinically similar photosensitive disorders. bartholomew's hospitals, london. patients with mpd have an increased incidence of both thrombosis and haemorrhage suggesting a pivotal role for; platelets in these conditions. this study aimed to examine platelet activation antigen expression in stable patients with mpd and to examine the predictive value of these antigens prospectively. patients with mpd had p selectin and gp measured using a refined minimally manipulative flow cytometric technique. expression of p selectin -median . % (inter quartile range . - . ), control . % ( . .- . ) and gp -median . % ( . - . ), control- . % ( . - . ), were significantly elevated p< . . patients were followed for a median of months. % experienced thrombosis and % bleeding during follow up. at entry to the study % of patients had previously experienced thrombosis, median disease duration deaths, of which were caused by thrombotic events in which the mpd was a major risk factor. increased expression of p selectin or gp expression failed to predict thrombosis or bleeding in this study. nor was any significant retrospective relationship demonstrated. however, previous thrombotic events were strongly associated with future events (p< . ). this association was independent of disease, duration, age and medication. not surprisingly disease duration was also correlated with thrombotic/bleeding events. taurine levels fall in gut mucosal cells during critical illness. however, taurine transport into human intestinal cells is poorly understood. the aim was to establish the efficiency of taurine uptake by enterocytes, and to examine uptake under stressful conditions. to investigate efficiency of taurine uptake, confluent caco- cells were incubated for time points up to h. in a second study, cells were incubated for h with medium containing dexamethasone and / or cytokines. media for both studies was supplemented with [ h]-taurine. radioactivity was related to mg/ml protein to calculate rate of taurine uptake for each time point. study : uptake exhibited a steady linear response which approached saturation at h. maximal uptake occurred at h after which the rate levelled off. study : dexamethasone alone reduced taurine uptake by . % (p< . ) and in combination with tnf-c~ and ifn ~/ it decreased transport by . %. (p< . ). lps alone impaired uptake by % (p< . ). conclusion: we have established the time course over which taurine transport reaches its maximum rate in caco- cells, and that corticosteroids and cytokines significantly impair uptake of taurine in these cells. elderly individuals have an increased risk of infection suggesting that immune responsiveness is altered with age. changes in the level of proinflammatory cytokine production may be an important indication of any such age related change. using flow cytometry we examined intracellular tnfet, il-lf~ and il- in pbmcs from normal healthy volunteers of different ages ranging from up to yr (n= ). tnf and il levels from pma stimulated cd positive cells (t cells) were shown, using this technique, to increase in an age dependent manner (p< . ). no il- was detected in any t cell sample. no significant differences were observed between the different age groups for tnfa, il- g or il- in cd + cells (monocytes). the age related changes detected by flow cytometry have been confirmed using conventional elisas. this novel method of proinflammatory cytokine detection has detected increased tnf and il levels in t cells from elderly healthy volunteers which may help explain some of the exaggerated inflammatory responses seen in elderly patients. detection of proinflammatory cytokines by conventional elisa or bioassay is problematic due to the presence of naturally occurring biological inhibitors. flow cytometry allows the simultaneous detection of both intra and extracellular antigens thus intracellular cytokine levels can be quantified while cell surface markers allow cell type identification. a range of monoclonal antibodies were examined for tnfcx, il-lg and il- using saponin permeabilisation oft cells (cd ), monocytes (cd ) and epithelial cells (ber-ep ). t cells and monocytes were grown in ~culture, t~p :to hr with or without pma activation, and intracellular cytokine levels were shown to increase with time, with the stimulated samples producing more cytol<ine than the spontaneous.samples (p< . ). elisas performed on these same samples (n--: ) correlated well with the flow cytometric results (r= . ). tnf~x, il- and il- are detectable !n monocytes while only tnfa and il- have been detected in tcells and epithelial cells using this methodology. this novel technique will allow us to identify the cytokine producing cell while also avoiding the influence of the extracellular millieu, thus being useful for a number of research and diagnostic applications. all the viral hepatitides are said to be common in the middle east. hepatitis a viru s (hav) seroprevalence is said to be % in the adult population of gulf countries. hepatitis b, c and d seroprevalence are taken from blood banks data which are biased by selection criteria and the background of blood donors. very little is known about hev in the uae. vol. , supplement no. irish journal of medical science the aim was to investigate the seroprevalence of hav, hbv, hcv, hdv & hev in the uae. we prospectively recruited volunteers drawn from all the uae, socioeconomic classes and from different age groups in both sexes. each donated ml. of venous blood and using an indirect enzyme immunoassay were tested for total ig to hav; hbsag using monoclonal antibody t.o hbsag; for hcv antibodies using recombinant antigens hc- , hc- , c- and ns- , ( rd generations); for total ig to hdv and for total ig to hev. positive samples were retested in duplicate for confirmation. this large sero-epidemiological study clearly shows that hav is decreasing in the uae. hbv is low but significant, hcv is similar to southern european countries, hev is lower than expected and hdv does not exist in hbsag carriers in the uae. reactive oxygen and nitrogen species produced by inflammatory cells plays a role in tissue injury and inflammation. taurine, a bamino acid present in high concentrations in leukocytes functions as an anti-oxidant by scavenging the mpo derived oxidant, hypochlorous acid, to form taurinechloramine. experimental colitis was induced in male sprague-dawley rats and randomised into two groups. one group received tap water whilst the second group was supplemented with taurine ( %). these groups were sub-divided into those receiving tnbs/ ethanol (n= ) or saline (n= ). an increase in wcc count, particularly neutrophils was found in colitis. tat~rine was found to reduce the severity of the disease (ns) usin.g a colon macroscopic score. in the first group, respiratory burst activity was reduced in animals that had developed colitis (p< . ) although mpo activity was augmented (p< . ). nitric oxide production was . and . fold respectively higher in inflamed and non-inflamed areas of the colon than that by normal colonic mucosa. animals supplemented with taurine demonstrated attenuated m po activity (p= . ). administration of taurine did not lead to a reduction in nitric oxide production. thus, taurine appears to have a beneficial role in reducing the severity of disease. a reduction in mpo and respiratory burst may be beneficial in reducing tissue damage. to women subsequently identified by a national screening programme which revealed to be positive for rna of hepatitis c, type lb. were referred to this centre for evaluation. had moderate activity by modified knodell index, mean (_+ . ), bridging fibrosis and a mean alt of ( - ). six months dual therapy was administered. two patients withdrew due to depression ( ) and hyperthyroidism ( ). one patient di d not respond. seven responded with normalisation of alt and negative pcr. eight patients showed improved histology (kai +_ . ), fibrosis was unchanged. adverse reactions in the treated group included marrow suppression ( ) and thyrotoxicosis ( ) . at six months follow up four remain in remission with normal alt negative pcr, three have relapsed, (pcr positive, mean alt ( - ). we conclude that despite adverse prognostic indicators in this homogenous group with chronic hepatitis c, lb and long disease duration, dual therapy with interferon and ribavirin achieved results comparable to prolonged high dose interferon in patients with more favourable initial prognoses. the aim of the study is to evaluate a day course of azithromycin in combination with omeprazole in eradication of h.pylori. twenty-four patients with proved h.pylori infection and peptic ulcer disease were treated with omeprazole mg daily for months plus azithromycin mg daily for days. h.pylori was diagnosed on the basis of clo test and histology. this was done at the beginning and months after the treatment. eradication was defined as negative clo test as well as negative histology. out of patients, patients were lost to follow-up. only patients were analysed. sixteen patients responded to treatment and had successful eradication as well as healing of their ulcers. these included patients with gastric ulcers ( . %), patients with duodenal ulcers, patients with gastric and duodenal ulcers and patients with non-ulcer dyspepsia. five patients did not respond including with non-ulcerative dyspepsia. duodenal ulcers and gastric ulcer. however their ulcers have healed and symptoms relieved and they were treated with other h.pylori eradication regimes. conclusion. in this small study we have shown that azithromycin mg oncedaily for days is an alternative safe effective and well tolerated monotherapy for eradication of h.pylori. it has been suggested that hepatitis c virus (hcv) infection is associated with a high prevalence of autoimmune disease, but that this risk may be diminished in patients who received a low viral load at initial infection. the aim was to determine the prevalence of symptoms, signs and markers of autoimmune disease in a group of women vol. , irish journal of supplement no, medical science infected with hcv contaminated anti-d immunoglobulin. patients, mean age . years (range - yrs) were examined. patients ( %) were rheumatoid factor positive. ana was positive in patients ( %) but titres were / in / . thyroid globulin antibodies were found in patients ( %) with also being tm positive. further patients were positive for thyroid microsomal ab. prevalence of apa, ara, sm were %, % and % respectively. one patient had cryoglobulins detected while antibodies against mitochondria and lkm-i were not found only one patient of was symptomatic. these levels of antibodies are no greater than levels in the general population and similar to other groups infected with hcv contaminated anti-d. these results further support the hypothesis that either low viral innoculum or long duration of infection may in some way result in a low level of autoimmunity. a previous study (ilan et al, arch int. med., ; : - ) suggested that lga deficiency disposes towards hepatitis c virus (hcv) infection in some patients. it has also been suggested.that iga deficiency may occur as a result of the viral infection as a secondary event. the aim was to determine the prevalence of selective and partial lga deficiency in patients with hcv infection using a radio-immunodiffusion technique. serum lgg and igm levels were also examined. immunoglobulin levels were measured in patients, women and men, mean age . years (range - years) infected with blood between and . none of the patients were found to have selective or partial iga deficiency. mean iga = . g/l (range . g. - . g/l). furthermore no patient exhibited deficiency of igg or igm. conclusion : no evidence of iga or other immunoglobulin deficiency exists among this group of patients with hcv infection. while it may be possible that lga levels decrease following infection, no evidence exists that this phenomenon persists in the long term. hereditary non-polyposis colorectal cancer (hnpcc) is the most common form of hereditary colon cancer and accounts for up to % of the overall cancer incidence. the disease is inherited in an autosomal dominant manner andis characterised by predominantly right-sided tumours. hnpcc is caused by mutations in one of four mismatch repair genes. these genes are homologues of the bacterial mismatch repair systerfi and their function is to detect and correct defects in dna. the hmsh gene is reported to account for up to % of cases, hmlhi, hpms and hpms account for %, % and % of cases respectively. we have collected eighteen irish hnpcc families which satisfy the amsterdam criteria for the disease. we have screened the entire hmsh gene in eight of these families. a combination of single strand conformational analysis (sscp) followed by dideoxy sequencing was used to detect defects in the dna. sequence analysis revealed the presence of a novel polymorphismin exon in one family and a possible splice site mutation in the remaining ten families, using a novel mutation detection technique. departments of medicine and immunology, university college hospital, galway. serial iga gliadin antibody (igaga) studies are used as a surrogate for repeated small intestinal biopsy to monitor dietary compliance in treated coeliac patients. we have correlated igaga levels with indices of hyposplenism (platelets and erythrocyte "pits") to determine if either index can be used to monitor adherence to a gluten free diet. the study population of biopsy proven coeliacs was stratified according to dietary status into gluten free diet (groups a) and normal diet (group b). the iga gliadin antibody titres were significantly lower in group a than group b ( . : units p = . ). in group a gliadin antibody titres showed a statistically significant correlation with both platelet (p = . ) and pits count (p -- . ) ; in group b gliadin antibody titres correlated with platelet counts only (p - . ). those ona gluten free diet were further subdivided according t ~ reported degree of adherence to the diet (strict diet -sgfd, lax'diet -lgfd). significantly higher gliadin antibody titres were found in lgfd than sgfd ( . ; . p = . ), but the ptatelet and.pit counts were similar in the two groups. igaga is superior to platelet or pit coums in monitoring dietary'compliance in coeliac disease. hospital, galway. iga endomysial antibodies (igaea) are said to be more specific and sensitive indicators of coeliac disease than iga gliadin antibodies (igaga). initially the igaea substrate was monkey oesophagus and more recently umbilical cord. a new commercial product has becomeavailable using primate smooth muscle (accu track -diagnostic slides). we have undertaken a preliminary study to compare the performance of this lgaea assay and igaga elisa in treated and untreated coeliacs, coeliac relatives and controls. the study population consisted of biopsy-proven coeliacs who were stratified according to "reported" dietary status into good gluten free diet (group a), poor gluten free diet (group b) normal diet (group c) and unaffected coeliac relatives (group d) and controls (group e). elevated igaga (> units) were found in the following groups : a %, b %, c %, d %. igaea were positive in a %, b %, c %. with respect to igaga positivity, there were false positive igaeaa %, b . %, c % and false negative -a %, b %, c %, d %. in this preliminary study in untreated coeliac patients the performance of the igaea test was on a par with the igaga assay. alt and fibrosis may be associated with a non-alcohol steatohepatitis and these processes may be synergistic. finally, number and type of riba bands is not a predictor of inflammatory activity. ~stepping hill hospital, stockport, uk sk je. royal oldham hospital, rochdale rd., oldham ol jh. we investigated upper gut bleeding in patients aged years and over. a proforma addressing demography, drug therapy, clinical status, timing of endoscopy / surgery, and outcome was used. consecutive patients (median age years, range - ) were studied over months. ( %) underwent gastroscopy with a % diagnostic yield. patients had severe oesophagitis, had oesophageal malignancy, had gastric ulcers - of which are malignant and had duodenal ulcers. ulcerogenic drugs were implicated in patients. patients were referred for surgery, operated upon with one postoperative death. had haemoglobins of g/dl or less. all malignant lesions were inoperable. the overall mortality was % reducing to % if neoplasla were excluded. co-morbidity influenced mortality. patients were discharged with a median hospital stay of days. information on cause of bleeding greatly influenced management. the prognosis of gut haemorrhage in the very old need not be so poor. a few require surgery but the majority respond to active medical resuscitation which is a key factor in determining outcome. we advocate low threshold for endoscopy, judicious use of ulcerogens and adherence to guidelines on management of upper gut haemorrhage. haemochromatosis (hh), a common recessively inherited disorder of iron metabolism is closely linked to the hla-a locus on chromosome . linkage studies have demonstrated a close association between (hh) and the hla alleles, a and b ("ancestral haplotype"). heterogeneity at the molecular level may account for the variance in clinical phenotypic expression. the aim was to evaluate phenotypic expression of hh in the presence/absence of the a -b ancestral haplotype. probands ( m: f) from unrelated irish families were investigated. phenotypic variability was assessed with regard to l) age; ) % trans.sat.; ) serum ferritin; ) liver bx iron grade; ) body iron stores and )symptomatology. three males were homozygous for a b , were heterozygous for a b and were non-a b . symptomatology, trans, sat., serum ferritin and liver bx grade were not influenced by homozygousity or heterozygousity for a b . conclusion: there were no significant differences in phenotypic expression on comparison of the three haplotype groups. no predominant genotype appears to be responsible for phenotypic severity in irish families indicating the possibility of multiple mutagenicity of the hh gene. of riba positive anti-d associated chronic hepatitis c patients, were pcr positive but had surprisingly mild disease. the disease status of the pcr negative patients was hitherto uncertain and is the subject of this study. / riba positive patients referred to this centre were biopsied because of elevated alt ( ) or florid symptoms which dated from inoculation ( ). histological activity index * , , , , , (f), (f), , (f), , , , , , , , no bile duct damage, lymphoid follicles or aggregates was observed. / had mild periportal fibrosis (f), of these had steatohepatitis with obesity ( ) and impaired glucose tolerance ( ) suggesting dual pathology. we conclude that riba positive, pcr negative patients have minimal disease activity. elevated the association between the hla locus and haemochromatosis (hh) has allowed early identification of affected siblings. it is unclear what proportion of subjects who are predicted to be homozygous or heterozygous for the disease by hla typing develop the disease. studies correlating clinical features with hla type in families from ireland -a putative source of this celtic trait have not been described. the aim was to correlate clinical, biochemical and pathologic features of hh with hla typing in first degree relatives of probands. initial analyses identified homozygous (hh), heterozygous (hn) and normal (nn) individuals. however, / hn individuals had stainable iron on liver biopsy, confirming hh. further hla analysis revealed homozygous x heterozygous matings and identification of all disease haplotypes within each pedigree allowed final classification of hh, hn and nn individuals. vol. . supplement no. conclusion: this study demonstrates the importance of hla typing in the clinical management of families with hh, furthermore, in multiply affected families the incidertce of homozygous x heterozygous matings is high indicating the high degree of "pseudodominance" in the irish population. the degree of acute hepatic failure after severe trauma and sepsis is related to the extent of hepatocyte (hc) damage and cell death resulting from either necrosis or apoptosis. we have previously demonstrated that tnf-ct and lps can directly lead to hc necrosis, but not apoptosis. recent, studies have shown that reactive oxygen intermediates (roi) and nitric oxide (no) are capable of inducing apoptosis in eukariotic cells. however, it is unclear whether roi or no are involved in hc cell death. the aims of this study were to evaluate the role of no and roi in hc cell death (apoptosis vs necrosis). hcs were isolated from sprague-dawley rats, and cultured with the no donor, sodium nitroprusside (snp) or the roi generation system, hypoxanthine-xanthine oxidase (hx-xod) and h . the effect of lps, tnf-t~, and ifn-y alone or in combitmtion with different antioxidants and the no synthase inhibitor, n-methyl arginine (nma) on hcs was also assessed. snp caused a dose-dependent increase in hc apoptosis. roi generated by hx-xod and h did not induce hc apoptosis, but were responsible for hc necrosis. tnf-ct alone failed to induce hc apoptosis, but when ~combined with antioxidants resulted in increased hc no production and apoptosis. this effect was attenuated by nma. snp also induced hc damage and hc necrosis. moreover, tnf-ct-mediated hc damage and necrosis could be further reduced by the combination of antioxidants and nma. these results indicate that roi preferentially induce hc necrosis, but not apoptosis. induction of no resulted in both hc apoptosis as well as hc necrosis, which suggest that overproduction of no may be detrimental during the sirs. irish journal of medical science intervention was not uniform. mean albumin was . g/ . mean weight was . kg. poor cognitive status greatly increased the requirement for dietetic consultation time. lack of dietetic resources results in inadequate monitoring of these patients following discharge. this study highlights the need for a dedicated clinical nutrition service, for medical services for older people. periconceptual consumption of folic acid has been shown to decrease the incidence of neural tube defects. the preventative strategy of universal food fortification with folic acid presents the possible risk of masking the diagnosis of cobalamin deficiency in pernicious anaemia. in addition, the ultimate longterm effect of universal exposure of adult or foetal cells to a synthetic substance, ie. folic acid, is unknown. in this study, the threshold oral dose of folic acid in a number of foods above which metabolically-unaltered vitamin appeared in serum postprandially was determined in a young and elderly population by microbiological assay of serum pre-fractionated by hplc. subjects on a five-day regime of fortified cereal and bread along with their normal unfortified diet. were shown to have a threshold level of ~g/d, abovewhich unaltered folic acid appeared in the serum. individuals given folic acid in either isotonic saline, milkor white bread exhibited a threshold level of ~tg per serving. from patterns of food consumption in ireland, even moderate levels of fortification are likely to lead to some population groups being exposedto excessive amounts of un-altered folic acid in serum. many older people are nutritionally compromised. there is clear evidence that: nutrition intervention reduces morbidity m and mortality in older patients. to identify the spectrum of nutritional abnormalities referred for dietetic intervention and the problems associated with nutritional assessment, elderly patients were alphanumerically selected from files of the department. of nutrition and dietetics. the most common dietetj.c interventions were: use of supplements %; high protein high calorie diet %; nasogastric feeding %; reduction fat %; iron/thiamine assessment %; high fibre diet %; diabetic diet %; nutrition swall w programme %; lipid lowering diet %. some % referred required nutritional supplements, but the profile of an increase in oxidative stress in cystic fibrosis patients has been suggested. activated neutrophjls in the presence of chronic lung inflammation in addition to increased activity of the electron transport chain in cfmay. increase free radical generation. antioxidant protection against free radical attack is likely to be compromised as a i'esult of deficiencies in fat soluble antioxidants vitamins. in the present study stimulated thiobarbituric aoid reacting substances (tbars) were measured to determine the ability of plasma to withstand lipid peroxidation. copper was used to in!tiate the breakdown of lipids to lipid hydroperoxides and eventually to aldehydes, mainly malonyldialdehyde (mda). pooled cf plasm a and pooled control plasma were incubated for , , , , and min. mda complexes with thiobarbituric acid which absorbs at approximately rim. there is a lag phase where antioxidartts vol. , irish journal of supplement no. medical science in the plasma or tissue protect against lipid peroxidation, then a log phase where the protective effect is overcome and finally the reaction reaches a plateau when lipid peroxidation is complete. absorbance at nm was measured in all samples and zero order and first derivative spectra were obtained. the lag phase appears to be longer in the pooled cf plasma compared with controls. plasma t~-tocopherol levels were within the normal range in both groups, indicatin~ an alternative protective effect in cf. mild hyperhomocysteinaemia is an established risk factor for heart disease. a source of homocysteine in humans is the essential amino acid methionine found in protein of animal origin. in an -week study weekly fasting plasma homocysteine levels were examined in a group of healthy male subjects (n= ) under normal dietary conditions (weeks to ) and in response to graded increased methionine intakes (weeks , , ). nutrient intakes, including methionine, were calculated from x -day food records. under normal dietary conditions weekly mean plasma homocysteine levels were not significantly different (anova) from each other ranging from . + . to . + . ~tm/ . doubling daily methionine intakes (supplementing with mg/ kg/d) did not result in a significant increase in plasma homocysteine ( . + . ~m/ ), however, significant increases were achieved when diets were supplemented with methionine at levels of and mg/kg/d resulting in mean plasma homocysteine levels of . + and . am/ + . , respectively. mean plasma homocysteine levels returned to baseline ( . + . ~tm/l) days post supplementation. we conclude that supplementary methionine results in a significant increase in plasma homocysteine only when levels of five times the normal dietary intake are reached. this study is evaluating the use of a synthetic construct which encompasses primer binding sites for lpl and a variety of cytokine and other transcripts, to quantify lpl expression in cultured human monocyte-derived macrophages. following isolation of total rna at various times during cell culture, its reverse transcription (rt) using random hexamer primers generating first strand cdna and specific amplification of lpl cdna targets by polymerase chain reaction (pcr), generates products identifiable on gel electrophoresis. quantitation of message is obtained by incorporating the pawl construct in the rt assay in varying quantities as an internal standard with known amounts of monocyte-macrophage rna (l~tg). pcr amplification of this construct yields size distinguishable products from that produced by the monocyte-macrophage lpl cdna transcript. pcr conditions for the assay have been optimised at cycles of denaturation (tmin @ ~ annealing ( . min@ ~ and extension (lmin@ ~ lpl mrna has been detected in cultured monocytes and macrophages throughout their differentiation. also increased expression of monocyte lpl mrna has been observed following hr incubations with chylomicrons ( t~g/ x mononuclear cells/ ml)-when compared with controls. interestingly, little or no lipase mrna was detectable in circulating monocytes using identical pcr conditions to preparations of mrna from the day and day cultured cells. this methodology will now permit investigation of the factors controlling lpl expression in cultured human monocytic cells. replacement growth hormone (gh) therapy in adult hypopituitarism is attracting increasing interest. in markussis (l) detected premature atherosclerosis by ultrasonography in the untreated patient we have shown plaque regression with patients on replacement gh (norditropin) in a -month trial. females and males were recruited, mean age . years. at each timepoint plaque characteristics were measured by duplex ultrasound. patients showed a large reduction in plaque size (mean %) after four months treatment (p value < ~ l). similarly, highly significant values in cholesterol, hdl and ldl and apo a are achieved. chol hdl ldl apo a pretreatment . . . posttreatment . w . w . w ~ w achieve a high degree of statistical significance (p< . ). the significant reductions achieved in plaque characteristics in six patients studied who showed plaque formation correlates with other parameters traditionally accepted as reducing cardiovascular risk. hypertension is found in approximately % of patients with cushing's syndrome, but the mechanism is poorly understood. previous studies in our unit have examined levels of exchangeable sodium, plasma renin and angiotensin ii and cardiac sensitivity to phenylephrine. one previous study has demonstrated enhanced pressor responsiveness to noradrenaline in a group of patients with cushing's syndrome due to adrenal adenoma. we have investigated the blood pressure response to noradrenaline in patients with pituitary dependent cushing's syndrome and in controls matched for age, sex and bmi. noradrenaline was infused for minute intervals at five different concentrations between . and . mcg.kg.min -~ multiple systolic and diastolic readings were recorded and the infusion was stopped if the systolic pressure became > mmhg, diastolic _> l mmhg or the systolic pressure rose > mmhg. basehne blood pressure in the patients with cushing's disease (cd) was / + / compared with / + / mmhg in the normal controls (nc). in of the patients with cushing's disease, the test had to be stopped before completion of the protocol, whereas this was necessary in only one control subject the change in blood pressure from basehne to the blood pressure value recorded either at the time the test was stopped or at the peak blood pressure reading during equivalent noradrenaline infusions was compared between the matched pairs. the mean change in diastolic pressure was + mmhg in cd compared with + in nc (p< . ). there was no statistically slgmficant difference in either systolic pressure ( + vs + mmhg) or mean arterial pressure ( + vs + mmhg). these results demonstrate an increased diastolic pressor response to noradrenahne tn cushing's disease. increased pressor sensitiwty to uoradrenaline may contribute to the elevated blood pressure seen in cushing's disease. increased plasma homocysteine (thcy) is an independent risk factor for premature vascular disease. patients with insulindependent diabetes have an increased prevalence of cardiovascular disease. accordingly, we measured plasma thcy concentration in such patients ( - y), randomly selected, and in control subjects. in controls, thcy was higher in males than in females (supine: geometric mean ( % ci): , . ( . , ) v . ( . , . ) i.tmol/l, p< . ), as previously described, but there was no gender difference in patients. male patients, without microvascular complications, had lower thcy than controls (supine: . ( . , . ) v . ( . , ) ~mol/l, p< . ), but values in female patients without complications were similar to those of female controls, thcy significantly correlated with age in diabetics but not in controls, thcy increased in patients with increased severity of microvascular complications, partly due to the effect of age. thcy was higher when standing.than when supine in both controls ( ( . , . ) v ( . , . )lalmol/l, p< . ) and patients ( . ( . , . ) v . ( . , . )l.tmol/l, p< . ). the absence of gender difference, the association between thcy and age, and higher levels with increasing microvascular complications suggest thcy could be of pathogemc significance in iddm patients, despite unexpectedly low levels in male patients without complications. differences.between supine and erect samples may be due to haemodilution of albumin-hound thcy in the latter a review of the treatment outcome of thyrotoxicosis with standard dose/doses of radio-active iodine (sdrai) in consecutive patients presented to the endocrinology department, uchg, from december -december was analysed. the mean pre-treatment levels of free thyoxine (ft ) was correlated with the treatment outcome. there was statistically significant difference in the pre-treatment ft between responders and nonresponders to the first dose rai (p = . ). response with hypothyroidism and/or euthyroidlsm was considered successful treatment / ( %) responded to a single dose rai; / ( . %) and / ( %) responded with euthyroldism and hypothyroidism respectively / ( . %) and / ( . %) responded to the second and third doses of rai respectively giving a total response rate of . % and % respectively. interestingly, / ( . %) patients failed to respond even to the fourth dose rai / ( . %) patients with t toxicosis ( females and male). two responded to the first dose (one with hypothyroidism and the other with euthyroidism), the remaining required a second dose, which produced the same results. no statistically significant difference in the response rate between t and t toxicosis (p = . ) was observed. inherent in the st. vincent declaration targets is the need for continuous data collection and audit. we present preliminary information from the mater database, the first prospective audit of patients from a homogenous irish population. , iddms ( m: f) were identified with the following characteristics (mean + sd), age . + . years, duration of dm + . years, bmi . + . (males) and . + . kg/m (females), hbalc . + . % (n< . %). no male:females differences existed in the above nor in macrovascular complication rate . % (predominantly peripheral vascular &sease and lschaemic heart disease). however, males were more likely to be current smokers ( % vs %, p = ). hypertension rates ( . m vs . %f), cholesterol > . mmol/ ( . m vs . %f) were similar but more males had cholesterol < . mmol/l ( . m vs . %f, p < . ). clinical nephropathy was present in . % of males vs . % in females (p< ) . % had clinical peripheral neuropathy. retinopathy will be described elsewhere. . % of females and . % of males had a history of hyperthyroidism and . % of females vs . % of males of hypothyroidism. . % had history of psychiatric disease. conclusion although not a population based study, care of iddm in ireland is almost totally hospital clinic based cigarette smoking is identified as the major problem to be addressed patients with diabetes meltitus (dm) are at a higher risk of developing vascular complications, including coronary artery disease (cad). we performed a detailed analysis of predictors of cad and its seventy in patients with dm and chest pain patients in total single vessel cad (svd) in , double vessel (dvd) in , and triple vessel (tyd) in on cine contrast angiography clinical, biochemical and dobutamme stress echocardiographic findings are tabulated below for patients with angiographically proven coronary artery disease. patients with tvd had a longer duration of dm ( years) and were more likely to have retinopathy ( %) the sensitivity of dse was excellent for severe disease. conclusion' duration of dm, retinoapthy, and a positive dse were the best predictors of severe cad in a diabetic population with chest pain the haemodynamic hypothesis for the pathogenesis of diabetic microangiopathy argues that an initial increase in microvascular flow leads to sclerosis and disturbed microvascular autoregulation. we have recently demonstrated impairment of vasoconstrictor responses to endothelin- , a potent endothelium-derived constrictor substance, in niddm and have suggested that this could contribute to the initiation of microangiopathy the purpose of this study was to determine whether responsiveness to endothelin-i is also impaired in iddm. non-specific vascular smooth muscle contraction was assessed using high dose serotonin eleven patients with iddm and control subjects underwent forearm blood flow (fbf) measurement by venous occlusion plethysmography in response to local infusions of endothelin- ( pmol/min for minutes) and serotonln ( la g/min for minutes) control subjects showed slow onset vasoconstriction in response to endothehn- reaching maximum at minutes (p< . ) the diabetic group did not respond to endothelin-i group differences were significant (p= . ). the two groups showed similar vasoconstriction in response to serotonln. in conclusion, vasoconstriction in response to endothelin-t is impaired m iddm non-specific vascular smooth muscle contraction is preserved. impaired vascular responsiveness to endothehn-i is a possible common mechanism for the pathogenesls of microanglopathy in ddm and niddm. we measured total corrected (tca) and standardised ionised calcium (lca) in a population of intensive care ( cu) patients (with a mean age of + years, % male) to determine the prevalence of abnormalities in circulating calcium and its possible determinants severity of illness was measured by the apache ii score (acute physiological and chronic health evaluation). for comparison of ica we examined subjects undergoing arterial gases which proved to be normal and non-critically hypoxlc subjects ica was measured on arterial gas samples and corrected for ph % of icu patients had a total ca (unadjusted) of < mmol/l. after adjustment for serum albumin, % of icu patients had an tca < mmol/ % of icu patients had a serum phosphate of < . mmol/ ica in controls was . + . mmol/ and . + . mmol/ in hypoxlc non icu patments (ns) ica in icu was lower: . + . (< . ). tca and lca were not slgmficantly related. tca and ica did not sigmficantly differ between patients who died and who survived in the icu, and they were not related to apache ii score. belfield, dublin . from july to june there was a -fold increase m the annual number of specimens submitted to the virus reference laboratory because of a perceived risk of contracting hiv through a needlestick injury, blood splash, human bite, or through occupational exposure. needlestick-associated specimens also comprise an increasing proportion of 'at-risk' specimens, rising from . % m the year july -june to . % in the year july -june . between july and june a total of patients had specimens submitted for hiv antibody testing after a perceived'exposure to hiv of these only patients had more than specimen taken. although the time of putative exposure is rarely avadable, the median interval between st and nd postexposure specimens for these patients is months with / ( %) lying between to months. if the risk of hiv refection from a needlestlck injury is assessed as sufficient to warrant serological investigation, the timing and number of blood samples are important. a negative report from a single early specimen may not indicate an absence of infection a basehne specimen and follow-up specimens at weeks, months and at a minimum of months post-exposure are recommended appropriate serology for other viral mfections (hepatitis b and hepatitis c) should also be considered. since the beginning of a significant sustained increase in the numbers of hepatitis a cases has occurred. the number of cases m was against in . this reverses the continuous fall observed over previous years. the reason for this increase remains unidentified at present this increase has occurred following a dramatic increase in the total number of hepatitis a igm tests carried out by the vrl since february/ march the increase in the number of tests carried out since this time is primarily attributable to increased hepatitis testing following receipt of hepatitis c-contaminated rhesus anti-d immunoglobuhn. analysis of the age profiles of the positive patients and of all the referrals indicates that . % of positive results were found m patients aged yr or less whdst % of all hav igm tests were performed on individuals aged yr or greater. this raises the question whether greater selectivity should be employed when requesting hepatitis a tests studies report compliance rates ranging from to % in hiv negative patients there has been no comprehensive study of compliance in hiv positive patients. accurate measurements of compliance are not easy; easy measurements of comphance are not accurate "). to determine the compliance rate in hiv posmve patients attending st. james hospital, dublin, one hundred consecutive patients attending the service were interviewed (homosexual , ivdu , heterosexual ). the questionnaire was divided into three sections. firstly, a medical review was completed by the clinicmn which included demographic data, cd count, cdc staging, karnofsky index and prescribed medication. secondly, the pharmacy detailed the medication dispensed to each patient. the third section comprised a patient interwew to determine adherence to, and understanding of prescribed drug therapy. we report an overall comphance rate of %. this was unevenly distrtbuted between the two main patient groups ( % in homosexuals, % in ivdu) the following factors were found to mfluence compliance: number of medications, cdc stage, karnofsky index, dysphagia, educational and socio economic factors. we also found that poor patient understanding of the prescribed therapy significantly affected compliance. the aim was to determine the incidence of stds in patients presenting for hiv testing at the department of genito-urinary medicine, saint james's hospital. a retrospective analysis of all patient notes who presented for hiv testing between july ' and december ' was undertaken. according to clinic policy all patients had been screened for the following stds; neisseria gonorrhoea, chlamydia trachomatls trlchomonas vagmalis, candida, human papilloma virus, herpes simplex virus syphilis and hepatitis b in addition intravenous drug users (ivdus) were also screened for hepatttls c all patients underwent pre test counselhng. sex, age, risk groups and diagnoses were noted. patients presented for hiv testing, of whom % were male, % female, wtth an average age of . years. of the total, % were, or had previously been ivdus. of the total ivdus, were heterosexual males were bisexual and were females . % of the male patients were homosexual and % btsexual there were posmve hiv tests ( % of total); males and female. in this group there were patients with hepatitis c, all of whom were ivdus. no other stds were detected in the hiv negative group hepatitis c was diagnosed in , hepatttis b in , anogenital warts in , herpes gemtalis in , syphilis in , n. gonorrhoea in , t. vagmalis in i, c. trachomatls m , g vaginalis in and candldlasis in . this study confirms the importance of std screening in all patients requesting a hiv test. of the total testing for hiv, % had a concurrent std diagnosis although no stds were identified in the hiv positive group this may be more a reflection on the makeup of the irish hiv positive population, the majority being ivdus, rather than a difference m the mode of sexual toxoplasmosis is the most common opportumstlc infection of the central nervous system in aids patients. in clinical practice the diagnosis depends on clinical, radiographic and serological findings coupled to chnical response to therapy brain biopsy is not routinely performed. in this retrospective review, we describe our experience with diagnosing toxoplasmosis we examined (a) the clinical demographics and presentation, radiographic findings, response to therapy and patient outcome (b) role of polymerase chain reaction (pcr) in detecting toxoplasma gondii from blood and csf samples (c) the usefulness of serology in diagnosing acute infection. all cases diagnosed as toxoplasmosls based on the above criteria were reviewed. pcr to detect toxoplasma gondn dna used primers to the b gene giving a bp amphficatton product serological tests used were sabln-feldmen dye test and latex agglutination. there were cases diagnosed ( m, f, cd - ; cdc iv ). panents unknown to be hiv posmve presented with cerebral toxoplasmosis patients were not receiving continuous systemic prophylaxis against pce diagnostic value oft gondii pcr in blood and csf showed a sensmvlty of %, specificity of %, ppv was % and npv was % determination of lg subtype was of limited value % ( ) of patients were seronegatlve of whom % ( ) had histologically proven disease of these latter cases were pcr negative the dye test was of poor predictive value this review confirms the need to combine all parameters in making a diagnosis of toxoplasmosis in lmmunocompromlsed hosts. the performance of the newly developed, rapid and fully automated m~croparticle enzyme immunoassay abbott imx hiv- /hiv- rd generation plus assay for the detection of antibody to hiv- and hiv- including subtype o m human serum or plasma was assessed the assay was evaluated by testmg specimens from blood donors, diagnostic populations and hospitalized patients, hiv seroconverslon panels confirmed hiv-positive specimens, and potentially interfering specimens. the abbott imx hiv- /hiv- rd generation plus assay showed an overall apparent specificity of . % (lower limit of % ci . %) in the tested blood donor populations (n= t). this comparable to the specificity found for the abbott imx hiv-i/hiv- rd generation plus eia ( %) and the axsym h v- /hiv- assay ( %). the apparent sensitivity of the abbott imx hiv-i/hiv- rd. generation plus assay is at least equivalent to that of the abbott imx hiv-i/hiv- rd generation plus eia and the axsym hiv-i/hiv- assay. of hiv-i seroconversion panels tested, the abbott imx hiv- /hiv- rd generation plus assay detected seroconverslon earlier on up to panels, depending on the comparison assay. among specimens from asymptomatic and symptomatic hiv patients, the abbott imx hiv- /hiv- rd generation plus assay detected ( ) including specimens characterized as hiv- subtype o. the abbott imx hiv- /hiv- rd generation plus assay is an extremely sensltlve and highly specific assay for the early detection of antibody to hiv- /hiv- and shows at least an equivalent performance to the abbott imx hiv-i/hiv- rd generation plus eia and the axsym hiv- /hiv- assay. the fully automated imx instrument system offers ease of use and rapid results on a widely accepted and reliable platform. streptokinase (sk), a kd protein produced by group c b hemolytic streptococci, is a widely used thrombolytic agent. anti-sk antibodies arise either as a result of therapeutic administration of sk or following natural mfection with streptococci although the clinical significance of antl-sk antibodies is not clear, there is evidence that some anti-sk antibodies arising from natural infections can interfere with sk activity tn vtvo, resulting in thrombolytlc failure. to facilitate further investigations of these antibodies, we have developed and validated a highly sensitive functional assay, which measures sk neutralisatlon activity of serum independently of other circulating inhibitory factors in the sample, and a rapid and convenient enzymeimmunoassay for the detection of anti-sk antibodies. analysis of over random serum samples from the local blood bank with the enzymeimmunoassay showed the prevalence of antl-sk antibodies to be approximately %. all the positive samples and an equal number of the negative samples randomly selected were analysed by the functional assay the agreement between the results of the two assays was excellent indicating that our enzymelmmunoassay was a convement method for detection of anti-sk antibodies which could neutrahse sk activity m vitro irish journal of medical science tuberculosis drug therapy, isolation precautions and prophylaxis. conventional methods of detection such as microscopy and culture either lack sensitivity and specificity or are timeconsuming. in this study we investigated the use of a pcr based diagnostic assay for the detection of m. tuberculosis in sputum samples this assay has been developed by bioresearch ireland (bri) and raggio italgene. sputum samples were lysed and pcr amplified using an m. tuberculosis complex-specific primers. the results obtained using the bri/c-trak tm technology were initially compared to the amplicor system (hoffman la roche). both probe detection methods represent fast and reliable methods for the detection of m. tuberculosts in clinical samples. this test is designed to eliminate the possibility of obtaining false negatives. strongyloldes stercoralis infection in humans ts endemic in the tropics. as travel is becoming more common, it will be seen more frequently. two cases of this infection in irish people are described. case i. a year old women had travelled and worked in poor rural areas of mexico for one month, three years before presentation. two and a half years later she developed abdominal discomfort, anorexia and sore throat. myalgia, arthralgta and a transient skin rash began to appear in the next month. eosinophilia, mild anaemia and raised liver blood tests were noted. elisa test for strongyloides was positive but parasites were not seeri in the faeces. ivermectin was given and the patient feels better. case ii. a year old nurse had arthralgia, fatigue and some weight loss for months'. on two occasions in the last four years, she had been travelling extensively in s.e. asia for a total of four months. she was admitted to hospital because of acute fever and loin pain. a urinary tract infection was diagnosed. absolute eosinophil count was i'aised . x ^ / . esr was mm/hr. strongyloides elisa was positive and treatment administered as above. strongylotdes is the most important nematode in the returned tropical traveller. it can multiply and persist within the body for long periods of time and it can cause hypertnfection syndrome, a protean fulminating infection of bowel, lungs, blood stream and brain, in those who are lmmunocompromised. diagnosis can be difficult by stool microscopy. thlabendazole has side effects but ivermectin is safe and effective. june , there was an outbreak of c.difficde-associated diarrhoea (cdad) at st. james's hospital. the aims of this study were to determine the incidence and outcome of cdad in hiv positive and negative patients we prospectively reviewed all patients with diarrhoea, a positive c.difftcile cytotoxin assay, and in whom no other infectious cause for diarrhoea was identified demographic data, history of diarrhoeal episodes, risk factors and outcome were recorded. the incidence of cdad in hiv negative patients was . per hospital admissions, compared to per admissions m hiv positive patients. the average number of courses of antibtotlcs received, in hiv negative patients prior to the onset of symptoms was . , and % of this group were exposed to third generation cephalosporins. hiv positive patients received an average of . courses of antibiotics and no patients received third generation cephalosporins. there were no deaths due to cdad in hiv positive patients however hiv negative patients died from severe pseudomembranous colitis in conclusion we documented a unexpectedly low incidence and complication rate of cdad in hiv positive patients this is surprising considering their multiple hospital admissions and exposure to ant~microbial and chemotherapeutic agents. the number of new positive hiv specimens detected at the virus reference laboratory has risen from a cumulative total of m july of to in september of . we examined our data to determine the proportional make up of these positives by major risk group. in august . % of positive specimens were from intra venous drug abusers (ivda) by september ivda made up % of the total positive hiv specimens. positive specimens from homosexual individuals rose fro.m l % of total positives in august to . % in september there were no recorded positive specimens from heterosexual exposure in august but in september . % of positive specimens recorded heterosexual exposure. a further category which includes blood donors, haemophiliacs, transplant patients and organ donors made up % of total positives in august and in september made up . % of total positives. we further examined our data in order to show when these changes occurred by ascertaining how many new positive patients have been discovered per year in each of the main risk groups. see in the united kingdom echovlrus type (echo- ) is regularly isolated, with nearly reports annually to the central public health laboratory. reports increased during - and overall it is the second most commonly reported echovlrus in the u.k. echo- epldemiology is different to that of other enteroviruses; over % of patients with echo- tsolated are less than years of age echo- shows distinctive and unique cytopathogenic features in tissue culture, and based on sequence analyses, it seems to belong to a separate subgroup of picornaviruses. echo- has been associated with respiratory symptoms in premature infants, myocarditis and severe encephalitis. in an outbreak of acute flaccid paralysis associated with echo- was described in jamaica in six patients, four of whom died. we describe three cases of sudden death in infants associated with echovirus type infection case i: s d. born / / ; birth asphyxia and death at two days of age; echo- isolated on from spleen. this study assessed the antibiotic sensitivity of organisms causing urinary tract infections (uti) among genito-urinary medicine (gum) clinic attenders in order to determine whether it is worthwhile giving tetracycline for dipstick (nitrite) positivity, even in the absence of clinical features of uti. we looked retrospectively at laboratory confirmed uti's diagnosed among gum clinic attenders over a period of eight months. we assessed antibiotic sensitivities of the organisms involved, and determined how many dipstick positive urines which were left untreated turned out to be real uti's. % of uti's were due to coliforms and % of these were sensitive to tetracycline. % of uti's were due to staphylococcus saprophyticus, % due to beta haemolytic streptococcus group b, % due to enterococcus, % due to proteus species and % due to coagulase negative staphylococci. % of nephur positive urines were left untreated. % of these were nitrite positive. failure to treat a positive urine dipstick which turned out to be a uti necessitated a further clinic visit for adequate treatment. nitrite positive urines should be treated as a uti, even in the absence of clinical features of uti, either with trimethoprim or tetracycline. the number of untreated uti's and unnecessary extra visits to gum clinics would have been reduced with the use of judicious antibiotic therapy for nitrite positive urines. strains of enterococci resistant vancomycin have been reported with increasing frequency. in , we investigated an increase in the frequency of vancomycin-resistant enterococcus faecium (vref) among patients in the haematology/oncology unit using pulse-field gel electrophoresis (pfge) to genotype these isolates and to assist in establishing the source of these vref. eighteen clinical isolates of vref from blood, urine sputum and-faeces and two environmental isolates were collected from separate patients between march and july . minimum inhibitory concentrations (mics) to several antibiotics including teicoplanin and vancomycin were determined by agar dilution. pfge were performed following smal restriction endonuclease digestion. antimicrobial susceptibility testing revealed high level resistance to vancomycin and teicoplanin; mics > mg/ l and > mg/l respectively. this antibiogram is consistent with the van a phenotype. pfge of all isolates revealed identical patterns indicating clonal spread of vree subsequent implementation or infection control measures reduced the frequency of vref isolation. pfge proved useful in demonstrating clonal spread of vref and.in emphasising the need for infection control measures. a prospective audit of baeteraemia in our bed teaching hospital was carried out from february to march . clinical and microbiological data were collected on episodes of bacteraemia in patients. of these ( %) were hospital acquired and ( %) community acquired. urinary tract and respiratory tract sources were implicated in % and % of community acquired episodes, making e. coli and s. pneumoniae the commonest community acquired isolates ( % and % respectively). other gram negative bacilli accounted for % and s. aureus for %. coagulase negative staphylococci were the commonest hospital acquired isolate ( %) followed by s. aureus ( %), e. coli (i %) and enterococcus spp. ( %). enterobacter spp. were the second commonest gram negative isolate ( %). central venous cannulae were implicated in % of hospital acquired cases. urinary tract infections accounted for %. % of which were catheter related. invasive diagnostic procedures (angiography, prostate and liver biopsies, sinography) were implicated in t episodes. gentamicin resistance was found in % of hospital acquired aerobic gram negative bacilli and mrsa accounted for % of hospital acquired s. aureus. these figures are higher than expected but may be explained by outbreak of mrsa and gentamicin resistant entercobacter spp. which occurred during the study period. the past severalyears have seen a significant increase in the recognition of moraxella (branhamella) catarrhalis as a respiratory pathogen (~). the pathogenic mechanisms employed by the organism are largely unknown, but adherence may play a role ~ ~. in our investigation the haemagglutinating ' activity of isolates of m. catarrhalis was determined by a microtitre method. no isolate agglutinated horse, chick or sheep red blood cells (rbc). seventeen isolates agglutinated human rbc, x~hile of these isolates also agglutinated rabbit red. blood cells. haemagglutination of human and rabbit red blood cells was inhibited by porcine mucin. galactose inhibited the haemagglutiriating activity of the isolates which agglutinate both human and rabbit rbc and yet bad no effect on the haemagglutinating activity of the isolates which haemagglutinate human rbc alone. .electron microscopy studies of the bacteria demonstrated a diffuse outer fibrillar layer on the surface of haemagglutinating positive isolates, thislayer was subsequently removed following trypsin treatment, as was the haemagglutinating activity. a kda trypsin sensitive protein appears to be associated wfth haemagglutinating properties. mrsa is an increasingly important cause of morbidity, and is spreading from large hospitals to smaller community-based facilities and nursing homes. the objective of this survey was to obtain an indication of the size of the mrsa problem in ireland prior to introducing national mrsa control guidelines. a survey of all microbiology laboratories in ireland was carried out over two weeks in spring . for patients from whom mrsa was isolated during the study period standard demographic and clinical data were requested and period prevalence/ discharges was calculated. all microbiology laboratories surveyed responded. mrsa was isolated from patients during the week period. the period prevalence of mrsa/ , discharges was . . males aged + had the highest rate of infection ( / discharges). half of all isolates were from patients in surgical or medical wards, but % were from community-based sources e.g. gps, nursing homes, hospices. thirty-two percent of mrsa patients were infected rather than colonised. mrsa is clearly a substantial problem in ireland. while it is largely a hospital problem at present, the increasing trend for day procedures and shorter stays means that infection will increase in the community. a survey in a university hospital in the usa revealed % of mrsa cases to be communityacquired. tonsil core specimens were cultured for bacteria including mycoplasma, chlamydia and ureaplasma urealyticum in children undergoing tonsillectomy for recurrent acute tonsillitis. serology for chlamydia and mycoplasma pneumoniae was obtained in of the children. the polymerase chain reaction (pcr) was used to investigate the presence of chlamydia pneumoniae in core tonsil tissue. ureaplasma urealyticum was cultured in three children ( . %) and mycoplasma salivarium in two children ( . %). culture was negative for chlamydia pneumoniae and mycoplasma pneumoniae. the complement fixation test for chlamydia species was positive in / children ( %) indicating previous infection. specific immunofluorescence testing for c. pneumoniae was positive for lgg (titre> ) in / ( %). igm antibody to c. pneumoniae and antibodies to c. trachomatis and c. psittaci were not detected. ninechildren ( %) had titres > to m. pneumoniae. pcr failed to demonstrate c. pneumoniae. aerobic and anaerobic bacteria were cultured from all specimens. the culture of ureaplasma urealyticum in . % of our patients indicates a higher rate of colonisation then previously thought. this study irish journal of medical science demonstrates past infection with c. pneumoniae in % and with m. pneumoniae in % of children with recurrent tonsillitis. however c. pneumoniae and m. hominis do not play a significant role in childhood recurrent tonsillitis. multiply resistant enterococci are increasingly common causes of serious infection in hospitalized patients. high level gentamicin resistance (mic > mga) in enterococci further compromises the therapy of such infections. we have identified seven clinical isolates of enterococcus hirae demonstrating high-level gentamicin resistance (hlgr: mic > i mg/ ). to our knowledge this is the first report of hlgr for this enterococcus species. plasmid analysis has demonstrated the presence of a single, large plasmid in all seven isolates, as well as several smaller plasmids in some of the isolates. filter mating experiments have revealed that in all seven cases, hlgr was transferred to a laboratory recipient e. faecalis jh- by conjugation. plasmid analysis of transconjugant strains confirmed transfer of the large plasmid in all cases. based on restriction enzyme profiles, two distinct conjugative plasmids were identified for the e. hirae isolates investigated. at present we are using southern blot techniques with oligonucleotide probes designed to hybridise to the hlgr determinant found in other species of enterococcus. the results will confirm whether or not the same resistance determinant is responsible for the dissemination of hlgr in the genus enterococcus. dublin . aminoglycosides remain commonly used in the treatment of severe gram negative infection and have conventionally been given on a twice or thrice daily basis. single daily dosing offers advantages with respect to less nephrotoxicity, better bactericidal activity, convenience, nursing time, cost and should avoid subtherapeutic dosing which has a significant impact on outcome. we reviewed serum gentamicin assays from january to december to assess potential toxicity and subtherapeutic dosing in patients who received once daily gentamicin and those who received multiple daily dosing. assays were performed in the study period. of those were random assays and not included. there was a trend towards significantly less potentially toxic levels in the once daily group compared to the multiple daily group (p< . ). once daily dosing produced significantly less subtherapeutic dosing (p< . ). over % of peak assays in the once daily group were in the recommended range. we conclude that current practice of multiple daily dosing of gentamicin leads to significant underdosing and more potentially toxic trough levels. measurement of trough assays only in patients who are treated with once daily aminoglycosides is sufficient and will have considerable cost savings. respiratory, syncytial virus (rs virus) is a major respiratory pathogen of infants less than year old. it occurs in annual epidemics during the winter and early spring in temperate climates. during rs virus epidemics a significant number of infants less than months old are hospitalised with symptoms of bronchiolitis and pneumonia. rs virus exists in two antigenically distinct subgroups, a and b which are known to cocirculate in the same community during the same rs virus season. there is much debate regarding the virulence of one strain over the other. using a panel of monoclonal antibodies specifically directed against the two rs virus strains, rs virus isolates from specimens sent to the virus reference laboratory, university college dublin, over seven consecutive rs virus seasons ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were typed and the rs virus subgroup predominance monitored. subgroup a was the most predominant of the rs virus isolates accounting for . % of the total and was found to be the predominant rs virus strain in six out of the seven rs virus seasons studied. subgroup b predominated in a season in which the number of rs virus detections peaked much later than normal. treatment of fungal infections in patients in intensive care unit (icu.) is usually empiric. the aim of this study was to identify candida species isolated from i.c.u. patients and to test their susceptibility to antifungal agents to enable more directed therapy. forty candida sp. from patients in i.c.u. were isolated from the following sites, blood culture ( ), central venous catheter ( ), chest drain fluid ( ), wounds ( ), catheter urine ( ), bronchial lavage ( ), sputum ( ). strains were identified by standard procedures. minimum inhibitory concentrations of amphotoeracin b, -flucytosine and fluconazole were obtained by agar dilution test and e test. the isolates were identified as c.albicans ( ), c.glabrata ( ), c.krusei ( ), c.tropicalis ( ), c.parapsilosis ( ), c.kefyr (i). all the candida species were sensitive to amphoteracin b (mic = -< mg/l), and flucytosine (mi c= < mg/l). c.albicans and c.parapsilosis were fluconazole sensitive (mic = - mg/l). four of eight c.glabrata were fluconazole resistant (mic = > mg/l). c.krusei, c.tropicalis and c.kefyr were also resistant (mic = > mg/l). wbilst there were no major discrepancies between the agar dilution test and the e test, the agar dilution test was laborious and required a high degree of skill. the e test was easier to read and more reliable results were obtained provided the inoculum was carefully standardised. this study shows that azole antifungals should not be ganglion is probably the commonest tumour encountered in the hand and the wrist. it often arises from tendon sheath or lining of a joint capsul. the treatment can be surgical or nonsurgical, the latter includes aspiration with or without injection of steroids. surgical treatment of ganglion can pose a difficult situation to deal with. it requires hand surgeon to deal with one such problem, we present a year old man with tender mass in the hypothenar eminence. during surgical exploration it was obvious that the ganglion was infiltrating the wall of the ulnar artery, and the histology proved this later. the clinical features, management arid the outcome of this unusual case are discussed. ischaemic preconditioning (ipc) of the myocardium with repeated brief periods of ischaemia and reperfusion (i-r) prior to prolonged ischaemia significantly reduces subsequent infarction. following ipc two "windows of opportunity" (early and late) exist during which prolonged ischaemia can occur with reduced myocardial infarction, we investigated if ipc of skeletal muscle prior to flap creation improved subsequent flap survival and perfusion in either early or late windows. the latissimus dorsi muscles (ldm) of sprague-dawley rats were used. group : (control, n= ). the ldm was elevated as a thoracodorsatly based island flap group : (early ipc, n= ). the ldm was preconditioned with two minute episodes of normothermic global ischaemia with intervening minute episodes of reperfusion prior to elevation. group : (late ipc, n= ). the ldm was elevated hours after ipc ischaemia was created by occlusion of the thoracodorsal artery and vein and the intercostal perforators having previously isolated the muscle on these vessels. muscle perfusion was assessed by a laser doppler perfusion imager. one week after flap elevation the percentage of muscle necrosis was measured by computer-assisted planimetry ipc significantly reduced muscle flap necrosis (table) in both early and late windows. muscle flap perfusion was similar in all groups. this study compares the biochemical, serological and histopathological findings in women with chronic hepatitis c virus (hcv) infection with age-matched women with established autoimmune hepatitis (aih). there is increasing evidence of autoimmunity in hcv liver pathology. because of different treatment regimens for hcv (d-interferon) and aih (steroids, immunosuppression), clear distinction between the two diseases is desirable. liver enzymes (alt, ast, alkaline phosphatase), antinuclear factor (anf), anti-smooth muscle (asm) and antimitochondrial (ama) antibodies are compared for both groups of patients. liver biopsies from all women were compared using the grading and staging system of ishak et al ( ) . the results show that while some women in both groups show elevated liver enzymes, positive anf and asm autoantibodies, the values are much higher in aih. similarly aih patients show overall more severe disease on liver histology. both groups demonstrate poor correlation between histological features and autoantibody titre. we conclude that distinction between hcv and a!h is usually possible with liver histology and serology. some women with chronic hcv and positive autoantibodies however," demonstrate a histological and serological picture suggesting that chronic hcv may be mediated by an immunopathogenic mechanism. irish journal of medical science terminal changes only, showed lymphocytic meningitis and of these also had perivascular lymphocytic inflammation (cd positive) in the subependymal regions, brainstem and choroid plexus. two brains showed purulent meningitis and one case had central pontine myelinolysis probably related to profound metabolic disturbance. basal ganglia mineralization, hiv encephalitis (hive) or hiv leucoencephalopathy (hivl) were not present. these findings differ considerably from those described in us cases, in whom the majority have evidence of hiv within the cns. relatively early death from systemic infection may account for the lack of hive/hivl in these cases. the lymphocytic meningitis and perivascular inflammation may represent an immuno-allergic reaction, previously reported as "early" changes, regarded as important in inducing vascular damage which allows subsequent entry of h v into the brain. the aim of this study is to assess apoptosis in areas of interface hepatitis and spotty necrosis in hepatitis c virus (hcv) infected liver biopsies, and to correlate the degree of apoptosis with severity of histological activity. liver biopsies were randomly selected from a group of type lb hcv positive women. these patients were diagnosed by recombinant immunoblot assay (riba) test and the presence of hcv rna was confirmed using the polymerase chain reaction (pcr). apoptosis was demonstrated in the biopsies by the oncor apoptag in-situ hybridisation technique. the average number of apoptotic hepatocytes per portal tract, and within the parenchyma per x objective, was determined. the modified histological activity index (h.a.i.) was used to score each biopsy. comparison of the results shows that increasing numbers of apoptotic hepatocytes are consistently associated with increasing scores for interface hepatitis and spotty necrosis. it is concluded that apoptosis occurs in hcv infected livers and that it correlates with increasing histological activity .indicating a significant role for apoptosis in the pathogenesis of hcv liver disease. and university of edinburgh, scotland. paediatric aids represents only % of cases worldwide, in most published series parental iv drug use was the main risk factor, cns pathology was a late feature of the disease and antiretroviral treatment had been given. we studied eleven brains from romanian children with probable postnatal hiv infection using standard neuropathological stains and immunostains for lymphocyte markers and hiv p antigen. death was due to systemic infection, mostly pneumonia or gastroenteritis; antiretroviral treatment had not been given. three cases showed we studied the role of the p and bcl- genes in the pathogenesis of post-transplant lymphoproliferative disease (ptl). ten cases were examined by immunohistochemical and molecular methods. immunohistochemistry was performed using standard and antigen retrieval methods, with the p do- and the bcl- oncoprotein clone antibodies. dna was extracted from paraffin blocks and subjected to pcr, and single-strand conformation polymorphism (sscp) analysis searching for mutated p genes. samples showing any evidence of aberrant migrations were further analysed by direct sequencing. pcr was also used to detect bcl- gene rearrangements. we employed a technique called representational difference analysis to search for previously undescribed translocations or deletions which may be involved in breast carcinogenesis. dna was isolated from both invasive ductal carcinoma of the breast and normal tissue from the same patient. following restriction enzyme digestion and tigation of oligonucleotide linkers, pcr was carried out on both tumour and normal dna using oligonucleotide specific primers to obtain a representation of each genome (amplicons). digestion with the same restriction enzyme removed the original linkers, and a second set of oligonucleotides were ligated to normal amplicons only. the tumour derived amplicons were subtractively hybridised to the normal and subsequent pcr was used to isolate fragments unique to the normal dna (difference products). this was possible since oligonucleotides were ligated to normal dna only. following a series of further subtractive hybridisations and subsequent amplifications, purified difference product was obtained. difference products in the size range - bp were obtained. following further rounds of subtractive hybridisation and amplification, purified difference product will be sequenced and characterised by comparison with known gene sequences. the chromosomal location of the affected gene will be established by in-situ hybridisation and somatic ceil hybridisation, using the difference product as probe. protein s is secreted by osteoblasts and case reports of reduced bone mineral density in patients with total protein s deficiency have lead to the hypothesis that this inherited disorder is associated with generalised osteoblast dysfunction predisposing to osteoporosis ~. we have assessed bone formation in patients ( male and female) with total protein s deficiency and controls ( male and female) using two recently available sensitive markers; serum osteocalcin (oc) and serum procollagen carboxyterminal peptide (p cp), both secreted by the osteoblast. the mean total protein s level amongst the patients was _+ % (ref range - %), mean oc was . ng/ml (ref ~'ange . - ng/ml) and the mean picp was . + uga (ref range - ug/ ). in the control group, mean oc was . _+ ng/ml and the picp was + ug/ . there was no statistical difference between both groups using either marker. in conclusion bone formation as assessed by serum osteocalcin and p cp appears to be normal in patients with total protein s deficiency. hereditary spastic paraparesis (hsp) is a variably expressed neurodegenerative disorder which exhibits clinical and genetic heterogeneity. hsp can be inherited in an autosomal dominant (ad), autosomal recessive (ar) or x-linked manner. ad-hsp has been linked to a number of loci. we have ruled out linkage to these loci in a large irish family affected with ad-hsp. the aim of this study was to determine whether ad-hsp is linked to spinocerebellar ataxia loci (sca), sca-i & sca-ii. ad-hsp can be clinically similar to sca. dna was extracted from blood taken from co-operating family members. microsatellite markers spanning the sca-i and sca-ii loci were amplified by pcr. individuals were genotyped and linkage analysis was carried out using the linkage set of programs. significantly negative lod scores were obtained for both sca-i and sca-ii loci. d s gave maximum exclusion of cm on either side of the sca-i locus with a lod of - . at a recombination fraction of . . d s gave maximum exclusion of cm on either side of the sca-ii locus with a lod of - . at a recombination fraction of . . other markers examined also outruled linkage to these loci. we conclude that the gene for ad-hsp is unlinked to the major sca loci. serum vitamin b is frequently measured in the investigation of anaemia, and in screening neurological and other disorders. frequently, patients are found with low serum vitamin b level with a normal hb and without clinical abnormalities relevant to vitamin b deficiency. this study was carried out to determine the significance of a low serum vitamin b level. vitamin b measurements were carried out over an month period using a chemiluminescence method (abbott imx). clinical data was obtained retrospectively. of the samples ( . %) representing patients had a low serum vitamin b level (> pg/ml) with a mean of pg/ml ( - ). data was available on patients. ( %) had a hb below the normal range with median serum vitamin b level of pg/ ml ( - ). ( %) had a normal hb, mcv and mchc with a median serum vitamin b of pg/ml ( - ), and had a normal hb with an abnormal mcv or mch. lft's, autoantibodies, schillings test and bone marrow examination data will be presented. in conclusion in patients with a low serum vitamin b level, there was no significant difference in the b levels in those patients with a normal or a low hb concentration. it would appear that serum vitamin b is a poor discriminatory test but that changing the normal range may not help in screening. low serum vitamin b on its own may not appear to provide adequate grounds for lifelong replacement therapy. of in for males and in for females. as expected the overall incidence of hodgkin's was lower with one third of male and one quarter of female lymphoma cases affected by the disease. a distinct age specific pattern is evident depending on lesion type. marked variation in incidence levels were noted throughout the study region. an extremely varied pattern is evident in the survival rates for lymphoma patients. the cork and kerry rates for malignant lymphoma are relatively low when compared with international levelstzl obstetric complications and schizophrenia: methodology and mechanisms perinatal complications and clinical outcome within the schizophrenia spectrum ) negative symptoms, cognitive impairment and duration of initially untreated psychosis in schizophrenia davnet's hospital, monaghan, and royal college of surgeons in ireland retinal pathology in kearns sayre syndrome mitochondrial dna and disease mitochondrial myopathies: clinical features, investigation, treatment and genetic counselling phenytoin induced pseudolymphoma. a report of a case and review of the literature cutaneous reactions in head injured patients receiving phenytoin for seizure prophylazis hydantoin induced pseudopseudolymphoma branhamella catarrhalis: an organism gaining respect as a pathogen correlation between branhamella catarrhalis adherence to oropharyngeal cells and seasonal incidence of lower respiratory tract infections ) epidem ology and survival rates for all lymphoma patients registered in cork and kerry over the eight year period an indepth review of all lymphoma (icd -o code ) patients registered by the sourthern tumour registry during the eight year period / ~l annual age adjusted rates of . . and . per , were seen for males and females respectively. these levels indicate a lifetime (up to yr) risk references . cancer, the irish experience cancer incidence in five continents volume v immunohistochemistry for bcl- oncoprotein without antigen retrieval gave negative results, but with antigen retrieval, showed positive staining in out of cases. no bcl- rearrangements were detected by pcr. the combination of sscp and sequencing confirmed only wild type dna in all cases, p immunohistochemistry by standard methods revealed positive staining in only one out of nine samples analysed. when the antigen retrieval method was employed for this antibody, positive staining was seen in > % of tumour cells in four further cases.our results suggest that p does not play major role in ptld. bcl- overexpression but not rearrangement may contribute to the development of ptld. transplant arteriopathy (ta) is the major cause of death in cardiac allograft recipients. the pathogenesis is unclear. we have previously shown a plasma cell predominance in the infiltrate of ta, leading us to hypothesise a role for epstein-barr virus (ebv) infection in its pathogenesis. an association between cytomegalovirus (cmv) and ta has previously been suggested. the aim of the study was to investigate the role of epstein-barr virus (ebv) and cytomegalovirus (cmv) in the pathogenesis of ta.we performed pcr for cmv and ebv dna and protein (lmp) in seven cases of ta, involving cardiac allografts. restriction mapping was used to confirm that pcr products were either cmv or ebv dna respectively.cmv dna was found in four cases. ebv dna was found in six of the seven cases and ebv lmp staining was present in six cases. ebv was detected in all cases by either pcr or ihc.our results suggest that ebv infection may play a pathogenic role in transplant arteriopathy. the evidence for a similar role for cmv ~s less strong. st. vincent's hospital. hereditary spastic paraplegia (hsp) is a neurodegenerative disorder characterised by progressive spasticity, primarily of the lower limbs. it can be inherited in an autosomal dominant (ad), autosomal recessive or x-linked manner~ we have identified a large irish family (family a) affected with ad hsp that cosegregates with dementia. three. loci have previously been identified that are linked to ad hsp in families of different ethnic origin. the locus on chromosome is reported to be the major hsp locus. the aim of the present study was to examine family a for linkage to the chromosome hsp locus.dna has been extracted from blood taken from all co-operating family members for genotyping. polymorphic microsatellite markers from chromosomal region p - have been amplified by pcr, electrophoresed on a denaturing polyacrylamide gel and detected by silver staining. linkage analysis was carried out using the linkage series of programs. linkage analysis excluded the hsp gene from the chromosome p locusl the most significant marker was d s , with a lod score of- . for recombination fraction . , thereby excluding approximately cm either side of this marker. negative lod scores were also obtained for the other markers chosen (d s , d s , d s , d s ) excluding cm, cm, cm and cm respectively.the current study has therefore successfully excluded linkage of ad hsp in family a to the major locus on chromosome p. further studies are underway to exclude linkage of hsp in this family from other candidate loci, prior to carrying out a genome wide search. the presence of dementia in this family in association with hsp suggests that a new and as yet unidentified gene is responsible. vincent's hospital. eye and ear hospital, dublin. ched is a corneal endothelial dystrophy characterised by diffuse bilateral corneal opacities resulting in impaired vision. both autosomal dominant and autosomal recessive modes of inheritance have been described. another endothelial dystrophy, posterior polymorphous dystrophy (ppmd) has been linked to qll.we have used homozygosity mapping to analyse a pedigree with autosomal recessive ched for linkage to ql. . all affected individuals are offspring of consanguinous matings. homozygosity mapping is based on the principle that these offspring would be homozygous for genetic markers near the disease gene. homozygous regions would be random between different offspring of these matings, except at the di-sease locus shared by affected offspring.dna was extracted from blood taken from family members, of which have ched. allele frequencies were determined in pooled dna from affected individuals. pooled dna from unaffected individuals was used as a control. at the disease locus, a shift in allele frequencies towards a single homozygous allele would be observed in the affected dna pool. pooled dna was genotyped by pcr for polymorphic microsatellite markers in the region of qll. pcr products were separated on a polyacrylamide gel and visualised by silver staining. similar allele frequencies were observed at these loci in both dna pools demonstrating independent assortment of alleles. in addition, affected and unaffected family members were individually genotyped at these loci and no significant loss of heterogeneity in the affected individuals at these loci was observed. these data indicate exclusion of linkage of the ched gene to qll. key: cord- - twm hmc authors: vourc’h, gwenaël; plantard, olivier; morand, serge title: how does biodiversity influence the ecology of infectious disease? date: - - journal: new frontiers of molecular epidemiology of infectious diseases doi: . / - - - - _ sha: doc_id: cord_uid: twm hmc over the past years, biodiversity has been reduced on an unprecedented scale, while new infectious diseases are emerging at an increasing rate. greater overall biodiversity could lead to a greater diversity of hosts and thus of pathogens. yet disease regulation – due to the buffering role of host diversity – is considered to be one of the services provided by biodiversity. in this chapter, we ask how biodiversity is linked to infectious disease. first, we investigate the influence of the biodiversity of pathogens. we highlight that the number of pathogen species is not well known but that new findings are facilitated by the rapid expansion of molecular techniques. we show that, although there is a trend to find higher pathogen richness toward the equator, identifying a global pattern between the richness of all pathogen species and their latitudinal distribution is challenging. we emphasize that pathogen intraspecific diversity is a crucial factor in disease emergence and allows pathogens to adapt to the selective pressures they face. in addition, the selective pressure acting on hosts due to parasite, and reinforced by parasite diversity within hosts seems to be a major evolutionary and ecological force shaping hosts biodiversity. second, we investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing (“dilution effect”) or increasing (“amplification effect”) the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. investigate how the diversity of hosts influences infectious disease ecology. for multi-host diseases, a change in host species richness or abundance can modify the dynamics of local infectious diseases by either reducing ("dilution effect") or increasing ("amplification effect") the risk of transmission to the targeted host species. the underlying hypothesis is that, the competence of reservoirs varies according to the host species. the dilution effect has been demonstrated mainly through theoretical work and there have been only few case studies. regarding the genetic diversity of host, an important issue is: to what extent does a reduction of this diversity impact the ability of the host population to response to infectious diseases? third, we rapidly examine the role of biodiversity in the treatment of infectious diseases. to conclude, we consider that the consequences of the loss of species biodiversity on infectious diseases is still largely unknown, notably due to the lack of knowledge on the dynamics of host-pathogen relationships, especially at the population and at the community level.. we highlight that work on multi-host/ ulti-pathogen systems should be fostered and that new approaches, such as metagenomic investigations that does not require a priori assumptions, are promising to describe a community of pathogens and their interactions. over the past years, human activity has altered habitats and reduced biodiversity on an unprecedented scale that comes close to mass extinction (mea ) . at the same time, new infectious diseases seem to be emerging at an increasing rate (wilcox and gubler ) . during this period, there has been a dramatic spread of highly pathogenic diseases such as aids and multi-drug resistant bacterial infections, and in more recent years sars, west nile in north america, and highly pathogenic influenza viruses (jones et al. ) . habitat loss, largely a result of the conversion of forests and savannas into agricultural land, cities, and industrial sites, is the major cause of change in biodiversity. biodiversity represents the diversity of life at all levels of biological organization, from the genes within populations, the species that compose a community, to the communities that compose ecosystems. intuitively, one might assume that greater overall biodiversity would lead to a greater diversity of pathogens and hosts, and thereby increase the incidence of infectious diseases (dunn et al. ). yet disease regulation is said to be one of the services provided by biodiversity because a high level of species diversity creates a buffer that reduces the risk of transmission (mea ; walpole et al. ). scientific evidence supporting both of these views is beginning to emerge, but the core question remains: how is biodiversity linked to infectious disease? this is the question addressed in this chapter. pathogens are organisms that have a negative impact on the fitness of their host(s), often, if now always, by producing visible symptoms (e.g. a disease). such trophic interaction between two organisms, a host and a parasite, is just one of several interactions that take place within communities and ecosystems, others being those of prey-predator and plant-phytophagous for instance (begon et al. ) . to date, more attention has been paid to these other interactions, and their roles in ecosystem functioning (e.g. steffan and snyder ) , than to pathogen-host interactions, and food web studies only recently have begun to take parasites into consideration (arias-gonzález and morand ; lafferty et al. ) . studies incorporating pathogens are scarce (hudson et al. ) , probably due to the difficulties of surveying pathogens (using intrusive or even destructive sampling methods…). moreover, the systematics and even basic aspects of parasite biology often are unknown. however, although numerous species of pathogens still need to be described (dobson et al. ) , there is no doubt that pathogens represent a large part of biodiversity on earth. given that each free living species is host to numerous pathogens, and that pathogens of pathogens also exist (consider, for example, phages that are virus affecting bacteria), several authors believe that pathogens may be the most diverse living group on earth (windsor ) . the link between biodiversity and the ecology of infectious diseases is not simple. in this chapter, we investigate how biodiversity influences the ecology of infectious diseases at the intraspecific level (genetic variability of pathogens and hosts) and at the level of communities (species composition). although we mainly provide examples from human and animal diseases, we also use some illustrations from plants. we describe patterns of biodiversity and the consequences of changes in biodiversity on the ecology of infectious diseases. lastly, we rapidly examine the role of biodiversity in the treatment of infectious diseases. infectious disease ecology? we shall consider infectious diseases caused by bacteria, virus, fungi, protozoa and endo-parasites, and exclude from our analysis ecto-parasites that are considered here as disease vectors. in the light of the discussion above, the pathogen status of a given living organism clearly is not a straightforward question (consider, for example, the case of some rickettsia species that are considered to be not only blood vertebrate pathogens, but also tick symbionts, perlman et al. ). this status is related to the host, and varies with individual hosts and species as well as in space and in time (different hosts, for example, can have different resistance or susceptibility). when pathogens have complex life-cycles, some stages may have a different biology (such as biotrophic or necrotrophic plant pathogens, morris et al. ). furthermore, horizontal gene transfer is so extensive in bacteria that many microbio logists question the existence of species in bacteria, preferring to consider bacteria as populations that exchange genes. however, the existence of core genes responsible for the maintenance of species-specific phenotypic clusters is an argument supporting the identification of bacterial species (riley and lizotte-waniewski ) . for these reasons, combined with the limited knowledge available of the systematics of many pathogens (brooks and hoberg ) , it is difficult to accurately estimate the number of pathogen species. estimations of pathogen species richness vary from % to % of living beings (de meeûs et al. ; poulin and morand ) . in estuaries, the biomass of macro and micro-parasites has been estimated as exceeding that of top predators . although the existence of pathogens has been known for a long time, lists of species only were compiled recently for human and animals (ashford and crewe ; cleaveland et al. ; taylor et al. ) , with an update on human pathogens completed in (woolhouse and gaunt ) . approximately , human pathogens were reported, livestock pathogens (cattle, sheep, goats, pigs and horses), and domestic carnivore pathogens (dogs and cats). no clear figure was given for wildlife (but see the global mammal parasite database at http://www.mammalparasites.org/). on average, over two new species of human viruses also are discovered each year (woolhouse et al. ) . pathogens affecting humans have received more attention than those affecting other species. if one assumes that other animal species are affected in a proportional manner, huge numbers of pathogens remain to be discovered. altogether, , , , and , of fungi, viruses and bacteria respectively have been described, which represent only %, %, and - % of the total estimated number of species of fungal, viral and bacterial species. it is difficult to know the number of plant pathogens, but a significantly proportion of the fungal, viral and bacterial species are likely to be plant pathogens (ingram ) . until recently, many new pathogen discoveries relied on the investigation of atypical symptoms. today, new findings are facilitated by molecular techniques that render it possible to detect and characterise unculturable pathogens and to investigate the presence of genes and genomes independently of individuals (metagenomics). although multi-host pathogens are more numerous than single hosts, interactions between pathogens and hosts can evolve towards the specialisation of pathogens on a given host species (cleaveland et al. ; huyse et al. ; pedersen et al. ) . such a specialisation can lead to speciation, id est the birth of a new pathogen species. co-cladogenesis, a process of parallel diversification in hosts and pathogens, also can give birth to numerous pathogen species (page ) . until the development of molecular tools, these species were very difficult to distinguish (cryptic species). systematic investigations using molecular tools have made it possible, however, to reveal a high diversity of pathogens. for instance, in a systematic inventory of viruses in various vertebrate hosts conducted over a year period in the central african republic, different viruses were isolated, including new ones (saluzzo et al. ) . two species of plasmodium, p. falciparum, infecting humans, and p. reichenowi, infecting chimpanzees, were long considered to be within the clade that includes humans and the great apes. however, recent studies of apes in their natural habitat have revealed a much higher diversity of species infecting great apes; in addition, it has been found that p. falciparum also infect gorillas (liu et al. ; prugnolle et al. ) and are at the origin of human malaria. metagenomic studies in ecosystems such as human faeces (zhang et al. ) and marine sediments (breitbart et al. ) also have revealed that the majority of viral sequences found did not match in the databanks. finally, new investigations have been launched to monitor people, animals and animal die-offs in areas where people have a high exposure to wildlife. generic, broad screening tools will be used to detect pathogen species (wolfe et al. ). to our knowledge, a similar approach has not yet been implemented for pathogens of animals or plants. in addition to the inventories of pathogen biodiversity, scientists have investigated which part of the world holds the highest diversity of pathogen species. many studies on plants and animals have shown that species richness decreases the further one moves away from the equator. the reasons most likely are linked to the area, energy, time and habitat heterogeneity, and geometric constraints (gaston and blackburn ) . comparative studies exploring pathogen species richness in the tropics compared to temperate zones are scarce and have produced discrepant results. guernier et al ( ) studied the worldwide distribution of human pathogens (bacteria, virus, fungi, protozoa, and helminths) according to environmental, demographic and economical factors. they found that parasite species richness decreased with latitude and had a spatially nested organization; i.e. some species are widely distributed and occur in many communities while others have more restricted distributions and occur only in a subset of locations. such findings were confirmed by the analysis of dunn et al. ( ) , who showed that human pathogen diversity was strongly related to both mammal and bird species richness. diseases that occur in temperate zones also tend to occur in the tropics, while some tropical diseases are restricted only to the tropics. in primates, the number of protozoa species, which primarily are vector-borne transmitted, increase as one approaches the equator, however, the same trend was not found for viruses and helminths (nunn et al. ). lindenfors et al ( ), who examined the parasite richness of parasite species and terrestrial carnivore species, found that helminth parasite species richness increased the further away one moved from the equator. the reason for this finding is unknown and may be related to a bias in sampling because carnivores inhabiting areas of industrialized countries in the northern hemisphere may have been sampled more intensely. poulin ( ) and bordes et al ( ) did not find any correlation between helminth species richness at intra or interspecific levels and latitude. some studies have shown higher tick species richness at lower latitudes (cumming ) . however, this is not the case for flea species, which have been found to have low richness at lower latitudes (krasnov et al. ) . a final example is ichneumonid parasitoid hymenoptera. although a higher specific host diversity is found in the tropics, the number of species of this parasitoid group is similar in both tropical and temperate regions. it has been hypothesised that this is due to habitat fragmentation (leading to a lower density of hosts); lack of seasonality (and thus of a host population dynamics with peaks and high density of hosts), or the higher content in toxic compounds of tropical plants and thus in phytophagous insects (the "nasty hypothesis") (gauld et al. ) . a meta-analysis of parasite-associated host mortality (robar et al. ) revealed that host mortality risk declined as one moves away from the equator, indicating that, in addition to parasitic load, the effect of parasites on host mortality might be determined by some abiotic factors. thus, although there is a trend to find higher pathogen richness as we move toward the equator, it is thus challenging to identify a global pattern between the richness of all pathogen species and their latitudinal distribution. however, it should be noted that of the pathogens that have been discovered since , most have a global distribution (woolhouse and gaunt ) . pathogens generally are characterised as having higher mutation rates and generation times than those of their hosts (hamilton et al. ). genetic variability also results from recombination during sexual reproduction of eukaryotic pathogens, and any other genetic exchange mechanisms such as bacterial conjugation or viral recombination. in addition, many animal and plant pathogens use a vector to increase gene flow among populations and to reach a new individual host. this genetic diversity is a crucial factor in disease emergence (cleaveland et al. ) and allows pathogens to adapt to the main selective pressures they face: hosts' immune systems, the need to be transmitted, and treatments or vaccines used to counter infections. the capacity of some pathogens to genetically diversify facilitates their ability to evade host immune systems. one of the best examples is the human immunodeficiency virus (hiv), which is able to change its appearance faster than the time its takes for the immune system to reply (drosopoulos et al. ). another example is p. falciparum, which generates high levels of variability in genes involved in antigenic variability and virulence (var genes) by producing frequent recombination events between heterologous chromosomes (freitas-junior et al. ) . high genetic variation of pathogens also is involved in the interspecies infection process as it facilitates the infection of a broader range of host species, which is another characteristic of emerging pathogens (cleaveland et al. ; woolhouse and gowtage-sequeria ) . the evolutionary potential of pathogens allows them to respond quickly to the directional selective pressure provided by the massive use of drugs (palumbi ) . in areas where selective pressure is important, such as in hospitals, multiresistant bacteria are very frequent (levy and marshall ) . for bacteria, resistant genes probably originated from environmental organisms with which they shared their ecological niche (aminov and mackie ) . these genes can be transferred between different species of bacteria and even between species that colonize different hosts (nikolich et al. ) . although vaccination is a major advance of modern medicine, it thus far has contributed to the eradication of only one infectious disease in humans (small pox, www.who.int/mediacentre/factsheets/smallpox/en) and one in cattle, buffalo and wildebeest (rinderpest, normile ) . as many vaccines do not totally block transmission, vaccination modifies the selective pressure on pathogens. depending on how vaccines act on the pathogen, the epidemiology consequences can differ (gandon and day ) . for instance, theoretical work has shown that vaccines that reduce the growth rate or toxicity of pathogens also reduce selection pressure against virulent pathogens, leading to higher intrinsic virulence (gandon et al. ). in the poultry industry, an increase in virulence of avian tumour viruses has followed the use of vaccines that reduce virus growth rates (witter ) . although plants lack an adaptive immune system, through evolution they have developed various strategies to stop plant pathogen infections. an induced or acquired systemic resistance occurs following host recognition of a pathogen, which triggers the production of a hypersensitive reaction (jones and dangl ) . through this mechanism, the plant provides itself protection from secondary infection in distal tissues, even if the plant faces a pathogen for which it does not have the resistance gene (durrant and dong ). the immune system of vertebrates acquires its efficiency by being exposed to a diverse array of pathogens. the striking increase in hygiene standards that began in the early twentieth century has considerably lowered humans' exposure to pathogens, at least in developed countries. the immune response triggered by a pathogen can provide some cross protection against other pathogens (e.g. huang et al. ) . a low exposure to a diversity of pathogens has had immediate consequences in decreasing the risk of disease. but could this low exposure also induce evolutionary change in the ability of a host to respond to infection? due to a trade off between investment in disease resistance and other traits linked to fitness, low exposure could decrease the frequency of resistance down through the generations, setting the stage for a potentially devastating outbreak (altizer et al. ; graham et al. ) . domestic species that are bred to be protected from pathogens might be more susceptible to infectious diseases (lyles and dobson ) . furthermore, it has been suggested that on islands, where some pathogens may be absent, hosts may have lower immune response abilities (island syndrome) (lee and klasing ) . however, studies that have tested this hypothesis, both using experimental and theoretical approaches, have had contrasting results (beadell et al. ; hochberg and møller ; matson ) . infections by different species of pathogens or by different strains of the same species within the same individual host or vector are common (abbot et al. ; cox ) . in fact, parasite diversity in hosts seems to be a major evolutionary and ecological force for hosts (bordes and morand ). these concomitant infections can trigger cross-effective immune responses between pathogens that are antigenically similar, having thus an impact on the issue of the infection (lee et al. ). an infection also can enhance susceptibility to subsequent infection (cattadori et al. ). in particular, individuals with already are in poor physical condition may be more susceptible to multiple infections (beldomenico and begon ; telfer et al. ) . furthermore, concomitant infection may allow the exchange of genetic material between strains of a given pathogen species or even between species through horizontal gene transfer (see sect. . . above), allowing the emergence of new virulent strains. an extreme case is one in which a pathogen drives the extinction of a population or species. such scenarios are rare but do occur, generally due to a conjunction of pathogens and other causes. for instance, the decline of amphibian populations around the world is thought to be linked to a fungal pathogen batrachochytrium dendrobatidis causing chytridiomycosis (crawford et al. ). amphibians could have an increased susceptibility to the fungus due to changes in temperature variability (rohr and raffel ) . another example is the dramatic decline of the native red squirrel in the uk that has been attributed to a combination of direct competition with the grey squirrel and disease-mediated competition because the grey squirrel is a reservoir host of the squirrelpox virus that causes disease in the red squirrel (tompkins et al. ) . the local extinction of a host also may have tremendous consequences on an entire ecosystem (see for example the case of the wildebeest /rinderpest interactions in the serengeti, holdo et al. ). disease ecology? a change in species richness or abundance can modify the dynamics of local infectious diseases by either reducing or increasing the risk of transmission to the targeted species. the first outcome has been named, the "dilution effect", the second, the "amplification effect". the term "dilution effect" has conveyed different meanings since its first use in disease ecology literature (see box in the paper keesing et al. ) . the broad definition of the dilution effect refers to "the phenomenon -the net effect -when increased species diversity reduces disease risk" that is produced by a variety of mechanisms ("amplification effect" refers to the opposite phenomenon) (keesing et al. ) . this applies to vector-borne and directly transmitted diseases, although the concept of dilution has been developed most with regards to the tick-borne lyme disease (allan et al. ; logiudice et al. logiudice et al. , . the hypothesis underlying the amplification and dilution effect is that for many diseases, the competence of reservoirs, i.e. the ability to become infected and retransmit the pathogen, varies according to the host species (haydon et al. ) . the composition of the host community thus can influence the transmission dynamic of the disease. similarly, since vectors have different competence to transmit pathogens, the composition of the vector community likely influences transmission dynamics. different mechanisms are thought to be involved, but they are difficult to differentiate (begon ; keesing et al. ) . one is the modification of the encounter rate (when reduced, this corresponds to the "dilution effect" sensu stricto). in the presence of species that are poorly competent, the transmission event that should link an infectious individual to a susceptible individual instead links infectious individuals to non-competent individuals. for vector-borne diseases, the increased diversity of a poorly competent host species on which the vector feeds increases the proportion of vector bites that are wasted. for direct transmission, the addition of non competent hosts can decrease transmission if these hosts remove infectious particles (begon ) . a second mechanism at work is that a high diversity of host species regulates the abundance of the competent host population. this regulation can be mediated by interspecific competition for limiting resources or by predation upon competent hosts. this typically is the idea that underlies biological controls, where organisms prey upon reservoir hosts, vectors or intermediate hosts (straub and snyder ) . a third mechanism is based on the link between species richness and host mortality. this is the case when predators modify the mortality rate of a host and lower pathogen transmission by feeding on a heavily diseased individual (packer et al. ) . two other mechanisms are cited by keesing et al ( ) , but they are difficult to demonstrate: (i) the modification of recovery when species added to a community facilitate the recovery of infected individuals by, for instance, providing resources, and (ii) the modification of transmission once the contact has occurred, for instance, when adding a species modifies the pathogen load within the host. the dilution effect has been demonstrated mainly through theoretical work; there have been few case studies. one of the main examples is lyme disease in the usa that is caused by pathogenic bacteria transmitted by ticks. these ticks feed readily on many species of vertebrates and these species vary in their degrees of reservoir competence. the white-footed mouse (peromyscus leucopus) is thought to be the most competent host and dominates in fragmented forests. in native forests, which harbour a higher diversity of species than fragmented forests, ticks have a higher probability to dilute their bite by feeding on a less competent host (allan et al. ; logiudice et al. logiudice et al. , . however, such a dilution effect has not been demonstrated in europe, probably because of the complexity of the disease ecology which involves numerous reservoir host and bacteria species (halos et al. ) . another example is the west nile virus, where an increased diversity of non passerine birds, which are less competent reservoir hosts compared to passerines, was associated with decreased west nile virus infection in mosquitoes and humans (ezenwa et al. ; swaddle and calos ) . to date, there have been few examples of directly transmitted diseases, but studies on hantaviruses have shown that higher diversity of small mammals appears to regulate reservoir host populations through competition or predation. high small-mammal diversity also might inhibit intraspecific aggressive encounters between reservoir hosts that result in hantavirus transmission (suzán et al. ). in plants, crop diversity can reduce the total burden of disease in agricultural systems. this results from the combined effects of (i) the limitation of pathogen dispersal thanks to the physical barriers provided by the presence of non-host plants (burdon and chilvers ) , (ii) induced systemic resistance, and (iii) competition among pathogens. the efficiency of crop mixtures is linked to the size of the area on which this method is used: a high level of success has been observed in a field trial with susceptible and resistant varieties of rice conducted on a large scale ( , ha) in china (zhu et al. ) . illustrations of amplification effects are typically the consequences of species introduction that radically modifies encounter rates. the added species can introduce new pathogens that infect native hosts (spillover) (bruemmer et al. ) or amplify the circulation of local pathogens (spillback) (kelly et al. ). the introduction of additional species also can provide sources of vector meals and increase vector numbers or activity (saul ) . for instance, the introduction of siberian chipmunks (tamias sibiricus) in suburban forests could increase the risk of lyme disease because this host seems to be more competent than native hosts (vourc'h et al. ). the introduction of the mosquito aedes albopictus in many parts of the world has facilitated the transmission of the chikungunya virus (benedict et al. ; charrel et al. ) . theoretical works based on deterministic modelling have looked at the conditions in disease transmission dynamics that are needed for the amplification or the dilution effect to occur (begon ; dobson ) . when there is a relationship between the risk of a disease, the abundance of the reservoir host, and the abundance of an additional host, the addition of a species does not necessarily decrease the risk. in the case of lyme disease, for example, tick abundance mainly is determined by the abundance of deer, which are in fact a non competent reservoir. an increased abundance of deer may reduce infection prevalence when immature ticks are feeding on the deer. at the same time, however, the overall number of adult ticks increase proportionally with the number of deer (begon ) . further research in this field are relying on the modelling of the global community competence of hosts and vectors (roche ) . scientists and societies are increasingly interested in the dilution effect (mea ) due to the link between habitat disturbance, generalist host characteristics, and their competence in disease transmission. disturbance seems to favour generalist hosts (hosts that use different types of habitats) (devictor et al. ; marvier et al. ) , and these hosts often have a broad geographical distribution (mckinney and lockwood ; smart et al. ). crucially, these species also seem to have a higher competence reservoir or vector reservoir than species that are not favoured by disturbance (mills ; molyneux et al. ; vittor et al. ) . for example, many murid rodents that are recognized hosts of hemorrhagic fever viruses are opportunistic species that seem to be favoured in disturbed environments. the question is whether there is a causal link between a species' generalist and opportunist character and its disease competence. why are murid species associated with hemorrhagic fever more generalist than those which are not? could it be possible that specialist species also carry hemorrhagic fever viruses, only these viruses have not yet been identified? or is there something intrinsic in opportunistic species that makes them more likely to evolve and maintain hemorrhagic fever viruses (mills )? only a very small subset of plant and animal species have been domesticated (diamond ) . many species of that small subset, for example, cattle (in animals) and maize (in plants), have seen their genetic diversity considerably reduced for the purpose of intensive production (the bovine hapmap consortium, matsuoka et al. ) . in the wild, small populations of endangered species often have a very reduced genetic diversity (keller and waller ) . this then raises the following question: to what extent does a reduction of the genetic diversity in a host species impact the ability of the host population to response to infectious diseases (may ) ? genetic loci associated with the major histocompatibility complex (mhc) plays a key role in the acquired immune response of vertebrates (altizer et al. ) . mhc genes code for molecules that recognize foreign peptides (antigens) and display them on the cell surface. when the mhc-protein is displayed, it can be presented to immune cells, such as t lymphocytes or natural killer cells, which subsequently can trigger an adaptive immune response. because mhc genes are faced with an important diversity of antigens, they must themselves be diverse. the measure of the genetic diversity of mhc based on an analysis of polymorphism sequences of mhc among individuals in populations has been widely used in conservation biology as a proxy to estimate the fitness of populations confronted by pathogens (alcaide et al. ; bernatchez and landry ; sommer ) . however, the level of genetic variation at mhc loci results from different evolutionary forces (selection, gene flow, mutation) varying both in space and time in co-evolutionary systems involving both hosts and pathogens, making conservation genetics of non-model organism a challenging task (stockwell et al. ) . we already have many examples where low genetic diversity of species has favoured the diffusion of, and/or susceptibility to, pathogens. for example, the low genetic diversity of the tasmanian devil could be involved in its susceptibility to facial tumor disease (mccallum ) . the low genetic diversity found in commercially traded bee queens has been hypothesised as being one of the factors explaining colony collapse disorder (le conte and navajas ). the problem is even more critical in intensive crops in which disease resistance has relied on the use of a very small number of genes. this selection strategy has proven to be ineffective as pathogens manage to overcome the resistance. for example, the resistance of brassica napus (canola, oilseed rape and colza) to leptosphaeria maculans (causing the blackleg disease) due to a major resistance gene was overcome in an area covering approximately , ha in south australia in a period of years (sprague et al. ). even with advances in synthetic chemistry, which provides many biologically active molecules, pharmaceuticals derived from nature remain an important part of pharmaceutical practice today (newman et al. ). all organisms have developed compounds to protect themselves against infectious diseases and to interact with individuals of their own species or other species (e.g. rogerio et al. ). these molecules, coming from all organisms (bacteria, fungus, animals, plants) in terrestrial, marine and extreme ecosystems, represent an amazing diversity that has been tested in the field for millions of years by involving millions of individuals. however, only a very small subset of plants and marine organisms has been investigated for novel bioactive compounds. furthermore, it is estimated that less that % of bacterial species and only % of fungal species are known. those which have not yet been identified could be sources of novel molecules (cragg and newman ) . observations of natural medicine practices used by indigenous people have led to the discovery of many drugs. the most well known and widely used pharmaceuticals are quinine, used as a model to synthesize anti-malarial drugs (chloroquine and mefloquine), and artemisinin, indentified as a potent anti-malarial drug by chinese scientists (newman et al. ) . animals also are a source of inspiration for drugs against infectious diseases. for instance, compounds of the sponge cryptotethya crypta inspired the elaboration of antiviral medication such as azt used in the treatment of hiv/aids (cragg and newman ) . observing great apes medicate themselves through the plants they eat also could help to reveal new active compounds (krief et al. ). pathogens constitute a large part of biodiversity on earth and are present in all ecosystems and at all trophic levels, where they have a large impact on ecosystem functioning and on the population dynamics and evolution of their hosts. the recent acceleration of biodiversity loss due to human activities deeply impacts host-pathogen dynamics. pathogens and hosts form co-evolving systems exercising major selective pressures on each other. furthermore, the virulence or pathogenicity of a given species depends on its environment -which includes the hosts -that is highly variable in space and time. in such a context, human beings will never be able to completely control or eradicate every pathogen species; rather, we should accept that we must coexist with pathogens. to better understand and predict the evolution of pathogens and the impact of human activities on them, more in-depth studies are needed on how pathogens interact with host communities within different ecosystems. to understand human, animal, and plant epidemics, two-species systems involving only a single host and a single pathogen species are no longer appropriate. the approach considering multi-host/multi-pathogen systems in their environment is the framework that now needs to be used to deepen our understanding of disease dynamics woolhouse et al. ) (fig. . ) . however, these dynamics are very complex and difficult to study because precise knowledge regarding the diversity of pathogens and of interactions taking place on several scales is lacking (lloyd-smith et al. ). in addition to intensive fieldwork to collect adequate data and modeling to understand the main processes, the use of molecular tools in a multi-host/multi-pathogen framework will facilitate investigations into pathogen-host community interactions. in particular, new generation sequencing techniques render it easier to characterize the genetic diversity of pathogens and hosts. for instance, metagenomic investigations allow an approach that does not require a priori assumptions that is useful to describe a community of pathogens and their interactions. molecular techniques also may be used to clarify the taxonomic status of pathogens, revealing cryptic species or host races. with suitable molecular fig. . schematic representation of the link between biodiversity and the ecology of infectious diseases. diseases results from the complex interactions between the three compartments corresponding to pathogens, hosts and vectors (in the case of vector-borne diseases). the biodiversity of these three compartments can be considered at the community level (each circle corresponding to a species) or at the intraspecific level (each circle corresponding to a population or an individual within a population). the gray shading off of each unit considered (species, population or individual) illustrates its genetic or phenotypic variability in space and time, while variations in size illustrate frequency or density differences within the ecosystem. interactions within each compartment can be direct (competition) or indirect (apparent competition…), synergic or antagonistic as illustrated by the different arrows markers (producing a high level of polymorphism), the analysis of genetic variability within a spatially explicit framework renders it possible to identify the routes followed by a given pathogen. moreover, molecular techniques can be used to identify genes involved in important life history traits of a pathogen such as virulence and transmission. better knowledge of the mechanisms involved in host-pathogen interactions, and the extent of their variability, will significantly advance our understanding of outbreaks. although our knowledge of the number and variety of pathogens is not complete, it appears that their diversity, like that of their hosts and vectors, is greater in tropical areas than in temperate ones, and in undisturbed habitats than disturbed ones (chaisiri et al. ; friggens and beier ) . the reason we are so concerned by the loss of species biodiversity is because a reduction of biodiversity seems to favour opportunistic species that are highly competent as pathogen reservoirs and vectors. however, this observation was derived from only a few studies and theoretical works, mainly undertaken in the temperate zones. further investigations should be launched to verify the link between, and understand the process involved in, biodiversity loss and disease dynamics. this especially should be done in the tropics to understand whether high levels of biodiversity create a buffer reducing the risk of disease 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biology of multihost pathogens temporal trends in the discovery of human viruses rna viral community in human feces: prevalence of plant pathogenic viruses genetic diversity and disease control in rice key: cord- -yog sfvi authors: liu, xiaobo; sun, jimin; liu, xiaodong; fan, jingchun; niu, yanlin; lang, lingling; liu, qiyong title: ambient temperature and major infectious diseases in china date: - - journal: ambient temperature and health in china doi: . / - - - - _ sha: doc_id: cord_uid: yog sfvi infectious diseases are a group of diseases which have complex transmission ways and various influencing factors. clarifying the correlation between ambient temperature and major infectious diseases in china is a crucial step toward the successful control of infectious diseases including vector-borne diseases, water-borne diseases, food-borne diseases, respiratory infectious diseases, etc. and the implementations of climate change adaption strategy and measures in china. however, no study has systematically reviewed the available evidences on the impact of ambient temperature on the incidence of major infectious diseases, and such information is essential for policymakers and stakeholders to take specific actions to control infectious diseases and protect the vulnerable population in the future. in order to fill this gap, we systematically review the current evidence for the effect of ambient temperature on major infectious diseases in china. the findings could provide explicit information for the scientific prevention and control of infectious diseases in china. infectious diseases continue to be the major health threats in most regions globally. as one type of important infectious diseases, vectorborne diseases (vbds) are expected to affect about % of the world's population. according to who's report, % of the global burden of communicable diseases was due to vector-borne diseases. in china, of the notifiable infectious diseases are vector-borne diseases. great achievements have been made in the control of infectious diseases in the past decades, and five notifiable infectious diseases were eradicated or nearly eradicated at present, including polio, filariasis, severe acute respiratory syndrome (sars), plague, and diphtheria [ ] . the incidences of some notifiable infectious diseases were reduced, including cholera, hepatitis a, bacterial dysentery, amoebic dysentery, typhoid, paratyphoid, gonorrhea, pertussis, epidemic cerebrospinal meningitis, epidemic hemorrhagic fever, rabies, leptospirosis, anthrax, typhus, encephalitis, malaria, tuberculosis, and tetanus. by contrast, an increasing trend was observed for notifiable infectious diseases, including human immunodeficiency virus (hiv), brucellosis, hepatitis c, hepatitis e, syphilis, scarlet fever, dengue, influenza, infectious diarrhea, hydatid disease, leishmaniasis, and schistosomiasis. among these diseases, dengue fever (df), malaria, hemorrhagic fever with renal syndrome (hfrs), plague, and severe fever with thrombocytopenia syndrome (sfts) were the most prevalent vector-borne diseases in china. according to literature review, these diseases mentioned above are all sensitive to the variations of meteorological factors, especially for ambient temperature [ , ] . water-borne diseases are conditions caused by pathogenic microorganisms that are transmitted in water. the infection would occur when people ingested contaminated water or contacted with infected water containing various forms of intestinal pathogens during bathing or swimming. as a type of old diseases, the occurrence of schistosomiasis and its vector could also be impacted by ambient temperature [ ] . at present, the epidemic of hand-foot-mouth disease (hfmd) is very serious in china, and it is believed that heat could facilitate the spread of hfmd [ ] . regarding respiratory infectious diseases, influenza is surely a representative infectious diseases sensitive to ambient temperature in china [ ] . therefore, to better understand the relationship between meteorological factors and infectious diseases in china, it is essential to carry out prediction and projection of important infectious diseases under the context of climate change which will be of great significance to the prevention and control of infectious diseases in future. however, little information was available regarding the effect of ambient temperature on infectious diseases in china. in this chapter, we extensively reviewed the available evidence on the effect of ambient temperature on major infectious diseases in china, in order to make up this gap, and provide scientific guidance for policymakers to implement corresponding actions to reduce the incidence of major infectious diseases. ambient temperature and vector-borne diseases in this chapter, some representative mosquitoborne diseases with the biggest diseases burden or public health significance were selected. literatures concerning these themes are reviewed to clarify the relationship between ambient temperature and mosquito-borne diseases. df is the most rapidly spreading mosquito-borne viral disease, showing a -fold increase in global incidence over the past years. three quarters of the population exposed to dengue are in the asia-pacific region. since the first recorded outbreak of dengue in foshan, guangdong, in , df occurs frequently in southern china and becomes a major public health threat in china [ ] . in , df cases occurred in china with two of these dead. the means by which ambient temperature affects df are via the following bioecological aspects: dengue virus, aedes mosquitoes, human population, and its transmission environment [ ] . the ambient temperature could not only impact the reproductive of dengue virus in aedes mosquitoes but also the distribution of aedes mosquitoes. in china, regarding the impact of ambient temperature on dengue vector distribution, wu et al. found that ae. albopictus have expanded their geographic range to areas with an annual mean temperature below °c and a january mean temperature below − °c using climex model [ ] . temperature plays an important role in vector competence. liu et al. found that temperature increase enhances the competence of ae. albopictus to transmit dengue virus [ ] . most studies in china believed that temperature can drive dengue transmission [ , ] , showing a nonlinear effect [ , ] . among those studies, different models are employed such as generalized additive model (gam), autoregressive integrated moving average (arimax) model, ecologically based model, etc. in recent years, most studies were carried out in guangdong and yunnan provinces considering the higher frequency of df outbreak. local df cases were positively associated with temperature with different time lags in guangzhou [ ] . the minimum temperature at a lag of month was positively associated with dengue incidence in guangzhou [ ] . the mean and minimum temperatures were positively associated with increased df risk, while the maximum temperature was negatively associated with df transmission [ ] . xiang et al. demonstrated that a reversed u-shaped nonlinear association was found between ambient temperature and df in guangzhou. the optimal maximum temperature (tmax) range for dengue transmission was . - . °c and . - . °c for minimum temperature (tmin) [ ] . in guangzhou, a negative binomial regression model was adopted, and the results showed that average temperature (tave) and previous month's minimum temperature (tmin) were positively associated with df incidence. a threshold of . °c was found in the relationship between the current month's tmin and df incidence [ ] . regarding the comparative studies of the influence of ambient temperature on df in the domestic and abroad, the current studies in china mainly focus on the influence of ambient temperature on the distribution and spread of df and its vectors via the application of different models. and this is consistent with the international researches. however, at present, very little information is available regarding the impact mechanism of the ambient temperature on dengue virus, vectors, and ultimately dengue transmission mechanism, and limited literatures could be found regarding the impact of social and economic factors on df, and these field should be the focus in future study. as a representative vector-borne disease, malaria is due to parasites of the genus plasmodium and transmitted by four species of female anopheles mosquitoes in china, including an. sinensis, an. lesteri, an. minimus, and an. dirus, respectively. despite dramatical reductions in the incidence of malaria observed at present, imported cases still constitute a threat to the malaria elimination commitment of the chinese government to who. most studies found that temperature was a fundamental meteorological factor related to malaria incidence in china [ ] . extreme high temperature restricts the development of mosquitoes and reduces the transmission of malaria since higher temperature increases mosquito growth, virus replication, and biting frequency of vectors. generally speaking, temperatures lower than °c or higher than °c are not conductive to mosquitoes development [ ] . daily variations on temperature could impact necessary mosquito and parasite characteristics that help to malaria transmission intensity [ ] . in jinan, maximum temperature with a °c rise may be relevant to a . - . % increase in malaria cases, while minimum temperature with a °c rise might lead to about . - . % increase in malaria cases [ ] . li et al. reported that each °c rise in temperature amounts to an increase of . % in the monthly malaria cases in guangzhou [ ] . however, the correlation between temperature indexes and incidence of malaria may not be throughout the year. tian et al. found an increased risk of transmission due to a high temperature in warmer winters [ ] . the effect of ambient temperature concerning malaria frequently leads to a lag-time effect because of the life cycle of anopheles and the parasite [ ] . zhou et al. revealed that a . % variation on monthly malaria incidence was related to the average temperature in last months in central china [ ] . in four counties of yunnan, an increasing temperature resulted in increased malaria risk the varied lag periods for these associations [ ] . almost all studies mentioned above identified an association between ambient temperature indicators and transmission of malaria in different sites and periods lag in china. this is coherent to the similar studies abroad. however, very few studies focus on the impact mechanism of the ambient temperature on malaria parasites, vectors, and its transmission. plague, caused by yersinia pestis, is one of the most devastating infectious diseases in human history. the three plague pandemics have caused numerous deaths worldwide and changed human civilization [ , ] . in china, plague is belonging to category a of notifiable infectious diseases and once constituted a great burden during the initial stage of new china. at present, the disease was well controlled, and the incidence of plague keeps very low level. climate affects the plague intensity through its effect on maintenance and replication of the pathogen, host, and vector populations by affecting temperature and precipitation in some studies [ ] [ ] [ ] . suitable climate could increase the reproductive rate of pathogenic microbes [ ] and contribute to form stable plague foci [ ] . rodent populations and flea survival respond rapidly to climate variations [ ] . epizootics are likely to occur when rodent and flea number excess a certain threshold [ ] [ ] [ ] . the high abundance of rodents and fleas would also increase the human plague risk [ ] . temperature seems to determine the distribution of yersinia pestis. nearly % of human plague occur in regions with an average annual temperature greater than °c, and most large plague outbreaks occur within areas with an annual temperature of - °c [ ] . additionally, outbreaks of historical plague pandemics also seemed to be driven by climate change. the introduction of the black death is associated with climate fluctuations in central asia, with an approximate delay of years [ ] . the abovenormal warmer and wetter climate during and may be responsible for extensive plague outbreaks in china during the third pandemic [ ] . the associations between climate and plague can vary by regions [ , ] . regarding the comparative studies, most studies have focused on the individual effect of precipitation or temperature, or the mixed effect of two climate variables. as for the high tem-perature, plague would increase under this condition in western united states [ , ] . in arizona and new mexico, plague would increase if the number of days above a certain temperature threshold increased, with °f for new mexico and °f for arizona, respectively [ ] . however, the transmission between fleas and mammalian hosts was significantly reduced when temperature excess . °c [ ] . the mixed effect of precipitation and temperature also revealed a regional difference. yersinia pestis prevalence was shown to increase with warmer springs and wetter summers in gerbils in kazakhstan [ ] . similarly, the rodent and flea densities, as well as the presence of plague in mongolian gerbils, are positively correlated with the precipitation and temperature in china [ ] . high risk of human plague tends to follow the plague outbreaks in hosts [ ] . however, an increased plague incidence can be expected in vietnam during the hot, dry seasons [ ] . besides, plague outbreaks in preindustrial period were more frequent in cold and arid climate in europe [ ] . plague is also associated with large-scale weather events [ ] . el nino southern oscillation (enso) and the pacific decadal oscillation (pdo) are related to precipitation and temperature which could in turn affect plague dynamics [ , ] . the increasing rate of human plague was closely associated with southern oscillation index (soi) and sea surface temperature of east pacific equator (sst) in china. ari et al. found pdo explained much of the human plague variation in western united states. however, as the association between large-scale climate and plague was nonstationary and nonlinear in madagascar and europe [ , ] , we should pay more attention to the scale of the climate and the complexity of the interaction in the ecosystem when analyzing large-scale climate and plague systems. hfrs caused by the hanta virus and results in about , hospitalized cases yearly. it is generally thought that ambient temperature is one of the most key meteorological factors exerting the impact of endemic intensity of hfrs [ ] , because warm temperature is in ideal condition for the growth of crops and increase of rodent. furthermore, it is probably that surface air temperature can increase more obviously in winter and spring than in other seasons, particularly in the northern china [ ] . as the temperature increases, areas with relatively lower temperature could become beneficial to rodent breeding then extend the breeding seasons, which have a potential impact on the number, scale, and emergence of new natural foci of hfrs. in china, most studies showed that hfrs is sensitive to ambient temperature. temperatures from to °c are most beneficial to rodent growth, and breeding rates for both a. agrarius and r. norvegicus would decrease when temperatures were out of this range [ ] . temperature of °c as a threshold was identified in a study, below which there was a positive association, while above the threshold there was an inverse association [ ] . likewise, it is reported that the highest incidence of hfrs is with temperatures about . °c [ ] . temperature above . °c and below °c revealed positive effects on the hfrs incidence of months later; in contrast, temperature between . °c to . °c was negative with the incidence of hfrs [ ] . however, it is difficult to compare findings across different studies given the range of metrics and methodologies used. a recent comprehensive review suggested that many studies that quantified hfrs-ambient temperature associations have drawn different conclusions (either positive [ ] [ ] [ ] [ ] [ ] [ ] [ ] or negative [ , , ] ), possibly because of the use of different statistical methods and climate characteristics of the study regions. variables from different studies included mean, minimum, maximum ambient temperature and land surface temperature. land surface temperature mainly impacts the distribution and abundance of populations of a. agrarius [ ] , while ambient temperature mainly affects the behavior of rodent and human. sfts is an emerging infectious disease which was firstly identified in . although sfts is considered to be transmitted by air or direct contact with secretion or blood of sfts patients, the current evidences reveal that the majority of sfts cases were infected via tick bites. the majority of cases were happened from april to october. meteorological factors may exert some impact on ecology of sftsv directly or indirectly by affecting tick in itself, tick-human interactions, and virus replication. table . listed the related studies of climatic factors and sfts in china. sun et al. conducted an analysis concerning ambient temperature and sfts in counties in henan and hubei via distributed lag nonlinear models (dlnms). they reported that the effects of temperature on incidence of sfts were nonlinear, with larger relative risk (rr) at the higher temperature on lag . the high temperatures had acute and short-term effects, while the effects in low-temperature ranges were persistent over longer lag periods. higher temperatures such as . °c and . °c had the maximum rr for sfts cases on the current week, which decreased quickly in the next weeks. low temperatures including . °c and . °c had the minimum rr on the current week and had the maximum rr at lag weeks, which decreased slowly in the next weeks. the effects of lower temperatures could last weeks, but the effect of . °c was not significant at lag weeks [ ] . a negative binomial regression model (nbm) established by sun et al. revealed that the occurrence of sfts would increase by . % and . %, respectively, if monthly maximum temperature and mean relative humidity increase one unit [ ] . du et al. studied geographic distribution and related factors of sfts and found that temperature is one of the key environmental factors affecting the occurrence of sfts [ ] . similarity, cattle density, rain-fed cropland, builtup land, temperature, and relative humidity were independent risk factors for the distribution of sfts [ ] . the risk of sfts increased when reached a threshold with monthly average temperature higher than . °c, or monthly average relative humidity higher than . %, or ( % ci) were schistosomiasis, caused by a trematode worms belonging to the genus schistosoma [ ] , is a kind of water-borne diseases, bringing a heavy burden on the residents of the endemic areas [ ] . the parasite species which can infect human [ ] . schistosomiasis japonica that caused by s. japonicum is widely spread in china since , particularly in areas along the yangtze river and further south [ ] . oncomelania hupensis, the sole intermediate host of s. japonicum, plays an important role in the transmission of s. japonicum in china and correlates closely with the distribution of this disease [ , ] . s. japonicum completes its life cycle through a sexual generation in the vascular system of the definitive host (i.e., mammals) and an asexual generation in o. hupensis. ambient temperature is an important ecological factor for growth and development of s. japonicum and the presence of o. hupensis, which can influence the prevalence and distribution of schistosomiasis in china [ ] [ ] [ ] [ ] [ ] [ ] . in the process of transmission of schistosomiasis, ambient temperature plays a vital role in the biological activity of o. hupensis and the development of s. japonicum within the intermediate host. the optimal temperature range for miracidia infecting o. hupensis is between °c and °c. there is no significance in infection rates when temperature ranges between °c and °c and low infection rates under °c. however, miracidia can never infect o. hupensis when temperature drops to . °c [ , ] . after the invasion, s. japonicum arrested their development in snails when temperature was kept at about . °c or above °c, while the optimum development occurred at °c- °c, which means within a temperature range of . °c- °c, the higher the temperature is, the shorter the pre-patent period of s. japonicum within o. hupensis will be [ , ] . temperature could also influence the cercaria effusion, where °c- °c were the optimal range [ ] . the optimal temperature for o. hupensis ranges between °c and °c, which means any temperature out of this range would result in delayed or arrested development and reproduction of o. hupensis [ , , ] . physiological functions of o. hupensis declines as environmental temperature drops [ ] . when the temperature drops to . °c- . °c, half of the snails were in hibernation (et ) [ , ] . in yunnan, it is concluded that the optimal lst for o. hupensis was ≥ . °c after considering land surface temperature (lst) as the most suitable environmental factor for snail habitat prediction [ ] . in dongting lake region, the mean snail density increased gradually when the temperature was between . and . °c, while mean snail density decreased gradually when the temperature was from . to . °c in the gwr (geographically weighted regression) model. a possible suitable range of temperature was from . to . °c estimated by interquartile range in high-high clustered areas. [ ] . the accumulated degree-days (add) are considered to be similar to growing degree-days (gdd), which both reflects the heat accumulation during the development of the organism. it was estimated that the mean add for the development of s. japonicum in its intermediate host snail was . - . degree-days, and the same index for the development of a generation of o. hupensis was . degree-days [ , ] . several studies conducted in yunnan, jiangxi, anhui, and hunan have drawn the similar conclusion that the mean lst, the median night-time lst, the maximum lst at daytime, the maximum and minimum lst at night, and average temperature in june were positively associated with the prevalence of s. japonicum, which showed the importance of lst [ , [ ] [ ] [ ] [ ] [ ] [ ] . from another perspective, a spatio-temporal kriging model suggested seasonal variation of lst at daytime were negatively associated with the risk of schistosomiasis [ ] , for the possible reason that large seasonal temperature differences would impede the development of s. japonicum [ ] . the january temperature is a significant determinant to the distribution of schistosomiasis. when the january mean minimum temperature is below − °c or the annual extreme low temperature drops under − . °c, it is not suitable for o. hupensis to survive, which is the main reason that restricts transmission of s. japonicum shifting toward to north [ , ] . however, the northern limit of the schistosome-endemic zone has shifted due to climate change. yang et al. [ ] found that the distribu-tion limits of o. hupensis have shifted from ° 'n to ° 'n due to an increase of . °c of january temperature in the past years. in other words, the potential transmission area have expanded by more than , km , which resulted in an additional . million people at risk of schistosomiasis [ ] . the average minimum temperature in january and in winter had predominant influence on oncomelania density and frame occurrence rate of living oncomelania, respectively. the variation of average minimum temperature in january by °c would lead to the change of oncomelania density by . - . %. the variation of average minimum temperature in winter by °c would lead to the change of frame occurrence rate by . - . % [ ] . what's more, o. hupensis can only exist in areas with an annual mean temperature of - °c [ ] . yang et al. found that the lowest air temperature in a year was one of the factors that significantly affect the occurrence of snails in hunan, china. when the lowest air temperature in a year ranges from − . to − . °c, the snails could exist, while no snail can survive when the range was between − . and − . °c [ ] . a predictive model based on distribution of schistosomiasis in eastern china has been constructed to estimate the probability that schistosomiasis occurs in a target area, which showed a mean temperature of coldest quarter was of significance in model [ ] . however, air temperature is less suitable for predicting snail density compared to soil temperature [ ] . having realized the ambient temperature is one of the most principal elements affecting distribution and transmission of schistosomiasis, researchers in china focused on related studies from different perspectives. generally, the conclusions that we had drawn are in line with those of other countries, while the subtle difference is probably due to geographic variation of the temperature. though large number of studies that involving different temperature-related variables have been conducted, the question that which one is the most closely related to the schistosomiasis is still unknown. in addition, widespread use of gis/rs promotes the multiscale studies in china, but there is not many studies carried out on national level yet where the trend of temperature variation is more stable. that's the direction we should focus on in future. hand-foot-mouth disease (hfmd) is an infectious disease of infants and children [ ] caused by viruses from the group called enteroviruses. according to who's report, outbreaks of hfmd occur every few years in different parts of the world, but in recent years these have occurred more in asia. countries with recent large increases in the number of reported hfmd cases in asia include china, japan, hong kong (china), republic of korea, malaysia, singapore, thailand, and vietnam. in china, hfmd is one of the most common infectious diseases [ ] . it tends to occur in outbreaks during spring, summer, and autumn seasons. there has been a substantial increase of hfmd in many parts of the country in recent years [ ] . most of literatures reported that hfmd is a climate-sensitive disease, and it positively correlates with temperature with some days lag. a study in beijing revealed that mean temperature was positively associated with hfmd [ ] . in jiangsu, average temperature was positively correlated to hfmd incidence, while low temperature or high temperature was negatively related [ ] . in zhengzhou, average atmospheric temperature with or weeks lagged were identified as significant predictors for the number of hfmd and the pathogens [ ] . using meta-analysis, cheng et al. analyzed the relationship between ambient temperature and hfmd in east and southeast asia and found that ambient temperature could increase the incidence of hfmd in asia-pacific regions. it was revealed that °c increase in the temperature was significantly correlated to the increasing of the incidence of hfmd [ ] . as to the specific threshold, when the temperature was above . °c and the relative humidity was between . and . %, the rr of hfmd was . relative to monthly average incidence [ ] . regarding the relationship between hfmd and ambient temperature, most studies in other countries focus on the temperature threshold for the risk of hfmd and the quantitative relationship between temperature increase and hfmd. and this is consistent to china's study. in japan, sumi a revealed that the average temperature data indicated a lower threshold at °c and a higher threshold at °c for risk of hfmd infection. maximum and minimum temperature data indicated a lower threshold at °c and a higher threshold at °c [ ] , and the threshold is higher than that in du et al.'s study in china in . in south korea, at an average temperature below °c, the hfmd rate increased by . % for every °c rise in average temperature ( % confidence interval (ci), . , . %) [ ] . in vietnam, a °c increase in average temperature was associated with . % increase in hfmd rate at lag days ( % ci . - . ) [ ] . however, very little information is available regarding the relationship between hfmd and socioeconomic factors and demographic features. therefore, more studies are needed to clarify the relationship between ambient temperature and incidence of hfmd in various settings with distinct climate, socioeconomic, and demographic features. respiratory infectious diseases are a group of commonly and frequently occurring diseases, the lesion mainly in the trachea, bronchus, lungs, and thoracic cavity. climatic conditions may have affected the incidence of respiratory infectious diseases. influenza, commonly known as "the flu," is a representative respiratory infectious disease caused by some influenza virus. regarding the virus classification, influenza viruses belong to rna viruses that include three of the five genera of the family orthomyxoviridae, that is, influenza a virus, influenza b virus, and influenza c virus. a fourth family of influenza viruses has been proposed -influenza d. the type species for this family is bovine influenza d virus which was first isolated in . the influenza a virus can be subdivided into some serotypes on the basis of the antibody response to these viruses. the serotypes confirmed in humans and ordered by the number of known human pandemic deaths include h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , h n , and h n , respectively. climate change may alter the incidence and severity of respiratory infections by affecting vectors and host immune responses [ ] . most literatures revealed that influenza is a climatesensitive disease [ ] . climate change may affect the distribution and migration of the host of influenza virus and will eventually affect the transmission cycle, prevalence, and intensity of influenza. most of studies revealed that ambient temperature was correlated to influenza risks with possible nonlinear, interactive, and lagged effects [ ] . in china, the majority of studies focus on the influenza a virus, and studying the relationship between ambient temperature and h n , h n , few studies focus on avian influenza virus (aiv) and influenza b virus. lower temperature was the climatic factor facilitating local transmission of pandemic influenza a (h n ) in mainland china after correction for the effects of school summer vacation and public holidays, as well as population density and the density of medical facilities [ ] . in changsha, the sensitive climatic factors did have a "driving effect" on the incidence of influenza a (h n ). in the initial stage of the disease, a -day lag was found between the incidence and the daily minimum temperature. in the peak period of the disease, the daily minimum temperature was negatively relevant to the incidence [ ] . the outbreak of human infections with an emerging avian influenza a (h n ) virus occurred in china in early . a boosted regression tree (brt) models revealed that temperature significantly contributed to the occurrence of human infection with h n virus [ , ] . in shanghai, h n incidence rate was significantly associated with fortnightly mean temperature (relative risk (rr), . ; % cred-ible interval (ci), . - . ) [ ] . mean monthly temperature was significantly associated with the occurrence of human h n infection [ ] . zhang et al. found that both daily minimum and daily maximum temperature contributed significantly to human infection with the influenza a h n virus [ ] . models incorporating the nonlinear effect of minimum or maximum temperature on day prior to disease onset were considered to have the best predictive effect. liu et al. investigate the independent and interactive effects of ambient temperature (tm) and absolute humidity (ah) on h n risks in china. significantly nonlinear negative associations of tm and vp with h n risks were observed in all cases, and in cases from northern and southern regions. different risky windows of h n infection exist in the northern (tm, - °c; vp, mb) and southern areas (tm, - °c; vp, - mb) [ ] . temperature was correlated to the avian influenza virus (aiv) invasion in the destination to some degree [ ] . since the end of , highly pathogenic avian influenza viruses (hpai) h n have caused lots of outbreaks in poultries and wild birds from east asia and have spread to at least countries. liu et al. developed a new climatic approach for early predicting future hpai outbreaks and preventing pandemic disasters. the results demonstrate a temperature drop shortly before these outbreaks in birds in each of the eurasian regions stricken in and . dust storms, like those that struck near china's lake qinghai around may , , exacerbated the spread of hpai h n virus, causing the deaths of a record number of wild birds and triggering the subsequent spread of h n [ ] . climate factors were the strongest predictors of influenza b seasonality, including minimum temperature [ ] . in comparison with the incidence of influenza to climatic factors during - in five countries, tang et al. found that the mean temperature was the key climate variable associated with the incidence of influenza b in hong kong, brisbane, melbourne, and vancouver [ ] . furthermore, internet search metrics in conjunction with temperature [ ] could be adopted to predict influenza outbreaks, which can be regarded as a prerequisite for establishing early-warning systems using search and temperature data. currently, most studies focus on influenza a virus, and very few studies are available regarding the correlation between ambient temperature and influenza b, c, and d virus. there is still lack of enough evidences concerning the independent and interactive effects of ambient temperature and other complicated climatic factors on the risk of influenza in china and comparative studies in different countries [ ] . in addition, most studies in china consider the effect of specific temperature on influenza rather than a decrease of temperature; this is also important. jaakkola k et al. found that a decrease rather than low temperature increases the risk of influenza epidemic in a cold climate [ ] . in china, there are notifiable infectious diseases including from category a, from category b, and from category c. besides the major infectious diseases mentioned above, there are still some other infectious diseases that correlate to ambient temperature such as japanese encephalitis (je) and chikungunya. je is an important mosquito-borne disease and is commonly transmitted via the bite of culex tritaeniorhynchus with pig as a reservoir host and source of infection. at present, the morbidity and mortality of je has declined gradually year by year. however, je is still one of the threats to the public, and it has recently spread to new territories. in china, some studies revealed the positive relationships between ambient temperature and je when controlling for non-climatic factors. using arima models, monthly average temperature was positively associated with incidence of je in linyi, shandong after adjusting for mass vaccination in this area [ ] . correlation analy-sis and back propagation artificial neural work were applied; the annual je incidence was considered to be positively correlated with maximum temperature and extreme maximum temperature [ ] . in areas close to the three gorges dam, a significant positive association between temperature with a lag of and months and je incidence was found [ ] . few studies believed that temperature has a threshold effect on je cases. in , bi et al. have clarified positive relationships between monthly maximum temperature, minimum temperature, and je transmission in a rural region of anhui and a metropolitan area of shandong with no rice plating and the uncertain role of pigs in je transmission [ ] . in jinan, an obvious increase in je cases occurred when the monthly mean maximum temperature was higher than . °c or the minimum temperature was over . °c [ ] . these findings mentioned above are consistent with the threshold temperature detection in linyi [ ] . chikungunya fever is an emerging infection of chikungunya virus (chikv), constituting a serious public health problem. it is transmitted mainly by mosquitoes of the genus aedes although other ways of transmission by blood transfusions and vertical transmission have also been reported [ ] . it is a climate-sensitive mosquitotransmitted viral disease which was first identified in africa; now its distribution spread to asia. in china, the first outbreak of chikungunya happened in dongguan, guangdong, . after that, the outbreak happened in zhejiang in . however, few studies are available at present concerning the correlation between ambient temperature and chikungunya fever in china. projection of important infectious diseases under the context of climate change is beneficial to the prevention and control of infectious disease in the future. in this section, we summarized the projection of representative vector-borne diseases including rodent-borne diseases, waterborne diseases, intestinal infectious diseases, and respiratory infectious diseases. scenario, the high-risk area of df will further expand, and it will increase to counties (districts) ( million population) in . in china, very little literatures are available regarding the distribution of malaria in the future. using maxent species distribution model, the environmentally suitable area (esa) of a. dirus and a. minimus will increase by and % in the context of three climatic scenarios (rcp . , rcp . , and rcp . ) in the s. in the s, the esa of a. lesteri and a. sinensis under two scenarios (rcp . and pcp . ) will increase % and %, respectively. meanwhile, considering the level of land use and urbanization, the population of exposed to four anopheles mosquitoes in the s and the s showed a significant net increase [ ] . regarding the trend of malaria incidence in different scenarios in the future, it will show the similar trend with anopheles mosquitoes [ ] . in counties in henan and anhui from to , b low emission scenario, a b intermidiate scenario, and a high emission scenario, that are nearly consistent to the scenarios rcp . , rcp . , rcp . , and rcp . , respectively, were used, and gp-based model was adopted to describe the nonlinear relationship among incidence, temperature, and humidity and then project the change of incidence under different years. it is demonstrated that malaria incidence will increase markedly, and the distribution area of malaria will expand markedly in the future under the scenario of no malaria control. specifically, the malaria incidence in north china will increase - % in s. based on literature review, at present, no information is available regarding the projection of rodent-borne diseases such as hfrs, plague under different climatic scenarios. in future, the research in this field should be strengthened. with the development of new technologies and methods, accurate and stable prediction of ambient temperature-based schistosomiasis transmission becomes possible. based on the historical data, some researchers applied the future temperature data into the transmission model for schistosomiasis projection [ ] . according to historical data of temperature from to in china, a prediction that the mean january temperature will increase by . °c in and by . °c in . based on biologic model, for these temperature increases, potential risk areas for schistosomiasis transmission will increase an additional , km and , km by and , respectively. disease transmission is thus likely to occur in previously non-endemic areas, such as the southern parts of shandong and henan. under the cir-cumstances, the transmission intensity is possible to increase in areas already endemic for schistosomiasis [ ] . to some extents, the predictions might explain the recent observations of reemergence of this disease in areas where up to the criteria for transmission control, or even interruption [ , , ] . based on the data of mean temperature and monthly minimum temperature in january in china, the impact of warming climate in winter to the scale of schistosomiasis spreading was assessed using the indexes of °c mean temperature and − °c monthly minimum temperature in january. results showed the possibility that o. hupensis moves northward [ ] . through comparison in january average temperature °c, january average minimum temperature − °c, and january average temperature . °c, peng et al. found the last one fitted the schistosomiasis endemic areas best. by this standard, the potential epidemic areas moved toward north, and endemic areas would significantly increase in compared to that in [ ] . zhu et al. utilized fine-tuned maxent (fmaxent) and ensemble models to anticipate potential distributions of o. hupensis under future climate change scenarios on the background of snwdp in china. results indicated increased suitability and range expansion in o. hupensis in the future. the southern central route of snwdp will coincide with suitable areas for o. hupensis in - . its suitable areas will also expand northward along the southern eastern route in - [ ] . modeling with application of gis and rs that integrated ambient temperature (i.e., lst) resulted in a good predictive accuracy for the presence of o. hupensis in recent years [ ] . zhou et al. applied the mean monthly temperature and other environment variables in gis model to predict the transmission of schistosomiasis in the southern part of china. s. japonicum-endemic areas were restricted to settings with a transmission risk index exceeding , which is mostly consistent with the − °c average minimum temperature isotherm in china, while an improved model conducted by zhao et al. supposed isotherm was at − °c [ ] [ ] [ ] . after that, an improved gis forecast model combined with mean minimum temperature in january revealed hotspots of high transmission intensities in jiangsu and adjacent areas in different transmission seasons, which showed a high sensitivity of . % [ ] . by using the results from precis on reference years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , more than meteorological indexes including the highest and lowest temperature of the day in s ( - ) and s ( - ) were estimated under a and b scenario, which were developed in the ipcc (intergovernmental panel on climate change) special report on emissions scenarios (sres) to reflect the extent of climate change. the biology-based model is used to calculate the corresponding risk areas and potential transmission index in china in response to different climate scenarios. the transmission areas of schistosomiasis are supposed to extend to north both in s and s under a and b scenario, especially in jiangsu and anhui, and the extended areas in s are larger than that in s. north boundary of the transmission areas will extend further north under a than that under b in s, which has reached shandong. compared with the year of , the high-risk areas with potential transmission index> increased by . % and . % under a and b in s, respectively, which further increased in the s [ ] . based on literature review, the association between temperature and hfmd varies across china and that the future impact of climate change on hfmd incidence will vary as well. zhao et al. projected the change in hfmd cases due to projected temperature change by the s [ ] . they found that the projected incidence of hfmd increased by . % and . % by the s under the rcp . and . scenarios, respectively. however, regional projections suggest that hfmd may decrease with climate change in temperate areas of central and eastern china. wang et al. adopted the spatial regression model to project the incidence of hfmd according to projected climatic factors and population under different emission scenarios. there was not significant variation of the average incidence of hfmd from s to s under rcp . scenario. the incidence of the disease was also increased under rcp . and rcp . scenarios. however, the average incidence of hfmd would increase linearly under rcp . scenario with the fastest growth rate (unpublished). regarding the trend of hfmd in various major administrative regions under different climate scenarios, the incidence of hfmd in the northeast and northwest regions declined continuously in the future, while the increase of the incidence of the southwest region was under rcp . scenario. under the scenario of rcp . , the incidence of hfmd in north china, east china, central china, southern china, and southwest increased continuously. in the context of rcp . , the incidence of the disease in north china, east china, central china, and southern china increased continuously. and in the context of rcp . , the incidence of the disease in other regions, except in the northeast, increased. according to the trend of hfmd in various climate zone under different climate scenarios, the trend of the incidence of hfmd is increased in the qinghai tibet plateau and the middle subtropical region while that decreased in the middle temperate zone under the scenario of rcp . . under the rcp . scenario, the incidence of hfmd in the warm temperate zone, the northern subtropical zone, the middle subtropical, and the south subtropical regions is increased continuously while that decreased in the moderate temperate zone and the cold temperate zone; and the incidence of hfmd in warm temperate, northern subtropical, south subtropical, and marginal tropics is increased continuously while that decreased in middle temperate zone and cold temperate zone under rcp . scenario; and the incidence of hfmd in other regions increased except that in the middle temperate zone and cold temperate zone under rcp . scenario. regarding the projection of influenza in china, at present, very little information is available. take h n avian influenza for example, based on global climate projection models under the future scenarios, chen et al. obtained seasonal distribution of migratory birds in the context of different climate models and the seasonal distribution of h n avian influenza in migratory birds. the results show that japan and southern china will become high-risk areas of h n highly pathogenic avian influenza in january and february. northern africa, western asia, and central asia entered a high-risk period from april to june. the west coast of africa, west asia, india, and southern china become areas high risk of outbreak after october. compared with the current situation in high-risk areas, high-risk areas in africa will move northward from the central part of the continent. in addition, the high-risk area of h n avian influenza in winter in future will spread from low latitudes to high latitudes. in this chapter, the relationship between ambient temperature and infectious diseases were systematically summarized in china. we focused on not only the impact of 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mainland china key: cord- -aods rf authors: lessenger, james e. title: diseases from animals, poultry, and fish date: journal: agricultural medicine doi: . / - - - _ sha: doc_id: cord_uid: aods rf nan a key problem is the lack of foot protection so that the unprotected feet of workers come in contact with feces of the animals. the fecal-hand route of transmission is also critical. perhaps the most insidious and difficult to control is the consumption of raw poultry and meat products by workers in farms and processing plants. many people in agriculture are living on subsistence or below-subsistence wages and consume products off the processing lines. many of these products are not fully processed and may contact pathogens that have not been killed through cooking or irradiation (see chapter ) (table . ) ( , ) . the improper handling of manure is a major source of disease, including the use of manure on food crops, the discharge of manure into community water sources, and the spread of manure onto areas where children play. in canada, an outbreak of escherichia coli o :h was traced to organic growers who contaminated their produce with cow manure containing e. coli. also in canada, an outbreak of citrobacter freundii infections was associated with parsley originating from an organic garden in which pig manure was used. other documented infections of humans from manure-contaminated foods includes listeria monocytogenes in cabbage contaminated by sheep waste, cryptosporidium spread by municipal water contaminated by cattle, salmonella hartford in food prepared by contaminated water from a shallow well polluted with poultry manure, and pleisomonas shigelloides infection associated with well-water contaminated by poultry manure ( ) . j.e. lessenger guan and holley ( ) , and weber and rutala ( ) . workers, visitors, inspectors, veterinarians, and people who live on or adjacent to farms, ranches, feedlots, processing plants, and other agricultural endeavors are at risk for contracting diseases from animals, poultry, or fish. one needs only to follow the animals from the farm to the feedlots, slaughter house, processing and sorting lines, and packaging plants to appreciate the large number of people who are at risk due to contact with animals and animal products. physicians and other health care professionals are also at risk as they visit farms and plants for inspections or orientations ( ) . methods of preventing the transmission of infectious material from animals and poultry to agricultural workers mirror in many ways the safety techniques for protection from chemicals, trauma and other hazards (see chapter ) . the methods are summarized in table . . key to the prevention of the transmission of animal disease to humans is the proper processing of food products. this includes proper cook times and temperatures, adequate refrigeration, and appropriate transportation, processing, and stocking in stores. personal protective equipment includes hats or head coverings and protective coats or uniforms that can be laundered and left at the plant or farm. boots should also be cleaned and left at the farm or plant. especially in poultry operations, protective particulate masks may be necessary. in some . diseases from animals, poultry, and fish protective physical barriers in farm, ranch, or plant design allow for the raising or processing of food products without actual contact of humans with the animals or products. built-in barriers, changing rooms, boot baths, and hand-free handling techniques allow for the safe and efficient handling of food. in british chicken hatcheries, an aggressive combination of egg sanitization and handling methods was successful in decreasing zoonotic infections and diseases spread through flocks. procedures included: . design changes in incubators . whole building ventilation systems . control of dust, fluff, and aerosol production . disinfection of surfaces and equipment . improved handling of wastes ( ). policies and procedures to limit or prevent physical contact with animals, feces, or urine prevent transmission. rules prohibiting the consumption of food products on farms and ranches or on production lines are especially important. not only can the production food product be infectious to workers, but food brought in by workers can become contaminated, which mandates eating areas for workers away from the livestock ( ) . aggressive veterinary monitoring of livestock can detect early evidence of disease outbreaks in herds. similarly, public health monitoring of disease in humans can detect and appropriately treat epidemics of food-borne disease in humans and trace the source to the food-processing breakdown that caused the disease. hazard analysis of critical control points (haccp) is crucial to the prevention of infections in herds. low cost, ease of performance, and rapidity of results are the key criteria for the tests, and are sometimes more important than the performance characteristics of sensitivity, specificity, and reproducibility. field test kits are available for bacterial, protozoa, antibiotic residue, and other parameters of animal health ( , , ) . medical monitoring can detect early disease and prevent its spread to other employees, the food product, and family members. pre-placement medical monitoring can identify people who are susceptible to infection, for example people with diabetes or immune diseases. in parts of the world where bovine tuberculosis is common, tb skin test monitoring can detect early infections and allow early treatment ( , ) . immunizations are expensive, unavailable in many parts of the world, and only recommended for areas of high infectivity or occupations of high risk such as veterinarians. three critical immunizations are tetanus, rabies, and influenza (see chapter ) . vaccines against salmonella, shigella, and other pathogens are in development or testing. training and education in proper handling techniques are important. proper ways of herding, handling, and caring for animals and poultry can prevent infection and the transmission of infectious material. see chapter for details of education and training. research and the development of new techniques to prevent transmission are critical. for example, airborne dust has been discovered to be a carrier of pathogens in broiler breeder pullets (chicken pens). the use of an electrostatic space charge system has decreased the particle concentration and, in the process, decreased the potential of disease transmission to other chickens and to poultry workers ( ) . hygiene, both in the person and in the workplace, is essential in preventing the transmission of disease. for example, in many german piggeries workers must shower and change clothing when they enter and leave the buildings. this technique prevents the infection of the pigs with outside pathogens, the transfer of pathogens from one piggery to another, and the transfer of pathogens to the home environment. especially important are the cleaning of machinery and the timely cleaning of animal and poultry urine and feces. not only can urine and feces be infectious but they can attract insects that can spread pathogens. as in medicine, the most important hygiene procedure is aggressive hand washing for all persons handling food products. in louisiana, for example, alligator farmers must wear rubber boots and waders to protect themselves from pathogens (but not from bites, which can go right through the protective ensembles). each day, the pens must be flushed and hosed off to remove the wastes that could harbor pathogens dangerous to the alligator colonies and workers. governmental regulations and oversight are important in providing standardization and systemization of methods and procedures to reduce the risk of infection to agricultural workers. good regulations and oversight are evidence-based and consistent with sound agricultural methods (see chapter ). it is not enough to have rules, regulations, equipment and techniques to prevent the spread of pathogens from animals and poultry to workers. fair and consistent supervision by knowledgeable managers is critical to see that the proper equipment and supplies are used and that handling and hygiene rules and regulations are carried out. game are mammals killed or captured in the field for human consumption or for their hides, including elk, boars, bison, and deer. production animals include cattle, pigs, goats, sheep, horses, dogs, deer, and other animals grown in small to large farms and ranches for human consumption. typically the animals are slaughtered and dressed in various cuts made from the different parts of the animal. in addition, many animals are raised and kept as pets. rabies is a common viral infection in children who live in rural areas and in people who handle un-immunized mammalian animals. the prophylaxis for rabies is discussed in chapter . with the exception of four cases where the disease was treated with intensive therapy, the disease is considered universally fatal. therefore, immunizations and prophylaxis are critical. during may and june , the first cluster of human monkeypox cases in the united states was reported. most patients with this febrile, vesicular rash illness presumably acquired the infection from prairie dogs. monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with lesser amounts in adjacent macrophages, fibroblasts, and connective tissues. viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus among rodents and to humans. prairie dogs can be studied for insights into transmission, pathogenesis, and vaccine and treatment trials, because they are susceptible to severe monkeypox infection ( ) . chronic wasting disease (cwd) in north american deer and elk has been associated with creutzfeldt-jakob disease (cjd) in hunters who killed, prepared, and ate their own game. an absolute association was not established, but further monitoring is ongoing (see chapter ) . creutzfeldt-jakob disease does not appear to be a problem with workers who raise cattle or dairy cows ( ) . champylobacter chlamydophila abortus is a well recognized pathogen causing abortions in cattle and goats. a recent report from germany cites a case where a pregnant woman became infected from farm animals and aborted. this rare zoonotic infection underlines the insidious and widespread problem of zoonotic infections on farms ( , ) . campylobacter jejuni and c. coli have recently become recognized as common bacterial causes of diarrhea. infection can occur at any age. sources of infection are typically mammalian and avian hosts. the usual incubation period of campylobacter enteritis is to days. fever, diarrhea and abdominal pain are the most common clinical features. the stools frequently contain mucus and, a few days after the onset of symptoms, frank blood. significant vomiting and dehydration are uncommon. a rapid presumptive laboratory diagnosis may be made during the acute phase of the illness by direct phase-contrast microscopy of stools. isolation of the organism from stools requires culture in a selective medium containing antibiotics and incubation under reduced oxygen tension at ˚c. the organism persists in the stools of untreated patients for up to weeks following the onset of symptoms. erythromycin may produce a rapid clinical and bacteriologic cure and should be used to treat moderately to severely ill patients as well as patients with compromised host defenses ( ) . salmonellosis is one of the most important public health disease problems, affecting more people and animals than any other single disease in agriculture. in canada, for example, there were , cases of food-borne salmonellosis in humans during . the native habitat of members of the genus salmonella is the intestinal tract of warm-blooded and many coldblooded vertebrates. in humans, the incubation period is to hours and produces headache, malaise, nausea, fever, vomiting, abdominal pain, and diarrhea (with and without blood). salmonella is also capable of invading the intestinal mucosa, entering the blood stream, and causing septicemia, shock, and death. the diagnosis is made through the clinical presentation and confirmation with blood and stool cultures and serology. treatment is first started empirically pending culture results and then adjusted if necessary. multi-drug resistant s. typhimurium bacteria have been documented to be present in milk after pasteurization ( , ) . listeria monocytogenes is a zoonotic food-born pathogen that is responsible for % of food-related deaths in the united states annually and that is a major cause of food recalls worldwide. agricultural exposure is through drinking unpasteurized milk or direct contact with the animal or manure. the disease pattern is similar to salmonella ( ). tuberculosis (tb) continues to be a worldwide infectious problem for humans. while human-to-human infection is of greatest concern, one infected dairy herd can infect hundreds, if not thousands, of people. potentially, tuberculosis can infect any mammal, although production cattle, especially dairy cattle, are at greatest risk. complicating efforts to combat the disease is the fact that deer, badgers, elk and other wild species have been found to harbor the mycobacterium. in england, badgers were found to be spreading the infection to herds of cattle. also, in england and ireland, herds of sheep were found to be infected. in new zealand, wild brush tail possums (trichosurus vulpecula) were discovered to be the main source of infection in livestock, including deer herds. in tanzania, tuberculosis-infected herds were found more often in small, pastoral farms that have little veterinary monitoring, as opposed to the large, commercial enterprises ( ) ( ) ( ) ( ) . in a los angeles zoo, tb was found in two asian elephants, three rocky mountain goats, and one black rhinoceros. an investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training the elephants and attending an elephant necropsy ( ) . human-to-animal transmission of tb has been documented. in an exotic animal farm in illinois, three elephants died of mycobacterium tuberculosis and a fourth tested culture-positive. twenty-two handlers were screened for tb; eleven had positive reactions to intradermal injection with purified protein derivative. one had a smear-negative, culture-positive active tb. dna comparisons by is and tbn typing showed that the isolates from the four elephants and the handler with active tb were the same strain, thus documenting that the infection of the elephants came from the handler ( ) . mycobacterium (tuberculosis) can infect agricultural workers in a number of ways: . human-to-human contact with co-workers through the inhalation of respiratory droplets . drinking contaminated, unpasteurized milk . direct contact with infected animals . direct contact with the secretions of infected animals such as respiratory droplets, milk, manure, urine, semen, and vaginal secretions . direct contact or inhalations of respiratory droplets during necropsy, slaughter, or processing of meat or dairy products ( ) ( ) ( ) ( ) ( ) . the clinical presentation is that of weight-loss, night sweats, a chronic cough, and hemoptysis. asymptomatic workers are typically discovered through public health surveys. diagnosis is through the purified protein derivative (ppd) skin test, smears of respiratory secretions demonstrating acid-fast bodies, cultures of respiratory secretions and other body fluids, radiographs demonstrating caseating granulomas, and other typical findings. treatment is by multidrug therapy, complicated by regional drug resistance patterns ( ) ( ) ( ) ( ) ( ) . giardia infections have been associated with contaminated sewage and water in agricultural environments, producing gastroenteritis. in the sierra foothills of california, cattle drink water contaminated by infected beavers. beaver-and cattle-contaminated water is then consumed by unsuspecting tourists who develop crampy abdominal pain, fevers, and a profuse bloody diarrhea. the giardia infections are easily treated with metronidazole ( ). fowl are birds that grow in the wild. nearly every bird found in the wild can be prepared for human consumption. poultry are birds grown in farm environments for human consumption. common poultry include: chickens, turkeys, ducks, pigeons, game hens, geese, doves, and peacocks. avian influenza a (h n ) first infected humans in , in hong kong. the virus was transmitted directly from birds to humans. eighteen people were admitted to hospitals, and died. in , cases of avian influenza a (h n ) infection occurred among members of a hong kong family, of whom had traveled to mainland china. one person died. how or where these people became infected was not determined. influenza a has the potential to cross species and has been implicated in the flu pandemics in the th century ( , and ). pandemics occur when conditions are met: . the emergence of influenza a virus with a hemagglutinin subtype is completely different from that of strains circulating in humans for many preceding years. . there is a high proportion of susceptible people in the community (i.e., a population with low antibody titers to the new strain). . efficient person-to-person transmissibility of the new virus is possible with accompanying human disease ( ) ( ) ( ) . the reported signs and symptoms of avian influenza in humans include: . typical flu-like symptoms such as fever, cough, sore throat, and muscle aches . eye infections . pneumonia . acute respiratory distress syndrome (ards) . multiple organ failure . lymphopenia . elevated liver enzyme levels . abnormal clotting profiles. physicians are advised to isolate the patient, initiate droplet precautions, and contact their local medical officer for further discussions if an epidemiological link is suspected. the world health organization (who) is moving to rapidly produce a new influenza vaccine capable of protecting people against the h n strain of avian influenza a. preliminary genetic tests conducted in cdc laboratories in atlanta, london, and hong kong suggest that the h n strain is resistant to amantadine and rimantadine but is believed to be susceptible to neuraminidase inhibitors. the who has recommended urgent, rapid culling of infected and exposed bird populations to eliminate the reservoir of the h n strain. in addition, who has discouraged the practice of marketing live poultry directly to consumers in areas currently experiencing outbreaks of avian influenza a (h n ). some countries have introduced trade restrictions to protect animal health. however, available data do not suggest that processed poultry products (i.e., refrigerated or frozen carcasses and products derived from them) or eggs from affected areas pose a public health risk. the virus is killed by cooking ( ) ( ) ( ) . newcastle disease is caused by virulent strains of apmv. death rates among naive bird populations can exceed %. the virus responsible for newcastle disease has been known to cause conjunctivitis and upper respiratory infections in humans since the s. the disease is self-limiting and does not have any permanent consequences ( ) . in , wisconsin public health officials were notified of two cases of febrile illness in workers at a commercial turkey breeder farm. a high prevalence of west nile virus antibody was found among workers and turkeys. an associated high incidence of febrile illness among farm workers also was observed. possible non-mosquito transmission among birds and subsequent infection of humans was postulated, but the mode of transmission was unknown ( ) . avian tuberculosis was diagnosed in two mature rheas on different ratite farms over a -year period. both birds died after weight loss and development of granulomas in the lungs of one bird and bilaterally in the cubcutis cranial to the shoulder in the other. smears and cultures of the granulomas were positive for acid-fast bacilli and tuberculosis ( ) . chlamydophila (chlamydia) psittaci, c. trachomatis, and c. pneumoniae can be passed from birds of all species to humans. wild pigeons and pheasants have been demonstrated to be a source. wild birds in captivity, pets (usually cockatiels, parakeets, parrots, and macaws), and production animals can infect workers, and there are reports of customs and health inspection workers becoming infected. infection is through contact with feces, urine, and oral secretions ( ) . mild infection produces a tracheobronchitis with flu-like symptoms of cough, congestion, myalgias, fatigue, and fever. in severe infections, untreated workers, and immunocompromised workers, pneumonia, sepsis, shock, and death can occur. radiographs reveal a lobar infiltrate ( ) . diagnosis is by detection of the s rrna gene of c. psittasi in sputum with a pcr analysis, and a typical radiographic appearance and culture. tetracyclines and erythromycin are effective for treatment. prevention is through close monitoring and culling flocks and pet birds and personal protection equipment ( ). raising poultry at home is common in low-income countries. studies demonstrate that proximity to free-range domestic poultry increases children's risk of infection with diarrhea-causing organisms such as campylobacter jejuni. corralling might reduce the risk, but research on the socioeconomic acceptability of corralling is lacking. many people report that home-grown poultry and eggs taste better and are more nutritious. they enjoy living around animals and want to teach their children about raising animals. to prevent theft, some residents shut their birds in provisional enclosures at night but allege that birds are healthier, happier, and produce better meat and eggs when let loose by day. many rural peoples view bird feces in the house and yard as dirty, but few see a connection to illness. residents consider chicks and ducklings more innocuous than adult birds and are more likely to allow them inside the house and permit children to play with them. additional food and water costs with corralling are a significant obstacle for some. adequate space and corral hygiene must also be addressed to make this intervention viable. developing a secure, acceptable, and affordable corral remains a challenge for rural populations ( , ) . although approximately % of disease caused by non-typhoidal salmonella is transmitted by food-borne vehicles, four documented salmonella outbreaks in the s have been traced to contact with young poultry. no environmental studies of source hatcheries were completed. a case-control study was performed by comparing culture-confirmed salmonella infantis in michigan residents, identified between may and july , with two age-and neighborhood-matched controls. eighty environmental and bird tissue samples were collected from an implicated hatchery; all salmonella isolates underwent pulsed-field gel electrophoresis (pfge) analysis. the study included case-patients sharing the same pfge subtype and matched controls. within days before illness onset, % of case-patients resided in households raising young poultry compared with % of controls (matched or . ; % ci . , . ). eight hatchery samples yielded s. infantis with pfge subtypes matching the patients' isolates. this investigation identified birds from a single hatchery as the source of human illness and confirmed the link by matching pfge patterns from humans, birds and the hatchery environment. subsequent public health interventions reduced, but did not eliminate, transmission of poultry-associated salmonellosis. five additional pfge-linked cases were identified in spring , necessitating quarantine of the hatchery for depopulation, cleaning and disinfection ( ) . fish farming, or aquaculture, for fish and shellfish is becoming more common and more internationalized with every passing year. in the united states, more than half the seafood consumption is imported, much of it from fish farming. the world's seafood trade is very complex, and if is often difficult or impossible to determine where the seafood is raised or harvested. for example, the united states imports salmon from switzerland and panama though neither country is known for large salmon fisheries ( ) . in general, farmed fish is as safe and nutritious as wild-caught species, but there are public health hazards associated with ignorance, abuse, and neglect of aquaculture technology. numerous small fish ponds increase the shoreline of ponds causing higher densities of mosquito larvae and cercaria, which can increase the incidence and prevalence of lymphatic filariasis and schistosomiasis. especially dangerous is the use of human waste draining to fertilize or create ponds. technology abuse includes the misuse of therapeutic drugs, chemicals, fertilizers and natural fish habitat areas. technology neglect includes the failure to pay attention to mosquito habitats and the concomitant increase in malaria, as well as the propagation of other organisms ( ) . human exposure can be through direct skin contact with fish or the consumption of contaminated fish or shellfish products or contaminated water. the main pathogens acquired topically from fish (through spine puncture or open wounds) are aeromonas hydrophila, edwardsiella tarda, erysipelothrix rhusiopathiae, mycobacterium marinum, streptococcus iniae, vibrio vulnificus, and vibrio damsela. s. iniae has recently emerged as a public health hazard associated with aquaculture, and m. marinum often infects home aquarium hobbyists. common zoonoses contracted through the consumption of contaminated products or water include salmonella, leptospirosis, yersiniosis, and tuberculosis ( ) . salmonellae species have been found associated with all of the poikilothermic vertebrate species studied, as well as the mollusks and crustaceans ( ) . leptospirosis does occur in the poikilothermic vertebrates, as evidenced by positive serological reactions and by the isolation of pathogenic leptospiral serovars. the finding of leptospirosis species in fish, mollosks and other aquatic species are of special importance in view of the increased worldwide interest in aquaculture farming. since , of the ( . %) reported human cases of leptospirosis in hawaii have been associated with aquaculture industries (taro farms, prawn farms and watercress farms) ( ) . species of yersinia are a particular problem in fish and in people involved in fish farming. workers who wade in fish ponds or drink drainage water are especially at risk. yersinia enterocolitica has been demonstrated to be a causative agent in acute diarrhea illness in humans after workers become infected through the feces-hand-oral route ( ) . tuberculosis has also been reported in freshwater and marine fish species (piscine tuberculosis), especially in those grown on fish farms. mycobacterium marinum and m. celonae have been demonstrated in fish farms ( ) . turtles, lizards, snakes, green iguanas (iguana iguana), alligators, and crocodiles are grown from eggs in farms for their hides and meat. some species are also grown for sale as pets. salmonella infections in persons who had contact with reptiles usually cause gastroenteritis but can result in invasive illness, including septicemia and meningitis, especially in infants and immunocompromised persons. for decades, reptiles have been known to be a source for salmonellosis; however, numerous reptile owners remain unaware that reptile contact places them and other household members, including children, at greater risk for infection. ( ) captive reptiles (such as iguanas) are routinely identified as reservoirs of salmonella and the number of reports about reptile-associated salmonellosis is increasing. in germany and austria, salmonella was detected in . % of fecal reptile samples cultured. the percentage of salmonella-positive samples was significantly lower in turtles as compared with lizards and snakes, as salmonella was only detected in one sample from a single turtle out of turtles investigated. in all, different salmonella serovars were found. all isolated salmonella belonged to the species enterica, predominantly to the subspecies i (n = ) and iiib (n = ) but also to subspecies ii (n = ), iiia (n = ), and iv (n = ). all isolates were sensitive to the antimicrobials examined. a significantly higher percentage of salmonella-positive reptiles was detected in the group of owners who purchased reptiles in comparison with pure breeders. the high percentage of salmonella in reptiles in the study confirms the risk for the transmission of the infection to humans ( ) . zoonoses as a risk when associating with livestock or animal products social and environmental risk factors in the emergence of infectious diseases public health implications related to spread of pathogens in manure from livestock and poultry operations occupational bio hazards: current issues pathogen survival in swine manure environments and transmission of human enteric illness: a review zoonotic infections an approach to reduction of salmonella infection in broiler chicken flocks through intensive sampling and identification of cross-contamination hazards in commercial hatcheries maff statuatory incident reports in surveillance, prevention, and control of human salmonella typhimurium infection surveillance and control of emerging zoonoses testing to fulfill haccp (hazard analysis critical control points) requirements: principles and examples effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust veterinary monkeypox virus working group. monkeypox transmission and pathogenesis in prairie dogs wild game feasts and fatal degenerative neurological illness abortion in humans caused by chlamydophila abortus chlamydophila abortus infection in a pregnant woman associated with indirect contact with infected goats human salmonellosis associated with exotic pets multidrugresistant salmonella typhimurium infection from milk contaminated after pasteurization dairy farm reservoir of listeria monocytogenes sporadic and epidemic strains a study of the foodborne pathogens: campylobacter, listeria and yersinia, in faeces from slaughter-age cattle and sheep in australia factors influencing the incidence and scale of bovine tuberculosis in cattle in southwest england diagnostic strategies and outcomes on three new zealand deer farms with severe outbreaks of bovine tuberculosis prevalence of bovine tuberculosis in cattle in different farming systems in the eastern zone of tanzania human exposure following mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants avian influenza: recent developments human health implications of avian influenza viruses and paramyxoviruses avian influenza outbreak: update phylogenetic relationships among virulent newcastle disease virus isolates from the - outbreak in california and other recent outbreaks in north america center for disease control and prevention. west nile virus infection among turkey breeder farm workers: wisconsin tuberculosis in farmed rheas (rhea americana) a flu like syndrome a woman with a lobar infiltrate due to psittacosis detected by polymerase chain reaction campylobacter, from obscurity to celebrity campylobacter enteritis human salmonellosis associated with young poultry from a contaminated hatchery in michigan and the resulting public health interventions public, animal, and environmental health implications of aquaculture topically acquired bacterial zoonoses from fish: a review epidemiologic aspects of salmonellosis in reptiles, amphibians, mollusks and crustaceans-a review leptospirosis in poikilothermic vertebrates. a review reptile-associated salmonellosis-selected states salmonella enterica in reptiles of german and austrian origin systemic infection with alaria americana (trematoda) amphibians include frogs, toads, newts, and salamanders that are caught in the wild or grown on farms for use as food or as pets. frogs are caught in the wild and grown in farms for their meat, primarily frog legs. eating inadequately cooked frog legs can lead to an infection of alaria americana, a trematode. increasing evidence suggests that amphibians can pose risks for salmonellosis in humans ( ) . key: cord- -pyb pt authors: newell-mcgloughlin, martina; re, edward title: the flowering of the age of biotechnology – date: journal: the evolution of biotechnology doi: . / - - - _ sha: doc_id: cord_uid: pyb pt nan the significance of developing genetic and physical maps of the genome, and the importance of comparing the human genome with those of other species. it also suggested a preliminary focus on improving current technology. at the request of the u.s. congress, the office of technology assessment (ota) also studied the issue, and issued a document in -within days of the nrc report -that was similarly supportive. the ota report discussed, in addition to scientific issues, social and ethical implications of a genome program together with problems of managing funding, negotiating policy and coordinating research efforts. prompted by advisers at a meeting in reston, virginia, james wyngaarden, then director of the national institutes of health (nih) , decided that the agency should be a major player in the hgp, effectively seizing the lead from doe. the start of the joint effort was in may (with an "official" start in october) when a -year plan detailing the goals of the u.s. human genome project was presented to members of congressional appropriations committees in mid-february. this document co-authored by doe and nih and titled "understanding our genetic inheritance, the u.s. human genome project: the first five years" examined the then current state of genome science. the plan also set forth complementary approaches of the two agencies for attaining scientific goals and presented plans for administering research agenda; it described collaboration between u.s. and international agencies and presented budget projections for the project. according to the document, "a centrally coordinated project, focused on specific objectives, is believed to be the most efficient and least expensive way" to obtain the -billion base pair map of the human genome. in the course of the project, especially in the early years, the plan stated that "much new technology will be developed that will facilitate biomedical and a broad range of biological research, bring down the cost of many experiments (mapping and sequencing), and finding applications in numerous other fields." the plan built upon the reports of the office of technology assessment and the national research council on mapping and sequencing the human genome. "in the intervening two years," the document said, "improvements in technology for almost every aspect of genomics research have taken place. as a result, more specific goals can now be set for the project." the document describes objectives in the following areas mapping and sequencing the human genome and the genomes of model organisms; data collection and distribution; ethical, legal, and social considerations; research training; technology development; and technology transfer. these goals were to be reviewed each year and updated as further advances occured in the underlying technologies. they identified the overall budget needs to be the same as those identified by ota and nrc, namely about $ million per year for approximately years. this came to $ billion over the entire period of the project. considering that in july , the dna databases contained only seven sequences greater than . mb this was a major leap of faith. this approach was a major departure from the single-investigator-based gene of interest focus that research took hitherto. this sparked much controversy both before and after its inception. critics questioned the usefulness of genomic sequencing, they objected to the high cost and suggested it might divert funds from other, more focused, basic research. the prime argument to support the latter position is that there appeared to be are far less genes than accounted for by the mass of dna which would suggest that the major part of the sequencing effort would be of long stretches of base pairs with no known function, the so-called "junk dna." and that was in the days when the number of genes was presumed to be - , . if, at that stage, the estimated number was guessed to be closer to the actual estimate of - , (later reduced to - , ) this would have made the task seem even more foolhardy and less worthwhile to some. however, the ever-powerful incentive of new diagnostics and treatments for human disease beyond what could be gleaned from the gene-by-gene approach and the rapidly evolving technologies, especially that of automated sequencing, made it both an attractive and plausible aim. charles cantor ( ) , a principal scientist for the department of energy's genome project contended that doe and nih were cooperating effectively to develop organizational structures and scientific priorities that would keep the project on schedule and within its budget. he noted that there would be small short-term costs to traditional biology, but that the long-term benefits would be immeasurable. genome projects were also discussed and developed in other countries and sequencing efforts began in japan, france, italy, the united kingdom, and canada. even as the soviet union collapsed, a genome project survived as part of the russian science program. the scale of the venture and the manageable prospect for pooling data via computer made sequencing the human genome a truly international initiative. in an effort to include developing countries in the project unesco assembled an advisory committee in to examine unesco's role in facilitating international dialogue and cooperation. a privately-funded human genome organization (hugo) had been founded in to coordinate international efforts and serve as a clearinghouse for data. in that same year the european commission (ec) introduced a proposal entitled the "predictive medicine programme." a few ec countries, notably germany and denmark, claimed the proposal lacked ethical sensitivity; objections to the possible eugenic implications of the program were especially strong in germany (dickson ) . the initial proposal was dropped but later modified and adopted in as the "human genome analysis programme" (dickman and aldhous ) . this program committed substantial resources to the study of ethical issues. the need for an organization to coordinate these multiple international efforts quickly became apparent. thus the human genome organization (hugo), which has been called the "u.n. for the human genome," was born in the spring of . composed of a founding council of scientists from seventeen countries, hugo's goal was to encourage international collaboration through coordination of research, exchange of data and research techniques, training, and debates on the implications of the projects (bodmer ) . in august nih began large-scale sequencing trials on four model organisms: the parasitic, cell-wall lacking pathogenic microbe mycoplasma capricolum, the prokaryotic microbial lab rat escherichia coli, the most simple animal caenorhabditis elegans, and the eukaryotic microbial lab rat saccharomyces cerevisiae. each research group agreed to sequence megabases (mb) at cents a base within years. a sub living organism was actually fully sequenced and the complete sequence of that genome, the human cytomegalovirus (hcmv) genome was . mb. that year also saw the casting of the first salvo in the protracted debate on "ownership" of genetic information beginning with the more tangible question of ownership of cells. and, as with the debates of the early eighties, which were to be revisited later in the nineties, the respondent was the university of california. moore v. regents of the university of california was the first case in the united states to address the issue of who owns the rights to an individual's cells. diagnosed with leukemia, john moore had blood and bone marrow withdrawn for medical tests. suspicious of repeated requests to give samples because he had already been cured, moore discovered that his doctors had patented a cell line derived from his cells and so he sued. the california supreme court found that moore's doctor did not obtain proper informed consent, but, however, they also found that moore cannot claim property rights over his body. the quest for the holy grail of the human genome was both inspired by the rapidly evolving technologies for mapping and sequencing and subsequently spurred on the development of ever more efficient tools and techniques. advances in analytical tools, automation, and chemistries as well as computational power and algorithms revolutionized the ability to generate and analyze immense amounts of dna sequence and genotype information. in addition to leading to the determination of the complete sequences of a variety of microorganisms and a rapidly increasing number of model organisms, these technologies have provided insights into the repertoire of genes that are required for life, and their allelic diversity as well as their organization in the genome. but back in many of these were still nascent technologies. the technologies required to achieve this end could be broadly divided into three categories: equipment, techniques, and computational analysis. these are not truly discrete divisions and there was much overlap in their influence on each other. as noted, lloyd smith, michael and tim hunkapiller, and leroy hood conceived the automated sequencer and applied biosystems inc. brought it to market in june . there is no much doubt that when applied biosystems inc. put it on the market that which had been a dream became decidedly closer to an achievable reality. in automating sangers chain termination sequencing system, hood modified both the chemistry and the data-gathering processes. in the sequencing reaction itself, he replaced radioactive labels, which were unstable, posed a health hazard, and required separate gels for each of the four bases. hood developed chemistry that used fluorescent dyes of different colors for each of the four dna bases. this system of "color-coding" eliminated the need to run several reactions in overlapping gels. the fluorescent labels addressed another issue which contributed to one of the major concerns of sequencing -data gathering. hood integrated laser and computer technology, eliminating the tedious process of information-gathering by hand. as the fragments of dna passed a laser beam on their way through the gel the fluorescent labels were stimulated to emit light. the emitted light was transmitted by a lens and the intensity and spectral characteristics of the fluorescence are measured by a photomultiplier tube and converted to a digital format that could be read directly into a computer. during the next thirteen years, the machine was constantly improved, and by a fully automated instrument could sequence up to , , base pairs per year. in three groups came up with a variation on this approach. they developed what is termed capillary electrophoresis, one team was led by lloyd smith (luckey, ) , the second by barry karger , and the third by norman dovichi. in molecular dynamics introduced the megabace, a capillary sequencing machine. and not to be outdone the following year in , the original of the species came up with the abi prism sequencing machine. the is also a capillary-based machine designed to run about eight sets of sequence reactions per day. on the biology side, one of the biggest challenges was the construction of a physical map to be compiled from many diverse sources and approaches in such a way as to insure continuity of physical mapping data over long stretches of dna. the development of dna sequence tagged sites (stss) to correlate diverse types of dna clones aided this standardization of the mapping component by providing mappers with a common language and a system of landmarks for all the libraries from such varied sources as cosmids, yeast artificial chromosomes (yacs) and other rdnas clones. this way each mapped element (individual clone, contig, or sequenced region) would be defined by a unique sts. a crude map of the entire genome, showing the order and spacing of stss, could then be constructed. the order and spacing of these unique identifier sequences composing an sts map was made possible by development of mullis' polymerase chain reaction (pcr), which allows rapid production of multiple copies of a specific dna fragment, for example, an sts fragment. sequence information generated in this way could be recalled easily and, once reported to a database, would be available to other investigators. with the sts sequence stored electronically, there would be no need to obtain a probe or any other reagents from the original investigator. no longer would it be necessary to exchange and store hundreds of thousands of clones for full-scale sequencing of the human genome-a significant saving of money, effort, and time. by providing a common language and landmarks for mapping, sts's allowed genetic and physical maps to be cross-referenced. with a refinement on this technique to go after actual genes, sydney brenner proposed sequencing human cdnas to provide rapid access to the genes stating that 'one obvious way of finding at least a large part of the important [fraction] of the human genome is to look at the sequences of the messenger rna's of expressed genes' (brenner, ) . the following year the man who was to play a pivotal role on the world stage that became the human genome project suggested a way to implement sydney's approach. that player, nih biologist j. craig venter announced a strategy to find expressed genes, using ests (expressed sequence tag) (adams, ) . these so called ests represent a unique stretch of dna within a coding region of a gene, which as sydney suggested would be useful for identifying full-length genes and as a landmark for mapping. so using this approach projects were begun to mark gene sites on chromosome maps as sites of mrna expression. to help with this a more efficient method of handling large chunks of sequences was needed and two approaches were developed. yeast artificial chromosomes, which were developed by david burke, maynard olson, and george carle, increased insert size -fold (david t. burke et al., ) . caltech's second major contribution to the genome project was developed by melvin simon, and hiroaki shizuya. their approach to handling large dna segments was to develop "bacterial artificial chromosomes" (bacs), which basically allow bacteria to replicate chunks greater than , base pairs in length. this efficient production of more stable, large-insert bacs made the latter an even more attractive option, as they had greater flexibility than yacs. in in a collaboration that presages the snp consortium, washington university, st louis mo, was funded by the pharmaceutical company merck and the national cancer institute to provide sequence from those ests. more than half a million ests were submitted during the project (murr l et al., ) . on the analysis side was the major challenge to manage and mine the vast amount of dna sequence data being generated. a rate-limiting step was the need to develop semi-intelligent algorithms to achieve this herculean task. this is where the discipline of bioinformatics came into play. it had been evolving as a discipline since margaret oakley dayhoff used her knowledge of chemistry, mathematics, biology and computer science to develop this entirely new field in the early sixties. she is in fact credited today as a founder of the field of bioinformatics in which biology, computer science, and information technology merge into a single discipline. the ultimate goal of the field is to enable the discovery of new biological insights as well as to create a global perspective from which unifying principles in biology can be discerned. there are three important sub-disciplines within bioinformatics: the development of new algorithms and statistics with which to assess relationships among members of large data sets; the analysis and interpretation of various types of data including nucleotide and amino acid sequences, protein domains, and protein structures; and the development and implementation of tools that enable efficient access and management of different types of information. paralleling the rapid and very public ascent of recombinant dna technology during the previous two decades, the analytic and management tools of the discipline that was to become bioinformatics evolved at a more subdued but equally impressive pace. some of the key developments included tools such as the needleman-wunsch algorithm for sequence comparison which appeared even before recombinant dna technology had been demonstrated as early as ; the smith-waterman algorithm for sequence alignment ( ); the fastp algorithm ( ) and the fasta algorithm for sequence comparison by pearson and lupman in and perl (practical extraction report language) released by larry wall in . on the data management side several databases with ever more effective storage and mining capabilities were developed over the same period. the first bioinformatic/biological databases were constructed a few years after the first protein sequences began to become available. the first protein sequence reported was that of bovine insulin in , consisting of residues. nearly a decade later, the first nucleic acid sequence was reported, that of yeast alanine trna with bases. just one year later, dayhoff gathered all the available sequence data to create the first bioinformatic database. one of the first dedicated databases was the brookhaven protein databank whose collection consisted of ten x-ray crystallographic protein structures (acta. cryst. b, ) . the year saw the creation of the genetics computer group (gcg) as a part of the university of wisconsin biotechnology center. the group's primary and much used product was the wisconsin suite of molecular biology tools. it was spun off as a private company in . the swiss-prot database made its debut in in europe at the department of medical biochemistry of the university of geneva and the european molecular biology laboratory (embl). the first dedicated "bioinformatics" company intelligenetics, inc. was founded in california in . their primary product was the intelligenetics suite of programs for dna and protein sequence analysis. the first unified federal effort, the national center for biotechnology information (ncbi) was created at nih/nlm in and it was to play a crucial part in coordinating public databases, developing software tools for analyzing genome data, and disseminating information. and on the other side of the atlantic, oxford molecular group, ltd. (omg) was founded in oxford, uk by anthony marchington, david ricketts, james hiddleston, anthony rees, and w. graham richards. their primary focus was on rational drug design and their products such as anaconda, asp, and chameleon obviously reflected this as they were applied in molecular modeling, and protein design engineering. within two years ncbi were making their mark when david lipman, eugene myers, and colleagues at the ncbi published the basic local alignment search tool blast algorithm for aligning sequences (altschul et al., ) . it is used to compare a novel sequence with those contained in nucleotide and protein databases by aligning the novel sequence with previously characterized genes. the emphasis of this tool is to find regions of sequence similarity, which will yield functional and evolutionary clues about the structure and function of this novel sequence. regions of similarity detected via this type of alignment tool can be either local, where the region of similarity is based in one location, or global, where regions of similarity can be detected across otherwise unrelated genetic code. the fundamental unit of blast algorithm output is the high-scoring segment pair (hsp). an hsp consists of two sequence fragments of arbitrary but equal length whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score. this system has been refined and modified over the years the two principal variants presently in use being the ncbi blast and wu-blast (wu signifying washington university). the same year that blast was launched two other bioinformatics companies were launched. one was informax in bethesda, md whose products addressed sequence analysis, database and data management, searching, publication graphics, clone construction, mapping and primer design. the second, molecular applications group in california, was to play a bigger part on the proteomics end (michael levitt and chris lee). their primary products were look and segmod which are used for molecular modeling and protein design. the following year in the human chromosome mapping data repository, genome data base (gdb) was established. on a more global level, the development of computational capabilities in general and the internet in specific was also to play a considerable part in the sharing of data and access to databases that rendered the rapidity of the forward momentum of the hgp possible. also in edward uberbacher of oak ridge national laboratory in tennessee developed grail, the first of many gene-finding programs. in the first two "genomics" companies made their appearance. incyte pharmaceuticals, a genomics company headquartered in palo alto, california, was formed and myriad genetics, inc. was founded in utah. incyte's stated goal was to lead in the discovery of major common human disease genes and their related pathways. the company discovered and sequenced, with its academic collaborators (originally synteni from pat brown's lab at stanford), a number of important genes including brca and brca , with mary claire king, epidemiologist at uc-berkeley, the genes linked to breast cancer in families with a high degree of incidence before age . by a low-resolution genetic linkage map of the entire human genome was published and u.s. and french teams completed genetic maps of both mouse and man. the mouse with an average marker spacing of . cm as determined by eric lander and colleagues at whitehead and the human, with an average marker spacing of cm by jean weissenbach and colleagues at ceph (centre d'etude du polymorphisme humaine). the latter institute was the subject of a rather scathing book by paul rabinow ( ) based on what they did with this genome map. in , an american biotechnology company, millennium pharmaceuticals, and the ceph, developed plans for a collaborative effort to discover diabetes genes. the results of this collaboration could have been medically significant and financially lucrative. the two parties had agreed that ceph would supply millennium with germplasm collected from a large coterie of french families, and millennium would supply funding and expertise in new technologies to accelerate the identification of the genes, terms to which the french government had agreed. but in early , just as the collaboration was to begin, the french government cried halt! the government explained that the ceph could not be permitted to give the americans that most precious of substances for which there was no precedent in law -french dna. rabinow's book discusses the tangled relations and conceptions such as, can a country be said to have its own genetic material, the first but hardly the last franco-american disavowal of détente (paul rabinow, ) . the latest facilities such as the joint genome institute (jgi), walnut creek, ca are now able to sequence up to mb per day which makes it possible to sequence whole microbial genomes within a day. technologies currently under development will probably increase this capacity yet further through massively parallel sequencing and/or microfluidic processing making it possible to sequence multiple genotypes from several species. nineteen ninety-two saw one of the first shakeups in the progress of the hgp. that was the year that the first major outsider entered the race when britain's wellcome trust plunked down $ million to join the hgp. this caused a mere ripple while the principal shake-ups occurred stateside. much of the debate and subsequently the direction all the way through the hgp process was shaped by the personalities involved. as noted the application of one of the innovative techniques, namely ests, to do an end run on patenting introduced one of those major players to the fray, craig venter. venter, the high school drop out who reached the age of majority in the killing fields of vietnam was to play a pivotal role in a more "civilized" but no less combative field of human endeavor. he came onto the world stage through his initial work on ests while at the national institute of neurological disorders and stroke (ninds) from to . he noted in an interview with the scientist magazine in , that there was a degree of ambiguity at ninds about his venturing into the field of genomics, while they liked the prestige of hosting one of the leaders and innovators in his newly emerging field, they were concerned about him moving outside the nind purview of the human brain and nervous system. ultimately, while he proclaimed to like the security and service infrastructure this institute afforded him, that same system became too restrictive for his interests and talent. he wanted the whole canvas of human-gene expression to be his universe, not just what was confined to the central nervous system. he was becoming more interested in taking a whole genome approach to understanding the overall structure of genomes and genome evolution, which was much broader than the mission of ninds. he noted, with some irony, in later years that the then current nih director harold varmus had wished in hindsight that nih had pushed to do a similar database in the public domain, clearly in venter's opinion varmus was in need of a refresher course in history! bernadine healy, nih director in , was one of the few in a leadership role who saw the technical and fiscal promise of venter's work and, like all good administrators, it also presented an opportunity to resolve a thorny "personnel" issue. she appointed him head of the ad hoc committee to have an intramural genome program at nih to give the head of the hgp (that other larger than life personality jim watson) notice that he was not the sole arbitrator of the direction for the human genome project. however venter very soon established himself as an equally non-conformist character and with the tacit consent of his erstwhile benefactor. he initially assumed the mantle of a non-conformist through guilt by association rather than direct actions when it was revealed that nih was filing patent applications on thousands of these partial genes based on his ests catalyzing the first hgp fight at a congressional hearing. nih's move was widely criticized by the scientific community because, at the time, the function of genes associated with the partial sequences was unknown. critics charged that patent protection for the gene segments would forestall future research on them. the patent office eventually rejected the patents, but the applications sparked an international controversy over patenting genes whose functions were still unknown. interestingly enough despite nih's reliance on the est/cdna technique, venter, who was now clearly venturing outside the ninds mandated rubric, could not obtain government funding to expand his research, prompting him to leave nih in . he moved on to become president and director of the institute for genomic research (tigr), a nonprofit research center based in gaithersburg, md. at the same time william haseltine formed a sister company, human genome sciences (hgs), to commercialize tigr products. venter continued est work at tigr, but also began thinking about sequencing entire genomes. again, he came up with a quicker and faster method: whole genome shotgun sequencing. he applied for an nih grant to use the method on hemophilus influenzae, but started the project before the funding decision was returned. when the genome was nearly complete, nih rejected his proposal saying the method would not work. in a triumphal flurry in late may and with a metaphorical nose-thumbing at his recently rejected "unworkable" grant venter announced that tigr and collaborators had fully sequenced the first free-living organism -haemophilus influenzae. in november , controversy surrounding venter's research escalated. access restrictions associated with a cdna database developed by tigr and its rockville, md.-based biotech associate, human genome sciences (hgs) inc. -including hgs's right to preview papers on resulting discoveries and for first options to license products -prompted merck and co. inc. to fund a rival database project. in that year also britain "officially" entered the hgp race when the wellcome trust trumped down $ million (as mentioned earlier). the following year hgs was involved in yet another patenting debacle forced by the rapid march of technology into uncharted patent law territory. on june , hgs applied for a patent on a gene that produces a "receptor" protein that is later called ccr . at that time hgs has no idea that ccr is an hiv receptor. in december , u.s. researcher robert gallo, the co-discoverer of hiv, and colleagues found three chemicals that inhibit the aids virus but they did not know how the chemicals work. in february , edward berger at the nih discovered that gallo's inhibitors work in late-stage aids by blocking a receptor on the surface of t-cells. in june of that year in a period of just days, five groups of scientists published papers saying ccr is the receptor for virtually all strains of hiv. in january , schering-plough researchers told a san francisco aids conference that they have discovered new inhibitors. they knew that merck researchers had made similar discoveries. as a significant valentine in the u.s. patent and trademark office (uspto) grants hgs a patent on the gene that makes ccr and on techniques for producing ccr artificially. the decision sent hgs stock flying and dismayed researchers. it also caused the uspto to revise its definition of a "patentable" drug target. in the meantime haseltine's partner in rewriting patenting history, venter turned his focus to the human genome. he left tigr and started the for-profit company celera, a division of pe biosystems, the company that at times, thanks to hood and hunkapillar, led the world in the production of sequencing machines. using these machines, and the world's largest civilian supercomputer, venter finished assembling the human genome in just three years. following the debacle with the then nih director bernine healy over patenting the partial genes that resulted from est analysis, another major personality-driven event in that same year occurred. watson strongly opposed the idea of patenting gene fragments fearing that it would discourage research, and commented that "the automated sequencing machines 'could be run by monkeys.' " (nature june , ) with this dismissal watson resigned his nih nchgr post in to devote his full-time effort to directing cold spring harbor laboratory. his replacement was of a rather more pragmatic, less flamboyant nature. while venter maybe was described as an idiosyncratic shogun of the shotgun, francis collins was once described as the king arthur of the holy grail that is the human genome project. collins became the director of the national human genome research institute in . he was considered the right man for the job following his success (along with lap-chee tsui) in identifying the gene for the cystic fibrosis transmembrane (cftr) chloride channel receptor that, when mutated, can lead to the onset of cystic fibrosis. although now indelibly connected with the topic non-plus tout in biology, like many great innovators in this field before him, francis collins had little interest in biology as he grew up on a farm in the shenandoah valley of virginia. from his childhood he seemed destined to be at the center of drama, his father was professor of dramatic arts at mary baldwin college and the early stage management of career was performed on a stage he built on the farm. while the physical and mathematical sciences held appeal for him, being possessed of a highly logical mind, collins found the format in which biology was taught in the high school of his day mind-numbingly boring, filled with dissections and rote memorization. he found the contemplation of the infinite outcomes of dividing by zero (done deliberately rather than by accident as in einstein's case) far more appealing than contemplating the innards of a frog. that biology could be gloriously logical only became clear to collins when, in , he entered yale with a degree in chemistry from the university of virginia and was first exposed to the nascent field of molecular biology. anecdotally it was the tome, the book of life, penned by the theoretical physicist father of molecular biology, edwin schrodinger, while exiled in trinity college dublin in that was the catalyst for his conversion. like schrodinger he wanted to do something more obviously meaningful (for less than hardcore physicists at least!) than theoretical physics, so he went to medical school at unc-chapel hill after completing his chemistry doctorate in yale, and returned to the site of his road to damascus for post-doctoral study in the application of his newfound interest in human genetics. during this sojourn at yale, collins began working on developing novel tools to search the genome for genes that cause human disease. he continued this work, which he dubbed "positional cloning," after moving to the university of michigan as a professor in . he placed himself on the genetic map when he succeeded in using this method to put the gene that causes cystic fibrosis on the physical map. while a less colorful-in-your-face character than venter he has his own personality quirks, for example, he pastes a new sticker onto the back of his motorcycle helmet every time he finds a new disease gene. one imagines that particular piece of really estate is getting rather crowded. interestingly it was not these four hundred pound us gorillas who proposed the eventually prescient timeline for a working draft but two from the old power base. in meetings in the us in , john sulston and bob waterston proposed to produce a 'draft' sequence of the human genome by , a full five years ahead of schedule. while agreed by most to be feasible it meant a rethinking of strategy and involved focusing resources on larger centers and emphasizing sequence acquisition. just as important, it asserts the value of draft quality sequence to biomedical research. discussion started with the british based wellcome trust as possible sponsors (marshall e. ) . by a rough draft of the human genome map was produced showing the locations of more than , genes. the map was produced using yeast artificial chromosomes and some chromosomes -notably the littlest -were mapped in finer detail. these maps marked an important step toward clone-based sequencing. the importance was illustrated in the devotion of an entire edition of the journal nature to the subject. (nature : - ) the duel between the public and private face of the hgp progressed at a pace over the next five years. following release of the mapping data some level of international agreement was decided on sequence data release and databases. they agreed on the release of sequence data, specifically, that primary genomic sequence should be in the public domain to encourage research and development to maximize its benefit to society. also that it be rapidly released on a daily basis with assemblies of greater than kb and that the finished annotated sequence should be submitted immediately to the public databases. in an international consortium completed the sequence of the genome of the workhorse yeast saccharomyces cerevisiae. data had been released as the individual chromosomes were completed. the saccharomyces genome database (sgd) was created to curate this information. the project collects information and maintains a database of the molecular biology of s. cerevisiae. this database includes a variety of genomic and biological information and is maintained and updated by sgd curators. the sgd also maintains the s. cerevisiae gene name registry, a complete list of all gene names used in s. cerevisiae. in a new more powerful diagnostic tool termed snps (single nucleotide polymorphisms) was developed. snps are changes in single letters in our dna code that can act as markers in the dna landscape. some snps are associated closely with susceptibility to genetic disease, our response to drugs or our ability to remove toxins. the snp consortium although designated a limited company is a nonprofit foundation organized for the purpose of providing public genomic data. it is a collaborative effort between pharmaceutical companies and the wellcome trust with the idea of making available widely accepted, high-quality, extensive, and publicly accessible snp map. its mission was to develop up to , snps distributed evenly throughout the human genome and to make the information related to these snps available to the public without intellectual property restrictions. the project started in april and was anticipated to continue until the end of . in the end, many more snps, about . million total, were discovered than was originally planned. by the complete genome sequence of mycobacterium tuberculosis was published by teams from the uk, france, us and denmark in june . the abi prism sequencing machine, a capillary-based machine designed to run about eight sets of sequence reactions per day also reached the market that year. that same year the genome sequence of the first multicellular organism, c. elegans was completed. c. elegans has a genome of about mb and, as noted, is a primitive animal model organism used in a range of biological disciplines. by november the human genome draft sequence reached mb and the first complete human chromosome was sequenced -this first was reached on the east side of the atlantic by the hgp team led by the sanger centre, producing a finished sequence for chromosome , which is about million base-pairs and includes at least genes. according to anecdotal evidence when visiting his namesake centre, sanger asked: "what does this machine do then?" "dideoxy sequencing" came the reply, to which fred retorted: "haven't they come up with anything better yet?" as will be elaborated in the final chapter the real highlight of was production of a 'working draft' sequence of the human genome, which was announced simultaneously in the us and the uk. in a joint event, celera genomics announced completion of their 'first assembly' of the genome. in a remarkable special issue, nature included a -page article by the human genome project partners, studies of mapping and variation, as well as analysis of the sequence by experts in different areas of biology. science published the article by celera on their assembly of hgp and celera data as well as analyses of the use of the sequence. however to demonstrate the sensitivity of the market place to presidential utterances the joint appearances by bill clinton and tony blair touting this major milestone turned into a major cold shower when clinton's reassurance of access of the people to their genetic information caused a precipitous drop in celera's share value overnight. clinton's assurance that, "the effort to decipher the human genome will be the scientific breakthrough of the century -perhaps of all time. we have a profound responsibility to ensure that the life-saving benefits of any cutting-edge research are available to all human beings." (president bill clinton, wednesday, march , ) stands in sharp contrast to the statement from venter's colleague that " any company that wants to be in the business of using genes, proteins, or antibodies as drugs has a very high probability of running afoul of our patents. from a commercial point of view, they are severely constrained -and far more than they realize." (william a. haseltine, chairman and ceo, human genome sciences). the huge sell-off in stocks ended weeks of biotech buying in which those same stocks soared to unprecedented highs. by the next day, however, the genomic company spin doctors began to recover ground in a brilliant move which turned the clinton announcement into a public relations coup. all major genomics companies issued press releases applauding president clinton's announcement. the real news they argued, was that "for the first time a president strongly affirmed the importance of gene based patents." and the same bill haseltine of human genome sciences positively gushed as he happily pointed out that he "could begin his next annual report with the [president's] monumental statement, and quote today as a monumental day." as distinguished harvard biologist richard lewontin notes: "no prominent molecular biologist of my acquaintance is without a financial stake in the biotechnology business. as a result, serious conflicts of interest have emerged in universities and in government service (lewontin, ) . away from the spin doctors perhaps eric lander may have best summed up the herculean effort when he opined that for him "the human genome project has been the ultimate fulfilment: the chance to share common purpose with hundreds of wonderful colleagues towards a goal larger than ourselves. in the long run, the human genome project's greatest impact might not be the three billion nucleotides of the human chromosomes, but its model of scientific community." (ridley, ) . gene therapy the year also marked the passing of another milestone that was intimately connected to one of the fundamental drivers of the hgp. the california hereditary disorders act came into force and with it one of the potential solutions for human hereditary disorders. w. french anderson in the usa reported the first successful application of gene therapy in humans. the first successful gene therapy for a human disease was successfully achieved for severe combined immune deficiency (scid) by introducing the missing gene, adenosine deaminase deficiency (ada) into the peripheral lymphocytes of a -year-old girl and returning modified lymphocytes to her. although the results are difficult to interpret because of the concurrent use of polyethylene glycol-conjugated ada commonly referred to as pegylated ada (pgla) in all patients, strong evidence for in vivo efficacy was demonstrated. ada-modified t cells persisted in vivo for up to three years and were associated with increases in t-cell number and ada enzyme levels, t cells derived from transduced pgla were progressively replaced by marrow-derived t cells, confirming successful gene transfer into long-lived progenitor cells. ashanthi desilva, the girl who received the first credible gene therapy, continues to do well more than a decade later. cynthia cutshall, the second child to receive gene therapy for the same disorder as desilva, also continues to do well. within years (by january ), more than gene therapy protocols had been approved in the us and worldwide, researchers launched more than clinical trials to test gene therapy against a wide array of illnesses. surprisingly, a disease not typically heading the charts of heritable disorders, cancer has dominated the research. in cancer patients were treated with the tumor necrosis factor gene, a natural tumor fighting protein which worked to a limited extent. even more surprisingly, after the initial flurry of success little has worked. gene therapy, the promising miracle of failed to deliver on its early promise over the decade. apart from those examples, there are many diseases whose molecular pathology is, or soon will be, well understood, but for which no satisfactory treatments have yet been developed. at the beginning of the nineties it appeared that gene therapy did offer new opportunities to treat these disorders both by restoring gene functions that have been lost through mutation and by introducing genes that can inhibit the replication of infectious agents, render cells resistant to cytotoxic drugs, or cause the elimination of aberrant cells. from this "genomic" viewpoint genes could be said to be viewed as medicines, and their development as therapeutics should embrace the issues facing the development of small-molecule and protein therapeutics such as bioavailability, specificity, toxicity, potency, and the ability to be manufactured at large scale in a cost-effective manner. of course for such a radical approach certain basal level criteria needed to be established for selecting disease candidates for human gene therapy. these include, such factors as the disease is an incurable, life-threatening disease; organ, tissue, and cell types affected by the disease have been identified; the normal counterpart of the defective gene has been isolated and cloned; either the normal gene can be introduced into a substantial subfraction of the cells from the affected tissue, or the introduction of the gene into the available target tissue, such as bone marrow, will somehow alter the disease process in the tissue affected by the disease; the gene can be expressed adequately (it will direct the production of enough normal protein to make a difference); and techniques are available to verify the safety of the procedure. an ideal gene therapeutic should, therefore, be stably formulated at room temperature and amenable to administration either as an injectable or aerosol or by oral delivery in liquid or capsule form. the therapeutic should also be suitable for repeat therapy, and when delivered, it should neither generate an immune response nor be destroyed by tissue-scavenging mechanisms. when delivered to the target cell, the therapeutic gene should then be transported to the nucleus, where it should be maintained as a stable plasmid or chromosomal integrant, and be expressed in a predictable, controlled fashion at the desired potency in a cell-specific or tissue-specific manner. in addition to the ada gene transfer in children with severe combined immunodeficiency syndrome, a gene-marking study of epstein-barr virus-specific cytotoxic t cells, and trials of gene-modified t cells expressing suicide or viral resistance genes in patients infected with hiv were studied in the early nineties. additional strategies for t-cell gene therapy which were pursued later in the decade involve the engineering of novel t-cell receptors that impart antigen specificity for virally infected or malignant cells. issues which still are not resolved include nuclear transport, integration, regulated gene expression and immune surveillance. this knowledge, when finally understood and applied to the design of delivery vehicles of either viral or non-viral origin, will assist in the realization of gene therapeutics as safe and beneficial medicines that are suited to the routine management of human health. scientists are also working on using gene therapy to generate antibodies directly inside cells to block the production of harmful viruses such as hiv or even cancer-inducing proteins. there is a specific connection with francis collins, as his motivation for pursuing the hgp was his pursuit of defective genes beginning with the cystic fibrosis gene. this gene, called the cf transmembrane conductance regulator, codes for an ion channel protein that regulates salts in the lung tissue. the faulty gene prevents cells from excreting salt properly causing a thick sticky mucus to build up and destroy lung tissue. scientists have spliced copies of the normal genes into disabled adeno viruses that target lung tissues and have used bronchioscopes to deliver them to the lungs. the procedure worked well in animal studies however clinical trials in humans were not an unmitigated success. because the cells lining the lungs are continuously being replaced the effect is not permanent and must be repeated. studies are underway to develop gene therapy techniques to replace other faulty genes. for example, to replace the genes responsible for factor viii and factor ix production whose malfunctioning causes hemophilia a and b respectively; and to alleviate the effects of the faulty gene in dopamine production that results in parkinson's disease. apart from technical challenges such a radical therapy also engenders ethical debate. many persons who voice concerns about somatic-cell gene therapy use a "slippery slope" argument. it sounds good in theory but where does one draw the line. there are many issues yet to be resolved in this field of thorny ethics "good" and "bad" uses of the gene modification, difficulty of following patients in long-term clinical research and such. many gene therapy candidates are children who are too young to understand the ramifications of this treatment: conflict of interest -pits individuals' reproductive liberties and privacy interests against the interests of insurance companies or society. one issue that is unlikely to ever gain acceptance is germline therapy, the removal of deleterious genes from the population. issues of justice and resource allocation also have been raised: in a time of strain on our health care system, can we afford such expensive therapy? who should receive gene therapy? if it is made available only to those who can afford it, then a number of civil rights groups claim that the distribution of desirable biological traits among different socioeconomic and ethnic groups would become badly skewed adding a new and disturbing layer of discriminatory behavior. indeed a major setback occurred before the end of the decade in . jesse gelsinger was the first person to die from gene therapy, on september , , and his death created another unprecedented situation when his family sued not only the research team involved in the experiment (u penn), the company genovo inc., but also the ethicist who offered moral advice on the controversial project. this inclusion of the ethicist as a defendant alongside the scientists and school was a surprising legal move that puts this specialty on notice, as will no doubt be the case with other evolving technologies such as stem cells and therapeutic cloning, that its members could be vulnerable to litigation over the philosophical guidance they provide to researchers. the penn group principal investigator james wilson approached ethicist arthur caplan about their plans to test the safety of a genetically engineered virus on babies with a deadly form of the liver disorder, ornithine transcarbamylase deficiency. the disorder allows poisonous levels of ammonia to build up in the blood system. caplan steered the researchers away from sick infants, arguing that desperate parents could not provide true informed consent. he said it would be better to experiment on adults with a less lethal form of the disease who were relatively healthy. gelsinger fell into that category. although he had suffered serious bouts of ammonia buildup, he was doing well on a special drug and diet regimen. the decision to use relatively healthy adults was controversial because risky, unproven experimental protocols generally use very ill people who have exhausted more traditional treatments, so have little to lose. in this case, the virus used to deliver the genes was known to cause liver damage, so some scientists were concerned it might trigger an ammonia crisis in the adults. wilson underestimated the risk of the experiment, omitted the disclosure about possible liver damage in earlier volunteers in the experiment and failed to mention the deaths of monkeys given a similar treatment during pre-clinical studies. a food and drug administration investigation after gelsinger's death found numerous regulatory violations by wilson's team, including the failure to stop the experiment and inform the fda after four successive volunteers suffered serious liver damage prior to the teen's treatment. in addition, the fda said gelsinger did not qualify for the experiment, because his blood ammonia levels were too high just before he underwent the infusion of genetic material. the fda suspended all human gene experiments by wilson and the university of penn subsequently restricting him solely to animal studies. a follow-up fda investigation subsequently alleged he improperly tested the experimental treatment on animals. financial conflicts of interest also surrounded james wilson, who stood to personally profit from the experiment through genovo his biotechnology company. the lawsuit was settled out of court for undisclosed terms in november . the fda also suspended gene therapy trials at st. elizabeth's medical center in boston, a major teaching affiliate of tufts university school of medicine, which sought to use gene therapy to reverse heart disease, because scientists there failed to follow protocols and may have contributed to at least one patient death. in addition, the fda temporarily suspended two liver cancer studies sponsored by the schering-plough corporation because of technical similarities to the university of pennsylvania study. some research groups voluntarily suspended gene therapy studies, including two experiments sponsored by the cystic fibrosis foundation and studies at beth israel deaconess medical center in boston aimed at hemophilia. the scientists paused to make sure they learned from the mistakes. the nineties also saw the development of another "high-thoughput" breakthrough, a derivative of the other high tech revolution namely dna chips. in biochips were developed for commercial use under the guidance of affymetrix. dna chips or microarrays represent a "massively parallel" genomic technology. they facilitate high throughput analysis of thousands of genes simultaneously, and are thus potentially very powerful tools for gaining insight into the complexities of higher organisms including analysis of gene expression, detecting genetic variation, making new gene discoveries, fingerprinting strains and developing new diagnostic tools. these technologies permit scientists to conduct large scale surveys of gene expression in organisms, thus adding to our knowledge of how they develop over time or respond to various environmental stimuli. these techniques are especially useful in gaining an integrated view of how multiple genes are expressed in a coordinated manner. these dna chips have broad commercial applications and are now used in many areas of basic and clinical research including the detection of drug resistance mutations in infectious organisms, direct dna sequence comparison of large segments of the human genome, the monitoring of multiple human genes for disease associated mutations, the quantitative and parallel measurement of mrna expression for thousands of human genes, and the physical and genetic mapping of genomes. however the initial technologies, or more accurately the algorithms used to extract information, were far from robust and reproducible. the erstwhile serial entrepreneur, al zaffaroni (the rebel who in founded alza when syntex ignored his interest in developing new ways to deliver drugs) founded yet another company, affymetrix, under the stewardship of stephen fodor, which was subject to much abuse for providing final extracted data and not allowing access to raw data. as with other personalities of this high through put era, seattle-bred steve fodor was also somewhat of a polymath having contributed to two major technologies, microarrays and combinatorial chemistry, the former has delivered on it's, promise while the latter, like gene therapy, is still in a somewhat extended gestation. and despite the limitations of being an industrial scientist he has had a rather prolific portfolio of publications. his seminal manuscripts describing this work have been published in all the journals of note, science, nature and pnas and was recognized in by the aaas by receiving the newcomb-cleveland award for an outstanding paper published in science. fodor began his industrial career in yet another zaffaroni firm. in he was recruited to the affymax research institute in palo alto where he spearheaded the effort to develop high-density arrays of biological compounds. his initial interest was in the broad area of what came to be called combinatorial chemistry. of the techniques developed, one approach permitted high resolution chemical synthesis in a light-directed, spatially-defined format. in the days before positive selection vectors, a researcher might have screened thousands of clones by hand with an oligonucleotide probe just to find one elusive insert. fodor's (and his successors) dna array technology reverses that approach. instead of screening an array of unknowns with a defined probe -a cloned gene, pcr product, or synthetic oligonucleotide -each position or "probe cell" in the array is occupied by a defined dna fragment, and the array is probed with the unknown sample. fodor used his chemistry and biophysics background to develop very dense arrays of these biomolecules by combining photolithographic methods with traditional chemical techniques. the typical array may contain all possible combinations of all possible oligonucleotides ( -mers, for example) that occur as a "window" which is tracked along a dna sequence. it might contain longer oligonucleotides designed from all the open reading frames identified from a complete genome sequence. or it might contain cdnas -of known or unknown sequence -or pcr products. of course it is one thing to produce data it is quite another to extract it in a meaningful manner. fodor's group also developed techniques to read these arrays, employing fluorescent labeling methods and confocal laser scanning to measure each individual binding event on the surface of the chip with extraordinary sensitivity and precision. this general platform of microarray based analysis coupled to confocal laser scanning has become the standard in industry and academia for large-scale genomics studies. in , fodor co-founded affymetrix where the chip technology has been used to synthesize many varieties of high density oligonucleotide arrays containing hundreds of thousands of dna probes. in , steve fodor founded perlegen, inc., a new venture that applied the chip technology towards uncovering the basic patterns of human diversity. his company's stated goals are to analyze more than one million genetic variations in clinical trial participants to explain and predict the efficacy and adverse effect profiles of prescription drugs. in addition, perlegen also applies this expertise to discovering genetic variants associated with disease in order to pave the way for new therapeutics and diagnostics. fodor's former company diversified into plant applications by developing a chip of the archetypal model of plant systems arabidopsis and supplied pioneer hi bred with custom dna chips for monitoring maize gene expression. they (affymetrix) have established programs where academic scientists can use company facilities at a reduced price and set up 'user centers' at selected universities. a related but less complex technology called 'spotted' dna chips involves precisely spotting very small droplets of genomic or cdna clones or pcr samples on a microscope slide. the process uses a robotic device with a print head bearing fine "repeatograph" tips that work like fountain pens to draw up dna samples from a -well plate and spot tiny amounts on a slide. up to , individual clones can be spotted in a dense array within one square centimeter on a glass slide. after hybridization with a fluorescent target mrna, signals are detected by a custom scanner. this is the basis of the systems used by molecular dynamics and incyte (who acquired this technology when it took over synteni). in , incyte was looking to gather more data for its library and perform experiments for corporate subscribers. the company considered buying affymetrix genechips but opted instead to purchase the smaller synteni, which had sprung out of pat brown's stanford array effort. synteni's contact printing technology resulted in dense -and cheaper -arrays. though incyte used the chips only internally, affymetrix sued, claiming synteni/incyte was infringing on its chip density patents. the suit argued that dense biochips -regardless of whether they use photolithography -cannot be made without a license from affymetrix! and in a litigious congo line endemic of this hi-tech era incyte countersued and for good measure also filed against genetic database competitor gene logic for infringing incyte's patents on database building. meanwhile, hyseq sued affymetrix, claiming infringement of nucleotide hybridization patents obtained by its cso. affymetrix, in turn, filed a countersuit, claiming hyseq infringed the spotted array patents. hyseq then reached back and found an additional hybridization patent it claimed that affymetrix had infringed. and so on into the next millennium! in part to avoid all of this another california company nanogen, inc. took a different approach to single nucleotide polymorphism discrimination technology. in an article in the april edition of nature biotechnology, entitled "single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips," nanogen describes the use of microchips to identify variants of the mannose binding protein gene that differ from one another by only a single dna base. the mannose binding protein (mbp) is a key component of the innate immune system in children who have not yet developed immunity to a variety of pathogens. to date, four distinct variants (alleles) of this gene have been identified, all differing by only a single nucleotide of dna. mbp was selected for this study because of its potential clinical relevance and its genetic complexity. the samples were assembled at the nci laboratory in conjunction with the national institutes of health and transferred to nanogen for analysis. however, from a high throughput perspective there is a question mark over microarrays. mark benjamin, senior director of business development at rosetta inpharmatics (kirkland, wa), is skeptical about the long-term prospects for standard dna arrays in high-throughput screening as the first steps require exposing cells and then isolating rna, which is something that is very hard to do in a high-throughput format. another drawback is that most of the useful targets are likely to be unknown (particularly in the agricultural sciences where genome sequencing is still in its infancy), and dna arrays that are currently available test only for previously sequenced genes. indeed, some argue that current dna arrays may not be sufficiently sensitive to detect the low expression levels of genes encoding targets of particular interest. and the added complication of the companies' reluctance to provide "raw data" means that derived data sets may be created with less than optimum algorithims thereby irretrievably losing potentially valuable information from the starting material. reverse engineering is a possible approach but this is laborious and time consuming and being prohibited by many contracts may arouse the interest of the ever-vigilant corporate lawyers. over the course of the nineties, outgrowths of functional genomics have been termed proteomics and metabolomics, which are the global studies of gene expression at the protein and metabolite levels respectively. the study of the integration of information flow within an organism is emerging as the field of systems biology. in the area of proteomics, the methods for global analysis of protein profiles and cataloging protein-protein interactions on a genome-wide scale are technically more difficult but improving rapidly, especially for microbes. these approaches generate vast amounts of quantitative data. the amount of expression data becoming available in the public and private sectors is already increasing exponentially. gene and protein expression data rapidly dwarfed the dna sequence data and is considerably more difficult to manage and exploit. in microbes, the small sizes of the genomes and the ease of handling microbial cultures, will enable high throughput, targeted deletion of every gene in a genome, individually and in combinations. this is already available on a moderate throughput scale in model microbes such as e. coli and yeast. combining targeted gene deletions and modifications with genome-wide assay of mrna and protein levels will enable intricate inter-dependencies among genes to be unraveled. simultaneous measurement of many metabolites, particularly in microbes, is beginning to allow the comprehensive modeling and regulation of fluxes through interdependent pathways. metabolomics can be defined as the quantitative measurement of all low molecular weight metabolites in an organism's cells at a specified time under specific environmental conditions. combining information from metabolomics, proteomics and genomics will help us to obtain an integrated understanding of cell biology. the next hierarchical level of phenotype considers how the proteome within and among cells cooperates to produce the biochemistry and physiology of individual cells and organisms. several authors have tentatively offered "physiomics" as a descriptor for this approach. the final hierarchical levels of phenotype include anatomy and function for cells and whole organisms. the term "phenomics" has been applied to this level of study and unquestionably the more well known omics namely economics, has application across all those fields. and, coming slightly out of left field this time, the spectre of eugenics needless to say was raised in the omics era. in the year american and british scientists unveiled a technique which has come to be known as pre-implantation genetic diagnosis (pid) for testing embryos in vitro for genetic abnormalities such as cystic fibrosis, hemophilia, and down's syndrome (wald, ) . this might be seen by most as a step forward, but it led ethicist david s. king ( ) to decry pid as a technology that could exacerbate the eugenic features of prenatal testing and make possible an expanded form of free-market eugenics. he further argues that due to social pressures and eugenic attitudes held by clinical geneticists in most countries, it results in eugenic outcomes even though no state coercion is involved and that, as abortion is not involved, and multiple embryos are available, pid is radically more effective as a tool of genetic selection. the first regulatory approval of a recombinant dna technology in the u.s. food supply was not a plant but an industrial enzyme that has become the hallmark of food biotechnology success. enzymes were important agents in food production long before modern biotechnology was developed. they were used, for instance, in the clotting of milk to prepare cheese, the production of bread and the production of alcoholic beverages. nowadays, enzymes are indispensable to modern food processing technology and have a great variety of functions. they are used in almost all areas of food production including grain processing, milk products, beer, juices, wine, sugar and meat. chymosin, known also as rennin, is a proteolytic enzyme whose role in digestion is to curdle or coagulate milk in the stomach, efficiently converting liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer periods in a neonate's stomach. the dairy industry takes advantage of this property to conduct the first step in cheese production. chy-max™, an artificially produced form of the chymosin enzyme for cheese-making, was approved in . in some instances they replace less acceptable "older" technology, for example the enzyme chymosin. unlike crops industrial enzymes have had relatively easy passage to acceptance for a number of reasons. as noted they are part of the processing system and theoretically do not appear in the final product. today about % of the hard cheese in the us and uk is made using chymosin from geneticallymodified microbes. it is easier to purify, more active ( % as compared to %) and less expensive to produce (microbes are more prolific, more productive and cheaper to keep than calves). like all enzymes it is required only in very small quantities and because it is a relatively unstable protein it breaks down as the cheese matures. indeed, if the enzyme remained active for too long it would adversely affect the development of the cheese, as it would degrade the milk proteins to too great a degree. such enzymes have gained the support of vegetarian organizations and of some religious authorities. for plants the nineties was the era of the first widespread commercialization of what came to be known in often deprecating and literally inaccurate terms as gmos (genetically modified organisms). when the nineties dawned dicotyledonous plants were relatively easily transformed with agrobacterium tumefaciens but many economically important plants, including the cereals, remained inaccessible for genetic manipulation because of lack of effective transformation techniques. in this changed with the technology that overcame this limitation. michael fromm, a molecular biologist at the plant gene expression center, reported the stable transformation of corn using a high-speed gene gun. the method known as biolistics uses a "particle gun" to shoot metal particles coated with dna into cells. initially a gunpowder charge subsequently replaced by helium gas was used to accelerate the particles in the gun. there is a minimal disruption of tissue and the success rate has been extremely high for applications in several plant species. the technology rights are now owned by dupont. in some of the first of the field trials of the crops that would dominate the second half of the nineties began, including bt corn (with the bacillus thuriengenesis cry protein discussed in chapter three). in the fda declared that genetically engineered foods are "not inherently dangerous" and do not require special regulation. since , researchers have pinpointed and cloned several of the genes that make selected plants resistant to certain bacterial and fungal infections; some of these genes have been successfully inserted into crop plants that lack them. many more infection-resistant crops are expected in the near future, as scientists find more plant genes in nature that make plants resistant to pests. plant genes, however, are just a portion of the arsenal; microorganisms other than bt also are being mined for genes that could help plants fend off invaders that cause crop damage. the major milestone of the decade in crop biotechnology was approval of the first bioengineered crop plant in . it represented a double first not just of the first approved food crop but also of the first commercial validation of a technology which was to be surpassed later in the decade. that technology, antisense technology works because nucleic acids have a natural affinity for each other. when a gene coding for the target in the genome is introduced in the opposite orientation, the reverse rna strand anneals and effectively blocks expression of the enzyme. this technology was patented by calgene for plant applications and was the technology behind the famous flavr savr tomatoes. the first success for antisense in medicine was in when the u.s. food and drug administration gave the go-ahead to the cytomegalovirus (cmv) inhibitor fomivirsen, a phosphorothionate antiviral for the aids-related condition cmv retinitis making it the first drug belonging to isis, and the first antisense drug ever, to be approved. another technology, although not apparent at the time was behind the second approval and also the first and only successful to date in a commercial tree fruit biotech application. the former was a virus resistant squash the second the papaya ringspot resistant papaya. both owed their existence as much to historic experience as modern technology. genetically engineered virus-resistant strains of squash and cantaloupe, for example, would never have made it to farmers' fields if plant breeders in the 's had not noticed that plants infected with a mild strain of a virus do not succumb to more destructive strains of the same virus. that finding led plant pathologist roger beachy, then at washington university in saint louis, to wonder exactly how such "cross-protection" worked -did part of the virus prompt it? in collaboration with researchers at monsanto, beachy used an a. tumefaciens vector to insert into tomato plants a gene that produces one of the proteins that makes up the protein coat of the tobacco mosaic virus. he then inoculated these plants with the virus and was pleased to discover, as reported in , that the vast majority of plants did not succumb to the virus. eight years later, in , virus-resistant squash seeds created with beachy's method reached the market, to be followed soon by bioengineered virus-resistant seeds for cantaloupes, potatoes, and papayas. (breeders had already created virusresistant tomato seeds by using traditional techniques.) and the method of protection still remained a mystery when the first approvals were given in and . gene silencing was perceived initially as an unpredictable and inconvenient side effect of introducing transgenes into plants. it now seems that it is the consequence of accidentally triggering the plant's adaptive defense mechanism against viruses and transposable elements. this recently discovered mechanism, although mechanistically different, has a number of parallels with the immune system of mammals. how this system worked was not elucidated until later in the decade by a researcher who was seeking a very different holy grail -the black rose! rick jorgensen, at that time at dna plant technologies in oakland, ca and subsequently of, of the university of california davis attempted to overexpress the chalcone synthase gene by introducing a modified copy under a strong promoter.surprisingly he obtained white flowers, and many strange variegated purple and white variations in between. this was the first demonstration of what has come to be known as post-transcriptional gene silencing (ptgs). while initially it was considered a strange phenomenon limited to petunias and a few other plant species, it is now one of the hottest topics in molecular biology. rna interference (rnai) in animals and basal eukaryotes, quelling in fungi, and ptgs in plants are examples of a broad family of phenomena collectively called rna silencing (hannon ; plasterk ) . in addition to its occurrence in these species it has roles in viral defense (as demonstrated by beachy) and transposon silencing mechanisms among other things. perhaps most exciting, however, is the emerging use of ptgs and, in particular, rnai -ptgs initiated by the introduction of double-stranded rna (dsrna) -as a tool to knock out expression of specific genes in a variety of organisms. nineteen ninety one also heralded yet another first. the february , issue of science reported the patenting of "molecular scissors": the nobel-prize winning discovery of enzymatic rna, or "ribozymes," by thomas czech of the university of colorado. it was noted that the u.s. patent and trademark office had awarded an "unusually broad" patent for ribozymes. the patent is u.s. patent no. , , , claim of which reads as follows: "an enzymatic rna molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded rna in a separate rna molecule, and causing cleavage at said cleavage site by a transesterification reaction." although enzymes made of protein are the dominant form of biocatalyst in modern cells, there are at least eight natural rna enzymes, or ribozymes, that catalyze fundamental biological processes. one of which was yet another discovery by plant virologists, in this instance the hairpin ribozyme was discovered by george bruening at uc davis. the self-cleavage structure was originally called a paperclip, by the bruening laboratory which discovered the reactions. as mentioned in chapter , it is believed that these ribozymes might be the remnants of an ancient form of life that was guided entirely by rna. since a ribozyme is a catalytic rna molecule capable of cleaving itself and other target rnas it therefore can be useful as a control system for turning off genes or targeting viruses. the possibility of designing ribozymes to cleave any specific target rna has rendered them valuable tools in both basic research and therapeutic applications. in the therapeutics area, they have been exploited to target viral rnas in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. most notably, several ribozyme gene therapy protocols for hiv patients are already in phase trials. more recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. however, targeting ribozymes to the cellular compartment containing their target rnas has proved a challenge. at the other bookend of the decade in , samarsky et al. reported that a family of small rnas in the nucleolus (snornas) can readily transport ribozymes into this subcellular organelle. in addition to the already extensive panoply of rna entities yet another has potential for mischief. viroids are small, single-stranded, circular rnas containing - nucleotides arranged in a rod-like secondary structure and are the smallest pathogenic agents yet described. the smallest viroid characterized to date is rice yellow mottle sobemovirus (rymv), at nucleotides. in comparison, the genome of the smallest known viruses capable of causing an infection by themselves, the single-stranded circular dna of circoviruses, is around kilobases in size. the first viroid to be identified was the potato spindle tuber viroid (pstvd). some species have been identified to date. unlike the many satellite or defective interfering rnas associated with plant viruses, viroids replicate autonomously on inoculation of a susceptible host. the absence of a protein capsid and of detectable messenger rna activity implies that the information necessary for replication and pathogenesis resides within the unusual structure of the viroid genome. the replication mechanism actually involves interaction with rna polymerase ii, an enzyme normally associated with synthesis of messenger rna, and "rolling circle" synthesis of new rna. some viroids have ribozyme activity which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates. it has been proposed that viroids are "escaped introns". viroids are usually transmitted by seed or pollen. infected plants can show distorted growth. from its earliest years, biotechnology attracted interest outside scientific circles. initially the main focus of public interest was on the safety of recombinant dna technology, and of the possible risks of creating uncontrollable and harmful novel organisms (berg , ) . the debate on the deliberate release of genetically modified organisms, and on consumer products containing or comprising them, followed some years later (nas, ) . it is interesting to note that within the broad ethical tableau of potential issues within the science and products of biotechnology, the seemingly innocuous field of plant modification has been one of the major players of the 's. the success of agricultural biotechnology is heavily dependent on its acceptance by the public, and the regulatory framework in which the industry operates is also influenced by public opinion. as the focus for molecular biology research shifted from the basic pursuit of knowledge to the pursuit of lucrative applications, once again as in the previous two decades the specter of risk arose as the potential of new products and applications had to be evaluated outside the confines of a laboratory. however, the specter now became far more global as the implications of commercial applications brought not just worker safety into the loop but also, the environment, agricultural and industrial products and the safety and well being of all living things. beyond "deliberate" release, the rac guidelines were not designed to address these issues, so the matter moved into the realm of the federal agencies who had regulatory authority which could be interpreted to oversee biotechnology issues. this adaptation of oversight is very much a dynamic process as the various agencies wrestle with the task of applying existing regulations and developing new ones for oversight of this technology in transition. as the decade progressed focus shifted from basic biotic stress resistance to more complex modifications the next generation of plants will focus on value added traits in which valuable genes and metabolites will be identified and isolated, with some of the later compounds being produced in mass quantities for niche markets. two of the more promising markets are nutraceuticals or so-called "functional foods" and plants developed as bioreactors for the production of valuable proteins and compounds, a field known as plant molecular farming. developing plants with improved quality traits involves overcoming a variety of technical challenges inherent to metabolic engineering programs. both traditional plant breeding and biotechnology techniques are needed to produce plants carrying the desired quality traits. continuing improvements in molecular and genomic technologies are contributing to the acceleration of product development in this space. by the end of the decade in , applying nutritional genomics, della penna ( ) isolated a gene, which converts the lower activity precursors to the highest activity vitamin e compound, alpha-tocopherol. with this technology, the vitamin e content of arabidopsis seed oil has been increased nearly -fold and progress has been made to move the technology to crops such as soybean, maize, and canola. this has also been done for folates in rice. omega three fatty acids play a significant role in human health, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), which are present in the retina of the eye and cerebral cortex of the brain, respectively, are some of the most well documented from a clinical perspective. it is believed that epa and dha play an important role in the regulation of inflammatory immune reactions and blood pressure, treatment of conditions such as cardiovascular disease and cystic fibrosis, brain development in utero, and, in early postnatal life, the development of cognitive function. they are mainly found in fish oil and the supply is limited. by the end of the decade ursin ( ) had succeeded in engineering canola to produce these fatty acids. from a global perspective another value-added development had far greater impact both technologically and socio-economically. a team led by ingo potrykus ( ) engineered rice to produce pro-vitamin a, which is an essential micronutrient. widespread dietary deficiency of this vitamin in rice-eating asian countries, which predisposes children to diseases such as blindness and measles, has tragic consequences. improved vitamin a nutrition would alleviate serious health problems and, according to unicef, could also prevent up to two million infant deaths due to vitamin a deficiency. adoption of the next stage of gm crops may proceed more slowly, as the market confronts issues of how to determine price, share value, and adjust marketing and handling to accommodate specialized end-use characteristics. furthermore, competition from existing products will not evaporate. challenges that have accompanied gm crops with improved agronomic traits, such as the stalled regulatory processes in europe, will also affect adoption of nutritionally improved gm products. beyond all of this, credible scientific research is still needed to confirm the benefits of any particular food or component. for functional foods to deliver their potential public health benefits, consumers must have a clear understanding of, and a strong confidence level in, the scientific criteria that are used to document health effects and claims. because these decisions will require an understanding of plant biochemistry, mammalian physiology, and food chemistry, strong interdisciplinary collaborations will be needed among plant scientists, nutritionists, and food scientists to ensure a safe and healthful food supply. in addition to being a source of nutrition, plants have been a valuable wellspring of therapeutics for centuries. during the nineties, however, intensive research has focused on expanding this source through rdna biotechnology and essentially using plants and animals as living factories for the commercial production of vaccines, therapeutics and other valuable products such as industrial enzymes and biosynthetic feedstocks. possibilities in the medical field include a wide variety of compounds, ranging from edible vaccine antigens against hepatitis b and norwalk viruses (arntzen, ) and pseudomonas aeruginosa and staphylococcus aureus to vaccines against cancer and diabetes, enzymes, hormones, cytokines, interleukins, plasma proteins, and human alpha- -antitrypsin. thus, plant cells are capable of expressing a large variety of recombinant proteins and protein complexes. therapeutics produced in this way are termed plant made pharmaceuticals (pmps). and non-therapeutics are termed plant made industrial products (pmips) (newell-mcgloughlin, ) . the first reported results of successful human clinical trials with their transgenic plant-derived pharmaceuticals were published in . they were an edible vaccine against e. coli-induced diarrhea and a secretory monoclonal antibody directed against streptococcus mutans, for preventative immunotherapy to reduce incidence of dental caries. haq et al. ( ) reported the expression in potato plants of a vaccine against e. coli enterotoxin (etec) that provided an immune response against the toxin in mice. human clinical trials suggest that oral vaccination against either of the closely related enterotoxins of vibrio cholerae and e. coli induces production of antibodies that can neutralize the respective toxins by preventing them from binding to gut cells. similar results were found for norwalk virus oral vaccines in potatoes. for developing countries, the intention is to deliver them in bananas or tomatoes (newell-mcgloughlin, ) . plants are also faster, cheaper, more convenient and more efficient than the principal eukaryotic production system, namely chinese hamster ovary (cho) cells for the production of pharmaceuticals. hundreds of acres of protein-containing seeds could inexpensively double the production of a cho bioreactor factory. in addition, proteins can be expressed at the highest levels in the harvestable seed and plant-made proteins and enzymes formulated in seeds have been found to be extremely stable, reducing storage and shipping costs. pharming may also enable research on drugs that cannot currently be produced. for example, croptech in blacksburg, va., is investigating a protein that seems to be a very effective anticancer agent. the problem is that this protein is difficult to produce in mammalian cell culture systems as it inhibits cell growth. this should not be a problem in plants. furthermore, production size is flexible and easily adjustable to the needs of changing markets. making pharmaceuticals from plants is also a sustainable process, because the plants and crops used as raw materials are renewable. the system also has the potential to address problems associated with provision of vaccines to people in developing countries. products from these alternative sources do not require a so-called "cold chain" for refrigerated transport and storage. those being developed for oral delivery obviates the need for needles and aspectic conditions which often are a problem in those areas. apart from those specific applications where the plant system is optimum there are many other advantages to using plant production. many new pharmaceuticals based on recombinant proteins will receive regulatory approval from the united states food and drug administration (fda) in the next few years. as these therapeutics make their way through clinical trials and evaluation, the pharmaceutical industry faces a production capacity challenge. pharmaceutical discovery companies are exploring plant-based production to overcome capacity limitations, enable production of complex therapeutic proteins, and fully realize the commercial potential of their biopharmaceuticals (newell-mcgloughlin, ) . nineteen ninety also marked a major milestone in the animal biotech world when herman made his appearance on the world's stage. since the palmiter's mouse, transgenic technology has been applied to several species including agricultural species such as sheep, cattle, goats, pigs, rabbits, poultry, and fish. herman was the first transgenic bovine created by genpharm international, inc., in a laboratory in the netherlands at the early embryo stage. scientist's microinjected recently fertilized eggs with the gene coding for human lactoferrin. the scientists then cultured the cells in vitro to the embryo stage and transferred them to recipient cattle. lactoferrin, an iron-containing anti-bacterial protein is essential for infant growth. since cow's milk doesn't contain lactoferrin, infants must be fed from other sources that are rich in iron -formula or mother's milk (newell-mcgloughlin, ) . as herman was a boy he would be unable to provide the source, that would require the production of daughters which was not necessarily a straightforward process. the dutch parliments permission was required. in they finally approved a measure that permitted the world's first genetically engineered bull to reproduce. the leiden-based gene pharming proceeded to artificially inseminate cows with herman's sperm. with a promise that the protein, lactoferrin, would be the first in a new generation of inexpensive, high-tech drugs derived from cows' milk to treat complex diseases like aids and cancer. herman, became the father of at least eight female calves in , and each one inherited the gene for lactoferrin production. while their birth was initially greeted as a scientific advancement that could have far-reaching effects for children in developing nations, the levels of expression were too low to be commercially viable. by , herman, who likes to listen to rap music to relax, had sired calves and outlived them all. his offspring were all killed and destroyed after the end of the experiment, in line with dutch health legislation. herman was also slated for the abattoir, but the dutch public -proud of making history with herman -rose up in protest, especially after a television program screened footage showing the amiable bull licking a kitten. herman won a bill of clemency from parliament. however, instead of retirement on a comfortable bed of straw, listening to rap music, herman was pressed into service again. he now stars at a permanent biotech exhibit in naturalis, a natural history museum in the dutch city of leiden. after his death, he will be stuffed and remain in the museum in perpetuity (a fate similar to what awaited an even more famous mammalian first born later in the decade). the applications for transgenic animal research fall broadly into two distinct areas, namely medical and agricultural applications. the recent focus on developing animals as bioreactors to produce valuable proteins in their milk can be catalogued under both areas. underlying each of these, of course, is a more fundamental application, that is the use of those techniques as tools to ascertain the molecular and physiological bases of gene expression and animal development. this understanding can then lead to the creation of techniques to modify development pathways. in a european decision with rather more far-reaching implications than hermans sex life was made. the first european patent on a transgenic animal was issued for a transgenic mouse sensitive to carcinogens -harvard's "oncomouse". the oncomouse patent application was refused in europe in due primarily to an established ban on animal patenting. the application was revised to make narrower claims, and the patent was granted in . this has since been repeatedly challenged, primarily by groups objecting to the judgement that benefits to humans outweigh the suffering of the animal. currently, the patent applicant is awaiting protestors' responses to a series of possible modifications to the application. predictions are that agreement will not likely be forthcoming and that the legal wrangling will continue into the future. bringing animals into the field of controversy starting to swirl around gmos and preceding the latter's commercialization, was the approval by the fda of bovine somatotropin (bst) for increased milk production in dairy cows. the fda's center for veterinary medicine (cvm) regulates the manufacture and distribution of food additives and drugs that will be given to animals. biotechnology products are a growing proportion of the animal health products and feed components regulated by the cvm. the center requires that food products from treated animals must be shown to be safe for human consumption. applicants must show that the drug is effective and safe for the animal and that its manufacture will not affect the environment. they must also conduct geographically dispersed clinical trials under an investigational new animal drug application with the fda through which the agency controls the use of the unapproved compound in food animals. unlike within the eu, possible economic and social issues cannot be taken into consideration by the fda in the premarket drug approval process. under these considerations the safety and efficacy of rbst was determined. it was also determined that special labeling for milk derived from cows that had been treated with rbst is not required under fda food labeling laws because the use of rbst does not effect the quality or the composition of the milk. work with fish proceeded a pace throughout the decade. gene transfer techniques have been applied to a large number of aquatic organisms, both vertebrates and invertebrates. gene transfer experiments have targeted a wide variety of applications, including the study of gene structure and function, aquaculture production, and use in fisheries management programs. because fish have high fecundity, large eggs, and do not require reimplantation of embryos, transgenic fish prove attractive model systems in which to study gene expression. transgenic zebrafish have found utility in studies of embryogenesis, with expression of transgenes marking cell lineages or providing the basis for study of promoter or structural gene function. although not as widely used as zebrafish, transgenic medaka and goldfish have been used for studies of promoter function. this body of research indicates that transgenic fish provide useful models of gene expression, reliably modeling that in "higher" vertebrates. perhaps the largest number of gene transfer experiments address the goal of genetic improvement for aquaculture production purposes. the principal area of research has focused on growth performance, and initial transgenic growth hormone (gh) fish models have demonstrated accelerated and beneficial phenotypes. dna microinjection methods have propelled the many studies reported and have been most effective due to the relative ease of working with fish embryos. bob devlins' group in vancouver has demonstrated extraordinary growth rate in coho salmon which were transformed with a growth hormone from sockeye salmon. the transgenics achieve up to eleven times the size of their littermates within six months, reaching maturity in about half the time. interestingly this dramatic effect is only observed in feeding pins where the transgenics' ferocious appetites demands constant feeding. if the fish are left to their own devices and must forage for themselves, they appear to be out-competed by their smarter siblings. however most studies, such as those involving transgenic atlantic salmon and channel catfish, report growth rate enhancement on the order of - %. in addition to the species mentioned, gh genes also have been transferred into striped bass, tilapia, rainbow trout, gilthead sea bream, common carp, bluntnose bream, loach, and other fishes. shellfish also are subject to gene transfer toward the goal of intensifying aquaculture production. growth of abalone expressing an introduced gh gene is being evaluated; accelerated growth would prove a boon for culture of the slowgrowing mollusk. a marker gene was introduced successfully into giant prawn, demonstrating feasibility of gene transfer in crustaceans, and opening the possibility of work involving genes affecting economically important traits. in the ornamental fish sector of aquaculture, ongoing work addresses the development of fish with unique coloring or patterning. a number of companies have been founded to pursue commercialization of transgenics for aquaculture. as most aquaculture species mature at - years of age, most transgenic lines are still in development and have yet to be tested for performance under culture conditions. extending earlier research that identified methylfarnesoate (mf) as a juvenile hormone in crustaceans and determined its role in reproduction, researchers at the university of connecticut have developed technology to synchronize shrimp egg production and to increase the number and quality of eggs produced. females injected with mf are stimulated to produce eggs ready for fertilization. the procedure produces percent more eggs than the traditional crude method of removing the eyestalk gland. this will increase aquaculture efficiency. a number of experiments utilize gene transfer to develop genetic lines of potential utility in fisheries management. transfer of gh genes into northern pike, walleye, and largemouth bass are aimed at improving the growth rate of sport fishes. gene transfer has been posed as an option for reducing losses of rainbow trout to whirling disease, although suitable candidate genes have yet to be identified. richard winn of the university of georgia is developing transgenic killifish and medaka as biomonitors for environmental mutagens, which carry the bacteriophage phi x as a target for mutation detection. development of transgenic lines for fisheries management applications generally is at an early stage, often at the founder or f generation. broad application of transgenic aquatic organisms in aquaculture and fisheries management will depend on showing that particular gmos can be used in the environment both effectively and safely. although our base of knowledge for assessing ecological and genetic safety of aquatic gmos currently is limited, some early studies supported by the usda biotechnology risk assessment program have yielded results. data from outdoor pond-based studies on transgenic catfish reported by rex dunham of auburn university show that transgenic and non-transgenic individuals interbreed freely, that survival and growth of transgenics in unfed ponds was equal to or less than that of non-transgenics, and that predator avoidance is not affected by expression of the transgene. however, unquestionably the seminal event for animal biotech in the nineties was ian wilmut's landmark work using nuclear transfer technology to generate the lambs morag and megan reported in (from an embryonic cell nuclei) and the truly ground-breaking work of creating dolly from an adult somatic cell nucleus, reported in february, (wilmut, ) . wilmut and his colleagues at the roslin institute demonstrated for the first time with the birth of dolly the sheep that the nucleus of an adult somatic cell can be transferred to an enucleated egg to create cloned offspring. it had been assumed for some time that only embryonic cells could be used as the cellular source for nuclear transfer. this assumption was shattered with the birth of dolly. this example of cloning an animal using the nucleus of an adult cell was significant because it demonstrated the ability of egg cell cytoplasm to "reprogram" an adult nucleus. when cells differentiate, that is, evolve from primitive embryonic cells to functionally defined adult cells, they lose the ability to express most genes and can only express those genes necessary for the cell's differentiated function. for example, skin cells only express genes necessary for skin function, and brain cells only express genes necessary for brain function. the procedure that produced dolly demonstrated that egg cytoplasm is capable of reprogramming an adult differentiated cell (which is only expressing genes related to the function of that cell type). this reprogramming enables the differentiated cell nucleus to once again express all the genes required for the full embryonic development of the adult animal. since dolly was cloned, similar techniques have been used to clone a veritable zoo of vertebrates including mice, cattle, rabbitts, mules, horses, fish, cats and dogs from donor cells obtained from adult animals. these spectacular examples of cloning normal animals from fully differentiated adult cells demonstrate the universality of nuclear reprogramming although the next decade called some of these assumptions into question. this technology supports the production of genetically identical and genetically modified animals. thus, the successful "cloning" of dolly has captured the imagination of researchers around the world. this technological breakthrough should play a significant role in the development of new procedures for genetic engineering in a number of mammalian species. it should be noted that nuclear cloning, with nuclei obtained from either mammalian stem cells or differentiated "adult" cells, is an especially important development for transgenic animal research. as the decade reached its end the clones began arriving rapidly with specific advances made by a japanese group who used cumulus cells rather than fibroblasts to clone calves. they found that the percentage of cultured, reconstructed eggs that developed into blastocysts was % for cumulus cells and % for oviductal cells. these rates are higher than the % previously reported for transfer of nuclei from bovine fetal fibroblasts. following on the heels of dolly, polly and molly became the first genetically engineered transgenic sheep produced through nuclear transfer technology. polly and molly were engineered to produce human factor ix (for hemophiliacs) by transfer of nuclei from transfected fetal fibroblasts. until then germline competent transgenics had only been produced in mammalian species, other than mice, using dna microinjection. researchers at the university of massachusetts and advanced cell technology (worcester, ma) teamed up to produce genetically identical calves utilizing a strategy similar to that used to produce transgenic sheep. in contrast to the sheep cloning experiment, the bovine experiment involved the transfer of nuclei from an actively dividing population of cells. previous results from the sheep experiments suggested that induction of quiescence by serum starvation was required to reprogram the donor nuclei for successful nuclear transfer. the current bovine experiments indicate that this step may not be necessary. typically about embryos needed to be microinjected to obtain one transgenic cow, whereas nuclear transfer produced three transgenic calves from reconstructed embryos. this efficiency is comparable to the previous sheep research where six transgenic lambs were produced from reconstructed embryos. the ability to select for genetically modified cells in culture prior to nuclear transfer opens up the possibility of applying the powerful gene targeting techniques that have been developed for mice. one of the limitations of using primary cells, however, is their limited lifespan in culture. primary cell cultures such as the fetal fibroblasts can only undergo about population doublings before they senesce. this limited lifespan would preclude the ability to perform multiple rounds of selection. to overcome this problem of cell senescence, these researchers showed that fibroblast lifespan could be prolonged by nuclear transfer. a fetus, which was developed by nuclear transfer from genetically modified cells, could in turn be used to establish a second generation of fetal fibroblasts. these fetal cells would then be capable of undergoing another population doublings, which would provide sufficient time for selection of a second genetic modification. as noted, there is still some uncertainty over whether quiescent cells are required for successful nuclear transfer. induction into quiescence was originally thought to be necessary for successful nuclear reprogramming of the donor nucleus. however, cloned calves have been previously produced using non-quiescent fetal cells. furthermore, transfer of nuclei from sertoli and neuronal cells, which do not normally divide in adults, did not produce a liveborn mouse; whereas nuclei transferred from actively dividing cumulus cells did produce cloned mice. the fetuses used for establishing fetal cell lines in a tufts goat study were generated by mating nontransgenic females to a transgenic male containing a human antithrombin (at) iii transgene. this at transgene directs high level expression of human at into milk of lactating transgenic females. as expected, all three offspring derived from female fetal cells were females. one of these cloned goats was hormonally induced to lactate. this goat secreted . - . grams per liter of at in her milk. this level of at expression was comparable to that detected in the milk of transgenic goats from the same line obtained by natural breeding. the successful secretion of at in milk was a key result because it showed that a cloned animal could still synthesize and secrete a foreign protein at the expected level. it will be interesting to see if all three cloned goats secrete human at at the identical level. if so, then the goal of creating a herd identical transgenic animals, which secrete identical levels of an important pharmaceutical, would become a reality. no longer would variable production levels exist in subsequent generations due to genetically similar but not identical animals. this homogeneity would greatly aid in the production and processing of a uniform product. as nuclear transfer technology continues to be refined and applied to other species, it may eventually replace microinjection as the method of choice for generating transgenic livestock. nuclear transfer has a number of advantages: ) nuclear transfer is more efficient than microinjection at producing a transgenic animal, ) the fate of the integrated foreign dna can be examined prior to production of the transgenic animal, ) the sex of the transgenic animal can be predetermined, and ) the problem of mosaicism in first generation transgenic animals can be eliminated. dna microinjection has not been a very efficient mechanism to produce transgenic mammals. however, in november, , a team of wisconsin researchers reported a nearly % efficient method for generating transgenic cattle. the established method of cattle transgenes involves injecting dna into the pronuclei of a fertilized egg or zygote. in contrast, the wisconsin team injected a replication-defective retroviral vector into the perivitelline space of an unfertilized oocyte. the perivitelline space is the region between the oocyte membrane and the protective coating surrounding the oocyte known as the zona pellucida. in addition to es (embryonic stem) cells other sources of donor nuclei for nuclear transfer might be used such as embryonic cell lines, primordial germ cells, or spermatogonia to produce offspring. the utility of es cells or related methodologies to provide efficient and targeted in vivo genetic manipulations offer the prospects of profoundly useful animal models for biomedical, biological and agricultural applications. the road to such success has been most challenging, but recent developments in this field are extremely encouraging. with the may announcement of geron buying out ian wilmuts company roslin biomed, they declared it the dawn of an new era in biomedical research. geron's technologies for deriving transplantable cells from human pluripotent stem cells (hpscs) and extending their replicative capacity with telomerase was combined with the roslin institute nuclear transfer technology, the technology that produced dolly the cloned sheep. the goal was to produce transplantable, tissue-matched cells that provide extended therapeutic benefits without triggering immune rejection. such cells could be used to treat numerous major chronic degenerative diseases and conditions such as heart disease, stroke, parkinson's disease, alzheimer's disease, spinal cord injury, diabetes, osteoarthritis, bone marrow failure and burns. the stem cell is a unique and essential cell type found in animals. many kinds of stem cells are found in the body, with some more differentiated, or committed, to a particular function than others. in other words, when stem cells divide, some of the progeny mature into cells of a specific type (heart, muscle, blood, or brain cells), while others remain stem cells, ready to repair some of the everyday wear and tear undergone by our bodies. these stem cells are capable of continually reproducing themselves and serve to renew tissue throughout an individual's life. for example, they continually regenerate the lining of the gut, revitalize skin, and produce a whole range of blood cells. although the term "stem cell" commonly is used to refer to the cells within the adult organism that renew tissue (e.g., hematopoietic stem cells, a type of cell found in the blood), the most fundamental and extraordinary of the stem cells are found in the early-stage embryo. these embryonic stem (es) cells, unlike the more differentiated adult stem cells or other cell types, retain the special ability to develop into nearly any cell type. embryonic germ (eg) cells, which originate from the primordial reproductive cells of the developing fetus, have properties similar to es cells. it is the potentially unique versatility of the es and eg cells derived, respectively, from the early-stage embryo and cadaveric fetal tissue that presents such unusual scientific and therapeutic promise. indeed, scientists have long recognized the possibility of using such cells to generate more specialized cells or tissue, which could allow the generation of new cells to be used to treat injuries or diseases, such as alzheimer's disease, parkinson's disease, heart disease, and kidney failure. likewise, scientists regard these cells as an important -perhaps essential -means for understanding the earliest stages of human development and as an important tool in the development of life-saving drugs and cell-replacement therapies to treat disorders caused by early cell death or impairment. geron corporation and its collaborators at the university of wisconsin -madison (dr. james a. thomson) and johns hopkins university (dr. john d. gearhart) announced in november the first successful derivation of hpscs from two sources: (i) human embryonic stem (hes) cells derived from in vitro fertilized blastocysts (thomson ) and (ii) human embryonic germ (heg) cells derived from fetal material obtained from medically terminated pregnancies (shamblott et al. ) . although derived from different sources by different laboratory processes, these two cell types share certain characteristics but are referred to collectively as human pluripotent stem cells (hpscs). because hes cells have been more thoroughly studied, the characteristics of hpscs most closely describe the known properties of hes cells. stem cells represent a tremendous scientific advancement in two ways: first, as a tool to study developmental and cell biology; and second, as the starting point for therapies to develop medications to treat some of the most deadly diseases. the derivation of stem cells is fundamental to scientific research in understanding basic cellular and embryonic development. observing the development of stem cells as they differentiate into a number of cell types will enable scientists to better understand cellular processes and ways to repair cells when they malfunction. it also holds great potential to yield revolutionary treatments by transplanting new tissue to treat heart disease, atherosclerosis, blood disorders, diabetes, parkinson's, alzheimer's, stroke, spinal cord injuries, rheumatoid arthritis, and many other diseases. by using stem cells, scientists may be able to grow human skin cells to treat wounds and burns. and, it will aid the understanding of fertility disorders. many patient and scientific organizations recognize the vast potential of stem cell research. another possible therapeutic technique is the generation of "customized" stem cells. a researcher or doctor might need to develop a special cell line that contains the dna of a person living with a disease. by using a technique called "somatic cell nuclear transfer" the researcher can transfer a nucleus from the patient into an enucleated human egg cell. this reformed cell can then be activated to form a blastocyst from which customized stem cell lines can be derived to treat the individual from whom the nucleus was extracted. by using the individual's own dna, the stem cell line would be fully compatible and not be rejected by the person when the stem cells are transferred back to that person for the treatment. preliminary research is occurring on other approaches to produce pluripotent human es cells without the need to use human oocytes. human oocytes may not be available in quantities that would meet the needs of millions of potential patients. however, no peer-reviewed papers have yet appeared from which to judge whether animal oocytes could be used to manufacture "customized" human es cells and whether they can be developed on a realistic timescale. additional approaches under consideration include early experimental studies on the use of cytoplasmic-like media that might allow a viable approach in laboratory cultures. on a much longer timeline, it may be possible to use sophisticated genetic modification techniques to eliminate the major histocompatibility complexes and other cell-surface antigens from foreign cells to prepare master stem cell lines with less likelihood of rejection. this could lead to the development of a bank of universal donor cells or multiple types of compatible donor cells of invaluable benefit to treat all patients. however, the human immune system is sensitive to many minor histocompatibility complexes and immunosuppressive therapy carries life-threatening complications. stem cells also show great potential to aid research and development of new drugs and biologics. now, stem cells can serve as a source for normal human differentiated cells to be used for drug screening and testing, drug toxicology studies and to identify new drug targets. the ability to evaluate drug toxicity in human cell lines grown from stem cells could significantly reduce the need to test a drug's safety in animal models. there are other sources of stem cells, including stem cells that are found in blood. recent reports note the possible isolation of stem cells for the brain from the lining of the spinal cord. other reports indicate that some stem cells that were thought to have differentiated into one type of cell can also become other types of cells, in particular brain stem cells with the potential to become blood cells. however, since these reports reflect very early cellular research about which little is known, we should continue to pursue basic research on all types of stem cells. some religious leaders will advocate that researchers should only use certain types of stem cells. however, because human embryonic stem cells hold the potential to differentiate into any type of cell in the human body, no avenue of research should be foreclosed. rather, we must find ways to facilitate the pursuit of all research using stem cells while addressing the ethical concerns that may be raised. another seminal and intimately related event at the end of the nineties occurred in madison wisconsin. up until november of , isolating es cells in mammals other than mice proved elusive, but in a milestone paper in the november , issue of science, james a. thomson, ( ) a developmental biologist at uw-madison reported the first successful isolation, derivation and maintenance of a culture of human embryonic stem cells (hes cells). it is interesting to note that this leap was made from mouse to man. as thomson himself put it, these cells are different from all other human stem cells isolated to date and as the source of all cell types, they hold great promise for use in transplantation medicine, drug discovery and development, and the study of human developmental biology. the new century is rapidly exploiting this vision. when steve fodor was asked in "how do you really take the human genome sequence and transform it into knowledge?" he answered from affymetrix's perspective, it is a technology development task. he sees the colloquially named affychips being the equivalent of a cd-rom of the genome. they take information from the genome and write it down. the company has come a long way from the early days of venter's ests and less than robust algorithms as described earlier. one surprising fact unearthed by the newer more sophisticated generation of chips is that to percent of the non-repetitive dna is being expressed as accepted knowledge was that only . to percent of the genome would be expressed. since much of that sequence has no protein-coding capacity it is most likely coding for regulatory functions. in a parallel to astrophysics this is often referred to in common parlance as the "dark matter of the genome" and like dark matter for many it is the most exciting and challenging aspect of uncovering the occult genome. it could be, and most probably is, involved in regulatory functions, networks, or development. and like physical dark matter it may change our whole concept of what exactly a gene is or is not! since beadle and tatum's circumspect view of the protein world no longer holds true it adds a layer of complexity to organizing chip design. depending on which sequences are present in a particular transcript, you can, theoretically, design a set of probes to uniquely distinguish that variant. at the dna level itself there is much potential for looking at variants either expressed or not at a very basic level as a diagnostic system, but ultimately the real paydirt is the information that can be gained from looking at the consequence of non-coding sequence variation on the transcriptome itself. and fine tuning when this matters and when it is irrelevant as a predicative model is the auspices of the affymetrix spin-off perlegen. perlegen came into being in late to accelerate the development of high-resolution, whole genome scanning. and they have stuck to that purity of purpose. to paraphrase dragnet's sergeant joe friday, they focus on the facts of dna just the dna. perlegen owes its true genesis to the desire of one of its cofounders to use dna chips to help understand the dynamics underlying genetic diseases. brad margus' two sons have the rare disease "ataxia telangiectasia" (a-t). a-t is a progressive, neurodegenerative childhood disease that affects the brain and other body systems. the first signs of the disease, which include delayed development of motor skills, poor balance, and slurred speech, usually occur during the first decade of life. telangiectasias (tiny, red "spider" veins), which appear in the corners of the eyes or on the surface of the ears and cheeks, are characteristic of the disease, but are not always present. many individuals with a-t have a weakened immune system, making them susceptible to recurrent respiratory infections. about % of those with a-t develop cancer, most frequently acute lymphocytic leukemia or lymphoma suggesting that the sentinel competence of the immune system is compromised. having a focus so close to home is a powerful driver for any scientist. his co-founder david cox is a polymath pediatrician whose training in the latter informs his application of the former in the development of patient-centered tools. from that perspective, perlegen's stated mission is to collaborate with partners to rescue or improve drugs and to uncover the genetic bases of diseases. they have created a whole genome association approach that enables them to genotype millions of unique snps in thousands of cases and controls in a timeframe of months rather than years. as mentioned previously, snp (single nucleotide polymorphism) markers are preferred over microsatellite markers for association studies because of their abundance along the human genome, the low mutation rate, and accessibilities to high-throughput genotyping. since most diseases, and indeed responses to drug interventions, are the products of multiple genetic and environmental factors it is a challenge to develop discriminating diagnostics and, even more so, targetedtherapeutics. because mutations involved in complex diseases act probabilisticallythat is, the clinical outcome depends on many factors in addition to variation in the sequence of a single gene -the effect of any specific mutation is smaller. thus, such effects can only be revealed by searching for variants that differ in frequency among large numbers of patients and controls drawn from the general population. analysis of these snp patterns provides a powerful tool to help achieve this goal. although most bi-alleic snps are rare, it has been estimated that just over million common snps, each with a frequency of between and %, account for the bulk of the dna sequence difference between humans. such snps are present in the human genome once every base pairs or so. as is to be expected from linkage disequilibrium studies, alleles making up blocks of such snps in close physical proximity are often correlated, resulting in reduced genetic variability and defining a limited number of "snp haplotypes," each of which reflects descent from a single, ancient ancestral chromosome. in cox's group, using high level scanning with some old-fashioned somatic cell genetics, constructed the snp map of chromosome .the surprising findings were blocks of limited haplotype diversity in which more than % of a global human sample can typically be characterized by only three common haplotypes (interestingly enough the prevalence of each hapolytype in the examined population was in the ratio : : . ).from this the conclusion could be drawn that by comparing the frequency of genetic variants in unrelated cases and controls, genetic association studies could potentially identify specific haplotypes in the human genome that play important roles in disease, without need of knowledge of the history or source of the underlying sequence, which hypothesis they subsequently went on to prove. following cox et al. pioneering work on "blocking" chromosome into characteristic haplotypes, tien chen came to visit him from university of southern california and following the visit his group developed discriminating algorithms which took advantage of the fact that the haplotype block structure can be decomposed into large blocks with high linkage disequilibrium and relatively limited haplotype diversity, separated by short regions of low disequilibrium. one of the practical implications of this observation is as suggested by cox that only a small fraction of all the snps they refer to as "tag" snps can be chosen for mapping genes responsible for complex human diseases, which can significantly reduce genotyping effort, without much loss of power. they developed algorithms to partition haplotypes into blocks with the minimum number of tag snps for an entire chromosome. in they reported that they had developed an optimized suite of programs to analyze these block linkage disequilibrium patterns and to select the corresponding tag snps that will pick the minimum number of tags for the given criteria. in addition the updated suite allows haplotype data and genotype data from unrelated individuals and from general pedigrees to be analyzed. using an approach similar to richard michelmore's bulk segregant analysis in plants of more than a decade previously, perlegen subsequently made use of these snp haplotype and statistical probability tools to estimate total genetic variability of a particular complex trait coded for by many genes, with any single gene accounting for no more than a few percent of the overall variability of the trait. cox's group have determined that fewer than total individuals provide adequate power to identify genes accounting for only a few percent of the overall genetic variability of a complex trait, even using the very stringent significance levels required when testing large numbers of dna variants. from this it is possible to identify the set of major genetic risk factors contributing to the variability of a complex disease and/or treatment response. so, while a single genetic risk factor is not a good predictor of treatment outcome, the sum of a large fraction of risk factors contributing to a treatment response or common disease can be used to optimize personalized treatments without requiring knowledge of the underlying mechanisms of the disease.they feel that a saturating level of coverage is required to produce repeatable prediction of response to medication or predisposition to disease and that taking shortcuts will for the most part lead to incomplete, clinically-irrelevant results. in hinds et al. in science describe even more dramatic progresss. they describe a publicly available, genome-wide data set of . million common singlenucleotide polymorphisms (snps) that have been accurately genotyped in each of people from three population samples. a second public data set of more than million snps typed in each of people has been generated by the international haplotype map (hapmap) project. these two public data sets, combined with multiple new technologies for rapid and inexpensive snp genotyping, are paving the way for comprehensive association studies involving common human genetic variations. perlegen basically is taking to the next level fodor's stated reason for the creation of affymetrix, the belief that understanding the correlation between genetic variability and its role in health and disease would be the next step in the genomics revolution. the other interesting aspect of this level of coverage is, of course, the notion of discrete identifiable groups based on ethnicity, centers of origin and such breaks down and a spectrum of variation arises across all populations which makes the perlegen chip, at one level, a true unifier of humanity but at another adds a whole layer of complexity for hmos! at the turn of the century, this personalized chip approach to medicine received some validation at a simpler level in a closely related disease area to the one to which one fifth of a-t patients ultimately succumb when researchers at the whitehead institute used dna chips to distinguish different forms of leukemia based on patterns of gene expression in different populations of cells. moving cancer diagnosis away from visually based systems to such molecular based systems is a major goal of the national cancer institute. in the study, scientists used a dna chip to examine gene activity in bone marrow samples from patients with two different types of acute leukemia -acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all). then, using an algorithm, developed at the whitehead, they identified signature patterns that could distinguish the two types. when they cross-checked the diagnoses made by the chip against known differences in the two types of leukemia, they found that the chip method could automatically make the distinction between aml and all without previous knowledge of these classes. taking it to a level beyond where perlegen are initially aiming, eric lander, leader of the study said, mapping not only what is in the genome, but also what the things in the genome do, is the real secret to comprehending and ultimately curing cancer and other diseases. chips gained recognition on the world stage in when they played a key role in the search for the cause of severe acute respiratory syndrome (sars) and probably won a mcarthur genius award for their creator. ucsf assistant professor joseph derisi, already famous in the scientific community as the wunderkind originator of the online diy chip maker in pat brown's lab at stanford, built a gene microarray containing all known completely sequenced viruses ( , of them) and, using a robot arm that he also customized, in a three day period used it to classify a pathogen isolated from sars patients as a novel coronavirus. when a whole galaxy of dots lit up across the spectrum of known vertebrate cornoviruses derisis knew this was a new variant. interestingly the sequence had the hottest signal with avian infectious bronchitis virus. his work subsequently led epidemiologists to target the masked palm civet, a tree-dwelling animal with a weasel-like face and a catlike body as the probable primary host. the role that derisi's team at ucsf played in identifying a coronavirus as a suspected cause of sars came to the attention of the national media when cdc director dr. julie gerberding recognized joe in march , press conference and in when joe was honored with one of the coveted mcarthur genius awards. this and other tools arising from information gathered from the human genome sequence and complementary discoveries in cell and molecular biology, new tools such as gene-expression profiling, and proteomics analysis are converging to finally show that rapid robust diagnostics and "rational" drug design has a future in disease research. another virus that puts sars deaths in perspective benefitted from rational drug design at the turn of the century. influenza, or flu, is an acute respiratory infection caused by a variety of influenza viruses. each year, up to million americans develop the flu, with an average of about , being hospitalized and , to , people dying from influenza and its complications. the use of current influenza treatments has been limited due to a lack of activity against all influenza strains, adverse side effects, and rapid development of viral resistance. influenza costs the united states an annual $ . billion in physician visits, lost productivity and lost wages. and least we still dismiss it as a nuisance we are well to remember that the "spanish" influenza pandemic killed over million people in and , making it the worst infectious pandemic in history beating out even the more notorious black death of the middle ages. this fear has been rekindled as the dreaded h n (h for haemaglutenin and n for neuraminidase as described below) strain of bird flu has the potential to mutate and recognise homo sapiens as a desirable host. since rna viruses are notoriously faulty in their replication this accelerated evolutionary process gives then a distinct advantage when adapting to new environments and therefore finding more amenable hosts. although inactivated influenza vaccines are available, their efficacy is suboptimal partly because of their limited ability to elicit local iga and cytotoxic t cell responses. the choices of treatments and preventions for influenza hold much more promise in this millennium. clinical trials of cold-adapted live influenza vaccines now under way suggest that such vaccines are optimally attenuated, so that they will not cause influenza symptoms but will still induce protective immunity. aviron (mountain view, ca), biochem pharma (laval, quebec, canada), merck (whitehouse station, nj), chiron (emeryville, ca), and cortecs (london), all had influenza vaccines in the clinic at the turn of the century, with some of them given intra-nasally or orally. meanwhile, the team of gilead sciences (foster city, ca) and hoffmann-la roche (basel, switzerland) and also glaxowellcome (london) in put on the market neuraminidase inhibitors that block the replication of the influenza virus. gilead was one of the first biotechnology companies to come out with an anti-flu therapeutic. tamiflu™ (oseltamivir phosphate) was the first flu pill from this new class of drugs called neuraminidase inhibitors (ni) that are designed to be active against all common strains of the influenza virus. neuraminidase inhibitors block viral replication by targeting a site on one of the two main surface structures of the influenza virus, preventing the virus from infecting new cells. neuraminidase is found protruding from the surface of the two main types of influenza virus, type a and type b. it enables newly formed viral particles to travel from one cell to another in the body. tamiflu is designed to prevent all common strains of the influenza virus from replicating. the replication process is what contributes to the worsening of symptoms in a person infected with the influenza virus. by inactivating neuraminidase, viral replication is stopped, halting the influenza virus in its tracks. in marked contrast to the usual protracted process of clinical trials for new therapeutics, the road from conception to application for tamiflu was remarkably expeditious. in , gilead and hoffmann-la roche entered into a collaborative agreement to develop and market therapies that treat and prevent viral influenza. in , as gilead's worldwide development and marketing partner, roche led the final development of tamiflu, months after the first patient was dosed in clinical trials in april , roche and gilead announced the submission of a new drug application to the u.s. food and drug administration (fda) for the treatment of influenza. additionally, roche filed a marketing authorisation application (maa) in the european union under the centralized procedure in early may . six months later in october , gilead and roche announced that the fda approved tamiflu for the treatment of influenza a and b in adults. these accelerated efforts allowed tamiflu to reach the u.s. market in time for the - flu season. one of gilead's studies showed an increase in efficacy from % when the vaccine was used alone to % when the vaccine was used in conjunction with a neuraminidase inhibitor. outside of the u.s., tamiflu also has been approved for the treatment of influenza a and b in argentina, brazil, canada, mexico, peru and switzerland. regulatory review of the tamiflu maa by european authorities is ongoing. with the h n birdflu strain's relentless march (or rather flight) across asia, in through eastern europe to a french farmyard, an unwelcome stowaway on a winged migration, and no vaccine in sight, tamiflu, although untested for this species, seen as the last line of defense is now being horded and its patented production right's fought over like an alchemist's formula. tamiflu's main competitor, zanamivir marketed as relenza™ was one of a group of molecules developed by glaxowellcome and academic collaborators using structure-based drug design methods targeted, like tamiflu, at a region of the neuraminidase surface glycoprotein of influenza viruses that is highly conserved from strain to strain. glaxo filed for marketing approval for relenza in europe and canada. the food and drug administration's accelerated drug-approval timetable began to show results by , its evaluation of novartis's gleevec took just three months compared with the standard - months. another factor in improving biotherapeutic fortunes in the new century was the staggering profits of early successes. in , $ . billion of the $ . billion in revenue collected by genentech in south san francisco came from oncology products, mostly the monoclonal antibody-based drugs rituxan, used to treat non-hodgkin's lymphoma, and herceptin for breast cancer. in fact two of the first cancer drugs to use the new tools for 'rational' design herceptin and gleevec, a small-molecule chemotherapeutic for some forms of leukemia are proving successful, and others such as avastin (an anti-vascular endothelial growth factor) for colon cancer and erbitux are already following in their footsteps. gleevec led the way in exploiting signal-transduction pathways to treat cancer as it blocks a mutant form of tyrosine kinase (termed the philadelphia translocation recognized in 's) that can help to trigger out-of-control cell division. about % of biotech companies raising venture capital during the third quarter of listed cancer as their primary focus, according to online newsletter venturereporter. by according to the pharmaceutical research and manufacturers of america, medicines were in development for cancer up from in . another new avenue in cancer research is to combine drugs. wyeth's mylotarg, for instance, links an antibody to a chemotherapeutic, and homes in on cd receptors on acute myeloid leukemia cells. expertise in biochemistry, cell biology and immunology is required to develop such a drug. this trend has created some bright spots in cancer research and development, even though drug discovery in general has been adversely affected by mergers, a few high-profile failures and a shaky us economy in the early 's. as the millennium approached observers as diverse as microsoft's bill gates and president bill clinton predicted the st century wiould be the "biology century". by the many programs and initiatives underway at major research institutions and leading companies were already giving shape to this assertion. these initiatives have ushered in a new era of biological research anticipated to generate technological changes of the magnitude associated with the industrial revolution and the computerbased information revolution. complementary dna sequencing: expressed sequence tags and human genome project basic local alignment search tool high-tech herbal medicine: plant-based vaccines asilomar conference on recombinant dna molecules potential biohazards of recombinant dna molecules hugo: the human genome organization chimeric plant virus particles administered nasally or orally induce 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oral immunogenicity in mice rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain fv epitopes in tobacco plants generation and analysis of , human expressed sequence tags national academy of sciences. introduction of recombinant dna-engineered organisms into the environment: key issues functional foods and biopharmaceuticals: the next generation of the gm revolution in let them eat precaution biotechnology: a review of technological developments, publishers forfas vitamin-a and iron-enriched rices may hold key to combating blindness and malnutrition: a biotechnology advance french dna: trouble in purgatory genome: the autobiography of a species in chapters harper collins derivation of pluripotent stem cells from cultured human primordial germ cells production of correctly processed human serum albumin in transgenic plants high-yield production of a human therapeutic protein in tobacco chloroplasts the common thread: a story of science, politics, ethics and the human genome capillary gel electrophoresis for dna sequencing. laser-induced fluorescence detection with the sheath flow cuvette production of functional human alpha -antitrypsin by plant cell culture genetic modification of oils for improved health benefits, presentation at conference, dietary fatty acids and cardiovascular health: dietary recommendations for fatty acids: is there ample evidence? stable accumulation of aspergillus niger phytase in transgenic tobacco leaves antenatal maternal serum screening for down's syndrome: results of a demonstration project viable offspring derived from fetal and adult mammalian cells key: cord- -qt worsl authors: nan title: abstract date: - - journal: virchows arch doi: . /s - - -z sha: doc_id: cord_uid: qt worsl nan bladder cancer has served as one of the most important sources of information about the events that underlie the development of human solid malignancies. although "field effects" that alter the entire bladder mucosa appear to initiate disease, tumors develop along two distinct biological "tracks" referred to as papillary and non-papillary that present different challenges for clinical management. more recently, whole organ mapping combined with genomic platforms have identified a novel class of candidate tumor suppressors (forerunner genes) that localize near more familiar tumor suppressors but are disrupted at an earlier stage of cancer development. these studies suggested three steps for the involvement of the model tumor suppressor locus, rb , in tumor development. in the first step, one allele of forerunner (fr) gene and rb is inactivated by deletions. in the second step, homozygous inactivation of the fr genes is accomplished by hypermethylation or mutations. the inactivation of fr genes is associated with the initial clonal expansion of preneoplastic urothelial cells. in the third step, the contiguous tumor suppressor, rb , is inactivated by a mutation, which is associated with clonal evolution into carcinoma in situ progressing to invasive cancer. furthermore, we have discovered that aggressive muscle-invasive tumors express molecular markers characteristic of a developmental process known as "epithelial-to-mesenchymal transition." emerging evidence indicates that urothelial cancers contain subpopulations of tumor-initiating cells (cancer stem cells), but the phenotypes of these cells in different tumors may be heterogeneous, raising questions about whether or not the two major subtypes of cancer share a common precursor. pathology as a medical specialty has been around for about a century and a half, even though the interest in understanding disease, the focus of pathology as an academic discipline, has inspired physicians since the dawn of mankind. understanding disease remains the primary focus of pathology. in the practice of diagnostic pathology, this knowledge, notably through its morphological expression, is applied to the diagnosis of disease through the examination of cells and tissues. relatively new in this field is the notion of pathology as 'the science behind the cure'. this phrase, coined by the pathological society of great britain and ireland, refers to a widening of the scope of pathology: 'understanding disease' continues to be applied to diagnosis and classification, but now also to conceiving of new ways to treat and of new diagnostic tools assisting the physician in choosing the appropriate treatment. targeted therapy, the target being a molecular pathway involved in the pathogenesis of the lesion, along with biomarkers that are predictive for response to treatment, are in the forefront of pathology. targeted drugs that require 'companion' diagnostics, a combination also called 'theranostics', are reshaping the practice of pathology. it is this paradigm shift in pathology that makes it one of the most exciting medical specialties of today. i would love to be a resident in pathology again. the prospects for this mother of disciplines in modern medicine have never been as bright. is this entirely new? certainly, the molecular revolution with 'high-throughput' technologies, array diagnostics such as 'mammaprintr' and 'deep sequencing' to name just a few technologies we have had to become familiar with, have had and will continue to have in the years to come a profound impact on the way we practice pathology. but guiding the surgeon's hand or the pen of the oncologist in prescribing drugs has been a primary responsibility for pathologists ever since examination of cells and tissues was being applied to the practice of medicine. prognostic factors have interested pathologists long before the onset of the molecular revolution. grading and staging of neoplastic disease based upon morphological characteristics remains the mainstay of stratification of patient groups for treatment optimization. and predictive value is not limited to molecular markers: small cell carcinoma of the lung, to name just one example, will not be treated surgically, but chemotherapy is effective, even though as a rule not to the extent that the patient can be cured. we dispose of numerous morphological parameters that are prognostic and predictive. that pathological parameters have predictive value is therefore nothing new. this has become more so with the discovery that steroid hormone receptor expression predicts the response to anti-hormonal treatment and that her /neu amplification predicts the response to her /neu antibodies in breast cancer. it has become clear that the type of c-kit mutation in a gist predicts whether or not the patient will respond to a tyrosine-kinase inhibitor (imatinib), which dose of the drug would be appropriate or which second-generation inhibitor might be used. k-ras mutations will tell the oncologist that the patient will not respond to egfr inhibitors in the form of small molecules (gefatinib) or monoclonal antibodies (cituximab). the latter is also a striking example of the limitations of many of the tools that we dispose of: the k-ras mutation status will tell us who will not respond to the therapy, but not who will. based upon detailed knowledge of the molecular characteristics of many types of cancer, pharmaceutical companies are now developing a host of new drugs and more often than not are teaming up with pathologists to develop diagnostic tests that might predict which patient groups might profit from the new drug. this should not become a hype, the type of buzz that went around the h n pandemic which in hindsight was not justified by what happened in the field. let us remain modest. first of all, the number of diseases for which the combination of new drug and predictive molecular test exists remains limited. secondly, the impact of this new and promising approach to final outcome should keep us modest. for colorectal cancer, new therapies have pro-longed survival, but not cured more patients. thirdly, expectations that we will be able to predict at the level of the individual patient what the best therapy would be and how the disease will respond or progress is an illusion. 'individualized' medicine is still based upon studies on large patient groups with as outcome parameters that with a certain level of probability will indicate what might happen. a lot remains to be studied and developed. this implies also a new role for pathology. we should team up with clinical trials, using this context to conduct biomarker studies that can be implemented in daily practice. we should collaborate with drug developers to get to the desired 'companion' diagnostic tools. for pathology, exciting times indeed. breast lesions mimicking malignancy z. varga* *switzerland cell types arising normally in the breast can occasionally exhibit peculiar overgrowth or nuclear features mimicking malignant cells and thus representing potential diagnostic pitfalls. myoepithelial cell hyperplasia can reach an extensive mass and imitate lobular neoplasia or ductal carcinoma in situ with pagetoid spread. ductal epithelial cells with apocrine phenotype can appear, with highly worrisome nuclear features making the distinction between a high-grade ductal carcinoma in situ and a banal apocrine change at times quite challenging. the evidence of a preserved myoepithelial layer is a commonly accepted morphological criterion for noninvasive processes in the breast. the distinction between adenosis, highly differentiated ductal carcinomas and cancerisation of the lobules through in situ ductal carcinoma can be easily done by decorating myoepithelial cells in immunohistochemical reactions. an important exception to this rule is, however, the benign microglandular adenosis, which completely lacks the outer myoepithelial layer. intraductal epithelial proliferations can be quite florid in their extension. the question whether they represent atypical hyperplasia resp. ductal carcinoma in situ or appear simply within the scope of a florid intraductal hyperplasia occurs often in daily diagnostic service. the use of a distinct and technically reliable diagnostic panel (e-cadherin, basal cytokeratins, hormone receptors, her immunohistochemistry and in situ hybridisation, collagen iv reaction, ki- , s- and myoepithelial markers) can clear the situation in most of these difficult cases and thus can lead to the correct diagnosis. immunohistochemical differentiation of intraductal breast lesions w. p. olszewski* *center of oncology, dept. of pathology, warsaw, poland objective: immunopathological methods, particularly immunohistochemistry (ihc), are now routinely used in all invasive breast carcinoma cases as part of the evaluation of predictive factors (er, pgr, and her ). other ihcdetected antigens are used for the determination of invasiveness (e.g. p and sma), searching of primary (e.g. gcdfp and mammaglobin) or in scientific field as surrogates of genetic subtyping (er, pgr, her , egfr, lmwck, hmwck). increasing breast cancer screening on one hand and advanced surgical methods of biopsy (e.g. core biopsy, vacuum-assisted core biopsy) on the other produce a growing number of pathology material in which pathologists must diagnose on the basis of scarce tissue. most of these cases are noninvasive. however, the spectrum of possible therapies for noninvasive lesions includes observation on one side and mastectomy with radiotherapy on the other. method: therefore, determination of the exact nature of intraductal breast lesions has to be precise and sure. it generates the use of ihc antibodies in intraductal breast lesions. results: in some cases, results of such approaches create new entities. an example of such situation is differential diagnosis between clis and dcis. in a small number of cases, the interpretation of obtained stains (e-cadherin and ck be ) adds entities like positive hybrid lesion and negative hybrid lesion. conclusion: the aim of the lecture was the presentation of increasingly used ihc antibodies in intraductal breast lesions and its correlation with histological features of usual ductal hyperplasia, atypical ductal hyperplasia, lobular neoplasia, ductal carcinoma in situ, and columnar lesions (ccc, ccc+atypia, cch, cch+atypia) . the other topic is ihc expression of surrogates of genetic subtyping in carcinoma in situ. low-grade and high-grade breast cancer pathways i. ellis* *united kingdom tnm g. cserni* *hungary background: the tnm, i.e. the tumour node metastasis classification of malignant tumours, is about years old and was last updated in . although the oncology and pathology communities are expected to use this modified version from , the breast working group and the organizing committee has considered including a brief review of the topic in keeping with the educational aspects of the intercongress meetings of the european society of pathology. method: the lecture will briefly summarize the basic concepts of the tnm classification in general and will focus on the new aspects which are of interest to pathologists dealing with breast cancer specimens. some former proposals to modify the tnm will also be discussed, including the european institute of oncology proposal to abolish the present categories and replace them with coded prognostic data reporting and the replacement of the nodal categories based on the number of involved lymph nodes by categories based on the lymph node ratio (proportion of involved and examined nodes). results: na. conclusion: the tnm has been criticized for not providing enough prognostic information to guide treatment. however, it must be kept in mind that the tnm was not devised for making treatment decisions solely on the basis of the information it provides but for reporting the anatomic extent of disease in a categorical format. tnm still fulfils this aim. no system is perfect, and tnm is not an exception to this. should we use it, let us use it as correctly and uniformly as possible. wednesday, september , . - . , aula duża b gastrointestinal stromal tumours results: gist is defined as kit or pdgfra-driven mesenchymal tumor of the gi-tract, with a characteristic set of histologic features (spindle cell or epithelioid), and generally by immunohistochemical positivity for kit (cd ). dog /ano is a helpful additional marker for the positive identification of gist. current prognostication is based on mitotic activity, expressed as mitotic figures per high-power fields in an area of mm and on tumor size. different criteria are applied for gastric and intestinal gists because the latter, even with the same parameters, have a more malignant behavior. in the differential diagnosis of gist, one has to consider the following facts: wide histologic variation (especially in gastric gists), the fact that gists are the most common mesenchymal tumor in the gi tract and whole abdomen, and occurrence of gists virtually anywhere in the abdomen and rarely in distant peripheral metastatic sites. tumors that have to be considered in the differential diagnosis especially include true smooth muscle tumors, glomus tumor, gastrointestinal schwannoma, clear cell sarcoma, synovial sarcoma, desmoid, inflammatory fibroid polyp, inflammatory myofibroblastic tumor, and the newly described plexiform fibromyxoma and gastroblastoma. conclusion: detailed histologic examination supported by immunohistochemistry and kit/pdgfra mutation analysis will yield optimal diagnostic accuracy. clinical significance of kit and pdgfra mutations in gastrointestinal stromal tumours, inflammatory fibroid polyps and mucosal malignant melanomas j. lasota* *usa gists are the most common mesenchymal neoplasms of the gastrointestinal tract and represent a morphological and biological continuum from small, microscopically detected benign tumors to large sarcomas. kit or platelet-derived growth factor receptor α (pdgfra) oncogenic mutations are a typical molecular feature of gist. kit and pdgfra genes map to chromosome q and encode highly homologous transmembrane glycoproteins. activation of kit regulates important cell functions, including proliferation, apoptosis, chemotaxis and adhesion. imatinib mesylate that specifically inhibits abl, kit, and pdgfra receptor tyrosine kinases has been successfully used in the treatment of clinically advanced, unresectable, and metastatic gists. majority of kit exon mutants respond well to imatinib, while kit exon mutants are less sensitive to imatinib and require more aggressive treatment. also, tumors with the pdgfra asp val substitution, which corresponds to imatinib-resistant kit asp val mutation reported in human mastocytosis, are resistant to imatinib. although many patients benefit from imatinib treatment, resistance often develops due to secondary kit or pdgfra mutations or kit locus amplification. sunitinib mesylate, a multi-targeted inhibitor of kit, pdgfrs, vegfrs, flt , and ret receptor tyrosine kinases and other kinase inhibitors have been used for the treatment of imatinib-resistant gists. the type of secondary kit mutation can indicate response to sunitinib treatment. kit mutations were identified in mucosal mms and pdgfra mutations in gastric and intestinal ifps. although the presence of kit mutation does not necessarily correlate with kit expression in mm, an inhibition of kit receptor tyrosine kinase might still be beneficial in the treatment of selected cases. the histopathology of primary non-scarring and scarring alopecias may be complex. the reason for this is that often, the scalp biopsy provided is inadequate, and the clinical history and pattern of the alopecia are not provided to the dermatopathologist. increased diagnostic yield may be achieved by the combined use of transverse and vertical sections. this requires good communication between the dermatologist performing the biopsies, the dermatopathology laboratory and the dermatopathologist. common forms of scarring alopecias include the lymphocytic, (discoid lupus erythematosus, lichen planopilaris, central centrifugal cicatricial alopecia, pseudopelade of brocq), the neutrophilic, (folliculitis decalvans, dissecting folliculitis), and the mixed, (acne keloidalis) entities. among the non-scarring alopecias are androgenic alopecia, telogen effluvium, alopecia areata, trichotillomania and traction alopecia. in all cases of primary alopecia, a multi-team approach involving the dermatologist for adequate tissue sampling, the laboratory for appropriate laboratory processing and the dermatopathologist, together with pertinent clinical information affords the best opportunity of arriving at the correct diagnosis. graft versus host disease: an update d. massi* *university of florence, dept. of critical care medicine, italy our current understanding of histopathological features of cutaneous graft versus host disease (gvhd) is largely based on experience gained during decades of ablative hsct, in which day was regarded as the temporal cutoff for the separation of acute vs. chronic gvhd, and traditionally, acute and chronic gvhd were considered to be distinct histopathological entities. acute gvhd in the skin has been generally described as an interface dermatitis, with vacuolar basal alteration, dyskeratotic (apoptotic) keratinocytes in the epidermis and/or adnexal structures, scant dermal lymphoid infiltrate, and lymphocytic satellitosis. in contrast, chronic gvhd has commonly been reported as either lichenoid or sclerodermoid gvhd, characterized by homogenization of dermal collagen bundles, with little or no epidermal involvement. at variance with this dichotomic picture, histology of cutaneous gvhd following reduced intensity conditioning regimens is protean, displaying a variable constellation of histopathological alterations. these changes frequently blur the distinction between clear-cut clinical gvhd categories, as known in the classical ablative hsct setting. sampling errors and other technical issues may occur and may significantly affect the number of false negative cases. the utility of skin biopsies varies according to the pathologist's experience and to the prevalence of gvhd. clinicians' overall perception is that undertreatment of gvhd is much worse than overtreatment of patients without gvhd. therefore, early immunosuppressive therapy in patients with a skin eruption often precedes the results of the histopathological analysis. the pathogenesis and pathology of gvhd will be illustrated by elucidating the selective epithelial targets in the afferent stage and cellular response in the efferent stages. method: our practice in a large cancer center sees drug eruptions in the context of cancer treatment and must often be evaluated within a differential diagnosis with graft versus host disease on our bone marrow transplant service. results: with the advent of specific, targeted therapy for cancer, a new spectrum of interesting and unusual drug eruptions has emerged related to the mechanism of action of these agents. conclusion: this presentation will focus on the unique cutaneous effects arising in association with targeted therapy for cancer. wednesday, september , . - . , aula Średnia b background: the adult onset of nephrotic syndrome has been reported to be associated with underlying cancer in about % of cases. it is believed that the glomerular injury in many instances is due to the deposition of immune complexes in the glomerular capillaries. on the other hand, the course of illness in many cancer patients is complicated by infections, which may also be involved in the pathogenesis of glomerular injury. the malignancy is usually found simultaneously with, shortly before, or within a short time after the diagnosis of glomerulopathy. the glomerular lesion of paraneoplastic nephrotic syndrome usually presents as membranous nephropathy in patients with solid tumors, particularly adenocarcinomas of the lung and gastrointestinal tract. minimal change disease is strongly associated with hodgkin's lymphoma, and the most common lesions observed in patients with chronic lymphocytic leukemia are membranoproliferative glomerulopathy and membranous nephropathy. extracapillary crescentic glomerulopathy, iga nephropathy and focal segmental glomerulosclerosis are rarely associated with neoplasia. method: we present six cases of nephropathies associated with malignancies: lung, gastric and colon cancer, as well as hodgkin's lymphoma and chronic lymphocytic leukemia. all patients were over the age of and were presenting nephrotic syndrome. results: membranous glomerulopathy, minimal change disease and membranoproliferative glomerulopathy were established on the basis of light microscopy, immunofluorescence and electron microscopy. the renal lesions in paraneoplastic nephropathies did not differ from idiopathic forms of glomerulopathies. the clinical and morphologic spectrum of plasma cell dyscrasia continues to expand. while in the most advanced stage of this disease patients are diagnosed with overt multiple myeloma, at the opposite end of the spectrum, mgus is more typical. not infrequently, the underlying disease is clinically silent or can be associated with renal symptoms that prompt further investigation and thus underscore the role of renal pathologists in the early detection of the underlying process. while light chain cast nephropathy affecting distal tubules and light chain amyloid or non-amyloidotic lcdd are well recognized, in recent years, attention has focussed on the pathology affecting the proximal tubules and interstitium. proximal tubulopathy associated with an underlying plasma cell dyscrasia may be very subtle and associated with the formation of crystals or only with light chain restriction detectable in the lysomes within proximal tubules. interstitial nephritis may be associated with a rather nonspecific morphology, and only an enhanced index of suspicion may lead to the discovery of an associated light chain restriction. thus, renal pathologists are in an advantageous position to diagnose the underlying process at an early stage. therefore, it is critical to perform a full investigation of the kidney biopsy, including immunofluorescence studies that employ testing for immunoglobulin light chains, as well as electron microscopy. renal pathologists should be alerted particularly by biopsies that yield seemingly nonspecific or non-diagnostic results. investigations with congo red stain, careful evaluation of immunofluorescence studies, and electron microscopic analysis of organized and related deposits are necessary. non-hodgkin lymphomas and ptld in renal biopsies a. c. feller* *germany cytostatic drug and radiation-associated renal lesions h. regele* *austria malignant tumors might impair renal function in multiple ways. in addition to a direct impact of the tumor or its derivatives like light chains in the case of multiple myeloma, renal injury might also be a consequence of therapeutic interventions. certain cytostatic drugs (especially platin-based compounds), but also irradiation, are long known to cause renal tissue injury. the damage might affect both the tubulointerstitial space and the vasculature. in addition, it was shown that recently introduced biological agents might lead to additional patterns of drug-induced injury. blocking the action of vegf in order to interfere with tumor-induced neoangiogenesis might for example cause renal thrombotic microangiopathy. in my talk, i will discuss the histological patterns of injury caused by the different treatment modalities and the underlying pathogenic mechanisms. the knowledge of these mechanisms might help prevent renal damage. background: the morphological presentation of lung problems in infants and children is not always pathognomonic for a specific diagnosis. basic patterns are not always easily recognized and may be different from the adult situation, even with the same classifying diagnosis. method: as lung neoplasms are seldom seen in children, this overview will focus on non-neoplastic lung disease. results: a main category is interstitial lung disease. the main adult patterns of interstitial disease can also be observed in children, although uip, dip and nsip are rather rare and not always with similar aetiology (e.g. desquamative interstitial pneumonia, associated with smoking in adults). the distribution of the interstitial changes is in those cases similar to adults, but morphological presentation may be different because of a different lung structure. some interstitial patterns are specific for childhood: for example, chronic pneumonitis of infancy, pulmonary interstitial glycogenosis (formerly cellular interstitial pneumonitis) and neuroendocrine cell hyperplasia of infancy, but even so, these are not always easy to discern form the other patterns. if a (wedge) lung biopsy is taken at late stage or only from the most severe abnormalities, a characteristic pattern may be obscured by nonspecific common final pathway changes like fibrosis. conclusion: all diffuse lung diseases in infancy can be caused by many etiologic factors. this means that although there are several hallmarks that can aid in making a proper pathological diagnosis, similar as in adults, proper clinical and radiological information is essential. objective: tobacco and cigarettes are implicated in multisystemic carcinogenesis due to carcinogenic components that induce maintained inflammation, endocrine deregulation and genetic alterations in continuum. as ambience influence acts through a multifactorial behaviour gathering smoking, obesity and dietary mistakes, being sedentary, alcoholism, sexual promiscuity, toxic addition and general pollution, the isolated influence of smoking is reinforced. smoking-related non-tumoral lung diseases comprise copd, rb-ild, dip, acif/ibip, hx (langerhans cell histiocytosis), sarcoidosis (not consistent), eosinophilic lung, constrictive and lymphocytic bronchiolitis, emphysema together with sub-pleural and interstitial fibrosis uip-like or nsip-like, as well as possible combinations of all these diseases and patterns. smoking also increases or exacerbates infections (tp and other bacterial pneumonias, varicella pneumonia and influenza), pulmonary hemorrhage and pneumothorax and vascular walls fibrosis. conclusion: as smoking-related interstitial lung disease (srild) has been proposed to encompass clinical and radiological coordination, it means that molecular pathology achieves its role to define therapy as it is being tried in cancer. despite observed morphology, it is possible to localize the defence response along the bronchial-bronchiolar-alveolar pathway as epithelial and mesenchymal cells develop particular ways of adaptation, either by emt and met. as inflammation stands through fibroblasts, cellular mtor and macrophage activation, also epithelial hyperplasia and metaplasia, install both at bronchial and pulmonary stem cells pool. smoking-related lung diseases are not consistent with a spectrum but rather with expertise to report delicate inflammatory changes in morphology that diverge to interstitial (and vascular) incapacities and/or to pre-neoplastic epithelial lesions and bronchial-pulmonary carcinoma. pitfalls in interstitial pneumonias h. popper* *medical university of graz, institute of pathology, austria interstitial pneumonias can be classified into uip, dip, nsip, op, and dad. whereas dad as an acute interstitial pneumonia is easily separated and diagnosed, due to the presence of alveolar damage and hyaline membranes, the other entities might cause problems in diagnosing. the most important distinction is between uip and the other entities because uip is a rapid progressive and deteriorating disease with a high mortality rate. usual interstitial pneumonia is histologically characterized by the presence of fibroblastic foci, cystic changes called honeycombing, areas of fibrosis and scars, and a temporal heterogeneity, which results in areas of early inflammatory changes, late changes, and also areas of uninvolved lung parenchyma. the disease usually starts from the lung periphery in a patchy distribution pattern. the lower lobes are usually more affected than the upper lobes. the disease starts with tiny lesions where the surface epithelium is destroyed. underneath myofibroblasts are proliferating and the stroma is changed into a myxoid stroma composed of acidic mucopolysaccharides. these lesions are called fibroblastic focus. this repair process is accompanied by a regeneration of pneumocytes, which usually undergo metaplastic changes (bronchiolization, cuboidal metaplasia, stem cell proliferation). finally, this process results in fibrosis. several fibrotic areas might finally confluent into a scar tissue. fibroblastic foci are not only seen in alveolar septa but also in bronchioles and small bronchi. scarring in these airways results in the obstruction of the airways, and the periphery of these airways undergoes a cystic dilation, accompanied by metaplasia of the epithelium; again, bronchiolization and metaplasia, even squamous metaplasia, can occur. mucus produced by the epithelium cannot escape, and therefore, the cystic spaces increase in size, being finally visible at ct magnification. these cystic air spaces form the honeycomb lesions. in uip, fibroblastic foci can be seen in the different stages of development: early stages with lots of myxoid matrix, immature collagen, and many proliferating myofibroblasts and old stages with lots of mature collagen and fibrocytes. honeycomb lesions may present in a florid stage where some fibroblastic foci can be seen within the cysts, or older lesions with lots of mucus, or old lesions with secondary bacterial pneumonia overlying uip. also, the scars can be old, devoid of inflammatory infiltrates, or younger with scattered fibrocytes and lymphocytes. this together forms the temporal heterogeneity of uip. fibroblastic foci are often used as the dominant and even sole diagnostic criterion for the diagnosis of uip. this can result in an incorrect diagnosis since other interstitial diseases can also present with fibroblastic foci. in the presentation, we will review several cases presenting with fibroblastic foci, which can closely mimic and might give rise to an incorrect diagnosis of uip. pitfalls in drug-induced lung pathology b. murer* *italy the role of pathologists in translational research for patients and industry m. dietel* *universitätsmedizin charité, berlin, germany background: due to continuous technical developments and new insights into the complexity of many diseases, e.g. cancer, molecular pathology is rapidly growing gaining center stage in the clinical management of tumors and pharmaceutical development of new anti-cancer drugs. activated signaling components are the targets for the newly developed inhibitors, e.g. small molecules (gefitinib, erlotinib) and therapeutic antibodies (panitumumab, cetuximab). however, the application of the compounds in clinical trials has revealed promising results only when predictive procedures have been available for determining which patients will benefit from targeting therapy, so-called eligibility or predictive tests, e.g. her in breast cancer, kras and egfr mutations in colorectal cancer and non-small cell lung cancer. for the pharmaceutic industry, predictive tissue-based assays are of increasing importance in the development of new targeted drugs. fda and emea stressed this issue several times during the process of approval, and pathology based analyses will become an essential factor in drug development. conclusion: for pathology, the situation to become a partner in the clinical decision, which drug shall be given to the patient and which assay can be made available for a new, is a chance and a challenge in one. the issues to be solved are: ( ) morphology, in particular immunohistochemistry and in situ hybridization, must become measurable, e.g. by virtual microscopy. ( ) the molecular assays must be done absolutely reliably and be reproducible. ( ) quality control, i.e. ring trials/round robin tests, must be passed by all active labs. if these challenges (and some others) are met, molecular pathology is facing an excellent development. the role of biobanking and translational research for the pathologist f. carneiro* *ipatimup, porto, portugal among biobanking initiatives, tumour banks play a pivotal role in biomedical research. the general aim of a tumour bank is to acquire neoplastic and control non-neoplastic samples in standardized conditions for research (basic, clinical or translational) . a tumour bank is a vital new resource for cancer research providing high-quality, wellcharacterized tissue. it is possible for pathologists to collect fresh tissue prospectively during their routine dissection procedures. in this way, the specimens can be optimally sampled and stored for both diagnosis and research purposes. ideally, specimens are sampled immediately after surgery, prior to fixation, to ensure optimal preservation of proteins and nucleic acids. retrospective collection of tumour tissue for study and banking purposes is feasible also because in most countries, pathology laboratories have been legally obliged to file, for at least some years, the formalin-fixed and paraffin-embedded samples that were analyzed. over the last decade, tumour banks acquired a pivotal role in translational research in the field of oncology, providing tools for the evaluation of new predictive factors; evaluation of the value of a known target in a new entity; search for new therapeutic targets; validation of new diagnostic markers; and implementation of new diagnostic procedures, namely development of tissue-based diagnostic tests for guidance of therapy with new drugs introduced in clinical practice. in this scenario, it is a priority to emphasize the central role that pathologists play in translational research, specifically in tumor banking, by the establishment of a bridge between clinicians and basic researchers. limits and perspectives for standardization of tissue processing g. bussolati* *italy the paper by pupo and colleagues is a well-conducted study on the role of the gasotransmitter hydrogen sulphide in angiogenesis. the increased effect of hydrogen sulphide in tumor-derived endothelial cells and its possible effect as a vegf second messenger are of interest in the field of tumor angiogenesis. criticisms: (a) it would be important to show the specificity and non-toxicity of hydrogen sulphide synthesis inhibitor on endothelial cells. ( ) experiments using the cystathionine lyase inhibitor alone on tec proliferation and cytotoxicity are required. ( ) standardization and controls of methods for molecular analysis in archive tissues g. hoefler* *medical university of graz, institute of pathology, austria background: formalin-fixed paraffin-embedded (ffpe) samples represent the vast majority of tissue specimens available from tissue archives, available routine diagnostics, as well as research purposes. these tissues vary among institutions with respect to pre-fixation time, formalin fixation time, concentration and buffer systems used. therefore, it is of utmost importance to standardize the methods for molecular analyses to obtain consistent and comparable results. method: the first and maybe the most important step is the isolation of nucleic acids (dna, rna) from ffpe samples. in a recent multi-centric study performed by european laboratories of the impacts group, the results obtained by different laboratories varied significantly, even when the same commercial kit was used. the dna extraction protocols used by the laboratories ranged from homemade protocols with and without purification steps to commercially available kits. the extractions were performed using the same ffpe specimens. results: for array applications or tests that require accurately determined dna input, silica-based adsorption columns for dna recovery are recommended. for rna extractions, the best results were obtained for chromatography column-based commercial kits, with respect to quantity and quality. quality testing resulted in the successful amplification of -to -bp pcr products from most tissues. modifications of the protocol, especially with regard to proteinase-k digestion, led to significant improvements, also for the performance of commercial kits. the results emphasize the need for standardization and control of methods for molecular analysis in archive tissues to allow the generation of valid and comparable results in both diagnostic and in research settings. approaches to the development of a european network of archive tissues g. stanta* *university of trieste, a.c.a.d.e.m., italy ffpe tissues taken from patients are stored, sometimes for a very long time, in the pathology archives. with this material, it is possible to do very important translational research. any strategy is possible, such as that of large number analysis or rare entity collections. there is the necessity to transform our pathology biorepositories into a biobanking network. this can be done through a european organization that could be developed within the esp and starting from the "impacts" groups, which gained a large experience in molecular analysis validation and standardization of this kind of tissues. a european archive tissue biobank network depends on the willingness of the pathologists to participate, considering that this can significantly improve the diffusion of molecular methods, translational research and consequently acceleration of clinical application of molecular methods among pathologists. this can be done by a voluntary and collaborative participation of pathologists in specific projects, as scientific collaborators. the pathology archives will always be clinical biorepositories, and their function is going to change into a biobank function after accepting participation in a specific project, with the anonymization of the personal data of the patients. the networking system is based on low-cost activities with an easy governance and management of the biobanking system and with guarantees of protection of human material and data. qualification, education and training will be implemented by the network organization and with the collaboration of the esp. with cisplatin mg/m every weeks for four cycles, followed by mastectomy and then conventional chemotherapy. surgery occurred weeks after the final dose of cisplatin. the excised breast tissue and lymph nodes were examined for the presence of residual disease. pathologic complete response was determined by a review of surgical specimens. results: forty patients were enrolled in the study. twentyfour patients had tumors of > cm and patients had positive lymph nodes at diagnosis. thirty-seven patients completed four cycles of cisplatin and three patients completed two cycles. clinical complete response was observed in patients ( %). pathologic complete response was observed in patients ( %). conclusion: platinum-based chemotherapy is effective in a high proportion of patients with brca -associated breast cancers. clinical trials are warranted to determine the optimum treatment for this subgroup of breast cancer patients. clinical outcome of ovarian cancer in respect to polymorphism of low-penetrance genes e. grzybowska*, k. tcza, j. pamua-piat, s. jdru, e. telka, z. kosza, b. zema *centre of oncology-msc memoria, dept. of molecular biology, gliwice, poland objective: we wanted to analyze the influence of modifying genetic factors on the risk of brca(+) and brca(−) breast and ovary cancers and on clinical parameters as the age of onset and survival. method: the study includes three hundred forty-eight anonymous healthy women (control group), persons with ovary cancer, persons with breast and ovary cancer (case group). the patients under study developed breast cancer prior to ovary cancer. rflp and asa pcr were used to analyze mutations in brca genes and polymorphisms in pgr, mdr and tp genes. results: the presence of allele t of pgr significantly decreased the risk of second malignancy (breast cancer; or= . , p= . ). heterozygote ct of mdr gene c t and genotypes ag and at of g t/a were at lower risk of developing ovary cancer. genotype ct of c t polymorphism had protective effect against developing the second malignancy, while allele tt increased the risk of breast cancer. carriers of allele c for tp arg pro polymorphism had a significantly lower risk of developing ovarian cancer (or= . , p= . ) the best survival was found for patients carrying germline mutation in brca gene; the worst survival was observed for the group of patients with breast and ovary cancer and without germline mutation in brca gene. alleles of tp arg pro significantly modified the survival of the group of patients who were diagnosed with breast cancer prior to ovarian carcinoma. the worst survival was connected with rare allele c. conclusion: polymorphic variants of pgr, mdr and tp genes under study modified more the clinical course and the risk of second malignancy than the risk of developing ovary cancer alone. dna testing for high risk of cancers b. górski* *poland genetics of colorectal cancers r. scott* *discipline of medical genetics, school of biomedical sciences, newcastle, australia the study of rare inherited conditions with high penetrance that predispose to colorectal cancer has significantly advanced our knowledge of the genetic basis of disease and represents a paradigm that has served to better understand the molecular mechanisms that underlie the development of disease in persons who have no apparent genetic predisposition. inherited predispositions to colorectal cancer can be divided into essentially two groups: those that present with a premalignant phenotype (such as familial adenomatous polyposis (fap) and the hamartomatous polyposis syndromes) and those like hereditary non-polyposis colorectal cancer, lynch syndrome and muir-torre syndrome. for predispositions where the underlying genetic basis of the disease is known, much has been learned about disease penetrance and the expected spectrum of disease as increasing numbers of mutation carriers are characterized. this has lead to the reclassification of some defined syndromes as a result of the inherited predisposition being identical. improved knowledge about disease penetrance has also resulted in a number of questions being raised with respect to what factors influence disease expression in patients known to harbour a mutation in a colorectal cancer susceptibility gene. of particular interest are additional genetic factors (termed modifier genes) that have been correlated with disease manifestation. more recently, the focus of attention has shifted from familial aggregations of disease where there is a well-defined inherited component to population studies aimed at identifying common low-penetrance disease alleles that are associated with small affect sizes. these studies are significant not only because they are identifying genetic factors associated with colorectal cancer risk but also potential modifier genes that are important in modulating disease expression in persons who harbour a genetic predisposition. eosinophil functions have been associated for a long time with effector activity in adaptive immune responses during parasitic infections and inflammatory processes in allergic manifestations as well as in mucosal responses. the production by eosinophils of a vast array of cytokines as well as their expression of innate receptors and mediators confer to eosinophils a unique contribution both in inflammatory and adaptive responses but also in immunoregulation and innate immunity. eosinophils are customary inhabitants of the gastrointestinal tract, except for the oesophagus. beneficial function of intestinal eosinophils was their ability to defend host against helminths. however, eosinophil accumulation in the gastrointestinal tract is a common feature of numerous gastrointestinal disorders, including inflammatory bowel diseases, intestinal vasculitis, gastroesophageal reflux disease and food allergy. primary eosinophilic gastrointestinal disorders, defined as disorders that selectively affect the gastrointestinal tract with eosinophil-rich inflammation of unknown etiology, include eosinophilic esophagitis, eosinophilic gastroenteritis and eosinophilic colitis and are occurring with increasing frequency. decrease in the rate of parasitic infections in the developed world is associated with a rise in atopic/ allergic disorders and increased hypersensitivity responses to allergens and may represent a driving factor towards the recruitment and activation of gut eosinophils in those disorders. although the pathogenesis of those disorders is still poorly understood, new findings on gastrointestinal eosinophil proliferation and migration mechanisms involving il- and eotaxins provide a rationale for specific disease therapy. the present review will summarize knowledge on the physiology of gut eosinophils and will illustrate some aspects of eosinophilic disorders of the gastrointestinal tract. gastro-oesophageal reflux disease (gerd) and eosinophilic oesophagitis (ee) are the two main non-tumoral diseases involving the oesophagus and can develop in both children and adults. the former, the more frequent, is present in up to % of the western population. it is the consequence of a massive flow of gastric contents back into the oesophagus, leading to symptoms or organ damage. eosinophilic oesophagitis, first reported in and defined as a specific clinicopathological entity in , is characterized by eosinophilic infiltration of the oesophagus. its cause or causes are unknown despite its frequent association with an allergic setting. in , a consensus meeting of the american gastroenterological association institute and the north american society for paediatric gastroenterology, hepatology and nutrition recommended diagnostic criteria for ee. despite this increased knowledge of both pathologies, gerd and ee present considerable clinical and pathological overlap. their distinction remains, however, crucial as their clinical outcome and treatment modalities are quite different. the pathologist has to be aware that the diagnosis cannot be based solely on pathological features. the aim of this presentation was to provide pathologist both clinical and histological diagnostic clues in order to propose the most accurate diagnosis and treatment. eosinophilic enterocolitis a. driessen* *the netherlands eosinophilic gastro-enterocolitis, consisting of eosinophilic gastroenteritis (eg) and eosinophilic colitis (ec), belongs to eosinophilic gastrointestinal disorders whose diagnosis is based on gastrointestinal symptoms, an eosinophilic infiltration in the gut and the exclusion of secondary causes of eosinophilia (collins mh , furuta gt , shifflet a . eg and ec are both rare entities. eg is a predominantly benign disease occurring at any age (peak incidence, third-fifth decade). it may affect any part of the gastrointestinal tract, most commonly the stomach ( - %) and small intestine ( - %). ec is more uncommon, involving the colon and less frequently the rectum. symptoms vary in function of the affected layer of the wall: mucosa ( - %): abdominal pain, diarrhea; muscular layer ( - %): intestinal obstruction; serosa ( - %): ascites. the ethiopathogenesis is not fully clear, but is sometimes related to a food allergy. diagnosis requires a panendoscopy with biopsy. histologic examination may, however, be hampered by the patchy distribution of the mucosal inflammation and the presence of a normal mucosa in association with muscular/serosal involvement. in this case, a full-thickness biopsy may be necessary for diagnosis. microscopic features are the presence of numerous eosinophils in the lamina propria, extending into the epithelium (crypt abscesses) and the submucosa, associated with architectural abnormalities. in mural or serosal eg/ec, eosinophils are predominantly situated in the muscular or peritoneal layer. differential diagnosis includes other disorders with eosinophilia, e.g. inflammatory bowel disease, celiac disease, parasitic infection, vasculitis. several treatment modalities have been described in literature, such as restriction diets, corticosteroids or antihistamines. surgery is restricted to the resection of stenosed segments in mural enterocolitis. systemic eosinophilic disorders and the gi tract a. hoorens* *uz brussel, dept. of pathology, belgium secondary eosinophilic disorders, including infectious, inflammatory, hypersensitivity and neoplastic illnesses, always require exclusion before making the diagnosis of eosinophilic oesophagitis/gastroenteritis/colitis, particularly in case of peripheral blood eosinophilia. parasite infections are well known to present with eosinophilia of the gastrointestinal mucosa. a drug-induced aetiology should also always be considered. gastrointestinal eosinophils may be a feature of connective tissue disease, especially scleroderma, and can accompany vasculitis in polyarteritis nodosa and churg-strauss syndrome. with very pronounced peripheral eosinophilia, hypereosinophilic syndrome (hes) with gastrointestinal involvement, clonal eosinophilia and lymphocytic variant hypereosinophilia should be considered. hes is defined as eosinophilia (≥ . × /l) for at least months, no known cause of eosinophilia, and evidence of organ involvement. the gastrointestinal tract is affected in %. when only the digestive tract is involved, it may prove difficult to distinguish hes and eosinophilic gastroenteritis. involvement of the intestinal tract in hes has been associated with limited prognosis and, in some, a fatal outcome. eosinophilic infiltration of the gi tract in hes should be distinguished from eosinophilic infiltration of the gi tract in lymphocytic variant hypereosinophilia where eosinophilia is associated with phenotypically abnormal and/or clonal t lymphocytes. clonal eosinophilia is characterized by neo-plastic proliferation of eosinophils as part of an underlying myeloid malignancy and can accompany any one of the myeloid malignancies. two distinct subcategories of clonal eosinophilia are recognized: chronic eosinophilic leukaemia, nos and myeloid/lymphoid neoplasms with abnormalities involving pdgfra/pdgfrb or fgfr . accurate diagnosis of all these conditions requires the correlation of endoscopic and biopsy findings together with a careful clinical examination. gi eosinophilia in paediatrics m. walker* *united kingdom eosinophils are powerful innate immune cells home to the gastrointestinal tract and play a major role in both host immunity to luminal pathogens and maintenance of homeostasis of intestinal epithelium in the normal gastrointestinal tract (git) . normal numbers at different git sites are defined in children. however, if in excess, eosinophils may play a key role in the pathogenesis of disease of the git, including primary eosinophilic gastrointestinal disease (egids). data from the world wide web-based registry of egids show that these have a strong genetic and allergic component, % having coexistent atopic disease, % food sensitisation and % with a family member with similar disorders. the most studied egid in children is eosinophilic oesophagitis; symptoms include feeding intolerance and gerd symptoms. endoscopy shows a characteristic linear furrowing, and histological features include ≥ eosinophils/ hpf (peak count). there is a male preponderance and an allergic and genetic component. around - % of children are affected by food allergy, most commonly cow's milk allergy and egg and peanut allergies which may manifest as eosinophil-induced gi disorders. eosinophilic gastroenteritis is manifest as allergic eosinophilic gastroenteritis, allergic proctocolitis and food protein-induced enterocolitis syndrome (fpies) . eosinophilia is also seen in helminth infection, inflammatory bowel disease, coeliac disease and graft vs. host disease where eosinophil density can correlate with disease severity. recent work has implicated duodenal eosinophilia in functional conditions, particularly paediatric dyspepsia, with success in treatment aimed at the eosinophilmast cell axis. parasites and eosinophils in the gi tract g. de hertogh* *uz leuven, dept. of pathology, belgium background: human endoparasites belong to four groups: the protozoa and the nematodes, cestodes and trematodes. about protozoan and helminthic species can cause gastrointestinal (gi) pathology. eosinophils are frequently associated with the lesions present in these conditions. method: pubmed-based review of the associations between gi parasites and eosinophils. results: protozoan parasites may be located on the mucosal surface or in the bowel wall in an intra-or extracellular position. eosinophils can be increased in blood and tissues, notably with isospora belli. dientamoeba fragilis infection may even masquerade as allergic colitis. invasive amoebiasis (entamoeba histolytica) on the contrary has been associated with a decreased number of tissue eosinophils. worms may be observed in the egg, larval or adult stage buried in or attached to the bowel wall. helminthic infections are classically associated with blood and tissue eosinophilia, which can be limited to the place of attachment of the organisms. the number of eosinophils in biopsies can be very high and even suggestive of primary eosinophilic enteritis, e.g. in trichuriasis, anisakiasis and enterobiasis, and with ancylostoma caninum and angiostrongylus infections. alternatively, chronic inflammation with eosinophilia may be confused with ulcerative colitis or crohn's disease, the latter especially if granulomas are present (as with schistosoma mansoni infection and more rarely in strongyloidiasis, anisakiasis and enterobiasis). conclusion: eosinophils are involved in the defense against many protozoan and helminthic gi parasites. the resulting histological picture may at times be confused with other disorders such as primary eosinophilic enteritis and the idiopathic chronic inflammatory bowel diseases. the diagnosis of invasive melanoma is straightforward in most cases. it is based on the recognition of a malignant junctional, in situ component in addition to an invasive dermal component. classical diagnostic criteria are established mainly for superficial spreading melanoma, being the most common melanoma subtype. the morphological spectrum of melanoma is, however, wide, and distinction from both melanocytic naevi as well as non-melanocytic tumours may be challenging in individual cases. in particular, some benign melanocytic naevi may show concerning histological features overlapping with those of melanoma, resulting in significant potential for overdiagnosis. melanocytic lesions notoriously difficult to separate from melanoma include dysplastic naevi, naevi of special sites, halo naevi, recurrent naevi, mitotically active naevi and deep penetrating as well as blue naevi. nonmelanocytic tumours may mimic melanoma by recapitulating a junctional in situ as well as a dermal invasive component showing similar cytological and architectural features, including epithelioid, spindle cell, small cell or desmoplastic differentiation, in addition to immunohistochemical expression of the so-called melanoma markers such as s , hmb- and melan a. these tumours may be of epithelial, haematolymphoid as well as mesenchymal lineage ranging from benign to outright malignant. awareness of the critical entities in the differential diagnosis of melanoma and their differentiating features and diagnostic clues is important to avoid misdiagnosis, especially as the treatment modalities and clinical behaviours may differ significantly. a. batistatou* *greece molecular studies of melanocytic lesions are necessary in order to identify additional prognostic and predictive biomarkers and establish the pathways of relevance for targeted therapy. the advent of biotechnology has also enabled the utilization of molecular testing as a diagnostic adjunct in the microscopic evaluation of difficult melanocytic lesions. Τhe molecular multistep process of melano-magenesis has been correlated to the clark model for melanoma development. the first step of melanocytic hyperplasia and nevus formation has been linked to constitutive activation of the erk-mapk pathway as a result of somatic mutations of braf or n-ras (or h-ras or gnaq). the development of cytologic atypia (dysplastic nevi) is related to alterations in cell growth, apoptosis and dna repair; inactivation of cdkn a; and pten pathways and tp . radial growth phase has been associated with decreased differentiation and deregulated expression of mitf, as well as deficiency in the p ink a-rb pathway. alterations in cell adhesion, such as reduction/loss of e-cadherin, increase of n-cadherin, αvβ integrin, mmp- and increased expression of osteopontin are associated with the development of radial growth phase and metastatic melanoma. alternatively, some melanomas (including acral and mucosal) arise de novo and harbor mutations/ amplifications of kit and amplifications of cyclin d or cdk genes. other less frequent, pathogenetic pathways have also been proposed. in the near future, distinct molecular signatures of gene expression may be useful in identifying melanocytic lesions as melanomas or nevi. for melanomas, refined classification systems based on molecular analysis could provide more accurate prognostic markers and enable targeted therapy. novel targeted therapies in melanoma and the pathologists' role in therapeutic selection a. lazar* *usa background: when melanoma metastasizes, it is a deadly disease with unsatisfactory treatment options. it has become clear that melanoma is composed of multiple genetic subsets of disease that have different oncogene additions and thus distinct therapeutic vulnerabilities. method: many clinical trials and application of targeted therapies outside of the trial setting depend on melanoma subtype and the genetic features of an individual case. pathologists are absolutely critical in this area of testing and in therapeutic selection to enable precision medicine. results: targeted therapies based on the molecular genetic features of individual melanoma cases is beginning to drive targeted therapy selection and can greatly enrich the rate of response to particular treatments. conclusion: this lecture will focus on the genetic features of melanoma currently most relevant to treatment and how testing drives therapeutic selection. in the very near future, such testing will be employed for treatment in virtually all cases of metastatic melanoma. nephron number as determined during nephrogenesis may be one determinant of renal disease and hypertension in later life. various genetic and epigenetic factors, but also maternal or environmental causes, are known to influence nephron number. an involvement of the kidney in the development of hypertension has been postulated for a long time and supported by experimental studies in rats and sheep. brenner and colleagues supposed a direct association between nephron number and blood pressure in humans. their so-called brenner hypothesis postulated that any reduction in nephron endowment leads to hyperfiltration of the remaining glomeruli, followed by glomerular enlargement with glomerular and then systemic hypertension, resulting in glomerulosclerosis, thereby establishing a vitious circle. in line with this theory, an association of low nephron number and development of hypertension was shown in different animal models as well as in two autopsy studies in humans. keller et al. found in caucasian patients with essential hypertension a significantly lower number of glomeruli per kidney than in matched controls. in parallel, mean glomerular volume was more than twice as high in hypertensive patients as in normotensive control patients, indicating compensatory glomerular enlargement. these results were confirmed in a caucasian american population, whereas in african americans, there was no association between glomerular number and blood pressure. the pathomechanisms linking low nephron number and hypertension are only partly understood. among others, inappropriate activation of the renin-angiotensin system, impaired tubular salt handling leading to salt and volume retention, or post-glomerular structural changes have been discussed. the new classification of lupus nephritis: a critical reappraisal a. halon* *wroclaw medical university, poland background: systemic lupus erythematosus (sle) is the prototype of human autoimmune multisystemic disease and has a wide spectrum of clinical manifestations, of which renal failure is the most common and severe. about - % of patient with sle suffer from lupus nephritis (ln), and it is one of the major causes of morbidity and mortality. ln manifests as diverse patterns of immune complexmediated renal disease involving all tissue compartments: glomerular, vascular and tubulointerstitial. iga nephropathy is a disease of a diverse course and outcome. there were several attempts to create a morphological classification that would serve as a tool efficiently defining the potential responsiveness to the immunosuppressive treatment as well as prognosis in individual cases. the last of these proposals, the oxford classification of iga nephropathy, was published in july . the aim of our study was to compare the utility of few functioning classification systems, including oxford and hass classifications and japanese histological grading, as well as our own morphological index of biopsies with iga nephropathy. on the basis of data collected in polish renal biopsy registry, we selected iga nephropathy cases that were characterized by at least years of post-biopsy follow-up, as well as satisfactory tissue material for light microscopy evaluation. microscopical grading was performed by a group of experienced nephropathologists. the lecture will be devoted to the results and conclusions of this study. the myofibroblast. from wound healing to neoplasia. with special emphasis on tissue fibrosis and fibrocontractive conditions. twenty-five years of reflexions w. schürch* *canada the myofibroblast (mf) was discovered in in electron micrographs from experimental granulation tissue. this cell was shown to share features of fibroblasts and smooth muscle cells, hence its name. in due course, it was found to be the principal cell to effect wound contraction, i.e., wound healing. this unique cell was later defined at the histologic, ultrastructural, immunohistochemical, biochemical and pharmacological levels. for the surgical pathologist, the mf is best defined by its ultrastructure. immunohistochemical studies demonstrate a heterogeneous pattern of cytokeletal phenotypes regarding actin isoforms and intermediate filaments. five immunophenotypes were identified. ultrastructural features that define the mf include: (a) stress fibers, i.e., bundles of microfilaments with interspersed dense bodies; (b) well-developed cell-to-stroma attachment sites, i.e. fibronexi, intercellular intermediate and gap junctions; and (d) abundant production of extracellular matrix. mfs have been observed in normal tissues, in granulation tissue and in several pathologic settings which will be discussed in detail. numerous cells can modulate into a mf phenotype: foremost, the local resident fibroblasts, followed by pericytes, vascular smooth muscle cells, liver perisinusoidal stellate cells, kidney mesangial and tubular epithelial cells, bone marrow stromal cells and mesothelial cells. for the development of the mf phenotype, a two-stage model was proposed. following tissue injury, complex changes in the microenvironment occur with the release of cytokines and the concerted action of a permanent feedback between intra-and extracellular tension, transforming progenitor cells into proto-mfs and then into differentiated mfs. three types of pulmonary preneoplastic changes (ppc) are accepted: squamous dysplasia and carcinoma in situ (sd/ cis), atypical adenomatous hyperplasia (aah), and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (dipnech). sd/cis are associated with the development of squamous cell carcinoma (scc). aah is a pre-invasive lesion for adenocarcinoma (adc), and dipnech may progress to carcinoid. recently, adenocarcinoma in situ (ais, formerly bronchioloalveolar carcinoma (bac)) was included among ppc. pathogenesis of scc and adc is a multistep and multicentric process involving the transformation of the normal bronchial mucosa and alveolar lining cells through a continuous spectrum of lesions. the sequence of ppc for scc includes: hyperplasia > metaplasia (sm) > sd > cis > scc; for adc, it is hyperplasia > aah/ bac > adc; and for carcinoid, it is dipnech > tumorlet. numerous genetic and molecular abnormalities occur in the very early stages of lung carcinogenesis, including hyperplasia and metaplasia and even in normal-appearing bronchial epithelium. it is not known which genetic changes are the most important or at what stage the process is irreversible. sm and hyperplasia can be either a repair or a reactive and reversible process of injured bronchial epithelium. it is found in purely inflammatory settings. these reactive processes usually do not progress to dysplasia and carcinoma. that is, morphology is a gold standard in the diagnosis of both reactive and ppc, and no ancillary studies can be used as a diagnostic aid. the strict histologic criteria are used to assist in the recognition and grading of mucosal lesions. handling the problematic pleural biopsy in suspected mesothelioma cases s. anttila* *huslab jorvi, dept. of pathology, espoo, finland the most challenging diagnostic problems in pleural biopsies include distinguishing between benign mesothelial hyperplasia and epithelioid malignant mesothelioma and between reactive pleural fibrosis and sarcomatoid or desmoplastic mesothelioma. in addition, sometimes rare tumor types, such as lymphohistiocytoid malignant mesothelioma or epithelioid hemangioendothelioma, may mimic reactive processes. the first question to be considered when handling a problematic pleural biopsy is: is the biopsy material adequate and representative as can be judged from clinical, imaging and thoracoscopic findings? if in doubt, a new biopsy should be recommended. the distinction between benign and malignant mesothelial proliferation can be made on morphological grounds only, although immunostains, e.g., for ema and desmin, may favor either a benign or malignant lesion. the most reliable criterion of malignancy is invasion. immunostains for broad-spectrum cytokeratins may aid in recognizing invasion both in epithelioid and spindle cell lesions. in spindle cell lesions, cytokeratins also help recognize the growth patterns of spindle cells. in reactive pleural fibrosis, a layer of cytokeratin-positive cells may be seen parallel to the pleural surface, whereas in sarcomatoid or desmoplastic mesothelioma, the pattern of spindle cells is haphazard or storiform. negativity of immunostaining for cytokeratins in a spindle cell lesion should not automatically lead to a conclusion of a benign process as, e.g., sarcomas and about a quarter of sarcomatoid or desmoplastic mesotheliomas are cytokeratinnegative. correlation of histological and immunochemical findings with clinical and imaging findings is of utmost importance for a correct diagnosis without unnecessary delay. these rare tumours stem from mononuclear phagocytes or antigen-presenting dendritic cells. the latter belong to different cell lineages (either haematopoietic or mesenchymal). histiocytic sarcoma (hs) affects adults and is characterised by an aggressive clinical course in most instances. hs more often occurs at extranodal sides and consists of pleomorphic, atypical, large cells that are cd + , cd + , lysozyme + , cd + , cd r + , and hla-dr + . partial expression of s- is recorded. tumours derived from langerhans cells (lc). they maintain the phenotypic profile (s + , cd a + , langerin + ) and ultrastructural features (birbeck granules + ) of lc. they include lc histiocytosis and lc sarcoma. the former represents a well-known entity, more commonly observed in children: its behaviour varies according to the stage. the latter is a very aggressive disease of adulthood with overt cytological atypia and dismal prognosis. interdigitating cell sarcoma (idcs) is a very rare neoplasm of adulthood that cannot be differentiated from follicular dendritic cell sarcoma morphologically, both being composed of oval fusiform cells growing in fascicles, storiform pattern or °whorls. the diagnosis is based on s- and vimentin positivity in the absence of other lineage markers. follicular dendritic cell sarcoma (fdcs) occurs in adults and more often presents in the lymph node, at times within the context of castleman disease. in % of cases, it has an indolent behaviour. hodgkin lymphomas (hl) consist of two main categories, classical hl (chl) and nodular lymphocyte predominant hodgkin lymphoma (nlphl). the latter occasionally shows large diffuse areas. in particular in small biopsies, this generates a difficult differential diagnosis of t cell/ histiocyte-rich large b cell lymphoma (thrlbcl). immunohistochemistry with a large panel of antibodies may be of help, but cannot solve all problems, and clinical aspects should always be taken into account as well. in view of the important clinical (therapeutic and prognostic) consequences, such cases should also be evaluated by an expert hematopathologist. the second problem is the distinction between chl and primary mediastinal large b cell lymphoma (pmbl) or diffuse large b cell lymphoma (dlbcl) nos. there may be various situations: a mediastinal mass with a synchronous composite chl and pmbl with divergent morphology and immunophenotype. this situation may be more frequent than thought since usually, only a small sample from the mediastinal mass is taken. both lymphomas may also develop metachronously. the coincidence of both lymphoma types is explained by the fact that both lymphomas likely have a common (thymic) origin. indeed, they share many genetic and molecular features as well. more difficult to understand and diagnose are the cases that arise outside the mediastinum with mixed features of chl and dlbcl-nos and cases of otherwise chl that strongly express cd and/ or other b cell markers. in such cases, other diagnoses should be considered as well, such as ebv + dlbcl of the elderly. several cases will be discussed during the session. peripheral t cell lymphomas: a new frontier s. pileri*, c. agostinelli, p. p. piccaluga *bologna university, dept. of haematology and oncology, italy peripheral t cell lymphomas (ptcls) account for about % of lymphoid tumours worldwide. almost half of them show such a morphologic and molecular variability as to hamper any further classification and to justify their inclusion in a wastebasket category termed "not otherwise specified (nos)". the latter corresponds to neoplasms with aggressive presentation, poor response to therapy and dismal prognosis. conversely to b cell lymphomas, ptcls have so far been the object of a limited number of studies aiming to elucidate their pathobiology and identify novel pharmacologic approaches. herewith, the authors revise the most recent contributions on the subject based on the experience they gained in the extensive application of microarray technologies. ptcls/nos are characterised by the erratic expression of t-cell-associated antigens, including cd and cd , recently proposed as targets for ad hoc immunotherapies. they also show variable ki- marking, rates > % heralding a worse prognosis. gene expression profiling (gep) studies reveal that ptcls/nos derive from activated t lymphocytes, more often of the cd + type, and bear a signature composed of genes and related products that play a pivotal role for cell signalling transduction, proliferation, apoptosis and matrix remodelling. this observation seems to pave the way to the usage of innovative drugs, such as tyrosine kinase and histone deacetylase inhibitors whose efficacy has been proven in ptcl primary cell cultures. gep does also allow better distinction of ptcl/ nos from angioimmunoblastic t cell lymphoma, the latter being characterised by follicular t-helper lymphocyte derivation and cxcl , pd and vegf expression. the south saharan african countries are low-resource settings where the histological diagnosis of diseases and consequently the correct managing of the patient are difficult to obtain. telepathology allows doctors working in remote locations to obtain a definite diagnosis through the transmission of tissue specimen via remote telecommunication. in particular, the "virtual slide" system allows capturing a visual image of an entire slide of a specimen which is then forwarded to another location for diagnosis. association pathology beyond borders (apof) in , in a small rural hospital in zambia, to supply to the lack of pathology skilled personnel, started a project to train local staff to become technicians, able to prepare histological and cytological slides and to screen conventional pap smears. the two zambian technicians signed out negative pap smears, and in the presence of abnormal findings, they took pictures of significant diagnostic fields. then the images were sent through internet to experts in italy who, on a rotating roster, were responsible of the final diagnosis. since , the technicians were also able to prepare histological slides from the surgical specimen: digital images of the slides were taken through a scanner and saved in a database in a local server. through a satellite connection and a made-to-measure archival software and web site, pathologists in italy were able to examine the specimen, record their diagnosis and transmit it directly to zambia. in low-income countries where no other possibilities are available, telepathology seems to be a reliable and secure diagnostic tool. leave a mark: organizational models in building and managing a pathology service in developing countries p. giovenali* *ospedale s. maria, della misericordia, perugia, italy the main aim of the ngo association pathologist beyond borders (associazione patologi oltre frontiera, apof) is the improvement of activities related to anatomical pathology in developing countries to raise the health standard in those areas through the performing of histological and cytological diagnoses for therapeutic and preventive purposes, such as screening programs. an essential part of every project managed by apof consisted in the construction and organization of new pathology services or in the implementation of already existing laboratories. while taking into account every particular local environment, needs and available resources, apof decided to export the same models that have proven effective in our context: the technical histological and cytological process, from the surgical sampling to the final report, was set in detail trough precise guidelines with the inclusion of procedures dedicated to process control. similar guidelines were used to update and adjust the diagnostic parameters according to international consensuses. the allocated funds were mainly used for the purchase of equipment and consumables and for the organization of updating courses dedicated to the technical and medical staff. in places where no skilled personnel were available, longer and more in-depth classes were organized for local staff, both in the place or in italy. the staff, once trained, passed also an examination to obtain an official and specific degree. the main purpose of this kind of management was to build a reliable system that could then be directly managed by local staff at the end of the project. the great debate about screening models in developing countries: matching needs and opportunities s. guzzetti* *ospedale evangelico valdese, asl to , turin, italy although in the developed world the importance of the correct diagnosis is appreciated as a critical issue, this is still an evolving concept in some of the developing countries, especially in africa. in particular, there are striking differences in the turnaround time for histopathological diagnosis, in the accuracy of diagnosis that has a profound impact on patients' management and ultimate outcome. the current problems in practice of lymphoid/lymphoma diagnosis include-basing treatment decisions on fine needle aspiration cytology in a large proportion of cases, poor quality histology in a minority of cases where biopsies are performed, complete lack of immunohistochemistry and other supportive investigations, and lack of an update on the current criteria for the diagnosis of various lymphoid pathologies. in africa, a majority of the laboratories still use the working formulation for clinical usage, a lymphoma classification from the early s which is based on morphology alone and does not include many entities recognized in the last years. without accurate diagnosis, any research project and effective patient management cannot be instituted. though there are no magic answers for an issue of this magnitude, on which other aspects are critically dependent, twinning between institutions in the developed countries and developing countries seems to be the most likely longterm approach. examples of twinning approach to childhood cancer diagnosis and treatment have been africa. these programmes have led to improvements in the diagnostic accuracy through capacity building and joint research projects with both direct and indirect technology transfer. the role of apof in low-resource settings: present and future projects for the development of surgical pathology in developing countries v. stracca-pansa* *italy in the last years, the cancer issue in most african countries is more and more dramatic. the who afro and the resolution of the th world health assembly made cancer one of the health priorities in developing countries and pushed african countries to formulate national cancer control programmes. once considered a disease of the rich, the pendulum has swung dramatically, and some % of new cancer cases in the next decade will be in the developing world. many poor countries are unable to cope with the accelerating burden of cancer. the essential purpose of the breast cancer pathology report is to communicate pathologic findings that aid in prognostication and guide the selection of appropriate local and systemic treatment for patients with breast cancer. to be clinically useful, the content of the report must change as new prognostic and predictive factors are validated and others become obsolete. in , the list of useful featuresthose which are grounds for specific therapeutic actions-is relatively short: the anatomic extent of disease in the breast and axilla (ajcc/uicc stage), the specimen margin status, and measures of certain cancer cell proteins (hormone receptors, her- /neu) that predict the likelihood of response to specific adjuvant therapies. histological subtype, carcinoma grade, the presence of lymphatic tumor emboli, the finding of paget disease in a mastectomy specimen are also often reported, but do not inform specific treatments the way that stage, margin status and predictive markers do. in patients with node-negative, estrogen receptor-positive breast cancer, the pathologist selects a paraffin-embedded tumor sample for the oncotype dx assay, which is used to predict the risk of recurrence in patients treated with tamoxifen and may addend the recurrence score to the surgical pathology report. the breast cancer pathology report in is vital to appropriate breast cancer treatment, but can be expected to evolve. in order to assure the quality of pathology in all of sweden, the swedish society of pathology has instituted organspecific quality assurance and standardization groups. these groups consist of dedicated pathologists from many subspecialty areas and are called kvast groups. they meet regularly and their principal work is to formulate and maintain a document with guidelines which has a common framework for all the organ-specific groups. our group prepares the guidelines for breast pathology. the mandatory sections in all the guidelines include clinical background information, instructions to clinicians as how to handle the specimens, what information is needed on the requisition form, gross description, analyses, what information must be reported by the pathologist (gross and microscopic), recommended classification system, administration and miscellaneous. sweden has a national cancer strategy programme emphasizing a patient perspective by focusing the patient process instead of piecing the treatment process together through independent specialities, as has been done previously. in , a group of breast cancer clinicians established a national quality register for breast cancer patients called inca which also include a pathology section. in collaboration with the swedish breast cancer group and the breast kvast group, another group called sweqa works with quality assurance programmes of the biomarkers used in routine pathology; estrogen and progesterone receptors, her status, and histologic grade. the guidelines for breast pathology will require the breast pathologists to participate in the breast cancer patient process by focusing on the standardisation of requisition and pathology reports and quality assurance of biomarkers. working on national guidelines: hungarian guidelines g. cserni* *hungary background: all pathologists feel that the establishment of pathological diagnoses is somewhat subjective. writing national guidelines is useful in order to achieve better consistency in reporting at the level of a geographic area, hungary. as not all countries have their national guidelines on breast reporting, it was thought useful to provide some examples of how guidelines can be constructed and/or adopted. method: the text of the hungarian guidelines on breast pathology reporting was written by a committee of pathologists with expertise in breast pathology. the basis of the new text was a consensus document from years ago. this was rewritten, circulated, and modified several times, and the updated text, ready for wider discussion, was put on the web (internet) along with four other texts (diagnostic radiology/imaging including nuclear medicine; surgery including reconstructive surgery; radiotherapy; and systemic therapy). the url allowing access to the texts was widely circulated on discipline-specific web sites. a consensus conference was organized for live discussion, and written comments were also welcome both from the writing committee members of the other texts and from the wider medical community (the other texts were discussed similarly to allow better congruence). all relevant comments were incorporated and the pre-final text was discussed at the consensus conference. the writing committee finalized the text on the basis of the relevant comments discussed at the conference. the pathology guidelines were scheduled for publication with the other texts in the national oncology journal, magyar onkologia (hungarian oncology). results: na. conclusion: na. working on national guidelines: polish guidelines e. chmielik *, w. p. olszewski, j. rys *oncology institute, dept. of pathology, gliwice, poland objective: the pathologists from the three major oncology centers have worked out the guidelines for breast cancer pathology reporting. method: the proposed report was mainly based on the european and american guidelines as well as on the seventh version of tnm classification and was prepared in cooperation with radiologists and surgeons. guidelines of the pathological reporting of breast cancer were accepted by the multidisciplinary board of the main national specialists representing different medical professions such as pathology, oncologic surgery, medical oncology, chemotherapy and radiotherapy, and afterwards, they were described in the supplement of the polish journal of pathology ( , vol , issue ) the guidelines include the rules of interpretation of needle core biopsies and other diagnostic procedures, as well as the recommendations of gross description and processing of surgical specimens depending on the type of surgical treatment. especially, pathology report of breast carcinoma after neoadjuvant chemotherapy was proposed. special techniques used for diagnosis of breast lesions, their performance and interpretation are also included. results: finally, the histopathological evaluation form of breast cancer has been proposed. in a year after the supplement publication, it is planned to conduct a survey to find out the practical use of those guidelines. on the basis of both the answers to those questions and medicine-based evidence, polish guidelines are going to be compiled. the transplant nephrologist decides to biopsy a given patient on the basis of clinical symptoms and laboratory data. the renal pathologist then makes a histological diagnosis which allows an evidence-based approach to specific therapy, which again will be decided on by the clinician. renal transplant diagnoses generally fall into one of five categories: preexisting donor-related diseases, rejection, drug toxicity, infection, recurrent or de novo renal diseases. although many diagnoses are typically encountered during a certain phase after transplantation, clinical information alone will often not suffice to differentiate between diagnoses, which require a totally opposite treatment regimen. the timing of the biopsy is most important to achieve a successful treatment and to avoid irreversible damage to the transplant. the rule of thumb at our center is to biopsy early in delayed graft function if there is a change in serum creatinine of more than . mg/dl or there is a newly diagnosed or increased proteinuria. sufficient biopsy material, adequate workup including c d and sv immunohistochemistry, and an experienced pathologist are the other prerequisites. a close collaboration between nephrologist and pathologist with regular case discussions not only helps understand the viewpoint of the clinical/pathological partner but will also aid in patient management. pathology in zero-hour biopsies and clinical consequences: hungarian and polish experience a. perkowska-ptasinska*, e. kemeny *poland at the university of szeged, hungary, we carried an original clinicopathological study to assess what type of vascular changes-if any-are associated with late graft dysfunction. we examined in zero-hour biopsies the frequency and severity of nonspecific morphological lesions semiquantitatively. the wall thickness/lumen (w/l) ratio of each artery present in the biopsy was determined by morphometry. among the arterial changes studied, only the intimal fibroelastosis (ife) of moderate degree (frequency, . %) revealed a significant correlation with serum creatinine at months (p< . ). in ife, there was also a significant correlation with the frequency and severity of tubular atrophy and interstitial fibrosis (p< . ). by morphometry, a significant association was found between the mean w/l ratios of arteries and the degree of ife (p< . ). according to hungarian experience, donor kidneys with a moderate degree of ife do indeed have a higher risk of late graft dysfunction. the analogically defined polish retrospective study revealed that the presence of arteriolar hyalinisation in the implantation biopsy was associated with more profound reduction of gfr at rd, th and th months after transplantation in those patients who experienced episodes of an acute rejection (p= . ) within first post-transplant months. this suggests that donor-derived arteriolar hyalinisation predisposes to more severe acute rejection-associated graft damage. new techniques for the identification of hla antibodies g. boehmig* *austria background: it has become evident that antibody-mediated immunity plays a critical role in acute and chronic allograft rejection. according to the banff scheme, serological alloantibody detection represents one of the major diagnostic criteria for antibody-mediated rejection (amr). the design of sophisticated diagnostic tests for prediction and monitoring of amr has become a major goal in transplant medicine. method: an important innovation has been the establishment of solid phase hla antibody detection using flow cytometry, elisa or luminex technology. in this context, luminex-based bead array technology represents an attractive strategy for a detailed analysis of anti-hla reactivity patterns. salivary and mammary glands share an identical ductuloacinar architecture, and thus, it is not surprising that lesions and tumours arising in both organs share considerable histologic similarities. salivary-type tumours are well known in breast; in contrast, breast-like lesions are rarely described in salivary glands. one of the commonest breast conditions, benign fibrocystic disease, was not thought to have a salivary counterpart, but recently, sclerosing polycystic adenosis (spa) was described as a distinctive neoplastic lesion of the major salivary glands. although spa has many histologic similarities to its mammary counterpart, it represents a true neoplastic condition characterized by clonality, focal dysplasia, and a tendency to recur. up till now, secretory carcinoma (sc) has been considered to occur only in the breast. recently, we published a series of salivary gland tumors with histomorphological and immunohistochemical features reminiscent of sc of the breast. this is an unusual, hitherto undescribed, distinctive salivary gland tumor, with some morphological features of both salivary acinic cell carcinoma (acicc) and mammary sc, characterized immunohistochemically by strong vimentin and s- protein positivity. microscopically, the tumours exhibit a lobulated growth pattern, and they are composed of microcystic and glandular spaces with abundant eosinophilic homogenous or bubbly secretory material, which is positive for pas, mucicarmine, muc , muc , and mammaglobin. the decision to undertake the sequencing of the entire human genome stemmed from many sources, in particular rrenato dulbecco's seminal article in science ( ; : - . extraordinary technical advances were required to facilitate this, but few could have predicted the attendant conceptual revolutions. since the publication in of the first draft of the human genome (and the parallel reports of the sequences of many other genomes), our view of the complexity of the organisation and regulation of genomes has increased. the encode project (genome res ; : - ) further highlighted the extraordinary diversity of transcription and demonstrated that the protein coding regions are but a fraction of the transcriptome. the importance of non-coding regions in genome regulation has become manifest, and the diversity of splicing events was previously unsuspected. the previous drip of new information has become a torrent, and 'next-generation' sequencing technologies promise to increase this driving cost of sequencing entire genomes to sub-$ , levels. the timescale of generating data similarly collapses to a mere fraction of that required even years ago. the information load will be immense and new approaches will be needed to deal with the impact of this on clinical decision making. the consequences for these developments are perhaps unfathomable at present, but we are challenged to find ways to take advantage of these developments lest pathology be bypassed by other disciplines. central to this will be educational programmes at the undergraduate and postgraduate level to 'future proof' tomorrows clinicians, including pathologists. new approaches to molecular classification of lymphomas l. leoncini*, s. lazzi, g. de falco, c. bellan, a. onnis, v. mourmouras *university of siena, italy background: the era of molecular diagnostics of lymphoid malignancies started with the cloning of the immunoglobulin and t cell antigen receptor genes. southern blot analysis was applied in clinical laboratories to establish clonality of lymphoid proliferation. this was followed by the cloning of a number of translocation breakpoints in the more common lymphomas. method: the advent of polymerase chain reaction provided an alternative to southern blot analysis as it is simpler and faster. in addition, the amount of clinical material required is much smaller, and it can be performed on archival paraffin-embedded materials. results: gene expression (ge) analyses by use of microarrays (mas) have become an important part of biomedical and clinical research. the resulting data may provide important information regarding pathogenesis and may be extrapolated for the diagnosis and prognosis of non-hodgkin lymphoma (nhl)( ). this genomic technology has revealed that existing diagnostic categories of nhl comprised multiple molecular and clinically distinct diseases. in addition, gene expression profiling studies may lead to the identification of novel targets for the development of new therapeutic agents for nhl. conclusion: more recently, the discovery of a novel class of small non-coding rnas, the micrornas, has opened a new scenario in understanding the regulation of gene expression. mirnas control gene expression at the posttranscriptional level, and deregulation of their physiological function has been revealed to be crucial in cancer. mirna expression profile can be obtained by microarray and is even more informative than gep as a few mirna alterations can specifically identify a tumor (histo) type. egfr mutation analyses in nsclc-experiences of a nationwide ring trial in germany m. dietel* *universitätsmedizin charité, berlin, germany background: since non-small cell lung carcinoma (nsclc) is being predominantly diagnosed at advanced stage, the option of a curative therapy no longer exists in most instances. roughly speaking, the first therapeutic standard measure to treat nsclc consists of a platinumbased chemotherapy, reaching a response rate of - %. recently, new substances have been introduced to treat nsclc, i.e. erlotinib (tarceva®) and gefitinib (iressa®). both inhibit the epidermal growth factor receptor (egfr ). results: for the egfr-inhibitors, it is possible to predict tumor response by egfr mutation analyses. this is the reason why the emea has approved iressa treatment only after such analysis, which has to be performed pretherapeutically. the test has to be done by pathologists who ( ) should reconfirm the diagnosis on an h&e slide; ( ) he then should identify and mark the tumor area, ( ) followed by manual microdissection to assure that at least % of the material is indeed nsclc. ( ) results: molecular tests and gene expression profiles that purport to predict patient outcomes and drive therapeutic decisions are currently favored by oncologists. however, ihc is the only laboratory venue that supplies molecular morphology that may be directly visualized and interpreted. in addition to diagnostic ihc, theranostic and genomic applications are also now in the menu of the pathologist. conclusion: pathologists must be able to emphasize the molecular morphology of ihc and how it can supply theranostic and genomic information in addition to diagnostic applications. our ihc challenges include standardization of the total test and the ability to quantitate results for patient care. most common laboratory pitfalls in ihc s. nielsen* *aalborg hospital, dept. of pathology, denmark most common laboratory pitfalls in ihc immunohistochemistry (ihc) is a well-established technique and used daily in virtually all departments of surgical pathology as a diagnostic, predictive and prognostic tool. however, ihc is an assay influenced by multiple parameters and the final result is highly dependent on the choice and performance of these variables. in the protocol setup for ihc, both the preanalytical, analytical and post-analytical parameters will affect ihc staining, and it is of utmost importance to be familiar with these technical aspects in order to use ihc as a diagnostic tool. in the pre-analytical phase, fixation still is the key element for a reliable result, and it is essential that fixation is standardized with respect to the choice of fixative, time to and time in fixative in order to get consistent results and to avoid false negative or false positive reactions. the implementation of new tissue processing techniques based on, e.g. modified reagents, can also affect the staining result. regarding the analytical phase, the three key elements are: ( ) appropriate epitope retrieval, ( ) a sensitive and specific primary antibody, and ( ) a robust detection system. in external quality programs for ihc, it has been shown that most errors in ihc are related to epitope retrieval and/or the primary antibody. to validate the performance and consistency of ihc, it is necessary to use internal and external controls. especially the use of multi-tissue blocks containing tissues with different levels of the antigen is superior to single control blocks. most common interpretation pitfalls in ihc j. klos* *norway background: the real frequency of interpretation pitfalls in immunohistochemistry is difficult to assess and may vary greatly between laboratories and pathologists. the risk of misinterpretation increases with the greater use of immunostaining as an integral part of theranostic workup, the increasing number and varying quality of available antibodies, as well as the rapidly expanding body of information regarding the complexity of ihc profiles of tumours. growing clinical expectations for pathological evaluation are an additional challenge. method: the presentation will address the most important elements regarding the correct interpretation of immunostaining using aaaspin as a simple algorithm for safer approach. results: a-adequate antibody panel: selection of antibodies is often a consequence of considered differentials, but results of immunostaining may also influence the choice of antibodies in subsequent analyses. a-antibody clone: the spectrum of reactivity and cross-reactivity should be known. according to the banff classification, antibody-mediated renal allograft rejection (abmr) is defined by circulating donor-specific antibodies (dsa), c d deposition in peritubular capillaries (ptc), and histologically detectable graft injury. these diagnostic criteria had primarily been developed and validated in biopsies from patients with early post-transplant dysfunction. results from studies in patients without clinically detectable graft dysfunction, however, cast doubt on the universal applicability of these criteria. in my talk, i will address the following issues: ( ) c d deposits but no abmr? findings in protocol biopsies from patients with stable renal function indicate that c d deposition is not necessarily always associated with renal dysfunction. the low predictive value of c d in these patients might be due to the accommodation of grafts and highlights the fact that the diagnostic impact of c d strongly depends on the clinical context. ( ) abmr but no c d? complement activation likely is not only of diagnostic importance but also part of the pathogenesis of abmr. results from gene expression profiling studies in human graft biopsies and from animal models, however, suggest that there also might be complement-independent mechanism of abmr. especially the lesions of chronic rejection might at least in part result from antibodymediated complement-independent endothelial injury. ( ) are "atypical" staining patterns of diagnostic relevance? c d can, in addition to the diagnostically relevant linear deposition in ptc, also be found in other locations and different staining patterns. it, however, appears that these other/additional staining patterns are of limited diagnostic value, at least for the detection of ambr. during gestation, several microorganisms can infect the foetus and damage the developing nervous system, leading to severe neurodevelopmental sequelae. the acronym torch was introduced to refer to these organisms including toxoplasmosis, other microorganisms, rubella, cytomegalovirus and herpes simplex virus. torch infections share similar clinical features: the maternal infection is regularly asymptomatic, the clinical presentation of infected fetuses and neonates is quite identical, and, sometimes, a clinically silent infection in the neonatal period will be responsible for permanent neurological deficits occurring later in infancy. extensive immunization programs, in developed countries, have led to an impressive decrease of congenital rubella syndrome, and the development of strategies of prenatal diagnosis and antimicrobial therapies has improved the management of congenital infections. despite these significant advances, torch infections remain a worrying cause of vision loss, hearing loss, and neurological disabilities in both developed and developing countries, and new syndromes have emerged such as congenital infections due to human immunodeficiency virus (hiv) and the human parvovirus b . in addition, in the two last decades, growing evidence has supported the hypothesis of a significant association between choriamnionitis and cerebral palsy: brain lesions would result from a production of cytokines through a mechanism of toxicity. a wealth of literature has summarized current knowledge concerning epidemiology, microbiology, diagnosis and the management of congenital infections. in this presentation, we will focus on the neuropathological features of the most frequent intrauterine infections and underscore the invaluable contribution of neuropathological studies in understanding the pathogenesis of developing brain lesions. development and functions of the dura mater w. squier* *united kingdom the dura is more than just a fibrous covering for the brain; it has other functions, including control of venous outflow and uptake of csf. dural development is not complete at birth; it undergoes considerable remodelling in early life. the meninges develop from primitive mesenchyme derived from the neural crest. the dura has two leaflets, the outer periosteal and the inner meningeal which forms the falx and tentorium. between them are the venous sinuses, which drain the entire venous outflow of the brain. bridging veins are few and carry high blood flow. a muscular sphincter at their junction with the dura regulates blood flow when intracranial pressure increases. a venous plexus, most extensive at birth, is found in the posterior falx, tentorium and the parasagittal regions. arachnoid granulations develop at months of postnatal life. originally thought to be responsible for drainage of csf, they more likely monitor csf homeostasis and pressure. fluid channels in the dura may represent a system for the uptake of csf. in early life, blood may reflux into these channels from the venous sinuses, causing intradural bleeding which is common in immature infants. dural bleeding causes symptoms such as seizures and vomiting difficulties and is associated with age-related imaging changes in the underlying brain parenchyma. we do not yet understand the pathophysiology of these observations. mitochondrial encephalomyopathies (me) represent a heterogeneous group of diseases, secondary to respiratory chain dysfunction, impaired atp production and energy crisis in the affected cells. me could be associated with defective nuclear (ndna) or mitochondrial (mdna) genome, resulting in autosomal or maternal inheritance, respectively. although multiple organ involvement is very common, the most affected are tissues with high oxidative metabolism such as cns, myocardium and striated muscle. myoclonus epilepsy with ragged red fibres (merrf), mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (melas), leber's hereditary optic atrophy (lhoa), kearns-sayre syndrome and leigh's disease represent the classical examples of me. precise phenotype/genotype correlations are very difficult due to the presence of overlap syndromes and atypical cases. the topography of lesions in the cns determines symptomatology and varies in these conditions. morphologically, me are characterized by variably expressed mitochondrial abnormalities such as intra-mitochondrial crystalloid inclusions and ragged red fibres (rrf) in muscle biopsies, as well as selective neuronal loss in different cns structures, laminar cortical necroses, microinfarctions and spongy degeneration in the grey or white matter. however, the common and diagnostically useful feature is the presence of cox-negative fibres, abnormal ultrastructure of muscle mitochondria and variably expressed vascular abnormalities. although clinicopathological correlations are often problematic, the involvement of small caliber vessels may explain many focal cns manifestations, while multisystem disease is very likely secondary to mitochondrial dysfunction in the neuronal component. results: kuru in the fore language means to shiver or to shake from fever and cold. ritualistic endocannibalism (eating of the relatives as a part of a mourning ritual, but not as an alimentary habit) was practiced not only in the kuru area but in many surrounding eastern highland groups which never developed kuru. the most striking neuropathologic feature of kuru was the presence of numerous amyloid plaques. conclusion: we may also speculate what would happen if kuru had not been discovered or did not exist. the infectious nature of creutzfeldt-jakob disease would probably not have been suspected until the beginning of the variant creutzfeldt-jakob disease (vcjd) outbreak in the uk. creutzfeldt-jakob disease and gerstmann-sträussler-scheinker disease would have remained for decades as obscure neurodegenerations of merely academic interest. the familial forms of creutzfeldt-jakob disease would not have benefited from prnp gene analysis, but only later would have been studied by linkage analysis and reverse genetics probably. the whole field would have probably remained of only arcane interest to veterinarians until the bse epidemic began to exert its devastating effect. the discovery of vcjd would have been delayed as no surveillance would have been initiated for creutzfeldt-jakob disease. and, perhaps most importantly, the sea change in mentality that has led to the conception of 'protein misfolding diseases', including not only the neurodegenerative but also an increasing number of nonneurological disorders, would have been delayed by decades. cell autocannibalism in central nervous system. the role of autophagy in neurodegeneration and nervous system tumours b. sikorska* *poland background: autophagy is a process by which subcellular constituents and organelles are targeted for degradation in lysosomes. in macroautophagy, proteins and organelles are sequestrated into a double membrane-bound vacuole called autophagosome, formed by the er membranes, under the direction of various proteins including map-lc (a microtubule-associated protein, light chain ). in addition to maintaining cellular homeostasis, autophagy may also contribute to cell damage. recently, autophagy was reported to be involved in many pathological processes including neurodegeneration, inflammatory processes and cancer. the role of autophagy in neurodegeneration is not only in removing protein aggregates but also in inducing the neuronal death. method: autophagy in human neurodegenerative diseases and tumours of the cns was studied using electron microscopy, immunohistochemistry and confocal laser microscopy. results: the presence of autophagy in prion diseases and other neurodegenerative disorders was shown. the extent of autophagy in tumours varied depending on histological type and malignancy. conclusion: although the role of autophagy in neurodegenerative diseases remains unknown, at least three hypotheses must be taken into consideration: ( ) removing protein aggregates, ( ) one of the ways of neuron death, and ( ) formation of spongiform change in prion diseases. the role of autophagy in cancer seems to be dual: on one hand, there is a growing body of evidence supporting the idea that autophagy may represent a tumor suppressor mechanism by reducing intratumoral necrosis, restricting oxidative stress and limiting chromosomal instability; on the other hand, autophagy may be an important process used by tumor cells to escape various types of stress and even therapeutic agents. advances in biology of gliomas w. biernat* *medical university of gdansk, dept. of pathology, poland objective: gliomas are the most common neoplasms of the central nervous system. the treatment of gliomas has been slowly changing for a few decades, and understanding of the glioma biology makes an important basic for the introduction of new treatment modalities. method: the most unfavorable prognosis concerns the group of diffuse gliomas which, due to the infiltrative growth, cannot be cured by surgery. the most common type of diffuse glioma is glioblastoma; this tumor may develop de novo (without preceding lower grade precursor lesion) or as a consequence of progression of malignancy. results: the molecular pathways most commonly seen in primary and secondary glioblastomas have been described, but recently, a new marker, isocitrate dehydrogenase (idh ), was defined as mutated early in the development of low-grade gliomas. the gene encoding this protein is located on chromosome q . idh catalyzes oxidative carboxylation of isocitrate to alphaketoglutarate. nicotinamide adenine dinucleotide phosphate (nadph) is the result of this reaction. idh is somatically mutated in low-grade gliomas and glioblastomas. on the other hand, alterations of the erk/mapk intracellular pathway occur in circumscribed gliomas, e.g. pilocytic astrocytoma. conclusion: all these informations suggest that differential utility of these markersand will be presented. toward "fingerprinting" of brain tumours based on the synchrotron radiation x-ray fluorescence, fourier transform infrared microspectroscopy (ftirm) and discriminant analysis d. adamek*, m. szczerbowska-boruchowska, m. lankosz *medical college krakow, dept. of neuropathology, poland objective: the neuropathologic diagnosis of brain tumours remains burdened by their well-known heterogeneity and difficulty to eliminate subjectivity in diagnosing. method: synchrotron radiation-based techniques were applied to biochemical micro-imaging of brain tumours of different types and various grades of malignancy. the specimens were cryosectioned, mounted on appropriate sample supports and freeze-dried. synchrotron radiation x-ray fluorescence was used for elemental analysis of samples. the level and distribution of p, s, cl, k, ca, fe, cu, zn, br and rb was determined. the biomolecular composition of neoplastic tissues was determined by fourier transform infrared microspectroscopy (ftirm). the composition of the tissues was used to construct a diagnostic classifier for brain tumours using discriminant analysis (da). results: it was found that cu, s, cl, k, and zn are the elements of the highest importance for the discrimination of tumor grade, as well as the tumor type. elemental analysis allowed % accordance with histological type and grade of tumor. the da applied to infrared absorption spectra indicated that lipids, amide i and amide ii, as well as phosphate group are of the highest importance for the discrimination of tumor type and tumor grade. the model obtained allowed differentiation between all investigated tumours and control samples as well as correct group classification in %. conclusion: it is difficult to speculate on the meaning of our findings with the biochemical perspective; however, the da based on elemental and biomolecular composition of tissue may be a potentially valuable method assisting the recognition and maybe grading of brain tumours. ( ) they are benign in the majority of cases; and ( ) the diagnosis of malignancy is based on cytologic features (papillary carcinoma) or on the presence of invasion of the tumor capsule or of blood vessels (follicular carcinoma). the common occurrence of benign thyroid nodules mandates an effective method for preoperative screening. the diagnosis of papillary thyroid carcinoma, by far the most common thyroid malignancy, is based on the identification of specific cytologic features. therefore, fine needle aspiration biopsy (fnab) has easily emerged in the past years as the most accurate and cost-effective tool, indeed a true cornerstone, for the preoperative management of thyroid nodules. standardized terminology to report diagnoses is highly recommended and is being implemented worldwide. the type of genetic alterations in thyroid cancer and the very nature of fnab samples make them ideally suited for molecular analysis. on the other end, the importance of intraoperative frozen section diagnosis has been constantly decreasing over the years as a direct consequence of the widespread application of fnab. it is now usually performed with cases that are suspicious after fnab and may be useful in some cases with indeterminate cytologic diagnosis. how to separate follicular adenoma from follicular carcinoma and follicular variant ptc? m. sobrinho-simoes* *portugal background: the differential diagnosis between fa, fc and fvptc is, at present, the most frequent reason for any consultancy practice on thyroid tumours. we will review this differential diagnostic problem taking into consideration that it only concerns the encapsulated type of fvptc. the poorly circumscribed/infiltrative types of fvptc, as well as the multinodular/diffuse type, do not create any major diagnostic doubts and are easily diagnosed, putting together the pattern of growth and the nuclear features. method: if one sticks to the differential diagnosis of fa, fc and encapsulated type of fvptc, the histopathological diagnostic hints are two: invasiveness (vascular invasion rather than pure capsular invasion) and presence of ptcnuclear features. results: we will discuss what to do whenever such histopathologic findings are not clear-cut enough to support a definitive diagnosis. we will review, in this context, the diagnostic value of immunohistochemical markers of differentiation and/or proliferation and that of molecular markers in a retrospective study of cases of well-differentiated carcinomas of the thyroid that have given rise to metastases and/or to local, clinically aggressive recurrences. the results obtained in this study will be used to determine the best diagnostic options and to support the conclusion that most, if not all, follicular tumours and welldifferentiated tumours of uncertain malignant potential, as well as minimally fc without vascular invasion and noninvasive encapsulated type of fvptc, carry a good prognosis even when treated by lobectomy alone. angioinvasive well-differentiated carcinoma, widely invasive follicular carcinoma, solid variant of papillary carcinoma and poorly differentiated carcinoma: from diagnosis to prognosis m. volante* *university of turin, clinic and biol. sciences, orbassano, italy malignant thyroid tumors are generally divided into welldifferentiated and undifferentiated (anaplastic) carcinomas, the former usually having a low malignant clinical behavior and good prognosis, and the latter being almost all very aggressive and rapidly fatal. diagnostic criteria rely on the recognition of cytological characteristics (i.e. nuclear alterations specific for papillary carcinoma), architectural patterns (i.e. papillary, follicular, solid, trabecular, insular or diffuse), high-grade features (i.e. increased mitotic index, necrosis) and invasive properties (i.e. vascular and/or capsular invasion). all of the aforementioned features are alternatively used as diagnostic hallmarks of specific histotypes, as peculiarities of specific variants, or as markers of aggressiveness, and their recognition is therefore essential to classify and stratify prognostically each individual tumor. the presence of vascular invasion is essential to recognize the malignant nature of a follicular tumor, but its extent is the major prognostic parameter in follicular carcinoma and draws a line between minimally invasive and widely invasive forms that are characterized by distinctive clinical outcomes. necrosis and high mitotic index usually occur in aggressive cases of papillary thyroid carcinoma, but together with the presence of solid/insular/ trabecular growth patterns represent the diagnostic features of poorly differentiated carcinoma, a specific tumor entity that shows a clinical behavior intermediate between welldifferentiated and undifferentiated carcinomas. at variance, the presence of a solid growth pattern in an otherwise typical papillary carcinoma depicts its solid variant, which is more often encountered in children and radiation-exposed individuals but has a clinical behavior usually similar to its conventional counterpart. as indicated by the world health organisation (who), histological grading is a way of predicting the biological behaviour of neoplasms. especially in neuropathological settings, tumour grade is often the key factor determining therapeutic decisions. clinical, radiological, macroscopic, histological and ultrastructural factors may already be summarized in a diagnosis that very well reflects the biology of the respective tumour entity, at least to a certain degree, but usually not in a personalized manner. this is exactly the stage where molecular biology comes into play. in recent years, much effort has been made on molecular characterization of brain tumours as there is an urgent need for specific and sensitive, highly reproducible tumour markers with a prognostic as well as predictive value. state of the art in modern neuropathology therefore ranges from basic immunohistochemical profiling, evaluation of genetic susceptibility, comparative genomic hybridization (cgh) approaches, fluorescence in situ hybridization (fish), screening for genetic hallmark mutations, evaluation of mrna expression of specific growth factor receptors, assessment of promoter methylation status and gene expression profiling to loss of heterozygosity (loh) analyses. the challenge now for pathologists confronted with brain tumours is to integrate knowledge derived from the latest molecular biological methods into the established panel of pure morphology-based investigations. objective: skeinoid fibres are globular brightly eosinophilic pas-positive extracellular collagen deposits seen commonly in gastrointestinal stromal tumours (gist) of the small bowel. ultrastructurally, they display similarity to "skeins of yearn", hence the name. however, hyaline globules are occasionally encountered in leiomyomatous gi neoplasms and may be mistaken for true skeinoid fibres leading, to an erroneous diagnosis of gist. method: we analyzed histologically and immunohistochemically well-characterized true smooth muscle neoplasms of the gi tract for the presence of hyaline globules and examined representative examples of them from formalin-fixed and deparaffinized tissue by electron microscopy. results: pas-positive intracellular and interstitial hyaline globules were detected in all benign paucicellular leiomyomas of the muscularis mucosae (n= ) and the muscularis propria (n= ) irrespective of tumour size and site, and rarely also in the adjacent muscularis propria, but in none of the leiomyosarcomas (n= ) and cellular leiomyoma (n= ). similar to surrounding tumour cells, hyaline globules expressed desmin, alpha-sma and h-caldesmon, but were negative for cd and cd . the mostly ovoid-shaped structures displayed at ultrastructural examination variably oriented bundles of filaments with a diameter of approximately - nm. at the periphery of these inclusions, altered filamentous material was recognized in different stages of degeneration with variable condensed matrix and occasional peripheral condensation suggestive of calcification. true skeinoid fibres were not detected. conclusion: the above findings are consistent with a multistep degenerative phenomenon affecting individual smooth muscle cells in paucicellular gi leiomyomas. awareness of this finding should prevent misinterpretation as gist, particularly in small biopsies. pseudotumours of the kidney j. lloreta-trull* *hospital del mar, dept. of pathology, barcelona, spain several clinicopathologic entities are grouped under the concept of pseudotumour: all of them usually result in a mass effect, therefore mimicking a neoplastic process grossly and sometimes microscopically. in the kidney and the urinary tract, the diseases most often presenting as a pseudotumour are malakoplakia and xantogranulomatous pyelonephritis. in fact, both of them can be considered inflammatory pseudotumours, i.e. inflammatory processes producing a mass effect. a more restrictive use of the term inflammatory pseudotumour applies to a subset of lesions that was initially known as myofibroblastic inflammatory pseudotumour and is currently referred to as inflammatory myofibroblastic tumour. ultrastructural examination reveals the pathogenesis of malakoplakia as a defect in the mechanisms of lysosomal extrusion from histiocytes. thus, the profusion of secondary lysosomes constitutes an optimal milieu for the deposition of calcium salts in typical michaelis-gutmann bodies. on the other hand, the foamy histiocytes of xantogranulomatous pyelonephritis are characterised by a profusion of myelin bodies due to the predominant phospholipid composition of the lysosomes, resulting from chronic destruction of renal tissue and bacterial degradation. inflammatory myofibroblastic tumour is a true neoplastic lesion with myofibroblastic differentiation and a prominent inflammatory background. this is an important differential diagnosis for both malakoplakia and xantogranulomatous pyelonephritis that may have an extensive myofibroblastic component. similar to its crucial role in kidney tumours, electron microscopy is a useful adjunct in the diagnosis of renal pseudotumours and has been essential in the elucidation of their pathogenesis. friday, september , . - . , sala wystawowa b objective: infectious complications have essential value at the conditions caused by primary and secondary immunodeficiencies, including connected with a hiv or immunosuppression therapy. laboratory diagnostics of opportunistic infections in conditions inadequate formation of antibodies is directed on revealing of antigenes of the activator. method: one hundred fifty samples of washing waters of bronchial tubes are investigated at diagnostic or medical bronchoscopy of patients (children and adults) with acute lymphoid ( . %) and myeloid ( . %) leukemia, chronic lymphoid and myeloid leukemia, hodgkin's and non-hodgkin's lymphomas. patients have been surveyed in different terms after transplantation of a bone brain. cytologic preparations were painted h&e, ziehl-neelsen and pas, ich. the cellular structure, condition epithelial cells, a degree of expressiveness and character macrophages activity epithelial and non-epithelial cells, a degree of expressiveness and character inflammations and also the presence of specific activators were estimated. results: data obtained by us testify to the prevalence bacteria ( . %) and viruses ( . %), inflammatory processes in respiratory ways. the changes connected with specific (tumoral), mycoses (aspergillosis, zygomycosis, a candidiasis) and pneumocystosis were less often observed by defeat. in one сase, it was documented as mycobacteriosis. in . % of cases of pathological changes, it is not revealed; in . % of cases, the changes connected with natural return development of pathological process were observed. conclusion: carrying out complex research of a biological material for the definition of character and a degree of expressiveness of pathological process and also the proof of a role of the infectious agent in the development of pathological process is expedient. diagnosis of infections in fine needle aspiration (fna) in immunocompetent and immunocompromised patients b. bode* *universitätsspital zürich, institute of zytology, switzerland fine needle aspiration (fna) is a well-established method for the evaluation of the aetiology of superficial and deep masses. an infectious process has to be considered as a differential diagnosis in all cases, especially in the setting of immunosuppression (hiv infection, transplantation, chemotherapy, inborn defects). fna allows a rapid distinction between neoplastic and inflammatory/infectious lesions. a wide range of pathogens including various viruses (cmv, ebv), bacteria (grampositive cocci, actinomyces, spirochetes, mycobacteria), fungi (aspergillus, cryptococcus) and parasites (toxoplasma, echinococcus) may be identified cytologically. infectious agents may be visualised either direct in standard stainings (cmv, fungi), in special stainings (mycobacteria) or immunohistochemically (spirochetes), either on direct smears or in cell blocks. molecular methods (in situ hybridization, pcr with sequencing) applied to fna specimen help identify and often subtype the pathogen if required (ebv, mycobacterium tuberculosis vs. atypical mycobacteriosis). fna is a particularly convenient method for fresh, sterile sample collection for microbiological examination with culture and drug resistance studies, playing an important role in treatment decision, e.g. in therapy of multiresistant tuberculosis. in some infectious diseases, the final diagnosis may be made by serology following indirect cytological hints in a fna specimen (e.g. toxoplasmosis, hiv). interestingly, infectious agents have been identified as aetiologically relevant factors in several tumor types (ebv in burkitt lymphoma or lymphoepithelial carcinoma, hhv in kaposi sarcoma, hpv in oropharyngeal carcinoma), so that the identification of the pathogens in fnas may play an important role in the precise diagnosis of some neoplasias. usefulness of oral cytopathology in the diagnosis of infectious diseases in immunocompromised patients p. hofman* *france background: cytology is an accepted and widely employed diagnostic methodology used in the early diagnosis of oral cancer. however, the diagnosis of many other specific clinical conditions of the mouth can be made from examination of smears, in particular infectious diseases. method: in recent years, the spectrum of infectious diseases of the mouth has changed. firstly, oral infections observed in immunocompromised patients have dramatically increased owing to the widespread implementation of solid organ and bone marrow transplantation and in the increasing prevalence of hiv infections. secondly, while the occurrence of many oral lesions has decreased significantly since the advent of highly active antiretroviral therapy, the incidence of oral warts has increased. results: in this regard, cytology can be used as a rapid, noninvasive, inexpensive and simple routine procedure in diagnosing infectious diseases of the mouth. the role of the cytopathology laboratory is diagnostic, i.e., to suggest or identify the presence of an infectious agent. however, exogenous structures that can mimic a variety of pathogens can pose a serious challenge. conclusion: the contaminants can be distinguished from microorganisms by their haphazard arrangement and lack of internal structure. moreover, the absences of acute or chronic inflammation, of macrophages or multinucleated giant cells with or without granulomas, and the absence of necrosis are features that should alert the cytologist to the possibility of contamination. finally, ancillary methods can be developed from cytological samples, which increases the specificity and the sensitivity for the diagnosis of infectious diseases in mouth. the the essentials in making a pathology diagnosis a. batistatou* *greece pathology is the discipline concerned with understanding the nature of human disease. pathologists' interpretations of tissue lesions become data, guiding decisions for patient management. cognition, the sum of processes by which the visual input is transformed, reduced, elaborated, stored, recovered and used, is very important in pathology. looking through the microscope at the stained tissue section (global impression and focal search) leads to perception of forms and colours. various regions are examined under low and high magnifications. then the pathologist puts the observed pieces of the colourful puzzle in place. an experienced pathologist proceeds promptly to pattern recognition and probably to diagnosis, while a novice takes more time, usually by not focusing on the significant areas but rather via an exhaustive search of the whole slide. non-verbalized pattern recognition consists of short sequences which result from conversion of longer series of specific features. alternative cognitive methods are diagnostic algorithms and the hypothetico-deductive strategy. experienced pathologists perceive each case as a whole, constituted by parts varying in importance and relevance. and while novices prefer to use analytic reasoning strategies, which are conscious and controlled, experts often use implicit reasoning, which is unconscious and automatic and relies largely on tacit knowledge. pathologists have faith in the analysis of morphology whose power has been appreciated even in the current molecular era. the use of virtual microscopy has a great advantage over the exchange of static images for diagnosis and training since it also allows for the initial steps to diagnosis, i.e. search, detection and perception. pathology diagnoses were asked for in the context of quality control of cervical cytology and dermatopathology. conclusion: in patient care, the dutch pathologists profit from the historical pathology diagnoses dating back to . for quality control of the lcpl, the diagnoses of other pathology laboratories of the hospitals where the patients of the general practitioners were operated provided by the palga are of paramount importance. in , the palga system will also be employed for the nationwide evaluation of the early detection of cervix, breast, and colon carcinoma. virtual microscopy in routine diagnostic procedure of pathology m. dietel* *universitätsmedizin charité, berlin, germany background: virtual microscopy (vm) is now widely applied in pathology. many studies underline the diagnostic security, the technical robustness as well as the versatility of this method. the standardization organizations dicom, hl and ihe call virtual slides as whole slide images (wsi) now and integrate the technology into their standardization strategy. in contrast to the increasing application in educations, the virtual microscopy is far from routine use in surgical pathology. this goes back to several reasons concerning technical and personal requirements: costs (scanning devices and storage), scanning time (between and min for a biopsy and between and min for a surgical specimen) and speed of virtual microscopes in comparison to conventional microscopy. results: caching and prefetching may speed up image loading, the bottleneck in virtual microscopy. the positive effects of different prefetching and caching technologies which depend on the user's behaviour will be discussed. further, the process of secondary diagnostic was evaluated using the "t. konsult pathologie" service of the professional association of german pathologists within the german breast cancer screening program. conclusion: in summary, it could be shown that the safety of diagnostic on wsi is comparable to the conventional diagnostic based on glass slides and a classical microscope. discrepancies go back to problems with the difficulty of the case itself and not to technical problems with virtual microscopy. virtual microscopy in teaching pathology in pomeranian medical university a. kram*, w. domagała *pomeranian medical university, szczecin, poland objective: microscopy is an important way of understanding the morphologic basis of diseases, and a slide seminar is part of the pathology program in medical schools worldwide. virtual pathology which allows seeing the entire digitized microscopic slide on the computer screen has been introduced recently. here, we share our experience in using digitized slides in teaching pathology and also discuss student's survey results. this has been the first implementation of a complete virtual pathology course for students of medicine in poland. method: two hundred twenty-four virtual slides for pathology course for medical students were produced by the use of aperio scanscope cs scanner and hosted in spectrum database. starting from the year , a group of polish-and english-speaking students used glass slides and microscopes in the first year and then virtual slides in the second year. at the end of the second year, on completion of the pathology course, the students were given a questionnaire to evaluate both systems. results: eighty-five percent of students preferred the screen over the microscope. continuous and easy access to virtual slides via the internet was indicated as one of the major advantages of virtual microscopy. some technical problems having an adverse impact on learning efficacy were noted. conclusion: the move to virtual microscopy and computer-assisted pathology teaching appears to be well received by students and enhance their learning ability in the field of pathology. objective: since years, there is an increase of incidence and mortality of adenocarcinoma as compared to epidermoid carcinoma. this increase is particularly notable for women younger than years. adenocarcinoma equals one fourth of cancers of the cervix in some countries like the uk where there is an organized screening program. method: the bethesda terminology has introduced for the first time the cytological diagnosis of adenocarcinoma in situ, the glandular counterpart of high-grade squamous intraepithelial lesion (hsil). results: it is a rare lesion that corresponds to % of abnormal pap smears and . % of all pap smear diagnoses. adenocarcinoma in situ is characterized by periphery radial arrangement of feather-shaped nuclei, cigar stack-shaped pseudostratified strips, rosettes images, hyperchromatic nuclei with granular structure and clean background without necrosis. on the contrary, invasive endocervical adenocarcinoma is characterized by papillary groups, lost of cellular cohesion, polymorphism of the nuclei with one or two prominent nucleoli and tumoral diathesis with dirty background, cellular debris and fragmented red blood cells. the bethesda terminology has maintained the grey zone for glandular cells abnormalities not well defined, included in the diagnosis of atypical glandular cells of endocervical, endometrial or undetermined. conclusion: the efficacy of cytological screening for detecting ais will lead to the increase of the rate of detection of ais. this will have an impact on the incidence and mortality of invasive adenocarcinoma in the coming years. the protective effect is beginning to be seen only recently for women younger than years in australia and in england. squamous lesions of the uterine cervix j. bulten* *radboud university, dept. of pathology, nijmegen, the netherlands cervical cancer is a major cause of death in women and the second most frequent cancer throughout the world, accounting for almost % of all malignancies in women. in this course, an overview is presented of the precursors of squamous cell carcinoma, its mimics, microinvasive carcinoma and cervical squamous cell carcinoma. the cin terminology (cervical intraepithelial neoplasia, grades - ) of richart is most widely used for cervical cancer precursors. nowadays, it is evident that cin is not a continuum, but reflects merely a low-grade entity (koilocytosis, flat condyloma and cin ) and conversely a true intraepithelial neoplastic process (cin - and carcinoma in situ). in diagnosing cin, hpv testing is not recommended. on the contrary, post-treatment hpv testing can predict treatment failure and thus residual cin. in grading cin and to differentiate cin from its mimics as atrophy, immature metaplasia or reserve cell hyperplasia immunohistochemistry (mib and p ) can be applied. due to preceding biopsies, microinvasive carcinoma is frequently overdiagnosed. colposcopy, cytology and immunohistochemistry are not very helpful in diagnosing microinvasive growth. as the diagnosis of (micro-)invasive growth has great clinical implications, cervical excision biopsies or cones should be totally embedded and serially sampled. there are three types of invasive growth, and the invasion depth should always be measured between the last cell of the deepest invasive focus perpendicular to the (expected) site of origin. finally, the several types of squamous cell carcinoma and its mimics are presented. , and unfavorable prognosis (alveolar rhabdomyosarcoma). immunohistochemistry can be used to separate between embryonal and alveolar rms. ap and pcadherin are almost exclusively expressed in alveolar rms, whereas egf receptor and fibrillin are found in embryonal rms. in addition, the myf- expression of alveolar rms is usually much stronger than in embryonal rms. genetic changes have been found for both embryonal and alveolar rms. most alveolar rms have a balanced reciprocal translocation, t( ; )(q ;q ). a smaller subgroup of alveolar rms has a t( ; ) translocation. two other translocations, a t( ; )(q ;p ), which generates a fusion protein composed of pax and ncoa , and a t( ; x) resulting in a fusion protein pax /afx, have been described in single cases. in embryonal rms, a loss of heterozygosity (loh) on chromosome p . has been shown. the involved gene or genes have not yet been clearly identified, but several interesting genes are located in this area like the myod gene, the igf- gene, the ldh (muscle subunit) gene, and the wt gene. a possible imprinting of this gene has also been postulated. infantile fibrosarcoma and malignant fibrous tumours r. alaggio* *anatomia patologica, scienze diagnostiche e terapie, padova, italy background: fibroblastic/myofibroblastic lesions account for % of pediatric soft tissue tumours and include benign neoplasms, reactive, pseudoneoplastic proliferations and, rarely, malignant tumours. the malignant lesions, registered in the italian protocol rms- , are reported to define their clinicopathologic and molecular features. method: forty-eight patients with fibrosarcoma (congenital and adult) were reviewed (table) . four cases, originally diagnosed as cifs, with negative etv -ntrk transcript, were reclassified as composite fibromatosis (two), undifferentiated sarcoma (one) and rhabdomyofibrosarcoma (one) and excluded from this study. results: see some of these lesions are genuine benign neoplasms; others have a reactive nature. nodular fasciitis is a fibroblastic/ myofibroblastic proliferation mostly seen in the subcutaneous tissue, morphologically presenting a loose and culture-like growth pattern. proliferative fasciitis (and proliferative myositis) is, likewise, nodular fasciitis, a fibroblastic/myofibroblastic proliferation with the presence of large ganglionlike cells. myositis ossificans is a benign and reactive lesion. fibrous hamartoma of infancy is a relatively common benign tumour of early childhood which presents three components: fibrocollagenous tissue, immature-primitive mesenchymal cells and mature fat tissue. myofibroma/myofibromatosis is a benign tumour observed in newborns and children within the first years of life, as well as in elderly patients. fibromatosis colli is a rare benign lesion typically seen in the sternocleidomastoid muscle in infants. microscopically, it is a proliferation of fibroblasts in between the muscle fibres forming a "scar-like" pattern. juvenile hyaline fibromatosis is a very uncommon, non-neoplastic lesion. fibroma of tendon sheath is a benign (possibly reactive) lesion related to tendons. calcifying aponeurotic firbroma is the tumour more frequently seen in children. gardner fibroma is an uncommon benign tumour mostly seen in infants, children and adolescents. calcifying fibrous tumour is a rare benign tumor more frequently observed in children and young adults. background: a standard clinical terminology like snomed ct is important not only for pathology information systems but also to integrate these systems with many other hospital information systems (his, pacs, departmental systems, encoding, database patients) and primary care information systems. method: snomed ct includes two hierarchies (morphologic abnormality and disorder) with similar terms. a snomed ct implementation was designed taking into account both the difference between these hierarchies and the relationship between terms located in different hierarchies. this is especially necessary in the postcoordination of clinical expressions and the creation of subsets. results: the january edition of the subset of pathology, which distributes the college of american pathologists (cap), contains , terms, but this subset needs to be improved to give it a clinical use. in sescam, , morphological concepts and specimen concepts were selected to create a minimum basic set for use in pathology information systems. snomed ct is also used in the sescam telepathology portal (project serendipia), developed according to the ihe-anatomic pathology initiative and dicom supplement- for medical imaging. conclusion: a snomed ct implementation strategy should not only include a list or subset of terms, but it must also consider the efficient use of relationships between terms and concept hierarchies, a mechanism for extension and update, and a plan for the coexistence or migration of legacy snomed ii codes. latest developments in data storage devices applicable for virtual slides j. görtler* *belgium structured reports in tumor pathology-how and why g. haroske* *germany objective: among all medical documents, pathology reports are often highly critical for patient care. in terms of information technology and knowledge engineering, presently, these reports are widely lacking adequate structure to support their usage in knowledge retrieval technologies for medical decision making, research, epidemiology, quality management and medical education. method: by means of literature surveys and by a questionnaire sent to vendors of pathology management systems (pms), the degree of structure in pathology reports in pms sold in germany was analyzed and compared with user needs expressed by pathologists. results: pathology reports are widely displayed, stored and exported as free text, identifiable by human beings, but without "observation identifiers" of a coding system not readable by a computer. a few systems allow textual information to be structured in sections partly identified by coding systems as snomed ct, etc. within the sections, the information is still unstructured. at the very beginning are attempts to structure the report as a list of coded items based on templates, which are controlled for identifiers, version, and the underlying concepts and representations. this is by far the most granular structure for computer readability and knowledge engineering. conclusion: there are very different opinions about the aim and degree of structured reporting. although standardization efforts have led to a series of reporting minimum requirements in tumor pathology, their implementation in structured data entry systems is still lacking. tools for data mining in routine pathology will be decisive for all important progress in pathology informatics at all. experiences of virtual microscopy in tma application g. kayser* *germany how to scale image information in virtual slides? k. kayser*, s. borkenfeld, j. görtler, g. kayser *heidelberg, germany objective: the application of virtual slides requires unequivocal definition and scaling of image information. scaling of image information offers the evaluation of diagnosis and content-based image retrieval (cbir). method: theory in principle-image information can be extracted with or without knowledge of its content. knowing its content (for example organ, potential diseases), we can segment biological meaningful objects (nuclei, membranes, etc.) and measure their features (size, gray values, derived parameters). the obtained results can be scaled and associated with the underlying disease. in reverse, knowing the applied algorithms and the object (and structure) features, cbir can be applied. without predefined knowledge, basic image information is mapped on spatial image units (pixels), called texture. they can be quantified by the application of stochastic geometry: each biological meaningful object is composed of so-called primitives (points, fibers, rings, plateaus). their mapping on an object can be used to detect knowledgeindependent content and scalable image information. results: object-and structure-based ( ) and the texturebased ( ) algorithms have been implemented and applied on more than , images obtained from h&e-stained glass slides of different organs (colon/rectum, lung, pleura, thyroid) in search for disease-significant areas (fields of view) and disease classification. the accuracy of the methods in field of view selection was computed to > %, that of prospective disease classification > %. hyperplastic lesions of the appendiceal mucosa are classically divided into two groups: diffuse hyperplasia and localized polyps. localized lesions were usually diagnosed as hyperplastic polyps, but some of these lesions are now better classified as sessile serrated adenomas. both diffuse hyperplasia and polyps are in most cases incidental findings on appendectomy specimens (performed for appendicitis or systematically sampled in a colectomy specimen). however, these lesions are significantly associated with colon adeno-carcinoma, and therefore, their discovery in an appendectomy specimen is an indication of further examination to exclude colorectal neoplasia. diffuse mucosal hyperplasia extends to more than one half of the circumference of the appendiceal mucosa. it exhibits elongated crypts with serrated architecture. the epithelium is composed of columnar cells alternating with large goblet cells in various proportions. the crypt bases are not serrated and are lined by regular cells with no atypia. hyperplastic polyps are supposed to be rare in the vermiform appendix. they are localized lesions, with a histological aspect similar to the corresponding colorectal lesions. similarly to diffuse mucosal hyperplasia, they are characterized by a serrated architecture restricted to the upper part of the crypts and by the lack of any dysplastic features. when lesions associate hyperplastic serrated features in one area and adenomatous (tubular and/or villous) features in another, they are designated as mixed adenomatous and hyperplastic polyps, as tumours of the colon and rectum. however, it is probable that at least part of these mixed lesions represent hyperplastic serrated polyps that have progressed to dysplasia. sessile serrated adenoma (or sessile serrated polyp or serrated polyp with abnormal proliferation) is a more recently described type of colorectal polyp with a serrated architecture. they occur frequently in the right colon, and although they resemble to hyperplastic polyps, they are characterized by subtle architectural and cytological features, especially dilation and serration of the basal part of the crypts. they can be dysplastic or not. the relative frequency of sessile serrated adenomas and other serrated lesions of the appendix is not well established, but it is highly probable that many appendiceal lesions previously designated as diffuse hyperplasia or hyperplastic polyp represent cases of sessile serrated adenomas. both types of lesions have been proven to show decreased expression of mlh and mgmt in a majority of cases and also braf mutation in a significant proportion of cases. these genetic and epigenetic alterations are similar to those observed in sessile serrated adenoma of the colon, which is now considered as a precursor lesion for a subgroup of colorectal cancer. the differential diagnosis of serrated lesions of the appendix also includes adenomas of colorectal type (more often showing villous architecture when compared to colorectal lesions) and low-grade mucinous neoplasms, often referred to as cystadenomas. adenocarcinoma, mucinous neoplasm of the appendix and pseudomyxoma peritonei a. jouret-mourin* *cliniques universitaires st luc, brussels, belgium primary adenocarcinoma of the appendix is unusual. some resemble typical colonic adenocarcinoma in their appear-ance and behaviour, but others are well-differentiated neoplasms or adenoma-like that may be associated with the pseudomyxoma peritonei syndrome without distant metastases. this pathological spectrum is confused because of terminological inconsistencies and the large variety of diagnostic criteria. therefore, there remains a "grey area" between benign and malignant lesions. some authors have recently proposed a new terminology based on the biological potential of the different lesions. lowgrade appendiceal mucinous neoplasm (lamn) is defined as a subset of lesions that have low-grade cytological atypia and minimal architectural complexity. these lesions penetrate into or through the appendiceal wall often on a broad front, without overt invasion and may be associated with pseudomyxoma peritonei. consequently, such lamn category includes the old terms of "tumours of uncertain malignant potential, "pseudomyxoma with adenoma-like histology" and the "low-grade mucinous adenocarcinoma" which can also produce pseudomyxoma. invasive mucinous adenocarcinoma (maca) includes a destructive invasion of the appendiceal wall with high-grade atypia, complex proliferation (i.e. cribriform pattern), desmoplastic stroma, signet ring cells differentiation or undifferentiated tumours. lymph node metastases occur lately, but peritoneal spreading is more frequent. some recent publications have demonstrated that protein immunoexpression profiles offer predictive factors of adverse clinical outcomes, which should further facilitate the classification of appendiceal mucinous neoplasms. appendiceal mucinous tumours can occur in patients who have suffered long-standing ulcerative colitis, familial polyposis and hnpcc syndrome. most of them show microsatellite instability probably given the high frequency of serrated precursor. pseudomyxoma peritonei syndrome is defined as the presence of abundant mucinous material on the peritoneal surface. this entity should be retained as a clinical term. besides exceptional cases of pseudomyxoma reported in association with mucinous carcinomas at other sites, all cases of pseudomyxoma peritonei are related to appendiceal rupture with subsequent transcelomic spread of tumoral cells. some authors have proposed that the same terminology should be used for the appendiceal primaries and the peritoneal lesions since lamn lesions confined to the appendix have the same appearance as those that have spread to the peritoneum. in addition, any tumour capable of producing pseudomyxoma peritonei should be considered as adenocarcinoma. therefore, the classification of pseudomyxoma perotonei includes low-grade and high-grade mucinous adenocarcinoma. the acellularity of the extra-appendicular mucin, based on extensive sampling, must be described in the pathological report because of its prognostic implications. mixed mucus-secreting and endocrine tumours of the appendix j.-y. scoazec* *hospices civils de lyon, hôpital edouard herriot, france vermiform appendix is one of the most frequent sites of mixed endocrine-exocrine tumours in the gastrointestinal tract. one histologic type of mixed mucus-secreting and endocrine tumor is specific for the appendix: this distinctive tumour is known as goblet cell carcinoid. the others are mixed endocrine-exocrine carcinomas raising the same problems of diagnosis and management than in the other digestive locations. goblet cell carcinoid is a highly distinctive clinical and histological entity. in contrast to the much more common pure neuroendocrine tumours of the appendix, which are usually discovered incidentally in young patients, goblet cell carcinoids present a peak of incidence in the fifth decade and are usually symptomatic. at macroscopic examination, goblet cell carcinoids present a very particular pattern of growth, resulting in the thickening of the whole appendicular wall and in a marked reduction in the calibre of the lumen, in the absence of visible tumour mass. this aspect is due to the predominant infiltration of the deepest part of the mucosa and the submucosa by tumour cells, which result in their fibrosis and thickening. at a higher magnification, tumor cells are grouped in small nests formed by large cells, with an abundant, pale, mucus-laden cytoplasm and an eccentric nucleus. these cells are strongly reactive with alcian blue and pas stainings. small endocrine cells are closely intermingled with mucus-laden cells; they are usually difficult to identify by conventional stainings; they are much more easily demonstrated through the immunodetection of endocrine and neuroendocrine markers such as chromogranin a and synaptophysin. finally, some amphicrine cells, with a combination of exocrine and endocrine markers, may be present. the number of endocrine cells admixed with mucus-laden cells is highly variable from one case to another and even from one region to another in the same tumour. there is no minimal amount of endocrine cells required for the diagnosis of goblet cell carcinoid, which is achieved by the combination of a typical macroscopic aspect, the presence of characteristic mucus-laden cells and the demonstration of even a few endocrine cells intermingled with the predominant mucus-producing population. from the molecular point of view, goblet cell carcinoids frequently harbour loss of heterozygosity in q, q and q. goblet cell carcinoids carry a high risk of peritoneal dissemination, but a low risk of distant metastasis. their prognosis is therefore much poorer than that of typical neuroendocrine tumours of the appendix: the -year survival is only % instead of %, all stages included; even for patients with localized disease, it is % instead of %. importantly, the tnm classification of goblet cell carcinoid must follow the criteria used for the staging of appendicular carcinomas, not those recently proposed for appendicular "carcinoids". mixed endocrine-exocrine carcinomas of the appendix combine an exocrine component, usually corresponding to a mucus-secreting adenocarcinoma of variable differentiation and an endocrine component, which may be well or poorly differentiated. the diagnosis of mixed tumour must be made with caution, only if the endocrine component is clearly neoplastic and represents at least % of the whole tumour. the prognosis and treatment are those of the most aggressive component. electron microscopy has been relatively surpassed by immunohistochemistry in pathology, especially for the diagnosis of neoplastic processes. however, its use is mandatory in neurodegenerative and neuromuscular disorders of non-neoplasic origin. ultrastructural examination has been proven to be useful in the following issues: ( ) to understand the physiopathology of muscle disorders. muscular dystrophies are produced by mutations in genes codifying proteins located at different subcellular levels. ultrastructural alterations have been observed at the level where the defective protein is expressed, i.e. plasma membrane alterations in dysferlinopathies, caveolinopathies and sarcoglycanopathies, or nuclear envelope membrane defects in emery dreifuss muscular dystrophy and laminopathies. ( ) to identify structures not visible with light microscopy. this is the case of intranuclear inclusions in oculo-pharyngeal muscular dystrophy, in inclusion body myositis, or tubulo-reticular inclusions seen in several autoimmune disorders and in aids. ( ) to determine the origin of specific anomalies depicted with light microscopy. ultrastructure of tubular aggregates, nemaline bodies, central cores or paracrystalline mitochondrial inclusions has been crucial to define the origin and organization of such changes at the subcellular components of the muscle fiber. ( ) to study in depth lysosomal storage disorders. the identification of different types of inclusions in neuronal ceroid lipofuscinosis according to the phenotype-genotype form of the disease has been especially useful, and so it was for the diagnosis of late-onset forms of glycogenosis type ii, fabry disease or others. in conclusion, electron microscopic examination is crucial to identify the subcellular structural involvement in some muscle disorders and to understand the underlying pathological mechanisms. electron microscopy of peripheral nerve a. vital* *bordeaux university hospital, dept. of pathology, france objective: electron microscopic examination (eme) of a peripheral nerve biopsy (pnb) is particularly valuable to assess the type of nerve fiber lesions, endoneurial deposits and storage materials. method: a well-done pnb, as well as a reliable methodology for fixation and embedding of specimens, constitute a prerequisite for an informative eme. results: if most cases of hereditary peripheral neuropathies are now diagnosed by molecular biology, nerve lesion analysis can direct the search for mutations in specific genes. prominent "onion bulb" formations are associated with pmp duplication. "tomaculae" correspond to pmp deletion, but can also be observed in certain cases of p mutation. other p mutations are mainly associated with irregularly uncompacted myelin lamellae. discrete "onion bulb" formations in association with "pseudo-onion bulb" formations surrounding clusters of regeneration are much suggestive of connexin mutation. abnormal mitochondria point to a mitofusin gene mutation. ultrastructural evidence of macrophageassociated demyelination is very supportive in atypical cases of inflammatory demyelinating polyneuropathy. in some cases of paraproteinemic neuropathy, myelin lamellae may exhibit a regular spacing corresponding to the presence of immunoglobulin m. in others, granular immunoglobulin deposits are identified in the endoneurium. occasionally, the endoneurial deposits have the tubular ultrastructure of a monoclonal cryoglobulin. the presence of osmiophilic lamellar inclusions in schwann cells points to treatment intolerance. at last, eme of a pnb may help in the diagnosis of a hereditary storage disease. conclusion: eme can be decisive to establish the etiological diagnosis of a peripheral neuropathy and must be supported by a reliable methodology. electron microscopy of metabolic storage diseases i. ferrer* *hospital universitari de bellv, institut neuropatologia, spain metabolic diseases of the nervous system in infancy constitute a very complex group of variegated diseases with particular clinical, morphological, biochemical and genetic characteristics. diagnosis of such disorders usually demands the collaboration of different specialists, methods, and skills. this is particularly important in order to avoid unnecessary probes and superfluous additional suffering related with inappropriate complementary examinations and visits. in recent years, molecular and genetic studies have facilitated a correct diagnosis during life, and improvements in therapy have permitted the application of new therapeutic tools in some cases. morphological visualization of determinate lesions in skin, conjunctival, appendicular and, rarely, rectal mucosa, is extremely useful to support a clinical diagnosis. lipidosis, mucopolysccharidosis, mucolipidosis, polyglucosan storage diseases and glycogenosis affect several cell types outside the central nervous system, including axons and schwann cells, endothelial cells, pericytes, smooth muscle fibres, sebaceous and eccrine glands and ducts, and striate muscle. deposits are usually too small to be visible with an optical microscope. yet electron microscopy permits the observation of lysosomal inclusions filled with specific inclusions and deposition of abnormal material in different cells. in addition to metabolic diseases, other degenerative diseases affecting the nervous system in which the diagnosis is mainly based on key pathological markers are also subject to electron microscopy analysis during life. nuclear neuronal inclusion body disease and infantile neuroaxonal dystrophy, among others, can be diagnosed by means of appendicular (or rectal) biopsies and by examining the nerve terminals and skin and conjunctiva, respectively. this presentation will cover a range of sinonasal inflammatory diseases, focussing on those where an improved understanding of pathogenesis illuminates diagnostic histopathology. the categorisation of sinonasal fungus-related diseases is important for prognosis and treatment. fungi are common airborne allergens and can be cultured from the nasal secretions of most healthy individuals. the diagnosis of fungal sinusitis relies on the histological recognition of fungi in sinus tissue, although the morphology of fungal hyphae is of limited value in distinguishing between fungal species. invasive fungal sinusitis is almost always encountered in patients whose immune system is compromised and is associated with necrosis and vascular invasion by fungi. noninvasive fungal sinusitis includes sinus fungal balls and allergic fungal sinusitis, a disease characterised by socalled allergic mucin and scanty fungal hyphae. the pathogenesis of chronic rhinosinusitis involves a complex interplay between environmental factors and genetically influenced immune responses; eosinophils may be seen in both allergic rhinosinusitis and in patients with no evidence of allergy. the differential diagnosis of inflammatory polyps will be discussed. pathologists should be aware of the many sinonasal diseases that may include a component of granulomatous inflammation including mycobacterial infection, cocaine-induced midline destructive disease and wegener's granulomatosis, and of the value of ancillary investigations such as autoantibodies in their differential diagnosis. autoimmunity to type ii collagen is involved in relapsing polychondritis, a multisystem disorder in which destruction of the cartilages of the nose, ears and upper airway is prominent. classification and diagnosis of sinonasal adenocarcinomas i. leivo* *haartman institute, dept. of pathology, helsinki, finland salivary gland-type sinonasal adenocarcinomas can occur in the sinonasal tract, and they resemble the corresponding tumours in salivary glands. the most common types are adenoid cystic carcinoma and adenocarcinoma, not otherwise specified. intestinal-type sinonasal adenocarcinomas mimic the histological appearances of intestinal adenomas, carcinomas, or rarely even the normal intestinal mucosa. occurrence of the intestinal-type sinonasal adenocarcinoma has been associated with long-term exposure to hardwood (beech, oak) dusts. in the woodworking industry, these tumours can be , times more common than in the general population. the average exposure time is years. histologically, the most common appearances resemble colonic adenocarcinomas. if the resemblance is striking, the rare possibility of a metastasis from the gastrointestinal tract should be considered. similar to colonic adenocarcinomas, intestinal-type sinonasal adenocarcinomas stain for ck , cdx- , and muc , but they also stain for ck . thus, the differential diagnosis between a primary intestinal-type sinonasal adenocarcinoma and a metastatic colonic adenocarcinoma cannot be based on histomorphology or immunophenotype alone. then, the recommended approach is colonoscopy. the intestinal-type sinonasal adenocarcinoma is a high-grade malignancy. however, the prognosis of patients with hardwood dust exposure is better than in sporadic tumours. in addition, papillary growth patterns relate to a more favourable outcome. low-grade sinonasal adenocarcinomas have not shown occupational or environmental associations. histologically, they are papillary or glandular, with a single layer of uniform epithelial cells. cytologically, they are bland, but malignancy is revealed by the complexity of architecture and invasive growth. lowgrade sinonasal adenocarcinomas do not express intestinal immunophenotypic markers. background: computer-assisted microscopy (cam) offers proven solutions in immunohistochemical assessments, sharing digital slides with collaborators in different places, storing and organize pathological and clinical data and research. method: we initially developed an in-house system for the creation of digital slides and for remote diagnoses on frozen sections, suggesting that digital pathology would become the new standard in surgical pathology. in our institution, we adopt cam using the aperio scanscope xs for several applications: results: ( ) detection and semiquantitative measurement of equivocal her immunoreactivity and of ki in breast cancer-our data on over cases with comparison between cam and human evaluation and with fish analysis strongly support its feasibility and reliability in routine practice; ( ) to store and analyze tissue microarrays with all associated experimental and follow-up data, permitting fast access to adjacent sections and comparing stains across multiple slides; ( ) to store clinical-pathological data of patients for discussion in multidisciplinary meetings and courses. conclusion: in our opinion, cam provides a unique opportunity for image visualization and analysis, archival and retrieval, which in the near future will probably replace the usual microscopy in routine practice. bibliography . objective: an automated method for immunohistochemical assessment of ki- proliferation index was developed and evaluated. using an open source image manipulation tool, imagej, a macro called ihcj was created for the assessment of immunohistochemistry stainings. the performance of the ihcj macro was evaluated in a tissue microarray series of breast cancer specimens from , patients. the specimens were stained with an anti-ki- antibody, counterstained with hematoxylin and digitized. the ki- proliferation index was calculated both visually and automatically with the ihcj macro ( fig. ) . the prognostic value of the ki- proliferation index was evaluated in uniand multivariate survival analyses. results: in a univariate survival analysis, the hazard ratio of distant recurrence during a median follow-up of . years for the automated ki- medium area fraction was . during the past years, we have completed a transition of the basic and oral pathology practical courses from light to virtual microscopy. after a pilot feasibility study, the entire training set of glass slides was digitized and located on webmicroscope server. giving access to the web, computers have become perfect companions of the students. the study material consists now of over fully digitized slides covering entities in basic pathology and entities in oral pathology. digitized slides are linked with still macro-and microscopic images, organized with clinical information into virtual cases. we undertake a comprehensive evaluation of this new approach at the end of every academic year. in these surveys, students rate the software, the quality of the images, handling of the images, and effective use of virtual slides during the time of the practical. satisfaction surveys demonstrate a steady improvement over the past years as various student suggestions were implemented. an overwhelming majority of our students considered using digitized slides at their convenience as highly desirable. overall, the quality of the images was rated as very good. however, due to the limitation of resolution of few primary scans, we have rescanned these slides at × objective and a , × , -pixel camera to alleviate these problems. our students and faculty consider the virtual microscope as a significant improvement. the administrators of our department consider virtual microscopy as innovative and costeffective because there is no longer the expense of the maintenance and replacement of microscope and glass slides. a web-based examination system for students using webmicroscopy for learning oral pathology j. szymas*, m. lundin, j. lundin *university of medical sciences, dept. of clinical pathology, poznan, poland we have developed and evaluated a user-friendly online interactive teaching and examination system for pathology courses. already in , at the end of the first course, students take a multiple-choice question exam (one correct answer per question). every exam question is linked with a proper virtual slide and the time for the answer is unlimited. this examination system uses advanced html features, and the web browser that can handle frames, javascript and cookies is required. setting the screen to a resolution of , × or higher and decreasing the size of the menu bars if necessary is helpful in viewing the digitized histological slide and the question at the same time. specific instructions about answering the questions are also given at the beginning of the examination. a score is generated for questions which are correctly answered. the scores are accumulated until the student decides to quit the web browser. in the last years, an overall % concordance rate has been achieved on practical examination based on virtual slides connected with multiple-choice questions per student. the online practical examination system was evaluated by the students using a questionnaire. students were asked to reply to a survey to evaluate the usefulness of digitized slides for practical examination. all students preferred the online examination to a traditional microscope and paper-and-pencil examination and all felt that the quality of digitized slides was superior to that of classical glass slide and allowed to make a more accurate diagnosis (rating . out of ). use of gray level co-occurrence matrix in detecting corresponding fragments and evaluating quality of virtual slides s. walkowski*, j. szymas *poznan university of technology, faculty of computing science, poland virtual slides may be acquired using various scanners, produced by different companies. pathologists who examine digital images captured by different scanners may subjectively assess their quality. in this work, an algorithm which may objectively compare the quality of virtual slides is proposed and implemented. in the first step, the algorithm selects some fragments of the slide captured by one scanner. then, it finds these fragments in virtual slides acquired by scanning the same glass slide using other devices. this is done by creating overviews of the whole slides (by zooming them out) and calculating gray level cooccurrence matrices (glcm) for each selected fragment and for the overviews of other slides using windows of adequate size. comparing some of the haralick features of the matrices (especially measures related to orderliness) allows estimating the positions of the fragments in other virtual slides. detected areas, in original resolution, are used to calculate glcm and its haralick features again. this time, measures for evaluating the quality (like contrast) are used. aggregation of the calculated values allows comparing the relative quality of the virtual slides. this method is tested on two sets of ten virtual slides. they were created by capturing the same set of glass slides using two different devices: robotic microscope axioscope (zeiss) equipped with axiocam hrc ccd camera and deskscan (zeiss) with standard equipment. acquired images were stitched and converted by specialized software based on advances in aerial and satellite imaging. color/image quality standardization in whole slide imaging y. yagi* *usa conclusion: vancb in breast lesions assisted the therapeutic decisions. a close interdisciplinary approach assured optimal results ( ). careful initial clinical-radiologic-pathologic correlation should dictate clinical management of equivocal fibroepithelial lesions diagnosed on core biopsy e. resetkova*, c. albarracin, e. arribas, n. sneige *mdacc, dept. of pathology, houston, usa objective: equivocal fibroepithelial lesions (fel) diagnosed on core biopsy (cb) may prove to be either fibroadenoma (fa) or phyllodes tumor (pt) at excision, due to which their management is controversial. method: ( ) to study the outcome of patients with fel on cb over a -year period at our institution. consensus on initial patient's clinical management was reached during a multidisciplinary conference based on correlation of clinical data, radiology, and pathology. ( ) to examine if any clinical, radiologic or histologic parameters could predict final classification, we retrospectively reevaluated imaging findings (mammography and sonography) as well as multiple histologic criteria (e.g. pattern, stromal cellularity, stromal atypia, mitosis, etc.) on cb and correlated these with diagnosis at excision. results: ( ) of excised lesions, were classified as pts, as benign cfels and as fas. in patients with no excision, no evidence of disease progression was observed ( ± months, mean ± sd). fels with indeterminate or suspect imaging findings, larger size, and an equivocal comment such as "cannot rule out pt" in the pathology report were excised more frequently (p= . , p= . and p= . , respectively). ( ) final diagnoses did not correlate with retrospective evaluation of any clinical data, imaging findings or histologic parameters. conclusion: careful initial clinicopathologic and radiologic correlation may select majority of clinically significant lesions for proper surgical management. follow-up may be an appropriate alternative for a subset of patients, given a good correlation. no radiologic or histologic parameters or clinical data on their own are distinctive enough to predict final classification of fels. objective: inconclusive results of the role of topoisomerase a (topa a) in breast carcinoma biology and therapy are partially due to numerous thresholds of positivity measured by immunohistochemical (ihc) analysis. in the presented study, different criteria found in literature of the subject were compared with results of topa a ihcevaluated material ( cases). in our study, we have the possibility to evaluate topa a in breast cancer cells using two methods (immunohistochemistry (ihc) and fluorescence in situ hybridization (fish)) in all cases. as a part of a broader study, we also were able to correlate the results of topa a in in situ carcinoma, benign lesions and breast tissue without pathological changes. we have also analyzed other prognostic and predictive factors as well as clinical outcome. method: using ihc and fish methods, we compared the percentage of stained cells, strength of nuclear stain, presence of co-expressed cytoplasmatic and membranous stain, amplification of topa a gene and also response to anthracyclin-containing chemotherapy from clinical data. results: analyzing the obtained data, we set our own cutoff points. we assumed that the best correlation both with genomic aberrations and other data listed above, which we considered important in this study, is when both the percentage and strength of stain are taken into account. in our study, a cutoff point for positive topa a status evaluated by ihc method is at least % of carcinoma cells with strong ( +) nuclear stain or at least % of carcinoma with intermediate ( +) nuclear stain. conclusion: the presented criteria as defined above correlate with amplification (ratio ≥ . ) and response to objective: the expression of tyrosine kinases in endometrial stromal tumors is controversial. several sporadic responses to imatinib have been observed in metastatic endometrial stromal sarcomas (ess). we therefore aimed to perform a retrospective analysis of possible molecular targets in ess: c-kit, pdgfra and egfr. method: paraffin blocks from patients with previous diagnosis of low-grade ess and high-grade endometrial sarcomas from nine different institutions were examined and reviewed. exons , , , and of the c-kit gene and exons and of the pdgfra gene were amplified by pcr and sequenced. the incidence and distribution of the kit, pdgfra, cd and calponin expression were examined by immunohistochemistry, and egfr amplification was assessed by fluorescence in situ hybridization (fish). results: no mutations in c-kit and pdgfra genes were detected. we observed several polymorphisms: i i, exon ( / ); v v, exon ( / ); p p, exon ( / ); and one case with a double polymorphism v v/ g g. overexpressions of kit, pdgfra, cd and calponin was detected in ( %), ( %), ( %) and ( %) cases, respectively, whereas amplification for egfr gene by fish was not detected. conclusion: expression of kit in cases of ess is infrequent and always weak. pdgfra expression was observed in % of the cases studied. we did not observe any mutations of c-kit and pdgfra or amplification of the egfr gene. so it is unlikely that patients with ess can benefit from therapies with anti-egfr and/or c-kit tyrosine kinase inhibitors. uterine tumors resembling ovarian sex cord tumors. a study of five cases e. bakula-zalewska*, a. nasierowska-guttmejer, a. daska-bidziska, m. bidziski *institute of oncology, dept. of pathology, warsaw, poland objective: uterine tumours resembling ovarian sex cord tumours (utrosct) cause confusion with respect to nomenclature as well as diagnostic difficulties. these tumours belong to the group of low-grade malignant neoplasms, and their clinical course likely depends on the percentage of the sex cord-like component. morphologically, they can be divided into two groups: those with < % sex cord-like areas (type ) and those with more than % (type ). method: five patients with primary utrosct underwent treatment in the cancer center, warsaw, between and . biopsies or excisions from all tumours were examined microscopically and immunohistochemically (ih). treatment and follow-up were correlated to histopathological diagnosis. results: patients ranged from to years of age and tumour size from to cm. tumours contained % to % of sex cord component (two tumours of type and three of type ). by ih examination, the sex cord-like component was keratin-positive in all five cases, while the stromal component was positive for cd and negative for h-caldesmon. in addition, pgr positivity was found in all cases, while sma, ckae / and inhibin was positive in three. four patients were treated with gestagens in addition to surgery. no recurrences were noted in any of these four patients over a -to -year period of follow-up. conclusion: a correct subclassification of sarcomas of utrosct type is of crucial importance since most patients with these rare neoplasms respond well to gestagen therapy and have a good prognosis compared to other endometrial stromal sarcomas. cd expression in serous invasive ovarian cancer and serous ovarian borderline tumors w. pokieser*, s. hauptmann *wilhelminenspital wien, dept. of pathology, austria objective: cd is a cell surface glycoprotein that plays a critical role as adhesion molecule for the guidance of migration of neuronal cells during embryofetal development. it is also expressed on adult neuroendocrine cells, hematological neoplasms, as well as in various carcinomas. method: in this study, we retrospectively investigated the expression of cd in a series of serous ovarian carcinomas and serous borderline tumors and analyzed the significance of this marker with regard to patient outcome. neuroendocrine cells were determined by immunohistochemical positivity of highly specific chromogranin a. patients were treated homogenously by use of radical surgery and combined chemotherapy (carboplatin and taxol) in the case of carcinoma and of surgery alone for borderline tumors. follow-up data were available for all patients. results: sixty-seven cases ( %) of invasive carcinomas, but only of ( %) borderline tumors, were positive for cd (p> . ), indicating that this phenomenon is significantly more present in invasive lesions. cd expression in the borderline tumor was accompanied by the presence of single scattered chromogranin a-positive neuroendocrine tumor cells, whereas in serous ovarian carcinomas, coexpression was only seen in less than a quarter of cases. conclusion: our results demonstrate that cd positivity is much more frequently found in serous ovarian carcinomas than in serous borderline tumors; however, expression of cd had no prognostic effect. on the other hand, cd should not be used as a marker for neuroendocrine differentiation in serous neoplasm. because of different chemotherapies in neuroendocrine ovarian carcinomas, specific neuroendocrine markers like chromogranin a or synaptophysin are recommended in these cases. are the cancer-testis (ct) antigens reliable markers of stem cells in squamous cell carcinoma of vulva? j. sznurkowski*, a. zabrocki *medical university of gdadsk, dept. of gynaecological oncology, poland objective: vulvar cancer represents - % of all gynecological malignancies. squamous cell carcinoma is a predominating malignancy at this site as it accounts for approximately - % of vulvar cancers. analyses of humoral and cellular immune responses to autologous cancer determined cancer-testis (ct) antigens as potential targets for immunotherapy. as their expression has been identified in a variety of malignant neoplasms, they appeared as potential candidates for target therapy, i.e. t cell-mediated immunotherapy of cancer. method: seventy-six patients with verified histopathological diagnosis and full clinical history were included into the study. the slides were incubated with the monoclonal antibody against mage-a , mage-a and ny-eso- . all statistical analyses were performed with the statistical software statistica . the expression of ny-eso- was revealed in one case. the expression of mage-a and magea- was identified in % and % primary tumors, % and % lymph node metastases and % and % local recurrences, respectively. cytoplasmatic expression of these antigens was identified in suprabasal and squamoid cells of scc ( fig. objective: the objective was to investigate the expression of c-kit protein in salivary gland carcinomas and to correlate it to prognosis. method: immunohistochemistry for c-kit protein was performed in carcinomas using formalin-fixed paraffinembedded sections. for the evaluation of reactivity of tumor cells, a combination of the cytoplasmatic and/or membranous staining and the percentage of positive cells were applied. only cases without any staining pattern were considered negative. for prognostic correlation, univariate disease-specific survival curves were calculated by the kaplan-meier method and distributions were compared using the log-rank test. results: of all cases, only six were c-kit-negative, including three low-grade mucoepidermoid carcinomas, two carcinomas ex pleomorphic adenoma and one salivary analogon of secretory mammary carcinoma. the group of ( %) positive tumors was dominated by nine adenoid cystic carcinomas (all but one revealing strong reaction), followed by five acinic cell, three each mucoepidermoid and salivary duct carcinoma, two carcinomas ex pleomorphic adenoma and five other tumors, in all of which positive staining ranged from % to %, with the immunoreaction varying from weak to strong. diseasespecific survival in c-kit-negative (n= ) carcinomas did not differ from that in positive (n= ) cases (χ = . ). conclusion: of all salivary gland carcinomas, only adenoid cystic carcinoma was regularly associated with c-kit expression. immunoreactivity in other subtypes, considering our as well as published data, greatly varies. c-kit expression harbours no significant prognostic information. results: aspirates were cellular showing groups with stroma and single cells. groups had a variable configuration with two major forms. some were large, three-dimensional and puzzle-like, with little ramifications. cells were densely packed and intimately related to metachromatic stroma. others were of medium size with prominent prolongations consisting of an inner core of hyaline stroma surrounded by tumoral cells. ten cases showed cylindromatous stromal structures. seven aspirations were cystic. stroma was homogeneous with non-fibrillary morphology. neoplastic cells showed a variable morphology from small, basaloid to larger ones with epithelioid morphology. moderate pleomorphism was seen in five cases. there were two misdiagnosis of acinic cell carcinoma, and two cases were considered as suspicious of malignancy. after a revision of these cases, two showed features of bca, while in the remaining two, the suspicious of malignancy persisted because of minimal amount of stroma, predominant epithelioid cell morphology and cellular density. conclusion: bca shows characteristic features that allow in many cases a precise diagnosis. it may be difficult to differentiate from epithelial-rich pleomorphic adenoma, and in these cases, a diagnosis including both possibilities seems preferable. in our series, the absence of stroma was responsible for the misdiagnosis with acinic cell carcinoma in two cases. objective: pheochromocytoma and paraganglioma are rare tumors arising from chromaffin cells. almost % of them are part of typical familial syndromes: von hippel-lindau disease (vhl), neurofibromatosis type (nf ), multiple endocrine neoplasia type (men ) and type (men ), pheocromocytoma-paraganglioma syndromes (pgls; sdhb, sdhc, and sdhd) and a newly described syndrome related to tmem gene mutations. recently, sdhb immunohistochemical analysis has been proposed as a promising molecular marker for succinate dehydrogenase mutation-related neoplasms (i.e. pgls). method: all cases of reported pheochromocytomas (n= ) and paragangliomas (n= ) between and were retrieved from the archives of the department of pathology of padova university. ffpe specimens were genetically characterized for familial syndromes. syndromic cases and a control group were semiquantitatively ( , +, +) evaluated for sdhb immunohistochemical expression. results: out of cases, cases showed sdhd (n= ), tmem (n= ), ret (n= ), men (n= ), vhl (n= ), or nf (n= ) germline mutations. the other six sporadic cases were evaluated as control. the three sdhd-mutated cases showed either negative (n= ) or + (n= ) sdhb immunostaining. completely negative staining was also observed in a sporadic case. a strong sdhb immunoreaction was observed in and a weak immunoreaction in four of the remaining cases. conclusion: sdhb immunohistochemical analysis, even not pgl-specific, can be used to triage genetic testing in pheochromocytoma/paraganglioma patients. further multiinstitutional studies should investigate the diagnostic power of this remarkable novel diagnostic tool. objective: inhibition of dipeptidyl peptidase- (dpp- ) activity by sitagliptin has been shown to improve glycemic control in patients with type diabetes mellitus (t dm) by prolonging the actions of incretin hormones, but the real impact of low-dose sitagliptin treatment on pancreatic lesions is almost unknown. this study aimed to evaluate the effects of sitagliptin on the biochemical and histological (pancreatic) parameters of zucker diabetic fatty (zdf, fa/fa) rats, an animal model of t dm. method: diabetic (fa/fa) zdf male rats were treated with vehicle or sitagliptin ( mg/kg body weight per day) during weeks (n= each). the following parameters were assessed: serum glycaemia, hba c, insulin and lipid profile; serum and pancreas oxidative stress (mda) and endocrine and exocrine pancreas histology, estimating and rating inflammatory infiltrate, fibrosis, vacuolization and congestion in a semiquantitative score ranging from (minimal) to (severe and extensive damage). results: sitagliptin in diabetic zdf rats promoted beneficial effects on dysglycaemia, dyslipidaemia, inflammatory profile and pancreatic oxidative stress. endocrine and exocrine pancreas presented a reduction/amelioration of fibrosis severity, inflammatory infiltrate, intra-islet vacuolation, and congestion vs the vehicle-treated diabetic rats. conclusion: the favourable biochemical profile promoted by sitagliptin in the diabetic rats, together with protection against endocrine and exocrine pancreatic lesions, might represent a further advantage of low doses of sitagliptin in the management of t dm. objective: several studies have suggested an increase in the incidence of right-sided colorectal carcinomas (crc). if the crc right shift is true, it is also likely that the incidence of right-sided advanced adenomas (rsaa) should increase in time. method: two large endoscopic datasets (retro and pbp) have been compared. retro contains data from the department of gastroenterology between and . pbp covers data from the polish national colorectal cancer screening program ( ) ( ) ( ) ( ) ( ) . only patients with ad-vanced adenomas who underwent total colonoscopy were included into the analysis. advanced adenoma was defined as: villous or tubulo-villous, adenoma with high-grade neoplasia or ≥ -cm lesion. two definitions of proximality were applied. the first defined right colon is proximal to the splenic flexure and the second, additionally, the descending colon. a logistic regression analysis was used to compare the incidence of rsaa between groups, adjusted for patients' age and sex. results: two hundred and , patients with advanced adenomas were found in two datasets, respectively. both groups differed significantly according to age and sex. to adjust for possible confounders, the multivariate model was fitted. conclusion: there was no statistically significant increase in the proportion of patients with rsaa between both groups when adjusted for patients' age and sex. objective: the aims of this study were to characterize the immunological microenvironment of tumour buds and evaluate the prognostic effect of immune markers and their ratios with tumour budding stratified by mismatch repair (mmr) status. method: paraffin-embedded tumour blocks from patients with colorectal cancer were cut in series and double-immunostained for ck with anti-cd , -cd , -cd , -cd , -cd , -cd , -cd , -foxp , -granzyme b, -mast cell tryptase, -cd , and -tia- and stratified into mmr-proficient and -deficient cases. tumour buds, immune cells and the ratio of immune cells/tumour buds was obtained. results: mmr-deficient tumours showed significantly less tumour budding (p< . ) and greater amounts of cd + , cd + , and granzyme b + cells (p< . ). high amounts of cd + , cd + , cd + , cd + , foxp + , granzyme b + and cd + were linked to improved survival (p< . ). prognostic effects differed between proficient and deficient tumours. marker/budding ratios showed stronger relative risks of death when compared to most markers alone. in lymph node-negative patients, high marker/budding ratios of cd (hr, . ( . - . )), cd (hr, . ( . - . )), cd (hr, . ( . - . )), foxp (hr, . ( . - . )) and granzyme b (hr, . ( . - . )) were independently linked to improved outcome. conclusion: marker/budding ratios for cd + , cd + , foxp + , granzyme b + and cd + are stronger prognostic features than the assessment of immune markers alone. these ratios are independent prognostic factors, particularly in lymph node-negative patients. moreover, our findings underline the considerable confounding effect of mmr status in the evaluation of immune response in colorectal cancer. human crypt stem cell dynamics in juvenile polyposis (jps) and peutz-jeghers (pjs) patients d. langeveld*, f. morsink, v. de boer, j. offerhaus, w. de leng *umc utrecht, dept. of pathology, the netherlands objective: pre-tumour progression is a process that precedes the adenoma-carcinoma sequence; colorectal mucosa appears normal, but cell kinetic and stem cell abnormalities suggest the existence of molecular alterations. pre-tumour progression can be made visible by studying methylation patterns, or tags, in stem cells of the crypt. shibata et al. ( ) showed higher crypt methylation heterogeneity in normal-appearing colonic crypts of familial adenomatous polyposis (fap) patients compared to normal crypt mucosa of non-fap colons. this indicates enhanced stem cell survival, which may be a predictive marker of increased neoplastic outgrowth since longer lived stem cells have the potential to gain more mutations. the objective of this study was to investigate stem cell survival in juvenile polyposis syndrome (jps) and peutz-jeghers syndrome (pjs), a biomarker predictive of pre-tumour progression. method: dna was isolated from laser-microdissected crypts of normal colonic jps and pjs tissue and agematched controls. dna was bisulphite-converted and methylation tags were created by amplifying the cpg islands in the csx gene. sequence analysis of this pool of methylation tags allows characterisation of the heterogeneity among the different tags recovered from multiple stem cell pools. objective: lymph node metastasis is an important prognostic factor in esophageal cancer. vascular endothelial growth factor c (vegf-c) and vascular endothelial growth factor d (vegf-d) are considered to induce both lymph node metastasis and lymphatic involvement in various cancers related to lymphangiogenesis. in many types of cancer, podoplanin (d - ) immunostaining has recently been used to detect lymphatic vessel invasion (lvi), but invasion detected using d - immunostaining for a predictor of nodal metastasis was controversial. we examined the relationship between vegf-c, vegf-d and lvi and clinicopathological features in patients with esophageal cancer who underwent esophagectomy. method: vegf-c, vegf-d and podoplanin expression was examined using immunohistochemical staining in surgically resected tissues. differences in categorical data were assessed by a chi-square test or fisher's exact test. results: podoplanin expression was observed in the endothelial cells of lymphatic channels. none of podoplanin-positive vessels were blood vessels. intratumoral lymph vessels were small and collapsed, while peritumoral lymphatic vessels with lvi were dilated and large. the expression of vegf-c and vegf-d protein was recognized in the cytoplasm of cancer cells, but not in the nucleus. we found that peritumoral lvi significantly correlated with lymph node metastasis (p= . ), pathologic stage (p= . ), tumor depth (p= . ) and tumor size (p= . ). the expression levels of vegf-c and vegf-d were associated with lvi. conclusion: vegf-c, vegf-d and peritumoral lvi may play an important role in lymphangiogenesis and the process of lymphatic metastasis of esophageal cancer. objective: for several anatomical localisations of extranodal marginal zone lymphoma of malt type (emzcl), an association with chronic inflammation caused by microbiological agents (e.g. helicobacter pylori in the stomach) has been described. in the lung, a link between lymphomagenesis and a defined causative organism is still missing. method: a comprehensive diversity survey using s rdna library construction followed by restriction fragment length polymorphism (rflp) analysis, sequencing and phylogenetic tree construction was employed on nine cases each of balt lymphoma and control lung tissues (normal fetal lung, pneumonitis, cancer). this highly sensitive method, hereafter termed "sharp-screening", allows for the identification of the entire bacterial population in the tissue in a cultivation-independent manner. following the results of sharp screening, an alcaligenaceae-specific pcr assay was employed on additional balt lymphoma cases and control lung tissues. results: in eight of the nine cases of balt lymphoma, bacteria of the alcaligenaceae family (alcaligenes, achromobacter, akiw ) were detected, whereas none of the control cases showed the presence of these clades. the subsequently performed alcaligenaceae-specific pcr assay detected bacteria of the alcaligenaceae family in of the independently investigated balt lymphoma cases and in only one of control lung tissues. conclusion: s rdna library construction in combination with rflp screening and phylogenetic analyses (sharp-screening) is a novel, molecular and cultivationindependent tool for the analysis of the microbial environment in chronic inflammation processes. betaproteobacteria of the alcaligenaceae family may be associated with and possibly involved in the lymphomagenesis of balt lymphomas. objective: diffuse large b cell lymphoma (dlbcl) is the most common type of non-hodgkin lymphoma. recently, major advances in the molecular characterization of dlbcl have been made through gene expression profiling studies that provided more insight into the biological heterogeneity within dlbcl. one of the identified molecular subgroups has been termed the "b-cell receptor (bcr)/proliferation cluster" due to the increased expression of bcr signaling cascade components including the spleen tyrosine kinase (syk), which has emerged as an important therapeutic target within tumors of this subgroup. in fact, a recent clinical trial with a syk inhibitor on uncharacterized dlbcls has shown promising results, but only in a subset of patients. method: we employ a novel quantitative tissue-based immunohistochemical (ihc) methodology using markers of activated bcr signaling to assess the activity of the bcr pathway in formalin-fixed paraffin embedded primary dlbcls. results: antibodies to phosphorylated forms of lyn, syk, btk, blnk and akt as well as foxo antibody represent attractive ihc markers to evaluate the activation of bcr signaling and comprise the basis of a biological signature of activated bcr signaling in dlbcl. conclusion: we report on the development of a quantitative tissue-based immunohistochemical (ihc) methodology employing activation-specific antibodies against multiple components of the bcr signaling pathway that will assess the activity of the bcr pathway in formalin-fixed paraffinembedded primary dlbcls. this approach will identify the subset of patient tumors that are actively signaling through the bcr pathway and, therefore, will predict therapeutic responsiveness to targeted inhibition of bcr signaling. enteropathy-associated t cell lymphoma: a clinicopathologic study focussing on prognostic factors and the frequency of defective t cell receptor chains p. czapiewski*, p. fisch, a. schmitt-graeff *medical university of gdansk, dept. of pathology, poland objective: enteropathy-associated t cell lymphoma (eatl) is a rare highly aggressive t cell neoplasm divided into type i (cd − , cd − ) and type ii (cd + , cd + the presence of intra-tumoral neutrophils is an independent prognostic factor in early-stage non-small cell lung carcinomas m. ilie*, v. hofman, c. butori, c. marquette, n. vénissac, j. mouroux, p. hofman *chu de nice, lpce, france objective: cancer-related inflammation has emerged as the seventh hallmark of cancer. in the tumor microenvironment, inflammatory cells, including neutrophils, influence almost every aspect of cancer initiation and progression, including tissue invasion and metastatic potential. however, the influence of intra-tumoral neutrophils in non-small cell lung carcinoma (nsclc) progression is currently unknown. this study was conducted in order to correlate the number of tumor-associated neutrophils (tans) and the prognostic of stage i nsclc. method: immunostainings on stage i ( t n and t n ) nsclc included on tissue microarrays were performed with anti-myeloperoxidase and anti-cd b anti- results: the patients comprised ten women and one man (mean age . ± . years, age range - years). immunofluorescence microscopy showed intense linear staining for igg and c along glomerular and distal tubular basement membrane in all patients. by light microscopy, irregular diffuse or focal extracapillary crescentic glomerulonephritis was found in all patients, associated with necrosis of varying extent in nine. the percentage of crescents was % to %. extraglomerular necrotizing vasculitis of small arteries was present in six patients, and in one, elastica destruction indicative of previous vasculitis was observed. an assessment of the age of histopathologic lesions showed lesions of varying age in all biopsies. active lesions were predominant in four and chronic in three, while active and chronic lesions of equal distribution were seen in four biopsies. conclusion: all our patients with coexisting anti-gbm antibodies and anca were older and all had renal histopathologic lesions of varying age, active and chronic. these results confirm a hypothesis that anti-gbm antibody production may start after injury to the glomerular basement membrane by anca. renal amyloidosis secondary to behçet's disease: clinicopathologic features of cases k. kosemehmetoglu*, d. baydar *ankara ataturk eah, dept. of pathology, turkey objective: amyloidosis is a rare complication of behçet's disease (bd). we describe ten patients with bd and renal amyloidosis. method: kidney biopsy slides were culled from the archives and reviewed. intensity and relative distribution of amyloid deposition in renal compartments were noted. glomerular deposits were classified as hilar (pattern i), mesangial nodular (pattern ii) and mesangiocapillary (pattern iii). chronic tubulointerstitial damage was graded from to ( %, -< %, -< % and ≥ %). amyloid typing was performed by immunohistochemistry. clinical information was gathered from hospital records and computer-based patient data system. results: all patients were male. their age ranged from to . full-blown nephrotic syndrome was present in six cases. duration of bd before diagnosis of amyloidosis differed from to months (mean ), and mean follow-up period was . months. immunohistochemical typing revealed aa amyloid in all ten. four cases showed glomerular-dominant and two cases vascular-dominant amyloid deposition; the rest were co-dominant glomerular and vascular. the frequencies of glomerular amyloid patterns were % for hilar, % for mesangial nodular and % for mesangiocapillary. hilar pattern was associated with vascular-dominant amyloid deposits. severe proteinuria correlated with the presence of mesangiocapillary pattern. grade iii chronic tubulointerstitial injury was detected in three cases. patients were treated with steroids and/or different immunosuppressives. among them, two progressed to endstage kidney failure, one showed remission, two were lost to follow-up, and rest had persistent proteinuria. conclusion: renal amyloidosis in bd has a diverse pathology in terms of preferential location of amyloid deposition and its intensity. patients follow variable clinical courses accordingly. objective: kras is an oncogene that is commonly mutated in various malignancies. mutations in kras are associated with lack of response to anti-egfr treatment in colon and lung carcinomas, and patients must undergo genetic testing to be eligible for treatment. several genetic tests for kras mutation analysis exist, but most are either too expensive or lack the sensitivity to identify a mutation if the percentage of tumor cells in the sample is small. the purpose of the present study was to develop a novel kras mutation detection method that is both cost-effective and sensitive. method: we designed a locked nucleic acid (lna)containing probe that inhibits the amplification of wildtype kras, thereby giving an advantage to mutated kras in pcr reaction (pcr clamping). the sensitivity of this method was evaluated using serial dilutions of plasmids containing wild-type and mutated kras fragments. the method was further evaluated on archived tissue samples of colon carcinoma and compared to direct sequencing and high resolution melting (hrm) methods. results: lna-based probe successfully inhibited the amplification of wild-type kras. the method enabled the detection of as little as % mutated dna in the sample analyzed. the method was superior to direct sequencing when the percentage of malignant cells in the sample was % or less. there was % correlation between the results of pcr clamping and hrm. objective: a newly discovered gene, jk- (fam b), located in chromosome p . plays an important role in the molecular pathogenesis of esophageal cancer. this study aims at examining the role of this gene in colorectal tumors by analyzing the differences in gene mrna expression in both benign and malignant colorectal tumors. the study was performed on colorectal adenocarcinoma, benign adenoma and nonneoplastic colorectal tissues. gene expression levels were determined using real-time pcr. the expression levels were calculated as a ratio of the ct value for jk- to gapdh and expressed as inverse ratio. results: the jk mrna was detected in all colorectal tissues. the mean inverse ratio for jk- gene in adenoma samples was . ± . , which was significantly higher than in non-tumor samples which was . ± . (p= . ). on the other hand, cancer samples had a mean inverse ratio of . ± . , which was significantly lower than in non-tumor tissues (p< . ). the under-expression of the gene in cancer suggests that loss of gene expression may promote tumor progression, that it may act as a tumor suppressor gene, or that it relates to certain genetic events in tumor pathogenesis. however, over-expression of jk- in adenoma samples may indicate that jk- has a functional role in dysplastic benign colon tumors, a function that is disrupted once the tumor progresses to a more invasive form. wednesday, objective: invasive breast carcinoma represents are characterized by invasion of adjacent tissues and a marked tendency to metastasize to distant sites. in order to determine specific prognostics factors, we established histopathologic, immunohistochemical and morphometric profiles of these types of carcinoma. method: in our study, we investigate cases diagnosed with invasive breast carcinoma in which we established microscopic characterization, immunohistochemical profiles (expression of proliferation markers, steroid receptors and her ) and computer-assisted morphometric profiles by determining the mean values for nuclear area, cellular area and n/c ratio with lucia net software. the distribution of markers and histological grade among types of carcinoma was statistically analyzed using the χ test. results: the histological type of invasive carcinoma was: . % ductal carcinoma, % mixed ductal and lobular carcinoma, and . % lobular carcinoma. according to histological differentiation, % was g , % was g and % was g . immunohistochemical expression of er was positive in % and negative in %, for pr was positive in % and negative in % and the expression of her was positive in % and negative in %. ki was intensely positive in %, moderately positive in % and intensely positive in %. p was positive in % cases and negative in % of cases. morphometric analysis revealed that mean nuclear area and n/c ratio increase are concordant with grading, but mean cytoplasmatic area has no characteristic variation. conclusion: the expressions of proliferation markers correlate with carcinoma grading. poorly differentiated and triple-negative carcinoma (er, pr and her ) with intense expression of proliferation markers and increase mean nuclear area have negative prognosis. objective: mapping tumour cell protein networks in routinely processed clinical samples, like formalin-fixed and paraffin-embedded (ffpe) tissues, will be critical for realizing the promise of personalized molecular therapy as only a subset of patients will respond. therefore, techniques being able to detect the entire spectrum of deregulated pathways in tumors before, during, and after treatment are required to assess success or failure of targeted therapies. the aim of our study was to monitor protein networks in ffpe breast cancer tissues with special emphasis on epidermal growth factor receptor (her )mediated signaling pathways to identify and validate new disease markers in order to predict response to current treatments. method: using a recently developed technology for the extraction of full-length proteins from ffpe tissues, we analyzed molecules involved in her -related signaling by reverse phase protein microarray (rppa) in a series of ffpe breast cancer patients. to this end, we evaluated > commercial antibodies for specificity in lysates from ffpe breast cancer samples in western blots and rppa. results: we found her (r's= . ), the egfr (r's= . ) and the receptor of upa (r's= . ) correlating with her . however, we could not demonstrate any significant correlation between her expression and clinicopathological parameters. conclusion: our results uncover molecules linked to her signaling and cancer progression. thus, these finings provide possible new targets for cancer treatment and may assist in optimal patient selection. objective: the pi k/akt/mtor pathway has a fundamental role in signal transduction, and its alterations are of pathogenetic role in human cancer, including breast carcinomas (bc). several drugs may interfere with the altered activation of this pathway, including the recently developed mtor inhibitors. however, few data are available concerning mtor levels in human breast cancer. method: the aim of the present study was to analyze the immunohistochemical expression of mtor and p s k in a consecutive series of bc, with long-term follow-up which had been studied for er, pgr, her , p and mib expression and for pi k and akt mutations. results: mtor and p s k expression were positively associated (p = . ), but were not related to other molecular markers with the exception of high p s k being associated with pi k mutations (p= . ). in the whole series of cases, mtor and p s k are not associated with clinical outcome. however, high mtor are associated with prolonged dfs and os in node-positive cases, in st. gallen high-risk group, and in patients treated with chemotherapy. high p s k expression was associated with prolonged dfs and os in st. gallen high-risk group. conclusion: our data show that mtor and p s k are promising biomarkers in bc whose level of expression may be of prognostic value. objective: lymphangiogenesis occurrence and the biomechanisms in lymph nodes are still unknown. the immunohistochemical application of the usually known lymphatic markers vegfr- , lyve- , prox- , and podoplanin was explored to study lymphatic vessels in tumour metastasis and non-metastatic lymph nodes of breast cancer cases to understand the metastization pathway. method: lymph nodes without and with metastases from cases of invasive breast cancer were selected. serial sections were immunostained for vegfr- , lyve- , prox- , and podoplanin using immunohistochemical protocols in accordance with the respective recommendations. the microanatomical distribution of each antibody in the lymph node, different cell type expression, adjacent mesenchymal structure positivity and neoplastic cell positivity were validated and reported as negative and positive (with the cutoff +, ++, +++ for the neoplastic cells). results: no markedly or obvious staining was obtained for lymphatic vessels. positive staining of metastatic neoplastic cells, lymphoid cells of germinal centres and node pericapsular fatty tissue has been observed. immunostained neoplastic cells revealed a predominant subcapsular and perihilar location. conclusion: the applied primary antibodies were unsuccessfully used as lymphatic markers. the spread of neoplastic cells expressing these markers may have acquired particular characteristics to develop lymphatic dissemination until reaching lymph nodes. lymphangiogenesis in breast cancer continues without demonstration in lymph nodes, while the fatty tissue can be implicated in epithelial mesenchymal transition (emt). this thesis turned up by the proximity of the positive neoplastic cells to the mesenchymal components of surrounding lymph node tissue as adipocytes were immunoreactive. objective: there is a significant percentage of patients with metastatic breast cancer and her receptor overexpression that do not respond to therapy directed with trastuzumab. in these patients, overall survival and diseasefree time are significantly shorter. several mechanisms that explain the resistance to therapy with trastuzumab have been reported. this paper aims to address the study of alterations in one of the signalling pathways of her related trastuzumab in response to breast cancer in order to incorporate into the clinical routine of other predictive biomarkers of response to therapy. method: the study comprised cases of metastatic breast carcinoma with her overexpression (herceptest +) or gene amplification (fish). all patients were treated with trastuzumab. the assessment of pten and pakt expression was based on staining intensity and percentage of stained cells. results: our series showed a statistically significant association between a positive expression of pten and a reduced expression of pakt (p = . ). the positive expression of pten was associated with a better treatment response rate (p = . ). it was also noted that the reduction of pten was associated with a shorter time of progression (p= . ) and a lower overall survival (p= . ). pakt overexpression was associated with a lower overall survival (p= . ). conclusion: our study shows that immunohistochemical expression of pten and pakt could be used as both prognostic and predictive biomarkers in breast cancer. objective: vascular endothelial growth factor (vegf) has been shown to play a major role in tumor angiogenesis in many studies. this study aimed at evaluating the relationship between vegf expression and lymph node metastasis in patients with invasive ductal carcinoma (idc) of the breast. method: in a case-control setting, we evaluated specimens of idc in patients with and without axillary lymph node metastasis. the immunohistochemistry staining was employed for evaluating the expression of vegf in the specimens of both groups. the rate of positive specimens was then compared between the groups. the relationship between the vegf expression and the size and grade of tumors were assessed. results: eighty women with idc of breast, cases with axillary lymph node metastasis and controls without any evidence of axillary lymph node involvement, were enrolled. there were ( %) and ( %) cases with positive expression of vegf in the case and control groups, respectively (p= . ). the mean size of tumor was significantly higher in patients with positive expression of vegf ( . ± . cm vs . ± . cm, p= . ). the current study showed that there is no significant relation between the rate of expression of vegf in cases of idc of the breast and the axillary lymph node metastasis; however, there may be a relation between vegf and the size of the tumor. abnormal membrane cytoskeletal cross-linker ezrin cellular distribution is associated with clinicopathological features in invasive breast cancer a. halon*, p. donizy, l. halon, r. matkowski *wroclaw medical university, poland objective: ezrin is member of the erm (ezrin/radixin/ moezin) protein family which acts as a membrane organizer and linker between the plasma membrane and cytoskeleton and plays a key role in the control of cell morphology, adhesion, polarity, motility and cell survival. the aim of the present study was to elucidate the clinical significance of subcellular expression pattern of ezrin in patient with invasive breast cancer (bc). method: immunohistochemistry for ezrin monoclonal antibody was performed on paraffin-embedded specimens from patients with bc. immunoreactivity was semiquantitatively scored according to remmele immunoreactive score (irs). results: expression patterns of ezrin were divided into cytoplasmic ( , . %), membranous ( , . %), nuclear ( , . %) and apical ( , . %). cytoplasmic pattern was the only one observed in cases ( %) and mixed immunotopography in the others. there were significant positive associations between apical and membranous ezrin localization and favorable tumor characteristics and lack of lymph node metastases. cytoplasmic ezrin staining was associated with adverse clinical outcome. lack of apical ezrin was likely to present with bone metastases in the absence of visceral disease. membranous ezrin localization was correlated with good prognosis both for csos (p= . ) and dfs (p= . ). a multivariate analysis demonstrated that membranous ezrin distribution is an independent prognostic factor (p= . ). conclusion: the switch of ezrin localization from the apical and the membrane to the cytoplasm is correlated with dedifferentiation and adverse features in bc. cellular ezrin distribution contributed to discriminating between patients with lymph node and distant metastases and those with better prognosis. objective: epidermal growth factor receptor (egfr) is a tyrosine kinase growth factor receptor expressed in a variety of human cancers and related with poor prognosis. recently, the astounding development of molecule-targeting drugs such as anti-egfr-targeting drug has attracted considerable interest of oncologists. the objective of this study was to evaluate the expression of egfr in breast carcinomas and its possible relationship with different clinical-pathological parameters. method: one hundred and seventeen breast carcinomas were included in this study. using a standard immunohistochemical technique, the expression of egfr, her , and hormone receptors was investigated. in addition, haematoxylin and eosin-stained sections of these tumors were studied for several morphological parameters. results: egfr-positive expression was detected in nine tumors ( . %). expression of egfr was positively associated with high histological grade (p= . ), highgrade comedo-type necrosis (p= . ), high degree of stroma lymphocyte infiltration (p= . ), pushing tumor margin (p= . ), and hormone receptor-negative her negative status (triple-negative phenotype, p= . ). no significant difference of egfr expression according to age was recognized. conclusion: egfr expression is more often present in the group of triple-negative breast carcinomas, and its expression is associated with features of poor prognosis. egfr expression might be important as a potential target for molecular therapy in breast cancer. objective: the purpose of the study was to assess morphometric parameters of nuclei and nucleoli in invasive ductal breast carcinomas and to compare them with axillary lymph node status. method: histologic sections from tumors were stained with methyl green-pyronin y and measurements were made using a computerized image analysis. the following seven parameters were evaluated: the nucleolar area, long-to-short nucleolar axis ratio, nucleolar shape parameter assessing the degree of nucleolar roundness, the nuclear area, long-toshort nuclear axis ratio, number of nucleoli in the nucleus and the percentage of the nuclear area occupied by nucleoli. results: a statistically significant association between a nuclear area, long-to-short nuclear and nucleolar axis ratio and bloom-richardson (b-r) histologic grade of breast tumors was found in relation to the axillary lymph node status. in b-r grade tumors, the nuclear area was significantly larger in tumors with negative versus positive axillary lymph nodes (p< . ). long-to-short nuclear and nucleolar axis ratios were higher in b-r grade tumors with negative versus positive lymph nodes (p< . ), i.e. the shape of nuclei and nucleoli of tumor cells from node-negative cancers was more elliptic as compared to node-positive ones. objective: the urokinase-type plasminogen activator (upa) and its main inhibitor pai- facilitate migration and invasion of cells in physiological and pathological contexts. both factors are clinically applicable predictive markers in node-negative breast cancer patients used to stratify patients for adjuvant chemotherapy. besides their classical functions within the plasminogen activation system, these factors are shown to play important roles in cell signaling, thus providing new possibilities to interfere with cancer progression and metastasis. although such networks are well described in cell culture systems, their analysis in tissues was hindered because protein extraction and direct analysis was possible only from fresh or frozen material, which may be not available in routine laboratory setting. method: we extracted proteins from formalin-fixed paraffin-embedded tissues, being the main source of archived patient samples in pathology institutes worldwide, by a new protocol developed in our laboratory and analyzed the extracts for interactions of upa and pai- with molecules belonging to signaling cascades relevant in cancer progression in a collective of breast cancer patients using protein microarrays. results: a substantial correlation of upa with erk and stat expression was ascertained, while pai- correlates with akt activation and egfr-related signaling. statistical analysis of upa and pai- protein expression did not reveal a significant correlation of upa or pai- with staging, grading or age of the patients. objective: breast cancer is one of the most common human neoplasms accounting for approximately one quarter of all cancers in females. in young women, the risk is low, but because of the density of breast tissue, by the time a lump can be felt, the cancer is often advanced. fine needle aspiration of breast lesions is routinely performed in our hospital. we reviewed our archives and found the cases of breast cancer in women under years old. method: between january and march , fine needle aspirates in breast lesions were performed in our institution. the aspiration was done using a -gauge needle. conventional and thinprep smears were prepared. estrogen and progesterone receptor studies were part of the routine workup of a breast cancer as well as other markers (c-erb-b , p , e-cadherin, gcdfp- ). results: one hundred and fifty-eight of the cases were carcinomas. ten of these patients were under years old (mean age . ). in eight of these patients, cytology correctly diagnosed seven ductal and a tubular carcinoma. in two cases, the cytologic diagnosis was suggestive of carcinoma, and the excisional biopsy revealed a lobular and a mixed carcinoma. conclusion: it is mandatory in young women that a breast lesion be identified using mainly ultrasound and fine needle aspiration, which are simple and well-tolerated diagnostic tools. however, mammogram may also be recommended for younger women when a family history of breast cancer or other risk factors exist. mgmt protein expression as a potential prognostic marker in breast cancer j. objective: sentinel lymph node biopsy (slnb) is rapidly becoming the standard of care in staging the axilla for early-stage, clinically node-negative breast cancer. this audit reviews the laboratory handling and pathological reporting of sentinel nodes (sns) across kent and has been performed at the request of the kent cancer network breast disease oriented group (dog). method: a retrospective analysis of consecutive breast sn cases from each of the five kent hospitals was carried out. all reports and slides of positive sns were reviewed. detailed structured questionnaire-based enquiries to lead breast pathologists were made. data were entered in microsoft excel and analysed in relation to the standards. all aspects of the pathology service were examined, including macroscopic data (number of sns/case, dimensions, sampling and slicing details) and microscopic data (performance of levels and immunohistochemistry, tnm staging and snomed coding). results: one site was unaware of radioactivity guidelines while another was unaware of national guidelines. twenty-eight percent to % of sns were bisected longitudinally and levels were sparingly used, except at site. tnm staging and snomed coding in the reports were variable. conclusion: this audit demonstrates a substantial variation in sn handling and reporting practice across kent. since the quality of the pathology service in breast cancer is of paramount importance, it is imperative that a standardised approach is achieved in the handling and reporting of sns. to achieve this, recommendations have been made and agreed upon, the success of which will be measured in a reaudit next year. objective: background: the study analyzes the positivism of the p protein and the oncoprotein c-erbb and their implication in the appreciation of prognostic invasive mammary carcinomas. method: in invasive mammary carcinomas, we have realized the immunohistochemical determination of the p protein with the avidine-biotine complex method (abc) using the dako p mouse monoclonal antibody (dilution : ). in invasive mammary carcinomas, we have determined immunohistochemically the c-erbb oncoprotein with the dako rabbit polyclonal antibody, human anti-protein c-erbb (dilution : ). the tumoral tissues were fixed in % formaldehyde and included in paraffin. results: out of invasive mammary carcinomas, ( %) have been p -positive, and out of carcinomas, ( . %) overexpressed the c-erbb oncoprotein. in the studied casuistic, we did not remark any connection between the p expression and the overexpression of c-erbb . conclusion: the presently best-known mutant gene in invasive mammary cancer seems to be the p tumor suppressor gene. we did not remark any connection between the p protein expression and the tumors' sizes, patients' ages, the clinical stage of the disease and the overexpression of c-erbb . the usage of the c-erbb status (her- /neu) in patients suffering invasive mammary carcinomas is a predictor of therapy response. objective: there are only a few entities in the breast which show a chondroid differentiation. the differential diagnosis varies from the harmless choristoma to the also benign pleomorphic adenoma, to the syringioma and phylloides tumour with uncertain dignity to the high malignant metaplastic carcinoma. the differentiation between these different neoplasias is essential for the patient because of the following therapeutical procedures and the prognostic outcome. the final diagnosis in a mammary punch biopsy may be difficult or impossible because of the small specimen and the heterogeneity of the tumour. method: our specimen is taken from a -year-old female. with mammography, new polymorph calcifications were found in the left breast. the results were classified as birads and surgical excision was recommended. one month after mammography, a stereotactic wire-marked diagnostic excision was performed. results: in histological examination, a chondroid proliferation appears with a beginning metaplastic ossification and two small foci of spindle cells and myxoid appearance. no suspicion for malignancy was found in the whole specimen. a chondroid choristoma in the left breast was diagnosed. in mammographic control months later, no calcifications were detectable. so the tumour was totally removed. conclusion: a choristoma of the breast is very rare. in literature, it is described as choristoma (tavassoli ) or as true chondroma (rosen and oberman ). the secured demarcation between the differential diagnosis is only possible at the in whole removed and nearly totally examined tumour. a chondroid differentiation in a mammary punch biopsy should be classified in the category b . a diagnostic excision should be always performed. objective: vascular tumors of the breast are very uncommon, consisting of variants of hemangioma and angiosarcoma. although angiosarcoma related to chronic lymphoedema is the most frequent, it only represents . % of breast tumors. usually, primary angiosarcoma involves young pregnant females, with only six cases occurring in males reported to date. epithelioid angiosarcoma is very unusual; their presence in males has been reported only once before in a young patient. method: this is the second case report of an epithelioid angiosarcoma in a male breast but the first one of poorly differentiated type. it developed in a -year-old patient, unrelated to hormonal dysfunction or treatment. results: the tumor located in the breast parenchyma had a -cm diameter and solid firm multinodular grey colour areas admixed with cystic spaces with hemorrhagic content. microscopically, the tumor was composed of mainly polygonal epithelioid tumor cells with abundant eosinophilic or pale cytoplasm and pleomorphic nuclei, arranged in solid areas and admixed with few spindle cells. less than % of the tumor was composed of more differentiated areas. the tumor cells in both solid and cystic areas were positive for cd and vimentin but negative for cd , er, pr, hmb- , and s- protein. pan-ck was positive in both epithelioid and spindle tumor cells. conclusion: immunohistochemistry is very useful in order to establish a correct diagnosis; factor viii, cd and cd can be used singly or in combination, although they may be lost in high-grade tumors. epithelial markers may be also positive in epithelioid angiosarcoma. wednesday, september , basement the semiquantitative immunohistochemical evaluation of the lymphangiogenesis density at cutaneous melanoma and its correlation with the sentinel lymph node status p. buzrla* *faculty hospital ostrava, institute of pathology, czech republic objective: lymphangiogenesis is a predictor of the sentinel lymph node status at cutaneous malignant melanoma. the aim of our investigation was the immunohistochemical semiquantitative evaluation of the lymphangiogenesis density and its correlation with the sentinel lymph node status. the file comprises patients with cutaneous melanoma, of them having the positive sentinel lymph node. the results of the investigation are partially in harmony with another study. method: for this investigation, the files of patients with cutaneous melanoma radically excised and the sentinel lymph nodes were extirpated. the lymphangiogenesis density was immunohistochemically evaluated using cd and d - (podoplanin). the localization of the lymphangiogenesis in cutaneous melanomas was determined and the lymphatic vessels were counted in mm . after that, we evaluated the correlation between the lymphangiogenesis localization and the invasion depth according to clark, following the correlation of the lymhangiogenesis density with the sentinel lymph node status. results: lymphangiogenesis was dominant in the peritumorous localization of cutaneous melanomas. the lymphangiogenesis density and its localization have no influence upon the lymph node status. the correlation of the depth invasion with the lymph node status was not confirmed. the results of the lymphangiogenesis localization are according to other study papers. we did not confirm the correlation between the lymphangiogenesis density and the sentinel lymph node status. it is necessary to extend the set of investigated patients with cutaneous melanoma and to use the lyve- antibody. the extension of the count of patients with cutaneous melanoma and using other antibodies such as lyve- are necessary in consecutive evaluations. objective: melanoma is a malignant tumor of the neural crest-derived cells named melanocytes. it is an aggressive neoplasm due to its high rate of proliferation and invasion. disruptions in the cellular signaling pathways promote melanoma development and progression. aberrant notch activation has been related to a variety of human neoplasms. we investigate the potential involvement of notch signaling in melanoma development and progression. for this purpose, we analyzed the expression of three essential components of the notch pathway: notch , adam and adam /tace. method: melanoma samples from seven patients were tested and compared with normal skin samples. immunofluorescent staining was performed on -μm-thick cryosections. primary antibodies were applied and incubated overnight at °c. to detect the primary immune reaction, we used fitc-labeled goat anti-mouse secondary antibodies. the samples were examined using light and uv microscopy. for the western blot technique, the western-breeze kit was used. results: notch expression in normal skin was detected in the epidermis and also in hair follicles, sweat glands, sebaceous glands and blood vessels. melanoma cells showed moderate to high levels of expression of notch . adam expression was detected in the epidermis and also in hair follicles. in some melanoma samples, adam over-expression was observed. tace/adam expression was detected only in the epidermis. western blot analysis confirmed the presence of these proteins in cytoplasmatic and nuclear lysates. conclusion: we observed that the notch signaling pathway is activated in melanoma cells. critical members of the notch pathway are potential therapy targets. objective: amelanotic melanoma is a subtype of cutaneous melanoma characterized by little or no pigment on visual inspection; it represents - % of all melanomas. the lack of pigment makes it a great mimic of other benign and malignant lesions, a fact that can mislead the correct diagnosis. method: the study included a group of cases of amelanotic melanomas, aged - years. most of the tumors were thinner than mm breslow and clark levels i to iv. the diagnostic was formulated based on special coloration for the melanic pigment (masson-fontana) and using immunohistochemical techniques (hmb- , vim, s- ). the simultaneous positivity of these markers has shown the melanic aspect of the tumor. bcl- , ki- and p- was performed by standard immunohistochemical staining using monoclonal antibodies. results: bcl- expression was positive in % cases; ki- expression was positive in most of the cases, values between % and %; and p- expression was positive in most of the cases, values between % and %. conclusion: our frequency detection is not related with the size of the tumor, depth, ulceration or mitotic activity. most lesions present expression of bcl- in % of the cases and were associated with good prognosis; p- expression is between % and % and was associated with poor survival. although ki- expression is between % and %, it is not related with the prognosis. it was suggested that p- and bcl- expression could be useful in predicting the biological behaviour. neoplastic embolisations were identified in two cases of the study group. objective: desmoplastic melanomas represent an extreme degree of fibroblastic or myofibroblastic metaplasia accompanied by abundant collagen synthesis. those tumors are in general associated with high incidence of recurrence and metastasis and a poor prognosis. method: a patient p.c., male, years old, presents at the clinical exam a right calcaneo-plantar ulcero-proliferative and multinodular lesion ( / , / , cm), with a few months' course; there is local discomfort and functional impairment. clinical diagnostic was that of kaposi sarcoma and for the histopathology was sent a cutaneous specimen of / / cm that contains the lesion. the specimens were examined using basic histological techniques and immunohistochemistry with antibodies anti-s protein, anti-cd , anti-desmin, melan a, hmb- and pan ck. results: microscopically, it was identified as tumoral proliferation with spindle cells, uniform nucleus, some of them hyperchromatic, prominent nucleoli, some tumoral cells with brown melanic pigment and a rich mitotic activity. immunohistochemical expression was intensely positive for s protein and melan a and negative for hmb , desmin, cd , pan ck, associated with focal lymphocytic inflammatory infiltrate. tumoral proliferation includes adnexal structures of the skin and is ulcerated. the histopathological aspects lead to a desmoplastic melanoma. in spite of the large excision margins sidewise and in the depth, the tumor has reappeared locally after months from excision and treatment, with inguinal ipsilateral metastasis. conclusion: the presented facts suggest a more close attention to the clinical and histopathological differential diagnosis of the tumoral cutaneous lesions in the lower limb extremities. this could influence the surgical therapeutic act. was /nmc. histological exam of skin biopsies revealed in dermis a significant proliferation of irregular, small vascular channels, covered by a single layer of atypical endothelial cells. these channels were predominately arranged around preexistent mature vessels and adnexal structures. they were placed parallel to the epidermis and were marked by cd antibody. with this antibody, we observed that majority of these vessels were intermediary vessels whose proliferation could be suppressed by anti-angiogenic therapy. based on clinical, histological and immunohistochemical aspects, diagnosis of kaposi's sarcoma in early stage was made. chemotherapy was initiated (haart scheme) for months. after this treatment, skin plaques have disappeared and serum level of cd increased at /nmc. conclusion: it is very important to recognize early phase of kaposi's sarcoma and to initiate the treatment in order to improve prognosis of this lesion in hiv-infected and also immusdepressed patients. the objective: skin cancers are one of the most common human malignancies. although the prognosis of these patients has improved in the last decades, there is still a need for novel treatment modalities and prevention from recurrence after the treatment of skin tumors. p is a tumor-inhibiting gene which is believed to be defective in many malignant situations. ki is a non-histonic protein which mainly interferes with the proliferation and has many controlling effects during the cell cycle. this study aimed at evaluating p and ki- expression in skin epithelial tumors by immunohistochemical method. method: in a descriptive setting, biopsy samples- basal cell carcinomas (bccs), ten squamous cell carcinomas (sccs), eight keratoacanthomas (kas) and two tricoepitheliomas (tes)-were immunohistochemically evaluated for p and ki expression during a -month period. the incidence and expression rate of these two variables were separately reported in each group of samples. objective: the coexistence of anti-neutrophil crescentic glomerulonephritis with membranous glomerulopathy is very rare. little is known about the clinical course, treatment and outcome of patients with concurrent coexistence of these nephropathies. method: we present the case of a -year-old man with rapidly progressive renal failure, anti-neutrophil perinuclear antibodies positivity, pulmonary infiltrates and vasculitis. results: the patient was admitted to the pulmonary department due to febrile state, weakness and weight loss. physical examination revealed fine crackles over the left lung, leg edema, and vascular skin lesions on inferior extremities. urinalysis showed proteinuria and hematuria with many rbc casts, and in a -h urine collection, the proteinuria of g was observed. serologic workup was positive for p-anca, negative for c-anca, ana, and anti-gbm antibodies and showed normal levels of c and c . hcv, hbv and hiv infection were excluded. in lab tests, the rapid increase of serum creatinine from . to . mg/dl over weeks was observed. chest ct revealed pulmonary infiltrates of left lung upper lobe. the patient underwent a kidney biopsy which showed necrotizing crescentic glomerulonephritis and membranous glomerulopathy. in light microscopy examination, glomeruli with necrotic lesions and cellular crescents were seen. electron microscopy revealed epimembranous and intramembranous deposits. the patient was treated with steroids; cyclophosphamide and seven plasmapheresis were performed with improvement in renal function. on -year follow-up, his proteinuria had decreased to mg/day and his creatinine had stabilized at . with egfr of ml/min. objective: in the last years, the study of predictive and prognostic markers of non-small cell lung carcinomas (nsclc) has been a continuous challenge. it is well known that the imbalance between akt and pten expressions plays an important role in cell proliferation, angiogenesis and tumor cell invasiveness in various solid malignancies. the aim of our study was to analyze the akt and pten expressions in primitive non-small cell lung carcinomas and, also, in their metastases. method: we have examined akt and pten immunohistochemical expressions in primitive non-small cell lung carcinomas with different stages and in ten metastatic nonsmall cell lung carcinomas: brain, adrenal glands and lungs. results: akt expression was higher in metastatic than in primitive non-small cell lung carcinomas, while pten was over-expressed in % of all tumors. there were no obvious associations between tumor stage and akt/pten expression. there were no correlations between pten over-expression and akt expression. conclusion: akt activation may play an important role in tumor cell invasiveness in non-small cell lung carcinomas, but pten suppressor gene may not be involved in akt activation. objective: the role of mirnas in cancer is a rapidly emerging area of investigation. expression profiling has identified mirna signatures in cancers that associate with the diagnosis, staging, progression, prognosis, and response to treatment. mirnas are ideal biomarkers in ffpe tissue because unlike mrna, mirna integrity is affected very little by formalin fixation. previous studies have shown that mir- overexpression correlated with poor prognosis in nsclc patients. in this study, we investigated the expression of mir- in primary carcinoma and metastasis and near non-tumor parenchyma. method: ffpe samples from surgical specimens and biopsies of seven pulmonary adenocarcinomas and five squamous cell carcinomas and respective metastasis together with normal lung tissue (alveolar and bronchial) from the same case; it was imperative to separate these well-characterized areas by lasercapture microdissection (lcm) prior to rna analysis. results: the expression level of mir- by qrt-pcr was significantly higher in tumor tissues than in adjacent normal tissues (p= . ). the overexpression in the metastasis samples compared to adjacent normal tissue was almost statistically significant (p= . ). conclusion: mir- was overexpressed in tumor tissues relative to adjacent non-tumor tissues. we found an increase in mir- expression in primary carcinoma and metastasis in pulmonary adenocarcinomas when compared with mir- lower expression in squamous cell carcinoma. despite the small sample studied, further investigation may indicate the therapeutic and prognostic relevance of this determination as previous studies suggest that mir- acts as an oncogene and has a role in tumorigenesis through the regulation of tumour suppressor genes. objective: routine diagnosis and prognostic methods may also be useful to add data to clinical information in advanced primary carcinomas if conveniently explored. the evaluation of the potential of h nmr spectroscopy for providing biochemical information of the different histological types and for discriminating between tumour and pulmonary parenchyma as well as biochemical information about different histological types was tried as a preliminary approach. method: small frozen samples of pulmonary tumours were collected together with macroscopically normal parenchyma from surgical specimens before histological classification and mirror tissue was ffpe. paired cases were directly analysed by high-resolution magic angle spinning (hrmas) h nmr spectroscopy ( mhz). the spectral profiles obtained were subjected to multivariate analysis: principal component analysis (pca) and partial least squares regression discriminant analysis (pls-da) with the predictive ability of the statistical models. results: tumor and control tissues were clearly discriminated in the pls-da model with high level of sensitivity ( % of tumor samples correctly classified) and % specificity (no false positives). the metabolites giving rise to this separation were mainly lactate, gpc, pc, taurine, glutathione and udp/utp (elevated in tumors), and glucose, phosphoethanolamine (pe), acetate, lysine, methionine, glycine, myo-and scyllo-inositol (reduced in tumors compared to control tissues). furthermore, pls-da allowed carcinoids to be discriminated from adenocarcinomas and epidermoid carcinomas. conclusion: this preliminary study raised the nmr profile between the different histological types of bronchialpulmonary carcinomas through multivariate analysis and scaled metabolic profiles that may be delineated to improve clinical decisions in future. objective: the rna binding protein hur can stabilize and/or regulate the translation of target mrnas affecting cellular responses. the expression of hur is increased in several cancers, and cytoplasmic immunoreactivity was found to be closely related to poor outcomes. method: we analyzed, by quantitative immunohistochemistry, the expression of hur in lung adenocarcinoma of mixed histologic type from stage i and ii patients. moreover, we evaluated the level of hur expressed both in the nucleus and cytoplasm of tumor cells (t-hur) and evaluated the impact of nuclear/cytoplasmic ratio (n/c) on clinical outcome. results: both t-hur and n/c were not associated with age, tumor diameter and histopathological grading, whereas high t-hur and low n/c were associated with lymph node involvement at presentation. cox's regression analysis, using either t-hur or n/c as continuous covariates, showed that high t-hur and low n/c were associated with the risk of death and metastasis. the plots of the estimates, at -year follow-up, of metastasis-free and overall surviving as a function of t-hur and n/c levels, showed that the increase of t-hur and the decrease of n/c were associated with an increase of risk. in the multivariate analysis, both t-hur and n/c retained an independent prognostic significance relative to metastasis-free and overall survival. conclusion: in conclusion, these data indicate that the over-expression of hur in lung adenocarcinoma cells is an independent prognostic marker of a poor clinical outcome. moreover, the cytoplasmic localization of hur expression is more relevant than the nuclear one for the tumor cell behavior. objective: the eml -alk fusion gene has been detected in - % of non-small cell lung cancers. we evaluated the incidence, prognosis and the characteristics of alkrearranged non-small cell lung cancers and the optimal diagnostic modality to detect alk rearrangements in routine clinical practice. method: seven hundred seventy-nine surgical specimens of non-small cell lung cancer from two institutions were analyzed. to identify alk rearrangements, immunohistochemistry and fluorescent in situ hybridization (fish) examination were performed and compared. the clinicopathologic characteristics of tumors with and without alk rearrangements were analyzed. egfr and kras mutations were determined by dna sequencing. results: we identified ( . %) non-small cell lung cancer with alk rearrangements within our cohort by immunohistochemistry. forty-three ( . %) of the eml -alk tumors were adenocarcinomas. alk rearrangement was associated with never smoking (p< . ) and female (p < ). patients in the eml -alk fusion gene in adenocarcinoma had no effect on progression-free survival and overall survival. immunohistochemical results were correlated with fish results. conclusion: the patients most likely to harbor eml -alk are never smokers with adenocarcinoma. but eml -alk was not an independent prognostic factor. immunohistochemical staining for alk could be used as a screening method to detect alk gene rearrangement. objective: lung cancer is one of the most common malignant tumors worldwide, with a growing incidence. in romania, lung cancer has a prevalence of % and an incidence of %; in terms of deaths from cancer, it ranks first place in males and fourth place in females. method: we analyzed the malignant lung tumors from january to february using information obtained from observation sheets of thoracic surgery clinic and the database of the pathology department of the university emergency hospital bucharest. distribution of cases was followed depending on age, environment, gender, living and working conditions (smoking, toxic, radiation), tumor stage and histological appearance. the analysis of these data shows that most lung tumors occurred in the fifth decade of age and is more common in men. regarding histological type and age, the most common tumors were moderately differentiated squamous carcinomas (the decade iv, v, vi and vii). four patients were hospitalized for another disease (inguinal hernia, uterine fibroma) and were subsequently transferred to the clinic for thoracic surgery for specialist treatment. conclusion: physical examination, identifying the factors contributing to lung cancer appearance and its diagnosis in early stages, as well as an adequate communication between specialists, may contribute to the decrease of mortality as well as socioeconomical costs. objective: lung damage in emphysema has been traditionally attributed to an imbalanced proteases/antiproteases, especially in α -antitrypsin deficiency (aatd). however, activation of lymphocytes with organisation in lymphoid follicles (lfs) seems to play a crucial role in disease pathogenesis. the aim of this study was to quantify the number and distribution of lfs in native lungs from patients transplanted for emphysema. method: we compared two groups of patients with either aatd (n= ) or normal levels (aatn, n= ). bronchiolar, perivascular and parenchymal lfs were evaluated on sections immunostained with anti-cd .results were expressed as lf number per square centimeter of examined tissue and, for bronchiolar lfs, as percentage of airways with lfs. results: lfs, almost absent in normal lungs from donors, were frequent in subjects with severe emphysema both with and without aatd. lf total number was increased in aatd patients as compared to aatn, and this was mainly due to a higher number of parenchymal lfs (median, range: , - vs , - lfs/cm , p= . ). perivascular and bronchiolar lfs were numerically increased in patients with aatd ( , - vs , - lfs/cm ; , - vs , - %), but did not reach the levels of statistical significance. conclusion: lf number is increased in patients with end-stage emphysema, particularly in those with aat deficiency. these results extend our knowledge of disease pathogenesis, suggesting that lymphocyte activation is a crucial event even in aatd-related emphysema, and the excessive lung damage observed in these patients may trigger the persistence of the immune response, possibly with autoimmune mechanisms. objective: (lyg) is a rare angiocentric lymphoproliferative process predominantly affecting the lung. the diagnosis of this condition is often difficult as the physical signs, history, chest x-ray, and routine laboratory investigations are usually nonspecific. nevertheless, it is important to establish a tissue diagnosis as this lymphoproliferative disorder can be refractory to treatment and even progress to overt lymphoma. method: one case of pulmonary lyg in a -year-old nigerian man of ibo extraction treated in our centre in and followed up for a year is presented. the difficulty in making diagnosis is highlighted and treatment modality discussed. results: our patient received two courses of cyclophosphamide and prednisolone before defaulting. there was evidence of some remission, even though incomplete, following the first course of chemotherapy (fig. ) . however, his representation a year later with evidence of relapse was a pointer to the ongoing process of the lesion. whether this case had progressed to lymphoma was difficult to say as he declined a readmission and further investigation. conclusion: a subset of angiocentric lymphoproliferative process including lyg shows clinical response to cytotoxic chemotherapy and steroid when commenced early, as is partly true here. lymphomatoid granulomatosis should be considered in long-standing nodular pulmonary lesion and pleural effusion of uncertain aetiology. immunohistochemical study about pathogenesis of acute respiratory distress syndrome g. simona*, i. jung, l. azamfirei, d. demian, r. solomon, b. grigoroiu *umf, dept. of pathology, targu-mures, romania objective: despite modern treatments, the prognosis of acute respiratory distress syndrome (ards) remains unfavorable and its pathogenesis is not explained enough. in few recent studies, the pathogenetic and prognostic role of vascular endothelial growth factor (vegf) and its receptors was investigated in ards, but the obtained results were controversial. method: we have compared the immunohistochemical expression of cd , vegf-a and its receptors (veg-fr and vegfr ) in normal lung (n= ) and also in early and late ards (within h, respectively, after days). cases with ards were necroptic cases (n= ), and also lung biopsies (n= ). labvision antibodies were used. serum level of vegf was also determined. results: in normal lung, all antibodies marked the endothelial and alveolar cells and also macrophages. in early ards, the intensity of vegf decreased in both endothelial cells and alveolocytes, but increased in hyaline membranes (hm) after - days. vegr marked the preserved alveolar cells and vegfr marked alveolar macrophages. hm were negative for both receptors. in late ards, down-regulation was more prominent for all antibodies. serum level of vegf was up-regulated at the same time with decreasing vegf intensity in lung cells, and also with hm appearance. all dyed patients presented hm, independently by phases of ards. cd marked alveolar macrophages and sometimes hm. conclusion: the formation of hm in ards showed an unfavorable prognosis. strong intensity of vegf and the inconstant presence of cd in hm proved that both destroyed alveolar cells and macrophages are responsible for vegf production, but the increased serum level of vegf also proved its serum extravasations in hm. optimalization of flow cytometric assessment of tnf in alveolar macrophages using multiparametric analysis e. stránská*, p. mandáková, m. vašáková, r. matej *university hospital motol, dept. of pathology, praha, czech republic objective: alveolar macrophages (ams) contribute to the pathogenesis of different lung diseases by producing proinflammatory cytokines. within the frame of the project, which is mainly concerned with cytokine expression in bronchoalveolar lavage fluid (balf) of patients with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, we optimized the flow cytometric (fc) examination of tnfα expression in ams. the aim was to determine spontaneous tnfα production, the production during the culture and after stimulation with lipopolysaccharide. furthermore, we focused on antibody selection since ams displayed high level of autofluorescence. method: we examined balf obtained from control patients. tnfα production was assessed immediately after balf delivery, after -h stimulation with lipopolysaccharide (monensin, protein transport inhibitor was added after h of incubation) and also after incubation with monensin only. simultaneously, various concentrations of lipopolysaccharide were tested ( . , and μg/ml). fc detection of ams was simplified using multiparametric analysis, a combination of optical parameters along with the expression of surface markers cd and cd . results: concentration of μg/ml of lps was found to be optimal for ams activation, although all tested concentrations are suitable. the high background autofluorescence of ams can be efficiently reduced by the use of antibody conjugated with long-wave emitting fluorochrome, e.g. apc. objective: the aim of the present study was to contribute to the understanding of the importance of evaluating bone marrow biopsy (bmb) by computer-assisted digital image analysis (cia). moreover, the intention was to investigate the frequency of marrow fibrosis in multiple myeloma (mm) and to correlate it with plasma cell infiltration during the follow-up of patients. method: bone marrow biopsy from patients diagnosed with mm were analyzed at the time of diagnosis and reevaluated after the completion of the therapy. the percentage of plasma cells was estimated on immunohistochemically stained paraffin sections with anti-cd and ig light chains by alphelys spot browser integrated system. the fibrosis was analyzed according to the european consensus on grading bone marrow fibrosis. results: plasma cell percentage at the time of diagnosis ranged from % to % (median %) and in follow-up from % to % (median %). increased fibrosis, which appeared to be restricted to areas of severe plasma cell infiltration, was seen in % of bmb at first diagnosis. in a follow-up study, the percentage of plasma cell infiltration decreased in of , increased in of and remained nearly equal in of mm patients. all patients with disease progression after first-line therapy had increased fibrosis in bmb. the degree of fibrosis correlated with plasma cell percentage, clinical stage and response to therapy. conclusion: in all cases of mm, a bmb is essential at diagnosis as it helps identify a subset of myeloma patients with increased marrow fibrosis and response to therapy. objective: lymphoma is the commonest haematological malignancy in the developed world. we undertook an audit of all laboratory reports from the pathology department of beaumont hospital that confirmed or suspected a diagnosis of lymphoma over an -year period, assessing the following: incidence of lymphoma by age at presentation and gender, site of presentation, type of lymphoma diagnosed, number of cases utilising immunohistochemistry, number of cases where bone marrow staging was performed and the results. method: six hundred forty-seven cases were retrieved from the laboratory database where the histology report provided a definitive diagnosis of lymphoma or strongly suspicious for lymphoma. seventy-seven were diagnosed as hodgkin's lymphoma and diagnosed as non-hodgkin's lymphoma, with cases strongly suspicious for lymphoma. results: the study showed an average age of . years at presentation, with incidence of lymphoma increasing with age and peaking at an age range of - years, reducing thereafter. the highest incidence of hodgkin's lymphoma occurred in the age ranges - and - years. incidence of lymphoma was similar in both genders. b cell lymphoma had the highest incidence, followed by hodgkin's lymphoma. three hundred thirty-one cases had a nodal site of presentation, were extranodal, with the remaining from uncertain site (site labelled, for example, mediastinal mass). brain was the most common extranodal site with cases. immunohistochemistry was performed in % of cases. six percent of cases had bone marrow involvement. conclusion: the incidence of lymphoma for years in beaumont hospital is mostly consistent with the epidemiology established by the who. objective: mpns consist of polycythemia vera (pv), essential thrombocytosis (et), and primary myelofibrosis (pmf). though a number of clinical and diagnostic factors have been determined, the current classification in mpns is still not satisfactory. all mpns are characterized by jak tyrosine kinase (v f) mutation related to the deregulation of intracellular signalling pathways, e.g. ras-erk and pi k-akt, while bcl negatively regulates the pi k-akt pathway. the aim of our study was to attempt to indentify the presence and distribution of erk and bcl expression as factors which might distinguish each mpn. method: expressions of erk and bcl were investigated (immunohistochemistry with envision and dab) on trephine samples in pv, et and pmf patients. anti-erk antibodies were directed to the epitope corresponding to a site near the c-terminal of these molecules. bcl antibodies bind an epitope located between amino acids - of human bcl . finally, sections were studied under light microscope and evaluated using a semiquantitative method. results: there were similarities and differences in terms of erk expression among three mpn disorders. there was a widespread occurrence of anti-erk in the cytoplasm as well as in the nucleus. observed differences concern the levels of expression of this antigen in the nucleus. objective: there is a wide overlap of symptoms and signs among gastrointestinal (gi) graft versus host disease (gvhd) and other gi diseases (infections, drug toxicity, etc.). the diagnosis is based upon histologic and endoscopic findings. accurate diagnosis is important as it determines a specific treatment and contributes significantly to the morbidity and mortality of post-allogeneic stem cell transplant (sct) patients. persistence of gi symptoms despite initial treatment is frequent, and ulterior histologic evaluation may be helpful to address therapy. method: we performed a retrospective study of patients with persistent diarrhea despite treatment based on the first biopsy diagnosis in order to determine the diagnostic value of serial gi biopsy evaluation in sct patients. simultaneous microbiologic analyses of stools and cmv antigenemia monitoring were performed. results: of the first rectosigmoid biopsies, ( %) were diagnosed as gvhd, two ( %) as gvhd with cmv infection, four ( %) as non-cmv infection, and five ( %) as normal or unspecific. second gi biopsies were diagnostic of active gvhd in six ( %) cases, gvhd with cmv infection in four ( %), regenerative changes post-gvhd in eight ( %), cmv infection in three ( %), and normal or unspecific in eight ( %). in out of ( %) patients, the histologic findings of the second biopsy were different compared to the first biopsy, leading to a therapy change in ( %) patients. conclusion: our results showed a reliable diagnostic value for serial gi histologic evaluation that could impact on the therapeutic decision in patients with persistent diarrhea after allo-sct. thymoma with subtotal loss of keratin expression: a potential diagnostic pitfall p. adam* *medical university of tübingen, institute of pathology, germany objective: the differential diagnosis between thymoma and t lymphoblastic lymphoma poses major difficulties in small needle biopsies since both entities contain immature and highly proliferating t cells. one essential diagnostic criterion favouring thymoma is the demonstration of increased numbers of cytokeratin-positive epithelial cells. we describe a series of type b /b thymomas with substantially reduced cytokeratin expression. method: eight thymoma cases were analyzed for the expression of various cytokeratins and other epithelial markers (panck ae / , ck , ck / , cam . , ck / , p , beta-catenin, e-cadherin). as controls, type b and b thymomas and thymic carcinomas arranged on a multi-tissue histoarray were analyzed. results: with regard to cytokeratin expression, three b thymomas were completely negative for panck, ck , ck / and cam . . with regard to the other epithelial markers, seven of seven cases showed a strong nuclear expression of p and strong membranous staining for e-cadherin and beta-catenin. analysis of control cases on a multi-tissue array showed the expression of panck, ck , ck / , cam . and ck / in %, %, %, % and %, respectively. the other epithelial markers p , e-cadherin and beta-catenin were found in %, % and %. conclusion: loss of cytokeratin expression in type b /b thymomas is a potential diagnostic pitfall in the differential diagnosis with t lymphoblastic lymphoma and can be expected in - % of cases. interestingly, thymomas with loss of keratin expression seem to upregulate e-cadherin and beta-catenin. an unusual uterine tumour with signet ring cell features s. rossella*, m. fiche, c. achtari, n. ketterer, l. de laval *chuv lausanne, dept. of pathology, switzerland objective: lymphomas with signet ring cell features are rare, as is uterine dissemination of lymphomas. we report an exceptional case of a uterine tumor combining these two characteristics. method: a -year-old female was diagnosed in with localized nodal grade follicular lymphoma (stage ia). she received local radiation therapy, experienced total remission, and did well until when a systematic ct scan evidenced a pelvic anterior-lateral mass. total enlarged hysterectomy was performed. results: the anterior uterine wall contained a . -cm fish flesh well-delineated mass corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells with extensive signet ring cell changes. tumor cells were cd -, cd -, bcl -, and bcl -positive with a low proliferation rate (< - %); cd underlined a focal follicular architecture. the vacuoles were pas-negative and did not stain for immunoglobulin; ultrastructural analysis revealed nonspecific degenerative vacuoles. no lymph nodes were identified isolated from the surgical specimen. objective: upregulation of the embryonic notch signaling pathway has been observed in several different malignancies, among which is pancreatic ductal adenocarcinoma (pdac). for lack of a receptor-specific blocking antibody, less specific methods were used to suppress notch signaling in earlier research. with use of a receptorspecific monoclonal blocking antibody against the notch receptor (n -ab), we determined the exact effects of notch receptor inhibition on different pdac cell lines. method: after confirming active notch signaling, n -ab activity and specificity were tested with immunofluorescence and qrt-pcr. cells were treated with n -ab and different assays were performed to establish the effects of notch receptor inhibition on growth rate and colony forming potential. in vivo experiments were performed: mice were injected with treated or untreated cells subcutaneously and tumor growth was monitored. results: treatment with n -ab did not influence the growth rate of the cells. colony forming potential did decline upon treatment with notch -ab, although this decline was only twofold. in vivo experiments did not show any differences between the untreated and treated group. conclusion: although notch receptor inhibition did diminish anchorage-independent growth in pdac, this effect was much less profound than previously reported. no effect on growth rate was observed upon treatment with n -ab, which conflicts with the general statement that notch receptor inhibition results in a decline in growth rate. notch should still be looked upon as a potential treatment target, but further determination of the different components of the notch pathway and their role in tumorigenesis seems advisable. objective: due to adverse side effects, efforts are made to reduce the time of daily lateral electrical surface stimulation (less) therapy in the treatment of idiopathic scoliosis in children and adolescents. method: studies were carried out on ten male rabbits, new zealand purebred, aged . months, with b.m. of , - , g. animals were divided into two groups (n= ): ( ) short-term less was applied for h daily during months and ( ) control group without less, but with other experimental parameters as in group . stimulation was performed with scol- (elmech). clinical, macroscopic and microscopic observations were performed. results: similar growth of b.m. was observed in the rabbits from both groups during the first months of the experiment. in month , a slightly smaller increase was observed in the animals from group than in those from group . the mean mass of the suprarenal glands in the stimulated rabbits was . ± . g, while those from the group were . ± . g. a microscopic examination revealed hypertrophy of zona fasciculata with visible overgrowth of glandular cells in rabbits from group . the results of clinical observations as well as morphological lesions indicate the presence of adaptive stress in rabbits stimulated with short-term less. objective: basal cell carcinoma (bcc) is considered a skin cancer with long evolution and low metastatic risk. method: we selected for presentation three cases with bcc operated between and , with a special history and evolution. results: the first case ( -year-old female) developed a small occipital tumour, incompletely explored and excised in another surgical department. we performed ten serial excisions (soft tissue and invaded bone) during years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and used complex reconstructive techniques (local/muscular/free omentum flaps). the second case ( -year-old male) had a neglected frontal tumour invading the orbital region; during a period of years, we performed three excisions and reconstructed the region with skin graft and temporal flap, trying to save the eye. finally, we had to excise the invaded eyeball and we used frontal flap for reconstruction ( ) . the third case ( -year-old female) had a small bcc on the left buttock, which was excised in correct limits and reconstructed with a skin graft ( ). fiveyears later, a new ulcer appeared. the patient neglected the tumour during a period of years. the bcc progressively invaded with a buttock, the upper third of posterior left thigh and the left gluteus maximus muscle, inducing severely altered general status and anemia. we performed a wide excision (including the muscle) and we reconstructed with a fascio-cutaneous flap and skin grafts ( ). conclusion: bcc can be a cancer with a high local aggressivity if the patient or the surgeon underestimates it. objective: eyelids represent a special location of the basal cell carcinoma (bcc) due to the proximity of the eyeball. the aim of this study was to review the clinical and histopathologic features and outcomes of eyelid basal cell carcinomas. the clinical records and histopathologic specimens of patients with eyelid basal cell carcinomas were reviewed and analyzed retrospectively. the main outcome measures are clinical characteristics, lesion size, histologic subtypes, severity of peritumorous inflammation, recurrence rate and prognostic features. results: the most common histologic subtypes were infiltrative, nodular, and basosquamous basal cell carcinomas. of the patients, . % were previously recurrent. recurrent basal cell carcinomas were larger, with longer duration of lesion. basosquamous basal cell carcinomas were more likely to have prior recurrences, larger lesion size, and the highest rate of orbital invasion. perineural invasion was most frequent in morpheaform and basosquamous subtypes. peritumorous inflammation differed between subtypes and was highest in the superficial subtype. the recurrence rate was . %. conclusion: the outcomes were worse than previously reported due to the delay in treatment. the prognostic factors associated with secondary orbital invasion are previous recurrences, aggressive histologic subtypes, longer duration of lesion and larger lesion size. histological assessment of small bowel hypoperfusion lesions in the pig a. l. oliveira*, d. ferreira, h. vala *agrarian superior school, viseu, portugal objective: authors propose the use of a quantitative morphological assessment for helping in studies concerning intestinal hypoperfusion. the method was applied to small intestine mucosa stained with standard hematoxylin and eosin in pigs that underwent severe hypotension due to acute hemorrhage. method: six large white pigs underwent total intravenous anesthesia with propofol and remifentanil. arterial blood ( ml/kg) was passively removed from the femoral artery over min. volume was replaced using ringer lactate in group and hydroxyethyl starch / . in group , with a delay of min after bleeding. onehour after the volume replacement, pigs were euthanized and small intestine samples were taken for histopathological examination. parameters were classified using two specific scales (chiu ; Çetin ; kaplan ) . mucosal loss (ml) percentage and crypt/ interstitium ratio were obtained (faleiros ). results: inflammatory infiltrate was present in all animals, varying from grade to grade . hydropic cellular degeneration and epithelial detachment were more pronounced in duodenum and more noticeable in group . in group , ml percentage was . ± . % in duodenum, . ± . % in jejunum and . ± . % in ileum. in group , ml percentage was . ± . in duodenum and ± % in the other intestinal segments. in the whole small intestine was . ± . % in group and . ± . % in group . crypt/interstitium ratio did not present significant differences between the groups. conclusion: quantitative morphological assessment may be useful in quantifying the degree of mucosal lost in small intestine stained with hematoxylin and eosin from pigs submitted to acute severe bleeding. results: case : the tumor in the thoracic aorta was found at autopsy. histologically, the tumor consisted of isolated atypical cells, suggesting sarcoma. immunohistochemically, tumor cells were positive for cd . the same atypical cells were seen in the intrarenal arteries as emboli accompanied with vasculitides. case : autopsy revealed lymphoplasmacytic lymphoma (lpl) involving kidneys. and more, amyloid deposition in the lungs and mpo-ancaassociated crescentic glomerulonephritis, i.e. mpa, were noted. conclusion: aortic angiosarcoma is an extremely rare lesion, and it is known that the tumor often causes distal emboli and presents vascilitis syndrome. the second case was diagnosed as lpl, mpa and amyloidosis. mpa is known to occur in some patients with malignancy. it has been reported that some malignancies produce vasculitidesinducing factors. in our cases, such factors produced by malignant cells might induce vasculitides. monoclonal antibodies in the diagnostic of spinal metastases g. kropczydski* *zabrze, poland the skeletal system is the third most frequent seat of metastases, and metastatic tumours are the most common bone malignancies. the diagnostic workup of spinal metastases begins with the identification of the primary neoplastic site. the aim of the study was to determine the utility of monoclonal antibodies in the diagnostic workup of spinal metastases. materials and methods: this is a retrospective analysis of patients whose histopathologic examination confirmed the presence of neoplastic foci in the spine. samples of metastatic tumours of patients whose primary tumour sites had not been identified were subjected to an immunohistochemical analysis based on monoclonal antibodies and assays for antigens associated with tumours most often producing bony metastases. results: the monoclonal antibodies and assays were shown to be useful aids for the identification of the histology and location of the primary tumour in patients in whom routine histological assessments had failed to determine the histological type of tumour. conclusions: the length of survival of patients with malignant spinal metastases is influenced by the type of neoplasm and locally radical surgery combined with palliative radiation therapy. in many cases, effective immunohistochemical workup can contribute to halting the progression of the tumour by enabling qualification for appropriate surgical and oncological treatment. origine and frequency of pulmonary bone fragment embolism: an autopsy study n. willi*, t. thiesler, p. ochsner, g. cathomas *kantonsspital liestal, institute of pathology, switzerland objective: pulmonary bone fragment embolism (pbfe) is a rare event observed at autopsy. pulmonary bone marrow embolism (bme) is more common, but both types of embolisms are considered to have little clinical relevance. the aim of this study was to analyze the frequency of pbfe and bme and correlate these events with clinical findings. method: in an autopsy cohort, the frequency of embolic particles of pbme and bme was evaluated by reanalyzing all pulmonary h&e-and elastin-stained (evg) sections. results: in a total of , autopsies, ( . %) pbfe and ( . %) bmes were detected. compared to bme, pbfe were significantly more common in patients with osteomyelitis [ of ( %) versus of , p< . ). in contrast, bme was more common in patients with costal fractures ( of versus of , p< . ), but no patient showed both types of embolism. in addition, pbef and bme were observed in patients with bone metastasis and orthopaedic devices. the highest density ( . particles per square centimeter) was observed in a patient who died immediately following femoral medullary nailing, presenting with the clinical picture of pulmonary thromboembolism. conclusion: in the autopsy cohort analyzed, an incidence of ‰ for pbfe was considerably higher as previously reported and pbef showed a strong correlation with osteomyelitis, and the application of orthopaedic devices and extensive pbfe may lead to death. bme are correlated with rib fracture, but no association between pbfe and bme was observed, suggesting a different pathogenetic mechanism. ( )). results: we performed correct oncological excision in % of the cases; ten patients with bcc needed re-excision. in the same surgical stage, we reconstructed the defect using local flaps for patients ( . %), local flaps and skin grafts for six patients and skin grafts only in the seven cases with mm. the main techniques used were: frontal or temporal flaps for the orbital /nasal regions, naso-genian or fan flaps for the lips and cervico-facial flaps for the cheek. when necessary, full-thickness skin grafts taken from preor retroauricular donor zones were performed ( cases). after surgery, all the patients were examined by the oncologist; only patients diagnosed with squamous cell carcinoma or malignant melanoma accepted the excision of the local regional lymph nodes. conclusion: correct tumoural excision in the face is difficult to perform. the skin graft is the best reconstruction technique from the oncological point of view, but to respect the aesthetic and functional rules, we have to perform flaps. indian ink vs tissue marking dye: a quantitative comparison of two widely used macroscopical staining tool k. kosemehmetoglu*, g. guner, d. ates, e. soylu *ankara ataturk eah, dept. of pathology, turkey objective: the evaluation of the surgical margins is a major concern in surgical pathology and marking of surgical margins with substances; indian ink and tissue marking dyes are widely used. as there is no systematic study comparing tissue marking dyes and indian ink, the most common substances used for the purpose, this study was conducted to compare the two. method: unit price, penetration into the tissue, the spreading area of one drop of dye on tissue paper, brightness under the microscope and the intensity of colors on image analysis program were compared for each of the five colors of rotring's indian ink and thermo-shandon's tissue marking dyes applied on reduction mammoplasty specimens. results: indian ink seemed to be slightly less smudgy than tissue marking dye. indian ink tended to remain on the surface and showed less tissue penetration compared to tissue marking dye with respect to all colors, except yellow. blue color of thermo-shandon and yellow color of both rotring and thermo-shandon had the deepest penetration in fatty tissues. red color of rotring was lost nearly totally after processing. rotring has revealed comparable results to thermo-shandon; even the black and green colors of rotring were more intense. on the other hand, thermo-shandon was better when red, blue and yellow colors are concerned. conclusion: rotring's indian ink is proven to be just as effective as thermo-shandon's tissue marking dye and bares majority of the characteristics of a perfect staining substance, which are easily applied, quickly fixed, durable and cheap, contain no potential contaminants, is worksafe, would not smudge/stain surrounding tissues, and look bright under the microscope without obscuring the view. objective: during the development of anatomy since the fifteenth century, a phenomenon of anatomical museums appeared. method: we present a short history of this phenomenon in europe and give insight into the form of acquisition and exhibition of the preparations. results: the prototypes were "curiosities of nature" collections with one of the first founded by aldrovandi in bologna (sixteenth century). the next famous and focused on the human body were the collections of f. ruysch in the netherlands (seventeenth century). he was performing autopsies in public and after that presented preserved parts or whole bodies and osteological preparation sets as artistic dioramas in a special exhibition house. his collection, sold to peter the great, created the foundations for kunstkamera in sankt petersburg. in the eighteenth century, teachers in surgical and anatomical schools (beginning of anatomopathology) realized the educational power of such collections. museums like the one by h. fragonard in paris, france and hunterian in glasgow were established. the next big collection-narrenturm in vienna-had many exhibits obtained from algemeine krankenhaus. the first typical anatomopathological museum was created by r. virchow in berlin (nineteenth century). virchow's motto was "no day without a preparation". in polish universities, l. bierkowski (cracow) and a. bielkiewicz (vilnius) founded such museums. that time in all europe, anatomopathological collections became popular, gathering preparations: osteological, wet (alcohol, formaldehyde, other preservatives), dry and mummified. the fall of phenomenon started in the second half of the twentieth century. nowadays, we face the era of digitalisation of images but, on the other hand, collections of "plastinated bodies". the ethical and legal aspects become more important. results: macroscopic examination revealed the absence of traumatic lesions and the presence of cardiomegaly, threeelectrode pacemaker inserted in the right ventricle, anomalously coloured myocardium, lung oedema and congestion, chronic stasis liver and cerebral oedema. microscopic examination showed lesions of severe cellular acute cardiac rejection (grade r) in a heart with graft vascular disease. conclusion: the medico-legal autopsy allowed the confirmation of a death of natural cause ( ); the exclusion of infection and neoplasia-frequent and severe causes of morbidity in the heart-transplanted population ( ); the documentation of graft vascular disease, which is related to the time span after transplantation ( ); and the diagnosis and characterization of the acute rejection ( ) as adequate cause of unexpected death, without prodromic signs and symptoms. finally, it allowed the correlation of acute rejection and the suspension of immunosuppressive therapy by the patient-data obtained through the family ( ). all these conclusions may provide information about death causes of the heart-transplanted population dying outside the hospital, which will complement that of transplanted inpatient deaths. objective: sudden death is a possible major complication of congenital cardiac malformations. method: the authors report the case of a -year-old male that unexpectedly died at work while performing a minor effort task. he was apparently healthy, apart from the occurrence of very rare "epileptic attacks". a medico-legal autopsy was performed. results: the autopsy-in the setting of a forensic postmortem examination-revealed heart malformations that are consistent with ebstein's disease of the tricuspid valve, associated (as seldom may occur) to mitral valve dysplasia and patent foramen ovale. the relevance of this case lies upon three facts: ( ) the rarity of the pathological entity, ( ) the survival of the victim until the adult age without surgical intervention and ( ) the fact that the symptomatic announcing event was sudden and unexpected death. objective: the clinical use of doxorubicin (dox) is limited by dose-related cardiotoxicity. one-electron redox cycling of the quinone moiety has long been known to form reactive oxygen species. tirapazamine is a relatively new cytostatic compound most efficient in areas of hypoxia, however under aerobic conditions causing significant oxidative stress. aim: the aim of this study was to assess morphological changes in the myocardium of rats treated with doxorubicin and tirapazamine and evaluate changes in plasma level of a cardiac damage marker, ctni. method: rats were weakly treated i.p. with . mg/kg b.w. of doxorubicin and or mg/kg b. w. of tirapazamine for weeks. control group was given saline. samples of myocardium and plasma were taken a week after last dose. tissue samples were stained with hematoxylin and eosin, van gieson method and selye method and examined under light microscope. results: myocardium of rats receiving a combination of doxorubicin and lower dose of tirapazamine exhibited similar changes as rats administered with doxorubicin solely. animals treated with doxorubicin and higher dose of tirapazamine displayed vacuolization of sarcoplasm, wavy cardiomyocytes and fuxin-positive areas in selye method as well as signs of minor interstitial fibrosis. all described alterations in this group were accompanied by significantly higher levels of plasma ctni. conclusion: both drugs caused cardiac changes in rats, including necrosis, mononuclear infiltrations and minor fibrosis. combination of doxorubicin and higher dose ( mg/kg) of tirapazamine seems to have a synergic effect on changes in myocardium. objective: disturbed glucose metabolism, particularly in diabetes, is an important factor leading to the formation of advanced glycation end products (age). the amount of age and its distribution in the cardiocytes of a diabetic myocardium and in cardiopathies is not yet well understood. studies were performed to assess age deposits in cardiocytes in chronic heart failure. method: the diabetic groups consisted of autopsy cases with diagnosed diabetes, hearts explanted during transplantation with ischemic cardiopathy and diabetes, and eight hearts from dilated cardiopathy with diabetes. the two non-diabetic groups comprised hearts explanted from non-diabetic subjects: with ischemic cardiopathy and with dilated cardiopathy. the control group consisted of samples from healthy heart donors. immunohistochemical localization of age was applied, and a semiquantitative scale was used. results: positive staining was present in both cardiopathic groups with diabetes ( % of cases), then gradually decreased to % in diabetes, nearly % in cardiomyopathy, and to % in the control group. negative staining seemed to be characteristic of control group and nondiabetics, whereas granular and diffuse staining pattern (mixed pattern) was most frequent in diabetic groups. the semiquantitative results supported an increased age accumulation in cases with cardiomyopathy and in diabetes. age staining showed no significant correlation with patient age, bmi or diabetes duration. objective: desmoplastic small round cell tumor (dsrct) is a rare malignant serosa-related tumor involving mostly the pelvic and abdominal cavities, but very uncommon in the paratesticular region. dsrct affects young adults, mainly men, and is classically associated with a bad prognosis. method: we report the case of a -year-old man admitted to the hospital for a progressive enlargement of the right hemiscrotum with a -month history. a preoperative diagnosis of testicular tuberculosis was set and consecutively a right high orchiectomy was performed. at macroscopy, the testicular parenchyma showed no abnormal areas, but the tunica vaginalis was irregular and diffusely enlarged by a whitish to gray firm tumoral mass. microscopically, the tumor consisted of nests of small, round to spindle cells, with scant cytoplasm, surrounded by a mild desmoplastic stroma. an immunohistochemical study was performed for the differential diagnosis. results: immunohistochemically, the tumor cells were positive for ae /ae and nse and negative for lca, chromogranin, synaptophysin and cd antibodies. positivity and a very peculiar staining aspect were noticed with desmin antibody: perinuclear and dot-like, suggesting a dsrct. fluorescence in situ hybridization (fish) was accomplished, demonstrating ewsr rearrangement, which in this immunophenotypical context pleads a diagnosis of dsrct. conclusion: the awareness and the knowledge of dsrct in the paratesticular area are needed for a correct diagnosis. the differential diagnosis of this tumor with other malignancies involving this region is mandatory as it has a different clinical management. comparison of prognosis of gastrointestinal stromal tumors depending on a primary site and a type of mutation w. michej*, p. rutkowski *cancer centre instytute, dept. of pathology, warsaw, poland objective: majority of primary gists originate from the stomach (g) or small bowel (sb). the aim of the study was the analysis of the pathologic and molecular factors influencing the patients' prognosis after resection of primary gists, deriving from the stomach and intestinum, omentum (ng). method: in our department, a group of registered prospectively primary gists cd (+) comprising g-gists ( . %) and ng-gists ( . %) was analyzed; molecular examination was also made using pcr. surgeons from our institute compared our results, observing diseasefree survival (dfs). results: median size of g-gists as compared to ng-gists was significantly lower ( vs. . cm,) and g-gists were characterized by lower mitotic activity (median, / vs. / hpf). in g-gist, the most common detected mutations were exon kit ( %) and exon pdgfra, but in ng-gist exon and kit. prognosis of g-gists was significantly better as compared to ng-gist: -year dfs rate, % vs. %. in ng-gists, the following factors showed negative impact on dfs based on analysis: mitotic index > / hpf, primary tumor size > cm, pathological subtype: spindle. the high prognostic significance of ajcc staging system classification in g-and ng-gists was confirmed also in multivariate analysis. conclusion: reliability of ajcc risk classification after resection of primary gist was confirmed. patients with primary gist, originating from the stomach, have better prognosis as compared with gists located in other places. in both groups, primary tumor size and mitotic activity are still the most important prognostic factors. objective: malignant peripheral nerve sheath tumors (mpnst) are highly aggressive sarcomas which may be encountered either sporadically or in the context of the inherited tumor syndrome, neurofibromatosis type (nf ). epidermal growth factor receptor (egfr) overexpression has been reported in mpnst, which has been implicated in their pathogenesis. we present three cases that have different pathogenetic origins. method: the first patient was a -year-old woman who received radiotherapy to the axillary region following radical mastectomy for breast cancer years ago. she presented with a mass in the axillary region that has been growing during the last . years. second case was a -year-old man who was admitted with a . -cm mass in the right anterior femoral region. the tumor was low-grade and consisted of cartilaginous heterologous component. third case was a -year-old man with congenital café-aulait spots and soft tissue swellings all over his body. he was admitted for soft tissue swellings in the left axilla and in the neck region and years ago, respectively. both of the lesions were diagnosed as plexiform neurofibroma. currently, the patient attended with -cm mass in the gluteal region. pathologic diagnosis was consistent with mpnst. results: the first and the third cases showed a higher egfr expression than the second case. conclusion: we aimed to describe the morphological features and discuss the clinical background and different pathogenetic origins of these three cases with mpnst. the role of egfr overexpression was also evaluated in this case series. results: the cases reviewed belonged to different subtypes of sarcoma, with leiomyosarcoma of greatest incidence ( %). the most affected age at presentation was - . gender distribution was almost equal (m/f : ). the majority ( %) of cases were located in the trunk. ten commonly used immunohistochemical markers were identified, with vimentin being the most common ( %). over surgeons were responsible for the care of these sarcomas. less than half the cases were graded at diagnosis. of those graded, % were high grade, % intermediate grade and % low grade. the size at presentation ranged from to cm (mean . cm). conclusion: soft tissue sarcomas are a widely diverse group of uncommon tumours. we confirmed that our incidence rates are comparable with those published in the usa, with leiomyosarcoma being the most common subtype. of note is that over surgeons were involved in the management of these cases over years. this would suggest a need for a nationalized specialist centre with surgeons experienced in the treatment of these rare tumours. objective: calcifying aponeurotic fibroma is a rare soft tissue tumor of children and adolescents. these lesions are presented as slow-growing painless masses that usually affect the distal part of the extremities. they are locally aggressive tumors that infiltrate the surrounding muscle and fascia. recurrence rate is over %, but malign transformation is very rare. we report a -year-old boy with calcifying aponeurotic fibroma. method: we present a -year-old boy with non-movable firm mass on his leg. it was represented as a × . -cm diameter mass which surrounded the peroneal tendon with an infiltrating contour on x-ray and mri. fibroblastic proliferation areas, dense collagenous stroma, calcification and osteoid matrix were observed histopathologically. the tumor infiltrated peripheral muscle tissues and surrounding vessels and nerves. there were no mitotic figures and evidence of atypia. overall, histopathologic and radiologic features exhibited the same characteristics of calcifying aponeurotic fibroma. conclusion: calcifying aponeurotic fibroma is a locally aggressive tumor with a high rate of recurrence. especially in young patients, those under years of age had higher risk of recurrences. very few cases of malignant transformation have been reported. surgical total excision is suggested if ever possible and is the mainstay of the treatment approach. however, it is not possible in most of the cases because of its locally infiltrative behaviour. differential diagnoses include aggressive fibromatosis, nodular fasciit, fibrosarcomas, monophasic synovial sarcoma and some other malignant spindle cell tumors. surgical management should be conservative; excision and re-excision, if necessary, are preferable to radical or mutilating surgical procedures to maintain the function of the extremity. elastofibroma dorsi: case report. clinicopathological findings s. papaemmanouil* *g. papanikolaou general hosp., dept. of pathology, thessaloniki, greece objective: elastofibroma is a tumor-like lesion of soft tissue characterized by a large number of coarse, enlarged elastic fibers and fatty tissue. its usual location, in the scapular area, deep in the latissimus dorsi and rhromboid muscles, led to the name "elastofibroma dorsi". most of the elastofibromas are usually asymptomatic, unilateral and occur mainly in middle-aged and elderly women. clinically, these lesions may simulate an aggressive soft tissue tumor. method: design: a -year-old woman presented with a slow-growing subcutaneous mass in the right lateral chest wall. ct scan revealed an ill-defined solid mass, lying deep within the latissimus dorsi. the diagnosis of elastofibroma was suspected and the patient underwent a simple excision of the mass. the surgical specimen consisted of a poorly circumscribed mass, measuring × × cm, with gray-white cut surface and elastic consistence. the sections were examined with h+e and elastic stains and followed by immunohistochemical study for a-sma, cd , and h-caldesmon. results: the lesion was composed of a mixture of collagenous stroma, few spindle cells, mature adipose cells and large coarse eosinophilic elastic fibers, which stain with verhoeff-elastica and masson-trichrome. the spindle cells were positive for cd and negative for a-sma and h-caldesmon. the microscope findings confirmed the preoperative diagnosis. conclusion: elastofibroma is an unusual benign lesion cured by simple excision. the irregular elastic fibers may be the result of abnormal elastogenesis. the familial occurrence supports a genetic predisposition to this lesion. the most important differential diagnosis consideration is desmoid fibromatosis. objective: soft part sarcoma (sps) represents a difficult working area, either for the pathologist and the oncologist. prognostic factors are limited in these tumors, and the search for clinically relevant molecular markers is relatively incipient. we have proposed the present study based on previous results by our group on differential gene expression of soft tissue tumors, which showed significant overexpression of the top a gene in sarcomas. as top a is coded by a gene sited in chromosome , we have also included her- -neu. survivine was also included as previous studies have shown a significant correlation with survival in sps. our aim was to evaluate immunohistochemical protein expression of the three genes and also assess the gene copy numbers of top a and her- -neu by fish analysis in sps in order to verify the existence of prognostic value on these parameters. method: paraffin-embedded tissues from patients with sps were included in the study. results: analysis of multiple parameters showed that combined immunohistochemical expression of top a and survivine were significantly associated with overall survival among sarcomas. this association was also valid when only high-grade sarcomas were analysed. there was no correlation between gene amplification and protein expression of top a, or with her- -neu. objective: the inflammatory amelioration of affected joints was characterized by aa-pas and herovici methods in a k/bxn mouse model of spontaneous chronic arthritis that shares many similarities with rheumatoid arthritis (ra) and was used to study the potential of cd + t cell depletion with monoclonal antibodies (mab) to stop and reverse experimental arthritis progression. method: five groups of mice (including control) were treated with specific anti-cd mab (yts and yts . ) with and without thymectomy. cd + t cells from the blood and articular infiltrate of k/bxn mice were characterized for cell surface phenotypic markers and for cytokine production. affected joints were submitted to routine decalcification and paraffin inclusion. results: mice receiving anti-cd mab improved the arthritis score days after treatment. recovery of the cd + t cells was associated with a new increase of the arthritis score after days. thymectomized and anticd mab-treated mice improved arthritis score permanently. anti-cd mab-treated animals normalized the serological levels of tnf-a, ifn-, il- and il- , and histological analysis showed an absence of inflammatory infiltrate. conclusion: to the best of our knowledge, it was shown for the first time that k/bxn mice activated and effector memory cd + t cells are present in the peripheral blood and joints and that they play an important role in arthritis maintenance since treatment with specific anti-cd mabs significantly improved disease signs, supported by histological observations. these results document that cd + t cells should be regarded as major players in the k/bxn model of experimental arthritis alongside cd + t cells and b cells. method: a -year-old woman presented with postmenopausal bleeding. the diagnosis was mixed mullerian tumoral proliferation in the probe cüretage material. later on, total abdominal hysterectomy and bilateral salpingo-oopherectomy was performed. macroscopically, the specimen had a . × × -cm polypoid mass in the uterine cavity. microscopically, there was a tumoral proliferation of swollen epithelioid cells that formed sex cord-like tubular formations and showed trabecular and insular patterns. one-layered columnar cells laid the endometrial glands peripherally of this proliferation. the neoplastic cells had mild hyperchromatism and pleomorphism and mitosis. there was no necrosis. ki- proliferation index was - %; the neoplastic cells were positive for vimentin, estrogen receptor, progesterone receptor, smooth muscle actin and bcl- , but negative for pancytokeratin, cd , hmb- and alpha-inhibin immunohistochemically. results: by the help of these findings, we reported the case as 'pecoma-like epithelioid-type endometrial stromal sarcoma', though hmb- was negative, but the morphological findings were similar. conclusion: we present the case here because of its rareness and to discuss it with the description of additional cases; more insight into their behavior and predictive morphological parameters may be achieved. intestinal-type adenocarcinoma in a background of mature cystic teratoma of the ovary: case report e. kimiloglu sahan*, s. tettkurt, n. erdogan, y. t. ayanglu, u. kafrinoglu *taksim training hospital, dept. of pathology, istanbul, turkey objective: mature cystic teratoma of the ovary, frequently called a dermoid cyst, is the most common of all ovarian neoplasms and represents the majority of benign ovarian neoplasms in women < years of age. malignant transformation of mature cystic teratoma of the ovary occurs in approximately % of all cases. patients found to have malignant transformation in a mature cystic teratoma are likely to be postmenopausal. adenocarcinoma has been reported to occur in . % of cases of mature cystic teratoma. method: the operation of total abdominal hysterectomy and bilateral salpingo-oophorectomy was performed on a postmenopausal woman of years. in the macroscopical sections of the ovary, there were multiple cysts which had . cm of greatest diameter. there were sebum-like material and hair in some of these cysts. microscopically, there was a malignant proliferation of epithelial type that formed tubular, cribriform and polyadenoid formations. the cells of this proliferation were large and pleomorphic and had a high mitotic index. there was necrosis widely. columnar epithelium was lying in the cystic spaces. there were lipid-laden macrophages and histiocytic multinuclear cells, lymphoplasmositer inflamatuar cell infiltration around them. there were hair follicles focally. immunohistochemically, tumor cells were positive for cytokeratin and cdx- diffusely and negative for cytokeratin . results: the diagnosis was intestinal-type adenocarcinoma in a background of cystic teratoma. endoscopically and colonoscopically, there was no tumor in intestines. conclusion: although gastrointestinal epithelium is found in mature cystic teratoma, intestinal adenocarcinoma is uncommon in the ovary. because of its rareness, we present the case here. objective: cervical carcinoma metastasises mainly via the lymphatics. attempts to correlate the expression of various lymphatic markers with lymph node metastasis have provided contradictory results. we investigated the expression of lymphangiogenesis-associated molecules in human cervical squamous cell carcinoma (scc). method: the expression of vegf-d, prox- and d - were evaluated by immunohistochemistry in paraffinembedded tissue samples from cases of invasive cervical scc. correlations with tumour grade, figo stage, lymph node metastases, lymphatic emboli and inflammation were also examined. results: cytoplasmic and/or nuclear vegf-d expression in the tumor cells was found in of ( %) cases and correlated significantly with ln metastasis (p= . ), lymphatic emboli (p= . ) and figo stage (p= . ). d - and prox- were expressed in the lymphatic endothelium, while in of (%) cases, nuclear staining for prox- in the tumor cells was also observed. there was a significant correlation between d - peritumoral lymph vessel density (lvd) and the presence of lymphatic emboli (p= . ) as well as between both intratumoral and peritumoral prox- lvd and the degree of inflammation (p< . ). a significant correlation of vegf-d expression with peritumoral d - and prox- lvd (r= . , p= . and r = . , p = . , respectively) was also noticed. our results indicate that vegf-d seems to be implicated in lymphangiogenesis and in the progres-sion of cervical scc, and d - may be a useful diagnostic tool in the assessment of lymphatic emboli. inflammation also probably plays a critical role in promoting lymphangiogenesis. objective: the protein p (ink a) is overexpressed in cervical lesions associated with high-risk human papillomavirus (hpv) subtypes and , but not in low-risk hpv subtypes and or non-hpv-associated cervical lesions. distinction between low-and high-grade cervical lesions is very important for the management of patients with cervical intraepithelial neoplasia. in this study, we wanted to determine the usefulness of p (ink a) expression in biopsy specimens with indistinct initial histologic diagnosis (cin - ) . method: we applied immunostaining for p (ink a) in consecutive cervical biopsy specimens with equivocal initial histologic diagnosis (cin - ) during the period between january , and december , . results: diffuse positive expression in the lower third of epithelium was found in % ( / ) of cervical biopsies; in % ( / ), the positive expression extended to two thirds of the epithelium, suggesting the diagnosis of cin . fifteen biopsy specimens ( %) were negative or only focally positive for p (ink a), indicating the diagnosis of immature squamous metaplasia. conclusion: the data support the routine use of p (ink a) immunohistochemical evaluation of cervical biopsy specimens for better discrimination of negative, low-grade and high-grade cervical lesions. cervical adenocarcinomas concomitant with cin or squamous cell carcinoma lack gastric phenotype y. wani*, k. notohara *kurashiki central hospital, dept. of pathology, japan objective: uterine cervical adenocarcinoma with gastric phenotype was recently proposed and shows an aggressive clinical course. the aim of the study was to examine whether the gastric phenotype is present in cervical adenocarcinoma in situ (ais) or invasive adenocarcinoma (ac) concomitant with cervical intraepithelial neoplasia (cin) or invasive squamous cell carcinoma (sc). method: fifteen cases of cervical glandular neoplasms concomitant with cin or sc were retrieved from kch files and subclassified by ais+cin (n= ), ac+cin - (n= ), ac+cin (n= ), ac+sc (n= ). of those, ac and sc were independently present and the so-called adenosquamous carcinoma was excluded. in addition, no cases of minimal deviation adenocarcinoma were observed. we evaluated the presence of gastric morphology based on the criteria previously described by kojima et al. (ajsp ) . in immunohistochemistry, the staining of muc and hik was performed and scored as negative; < %, focal; % to < %, diffuse; % or more. ( ) cd expression is increasing in hyperplastic processes and ec, but there are no differences of cd in atypical hyperplasia and endometrial carcinoma. ( ) pten protein expression in eh and ec was variable, but with tendency towards its diminution in cystic glands, atypical hyperplasia and ec. the association between neoplasia of the endometrium and others benign genital lesions: independent or similar mechanism? m. sajin* objective: schwannoma of the uterus is a benign but very rare neoplasia. we report a case of uterine schwannoma occurring in a -year-old woman with a recent history of abnormal uterine bleeding. method: the curettage material was routinely processed and stained with h&e. in addition, immunohistochemistry was performed with antibodies against s -protein, sma, cd , caldesmon, melan a, hmb , cd and mib- . results: in histological investigation, very few preexisting non-neoplastic glands and a tumor with dense fibrillary matrix was seen. the nuclei were oval to bipolar and cells showed no borders. few psammoma bodies were seen in the tumor, also incipient verocay bodies. the tumor cells were intensely positive for s- protein; no reaction with all other antibodies was seen. less than % of tumor cells were positive for mib- . the histopathology and the immunohistochemistry in this case revealed an exceedingly rare example of a uterine (subendometrial) schwannoma. expression of pten, ß-catenin and estrogen receptor in endometrial hyperplasia: an immunohistochemical study s. wojtylak*, b. pieczynska, k. klonowska, a. zawrocki, w. biernat *medical university of gdansk, dept. of pathology, poland objective: type i endometrial carcinomas with endometrioid and mucinous morphology develop in the patients with chronic hyperestrogenism. these tumors show frequent pten ( - % of cases) and β-catenin mutations ( - % of cases). method: the study group consisted of cases of endometrial hyperplasia (eh) and cases of endometrial atypical hyperplasia (eah). as a control group, cases of anovulatory cycle endometria (ace) were selected. the immunohistochemical analysis was performed on the endometrial curettings by means of tissue microarray. for the objective evaluation of analysed proteins, expression h scores (percentage of positive cells) have been used. conclusion: expression of analysed proteins significantly distinguishes endometrial hyperplasia from disordered endometrial proliferation. the most extreme level of abnormal expression of the studied proteins was seen in atypical hyperplasia, confirming its close relationship to endometrial carcinoma. analysis of pten and β-catenin expression may be a useful adjunct in small endometrial biopsies from patients with suspected precursor states for endometrial carcinoma. objective: cotyledonoid-dissecting leiomyoma of the uterus (cdlu, also known as sternberg tumor) is an extremely rare variant of smooth muscle neoplasm. less than cases of cdlu have been reported so far. cdlu is characterized by alarming distinctive gross appearance suggestive of a malignant neoplasm, but paradoxically universally benign clinical behavior. although the histopathological picture of cdlu was well described, there are few if any data which may explain extrauterine overgrowth of the tumor. method: four previously not described cases of cdlu were submitted to histopathologic examination and selected immunohistochemical assays (p , ki , bcl- , wt- , p ). results of immunohistochemical stainings were compared to those previously reported in benign and malignant uterine smooth muscle neoplasms. results: the age of patients with cdlu ranged from to years (median years). grossly, all the tumors were exophytic and composed of irregular nodular protrusions. median diameter of tumors was cm (range - cm). microscopically, the cases were composed of smooth muscle fascicles dissecting the extratumoral myometrium and forming swirled nodules, similarly to previous cdlu reports. nuclear p expression was seen in < % of tumor cells. ki- proliferative index in all cdlu cases was below %. bcl- was expressed focally in a single case and diffusely in three other cases. wt- diffuse nuclear expression was seen in all cases. all cases were p -negative. conclusion: p /ki- /bcl -/wt- /p immunohistochemical expression patterns in cdlu resembled those previously reported in uterine usual type leiomyomas and did not explain the distinctive gross appearance of these tumors. objective: epithelial ovarian carcinoma (eoc) is subdivided into five histological subtypes: serous, mucinous, endometrioid, clear cell, and transitional. the differential diagnosis between these subtypes is mostly unproblematic, but is getting difficult in poorly differentiated tumors. the aim of the present study was to establish an immunohistochemical marker panel in a series of unequivocal highly differentiated eocs that simplify the differentiation in difficult eocs. method: we performed an immunostaining study in a small series of highly differentiated eocs ( cases). after examining the cases with markers, we identified antibodies as useful markers (ca - , cd , ca cd , cd v , cd h, claudin , egfr, fak, opn, p , p , and pr). the percentage of the antibody-marked tumor cells per tumor was observed counted. the f test was used to determine differences between the means of percent expressions of the antibodies. for predicting the histological subtype from the antibodies data, a multinominal logistic regression model was performed. we additionally accomplished a leave-one-out method cross-validation experiment to mimic real-life situation of predicting an unknown histological type. results: in % of the cases, the initial multinomial regression model (using all observations) was able to predict the correct subtype. however, this number reduced to % in the leave-one-out experiment. conclusion: the match of % correct predictions in the full data set is in agreement with another publication ( %) using a similar panel. however, the low number of correct predictions in the leave-one-out experiment affirms that this is no valid panel to distinguish the histological types of eocs by immunohistochemistry. objective: teratomas usually manifest as masses in descended or undescended testes, in extratesticular tissues where germ cells tumors occur and in ovary in females. teratomas are germ cell tumors composed of an array of tissues derived from two or three embryonic layers in any combination. method: our report presents two cases of immature teratomas. the first patient is a -year-old man admitted in surgical department of county hospital of constanta for increased size in the left testicle in last months, and the second case is a -year-old girl hospitalized in the same department for retroperitoneal tumoral mass. ct scan shows in both cases testicular and ovarian mass with nonhomogeneous structure, surrounded by liquidian areas. results: histological exam revealed in both situations immature teratoma with mesenchymal, epithelial adenocarcinoma and neuroepithelial features and mature zones with condroid tissues. there are no specific immunohistochemical markers for teratoma, but individual antibodies can be used to identify particular tissues. in both cases, chromogranin, synaptophysin and afp were positive. there was no correlation found for the rest of ihc markers used for these cases, markers that were positive or negative (plap, beta-hcg, cd , s ). prognosis was favorable after surgery on the young man, but the young woman was given chemotherapeutical support. conclusion: immature teratomas are rare germ cell tumors. the incidence in children is % to % of ovarian and testicular tumors. in both cases, the ihc confirms immature teratoma diagnosis. immature intestinal type of glands were positive for afp, and neuroepithelial structures were positive for chromogranin and synaptophysin that sustain immature teratoma. objective: vascular tumors of the ovary are rare, with only lymphangiomas published in the literature so far. they are usually asymptomatic and represent an incidental finding. histologically, most lymphangiomas are of cavernous type and do not exceed - cm in diameter. we report two unusually large lymphangiomas measuring and cm in diameter, respectively, both of them presenting with ultrasonographical images of malignancy due to the large size and multicystic appearance. method: patients were and years old, respectively, the latter with a clinical history of pregnancies and associated leiomyoma and endometrial polyp. the former was operated for a large pelvic cystic mass which was on microscopic examination diagnosed as a borderline serous tumor of the left ovary. results: macroscopically, they were poorly defined multicystic tumors that on cut section were dramatically reduced in size (deflated), oozing a clear, thin fluid. microscopically, there was an anastomosing network of empty-appearing cystic spaces that were lined by flattened cuboidal cells without atypia. immunohistochemically, their vascular nature was demonstrated as the cells lining the cystic network were positive for cd , cd and d - (podoplanin) and negative for epithelial markers. conclusion: lymphangiomas are benign masses and their histogenesis is uncertain. they may represent a reactive process occurring after impaired or blocked regional lymphatic drainage related to conditions such as chronic follicular salpingitis, radiation therapy or various neoplastic processes. one of our cases lacked any association and the other one was associated with a surface, noninvasive tumour. we thus conclude that our cases are likely to be true neoplasms. placental mesenchymal dysplasia (pmd) with mosaic triploidy and chorangiomas presenting as a partial hydatidiform mole (phm) j. stanek*, m. khalequzzaman *cincinnati children's hospital, dept. of pathology, usa objective: phm may show radiologic, gross, and histological features overlapping with pmd. pmd with aneuplodies has been reported, but not with a mosaic triploidy. method: a , -g male fetus without hemihypertrophy or congenital malformations was born at weeks gestation. the -g dysmature and focally hydropic placenta showed varicose dilatation of focally thrombosed chorionic vessels, and two translucent thin-walled septal cysts grossly suggestive of phm. the stem villi showed myxoid stromal change. there was no trophoblastic hyperplasia or pseu-doinclusions. the chorionic and stem vessels were dysplastic. two . -and . -cm glut- positive capillary chorangiomas were found. results: ,xxy/ ,xy karyotype was revealed by fish. amplification of three microsatellite loci on chromosome p . showed that the predominant cell line was ,xy. there was no evidence of paternal isodisomy of beckwith-wiedemann syndrome. conclusion: this unique pmd case with mosaic diandric triploidy and chorangiomas was grossly suspicious for phm because of the presence of thin-walled translucent "hydatid" vesicles admixed with placental tissue. however, a large for gestational age and dysmature fetus without anomalies and characteristic gross and microscopic placental findings were diagnostic of pmd. the distinction between phm and pmd is clinically valid as phm can be complicated by persistent gestational trophoblastic disease or trophoblastic tumors. the presence of diandric triploidy is not automatically equivalent to phm. objective: the biological role of phosphorylation of the p protein in tumour cells is still investigated. the study evaluated p protein overexpression, p phosphorylated at serine (ser ), serine (ser ), and expression of bax, cas proteins in ovarian neoplasms and their association with clinicopathological parameters. method: the expression of analyzed proteins was examined on malignant and benign ovarian neoplasms using immunohistochemistry. results: differences between the expression of studied proteins in benign and malignant ovarian neoplasms were significant (p< . ). p -ser phosphorylation was associated with advanced stage (p= . ). p protein overexpression was associated with poor differentiated tumour (p= . ), while the differences between cas expression were observed in moderate and poor tumour grade (p= . ). the correlations between bax/p -ser , cas/p -ser and p -ser expression were found in ovarian carcinomas (p< . ). p /cas-positive cancers were associated with poor differentiated (p= . ) and advanced tumours (p= . ). p /bax-positive cases were found in poorly differentiated cancers (p= . ). these immunophenotypes revealed phosphorylation of p at ser and in . % of cases. conclusion: our results suggest that the expression of p protein phosphorylated at ser might depend on the morphological maturity of tumour cells. the revealed associations between bax, cas and p protein phosphorylated at serine and expression indicate that phosphorylation of p protein at ser and might play a role in the regulation of apoptosis-related protein expression in ovarian carcinomas. experimental model of female hormonal sterility received by influence on a bitch rat of long constant illumination s. abuzaid*, i. kuzmina, g. gubina-vakulik, n. kolousova *national medical university, kharkov, ukraine objective: female hormonal sterility is successfully treated. the experimental model of this morbid condition of ovaries is necessary for the approbation of various methods of treatment of female hormonal sterility. in this connection, we have estimated histologically the structural changes of ovaries of a rat after the long maintenance in the conditions of constant illumination. method: two groups of females vistar rats at the age of months are generated: control group (Сgr), six individuals, and basic group (Вgr), six individuals. rats of Вgr within months were contained in the conditions of constant illumination and rat Сgr in other premise, in conditions of usual change of day and night. ovaries are studied histologically. results: the ovary of rats Вgr has reduced dimensions. absence of normal follicles of any degree of maturing and yellow bodies, presence of cystically variated follicles, a considerable quantity of atretic follicles, and presence of white bodies are microscopically observed. it is possible to assume that constant illumination of animals causes stimulation of the production of serotonin and inhibition of the production of a melatonin by a cone-shaped gland. in the beginning, there is a stimulation of a hypothalamus-hypophysial-ovarian axis and then exhaustion of compensatory mechanisms and development of hormonal sterility. objective: the expression of p protein is associated with a high risk of hpv infection. the purpose of this study was to evaluate if overexpression of p ink a protein can be a useful biomarker to detect dysplastic cells in cervical smears of women with normal colposcopic findings but hgsil cytology. method: cases were retrospectively searched in cytology data of the past years. one hundred thirty-nine cervical smears from patients with hgsil were reviewed by two cytologists. the colposcopic results concerning those cases were also obtained from the records. forty-three of ( . %) women had no pathologic colposcopic findings. immunocytochemistry for p was performed on all cases with normal colposcopy and hgsil cytology. the immunocytochemical staining results were estimated and classified into four grades: , ( - % positive cells), ( - % and patchy), (> % and diffuse). results: the p protein expression was not observed in two cases ( . %), whereas it was strong and diffuse in nine patients ( . %). strong and patsy p expression was detected in patients with hgsil cytology and normal colposcopy ( . %). rare positive cells ( - % cells with nucleus stained with anti-p antibody) were present in cervical smears ( . %). conclusion: colposcopy with guided biopsy detects approximately two thirds of cin cases (gage et al., national cancer institute, usa, ) . increased expression of p ink a reflects the increasing expression of viral oncogenes in dysplastic cervical cells. p ink a is a specific biomarker to identify hgsils, so it could be a useful adjunct for the reevaluation women with hgsil cytology and normal colposcopy in order to correct the underdiagnosed hgsils and prevent delay in therapeutic managements. objective: prior to the introduction of computer-assisted screening in the netherlands, it was agreed upon to first validate this method with an a priori agreed upon uniform protocol for validation. aim was a kappa value of . or higher. method: a validation sample set was made of anonymized routine screen samples, enriched for abnormalities (to %). two screeners from one laboratory scored these with either computer-assisted screening or conventional thin-layer cytology. slides were scored dichotomously independently for either mode of screening as either within normal limits or outside normal limits. the procedure was supervised by an external quality pathologist. after a pilot (n= ), the 'set' was transported to other laboratories for the application of a similar protocol. all scores were computed from six different laboratories (n= , ). results: this nationwide validation study required on average weeks in the participating laboratories. first two kappa scores were . ( % ci . - . ) and . ( % ci . - . ) for the two techniques. after months, four additional laboratories had accomplished the validation procedure. cumulative kappa score for six laboratories was . ( % ci . - . , n= , ) all average scores were above the preset norm of . . conclusion: a nationwide validation protocol could be accomplished without complications. the results showed good agreement between computer-assisted screening and conventional screening on thin-layer technique. the validation protocol allows for the implementation of computerassisted screening in programme-based screening in the netherlands since detection rates will not alter in a significant manner. diagnosis of malignant mesothelioma by fine needle aspiration cytology m. almeida*, p. campos *hospital santa maria, patologia morfologica, lisbon, portugal objective: cytopathologic descriptions of mesotheliomas are limited. this study shows the results of fine needle aspiration (fna) cytology in ten cases of malignant mesothelioma. method: material was obtained by ct-guided fine needle aspiration (fna). smears were stained with giemsa and papanicolaou stain, and cell block sections, when done, were stained with hematoxylin and eosin. immunocytochemical (icc) studies were done in smears/cell block using adenocarcinoma markers (cea, ema, cd , ck and ttf ) and mesothelial markers (calretinin, wt , ck / ). results: all mesotheliomas except three of peritoneal origin were from the pleura. there were nine epithelial malignant mesotheliomas (emm) and one sarcomatous mesothelioma (smm). all mesotheliomas except three of peritoneal origin were from the pleura. there were nine epithelial malignant mesotheliomas (emm) and one sarcomatous mesothelioma (smm). in fna, all emms were highly cellular, with a large number of small sheets, and tridimensional clusters with smooth or lobulated contours as well as single cells. the cells were round to polygonal with abundant cytoplasm and well-defined cell borders and the nuclei were predominantly centrally located, round to oval, medium to large with fine chromatin and small but prominent nucleoli; occasional binucleation was noted. the smm showed malignant spindle-shaped cells with scant, ill-defined cytoplasm singly and in loose clusters, in a bloody background. immunocytochemical staining on the smears/cell block were positive for calretinin, ck / ,ck and wt and negative for cd ,cea and ttf . conclusion: cytologic findings coupled with immunocytochemical studies and in combination with radiologic information and clinical history are highly accurate in the diagnosis of malignant mesothelioma. solid pseudopapillary tumor of the pancreas diagnosed on endoscopic ultrasound-guided fine needle aspiration material a. demir*, r. sadik, p. saksena *sahlgrenska university hospital, dept. of clin. pathology and cytology, gothenburg, sweden objective: solid pseudopapillary tumor (sppt) of the pancreas is a rare, low-grade, epithelial neoplasm that is usually discovered incidentally in young women. method: we present a -year-old woman with × -cm round tumor in the caput pancreatis. endoscopic ultrasound-guided fine needle aspiration (eus-guided fna) was performed. smears were stained with giemsa and mc manus. cell block was used for eosin, mc manus and immunohistochemical stainings. results: cell-rich smears showed single cells and aggregates of uniform cells forming branching papillary clusters with delicate myxoid fibrovascular cores. the nuclei of the cells were round or oval; nucleoli were small or inconspicuous. cytoplasms were usually vacuolated. intra-and extracytoplasmic myxoid globules were noticed. tumor cells showed diffuse positivity for cd , progesterone receptor and cd and focal positivity for synaptophysin and ae /ae . chromogranin a, ck / , ck , tag were negative. ki showed low (< %) positivity. the case was diagnosed as sppt based on morphological and immunohistochemical features, which was confirmed on the surgical material. conclusion: distinguishing sppt from other pancreatic tumors, especially pancreatic endocrine tumors, can be challenging. clinical setting, cytomorphologic features, and immunostains of the cell block help distinguish sppt from pancreatic endocrine tumors, acinar cell carcinoma, and papillary mucinous carcinoma. the objective: most countries, including greece, have adopted the triple assessment approach to breast diagnosis, with fnac as the first-line pathological investigation with the exception of microcalcifications. the majority of european countries use similar reporting systems for breast fnac (c -c ), in keeping with the european guidelines for quality assurance in breast cancer screening and diagnosis. method: in order to explore current practice in our hospital, we reviewed breast fnas within a -year period, collected and interpreted by both the cytopathologists and less by the surgeons, where no clinical data were available. results: there were a total of cancers ( %), of which were diagnosed by fna ( as c and as c ). overall, were diagnosed as c ( . %), as c ( . %), as c ( . %) and as c ( . %). of the c , were confirmed histologically (mastectomy specimens), whereas in patients, there was no feedback information. there was one false positive case concerning two separate fibroadenomas, one of which in the mammary tail, sent to the lab as axillary lymph node. in the c category, there were carcinomas, two fibroadenomas, one fibrocystic disease, and had no confirmatory biopsy. in the c category, there were six carcinomas (four ductal, one lobular, one metaplastic), two columnar cell change and one fat necrosis. our results confirm the accuracy of fna in the diagnosis of malignancy and show the major importance of a multidisciplinary approach and knowledge of the clinical and radiologic data by the interpreters of the fnas. benign renal tumors mimicking malignancy in fine needle aspiration biopsy samples: report of three cases h. gutnik*, m. strojan fležar, z. ovcak, j. pižem *medical faculty ljubljana, institute of pathology, slovenia objective: fine needle aspiration biopsy (fnab) is a wellestablished method to diagnose renal tumors. however, it is challenging to diagnose rare benign renal tumors with unusual cytomorphology mimicking malignant tumors. we report three such cases. method: all three kidney tumors were discovered by ultrasound examination, and ultrasound-guided fnab was performed. the first case was a multicystic renal tumor in the right kidney of a -year-old woman. the second case was an atypical cyst in the left kidney of a -year-old man. the third case was a solid tumor in the left kidney of a -year-old woman. results: case : papillary-like structures and macrophages, supplemented by immunocytochemistry, rendered a diagnosis of renal carcinoma, papillary type, while disregarding stromal fragments. histological diagnosis was a mixed epithelial and stromal tumor (mest). case : similar morphology led to the same cytological diagnosis as in the first case with subsequent histological diagnosis of a cystic nephroma. case : small groups of severely atypical cells were suspicious of a malignant tumor, possibly a metastatic one. the histological diagnosis was juxtaglomerular tumor, confirmed immunohistochemically and by electron microscopic examination only after additional clinical data about unexplained arterial hypertension from the age of were obtained. conclusion: cytologic samples of cystic renal tumors should be evaluated cautiously, including the possibility of mest/cystic nephroma group of tumours. the case of juxtaglomerular tumour shows the utmost importance of reporting the relevant clinical data to the pathologist. objective: in some meningiomas, primarily with a large vascular supply, preoperative embolization is performed a few days before surgery in order to soften the tumor and minimize intraoperative bleeding. post-embolization histological changes consist mainly of necrosis, ischemic cellular changes, vascular fibrinoid necrosis and increased proliferative index. these changes may result in overgrading. we have found no description of such changes in the cytological literature. method: we reviewed cases of meningiomas with prior embolization. in of them, relevant cytological changes induced by embolization were present. cytological material was obtained during intraoperative consultation, either by scrapping or squashing of tissue samples. on histology, all of them were grade i meningiomas and showed intravascular embolic material. results: cytology revealed relevant cellular dissociation with frequent single cells. ischemic cellular changes were a common finding and consisted of cell shrinkage, nuclear piknosis and ill-defined cytoplasmic limits. ischemic changes were better appreciated in single cells. eight cases showed histiocytes and neutrophils. confluent areas of necrosis were seen in one case. none of the cases showed cellular atypia or significant mitotic activity. embolic material was seen cytologically in four cases. of the embolized meningiomas, nine of them showed no significant cytological abnormalities. conclusion: embolization of meningiomas induces cytolomorphological changes that may be relevant in some cases. greater cellular dissociation and ischemic changes, as well as inflammation, may result in a worrisome cytological image. when faced with such changes, the pathologist should always consider the possibility of embolization, avoiding overgrading or misdiagnosis. the statistical comparison of different parameters was made using mann-whitney u test and spearman rank order correlation, with the aim of establishing statistically relevant differences and spearman correlation coefficients (r) between glucose in blood and periodontal disease, ageand sex-related, the length of the diabetes disease, the cytological periodontal stage and the length of the periodontal disease, age-related. results: the statistical calculation has revealed important differences of the blood glucose between the cytological periodontal stages and (p< . ), and (p< . ), and and (p> . ). in a similar way, there were also differences between stages and (p< . ), and (p> . ), and and (p< . ). conclusion: the neutrophils that have been drawn by the presence of bacteria or by the erosion areas of the oral and gastric mucosa can promote an increased gravity of the epithelial lesions, suggesting a particularity of the microbial infection at the diabetic patient who has hyperglucidic stress with the modification of the cellular and humoral immunity in the background. objective: treatment of malignant peritoneal effusions is generally palliative; therefore, quality of life issues, as well as the risks and benefits of the therapeutic options, become more critical. cytomorphologic examination alone provides only limited sensitivity for the detection of metastatic carcinoma cells in many cases of serous effusions. early diagnosis and management of peritoneal metastases from cancer patients represent new directions of researches. method: the current study was aimed at differentiating peritoneal liquids encountered in available cases, chosen to show both biochemical patterns (benign and malignant) and in this way to achieve a diagnostic value of the biochemical method. a panel of biochemical markers (tp, alb, ldh, tc, gl, tl, tg, aa, alp, u, tb, db, ast, alt, mg, fe, k) were determined from the resulted supernatant after centrifugation in blood and peritoneal fluid using automatic and semi-automatic biochemistry analyzer. results: thus, of all measured parameters, the highest accuracy in the differential diagnosis between malignant and the benign cases (cirrhosis) was obtained by measuring peritoneal effusions tc ( %), peritoneal fluid ldh ( . %) and saag ( %). conclusion: it is concluded that a suitably chosen panel, consisting of the best specific markers found, can be of great value for the initial differentiation and subsequent guidance in the diagnosis. abbreviations: tp total proteins, alb albumin, ldh lactate dehydrogenase, tc total cholesterol, gl glucose, tl total lipids, tg trglycerides, aa alpha amylase, alp alkaline phosphatase, u urea, tb total bilirubin, db direct bilirubin, ast aspartate aminotransferase, alt alanine aminotransferase, mg magnesium, fe iron, k potassium, pe/s effusions/serum ratio, saag serum-ascitic albumin gradient, sag serumeffusion gradient. objective: hyalinizing trabecular tumor (htt) is an unusual type of thyroid follicular neoplasm that may represent a major diagnostic problem in routine thyroid practice. method: we present the case of a -year-old woman in which a fine needle aspiration (fna) was performed for a . -cm unique nodule, incidentally discovered in the left thyroid lobe. a hemi-thyroidectomy was performed as the aspiration specimen was reported suspicious for papillary thyroid carcinoma (ptc). macroscopically, the nodule was light tan and well circumscribed. in microscopy, it had prominent trabecular architecture and cytological features strongly suggesting a ptc: nuclear enlargement with particularly abundant nuclear grooves and cytoplasmic invaginations. an intratrabecular abundant, homogenous and eosinophilic hyaline material was also noticed. immunohistochemistry, including anti-thyroglobulin, anti-ttf , anti-hbem , anti-ck and anti-mib antibodies, was performed. results: the tumor cells were strongly positive for thyroglobulin and ttf and were negative for hbem , ck- and chromogranin. a characteristic membranous and peripheral cytoplasmic staining was noticed with mib antibody. conclusion: despite the cytological aspect, a diagnosis of htt was made based on the well-circumscribed character of the tumor and the negativity for hbem and ck- . moreover, the unique reactive pattern of mib was an important clue to the diagnosis. careful evaluation is needed in such cases in which cytological criteria are too obviously suggesting a ptc. the differential diagnosis is essential because unlike ptc, htt is considered a benign tumor and has a different behavior and therapeutic approach. objective: thyroid nodules are a common clinical problem and the fine needle aspiration (fna) has become a standard procedure for their clinical triage. its widespread use allows a better selection of patients in need of surgical treatment. method: four thousand three hundred fifty-nine fna were performed between and , always by the same pathologist using a -gauge needle and a cameco suction device. at least three aspirations were carried out, and three to five slides were obtained for each nodule. they were immediately fixed in alcohol and stained with hematoxylin-eosin or with papanicolaou. the cytological diagnosis was classified in four categories: inadequate, benign, suspicious and malignant. six hundred thirty-eight cases had a histological follow-up. the benefits of the cytologic diagnosis were estimated by statistical analysis (epiinfo software version . . ). results: the sensitivity of fna was . % and the specificity . %. there were . % false negative and . % false positive findings. the p value was < . , considered extremely significant. the main causes of diagnostic errors were sampling errors, similar cytological features for different entities, failure to recognize the follicular variant of papillary carcinoma, and coexistence of non-neoplastic and neoplastic processes in the same gland. conclusion: although the sensitivity and the sensibility does not attain %, fna proves to be the most reliable and cost-effective method for distinguishing benign from suspicious or malignant thyroid lesions. limitations of the method included false negative, non-diagnostic and indeterminate or "suspicious" results. nevertheless, the routine use of fna reduces the rate of unnecessary surgery for thyroid nodules. objective: medullary (mtc) and papillary thyroid carcinoma (ptc) have always been considered different from each other, in their cell origin, histopathological features and incidence. we describe one rare case with simultaneous multicentric ptc, classic or oncocytic type, in one lobe and "composite thyroid carcinoma" with mixed features of mtc and ptc in the other one. these tumors were associated with graves' disease. method: surgical specimens were routinely processed, paraffin-embedded, and he-, van gieson-and grimeliusstained. immunohistochemical study included antibodies anti-cytokeratin (ck ), chromogranina (cga), thyroglobulin (tg), calcitonin (ct), and hbme . results: in the right lobe, there were two, tan brown, nodular lesions measuring . and . cm, respectively, consisting of ptc oncocytic type with follicular and focal papillary pattern and a classic variant of ptc. in the lower pole of the left lobe, a grayish nodule of . cm, with trabecular, follicular and focal papillar architecture was detected, presenting small tumor cells, pleomorphic nuclei and occasional cytoplasmic pseudoinclusions. tumor cells showed intense positive expression for tg, ck and moderate reactivity for cga and ct. hbme was focally positive with membrane staining along lateral and abluminal surfaces. conclusion: our case report emphasizes the role of detailed histopathological analysis, especially when thyroid nodules harbor various aspects, and the use of immunohistochemistry in the identification of rare types of thyroidian neoplasm. this case might be explained by the possibility of activating a common tumorigenic pathway for both follicular and parafollicular thyroid cells or a common stem cell. thyroid metastasis of clear cell renal cell carcinoma: a series of three cases l. bejan* *emergency county hospital, dept. of pathology, târgu mures, romania objective: thyroid metastases are extremely rare, the kidney being the most frequent primary tumor site. method: we report three cases of thyroid metastasis of clear cell renal cell carcinoma (cc-rcc), one of them being the primary manifestation of a renal tumor and the other two with a previous -and -year, respectively, history of renal neoplasm. the first case, a -year-old man, was admitted to the hospital with an initial diagnosis of bilateral goiter, while for the other two, a -year-old man and an -year-old woman, the discovery was incidental. results: on gross examination, white grayish illcircumscribed solid nodules were described in the right lobe (one nodule, first case), in the left lobe (two nodules, second case) and in both lobes (multiple nodules, third case). all three cases presented similar microscopic tumoral aspects: a solid architecture, polygonal cells with abundant clear or eosinophilic cytoplasm, distinct cell borders and moderately enlarged nuclei with proeminent nucleoli. since the microscopic aspect did not correspond to any primary tumor of the thyroid, immunohistochemistry was performed. in all cases, the tumor cells stained positive for cd and negative for thyroglobulin, supporting the diagnosis of a thyroid metastasis of cc-rcc. conclusion: thyroid metastases of cc-rcc are uncommon and may represent the first sign of a yet undiagnosed asymptomatic renal tumor or a metastasis from a tumor surgically removed years before (up to years). in setting a correct diagnosis, immunohistochemical studies together with the medical history are extremely important. primary angiosarcoma of the thyroid gland. a case report k. bednarek-rajewska* *poznan, poland objective: primary angiosarcoma of the thyroid is a rare and controversial entity most commonly seen in patients from the alpine regions with a long-standing history of colloid goiter. purpose: we describe a case of primary, poorly differentiated angiosarcoma in a -year-old male who presented with a rapidly enlarging thyroid mass and mild dysphagia during the preceding month. method: a ct scan of the neck revealed a large tumor mass in the upper pole of the left lobe of the thyroid measuring . × . × cm extending into the surrounding adipose tissue and infiltrating the left internal jugular vein. lymphadenopathy was identified in the left submandibular region and lower neck. cytomorphologically, the aspirate was cellular, with variably cohesive clusters of atypical spindle cells and necrotic masses. thyreoidectomy procedure was performed. histologically, the tumor was composed of spindle, polygonal and epithelioid cells with vesicular nuclei and prominent nucleoli growing in solid sheets and occasionally forming cleft-like vascular spaces filled with erythrocytes. extensive necrosis was present. mitotic activity was mitoses/ hpf. immunohistochemical stains showed tumor positivity for cd , vimentin, factor viii, wt- , m-act and cd . conclusion: the prognosis in primary angiosarcoma of thyroid is generally poor especially if the tumor penetrates the capsule of the thyroid or if there are metastases present. the patient developed further metastases in the cervical lymph nodes within few weeks after thyroidectomy despite radio-and chemotherapy. due to the rapid progression of the disease, the patient was scheduled for palliative radiotherapy. the patient succumbed to the disease months after diagnosis. objective: pheochromocytoma can be defined as a paraganglioma of the adrenal medulla and has been called the " % tumor" because approximately % are bilateral, % are extra-adrenal, % occur in children, and % are malignant. aim: in this report, we described the histopathological aspects of pheochromocytoma in correlation with immunohistochemical reaction. method: the study group included cases of pheocromocytoma diagnosed by the chromaffin reaction, he and van gieson's reaction. immunohistochemistry included monoclonal antibodies: chromogranin a, neuron-specific enolase and s protein. results: the patients have encapsulated, usually soft, and, on section, yellowish-white to reddish-brown tumors. the larger tumors had areas of necrosis, hemorrhage and cyst formation. histopathologically, the tumor cells are characteristically arranged in well-defined nests (zellballen) bound by a delicate fibrovascular stroma, which may contain amyloid. the cells vary considerably in size and have a finely granular basophilic or amphiphilic cytoplasm. the nuclei, usually round or oval with prominent nucleoli, may contain inclusion-like structures. lipid accumulation may develop in the cytoplasm and lead to confusion with adrenal cortical tumors. giant, hyperchromatic nuclei are common and are not an expression of malignancy. chromogranin a and nse were positive. sustentacular cells form a peripheral coat around the "zellballen" and were strongly reactive for s protein. conclusion: all our cases were benign, although % of pheochromocytomas are malignant and have a marked tendency to metastasize to skeletal system, particularly the ribs and spine. the sustentacular cells are more numerous in pheochromocytomas associated with men than in sporadic cases. primary carcinoid tumor of gallbladder c. comingle*, Ü. sekmen, g. altaca, b. bilezikçi *baskent university, faculty of medicine, istanbul, turkey objective: primary carcinoid tumor of gallbladder is a rare neoplasm. they usually present as a polypoid lesion. method: we report an incidentally found carcinoid tumor of gallbladder located in the cystic duct occurring in a -year-old male patient detected in cholecystectomy material. results: histopathologically, in cystic duct, an incidental neoplasm measuring mms in maximum diameter was detected, which was grossly unremarkable. the tumor constituted glandular structures. it was not invading muscular layer. immunohistochemically tumor was positive for synaptophysin. patient is alive and well after months. conclusion: definite diagnosis of carcinoid tumor of gallbladder is usually made on histopathological examination after surgery. our case is unique in that it was found incidentally, which was grossly unremarkable. it seems so that surgery alone is a reliable definite management for these small carcinoid tumors. objective: accurate identification of kras mutations has great importance for target therapy. reproducibility and cheapness of different methods are crucial to be safely applied in colorectal routine diagnostic setting. method: one hundred twelve ffep specimens, previously studied by arms/scorpions real-time pcr (therascreen, roche, italy) designed as reference method, were in parallel analysed by other two methods: restriction fragment length polymorphism-pcr (ampli-set-k-ras, bird, italy) and pcr/reverse hybridization tests (k-ras stripassay, viennalab, austria), respectively, called methods a and b. the methods were compared considering the results, costs, and working times. regarding kras genotype, selected samples were: wild type (wt), mutated in codon , and in codon . results: six of wt samples showed mutation on codon : three were false positives of method a and three of method b. two of samples mutated in codon resulted also positive on codon by method b. since method a is based on cleavage action of the restriction enzyme, we have increased both restriction enzyme concentration and digestion time, abolishing the false positives. on the contrary, there was no way of acting on method b. conclusion: although method a does not define the codon mutation type and is time-consuming, it leads to the same results of the reference method operating our slight methodological modifications. method a resulted cheap ( % of the reference kit price), useful and reproducible for routinely kras testing in colorectal target therapy. work was supported by fondazione cassa di risparmio di puglia, italy. objective: colorectal carcinoma (crc) is a worldwide common malignancy; young patients represent a third of all cases, with a rise over the last years. fifteen percent of crc have microsatellite instability (msi) due to alterations in mismatch repair (mmr) genes. familiar msi cases ( %) have germline mutations. sporadic msi crc ( %) have hypermetilated mlh- promoter. braf v e mutation exclusively occurs in sporadic msi crc. immunohistochemistry detects msi cases with high concordance with microsatellite analysis. the objective was to compare histopathology and patterns of expression of mmr proteins and braf v e mutation in patients below and over years old and determine if age is a risk factor for defective mmr protein expression and braf mutations. method: one hundred six patients < years were retrieved. age, sex, location, histologic type, tnm, infiltration and metastatic lymph nodes data were collected. mlh and msh immunohistochemistry and braf rt-pcr mutation was performed in < years and patients > years as control group. student's t, χ and logistic regression analysis were carried out. results: medullary and mucinous types were more frequent among young patients and intestinal type in older patients (p= . ). no differences were noticed regarding clinicopathological stages between groups. mmrp expression was absent more frequently in < and msh was negative in . % of these patients. no braf mutations were detected in any group. conclusion: we found an association between young age and defective mmr expression (or . ). msh lack of expression is more frequently due to a germline mutation. the fact that none of our patients had braf mutation could be partly due to the small sample size or alternate v e or k-ras mutations. objective: angiogenesis plays an important role in the progression of colorectal cancer (crc). evidence from preclinical and clinical studies indicates that vascular endothelial growth factor (vegfa) is the predominant angiogenic factor in crc. vegfa is expressed in approximately % of crcs, with minimal to no expression in normal colonic mucosa. therefore, the aim of this study was to analyze the prognostic impact of vegfa amplification in crc. method: vegfa gene amplification was evaluated in a large series of sporadic crc resections (n= , ) by fish analysis using the tissue microarray technique. spectrum green-labeled probes originated from the fish clone rp - g were used together with spectrum orange-labeled probes for the respective centromere as a reference. the vegfa amplification status was compared to relevant clinicopathological features. results: vegfa amplification was detected in patients ( %) and was significantly associated with higher t stage and higher tumor grade, presence of vascular invasion, right-sided location (p < . ) and b-raf mutation (p= . ). additionally, vegf amplification was associated with worse survival in univariate (p < . ) and multivariable analysis (p< . ; hr ( % ci), . ( . - . )). conclusion: vegfa amplification seems to highlight a small subset of crcs with aggressive phenotype. therefore, fish analysis of vegfa could represent an alternative evaluation system for identifying patients with poorer clinical outcome who could be candidates for anti-vegfa therapies. objective: k-ras oncogene is frequently mutated in colorectal cancer and is currently established as a predictive biomarker for anti-egfr targeted therapy. b-raf is a serine/threonine kinase of the ras/raf/mek /erk signal transduction pathway which is mutated in a subset of k-ras wild-type patients with colorectal cancer. the aim of this study was to examine the mutational status of k-ras and b-raf genes in correlation with tumor clinicopathological characteristics. method: dna was extracted from microdissected formalin-fixed paraffin-embedded tissues. three hundred forty-four cases were screened for k-ras and b-raf mutations at codons and and exon respectively using high-resolution melting analysis, sequencing and/or pyrosequencing. statistical analysis was performed using stata for windows. results: k-ras mutations in codons and were present in out of cases. the most frequent types of mutation were pg d and pg v at codon and pg d at codon . b-raf mutations in exon (pv e) were present in . % of analyzed samples. b-raf mutations were observed in a marginally higher frequency in women, whereas k-ras mutations were positively correlated with patients' age. moreover, there was a higher prevalence of b-raf mutations in low-grade carcinomas (p= . ). conclusion: the detected mutation frequency as well as the prevalence of specific mutation types is in accordance with previous studies. the observed correlation of b-raf mutation with tumor grade implies its importance as a marker of tumor aggressiveness. the applied methods (hrm and pyrosequencing) were proven to be sensitive, fast and accurate for mutation detection in a clinical setting. objective: aberrant activation of the phosphatidylinositol- -kinase (pi kinase)-akt pathway is frequently observed in a wide range of cancers such as breast, colorectal, ovarian and lung. several components of this pathway such as pi k and akt can constitute potential therapeutic targets, and many small molecule inhibitors are in development on early clinical trials. the aim of this study was to examine the possible significance of somatic point mutations in pik ca and akt genes as biological markers or therapeutic targets in primary urothelial bladder carcinoma. method: one hundred twenty-two urinary bladder cancer specimens were screened for activating mutations in exons , of the pik ca gene as well as in exon of akt gene by pcr-sscp and high-resolution melting analysis. mutations were identified for pik ca gene with sequencing and for akt gene with pyrosequencing and statistical analysis was performed. results: four different mutations were detected in exon of pik ca gene ( . %). the mutations were identified as p.a t, p.a v, p.h r and p.g r. one out of cases was mutant in exon of pik ca gene ( . %, p.e k). the akt p.e k mutation was identified in out of bladder tumours ( . %). statistical analysis did not reveal any correlation with clinicopathological parameters (p> . ). conclusion: mutations in pik ca gene are quite frequently observed in bladder cancer, implying their importance as potential targets for anticancer treatment. on the other hand, mutations in akt gene are not a common event in this type of cancer. analysis of dna integrity, morphology, antigenicity with different preservation methods in brain tissue a. saetta*, g. agrogiannis, p. korkolopoulou, a. gavriil, n. kavantzas, e. patsouris, e. michalitsi, a. stamatelli, n. prekete, e. palliou *university of athens, st dept. of pathology, greece objective: due to the potential carcinogenicity of formaldehyde, its replacement by substitute fixatives is recommended and could increase the possibility for application of molecular biology approaches. the aim of this study was to examine dna preservation, morphology and antigenicity of brain tissues after fixation with a formalin-free fixative in comparison with formalin-fixed tissues as well as frozen ones. method: calibrated specimens from six different areas (frontal, parietal, occipital lobes, hippocampus, cerebellum and basal ganglia) of brains from elderly individuals (age > ) were fixed for h in two different fixatives (formalin, formalin-free fixative-rcl ) at room tempera-ture. tissues were stained with hematoxylin-eosin for histological examination and their antigenicity was determined by immunohistochemistry. the integrity of dna extracted from the samples with formalin fixation or formalin-free fixation was compared with that of cryosections with gel electrophoresis and the bioanalyzer. furthermore, pcr-pflps and real-time pcr (high-resolution melting analysis) were performed in order to assess the suitability of the extracted dna for downstream molecular pathological applications. results: immunohistochemistry with antibodies against bamyloid and tau did not reveal any significant differences in reactivity for both fixatives. on the other hand, dna extracted from frozen samples was of comparable integrity with that from formalin-free fixation. amplification efficiency for short dna fragments ( - bp) was similar between all preservation methods; however, long fragments ( bp) could not be amplified from formalin-fixed tissues. the formalin substitute fixative used in this study provided good histopathological quality, and also the extracted dna performed better at the subsequent molecular biological procedures. objective: the molecular mechanisms underlying the development of esophageal cancer remain elusive. the aim of this study was to examine the simultaneous presence of alterations in two main signal transduction pathways, ras/raf/mek/erk and pi k/akt, which are implicated in carcinogenesis. the prognostic significance of perk expression was also investigated. method: genomic dna was extracted from paraffinembedded tissue blocks ( squamous-cell, adenosquamous cell and adenocarcinomas). additionally, in cases, tumor cells were collected using laser microdissection in order to minimize contamination with stromal and normal cells. we searched for somatic mutations in kras, b-raf, egfr, akt- and pik ca genes by performing high-resolution analysis and pyrosequencing. the expression of activated erk protein was assessed by immunohistochemistry. results: k-ras mutation at codon was detected in one lasermicrodissected adenocarcinoma out of specimens ( %), whereas no mutations were found in exon of b-raf gene, in exons and of egfr gene, in exon of akt- gene and in exon of pik ca gene. perk nuclear expression was positively correlated with disease grade and stage and nuclear staining intensity with grade. furthermore, perk cytoplasmic immunopositivity was correlated with tumor grade. conclusion: mutations in k-ras gene are not frequently detected in esophageal cancer, but do exist. the lack of mutations in b-raf, egfr, akt- genes implies that these may not play an important role in the pathogenesis of esophageal cancer. the correlation of perk nuclear expression with stage and grade implicates perk as a marker of local tumor aggressiveness. objective: mutation analysis in ras/raf signaling pathway plays a key role in treatment decision in patients with metastatic colorectal cancer (mcrc). at this time, several methods are available for kras mutation detection; there are also commercially available kits, but validated methods and standardized testing procedures are still lacking. method: genomic dna was extracted from formalin-fixed paraffin-embedded (ffpe) tumor tissues after the evaluation of material by pathologists. for mutation analysis of kras gene, we used three methods: direct sequencing, allelic discrimination and high-resolution melting (hrm). results: we tested tumor samples (primary tumors and metastasis) from patients with mcrc. we identified kras mutation in of ( %) patients using allelic discrimination, while hrm detected mutations in of ( %) patients. due to low burden of tumor cells in two cases and limited sensitivity of sequencing, only patients were evaluated by this method. out of them, four ( %) patients showed positivity. we performed sensitivity testing: allelic discrimination ( % of mutant dna), hrm ( %) and sequencing ( %). conclusion: allelic discrimination is a convenient, fast and sensitive method designed for the detection of specific mutations. test costs are proportional to the number of tested mutations since a specific assay has to be used for each mutation. sequencing has a limited application due to low sensitivity, while hrm represents a sensitive, rapid and cost-limited methodology, but displays higher rate of false positive results if dna is in poor quality. this technique is suitable for routine screening; however, precise identification of mutations is not possible. therefore, a combination of at least two methods is highly recommended. objective: mgmt repairs dna damages and act as a tumor suppressor gene in normal cells, preventing dna mutation. several different approaches for mgmt immunohistochemical (ihc) testing have been used, resulting in a not universally accepted standard. we evaluated the ihc expression of mgmt using five different primary antibodies in invasive breast carcinomas in order to establish the most reliable marker for this protein. the results of immunostaining were compared to qrt-pcr, used as a parameter of reliability of gene expression. method: cases were randomly selected to build a tissue microarray. the five primary antibodies to mgmt were provided by: mt . (neomarkers, genetex and santa cruz), spm (santa cruz) and mt . (zymed). heatinduced antigen retrieval in citrate and the advance™ (dako) detection system were used. ihc was visually analyzed by microscope and automated analyzed by software applied to digital slides. qrt-pcr was performed in all tumors for transcript expression quantification. results: antibody spm (santa cruz) showed the highest sensitivity (p< . ), and antibody mt . (santa cruz) showed the least sensitivity (p< . ). antibody mt . (zymed) showed higher levels of cytoplasm staining, which was not observed in the other antibodies tested (p< . ). qrt-pcr results showed that . % of the samples showed hypoexpression of mgmt when compared to normal breast (p< . ). spm (santa cruz) was the only antibody which showed a positive and significant correlation with the results obtained by qrt-pcr (p= . ). conclusion: this antibody seems to be reliable and effective for research and clinical practice in breast cancer. (supported by fapesp and cnpq). objective: mgmt repairs dna damages and act as a tumor suppressor gene in normal cells, preventing dna mutation. several different methods for mgmt immunohistochemical (ihc) testing have been used, resulting in no universally accepted standard. we evaluated the ihc expression of mgmt of five different primary antibodies in invasive breast carcinomas. method: fifty-nine breast carcinomas were randomly selected for a tma construction. five different primary antibodies against mgmt were used for the ihc study: clone mt . (neomarkers, genetex and santa cruz), spm (santa cruz) and mt . (zymed). heat-induced antigen retrieval in citrate and advance™ ihc detection system were used. ihc was visually analyzed by microscope and automated analyzed by software applied to digital slides. qrt-pcr was performed in all tumors for transcript expression quantification. results: antibody spm (santa cruz) showed the highest sensitivity (p< . ), and antibody mt . (santa cruz) showed the least sensitivity (p< . ). antibody mt . (zymed) showed higher levels of cytoplasm staining, which was not observed in the other antibodies tested (p< . ). fifty-nine samples ( . %) showed hypoexpression of mgmt when compared to normal breast evaluated by qrt-pcr (p< . ). spm (santa cruz) was the only antibody which showed a positive and significant correlation with the results obtained from qrt-pcr (p= . ). conclusion: antibody spm (santa cruz) presented to be the most sensitive and specific antibody for the ihc evaluation of mgmt. this antibody seems to be of reliable and effective use for research and clinical practice in breast cancer. mgmt as a potential prognostic marker in breast cancer j. cirullo-neto*, k. carvalho, l. kagohara, e. olivieri, d. carraro, i. cunha, j. vassallo, f. soares, r. rocha *cancer hospital a c camargo, são paulo, brazil objective: mgmt repairs dna damages, via alkylation, by removing a methyl group from the o position of guanine. it acts as tumor suppressor gene in normal cells and prevents dna mutation. we evaluated mgmt expression in breast tumors, correlating it with other prognostic factors. method: sixty-four cases of invasive breast carcinomas were randomly selected for a tma construction. immunohistochemistry (ihc) was performed for mgmt and also for er, pr, her , ki , p , p , e-cadherin, ck and ck for luminal and basal phenotype classification. ihc was evaluated following the guidelines for each marker most recommended in the literature. fluorescent in situ hybridization (fish) was performed in those cases considered + in order to assess her gene amplification status. qrt-pcr was performed in frozen tissue from our tumor bank for all cases in order to evaluate mrna expression of mgmt. results: fourteen cases were triple-negative ( . %), and among those, seven cases were basal-like carcinomas ( . %). twenty-five cases ( %) were luminal-like type a, four cases were ( . %) luminal-like type b, and one case ( . %) was her -like type. mgmt showed significant lower expression in the basal-like tumors when compared to the luminal-like ones (p= . ). basal-like phenotype tumors presented higher positivity for p and ki than the luminal types (p= . and p= . , respectively). positive p and high ki tumors showed significant lower expression of mgmt (p= . and p= . , respectively). conclusion: mgmt assessment by ihc or molecular biology techniques may represent an important prognostic factor in breast cancer. objective: the col a gene codifies a protein expressed during cartilage and osteomorphogenesis which is present in the setting of several types of infiltrating malignant desmoplasia. our team generated a monoclonal antibody against procol a which was tested for infiltrating ductal pancreatic carcinoma versus chronic pancreatitis differential diagnosis and infiltrating ductal carcinoma of breast versus sclerosing adenosis differential diagnosis. method: to investigate its usefulness in order to assess the presence of invasion in bronchioloalveolar lung carcinomas (bac), we carried out immunostaining with the monoclonal antibody anticol a in a series of nine bac and six lung adenocarcinomas with bronchioloalveolar pattern. the results show lack of staining in seven of nine bac, whereas all six adenocarcinomas with bronchioloalveolar pattern did stain (significance, p= . ). conclusion: our conclusion is that the monoclonal antibody anticol a could be a useful marker of invasion in bac when invasion is doubtful. objective: poly(adp-ribose) polymerase- (parp- ) is a nuclear enzyme that plays a role in dna repair, differentiation, proliferation, and cell death. the polymorphisms of parp- have been associated with the risk of various carcinomas, including breast, lung, and prostate. we investigated whether parp- polymorphisms are associated with the risk of non-hodgkin lymphoma (nhl). method: subjects from a korean population consisting of nhl patients and controls were genotyped for five parp- polymorphisms (asp asp, ala ala, lys lys, ivs + a>g, and val ala) using highresolution melting polymerase chain reaction (pcr) and an automatic sequencer. results: none of the five polymorphisms were associated with overall risk for nhl. however, the val ala polymorphism was associated with reduced risk for nhl in males [odds ratio (or), . ; % confidence interval (ci), . - . for cc genotype and or, . ; % ci, . - . for tc genotype], with a trend toward a gene dose effect (p for trend, . ). asp asp (p for trend, . ) and lys lys (p for trend, . ) polymorphisms revealed the same trend. in an association study of parp- haplotypes, haplotype acaac was associated with decreased risk of nhl in males (or, . ; % ci, . - . ). the present data suggest that val ala, asp asp, and lys lys polymorphisms and the haplotype-acaac in parp- are associated with reduced risk of nhl in korean males. objective: the orphan leucine-rich repeat containing gprotein-coupled receptor lgr (gpr /hg /fex) is a novel marker for adult stem cells in small intestine, colon and hair follicles. it is suspected to have a pivotal role in cell proliferation and tumor formation and seems to be a potential therapeutic target. lgr expression is poorly investigated and neither its function in stem cells nor during tumor formation is known. method: our aim was to identify a lgr antibody with a high specificity among different providers (epitomics®, mbl®, sigma®, biosite®, abgent®) for screening selected human formalin-fixed and paraffin-embedded (ffpe) tissue samples by automated immunohistochemical staining. for proving antibody specificity, in situ hybridization with a crna probe of lgr is carried out on the same tissue samples used for immunohistochemistry. as positive control, we generate a lgr expressing human keratinocyte cell line (hacat) by retroviral transduction. results: the lgr antibody from epitomics® stains the outer root sheath of human hair follicle in anagen, whereas sigma-aldrich®'s anti-lgr antibody recognizes lgr in the inner root sheath. nerve tissue is completely negative using the epitomics® antibody, but contrarily, the anti-lgr antibody from mbl® shows strong staining of purkinje cells as well as of other nerve cells in the cortex and spinal cord. the sigma-aldrich® antibody also shows slight nerval staining, but nuclear. anti-lgr antibodies from biosite® and abgent® are all-around negative under the tested conditions. conclusion: all antibodies were optimized but the ihc results are quite incoherent; therefore, further investigations need to be done. objective: glioblastoma multiforme (gbm) and anaplasic astrocytoma (aa) are astrocytic neoplastic entities of the central nervous system that appear in adults, with high biological and clinical aggressiveness. despite the definition of new neoplasm genetic subgroups, the most relevant information in the prognostic of the patient still comes from factors as patient age, localization and size of the tumour, oedema and mass shifting. metabolic phenotyping may provide new information for a better management of this disease. method: we collected molecular profiles based on hrmas spectra for high-grade glioma biopsies ( gbm and aas). the amount of human tumor tissue analyzed for each subject ranged from to mg. all samples were analyzed by post-hrmas histopathology to assess the tissue integrity and double validate histological diagnosis. results: two major metabolic groups were detected, which included and samples, respectively. most aas were located in the same group. the phospholipid patterns and the glutamine/glutamate metabolic relatives seem to be the most relevant contribution to this grouping pattern. conclusion: hrmas provides high-resolution glioma molecular profiles. one of the groups detected, which include most aa samples, seems to reflect a less aggressive type of tumor. metabolic discrimination between these subgroups includes the levels of some metabolites which can be seen by mrs 'in vivo'. objective: hpv is one of the best-known etiologic agents for squamous epithelia-related neoplastic lesions including the cervix, larynx and skin. in recent years, some of nonsquamous epithelia-driven tumors like stomach, colon and lung adenocarcinomas were reported to be containing hpv. the prevalence of hpv in gastric adenocarcinomas, which is reasonably related to chronic gastritis and gastric intestinal metaplasia, is varied between % and % in large-scale studies. regardless of being 'passenger' or 'driver', to show hpv prevalence in chronic gastritis with metaplasia, the well-known two predisposing lesions of gastric cancers was our aim in the present study. method: dna extracted from formalin-fixed paraffinembedded gastric biopsies of cases was tested for hpv-dna by pcr assay. consensus primers were used. the presence of a -bp fragment was accepted as hpv positivity. results: eight out of biopsies were found hpv-dnapositive ( %). conclusion: hpv may accompany gastritis and metaplasia. hpv deserves more studies for pre-neoplastic and neoplastic gastric lesions to enlighten the 'passenger-driver' dilemma. objective: adamantinoma of long bones (alb) and osteofibrous dysplasia (ofd) are rare, osteolytic primary bone tumours of uncertain origin. to investigate the nature of the proliferating fibroblastic stromal cells in alb and ofd and to determine cellular mechanisms of osteolysis in these tumours, we carried out cell culture and molecular studies on two cases of alb and two cases of ofd. method: alb and ofd cells were cultured on coverslips and dentine in the presence and absence of macrophage colony-stimulating factor (mcsf)-primed cd + monocytes + mcsf + receptor activator of nuclear factor kappa b ligand (rankl). cultures were examined for the osteoclast formation markers tartrate-resistant acid phosphatase (trap), cd and lacunar resorption. alb and ofd cells were examined for osteoblast markers (mineralisation nodule formation, alkaline phosphatase). results: few cultured alb and ofd stromal cells expressed epithelial markers (epithelial membrane antigen [ema] and cytokeratin). ofd cells expressed osteoblast markers. cultured alb and ofd stromal cells showed no ultrastructural evidence of epithelial differentiation. alb and ofd cells were not capable of lacunar resorption, but co-cultures of alb or ofd stromal cells and monocytes resulted in the formation of multinucleated trap + and cd + cells capable of lacunar resorption. our findings indicate that ofd and alb stromal cells express epithelial and osteoblast markers. these cells support osteoclast formation from mononuclear phagocytes via a rankl-dependent mechanism, which may contribute towards the aggressive bone destruction associated with this tumour. objective: supratentorial primitive neuroectodermal tumor (spnet) is an embryonal tumor usually developing supratentorially in children or young adults and composed of undifferentiated or poorly differentiated neuroepithelial cells. it is a relatively rare tumor in middle-aged people and extremely rarely develops after radiotherapy. we report an extraordinary case of spnet in a -year-old female that developed years after radiotherapy of oligoastrocytoma. results: a -year-old female was regularly followed after treatment of oligoastrocytoma of the left frontal lobe. the tumor was subtotally resected and treated with radiotherapy years ago. the patient has persistent mild aphasia and mild right hemiparesis. ct performed due to increased incidence of headaches revealed a cystic lesion of the left frontal lobe, cm in diameter, located below the previous tumor. the recurrence of oligoastrocytoma was suspected; however, the diagnosis of spnet was rendered. conclusion: long survival in patients successfully treated for primary brain tumors may yield in independent treatment-induced primary malignancies of the central nervous system. objective: the interaction between different proteins plays an important role in the metastatic ability of brain tumor cells. the purpose of this study was to evaluate the expression of mmp- , mmp- , cd v , and ki- in gliomas of different grades of malignancy and correlate the expression between the studied proteins in tumors. method: expression of mmp- , mmp- , cd v , ki- was evaluated on formalin-fixed paraffin-embedded tissue blocks divided into: glioblastomas multiforme (n= ), anaplastic astrocytomas (n= ), anaplastic oligodendrogliomas (n= ) and normal brain tissue (n= ) using immunohistochemistry. results: mmp- , mmp- , cd v and ki- expression was found in . %, . %, . %, . % of gliomas, respectively.mmp- immunopositivity was significantly higher in glioblastoma multiforme than anaplastic astrocytomas (p= . ). no immunoreaction for all proteins was found in normal brain tissues. mmp- , mmp- , cd v , ki- immunoreactivity was significantly higher in glioblastomas when compared with anaplastic gliomas (p= . ). in gliomas, the percentage of positive cells and the intensity of the immunostaining were proportional to the degree of malignancy. positive correlations between ki- and mmp- (p= . ), mmp- and mmp- (p= . ), mmp- and cd v (p= . ) were found in malignant brain tumors. objective: gliomas are most common among primary brain tumors, and glioblastomas (gbl) are most fatal among gliomas. surgical operation concomitant with chemoradiotherapy is adopted to treat gliomas. temozolomide (tmz) is a new oral cytotoxic agent with less myelosuppression. epigenetic silencing of o -methylguanine-dnamethyltransferase (mgmt) by promoter methylation is associated with improved survival in gbl treated with tmz. in this study, we investigated mgmt promoter methylation in gliomas treated with concomitant chemoradiotherapy (ccrt) following operation. method: in cases of gliomas including gbl, six anaplastic astrocytomas (aa) and three anaplastic oligo-dendrogliomas (ao), formalin-fixed, paraffin-embedded archival samples were used to evaluate the methylation status of mgmt promoter via methylation-specific pcr. results: mgmt promoter methylation was detected in cases ( . %) of gliomas. mgmt promoter methylation was detected in . % of gbl, in . % of aa, and in . % of ao. gbl with methylation had significantly more prolonged overall survival (mean . months) compared with unmethylated gbl (mean . months, p= . ). methylated gbl also had more prolonged progression-free survival (mean . months) compared with unmethylated gbl (mean . months, p= . ). conclusion: the frequency of mgmt promoter methylation in korean glioma patients was similar to those in western countries. these data indicate that mgmt promoter methylation is prognostically significant in gbl given ccrt. mgmt promoter methylation status is considered to be a valuable predictive factor in the routine clinic for gbl. histopathological examination showed a mixed glioneuronal tumor composed of oligodendrocyte-like cells constituting neurophil-like islands, astrocytic cells and neurons scattered in glial tissue. immunohistochemically, glial tissue was gfappositive and neurophil-like areas and big neurons were synaptophysin-positive. in astrocytic component ki- , the proliferation index was below %. p was negative. results: consequently, the final diagnosis was spinal cord glioneuronal tumor with rosetted neurophil-like islands-who grade . these cases can be confused with oligodendrogliomas, astrocytomas and ependymomas. oligodendrogliomas are distinct with their molecular genetic features. conclusion: our case is unique in that it is the youngest reported patient having this tumor located in the spinal cord. proliferative activity in all subtypes of the glioblastoma b. jarosz*, m. jarosz, r. rola, t. trojanowski *lublin, poland objective: glioblastoma is the most malignant and the most frequent primary brain tumour in adults. it accounts for - % of astrocytic tumours. the tumour has two variants: giant cell glioblastoma and gliosarcoma. the purpose of the study was to show the differences between values of the mib- proliferative indexes of glioblastoma, giant cell glioblastoma and gliosarcoma. method: surgical specimens from patients were formalin-fixed, paraffin-embedded immunostained using mib- antibody. next, proliferative index (pi) was calculated. analysis of variance (anova) was used to test the hypothesis that mean values of mib- pi were equal for each diagnosis group. the mean values of mib- pi were as follows: glioblastoma, . % ( . - , %); giant cell glioblastoma, . % ( . - . %) and gliosarcoma, . % ( . - . %), but there were no statistically significant difference in the mib- pi mean value between diagnostic groups. we observed that proliferative activity shows regional variation and was the most prominent in glioblastoma with small cells. in giant cell glioblastoma, it was much lower. because of the absence of significant differences of mib- pis in our study, in the future, the study will be repeated on cases in each group of that tumour. the extracellular matrix and diffusion barriers in the focal cortical dysplasias of brain-an immunohistochemical study j. zamecnik*, l. vargova, a. homola, p. marusic, p. krsek, k. kuncova, e. sykova *charles university, dept. of pathology and mol. m, prague, czech republic objective: changes in the geometry and composition of the extracellular space (ecs) may influence the epileptogenesis in focal cortical dysplasias (fcd) of the brain. method: tissue ecs volume and geometry (the geometrical factor "tortuosity", reflecting various ecs diffusion barriers) were studied in cortical samples of patients surgically treated for epilepsy, including nine patients with fcd type i and of six patients with fcd type ii, by realtime iontophoretic method. consequently, the samples were subjected to immunohistochemical analysis of the composition of the extracellular matrix (ecm) and morphology of the gfap + glial cell processes. results: in both fcd type i and fcd type ii, the tortuosity of the ecs was significantly increased: . ± . and . ± . , respectively; control = . ± . (mean ± sem). the ecs volume fraction was not significantly changed. when compared to controls, no significant changes in ecm composition were noted in fcd type i. however, we observed an increase in gfap + glial processes in both types of fcd and pathological accumulation and distribution of some ecm molecules (tenascins c and r, hyaluronate, chondroitin sulphate, reelin) in the ecs of fcd type ii. conclusion: the ecs of fcd has increased tortuosity, reflecting the increase of diffusion barriers in the ecs due to gliosis and pathologic accumulation of some ecm molecules. we propose that disturbed extrasynaptic transmission, mediated by the diffusion of neuroactive substances through the ecs of such cortex, represents another factor of epileptogenicity in fcd. support: iga mzcr ns - and mzofnm . hemangiopericytoma of the pineal body: case report s. uyar bozkurt*, n. comingle, b. oz *marmara university, istanbul, turkey objective: hemangiopericytomas are rare mesenchymal neoplasms that presumably derived from pericytes. although hpc mas generally attached to the cranial and spinal dura, intraparenchymal and intraventricular rare tumors have been reported. method: a -year-old male patient presented with headache, nausea and blurred vision months ago. cranial mri revealed contrast-enhanced lesion × cm in diameter in pineal gland location. given the findings of the preoperative imaging studies, the lesion was consistent with parenchymal tumors of the pineal gland. a right occipital craniotomy and resection of the lesion was performed. histologically, the tumor was composed of clusters of monotonous, closely packed, small neoplastic cells. nuclei are oval to round with granular chromatin and inconspicuous nucleoli. mitosis was rare and necrosis was not found. stroma contained dilated, slit-like vascular channels lined by flattened endothelial cells. reticulin stain showed increased reticulin network of investing group of neoplastic cells. neoplastic cells were diffusely immunoreactive with bcl- , but were negative with epithelial membrane antigen, chromogranin, synaptophysin, pgp . , s- protein, and cd . a mib- labeling index was %. results: a diagnosis of hemangiopericytoma was made based on light microscopy and conventional histochemical and immunohistochemical studies. differential diagnosis included pineal parenchymal tumors and meningiomas. the present case provides one unique example of a rare entity to the diverse spectrum of the pineal region neoplasms encountered in neuropathology. objective: neurofibroma is the most frequent neurogenic tumor in the first year of life. it is a benign proliferation of schwann cells, perineurial cells, and fibroblasts, which may occur in association with neurofibromatosis type or sporadically. the most common sites of laryngeal involvement are the aryepiglottic folds and the arytenoids, areas of the larynx rich in terminal nerve plexuses. clinical findings: a -year-old girl presented with airway obstruction and stridor with a progressive nature since birth. direct laryngoscopy: laryngeal tumoral mass, located in the supraglotic area. complete surgical excision was performed. method: all anatomical pieces were fixed in % formaldehyde solution, paraffin-embedded, cut and stained (hematoxylin-eosin and van-gieson). results: macroscopically: -cm whitish multinodular mass, firm consistency, myxoid cut surface microscopically: the submucosal nerve fibers were expanded in a plexiform pattern, infiltrating between the submucosal glands due to a proliferation of small, delicate, wavy, spindle cells with markedly elongated, wavy nuclei with pointed ends and coarse collagen fibres, embedded in a myxoid matrix. immunohistochemistry: positivity of the nervous fibers for cd and s protein. act was negative in the nervous fibers and positive in the blood vessels. ki was negative. histopathology diagnosis: plexiform neurofibroma of the larynx. conclusion: neurofibroma of the larynx is a rare condition that should be considered in the differential diagnosis of children presenting with a submucosal laryngeal mass even without the other clinical signs of nf or nf . it has a high rate of recurrence or residual disease due to the possibility of malignant transformation. objective: brain lesions are highly heterogeneous both clinically and morphologically. the morphological study of these lesions is based in their cellular characteristics, different tissue organization patterns, vascular network, collagen distribution, presence of calcium, etc. mr imaging of excised tissue samples may provide valuable information about lesion structure and microarchitecture, increase the diagnosis specificity and help for better tissue characterization. method: fifteen brain lesions biopsies from the hospital clinico universitario de valencia were fixed with formalin and embedded in a gel matrix for minimizing motion artifacts. mr microscopy images and optical microscopy images were obtained for all of them. mr microscopy images included t , t , t * and dwi. images were subsequently analyzed by hierarchical clustering. results: mr microscopy of brain biopsies shows high resolution and quality. major histological findings (for example, healthy, vascular, proliferative or necrotic tissue) exhibit differential mr parameters values (t , t and diffusion coefficient among others). hierarchical cluster analysis of these parameters revealed different mr patterns correlating with relevant histopathological and immunohistochemical features. conclusion: mr microscopy provides functional, microstructural and biophysical information complementary to that obtained by conventional histopathology. our approach and findings may help in multidisciplinary groups for better integration of histopathology and mri in brain lesion diagnosis. interaction of vesicular monoamine transporter (vmat ) and neuromelanin pigment among the midbrain dopaminergic neurons in man p. pasbakhsh*, d. german *tehran university of, medical science, iran objective: neuromelanin (nm) pigment accumulate with age catecholaminergic neurons in man, and ventral substantia nigra dopaminergic neurons that are most vulnerable to degeneration in parkinson's disease (pd) contain the greatest amount of this pigment. in vitro data indicate that nm pigment is formed from the excess cytosolic catecholamine that is not accumulated into synaptic vesicles via the vesicular monoamine transporter (vmat ). method: using semiquantitative immunohistochemical methods in human postmortem brain, we sought to examine the relationship between the contents of vmat and nm pigment. the immunostaining intensity (isi) was measured for vmat in two regions of the midbrain dopaminergic cell complex. the isi of the cells was related to the density of nm pigment within the cells. we also measured the isi for tyrosine hydoxylase (th) and examined the noradrenergic neurons in the locus coeruleus (lc) in brain -- years of age. objective: human mitochondrial diseases have a relatively high incidence, multiple genetic causes and quite diverse clinical, biochemical and morphological phenotypes requiring a multidisciplinary approach for diagnosis. the study of protein aggregation in muscle fibres in these disorders may offer new insights on their complex pathogeny. method: we studied morphological aspects of the "ragged red" fibres-targeting our interest on the types and location of different protein aggregates. we selected diagnostic muscle biopsies from seven unrelated patients and used histological, histochemical, enzyme histochemical, ultrastructural and immunohistochemical techniques. results: the expression of cytoskeletal, transsarcolemmal, sarcomeric, chaperone type and nuclear proteins revealed that "ragged red" fibres and "ragged red" regions of the fibres accumulate diverse proteins, of which desmin, alpha b crystallin, as well as n-cam, dysferlin and heat shock protein appear to be regular ones. none of our cases had any accretion of plectin, actinin, beta and gamma dystroglycan. other proteins showed variable expression. conclusion: as the list of members in the "protein aggregate myopathies" family is constantly enlarging, the study of protein accumulation in mitochondrial myopathies is of clear interest for the future. note: zurac objective: prostate biopsy is performed to detect carcinoma in those at high risk on the basis of elevated psa and abnormal digital rectal examination. re-biopsy is often performed following an initial atypical diagnosis or rising psa. method: all histopathological reports of prostate biopsies at beaumont hospital, dublin, during the time period january to january were analysed. a total of , reports were retrieved and the data were recorded and analysed. results: all prostate biopsies were grouped into initial biopsy (no previous biopsy recorded) and repeat biopsy (at least one previous biopsy recorded). multiple report parameters were recorded for each biopsy including diagnosis, number of cores taken, number of positive cores and gleason grade, and these were compared for the two groups. in the original biopsy group, all biopsies reported as atypical (but not meeting criteria for carcinoma) were further analysed for changes on follow-up biopsy. in the repeat biopsy group, repeat biopsies performed for increased psa and for previous atypical diagnosis were analysed separately and compared. conclusion: repeat prostate biopsy is often performed for persistently elevated or rising psa and previous diagnosis of atypical changes. our study assessed the likelihood of finding carcinoma in a follow-up biopsy performed for these two reasons. our study also compared the biopsy adequacy and extent and grade of carcinoma found in both initial and repeat biopsies. the findings are significant in determining the likely clinical course and appropriate management of patients with atypical diagnoses and elevated psa. objective: since atypical adenomatous hyperplasia (aah), post-atrophic hyperplasia (pah) and proliferative inflammatory atrophy (pia) of the prostate demonstrate both overlapping histological features and generality of biological properties with prostatic intraepithelial neoplasia (pin) and atypical small acinar proliferation (asap), these processes were proposed in probable precancerous lesions of the prostate. method: we studied the cytological features with the use of morphometry. the expression of amacr and cocktail hwc+p was estimated with the use of immunostaining. for the image analysis, the software wsif imagej and imagescope was used. results: bc layer was fragmented in most of cases pah, pia, aah. the bc loss in pah-pia was % and % accordingly and was below that in pin- %, aah- %, and asap- %. analysis of cytological features and nucleolus frequency showed that pah-pia was similar to pin-asap and above that in aah. prominent nucleolus was identified in atrophy, precancerous lesions and pca. frequency of prominent nucleolus in pah ( % cases, % in cases) and pia ( % and % accordingly) was below that in pin ( % and %), asap ( % and %) and pca ( % and %). amacr expression was moderately strongly positive in precancerous lesions and pca and weakly positive in other groups. area of amacr expression in pin and asap was above that in aah, pah, pia and below that in cases pca. objective: presentation of rare lesions of the prostate. method: a -year-old man, with psa , underwent a needle biopsy of the prostate. a histological examination of biopsique carrots showed an infiltrating tiny lesion within adipose periprostatic tissue at the level of one biopsy of the left lobe nodule. the lesion consisted of little irregular glands and solid nests of atypical cells psa − and ck + , and ck + . we proposed a diagnostic of a metastasis/infiltration of unknown primary origin, likely from the urinary tract. this was confirmed by a cystoscopy revealing a tumor which had a histologic appearance of a choriocarci-noma, finally classed as a poorly differentiated urothelial carcinoma with a choriocarcinoma differentiation. results: the second case concerns a -year-old patient with decompensated cirrhosis admitted to the hospital for a gastroenterology bilan. rectal examination revealed an enlarged prostate. the hemostase was necessary after a core needle biopsy of the prostate. the histological examination demonstrated a highly vascular tumor composed of chief and sustentacular cells arranged in an alveolar pattern. immunohistochemical study confirmed a paraganglioma. there were no signs of malignancy; however, we did not observe any prostatic parenchyma at the tumoral level. conclusion: many significant non-epithelial tumors may arise in the prostate gland; although they are rare, their recognition by the pathologist is essential as their treatment and prognosis are quite variable. the core needle biopsy allows for a diagnostic of a variety of tumors, and one should not ignore, in spite of the absence of clinical symptoms and complementary information, the risk of metastatic lesion in the prostate. latent prostate cancer in association with benign prostatic hyperplasia after the chernobyl accident in ukraine a. romanenko*, a. chekalova, p. harkonen, a. vozianov *institute of urology, kiev, ukraine objective: tumor volume is a powerful predictor of patient outcome in prostatic adenocarcinoma. preoperative assessment of prostate cancer tumor volume is still a big challenge. the present study attempted to identify the most predictive biopsy variables for final tumor volume in radical prostatectomy specimens. method: we reviewed prostate needle biopsies in patients who subsequently underwent radical prostatectomy. the preoperative data collected were: psa, preoperative ultrasound, total prostate volume, number of positive biopsies, total percentage of cancer in the biopsy and gleason score. prostatectomy specimens were entirely embedded and mounted whole. tumor volume was measured using the grid method. preoperative data were compared with final tumor volume in radical prostatectomy specimens by univariate and multivariate analysis. results: the number of positive biopsies (p< . ), total percentage of cancer in the biopsy (p< . ) and gleason score (p< . ) were significant predictors of tumor volume on linear regression analysis. psa value (p= . ) and preoperative ultrasound total prostate volume (p= . ) were not significant predictors of tumor volume. on multivariate logistic regression analysis, we designed a model to predict final tumor volume. this model contains all the predictors mentioned above and significantly correlates with final tumor volume (p< . ). conclusion: preoperative data (gleason score, number of positive biopsies and total percentage of cancer in the biopsy) can be used to predict the volume of prostate cancer with acceptable accuracy. these variables must be mentioned to the clinician in order to help in the therapeutic decision. "negative" histopathology (stage pt ) in a radical prostatectomy specimen after a preoperative diagnosis of prostatic adenocarcinoma m. ligaj*, k. sikora, r. sosnowski *warsaw cancer center, dept. of pathology, poland objective: in rare cases, histologic assessment of the entire radical prostatectomy specimen (rps) after a positive biopsy does not reveal residual tumour. we analyze the incidence of "vanishing prostatic carcinoma" in our institution and present algorithm of pathologic workup of "negative" rpss. method: among rpss from patients treated for prostatic adenocarcinoma (january -december ), ten cases ( . %) showed no residual carcinoma despite complete sampling. a five-step protocol of additional pathologic workup included: ( ) preoperative biopsy review; ( ) "second look" review of the rps; ( ) immunohistochemistry ( betae , p ) performed on suspicious foci; ( ) deeper levels of each block with pin high grade and/or of blocks with areas where cancer was seen in the biopsy; and ( ) block-flipping of regions specified above with deeper levels. results: cancer diagnosis had been made in transurethral resection (turp) specimen (two patients) and core biopsy (eight patients). the turp cases were excluded on the assumption of complete pre-prostatectomy carcinoma resection. biopsy review (protocol step ) confirmed prostatic carcinoma in seven core biopsy specimens ( . %). in one case, diagnosis of adenocarcinoma made in another institution was not confirmed. steps - revealed residual carcinoma in a further four rps. despite full workup, no residual tumour was seen in three rp specimens ( . %). all three "negative" cases had undergone mab prior to rp and showed no clear evidence of residual carcinoma. conclusion: "vanishing prostate carcinoma" is a rare phenomenon, with higher incidence after neoadjuvant hormone therapy. extensive histologic workup of rp specimen reduced the rate of pt rp after a positive core biopsy from . % to . %. objective: large cell neuroendocrine carcinoma (lcnec) of the urinary bladder is a rare tumor with only a few cases reported. the diagnosis is based on the criteria established in lung tumors. we report the clinical and histological data of six patients with lcnec in urinary bladder. method: the clinical charts and histological slides of six patients with the lcnec diagnosis in urinary bladder from a single institute were reviewed. the diagnosis was confirmed by the use of immunohistochemistry with at least two positive neuroendocrine markers (synaptophysin, chromogranin and/or cd ). results: all patients were male. their age ranged from to . the most frequent clinical presentation was gross hematuria. their histology had overlapping features with urothelial carcinoma and was difficult to differentiate without immunohistochemistry. the most useful features were extreme hypercellularity ( / ), lack of exophytic papillary component ( / ), nuclear molding ( / ), finely stipled chromatin ( / ) and exuberant mitotic rate (most > /hpf) with innumerable apoptotic figures. in three cases, there were foci of admixed invasive or in situ urothelial carcinoma, in one case admixed adenocarcinoma. all tumors were infiltrative to muscularis propria at the time of diagnosis, except the most recent one which did not have muscle sampled in resection. distant metastases were found in liver, spleen and bones of one patient. three patients underwent radical cystoprostatectomy; all patients received adjuvant or neoadjuvant chemotherapy, and one was given additional radiotherapy. conclusion: distinction between lcnec and urothelial cancer is important because of likely different therapeutic options. lcnecs are highly aggressive tumors with at least pt disease at diagnosis. objective: bladder cancer's incidence is increasing worldwide. to improve the results of current bladder cancer treatments, new drugs are being tested in single use or in associations. gemcitabine is metabolized to the active diphosphate and triphosphate, which is integrated into the dna and causes cessation of dna polymerization. sirolimus inhibits mtor protein kinase. our aim was to study and compare the effect of gemcitabine and sirolimus in bladder cancer chemically induced in mice. method: n-butyl-n-( -hydroxybutyl) nitrosamine was administered to icr male mice in drinking water for weeks. one group was treated with gemcitabine ( . mg/kg, i.p.) and the other with sirolimus ( . mg/kg, i.p.). all animals were killed at the end of the experiment. results: animals not treated showed a higher incidence of neoplastic and pre-neoplastic lesions than animals treated ( % simple hyperplasia, % nodular hyperplasia, dysplasia %, cis % and invasive carcinoma % objective: the accurate diagnosis of poorly differentiated invasive carcinomas in transurethral resection (tur) specimens using only h&e-stained sections continues to be a challenge for the pathologist. the aim of this study was to evaluate and select an optimal immunohistochemical (ihc) panel of antibodies to establish the urothelial or prostatic origin of poorly differentiated tumors in tur specimens. method: ihc was performed to detect cytokeratin (ck) , ck , high-molecular-weight ck (hmwck), p , prostate-specific antigen (psa) and p s/amacr in cases of poorly differentiated invasive tumors: poorly differentiated prostatic adenocarcinomas (gleason score ≥ ) and high-grade urothelial carcinomas. the youngest patient was while the oldest was years of age. results: the sensitivities for labeling prostate cancers were: psa ( %), amacr ( %), ck ( %), ck ( %), p ( %), and hmwck ( %), while urothelial highgrade carcinomas demonstrated the following rates of positivity: hmwck ( %), ck ( %), p ( %), ck ( %), amacr ( %), and psa ( %). co-expression of ck / was noticed in % of the urothelial carcinoma cases, but it lacked in prostate carcinomas. objective: the authors evaluated the possible influences of ageing on the morphology of testicular intralobular interstitial tissue (tiit). method: the studied material consisted of surgical samples of testicular tissue from cases with orchiectomy for prostate adenocarcinoma. tissue samples were fixed in neutral buffered formalin embedded in paraffin, stained with hematoxylin-eosin and goldner trichrome and immunomarked for cd . images were acquired and measurements were performed with a specialized image analysis software after previous calibration. the assessed parameters were tiit amount (p-tiit) and intralobular vascular network (ivn). ten fields were randomly selected for each case, with × objective. mean p-tiit (mp-tiit)/field, mp-tiit/case and mp-tiit/age group were determined. regression line (rl), slope (m) and significance test for slope (p) were determined in order to assess the correlation of tiit amount changes with ageing. results: intralobular septae showed variable amount of collagen fibers, from simple linear fibrillary assemblies to marked thickening with obvious septal enlargement. the latter were seen mostly in the outer region of the lobules, but without any significant spread. tiit amount had a mean value of % of intralobular parenchyma. rl showed a discrete decreasing trend with ageing (m=− . ) confirmed by "p" value (p= . ). arteriolar wall presented degenerative changes with limited extension and variable intensity. peritubular and intramural capillaries were present in all age groups. endothelial cells revealed no significant changes. objective: angiomyolipoma is a rare neoplasm composed of varying admixtures of blood vessels, smooth muscle cells and adipose tissue. there is a strong association with tuberous sclerosis. we describe the first case report of multifocal renal angiomyolipoma developed on tuberous sclerosis and associated with endometriosis. method: imaging studies in an asymptomatic -year-old woman, affected by tuberous sclerosis with tsc gene mutation, revealed a partially solid and cystic mass measuring cm involving the left kidney and a solid mass measuring . cm in the right kidney. results: the surgical specimen consisted of two left nephron-sparing procedures and one at the right side with firm, pale nodules measuring . , . and . cm. the tumors were composed of spindle and epithelioid cells with an abundant pale eosinophilic or clear cytoplasm with round nuclei, mild atypia and low mitotic activity. rare islands of adipose tissue were observed. in the two left tumors, the predominant smooth muscle cells were associated with some foci of endometriosis. the spindle and epithelioid cells were immunoreactive with hmb and smooth muscle actin. the estrogen and progesterone receptors were positive in stromal and epithelial cells of endometriosis. the spindle cells of angiomyolipoma were positive with progesterone receptors. conclusion: multifocal pecoma of the female genital tract associated with diffuse endometriosis was described by froio et al in , but to our knowledge, this is the first case report of renal angiomyolipoma associated with endometriosis. this association represents a supplementary proof of potential role for hormones in the pathogenesis of angiomyolipoma. translocation-associated renal cell carcinoma ( objective: gastric malt (mucosa-associated lymphoid tissue) lymphoma is closely related with helicobacter pylori (hp) colonization and represents a multistage process from gastritis to lymphoma. for the differential diagnosis of malt lymphoma and reactive inflammation, wotherspoon proposed a histomorphological scoring with two intermediate categories, grades and . genetic studies (ig clonality and/or translocations and chromosomal aberrations) have not brought so far definite arguments to confirm or discard lymphoma in these problematic scores. objective: in gastric adenocarcinoma, overexpression of her has been correlated to tumour location and histologic type and varies considerably in the few published studies, probably due, at least in part, to the racial heterogeneity of patients included. the aim of the present study was to investigate the immunohistochemical expression of her protein in gastric adenocarcinomas of greek patients who live in the area of epirus. method: a total of primary gastric adenocarcinomas were retrieved from the archives of the department of pathology ( men and women). they were all located in the stomach, away from the oesophageal-gastric junction. forty-eight were intestinal type, diffuse type and mixed (lauren classification). immunohistochemical analysis of samples was performed using the polyclonal rabbit anti-human c-erb- oncoprotein (dako) antibody on paraffin sections. her scoring was performed according to published consensus panel recommendations. results: overexpression of her (ihc + and +) was detected in eight adenocarcinomas ( . %). specifically, score + was assigned to five cases ( . %, four intestinal type, one diffuse type) and + to three cases ( . %, two intestinal type, one diffuse type). score + was detected in ten cases ( . %, eight intestinal type, one diffuse type and one mixed), and in cases ( %). in two cases with score +, adjacent dysplastic epithelium was noted, which was also +. the results of the current study are comparable with those of the literature, indicating that assessment of immunohistochemical expression of her in gastric adenocarcinomas could lead to the stratification of patients, the goal being the optimal personalized treatment. the prognostic value of her- /neu expression in gastric cancer patients from lower silesia in poland a. halon*, p. donizy, l. halon, r. matkowski, j. rudno-rudzinska, w. kielan, j. rabczynski *wroclaw medical university, poland objective: overexpression of her- /neu was most widely explored in breast cancer. her- positivity is reported as - % in gastric cancer (gc). the aim of the study was to assess the prognostic significance of her- in gc patients from lower silesia, the southwestern region of poland. method: immunohistochemistry (ihc) for her- monoclonal antibody was performed on paraffin-embedded specimens. ihc expression of her- was evaluated using the remmele scale (irs) and standardised herceptest modified for gc. results: the status of her- protein expression was observed as the following: ( ) objective: breakage or degradation of extracellular matrix (ecm) and basement membrane (bm) is a critical step in tumor progression. matrix metalloproteinases (mmps) and their tissue inhibitors (timps) take important roles in these processes acting in a coordinated manner to form an integrated system. the aim of the study was the evaluation of mmp- , mmp- , timp- and timp- expression and the correlation with clinicopathological parameters and survival in gc. method: formalin-fixed paraffin-embedded tissue sections from gastric cancers were studied for the expression of mmp- , mmp- , timp- and timp- protein by immunohistochemistry. objective: amacr plays role in beta oxidation of branched fatty acids. it is expressed in several neoplasms like prostatic, hepatic and thyroid cancers. it is also believed to be expressed in gastric carcinoma (gc), particularly in better differentiated and less advanced lesions. the aim of the study was to assess amacr expression in gc and its correlation with clinical and histological prognostic factors. objective: brunner's gland hyperplasia is a rare lesion, with fewer than cases having been reported in the literature. it is considered to be a hamartoma, developing from the submucosal glands, usually in the first or second part of the duodenum. most of the lesions described were polypoid and < cm in greatest diameter. often, there is an association with peptic ulcer disease. method: we report the case of a -year-old man who presented with abdominal pain, vomiting and upper gastrointestinal bleeding. the anamnestic data showed chronic ulcer disease. he also was anaemic, with a slight raise in blood eosinophilia. results: the radiological examination of the upper gastrointestinal tract revealed pyloric stenosis due to a polypoid mass measuring cm. preoperative diagnosis of the mass could not be established. the mass was excised and at the same time a vagotomy with pyloroplasty (finney) was performed. brushing cytology of the surgical specimen revealed a great number of aggregated brunner's glands without any atypia, as well as many lymphocytes. a frozen section was negative for malignancy. the histologic diagnosis was that of diffuse nodular brunner's gland hyperplasia (hamartoma) accompanied by lymphocytic infiltration. the rate of cellular proliferation was low. during the postoperative period, there were no complications. a subsequent gastroscopy showed no pathologic findings, and the patient is well at present. conclusion: our case is reported because of its rarity and the difficulties in preoperative diagnosis. the literature is also reviewed. objective: in the differential diagnosis of gist, associated mesenchymal lesion represents an additional challenge. method: a -year-old woman with intestinal occlusion at surgery showed an ileal invagination on polypoid mass of cm and an ileal suberosal nodule of cm. results: the polypoid lesion, ulcerating the mucosa and growing in the muscular wall with subserosal hourglass-like expansion, showed a monotonous hypocellular mesenchy-mal proliferation (cd + , cd − ) with prominent vascular network in myxoid inflammatory stroma. considering the peculiar degenerative, inflammatory pattern of the polyp due to ischemia and the existence of a subset of gist c-kit negative, differential diagnosis was gist (c-kit − ) or inflammatory fibroid polyp (cd + ). after a careful review of the clinical and immunohistochemical and pathological findings, final diagnosis was inflammatory fibroid polyp (ifp). the minor nodule showing a spindle cell proliferation (cd + ) was diagnosed as gist. to our knowledge, no other studies report ifp and gist occurring synchronous in the ileum. conclusion: the correct definition of these lesions is crucial: defining both tumors as gists, prognostic and therapeutic assessment becomes poor (multiple familial or advanced metastatic gist). the recent discovery of pdgfr mutation (the mutually exclusive mutation of wild-type ckit gists) in the ifp questions his reactive nature and raises the possibility of a neoplastic process. then, the questions arising are whether the two tumors represent a fortuitous coexistence or involve the same carcinogenic agents on the same gene (located in the chromosome q) and if the proliferation can be inhibited by sti . objective: melanoma is a rare tumor of the anorectal area and accounts . % to . % of all anal tumours and approximately % of all primary melanomas. besides the positivity with conventional melanocytary immunohistochemical markers s , hmb , melan a are primary anorectal melanomas positive also with marker cd and negative with monoclonal antibody cea. the primary anorectal melanoma is a typical braf mutation (exon + ) and the allelic loss nf associated with neurofibromatosis. method: the melanocytary lesion of a -year-old woman in the anorectal canal was examined with immunohistochemical antibodies: s , hmb , ck ae /ae , cd and monoclonal cea (dako), and fish method with the use of combined sound vysis preb /lsi myb/lsi ccnd /cep (abbot, usa). results: our examination stated positivity with s , hmb , melan a and sporadic positivity with cd and negativity with monoclonal antibody cea. further, amplification of the gene rreb (locus p ) and ccnd (locus q ) was found. the results of our analysis confirm genetic abnormalities associated with melanoma, and our immunohistochemical analysis prefers primary melanoma before metastatic melanoma of the anorectal canal. objective: tumor budding or dedifferentiated single cells/small clusters at the invasive front of colorectal cancer is a histological feature highly predictive of lymph node and distant metastasis and an independent prognostic factor. we hypothesized that the evaluation of tumor budding could complement k-ras analysis to improve the individualized prediction of response to anti-egfr-based therapies in metastatic colorectal cancer (mcrc) patients. method: forty-three patients with mcrc treated with cetuximab or panitumumab were entered into this study. according to the recist criteria, patients had stable or progressive disease (non-responsive, nr), while patients had a partial response (pr). tumor buds were evaluated from whole tissue sections stained for pan-cytokeratin, evaluated in the densest region using a × objective, and "high-grade" tumor budding was defined as buds/highpower field. results: tumor buds and k-ras mutation both correctly classified % of patients. all patients with k-ras mutation (n = ) or high-grade tumor budding (n = ) were nr, of which four patients had both features. all pr were k-ras wild type with low-grade tumor budding. combined, the predictive value of k-ras and tumor budding was %. additionally, high-grade tumor budding was significantly related to worse progression-free survival (hr ( %ci), . ( . - . , p = . )). method: inter-institutional and interdisciplinary cooperation (of pathologists, geneticians, molecular biologists and oncologists of all three centers) during the last months based on mutual exchange of ideas, materials and laboratory protocols and common meetings and seminars. in the centres, the following three methodical options were tested-sequencing, rt-pcr (therascreen®) and snapshot multiplex analysis-and the results of the examinations were retested using another method to verify specificity and sensitivity of the testing. results: k-ras mutations were present in . % of the cases, seven adenocarcinomas and one liver metastasis. egfr was positive in ten cases in tumor cells and in seven cases in the vessels. immunohistochemical overexpression of ras protein was detected in samples. the relationship between the positivity of the k-ras mutation and the positive immunohistochemical reaction for egfr and the ras protein did not reach statistical significance. conclusion: there were no significant correlations between the mutational status of the k-ras gene and the ihc reaction for egfr and ras p protein, proving once more the major role of molecular analyses in colorectal carcinoma anti-egfr therapy. objective: hepatic pedicle triad occlusion is often used to prevent bleeding during hepatectomy despite the ischemia and reperfusion liver injury iatrogenically induced. this study aimed to estimate the impact of selective clamping of the hepatic artery (scha) in hepatocellular function. method: ( ) forty wistar rats were divided into four groups: three groups were subjected to a scha ischemia period for min: (a (n= ) submitted to a continuous scha; b (n= ) underwent an intermittent scha for min with min of reperfusion; c (n= ) underwent an intermittent scha for min with min of reperfusion)) and group d (n= ) without scha. ( ) determination of liver blood markers and hepatic extraction function (hef) using m tc-mebrofenin days before and after surgery. ( ) isolation of hepatocytes from the biopsy performed after surgery to evaluate oxidative stress (dcfh -da), characterization of cell death (annexin-v/propidium iodide) and assessment of mitochondrial membrane potential (jc- probe) by flow cytometry. results: ( ) there was significant increase of blood markers before and after scha, but without differences between groups (ns). ( ) hef maintained normal values without differences between groups (ns). ( ) there were no significant differences in viability and in the type of cell death, as well as in the production of reactive oxygen species between groups (ns). the scha compared to previous studies performed by us where total hepatic pedicle triad was clamped shows an increase of cell viability with a decrease of hepatocyte necrosis and/or apoptosis. scha is a potential alternative to decrease preoperative bleeding, maintaining hepatocellular function. objective: the selective portal vein clamping (scpv) is a potential alternative to decrease preoperative bleeding, maintaining hepatocellular function. this study aimed to evaluate the effect of scpv in hepatocellular function. method: ( ) fifty-two wistar rats were divided into four groups: three groups of animals were submitted to a scpv ischemia period for min (a (n= ) was submitted to a continuous scpv, b (n= ) underwent an intermittent scpv for min with min of reperfusion, c (n= ) underwent scpv for min with min of reperfusion)) and group d without scpv (n= ). ( ) determination of liver blood markers and hepatic extraction function (hef) using m tc-mebrofenin days before and after surgery. ( ) isolation of hepatocytes from the biopsy performed after surgery to evaluate oxidative stress (dcfh -da), characterization of cell death (annexin-v/propidium iodide) and assessment of mitochondrial membrane potential (jc- probe) by flow cytometry. results: ( ) mortality: a- %, b- %, c- % and d- % (p< . ). ( ) there was a statistically significant increase of the ast values (p< . ) and ldh (p< . ), but without differences between groups (ns). ( ) hef significantly decreased (p< . ), but without differences between groups (ns). ( ) there were no significant differences in viability and in the type of cell death or in the production of reactive oxygen species between groups (ns). conclusion: the scpv compared to previous studies performed by us where total hepatic pedicle triad was clamped shows an increase of cell viability with a decrease of hepatocyte necrosis and/or apoptosis. however, scpv above ' should be avoided given the high mortality observed. oxidative stress and liver morphology of rats received doxorubicin and tirapazamine e. korobowicz*, i. syroka, j. dudka, a. korga, r. gieroba *uniwersytet medyczny w lublini, katedra i zakbad patomorfologi, poland objective: tirapazamine (tpz) selectively kills hypoxic tumor cells. doxorubicin (dox) is one of the most effective drugs against a wide variety of cancers. it is widely accepted that oxidative stress is involved in dox organ toxicity. tpz, similarly to dox, is activated to tpz free radical by the action of several nadphdependent reductases and generate reactive oxygen species. nadph is also an essential cofactor for glutathione-dependent enzymes that constitute major cellular defenses against oxidative damage. aim: the aim of the study was to evaluate oxidative stress and liver morphology changes in rat that received tpz in addition to dox administration. method: male wistar rats were treated i.p. with dox ( . mg/kg b.w.) and tpz ( or mg/kg b.w.) weekly for weeks. sevendays after the last treatment, animals were killed and liver samples were analyzed for nadph, mda, gsh levels and histopathological changes. blood samples were analyzed for alt, ast activities and bilirubin concentration. results: anova test revealed significant higher levels of nadph and mda in liver in group receiving a higher dose of tpz with dox vs. dox group. there were no differences between group tpz+dox vs. dox of liver gsh concentration and plasma alt activity, but levels of gsh and alt in all these groups were significantly higher compared to control. microscopically, hydropic degeneration, necrosis, inflammatory cell infiltration and vascular lesion were observed in tpz+dox group. conclusion: tirapazamine causes oxidative stress in liver of rats receiving dox. this study suggests enhanced toxicity by tpz in rat livers which received dox. the influence of tetraiodothyronine-supplemented diet on selected redox equilibrium markers and liver morphology in rats treated with doxorubicin e. korobowicz*, a. korga, j. dudka *uniwersytet medyczny w lublini, katedra i zakbad patomorfologi, poland objective: the toxicity of doxorubicin results from reactive oxygen species generation. ros production depends on tissue-specific enzymatic performance. liver is an organ that is characterized by very intensive activity of these enzymes. as a result, redox equilibrium disorders may appear. moreover, changes in thyroid hormone concentrations are accompanied by ros production. thus, disorders in iodothyronine hormones status may result in the intensification of doxorubicin-induced oxidative stress. aim: the aim of this study was to evaluate the influence of dox and t -associated treatment on selected redox equilibrium markers and histopathological evaluation of the liver. method: rats were treated with dox (. mg/kg, i.p.) once a week for weeks. apart from dox, thyroxin was simultaneously given in drinking water . and . mg/l, respectively. the concentration of lipid peroxidation products-malondialdehyde (mda) and total glutathionewere measured in liver homogenates. liver morphology was evaluated in h+e and pas diastase staining. results: higher levels of mda in liver of all tested groups and at the same time in rats treated with dox plus t lower concentrations of total glutathione compared to control were observed. morphological evaluation of liver did not show any symptoms of necrosis and steatosis, but a decrease in glycogen content in dox+t group compared to dox treatment was noticed. conclusion: thyroxin supplementation causes redox equilibrium disorders and oxidative stress in liver of rats receiving dox. the study revealed the normalizing influence of thyroxin on glycogen deposits that were observed after doxorubicin treatment. objective: dysadherin is a cancer-associated cell membrane glycoprotein that inhibits cell-cell adhesion. in many types of cancer, dysadherin is related to decreased e-cadherin expression, promotes metastasis and is an independent predictor of poor patient survival. dysadherin expression in human hepatocellular carcinoma (hcc) has not been studied to date. method: we studied by immunohistochemistry hcc (grade i, ; ii, ; iii, ; and iv, ) from patients (m/f . , mean age . range - years) with monoclonal antibodies specific for dysadherin (clone ncc-m ) and Εcadherin (clone /e-cadherin). immunostaining of inflammatory and endothelial cells was used as internal positive control for dysadherin (dys) expression. results: non-neoplastic hepatocytes and cholangiocytes were dys-negative. in neoplastic cells (nc), dys-specific immunostaining was mainly membranous, while some cases showed additional nc cytoplasmic positivity. the majority of hcc ( / , . %) had - . % dys (+) Νc, . % ( / ) showed - % (+) Νc, while % ( / ) had > % (+) Νc. dys expression was negatively correlated with e-cadherin expression (p< . ), while a positive statistically significant correlation was observed between strong dys immunostaining (> %+Νc) and tumour grade (p< . ). there was no correlation with tumour size, vascular invasion or other clinicopathological parameters examined. conclusion: normal hepatocytes and cholangiocytes do not express dysadherin. in hcc, increased dysadherin expression is observed in approximately one third of the cases; it is more extensive in high-grade tumours and is related to decreased e-cadherin expression. klippel-trenaunay syndrome with clinical presentation as metastatic liver disease: a case report s. stadlmann*, d. lenggenhager, r. a. kubik-huch, j. h. beer, g. singer *kantonsspital baden, institute of pathology, switzerland objective: klippel-trenaunay weber syndrome (ktws) is a rare congenital malformation characterized by hemangiomata, varicose veins and bone and soft tissue hemihypertrophy. ktws can include additional vascular and lymphatic system abnormalities in various organs, but involvement of the liver is rare. we report a case of ktws simulating metastastic liver disease. method: a -year-old male with ktws presented with a tumor mass in the right abdomen. ultrasonography revealed a -cm cystic abdominal tumor and additional hypodense lesions in liver segments i, ii, and viii. a ct scan additionally showed multiple hypodense lesions in the spleen, peritoneum, lung, right scrotum, soft tissue and enlarged abdominal lymph nodes, highly suspicious for metastatic disease. results: laboratory data values were: gammagt u/ l (normal - ), ldh u/l (normal - ), albumin g/l (normal - ), and alpha feto-protein . μg/ l (normal < ). histopathology of the cystic abdominal tumor mass showed a complex vascular malformation of lymphangioma-hemangioma type. evaluation from two different tumorous liver lesions revealed nodular hepatic tissue separated by fibrous septa with reactive ductules and atypical vessels, consistent with multiple focal nodular hyperplasia (fnh) in the setting of ktws. conclusion: the spectrum of ktws can include arterial and lymphatic system abnormalities beyond the classical manifestation. our findings support the concept that multiple fnh characteristically occurs in a syndromic form and is induced by an irregular blood supply in the liver, with localized hyperperfusion leading to reactive nodular proliferation of liver tissue. microscopic evaluation of metaplastic and dysplastic changes in the mucous of the gallbladder d. lacka*, a. nasierowska-guttmejer *csk mswia, zaklad patomorfologii, warsaw, poland objective: in the intra-and extrahepatic biliary ducts, precancerous changes such as hyperplasia, metaplasia and dysplasia are diagnosed. in the gallbladder, cholecystitis chronica is diagnosed often with a disregard of the features of hyperplasia and metaplasia. generally accepted markers of precancerous changes of the gallbladder and biliary ducts are still to be found. aim: the aim was to define the accumulation of tp protein and mib activity in hyperplasia, metaplasia and dysplasia of the mucous membrane of the gallbladder. method: the material comprises gallbladders operated by laparoscopy and fixed in formalin. specimens were embedded in paraffin. in the microscopic sections with hyperplasia, metaplasia and dysplasia immunohistochemical stainings for tp and mib were done along with mucycarmin and pas with alcian blue stainings (mucous presence). results: the histopathologic diagnosis revealed cases ( %) of chronic calculous cholecystitis, cases of acute inflammation and secen cases of cancer. hyperplasia, metaplasia or dysplasia were found in cases ( . %) with chronic inflammation and accounted for hyperplasia without metaplasia- cases, pseudopyloric or intestinal metaplasia- cases, dysplasia- cases. mucous stain-ings confirmed the presence of pseudopyloric and intestinal metaplasia, which helped distinguish them from adenomyosis focuses. ihc confirmed the accumulation of tp protein and proliferative activity in metaplasia and dysplasia of the mucous membrane. conclusion: mucous stainings and positive tp and mib reactions suggest that metaplasia and dysplasia of the mucous membrane of the gallbladder can be classified as intraepithelial neoplasia changes. more research should be done. objective: we aimed to investigate the relationship between the findings in total or partial hepatectomy specimens. method: one hundred six cases were collected who had total transplantation or partial hepatectomies because of liver cirrhosis (lc), hcc or other hepatic disorders from the archives. then, clinical, histopathologic features and serologic findings of the cases were reviewed. the relationship between the histopathologic features and endoscopic, clinical and serologic results were investigated statistically using comparison tests. results: for the last -year period, data from patients who underwent transplantation or hepatectomy were collected. ninety-five had total, ten had partial hepatectomies, and one had metastasectomy. after the gross and microscopic examination, cases were diagnosed as lc, had hcc, three had parasitic cystic disease and ten had other unusual diseases. among lc cases, had hepatitis b virus (hbv), seven had hepatitis c virus (hcv), three had mixed virus etiology, six were alcoholic and were due to other causes. among lc patients, had hcc. fourteen cases were found to have one, seven had two and had more than two tumors. twenty-two of the hccs were located in the right, while nine had tumors in the left and right; one case had tumors in the right, left and caudate lobe. the results demonstrated that the most common hepatic disorder was cirrhosis due to hbv in the hepatectomy specimens and hcc seen in one third of them. studies with follow-up and response to therapy will form the basis of our future projects. objective: solid pseudopapillary tumor of the pancreas (sptp) represents - % of pancreatic tumors, it affects young women, and surgery is the treatment of choice. histologic features include loosely cohesive, relatively uniform polygonal cells surrounding delicate capillarysized blood vessels. most tumors are positive for betacatenin, cd , progesterone receptors and neuroendocrine markers. mutations in the beta-catenin gene are frequent. although considered benign, they are currently classified as low-grade malignant epithelial neoplasms. method: a -year-old woman presented with abdominal pain. a tumor in the pancreatic head with liver metastasis is diagnosed by tc. fine needle aspiration cytology is diagnostic of sptp. we reviewed four more cases of sptp. results: all five tumors occurred in women < years old that presented with abdominal pain or asymptomatic. tumors ranged from . to cm. four were located in the tail and one in the pancreatic head. all patients underwent surgery with negative margins. one patient had hepatic and nodal metastases as well as lymphatic and perineural invasion. all tumors were positive for cd and cd , three for progesterone receptors. none of the patients presented tumor recurrence ( months to years). conclusion: solid pseudopapillary tumor of the pancreas should be included in the differential diagnosis of pancreatic masses. surgery is usually curative and should also be attempted in rare cases with an aggressive behaviour. despite the characterization of the morphologic and molecular features of this enigmatic neoplasm, more work is needed to uncover its cell of origin and true histogenesis. objective: serous cystic neoplasms (scn) are rare pancreatic tumors. the aim of the study was to describe histopathological characteristics of scn diagnosed in a single center during a -year-long period. method: pathological reports including macro-and microscopic descriptions of scn cases as well as routine slides were retrieved from institutional databases and retrospectively reevaluated. only cases with available original slides were included in the study. results: several clinicopathologic scn subtypes were distinguished: serous microcystic adenomas ( . %), five serous oligocystic and ill-demarcated adenomas ( . %) and a single case of microcystic adenoma coexisting with multiple serous cysts in von hippel-lindau patient ( . %). there were females ( . %) and six males ( . %). median patients' age was years (range - years). fifteen cases ( . %) were localized in the head of the pancreas, ( . %) in the pancreatic body, and five ( . %) in the tail. median tumor diameter was . cm (range - cm, the diameter of three tumors was not known). eleven cases ( . %) were diagnosed in pancreaticoduodenectomy specimens, six ( . %) in middle segment pancreatectomy specimens, and four ( . %) in distal pancreatectomy specimens. six cases ( . %) were enucleated, two cases ( . %) were diagnosed in open biopsy and two ( . %) cases were found during autopsy. conclusion: scn is found in middle-aged or older patients, mainly women. scn may be localized in each pancreatic segment and grow to large masses. the reasons for preferential existence of scn in females remain unknown. p , ki- , cd expressions in gastrointestinal and pancreatic neuroendocrine tumors and the evaluation of these tumors with clinicopathological and prognostic parameters e. kimiloglu sahan*, n. erdogan, s. ceylan, i. ulusoy *taksim training hospital, dept. of pathology, istanbul, turkey objective: gastrointestinal and pancreatic neuroendocrine tumors (gepnets) originate from the cells of the diffuse endocrine system. their molecular genetic mechanism of development and progression is complex and remains largely unknown. the purpose of this study was to review the gastrointestinal and pancreatic neuroendocrine tumors and to investigate these tumours with an emphasis on their clinicopathological characteristics. postoperative period. another patient survived for years and died of unrelated cause. conclusion: pa occurs infrequently in young patients. although some cases are diagnosed early and may be treated with potentially curable surgery, long-term survivals are rather exceptional. granulomatous pancreatitis in a patient with lada type diabetes mellitus v. mandys*, m. kheck, m. andel * rd faculty of medicine, dept. of pathology, prague, czech republic objective: the aim of this presentation was to demonstrate an unusual case of granulomatous pancreatitis discovered in a patient suffering from the lada type diabetes mellitus. method: samples of pancreatic tissue obtained during the postmortem examination were processed by a routine histological technique. the slides were stained with h&e, trichrome and van gieson method. immunohistological methods were used to detect markers of endocrine cells of islets of langerhans and of macrophages. results: a -year-old female patient suffering from diabetes mellitus lada type, generalized atherosclerosis and hypertension died due to pulmonary embolism. lipomatosis of pancreatic tissue was observed during the postmortem examination. histologic examination of pancreatic tissue discovered multiple small non-caseating epithelioid cell and giant cell granulomas replacing the islets of langerhans. conclusion: to our knowledge, our case represents the first description of non-infectious granulomatous pancreatitis associated with diabetes mellitus of lada type. objective: evaluation of crohn's disease (cd) activity is crucial to optimize therapeutic strategies. differentiation between inflammatory and fibrostenotic lesions is decisive since the former are treated with drugs, while the latter require surgery. cd activity is based on a combination of clinical, biochemical, endoscopic and radiologic findings, which cannot reliably distinguish between both entities. in patients with refractory symptoms, therapy decisions rely heavily on histologic diagnosis. method: preoperative mr imaging was performed in cd patients undergoing elective bowel resection. mr evaluated wall thickness, pre-and post-contrast wall signal intensity, relative contrast enhancement, presence of edema, and luminal stenosis. matched histological sections of the mr images were stained with hematoxylin and eosin (he). evaluation of wall and submucosa thickness, percentage of inflammatory and fibrous components in the mucosa and submucosa (masson's trichromic and reticulin stains), submucosal edema, and vascularity (cd stains). results: cases assessed as inflammatory by he had higher early post-contrast signal intensity by rm than fibrostenotic cases (p= . ). submucosal thickness correlated with early fibrosis (reticulin, p= . ), but only a trend with high vascularity (cd ). fibrostenotic cases had submucosal thickness inversely correlated with early signal capture and with relative contrast enhancement. mucosal inflammation was inversely correlated with submucosal fibrosis and thickness (p= . ). established fibrosis (trichromic) had no delayed signal capture (p= . ). conclusion: there is a good correlation between histologic and mr findings. mr may reliably differentiate between inflammatory and fibrostenotic lesions, and it may have an important role as a new pre-surgical biomarker in the management of cd. the role of standard endoscopic biopsy in diagnosis of gastric gastrointestinal stromal tumors managed in the holycross cancer centre, kielce, poland, in years - w. rezner* *holycross cancer centre, dept. of neoplasm pathology, kielce, poland objective: intramural character of growth makes gastrointestinal stromal tumor (gist) difficult or impossible to access by endoscopic biopsy forceps, and therefore, the diagnostic yield of the biopsy does not exceed %, even with the use of special techniques. the aim of the study was to evaluate the role of standard endoscopic biopsy in preoperative assessment of gastric gists. method: from among cases of all gastric gists managed in our institution in the years - , we selected cases where standard forceps biopsy from subepithelial mass was obtained. we excluded five cases with no subepithelial mass visible endoscopically and two cases of small incidental gists in patients operated for other neoplasms. biopsies diagnosed as negative for gist were analysed retrospectively with serial sectioning and immunostains including, where appropriate, cd- , cd- , sma, s- and calponin in search for neoplastic tissue which could have not been apparent in original he-stained slides. results: none of the biopsy samples analysed retrospectively contained neoplastic tissue. material which enabled histopathological and immunohistochemical diagnosis of gist was obtained in out of cases. mucosal ulceration was present in all cases of diagnostic biopsies and in out of non-diagnostic biopsies. conclusion: no gist tissue was originally missed in biopsy material, so the diagnostic yield of standard endoscopic biopsy in diagnosis of gastric gists approached one third of the cases. immunohistochemical markers utilized in differential diagnosis between primary ovarian carcinoma from metastatic colorectal carcinoma a. iliesiu*, m. aschie, m. anca, n. anca, i. poinareanu, a. chisoi, p. corici, v. sarbu, i. aschie *clinical emergency hospital, dept. of pathology, constanta, romania objective: colorectal adenocarcinoma is the most common tumor that metastasizes to the ovary and is often difficult to distinguish from primary ovarian mucinous carcinoma. an immunohistochemical marker cdx- was found to have a diagnostic value in establishing the gastrointestinal origin of metastatic tumors. obtaining the correct diagnosis is difficult but crucial to treatment and prognosis. method: in our study realized in clinical county emergency hospital constanta, we evaluated the immunohistochemical expression of cdx and also the expression of cytokeratin , cytokeratin , cea, muc in cases representing ovarian adenocarcinoma and metastatic colorectal adenocarcinomas involving ovaries. results: searching the tumor registry and pathology database of our hospital, we found that the median age of the study group was years (range, - ). metastatic colorectal adenocarcinoma were almost always negative for muc ( . %), often negative for ck ( . %), focal or diffuse positive for cdx ( . %), diffuse positive for ck ( . %), focal or diffuse positive for muc ( . %), and diffuse positive for cea ( . %). almost all of the primary ovarian carcinomas lacked immunoreactivity for cdx- . in contrast, metastases to the ovary from colorectal primaries showed cdx- immunoexpression. conclusion: cdx- is a useful marker for differentiating primary ovarian carcinoma from carcinomas metastatic to the ovary. ck , ck , cdx and muc ihc staining is a useful adjunctive diagnostic tool to differentiate metastatic colonic tumours from primary ovarian tumours, in addition to clinical history and gross and microscopic findings. multicystic mesothelioma-a case report j. jeruc* *faculty of medicine, institute of pathology, ljubljana, slovenia objective: multicystic mesothelioma or multilocular peritoneal inclusion cyst is a rare tumor with a predilection for pelvic surfaces of the peritoneum. most frequently, it occurs in women of reproductive age who have a history of previous pelvic surgery or infection. results: a -year-old man with no significant past medical history presented with abdominal pain lasting days in the ileocecal region. despite normal white cell count, acute appendicitis was suspected and the patient underwent surgery, during which a multicystic mass measuring . cm in the longest diameter was found. cysts were thin-walled, translucent, with a smooth outer surface, and mostly filled with clear and some with hemorrhagic fluid. histologically, they were lined by a single layer of cuboidal polygonal and focally hobnailshaped cells with small uniform nuclei without mitotic activity. cells stained positive for cytokeratin, calretinin and wt- , but were negative for endothelial markers, cea and estrogen and progesterone receptors. the loose connective tissue between cysts was infiltrated by lymphocytes and granulocytes. the diagnosis of multicystic mesothelioma was made. threemonths after surgery, the patient is doing well. conclusion: the majority of investigators considers this entity to be an unusual type of mesothelial neoplasm that has a tendency to recur locally and may rarely transform into a conventional mesothelioma. some, however, consider the lesion to be a non-neoplastic reactive mesothelial proliferation. multicystic mesothelioma has an indolent course, but approximately one half of cases recur. the differential diagnosis includes malignant mesothelioma, cystic lymphangioma, pseudomyxoma peritonei and mesenteric and omental cysts. collagen iv expression, as staining of the continuous basement membranes of vessels, varied from % in lr gists to % to % in ir and hr tumors, respectively. gamma's correlation test revealed correlation between microhemorrhages and grade (r= . , p= . ), tumor necroses and p (r= . , p= . ), tumor necroses and collagen iv (r=− . , p= . ) and vegf and collagen iv (r=− . , p= . ). conclusion: this study showed that both high p expression and the presence of tumor microhemorrhages are possibly related with unfavorable prognosis. in addition, we found that a tumor necrosis was correlated with a p and collagen iv. smooth muscle tumors with cd /cd positivity cells s. rjabceva* *belarusian medical academy, dept. of pathology, minsk, belarus objective: immunohistochemically, smooth muscle cells are characterized expression of sma and desmin. the aim of this study was to describe the clinicopathological and immunohistochemical features of smooth muscle tumors (smt) with cd /cd positivity cells. method: eleven cases diagnosed as smt were examined by both light microscopy and immunohistochemistry (ihc). i used ihc markers such as cd , cd , smooth muscle actin, desmin, vimentin, nse and s . results: the patients (m/f= : ), with age ranging between and years (mean = . ), had smt. a tumor size varied from . to cm (mean = . cm). tumors were localized in the esophagus ( / . %), stomach ( / . %), colon ( / . %), retroperitoneum ( / . %) and in the pelvis ( / . %). histologically, tumors are composed of elongated or ovoid cells with pale nuclei and eosinophilic cytoplasm which is distinctly fibrillar. immunohistochemically, cells of smt were positive for sma ( %), desmin ( %), vimentin ( %) and nse ( . %), but they were negative for cd , cd and s . i found that the scattered cells of the tumors were characterized by the expression of cd ( / %), cd ( / . %) and s ( / . %). these cells were located diffusely or focally in the smt. the immunophenotype of these cells was similar to immunohistochemical features of the interstitial cells of cajal or the gastrointestinal stromal tumor (gist). conclusion: although the scattered tumor cells revealed cd positivity, the histological finding was similar to smt and was different from gist. smt colonized by cd /cd -positive cells should not be confused with gists. updates of tnm classification: for the better or worse? b. karabulut*, a. m. sonmez, d. turan, n. kursun, a. ensari *ankara university med school, turkey objective: for the prognostic evaluation of tumours, the tnm classification is updated periodically. the use of this classification for colorectal cancer (crc) has caused some problems in the assessment of metastatic lymph nodes. in this study, we have investigated the effect of the updates on metastatic lymph node interpretation and, hence, staging. method: h&e slides of cases of crc were reevaluated microscopically to assess the number of metastatic lymph nodes according to the criteria of tnm , tnm and tnm . clinical parameters such as gender and age of the patients, tumour location, grade, and stage were retrieved from the patients' files. mann-whitney, kruskal-wallis, wilcoxon signed ranks and multiple comparison tests were used for statistical analysis. results: there were men ( . %) and women ( . %) with a mean age of . years (ranging from to ). the tumour was located in the right colon in cases ( . %) and cases in the left colon ( . %). there were three grade ( . %), grade ( . %), and grade tumours ( . %). the number of metastatic lymph nodes was significantly higher in grade tumours compared to grade tumours in tnm , tnm and tnm . according to tnm , significantly more metastatic lymph nodes were determined than tnm and tnm (p< . and p < . , respectively), whereas tnm revealed significantly more metastatic lymph nodes in comparison to tnm (p< . ). conclusion: our results show that there is significant variation between updates of tnm in terms of metastatic lymph node yield which will inevitably cause variation in the tnm classification. objective: metastatic lesions to the mandible are rare, comprising < % of all malignancies. an -year-old woman presented with a painful swelling in the right mandible growing progressively for months. method: computed tomography (ct) showed intraosseous expansile and destructive lesion in the corpus of the right mandible × mm in size expanding to the soft tissue laterally. after administration of contrast material, moderate enhancement was seen throughout the mass. the biopsy showed stromal tumor immunopositivity for cd . the patient had a medical history of ileal tumor resection years ago at another institute which was diagnosed as gastrointestinal stromal tumor (gist) and classified as intermediate risk group for potential of malignancy. results: the mandibular tumor was considered as a metastatic gastrointestinal stromal tumor. no tumor was seen on positron emission tomography (pet)/ct other than the mass in the right mandible. the histopathology of the ileal tumor was seen and the diagnosis was confirmed. imatinib treatment was started at a dose of mg daily. resection of the mandibular mass was planned by plastic and reconstructive surgeons. conclusion: liver and peritoneum are usual metastatic sites for gastrointestinal stromal tumors. to the best of our knowledge, metastasis of ileal gastrointestinal stromal tumor to the mandible has not been previously reported in the english literature. objective: warthin's tumor is the second most common neoplasm of the parotid gland accounting for - % of parotid neoplasms. however, parotid gland is an extremely rare site for extrapulmonary tuberculosis. objective: the purpose of this study was to assess the immunohistochemical (ihc) status of androgen receptor (ar) in various salivary gland cancers. the study group consisted of cases of primary salivary gland cancers from patients (f/m= : , age - years). these were adenoid cystic carcinomas (adcc), adenocarcinomas nos (adc), mucoepidermoid carcinomas (mec), acinic cell carcinomas (acc), carcinomas ex pleomorphic adenomas (caexpa), salivary duct carcinomas (sdc), basal cell adenocarcinoma (bcac) and large cell neuroendocrine carcinoma (lcnec). ihc reactions with monoclonal mouse antihuman androgen receptor antibody (dako, clone ar ) were done on -μm-thick sections cut from representative archival paraffin block. in each positive case, the revealed strength of ar expression was assessed as total score (ts) according to allred score. results: the ar expression was found in five adc nos, four caexpa, four sdc, four acc, three mec, and one adcc and bcac, and calculated ts ranged from three to eight points. ts had eight points in six cases, seven in three cases, six in one case, five in three cases, four in two cases and three in four cases. conclusion: ar expression was found in of ( %) primary salivary gland cancers. the strength of ar expression differed in various salivary gland cancers, and it was most frequent in caexpa ( / ) and sdc ( / ) and less frequent in adc ( / ) and acc ( / ), whereas only found incidentally in others. mena expression in normal and neoplastic salivary glands g. simona* *umf, dept. of pathology, targu-mures, romania objective: recently, studies revealed that human orthologue of murine mena (mammalian ena), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during breast, colon and pancreatic carcinogenesis. in our previous studies, we observed that mena was modulated during colon and cervix carcinogenesis. in this study, we analysed mena expression in lesions of salivary glands (sg). previous studies regarded only maspin expression in these lesions, a protease inhibitor which is increased in benign tumors but decreased in sg carcinomas. method: we have analyzed mena expression in normal sg (n= ) and also benign (n= ) and malignant (n= ) lesions of sg. for the immunohistochemical staining, we used the murine mena antibody, provided by bd biosciences. mena expression was quantified in cytoplasma of tumor cells. results: all normal sg and their benign lesions were mena-negative. it included ten pleomorphic adenomas and ten warthin's tumors. ductal adenocarcinomas (n= ), independently by their histological grade and also carcinomas with acinary cells (n= ) and squamous carcinomas (n= ), were positive. no difference of mena intensity in positive cases was observed. all lymphomas (n= ) were mena-negative. conclusion: this is the first study in literature about mena expression in sg tumors. our results prove that mena plays a role in carcinogenesis of different organs, including sg, but the exact mechanism is not yet known. in accordance with different previous studies about maspin expression in sg lesions, it seems that these two antibodies are reversely correlated. future studies are necessary in order to elucidate their role in sg tumors. secondary tumors of the salivary glands a. c. faur* *university of medicine, 'v babes' timisoara, romania objective: secondary tumors of salivary glands are rare and constitute about % of all malignant salivary neoplasm. method: a study has been carried out for years on cases of salivary gland tumors, and only one case of salivary metastatic lesion was diagnosed. a -year-old female with a history of a right temporal malignant melanoma was admitted at the plastic surgery department of county hospital of timisoara. sixmonths after the surgery, the patient returned to this clinic with a rapidly growing mass in the right temporal region and right preauricular region. a × -cm diameter tumor was excised with adjacent lymph nodes. a superficial parotidectomy was performed also, preserving the facial nerve and its branches. formalin-fixed paraffin-embedded tissue samples were cut at μm and stained using hematoxylin and eosin (he). results: on he stain, the periparotid and temporal region lymph nodes were infiltrated by malignant melanoma cells. the parotid tissue also had metastatic lesions. the metastatic malignant melanoma lesion consisted of epithelioid neoplastic cells with cytologic and nuclear atypia, large nucleoli, atypic mitotic figures and abundant melanin pigmentation. necrosis and hemorrhage were also seen. conclusion: the parotid gland and its lymph nodes are possible sites of metastasis from head and neck tumors, especially squamous cell carcinoma or melanoma. the rarity of salivary secondary tumors prompted us to report this case. objective: primary intraosseous mucoepidermoid carcinoma of the jaws is a rare lesion accounting for < % of all mucoepidermoid carcinomas reported in the literature. it is a neoplasm of adult life and affects females twice more frequently than males. histologically, it is a low-grade carcinoma usually affecting the mandible. the radiologic presentation is that of uni-or multilocular lesions. their origin still remains controversial. method: we report the case of a -year-old female who presented with a painful swelling in the left maxilla of month's duration. there was no history of previous surgical intervention. clinical examination revealed an ulcerated swelling. cervical lymphadenopathy was absent. results: the radiological examination showed a unilocular compact lesion in the maxilla, not unequivocally diagnostic of malignancy. surgical excision of the lesion together with a small part of the maxilla was performed. grossly, it was a solid mass of relatively soft and only focally bony consistence measuring . cm. on brushing cytology of the surgical specimen, groups of mucous cells without any significant atypia intermingled with aggregates of squamous cells. the histologic diagnosis, confirmed by histochemical and immunohistochemical methods, was that of low-grade mucoepidermoid carcinoma of the maxilla. the postoperative period was uneventful. the patient is on regular follow-up and is disease-free after years. conclusion: our case is reported because of its rarity, and at the same time, the literature is reviewed and speculations about the pathogenesis of mucoepidermoid carcinomas are attempted. odontogenic myxoma (om) in the maxilla: a case report and review of the literature s. papaemmanouil*, n. pastelli *g. papanikolaou general hosp., dept. of pathology, thessaloniki, greece objective: odontogenic myxoma is an intraosseous tumor of the jaws, relatively benign with locally aggressive behaviour. most of the oms are slowly growing with no symptoms. large tumors cause painless expansion. it occurs in patients over the age of and is mostly located in the mandible. method: a -year-old man was presented with a progressively enlarging mass in the maxillary area. the imaging findings revealed a large, rather wellcircumscribed tumor mass in the body of the maxillary bone. the mass was excised by peripheral ostectomy. results: the gross examination revealed a grey-white mass with translucent mucinous appearance measuring × . × cm. the sections were examined with h+e and mucin stains and followed by ihc study for mib- (ki- ). the morphologic, histochemical and ihc data were consistent with an odontogenic myxoma. conclusion: our study comments on the imaging findings of macroscopic and microscopic features and the differential diagnosis of this tumor. the current literature is reviewed. objective: the role of interactions between mmp- , mmp- and kai protein expression as markers involved in aggressive behaviour of tumours is still under investigation. the study aimed to evaluate and compare mmp- , mmp- and kai expression in primary oral squamous cell carcinomas (oscc) and adjacent marginal normal squamous cell epithelium and to estimate their association with each other in relation to clinical variables. method: mmp- , mmp- , and kai expression was evaluated on primary oscc (n= ) and adjacent marginal normal tissues (n= ) using immunohistochemistry. results: significant differences between mmp- and kai expression was found in oscc and marginal normal tissue (p= . , p= . , respectively). in normal tissue, mmp- , mmp- expression was found in basal cell layers and also frequently was observed in stromal tissues with inflammatory component. kai protein was not observed in stromal tissues. no significant differences were observed between mmp- , mmp- , kai expression and tumour grade and stage. inverse correlation between mmp- and kai expression was found in oscc (p= . ). kai -negatve/ mmp- -positive cases were observed mainly in high tumour grade and in advanced stage of tumours. conclusion: inverse correlation revealed between kai protein and mmp- expression in oscc could affect the function of kai suppressor protein and influence the metastatic potential of the tumour cells. our results suggest that accumulation of mmp- and mmp- in the surrounding tissue may enhance local invasion in oscc. expression of mmp- and pten in laryngeal squamous cell carcinoma-biological predictors? m. bodnar*, p. burduk, w. kazmierczak, a. marszalek *collegium medicum, bydgoszcz, poland objective: reliable predictors of laryngeal squamous cell carcinoma (lscc) biology are still missing. the aim of this study was to investigate two newly discovered factors (mmp- and pten) in lscc patients with correlation to the stage of the disease. method: we used specimens from surgical resections selected from patients and divided into controls ( samples) and lscc ( samples). we used standard immunohistochemistry for mmp- and pten expression studies with envision system and dab as a chromogen. antigen expression was classified as followed: -no positive cells, -< %, - - %, -above % positive cells. all results were statistically evaluated using mann-whitney u test and anova, with statistical significance at p< . . results: in patients with lscc, the expression pten was detected in the cytoplasm of % tumor cells and % stromal area (p< . ) while in controls only in % of stromal area (p< . vs lscc). pten expression was decreased in tumor cells in n+ vs n cases ( % vs %). in lscc, mmp- was found in . % of the tumor cells, but in % of stromal area (p< . ). there was no mmp- expression in controls, while expression level ( - scale) was higher in tumor cells versus stromal compartment. mmp- stromal expression in n and n+ cases was % versus % of the area, respectively. conclusion: we might conclude that decreased pten expression in tumor cells predicts lymph node involvement, which then is accompanied by decreased expression of mmp- in stromal area in the main lesion. mucosal large cell neuroendocrine carcinoma (mlcnec) of the head and neck regions: a new clinicopathologic entity k. kusafuka*, m. abe, y. iida, t. onitsuka, t. nakajima *shiuzoka cancer center, pathology division, nagaizumi, japan objective: large cell neuroendocrine carcinoma (lcnec) is well known as a subtype of lung cancer, but it is extremely rare in the head and neck regions. our objective was to establish mucosal lcnec (mlcnec) as a new entity in the head and neck regions. method: we reestimated surgically resected specimens of the primary mucosal carcinoma in the head and neck regions, including basaloid squamous cell carcinoma, during - . the immunostainings for neuroendocrine (ne) markers such as cd , chromogranin-a and synaptophysin were performed. in the cases which were positive for two or three ne markers, we re-diagnosed as "mlcnec". results: only eight cases ( . %) were re-diagnosed as mlcnec. all cases were male and their mean age was . years. three cases occurred in the tongue base, four cases in the larynx and one case in hypopharynx. although seven cases showed numerous regional lymph node metastases, only one case showed death of disease. histologically, mlcnec showed the sheet-like, trabecular, organoid pattern growth of relatively large basaloid cells in which the central necrosis, rosette formation, peripheral palisading, and high mitotic figures were sometimes seen. immunohistochemically, mlcnec indicated to be positive for two or three ne markers. only three cases of mlcnec were immunopositive for ttf- , whereas all cases except one case were only focally immunopositive for p . all cases showed high proliferating activity. conclusion: we propose that mlcnec can occur in the head and neck regions and is a new clinicopathological entity. the prognosis of mlcnec remains unclear. objective: massive ovarian edema is an unusual cause of ovarian enlargement in young women. it is due to the accumulation of edema fluid and in some cases is associated with mature cystic teratoma. the patients are young (average age), with present abdominal pain, menstrual abnormalities or evidence of hypergonadism. the ovarian enlargement is unilateral in % of cases. method: we report a case of a -year-old girl with tumor of the left ovary. grossly, the enlarged ovary's greatest dimension was × cm ( , g), composed of solid mass with large gelatinous and hemorrhagic foci. paraffin sections from samples were stained with heeo and immunostained for vimentin, s protein, sma, desmin and cd . results: histological findings were identical in all slides. ovarian stroma cells were scattered within abundant edematous fluid, and in some foci, hypocellular stroma surrounded follicles. in the cortex, follicles were dilated in fibromatous stroma with rare luteinized cells. cd confirmed the presence of many vessels with thrombi composed of fibrin-causing hemorrhagic necrosis. in some areas of necrosis, we found foci of dystrophic calcification suggesting an older process. conclusion: massive ovarian edema is a lesion which can be misdiagnosed as a neoplasm. the cause is unclear. it has been attributed to intermittent torsion of the ovarian pedicle and interference with its lymphatic drainage. the fibromatosis and massive edema represent two ends of the same disorder. objective: intimal fibroplasia is a variant of fibromuscular dysplasia (fmd), a non-inflammatory and nonatherosclerotic arterial segmental disease, resulting in narrowing of the lumen. fmd of coronary arteries is a rare pathology associated with cardiac and sudden death occurring during infancy, childhood and young adulthood. method: we describe the case of a female infant born prematurely (gestational age + / ) presenting a mild supravalvular aortic stenosis associated with an atrial septal defect and cardiac valvular dysplasia. during the first weeks of life, she developed a progressive obstructive cardiomegaly and she died at day of a cardiogenic shock. results: the autopsy confirmed a marked cardiac hypertrophy and the aforementioned congenital cardiac anomalies. histologically, we observed a myxomatous thickening of the aortic, pulmonary and mitral valves and a significant segmental intimal fibroplasia of all coronary arteries, producing subtotal obstruction of the right coronary artery. the myocardium presented multiple areas of infarction of various ages and foci of interstitial fibrosis with calcifications. sampling of the aorta and its major branches showed mild to moderate intimal fibroplasia of the aorta, celiac trunk, superior mesenteric and left renal arteries. the pathogenesis of fmd is unclear: mural ischemia, hormonal, genetic and mechanical factors have been suggested. this case supports the theory of a congenital origin and emphasizes the importance of an extensive examination of coronary arteries when performing autopsy in paediatric patients as fmd is a segmental disease, and it must be considered in the etiologic differential diagnosis of cardiac and sudden death in paediatric age. increased amount of chorionic disc extravillous trophoblasts (evt) in placental hypoxia j. stanek* *cincinnati children's hospital, dept. of pathology, usa objective: increased amount of evt in the maternal floor was found in preeclamptic placentas. this analysis intends to retrospectively prove that the evt is increased in the whole chorionic disc also in other than preeclampsia clinical conditions at risk for fetal and placental hypoxia. method: frequencies of clinical and placental parameters of consecutive cases with more than five cell islands per full-thickness paracentral section of grossly unremarkable placenta (study group, sg) were compared to all remaining , placentas (control group, cg). results: the numbers of placental septa/cell islands were statistically significantly (p< . ) higher in the sg than in the cg in association with preeclampsia, chronic hypertension, diabetes mellitus, oligohydramnios, intrauterine growth restriction, induction of labor, cesarean sections, low -min apgar score, small placentas, placental infarction, massive perivillous fibrin deposition, decidual arteriolopathy, diffuse placental hypoxia, microscopic chorionic pseudocysts, and maternal floor clusters of multinucleate trophoblastic giant cells. the reverse was seen in the premature rupture of membranes, perinatal mortality, abnormal umbilical cord, acute chorioamnionitis, chronic villitis of unknown etiology, and histological placental meconium staining. conclusion: the amount of chorionic disc evt is increased in association with clinical conditions and placental lesions known to be associated with hypoxia, but is decreased in "non-hypoxic" clinical conditions and placental lesions. counting the placental septa and cells islands can serve as a surrogate test of placental hypoxia, not only in preeclampsia. pseudo-torch and the cerebro-costo-mandibular syndrome j. stanek*, a. oestreich *cincinnati children's hospital, dept. of pathology, usa objective: while the posterior rib gaps and pierre robin sequence are well documented, components of the cerebrocosto-mandibular syndrome (ccms), the "cerebral" part thereof is more variable, including mental retardation in survivors, hydrocephalus, severe hypoplasia of frontal and occipital lobes, abnormal olfactory bulbs, cerebral heterotopias, or gliosis. method: this is a case report of unusual brain findings in ccms, to our knowledge found for the first time at postmortem examination. results: because of fetal hydrops and congenital anomalies found on prenatal ultrasound, a -week pregnancy was terminated to reveal a fetus with multiple rib gaps, dysmorphic facial features, prominent micrognathia, higharched palate, club feet, dysplastic organ of corti, incomplete visceral rotation, pseudoglandular transformation of the pituitary gland, mucosal eosinophilia of the stomach and intestines, microcalcifications of liver, and extensive, diffuse, predominantly perivascular and not only periventricular brain calcifications. there was no laboratory evidence of intrauterine infection either clinically or by in situ hybridization. conclusion: this case illustrates a not yet described pseudo-torch, congenital infection-like, presentation of the ccms with extensive brain calcifications and mucosal eosinophilia of the gastrointestinal tract. extensive brain calcifications were described in other genetic syndromes such as baraitser-reardon syndrome, aicardi-goutieres syndrome, and mitochondrial encephalopathies, but not the ccmc. calcium metabolism can have genetic background in the ccms and explain cerebral dysfunction in at least a subset of the syndrome. objective: alveolar capillary dysplasia (acd) with or without misalignment of pulmonary veins (mpv) is an uncommon congenital cause of persistent pulmonary hypertension of the newborn characterized by a lack of alveolar and vascular development. most are sporadic, but familial acd/mpv has been reported, linked to the gene foxf on chromosome q . -q . . it is universally fatal and diagnosis is entirely dependent on the pathological examination of surgical lung biopsy specimen. four cases of acd, three with mpv, are reported. method: the four neonates were full term, without associated congenital anomalies. they were admitted to the paediatric intensive care unit for hypoxia and severe pulmonary hypertension unresponsive to maximal cardiorespiratory support, including high-frequency ventilation, inhaled nitric oxide and extracorporeal membrane oxygenation. results: surgical lung biopsies performed in three cases and postmortem lung examination in one case showed pathological findings of acd, characterized by poor capillary apposition and density and medial arterial hypertrophy. these lesions were associated with misalignment of pulmonary veins in three cases. the four infants died of refractory hypoxemia during the first months of life. conclusion: these observations emphasize the importance of considering acd with or without mpv in all newborn infants who present a persisting severe pulmonary hypertension without anatomical cause. histological diagnosis based on early surgical lung biopsy may prevent from using costly, invasive and ineffective treatments and procedures such as extracorporeal membrane oxygenation. a case of complete trisomy : autoptic, cytogenetic and radiologic findings e. gradhand*, s. becker, c. richter, d. wand, c. kunze, s. hauptmann *medizin. universität halle, institute of pathology, germany objective: an autopsy of a female fetus of the + nd gestational weeks was performed after an induced abortion due to multiple intrauterine malformations suspecting a chromosomal disorder. method: prior to the autopsy, the -g female fetus was extensively x-rayed. we performed an autopsy including histological examination of all organs. cytogenetic analysis was performed using both chorionic trophoblast and tendon fibroblasts. results: the fetus was characterized by pronounced global hydrops, craniofacial dysmorphy and malformations of both hands and feet. we found a complex cardiac malformation, unilobular lungs, dysmorphic liver and spleen, malrotated and doubled right kidneys, and an aplasia of the left kidney. the parenchymatous organs showed inconspicuous histomorphological architecture. in the cerebrum, no corpus callosum was found and immature neuronal rosettes in both optical nerves were detected. xray showed vertebral clefts, a slight hypertelorism, skeletal dysplasias of both hands, and confluent frontal and occipital fontanelles. cytogenetics turned out a complete trisomy (gtg-banding). conclusion: we present radiological, autoptic and cytogentic findings in a case of a very rare chromosomal aberration which mostly occurs as a mosaic or partial trisomy . this chromosomal abnormality usually induces early abortion. therefore, a fetus with trisomy and a gestational age of + weeks is very uncommon. we found multiple malformations of the skeleton and organs, mostly located in the midline. holoprosencephaly with cyclopia: presentation of two fetuses with multiple congenital malformations and investigation of sonic hedgehog (shh) expression p. papanastasopoulos*, j. coulouras, p. aroukatos, m. repanti, e. farri-kostopoulos, h. papadaki *general hospital agios andrea, dept. of pathology, patras, greece objective: holoprosencephaly is the commonest forebrain developmental anomaly occurring in of , live borns. sonic hedgehog is the major gene implicated in holoprosencephaly. method: two fetuses of -week and -week pregnancies, terminated due to multiple congenital malformations, were autopsied, and paraffin-embedded tissue sections were examined microscopically. shh expression was investigated immunohistochemically in orbital and cns sections. results: twenty-seven-week fetus: karyotype xx; alobar holoprosencephaly, true cyclopia, proboscis formation, pituitary gland anterior lobe agenesis, atretic anus and vagina, hypertrophic clitoris, female internal genitalia, right hand oligosyndactyly, left-sided overriding fourth and fifth toes, ventricular septal defect, hypoplastic left heart complex, truncus arteriosus, asplenia, irregular liver lobation, left kidney agenesis, right kidney cystic dysplasia, hypoplastic right ear auricle, external auditory meatus atresia. thirty-two-week fetus: karyotype xy; alobar holoprosencephaly, synophthalmia, proboscis formation, hypoplastic right heart complex, patent foramen ovale, ostium secundum defect, tricuspid valve and pulmonary artery atresia, pulmonary arteries analogous branching from the aorta. no teratogenetic factors and consanguinity were reported. histological examination revealed scattered rosette-like structures in areas of dysplastic retina in both cases. immunohistochemical expression of shh was negative. conclusion: the absence of shh immunostaining suggests a potential causative role in the pathogenesis of the above phenotypes. even though both karyotypes were normal, the authors could not exclude the possibility of the existence of chromosomal alterations not detectable by routine karyotypic analysis. finally, to our best knowledge, the above phenotypes do not seem to match any known clinical syndromes, and we therefore consider their origin unknown at present. the impact of nutritional colourant tartrazine (e ) on morphological state of thymus of rat descendants i. goryanikova*, i. sorokina *lugansk state medical university, dept. of pathomorphology, ukraine objective: synthetical colourant of yellow colour tartrazine (e ) whose application is prohibited in some countries of eu got widespread use. the aim of the work was the assessment of influence tartrazine to thymus of infant rat descendants month from birth from mothers who had been taking tartrazine during gestation and feeding. method: it was histologically discovered that density of cortical and medullar thymocytes was much higher than in the control group. mitosis is often diagnosed in the subcapsular zone. the most part of thymocytes of this zone is apoptosis amenable, cells with low concentration of dna in the nucleus, with the sings of dystrophy and necrosis often met. thymus medullary substance generally consists of fine lymphocytes. the small bodies of thymus, which are situated in medullary substance, are few in number and fine. results: immunohistochemical investigation with mca defined the signs of thymocyte-interrupted maturation of depletion of cd and cd population and macrophage ed and b lymphocytes cd ra amount rising. conclusion: thymus morphological specification of infant rats with tartrazine intoxication (during month) indicates frank hyperplasia of thymus lymphoid with underlying maturation retention, and component lymphocyte immunological differentiation points to antigenic activation, which influenced the test animals. objective: pleuropulmonary blastoma (ppb), a rare tumor arising during fetal lung development, can be part of an inherited cancer syndrome. about % of children with ppb have a family history of cystic nephroma and rhabdomyosarcoma. it is related to dicer mutations on q and consists in malignant mesenchymal cells and cysts lined by benign epithelium. method: we report our experience in a paediatric pathology center. two girls of and months presented pulmonary lesions, the first purely cystic and the second purely solid, corresponding histologically to early type i and type iii ppbs. results: the first patient underwent an upper lobectomy with an unremarkable postoperative outcome. the second patient presented a thoracic relapse years after initial treatment consisting in upper lobectomy, pre-and postoperative chemotherapy. tumoral karyotype revealed complex clonal chromosomal aberrations, dominated by interstitial q , q deletion and trisomy . no familial history of neoplasia. she is disease-free years after presentation. conclusion: ppb must be taken into account in the evaluation of cystic and even solid pulmonary lesions in children under years. a correct histologic diagnosis is needed to identify early ppb, which may be difficult to differentiate from congenital pulmonary airway malformation (cpam), to appropriately manage patients and to identify an inherited predisposition to ppb. recent data showed that dicer on q , a gene implicated in mirna synthesis, plays a crucial role in lung development. loss of dicer alters mirna-dependent regulation of diffusible growth factors that induce mesenchymal cell proliferation. results: pi ki ranging - % (median %) and topo a ranging - % (median %) were lower in children older than months (> m, p= . and p= . , respectively). the two markers were strongly interrelated (r= . ). higher ki- and topo a correlated with higher mki and adrenal location and inversely with increasing tumor differentiation. the cutoff values of pi ki ≥ % and topo a ≥ % correlated with fatal outcome of the disease. in the subgroup of patients, > m significant correlations between higher values of both pi markers and metastatic stage, unfavorable histology, high mki, mycn amplification, and adrenal localization were observed. at cutoffs ki ≥ % and topo a ≥ %, the markers predicted long-term unfavorable outcome by kaplan-meyer analysis. cox regression analysis indentified pi (assessed jointly as ki ≥ % + topo a ≥ %) as the independent prognostic factor. conclusion: ki and topo a pi markers have prognostic significance and clinicopathological correlations in nb. we propose to include pi to the standard histological assessment protocol of nb tumors. we decided to test the hypothesis that fatal cases of influenza occur mainly in association with important comorbidities as was stated based in previous studies. method: we included in our study patients with fatal ah n influenza. during autopsy, we largely sampled vital organs-brain, lungs, heart, liver, kidney, lymphoid organslymph nodes and spleen and any other diseased organ. results: in our group, sex ratio was male/female = . : ; patients were and years old (mean age . ). nine women were pregnant/parturient ( . %). other patients presented obesity, malignant neoplasia, diabetes mellitus, cardiac diseases and tuberculosis. we analyzed the prevalence of these comorbidities in the general population (see table) . however, patients did not have any comorbidities or any known cause of immunosuppression. conclusion: our study group was heterogeneous with a large age distribution and different associated diseases. we identified new risk factors for fatal outcome in ah n patients (obesity and/or pregnancy) and exclude other diseases as risk factor for severe evolution (diabetes mellitus). we have to emphasise that in half of the cases, there were no comorbidities or special status to actuate the severity of the disease. these findings are reinforcing the statement that the novel influenza has a different behavior than common influenza. objective: we present the first case reporting the exceptional association of whipple's and crohn's disease. method: a -year-old man developed since diarrhea, abdominal pain, weight loss, and polyarthralgia. endoscopy showed aphthous ulcers in terminal ileum and colon. histology revealed a rich collection of epithelioid granulomas in the lamina propria with chronic inflammation. a diagnosis of crohn's disease (cd) was given and steroid therapy was administered. diarrhea and abdominal pain quickly subsided. perduring a severe weight loss, a gastroduodenal endoscopy was performed. oedema with whitish spots in the duodenum was observed. histology demonstrated lymphangiectasia and an infiltration of pas positive foamy histiocytes in the lamina propria. immunohistochemistry for tropheryma whipplei was positive. results: whipple's disease (wd) diagnosis was made and an antibiotic therapy was given. in respect to symptomatic improvement, persistence of the bacterium was demonstrated in the following performed gastric and duodenal biopsies. immunohistochemistry in previous ileal and bowel specimens was positive in the ileum. conclusion: in wd, the macrophages seem to be of central importance in the development of the disease. this infection shares with cd the pathogenetic hypothesis that a defect in cellular immune response contributes to the development of the disorder. evidences also suggest that bacteria play a role in the onset and perpetuation of cd. this case strengthens the immunological hypothesis in the pathogenesis of the disorders. the functional defect of the macrophages caused by wd can explain the amazing number of granulomas in crohn's-affected specimens. the double immunological defect due to cd and cd may contribute to the lack of eradication of the bacteria. objective: the aim of the study was to analyze the experience obtained during pathological diagnosis using digital microscopy and telepathology. method: there were microscopic examinations performed over the internet using a dynamic digital microscope nicon-coolscope, scanners aperio-scanscope and olympus dotslide, between january and march . the study included intraoperative studies, cytology specimens and oligobiopsies. the specimens were prepared and sent electronically by four cytotechnicians and assessed by five pathologists. all diagnoses were verified through routine, light microscopy assessments. results: in cases of intraoperative, frozen sections studies as well as oncologically positive cytological diagnoses, above % agreement was noted between the diagnoses made due to evaluation over the internet and the routine assessment. the agreement of % was achieved in cases of benign lesions and the assessment of bronchial surgical margins. conclusion: ( ) the efficiency of histological slide examination by sending digital images via the internet in cases of intraoperative frozen sections during thoracic surgery or to confirm "positive" results is comparable to the traditional assessment in a light microscope. ( ) application of this method enables surgeons at the hospitals which do not have a readily available pathologist to perform the surgical procedures. objective: optimum diagnosis of glomerulopathies requires light microscopy, immunofluorescence and electron microcopy. in fact, electron microscopy has a confirmatory role in glomerular diseases. in this study, the value of electron microscopy in the diagnosis of glomerulopathies in children was investigated. method: the contribution of electron microscopy to the final diagnosis was graded as necessary-diagnosis could not be reached without it; supportive-it increased the level of confidence in the final diagnosis; and noncontributorythe diagnosis does not need electron microscopy for confirmation. results: one hundred thirty-four cases of renal biopsy with some clinical data are reviewed. the contribution of electron microscopy to the final diagnosis was necessary in cases ( %), supportive in cases ( %) and noncontributory in cases ( %). conclusion: it is concluded that ultrastructural study was an essential tool in the study of renal biopsy in childhood glomerulopathies, suggesting that electron microscopy still remains a useful tool in the diagnosis of glomerular disease. ki- , p , p kip- , p and bcl- proteins were ihc-investigated in bph from all patients. results: the incidences of lpc (gleason score ), chronic prostatitis, proliferative inflammatory atrophy (pia) and prostatic intraepithelial neoplasia (pin) were . %, . %, %, and . % in group and . %, . %, . %, and . % in group , respectively. greatly elevated levels of p , ki- , and bcl- associated with decreased levels of p kip- and p in areas of pia and less lpc and pin in group compared with group patients were obtained with statistically significant differences. conclusion: our study suggests that chronic long-term low-dose radiation exposure might result in the increase of chronic inflammation, and it is now found to be associated with increased incidences of pia and pin in bph accompanied by p , p kip- , and bcl- alteration, which in turn could lead to prostate carcinogenesis. mixed peripheral ductal prostatic adenocarcinoma in radical prostatectomies: a five-year experience in a small spanish community hospital c we performed immunohistochemistry to characterise the expression of the immunoreactivity for synaptophysin, chromogranin, p , ki and cd . results: mean age of the patients was . ± . . thirteen ( . %) of the patients were males and eight ( . %) of the patients were females. the method of statistics we used was spss . . according to spearman's correlation test, there was a good correlation between ki- expression and tumor type (p< . , r = . ). also, there was a good correlation between p expression and tumor type ck and ki- immunohistochemical staining in gastroenteropancreatic neuroendocrine tumours of ( %) were g , of ( %) were g , and of ( %) g . four pancreatic tumours and six gi tumours had distant metastases. of the four pancreatic tumours, two were g , one was g , and one was g ; two stained diffusely with ck , while the remaining two showed weaker staining. three metastatic gi tumours were g and three were g ; allred score for ck was - in five of six ( %) pancreatic adenocarcinoma (pa) is a very rare lesion in young patients. patients in the age of or younger reported previously constituted < % of all patients with pa. the clinical and histopathological data concerning these patients are therefore scanty. the aim of the study was to describe characteristics of young patients with pa treated and diagnosed in a only cases with available original slides were included in the study. results: there were ( . %) cases of pancreatic ductal adenocarcinomas (not otherwise specified), two ( . %) cases of adenocarcinoma associated with intraductal papillary mucinous neoplasm and a single ( %) case of adenosquamous carcinoma. twenty ( . %) patients were males and ( . %) were females. median age of patients was . years (range - ). twenty-one ( . %) patients were treated with pancreaticoduodenectomy, three ( . %) with distal pancreatectomy, and two ( . %) with total pancreatectomy. in seven ( . %) patients, pa was diagnosed in open surgical biopsy specimen (the majority of these patients were treated with palliative surgery) objective: we analysed a series of upper gastrointestinal (gi) adenocarcinomas treated by neoadjuvant chemotherapy (ctx) between and , which all were consistently worked up and evaluated by a standardized tumor regression grading system (trg) in our institution. method: one hundred eight esophageal adenocarcinomas (ea), adenocarcinomas of the esophagogastric junction (aej) and gastric carcinomas (gc) treated by a cisplatin/ -fu-based ctx were included. trg was determined using a four-tiered system based on the estimation of the percentage of residual tumor in relation to the previous tumor bed. results: in total, patients ( %) had a complete tumor regression (trg a) and patients ( %) had a subtotal regression (trg b, - % residual tumor). partial tumor regression (trg , - %) was observed in cases ( %); patients ( %) had minimal or no regression (trg , > %). tumor regression was significantly associated with post-treatment ypt, ypn, ypl category, r status and survival (p< . ) and was shown to be an independent prognostic factor for survival (p= . ). some characteristics relating to the tumor site were, in particular, the higher frequency of total or subtotal tumor regression in ea and aeg (p< . ) and the association of pretherapeutic tumor grading and lauren's classification with tumor regression (p= . ) in gc. conclusion: we present our year's experience of a highly standardized evaluation of upper gi adenocarcinomas after neoadjuvant ctx demonstrating highly objective and prognostic relevant information from a very large collective. we recommend the implementation of a standardised trg system in every pathological report of these tumours. objective: fibroelastotic changes (fec) of the gastrointestinal tract (git) are rare lesions. only cases have been reported in the literature. their role as an own entity is unclear and is not generally accepted. method: after initial recognition of an elastotic lesion in a hemicolectomy specimen, special attention was paid to amorphic accumulations in h&e-stained slides of git specimens. elastica-van-giesson staining was performed to verify the elastotic origin. results: within years, a total number of polypoid lesions were collected. one lesion was found in the ileum and six lesions occurred in the stomach. the remaining cases were colon specimens. in five cases, fec were associated with neoplastic lesions. in cases, however, the accumulation of elastotic fibres was the only histomorphological finding. one patient received intensive short intervals surveillance because of a highly suspicious endoscopic finding in the stomach. in two cases, a clinical relevant stenosis was caused by a submucosal thickening of the bowl wall with massive accumulation of elastotic fibres. right hemicolectomy was performed in both cases. conclusion: fibroelastotic changes are not as exceedingly rare as thought before. they are the only histological finding in a certain number of polypoid lesions in the git. expression of p , vegf and collagen iv in gastrointestinal stromal tumors and its relationship with clinicopathological parameters s. rjabceva*, y. rogov, i. dulinez, m. vozmitel, a. krilov *belarusian medical academy, dept. of pathology, minsk, belarusobjective: gastrointestinal stromal tumors (gists) have a spectrum from minimal indolent tumors to sarcomas. the aim of this study was to assess the expression of p , vegf and collagen iv in gists and to establish immunohistochemical correlations with clinicopathological parameters. method: twenty-four cases diagnosed as gist were examined by both light microscopy and immunohistochemistry (ihc). we used ihc markers such as p , vegf and collagen iv. all tumors were classified by the risk grade system by miettinen et al. results: of the tumors, / . % were classified into low risk grade (lr), / % in the intermediate risk (ir) and / . % in the high risk group (hr). focal necroses ( %, % and %, respectively) and microhemorrhages ( . %, % and %, respectively) were present in gists of all groups. a p expression was correlated with a high risk grade (r= . , p= . ). gists of all groups showed vegf expression ( . %, . % and %, respectively).objective: novel influenza is an acute viral infection of the respiratory tract caused by type a influenza virus, affecting by now the entire world. although the illness is mild and self-limited in a great majority of cases, few patients may have a severe clinical course eventually leading to death. method: we studied necroptic tissue samples from patients submitted over a -month period from a total of , positive cases identified in romania since the outbreak of this type of influenza. data collected included demographic and clinical information. results: most of the patients ( . %) were adults between and years (median age, . years), male/female ration . : . the vast majority of the cases revealed diffuse alveolar damage, interstitial pneumonia and bronchopneumonia, all of them associating severe cytopathic effect in the bronchial and alveolar epithelial cells; cases presented pulmonary ischemic lesions. more than half of the cases ( . %) presented myocardial interstitial inflammatory infiltrate with full-blown picture of myocarditis in patients. of the patients, . % had lymphocytic depletion in lymphoid organs. lymphocytic meningitis, meningothelial hyperplasia, acute tubular necrosis, and liver steatosis were identified in some cases. objective: hpv dna has been identified in almost all cervical cancers, and women with active hpv infection (hpvi) express e /e oncogenes. as only a small proportion of infections progress towards cancer, it is important to distinguish transient hpvis from persistent or progressive ones. method: one hundred ninety-six samples were tested by conventional pap smear, hpv-dna test and typing, e /e -mrna expression from hpv types , , , , . kstatistic value was used in order to identify possible significant associations. results: pap-smear: negative (neg), ( . %); atypical cells of undetermined significance (ascus), ( . %); low-grade squamous intraepithelial lesion (lsil), ( . %); high-grade squamous intraepithelial lesion (hsil), ( . %). one hundred ninety-two of samples ( . %) were positive to the hpv-dna test and of ( objective: etiology of multiple sclerosis (ms) includes the virus infection which plays probably an important cofactor role in the pathogenesis of disease. clinical and morphological analysis of the cases of chronic herpetic meningoencephalitis (chme) and ms permitted to discover two cases of a combination of diseases: man, age years (onset of ms since years), and woman, age years (onset of ms since years). results: autopsy revealed the small plaques in white matter of brain and cord and some foyers of necrosis in frontal lobe and brain stem. the histological signs of chronic inflammation were established in both cases. leptomeninges and brain were infiltrated by lymphocytes and macrophages (perivascular cuffs). the progressive lesions of cortical neurons and proliferation of macroglial cells were constant histological findings. etiological diagnosis of chme was established on the base of typical intranuclear viral inclusion types i and ii in the cells of brain as well as on the revealing of hsv antigens in histological slice. laminar nonischemic necrosis was found in the cortex of frontal and parietal lobes. in the brain stem, necrosis appeared as the small foci of destruction of nervous tissue. in one case, laminar necrosis were detected in white matter of hippocampe. conclusion: thus, the morphological investigation on the light microscopic level permits correctly to recognize the combination of ms and chme. key: cord- -oatjcmy authors: arata, andrew a. title: old and new pestilences date: journal: understanding the global dimensions of health doi: . / - - - _ sha: doc_id: cord_uid: oatjcmy a. “any fatal epidemic disease, affecting man or beast, and destroying many victims.” the oxford universal dictionary, (rd) edition, , oxford press, pp. b. “a contagious or infectious epidemic disease that is virulent and devastating.” webster’s seventh new collegiate dictionary, . g. & c. merriam co., springfield, mass., pp. accordingly, a pestilence should be an infectious disease, devastating (killing) a large number of people (or animals). there has been much popular interest in, as well as technical concern over, newly emerging diseases, and there is a fear that heretofore unknown virulent pathogens will create new, global epidemics. at the time of this writing, two such pathogens are active, warranting such concern: a) cases of sars (severe acute respiratory syndrome, caused by a coronavirus) appeared in china in november, , and has spread to western and central europe and north america; b) a strain of avian influenza virus (n h ), first identified in hong kong in , reemerged in in southeast asia. other avian flu strains found simultaneously in poultry in north america have underscored the concern of local and international health authorities. both sars and avian flu demonstrate high mortality rates, but, to date, the number of cases has been only in the hundreds. so, are these pestilences? what constitutes a pestilence? is the term synonymous with newly emerging diseases? two definitions of pestilence have near unanimity, but are not very specific: a) "any fatal epidemic disease, affecting man or beast, and destroying many victims." the oxford universal dictionary, rd edition, , oxford press, pp. b) "a contagious or infectious epidemic disease that is virulent and devastating." webster' s seventh new collegiate dictionary, . g. & c. merriam co., springfield, mass., pp. accordingly, a pestilence should be an infectious disease, devastating (killing) a large number of people (or animals). the truly epidemic diseases are usually of viral or bacterial origin (although we will make a case for some other types of pestilences). the classification of that may have been bubonic plague continuing until a.d. ). the "black death" of the fourteenth century, which continued to appear in chronic pockets of europe and the middle and near east for centuries thereafter, is by far the best-known plague, and the one that produced the greatest mortality and social impact on the affected populations. consider that as a conservative estimate, − % of the european population died, and maybe more. this death rate reduced the available work force so that, for the first time, peasants and landless people could sell their labor, which introduced freedom of movement and resulted in economic changes that eventually contributed to the decline of the feudal system. the rd pandemic of plague began in the s and continues to this date, although reduced in more recent years. a major characteristic of this epidemic has been the dissemination of plague from its traditional homes in africa and asia into areas previously plague free, especially north and south america, by the inadvertent transport of rats and their fleas by boat. the infection is now well established in africa (gerbils); central asia (gerbils, ground squirrels or "susliks," and marmots); southeast asia (various rattus species); north america (ground squirrels and some native field mice); and south america (introduced rattus). the last major urban outbreak was in surat, india in : more than , cases and deaths were reported. however, the impact of this outbreak was also seen in the number of people who fled the plague zone, and the over two billion dollar loss that ensued. only a few outbreaks are reported to who each year. indochina and burma frequently report, as well as sites in africa (ovamboland on the frontier between namibia and angola); the united states has a vast infected area in the west of the country, but only - cases per year are reported, with - deaths on average every years. many other sites of infection are known and should be monitored, as some rodent species are highly susceptible to serving as effective amplifying reservoirs, whereas others maintain low-level infections for long periods of time, allowing much time to pass between outbreaks. environmental measures (rat-proofing, rodent and flea control, etc.,) are the first measures of control. surveillance, prompt diagnosis, and treatment with antibiotics (e.g., streptomycin and tetracycline) are recommended. yellow fever is the best known of the arboviral (arthropod-borne virus) diseases. there are some known arboviruses, of which about , produce disease in man. both the yellow fever virus and the primary mosquito vector, aedes aegypt, are of african origin -the species name, 'aegypti,' refers to classical africa in general, not, specifically, modern egypt. most cases in africa occur east to west along the transition zone (ecotone) between the savannas and the rain forests inhabited by numerous aedine vectors as well as a. aegypti. the disease has two cycles: a 'jungle' cycle involving various tree dwelling mosquitoes and nonhuman primates as reservoirs, and an 'urban' cycle, with a. aegypti as the vector and humans as reservoirs. the last reported major african outbreak vectored by a. aegypti occurred in nigeria and involved some , cases and over , deaths between and . yellow fever was introduced into the americas one or more times most likely during the age of sail: the virus can be transmitted vertically (transovarian passage) in a. aegypti. the mosquito' s eggs can easily be laid in water barrels and withstand desiccation for months, only to hatch and develop when submersed at a later date. epidemics of yf raged throughout the caribbean and tropical america until the end of the s, when the transmission cycle was elucidated by the team led by walter reed, confirming the role of a. aegypti which had been proposed by, but not confirmed by, carlos finlay. epidemics occurred as far north as philadelphia in the united states and the last epidemic in north america occurred in new orleans as late as , with over , deaths. cases (with - %) mortality continue to occur sporadically in brazil and in the foothills of several andean countries (bolivia, peru, ecuador, and colombia) . often the victims are young, indigenous males from the highlands who were temporarily working in the coca processing plants in the forests. these infected areas are only kilometers from large cities (with populations of more than million people) such as santa cruz, bolivia, which are accessible by public transportation and are heavily infested with a. aegypti. although the yf vaccine is one of the oldest, safest, and most effective available, and immunological protection is rated for at least years, vaccination coverage in many of the affected areas of africa and south america is low. the cholera pathogen, vibrio cholera, originally described by robert koch, was one of the first human pathogens (along with anthrax and tuberculosis) to be identified, in the late s, shortly after pasteur' s publication of the "germ theory." koch and his students studied material they collected in alexandria, egypt, during an outbreak. it was difficult to determine the origin of cholera and/or to distinguish it historically from other diarrhetic diseases except by the severity and rapidity of onset. health historians such as mcneill suggest an origin on the indian sub-continent, associated with dense populations, poor hygiene, and certain religious practices such as communal bathing; thus the term "asiatic cholera," by which the disease became known in europe in the s. the disease' s appearance in europe and the americas (london and new york in , and again in ) were clearly associated with intercontinental traffic. it was during the epidemic in london that a physician, john snow, noted the clustering of cases and deaths in people using the same water source, and proposed what turned out to be the correct action to stop the epidemic ("take the handle off the broad street pump!!"), although he had no idea of the actual cause of the disease. however, it was such observations, along with structural, hygienic, and administrative changes in major cities, particularly in europe and north america, that established the public health measures that we tend to take for granted in this early part of the st century. cholera is still with us: various serotypes of the vibrio have spread since the early s, affecting over countries in asia, in the americas, and into the west pacific. in , approximately , cases and , deaths from cholera were reported. in , the el tor strain of cholera was reported in lima, peru; by , almost a million cases had been reported in the western hemisphere. measles is one of the oldest known and most widespread infections of man: epidemics ascribed to measles appear in the oldest literature, although they are often confused with smallpox. however, in a.d., ad ahrun, a christian priest living in alexandria, egypt, described the pox lesion, and in a.d. the arab physician al-razi distinguished between the two diseases. prior to widespread immunization, measles was common in childhood-more than % of people were infected by age . although endemic in large communities, measles became epidemic every several years, with the severity of infection decreasing with the frequency of the epidemics. in his study of the history of plagues, mcneill makes mention of the importance of animal husbandry and zoonotic diseases in the area. measles, he claims is probably related to both rinderpest (in hoofed-mammals) and canine distemper. because dogs, sheep, and goats have been domesticated for at least , years, measles may have been among the first viral diseases to have "jumped the species barrier." as we will see, most, if not all, of the new pestilences are, or may be, derived from animal wild or domesticated reservoirs. measles was responsible for (or contributed to, along with smallpox) the decimation of the indigenous amerindian populations, first in central and south america at the time of the spanish conquest ( s), and later ( s and s), in north america. amerindian populations lacked immunological protection from these and other imported infectious diseases. some attribute this immunological naïveté to the comparatively small number of domesticated animal species-dogs, ducks and turkeys, guinea pigs, and cameloids (llamas and relatives) in the andes, and few, if any, in large number prior to the european invasion. in any case, the attack and mortality rates were staggering. by one estimate, a pre-conquest amerindian population of perhaps thirty million by was reduced by %, down to only million. this catastrophe occurred in less than years after the spanish entered the american mainland. influenza is another viral disease that has many unstable varieties infecting a host of mammalian and avian species, both wild (sylvatic) and domestic. epidemics with symptoms similar to modern influenza were noted by hippocrates as early as b.c., and later, in rome, by livy. various medieval and renaissance writings describe influenza-like illnesses. robert johnson of philadelphia is credited with the first "modern" description of an influenza epidemic, which occurred in that city in . his description was applied to subsequent epidemics in , , , - , and . antigenic shifts in the structure of the influenza virus may change the virulence of the strains, increasing the likelihood of epidemics. the most severe flu epidemic ever recorded ( ) ( ) -also known as the spanish flu (although it did not originate there)-first struck world war i troops of all combatant nations while in northern france, and it continued on to become a global pandemic. conservative estimates of mortality range between twenty and forty million persons, and other estimates more than double these figures. the ease with which the various influenza strains infect domestic mammals, pigs, and poultry (chickens and ducks) producing huge reservoirs of potentially infectious material, often proximate to human habitations, is a major public health concern. especially worrisome are the conditions under which millions of such animals are raised and brought to market. the 'old' diseases examined above are only a few of those which might be used as examples of the old pestilences: others might prefer to include schistosomiasis, typhus (murine and/or louse-borne), and several of the classic childhood diseases (diptheria, pertussis, tetanus, rubella), as well as leprosy, yaws, the leishmaniases, and, certainly, smallpox. fortunately many of those mentioned here (schisto and others) are being controlled rather well in some areas by vaccines, specific drugs, and/or antibiotics when applicable, at least in the more developed countries. even polio, which had been a major epidemic threat for centuries, has been virtually eliminated as a threat in areas where the politics and health infrastructure allow the efficient application of this very effective vaccine. much of the fear engendered by specific diseases depends on the time, place, and severity of the local outbreaks, as well as the knowledge and perception of the community. for example, i was raised in new orleans, in the southeast of the united states, during the s. although i and my brothers were normal, well nourished children, our parents were fearful of dogs (rabies), cuts on unshod feet (tetanus), and any summer colds or stiffness/weakness of the extremities (polio), and they preached cleanliness as a means to prevent anything bad happening. these diseases are old, but at present each has developed certain new characteristics that make their modern expression different from their historic ones, and decreases our ability to control them. in the last years, malaria parasites have developed resistance to chloroquine, the most common, globally used anti-malarial drug; at the same time, the anopheline mosquito vectors of malaria have progressively developed a parallel resistance to the insecticides used to control them. dengue, and dengue hemorrhagic fever (dhf), have spread globally, infecting vast new areas, especially urban areas where the human living conditions are substandard, but readily suited for vector breeding. finally, tuberculosis, whose incidence was slowly reduced in the late s and early s by improved public health, housing conditions, and nutrition, has again surfaced as a secondary infection to immuno-compromised persons, especially those suffering from hiv infections. at the same time, the causative agent, mycobacterium tuberculosis, continues to develop resistance to the most economic and readily available antibiotics. malaria is caused by blood parasites of the genus plasmodium and vectored by anopheline mosquitoes. there are four species of human malaria parasites: p. falciparum, p. malariae, p. vivax, and p. ovale, as well as a number of related species infecting other mammals (non-human primates, rodents, etc.). historians note that malaria-like symptoms were discussed in the chinese canon of medicine ( b.c.) and malaria-like illnesses were described in th -century b.c. cuneiform literature from nineveh (now part of iraq). hippocrates made a connection between stagnant water and fevers in the local population. it is estimated that there are still several hundred million unreported cases each year resulting in - million deaths per annum, mostly children. although malaria is still endemic in asia, latin america, and africa, % of the cases are found in africa, where p. falciparum is the most common malaria parasite. such huge figures mask the focal, and sometimes epidemic, nature of malaria, which may be brought about by natural or man-made environmental conditions. some of the human activities that may enhance malaria transmission may be development projects for agriculture (e.g., irrigation schemes), other water and land use projects (as in the amazon basin, converting forest areas through resource extraction such as mining and logging) into marginal livestock and farming areas. often such environmental changes bring about changes in malaria transmission from 'stable' (endemic) to 'unstable' (epidemic). in highly endemic areas, severe malaria and death is concentrated in the younger age groups, whereas in the areas of unstable (epidemic) transmission, severe malaria and death is more evenly distributed throughout all age groups. needless to say, prevention and /or case control strategies must be different for each transmission type. in many parts of the world the anopheline vectors of malaria have developed resistance to the insecticides used for their control. frequently, this is due to the use, often excessive, of the same or similar insecticides for control of agricultural pests in the same geographic areas. such resistance not only hinders control operations directly, but also indirectly, by increasing the need for greater quantities and/or more costly insecticides. broadscale usage of insecticides has also become limited on environmental grounds, because some donors have reduced funding insecticide purchases. by far the most serious setback to malaria control in recent decades has been the emergence and spread of chloroquine-resistant strains of p. falciparum, the causative agent of the most severe form of malaria, and the most common in africa. emerging in the s in southeast asia and south america, resistance spread rapidly from these focal points. it was not noted in africa until - but spread rapidly in the ensuing ten-fifteen years. chloroquine-resistant strains of p. vivax have been identified in some areas of southeast asia, new guinea, and indonesia. efforts to produce a malaria vaccine(s) have been under way for over years. a number of candidate vaccines have been produced, but none are operational in humans as yet. like yellow fever, described earlier, dengue and dengue hemorrhagic fever are vector-borne diseases transmitted (primarily but not exclusively) by the mosquito aedes aegypti. "classical" dengue is caused by infection with one of the four serotypes of the dengue virus. dhf may occur following a subsequent infection with a different serotype. the following quotation is from an article written by the author in (r. lennox and a. arata, dengue fever: an environmental plague for the new millennium. capsule report, environmental health project/usaid. pp.): with . billion people at risk and estimated cases in the tens of milllions, dengue is considered by many to be the second most important vector-borne disease in the world (surpassed only by malaria). classical dengue and its more lethal form, dengue hemorrhagic fever (dhf), now circle the world with endemic illness and continuing threats of epidemics. dengue is very much an environmental disease, affecting urban and periurban settlements in more than countries. it is characterized by seasonal outbreaks of illness carried by mosquitoes that thrive in household containers which collect water (such as flowerpots and washtubs) and in the detritus of human consumption, such as bottles, tin cans, and old bottles. children, specially in asia, are most frequently and seriously affected by the severe form of the infection, dhf. mosquito control is the only effective approach to prevention, although effective case management will reduce mortality. insecticides targeted at larval mosquitoes are effective, but resistance of mosquitoes to affordable and environmentally safe chemicals as well as declining will and infrastructure have all but eliminated this approach in most countries. vaccines are in the pipeline, but a system which could deliver them to half the world' s population is probably at least a decade away. community action-to protect containers from becoming havens for mosquito breeding and to dispose of empty containers and trash, along with surveillance and personal protection-is the best hope for transmission risk reduction. tuberculosis is another ancient disease that has bridged the old to new definition: the tb bacillus, mycobacterium tuberculosis, was among the first to be scientifically identified and described (by robert koch, in ). the disease is transmitted by airborne droplets from people with pulmonary or laryngeal tuberculosis. this mode of transmission is most effective in dense populations, and hence tb became widespread with the development of urban centers in the middle ages (europe), and was very common from the th century until recently in europe. with improvements in housing and nutrition tb rates continued to decline (except for periods of war) until the first half of the th century. at that time, two conditions emerged: the development of multiple drug resistant tb (mdrtb) and the emergence and spread of acquired immune deficiency syndrome (aids) upon which tb is an opportunistic infection. prior to , about % of tb bacilli isolated from patients in the u.s. were resistant to even one antibacterial drug: in , % were resistant to at least one drug, and % were resistant to more than one drug. in the u.s. the cost of treatment of ten cases of mdrtb in texas in was us$ , . who lists tb as one of the major causes of mortality in the world. a new major funding effort (who and world bank and various bilateral donor groups) is focusing on hiv/aids, tb, and malaria as the most serious, and intractable, causes of death. other forms of tb, including non-pulmonary cases and those associated with other species of mycobacterium sp. (e.g. m. bovis), are sporadic, but suggest the possible very early animal origin of the pathogen group. diseases such as chagas disease and schistosomiasis are examples of diseases that do not easily fit the epidemic definitions of a pestilence mentioned earlier in this chapter, but they do heavily impact the affected populations, not only through mortality rates, but especially through morbidity/disability. there are several forms of schistosomiasis caused by different species of schistosoma, a blood fluke (trematode)-this is an ancient illness, known from egyptian antiquity. infections occur in fresh water where people work and/or wash and children play. larval worms, known as cercaria, developed in a snail intermediate host, pass through the skin and penetrate diverse organs according to species. the most important effects are those that arise from chronic, and cumulative, infection. chagas disease has a very different etiology, mode of transmission, and pathology than does schistosomiasis. by definition it could be new because it was first described in by the brazilian carlos chagas, who subsequently described the pathogen, a flagellate protozoan, trypanosoma cruzi, and the vectors, bloodfeeding triatomine bugs. the disease is also know as american trypanosomiasis, and occurs only in the western hemisphere, from mexico to argentina-a few cases have been reported in north america. this form is very different from african trypanosomiasis (sleeping sickness). the initial (acute) phase of the disease usually occurs in children; there is then a long latent phase (∼ years or more), culminating later in life in a chronic phase which may include irreversible cardiac and/or intestinal manifestations and shortened life spans in the victims. paho and who consider chagas disease to be the most serious parasitic disease in latin america and the main cause of heart disease in the region. there is no adequate medical intervention. the infection can be transmitted by vectors, congenitally, or by transfusion of blood or blood products. an estimated million persons in the region are at risk, and in some countries (e.g., bolivia) % of the million inhabitants have been shown to be seropositive. in addition, in bolivia, one study demonstrated that the burden of chagas disease, in terms of disability adjusted life years (dalys), was million dalys, or estimated loss of million bolivianos: equal to more than million us dollars at the time of the report ( ). the purpose of this brief segment is to emphasize that pestilences need not carry with them only high mortality. very high morbidity and sustained disability with all the concurrent social and economic implications can be a tremendous burden on a population-or a nation. puerperal fever, a forgotten pestilence, is caused by a streptococcal infection and is an iatrogenic disease (induced by a physician) that was once the scourge of pregnant women, before physicians learned to wash their hands before examining pregnant women and/or assisting at childbirth. improved hygiene in hospitals was concurrent with the development of the germ theory and mortality rates dropped quickly. this disease, also called childbirth fever, was never reported as one of the great pestilences, however a few figures reveal the state of scientific knowledge regarding any infectious diseases, both endemic and epidemic. it is frightening that not only was the incidence of puerperal fever higher in the hospitals, but so was the associated mortality: % of the patients died if the disease occurred after a home delivery, but - % died if the disease was contracted in a hospital. although we have no crystal ball to predict what, if any, new pestilences are in store for mankind in the future, several groups of zoonotic viruses include likely candidates (table ) . also included is hiv/aids, truly a new pestilence that already, in a relatively brief period, has taken its place among the worst pestilences ever known to man. as mentioned above, there are over arboviruses isolated and characterized-about are capable of infecting humans, from nonapparent infections to very severe ones. two of these have already been mentioned above (dengue and dhf and yellow fever), but the arboviruses as a group represent the source of many potentially new diseases-or, put more correctly, existing zoonotic diseases that emerge when humans accidentally become involved in their cycles. a good example is the recent outbreak of west nile encephilitis in the u.s. in and , the virus was isolated from/around new york city from large numbers of dead birds (especially crows and jays): human cases and two deaths were confirmed. by , the disease moved west toward the mississippi river, infecting people and killing nine. in , there were over , cases ( fatal); by , the virus, and human cases, were found in all contiguous states (excepting alaska and hawaii). the virus has been found in mammals, birds, and mosquitoes throughout the u.s. but is this a new disease, or just a disease new to us? west nile virus has been found in over countries since its discovery in in uganda, and has been most of the above are mosquito-borne, and the major mosquito vector genera, culex, aedes, and anopheles, have global representatives from which a competent vector might be found. the same is true of ticks, sandflies, and other potential vectors. rodents, or other local vertebrates, may serve as reservoir hosts while infected migratory birds may provide distribution of the infection. although many arboviral infections have broadly similar transmission cycles, the ecology and dynamics of each may differ widely. arboviruses do not belong to a single viral family, but rather, to several,which increases their diversification. although the potential for increased arboviral epizootics or epidemics is high, the most recent episodes have not been high on the pestilence scale; rather, the most severe arboviral epidemics have been yf and dengue/dhf, the oldest of the group. the arenaviruses were thought for years to be monotypic, a single species, lymphocytic choriomeningitis (lcm), occurring primarily in the house mouse/ laboratory mouse, mus musculus. the virus (first described in ) has been isolated in numerous locations, but human disease is known only from europe and the americas. a second arenavirus was isolated from a phyllostomatid (fruit-eating) bat from trinidad, but there was no associated human disease. severe hemorrhagic cases in argentina and later in bolivia in the s and s resulted in the discovery of new viruses and diseases in these countries-junin virus/argentine hemorrhagic fever (ahf) and machupo virus/bolivian hemorrhagic fever (bhf). more recently, additional arenaviruses found in brazil (sabia virus) and venezuela (guanarito virus) produce similar hemorrhagic symptoms. ahf is the most common, - , recorded annually between and -the others are only sporadic, but mortality rates are high in all these diseases. in each of these, transmission is by contact with infected rodent excreta, dust, and other substances associated with grain harvesting and storage. there are another five arenaviruses in the americas that are not known to cause any illness in humans or their rodent hosts. all of the rodents associated with these viruses belong to only one of the rodent families currently inhabiting south america. these rodent genera (calomys, sigmodon, oryzomys, et al) , are very closely related, and share a common ancestry. paleontological evidence indicates that the isthmus of panama was a bridge connecting north and south america more than - million years ago, allowing a faunal interchange. the sigmodont rodent progenitors entered south america at that time, and rapidly evolved into the modern genera and species. presumably the "ancestor virus" tagged along, co-evolving into the situation that now exists. by far the most important arenaviral disease is lassa fever: discovered in nigeria in , it is known from african countries, mostly in west and central africa, but also zimbabwe and mozambique. the natural host of lassa virus is the multi-mammate rat, mastomys natalensis, one of the most common and widely distributed african field rats. like their south american counterparts, the ahf and bhf hosts, mastomys, is basically a grassland species, easily adapting to the man-made grasslands of maize, sorghum, millet, sugarcane, and other cultivated grasses. cases of lassa are generally associated with agricultural activities and food storage: transmission is by contact with excreta of infected rodents. without laboratory facilities for confirmation, it is difficult to distinguish lassa fever from ebola, yf, or even severe cases of malaria. there are an estimated , cases a year, with more than % mortality rate in hospitalized cases. the disease is more severe in pregnancy, with fetal mortality reported at more than %. in the early s (and before ebola outbreaks occurred), lassa caused great consternation in europe and the americas over the possibility of introduction of this disease. these concerns still exist and have been heightened after the appearance of these other groups of viral hemorrhagic diseases. the hantaviruses are comprised of two large groups of viruses, all transmitted by rodents and producing a range of hemorrhagic, renal, and/or pulmonary complications. the old world hantaviruses are comprised of over different viruses, several known for some time under a different classification (e.g., hanta virus is the cause of korean hemorrhagic fever with renal syndrome, an important military disease in the s). most cases still occur in agrarian and military populations and occur in over countries in asia, africa, and europe: each year approximately , cases occcur in eurasia, with more than % of these reported in china. case fatalities range from . % to . % depending on the virus. the or so new world hantaviruses produce a pulmonary, rather than a renal, syndrome. since being described as a group in , approximately , cases have been reported in the americas, with a high case fatality rate ( - %). the natural hosts/reservoirs for the hantavirus groups are mostly muroid rodents (old world group), and cricetid rodents (new world group). this is not surprising, as these two are amongst the largest and most widely distributed mammalian families. however, the manner and zones of transmission are similarrodent contamination of grain crops in the field and storage where people come in contact with rodent excreta. the two closely related filoviruses (marburg and ebola) are among the most virulent viruses yet described with an overall fatality rate of more than %, and higher in several outbreaks (possibly augmented by use of dirty syringes and needles to give injectable chloroquine (an anti-malarial drug) to the patient' s friends who carried him/her to the hospital. marburg virus was first described ( ) among monkeys sent from east africa to european laboratories, there killing laboratory technicians. subsequent outbreaks have occurred in africa. ebola virus appeared in in simultaneous outbreaks in zaire (democratic republic of congo). (barry, ) one ebola strain was implicated in an outbreak in an animal holding facility in reston, virginia, u.s.a. several humans seroconverted but showed no disease symptoms. the repeated outbreaks of ebola and marburg virus, mostly in central africa, have been described as commencing with "rapidity and devastation." during an epidemic, transmission is generally by contact with contaminated blood or other tissues from infected persons. most outbreaks have been in rather remote areas with poor health care facilities, so that patients are seen only with advanced symptoms. we have not been able to find reservoir organisms (there have been subsequent, better equipped expeditions than the one described in the footnote, but none have been successful), nor do we know the mechanism(s) of transmission in the wild. one distinct ebola virus strain from ivory coast was isolated from a chimpanzee: primates are hunted and eaten by humans in parts of africa and this may serve as the 'link' at which the virus(es) are able to "cross the species barrier" and enter the human population. (barry, ) the government of sudan requested who assistance, and the government of zaire requested the same from the u.s. government (cdc). representatives of who and cdc met in the next few days at the london school of hygiene and tropical medicine to work out details and coordination (who was represented by dr. paul bres and the author, and cdc by dr. karl johnson). we had all thought of lassa and marburg viral fevers, and were surprised when dr. johnson said it was neither: he then showed us electron photomicrographs of tissue taken from an early case-the stringlike " and " figures were just like marburg. but, he explained, this one was serologically distinct from marburg, and they proposed to name it after a river in the area, the 'ebola'. we agreed that i (aaa) and a virologist (dr. bruce johnson) from the lshtm would go to the site in sudan to sample potential reservoirs and/or vectors. bruce would bring the supplies needed for taking tissue samples and the liquid nitrogen containers needed to return the samples to the uk. i was to gather the animal collecting materials. who had no such equipment in geneva, of course, so i borrowed 'mist' nets for collecting bats from the british musum (natural history) and the museé d' histoire naturelle in geneva and borrowed sample rodent traps from the swiss agricultural research station in nyon, near geneva. we had the traps made in nzara, one of the sites of the outbreak in sudan. to autoclave the dissecting instruments we purchased two household 'pressure cookers' at the local super market (migros) in geneva. placed on stones over an open fire, they served well. an experimental ebola vaccine has been reported to be successful in trials with non-human primates. human trials will be conducted soon. two previously unknown and unrelated human viral infections, severe acute respiratory syndrome (sars) and an asian avian influenza (strain h n ), originating in southeast asia, have received a great deal of popular attention and public health concern. in november , cases of a respiratory illness, subsequently labeled sars, appeared in china. a delay in timely reporting of the initial cases allowed it to spread to other southeast asian countries, australia, the americas, and at least european countries. reports of the actual number of persons infected varied, but cases numbered in the thousands, and mortality rates of up to % were indicated. surveys of wild animals captured for human consumption quickly showed that ferrets, civets (related to mongooses), and raccoon dogs (shaggy fox-like carnivores) were positive for harboring the virus, but it is not known if any of these are the true reservoir in nature. the who has reported that the chain of transmission may have been broken (no new cases reported in a period of time equal to two consecutive day incubation periods). this is clearly a case of a virus "species jumping". in the world' s largest, most densely populated country this could spell disaster, especially if the reporting network is compromised. the asian avian influenza strain initially appeared in poultry in hong kong in , when it jumped the species barrier and killed out of infected persons. this recent outbreak spread to korea (december, ) , then japan and vietnam (january ). hong kong reportedly slaughtered . million chickens and ducks, and as many as three million slaughtered through the southeast asia region, but other reports indicate that there are nonspecific wild variants of this strain in wild birds that serve as natural reservoirs. of major concern is that outbreaks of highly pathogenic avian influenza are increasing in frequency and severity. reportedly, in the years from to , there were only outbreaks, but in the past six years, from to , there have been six, not including the most recent incidents. if bubonic plague was the quintessential pestilence of the ancient and medieval worlds, acquired immunodeficiency syndrome, caused by the human immunodeficiency virus (aids/hiv) is the chief pestilence of the modern world; and it is still growing, not receding. there is also a vast literature that will not be reviewed here, but the following data points describe the severity of the pandemic pestilence: r aids is gaining a firmer foothold in the large populations of india and china; r world wide, million people are infected with hiv; r - million of these infected people live in sub-saharan africa; r million persons became infected this year, , are children; r million persons died of aids this year, , of them less than years old; r existence of simian immunodeficiency virus (siv) suggests animal origin. the social damage accompanying this pandemic is not reflected in the bare figures given above; especially the orphaned children, destroyed family structures, and so forth. it has been estimated that billion dollars us, per annum, is required to provide the prevention and treatment facilities and services needed: to date, less than one-half ($ . billion per annum) has been made available. some of the old category diseases are still strongly with us (e.g., malaria, tb, influenza), and, by adapting traits such as drug-resistance and crossing or jumping species, they expand their reservoir-host base. as such, they could be considered new. some other old diseases are rather well controlled in the developed countries where the surveillance systems are efficient and vaccination and other preventive services are readily available and properly used. these would include smallpox (eradicated), polio (eradicated in some areas), and childhood illnesses such as pertussis, diptheria, tetanus, measles, and so on. even bubonic plague could be characterized as being under control-it is widespread, but also well understood, and with vector control and appropriate antibiotics, outbreaks are not severe and mortality is low. on the other hand, some of the new (most recently discovered) diseases like ebola and hiv/aids are hard to handle. we know little about the natural history of ebola, lassa, or the south american hemorrhagic fevers, and our knowledge of hiv/aids in the laboratory probably exceeds our understanding of the socioeconomic impacts it is having on whole cultures. when lassa virus "jumped" from the field rat, mastomys, to humans it was dreadfully virulent, and it seemed to come from nowhere. but, after a few years, we know that (with one exception from a bat) all arenaviruses are well adapted to particular rodent groups; most rodents are grass eaters, and lots of crops are grasses (wheat, maize, sugarcane, rice, etc.); therefore, the arenaviral fevers are seen primarily in agricultural settings and with stored grain. yet, for the more recently known hantavirus group, or even less with the multiferous arboviruses, we do not have good data on ecological determinents, or even host-reservoir relationships. at the same time, people are modifying environmental conditions rapidly and extensively, and we have little information indicating whether such changes will eliminate potential disease cycles or exacerbate them. this may be even more important for diseases like influenza. if they have obligate or opportunistic vertebrate hosts and these are coincidentily reduced in number or eliminated, what selection pressures are set in action on the virus population to select new hosts? and when it comes to modifying environments, man has no equal. yet we know that this microbial evolution is going on at a rapid pace-just look at how fast drug-resistance develops and spreads! in reading articles and researching references for this document, i was amazed to discover again how many human illnesses have their direct animal (zoonotic) counterparts, or were vectored/hosted by arthropods, rodents, or snails, and how an avian influenza can become a mammalian influenza very quickly, and how a bat or an oppossum can do the same for the chagas disease trypanosome. it is in this context that i feel that we know very little of the natural history or the ecological dynamics of the disease transmission cycles we teach. especially disturbing is to read of a new strain of asian avian influenza and the necessity, around the world, to kill millions of birds. if one was to dream up a model pathogen incubator and dissemination engine, the perfect model would be a modern chicken farm of , birds, defecating as birds do, and that at a constant temperature and with residues of organic chicken feed all about. and we wonder why new diseases emerge? any farmer worth his/her salt knows that monoculture breeds pests. this is a good place to bring up one other difficult subject-bioterrorism. it is difficult for one dedicated to public health principles to imagine why anyone would even consider using infectious diseases as a weapon, but it is being done, and we need to be able to distinguish between a natural epidemic and one orchestrated by man. again, knowledge of the natural history of the organisms, their natural hosts and reservoirs, will help. already the u.s.a. is stockpiling smallpox and anthrax vaccines in large quantities. one final point; most people concerned with new versus old pestilences work as epidemiologists, infectious disease specialists, hospital officials, and so forth. but public health work is broader than the study and treatment of infectious diseases, and the study the global burden of disease, sponsored by the who, world bank, and harvard university, based on measuring dalys, predicts that fewer infectious disease will be as important in the future as they are at present. for example, "the next two decades will see dramatic changes in the health needs of the world' s populations, and non-communicable diseases such as depression and heart disease . . . are replacing the traditional enemies, such as infectious diseases and malnutrition." maybe toxic smog and non-communicable diseases will replace pestilences, both old and new! the great influenza control of communicable diseases manual exotic viral diseases death by migration plague: an ancient disease in the twentieth century (rev emerging infections plagues and people the global burden of disease the doctor's plague viruses, plagues, and history emerging infectious diseases (vol. - ). www.cdc.gov/eid or hard copy from cdc key: cord- -r usk authors: nan title: research communications of the th ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: r usk nan saccharomyces boulardii (sb)is a non-pathogenic yeast used in the prevention and treatment of gastrointestinal disorders in human beings and horses. the aim of this study was to evaluate the effect of sb in healthy dogs and dogs with chronic enteropathies (ce). sb was formulated in x cfu capsules. its concentration and viability within the capsules was controlled by yeast culture in subsequent steps until expiration date. four healthy dogs (hd) and dogs with ce ( inflammatory bowel disease -ibd, protein losing enteropathy -ple) were included. in hd sb was administered for days ( x cfu/kg bid); daily clinical evaluation was performed to assess possible adverse effects and quantitative stool cultures for yeasts were performed before, during and after the administration. in dogs with ce a randomized double blind placebo-control study was performed, administering sb ( x cfu/kg bid) or placebo (pl). sb or pl administration was added to standard therapeutic protocols (diet, antibiotics and immunosuppressive drugs), to evaluate its efficacy for the treatment of ibd and ple. complete blood work, abdominal ultrasonography, gastro-duodenal and colon endoscopy and histopathological evaluation of intestinal samples were performed at diagnosis and after days of treatment. validated score system for the clinical signs (ceccai), ultrasonography, endoscopy and histopathology were applied. significance was set for p < . . results in hd showed the absence of sb in the faeces before treatment, its presence after one day, its steady state ( x cfu/g) after days and its complete elimination days after withdrawal of treatment. no adverse effects were reported. in ce dogs the clinical score improved significantly in dogs receiving sb compared to dogs receiving pl (p = . ). in ple dogs the albumin concentration increased significantly (p = . ) in the group receiving sb with respect to pl. the daily frequency of defecation in the sb group was significantly lower with respect to pl after (p = . ) and (p = . ) days of treatment. no statistical differences were found between dogs receiving sb and pl after treatment, based on the endoscopic evaluation of duodenum and colon. no statistical differences were found between the two groups on the duodenal ultrasonographic and histological evaluation after treatment. in conclusion, sb can be safely used in dogs with ce, in addition to standard treatment, to achieve a better control of clinical signs and a significant increase in albumin concentration compared to the standard therapy alone. no conflicts of interest reported. canine inflammatory bowel disease (ibd) is an immune-mediated enteropathy likely triggered by environmental and immunoregulatory factors in genetically susceptible dogs. previous studies suggest a pivotal role for gut bacteria in disease pathogenesis since luminal microbial composition is markedly altered (ie, dysbiosis) at diagnosis. probiotic bacteria appear to be therapeutically effective in some forms of human ibd. controlled studies evaluating the efficacy of probiotic therapy for canine ibd have not been previously reported. the aim of the present study was to characterize the mucosa-associated microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of orally administered vsl# probiotics in dogs with ibd. twenty dogs diagnosed with moderate-to-severe ibd (cibdai score > ) were randomized to receive standard therapy (ie, elimination diet and glucocorticoids) with or without probiotic vsl# . the mucosal microbiota from endoscopic intestinal biopsies of ibd dogs and controls was evaluated by fluorescence in situ hybridization (fish) targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. epithelial tight junction protein (tjp) expression was studied using immunohistochemistry. clinical signs and changes in mucosal microbiota and tjp expression were assessed before and after probiotic vsl# therapy. ibd dogs showed a reduction in gi signs following weeks of probiotic therapy compared with baseline cibdai scores (p < . ). adherent and sporadic invasive bacteria (eub) were observed in the small intestines and colon of healthy dogs. the diseased canine duodenum was nearly bacteria-free. ibd dogs given probiotic vsl # had altered spatial redistribution of most bacterial groups in the mucus and adherent compartments of the colon. subset analysis showed that lactobacilli were significantly (p < . ) increased in the lumen and mucus post-vsl # , while the number of mucus laden bifidobacteria approached significance (p = . ). expression of tjp showed that occludin was significantly lower in control intestines as compared to duodenal and colonic mucosa obtained from ibd dogs that received probiotic (p = . and p = . , respectively). in contrast, claudin- expression in the colon was significantly higher (p < . ) in control dogs versus vsl # treated ibd dogs. our data demonstrate that probiotic vsl# alters some of the mucosa-associated microbiota in dogs with ibd. these probiotic changes in bacterial composition are associated with up-regulated tjp expression indicative of enhanced epithelial barrier integrity, similar to vsl# -induced disease protection seen in human ibd. the probiotics used in the trial were supplied free of charge by the manufacturer. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb e (ef) on intestinal microbiome composition. dogs were recruited to receive ef at x e cfu in a double-blinded, placebo-controlled manner in addition to an exclusion diet (hydrolysed protein). seven dogs were included in the probiotics group and dogs in the placebo group. all dogs improved clinically after treatment, however, there was no obvious effect on clinical severity in those that received probiotics. fresh naturally voided faecal samples were collected from all dogs before and after treatment, snap-frozen in liquid nitrogen and stored at - °c until further analysis. genomic dna was extracted from each faecal sample using the mobio power soil dna isolation kit (mobio laboratories), as recommended by the manufacturer. next generation sequencing was performed on the ion-torrent[trademark] (life technologies) platform based upon the v -v region (e. coli position - ) of the s rrna gene with the following primers: forward f: gag-tttgatcntggctcag and reverse r: gtnttacn gcggckgctg. raw sequence data were screened, trimmed, filtered, and chimera depleted with default settings using the qiime pipeline version . and uchime software, in which microbiome composition between treatment groups before and after treatment was compared. microbiota composition was not significantly different between probiotic and placebo treatment groups, and did not change significantly before and after treatment. however, there was large individual variability in the microbiome composition. species richness of faecal samples increased after treatment in both groups, but was only statistically significant in the probiotic treatment group. in conclusion, probiotic treatment in dogs with ibd leads to a significantly increased richness of the faecal bacterial microbiome. a possible additional effect of the change of diet cannot be excluded. further studies should investigate microbiomic changes in healthy dogs fed the same diet to assess if similar changes in the fecal microbiome occur due to dietary changes alone. this study is based on a phd supported by probiotics ltd., somerset, uk (the manufacturer of the probiotic product enterococcus faecium mentioned in this study). the aim of this study was to assess the prevalence and risk factors for faecal carriage of extended-spectrum beta-lactamases (esbl) and plasmidic ampc beta-lactamases (pampc) e. coliproducers in healthy dogs. a -month cross-sectional study was conducted at a private hospital in lisbon, portugal and rectal swabs were obtained from healthy dogs. the dogs included in the study were healthy with no history of antimicrobial consumption in the previous month. esbl and pampc genes were detected by pcr and were sequenced. potential risk factors for esbl-and pampc-producing e. coli faecal carriage were obtained through a questionnaire to the owner regarding reason for veterinary visit, hospitalisation and antimicrobial treatment within the last year, habitat (shelter, dog breeders and private owner), cohabitation with other animals, street access, kennel/hotel access, age and gender. data were analysed by sas software (version . ; sas institute inc., cary, n.c.) and logistic regression models were used. rectal swabs obtained from healthy dogs yielded positive samples for e. coli. about % of the isolates carried esbl genes (bla ctx-m- n = , bla ctx-m- n = , bla ctx-m- n = , bla ctx-m- -like n = ) and % carried pampc genes (bla cmy- n = , bla cmy- -like n = , bla dha- n = ). thirteen dogs carried an e. coli isolate with both an esbl and a pampc gene. dogs previously treated with antimicrobials within the last year were at higher risk of carrying at least one ß-lactamase (p = . ; or = . ; ci %: . - . ) or both ß-lactamases (p = . ; or = . ; ci %: . - . ) than non-treated dogs. dogs in shelters/breeders tended to show a higher incidence of esbl-producing e. coli (p = . ; or = . ; ci %: . - . ) or at least one ß-lactamase producing e. coli (p = . ; or = . ; ci %: . - . ) than dogs from private owners. males tended to be less likely to carry at least one ß-lactamase (esbl or pampc) (p = . ; or = . ; ci %: . - . ) or a pampc enzyme (p = . ; or = . ; ci %: . - . ) than females. this study suggests that dogs may act as reservoirs for resistant bacteria, namely for cephalosporin-resistant e. coli. three potential risk factors associated with the carriage of esbl-and/or pampc-producing e. coli by dogs were identified, which is important for the implementation of effective control measures and judicious antimicrobial therapy. conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party there are few reports in the literature reporting long-term relapse rate, owner compliance and clinical severity of dogs with chronic enteropathies. the goal of this study was to compare clinical activity index (ccecai), number of relapses and compliance rates - years after diagnosis. food-responsive disease (frd) was defined as dogs that responded to elimination diet alone within weeks after initiating therapy, whereas antibiotic-responsive disease (ard) dogs had an unsuccessful dietary trial before and responded to metronidazole within weeks after initiation of therapy, and steroidresponsive disease (srd)dogs had an unsuccessful dietary and antimicrobial trial before, and required immunosuppressive therapy to control their clinical signs. ccecai was extracted from the medical record database at - years after diagnosis. relapse rate was obtained by requesting the medical records of the referring veterinarians and defined as number of return visits to the referring practice after diagnosis. compliance data was obtained by telephone questionnaire to the owners. the frd group consisted of / dogs ( %), whereas the ard and srd groups consisted of ( %) and dogs ( %), respectively. there was a significant difference in ccecai at follow-up between frd and ard, and frd and srd (median ccecai . (range - ) for frd, (range - ) for ard, and . (range - ) for srd, p = . ). for the frd dogs, % of owners stated that they deviated from the prescription diet on a daily basis, % once a week, and % once a month, with a median ccecai at the time of deviation from the diet of . (range - ). relapse rate was highest for the ard group, when compared to frd and srd ( for ard, . for frd, and . for srd, p = . ). in the frd group, / dogs had been kept on the prescribed diets, and dogs had been changed to supermarket brands. all of the ard dogs had been given immunosuppressive treatment in addition to antibiotics at the time of follow-up, while / srd dogs were still on immunosuppressive treatments, with one dog being in remission with dietary treatment alone. in conclusion, this pilot study indicates that compliance rate for frd dogs is the lowest, with owners willing to tolerate the highest severity of clinical signs related to deviation from the prescription diet. ard dogs had the highest relapse rate in this cohort, indicating poor response to treatment in the long-term. conflicts of interest: dr allenspach has received research funding from bbsrc, american kennel club, comparative gastroenterology society, probiotics ltd uk, laboklin gmbh germany, and bioiberica sp. she has also undertaken paid consultancy work for bioiberica spain and hoffmann-laroche, switzerland. despite the high prevalence of canine pancreatitis in postmortem studies and the introduction of new diagnostic tests, it is believed that the disease, particularly in its chronic form, remains under recognised due to the non-specific nature of presenting signs. histology is considered to be the gold standard for diagnosis of canine pancreatitis, however, most clinicians are reluctant to take pancreatic biopsies due to significant risks to the patient. numerous serum markers have been reported to be elevated in canine pancreatitis, although most lack sensitivity or specificity. consequently, confirmed diagnosis requires results from a range of tests including imaging, serum biochemistry and physical examination. we and others have previously shown in other diseases that performance of individual low specificity markers can be dramatically improved by combining data from multiple markers with clinical information using analytical algorithms. we therefore applied this approach to the detection of pancreatitis in dogs. the activity of two non-specific biomarkers, amylase and lipase, was determined in serum samples from dogs suspected of having pancreatitis by their veterinarian. of these samples were positive by virtue of their pancreatic lipase (cpli) results (cpli > ug/l). the amylase and lipase data was then used to develop a series of algorithms using mathematical data mining and classification techniques. additional algorithms were developed using extra parameters including age, sex, vomiting, diarrhoea and abdominal pain in addition to the two enzyme levels. the performance of the algorithms was assessed using separate blinded serum samples taken from dogs which were scored clinically for acute pancreatitis according to the system described by mccord et al (j vet intern med ; : - ) . these cases presented for evaluation with vomiting, diarrhoea, inappetance and abdominal pain and were included if a clinical history, results of routine haematology, biochemistry, cpli assay and abdominal ultrasound were available. the results of the multifactorial analysis and cpli assay results were compared to the clinical scores. using amylase and lipase data alone, the algorithm gave a sensitivity of . % and specificity of . %, compared to cpli results for the same samples of . % and . % respectively when both methods were referred to clinical scoring. when the presence of additional clinic data was also included into the algorithm, the sensitivity increased to . % with specificity of %. the data suggests that test performance for canine pancreatitis can be dramatically improved when multiple diagnostic parameters are combined using disease specific algorithms. the author receives a salary as editor of the bsava journal companion, and has undertaken unrelated paid consultancies for bayer and merial. the author also receives a salary from avacta animal health, and duties involved working directly on this project. canine chronic enteropathy (cce) can cause significant long-term morbidity. in some cases this is due to intestinal inflammation, resulting from idiopathic inflammatory bowel disease (ibd). currently, the diagnosis of idiopathic ibd and assessment of disease severity relies on results of subjective clinical indices, laboratory data, diagnostic imaging and intestinal histopathology, whilst ruling out known causes of inflammation. in humans with ibd, a number of faecal biomarkers including lactoferrin, aid with diagnosis and determining disease activity. it may therefore be valuable to develop similar non-invasive objective methods to aid diagnosis and clinical assessment of disease severity in dogs with intestinal inflammation due to idiopathic ibd. this pilot study aimed to measure faecal lactoferrin concentration (flc) in dogs with cce and histologically confirmed intestinal inflammation (hcii) and to compare this with control dogs. in addition, the flc in dogs with hcii would be compared with the canine inflammatory bowel disease activity index (cibdai) and wsava standard histopathological criteria for intestinal inflammation to determine whether there was correlation between these methods when assessing disease severity. faecal samples were obtained from dogs with hcii (n = ) having undergone investigation for cce (serum biochemistry, complete blood count, full faecal and urinalysis, serum cobalamin, quantitative cpli, abdominal ultrasound and intestinal biopsies). the control population were dogs presented for reasons unrelated to cce (n = ). analysis was carried out using a faecal lactoferrin elisa previously validated in dogs (techlab, usa). the flc in dogs with hcii (median . lg/g -range . to . ) was significantly higher than control dogs (median . lg/g -range . - . ) (p < . ). a cut-off flc of . lg/g correctly identified / ( %) of dogs with hcii. using this cut-off, there was no overlap between non-cce dogs flc and the hcii group; giving a sensitivity of % and specificity of %. neither the presence of neutrophils nor the extent of inflammation on histopathology showed significant correlation with flc. the cibdai showed moderate correlation with flc in dogs with hcii (r = . , p = . ). the results of this pilot study suggest that flc is able to discriminate between dogs with cce due to hcii and dogs without cce. it is possible that incorporating flc into a panel of faecal biomarkers will enable non-invasive assessment of hcii and could serve as an adjunct to current measures of disease severity in dogs with idiopathic ibd. ryan bettencourt and james boone are employees of techlab, usa. they provided the elisa kits free of charge for this work. there was no other incentive provided and the results have been openly discussed between all parties. there has been no censorship placed on the results by tech-lab and they have been supportive of the work and submission of this abstract. there are no other conflicts to disclose. fecal s a and fecal calprotectin concentrations have been described as biomarkers in dogs with chronic enteropathies [ ]. however, to date there has been no direct comparison of these two markers in dogs with chronic diarrhea. the aim of this study was to evaluate the performance of these two markers in this situation. thirty one dogs presented for a history of chronic diarrhea were prospectively enrolled. the initial diagnostic workup for all patients included a serum biochemistry profile, fecal parasitology, abdominal ultrasound examination, and gastrointestinal endoscopy with collection of endoscopic biopsies. the severity of clinical signs was evaluated using the ccecai scoring index and patients were grouped by having a ccecai of < or ≥ . fecal calprotectin and s a were quantified as previously described [ ] . correlations were evaluated with the spearman rank correlation test. for both markers a receiver operating characteristics (roc) curve was used to select cut-off value that allowed the best discrimination between dogs with a ccecai< and dogs with a ccecai ≥ . sensitivity and specificity were calculated. correlation analyses revealed a significant positive correlation between s a and calprotectin (r = . ; p < . ). the optimal cut-off value for fecal calprotectin concentration was . lg/g, which was associated with a sensitivity of . % and a specificity of . % (auc= . ; p = . ). the optimal cutoff value for fecal s a concentration was . ng/g, which was associated with a sensitivity of . % and a specificity of % (auc= . ; p = . ). the sensitivity for fecal s a was higher than that for fecal calprotectin (p = . ). no significant difference was observed for the specificity of these two markers (p = . ). out of the dogs ( %) had concordant results for s a and calprotectin tests. among these dogs, presented with a ccecai < and of these dogs had both markers below their cut-off values. among the dogs with a ccecai≥ , dogs had both markers above their cutoff values. % of dogs ( / ) presented histologic signs of inflammation. sensitivities for fecal calprotectin and s a concentrations for histopathological intestinal inflammation were % and %, respectively, and specificities were % and %, respectively. at least in this group of patients fecal s a concentration was more sensitive (but less specific) to detect dogs with a cce-cai ≥ or histopathologic intestinal inflammation than fecal calprotectin concentration. weight loss and malabsorption of fat, protein, cobalamin and tocopherol in the face of normal exocrine pancreatic function have been reported in up to - % of cats older than years of age fed a variety of nutritionally balanced dry and wet foods (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, focus on gastroenterology, [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . the objectives of this study were to determine if serum cobalamin concentrations increased after oral administration of a cobalamin supplement to affected cats, and the duration of any positive response following cessation of supplementation. the study evaluated cats older than years of age with fat malabsorption demonstrated by either increased fecal fat (> %) or subnormal fat digestibility (< %), but without exocrine pancreatic insufficiency (epi) as assessed by assay of serum trypsin-like immunoreactivity (ftli). a commercially available solution of cobalamin containing mg mixed with ml of a liquid flavor enhancer was added to the food of each cat in a single dose each day for months, after which supplementation ceased. serum cobalamin (assayed by competitive binding assay performed through the gi laboratory at texas a&m university and evaluated using reference ranges derived by that laboratory) was determined immediately prior to initiation of supplementation, then weekly for weeks, then monthly for months. at the start of the study serum cobalamin was subnormal (< ng/l) in of the cats (range < to ng/l) and within the reference range ( to ng/l) in the remaining cats ( to ng/l). serum cobalamin was above the reference range in every cat ( to ng/l) after one week of supplementation and remained above the reference range in every sample collected during the supplementation period, with the exception of two cats with values within the reference range when supplementation was stopped. serum cobalamin , and months after cessation of supplementation ranged from < to , < to , and < to ng/l, respectively. at the end of the study serum cobalamin was subnormal in of the cats. it is concluded oral cobalamin supplementation can effectively increase serum cobalamin concentrations in geriatric cats with idiopathic chronic enteropathy, but that following cessation of supplementation concentrations decrease rapidly and can become subnormal again within as few as weeks. the primary author collaborated with nestle-purinaon the work reported in this abstract, and a co-authoris an employee of nestle-purina. the primary author has previously received funding from iams, mars, hills and nestle-purina. the author also acts as a paid consultant for the gi-lab, texas a&m university. left atrial measurements are crucial in assessing severity of cardiac disease in dogs with myxomatous mitral valve disease (mmvd). however, linear and area dimensions might not provide a comprehensive assessment of patient status, and cannot differentiate between severe subclinical (b ) and clinical disease (chf). estimates of left atrial function could provide additional information to help categorize these patients. we examined dogs with mmvd ( normal, b , b and c) presented for cardiac evaluations by d echocardiography. left atrial linear and area dimensions in right parasternal short and long axis views were obtained at time points -early diastole (la max ), just prior to mitral valve opening, at the onset of atrial systole (la p ) and just prior to mitral valve closure (la min ). we calculated indices of la function: total la emptying fraction (la tef ), active la emptying fraction (la act ), passive la emptying fraction (la pas ) and la reservoir function (la res ) for all sets of measurements. we examined the differences in selected la function indices between different disease stages with a kruskal wallis test with post-hoc multiple comparisons. we also examined the diagnostic accuracy of selected indices of la function in differentiating dogs in stage b and stage c (chf) using roc analysis. three functional indices consistently differed across the various stages of mmvd -la tef , la act and la res . these differences were most apparent in the rpla view for linear measurements and rpsa view for area measurements. dogs with chf had worse function than all other groups, which differed variably depending on the functional index being examined. laarea act showed the best ability to discriminate between b and chf dogs, with a % specificity, % sensitivity and an auc of . , but this was no better than use of la:ao measurements. our data suggest that la function differs between dogs with differing severities of mmvd, but does not provide a clear distinction between dogs with subclinical disease and chf. no conflicts of interest reported. materials and methods: de was used to evaluate consecutive, non-sedated dogs that weighed more than kg. the study population for the morphologic study included normal dogs, and dogs with acvim stages b or b -c mmvd. de image data were digitally recorded and then analyzed offline, using commercially available software. results: de image acquisition was feasible in / ( . %) consecutive dogs. patient anxiety ( ), arrhythmias ( ) and panting ( ) explained failure to obtain a de dataset. fortyone of ( . %) datasets were of analyzable quality. body weight and heart rate were significantly lower in dogs for which it was possible to perform de. dogs with analyzable de datasets were significantly older and weighed less than dogs in which de could not be analyzed. the mitral valve of normal dogs is saddle shaped (annulus height to commissural width ratio (ahcwr): . ae . [mean ae sd]) and has an elliptical annulus (sphericity index (si): . ae . ). the following measurements were significantly related to body surface area (bsa): antero-posterior diameter (apd) (r = . , p < . ), anterolateral-posteromedial diameter (alpmd) (r = . , p < . ), annulus area (aa) (r = . , p < . ), anterior leaflet length (all) (r = . , p < . ), anterior leaflet area (ala) (r = . , p < . ). these variables were indexed (i) to bsa for subsequent statistical analyses. dogs with mmvd had a significantly greater si, non-planar angle, apdi, alpmdi, alai and alli, while having a significantly lower posterior leaflet area (pla), posterior leaflet length (pll), annulus height (ah), tenting height (th), tenting volume (tv), tenting area (ta), and ahcwr compared to normal dogs. ah, tv and ta were significantly greater in normal dogs, compared to dogs with mmvd. si, apdi, al-pmdi, aai, alai and alli were significantly greater in dogs with stages b -c mmvd, compared to normal dogs and those in stage b . pll and pla were significantly lower in b -c dogs, compared to normal dogs. th was significantly different between the three groups; greatest in normal dogs and lowest in dogs in stages b -c, suggesting that flattening of the mv occurs with disease progression. conclusions: de assessment of the canine mv is feasible. morphologic changes associated with mmvd progression are presented. effective regurgitant orifice area (eroa), calculated from a dimensional measurement of the width of vena contracta (vc) as the narrowest portion of the proximal regurgitant jet, might be used to estimate severity of mitral regurgitation (mr). however, this simplified assumption only holds when the eroa is circular, which might not be true in dogs with myxomatous mitral valve disease (mmvd). the aim of the study was to compare measured eroa using color doppler real-time dimensional echocardiography (rt d) with calculated eroa estimated by dimensional echocardiography ( d) in chamber ( ch) and chamber ( ch) views of the left ventricle (lv) in dogs with mmvd. ninety-three privately owned dogs of breeds diagnosed with naturally acquired mmvd were examined using d and rt d. according to the acvim classification of congestive heart failure (chf), dogs were classified with chf ( in class c and in class c ) and dogs without chf ( dogs in class b and dogs in class b ). age ranged from to years (median years), and body weight ranged from . to kg (median kg). fifty-nine males ( %) and females ( %) were included, and heart rate ranged from to beats/minute (median b/min). eroa was calculated from d measurements of vc diameter, in the ch view only (assuming a circular regurgitant orifice), and from measurements of vc diameter in both ch and ch views (assuming an eliptical regurgitant orifice) of lv. bland-altman plots were used to compare eroa measured by rt d with calculated eroa obtained from d ch and ch/ ch lv views. none of the d estimations of eroa showed good agreement with the measured rt d eroa when corrected for bsa, and the difference between methods increased with increasing eroa. the difference between rt d and d methods normalized to the mean eroa value did not increase with increasing eroa, but showed a systematic underestimation of eroa by % ( ch) and % ( ch/ ch), respectively, compared to rt d. the beat-to-beat variation of eroa assessed by rt d (n = ) had a coefficient of variation ranging from . % to % (median %). in conclusion, substituting assessment of eroa with a measurement of vc in or dimensions might underestimate the mr severity in dogs with mmvd. in some dogs, the beat-tobeat variation of the eroa was large, thereby necessitating the need for several consecutive measurements. no conflicts of interest reported. micrornas (mirna) are short ( - nucleotides), singlestranded, non-coding rnas that specifically anneal with complementary sequences in multiple mrna targets, and they silence mrnas and suppress downstream protein translation. a mirna can act as a fine-tuner of gene expression or an on/off switch. these features highlight the potential of mir-nas as therapeutic targets. the role of mirnas in myocardial fibrosis and hypertrophic cardiomyopathy has been widely studied in human patients. however, there is no data available for canine and human myxomatous mitral valve disease (mmvd). the aim of this study was to investigate mirna transcriptomics in canine mmvd by using global transcriptional profiling, mirna target prediction software (diana tool, targetscan . ) and network analysis software (biolayout express d ). four myxomatous mitral valves (ckcs) and controls valves were profiled using the affymetrix canine gene . st array. in total out of mirnas were found to be statistically significantly differentially expressed (down-regulated) based on the false discovery rate, p-value, and fold-change. expression of three mirna (cfa-mir- b, cfa-mir- c, cfa-mir- ) were also validated by quantitative polymerase chain reaction (q-pcr, taqman), and the results were in agreement with the microarray findings. for network analysis and visualization, markov clustering algorithms were conducted in bio-layout express d , and major clusters of mirnas were exported and uploaded to the diana-mirpath (kegg pathway) web-server. the pathways identified in the main cluster were attributed to the biological functions of focal adhesion, cytoskeleton (actin) regulation, tgf-b signalling, glycosaminoglycan biosynthesis, osteoclast differentiation, notch signalling and vegf signalling. the most significantly down-regulated mirna in mmvd was cfa-mir- , which is an endothelial specific mirna shown to regulate endothelial migration and vessel patterning. the top predicted target of cfa-mir- is glucuronic acid epimerase (glce) which is the main enzyme controlling heparan sulphate biosynthesis. other interesting findings were down-regulation of cfa-mir- and members of the cfa-mir- family. cfa-mir- targets multiple extracellular matrix transcripts, such as collagens, elastin, integrin, laminin, mmp (matrix metalloproteinase) and adamts (a disintegrin and metalloproteinase with thrombospondin motifs), whereas cfa-mir- targets hyaluronic acid synthase (has ). since the major pathology of mmvd is aberrant turnover of extracellular matrix proteins, this may be linked to mir-na regulation. dysregulation of valve mirnas might be potential therapeutic targets in the treatment of canine mmvd. no conflicts of interest reported. mitral regurgitation (mr) progresses slowly, but dogs living long enough often develop congestive heart failure (chf). however, tools to predict onset of chf are sparse. echocardiographic examinations in dogs were performed in a longitudinal, multicenter study with a surveillance time of up to . years. client-owned dogswere enrolled at the university hospitals in finland, sweden and denmark (subset to the svep study). left ventricular end diastolic (lvidd) and systolic (lvids) diameters, fractional shortening (fs), left atrial (la) and aortic root (ao) diameters were estimated. values were normalized for body size (nlvidd, nlvids, and nla, respectively) and, for comparison, ratios to aortic root were calculated (lvidd/ao, lvids/ao and la/ao, respectively). a cox's proportional hazard analysis with a counting process approach was used. spline smoothed graphical models were constructed to evaluate linearity of hazards. curves were then used to find cut-off values for interval hazard ratios (hrs). the hr for nlvidd, nlvids and nla (per . unit, % confidence intervals), were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hrs for lvidd/ao, lvids/ao and la/ao ( . unit increase) were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hr for fs was . ( . - . , p = . ). the relative hazard plot presented a steep increase for fs values above %. hrs for intervals < %, < %, and ≥ % were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hr for nlvidd increased linearly. hrs for intervals . < . , . < . , . < . and ≥ . were . ( . - . , p = . ), . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. in contrast, the hazard for nlvids remained stable until . , whereafter it increased. the hrs for nlvids ( < . , . < . , . < . and ≥ . ) were . ( . - . , p = . ), . ( . - . , p = . ), ( . - , p = . ), and . ( . - , p = . ), respectively. hrs for values normalized to ao diameter behaved in a parallel way. we conclude that fs, left ventricular and atrial size may be used to predict chf. however, because the value of a hr is dependent on the unit used and, more essentially, does not account for nonlinear change in hazard, interpretation of hazards is challenging. in contrast, interval hazards are only dependent on the reference interval used. therefore they are easier to implement in every day clinical work. no conflicts of interest reported. systemic arterial hypertension is not frequently recognized in dogs with mitral valve degeneration (mvd), although borderline hypertension is difficult to assess, mainly because of different measurement techniques, inter-operator variability and, most importantly, examination-related stress. the object of this study was to evaluate systolic arterial blood pressure (sbp) at initial presentation and at regular intervals in dogs with various clinical stages of mvd. fifty six dogs with mvd that had not received any heart medication prior to admission, were included in the study. based on the isachc staging system, were assigned to class i (group a), to class ii (group b) and to class iii. small-breed dogs and miniature poodles, in particular, were overrepresented. comorbidities that could affect sbp were ruled out prior to enrollment. sbp was measured using a commercially available veterinary oscillometric device, by applying the proper cuff on the cephalic artery. dogs were left to acclimate for - minutes and measurements were always taken by the same investigator, before any other examination was performed, with the dog sitting on the owner's lap. a total of readings were taken, outlier values were discarded and the mean of the remaining measurements was documented. after initial consultation, treatment was customized according to the clinical stage. sbp was then measured every months, up to months after initial admission. at presentation, all class i dogs had sbp > mm hg, with only / having spb ≥ mm hg, whereas all class ii dogs had sbp < mm hg. of class iii dogs, had sbp > mm hg, and had sbp ≥ mm hg. a linear mixed effects model was used to assess the temporal variability of the measured parameters between groups. groups were matched for gender, age and body weight. blood pressure measurements, for the duration of the study, were higher in group a dogs, compared to groups b and c (p < , ). at the same time, group c had significantly higher sbp values than group b dogs (p < , ). asymptomatic mvd dogs seem to have higher sbp measurements, compared to those with clinical evidence of heart failure. whether this difference is stress-related, a maladaptive mechanism of sympathetic and raas activation to mvd or idiopathic remains to be elucidated. no conflicts of interest reported. sarcoplasmic reticulum (sr) ca + -atpase and its regulatory proteins are pivotal determinants of myocardial active relaxation via calcium uptake against the sr-cytoplasmic gradient. the lowered density of the sr ca + -atpase has been well demonstrated in many species during chronic hemodynamic overload. the genes linked to sr calcium uptake were reported not only being expressed in peripheral blood but serving as potential cardiac biomarkers in dogs with chronic mitral regurgitation, such as sr ca + adenosine triphosphatase isoform a (ser-ca a), phospholamban (pln), and hs- associated protein x- (hax- ). the aim of this study is to determine whether the target genes expressed in the blood will be translatable to the myocardial setting as cardiac biomarkers. the mrna expression levels of the target genes (serca a, pln, hax- ) from biopsied left ventricle (lv) and peripheral white blood cells (pwbc) in surgical mitral valve repair cases were estimated with quantitative real-time pcr using comparative ct method with gapdh. the gene expression levels in lv and pwbc were compared and their clinical relationships were evaluated. the diagnostic power of the genetic expressions in pwbc was analyzed by comparing to those of normal dogs. the levels of all target genes expressed in lv and pwbc were highly correlated each other in linear regression analysis (p < . ; serca a, r = . , r = . ; pln, r = . , r = . ; hax- , r = . , r = . ), although lv and pwbc showed different expression levels in a paired comparison (p < . ). according to the severity of the heart failure (isachc), the expression levels of all genes were gradually and significantly reduced in both lv and pwbc (p < . ). especially, the serca a and pln expressed in pwbc could clearly discriminate all isachc groups from the control (p < . ). multivariate regression adjusted by age and body weight revealed that serca a and pln in lv were negatively associated with lv internal systolic dimension (p = . , adjusted r = . and p = . , adjusted r = . , respectively). pln was also negatively related with lv internal diastolic dimension (p = . , adjusted r = . ). additionally, receiver-operating characteristic analysis using pwbc showed high area under the curve (auc) values for all target genes on overall isachc groups (p < . ; serca a, auc= . ; pln, auc= . ; hax- , auc= . ). in conclusion, the transcriptional changes of the calcium uptake related genes in pwbc may be able to reflect myocardial hemodynamic stress as well as to be utilized as promising cardiac biomarkers. no conflicts of interest reported. the aim of this study was to estimate heart-rate normalized pulmonary transit times (nptt) in cardiomyopathic cats with or without congestive heart failure (chf), to assess potential associations of echocardiographic variables and nptt, and to evaluate nptt as a test for presence of chf. privately owned cats were included. nptt was measured using echocardiography and the ultrasound contrast media sonovue â in groups of cats: healthy cats (group ), cats with cardiomyopathy (cm) but without chf (group ), and cats with cm and chf (group ). receiver operating characteristic curves (roc) were created for nptt, left atrial diameter (lad) and the left atrial to aortic root ratio (la:ao) to assess and compare their usefulness as tests for presence of chf. interrelations between pulmonary blood volume (pbv), nptt, stroke volume (sv) and echocardiographic variables were investigated by means of uni-and multivariate analysis. nptt values in group , group and group were . (interquartile range (iqr) . - . ), . (iqr . - . ), and . (iqr . - . ), respectively. values were significantly different between all groups. pulmonary blood volumes in group , group , and group were . ml (iqr . ml- . ml), . ml (iqr . ml- . ml) and . ml (iqr . ml- . ml). sv, pbv and shortening fraction < % were significant predictors of nptt. nptt and la:ao ratio, not sv were the main predictors of pbv. analyzing roc for nptt as a clinical test for chf yielded an auc of . which was similar for la:ao ratio. nptt may be useful test for the presence of chf in cats with cm and as a measure of cardiac performance. nptt and la: ao ratios predict chf with equal accuracy. increased pbv is significantly associated with higher nptt and la:ao ratios. both decreased sv and increased pbv explain the increased nptt in cardiomyopathic cats. the author received a travel scholarship from zoetis to attend this congress. acute arterial thromboembolism (aate) occurs commonly in cats, and less frequently in dogs, mostly resulting in limb paresis or paralysis. diagnosis is based typically on physical examination and advanced imaging. diminished affected-limb peripheral blood flow induces changes in several analytes concentrations in affected limb venous samples, compared to their peripheral venous concentration. we hypothesized that in aate, local, affected-limb venous glucose concentration decreases below reference interval, while systemic glucose concentration remains unaffected. the study included groups for each species: paralytic aate cases, non-ambulatory controls with limb paralysis of orthopedic or neurologic disorders, and ambulatory controls diagnosed with various diseases. systemic and peripheral, affected-limb blood glucose concentrations were measured. group absolute (dglu) and relative (% dglu) differences were compared. no procedure-associated complications or pain were noted. peripheral blood glucose concentrations were decreased (p ≤ . ) only in cats and dogs with aate. dglu and %dglu were higher in the aate groups in both cats and dogs compared to their respective control groups (p < . , p < . , respectively), with no differences between the control groups. receiver operator characteristics analysis of dglu and %dglu as predictors of aate in cats had areas under the curve of . and . , respectively, and . and . , in dogs, respectively. dglu cutoffs of mg/dl and mg/dl, in cats and dogs, respectively, corresponded to sensitivity and specificity of % and % in cats, respectively, and % in dogs. dglu and %dglu are extremely accurate, readily-available, simple diagnostic markers of aate in cats and dogs. no conflicts of interest reported. glycaemia determination is usually included in routine biochemisty panels. no works are devoted to the evaluation of pheripheral glycaemia in animals suffering from arterial thrombosis. the aim of this study was to document the pheripheral glycaemia variations in hypoperfused limbs of patients affected by mriconfirmed arterial thrombosis. eleven dogs referred for monoparesis or paraparesis were recruited. inclusion criteria were a clinical examination supportive of limb hypoperfusion and availability of blood cell count, biochemical profile and urine analyses. before mri examination, peripheral glycaemia was tested. two blood samples were obtained, one from the affected limbs and one from a healty limb.plasmatic glycaemia was measured using an automated glucose analyser. all the patients underwent a total body mri (mri intera . t, philips medical systems) that provided the final diagnosis. the arterial thrombosis location was documented and the entity was scored. all the eleven patients were diagnosed with a peripheral thrombosis involving an arterial vessel and in some cases the relative branches. the thrombus was located: in the abdominal aorta ( / ), in the subclavian artery ( / ), in the axillary artery ( / ), in the iliac arteries ( / ). of the total amount of abdominal aortic thrombosis, / involved also the internal iliac arteries, / the external ones and / both internal and external. the extent of the thrombosis was classified as grade (g ), when the greatest portion of the thrombus did not reach half of the vessel lumen ( / patients); grade (g ), when the greatest portion of the thrombus was between / and / of the vessel lumen ( / ); grade (g ), when the thrombus exceded / of the lumen ( / ). a substantial decrease in pheripheral glycaemia values was found in sampling arising from the thrombosisaffected limbs. comparing thrombosis-affected limbs values with healthy limbs measurements from the same patient, the reduction was found from . % to . %. accounting only the g scored patients, the percentage of reduction was found up to the . % suggesting a proportional decrease related to the grade of occlusion. results from this study suggest that peripheral glycaemia values are affected by limb hypoperfusion disorders. if an arterial thrombosis is suspected, samples from the affected limbs and the healthy ones could be used to compare glycaemia values and to support the early stage therapy in anticipation of diagnostic imaging. further studies are needed to confirm the proportional relation of the decrease with thrombus entity. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwt). pulmonary hypertension (ph) may develop secondary to hypoxic vasoconstriction and/or pulmonary parenchymal infiltration. in the absence of measurable tricuspid regurgitation (tr), this co-morbid condition may be difficult to diagnose non-invasively. the degree of cardio-pulmonary impairment in cipf dogs can be evaluated through blood gas analysis (bga) and minute walking test ( mwt). a new echocardiographic index, the right pulmonary vein to pulmonary artery ratio (pv/pa) has been described for the detection of pulmonary venous hypertension. the aim of this study was to investigate pv/pa in cipf in order to determine its utility in the detection of ph and in the assessment of cardio-pulmonary disease severity. this prospective clinical cohort study included whwt with cipf (group a), healthy whwt (group b) and healthy dogs from other breeds (group c). diameters of right pv and pa were measured, in bi-dimensional (bd) and m-modes (mm), in a parasternal right long axis view, at the end of the t wave. other echocardiographic parameters for evaluation of ph were also measured: speed of tr, acceleration time to ejection time ratio of the pulmonary flow (at:et) and pulmonary artery to aorta ratio (pa/ao). bga was performed in dogs ( , and in groups a, b and c) and mwt in dogs ( , and ). values are given as meanaesd. in bd and mm mode, the pv/pa ratio was lower in group a (mm: . ae . , bd: . ae . ) compared to group b (mm: . ae . , bd: . ae . , p ≤ . ) and group c (mm: . ae . , bd: . ae . , p ≤ . ). the changes in pv/pa were both due to an increase of pa (p ≤ . ) and a decrease of pv (p ≤ . ). tr was found in % of dogs with cipf; mean pressure gradient was . ae . mmhg. at:et was lower in group a ( . ae . ) compared to group c ( . ae . , p = . ) and tended to be lower compared to group b ( . ae . , p = . ). pa/ao was not statistically different between groups. pv/pa was correlated with arterial po values (b mode: r = . , p = . ) and results of the mwt (b mode: r = . , p = . ). pv/pa was also correlated with at:et and the speed of tr, but not with pa/ao. in conclusion, in whwt affected by cipf, pv/pa is a useful indicator of ph and could serve in the assessment of disease severity. no conflicts of interest reported. ventricular septal defect (vsd) is the fourth most common congenital cardiac defect in dogs and the most common in cats. the aim of this study was to evaluate the long-term outcome in vsd patients. case records of animals were reviewed, of these re-evaluated echocardiographically and followed up by phone interview only. out of dogs pug was the most common breed ( %) followed by border terrier ( %). out of cats domestic short hair was most common ( %) followed by main coon ( %). isolated vsds were present in dogs and cats. complex defects (cds) were present in cases, most frequent anomalies being sub-aortic stenosis ( dogs, cat), pulmonic stenosis ( dogs, cats), tetralogy of fallot ( dogs, cats), cushion defects ( cats) and double-chambered right ventricle (dcrv) ( dogs, cats; in / dogs not present initially supporting the cause-and-effect theory). eisenmenger was observed in dog and cats. aortic insufficiency, not considered a cd, was noted in dogs. in dogs and cats ( % of isolated vsds and cat with a dcrv) the defect closed spontaneously. nine dogs and cats ( %) died of non-cardiac causes with an age at death of to (mean . ) months; dogs and cats died due to cardiac causes with an age at death of . to (mean . ) months. cardiac deaths were sudden ( dogs with cds) or euthanasia for left sided congestive heart (chf) failure associated with cds ( dogs, cat); right sided chf associated with cds ( cats); biventricular failure ( cat with cd); weakness (eisenmenger, dog and cat; fallot dog; cd cat). two cats developed chf due to unrelated hcm. only one dog with an isolated vsd was euthanized for chf. these results indicate that spontaneous vsd closure occurs more often than previously thought, most patients with isolated restrictive vsds live a normal life span without surgical intervention, but non-restrictive vsds or complex defects can be associated with significant morbidity and mortality. echocardiography early in life is crucial to identify the anomaly and cds, as well as useful to prognosticate long-term outcome and to identify patients where a surgical intervention should be considered if available. follow-up echocardiography is indicated to corroborate the prognosis, to detect complications due to the vsd and to detect unassociated acquired cardiac diseases. no conflicts of interest reported. centronuclear myopathy (cnm) is the most prevalent congenital inherited disorder affecting skeletal muscles in labrador retrievers. this disabling condition segregates worldwide and a recessive loss-of-function founder mutation was identified in the protein tyrosine phosphatase-like, member a gene (ptpla/ hacd ). the objectives of this study were ) to describe ptpla expression pattern in hearts from homozygous wild type (wt), heterozygous (het) and homozygous mutated (cnm) dogs, ) to assess and compare the left myocardial function in aging wt, het and cnm dogs. for this purpose, seven wt, four het and eleven cnm dogs were included in the study. ptpla mrna levels were assessed by rt-pcr and rt-qpcr. all dogs were examined using conventional echocardiography, d color tissue doppler imaging (tdi) and tdi-derived strain imaging. we found that the expression of the two wild type ptpla splice isoforms increased post-natally in wt dogs. their levels were halved in het dogs and drastically reduced in cnm dogs. in both het and cnm dogs, a slight left ventricular hypertrophy was detected using conventional echocardiography. tdi and strain imaging revealed that the left ventricular myocardial function was significantly altered in both het and cnm dogs compared to wt dogs. moreover, these functional defects were associated with significantly higher values of systemic arterial blood pressure, although maintained within normal ranges. in conclusion, subclinical myocardial alterations were detected in both het and cnm aging dogs from our french pedigree, suggesting a role for ptpla in long-term cardiovascular homeostasis. these findings prompt globalized confirmation in additional ptpla"'deficient dogs, which may thus be considered as a new large-size model for human left ventricular sub-clinical myocardial dysfunction. no conflicts of interest reported. mitral regurgitation (mr) secondary to degenerative mitral valve disease (dmvd) is the most common heart disease in dogs. in dogs with mr, mitral valve prolapse caused by degeneration of the mitral valve leaflet, chordae tendinae extension and/or rupture and mitral annulus dilation are observed. however, limited data are available on morphological changes in dogs with mr. currently, there are no studies confirming the anomaly of the mitral complex via direct observation in living dogs with mitral regurgitation. at our institution over the last ten years, approximately dogs have undergone mitral valve repair. during surgery, the anomaly of mitral complex can be observed macroscopically (directly visualized). to our knowledge, this is the first study evaluating the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. animals: dogs that underwent mitral valve repair with cpb at nihon university between february and june were included in this study. methods: confirmation of chordae tendineae rupture was visually confirmed during surgery. the sites of chordae tendineae rupture were also recorded at that time. septal chordae and mural chordae were divided three division depend on the site (s , s , s and m , m , m respectively). results: ninety eight dogs were included in this study. the mean age and body weight were . ae . years and . ae . kg, respectively. of the dogs, ruptured chordae was observed in dogs ( . %). septal leaflet chordae was ruptured in dogs ( . %) and mural leaflet chordae was ruptured in dogs ( . %). chordae of both leaflets were ruptured in dogs ( . %). no chordal rupture was observed in dogs ( . %). in the dogs with ruptured septal chordae, the chordae between s and s was most often ruptured (n = , %). in this study, rupture of the septal chordae tendineae was most commonly observed. this is the first pilot study to visually evaluate the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. future studies comparing pathological changes and molecular biological analysis to gross findings of mitral chordae tendineae in dogs undergoing mitral valve repair may be useful in advancing the understanding of the disease. no conflicts of interest reported. echocardiographic aortic valve (ao) measurements are routinely obtained during cardiac evaluation of patients. cardiologists commonly use diastolic ao measurements to obtain ratiometric weight-independent estimates of dimensions of other cardiac structures, most commonly the left atrium (la). however, no consensus exists about the point in diastole at which ao measurements should be obtained -immediately after closure of the aortic valve, when la size is largest (ao max , but often with least distinct margins), during the p-wave of the ecg (ao p ) and at the onset of ventricular electrical systole, when la size is smallest (ao min ). we examined the linear and area dimensions of the ao (aod and aoa) to determine if clinically significant differences exist at distinct diastolic time-points, or if these measurements could be interchangeable. we examined patients ( dogs and cats) presented for cardiac evaluations by d echocardiography. three replicates of each time-point linear and area measurement (ao max , ao p , ao min ) were obtained in each patient and averaged for analysis. only patients with aortic valve disease and those with atrial fibrillation were excluded from analysis. beat-to-beat variability of the ao measurements was determined. standard and normalized limits of agreement (loa) plots were generated for each pairwise comparison. the frequency of each ao measurement being the largest or smallest within-patient measurement was determined, and compared via repeated measures anova. all pairwise agreement plots of both aod and aoa demonstrated heteroscedasticity; normalized aod plots showed % loa to be % of the mean aod measurement, with a bias of approximately . % for aod max -aod min , % for aod max -aod p , and % for aod p -aod min . normalized aoa plots showed %loa to be % of the mean aoa measurement, with a bias of approximately % for aoa max -aoa min , % for aoa max -aoa p , and % for aoa p -aoa min . aod max was the largest measurement in / ( %) patients and aod min was the smallest measurement in / ( %) patients; aoa max was the largest measurement in / ( %) patients and aoa min was the smallest measurement in / ( %) patients. rmanova confirmed that ao max >ao p >ao min (p < . ). median within-patient within-measurement variability was % for aod and % for aoa measurements. our data suggest that ao measurements differ throughout diastole, with ao max >ao p >ao min . the disparity is greater for area than linear estimates. the degree of disagreement between ao max and ao p is small and similar to the within-measurement variability. thus, using either ao max or ao p measurements should result in similar ratiometric estimates of cardiac dimensions. no conflicts of interest reported. feline hypertrophic cardiomyopathy (fhcm) is the most common heart disease in cats. hcm is considered an inherited disease of the sarcomere and fhcm has been linked to mutations in one sarcomere protein i.e. mybpc . however, the pathophysiologic mechanisms behind disease development and progression are largely unknown. in this study we investigate whether mitochondrial morphological changes in the myocardium accompany mitochondrial dysfunction and enhanced oxidative stress formation that we recently found in fhcm. myocardial tissue from the left ventricle (lv) was obtained immediately after euthanasia from cats diagnosed with primary hcm on echocardiography ( maine coon, british shorthair, exotic shorthair, norwegian forest cat) and age-matched control cats ( maine coon, norwegian forest cats). ultrastructural examination was performed by the use of transmission electron microscopy. in hcm cats, marked ultrastructural changes of the cardiomyocytes were observed. the population of subsarcolemmal mitochondria (ssm) was absent in large cellular areas in cats with moderate and severe lv hypertrophy. flattening of the sarcolemma was a common finding, causing disorganization of the t-tubular system. interfibrillar mitochondria (ifm) were disorganized but not depleted. additional changes in cardiomyocytes from cats diagnosed with fhcm included remodeling of sarcomeres, disorganization of myofibrils, convolution of gap junctions, accumulation of intracellular z-disc material, perinuclear lipofuscin granula and extensive extracellular deposits of collagen. in healthy mammalian cardiomyocytes, the t-tubular system upholds cellular structure, prevents mitochondrial reticulum formation and provides calcium, oxygen and substrates, necessary for normal functioning muscle. disorganization of the sarcolemma and t-tubular system may cause the depletion of ssm. possible mechanisms are atrophy or disruption of the mitochondria or altered fusion-fission dynamics. calcium cycling and substrate supply are likely to be compromised by the observed structural changes. we propose this to be related to mitochondrial dysfunction and oxidative stress formation that occurs in fhcm, however a causative relationship remains unknown. in conclusion, morphological changes of mitochondria and extra-sarcomeric structures are common in fhcm, regardless of breed, genotype and phenotypic disease expression. moreover, mitochondrial subpopulation-specific changes occur in fhcm with depletion of ssm. ultrastructural and functional changes of cardiac muscle mitochondria are considered important molecular mechanisms, responsible for the development and progression of fhcm and may be relevant future treatment targets. no conflicts of interest reported. patent ductus arteriosus (pda) is one of the most common congenital cardiac defect in the dog. ductal patency is associated with pulmonary overcirculation, left ventricular volume overload and can rapidly determine congestive heart failure if untreated. several devices to close the pda have been used, with amplatzer canine duct occluder (acdo©) being considered the safer device with lowest complication rates. echocardiography represents the cornerstone of pda diagnosis, but its role has been recently expanded to wider field of application: device sizing and intraoperative monitoring, as well as a tool to quantify cardiac morphology and function. speckletracking echocardiography (ste) has been used to evaluate cardiac function in a wide variety of diseases in human and veterinary patients, however no study has evaluated its usefulness in dogs affected by pda both before and after percutaneous closure of pda. the aim of our study was therefore to assess standard m/bmode derived parameter of cardiac function and ste derived longitudinal, radial and circumferential strain and strain rate before and after pda closure. twenty-five dogs of different breeds, age and weight were prospectively recruited and a complete echocardiographic evaluation was performed before and hours after pda closure. end diastolic and systolic diameters indexed for body surface area (edvi/esvi) both derived by m-mode and b-mode views, allometric scaling derived allod and allos, sphericity index (si) and pulmonary to systemic flow ratio (qp/qs) were assessed both pre and postoperatively. ste derived parameters assessed were longitudinal, radial and circumferential strain and strain rate. a statistically significant difference was found in all standard parameters of cardiac function before and after pda closure (p < . ), with a general decrease in values hours postoperatively. ste derived parameters of cardiac function showed a trend toward a decrease back to normal values, which was statistically significant (p < . ) for circumferential and radial strain and strain rate, while longitudinal strain and strain rate did not reach statistically significance. based on our results, no cardiac dysfunction was identified by the use of ste derived parameters both before and after pda closure, with an increased contractility as identified by higher than normal ste values before pda closure and a decrease back to normal strain and strain rate values for both circumferential and radial immediately after percutaneous closure. longitudinal strain persists on higher than normal values, refusing the hypothesis of systolic dysfunction after pda closure and suggesting a longer reverse remodeling process after pda closure. dr bussadori receives royalties from esaote (florence, italy) related to an european patent (nr ) he developed for xstrain software. the study was not funded by a research grant. cardiac cachexia which is characterized by progressive weight loss and depletion of lean body mass, is an independent predictor of survival in human patients with congestive heart failure. chronic degenerative mitral valve disease (cdmd) is one of the most common cardiac diseases in dogs. the aims of this study were to evaluate the prevalence and the effects of cardiac cachexia in survival of dogs with cdmd. medical records of client-owned dogs with cdmd were reviewed. the mean age at entry was . ae . years; were females, and were males. data obtained from the records including breed, sex, body weight, age at diagnosis, complete blood counts, biochemical profiles, urinalysis, systemic blood pressure, thoracic radiographs, electrocardiograms, ultrasonography and echocardiographic examinations at initial visit and survival time. diagnosis of cdmd was based on echocardiographic characteristics and categorized by modified new york heart association (nyha) functional classification. cardiac cachexia was defined as presence unintentional weight loss (> % within months after diagnosis) together with anorexia and muscle weakness, anemia (red cell count < . /ll, hemoglobin < g/dl, or both), hypoalbuminemia (plasma albumin < . g/dl), and azotemia (blood urea nitrogen > g/dl, creatinine > . g/dl, or both). dogs with other cardiac disorders and other systemic disorders those would cause anemia and hypoalbuminemia were excluded from this study. prevalence of cardiac cachexia, anemia and azotemia was . %, . % and . %, respectively. these conditions were the most prevalent in nyha class , followed by nyha classes and . the prevalence of hypoalbuminemia was not significantly different among classes. the one-year body weight change was found in the nyha classes (increased . ae . %), (decreased . ae . %) and (decreased . ae . %). the difference between classes and was significant. results of the cox proportional hazard model indicated that survival time was significantly positively associated with nyha functional severity at diagnosis (p < . ), presence of cardiac cachexia, weight loss, anemia, hypoalbuminemia and azotemia (p < . , p = . , p = . , p = . and p = . , respectively). the prevalence of cardiac cachexia was common in advanced cdmd dogs, and the parameters of cardiac cachexia, namely weight loss, anemia, hypoalbuminemia and azotemia were strong prognostic factors associated with survival. no conflicts of interest reported. mitral valve disease (mvd) is the most common cardiovascular disease in dogs. it's characterized by myxomatous degeneration, which causes mitral valve prolapse (mvp), mitral regurgitation (mr) and a left apical systolic murmur (lasm). mvd affects small breed dogs with a very high prevalence in cavalier king charles spaniels (ckcs). the main goal of this study was to determine the prevalence of lasm, mvp and mr in the maltese, the most presented breed among dogs with mvd in taiwan. the correlation between these measurements and the influence of age, gender, reproductive state, and body weight were also investigated. study results were compared to other mvd prevalence studies in europe and north-america. client-owned maltese dogs ( males and females; body weight . - . kg; age - yrs) with no signs of heart failure were recruited. the intensity (grade - ) of lasm was recorded. grade of mvp (mild/severe) and mr severity (mild/moderate/ severe) were evaluated by echocardiography. logistic regression was used to determine the correlation between age and presence of lasm, mvp and mr. a chi-square test was used to evaluate whether sex and reproductive-status were related to prevalences of lasm, mvp and mr. spearman's correlation coefficient was used to assess the relationships between age, body weight, lasm intensity, grade of mvp and severity of mr. the prevalence of lasm, mvp and mr were . %, % and . %, respectively. all have positive correlation with age (p = . ). the age at which % of the dogs had lasm, mvp and mr was . , . , and . years, respectively. the lasm intensity, mvp grade and mr severity were all positively correlated to age (all p = . ) and had no correlation with bw and reproductive status. females had a significantly higher prevalence of lasm than males ( % vs. . %, p = . ). maltese dogs in taiwan have a very high prevalence and an early development of mvd as compared to other small breed dogs, similar to mvd in ckcs in other countries. since we only recruited asymptomatic dogs, this study may underestimate the prevalence of mvd in the whole maltese population. to our knowledge, this is the first report to document the high prevalence of mvd in taiwanese maltese.the maltese may be a new canine model for genetic, pathology, and natural history studies in mvd. boehringer-ingelheim sponsored the author's accommodation costs for this congress. esvc-o- cardiorenal syndrome in dogs with chronic valvular heart disease: a retrospective study. e. martinelli , p. scarpa , c. quintavalla , c. locatelli , p. brambilla . university of parma, parma, italy, university of milan, milan, italy in human medicine, primary disorders of the heart often result in secondary dysfunction or injury to the kidneys. the coexistence of the two problems in the same patient is referred as cardiorenal syndrome (crs). just little information about crs is available in veterinary medicine. the aim of this study was to define the prevalence of chronic kidney disease (ckd) complicating chronic valvular heart disease (cvhd) in dogs and to investigate the relationship between class of cardiac insufficiency (acvim) and class of renal insufficiency (iris). medical records of dogs presented at the cardiology service of the department of veterinary science and public health, university of milan, between january and december were retrospectively evaluated. dogs with a complete physical examination, thoracic radiographs, a cvhd diagnosis based on echocardiographic examination, and a serum biochemical panel, including assessment of serum creatinine (scr) and serum urea (bun), were included in the study. dogs with other heart disease, neoplasm or systemic diseases were not included in the study. one hundred eighteen dogs of both genders ( males and females), to years of age ( . ae . years), to kg of bodyweight ( . ae . kg) fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( . %), york shire terrier ( . %), shih -tzu ( . %), pinscher ( . %) and dachshund ( . %). dogs were classified as follow: % acvim a, % acvim b , % acvim b , % acvim c and % acvim d. while the % of the dogs were normoazotemic (scr < , mg/dl), % were staged in iris , % in iris and %in iris . statistical analysis was performed using jmp . (sas institute inc.). a p value < , was considered significant. the prevalence of ckd associated with azotemia in dogs affected by cvhd was %. there was a statistically significant direct correlation between acvim and iris class (pearson test p = . ). unexpectedly, the % of dogs receiving drugs for medical management of heart failure (acvim class c and d) were normoazotemic. despite a definite conclusion about the role of cvhd on the induction and/or progression of ckd cannot be drawn from this cross-sectional study, these results suggest that there is a direct correlation between the severity of ckd and cvhd. no conflicts of interest reported. there is growing evidence of breed differences in concentrations of several blood variables in dogs. the aim of the study was to investigate breed differences in plasma concentrations of components of the renin-angiotensin-aldosterone system (raas), endothelin- (et- ) and serum cortisol concentration in healthy dogs. healthy, privately-owned dogs of nine breeds were examined at five centers as part of the european lupa-project. absence of cardiovascular or other clinically relevant organrelated or systemic disease was ensured by thorough clinical investigations. plasma concentrations of et- and aldosterone, renin activity, and serum concentration of cortisol were measured by ria or elisa assays. overall significant breed differences were found (p < . for all variables). bonferroni-corrected pair-wise significant differences between breeds were found in % of comparisons for et- , % for cortisol, % for renin and % for aldosterone. for et- , the highest median concentration was found in newfoundlands with values > times higher than most other breeds, while renin was highest in dachshunds, > times higher than in newfoundlands and boxers, which had the lowest concentrations. aldosterone was especially low in belgian shepherds with median concentration < times than the other breeds. cortisol was highest in finnish lapphunds, almost times higher than boxers with the lowest concentration. in conclusion, considerable inter-breed variation in concentrations of et- , components of raas and cortisol was found in healthy dogs. these differences are likely influenced by genetic factors and should be taken into account when designing clinical trials and tests. breed-specific reference ranges might be necessary. no conflicts of interest reported. most studies that assess weight management in obese dogs only examine the early stages of weight loss, and this may not properly reflect a complete weight management regime. the aim of the current study was to examine the kinetics of a complete weight management cycle in obese client-owned dogs. dogs referred to the royal canin weight management clinic, university of liverpool, for the management of obesity, were eligible for inclusion. all dogs were followed until they had either completed (i.e. reached target weight) or the programme was discontinued. rate of weight loss, percentage weight lost, and energy were assessed at different time points. a total of dogs were included, with a range of breeds, ages and sexes represented. rate of weight loss steadily decreased throughout the weight loss period (d : . ae . %/wk; d : . ae . %/wk; d : . ae . %/wk; d : . ae . %/wk; d . ae . %/wk; d : . ae . %/wk; p < . ). the energy intake required to maintain weight loss also progressively decreased (p < . ). by day , mean aesd weight loss was ae . %, and compliance was good, but most had not com-pleted ( % completed, % ongoing, % discontinued). thereafter, more dogs completed, but the number of discontinuing also increased (d : ae . % weight loss, % completed, % ongoing, % discontinued; d : ae . % weight loss; % completed, % ongoing, % stopped). initial weight loss is good in obese dogs but, thereafter, steadily worsens. thus, studies examining only the first few months of weight loss are not fully representative of the entire weight loss process. conflicts of interest: the following conflicts of interest apply: the diet used in this study is manufactured by royal canin.whilst vb is employed by royal canin. vb and ss are employed by royal canin. ajg's readership is funded by royal canin. obesity and obesity-related metabolic dysfunctions are increasing in humans as well as in dogs. obese dogs become affected by chronic diseases at young age, have a decreased quality of life and a shorter life-span. the aim of the study was to describe the metabolic and hormonal response to a feed-challenge test in lean and overweight dogs. twenty-eight healthy intact male labrador retrievers aged . ae . years with varying body condition score (bcs, scale - ) were included. twelve dogs were classified as lean (bcs - ), ten as slightly overweight (bcs ) and six as overweight (bcs . - ). an overnight fasting period and blood sample collection was followed by a high fat meal. after food intake, blood samples were collected hourly for four hours. a glucagon elisa was validated for use in dogs. the assigned bcs was supported by positive association with serum leptin concentrations. postprandial triglyceride concentration was significantly higher in the overweight group. a tendency to higher cholesterol concentration was seen in the overweight group but cholesterol was not affected by food intake. glucagon concentration rose after food intake and resembled the response seen in humans after a mixed meal. glucose and insulin concentrations followed the same pattern while free fatty acids had declined one hour after the meal. in this study, the metabolic and hormonal response to a high fat meal was similar between lean and slightly overweight dogs, whereas the response of overweight dogs differed. studies on the health significance of postprandial hypertriglyceridemia in dogs are warranted. conflicts of interest: the study was financially supported by the swedish veterinarian federation, the companion animal research foundation, and the foundation of thure f. & karin forsberg. feline weight-loss programs are often hindered by compliance issues and sedentary lifestyle. the purpose of this study was to assess the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned cats. the objectives were ) to evaluate weight loss parameters in cats fed the ndwmf* and ) to describe the owner's perception of the cat's quality of life. overweight/obese, otherwise healthy, client-owned cats (> / body condition score -bcs) were enrolled in the study (n = ). initial veterinary evaluation comprised a physical examination, nutritional assess-ment, determination of ideal body weight (ibw), and development of weight loss feeding plan. daily energy requirement (der) for weight loss was calculated as der = . x ( x ibw kg . ). initial and follow-up evaluations (monthly for months) consisted in determination of body weight (bw), bcs, body fat index (bfi), muscle condition score (mcs), and current feeding practices. quality of life assessment by owners included cat's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis encompassed scatterplots, regression analysis, summary statistics as appropriate for the type of analyses (continuous or categorical variables, distribution), a mixed model anova was used to assess changes over time (statistical significance at p < . ). eighty three percent of the cats (n = ) lost weight with an average weight loss of % (sem, . %) over months and an average weekly weight loss rate of . % (sem, . %). a significant decrease in bcs from week - and in bfi from week - compared to baseline was observed. mcs did not change. average duration of weight loss was days (sem, . days) with days (sem, . days) between visits. fourteen percent of cats achieved ibw ( . , ci: . - . ). seventy nine percent of cats ate more than the recommended der (median fed above der= %), and the majority of these cats still lost weight. owners perceived a significant increase in energy and happiness (>week ) compared to baseline in the cats that lost weight without changes in appetite or begging behavior. no significant changes were seen in scores for flatulence, stool volume, and fecal score. in conclusion, this clinical study showed that feeding the ndwmf* to client-owned, overweight/obese cats resulted in weight loss. owners reported significant improvements in cat's quality of life without negative side effects. * porphyrias are a group of inborn errors of metabolism resulting from accumulation of porphyrins due to deficient activities of specific enzymes in heme biosynthesis. in humans, they are clinically classified as either erythroid with cutaneous involvement or hepatic with acute neurovisceral attacks. here we describe the clinical, biochemical, and molecular genetic studies in porphyric cats from new brunswick, canada. from to , three separately identified adult domestic shorthair cats from the city of saint john in new brunswick were found to have erythrodontia (brown discolored teeth which fluoresced pink) and pigmenturia. a mild compensated hemolytic disorder with numerous small dark blue irregularly shaped erythrocyte inclusions was noted. there was no evidence of acute lifethreatening neurovisceral attacks or cutaneous lesions. necropsy of one cat revealed massive deposition of porphyrins in all bones and teeth. urine and edta blood samples from one cat were metabolically studied, while molecular genetic studies were performed in all cats either from edta blood or a formalinized splenic tissue block. urinary d-aminolevulinic acid, porphobilinogen, uroporphyrin i, and coproporphyrin i concentrations were increased in the cat studied, suggesting an acute intermittent porphyria (aip). the erythrocytic hydroxymethylbilane synthase (hmbs) activity in erythrocytes was approximately half normal suggesting a dominant enzymopathy, while the erythrocyte uroporphyrinogen iiisynthase activity was normal. sequencing the feline hmbs gene revealed a heterozygous intronic base deletion (c. - _- del) which results in an insertion in the mrna and would predict a truncated protein. in conclusion, these three domestic shorthair cats had the same hmbs mutation causing an autosomal dominantly inherited aip. cats with discolored teeth and normal or mild hemolysis may have either acute intermittent porphyria or congenital erythroid porphyria. interestingly, seven disease-causing mutations have now been found by us in the hmbs gene -more than in any other gene in cats. the biochemical and molecular characterization facilitates clinical screening of affected cats to reach a specific diagnosis. supported in part by nih od . urs giger and raj karthik are also part of the laboratory that offers dna testing for this mutation. fibrinogen decreases when coagulation is activated to form fibrin, while fdps and d-dimers represent the products of fibrinolysis. in humans, activation of coagulation and fibrinolysis develops in all type of ascites and it is also associated with signs of systemic fibrinolysis.these results have lead to the suggestion that ascitic fluid is inherently fibrinolytic. preliminary studies showed similar results also in dogs (javma nov. , ecvim proceedings . in addition, in an old experimental study conducted in dogs, inoculation of blood or of a solution containing fibrinogen and thrombin into the pleural cavity resulted in the activation of the coagulation system followed by fibrinolysis. therefore, the objective of the present study was to determine whether the activation of coagulation and fibrinolysis (i.e. low fibrinogen and elevated fdps and ddimer) occurs not only in the ascitic fluid, as alredy been demonstrated, but also in all type of pleural effusions in dogs. thirty-three dogs referred to the san marco veterinary clinic with pleural effusion, but without ascites, were studied. fibrinogen, fdps, and d-dimer concentrations were measured and then compared in both pleural fluid and venous blood via wilcoxon signed ranks test. the dog's pleural effusions were categorized based on pathophysiology of fluid formation into dogs with transudate ( due to increased hydrostatic pressure and due to decreased osmotic pressure), with an exudate (of which due to septic causes), with a haemorrhagic pleural effusion, and with a chylous effusions. the fibrinogen concentration in the pleural effusion (median: mg/dl; range: - ) was significantly lower (p < . ) than the plasma fibrinogen concentration (median: mg/dl; range: - ). in all dogs, the fibrinogen pleural fluid concentration was lower than the plasma concentration. the fdp concentration in the pleural effusion (median: mg/dl; range: . - ) was significantly (p < . ) higher than plasma fdps concentrations (median: . mg/dl; range: . - . ). in case, the fdps pleural fluid concentration was lower than the plasma concentration and in cases the pleural fluid concentration was higher. the d-dimer concentrations were significantly(p < . ) higher in the pleural effusion (median: . lg/ml; range: . - . ) than in the plasma (median: . lg/ml; range: . - . ). in one case, the d-dimer pleural fluid concentration was lower than the plasma concentration and in cases was higher. these findings support the hypothesis that activation of coagulation followed by fibrinolysis occurs in all type of pleural effusions. no conflicts of interest reported. during primary hyperfibrinogenolysis (phf), fdps production is increased but production of d-dimer is not. therefore, elevated fdps and normal d-dimer are considered an indicator of phf. in humans and dogs, activation of coagulation and fibrinolysis develops in all type of ascites and it is associated with systemic phf, suggesting that ascitis is inherently fibrinolytic. preliminary data have shown that activation of coagulation followed by fibrinolysis occurs also in all type of pleural effusions (pe). the objective of this study was to determine if systemic phf occurs also in dogs with pe. thirty-three dogs referred to the san marco veterinary clinic with pe, but without ascites, were studied (group ). from the electronic data-base of the clinic dogs for inclusion in control groups (healthy dogs) and (sick dogs without pe or ascites) were randomly selected and individually matched to group dogs for age, sex, and breed. fibrinogen, fdps, d-dimers, c-reactive protein (crp), fibrinogen/crp ratio, and prevalence of phf (i.e., dogs with elevated plasma fdps and normal d-dimer) were determined. differences between the groups were analyzed using anova (fibrinogen), chi-square (fdps and prevalence of phf) and kruskal-wallis test (crp, fibrinogen/crp ratio, and d-dimer). post-test analysis were performed by tamhane and mann-whitney test. fibrinogen concentration in group was significantly increased compared to group (p < . ), but not compared to group (p = . ). fdps concentration in group was significantly increased compared to groups (p < . ), but not compared to group (p = . ). d-dimers concentration in group was significantly increased compared to group (p < . ), but not compared to group (p = . ). crp was significantly increased in group compared to group and (p < . for both comparison). fibrinogen/crp ratio was significantly decreased in group compared to group and (p < . for both comparison). prevalence of phf was significantly higher in group compared to groups (p = . ), but not compared to group (p = . ). these results support the hypothesis that phf occurs significantly more often in dogs with pe compared to healthy dogs. despite there was a trend of increased phf also in dogs with pe compared to sick dogs, this difference did not reach significance. nevertheless, the decreased in fibrinogen/crp ratio in group compared to group , in the face of a similar d-dimer concentration, would suggest that phf is also more prevalent in dogs with pe compared to sick control dogs. no conflicts of interest reported. the systemic inflammatory response syndrome (sirs) refers to clinical signs of systemic inflammation in response to (non-) infectious insults. current diagnosis of sirs is based on clinical and basic laboratory data and is a sensitive screening to identify patients at risk. c-reactive protein (crp) is a major canine acute phase protein with concentrations related to disease severity and underlying cause. crp rises in response to proinflammatory cytokines, mainly interleukin (il)- and tumor necrosis factor (tnf)-a, which are considered the main triggers of sirs. we therefore evaluated crp, il- and tnf-a kinetics in canine emergency sirs patients hypothesizing that crp is ( ) increased in dogs with a clinical sirs-diagnosis, ( ) correlated with il- and tnf-a concentrations, ( ) influenced by the underlying etiology, and ( ) a prognostic marker. canine emergencies with clinically diagnosed sirs were prospectively included. serum and plasma were immediately stored at - °c after sampling at presentation, after (t ), (t ), (t ) and (t ) hours, and at a control visit (t m) over one month after discharge. serum crp was measured with a caninespecific immunoturbidimetric crp assay. plasma il- and tnfa were measured using a bioassay measuring biologically active cytokine concentrations. disease categories were infection (i), neoplasia (n), trauma (t), gastric-dilation and volvulus (gdv), other gastrointestinal (gi), renal (r) and miscellaneous (m) diseases. statistical analysis was performed with sas. concentrations of inflammatory cytokines were expressed logarithmically, with univariate analysis confirming normal distribution. a correlation procedure, mixed procedure on a linear model and a logistic procedure were performed (p-value < . ). sixty seven dogs (i = , n = , t = , gdv= , gi= , r = , m = ) were included. forty-three patients survived (seven died, seventeen were euthanized). twenty patients had a control visit. crp was elevated in . % of dogs at presentation, and only remained within reference range ( - . mg/l) throughout hospitalization in four dogs ( . %). crp concentrations were significantly higher from t ( . ae . mg/l) to t ( . ae . mg/l) decreasing at t ( . ae . mg/l), and returning within reference range at t m ( . ae . mg/l) in all but one dog ( . mg/l). crp was significantly correlated with logarithmical concentrations of il- and tnf-a, however, these did not change significantly over time. none of the evaluated parameters was associated with disease category, nor outcome. crp appears useful to diagnose sirs in emergency patients, and tends to decrease during hospitalization. however, crp, neither il- nor tnf-a concentrations appear useful to predict the underlying disease and outcome in sirs patients. no conflicts of interest reported. calprotectin (s a /a complex) belongs to the s /calgranulin family, and is primarily released from activated neutrophils and macrophages. serum calprotectin concentrations (cp) were shown to be increased in dogs with inflammatory diseases such as inflammatory bowel disease, pancreatitis, systemic inflammatory response syndrome, and sepsis. canine cp thus appears to be a biomarker of inflammation. considerable day-today variation of fecal canine cp was found in both healthy dogs and dogs with chronic gastrointestinal disease. however, the biological variation of canine cp in serum has not been reported. the aim of this study was to determine the biological variation of serum canine cp and its minimum critical difference (mcd). eleven healthy dogs were used for this study. biological variation of serum canine cp was evaluated over a . -months period. tests for outliers were carried out at levels (within-run analytical variance, intra-, and inter-individual variation). a nested analysis of variance (anova) model was used to calculate analytical (cv a ), intra-individual (cv i ), inter-individual (cv g ), and total variation (cv t ), and to determine the index of individuality (ii), index of heterogeneity (ih), and mcd. a total of serial specimens were collected from dogs, serial samples from dogs, and serial samples from dogs. four within-subject outliers were detected and excluded from further analysis, yielding a total of serum samples and slightly right-skewed data. no outlying observations (cochrane test) or outliers among mean concentrations of subjects (reed's criterion) were detected. cv a was calculated as . %, cv i as . %, and cv g as . %, resulting in a cv t of . %. index of individuality (ii) was determined to be . and ih was . , yielding a one-sided mcd of . mg/l. the analytical goal of cv a ≤ ½ cv i was satisfied. although serum canine cp remained within a relatively narrow concentration range in healthy dogs, moderate individuality was detected. moderate changes in serum canine cp ( . mg/l) between sequential measurements are needed to be considered clinically relevant, and using a population-based reference interval may or may not be appropriate for serum canine cp. using the mcd with the previously determined median canine cp concentration ( . mg/l) for the reference sample group yielded a serum canine cp concentration close to the upper limit of the previously established reference interval ( . mg/l), showing that the reference interval for serum ccp ( . - . mg/l) is within reasonable limits. the assay used in the study was developed at the gi laboratory, texas a&m university. most authors also work at the gi laboratory, texa a&m university. canine leishmaniasis (canl) is a multisystemic disease that is endemic in the mediterranean region. in the past, concentrations of acute phase proteins (apps), and specifically c-reactive protein (crp), haptoglobin (hp), ceruloplasmin (cp), serum amyloid a (saa) and albumin (alb), have been reported to change in dogs with leishmaniasis, and revert to normal after successful treatment, highlighting the intrinsic inflammatory reaction of the host to the parasite. since the spectrum of clinical and laboratory derangements is broad, it is possible that apps are increased specifically because of certain clinicopathological syndromes associated with canl. a total of dogs with canl, diagnosed on the basis of cytological amastigote identification and ifat serology, were retrospectively included in the study. in all of them, crp, saa, hp and alb were measured at interlab-umu, murcia, spain, in aliquots of serum, which were stored in - °c for - years (median: years). results for each of the apps were correlated to laboratory and clinical parameters (n: ), clinical and parasitological scoring (n: ), ehrlichia and leishmania serology (n: ), and clinical staging according to leishvet (n: ), using an array of linear and ordinal regression models, as well as one-way anova, t-test and fisher's lsd test. crp and alb were by far the apps most frequently correlated with clinical and laboratory abnormalities such as nutritional status, lethargy and skin ulcers (p < , ), as well as urinary protein to creatinine ratio (upc), total serum protein, and urine specific gravity (p < , ). there were limited associations between hp, cp, saa and clinicopathological parameters. a minor linear relationship was observed between crp and clinical scoring. crp and alb were also correlated with parasitological scoring in bone marrow, but not lymph node cytology (p < . ). dogs with ehrlichia titers had higher crp, cp and lower alb concentrations. finally, crp concentrations were higher in later compared to earlier stages of the infection, as defined by the leishvet criteria. the inflammatory component to leishmania infection doesn't seem to be exemplified by the reaction of a particular tissue, with the possible exception of glomerulonephritis. the magnitude of increase in crp and decrease in albumin is correlated with clinical staging and bone marrow parasitological scoring. no conflicts of interest reported. the consequences of abnormal platelet function in dogs and cats can be devastating and the use of anti-thrombotic therapy to prevent thrombotic events is increasingly common. the ability to measure platelet function and the efficacy of anti-thrombotic therapy is difficult due to limited availability of equipment and inability to delay platelet function analysis. the aim of this study was to adapt and validate test procedures and protocols previously developed for humans for use in dogs and cats. residual samples of citrate anticoagulated blood were used from dogs and cats presented to a specialist referral centre for various reasons unrelated to clotting abnormalities. initially the blood was stimulated using specific combinations of either arachidonic acid/epinephrine (aa/epi) or adp/u , designed to assess the effects of the anti-thrombotic agents aspirin and clopidogrel respectively. after minutes stimulation, the blood samples were fixed using a patented platelet fixative solution developed for human platelets, which allows the delayed analysis of p-selectin an established marker of platelet activation. all analysis was performed by flow cytometry. in order to do this, specific antibodies were selected for the recognition of both canine and feline platelets. cd was used as a platelet identifier antibody while appropriate cd p (p-selectin) antibodies for each species were chosen. fixed samples were repeatedly analysed at time points between to days following fixation to establish the stability of the fixed samples. thirteen dogs and three cats were analysed. high p-selectin expression was detected following stimulation with aa/epi and adp/u in both dogs and cats following fixation. this was significantly different to unstimulated blood (p < . ). there was no significant difference in detectable pselectin expression following storage of the fixed samples at any time-point up to days. this confirmed the fixative was suitable as a preservative of canine and feline platelets. a limited number of dogs were evaluated whilst receiving antithrombotic medication. there was a significant difference in the activation of platelets in the dogs treated with either aspirin (p < . ) or clopidogrel (p < . ) compared with untreated dogs following stimulation with aa/epi (aspirin group) or adp/ u (clopidogrel group). our results show that fixation and delayed analysis of platelet function in dogs and cats is possible for up to days. this demonstrates an exciting opportunity to analyse platelet function remotely and to determine the efficacy of thromboprophylaxis in animals presenting to clinics that do not have on-site platelet analysers. no conflicts of interest reported. several authors consider thyroid hormone supplementation as a valid initial treatment option for dogs with aggression related problems. indeed, mood and behaviour modulating properties of thyroid hormones may, in part, be mediated through the interaction of thyroid hormones with neurohormones such as serotonin and prolactin. at present, prospective trials evaluating neurohormonal status or behaviour in hypothyroid dogs before and after thyroid supplementation are lacking. therefore, the aims of this study were to assess behaviour and measure serum serotonin and prolactin concentrations in dogs with spontaneous hypothyroidism before and after treatment.twenty three client-owned dogs diagnosed with spontaneous primary hypothyroidism were prospectively included in our study. after diagnosis all dogs were treated with levothyroxine ( micrograms/kg bid). behaviour of dogs was screened at initial presentation, at weeks and months after initiation of therapy. owners had to fill in a hard copy of the standardized canine behavioural assessment and research questionnaire (c-barq) consisting of scored questions evaluating seven behavioural categories. the average score on all questions was calculated for each dog at each of the three time periods and a paired t-test was used for comparison. serum serotonin and prolactin concentrations were evaluated at each time period using a commercially validated elisa kit and heterologous ria, respectively.results of the c-barq after six weeks of thyroid hormone supplementation when compared with the time zero demonstrated a significant increase (p < . ) in excitability, activity and aggression, which most likely became unmasked owing to improved overall activity of dogs. conversely, at six months period when compared with the time zero no significant changes in any of the behavioural symptoms were observed. serum serotonin was measured in / dogs colorimetrically at nm. at time zero, weeks and months serum serotonine was . (range, . - . ), . (range, , - . ) and . (range, . - . ). no significant difference was noted between week and month period comparing to time zero (p = . and p = . ). serum prolactin concentration measured in / dogs at time zero, weeks and months was . ng/ml (range, . - . ), . ng/ml (range, . - . ) and . ng/ml (range, . - . ) and did not differ significantly in either time period when compared with time zero (p = . and p = . ).altogether, results of this study failed to demonstrate a significant role of thyroid supplementation on the majority of evaluated behavioural symptoms as well as neurohormonal status of hypothyroid dogs during months of therapy. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment of hyperthyroidism in cats, but no prospective studies of the prevalence, clinical features, routine laboratory findings, or results of thyroid function tests have been reported in a series of hypothyroid cats. in this study, we describe the features of hypothyroidism in cats treated with radioiodine over a -month period (october -march ). during this same period, we treated % hyperthyroid cats with radioiodine, providing a prevalence rate of %. hypothyroidism was diagnosed - days (median, days) after i treatment, with doses ranging from - mbq (median, mbq; median pretreatment t , nmol/l). the hypothyroid cats ranged in age from - years (median, years). all were dsh/dlh; ( %) were female and were males (p = . ). clinical signs in these cats included overweight/obesity in ( %), lethargy/dullness in ( %), poor appetite in ( . %), and polyuria/polydipsia in ( %). abnormalities on physical examination included dermatologic signs (dry coat, seborrhea, matting) in ( %) and bradycardia (< bpm) in . twenty-two cats ( %) had no noticeable clinical features of hypothyroidism. routine laboratory abnormalities included hypercholesterolemia (> mmol/l) in ( %) and new or worsening azotemia (> lmol/l) in ( %) and ( %) cats, respectively. median serum concentrations of total t ( . nmol/l; reference interval [ri], - nmol/l), t ( . nmol/l; ri, . - . nmol/l), and ft ( pmol/l; ri, - pmol/l) were all in the low end of the ri. normal ri values for t and ft were maintained in ( %) and ( %) of the cats, respectively. serum ctsh values were high in all cats (median, . ng/dl; range, . - . ng/dl; ri, . - . ng/ml). thyroid scintigraphy showed less-than-normal amounts of residual tissue, as well as low values for thyroid-to-salivary ratio and %-uptake of pertechnetate, in ( %). of those cats with normal scintiscans, serum ctsh decreased into the ri without treatment when retested - months later. in conclusion, this study confirms that i-induced hypothyroidism is not uncommon, with an apparent female sex predilection. serum t and ft remain normal in most cats, but high serum ctsh values and thyroid scintigraphy aid in diagnosis. unless cats have overt, long-standing hypothyroidism, most cats with subclinical disease are relatively asymptomatic, other than worsening azotemia. subclinical hypothyroidism will be transient in some cats, with normalization of ctsh values within a few months. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment for hyperthyroidism in cats. at our clinic where we use a variable -i dosing protocol (based on tumor volume and severity of hyperthyroidism), the prevalence of overt or subclinical hypothyroidism is at least %. during the -month period from october to march , we treated cats with iatrogenic hypothyroidism, which had developed - days (median, days) after treatment with radioiodine (median dose, mbq). these cats ranged in age from - years (median, years); all were dsh/dlh; ( %) were female and were males. new or worsening azotemia (> lmol/l) was documented in ( %) and ( . %) cats, respectively. diagnosis of hypothyroidism was based on the following: ) low to low-normal serum concentrations of t , ft , and t ; ) high serum tsh concentration (> . ng/dl); and ) less-than-normal amounts of residual tissue on thyroid scintigraphy. all cats were given thyroid hormone replacement as a liquid l-t preparation (leventa; merck animal health). cats were monitored at - month intervals by repeating serum t and tsh concentrations - hours after the morning l-t dose. ten of the cats were started on a once-daily l-t regimen ( lg); of these, only ( %) had suppression of high serum tsh values into the reference interval (ri). of the cats that had persistently high tsh values, were switched to twice-daily administration ( - lg, bid), which successfully lowered high tsh concentrations in cats. the remaining cats were started on twice daily l-t ( lg, bid); of these, normalization of tsh occurred in cats. overall, l-t treatment was successful in normalizing tsh concentrations in ( %) cats, with once-daily and with twice-daily dosing. peak serum t concentrations of ≥ nmol/l were needed in most cats to normalize tsh values. higher serum t and lower tsh concentrations were achieved when l-t was administered on an empty stomach rather than given with food. a significant decrease (p < . ) in serum creatinine occurred after treatment with l-t . in conclusion, our results indicate that twice-daily administration of l-t is needed in most cats with iatrogenic hypothyroidism to normalize high serum tsh concentrations. many cats appear to absorb l-t rather poorly, which can be enhanced by giving the drug on an empty stomach. the azotemia that commonly develops in cats with hypothyroidism improved or stabilized with adequate l-t supplementation. no conflicts of interest reported. congenital hypothyroidism (ch) has been reported in many species; the hereditary forms can be divided into thyroid dysmorphogenesis and dyshormonogenesis. while thyroid hypoplasia has been described in dogs and cats, the molecular basis remains unknown. in contrast few breeds of dogs with goiterous ch were found to have deficient thyroid peroxidase (tpo) activity. the purpose of our study was to characterize a family of domestic shorthair cats with goiterous ch and disease-causing tpo gene mutations. clinical features included dwarfism and dullness, known as cretinism and seen with ch in all species, but also constipation and megacolon which are unique to cats with ch. pedigree analysis documented an autosomal recessive mode of inheritance. affected kittens developed a goiter and had low serum thyroxine (t ) and triiodothyronine (t ) when compared to controls, but high thyroid stimulating (tsh) hormone levels indicating thyroid dyshormonogenesis. oral thyroid supplementation corrected the progression of clinical signs and prevented further constipation and reversed the megacolon. the tpo enzyme activity was extremely low in hypothyroid cats when compared to that of normal cats. genomic dna and cdna from affected, carrier, and normal cats were extracted and sequenced based upon primers developed from the feline genome database. a homozygous missense point mutation (c. g>a) in tpo, which results in an amino acid change (p.ala thr), was discovered in affected cats and the mutant allele segregated within the family with goiterous ch. this is the first report of a tpo deficiency in cats. other unrelated domestic shorthair cats with goiterous ch did not have this same tpo mutation. the prevalence of this tpo mutation in the domestic cat population seems low, but ch is likely underreported in cats. supported in part by nih od . some of the authors are members of diagnostic laboratories (penngen). supported in part by the nih od # . glucagon-like peptide- (glp- )is a gastrointestinal hormone released in response to food intake that increases insulin secretion, inhibits glucagon secretion, slows gastric emptying and induces satiation. it is also assumed to stimulate beta-cell proliferation. glp- agonists are successfully used in humans with type diabetes mellitus usually either in combination with insulin or other anti-diabetic drugs. in healthy cats twice daily (exenatide) as well as once weekly (exenatide extended-release (er)) application of glp- agonists induced pronounced insulin secretion. benefits of exenatide er are the regimen of once weekly injection and less side effects. the objective of the study was to assess whether administration of exenatide er in addition to standard treatment leads to improved glycemic control and higher remission rates in cats with newly diagnosed diabetes. the study was designed as a prospective, placebo-controlled clinical trial. cats were randomly assigned to two groups receiving exenatide er (group : bydureon â , mg/kg, q d, sc) or . % saline solution (group : q d, sc). both groups additionally received insulin glargine (lantus â , initial dose: ≤ kg: . iu, q h; > kg . - . iu, q h) and diet (purina dm â ). exenatide er was applied over weeks or, in case of remission, for additional weeks after cessation of insulin application. cats were rechecked , , , and weeks after starting therapy. remission of diabetes was defined as absence of clinical signs of diabetes and normal blood glucose and fructosamine concentrations for at least weeks after discontinuing insulin injections. so far cats have completed the study. mild and transient side effects in group (n = ) were reduced appetite (n = ), nausea (n = ), vomitus (n = ), tiredness (n = ) and hiding in dark spots of the house (n = ). in group remission was achieved in / ( %) cats and good metabolic control in / ( %) nonremission cats. in group remission was achieved in / ( %) cats and good metabolic control in / ( %) non-remission cats. median insulin dose given during the study period was . iu/ kg/day in group and . iu/kg/day in group . the preliminary results suggest that exenatide er can be used safely in diabetic cats. a tendency for higher remission rate, better metabolic control and lower insulin requirement was seen when exenatide er was added to the standard treatment regimen. further cases need to be evaluated to verify the potential beneficial role of exenatide er. no conflicts of interest reported. feline diabetes mellitus shares many similarities with human type diabetes mellitus (t dm), including clinical, physiological and pathological features of the disease. domestic cats spontaneously develop diabetes associated with insulin resistance in their middle age or later, with residual but declining insulin secretion. humans and cats share the same environment and risk factors for diabetes, such as obesity and physical inactivity. moreover, amyloid formation and loss of beta cells are found in the diabetic cat pancreas, as in humans. subsequently, studying the molecular mechanisms in the failing beta cells may contribute to a better understanding of the pathophysiology of t dm in both cats and humans. the aim of the present study was to develop a method to study mrna expression of islet-specific genes in healthy and diabetic cats. previous attempts in isolating feline islets with different collagenase-based protocols have led to damaged islets or islets coated with exocrine acinar cells, which either way compromise the results obtained from gene expression studies. by using the laser microdissection technique, we were able to sample islets that were not contaminated with exocrine tissue, from both healthy and diabetic cats. high rna quality was confirmed with gel electrophoresis. by quantitative real-time pcr (qrt-pcr), mrna levels of the islet-specific genes insulin, pdx- , iapp, chga and ia- were detected in both healthy and diabetic cats. we used actin b, gapdh and rps as internal reference genes for normalizations of our qrt-pcr data. the laser microdissection technique allows studies of islets without contamination of acinar cells, as shown in this study, and is of great advantage since it is difficult to get pure feline islets from collagenase-based isolation. differences in gene expression in healthy and diabetic cats may reveal underlying mechanisms for beta cell dysfunction and decreased beta cell mass in human and feline type diabetes. conflicts of interest: the study was financially supported by the swedish juvenile diabetes foundation, the fredrik and ingrid thuring foundation, the magnus bergvall foundation, the lars hierta memorial foundation, and the foundation for research, agria insurance company. feline acromegaly is an increasingly recognised endocrinopathy among diabetic cats, caused by chronic excessive growth hormone secretion by a functional somatotrophinoma in the pars distalis of the anterior pituitary gland. the majority of human somatotrophinomas are sporadic, however up to % of familial isolated pituitary adenomas are caused by germline mutations of the aryl-hydrocarbon-receptor interacting protein (aip). feline acromegaly has phenotypic and biochemical similarities to human familial acromegaly with aip mutations, such as male predominance, somatotroph macroadenoma and resistance to octreotide therapy. the objective of this study was to identify the feline aip gene, identify single nucleotide polymorphisms (snps) within this gene and compare any snps with reported human aip snps. stored pituitary tissue from an acromegalic cat was used to create feline aip cdna using feline specific aip primers. stored edta blood from acromegalic cats (diagnosis of insulin resistant diabetes mellitus, serum igf- > ng/ml and pituitary mass > mm identified using pituitary computed tomography or necropsy) and control cats (no history of diabetes mellitus and greater than years of age) were selected, dna extracted and genotyped using pcr, agarose gel electrophoresis and sanger sequencing. the feline aip gene was identified, encoding a amino acid protein with % homology to the human aip protein. a blast search revealed this gene contained exons and exon specific primers were created to enable sequencing. a single nonconservative snp was identified in exon (aip:c. g>t), encoding for an amino acid change from aspartic acid to glutamic acid in / acromegalic patients and / control cats. two additional conservative snps were also identified (aip:c. t>c and aip:c. t>c). exon encodes for a region of the aip protein considered essential for aip-aip receptor interaction. although different human aip mutations have been identified to date, a human aip:c g>t mutation has not yet been identified. the aip n-terminal is required for the stability of the aip protein-aip-receptor complex, and essential for the regulation of translocation into the nucleus, where it binds to aryl hydrocarbon receptor nuclear translocator leading to activation of genes thought to act as tumor suppressors. loss of normal aip activity is thought to promote somatotrophinoma development. it is therefore possible that the detected aip:c. g>t mutation predisposed to somatotrophinoma tumorigenesis in the two affected patients, and a study containing a larger number of cases is indicated. no conflicts of interest reported. hypersomatotrophism (hs) is an important cause of feline diabetes mellitus (dm). in humans surgical removal of the somatotrophinoma is generally recommended, though hypophysectomy programs have suffered from significant initial morbidity and mortality given a documented steep learning curve in newly established programs. hypophysectomy as treatment for feline hs has thus far only been described in a handful of cases, all having been treated by one single experienced hypophysectomy team. this study's aim was to evaluate the learning curve of a de novo established hypophysectomy program, through analysis of peri-and post-operative morbidity and mortality, and endocrine outcomes in the first cohort of cats with hs treated. from owners of diabetic cats with confirmed hs (igf- > ng/ml, pituitary mass) presented at the royal veterinary college were offered hypophysectomy. all cats undergoing surgery were operated by one neurosurgeon with previously only cadaveric experience of the procedure, through an adapted transsphenoidal approach referencing bony landmarks to computed tomographic scans reconstructed on neuronavigation software. the somatotrophinoma was extirpated using fine surgical tools. all cats received intense electrolyte and blood pressure monitoring, peri-and post-operative ddavp and intravenous insulin and hydrocortisone infusion, transitioning to subcutaneous glargine, conjunctival ddavp, oral hydrocortisone and levothyroxine. between april -february , cats underwent hypophysectomy (median + range age: . years, . - . ; igf- : ng/ml, -> ; pituitary height . mm, . - . ). all displayed uncontrolled dm due to hs (median fructosamine: umol/l); none displayed overt central neurological deficits. two cats ( %, cats and ; pituitary height (mm): . and . ) required mechanical ventilation post-operatively and both were euthanized. post-mortem magnetic resonance imaging revealed brain herniation and cerebral ischaemia was suspected. one cat suffered cardiac arrest post-operatively at time of jugular catheter placement, though made an uneventful recovery. four other cats developed congestive heart failure within days, which was successfully treated not necessitating ongoing therapy. temporarily diminished tear production was seen in cats. seven of the ten surviving cats went into diabetic remission within a median of . days ( - ); others saw reduction of insulin needs by %. serum igf- normalised rapidly and significantly in all but one cat (median serum igf- ng/ml within days). persistent neurological deficits or palatal wound breakdown were not encountered. starting a hypophysectomy program to treat feline hs was associated with some risk of mortality, though surviving cases benefited from the procedure with a high incidence of diabetic remission. no conflicts of interest reported. pituitary dependent hypercortisolism (pdh) in dogs is frequently associated with high serum phosphate and parathormone concentrations. the pathogenesis of such abnormalities remains unknown and the evaluation of the urinary fractional excretion of phosphate and calcium in pdh dogs might be helpful in enhancing the knowledge regarding this issue. the aim of the present study was to evaluate the serum and urinary concentrations and the urinary fractional excretion of phosphate and calcium in dogs with pdh. medical records from one referral center were retrospectively evaluated between and . the diagnosis of pdh was confirmed using the cortisol to creatinine ratio, the ldds test and/or acth stimulation test, the plasma acth concentration, ultrasonography of the adrenal glands and computer tomography (ct) of the pituitary and the adrenal glands in dogs with consistent clinical signs. only newly diagnosed dogs, before treatment for pdh, were evaluated. two control groups were included: one healthy and one sick control dog (without pdh) for each dog with pdh were included. healthy control dogs (hcd) and sick control dogs (scd) were matched for age (ae months), breed, sex and sexual status. data were analysed using non-parametric tests and expressed as median and ranges. significance was set at p < . . one-hundredsixty-seven dogs with pdh were eligible for inclusion in the study. the median age at diagnosis was years (range: - ) and the median body weight was . kg (range: . - . ). there were female ( spayed) and male ( castrated). serum phosphate concentration ( . mg/dl, . - . ) was significantly (p < . ) higher compared to hcd ( . mg/dl, . - . ) and scd ( . mg/dl, . - . ). serum calcium concentration ( . mg/dl, . - . ) was significantly higher compared to scd ( . mg/dl, . - . ) but not different compared to hcd ( . mg/dl, . - . ). urinary fractional excretion of phosphate ( . %, . - . ) was significantly lower compared to hcd ( . %, . - . ) and scd ( . %, - . ). urinary fractional excretion of calcium ( . %, - . ) was significantly higher compared to hcd ( . %, . - . ) and scd ( . %, - . ). urinary calcium to creatinine ratio ( . , - . ) was significantly higher compared to hcd ( . . - . ) and scd ( . , - . ), while urinary phosphate to creatinine ratio were not significantly different in pdh dogs, hcd and scd. in conclusion pdh dogs have lower phosphaturia and higher calciuria compared to control dogs. this findings suggest that, at least in part, the high serum phosphate concentrations are related to the renal retention of phosphate. no conflicts of interest reported. four cortisol-based methods of monitoring trilostane treatment of canine hyperadrenocorticism were compared to the results of a clinical scoring scheme based on an owner questionnaire. cases of canine hyperadrenocorticism that had received a consistent dose of trilostane for more than one month were recruited from first opinion and referral practice. each dog was used only once. owners were asked to complete a questionnaire that assessed clinical control. the dogs were then categorised as being over-controlled, well-controlled, moderately-controlled and poorly-controlled. cortisol was measured in serum samples taken pre-trilostane (peak), hours post-trilostane (trough) and hour post-acth injection. dogs that had an increase in cortisol after trilostane administration were excluded. a scoring system was developed for each of these measurements. a fourth scoring system was developed using a novel algorithm that combined the peak and trough cortisol (peak-trough). the results of each of the scoring systems categorised the dogs into those that would be expected to be over-controlled, well-controlled, moderately-controlled and poorly-controlled. weighted kappa was calculated to assess the agreement between the categorisation according to each of the methods compared to the categorisation using the owners score. the pearson correlation coefficient was calculated to assess relationships between the various parameters. in total tests were analysed. when compared to the results of the owner's questionnaire , , and dogs were correctly categorised using the peak-trough, peak alone, post-acth and trough alone respectively. amongst the miscategorised results , , and dogs were incorrect by category and , , and dogs by categories using the peak-trough, peak alone, post-acth and trough alone respectively. all methods correctly recognised the over-controlled dog that had been identified by the owner's score. the weighted kappas for post-acth and trough cortisol categories compared to the owner score categories were . and . respectively (defined as slight agreement). in contrast the weighted kappas for the peak and peak-trough categories were . and . respectively (defined as fair agreement). there were no significant correlations between the absolute clinical scores and cortisol concentrations. there were significant correlations between the cortisol measurements. the novel methods of peak-trough and peak cortisol better reflected the level of clinical control of hyperadrenocorticism identified by the owners' questionnaire than either post-acth stimulation or trough cortisol. peak-trough and peak cortisol concentrations should be further investigated as monitoring methods for trilostane. financial support from dechra pharmaceuticals. the prognosis of canine adrenocortical insufficiency is generally regarded to be excellent. however, there is paucity of sur-vival analyses in the literature. the aim of the present study was to evaluate the survival of dogs with the diagnosis adrenocortical insufficiency based on data from a cohort of , swedish client-owned dogs insured in one insurance company (agria pet insurance, stockholm, sweden) during the time period - . dogs were identified by search for insurance claims with the register code for adrenocortical insufficiency. dogs were excluded from analysis if they had a previous history of hypercortisolism, and if they were born before begin of the study period. kaplan-meier survival analysis was performed. dogs were regarded as censored when the registered cause of death was other than adrenocortical insufficiency or hypercortisolism that was registered after the first claim for adrenocortical insufficiency. data from dogs was included. one hundred twenty-four dogs were registered to be dead. in dogs the cause of death was related to the adrenocortical insufficiency. the -year estimated survival-rate was % ( % ci, - %). the -year estimated survival-rate was % ( % ci, - %). the -year estimated survival-rate was % ( % ci, - %). twelve dogs ( . %) were still alive after years. in conclusion, the long-term survival of dogs with adrenocortical insufficiency was reasonably good. however, the diseases-related mortality was higher than expected, and occurred mainly during the first years after diagnosis. conflicts of interest: this study was supported by grants from the swedish research council and the foundation for research, agria insurance company. the concomitant occurrence of two or more endocrine tumors and/or hyperplasias, known as multiple endocrine neoplasia (men) is a well-known entity in humans. multiple gene mutations have been identified. the two major forms are men and men . in men , the main affected organs are parathyroid, pancreas and pituitary gland. men occurs in clinical variants: men a, characterized by medullary thyroid carcinoma (mtc), pheochromocytoma and primary hyperparathyroidism; men b, characterized by mtc, pheochromocytoma and additionally abnormalities; familial medullary thyroid carcinoma. in dogs and cats only a few cases have been reported and it is unknown whether hereditary men-like syndromes exist in these species. the aim of this study was to evaluate the prevalence of multiple endocrine tumors in dogs and cats at our institution, to identify possible breed and sex predispositions and to investigate similarities with the human men syndromes. autopsy reports of dogs and cats from until were reviewed. animals with at least two endocrine tumors/hyperplasias (eth) were included. autopsy reports of dogs and cats were examined. dogs had eth affecting a single organ, had multiple eth; cats had single eth, had multiple eth. in dogs with multiple eth, the most common breeds were west highland white terrier (whwt, / ), poodle, golden retriever, mixed-breed dogs (each / ). / were male ( intact); / were female ( neutered). median age was years (range - ). the most common combination was multiple testicle tumors of various types ( / ). the most common affected organs were the adrenals ( / ). adrenal cortical adenomas/carcinomas/hyperplasias were mainly associated with pheochromocytomas ( / ), testicle tumors ( / ) and insulinomas ( / ). all whwts had adrenal adenomas. both poodles had pheochromocytoma associated with pituitary adenoma or adrenal hyperplasia. dogs showed tumor combinations similar to the human men syndrome: pituitary adenoma and insulinoma; pituitary adenoma and parathyroid hyperplasia. / cats were domestic short/long hair, / were persians. / were male ( castrated); / were female ( neutered). the median age was . years (range - ). the most common affected organs were thyroid glands ( / ), combined mostly with lesions of parathyroid ( / ) and adrenal glands ( / ). none of the cats had combinations similar to the human men syndromes. the prevalence of multiple eth in dogs and cats was . % and . %. men-like syndromes were extremely rare in dogs and non-existing in cats. no sex predisposition was observed. possible breed predispositions need further investigations. no conflicts of interest reported. canine angiostrongylosis is an increasingly reported disease worldwilde, including many european countries, possibly due to climatic factors, presence of foxes (acting as reservoir) or more simply, to the availability of more accurate diagnostic methods. although detection of the first-stage larvae (l ) using the baermann technique on faecal samples (preferably collected over three consecutive days) remains the gold standard, recently developed serological and molecular tests (quantitative polymerase chain reaction, qpcr) are now available. until now, the prevalence of canine angiostrongylosis among healthy and coughing dogs in belgium was unknown. the aims of the present study were ( ) to describe a clinical series of recent autochtonous cases and ( ) to retrospectively assess angiostrongylus vasorum qpcr in bronchoalveolar lavage fluid (balf) samples, collected over the last years from a larger series of dogs, healthy or with other respiratory conditions, in order to investigate the past prevalence of the disease in belgium. seven dogs, living in southern or eastern belgium, were recently diagnosed as having angiostrongylosis (mean age= . y, mean body weight= . kg). they all presented with respiratory signs of variable severity. in dogs, balf was obtained and qpcr was positive in all of them, at moderate or high level (ct from , to , ) while larvae were detected in the faeces of only animals. in the remaining two dogs, no balf was obtained, but coproscopy was positive. all dogs responded to medical treatment, consisting in a -week course of fenbendazole and/or two spot-on application of moxidectin at -month interval. balf samples were collected between and from asymptomatic client-owned dogs and dogs with various respiratory conditions, including dogs with confirmed bordetellosis, dogs with eosinophilic bronchopneumopathy (ebp), dogs with chronic bronchitis and dogs with bacterial bronchopneumonia, and were retrospectively assessed with a a. vasorum qpcr assay. amongst those dogs, only one balf, from a dog with ebp, yielded a positive qpcr result. in this dog, faecal analysis was negative. the present data show that, based on balf qpcr and coproscopy, presence of angiostrongylosis in healthy and coughing dogs was negligible in belgium until the last months. it is now considered as an emerging condition and must be included in the differential diagnosis in coughing dogs. the present results also support that qpcr detection of a. vasorum in balf, when available, is an adequate and reliable detection technique. no conflicts of interest reported. ligneous membranitis is a rare chronic inflammatory disease associated with congenital plasminogen deficiency. it has only been described in six unrelated dogs. the objective of this study is to report the presentation, clinicopathological and post mortem findings in three related scottish terrier puppies with ligneous membranitis. ligneous membranitis is well described in humans, where it is inherited in an autosomal recessive manner. patients commonly present as infants. ocular, oral and genital lesions are most common, but other organs are occasionally involved and congenital obstructive hydrocephalus is reported in some individuals. numerous mutations and polymorphisms in the plasminogen gene have been identified in affected individuals. the affected scottish terriers (two male and one female) presented at months of age with severe proliferative and ulcerative conjunctivitis and gingivitis/stomatitis; biopsy confirmed ligneous membranitis. other clinical signs included increased upper respiratory tract noise, nasal discharge and lymphadenopathy. one male was cryptorchid. clinical pathological findings included neutrophilia, proteinuria and hypoalbuinaemia. serum plasminogen activity was measured in two dogs, and was low in one. the dam and sire of the affected dogs had normal serum plasminogen activity and no history or clinical signs consistent with ligneous membranitis. no significant clinical improvement was evident following treatment with antibiotics, glucocorticoids, topical ciclosporin or heparin. one dog died of cardiopulmonary arrest in the hospital and the two other dogs were euthanized due to progressive clinical signs. post-mortem evaluation of the affected dogs revealed multiple abnormalities including severe proliferative fibrinous lesions affecting the trachea, larynx and epicardium, and multiple fibrous adhesions throughout the thoracic and abdominal cavities. the male dog had internal hydrocephalus and lacked a cerebellar vermis. this is the first report of ligneous membranitis in related dogs and the first report in scottish terriers. sequencing the plasminogen gene in the affected dogs, their parents and unrelated control dogs to identify polymorphisms or mutations that may be associated with ligneous membranitis in dogs is ongoing. the author received a travel scholarship from zoetis to attend this congress. health screening of elderly dogs is often recommended, but scientific information on clinical and laboratory abnormalities in senior and geriatric dogs is scarce. this study was undertaken to describe blood pressure measurement, physical examination (pe) abnormalities and routine laboratory test results in senior and geriatric dogs that were apparently healthy for the owner. because life expectancy in dogs is related to body size, the inclusion of dogs was based on a human/pet analogy chart to determine whether a dog was senior (n = ) or geriatric (n = ). to verify health status, owners were asked to complete an extensive questionnaire. systolic blood pressure (sbp) was measured using the doppler technique according to the acvim guidelines. subsequently a thorough pe was performed, including body and muscle condition scoring, orthopedic examination, neurologic evaluation, indirect fundoscopy and bilateral schirmer tear test. complete blood count, serum biochemistry and urinalysis (including urinary sediment, urinary protein:creatinine ratio (upc) and bacterial culture) were evaluated. in of dogs sbp exceeded mmhg, none of the dogs had fundoscopic lesions secondary to hypertension. body condition score was abnormal in animals, were overweight or obese. physical examination revealed a heart murmur in , submandibular lymphadenopathy in , moderate to severe dental plaque in and one or more (sub)cutaneous masses in dogs. twenty-three dogs were leukopenic, had a decreased phosphorus, an increased serum creatinine and one dog a decreased total thyroxine (with concurrent increased thyroid stimulating hormone). crystalluria was commonly detected ( / ) and mostly due to low numbers (< /high power field) of amorphous crystals ( %). struvite crystals were present in % of the crystalluric dogs. overt and borderline proteinuria were detected in and of dogs, respectively. four dogs had a positive urinary culture. sbp was not significantly different between the senior and geriatric group. there was no significant effect of obesity or gender on sbp. the platelet count (p = . ), total thyroxine concentration (p = . ) and the frequency of orthopedic problems (p = . ) and cutaneous masses (p = . ) were significantly higher in the geriatric compared to the senior dogs. hematocrit (p = . ) and body temperature (p = . ) were significantly lower in the geriatric group. these findings indicate that physical and laboratory abnormalities are common in apparently healthy senior and geriatric dogs. this underlines the necessity for regular health screening in elderly dogs and the urgent need for reliable and maybe age specific reference intervals in veterinary medicine. the cost of examinations reported in this study were covered by hill's pet nutrition belgium. systemic lupus erythematosus, sle, is a chronic autoimmune disorder with varying clinical manifestations and diagnosis is based on both clinical signs and laboratory findings. other systemic rheumatic diseases, referred to as sle-related diseases or immune-mediated rheumatic disease (imrd), are also described. the most common clinical signs in dogs are stiffness and pain from varying joints. one hallmark of sle and sle-related diseases in both dogs and humans is high titres of circulating antinuclear antibodies (ana), which can be demonstrated by the indirect immunofluorescence (iif) ana test. earlier studies have shown that canine iif ana positive samples may be divided into two main subgroups: homogenous (ana h ) and speckled (ana s ) iif ana fluorescence pattern. in humans, further determination of the specificity of ana positive sera is frequently employed to characterize the ana reactivity. some of these ana specificities have been demonstrated in man to strongly associate with different systemic autoimmune diseases and also with different iif ana staining patterns. presence and character of antinuclear antibodies in canine sle-related diseases are not well described. the aim of this work was to further characterize the ana specificity in dogs with sle-related disease/imrd. sera from anapositive dogs, including different breeds, were analyzed with elisa and line blot techniques (elisa and euroline ana profile, euroimmun, germany). the five most prevalent breeds were german shepherd dog, nova scotia duck tolling retriever, cocker spaniel, crossbreed and golden retriever. sera displayed a homogenous and a speckled iif ana fluorescence pattern. several specific ana-reactivities earlier characterized in human patients were identified. the majority of ana h , n = , %, showed reactivity against nucleosomal antigens and ( %) against dsdna when conducted on line blot. these sera also reacted against nucleosomes and dsdna on the elisa. there were some additional positive with the elisa, so in total the elisa identified % with nucleosomal and % with dsdna reactivity. in few cases, other reactivities identified were against histones, pcna, jo- and rnp. in the ana s subgroup, the sm+rnp antigen evoked the most frequent reactivity, n = , % with both line blot and elisa. in few cases, reactivity against dsdna, pcna, jo- , pmscl kd, scl- , ssa and ssb were identified. in several dogs no specific antigen was identified. further studies are in progress in order to in more detail characterize and identify subtypes of already known and unknown antigens with clinical importance in canine autoimmunity. one of the authors, erik lattwein, is employed by euroimmun where the analyses were performed. chronic kidney disease (ckd) has a high prevalence in cats. routine renal markers, serum creatinine (scr) and urea are not sensitive or specific enough to detect early ckd. serum cystatin c (scysc) has advantages over scr for the detection of early kidney dysfunction, both in humans and dogs. a significant higher scysc concentration in ckd cats has been demonstrated. the objective of this study was to determine the effect of age, gender and breed on feline scysc and to establish a reference interval for feline scysc. in total, healthy cats between one and years were included. serum cysc was determined with a validated particleenhanced nephelometric immunoassay (penia). serum cr, urea, urine specific gravity (usg), urinary protein: creatinine ratio (upc) and systolic blood pressure (sbp) were also measured. to test for difference between the groups, the f-test was used. the lower and upper value of the % reference interval were obtained as the . % and . % quantiles of the scysc observations. no significant differences in scysc concentration were observed between young, middle-aged and old cats; between female, female neutered, male and male neutered cats; and between purebred and domestic short-or longhaired cats. the % reference interval for feline scysc was determined as [ . - . mg/l]. there was a significant difference in scr concentration between domestic short-or longhaired cats and purebred cats. the sbp was significantly influenced by gender as well as age, while urea was influenced by both age, gender and breed. this study showed that the biological factors age, gender and breed have little or no impact on feline scysc, in contrast to scr and serum urea, making it an interesting marker. therefore, further studies are warranted to evaluate the diagnostic value of scysc as a renal marker in cats. this study recieved support from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l. ghys. assessment of renal function is often needed, however existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. in this pilot study the feasibility of a transcutaneous glomerular filtration rate measurement was investigated. the transcutaneous disappearance rate (expressed as half-life) of fluorescein-isothiocyanatelabelled sinistrin (fitc-s) was measured in three healthy research dogs and three healthy research cats. plasma clearance of sinistrin ( data points) was performed in both species as previously described (res vet sci ; : - and j fel med surg ; : - ) and half-life was calculated using a -compartment model with a freely available pharmacokinetic calculator (comput meth prog bio ; : - ) . renal elimination of fitc-s was measured transcutaneously for hours ( - data points) using a miniaturized device as described previously for the same purpose in rats (kidney int : - ). the procedures were performed in awake, freely moving animals using escalating doses of fitc-s ( mg/kg, mg/kg, mg/kg) with a wash-out period of at least h in each animal. to find the best position for the device, multiple devices were placed on each animal. the resulting fitc-s disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. in both species mg/kg were adequate for kinetic calculation. the most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. transcutaneous fitc-s clearance was then repeated using the optimal dose and location and in parallel with the plasma sinistrin clearance. plasma sinistrin clearances [ml/kg/min] were . , . and . in the three dogs, respectively. corresponding plasma elimination half-lives [min] were , and , and corresponding transcutaneous elimination half-lives [min] were , and , respectively. plasma sinistrin clearances [ml/kg/min] were . , . and . in the three cats, respectively. corresponding plasma elimination half-lives [min] were , and , and corresponding transcutaneous elimination half-lives [min] were , and , respectively. in conclusion, transcutaneous fitc-s clearance is a feasible method for assessment of gfr in awake dogs and cats. it is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. a dose of mg/kg of fitc-s seems adequate for kinetic assessment. further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions. conflicts of interest: zhp and sg are supported by the ec fp marie-curie programme: nephrotools. the device development was supported by the fp activity: place-it.ng is owner of a patent covering fitc-sinistrin and the technology for its measurement. excretion of urinary biomarkers of renal damage should occur at an early stage of chronic kidney disease (ckd), thus facilitating earlier diagnosis of renal disease. albumin and cystatin c in the renal ultrafiltrate are mostly reabsorbed by the proximal tubular cells, therefore increased urinary excretion of albumin and cystatin c (uac and ucysc) would be expected to correlate with the presence of renal tubular damage and ckd. the aim of this study was to establish biological validity of two particle enhanced turbidimetric assays (petias) for the measurement of albumin and cystatin c (previously validated for use in feline urine) by comparing the uac and ucysc between non-azotaemic cats and cats with azotaemic ckd. blood and urine samples were obtained from cats at three uk first opinion practices as part of a geriatric screening programme. haematology, serum biochemistry (including total thyroxine concentration (tt )) and urinalysis (including urine protein:creati-nine ratio (upc)) were performed. dental disease score (calculus and gingivitis) and body condition score (bcs) were recorded. cats with tt > nmol/l, evidence of pyuria or bacteruria, or significant systemic disease were excluded. uac and ucysc were determined in non-azotaemic cats (n = ) and cats with azotaemic ckd (n = , defined as a serum creatinine concentration > lmol/l and concurrent urine specific gravity < . ). comparisons between the non-azotaemic and azotaemic ckd groups were made using the mann whitney u test. correlations were assessed by spearman's correlation coefficient. data are presented as median [ th , th percentile] and statistical significance was defined as p < . . uac was significantly higher in the azotaemic group than the non-azotaemic group ( . [ . , . ]x - vs. . [ . , . ]x - ; p = . ), whereas upc was not significantly different between the groups (p = . ). unexpectedly, ucysc tended to be lower in azotaemic cats than non-azotaemic cats ( . [ . , . ]x - vs. . [ . , . ]x - ; p = . ). uac was weakly positively correlated with serum urea concentration (r s = . , p = . ), but was not correlated with serum creatinine concentration. ucysc was not significantly correlated with serum concentrations of urea, creatinine or tt . uac was also weakly negatively correlated with dental calculus score (r s = - . ; p = . ) and bcs (r s = - . ; p = . ). uac appears to be a more sensitive test for azotaemic ckd than upc, however the apparent low specificity may limit the utility of uac as a urinary screening test for ckd. increased ucysc (determined by petia) would not appear to be a marker of azotaemic ckd in cats. no conflicts of interest reported. cystinuria is an inherited metabolic disorder that causes defective tubular reabsorption of the aminoacids cystine, ornithine, lysine and arginine (cola). the low solubility of cystine in acidic urine promotes formation of cystine crystals and uroliths in the urinary tract resulting in the clinical signs of stranguria, urinary obstruction and renal failure in affected individuals. cystinuria occurs in > breeds of dog and has been classified into types ia (newfoundland, landseer, labrador), iia (australian cattle dog), ib (miniature pinscher) and iii (androgen-dependent; e.g. mastiff, irish terrier). the kromfohrl€ ander is a medium-sized companion dog, bred initially as a cross between a wire fox terrier and a grand griffon vend een, first recognised internationally in . cystinuria has been suspected in this breed but no cases have been reported in the literature to date. we determined urinary cola concentrations in adult kro-mfohrl€ ander dogs aged - years comprising intact and castrated males, and intact and spayed females. a total of ( %) intact males aged . to . years had cola values > lmol/g creatinine and several developed cystic calculi. furthermore, intact male dogs had increased cola but normal cystine levels. all castrated males had normal cola concentrations. no females had increased cola and cystine concentrations or formed any cystine calculi. we conclude that cystinuria with cystine calculi occurs frequently in adult intact male kromfohrl€ ander dogs but neither is seen in females. this appears to be an androgen dependent type iii cystinuria, as seen in mastiff-type dogs and irish terriers. thus, castration may resolve the increased urinary cola excretion and risk for cystine calculi formation and obstruction. the precise mode of inheritance is still unclear. all adult intact male kromfohrl€ ander dogs should be screened by urinary cola testing. work carried out at the author's previous place of employment (university children's hospital, frankfurt, germany). acs, rk, em, md and ug provide a diagnostic service for cystinuria and other inborn errors of metabolism in companion animals. ureteral urolithiasis is an emerging medical concern in cats. there are few reports on epidemiology, diagnosis or medical management of ureteral calculi in cats, particularly in europe. cats diagnosed with ureteral urolithiasis in the teaching hospital of the veterinary school of alfort from to were included in this study. diagnosis was confirmed with radiographs, ultrasound scan and/or laparotomy. signalment, clinical signs, clinicopathologic and diagnostic imaging findings, medical treatment and outcome were recorded. epidemiological data were compared to a reference population of cats. eighty three cats were included in the study. the occurrence of ureteral urolithiasis was significantly higher in birman to the author knowledge, it is the first time that a higher prevalence of ureteral calculi in birman cats is reported in europe. spontaneous elimination of calculus is associated with a small size (< . mm). if the size of calculi tends to be bigger in cats with no improvement of renal function after medical treatment, prospective studies are still needed to determine the best medical treatment. no conflicts of interest reported. feline immunodeficiency virus (fiv) infection has been associated with kidney disease, mainly characterised by an increased prevalence of proteinuria in fiv-infected cats. however, studies evaluating renal variables in fiv-positive cats are scarce. recently, a higher systolic blood pressure (sbp) was reported in a small number of fiv-infected cats. hypertension is an important cause of proteinuria and a frequent cause of renal disease in human immunodeficiency virus (hiv) positive patients. therefore, our main objective was to describe sbp in clinically ill fivpositive cats. secondly we aimed to evaluate routine renal variables in this population. naturally infected clinically ill fiv-positive cats were prospectively included. the doppler ultrasonic technique was used to measure sbp according to acvim guidelines. serum creatinine (screat) and urea (surea) concentrations, urine specific gravity (usg) and urinary protein:creatinine ratio (upc) were determined. the study included cats, with a mean age of . ae . years and a mean body condition score of . ae . on a nine-point scale. the sbp ranged from to mmhg, with a mean of ae mmhg. only two cats were hypertensive (sbp > mmhg). both had isosthenuric urine, were borderline proteinuric (upc . - . ) and one of them was mildly azotemic. mean screat was . ae . lmol/l (reference interval (ri) . - . lmol/l) and mean surea concentration . ae . mmol/l (ri . - . mmol/l). thirteen cats showed increased screat levels, with decreased usg (< . ) in eight, proteinuria (upc > . ) in seven and increased surea concentrations in ten of them. five out of ten azotemic cats were proteinuric with a decreased usg. mean upc was . ae . , with a wide range from . to . . borderline proteinuria was present in / ( . %) and proteinuria in / ( . %). half of the proteinuric cats had a decreased usg. mean usg was . ae . . one third of all cats had a decreased usg, with isosthenuria in seven of them. these results demonstrate that proteinuria and poorly concentrated urine are common in naturally infected clinically ill fivpositive cats, confirming previous reports in cats and humans. however, longitudinal studies of (borderline) proteinuric patients are needed to elucidate the clinical relevance. the low number of hypertensive patients and low mean sbp in our study indicate that hypertension is uncommon and unlikely to be the cause of renal damage in clinically ill fiv-infected cats. aratana therapeutics nv financially supports a clinical trial on the use of antivirals in fiv cats at our university. screeninng examinations repoorted in this trial were part of the required pre-trial investigations for that study. the presenting author is also funded by a scholarship from aratana therapeutics av. the objective of this study was to identify the prevalence of bacterial species and the susceptibility of isolates to doxycycline, trimethoprim-sulfamethoxazole (tms), ampicillin, amoxicillinclavulanic acid (amc), cephalothin, and enrofloxacin in cats with urinary tract infections (uti) with and without predisposing comorbidities. a retrospective analysis of case records between and was performed and resulted in inclusion cats into the study: cats with a systemic comorbidity, cats with indwelling urinary catheters, cats with other local comorbidities, and cats with no comorbidity. the most commonly isolated bacteria were escherichia coli (e. coli), streptococcus species (spp.), staphylococcus spp., and enterococcus spp.. the proportion of gram-negative isolates was significantly higher in the cats with systemic comorbidities than in cats with indwelling urinary catheters (p < . ) and cats with other local abnormalities (p < . ), whereas gram-positive isolates were significantly more commonly isolated from cats with indwelling urinary catheters and other local comorbidities than in cats with systemic comorbidities (p < . ). the proportions of isolates susceptible to amc, enrofloxacin, and tms and the antimicrobial impact factors (if) were higher than the proportions of isolates susceptible to doxycycline, ampicillin, and cephalothin and the respective if. based on these findings, amc and tms would be the firstchoice antimicrobial agents for empiric treatment of bacterial the increasing rates of resistance exhibited by uropathogens represent a serious problem for the selection of an appropriate antibiotic. the aim of this study was to determine secular trends of companion animal urinary tract infection (uti) that involve extended-spectrum b-lactamase (esbl)-and carbapemenase-producing gram negative bacteria (namely, escherichia coli, klebsiella pneumoniae, proteus mirabilis, acinetobacter baumannii), methicillin-resistant-staphylococci (mrs) and ampicillin and high-level-gentamicin-resistance (hlgr) enterococci. nine hundred and twenty two uropathogenic bacteria were isolated from dogs and cats, between january and march , at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. isolates were identified using standard commercial systems. susceptibility testing was performed using the disk diffusion and broth microdilution methods. clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) production was screened by double-disk synergy test. the esbl, plasmid-mediated ampc, carbapemenases, meca and aac( ')-ieaph( '')-ia genes were detected by pcr and gene enzymes were sequenced. among enterobacteriaceae . % were dhaproducers, . % were esbl-producers and . % were cmyproducers. all isolates were also multidrug-resistant. cefalosporinases-producer enterobacteriaceae were detected in , the first being a cmy- -producer e. coli. all the esbl-producers were e. coli or k. pneumoniae producing ctx-m-group enzymes. ampicillin-resistance in enterococci was present throughout the years ( , %, n = ). hlgr appeared in enterococci in and was confirmed by the detection of the bifunctional enzyme that confers high level resistance to aminoglycosides ( out of isolates). in this study we showed that in the last decade the emergence of resistance to critically important antimicrobials among uropathogens from companion animals is a concerning fact. the multidrug-resistant enterobacteriaceae may compromise effective therapeutic options, namely third and fourth generation cephalosporins, fluoroquinolones, trimethoprim/sulpha combinations. the emergence of mrsa/mrsp and hlgr among uropathogens is also a therapeutic challenge. the detection of uropathogens with antimicrobial resistance is not only an animal health issue but also a matter of public health, since companion animals may act as reservoirs of antimicrobial resistant bacteria or resistance genes for humans. the author currently receives a phd grant funded by the portuguese foundation for science and technology. in the past, the author received once research support and honoraries from portuguese merial for a project on canine vector borne diseases. the aim of the present study was to use insurance data to estimate morbidity and mortality related to kidney disease in the swedish dog population. insurance company data from veterinary care-insured and lifeinsured dogs during the years - were studied retrospectively. incidence-and mortality rates were calculated for the whole group of dogs as well as divided by sex and breed. for the breeds with the highest incidence-and mortality rates, respectively, the reasons for kidney disease were investigated by dividing the diagnoses into ethiology groups. the total number of veterinary care-insured dogs was , . the total incidence rate of kidney disease in this group of dogs was . ( . - . ) cases/ , dog-years at risk. the number of dogs in the life insurance was , and in this group the total kidney-related mortality rate was . ( . - . ) deaths/ , dogyears at risk. the most commonly reported ethiologies of kidney disease were "ethiology not determined"and "infectious/ inflammatory". the breeds with the highest incidence rate of kidney disease were the bernese mountain dog, miniature schnauzer and boxer. the breeds with the highest mortality caused by kidney disease were the bernese mountain dog, shetland sheepdog and flatcoated retriever. in conclusion, the epidemiological information provided in this study concerning kidney disease in dogs can assist clinicians in establishing diagnoses, and can assist breeders in defining priorities for preventative measures. it can also provide valuable information for future research. the senior author has received money from the insurance company we have used data from to write our study, for another project. jens h€ aggstr€ om and ingrid ljungvall have received financial support for research from sante animale, agria insurance ltd, sveland insurance ltd, forsgren research foundation. both of these authors have also undertaken paid consulatcny work for boehringer-ingelheim, ceva sante animale. mast cell tumours represent the most common cutaneous tumour in the dog. diagnosis of a mast cell tumour can be achieved through cytological examination of fine needle aspirate. however the grade of the tumour is an important prognostic marker and requires so far histologic assessment. a -tier histologic grading system based on number of mitoses, multinucleated cells, bizarre nuclei and karyomegaly was recently proposed by kiupel et al. the aim of this study was to assess if the cytomorphological criteria proposed in the -tier histologic grading system are applicable on cytology specimens. ninety-three mast cell tumour specimens of grade i or grade iii according to patnaik with both histological specimens and fine needle aspirates were retrospectively taken from a data set and histologically and cytologically re-evaluated. according to the kiupel grading system thirty-six were diagnosed histologically as high grades and fifty-seven were considered low-grade mast cell tumours. the cytologic examination of the corresponding specimens revealed thirty-one high grade and fifty-five low-grade tumours. an agreement between histologic and cytologic diagnosis based on the kiupel grading system was achieved in eighty-six cases (accuracy . %, specificity . %, sensitivity . %). five high-grade tumours ( . %) were considered as low grade on cytology. cytologic grading of mast cell tumours in the dog has satisfactory accuracy, sensitivity, and specificity. histologic grading of canine mast cell tumours still remains the gold standard, but cytology already gives reliable information. no conflicts of interest reported. in canines mastocytomas are among the most frequently diagnosed neoplasms of the skin. high grade mastocytomas (grade iii, patnaik classification) are characterized by an uncontrolled growth of neoplastic mast cells (mc) and a poor prognosis. recently, the kit-targeting tyrosine kinase inhibitors masitinib and toceranib have been approved for the treatment of canine mc tumors. these drugs are able to induce responses in mastocytoma patients. however, in many patients, relapses are seen. therefore, research is focusing on new drug targets. recently, the transcription factor stat has been reported to play an important role in the proliferation and survival of human neoplastic mc. the aim of the present study was to evaluate the jak -stat pathway in canine mastocytomas. to address this aim, the canine mastocytoma cell lines c and ni- as well as inhibitors directed against jak or stat were employed. as assessed by immunocytochemistry, c cells and ni- cells were found to express pstat in their cytoplasm and nuclei. intracellular expression of pstat was confirmed by flow cytometry. interestingly, c cells were found to express higher levels of pstat compared to ni- cells. next, we treated c cells and ni- cells with various concentrations of the stat inhibitors piceatannol and pimozide and the jak inhibitors azd and tg . as assessed by h-thymidine uptake, all compounds were found to inhibit the proliferation of canine mc in a dose-dependent manner. drug effects were found to vary in different cell lines, with the following rank-order of potency (ic values): tg : . - . lm; pimozide: . - . lm; azd : - lm; piceatannol: - lm. to further explore the mechanism of drug-induced inhibition of proliferation, we examined cell cycle progression and apoptosis in drug-exposed cells. whereas all drugs tested induced only moderate cell cycle arrests in the g phase, these drugs were found to induce substantial apoptosis in c cells and ni- cells as evidenced by microscopy and annexin-v/pi staining. together, our data show that jak -and stat -targeting drugs exert anti-proliferative and apoptosis-inducing effects in canine mastocytoma cells suggesting that this signaling pathway may be a promising new therapeutic target in canine mastocytomas. the clinical relevance of this observation remains to be determined. no conflicts of interest reported. subcutaneous mast cell tumours (sqmct) in dogs are relatively uncommon compared to their cutaneous counterparts. the veterinary literature describes these tumours as a specific pathological entity with, in general, a low probability of aggressive progression. surgery is considered the main treatment modality, while medical treatment has not been described. the purpose of this study was to determine progression free survival (pfs) for a chemo na€ ıve cohort of dogs presented with non-resectable and/ or metastasized sqmct, which all underwent masitinib-based therapy. data were collected for patients with sqmct presented to participating centres in the netherlands and the uk from / / to / / , which received masitinib-based therapy. treatment protocols employed, included masitinib alone (m), masitinib and prednisolone (mp), masitinib plus vinblastine and/ or lomustine and prednisolone (mpc). response to therapy was measured conforming to recist . . adverse events were graded using vcog-ctcae . . patients were grouped according to presence or absence of metastasis, treatment protocol used, previous surgery, and remission status achieved; simple comparisons were made to evaluate possible significance. twenty-five cases were identified. / were female. median age of occurrence was years ( - ). diagnosis was made by histology in / ; proliferation indices were defined in only dogs. fourteen cases exhibited metastasis at initiation of therapy. pfs for all cases ranged from - days. median/mean pfs (days) according to treatment was m: / d (n = ), mp: / d (n = ), mpc: / d (n = ). median/mean pfs according to metastasis status was m : / d (n = ) and m : / d (n = ). dogs who underwent previous surgery (n = ) had a median/mean pfs of / d compared to those who had no surgery / d. looking at remission status, median pfs of patients who achieved a complete remission was not reached, with a mean pfs of d (n = ). median/mean pfs of patients with partial remission was / d (n = ), stable disease / d (n = ), and progressive disease / d (n = ). / cases experienced suspected adverse events. three dogs, two of which ultimately died, had seven grade - adverse events (anaemia (n = ), hepatotoxicity (n = ), gastrointestinal toxicity (n = )). masitinib-based treatment is effective in the management of sqmct perceived to be aggressive. patients do not appear to benefit from prior surgery. metastatic status did not influence outcome. adding chemotherapy negatively influenced pfs. complete remission is a very favourable prognostic development. the authors have received financial support from ab science to help with the costs of statistical analyses in an unrelated project advances in distinction between morphological subtypes of canine non-hodgkin's lymphomas (nhl) have provided a better understanding of this cancer in dogs. diffuse large b-cell lymphomas (dlbcl) are the most frequent form of nhl in dogs including some distinguished morphological subtypes (mainly centroblastic polymorphic and immunoblastic) according to the who classification. few clinical studies reported dlbcl clinical outcomes under treatment while survival times of the centroblastic polymorphic subgroup were reported. the aim of this retrospective study was to evaluate the response of dlbcl to a standardized multi-agent chemotherapy protocol. medical records from dogs with a diagnosis of dlbcl between and were retrospectively reviewed. inclusion criteria were the availability of complete initial and follow-up information and the application of a standardized multi-agent l-cop chemotherapy protocol as previously described. dogs which received corticosteroids before the initiation of treatment and dogs which died for other reasons than their related disease before the end of the induction period ( d) were excluded. response to chemotherapy was evaluated every week during the induction of treatment, then every to weeks. statistical analysis was performed using kaplan-meier analysis. thirty cases of dlbcl meeting all inclusion criteria were included from the initial population. seven dogs were in clinical stage iii according to the who classification, in stage iv and in stage v. nineteen dogs were in substage a, and in substage b. on dogs which have an adequate response evaluation, dogs ( . %) achieved a complete remission. the median and mean duration of first remission were d and d, respectively (range - d). the median and mean durations of survival time were respectively d and d (range - d). one year and -years survival rates were % and % respectively. according to the statistical analysis, neither clinical stage (p = . ) nor substage at presentation (p = . ) or morphological subtype (immunoblastic vs centroblastic polymorphic, p = . ) were considered as a significant prognostic factor regarding the duration of the first remission and the overall survival time. a complete response was significantly associated with longer survival times (p = . ). to conclude, the dlbcl displayed a good clinical response to l-cop protocol, with a median survival time of days. the only significant prognostic criterion identified was a complete clinical response to the treatment. a prospective controlled study on a larger population is warranted to confirm these results. no conflicts of interest reported. canine histiocytic sarcoma (hs) is an aggressive round cell neoplasm with a poor prognosis. both lomustine and doxorubicin have been evaluated as first line chemotherapy agents with response rates of up to % and median survival times around - months. the aim of this study was to evaluate the response to epirubicin in a population of dogs with hs pre-treated with lomustine. medical records of dogs with a diagnosis of hs that were treated with lomustine and subsequently epirubicin were retrospectively evaluated. fifteen dogs received lomustine followed by epirubicin. there was a measureable response to lomustine in seven of dogs with evident disease, % ( cr & pr). an additional dogs achieved stable disease for an overall biological effective response of %. median ttp following lomustine could be assessed in dogs and was days (range - ). all fifteen dogs received epirubicin as a rescue agent: nine following progressive disease and three with stable disease on lomustine. one dog received epirubicin after completing six doses of lomustine in complete remission and two dogs in partial remission changed to epirubicin due to hepatotoxicity associated with lomustine. response rate to epirubicin was % and biologic effective response was % ( cr, pr, sd, pd) in dogs. one dog was euthanized due to epirubicin associated gastro-intestinal toxicity and dog stopped treatment with no assessment of response. median duration of response to epirubicin was days (range: - ) and dog is still alive and in remission ( days). overall median survival time for dogs receiving epirubicin following lomustine was days (range: - ). single agent epirubicin is a valid rescue therapy after lomustine for canine histiocytic sarcoma and results in modestly improved overall survival times in responding patients. the author received a travel scholarship from zoetis to attend this congress. the incidence of melanocytic lesions is increasing among canine population. canine malignant melanoma could have an aggressive behavior, metastasize early in the course of the disease and be resistant to most current therapeutic regimens leading to the need of finding markers with potential as therapeutic targets. the overexpression of cox- seems to play a key role in melanocytic tumours, having being described an association between high cox- immunoexpression and the malignant behavior. in order to contribute to the understanding of the role of cox- in melanocytic tumours, three main pathways were investigated: angiogenesis, tumour cell proliferation and inflammatory microenvironment (t-lymphocytes and macrophages). fifty one ( ) melanocytic tumours [ cutaneous ( malignant melanomas and melanocytomas) and oral malignant melanomas] were included. all the samples were submitted to immunohistochemical staining carried out by the streptavidinbiotin-peroxidase method, with a commercial detection system with or without melanin blanching, for detection of the following markers (cox- , ki- , factor viii, vegf, cd and mac ). in melanocytic tumours (n = ), both cox- labelling extension and intensity revealed a statistically significant association with angiogenesis by factor viii (p < , ), vegf (p < , ); ki- (p < , ), cd + t-lymphocytes (p < , ) and mac (p < , ). considering only malignant melanomas (n = cases), cox- labelling extension revealed a statistically significant association with angiogenesis (p = , ) and cd + t-lymphocytes (p = , ). cox- intensity was also positively associated with angiogenesis (p = , ) and with mac (p = , ). present study demonstrated a link between high cox- immunoexpression and increased angiogenesis and tumoural t-lymphocyte and macrophage infiltration in malignant melanomas. these findings reinforce the usefulness of using selective cox- inhibitors as a valuable therapeutic tool in malignant melanocytic tumours. this study received financial support from a company (merial). neuter status and risk of malignant neoplasia is not well evaluated in the canine population, when excluding neoplasia not normally believed to be sex-hormone dependent. denmark and the scandinavian countries have a high proportion of intact dogs compared to populations from other parts of the world. in the present study it was hypothesized that there would be no difference in gender and neuter status between the population of canine patients with a non-sex-hormone dependent malignant neoplasia reported to the danish veterinary cancer registry and a general population. from august to march , canine neoplasms were reported to the danish veterinary cancer registry. the total number of malignant ( ) and benign ( ) were comparable ( % and %). malignant neoplasms totalled , when tumors from areas of distribution with known sex-hormone dependency (reproductive organs, mammary gland, perineal), and cases with unknown area of distribution were excluded. the overall distribution of malignant neoplasia was ( %) intact male dogs, ( . %) neutered male dogs, ( %) intact female dogs and ( . %) neutered female dogs. the distribution was even between male and female dogs ( . % and . %). compared to a known standard population of dogs, there was an overall statistically significant association of malignant neoplasia with neuter status in both sexes. for both genders this was significant for lymphoma, mast cell tumors and osteosarcomas,. for neutered females, but not males, there was increased risk of hemangiosarcoma, squamous cell carcinoma and malignant melanoma. these findings indicate that there might be an association between neuter status and development of malignant neoplasia but larger prospective studies are needed to evaluate the risk of non-sex hormone dependent cancers in neutered dogs. no conflicts of interest reported. serum acute phase proteins (apps) are considered biomarkers of the acute phase reaction, and are being increasingly used in human and veterinary medicine in diagnosis and monitoring of neoplastic diseases. in the cat, serum amyloid a (saa) is considered a positive major app, haptoglobin (hp) a moderate app, and albumin and insulin-like growth factor- (igf- ) negative apps. the aim of the present study was to characterize the apps response in cats with mammary tumours. for that purpose, saa, hp, igf- and albumin serum concentrations were determined in female cats with malignant mammary tumours. cats with history of previous tumours or with concomitant tumours or other diseases were excluded. information on cats age, gender, breed, tumour type, histological grade, tumour size and location, skin ulceration, vascular neoplastic infiltration, necrosis, metastasis to regional lymph nodes, thoracic or abdominal organs, and survival time from diagnosis was assessed. blood samples were collected before surgery in all cats, and whenever possible, serial samples collected on control visits. owners gave informed consent. studied population included domestic short-haired cats with ages ranging from eight to years ( , + /- , ). all had carcinomas, including solid carcinomas (n = ), tubulopapillary carcinomas (n = ), one cribiform carcinoma and one carcinosarcoma. at the time of diagnosis, % of cats had an increase in serum concentration of hp and % of saa, and % had a decrease in concentration of albumin. mean and standard deviation values were of , + /- , g/dl for albumin (reference range , - , g/ dl), , + /- , lg/dl for igf- , , + /- , lg/ml for saa (reference value ˂ lg/ml), and , + /- , g/l for hp (reference value ˂ g/l). a positive correlation (r = , ) was detected between increases in serum concentrations of saa and hp. the increase in the size of tumour was significantly associated with the concentration of saa (p˂ , ). serum hp concentrations were significantly increased in tubulopapillary carcinomas (p˂ , ), and igf- decreased in solid carcinomas (p˂ , ). in the cats where serial determinations were performed, development of thoracic metastasis was significantly associated with a decrease of serum concentration of albumin (p˂ , ), and with an increase of saa (p˂ , ). this study suggests that feline mammary tumours are associated with an acute phase response. according with the results obtained, saa, hp, albumin and igf- might be important serum biomarkers in diagnosis and monitoring of the evolution of feline malignant mammary neoplasias. no conflicts of interest reported. histiocytic sarcoma (hs) is a neoplastic proliferation of interstitial dendritic cells or tissue macrophages. dogs with hs can present with local disease or with multifocal (disseminated) involvement. disseminated hs is poorly responsive to therapy and almost always fatal. little is established regarding the aetio-pathology of histiocytic sarcoma in dogs. the purpose of this study was to establish and characterise a hs cell line from fresh tumour samples obtained from a dog with disseminated hs in order to further clarify disease pathogenesis and behaviour. with owner consent, treatment-na€ ıve tumour sections were collected from a dog with disseminated hs that was euthanased. tumour tissue was assessed with immunohistochemistry (ihc) using antibodies against canine cd , cd , and pax- to support the diagnosis of histiocytic sarcoma. primary cell cultures (hscs), established from the tumour were cultured and maintained in modified eagle's medium with % fetal bovine serum, l-glutamine, penicillin and streptomycin, in standard conditions. hscs were characterised by alpha naphthyl acetate esterase (anae) and lysozyme staining while pcr was used to detect cell markers cd a, cd c, mhc ii, cd , ccr , e-cadherin, and cd . cell surface markers were compared to an established canine hs cell line (dh ). phagocytic activity of hsc cells was assessed using cellular uptake of carboxylated fluorescent beads and documented using flow cytometry and fluorescent microscopy. tumour tissue was strongly cd positive and negative for cd and pax- . cultured cells exhibited morphological characteristics consist with dendritic cells, such as projections and pleomorphism. hsc cells stained positively for non-specific esterase (anae) and lysozyme, and pcr indicated cells were positive for cd a, cd c, mhc ii and cd and negative for cd and e-cadherin. hsc cells were positive for mhc ii and ccr while dh cells were negative. phagocytic activity was evident. a novel hs cell line (hsc) was established and characterized from primary tumour tissue collected from a dog with disseminated disease. hsc cells were most consistent with interstitial dendritic cell origin based on cd a, cd c, and mhc ii staining as well as demonstrable phagocytic activity. hsc cells also displayed expression of ccr , unlike the established dh line, supporting a notion that hs consists of a variety of subtypes. ccr has been linked to hs growth and metastasis, suggesting it may represent a possible therapeutic target. further studies establishing and characterising canine hs cells may contribute to the elucidation of mechanisms of tumourigenesis. no conflicts of interest reported. virulent-systemic (vs)-fcv that induce cutaneous edema, ulcerations of the head and feet, and occasionally jaundice have been described in the usa and europe. here we characterize for the first time vs-fcv outbreaks in cats in switzerland and liechtenstein. the four outbreaks occurred in three geographically separated locations: schaan (liechtenstein, shelter ), zurich (switzerland) and lausanne (switzerland, shelter ) between november and january . pcr (fcv and feline herpesvirus- , fhv- ), virus isolation and felv/fiv testing were performed on saliva and blood samples collected from clinically affected cats. furthermore, saliva for pcr was collected from additional cats in shelter . phylogenetic analyses were performed based on the capsid (vp ) gene sequence of fcv. vs-fcv isolates were tested for virus neutralization with sera raised against common fcv vaccine strains. outbreak occurred in a cattery in liechtenstein and involved five non-vaccinated, -months old siblings with fever, edema, skin and tongue ulcerations. outbreak occurred in a small animal clinic in zurich. a -year old cat presented with severe paw edema, fever, tongue and skin ulcerations, progressive hypoproteinemia and hyperbilirubinemia. outbreaks and happened in a cattery in lausanne five months apart and involved two litters of non-vaccinated, -to -months old kittens. the cats presented with fever, nasal discharge, edema and skin and oral ulcerations. all affected cats tested fcv-positive but negative for fhv- , felv and fiv, except for one kitten from outbreak (fiv-positive). all cats in outbreaks and recovered, whereas all cats in outbreaks and died or were euthanized because of clinical deterioration. each outbreak was caused by a phylogenetically distinct vs-fcv strain. in shelter , the queen and three in contact cats remained asymptomatic although infected with the same vs-fcv strain. furthermore, / other cats of shelter were infected with closely related, but distinct fcv strains. the vs-fcv isolates from the two outbreaks in shelter were distinct but phylogenetically related. all vs-fcv isolates from cats from the same outbreak showed a similar virus neutralization pattern, but neutralization differed between different outbreaks. in conclusion, all vs-fcv outbreaks involved multi-cat environments. the same vs-fcv strains with similar virus neutralization patterns were isolated from cats from the same outbreak. not all cats infected with a vs-fcv strain developed disease and mortality varied between the outbreaks. the sera for virus neutralization were provided by merial, france. defined herein as presence of sneezing, nasal-and/or ocular discharge, conjunctivitis and/or keratitis), but also oral cavity lesions, chronic stomatitis, limping syndrome and, rarely, virulent systemic disease. the aims of the present study were to compare cats suspected of fcv (fcv-sc) based on clinical symptoms and healthy controls (controls) and to investigate potential risk and protective factors, such as co-infection with feline herpesvirus- (fhv- ), mycoplasma felis, chlamydophila felis, bordetella bronchiseptica and feline retroviruses, vaccination, gender, age, breed, housing and corticosteroid and antibiotic treatment. oropharyngeal, nasal and conjunctival swabs from fcv-sc and controls were collected into transport medium, processed within hours after collection and analyzed for fcv by virus isolation and for all tested pathogens using molecular assays. the samples were collected by randomly selected veterinary practices in different areas of switzerland ( fcv-sc and controls/ area). to record clinical data, retroviral status and vaccination history of the cats, a questionnaire was filled out by the private veterinarian. the seven tested pathogens were found in the investigated population. the prevalence (fcv-sc vs. controls) was: fcv % vs. %; fhv- % vs. %, c. felis % vs. %, b. bronchiseptica % vs. %, m. felis % vs. %, feline leukemia virus % vs. % and feline immunodeficiency virus % vs. %. fcv-sc were positive for fcv significantly more often compared with controls (or . ) and shed more fcv. co-infections with up to four pathogens were detected; fcv-sc were significantly more frequently co-infected ( %) compared with controls ( %). gingivostomatitis and oral ulceration but not urtd were highly associated with fcv infection. in contrast, c. felis was associated with urtd; fhv- was associated with nasal and ocular discharge and m. felis with conjunctivitis and ocular discharge. risk factors for fcv infection were housing in groups (especially ≥ cats), an intact gender, maine coon breed and corticosteroid therapy. fcv-positive cats with gingivostomatitis were older and more commonly vaccinated than fcv-positive cats without gingivostomatitis. moreover they shed more fcv than cats with urtd. vaccination and primary immunization defined as two vaccinations - weeks apart with the same vaccine brand were protective factors against fcv but not fhv- infection. vaccination was associated with a decreased incidence of urtd in fcv-infected cats (or . ). further analyses will investigate cross-neutralization patterns of the prevailing fcv isolates. conflicts of interest: the study was partially funded by merial, france, and biokema, switzerland. antibody preparations are commonly used for the treatment of feline upper respiratory tract disease (furtd), although their efficacy has not been proven. the aim of this study was to evaluate efficacy of a commercial serum containing antibodies against feline herpesvirus- (fhv- ) and feline calicivirus (fcv) in cats with acute viral furtd. this prospective, randomized, placebo-controlled, double-blind study included cats with acute (< days) clinical signs of fhv- and/or fcv infection (confirmed by quantitative pcr). all cats received symptomatic treatment and either hyperimmune serum (n = ) (≤ weeks ml, > weeks ml, subcutaneously q h, topically into eyes, nostrils, and mouth q h) or saline (n = ) for three days. clinical signs, including a 'furtd score' and general health status, were recorded daily (day to and on day ). fcv shedding was determined on day and . statistical analyses included one-way analysis of variance, mann-whitney u, and student's t-test (improvement of clinical signs), fisher's exact test (fcv shedding), and spearman analysis (correlation clinical signs with virus load). clinical signs and general health status improved significantly in both groups. however, while placebo-treated cats had only improved significantly by day , cats receiving antibodies already significantly improved in their 'furtd score' (p = . ) and general health status (p = . ) by day . there was no significant difference in the number of cats shedding fcv and no correlation between viral load and clinical manifestation. administration of antibodies lead to faster improvement of clinical signs in cats with acute viral furtd, but did not influence fcv shedding. no conflicts of interest reported. different viral and bacterial pathogens can be involved in feline upper respiratory tract disease (furtd). although some clinical signs have been associated with certain pathogens, clinical signs can be variable and non-specific. aim of the study was to compare detection rates of feline herpesvirus- (fhv- ), feline calicivirus (fcv),and chlamydophila felis (c. felis) in cats with furtd on different sampling sites, and to correlate test results and clinical signs. swabs of nose, oropharynx, tongue, and conjunctiva were taken from cats with signs of furtd. on all samples, reverse transcription polymerase chain reaction (rt-pcr) was performed for detection of fcv, and polymerase chain reaction (pcr) for detection of fhv- and c. felis. fisher's exact test was used for all comparisons. the level of significance was p < . . pathogens were detected in . % of cats. of these, . % were positive for fhv- , . % for fcv, and . % for c. felis. fcv was isolated significantly more often from oropharynx ( . % of fcv-positive cats) and tongue ( . %) compared to conjunctiva ( . %) (p < . ). there was no significant difference between the sampling sites for detection of fhv- and c. felis. in addition, there was no preferred sampling site in cats with respective clinical signs, including oral ulceration, conjunctivitis, and keratitis. in cats with furtd, the oropharynx can be recommended as the preferred sampling site for detection of fcv, fhv- , and c. felis. based upon clinical signs it cannot be determined which sampling site should be selected for detection of the pathogens. no conflicts of interest reported. pulmonary haemorrhage syndrome (lphs). s. schuller , s. callanan , s. worrall , t. francey , a. schweighauser , j.e. nally . bern university, bern, switzerland, university college dublin, dublin, ireland leptospiral pulmonary haemorrhage syndrome (lphs) is a severe form of leptospirosis, which has been increasingly recognised in humans and many animal species in the past years. patients with lphs may develop rapidly progressive intra-alveolar haemorrhage, leading to high mortality. the pathogenic mechanisms of lphs are poorly understood hampering the application of effective treatment strategies. studies in humans and experimentally infected guinea pigs have demonstrated deposition of immunoglobulin and complement c in lphs lung tissue in the absence of significant numbers of leptospires, suggesting that lphs is, in part, caused by autoimmunity. the aim of this project was to describe the histopathologic features of lphs in dogs and to investigate whether igg and igm deposition is present in affected canine lung tissue. single-step immunohistochemistry (ihc) for dog igg, igm and leptospiral outer membrane vesicles was performed on lung tissues from dogs with lphs, dogs with pulmonary haemorrhage due to other causes and healthy dog lungs. acute intra-alveolar haemorrhage and oedema in the absence of significant inflammatory infiltrates were present in all lphs lung tissues. three ihc staining patterns were observed in lphs lung tissue: alveolar septal wall staining with (igg n = /igm n = ) and without intra-alveolar staining (igg n = /igm n = ) and staining of intra-alveolar fluid only (igg n = /igm n = ). intra-alveolar staining appeared to favour alveolar surfaces in some cases (igg n = /igm n = ). healthy control lungs showed no staining, whereas haemorrhagic lung showed staining of intraalveolar fluid (igg/igm n = )) and occasional, mild and discontinuous staining of alveolar septa (n = ). leptospiral antigens were not detected in any of the tissues. results indicate that histopathologic features of canine lphs are similar to what has been described in other species. ihc demonstrated that alveolar septal deposition of igg/igm is present in most dogs with naturally occurring lphs. while these findings support a role of the humoral immune response in the development of lphs, our findings do not indicate whether autoimmunity is a primary or secondary event in the pathogenesis of lphs. no conflicts of interest reported. leptospirosis, a zoonotic bacterial disease with a worldwide distribution, is a re-emerging disease in humans and dogs. acute renal and hepatic failure are the most frequently reported clinical manifestations of canine leptospirosis. the aim of this study was to describe clinical, laboratory and radiological features, the outcome as well as the distribution of leptospira serogroups in dogs with leptospirosis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . medical records of dogs diagnosed with leptospirosis were evaluated retrospectively. diagnoses were based on microscopic agglutination testing (mat), blood/urine pcr, and histopathology (levaditi staining). mat-titers ≥ : against non-vaccine and ≥ : against vaccine serovars or a -fold rise of titers within - weeks were considered diagnostic. dogs met the inclusion criteria. in dogs diagnostic mat-titers were present (mainly against serogroups grippotyphosa ( %), australis ( %), and pomona ( %). at initial presentation, the most common clinical signs were lethargy ( %), anorexia ( %), vomitus ( %), a painful abdomen ( %), diarrhea ( %), oliguria ( %), tachypnea ( %), delayed capillary refill time ( %), pale mucous membranes ( %), fever ( %), hypothermia ( %), and icteric mucous membranes ( %). abnormal findings of the cbc included anemia ( %), thrombocytopenia ( %) and leukocytosis ( %). biochemistry abnormalities included increased creatinine concentrations ( %), increased liver enzyme activities ( %), hyperbilirubinemia ( %), hyperphosphatemia ( %), hyponatremia ( %), and hypoalbuminemia ( %). urinalysis often revealed glucosuria ( %) and an elevated urine-protein/creatinine-ratio ( %). radiological pulmonary changes were detected in % of the dogs initially or during the course of disease. dogs died or were euthanized, of them due to "leptospiral pulmonary hemorrhage syndrome". in this study, non-vaccine serogroups were the most common serogroups detected by mat. in the majority of patients renal ( %) and/or hepatic ( %) disease was detected. a pulmonary form of leptospirosis was present in % of the dogs. lung involvement represented a severe complication causing increased mortality depending on the severity of respiratory signs. no conflicts of interest reported. leptospirosis is a zoonotic disease that can affect multiple organs with renal and hepatic involvement being considered to be the most common. the aim of this study was to evaluate a large number of dogs with leptospirosis for cardiac and/or exocrine pancreatic involvement. a total of dogs were diagnosed with leptospirosis based on clinical signs and either microscopic agglutination test, blood/ urine polymerase chain reaction, and/or histopathology. at the time of admission and, in most patients, after an average of two weeks canine pancreatic lipase immunoreactivity (cpli, as measured by spec cpl â ), ultrasensitive cardiac troponin i (ctni), and c-reactive protein (crp) were analyzed. data were analyzed with non-parametric statistics. the level of significance was set at p < . . upon admission, common clinical signs reported included lethargy (n = ), vomiting (n = ), abdominal pain (n = ), dyspnea (n = ), pale mucous membranes (n = ), oliguria (n = ), hypothermia (n = ), and fever (n = ). anemia (n = ), thrombocytopenia (n = ), leukocytosis (n = ), were frequently reported hematology findings. increased concentrations of creatinine (n = / ), phosphorus (n = / ), alt (n = / ), sap (n = / ) and bilirubin (n = / ) were also frequently recorded. crp (median: . mg/l; range: . - . mg/l, reference interval (ri): . - . mg/l), ctni (median: . ng/l; range: . - . ng/l, ri: - . ng/l), and cpli (median: lg/l; range: - lg/l, ri: - lg/l) concentrations were above the upper limit of the reference intervals in / ( %), / ( %), and / ( %) dogs, respectively and serum cpli concentration was above the suggested cut-off value for a diagnosis of pancreatitis in / ( %) dogs. crp and ctni, but not cpli were higher upon admission compared to the re-check measurement (p = . and . , respectively). dogs with increased serum cpli concentrations also showed a higher proportion of dogs with increased serum ctni concentrations (p = . ). there was no statistically significant correlation of cpli concentrations with a history of abdominal pain and/or vomiting. biochemical results were compatible with multiple organ impairment with involvement of kidneys, liver, heart, and exocrine pancreas where at least two organs were affected in / ( %) dogs. forty ( %) of dogs recovered, ( %) died, and ( %) were euthanized. ctni and cpli were higher in non-survivors, but these differences did not reach statistical significance. however, the number of organs affected and outcome were significantly correlated (p = . ). our data suggest that infection with leptospira is characterized by a systemic inflammation with variable multiple organ involvement and damage, often including the heart and also the exocrine pancreas. the study was funded by texas a&m university. the primary author and two co-authors work at the gi laboratory, texa a&m university. canine bartonellosis is increasingly recognized worldwide and may be associated with diverse clinical manifestations. recent evidence suggests that bartonellosis also causes lameness and polyarthritis in dogs. however, pcr amplification of bartonella dna and isolation of bartonella species from canine synovial fluid (sf) samples have rarely been reported. canine leishmaniosis (canl) due to leishmania infantum is a multisystemic disease commonly associated with polyarthritis. based on the hypothesis that concurrent bartonella infection may be a contributing factor for the development of arthritis in dogs with canl, the main objective of this study was to investigate the microbiological and molecular prevalence of bartonella spp. in dogs with naturally-occurring canl, with or without cytologically documented arthritis. from a previous study, dogs with canl were retrospectively studied for bartonella spp. infection. diagnosis of canl was based on compatible clinical and clinicopathological abnormalities, positive serology, and lymph node or bone marrow (bm) cytology. dogs with serological evidence of other vector-borne infections (anaplasmosis, borelliosis, dirofilariosis and ehrlichiosis) and dogs recently vaccinated or medicated were excluded from the study. arthritis defined as a neutrophil percentage in excess of % of nucleated cells in sf cytology was documented in / ( . %) of dogs. a total of archived specimens from dogs, including edta-anticoagulated blood samples, bm and sf aspirates were tested for bartonella spp. dna using a bartonella alpha proteobacteria growth medium (bap-gm) diagnostic platform. eight ( . %) dogs were infected with one or two bartonella species, including candidatus bartonella merieuxii(n = ), b. henselae sa (n = ) and b. rochalimae (n = ). bartonella spp. dna was amplified from bm in dogs and from blood in dogs but was not amplified from any sf sample. overall, ( . %) dogs with and ( . %) dogs without arthritis were infected with a bartonella species. the prevalence of bartonella spp. dna in the dogs with or without arthritis did not differ (v test for independence, p = . the prevalence of giardia in dogs ranges between . % and . %, with a higher prevalence in puppies. however, the risk factors for giardia infection around weaning have been poorly described. the aim of the study was to evaluate risk factors for giardia infection in puppies during the first weeks of life and to determine an impact of this parasite on feces quality. puppies from litters living in a breeding kennel were followed between and weeks of age. each puppy was treated with fenbendazole (panacur â , msd, france, mg/kg, per os, q h) for consecutive days at , , and weeks of age. for each puppy, fecal consistency was evaluated using a -point scale. excretion of enteropathogens was evaluated by qpcr for canine parvovirus type (cpv ), qrt-pcr for canine coronavirus (ccv), coproantigens quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for any eggs and oocysts. a generalized linear mixed model (proc glimmix) with giardia infection as a binary outcome was used to assess the following effects: breed size, age, and cpv , ccv and isospora ohioensis infections. a linear mixed model (proc mixed) with fecal score as outcome was used to determine the following effects: breed size, age, and giardia, cpv , ccv and i. ohioensis infections. a total of fecal samples were collected; cpv , giardia, i. ohioensis and ccvwere detected in respectively . %, %, . % and . % of the samples. the risk of giardia infection increased with age (odd ratio= . ; %ci= . - . ; p = . ). neither breed, nor cpv , ccv and i. ohioensis infections influenced risk of giardia infection (p = . ; p = . ; p = . ; p = . respectively). giardia infection did not impact feces quality (p = . ), whereas a significant influence of cpv (p < . ), ccv infection (p = . ) and breed size (p < . ) was evidenced. this study underlines that even with an adapted deworming program the eradication of giardia is difficult to obtain in large dog packs. the higher prevalence of giardia in puppies of weeks and older could be linked with the immunity gap during this period. giardia was not associated in our study with an increased risk of diarrhea. the lack of pathogenicity of the parasite per se could be hypothesized, but also an efficacy of the treatment for the prevention of the clinical signs or a local and systemic immunity limiting clinical signs. financial support from royal canin. hepatic encephalopathy (he) can occur in dogs as a complication of primary liver disease or as consequence of congenital portosystemic shunts (pss). the aim of this study was to assess magnetic resonace spectroscopy (mrs) usefulness in he diagnosis. twenty dogs with a presumptive diagnosis of he were enrolled. inclusion criteria were: a clinical examination, a blood cell count and biochemical panel, at least one plasmatic ammonia determination higher than lmol/l, optional hystopatology of the liver, requirement of magnetic resonance imaging (mri) investigation by the clinician. even though mrs represents a non-invasive procedure, informed consent was obtained from dogs'owners. he diagnosis was confirmed in / : / had a pss, / showed hepatic microvascular dysplasia and / had hepatic cirrhosis histologically confirmed. the control-group was made of patients retrieved from our database that underwent mri without showing any abnormalities on brain scans. the mr protocol included t -weighted fast spin-echo, t -weighted sequences, proton density, and radio-frequency pulses gradient-echo. on mri examination cerebral atrophy was evident in all the patients ( / ), ranged from mild to severe. in / patients symmetrical bilateral hyperintensities in the globus pallidus on t -weighted images were evident. mrs was performed using a short te ( ms) and it was defined a volume of interest in the parieto-occipital region by -mm side cube ( cc of volume). glutamine-glutamate (gln), n-acetylaspartate and n-acetyl aspartyl glutamate (naa), choline derivatives (cho), and myo-inositol (ins) were analyzed. comparing spectra from the he affected dogs with those from the control-group decreased values of ins, cho, naa as well as high field mri combined with brain mrs provide accurate and non-invasive diagnosis of canine he. in accordance with human medicine publications, it could be state that mrs has a role in he diagnosis and follow-up with particular mention monitoring. no conflicts of interest reported. diagnosis of hepatobiliary diseases often requires hepatic tissue sampling for histologic evaluation. the laparoscopic technique is a safe method and allows acquisition of tissue by wedge and needle biopsies from different liver lobes in a minimally invasive way.specimens obtained with an -gauge biopsy needle must be interpreted with caution due to considerable variability in tissue involvement with certain disease processes. we hypothesized that needle biopsy specimens would produce findings divergent from those produced by wedge biopsy specimens. the goal of the study was to compare histological findings from two laparoscopic biopsy methods (wedge and needle) and assess which sampling technic can represent the overall disease process. procedure: all dogs included in this prospective study study were suspected diffuse hepatic disease and underwent laparo-scopic hepatic biopsy (wedge and needle - g) between and . all biopsy specimens were examined on the basis of morphologic criteria and a comparison was made between the two types of biopsies procedures according to wsava liver standardization group morphologic criteria. results: twenty-two dogs were included. no complications were reported during the laparoscopic procedure. the median number of portal triads per needle biopsy specimen was (range, to ) compared to (range ; ) with wedge biopsy specimen. the median length of needle biopsy specimens was mm; (range, to mm) and mm for all wedge biopsies. on the basis of biopsy interpretation, the diagnosis was overall similar with the two methods: dogs had vacuolar hepatopathy, acute cholangitis, non-specific acute form in dogs. chronic hepatitis with cirrhosis was found in cases, dogs had diffuse neoplasia and miscellaneous hepatic disorders. the fibrosis was considered to be severe in dogs, moderate in dogs and mild in dogs. no quantitative and qualitative difference was observed between the two types of biopsies specimen. this study demonstrates that the biopsies with a needle length of at least mm brings satisfactory information for the evaluation of most of the inflammatory, vacuolar hepatopathies, fibrosis and diffuse tumoral infiltrations. wedge biopsies allow to examine the largest number of portal triad, more contributory for certain forms of cholangitis affecting larger canals and for a single case, images of peri-hepatitis were counted at the level of the capsule. fibrosis does not seem to be more important in the sub-capsular zone contrary to what is observed in human pathology. no conflicts of interest reported. indocyanine green (icg), a fluorescence dye, is excreted solely by the liver without enterohepatic re-circulation. hence it has been used for decades as an ideal albeit invasive marker of hepatic function and blood flow in cats and dogs. here we evaluated the feasibility of a minimally invasive transcutaneous icg clearance to assess hepatic function instantaneously. transcutaneous icg clearance was performed in healthy research cats and healthy research dogs with normal liver function (bile acid stimulation test, ammonia tolerance test) using a modified device (kidney int : with an excitation wave length of nm and an emission wave length of nm. the devices were placed on different locations (lateral thoracic wall, ventrolateral abdomen, metatarsus and antebrachium) and fixed with a light bandage. to find a suitable dose to reach adequate transcutaneous peak concentrations escalating doses of icg ( . , . and . mg/kg) were injected intravenously with a wash out period of at least h in-between. measurement was continued for hour after injection in awake animals moving freely. the resulting icg disappearance curves were visually inspected to find best location (minimal artifacts, acceptable background noise) and dose. in all animals a dose of . mg/kg was deemed ideal and the thoracic and abdominal wall gave consistent results. half-life of icg clearance was calculated using a one-compartment model. half-lives in cats were . , . and . minutes, respectively; in dogs: . , . and . minutes, respectively. in conclusion, the transcutaneous assessment of icg clearance is feasible in a clinical setting. results are obtained within one hour and can be assessed instantaneously. the procedures are minimally invasive and well tolerated by the animals. given that most patients with a presumed liver problem undergo abdominal ultrasound no further clipping of hair is necessary as the device might be placed in this area. further studies are necessary to obtain reference values in healthy pets and those with various conditions leading to impaired hepatic function. no conflicts of interest reported. gallbladder diseases like gallbladder mucocele and cholecystitis can reduce gallbladder motility and may lead to cholestasis. since impaired gallbladder emptying contributes to sludge and gallstone formation, the evaluation of gallbladder motility requires accurate and appropriate methodology. three-dimensional ( d) ultrasonography has been shown to be accurate and appropriate tool for measurement of gallbladder volume in humans. therefore, we applied this novel technique for the first time to study preprandial and postprandial gallbladder volume in healthy mixed-breed dogs and compared the results to twodimensional ( d) ultrasonography. the dogs were placed in dorsal recumbency to obtain ultrasonographic measurements of the gallbladder. measurements by both d and d ultrasonography were recorded in preprandial state and after ingestion of full-fat milk. the preprandial and postprandial gallbladder volumes determined by d ultrasonography were significantly higher than corresponding volumes by d ultrasonography ( . ae . vs . ae . and . vs . ml/kg, respectively, p < . ). in d ultrasonography, most dogs ( / [ %]) had a preprandial gallbladder volume ≤ . ml/kg. however, in d ultrasonography, / ( %) of dogs had a preprandial gallbladder volume ≥ . ml/kg. gallbladder contraction index was higher in d ultrasonography than d ultrasonography, however, it did not reach statistical significance (p = . ). in conclusion, d ultrasonography showed larger gallbladder volumes than d ultrasonography in healthy dogs. it seems that d ultrasonography is appropriate adjunct device to d ultrasonography to estimate gallbladder volume when d ultrasonography could not detect whole gallbladder volume. more research is needed to determine clinical value of d ultrasonography in canine gallbladder imaging. no conflicts of interest reported. the aim of our study was to compare high-definition oscillometry (hdo) and doppler ultrasonographic measurements with direct blood pressure measurements in conscious dogs. the doppler study was performed by three investigators and by using different sphygmomanometers with different sized cuffs. devices and measurement sites were changed randomly among the investigators. cuffs were wrapped around the antebrachium in the forelimb or around the mid-metatarsus in the hind limb. in addition to the limb sites, cuffs were also placed around the base of the tail in case of the hdo method. cuff sizes were - % of the measured limb circumferences during the doppler measurement. for the hdo method all measurements were performed by the same investigator and the cuffs provided by the manufacturer were used: the smallest cuff was used for the hind limb and tail, and the medium cuff was used for the forelimb measurements. dogs were gently held in lateral recumbent position, measurement were performed on the nondependent limbs. radio-telemetry transducers were implanted to the right femoral artery some months to a year preceding the blood pressure measurements for reasons unrelated to our study. direct blood pressures varied - mmhg and - mmhg during the doppler and hdo measurements, respectively. two-hundred paired simultaneous doppler and direct measurements from dogs and paired simultaneous hdo and direct telemetric measurements from dogs were obtained. at least successful consecutive measurements could be obtained by the same investigator at the same site during the doppler or hdo measurement in and cases, respectively. thus, the mean of these measurements could be calculated similarly to the established everyday clinical practice. bias (mean difference), precision (standard deviation) and limits of agreements were calculated both from the individual paired measurements and from the means of the consecutive measurements using bland-altman spot analysis. systolic measurement performed on the tail with the hdomethod yielded the smallest bias and deviation and the best limits of agreement during this study. both doppler and hdo-measurements performed on the forelimb overestimated, while hind limb measurement underestimated the direct telemetric pressures. results of all three measurement sites by hdo performed better than forelimb or hind limb doppler-measurements, however hdo-measurements were more difficult to obtain and more often resulted with measurement failure compared to the doppler technique. cuff size above % of the measured limb circumference showed better results than smaller cuff sizes during the doppler measurements. no conflicts of interest reported. the doppler technique is considered the most repeatable indirect method to measure systolic arterial pressure (sap) in dogs. however, recent studies emphasized the effect of body position and used limb on sap measurement. the aim of this study was to determine whether a difference existed in sap measured simultaneously in dogs using different limbs, with two doppler units by two different operators. sixty clientowned dogs, admitted to the veterinary hospital for different reason, were enrolled. they were divided in groups based on body size: small breed dogs (< kg); medium breed ( - kg); large breed (> kg). for each dog the anxiety status was recorded. sap was measured via doppler technique when dogs were in right lateral recumbency in a quite environment. right and left forelimb sap and left forelimb and left hindlimb sap were recorded simultaneously, with two identical doppler units equipped with headphones, by two operators. measurement was performed based on the acvim guidelines. five measurements were recorded, the higher and lower values were discarded from the analysis. the relationship of mean sap for each limb with body weight, sex, anxiety status and sap value was evaluated. mean ae sd sap was significantly higher for the right forelimb ( . ae . ) compare to the left forelimb ( . ae . ) on overall population. the difference was significant for large breed dogs, males and dogs with sap ³ mmhg. sap was higher for the left forelimb ( . ae . ) compare to the left hindlimb ( . ae . ) on overall population. the difference was significant for medium and large breed dogs, females, calm animals and dogs with sap ³ mmhg. the mean sap from the left forelimb recorded by two different operators at two different moments, were compared and no difference was evident. in conclusion, sap measurement from different limbs, in dogs in right lateral recumbency, is poorly correlated. measurement of sap from the left forelimb is more repeatable during time and between different operators. sap trend monitoring should be done using the same measurement site for any animal. no conflicts of interest reported. amlodipine has been considered the treatmenf of choice for hypertension in cats for more than a decade. there is, however, an unmet need for a cat-specific formulation. the aim of the study was to assess the efficacy of chewable amlodipine tablets in reducing systolic blood pressure (sbp) in cats diagnosed with hypertension. seventy-seven client-owned cats were included in the study (mean age years). the study was randomised, double-blind, placebo controlled, and consisted of two phases. in the blinded phase, cats received . mg/kg amlodipine once daily for days. if they responded the dose remained the same to day . for non-responders, the dose was increased to . mg/kg. thirty-five cats received placebo following the same protocol. arterial blood pressure was measured using a high definition oscillometry method. at day a responder was defined as a cat showing a decrease of sbp to ≤ mmhg or a decrease from baseline of at least %. after days all cats continued with amlodipine for - months in an open phase with the placebo cats repeating the same dose escalation protocol as in the blinded phase. the responder rate was % in the amlodipine group and % in the placebo group following the dose escalation from day being applied to % and % of cats receiving amlodipine and placebo respectively. cats receiving amlodipine were . ( % ci . to . ) times more likely to be classified as responders when compared to those receiving placebo (logistic regression model, p = . ). from a baseline value of . ae . and . ae . mmhg the mean sbp decreased to . ae . mmhg with amlodipine and to . ae . mmhg with placebo (repeated measures analysis of covariance model, p < . ) by day . the responder rate was not influenced by factors other than amlodipine treatment (e.g. baseline blood pressure, concomitant ace inhibitor therapy, renal disease). there were no differences between the amlodipine and placebo groups in the frequency of adverse events reported during the -day blinded phase. likewise, there were very few changes in the laboratory values over time in either group. the present study is the first large clinical trial to show that amlodipine is clearly superior to placebo in the treatment of cats with hypertension. the chewable amlodipine formulation effectively reduced sbp, had a good palatability and was well tolerated. it can be used concomitantly with ace inhibitors and in cats with renal disease. conflicts of interest: m. huhtinen and j. aspegr en are employees of the sponsor j. elliott has the following information to disclose: amlodipine is the treatment of choice for feline hypertension. limited published data exist on serum concentrations achieved in hypertensive cats. the aim of the study was to assess serum amlodipine concentrations in cats treated with a new formulation of amlodipine and relate these to the blood pressure reduction achieved. seventy-seven client-owned hypertensive cats were enrolled into a randomized, double-blind and placebo controlled study consisting of two phases. in phase one, cats (group a) received . mg/kg amlodipine once daily for days. if they were deemed to have responded (see below) the dose remained the same to day . for non-responders, the dose was increased to . mg/kg. thirty-five cats (group b) received placebo following the same protocol. blood pressure was measured using high definition oscillometry. a responder was defined as a cat showing a decrease of systolic blood pressure (sbp) to ≤ mmhg or a decrease from baseline of ≥ %. following day (phase ), group a continued on amlodipine and group b switched to amlodipine and the dose was adjusted as per phase . both groups were followed for days on amlodipine. blood was collected at days (group a) and (both groups) and serum [amlodipine] measured by liquid chromatography mass spectrometry. the sbp measured on treatment was calculated as percentage of the baseline sbp and plotted against serum [amlodipine] using a sigmoidal emax model (winnonlin software). data are expressed as mean ae se. the serum concentrations of group a cats that remained on . mg/kg were . ae . ng/ml whereas those switched to . mg/kg were . ae . ng/ml. when data from groups a and b were pooled, a sigmoidal relationship between percentage baseline sbp and serum [amlodipine] was found. estimated values of lowest percentage baseline blood pressure on treatment (emax) was . ae . %, with an ec value of . ae . ng/ ml and a slope function of . ae . . the serum concentration required to reduce blood pressure by % was estimated to be ng/ml. the present study related blood pressure reduction to serum [amlodipine] in feline clinical hypertension. the limitations of this study were the limited number of blood samples collected and lack of information relating to the exact timing of blood sampling relative to dosing in some cats. however, these data could be used to define appropriate therapeutic serum [amlodipine] in hypertensive cats. m huhtinen is an employee of the sponsor; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd.; l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health. commercial assays for the measurement of canine and feline pancreatic lipase immunoreactivity (spec cpl â and spec fpl â , respectively; idexx laboratories, westbrook, me, usa) have been available for a few years and have previously been analytically and clinically validated. recently, new commercial assays for the measurement of these parameters have become available, though neither one of these assays have been analytically or clinically validated in the literature. thus, the goal of this study was to compare these newly available assays to the established assays. leftover serum samples from diagnostic submissions to the gi laboratory were collected based on certain parameters (e.g., results throughout the working range of the assay, good quality sample or hemolytic, lipemic, or icteric sample) and were assigned random sample id numbers. the samples were evaluated by spec cpl â or spec fpl â , sent on dry ice to the klinik am hochberg, and one aliquot of each sample was blindly submitted to laboklin for measurement of cpli and fpli by their newly released in-house assay and also to the gi lab at texas a&m university for repeated analysis by spec cpl â and spec fpl â to exclude any effect of shipping. there was no significant difference between serum cpli or fpli concentrations before or after shipping at the gi lab (pvalues for wilcoxon matched-pairs signed rank tests: . and . , respectively). in contrast, there was a significant difference between serum cpli or fpli concentrations between the newly released assays and the previously established assays (pvalues < . and . , respectively). while there was a significant correlation between the newly released and the previously released assays (spearman r: . and . , respectively), this correlation was very poor for assays that supposedly measure the same analyte. also, the interpretation for serum cpli and fpli results between the previously developed assays and the new assays did not agree for many of the samples. finally, both newly developed assays showed some erratic results. in conclusion, the newly released assays for the measurement of cpli and fpli do not agree with previously established and validated assays, provide different interpretations, and show erratic results. thus, further research is needed before these newly released assays could be recommended for clinical use. dr. steiner serves as director and dr. suchodolski serves as associate director of the gastrointestinal laboratory at texas a&m university. dr. steiner also serves as a paid consultant to idexx laboratories, westbrook, me, usa. both the gastrointestinal laboratory and idexx laboratories offer cpli and fpli testing on a fee-for-service basis. digestive health is a main concern for growth, morbidity and mortality in weaning puppies. fecal immunoglobulin a (iga) has been suggested as a useful noninvasive biomarker for mucosal immunity. the purpose of this study was to evaluate the effect of infection with enteropathogens on fecal iga concentrations in puppies and that of physiological factors such as age and breed size. puppies from breeding kennels were included in the study. puppies were between and weeks of age (meanaestandard deviation (sd): . ae . weeks). depending on the mean adult body weight of their respective breed, the puppies were divided into small (if mean adult body weight < kg) or large (> kg) breed puppies. for each puppy, fecal consistency was evaluated using a -point scale and feces were collected for the evaluation of presence of fecal enteropathogens and fecal iga concentrations. the presence of enteropathogens in fecal samples was evaluated by qpcr for canine parvovirus type (cpv ), qrt-pcr for canine coronavirus (ccv), coproantigen quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for other parasite eggs and oocysts. fecal iga concentrations were measured by an elisa test. statistical analyses were performed using sas software. a linear mixed model (proc mixed) with fecal iga concentration as outcome was used to determine the following effects: enteropathogen infection, breed size, age, and fecal score. the respective influence of litter and breeding kennel as random effects was also determined. data is presented as mean ae sd. small breed dogs represented . % ( / ) of the total number of dogs included. at least one enteropathogen was identified in . % of puppies ( / ). fecal iga concentration was significantly influenced by fecal enteropathogens (p = . ). puppies infected with at least one enteropathogen had significantly lower fecal iga concentrations than puppies without any enteropathogens ( . ae . lg/g vs. . ae . lg/g). breed (p = . ), but not age (p = . ), influenced iga concentration. small breed puppies had significantly higher fecal iga concentrations than large breed puppies ( . ae . lg/g vs. . ae . lg/g). no significant relationship between fecal iga concentration and feces quality was evidenced (p = . ). this study suggests that fecal iga concentration is a promising marker for subclinical infection by at least one enteropathogen and confirms that digestive physiology varies with the breed size. a link between lower digestive immunity and higher susceptibility to enteropathogen infection needs further investigation. conflicts of interest: financial support of royal canin. canine chronic enteropathies (cce) include diet-responsive, antibiotic-responsive, and immunosuppressive-responsive enteropathies (ire). this prospective study was designed to evaluate a commercial hypoallergenic dry diet a containing oligopeptides as the only protein source for the management of dogs with ire and as an alternative to immunosuppressive therapy over a week period. nineteen dogs across france and quebec entered the study. dogs with food or antibiotic-responsive chronic enteropathy, hypoproteinemia, or treated with immunomodulating drugs were excluded from the study. dogs were included in the study after complete clinical, ultrasonographic, endoscopic evaluation and histopathological evaluation of intestinal biopsies showing signs of intestinal inflammation. the owners were instructed to feed exclusively the study diet a . canine inflammatory bowel disease activity index (cibdai) scores, fecal scores as observed by the dog-owners, and body weight were evaluated at baseline, , and weeks after inclusion. dietary treatment was regarded successful if the cibdai score was reduced by at least %. the protocol has been reviewed and accepted by royal canin ethics committee and owners completed an informed consent. results are presented as meanaesd (range). statistical comparisons were performed with a wilcoxon test. thirteen dogs ( intact males, neutered and intact females) completed the trial. seven dogs were excluded ( diagnosed with giardia, s with no histological evidence of inflammation, with hypoadrenocorticism). mean age was . years ae . ( . - . ), mean body weight . kg ae . ( . - . ). cibdai score was . ae . ( - ) at inclusion, was . ae . ( - ) after weeks and was . ae . ( - ) after weeks (p = . and p = . vs inclusion, respectively). fecal scores after [ . ae . ( - )] and weeks [ . ae . ( - ) ] were improved compared to inclusion scores . ae . ( - ) (p = . and p = . , respectively). the low molecular weight poultry feather hydrolyzed proteinbased dry extruded diet a appears to be effective in the management of idiopathic ibd without any concurrent immunosuppressive drug over the week period of this pilot study. these preliminary findings should be confirmed by a prospective, randomized double blind study. feline pancreatitis is the most common exocrine pancreatic disorder with varied mortality. however, there is no available and reliable method to evaluate the severity and prognosis of the disease. ninety-two cats diagnosed as pancreatitis with acute onset of compatible clinical signs and a positive snap â fpl tm test between october and september were enrolled in this study. all cats were divided into survival (n = ) and nonsurvival (n = ) groups. fifty-two parameters including signalments, clinical signs, physical examinations, clinicopathological examinations, diagnostic images, complications and concurrent diseases were analyzed and compared between the two groups. parameters with p ≤ . were considered for further analyses. the mortality in this study was . %. hematocrit, albumin, bun, creatinine, total bilirubin, calcium, phosphorous, body temperature, systolic blood pressure, the body cavity fluids, complications, e.g. systemic inflammatory response syndrome (sirs) and acute renal failure (arf) were found to be significantly associated with disease severity and prognosis, and were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each cat by summing all weighting factors. the total scores of each cat ranged from to . the severity scores in this study achieved an area under receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with the sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was significant difference (p < . ) between the two groups of the cut-off point. furthermore, the mortality reached to % when the score more than . the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in cats with pancreatitis. no conflicts of interest reported. convincing evidence for the role of clostridium (c.) perfringens as a primary pathogen in acute haemorrhagic diarrhoea syndrome (ahds) in dogs was recently found. it is suspected that clostridial toxins, especially c. perfringens enterotoxin, play a relevant role in the disease process. however, to date enterotoxigenic c. perfringens strains have only been described in single case reports. thus, the aim of this study was to indentify the specific c. perfringens genotype involved in adhs. small intestinal biopsies were collected with a sterile single-use biopsy forceps from ten dogs with ahds and immediately cultured. in / dogs, clostridial strains were isolated and identified as c. perfringens by mass spectrometry using maldi-tof ms. c. perfringens colonies from each dog were submitted for specific detection of the four major toxin genes (alpha, beta, epsilon, and iota), the enterotoxin gene, and the beta toxin by multiplex pcr. every clostridial isolate was typed as c. perfringens type a based on the detection of the alpha toxin encoding gene. in / isolates, additionally the beta toxin gene was identified, however, none of clostridial strains encoded for the c. perfringens enterotoxin gene. the results of this study suggest that c. perfringens type a is the most important c. perfingens genotype involved in the disease process of dogs with ahds. although c. perfringens enterotoxinhas been associated with intestinal diseases in humans, dogs, horses, pigs, and other animal species, this enterotoxin is most likely not responsible for the intestinal lesions in dogs with ahds. no conflicts of interest reported. p < . ). lack of treatment response was significantly associated with il (il: / , sre: / , fre: / , are: / ; p < . ). anemia, thrombocytopenia, and increased plasma urea were significantly associated with il (anemia: il: / , sre: / , fre: / , are: / , p < . ; thrombocytopenia: il: / , sre: / , fre: / , are: / , p = . ; increased urea: il: / , sre: / , fre: / , are: / , p = . ). hypoalbuminemia (< g/l) and hypocobalaminemia (< pg/ml) occurred significantly more frequently in dogs with sre (hypoalbuminemia: il: / , sre: / , fre: / , are: / , p < . ; hypocobalaminemia: il: / , sre: / , fre: / , are: / , p = . ). results of this study show that elderly and large breed dogs were more frequently affected with il and sre compared to other etiologies and both il and sre were associated with greater disease severity and/or a negative outcome. in comparison, anemia, thrombocytopenia, and increased plasma urea were most frequently detected in il whereas severe hypoalbuminemia and hypocobalaminemia were significantly associated with sre. no conflicts of interest reported. alpha -proteinase inhibitor (a -pi) is a proteinase-resistent protein that can be quantified in fecal, urine, and serum samples from dogs. recently, increased fecal and urinary canine a -pi (ca -pi) concentrations have been described in dogs with gastrointestinal diseases (e.g., inflammatory bowel disease [ibd] , but also in dogs with exocrine pancreatic insufficiency) and in dogs with chronic hepatitis or chronic kidney disease, respectively. decreased serum ca -pi concentrations have been reported in dogs with ibd, protein-losing enteropathy (ple), and hypocobalaminemia. treatment protocols for dogs with ibd and/or ple commonly include corticosteroids, but the effect of corticosteroid therapy on serum ca -pi concentrations have not yet been reported. the aim of this study was to evaluate the effect of hydro-cortisone on serum ca -pi concentrations in healthy dogs. twelve healthy beagle dogs were randomly allocated to a placebo-group (n = ) and to a treatment group (n = ; hydrocortisone-group). the placebo-group received an empty gelatin capsule po q h, whereas the hydrocortisone-group was treated with hydrocortisone at a dose of . mg/kg po q h. serum samples were obtained at baseline and on day , , , , and during treatment as well as day , , , , and post-treatment for all dogs. serum ca -pi concentrations were measured at all time points using an in-house radioimmunoassay. a mann-whitney u test was used to compare the baseline measurements of both groups. the effect of hydrocortisone-treatment on serum ca -pi concentrations was evaluated by comparing ca -pi at baseline and during treatment and between baseline and posttreatment period using a manova. baseline serum ca -pi concentrations did not differ between the hydrocortisone-and the placebo-group (p > . ). serum ca -pi concentrations increased significantly (p = . ) during the treatment period in the hydrocortisone-group (baseline [median in mg/l: , ] ). in contrast, no difference was observed between both groups when comparing serum ca -pi concentrations at baseline and during the post-treatment period (p > . ). this study showed that hydrocortisone-treatment over weeks did affect serum ca -pi concentrations in healthy dogs. whether corticosteroid therapy has any effects on fecal or urine ca -pi concentrations in healthy dogs remains to be determined. the author works at texas a&m university, whose gi lab currently offer a commercial assay for faecal alpha -proteinase inhibitor. canine chronic enteropathy (ce) is a common, but poorly understood syndrome, with variable response to therapy and prognosis. there is a need for novel biomarkers that are specific for intestinal disease and that provide objective measures of disease severity, progression, and prognosis. serum citrulline is a useful biomarker in human intestinal disease as it is specific to the small intestine and indicates globally reduced enterocyte mass and absorptive function in various disease states. it is used to determine, quantitatively, intestinal integrity at the enterocyte level and is not influenced by nutritional or inflammatory status. the aim of this study was to determine whether serum citrulline can be used as a biomarker for ce in dogs. in this retrospective study, computer records from the university of liverpool small animal teaching hospital were used to identify dogs with ce. disease severity was quantified by cibdai. controls were age-and breed-matched dogs without gastrointestinal disease. serum citrulline was measured by ultra-high performance liquid chromatography with tandem mass spectrometry. in dogs with ce, serum citrulline concentration was measured at presentation and at various time points after starting treatment. serum citrulline was measured in dogs with ce and controls. dogs responded to dietary manipulation (food-responsive enteropathy, fre) and responded to antibacterials (antibiotic-responsive diarrhoea, ard), with a further having invasive mucosal bacteria, of which one responded to antibacterials and one was refractory. dogs were diagnosed with idiopathic ibd (on the basis of exclusion of known causes and failure to respond to therapeutic dietary and antibiotic trials), of which responded to immunosuppressive therapy, were refractory, and were lost to follow-up. serum citrulline concentration did not differ between dogs with ce (median . lg/ml, range . - . ) and controls (median . lg/ml, range, . - . , p = . ). there was also no difference in serum citrulline concentration amongst dogs with fre, ard, ibd, and controls (p = . ). serum citrulline did not differ between dogs that responded well, or were refractory to treatment (p = . ), between dogs with and without protein-losing enteropathy (p = . ), or between dogs that survived and that were euthanased because of ce (p = . ). serum citrulline did not correlate with cibdai (r = . ). these findings do not support the use of serum citrulline as a biomarker in determining diagnosis, prognosis, or quantifying severity in dogs with ce. one of the co-authors (marco caldin) has a diagnostic laboratory offering citrulline assays. chronic enteropathy (ce) is a multi-factorial disease, which involves aberrant immune responses to commensal bacteria or dietary antigens. macrophages have an important role in human disease but little information is available in canine intestine. data to date have relied solely on macrophage identification using mac , an antibody directed against calprotectin, which recognizes both macrophages and neutrophils. in this study an alternative antibody for macrophages, am- k, directed against a scavenger receptor (cd ) was used and distribution of both markers was compared. this antigen is of interest as positive cells accumulate in intestine of humans with ce. endoscopic duodenal biopsies were obtained from seven crossbreed dogs. serial histologic sections were stained with mac or am- k. positively-stained cells were counted from random areas from both villous and crypt regions. stained cell localisation was subjectively evaluated and the percentage of positively stained cells from the total nucleated cells per , lm in the villus or crypt was compared between both antibodies using a wilcoxon signed-rank test. mac and am- k did not co-localize on serial sections. there were significantly more am- k positive cells than mac in the crypts ( . % [ - . ] versus . % [ - . ], p = . ). in contrast there was no difference in expression of either markers in the villi ( . % [ - . ] versus . % [ - . ], p = . ). this study reports for the first time the existence of two populations of macrophages in canine intestine. these results in normal dogs will be used to explore further the distribution and function of macrophages in dogs with ce. no conflicts of interest reported. chronic diarrhea and vomiting are common clinical signs in dogs. primary (e.g., inflammatory, infectious, neoplastic, mechanical, or other) and secondary gastrointestinal diseases (e.g., exocrine pancreatic, hepatic, renal, or endocrine disease) are possible underlying causes. the aim of this study was to evaluate the final diagnoses in dogs with chronic diarrhea and/or vomiting and to determine the prevalence of various primary and secondary gastrointestinal diseases in dogs with these gastrointestinal signs. medical records of dogs presented between july and august with chronic diarrhea (d), vomiting (v) or both (diarrhea and vomiting [vd]) were retrospectively reviewed. dogs were included if a minimum work-up (hematology, plasma biochemistry profile, and fecal parasitology) had been performed and if a final diagnosis was recorded ( / ). a primary gastrointestinal disease was recorded in % of the cases ( / ) and included inflammatory diseases ( / , exocrine pancreatic insufficiency, hypoadrenocorticism, polyendocrinopathy, dilated cardiomyopathy, and leukemia in one dog each). in total, % of the dogs were presented with d ( / ) followed by % with vd ( / ), and % with v ( / ). d and vd were significantly more frequent in dogs with primary gastrointestinal disease (d: / , vd: / ), compared to dogs with secondary gastrointestinal disease (d: / ; vd: / ; p = . , chi square test). v was significantly more common in dogs with secondary gastrointestinal disease ( / ) as compared to dogs with primary gastrointestinal disease ( / ; p = . ). in this study, food responsive enteropathy ( %) was the most commonly diagnosed cause of chronic gastrointestinal signs. chronic pancreatitis was the most frequent cause of secondary gastrointestinal disease ( %). diarrhea was significantly associated with primary and vomiting with secondary gastrointestinal disease. no conflicts of interest reported. matrix metalloproteinases (mmps) and are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. intestinal mucosal levels of mmp- and - have been shown to be increased in animal models and human ibd. to our knowledge, the presense of mmp- and - has not been studied in the intestinal mucosal samples of healthy dogs as well as in canine spontaneous ibd. thus, the main aim of this study was to identify the presence of mmp- and - in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava histology standards, recorded findings of all samples were considered insignificant. pro-mmp- and - activities were found in / ( %) and / ( %) of the samples, respectively. among four different parts of the intestine of dogs, the ileum had the highest positivity rates of / ( . %) and / ( . %) for pro-mmp- and - activities, respectively. however, statistical analysis showed no significant difference of pro-mmp- and - activities between the separate parts of the intestine (p > . ). the enzyme activities ranged for pro-mmp- between . and . arbitrary units (au) and for pro-mmp- between . and . au. none of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active mmp- and mmp- . this study showed that pro-mmp- and - could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of mmp- and - in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases. no conflicts of interest reported. digestive perforation is called spontaneous when it arises in the absence of foreign body ingestion, gastric dilatation and volvulus, external trauma, or previous digestive surgery. in dogs many predisposing factors have been identified, including antiinflammatory administration, severe hepatic or renal disease, stress, shock, gastric hyperacidity, neoplasia and idiopathic inflammatory bowel disease. spontaneous digestive perforation has been uncommonly reported in cats. the objectives of this study were to describe the clinical characteristics of spontaneous digestive perforation in cats, and to compare the frequency of malignant versus non-malignant causes for these perforations. to be included in this study, the perforation had to be spontaneous and confirmed by exploratory surgery. the medical records of cats diagnosed as having spontaneous digestive perforation between and were reviewed. the mean age of cats was . years ( months to years). five cats had concurrent illnesses including viral upper respiratory tract disease, pancreatitis and chronic kidney disease. the most frequently reported signs included anorexia ( %), vomiting ( %), and lethargy ( %). histological examination was performed in cats and diagnosed alimentary lymphoma in % and inflammatory lesions in the other % of them. six cats had received anti-inflammatory within the previous months. half of them were finally diagnosed with lymphoma. five cats with lymphoma received chemotherapy.three cats died early in the postoperative recovery period, cats were euthanized to days after surgery, and cats were still alive at the end of this study. in the absence of pneumoperitoneum, clinical signs and clinicopathological abnormalities are not specific enough to allow differentiation between cats with gastrointestinal ulceration and those with perforation. in most cases, there is no diagnostic test that individually determine whether perforation has occurred or is impending, and clinicians should use of multimodality diagnostic procedures such as radiography, ultrasonography, endoscopy, and abdominal fluid cytopathology to avoid delay in diagnosis of digestive perforation. histological examination of ulceration is essential as lymphoma should be suspected in all cats presented with spontaneous perforation. the link between anti-inflammatory administration and spontaneous perforation in cats is not established. no conflicts of interest reported. campylobacter species are commonly isolated from faeces of dogs and cats with c. upsaliensis (cu) and c. helveticus (ch) being the most frequently isolated. these two species are usually not considered pathogenic in dogs and cats and are closely related to each other and to c. jejuni, the most common cause of bacterial gastroenteritis in humans in the developed world. interestingly, despite their close genetic relationship, in humans cu is considered a pathogen while ch is not. this study aimed to describe whole genomes of cu and ch isolated from dogs and cats and to in silico investigate their pathogenic potential with comparison to several published genomes of c. jejuni and c. coli. genomic dna was extracted from three isolates of each of cu and ch recovered from the faeces of healthy dogs and cats. sequencing was performed using an illumina miseq to generate base paired reads. reads were trimmed for both length and quality. contigs were assembled using the velvet assembler. the concatenated contigs generated for each assembly were quality ranked (by number, size, maximum length and n ) and the three top ranked assemblies were annotated using the prokka annotation tool. ribosomal mlst nucleotide sequences were used as a proxy for the core genome to compare the phylogeny of cu and ch with other species in the campylobacter genus and visualised as a neighbornet using splitstree. annotated draft genomes were clustered using orthomcl and pathogenic traits were investigated in silico using pathogenfinder and viru-lentpred software. the cu and ch draft genomes were~ . mb and . mb in size, and comprised on average and contigs, and on average and predicted genes, respectively. of these cu had on average and ch hypothetical proteins. using orthomcl, a core genome of and genes resulted for cu and ch, respectively. neighbornet trees based on ribosomal mlst nucleotide sequences and the core genome confirmed the close phylogenetic relationship of ch and cu within the campylobacter genus. pathogenfinder predicted all isolates as human pathogens with probabilities of . - . %. both pathogenfinder and virulentpred identified many pathogenic proteins in cu and ch of different functions (e.g. chemotaxis, transporter and motility systems) but considerably fewer than in c. jejuni and c. coli. this study provides many insights into the pathogenic potential of pet-associated emerging campylobacter pathogens and is to our knowledge, the first to report a draft genome of ch. no conflicts of interest reported. there are only few laboratory markers being evaluated for diagnosing and/or monitoring canine chronic enteropathies, including inflammatory bowel disease (ibd). s a belongs to the s /calgranulin-protein family and has been proposed to play a central role in both innate and acquired immune responses. it has been reported to be increased in stool samples, serum and/or intestinal mucosa in human patients with ibd. myeloperoxidase (mpo) is an enzyme found mostly in granulocytes. intestinal mucosal levels of mpo have been shown to be increased in animal models and human ibd. to date, s a and mpo levels in intestinal mucosal samples have been reported neither from healthy dogs nor from dogs suffering from ibd. to start investigating this aspect in dogs, the objective of this study was to evaluate mucosal s a and mpo levels in the small and large intestines by using enzyme-linked immunoassay (elisa) and spectrophotometric methods, respectively. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava standards the histologic findings of all samples were considered insignificant. s a concentrations were from the highest to the lowest: ileum, . ( . - . ) lg/l; colon, . ( . - . ) lg/l; duodenum, . ( . - . ) lg/l; and jejunum, . ( . - . ) lg/l. the concentration in the ileum was significantly higher than in all other segments (p < . ), and the colonic mucosal concentration was higher than the jejunal (p < . ). the highest mpo activity was found in the ileum ( . [ . - . ] da/min), followed by jejunum ( . [ . - . ] da/min), duodenum ( . [ . - . ] da/min), and colon ( . [ . - . ] da/min). mpo activity was significantly higher in ileal and duodenal than in colonic mucosal samples (p < . ). the jejunal mpo activity was higher than the colonic and duodenal activity (p < . ). this study showed that using elisa and spectrophotometry allow the detection of canine intestinal mucosal s a and mpo, respectively. the levels on s a and mpo seem to differ between certain parts of the intestinal mucosa of healthy dogs. both assays appear to be useful to further evaluate the role of s a and mpo in the pathogenesis of canine chronic enteropathies, including ibd. dr. heilmann, dr. suchodolski, and dr. steiner have a patent pending that includes the canine s a assay used in this study. the authors declare that they have no further conflicts of interest. the aim of the present study was to establish the incidence of innocent cardiac murmurs in a fairly large number of clinically healthy puppies. a second aim was to evaluate a possible correlation between the presence of an innocent cardiac murmur and a lower hematocrit value. puppies of certain breeds are routinely screened for the presence of congenital porto-systemic shunts in the netherlands. breeders bring their nests to our clinic for individual measurement of blood ammonia concentration. in one year time (from february until january ) dogs of different breeds were examined, with of them being cairn terriers. the age of the dogs varied from to days (mean days). while the breeders were waiting for the blood results, the cardiac auscultation was performed by a single board-certified cardiologist (vsz). hematocrit was measured with an automatized hematology analyzer system from the surplus blood sample. cardiac murmur was found in dogs ( %). in all cases this was a soft ( - out of ) systolic murmur, most of the time with a musical character and with the point of maximal intensity on the left hemithorax, compatible with the description of an innocent cardiac murmur. no murmurs were found that could be compatible with a congenital cardiac anomaly. the hematocrit was significantly (p = . ) lower in the group of dogs with a murmur (mean . %, standard deviation . %) compared to the group without a murmur (mean . %, standard deviation . %). multivariate analysis shows that the presence of murmur is correlated with the hematocrit, but not with the age of the dogs. physiologic anemia has been long suspected to be one of the possible causes of innocent cardiac murmurs in young animals and children. however, according to the authors knowledge, no published reports exist that looked for a possible correlation. which other factors contribute to the presence of an innocent murmur is largely unknown. because of a large overlap between the hematocrit values of dogs with and without a cardiac murmur, measuring hematocrit in a particular pup would not help a less experienced first line veterinary practitioner to decide whether a murmur is innocent or the result of a congenital cardiac anomaly. limitation of this study is that no echocardiography was performed to rule out congenital cardiac anomalies as the cause of the murmurs. neither were the dogs re-checked for spontaneously disappearance of the murmur. no conflicts of interest reported. low intensity systolic murmurs, with point of maximal intensity over the outflow tract on the left side of thorax, are not uncommonly heard during cardiac auscultation of apparently healthy siberian husky dogs, often with excellent exercise tolerance. the origin of these murmurs in athletic dogs such as huskies is unlikely to be due to heart disease but more likely due to turbulent blood flow in the outflow tract caused by a large stroke volume and forceful cardiac contractility in early systole. the differentiation of these murmurs from "pathological"significant murmurs can however be problematic in general practice. the aim of the present study was to investigate the prevalence of murmurs in a sample of successfully racing siberian husky dogs and furthermore to study the phonocardiographic characteristics of these murmurs. phonocardiograms and ecgs were recorded in actively racing siberian husky dogs, with normal or excellent exercise tolerance. normal stamina was confirmed by successful racing. phonocardiograms were easy and rapid to record on a pc laptop connected to the meditron stethoscope in ambulant "field"practice. systole was measured as the duration measured from the onset of the first heart sound to the onset of the second heart sound and the murmur duration from the onset the first heart sound to the end of the murmur. the duration of the first heart sound plus the murmur was measured and calculated as a percentage of the duration of systole. cardiac murmurs of grade - were heard in % of dogs examined. phonocardiogram from these dogs revealed early systolic crescendo-decrescendo or decrescendo murmurs with a duration of maximally % into systole. all dogs with murmurs had a silent pause at the end of systole. ecg was normal in all dogs. murmurs in adult athletic dogs should not be regarded as a definite sign of heart disease. physiological flow murmurs of up to % of systole is a common finding in active siberian husky dogs (prevalence % in the examined sample). phonocardiography is a rapid and practical method for differential diagnoses between pathological murmurs and physiological flow murmurs. no conflicts of interest reported. nt-probnp has a degree of overlap with clinically normal animals, particularly those with mild or subclinical heart disease. prior studies have evaluated the sensitivity and specificity of a point-of-care second generation elisa that utilizes snap technology. the snap feline probnp test uses the same biological reagents as the cardiopet probnp test but provides results in minutes. we sought to prospectively validate the assay in a population of clinically normal cats. cats were recruited based upon the absence of a heart murmur, gallop, and/or arrhythmia. all cats received physical examination, non-invasive blood pressure measurement, complete biochemical analysis including a t , urinalysis and echocardiogram. only cats considered free of underlying cardiac or systemic disease were enrolled. sixteen adult cats were enrolled and blood samples were obtained for nt-probnp concentrations at , hr, hr, hr, hr, hr. samples were placed in edta tubes and centrifuged within one hour and split into two tubes for duplicate samples at each time point and stored at - °c. once all samples were collected, they were shipped on dry ice overnight and run in one batch (idexx laboratories) for measurement of nt-probnp concentrations. snap tests were visually evaluated by one blinded reader. comparison of snap assay vs. quantitative elisa revealed a . (auc) degree of correlation between assays, and that a positive snap test result was associated with a nt-probnp concentration of . pmol/l or greater. the average bnp concentration of abnormal cats ( . ae . ) determined by the snap assay was significantly greater than the normal ( . ae . ). this study was funded through idexx and the university of florida college of veterinary medicine resident grant competition. we acquired d echocardiographic cineloops from the left apical -chamber view optimized for the la, and analyzed atrial longitudinal strain (st) and strain rate (sr) in dogs ( healthy dogs and dogs with mmvd - acvim stage b , stage b and stage c). endocardial la ste curves were obtained and peak atrial longitudinal strain (pals), peak atrial contraction strain (pacs), conduit atrial longitudinal strain (cals); pals-pacs) and contraction strain index (csi -pacs/pals* ) were calculated. la sr curves were similarly obtained to determine the peak positive strain rate (srs) during left ventricle systole, the first negative peak strain rate (sre) during early diastole and the second negative peak strain rate (sra) during atrial contraction. for all variables, a mean of measures was used for the statistical analysis. we compared each of these variables between each acvim stage by kruskal-wallis tests and post-hoc pairwise comparisons, with comparison-wise a= . . normal dogs had higher pals and cals than dogs with mmvd (p < . and p = . ); stage c dogs had lower pals, pacs and cals than all other dogs (p < . , p = . and p = . ), but csi did not differ between groups (p = . ). stage c dogs had lower srs (p = . ), higher sre (p = . ) and sra (p = . ) than other dogs. normal dogs had lower sre and sra than dogs with mmvd (p < . ). our data suggest that ste might be useful in assessing la function in dogs with mmvd, and might potentially differentiate dogs with severe subclinical disease from dogs with congestive heart failure. no conflicts of interest reported. sighthounds are athletic dogs and they have been claimed to have larger hearts compared to similar sized breeds. the left ventricle (lv) may enlarge in response to cardiac disease, but also in response to training, so called athlete's heart syndrome, which is a benign condition. to distinguish abnormal echocardiographic measurements from normal, breed-specific reference values are needed. the aim of this study is to establish normal reference ranges for echocardiographic measurements in the saluki breed. the study comprised clinically healthy salukis ( males and females), mean age months (ae sd months), bodyweight (bw) , kg (ae , kg). case history was ascertained and dogs underwent physical examination, complete blood count, serum biochemistry profile, thyroid profile, blood pressure measurement and -min ecg. standard m-mode and d echocardiographic measurements were obtained. dogs with systolic murmur / , and dogs with mitral valve regurgitation (mr) < % (mr color flow jet area/left atrium areax % in apical view) were considered normal. linear regression models were used to establish reference ranges. heart rate (hr) varied from to bpm ( ae bpm). bw was a significant predictor for lv dimensions, i.e. m-mode lv diameter and d volume in diastole (lvidd and lvedv) and systole (lvids and lvesv), and mitral valve end point septal separation (epss). hr was a significant predictor for fs % (fractional shortening). predicted values ( % prediction intervals) were calculated from regression models where mean bw ( , kg) and age ( months), and median hr ( bpm) were used. normal reference ranges were: lvidd , mm ( , - , ), lvids , mm ( , ) , lvedv , ml ( , - , ), lvesv , ml ( , - , ), fs%: , % ( , - , ), ejection fraction ef%: , % ( , - , ), epss , mm ( , - , ), sphericity index , ( , - , ), interventricular septum in diastole , mm ( , - , ) and systole , mm ( , - , ), lv free wall in diastole , mm ( , - , ) and systole , mm ( , - , ), left atrial (la) diameter , mm ( , ) , aortic (ao) diameter , mm ( , - , ) , la/ao , ( , - , ), and aortic and pulmonic flow velocity , m/s ( , ) and , m/s ( , - , ), respectively. this study provides echocardiographic values for normal salukis which can be used as a reference values. no conflicts of interest reported. in mitral valve disease, atrial remodeling is an indicator of evolution and prognosis, the duration of the p wave being considered suggestive of the dilatation of the left atrium. in humans' studies, neurological conditions have a significant impact on cardiac electrophysiology by altering the electrical impulse conductibility. the aim of this study is to examine the duration of the p wave in dogs suffering from mitral valve disease in comparison to dogs diagnosed with different neuropathies without cardiac abnormalities. we analyzed standard electrocardiograms ( min of ecg, on peripheral leads) performed on three polymorphic groups of dogs (different age, weight and breed): group (n = ) healthy dogs, group (n = ) dogs diagnosed with mitral valve disease and group (n = ) dogs suffering from different neuropathies (without any associated or previously diagnosed cardiovascular disease). the duration of the p wave was measured for all dogs (five consecutive p waves without anomalies or artifacts) and reported to the degree of atrial remodeling, assessed by left atrium/ aorta ratio on echocardiography. the interpretation of the ecg and echocardiography was made by the same examiner (md). the results were statistically evaluated in a specialized program (ibm spss vs. ). the p wave recorded average values was . ae . seconds for the mvd group with significant differences between the stages of heart failure (p = . ). no correlations were found between its increase and the dilation of the left atrium (r = . ). there was no statistically significant difference regarding p wave duration when compared dogs of the neuropathy group and those of the mitral valve disease group (the p wave recorded average values = . ae . sec.). both, atrial tissue lesions (as in mitral valve disease) and autonomic nervous system anomalies (secondary to a neurological condition), may change the conductibility of the electrical impulse in the left atrium. the conductibility of the electrical impulse at this level does not seem to be influenced by its actual dilation, but by the impairment of the intra-atrial and inter-atrial conduction pathways. caution must be given when p wave is analyzed in dogs with concurrent cardiologic and neurologic condition. no conflicts of interest reported. newfoundland dogs were prospectively recruited among those undergoing screening for congenital and acquired heart disease. screening includes patient history, physical examination, and systemic arterial pressure measurement by doppler flow meter and transthoracic echocardiography (m-mode, d and echo-doppler). screening is performed on conscious dogs of at least year of age. dogs without historical, clinical, electrocardiographic and echocardiographic signs of cardiovascular disease were included in the study. unpaired, two-tailed student's t-test and linear regression were performed to evaluate the influence of gender, age and body weight (bw) on echocardiographic parameters. echocardiographic measurements were compared to previously reported reference values. the reference limits of echocardiographic parameters in the newfoundland dogs were calculated. forty-six healthy adult newfoundland dogs of both genders ( males and females), to years of age (mean . ae . years), to kg (mean . ae . kg) fulfilled the inclusion criteria. significant but weak correlations were detected between aortic diameter (ao) and age (p = . , r = . ), left atrial to aortic ratio (la/ao) and age (p = . , r = . ), e-point to septum separation (epss) and bw (p = . , r = . ), m-mode left ventricular internal diameter (lvid) in diastole (d) and systole (s) and bw (respectively p = . , r = . and p = . , r = . ), and between ao and bw (p = . , r = . ). none of the echocardiographic measurements was statistically different between males and females. left ventricular internal diameter in diastole, lvids, ao, epss increased with bw, as expected. the aorta appears to become wider with advancing age. a proportion of the studied population had m-mode parameters below the allometric scaling reference range, suggesting that this method can over-estimates m-mode parameters in this breed. these findings stress the importance to report newfoundland breed specific normal ranges for echocardiographic parameters. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease usually diagnosed by thoracic ct-scan that mainly affects west highland white terriers (whwt). pulmonary hypertension (ph), a severe co-morbid condition with a challenging diagnosis, may develop in cipf dogs. the ratio between the right pulmonary vein and pulmonary artery (pv/ pa) has been described as an echocardiographic indicator of ph in cipfdogs. this study was intended to investigate whether ct-scan angiography cardiac findings are ) altered in dogs with cipf compared to healthy control dogs and ) correlated with pv/pa measured by echocardiography (pv/pa us ). thoracic cta images from whwt with cipf (group a) and healthy controls from various breeds (group b) were retrospectively reviewed by one observer. all measurements were obtained in transverse post-contrast images displayed in a soft tissue window. pv and pa were measured dorsal to the right atrium, perpendicular to the long axis of these vessels. in addition, pulmonary trunk (pt) was assessed just ventral to the division of pulmonary arteries, perpendicular to its long axis. ascending aorta (ao) was also measured perpendicular to its long axis. transverse reformatted images were obtained to have a view equivalent to the standard chambers-echocardiographic view where right ventricle (rv) and left ventricle (lv) were measured. three ratios were calculated pv/pa ct , pt/ao and rv/lv, compared between groups and correlated with pv/pa us in both bi-dimensional (bd) and m-modes (mm). statistical analyses were performed with xlstat â software. values are given as mean ae sd. statistical significance was set at a p ≤ . . pv/pa ct was lower in group a ( . ae . ) in comparison to group b ( . ae . , p = . ) and correlated with pv/pa us (bd: r = . , p = . ; mm: r = . , p = . ). pt/ao was higher in group a ( . ae . ) compared to group b ( . ae . , p = . ) and correlated only with pv/pa us measured in bd mode (r = - . , p = . ). the rv/lv ratio was increased in group a ( . ae . ) in comparison to group b ( . ae . , p = . ) and a correlation between rv/lv and pv/pa us was found (bd: r = - . , p = . ; mm: r = - . , p = . ). in conclusion, in whwt with cipf, pv/pa ct , pt/ao and rv/lv ratios measured on thoracic cta images are correlated with pv/pa us and may serve in the assessment of ph. no conflicts of interest reported. companion animals presenting to the emergency room in distress need to be assessed rapidly and accurately to implement life-saving therapies. focused cardiac ultrasound (focus) can be a useful adjunct to the physical examination in assessing dyspneic animals in the emergency room. rapid bedside ultrasound evaluations performed by ec are commonly used in human medicine, however feasibility and utility of focus by ec in veterinary medicine has not been fully evaluated. the purpose of this study is to determine the baseline accuracy of focus performed by ec and whether or not a basic training session could improve accuracy compared to evaluation by a cardiology specialist. fifteen ec including boarded emergency-critical care specialists and emergency res-idents performed focus on four animals; a normal cat and dog, and a cat and dog with severe valvular and myocardial heart disease, respectively. ec semi-quantitatively assessed thoracic and echocardiographic parameters including left atrial dimension, left ventricular systolic function and wall thickness, right heart dimension, and presence or absence of pleural or pericardial effusion before and after a structured didactic lecture and hands-on practical session. primary outcome was the level of agreement with examination performed by a cardiologist. level of agreement regarding ec assessment of all parameters improved from . to . after training (p < . ). level of agreement concerning left atrial diameter improved from . to . (p < . ). ec confidence in their overall focus evaluation and findings improved from % to % (p < . ). in summary, ec accuracy and confidence in semi-quantitatively assessing basic cardiac parameters using focus were improved following a simple structured training session. focus might be a valuable tool to rapidly assess simple thoracic and cardiac parameters in the emergency setting. no conflicts of interest reported. obesity is an increasing health problem in dogs. success of weight-loss programs is often limited by compliance issues. the purpose of this study was to determine the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned dogs, under typical household conditions. the objectives were ) to evaluate weight loss parameters in dogs fed a ndwmf* and ) to assess the owner's perception of the dog's quality of life. overweight/obese, otherwise healthy, client-owned dogs (> / body condition score -bcs) were enrolled in the study (n = ). initial veterinary evaluation included physical examination, nutritional assessment, determination of ideal body weight (ibw), and development of weightloss feeding guidelines. daily energy requirement (der) for weight loss was calculated as der = x ibw kg . . initial and follow-up evaluations (monthly for months) encompassed determination of body weight, bcs, body fat index (bfi), muscle condition score (mcs), and feeding practices. quality of life assessment by owners included dog's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis comprised scatterplots, regression analysis, summary statistics as appropriate for the type of analyses performed (continuous or categorical variables, distribution), and a mixed model anova to assess changes over time (with statistical significance at p < . ). ninety four percent of the dogs lost weight (n = ) with an average weight loss of . % (sem, . %) over months and an average weekly weight-loss rate of . % (sem, . %). the mean duration of weight loss was days (sem, . days) with an average of days (sem, . days) between rechecks. thirty nine percent of dogs achieved ibw ( . , ci: . - . ). fifty five percent of dogs ate more calories from ndwmf* than the recommended der for weight loss (median fed above der= %) and % of these dogs ( . , ci: . - . ) still lost weight. thirty six percent of dogs received treats. bcs and bfi decreased significantly over time compared to baseline. owners perceived a significant increase in energy and happiness in the dogs that lost weight without changes in appetite or begging behavior. in conclusion, this clinical study confirmed the effectiveness of the ndwmf* in achieving weight loss in overweight/obese client-owned dogs in spite of higher than recommended caloric intake. owners reported significant improvements in dog's quality of life without negative side effects. * haptoglobin is a moderate acute phase protein in cats. as a part of the innate immune system its concentration rises within - hours after tissue damage. aim of the study was to validate an elisa which was recently developed for the measurement of feline haptoglobin and to compare it with a commonly used spectrophotometric assay. the concentration of haptoglobin was measured in healthy and sick cats using a sandwich-elisa (tecomedical group, rheinbach, germany). the validation included the detection of intra-assay and inter-assay variation, dilution linearity, spike recovery and lower detection limit. a spectrophotometric assay (tridelta development ltd, maynooth, ireland) was used as a reference method. all samples were measured in duplicate. statistical analysis was performed using ibm â spss â statistics (ibm corporation â ) and included descriptive statistics, spearman correlation (rs) and coefficients of variation (cv). the coefficients of variation were . %, . % and . % for intra-assay variability and . %, . % and . % for inter-assay variability. the ratio of observed to expected dilutional parallelism of serum samples diluted times ranged from to %. the ratio of observed to expected spike recovery of serum samples ranged from % to %. the lower detection limit was . mg/ml. the correlation between the assays was significantly strong (rs = . , p < . ). the recently available sandwich-elisa provides a high accuracy and precision and can therefore be used for the measurement of feline haptoglobin. the rd and th author (m. hennies and c. wienen) work for the company tecomedical group that developed the elisa which was evaluated in the study. they provided the kits and they helped with performing the tests, but they did not have any influence on the results and the interpretation of the data. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease that mainly occurs in the west highland white terrier (whwt) breed. the cipf diagnosis commonly relies on thoracic high-resolution computed tomography (hrct) findings and ultimately on histopathology. as those tests are not easily performed in practice, identification of measurable markers of fibrosis, that might help to diagnose and/or monitor the course of cipf, is helpful. vegf is an angiogenic regulator involved in a variety of physiological and pathological processes. in human ipf, serum vegf concentration has been shown to be higher in ipf patients compared to healthy volunteers and may reflect the severity of the lung disease. the aims of the present study were ( ) to investigate the potential role of vegf as a peripheral blood biomarker in cipf; and ( ) to investigate possible breed-related differences in basal vegf concentration, that might explain the high predisposition of the whwt breed for cipf.therefore, vegf was determined by elisa (canine vegf quantikine elisa kit, r&d systems) in the serum of whwt with cipf confirmed by hrct and/or histopathology (median age years, range - ), healthy whwt ( , - ), and healthy dogs of other breeds, including: scottish terrier (st) ( , - ), jack russell terrier (jrt) ( , - ), maltese ( , - ), king charles spaniel (kcs) ( , - ), labrador retriever (lr) ( , - ) and malinois belgian shepherd ( , - ). health status was based on clinical examination, serum biochemistry and haematology in all healthy dogs and a thoracic hrct was performed in / healthy whwt. the khi² test with the threshold % was used for the statistical analysis (xlstat â software). eight cipf whwt ( %) have serum vegf concentrations above the kit detection limit ( . pg/ml) compared to whwt ( . %) in the group of healthy dogs (p = . ). concerning inter-breed differences in healthy dogs, most values obtained were below the kit detection limit with only kcs ( %), jrt ( %), lr ( %) and st ( %) having vegf serum levels above . pg/ml (p = . ). results of the present study show that ( ) vegf might be an interesting blood biomarker for cipf; ( ) canine vegf quantikine elisa kit is not appropriate for measurement of serum vegf levels in healthy canine populations. no conflicts of interest reported. the total protein (tp) concentration and cell count of pleural and abdominal fluid is used to differentiate a transudate from an exudate. tp can be measured by automated wet chemistry analyser or more easily using a refractometer. the aim of this study was to assess if refractometer values of tp are useful for this purpose. retrospectively samples from canine pleural and abdominal effusions in which tp concentration was measured both with a refractometer as well using pentra (abx horiba, montpellier) were included. samples were collected into heparinized tubes and analysed within hours. bland-altman diagrams were created and correlation between both measurements was calculated by spearman s nonparametric correlation. over a -months period, pleural and abdominal effusion samples were analysed with both techniques. median (range) tp concentrations in pleural effusion measured by refractormeter or by pentra was ( - ) g/l and ( - ) g/l, respectively. median (range) tp concentrations in abdominal effusions measured by refractometer or pentra was ( - ) g/l and ( - ) g/l, respectively. tp measurement between refractometer and pentra values were significantly correlated in pleural (r = . , p < . ) and abdominal (r = . , p < . ) effusion. the bland-altman graph showed a bias in the thorax and abdomen of . and . . the refractometer is an acceptable, rapid and efficient method for determination of total protein concentration in pleural and abdominal effusions in dogs to differentiate transudates from exudates. no conflicts of interest reported. coagulation factor vii (fvii) deficiency has been reported in beagles since the 's. deficient dogs show a mild hemorrhagic tendency, but often remain asymptomatic and are incidentally discovered by an isolated prolonged prothrombin time due to < % plasma fvii activity. factor vii deficiency occurs commonly in beagles, alaskan klee kais and scottish deerhounds. in these breeds it is caused by a single missense mutation (c. g>a, p.gly glu) in the second epidermal growth factorlike domain of fvii, which drastically reduces the secretion and activation of fvii. research beagles were also commonly affected which may have pharmaco-toxicologically affected studies but specific dna screening programs have been established. we report here on the discovery of fvii deficiency in welsh springer spaniels (wss) in finland based upon a novel screening panel for~ known mutations underlying inherited disorders in different canine breeds (www.mydogdna.com). among wss initially tested, were heterozygously ( %), and homozygously affected for the same fvii mutation, which was confirmed by sequencing in all dogs. in order to determine whether the mutation causes fvii deficiency also in this breed, we recruited littermates and their mother. none of these wss had shown an increased hemorrhagic tendency, but affecteds bled excessively following blood collection. we found that the homozygous affected dogs of the litter exhibited markedly prolonged prothrombin time but normal partial thromboplastin time. they also had drastically reduced fvii activities but normal to high fviii and fix activities compared to their littermate controls. the heterozygous carriers tested did not show any prolongations in their prothrombin time, but had half normal fvii activity. in conclusion, we document here the presence of fvii deficiency in wss based upon dna and coagulation activity testing. the common gly glu mutation must have arisen prior to the separation of the very different fvii deficient breeds. there is no knowledge of an advantage of the heterozygote state. while there is only a mild hemorrhagic tendency, bleeding dogs could be treated with fresh frozen or cryo-poor plasma or human recombinant fviia. this preliminary study indicates a high carrier frequency in wss. screening by new platform dna methods for this and other ancestral defects is helpful to detect additional hereditary diseases and genetic predispositions in different breeds, while other mutations are new and restricted to one or related breeds. authors are affiliated with genetic disease screening test laboratory. remarkably little has been published on haematological and serum biochemical phenotypes of the domestic dog. information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database; similar information was collected from all dogs with normal serum biochemical profiles, considering all parameters other than glucose as inclusion criteria. normal haematological profiles were available for dogs, of which also had machine platelet concentrations within the reference interval; normal serum biochemical profiles were available from dogs, of which also had accompanying normal serum glucose concentrations. for the haematological data, pure breeds plus a mixed breed control group were represented by or more dogs, while for the serum biochemical data, pure breeds plus a mixed breed control group were represented by or more individuals. all measured haematological parameters except mean corpuscular haemoglobin concentration (mchc), and all serum biochemical analytes except sodium, chloride and glucose, varied with age. concentrations of white blood cells (wbcs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex, as did total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, and activities of alanine aminotransferase (alt), creatine kinase (ck), amylase and lipase. neutering status had an impact on haemoglobin concentration, mean corpuscular haemoglobin (mch), mchc, and concentrations of wbcs, neutrophils, monocytes, lymphocytes and platelets, as well as all serum biochemical analytes except albumin, sodium, calcium, urea and glucose. principal component analysis (pca) of haematological data revealed pure breeds with distinctive phenotypes, while pca of serum biochemical data revealed over pure breeds with distinctive phenotypes. furthermore, all haematological parameters except mchc and all serum biochemical analytes except urea and glucose showed significant differences between specific individual breeds and the mixed breed group. twenty-nine breeds had distinctive haematological phenotypes and breeds had distinctive serum biochemical phenotypes when assessed in this way. tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. this study represents the first large-scale analysis of haematological and serum biochemical phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of haematological and biochemical traits, triangulating phenotype, breed and genetic predisposition, as well as the urgent need for breed-specific reference intervals in clinical practice. the author has received funding from bbsrc, petplan charitable trust, and cruk), but none of thesegrants were for this study. for each cat aged years or older, irrespective of their suspected thyroid status, presented to eight veterinary practices in portugal, the veterinarian and the pet owner had to complete a questionnaire and the veterinarian had to take a venous blood sample (into a plain tube) from the cat, after obtaining signed owner consent. the veterinary questionnaire included history, attitude, activity, heart rate and thyroid palpation. cats aged < years and those diagnosed previously with hyperthyroidism were excluded. blood samples were centrifuged and the serum harvested and stored frozen until collection by the laboratory within days of sampling. total t was measured using a chemiluminescent method (immulite , siemens). cats were classified as hyperthyroid, equivocal or euthyroid based on a total t concentration of > nmol/l, - nmol/l or < nmol/l, respectively. repeat measurement of total t after - weeks was recommended for all equivocal cases. the individual cat was the statistical unit. descriptive statistics was used to summarise the data and associations between different clinical signs analysed using chi-square, fisher's exact test or the mann-whitney u test. the level of significance was set at . . thirty cats were excluded from the prevalence analysis because they were aged < years (between and years, n = ) or their age was not stated (n = , four of these cats were hyperthyroid). by the end of february , samples had been submitted from cats that met the inclusion criteria. based on the thyroid hormone analysis, there were / ( %) hyperthyroid, ( %) equivocal and ( %) euthyroid cats. very few follow-up blood samples were taken. hyperthyroidism appears to be not uncommon in portuguese cats. getting owners to return for follow-up blood sampling appears to be problematic. under-reporting of hyperthyroidism appears to be a significant problem in portugal, as has been reported for some other countries. thyroid palpation should form part of routine physical examinations, especially of middle aged and older cats. older cats in portugal should be screened for hyperthyroidism even in the absence of a detectable thyroid nodule. both authors are employees of msd animal health. msd animal health funded the study. msd animal health has an approved veterinary medicinal product for the treatment of feline hyperthyroidism. this product is available commercially in some eu markets but not in portugal. diabetes mellitus is one of the most commonly encountered endocrinopathies in cats and its prevalence has increased in the past. similar to human type diabetes, feline diabetes is associated with comparable lesions occurring in the pancreatic islets, namely islet amyloidosis and beta-cell loss. studying the pathophysiology of feline diabetes and the molecular mechanisms through which glucose metabolism is disturbed is largely hampered by the lack of a method for the isolation of pure pancreatic islets. the aim of this project was to improve a previously established method for the isolation of pancreatic islets; in particular enhancing the purity of isolated islets in this species. cats that died or were euthanized due to severe illness other than pancreatitis or other pancreatic disease were enrolled. pancreata were perfused post-mortem with ml collagenase type iv ( . mg/ml) through the pancreatic duct. the perfused organ was then digested for ', ' and ' at °c in a water-bath and purified using a filtration method. islet cell viability and purity were determined by thiazolyl blue tetrazolium bromide (mtt assay) and dithizone staining, respectively. perfusing the pancreas through the pancreatic duct allowed collagenase to access the islets using anatomical structures and to improve islet yield compared to previously established protocols in this species. the digestion time of ' provided the best islet yield. after digestion, feline pancreatic islets remained satisfactorily viable for days in the culture system following regular media changes. the current study has successfully optimized the isolation, purification and culture maintenance of feline islets. the successful yield and viability of islets isolated through the suggested protocol may provide promising potential as a source of islets for diabetes research in cats. no conflicts of interest reported. nesidioblastosis describes a syndrome of acquired hyperinsulinaemia and associated hypoglycaemia secondary to focal or diffuse (non-neoplastic) beta cell hyperplasia within the pancreas. beta cell dysregulation is thought to occur secondary to pancreatic injury. this syndrome has been reported in humans with increasing frequency, but it has not previously been described in domestic pets. a six year old, de-sexed female british shorthair cat presented with acute onset weakness and mental dullness. upon initial presentation the cat was mildly hyperglycaemic ( . mmol/l; . - . mmol/l). over the following hours the cat developed central blindness, tremors, intermittent seizures and opisthotonus. repeat blood sampling revealed a marked hypoglycaemia ( . mmol/l). an insulin level (performed on serum obtained while the cat was hypoglycaemic) was inappropriately elevated ( pmol/l; reference range - pmol/l). an intravenous bolus of % glucose resulted in rapid resolution of all clinical signs and mild transient hyperglycaemia ( . mmol/l). despite frequent feeding, the hypoglycaemia ( . mmol/l) recurred, so an intravenous glucose continuous rate infusion was commenced. an abdominal ultrasound was unremarkable, although three cranial mesenteric lymph nodes were noted to be prominent ( mm in width). an exploratory laparotomy revealed a firm and erythematous left limb of the pancreas. the body and right limb of the pancreas appeared grossly normal. following surgical resection of the left limb of the pancreas, the cat returned to a euglycaemic state after a brief rebound hyperglycaemia. histopathology revealed pancreatic fibrosis with marked multifocal micronodular hyperplasia of exocrine and endocrine cells, mild lymphoplasmacytic inflammation and ductular ectasia. synaptophysin immunohistochemistry confirmed nodular beta cell hyperplasia. mild granulomatous lymphadenitis and hydropic change within hepatocytes was also noted. the cat recovered uneventfully without any further intervention. it gained weight and remained euglycaemic over the following six months. while beta cell hyperplasia has been reported as an incidental histopathological finding in euglycaemic young beagles, this is the first reported case of clinically significant hypoglycaemia secondary to nesidioblastosis in a domestic pet. while this condition is rare, nesidioblastosis is being increasingly recognised in the human field and it is an important differential to consider when investigating hypoglycaemia as it cannot be differentiated from insulinoma without histopathological evaluation. age of onset may provide a clue to this non-neoplastic disease, as this cat was much younger than all previously reported cases of feline insulinoma (all > years of age at diagnosis). while recurrence has been reported in humans, a favourable outcome is anticipated following partial pancreatectomy. no conflicts of interest reported. hyperthyroidism is the most common feline endocrinopathy of geriatric cats worldwide. nonetheless, data concerning the accurate prevalence of feline hyperthyroidism (fh) is scarce; and apparently exhibit geographical variation, which can be an important instrument in investigating risk factors through the analysis of exposure to different factors in areas of high and low prevalence. in europe, fh is considered more frequent in the northern than in the southern countries. the aims of this study were to determine the occurrence of fh in a region of portugal, to characterize clinical presentation and potential risk factors. during an -month period, geriatric cats ≥ eight years from aveiro (central region of portugal) were selected. cats were excluded if presented in shock or moribund, or in treatment with drugs that might affect total t (tt ) serum concentration. the tt concentration was determined through chemiluminescence (immulite â , siemens), and diagnosis of fh established if tt serum concentration ≥ . lg/dl (reference values . - . lg/dl) associated with compatible clinical signs. information on age, gender, breed, weight, housing conditions (indoor vs outdoor), use of external parasiticides, food (dry vs canned food and flavor), use of litter box, environment, clinical signs and laboratory data was collected. all owners gave informed consent. population studied included males ( . %) and females ( . %), mainly domestic short-haired cats ( . %). ages ranged from eight to years old ( . + /- . ). eight ( . %) cats were diagnosed with hyperthyroidism. if only cats ≥ years of age were considered (n = ), prevalence raised to . %. hyperthyroid population comprised four males and four females, ranging from to years old ( . + /- . ). increasing age (p = . ), polyphagia (p˂ . ), weight loss associated with increased (p˂ . ) or normal appetite (p˂ . ), presence of thyroid uni or bilateral nodules (p˂ . ), vomiting (p = . ) and hyperactivity (p = . ) were significantly associated with hyperthyroidism in the geriatric population studied. environment was also significantly associated with development of fh (p˂ . ), with cats from urban or semi-rural areas at higher risk of developing the disease than cats living in a rural environment. no other significant associations were found between hyperthyroidism and other factors analyzed. in our knowledge, no epidemiologic studies on fh have been performed in portugal, a country where the occurrence is believed to be low, but in which the population of pet cats, the feline geriatric population and the clinical cases diagnosed have been increasing. conflicts of interest: the study was partially supported by laborat orio segalab s.a. and dechra veterinary products. canine diabetes mellitus (cdm) has been proposed to be a spontaneous animal model of human autoimmune diabetes, and comparative research can be undertaken to investigate the interaction between genetic and environmental factors. most epidemiological studies of cdm have been performed in northern european and north american populations. our aim was to evaluate the epidemiology and clinical features of the diabetic dog population from the canary islands, with special focus on immune-mediated disease. dogs attending our veterinary teaching hospital were included from january to january . previously diagnosed and new cases were considered. prevalence was calculated as number of cdm/total number of dogs attending the hospital and incidence as newly diagnosed cases divided by the same value per year. anti-insulin antibodies were assessed by elisa. genotyping for dog leukocyte antigen (dla) and measurement of canine anti-gad and anti-ia antibodies by radio-immunoprecipitation assay were performed in dogs with suspected immune-mediated diabetes. twenty-nine dogs with cdm were identified from a mean population of ( - ) dogs per year (mean prevalence . % and mean incidence cases per year per , ). age at diagnosis was . years (range: . - y). most dogs were not neutered ( % females; % males). nine breeds were represented, including poodle ( %) and andalusian wine-cellar rathunting dog ( %). seasonality was observed in the diagnosis with peaks in december and march-april. diabetes was classified as dioestrus diabetes ( %), idiopathic/immune-mediated ( %), iatrogenic ( %) and secondary to pancreatitis ( %) or other endocrine disorders ( %). insulin-treated dogs were negative for anti-insulin antibodies (n = ). from the suspected immune-mediated cases (n = ), autoantibody reactivity was shown in two cases (anti-gad , n = ; anti-ia , n = ). no previously described, diabetes-risk dla-types were identified. although age, prevalence and incidence did not differ from previous studies, the high proportion of entire females likely explained the high frequency of dioestrus diabetes. the andalusian wine-cellar rat-hunting dog was identified as a high-risk breed for cdm. most of the dla-types seen have not been previously described, but at least two have been associated with increased risk of autoimmunity in dogs. further population-based studies are needed in different regions, to assess the heterogeneous nature of this disease. no conflicts of interest reported. the cortisol-dehydroepiandrosterone (dhea)-ratio is widely used in human medicine as a marker for stress however it is not clear whether it could also help in distinguishing hyperadrenocorticism (hac) from other diseases which might have a negative impact on the outcome of a dexamethasone low dose test. therefore the aim of the study was to evaluate the cortisol-dhea-ratio as an additional diagnostic marker for hac in dogs. to achieve this aim, a reference range of this ratio depending on the sex should be evaluated in healthy dogs and compared with dogs having a hac. in healthy dogs (age: - . years) and in dogs with hac (age: . - . years) of different breeds the plasma concentration of cortisol (immulite system, siemens healthcare diagnostics) and dhea (beckman coulter) was measured and the ratio was calculated. all dogs were patients of the small animal clinic except five of the healthy dogs which were recruited from the institute of pharmacology, toxicology and pharmacy of the university. with these data the cortisol-dhea-ratio was calculated for male dogs (healthy dogs n = ; dogs with hac n = ), neutered males (healthy dogs n = ; dogs with hac n = ), female dogs (healthy dogs n = ; dogs with hac n = ) and spayed females (healthy dogs n = ; dogs with hac n = ). the statistical analysis was performed with sigma stat. the plasma cortisol-dhea-ratio of healthy male dogs was the lowest ratio of all sexual categories (mean average . ae ) and it differed significantly to all other sexes (neutered males = ae , p = . ; females = ae , p < . and spayed females ( ae . , p = . ). the cortisol-dhea-ratio showed no significant difference between male and female dogs with hac. spayed females with hac had significantly higher cortisol-dhea-ratios ( ae ) than healthy spayed females (p = . ) but no significant differences were found in other sexual categories. this preliminary data indicates that the cortisol-dhea-ratio might not be a very promising tool for the diagnosis of hac. in addition, the significant gender-dependency of this parameter has to be considered and may generally limit its clinical usefulness. this study is financially supported by the bruns-stiftung. no conflicts of interest reported. hyperthyroidism is common in older cats. the aim of this study was to assess the prevalence of feline hyperthyroidism and potential intrinsic risk factors in a hospital population in southern germany. total thyroxine (t ) was prospectively measured by enzyme immunoassay (eia) in sera of cats older than years that were presented to the clinic of small animal medicine. a standardized physical examination was performed, and body condition score (bcs) and thyroid palpation score (tps) were assessed. association between signalement, bcs and tps was analyzed by student s unpaired t-test, chi-square, and mann-whitney test. level of significance was set at . . fifty nine cats were diagnosed with hyperthyroidism leading to a prevalence of . % (ci . - . ). hyperthyroid cats were older than non-hyperthyroid cats (p < . ) and more often female (p = . , odds ratio . ). domestic short or long hair cats were more often affected than pedigrees (p = . ). hyperthyroid cats had higher tps (p < . ) and lower weight than non-hyperthyroid cats (p < . ) although bcs was not different (p = . ). in ( . %) cats, the elevated t was an incidental finding. in of those, the disease was confirmed later (the others were dead due to unrelated diseases). in patients, hyperthyroidism was considered a differential diagnosis and was confirmed in ( . %) cats although in cats additional diagnostic means were necessary. older female domestic cats are predisposed to hyperthyroidism which is frequently diagnosed after the initial clinical suspect. in a few affected cats an elevated t is not present or can precede clinical signs. conflicts of interest: the study was partially funded by msd intervet. the main endocrinopathy affecting both humans and pet felines is diabetes mellitus. accurate diagnosis is the most important aspect in the future outcome of the disease. a computer based decision support system (dss) is targeted on assisting clinicians with one or more steps of the diagnostic process. the novelty of our dss emerges from the possibility of assisting both clinical and paraclinical diagnosis stages of diabetes mellitus and all common combination of disorders associated with this endocrinopathy. the motivation behind the development of such system is the desire to maximize the reliability of clinical decisions. the design of our feline diabetes mellitus dss emerges from the syndrome of polyuria-polydipsia, with the possibility of spotting the accompanying pathologies. fuzzy logic is used for dealing with knowledge representation and uncertainty. the fuzzy rules proposed to represent this knowledge emerge from anamnesis, clinician's input, clinical and paraclinical description, and confirmation diagnostic tests. clinical signs such as polyuriapolydipsia, persistent hyperglycemia, polyphagia, weight fluctuations, administration of drugs with a diabetogenic potential, were considered decisive in the pattern of diagnosis establishment. registered medical records of cats, males and females, whit ages from to years old, were analyzed in order to validate the dss. using matlab software, the dss was implemented and tested. for any case with polyuria-polydipsia the system provides, via a friendly graphical user interface, the diagnosis with the highest probability. the set of diagnoses which can be generated by the dss consists in: a) diabetes mellitus; b) diabetes mellitus induced by (b. ) hypersomathotropism, (b. ) hyperthyroidism, (b. ) hyperadrenocorticism and (b. ) diabetogenic medication; c) diabetes mellitus in association with (c. ) chronic kidney failure and (c. ) heart failure; d) ketoacidodic diabetes mellitus; e) pancreatitis. the dss was applied with success on all cases, revealing the following diagnoses / no of cases: (a) - , (b. ) - , (b. ) - , (b. ) - , (c. ) - , (c. ) - , (d) - . an adequate treatment protocol requires an accurate and complete diagnosis. advanced computational systems accompany clinicians in their decision making, leaving a reduced space for medical errors and superfluous, expensive and time consuming tests. future work will be targeted on exploring the possibilities of combining the dss with an artificial neural network model for diabetes mellitus. this can be the foundation of a complete case oriented management system for feline diabetes mellitus and associated disorders. no conflicts of interest reported. activins are cytokines belonging to the transforming growth factor (tgf)-b superfamily. it is thought that activins may be the key intermediary in tgf-b mediated fibrotic response. activin a has been suggested to participate in the pathogenesis of human idiopathic pulmonary fibrosis (ipf), but studies regarding the role of activin b are still spares. canine ipf (cipf) is a chronic, incurable interstitial lung disease occurring particularly in west highland white terriers (whwts). during the disease course, acute exacerbations (aes), with poor prognosis, can occur. histopathologically aes of cipf are featured by diffuse alveolar damage, which is also a key feature in acute respiratory distress syndrome (ards). our objective was to study the expression of activin a and b by immunohistochemistry in the lung tissue of cipf whwts (n = ), cipf whwts with concurrent ae (n = ), and dogs of various breeds with ards (n = ), and to compare these findings to healthy whwts (n = ). in addition, western blot analysis of activin b from bronchoalveolar lavage fluid (balf) of cipf whwts (n = ) and healthy whwts (n = ) was conducted. we demonstrated that activin b, but not activin a, is strongly expressed in the altered alveolar epithelium in lungs of diseased whwts as well as in ards lungs. furthermore, activin b was detected in balf of cipf whwts, most notably in samples from dogs with ae, but not in balf of healthy whwts. this novel finding suggests that activin b participates in the pathophysiology of cipf and might act as a potential marker of alveolar epithelial damage. no conflicts of interest reported. dogs of the breed nova scotia duck tolling retriever (nsdtr) are affected by several immune-mediated diseases, in particular steroid-responsive meningitis-arteritis (srma) and an immune-mediated rheumatic disease (imrd). imrd is a systemic lupus erythematosus-related disease characterized by chronic stiffness and pain in several joints. the aim of this study was to investigate the morbidity in nsdtrs and to test the hypothesis that nsdtrs are predisposed to srma and imrd. insurance data from a swedish insurance company (agria insurance company, stockholm, sweden) from - was used for the study. approximately one third of swedish dogs are insured by agria and the insurance database is a validated tool for epidemiological studies. assessment of morbidity was based on veterinary care events. disease diagnoses were grouped in both general and specific disease categories. individual diagnoses that were likely to represent imrd were combined. morbidity was defined as incidence rates and presented as number of cases per dog years at risk (dyar). relative risk (rr) for nsdtrs compared to other breeds combined was calculated. the study included dogs, were nsdtrs. the most common general causes of veterinary care for nsdtrs were injuries followed by gastrointestinal and musculoskeletal disorders with significant increased risk (rrs between . and . ) for nsdtrs compared to other breeds. the highest relative risk for nsdtrs was for systemic lupus erythematosus (rr . ). compared to other breeds, nsdtrs had an increased risk for srma (rr . ) and imrd (rr . ) with an incidence rate of . cases per dyar for srma and . cases per dyar for imrd. the incidence rate for srma and imrd in nsdtrs were also compared to dogs of other retriever breeds. the comparison revealed that nsdtrs also had a significant increased risk for both srma (rr . ) and imrd (rr . ) when compared to other retrievers only. this study is the first to investigate the morbidity for imrd in nsdtrs, which is important for further research and breeding practice. for several reasons the incidence rates might be underestimated and exact numbers should be interpreted with caution. however underestimation of incidence rates should not differ between dogs of different breeds, therefore not affecting the risk calculations. it can be concluded that nsdtrs are predisposed to the diseases srma and imrd with an increased risk compared to other breeds and to other retrievers. brenda n. bonnett consults with agria insurance company on various projects. agria insurance company has also funded work leading to the development of the insurance data base that my study was based on. canine infectious respiratory disease (cird) is a multifactorial contagious disease caused by respiratory viruses and selected bacterial pathogens. cird has been shown to be a predisposing factor in the development of bacterial pneumonia (bp) in dogs housed in dense populations such as kennels and rehoming centers. the aim of this study was to determine the prevalence of viral co-infection and to assess its effects on disease severity in household dogs diagnosed with bp. a prospective cross-sectional observational study was conducted and dogs diagnosed with bp caused by opportunistic bacteria were included. dogs with chronic (> days) tracheobronchitis caused by bordetella bronchiseptica were included as controls for virus analysis. diagnosis was confirmed by thorough clinical examinations as well as with cytological and bacterial analysis of bronchoalveolar lavage (bal) or transtracheal wash (ttw) samples. canine parainfluenssavirus (cpiv), canine adenovirus, canine herpesvirus, canine distempervirus, canine respiratory coronavirus (crcov) and canine pneumovirus were analysed in bal or ttw samples using rt-pcr assay. cpiv was detected in / ( %) and crcov in / ( %) respiratory samples in dogs with bp. respiratory viruses were not detected in dogs with chronic tracheobronchitis. there were no significant differences in the duration of hospitalization (p = . ) or arterial pao at presentation (p = . ) between bp dogs with and without a viral co-infection. these results indicate that co-infections with respiratory viruses are common also in household dogs with bp. additionally, viral co-infections did not cause a more severe course of bp. the author's researcjh is financially supported by the finnish foundation of veterinary r and the finnish veterinary foundation. esvim-p- causes of canine anemia in taiwan: a five-year retrospective survey. e.c.y. lin , p.c. liu , l.l. chueh , b.l. su . graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, institute of veterinary clinical sciences, national taiwan university, taipei, taiwan anemia is a common hematologic disorder in dogs, however, few data are available regarding epidemiology and causes in taiwan. to investigate the causes of anemia, anemic cases (pcv< %) collected between january and december at national taiwan university veterinary hospital (ntuvh) were analyzed. most dogs ( . %, n = ) presented with a mild form ( %≦pcv< %), which was followed by a moderate form ( %≦pcv< %; . %, n = ) and a severe form (pcv< %; . %, n = ). among the dogs with identifiable causes, . % ( dogs) were induced by single cause, whereas . % ( dogs) by multiple causes. neoplasia-related anemia (n = ), infectious pathogens-related anemia (n = ), renal disease-related anemia (n = ) and post-surgery/ traumarelated anemia (n = ) account for . , . , . and . % of single-cause cases, respectively. furthermore, of them ( . %) presented with severe anemia. severe anemia primarily resulted from infectious disease-related anemia ( . %), followed by imha ( . %), and tumor-related anemia ( . %). of the infectious disease-related severe anemic dogs, the most common diagnosed pathogen was babesia gibsoni ( . %, n = ), followed by ehrlichia canis ( . %, n = )and babesia canis ( . %, n = ). taken together, tumors, infectious diseases, and renal failure are the most frequently causes of canine anemia in taiwan, furthermore, b. gibsoni appeared to be the most important infectious pathogen causing severe anemia which may be associated with the climate in this geographical area. no conflicts of interest reported. bordetella bronchiseptica (bb) is one of the primary causative agents of canine infectious respiratory disease (cird). this contagious disease, commonly seen in young dogs, is often self-limiting, although a wide range of respiratory signs can be found, from mild illness to severe pneumonia leading to death. although mycoplasma cynos (m. cynos) was recently identified as an emerging and possibly lethal pathogen in cird , the role of m. canis and m. cynos as primary respiratory pathogens still remains unclear. detection of these bacteria is now improved by quantitative polymerase chain reaction (qpcr). in dogs with cird due to bb, the frequency of co-infection with mycoplasma spp, in par-ticularm. cynos, and their possible role in the severity of the clinical signs are unknown. the aim of the present study was to investigate the presence of m. canis and m. cynos in a population of dog infected with bb, compared with other populations: healthy dogs and dogs with bacterial bronchopneumonia where bb was not involved (bbp). therefore, bb, m. canis and m. cynos were detected by qpcr in the bronchoalveolar lavage fluid (balf) sample in dogs with bb (mean age = . y, mean bw = . kg), dogs with bbp ( . y, . kg), and healthy dogs ( . y, . kg). bordetellosis was diagnosed based on clinical findings together with demonstration of pleiomorphic cocci/coccobacilli adhering to the cilia of the epithelial cells on cytospin balf preparations, and positive qpcr on balf. a clinical severity index (csi to ) was assigned based on clinical signs (cough - , dyspnea - , lethargy - , fever - ), thoracic radiographic pattern ( - ), and balf score ( - ). bbp was diagnosed based on clinical findings, balf cytology and culture. m. canis was indifferently detected in healthy ( / , %), bbp ( / , %) and bb dogs ( / , %) while m. cynos tended to be more frequently detected in bb group ( / , %) than in healthy ( / , %) and bbp dogs ( / , %) (khi² test, p = , ). in bb dogs, no correlation could be detected between csi and presence of m. cynos (khi² test p = , ). in conclusion, the present data suggest that, in cird, coinfection with bb and m. cynos is frequent, but is not correlated with clinical disease severity. further studies are required to investigate whether coinfection of bb and m. cynos deserves specific therapeutic considerations. no conflicts of interest reported. canine idiopathic eosinophilic bronchopneumopathy (ebp) is a disease characterized by eosinophilic infiltration of the lung and bronchial mucosa in young adults. aetiology remains unclear although immunologic hypersensitivity is clearly suspected, while inciting antigens are generally unidentified. in humans as in cats, infections with mycoplasma spp. have been discussed as potential triggers in inflammatory bronchial disease , . bordetella bronchiseptica (bb) is a recognized pathogen agent of canine infectious tracheobronchitis. detection of bb and mycoplasma spp, especially mycoplasma cynos (m. cynos), and their potential role of in canine inflammatory bronchitis, have not been investigated. the aim of the present study was to investigate the frequency of bb, mycoplasma canis (m. canis) and m. cynos in canine ebp. therefore, presence of bb, m. canis and m. cynos were retrospectively assessed by quantitative polymerase chain reaction (qpcr) in bronchoalveolar lavage fluid (balf) samples from dogs with ebp (mean age = . y, mean body weight = . kg) as well as in dogs with aspecific chronic bronchitis ( . y, . kg). based on clinical signs, a clinical severity score (css, - ) was assigned each ebp dog. although all balf culture and cytology were negative for this bacteria, bb was more frequently detected by qpcr in ebp dogs ( / , %) than in cb dogs ( %) (khi² test, p = , ). presence of bb in ebp dogs was independant of age but significantly associated with css (khi² test, p = , ). results of qpcrwere positive for m. canis and m. cynos in ( %) and ( %) ebp dogs and in ( %) and ( %) cb dogs, respectively. there was no difference between the groups for any of the organisms. any relation between age or css and presence of m. spp in ebp dogs was observed. in conclusion, m. canis and m. cynos do not seem to be predominantly involved in the pathogenesis of canine ebp. however, bb is more frequently detected in balf from ebp dogs than from dogs with aspecific cb and its presence is associated with clinical severity. whether bb is able to trigger eosinophilic inflammation or is only more easily collected in an inflamed environment is unclear. but ebp dogs could potentially act as bb carriers and source of infection. therefore, bb should be systematically searched for in canine ebp cases and treated accordingly. no conflicts of interest reported. distal renal distal renal tubular acidosis (drta) was recently reported in three dogs with imha. the purpose of this study was to explore the hypothesis that drta is an underdiagnosed concurrent disorder in dogs with imha. we report the clinical presentation and outcome of three dogs where the combination of imha and drta was strongly suspected. the medical records of dogs diagnosed with imha at the university of edinburgh hospital for small animals between january and may were reviewed to identify cases where venous blood gas analysis and urinalysis had also been carried out. for the purpose of this retrospective study imha was defined by the presence of anaemia with pcv < %, and one or more of the following criteria; a positive slide agglutination test, positive coombs' test or moderate to marked spherocytosis. the criteria for diagnosis of drta included moderate to marked hyperchloremic metabolic acidosis with a normal anion gap; urine ph (> . ) in the face of metabolic acidosis; hypokalaemia (< . mmol/l). fifty-seven records were evaluated, with cases being excluded due to insufficient clinical information, including inability to determine urinary ph due to the severity of pigmenturia in four cases. of the cases where there was sufficient clinical data to assess the likelihood of drta only one case fulfilled all the criteria; two cases fulfilled all but one of the criteria and drta was strongly suspected based on clinical progression and persistence of urine ph > in the face of severe metabolic acidosis. of the three cases where concurrent imha and drta was suspected, two survived to discharge; one was still alive at the time of writing ( months after discharge) and the other was euthanased months after discharge following the development of multiple joint effusions and skin lesions suggestive of sle. venous blood gas analysis and assessment of urine ph should be considered in all cases of imha to exclude the possibility of concurrent drta, particularly where persistent hypokalaemia is detected. prospective evaluation of a larger cohort of imha cases is required to determine the actual incidence of concurrent drta. no conflicts of interest reported. persistent renal proteinuria is considered an early marker of chronic kidney disease (ckd) and it is listed among the initiation factors and progression factors according to kdoqi guidelines. nevertheless, few data are available about the prevalence of proteinuria in cats affected with ckd, in which it is assumed that nephropathy is mainly characterized by tubulointerstitial damage. the aim of this study is to determine the prevalence of proteinuria in cats affected with ckd and to valuate the relations between urine protein to creatinine ratio (upc) or iris substaging by proteinuria, towards purebred, sex, age, haematology, biochemistry and urinalysis. wilcoxon test, linear regression and chi-square test were used for the statistical analysis. data from cats were considered. non-renal proteinuria was an exclusion criterion. proteinuric cats (upc> . ) were . % in ckd cats, while . % could be substaged as bordeline proteinuric ( . ) in cats, proteinuria tends to increase with aging (p < . ) and with worsening of the nephropathy (p = . ). proteinuria was related to the anaemic state in ckd cats: upc significantly increases with rbc count, hb, ht and mch decreasing (p < . and p = . respectively). proteinuria tends to increase with wbc count (p = . ) and neutrophils increasing (p = . ), while tends to decrease with lymphocytes increasing (p = . ). furthermore, upc significantly increases in presence of an inflammatory serum protein electrophoretic pattern. upc tends to increase with phosphorus and alp increasing (p < . and p = . respectively); while the role of phosphorus in ckd is well known, the increase of alp is questionable: it has been hypothesized that higher alp levels in ckd could be related to b-alp increase due to bone remodelling in secondary renal hyperparathyroidism. considering urine parameters, upc increases when urinary specific gravity and ph decrease (probably related to worsening of ckd and development of a metabolic acidosis) and when glicosuria is present, regardless of the cause. furthermore, proteinuria increases in presence of rbc in urinary sediment and in samples where casts were observed, in particularly when rbc casts (considered always pathological and indicative of glomerular damage) were present. upc values assessed in proteinuric cats and data analysis suggest the need of deepen the analytical variability of upc and the opportunity to reconsider the intervals of substaging by proteinuria in cats. no conflicts of interest reported. the aim of this retrospective study was to evaluate: a) the relations between urine culture results and urinalysis parameters; b) the results of the antimicrobial susceptibility tests. urine samples were collected by cystocentesis from dogs and cats, whose diagnostic workup included a differential diagnosis of uti: all samples underwent a complete urinalysis, upc ratio assessment and urine culture. infected vs sterile results were related to urine physical, chemical parameters and observations from urinary sediment analysis. statistical analysis was performed using jmp . (sas institute inc.). a p value < , was considered significant. urine culture resulted positive in dogs ( %) and cats ( %). the presence of uti was significantly related to urine physical properties (color and turbidity), usg and leukocyturia: infections tended to be more frequent in urine samples characterized by a light yellow color, cloudy or sub-limpid aspect and low usg. nevertheless, urine was limpid in % of infected samples, and a normal usg was found in . % of dog's uti but only in . % of cats. although leukocyturia tends to become higher in infected samples both in dogs and cats (p < . ), in . % of infected sediments wbc count was normal. haematuria detected by dipstick was significantly related to uti in dogs but not in cats, nevertheless the rbc count in sediment was not related to infection in both species: rbc count was normal in . % of infected feline samples and in . % of canine samples. no significant relation between presence or absence of uti and albuminuria, bilirubinuria, glycosuria was detected, while upc tends to become significantly higher in dogs. although the chi-square test showed a significant relation between infection and the detection of bacteria in urinary sediment, a pseudobacteriuria was found in . % of samples; furthermore bacteria weren't observed in . % of infected samples (usg< . ). e. coli was isolated in the majority of samples ( , %), compared to other species: staphylococcus( . %), proteus ( . %) and streptococcus. ( . %). the urinalysis pitfalls and the high antibiotic resistance verified towards the most widely used molecules (penicillins, cephalosporins, quinolones) strongly indicates the importance to perform antimicrobials susceptibility tests to avoid the risk of failure associated with the use or abuse of empiric therapies in utis. no conflicts of interest reported. azotemia in dogs with chronic heart failure may reflect impaired renal function not only because of inadequate renal perfusion, but also due to organic renal injury. impaired renal function is observed in % of dogs with heart failure. altered renal hemodynamics due to decreased cardiac output results in renal hypoperfusion, and resultant elevation of blood urea nitrogen and creatinine, defined as azotemia. azotemia is a prognostic factor in dogs with mitral regurgitation, therefore, preservation and/or restoration of renal function is thought to improve prognosis. medical treatment for heart failure, however, includes angiotensin converting enzyme inhibitors and loop diuretics, which has been shown to increase the risk of developing azotemia. we hypothesized that mitral valve repair surgery ameliorates renal function by improvement of systemic hemodynamics. the change in renal function in dogs with mitral regurgitation was assessed by evaluating time-dependent changes in glomelular filtration rate by inulin clearance before and after cardiac surgery. eighteen dogs with severe mitral regurgitation with azotemia (plasma urea nitrogen level > mg/dl, plasma creatinine level > . mg/dl) were included in this study. the glomerular filtration rate in all dogs were evaluated by determining inulin clearance before and months after surgery. serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, and plasma creatinine concentration were measured at each time point as well as during the initial staging of heart failure based on the international small animal cardiac health council (isachc). left atrial/aorta ratio by echocardiography and vertebral heart size by thoracic radiographs were also measured. glomerular filtration rate significantly increased months after surgery ( . ml/min/m [ . - . ], . ml/min/kg [ . - . ]) compared to before surgery ( . ml/min/m [ . - . ], . ml/min/kg [ . - . ]) (p < . ). the isachc stage of heart failure was improved at months after surgery compared to before surgery. in addition, serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, la/ao and vhs significantly decreased after surgery (p < . ). the use of diuretics decreased after mitral valve repair surgery and consequently, a decrease in plasma urea nitrogen and creatinine levels were observed. therefore, this suggests that the main cause of azotemia in dogs with mitral regurgitation may be due to inadequate renal blood flow and exacerbation by the use of diuretics. no conflicts of interest reported. glomerular filtration rate (gfr) is generally considered to be the gold standard measurement of kidney function. gfr can be calculated by measuring serum iohexol clearance using concentrations at , and hours following a bolus injection. for validation, serum samples were spiked at low ( . mg/ ml), medium ( . mg/ml) and high ( . mg/ml) iohexol concentrations. they were analysed, along with standard calibration curves ( concentrations ranging from . to . mg/ml), using deltadot's label-free high performance capillary electrophoresis (hpce) system. data were analysed using deltadot's general separation transform (gst). clinical and spiked serum samples were also sent for analysis by mass spectrometry (ms) at a reference laboratory ( samples for comparison). concentrations obtained by hpce and ms were compared in a bland altman plot. gfr for clinical samples was calculated from the measured iohexol concentrations using the method reported by bexfield ( ) . a validated method was produced, with a lower limit of detection of . mg/ml and an lower limit of quantification of . mg/ml. the upper limit of quantification was . mg/ml. the standard curve had excellent linearity (r = . ). maximum inaccuracy was less than . % of the true value, except at lloq, where it was within . %. average within day variability was less than . % at all levels, while between day variability was less than . %, except at lloq, where it was less than . %. agreement between the results obtained by measurement with hpce and ms was good (bias . %, lower and upper limits of agreement of - . and . %, respectively). method specificity was confirmed by the absence of matrix effect in six serum specimen obtained from clinical dogs. clinical samples were analysed and gfr reported with a day turnaround time. in conclusion, hpce provides an accurate and precise method for measuring iohexol in canine serum. conflicts of interest: l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health; s williams is an employee of the rvc and works in collaboration with deltadot ltd; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd. esvonc-p- cystic pancreatic neoplasia in cats. c.m. borschensky , k. steiger , a. staudacher , m. schlitter , i. esposito , h. aupperle . laboklin gmbh&co. kg, bad kissingen, germany, tu m€ unchen, institute of pathology, m€ unchen, germany, veterinary clinic dr. staudacher, aachen, germany pancreatic neoplasms in the cat mostly exhibit a solid growth pattern and are diagnosed as carcinomas. in contrast, only few reports about cystic pancreatic lesions exist. until now, only benign cystic pancreatic lesions are described in the literature. according to the histological pattern, they have been termed as cysts, (acinar) cystadenoma or pseudocysts. in man, cystic pancreatic neoplasms are classified according to the localisation (intra-/extraductal), growth pattern and differentiation (mucinous, (tubulo)papillary, serous, acinar). the aim of this study was to characterise feline pancreatic neoplasms in more detail, based on the human classification system with a special view on cystic lesions. pancreatic masses sent to laboklin from domestic cats ( - years) were investigated routinely macroscopically and by histological methods (h.&e. stain). the neoplasms showed a cystic (n = ) or solid (n = ) pattern. cystic pancreatic tumors were up to cm in diameter and were classified as benign variants in five and malignant variants in three cases. based on the human classification system, they were classified as tubulopapillary (n = ), acinar (n = ) and mixed (n = ) adenomas and mixed carcinomas (n = ), respectively. solid pancreatic nodules were diagnosed as carcinomas with a tubular (n = ) or acinar (n = ) differentiation pattern. in summary, the gross structure (solid versus cystic) seems to be of prognostic relevance. in contrast to solid tumors, cystic pancreatic lesions in the cat behave benign in a higher percentage of cases, resulting in a better prognosis. therefore, surgical excision of these cystic masses can be recommended. with respect to the human classification system, three different subtypes of cystic pancreatic neoplasms were detected in the cat that have not been described before in veterinary medicine: tubulopapillary, acinar and mixed. to best of our knowledge, this is the first report of cystic adenocarcinomas in feline pancreas. further corresponding clinical and histological investigations are needed for a better diagnostic (ultrasound, mri) and prognostic characterisation of cystic lesions in feline pancreas. no conflicts of interest reported. the immunohistochemical detection of cyclooxygenase- (cox- ) expression in canine mast cell tumours was recently described by our team (prada et al., ) . however its prognostic value needs to be established. the aim of the present work was study the prognostic value of cox- expression by investigating the relationship with several clinical and pathological variables including the overall survival (os) time. we included dogs with mast cell tumours ( grade i; grade ii and grade iii). cox- immunohistochemical expression was carried out by a streptavidin-biotin method. for the cox- immunoreactivity evaluation were considered the number of positive cells (cox- extension), the intensity and the score of cox- . the following clinical and pathological features were considered: animal age, sex, tumour anatomical location, tumour size, skin ulceration, histological grade, histological safety margins and number of mitosis. cox- expression was correlated with the clinical and pathological data and with the overall survival. cox- intensity was statistical significantly associated with skin ulceration (p = , ); histological grade (p = , ) and absence of histological safety margins (p = , ), high mitotic number (p = , ) and with overall survival (p = , ). both cox- extension and cox- immunohistochemical score present no statistical relationship with the variables considered neither with the overall survival. our results suggest that cox- have an important role in dog mast cell tumours progression and could constitute a promising therapeutic target in this neoplasia. however, our study also demonstrated that in mcts, is the cox- intensity that has the prognostic value, not the number of cox- positive cells (cox- extension) and not the cox- immunohistochemical score. consequently, cox- intensity should be elected for evaluating the cox- positivity in mcts immunohistochemical studies. merial provided financial support for immunohistochemical analayis. the research centres has also received financial suppoprt from cecav, ceca and citab. dogs which were radiated for a subcutaneous sarcoma between and were included. medical records were reviewed and patient characteristics, treatment protocols, adjuvant therapies and outcome were analysed. follow-up information was obtained from medical records and by phone conversations with veterinarians or pet owners. thirty-two dogs were included into this study. mean age was years and mean body-weight was kg. male dogs were slightly overrepresented ( . %). curative intent radiotherapy was applied in dogs and palliative intent in dogs with a mean total dose of and . gray, respectively. in dogs microscopic disease was radiated. five dogs received liposomal doxorubicin concomitantly with radiotherapy, two received adjuvant doxorubicin and one intralesional cisplatin. overall median survival time was days with curative and days with palliative treatment. overall median survival time in dogs with macroscopic disease was days and in patients with microscopic disease it was not reached. radiotherapy was generally accepted as new treatment modality by pet owners and referring veterinarians. comparable to the literature, best outcome was achieved for dogs radiated with microscopic disease conflicts of interest: no conflicts of interest reported. oncept â , is indicated for the treatment of stage ii or iii oral melanoma after local control with survival times significantly increased following vaccination. a similar improvement in survival times has also been reported with digit melanoma. medical records of dogs diagnosed with melanoma between march and december were retrospectively evaluated. inclusion criteria were a histopathological diagnosis of melanoma, surgical excision of the tumour, and vaccination using the oncept â vaccine. dogs met the inclusion criteria. nificant renal involvement in dogs with cvl. this result indicates staffordshire terrier ( ), bouvier, giant schnauzer, maltese, irish setter, kerry blue terrier, golden retriever, scottish terrier, and great dane ( of each). dogs had stage ii digit melanoma with an equal sex distribution and a median age of . years (range - ). currently still alive and one dead, the latter following surgery for resection of a rib osteosarcoma the median survival time of the dogs still alive is . months (range - ) versus . months (range - ) for the dogs that have died. none of the dogs showed any adverse effect to the vaccine infected macrophages can cause injury in different organs, including the kidney. cvl is known as a common cause of glomerulonephritis. thus, this study aimed to investigate and characterize the renal lesions in dogs seropositive for leishmania sp. in brazil. this project was approved by the animal ethics committee of uece, brazil. twenty adult dogs seropositive for cvl from center for zoonosis control were randomly selected for this experiment. cvl was diagnosed by immunofluorescence and elisa. urine and blood sampling and kidney harvesting were performed immediately after euthanasia that the glomerulonephritis is a common sequelae related to leishmaniasis infection. even dogs in stage of ckd showed significant renal histopathological changes. animals infected with leishmania sp. may have severe renal damage and risk of progressive chronic kidney disease even when no increase of creatinine levels or proteinuria is detected.conflicts of interest:the authors received funding to pay the phd scholarship of one student (conselho nacional de pesquisa e desenvolvimento -cnpq-brazil) and received a research grant from fundac ßão cearense de apoio ao desenvolvimento cient ıfico e tecnol ogico -funcap. anaplasma phagocytophilum, the causative agent of canine granulocytic anaplasmosis, is an obligatory intracellular bacterium transmitted by ixodes ticks. transmission via blood transfusion has rarely been described in human medicine and once in a dog. in the berlin/brandenburg area the seroprevalence rate in dogs was % regardless of health status.the aim of this study was to evaluate pcr screening results for a. phagocytophilum in canine blood donors between - in order to estimate the risk of transfusion-transmitted infection. edta blood samples from dogs were submitted for a. phagocytophilum real-time pcr testing (targeting the msp gene). altogether dogs were tested up to times. clinical and laboratory data were examined before each donation. statistical analysis was performed using spss . .the pcr test was positive for of the samples. none of the dogs tested pcr positive more than once. positive results were most often detected in june ( ), may ( ), and july ( ), but also in five other months. in three dogs a mild increased in rectal temperature (≥ , °c) was documented. mild laboratory abnormalities were noted in dogs: thrombocytopenia ( ), leukocytosis ( ), leukopenia ( ), anemia ( ) and hyperproteinemia ( / ); four dogs had more than one abnormality. there was no significant difference between the pcr negative and positive blood samples in regard to laboratory abnormalities.altogether, . % of blood samples from healthy canine blood donors were pcr positive for a. phagocytophilum. therefore, blood donors should be screened by pcr in endemic areas all year round. no conflicts of interest reported. the study population consisted of cats, including control cats recruited from veterinary practices across the country. among the disease cats, cats presented urtd, cats had conjunctivitis and cats suffered chronic gingivostomatitis, many of them presenting more than one clinical sign. pcr for the above-mentioned pathogens was performed from pooled conjunctival and oropharyngeal swabs for each cat. a questionnaire regarding signalment (age, breed, sex, neuter status), environment (indoor, number of cats in household) and vaccination history was obtained. data was analysed by multivariable logistic regression with alpha equal to < . .the prevalence for the four pathogens has been previously reported in detail. briefly, the prevalence among the four groups (including controls) ranged from to % for fhv- , - % for fcv, - % for chlamydophila felis and - % for mycoplasma felis.in the univariate analysis, age, neutering status, being purebred, indoor keeping, number of cats in the household and body weight were variably associated with the different groups of disease and the presence of the pathogens. in the multivariable analysis, only the following factors remained significant. in the multivariable analysis, only the following factors remained significant: urtd was significantly associated with positive results for fhv- , chlamydophila felis and mycoplasma felis (in addition to being male and not castrated); conjunctivitis was significantly associated to positive results for fhv- and chlamydophila felis (in addition to being young, not castrated and purebred) and cgs was significantly associated to positive results for fcv (in addition to being young, male and purebred). not being properly vaccinated was a significant risk factor only when all three groups were analyzed together. the number of cats in the household was an independent risk factor for detecting each of the pathogens studied. the age was also a significant factor in cats with fcv and chlamydophila felis, being older cats predisposed to fcv and younger cats predisposed to chlamydophila felis.the present study describes important epidemiological data for cats presenting urtd, conjunctivitis and/or cgs, and emphasizes the complex interrelationships occurring among the different pathogens. our results also support the role of fcv in cats with chronic gingivostomatitis.conflicts of interest:the study was funded and designed by merial laboratories. reports of methicillin-resistant staphylococci (mrs) in animals have become more frequent in last years. various studies have demonstrated the transmission of mrsa between animals and humans in daily contact with animals, however there is only limited data so far available on the transmission of methicillinresistant coagulase-negative staphylococci between animals and humans. the objective of this study was to investigate the frequency of methicillin-resistant staphylococci (mrs) carriage in healthy veterinarians, veterinary nurses, veterinary assistants, veterinary students and farm workers from several veterinary hospitals, clinics and farms.nasal swabs were collected from veterinarians ( small animal veterinarians and pig veterinarians), veterinary nurses, veterinary students, veterinary assistants and farm workers. mrs were screened on brilliance tm mrsa agar (oxoid) or chromid tm mrsa (biom erieux). after - h of incubation at °c, suspected colonies on both media were subcultured onto blood agar plates. species identification was obtained by species-specific pcr. methicillin-resistance was confirmed by pcr amplification of the meca gene. mrsa isolates were characterized by mlst.thirty-nine mrs were identified in humans ( veterinarians, veterinary nurses, veterinary students, veterinary assistants and farm workers). the mrs isolates were identified as staphylococcus aureus (mrsa, n = ), s. epidermidis (mrse, n = ), s. pseudintermedius (mrsp, n = ), s. haemolyticus (mrsh, n = ) and mrs coagulase-negative staphylococci. the frequency of colonization by mrs was similar in both small animals and pigs veterinarians (ae %). one veterinary student was colonized simultaneously with an mrse and an mrsh. the predominant st in humans in contact with small animals was st and in humans in contact with pigs was st .in our study the frequency of colonization by mrsa was high, but the frequency of mrse should not be underestimated. mrsa isolates in this work belonged mainly to the st lineage which is the most frequent in small animals and humans in europe. humans in daily contact with animals can become colonized by mrs of animal origin and thus are important keys for infection control programs in veterinary hospitals and farms.conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party (awp) and member of the antimicrobial advice ad hoc expert group (ameg) of the european medicines agency (ema). , species not determined [ ]) did not. all cats underwent antibiotic treatment (doxycycline or a fluoroquinolone); cats received blood transfusions and/or oxyglobin â . three cats were euthanatized within days due to concurrent disease (fiv, pancreatitis/cholangitis) or financial constraints, one cat due to persistent anemia after weeks. four cats were lost to follow-up. the remaining cats underwent follow-up for a period of - weeks (median ). hemoplasma pcr analysis was conducted - times on blood samples at variable time points from of the follow-up cats. the first negative pcr in cases occurred after (cmhm, during antibiotic treatment), (cmhm, during antibiotic treatment), (cmhm, during antibiotic treatment) and (mhf, after completion of antibiotics) weeks. one cat remained pcr positive (cmhm) at , , and (all during antibiotic treatment) weeks, and another cat (cmhm) was pcr positive at weeks. reactivation of the hemoplasma species (documented by hemolysis and positive pcr) occurred in cats (both cmhm) and times, respectively, up to weeks after initial presentation. reactivation was suspected (no pcr testing available) in additional cases (cmhm [ ], mhf [ ]). four of the follow-up cats were euthanatized after - weeks (median ) due to concurrent disease (cardiomyopathy, immune-mediated thrombocytopenia, postoperative complications, diabetes mellitus). infection with hemoplasmas is often chronic, can reactivate months later and is rarely the reason for euthanasia. no conflicts of interest reported. canine distemper (cd) is a worldwide occurring infectious disease caused by a morbillivirus of the family paramyxoviridae. cdv infection can result in a systemic infection. dogs presented with neurologic signs revealed the terminal stage of the disease and usually failed to therapy. additional passive immunotherapy is hypothesized to be beneficial in the early stage of cdv infection. porcine anti-cdv antibody subunit f (ab') [f(ab') ] was produced by animal technology laboratories, agriculture technology research institute. eighteen cdvnaturally infected dogs showing respiratory signs but no neurological signs were treated with the combination of f(ab') and supportive therapy (group ). group included dogs in a similar clinical signs (without neurological signs) that received only supportive therapy. the survival rate was . % ( / ) in group and . % ( / ) in group , respectively, with a significant difference between the two groups (p < . ). the progressive rates of developing neurological signs during therapy of group and group were . and . %, respectively. there was no significant difference between the two groups. the survival rates of dogs developing neurological signs during therapy were % ( / ) in group and . % ( / ) in group , respectively, with a significant difference between the two groups. in conclusion, additional administration of porcine anti-cdv antibody subunit f(ab') before developing of neurological signs could decrease the mortality and furthermore reduce the rate of developing neurological signs. no conflicts of interest reported. key: cord- -h o yqv authors: nan title: oral communications and posters date: - - journal: inflamm res doi: . /bf sha: doc_id: cord_uid: h o yqv nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : ( ) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? ( ),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; ( ) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd + tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp( - ) peptide in complex with hla-dr b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr b gene and a human t-cell receptor (tcr) that recognises the mbp - peptide in the context of hla-dr b either spontaneously or after immunization with mbp - develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far genes in this concert have been identified; the mhc class ii ab gene in the mouse ( ) and the ncf gene in the rat ( ) and in the mouse ( ) . the ncf protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf deficiency linking the ncf pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats ( ). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to % of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry- (azd ) is an inhibitor of mek / currently in development for cancer. phase determined the msd ( mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (- %) and proliferative index (- %). the trough plasma concentration ( ng/ml) corresponded to~ % inhibition of perk. about % of pts had stable disease after months. these results demonstrate that arry- (azd ) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase studies, in melanoma, colorectal, lung and pancreatic cancer. arry- is another potent, selective mek / inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry- inhibited tnfa, il- b and il- (ic s of , and nm, respectively). % inhibition of perk required nm. arry- was highly efficacious in cia and aia rat models, with ed s of and~ mg/ kg, respectively. in normal volunteers arry- was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry- monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry- in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock- isoform.these rock- inhibitors, typified by slx- and slx- , are potent (ic < nm), selective for rock- (> fold selectivity for rock- over rock- ) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx- inhibits cell proliferation and migration in several tumor cell lines including ht- , panc- and mda-mb- . moreover in xenograft studies using nude mice, slx- significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx- prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx- is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock- selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx- and slx- will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox- is constitutively expressed.the cox- gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox- are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox- can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox- plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox- and tumorigenesis come from genetic studies.overexpression of cox- has been observed to drive tumor formation whereas cox- deficiency protects against several tumor types.selective cox- inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox- inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox- -derived pge are found in both inflamed and neoplastic tissues.the fact that pge can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox- contributes to both wound healing and tumor growth.taken together, it seems likely that cox- induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. ( ), k hagihara( ), t nishikawa( ), j song( ), a matsumura ( ) ( ) health care center, osaka university, japan ( ) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il- in the inflammatory status. to know the pathogenic role of il- and the efficacy of il- blockade in inflammation, a humanized anti-il- receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il- blocking therapy improved the clinical symptoms and the laboratory findings, the il- function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il- , il- and tnf-alpha. in result, il- was an essential cytokine in induction of saamrna through the activation of stat which constructed the complex with nf-kappab p and a cofactor p . although there was no stat consensus region on saa promoter, stat bound at the site of nf-kappab re. the above research proved that il- signal is essential on the synergistic induction of saa via newly discovered stat transcriptional mechanism, suggesting the presence of this stat mechanism in inflammation, and confirming the normalization of serum saa level by il- blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada( ), k matsushima( ), s kaneko ( ) ( ) kanazawa university, japan ( ) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)- / monocyte chemotactic and activating factor (mcaf)/ ccl , a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. ( ), p lacamera ( ), b shea ( ), g campanella ( ), b karimi-shah ( ) , n kim ( ), z zhao( ), v polosukhin ( ), y xu( ), t blackwell ( ) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa , are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa -deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r receptor. we have shown that tnf-r is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor- (par- ) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par- is implicated in innate defense responses associated with lung inflammation. we showed that par- is expressed by human alveolar (a ) and bronchial ( hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh . in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par- , but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh . preincubation of a par- transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par- , generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par- in the respiratory tract, thereby altering the host innate defense mechanisms. caspase- -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor (par ) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars , and , suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (> %), which is not seen in dizygotic twins (< %) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before . incidence has increased since now leading to a lifetime prevalence of up to . % in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome being most consistent between different populations. in three coding variations in the card gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card gene. variations in the card gene do not fully explain the linkage finding in the pericentromeric region of chromosome . after stratification for card variants, the broad linkage peak is reduced to two more defined peaks on p and q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes and , respectively). dlg- is the example of a low-risk susceptibility gene with a modest associated odds ratio ( . - . ) . interestingly, the associa-tion signal appears to be confined to young males. slc a / which encode the kation-transporters octn and have been suggested to represent the disease gene in the + kb haplotype block on chromosome q . mdr has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a k illumina scan has been published identifying il- r as a further disease gene. we used a genome wide candidate gene approach (with appr. . csnps) to identify atg l as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr , teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr +/ cd + t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx -b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr .the compound proved to be highly efficacious in the tnf-aare and mdr a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx -b was well-tolerated, and no drug-related saes were reported.a -day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx -b was shown to be both safe and to have encouraging clinical results: % of patients on ccx -b (cdai ! , crp > . mg/l) exhibited a -point drop in cdai compared to % on placebo. furthermore, a crp decrease of mg/l was seen in the ccx -b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il- p , ifn-gamma, and the chemokine rantes was shown in the ccx -b group while levels remained stable in the placebo group. ccx -b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg d in a model of inflammatory bowel disease, where cd +cd -t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg d antibody therapy. the expression of nkg d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg d on the cell surface as well as increased levels of nkg d mrna in cd + t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg d antibody (cx ) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx treatment decreased the cellsurface expression of nkg d and prophylactic administration of cx attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx administration to mildly colitic animals, whereas cx did not attenuate severe colitis. thus, nkg d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg d in cd + t cells increased markedly during the development of disease and since administration of cx early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than have gained approval. the pipeline contains many more potential biopharmaceuticals and at present in new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled % human biologicals are potentially immunogenic ( ) ( ) . i shall discuss two examples: ) recombinant human cytokines (ifn-beta- a and - b), and ) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used ( - ). it took more than years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized ( , ). ) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, - % of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnß antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnß in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnß antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit , was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, : - , ) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par and par also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par activation in joint inflammation and pain. male c bl/ mice received an intra-articular injection of either the par activating peptide aypgkf-nh or the inactive peptide yapgkf-nh ( mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par activating peptide caused knee joint blood flow to gradually increase by up to % over the succeeding hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par antagonist pepducin p pal ( mg i.p.). the control peptide yapgkf-nh had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par receptors in mice knees causes joint inflammation and pain. vincent lagente ( ), e boichot ( ) ( ) air liquide, centre de recherche claude-delorme, jouy en josas, france ( ) inserm u , universitØ de rennes matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp- ) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp- /timp- ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that members of the par family (par , par and par ) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par and par in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par and par -dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par and par dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par -dependent mechanism.in contrast, activation of par on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled consecutive dogs (group b). results: dogs were excluded from group a where / dogs ( %) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in , a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including consecutive dogs with a final diagnosis known to predispose for dic ( % were finally diagnosed with dic). outcome was -day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim : ; - ) . relative risk according to bpw (rr [ % confidence interval]) for (a) a dic diagnosis and (b) -day mortality, were assessed. -day mortality in the study population was %. % were bpw positive. bpw was not a significant diagnostic factor for dic (rr= . [ . ; . ] ), but strongly so for outcome (rr= . [ . ; . ] ) with a % ( / ) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. ( ), b hideo ( ) ( ) department of molecular pathology, kumamoto university, japan ( ) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin- expression (graÅa-souza et al., ) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., ) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. ( ) ( ) university of melbourne, victoria, australia ( ) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx ), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx -/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx -/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx -/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx -/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase- (mmp ) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx -/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp . chris bolton( ), c paul( ), s barker ( ), r mongru ( ) ( ) william harvey research institute, london, uk ( ) university west of england, bristol ( ) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a subunit (glyr a ) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a , the reduction of glyr a was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a , while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a may be a target molecule for pkg to induce allodynia. ( ), r leite( ), ys bakhle ( ) ( ) federal university of minas gerais, belo horizonte, brazil ( ) medical college of georgia, augusta, usa ( ) imperial college, london, uk selective cyclooxygenase inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats ( - g; - animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and min later given cx ( mg/kg, s.c.). after a further min, rats were injected (ipl) with the inflammatory stimulus, carrageenan ( mg/paw). mechanical pain threshold was hourly measured over the next h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin % reversal = . nanomoles), higher doses of microtubule inhibitors (taxol % reversal = . nanomoles) with no effect of acrylamide ( up to nanomoles).we conclude that ) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and ) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox- in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox- inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for hrs daily for a period of seven days. nimesulide ( . mg/kg, i.p.) was administered daily for days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in . ul. ngf ( . , and ug) injected in the tmj challenged h prior by cg ( ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k a ( and ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production and h after the tmj cg ( ug) injection. the tmj injection of ngf ( ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf ( ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd +/f / -ve/cd c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam- , although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl /mcp- , and its receptor ccr , and in response to anti-cxcr .this was supported by in vitro experiments showing that mif induced ccl /mcp- release from cultured murine endothelial cells.finally, mice lacking cd , the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd .this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf- ) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap- activities. gm-bmm preferentially produced tnfa, il- , il- and il- while, conversely, bmm generated more il- and ccl ; strikingly the latter population could not produce detectable il- and il- . following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap- activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il- following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type diabetes mellitus (t dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad , ia- and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd and cd t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th , tc ) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b: - cause diabetes . knockouts of the insulin gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin gene (islet expression) prevents % of diabetes . dual insulin knockout with transgenic insulin with altered peptide (b :a) prevents all diabetes . islets with native b: - sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells .anti-b - t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain . alpha chain as transgene sufficient to engender anti-insulin autoantibodies . kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as % of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a -step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam- and lfa- , have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa- antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, düsseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il- and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin- , tnf-µ, p mapk, msk / , mk , nf-kappab and ap- . histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h receptor (h r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h r deficient mice and mice treated with h r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th responses. ex vivo restimulation of primed t cells showed decreases in th cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h r was responsible for the the t cell effects. the influence of h r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h r deficient mice. the h r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h r was minor. surprisingly the h r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h r antagonists. ( ), bsp reddy ( ) ( ) nizams institute of medical sciences, hyderabad; india ( ) genix pharma, india rupatidine, carries the majority of the histamine h receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either mg rupatidineformulation after overnight fast. washout was days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc - mmsq/hr and auc - %/hr) both for untransformed and log transformed data were found to be within - % of % ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet ( ), h yoshimura( ), k ohara ( ), y mastui ( ), h hara ( ), h inoue ( ), h kitasato ( ), c yokoyama( ), s narumiya ( ), m majima ( ) ( ) kitasato university school of medicine, kanagawa, japan ( ) tokyo dental medical colledge, tokyo, japan ( ) kyoto university school of medicine, kyoto, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitorsconstitute revascularization. we hypothesized that txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggrega- inflamm. res., supplement ( ) tion.to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out(ipko) and thromboxane (tx) a receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf- and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. purpose: the s calcium-binding proteins, a , a and a are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a /a is anti-microbial; a has oxidant-scavenging functions. a is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys to ala a mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s s in asthmatic lung. the s s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a . a , a or the a /a complex had relatively low ability to induce il , tnf, il , and chemokines mrna from pbmc compared to a . only a induced significant levels of il ; none induced il or gm-csf mrna compared to lps. in contrast to a which is activating, a significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys . conclusions: the cytokine profile generated by a in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a in oxidant defence, particularly to hypobromite generated by activated eosinophils. ( ), d mankuta( ), g gleich ( ), f levi-schaffer ( ) ( ) hebrew university of jerusalem, israel ( ) hadassah university hospital, israel ( ) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson ( ), d kashanin ( ), f odowd( ), v williams( ), g walsh ( ) ( ) cellix ltd, institute of molecular medicine, dublin, ireland ( ) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam- and vcam- , is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. - dynes/cm ). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam- , icam- , and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam- are high ( - %, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam- , with pma-stimulated cells showing a higher affinity for icam- than nonstimulated cells (approx. % and %, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph . ). male c bl/ were instilled with ml/kg of gj. after or h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. . ae . , sg h: . ae . ; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il- ratio in balf at the nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il- ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c bl/ mice were subjected to min of intestinal ischemia and challenged with . mg/kg of intranasal lps at the th hour of reperfusion (two-hit). balf and culture of lung explants were performed h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r ( . ae . vs . ae . , mg/mg). lung mpo was increased ( . ae . vs . ae . ; od nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group ( . ae . vs . ae . ; x e cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf ( . ae . vs . ae . ; mm) and in lung explants ( . ae . vs . ae . ; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. ( ) ( ) novartis institutes for biomedical research, horsham, uk ( ) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over % (p< x - ) for exercise capacity, measured by running on a treadmill.after months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p< . ); however there was a - -fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p< . ).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p< . ). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. ( ), ap ligeiro-oliveira( ), jm ferreira-jr( ), sr almeida( ), w tavares de lima( ), shp farsky ( ) ( ) university of s¼o paulo, brazil ( ) regional integrated university of alto uruguai and missðes, brazil methods: male wistar rats were exposed to vehicle or hq ( mg/kg; ip.;daily, days, two-day interval every five days). on day , animals were ip sensitized with ovalbumin (oa). assays were performed on day . results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound / . in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd and cd on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missðes / university of s¼o p, department of clinical and toxicological analyses, s¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br ( ) ( ) radboud university nijmegen, medical centre, nijmegen, the netherlands ( ) university hospital, zürich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr and tlr was associated with the expression of ifna, tnfa, il- b, il- , il- , and il- and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr / tlr expression in synovial tissue was associated with the presence of ifna, il- b and il- , but not tnfa, il- and il- . to investigate whether ifna, il- b and il- could induce tlr / tlr upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr / mrna expression. ifna incubation resulted in significant tlr /tlr upregulation, whereas il- b and il- did not. pre-incubation with ifna and subsequent stimulation of tlr /tlr significantly enhanced il- , tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il- b were produced upon stimulation with atp, but not upon tlr / tlr stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il- b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il- b / il- in synovial tissue. first, involvement in tlr /tlr regulation and second, involvement in atp-induced production of biologically active il- b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod (nucleotide/ binding oligomerization domain) and nod in model of acute joint inflammation induced by intraarticular injection of tlr (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod and nod occurs also in humans, we exposed pbmcs carrying either nod frameshift or nod frameshift mutation with scw fragments in vitro. both tnfa and il- b production was clearly impaired in pbmcs carrying the nod fs compared to pbmcs isolated from healthy controls. in line with the nod gene deficient mice, human pbmcs bearing the nod mutation produced enhanced levels of proinflammatory cytokines after h stimulation with scw fragments. these data indicated that the nlr family members nod and nod have a different function in controlling tlr -mediated pathways. we hypothesize that intracellular nod -nod interactions determine the cellular response to tlr triggers. whether lack of controlling tlr -driven pathways by nod signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp- cells with vitamin-d . lilra expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra expression (p= . ). treatment of macrophages with lps, tnf-a, il- b and ifn-g but not il- caused significant down-regulation of lilra (p< . ). function of lilra was assessed by cross-linking with plate-immobilised lilra -specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at h. correlation between lilra expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra could potentially provide therapeutic benefit. ( ) ( ) university of valencia, spain ( ) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm- ) on an animal model of human rheumatoid arthritis. dba- /j mice were treated with corm- ( , or mg/kg/day, i.p.) from day to after collagen-induced arthritis (cia) and sacrificed on day . administration of corm- resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm- treatment with the most pronounced protective effect observed at mg/kg. the levels of pro-inflammatory mediators (pge , il- beta, tnfalpha, il- and il- ) in the hind paw homogenates were significantly inhibited by corm- . in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm- alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm- appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without ng/ml il- a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a - were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < . ). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il- a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr and tlr activation in the expression of arthritis was studied using interleukin- receptor antagonist deficient (il- ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il- ra-/-tlr -/-mice developed more severe arthritis compared to il-ra-/-tlr +/+ littermates; whereas, tlr -/-il- ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr and tlr differentially regulated the disease expression, we studied the role of these tlrs in il- production and th development, both important in il- ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il- upon stimulation with tlr and tlr ligands; however, il- ra-/-bmdcs produced less il- than wild type dcs upon tlr stimulation and more il- than wild type dcs upon tlr stimulation. furthermore, il- ra-/-t cells produced lower amounts of il- when cultured with tlr -activated apcs and higher amounts of il- when cultured with tlr -stimulated apcs, both in combination with cd stimulation. facs analysis of th (cd +/il- +) cells from both spleen and draining lymph nodes revealed % reduction in il- ra-/-tlr -/-mice compared to il- ra-/-tlr +/+ littermates. specific cd /cd stimulation of non-adherent splenocytes confirmed lower il- production in il- ra-/-tlr -/-. these findings suggest important roles for tlr and tlr in regulation of th development and expression of arthritis. prostaglandine (pge ) stimulates the transactivational activity of p through p map kinase-dependent ser phosphorylation (jbc ) .p controls cell-cycle progression, in part, by differential regulation of ap- proto-oncogenes (jun/fos).currently we studied pge control of cyclin d promoter activity with particular attention to the role of ap- oncogenes.pge induced a . fold increase in junb mrna expression (northern blot), a . -fold increase in junb promoter activity (luciferase assay), and increased ser junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra / , and fosb expression were upregulated by pge .in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g v) resulted in a . fold increase in cyclin-d promoter activity, cyclin-d synthesis, thr /tyr phosphorylation of erk / ( . fold) and ap- (c-jun)-dependent transactivity ( . fold); cyclin d /cdk - inhibitor p ink a synthesis was suppressed. addition of excess rass n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d promoter activity/protein synthesis, blocked p ink a synthesis; the latter effects were reversed by the addition of excess junb.pge exerted temporal and bi-phasic dose-dependent control of the cyclin d promoter activity, largely through differential ap- activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla / cox/pges biosynthetic axis and highlight the complexity of pge action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., , inflammation res. (s ) s ) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider ( , pnas. : - ) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha ( mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c bl/ mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day . hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic < . mm). diga ( ml, mm) i.a. prevented hour aia (- . +/- . mm).diga at mg/kg reduced aia when administered i.v. (- . +/- . mm, p< . ) and i.p. (- . +/- . mm, p< . ), but is hydrolysed p.o. ( . +/- . mm ns). polysulphated diga (diga s) was unstable, but stabilised by n-acetylation (nacdi-ga s).tnf synthesis was potently inhibited by both nacdiga and nacdiga s (ic < . mm).nacdiga s ( mg/kg p.o.) inhibited aia (- . +/- . mm), and nacdiga s with lower degrees of sulphation (mw and kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on persons, divided into groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: ) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. ) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs expression following exposure of murine macrophages to lps implicated socs in the control of lps-mediated signaling. socs regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs in regulating the production tnf by lps and pam csk -activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs (adv-socs ), control vector (adv-gfp) or left untreated. adv-socs monocytes were exposed to tlr and tlr ligands, lps ( ng/ml) or pam csk ( ng/ml). facs analysis demonstrated infection efficiencies of ae % and ae % (n= , mean ae sem) of monocytes expressing adv-gfp or adv-socs at moi . adv-socs blocked lps and pam csk induced tnf mrna and protein production in a dose-dependent manner. in contrast, il- and il- production by adv-socs -infected monocytes was not blocked. adv-socs also blocked lps and pam csk induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of ae % or ae % were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p activation. furthermore, the kinetics of lps and pam csk induced ikba phosphorylation and degradation in adv-socs monocytes remained unaffected (n= and donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p or erk mapk pathways. these data suggest that socs regulation of lps and pam csk -induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels ( - mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: ) measuring cytosolic free calcium ([ca +] i) in fura- loaded hdsmc using spectrofluorometry and ) force measurements in - mg detrusor strips. hdsmc responded to beta-nad+ ( - mm) with an immediate and transient increase in [ca +] i. the ca + transient was followed by one or two much slower and transient increases in [ca +] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca + responses persisted in the absence of extracellular calcium. the ca + responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p x purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp -induced neurotoxicity. we recently showed that tat induces the overexpression of il- via cellular kinase pkr and activation of transcription factor ets- . in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat tyrosine phosphorylation, but had no effects on the serine residue of stat and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat was abrogated by tat. the inhibition of phospho-stat led to the deformation of stat homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and , oligoadenylate synthetase ( , oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of , oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase- belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin- (il- ), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase- in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase- and il- . interestingly, increased caspase- activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p mapk dependent increased secretion of procaspase- and active caspase- . furthermore, anisomycin increased the mrna expression of il- through a p mapk dependent but caspase- independent mechanism, reaching a maximum level after hours of stimulation. finally, anisomycin caused a rapid ( hours) increase in the secretion of proil- and active il- . secretion of active il- was mediated through a p mapk/caspase- dependent mechanism, whereas secretion of proil- was mediated by a p mapk dependent but capsase- independent mechanism. these data demonstrate that the activity of caspase- is increased in psoriatic skin and that il- secretion is regulated by a p mapk/caspase- dependent mechanism, making caspase- a potential target in the treatment of psoriasis. prostaglandin e (pge ) regulates the stability of cyclooxygenase- (cox- ) mrna through adenylate/uridylate-rich elements (ares) in the untranslated region ( utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge dependent stabilization of cox- in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox- utr] reporter fusion mrna ( ae . % decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il- b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~ kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox- gene transcription, thus enabling ttp to regulate cox- gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox- mrna stability and transcription and whose localization and interaction with other factors is regulated by pge . these data can provide important insight into deciphering the role of pge in fine-tuning physiological and pathophysiological gene regulation. ( ) ( ) chinese academy of sciences, shanghai, pr china ( ) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)- is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp- has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp- is regulated is still not fully understood. here, we show that inhibition of p map kinase by specific inhibitor sb or rna interference (rnai) markedly reduced the expression of mkp- in lps or pgn-treated macrophages, which is correlated with prolonged activation of p and jnk. depletion of mapkap kinase (mk ), a downstream substrate of p , by rnai also inhibited the expression of mkp- . the mrna level of mkp- is not affected by inhibition of p , but the expression of mkp- is inhibited by treatment of cycloheximide. thus, p mapk plays a critical role in mediating expression of mkp- at a posttranscriptional level. furthermore, inhibition of p by sb prevented the expression of mkp- in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p -mk -dependent induction of mkp- in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il- and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd /cd mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th cytokine il- is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar -/-and ifnar -/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p x receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin- beta (il- b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin- , a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin- is required for processing of caspase- and release of mature il- b induced by p x receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il- b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin- -independent phase induced by p x receptor activation, and this was unaltered by pannexin- inhibition.nigericin did not induce dye uptake.inhibition of pannexin- blocked caspase- and il- b processing and release in response to this two stimuli.thus, while pannexin- is required for il- b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin- hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il- . here we verify the effect of saa on the mrna expression and release of mip- alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip- alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa ( ug/ml) induced the expression of mip- alpha mrna at , and hours. mip- alpha protein was found in the suppernatant of and hours cultures (p< , ) and the addition of sb (p inhibitor) and pd (erk / inhibitor) completely abolished the release of mip- alpha. conclusions: saa is an inducer of mip- alpha expression in pbmc and p and erk / are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p decrease mediator production and therefore can produce profound anti-inflammatory effects. arry- is a potent inhibitor of p enzyme (ic < nm) with a novel pharmacophore and physiochemical properties distinct from those of other p inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic < nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed ~ mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of , , , or mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry- was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il , pge and tnffz with > % inhibition observed at the mg dose level. the plasma concentrations of drug peaked at~ ng/ml at the mg dose and cytokine inhibition was sustained for > hours, showing that low doses of arry- produced profound effects on clinical biomarkers. further evaluation of arry- in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, mg/kg; oral route; days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam- on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il- b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name ( mg/kg; oral route; days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained hours after i.p. injection of oyster glycogen ( %; ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of x cultured cells before and hours after lps stimulation ( m;g/ml). results: levels of no, tnf and il- b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il- b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin- (il- )-induced mmp- expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il- beta alone or in combination. mmp- mrna was analyzed by rt-pcr. mmp- protein, phospho-stat and p / mapk levels were measured by western blotting. mmp- promoter-luciferase, cmv-cbp/p plasmids and stat sirna were transfected by calcium phosphate method. ap- activity was monitored by elisa. stat -cbp/p interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il- -induced expression of mmp- and promoter activity. blockade with neutralizing ifn-gr antibody revealed that mmp- inhibition by ifn-¼ was mediated by the ifn-¼ receptor. ifn-beta-stimulated activation of stat was directly correlated with mmp- suppression. knockdown of stat gene by specific sirna or its inhibition with fludarabine partially restored the il- induction of mmp- expression and promoter activity. ifn-g did not alter activator protein (ap- ) binding ability but promoted physical interaction of stat and cbp/p co-activator. p overexpression reversed ifn-g inhibition of endogenous mmp- mrna expression and exogenous mmp- promoter activity. conclusions: ifn-g through its receptor activates stat , which binds with cbp/p co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp- gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for ( ), p asmawidjaja ( ), r hendriks( ), erik lubberts ( ) ( ) erasmus medical center, department of rheumatology, rotterdam, the netherlands ( ) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il- in th polarization in the prone autoimmune dba- mice with and without collagen-induced arthritis and to evaluate th specific cytokine and transcription factor expression. il- induced th cells in vitro from spleen cells of naïve and collagen-type ii (cii) immunized dba- mice. the percentage of th cells is markedly higher in cii-immunized versus naïve dba- mice. adding il- to tgf-beta/il- stimulated cd + t cells did not significantly increase the percentage of th cells. tgfbeta/il- in contrast to il- induced a relatively high percentage of il- +/ifn-gamma-cells and low il- -ifn-gamma+ cells. tgf-beta/il- did not increase il- receptor expression, which may explain why adding il- directly or two days after tgf-beta/il- did not result in an increase in the percentage of th cells. elevated expression of il- a and il- f as well as the th specific transcription factor rorgammat was found under il- as well as tgfbeta/il- conditions. interestingly, il- but not tgf-beta/il- is critical in the th cytokine il- expression in t cells from ciiimmunized dba- mice. these data show that il- was more pronounced in inducing il- +/ifn-gamma-(th ) cells under cii-immunized conditions. furthermore, il- did not markedly increase the percentage of th cells induced by tgf-beta/il- . however, il- is critical for the induction of il- expression, suggesting a unique role for il- in the induction of specific th cytokines ebi was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p of il- . ebi protein has been shown to form heterodimers with p . p /ebi termed il- , can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi and p is very limited. these data lead to the hypothesis that ebi may play a role independently from its association with p . thus, to define the function of ebi , we generated ebi transgenic (tg) mice expressing in multiple tissues. ebi tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd +, cd + t cells, b cells, nk cells and nkt cells. cd +t cells isolated from spleens of ebi tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd and anti-cd antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi tg mice were recovered with anti-ebi polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi antibody. in contrast, anti-p antibody suppressed chs responses in wt mice. these data suggest that ebi acts in different from il- , and reduces th responses. ( ), o thaunat( ), x houard ( ), o meilhac ( ), g caligiuri( ), a nicoletti ( ) ( ) inserm u and university denis diderot-paris , chu xavier bichat, paris, france ( ) inserm umr s , universitØ pierre et marie curie-paris , centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep -i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk / m and vascular inflammation leads to formation of leukotrienes through the -lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b (ltb ) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb -induced atherogenesis. targeting ltb -induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox- ). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox- ) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox- and the inducible enzyme responsible for pge synthesis (mpges- ) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi is the major biological active prostanoid produced in the normal physiological conditions. however, when cox- is induced, pgi and pge are equally produced. the role of cox- , cox- and mpges- activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep , ep , ep or ep ) preferentially stimulated by pge in the vascular wall. for these reasons, we have characterized the receptors activated by pge in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin- â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox- inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn (a gene that regulates treshold of lumphocyte activation) the allele c t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in dutch ra cases and unrelated dutch controls. carrier analysis of the snp (rs ) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p = . and or = . ). in a meta-analysis of all studies comparing individuals, the or for the cc genotype to develop ra was . and the p-value < . . in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd / q , card /nod and il r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card /nod is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card appear to show opposite effects to other models and human systems. in humans, card mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp /cryopyrin/ cias gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp protein associates with asc and caspase- (thereby forming a molecular machine termed inflammasome that displays high proil- beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il- beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp -dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. ( ) ( ) erasmus mc, rotterdam, the netherlands ( ) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd c-diphtheria toxin (dt) receptor (cd cdtr) transgenic mice by airway administration of dt h before ova aerosol ( x) challenge or during an ongoing inflammation (depletion after x ova aerosols continued with additional ova aerosols). numbers of balf eosinophils, th cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th -type cytokines (e.g. il- and il- ), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th -type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi -kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd r, cd r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a (spla ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla , hdl and ldl and of their hydrolysis products on mitogenesis and pge and ltb release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla . spla hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge from vsmc was enhanced by group x spla s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla .native ldl and its spla hydrolysis products enhanced the release of pge in order x>v>iia.the release of ltb from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla .migration of vsmc was significantly enhanced by spla iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca ( ) ( ) ipmc-cnrs umr , valbonne, france ( ) university of washington, seattle, usa ( ) inserm umrs , paris, france ( ) university of naples, italy the superfamily of phospholipase a comprises at least intracellular enzymes and up to secreted pla s (spla s). elucidating the biological roles of each pla member is currently the most challenging issue in the pla field. the different spla s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla can trigger secretion of tnf alpha, il and il in a non-enzymatic manner. on colorectal cancer cells, hgx spla stimulates cell proliferation, produces potent eicosanoids including pge , and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla s, we have examined fatty acid release by spla siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla was observed when the residual sm/ pdcho molar ratio had reached . compared to a starting ratio of . .group v spla showed preferential release of linoleate at residual sm/ptdcho molar ratio . , while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla , possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla . the fatty acid specificity of pla is significantly affected by sm. thus spla x exhibited enhanced specificity for the release of arachidonic acid ( : ) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of : by spla v. ceramide selectively stimulated the release of : by both enzymes. only the long chain ceramides (> carbons) were effective, while ceramide phosphate did not stimulate spla activity. sm-deficient cells released more : in response to spla -treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a (spla ) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a- , an inhibitor of spla enzymes, on atherosclerosis and cholesterol levels over weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study ( % fat; . % cholesterol, . % casein) and were treated with vehicle or a- bid at mg/kg or mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a- significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for weeks, oral dosing of a- ( mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a- is a potential novel therapeutic agent for the treatment of atherosclerosis. ( ), s doty( ), c antonescu ( ), c staniloae ( ) ( ) saint vincents hospital manhattan, new york, usa ( ) hospital for special surgery, new york, usa ( ) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene (tsg- ) is induced by tnf-a during inflammation and its secreted product tsg- glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox- and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg- is expressed in atherosclerotic plaques and investigated tsg- protein expression and cellular distribution on superficial femoral artery endarterectomy specimens from diabetic and non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg- antibody. tsg- expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg- expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg- stained the foamy macrophages. tsg- expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg- gene (more that fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg- in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg- is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. ( ), r krohn ( ), h lue ( ), jl gregory( ), a zernecke ( ), rr koenen ( ), t kooistra ( ), p ghezzi( ), r kleemann ( ), r bucala( ), mj hickey ( ), c weber ( ) ( ) university hospital of the rwth aachen, germany ( ) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr , inducing rapid integrin activation. mif directly bound to cxcr with high affinity (kd of . nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr as well as cd , a recently identified membrane receptor moiety for mif. accordingly, cxcr and cd were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr , serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. ( ), r toes ( ), h van bockel( ), paul quax ( , ) ( ) tno bioscienses, leiden, the netherlands ( ) department of vascular surgery, leiden university medical center, the netherlands ( ) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c bl/ mice depleted for natural killer (nk)-cells by anti-nk . antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jµ knockout mice that lack nk . + natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c bl/ mice depleted for cd + tlymphocytes by anti-cd antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd + t-lymphocytes. this impairment was even more profound in anti-nk . treated mhc-class-ii-deficient mice that lack both nkand cd + t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c bl/ mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd -positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd + t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf: u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by % (p= . ), when compared to saline treated controls (n= ).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c convertases). crry-ig ( mg every other day) led to % decrease in vein graft thickening (p= . ) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. ( ), m-c koutsing tine( ), p borgeat( ), h ong ( ), sylvie marleau ( ) ( ) universite de montreal, quebec, canada ( ) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep , a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd , elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from weeks of age. at weeks old, mice were treated daily with a s.c. injection of ep ( mg/kg) for days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for minutes, followed by minutes reperfusion. our results show that ep significantly reduced leukocyte accumulation by % in the lungs, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice (n = - per group), as assessed by myeloperoxidase assay. this was associated with a % reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd -/-deficient mice, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice. we conclude that ep protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd -mediated pathway. glycogen synthase kinase beta (gsk- beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk- beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk- beta, tdzd- and sb , on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to mmhg for min) and subsequently resuscitated with shed blood for h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd- ( mg/kg, i.v.) or sb ( . mg/kg, i.v.) min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd- , but not sb , attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin . neither of the gsk- beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk- beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. ( ), y ito ( ), h yoshimura ( ), h inoue ( ), n kurouzu ( ), h hara ( ), y mastui ( ), h kitasato ( ), s narumiya( ), c yokoyama ( ), m majima ( ) ( ) kitasato university school of medicine, japan ( ) kyoto university school of medicine, japan ( ) tokyo medical and dental university, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out mice (ipko) and thromboxane (tx) a receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf- and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from patients with synovitis of "t months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from patients undergoing vitrectomy for vitreoretinal disorders. one dimensional h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il- , ccl ), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. ( ), am artoli( ), a sequeira( ), c saldanha ( ) ( ) instituto de medicina molecular,faculdade de medicina de lisboa, portugal ( ) cemat, instituto superior tØcnico, universidade de tØcnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn- , slfn- , and slfn- were maximally induced after hours. the maximal induction of slfn- and slfn- was observed after hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn- , slfn- , slfn- and slfn- were elevated in joints affected by cia. to investigate the role of slfn- , we have generated a transgenic mouse line, which over expresses slfn- specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal . further characterisation of the slfn- over expressing mouse line will be used to assess the function of slfn- in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin , lcn- , is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn- determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn- is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn- . ( ), y cao ( ), s adhikari ( ), m wallig ( ) ( ) national university of singapore, department of pharmacology, singapore ( ) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein ( mg/kg) for , and hours respectively. neutralizing monoclonal anti-il- antibody ( . mg/kg) was administered either with or without crambene ( mg/kg) hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp- , tnf-a and il- â and up-regulating anti-inflammatory mediators like il- . phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd .the anti-inflammatory mediator il- plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il- is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. ( , ) ( ) northern ontario school of medicine, thunder bay, ontario, canada ( ) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, , : l -l ). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn delivery system.using several fluorescence-based detection systems, we established that the natural beta integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a a receptor (a ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b formation while potentiating that of prostaglandin e through the up-regulation of the cyclooxygenase (cox)- pathway. moreover, our laboratory determined that a ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a ar, migrated neutrophils expressed less cox- than their wild type counterpart while displaying higher mrna levels of tnf-and mip- . mononuclear cells from a ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p and pi- k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a amino acid protein that contains a s domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr (tandem repeat unit b), rhrnr (tandem repeat unit a) and rhrnr (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. ( ), n lu( ), r jonsson( ), d gullberg ( ) ( ) department of biomedicine, university of bergen, norway ( ) the gade institute, university of bergen, norway a ß is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a ß expression. specifically, we have analyzed the influence of cell passage, growth factors and the -d microenvironment. using sv immortalized as well as primary fibroblasts, we show that a ß integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a ß is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a ß that need to be to taken into consideration when evaluating the role of a ß in wound healing and fibrotic disorders. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par in the process of inflammation in the gut.colonic inflammation in c bl wildtype and par -/-mice was induced by treatment with . % dss (in drinking water) or tnbs ( mg or mg in ul of % ethanol, single intracolonic injection) or pre-sensitizing mice with % oxazolone (in olive oil) applied to the skin of the abdomen, and days later, a single intracolonic injection of % oxazolone (dissolved in % ethanol).intravital microscopy was performed, days (tnbs/dss) or days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par -/-.our evidences indicate that deficiency in par attenuates inflammatory responses in the experimental models of colitis associated with either th (tnbs/dss) or th (oxazolone) cytokine profile.therefore, par deficiency in the gut exerts antiinflammatory properties that are independent of th or th cytokine profile.the present study further highlights par as a potential target for inflammatory bowel diseases. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par (par -/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par deficiency. aim of this study was to assess the effects of par deficiency (via par -/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp ( mm) or paf ( nm) or by intraperitoneal injection of tnf-a; ( . mg -given hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par -/-versus par +/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par -/-mice compared to wild types as well. no difference was observed between par -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par activation in mouse colon leukocyte trafficking highlights par as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. ( ), kk hansen( ), k chapman( ), n vergnolle ( ), ep diamandis ( ), md hollenberg ( ) ( ) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada ( ) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks and to activate pars , and in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk is able to activate both of pars and and to prevent thrombin from activating par . on the other hand, klk was a specific activator of par . kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars and and by inactivating par , kallikreins, like klks and , may play a role in regulating the inflammatory response and perception of pain. ( ), d park ( ), b short( ), n brouard( ), p simmons( ), s graves ( ), j hamilton ( ) ( ) melbourne university, melbourne, victoria, ( ) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p subunit and nrf into hepg or other cells, as well as sirna technique to knockdown endogenous p in cells, we found that nf-kb p subunit repressed the anti-inflammatory and anticarcinogenetic nrf -are pathway at transcriptional level. in p -overexpressed cells, the are-dependent expression of heme oxygenase- was strongly repressed. in the cells where nf-kb and nrf were simultaneously activated, p unidirectionally antagonized nrf transcriptional activity. the p -mediated are inhibition was independent of the transcriptional and dna-binding activities of p . co-transfection and rna interference experiments revealed two mechanisms which coordinate the p -mediated repression of are: ( ) p selectively deprives creb binding protein (cbp) from nrf , but not mafk, by competitive interaction with the ch -kix domain of cbp, resulting in inactivation of nrf transactivation domain and concomitant abrogation of the nrf -stimulated coactivator activity of cbp; ( ) p promotes recruitment of histone deacetylases (hdac ) to are by enhancing the interaction of hdac with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p subunit. since various inflammatory and tumor tissues constitutively overexpresses p in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd l, only lps-matured dc expressed par and par on their surface. stimulation of dc with thrombin, par -or par -activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk / and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain (mlc ). the erk / -and rock-i-mediated phosphorylation of mlc was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine- ( f) by either nh -reactive n-succinimidyl- -[ f]fluorobenzoate or sh-reactive n-[ -( -[ f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, - %; specific radioactivity, - gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of f-nldl and f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of f-nldl and f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-Þb (nf-Þb), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p mapk and the down stream kinases msk and in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il- b before kinase activation was determined by western blotting. a significant inhibition of both msk and activations was seen after pre-incubation with dmf and stimulation with il- b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p (ser ), which is known to be transactivated by msk . furthermore, incubation with dmf before stimulation with il- b resulted in a significant decrease in nf-kb binding to the il- kb and the il- kb binding sites as well as a subsequent decrease in il- and il- mrna expression. our results suggest that dmf specifically inhibits msk and activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki- and ck- . epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol ; , - ) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer ( ), e hasselager( ), j clausen( ), l iversen ( ), k kragballe ( ) ( ) aarhus university hospital, denmark ( ) novo nordisk a/s, denmark interleukin- (il- ) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il- are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il- is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il- expression in non-lesional psoriatic skin ex vivo. mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il- beta ( ng/ml) for , , , , , and hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il- beta rapidly induced il- mrna expression in the biopsies. the highest level of il- mrna was detected after hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il- mrna. increased levels of il- protein were detected in the supernatant of the il- beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il- protein in the keratinocytes, whereas the il- protein was present in epidermal cd a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il- expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il- protein in epidermal dendritic cells whereas no il- was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il- . one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp- was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp- activity and mrna levels induced by tnf was inhibited by~ % following h treatment with calcitriol. using specific inhibitors we established that the induction of mmp- was dependent upon the erk pathway, while p -mapk and pkc inhibited, and jun-kinase, pi- -kinase and src did not affect it. levels of c-fos, a component of ap- transcription complex known to mediate mmp- induction, were elevated by tnf and further increased by calcitriol. the induction of mmp- by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp- by egf. we conclude that calcitriol inhibits the induction of mmp- gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. ( ), p verzaal ( ), t lagerweij ( ), c persoon-deen ( ), l havekes ( ), a oranje ( ) ( ) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands ( ) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd + t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th -mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. ( ), y sawanobori ( ), u bang-olsen ( ), c vestergaard( ), c grønhøj-larsen ( ) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il- mrna has been shown. also, transgenic mice over-expressing il- exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il- antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il- ratanti-mouse mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il- as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il- antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il- antibody treatment in the time interval day - . our results suggest an anti pruritic role for il- antibody in an atopic dermatitis-like animal model. anti il- antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. ( ), p ferro ( ), hm asnagli ( ), v ardissone ( ), t ruckle ( ), f altruda ( ), ch ladel ( ) ( ) rbm merck serono/university of torino, italy ( ) merck serono pharmaceutical research institute, geneva, switzerland ( ) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide -kinases (pi ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi k-g inhibitor as on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose , mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi k-g are demonstrable after oral administration of selective inhibitors like as in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box (hmgb ) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb absorber. the purpose of this study was to test the hypothesis that hmgb removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb absorber group (group i),hmgb nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at hours after lps injection. the rats were sacrificed hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb with anova. results: survival rate was % in the group i at hours after lps injection, as compared with % in the group ii and % in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb therapeutically reverses lethality of established sepsis indicating that hmgb inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp ( ) ( ) department of pharmacology, national university of singapore, singapore ( ) dso national laboratories, singapore hydrogen sulfide (h s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, mg/kg i.p.), an inhibitor of h s formation or sodium hydrogen sulfide (nahs) ( mg/kg, i.p.), a h s donor. pag was administered either hour before or hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam- , p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs ( mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-x×b inhibitor, bay - . h s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay - . moreover, expression of cxcr in neutrophils obtained from h s treated mice was significantly upregulated leading to an obvious elevation in mip- directed migration of neutrophils. therefore, h s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid (trpv ) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv antagonists are not available for use in the mouse in vivo, thus established trpv knockout (-/-) mice were used. c bl wt and trpv -/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv -/-mice, with trpv -/-mice showing an enhanced drop at h. in a separate group temperature, a proposed pre-mortality marker was also reduced by h, again with a significantly increased drop in trpv ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv -/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv -/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv plays a protective role in sepsis. trpv is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. ( ), da souza-junior( ), l de paula ( ), mc jamur ( ), c oliver ( ), sg ramos ( ), cl silva ( ), lucia helena faccioli ( ) ( h rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il- , kc, mcp- and mip- were detected in the lungs by day . in vivo degranulation of mast cells by c / led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c / . histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c / . of interest, the number of mycobacterial bacilli recovered from the lungs was log higher after treatment of infected mice with c / . these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. ( ), ms chadfield ( ), db sørensen ( ), h offenberg ( ), m bisgaard ( ), he jensen ( ) ( ) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark ( ) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: female balb/ c-j mice (app. g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp ) and porcine (p ) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp- and metalloproteinase inhibitor timp- . results: all mice infected with the avian strain were euthanized after hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after , and h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp- and timp- between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp- and timp- . ( ), r molinaro ( ), a franÅa ( ), m bozza ( ), f cunha( ), s kunkel ( ) ( ) universidade do rio de janeiro, brazil ( ) universidade de s¼o paulo, brazil ( ) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr knockout mice (ccr -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr -/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c /bl mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since h after and until days. ccr -/-mice subjected to clp presented an increase in survival rate ( %) compared with wildtype mice ( %), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr -/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr -/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr -/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -µ and il- â) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa ( ), commiphora mukul ( ) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il- , cxcl and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl , which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr , the receptor for cxcl . while plasmablasts migrate in response to cxcl , plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr , the receptor for the interferon-gamma-induced chemokines cxcl , and , which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, ( ), h lee ( ) c a is a potent inflammatory mediator produced during complement activation. unregulated c a signalling through its receptor (c ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c ar antagonists for human therapy. we took neutrophils from genetically modified human c ar knock-in mice in which the mouse c ar coding region was replaced with human c ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c ar. these mabs inhibit c a-induced neutrophil migration and calcium-flux, and bind to a region of the nd extracellular loop of c ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c ar knock-in mice were given - mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single mg/kg dose of anti-c ar mab given days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g arrest in macrophage cell line raw . , their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba- mice by immunisation with bovine type ii collagen/adjuvant on day and . cor , synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day . etanercept ( mg/kg ip q d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry- is a potent (ic = nm), selective, atp-uncompetitive mek / inhibitor.arry- is highly efficacious in cia and aia rat models, with ed s of and mg/kg, respectively, equal to or better than standard agents. addition of arry- to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il- and il- production (ic s of , and nm, respectively). in contrast, inhibition of perk required nm to achieve a % reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of , , , or mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry- was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il and tnffz with > % inhibition observed at plasma concentrations of and ng/ml, respectively. similar inhibition of perk required ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry- and helped define tolerability. clinical evaluation of arry- in combination with methotrexate in patients with rheumatoid arthritis is on-going. p (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il- a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p activity is expected to regulate the levels of tnf-a and il- b thereby alleviating the effects of inflammation in ra. a new class of p inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. ( ), h aaes ( ), w-h boehncke( ), j pfeffer( ), t skak-nielsen( ), i teige ( ), k abell ( ), ph kvist ( ), e ottosen ( ), tk petersen ( ), lars svensson ( ) ( ) discovery, leo pharma, industriparken, ballerup, denmark ( ) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il- b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately % of psoriasis patients develop psoriatic arthritis. leo is a member of newly developed class of selective p map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice ( % reduction in mice and % in rats at mg/kg p.o.). in the humanised psoriasis model, leo at a dose of mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by %) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx- is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock- . its ic for rock- and rock- inhibition is nm and > mm respectively. the ability of slx- to inhibit septic liver injury was investigated in c bl/ j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps ( mg/mouse) and d-gal ( mg/ mouse) i.p and . hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a > fold increase in serum alt and ast levels. this increase was reduced by > % in mice pretreated with slx- either orally ( mg/kg, and hrs prechallenge) or i.p. ( - mg/kg, min pre-challenge). slx- inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by > %. to assess the kinetics of slx- s benefit, slx- ( mg/kg i.p.) was given min prior to, or or min after the lps/ d-gal. slx- was effective at inhibiting the rise in alt and ast levels at all time points suggesting that inhibiting rock- even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, out of mice given lps/d-gal were dead by hrs whereas in mice given slx- ( mg/kg orally) animal died at hours and the remaining mice were alive hrs later. these results show that specific inhibitors of rock- may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il- secretion in human purified t-cells stimulated with anti-cd and anti-cd or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. ( ), j zhang ( ), k henley ( ), m white ( ), d hilton ( ), b kile ( ) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c bl/ control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments . f(ab) or iv. f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il- beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt ( ), u hüffmeier ( ), i kçnig ( ), j lacorz ( ), a reis ( ), k reich ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*- a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*- t was detected (or= . , % ci . - . ; pcorr= . ) also in patients negative for the psors risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*- a and psoriasis seems to primarily reflect ld with psors , tnf*- t may represent a risk factor for psa independent of psors . ( ), n modi ( ), m stanford ( ), e kondeatis ( ), r vaughan ( ), f fortune ( ), w madanat ( ), c kanawati( ), p murray ( ) methods: dna was obtained from patients with bd, from the uk and from the middle east (me), and controls individuals, from the uk and from me. dna was prepared by proteinase k digestion, and salt extraction and - and snp were detected by a pcr-ssp. results: there was no significant difference in expression of - c/t or a/g when all bd patients were compared to all control individuals, (p= . and . , respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p= . , p= . ) or me bd patients (p= . , p= . ) when compared to the appropriate controls. ( ), da brown ( ), h johnen ( ), mr qiu ( ), t kuffner ( ), pgm curmi ( ), l brown ( ), m mazzanti ( ), sn breit ( ) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il- gene is single copy gene located on chromosome q . - in humans. interleukin- is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin- concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il - snp in the children with the cp. methods: dnas obtained from peripheral blood of cp patients and unrelated healthy volunteers were genotyped for the il - snp pcr-rflp method. results and conclusions: il - genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il fngene polymorphisms and cerebral palsy has to be investigated in the future studies. ( ), d delbro ( ), e hansson ( ) ( ) kalmar university, sweden ( ) gçteborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: . the expression of alpha nachrs and alpha nachrs by immunofluorescence and western blot. . intracellular ca +-transients spread within the astroglial networks after stimulation with nicotine. . pro-inflammatory cytokines, il- b, il- , and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca + transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca + responses were blocked by the alpha nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database ). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at mg/kg dose, and above. in the th test weak % effect was seen at side effect free dose ( mg/kg). in the arthritis model b.i.d. mg/kg dose of tramadol given on days - after cfa caused a maximum % effect on weight bearing incapacitance (day ). however, its effect seemed to diminish ( % on day ) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at but not at mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge and pgd in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk- .biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge and pgd levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk- was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo( ), lm yshii ( ) ( ), sk costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college, london, uk ( ) butantan institute, s¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr , a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin ( %, h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il- b and il- , but not tnfa) and anti-inflammatory (il- ) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr ( nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. ( ) ( ) university of s¼o paulo, s¼o paulo, brazil ( ) butantan institute, s¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or , -naphthoquinone ( , -nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and , -nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep ( mg/kg) and , -nq ( nmol/kg). after min, dep and , -nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following h treatment with pollutants. this treatment contributes to augment the ach ( - - - m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: , -nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and , -nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv ( , , mm) dose-dependently inhibited ng/ml lps induced tnfa, il- b, il- , mcp- and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk / and jnk but not p . nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk / , jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. ( ), cs patil( ), sv padi ( ), vp singh ( ) ( ) university institute of pharmaceutical sciences, panjab university, chandigarh, india ( ) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia days after the nerve ligation. sildenafil ( and fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) and glyt are expressed in glia and neurons, respectively. glyt make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt (sarcosine and org ) and glyt (alx and org ) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt and glyt inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp ff mouse to assess the effect of the gp ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il- family cytokine receptor gp preventing shp /ras/erk signaling and leading to constitutive stat transcriptional activation. tumour development is inflammation dependent, there is a requirement for il- , and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s inflamm. res., supplement ( ) posters dispensable for tumorigenesis. in gastric antrum the gp ff mutation results, decreased erk / activation and constitutive phosphorylation of stat , abnormal signaling is replicated in macrophages. antral stat activation is unaffected by depletion of il- . however in macrophages an absence of il- results in higher stat activation demonstrating the anti-stimulatory role of il- on macrophages. the gp ff mutation results in macrophages with decreased il- and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp ff mice display aberrant gp signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. ( ), y riffo-vasquez( ), s brain( ), s costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and , -naphthoquinone ( , -nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv knockout (ko) mice exposed to dep and , -nq. dep ( mg/kg) and , -nq ( nmol/kg)-induced airway inflammation was assessed via i-labelled albumin h post pollutants injection. mpo assay and histopathology were performed h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and , -nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv ko, suggesting these pollutants act via a trpv -mediated mechanism. in contrast, mpo activity in the airways of trpv ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv ko, indicating that the lack of trpv receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv receptors. acknowledgements: capes, cnpq, fapesp. ( ), n sato( , ), y endo ( ), s sugawara ( ) ( ) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan ( ) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j . . female balb/c mice ( weeks old) received a basal diet or a biotin-deficient diet for weeks.ten days after sensitization with intraperitoneal injection of lps and nicl , the mice were challenged with intradermal injection of nicl into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j . cells were cultured in biotin-sufficient or deficient medium for weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il- beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j . cells produced il- beta significantly higher than biotinsufficient j . cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il- beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il- , icam- and e-selectin were studied in patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in patients and severe -in patients. the highest levels of il- were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il- and adhesion molecules and severity of pancreatitis. during first week the levels of il- gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam- levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il- and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il- and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for â-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd and dnam- on mc, and b and nectin- on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells ( - weeks with scf, interleukin- and prostaglandin e , cbmc). cbmc were activated, after their culture for days with myeloma ige ( mg/ml), by rabbit anti-human ige antibodies ( mg/ml), for , and h at c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl- , bcl- , bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl- that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl- and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl- family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. ( ), c armishaw( ), z yang ( ), h cai ( ), p alewood( ), c geczy ( ) ( ) university of new south wales, faculty of medicne, sydney, australia ( ) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s a , s a and s a are closely related proteins associated with inflammation. s a is expressed in the human genome, but not in the mouse. s a is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s a and human (h) s a share % structural identity, but the hinge domains are more divergent. the ms a hinge (ms a - ) is a potent chemoattractant for leukocytes whereas this sequence in hs a (hs a - ) is inactive. methods: s a hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp cells or murine mc in vitro and mouse footpad injection. results: s a hinge domain (s a - ) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s a , the hinge domain only weakly induced cytokine production (il , il ) by macrophages. residues essential for oedema were hydrophobic in nature (leu , isoleu , isoleu and isoleu ). n and i were essential for responses provoked by - m s a - whereas mutation of k , l , n , i , d , k and i significantly reduced migration with - m s a - . conclusions: s a and ms a may be functional chemattractant equivalents; s a may have arisen by duplication of human s a . isoleucine residues in s a hinge domain are essential for its proinflammatory properties. ( ), g kiriakopoulou ( ), e tsimara( ), a voultsou( ), k zarkadis ( ) ( ) general hospital of zakynthos, greece ( ) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes . eo . %, hb . g/dl, ht . %, mcv , fl, mch . pg, thrombocytes . , blood sedimentation mm, fe mg/ml, ferritin . ng/ml. normal biochemical examinations. u/s: livers size lightly increased . mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules ( - mm) with a thin wall and quills on the side, as well as three imago worms (> mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites . , eo . %, hb , g/dl, ht . %. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement ( ) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average . mg/ l; p < . ) and with attacks (average . mg/l; p < . ) were significantly higher than those of normal controls (average . mg/l). serum hs-crp levels were inversely correlated with fev . % in asthmatic patients (r = - . , p < . ). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was % hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. ( ), sk bk( ), v sharma( ), rp bhandari ( ) ( ) nepal medical college teaching hospital, nepal ( ) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s inflamm. res., supplement ( ) posters from january to december . demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was , , giving an incidence of . per deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over % were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were . and . per deliveries (p <. );the corresponding cases of deepvenous-thrombosis diagnosed were . and . per deliveries, respectively (p <. ). the majority ( %) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of < weeks, a bmi of , or more and maternal-age of or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. ( ), ch ladel ( ), t ruckle( ), c rommel( ), r cirillo ( ) ( ) rbm-merck serono, colleretto giacosa, italy ( ) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi k signalling inhibition resulted in clinical amelioration of disease symptoms. as , a novel isoform non-selective, yet specific class-i-pi k inhibitor, administered at mg/kg twice-a-day for days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as was able to decrease its levels down to values comparable with naïve animals. controls and as treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi kg) and immunosuppressive (via pi kd/a/b) activity. ( ) ( ) institute of biomedical science, university of sao paulo, brazil ( ) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol ( ìg/kg) or progesterone ( ìg/kg) h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement ( ) s the release of il- and reduced that of il- and tnf. the release of il- by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il- and increased il- production in bal and increased that of il- and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam- and mac- expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after (ovx- ) or days (ovx- ) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, %; aerosol) and sacrificed h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam- , mac- and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day , e-selectin expression increased(ovx- = , ae , ) and at day , decreased (ovx- = , ae , ) as compared to respective controls (sh- = , ae , ; sh- = , ae , ). estrogen treatment reverted this profile in both groups (ovx- +e= , ae , ; ovx- +e= , ae , ). mean fluorescence intensity of bal cells showed increase of mac- expression (ovx- = ae , vs sh- = , ae , ), icam- (ovx- = , ae , vs sh- = , ae , ) and l-selectin (ovx- = , ae , vs sh- = , ae , ) at day i.e., ovx- group. on the other hand, we observed a decrease in icam- (ovx- = , ae , vs sh- = ae , ) and mac- expression (ovx- = , ae , vs sh- = , ae , ) was seen in ovx- group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx- ) and decreased (ovx- ) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell ( ), s lim ( ), y lin ( ), bp leung ( ), c thiemermann ( ), wsf wong ( ) ( ) national university of singapore ( ) the william harvey research institute, london, uk glycogen synthase kinase b (gsk- b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk- b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd- , a non-atp competitive gsk- b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd- significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il- , il- and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd- also suppressed the mrna levels of icam- , vcam- and chitinase proteins in the lung. histological studies revealed that tdzd- substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd- also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd- . our findings suggest that inhibition of gsk- b may have therapeutic potential for the treatment of allergic airway inflammation. ( ), a yildirim ( ), f ercan ( ), n gedik ( ), m yuksel( ), inci alican ( ) ( materials and methods: sprague-dawley rats ( - g) were exposed to oc (burn group) or oc water bath (control group) for s under ether anesthesia. adm ( ng/kg; s.c) was administered min before burn and all rats were decapitated h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p< . - . ) and mpo activity showed a marked increase in all tested tissues (p< . ) of the burn group. adm reversed these parameters effectively (p< . - . ). luminol chemiluminescence levels showed increases in both ileum and lung (p< . - . ) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p< . ) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p< . ). adm reduced plasma tnf-alpha level (p< . ) which showed a significant increase in burned animals compared to controls (p< . ). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday ( ), sd joshi ( ), kr reddy ( ) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg +, and an inhibitor, hql- , with anti-inflammatory activities in vivo, at . resolution. hql- was found to reside within the catalytic cleft between trp and gsh in the quaternary complex. hql- inhibited h-pgds competitively against the substrate pgh as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql- bound to h-pgds with an affinity that was -fold higher in the presence of gsh and mg + than in their absence. hql- inhibited selectively pgd production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql- inhibited antigen-induced production of pgd , and airway inflammation in mice without affecting the production of pge and pgf f¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats ( - g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for h ( breath/min). one lung was subjected to hyper-ventilation ( ml/kg/lung) and the other was ventilated with a normal volume ( ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip- was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip- expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip- between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. ( ), j-y gillon( ), v lagente ( ), e boichot ( ) ( ) university of rennes , rennes, france ( ) serono international s.a., geneva, switzerland macrophage elastase (mmp- ) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp- in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp- (rhmmp ) on il- /cxcl release from alveolar epithelial cell line a and we explored the underlying mechanisms. a cells were stimulated with rhmmp- ( . - - . - u/ml) during hours and il- /cxcl level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p kit. we observed that mmp- elicited il- /cxcl release in a dose-dependent manner. this production could be prevented by a pretreatment for hour with a selective mmp- inhibitor (as - mm) or with the nonselective inhibitor batimastat ( - mm) . the il- /cxcl production was also inhibited by actinomycin d ( mg/ml), erk / inhibitors (u mm and pd mm) and nfkb inhibitors including bay - ( mm) and nfkb activation inhibitor ( nm), whereas p kinase inhibitor (sb ) had no effect. stimulation with mmp- was rapidly followed by a phosphorylation of erk / ( min) and an nfkb nuclear translocation and activation ( h). the nfkb activation was not inhibited by a treatment with u . these data suggest that alveolar epithelium is a target of mmp- since it upregulates gene expres-s inflamm. res., supplement ( ) posters sion and release of il- /cxcl , via erk / and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)- , in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h cells were exposed to serial dilutions of test agents in serum-free medium for hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il- , matrix-metalloprotease- (mmp- ), the gel-forming mucin muc ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p s cyp a and cyp b were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il- alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th ) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase- (cox- ) and cox- dependent phases of prostaglandin d (pgd ) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic values of . mm and . mm, respectively. this compound inhibited cox- and -dependent conversion of the exogenous arachidonic acid to pgd in a dose-dependent manner with an ic values of . mm and . mm, respectively. however, dpt did not inhibit cox- protein expression up to a concentration of mm in the bmmc, indicating that dpt directly inhibits cox- activity. furthermore, this compound consistently inhibited the production of leukotriene c (ltc ) in a dose dependent manner, with an ic value of . mm. these posters inflamm. res., supplement ( ) s results clearly demonstrate that dpt has a dual cox- / -lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf- , could block the inflammation induced by an acute exposure to cigarette smoke in strains of mice, balb/c and c /bl .animals were administered the compound ( - mg/kg) either per os (p.o.) or intranasally (i.n.) hour before and hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf- dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~ % at mg/kg; p < . )or i.n. (~ % at mg/kg; p < . ).however, the compound had no clear effect on bal neutrophil infiltration in c /bl mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < . ). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il- beta, kc, and mip- , pkf- had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table ) cytokine analysis showed significant increases in tnfalpha, il- , kc, gm-csf, mcp- , il- beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in ( ) tnfa + gm-csf ( ) gm-csf ( ) il- + gm-csf ( ) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst- assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd a low cd b + cd c + cd + cd + cd low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd + t cell proliferation, il- production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after weeks. the hmscs were cultured and in passage ,the cell surface antigens of both groups were analyzed by flow cytometory.in passage , in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after weeks, alp staining and activity were measured in each group.after weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for weeks and weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal høgh ( ), hm lindegaard ( ), gl sorensen ( ), a høj ( ), c bendixen ( ), p junker ( ), u holmskov ( ) ( cytosolic phospholipase a (cpla ) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla -deficient mice are resistant to cia. thus we aimed to study whether cpla is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla antibodies revealed low positive cpla protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla were detected. in addition, robust cpla protein expression in the skeletal muscles surrounding the joints and strong cpla positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla protein, due to an inflammatory infiltrate and increased cpla expression, suggests an important role of cpla in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b .q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome a disease protective locus (cia ) and another on chromosome a disease-promoting locus (cia ). nod.q allele at cia promotes arthritis whereas cia , has a protective effect in contrast to the b .q allele on chromosome . a promising candidate gene in cia locus is complement factor (c ) as the nod.q allele produced defective c protein. cia locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf , fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf mutated and wildtype mice. we could show that ncf mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf wildtype dc. in addition, ncf mutated dc induced higher levels of il- production by hybridoma t cells. to analyze the role of ncf in dc on arthritis, mice were developed expressing functional ncf restricted to dc (b .qdcn). these mice are characterized for burst, ncf expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf deficient mice. this suggests that ncf plays a role in the uptake and processing of posters inflamm. res., supplement ( ) s antigens, probably by dc. this will be further investigated with the b .qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days and in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day and % incidence was observed on day . mif -/-mice exhibited delayed arthritis, with onset on day and % incidence on day . mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo ( ), a denadai-souza ( ), lm yshii ( ), a schenka ( ), ma barreto ( ), d boletini-santos ( ), c lima ( ), v rioli ( ), mn muscar ( ), e ferro ( ), sk costa ( ) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin ( or ìg/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day . results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day , there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag , we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo ( ), r saldanha-gama ( ), ja moraes ( ), r zingali ( ), c marcienkewicz ( ) ( ) universidade do estado do rio de janeiro, rio de janeiro, brazil ( ) universidade federal do rio de janeiro, rio de janeiro, brazil ( ) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb , a b and/or avb ) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a /a b integrins, vlo , induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo interaction with a b integrin, activating the focal adhesion cascade. vlo effects on the delay of neutrophil is modulated by pi k, erk- mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl- family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a b integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) ( ), h serezani ( ), m peters-golden( ), sonia jancar ( ) '( ) university of s¼o paulo, brazil ( ) university of michigan, usa it is been shown that leukotriene (lt) b and cysteinyl lt (ltc , ltd and lte ) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin - um), pkc-a (ro- - - nm), pi k (ly - um and wortmannin- nm), erk / (pd - um), cpla (aacocf - um), p mapk (sb - um) and ca++ (bapta/am- um), before stimulation with ltb or ltd and addition of igg-opsonized red blood cells for min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb -enhanced phagocytosis is dependent on pkc-a, while ltd effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin- mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin- is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin- as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin- at gene and protein level in monocytic thp- cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin- on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin- and - expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin- and- expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc was incubated with pap ( ng/ml) and/or tnfa ( ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p . results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u macrophages. methods: ha was added to u macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule- (icam- ) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)- b, and il- were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il- b, and il- . in contrast to kda ha, kda ha at mg/ ml inhibited lps-induced cytokine production. anti-icam- antibody blocked the effects of ha on the lps actions on u cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam- antibody reversed the inhibitory effects of ha on phosphorylation of p nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam- through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox- is prostacyclin (pgi ), which is a potent vascular patency factor. induction of endothelial cox- under hypoxic conditions is well documented. this response, along with associated pgi release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox- expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox- promoter region in huvec. cox- induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox- expression than those for nfkb. furthermore, expression of cox- was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi but not vaso-occlusive thromboxane a was seen. thus the putative protective induction of cox- in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th -predominant immune responses remains to be investigated. therefore, the effects of a th selective inhibitor tak- were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak- was administered orally, starting at the time of induction of glomerulonephritis. in group , the drug was administered daily for the initial days. tak- was administered on day only in group , and from day to in group . in each group, nephritic rats were killed on days and . results: in group , glomerular damage, including crescent formation, was improved on day , with reductions in the numbers of cd , cd and ed- positive cells, as well as in urinary protein excretion. protein and transcript levels of th cytokines in the diseased kidneys were markedly decreased by tak- treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group , in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group , partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s inflamm. res., supplement ( ) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd- mice were injected intraperitoneally with mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline ( mg/kg), lisofylline ( mg/kg) or prednisolone ( mg/kg). the tissues were harvested , , , , and h following lps administration and stored at - c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin- beta (il- beta), interleukin- (il- ), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at or h after lps administration, whereas the expression of tnf-alpha and il- beta in lungs and il- in liver reached the peak values at h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately %), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz( ), m losada ( ), c company ( ), c lope-gines( ), l piqueras( ), j cortijo ( ) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx cl (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii microm or with tnfalpha ( ng/ml) for , and h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after h and h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for and h. these effects were caused by the interaction of aii with its at receptor since they were abolished by losartan (at receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for and h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th -type cytokine profiles, i.e. m. globosa induced il- , il- , and il- secretion from the keratinocytes, whereas m. restricta induced il- secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il- and il- , respectively. the m. globosainduced increase in il- secretion was inhibited by mabs against cd and cd . in case of m. restricta, mabs against toll-like receptor (tlr ) and cd suppressed significantly il- secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl ) and tarc (thymus and activation-regulated chemokine/ccl ) are th type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e sub-fraction showed inhibitory activity on the stat protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il- family now consists of members, most of which have assigned functions.there are members of the il- receptor family (including the decoy receptor type ii il- r).many of the il- family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il- family members il- f , f and f signal through a complex of the il- r family member rp in association with il- r acp to activate common inflammatory pathways.the specific activity is is low, on the order of - ug/ml ec .we have found that removal of a few n-terminal amino acids from il- f , f , f and f can increase their bioactivity approximately -fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il- f is capable of antagonizing signaling via il- rrp , but full-length f is inactive. ( ) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr (hvem/tnfrsf ), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf ), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr pathway. c bl/ tr knockout mice and their wild-type control were fed a high-fat diet for weeks and the obesity phenotypes were determined in the obese tr knockout mice and the control. the obese tr mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, mg/kg, i.v.) and controls were sensitized with ova ( ìg) and al(oh) ( mg, s.c.) days after alloxan or saline injection. the animals were challenged days later by the intratracheal instillation of ova ( mg/ . ml). the following analyses were performed h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il- beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils ( %) and mononuclear cells ( %); reduced levels of tnf-alpha ( %) and il- beta ( %), and reduced p-selectin expression ( %) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin ( iu, s.c.), h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il- beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il- and the adhesion molecule icam- by real-time pcr. icam and il- were induced rapidly peaking after h, their mrna levels increased again from h to reach a plateau between h to h after exposure to tnf, whereas tnf mrna levels increased steadily between h and h. h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il- but did not affect that of icam- or tnf h following exposure while calcitriol markedly inhibited the induction of all pro-inflammatory genes h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p by tnf. this action was mimicked by the posters inflamm. res., supplement ( ) s jnk inhibitor sp and the p inhibitor sb .the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p cascades. ( ), cm lotufo ( ), p borelli ( ), zs ferreira ( ), rp markus ( ), shp farsky ( ) ( ) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of s¼o paulo, brazil ( ) department of physiology, bioscience institute,university of s¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru -treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru -treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam- , vcam- and pecam- . participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru -treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. ( ), y naito( ), t okuda ( ), k mizushima ( ), t okayama ( ), i hirata ( ), h tsuboi ( ), t suzuki ( ), o handa ( ) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co( ppm- ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc- mrna/protein and p-p mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc- mrna/protein and p-p mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero ( ), m maróstica ( ), m saad( ), j pedrazzoli jr ( ) ( ) s¼o francisco university medical school, brazil ( ) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass ( . +- . and . +- . % of body weight for colitis and control, respectively; p< . ) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter ( . +- . and . +- . um for colitis and control, respectively; p< . ), higher basal lipolysis ( . +- . and . +- . ug.mg- for colitis and control, respectively; p< . ) and tnf-alpha production ( . +- . and . +- . ng.mg- for colitis and control, respectively; p< . ). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis ( . +- . and . . +- . ug.mg- for colitis and control, respectively; p< . ), increased tnfalpha ( . +- . and . +- . ng.ml- for colitis and control, respectively; p< . ), leptin ( . +- . and . +- . pg.ml- for colitis and control, respectively; p< . ) and adiponectin production ( +- and +- ng.ml- for colitis and control, respectively; p< . ). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il- â, il- , il- , kc, cox- , igg heavy chain, and phospho-stat in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p mapk, the activation of the p mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p mapk and of mk and hsp , two down-stream targets, were not increased in dss treated animals compared to controls. leo , a potent inhibitor of p activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr + cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/- ng/ml lps. for mlr, balb/c tcells were added for days. cells were incubated with sb ( -( -fluorophenyl)- -( -ethylsulfinylphenyl)- -( -pyridyl) h-imidazole, sb) or ml ((rs)-{ -[ -( -fluorophenyl)- -methylsulfanyl- h-imidazol- -yl]pyridine- -yl}-( -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il- production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic spdc . mm, bmdc . mm) and ml (ic spdc . mm, bmdc . mm). preincubation with dcs had no effect, despite reduced lps stimulated il- and tnfa synthesis by sb (ic il- . mm, tnf . mm) and ml (ic il- . mm, tnf . mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p mapk in the mlr is under investigation. p inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. ( ), s luik( ), s laufer( ), m seed ( ), v holan( ), s fiorucci ( ) ( ) synovo gmbh, tübingen, germany ( ) university of tübingen, germany in vivo anti-inflammatory activity of certain p kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically ( d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at mmolkg- d- . we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il- , il- ) and adhesion molecules (e-selectin and icam- ) were measured in patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the , , and day. all patients were divided on two groups: first group compiled patients, in which dexamethasone ( mg/day during - days) was applied in the complex management of acute pancreatitis, and control group - patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il- , il- , il- , icam- , and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th transcription factor gata- can inhibit th /th cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day . at day , t cell specific gata- tg mice did not show any difference in arthritis score compared to wild type mice. however, at day , wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata- tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata- protects against development of severe joint inflammation. facs analysis revealed low levels of il- +/ifn-gammacells in wild type as well as in gata- tg mice at day . as expected, il- positive cells were higher in gata- tg mice compared with wild type mice. interestingly, at day , the percentage of il- +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata- tg mice, suggesting prevention of th expansion under gata- overexpression in vivo. these data revealed that t cell specific overexpression of the th transcription factor gata- protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il- +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata- overexpression in t cells prevents expansion of the il- +/ifn-gamma-t cell subset. pingping jiang ( ), pt sangild( ), t thymann ( ), hh-y ngai ( ), w-h sit ( ), k-l chan ( ), jm-f wan ( ) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis ( d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to days of parenteral feeding followed by day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = ) were compared with corresponding pigs remained healthy (n = ). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase and chain a, medium-chain acyl-coa dehydrogenase with -thiaoctanoyl-coa etc.), inflammation (peptide-binding protein (pbp ) and snail homolog ), signal transduction proteins (thyroid hormone binding protein precursor, park protein and chain b, structure of the rho family gtp-binding protein cdc in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli :b ) by inhalation. the lungs were lavaged hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of mg ( ml/animal) of lps (p.aeruginosa) to c bl/ j mice. control animals received a single dose of sterile ml . % saline/animal. the mice were terminated , , , and h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by %). treatment with daivonex / dovobet also showed an anti psoriatic effect with a % reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res ( ): ) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or % protein diet (c-group) during the first days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o -, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin (k ) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k /vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with -o-tetradecanoylphorbol- -acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone- -valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd -positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone- -valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type diabetes (t d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t d. we aim to detect how gimap affects the lifespan of t cells. results: overexpression in c cells of both wt gimap and gimap with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap causes apoptosis in jurkat cells and primary naïve t cells but not in activated t cells. finally, gimap -mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap causes rapid death of the cells. this suggests that gimap is pro-apoptotic. the results with human wt gimap support this conclusion: recently, much focus has been on the cellular cd / cadpr signaling system during inflammatory processes. the cd /cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il- . to our knowledge, the expression and function of the cd /cadpr signaling system in the human detrusor muscle have not been described. cd protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca + concentration ([ca +] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd which could be upregulated by il- ( ng/ml). in hdsmc briefly activated with il- ( ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca +]i. cyclic adpr-mediated ca + increase was greatly reduced in ca + free medium suggesting ca + entry as well as ca + release. cyclic adpr -elicited ca + increase was mimicked by -deaza-cadpr, and blocked by -bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il- , cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that ) hdsmc express cell surface cd , ) the expression and function of cd are augmented by il- , ) externally added cadpr elicited a rapid, il- -dependent, and -bromo-cadpr-inhibitable ca + mobilization, ) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd /cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for days before induction of arthritis and days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin ( mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration ( mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t ), and compared with the original formula (t ) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t cream (n= ) or no cream (n= ) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment ( . ml ; stirred continuously at c). min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ,) or not in the presence of % t , % t , or no treatment. hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < . ). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t with zinc only was more effective than t (treat:f , = . , p < . ; lps:f , = . , p < . ; treat by lps:f , = . , p < . ). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. ( ) ( ) osprey pharmaceuticals limited, canada ( ) probetex, inc., texas, usa the ccl /ccr chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl chemokine fused to a truncated form of the enzymatically active a domain of shiga toxin has been developed. the ccl portion binds specifically to ccr -bearing leukocytes and enters the cells, where the sa portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day and treated intravenously with vehicle, or mg/kg of the recombinant protein q d from day until day . urine and blood collections were made prior to ats injection and on days and . animals were sacrificed on day . no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of , , , and % for glomerular lesions, m/m count, fibronectin and µ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement ( ) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning ( h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd b expression on resting monocytes harvested from h blister was lower in patients as compared to controls (p= . ). lower expression of cd b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p= . and p= . , respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip- µ was higher among patients (p= . ) and similar levels were seen in blister fluids. concentration of mcp- was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru inhibited no production, and inos protein and mrna expression in murine j macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac selective mc . hdac inhibitors reversed the effects of dexamethasone and ru on inos expression or no production. stably transfected a / cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv -dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd mice ( - g) were given intraplantar injections (i.pl.) of tnfa ( pmol/ microl) and tyrode (as vehicle, contralateral paw; microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and h after injection. indomethacin ( mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle ( % nahco ). another group of mice were injected with tnfa i.pl. plus % nahco with the contralateral paw injected with tyrode plus indomethacin ( mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa ( pmol) leads to significantly reduced (p< . compared to baseline values) paw withdrawal latency in both paws h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin ( mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox- inhibitor, nimesulide. in conclusion, cox- derived prostaglandins are important in the development of hyperalgesia. local cox- inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats ( - g) were administered dexamathasone ( mg/kg, i.p.) either h before or h after lps ( mg/kg, i.p.) injection. animals were killed h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il- b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h s concentration and liver and kidney h s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h s and tissue h s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps ( ng/ml, h, oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone ( nm) significantly (p< . ) reduced expression of both cse and inos. these data provide further evidence that h s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h s production. ( ), u jalonen ( ), h kankaanranta ( ), r tuominen( ), e moilanen ( ) ( ) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland ( ) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup , tis , g s and zfp , is a factor that binds to utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s inflamm. res., supplement ( ) posters concentration of pma for h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro (inhibits pkcb, & and e), gÖ (inhibits pkca, b and &) and cgp (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp did not affect ttp mrna half-life and therefore we measured the effects of cgp on the activation of transcription factors involved in ttp expression. cgp had no effect on the activation of nf-kb, egr or sp . in contrast, cgp reduced the activation of transcription factor ap- , which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap- . the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il- , il- and no) in raw . cells. among them, -oxooctadec- -enoic acid and -oxooctadec- -enoic acid inhibited the production of tnf-a, il- , il- and no at the concentration of mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il- , il- and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen( ), p hjarnaa ( ), j fensholdt ( ), p-p elena( ), k abell ( ), tk petersen ( ) ( ) discovery, leo pharma, ballerup, denmark ( ) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr- . additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd induced il- response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to mg/kg i.p. or mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi kg gene depletion and pi kd kinase-inactive mutation, pik cgd koi, exhibited thymus atrophy, similar to previously reported pi kg and d double knockout (p g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg / igg a ratio and ige level were elevated in pik cgd koi mice corresponding to a skewed th profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi k d or together with pi k g selective inhibitors, while th cytokines were suppressed th cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th reaction but attenuate th medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, h before i.d. injection of . ml pbs containing des-arg -bradykinin (dapk, nmol right hind paw) and . ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) - minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk / . dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp and erk / pd inhibitors. western blot analysis shows that phosphorylation of jnk and erk / is important in the up-regulation of b receptors. our results clearly show that the phosphorylation of both erk / and jnk mapkinases is an important step for the in vivo up-regulation of b receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b receptor require further investigation. continued interest in development of small molecule inhibitors of p mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p leads to increase production of pro-inflammatory cytokines such as tnf-a and il- b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified -( -chlorophenyl)- -(( -methoxyphenoxy)methyl)- [ , , ] triazolo [ , -b] [ , , ] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the -substituted- -amino- -mercapto- h- , , -triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position and . synthesis and sar will be presented. cytokines like il- b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il- b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il- b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p mapk inhibitor sb and a highly active compound developed in our lab. a data set was generated by determining whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il- and tnf a. inhibition of p map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p map kinase. scope of this work was to investigate -thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised -alkylsulfanyl, -( -fluorophenyl), -( -aminopyridin- -yl) substituted imidazoles as p map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the -aminopyridin- -yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the -thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met , for inhibitory activity. less clear is the importance of the hydrogen bond between n of the imidazole ring and lys of p map kinase.to investigate the role of lys in interacting with the scaffold we prepared two sets of , diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys , contrary to the observation on the compound vk- and p map kinase, that a nitrogen atom bearing a lone pair in position of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys rather than to form an attractive interaction with p map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of -substituted and unsubstituted , -diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p . these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw . cells. treatment of c bl/ mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il- b in the serum. the serum level of the anti-inflammatory cytokine il- was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk / and p mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw . cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide mg for days followed by mg daily for weeks. at week all patients started infliximab mg/kg, and received a further four infusions at weeks , , and . results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a , the active metabolite of leflunomide, and adverse events. the mean disease activity score (das ) fell from . at week to . (p< . ) at week and remained between . and . up to week . in those patients remaining on treatment, more than % achieved an acr response from week to week , and up to % achieved an acr response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il- those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of anticollagentype ii mabs on day , followed by i.p. injection of lps on day to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide ( mg/kg/day) starting on the day of first immunization (day ) in prophylactic treatment group and after the onset of arthritis (day ) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il- b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group ( . ae . ) and therapeutic treatment group ( . ae . ) compared to untreated group ( . ae . , p = . and p = . respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p< . ). steady state mrna levels of il- b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p< . ). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine ( ìm) to detect oxygen free radical production. the cells ( , x cell/ml) were then incubated with a range of concentrations of fibrinogen ( - mg/dl) for minutes. our results show that posters inflamm. res., supplement ( ) s fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations ( - mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox- and cox- ) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox- is mainly involved in homeostatic processes, while the inducible isoform (cox- ) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox- and cox- of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox- and anti-cox- effects by prostaglandin e (pge ) measurement after a (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing reference nsaids, selective or not for cox- or cox- . fifty % inhibitory concentration (ic ) values against cox- and cox- and cox- :cox- ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= . ). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox- and cox- . for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day . the inflammatory response was measured after , and hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at / hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp- were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd b, were cd lo, ssclo and rp- + (granulocyte marker). in humans, transmigrated granulocytes expressed cd and cd b. these cells had a significantly higher cd b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated consecutive patients with mean age ae . years who were admitted within h after ischemic stroke. a control group of patients with mean age ae . years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: . ae . and ae . nmol/l. correlation analysis revealed p< . . discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p< . ) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from ffae in the control to ffae at the concentration of ffmg/ml (p< . ). in the ischemic phase, cynodon dactylon ( ffmg/ml) also decreased the incidence of vt from % (control) to %. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p< . for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during min followed by h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox- (hypothalamus) associated to a marked reduction of ca +-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca +-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson ( ), h qui ( ), m wang ( ), i vedin ( ), jz haeggstrçm ( ), j palmblad ( ) ( ( ), r carnuccio( ), p romagnoli ( ), f rossi ( ) ( ) second university of naples, italy ( ) university of naples, italy ( ) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury ( ) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox- , nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone ( mg/kg/ die, by gavage) for days before carotid injury and days after injury. control rats received physiological solution. days after injury cox- expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p< . ). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by % in treated versus control carotids (p< . ). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h o production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h o production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( ( ), b sehnert ( ), h lanig( ), s päßler( ), r holmdahl ( ), h burkhardt ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander university of erlangen-nuremberg, germany ( ) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic that is highly somatically mutated and its epitope on type ii collagen (cii, aa - ). methods: the establishment of a dynamic simulation modelling of a ciic single-chain fragment (scfv) in complex with the triple helical ciic epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c scfvs and the respective ciic epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr and cdr regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c scfv sequence into the respective germline does not affect cii binding affinity (kd x - ) could be confirmed experimentally by the mutagenesis of (!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd + t-cell proliferation and active brain lesions, on the one hand, and il- , il- and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il- production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il- , but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il- responses, and disease activity is associated with mbp-induced proliferation of cd + t cells. ( ), k goula ( ), p georgakopoulos ( ) ( ) renal unit, st. anrdew hospital, patras, greece ( ) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men ( %). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of maintenance haemodialysis patients at our center over the past years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture. patients ( . %) from the study group were diabetic. results: cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli ( cases), enterobacter aerogenes ( case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone ( mg/kg/day). thereafter the amount of steroid was reduced to . mg/kg/day and a low dose of paquinimod ( . mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd +, cd + and on cd -cd -t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at : . mp were separated on -d gels and tested in wb ( : ) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around , , , kda ( total positive/ tested patients). the dominant antigen was at kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m muscarinic receptor was overcome. proteins were separated on d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for h, exposed to uvb and further cultured for another h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase (msk ) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin- â (il- ß) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase- (mmp- ) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp- activity and mmp- expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp- with the ic s of . - . ìm, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp- induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin ( . - . ìm) strongly inhibited mmp- induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap- , whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p mapk activation, and kaempferol inhibited the p mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp- inhibition and mmp- down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. ( ) ( ) university of valencia, spain ( ) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol- -thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-Þb activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta ( mg/ml) produced a % inhibition of oedema and a strong reduction of pge ( %), ltb ( %) and mpo activity ( %) in skin homogenates. the inhibitory effect of avarol at the same dose was % for oedema, % for pge , and % for ltb and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-Þb nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-Þb activation and can be mediated by the downregulation of intracellular signal-transduction ( ), ams silva( ), cmm santos( ), dcga pinto( ), jas cavaleiro( ), jlfc lima ( ) ( ) requimte, departamento de química-física, faculdade de farmµcia da universidade do porto, porto, portugal ( ) departamento de química, universidade de aveiro, aveiro, portugal -styrylchromones are a novel class of chromones, vinylogues of flavones ( -phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of -styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic -styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several -styrylchromones on the proinflammatory enzymes cyclooxygenase (cox- ), cyclooxygenase (cox- ) and -lipoxygenase ( -lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied -styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested -styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of -styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers f-fluorodeoxyglucose (fdg), -o-methyl- - f-fluoro-l-dopa (omfd), and f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp- was compared with that by squamous cell carcinoma (fadu), mamma (mcf- ) and colorectal adenocarcinoma (ht ) cell lines (without or in the presence of specific inhibitiors). several thp- phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp- cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp- cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp- phenotypes and between thp- phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a -end sage ( sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by sage in colon cancer cell lines treated with epigenetically affecting agents, -aza- deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin- (il- ) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il- is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il- contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il- . serum levels of il- are elevated in patients with colorectal cancer, however serum levels of il- were found to be independent of il- mrna expression in tumor tissue. in this study we analyzed il- mrna expression by real-time pcr in sporadic colon cancer tissue as well as corresponding normal mucous tissue. il- mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p= , ). there was no correlation betweenil- mrna expression and tumor grade or stage. thus we can conclude that il- produced at the tumor site is not involved in sporadic colon cancer progression. ( ), t aiamsa-ard ( ), v chinswangwatanakul ( ), ki techatraisak ( ), s chotewuttakorn ( ), a thaworn ( ) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with % of serum and incubated for hours. cells were analyzed by using [ h] thymidine and immunoblotting assay for cell proliferation and cox- /nos- protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox- but not nos- in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox- . conclusions: this result suggested that some factors in serum of cancer patients could induce cox- protein expression in huvec. the increasing of cox- might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok- @on neutrophil-derived superoxide production and tumor growth. methods: ah a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok- , a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok- on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok- . ( ), m jokic ( ), v zjacic-rotkvic( ), s kapitanovic ( ) ( ) university hospital sestre milosrdnice, bucharest, romania ( ) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il- is a pleiotropic cytokine mapped to chromosome p - . its promoter snp - g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from patients diagnosed with gep-net and age and sex-matched volunteers were analyzed for - g/c snp of the il- gene. to analyze il - c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis Ä test and fishers exact test were used. the level of significance was . . results: there were no differences observed in the frequencies of the - high expression (gc and gg) genotypes between the patients and healthy volunteers (p= . ), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p= . ). - g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p= . ). conclusions: high expression genotypes of il- - snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type receptor (nts ). here we show that hmc- human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc- cell rna yielded a band with % sequence identity to pront and a band corresponding to the pront processing enzyme, pc a.immunocytochemistry on hmc- cells showed specific staining for pront. stimulation of hmc- cells with a + pma, pge , c / or mastoparan released immunoreactive nt.rt-pcr on hmc- cell rna yielded a band with % sequence identity to human nts . western blotting gave bands corresponding to unglycosylated ( kda) and glycosylated ( kda) nts .immunocytotochemistry on hmc- cells showed specific staining for nts . these finding have significance for the role of mast cells in tumor growth. ( ), j buddenkotte ( ), mp schçn( ), m steinhoff ( ) ( ) university hospital münster, germany ( ) university hospital würzburg, germany the proteinase-activated receptor par- has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par- in cutaneous cancerogenesis is still unknown. here we could show a protective role of par- in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par- -deficient and wild-type mice were painted once with dmba ( , -dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate ( -o-tetradecanoylphorbol- -acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after weeks, par- -deficient mice showed a significantly increased number of skin tumors ( per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par- stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase / (erk / ) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta (tgf-â ). thus, our results provide the first experimental evidence for a tumor-protective role of par- . ( ), ma arbós ( ), a fraga ( ), i de torres( ), j reventós ( ), j morote ( ) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf /cxcr and mcp /ccr chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf /cxcr and mcp /ccr expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b f (sc). paf-r antagonist, web ( mg/kg, ip) was given daily for days. we found that eat growth was significantly reduced by pretreatment with web , and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web pretreatment. eat growth was accompanied by increased production of prostaglandin e , vegf and no which was reduced significantly by web treatment. in b f melanoma, web , alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web- reducedactive caspase- expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. ( ), mt quiles ( ), a figueras( ), r mangues( ), f vinals( ), jr germa( ), g capella ( ) ( ) institut de recerca vall de hebron, barcelona, spain ( ) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl (sdf- )/ cxcr chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys and asp nih t transfectants. cys tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with µ-smooth muscle actin positives cells surrounded by f / macrophages were only observed in asp tumors associated with a shorter growth period. asp tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp- protein levels were similarly diminished in both transfectants. higher mmp and mmp activities and expression were observed in asp tumors probably produced by macrophages or stromal cells. total and active mmp levels were higher in cys tumors. the expression of sdf- and cxcr remained unchanged while sdf- g isoform was selectively induced in cys tumors, suggesting sdf- a or b are induced in asp tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf- expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase- and substrates; tnf-alpha; il- ). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher ( -fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ and gencat_agaur_ xt_ ). ( ), m spinola( ), c pignatiello( ), w cabrera ( ), og ribeiro ( ), n starobinas( ), t dragani ( ) ( ) butantan institute, sao paulo, brazil ( ) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c bl/ and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing , mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin (vnn ) and major histocompatibility antigen e alpha (h -ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome ( . cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr- and - , pdgfr, kit, csf- r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability ( %) has translated into superior in vivo anti- inflamm. res., supplement ( ) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk . thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a and du- ) or low passage patient derived tumors (cxf ; colon cancer, rxf ; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. ( ), p bobrowski( ), m shukla ( ), t haqqi ( ) ( ) albany medical college, usa ( ) rainforest nutritionals, inc, usa ( ) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol # . proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il- b ( ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado ( - mg/ml) was tested for its ability to maintain optimal igf- transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity ( mg/kg po) leading to gsh safety rating of /unclassifiable. oligomeric proanthocyanidin content was high ( mg/kg) with the majority of oligomers > mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il- b induced glycosaminoglycan release from human cartilage explants. progrado prevented il- b induced suppression of igf- production from human cartilage explants as well as stimulating basal igf- production (p< . ). comparable changes in igf- gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf- . this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h o -induced cytotoxicity and oxidative dna damage in the nih/ t fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of , -diphenyl- picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h o -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h o , without cytotoxicity except for the hexane extract at high concentrations (> mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk project from korea research foundation). sirt is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt activity, comparing heart (h) and adipose (a) tissue of sedentary young (n ), sedentary old (n ) and trained old (n ) rats. the trained old rats performed a -weeks moderate training on treadmill. on h and a tissue of all rats sirt activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts -hydroxynonenal ( -hne), mnsod, catalase and foxo a by western blot, and gadd a, cyclin d and foxo a mrna by rt-pcr. aging reduced sirt activity in h (p< . ) without effects in a, producing an increase of mda (h, p< . ; a, p< . ) and -hne (h, p< . ; a, p< . ), and a decrease of mn-sod (p< . ) and catalase (p< . ) expression in both h and a. aging did not affect foxo a protein expression in h, and foxo a mrna in a. exercise produced an increase in h foxo a protein expression (p< . ) and in a foxo a mrna, associated to higher mn-sod (h, p< . ; a, p< . ) and catalase (h, p< . ; a, p= . ) levels in both h and a of aged rats. in heart exercise-induced higher sirt activity bring on decrease in cyclin d and increase in gadd a mrna expression. in a we found a similar decrease in cyclin d , without changes in gadd a mrna expression. these findings suggest that exercise is able to increase sirt activity in aged rats. ( ), t horiguchi( ), k abe( ), h inoue( ), t noma ( ) ( ) institute of health biosciences, the university of tokushima graduate school, tokushima, japan ( ) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl -induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with f×l/ kg bw of ccl w/wo mg/ kg bw of gl. after hours and hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between hour-treated-livers (ccl or ccl and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at hours compared to that in ccl treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a , hsp , and procollagen were still very high in gl-treated liver. after hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl -treated liver. then, we screened , genes by microarray and found that genes were up-regulated and genes were down regulated in ccl +gl compared to ccl treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn ). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp- monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp- cells. we also demonstrate that such modulation involves both erk / and p mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. ( ), b arnold( ), g opdenakker ( ) ( ) jagiellonian university, department of evolutionary immunobiology, krakow, poland ( ) german cancer research center, department of molecular immunology, heidelberg, germany ( ) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase (mmp- -/-) at least one compensatory mechanism operates as there are elevated levels of pge of cox- origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp- -/-mice as at hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp- -/-mice. for this numbers of apoptotic (annexin v) and necrotic ( -aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase were significantly lowered in mmp- -/-mice while levels of caspase , and were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp- -/mice during zymosan peritonitis and it is due to the decreased levels of active caspase . the increased activity of other examined caspases is most probably independent of apoptosis. ( ), h james ( ) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), and mg/kg produced a dose-dependent inhibition of paw edema ( % and % at h; % and % at h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), mg/kg( % at h; % at h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of mg/ kg( % at h; % at h) but not with a dose of mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin mg and mg/kg ( % and % at h; % and % at h respectively) and selective cox- inhibitor rofecoxib, mg/kg ( % and % respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. ( ), p hart( ), j edwards ( ), c quirk ( ) ( ) molecular pharmacology limited (usa), australian division, perth, western australia ( ) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il- â production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen ( %) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at min, or at hrs after injury. a third injury site was not treated. photographs were taken before, hrs, and weeks after injury. cytokines: human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of % or % active ingredient. hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at hrs after oxsoralen-uv, the min treatment site showed slight erythema, the hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. weeks after injury, the min site was normal, the hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il â secretion by . % and . %, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. ( ), p hart( ), j snowden ( ), maud eijkenboom ( ) ( ) molecular pharmacology limited (usa), australian division,perth, western australia ( ) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of %, . %, %, . %, %, % or % bov-zn (two pooled experiments). hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for %, %, %, % fcs against %, . %, %, % bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter ) in treated and untreated cell cultures over - hrs at intervals. the ic for tnf-a suppression was reached at . % bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by % bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-£\ and il- fÒ. these cytokines are known to be regulated by the stress-activated p fnfnmap kinase pathway. because of this, inhibition of p map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last years, numerous groups have reported on the development of p map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p vs other kinases, enabling the development of p selective molecules. inflamm. res., supplement ( ) posters anti-inflammatory activities. a series of labdane-type diterpenoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, and were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of and % were noted in the -o-tetradecanoyl-phorbol- -acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e , and tumour necrosis factor-alpha in bacterial endotoxin-activated raw . macrophage cells with ic in the range - mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase- , as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbß, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of mg/kg and substance ffw- Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect ( %) at a dosage of mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first min and hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw- Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th -response with increased levels of proinflammatory cytokines like tnfa and il , and mouse models of crohns disease show that the balance of il / is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il , il and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d , which were able to suppress the cocktail induced il secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p - inflammation and human incisional hernia pathophysiology maria antonia arbos via( ) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney ( ae vs. ae ae ; p< . ), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of s¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge mimetic combination therapy (dp- ) that inhibits basal and lps/tlr induced tnf-?, il- ß, and mmp- , , , in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp dramatically inhibited il- ß, tnf-?, mip , mcp- and il- expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il- ß, mmp- and to a lesser extent mmp- expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data ( , anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb from the -lipoxygenase pathway, pge through the inducible cyclooxygenase (cox- ) pathway and cytokines/chemokines as tnf-alpha, il- beta, il- , mip- alpha, mip- beta, mip- alpha and mip- alpha. engagement of the adenosine a a receptor (a ar) blocks the in vitro synthesis of ltb while it potentiates the cox- pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip- alpha, mip- beta, mip- alpha and mip- alpha in toll-like receptor- -stimulated human neutrophils. little effect was observed on il- beta and il- . using the murine air pouch model of inflammation with a ar-knockout mice, we observed that the activation of a ar positively impacts the expression of cox- in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a a receptor, neutrophils that migrated into the air pouch h following lps injection expressed higher mrna levels of tnf-alpha, mip- alpha and mip- beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a ar-knockout mice and in view of the potent inhibitory actions of pge on inflammatory cells, an increased cox- expression and a prevented release of tnf-alpha, mip- alpha and mip- beta caused by a ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis ( d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw . after challenged with lps (escherichia coli :b ) for , and hours. we identified proteins from approximately detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein , capg protein, poly(rc) binding protein , isocitrate dehydrogenase (nad+) alpha, lactate dehydrogenase , a chain, guanine nucleotide binding protein (g protein), beta polypeptide like , triosephosphate isomerase and proteasome alpha subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p , malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified -amino- -benzyl- -( -bromophenyl)- -methyl- , -dihydropyrano[ , c] pyrazole- -c arbonitrile as a potential novel lead structure as p map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol- ( h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha -acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from . to . ìg/ml in male, and from . to . ìg/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at . - . ìg/ml or days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from to ìg/ml in male, and from to ìg/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at - , ìg/ml. despite a high value of , - , ìg/ml being observed for serum aag levels in pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid ( ìg/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi- , a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi- .the ability of pbi to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi- treated animals, n= ) were used to assess leukocyte infiltration and to measure by elisa tnf-µ, mcp- and pge production.in vitro, pbi- is able to significantly decrease by . % ae . % (p< . ), tnf-a production by human lpsactivated pmn.in vivo, pbi- significantly decreased the production of tnf-a ( . % ae . %; p< . ), mcp- ( . % ae . %; p< . ) and pge ( . % ae . %; p< . ) induced by lps injection.however, pbi- did not significantly inhibit leukocyte infiltration.these results show that pbi- is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. ( ), lj lowenstine,( ), aj norris( ), t spangler( ), lm woods ( ) ( ) zoological society of san diego, usa ( ) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n= each) were selected for parasitology and em (group ), and gross and histopathology (group ). all outbreak cases and group controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in % ( / ) of outbreak cases and in % ( / ) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in archived crows submitted to the vmth from - , suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk / by mek / is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as , a selective mek inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for days (twice daily), by oral route at the doses of , and mg/kg. as at and mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o -.), hydrogen peroxide (h o ), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. ( ), h spalteholz ( ), u reibetanz ( ), p salavei ( ), m fischlechner ( ), h-j glander( ), j arnhold ( ) ( ) university of leipzig, medical faculty, institute for medical physics and biophysics, germany ( ) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects - % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h o ) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor- (par ) in inflammation and innate immunity. par is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par . par is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation ( hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. ( ), k bendtzen ( ), f sellebjerg ( ), ch nielsen ( ) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from ms patients and healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd + monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il- , tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione( ), g tarantino( ), a spanò( ), p ladogana ( ), fl perrone( ), s padula( ), a riccio ( ) ( ) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy ( ) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: patients with arthritis were selected in a population of subjects affected by hcv infection. each patients was evaluated by clinical examination ( denoted poliarticular and mono-oligoarticualr involvement), by x-graphic aspects of joint involvement ( patients presented join erosions), by ana, rf and anti-ccp positiveness.results: , % of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in out of the patients with joint erosions, and only in out of the patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg d on cd + t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type diabetes. the monoclonal antibody cx recognizes murine nkg d and has been shown to block ligandbinding and mediate internalization of nkg d. furthermore, cx can inhibit and/or ameliorate disease in animal models of type diabetes and inflammatory bowel disease. thus, it is very likely that nkg d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb .scid mice. different doses of cx antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx elisa in serum) and flow cytometry (down-regulation of nkg d on cd b+ nk cells) for up to two weeks after administration. we found that cx very efficiently down-regulate nkg d on cd b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx antibody in vivo. ( ), k mehta( ), n deo( ), j chaudhary( ), p bobrowski ( ) ( ) albany medical college, usa ( ) vedic lifesciences, usa ( ) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n= ) were screened and randomized to receive glucosamine sulfate (n= , mg/day) or reparagen (n= , mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a % improvement in womac pain, with assessments at , , , and weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p< . ), with a similar, progressive improvement over the course of the week treatment protocol ( - % reduction in total womac or vas scores). the response rate was substantial for both glucosamine ( %) and reparagen ( %), which exceeded placebo responses ( %, p < . ) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p < . ), and serum igf- levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin- (il- ) and monocyte chemoattracting protein- , two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by % and %, respectively. corresponding reduction in ibuprofen group was % and %. protective mechanisms involved were assessed in human cultured u macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha ( % and % reduction by supplementation with nm and ibuprofen, respectively) and il- ( % and % in corresponding reduction). nm supplementation dramatically reduced prostaglandin e secretion by stimulated macrophages along with cyclooxigenase- (cox ) cellular protein expression. mrna levels forcox and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. ( ), hp kim ( ), kh son ( ) ( ) college of pharmacy, kangwon national university, south korea ( ) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw . cells. among the tested compounds, -methoxy- , -dichlorochalcone (ch ), -hydroxy- -methoxychalcone (ch ), -hydroxy- -bromo- -methoxychalcone (ch ) and -hydroxy- , -dimethoxychalcone (ch ) potently inhibited no production (ic s, . - . mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position ( or ) to carbonyl moiety with/without -(or -)hydroxyl group and -halogen substitution in b-ring. when the cellular action mechanisms of ch , ch and ch were further examined, it was revealed that ch and ch clearly down-regulated inos expression while ch did not. moreover, ch and ch were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)- activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox- expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox- activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, cyclohexylimino- -methyl- , -dihydro- h-benzo [ , ] oxathiol- -one (lyr- ) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw . . the lyr- reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr- could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr- inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys- to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser- and to glu). therefore, lyr- could inhibit lps-induced nf-kb activating pathway by targeting ser- and/or residues on the activation domain of ikkbeta. as pharmacological actions, lyr- prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox- , or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw . . furthermore, lyr- protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline ( mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, nm, , and mm, increased guinea pig plasma dao activity by , , and %, respectively, while sertraline increased it at mm (by %). at higher concentrations ( and mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at and nm amitriptyline the activity of hnmt increased by and %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider( , ), c macarthur ( ), d trune ( ), s mccoy ( ) ( ) oregon health and science university, portland, oregon, usa ( ) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p , for treatment of inflammation induced by either lps or bacteria.peptide p was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p inhibited lps-induced production of serum inflammatory mediators.moreover, p treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p .simultaneous injection of bacteria and peptide p resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p treatment.-subcutaneous and oral administrations of p , but not intravenous administration, were also efficacious in reducing inflammation. administration of p after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. ( ), c zhou( ), y zhang( ), m sun( ), x wan ( ), h yu( ), x yang( ), rd ye ( ), j-k shen ( ) formyl peptide receptor-like (fprl ) is a structural homologue of fpr, which binds chemotactic peptides of as small as amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl ligands include peptides of - amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl -transfected cell line. although substitution of d-met resulted in loss of activity, removal of val together with d-met produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl ligand. further substitution of lys with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl through aromatic interactions involving the side chains of trp and tyr , hydrophobic interaction of nle , and the thio-based hydrogen bonding of met , with the respective residues in fprl which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl -related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il- , tnf-a and il- levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects ( , and mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses ( . and mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin ( and mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin . mg/ mouse/day in combination with glycine or mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. ( ), p sambrook( ), k fukudome( ), m xue ( ) ( ) university of sydney, st leonards, nsw, australia ( ) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr . cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein- -induced chemotaxis of monocytes by approximately %. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than % in ra monocytes. the inhibitory effects of apc were reversed by rcr , indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord- -h hxroos authors: wielinga, peter r.; schlundt, jørgen title: one health and food safety date: - - journal: confronting emerging zoonoses doi: . / - - - - _ sha: doc_id: cord_uid: h hxroos many, if not most, of all important zoonoses relate in some way to animals in the food production chain. therefore food becomes an important vehicle for many zoonotic pathogens. one of the major issues in food safety over the latest decades has been the lack of cross-sectoral collaboration across the food production chain. major food safety events have been significantly affected by the lack of collaboration between the animal health, the food control, and the human health sector. one health formulates clearly both the need for, and the benefit of cross-sectoral collaboration. here we will focus on the human health risk related to zoonotic microorganisms present both in food animals and food derived from these animals, and typically transmitted to humans through food. some diseases have global epidemic—or pandemic—potential, resulting in dramatic action from international organizations and national agricultural- and health authorities in most countries, for instance as was the case with avian influenza. other diseases relate to the industrialized food production chain and have been—in some settings—dealt with efficiently through farm-to-fork preventive action in the animal sector, e.g. salmonella. finally, an important group of zoonotic diseases are ‘neglected diseases’ in poor settings, while they have been basically eradicated in affluent economies through vaccination and culling policies in the animal sector, e.g. brucella. here we will discuss these three different foodborne disease categories, paying extra attention to the important problem of antimicrobial resistance (amr). in addition, we present some of the one health inspired solutions that may help reduce the threat of several of the foodborne diseases discussed. people in ancient times already understood they could get sick from consumption of infected meat. by keeping their animals healthy, and by using dedicated methods of food preparation and conservation, ancient farmers learned how to improve health and prevent disease. probably the oldest written document about it is 'on airs, waters, and places', written by hippocrates, which describes how human health is influenced by its interaction with the environment. since then, our health situation has improved by applying these simple rules of thumb, and even more through improved technologies such as good animal management, hygiene and biosecurity, vaccination programs and prudent animal drug treatment. nowadays, some of the most feared zoonotic diseases such as anthrax or brucellosis are absent in many countries. however, there are still many important diseases that threaten human health and which have animals as their reservoir. these animal reservoirs range from wildlife to domestic animals, both in companionship and agricultural settings. by the obvious close contact and the sheer number of animals needed for consumption, the animals produced for food form the largest reservoir and production grounds for emerging zoonotic pathogens. actions of authorities to protect society from zoonotic diseases differ significantly according to socio-economic status and the zoonotic pathogen in question. basically, zoonotic diseases related to food animals can be separated into three groups. in the first group are diseases with a potential for global spread and with a dramatic public relations potential, often these diseases have a significant human reservoir showing human-human transmission, e.g. sars, avian influenza and certain types of amr bacteria. the second group is constituted by persistent zoonotic diseases related to the industrialized food production chain, such as salmonella and campylobacter, which are broadly distributed in the farm-to-fork chain. these human pathogens are often non-pathogenic in animals and seem to be distributed in all countries, both rich and poor. in the third group are the 'neglected zoonotic diseases'. they are zoonotic diseases which have been eradicated (or drastically reduced) in affluent economies through vaccination and culling policies, and through introduction of hygienic and animal biosafety management practices. however, in many poor settings these diseases are 'neglected diseases' and receive very little attention from national authorities or even international organizations. this group includes brucella, bovine tb (tuberculosis), i.e. mycobacterium bovis, and many parasitic diseases, e.g. leishmaniasis and cysticercosis. in addition to these traditional infectious diseases there is a new threat of antimicrobial resistant (amr) bacteria. caused by the use of antimicrobials both in human and veterinary medicine this problem has emerged and is now to be recognized as one of the most important threats to human health. although in the detail the control of these groups of diseases differ, they are all most efficiently prevented by a one health approach which considers the full farm-to-fork chain. such preventive and holistic approaches may reduce both the disease burden to human health and the economic burden to developing economies, and therefore represent a significant potential for improvement related to food safety as seen in a one health perspective. through food and feed, direct contact, and via the environment, the human-and the animal microbial flora are in contact with each other. figure . outlines the most important routes of transmission for infectious diseases between humans and animals. via these routes infectious diseases from (food-)animals may enter the human reservoir and vice versa. the foodborne transmission route is probably the most important gateway for this contact, and the vast majority of human infections with enteric zoonotic bacterial pathogens, such as salmonella enterica, campylobacter coli/jejuni, and yersinia enterocolitica, occur through this route. for other diseases there is evidence that transmission also occurs via direct contact between (food) animal and humans, e.g. live-stock associated methicillin resistant staphlylococcus aureus (mrsa) (graveland et al. ) . next, there is transmission via the environment (e.g. surface water or water used to irrigate plants) mainly as a result of spreading of manure into the environment (spencer and guan ; hutchison et al. ) . and though much less frequently reported, there may be transmission of pathogens from humans to animals, which most probably was the meat/milk/eggs etc. direct contact and products (eg.skin) veterinary medicine food & consumer authorities food environment indirect contact via water/air (faeces / urine / corpses) fig. . schematic presentation of important microbial transmission routes via which the human and (food) animals are in contact with each other. in blue control mechanisms are shown, and in red some of the transmission routes that are more difficult to control. via the environment transmission may take place of microorganisms present in excretion products, and in diseased animals and people. in addition, wildlife constitutes a risk, as it holds a broad spectrum of diseases, including many highly pathogenic diseases case for the staphylococcus aureus cc (price et al. ) . in many developing countries, wildlife forms an additional important reservoir for foodborne pathogens, not only through consumption of wildlife. because of often lower bio-safety levels in these countries, direct contact between humans and food animals is generally more frequent, and diseases from the wildlife community may cross over more easily to domestic animals. for instance, the general understanding now is that the sars epidemic in originated in direct human contact with and/or consumption of wildlife, or indirectly through contact between wildlife and domestic animals (guan et al. ; shi and hu ) . wildlife holds a broad spectrum of diseases including some of the most feared, such as ebola, rabies and anthrax and, and in contrast to other food sources, much of the consumption of wildlife goes undetected by food controlling agencies. for these reasons, and because of the global trade in wildlife derived food and other items (pavlin et al. ), consumption of wildlife animals, and the spillover of infectious diseases from wildlife to food/production animals, should not be overlooked. the spread of foodborne zoonoses through the food production chain is often referred to as the 'farm-to-fork ' (or 'farm-to-table' or 'boat-to-throat') chain. it should be noted that risk mitigation solutions under this framework typically have focused on a consideration of the full food production continuum, involving all relevant stakeholders, i.e. a typical one health framework invented before the one health paradigm was defined. figure . tries to capture a generalized picture of a farm-to-fork chain, starting with animal feed and ending in human consumption of animal food products. although a number of very important zoonoses are related to wildlife-and in some cases directly transmitted from wildlife animals-the vast majority of zoonotic disease cases in the world relate to animals that are bred for food purposes. such zoonotic pathogens include bacteria such as brucella, salmonella, campylobacter, verotoxigenic escherichia coli and leptospira; parasites, such as taenia, echinococcus and trichinella; and viruses, such as influenza a h n (avian influenza) and rift valley fever virus. next to these infectious diseases, derived agents such as (microbial) toxins and prions (prusiner ) form another important zoonotic subgroup. diseases originating on the farm can in many cases most efficiently be dealt with on the farm itself, thereby eliminating more complex measures or crosscontamination down the farm-to-fork chain. for example, brucellosis in animals (mainly cattle, sheep and goats) has been eliminated in many countries, thereby virtually eliminating the human disease burden (godfroid and käsbohrer ) . also, some of the main parasites can be effectively controlled at the farm level, and this could work for both taenia solium in pigs (defined by who/fao/oie as a 'potentially eradicable parasite'), as well as, trichinella spiralis which is found in many wild animals and importantly in pigs for human consumption. both parasites have essentially been eliminated from farmed pigs in most northern european countries (who/fao/oie ; gottstein et al. ). however, both diseases still form a serious 'neglected diseases' threat in settings where there is potential for contact between wild and domestic animals. it was primarily the outbreaks of sars and zoonotic influenza, amr (dealt with separately) and bse (bovine spongiform encephalopathy) which alerted the world to the need for a one health approach. outbreaks of viral diseases in humans, originating in or spreading through farm animals (avian flu-h n and swine flu-h n ) have caused major global alerts in the last decade. these influenza outbreaks spread very quickly, either in the animal population (h n ) or directly in the human population (h n ), and formed a global threat for human health. h n was therefore characterized by the world health organization (who) as a pandemic. although in total the human disease burden related to the endemic bacterial zoonoses is probably many fold higher than these influenza outbreaks, it is basically these relatively few but fast spreading outbreaks that have put one health on the global agenda. in addition, the failure to predict, monitor and control the spread of these diseases in animals presented regulators and politicians with a wake-up call, and made them demand (better) cross-sectoral collaboration between the animal and human health sectors. prions, non-living infectious agents, have been a significant burden of disease in animal and man. the most well-known zoonotic prion disease is probably the one causing bovine spongiform encephalopathy (bse) in cows, and new variant creutzfeldt-jakob disease in humans, as represented by the massive outbreak of 'mad cow disease' in the uk in the s and s. this agent, a mutant protein, which mainly sits in the brain, got into the (beef) food chain by the feeding of ruminant derived meat and bone meal to ruminants. prions (prusiner ) , scrapie (the disease in goats), spongiform encephalopathy of rocky mountain elk, transmissible mink encephalopathy, kuru and creutzfeldt-jakob disease were known before the large outbreak of bovine spongiform encephalopathy in the uk. it, however, took some time and great efforts, and an early one health approach, to establish the links between the different animal diseases and the human disease (hill et al. ; prusiner ; ghani et al. ). this insight created a background to efficiently stop the spread of this prion disease, by banning the use of animals in animal feed, and seeing a subsequent decrease of the disease in humans (hoinville ) . in the western world prion diseases have attracted much attention, and their control has resulted in a large economic burden to society. in developing countries, often with less strict rules about the re-use of dead animals, and more direct contact with wildlife, prion diseases may still be endemic though unrecognized. in contrast to the dramatic outbreaks discussed above, many food-related zoonoses are endemic in farm animals and some of the most important of these do actually cause disease in the animals. it should be realized that most countries-including most developing countries-produce large amounts of food animals, and most of the production takes place in some sort of industrialized setting. such settings are invariably linked to a number of important zoonotic pathogens. table . shows three lists of the most important food pathogens, as reported in studies published by the cdc in the usa and by rivm (havelaar et al. ) in the netherlands, as well as a list of pathogens recognized by ecdc as focus organisms for the eu. although widespread, these pathogens are often not recognized as important human pathogens because of their often mild disease syndromes in healthy persons (e.g. limited to diarrhea and vomiting) and because of the complexity of source attribution. however, they do form a serious threat to the vulnerable segments of our societies (i.e. the young, the elderly, the immune-compromised and recovering patients), and some patients may develop long-lasting chronic disorders (e.g. arthritis and neurological disorders) (mckenna ). these facts, together with the sheer number of infections they cause, results in a substantial total burden of disease for these pathogens as expressed in disability-adjusted life years (dalys) (see murray ) . for instance, the study of havelaar et al. ( ) showed that the total burden of the diseases he studied was , dalys, for a total of . million cases and deaths caused by these diseases (in million people). source attribution estimates showed that one-third could be attributed to foodborne transmission. similarly large numbers were reported for the usa (cdc ), where surveillance studies of known pathogens gave an estimated total of . million illnesses, , hospitalization and , deaths attributed to foodborne diseases (in million people). importantly, the latter report also showed that the pathogens studied make up only % of the foodborne diseases, and the majority of % is caused by unknown agents. this situation is probably not unique for the usa, and thus indicates that there is still much health to be gained from improved food safety. table . shows that an almost identical list of disability-adjusted life years (dalys), are a combined estimate of the burden of disease due to both death and morbidity. one daly can be thought of as year of healthy life lost and is often expressed in years of life lost on the population level, and can be thought of as a measure of the gap between current health status and an ideal situation where each individual in the population lives to old age, free from disease and disability (murray ) for the list of the cdc and the netherlands the ranking in terms of incidence, hospitalizations, deaths or daly is given. the list from ecdc was generated by expert consultation and for use as an eu focus list in future disease burden studies important foodborne pathogens are found in europe, and that toxoplasma gondii, listeria monocytogenes, campylobacter, rotaviruses, noroviruses and salmonella, should probably form the key targets for interventions. except for norovirus, these pathogens have been shown to be zoonotic, and find their way to humans via food and the environment ( fig. . ). a one health approach ensuring efficient crosssectoral collaboration and data-sharing, could lay the foundation for a realistic description of the situation, and could help implement sensible cross-sector solutions. building on the idea of one health to control these diseases, there are several countries (especially in northern europe and north america) that have instituted cross-sectoral data collection and collaboration. this is typically done through the construction of zoonosis centers or their equivalents. these centers aim to stimulate and facilitate the collaboration between human, veterinary and food institutes. some examples of such specialized centers are the us national center for emerging and zoonotic infectious diseases (http://www.cdc.gov/ncezid), the british national centre for zoonosis research (http://www.zoonosis.ac.uk/ zoonosis) and danish zoonosis centre which is part of the danish national food institute (http://www.food.dtu.dk/english/research/research_groups/zoonosis_ centre.aspx). two clear examples of what such centers can accomplish are: the reduction of salmonella in food animals in denmark, and the danish integrated approach to combat amr (described below in a separate section). in the danish salmonella reduction program, data sharing across animal, food and human health sectors has enabled science-based solutions, and has most noticeably resulted in significant reductions in human salmonellosis through lowering salmonella prevalence in animals (wegener et al. ) . in relation to laying hens the program started with a simple and inexpensive serological surveillance of egg producers. flocks found positive were either culled and repopulated, or used to produce heat-processed eggs, danish eggs are now considered free of salmonella. next to this arm, a program of surveillance and eradication of infected broiler flocks was setup. the effect of the whole program was measured in term of cases of human salmonellosis, which were found to be significantly reduced as the project progressed in time. the construction and solutions of this program clearly followed one health principles. food, veterinary and human health sciences worked together, using similar detection and (geno)typing techniques, which enabled comparison and sharing of data. this top-down selection of salmonella-free poultry could work in other countries with industrialized food animal production as well. in other countries-including most likely most developing countries, salmonella-positive animals have been imported from big producers in industrialized countries. one such documented example was the import into zimbabwe of salmonella enteritidis via live animals. salmonella entered the country through import of infected poultry in the commercial national production system around , and thereafter spread quickly to the communal sector (small-scale farming), as well as to the human population (matope et al. ). the most likely reason for the spread within zimbabwe was that old animals from the commercial sector were sold to small-scale communal production systems. as the trade of live animals is done on a global level and does not take into account whether the traded animals carry any of the diseases from table . , reducing the prevalence in the commercial sector in producing countries, may also lower the global spread and human disease burden in the rest of the world. the spectrum of neglected diseases is broad and includes diseases caused by bacteria, viruses and parasites. many are found world-wide but their prevalence in the human and animal populations varies according to the local agricultural, demographic and geographic conditions and tradition. for many of the neglected diseases solutions to dramatically decrease the disease burden are well-known, but action is lagging, this is the case for example for many of the parasitic zoonoses. this is the reason why the who refers to these diseases as 'neglected diseases' (who ; molyneux et al. ) . neglected diseases may be categorized into two (strongly overlapping) categories. in the first category are the neglected tropical diseases which include chagas disease, trypanosomiasis, leprosy, rabies, schistosomiasis and others, many of which are zoonotic and parasitic diseases. the second category are the neglected zoonotic diseases, covering many of the diseases above and also some bacterial diseases such as anthrax, bovine tuberculosis (tb), brucellosis, and also cysticercosis and echinococcosis. many of the neglected diseases are carried by wildlife and in poor and rural settings by livestock (e.g. brucellosis, anthrax, leptospirosis, q-fever and bovine tb). in addition, many are food-and waterborne (e.g. brucellosis, cysticercosis/ taeniasis and echinococcosis). in particular, the prevalence of bovine tb appears to be increasing in many poor settings and has been linked to hiv infections as an important factor for progression of a tb infection to an active tb disease (lobue et al. ) . brucellosis and bovine tb in cattle cause lowered productivity in the animal population, but seldom death, and both have largely been eradicated from the bovine population in the developed world by test-and-slaughter programs, which in effect has eliminated the human health problem (godfroid and käsbohrer ) . some of the parasitic diseases (e.g. schistosomiasis, cysticercosis, trematodiasis and echinococcosis) have high mortality rates and long-term sequelae including cancer and neurological disorders. cysticercosis is emerging as a serious public health and agricultural problem in poor settings (garcía et al. ) . humans acquire taenia solium tapeworms when eating raw or undercooked pork contaminated with cysticerci. the route of transmission is, pigs infected through taenia eggs shed in human faeces, and the disease is thus strongly associated with pigs raised under poor hygienic conditions. this means that the cycle of infection can be relatively easily broken by introducing efficient animal management, as has been done in most developed countries. given that % of the rural population in poor countries is dependent on livestock as working animals to survive (fao ) , the effect of these animals carrying a zoonotic disease can be dramatic, both relative to human health directly, but also as it affects the potential to earn an income. this also affects the potential mitigation action; for instance the large-scale culling of animals, which can be a viable solution in rich countries, might be problematic in the poorest countries. such solutions would not only mean loss of food, but also a serious socio-economic disruption, in some cases leading to national instability. some of the recent serious outbreaks of antibiotic resistant (amr) foodborne disease, such as ehec in germany (mellmann et al. ) , have shown us a new problem. there seems to be a global trend with the prevalence of amr rising (who ; danmap ; ecdc ; aarestrup ) . especially dangerous is the emergence of resistance against antimicrobials that are considered critically important in human medicine, and in multidrug resistant (mdr) infections (potron et al. ; kumarasamy et al. ). in the early s antibiotics were first introduced to control bacterial infections in humans. the success in humans led to their introduction in veterinary medicine in the s, where they were used in both production and companion animals. nowadays, antibiotics are also used with intensive fish farming and to control some infectious diseases in plants. their use is thus widespread. antibiotics in animals are mainly used in three ways: ( ) for therapy of individual cases, ( ) for disease prevention (prophylaxis) treating groups of animals, and ( ) as antibiotic growth promoters (agp) treating groups of healthy animals with sub-therapeutic concentrations to promote animal growth. when first introduced, the use of antibiotics led to improved animal health, and subsequently higher levels of both food safety and food security. all use, but in particular the use as agp, resulted in a dramatic rise in the use of antibiotics, and for instance, between and the use in the united states alone went from to , t (who ). however, the use of antibiotics in animals has over the years also resulted in a selective pressure for amr microorganisms, contributing significantly to the human health problem of amr bacteria. notably a number of bacterial strains that were previously susceptible to antibiotics are now in very high frequencies becoming resistant to various antibiotics, some of which are very important as last resort treatment potential for humans (bonten et al. ) . in particular the use as agp is questionable, as the concentrations used are sub-therapeutic which result in the selection for resistance but do not efficiently kill microorganisms. nowadays there are serious efforts by national authorities and some international organizations to reduce the antibiotic overuse in animals (food agricultural organization of the united nations (fao)/world organization for animal health (oie)/who ; who ; u.s. food and drug administration (fda) ( )), especially-but not only-through abolishing their use as agp. however, there seem to be major problems in ensuring cross-sectoral understanding, since the veterinary and medical professions are still in debate about how the amr problem has emerged (phillips et al. ; karp and engberg ; smith et al. ; price et al. ) . to achieve a science-based understanding of the problem, data on amr from both the animal and the human side should be compared, and both risk assessments and source attributions performed in an integrated way. in other words, a one health approach in which human and animal health sectors, including food and environmental sectors, work together, may help to deliver answers needed and suggest ways to reduce problems in both human and animal reservoirs (figs. . and . ). in addition to the factors described above, food production and food trade are now more and more global, and thus some of the food related problems are also global food problems. on the positive side, globalization has helped with some of the important global food issues: it raised food security, made our food more varied and tastier, and even including transport costs in the equation, still has global financial advantages. however, together with the food also the foodborne diseases now travel the globe. and if we do not stay on top of the problem, disease outbreaks might affect large parts of the global food sector negatively, in the end leading to negative health-but will also have financial and socio-economical consequences. a more holistic and pro-active approach to food safety may help prevent future food disasters and build healthy economies. one health approaches to combat zoonotic foodborne diseases need to consider at least three levels, the international level, national level and the farm level where the actual production takes place. to facilitate the work at all these levels, many countries have established specialized zoonosis centers. these centers focus their work on zoonotic diseases and promote collaboration between different sectors, and between different countries. they examine the prevalence of zoonotic diseases in humans and (food-)animals, their routes of transmission, the risk associated with their presence in our food chain, and the relation between human disease and zoonotic transmission. in addition, as our food production system has become increasingly dependent on global trade, the approaches taken by these zoonoses centers (should) also include a global angle. national zoonosis centers may also help tackle the global problems associated with zoonotic diseases. however, at the moment most of this work is done by international and global organization, such as the who, oie, and fao. these three international organizations have recognized that combating zoonoses is best achieved via a one health approach, as stated in their seminal paper 'a tripartite concept note' (fao/oie/who ), in which they express the need to collaborate for a common vision. given the impact zoonotic diseases have in socio-economical terms and on the vulnerable sectors in our societies, a one health vision is also endorsed by the world bank and the united nations children's fund (unicef) (world bank/who/unicef/oie/fao/unsic ). in their common vision, they say that a one health approach may lead to novel and improved solutions, including solutions that have not been considered before because of the high costs involved. for instance, while in some cases vaccination is the ultimate tool to prevent disease, it is not always considered because the costs of mass vaccination are higher than the public health benefit savings, or because of global trade regulations. under a one health approach sharing of costs, as well as other mitigation strategies could likely enable novel ways of reaching sensible solutions (narrod et al. ) . for global infectious disease safety national authorities report to who important outbreaks of human disease which have the potential of cross-border spread, under the auspices of the international health regulations (ihr) (who ) . these regulations also cover foodborne diseases associated with globally traded food. however, given the major impact that such announcements may have on global food trade, such as was the case with bse in the uk or the more recent trade barriers put up after the ehec outbreak in germany, national authorities may have become more careful and restricted in what they report. a global one health approach which both considers human health aspects and socio-economic consequences would therefore be a welcome improvement to the ihr of . next to who, other organizations are active in reporting global infectious disease outbreaks, most notably promed-mail (http://www.promedmail. org), which is an internet based program for monitoring emerging diseases worldwide, set up by the international society for infectious diseases. the program is dedicated to rapid global dissemination of information on outbreaks of infectious diseases and acute exposures to toxins that affect human health, including those in animals and in plants grown for food or animal feed, and thereby supports the one health principles. many of the (international) organizations and governing bodies named above have generated guidelines to control -and disseminate information about-food related zoonoses, such as for instance who's global foodborne infections network (gfn) (www.who.int/gfn), the european food safety authority (efsa) (www.efsa.europa.eu/en/topics/topic/zoonoticdiseases), foodnet from the us centers for disease control and prevention (www.cdc.gov/foodnet) and others. the goal of these networks is essentially the same: to help capacity-building and promote integrated, laboratory based surveillance and intersectional collaboration among human health, veterinary and food-related disciplines to reduce the risk of foodborne infections. the emergence of amr in food animals is a serious threat for modern human medicine. the risks exist that both (i) the overuse by mass prophylaxis and agp in animals, and (ii) the misuse of human critically important antibiotics in animals, will lead to the emergence of new amr organisms which may spread to the human reservoir, and via global food trade spread around the world. in the most critical scenario this will make our arsenal of antibiotics unfit to treat previously treatable infectious disease, and it might take us back to a situation as before world war ii, when antibiotics were not yet used in human medicine. one health principles may help mitigate this risk and deal with the amr problem in an efficient way. collaboration between the fao/who codex alimentarius commission and the oie have generated important guidance on how an integrated approach and the prudent use of antimicrobials may reduce the emergence of amr in (food-)animals and subsequently in humans. previous to this, in the who published the 'global principles for the containment of antimicrobial resistance in animals intended for food' (who ) which all countries should follow to reduce the risk of amr. the three major principles are: • use of antimicrobials for prevention of disease can only be justified where it can be shown that a particular disease is present on the premises or is likely to occur. the routine prophylactic use of antimicrobials should never be a substitute for good animal health management. • prophylactic use of antimicrobials in control programs should be regularly assessed for effectiveness and whether use can be reduced or stopped. efforts to prevent disease should continuously be in place aiming at reducing the need for the prophylactic use of antimicrobials. • use of antimicrobial growth promoters that belong to classes of antimicrobial agents used (or submitted for approval) in humans and animals should be terminated or rapidly phased-out in the absence of risk-based evaluations. these global principles have been supplemented with, ( ) guidance on the prudent use of antibiotics from the codex alimentarius commission together with oie, and ( ) six priority recommendations from who to reduce the overuse of antibiotics in food animals for the protection of human health (who ), being: ( ) require obligatory prescriptions for all antibiotics used for disease control in (food) animals; ( ) in the absence of a public health safety evaluation, terminate or rapidly phase out the use of antibiotics for growth promotion if they are also used for treatment of humans; ( ) create national systems to monitor antibiotic use in food-animals; ( ) introduce pre-licensing safety evaluation of antibiotics [intended for use in food animals] with consideration of potential resistance to human drugs; ( ) monitor resistance to identify emerging health problems and take timely corrective actions to protect human health; ( ) develop guidelines for veterinarians to reduce overuse and misuse of antibiotics in food animals. a recent publication (who ) covers the broader scope of amr in relation to both animals and humans. thus, a 'one health' approach has explicitly been proposed by these international organizations to mitigate the risk of amr. since the occurrence of amr in the food production sector, different programs to contain zoonoses and amr zoonoses have been developed following these principles and guidelines. the danish program to contain amr zoonoses, danmap, has in particular gained international attention and has been analyzed in different publications (who ; hammerum et al. ; aarestrup et al. ) . the reason for this was the early one health approach that the danish government and stakeholders proposed to combat amr. in , after publication of the finding that % of enterococci in all industrial produced chickens in denmark were highly resistant to vancomycin (a last resort drug for human therapy) the government decided that actions had to be taken (wegener et al. ) and set up the danish integrated antimicrobial resistance monitoring and research program (danmap). figure . shows the organization of danmap and how the animal health, food safety and public health sectors work together. the objectives of danmap are: ( ) to quantitatively monitor the consumption of antimicrobials used in (food) animals and humans, ( ) to quantitatively monitor the occurrence of amr in (zoonotic) bacteria in animals, food and humans, ( ) to study and describe the associations between antimicrobial consumption and antimicrobial resistance, and ( ) to identify routes of transmission and areas for further research. next to this an automated/ict program, called vetstat, was introduced to collect quantitative data on all prescribed medicine for animals from veterinarians, pharmacies and feed mills (stege et al. ) . vetstat data on drug usage proved important for understanding the different aspects of the antibiotic usage problem, and to provide tools to control the use. for instance, with the information from vetstat it has been possible for the danish veterinary and food authority (dvfa) to introduce "the yellow card initiative" (dvfa ) . this initiative works similarly to that in football, and farmers and veterinarians get a yellow card when their antimicrobial use is excessive as compared to similar farms. only by reducing the antibiotic use, which may be done for instance by adopting management practices from low users, the card can be retracted. this has not only worked as a stick, it also gives the farmers a sense of how they are doing compared to their colleagues. in the european union several countries have now also started to collect antibiotic usage data and to compare antibiotic use at country level (ema ). surveillance of foodborne infections, and infectious diseases in general, is important to understand the transmission of infectious diseases and identify risks. to do this efficiently data collection should be done in a harmonized way, so data can be compared and integrated. until now, this has been difficult because different human medical, veterinary medical, food and environmental laboratories have been using different techniques for surveillance, making it often almost impossible to compare data. with the introduction of whole genome sequencing (wgs) this problem may be solved. its unbiased way of detecting dna and its single platform (the dna code) for comparing genomic information gives wgs the potential to take disease diagnostics to a new level. some early uses showing the value of wgs for diagnostic and epidemiological purposes were the tracking of the massive cholera outbreak in haiti in (hendriksen et al. ) and by the ehec outbreak that was first detected in germany and later also found in other countries and which could be traced back to egyptian imported fenugreek seed using wgs (mellman et al. ). following up on these successes, an international group of scientists with representatives from oie, who, ec, usfda, us cdc, ecdc, universities and public health institutes, came together in brussels, september , to further discuss the possibilities of using wgs on a larger scale. their simple conclusion was that the technology to use wgs for diagnostic purposes is available, and its potential high, however, to make efficient use of the data, a global genomic database is needed (kupferschmidt ; aarestrup et al. ). such a database should be open to, and supported by, scientists from all fields: human health, animal health, environmental health and food safety, and should include genomic data for all types of microorganisms as well as meta-data to trace back the source of the microorganism. building such a database depends on a global one health approach, and in a one health manner both human health as well as other sectors will benefit from it. an important aspect of pursuing this initiative is that it will not only be beneficial for the developed world, but it may especially be beneficial for developing countries. for them genomic identification will mean a giant leap forward as they do not need to implement the wide variety of specialized methods that are nowadays used in the developed world. if set up in a sensible, inclusive, open-source framework wgs analysis will provide the world with a strong weapon in the fight to combat infectious diseases in all sectors. one health approaches may be synergistic in controlling foodborne zoonotic diseases to support both sufficient food safety, and sustainable food security. clearly, because of the unique situation of transmissibility between humans and animals, zoonoses control relies on the control of the microorganisms in ( ) animals, ( ) the food chain and in ( ) humans. in addition, as zoonoses originate in animals before being transmitted to humans, the most effective interventions may be achieved at the source, i.e. at the farm. to be most effective, approaches to reduce the risk of foodborne zoonoses should include all stakeholders from the human as well as the animal health side. at the transmission level, it will be of major importance to involve food and consumers authorities and related stakeholders (e.g. environmental specialist), to make sure the spillover from the animal reservoir is kept as low as possible. the exact solution will differ per country and type of disease (e.g. in many developing countries neglected diseases may still be of importance). given that % of the rural population in poor countries is still dependent on livestock as working animals to survive, the effect of these animals carrying a zoonosis will work out differently than in the industrialized settings. a number of the most important zoonoses relate directly to food production systems in poor settings which could be reduced dramatically through well-known interventions, such as has been the case for brucella, bovine tb and cysticercosis. furthermore, it is important to realize that much of the one health efforts until now have focused on zoonotic pathogens with a potential for dramatic global spread (such as avian influenza and bse). however, major health gains can be obtained with the endemic zoonotic pathogens. for instance salmonella causes a dramatic global disease burden because of the sheer number of cases and the global spread via food and live animal trade. for salmonella there are efficient methods to reduce the prevalence in food animals. one health approaches in the food sector are complex and involve both public and commercial stakeholders, which may put limitations on what can be done. on the one hand food should be nutritious and adding to one's health. on the other hand most food is commercially produced and traded. as food is a commercial product, one of the ways to make food producers (the supply-side) produce more healthy food, is if the public demands this (the supply-demand balance). therefore, educating the public to buy healthier food may be a way to make food manufacturers produce healthier food. next, there are other stakeholders and fields of science (e.g. industrial sciences or logistics) and policy (e.g. economics) that contribute to the food chain, but which may focus on other aspects than healthy food alone, and their conclusions may conflict with the food safety principles (e.g. the use of agp to improve animal growth). clearly food safety is a complex issue, and integration of all its problems and data is difficult and should best be limited to its essential components. for this reason, countries should learn from experiences abroad that have documented success. there are many examples of one health approaches that have helped lower the risk of zoonotic foodborne disease. key to all approaches has been surveillance of the farm-to-fork chain. surveillance should be done at relevant levels of the chain, at the farm level by the veterinary system, and at the food production stage by food-scientist. findings should be shared and compared with the findings in human medicine, to be able to make decisions about potential risks for human health. thus the animal, food and human sectors need working together, to collect and to share data in such a way that they may be compared. as there are still many different techniques used in all three fields, it is still difficult to compare data. an important development in infectious disease diagnostics will be the introduction of wgs techniques, and the construction of a global, open-access genomic database for microorganisms. in a one health manner, the latter would take diagnostics to a new level, and will greatly improve human, animal and food safety. sustainable farming: get pigs off antibiotics changes in the use of antimicrobials and the effects on productivity of swine farms in denmark integrating genomebased informatics to modernize global disease monitoring, information sharing, and response vancomycin-resistant enterococci: why are they here, and where 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union and norway at the turn of the twenty-first century epidemiology, diagnosis, treatment, and control of trichinellosis persistence of livestock associated mrsa cc in humans is dependent on intensity of animal contact isolation and characterization of viruses related to the sars coronavirus from animals in southern china danish integrated antimicrobial resistance monitoring and research program disease burden of foodborne pathogens in the netherlands population genetics of vibrio cholerae from nepal in : evidence on the origin of the haitian outbreak the same prion strain causes vcjd and bse decline in the incidence of bse in cattle born after the introduction of the 'feed ban' analyses of livestock production, waste storage, and pathogen levels and prevalences in farm manures comment on: does the use of antibiotics in food animals pose a risk to human health? a critical review of published data emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study outbreak detectives embrace the genome era tuberculosis in humans and animals: an overview salmonella enteritidis in poultry: an emerging zoonosis in zimbabwe food poisoning's hidden legacy prospective genomic characterization of the german enterohemorrhagic escherichia coli o :h outbreak by rapid next generation sequencing technology zoonoses and marginalised infectious diseases of poverty: where do we stand? quantifying the burden of disease: the technical basis for disability adjusted life years a one health framework for estimating the economic costs of zoonotic diseases on society risk of importing zoonotic diseases through wildlife trade, united states does the use of antibiotics in food animals pose a risk to human health? a critical review of published data european dissemination of a single oxa- -producing klebsiella pneumoniae clone staphylococcus aureus cc : host adaptation and emergence of methicillin resistance in livestock prion diseases and the bse crisis a review of studies on animal reservoirs of the sars coronavirus agricultural antibiotics and human health public health implications related to spread of pathogens in manure from livestock and poultry operations vetstat-the danish system for surveillance of the veterinary use of drugs for production animals food and drug administration (fda) ( ) years of resistance who global principles for the containment of antimicrobial resistance in animals intended for food: report of a who consultation with the participation of the food and agriculture organization of the united nations and the office international des epizooties who global strategy for containment of antimicrobial resistance impacts of antimicrobial growth promoter termination in denmark. the who international review panel's evaluation of the termination of the use of antimicrobial growth promoters in denmark international health regulations neglected tropical diseases: hidden successes, emerging opportunities tackling antibiotic resistance from a food safety perspective in europe guidelines for the surveillance contributing to one world, one health: a strategic framework for reducing risks of infectious diseases at the animal-human-ecosystems interface key: cord- -vx rgs r authors: nair, ranjit; patel, krina title: what the intensivists need to know about critically ill myeloma patients date: - - journal: oncologic critical care doi: . / - - - - _ sha: doc_id: cord_uid: vx rgs r multiple myeloma (mm) is a hematological malignancy characterized by an increase in aberrant plasma cells in the bone marrow leading to rising monoclonal protein in serum and urine. with the introduction of novel therapies with manageable side effects, this incurable disease has evolved into a chronic disease with an acceptable quality of life for the majority of patients. accordingly, management of acute complications is fundamental in reducing the morbidity and mortality in mm. mm emergencies include symptoms and signs related directly to the disease and/or to the treatment; many organs may be involved including, but not limited to, renal, cardiovascular, neurologic, hematologic, and infectious complications. this review will focus on the numerous approaches that are aimed at managing these complications. mm is a hematological malignancy characterized by an increase in aberrant plasma cells in the bone marrow leading to rising monoclonal proteins in the serum and urine. in general, it is a disease of older adults; however, mm may effect younger patients as well: % are < years, % are < years, and . % are < years in age [ ] . it accounts for about % of hematological malignancies in the united states, with an estimated , new cases in . patients often present with osteolytic bone lesions, fractures, bone pain, progressive anemia, hypercalcemia, renal insufficiency, recurrent infections, and/or bleeding. if not treated appropriately and urgently, any of these may lead to death. the introduction of novel agents to the traditional arsenal of drugs comprising of steroids, chemotherapy, and autologous stem cell transplantation has shifted the treatment platform from one which often required repeated acute interventions to that of a more chronic manageable disease. as the acute complications can be potentially fatal, early recognition and intensive care management is the key to successful outcomes. these patients who present with complications are often excluded from mm trials, and their prognosis in the modern era is uncertain. reports suggest that in-hospital mortality from acute complications of mm has decreased significantly over the past decade. continued risk factors for death in this group of patients are organ failure, poor chronic health status, and delayed intensive care management [ ] . despite having increased life expectancy now, the majority of mm patients ultimately develop resistant subclones, leading to disease progression and disease-related complications. with increasing complexity of mm treatments, patients are at risk for more challenging drug toxicities. recognizing and managing the potential side effects of present-day novel regimens is therefore a cornerstone in mm care. in this chapter, we will provide a systematic review of the key critical clinical events which cause medical emergencies in mm. this crucial, fundamental knowledge for all clinicians who routinely care for mm including intensivists will allow for optimal treatment outcomes and survival in mm. complications resulting from stem cell transplant or newer therapies including chimeric antigen receptor t cell therapy are beyond the scope of this review and will not be discussed here. renal insufficiency in mm is a medical emergency that needs immediate work-up and management. there is a wide variation in data reporting on the incidence and the degree of renal failure due to differences in the definitions of renal dysfunction, sample sizes of studies, and populations assessed. renal dysfunction occurs in more than a third to half of newly diagnosed patients (serum creatinine (scr) above the upper normal limit or mg/dl or egfr < ml/min/ . m ). table details the list of common causes of renal dysfunction encountered in clinical practice. as per the rifle risk category, approximately - % of patients develop renal failure, and half become dialysis dependent [ , ] . even after an initial renal recovery with appropriate myeloma-directed therapy, more than half of patients can still develop renal failure during the disease course. in the absence of renal recovery, mortality risk is reported to be higher in this population with shortened overall survival. with novel therapies in myeloma, reversibility of renal insufficiency (ri) is associated with improved survival with some studies demonstrating similar survival in patients who recover from acute kidney injury (aki) and those without renal impairment. therefore, achieving renal recovery is of priority and the main goal in these patients [ , , , , ] . mm kidney: in many cases (approximately - % of cases), renal damage is caused by the disproportionate free light chain (flc) excretion through kidney glomeruli, buildup of light chains in distal tubules, and cast formation which further leads to renal tubular obstruction, an entity referred to as mm kidney. the tubules eventually rupture and flc extravasates, causing interstitial inflammation and damage. this is usually compounded by renal injury from hypercalcemia, dehydration, poor intake, use of nephrotoxic analgesics, iv contrast, use of ace/arb blockade, underlying chronic kidney disorder, and/or underlying infection. early diagnosis and timely therapeutic intervention at the time of presentation to the emergency center are critical. therapy should include aggressive resuscitation with iv fluids in efforts to maintain normal urine output, treating concomitant hypercalcemia and other electrolyte imbalances, initiating treatment of underlying infection, and avoiding nephrotoxins. a multidisciplinary approach with hematology and nephrology consults is paramount to optimize care in these patients. suspicion of cast nephropathy in newly diagnosed myeloma patients with serum flc above mg/l should be high, especially if associated selective proteinuria composed of flc/bjp exists, as the degree of renal injury is usually related to tumor load [ ] . a percutaneous kidney biopsy with histology is not required to initiate treatment if myeloma has been confirmed but can be helpful to confirm the diagnosis, assess concomitant light chain or amyloid deposition disease, and predict prognosis. the need for a renal biopsy should be discussed for patients with lower levels of toxic flc, as these patients are more likely to have alternate etiologies for their ri. in patients with significant light chain burden, it is critical to begin treatment immediately with renal recovery as the major immediate goal. the only proven modality with the best chance of renal recovery is rapid cytoreduction to decrease light chain production. newer chemotherapy agents lower flcs quickly, especially with a combinatorial approach. many clinical trials have shown that a bortezomib-dexamethasone combination regimen achieves this goal [ ] . attaining a pre-dialysis serum-free light chain level below mg/l and a % reduction after the first cycle of chemotherapy are independent predictive factors of renal recovery and hemodialysis independence at year [ , ] . hemodialysis or tpe for the sole purpose of offloading tumor burden is currently investigational, as none of the randomized trials have shown a significant benefit. though it takes a few days to weeks to observe the impact of chemotherapy on light chain production, current evidence suggests using hemodialysis only for clinical indications of renal replacement therapy which may exist and not solely for reduction of tumor load. based on two randomized clinical trials, the use of high cutoff hemodialysis to rapidly reduce the load of nephrotoxic light chains may be an option in patients requiring dialysis when treated with bortezomib-based therapies, but the results may be stochastic at best [ , , ] . tpe can rapidly reduce flc loads; however its role in cast nephropathy comes from small retrospective trials which provide no data on the renal histology or sflc levels. in studies that demonstrated benefit, obtaining a biopsy and using a proteasome inhibitor (pi)-based treatment were not uniformly applied, making interpretation of the data difficult. especially in the era of novel drugs, the evidence in favor of tpe is limited [ , , ] unless there is evidence of igm or iga paraproteinemia-associated hyperviscosity syndrome. the utility of urinary alkalization is debated as this theoretically decreases tubular cast formation and, however, can cause risk of renal calcium precipitation, especially if the patient presents with concurrent hypercalcemia. acute tubular necrosis: atn is a consequence of an already compromised renal function in patients with cast nephropathy. hypercalcemiarelated vasoconstriction with subsequent reduced renovascular flow, dehydration, and use of loop diuretics can speed up cast formation. there are some reports of carfilzomib causing direct proximal tubular injury; however, the risk appears to be very small [ ] . monoclonal deposition disease: in contrast to myeloma kidney, midd (heavy chain, light chain, or heavy chain-light chain) and al amyloidosis result from gradual non-fibrillar (congo red negative) and fibrillar (congo red positive) protein deposition, respectively, in the glomeruli. midd is most commonly kappa subtype, while al amyloidosis tends to be lambda; both tend to be associated with predominant albuminuria unlike cast nephropathy. though the most characteristic presentation is nephrotic syndrome due to glomerular involvement, patients with midd can have rapid deterioration of renal function which could be fatal if diagnosis is not reached in a patient presenting with new-onset renal failure. life-threatening complications which need to be closely monitored include arrhythmias, congestive heart failure, and bleeding diathesis. to confirm the diagnosis, subcutaneous fat pad biopsy can be pursued initially, as it is less invasive and can potentially diagnose an al amyloid deposition disease. if negative, proceed with kidney biopsy to look for al amyloid, midd, or other potential renal glomerulopathy. fanconi syndrome: the presence of hypophosphatemia, hypokalemia, hypouricemia, renal tubular acidosis, and glycosuria should raise the suspicion of fanconi syndrome in mm. it is a rare wasting syndrome due to proximal renal tubule dysfunction more commonly seen in patients with mgus leading to loss of glucose, phosphate, amino acids, and uric acid. though it runs a benign course, it can complicate the concomitant electrolyte imbalance and renal failure. ironically, despite the glycosuria, the serum glucose is usually normal, which goes along with the defect in the proximal tubular transport defect [ ] . mm patients are at high risk for cardiac complications secondary multiple factors including an older age group of patients with underlying comorbidities, concurrent kidney involvement, mm-associated deposition disease, and/or anti-mm drug-related side effects. cardiac amyloid: approximately % of patients with mm can have concomitant light chain (al) amyloidosis, the most common type of systemic amyloidosis associated with plasma cell dyscrasias; often this association is missed at the time of mm diagnosis. in amyloidosis, heart involvement is seen in approximately half of the cases [ ] . cardiac amyloidosis can be clinically silent initially, and a mm patient presenting with progressive dyspnea, worsening edema with evidence of heart failure, or dysrhythmia presenting as syncope or hypotensive event requires a thorough work-up to rule out coexisting amyloid disease. detection of amyloid in any organ requires ruling out cardiac involvement. other symptoms which warrant amyloid work-up include easy bruising, periorbital purpura, macroglossia (which is better appreciated when associated with tooth indentation), and carpal tunnel syndrome. once significant symptoms of cardiac amyloid appear, most commonly related to heart failure, median survival shortens, especially in the event of a late diagnosis. amyloid deposition takes place extracellularly throughout the ventricles, atria, valves, conduction system, and in the perivascular regions of small vessels. this eventually leads to thickened, non-dilated ventricles as well as atrial thickening that eventually progresses to thinned dilated atria. the serum concentration of free light chains and cardiac enzymes (n-terminal pro-brain natriuretic peptide and cardiac troponins) are sensitive markers for systemic and cardiac involvement, respectively, in al amyloidosis [ ] . n-terminal pro-b-type natriuretic peptide levels are elevated in cardiac amyloidosis, even in the absence of heart failure, due to regional myocardial stress due to fibrillar extracellular deposition [ ] . cardiac troponins can be elevated, even with an unremarkable coronary angiography, due to predominant deposition in the small intramural vessels sparing the epicardial arteries, but associated with nonischemic destruction of cardiomyocytes. ecg findings suggestive of cardiac involvement include a pattern of low voltage (cardiac changes from amyloid infiltration rather than myocyte hypertrophy) defined as a qrs voltage amplitude of . mv in all limb leads or mv in all precordial leads (sensitivity - %). this may be described as a pseudo-infarct pattern ( - %). atrioventricular right-or left-bundle branch blocks may also suggest cardiac amyloid. echocardiography in advanced stages usually demonstrates a restrictive pattern due to the structural changes and increased echogenicity of the myocardium with a "granular speckling" (low sensitivity of - % but with a high specificity ( - %)). cardiac mri in advanced cardiac amyloidosis shows diffuse subendocardial heterogeneous enhancement in a nonvascular distribution on delayed contrast-enhanced mr images using inversion recovery technique. this, along with diffuse multichambered wall thickening, posterior right atrial wall thickening, or interatrial septal thickening > mm, is specific to amyloidosis [ , , ] . cardiac catheterization is not always required but used to obtain endomyocardial biopsy. it may help to provide important information for cardiac hemodynamics, which generally shows impaired ventricular filling with an elevated left ventricular end-diastolic pressure. demonstration of left ventricular diastolic pressure greater than right remains to distinguish restrictive cardiomyopathy from its differential diagnosis of constrictive pericarditis. fine-needle aspiration of abdominal fat is relatively sensitive for amyloid deposits (> % of patients with al amyloidosis); other sites of amyloid deposition include minor salivary glands, gingiva, and rectum [ , ] . in a patient with clinical suspicion of cardiac involvement, the presence of physical exam findings, blood markers, imaging, and a cardiac biopsy consistent with amyloid are adequate for diagnosis. patients presenting with acute heart failure in al amyloidosis require intensive care monitoring and aggressive supportive care. diuresis is the key and is probably the only treatment modality that can improve hemodynamics. optimal therapy usually requires using higher doses of diuretics or in combinations (furosemide, torsemide, spironolactone, and metolazone) to maintain adequate diuresis, especially in the setting of nephrotic syndrome. an intravenous route is likely preferred in the acute stage, due to the often-present gut edema and better bioavailability. aggressive supportive care includes maintaining strict intake output measurements, dietary salt restriction, measuring daily weights, and monitoring for arrhythmias. the uses of cardiac protective drugs such as beta-blockers should be reserved for cases of atrial fibrillation to maintain adequate filling pressure. in patients without af, its use should be discussed on a case-by-case basis, considering the significant risks including labile blood pressure due to autonomic neuropathy and risk of bradycardia due to coexistent conduction defects. caution should be maintained with the use of angiotensin-converting enzyme inhibitors and angiotensin ii inhibitors, due to similar risk of exacerbating hypotension. both digoxin and calcium blockers, especially nifedipine, have the tendency to bind to amyloid fibrils. this can precipitate congestive heart failure due to increased susceptibility to digoxin toxicity and enhance the negative ionotropic effect with calcium channel blockers [ , , ] . syncope can result from af with rapid heart rate, embolic phenomenon, conduction defects, bradycardia, and ventricular arrhythmias. the most common atrial arrhythmia ( - % of patients) is af and is associated with a very high incidence of thromboembolism. in a mayo clinic study, cardiac amyloidosis patients undergoing transesophageal echocardiography had a higher frequency of intracardiac thrombus ( %) irrespective of the presence of atrial fibrillation. factors associated with increased risk of intracardiac thrombosis include atrial fibrillation, poor left ventricular diastolic function (grade or ), lower left atrial appendage emptying velocity ( cm/s), increased rv wall thickness, low systolic blood pressure, and increased heart rate. this group of patients should be considered high risk for thromboembolic events and merits screening with tee. anticoagulation should be strongly considered in the presence of any of these risk factors due to the heightened risk for mural thrombus development and embolic events. this should be weighed against the risk of bleeding in these patients due to the potential for concomitant coagulopathy and fragile vasculature [ ] . pacemakers are a consideration in the presence of heart block or symptomatic bradycardia. the role of intracardiac defibrillators is controversial due to limited evidence showing a survival benefit both for primary and secondary prevention. several studies report ventricular arrhythmias in about a third of patients; however, this does not appear to be the leading cause of mortality in this patient group. once congestive heart failure occurs, the clinical course for patients rapidly deteriorates, with a median survival of - months from diagnosis. death in severe cardiac amyloid is often heralded by agonal bradycardia, followed by electromechanical dissociation or pea. nonetheless consensus guidelines do not support icd placement for primary prevention of scd in patients if life expectancy is < year. hence these devices have rarely been used in patients with cardiac al amyloidosis. with improvement in the therapeutic platform of cardiac amyloidosis and improved survival, it becomes a priority to be able to select the patients that benefit from antiarrhythmic prophylaxis and primary prevention icds. with the available data, icds should be considered in cardiac amyloid patients with recurrent episodes of non-sustained vt, unexplained syncope, young age at diagnosis, early stages of disease at diagnosis with low levels of cardiac biomarkers, and prognosis > year [ , , , ] . anti-myeloma drug-related cardiotoxicity: proteasome inhibitors are currently the main class of drugs used in front line and in the relapsed setting of mm. fda-approved therapies in this class include bortezomib, carfilzomib, and ixazomib [ ] . among them, carfilzomib has shown the highest rates of cardiotoxicity. in a meta-analysis looking at the cardiotoxicity of carfilzomib, all-grade and high-grade cardiotoxicity were seen in % and % of patients, respectively, higher than the control arm. the most significant events included heart failure ( %), arrhythmias ( . %), and ischemic events ( %). rare reports of carfilzomibassociated cardiac arrests also exist. risk can be higher in patients with comorbidities such as older age, obesity, anemia, kidney dysfunction, previous mediastinal radiation, high-dose corticosteroids, prior or concurrent anthracycline exposure, use of higher carfilzomib doses, short infusion times, and undiagnosed amyloidosis [ , , , , ] . anecdotal reports suggest bortezomib may lead to cardiotoxicity as well, although a recent meta-analysis did not find a significantly increased risk compared to the control arm [ , ] . the first intervention in suspected cardiotoxicity is to hold further therapy and follow expectant management. in cases of congestive heart failure, once the heart failure symptoms improve, it is reasonable to restart the chemotherapy at a lower dose, with a longer infusion time and limiting peri-treatment iv fluids. arrhythmic events would require careful consideration regarding rechallenging patients after considering the risk-benefit ratio. hyperviscosity syndrome: in a patient undergoing work-up for mm or with an established diagnosis, symptoms of headache, nose bleeds, mental status or visual changes, auditory symptoms, focal weakness, excessive lethargy, seizures, or coma should raise suspicion for hyperviscosity syndrome. though extremely uncommon in mm ( %) in comparison to waldenström's macroglobulinemia ( - %), a low threshold should be kept to work up a patient for hyperviscosity as this can be fatal if not recognized early. the viscosity tends to increase with increase in molecular size, shape, and weight of the involved paraprotein and abnormal polymerization. patients tend to develop symptoms when the serum viscosity reaches centi-poise (cp) (normal serum viscosity, . - . cp). this usually corresponds to an m spike of g/dl if igm, g/dl for igg, and - g/dl for iga paraproteinemia. however, these levels are arbitrary, and the relationship is nonlinear as patients can have symptoms even with a level lower or could be asymptomatic with a higher level [ , ] . in mm, igg (mostly igg ) or iga paraprotein tends to be reported more commonly with hyperviscosity than igm most likely due to the inherent rarity of igm mm [ , ] . due to increased serum viscosity, there is impaired blood flow and sludging throughout the microvasculature, leading to tissue ischemia, placing the patient at risk for thrombosis to major organs leading to myocardial, cerebrovascular, and retinal ischemia. with passive venous congestion, patients can often present with bleeding symptoms. thorough physical exam is paramount including a fundoscopic exam, which may reveal dilated, tortuous, engorged, "sausage" veins with associated hemorrhages. bleeding at sites involving mucosa is common which can include oral and nasal cavity, the uterus, and the gastrointestinal tract. purpura and skin bruising are also common. consensus mm guidelines do not recommend plasmapheresis in the absence of signs or symptoms for hyperviscosity. however, a low threshold should be maintained to initiate treatment at the earliest suspicion of symptoms or signs; it follows a two-prong strategy consisting of immediate plasmapheresis to lower the excess paraproteins and chemotherapy directed at the primary process of mm. unlike the response of igm-related paraproteinemia (> % intravascular), where a - % reduction of viscosity is expected with one treatment, iga or igg (< % intravascular) hyperviscosity can take several sessions to achieve the same result [ ] . if plasmapheresis is not readily available and the patient is having lifethreatening symptoms, phlebotomy may reduce hyperviscosity-related acute symptoms. in mm patients, who are often anemic at presentation, blood transfusions should be done only in emergent situations and with caution until after the initiation of plasmapheresis to prevent rise in serum viscosity [ , ] . venous thromboembolism: patients with mm are at an increased risk of venous thrombosis due to patient-related, disease-related, and treatment-related factors. patient-related factors include older age, recent surgery or immobilization, and decreased functional status; the major disease-related factor is increased pro-thrombotic status from heightened inflammatory markers, especially at diagnosis. there is a high level of factor viii and von willebrand factor as well as acquired resistance to activated protein c [ ] . in addition, mm patients treated with immunomodulators especially thalidomide or lenalidomide used in combination with steroids or other chemotherapeutic agents increase the risk further [ , ] . management of acute dvt or pe involves withholding the offending drug if any and starting immediate systemic anticoagulation with low-molecular-weight heparin or unfractionated heparin. lmwh is the preferred anticoagulation at the time of transition to outpatient therapy as evidence suggests better safety and a superior efficacy in stabilizing the clot in comparison to other anticoagulants [ ] . factors that make oral anticoagulation less ideal is drugdrug interaction and unreliable therapeutic levels resulting from patient-related malnutrition, infection, and drug holds from cytopenias. once full anticoagulation is attained, the immunomodulator can be reintroduced. in the presence of thrombocytopenia, which is commonly encountered in mm patients, the dose of lmwh needs adjustment and in general a recommendation of % dose reduction if platelet counts below , /μl, and discontinuation if platelet count < , /μl is followed. duration of anticoagulation should be a minimum of months; however in the presence of the offending drug, systemic anticoagulation should be continued for the total duration of therapy [ ] . arterial thromboembolic events, though rare, have also been reported with thalidomide, lenalidomide, and pomalidomide; rarely, this may present as myocardial infarction or stroke. the role of newer oral anticoagulants including factor xa inhibitors and direct thrombin inhibitors is currently investigational in the treatment of malignancy-associated thromboembolic events; however, they are a good alternative for patients with renal failure [ ] . sepsis/invasive infections: - % of newly diagnosed mm patients succumb to the disease in the first few months with infection contributing to more than half the cases [ , , ] . high incidence of early infection in mm is due to the suppressed humoral and t cell-mediated immunity by the disease itself. immunosuppression is mediated by disease-and treatment-related factors including decreased ratio of functional to dysfunctional immunoglobulins, defects in antibody opsonization, steroid-related t cell defects, secondary immunodeficiency related to chemotherapy, restricted pulmonary reserve from thoracic rib fractures and opiate use, mucosal damage, indwelling catheters, and presence of renal failure [ , , ] . bacterial infections, especially streptococcus pneumoniae, haemophilus influenzae, and escherichia coli, tend to be the predominant organisms causing infection at diagnosis, while staphylococcus aureus and other gram-negative infections are more common in the relapsed refractory setting [ , ] . the use of novel drugs combined with steroids has increased the rate of viral and fungal infections which can occur any time during the disease course [ , , ] . viral infections, especially herpes simplex virus (hsv) and varicella-zoster virus (vzv), have increased in incidence with the introduction of proteasome inhibitors such as bortezomib. viral prophylaxis with acyclovir or valacyclovir is recommended in all patients undergoing therapy with proteasome inhibitors or monoclonal antibodies. although invasive fungal infection (approximately - %) incidence is low, the mortality rate is high with approximately % of patients requiring icu management. the introduction of novel agents tends to be associated with later-onset invasive fungal infections compared to studies from the conventional chemotherapy era where an earlier onset was noted [ ] . furthermore the use of long-term and high-dose dexamethasone can cause persistent defect in t cell-mediated immunity which predisposes patients to mucosal candidiasis, p. jirovecii, hsv, and vzv infections. following sepsis guidelines with aggressive fluid resuscitation and broad-spectrum antibiotic coverage as well as growth factor support if neutropenic is warranted in a patient presenting with suspected sepsis/infection. in case of clinical deterioration with unclear source of infection, persistent febrile neutropenia, or hemodynamic compromise, consider computer tomography (ct) scan including sinus imaging. other investigative modalities for diagnosis should include bronchoscopy/bal with microscopy and bacteria, viral, and fungal culture. in suspected cases of fungal infection, aspergillus pcr and galactomannan testing on serum and bal should be performed. due to concomitant immunodeficiency, treatment with intravenous immunoglobulins is considered in patients with recurrent bacterial or viral infections; however its role in treating an acute infection is limited. direct involvement of pulmonary parenchyma in mm is extremely rare. pulmonary complications can arise from a broad range of different mechanisms both infectious and noninfectious. in majority of the cases presenting with respiratory failure, the etiology is infectious. patients with myeloma can present with lung infiltrates, ground glass opacities, nodules, masses, effusion, and lymphadenopathy. noninfectious causes that need to be ruled out include congestive heart failure, pulmonary embolism, pulmonary hemorrhage, and drug-induced pneumonitis. myeloma lung: case reports and case series describe the various ways mm can involve the lung. presentation of lung disease is variable and includes consolidative or interstitial pattern infiltrates on imaging, intraparenchymal plasmacytomas, intrapulmonary calcifications, or pleural effusions [ , , ] . presentations similar to ards have also been reported [ , , , , ] . a high index of suspicion is required as there is a broad differential of diagnoses with similar radiographic findings. patients with extramedullary mm tend to have more aggressive disease and can deteriorate rapidly due to disease progression if not recognized and treated early. if there is evidence of mm progression on blood tests or bone marrow, a biopsy (surgical, transbronchial) or bronchoscopy with aspiration and/or bronchoalveolar lavage (bal) should be strongly considered with microbiologic and flow cytometric analysis. once infection is excluded and evidence of progression is confirmed, prompt initiation of mm directed therapy is warranted [ ] . diffuse alveolar hemorrhage: in patients presenting with acute onset hypoxia, pulmonary opacities, and fevers, especially in the setting of thrombocytopenia and anemia, pulmonary hemorrhage is a possibility. diffuse alveolar hemorrhage (dah) has been rarely reported in mm patients, but there have been cases associated with bortezomib and in the posttransplant setting [ , , , ] . though rare, this is potentially a fatal event. a thorough microbiology screen, echocardiogram, ct imaging, autoimmune work-up, and coagulopathy work-up are warranted [ ] . bal showing presence of gross blood and the percentage of hemosiderin-laden macrophages (siderophages) > % at bal are suggestive of a diagnosis of dah [ , , ] . aggressive supportive care and empiric broad-spectrum antimicrobial therapy should be considered. the role of early initiation of steroids is currently based on retrospective series and case reports. an md anderson prospective study examined critically ill adult hematopoietic transplant patients treated for dah, and on multivariate analysis, an initial medium ( - mg/day) and high-dose ( mg/day) steroids were associated with a higher icu mortality (p = . ) as compared with the low dose (< mg/day). adjunctive treatment with aminocaproic acid in this study, which is commonly used in dah, did not produce differences in outcomes [ , ] . drug-induced interstitial pneumonitis (dip): dip manifesting as interstitial lung disease has been reported with lenalidomide, thalidomide, and bortezomib [ , , , ] . though mechanisms are not fully elucidated, oxidative stress, prostaglandin e (pge ) inhibition, fibroblast proliferation, and impairment of cell repair by epidermal growth factor receptor blocking may play a role. bal with culture and microscopy should be able to help rule out infectious causes. the lymphocytic preponderance typically seen in bronchoalveolar lavage (bal) is supportive of a hypersensitivity mechanism; however, this finding is nondiagnostic and requires meticulous exclusion of other causes. invasive procedures like biopsy usually do not provide any specific findings diagnostic for dip and, therefore, should be obtained only on a case-by-case basis. once dip is suspected, stopping the offending agent is the main therapeutic approach; steroids may be used in severe cases [ , ] . although no guidelines exist on the duration of steroids, in general, a slow taper is recommended [ ] . caution should be maintained on rechallenging the patients with the offending medication as the risk of recurrent reaction is extremely high. hypercalcemia: being one of the four diagnostic criteria for mm (crab criteriacalcium, renal insufficiency, anemia, and bone disease), hypercalcemia is defined as calcium level corrected for albumin mg/dl. almost a third of mm patients are diagnosed with hypercalcemia at diagnosis or during the disease course [ , ] . the mechanism is due to local osteolysis; at the cellular level, it is mediated by interleukin , interleukin , and tumor necrosis factor α with rankl being the final common mediator. most patients presenting with hypercalcemia have associated anemia, thrombocytopenia, kidney dysfunction, lytic lesions, and advancedstage disease. hypercalcemia is associated with aggressive disease biology and heavy disease burden and remains a poor prognostic feature even in the era of novel agents [ ] . clinically, hypercalcemia can be asymptomatic; however, patients often have symptoms which range from subtle signs of malaise or fatigue to severe consequences including renal failure, altered mentation, seizures, shortened qt, and risk of arrhythmias. gastrointestinal manifestations such as nausea, vomiting, ileus, anorexia, and episodes of pancreatitis have been reported [ ] . if not treated promptly, hypercalcemia is lethal. increased calcium promotes natriuresis which can further worsen an already compromised kidney function. this dehydration then leads to worsening hypercalcemia and renal function. though the threshold levels are arbitrary, patients with serum calcium level < mg/dl or minimal symptoms can be managed with fluid resuscitation which reverses the hypovolemia, repletes the intravascular volume deficit, and improves urinary calcium excretion. though the definitive approach is prompt mm treatment initiation, often due to patient frailty and concomitant organ damage, chemotherapy cannot be initiated until the patient is more stable. if the patient is on calcium, vitamin d supplements, thiazide diuretics, or any other drugs which promote hypercalcemia, they should be stopped immediately. in patients with levels > mg/dl or with significant symptoms, other adjunct therapies should be used as a temporizing measure. loop diuretics, which were previously commonly used for hypercalcemia, should not be routinely used. the utility of forced saline diuresis is not consistent; with lower doses, calciuresis is an indirect effect of the accompanying natriuresis. to cause a direct calciuretic effect, doses as high as mg/h is required, which can cause unwanted metabolic derangements and worsening renal cast formation with risk of worsening renal dysfunction [ ] . the use of calcitonin to treat patients with hypercalcemia is limited owing to its inability to dramatically lower serum calcium levels. however, it can work synergistically with other therapies without significant adverse side effects until chemotherapy can be initiated. calcitonin, by inhibition of bone resorption and increased renal calcium excretion, has rapid onset of action and can lower calcium levels by - mg/dl ( . mmol/l). the dose is iu/kg given subcutaneously or intramuscularly every - h. the efficacy in general wears off after - days due to the escape phenomenon (tachyphylaxis) of the calcitonin sensing receptors [ ] . preclinical studies suggest tachyphylaxis can be prevented with the addition of corticosteroids [ , , ] . with its anti-mm effect, adding corticosteroids can add to the synergy to this combination in the treatment of hypercalcemia. the role of steroids in hypercalcemia of malignancy comes from case reports and case series. corticosteroids decrease calcium absorption from the gut, decrease renal tubular reabsorption, and promote excretion [ , ] . prednisone (dose of - mg/day) or iv hydrocortisone (dose of - mg/day) for - days is usually used in this setting. this dose, which is effective in patients with chronic granulomatous diseases (e.g., sarcoidosis) and malignancies such as lymphoma, may be suboptimal for patients with mm due to the direct osteolytic action of mm cells. if clinical status permits, dexamethasone should be considered at mm treatment doses ranging from to mg weekly or to mg daily on a days on, days off schedule. intravenous bisphosphonates (pamidronate, zoledronic acid, ibandronate) work by blocking osteoclast-mediated bone resorption. intravenous zoledronic acid is more routinely used than other drugs in this category due to its higher potency, efficacy, and shorter infusion time ( min) , when compared to ibandronate and pamidronate [ , , , ] . bisphosphonates have an established role in severe or symptomatic hypercalcemia, but often it is challenging to administer them upfront for mm patients due to the renal function-based dose limitation [ ] . it also has slow onset of action and reaches peak in about - days; therefore it needs to be used in a combinatorial approach. denosumab, on the other hand, has a renally independent clearance mechanism and hence does not require kidney functionbased dosing adjustments. it is currently fda approved to treat hypercalcemia of malignancy refractory to bisphosphonate therapy and is dosed at mg subcutaneous injection every weeks with additional doses of mg on days and of the first month of therapy [ , ] . mithramycin and gallium nitrate are drugs of historical interest and are not currently used in the standard setting. in refractory cases of hypercalcemia seen in advanced mm and kidney involvement, with impending cardiovascular or cerebrovascular deterioration, hemodialysis ca + -free dialysate should be strongly considered. tumor lysis syndrome: due to the low proliferative activity of plasma cells which is < % in majority of patients, tls is extremely uncommon in mm [ , ] . mm patients who are likely to be at risk are patients with high tumor burden (as noted by an increased serum lactate dehydrogenase and beta- microglobulin), diffuse bone marrow disease (plasma cells > % of all nucleated cells as per a case series), extensive skeletal involvement, high-risk cytogenetics, and plasmablastic morphology [ , ] . there are reports of bortezomib, thalidomide, and steroid associated tls [ , ] . tumor lysis syndrome manifests due to lysis of massive numbers of tumor cells releasing intracellular potassium and phosphorous causing hyperkalemia and hyperphosphatemia. this causes calcium to precipitate as calcium phosphate in tissues causing secondary hypocalcemia. hyperuricemia results from the breakdown of nucleic acids in the tumor cells. the resultant metabolic derangement, if left untreated, can progress to kidney failure, mental status changes, seizures, arrhythmias, and even death. diagnosing tls can be challenging in patients with mm due to its uncommon association and often seen associated renal insufficiency [ ] . it is imperative for clinicians to be aware of this complication in mm patients undergoing treatment with novel combinations. tls is managed with aggressive intravenous hydration, rasburicase, allopurinol, and at times sodium bicarbonate. timely initiation of renal replacement therapy is paramount in patients with lifethreatening electrolyte disturbances. epidural spinal cord compression (escc): acute emergencies related to the nervous system in mm are mostly related to escc. vertebral involvement is prevalent in - % of mm patients, and escc is encountered in approximately - % of patients either at presentation or during the course of disease [ , ] . the compression usually results from vertebral fractures caused by osteolytic tumors or malignant osteoporosis. very rarely, it has been reported due to extraosseous epidural mm or from extradural extension of plasmacytoma [ ] . in majority of the cases, thoracic or lumbar spinal cord is involved (> %) [ , ] . it is vital to recognize the symptoms early, with prompt imaging since the strongest predictor of neurologic outcome with treatment is the neurologic status when treatment is initiated [ ] . clinical features depend on the site and degree of involvement. a new-onset back pain or a worsening of an existing pain in a mm patient should prompt a work-up to rule this out. pain can be localized or radiating which usually worsens with lying down. associated bowel and bladder dysfunction is present in about half of the cases [ ] . if treatment is delayed, myelopathy progresses to loss of sensory and motor function leading to paraplegia. spinal cord injury at the cervical level is uncommon; however, if affected above c -c , it results in respiratory, cardiac, and autonomic decompensation [ ] . gadolinium-enhanced mri is the initial gold standard imaging modality as it can aid in the thorough evaluation of spine, degree of tumor or fracture involvement, and for radiation planning [ ] . the safety of mri should be discussed in the presence of pacemakers and defibrillators in a multidisciplinary fashion, and if contraindicated, ct myelography should be performed. there is a lack of consensus guidelines for treatment and hence warrants an emergent multidisciplinary team consult involving the oncologist, neurosurgeon, and radiation oncologist. the optimal management depends on the extent of disease, performance status of the patient, and presence of spine instability. immediate therapeutic strategies involve achieving minimal mobility until stability of the spine is achieved. steroids should be initiated even while awaiting diagnostic study results (dexamethasone at doses mg iv followed by mg daily in divided doses with a taper over weeks) [ ] . doses as high as mg initial bolus were used in the past, but are not used any more due to risk of significant side effects and limited evidence to suggest benefit [ ] . spine stabilization by reducing the mechanical load can be achieved with the use of a neck collar (e.g., philadelphia collar) in case of cervical spine or braces at other sites and is not generally recommended for longer than months [ ] . a thorough baseline neurological exam followed by serial neurological exams should be done to assess clinical status. radiotherapy should be started as soon as possible as mm is extremely radiosensitive, unless in very late in the disease. in a retrospective study which included patients with mm/lymphoma treated with rt alone, % of the patients experienced an improvement in motor function, % had no change, while % deteriorated. in this group, patients who experienced slowly progressive motor deficit defined as time of onset > days had better response rates than patients who had fast onset deficit defined as time of onset < days. the improvements in motor function in these groups were % and %, respectively. no patients who received radiotherapy progressed in the slow onset motor weakness group [ , ] . current evidence does not support resorting to surgery in patients with escc, as most mm cases respond to radiation. the need for surgical stabilization with fixation or by percutaneous repair is in general reserved for cases with spine instability or spine compression from a vertebral body. treatment of the underlying malignancy with systemic chemotherapy or novel agents, bone-modifying agents, and neurorehabilitation can help in recovery of the vertebral and spinal damage [ , ] . cns myelomatosis: neurological symptoms in mm are most commonly due to hypercalcemia, cord compression, anti-mm drug related, hyperviscosity, or amyloid-related neuropathy. direct involvement by malignant cells of the central nervous system (cns) or peripheral nervous system, unlike lymphoma, is uncommon in mm. direct cns involvement in mm often presents as leptomeningeal myelomatosis (lmm), intraparenchymal metastasis, cranial nerve involvement, or radiculopathy [ , , ] . though conventional wisdom would suggest cns involvement is seen later during the disease, most patients present relatively early with median time of diagnosis of lmd around months from mm diagnosis. hence the risk is unlikely only related to improved survival in mm patients but also due to an aggressive early biology. supporting this contention, cns involvement is reported mostly in patients with high-risk mm with heavy tumor burden, plasmablastic morphology, or plasma cell leukemia [ ] . patients often present with focal weakness, cranial nerve palsies, mental status changes, speech and gait disturbances, symptoms of increased intracranial pressure, and/or seizures. csf examination reveals pleocytosis, increased protein levels higher than g/dl, presence of monoclonal plasma cells, and in some cases an increased opening pressure. the diagnosis is usually reached with the first analysis of csf [ ] . mri imaging with and without contrast would aid in identifying the presence and extent of the disease. in the presence of symptoms, steroids can help in alleviating the symptoms and reducing intracranial pressure. therapy involves strategies similar to that used for other hematological malignancies with cns involvement, including intrathecal chemotherapy and cranial irradiation with systemic chemotherapy. despite majority of patients achieving csf clearance with cns-directed therapy showing significant potential for symptom improvement, the overall prognosis is grim with the currently approved therapies in mm. the median survival ranges from to months, with a small fraction surviving past years [ ] . hence it is paramount to have a thorough discussion on the goals of therapy once cns involvement is diagnosed [ , ] . anemia: hematological emergencies in mm most commonly arise due to disease-or treatment-related myelosuppression. anemia (hb > g/dl below the lower limit of normal or a hemoglobin value < g/ dl) is a common complication seen in - % mm patients at presentation and in nearly all patients at some point in the disease course [ ] . several factors drive anemia in mm patients which include bone marrow involvement, low serum erythropoietin level due to kidney dysfunction, hemolysis, impaired availability of storage iron, and anti-mm treatment-related bone marrow suppression. an aggressive anti-mm treatment approach despite the severity of anemia is warranted in patients with anemia suspected to be from mm. this is likely encountered in most cases of newly diagnosed mm. myeloma therapy often worsens the anemia, before it improves once myeloma responds. these patients should be closely monitored for transfusion requirement. no universal guidelines exist for transfusion goals. in general, leukoreduced and irradiated blood is used for transfusion for hemoglobin level < - g/dl or symptomatic anemia. the transfusion goal can vary in the presence of other comorbidities such as cardiac ischemia, active bleeding, or if symptoms due to hypoperfusion are present. a major proportion of patients with mm who have chronic anemia are those with long-standing disease in partial remission or stable response. erythropoiesis-stimulating agents are not routinely used, except in this population after careful selection, to improve the ongoing anemia despite chemotherapy. anti-mm drug-induced tma: in patients who present with new-onset anemia and thrombocytopenia, more than often, it is ascribed to myelosuppressive treatment or for mm progression. in the setting of worsening aki, it is imperative to check for evidence of hemolysis with a haptoglobin level, ldh level, and peripheral blood smear looking for schistocytes. one should strongly consider ruling out tma as it has been reported with novel drugs and with older chemotherapeutic agents; two notable novel drugs are bortezomib and carfilzomib, and a chemotherapeutic agent is cisplatin. prompt discontinuation of these medications is warranted if the medication is suspected to be the likely culprit. plasmapheresis is initiated in most of patients, and eculizumab has been tried in a small number of patients. the role of these two therapies in malignancy-associated tma is controversial and should be discussed on a case-by-case basis [ , , , ] . the novel drug combinations with manageable adverse profile hold great promise in mm, which has turned mm into a chronic disease with an acceptable quality of life for patients. it is also crucial to recognize the complications of mm or its treatment and understand the management strategies, which could help prevent excess morbidity or mortality associated with this incurable disease. multiple myeloma and light chain-associated nephropathy at end-stage renal disease in the united states: patient characteristics and survival diffuse alveolar hemorrhage in 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lymphoma and myeloma management of tumor lysis syndrome in patients with multiple myeloma during bortezomib treatment thalidomidedexamethasone as primary therapy for advanced multiple myeloma carfilzomib-associated cardiovascular adverse events: a systematic review and meta-analysis multiple myeloma presenting as interstitial lung disease into the heart: the emerging role of the ubiquitin-proteasome system incidence and risk of cardiotoxicity associated with bortezomib in the treatment of cancer: a systematic review and metaanalysis acute spinal cord dysfunction. in: the washington manual of medical therapeutics retracted: overview of recent trends in diagnosis and management of leptomeningeal multiple myeloma multiple myeloma involving skin and pulmonary parenchyma after autologous stem cell transplantation proteasome inhibitor associated thrombotic microangiopathy hypercalcemia remains an adverse prognostic factor for newly diagnosed multiple myeloma patients in the era of novel antimyeloma therapies conflicts of interest kp = research funds from poseida, intrexon/ziopharm, takeda. advisory board/consultancy in takeda, dava oncology, celgene, janssen, amgen, bms, and oncopeptides. key: cord- -yl vanuh authors: herberg, jethro; pahari, amitava; walters, sam; levin, michael title: infectious diseases and the kidney date: journal: pediatric nephrology doi: . / - - - - _ sha: doc_id: cord_uid: yl vanuh the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. in this chapter, a microbiologic classification of the organisms affecting the kidney is adopted. although they are important causes of renal dysfunction in infectious diseases, urinary tract infections and hemolytic uremic syndrome (hus) are not discussed in detail because they are considered separately in chapters and respectively. elucidation of the cause of renal involvement in a child with evidence of infection must be based on a careful consideration of the geographic distribution of infectious diseases in different countries. a history of foreign travel; exposure to animals, insects, or unusual foods or drinks; outdoor activities such as swimming or hiking; and contact with infectious diseases must be sought in every case. the clinical examination should include a careful assessment of skin and mucous membranes and a search for insect bites, lymphadenopathy, and involvement of other organs. a close collaboration with a pediatric infectious disease specialist and hospital microbiologist will aid the diagnosis and management of the underlying infection. a tantalizing clue to the pathogenesis of glomerular disease is the marked difference in the incidence of nephrosis and nephritis in developed and underdeveloped areas of the world. in several tropical countries, glomerulonephritis (gn) accounts for up to % of pediatric hospital admissions; the incidence in temperate climates is -to -fold less. this difference might be explained by a complex interaction of several different factors, including nutrition, racial and genetically determined differences in immune responses, and exposure to infectious diseases. a growing body of evidence, however, suggests that longterm exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. renal involvement in infectious diseases may occur by a variety of mechanisms: direct microbial invasion of the renal tissues or collecting system may take place in conditions such as staphylococcal abscess of the kidney as a result of septicemic spread of the organism or as a consequence of ascending infection; damage to the kidney may be caused by the systemic release of endotoxin or other toxins and activation of the inflammatory cascade during septicemia or by a focus of infection distant from the kidney; ischemic damage may result from inadequate perfusion induced by septic shock; the kidney may be damaged by activation of the immunologic pathways or by immune complexes resulting from the infectious process. in many conditions, a combination of these mechanisms may be operative. in the assessment of renal complications occurring in infectious diseases, the possibility of druginduced nephrotoxicity caused by antimicrobial therapy should always be considered. the nephrotoxic effects of antibiotics and other antimicrobial agents are not addressed in this chapter but are covered in chapter . bacterial infections associated with renal disease and the likely mechanisms causing renal dysfunction are shown in > impaired renal function is a common occurrence in systemic sepsis ( ) . depending on the severity of the infection and the organism responsible, the renal involvement may vary from insignificant proteinuria to acute renal failure requiring dialysis. the organisms causing acute renal failure as part of systemic sepsis vary with age and geographic location and also differ in normal and immunocompromised children. in the neonatal period, group b streptococci, coliforms, staphylococcus aureus, and listeria monocytogenes are the organisms usually . responsible. in older children, neisseria meningitidis, streptococcus pneumoniae, and s. aureus account for most of the infections. in people who are immunocompromised, a wide range of bacteria are seen, and, similarly, in tropical countries other pathogens, including haemophilus influenzae, salmonella species, and pseudomonas pseudomallei, must be considered. where h. influenzae type b vaccine has been introduced, however, the incidence of severe systemic infections due to this organism has shown a sharp fall. systemic sepsis usually presents with nonspecific features: fever, tachypnea, tachycardia, and evidence of skin and organ underperfusion. the pathophysiology of renal involvement in systemic sepsis is multifactorial ( , ) . hypovolemia with diminished renal perfusion is the earliest event and is a consequence of the increased vascular permeability and loss of plasma from the intravascular space. hypovolemia commonly coexists with depressed myocardial function because of the myocardial depressant effects of endotoxin or other toxins. the renal vasoconstrictor response to diminished circulating volume and reduced cardiac output further reduces glomerular filtration, and oliguria is thus a consistent and early event in severe sepsis ( , ) . a number of vasodilator pathways are activated in sepsis, including nitric oxide and the kinin pathways. this may lead to inappropriate dilatation of vascular beds. vasodilation of capillary beds leading to warm shock is common in adults with sepsis due to gram-negative organisms but is less commonly seen in children, in whom intense vasoconstriction is the usual response to sepsis. if renal underperfusion and vasoconstriction are persistent and severe, the reversible prerenal failure is followed by established renal failure with the characteristic features of vasomotor nephropathy or acute tubular necrosis. other mechanisms of renal damage in systemic sepsis include direct effects of endotoxin and other toxins on the kidney, and release of inflammatory mediators such as tumor necrosis factor (tnf) and other cytokines, arachidonic acid metabolites, and proteolytic enzymes ( ) . nitric oxide (no) is postulated to play a key role in the pathophysiology of renal failure in sepsis. whether the renal effects of increased no are beneficial or harmful remains unclear. trials of selective no synthetase inhibition did not offer any advantages over saline resuscitation ( ) . no in endotoxemia is possibly beneficial because it maintains renal blood flow and glomerular filtration. activation of coagulation is an important component of the pathophysiology of septic shock and may contribute to renal impairment. activation of multiple prothrombotic and antifibrinolytic pathways occurs, together with downregulation of antithrombotic mechanisms such as the protein c pathway. treatment with activated protein c has been shown to improve outcome of adult septic shock, but has not been confirmed to have benefit in pediatric sepsis, and may carry a risk of bleeding particularly in infants ( ) . the renal findings early in septic shock are oliguria, with high urine/plasma urea and creatinine ratios, low urine sodium concentration, and a high urine/plasma osmolarity ratio. once established, renal failure supervenes, and the urine is of poor quality with low urine/ plasma urea and creatinine ratios, elevated urine sodium concentration, and low urine osmolarity. proteinuria is usually present, and the urine sediment may contain red cells and small numbers of white cells ( ) . the management of acute renal failure in systemic sepsis depends on early diagnosis and administration of appropriate antibiotics to cover the expected pathogens. . in addition, management is directed at improving renal perfusion and oxygenation. volume replacement with crystalloid or colloid should be undertaken to optimize preload. central venous pressure or pulmonary wedge pressure monitoring is essential to guide volume replacement in children in severe shock ( , ) . the use of low-dose ( - pg/kg/min) dopamine to reduce renal vasoconstriction together with administration of inotropic agents such as dobutamine or epinephrine to improve cardiac output may reverse prerenal failure. early elective ventilation should be undertaken in patients with severe shock. if oliguria persists despite volume replacement and inotropic therapy, dialysis should be instituted early, because septic and catabolic patients may rapidly develop hyperkalemia and severe electrolyte imbalance. in most children who develop acute renal failure as part of systemic sepsis or septic shock, the renal failure is of short duration, and recovery can be expected within a few days of achieving cardiovascular stability and eradication of the underlying infection. occasionally, renal cortical necrosis or infarction of the kidney may result in prolonged or permanent loss of renal function. meningococcus n. meningitidis continues to be a major cause of systemic sepsis and meningitis in both developed and underdeveloped parts of the world ( ) . in developed countries, most cases are caused by group b and y strains, particularly after introduction of meningococcal c vaccination, whereas epidemics of meningococcus groups a, c and w continue to occur in many underdeveloped regions of the world ( , ) . infants and young adults are most commonly affected, but cases in adolescents and young children are also common. there are two major presentations of meningococcal disease ( ) : meningococcal meningitis presents with features indistinguishable from those of other forms of meningitis, including headache, stiff neck, and photophobia. lumbar puncture is required to identify the causative agent and distinguish this from other forms of meningitis. despite the acute nature of the illness, the prognosis is good, and most patients with the purely meningitic form of the illness recover without sequelae. meningococcemia with purpuric rash and shock is the second and more devastating form of the illness. affected patients present with nonspecific symptoms of fever, vomiting, abdominal pain, and muscle ache. the diagnosis is only obvious once the characteristic petechial or purpuric rash appears. patients with a rapidly progressive purpuric rash, hypotension, and evidence of skin and organ underperfusion have a poor prognosis, with a mortality of - %. adverse prognostic features include hypotension, a low white cell count, absence of meningeal inflammation, thrombocytopenia, and disturbed coagulation indices ( ) . renal failure was seldom reported in early series of patients with meningococcemia, perhaps because most patients died rapidly of uncontrolled septic shock. with advances in intensive care, however, more children are surviving the initial period of profound hemodynamic derangement, and renal failure is more often seen as a major management problem. approximately % of children with fulminant meningococcemia develop renal failure, which usually occurs - h after the onset of illness ( ) . the pathophysiology of meningococcal septicemia involves the activation of cytokines and inflammatory cells by endotoxin ( , ) . mortality is directly related to both the plasma endotoxin concentration and the intensity of the inflammatory response, as indicated by levels of tnf and other inflammatory markers ( ) . patients with meningococcemia have a profound capillary leak leading to severe hypovolemia. loss of plasma proteins from the intravascular space is probably the major cause of shock ( ) , but myocardial suppression secondary to il- production is also important ( ) . intense vasoconstriction further impairs tissue and organ perfusion, and vasculitis with intravascular thrombosis and consumption of platelets and coagulation factors is also present ( ) . oliguria is invariably present in children with meningococcemia during the initial phase of the disorder. this is prerenal in origin and may respond to volume replacement and inotropic support. if cardiac output cannot be improved and renal underperfusion persists, established renal failure supervenes. occasionally, cortical necrosis or infarction of the kidneys occurs. children with meningococcemia should be aggressively managed in a pediatric intensive care unit, with early administration of antibiotics (penicillin or a third-generation cephalosporin), volume replacement, hemodynamic monitoring, and the use of inotropic agents and vasodilators. if oliguria persists despite measures to improve cardiac output, elective ventilation and dialysis should be instituted early ( , ) . because activation of coagulation pathways occurs, severe acquired protein-c deficiency may result and is usually associated with substantial mortality ( ) . protein c is a natural anticoagulant which also has important antiinflammatory activity. despite evidence for impaired function of the activated protein c pathway in meningococcal diseases ( ) , and adult trials suggesting benefit of activated protein c administration in septic shock (prowess trial) ( ) , pediatric trials of activated protein c showed no clear benefit, and were associated with increased risk of intracranial bleeding in very young infants ( ) . the role of apc therapy in pediatric sepsis remains unclear. most patients who survive the initial - h of the illness and regain hemodynamic stability will ultimately recover renal function even if dialysis is required for several weeks. the least common presentation of meningococcal sepsis is chronic meningococcemia. patients with this form of the illness present insidiously with a vasculitic rash, arthritis, and evidence of multiorgan involvement. the features may overlap those of henoch-schonlein purpura or subacute bacterial endocarditis (sbe), and the diagnosis must be considered in patients presenting with fever, arthritis, and vasculitic rash, often accompanied by proteinuria or hematuria. response to antibiotic treatment is good, but some patients may have persistent symptoms for many days resulting from an immunecomplex vasculitis. staphylococcal infections may affect the kidneys by direct focal invasion during staphylococcal septicemia, forming a renal abscess; by causing staphylococcal bacteremia; or by toxin-mediated mechanisms, as in the staphylococcal toxic shock syndrome. staphylococcal abscess. staphylococcal renal abscess presents with fever, loin pain and tenderness, and abnormal urine sediment, as do abscesses caused by other organisms ( ) . the illness often follows either septicemia or pyelonephritis. the diagnosis is usually considered only when a patient with clinical pyelonephritis shows an inadequate response to antibiotic treatment. the diagnosis is confirmed by ultrasonography or computed tomographic scan, which shows swelling of the kidney and intrarenal collections of fluid. antibiotic therapy alone may result in cure, but if the patient remains unwell with evidence of persistent inflammation despite use of appropriate antibiotics, surgical intervention may be required. percutaneous drainage under ultrasonographic or computed tomographic scan guidance is often effective and may avoid the need for a more direct surgical approach ( , ) . staphylococcal toxic shock syndrome. the staphylococcal toxic shock syndrome is a systemic illness characterized by fever, shock, erythematous rash, diarrhea, confusion, and renal failure. the disorder was first described by todd et al. in in a series of seven children ( ) . during the s, thousands of cases were reported in the united states. most cases were in menstruating women, in associated with tampon use. although most cases worldwide are seen in women and are associated with menstruation, children of both sexes and of all ages are affected ( ) . the illness usually begins suddenly with high fever, diarrhea, and hypotension, together with a diffuse erythroderma ( ) . mucous membrane involvement with hyperemia and ulceration of the lips and oral mucosa or vaginal mucosa, strawberry tongue, and conjunctival injection are usually seen. desquamation of the rash occurs in the convalescent phase of the illness. confusion is often present in the early stages of the illness and may progress to coma in severe cases. multiple organ failure with evidence of impaired renal function, elevated levels of hepatic transaminases, thrombocytopenia, elevated cpk and disseminated intravascular coagulation (dic) is often seen. according to cdc criteria, the diagnosis is made on the basis of the clinical features of fever, rash, hypotension, and subsequent desquamation along with deranged function of three or more of the following organ systems: gastrointestinal (gi), mucous membranes, renal, hepatic, hematologic, central nervous system, and muscle. other disorders causing a similar picture, such as rocky mountain spotted fever, leptospirosis, measles, and streptococcal infection, must be excluded. the staphylococcal toxic shock syndrome is now known to be due to infection or colonization with strains of s. aureus that produce one or more protein exotoxins ( ) . most cases in adults are associated with toxic shock toxin i; in children, many of the isolates associated with the syndrome produce other enterotoxins (a to f). the staphylococcal enterotoxins appear to induce disease by acting as superantigens ( ) , which activate t cells bearing specific v beta regions of the t-cell receptor; this causes proliferation and cytokine release ( ) . the systemic illness and toxicity are believed to result largely from an intense inflammatory response induced by the toxin. the site of toxin production is often a trivial focus of infection or simple colonization, and bacteremia is rarely observed. renal failure in toxic shock syndrome is usually caused by shock and renal hypoperfusion. in the early stages of the illness, oliguria and renal impairment are usually prerenal and respond to treatment of shock and measures to improve perfusion. in severe cases and in patients in whom treatment is delayed, acute renal failure develops as a consequence of prolonged renal underperfusion, and dialysis may be required. in addition to underperfusion, direct effects of the toxin or inflammatory infectious diseases and the kidney mediators may also contribute to the renal damage. recovery of renal function usually occurs, but in severe cases with cortical necrosis or intense renal vasculitis, prolonged dialysis may be required. the management of staphylococcal toxic shock syndrome depends on early diagnosis and aggressive cardiovascular support with volume replacement, inotropic support, and, in severe cases, elective ventilation. if oliguria persists despite optimization of intravascular volume and administration of inotropic agents, dialysis should be commenced early ( ) . anti-staphylococcal antibiotics should be started as soon as the diagnosis is suspected and the site of infection identified. initial empiric antimicrobial therapy should include an anti-staphylococcal antibiotic effective against betalactamase-resistant organisms and a protein synthesisinhibiting antibiotic such as clindamycin to stop further toxin production ( ) . if there is a focus of infection such as a vaginal tampon, surgical wound, or infected sinuses, the site should be drained early to prevent continued toxin release into the circulation. the intravenous administration of immune globulins may be considered when infection is refractory to several hours of aggressive therapy, an undrainable focus is present, or persistent oliguria with pulmonary edema occurs ( ) . with aggressive intensive care, most affected patients survive, and renal recovery is usual, even in patients who have had severe shock and multiorgan failure. relapses and recurrences of staphylococcal toxic shock syndrome occur in a proportion of affected patients because immune responses to the toxin are ineffective in some individuals. panton valentine leucocidin (pvl) producing staphylococcal infection: in recent years there have been increasing reports of severe staphylococcal disease, associated with shock and multiorgan failure, caused by strains of staphylococci producing the pvl toxin. panton-valentine leukocidin (pvl) is a phage-encoded toxin, which profoundly impairs the host response due to its toxic effect on leucocytes (see review ( ) ). pvl producing strains are associated with tissue necrosis and increased propensity to cause abscesses in lung, bone, joint, and soft tissue infections. perinephric abscesses have been reported ( ) . there are increasing numbers of children and adults admitted with fulminant sepsis, and shock due to pvl producing strains, and renal failure is a significant component of the multiorgan failure. in addition to intensive care support, antibiotic treatment of pvl strains should include antibiotics which reduce toxin production, such as clindamycin, linezolid or rifampicin, as well as vancomycin if the strain is resistant to methicillin. beta-lactam antibiotics should be avoided, as there is some data to suggest that pvl toxin production can increased by these antibiotics under some conditions ( ) . immunoglobulin infusion may also be of benefit. aggressive surgical drainage of all collections requires close consultation with orthopedic and surgical teams. the group a streptococci (gas) are a major worldwide cause of renal disease, usually as poststreptococcal nephritis. however, in addition to this post-infection immunologically mediated disorder, in recent years there have been increasing reports of gas causing acute renal failure as part of an invasive infection with many features of the staphylococcal toxic shock syndrome ( ) . acute poststreptococcal glomerulonephritis. acute poststreptococcal gn (apsgn) is a delayed complication of pharyngeal infection or impetigo with certain nephritogenic strains of gas. different strains can be serotyped according to the antigenic properties of the m protein found in the outer portion of the bacterial wall. apsgn after pharyngeal infection is most commonly associated with serotype m . in contrast, in apsgn after impetigo, serotype m is most commonly identified ( ) . on occasions, other serotypes and non-typeable strains have been described as causing gn. the pathology and pathogenesis of the disorder is discussed in detail in chapter . apsgn has a worldwide distribution. epidemiologic differences are observed between pharyngitis-associated and impetigo-associated streptococcal infections. pharyngitis-associated apsgn is most common during school age and has an unexplained male/female ratio of : . it occurs more often in the cooler months, and familial occurrences are commonly described. the latent period is - weeks, in notable contrast to impetigo-associated cases, which have a latent period of - weeks. in many developing countries, children have chronic skin infections, and it may be difficult to establish the latent period with accuracy. impetigoassociated cases are more common in the warmer months, sex distribution is equal, and children tend to be younger. introduction of a nephritogenic strain into a family often results in the occurrence of several cases within that family, and in some cases, attack rates of up to % have been described ( ) . the incidence is linked to poor socioeconomic conditions. renal involvement in apsgn can be mild, and in many patients, the disease may not be manifested clinically. studies of epidemics with nephritogenic strains of streptococci have shown that up to % of those infected had subclinical evidence of renal disease ( , ) . in a typical case a sudden onset of facial or generalized edema occurs. hypertension is usually modest but is severe in % of cases, and occasionally may lead to encephalopathy or left ventricular failure. the urine is smoky or tea colored in - % of cases. pallor, headache, backache, lethargy, malaise, anorexia, and weakness are all common nonspecific features. the urine volume is decreased. proteinuria is present (up to mg/dl), and microscopy shows white cells, red cells, and granular and hyaline casts. urea, electrolyte, and creatinine levels are normal in subclinical cases but show features of acute renal failure in severe cases. it may be possible to culture gas from the skin or the throat in some patients. other evidence of infection with a gas can be obtained through the antistreptolysin-o titer (asot), which is increased in - % of cases. early antibiotic treatment can reduce the proportion of cases with elevated asot to %. anti-deoxyribonuclease b and anti-hyaluronidase testing has been shown to be of more value than asot in confirming group a streptococcal infection in impetigo-associated cases. measurement of anti-m protein antibodies is of more value for epidemiologic purposes than for the diagnosis of individual cases ( ) . decreased c and total hemolytic complement levels are found in % of cases during the first weeks of illness and return to normal after - weeks. penicillin should be given to eradicate the gas organisms. erythromycin, clindamycin, or a first-generation cephalosporin can be given to patients allergic to penicillin. antibiotic treatment probably has no influence on the course of renal disease but will prevent the spread of a nephritogenic strain ( ) . close contacts and family members who are culture-positive for gas should also be given penicillin, although antibiotic treatment is not always effective in eliminating secondary cases. recurrent episodes are rare, and immunity to the particular nephritogenic strain that caused the disease is probably lifelong. antibiotic prophylaxis is therefore unnecessary. most studies suggest that the prognosis for children with apsgn is good, with more than % making a complete recovery. however, % of cases may have a prolonged and more serious course with long-term chronic renal failure ( ) . other streptococci. apsgn has also been described after outbreaks of group c streptococcus infection ( ) . this has occurred after consumption of unpasteurized milk from cattle with mastitis. patients developed pharyngitis followed by apsgn. endostreptosin was found in the cytoplasm of these group c strains, and during the course of the illness, patients developed anti-endostreptosin antibodies. this antigen has been postulated to be the nephritogenic component of gas. in addition, strains of group g streptococci have been implicated in occasional cases of apsgn ( ) . isolates possessed the type m protein antigen identical to the nephritogenic type m antigen of some group a streptococcal strains. streptococcal toxic shock syndrome and invasive group a streptococcal infection. since , there have been several reports of an illness with many similarities to the staphylococcal toxic shock syndrome, occurring in both children ( ) and adults, associated with invasive group a streptococcal disease ( , , ) . patients with this syndrome present acutely with high fever, erythematous rash, mucous membrane involvement, hypotension, and multiorgan failure. unlike staphylococcal toxic shock syndrome, in which the focus of infection is usually trivial and bacteremia is seldom seen, the streptococcal toxic shock syndrome is usually associated with bacteremia or a serious focus of infection such as septic arthritis, myositis, or osteomyelitis ( , ) . laboratory findings of anemia, neutrophil leukocytosis, thrombocytopenia, and dic are often present, together with impaired renal function, hepatic derangement, and acidosis. acute renal failure requiring dialysis occurs in a significant proportion of cases. it is not clear why there are increasing numbers of cases with invasive disease caused by gas, nor why there has been an emergence of streptococcal toxic shock syndrome, and indeed a similar syndrome caused by some pseudomonas and klebsiella strains. the most common antecedent of invasive gas disease is varicella infection, with the streptococcal infection developing after the initial vesicular phase of the disease is subsiding. strains causing toxic shock syndrome and invasive disease appear to differ from common isolates of gas in producing large amounts of pyrogenic toxins that may have superantigenlike activity. another important mechanism is the production by invasive gas of an il protease. il serves as a molecular bridge between receptors on neutrophils and the vascular endothelium. cleavage of this protein prevents neutrophil attachment to the endothelium, and results in uncontrolled spread of the bacteria through the tissues ( ) . in severe cases necrotizing fasciitis occurs with extensive destruction of the subcutaneous tissues, and is often associated with multiorgan failure. the pathophysiology of streptococcal toxic shock syndrome and that of invasive disease is similar in that superantigen toxins that induce release of cytokines and other inflammatory mediators play a role in both conditions. however gas toxic shock is usually more severe, carries a higher mortality, and is more often associated with focal collections or necrotizing fasciitis. treatment of streptococcal toxic shock syndrome depends on the administration of appropriate antibiotics, aggressive circulatory support, and treatment of any multiorgan failure. surgical intervention to drain the infective focus in muscle, bone, joint, or body cavities is often required. antibiotic therapy with beta-lactams should be supplemented by treatment with a protein synthesisinhibiting antibiotic, such as clindamycin, and it is suggested that this limits new toxin production ( , ) . a number of new therapies are in development. firstly, pooled intravenous immunoglobulins are now in widespread use in the treatment of toxic shock, particularly when caused by streptococcus ( , ) . the role of steroids remains unclear, with their hemodynamic benefit set against the detrimental effects of hyperglycemia secondary to gluconeogenesis. ( ) . the benefit of insulin therapy to control hyperglycemia is unclear. a recent study in adults found that intensive insulin therapy increased the risk of serious adverse events ( ) . in contrast to adult patients, in children with severe sepsis, the use of activated protein c (drotrecogin) cannot be recommended, as in a multicenter trial, fatality was increased in the treatment group ( ) . recovery of renal function occurs in patients who respond to treatment of shock and the eradication of the infection. leptospirosis is an acute generalized infectious disease caused by spirochetes of the genus leptospira ( ) . it is primarily a disease of wild and domestic animals, and humans are infected only occasionally through contact with animals. most human cases occur in summer or autumn and are associated with exposure to leptospiracontaminated water or soil during recreational activities such as swimming or camping. in adolescents and adults, occupational exposure through farming or other contact with animals is the route of infection. the spirochete penetrates intact mucous membranes or abraded skin and disseminates to all parts of the body, including the cerebrospinal fluid (csf). although leptospires do not contain classic endotoxins, the pathophysiology of the disorder has many similarities to that of endotoxemia. in severe cases, jaundice occurs because of hepatocellular dysfunction and cholestasis. renal functional abnormalities may be profound and out of proportion to the histologic changes in the kidney ( ) . renal involvement is predominantly a result of tubular damage, and spirochetes are commonly seen in the tubular lesions. the inflammatory changes in the kidney may result from either a direct toxic effect of the organism or immunecomplex nephritis. however, hypovolemia, hypotension, and reduced cardiac output caused by myocarditis may contribute to the development of renal failure. in severe cases, a hemorrhagic disorder caused by widespread vasculitis and capillary injury also occurs ( , ) . the clinical manifestations of leptospirosis are variable. of affected patients, % have the milder anicteric form of the disorder, and only - % have severe leptospirosis with jaundice. the illness may follow a biphasic course. after an incubation period of - days, a nonspecific flu-like illness lasting - days occurs, associated with septicemic spread of the spirochete. the fever then subsides, only to recur for the second, ''immune,'' phase of the illness. during this phase, the fever is low grade and there may be headache and delirium caused by meningeal involvement, as well as intense muscular aching. nausea and vomiting are common. examination usually reveals conjunctival suffusion, erythematous rash, lymphadenopathy, and meningism. the severe form of the disease (weil's disease) presents with fever, impaired renal and hepatic function, hemorrhage, vascular collapse, and altered consciousness. in one series the most common organs involved were the liver ( %) and kidney ( %). cardiovascular ( %), pulmonary ( %), neurologic ( %), and hematologic ( %) involvements were less common ( ) . vasculitis, thrombocytopenia, and uremia are considered important factors in the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis ( ) . urinalysis results are abnormal during the leptospiremic phase with proteinuria, hematuria, and casts. uremia usually appears in the second week, and acute renal failure may develop once cardiovascular collapse and dic are present ( ) . the clinical features of leptospirosis overlap with those of several other acute infectious diseases, including rocky mountain spotted fever, toxic shock syndrome, and streptococcal sepsis. the diagnosis of leptospirosis should be considered in febrile patients with evidence of renal, hepatic, and mucous membrane changes and rash, particularly if a history of exposure to fresh water is found. diagnosis can be confirmed by isolation of the spirochetes from blood or csf in the first days of the illness or from urine in the second week ( ) . the organism may be seen in biopsy specimens of the kidney or skin or in the csf by dark-field microscopy or silver staining. serologic tests to detect leptospirosis are now sensitive and considerably aid the diagnosis. immunoglobulin m (igm) antibody may be detected as early as - days into the illness, and antibody titers rise progressively over the next - weeks. some patients remain seronegative, and negative serologic test results do not completely exclude the diagnosis. in one series levels of igm and igg anticardiolipin concentrations were significantly increased in leptospirosis patients with acute renal failure ( ) . leptospirosis is treated with intravenous penicillin or other beta-lactam antibiotics. the severity of leptospirosis is reduced by antibiotic treatment, even if started late in the course of the illness ( ) . supportive treatment with volume replacement to correct hypovolemia, administration of inotropes, and correction of coagulopathy is essential in severe cases. dialysis may be required in severe cases and may be needed for prolonged periods until recovery occurs. infection with s. pneumoniae is one of the most common infections in humans and causes a wide spectrum of disease, including pneumonia, otitis media, sinusitis, septicemia, and meningitis. despite the prevalence of the organism, significant renal involvement is relatively rare but is seen in two situations: pneumococcal septicemia in asplenic individuals or in those with other immune deficiencies presents with fulminant septic shock in which renal failure may occur as part of a multisystem derangement. the mortality from pneumococcal sepsis in asplenic patients is high, even with early antibiotic treatment and intensive support. the second nephrologi syndrome associated with s. pneumoniae is a rare form of hus. in , gasser and colleagues described hus as a clinical entity in children, and they included two infants with pneumonia among the five patients they described ( ) . hus associated with pneumococcal infection is induced by the enzyme neuraminidase released from s. pneumoniae ( , ) . thomsen-friedenrich antigen (t antigen) is present on the surface of red blood cells, platelets, and glomerular capillary endothelia against which antibodies are present in normal serum. neuraminidase causes desialation of red blood cells, and possibly other blood cells and endothelium, by the removal of terminal neuraminic acid, which leads to unmasking of the t antigen. the resultant widespread agglutination of blood cells causes intravascular obstruction, hemolysis, thrombocytopenia, and renal failure. results of the direct coombs test are frequently positive, either from bound anti-t igm or from anti-t antibodies. the diagnosis of thomsen-friedenrich antibody-induced hus should be suspected in patients with acute renal failure, thrombocytopenia and hemolysis after an episode of pneumonia or bacteremia caused by s. pneumoniae. fragmented red blood cells will usually be present on blood film. association with s. pneumoniae is defined by culture of pneumococci from a normally sterile site within a week before or after onset of signs of hus. clues to a pneumococcal cause, in addition to culture results, include severe clinical disease, especially pneumonia, empyema, pleural effusion, or meningitis; hemolytic anemia without a reticulocyte response; positive results on a direct coombs test; and difficulties in abo crossmatching or a positive minor crossmatch incompatibility ( ) . however, when renal disease is seen in the context of severe pneumococcal infection, it is important to maintain a broad diagnostic perspective, because the occurrence of acute tubular necrosis due to septic shock and dic is well described ( , ) . therapy for this syndrome should be with supportive treatment and antibiotics (usually a third generation cephalosporin); dialysis may be required if renal failure occurs. because normal serum contains antibodies against the thomsen-friedenrich antigen, blood transfusion should be undertaken with washed red blood cells resuspended in albumin rather than plasma ( , ) . exchange transfusion and plasmapheresis have been used in some patients, with the rationale that these procedures may improve outcome by eliminating circulating neuraminidase ( , , ) . intravenous igg has been used in a patient and was shown to neutralize neuraminidase present in the patient's serum ( ) . in comparison to patients with the more common diarrhea-associated hus, s. pneumoniae-induced hus patients have a more severe renal disease. they are more likely to require dialysis. their long-term outcome maybe affected by the severity of the invasive streptococcal disease itself, and a significant proportion of surviving patients ( - %) develop end-stage renal failure ( , ) . a recent review of uk cases found an eightfold increase in early mortality as compared to diarrhoea-induced hus ( ) . gastrointestinal infections (escherichia coli, salmonella, campylobacter, yersinia, shigella, vibrio cholerae) the diarrheal diseases caused by escherichia coli, salmonella, shigella, campylobacter, vibrios, and yersinia remain important and common bacterial infections of humans. although improvements in hygiene and living conditions have reduced the incidence of bacterial gastroenteritis in developed countries, these infections remain common in underdeveloped areas of the world, and outbreaks and epidemics continue to occur in both developed and underdeveloped countries. renal involvement in the enteric infections may result from any of four possible mechanisms. regardless of the causative organism, diarrhea results in hypovolemia, abnormalities of plasma electrolyte composition, and renal underperfusion. if severe dehydration occurs and is persistent, oliguria from prerenal failure is followed by vasomotor nephropathy and established renal failure. e. coli, shigella, and salmonella (particularly salmonella typhi) may invade the bloodstream and induce septicemia or septic shock. acute renal failure is commonly seen in infants with e. coli sepsis but is also reported with klebsiella, salmonella, and shigella infections. its pathophysiology and treatment were discussed previously. enteric infections with e. coli, yersinia, campylobacter, and salmonella have been associated with several different forms of gn, including membranoproliferative gn (mpgn), interstitial nephritis, diffuse proliferative gn, and iga nephropathy ( ) ( ) ( ) . in typhoid fever, gn ranging from mild asymptomatic proteinuria and hematuria to acute renal failure may occur ( , ( ) ( ) ( ) . renal biopsy findings show focal proliferation of mesangial cells, hypertrophy of endothelial cells, and congested capillary lumina. immunofluorescent studies show igm, igg, and c deposition in the glomeruli, with salmonella antigens detected within the granular deposits in the mesangial areas. in the iga nephropathy after typhoid fever, salmonella vi antigens have been demonstrated within the glomeruli. yersinia infection has been reported as a precipitant of gn in several studies ( , ) . transient proteinuria and hematuria are found in % of patients with acute yersinia infection, and elevated creatinine levels in %. renal biopsy reveals mild mesangial gn or iga nephropathy. yersinia antigens, immunoglobulin, and complement have been detected in the glomeruli. yersinia pseudotuberculosis is well recognized as one of the causes of acute tubulointerstitial nephritis causing acute renal failure, especially in children; patients have histories of drinking untreated water in endemic areas ( ) ( ) ( ) . the illness begins with the sudden onset of high fever, skin rash, and gi symptoms. later in the course, periungual desquamation develops, mimicking kawasaki disease. elevated erythrocyte sedimentation rate, c-reactive protein level, and thrombocytosis are noticeable, and mild degrees of proteinuria, glycosuria, and sterile pyuria are common. acute renal failure, which typically develops - weeks after the onset of fever, follows a benign course with complete recovery. renal biopsy mainly reveals findings of acute tubulointerstitial nephritis. antibiotic therapy, although recommended, does not alter the clinical course, but reduces the fecal excretion of the organism ( , ) . hus is characterized by three distinct clinical signs: acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. it was first described in and was associated with infection by shiga toxin-producing shigella dysenteriae. a major breakthrough in the search for the cause of hus occurred in the s when karmali et al. reported that of children with diarrheaassociated hus had evidence of infection with a strain of e. coli that produced a toxin active on vero cells ( ) . in diarrhea-associated hus in the united states and most of europe, e. coli :h is the most important of these strains. e. coli :h occurs naturally in the gi tract of cattle and other animals, and humans become infected through contaminated food products. most outbreaks have been associated with consumption of undercooked meat, but unpasteurized milk and cider, drinking water, and poorly chlorinated water for recreational use have also been implicated as vehicles for bacterial spread. hus is discussed in detail in chapter . the global epidemic of mycobacterium tuberculosis is growing. several factors have contributed to this increase, including the emergence of the human immunodeficiency virus (hiv) infection epidemic, large influxes of immigrants from countries in which tuberculosis (tb) is common, the emergence of multiple-drug-resistant m. tuberculosis, and breakdown of the health services for effective control of tb in various countries. it is generally estimated that, overall, one-third of the world's population is currently infected with the tb bacillus. there are more than million cases of tb, which result in the death of approximately million people each year. furthermore, - % of people who are infected with the tb bacillus develop tb disease or become infectious at some time during their lives ( , ) . after respiratory illness in children, mycobacteria are widely distributed to many organs of the body during the lymphohematogenous phase of childhood tb ( ) . tubercle bacilli can be recovered from the urine in many cases of miliary tb. hematogenously-spread tuberculomata develop in the glomeruli, which results in caseating, sloughing lesions that discharge bacilli into the tubules. in most cases, the renal lesions are asymptomatic and manifest as mycobacteria in the urine or as sterile pyuria. tuberculomata in the cortex may calcify and cavitate or may rupture into the pelvis, discharging infective organisms into the tubules, urethra, and bladder. dysuria, loin pain, hematuria, and pyuria are the presenting features of this complication, but in many cases, the renal involvement is asymptomatic, even when radiologic and pathologic abnormalities are very extensive. continuing tuberculous bacilluria may cause cystitis with urinary frequency and, in late cases, a contracted bladder ( ) . the intravenous urogram is abnormal in most cases. early findings are pyelonephritis with calyceal blunting and calyceal-interstitial reflux. later, papillary cavities may be seen, indicating papillary necrosis. ureteric strictures, focal calcification, hydronephrosis, and cavitation may also be seen. renal function is usually well preserved, and hypertension is uncommon. in some cases, either the infection itself or reactions to the chemotherapeutic agents may result in renal failure with evidence of an interstitial nephritis ( ) ( ) ( ) . classic symptomatic renal tb is a late and uncommon complication in children, rarely occurring less than or years after the primary infection, and therefore is most commonly diagnosed after adolescence ( , ) . adult studies have shown that - % of renal tb coexists with active pulmonary tb and - % of screened sputumpositive pulmonary tb patients have renal involvement. the diagnosis is established by isolation of mycobacteria from the urine or by the presence of the characteristic clinical and radiographic features in a child with current or previous tb. renal tb is treated with drug regimens similar to those used for other forms of tb, with isoniazid, rifampicin, pyrazinamide and ethambutol administered initially for months, and isoniazid and rifampicin then continued for a further - months. late scarring and urinary obstruction may occur in cases with extensive renal involvement, and such patients should be followed by ultrasonography or intravenous urogram. mycobacteria, both m. tuberculosis and atypical mycobacteria, have also emerged as important causes of opportunistic infection in immunocompromised patients undergoing dialysis and in patients undergoing renal transplantation. the possibility of mycobacterial disease must be considered in patients with fever of unknown origin or unexplained disease in the lungs or other organs. results of the mantoux test are often negative, and diagnosis depends on maintaining a high index of suspicion and isolating the organism from the infected site. renal involvement has been well documented in both congenital and acquired syphilis, with an estimated occurrence of . % in patients with secondary syphilis and up to % in those with congenital syphilis ( , ) . the most common manifestation of renal disease in congenital syphilis is the nephrotic syndrome, with proteinuria, hypoalbuminemia, and edema. in some patients, hematuria, uremia, and hypertension may be seen. the renal disease is usually associated with other manifestations of congenital syphilis, including hepatosplenomegaly, rash, and mucous membrane findings. nephritis in congenital syphilis is usually associated with evidence of complement activation, with depressed levels of clq, c , c , and c . histologic findings are a diffuse proliferative gn or a membranous nephropathy. the interstitium shows a cellular infiltrate of polymorphonuclear and mononuclear cells ( ) . immunofluorescent microscopy reveals diffuse granular deposits of igg and c along the glomerular basement membrane (gbm). mesangial deposits may also contain igm. on electron microscopy, scattered subepithelial electron-dense deposits are seen, with fusion of epithelial cell foot processes ( ) . good evidence exists that renal disease is due to an immunologically mediated reaction to treponemal antigens. antibodies reactive against treponemal antigens can be eluted from the glomerular deposits, and treponemal antigens are present in the immune deposits. treatment of both congenital and acquired syphilis with antibiotics results in rapid improvement in the renal manifestations ( , ) . renal involvement is surprisingly rare in mycoplasma pneumoniae infection considering the prevalence of this organism and its propensity to trigger immunologically mediated diseases such as erythema multiforme, arthritis, and hemolysis. acute nephritis associated with mycoplasma infection may occur - days after the respiratory tract infection ( , ) . renal histopathologic findings include type mpgn, proliferative endocapillary gn, and minimal change disease ( ) . antibiotic treatment of the infection does not appear to affect the renal disease, which is self-limited in most cases ( , ) . since its recognition in , legionnaires' disease, caused by legionella pneumophila, has emerged as an important infectious diseases and the kidney cause of pneumonia. the disease most commonly affects the elderly but has been reported in both normal and immunocompromised children ( , ) . renal dysfunction occurs in a minority of patients ( ) . patients who develop renal impairment present with oliguria and rising urea and creatinine levels. they are usually severely ill, with bilateral pulmonary infiltrates, fever, and leukocytosis. shock may be present, and the renal impairment has been associated with acute rhabdomyolysis with high levels of creatine phosphokinase and myoglobinuria. renal histologic examination usually shows a tubulointerstitial nephritis or acute tubular necrosis ( , ) . the pathogenesis of the renal impairment is uncertain, but the organism has been detected within the kidney on electron microscopy and immunofluorescent studies, which suggests a direct toxic effect. myoglobinuria and decreased perfusion may also be contributing factors, however. mortality has been high in reported cases of legionnaires' disease complicated by renal failure. treatment is based on dialysis, intensive care, and antimicrobial therapy with erythromycin ( ) . steroid therapy may be effective for tubulointerstitial nephritis ( ) . the rickettsial diseases are caused by a family of microorganisms that have characteristics common to both bacteria and viruses and that cause acute febrile illnesses associated with widespread vasculitis. with the exception of q fever, all are associated with erythematous rashes. there are four groups of rickettsial diseases: . the typhus group includes louse-borne and murine typhus, spread by lice and fleas, respectively. . the spotted fever group includes rocky mountain spotted fever, tick typhus and related mediterranean spotted fever and rickettsial pox, which are spread by ticks and mites, with rodents as the natural reservoir. . scrub typhus, which is spread by mites. . q fever, which is spread by inhalation of infected particles from infected animals. rickettsial diseases have a worldwide distribution and vary widely in severity, from self-limited infections to fulminant and often fatal illnesses ( ) . in view of the widespread vasculitis associated with these infections, subclinical renal involvement probably occurs in many of the rickettsial diseases. however, in rocky mountain spotted fever, tick typhus, and q fever, the renal involvement may be an important component of the illness. rocky mountain spotted fever is the most severe of the rickettsial diseases ( , ) . the onset occurs - days after the bite of an infected tick. high fever develops initially, followed by the pathognomonic rash, which occurs between the second and sixth days of the illness. the rash initially consists of small erythematous macules, but later these become maculopapular and petechial, and in untreated patients, confluent hemorrhagic areas may be seen. the rash first appears at the periphery and spreads up the trunk. involvement of the palms and soles is a characteristic feature ( ) . headache, restlessness, meningism, and confusion may occur together with other neurologic signs. cardiac involvement with congestive heart failure and arrhythmia are common. pulmonary involvement occurs in - % of cases. infection is associated with an initial leucopenia, followed by neutrophil leukocytosis. thrombocytopenia occurs in most cases. histopathologically, the predominant lesions are in the vascular system ( ) . rickettsiae multiply in the endothelial cells, which results in focal areas of endothelial cell proliferation, perivascular mononuclear cell infiltration, thrombosis, and leakage of red cells into the tissues. the renal lesions involve both blood vessels and interstitium, and acute tubular necrosis may occur. acute gn with immune-complex deposition has been reported ( ) , but in most cases the pathology appears to be a direct consequence of the invading organism on the renal vasculature ( , ) . renal dysfunction is an important complication of rocky mountain spotted fever. elevation of urea and creatinine levels occurs in a significant proportion of cases, and acidosis is common. prerenal renal failure caused by hypovolemia and impaired cardiac function may respond to volume replacement and inotropic support, but acute renal failure may subsequently occur, necessitating dialysis. rocky mountain spotted fever is diagnosed by the characteristic clinical picture, the exclusion of disorders with similar manifestations (e.g., measles, meningococcal disease, and leptospirosis), and detection of specific antibodies in convalescence. culture of rickettsia rickettsii, immunofluorescent staining, and polymerase chain reaction (pcr) testing of blood and skin biopsy specimens are available only in reference laboratories. antibiotics should be administered in suspected cases without awaiting confirmation of the diagnosis ( ) . doxycycline is the drug of choice for children of any age. chloramphenicol is also effective ( ) . intensive support of shock and multiorgan failure may be required in severe cases, and peritoneal dialysis or hemodialysis may be required until renal function returns. before the advent of specific therapy, mortality was %. today the overall mortality in the united states is still - %. death predominantly occurs in cases in which the diagnosis is delayed. q fever is caused by coxiella burnetii and has a worldwide distribution, with the animal reservoir being cattle, sheep, and goats. human infection follows inhalation of infected particles from the environment. the clinical manifestations range from an acute self-limited febrile illness with atypical pneumonia to involvement of specific organs that causes endocarditis, hepatitis, osteomyelitis, and central nervous system disease ( ) . proliferative gn may be associated with either q fever endocarditis, rhabdomyolysis or a chronic infection elsewhere in the body ( ) . renal manifestations range from asymptomatic proteinuria and hematuria to acute renal failure, hypertension, and nephrotic syndrome. renal histologic findings are those of a diffuse proliferative gn, focal segmental gn, or mesangial gn. immunofluorescent studies reveal diffuse glomerular deposits of igm in the mesangium, together with c and fibrin. c. burnetii antigen has not been identified within the renal lesions. treatment of the underlying infection may result in remission of the renal disease, but prolonged treatment may be required for endocarditis. tetracycline has been used in conjunction with rifampicin, co-trimoxazole, or a fluoroquinolone. nephritis has been reported in association with the presence of a wide range of microorganisms that cause chronic or persistent infection (> table - ) ( , ). it is likely that any infectious agent that releases foreign antigens into the circulation, including those of very low virulence, can cause renal injury either by deposition of foreign antigens in the kidney or by the formation of immune complexes in the circulation, which are then deposited within the kidney. nephritis is most commonly seen in association with intravascular infections such as sbe or infected ventriculoatrial shunts, but it is also seen after focal extravascular infections; ear, nose, and throat infections; and abscesses. renal involvement is one of the diagnostic features of bacterial endocarditis. virtually all organisms that cause endocarditis also produce renal involvement ( > table - ). although endocarditis caused by bacteria is the most common and is readily diagnosed by blood culture ( ), unusual but important causes of culture-negative endocarditis include q fever ( ) and legionella infection ( ) . in the immunocompromised individual, opportunistic pathogens such as fungi and mycobacteria are important causes. the usual renal manifestations of sbe are asymptomatic proteinuria, hematuria, and pyuria. loin pain, hypertension, nephrotic syndrome, and renal failure may occur in more severe cases. the renal lesions occurring in endocarditis are variable, and focal embolic and immune-complex-mediated features may coexist ( , , ) . embolic foci may be evident as areas of infarction, intracapillary thrombosis, or hemorrhage. more commonly, there is a focal necrotizing or diffuse proliferative gn. immunofluorescent studies show glomerular deposits of igg, igm, iga, and c along the gbm and within the mesangium. electron microscopy reveals typical electron-dense deposits along the gbm and within the mesangium ( , , ) . early reports suggested that the renal lesions were caused by microemboli from infected vegetations depositing in the kidney, a hypothesis supported by the occasional presence of bacteria within the renal lesions. most subsequent evidence, however, indicates that immunologic mechanisms rather than emboli are involved in the pathogenesis in most cases: bacteria are rarely found within the kidney, and renal involvement occurs with lesions of the right side of the heart, which would not be likely to embolize to the kidney. immune complexes containing bacterial antigens are present in the circulation, and both bacterial antigens and bacteria-specific antibodies can be demonstrated within the immune deposits in the kidney. serum c level is usually low, and complement can be found within both the circulating and the deposited immune complexes. these features all support an immune-complex-mediated pathogenesis of the renal injury ( , , ) . treatment of the endocarditis with antibiotics usually results in resolution of the gn and is associated with the disappearance of immune complexes from the circulation and return of c levels to normal. the prognosis of the renal lesions in sbe generally depends on the response of the underlying endocarditis to antibiotics or, in cases of antibiotic failure, to surgical removal of the infective vegetations ( ) . in patients previously treated by shunting for hydrocephalus, there is a well-documented association of gn with infected ventriculoatrial shunts. this condition is another example of an immune-complex nephritis similar to that seen in endocarditis ( ) . coagulase-negative staphylococci are the causative organisms in % of cases. the clinical and pathologic findings are similar to those in sbe. presenting features are proteinuria, hematuria, and pyuria, and they may progress to renal failure. immune complexes containing the bacterial antigens and complement are present in the serum, and c is depressed. histologic findings are those of a diffuse mesangiocapillary gn. immunofluorescent microscopy demonstrates deposits of immunoglobulin and c along the gbm, and bacterial antigen can be demonstrated in the renal lesions ( ) . the prognosis for the renal lesion is good if the infection is treated early. this usually involves removal of the infected shunt and administration of appropriate antibiotics ( , ) . the possible progression to end-stage renal disease requires frequent nephrologic monitoring of patients with ventriculoatrial shunts ( ) . there are a few reports in the literature of a similar renal complication occurring in chronic infection of ventriculoperitoneal shunts. gn has been reported after chronic abscesses ( ), osteomyelitis, otitis media, pneumonia, and other focal infections ( > table - ). acute renal failure has been the presenting feature of focal infections in various sites, including the lung, pleura, abdominal cavity, sinuses, and pelvis. many different organisms have been responsible, including s. aureus, pseudomonas, e. coli, and proteus species. this is probably another example of immunecomplex gn. c level is decreased in approximately onethird of reported cases, and immunofluorescent studies reveal diffuse granular deposits of c in the glomeruli of all reported instances, with a variable presence of immunoglobulin. the renal lesion is that of mpgn and crescentic nephritis. the renal outcome is reported to be good with successful early treatment of the underlying infection. the role of viral infections in the causation of renal disease has been less well defined than that of bacterial infections. clearly defined associations of renal disease have been made with hepatitis b virus (hbv), hepatitis c virus (hcv), hiv, and hantaviruses, but the role of most other viruses in the pathogenesis of renal disease is not clearly defined. most viruses causing systemic infection may trigger immunologically mediated renal injury. with increasing application of molecular techniques, it may be that a significant proportion of gns currently considered to be idiopathic will ultimately be shown to be virus induced. in children with immunodeficiency states and those undergoing renal transplantation, viruses such as cytomegalovirus (cmv) and polyoma virus have been recognized to be associated with nephropathy. since the discovery of hepatitis b surface antigen (hbsag) in , hepatitis virus has been shown to infect more than % of the world's population and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. some million people have hbsag in the circulation (who figures). the infection is most common in africa and the orient, where it is acquired in childhood by vertical transmission from infected mothers or by horizontal transmission from other children or adults. in developed countries, transmission in adults occurs more often by blood product exposure, sexual contact, or intravenous drug use. the epidemiology of hbv infection in children is changing following the widespread use of effective vaccination at birth, in both developed and developing countries. hbv is a complex dna virus with an outer surface envelope (hbsag) and an inner nucleocapsid core containing the hepatitis b core antigen (hbcag), dna polymerase, protein kinase activity, and viral dna. incomplete spherical and filamentous viral particles consisting solely of hbsag are the major viral products in the circulation and may be present in concentrations of up to particles per ml of serum. hepatitis b e antigen (hbeag) can be released from hbcag by proteolytic treatment and may be found in the circulation either free or complexed to albumin or igg antibodies. the presence of hbeag correlates with the presence of complete viral particles and the infectivity of the individual ( ) . infection with hbv may result in either a self-limited infectious hepatitis followed by clearance of the virus and complete recovery, or a chronic or persistent infection in which the immune response is ineffective in eliminating the virus. chronic hbv infection with continued presence of viral antigens in the circulation caused by an ineffective host immune response provides the best-documented example of immunologically mediated renal injury caused by persistent infection ( ) . development of chronic hbv infection is positively associated with infection at a younger age, particularly in infancy, where the rate of chronic infection is up to %. in contrast, the likelihood of an acute, symptomatic illness increases with age, to a level where approximately % of infections are symptomatic in children above years. in the early prodromal phase of hbv hepatitis, before the onset of jaundice, some patients develop fever, maculopapular or urticarial rash, and transient arthralgias or arthritis. occasionally, proteinuria, hematuria, or sterile pyuria are observed. the syndrome usually lasts - days and often resolves before the onset of jaundice ( , ) . there have been no histologic studies of the renal changes during this prodromal period. since , numerous reports have linked hbv infection with polyarteritis nodosa (pan). most of these cases have been in adults, but the disorder has also been reported in children ( , ) , where it is estimated that approximately one third of pan cases are caused by hbv ( ) . hbv pan appears to be uncommon in africa and the orient, where infection is usually acquired in childhood, and has declined in incidence following the introduction of hbv vaccination ( ) . hbv pan presents weeks to months after a clinically mild hepatitis but may occasionally predate the hepatitis. after a prodromal illness, frank vasculitis affecting virtually any organ appears. abdominal pain, fever, mononeuritis multiplex, and pulmonary and renal involvement may occur. the renal involvement may appear as hypertension, hematuria, proteinuria, or renal failure (see chapter ) . laboratory investigations reveal a florid acute-phase response, leukocytosis, and anemia. transaminase levels are usually elevated, and hbsag is present in the circulation. the pathology consists of focal inflammation of small and medium-sized arteries, with fibrinoid necrosis, leukocyte infiltration, and fibrin deposition. renal pathology may be limited to the medium-sized arteries or may coexist with gn ( , ) . circulating immune complexes containing hbsag and anti-hbs antibodies are usually present in the circulation ( , ) . c , c , and total hemolytic complement levels are depressed. hbsag, igg, and igm antibodies to hbv and c have been identified by immunofluorescence in the blood vessels ( ) . a positive anca excludes hbv-pan ( ) . although most evidence suggests that the pathogenesis involves an immunecomplex-mediated vasculitis, autoantibody or cell-mediated vascular injury may coexist. if the condition is untreated, the mortality is high ( ) . most studies suggest that steroids or immunosuppressants help to suppress the vasculitis but potentially predispose to chronic infection . ( , ) . successful treatment of hepatitis b-associated pan with nucleoside analogues such as lamivudine or newer anti-viral drugs, either alone or in combination with interferon-alpha and conventional immunosuppressive therapy, has been reported ( ) ( ) ( ) . hbv is now the major cause of membranous gn (mgn) in children worldwide. the proportion of patients with mgn caused by hbv is directly related to the incidence of hbsag in the population, with - % of all cases of mgn in some african and oriental countries being associated with hbv ( , ) (see chapter ) . hbv mgn usually presents in children aged - . there is a striking male predominance; in the united states, % of patients are males ( ) . the virus is usually acquired by vertical transmission from infected mothers or horizontally from infected family members. unlike adults with hbv mgn, children do not usually have a history of hepatitis or of active liver disease, but liver function test results are generally mildly abnormal. liver biopsy specimens may show minimal abnormalities, chronic persistent hepatitis, or (occasionally) more severe changes ( ) . the renal manifestations are usually of proteinuria, nephrotic syndrome, microscopic hematuria, or (rarely) macroscopic hematuria. hypertension occurs in less than % of cases, and renal insufficiency is rare. hbsag and hbcag are usually present in the circulation, and hbe antigenemia is seen in a high proportion of cases. occasionally, hbsag may be found in the glomeruli but is absent from the circulation. c and c levels are often low, and circulating immune complexes are found in most cases. immunohistologic study reveals deposits of igg and c and (less commonly) igm and iga in subepithelial, subendothelial, or mesangial tissue. hbv particles may be seen on electron microscopy, and all the major hepatitis b antigens, including hbsag, hbcag, and hbeag, have been localized in the glomerular capillary wall on immunofluorescence. immunologic deposition of hbv and antibody in the glomerular capillary wall is clearly involved in the glomerular injury, but the underlying immunologic events are incompletely understood ( , ) . passive trapping of circulating immune complexes may be involved, but the circulating immune complexes containing hbsag are usually larger than would be expected to penetrate the basement membrane. hbsag and hbcag are anionic and are therefore unlikely to penetrate the glomerular capillary wall. in contrast, hbeag forms smaller complexes with anti-hbe antibodies and may readily penetrate the gbm. this may explain the observation that hbeag in the circulation frequently correlates with the severity of the disease ( ) . an alternative mechanism for immunemediated glomerular injury is the trapping of hbv antigens by antibody previously deposited in the kidney. anti-hbe antibodies are cationic and may readily localize in the glomerulus and subsequently bind circulating antigen and complement. the third possibility is that the depositions of hbv and antibodies are consequences of glomerular injury by cellular mechanisms or autoantibodies. little evidence supports this view at present ( ) . a transgenic mouse model of hbv-associated nephropathy has been developed, in which hbsag and hbcag is expressed in liver and kidney, particularly tubular epithelial cells, without viral replication. in these mice, gene expression analysis revealed upregulation of acute-phase proteins, particularly c , although measurable serum c levels were reduced. this supports the notion that local persistent expression of hbv viral proteins contributes to hbv-associated nephropathy ( ) . hbv infection has been associated with a variety of other forms of gn in both adults and children. in one small series in children, mpgn was found to be equal in incidence to mgn in the spectrum of hbv-associated gns ( ) . both mpgn and mesangial proliferative gn may be triggered by hbv. in several countries where hbv is common, the proportion of patients with these forms of nephritides who test positive for hbv greatly exceeds the incidence of positivity in the general population ( ) . as with mgn and hbv-associated pan, circulating immune complexes and localization of hbv antigens in the glomeruli have been reported in both mpgn and mesangial proliferative gn, and it is likely that similar mechanisms are occurring ( , ) . several other forms of gn have been associated with hbv, including iga nephropathy, focal glomerulosclerosis, crescentic nephritis, and systemic lupus erythematosus, but the evidence for these associations is less consistent than for the entities discussed earlier ( ) . hbv is normally cleared as a result of cell-mediated responses in which cytotoxic t cells and natural killer cells eliminate infected hepatocytes. it is not surprising, therefore, that the administration of steroids and immunosuppressive agents either may have no effect on hbv disease or may increase the risk of progressive disease ( ) . children with hbv mgn have a good prognosis, and two-thirds undergo spontaneous remission within years of diagnosis. steroid therapy does not appear to provide any additional benefit ( , , ) . antiviral therapy with pegylated interferon-alpha and lamivudine shows promise in facilitating clearance of hbv, and in some cases, elimination of the infection with antiviral therapy in both children and adults is associated with improvement or resolution of the coexisting renal disease. there is considerable effort being put into the development of newer anti-viral agents which avoid the common problems of resistance associated with lamivudine ( - ). hcv is an enveloped, single-stranded rna virus of approximately . kb in the flaviviridae family. there are six major hcv genotypes. hepatitis c is a common disease affecting approximately million people worldwide. in the united states, . million persons are estimated to be anti-hcv positive, and . million may be chronically infected ( ) . an estimated , children in the united states have antibody to hcv and , - , are chronically infected ( ) . children become infected through receipt of contaminated blood products or through vertical transmission. the risk of vertical transmission increases with higher maternal viremia and maternal co-infection with hiv. acute hcv infection is rarely recognized in children outside of special circumstances such as a known exposure from an hcv-infected mother or after blood transfusion. most chronically infected children are asymptomatic and have normal or only mildly abnormal alanine aminotransferase levels. although the natural history of hcv infection during childhood seems benign in the majority of instances, the infection can take an aggressive course in a proportion of children, leading to cirrhosis and end-stage liver disease during childhood. the factors responsible for this more aggressive course are unidentified ( ) . even in adults, the natural history of hcv infection has a variable course, but a significant proportion of patients will develop some degree of liver dysfunction, and - % will eventually have end-stage liver disease as a result of cirrhosis. the risk of hepatocellular carcinoma is significant for those who have established cirrhosis. hepatitis c is currently the most common condition leading to liver transplantation in adults in the ''western world.'' gn has been described as an important complication of chronic infection with hcv in adults. the clinical presentation is usually of nephrotic syndrome or proteinuria, hypertension, or hematuria, with or without azotemia ( ) . mpgn, with or without cryoglobulinemia, and mgn are most commonly described. isolated case reports of other, more unusual patterns of glomerular injury, including iga nephropathy, focal segmental glomerulosclerosis, crescentic gn, fibrillary gn, and thrombotic microangiopathy, have also been associated with hcv infection ( , ) . glomerular deposition of hepatitis antigens and antibodies has been described and is believed to play a role in pathogenesis. cryoglobulinemia is a common accompaniment of gn that is associated with the depression of serum complement levels ( ) . renal failure may develop in - % of patients who have mpgn ( , ) . the presence of virus-like particles as well as viral rna within the kidney sections of patients with hcv-associated glomerulopathies has been reported ( ) . the diagnosis should be suspected if glomerular disease is associated with chronic hepatitis, particularly with the presence of cryoglobulins, but renal biopsy is necessary to establish a definitive diagnosis. hcv infection is relatively common in children with end-stage renal disease and is an important cause of liver disease in this population. acquisition of hcv infection continues to occur in dialysis patients because of nosocomial spread ( ) . elevation of transaminase level is not a sensitive marker of infection in children and hcv enzymelinked immunosorbent assay or pcr testing should be used to increase sensitivity ( ) . hcv-infected renal transplant recipients had higher mortality and hospitalization rates than other transplant recipients ( ) , and hcv infection has been reported to be associated with de novo immune-mediated gn, especially type mpgn, in renal allografts, resulting in accelerated loss of graft function ( , ) . no large randomized, controlled trials of treatment of children with chronic hepatitis c have been performed, although one study (peds-c) is currently recruiting patients into a trial of pegylated interferon +/À ribavirin ( ) . small heterogeneous studies of interferon monotherapy have reported sustained virologic response rates of - % ( ) . in adults, improvement of proteinuria and renal function often follows interferon-alpha treatment ( , ) , but relapses are common after cessation of treatment. combination of interferon with ribavirin in infectious diseases and the kidney patients with chronic liver disease has been shown to increase the rate of sustained response in these patients ( ) . as yet, however, there are few data regarding the use of combination therapy with interferon and ribavirin in children. moreover, interferon-alpha therapy is associated with acute or subacute renal failure in more than one-third of the patients with renal transplants ( ) . hepatitis c may be complicated by systemic mixed cryoglobulinemic (mc) vasculitis, and in some cases by a polyarteritis nodosa (pan)-type non-cryoglobulinemic vasculitis ( ) . treatment with interferon-a (ifn-a) and ribavirin mostly is associated with an improvement of vasculitic symptoms. in some cases, exacerbation and rarely new onset of vasculitis of the peripheral nervous system have been described after this treatment. in fulminant cases immunosuppressive therapy with steroids, and cyclophosphamide, or rituximab may be needed to control life threatening vasculitis prior to antiviral treatment ( ) . cytomegalovirus cmv is one of the eight human herpes viruses. transmission of the virus requires exposure to infected body fluids such as breast milk, saliva, urine, or blood. individuals initially infected with cmv may be asymptomatic or display nonspecific flu-like symptoms. after the initial infection cmv, like all herpes viruses, establishes latency for life but will be periodically excreted by an asymptomatic host. cmv replicates within renal cells, and on biopsy samples from immunocompromised hosts, viral inclusions can be visualized by light microscopy in cells of the convoluted tubules and collecting ducts ( ) . glomerular cells and shed renal tubular cells may have characteristic inclusions, but clinically evident renal disease is rare and is seen virtually only in immunocompromised or congenitally infected children ( , ) . the clinical manifestations of cmv-induced renal disease in congenitally infected infants are variable and range from asymptomatic proteinuria to nephrotic syndrome and renal impairment. in congenital cmv infection, histologic changes of viral inclusions commonly occur in the tubules. in addition, proliferative gn has been reported, with evidence on electron microscopy of viral immune deposits in glomerular cells ( , ) . in cmv-infected immunocompromised patients, immunecomplex gn has been documented with mesangial deposits of igg, iga, c , and cmv antigens within glomeruli. eluted glomerular immunoglobulins have been shown to contain cmv antigens ( ) . cmv is the most common viral infection after kidney transplantation. experience with pediatric kidney transplant recipients suggests a % incidence of cmv infection ( ) . the direct and indirect effects of cmv infection result in significant morbidity and mortality among kidney transplant recipients. cmv-negative patients who receive a cmv-positive allograft are at risk for primary infection and graft dysfunction. patients who are cmv seropositive at the time of transplantation are also at risk of reactivation and superinfection. tubulointerstitial nephritis is a well-characterized pathologic feature of renal allograft cmv disease, which can be difficult to distinguish from injury caused by rejection. histologic evidence of endothelial cell injury and mononuclear cell infiltration in the glomeruli has been reported ( ) . cmv glomerular vasculopathy in the absence of tubulointerstitial disease, causing renal allograft dysfunction, has also been reported ( ) . beyond the acute allograft nephropathy associated with cmv viremia, cmv is known to cause chronic vascular injury. this may adversely affect the long-term outcome of the allograft and may be the explanation for the observed association with chronic allograft nephropathy ( ) . newer techniques for rapidly diagnosing cmv infection are becoming widely available and include shell vial culture, pp antigenemia assay, pcr, and the hybridcapture rna-dna hybridization assay for qualitative detection of cmv pcr. quantitative plasma pcr testing (pcr viral load) is increasingly used for diagnosis and monitoring of cmv viremia in renal transplant recipients. antiviral agents that have been shown to be effective against cmv include ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir remains the drug of choice for treating established disease. intravenous ganciclovir therapy is preferred in children because of the erratic absorption of oral ganciclovir. major limitations of ganciclovir therapy are the induction of renal tubular dysfunction and bone marrow toxicity, principally neutropenia and thrombocytopenia. dosage adjustments are necessary for recipients with renal dysfunction. oral valganciclovir is now used for cmv prophylaxis post-transplant ( ) . use of other antiviral agents such as foscarnet and cidofovir is limited because of nephrotoxicity and difficulty of administration. a number of reports have demonstrated the effectiveness of high-titer cmv immune globulin therapy in reducing severe cmv-associated disease when used in combination with ganciclovir ( , ) . the association of varicella with nephritis has been known for more than years since henoch reported on four children with nephritis that occurred after the appearance of varicella vesicles. varicella, however, is rarely associated with renal complications ( ) . in fatal cases with disseminated varicella and in the immunocompromised individual, renal involvement is more common. cases in which varicella infection caused gn in renal transplant recipients have been reported ( ) . histologic findings in fatal cases include congested hemorrhagic glomeruli, endothelial cell hyperplasia, and tubular necrosis. in mild and nonfatal cases and in non-immunocompromised individuals, varicella is occasionally associated with a variety of renal manifestations, ranging from mild nephritis to nephrotic syndrome and acute renal failure ( ) . histologic findings include endocapillary cell proliferation, epithelial and endothelial cell hyperplasia, and inflammatory cell infiltration ( ) . rapidly progressive nephritis has also been reported. immunohistochemical studies reveal glomerular deposition of igg, igm, iga, and c . on electron microscopy, granular electron-dense deposits have been found in the paramesangial region, and varicella antigens may be deposited in the glomeruli. the features suggest an immune-complex nephritis. elevated circulating levels of igg and iga immune complexes and depressed c and c levels support this possibility ( ) . fulminant disseminated varicella and varicella in immunocompromised patients should be treated with intravenous acyclovir. renal involvement is common during acute infectious mononucleosis, usually manifesting as an abnormal urine sediment, with hematuria in up to % of cases. hematuria, either microscopic or macroscopic, usually appears within the first week of the illness and lasts for a few weeks to a few months. proteinuria is usually absent or low grade. more severe renal involvement with proteinuria, nephrotic syndrome, or acute nephritis with renal failure is much less common. acute renal failure may be seen during the course of fulminant infectious mononucleosis with associated hepatic failure, thrombocytopenia, and encephalitis. it is usually caused by interstitial nephritis that is likely the result of immunopathologic injury precipitated by epstein-barr virus (ebv) infection. however, the identification of ebv dna in the kidney raises the possibility that direct infection might play a role ( ) . the renal involvement must be distinguished from myoglobinuria caused by rhabdomyolysis, which may occur in infectious mononucleosis, and from bleeding into the renal tract as a result of thrombocytopenia. renal histologic findings in ebv nephritis are an interstitial nephritis with mononuclear cell infiltration and foci of tubular necrosis. glomeruli may show varying degrees of mesangial proliferation. on immunohistochemical study, ebv antigens are seen in glomerular and tubular deposits. the prognosis for complete recovery of renal function is good. treatment with corticosteroids may have a role in the management of ebv-induced acute renal failure and may shorten the duration of renal failure ( ) . ebv-associated post-transplantation lymphoproliferative disease is a recognized complication in renal transplant recipients. latent infection of ebv in renal proximal tubular epithelial cells has recently been described as causing idiopathic chronic tubulointerstitial nephritis ( ) . the herpes simplex virus (hsv) causes persistent infection characterized by asymptomatic latent periods interspersed with acute relapses. as with other chronic and persistent infections, immunologically mediated disorders triggered by hsv are well recognized, and it is perhaps surprising that hsv has rarely been linked to nephritis. acute nephritis and nephrotic syndrome have been associated with herpes simplex encephalitis. renal histology shows focal segmental gn with mesangial and segmental deposits of igm, c , and hsv antigens. as with other herpes viruses, hsv has been suggested as a trigger for iga nephritis, mpgn, and membranous nephropathy. elevated levels of hsv antibodies have been reported in patients with a variety of forms of gn, but no conclusive evidence exists of an etiologic role for hsv ( ) . adenovirus and enterovirus, are unrelated ubiquitous pathogens that infect large proportions of the population annually and yet are rarely associated with renal disease. the literature contains scattered reports of acute nephritis after infection with each of these viruses. adenovirus is a major cause of hemorrhagic cystitis and was implicated as the cause of hemorrhagic cystitis in - % of children with this disorder ( ) . boys are affected more often than girls, and hematuria persists for - days. microscopic hematuria, dysuria, and frequency may occur for longer periods. adenovirus types and are the usual strains isolated. picornaviruses, including enteroviruses, echovirus and coxsackieviruses, have been linked with acute nephritis and acute renal failure associated with rhabdomyolysis. coxsackie b virus can be isolated in urine. direct infection of kidney cells is supported by in vitro work demonstrating lytic infection of human podocyte and proximal tubular epithelial cell cultures, although different strains exhibit variable degrees of nephrotropism. renal damage in vivo may have both a direct lytic mechanism and an immune-complex basis ( ) . in the newborn, enteroviruses cause fulminant disease with dic, shock, and liver failure, and acute renal failure may occur. renal involvement from measles virus is uncommon, although measles virus can be cultured from the kidney in fatal cases. an acute gn has been reported to follow measles with evidence of immune deposits containing measles virus antigen within the glomeruli. the nephritis is generally self-limiting ( ) . mild renal involvement is common during the acute phase of mumps infection. one-third of children with mumps have abnormal urinalysis results, with microscopic hematuria or proteinuria. mumps virus may be isolated from urine during the first days of the illness, at a time when urinalysis findings are abnormal. plasma creatinine concentrations usually remain normal, despite the abnormal urine sediment, but more severe cases in adults have been associated with evidence of acute nephritis with impaired renal function. renal biopsy specimens demonstrate an mpgn with deposition of iga, igm, c , and mumps virus antigen in the glomeruli, which suggests an immune-complex-mediated process ( ) . despite the increasing availability of interventions to limit vertical hiv transmission, an estimated , children renal involvement in hiv infection was first described in in adults ( ) ( ) ( ) and in children ( ) , and renal involvement occurs in - % of hiv-infected children in the united states ( ) ( ) ( ) . since the development of highly active antiretroviral therapy (haart), however, the incidence of end-stage renal disease in hiv infection in both adults and children in industrialized countries has declined, but it is predicted that the dramatic decline in aids-related deaths will lead to an ageing population of hiv-infected individuals who will be at risk of non-hiv related renal problems, such that the numbers of hiv-positive esrd patients will increase in the united states ( ) . hiv infection is associated with a number of renal pathologies. hiv-associated nephropathy (hivan) is a syndrome of glomerular and tubular dysfunction, which can progress to end-stage renal failure. it is discussed more fully below. glomerular syndromes other than hivan include mgn that resembles lupus nephritis and immune-complex gn, with iga nephropathy and hcv-associated mpgn being the most common forms. there have also been several case reports of amyloid kidney ( , , ) . the kidneys may be affected by various other mechanisms. opportunistic infections with organisms such as bk virus (bkv) that give rise to nephropathy and hemorrhagic cystitis have been reported in association with hiv infection ( ) . systemic infections accompanied by hypotension can cause prerenal failure leading to acute tubular necrosis. acute tubular necrosis has also been reported in hiv patients after the use of nephrotoxic drugs such as pentamidine, foscarnet, cidofovir, amphotericin b, and aminoglycosides. intratubular obstruction with crystal precipitation can occur with the use of sulfonamides and intravenous acyclovir. indinavir is well recognized to cause nephropathy and renal calculi ( ) . mpgn associated with mixed cryoglobulinemia and thrombotic microangiopathy/atypical-cal hus in association with hiv infections have been reported ( , ) . hivan is characterized by both glomerular and tubular dysfunction, the pathogenesis of which is not entirely known. hivan is a clinico-pathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease ( ) . in adults in the united states, there is a markedly increased risk of nephropathy among african american persons with hiv infection. this appears to be true in children as well, but the data are sparse. the spectrum of hivan seems to be coincident with the degree of aids symptomatology. it is thought that hivan can present at any point in hiv infection, but most patients with hivan have cd counts of less than  / cells/ml, which suggests that it may be primarily a manifestation of late-stage disease ( ) . although a spectrum of clinicopathologic entities including mesangial hyperplasia, focal segmental glomerulosclerosis, minimal change disease, and systemic lupus erythematosus nephritis has been described, the classic pathologic feature of hivan is the collapsing form of focal and segmental glomerulosclerosis ( ) . in the affected glomeruli, visceral epithelial cells are hypertrophied and hyperplastic, and contain large cytoplasmic vacuoles and numerous protein resorption droplets. there is microcystic distortion of tubule segments, which contributes to increasing kidney size. podocyte hyperplasia can become so marked that it causes obliteration of much of the urinary space, forming ''pseudocrescents'' ( ) . capillary walls are wrinkled and collapsed with obliteration of the capillary lumina. the interstitium is edematous with a variable degree of t-cell infiltration ( ) . the bowman capsule can also be dilated and filled with a precipitate of plasma protein that represents the glomerular ultrafiltrate. one of the most distinctive features of hivan, however, is the presence of numerous tubuloreticular inclusions within the cytoplasm of glomerular and peritubular capillary endothelial cells ( ) . immunofluorescence testing is positive for igm and c in capillary walls in a coarsely granular to amorphous pattern in a segmental distribution ( , ) . the presence of the hiv genome in glomerular and tubular epithelium has been demonstrated using complementary dna probes and in situ hybridization. proviral dna has been detected by pcr in the glomeruli, tubules, and interstitium of micro dissected kidneys from patients who had pathologic evidence of hivan, but it has also been detected in the kidneys of hiv-positive patients with other glomerulopathies ( ) . a combination of both proliferation and apoptosis of renal cells may cause the loss of nephron architecture. apoptosis has been demonstrated in cells in the glomerulus, tubules, and interstitium of biopsy specimens from hiv-positive patients with focal segmental glomerulosclerosis. in addition, the role of various cytokines and growth factors, specifically transforming growth factor beta (tgf-beta), in the development of sclerosis has been studied ( , ) . transgenic murine models provide some of the strongest evidence for a direct role of hiv- in the induction of hivan. these mice do not produce infectious virus but express the hiv envelope and regulatory genes at levels sufficient to re-create the hivan that is seen in humans ( ) . serial deletion experiments have concluded that the nef and vpr genes are necessary though not sufficient for hivan pathogenesis. additional factors such as genetic predisposition are thought to explain the fact that african americans have a far greater likelihood of developing hivan than other racial groups, and that hivan is more likely in patients with a family history of esrd. hivan can manifest as mild proteinuria, nephrotic syndrome, renal tubular acidosis, hematuria, and/or acute renal failure ( ) ( ) ( ) ( ) . nephrotic syndrome and chronic renal insufficiency are late manifestations of hivan. children with hivan are likely to develop transient electrolytic disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. early stages of hivan can be identified by the presence of proteinuria and ''urine microcysts'' along with renal sonograms showing enlarged echogenic kidneys. urinary renal tubular epithelial cells are frequently grouped together to form these microcysts, which were found in the urine of children with hivan who had renal tubular injury ( ) . advanced stages of hivan typically present with nephrotic syndrome with edema, heavy proteinuria, hypoalbuminemia, and few red or white blood cells in urinary sediments. hypertension may be present, but usually blood pressure is within or below the normal range. hivan in adults follows a rapidly progressive course, with end-stage renal disease developing within - months, but in children this rapid progression does not necessarily occur. definitive diagnosis of hivan should be based on biopsy results, and biopsy should be performed if significant proteinuria is present, because in approximately % of hiv-infected patients with azotemia and/or proteinuria (> g/ h) who undergo renal biopsy, the specimen will have histologic features consistent with other renal diseases ( ) . when available, haart should be given to children with symptomatic hiv disease. specific treatment of hivan remains controversial. several studies have looked at the role of haart, angiotensin i-converting enzyme (ace) inhibitors, steroids, and even cyclosporin with somewhat encouraging results. however, as yet no randomized case-controlled trials have been undertaken. most of the studies have been small and retrospective, and many have included patients both with and without renal biopsy-proven hivan. cyclosporin has been used to treat hivan in children with remission of nephrotic infectious diseases and the kidney syndrome ( ) . similar responses have been reported to treatment with corticosteroids in various studies ( ) ( ) ( ) ( ) . ace inhibitors have been used with encouraging results ( ) . the general regimen used to treat patients with hiv, including haart, should be applied to children with hivan. the dosages of some medications must be adjusted to the patients glomerular filtration. there are reports of spontaneous regression of hivan with supportive management and treatment with haart, particularly with regimes containing protease inhibitors ( ) ( ) ( ) ( ) . it should be emphasized that the improvement reported with other modalities of treatment such as corticosteroids, cyclosporin, and ace inhibitors always occurs when these agents are given in conjunction with antiretroviral therapy. the kidneys of transgenic mice have been found to have elevated levels of tgf-beta messenger rna and protein ( ) . furthermore, gene expression analysis on tubular epithelial cells from a patient with hivan found upregulation of several inflammatory mediator genes downstream of interleukin and of the transcription factor nfkb ( ) . several other therapeutic options have been suggested, aimed specifically at the presumed role of tgfbeta in the pathogenesis of hivan. treatment directed at its synthesis using gene therapy to block tgf-beta gene expression is being explored. therapy directed at decreasing the activity of tgf-beta using anti-tgf-beta antibodies or other inhibitory substances is also an area of investigation. in addition, blocking renal receptors for chemokines such as rantes (regulated upon derivation, normal t cell expressed and secreted), interleukin- , and monocyte-chemoattractant protein- has been proposed as another possible treatment alternative ( ) . in the haart era, the outlook for hiv patients with esrd has improved, but these patients fare worse than esrd patients without hiv ( ) . most reports of hivinfected patients on hemodialysis have shown poor prognosis, with mean patient survival times ranging from - months. mortality is therefore still close to % within the first year of dialysis. in general, improved survival is associated with younger age at initiation of hemodialysis and with higher cd counts. access complications such as infection and thrombosis tend to occur at a higher rate in hiv-infected hemodialysis patients. cross infection with hiv in dialysis patients is very rare. no patient-topatient hiv transmission has yet been reported in a hemodialysis unit in the united states, although several such cases have occurred in south america ( , ) . peritoneal dialysis is an alternative for hiv-infected patients. the incidence of peritonitis varies across studies, but some studies did report a higher incidence of pseudomonas and fungal peritonitis in the hiv-positive population ( ) . infections with unusual organisms such as pasteurella multocida, trichosporon beigelii, and mycobacterium avium intracellulare complex have also been reported. several studies, however, have suggested that there is no significant difference between the hiv-infected and non-hiv-infected populations. of note is that virus capable of replication in vitro has been recovered from the peritoneal dialysis effluent, and it can be recoverable for up to days in dialysis bags at room temperature and for up to h in dry exchange tubing ( ) . previously, long-term dialysis had been thought to be preferable to renal transplantation, primarily because of the concern that the immunosuppressive therapy required after transplantation could promote progression of hiv/ aids. a multicenter prospective study has been addressing these questions ( ) . data so far indicate that the outcome for liver and kidney transplantation is not considerably different from patients without hiv, with good graft persistence, and a low rate of development of opportunistic infections in those with well-controlled hiv and relatively high cd counts ( ) . the human polyoma viruses are members of the papovavirus family and have received increasing attention as pathogens in immunocompromised patients. they are nonenveloped viruses ranging in size from - nm, with a circular, double-stranded dna genome that replicates in the host nucleus. the best-known species in this genus are the bkv, the jc virus (jcv), and the simian virus sv . bkv was first isolated from the urine of a -year-old man who developed ureteral stenosis months after renal transplantation ( ) . the name of the virus refers to the first patients initials, which is also true of jcv. bkv establishes infection in the kidney and the urinary tract, and its activation causes a number of disorders, including nephropathy and hemorrhagic cystitis. bkv-associated nephropathy has become an increasingly recognized cause of renal dysfunction in renal transplantation patients ( ) ( ) ( ) ( ) ( ) . jcv establishes latency mainly in the kidney, and its reactivation can result in the development of progressive multifocal leukoencephalopathy. there are a few reports of nephropathy in association with jcv infection (see references in ( ) ), but bkv poses a much bigger problem in this regard. recent studies have reported sv in the allografts of children who received renal transplants and in the urine, blood, and kidneys of adults with focal segmental glomerulosclerosis, which is a cause of end-stage renal disease and an indication for kidney transplantation ( ) . seroprevalence rates as high as - % have been reported among adults in the united states and europe. the peak incidence of primary infection (as measured by acquisition of antibody) occurs in children - years of age. bkv antibody may be detected in as many as % of children by years of age, and in - % of children by or years of age; antibodies wane thereafter. bkv infection may be particularly important in the pediatric transplantation population, in whom primary infection has a high probability of occurring while the children are immunosuppressed ( ) . primary infection with bkv in healthy children is rarely associated with clinical manifestations. mild pyrexia, malaise, vomiting, respiratory illness, pericarditis, and transient hepatic dysfunction have been reported with primary infection. investigators hypothesize that after an initial round of viral replication at the site of entry, viremia follows with dissemination of the virus to distant sites at which latent infection is established. the most frequently recognized secondary sites of latent infection are renal and uroepithelial cells. secondary infection has been reported to cause tubulointerstitial nephritis and ureteral stenosis in renal transplantation patients. it may be that renal impairment in immunocompromised patients and in non renal solid organ transplant recipients is found to be frequently associated with bkv infection. the reported prevalence of bkv nephropathy in renal allografts is between and % ( , , , , ) . asymptomatic infection is characterized by viral shedding without any apparent clinical features. viruria, resulting from either primary or secondary infection, can persist from several weeks to years. tubulointerstitial nephritis associated with bkv in renal transplant recipients is accompanied by histopathologic changes, with or without functional impairment. ''infection'' and ''disease'' must be differentiated carefully. bkv infection (either primary or reactivated) can progress to bkv disease, but will not always do so ( ) . furthermore, not all cases of bkv disease lead to renal impairment. however, infection can progress to transplant dysfunction and graft loss, although the diagnosis may be complicated by the coexistence of active allograft rejection. bkv nephritis is reported to have a bimodal distribution, with % of bkv-related interstitial nephritis cases occurring - weeks after transplantation and the remainder of patients developing disease months to years after transplantation ( ) . allograft failure is due mainly to extensive viral replication in tubular epithelial cells leading to frank tubular necrosis ( ) . although damage is potentially fully reversible early in the disease, persisting viral damage leads to irreversible interstitial fibrosis. tubular atrophy and allograft loss has been observed in % of affected patients ( , ) . in most cases, bkv nephropathy in adult renal transplant recipients represents a secondary infection associated with rejection and its treatment. in children, however, primary bkv infection giving rise to allograft dysfunction may occur ( ) . the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathologic features include severe tubular injury with cellular enlargement, marked nuclear atypia, epithelial necrosis, denudation of tubular basement membranes, focal intratubular neutrophilic infiltration, and mononuclear interstitial infiltration, with or without concurrent tubulins. this constellation of histologic features, particularly severe tubulitis, is often misinterpreted as rejection, even by the experienced pathologist. the presence of well-demarcated basophilic or amphophilic intranuclear viral inclusions, primarily within the tubular and parietal epithelium of the bowman capsule, can help distinguish bkv disease from rejection ( , , ) . additional tests such as immunohistochemistry, pcr analysis, or electron microscopy of biopsied tissue aimed at the identification of bkv may be required. a practical diagnostic approach for identifying bkv in renal transplant patients is summarized in > table - . bkv infection may cause ureteral obstruction due to ureteral ulceration and stenosis at the ureteric anastomosis. bkv-associated ureteral stenosis has been reported in % of renal transplant patients and usually occurs between and days after transplantation. ulceration due to inflammation, proliferation of the transitional epithelial cells, and smooth muscle proliferation may lead to partial or total obstruction. high-level bkv replication is implicated in acute, late-onset, long-duration hemorrhagic cystitis after bone marrow transplantation ( ) . there are two case reports in children of renal carcinomas arising in the transplanted kidney in association with bk virus nephropathy. it remains unclear whether infectious diseases and the kidney bk virus itself has oncogenic potential in the transplant setting, but this is possible given that the big t antigen (t-ag) expressed by polyomavirus family viruses has been shown to have the ability to disrupt chromosomal integrity ( , ) . whether patients with asymptomatic viremia or viruria need specific therapeutic intervention is not certain. review of the literature suggests that careful reduction of immune suppression, combined with active surveillance for rejection, will result in clinical improvement. reduction in immunosuppression may precipitate episodes of acute cellular rejection, which need to be judiciously treated with corticosteroids. the outcome of bkv nephropathy is unpredictable, and stabilization of renal function may occur regardless of whether maintenance immunotherapy is altered or not ( ) . some reports favor the use of cidofovir. cidofovir has important nephrotoxic side effects in the usual therapeutic dosage recommended for the treatment of cmv infection, and for bkv nephropathy a reduced dosage regime is generally used. the efficacy of cidofovir in reducing viremia has been demonstrated (see review in ( )). however, spontaneous clearance of viral infection after reduction of immunosuppression (without cidofovir) has also been reported. there are also case studies of the use of leflunamide. presence of bkv by pcr or decoy cells in urine signifies bkv replication. decoy cells are caused by infection of the urinary epithelial cells with human polyoma viruses. the nuclei are enlarged and nuclear chromatin is completely homogenized by viral cytopathic effect. positive pcr results for bkv viruria and presence of decoy cells have poor predictive value. specificity is increased if > cells/ cytospin along with presence of inflammatory cells. presence of antibody is usually indicative of previous infection; however, positive results for bkv dna pcr on serum signifies bk viremia. bkv pcr testing of plasma has proven to be a sensitive ( %) and specific ( %) means to identify bkv-associated nephropathy in adults. viral load has also been used to monitor infection and clearance. however, because primary infection occurs in childhood, it might not be applicable to the pediatric population. the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathology might mimic rejection or drug toxicity. however, characteristic findings have been described. electron microscopy and immune staining are helpful in confirming the diagnosis. pcr assays of viral load in tubular cells have been reported to be a sensitive marker for diagnosis and monitoring. viral hemorrhagic fever involves at least distinct rna viruses that share the propensity to cause severe disease with prominent hemorrhagic manifestations ( > table - ) . the viral hemorrhagic fevers, widely distributed throughout both temperate and tropical regions of the world, are important causes of mortality and morbidity in many countries. most viral hemorrhagic fevers are zoonoses (with the possible exception of dengue virus), in which the virus is endemic in animals and human infection is acquired through the bite of an insect vector. aerosol and nosocomial transmissions from infected patients are important for lassa, junin, machupo, and congo-crimean hemorrhagic fevers, and marburg and ebola viruses ( ) . viral hemorrhagic fevers have many clinical similarities but also important differences in their severity, major organs affected, prognosis, and response to treatment. in all viral hemorrhagic fevers, severe cases occur in only a minority of those affected; subclinical infection or nonspecific febrile illness occurs in the majority. fever, myalgia, headache, conjunctival suffusion, and erythematous rash occur in all the viral hemorrhagic fevers ( ) . hemorrhagic manifestations range from petechiae and bleeding from venepuncture sites to severe hemorrhage into the gi tract, kidney, and other organs. a capillary leak syndrome, with evidence of hemoconcentration, pulmonary edema, oliguria, and ultimately shock, occurs in the most severely affected patients ( ) . renal involvement occurs in all the viral hemorrhagic fevers, proteinuria is common, and prerenal failure is seen in all severe cases complicated by shock. however, in congo-crimean hemorrhagic fever and hemorrhagic fever with renal syndrome (hfrs), an interstitial nephritis, which may be hemorrhagic, is characteristic, and renal impairment is a major component of the illness. dengue is caused by a flavivirus that is endemic and epidemic in tropical america, africa, and asia, where the mosquito vector aedes aegypti is present ( ). classic dengue is a self-limited nonfatal disease; dengue hemorrhagic fever and dengue shock syndrome, which occur in a minority of patients, have a high mortality if not aggressively treated with fluids. after an incubation period of - days, the illness begins with fever, headache, arthralgia, weakness, vomiting, and hyperesthesia. in uncomplicated dengue the fever usually lasts - days. shortly after onset a maculopapular rash appears, sparing the palms and the soles, and is occasionally followed by desquamation. fever may reappear at the onset of the rash. in dengue hemorrhagic fever and dengue shock syndrome, the typical febrile illness is complicated by hemorrhagic manifestations, ranging from a positive tourniquet test result or petechiae to purpura, epistaxis, and gi bleeding with thrombocytopenia and evidence of a consumptive coagulopathy. increased capillary permeability is suggested by hemoconcentration, edema, and pleural effusions ( ) . in severe cases, hypotension and shock supervene, largely as a result of hypovolemia. renal manifestations include oliguria, proteinuria, hematuria, and rising urea and creatinine. acute renal failure occurs in patients with severe shock, primarily as a result of renal underperfusion. however, glomerular inflammatory changes may also occur. children with dengue hemorrhagic fever show hypertrophy of endothelial and mesangial cells, mononuclear cell infiltrate, thinning of basement membranes, and deposition of igg, igm, and c . electron microscopy shows viral particles within glomerular mononuclear cells ( ) . the diagnosis of dengue is made by isolation of the virus from blood or by serologic testing. there is no specific antiviral treatment, and management of patients with dengue shock syndrome or dengue hemorrhagic fever depends on aggressive circulatory support and volume replacement with colloid and crystalloid ( , ) . with correction of hypovolemia, renal impairment is usually reversible, but dialysis may be required in patients with established acute renal failure. yellow fever is caused by a flavivirus, and is transmitted by mosquito bites, typically aedes species. it remains an important public health problem in africa and south america. renal manifestations are common and include albuminuria and oliguria. over the next few days after first manifestation of infection, shock, delirium, coma, and renal failure develop, and death occurs - days after onset of symptoms. laboratory findings include thrombocytopenia and evidence of hemoconcentration, rising urea and creatinine levels, hyponatremia, and deranged liver function test results. pathologic findings include necrosis of liver lobules, cloudy swelling and fatty degeneration of the proximal renal tubules, and, often, petechiae in other organs. the oliguria appears to be prerenal and is due to hypovolemia; later, acute tubular necrosis supervenes. at present, there is no effective antiviral agent for yellow fever. . congo-crimean hemorrhagic fever, first recognized in the soviet union, is now an important human disease in eastern europe, asia, and africa ( ) . severely affected patients become stuporous or comatose - days into the illness, with evidence of hepatic and renal failure and shock. proteinuria and hematuria are often present. the disease is fatal in - % of cases. the virus is sensitive to ribavirin, but in one small trial of i.v. ribavirin versus supportive treatment only, there was no significant improvement in outcome in the treatment group ( ) . rift valley fever is found in many areas of sub-saharan africa. in humans, most infections follow mosquito bites or animal exposure. the infection may present as an uncomplicated febrile illness, with muscle aches and . ( ) . clinical entities include korean hemorrhagic fever, nephropathia epidemica in scandinavia, and epidemic hemorrhagic fever in japan and china. in general, hfrs due to hantaan, porogia, and belgrade viruses is more severe and has higher mortality than that due to puumala virus (nephropathia epidemica) or seoul virus. hantaan is predominant in the far east, porogia and belgrade in the balkans, and puumala in western europe; seoul has a worldwide distribution ( ) . the clinical features of the disease vary. the incubation period is - days. although hfrs occurs with the same clinical picture in children as in adults, both incidence rates and antibody prevalence rates are very low in children under years of age. men of working age make up the bulk of clinical cases ( ) . mild cases are indistinguishable from other febrile illnesses. in more severe cases, fever, headache, myalgia, abdominal pain, and dizziness are associated with the development of periorbital edema, proteinuria, and hematuria. there is often conjunctival injection, pharyngeal injection, petechiae, and epistaxis or gi bleeding. the most severely affected patients develop shock and renal failure. the disease usually passes through five phases: febrile, hypotensive, oliguric, diuretic, and convalescent. laboratory findings include anemia, lymphocytosis, thrombocytopenia, prolonged prothrombin and bleeding times, and elevated levels of fibrin degradation products. liver enzyme levels are elevated, and urea and creatinine levels are elevated during the oliguric phase. proteinuria and hematuria are consistent findings. the renal histopathologic findings are those of an interstitial nephritis with prominent hemorrhages in the renal medullary interstitium and renal cortex. acute tubular necrosis may also be seen. immunohistochemical analysis reveals deposition of igg and c , and the gbm, mesangial, and subendothelial deposits may be seen on electron microscopy ( ) . recovery from hantavirus-associated disease is generally complete, although chronic renal insufficiency is a rare sequela of hfrs. in mildly affected patients, the disease is self-limiting and spontaneous recovery occurs. however, in severe cases, with shock, bleeding, and renal failure, dialysis and intensive circulatory support may be required ( ) . mortality rates vary depending on the strain of virus; rates are - % for hemorrhagic fever and renal syndrome in china and significantly lower for the milder finnish form associated with the puumala virus strain. ribavirin is active against hantaan viruses in vitro, and clinical trials indicate that both mortality and morbidity can be reduced by treatment with this antiviral agent if it is administered early in the course of illness. dosages of mg/kg followed by mg/kg every h for days and then mg/kg every h for days have been used ( ) . lassa fever is a common infection in west africa, caused by an arenavirus, and usually manifests as a nonspecific febrile illness. in % of cases, a fulminant hemorrhagic disease occurs. in severe cases, proteinuria and hematuria are usually present, and renal failure may occur. ribavirin is effective in decreasing mortality. as in other hemorrhagic fevers, intensive hemodynamic support and correction of the hemostatic derangements are important components of therapy ( ) . junin and machupo viruses, the agents of argentine and bolivian hemorrhagic fever, respectively, cause hemorrhagic fevers with prominent neurologic features and systemic and hemorrhagic features similar to those of lassa fever. oliguria, shock, and renal failure occur in the most severe cases. marburg and ebola viruses have been associated with outbreaks of nosocomially transmitted hemorrhagic fever. both viruses cause fulminant hemorrhagic fever. onset is with high fever, headache, sore throat, myalgia, and profound prostration. an erythematous rash on the trunk is followed by hemorrhagic conjunctivitis, bleeding, impaired renal function, shock, and respiratory failure. the mortality rate is high. renal histopathologic findings in fatal cases are of tubular necrosis, with fibrin deposition in the glomeruli. there is no specific treatment for these disorders. the important role played by a number of other recently characterized viruses is only now being recognized, as improved molecular diagnostic techniques allow identification of hitherto unrecognized viruses. two examples of recently described viruses are metapneumovirus ( ) and bocavirus ( ) . while both have significant prevalence, and may make an important contribution to the burden of childhood viral infection, as yet there are no reports indicative of significant renal pathology in association with these infections. influenza virus has been linked with nephritis and acute renal failure. an emerging infectious disease is avian flu, caused by highly pathogenic h n strains which have hitherto been confined to an avian reservoir, and there have been several outbreaks of infection in humans, particularly in the first part of this decade. commonly, these patients develop a flu-like illness with prominent respiratory and gastrointestinal symptoms. renal failure may develop alongside multi-organ failure in the context of acute respiratory distress syndrome ( ) . as yet, there is no clear correlation of degree of initial renal insufficiency, and outcome ( ) . there is little data available on treatment, but based on the known resistance patterns of h n strains, oseltamivir and zanamivir are the preferred agents to be used for treatment of infection with h n . severe acute respiratory syndrome (sars) is a newlyemerged infectious disease which was first seen in south china in . it is caused by a sars coronavirus (sars cov). predominantly, it causes a viral pneumonia, with diffuse alveolar damage; it has considerable mortality ( ) . renal effects are not generally significant in the pathophysiology of sars. however, sars cov has been found in kidney tissue at post-mortem ( ) ( ) . sars cov enters cells via angiotensin converting enzyme (ace ) ( ) , and it is thought that the invasion of kidney tissue reflects the virus' tropism for ace , which is expressed on kidney cells. chronic exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. malaria is the best-documented parasitic infection associated with glomerular disease, but other parasitic infections including schistosomiasis, filariasis, leishmaniasis, and possibly helminth infections may also induce nephritis or nephrosis. malaria is estimated to cause up to million clinical cases of illness and more than million deaths each year ( ) . the association of quartan malaria and nephritis has been well known in both temperate and tropical zones since the end of the nineteenth century. epidemiologic studies provide the most conclusive evidence for a role of plasmodium malariae in glomerular disease ( , ) . chronic renal disease was a major cause of morbidity and mortality in british guiana in the s. the frequent occurrence of p. malariae in the blood of these patients led to detailed epidemiologic studies that implicated malaria as a cause of the nephrosis. after the eradication of malaria from british guiana, chronic renal disease ceased to be a major cause of death in that country ( ) . the link between malaria and nephrotic syndrome was strengthened by studies in west africa in the s and s that demonstrated a high prevalence of nephrotic syndrome in the nigerian population ( ) . the pattern of nephrotic syndrome differed from that in temperate climates, with an older peak age, extremely poor prognosis, and unusual histologic features. the incidence of p. malariae parasitemia in patients with the nephrotic syndrome in nigeria was vastly in excess of that occurring in the general population, whereas the incidence of plasmodium falciparum parasitemia was similar to that in the general population. the age distribution of nephrotic syndrome also closely paralleled that of p. malariae infection ( ) . in some affected patients, circulating immune complexes and immunoglobulin, complement, and antigens were present in the glomeruli that were recognized by p. malariae-species antisera. there is now a view that the patterns of childhood renal disease described in the last century may no longer be representative of the current situation. the variable patterns of renal disease throughout africa may no longer reflect a dominant role for ''malarial glomerulopathy,'' and the relative causative role of tropical infections in nephropathy remains an unanswered question ( ) . most patients have poorly selective proteinuria and are unresponsive to treatment with steroids or immunosuppressive agents. the characteristic lesions of p. malariae nephropathy are capillary wall thickening and segmental glomerular sclerosis, which lead to progressive glomerular changes and secondary tubular atrophy ( ) . cellular proliferation is conspicuously absent. electron microscopy shows foot-process fusion, thickening of the basement membrane, and increase in subendothelial basement membrane-like material. immunofluorescent studies show granular deposits of immunoglobulin, complement, and p. malariae antigen in approximately one-third of patients. in addition to the histologic pattern, termed quartan malaria nephropathy, p. malariae infection is associated with a variety of other forms of histologic appearance, including proliferative gn and mgn ( ) . although quartan malaria nephropathy has been clearly linked to p. malariae infection in nigeria, a number of studies from other regions in africa have not revealed the typical histopathologic findings described in the nigerian studies ( ) . furthermore, quartan malaria nephropathy may be seen in children with no evidence of p. malariae infection or deposition of malaria antigens in the kidney. this, together with the fact that antimalarial treatment does not affect the progression of the disorder, raises the possibility that factors other than malaria might be involved in the initiation and perpetuation of the disorder. although there is undoubtedly a strong association between p. malariae infection and nephrotic syndrome on epidemiologic grounds, the direct causal link is not proven. most likely, a number of different infectious processes, including malaria, hepatitis b, schistosomiasis, and perhaps other parasitic infections that cause chronic or persistent infections and often occur concurrently in malaria areas, may all result in glomerular injury and a range of overlapping histopathologic features. the prognosis for the nephrotic syndrome in most african studies has been poor, regardless of whether the histologic findings were typical of quartan malaria nephropathy or whether p. malariae parasitemia was implicated. treatment with steroids and azathioprine is generally ineffective, and a significant proportion of patients progress to renal failure. p. falciparum appears to be much less likely to cause significant glomerular pathology. epidemiologic studies have failed to show a clear association between p. falciparum parasitemia and the nephrotic syndrome. whereas renal failure appears to be a common complication of severe malaria in adults, it seldom occurs in children. renal biopsy specimens from adult patients with acute p. falciparum infections who have proteinuria or hematuria show evidence of glomerular changes, including hypercellularity, thickening of basement membranes, and hyperplasia and hypertrophy of endothelial cells ( ) . electron microscopy reveals electron-dense deposits in the subendothelial and paramesangial areas. deposits of igm, with or without igg, are localized mainly in the mesangial areas. p. falciparum antigens can be demonstrated in the mesangial areas and along the capillary wall, which suggests an immune-complex gn. the changes, generally mild and transient, are probably unrelated to the acute renal failure that may complicate severe p. falciparum infection ( ) . heavily parasitized erythrocytes play a central role in the various pathologic factors ( ) . renal failure occurring in severe p. falciparum malaria is usually associated with acidosis, volume depletion, acute intravascular hemolysis or heavy parasitic infection that leads to acute tubular necrosis. recent studies have confirmed an important role for volume depletion in children with severe falciparum malaria, who characteristically have evidence of tachycardia, tachypnoea, poor perfusion and in severe cases hypotension ( ) . volume expansion with either colloid or crystalloid results in improvement in hemodynamic indices and reduction in acidosis ( ) . there is growing evidence that volume expansion with albumin is associated with a better outcome than saline or synthetic colloids ( , ) . treatment with antimalarials, correction of hypoglycemia and infectious diseases and the kidney electrolyte imbalance, and volume expansion reduces mortality to less than %. although renal failure is usually associated with infection by p. falciparum, acute renal failure has been described with plasmodium vivax infection and mixed infections ( ) . the term blackwater fever refers to the combination of severe hemolysis, hemoglobinuria, and renal failure. it was more common at the start of the twentieth century in nonimmune individuals receiving intermittent quinine therapy for p. falciparum malaria. blackwater fever has become rare since , when quinine was replaced by chloroquine. however, the disease reappeared in the s, after the reuse of quinine because of the development of chloroquine-resistant organisms. since then, several cases have been described after therapy with halofantrine and mefloquine, two new molecules similar to quinine (amino-alcohol family) ( ) . renal failure generally occurred in the context of severe hemolytic anemia, hemoglobinuria, and jaundice. the pathophysiology of the disorder is unclear; however, it appears that a double sensitization of the red blood cells to the p. falciparum and to the amino-alcohols is necessary to provoke the hemolysis. histopathologic findings include swelling and vacuolization of proximal tubules, necrosis and degeneration of more distal tubules, and hemoglobin deposition in the renal tubules. recent studies indicates a better outcome with earlier initiation of intensive care and dialysis combined with necessary changes in antimalarial medications. schistosomiasis affects million people living in endemic areas of asia, africa, and south america ( ) . the infection is usually acquired in childhood, but repeated infections occur throughout life. schistosoma japonicum is found only in the orient, whereas schistosoma haematobium occurs throughout africa, the middle east, and areas of southwest asia. schistosoma mansoni is widespread in africa, south america, and southwest asia. human infection begins when the cercarial forms invade through the skin, develop into schistosomula, and move to the lungs via the lymphatics or blood. they then migrate to the liver and mature in the intrahepatic portal venules, where male:female pairing takes place. the adult worm pairs then migrate to their final resting site -the venules of the mesenteric venous system of the large intestine (s. mansoni) or in the venules of the urinary tract (s. haematobium). the females release large numbers of eggs, which may remain embedded in the tissues, embolize to the liver or lungs, or pass into the feces or urine. clinical manifestations may occur at any stage of the infection. cercarial invasion may cause an intense itchy papular rash. katayama fever is an acute serum sicknesslike illness that occurs several weeks after infection, as eggs are being deposited in the tissues. deposition of the eggs in tissues results in inflammation of the intestines, fibrosis of the liver, and portal hypertension. with s. haematobium, chronic inflammation and fibrosis of the ureters and bladder may lead to obstructive uropathy ( ) . renal manifestations of schistosomiasis occur most commonly in s. mansoni infection. schistosomal nephropathy usually presents with symptoms including granulomatous inflammation in the ureters and bladder, but glomerular disease (probably on an immune-complex basis) may also occur. renal disease usually occurs in older children or young adults with long-term infection, but serious disease may also occur in young children ( ) . the early renal tract manifestations of schistosomiasis are suprapubic discomfort, frequency, dysuria, and terminal hematuria. in more severe cases, evidence of urinary obstruction appears. poor urinary stream, straining on micturition, a feeling of incomplete bladder emptying, and a constant urge to urinate may be severely disabling symptoms. the fibrosis and inflammation of ureters, urethra, and bladder may be followed by calcification and may result in hydroureter, hydronephrosis, and bladder neck obstruction. renal failure may ultimately develop, and there is a suspicion that squamous cell carcinoma of the bladder may be linked to the chronic infective and inflammatory process. secondary bacterial infection is common within the obstructed and inflamed urinary tract ( ) . the hepatosplenic form of s. mansoni infection may be accompanied by a glomerulopathy in - % of cases, manifested in the majority as nephrotic syndrome ( ) . histopathologic findings include mesangioproliferative gn, focal segmental glomerulosclerosis, mesangiocapillary gn, mgn, and focal segmental hyalinosis ( ) . immune complexes may be detected in the circulation of these patients, and glomerular granular deposition of igm, c , and schistosomal antigens are seen on immunofluorescence. usually schistosoma-specific nephropathy is a progressive disease and is not influenced by antiparasitic or immunosuppressive therapy ( ) , but isolated case reports of remission after treatment with praziquantel have been reported ( ) . the diagnosis is confirmed by the detection of schistosoma eggs in feces, urine, or biopsy specimens. eggs are shed into the urine with a diurnal rhythm, and urine collected between am and pm is the most useful. urinary sediment obtained by centrifugation or filtration through a nuclepore membrane should be examined. in cases in which studies of urine and feces yield negative results in patients in whom the diagnosis is suspected, rectal biopsy specimens taken approximately cm from the anus have a high diagnostic yield for both s. mansoni and s. haematobium infection. biopsy of liver or bladder may be required to establish the diagnosis. antibodies indicating previous infection can be detected using enzyme-linked immunosorbent assay or radioimmunoassay. the tests are sensitive but lack specificity and may not differentiate between past exposure and current infection. praziquantel is the drug of choice for treatment of schistosomiasis. a single oral dose of mg/kg is effective in s.haematobium and s. mansoni infection and is usually well tolerated. the alternative drug for s. mansoni infection is oxamniquine. complete remission of urinary symptoms may occur in renal disease of short duration, but in late disease with extensive fibrosis, scarring, and calcification, obstructive uropathy and renal failure may persist after the infection has been eradicated. there are reports of a drastic decrease in the number of severe hepatosplenic forms of s. mansoni infection after mass treatment of the population in endemic areas with oxamniquine. this also reduced schistosomal nephropathy ( ) . visceral leishmaniasis is a chronic protozoon infection characterized by fever, hepatosplenomegaly, anemia, leukopenia, and hyperglobulinemia. proteinuria and/or microscopic hematuria or pyuria have been reported in % of patients with visceral leishmaniasis ( ) . acute renal failure in association with interstitial nephritis has also been reported ( ) . renal histologic analysis in patients with visceral leishmaniasis reveals glomerular changes, with features of a mesangial proliferative gn or a focal proliferative gn, or a generalized interstitial nephritis with interstitial edema, mononuclear cell infiltration, and focal tubular degeneration. immunofluorescence reveals deposition of igg, igm, and c within the glomeruli, as well as electron-dense deposits in the basement membrane and mesangium on electron microscopy ( ) . circulating immune complexes together with immunoglobulin and complement deposition in the glomeruli suggests an immune-complex cause. renal disease in leishmaniasis is usually mild and may resolve after treatment of the infection. renal dysfunction may be associated with treatment for visceral leishmaniasis with antimony compounds. proteinuria is more common in filarial hyperendemic regions of west africa than in nonfilarial areas. renal histologic analysis has shown a variety of different histopathologic appearances; the most common is diffuse mesangial proliferative gn with c deposition in the glomeruli ( ) . renal biopsy specimens also demonstrate large numbers of eosinophils in the glomeruli, and microfilariae may be seen in the lumen of glomerular capillaries. filarial antigens have been detected within immune deposits within the glomeruli. echinococcus granulosus causes chronic cysts within a variety of organs. in addition, nephrotic syndrome in association with hydatid disease has been reported. membranous nephropathy, minimal change lesions, and mesangiocapillary gn have been described in association with hydatid disease ( , ) . immunofluorescence reveals deposits of immunoglobulin, complement, and hydatid antigens within the glomeruli. remission of nephrotic syndrome has been reported with treatment by antiparasitic agents such as albendazole ( , ) . few reports have been published of renal disease occurring in patients with trypanosomiasis. the trypanosomal antigens can induce gn in a variety of experimental animals ( ) . nephrotic syndrome has occasionally been reported as a manifestation of congenital toxoplasmosis. dissemination of previously latent toxoplasma infection in patients undergoing treatment with immunosuppressive drugs has been increasingly recognized in recent years. reactivation of toxoplasmosis or progression of recently acquired primary infection should be considered in patients undergoing renal transplantation or immunotherapy for renal disease who develop unexplained inflammation of any organ. fungal infections of the kidneys and urinary tract occur most commonly as part of systemic fungal infections in patients with underlying immunodeficiency, as focal urinary tract infections in patients with obstructive lesions, or as a result of indwelling catheters. although candida infection is the most common fungal infection in both immunocompromised and non immunocompromised hosts, virtually all other fungal pathogens may invade the renal tract during severe immunocompromise. urinary infection with candida albicans is most commonly a component of systemic candidiasis in patients who are severely immunocompromised. systemic candidiasis is also seen in premature and term infants with perinatally acquired invasive candidiasis. presentation is usually with systemic sepsis, fever or hypothermia, hepatosplenomegaly, erythematous rash, and thrombocytopenia. systemic candidiasis may be seen on ophthalmologic investigation as microemboli in the retina. the first clue to the underlying diagnosis may be the presence of yeasts in the urine ( ) . candida involvement of the urinary tract may affect all structures including the glomeruli, tubules, collecting system, ureters, and bladder. microabscesses may form within the renal parenchyma, and large balls of fungi may completely obstruct the urinary tract at any level. acute renal failure caused by systemic candidiasis or obstruction of the renal tracts with fungal hyphae is a wellrecognized complication of systemic candidal infection ( , ) . indwelling catheters (which form a nidus for persistent infection) should be removed. successful treatment of non-obstructing bilateral renal fungal balls by fluconazole either alone or in combination with liposomal amphotericin b has been reported ( , ) . in the presence of obstruction, however, percutaneous nephrostomy to relieve the obstruction with antegrade amphotericin b irrigation, coupled with systemic antifungal therapy, is the mainstay of treatment ( ) . amphotericin b is the most effective antifungal agent, but it is not excreted in the urine. local irrigation via nephrostomy provides good results, however. for treatment of urinary tract candidiasis, it is usually combined with fluconazole or -flucytosine, both of which are excreted in high concentrations in the urine. treatment is required for weeks to months to ensure complete elimination of the fungus, and the ultimate outcome is largely dependent on whether there is a permanent defect in immunity. in , levin et al. first described hemorrhagic shock and encephalopathy, which appeared to be distinct from previously recognized pediatric disorders ( ) . other cases have subsequently been reported from several centers in the united kingdom, europe, israel, the united states, and australia, and the syndrome is now recognized as a new and relatively common severe childhood disorder ( ) . hemorrhagic shock and encephalopathy usually affects infants in the first year of life, with a peak onset at - months of age. a prodromal illness with fever, irritability, diarrhea, or upper respiratory infection occurs - days before the onset in two-thirds of cases. affected infants develop profound shock, coma, convulsions, bleeding and evidence of dic, diarrhea, and oliguria. laboratory findings include acidosis, falling hemoglobin and platelet levels, elevated urea and creatinine levels, and elevated levels of hepatic transaminases. despite vigorous intensive care, the prognosis is poor, and most affected infants die or are left severely neurologically damaged ( , ) . a small number of patients have been reported to survive without residual sequelae. the renal impairment appears to be largely prerenal in origin, and when aggressive volume replacement and treatment of the shock results in improved renal perfusion, rapid improvement in renal function is usually observed. in patients with profound shock unresponsive to initial resuscitation, vasomotor nephropathy supervenes and dialysis may be required. myoglobinuria in association with hemorrhagic shock and encephalopathy has been reported. following the description of the mucocutaneous lymph node syndrome by kawasaki in , kawasaki disease has been recognized as a common and serious childhood illness with a worldwide distribution. although the etiology remains unknown, epidemiologic features clearly suggest an infective cause. the disease occurs in 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therapy for the management of bk virus associated nephropathy in children and adults antiviral therapy and prophylaxis for influenza in children infectious diseases and the kidney key: cord- -c myq bi authors: iversen, patrick l. title: the threat from viruses date: - - journal: molecular basis of resilience doi: . / - - - - _ sha: doc_id: cord_uid: c myq bi infectious disease represent the most significant threat to human health. significant geologic cataclysmic events have caused the extinction of countless species, but these “wrath of god” events predate the emergence of homo sapiens. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic and are here to stay marking their presence with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but are never prepared. the long-term consequences often remain unrecognized and are always inconvenient including cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. viral success is based on rapid replication resulting in large numbers. single-stranded rna viruses with their high replication error rate represent a paradigm for resilience. contemplating recent career paths, i reviewed a broad range of scientific questions. most prominent was, why study an area of science that has no significant impact? in fact, why study anything that is not the most highly impactful area? this question demands a definition, what constitutes high impact? i decided to create a definition that impact and threat to life are related. further, threat to human life is the highest impact and it is likely that things threatening human life may also threaten all life. what presents the greatest threat to human life today? history should provide critical insights to answer this question. a comprehensive look would seek causes of mass extinctions over the past . billion years of life on earth. this perspective has been a trending subject in science with focus on five mass extinctions. there are inherent biases in focusing on mass extinctions such as these events fail to appreciate small living things such as single celled organisms or bacteria. another bias is in order to appreciate life in the past, the life form must occasionally produce a fossil when it dies. those concerns aside-geologic events have threatened the survival of living things (table . ). the actual events causing these extinctions are frequently debated, for example, the asteroid impact million years ago off the coast of mexico was accompanied by a massive tsunami that was responsible for mass extinctions in montana, the impact may have also triggered an enormous volcano in india resulting in ash and lava flow responsible for regional species extinctions, and the collective dust resulted in prolonged climate change which was probably responsible for even more species extinctions. the lesson remains the same, volcanic eruptions; climate change and asteroid impact threaten life in a highly significant way and are responsible for extensive selection pressure. unfortunately, these events are unpredictable and so enormous little can be done about them. a mass extinction may not represent a legitimate threat or selection pressure because large numbers of species are completely eradicated leaving few survivors to drive evolution. finally, outside of removing dinosaurs so that mammals could thrive, what impact did these events have on human life; humans were not yet on the earth when these events took place. refining the search to dramatic changes in human populations moves the period up to the last , years. mass migrations took place about every , years, which is in line with the precession of the earth leading to a possible linkage between earth's precession and human migration. this may have been due to regional climate change like ice ages in the northern hemisphere. however, human population changes on this timescale are not easily documented so estimates of climate and human population dynamics will be limited. time to refine the search. consider the exponential growth, outbreak, of tent caterpillars, malacosoma disstria, in montana reported by david quammen in his book spillover (quammen ) . the extensive caterpillar population consumed all of the leaves from the local elm and cottonwood trees in the summer of . the caterpillar activity produced a crackle sound, "like a distant brushfire." the city attacked these insects with a broad arsenal of modern countermeasures but to no effect. however, a nuclear polyhedrosis virus (npv), uses the caterpillar host density like a critical mass in a nuclear weapon to explode destroying the caterpillars in an epic battle. the caterpillar population retreated to undetectable levels in a single season as a result of npv. human populations changed over the past , years with particular emphasis on the most recent years. a human population curve over this segment of time shows an exponential growth period interrupted in the middle ages by the plague. hypothesis: the plague and other pandemic infectious disease events appear to be the greatest threat to human life. infectious disease kills million people each year, percent of the total million annual deaths in the global population (fauci and morens ) . a brief review of pandemic infections over the past years illuminates several events that reduced human populations. the plague of athens bce killed percent of the human population (table . ). both bacterial and viral pandemic yersinia pestis is a bacterium causing plague. fleas can be infected with y pestis which transmit the bacterium to rodents, the primary hosts. changes in the environment may lead to the movement of rats into populated areas where humans become infected. homer points to such an infection in the iliad in his description of the trojan war in bce. plague has returned several times since the trojan war imposing enormous loss of human life (table . ). the most recent plague epidemic killed over ten million people in india in the early th century. y pestis is still out there ready for favorable conditions to pounce on human populations but outcomes are likely to be less dramatic due to understanding of sanitation practices, quarantine, and availability of antibiotics. if infections are the greatest threat to human life, they should be critical drivers of evolution? clearly infections pose selection pressure on the human populations. origins of evolutionary thought did not include infection as darwin established key evolution concepts on the galapagos islands. these islands are isolated and an unlikely place for the spread of infections. the concepts speciation point to geographical separation of populations so infections would most likely be restricted to isolated populations. in many cases the survival selection pressure is not identified or ascribed to insufficient sources of food. unfortunately, common single-stranded rna viruses are so unstable that there are limited data for a viral fossil record. pandemic infections remain a threat to human survival in the presence of the information revolution, daily medical breakthroughs, and global travel. the human retrovirus hiv currently a global infection that infects up to % of the population in southern and eastern africa with a projected death toll of up to million by . measles killed million people in the last years and the development of an effective vaccine in reduced concerns for this infection but there were , deaths in the year . vaccination programs are frequently disrupted due to complacence resulting from vaccine success, conflicts that shift healthcare focus, and social crisis such as the recent ebola outbreak in west africa. smallpox is also an ancient infection causing fever, skin lesions, and at times death. king ramses v of egypt is thought to have died from smallpox around bce. introduced into mexico in , smallpox killed . million aztec indians or about half of the population in a period of years and then proceeded to decimate the population of south america. variola is a highly infectious virus killing - million people during the th century inspiring the eradication campaign in . variola was eradicated by december of (de cock , a rare triumph of public health. the who deserves acknowledgment for this unprecedented accomplishment and proof of concept that human suffering is not inevita-ble. however, variola is a dna virus with limited rate of mutation and a narrow host range so that animal reservoirs do not exist. the eradication of other viral infections will be more challenging. the world continues to confront a broad array of microbial threats. progress and preparedness make our engagement a likely success for those microbes that resurface and infections for which we have experience. medical and epidemiological uncertainties surround emerging infectious disease, those that challenge us with their novelty. pandemics dominate the infectious disease "fear factor" but each pandemic began as a much more frequent occurrence, an epidemic. most but not all epidemics come from emerging infectious agents, the most significant problem facing life on earth today. the concept of emerging is a human centric term as most of these infections are endemic in an animal host that serves as a viral reservoir. a report from the university of edinburgh identified human pathogens and are emerging or re-emerging of which % are zoonotic, that is jump from an animal host to human. numerous emerging infections caused by viral agents have imposed high impact on human survival (table . ). all the viral agents in table . have genomes based on single-strands of rna except hbv which should focus scientific attention on rna. there are numerous questions that strike investigators as they ponder a collection of viral agents like those in table . . the viral polymerase errors in replicating single-stranded rna genomes are not corrected so the species are constantly changing. the apparent success of these viruses is that as they move from reservoir hosts to humans and as humans become immune to the initial infection, the population of diverse genomes offers multiple chances to adapt by finding a "fit" genome version which can propagate until the next transition requiring adaption. acquired immunodeficiency syndrome (aids) is caused by human immunodeficiency virus (hiv- ), a retrovirus. these viruses have a single-stranded rna genome that is converted into dna, a paradigm shift in the flow of genetic information from dna to rna. the , , human deaths caused by hiv- (table . ) is accompanied by a spectrum of clinical signs; eg. fever, diarrhea, peripheral neuropathy, pelvic inflammatory disease, cervical cancer, cytomegalovirus retinitis, kaposi's sarcoma, lymphoma, mycobacterium avium infection, recurrent pneumonia, and wasting syndrome. hiv- genome diversity includes base substitution, insertion, deletion, recombination, and gain or loss of glycosylation sites which all arises from the limited fidelity of the viral reverse transcriptase. these mutations are found in clusters or hypervariable regions indicating the fit virus is selected for from vast numbers of less-fit genome sequences. hiv- emphasizes key observations: ( ) eid is a significant contemporary concern, ( ) we are not prepared for the novel characteristics introduced by eid, ( ) clinical signs can be diverse and often mimic symptoms of other diseases, and ( ) the replication mechanisms are often error prone resulting in an array of fit viruses. dengue is a flavivirus with a positive sense single-stranded rna (+ssrna) genome carried by mosquitos to man. dengue has been a tropical disease for hundreds of years, typically a disease of young children but in an emerging severity was recognized in manila (table . ), hemorrhagic fever (dhf) and dengue shock syndrome (dss). more than billion people are at risk of dengue infection but only a small fraction of those infected will develop dhf or dhs. infected people develop antibodies that can lead to antibody-dependent enhancement (ade) in subsequent infections and more severe dhf or dss outcomes. it appears ade events occur in people that produce immunoglobins (iggs) with enhanced affinity to the activating fc receptor due to the igg subclass and lack of a fucose glycan modification of the igg (wang et al. ) aedes aegypti is the primary vector transmitting dengue but a new vector, aedes albopictus, now carries dengue to the southern united states. emergence of dengue in the southern united states is likely due to used tires imported from japan which provided a place for the asian tiger mosquito to live. outbreaks of dengue fever have become more numerous and more severe over the past three decades. viral "fitness" is constrained by the requirements imposed by the natural host so that it is a low probability event for a virus to move from the natural host to a human. while an insect frequently plays the role of vector carrying a virus from an animal reservoir to humans, several zoonotic viruses are transmitted by placing humans near rodents. several arenaviruses, minus sense single-stranded rna viruses, jump from their rodent natural hosts directly to humans through contact with rodent urine or saliva. notable arenaviruses are named for the hemorrhagic fever (hf) region of their zoonosis; bolivian hf is caused by machupo (macv), argentine hf is caused by junin (junv) and venezuelan hf is caused by guanarito (gotv). these south american outbreaks are caused by new world arenaviruses in contrast to lassa hf (lasv), an african or old world arenavirus. lasv is an endemic disease of west africa (table . ) and can be confused with dhf or ebola infections. viruses that are transmitted by arthropod vectors are called arboviruses. in , only yellow fever of six known arboviruses caused disease in humans. the discovery of arbovirus caused human disease expanded beginning in the late s with western and eastern equine encephalomyelitis (weev, eeev) and st. louis encephalitis. both chickungunya and zika (table . ) are arboviruses that have emerged to become global infections in the twenty-first century. zika not only became infamous for causing microcephaly in newborns of infected mothers and guillain-barre syndrome but is now sexually transmitted between humans. i worked on a therapeutic for the treatment of ebola infections from to . the genome sequence recovered from each outbreak from to has been different. studies were conducted at usamriid in their bsl facility by expert ebola investigators. rhesus monkeys (macaca mulatta) were injected with plaque forming units (pfu) of zaire ebolavirus (zebov) kikwit into their thigh muscle. all of the untreated control monkeys died between days and following viral injection. we measured viral burden by quantitative polymerase chain reaction (qrt pcr) a measure of genome copies per milliliter (copies/ml) of plasma and plaque formation a measure of infectious viruses per ml (pfu/ml) plasma. we found an average of . x genome copies per ml on day post infection and . x pfu/ml on day . nearly genomes present for each successful virus in a nonhuvwas not observed, the dominant population of viral genome sequence did not change from one individual to another or from one time to another within the same individual. the existence of defective viral genomes may offer potential for rapid change but in a stable host, change was not rapid. when one considers the magnitude of differences in conditions as a virus jumps from a reservoir host to a human, rapid adaptability is a great advantage. a virus that kills all the hosts is not a successful virus. perhaps defective viral particles facilitate host immune responses giving them a chance to catch up to the rapidly proliferating virus. the ebola outbreak of illuminated the collateral damage that accompanies eid outbreaks. first, the first responders are local physicians and care givers are killed leaving a population lacking doctors and nurses. even physicians trained to exercise appropriate caution when interacting with patients such as dr. sheik humarr khan died of the ebola infection (bausch et al. ) . second, women are the main caregivers in their families and percent of ebola deaths in the outbreak were women. this leaves families lacking traditional structure. third, the outbreak disrupts production, labor markets and trade resulting in scarcity and inflation of food prices. food security and nutrition are diminished which preferentially affects poor people as they spend to % of their income on food. finally, public health measures are discontinued. before the outbreak, percent of children in west africa were receiving routine vaccinations but that figure fell below percent. the west africa loss of measles vaccinations was followed by measles out- coronaviruses are unique in their large single-stranded rna genome ( , bases) and are often associated with mild disease, bronchitis and gastroenteritis. a outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in china rapidly spread to cases in countries (table . ). retrospective analysis of this outbreak linked zoonosis to a culinary trend in which people sought out exotic animals to eat. these nontraditional food animals appeared in street markets in urban areas bringing people close to the reservoir host triggering the outbreak. the civet cat was identified as the source of the first human cases but this is not the natural host. the natural host is the fruit bat, which infects a variety of small mammals including the civet cat. a point of intervention has been removal of the civet cat from markets but the natural bat host remains in the environment maintaining the virus for the next outbreak. a second coronavirus outbreak in of middle east respiratory syndrome coronavirus (mers cov) rapidly involved countries including africa, western europe, and southeast asia. the number of cases is relatively low but the case fatality ratio is of great concern (table . ). the camel is reservoir host in this case but it may not be the natural host since camels often get sick as well. transition from animal-to-human transmission to efficient human-to-human transmission was observed quickly in the case of both sars-cov and mers-cov. this rapid adaptability of a highly infectious virus group should raise concern over endemic coronaviruses in companion animals since these viruses can clearly become pathogenic, can jump from animal to human and then become transmitted from human-to-human. an ongoing outbreak of yellow fever in africa (table . ) is a striking reminder that eid can re-emerge into virtually naïve populations. some people recover from the acute symptoms but liver damage causes yellow skin, the reason for the name for the disease. the liver damage leads to bleeding and kidney damage and occasionally death. yellow fever is a flavivirus that originated in africa but was distributed to the world on barges and sailing ships to tropical ports. the virus arrived in the americas on slave ships. this virus interrupted the building of the panama canal which attracted the attention of walter reed, the military physician responsible for demonstrating transmission by mosquitos. an effective vaccine is used worldwide with immunity lasting over years. the limitation of this vaccine like most is that populations need to be vaccinated regularly which in a world constantly changing due to political and economic drivers leads to vaccination gaps. cancer is one of the leading causes of human death but estimates are that percent of all human cancers are directly caused by viruses (table . ) (morales-sanchez and fuentes-panana ). the first tumor virus was identified by peyton rous in . he took cell lysates from a chicken sarcoma which he passed through a filter known to hold back bacteria and then injected the filtered lysate into chickens. a tumor developed at the injection site. the virus is now known as rous sarcoma virus (rsv), a single-stranded rna virus. rous won a nobel prize in physiology or medicine for the discovery in . rsv is a retrovirus that captured a tyrosine kinase, src gene, that triggers uncontrolled growth in infected host cells. the avian leukemia virus (alv) was discovered in denmark early in the twentieth century capable of causing disease in blood forming tissues of chickens. a strain of alv, avian myeloblastosis virus (amv) was described in that provided a convenient source of tissue for biochemical studies. during the s a strain of inbred mice was found to develop leukemia between and months of (burkitt's lymphoma ) . after that he saw a second case at the jinja district hospital on the shores of lake victoria this became more than a curiosity. a review of hospital case notes confirmed the prevalence of these tumors of the jaw and they were accompanied by swellings in kidneys, ovaries, adrenal glands and liver. he assembled data from patients and histological examination led to description of "lymphoma syndrome" or the "african lymphoma." burkitt received a grant in for ₤ to visit hospitals in eight african countries; uganda, kenya, tanzania, malawi, mozambique, zimbabwe, zambia, and republic of south africa to investigate african lymphoma. the tumor was found everywhere at the equator in areas where year-round temperature was above °c but not in areas over feet in elevation. burkitt further determined the tumors only occurred where the annual rainfall was above inches and not seen in the dry savannah of nigeria. burkitt contacted doctors in papua new guinea to discover these tumors were the most common childhood tumor in that country but only in wet coastal regions and not in the dry highland areas. burkitt's working hypothesis was that an insect transmitted virus infection was responsible for the lymphoma syndrome. he then observed adults with the lymphoma but only in individuals that moved into the "african lymphoma belt." these tumors were then observed in the united states and europe at a rate of - per million or about x less frequently than in africa. biopsy samples did produce viruses but none that came from insects so the working hypothesis was abandoned. a new hypothesis emerged between and in which p falciparum (a parasite causing malaria) was the causative agent for the lymphoma. it is believed that severe malaria does play a role in the development of burkitt's lymphoma. at the advice of peter clifford, burkitt administered methotrexate to treat patients which produced encouraging results. in burkitt gave a lecture at middlesex hospital, "the commonest children's cancer in tropical africa: a hitherto unrecognized syndrome." anthony epstein attended the lecture which changed his life and provides a punctuation mark in the history of science. epstein introduced himself after the lecture and the two sat down for tea. burkitt agreed to send tumor specimens to epstein and epstein received a grant from the us national institutes of health in for $ , which allowed him to employ two research assistants. yvonne barr was one of the research assistants that was able to propagate a virus after tries with tumor specimens. the other research assistant was bert achong, an electron microscopist, who was first to identify the virus as a member of the herpes virus family. the virus bears the name epstein barr virus or ebv and is a causative agent of burkitt's lymphoma. an interesting historical note involves werner and gertrude henle at the children's hospital of philadelphia. they created antibodies to ebv infected cells and their survey of blood samples from american adults revealed over % to be ebv positive in . they also showed that normal human lymphocytes could be made immortal by infecting them with ebv in . we now know ebv silently infects children in the western world but in those infected a bit later in life become susceptible to infectious mononucleosis (the kissing disease) as adolescents. in , a collaboration between george klein and the manolovs led to a discovery of a chromosome change that was specific to burkitt's lymphoma. this chromosome change was a translocation of : and rarely : or : (zech et al. ). the break points in chromosomes , , and contained immunoglobin genes that become active in b lymphocytes as they produce antibodies during infection. a nobel prize in to michael bishop and harold varmus provided the significance to the break point in chromosome , bringing active immunoglobin genes near the oncogene c-myc. in , a second cancer common in north and east africa as well as southern china was found to be ebv positive, carcinoma of the post-nasal space (nasal pharyngeal carcinoma or npc). the tumor is observed in epithelial cells not b-cells and is not geographically linked to endemic malaria regions. in this case, it appears that aerosol exposure to environmental carcinogens (possibly in preserved fish) may introduce mutations in nasal epithelial cells. the npc may be the result of silent ebv infection, carcinogen induced cellular mutations, and people carrying hla a* and b* antigens. in , several reports describing "fatal infectious mononucleosis" appeared. david purtilo described x-linked proliferative syndrome (xlp) based on an international registry of boys with fatal ebv infections (purtilo et al. ) . using genetic marker analysis from the international registry they narrowed xlp to a three million base pair segment on the x-chromosome. in , a -base pair segment encoding the xlp protein of amino acids was found to be lost or damaged in al xlp patients (nichols et al. ) . curiously, the xlp protein loss leads to a defect in nk and t lymphocytes which are necessary for recognition of ebv infected b lymphocytes. these individuals cannot make antibodies to ebv because their b cells require help from t lymphocytes. the development of potent immune suppressing drugs like cyclosporine a was linked to lymphoma after organ transplantation. two girls with acute lymphoblastic leukemia received bone marrow transplants from their hla-match brothers. the grafts were successful but they developed lymphoblastic leukemia from the transplant (male cells). these lymphomas do not present chromosomal translocation like burkitt lymphomas revealing a new path from ebv to lymphoma. the immune suppression upsets the ebv host homeostasis crippling t-cells that are required for keeping ebv under control. another situation where the virus can replicate without control is in hiv/aids patients and aids-lymphoma is yet another ebv induced casualty. ebv provides key insights into tumors associated with viral infections. first, the same virus is linked to multiple tumors in different populations including burkitt's lymphoma, hodgkin lymphoma, and nasopharyngeal carcinoma. second, tumorigenesis involves more than one mechanistic pathway but interest has focused on the latent membrane protein- (lmp ), ebv nuclear antigen (ebna ), and bamh -a reading frame- (barf ) genes. third, dissecting the association between infection with ebv and cancer has unfolded over a period of years and led to a greater understanding of cancer, the immune system, and methods for immortalizing cells to study specific cell clonal populations. ebv has been a boon to scientific research while imposing a challenge to human survival. approximately billion people have been infected with hbv and million suffer from chronic infection. acute hbv infections are usually self-limiting associated with . million cases a year. chronic hbv infections lead to complications in - percent of cases resulting in . to . million deaths each year. hb v causes - percent of the world hcc cases which represents percent of all cancers in the world. the hbx gene is considered key to oncogenesis. vaccination programs are effective in reducing mortality in infants and have reduced emerging prevalence of the disease. dietary exposure to aflatoxin enhances hbvrelated hcc (sun et al. ) as well as co-infection with hcv and excessive alcohol consumption. hepatitis b virus (hbv) was named due to differences in transmission: hepatitis a type virus follows fecal-oral transmission while b type virus is parenteral. the genome is circular dna that is not fully double-stranded with the end of the fulllength strand linked to dna polymerase. the genome is - nucleotides long (full length strand) and - nucleotides for the short strand. four genes are encoded including: ( ) c the core protein (hbcag) synthesized by uorf aug to make pre-core protein, ( ) p is the dna polymerase, ( ) s is the surface antigen (hbsag) which has three aug start sites that divide the gene into pre-s , pre-s , and s, and ( ) x is a gene that is not fully understood but may be a transcriptional transactivator. non-coding rna include hbv prealpha, hbv prebeta, and hbv rna encapsidation signal epsilon. there are known genotypes which differ by percent in sequence with distinct geographical distributions and are labeled a to j and at least subtypes. type f is the most divergent form and found in central and south america, predominantly brazil. human hbv has a narrow host range infecting humans and higher primates, eg. chimpanzees. in the s a woodchuck hepatitis virus (whv) was discovered which will not infect rodents. a ground squirrel form was identified (gshv) that is a distant relative of the marmot and woodchuck. indeed, dhbv infects ducks but not all species of duck, grey herons are infected with hhbv, the ross' goose with rghbv, the snow goose with sghbv, white storks with sthbv, and cranes with chbv. there are no hepadnaviruses in arthropods or insects. human t-cell lymphotropic virus (htlv- ) htlv- is a single-stranded rna retrovirus, defined by their use of reverse transcriptase, a polymerase, that makes a dna copy of the rna kb viral genome. the dna viral genome is integrated into the host genome where it is referred to as a provirus and is replicated along with the host genome during cell division. only percent of infected individuals will develop leukemia and this is observed to years after asymptomatic infection. the htlv- tax protein is likely to initiate cell transformation through interactions with transcription activators and cell cycle regulators. hepatitis c virus (hcv) hcv has infected approximately percent of the world's population (~ million) but screening of the blood supply has reduced prevalence. hcv is a flavivirus composed of a . kb single-stranded, positivesense rna. hcv is characterized by a single serotype but at least major genotypes. genotype b is the most common genotype seen in the united states and taiwan. hcv becomes a chronic infection by evading host immune defenses through a combination of: ( ) high replication rate ( virions/day) and ( ) lack of error proofreading by the viral polymerase leading to mutations in response to immune pressure. the genetic variability of hcv has limited efforts to design an effective vaccine. the polyomaviruses are small ( . kbp), double-stranded dna viruses. early studies with simian virus (sv ) led to identification of the large tumor antigen (large t; lt). lt is also found in bk and jc viruses which are more suspect human tumor viruses and merkel cell polyomavirus (mcv) which is now well established as a human tumor virus (feng et al. ) . the n-terminus of lt contains an lxcxe motif that interacts with the retinoblastoma protein rb while the c-terminus contains an atpase/dna helicase domain that can inactivate p . the ls-p complex activates insulin-like growth factor i (igf-i) which alone is capable of cell transformation. a recent report of morbidity and mortality reveals heart disease and cardiovascular events are the number one killer with neoplasia and infections following close behind. however, infections frequently cause neoplasia and cardiovascular disease leading to death. if we combine cardiovascular events and neoplasia caused by infection, then infectious disease is the most significant threat to human life and qualifies as the area of greatest impact. the picornavirus family are small (pico-), single-stranded, positive sense rna genome ( . kb) viruses that synthesize a single polyprotein that is cut into a small collection of functional proteins by virally encoded ( a and c) and cellular proteases. poliovirus is a picornavirus that has served as the prototype for the viral family. the enteroviruses are a group of picornaviruses that have been associated with cardiac disease. coxsackievirus b (cvb), coxackievirus a (cva), and echovirus infections lead to cardiac signs . % of the time. about one-third of patients with acute cardiac disease (inflammation of the heart) are antibody positive for enteroviruses. while acute myocarditis is often self-limiting, chronic cardiac disease often leads to dilated cardiomyopathy (dcm) which is present with no heart inflammation. dcm is associated with heart failure which can be lethal (table . ). these chronic infections lead to percent of all cardiac transplants worldwide. the enteroviral a protease can also degrade dystrophin in the heart leading to cardiac necrosis, reduced ejection fraction, and then to dilated cardiomyopathy. transmission of these viruses is from contaminated food and water. the human herpesviruses are large double-stranded dna genome viruses. the human herpesvirus- (hhv ) or cytomegalovirus (cmv) has a kbp genome encoding over two hundred genes. one problem in finding associations with cmv infections is that it is not an emerging disease, to % of the population has been infected making comparisons to a control group challenging. most infections are observed in children and in newborns serious clinical findings can be observed. hence, most adults carry latent infections that are reactivated when individuals become immune suppressed following solid organ transplantation, malignant hematological disease, and aids. reactivation in immune suppressed individuals is associated with increased mortality. the association with cardiovascular disease has been demonstrated in two recent studies. in one, cmv infections were detected in . percent of coronary artery samples from bypass operations compared to percent in of patients who needed cardiac surgery for reasons other than atherosclerosis hebar et al. ) . in the other, cmv reactivation (viremia) was detected in . % of immunocompetent patients admitted for major heart surgery (roa et al. ) . the incidence of coronary artery disease is the major contributor to death from heart disease and if to . % of this disease is associated with cmv infections, this infection is a significant human health hazard. recognizing an emerging infectious disease involves well established strategies of surveillance; ( ) identify unusual clusters of disease, ( ) evaluate the spread of an outbreak, ( ) estimate the magnitude of the problem, and ( ) if possible identify the atheroscleotic plaque infectious agent. the strategy has proven valuable for known infectious and noninfectious diseases but has limited capacity to detect emerging infectious diseases. however, deviation from the traditional approach to surveillance is not likely to gain support. al smith, a veterinary virologist, approached me after a seminar i presented in in the college of veterinary medicine at oregon state university. i had just joined avi biopharma as the head of their research and development program. al was a veterinarian and professor and had devoted his career to the caliciviridae family of viruses dating back to his time in the naval research station in california. he had isolated the nonhuman vesivirus group of caliciviruses not only from suffering sea lions in the channel islands, but from reptiles in the san diego zoo and whales held in captivity. al and his capable technician, doug skilling successfully propagated the virus isolates in cell culture, an accomplishment not shared by other laboratories at the time. al developed nucleic acid probes and induced antibodies to these viral isolates. over decades of research he assembled an extensive collection of vesiviruses which were held in redundant − ° freezers. his singular vesivirus focus gave the appearance of a zealot but his ability to cultivate viral isolates, his extensive collection of isolates, and his one of a kind detection reagents made him a one of a kind virologist. al was well beyond retirement age and was an "old school" virologist ready at a moment's notice to take his bag into the real world and collect swabs from ailing animals. he maintained careful records of the condition of his patients, their location, and the setting of the animal in the community. every field trip led to work in the lab propagating virus from his swabs. al wanted to know if i would be interested in finding an antiviral agent for these viruses. my prime directive at avi biopharma was to explore the capabilities of our proprietary antisense technology and i had yet to investigate targeting a virus. the caliciviruses have positive sense single-stranded rna genomes which express three genes from a single polyprotein. we identified an active agent following an investigation with small collection of candidates (stein et al. ) . the vesivirus group of caliciviruses are considered animal only and are not believed to infect humans but al began exploring human samples with his collection of detection reagents. after several years making incremental progress, we found an association between human blood samples seropositive for vesivirus and markers of liver disease (smith et al. ) . we felt this evidence of an emerging infectious disease in humans and its potential to cause liver disease would be welcomed by the medical community. al and i made a trip to washington dc at our own expense to relate the findings in person. we meet with virologists at the national institutes of health but were met with judicious skepticism. harvey alter had been instrumental in the discovery of hepatitis c virus (hcv) and felt all viral liver disease is already accounted for by hepatitis viruses. hence, no interest in our findings. we met with the american red cross blood banking group in shady grove maryland and were met with concern. blood supplies are scrutinized by the nucleic acid test (nat) to eliminate viral contamination and any further elimination of blood samples would threaten an already limited inventory. our findings simply add complication to the blood supply finding an emerging infectious disease business. they asked for more compelling and more extensive data to add robustness to our claims. the only problem is that they were not interested in providing financial support for the recommended studies. the conundrum is all too common, you need more data to convince granting agencies to support the work but there is no support to add to the limited data. life on the cutting edge is frequently discouraging. our strategy took two paths: one to seek commercial support and the other to create proof of concept data for an antiviral solution. the most logical commercial solution was to meet with michael houghton at chiron. he was the driving scientific force in finding hcv and chiron might like adding to their reputation by finding another previously unrecognized hepatitis virus. indeed, dr. houghton invited us to bay area to discuss the project. he reviewed our data and the quality of al's detection reagents. chiron provided modest support and their in-house laboratory help to confirm our observations, a glimmer of hope. al created a company, calicitech, so that he could accept support and license his reagent patents from the university. testing an antiviral agent in humans would be expensive but in the absence of natural history of the infection would make human proof of concept impossible. however, al had been contacted by a cat rescue facility in atlanta, mommy cat, describing an outbreak of feline calicivirus (fecv) in their cattery. these cats had all been vaccinated with an approved fecv vaccine which failed to protect these cats. a veterinarian can decide to use alternative medicines if there are no alternatives and the owner oc the cats can sign the equivalent of informed consent. we provided our fecv specific antiviral to mommy cat along with a detailed protocol for treatment. the infected kittens we treated survived while those not treated died providing encouraging data. we then discovered a similar outbreak in the humane society facility in eugene oregon, our neighbors. again, we reached an agreement to treat infected kittens and were successful ). this was the first time we treated an infection based on symptoms in an "outbread" population of cats. with over kitten patients and remarkable survival differences between treated and untreated, we had our proof of concept. there is no disease cluster to link to human vesivirus. norovirus is a calicivirus and is known to infect humans, in fact it is well known on cruise ships, day care centers, and extended care retirement facilities. our efforts to have human vesivirus recognized as an emerging infectious disease have failed. chiron was not impressed with our antiviral and no longer were interested in evaluating the detection reagents. if we are lucky, human vesivirus infections will remain mild clinical oddities. al smith has an interesting way of responding, "that virus is out there infecting humans. it won't go away." the existence of non-pathogenic viral infections led to the emergence of the study of the immune system, vaccination, gene therapy, and concern for future pandemics. we carry evidence of ancient retroviral infections in our genome from integration events that became vertically transmitted making up as much as percent of the human genome (meyer et al. ) . these genomic fossils are called endogenous retroviral genomes (erv). most erv sequences accumulate sufficient mutations over evolutionary time that horizontal transmission is unlikely. these viral genome segments are trapped but can still be transcribed and encode some viral proteins. the significance of these integrated genomes is a current topic of investigation. adeno-associated virus (aav) is a single stranded dna virus that infects humans but are not known to cause disease. the lack of pathology has led to their use as viral vectors for human gene therapy. aav can infect dividing and quiescent cells and will persist extra chromosomally without integrating into the genome of the host cell. aav is a member of the parvoviridae family in the genus dependoparvovirus. is an arenavirus closely related to lassa virus and shares reservoir hosts. mv is naturally attenuated and nonpathogenic in humans. infection of humans with mv protects against lethal challenge with lv (wulff et al. ) . flaviviridae) named after g. barker, a surgeon, first identified in . hgv infects one sixth of the world's population but does not cause human disease. a metaanalysis of publications investigating gbv-c infections in hiv-positive individuals indicate coinfection with gbv-c slows the progression of hiv disease in individuals that have been seropositive for years or more (zhang et al. a) . two of the studies investigated gbv-c years after documented hiv seroconversion estimate the hazard ratio of . ( % ci). is a retrovirus first identified in from a lymphoblastoid cell culture from a kenyan patient with nasopharyngeal carcinoma. hfv is homologous to primate foamy viruses and is most closely related to the chimpanzee foamy virus (sfvcpz). early studies raised alarm for association with autoimmune diseases but more extensive studies with more precise diagnostic reagents fail to find a disease associated with hfv. hfv is a rare human infection and concerns parallel sfv infections in humans. simian foamy virus (sfv; spumavirus-retroviridae) is a retrovirus infecting most primates born in captivity and people making contact with infected primates can become infected. human infections frequently occur in males probably requiring a bite from infected nonhuman primates but are harmless. the infected cells often fuse to form syncytia of giant foamy cells, which gives the virus its name. the error rate in sfv genomes is exceptionally low, . x − substitutions per site per year, compared to hiv − substitutions per site per year. since so-called crossspecies, infections have only been observed for a little over a decade the long term consequences are not known. these infections are watch and wait for everything from a zoonotic epidemic to identified disease clusters. perhaps this is exactly the sort of infection that will emerge as a significant human concern in the future. torque teno virus (ttv; alphatorquevirus) is a single-stranded, positive sense dna genome virus about . kb in size in the anelloviridae family. nearly percent of even healthy individuals are infected in some countries. the virus was discovered in as the "transfusion transmitted virus (ttv)" in a japanese patient. it is often found in patients with liver disease but does not cause hepatitis on its own. closely related torque teno mini virus (ttmv) were isolated in and found to have smaller genomes of . - . kb. ttmv infections are also common but do not cause any described human disease. human adenovirus type (rad ) is used to create an ebolavirus (ebov) vaccine encoding zaire ebolavirus glycoprotein failed to protect animals immune to ad . a replication defective chimpanzee-derived adenovirus (chad -ebo-z) provided protection against lethal ebov challenge in macaques but protection wane over several months. they boosted with a modified vaccinia ankara (mva-bn-filo) that led to durable protection (stanley et al. ). this vaccine progressed through phase i, single-blind, randomized human trials in mali between and . a single dose of the chad -evov-z is efficient as a prime vaccine strategy followed by mva-bn-filo as a boost was well tolerated in humans tapia et al. ) . is a base double-stranded dna virus infecting less than percent of the human population. wu, named after washington university, is found as a co-infection in various respiratory infections but wu does not cause disease on its own. wu is closely related to ki virus that also is not known to cause clinical disease. however, related polyomaviruses that are clinically relevant include bk virus associated with nephropathy, jc virus associated with progressive multifocal leukoencephalopathy, sv virus associated with mesothelioma, and merkel cell polyomavirus associated with cancer. vaccinia virus (orthopoxvirus) is a large double stranded dna virus closely related to smallpox. edward jenner, the father of immunology, found the milkmaids exposed to cowpox (vaccinia) were immune to smallpox in . this was the first vaccine (named after vaccinia) leading to the modern vaccine that has allowed for the eradication of smallpox. viral sequences are constantly mutating with no purpose other than seeking a survival/infectivity benefit. this means viruses with no current pathology represent a pre-mutation reservoir for the next catastrophic human pandemic. the popularity of rnaseq is likely to expand our catalog of nonpathogenic viral infections. however, the management of such information is in question. technology used to counter viral infections has resulted in over approved drugs for the treatment of nine different human viral infections in just years (de clercq and li ) . several different antiviral drug groupings have been reported, but the following arise from review of the mechanisms of action of antiviral drugs assembled in table . : ( ) inhibition of viral attachment and entry, ( ) inhibition of viral uncoating, ( ) viral polymerase inhibitors, nucleotide analogues (ntrti) and non-nucleotide reverse transcriptase inhibitors (nnrti) and dna polymerase inhibitors, nucleic acid synthesis inhibitors and nucleotide pool size agents, ( ) latency reversal agents, ( ) integrase inhibitors, ( ) protease inhibitors for both hiv and hcv, ( ) neuraminidase inhibitors, ( ) immune response modifiers, and ( ) antisense inhibitors. administration of hyperimmune sera from immunized animals or human donors was the first effective treatment for infectious diseases. the practice has limitations but is still used to treat bacterial toxins and viral infections caused by cmv, rsv, hav, hbv, rabv, vzv and mev (keller and stiehm ) . the development of human or humanized monoclonal antibodies (humabs) has created a feasible way to rapidly generate novel antiviral therapeutics. humabs have advantages over serum therapy in that they are chemically defined reagents with minimal variability, greater activity per mass of protein, and they have no immunological consequences related to serum sickness. several mabs have been approved for treatment of infectious diseases including viral and bacterial pathogens (table . ). the antiviral mab discovery field is exploding with activity particularly for hiv and hcv infections. a humanized mab targeting lymphocyte ccr receptors called pro has demonstrated potent and prolonged anti-hiv- activity and a large margin of safety (jacobson et al. a) . administration of pro by the subcutaneous route offers patients a way to self-administer the mab but importantly the mab is transported in the lymphatics providing enhanced access to binding to the cellular target (jacobson et al. b) . the next generation of antiviral therapeutics are likely to be dominated by mabs. perhaps the only way to clear a viral infection involves a host immune response (table . ). the innate immune response is particularly effective centered on a type -interferon pathway. unfortunately, many viruses carry mechanisms to evade host innate responses and innate immune effectors do not have the capacity for memory. the adaptive immune response can mitigate infection with antibodies, generally to surface antigens, which prevent the spread of the virus and t-lymphocytes, which can clear virus-infected cells. the first strategy will be to use an existing drug designed for another virus offlabel. this seems likely for antiviral drugs like cidofovir, foscarnet, and ganciclovir particularly for double-stranded dna viruses like ebv, hpv, and hhv . ribaviran has been used for a number of single-stranded rna viruses including polio, junin, and lassa fever. secondary strategies will require investment of time and effort beginning with vaccine development and creation of monoclonal antibodies. platform technology rna-based therapeutics offer rational design for an expansive number of new antiviral strategies. this advantage is superimposed on the theoretical advantages of selectivity, specificity and affinity provided by watson-crick base pairing. rna-based therapeutics are expected to provide a substantially more narrow range of pharmacokinetic properties and toxicities thus are easier to compare to each other and ultimately combine into multi-agent cocktails. however, the mechanism of action may vary from rnase h or risc mediated degradation of the targeted rna or steric inhibition of rna function. the objectives of our antiviral program have been to exploit the broad understanding of rna-based therapeutics for antiviral activity with a common chemical type, the phosphorodiamidate morpholino oliogmer (pmo) and their enhanced derivatives. in this way the mechanism of action is common to all agents, which is steric blockade of rna function. studies reported by zamecnik and stevenson introduced the first approach to identification of an antisense antiviral agent stephenson and zamecnik ) . they used a -mer targeted to rous sarcoma virus. since the rous sarcoma virus pioneering efforts, antiviral rna based therapeutics have involved multiple oligomer chemistries with a variety of different mechanisms of action. chemical approaches to oligomers directed to hiv have been plentiful. a nonionic methylphosphonate oligonucleotide targeted to the splice acceptor site of hiv tat inhibited splicing of viral rna inhibiting syncytia formation and p synthesis at um concentration. poor aqueous solubility limited the utility of the methylphosphonate chemistry. phosphoramidate chemistry was investigated for inhibition of the splice-donor and splice-acceptor of hiv tat agrawal et al. ) and were more potent but these agents were cytotoxic and poorly water soluble. phosphorothioate chemistry targeting hiv-rev (matsukura et al. ) and hiv-tat were shown to be effective in inhibiting hiv replication, were not cytotoxic and were very soluble. further, the hiv-rev phosphorothioate oligodeoxynucleotide was stable in vivo with an acceptable pharmacokinetic profile (iversen et al. ) . a -mer phoshorothioate called gem targeting the initiation site of hiv-gag was evaluated in clinical trials (agrawal ) but the trials were discontinued. i focused on phosphorodiamidate morpholino oligomer chemistry which is both stable and net-neutral in charge at physiological ph. single stranded rna viruses with positive sense (ssrna(+)) these viruses are the most simple in terms of genome size, number of potential translated viral proteins, their genomes are all linear and they enter the cell ready for translation. the design of steric blocking rna-based therapeutics involves preventing translation, disrupting rna secondary structure and masking recognition sites for rna dependent rna polymerase (rdrp). the targeting of either the ′-terminus and the first orf-aug are active. further, efficacy in animal challenge studies is observed with high fidelity when the most effective agent identified in vitro is employed. the family astroviridae with six different human astroviruses (huastv) responsible for - % of all gastroenteritis. we found the ′-terminus to be the most effective site to target. this was also the optimal site for caliciviridae including vesivirus (vev; martin-alonso et al. ; stein et al. ), norovirus (nov; bok et al. ) , and feline calicivirus (fcv; smith et al. ) ; the flaviviridae including dengue (den; kinney et al. ; holden et al. ) , and west nile virus (wnv; deas et al. ; zhang et al. ) , arteriviridae including agriculturally important equine arterivirus (eav; van den born et al. ) , and porcine respiratory and reproductive virus (prrsv; zhang et al. a, b) ; and the togaviridae exemplified by venezuelan equine encephalitis virus (veev; paessler et al. ). the family coronaviridae revealed a new active target site in the transcription regulatory sequence ( '-cgaac- ′) in both mouse hepatitis virus (mhv; neuman et al. ) and the severe acute respiratory syndrome virus (sars; neuman et al. ) . the picornaviridae active target site involved a highly conserved sequence in the internal ribosomal entry site (ires) in polio virus (pv; stone et al. ) , foot and mouth disease virus (fmdv; vagnozzi et al. ) , and the coxsackievirus (cvb ; yuan et al. ) . single stranded rna viruses negative sense (ssrna(−)) these viruses are generally more complex with respect to genome size, number of potential translated viral proteins, and multiple genome segments. the genome must be replicated prior to translation of viral proteins. the design of steric blocking rna-based therapeutics is similar to the positive sense rna genomes in that targets involve preventing translation and masking recognition sites for rna dependent rna polymerase (rdrp). we investigated different targets in measles virus (mev), a member of the paramyxoviridae, finding the translation initiation start site of n the optimal target (sleeman et al. ). studies with the human respiratory syncytial virus (hrsv) found the translation site for l to be most active (lai et al. ) . the orthomyxoviridae studies investigated influenza a virus probing each of the viral segments finding translation of pb active as well as the 'terminal of vnp for h n (ge et al. ) and h n (lupfer et al. ) but a combination of targets was required in animal studies with a high pathogenic viral h n isolate (gabriel et al. ). more extensive influenza a studies revealed a new target, the m-segment splice donor site. this target was evaluated in phase i clinical trials. the antisense platform technology has limitations in targeting viral sequences. inhibiting virally encoded proteins or blocking viral replication by interfering with the polymerase does not always work. the arenaviridae family proved difficult. we experienced some success with junin and lymphochoriomeningitis virus (lcmv) in cell culture observing log reductions in viral titer with a pmo targeting the highly conserved viral genome terminus. however, we failed to provide survival benefit to guinea pigs challenged with either junin or lassa virus. we then tried the mouse challenged with lcmv and failed again but we observed hemorrhagic disease in the mouse, a severe consequence of infection that mimics the worst aspects of arenaviral infection. we decided to try targeting host genes to mitigate disease in the mouse and found targeting il- provided a survival benefit (schnell et al. ) . the success in the face of failure puts targeting host genes at the center of attention for dealing with emerging infectious diseases. the antisense platform technology represents an excellent approach to rapid drug discovery for emerging infectious disease. rapid discovery demonstrated repeatedly but of course, the cost of advanced development is daunting. the application is best for immediate treatment of index cases, close contacts, and healthcare workers. we have an -pound gorilla in the room and everyone in the room considers it someone else's problem. the current course is to ignore the majestic creature until it starts tearing limbs from people and then the consensus is to kill the gorilla. agencies like the world health organization (who), the centers for disease control (cdc) and the national institutes of health (nih) can assemble highly skilled personnel and can confer with some of the greatest minds in the world. unfortunately, they are all aware of the potential problem that an emerging pandemic is likely to take us by surprise. significant speculation that a single-stranded rna virus will emerge killing tens of million people, costing hundreds of billions of dollars, and changing the course of human history. there is a high probability that the virus will be influenza a (h n or h n ) that will jump from a reservoir population of birds and establish human-to-human transmission. the pandemic will be a global event by the time an effective vaccine is available. neuraminidase (na) inhibitors as therapeutic and prophylactic agents in the setting of pandemic influenza a (flua) were called in to doubt in the past decade (michiels et al. ; jefferson et al. ) . indeed, % of isolates in the / a/h n pandemic were found to be resistant to adamantanes, and resistance to oseltamivir (tamiflu; osl) was observed in virus recovered from individuals taking osl therapeutically or prophylactically (dharan et al. ; cdc mmwr ) , while effect on duration of shedding was not impacted. a recent outbreak of an influenza a (h n ) virus caused cases and deaths in china revealed a novel na mutation r k resulting in high level resistance to osl (wang et al. ) . therapeutic options for treatment of individuals with complicated influenza a are severely limited, perhaps no options. pandemic strains such as a/h n pdm carried significant morbidity and mortality, particularly in those who had not experienced h -strain influenza in their lifetime. we are also witnessing more rapidly emerging highly pathogenic avian influenza strains that are resulting in human infection; some such as a/h n and a/h n appear to becoming more efficient in person-to-person transmission, and reports suggest osl resistance develops during treatment (de jong et al. ; lam et al. ) . we are not ready for an outbreak of avian flu or any other emerging single-stranded rna virus. why not? an estimate for the time and cost to develop a new drug is years and $ billion. the commercial use of a drug for an emerging infection is hard to estimate since by definition when you start development the infection has not emerged. most of us would be unlikely to use our retirement savings to invest in a drug development project with no reliable way to expect a return on our investment. it is a poor business model. when you consider there may be hundreds of emerging infectious diseases each times years and $ billion each the task is daunting. on which disease should we focus? consider the ebola drug avi- . the ebola therapeutic project began in following a laboratory accident at usamriid. we identified three compounds each with activity and when combined we observed unprecedented efficacy in a lethal challenge primate animal model (warfield et al. ). hundreds of experiments over the next years optimized these agents (swenson et al. ). we were able to obtain research grants from the transformational medical technologies (tmt) division of the defense threat reduction agency (dtra) within the department of defense (dod) and we completed proof of efficacy studies (warren et al. ) . this led to submission of an investigational new drug application (ind) to the food and drug agency (fda) and phase i safety and tolerability studies were conducted i healthy human volunteers (heald et al. ) . after years of continuous effort, we completed key aspects related to the fda approval process under the "animal rule" and we streamlined our treatment to a single agent, avi- (warren et al. ) . however, shortly before the ebola outbreak in western africa, the us government "budget sequester" cancelled our project and the most advanced therapeutic on the planet was not deployed to treat those infected during the outbreak. as the outbreak continued, we found no viral resistance of avi- unlike the monoclonal antibody therapy in use (khiabanian et al. ) . avi- sits on a shelf, a political casualty and an unfavorable business model. the global virosphere may contain up to virus/virus-like particles (suttle ) , the greatest reservoir of genetic diversity. the earth's atmosphere transports viruses all over the planet. viruses are found in soil at . x to . x particles per gram of dry soil (kimura et al. ) . the surface oceans carry approximately ten million viral particles in each milliliter of seawater, most of which are bacteriophages (phage). the small viral particles are easily carried into the upper environmental viral reservoirs atmosphere by up drafting winds. bacteria are deposited from the atmosphere at a rate of . to x per meter each day and viral deposition rates are - times greater (reche et al. ) . these phages influence bacterial lifecycles and play a role in natural energy and nutrient cycles fundamental to life on earth. the dynamics of phage-bacterial evolution drive changes in photosynthesis, phosphate, and nitrogen balance (breitbart ) . human accidental release of radioactive waste (discussed in chap. ) and disposal of chemicals including potent antiviral and antibacterial compounds (discussed in chap. ) may alter the eco-evolutionary dynamics producing unanticipated environmental consequences. the objective of this chapter has been to provide convincing evidence that infectious disease is the most significant threat to human health. the focus has been on viral infections because they rely on host ribosomes to produce their proteins, recent emerging infections have been from single-stranded rna genome viruses, and replication of rna viruses is error prone. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but have never prepared for these inevitable catastrophies. the long-term consequences often remain unrecognized and are always inconvenient such as cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. infectious disease will certainly continue to pose the most significant threat to human health in the age to cell phones, artificial intelligence, and global commerce. rapid replication of viral genomes combined with low fidelity polymerases provide the foundation for an unending source of new emerging infectious agents. these traits also make viral genomes sensitive to environmental contaminants in a way that may expand probabilities for zoonosis. infectious disease as part of our environment is not appreciated. the study of infectious disease is not a part of the curricula of students in environmental science/management. textbooks in in environmental studies do not include chapters in infectious disease. the integration of research at superfund sites focused on chemical contamination with infection and zoonosis would result in valuable insights into threat analysis. viruses with rna genomes lack sequence proofreading quality control during replication. the cumulative mutations in their genomes limits the genome size to under , bases. essentially, a larger genome would evolve out of existence, so called catastrophe evolution. the limited genome size makes these viruses exceptionally resilient to a changing environment. the virus must economize by combining functions. this means evolution and resilience are the same thing in the rna genome viruses. a unique insight is that in human evolution is restricted to the dna genome and resilience limited to rna, as it is in the rna genome viruses. antisense oligonucleotide-based therapy for hiv- infection from laboratory to clinical trials oligodeoxynucleotide phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus a tribute to sheik humarr khan and all the healthcare workers in west africa who have sacrificed in the fight against ebola virus disease: mae we hush inhibition of norovirus replication by morpholino oligomers targeting the '-end of the 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of rous sarcoma virus replication and transformation by a specific oligodeoxynucleotide characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with burkitt and non-burkitt lymphomas effect of early and late gb virus c viremia on survival of hiv-infected individuals: a meta-analysis suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions key: cord- -euzvhtax authors: janssens, wim; lehouck, an; decramer, marc; gayan-ramirez, ghislaine title: vitamin d and chronic obstructive pulmonary disease date: - - journal: vitamin d and the lung doi: . / - - - - _ sha: doc_id: cord_uid: euzvhtax vitamin d is an important regulator of calcium and bone homeostasis. it is also involved in the regulation of different genes and cellular functions, particularly in the context of inflammation, regeneration and immune control. conversely, vitamin d deficiency which is often found in chronic, infectious and inflammatory diseases is thought to drive or enhance uncontrolled inflammation. chronic obstructive pulmonary disease (copd) is characterized by chronic inflammation of the airways most often because of cigarette smoking. it has been recognized that repetitive airway infections and systemic consequences or co-morbidities also contribute to the progressive nature of copd. vitamin d deficiency is known to sneak in from the early stages of copd, to become highly prevalent at the more severe stages, and may thereby catalyse airway infection, inflammation and systemic consequences. undoubtedly, vitamin d deficiency enhances bone resorption and osteoporosis in copd for which appropriate vitamin d supplementation is recommended. however, conflicting evidence has emerged on the extra-calcemic effects of vitamin d in copd. a recent intervention trial with high-dose supplementation in copd was only able to reduce exacerbation frequency in the subgroup of patients with lowest baseline vitamin d levels. it confirms that severe vitamin d deficiency is a health hazard but that more clinical and experimental studies are needed to explore how vitamin d deficiency may affect airway biology and systemic effects in the context of smoke-induced lung diseases. over the last years, there is an increasing interest in the role of vitamin d and vitamin d de fi ciency in various chronic diseases. besides the well-known effect of vitamin d de fi ciency on bone loss in adults, accumulating evidence also links a low vitamin d nutritional status to highly prevalent chronic illnesses, including cancers, autoimmune diseases, infectious and cardiovascular diseases [ ] [ ] [ ] . vitamin d is now known to have an important in fl uence on immunoregulation and on the expression of antimicrobial peptides. since patients with chronic obstructive pulmonary disease (copd) often integrate all of these co-morbid diseases in one and given that most of copd exacerbations are triggered by bacterial and viral infection, vitamin d could play a key role in the pathogenesis of this disease. this chapter aims to discuss the prevalence and determinants of vitamin d de fi ciency in copd, the wellknown effect of vitamin d in the development and treatment of copd-associated osteoporosis and its potential role in the uncontrolled in fl ammatory cascade and systemic consequences of the disease. copd is a chronic disease characterized by air fl ow limitation that is progressive, not fully reversible and associated with an abnormal in fl ammatory response of the lungs to noxious particles or gases. in western countries, tobacco smoke is the major cause for copd, accounting for - % of the cases. since only % of smokers develop severe copd, other factors (biological, hereditary, environmental) must be involved [ ] . narrowing of the airways by in fl ammation, mucus production, irreversible remodelling and emphysema results in a limitation of expiratory air fl ow and disturbed gas exchange [ ] . currently, it is estimated that million people suffer from copd worldwide, a number that is still increasing. by , the world health organization predicts that copd will rise from the sixth to the third leading cause of death, next to only cardiovascular disease and cancer [ ] . according to the global obstructive lung disease (gold) de fi nition, diagnosis of copd should be based on spirometry measurements with a post-bronchodilator forced expiratory volume in s over forced vital capacity ratio (fev /fvc) below . . subsequently, copd can be categorized in different stages of severity (gold stages) going from mild, moderate and severe to very severe disease according to fev [ ] . it should be noted, however, that the majority of copd patients, especially in the early stages of the disease, report no complaints, do not perform spirometry and thus are unaware of their disease. treatment for copd focuses on minimizing symptoms and preventing exacerbations. with the exception of smoking cessation, so far, no treatment has clearly proven to signi fi cantly modify copd disease progression. recent data, however, indicate that pharmacological intervention with inhalation therapy may slow down the yearly decline of fev [ ] [ ] [ ] . the pathophysiology of copd is characterized by an increased in fl ammatory response in the airways and parenchyma. besides an increase in number of neutrophils, macrophages and t-lymphocytes, copd is associated with elevated concentrations of various cytokines, including interleukins (il- , il- and il- ), tumour necrosis factor alpha (tnf-a ), oxidative stress and the release of proteolytic enzymes. cigarette smoke is known to cause a direct injury of the airway epithelial cells leading to the release of endogenous intracellular molecules or danger-associated molecular patterns (damps). these signals are recognized by toll-like receptors on epithelial cells which initiate a non-speci fi c in fl ammatory response. as reaction on the release of early cytokines (tnf-a , il- , il- ), macrophages, neutrophils and dendritic cells are recruited to the site of in fl ammation [ ] . proteolytic enzymes and reactive oxygen species are released which may cause further damage to the lung. next, self-antigens and antigens coming from pathogens bound to dendritic cells can activate naive t cells into th cells [ ] and may lead to antibody producing b cells. this adaptive immune response may then further enhance the in fl ammatory cascade. in addition, the observed reduction of regulatory t cells (treg) in copd lungs against the rise of pro-in fl ammatory th cells is pointing towards an impaired immune regulation in copd [ ] . macrophages are believed to play a central role in the pathophysiology of copd [ ] . they are important sources of pro-in fl ammatory mediators but may also protect against infection by phagocytosis. in copd, alveolar macrophages appear to be resistant to the anti-in fl ammatory effects of corticosteroids by the reduced activity of histone deacetylase (hdac ), a nuclear enzyme that switches off in fl ammatory genes activated by the nuclear factor nf-k b [ ] . additionally, taylor and colleagues recently demonstrated that the phagocytotic capacity of macrophages of copd patients is impaired, which may lead to bacterial colonization and increased exacerbation frequency [ ] . overall, the complexity of the pathogenesis of copd with different mechanisms and risk factors is re fl ected in the broad variation of clinical phenotypes. with progression of the disease, as marked by a decline of forced expiratory volume in s (fev ), copd patients become more prone to exacerbations. an exacerbation of copd is an acute worsening of respiratory symptoms which is associated with increased symptoms and worsening of lung function. recent studies have shown that quality of life and health status of patients are mainly determined by the presence and frequency of such exacerbations which, in turn, may lead to a faster decline in fev [ ] . although viral and bacterial infections are assumed to be the major cause of exacerbations, other factors including environmental pollution and allergens have also been identi fi ed [ ] . the exact role of bacterial infection in copd exacerbations is often biased by bacterial colonization of the airways during stable state with the same organisms as those isolated at exacerbations: haemophilus in fl uenzae , streptococcus pneumoniae , moraxella catarrhalis , staphylococcus aureus and pseudomonas aeruginosa . different studies have shown that prevalence and load of organisms increases during an exacerbation [ ] , but recent evidence indicates that the acquisition of a new bacterial strain is likely the main trigger of an exacerbation [ , ] . besides bacterial infections, exacerbations may also be triggered by viral infections, including infections with rhinoviruses , coronavirus , respiratory syncytial virus , in fl uenza , parain fl uenza and adenoviru s. moreover, recently, a frequent exacerbator phenotype has been identi fi ed suggesting that the innate and adaptive immune defence of the host also contributes to exacerbation susceptibility and that individualized therapy may become important in the nearby future [ ] . copd is not restricted to the lungs but also associated with systemic in fl ammation and increased co-morbidities which are now considered as important targets in the therapeutic approach of copd. increased systemic levels of tnf-a , il- and c-reactive protein (crp) are commonly found in patients with copd [ ] . common co-morbidities of copd include lung cancer, cardiovascular disease, osteoporosis, diabetes and skeletal muscle dysfunction [ , ] . whether these co-morbidities are caused by the underlying disease or just co-exist because of common risk factors such as smoking, ageing and inactivity is far from understood. most likely, both mechanisms play together and the attractive hypothesis of in fl ammation in the lung "spilling over" into the systemic circulation and affecting other organs is corroborating this idea [ ] . whatever the mechanism may be, the presence of these co-morbidities clearly contributes to the poor outcome in copd [ , ] . vitamin d is generally obtained by photosynthesis in the skin but can also be derived from nutrition (fatty fi sh, fi sh liver oils and dairy products). ultraviolet light catalyses the fi rst step in the vitamin d biosynthesis, which is the conversion of -dehydrocholesterol into pre-vitamin d. the next step is a hydroxylation in the liver into -ohd, which then circulates in serum with a long half-life of days. next, -ohd is hydroxylated again into the active vitamin d metabolite , (oh) d by ahydroxylase (cyp b ) in the kidney which is controlled by serum levels of calcium and phosphate and regulating hormones such as parathyroid hormone (pth), calcitonin and phosphatonins. , (oh) d also induces the expression of a -hydroxylase (cyp a ) which catabolizes -ohd and , (oh) d into biologically inactive, water-soluble metabolites, thereby serving as its own negative feedback. the majority of -ohd and , (oh) d are bound to plasma proteins, of which more than % to the vitamin d binding protein (dbp), which carry out the delivery to their respective target organs [ ] . , (oh) d may thus bind to the nuclear vitamin d receptor (vdr) in the intestine, bone, kidney and parathyroid gland cells, resulting in the maintenance of normal serum calcium and phosphorus levels and their related effects on mineralization and turnover of bone [ , ] . since a -hydroxylase and the nuclear receptor vdr are widely present in cells of several extrarenal tissues such as skin, bone, prostate and immune cells, local , (oh) d concentrations can also exert different autocrine and paracrine functions. although the enzyme found here is identical to the one that is expressed in the kidney, its expression is regulated by immune signals instead of mediators of bone and calcium homeostasis [ , ] . , (oh) d mediates its effects by binding to the nuclear vdr the vitamin-vdr complex may then activate vitamin d response elements (vdre) on genes involved in different cellular processes. it is estimated that about % of the mouse/human genome is regulated by vitamin d [ ] . directly or indirectly, vitamin d controls many genes that are involved in the regulation of cellular proliferation, differentiation and apoptosis of healthy and pathological cells. because of its long half-life, -ohd is typically used to determine vitamin d status. it re fl ects vitamin d synthesized in the skin as well as that acquired from the diet and vitamin d degradation by catabolizing enzymes. when focusing on the calcemic effects, vitamin d insuf fi ciency is best de fi ned as a -ohd level below ng/ml ( nmol/l) [ , ] . a sensitive parameter to determine vitamin d de fi ciency is serum levels of pth. older data have clearly demonstrated that levels of -ohd below ng/ml are associated with an increase in pth expression [ ] . based on observational studies, several experts have suggested that, for non-calcemic effects, serum levels of at least ng/ml ( nmol/l) are required, but so far, intervention studies to support this are lacking. dietary sources of vitamin d are limited, and food forti fi cation is mostly inadequate or nonexistent. although sunlight is the most important source of vitamin d, several other factors can in fl uence the amount of vitamin d that can be synthesized: season, latitude, clothing, the use of sunscreen, darker skin pigmentation and age. for example, ageing is associated with decreased concentrations of -dehydrocholesterol in the skin, thereby reducing the capacity to synthesize vitamin d. even if regularly exposed to sunlight, elderly people produce % less cutaneous vitamin d than compared to young adults [ ] . due to cultural habits and clothing, even those who live in sunny climates are commonly found to be de fi cient in vitamin d. data from the third national health and nutrition examination survey (nhanes iii) revealed that in us adults, only % of the white population and % of the african americans had levels of vitamin d of at least ng/ml [ ] . according to the current de fi nitions, it is estimated that more than one billion people worldwide have impaired serum levels of vitamin d. as current supplementation regimens with a daily dose of - , iu of vitamin d restore de fi cient serum -ohd levels in a general adult population to concentrations above ng/ml, higher doses are probably required to increase -ohd levels to even higher levels that may be needed for non-calcemic diseases in a population at risk [ , ] . at present, we can only speculate on what such ideal target range of -ohd levels is to maximally exploit these extra-calcemic effects [ ] . we should also acknowledge that an extensive expert analysis of the potential effects of vitamin d supplementation on the health outcome of north-american subjects concluded that there is presently insuf fi cient evidence for extraskeletal bene fi ts of vitamin d therapy and that only new randomized controlled trials will be able to de fi ne such effect [ ] . copd patients should be considered at high risk to become vitamin d de fi cient for a variety of reasons: lower food intake, reduced capacity for vitamin d synthesis of the skin by ageing and smoking, the absence of outdoor activity and sun exposure, impaired activation by renal dysfunction and a lower storage capacity in muscles or fat by wasting may all contribute to a defective vitamin d status in copd [ ] . in , black and colleagues who examined spirometric data from the third national health and nutrition examination survey, a cross-sectional survey on , us civilians over years of age, discovered an important link between vitamin d and spirometric data [ ] . after adjustment for potential confounders, a strong relationship between serum levels of -ohd and pulmonary function, as assessed by fev and fvc, was found. although a signi fi cant correlation with airway obstruction could not be found, the observed dose-response relationship suggested a causal link [ ] . the observation that smoking african-americans more rapidly develop severe air fl ow obstruction as compared to caucasians is also in agreement with the idea that a presumed lower vitamin d status in african-americans correlates with an increased susceptibility to copd [ ] . furthermore, different genetic variants involved in the vitamin d signalling pathway are shown to determine -ohd levels [ ] , and some of these variants have repeatedly been associated with copd. for instance, variants of the dbp gene ( gc ) have been shown to be protective or risk factors for copd [ ] , and a more recent robust candidate gene study in two large data sets identi fi ed the gc genes as susceptibility loci for copd [ ] . in a study of forli and colleagues, vitamin d de fi ciency (< ng/ml) was found in more than % of a cohort waiting for lung transplantation [ ] , but they failed to compare vitamin d serum levels with a matched control group. however, we recently demonstrated in a group of smoking individuals that patients with copd were more likely to suffer from vitamin d de fi ciency than aged and gender-matched healthy control smokers without copd [ ] . in copd patients, we found a signi fi cant association between vitamin d serum levels and severity of disease assessed by fev . the prevalence of vitamin d de fi ciency de fi ned by -ohd levels below ng/ml increased to, respectively, % and % in severe (gold ) and very severe copd patients (gold ) (fig. . ) . interestingly, we also showed that -ohd levels were determined by genetic variants in the vitamin d binding gene ( gc) after correcting for age, gender, smoking history and disease severity and that homozygous carriers of the rs t allele with lowest vitamin d levels exhibited an increased risk for copd. although the risk effect of certain gc alleles may relate to a lower bioavailability of vitamin d, other authors have suggested that protein variants of dbp may directly affect in fl ammation in the airways [ ] . wood and colleagues, for instance, demonstrated that local dbp expression in sputum of copd patients correlates with macrophage phagocytosis capacity [ ] suggesting that the risk effect of dbp may be found in a different activation of macrophages [ ] . a recent population based study in , individuals living in hertfordshire (uk) found reduced vitamin d intake in the copd subgroup of individuals compared to the non-copd subjects but was not able to con fi rm the positive association between -ohd serum levels and fev or fvc [ ] . surprisingly, shaheen et al. found that patients with highest vitamin d levels were more likely to have copd. they concluded that in contrast to a prudent dietary pattern with the intake of high amounts of antioxidants [ ] , vitamin d was not an important determinant for adult pulmonary function and risk of copd. unfortunately, more than % of their population was taking dietary vitamin d supplements for unspeci fi ed reasons, which may have affected their analysis and biased their conclusions. finally, in a small sub-study of the lung health study iii conducted in selected individuals, mean -ohd levels of copd patients with a rapid fev decline were found to be similar to mean -ohd levels of patients with a slow decline, suggesting again that vitamin d de fi ciency does not contribute to copd progression [ ] . together, from these con fl icting data, it is clear that more prospective studies are needed to determine causal relationships between vitamin d de fi ciency and pulmonary function in copd. but even if causality cannot be found for pulmonary function variables, vitamin d de fi ciency may still alter the disease course by affecting other outcomes such as respiratory tract infections or co-morbidities. low levels of vitamin d result in low bioavailability of calcium which stimulates parathyroid glands to increase secretion of pth. in the kidneys, pth reduces the reabsorption of phosphate from the proximal tubule and increases calcium reabsorption in the distal tubule, resulting in a net increase in calcium/phosphate ratio. pth also induces renal a -hydroxylase expression which then leads to an increased production of active , (oh) d. , (oh) d enhances intestinal calcium absorption and acts on the immature osteoblastic cells to stimulate osteoclastogenesis through the rankl/rank regulatory system, with enhanced bone resorption and mobilization of calcium from the bone compartment, causing osteopenia, osteoporosis and increased risk for bone fractures [ ] . this results in higher levels of calcium and , (oh) d with a negative feedback on pth and a subsequent limitation of bone resorption. osteoporosis is a skeletal disorder which is characterized by compromised bone strength resulting in a higher susceptibility to fractures. bone strength is determined by the structural quality of the bone and by bone mineral density, the latter measured by dual x-ray absorptiometry (dxa) and used to de fi ne osteoporosis. osteoporosis is a major health problem as osteoporotic fractures are a frequent cause of signi fi cant and long-lasting morbidity in older individuals. hip fractures and other types of nonvertebral fractures account for most of the burden of osteoporosis, with increased mortality, functional decline, loss of quality of life and need for institutionalization [ ] . female gender, advancing age, a history of fragility fractures, current or former smoking, low body weight or weight loss and the use of systemic glucocorticoids are well-established risk factors for osteoporosis and osteoporotic fractures. as many of these risk factors are present in copd patients, especially at the more severe stages, it should be no surprise that osteoporosis and copd are strongly linked. a recent review of graat-verboom et al. con fi rmed low body mass, disease severity, use of corticosteroids, age and female gender to be independent risk factors for osteoporosis in copd [ ] . the majority of studies have reported an increased risk for osteoporosis with decreasing fev (fig. . ) [ ] [ ] [ ] . the prevalence of osteoporosis in copd varies between % and % depending on the diagnostic methods used, the population studied and the severity of the underlying respiratory disease [ ] . sin and colleagues used the nhanes iii data to demonstrate that air fl ow obstruction is independently associated with reduced bone mineral density [ ] . in their population-based cohort of , non-hispanic white participants, % of all women with severe copd had osteoporosis whereas almost all women with mild airway obstruction had osteopenia. in comparison, men were at lower risk than women but still, in men with severe copd, the prevalence of osteoporosis and osteopenia was % and %, respectively, which was approximately three times higher than expected. most studies looking at prevalence of osteoporosis have used dexa scans which measure bone mineral density but do not evaluate micro-architectural changes of the bone. it is known that these microarchitectural changes may equally cause fragility fractures, even with normal bone density, and should therefore be considered to be osteoporotic as well. when taking dexa measures and vertebral fragility fractures into account, graat-verboom et al. found that prevalence of osteoporosis almost doubled in copd outpatients [ ] . it indicates that prevalence of osteoporosis in copd might be much higher than the current prevalence data suggest [ ] . interesting relationships are also found with emphysema which is associated with reduced bone mineral density and lower body mass index and which may represent a clinical phenotype at risk for osteoporosis [ , ] . visual emphysema as assessed by ct scan was found to be an independent risk factor for osteopenia/osteoporosis [ ] , and different bone turnover markers are increased in copd. recent data also show that osteoprotegerin, which is critically involved in bone turnover by blocking rank-rankl interaction, may become a useful marker for parenchymal lung destruction in copd [ , ] . it is currently not known whether the vitamin d pathway is involved in the speci fi c development of emphysema, but studies in laboratory animals certainly corroborate this idea [ , ] . the consequences of osteoporotic fragility fractures in a copd population may be detrimental. in hip-fracture patients, mortality is close to % within year, and of those who do survive the fracture, again, some % will have to be institutionalized because of its functional consequences [ ] . the exact prevalence of hip fractures in copd patients has not been studied in detail, but it is probable that the impact of such events in disabled copd patients will be even worse. additionally, vertebral compression fractures can lead to back pain, functional impairments, increased kyphosis with reduced rib cage mobility and decline of pulmonary function [ ] [ ] [ ] . the impact of loss of vertebral height on pulmonary deterioration in copd has been demonstrated by leech and colleagues who found that vital capacity and total lung capacity incrementally declined as the number of thoracic vertebral fractures increased [ ] . in the eolo study, a large copd cohort of up to , participants, more than %, had one or more vertebral fractures and the prevalence signi fi cantly correlated with severity of disease [ ] . kyphosis related to osteoporosis may also cause limitation in rib mobility and inspiratory muscle dysfunction and was also correlated with loss of fev and fvc [ ] . there is no doubt that vitamin d protects against osteoporosis and osteoporotic fractures, and therefore, suf fi cient vitamin d supplementation should be encouraged. the fact that the majority of copd patients are of older age, have many common risk factors for osteoporosis and are more likely to be de fi cient in vitamin d supports standard supplementation, especially at the more severe stages of disease. a daily dose of - iu of vitamin d together with an adequate daily calcium intake ( , mg) is probably the best strategy to prevent fractures in older subjects. such supplementation is known to restore low serum -ohd levels in a general adult population to concentrations above the ng/ml ( nmol/l) threshold. patients with copd, particularly those on systemic corticosteroids, should also be considered for a dexa scan and if needed, osteoporosis medication [ ] . even though causality and therapeutic bene fi ts of vitamin d remain to be established for pulmonary in fl ammation and other co-morbidities, prevention of vertebral fractures will positively affect pulmonary function [ ] . along with a progressive loss of pulmonary function, copd patients become more prone to acute copd exacerbations which are an important cause of hospitalization, impaired quality of life and mortality [ ] . appropriate antimicrobial treatment is essential in the treatment of acute bacterial exacerbations whereas in case of colonization, repetitive and long-term antibiotic treatments are still avoided as they contribute to the multi-resistance of colonizing strains. anti-in fl ammatory treatments different from inhaled and systemic corticosteroids are currently validated in copd to reduce exacerbations on top of bronchodilator therapy. pde inhibitors and neo-macrolides are the most promising agents from this perspective and seem to be bene fi cial for a subgroup of patients with repetitive exacerbations [ ] [ ] [ ] . an attractive alternative approach might be the up-regulation of the innate immune defence system with vitamin d, particularly with regard to antimicrobial polypeptides [ ] . wang and colleagues demonstrated that in different cell types such as epithelial cells and white blood cells, the genes encoding for antimicrobial polypeptides such as cathelicidin (ll- ) and b -defensin are driven by vdr elements containing promoters [ ] . in human monocytes, tlr activation up-regulates expression of the vdr and the -a -hydroxylase genes, leading to induction of ll- and killing of intracellular mycobacterium tuberculosis [ ] . ll- is also found to be very effective in the killing of a number of antibiotic-resistant strains such as pseudomonas and s. aureus , different viruses and chlamydia [ , ] . as ll- is diffusely expressed in the surface epithelia of human airways, in the submucosal glands and in macrophages and neutrophils [ ] , substitution of local vitamin d insuf fi ciency may reduce bacterial load and concomitant airway in fl ammation [ ] . apart from its potential bene fi t on bacterial eradication, vitamin d may also down-regulate the complex in fl ammatory cascade at several levels. in vitro vitamin d can reduce the expression of tlrs which are critical in the induction of the early immune response [ ] . high levels of vitamin d also inhibit dendritic cell maturation with lower expression of mhc class ii molecules, down-regulation of co-stimulatory molecules and lower production of pro-in fl ammatory cytokines such as il- , il- , ifn-g and il- [ , ] . in several mouse models, vitamin d also leads to a switch from a th /th responses towards a th and regulatory t cell answer [ , [ ] [ ] [ ] . low serum levels of vitamin d have also been correlated with a decreased phagocytic activity of macrophages in patients with rickets [ ] whereas antimicrobial activity of macrophages against m. tuberculosis could be increased by vitamin d supplementation [ ] . overall, the potential of vitamin d in reducing proin fl ammatory processes of the innate and adaptive immune system, together with an increased bacterial eradication by self antimicrobial peptides and enhanced macrophage phagocytosis, may offer great potential in the treatment of exacerbations. indirect clinical evidence for such hypothesis may be found in the observation that exacerbations of copd are most common in winter, when -ohd levels are lowest. in addition, data from nhanes iii showed that upper respiratory tract infections were most frequent in patients with lowest vitamin d levels. to further investigate such intriguing hypothesis, a randomized placebo-controlled intervention trial was performed to evaluate the effect of vitamin d supplementation in copd patients, prone to exacerbations [ ] . one hundred and eighty-two patients with moderate to very severe copd and a history of recent exacerbations were supplemented with , iu vitamin d or placebo every weeks over one year. because of high-dose supplementation mean, -ohd serum levels increased signi fi cantly in the intervention arm and reached stable mean serum levels of ng/ml, which is in the therapeutic range of the hypothesized extra-calcemic effects [ ] . however, despite effective supplementation, no signi fi cant difference in time to fi rst exacerbation, time to fi rst hospitalization and exacerbation rate could be observed between the intervention and the control group. the absence of a therapeutic effect of vitamin d may relate to the fact that most of the patients presented with severe disease and were on maximal inhalation therapy. as all these treatments are known to reduce exacerbations [ , ] , it is likely that any additional effect of vitamin d on top of regular treatment is more dif fi cult to obtain. intervention within the more early copd stages taking less medications might therefore be more effective which is in line with the idea that such milder stages are also more sensitive to disease modi fi cation [ ] . interestingly, a signi fi cant baseline vitamin level by treatment interaction was observed for exacerbation rates. when performing a post hoc analysis in the subgroup of patients with very de fi cient vitamin d levels at baseline (< ng/ml), a signi fi cant % reduction of the number of exacerbations was observed in the intervention group. as one out of six copd patients in the trial presented with such asymptomatic low baseline -ohd levels which persisted during the entire course of the study, further focus and future studies on this important subgroup may be warranted, eventually resulting in better patient-tailored interventions. recently, a frequent exacerbator phenotype has been indenti fi ed suggesting that individualized therapy-including appropriate vitamin d substitution-may become important [ ] . we also assessed for the presence and load of pathogenic bacteria in cultures of morning sputa during the trial but found no difference in eradication between both study arms. however, monocyte phagocytosis capacity in peripheral blood monocytes of patients receiving vitamin d was signi fi cantly increased compared to the placebo group, an effect which was more pronounced in the subgroup with lowest baseline levels. it is therefore tempting to speculate that the signi fi cant reduction of exacerbations in the vitamin d de fi cient subgroup is explained by an important upregulation of impaired phagocytosis capacity [ ] . so far, hard evidence for this mechanism is lacking. finally, it should be noted that the lack of an overall effect could also relate to local vitamin d insensitivity because of epigenetic modi fi cations. in line with the known corticosteroid resistance in copd [ ] , recent studies in cancer have shown that epigenetic silencing of key enzymes of the vitamin d pathway may occur leaving tumour cells insensitive to vitamin d therapy [ , ] . at least, epigenetic modi fi cations especially in the context of smoking or poor diet may explain why many observations suggest causal relationships between vitamin d de fi ciency and in fl ammatory diseases whilst supplementation later on in the disease cannot reverse this process [ ] . skeletal muscle weakness is common in moderate to severe copd and is an independent predictor of respiratory failure and death [ ] . although the underlying mechanisms of skeletal muscle dysfunction in copd are not entirely understood, it is generally accepted that the combination of disuse because of respiratory limitation, with elevated oxidative stress, systemic in fl ammation, hypoxia and frequent steroid intake, is the main cause of deterioration [ ] . rehabilitation programmes in copd are proven successful, but there is still a large variability in training effectivity [ , ] . since muscle weakness is a prominent feature in rickets and chronic renal failure, and epidemiological studies found a positive association between -ohd levels and lower extremity function in older persons, vitamin d could be an important factor in muscle health [ ] . in elderly individuals, vitamin d status predicts physical performance and consequent decline during long-term follow-up [ ] . several double-blind randomized controlled trials demonstrated that vitamin d supplementation increased muscle strength and balance and reduced the risk of falling in elderly [ ] . although a recent meta-analysis looking at the effect of vitamin d supplementation on muscle strength was negative, positive effects were still found in very de fi cient patients [ ] . moreover, the cross-sectional analysis from nhanes indicated that muscle strength continued to increase throughout -ohd serum levels of - ng/ml, indicating that for obtaining bene fi cial effects on the muscle, higher dose supplementation might be necessary [ ] . on the pathological level, adults with vitamin d de fi ciency show predominantly type ii muscle fi bre atrophy [ ] with several muscle abnormalities such as enlarged inter fi brillar spaces, in fi ltration of fat, fi brosis and glycogen granules [ ] . conversely, increase in relative fi bre composition and type ii fi bre dimensions has been reported in elderly after treatment with vitamin d [ ] . these type ii fi bres are also the fi rst to be recruited to prevent falling [ ] , which may explain why vitamin d supplementation is shown to reduce falling [ ] . in copd, limb muscle adaptation leads to a decrease of the proportion of slow oxidative type i fi bres with a relative increase towards glycolytic type ii fi bres [ , ] , those fi bres that are preferentially affected by vitamin d de fi ciency. therefore, vitamin d de fi ciency may be of particular concern in copd patients with muscle weakness and dysfunction. the exact mechanisms by which vitamin d affect muscle function are not fully understood. however, , (oh) d may impair muscle function by altering calcium regulation. in particular, , (oh) d is responsible for the active calcium transportation into the sarcoplasmic reticulum by ca-atpase. it is known to regulate ca-atpase by phosphorylation of proteins in the sarcoplasmic reticulum membrane; it can increase phosphate transport across the membrane and interacts with calmodulin [ , ] . interestingly, calmodulin is highly sensitive to oxidative stress [ ] , a typical feature of copd, and , (oh) d may yield antioxidant properties. thus, both vitamin d de fi ciency and increased oxidative stress through, e.g. smoking may act synergistically impairing calmodulin function, muscle structure and contractility. , (oh) d has also an important role in protein synthesis in the muscle cell, mediated through nuclear receptor-mediated gene transcription [ ] . for example, it can affect actin and troponin c content, two major contractile proteins in skeletal muscle [ ] , or may up-regulate gene expression of muscular growth factors, for instance, igf-i [ ] . although it is tempting to extrapolate these vitamin d-mediated actions in skeletal muscles of healthy or elderly subjects [ , ] to a speci fi c population of copd patients, it is still to be shown that vitamin d de fi ciency contributes to the observed muscle weakness in copd. at present, there is no direct evidence for such causal relationship but the observation that vdr genotypes may in fl uence quadriceps strength in copd patients is in line with this assumption [ ] . recent data also show that vitamin d de fi cient copd patients referred for rehabilitation have a higher risk for dropout and reduced bene fi t on walking endurance [ ] . in a post hoc analysis in moderate to severe copd, we also found a signi fi cant effect of high-dose vitamin d supplementation on top of months of rehabilitation in terms of improved exercise capacity [ ] . therefore, randomized controlled trials investigating if supplementation of vitamin d de fi ciency may positively affect muscle force, muscle function and general copd outcomes are urgently needed. in the last years, many clinical studies have associated low vitamin d levels to prevalence and incidence of cancer, including lung cancer [ , ] . several studies associate vitamin d de fi ciency with autoimmune diseases like type i diabetes, multiple sclerosis and rheumatoid arthritis [ ] [ ] [ ] . de fi cient vitamin d levels have been linked to chronic infections such as tuberculosis and acute viral infections like in fl uenza or upper tract respiratory infections [ ] [ ] [ ] [ ] . similar data are also available which link vitamin d de fi ciency to cardiovascular diseases, arterial hypertension and even all cause mortality [ ] . it is beyond the scope of this book chapter to review all evidence on vitamin d in these different chronic diseases, but it is striking that many of them are currently considered to be co-morbid conditions of copd and accepted as important determinants of copd outcome and prognosis [ , ] . tackling vitamin d-mediated effects in these co-morbid conditions may therefore indirectly improve copd status [ ] . it should be stressed, however, that the above mentioned relationships between vitamin d de fi ciency and different chronic diseases are speculative and most often rely on cross-sectional and retrospective observations or on evidence of in vitro and animal research. in humans, placebo-controlled intervention studies and observational studies with prospective long-term follow-up speci fi cally designed to demonstrate causal relationships are often lacking. moreover, recent intervention studies with vitamin d supplementation in multiple sclerosis, diabetes, in fl uenza and tuberculosis have reported disappointing results, most often by their limitation of statistical power or insuf fi cient supplementation [ ] [ ] [ ] [ ] . recent expert analysis therefore concluded that there is yet insuf fi cient evidence for extra-calcemic bene fi ts of vitamin d therapy and that more randomized placebo-controlled interventions trials are needed to de fi ne such effects [ ] . for copd in particular, intervention studies targeting co-morbid conditions to improve copd-speci fi c outcomes will be needed and in analogy with the ongoing trial on statins in copd patients ( www.clinicaltrials.gov ), one may think about a large intervention study with vitamin d supplements. indirectly or directly, vitamin d is also believed to regulate extracellular matrix homeostasis in other tissues than bone, within particular lung and skin tissue via the control of transforming growth factor-b , matrix metalloproteinase and plasminogen activator systems [ ] [ ] [ ] [ ] . there is compelling evidence that vitamin d plays a key role in foetal lung growth, development and maturation [ , ] . although . (oh) d toxicity in klotho-null mice results in a phenotype of skin atrophy, osteoporosis and emphysema [ ] , recent data in mice show that severe vitamin d de fi ciency from early life also results in a impaired lung function by differences in lung volume and growth [ ] . evidence from human epidemiological studies also suggests that higher prenatal uptake of vitamin d protects against childhood wheezing [ ] . as impaired lung growth and childhood asthma are known risk factors for copd at later age [ , ] , the causal link between vitamin d de fi ciency and copd may therefore already exist from early childhood on. again, recent evidence from animal studies corroborates this idea. vdr knockout mice develop emphysematous airspace enlargement which is associated with the up-regulation of matrix metalloproteinases in the lung, an increased in fl ux of in fl ammatory cells and the development of typical lymphoid aggregates around the peripheral airways [ ] . the evidence that the vitamin d pathway plays a pivotal role in the biology of healthy and diseased lungs is compelling. as with many chronic diseases, vitamin d de fi ciency is highly prevalent in copd and occurs more frequently with increasing disease severity. such de fi ciency may not only enhance local airway in fl ammation but may also induce or accelerate ongoing co-morbid diseases and subsequently impair the general prognosis of the disease (fig. . ). so far, there is only hard evidence for a causal role of vitamin d de fi ciency in the pathogenesis of copdrelated osteoporosis. for extra-calcemic effects, a recent randomized controlled trial demonstrated no overall effect of high-dose supplementation on exacerbations but was supportive for an important effect in a limited subgroup of patients highly de fi cient for vitamin d. before embarking on new intervention trials with vitamin d in copd targeting subgroups, more fundamental research is needed to learn how vitamin d and its de fi ciency interact with 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edge: , -dihydroxyvitamin d is a direct inducer of antimicrobial peptide gene expression toll-like receptor triggering of a vitamin d-mediated human antimicrobial response human cathelicidin (ll- ), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity activities of ll- , a cathelin-associated antimicrobial peptide of human neutrophils the peptide antibiotic ll- /hcap- is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface fighting infections with vitamin d vitamin d down-regulates monocyte tlr expression and triggers hyporesponsiveness to pathogen-associated molecular patterns vitamin d signaling in immunemediated disorders: evolving insights and therapeutic opportunities prevention of autoimmune diabetes in nod mice by , dihydroxyvitamin d the coming of age of , -dihydroxyvitamin d( ) analogs as immunomodulatory agents immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a t helper (th) /th to a th and regulatory t cell pro fi le evaluation of phagocytosis in rickets control of mycobacterium tuberculosis through mammalian toll-like receptors high doses of vitamin d to reduce exacerbations in copd: a randomized trial a -year trial of tiotropium in chronic obstructive pulmonary disease combined salmeterol and fl uticasone in the treatment of chronic obstructive pulmonary disease: a randomised controlled trial treatment of copd: the sooner the better? decreased histone deacetylase activity in chronic obstructive pulmonary disease vdr microrna expression and epigenetic silencing of vitamin d signaling in melanoma cells dna methylation-related vitamin d receptor insensitivity in breast cancer vitamin d and susceptibility of chronic lung diseases: role of epigenetics peripheral muscle weakness contributes to exercise limitation in copd pulmonary rehabilitation in chronic obstructive 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dose-escalation trial of vitamin d and calcium in multiple sclerosis randomized trial of vitamin d supplementation to prevent seasonal in fl uenza a in schoolchildren vitamin d supplementation reduces insulin resistance in south asian women living in new zealand who are insulin resistant and vitamin d de fi cient-a randomised, placebo-controlled trial vitamin d as supplementary treatment for tuberculosis: a double-blind, randomized, placebo-controlled trial -dihydroxyvitamin d and its analogues down-regulate cell invasion-associated proteases in cultured malignant cells (oh) d is an autocrine regulator of extracellular matrix turnover and growth factor release via erp activated matrix vesicle metalloproteinases sputum matrix metalloproteases: comparison between chronic obstructive pulmonary disease and asthma upregulation of mmp- production by tnfalpha in keratinocytes and its attenuation by vitamin d childhood asthma may be a consequence of vitamin d de fi ciency the in utero effects of maternal vitamin d de fi ciency: how it results in asthma and other chronic diseases premature aging-like phenotype in fi broblast growth factor null mice is a vitamin d-mediated process maternal vitamin d intake during pregnancy and early childhood wheezing early life origins of chronic obstructive pulmonary disease risk factors for chronic obstructive pulmonary disease in a european cohort of young adults vitamin d de fi ciency and chronic obstructive pulmonary disease: a vicious circle key: cord- -naromr a authors: mcleish, caitriona title: evolving biosecurity frameworks date: - - journal: the palgrave handbook of security, risk and intelligence doi: . / - - - - _ sha: doc_id: cord_uid: naromr a the relationship between infectious disease and security concerns has undergone an evolution since the end of the cold war. what was previously seen as two separate domains – public health and national security – have, through various events and disease outbreaks in the last years, become intertwined and as a result biosecurity policies now need to address a spectrum of disease threats that encompass natural outbreaks, accidental releases and the deliberate use of disease as weapons. in the last decade of the twentieth century, particular concern began to be expressed that globalisation was facilitating the spread of infectious disease. in for example the us institute of medicine issued a report which warned "some infectious diseases that now affect people in other parts of the world represent potential threats to the united states because of global interdependence, modern transportation, trade and changing social and cultural patterns" (lederberg et al, , pv) . framing infectious disease in this way was part of a growing appreciation that a series of new security challenges, such as terrorism, drug trafficking, and environmental degradation, were supplanting the more traditional state centric national security concerns of the cold war era. (brower and chalk, ) as remarked upon by james woolsey during his nomination hearing for director of the central intelligence agency in : "in many ways today's threats are harder to observe and understand . . . yes, we have slain a large dragon, but now find ourselves living in a jungle with a bewildering number of poisonous snakes" (woolsey, , p ) . the intelligence community had first taken up the issue of the threat posed by infectious disease in the s in relation to hiv/aids (cia, ) . however a declassified national intelligence estimate from january expanded the scope of diseases that might pose security concerns. the report noted, for example, that since at least thirty previously unknown diseases had been identified and at least twenty older infectious diseases had re-emerged or spread geographically over the same period frequently in drug resistant form. the authors of the report believed that "the spread of infectious diseases results as much from changes in human behaviourincluding lifestyles and land use patterns, increased trade and travel, and inappropriate use of antibiotic drugs-as from mutations in pathogens" and suggested that, new and re-emerging infectious diseases will pose a rising global health threat and will complicate us and global security over the next years. these diseases will endanger us citizens at home and abroad, threaten us armed forces deployed overseas, and exacerbate social and political instability in key countries and regions in which the united states has significant interests. (nic, ) what prompted the release of this national intelligence estimate was the announcement by the then us secretary of state madeline albright that the first un security council session of the new millennium would be devoted exclusively to the threat to africa from hiv/aids. whilst this session is often remarked upon for ultimately leading to resolution on the responsibility of the security council in the maintenance of international peace and security: hiv/aids and international peace-keeping operations, it was the discussions within the session that did much to characterise the evolving nature of the relationship between infectious disease and security concerns. un secretary general kofi annan, for example, noted that the impact of aids in africa was "no less destructive than that of warfare itself and by some measures it was far worse" and went on: nowhere else had aids become a threat to economic, social and political stability on the scale that it now was in southern and eastern africa . . . in already unstable societies . . . that cocktail of disasters was a sure recipe for more conflict. and conflict, in turn, provided fertile ground for further infections. the breakdown of health and education services, the obstruction of humanitarian assistance, the displacement of whole populations and a high infection rate among soldiers . . . all ensured that the epidemic spread ever further and faster. (unsc, ) as president of the security council during this session, us vice-president al gore noted that the links being articulated between hiv/aids and insecurity presented an opportunity to recast the work of the security council for the new century. with echoes of woolsey's comments at his nomination hearing for cia director seven years earlier, gore is reported to have said that for the past years the security council: had dealt with a classic security agenda built upon common efforts to resist aggression, and to stop armed conflict. but while the old threats still faced the global community, there were new forces that now or soon would challenge the international order, raising issues of peace and war . . . includ[ing] the challenges of: the environment; drugs and corruption; terror; and new pandemics. (ibid) three months later, in april , president clinton took the unprecedented step of designating an infectious disease (aids) a threat to us national security (gellman, , pa ) taken together, these actions signalled a "securitization" (buzan et al, ) of infectious disease that resulted in greater political interest and access to larger economic resources so as to tackle to issue on a global scale. in line with the "securitization" thesis, political interest in hiv/ aids has remained high and superior financial resources have indeed been accessed. this included us president george w. bush promising $ billion over five years to international hiv/aids programmes in his state of the union speech. however, selgelid and enemark ( ) note that hiv/ aids is a disease of attrition, meaning that "the effects of these diseases are relatively familiar and slow-acting, they do not concentrate the minds of people and politicians as readily as an unfamiliar and sudden outbreak crisis." consequently it was growing anxiety over a perceived new type of terrorist that may deliberately use infectious disease to further their aims which gave further salience to the relationship between infectious disease and security concerns. the attacks on the world trade center and the pentagon on / fundamentally altered perceived societal vulnerability towards terrorist use of infectious disease. though the events themselves were quite unrelated to biological weapons (i.e. the hostile use of disease), the idea that non state actors, including terrorists, might seek to employ biological weapons to further their aims was lifted from (arguably) a niche concern to a mainstream security issue. calling it niche is not to say that bioterrorism had not been considered a security threat prior to many commentators had noted the potential (see for example stern, ; tucker, tucker, , moodie and roberts, ; smithson and levy, ) ; table top exercises had been conducted, domestic preparedness programmes initiated (guillemin, , p ) , and in countries such as the us, policy directives had been crafted that gave the highest priority to "developing effective capabilities to detect, prevent, defeat and manage the consequences of nuclear, biological or chemical materials or weapons use by terrorists" (united states, ) . however what the / attacks did was alter the global frame of reference about what terrorists writ large might now be prepared to undertake. the attacks appeared to suggest that what had been considered previously as restraining factors on terrorist actions, such as limiting casualties so as not to "risk of alienating the public especially their own supporters" were no longer valid (butler, , p ) . instead this new breed of terrorist and extremist appeared to want to cause casualties on a massive scale, and appeared undeterred by the fear of alienating the public, their own supporters, or indeed by considerations of personal survival. after the sheer destructiveness of / tucker ( ) notes that it was a logical next step for government officials to voice "fears that terrorists might unleash a devastating epidemic" as part of a second wave of attacks and in early october this hypothetical bioterrorism threat became a reality with the first death from inhalational anthrax in the us since . twenty-one others went on to be diagnosed with either inhalational or cutaneous forms of anthrax and five more people died. the source of the exposure was five letters containing anthrax spores anonymously posted to media outlets and members of the senate. coming so soon after the / attacks, these letters created a near hysterical atmosphere. tucker writes: cable news networks hyped the bioterrorism threat with apocalyptic scenarios; postal workers sorted mail wearing rubber gloves and surgical masks; thousands of senate staff members were put on prophylactic antibiotics; and letters addressed to government officials were irradiated with electron beams to kill lingering spores, delaying mail for weeks. meanwhile, tens of thousands of ordinary americans stockpiled ciprofloxacin (a potent antibiotic with potentially dangerous side effects), snapped up gas masks of questionable effectiveness from army supply stores and hoarded canned food and bottled water in anticipation of spreading epidemics and quarantines. (tucker, , p ) although the letters were only posted in the us, the anthrax letter attacks had global impact particularly because of the cognitive link that was made between biological weapons and the perpetrators of the / attacks. in europe for example, civil protection and security forces were put on alert, and public health systems had to deal with numerous items of mail containing powders suspected of being contaminated with anthrax. and at the political level, european countries acted at both the community level and national level. in october for example, the heads of state and government asked for a european level programme to be prepared to improve the cooperation between member states for the evaluation of risks, alerts, and intervention, and the collaboration in the field of research. at the national level many european countries re-examined their preparedness plans and strengthened or implemented new measures designed to prevent the misuse of the biological sciences. this included placing restrictions on physical access to, and work performed with, certain pathogens labelled as "dangerous." european states were not alone in re-examining their preparedness programmes: in the us for example, at least three new pieces of legislation were enacted in quick succession aimed at preventing the misuse of disease and they significantly increased their investment in bio-defences, including medical countermeasures. at the international level, the threat from the deliberate spreading of disease slotted neatly into the global "war on terror" that president bush had launched in the days following / . addressing the united nations general assembly in november bush described terrorists as searching for weapons of mass destruction, the tools to turn their hatred into holocaust. they can be expected to use chemical, biological and nuclear weapons the moment they are capable of doing so. no hint of conscience would prevent it. this threat cannot be ignored. this threat cannot be appeased. civilization, itself, the civilization we share, is threatened. (bush, ) consequently the global community also acted together to combat the threat from bioterrorism. this included a range of activities including "operational" initiatives such as the proliferation security initiative, the g global partnership against the spread of weapons and materials of mass destruction and the global health security initiative as well as broadening the mandate of international organisations such as the world health organisation such that they now had a role in responding to the "natural occurrence, accidental release or deliberate use of biological and chemical agents or radionuclear material that affect health." (wha, ) at the diplomatic level, the work of the biological weapons convention (bwc) now became focused on a broadened understanding of the threat posed by biological weapons, including the possibility of terrorist use of biological agents. the focus prior to had been state level adherence to the norms of the bwc. however if properly implemented at the national level, the convention addresses potential terrorist use by transferring the obligations that states agree tonot to develop, produce, manufacture or stockpile biological and toxin weapons or methods of delivery of such weaponsonto individuals in their territory or under their jurisdiction anywhere. when tabling a number of proposals for future work in late the us delegation noted that "many of these ideas will bear little resemblance to the traditional arms control measures of the past" including the negotiation of a legally binding verification protocol which had recently failed (us department of state, ). these alternative proposals eventually initiated an "intersessional process" where states parties to the bwc meet twice yearly to discuss, promote common understanding and achieve effective action on a number of topics related to this broadened understanding of biological threats. viewed within the bioterrorism/war on terror framing, the political significance of mitigating naturally occurring disease outbreaks was elevated by linking global health engagement with set of efforts to counter violent extremism and bring stability to conflict-prone areas (chreiten, ) . consequently, much of the engagement that took place was therefore focused on africa as home to a number of fragile states with porous borders and groups linked to al qaeda. concurrent with this terrorism-focused framing of the threats posed by infectious disease, another more human security focused framing of disease was forwarded in documents such as the united nations high level panel on threats challenges and change where the challenges of disease were presented as follows: the security of the most affluent state can be held hostage to the ability of the poorest state to contain an emerging disease. because international flight times are shorter than the incubation periods for many infectious diseases, any one of million international airline passengers every year can be an unwitting global disease-carrier. (anan, p ) part of the stimulus for framing of the threats from infectious disease as "without borders" came from the experiences of the severe acute respiratory syndrome (sars) outbreak. the sudden appearance of sars had, by the time the world health organisation (who) declared the outbreak contained in july , spread to countries on all continents, infected more than people and presented an % lethality rate. (who, ) unlike the apocalyptic "dread risk" scenarios for bioterrorism attacks in the same period, sars was a "dread reality": evidence showed sars to be a fast spreading disease that did not require a vector; symptoms appeared to begin an average of four days after exposure to an infected person and mimicked many common diseaseshigh fever, a dry cough and shortness of breath (who, ) and the disease showed no particular geographical affinity. indeed on this last point an association was made early on between sars and travel on commercial airlines (see for example olsen et al, ) which resulted in guidelines being issued regarding travel to and from areas affected by sars that focused on hand hygiene and specified that anyone suspected of having sars should wear a facemask. however, public perception of the risk of becoming infected with sars led to widespread use of facemasks whether on a flight or not (see for example hesketh, . fear of infection was therefore a potent ingredient in the sars epidemic: in toronto, canada, there were reports of "public bus drivers using face masks on routes near chinese communities and empty seats surrounding chinese university students" (schram, , p ) and at the height of the epidemic, despite only eight people in the us having laboratory evidence of sars, eichelberger ( ) notes that % of americans reported avoiding asian businesses. indeed across the us "restaurants, travel agencies and other businesses from new york to san francisco [reported] customer traffic is down by % or more" (hopkins, ) . as with the anthrax letters then, the effects of the sars epidemic were not confined to ill health, or to those countries directly affected. indeed, it was the economic repercussions of the outbreak that came to define the disease. one assessment for example, estimated the total cost of the epidemic to the asian regional economy at us$ billion in gross domestic product for , "that is, over us$ million per person infected by sars," with gross expenditure and business losses being estimated as high as us$ billion (rossi and walker, p - ) the authors also note that this was a shared economic burden whether the country reported infections or not because of the association between airline travel and infection. this is because as elbe ( ) notes the travel and tourism sectors in the region were heavily affected with "room and airline seat bookings to [the region] down in several cases by more than per cent compared to previous years." any lingering doubts about whether the trans-border spread of infectious diseases created security issues were removed by the sars outbreak. sars also drew attention to potential security implications of a wider set of emerging and re-emerging infectious diseases that could no longer be ignored. indeed quickly on the heels of sars epidemic, concern began to be expressed over the pandemic potential of h n avian influenza. sensitised to the potential of an influenza a type pandemic by the outbreak of h n , or "bird flu," fear was now being expressed that h n could mutate or combine with a human influenza virus to form a new virus, capable of sustained human-tohuman transmission (see for example lee and fidler, and who, ). writing in the new york times members of the senate committee on foreign relations, barak obama and richard lugar, framed the relationship between national security and an influenza pandemic as follows: when we think of major threats to our national security the first to come to mind are nuclear proliferation, rogue states and global terrorism. but another kind of threat lurks beyond our shores, one from nature not humansan avian flu pandemic. an outbreak could cause millions of deaths, destablize southeast asia . . . and threaten the security of governments around the world (obama and lugar, ) what h n did, elbe ( ) notes, was render the mere possibility of a future outbreak a sufficient condition for considering an infectious disease as a threat to security and so requiring investment and proactive pandemic preparedness. indeed in january , the international community pledged us$ . billion to fight avian influenza and prepare for a possible human pandemic (beijing declaration, ) . the un high level panel report quoted above also alludes to another vulnerability that was exposed during the sars outbreak, namely the deficiencies in the contemporary reporting system for infectious disease outbreaks. at the time, the who was prevented from responding to an outbreak until it had received official reports from governments (heymann, ) . in the case of sars, there was a three-month delay from onset until the who received official reports from the chinese ministry of health by which time there were over cases and the disease had spread to five countries. part of the inadequacy of that reporting system was the mismatch between the framework under which the who had to work, the international health regulations, and the tools that the who had at its disposal in . for example the who were unable to act despite having "epidemic intelligence networks" such as the global outbreak alert and response network (goarn) in place at the time of the sars outbreak that had picked up on an outbreak prior to the official notification. this intelligence had been gathered by goarn's early warning element which collects and verifies reports and rumours of epidemics from a wide variety of unofficial sources, including nongovernmental organisations, news media, electronic discussion groups such as the program for monitoring emerging diseases, and other official surveillance networks. when the who was eventually able to act, the response side of goarn was activated and within a period of weeks after the first recognised case, a virtual network of eleven leading infectious disease laboratories in nine countries had been established. connected by a secure website and daily teleconferences, the laboratories collaborated to identify the causative agent of sars and to develop a diagnostic test; similar groups were also created to pool clinical knowledge and compare epidemiological data on sars (knobler et al, ) . the who used this information to make recommendations on patient management which included issuing travel recommendations in an attempt to curb, and eventually stop, the international spread of this newly recognised virus (heymann, ) . perhaps the most important legacy of the sars epidemic, and to a lesser extent the h n outbreak, was the sense of urgency it gave to finalising the updates to the international health regulations (ihrs). begun in the mid- s, the revision process had two primary goals: to make use of modern communication technologies to understand where diseases were occurring and had the potential to spread, and to change the international norm for reporting infectious disease outbreaks so that countries were not only expected to report outbreaks, but also respected for doing so (heymann, ) the updates were completed in and went into effect in . amongst the many updates, the establishment of a global surveillance system for public health emergencies was critical. surveillance is defined in the revised ihrs as "the systematic on-going collection, collation and analysis of data for public health purposes and the timely dissemination of public health information for assessment and public health response as necessary" (who, ). the surveillance system operates from the local to the global level. at the national level each state party is now required to notify who of "all events which may constitute a public health emergency of international concern" including any unexpected or unusual public health event regardless of its origin or source and also requires state parties, as far as is practicable, to inform the who of public health risks identified outside their territories that may cause international disease spread. to assist in compliance with this obligation, the ihrs defines a public health emergency of international concern (pheic) as an extraordinary event which is determined [by the who director-general] . . . (i) to constitute a public health risk to other states through the international spread of disease and (ii) to potentially require a coordinated international response. (ibid) and defines disease as an illness or medical condition irrespective of origin or source, that presents or could present significant harm to humans that does or could threaten human health. (ibid) a decision-tree to assist state parties in defining whether a health related event is a pheic is included, so too a list of diseases for which a single case may constitute a pheic and so must be reported to the who immediately. this list consists of smallpox, poliomyelitis, human influenza caused by new subtypes, and sars. arguably, as a direct result of perceived reluctance on the part of the chinese authorities to be transparent in the early stages of the sars outbreak, the revised ihrs state that the who can collect, analyse and use information "other than notifications or consultations" including from intergovernmental organisations, nongovernmental organisations and actors, and the internet. furthermore the who can now act upon the information gathered by requesting "verification from the state party in whose territory the event is allegedly occurring." when so requested, the state party has hours to give an initial reply to the who, or acknowledge the request from them, and if possible provide the who with available information on the status of the event referred to in the request. this is done through the newly required national focal point for the ihrs, a role established to ease communication between the who and the state party. in permitting the who to act upon that information and requiring states to perform some form of action within hours of that request, the principle of national sovereignty became subordinate to the collective interests of global disease surveillance. this had stalled the revision process, but as katz and fischer ( ) note the "sudden fear of the consequences of a single nation's failure to report an emerging infectionwhether due to lack of will or capacityovercame many of the concerns about sovereignty." although nowhere in the revised ihrs is the word "intentional" or "deliberate" used the scope of the definition of disease within the revised ihrs and the newly expanded role of the who with regard to deliberate disease outbreaks mean that the ihrs do encompass communicable and non-communicable disease events, whether naturally occurring, accidentally caused, or intentionally created. in part, this is because whether deliberate, accidental or naturally occurring, the initial response to the outbreak would be the same, meaning that early warning systems, indeed in general strong public health systems, serve multiple purposes. at the time of writing the ihrs have been in force for nine years and there have been four declared public health emergencies of international concern, including the ebola virus outbreak in west africa, declared a pheic on august . between march , when the outbreak was first reported, and march when the the who director-general declared the pheic at an end the total number of reported cases in the three worst affected countries (guinea, liberia and sierra leone) was , . a small number of cases were also reported in nigeria and mali and a single case reported in senegal; however, these cases were contained, with no further spread in these countries. in addition there were a small number of exported cases in spain ( case); the united states ( cases); the united kingdom ( case) and italy ( case). a review of who's response to this ebola outbreak characterised it as "the most complex outbreak on record . . . [which] devastated families and communities, compromised essential civic and health services, weakened economies . . . isolated affected populations . . . [and] put enormous strain on national and international response capacities, including who's outbreak and emergency response structures" (who, ) . indeed, the strain was such that the international response to the outbreak included the establishment of the first ever united nations emergency health mission, the united nations mission for emergency ebola response or unmeer, after the unanimous adoption of general assembly resolutions / and / , and the adoption of security council resolution ( ) on the ebola outbreak. whilst the idea that health issues and security are linked was by now firmly embedded within the international political consciousness and that response to outbreaks were considered both a national and international responsibility, the ebola outbreak served to highlight a significant mismatch between those ideas and practical realities. the review of the who's response noted above was extremely critical of the response effort on a number of levels. regarding the actions of the who itself, the panel's assessment regarded there to have been "significant and unjustifiable delays" in declaring the ebola outbreak a public health emergency of international concern, despite early warnings about the outbreak from its own staff and from non governmental organisations such as médecins sans frontières, and that the "who does not currently possess the capacity or organizational culture to deliver a full emergency public health response" (who, , p ) . part of the reason for this is that there are no core funds for emergency response and the panel recommended the immediate creation of a contingency fund in support of outbreak response as well as the establishment of a who centre for emergency preparedness and response which would develop the necessary new structures and procedures to achieve full preparedness and response capacity. considering the outbreak in terms of the revised ihrs, the panel also noted that nearly a quarter of who's member states "in violation of the regulations," instituted travel bans and other additional measures not called for by who, "which significantly interfered with international travel, causing negative political, economic and social consequences for the affected countries" (ibid, p ). the panel went on to say that they consider the situation "in which the global community does not take seriously its obligations under the international health regulations ( )a legally binding documentto be untenable" (ibid). implementation statistics for the revised ihrs do indeed demonstrate that many states have had difficulties in implementing what is required of them in this new system. all states were to have the new national core surveillance capabilities in place by june ; however, by that deadline less than % of the who member statesthat is statesreported they had achieved the core capacities; countries requested and obtained an additional two year extension and countries neither submitted an extension request nor indicated that they are in compliance (katz and fischer, , p ) . at the end of the second two-year extension period the who executive board noted that only an additional states ( nations in total) reported that they had fully implemented the revised ihrs (world health organisation, ) . in part to redress these implementation difficulties, the us in partnership with about other countries, ios, ngos and public/private enterprises launched the global health security agenda (ghsa) in february . the ghsa has discrete action packages under the three cluster heading of "prevent, detect and respond" covering issue areas such as antimicrobial resistance, zoonotic diseases, real time surveillance and reporting. eight of these packages relate in whole to the revised ihrs and a package is also specifically dedicated to improving biosafety and biosecurity systems and preventing bioterrorism. the spectrum of issues being addressed by the global health security agenda reflects the evolution of biosecurity issues since the end of the cold war. what had previously been considered as two separate domainspublic health and national securityhave now become merged to create a spectrum of biosecurity issues that encompasses naturally occurring incidents, accidental outbreaks and deliberate use of infectious disease. this intertwining is reflected in both domains: in the public health domain, the who for example had its mandate extended to include responding to deliberate use of biological agents and in the traditional arms control arena, states parties to the biological weapons convention are creating synergistic relations with public health organisations to further their aims of mitigating the effects of a deliberate use should it occur. in addition, the global health security agenda also reflects a change in views regarding responsibility for responding to this spectrum of biosecurity issues: whereas in the security council viewed hiv/aids as posing a threat to a geographically defined area, the sars outbreak in and the potential of an influenza pandemic shortly thereafter illustrated the truly global interconnected nature of the threat and so the shared international responsibility of responding to them. to use an argument put forward by andrew lakoff and stephan collier ( ) , the issue for the future is not whether a disease outbreak can be characterised as a biosecurity threat which requires attention but what kind of biosecurity problem does it present, what kind of techniques are used to assess them and what is the most appropriate kinds of responses. notes . on this see for example central intelligence agency, unclassified report to congress on the acquisition of technology relating to weapons of mass destruction, st july- st december . available at https://www.cia.gov/ library/reports/archived-reports- /july_dec .htm#chemical. . for example: in the uk, the anti-terrorism crime and security act, created a list of "dangerous" pathogens which required additional security requirements and access restrictions. in addition, the secretary of state now had to be informed of any premises where any dangerous substances was kept and used. . in addition to political action, the scientific community also responded to the perceived heightened vulnerability, especially addressing what actions they might take to support national efforts to prepare against deliberate attacks using disease and what actions they needed to take to prevent their work from being deliberately misused and contributing to the development of biological weapons. for more on this see mcleish c ( ) "science and censorship in an age of bioweapons threat" science and culture : , - ; mcleish c and p nightingale ( ) "biosecurity, bioterrorism and the governance of science: the increasing convergence of science and security policy", research policy ( ) - . . for more information on these initiatives see http://www.psi-online.info; http:// www.nti.org/treaties-and-regimes/global-partnership-against-spread-weaponsand-materials-mass-destruction- -plus- -over- -program/ and http://www. ghsi.ca/english/index.asp. . at the time of writing three such intersessional processes have been completed which have focused on topics as diverse as strengthening national implementation of the convention; assistance and cooperation in the events of a biological weapons attack; reviewing relevant developments in science and technology; and awareness efforts amongst scientists. for more information on the biological weapons convention and the intersessional process see www part of the reason for the unprecedented scale of the outbreak was its spread to urban centres including the capital cities of the three worst affected countries foreword' a more secure world: our shared responsibility united nations high level panel on threats challenges and change interrogating bio-insecurities' the global threat of new and re-emerging infectious diseases: reconciling us national security and public health policy review of intelligence on weapons of mass destruction security a new framework for analysis us military global health engagement since / : seeking stability through health the global aids disaster unclassified report to congress on the acquisition of technology relating to weapons of mass destruction sars and new york's chinatown: the politics of risk and blame during an epidemic of fear pandemic security federal agency biodefense funding aids is declared threat to security: white house fears epidemic could destabilize world china in the grip of sars the international response to the outbreak of sars in communicating disease risk: then and now sars scare hurts business in chinatowns international pledging conference on avian and human pandemic influenza the revised international health regulations: a framework for global pandemic response moving forward to the public health response to sars, institute of medicine, forum on microbial threats biosecurity interventions: global health and security in question emerging infections: microbial threats to health in the united states, committee on emerging microbial threats to health, division, institute of medicine avian and pandemic influenza: progress and problems with global health governance science and censorship in an age of bio-weapons threat biosecurity, bioterrorism and the governance of science: the increasing convergence of science and security policy terrorism with chemical and biological weapons: calibrating risks and responses, alexandria, va: chemical and biological arms control institute. national intelligence council ( ) the global infectious disease threat and its implications for the united states grounding a pandemic transmission of the severe acute respiratory syndrome on aircraft assessing the economic impact and costs of flu pandemics originating in asia how popular perceptions of risk from sars are fermenting discrimination hiv/aids, security and ethics ataxia: the chemical and biological terrorism threat and the will terrorists turn to poison? chemical/biological terrorism: coping with a new threat toxic terror: assessing terrorist use of chemical and biological weapons scourge: the once and future threat of smallpox united nations general assembly resolution / ( ) measures to contain and combat the recent ebola outbreak in west africa united nations mission for ebola emergency response un security council holds debate on impact of aids on peace and security in africa on the responsibility of the security council in the maintenance of international peace and security: hiv/aids and international peace-keeping operations united nations security council resolution ( ) peace and security in africa united states department of state ( ) new ways to strengthen the inter-national regime against biological weapons global public health response to natural occurrence, accidental release or deliberate use of biological and chemical agents or radio nuclear material that affect health summary of probable sars cases with onset of illness from pandemic influenza preparedness and response: a who guidance document report of the ebola interim assessment panel world health organization executive board ( ) implementation of the international health regulations originally trained as an historian and philosopher of science, her work focuses on issues relating to governance of dual use technologies and the design of effective mechanisms to prevent misuse of legitimate science and technology key: cord- -xhzvp g authors: berencsi, györgy; szomor, katalin n. title: fetal and neonatal illnesses caused or influenced by maternal transplacental igg and/or therapeutic antibodies applied during pregnancy date: - - journal: maternal fetal transmission of human viruses and their influence on tumorigenesis doi: . / - - - - _ sha: doc_id: cord_uid: xhzvp g the human fetus is protected by the mother’s antibodies. at the end of the pregnancy, the concentration of maternal antibodies is higher in the cord blood, than in the maternal circulation. simultaneously, the immune system of the fetus begins to work and from the second trimester, fetal igm is produced by the fetal immune system specific to microorganisms and antigens passing the maternal-fetal barrier. the same time the fetal immune system has to cope and develop tolerance and t(reg) cells to the maternal microchimeric cells, latent virus-carrier maternal cells and microorganisms transported through the maternal-fetal barrier. the maternal phenotypic inheritance may hide risks for the newborn, too. antibody mediated enhancement results in dengue shock syndrome in the first month of age of the baby. a series of pathologic maternal antibodies may elicit neonatal illnesses upon birth usually recovering during the first months of the life of the offspring. certain antibodies, however, may impair the fetal or neonatal tissues or organs resulting prolonged recovery or initiating prolonged pathological processes of the children. the importance of maternal anti-idiotypic antibodies are believed to prime the fetal immune system with epitopes of etiologic agents infected the mother during her whole life before pregnancy and delivery. the chemotherapeutical and biological substances used for the therapy of the mother will be transcytosed into the fetal body during the last two trimesters of pregnancy. the long series of the therapeutic monoclonal antibodies and conjugates has not been tested systematically yet. the available data are summarised in this chapter. the innate immunity plays an important role in fetal defence. the concentration of interferon is relative high in the placenta. this is probably one reason, why the therapeutic interferon treatment of the mother does not impair the fetal development. at term, the amount of maternal igg antibody is higher in the neonate than in the mother. this pattern holds when the igg antibody is an anti-tnfa medication. a specific fc receptor neonate (fcrn) facilitates transfer of the igg antibodies across the syncytiotrophoblast into the fetal circulation (kane and acquah ). due to the high rate of igg transfer near term, babies have been found to have similar blood levels of infliximab to the mother. by discontinuing this drug - weeks prior to delivery, the baby will likely be born with no or minimal serum levels, thus avoiding immunosuppression in a young infant. though there are some suggestive data that certolizumab does not cross the placenta as easily as the igg derived drugs due to the pegylation of the molecules (clowse ) . in early human placenta the exchange tissue area (villi) is formed around the entire surface of the conceptus. this placental shape is called diffuse placenta. by the third month of pregnancy, only the villi near the initial site of implantation have persisted, leading to the formation of the disc-shaped placenta. although chorioallantoic placenta in humans begins functioning already by the end of the fourth week of pregnancy, this process is completed with the formation of diskshaped placenta (sadler ) . during the first trimester, the human fetus is surrounded by two fluid cavities, i.e., the inner amniotic cavity and the outer extra-embryonic coelomic cavity. the chorioallantoic placenta is only formed of fetal vessels' endothelium and trophoblastic layer, bathed directly in the maternal blood (van der aa et al. ). for out of monoclonal therapeutic compounds, for which toxicity studies (in a broad sense) were performed, no significant maternal, fetal, or neonatal toxicity was observed. for the remaining seven products, the most common adverse effects on reproduction and development were reduced fetal weight, increased abortion rates, and reduction in fertility, indicating the general toxicity of these compounds (pentsuk and van der laan ). twenty-four ( %) of the children had one or more congenital anomalies that are part of vacterl association, but only one child (with maternal etanercept administration) was diagnosed with vacterl association i.e. v: vertebral defects, a: anal atresia or imperforate anus, c: cardiac abnormalities [atrial septal defect, ventricular septal defect, and tetralogy of fallot], t: tracheoesophageal fistula or tracheal atresia/stenosis, e: esophageal atresia, r: radial and or renal abnormalities, and pre-axial l: limb abnormalities (carter et al. (carter et al. , ). fc-fcgr interactions play a critical role in the biological function of antibody and are likely to be instrumental in preventing or modulating lentiviral infections. antibody responses that depend on fc-fcgr interactions may help widen the spectrum and increase the potency of vaccine-induced antibodies (forthal and moog ) . maternofetal transmission of non-neutralising dengue virus specific antibodies result in the development of hemorrhagic fever of the newborns even in the case of the first infection with a heterotypic virus. the mechanism is the formation of non-neutralised neonatal virus and heterotypic maternal igg complexes, which will be taken up by endothelial and hematopoetic cells by (virus-abfc)-fcgr pinocytosis. the risk disappears after the th month of age of the children, when the maternal igg disappeared from the circulation of the children (libraty et al. ). this phenomenon has been observed in the case of other flaviviruses and enteroviruses, too (ferenczi et al. ; wang et al. b ). dengue serotype cross-reactive cytotoxic lymphocytes (ctl) clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. high avidity cross-reactive memory ctl may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. these cells appear to be subsequently deleted leaving a more serotype-specific memory ctl pool. antibody-dependent enhancement ade is neither a sufficient nor an absolutely necessary precondition for the development of severe shock syndrome or hemorrhagic disease. ctl response to a viral infection can be modulated by the infection history of an individual in a manner likely to contribute to disease severity l€ uhn et al. ). respiratory syncytial virus (rsv) enhancer activity of transplacental maternal antibodies have been observed first by osiowy et al. ( ) . rsv infection is also enhanced by non-neutralising antibodies (johnson and graham ) . vaccination using inactivated virus particles were caused immune enhancement in the case of rsv in contrast to other, for example influenza viruses (openshaw et al. ; ye et al. ) . cytotoxic t-cells, cytokines and interferon induction by toll-like receptors may potentiate this enhancement (mobbs et al. ; boukhvalova et al. ; tregoning et al. ; lee et al. a, b; nguyen et al. ; ubol and halstead ) . tumour necrosis factor b block of cell-replication enhances rsv-replication (gibbs et al. ). the evidence of the enhancer effect of non-neutralizing antibodies to rsv can be prevented by the treatment of children at increased risk with neutralizing monoclonal antibodies directed against the fusion (f) protein of rsv (motavizumab and/or palivizumab). this monoclonal antibodies were not found to cause harmful effects in connection with the preventive treatment of neonates and infants at risk (martin-mateos ; nieri et al. ; weisman ; groothuis et al. ). anti-rsv-f protein specific antibodies can prevent also the rsv-s. pneumoniae enhancement of respiratory infections (hament et al. ) . the blocking of influenza specific anti-neuraminidase antibodies using antiidiotypes indirectly enhanced the hemagglutination-inhibition titers of antisera (dowdle et al. ) . influenza h specific antisera were found to enhance virus replication in a macrophage-like cell line p d , when p d cells, previously had been treated with neuraminidase to remove the viral receptors (ochiai et al. ). maternal antibodies systemic lupus erythemadosus (sle), is characterised by antibodies towards dsdna and ro (e ligase regulating tlr signalling) which, are present several years before the onset of disease and the neonatal disease is caused by some of these transplacental antibodies (watson et al. ) . systemic lupus erythematosus (sle) is the most common autoimmune disease affecting women of reproductive age and is associated with poor maternal and fetal outcomes. cd (+)cd (+) t reg cells are a subset of t lymphocytes with potent immunosuppressive activity that play crucial roles in controlling immunological self tolerance. evidence suggests that they are augmented in pregnancy, especially in the first trimester, suggesting an important role in early placental development. the literature describing t reg cells in sle is conflicting, but sle is associated with reduced numbers and functionally defective t reg cells, which may predispose pregnant women with the disease to pregnancy complications. this article discusses the role of t reg cells in sle and pregnancy, and how these cells may contribute to poor pregnancy outcome in sleaffected women (blois et al. ; clark et al. ) . sle was induced by interferon a administration, and by a specific stimuli i.e. sunshine exposure and smoking. the hla-dr haplotype was also found to be a risk factor (klareskog et al. ) . alcohol consumption was shown to be protective in these illnesses. vaccinations in adult age was found to be innocuous concerning the risk of ra according to the results of a case-control study when common vaccinations years before onset of ra had been followed up. the effects of environment, however, are unknown for the neonatal heart block in p and ro positive women, but the active immunisation does not increase the risk of it (bengtsson et al. a, b) . in sle and sj€ ogren's disease pregnants with high antibody titers against the p epitope of ro are those who almost exclusively carry the risk that their fetuses will develop neonatal heart block between gestational weeks - . monitoring during these period the anti-ro antibodies, steroid treatment or preventive in utero pacemaker treatment may reduce the risk (wahren-herlenius ). foetal genes, maternal age and infectious agents may contribute to the risk of congenital heart block. especially inherited high level interferon production was also shown to be a risk factor for sle (niewold et al. ) . heparin treatment and intravenous gamma globulin (ivig) treatment and specific anti-idiotypes reduce the risk of the disease (clark et al. ) . ivig enhanced the anti-id antibody response in pregnant women with anti-la/ssb antibodies. the id:anti-id ratio was significantly higher in mothers whose offspring developed neonatal lupus compared to mothers who gave birth to a healthy child (p < . ). removal of anti-id antibodies substantially increased the reactivity against la( - ) in sera from five of seven mothers tested. ivig from batches administered to mothers who gave birth to a healthy child had an id:anti-id activity ratio of < , in contrast to that given to mothers who gave birth to a child with neonatal lupus (brucato et al. (brucato et al. , routsias et al. ) . the main pathomechanism of the development of neonatal lupus erythematosus is the transcytosis of maternal antibodies, and probably microchimeric cells. the pathogenesis of the maternal disease is extremely complex as summarised recently by perl ( ) and perl et al. ( ) . mitochondrial hyperpolarization underlies mitochondrial dysfunction, depletion of atp, oxidative stress, abnormal activation, and death signal processing in lupus t cells. nitric oxide production, expression of endogenous retroviral and repetitive elements such as hres- , (the long interspersed nuclear elements ), trex , interferon alpha (ifn-alpha), toll-like receptors and (tlr- / ), high-mobility group b protein, extracellular signal-regulated kinase, dna methyl transferase , histone deacetylase, spleen tyrosine kinase, proteasome function, lysosome function, endosome recycling, actin cytoskeleton formation, the nuclear factor kappa b pathway, and activation of cytotoxic t cells were shown to be components of the pathogenesis (varghese et al. ) . the hres- human endogenous retrovirus (erv) encodes a k nuclear autoantigen and a -kd small gtpase, termed hres- /rab . hres- /p is a target of cross-reactive antiviral antibodies, whereas hres- /rab regulates the surface expression of cd via endosome recycling. hres- /rab is overexpressed in lupus t cells where it correlates with increased recycling of cd and cd and contributes to downregulation of cd /tcrs via lysosomal degradation. erv proteins may trigger lupus through structural and functional molecular mimicry, whereas the accumulation of erv-derived nucleic acids stimulates interferon and anti-dna antibody production. erv proteins may trigger lupus through structural and functional molecular mimicry, whereas the accumulation of erv-derived nucleic acids stimulate interferon and anti-dna antibody production in sle (perl ; perl et al. ) . these complex of pathogenetic factors many of them influenced by pregnancy results the clinical disease of the newborns (ruiz-irastorza and khamashta ). neonatal lupus erythematosus (nle) is an inflammatory disorder of neonates characterized by transient cutaneous lesions and/or congenital heart block. the cutaneous lesions usually heal with minimal scarring within - months, but may be delayed for many months in occasional cases. the maternal antibodies disappear from the circulation of the newborns within - months, the long-lasting clinical symptoms indicate, that irreversible events, or impairment of fetal cells occur during the fetal life. photosensitivity is recognized as a component of this syndrome. u ribonucleoprotein (u -rnp) specific antibodies can be detected in the circulation of the newborns. skin and cardiac manifestations coexist in only % of patients. hepatic, hematological and, less commonly, pulmonary, neurological and gastrointestinal abnormalities may also be present (watson et al. ; perez et al. ) . neonatal lupus is a model of passively acquired autoimmunity in which a mother-, who may have systemic lupus erythematosus (sle) or sj€ ogren's syndrome (ss) or may be entirely asymptomatic-synthesizes antibodies to ssa/ro and/or ssb/ la ribonucleoproteins that enter the fetal circulation via trophoblast fcrn receptors and presumably cause tissue injury (lee ) as mentioned above. congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with ro/ssa autoantibodies. during pregnancy, the antibodies are transported across the placenta and affect the fetus. it has been previously demonstrated that antibodies directed to the - amino acid (aa) stretch of the ro component of the ro/ssa antigen correlate with the development of congenital heart block. the antibody recognition is dependent on a partly alpha-helical fold within the putative leucine zipper of the - aa stretch (ottosson et al. ). smith (sm) antigen, which is highly specific for sle is composed of at least nine different polypeptides with molecular weights ranging from to . kda, b (b , kda), b (b , kda), n (b , . kda), d ( kda), d ( . kda), d ( kda), e ( kda), f ( kda), and g ( kda). sle patients develop antibodies against the sm complexes of the small nuclear rnas u to u in the mother and recently these were also detected in neonatal le patient (ortiz-santamaria et al. ) . in neonatal lupus, mothers with high anti-idiotypic antibody activity against anti-la autoantibodies are at lower risk of giving birth to an unhealthy child, as compared with mothers without anti-idiotypic antibodies. usually anti-idiotypic antibodies may confer protection from the harmful effect of autoantibodies in certain autoimmune diseases (tzioufas and routsias ) . ivig enhanced the anti-id antibody response in pregnant women with anti-la/ssb antibodies. a high id:anti-id ratio in both the ivig preparation and the maternal serum may explain the absence of an effect of ivig in preventing recurrent neonatal lupus in some cases (routsias et al. ) . the children of the mothers suffering from sle and sj€ ogren's syndromes are at risk also for other rheumatic/autoimmune diseases without carrying antibodies reactive with ssa/ro or ssb/la antigens. among the siblings without neonatal lupus developed later juvenile rheumatoid arthritis, hashimoto thyroiditis, psoriasis and iritis, diabetes mellitus, congenital hypothyroidism and nephrotic syndrome (martin et al. ; winter and schatz ) . experimental systemic lupus erythemetosus could be induced in mice using immunisation with anti-idiotype antibodies (ab ) specific to the anti-dna-specific monoclonal antibodies (mendlovic et al. ) . identical twins of mothers suffering from sle had also transient neonatal bullous skin disease (nakajima et al. ). autoimmunity has been defined as a normal physiological state with control mechanisms that prevent autoimmunity from progressing to overt pathology. the onset of a subset of rheumatoid arthritis (ra) begins with appearance of citrullinated protein antigen (cpa) positivity when tolerance to certain citrullinated proteins/peptides is broken supported by certain hla-dr haplotypes (hla-dr and hla-drb in smokers). local expression of peptidylarginine deiminases and occasional elevation of inflammatory cytokines can be measured (klareskog et al. ) . methotrexate was significantly more effective than placebo in preventing progression to this disease state in anti-cpa-positive patients with undifferentiated arthritis (ua) whereas no difference between methotrexate and placebo was seen in the acpa-negative group of patients with undifferentiated arthritis (ua; arthritis without arthralgia). the risk of ra was lower in this group without treatment (ehrenstein et al. ; van dongen et al. ). the treatments using therapiesinfliximab (a tumour necrosis factor (tnf-a) blocker) and abatacept (t cell costimulation inhibitor; anti-cd and anti-cd ) did not result in significant improvement of the acpa patients with ua (saleem et al. ; emery et al. ) . the analysis of myeloid-related proteins in serum is an excellent tool for the diagnosis of systemic onset of juvenile idiopathic arthritis (jia), allowing early differentiation between patients with systemic-onset of jia and those with other inflammatory diseases. mrp- /mrp- and il- b represent a novel positive feedback mechanism activating phagocytes via two major signaling pathways of innate immunity (tlr ) during the pathogenesis of systemic-onset of jia (frosch et al. ). the rheumatoid arthritis was found to be improved during pregnancy probably due to galactosylation of igg molecules (f€ orger and Østensen ). women with ra can acquire the susceptibility allele through microchimeric cells. very high amounts ( . % of total pbmcs) of drb * microchimerism in patients with ra were observed. similarly, drb * microchimeric dna was observed in significantly higher quantities in women with ra compared with healthy control subjects. in contrast, there was no difference between women with ra and control subjects when microchimerism for non-ra-associated alleles (hla-dqb * and drb * / ) was analyzed (rak et al. ). by analogy to graft-versus-host disease (gvhd), pioneer studies on microchimerism in women with scleroderma proposed direct and indirect recognition mechanisms as underlying reactions of microchimeric cells (nelson ) . in gvhd, donor cells invade the recipient, which is not what is observed with microchimerism in patients with ra. however, the presence of drb * microchimerism ( . %) and drb * microchimerism ( . %) is not negligible, with frequencies among total host pbmcs similar to the frequencies of antigen-specific cd + t cells, which thus are sufficient to impact the host immune reactions. the haplotypes of the patients influence the progression of the rheumatoid arthritis, too (liu et al. ). autoimmune neonatal bullous skin disease caused by placental transfer of maternal igg autoantibodies is rare. it has been reported in neonates born to mothers with pemphigus vulgaris, pemphigus foliaceus, and gestational pemphigoid. vertically acquired congenital autoimmune blistering disorders appear to be self-limited and resolve with supportive therapy, concomitant with the presumed clearance of maternal autoantibodies from the neonate's circulation (abrams et al. ). antiphospholipid antibody syndrome (apas) is regarded as the most frequently acquired risk factor for thrombophilia. thrombophilia is the tendency to thrombosis. the antiphospholipid antibody syndrome (apas) is a disorder of recurrent vascular thrombosis, pregnancy loss and thrombocytopenia, associated with persistently raised levels of anti-phospholipid antibodies (apa). the apa are phospholipids (part of a cell's membrane) recognized by the body as foreign and antibodies are produced against them. maternal autoimmune diseases significantly reduce the pregnancy outcome of the women. the most frequent illnesses were antiphospholipid syndrome, antiphospholipid syndrome associated with a rheumatic disease (aps/rd), other rd patients, isolated autoantibodies (autoabs) in the absence of a definite autoimmune disease (aabs) and reactive arthritis or spondyloarthropathies. of these patients, . % had previous pregnancy complications with an anamnestic livebirth rate of . %. in these patients, . % of pregnancies resulted in preterm delivery and . % newborns had low weight at delivery. aps/rd patients had the worse outcome: . % resulted in miscarriage, . % resulted in growth restriction and % resulted in preterm delivery. this result was mainly due to patients with aps/systemic lupus erythematosus (sle) that had the lowest gestational age at delivery ( . ae . weeks) and the lowest newborn weight (canti et al. ) . antiphospholipid syndrome was suggested to be the result of antibodies, directed against cardiolipin as a result of antigenic mimicry. the suspected antigen is beta- -glycoprotein-i (b gpi) (sherer et al. ) . clinically significant are lupus anticoagulant, anticardiolipin antibodies and anti-b glycoprotein-i (anti-b gp-i) antibody. neonates born to mothers with primary aps are at risk of prematurity, being small for gestational age, and having thrombocytopenia (chou et al. ). antiphospholipid antibodies (apl) can impair the physiologic development of a fetus during pregnancy not only by causing thrombosis of the placental vessels, but also by directly binding throphoblast cells and modifying their functions (tincani et al. ). transplacentally transferred antiphospholipid antibodies act as a risk factor, but are not usually a sufficient condition for thrombosis and other thrombophilic risk factors should be systematically evaluated. long-term studies of children born to antiphospholipid-antibody-positive mothers provided the evidence of possible neurodevelopmental changes in these children and regular neuropsychological assessments are recommended. antiphospholipid-antibody-related thromboses in children are frequently associated with multiple antiphospholipid antibody positivity and concomitant presence of inherited prothrombotic disorders can be also detected in addition to nonthrombotic manifestations, particularly hematological, skin and neurological manifestations (avcin ) . treatment of pregnant women with apas results in marked improvement in the live birth rate ( . - . %). however, complications like preeclampsia and intrauterine growth restriction (iugr) occur even after treatment, requiring strict monitoring and timely delivery. aberrant concentrations of fetuin a and heat shock protein might have also role in the preeclamptic inflammation (molvarec et al. a, b; dadhwal et al. ). foetal/neonatal disease is due to transplacental thyrotrophin receptor stimulating antibodies (trab). it's extremely important recognising and treating graves' disease in mothers as soon as possible, because a thyrotoxic state may have adverse effects on the outcome of pregnancy and both on the foetus and newborn. neonatal grave's disease tends to resolve spontaneously within - weeks as maternal thyroid stimulating immunoglobulins are cleared from the circulation but subsequent development may be impaired by perceptual motor difficulties. hashimoto's thyroiditis is a very common autoimmune thyroid disease. in presence of maternal hashimoto's thyroiditis, there are usually no consequences on foetal thyroid, even if antitpo and antitg antibodies can be found in the newborn due to transplacental passage. however there are some reports describing foetal and neonatal hyperthyroidism in the affected mothers' offspring (radetti et al. ; hemminki et al. ) . a number of autoimmune diseases; especially autoimmune thyroid diseases, erythema nodosum and sarcoidosis parity might somehow be involved in maternal disease development (jørgensen et al. ) . maternal thyroid status assessment and treatment improves fetal outcomes and neuropsychological developmental of the newborn (staii et al. ). juvenile myasthenia gravis is associated with antibodies to the acetylcholine receptor (achr) in most patients. thymoma is rare, but often malignant in children. the frequency of juvenile myasthenia gravis with antibodies to the musclespecific kinase (musk) varies markedly in different countries. neonatal myasthenia gravis associated with musk antibodies is often a severe and protracted albeit transient disease (béhin et al. ; evoli ) . transient neonatal myasthenia gravis (mg) is a human model of passively transferring the disease. although all newborn babies of myasthenic mothers have anti-achr antibodies at birth (morel et al. ; tzartos et al. ) , only a small percentage of them ( - %) express the myasthenic syndrome (namba et al. ) . the myasthenic symptoms usually appear a few hours after birth and their average duration is about weeks. neonatal myasthenia gravis is transiently transferred from the mothers to the newborn. nicotinic acetylcholine receptor (achr) antibodies result in loss of achrs and also directly block the function of the remaining achr molecules, thereby causing a defect in neuromuscular transmission. the majority, though not all, of both myasthenic and non-myasthenic infants were found to have a repertoire of anti-achr specificities very similar to their mothers. no significant differences were observed between sera from the two groups of mothers. adequate treatment in mothers can reduce both frequency and severity of neonatal disease. neonatal disease will recover following ivig treatment (béhin et al. ; o'carroll et al. ). the absence of neonatal myasthenia gravis might be caused by the antigenic differences between the fetal and adult enzymes similar to those detected in rats (hall et al. ; hesselmans et al. ). the human fetal acetylcholine receptor (achr) is present until weeks gestation, when the fetal (g) subunit is replaced by the adult (e) subunit. the term "fetal acetylcholine receptor inactivation syndrome" has been proposed for the illness, when other developmental disorders were also caused by the maternal antibodies (oskoui et al. ). neonatal guillain-barré syndrome (gbs) was observed to occur - days postpartum in children born to mothers with gbs. serum from mother and infant depressed quantal content by approximately % and reduced the amplitude of postsynaptic currents by - % in mouse, newborn and juvenile rats. the antibody nature of the blockade could be confirmed by showing that monovalent fab fragments were similarly effective as purified immunoglobulin (ig) g. both cellular and humoral immune mechanisms are operative in guillain-barré syndrome (gbs). transplacentally transferred blocking antibodies may be specifically directed at epitopes of the mature but not the fetal neuromuscular junction (luijckx et al. ; buchwald et al. buchwald et al. , . guillain-barré syndrome and sydenheim's chorea are diseases, which were shown to be associated with the immune response after microbial infections. the occurrence of guillain-barré syndrome noted in infants whose mother had harmless autoimmune antibodies during pregnancy (buchwald et al. ; sladky ) . the syndrome's occurrence within families is also of interest. the mmp c(- )t and tnfa c(- )a snp were associated with severe weakness and poor outcome, indicating that these snps may be one of the factors predisposing to a severe form of gbs (geleijns et al. ). the associated features of er / ek carriers consist of favorable metabolic and body compositional conditions. in contrast, the n s polymorphism was reported to be associated with an enhanced sensitivity to glycocorticoids. haplotypes carrying the minor allele of the bcli polymorphism of the glycocorticoid receptor gene was related to the phenotype and outcome of gbs explaining the family dependence of the syndrome and other neonatal disorders (dekker et al. ). mutations in about a dozen of genes have been linked to the development of permanent neonatal diabetes mellitus (pndm). the most frequent causes of pndm are heterozygous mutations in the kcnj , ins and abcc genes. although pndm is a rare phenomenon (one case in about , live births), this discovery has had a large impact on clinical practice as most carriers of kcnj and abcc gene mutations have been switched from insulin to oral sulphonylureas with an improvement in glycemic control (aguilar-bryan and bryan ; rubio-cabezas et al. ). the majority of transient neonatal diabetes mellitus (tndm) cases have an abnormality in chromosome q . half of the ndm cases are transient (tndm) and the other most frequent causes of ndm are missense mutations in the pancreatic b-cell k atp channel genes kcnj , ins and abcc (chr ), and in the preproinsulin gene, ndm has been linked to numerous other genetic causes including point mutations in gck (chr ), glis (chr ), eif ak (chr ), pdx (chr ), ptf a (chr ), slc a (chr ), hnf b(chr ) or foxp (chrx) (aguilar-bryan and bryan ; bonnefond et al. ) . genetic mutations were identified in~ % of non-consanguinous probands with pndm/mdi, using sequential screening of kcnj , ins and abcc genes in infants diagnosed within the first months of age. this percentage decreased to % in those with diabetes diagnosed between and months. patients belonging to the latter group may either carry mutations in genes different from those commonly found in pndm/mdi or have developed an early-onset form of autoimmune diabetes. islet-cell antibodies (ica), glutamic acid decarboxylase autoantibodies (gada), tyrosine phosphatase-related proteins-islet antigen autoantibodies (ia- a), insulin autoantibodies (iaa), zinc transporter autoantibodies (znt a) were found in the sera of the children, suggesting autoimmune origin of their disease (russo et al. ). biliary atresia (ba) is a devastating disease of infants, invariably leading to cirrhosis, end-stage liver disease, and death if untreated. it has been shown using microarray technique, that the t-cell regulatory gene rras seems to be a key factor in the development of the disease (zhao et al. ) . a recent review reported that ba may involve a primary perinatal hepatobiliary reoviral or rotaviral infection and a secondary autoimmune-mediated bile duct injury (mack ). the maternal virus infections followed by maternofoetal microchimerism seems to be a very impressive explanation of the etiology (muraji et al. ) . in a mouse model, oral vaccination before mating with rotateq and rotarix prevented most rhesus rotavirus-induced ba (turowski et al. ). biliary atresia is probably the endresult of different aetiological factors, among which viruses and other agents may cross the placenta. otherwise it cannot be understand, why only one of the twins obtain the disease (morris et al. ) . the anti-idiotypes transcytosed by different efficiency into the circulation of the twins, might be an additional explanation for the asymmetric disease. maternal symptomless paraproteinemia was also found to be transmitted to the fetus. the paraprotein was detected after birth for months in the serum of the child and caused prolonged immunosuppression without later consequences (littlewood et al. ; littlewood and payne ) . transient neonatal acquired von willebrand syndrome (avws) has been observed peripartum. its clinical management is analogous to monoclonal gammopathy of undetermined significance (mgus) of the mother since it is the consequence of transplacental transfer of maternal igg antibodies (simone et al. ; nageswara rao et al. ). immune trombocytopenic purpura associated with pregnancy. fetal and neonatal alloimmune thromocytopenia (fmait) results from transplacental transfer of maternal antibodies that develop in response to alloimmunization against paternal human platelet antigens (hpas) expressed on fetal platelets. this thrombocyte loss could be prevented using modified igg molecules (ghevaert et al. ) . at present seven biallelic human platelet antigen (hpa) systems have been determined and can be typed using genomic dna. platelet genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patients' sera to complement the clinical history in the diagnosis of alloimmune platelet disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait). prenatal platelet typing of the fetus in suspected cases of fnait became also available (curtis ) . half of the infants were borne with low platelet counts and of required replacement therapy upon birth (ozkan et al. ; gasim ) . the maternal, transplacental igg binding to the fetal platelets was suggested to prevent their recirculation by fcgr binding to and phagocytosis by macrophages. the pathogenesis of immune trompcytopenic purpura and that associated to myelodysplasia and leukemia were shown to be different (psaila and bussel ; psaila et al. ). neonatal endarteritis has been diagnosed in the newborns of mothers suffering from endarteritis nodosa. fatal myocardial infarction in a neonate due to coronary arteries is compared with two lethal cases of mucocutaneous lymph node syndrome and/or infantile periarteritis nodosa (mlns/ipn). cutaneous polyarteritis was transmitted to the newborn, too (kitzmiller ; krapf et al. ; stone et al. ) . during the last decade no publication could be found on neonatal endarteritis. probably the pathogenesis of this disease has been reevaluated in the light of the molecular diagnostic findings. hereditary autoinflammatory syndromes are caused by monogenic defects of innate immunity and are classified as primary immunodeficiencies. these syndromes are characterized by recurrent or persistent systemic inflammatory symptoms and must be distinguished from infectious diseases, autoimmune diseases, and other primary immunodeficiencies. the sited review describes the epidemiological, clinical and laboratory features, prognosis, and treatment of the main autoinflammatory syndromes, namely: familial mediterranean fever; tnf receptor associated periodic syndrome; the cryopyrinopathies; mevalonate kinase deficiency; pediatric granulomatous arthritis; pyogenic arthritis, pyoderma gangrenosum and acne syndrome; majeed syndrome; and deficiency of interleukin receptor antagonist. the cryopyrinopathies discussed include neonatal-onset multisystem inflammatory disease (also known as chronic infantile neurologic, cutaneous and articular syndrome), muckle-wells syndrome, and familial cold autoinflammatory syndrome (jesus et al. ) . primary immunodeficiencies (pids) were analyzed to gain insight into the physiopathology of sle. some pids have been consistently associated with sle or lupus-like manifestations: (a) homozygous deficiencies of the early components of the classical complement pathway in the following decreasing order: in c q, % of affected patients developed sle; in c , %; inc r/s, %; and in c , up to %; (b) female carriers of x-linked chronic granulomatous disease allele; and (c) iga deficiency, present in around % of juvenile sle. mutations of the complement system (c -inhibitor; c q, c r, c s, c , c , c , c , c , c , c and c deficiencies were shown to facilitate the development of sle in addition to the facilitation of bacterial infections (c and c , encapsulated bacteria; c to c neisseria). other autoimmune diseases, i.e. polyendocrinopathy candidiasis ectodermal dystrophy (apeced), immune dysregulation polyendocrinopathy enteropathy x-linked (ipex), and autoimmune lymphoproliferative syndrome (alps), suggesting that mechanisms considered as critical players for induction and maintenance of tolerance to autoantigens, such as ( ) aire-mediated thymic negative selection of lymphocytes, ( ) foxp + regulatory t cell mediated peripheral tolerance, and ( ) deletion of auto-reactive lymphocytes by fas-mediated apoptosis, were not found to be associated with sle physiopathology (blois et al. ; carneiro-sampaio et al. ). behçet syndrome (bs) is a multisystem chronic inflammatory disorder, which is characterized by relapsing oral and genital ulceration and iridocyclitis. while being of unknown etiology, vasculitic changes of possible autoimmune origin are common to all involved organs, and thrombotic complications, which may adversely affect gestation, are frequently seen was shown to possess genetic etiology on the basis of the comparison of its incidence among monozygotic and dizygotic twins (masatlioglu et al. ). pregnancy does not have a deleterious effect on the course of bd and may possibly ameliorate its course. however, it seems that bd may adversely affect pregnancy. the miscarriage rate was higher, and the pregnancy complications and cesarean section rates were significantly elevated (jadaon et al. ) . overall, parity was associated with an % increased risk of female predominant autoimmune diseases. pregnancies resulting in liveborn children therefore seem to contribute only little to the general female predominance in autoimmune diseases. crohn's disease and ulcerative colitis; in idiopathic inflammatory bowel diseases (ibd), including crohn's disease and ulcerative colitis, there is pathogenic build-up of cd + t cells at sites of inflammation, mediated in part by il- , which provides (as described earlier) an anti-apoptotic signal to t cells through the induction of bcl- and bclxl and promotes th lineage differentiation. il- is upregulated in patients with both types of ibd. il- levels are elevated markedly in the serum of patients with ibd, decrease with treatment of inflammation and are predictive of ibd relapse. microbial pattern-recognition receptors, such as the tlrs and nod (nucleotide oligomerization domain ), which activate nf-kb, have been implicated in these conditions. neutralisation with an anti-il- r antibody largely prevented the colitis in mice (arad et al. ) . most women and men with ulcerative colitis (uc) and crohn's disease (cd) can expect a healthy child with neither preterm birth nor low birthweight. no neonatal forms have been described (ludvigsson and ludvigsson ; van assche et al. ) . later crohn's disease was shown to impair the fetal outcome in the case of the affected mothers (naganuma et al. ). colitis-associated-cancer (cac); tgf-b suppresses the formation of cancer by inhibiting il- trans-signaling and human samples of colon cancer have low-levels of il- r, as do samples of inflamed colon. adenomatous polyposis coli (apc) gene of mice with apc mutation develop cancer, genetic deletion of the tlr-adaptor protein myd decreased the number of cancers markedly. because myd activation in turn activates nf-kb, it is not surprising that il- production was decreased greatly in mice deficient in myd and supports prior data showing that il- is one of the effector signals in the tlr-nf-kb activation pathway (becker et al. ) . tgf-b was found to suppresses tumor progression in colon cancer by inhibition of il- trans-signaling. huntington disease was found to be of congenital origin, and the diagnosis can be obtained before implantation (hdcrg ; peciña et al. ) . the causal mutation is the expansion of a cag trinucleotide repeat tract in exon of a large gene on chromosome that results in the extension of a polyglutamine tract at the n-terminus of the encoded, ubiquitously expressed protein called huntingtin. from the maternal blood the dna from the fetal-maternal transport can be also applied for the prenatal diagnosis (bustamante-aragones et al. ). hypoparathyroidism with autoimmunity due to the q . deletion syndrome. the majority of patients acquired autoimmune antibodies, but without antiparathyroid antibodies (lima et al. ). wegener granulomatosis (wg) is systemic disease of unknown etiology characterized by necrotizing granulomatous inflammation, tissue necrosis, and variable degrees of vasculitis in small and medium-sized blood vessels. the classic clinical pattern is a triad involving the upper airways, lungs and kidneys. ninety percent of patients present with symptoms involving the upper and/or lower airways, and % will eventually develop renal disease (mubashir et al. ) . pregnancy in patients with wg requires preconceptional planning, careful clinical management, and vigorous treatment of active disease. the management is individualized and the pregnancy outcome is variable. antenatal management and therapeutic options are important (koukoura et al. ) . no neonatal disease have been described, and ivig treatment could be successfully applied in the steroid resistant patients (bellisai et al. ). kawasaki disease (kd) is an illness mostly of infants and young children, the majority being less than years old. the peak age of onset of illness is - months. it is unusual in very young infants. from to , only six instances of kd occurred in infants days of age or younger in japan, and infants days of age or younger accounted for only . % of all patients. the youngest infant of kd to date (a neonate week old) was reported by stanley and grimwood. the youngest reported from india is a -day-old infant (thapa et al. ). therapeutic monoclonal antibodies (mabs) are most commonly of the igg subclass, which is transported most efficiently to the fetus. in all animal species used for testing developmental toxicity, fetal exposure to igg is very low during organogenesis, but this increases during the latter half of gestation such that the neonate is born with an igg concentration similar to the mother. the therapeutic monoclonal antibodies might cause developmental and reproductive toxicity (dart) requiring testing of antibody-based therapeutics (pentsuk and van der laan ). guillain-barré syndrome during pregnancy can be also treated with ivig and plasmapheresis. (niklasson et al. ; goyal et al. ; bahadur et al. ; modi et al. ; ohlsson and lacy ) . polyclonal antisera, administered for passive immunization, are mixtures of different proteins, sharing binding activity against the antigen (ag) determinants of the same immunogen preparation. immunoadhesins (or immunoglobulin fusion proteins) are antibody-like molecules resulting from the fusion of a constant region (e.g., fc portion) of an immunoglobulin and the ligand-binding region of a receptor or an adhesive molecule. antibody fragments such as fab, scfv, diabodies, and minibodies are molecules devoid of part or whole of the fc portion. therefore, they have faster clearance and better tissue/tumor penetration than whole immunoglobulins and they will perform better than whole iggs in conditions where a short half-life is desirable, such as in radio-imaging and/or radio-therapy. they have no adcc and cdc triggering activity. the smallest proteins retaining antigen binding are a single variable domain antibody (nieri et al. ). ivig prevention improves the risk of heart block of sle patients (friedman et al. ) . several members of the idiotype-anti-idiotype network and antibody dimers including antigen-antibody complexes were found in the ivig preparations (luijten et al. ; osterhaus et al. ; clark et al. ). ad anti-idiotype monoclonal antibody can mimic the cd antigen. the molecular basis of ad mimicry has been identified with three cdr regions of ad showing similarity to three regions of cd . these regions have been analysed for potential t-cell epitopes, and sequences that are predicted to bind to hla/a , , and to hla/dr , , have been identified within the cdrh region of ad . ad can stimulate cd and cd responses in colorectal cancer patients with the appropriate haplotype. only a few patients produce a sustained memory response (durrant et al. ; reinartz et al. ) . most of these vaccines for the lymphoma therapy use the tumor b cell idiotype (the unique variable region of the surface immunoglobulin) as a tumor-specific antigen. in spite of several problems anti-idiotype vaccines prospect towards integration of this strategy in the therapeutic armamentarium for lymphoma (houot and levy ) . clinical studies have been published using different preparations and summarised at the end of table . . abagovomab (aca ) acts as an antigen mimic of the carbohydrate ovarian cancer antigen (wagner et al. ) . the frequency of peripheral t reg s was increased during abagovomab therapy in a high percentage of patients. despite higher t reg counts compared with baseline levels, the suppressive capacity of t reg s was reduced in a subset of patients. the data further indicate that the ability of t cells to proliferate in response to ca- in vitro could be associated with diminished t reg activity. importantly, ca- -specific immunity could not be enhanced by in vitro t reg depletion, as ca- induced cd + foxp + t reg s with suppressive capacity. abatacept (orencia) human igg fc domain ctla- ; anti-cd and anti-cd . abatacept (abt, orencia, bristol-myers squibb ltd) rheumatoid arthritis tumor necrosis factor inhibitor in phase clinical trial, but it was found effective only in very early phase of the disease (malottki et al. ; emery et al. ) . prophylactic withdrawal of drugs before pregnancy is mandatory (Østensen et al. ) . at present reports on abatacept, tocilizumab or anakinra are inconclusive therefore throughout pregnancy cannot be recommended (Østensen and f€ orger ) . abciximab (reopro) igg k-fab, chimera -integrina b (platelet-gp)haemostasis/trombosis (nieri et al. ). using immunohistochemistry, reopro was only detected attached to maternal and fetal platelets, and to the trophoblastic surface of the placental villi (miller et al. a, b) . the effect of abciximab to icam- was excluded, but an effect to monocytes could not be excluded (voisard et al. ) . adalimumab (ada, humira; abbott) human recombinant, igg k, anti-tnfa, as, psa, cd, pp, jia (van schouwenburg et al. ) tnf-alpha blocker therapy (adalimumab). the therapy of pregnants showed no increase in miscarriage, prematurity or structural malformations in neonates compared with non-exposed pregnancies (Østensen et al. ) . expression of hla-g on pmbcs is up-regulated in ankylosing spondylitis (as), correlates with acute phase reactants and decreases after tnf-alpha blocker therapy (chen et al. ) . anti-tnfa is used for the treatment of infectious bowel diseases even during pregnancy, which might influence the development of the fetal immune system (arsenescu et al. ) . anti adalimumab anti-idiotype antibodies (bartelds et al. malottki et al. ; nieri et al. ). ( ) ? teplizumab anti-cd t. diabetes herold et al. ( herold et al. ( , ? integrin a b direct exposure to anti-tnf treatment during pregnancy was not related to a higher incidence of adverse pregnancy outcomes than infectious bowell diseases overall (schnitzler et al. ). the anti-tnf agent was started prior to conception and continued until there was evidence of fetal cardiac activity. in women treated with a combination of anti-tnf therapy, anticoagulation therapy, and ivig, the live birth rates ( %) were greater than those in women treated with anticoagulation therapy alone ( %) or in those receiving a combination of anticoagulation therapy and ivig ( %). fetal outcomes, including gestational age and birth weight, were similar across the groups, and no congenital anomalies were reported after anti-tnf agent exposure (winger and reed ; vinet et al. ). rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, crohn's disease (Østensen and f€ orger ) . all users requested at least one time repeatedly the treatment in norway (mahic et al. ) . long term treatment did not resulted in reactivation of chronic hepatitis b virus infection (mori ) . the treatment caused the improvement in two bone formation markers -b-alkaline phosphatase and osteocalcin (veerappan et al. ) . alemtuzumab (mabcampath, genzyme) humanized, igg k, anti-cd anti-ca immunoglobulin directed against cd antigen expressed on t-and b lymphocytes, monocytes, macrophages, nk cells, and a subpopulation of granulocytes, but not on hematologic precursors. induction of cdc or adcc on an fcreceptor g-binding mechanism. pancreas transplant recipients on alemtuzumab maintenance therapy suffered frequently from red cell aplasia, and autoimmune hemolytic anaemia. (elimelakh et al. ) cord-blood-hematopoetic-stem-cell expansion and increase the availability of cord-blood units for transplantation (lim et al. ) . in contrast to ivig and rituximab the compound may be an effective therapy for complex immunohematologic disorders complicating hematopoietic stem cell transplantation. the paper emphasizes the importance of t-cells in transplant associated immune cytopenias (chao et al. ). b-ccl (nieri et al. ). alicaforsen is a human monoclonal antibody a b -integrin, also known as leukocyte function antigen (lfa)- , and its ligand, intercellular adhesion molecule- (icam- ), is important for the recruitment of leukocytes to inflammatory sites (bosani et al. ). anti-cd and anti cd intratumoral microvessel density (imvd) monoclonal antibodies. anti-cd was more effective against non-small cell lung cancer than anti-cd (tanaka et al. ) . anti-cd mab (bms- , bristol-myers squibb) - bb (cdw ), a member of tumor necrosis factor receptor (tnfr) superfamily stimulating t-cells, nk-cells and dcs . anti-cd mab enhances rituximabdependent cytotoxicity against the lymphoma cells (lee et al. ; kohrt et al. ) . anti-leu- b or anti-leu- c, igg a, marker of t-cell subpopulation (clement et al. ; champlin et al. ) . apomab (genentech) igg , against extracellular domain dr /tumor necrosis factor related (trail) receptor apoptosis inducing ligand (nieri et al. ). basiliximab (simulect) chimeric, igg k, anti-cd , il r antagonist (aktas et al. ) % reduced fetal body weight (pentsuk and van der laan ) . bavituximab (peregrine pharmaceuticals, inc., tustin, ca), anti-phosphatidylserine bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma (he et al. ). hcv therapy (immunostimulant) (dammacco et al. ; quer et al. ) . belatacept (ctla -ig) is a new recombinant molecule that interferes with the signal of t lymphocyte activation and prevents acute rejection after renal transplantation. hla-g acts as a naturally tolerogenic molecule in humans. patients treated with ctla -ig displayed significantly higher soluble hla-g (shla-g) plasma concentrations than patients treated with calcineurin inhibitors or healthy donors (bahri et al. ). ctla -ig-treated dc acted as tolerogenic apc through shla-g secretion as they suppressed t cell alloproliferation, which could be restored by using a neutralizing anti-hla-g ab (bahri et al. ). the use of anti tumour necrosis factor mabs are not recommended (partlett and roussou ) . cytotoxic t-lymphocyte-associated protein (ctla- ) is a negative regulator of t cell activation and may modulate peripheral self-tolerance (kaufman et al. ) . bevacizumab (avastin) humanized, igg k, anti-vascular endothelial growth factor-a (vegf-a) used for the treatment of non-small cell lung cancer, (nsclc) colorectal cancer (cc); (nieri et al. ). efd rabbit: dose-dependant decrease in maternal bodyweight, increase in fetal malformations and late resorptions; (pentsuk and van der laan ). intravitreal bevacizumab therapy during pregnancy for off-label ocular indications can result in significant visual improvement without adverse fetal events related to treatment (tarantola et al. ) . intravitreal . mg bevacizumab may reach the systemic circulation in plasma concentrations of ng/ml (csáky and do ) . two pregnants have lost their babies within days following intravitreal injections of bevacizumab (petrou et al. ). placenta gf levels are elevated in the plasma of colorectal and rectal carcinoma patients receiving bevacizumab (xu and jain ) . prevention of angiogenesis by msc in pancreatic cancer (beckermann et al. ) . blinatumomab (epcam, antigen -epithelial cell-adhesion molecule, -present on % of cancer cells) and mt- and mt present on all non-hodgin lymphoma cells (armstrong and eck ; nieri et al. ). cd /cd bispecific, single chain recombinant antibody topp et al. ) . catomaxomab (removab) bifunctional recombinant anti epidermal cell adhesion molecule epcam and anti-cd . it is inducing adcc in ovarian and stomach cancer. epcam is the ligand for human leukocyte immune-globulin like receptor (lair- ) (armstrong and eck ; nieri et al. ; bokemeyer ; r€ ussel et al. ) . ceavac (mimicking carcinoembryonic antigen) colon cc (foon et al. ). certolizumab (cimzia®; ucb) pegylated humanized antibody fab' fragment of tnf-a monoclonal antibody; ra, cd; (Østensen and f€ orger ) . certolizumab does not cross the placenta as easily as the igg derived drugs due to the pegylation of the molecules, thus reducing the harmful consequences to the fetus (clowse ) . cetuximab (erbitux) chimeric, igg k, anti-epidermal growth factor receptor- (her- ), apoptosis, cdcc colorectal cancer (cc); squamous cell cancer of head and neck (scchn) weight loss and reduced food consumption in high-dose group dose-dependent increase in abortion rates (not known, if could be associated with treatment) weight loss and reduced food consumption in high-dose group (powell et al. ; pentsuk and van der laan ; rech and vonderheide ) . chaglycd (cd -specific) a humanized antibody, an aglycosylated human igg antibody directed against cd was shown to reduce the insulin-requirement of the patients. residual beta-cell function was better maintained with chaglycd than with placebo (keymeulen et al. ) . cixutumumab (img- or cix) targeting insulin-like growth factor receptor (igf-ir) treatment for multiple cancers. human igg , blocks interaction between igf-ir and its ligands, igf-i and -ii, and induces internalization and degradation of igf-ir. its combination of cetuximab (mab against egfr) inhibited the growth of pancreatic cancer and promoted its regression. an antiangiogenic mechanism was associated with cix treatment. reviewed recently (quatrale et al. ) . hyperglycemia is a regular side effect, but the fetal consequences during pregnancy have not been evaluated yet (mckian and haluska ) . eyelash trichomegaly in adults (bouché et al. ; garrido et al. ) . cnto chimeric monoclonal antibody with high affinity for human il- myeloma multiplex sensitivity to glycocorticoid (voorhees et al. ). ct- (curetech) humanized anti-pd- igg mab that binds to mouse and human pd- , programmed death receptor . pd- but not ctla- blockage abrogates the protective effect of regulatory t cells in a pregnancy murine model. mkrtichyan et al. ; stagg et al. ; wafula et al. ). dacetuzumab (sgn- ; seattle genetics) humanized; and hcd fully human (novartis/xoma); cd , tumour necrosis factor receptor; b-cells, dcs, macrophages lymphomas . daclizumab (zenapax) humanized, igg k, anti-a-chain of cd , il- r antagonists (elimelakh et al. ; aktas et al. ) transplant rejection. eculizumab (soliris) humanized, igg / j, anti-human complement c ; paroxismal nocturnal haemoglobinuria (pnh); (thomas et al. ; nieri et al. ; danilov et al. ). there was no evidence of complement blockade from cord blood samples taken at delivery. eculizumab appears safe to use in this setting and is likely to prevent many of the complications usually observed (kelly et al. ) . edrecolomab (panorex) igg a, epcam antigen cdc-adcc cancer (nieri et al. ). efalizumab (raptiva) humanized, igg k, anti-integrin-cd a -psoriasis (nieri et al. ). progressive multifocal leukoencephalopthy was found to be a rare, but lethal disease associated with long term efalizumab therapy (kothary et al. ). epratuzumab, a humanized igg unconjugated anti-cd antibody effective against non-hodgkin lymphoma and follicular lymphoma (leonard et al. ; watanabe ) . consequences of the application during pregnancy has not been reported. etanercept (etn, enbrel, wyeth pharmaceuticals) human igg fc domain tnfr /p ; anti-tnfa as, psa, pp, jia; ng/ml cord blood. ng/ml week postpartum ng/ml; weeks postpartum; undetectable weeks postpartum (clowse ) . reduction of post-partum microchimerism (rak et al. ). complications of the therapy may be acute anterior uveitis (etanercept), psoriasis (infliximab > etanercept) and infectious bowel disease (ibd) (etanercept > infliximab) which were observed in association with the treatment using tnfantagonists. the paradoxical consequences, however, affected less than % of the treated patients (fouache et al. ). eternacept, which is a recombinant human p soluble receptor to tnf, failed in a phase ii trial with crohn's disease and the trial was discontinued. the recombinant receptor proved to be useful for the treatment of rheumatoid arthritis (malottki et al. ), but its use in pregnants has not been approved (Østensen et al. ) . etanercept treatment ( mg  /week) has been stopped weeks before pregnancy. the treatment had to be reinitiated from the th week of pregnancy and no fetal complication was observed (umeda et al. ). the etanercept treatment was initiated weeks before pregnancy ( mg/sq-m  weekly). the cord blood contained ng/ml etanercept in contrast to the maternal serum ( , - , ng/ml). no detectable etanercept was found in the newborn's blood th week after delivery, although the breast milk contained . ng/ml etanercept (murashima et al. ). all patients requested at least one times the repetition of the treatment according to a publication from norway (mahic et al. ) . fontolizumab used for the preventive treatment of newborns at risk for respiratory syncytial virus infection (rsv) resulted a significant decrease in c-reactive protein levels suggested a beneficial biological effect. (nieri et al. ; reinisch et al. ) . galiximab, a human-primate chimeric anti-cd antibody: galiximab is a human-primate chimeric anti-cd antibody with excellent tolerability and singleagent effectiveness for recurrent follicular lymphoma (fl), resistant to other therapeutical means (watanabe ) . gemtuzumab (mylotarg k ) igg k; humanised; cd -monocyte, myeloid cell drug targeting (nieri et al. ). golimumab (simponi®; centocor ortho biotech) human monoclonal igg antibody ra, as, psa (Østensen and f€ orger ) . hcd (novartis/xoma) cd -specific fully human igg mab with antagonistic activity that mediates adcc and blocks cd l-induced survival and proliferation of normal and malignant b cells (chatenoud and bluestone ) . ibritumomab tiuxetan (zevalin) murine, igg k, anti-cd ; radiol (yttrium ) imc-c (nieri et al. ). infliximab (ifx, remicade, schering-plough ltd) chimeric, igg k, anti-tumor necrosis factor alpha (tnfa) rheumatoid arthritis (ra) and crohn's disease (cd) (saleem et al. ; van schouwenburg et al. ) . fatal case of disseminated mycobacterial infection has been reported in an infant who received bcg vaccine at months of age. the mother had been treated with infliximab throughout her pregnancy. vaccination with live bacteria and viruses should be postponed in infants exposed to infliximab in utero, until serum levels are undetectable which may require more than months (djokanovic et al. ) . the fetal concentration of infliximab was found to be higher than that of the mother. this might be a risk for the postnatal development of the immune system (zelinkova et al. ). due to the high rate of igg transfer near term, babies have been found to have similar blood levels of infliximab to their mothers (clowse ) . all reported pregnancy outcomes under treatment with infliximab showed no increase in miscarriage, prematurity or structural malformations in neonates compared with non-exposed pregnancies (Østensen et al. ) . only the chimeric monoclonal anti-tnf antibody infliximab is currently available worldwide. the potency of this agent in moderate-to-severe ulcerative colitis (uc) and cd has been one of the most important advances in the care of inflammatory bowel disease (ibd) in the past decade (d'haens and daperno ) . anti-infliximab anti-idiotypes. no association was found between the patients' allotypes and the presence or concentration of anti-infliximab antibodies ) reduction in fertility (not known, whether related with male or female animals) (pentsuk and van der laan ). vacterl association? acute graft versus host disease (couriel et al. ) were described, but it proved to be useful for the treatment of rheumatoid arthritis (malottki et al. ) . inotuzumab ozogamicin (cmc- ), the calicheamicin-conjugated anti-cd monoclonal antibody and rituximab combination were used for the treatment of ankylosing spondilitis, psoriatic arthritis and ulcerative colitis; (nieri et al. ; smith et al. ) the concentration in the blood of the newborn was . mg/ml week post-partum and slowly declined over months (clowse ) . ipilimumab (fcgriib binding) overcoming tcla- -mediated immunosuppression, increasing anticancer immune-response (melanoma malignum; mdx- ; bristol-myers squibb/medarex); ctla- (cd ) t cells; t reg cells; colon and prostatic cancer; (nieri et al. ; houot et al. ) . iratumumab (sgn- and mdx- ) cd -specific igg used for the treatment of hodgkin's lymphoma. myelosuppression, fatigue, elevated liver enzymes were documented during therapy (klimm et al. ) . lfb-r , a fully human anti-rhesus d (rhd) antibody, for the prevention of feto-maternal allo-immunization in rhd-women, as a substitute for human polyclonal anti-rhd immunoglobulins (urbain et al. ). lfb-r , a monoclonal antibody directed against cd , for the treatment of b cell malignancies. antibody-dependent cellular cytotoxicity (adcc) activity and enhanced affinity to fcgriii (cd ), both correlated to a glycosylation pattern characterized by a low fucose content (urbain et al. ). mapatumumab; trail receptor activation (death receptor ) mediator of apoptosis in cancer cells (nieri et al. ). matuzumab (humanised anti-egfr monoclonal antibody; reviewed by seiden et al. ; and recently by quatrale et al. ) . melimmune: anti-idiotype antibody that mimic the high molecular weight chondroitin sulfate proteoglycan antigen of melanoma cells (pride et al. ; murray et al. ; ward et al. ) . mitumomab (bec , imclone systems) bec- anti-idiotype (giaccone et al. ; bottomley et al. ) bec is an anti-idiotypic antibody that mimics gd , a ganglioside that is expressed on the surface of tumor cells and is of neuroectodermal origin. ganglioside gd can be used as a vaccine against small cell lung cancer (sclc) (nieri et al. ). mln- anti-a b integrin antibody of igg type, humanised (reviewed by bosani et al. ) approved for the treatment of crohn's disease. motavizumab (humanised mouse monoclonal antibody). motavizumab targets a highly conserved epitope in the a antigenic site of the rsv fusion (f) protein, which is important in the invasion of rsv from cell to cell. motavizumab, which differs from palivizumab by just amino acids, has exhibited a -fold enhancement in binding to the rsv f protein compared with the first-generation mab, with an -fold faster association rate and sixfold slower disassociation rate (nieri et al. ; weisman ). muromonab, igg a, murine, t-cell cd blocade. cd -specific monoclonal antibodies can re-establish immune homeostasis in treated individuals. this occurs through modulation of the t-cell receptor (tcr)-cd complex (also termed antigenic modulation) and/or induction of apoptosis of activated autoreactive t cells, which leaves behind 'space' for homeostatic reconstitution that favours selective induction, survival and expansion of adaptive regulatory t cells establishing long-term tolerance. it is used for early treatment of diabetes type (chatenoud and bluestone ; nieri et al. ). natalizumab (tysabri) humanized, igg k, anti-a -integrin (vla- ), in the treatment of sclerosis multiplex (van schouwenburg et al. ) . natalizumab blocks both alpha- b integrin (vcam ) and alpha- b integrin (madcam ) interactions (rutgeerts et al. ). therapy of sclerosis multiplex will be more efficient in combination with interferon (miller et al. a, b; nieri et al. ). in animal experiments efd g. pig: reduced pregnancy rates in high-dose group; ppnd cyn: increased abortion and stillbirth rates (pentsuk and van der laan ). in cynomolgus monkeys, however, the abortion rate had not been increased, but hematopoetic changes were observed. natalizumab had no adverse effects on the general health, survival, development, or immunological structure and function of infants born to dams treated with natalizumab during pregnancy (wehner et al. a, b) . % of natalizumab therapy has been stopped because of pregnancy. three of patients treated at least for months developed progressive multifocal encephalopathy (pml; piehl et al. necitumumab (imc- f ) anti-egfr human monoclonal antibody (kuenen et al. ) . nimotuzumab (theracim) humanised, anti-egfr- /her- ; apoptosis, adcc; head and neck cancers (hncc), (spicer ; nieri et al. ; reviewed recently by quatrale et al. ) . omalizumab (xolair) humanized, igg k, anti-ige -asthma. causing marked reduction in serum levels of free ige and down-regulation of ige receptors on circulating basophils. effective in monozygotic twins (holgate et al. ; just et al. ; nieri et al. ) tolerability (corren et al. ). onercept, is a recombinant human p soluble receptor to tnf, failed in a phase ii trial with crohn's disease and the trial was discontinued (bosani et al. ). palivizumab (synagis) humanized, igg k, anti-respiratory syncytial virus "a" epitope of fusion protein. it is used for the prevention of respiratory syncytial virus infection of newborns with different risks for respiratory infections (martin-mateos ; nieri et al. ; weisman ) . panitumumab (vectibix) human, igg k, anti-human epidermal growth factor receptor binding the catalytic kinase domain of the receptor of colorectal cancer (cc; nieri et al. ). increased frequency of abortion/fetal death rates were observed in high-dose group (reviewed by pentsuk and van der laan ; nieri et al. ). eyelash trichomegaly in adults were seen (zhang et al. ; morris et al. ) . racotumomab( e ), an anti-idiotypic vaccine mimicking the n-glycolyl-gm ganglioside (guthmann et al. ; hernández et al. ) effective against breast and lung cancers. ngcgm is practically undetectable in healthy human tissues as a result of an alu-mediated inactivation of the gene, the ganglioside is highly expressed in several human cancer cells presumably due to incorporation of dietary ngc . ramucirumab (dc- ) (an antibody to the vegf receptor- ) (tonra et al. ; krupitskaya and wakelee ) . ranibizumab (lucentis, genentech) humanized, igg -fab, anti-human vasc. endothel. growth factor-a (vegf-a); for the treatment of choroidal neovascular (wet) age-related macular degeneration (armd) reviewed recently (ferrara et al. (ferrara et al. , ; neovascular acute myeloid leukemia (neovascular-aml); (csáky and do ; nieri et al. ). retuximab (epstein-barr virus) anti-cd- (sodani et al. ) . rituximab (rtx, mabthera k , roche) chimeric, igg k, anti-cd (sulesomab, leukoscan). murine fab, binds to surface granulocyte non-specific crossreacting antigen present on neutrophils. rhinitis, fever, chills and toxic laboratory findings occurred during the treatment (klimm et al. ) . hcv cryoglobulinaemia could be also treated (dammacco et al. ) . the treatment of pregnants because of bukitt's lymphoma resulted high rituximab concentrations and a transient complete b-cell depletion in the cord blood. b-cell recovery was fast, showing a regular immunophenotype without loss of cd antigen, no functional deficits and adequate vaccination igg titers (friedrichs et al. ) . administration in third trimester of pregnancy suppresses neonatal b-cell development, but without later neonatal consequences (klink et al. ) , in spite of these the prophylactic withdrawal has been recommended before pregnancy (Østensen et al. ) . human fetal b-cell depletion and lymphocytopenia in cynomolgus, were observed, too (vaidyanathan et al. ) . reduction of post-partum microchimerism was documented (rak et al. ). useful in rheumatoid arthritis therapy (malottki et al. ) in combination with chemotherapy depending on human concentrative nucleotide transporter (hcnt ) gene expression rate (rabascio et al. ) . non-hodgkin lymphoma, rheumatoid arthritis were the indications (nieri et al. ). cd is a costimulatory molecule expressed on a variety of immune cells after activation, including nk cells. cd stimulation by specific igg enhances the antilymphoma activity of anti-cd antibodies by enhancing adcc . of pregnancies with known outcomes, resulted in live births. twenty-two infants were born prematurely; with one neonatal death at weeks. eleven neonates had hematologic abnormalities; none had corresponding infections. four neonatal infections were reported (fever, bronchiolitis, cytomegalovirus hepatitis, and chorioamnionitis). two congenital malformations were identified: clubfoot in one twin, and cardiac malformation in a singleton birth. one maternal death from pre-existing autoimmune thrombocytopenia occurred. women should continue to be counseled to avoid pregnancy for months after rituximab exposure; however, inadvertent pregnancy does occasionally occur. practitioners are encouraged to report complete information to regulatory authorities for all pregnancies with suspected or known exposure to rituximab (chakravarty et al. ) . due to ongoing bleeding, rituximab was given in the th week of pregnancy. the platelet count rose to over  ( )/l after weeks. the neonatal b-lymphocyte count normalized at months after delivery. there were no neonatal complications of rituximab therapy (gall et al. ) . passenger lymphocyte syndrome has been described by lee et al. ( a, b) . siplizumab (cd or medi- ) is a humanised igglk monoclonal antibody that binds to human cd antigen. preclinical studies demonstrated that siplizumab kills target cells by adcc (fanale and younes ; watanabe ) . teplizumab (cd -specific, hokt g -ala-ala), a humanized fc mutated anti-cd monoclonal antibody induced tolerance, on the progression of type diabetes in patients with recent-onset disease even years after the first diagnosis (herold et al. (herold et al. , . tocilizumab (toc, roactemra, roche) against receptor of il- (mouse anti-human il- r antibody into human igg -k chain to create a human antibody with a human il- r binding site il- r a-chain or cd ; b-chain or cd ) at a low concentration of microg/ml, tocilizumab (anti-human il- receptor monoclonal antibody) inhibited the il- -induced matrix-metallo-proteinase (mmp) secretion which was shown to be stimulated in preterm premature rupture of membranes (pprm) (sato et al. ; mano et al. ; malottki et al. ; pham et al. ) . clinical phase trial for the treatment of rheumatoid arthritis has been approved. inherited autoinflammatory syndrome can be sometimes treated with anakinra and tocilizumab (goldfinger ). at present reports on abatacept, tocilizumab or anakinra are inconclusive therefore throughout pregnancy cannot be recommended (Østensen and f€ orger ) . normal pregnancy is characterised by elevated th activity and anti-inflammatory cytokines during the first trimester, followed by increased th activity and proinflammatory factors near term (challis et al. ). in contrast, preeclampsia (pe) is marked by an increase in proinflammatory tumor necrosis factor-a (tnf-a) and interleukin (il- ) cytokines as well as a decrease in the anti-inflammatory cytokines il- and il- . in cases of restricted fetal growth, tnf-a is also elevated when compared with normal pregnancy (dávila et al. ) . women living at , versus , m in colorado had higher proinflammatory (il- , tnf-a) relative to anti-inflammatory (il- ) cytokines during the second and third trimesters (coussons-read et al. ) . multigenerational andean versus shorter duration european high-altitude residents were found to be protected from altitudeassociated fetal growth restriction. higher il- b might play a role in protection from altitude-associated reductions in fetal growth (coussons-read et al. ; dávila et al. ) . tocilizumab treatment increased serum levels of il- and soluble il- r (sil- r; nishimoto et al. ). in combination with other drugs adult onset of still's disease can be improved using monoclonal antibodies (efthimiou and georgy ) . tositumomab anti-cd igg, b-cell lymphoma (armstrong and eck ; nieri et al. ). trastuzumab (herceptin) humanized, igg k, erbb , anti-her . induction of cdc or adcc on an fcreceptor g-binding mechanism. human anhydramnion and oligohydramnion will develop because of the caused fetal kidney insufficiency (watson ; robinson et al. ; katsumi et al. ; matsumoto et al. ). this decrease in amniotic fluid seems to be reversible with the discontinuation of trastuzumab (sukumvanich ) . transfer using aav-recombinant in mice does not induce anti-idiotypes (wang et al. a, b) . the mechanism of toxicity to the fetal kidneys is proposed to be associated with the different structure of egfr in the fetal renal-tubule epithelial cells (heterodimer of egfr and erbb in fetus vs. homodimer of egfr in adults). thus, trastuzumab will have a damaging effect on the fetal renal function, but it does not affect the kidneys of the adult (robinson et al. ) . anti-trastuzumab (ladjemi et al. ): anti-trastuzumab anti-id scfv , used as a therapeutic or prophylactic vaccine, protects mice from developing her -positive mammary tumors by inducing both anti-her ab antibody production and an anti-her th -dependent immune response. these results suggest that scfv could be used as an anti-id-based vaccine for adjuvant therapy of patients with her -positive tumors to reverse immunological tolerance to her . calmodulin inhibitors rescue trastuzumab sensitivity of breast tumours (kulkarni et al. ). the majority of these patients were able to tolerate therapy; however, oligohydramnios or anhydramnios occurred in out of the patients. this decrease in amniotic fluid seems to be reversible with the discontinuation of trastuzumab (sukumvanich ) . tremelimumab (cp- , ; pfizer); ctla- (cd ) t cells; t reg cells; colon and prostatic cancer; . triab d (triab) breast cancer (reece et al. (reece et al. , (reece et al. , . trigerm (disialoganglioside gd ) melanoma . tositumomab (iodine labelled), murine cd , cdc, adcc, radio-cytotoxicity non-hodgkin-lymphoma (nieri et al. ) veltuzumab is a humanized anti-cd antibody with structure-function differences from chimeric rituximab (watanabe ) . visilizumab (cd -specific) for the management of both crohn's disease (cd) and ulcerative colitis (uc). biologics under evaluation or approved for uc that are discussed include monoclonal antibodies to tumor necrosis factor ([tnf] infliximab), inhibitors of adhesion molecules (mln and alicaforsen), anti-cd antibodies (visilizumab), and anti-interleukin (il)- receptor antibodies (daclizumab). biologics under evaluation or approved for cd that are reviewed include three monoclonal antibodies to tnf (infliximab, adalimumab, and certolizumab pegol), monoclonal antibodies against il- , interferon-g, and il- receptors, inhibitors of adhesion molecules (natalizumab, alicaforsen), and growth factors. only the chimeric monoclonal anti-tnf antibody infliximab is currently available worldwide (d'haens and daperno ) . yttrium-ibritumomab tiuxetan and iodine-rituximab are anti-cd monoclonal antibodies combined with radioactive materials for diagnostic and/or therapeutic applications (watanabe ) . zalutumumab anti-egfr mab able to facilitate complement lysis of cancer cells (klausz et al. ) . reviewed recently (quatrale et al. ). expressed by antigen presenting myeloid cells (apc) (magnani et al. ). cd t-cell receptor (tcr)-cd complex resulting in the cells becoming 'blind' to antigen, a process that is also known as antigenic modulation (chatenoud and bluestone ) . cd over % (sato et al. ). cd ctla- negative regulator of t cell activation (kaufman et al. ). trail dr /tnf related receptor (nieri et al. interleukin (naugler et al. ; naugler and karin ; voorhees et al. ; reinartz et al. ). pd- programmed death protein ). complement factor thomas et al. ( ) breast cancer protein reece et al. ( reece et al. ( , reece et al. ( , ige immunoglobulin e, hypersensitivity (corren et al. ). anti-idiotypes carrier of the mimicry of epitopes (ab ) of antigens (k€ ohler ) . respiratory syncytial virus (nieri et al. ). cytotoxic t lymphocyte antigen- (ctla- ) and programmed cell death (pd- ) are members of the known t reg -associated molecules. blocking pd- abrogate the protective effect of t reg , resulting in a higher median abortion rate in comparison with the t reg / isotype-treated control while ctla- blockage did not interfere with the protective effect of t reg . pd- as an important mediator in t reg -induced fetal protection in the cba/ j · dba/ j murine model (wafula et al. ). ctla- was shown to interact with cd and cd resulting in termination of immune response (alegre et al. ) . mice genetically deficient in ctla- expression develop a lymphoproliferative disease which terminates in death by - weeks of age (tivol et al. ; waterhouse et al. ) . the cd possesses also role in the regulation of t-cells (sansom and walker ) . blockade of the interactions between cd and their ligands, cd and cd , has been shown to induce antigen-specific peripheral tolerance in organ transplantation. this knowledge has been successfully used in animal models to prevent allograft rejection by blocking cd and/or cd , thereby leading to long-term graft survival. cytokines favoring the maintenance of fetal survival mainly belong to the th -type (e.g. il- , il- , tgf-b), whereas pregnancy failure is associated with the th -type cytokines (e.g. ifn-g, tnf-a) at the materno-fetal interface and/or the absence of th -type cytokines. the combined use of anti-cd and anti-cd mabs in mice was effective in inducing maternal tolerance to the allogeneic fetus. blockade in vivo of cd and cd costimulation could prevent abortions by shifting cytokines from th predominance to th bias and expanding peripheral cd + cd + regulatory t cells (jin et al. ) . breakdown of immunologic self tolerance maintained by activated t cells expressing il- receptors (cd- ) results in the development of autoimmune diseases (sakaguchi et al. ; sakaguchi ) , which can be mitigated using anti-cd monoclonals. suppressive cd + cd t reg cells are elevated also during pregnancy (somerset et al. ) . intergins: progressive multifocal leukoencephalopathy was observed (pml) probably of polyomavirus etiology after natalizumab (anti-integrin-a ) therapy of crohn's disease (edula and picco ). tnfalpha blockers were shown to induce autoimmunity on ana and anti-dsdna antibodies in ra and spa patients. autoimmunity was induced more frequently with infliximab than etanercept and to a lesser degree to adalimumab therapy but, more importantly, this emergent autoimmunity was exceptionally associated to clinical manifestations of lupus (bacquet-deschryver et al. ) . the effect of infliximab, etanercept or adalimumab on spermatogenesis has been studied in patients with spondylarthritis (villiger et al. ) . sperm abnormalities were found in healthy controls. patients on anti-tnf therapy showed significantly better sperm motility and vitality than untreated patients (Østensen and f€ orger ). antibody products licensed for prevention or treatment of viral diseases include non-immune human immunoglobulin for use against hepatitis a and measles, virusspecific polyclonal human immunoglobulin against cytomegalovirus, hepatitis b, rabies, respiratory syncytial virus (rsv), vaccinia, and varicella-zoster, and the humanized monoclonal antibody palivizumab, fonolizumab and motavizumab (groothuis et al. ) . polyclonal immunoglobulin has also been used with various success for diseases caused by other human viruses including parvovirus b (pv b ), lassa virus, west nile virus, some enteroviruses, herpes simplex virus, crimean-congo haemorrhagic fever virus (cchfv), junin virus, severe acute respiratory syndrome-associated coronavirus (sars cov) and human immunodeficiency virus (hiv). serum polyclonal antibody preparations have been clinically effective in many cases, problems related to toxicity including a risk for allergic reactions, lot to lot variation and uncertain dosing have limited their use (casadevall (casadevall , . the use of rabies and tick-borne encephalitis virus-specific hyperimmune gamma globulins are used in several countries immediately following virus exposure (animal injuries or tick bites). cytomegalovirus-specific hyperimmune gamma globulin is used in the transplantation surgery (schmitz and essuman ) before the era of gancyclovir preventive therapy. sars cov surface glycoprotein, also called spike glycoprotein, (s protein or s glycoprotein) mediates viral entry into the host cell and has two functional domains s and s . the s domain is involved in the binding of the cellular receptor ace whereas the s domain facilitates the fusion between viral and host cell membranes. infections by many viruses, including coronaviruses, elicit potent neutralizing antibodies (nabs) that can affect the course of infection and help clear the virus; they can also protect an uninfected host exposed to the virus. an improved method for epstein-barr virus (ebv) transformation of human b cells has been developed based on cpg oligonucleotides that increases the b cell immortalization efficiency from - % to - %, and this method was used for selection of human abs specific for sars cov proteins. one of the selected antibodies, which was specific for the s glycoprotein on the viral spikes, was about -fold more efficient in neutralization than convalescent serum. nipah virus (niv) and hendra virus (hev) are closely related emerging paramyxoviruses that comprise the henipavirus genus. they are biological safety level- (bsl- ) pathogens, and are on the niaid biodefense research agenda as zoonotic emerging category c priority pathogens that could be used as bioterror agents (zhu et al. ). monoclonal antibodies of enzyme activity have been developed. these can be used in cancer therapy, but the application for the treatment of pregnant women is at present not yet approved (kulkarni et al. ; quatrale et al. ). immunotherapy offers a range of potential treatment options: drug treatment, as well as the treatment of overdose, prevention of brain or cardiac toxicity and fetal protection in pregnant drug abusers. clinical trials, cocaine and nicotine vaccines have been shown to induce antibody titers while producing few side effects (haney and kosten ) . plasmin may serve as a major driving autoantigen for some anticardiolipin (acl) in anti-phospholipid syndrome (aps) patients who are positive for igg anti-plasmin ab. one mab displayed the anti-cardiolipin (acl) and the lupus anti-coagulant (lac) activities and induced fetal loss when injected into pregnant mice ). molecular mimicry has been suggested to play a role in the pathogenesis of many autoimmune diseases, such as allergic encephalomyelitis, experimental myocarditis, and experimental autoimmune keratitis and uveitis antigenic molecular mimicry is characterising anti-dna antibodies. these are reacting with different proteins i.e. enzymes (blank and shoenfeld ) . in case of schizophrenia, the overall finding has been that, when a monozygotic twin has this serious neuromental disorder (nmd), the other identical twin has a % risk; whereas among dizygotic twins, the risk -when one is afflicted -is only %. neuromental disorders (nmd) might be caused indirectly by maternal transplacentally-acquired antibodies, to agents with epitope molecular mimicry with the developing nervous system, and cause alterations which will clinically manifest years later (nahmias et al. ) . serological evidence of previous exposure to ebv in children with ms supports a role for ebv infection early in ms pathogenesis, as already indicated by prospective studies in adults. higher antibody titers and t-cell responses to ebv in patients compared to healthy ebv carriers indicate possible continuous viral reactivation. ms patients have increased cd + and cd + t-cell responses to ebv antigens, particularly ebna . there is some evidence that ebv could break immune tolerance to myelin antigens through molecular mimicry. detection of ebv-infected b-cells in patients' brain raises the possibility that intrathecal b-cell abnormalities and t-cell-mediated immunopathology in ms are the consequence of a persistently dysregulated ebv infection. accordingly, targeting t-cells and/or b-cells with monoclonal antibody therapies ameliorates ms. whether ebv has a causative or pathogenic role in ms can now be addressed in relation to genetic, hormonal and other environmental influences that may affect ebv-host interactions (salvetti et al. ). functional suppression by cd + cd + regulatory t cells was also found to be impaired in ms patients (viglietta et al. ). newborns of pregnants suffering from multiple sclerosis (ms) were impaired by the disease. in case the father of the newborn was suffering from ms, no negative consequences could be documentet i.e. safe paternity characterises ms-patients. the results of mothers does not seem to have an impact on birth weight, however, ms may contribute to a reduced birth weight (hellwig et al. ) . the mothers suffering from ms are usually treated with long-term interferon (ifn) beta-therapy in spite of the pregnancy. the foetal exposure to subcutaneous interferon beta- a therapy before treatment discontinuation was at least days; most pregnancies ( / ; . %) were exposed for days. the rates of spontaneous abortion and major congenital anomalies in live births were in line with those observed in the general population (amato et al. ; sandberg-wollheim et al. ) . the in vitro susceptibility of bewo cells was increased for toxoplasma gondii following treatment with interferon gamma, interleukin and transforming growth factor -beta (barbosa et al. ), but similar consequences were not observed during pregnancy in vivo. congenital epidermolysis bullosa acquisita: 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cells secrete the immunosuppressive hla-g molecule upon ctla -ig treatment: implication in human renal transplant acceptance bewo trophoblast cell susceptibility to toxoplasma gondii is increased by interferon-gamma, interleukin- and transforming growth factor-beta anti-infliximab and antiadalimumab antibodies in relation to response to adalimumab in infliximab switchers and anti-tnf naive patients: a cohort study constitutive p promoter activation and il- production in the terminal ileum mediated by dendritic cells vegf expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma severe neonatal myasthenia due to maternal anti-musk antibodies pregnancy in wegener's granulomatosis: successful treatment with intravenous immunoglobulin effect of medication on microvascular vasodilatation in patients with systemic lupus erythematosus common vaccinations among adults do not increase the risk of developing rheumatoid arthritis: results from the swedish eira study 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pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic t-cells during the course of secondary dengue virus infection antiantibody enhancement of hemagglutination inhibition by influenza anti-neuraminidase a neoadjuvant clinical trial in colorectal cancer patients of the human anti-idiotypic antibody ad , which mimics cd an evidence-based review of natalizumab therapy in the management of crohn's disease pathogenesis and management of adult-onset still's disease compromised function of regulatory t cells in rheumatoid arthritis and reversal by anti-tnf alpha therapy red cell aplasia and autoimmune hemolytic anemia following immunosuppression with alemtuzumab, mycophenolate, and daclizumab in pancreas transplant recipients impact of t-cell costimulation modulation in patients with undifferentiated inflammatory arthritis or very early rheumatoid arthritis: a clinical and imaging study of abatacept (the adjust trial) acquired myasthenia gravis in childhood 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a multicenter, prospective, open-label clinical trial the effects of rituximab treatment during pregnancy on a neonate the myeloid-related proteins and complex, a novel ligand of toll-like receptor , and interleukin- beta form a positive feedback mechanism in systemic-onset juvenile idiopathic arthritis rituximab for management of refractory pregnancy-associated immune thrombocytopenic purpura t cells are crucial for the anti-metastatic effect of anti-epidermal growth factor receptor antibodies immune thrombocytopenic purpura in pregnancy: a reappraisal of obstetric management and outcome genetic polymorphisms of macrophage-mediators in guillain-barré syndrome developing recombinant hpa- a-specific antibodies with abrogated fcg receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia phase iii study of adjuvant vaccination with bec /bacille calmette-guerin in responding patients with limited-disease small-cell lung cancer (european organisation for research and treatment of cancer - b; silva study) cell cycle arrest by transforming growth factor beta enhances replication of respiratory syncytial virus in lung epithelial cells the inherited autoinflammatory syndrome: a decade of discovery acute inflammatory demyelinating polyneuropathy in patients with pregnancy cellular and humoral immune response to n-glycolyl-gm elicited by prolonged immunotherapy with an anti-idiotypic vaccine in high-risk and metastatic breast cancer patients a postnatal change in the immunological properties of the acetylcholine receptor at rat muscle endplates direct binding of respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal adherence to human epithelial cells and pneumococcal invasiveness in a murine model therapeutic vaccines for substance dependence antiphosphatidylserine antibody combined with irradiation damages tumor blood vessels and induces tumor immunity in a rat model of glioblastoma parenthood and immunomodulation in patients with multiple sclerosis the epidemiology of graves' disease: evidence of a genetic and an environmental contribution characterization of the antibody response against neugcgm ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody anti-cd monoclonal antibody in newonset type diabetes mellitus a single course of anti-cd monoclonal antibody hokt g (ala-ala) results in improvement in c-peptide responses and clinical parameters for at least years after onset of type diabetes development of innervation of skeletal muscle fibers in man: relation to acetylcholine receptors anti-immunoglobulin e treatment with omalizumab in allergic diseases: an update on anti-inflammatory activity and clinical efficacy immunomodulating antibodies and drugs for the treatment of hematological malignancies behçet's disease and pregnancy pediatric hereditary autoinflammatory syndromes blockade of cd and cd at the time of implantation inhibits maternal rejection to the allogeneic fetus in abortion-prone matings contribution of respiratory syncytial virus g antigenicity to vaccine-enhanced illness and the implications for severe disease during primary respiratory syncytial virus infection childbirths and risk of female predominant and other autoimmune diseases in a population-based danish cohort effectiveness of omalizumab in monozygotic twin sisters with severe allergic asthma placental transport of immunoglobulins: a clinical review for gastroenterologists who prescribe therapeutic monoclonal antibodies to women during conception and pregnancy trastuzumab activates allogeneic or autologous antibody-dependent cellular cytotoxicity against malignant rhabdoid tumor cells and interleukin- augments the cytotoxicity the ctla- gene is expressed in placental fibroblasts the management of pregnancy in paroxysmal nocturnal haemoglobinuria on long term eculizumab insulin needs after cd -antibody therapy in new-onset type diabetes autoimmune disorders: maternal, fetal, and neonatal risks prevention of autoimmune rheumatic disease: state of the art and future perspectives complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (egfr) and its variant iii (egfrviii) progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta- a for multiple sclerosis current treatment and immunotherapy of hodgkin's lymphoma rituximab administration in third trimester of pregnancy suppresses neonatal b-cell development regulation of immune response by anti-receptor antibody d stimulation enhances the antilymphoma activity of anti-cd antibodies progressive multifocal leukoencephalopathy associated with efalizumab use in psoriasis patients successful term pregnancy in a patient with wegener's granulomatosis: case report and literature review lethal vasculitis of coronary arteries in a neonate and two infants: possible neonatal variant of the mlns/ipn complex? ramucirumab, a fully human mab to the transmembrane signaling tyrosine kinase vegfr- for the potential treatment of cancer a phase i pharmacologic study of necitumumab (imc- f ), a fully human igg monoclonal antibody directed against egfr in patients with advanced solid malignancies calpain regulates sensitivity to trastuzumab and survival in her -positive breast cancer vaccination with human anti-trastuzumab anti-idiotype scfv reverses her immunological tolerance and induces tumor immunity in mmtv.f.huher (fo ) mice maternal autoantibodies and pregnancy. ii. the neonatal lupus syndrome administration of agonistic anti- - bb monoclonal antibody leads to the amelioration of inflammatory bowel disease rituximab for passenger lymphocyte syndrome associated with allogeneic sct ifn-gamma production during initial infection determines the outcome of reinfection with respiratory syncytial virus durable complete responses from therapy with combined epratuzumab and rituximab: final results from an international multicenter, phase study in recurrent, indolent, non-hodgkin lymphoma a prospective nested case-control study of dengue in infants: rethinking and refining the antibody-dependent enhancement dengue hemorrhagic fever model effect of anti-cd antibody alemtuzumab on ex-vivo culture of umbilical cord blood stem cells hypoparathyroidism and autoimmunity in the q . deletion syndrome placental transfer of igg paraprotein with prolonged immunosuppression placental transfer of an igg paraprotein associated with prolonged immunosuppression influence of hla-drb genes and the shared epitope on genetic susceptibility to rheumatoid arthritis in taiwanese increased frequencies of cd + cd (high) regulatory t cells in acute dengue infection guillain-barré syndrome in mother and newborn child monoclonal antibodies to p basic protein and their use in the investigation of some aspects of the guillain-barré syndrome the pathogenesis of biliary atresia: evidence for a virus-induced autoimmune disease killing of myeloid apcs via hla class i, cd and cd defines a novel mechanism of suppression by human tr cells prevalence, incidence and persistence of etanercept and adalimumab in norway adalimumab, etanercept, infliximab, rituximab and abatacept for the treatment of rheumatoid arthritis after the failure of a tumour necrosis factor inhibitor: a systematic review and economic evaluation tocilizumab inhibits interleukin- -mediated matrix metalloproteinase- and - secretions from human amnion cells in preterm premature rupture of membranes long-term followup of children with neonatal lupus and their unaffected siblings monoclonal antibodies in pediatrics: use in prevention and treatment a twin study in behçet's syndrome identification of predictive biomarkers for response to trastuzumab using plasma fuca activity and n-glycan identified by maldi-tof-ms the role of anti-idiotypic antibodies in the induction of experimental systemic lupus erythematosus in mice international natalizumab multiple sclerosis trial group ( a) a controlled trial of natalizumab for relapsing multiple sclerosis marginal transfer of reopro (abciximab) compared with immunoglobulin g (f ), inulin and water in the perfused human placenta in vitro clinicopathological features of malignant lymphoma in japan: the miyagi study anti-pd- synergizes with cyclophosphamide to induce potent anti-tumor vaccine effects through novel mechanisms cytokines in severe respiratory syncytial virus bronchiolitis guillain-barré syndrome in pregnancy: a rare complication of varicella preeclampsia is associated with decreased serum alpha( )-hs glycoprotein (fetuin-a) concentration circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia neonatal myasthenia gravis-a new clinical and immunologic appraisal on cases past hepatitis b virus infection in rheumatoid arthritis patients receiving biological and/or nonbiological disease-modifying antirheumatic drugs biliary atresia in a twin eyelash trichomegaly secondary to panitumumab therapy wegener granulomatosis: a case report and update maternal microchimerism in underlying pathogenesis of biliary atresia: quantification and phenotypes of maternal cells in the liver etanercept during pregnancy and lactation in a patient with rheumatoid arthritis: drug levels in maternal serum, cord blood, breast milk and the infant's serum fine specificity of high molecular weight-melanoma-associated antigen-specific cytotoxic t lymphocytes elicited by anti-idiotypic monoclonal antibodies in patients with melanoma conception and pregnancy outcome in women with inflammatory bowel disease: a multicentre study from japan transient neonatal acquired von willebrand syndrome due to transplacental transfer of maternal monoclonal antibodies the possible role of transplacentally-acquired antibodies to infectious agents, with molecular mimicry to nervous system sialic acid epitopes, as causes of neuromental disorders: prevention and vaccine implications neonatal lupus erythematosus in identical twins, showing transient bullous lesions neonatal myasthenia gravis: report of two cases and review of literature the wolf in sheep's clothing: the role of interleukin- in immunity, inflammation and cancer gender disparity in liver cancer due to sex differences in myd -dependent il- production microchimerism: incidental byproduct of pregnancy or active participant in human health? the synthetic bacterial lipopeptide pam csk modulates respiratory syncytial virus infection independent of tlr activation high serum ifn-alpha activity is a heritable risk factor for systemic lupus erythematosus could myocarditis, insulin dependent diabetes mellitus, and guillain-barré syndrome be caused by one or more infections agents carried by rodents mechanisms and pathologic significances in increase in serum interleukin- (il- ) and soluble il- receptor after administration of an anti-il- receptor antibody, tocilizumab, in patients with rheumatoid arthritis and castleman disease transient neonatal myasthenia gravis in a baby born to a mother with new-onset anti-musk-mediated myasthenia gravis infection enhancement of influenza a h subtype viruses in macrophage-like p d cells by cross-reactive antibodies intravenous immunoglobulin for suspected or subsequently proven infection in neonates immunopathogenesis of vaccine-enhanced rsv disease neonatal lupus erythematosus: a possible role for anti-sm antibodies antibody-dependent enhancement of respiratory syncytial virus infection by sera from young infants fetal acetylcholine receptor inactivation syndrome and maternal myasthenia gravis treatment with biologics of pregnant patients with rheumatic diseases update on safety during pregnancy of biological agents and some immunosuppressive antirheumatic drugs induction of anti-viral immune response by immunization with monoclonal anti-idiotype antibodies directed to private idiotopes structurally derived mutations define congenital heart blockrelated epitopes within the - amino acid stretch of the ro protein neonatal outcomes of pregnancy complicated by idiopathic thrombocytopenic purpura the treatment of rheumatoid arthritis during pregnancy one-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of huntington disease an interspecies comparison of placental antibody transfer: new insights into developmental toxicity testing of monoclonal antibodies pathogenic mechanisms in systemic lupus erythematosus endogenous retroviral pathogenesis in lupus early loss of pregnancy after intravitreal bevacizumab injection club rhumatismes et inflammation (cri) ( ) tocilizumab: therapy and safety management swedish natalizumab (tysabri) multiple sclerosis surveillance study partial reduction of human foxp cd t cells in vivo after cd -directed recombinant immunotoxin administration enhancement of cell-mediated immunity in melanoma patients immunized with murine anti-idiotypic monoclonal antibodies (melimmune) that mimic the high molecular weight proteoglycan antigen fc receptors in immune thrombocytopenias: a target for immunomodulation? differences in platelet function in patients with acute myeloid leukaemia and myelodysplasia compared to equally thrombocytopenic patients with immune thrombocytopenia nti-egfr monoclonal antibody in cancer treatment: in vitro and in vivo evidence new strategies for the treatment of hepatitis c virus infection and implications of resistance to new direct-acting antiviral agents expression of the human concentrative nucleotide transporter (hcnt ) gene correlates with clinical response in patients affected by waldenstr€ om's macroglobulinemia (wm) and small lymphocytic lymphoma (sll) undergoing a combination treatment with -chloro- -deoxyadenosine ( -cda) and rituximab foetal and neonatal thyroid disorders transfer of the shared epitope through microchimerism in women with rheumatoid arthritis clinical use of anti-cd antibody daclizumab to enhance immune responses to tumor antigen vaccination by targeting regulatory t cells use of the anti-idiotype antibody vaccine triab after autologous stem cell transplantation in patients with metastatic breast cancer interim analysis of the use of the anti-idiotype breast cancer vaccine d (triab) in conjunction with autologous stem cell transplantation in patients with metastatic breast cancer use of the anti-idiotype breast cancer vaccine d in conjunction with autologous stem cell transplantation in patients with metastatic breast cancer interleukin- fused to an anti-idiotype antibody in a vaccine increases the specific humoral immune response against ca + (muc- ) ovarian cancer fontolizumab in moderate to severe crohn's disease: a phase , randomized, doubleblind, placebo-controlled, multiple-dose study targeted treatment using monoclonal antibodies and tyrosine-kinase inhibitors in pregnancy association of the idiotype:anti-idiotype antibody ratio with the efficacy of intravenous immunoglobulin treatment for the prevention of recurrent autoimmuneassociated congenital heart block permanent neonatal diabetes mellitus -the importance of diabetes differential diagnosis in neonates and infants inflammatory lesions of the peritoneum mimic carcinomatosis after treatment with intravenous chemotherapy and intraperitoneal catumaxomab permanent diabetes during the first year of life: multiple gene screening in patients biological therapies for inflammatory bowel diseases langman's medical embryology, th edn cd as a molecular target for immunotherapy to treat acute myeloid leukemia with high evi expression naturally arising cd + regulatory t cells for immunologic self-tolerance and negative control of immune responses immunologic self-tolerance maintained by activated t cells expressing il- receptor alpha-chains (cd ) does the use of tumour necrosis factor antagonist therapy in poor prognosis, undifferentiated arthritis prevent progression to rheumatoid arthritis? preferential use of b . and not b . in priming of vaccinia virus-specific cd t cells epstein-barr virus and multiple sclerosis pregnancy outcomes in multiple sclerosis following subcutaneous interferon beta- a therapy natalizumab induction and maintenance therapy for crohn's disease the role of cd and cytotoxic t-lymphocyte antigen- (ctla- ) in regulatory t-cell biology reshaping a human antibody to inhibit the interleukin -dependent tumor cell growth comparison of the neutralizing and elisa antibody titres to human cytomegalovirus (hcmv) in human sera and in gamma globulin preparations outcome of pregnancy in women with inflammatory bowel disease treated with antitumor necrosis factor therapy a phase ii trial of emd (matuzumab), a humanized anti-egfr monoclonal antibody, in patients with platinum-resistant ovarian and primary peritoneal malignancies antiphospholipid syndrome (aps): where does it come from? acquired von willebrand's syndrome in systemic lupus erythematosus guillain-barré syndrome in children b-cell homeostasis requires complementary cd and blys/br survival signals purified t-depleted, cd + peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia normal human pregnancy is associated with an elevation in the immune suppressive cd +cd + regulatory t-cell subset technology evaluation: nimotuzumab, the center of molecular immunology/ym biosciences/oncoscience anti-erbb- mab therapy requires type i and ii interferons and synergizes with anti-pd- or anti-cd mab therapy hashimoto thyroiditis is more frequent than expected when diagnosed by cytology which uncovers a pre-clinical state cutaneous vasculitis in the newborn of a mother with cutaneous polyarteritis nodosa review of current treatment options for pregnancy-associated breast cancer evaluation of angiogenesis in non-small cell lung cancer: comparison between anti-cd antibody and anti-cd antibody intravitreal bevacizumab during pregnancy the huntington's disease collaborative research group (hdcrg) ( ) a novel gene containing a trinucleotide repeat that is expanded and unstable on huntington's disease chromosomes inhibition of complement activity by humanized anti-c antibody and single-chain fv loss of ctla- leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of ctla- combined antibody mediated inhibition of igf- r, egfr, vegfr for more consistent and greater anti-tumor effects targeted therapy with the t-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal residual disease in b-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival the role of t cells in the enhancement of respiratory syncytial virus infection severity during adult reinfection of neonatally sensitized mice preconceptional oral vaccination prevents experimental biliary atresia in newborn 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the effects of antitumour necrosis factor-a on bone metabolism in inflammatory bowel disease loss of functional suppression by cd +cd + regulatory t cells in patients with multiple sclerosis effects of tnf antagonists on sperm characteristics in patients with spondyloarthritis biologic therapy and pregnancy outcomes in women with rheumatic diseases effects of abciximab on key pattern of human coronary restenosis in vitro: impact of the si/mpl-ratio targeted inhibition of interleukin- with cnto sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death pd- but not ctla- blockage abrogates the protective effect of regulatory t cells in a pregnancy murine model immunological responses to the tumor-associated antigen ca in patients with advanced ovarian cancer induced by the murine monoclonal anti-idiotype vaccine aca immunological contribution to the pathogenesis of congenital heart block persistent expression of biologically active anti-her antibody by aavrh. -mediated gene transfer enterovirus infection of monocytes with antibody-dependent enhancement a glycoengineered anti-cd antibody with potent antibodydependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo treatment strategies for nodal and gastrointestinal follicular lymphoma: current status and future development lymphoproliferative disorders with early lethality in mice deficient in ctla- herceptin (trastuzumab) therapy during pregnancy: association with reversible anhydramnios neonatal lupus erythematosus. a clinical, serological and immunogenetic study with review of the literature postnatal development in cynomolgus monkeys following prenatal exposure to natalizumab, an alpha integrin inhibitor embryo/fetal development in cynomolgus monkeys exposed to natalizumab, an alpha integrin inhibitor motavizumab, a second-generation humanized mab for the prevention of respiratory syncytial virus infection in high-risk populations treatment with tumor necrosis factor inhibitors and intravenous immunoglobulin improves live birth rates in women with recurrent spontaneous abortion prevention strategies for type diabetes mellitus: current status and future directions down-regulation of placenta growth factor by promoter hypermethylation in human lung and colon carcinoma mhc class ii tetramers containing influenza hemagglutinin and ebv ebna epitopes detect reliably specific cd + t cells in healthy volunteers high intra-uterine exposure to infliximab following maternal anti-tnf treatment during pregnancy acquired trichomegaly and symptomatic external ocular changes in patients receiving epidermal growth factor receptor inhibitors rras: a key regulator and an important prognostic biomarker in biliary atresia exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody key: cord- -dnnkor o authors: georgiev, vassil st. title: tick-borne bacterial, rickettsial, spirochetal, and protozoal diseases date: journal: national institute of allergy and infectious diseases, nih doi: . / - - - - _ sha: doc_id: cord_uid: dnnkor o approximately tick species exist worldwide, parasitizing a broad array of mammals, including humans, and thereby playing a significant role in the transmission of infectious diseases ( ). in the united states, tick-borne diseases are generally seasonal and geographically distributed. they occur mostly during the spring and summer but can occur throughout the year. approximately tick species exist worldwide, parasitizing a broad array of mammals, including humans, and thereby playing a significant role in the transmission of infectious diseases ( ) . in the united states, tick-borne diseases are generally seasonal and geographically distributed. they occur mostly during the spring and summer but can occur throughout the year. these blood-feeding arthropods that parasitize all vertebrates can be classified into three families: (i) ixodidae (hard ticks) comprising approximately species and genera; (ii) argasidae (soft ticks), which consists of approximately species and genera; and (iii) nuttaliellidae, which is composed of only one species and is found only in africa ( , ) . ticks are major vectors of arthropod-borne infections and not only can transmit a wide variety of pathogens, such as rickettsia and other bacteria, viruses, and protozoa, but also may carry more than one infectious agent and thus transmit one or more infections to humans at the same time ( ) . infections transmitted by the ixodidae family (hard ticks) include (i) lyme disease (borreliosis); (ii) human ehrlichiosis; (iii) rocky mountain spotted fever; (iv) tularemia; (v) southern tick-associated rash illness; and (vi) babesiosis. infections transmitted by the argasidae family (soft ticks) include the tick-borne relapsing fever ( ) . ticks of the ixodidae family that transmit infections to humans are most often associated with the genera amblyomma, ixodes, dermacentor, and rhicephalus. they live in diverse but relatively humid habitats, and the infections they transmit are usually seasonal and geographically distributed. the hard ticks can attach securely to their hosts and feed slowly for prolonged periods, which will facilitate the transmission of infectious pathogens ( ) . in contrast, the only important disease-transmitting soft ticks belonging to the argasidae family are those of the genus ornithodoros, and they transmit spirochetes throughout their life cycle. ornithodoros feed rapidly, typically at night, and may transmit disease in as little as seconds ( ) . in the united states, ticks have been found in all regions of the country, and the incidence rates of tick-borne diseases have increased steadily over the past decade or so (http:// www .niaid.nih.gov/research/topics/lyme/introduction/htm). although lyme disease and rocky mountain spotted fever are well known to the general public, recently emerging infections, such as ehrlichiosis and anaplasmosis (formerly known as human granulocytic ehrlichiosis), have now also been firmly established in the country. the increasing reports of tick-borne diseases likely reflect improved awareness, surveillance and diagnosis, but the growing u.s. population and the spread of human communities into previously undeveloped environments also increase the regions of tick-human contact. since the identification of borrelia burgdorferi as the causative agent of lyme disease in , tickborne human bacterial pathogens have now been described throughout europe. these include five spotted fever rickettsiae, the etiologic agent of human granulocytic anaplasmosis (hga), four species of b. burgdorferi complex, and a new relapsing fever-causing borellia ( ) . if left untreated, the tick-borne infections can be associated with significant morbidity and even mortality. lyme disease (borreliosis) is the most prevalent tick-borne infectious disease in the united states. the disease is caused by a spiral-shaped bacterium borrelia burgdorferi and is spread by the deer tick ixodes scapularis. the likelihood for a person to be bitten by a deer tick is greater during the times of the year when ticks are most active. young deer ticks, called nymphs, are active from mid-may to mid-august and are approximately the size of poppy seeds. adult ticks, which are approximately the size of sesame seeds, are most active from march to mid-may and from mid-august to november. both nymphs and adult ticks can transmit lyme disease at any time when the temperatures are above freezing. during - , cdc received reports of , lyme disease cases from states and the district of columbia ( ); % of cases occurred among residents of the healthy people reference states (see table and the average annual rate in these reference states for the -year period was . cases per , population: . in , . in , and . in ( ) . not all deer ticks are infected with lyme disease. ticks can become infected only if they feed on small animals that are infected. in most cases, the tick must stay attached to humans for hours or more before the bacteria can be transmitted. there is no person-to-person spread of lyme disease. in extremely rare cases reported, the bacteria may be transferred from an infected pregnant woman to the fetus. even if successfully treated, a person may become re-infected if bitten later by another infected tick. the complex life cycle of b. burgdorferi, which passes through ticks and various intermediate hosts (mice and deer) before infecting humans, is still not completely understood ( , ) (http://www .niaid.nih.gov/research/topics/lyme). the outer surface protein a (ospa) of b. burgdorferi has been extensively studied, leading to a number of hypotheses regarding its role in conjunction with other cell surface proteins (ospb and ospc) in transmission of lyme disease ( ) . although b. burgdorferi depends on ixodes ticks and mammalian hosts for its life cycle, the search for the borrelial genes responsible for its parasitic dependence on these diverse hosts has been hindered by the difficulties in genetically manipulating virulent strains of the pathogen. nevertheless, there is strong evidence indicating that the inactivation and complementation of a linear plasmid- -encoded gene, bpta (formerly known as bbe ), is essential for the persistence of b. burgdorferi in i. scapularis ticks, and therefore, it must be considered to be a major virulence factor that is critical for b. burgdorferi's overall parasitic strategy ( ). early symptoms of lyme disease usually appear within to days after the bite of an infected tick. in % to % of cases, a rash resembling a bull's eye or solid patch (about inches in diameter) will appear and expand around or near the site of the bite. occasionally, multiple rash sites may also appear. in the early stage of the lyme disease, one or more of the following symptoms will appear: chills and fever, headache, fatigue, stiff neck, muscle and/or joint pain, and swollen glands. if not recognized or left untreated, symptoms become more severe, and include fatigue, a stiff aching neck, and tingling or numbness in the arms and legs, or facial paralysis. the most severe symptoms can occur even weeks, months, or years after the tick bite and include severe headache, painful arthritis, swelling of the joints, and heart and central nervous system problems. some evidence points to an autoimmune disease, perhaps triggered by the initial infection ( , ) (http://www .niaid.nih.gov/topics/ lymedisease/research/autoimmune.htm). early treatment of lyme disease involves the use of antibiotics, which in nearly all cases results in a complete cure. however, the likelihood of a full cure will decrease if treatment is delayed. and whereas in most infected individuals lyme disease can be easily treated with antibiotics, in a small percentage of patients it may lead to debilitating symptoms that may continue for years after treatment. as a result of dissemination through the bloodstream, b. burgdorferi can invade the central nervous system within days to a week after the initial skin infection. once in the central nervous system, the spirochete may affect the brain, most commonly causing a disturbance in thinking (cognition), known as lyme encephalopathy. other symptoms may include headache, mood swings, irritability, depression, and a high degree of fatigue. these symptoms comprise the typical features of neuropsychiatric lyme disease in adults (http://www.columbia-lyme.org/ flatp/lymeoverview.html). many of these symptoms are common manifestations in other disorders, such as mood or anxiety disorders, collagen vascular or autoimmune diseases, spinal cord compression, multiple sclerosis, metastatic diseases, endocrinologic disorders, fibromyalgia, chronic fatigue syndrome, and residual damage from past brain trauma or toxin exposure. nevertheless, knowing the typical cluster of symptoms can be helpful in diagnosing this condition. the majority of patients with lyme encephalopathy will not present with joint problems at the time that their cognitive symptoms have been recognized. it is important to emphasize that bedside neurologic examination does not usually disclose neurologic findings, and standard office-based cognitive screening test may not detect cognitive impairment. to detect cognitive disturbance, a more comprehensive neuropsychological examination would be needed. in addition, lumbar puncture, even though important in the differential diagnosis, should not be used to exclude neurologic lyme disease, as approximately % to % of patients with confirmed neurologic lyme disease may test negative in routine cerebrospinal fluid (csf) assays (http://www.columbia-lyme. org/flatp/lymeoverview.html). in general, the majority of patients who undergo early antibiotic therapy will not incur long-term central nervous system problems. the typical time course of the manifestations of lyme encephalopathy (http://www.columbia-lyme.org/flatp/lymeoverview.html) is as follows: (i) very early: erythema migrans (a red, round, expanding rash). (ii) one to months after infection: cardiac or early neurologic involvement (meningitis, encephalitis, cranial neuropathies) with mild to marked neuropsychiatric symptoms. (iii) six to months after infection: arthritis of multiple joints. (iv) two to years after infection: chronic cognitive problems. typical symptoms among adult patients with neuropsychiatric lyme disease (http://www.columbia-lyme.org/flatp/ lymeoverview.html) include: (i) mild to severe fatigue (a need for prolonged sleep), lowgrade fevers, night sweats, migrating arthralgias (joint pains) or arthritis (joint inflammation or swelling), muscle pains, sleep disturbances, and frequent and severe headaches. (ii) cranial nerve disturbance. though either facial nerve palsy or optic neuritis is not frequently manifested, patients may more commonly present with facial numbness and/or tingling. (iii) sharp, stabbing, deep/boring, burning, or lancinating (shooting) pains, as well as signs of peripheral neuropathy (multifocal numbness or tingling in hands or feet). (iv) cognitive problems may include problems of attention, memory, verbal fluency, and thinking speed. some patients may experience what is otherwise a normal environmental stimulation to be excessive, resulting in a cognitive "short-circuiting" (cognitive overload) where patients may start to feel confused, lose focus, stutter, or panic. (v) "brain fog," a term frequently used by patients with lyme disease to describe a syndrome characterized by lack of clarity in their cognitive processes similar to "depersonalization or derealization" in which the person's sense of self and place are altered. (vi) sensory hyperacuities characterized by heightened sensitivity to sound or to light, particularly in the early stages of neurologic lyme disease. (vii) spatial or geographic orientation problems where a patient may bump into the door jambs; try to place an object on a table only to have it fall to the floor due to a misjudgment of spatial distance; or get lost in a familiar place. (viii) less common neurologic syndromes include partial or complex seizures, multiple sclerosis-like illness, dementia-like illness, guillain-barré syndrome, strokes, and tullio phenomenon. most common symptoms of neuropsychiatric disorders in children suffering from lyme disease include headaches, disturbances of behavior or mood, fatigue (falling asleep in class), and problems with auditory and visual attention (some children could be mistakenly diagnosed as having attention deficit disorder). they may have fluctuating symptoms: worse on some days, remarkably better on others, without a clear cause (http://www.columbia-lyme.org/flatp/lymeoverview.html). as noted among adults, when lyme disease is treated early, few children will develop long-term cognitive or neuropsychiatric problems. prior vaccination with the licensed recombinant outer surface protein a (ospa) vaccine reduces the risk of developing lyme disease associated with tick bites ( ). administration of doxycycline ( mg, twice daily) or amoxicillin ( mg, times daily) for to days is recommended for the treatment of early localized or early disseminated lyme disease associated with erythema migrans, in the absence of neurologic involvement or third-degree atrioventricular heart block ( ) . in prospective studies, these two drugs have been shown to be effective in treating erythema migrans and associated symptoms. doxycycline has the advantage of being effective also for treating human granulocytic anaplasmosis (hga), another tick-borne infection that may occur simultaneously with lyme disease. however, doxycycline may be relatively contraindicated for pregnant women, during lactation, and for children aged years or younger (see also section . . . ). because of its higher cost, cefuroxime axetil ( mg orally, twice daily), which is as effective as doxycycline in the treatment of erythema migrans, should be reserved as an alternative agent for those patients who can take neither doxycycline nor amoxicillin ( ) . for children, the recommended dose of amoxicillin is mg kg − day − , divided into doses per day (maximum, mg/dose). cefuroxime axetil is an acceptable alternative given at a dose of mg kg − day − , divided into doses daily (maximum, mg/dose). macrolide antibiotics (azithromycin, erythromycin, and clarithromycin) are not recommended as first-line therapy for early lyme disease; when used they should be reserved for patients who are intolerant of amoxicillin, doxycycline, and cefuroxime axetil ( ) . intravenous ceftriaxone ( . g daily), although effective, is not superior to oral agents and is not recommended as a first-line agent for treatment of lyme disease in the absence of neurologic involvement or third-degree atrioventricular heart block. however, ceftriaxone is recommended for acute neurologic disease manifested by meningitis or radiculopathy ( ) . for children, the recommended dose of ceftriaxone is to mg kg − day − , in a single daily intravenous dose (maximum, . g), or cefotaxime ( to mg kg − day − ) divided into or doses (maximum, . g/daily) for to days ( ) . patients with first-or second-degree atrioventricular heart block associated with early lyme disease should be treated with the same antimicrobial regimens as patients with erythema migrans without carditis. the recommended treatment for patients with a third-degree atrioventricular heart block is parenteral antibiotics such as ceftriaxone in a hospital setting ( ) . although antibiotic treatment does not hasten resolution of seventh cranial nerve palsy associated with b. burgdorferi infection, antibiotics should be used to prevent further sequelae ( ). lyme arthritis usually can be treated successfully with antimicrobial agents administered orally or intravenously. thus, administration of oral doxycycline ( mg, twice daily) or amoxicillin ( mg, times daily), in each instance for days, is recommended for patients without clinically evident neurologic disease ( ) . for children, the recommended dose of doxycycline ( . to . mg/kg, twice daily; maximum, mg/dose) could be given to children age and older, or amoxicillin ( mg kg − day − , divided into doses per day; maximum, mg/dose) for days ( ) . whereas oral therapy is easier to administer than intravenous antibiotics, is associated with fewer adverse effects, and is significantly less expensive, its disadvantage is that some patients treated with oral antimicrobials have subsequently developed overt neuroborreliosis, which may require intravenous therapy for successful resolution ( ). the recommended therapy for patients with late neuroborreliosis affecting the central or peripheral nervous system is treatment with intravenous ceftriaxone ( . g, once daily for to weeks) ( ) . response to treatment is usually slow and may be incomplete. however, unless relapse is shown by reliable objective means, repeat treatment is not recommended ( ) . for children, the recommended treatment is a -to day course of ceftriaxone ( to mg kg − day − , in a single daily intravenous dose; maximum, . g) ( ). after an episode of lyme disease that is treated appropriately, some patients have a variety of subjective complaints, such as myalgia, arthralgia, or fatigue. such patients may then be classified as having either chronic lyme disease or post-lyme disease syndrome. however, both conditions are poorly defined because these patients represent a heterogenous group. because there have not been any randomized, controlled studies of patients who remain unwell after standard courses of antibiotic therapy for lyme disease, there are no convincing published data demonstrating that repeated or prolonged courses of either oral or intravenous antimicrobial therapy are effective for such patients ( ). the niaid has had a long-standing commitment to conduct research on lyme borreliosis, or lyme disease, beginning more than years ago when the cause of the disease was not yet known (http://www .niaid.nih.gov/ research/topics/lyme/research/). in , niaid-funded research efforts resulted in identifying borrelia burgdorferi, a spiral-shaped bacterium, or spirochete, as the causative agent of lyme disease ( r diagnosis, including the development and application of new technologies for rapid and sensitive diagnostic assays, as well as assessment, refinement, and standardization of improved diagnostic procedures. r treatment and prevention, including the development, application, and evaluation of novel and safe therapeutic approaches, as well as identification and characterization of candidate vaccines. r immune mechanisms, including understanding the development of protective immunity, characterizing the immunomodulatory properties of microbial antigens and evaluating their role in the pathogenesis, and characterizing the response of the host's immune system both during infection and after deliberate immunization. r pathogenesis, including the identification and characterization of virulence factors and the molecular basis for damage to host tissues during infection, and defining the role of cytokines and other immunomodulatory agents in the expression of disease. r epizootiology/ecology, including defining potential and established vectors and reservoirs, assessing the role of ticks and other vectors in transmitting the disease and maintaining virulence, relating the role of genetic variation in the incidence of disease in endemic areas, and defining effective measures for significantly reducing or eliminating populations of infected ticks in endemic areas. other developments of niaid-supported lyme borreliosis and tick-borne rickettsial disease research include: r the transmission of lyme disease r diagnostic procedures r co-infection r antibiotic therapy r the role of autoimmune reactivity r vaccine production lack of evidence of borrelia involvement in alzheimer's disease. because various published reports have suggested the possibility that b. burgdorferi may play a role in the etiology of alzheimer's disease, niaid intramural scientists have examined this issue in greater detail. the results of these studies, using a very sensitive polymerase chain reaction (pcr) assay capable of amplifying a borrelia-specific dna target sequence from all strains of b. burgdorferi sensu lato species known to cause disease in humans, have provided no evidence to indicate the presence of b. burgdorferi in the brains of patients with alzheimer's disease ( ). whereas early acute lyme borreliosis is easily cured by conventional antibiotic therapy, some patients who have been correctly diagnosed initially as having lyme disease may experience serious neurologic and musculoskeletal symptoms several months after receiving what appeared to have been successful antibiotic therapy. because it is unclear whether such symptoms are due to long-term persistent infections or other causes, the term posttreatment chronic lyme disease (ptcld) is often used to describe this condition, so as not to impose any judgment on the actual mechanism(s) that might be involved (see also section . . . ) . over the years, niaid has supported research regarding ptcld as well as other clinical issues (http://www . niaid.nih.gov/research/topics/lyme), including: this study, which was carried out in nemc in boston and completed in , was aimed at studying the clinical efficacy of antibiotic therapy for treating ptcld. it involved randomized, double-blind, placebo-controlled, multicenter trials to examine the safety and efficacy of ceftriaxone and doxycycline in patients with either seropositive or seronegative chronic lyme disease. the trials compared treatment with days of intravenous ceftriaxone followed by days of oral doxycycline to treatment with intravenous placebo followed by oral placebo for the same duration in patients who were either seropositive or seronegative at the time of enrollment. preliminary results from the trials showed that after days of continuous antibiotic therapy, there were no significant differences in the percentage of patients who felt that their symptoms had improved, gotten worse, or stayed the same between the antibiotic treatment and placebo groups in either trial ( ) . other results from the trials indicated that patients with ptcld did not show objective evidence of cognitive impairment and that days of continuous antibiotic therapy was not more beneficial for these patients than was administering a placebo ( ) . r state university of new york (suny) clinical study. in another placebo-controlled study conducted at suny at stony brook, patients with pctld were treated with either intravenous ceftriaxone or a placebo for days. they were then evaluated to determine whether there was significant improvement with respect to fatigue, cognitive function, and the clearance of ospa antigen that was present in the spinal fluid of only % of all enrolled patients. the results of the trial have shown that ceftriaxone therapy was associated with improvement in fatigue but not with the other primary outcome markers considered ( ) . because fatigue, which is a nonspecific symptom, was the only primary outcome measure affected and because the treatment examined was associated with adverse events, the results of the suny study do not support the use of additional antibiotic therapy with parenteral ceftriaxone in posttreatment, persistently fatigued ptcld patients (http://www .niaid.nih.gov/research/topics/lyme). r animal models. appropriate animal models also have provided considerable information on the transmission and pathogenesis of lyme borreliosis, as well as on the mechanisms involved in the development of protective immunity. niaid, in collaboration with the national institute of neurological disorders and stroke (ninds), has broadened these efforts to include comprehensive studies on non-human primate animal models for experimental research on the neuropathology associated with chronic lyme borreliosis ( ) . a major goal of these studies is to optimize the rhesus model of lyme borreliosis as well as to determine the pathogenesis of the disease with a focus on the neurologic manifestations. it is anticipated that these studies will expand the knowledge of those factors that contribute to the pathology associated with persistent infection of the central nervous system by b. burgdorferi and ultimately will enable scientists to devise more effective clinical approaches for treating chronic lyme borreliosis in humans. these studies will also supplement and enhance the results of current clinical research on the efficacy of antibiotic therapies for treating chronic lyme disease and provide precedents for use in designing future clinical studies and will ultimately enhance the results of current clinical studies on chronic lyme disease. inflammation of skeletal muscle is a consistent feature of lyme borreliosis, both in humans and in experimental animal models of infection. although several cytokines are expressed in muscle tissue, proinflammatory cytokines commonly associated with inflammation are not upregulated in borrelia-infected muscle. however, the expression of blymphocyte chemoattractant (blc), a chemokine implicated in the trafficking of b cells to tissues, is increased in borreliainfected muscles of non-human primates ( ) . using protein expression profiling, it has been shown that blc is upregulated in the spinal fluid of patients with neuroborreliosis but not in patients with noninflammatory and various other inflammatory neurologic diseases ( ) . because the upregulation of blc was found in every neuroborreliosis patient examined, it may be a valuable diagnostic marker for neuroborreliosis. other studies have shown that b. burgdorferi can be detected in mice for at least months after treatment with therapeutic doses of various antibiotics (ceftriaxone, doxycycline, or azithromycin). these surviving spirochetes could not be transmitted to healthy mice and some lacked plasmid genes associated with infectivity. by months, antibiotictreated mice no longer tested positive for the presence of b. burgdorferi, and even cortisone immunosuppression failed to alter this result; that is, it failed to activate infection. nine months after antibiotic treatment, low levels of borrelia dna still could be detected in some, but not all of the mice. these findings ( ) have indicated that noninfectious b. burgdorferi can persist for a limited time after antibiotic therapy. the implications of these findings to persistent infection and the nature of chronic lyme disease in humans remain to be assessed. results from recent studies have indicated that t cells from patients with chronic lyme disease were reactive not only against b. burgdorferi-specific antigens but also against various host (self) antigens ( ) . such antigenic mimicry might generate autoimmune inflammatory reactions that could be responsible for arthritic as well as neurologic symptoms associated with chronic lyme disease (http://www . niaid.nih.gov/research/topics/lyme/research/autoimmune/). in other studies, antibodies against the ospa epitopes of b. burgdorferi have also been shown to cross-react with neural tissue ( ) as well as myocin ( ) . such antigenic mimicry may have the potential to generate autoimmune inflammatory reactions that could be responsible for the neurologic symptoms associated with chronic lyme disease. in this context, it is interesting to note that homologies between proteins of b. burgdorferi and thyroid antigens have also been reported ( ) . in niaid-supported clinical studies, case subject patients with ptcld were compared with control subjects without such symptoms for the presence of several human leukocyte antigen (hla) class ii (drb and dqb ) genetic markers, some of which are known to be associated with the expression of autoimmune reactivity. the results obtained did not support the involvement of an autoimmune mechanism in ptcld ( ) . however, because not all autoimmune diseases are associated with specific hla haplotypes, these findings do not necessarily exclude that possibility. definitive proof would clearly involve demonstrating the presence of significant levels of relevant autoimmune antibodies and/or autoreactive t cells in patients with ptcld but not in treated control subjects without such symptoms. a greater frequency of drb * , which has been reported to be associated with antibiotic-treatmentresistant arthritis, was noted in the case subject patients; although this finding appeared to be nominally significant (p < . ), its biological significance is ambiguous because none of the case subjects considered had symptoms of inflammatory arthritis (http://www .niaid.nih.gov/ research/topics/lyme/research/autoimmune/). co-infection could represent a major potential problem, mainly because the ixodes ticks that transmit b. burgdorferi often carry-and simultaneously transmit-other emerging pathogens, such as anaplasma (ehrlichia) species, the causative agent of human granulocytic ehrlichiosis (hge), and babesia microti, which causes babesiosis (http://www .niaid.nih.gov/research/topics/lyme/ research/co-infection/). in europe and asia, ixodes ticks also are known to transmit tick-borne encephalitis viruses. fortunately, this tick-borne viral infection has not yet been reported in the united states, although co-infections with powasan virus and deer tick virus have been reported. co-infection by some or all of these other infectious agents may interfere with the clinical diagnosis of lyme borreliosis and/or adversely influence host defense mechanisms, thereby altering landmark characteristics of the disease and the severity of infection ( ) . niaid-supported studies have indicated that co-infection with hge increases the severity of lyme borreliosis ( ) . by contrast, when mice were co-infected with b. microti and b. burgdorferi, neither agent influenced the course of infection induced by the other as evidenced by the percentage of parasitemia, spleen weights, and hematologic and clinical chemistry parameters ( ) . in niaid-supported clinical studies on chronic lyme disease, patients with persisting symptoms were examined to determine if they might have been co-infected with other tick-borne infectious diseases at the time of their acute episode of lyme disease. among the tick-borne infectious diseases considered were babesiosis (babesia microti), granulocytic ehrlichiosis (anaplasma phagocytophilum), and tick-borne encephalitis virus infection. the seroprevalence rates for b. microti and a. phagocytophilum were found to be . % and . %, respectively, and no patient examined was found to be positive for tick-borne encephalitis viruses ( ) . thus, the persistence of symptoms in patients with "post-lyme syndrome" could not be attributed to co-infection with one of these pathogens. an examination of pathogen distributions in the tissues of mice infected with both b. burgdorferi and a. phagocytophilum, the bacterium that causes hge in humans, showed an increase in the numbers of b. burgdorferi in the ears, heart base, and skin of co-infected mice; however, the numbers of a. phagocytophilum remained relatively constant. the serum antibody response to a. phagocytophilum (but not to b. burgdorferi) decreased as a result of co-infection. these findings suggest that co-infection can influence not only pathogen burden but also host antibody responses ( ) . niaid intramural and extramural research programs have initiated clinical studies on chronic lyme disease. the intramural research program is conducting a comprehensive clinical, microbiologic, and immunologic assessment of patients with lyme disease. this involves multiple lines of investigation with emphasis on (i) defining various biological markers of infection; (ii) assessing clinical course and outcomes of patients with lyme borre-liosis; and (iii) characterizing the immune response generated in response to b. burgdorferi (http://www .niaid.nih. gov/research/topics/lyme/research/co-infection/). niaid is supporting various efforts to evaluate and improve existing diagnostic procedures. approximately % of its extramural lyme disease research portfolio is devoted to developing novel and more sensitive diagnostic procedures (http://www .niaid.nih.gov/ research/ topics/ lyme/ research/ diagnostics/). in , the fda granted approval to chembio diagnostic systems to market the wampole prevue borrelia burgdorferi antibody detection assay. the assay is a single-use, unitized immunochromatographic test that uses recombinant b. burgdorferi antigens for the qualitative presumptive (first step) detection of igg and igm antibodies to b. burgdorferi in human serum or whole blood. this test is to be used only in patients with history, signs, and symptoms that are consistent with lyme disease. it is intended for use in clinical and physicians' office laboratories. in collaboration with cdc, niaid is also playing a major role in encouraging the development of novel approaches to improve the diagnosis of lyme borreliosis in humans with various co-infections (e.g., ehrlichiosis or babesiosis), as well as in immunized people (http://www .niaid.nih. gov/research/topics/lyme/research/diagnostics/). for example, it has been shown in niaid-supported research that a synthetic peptide composed of amino acid residues (c ) derived from a variable surface antigen (vlse) of b. burgdorferi can be used in a new, rapid, and extremely sensitive elisa test (the c elisa) for diagnosing lyme disease. because this diagnostic test for lyme disease, which has been approved by fda, does not detect antibodies specific for recombinant ospa, it can be used even for those who have been immunized with the licensed ospa-based lymerix vaccine ( ) . although the lyme urinary antigen test (luat) is one of several diagnostic tests used routinely in niaid's clinical studies on chronic lyme disease, the results of independent quality control assessments of tests conducted by extramural and intramural scientists showed the luat to be unreliable because it yields an unacceptably high percentage of falsepositive reactions ( ) . a critical evaluation of urine-based pcr assays for the diagnosis of lyme borreliosis likewise affirmed that urine is not a suitable material for the diagnosis of lyme borreliosis ( ) . by contrast, the similar assessments confirmed a high degree of reproducibility and concordance (virtually %) for the results obtained using elisa and western blot assays ( ) . of great importance is the fact that decreases in the titer of antibodies against c can be used as an indicator of the efficacy of antibiotic therapy for patients with localized or disseminated lyme disease, but not for chronic lyme disease ( ) . this is indeed a major advancement, because no other laboratory test enables one to obtain such information ( ) . the results obtained with the c elisa assay are consistent with those obtained with other diagnostic tests and may eliminate the time and expense of conducting additional laboratory tests to confirm the diagnosis of lyme disease ( ) . niaid-supported investigators are now working closely with the cdc to determine if the c elisa can eventually replace the traditional two-tiered conventional elisa and western blot assays. the results of other studies confirmed that a decline in the anti-c antibody titer coincides with the efficacy of antimicrobial therapy in patients with early localized or early disseminated lyme borreliosis ( ) (see also . . ) . the b. burgdorferi-specific immune complex (ic) test in which polyethylene glycol (peg) is used to isolate ics from serum has been advocated by some investigators as an approach for the early diagnosis of active borreliosis. however, recent findings indicate that it may not be more effective in detecting early and active infections than other conventional tests in which unprocessed serum specimens are used ( ) . there is a great need to develop additional simple, sensitive, and rapid procedures to distinguish those persons who are actively infected with b. burgdorferi from those who have either recovered from a previous infection or have been immunized previously. because the genome of b. burgdorferi has now been completely sequenced, greater advances toward this goal are anticipated as this information is used in conjunction with microarray technology and proteomics to improve diagnosis, as well as to provide new insights on the pathogenesis of this disease and pathogenspecific host response mechanisms (http://www .niaid. nih.gov/research/topics/lyme/research/diagnostics/). there is no clear understanding about the molecular basis of how b. burgdorferi maintains itself in nature via a complex life cycle that involves passage through ticks and various intermediate hosts, such as mice and deer, before infecting humans. the outer surface protein a (ospa) of b. burgdorferi has been well studied, and there is much speculation about its role-in conjunction with other cell surface proteins (ospb and ospc)-in transmitting lyme disease ( ) . although b. burgdorferi depends on ixodes ticks and mammalian (rodent) hosts for its persistence in nature ( ), the search for borrelial genes responsible for its parasitic dependence on these types of diverse hosts has been hampered by limitations in the ability to genetically manipulate virulent strains of borrelia. despite this constraint, there is evidence to indicate that the inactivation and complementation of a gene (bbe ) encoded by a linear plasmid (lp ) plays a major role in the virulence, pathogenesis, and survival of b. burgdorferi during its natural life cycle ( ) . this gene, which has been renamed bpta (for borrelial persistence in ticks-gene a), potentiates virulence in mice and is essential for the persistence of b. burgdorferi in ixodes scapularis ticks. although bpta appears to be a lipoprotein expressed on the outer surface membrane of b. burgdorferi, the molecular mechanism(s) by which bpta promotes persistence within its tick vector remains to be elucidated. because bpta appears to be highly conserved (> % similarity and > % identity in amino acid sequence) in all b. burgdorferi sensu lato strains examined, it may be widely used to promote persistence in nature. given the absolute dependence on-and intimate association with-its tick and rodent hosts, bpta must be considered to be a major virulence factor that is critical for b. burgdorferi's overall infectious strategy ( ). strategies designed to block the synthesis or expression of bpta could be of great value in preventing the transmission of lyme disease. the potential role that differentially upregulated surface proteins play in the transmission of borreliosis and lyme disease pathogenesis have prompted investigators to conduct a comprehensive gene expression profiling analysis of temperature-shifted and mammalian host-adapted b. burgdorferi. the combined microarray analyses revealed that many genes encoding known and putative outer surface proteins are downregulated in mammalian host-adapted b. burgdorferi. however, at the same time, several different genes encoding at least seven putative outer surface proteins were found to be upregulated during the transmission and infection process. all seven proteins are immunogenic and generate the production of bactericidal antibodies in infected baboons ( ) . this suggests that these outer surface proteins might be excellent second-generation vaccine candidates. the above findings have been consistent with the results of published studies ( ) in which a novel experimental technique (xenodiagnosis by ticks) was used to determine whether b. burgdorferi can persist in mice long after antibiotic therapy. in these studies, an immunofluorescence assay and the pcr assay were used to demonstrate that b. burgdorferi could be detected in doxycycline-and ceftriaxonetreated mice for at least months (if not longer) after antibiotic therapy. however, the resulting surviving spirochetes were unable to infect other naïve mice because they lacked those linear plasmids (lp and lp ) that are essential for their ability to transmit infection ( ) . it is noteworthy that lp also encodes for a gene product (pnca or bbe ) that is essential for the survival of b. burgdorferi in a mammalian host ( ) . niaid-supported investigators have now been able to create various mutant strains of b. burgdorferi and have shown that although ospa and ospb are not required for infection of mice, they were essential for the colonization and survival of b. burgdorferi in ticks (http://www .niaid.nih.gov/ research/topics/lyme/research/transmission/). ixodes scapularis ticks have a receptor on the inner wall of their intestines to which b. burgdorferi is able to bind tenaciously by means of ospa, a cell surface protein. this receptor is called the tick receptor for ospa (trospa). attachment to trospa will enable b. burgdorferi to persist in the gut from the time they were ingested by ticks through a subsequent molt, thereby avoiding elimination; this would allow borrelia to be injected into a new host when the ticks take their next blood meal ( ) . when ticks take a blood meal, the production of ospa is downregulated in favor of the increased production of ospc. this results in gut-bound spirochetes becoming detached, which enables them to migrate to the salivary glands, where they can be injected into mammalian hosts. thus, trospa, in addition to other bacterial cell surface components, such as ospa, appears to play a key role in the transmission of lyme disease to humans. other studies have shown that if ticks are permitted to feed on mice that have been immunized previously with ospa, or have been treated with the antibody specific for ospa, the attachment and subsequent colonization of ticks by b. burgdorferi would be significantly impaired, if not prevented. this suggests the feasibility of developing oral-or vector-expressed transmission-blocking vaccines that involve the immunization of the intermediate hosts upon which ticks feed ( ) . several niaid-supported investigators are now examining and testing this approach under controlled laboratory conditions (http://www .niaid. nih.gov/research/topics/lyme/research/transmission/). results from other studies conducted by niaidsupported investigators ( ) have demonstrated that b. burgdorferi uses an immunosuppressive tick salivary protein (salp ) to facilitate the transmission of infection to mammalian hosts. this finding is based on observations that (i) the level of salp expression is enhanced by the presence of b. burgdorferi in infected ticks; (ii) salp adheres specifically to spirochete surface ospc both in vivo and in vitro, thereby increasing the ability of b. burgdorferi to infect mice; and (iii) the binding of salp protects b. burgdorferi from antibody-mediated killing in vitro, a factor that confers marked survival advantage. all of these observations suggest that salp and/or other tick salivary proteins might be excellent candidates for vaccines to block the transmission of lyme disease ( ) . in this context, prior and repeated exposure of experimental animals to uninfected ticks-and presumably their salivary proteins-has been shown to limit the capacity of infected ticks to transmit lyme disease ( ). two large pharmaceutical companies [glaxosmithkline (skb) and pasteur merieux connaught (pmc)] have devoted considerable effort to developing a vaccine for lyme disease. double-blind, randomized, placebo-controlled clinical trials, involving more than , volunteers from areas of the united states where lyme disease is highly endemic, have been completed for each of two b. burgdorferi recombinant ospa vaccines manufactured by skb and pmc. these vaccines were found to be % to % effective in preventing lyme disease after two injections and % to % effective in preventing lyme disease after three injections. the duration of the protective immunity generated in response to the skb vaccine (lymerix), which was licensed by the fda in december , is not known. consequently, the need for yearly booster injections remains to be established. researchers and health experts anticipate that the use of these vaccines in endemic areas would likely result in a significant reduction in the incidence of lyme disease in the future. niaid was not directly involved in the design and implementation of these particular vaccine trials; however, patents for cloning the genes used for the expression of recombinant ospa, as well as knowledge of the role of antibodies against ospa in the development of protective immunity, were derived from basic research funded by niaid (http://www . niaid.nih.gov/research/topics/lyme/research/vaccine/). in april , glaxosmithkline announced that even with the incidence of lyme disease continuing to increase, sales for lymerix declined from about . million doses in to a projected , doses in . although studies conducted by fda failed to reveal that any reported adverse events were vaccine-associated, glaxosmithkline has discontinued manufacturing the vaccine for economic reasons ( ) . niaid-funded investigators have developed an experimental bait delivery system for an ospa-based vaccine against b. burgdorferi in which mice were immunized orally (via gavage or bait feeding) with a strain of escherichia coli expressing the gene for ospa, which resulted in the appearance of serum antibody specific for ospa. when mice were exposed to ixodes nymphs carrying multiple strains of b. burgdorferi, oral vaccination was found to protect % of the mice from infection, and the resultant serum antibody response confirmed the presence of igg a/ b antibody specific for ospa (http://www .niaid.nih. gov/topics/lymedisease/research/vaccine.htm). this vaccination approach is able to generate a significant protective immune response against a variety of infectious strains of b. burgdorferi, thereby indicating that it can eliminate b. burgdorferi from a major host reservoir. it suggests that the broad delivery of an oral vaccine to wildlife reservoirs in an endemic area is likely to disrupt the transmission of lyme disease ( ) . these findings are consistent with the results reported by other investigators ( ) , thus affirming the utility of this approach. in other niaid-supported studies, scientists have developed a murine-targeted ospa vaccine using the vaccinia virus to interrupt the transmission of disease in reservoir hosts, thereby having the potential to reduce the incidence of human disease. oral vaccination of mice with a single dose of vaccinia virus expressing ospa resulted in high antibody titers to ospa, % protection of vaccinated mice from infection by b. burgdorferi, and a significant clearance of b. burgdorferi from infected ticks fed on vaccinated animals ( ) . these findings indicate that such a vaccine may effectively reduce the incidence of lyme disease in endemic areas. niaid is also funding preclinical studies to develop and test other candidate vaccines (e.g., decorin-binding protein a, or dbpa) for lyme disease. thus, medimmune, inc., and sanofi-aventis pharmaceuticals have reported that a combination vaccine composed of the dbpa and ospa of b. burgdorferi was more effective than either one given alone in preventing the development of borreliosis in experimental animals. on the basis of these encouraging findings, both companies have entered into an agreement to develop a new, more effective second-generation vaccine to prevent lyme disease in humans. although the results of previous studies indicate that dbpa induces the development of protective immunity in a murine model of lyme borreliosis when mice have been challenged (needle-inoculated) intradermally with in vitro-cultivated b. burgdorferi, such mice were not protected from infection transmitted by ticks carrying virulent b. burgdorferi. the principal mission of niaid is to study infectious diseases and host immune defense mechanisms; therefore, the institute conducts and supports most of the basic and clinical research on lyme disease funded by the national institutes of health (nih). however, because lyme disease affects different tissue and organ systems of the body, it is also a matter of great concern to other nih institutes and centers (http://www .niaid.nih.gov/research/topics/lyme/centers/). the national institute of arthritis and musculoskeletal and skin diseases (niams) is funding research on chronic lyme-induced arthritis, including the role of the immune system and genetic factors in contributing to its development. the national institute of neurological disorders and stroke (ninds) is funding research to characterize the neurologic, neuropsychological, and psychosocial manifestations of early and late lyme disease in both adults and children, as well as to characterize pathogenic mechanisms associated with the neurologic symptoms of chronic lyme disease. the national center for research resources (ncrr) provides resource support (non-human primates) for basic and clinical studies on both acute and chronic infection, as well as support for testing and developing candidate vaccines for lyme disease. in addition, the fogarty international center (fic) is funding research on lyme disease abroad, and the national institute on aging (nia) and the national institute of mental health (nimh) have focused on those aspects of lyme disease that relate to their specific missions. to facilitate cooperative interactions as well as to ensure that the research activities of all nih components are complementary, an nih lyme disease coordinating committee (ldcc) was established in . ldcc meets annually to review the results of current studies and recent advances in research on lyme disease. because the fda is responsible for evaluating the efficacy and safety of vaccines against lyme disease (e.g., the lymerix vaccine) and the cdc is especially interested in developing new and improved diagnostic procedures, representatives from fda and cdc have been invited to serve on the ldcc and to provide updates on their activities related to lyme disease. r c elisa diagnostic procedure. it has been shown that a synthetic peptide comprising amino acid residues (c ) derived from a variable surface antigen (v se) of b. burgdorferi can be used in a new, rapid, and extremely sensitive elisa test (the c elisa) for diagnosing lyme disease ( ) . the c elisa test is sensitive only to antibodies generated during an active infection (both early and late stages of lyme disease). another advantage of the test is its ability to detect antibodies specific for both north american and european strains of borrelia. of great importance is the fact that decreases in the titer of antibodies against c can be used as an indicator of the efficacy of antibiotic therapy for patients with localized or disseminated lyme disease, but not for chronic lyme disease. because the c elisa test would not detect antibodies specific for recombinant ospa, it can be used even for those patients who have been immunized with the licensed ospa-based lymerix vaccine ( ) . this is a major advance, because except for the c elisa no other laboratory test is capable of obtaining such information ( ). r an ecologic approach to preventing lyme disease. in a recently developed, ecologic approach to lyme disease prevention, researchers have intervened in the natural life cycle of b. burgdorferi by immunizing the wild whitefooted mouse (peromyscus leucopus), a reservoir host species, with either a recombinant antigen (ospa) of the spirochete or a negative control antigen in a repeated field experiment with paired experimental and control grids stratified by site ( ) . ospa vaccination significantly reduced the prevalence of b. burgdorferi in nymphal black-legged ticks (i. scapularis) collected at the sites the following year in both experiments. the magnitude of the vaccine's effect at a given site correlated with the prevalence of tick infection found on the control grid, which in turn correlated with mouse density. these data, as well as differences in the population structure of b. burgdorferi in sympatric ticks and mice, indicated that non-mouse hosts contributed more toward infecting ticks than previously expected. thus, where non-mouse hosts play a large role in the infection dynamics, vaccination should be directed at additional species ( ). r variable nature of antibodies specific for ospc influence virulence. the outer surface protein c (ospc) of b. burgdorferi, the spirochete that causes lyme disease, has been studied for its potential in the development of a vaccine ( ) . of the ospc types currently identified, a surprisingly large number (types a, b, c, d, k, n, and ) are associated with invasive disease. because a detailed knowledge of the antigenic structure of ospc would be essential for vaccine development, the antibody response against several different recombinant ospc proteins was examined in detail. the results have revealed a high degree of specificity, indicating that the immunodominant epitopes of ospc reside in the variable regions of the protein. to localize these epitopes, ospc fragments were generated and screened against serum collected from infected mice, thus allowing the identification of previously uncharacterized epitopes that define the type specificity of the ospc antibody response. the reported findings have provided valuable insights into the antigenic structure of ospc, as well as a basis for understanding the variable nature of the antibody response to this important virulence factor. the lyme disease spirochete, b. burgdorferi, is maintained in a tick-mouse cycle. evidence has demonstrated that the spirochete usurps a tick (i. scapularis) salivary protein, salp , to facilitate the infection of mice ( ) . the level of salp expression was selectively elevated by the presence of b. burgdorferi in i. scapularis. the salivary protein was shown to adhere to the spirochete and to specifically interact with b. burgdorferi's outer surface protein c. the binding of salp protected b. burgdorferi from antibody-mediated killing, thereby providing the spirochetes with a marked advantage when they were inoculated into naïve mice or mammals previously infected with b. burgdorferi. lyme disease. it has long been known that immunization of mice with outer surface protein a (ospa) will protect against transmission of b. burgdorferi infection and will reduce the carriage of this pathogen in feeding ticks. in a recent study, the development of a murine-targeted ospa vaccine using vaccinia virus to interrupt the transmission of disease in reservoir hosts has been reported ( ) . thus, oral vaccination with a single dose of the ospa-expressing vaccinia virus construct resulted in high antibody titers against ospa, % protection against infection by b. burgdorferi, and a significant clearance of b. burgdorferi from infected ticks that fed on immunized mice. the reported findings indicated that such a vaccine was effective and may provide a means to lower the incidence of human disease in endemic areas. acquisition and transmission of lyme disease by ticks. b. burgdorferi strains exhibit various degrees of infectivity and pathogenicity in mammals, which may be due to their relative ability to evade initial host immunity. innate immune cells recognize b. burgdorferi by toll-like receptors (tlrs) that use the intracellular molecule myeloid differentiation factor- (myd ) to mediate effector functions ( ) . in a mouse model of lyme disease using mutant strains of mice, the absence of myd was found to facilitate tick-transmission of strains of b. burgdorferi of both low and high infectivity ( ) . the reported data will broaden the understanding of factors that contribute the degree of pathogenicity observed between different clinical isolates of b. burgdorferi, as well as the genetic basis for host resistance or susceptibility to infection. tick-borne rickettsial diseases (tbrds) are caused by pathogens of the second main group (the spotted fever group) of the genus rickettsia (the other being the typhus group; see chapter ). tbrds continue to cause severe illness and death in otherwise healthy adults and children despite the availability of low-cost, effective antimicrobial therapy, and the reported incidence of tbrds has increased during the previous decade. the greatest challenge to clinicians is the difficulty of diagnosing these illnesses early in their clinical course when antibiotic therapy is most effective-early signs and symptoms are often nonspecific or mimic benign viral diseases, making diagnosis difficult ( ) . although clinically similar, the tbrds are epidemiologically and etiologically distinct diseases. in the united states, they include (i) human monocytotropic (or monocytic) ehrlichiosis (hme); (ii) human granulocytotropic (or granulocytic) anaplasmosis (hga; formerly known as human granulocytotropic ehrlichiosis or he); (iii) rocky mountain spotted fever (rmsf); (iv) ehrlichia ewingii infection; and (v) other emerging tbrds ( ) . additional diseases caused by the pathogenic members of the spotted fever group of rickettsia are the african tick typhus and rickettsial pox. it is interesting to note that the pathogenic tick-borne r. rickettsii, r. parkeri, and r. sibirica are phylogenetically distinct from the nonpathogenic species r. rhipicephali and r. montana. other species, such as r. felis and r. helvetica, are early diverging within the spotted fever group. the difference at the molecular level between the pathogenic and nonpathogenic species has not yet been completely elucidated. genome sequence of rickettsia conorii. complete genome sequence data has been generated for only one species of the spotted fever group, r. conorii ( ) . the genome of r. conorii is very small, only . mb, and similar to that of r. prowazekii (see chapter ) . the overall architecture of these two rickettsial genomes is essentially the same, with the exception of a few rearrangements near the terminus of replication. symmetric dna inversions at the regions surrounding the origins of replication and termination have been observed also in chlamydia ( ) . the symmetric nature of these rearrangements is thought to be the outcome of recombination events at the open replication forks. such translocation and inversion events have since been identified in a variety of genomes, suggesting that the replicating dna at the open replication fork is particularly vulnerable to recombination events ( ). appropriate antibiotic treatment should be initiated immediately after diagnosis is made based on clinical, laboratory, or epidemiologic findings ( ) . any delay in treatment may lead to severe disease and even a fatal outcome. because any of the tbrd pathogens is susceptible to tetracycline antibiotics (especially doxycycline), these drugs are considered the therapy of choice in nearly all clinical situations. fever typically subsides within to hours after doxycycline treatment is initiated during the first to days of illness ( ) . doxycycline is bacteriostatic against rickettsiae and is active in both children and adults. the recommended dose for adults is mg, twice daily (orally or intravenously). for children weighing less than lb ( . kg), the recommended dose is . mg/kg body weight, twice daily (orally or intravenously). intravenous administration is frequently indicated for hospitalized patients. the length of antibiotic therapy would be at least days after the fever subsides and until evidence of clinical improvement is noted (typically to days) ( ) . the tetracycline antibiotics are generally contraindicated for use in pregnant women because of risks associated with malformation of teeth and bones in the fetus and hepatotoxicity and pancreatitis in the mother ( , ) . however, tetracycline has been used successfully to treat hme in pregnant women ( ) , and its use may be warranted during pregnancy in life-threatening situations where clinical suspicion of tbrd is high. nevertheless, therapeutic choices for pregnant women with ehrlichiosis should be weighed cautiously, even when the benefits of doxycycline therapy generally outweigh its risks ( ) . chloramphenicol (no longer available as an oral formulation) is an alternative drug that has been used to treat tbrds such as rocky mountain spotted fever (rmsf) ( ) . however, the drug is associated with various side effects and may need monitoring of the patient's blood indices. moreover, epidemiologic studies using cdc case report data have suggested that patients with rmsf treated with chloramphenicol have a higher risk of dying than do patients who receive tetracycline ( , ) . whereas chloramphenicol is typically the preferred treatment for rmsf during pregnancy, care must be used especially when administering the drug late in the third trimester of pregnancy because of the risk of gray baby syndrome ( ). only the tetracycline antibiotics have demonstrated in vitro susceptibility and in vivo activity toward ehrlichia species. in spite of in vitro susceptibility against ehrlichia, the clinical effectiveness of rifampin is unknown ( ) . ehrlichia chaffeensis has demonstrated resistance to gentamicin, ciprofloxacin, penicillin, macrolides, and sulfacontaining drugs ( ). a substantial number of patients with tbrds may require hospitalization because of severe manifestations, including prolonged fever, renal failure, disseminated intravascular coagulopathy (dic), hemophagocytic syndrome, meningoencephalitis, and acute respiratory distress syndrome (ards) ( ) . a notable exception is anaplasmosis (hga), which has not been associated with meningoencephalitis. rocky mountain spotted fever frequently presents as a severe illness, during which patients commonly require hos-pitalization. up to % of untreated cases and % of treated cases have a fatal outcome, making rmsf the most often fatal rickettsial disease in the united states ( ) . host factors associated with severe or fatal rmsf include advanced age, male gender, black race, chronic alcohol abuse, and glucose- -phosphate dehydrogenase (g pd) deficiency ( ) . deficiency of g pd is a sex-linked genetic condition affecting approximately % of the u.s. black male population ( ) . deficiency of g pd is associated with a high proportion of fulminant cases of rmsf ( ) . fulminant cases follow a clinical course that is fatal within days of the onset of infection. long-term health effects of severe, life-threatening rmsf that may persist for more than year include partial paralysis of the lower extremities, gangrene requiring amputation (fingers, toes, arms, or legs), hearing loss, blindness, loss of bowel or bladder control, movement disorders, and speech disorders ( ) . similarly to rmsf, hme and hga can also cause serious or fatal illness, although at a lower frequency than that observed with rmsf. clinical conditions that may require hospitalization may include immunocompromised state, pain (headache, myalgia), mental confusion, cough, infiltrate in chest radiograph, abnormal spinal fluid findings, or specific acute organ failure ( ). it must be emphasized that during diagnosis, clinicians should be aware of the overlap of early symptoms of invasive meningococcal infection and tbrds. these conditions are difficult to distinguish early in the course of the illness. in patients for whom both conditions are included in the initial differential diagnoses, after performing blood cultures and lumbar puncture, empirical treatment for both diseases would be appropriate. such treatment could be accomplished by adding an appropriate parenteral penicillin (or cephalosporin) that has activity against neisseria meningitides to doxycycline therapy ( ). r. rickettsii, e. chaffeensis, e. ewingii, and a. phagocytophilum have specific and distinct cell tropism ( ) . r. rickettsii infects endothelial cells and, more rarely, underlying smooth muscle cells, where it multiplies freely in the cytoplasm. the rickettsiae cause a small-vessel vasculitis resulting in a maculopapular or petechial rash in the majority of patients. vasculitis, when occurring in organs (brain or lungs), could cause life-threatening complications ( ) . rickettsiae are not evident in blood smears and do not stain with the majority of conventional stains. ehrlichiosis and anaplasmosis are characterized by infection of leukocytes where the causative pathogens multiply in cytoplasmic membrane-bound vacuoles to form microcolonies known as morulae. e. chaffeensis most frequently infect monocytes, whereas a. phagocytophilum and e. ewingii demonstrate a predilection for granulocytes ( ) . morulae can be stained with conventional wright or giemsa stains and are occasionally observed in leukocytes in smears of peripheral blood, buffy coat preparations, or cerebrospinal fluid. although a routine blood smear can provide a presumptive clue for early diagnosis because of the visualization of morulae, still a confirmatory testing for ehrlichia or anaplasma species is required by serology, pcr, or immunostaining methods. also important to note is that the available methodology to demonstrate morulae in blood smears is not very sensitive, and a case of ehrlichiosis or anaplasmosis might be missed if the diagnosis relies solely on detecting morulae on blood smears. although the diagnostic sensitivity of blood smears is greater for hga than for hme, blood smears might only be positive in up to % of patients with hga ( ). since , the cdc has been tracking reported cases of several diseases collectively called human ehrlichiosis. however, the term "ehrlichiosis" is somewhat misleading, because when studied in detail it became clear that the etiologic agents of these emerging tick-borne infections are two different bacterial genera, ehrlichia and anaplasma. geographically, they have occurred primarily east of the rocky mountains ( , ) . in the united states, infections caused by ehrlichia spp. are typically transmitted by tick species of the genera amblyomma (a. americanum) and ixodes (i. scapularis and i. pacificus). both genera use small mammals and birds as their primary reservoirs ( ). morphologically, ehrlichia spp. are small intracellular gram-negative cocci that infect different hematopoietic cells, causing two etiologically and epidemiologically distinct forms of ehrlichiosis: human monocytic ehrlichiosis (hme) and human granulocytic anaplasmosis (hga). in the united states, most cases of both hme and hga occur in the spring and summer (april to september for hme, and may to august for hga), when ticks are at their peak ( ) . recently, the cdc has described a new group of diseases called "other and unspecified" human ehrlichiosis. these infections include diseases caused by a second ehrlichia species as well cases of previously mentioned illnesses that could not be definitely diagnosed as either hme or anaplasmosis ( , ) . the etiologic agent of human monocytic ehrlichiosis (hme) is ehrlichia chaffeensis (lone star tick), which infects the macrophages and monocytes. the pathogen is transmitted primarily by amblyomma americanum, but dermacentor variabilis (american dog tick) can also transmit the disease. the major reservoir for e. chaffeensis is the white-tailed deer, with most cases being reported in the south central and southeastern regions of the united states. hme has been mainly associated with males ( times more often than females), the elderly (over years of age), and immunocompromised hosts (hiv/aids patients, and those with asplenia or down's syndrome, and patients receiving immunosuppressive therapy) ( , , ) . clinical manifestations of hme include fever, headache, and rash presented as part of a prodrome consisting of abrupt, high-grade fever (> % of patients) often with an associated headache ( % to %), malaise ( % to %), nausea ( % to %), myalgia ( % to %), arthralgia ( % to %), lower back pain ( % to %), and gastrointestinal disorders ( % to %). the rash (on the trunk, extremities, and face, but rarely on the sole and palms) may be petechial, macular, maculopapular, or erythematous ( ) . the prodrome typically manifests itself to days (median days) after exposure to a tick. neurologic manifestations (symptoms of meningitis and encephalopathy) have been observed in approximately % of patients. laboratory findings of hme are characterized by reduction in the multiple hematopoietic cell lines (occurring early in the course of the disease), thrombocytopenia, and leukopenia. a large decline in the total lymphocyte count is often seen in the early stage of the disease, whereas lymphocytosis occurs later, during the recovery phase of hme. elevated liver enzyme levels (aspartate aminotransferase and alanine aminotransferase) are another characteristic laboratory finding of the disease and occur in % to % of patients ( ) . the manifestations of hme are typically moderate to severe and would require hospitalization of at least % of infected patients. if left untreated, hme may be fatal within the first weeks, especially in men, the immunocompromised, and the elderly ( ). the black-legged tick (ixodes scapularis) is the vector for anaplasma phagocytophilum in the new england and north central regions of the united states, whereas the western black-legged tick (ixodes pacificus) is the principal vector in northern california. because these ixodes species also transmit borrelia burgdorferi (the causative agent of lyme disease) and various babesia species, the preponderance of cases of hga occur in the same states that usually report high incidence of lyme disease and human babesiosis ( ) . simultaneous infection with a. phagocytophilum and b. burgdorferi has been reported ( , ) , and discerning such a mixed infection is vital because it might affect the choice of antimicrobial medication; whereas amoxicillin can be used to treat early stage of lyme disease, it is not effective against hga ( ) . in the absence of tick exposure, other modes of hga transmission have also been reported-butchers cutting fresh deer carcasses had contracted the disease ( ). this suggests blood as a potential source of transmission and represents a risk of occupational exposure. at-risk populations include the elderly, patients with chronic diseases (e.g., diabetes, collagen-vascular diseases), and patients on immunosuppressive therapy. hga is manifested as a constellation of nonspecific symptoms that occur after an incubation period of to days after tick exposure; generally to days elapse before a patient will seek medical care ( ) . the disease is commonly characterized by high-grade fever (over • c), rigors, nonspecific myalgia, severe headache, and malaise ( ). other symptoms may include nausea, nonproductive cough, arthralgia, and anorexia. although less common, % of patients with hga will present with rash ( ) , which is thought to be due to coinfection with lyme borreliosis ( ) . though associated with less morbidity and mortality than is hme, % of patients with hga will require hospitalization ( ). unlike patients with hme, those with hga may have normal blood cell counts ( ). nevertheless, approximately % of patients will have leukopenia and thrombocytopenia. increased levels of liver enzymes, in particular, hepatic transaminases and c-reactive protein, are also commonly observed. in general, laboratory abnormalities will reach their peaks within week after the onset of symptoms. during acute hga, morulae can be visualized (with microscopy) in the cytoplasm of leukocytes. this finding, if present, is diagnostic of hga. however, the absence of morulae does not exclude the diagnosis of hga ( ) . typically, the nonspecific disease presentation, lack of morulae, and the transient nature of the blood cell counts would make the diagnosis of hga difficult. as a result, the consensus approach for ehrlichiosis (cafe) society has developed a set of definitions to help clinicians with the diagnosis of hga ( , ) . although hme and hga are two distinct forms of human ehrlichiosis, the treatment is the same for both infections ( ). doxycycline is the primary agent recommended for treat-ment of hga. however, similarly to hme, doxycycline is contraindicated for pregnant women and children younger than age , posing a dilemma for clinicians treating these patients. data demonstrating the efficacy of rifampin in the treatment of ehrlichiosis in pregnant women are limited to just case reports ( ) . therapeutic choices for pregnant women with ehrlichiosis should be weighed cautiously, but the benefit of doxycycline therapy generally outweigh its risks ( ), and according to recommendations by the american academy of pediatrics and the cdc, doxycycline should be used in the treatment of children ( ) and neonates ( ) . it is recommended for children to start with oral doxycycline ( . mg/kg in divided doses) on day , followed by a single dose of . mg kg − day − ; the cdc is recommending the use of doxycycline . mg/kg in divided doses for children weighing less than kg, and mg twice daily (adult dose) for children weighing kg or more (http://www.cdc.gov/ncidod/dvrd/rmsf/treatment). in , rickettsia rickettsii was described in the blood vessels of infected patients and later identified as the etiologic agent of the rocky mountain spotted fever (rmsf). the rocky mountain spotted fever has long been established in the united states. in spite of its common name, it is relatively rare in the rocky mountain region but far more prevalent in the southeastern regions of the united states. most often the infection is transmitted by ticks of the genus dermacentor, which include the american dog tick (d. variabilis) in the eastern, central, and pacific coastal united states, and by the rocky mountain wood tick (d. andersoni) in the western united states. in , the common brown dog tick (rhipicephalus sanguineus), a vector in mexico, was also implicated in an arizona outbreak ( ) . the cayenne tick (amblyomma cajennense) is a common vector of rmsf in central and south america, and its range has extended into the united states in texas ( ) . a case report of rickettsia parkeri infection was recently published ( ) . the organism was first discovered in texas in amblyomma maculatum (gulf coast tick); before that, the disease had not been reported in humans. the main reservoirs for d. variabilis are small animals, such as mice and voles, and dogs and other large animals; and for d. andersoni, both small and large animals, typically wild rodents ( ). ticks become infected by feeding on infected animals, by transtadial and transovarian passage. humans are not a primary reservoir for r. rickettsii but are merely secondary hosts that enter the organism's life cycle tangentially through contact with arthropods. for humans to become infected with rickettsiosis, a tick may need to be attached for as little as to hours. however, attachment of hours or more is generally needed for transfer of the disease ( ). human infection may also result from contact with contaminated tick fluid and tissues during tick removal or from laboratory contact during culture and isolation. in cases reported to the cdc, approximately % occurred from april to september and % during the may to june period, although infections have occurred in every month ( ) . today, if left untreated, rmsf is the most fatal tick-borne infection in the united states, with an overall mortality of % ( ) . however, treatment with antibiotics has reduced the mortality rates to % to %, and this, in most cases, may be due to delay in the diagnosis of the disease. the causative agent of rmsf, r. rickettsii, has been included as a niaid category c biodefense priority pathogen. dogs are susceptible to rmsf, and they frequently develop the disease concurrently with other household members in an endemic area ( ) . the clinical and laboratory manifestations of rmsf are similar to those of hme and hga and generally appear within to days after a tick bite ( , ) . in rmsf, a rash typically appears to days after the onset of fever and will occur earlier in children than in adults; it is eventually observed in approximately % of children. the exanthema typically will begin with the appearance of small, blanching, pink macules on the ankles, wrists, or forearms that evolve to maculopapules. the classic centripetal spread of rash is typically not noticed by the patient and might be difficult to elicit from the clinical history ( ) . although the rash may expand to involve the entire body, its presence on the face is usually limited. patients with petechial rash are often severely ill, and although fever and organ dysfunction may resolve quickly with treatment, complete recovery can take longer. the rash progression of rmsf includes several critical exceptions and considerations as follows ( ): (i) a rash on the palms and soles is not pathognomonic and may occur in illnesses caused by drug hypersensitivity reactions, infective endocarditis, and a diverse group of other pathogens, including treponema pallidum, streptobacillus moniliformis, e. chaffeensis, and especially neisseria meningitides, as well as certain enteroviruses. (ii) the rash might be evanescent or localized to a particular region of the body. (iii) a rash might be completely absent or atypical in up to % of patients with rmsf. in certain cases, patients with rmsf (or ehrlichiosis) may seek medical attention for a febrile illness that mimics viral meningoencephalitis. focal neurologic deficits, including cranial or peripheral motor nerve paralysis or sudden transient deafness, may also be observed ( ) . laboratory findings, especially the complete blood cell count (cbc), are essential for the diagnosis of rmsf ( ) . the total white blood cell (wbc) count is typically normal in patients with rmsf, but increased numbers of immature bands are generally observed. thrombocytopenia, mild elevation in hepatic transaminases, and hyponatremia may be observed with rmsf. by comparison, leukopenia (up to % of patients), thrombocytopenia (up to % of patients), and modest elevation of liver transaminase levels are particularly suggestive for hme and hga ( ) . patients with rmsf may have various signs and symptoms that differ in degree of severity ( , ) . orally given antibiotics are adequate in cases of mild illness, whereas severely ill patients should be hospitalized and treated with intravenous antibiotics. in a retrospective study, information based on multivariate analysis has shown that only increased serum creatinine levels and neurologic symptoms were associated with mortality ( ) . the clinical outcome of rmsf is apparently strongly dependent on the time span between the patient's first visit and the start of therapy; if therapy had begun more than days after the first visit, the outcome is significantly poorer than if treatment had been initiated earlier. the tetracyclines are the cornerstone of therapy for rmsf, with doxycycline being the drug of choice ( , ) . however, as with ehrlichiosis, the use of doxycycline in pediatric and pregnant patients again poses a problem. generally, short courses of doxycycline may be administered in children younger than years of age. however, according to the guidelines of the american academy of pediatrics and the cdc, the empiric use of doxycycline in children and pregnant women, although possible, should be applied with caution and with careful consideration for maternal hepatotoxicity and permanent tooth discoloration. chloramphenicol has long been recommended as an alternative therapy for rmsf and is considered a suitable choice for patients who are pregnant or allergic to tetracyclines ( ). however, the adverse effects of chloramphenicol are well known: aplastic anemia, reversible bone marrow suppression, and gray baby syndrome ( ) . moreover, the chloramphenicol concentrations and reticulocyte counts should be monitored when the treatment exceeds days. clearly, the administration of either doxycycline or chloramphenicol in pregnant women is not without risks. amblyomma americanum is also the principal vector of the ehrlichial pathogen ehrlichia ewinglii ( ) . the ecologic features of e. ewinglii are not completely known. however, dogs and deer have been naturally infected. cases of granulocytotropic ehrlichiosis caused by e. ewinglii have been reported primarily in immunocompromised hosts. human infections with this pathogen have been reported throughout the range of the lone star tick ( ) . early clinical presentations in patients with e. ewinglii include fever, headache, myalgia, and malaise, and they are difficult to distinguish from other tbrds and noninfectious diseases ( ) . as in patients with hga, rash is rare in patients with e. ewinglii infection, and blood smears are useful for identifying patients with e. ewinglii. furthermore, evaluation of csf in patients with e. ewinglii has shown neutrophilic pleocytosis ( ) . appropriate antibiotic treatment should be initiated immediately when a diagnosis of e. ewinglii is made. doxycycline is the drug of choice for both children and adults, and as with the treatment of other tbrds, caution must be applied when doxycycline is used for the treatment of e. ewinglii ( ) . the first report of tularemia in the united states occurred in , in tulare county, california ( ) . one year later, the pathogen responsible for this outbreak was isolated and named bacterium tularense ( ) . the first report of tularemia in humans occurred in in two patients bitten by deerfly ( ) . the infection is transmitted by ticks and is passed transovarially among ticks. in , the organism was renamed francisella tularensis. f. tularensis is a highly contagious organism, which, in the context of biological weapons defense, is considered to be a potential threat. in fact, a tularemia outbreak in before the battle of stalingrad was the result of weaponized f. tularensis. there is no person-to-person transmission, but tularemia delivered as an aerosol could infect a large number of people. ecologically, tularemia is a disease of the northern hemisphere (north america, northern asia, scandinavia, europe, japan, and russia). in addition to transmission by ticks and other arthropods, f. tularensis can be transmitted by inhalation, ingestion of contaminated food or drinking water supplies, and animal bites ( ) . more than animal species are implicated as carriers of f. tularensis. consequently, in different regions of the world the disease is known by different names (rabbit fever, hare fever, deerfly fever, and lemming fever). in the united states, when transmitted by ticks, f. tularensis is primarily transmitted by a. americanum, d. andersoni, and d. variabilis ( ) . except for hawaii, all states have reported cases of tularemia, with the highest rates coming from arkansas, missouri, south dakota, and oklahoma. the disease has a predilection for males, especially native americans and alaskan natives, and children age to and adults age or older. most human outbreaks occur in spring and summer, which correlates with arthropod transmission ( , ). the bacteriology and taxonomy of f. tularensis is complex ( ) . it is a small, pleomorphic, aerobic, gram-negative coccobacillus that can be found both inside and outside of cells. the genus francisella is divided into three major biovars. biovar a (biogroup tularensis) predominates in north america and is the most virulent. biovar b (biogroup holarica) is found primarily in europe and asia, but also exists in north america. biovar c (biogroup novicida; formerly known as f. novicida) is found in parts of north america and has very low virulence ( ). the clinical course of tularemia is quite diverse, ranging from asymptomatic disease to septic shock and death ( ) . typically, tularemia is divided into six forms, reflecting the mode of transmission: (i) ulceroglandular; (ii) glandular; (iii) oculoglandular; (iv) oropharyngeal; (v) pneumonic (pleuritic); and (vi) typhoidal. tularemia is characterized by abrupt but nonspecific symptoms, such as fever, chills, headache, vomiting, fatigue, and anorexia, which make the disease difficult to diagnose. pulse-temperature disparity in which the patients may exhibit a high temperature without reflexive increase in pulse is a hallmark manifestation of the disease ( ) . ulceroglandular tularemia is the most common syndrome, accounting for % to % of f. tularensis infections. the pathogen enters through a scratch, abrasion, or tick and spreads via the proximal lymphatic system. as few as organisms can cause disease. this syndrome usually appears as a papule at the tick-bite site and progresses to a pustular, ulcerated lesion called an inoculation eschar ( ) . glandular tularemia is a relatively rare syndrome ( % of patients) with no ulcer present. the organism causes regional lymphadenopathy and is presumed to have gained access to the host through clinically unapparent abrasion ( ) . diagnosis may be difficult because the patient presents one or several enlarged lymph nodes with no skin lesion. oculoglandular tularemia occurs in approximately % of patients and results from inoculation of the eye by tularemiacontaminated fluids or fingers, perhaps after removal of the tick ( ). the clinical manifestations of oculoglandular tularemia are conjunctivitis with adjacent lymph node involvement, periorbital edema, and erythema. oropharyngeal tularemia accounts for less than % of all cases. this syndrome is not acquired by contact with ticks but results from ingestion of infected raw meats or contaminated water supplies ( ). symptoms include fever, exudative pharyngitis, or oropharyngeal ulcerations. because the manifestations mimic those of other upper respiratory infections, the diagnosis of oropharyngeal tularemia is based on exclusion from lack of response to antibiotics given for bacterial pharyngitis. pneumonic tularemia, the most severe form of the infection, may not be directly associated with tick exposure, but rather can develop through inhalation or secondarily by hematogenous spread ( ) . disease mortality is estimated to be around % ( ) . typhoidal tularemia is a rare syndrome ( ) manifested by fever, chills, and local findings to culture-negative septic shock. the syndrome may also be accompanied by pneumonia, elevated transaminase levels, and rhabdomyolysis, which leads to renal failure ( ) . the diagnosis of tularemia is confirmed when an antibody response occurs approximately weeks after the onset of disease. the preferred serologic methods are agglutination (latex or tube agglutination tests) or pcr. the latter is highly sensitive and safer, but its specificity is dependent on the dna sample and its purity ( ). treatment of tularemia is based solely on case reports and anecdotal experience. based on the latter, aminoglycosides, especially streptomycin and gentamicin, are regarded as the cornerstone of therapy ( ). a meta-analysis found that streptomycin was successful in % of patients, whereas gentamicin was successful in % ( ) . in addition, gentamicin was associated with a % relapse rate and an % failure rate. however, despite these drawbacks of gentamicin, its cure rate was equal to or greater than that of other classes of antimicrobials, thus making it an acceptable alternative to streptomycin. results from tetracycline therapy of tularemia have shown % success and no treatment failures; however, it was associated with % relapse rate ( ) . the high relapse rate of tetracycline may be the result of its bacteriostatic mode of action. in addition, tetracycline can be given only orally, which limits its use in patients with severe tularemia-the drug levels achieved with oral administration only minimally exceeded the minimum inhibitory concentration (mic) for f. tularensis. in patients treated with chloramphenicol, the success rate was % with a % relapse rate ( ) . like tetracycline, chloramphenicol is bacteriostatic. however, when compared with aminoglycosides and tetracycline, one advantage of chloramphenicol is its enhanced penetration into the central nervous system. this feature makes chloramphenicol a therapeutic option for treatment of meningeal tularemia. the fluoroquinolones are another therapeutic option for treating tularemia. ciprofloxacin and levofloxacin have been used in the treatment of pneumonic tularemia, with the former showing a low failure rate and fewer adverse effects ( ) , and no relapses (levofloxacin) year later. although data describing the efficacy of fluoroquinolones in the treatment of tularemia are still evolving, these agents have been as successful as other treatments of the disease ( ). in vitro data demonstrated that f. tularensis isolates have shown resistance to β-lactams and therefore they should not be recommended for treatment of tularemia ( ). erythema migrans, the characteristic rash associated with lyme disease, has been reported in patients living in the south central and southeastern united states. typically, it is associated with the bite of amblyomma americanum. however, the spirochete that causes lyme disease in north america, borrelia burgdorferi sensu stricto, has not been confirmed in these regions of the united states by culture from clinical specimens, and serum antibodies rarely indicate exposure ( ) . although amblyomma americanum is apparently not a vector for b. burgdorferi sensu stricto, the same ticks carry another spirochete, borrelia lonestari. in a case report ( ), a patient was described with erythema migrans and amblyomma americanum attachment. importantly, borrelia lonestari was identified both in the patient and the tick, and serology for b. burgdorferi sensu stricto was negative. therefore, this observation strongly suggested that amblyomma americanum can transmit the spirochete to humans, and the resulting rash, which resembled that seen with lyme disease, has become known as southern tick-associated rash illness (stari). borrelia lonestari (family treponemataceae) is a spirochete that has been detected in amblyomma americanum by dna analysis. unlike ixodes scapularis, a vector for lyme disease, amblyomma americanum is less likely to be infected with a spirochete, with only % to % of amblyomma americanum infected ( ) . in contrast, % to % of nymph stage ticks and % to % of the adult-stage ticks of i. scapularis are infected with a spirochete. the natural reservoir for b. lonestari has not been identified even though it was detected in white-tailed deer. in the only published case report of stari, the patient showed only mild symptoms, such as fatigue, cough, and right shoulder discomfort ( ) . fever and headache were absent, and results of musculoskeletal, neurologic, pulmonary, and cardiac examinations were normal. two erythematous lesions were also noted. the only abnormal laboratory finding was a slightly elevated serum alkaline phosphatase level. there is no specific serologic test for exposure to b. lonestari ( ) . in the only case reported, the patient underwent antibiotic therapy with doxycycline for weeks ( ); the skin lesions resolved in days, and the patient returned to health about days after therapy was initiated. babesiosis, a malaria-like disease caused by intraerythrocyte protozoa named babesia bigemina, was first described in ( ) . the parasite is also the cause of the texas cattle fever ( ). the first case of babesiosis in humans was reported in the former republic of yugoslavia in and in the united states in the late s ( ). the babesia protozoa may vary in size ( to μm) and can be oval, round, or pear shaped ( ) . more than different species have been identified, but only four have been reported to be pathogenic in humans. in the united states, infection is caused primarily by b. microti, and two new strains of babesia that can cause infection are designated as wa- and mo- . although human infections in the united states caused by b. divergens have not been reported, this protozoa is the primary cause of babesiosis in europe ( ). in the northeastern united states, the primary vector for b. microti is ixodes scapularis, and the primary reservoir is the white-footed mouse. although all stages of i. scapularis feed on humans, the nymph-stage tick is typically responsible for transmission of b. microti in humans. vectors and reservoirs for wa- and mo- have not been identified ( ) . while babesiosis infections have been observed in patients of all ages, it appears that the occurrence is higher in men, and persons older than years are prone to more severe infection. the infection is contracted most commonly during the summer (june to august) ( ). like malaria, the babesia species reproduce within the red blood cells and produce hemolysis, which is responsible for the clinical presentation of babesiosis ( ) . manifestations of the disease are diverse and may range from asymptomatic to fulminant, leading to prolonged illness and even death. although in the united states most cases of babesiosis are subclinical, when patients become symptomatic, manifestations usually appear after an incubation period of to weeks. the most common symptoms include fever ( % of patients), fatigue ( %), chills ( %), and headache ( %). less often, myalgia, anorexia, cough, nausea, vomiting, arthralgia, emotional liability, depression, sore throat, abdominal pain, conjunctival injection, photophobia, and weight loss have been reported ( ) . physical findings are generally nonspecific (high fever, mild splenomegaly, and hepatomegaly). unlike other tick-borne infections, rash is not common in babesiosis. the most common complications in patients with severe babesiosis are acute respiratory failure ( % of patients), disseminated intravascular coagulation ( %), heart failure ( %), coma ( %), and renal failure ( %). babesiosis is fatal in % to % of cases ( ) . laboratory findings may include a decreased hematocrit value, thrombocytopenia, and a normal or decreased white blood cell count. elevated levels of hepatic transaminases, bilirubin, and lactate dehydrogenase have also been observed. urinalysis may reveal proteinuria and hemoglobinuria ( ). most cases of human babesiosis in the united states are mild and may resolve without treatment ( ). however, therapy is required in those patients who have undergone splenectomy, are immunosuppressed, are elderly, or have significant symptoms. historically, the cornerstone of babesiosis therapy is a combination of clindamycin and quinine given for period of to days ( ). however, even though effective, the combination clindamycin-quinine has been associated with significant drug-related toxicities, such as hearing loss, tinnitus, vertigo, and diarrhea. atovaquone, an antiprotozoal drug, has been studied in combination with azithromycin for the treatment of b. microti infections ( ) . the atovaquone-azithromycin combination was compared with a -day oral treatment with clindamycin-quinine in immunocompetent adults with nonlife-threatening babesiosis ( ) . resolution of parasitemia and symptoms was similar in both groups; however, the adverse reactions were significantly less in patients receiving atovaquone-azithromycin ( %) than in those receiving clindamycin-quinine ( %). another drug combination found effective in the treatment of babesiosis was clindamycin-doxycycline-azithromycin in an aids patient who developed an allergy to quinine ( ) . the combination of sulfamethoxazole-trimethoprimpentamidine has been used successfully in the treatment of b. divergens ( ) . exchange transfusions, administered concurrently with antibiotic therapy, may be necessary for patients with severe babesiosis showing significant parasitemia (more than %), coma, hypotension, heart failure, pulmonary edema, or renal failure ( ) . exchange transfusions reduce parasitemia and will facilitate the removal of babesia-, erythrocyte-, and macrophage-produced by-products ( ). in , tick-borne relapsing fever (tbrf) was first described in west africa where it was transmitted by ornithodoros moubata soft ticks ( ) . tick-borne relapsing fever (tbrf) is a systemic borrelia infection caused by a group of closely related species of spirochetes: b. hermsii, b. turicatae, and b. parkeri. ( ) . tbrf is endemic in the western united states. it occurs sporadically, but several common source epidemics have been reported. as with other tick-borne diseases, tbrf is a seasonal illness; % of reported cases have occurred during the june to september period. however, in texas most episodes occur during the late autumn and early winter, with % reported from november to january ( ). this difference in seasonality may be related to differences in both organisms and habitats; in texas, cases typically represent b. turicatae infections acquired in caves, whereas in the northwestern united states, cases are generally b. hermsii infections acquired in mountainous regions ( ) . these spirochetes possess the unique ability to change outer surface proteins under pressure from the host immune system in a process known as antigenic variation, a phenomenon responsible for the recurring nature of the disease ( ) . thus, borreliae will sequester themselves in internal organs during afebrile periods and then will reappear with modified surface antigens to evade eradication. as borrelia organisms invade the endothelium, this can produce a low-grade, disseminated intravascular coagulation and thrombocytopenia. the relapse phenomenon occurs because of the antigenic variation-a genetically programmed shifting of outer surface proteins of borrelia that allows a new clone to avoid destruction by antibodies directed initially against the majority of the original infecting organisms. as a result, the patient will improve clinically until the new clone multiplies sufficiently to cause another relapse. the tick-borne illness tends to have more relapses (average of ) than does the louse-borne variety (often just one relapse). relapsing fever (rf) is an infectious disease transmitted to humans by two vectors, ticks and lice. the human body louse, pediculus humanus, is the specific vector for borreliae. pediculus pubis is not a vector. louse-borne relapsing fever is a more severe form than the tick-borne variety. regardless of the mode of transmission, a spirochetemia will develop. the louse-borne relapsing fever is caused by borrelia recurrentis. no animal reservoir exists. the lice that feed on infected humans acquire the borrelia organisms, which then multiply in the gut of the louse. when an infected louse feeds on an uninfected human, the organism gains access when the victim crushes the louse or scratches the area where the louse is feeding. b. recurrentis infects the person through either abraded or intact skin (or mucous membranes) and then invades the bloodstream. because ornithodoros ticks feed so rapidly, patients with tbrf are often unaware of the tick bite. a pruritic eschar may develop at the soft tick attachment site ( ). symptoms will appear abruptly on average days after tick exposure ( ) . common manifestations include fever, headache, myalgia, arthralgia, nausea, and vomiting. the primary febrile period when the temperature can rise as high as • f (or even higher) lasts about days (range, hours to days). patients then experience an afebrile period lasting about week, during which time they may experience malaise before symptoms suddenly recur. without treatment, several (three to five) relapses can be expected. however, the length and severity of the illness will typically decrease with each relapse ( ) . less common symptoms of tbrf include abdominal pain, confusion, dry cough, eye pain, diarrhea, dizziness, photophobia, and neck pain. rash (petechial, macular, or popular) occurs in about % of patients and develops as the fever subsides. other physical findings can be splenomegaly and hepatomegaly ( ) . neurologic complications (neuroborreliosis) will occur predominately in patients infected with b. turicatae ( % to %), but much less in patients infected with b. hermsii ( %). the most common manifestations of neuroborreliosis are cranial nerve palsies and meningisms ( ) . rare complications of tbrf are ocular disorders, myocarditis, and ruptured spleen ( ) . the most common hematologic abnormalities are thrombocytopenia ( % of patients), proteinuria ( %), and hematuria ( %). most patients with tbrf have a normal white blood cell count. no controlled studies have been published regarding treatment of tbrf. based on clinical experience, the tetracycline antibiotics have been the treatment of choice ( ( , ) . oral doxycycline, mg every hours for to days, is the typical treatment. in addition, penicillin, chloramphenicol, and erythromycin have all been used successfully to treat tbrf. tetracycline, erythromycin, and chloramphenicol are usually administered at dosages of mg every hours ( ) . patients with meningitis should receive intravenous therapy with penicillin g, cefotaxime, or ceftriaxone for days or more ( ). the jarisch-herxheimer reaction is a serious consequence of tbrf treatment. it is manifested as an acute exacerbation of the patient's symptoms that can occur with the start of the antibiotic treatment. it has been reported in more than % of patients treated for tbrf ( ) . the pathophysiology of the jarisch-herxheimer reaction has been studied most extensively in patients with louse-borne relapsing fever. the reaction is associated with transient increases in plasma concentrations of tumor necrosis factor-α (tfn-α), interleukin- , and interleukin- ( ) . anti-tnf-α antibodies prevented this reaction in patients with louse-borne relapsing fever ( ) . furthermore, meptazinol, an opioid partial agonist, reduced the severity of symptoms, whereas naloxone was ineffective ( ) . tick-borne bacterial, rickettsial, spirochetal, and protozoal infectious diseases in the united states: a comprehensive review ticks and tickborne bacterial diseases in humans: an emerging infectious threat tick-borne bacterial diseases emerging in europe lyme disease-united states the emergence of lyme disease temporal changes in outer surface proteins a and c of the lyme disease-associated spirochete 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pro-inflammatory cytokines, in muscle tissue in rhesus chronic lyme borreliosis the chemokine cxcl (blc): a putative diagnostic marker for neuroborreliosis detection of attenuated, noninfectious spirochetes of borrelia burgdorferi-infected mice after antibiotic treatment identification of candidate t-cell epitopes and molecular mimics in chronic lyme disease antibodies against ospa epitopes of borrelia burgdorferi cross-react with neural tissue evidence of borrelia autoimmunity-induced component of lyme carditis and arthritis homologies betwen proteins of borrelia burgdorferi and thyroid autoantigens a case-control study to examine hla haplotype associations in patients with posttreatment chronic lyme disease increased arthritis severity in mice coinfected with borrelia burgdorferi and babesia microti coinfection with borrelia burgdorferi and the agent of human granulocytic ehrlichiosis alters the murine immune responses, pathogen burden, and severity of lyme arthritis controlled trials of antibiotic treatment in patients with post-treatment chronic lyme disease, vector borne zoonotic dis cytolethal distending toxin is essential for colonization of helicobacter hepaticus in outbread swiss webster mice characterization of a borrelia burgdorferi vlse invariable region useful in canine lyme disease serodiagnosis by enzyme-linked immunosorbent assay intralaboratory reliability of serologic and urine testing for lime disease critical evaluation of urine-based pcr assay for diagnosis of lyme borreliosis pretreatment and post-treatment assessment of the c test in patients with persistent symptoms and a history of lyme borreliosis comparison of western immunoblotting and the c lyme antibody test for laboratory detection of lyme disease a decline in c antibody titer occurs in successfully treated patients with culturecontrolled early localized or early disseminated lyme borreliosis detection of immune complexes is not independent of detection of antibodies in lyme disease patients and does not confirm active infection with borrelia burgdorferi a plasmid-encoded nicotinamidase (pnca) is essential for infectivity of borrelia burgdorferi in a mammalian host identification of borrelia burgdorferi outer surface protein tro-spa, and ixodes scapularis receptor for borrelia burgdorferi an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle the lyme disease agent exploits a tick protein to infect the mammalian host and the tick-borne infection study group ( ) hypersensitivity to ticks and lyme disease risk adverse event reports following vaccination against lyme disease protective efficacy of an oral vaccine to reduce carriage of borrelia burgdorferi (strain n ) in mouse and tick reservoirs demonstration of ospc type diversity in invasive human lyme disease isolates and identification of previously uncharacterized epitopes that define the specificity of the ospc murine antibody 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nonendemia area tularemia and q fever tularemia: united states streptomycin and alternative agents for the treatment of tularemia: review of the literature tularemia epidemic in northwestern spain: clinical description and therapeutic response borrelia lonestari infection after a bite by an amblyomma americanum tick borrelia lonestari dna in adult amblyomma americanum ticks severe babesiosis in long island: review of cases and their complications atovaquone and azithromycin for the treatment of babesiosis treatment of transfusion-transmitted babesiosis by exchange transfusion tick-borne relapsing fever in north america antigenic variation of a relapsing fever borrelia species relapsing fever: a clinical and microbiological review tick-borne relapsing fever in the northwestern united states and southwestern canada the spectrum of relapsing fever in the rocky mountains neuroborreliosis during relapsing fever: review of the clinical manifestations, pathology, and treatment of infections in humans and experimental animals detection of plasma tumor necrosis factor, interleukins and , during the jarisch-herxheimer reaction of relapsing fever prevention of jarisch-herxheimer reactions by treatment with antibodies against tumor necrosis factor meptazinol diminishes the jarisch-herxheimer reaction of relapsing fever key: cord- - rm y authors: flower, darren r.; perrie, yvonne title: immunomic discovery of adjuvants, delivery systems, and candidate subunit vaccines: a brief introduction date: - - journal: immunomic discovery of adjuvants and candidate subunit vaccines doi: . / - - - - _ sha: doc_id: cord_uid: rm y mass vaccination, when coupled to profound improvements in general sanitation, has given rise to the most remarkable transformation in public health in human history. yet the development of vaccines remains largely trapped in the past, a hostage to the methodology of pasteur. infectious disease continues to threaten humanity, with new and renascent diseases emerging continually. the last two decades have seen a breath-taking revival in the commercial market for vaccines and the simultaneous emergence of a whole tranche of new technologies that promise to free vaccine development from the muddle of empirical thinking. in this short introduction, we set the scene for this renaissance, and explore how the combination of computational and experimental techniques promise so much for the future development of vaccines and the science of vaccinology. vaccination and sanitation are indisputably the most efficient, and thus costeffective, prophylactic treatments for infectious disease. together they are the firm bedrock upon which the modern world resides. the truth of this is often ignored by those intoxicated by the many distractions of life in the early twenty-first century, from burgeoning social media to the recondite discoveries of the large hadron collider. people say that the internet or the ipad or even facebook have transformed the world. if there is any truth in such assertions, then such transformations are at best shallow and superficial compared to the extraordinary reworking of lives that has transpired over hundreds rather than tens of years. the source of such change can be traced back, in part at least, to the discovery and exploitation of vaccines, vaccination, and vaccinology. for around the first years of the vaccine story, the story was solely that of smallpox. as recently as the late s, some - million cases of smallpox were recorded in countries, with annual deaths of million. yet today smallpox has, with the exception of a few well-guarded stockpiles, been completely eradicated: there have been no new cases for years. the story of smallpox is thus the high point of the vaccination story; no other disease has been eradicated. polio or poliomyelitis is the next nearest to full eradication, having long been targeted by a systematic, coordinated, worldwide eradication campaign. in , one such programme run by the pan american health organization effected a partial eradication of polio in the western hemisphere. subsequently, the global polio eradication program has radically reduced polio throughout the rest of the world, so that today we can count cases worldwide in the tens or hundreds instead of the hundreds-of-thousands or millions. yet, vaccine-preventable disease still kills millions. infectious and contagious disease cause approximately % of world mortality, particularly in children under five. while in developed countries, mortality for diseases such as diphtheria, polio, or measles is less than . %, in other parts of the world deaths from such infectious diseases is significant. pertussis, tetanus, influenza, hib, hepatitis b are all responsible for deaths that number in the hundreds-of-thousands. perhaps the most execrable situation is measles, which accounts for , (over ) and , (under ) deaths. however, the leading global causes of death worldwide remain tuberculosis; diarrhoeal illnesses, especially rotaviruses; hiv/aids; and malaria. in , . million contracted tb and . million died. disturbing though these numbers may seem, they nonetheless represent a significant reversal of a once ever-escalating trend. the number with latent tb peaked in at million, while deaths from tb reached their peak at . million in . however, these bald numbers are likely to be significant underestimates. let us also look at malaria. murray et al. have recently provided evidence that deaths from malaria over the thirty-year span to are much higher than previously believed [ ] . their epidemiological figures show a peaked distribution over this period, increasing from around a million in , peaking at approximately , , in , and then reducing to about , , in , with the greatest number dying in africa. these figures are roughly twice the values published by the who. it seems unlikely that the who's estimates for other major diseases are uniformly more accurate. why are these numbers so high? a principal reason is that there are no effective vaccines for either malaria or hiv, two of the who's big three diseases; nor is there expectation that such vaccines will appear in the near future, irrespective of the optimism of those working in the area. and as for tuberculosis-which is carried by around billion people worldwide-the only licensed vaccine has limited efficacy. many viral infections remain recalcitrant threats of the first order. about million people are infected with hepatitis b, million by hepatitis c, and million by human immunodeficiency virus type (hiv- ). this dire situation is further compounded by the threat from the new or previously unknown infectious diseases identified in the past years: hiv, west nile fever, ebola, dengue fever, sars, and the potentially pandemic h n influenza. every year, between and % of the global population becomes infected with a new influenza strain, causing upwards of half-a-million deaths. it is widely thought that there will be a continual emergence of new infectious diseases during the present century: emerging zoonotic infections and antibiotic-resistant bacteria prominent amongst them. in the face of declining birth rates, coupled to decades of ever-enhancing nutrition, advances in treatment regimens and medicines are leading the population of most developed and developing countries figuratively to age: that is for larger and larger proportions of the population to live to maturity and beyond. growth in life expectancy is matched by growth in the diseases of old age. these include neurodegenerative diseases, principally parkinson's or alzheimer's disease; cardiovascular diseases; and stroke. disease has altered significantly in the preceding century. it will continue to alter during the century to come. some alterations we can predict; others will escape the forecaster's eye. disease, particularly infectious disease, has been beaten, or, at least, severely restrained. many factors have conspired to effect this-improved water quality, better precautionary hygiene, improved nutrition, decreased overcrowding-as well as many interventionary measures, principally antibiotic therapy and vaccines. hitherto, vaccines have been an uncompromising success, yet, as we see, so much more needs to be done if the full potential of vaccines is to be achieved. although the licensing and use of vaccines varies between countries, - commonly licensed vaccines target a range of viral or bacterial infectious diseases, with approximately paediatric diseases targeted during the first few years of life. other than paediatric vaccination, most vaccines are used by travellers to tropical or subtropical regions; a significant minority fight infection in the developing world. vaccination also works to greatly reduce the morbidity of disease, often imbuing lifetime protection; this is particularly important for benign yet economically important infections, such as the so-called common cold. diverse sporadic or epidemic infections of the human respiratory track-as caused by an excess of distinct viruses, such as rsv or, more properly, respiratory syncytial virus, coronaviruses, influenza a and b, rhinoviruses, parainfluenza virus, and cytomegalovirus-remain a principal cause of hospitalisation and community morbidity with an estimated % of gp referrals associated with such infections, and cause the loss of enormous numbers of working days in developed countries. given the recalcitrance, and the immense investment in treatment and prophylaxis, it may be that many of the diseases alluded to above will never be eradicated, as smallpox was, and that vaccines alone will not be enough. we may instead need a complex network of prophylactic and therapeutic measures at least as complex as the diseases themselves in order to effectively reduce the prevalence of the disease and to treat those who become infected. whatever other countermeasures we may have recourse to-artemisinin-based drugs, genetically manipulated vectors, or insecticide-treated bednets-the clinical and cost effectiveness of vaccines means they remain the must-have component in the ongoing search for better means of combating endemic infectious disease. beyond infectious disease lies what is possibly the most underexplored area within the ever burgeoning field of vaccinology and vaccination: vaccines against allergy and allergic disease; vaccines that target so-called lifestyle diseases, such as those deriving from addiction; and vaccines that target chronic diseases, the most important of which is cancer. therapeutic vaccines against cancer are probably the best studied amongst the more novel, innovative, and underexplored areas at the forefront of vaccine discovery. the present overall whole-life risk from cancer, at least in developed countries, runs at or about %. the figure is currently rising. there are approximately · million new cancer cases in europe each year, resulting in around · million deaths. in the usa, over one and half million new cases are reported annually. clearly, this is an important disease burden, and thus a key target for the pharmaceutical and biotechnology industries. lifestyle vaccines are another innovation, of which much is expected in certain quarters. they target all kinds of medical problems, ranging from drug addiction through dental caries, all the way to major genetic and multifactorial diseases, including obesity. versatility and flexibility are major hallmarks of the vaccination concept. vaccines take many forms and work in many ways. this facet has been exploited in the development of life-style vaccines. let us look at serious addiction. during , the united nations office on drugs and crime estimated . - . % of the global population abused so-called illicit substances-a serious problem indeed with an enormous implicit health, economic, and behavioural burden, with the worst excesses coming from cannabis abuse and the abuse of amphetamine, cocaine, and opiates. anti-drug vaccines generate antibodies able to bind particular drugs; the drug-antibody complexes thus generated should have too high a molecular weight to penetrate the blood-brain barrier effectively, thus reducing the amount and rate of drug egress into the brain and so inhibiting psychoactive effects at the system level. anti-addiction vaccines have been with us for some time, beginning over years ago, when two proof-of-principle studies [ , ] demonstrated in rats and in rhesus monkeys that morphine could be used as a hapten in order to create an antibody against morphine addiction. today, addiction vaccines are being developed to target a range of major abused drugs, such as nicotine, cocaine, various amphetamines, and heroin. anti-allergy vaccination also offers great potential for successful commercial exploitation. the prophylaxis and treatment of allergy can now be addressed in many ways, including, notably, recombinant proteins and dna vaccines. vaccines against the common cold or anti-allergy vaccines are similar in mechanism to many lifestyle vaccines. these do not save lives directly but do help to greatly reduce the vast economic burden of disease morbidity. an array of interconnecting factors that have made the pharmaceutical and biotechnology industries re-evaluate the potential of vaccines as a commercially viable product. prior to , there were relatively few vaccines, most targeting major pandemic diseases of the developed or developing worlds. subsequently, partly as a result of enhanced technology as discussed at length in the current book, many vaccines have become available, most recently the cervical papillomavirus vaccine. likewise, there are hundreds upon hundreds of vaccines in trials. the growth rate in the sales of vaccines reflects this feverish and febrile activity: the rate of sales growth for vaccines is something like %, compared to the sluggish drugs market, meandering its desultory way at % per annum. there is profit in vaccines, clearly; what remains problematic for the profit-driven decision-making processes of big pharma is the haphazard and probabilistic nature of vaccine discovery. what the pharmaceutical industry needs is the capacity to apply the same systematic, automated, high-technology approaches used to identify new small-molecule drugs to the discovery and development of vaccines. no right-minded scientist, looking back across the last years, would wish to argue seriously with the contention that the design and development of vaccines is an innately labour-intensive process. the processes deployed to meet the objective of creating new and better vaccines are in desperate need of change. this change must be radical if we hope to simplify such processes. simple processes are hopefully also fast and efficient processes. in the search for subunit vaccine antigens, one technical development-reverse vaccinology-has proved the most profound and hopeful. just over a decade ago, rino rappuoli used the expression "reverse vaccinology" to describe development of vaccines using a genomic-based approach, rather than the ponderous empirical methods favoured then, and still in use today. reverse vaccinology seems about to deliver on its early potential: the european medicines agency is in the process of evaluating novartis's bexsero, the first commercial vaccine developed using the reverse vaccinology approach. the vaccine may become the first vaccine effectively to combat meningococcus b, a disease causing over % of global meningococcal meningitis. a decision on bexsero is expected shortly. during the development of bexsero, new protective protein antigens were identified using genomics: initially over surface-exposed proteins were predicted from the n. meningitidis proteome as molecules liable to host immune surveillance, of which about were then expressed in e. coli. this number was reduced by using these proteins to immunize sets of mice, identifying that could induce antibodies in vivo, of which killed n. meningitidis in vitro. by comparing the genomes of clinical strains, a further subset of proteins offering broad protection could be identified. reverse vaccinology has become perhaps the most famous well-developed approach amongst many advanced approaches now available within the discipline of vaccinology. indeed, a whole range of other, high-technology methods and techniques have been and are being developed to complement and optimise reverse vaccinology. capitalising on its success, these offer new hope in our constant struggle with infection; all we need is for this technology to be fostered, developed, and utilised. this book is intended to fill a gap, if not a void, in current thinking within vaccine design and development by attempting to draw together several disparate strands; and, by doing so, also identify and illuminate some important areas replete with potential. science, in much the same way that all human activities, from the most profound to the most trivial, follows fashion and progresses by tracking trends. whether we think of publically funded science or the pharmaceutical industry, similar phenomena are observed. science follows the money, and money follows consensus. the decision-making process underpinning the strategic direction that policy in both publically funded science and the pharmaceutical industry takes is only in part influenced by science. it is also regrettably in thrall to many, sometimes contradictory, voices: the fickleness of public opinion, vested interests of many hues and flavours, and the myopia of the profit margin, amongst many others. this is because the decision-makers in such organisations are seldom if ever scientists engaged in doing science directly; management and policy, at both the strategic and tactical levels, are often swayed by the prevalence of opinion. in the pharmaceutical industry, for instance, this is manifest as the next big thing: combinatorial libraries, genomics, high-throughput screening, antisense, even molecular modelling; all were hailed as transformative saviours that would remove happenstance and unpredictability from drug discovery-yet as the current parlous state of the pharmaceutical industry readily attests, while all promised much, none really delivered. no single technique can achieve everything, which is why we should always develop a large range of alternatives, both informaticsbased and experimental, all running in parallel. central to computational immunology is the capacity to make accurate predictions. yet, obtaining routes to prediction that are accurate, robust, and dependable continually eludes us. immunoinformatics deals with empirical, datadependent methods. the success and utility of such methods depends very much on the data used to propagate and parameterise them; they cannot escape the severe limitations imposed by the data used to create them. the data from which we build models forms a complex phase space of structural and property variation, which can be extremely multidimensional, with a high degree of interdimensional correlation. when the data we work with is reliable and our knowledge of it is complete, then we can create useful models by applying standard methods from computer science to build accurate and predictive models relating observed biological activity to underlying measurable or predictable properties. usually, such approaches are also much superior when used to interpolate than they are when used to extrapolate. we need complete and thorough data sets effectively and efficiently able to explore the complex relationships between structure and function, necessitating continuous improvement in all aspects of data quality. within the wider context of vaccine design and discovery, we shall in this book describe and explore a range of key alternative strategies and technologies, both informatics-based and experimental, which are, by degrees, both supportive and complementary to reverse vaccinology and more traditional approaches to vaccine discovery. this book looks in turn at reverse vaccinology and the identification of putative candidate antigens, at the discovery of a wide range of different types of adjuvants, and finally at the development of sophisticated new delivery mechanisms, such as liposomes and other applications of nanotechnology. in chap. , cafardi et al. review the present state of play with respect to reverse vaccinology, with particular emphasis on how completion of bacterial genomes impinges upon the vaccine discovery. they show how this approach allows the development vaccines that are difficult or near impossible to address with conventional approaches. they also highlight how advances in genome-based techniques and in so-called next-generation sequencing approaches and technologies will help to enhance reverse vaccinology, enabling timely identification of novel candidate antigens for new, emerging, or recrudescent infectious diseases. in chap. , flower et al. review the discovery of candidate vaccine antigens in more detail. placing their analysis in the context of emerging ideas about the possible nature of immunogenicity and how it may be propagated by elements of the immune response at the system level, the authors discuss the three main approaches to the identification of novel immunogenic antigens: sequence similarity-based approaches, whereby the antigen nature of a protein is inherited from similar sequences; methods based on identifying the subcellular location of microbial proteins, on the basis that proteins with only certain locations would be accessible to immune surveillance; and the use of empirical alignment-independent approaches to the prediction of antigens. usefully, the chapter also includes discussion of expert systems for antigen discovery. in chap. , vordermeier et al. review how genomics and the development of bioinformatics have radically transformed the cattle vaccinology of bovine tuberculosis. within the context of a generalised infrastructure of bioinformatic analytical techniques, the authors describe in detail how the application of comparative in silico transcriptome and genome analysis is able to undertake prospective prioritisation of immunogenic antigens for experimental testing, leading to the identification of candidate subunit vaccines. in chap. , he explores the use of epitope-focused immunoinformatic analysis in the prediction of optimal vaccine candidates when undertaking a genome-wide reverse vaccinology exercise. specifically, he describes the web-server vaxign, concentrating on a case study: vaccine design against the virulent bacterium francisella tularensis, where candidates were chosen using a combination of pertinent selection criteria. in chap. , dhillon et al. offer us a wide-ranging review of methods and strategies for two important areas of immunoinformatic analysis within the domain of vaccine discovery: predicting the immunogenic subcellular location of microbial proteins and identifying proteins encoded by so-called genomic islands. while in chap. , ansari et al. describe a variety of database systems that facilitate immunoinformatics and antigen selection. chapters and look at adjuvants and their discovery. in chap. , edwards describes the basis of adjuvant action, and the role played by macromolecular adjuvants. in chap. , flower explores and examines different varieties of molecular adjuvant and their discovery, concentrating on small molecule adjuvants, and their systematic identification using virtual screening technology. this topic is put into context by a thorough review of extant adjuvants, molecular mechanisms of adjuvant action, as well as macromolecular adjuvants and how various adjuvants engage pattern recognition receptors of the innate immune system. in addition to the characteristics of the antigen and the adjuvant independently, how the antigen and adjuvant are presented to the immune system has a major impact on the biological output of the vaccine. indeed, the co-delivery and continued association of antigen and adjuvant may be a prerequisite in effective immunisation. this is not a new idea; the ability of alum to promote an antigen depot effect at the site of action has been ascribed as one of its main mechanisms of action for several years. for example, since the who has recommended that over % of diphtheria toxoid needs to be adsorbed to alum for its effective use [ ] . whilst this does not ensure that the antigen remains adsorbed to alum after injection and exposure to interstitial fluid, it is thought to at least initially promote colocation of the antigen with the alum adjuvant. however, alum is not the only adjuvant able to promote the co-delivery of antigens and adjuvants in one system; a range of particulate delivery systems can offer this. examples of such delivery systems include lipid-based systems (e.g., liposomes, niosomes, iscoms) and microparticles. each of these systems offer a suite of advantages and disadvantages and when considering the choice of delivery system attributes including antigenloading capacity, antigen retention and protection both on storage and within the biological milieu, and the ability to incorporate adjuvants within the delivery system all require optimisation and this is without consideration of the ability of the delivery system to act as an adjuvant in its own right. in this book we aim to address these issues by considering several of the most commonly employed particulate vaccine delivery systems. out of these particulate delivery systems liposomes are one of the most established systems; liposomes were first reported as an effective immunological adjuvant for diphtheria toxoids by allison and gregoriadis in [ ] . since then, a large array of understanding on their design has been gathered and strong links between their formulation and function identified. of these parameters, the composition of the liposomes will play a pivotal role. for example, the surface charge of liposomes used for vaccine delivery can influence the interactions between liposomes and protein antigens, and affect how liposomes interact with cells. this manipulation of surface charge can range from the inclusion of anionic lipids such as phosphatidylserine, which may facilitate the targeting of antigen presenting cells through interaction with phosphatidylserine receptors. alternatively, the use of cationic lipids can improve the loading of anionic antigens to the liposomes and upon injection promote a depot effect at the site of injection, promoting the codelivery of antigen and adjuvant to dendritic cells (e.g., [ , ] ). however, this depot effect is more than electrostatically driven, with the choice of cationic lipids used in the formulation having an impact as explored in chap. . in addition to liposomes, there are a range of alternative surfactant-based delivery systems, such as niosomes. these are similar in many ways to liposomes; however, non-ionic surfactants form the main component of their bilayers. the most common composition of niosomes investigated for vaccine delivery is -monopalmitoyl glycerol, cholesterol, and dicetyl phosphate. one might argue this is not a niosome formulation due to the inclusion of anionic dicetyl phosphate. however, the addition of charged surfactants has proven to enhance the stability of these vesicles and their inclusion in these vesicles is common practice. generally, niosomes exhibit many similarities to liposomes; however, potential advantages cited include their lack of predisposition to oxidative degradation, as well potential lower cost of components and reduced variability compared to natural phospholipids. whilst with current manufacturing methods, the cost and reproducibility of phospholipids is less of an issue, niosomes still offer a useful alternative to liposome formulations. in particular, niosomes appear an attractive option for oral delivery of vaccines due to their ability to withstand the harsh gastrointestinal environment as outlined in chap. . with both liposomes and niosomes, immunostimulatory agents can be easily incorporated within the system and in some cases this can result in restructuring of the particulate delivery system as is the case with iscoms (chap. ). iscoms are prepared from a mixture of phospholipid, cholesterol, and a saponin (often quil a). whilst a phospholipid/cholesterol mixture would normally form liposomes, the addition of appropriate concentrations of saponin to the mixture can result in restructuring of the system to form spherical, open, cage-like structures around nm in size, as nicely shown in chap. . given that their structures are open, iscoms cannot incorporate hydrophilic antigens, and antigens need to display a degree of lipophilicity for inclusion into iscoms. if required, antigens can be modified through a range of methods to incorporate lipophilic regions within their structure, thereby promoting their incorporation into the structure. alternatively, similar to cationic liposomes, cationic iscoms (where cationic components are used to build their structure) can electrostatically bind a range of anionic antigens and enhance their delivery. in chap. the formulation, preparation, and application of iscoms as vaccine adjuvants is considered. however, lipid-based systems are only one group of particulate delivery systems, and polymeric systems have also been extensively studied. in particular the use of biodegradable polymers to formulate nano-and microparticulate delivery systems for vaccines has been widely investigated. much like the lipid-based systems, there is a wide selection of options to consider in the formulation of polymeric nanoparticles and microspheres for vaccine delivery, with polyester polymers offering advantages due to their clinical approved use in a range of medical products. as with the other systems considered, immunostimulatory agents can be incorporated within these polymer constructs and thus these systems can act as delivery systems and adjuvants for antigens. within chap. , the design of polymeric microspheres as vaccine adjuvants is considered from the choice of base polymer, through to optimisation of process parameters, and finally to considerations of their stability as a product. whilst much of current research into the development of vaccines has focused on the design of vaccines for administration of a particulate suspension, dry powder vaccines may hold considerable advantages. in chap. , the authors consider the design of dry powder vaccines. such vaccines may offer low-cost, temperaturestable products suitable for pulmonary delivery, with the added advantage that the pulmonary route avoids the use of needles (and their associated risks). furthermore, it can allow for the effective delivery of antigens to target cells of the immune system without the harsh conditions faced by orally delivered vaccines. the development of spray-drying methods outlined in chap. supports the ability of such powder vaccines to be delivered using conventional dry powder inhalers already clinically licensed for pulmonary delivery. therefore, by considering these vaccine delivery platforms in conjunction with the appropriate choices for antigen and adjuvant it is hoped that the threefold multicomponent nature of a vaccine can be considered more completely than before. when viewed conceptually, vaccines comprise an important triad. the first part of the vaccine, and in a sense the most important, is the biological component. this is the whole protein, or whole organism, or epitope-based part which confers the ability to be recognised by the immune system. it is this part which differentiates one vaccine from another, an anti-flu vaccine from an anti-tb vaccine. the second part of the vaccine is the adjuvant, which is one of many alternatives, that confers an immunogenicity to many vaccines that they would otherwise not possess. it often does this in a generic fashion, such as via agonising the innate immune system, so that the same adjuvant is quite capable of functioning in many different vaccine formulations. the final and third part of the vaccine is the delivery vehicle, as opposed to the delivery mechanism, such as oral vaccines versus injectable. the vehicle can be things as different as a viral vector or a liposome. an attenuated or heat-treated whole-organism vaccine can be thought of as combining all three parts of this triad in one supra-molecular moiety. of course, this is a gross simplification, and many other things go into deployable vaccine formulations, such as preservatives, contaminants, and other chemical or biological components that so exercise the anti-vaccine lobby. hopefully, this book will encourage us to think of vaccines in these terms, and provides the background necessary to engage with each of the three components of the vaccine triad. with this in mind, the anticipation inherent with this work is indeed simple and straightforward: to foster and foment interest in those areas of vaccinology that this far have not received the level of intense work that they richly deserve. to help achieve this, we have sought to balance optimistic positivity and cold, hard rationality. not all of the approaches described will ultimately bear fruit, but each should, nonetheless, be examined with equal diligence, sedulousness, and assiduity. global malaria mortality between and : a systematic analysis evidence for active immunity to morphine in mice changes in heroin selfadministration by a rhesus monkey after morphine immunisation world health organization. manual for the production and control of vaccines-tetanus toxoid. blg/undp/ . rev liposomes as immunological adjuvants liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen a liposome-based mycobacterial vaccine induces potent adult and neonatal multifunctional t cells through the exquisite targeting of dendritic cells key: cord- -n majqes authors: modrow, susanne; falke, dietrich; truyen, uwe; schätzl, hermann title: historical overview date: - - journal: molecular virology doi: . / - - - - _ sha: doc_id: cord_uid: n majqes “poisons” were originally considered as the causative agents of illnesses that we know as viral diseases today. at that time, there were no standard methods to detect pathogenic (disease-causing) organisms such as bacteria and protozoa in the supposed “poisonous materials”. only animal experiments performed by louis pasteur at the end of the nineteenth century, in which no dilution of the poisonous properties was achieved even after several passages, suggested that the disease-causing agent was able to multiply in the organism. therefore, there was talk of a reproducible “virus” (latin for “poison” or “slime”) in living organisms, and later also in cells. in st. petersburg in , dimitri i. ivanovski demonstrated that tobacco mosaic disease is caused by an “ultrafilterable” agent, whose size is significantly smaller than that of bacteria: tobacco mosaic virus (bacteria filters have a pore size of approximately . μm, however, most viruses are smaller than . μm). soon afterwards, martinus willem beijerinck came to the same conclusion: he developed, for the first time, the notion of a self-replicating, “liquid” agent (contagium vivum fluidum). the discovery of foot-and-mouth disease virus by friedrich loeffler and paul frosch in greifswald in was the first evidence of an animal pathogenic virus. since when have we known of viruses? "poisons" were originally considered as the causative agents of illnesses that we know as viral diseases today. at that time, there were no standard methods to detect pathogenic (disease-causing) organisms such as bacteria and protozoa in the supposed "poisonous materials". only animal experiments performed by louis pasteur at the end of the nineteenth century, in which no dilution of the poisonous properties was achieved even after several passages, suggested that the disease-causing agent was able to multiply in the organism. therefore, there was talk of a reproducible "virus" (latin for "poison" or "slime") in living organisms, and later also in cells. in st. petersburg in , dimitri i. ivanovski demonstrated that tobacco mosaic disease is caused by an "ultrafilterable" agent, whose size is significantly smaller than that of bacteria: tobacco mosaic virus (bacteria filters have a pore size of approximately . mm, however, most viruses are smaller than . mm). soon afterwards, martinus willem beijerinck came to the same conclusion: he developed, for the first time, the notion of a self-replicating, "liquid" agent (contagium vivum fluidum). the discovery of foot-and-mouth disease virus by friedrich loeffler and paul frosch in greifswald in was the first evidence of an animal pathogenic virus. however, it can be retrospectively documented that as long as , years agowithout knowledge of the nature of the pathogens -practices were implemented which today would be described as vaccinations against viral diseases. in ancient china, india and egypt, devastating smallpox epidemics must frequently have occurred; pharaoh ramses v -as his death mask shows -most likely died of an infection of smallpox virus. as observed at that time, people who had survived the disease were spared from the disease in further epidemics; therefore, they had to have developed some kind of protection caused by the first disease -they were immune. this protective status could also be induced artificially; when dried scabs of smallpox were transmitted to healthy people, they were at least partially protected against smallpox -a measure that we now denominate variolation (the medical term for smallpox is "variola"; ▶ sect. . ). historical descriptions indicate that smallpox was used as a biological weapon at that time. in the eighteenth century, it was discovered in england and germany that overcoming milker's nodule disease, which is triggered by a virus related to smallpox, confers protection against genuine smallpox. edward jenner must have been aware of these observations in when he transmitted swinepox and cowpox material, as a sort of vaccine, initially to his first-born son and later to james phipps, a young cowherd. both boys remained healthy following exposure to the human pathogenic smallpox virus by inoculation of smallpox pus; in fact, a protective effect was generated by this first deliberate "virological experiment". knowledge of this vaccination spread very rapidly from england to the european continent and the usa. the term "vaccination" is derived from the latin vacca, which means "cow". vaccinations were soon prescribed by law and this led to a gradual reduction of the dreaded disease. in the former german reich, a first vaccination law was enacted in . however, it still took about years until a human, ali maov maalin, was naturally infected with smallpox (in somalia) for the last time (in ), after the who had conducted a worldwide vaccination programme. today, the disease is considered eradicated. on a similar basis, i.e., without precise knowledge of the nature of the pathogen, louis pasteur developed a vaccine against rabies (▶ sect. . . ) in paris in . he transmitted the disease intracerebrally to rabbits in , seeing the causative agent rather in unknown and invisible microbes. as he demonstrated, the pathogen lost its disease-inducing properties by continuous transmission in these animals. in this way, pasteur achieved the basis for a vaccine virus (virus fixe), which, in contrast to the wild-type pathogen (virus de rue), was characterized by a constant incubation period. rubbed and dried spinal cord of rabbits that had been inoculated with the virus fixe was no longer infectious, but caused (initially in dogs) protection against rabies. for the first time, in , pasteur inoculated a -year-old alsatian boy named joseph meister with this material. the boy had been bitten by a rabid dog days before, and finally survived, by virtue of the protective effect induced by the vaccine. they especially noticed the striking ability of these agents to lyse bacteria and, therefore, called them bacteriophages -according to the greek word phagein, which means "to eat". the exploration of the nature of bacteriophages has provided virology with important findings and impulses both in methodological and in conceptual terms. many of the steps that characterize a viral infection were first discovered in experiments with bacterial viruses: such processes include attachment and penetration, the reproduction-cycledependent regulation of gene expression that results in early and late synthesized proteins, and lysogeny, which is associated with the existence of prophages. the study of viruses and their attributes was particularly difficult because they, in contrast to bacteria, could not be propagated in artificial culture media. however, it could be ascertained that some of the pathogens isolated from diseased people were transmissible to animals, in which they were able to reproduce. for example, human herpes simplex virus was transmitted from human skin blisters to the cornea of rabbits by wilhelm gr€ uter in marburg in . the extraordinary susceptibility of ferrets allowed the isolation of influenza a virus by christopher andrews, wilson smith and patrik laidlaw from pharyngonasal fluid of a sick person for the first time in . animal experiments also provided many insights into the pathogenesis of viral infections from another point of view. richard e. shope discovered rabbit papillomavirus in , and thus the first tumour virus, which -as was later shown -contains a dna genome. he suspected that such a virus could exist in a latent form as a provirus in the organism. in addition, the discovery that skin cancer can develop from benign skin papillomas is attributed to him. hence, malignant tumours develop in two or more steps -nowadays a universally accepted notion. shope further observed that the incidence of cancer differs in different rabbit breeds, and thus genetic traits of the host also influence the development of cancer. in the framework of animal experiments, erich traub made an important observation while studying the virus of lymphocytic choriomeningitis in princeton in : when pregnant mice were infected with the virus, the virus was transferred to the embryos; mother animals became sick from meningitis, and produced protective antibodies in the further course of the disease. by contrast, the newborn mice remained healthy, but secreted large quantities of the virus for life without developing a specific immune response against the pathogen. this discovery was the first example of an immune tolerance induced by a virus, but the general significance of this phenomenon was not recognized, and the now popular term was not coined (▶ sect. . . ). later, lymphocytic choriomeningitis was shown to be an immunologically related disease. the restriction of cytotoxic t lymphocytes by certain genetically determined types of mhc proteins was demonstrated for the first time in this model by rolf m. zinkernagel und peter c. doherty in . the above-mentioned experiments of traub evidenced also for the first time the intrauterine transmission of a virus. this raised the question of similar ways of infection in humans. in fact, after a severe rubella epidemic in australia in , sir norman gregg observed embryopathies when pregnant women were affected by the infection. as demonstrated later, these malformations were the result of intrauterine transmission of rubella virus. in , coxsackievirus was discovered after transmission of virus-containing stool extracts into newborn mice (coxsackie is a small town in the us state of new york). later in new york, ludwik gross isolated murine leukaemia virus from blood cells. besides the importance for tumour virus research, these observations aroused interest in the question concerning the basis of the high susceptibility of newborn animals to viral infections, and suggested investigations on the innate resistance of an organism to infections as well as the time and the causes of its formation. laborious and time-consuming experiments were initially the only way to prove the existence of viruses: therefore, there simpler methods were sought. one way involved the observation of so-called inclusion bodies in virus-infected tissues, which were soon judged as an indication for proliferation of the pathogen; as we now know, inclusion bodies are the accumulation of viral proteins and particles in the cytoplasm or the nucleus. the first inclusion bodies were discovered by dimitri i. ivanovski; at the same time, guiseppe guarnieri discovered similar deposits in cells infected with smallpox virus, then in , adelchi negri found inclusion bodies in ganglion cells of rabid animals, which were later named after him. thus, there were at least simple dye detection methods for some viral diseases. however, virus culture methods became available later. in the s, it was found that embryonated chicken eggs are appropriate for the propagation of some viral species. between and , a pandemic emerging viral disease, spanish flu, claimed more than million lives, i.e., more than in the first world war. after cultivation of the virus responsible in embryonated chicken eggs in , their haemagglutinating properties were discovered in (i.e., their ability to agglutinate red blood cells), thereby laying the basis for the development of haemagglutination tests to detect viruses. another important step in the history of modern virology was the development of the first ultracentrifuges, which became available at about the same time. they made possible the sedimentation and concentration of the minute virus particles. however, the breakthrough in the elucidation of the pathogenetic mechanism of influenza viruses was only possible by the development of molecular biological techniques that allowed the investigation of the genetic material of this pathogen, which exists in the form of singlestranded rna. its sequencing revealed the genetic reasons for the previously not understood ability of influenza viruses to change their antigenic properties at periodic intervals (▶ sect. . . ). however, it was particularly the donation-funded research of poliomyelitis (▶ sect. . . ) which provided crucial new insights. retrospectively, it represents the actual transition to molecular biological research of viral infections. the strong increase in the incidence of polio and the number of deaths -a result of enhanced hygiene standards and the shift of infection rates into later years of life -brought about in the usa the establishment of the national polio foundation by franklin d. roosevelt, himself a victim of this disease, in the early s. with the funds raised, a major research programme was initiated whose coronation was the discovery of the cytopathic effect by john f. enders, thomas h. weller and frederik c. robbins in . in , hugh b. and mary c. maitland had already introduced the method of tissue culture, in which the cells of small tissue pieces were cultivated in serumcontaining liquid media and infected with viruses. successful viral replication was then demonstrated in animal experiments or by detecting the presence of inclusion bodies. when antibiotics became available in the s, it was then possible to largely prevent bacterial contaminations in cultures, which led to much simpler handling in this method. polioviruses were cultivated in embryonic human cells of fixed kidney tissue fragments, and thereby cellular alterations were easily identifiable. this diagnostically valuable cytopathic effect drove the development of virology forwards. it was the basis for the plaque test that was developed by renato dulbecco and marguerite vogt in , which rendered possible, for the first time, the quantitative determination of the number of infectious particles in cell culture. the capability to cultivate polioviruses under controlled conditions in vitro was the basis for the development of the two polio vaccines: the inactivated vaccine developed by jonas e. salk and the live vaccine with attenuated, i.e., weakened polioviruses, developed by albert b. sabin. both vaccines are still in use today. later, vaccines against measles, rubella and mumps were also produced following the principle applied by sabin. by using the method of cell culture, it was possible to cultivate even yellow fever virus, vaccinia virus and rabies virus in vitro. wallace p. rowe isolated adenoviruses from cultures of human tonsil tissue after a long cultivation period in . a further development of viral cultivation in vitro provided the method of co-cultivation, which consists in the addition of indicator cells to the tissue cultures, which indicate viral replication by the occurrence of a cytopathic effect. in this manner, the existence of herpes simplex virus was verified in latently infected human dorsal root ganglia in , whereas direct virus detection was not possible at that time. until then, it had generally been assumed that in the course of viral infections the pathogen would be eliminated completely from the body by the resulting antibodies. the occurrence of herpes blisters as recurrent disease in people with antibodies -known as herpes immunological paradox -refuted that notion (▶ sect. . ). ernest w. goodpasture had previously suggested that the trigeminal ganglia should contain a "latent" form of the virus. after such a virus had been detected by co-cultivation, it was recognized that there are a number of infections with latent or persistent viruses, which -independently of the illness symptoms -are excreted either intermittently (e.g., herpes simplex virus) or permanently (such as epstein-barr virus). the primary isolation of human immunodeficiency virus (hiv) was also accomplished by co-cultivation of lymph node biopsy material from an aids patient with suitable t lymphocytes. concurrently with the rather practical benefits from developments in viral cultivation, interest in general biological issues became increasingly important. the crystallization of tobacco mosaic virus from liquid media by wendell stanley in california in stimulated discussions as to whether viruses are dead or living matter. the main question concerned, however, the nature and structure of the genetic material, which was found to be nucleic acid by the seminal experiments of oswald t. avery, colin mcleod and maclyn mccarty with pneumococci in . in , alfred d. hershey and martha chase proved that during bacteriophage t infections only dna, but not the protein shell of the virus, penetrates into the bacterial cell. this demonstrated that nucleic acids are the carrier of genetic information. a few years later, in , gerhard schramm and heinz fraenkel-conrat showed independently in tobacco mosaic virus that rna can also be infectious. schramm and his colleagues had already described in germany in that tobacco mosaic virus is composed of rna and proteins; however, these findings attracted initially only little attention. the base ratios in dna molecules (a ¼ t and c ¼ g) that were discovered and developed by edwin chargaff enabled james d. watson and francis h. crick, in connection with the rosalind franklin's x-ray structural analysis, to develop their model of the dna double helix in . in , matthew meselson and franklin w. stahl demonstrated that dna is replicated semiconservatively during cell division. these fundamental insights created the way for the elucidation of basic molecular biological processes which are nowadays generally familiar. the now common molecular genetic, biochemical and immunological methods allow the detection of viruses in the organs and the study of their spread in the organism. the function and effect of viral genes can be explored in isolation and in interaction with other viral or cellular components. today, many viruses can be cultivated in large quantities in vitro in order to resolve their morphology and particle structure as well as to sequence their genetic information. in the case of non-cultivable viruses, modern molecular biological methods are available which make possible the investigation of the pathogens. virus-cell interactions can be explored, and provide important insights into the mechanisms of viral replication. on the other hand, many of the molecular processes in eukaryotic cells have been elucidated by using viruses as cell research tools. in this way, the process of rna splicing was originally described in adenoviruses, in which widely separated gene segments are assembled into single messenger rna molecules after transcription. the fact that dna is arranged with histone proteins into nucleosome structures within the nucleus was also first discovered in a virus, simian virus . in addition, even enhancers were originally described in viruses, i.e., the specific dna regions that increase the expression of certain genes in a localization-and orientationindependent manner. several of these viral regulatory elements have been used for alternative purposes: for example, the most frequently used promoter/ enhancer sequences to control the expression of heterologous genes in commercially available vectors are derived from cytomegalovirus. this immediate-early promoter/enhancer region actually regulates the expression of early genes of the virus (see ▶ sect. . ). furthermore, the transfer of nucleic acid sequences and foreign genes by viral transduction, e.g., using vector systems based on the functions of adenoviruses or retroviruses, is today an indispensable constituent of molecular and cell biology, and has essentially contributed to the development of gene therapy procedures. what is the importance of the henle-koch postulates? the study of the epidemiology and pathogenesis of infectious diseases raises the fundamental question of how can it be proved that an illness is caused by infection with a bacterium or a virus. robert koch derived four postulates from his work with anthrax bacteria between and , which his teacher jacob henle had previously developed as a hypothesis from the study of so-called miasma and contagions, i.e., the animate or inanimate disease and infection agents: . a pathogen must be detected in all cases of a certain disease, but it must be absent in healthy organisms. . the pathogen must be cultivable on culture media or in suitable cell cultures in the form of pure cultures. . healthy animals must develop the same disease after inoculation of the pathogen. . the causative agent must be reisolated from the infected animals. koch noted that the postulates do not comply in all cases. he acknowledged that there are healthy and long-term carriers and that a normal flora of facultative pathogenic bacteria exists. in the realm of virology, not all pathogens comply with these postulates. positive examples are measles virus (▶ sect. . . ), human poxviruses (▶ sect. . . ), canine and feline parvoviruses (▶ sect. . . ) and classical swine fever virus (▶ sect. . . ). with regard to viral diseases, charles river proposed modifications of the postulates in . the exceptions concern preferentially latent or persistent viral infections and the fact that tissue and organ damage or tumour formation cannot always be reproduced as consequences of infection. taken from epidemiology, the "evans postulates" are a worthy supplement (▶ table . ). they show the aetiological importance of a pathogen for a clinical picture if, among others, the pathogen is significantly more frequent in an exposed group and the disease in this population is commoner than in a non-exposed group. similarly, an immune response should be detectable in the affected collectives. the evans postulates are valuable particularly for multifactorial infectious diseases, such as canine kennel cough and porcine circovirus infection (▶ sect. . . ). the further development of virological and immunochemical detection methods in recent years has allowed the use of additional criteria for the causal relationship between a virus and a disease. these include the detection of a specific humoral or cellular immune response against the pathogen, i.e., igm or igg antibodies and specific stimulatable lymphocytes, and the detection of viral proteins, enzyme activities, dna or rna by in vitro and in situ methods. the specific detection of viral nucleic acids in tissues is especially important for the aetiological correlation between persistent viral infections and cancer. the fulfilment of koch's postulates or their modifications is still essential for the development of aetiological relationships between the pathogen and the host. what is the interrelationship between virus research, cancer research, neurobiology and immunology? as early as in , it was demonstrated that rous sarcoma virus can cause cancer. in , margarete vogt and renato dulbecco observed the transformation from benign to malignant cells after infection with murine polyomavirus in vitro. after animals had been inoculated, these cells generated tumours. shortly afterwards, it was also discovered that rous sarcoma virus can transform cells in vitro. thus, tumour virus research became a driving force of virology. it has enriched the realm of cancer research with decisive impulses both in conceptual and in methodological terms. experiments with the oncogenic polyomavirus also showed that its dissemination within mouse populations can be followed by serological methods. this aroused the hope that it would be possible to reduce cancer development to viral agents and to study its nature using the classical methods of epidemiology such as virus isolation and antibody detection. in particular, the study of oncogenic retroviruses in animal systems provided seminal insights into the molecular processes that lead to carcinogenesis (▶ sect. . ). by investigating oncogenic retroviruses in , howard temin and david baltimore discovered reverse transcriptase -an enzyme that transcribes the single-stranded rna of retroviruses into double-stranded dna. after integration of the viral genetic information into the genome of the host, these viruses lose their individual existence. a few years earlier, temin had described that an inhibitor of dna synthesis prevents replication of rous sarcoma virus, which should not be the case in a typical rna virus. the integration of a viral genome, which is then present as a provirus, has been associated with tumour development. this event interrupts the continuity of the genome of the cell, as cellular and viral genes can be amplified and destroyed, or their expression can be activated by recombination with viral promoters. as mentioned above, shope had already described that carcinomas arise from papillomas by a two-stage or multistage process. the development of cervical carcinoma caused by human papillomaviruses and that of primary liver cancer caused by hepatitis c virus or hepatitis b virus are similar. even epstein-barr virus exerts its tumorigenic effect in a complex way: the viral dna is detectable in nasopharyngeal carcinoma tumour cells and in various lymphomas (african burkitt's lymphoma). the cells are infected and immortalized, but do not produce infectious virus particles. furthermore, chromosomal translocations are found in b-cell lymphomas (▶ sect. . ). however, malignant transformation develops in a multistep process by interaction with other factors, such as malaria, which contributes to a chronic stimulation of cells. research on the molecular processes that occur in infections with papillomaviruses, hepatitis b virus and retroviruses has led to the development of vaccines which induce protection against the respective viral infection and are capable of preventing the development of cancer as a long-term consequence. the vaccination strategy in southeast asia that was initiated and promoted by the who years ago has led to a significant decrease of primary liver carcinoma, which is caused by hepatitis b virus infections (▶ sect. . ). in veterinary medicine, vaccines against feline leukaemia virus have proved that cats are protected against infections by this exogenous retrovirus and that tumour formation can be prevented (▶ sect. . ). the detailed investigation of the molecular biology and pathogenesis of human papillomavirus infections by the research group of harald zur hausen at the german cancer research center (dkfz) paved the way for the development of appropriate vaccines. these have been available for several years and protect against infections with the highly oncogenic papillomaviruses: they prevent the possible development of cervical carcinoma -one of the commonest cancers in women (▶ chap. , ▶ sect. . ). in september , harald zur hausen was awarded the nobel prize in physiology or medicine for his work concerning the role of papillomaviruses in cervical cancer. the term "slow virus infections" was initially coined by björn sigurdsson for maedi disease of sheep in iceland in . maedi-visna virus causes respiratory symptoms in a slow and progressive disease after very long incubation and latency periods. maedi-visna virus thus became a model for a range of pathogens that cause diseases with a similar protracted course (▶ sect. . . ). the exploration of its pathogenesis revealed that most slow virus infections are caused by pathogens which are usually associated with other diseases. slow virus infections principally affect the central nervous system and are caused, for example, by measles virus and jc polyomavirus. subacute sclerosing panencephalitis is probably caused by mutations in measles virus genes, which lead to the emergence of defective virus particles (▶ sect. . . ) . in progressive multifocal leucoencephalopathy, which is triggered by jc polyomavirus, the virus seemingly enters the brain very early and persists there for a long time before the disease breaks out as a result of damage to the immune system (e.g., by infection with hiv; ▶ sect. . . ). infections with hiv can also be considered as a slow virus disease. similarly, prion diseases also progress along the lines of a slow virus infection, but they are caused by non-viral pathogens and have a fundamentally different pathogenesis (▶ chap. ). working on yellow fever virus, m. hoskins, g.m. findlay and f. maccallum discovered the phenomenon of interference in : if an avirulent virus was injected into an experimental animal, the animal was protected from the consequences of infection by a virulent strain when it was applied within the next h, i.e., before the onset of an immune response. in , alick isaacs and jean lindenmann showed that interferon is responsible for the interference effect. it is species-specific, inducible and belongs to a group of substances that are known as cytokines today. interferons play an important role in the primary, non-specific defence against viral infections and in stimulating the immune system. the observation that antiviral interferon preparations also have tumour-inhibiting effects was surprising. generally, cytokines are synthesized when a suitable inducer binds as a ligand to its receptor on the cell membrane, thus triggering specific signal transduction processes in the cell (▶ chap. ). attempts have been made to develop antiviral chemotherapeutic agents since about . in retrospect, this search can be divided into three stages: the first successful experiments for therapy of a viral infection were performed by josef wollensak and herbert e. kaufman around in herpetic keratitis. they used substances that inhibit viral replication in vitro and were known from experimental cancer therapy. however, the selectivity of these substances, i.e., their ability to selectively influence viral and not cellular processes, was only slight because of the high cytotoxicity of the compounds. after the discovery of virus-coded enzymes such as thymidine kinases, dna polymerases and proteases, it was possible to address the development of specific inhibitors. antiviral drugs such as amantadine against influenza a virus (▶ sect. . ) and adenine arabinoside and acyclovir (acycloguanosine) against herpes simplex virus (▶ sect. . ) were found by targeted empiricism, i.e., by attempting to find a compound that selectively influences viral reproduction among many compounds with similar effects. the achievement of gertrude elion and her staff to use acyclovir as a systemically applicable and selective antiviral drug in herpes encephalitis was an important milestone in chemotherapeutic research. she was awarded the nobel prize in physiology or medicine in for her work. after the development of dna sequencing techniques, the experimental chemotherapy of viral infections entered its third stage. virus-encoded enzymes were discovered such as the retroviral protease and neuraminidase of influenza viruses, and it was possible to build structural models of enzymes by comparison with proteins of similar functions and known three-dimensional structures. this allows one to identify potential active centres and to develop compounds, also known as "designer" drugs, which accommodate within the active centres and inhibit the viral enzymes. that means deviating from purely empirical research, and is a first step towards a more rational development of antiviral compounds (▶ chap. ). molecular virology has achieved significant successes in recent decades: many infectious diseases can be prevented through the use of modern vaccines or have been completely eradicated (▶ chap. ). this ultimately made possible the global elimination of infectious agents such as smallpox virus. poliovirus, which causes poliomyelitis, is no longer found on some continents, and is currently confined to fewer than ten countries worldwide. in cases in which no preventive vaccination is possible today, e.g., against hiv and some herpesviruses such as cytomegalovirus and herpes simplex virus, a large number of antiviral drugs are available. although these drugs do not provide a cure, they substantially allow the control of symptoms. these successes might tempt one to assume that virus research has become unnecessary. the assessment of the epidemiological situation by the who and the many sensationalistic headlines in newspapers and the media with which we are repeatedly confronted imply the opposite. because of their frequent and high rates of mutation, viruses are subject to continuous change and development: viruses are permanently compelled to cope with the infected organism and its immune defence systems, always trying to undermine and circumvent them. in particular, viruses that persist in the organism are capable of evading the host immune defence systems by very skilful strategies. the worldwide increase in travel leads not only to contact with new human pathogens, but also to their rapid dissemination. this is demonstrated, for example, by sars virus infections (▶ sect. . ), the pandemic with the new influenza a virus variant (mexican flu, "swine flu") and the threatening potential with regard to humans of new highly pathogenic influenza viruses (▶ sect. . ). new and novel viral diseases which have their origin in the animal kingdom (zoonoses) are also expected owing to increased environmental changes and their serious consequences. outbreaks of infection with ebola virus, nipah virus and hendra virus are examples. deforestation of rainforests has led to a change in living conditions for bats, which then infect horses and pigs and, via these intermediate hosts, also humans. birds carried west nile virus from africa to north america, and avian flu virus h n was transported from asia to europe by migratory birds. the aids pandemic that was induced by human immunodeficiency viruses was originally the result of a zoonotic transmission from monkeys to humans, followed by efficient further dissemination within the human population. the threat from both new and already well-known viral infections will not decrease because of reduced vaccination, particularly in industrialized countries; therefore, scientists who conduct research in the field of molecular virology will continue to have an ample sphere of activity. behbehani am ( ) the smallpox story in words and pictures. university of kansas medical center, kansas city causation and disease. the henle-koch postulates revisited helmut ruska and the visualisation of viruses mikrobenj€ ager. ullstein, frankfurt levine aj ( ) viruses. palgrave macmillan m€ uller r ( ) medizinische mikrobiologie. parasiten, bakterien, immunit€ at, th edn history of virology key: cord- -zhmnfd w authors: straif-bourgeois, susanne; ratard, raoult title: infectious disease epidemiology date: journal: handbook of epidemiology doi: . / - - - - _ sha: doc_id: cord_uid: zhmnfd w the following chapter intends to give the reader an overview of the current field of applied infectious disease epidemiology. prevention of disease by breaking the chain of transmission has traditionally been the main purpose of infectious disease epidemiology. while this goal remains the same, the picture of infectious diseases is changing. new pathogens are identified and already known disease agents are changing their behavior. the world population is aging; more people develop underlying disease conditions and are therefore more susceptible to certain infectious diseases or have long term sequelae after being infected. the following chapter intends to give the reader an overview of the current field of applied infectious disease epidemiology. prevention of disease by breaking the chain of transmission has traditionally been the main purpose of infectious disease epidemiology. while this goal remains the same, the picture of infectious diseases is changing. new pathogens are identified and already known disease agents are changing their behavior. the world population is aging; more people develop underlying disease conditions and are therefore more susceptible to certain infectious diseases or have long term sequelae after being infected. infectious diseases are not restricted to certain geographic areas anymore because of the increasing numbers of world travelers and a worldwide food distribution. the fear of a bioterrorist attack adds a new dimension in infectious disease epidemiology, and health departments enhance their surveillance systems for early detection of suspicious disease clusters and for agents used as weapons of mass destruction. improvements in laboratory techniques and mapping tools help to expand the knowledge of transmission of disease agents and enhanced surveillance techniques are feasible as a result of software progress and reporting of diseases via secure internet sites. surveillance and outbreak investigations remain the major responsibilities in public health departments. epidemiologic methods and principles are still the basis for these tasks but surveillance techniques and outbreak investigation are changing and adapting to improvements and the expanded knowledge. conducting surveys is a useful way to gather information on diseases where surveillance data or other data sources are not available, especially when dealing with emerging or re-emerging pathogens. program evaluation is an important tool to systematically evaluate the effectiveness of intervention or prevention programs for infectious diseases. infectious diseases are a major cause of human suffering in terms of both morbidity and mortality. in , out of an estimated total of million deaths, million were due to infectious diseases (who a,b) . the most common cause of infectious disease deaths were pneumonia ( million), diarrhea ( million) followed by tuberculosis, malaria, aids and hepatitis b. not surprisingly, there is a large imbalance in diseases between developing and industrialized countries (see table . ). morbidity due to infectious diseases is very common in spite of the progress accomplished in recent decades. even in industrialized countries, the prevalence of infection is very high for some infectious agents. serologic surveys found that by young adulthood the prevalence of antibodies was % against herpes simplex virus type , - % against type , % against human herpes virus, % against hepatitis a, % against hepatitis c, - % against hepatitis b, and % against chlamydia pneumoniae (american academy of pediatrics ; mandell et al. ) . annually, approximately , , episodes of diarrhea leading to , hospitalizations and deaths occur among adults in the united states (mounts et al. ). the center for disease control and prevention (cdc) estimates that each year million people in the us get sick, more than , are hospitalized and die as a result of foodborne illnesses (cdc ) . every year influenza circulates widely, infecting from % to % of the world population. the importance of infectious disease epidemiology for prevention it is often said that epidemiology is the basic science of preventive medicine. to prevent diseases it is important to understand the causative agents, risk factors and circumstances that lead to a specific disease. this is even more important for infectious disease prevention, since simple interventions may break the chain of transmission. preventing cardiovascular diseases or cancer is much more difficult because it usually requires multiple long term interventions requiring lifestyle changes and behavior modification, which are difficult to achieve. in , the american commission of yellow fever, headed by walter reed, was sent to cuba. the commission showed that the infective agent was transmitted by the mosquito aedes aegypti. this information was used by the then surgeon general of the us army william gorgas, to clean up the year old focus of yellow fever in havana by using mosquito proofing or oiling of the larval habitat, dusting houses with pyrethrum powder and isolating suspects under a mosquito net. this rapidly reduced the number of cases in havana from in to in (goodwin ) . a complete understanding of the causative agent and transmission is always useful but not absolutely necessary. the most famous example is that of john snow who was able to link cholera transmission to water contamination during the london cholera epidemic of by comparing the deaths from those households served by the southwark & vauxhall company versus those served by another water company. john snow further confirmed his hypothesis by the experiment of removing the broad street pump handle (wills a ). over the past three decades, more than new pathogens have been identified, some of them with global importance: bartonella henselae, borrelia burgdorferi, campylobacter, cryptosporidium, cyclospora, ebola virus, escherichia coli :h , ehrlichia, hantaan virus, helicobacter, hendra virus, hepatitis c and e, hiv, human herpesvirus and , human metapneumovirus, legionella, new variant creutzfeldt-jakob disease agent, nipah virus, parvovirus b , rotavirus, severe acute respiratory syndrome (sars) etc.. while there are specific causative agents for infectious diseases, these agents may undergo some changes over time. the last major outbreak of pneumonic plague in the world occurred in manchuria in . this scourge, which had decimated humans for centuries, is no longer a major threat. the plague bacillus cannot survive long outside its animal host (humans, rodents, fleas) because it lost the ability to complete the krebs cycle on its own. while it can only survive in its hosts, the plague bacillus also destroys its hosts rapidly. as long as susceptible hosts were abundant, plague did prosper. when environmental conditions became less favorable (lesser opportunities to sustain the host to host cycles), less virulent strains had a selective advantage (wills b) . the influenza virus is the best example of an agent able to undergo changes leading to renewed ability to infect populations that had been already infected and immune. the influenza virus is a single stranded rna virus with a lipophilic envelope. two important glycoproteins from the envelope are the hemagglutinin (ha) and neuraminidase (na). the ha protein is able to agglutinate red blood cells (hence its name). this protein is important as it is a major antigen for eliciting neutralizing antibodies. antigenic drift is a minor change in surface antigens that result from point mutations in a gene segment. antigenic drift may result in epidemics, since incomplete protection remains from past exposures to similar viruses. antigenic shift is a major change in one or both surface antigens (h and|or n) that occurs at varying intervals. antigenic shifts are probably due to genetic recombination (an exchange of a gene segment) between influenza a viruses, usually those that affect humans and birds. an antigenic shift may result in a worldwide pandemic if the virus can be efficiently transmitted from person to person. in the past three decades throughout the world, there has been a shift towards an increase in the population of individuals at high risk for infectious diseases. in industrialized nations, the increase in longevity leads to higher proportion of the elderly population who are more prone to acquiring infectious diseases and developing life threatening complications. for example, a west nile virus (wnv) infection is usually asymptomatic or causes a mild illness (west nile fever); rarely does it cause a severe neuro-invasive disease. in the epidemic of west nile in louisiana, the incidence of neuro-invasive disease increased progressively from . per , in the to age group to per , in the to year old age group and jumped to per , in the age group and older. mortality rates showed the same pattern, a gradual increase to . per , in the to age group with a sudden jump to per , for the oldest age group of and older. improvement in health care in industrialized nations has caused an increase in the number of immune-deficient individuals, be it cancer survivors, transplant patients or people on immuno-suppressive drugs for long term auto-immune diseases. some of the conditions that may increase susceptibility to infectious diseases are: cancers, particularly patients on chemo or radiotherapy, leukemia, lymphoma, hodgkin's disease, immune suppression (hiv infection), long term steroid use, liver disease, hemochromatosis, diabetes, alcoholism, chronic kidney disease and dialysis patients. for example persons with liver disease are times more likely to develop vibrio vulnificus infections than are persons without liver disease. some of these infections may be severe, leading to death. in developing countries a major shift in population susceptibility is associated with the high prevalence of immune deficiencies due to hiv infections and aids. in botswana which has a high prevalence of hiv (sentinel surveillance revealed hiv seroprevalence rates of % among women presenting for routine antenatal care), tuberculosis rates increased from per , in to per , in (lockman et al. ) while before the hiv|aids epidemics, rates above were very rare. changes in lifestyles have increased opportunities for the transmission of infectious disease agents in populations previously at low risk. intravascular drug injections have increased the transmission of agents present in blood and body fluids (e.g. hiv, hepatitis b and c). consumption of raw fish, shell fish and ethnic food expanded the area of distribution of some parasitic diseases. air travel allows people to be infected in a country and be half-way around the globe before becoming contagious. by the same token, insects and other vectors have become opportunistic global travelers. aedes albopictus, the asian tiger mosquito, was thus imported in to houston, texas inside japanese tires. subsequently, it has invaded us states. with the advent of nucleic acid tests, it has become possible to detect the presence of infectious disease agents in the air and environmental surfaces. for example, the use of air samplers and polymerase chain reaction analysis has shown that bordetella pertussis dna can be found in the air surrounding patients with b. pertussis infection, providing further evidence of airborne spread (aintablian et al. ) and thus leading to re-evaluate the precautions to be taken. however the presence of nucleic acids in an environmental medium does not automatically mean that transmission will occur. further studies are necessary to determine the significance of such findings. infectious disease agents, when used in bioterrorism events, have often been reengineered to have different physical properties and are used in quantities not usually experienced in natural events. there is little experience and knowledge about the human body's response to large doses of an infectious agent inhaled in aerosol particles that are able to be inhaled deep into lung alveolae. during the anthrax letter event, there was considerable discussion about incubation period, recommended duration of prophylaxis, and minimum infectious dose. this lack of knowledge base has led to confusion in recommendations being made. although the basics of infectious disease epidemiology have not changed and the discipline remains strongly anchored on some basic principles, technological developments such as improved laboratory methods and enhanced use of informatics (such as advanced mapping tools, web based reporting systems and statistical analytical software) have greatly expanded the field of infectious disease epidemiology. molecular techniques are being used more and more as a means to analyze epidemiological relationships between microorganisms. hence the term molecular epidemiology refers to epidemiologic research studies made at the molecular level. the main microbial techniques used, target plasmids and chromosomes. more specifically, plasmid fingerprinting and plasmid restriction endonuclease (rea) digestion, chromosomal analysis including pulse field gel electrophoresis (pfge), restriction fragment length polymorphism (rflp), multi-locus sequence type (mlst) and spa typing to name a few of these techniques. polymerase chain reaction (pcr) is used to amplify the quantity of genomic material present in the specimen. real-time pcr detection of infectious agents is now possible in a few hours. these techniques are becoming more widely used, even in public health laboratories for routine investigations. it is beyond the scope of this text to describe these methods in more detail. applications of molecular epidemiology methods have completely changed the knowledge about infectious disease transmission for many microorganisms. the main application is within outbreak investigations. being able to characterize the nucleic acid of the microorganisms permits an understanding of how the different cases relate to each other. molecular epidemiology methods have clarified the controversy about the origin of tuberculosis cases: is it an endogenous (reactivation) or exogenous (reinfection) origin? endogenous origin postulates that mycobacterium tuberculosis can remain alive in the human host for a lifetime and can start multiplying and producing lesions. on the other hand exogenous origin theory postulates that reinfection plays a role in the development of tuberculosis. the immunity provided by the initial infection is not strong enough to prevent another exposure to mycobacterium tuberculosis and a new infection leads to disease. in countries with low tuberculosis transmission, for example the netherlands, most strains have unique rflp fingerprints. each infection is unique and there are hardly any clusters of infections resulting from a common source. most cases are the result of reactivation. this is in contrast with areas of high endemicity where long chains of transmission can be identified with few rflp fingerprinting patterns (alland et al. ) . in some areas, up to % of tuberculosis cases are the result of reinfection. numerous new immunoassays have been developed. they depend on an antigenantibody reaction, either using a test antibody to detect an antigen in the patient's specimen or using a test antigen to detect an antibody in the patient's specimen. an indicator system is used to show that the reaction has taken place and to quantify the amount of patient antigen or antibody. the indicator can be a radioactive molecule (radioimmunoassay [ria]), a fluorescent molecule (fluorescent immunoassay [fia]), a molecule with an attached enzyme that catalyzes a color reaction (enzyme-linked immunoassay [elisa or eia]), or a particle coated with antigen or antibody that produces an agglutination (latex particle agglutination [la] ). the reaction can be a simple antigen|antibody reaction or a "sandwich" immunoassay where the antigen is "captured" and a second "read out" antibody attaches to the captured antigen. the antibody used may be polyclonal (i.e. a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope) or monoclonal (i.e. immunoglobulin molecules of single-epitope specificity that are secreted by a clone of b cells). it may be directed against an antigen on an epitope (i.e. a particular site within a macromolecule to which a specific antibody binds). plotting diseases on a map is one of the very basic methods epidemiologists do routinely. as early as john snow, suspecting water as a cause of cholera, plotted the cases of cholera in the districts of golden square, st. james and berwick, in london. the cases seemed to be centered around the broad street pump and less dense around other pumps. the map supplemented by other observations led to the experiment of removing the handle on the broad street pump and subsequent confirmation of his hypothesis (snow ) . geographical information systems (gis) have been a very useful tool in infectious disease research. gis are software programs allowing for integration of a data bank with spatial information. the mapping component includes physical layout of the land, towns, buildings, roads, administrative boundaries, zip codes etc. data may be linked to specific locations in the physical maps or to specific aggregates. a gis system includes tools for spatial analysis. climate, vegetation and other data may be obtained through remote sensing and combined with epidemiologic data to predict vector occurrence. however, these tools should be used with caution. they can be useful to generate hypotheses and identify possible associations between risk of disease and environmental exposures. because of potential bias, mapping should never be considered as more than an initial step in the investigation of an association. "the bright color palettes tend to silence a statistical conscience about fortuitous differences in the raw data" (boelaert et al. ) . for statistical methods in geographical epidemiology see chap. ii. of this handbook. web based reporting, use of computer programs and developments of sophisticated reporting and analytical software have revolutionized epidemiologic data collection and analysis. these tools have provided the ability to collect large amounts of data and handle large databases. however this has not been without risks. it remains crucial to understand the intricacies of data collected to avoid misinterpretation. for example, one should be aware that diseases and syndromes are initially coded by a person who may not be very software proficient, using shortcuts and otherwise could enter data of poor quality. what are the questions to be answered? too often one sees epidemiologists and statisticians preparing questionnaires, carrying out surveys, gathering surveillance information, processing data and producing reports, tables, charts and graphs in a routine fashion. epidemiology describes the distribution of health outcomes and determinants for a purpose. it is important to question the goals and objectives of all epidemiologic activities and tailor these activities to meet these objectives. the description of disease patterns includes analysis of demographic, geographical, social, seasonal and other risk factors. age groups to be used differ depending on the disease e.g. diseases affecting young children should have numerous age groups among children; sexually transmitted diseases require detailed age groups in late adolescence and early adulthood. younger age groups may be lumped together for diseases affecting mainly the elderly. gender categorization, while important for sexually transmitted diseases and other diseases with a large gender gap (such as tuberculosis), may not be important for numerous other diseases. geographical distribution is important to describe diseases linked to environmental conditions but may not be so useful for other diseases. surveillance, both active and passive, is the systematic collection of data pertaining to the occurrence of specific diseases, the analysis and interpretation of these data, and the dissemination of consolidated and processed information to contributors to the program and other interested persons (cdc b). in a passive surveillance system the surveillance agency has devised and put a system in place. after the placement, the recipient waits for the provider of care to report. passive case detection has been used for mortality and morbidity data for decades. it is almost universal. most countries have an epidemiology section in the health department that is charged with centralizing the data in a national disease surveillance system collecting mortality and morbidity data. in theory, a passive surveillance system provides a thorough coverage through space and time and gives a thorough representation of the situation. practically, compliance with reporting is often irregular and incomplete. in fact, the main flaws in passive case detection are incomplete reporting and inconsistencies in case definitions. the main advantages are the low cost of such a program and the sustained collection of data over decades. the purpose is to produce routine descriptive data on communicable diseases, generate hypotheses and prompt more elaborate epidemiologic studies designed to evaluate prevention activities. some conditions must be met to maximize compliance with reporting: . make reporting easy: provide easy to consult lists of reportable diseases, provide pre-stamped cards for reporting, provide telephone or fax reporting facilities. . do not require extensive information: name, age, sex, residence, diagnosis. some diseases may include data on exposure, symptoms, method of diagnosis etc. . maintain confidentiality and assure reporters that confidentiality will be respected. . convince reporters that reporting is essential: provide feedback; show how the data are used for better prevention. confidentiality of data is essential, particularly for those reporting health care providers who are subject to very strict confidentiality laws. any suspicion of failure of maintaining secure data would rapidly ruin a passive surveillance program. in an active surveillance system, the recipient will actually take some action to identify the cases. in an active surveillance program, the public health agency organizes a system by searching for cases or maintaining a periodic contact with providers. regular contacting boosts the compliance of the providers. providers are health agencies but also as in passive case detection, there may be day care centers, schools, long term care facilities, summer camps, resorts, and even public involvement. the agency takes the step to contact the health providers (all of them or a carefully selected sample) and requests reports from them at regular intervals. thus no reports are missing. active surveillance has several advantages: it allows the collection of more information. a provider sees that the recipient agency is more committed to surveillance and is therefore more willing to invest more time her|himself. it allows direct communication and opportunities to clarify definitions or any other problems that may have arisen. active surveillance provides much better, more uniform data than passive case detection but active case detection is much more expensive (see tables . and . ). active surveillance systems are usually designed when a passive system is deemed insufficient to accomplish the goals of disease monitoring. this type of surveillance is reserved for special programs, usually when it is important to identify every single case of a disease. active surveillance is implemented in the final phases of an eradication program: smallpox eradication, poliomyelitis eradication, guinea worm eradication and malaria eradication in some countries. active surveillance is also the best approach in epidemic or outbreak investigations to elicit all cases. in the smallpox eradication program, survey agents visited providers, asking about suspected cases and actually investigating each suspected case. in polio eradication programs, all cases of acute flaccid paralysis are investigated. in malaria eradication programs and some malaria control programs, malaria control agents go from house to house asking who has fever or had fever recently (in the past week or month for example). a blood smear is collected from those with fever. a case register is a complete list of all the cases of a particular disease in a definite area over a certain time period. registers are used to collect data on infections over long periods of time. registers should be population based, detailed and complete. a register will show an unduplicated count of cases. they are especially useful for long term diseases, diseases that may relapse or recur and diseases for which the same cases will consult several providers and therefore would be reported on more than one occasion. case registers contain identifiers, locating information, disease, treatment, outcome and follow-up information as well as contact management information. they are an excellent source of information for epidemiologic studies. in disease control, case registers are indispensable tools for follow up of chronic infections disease such as tuberculosis and leprosy. the contents and quality of a case register determine its usefulness. it should contain patient identifiers with names (all names), age, sex, place and date of birth, complete address with directions on how to reach the patient, name and address of a "stable" relative that knows the patient's whereabouts, diagnosis information with disease classification, brief clinical description (short categories are better than detailed descriptions), degree of infectiousness (bacteriological, serological results), circumstances of detection, initial treatment and response with specific dose, notes on compliance, side effects, clinical response, follow-up information with clinical response, treatment regimen, compliance, side effects, locating information; for some diseases contact information is also useful. updating a register is a difficult task. it requires cooperation from numerous persons. care must be taken to maintain the quality of data. it is important to only request pertinent information for program evaluation or information that would remind users to collect data or to perform an exam. for example, if compliance is often a neglected issue, include a question on compliance. further details concerning the use of registries in general are given in chap. i. of this handbook. sentinel disease surveillance for sentinel disease surveillance, only a sample of health providers is used. the sample is selected according to the objectives of the surveillance program. providers most likely to serve the population affected by the infection are selected, for example child health clinics and pediatricians should be selected for surveillance of childhood diseases. a sentinel system allows cost reduction and is combined with active surveillance. a typical surveillance program for influenza infections includes a selected numbers of general practitioners who are called every week to obtain the number of cases presented to them. this program may include the collection of samples for viral cultures or other diagnostic techniques. such a level of surveillance would be impossible to maintain on the national level. surveillance systems are evaluated on the following considerations (cdc b): usefulness: some surveillance systems are routine programs that collect data and publish results; however it appears that they have no useful purpose -no conclusions are reached, no recommendations are made. a successful surveillance system would provide information used for preventive purposes. sensitivity or the ability to identify every single case of disease is particularly important for outbreak investigations and eradication programs. predictive value positive (pvp) is the proportion of reported cases that actually have the health-related event under surveillance. low pvp values mean that non-cases might be investigated, outbreaks may be exaggerated or pseudo outbreaks may even be investigated. misclassification of cases may corrupt the etiologic investigations and lead to erroneous conclusions. unnecessary interventions and undue concern in the population under surveillance may result. representativeness ensures that the occurrence and distribution of cases accurately represent the real situation in the population. simplicity is essential to gain acceptance, particularly when relying on outside sources for reporting. flexibility is necessary to adapt to changes in epidemiologic patterns, laboratory methodology, operating conditions, funding or reporting sources. data quality is evaluated by the data completeness (blank or unknown variable values) and validity of data recorded (cf. chap. i. of this handbook). acceptability is shown in the participation of providers in the system. timeliness is more important in surveillance of epidemics. stability refers to the reliability (i.e., the ability to collect, manage and provide data properly without failure) and availability (the ability to be operational when it is needed) of the public health surveillance system. the major elements of a surveillance system as summarized by who are: mortality registration, morbidity reporting, epidemic reporting, laboratory investigations, individual case investigations, epidemic field investigations, surveys, animal reservoir and vector distribution studies, biologics and drug utilization, knowledge of the population and the environment. traditional surveillance methods rely on counting deaths and cases of diseases. however, these data represent only a small part of the global picture of infectious disease problems. mortality registration was one of the first elements of surveillance implemented. the earliest quantitative data available on infectious disease is about mortality. the evolution of tuberculosis in the us for example, can only be traced through its mortality. mortality data are influenced by the occurrence of disease but also by the availability and efficacy of treatment. thus mortality cannot always be used to evaluate the trend of disease occurrence. reporting of infectious diseases is one of the most common requirements around the world. a list of notifiable diseases is established on a national or regional level. the numbers of conditions vary; it ranges usually from to conditions. in general, a law requires that health facility staff, particularly physicians and laboratories, report these conditions with guaranteed confidentiality. it is also useful to have other non-health related entities report suspected communicable diseases such as day care centers, schools, restaurants, long term care facilities, summer camps and resorts. regulations on mandatory reporting are often difficult to enforce. voluntary compliance by the institution's personnel is necessary. reporting may be done in writing, by phone or electronically in the most advanced system. since most infectious diseases are confirmed by a laboratory test, reporting by the laboratory may be more reliable. the advantage of laboratory reporting is the ability to computerize the reporting system. computer programs may be set up to automatically report a defined set of tests and results. for some infectious diseases, only clinical diagnoses are made. these syndromes may be the consequences of a large number of different microorganisms for which laboratory confirmation is impractical. when public or physician attention is directed at a specific disease, reporting may be biased. when there is an epidemic or when the press focuses on a particular disease, patients are more prone to look for medical care and physicians are more likely to report. reporting rates were evaluated in several studies. in the us, studies show report rates of % for viral hepatitis, hemophilus influenzae %, meningococcal meningitis % and shigellosis %. it is important to have a standardized set of definitions available to providers. without standardized definitions, a surveillance system may be counting different entities from one provider to another. the variability may be such that the epidemiologic information obtained is meaningless. most case definitions in infectious disease epidemiology are based on laboratory tests, however some clinical syndromes such as toxic shock syndrome do not have confirmatory laboratory tests. most case definitions include a brief clinical description useful to differentiate active disease from colonization or asymptomatic infection. some diseases are diagnosed based on epidemiologic data. as a result many case definitions for childhood vaccine preventable diseases and foodborne diseases include epidemiologic criteria (e.g., exposure to probable or confirmed cases of disease or to a point source of infection). in some instances, the anatomic site of infection may be important; for example, respiratory diphtheria is notifiable, whereas cutaneous diphtheria is not (cdc ) . cases are classified as a confirmed case, a probable or a suspected case. an epidemiologically linked case is a case in which ) the patient has had contact with one or more persons who either have|had the disease or have been exposed to a point source of infection (including confirmed cases) and ) transmission of the agent by the usual modes is plausible. a case may be considered epidemiologically linked to a laboratory-confirmed case if at least one case in the chain of transmission is laboratory confirmed. probable cases have specified laboratory results that are consistent with the diagnosis yet do not meet the criteria for laboratory confirmation. suspected cases are usually cases missing some important information in order to be classified as a probable or confirmed case. case definitions are not diagnoses. the usefulness of public health surveillance data depends on its uniformity, simplicity and timeliness. case definitions establish uniform criteria for disease reporting and should not be used as the sole criteria for establishing clinical diagnoses, determining the standard of care necessary for a particular patient, setting guidelines for quality assurance, or providing standards for reimbursement. use of additional clinical, epidemiological and laboratory data may enable a physician to diagnose a disease even though the formal surveillance case definition may not be met. surveillance programs collect data on the overt cases diagnosed by the health care system. however these cases may not be the most important links in the chain of transmission. cases reported are only the tip of the iceberg. they may not at all be representative of the true endemicity of an infectious disease. there is a continuous process leading to an infectious disease: exposed, colonized, incubating, sick, clinical form, convalescing, cured. even among those who have overt disease there are several disease stages that may not be included in a surveillance system: some have symptoms but do not seek medical attention some do get medical attention but do not get diagnosed or get misdiagnosed some get diagnosed but do not get reported cases reported cases diagnosed but not reported cases who seek medical attention but were not diagnosed cases who were symptomatic but did not seek medical attention cases who were not symptomatic infectious disease cases play different roles in the epidemiology of an infectious disease; some individuals are the indicators (most symptomatic), some are the reservoir of microorganisms (usually asymptomatic, not very sick), some are amplifiers (responsible for most of the transmission), some are the victims (those who develop severe long term complications). depending on the specific disease and the purpose of the surveillance program, different disease stages should be reported. for example in a program to prevent rabies in humans exposure to a suspect rabid animal (usually a bite) needs to be reported. at the stage where the case is a suspect, prevention will no longer be effective. for bioterrorism events, reporting of suspects is of paramount importance to minimize consequences. waiting for confirmation causes too long of a delay. in the time necessary to confirm cases, opportunities to prevent co-infections may be lost and secondary cases may already be incubating, depending on the transmissibility of the disease. surveillance for west nile viral infections best rests on the reporting of neuroinvasive disease. case reports of neuro invasive diseases are a better indicator than west nile infection or west nile fever cases that are often benign, go undiagnosed and are reported haphazardly. for gonorrhea, young males are the indicators because of the intensity of symptoms. young females are the main reservoir because of the high proportion of asymptomatic infections. females of reproductive age are the victims because of pelvic invasive disease (pid) and sterility. a surveillance program for hepatitis b that only would include symptomatic cases of hepatitis b could be misleading. a country with high transmission of hepatitis b from mother to children would have a large proportion of infected newborn becoming asymptomatic carriers and a major source of infection during their lifetime. typically in countries with poor reporting of symptomatic hepatitis, the reporting of acute cases of hepatitis b would be extremely low in spite of high endemicity which would result in high rates of chronic hepatitis and hepatic carcinoma. most morbidity reporting collects data about individual cases. reporting of individual cases includes demographic and risk factor data which are analyzed for descriptive epidemiology and for implementation of preventive actions. for example, any investigation leading to contact identification and prophylaxis requires a start from individual cases. however, identification of individuals may be unnecessary and aggregate data sufficient for some specific epidemiologic purposes. monitoring an influenza epidemic for example, can be done with aggregate data. obtaining individual case information would be impractical since it would be too time consuming to collect detailed demographics on such a large number of cases. aggregate data from sentinel sites consists of a number of influenza-like illnesses by age group and the total number of consultants or the total number of 'participants' to be used as denominators. such data is useful to identify trends and determine the extent of the epidemic and geographic distribution. collection of aggregate data of the proportion of school children by age group and sex is a useful predictive tool to identify urinary schistosomiasis endemic areas (lengeler et al. ) without having to collect data on individual school children. epidemics of severe diseases are almost always reported. this is not the case for epidemics of milder diseases such as rashes or diarrheal diseases. many countries do not want to report an outbreak of disease that would cast a negative light on the countries. for example, many countries that are tourism dependent do not report cholera or plague cases. some countries did not report aids cases for a long time. case investigations are usually not undertaken for individual cases unless the disease is of major importance such as hemorrhagic fever, polio, rabies, yellow fever, any disease that has been eradicated and any disease that is usually not endemic in the area. outbreaks or changes in the distribution pattern of infectious diseases should be investigated and these investigations should be compiled in a comprehensive system to detect trends. while the total number of infectious diseases may remain the same, changes may occur in the distribution of cases from sporadic to focal outbreaks. for example the distribution of wnv cases in louisiana shifted from mostly focal outbreaks the first year the west nile virus arrived in the state in , to mostly sporadic cases the following year in (see fig. . ) . surveys are a very commonly used tool in public health, particularly in developing countries where routine surveillance is often inadequate (cf. chap. iv. of this handbook). survey data needs to be part of a comprehensive surveillance database. one will acquire a better picture from one or a series of well constructed surveys than from poorly collected surveillance data. surveys are used in control programs designed to control major endemic diseases: spleen and parasite surveys for malaria, parasite in urine and stools for schistosomiasis, clinical surveys for leprosy or guinea-worm disease and skin test surveys for tuberculosis. surveillance of microbial strains is designed to monitor, through active laboratory based surveillance, the bacterial and viral strains isolated. examples of these systems are: in the us, the pulsenet program is a network of public health laboratories that performs dna fingerprinting of bacteria causing foodborne illnesses (swaminathan et al. ). molecular sub-typing methods must be standardized to allow comparisons of strains and the building of a meaningful data bank. the method used in pulsenet is pulse field gel electrophoresis (pfge). the use of standardized subtyping methods has allowed isolates to be compared from different parts of the country, enabling recognition of nationwide outbreaks attributable to a common source of infection, particularly those in which cases are geographically separated. the us national antimicrobial resistance monitoring system (narms) for enteric bacteria is a collaboration between cdc, participating state and local health departments and the us food and drug administration (fda) to monitor antimicrobial resistance among foodborne enteric bacteria isolated from humans. narms data are also used to provide platforms for additional studies including field investigations and molecular characterization of resistance determinants and to guide efforts to mitigate antimicrobial resistance (cdc ) . monitoring of antimicrobial resistance is routinely done by requiring laboratories to either submit all, or a sample of their bacterial isolates. surveillance for zoonotic diseases should start at the animal level, thus providing early warning for impending increases of diseases in the animal population. rabies surveillance aims at identifying the main species of animals infected in an area, the incidence of disease in the wild animals and the prevalence of infection in the asymptomatic reservoir (bats). this information will guide preventive decisions made when human exposures do occur. malaria control entomologic activities must be guided by surveillance of anopheles population, biting activities, plasmodium infection to biting acivities and plasmodium infection rates in the anopheles population. surveillance for dead birds, infection rates in wild birds, infection in sentinel chickens and horse encephalitis are all part of west nile encephalitis surveillance. these methods provide an early warning system for human infections. the worldwide surveillance for influenza is the best example of the usefulness of monitoring animals prior to spread of infection in the human population. influenza surveillance programs aim to rapidly obtain new circulating strains to make timely recommendations about the composition of the next vaccine. the worldwide surveillance priority is given to the establishment of regular surveillance and investigation of outbreaks of influenza in the most densely populated cities in key locations, particularly in tropical or other regions where urban markets provide opportunities for contacts between humans and live animals (snacken et al. ). the rationale for selecting infectious diseases and an appropriate surveillance method is based on the goal of the preventive program. outbreaks of acute infectious diseases are common and investigations of these outbreaks are an important task for public health professionals, especially epidemiol-ogists. in , a total of foodborne outbreaks with , cases involved were reported in the us (cdc ) with norovirus being the most common confirmed etiologic agent associated with these outbreaks (see table . ). outbreaks or epidemics are defined as the number of disease cases above what is normally expected in the area for a given time period. depending on the disease, it is not always known if the case numbers are really higher than expected and some outbreak investigations can reveal that the reported case numbers did not actually increase. the nature of a disease outbreak depends on a variety of circumstances, most importantly the suspected etiologic agent involved, the disease severity or case fatality rate, population groups affected, media pressure, political inference and investigative progress. there are certain common steps for outbreak investiga-tions as shown in table . . however, the chronology and priorities assigned to each phase of the investigation have to be decided individually, based on the circumstances of the suspected outbreak and information available at the time. another way to detect an increase of cases is if the surveillance system of reportable infectious diseases reveals an unusually high number of people with the same diagnosis over a certain time period at different health care facilities. outbreaks of benign diseases like self-limited diarrhea are often not detected because people are not seeking medical attention and therefore medical services are not aware of them. furthermore, early stages of a disease outbreak are often undetected because single cases are diagnosed sporadically. it is not until a certain threshold is passed, that it becomes clear that these cases are related to each other through a common exposure or secondary transmission. depending on the infectious disease agent, there can be a sharp or a gradual increase of number of cases. it is sometimes difficult to differentiate between sporadic cases and the early phase of an outbreak. in the st. louis encephalitis (sle) outbreak in louisiana, the number of sle cases increased from to between week one and two and then the numbers gradually decreased over the next weeks to a total of cases (jones et al. ) . . after the initial report is received, it is important to collect and document basic information: contact information of persons affected, a good and thorough event description, names and diagnosis of hospitalized persons (and depending on the presumptive diagnosis their underlying conditions and travel history), laboratory test results and other useful information to get a complete picture and to confirm the initial story of the suspected outbreak. it also might be necessary to collect more biological specimens such as food items and stool samples for further laboratory testing. . based on the collected information the decision to investigate must be made. it may not be worthwhile to start an investigation if there are only a few people who fully recovered after a couple of episodes of a self-limited, benign diarrhea. other reasons not to investigate might be that this type of outbreak occurs regularly every summer or that it is only an increase in number of reported cases which are not related to each other. on the other hand, however, there should be no time delay in starting an investigation if there is an opportunity to prevent more cases or the potential to identify a system failure which can be caused, for example, by poor food preparation in a restaurant or poor infection control practices in a hospital or to prevent future outbreaks by acquiring more knowledge of the epidemiology of the agent involved. additional reasons to investigate include the interest of the media, politicians and the public in the disease cluster and the pressure to provide media updates on a regularly basis. another fact to consider is that outbreak investigations are good training opportunities for newly hired epidemiologists. sometimes lack of data and lack of sufficient background information make it difficult to decide early on if there is an outbreak or not. the best approach then is to assume that it is an outbreak until proven otherwise. . prevention of more cases is the most important goal in outbreak investigations and therefore a rapid evaluation of the situation is necessary. if there are precautionary measures to be recommended to minimize the impact of the outbreak and the spread to more persons, they should be implemented before a thorough investigation is completed. most likely control measures implemented by public health professionals in foodborne outbreaks are: recall or destruction of contaminated food items, restriction of infected food handlers from food preparation, correction of any deficiency in food preparation or conservation. . after taking immediate control measures, the next step is to know more about the epidemiology of the suspected agent. the most popular books for public health professionals include the "red book" (american academy of pediatrics ), the "control of communicable diseases manual" from the american public health association (apha ) or other infectious disease epidemiology books as well as the cdc website (www.cdc.gov). if the disease of interest is a reportable disease or a disease where surveillance data are available, baseline incidence rates can be calculated. then a comparison is made to determine if the reported numbers constitute a real increase or not. furthermore, the seasonal and geographical distribution of the disease is important as well as the knowledge of risk factors. many infectious diseases show a seasonal pattern such as rotavirus or neisseria meningitides. for example in suspected outbreaks where cases are associated with raw oyster consumption, the investigator should know that in the us gulf states vibrio cases increase in the summer months because the water conditions are optimal for the growth of the bacteria in water and in seafood. this kind of information will help to determine if the case numbers show a true increase and if it seems likely to be a real outbreak. . for certain diseases, numbers are not important. depending on the severity of the disease, its transmissibility and its natural occurrence, certain diseases should raise a red flag for every health care professional and even a single case should warrant a thorough public health investigation. for example a single confirmed case of a rabid dog in a city (potential dog to dog transmission within a highly populated area), a case of dengue hemorrhagic fever or a presumptive case of smallpox would immediately trigger an outbreak investigation. . sometimes an increase of case numbers is artificial and not due to a real outbreak. in order to differentiate between an artificial and a natural increase in numbers, the following changes have to be taken into consideration: alterations in the surveillance system, a new physician who is interested in the disease and therefore more likely to diagnose or report the disease, a new health officer strengthening the importance of reporting, new procedures in reporting (from paper to web based reporting), enhanced awareness or publicity of a certain disease that might lead to increased laboratory testing, new diagnostic tests, a new laboratory, an increase in susceptible population such as a new summer camp. . it is important to be sure that reported cases of a disease actually have the correct diagnosis and are not misdiagnosed. is there assurance that all the cases have the same diagnosis? is the diagnosis verified and were other differential diagnoses excluded? in order to be correct, epidemiologists have to know the basis for the diagnosis. are laboratory samples sufficient? if not, what kind of specimens should be collected to ascertain the diagnosis? what are the clinical signs and symptoms of the patient? in an outbreak of restaurant associated botulism in canada only the th case was correctly diagnosed. the slow progression of symptoms and misdiagnosis of the dispersed cases made it very difficult to link these cases and identify the source of the outbreak (cdc (cdc , . . the purpose of a case definition is to standardize the identification and counting of the number of cases. the case definition is a standard set of criteria and is not a clinical diagnosis. in most outbreaks the case definition has components of person, place and time, such as the following: persons with symptoms of x and y after eating at the restaurant z between date and date . the case definition should be broad enough to get most of the true cases but not too narrow so that true cases will not be misclassified as controls. a good method is to analyze the data, identify the frequency of symptoms and include symptoms that are more reliable than others. for example, diarrhea and vomiting are more specific than nausea and headache in the case definition of a food related illness. . what kind of information is necessary to be collected? it is sufficient to have a simple database with basic demographic information such as name, age, sex and information for contacting the patient. more often, date of reporting and date of onset of symptoms are also important. depending on the outbreak and the potential exposure or transmission of the agent involved further variables such as school, grade of student or occupation in adults might be interesting and valuable. . during an outbreak investigation it is important to identify additional cases that may not have been known or were not reported. there are several approaches: interview known cases and ask them if they know of any other friends or family members with the same signs or symptoms, obtain a mailing list of frequent customers in an event where a restaurant is involved, set up an active surveillance with physicians or emergency departments, call laboratories and ask for reports of suspected and confirmed cases. another possibility is to review surveillance databases or to establish enhanced surveillance for prospective cases. occasionally it might be worthwhile to include the media for finding additional cases through press releases. however the utility of that technique depends on the outbreak and the etiologic agent; the investigator should always do a benefit risk analysis before involving the media. . after finding additional cases, entering them in the database and organizing them, the investigator should try to get a better understanding of the situation by performing some basic descriptive epidemiology techniques such as sorting the data by time, place and person. for a better visualization of the data, an epidemic or "epi" curve should be graphed. the curve shows the number of cases by date or time of onset of symptoms. this helps to understand the nature and dynamic of the outbreak as well as to get a better understanding of the incubation period if the time of exposure is known. it also helps to determine whether the outbreak had a single exposure and no secondary transmission (single peak) or if there is a continuous source and ongoing transmission. figures . and . show "epi" curves of two different outbreaks: a foodborne outbreak in a school in louisiana, and the number of wnv human cases in louisiana in the outbreak, respectively. sometimes it is useful to plot the cases on a map to get a better idea of the nature and the source of an outbreak. mapping may be useful to track the spread by water (see john snow's cholera map) or by air or even a person to person transmission. if a contaminated food item was the culprit, food distribution routes with new cases identified may be helpful. maps, however, should be taken with caution and carefully interpreted. for example, wnv cases are normally mapped by residency but do not take into account that people might have been exposed or bitten by an infective mosquito far away from where they live. for outbreak investigations, spot maps are usually more useful than rate maps or maps of aggregate data. depending on the outbreak it might be useful to characterize the outbreak by persons' demographics such as age, sex, address and occupation or health status. are the cases at increased susceptibility or at high risk of infection? these kinds of variables might give the investigator a good idea if the exposure is not yet known. for typical foodborne outbreaks however, demographic information is not very useful because the attack rates will be independent of age and sex. more details on methods used in descriptive epidemiology are given in chap. i. of this handbook. . based on the results of basic descriptive epidemiology and the preliminary investigation, some hypotheses should be formulated in order to identify the cause of the outbreak. a hypothesis will be most likely formulated such as "those who attended the luncheon and ate the chicken salad are at greater risk than those who attended and did not eat the chicken salad". it is always easier to find something after knowing what to look for and therefore a hypothesis should be used as a tool. however, the epidemiologist should be flexible enough to change the hypothesis if the data do not support it. if data clues are leading in another direction, the hypothesis should be reformulated such as "those who attended the luncheon and ate the baked chicken are at greater risk than those who attended and did not eat the baked chicken". to verify or deny hypotheses, measures of risk association such as the relative risk (rr) or the odds ratio (or) have to be calculated (as described in chaps. i. , i. , and i. of this handbook). the cdc has developed the software program 'epiinfo' which is easy to use in outbreak investigations, and, even more importantly, free of charge. it can be downloaded from the cdc website (http:||www.cdc.gov|epiinfo|). measures of association, however, should be carefully interpreted; even a highly significant measure of association can not give enough evidence of the real culprit or the contaminated food item. the measure of association is only as good and valid as the data. most people have recall problems when asked what they ate, when they ate and when their symptoms started. even more biases or misclassifications of cases and controls can hide an association. a more confident answer comes usually from the laboratory samples from both human samples and food items served at time of exposure. agents isolated from both food and human samples that are identified as the same subtype, in addition to data results supporting the laboratory findings, are the best evidence beyond reasonable doubt. . as the last step in an outbreak investigation, the epidemiologist writes a final report on the outbreak and communicates the results and recommendations to the public health agency and facilities involved. in the us, public health departments also report foodborne outbreaks electronically to cdc via a secure web based reporting system, the electronic foodborne outbreak reporting system (efors). the "traditional" foodborne outbreak the "traditional" foodborne outbreak is usually a small local event such as family picnic, wedding reception, or other social event and occurs often in a local restaurant or school cafeteria. this type of outbreak is highly local with a high attack rate in the group exposed to the source. because it is immediately apparent to those in the local group such as the group of friends who ate at the restaurant or the students' parents, public health authorities are normally notified early in the outbreak while most of the cases are still symptomatic. epidemiologists can start early on with their investigation and therefore have a much better chance to collect food eaten and stool samples of cases with gastroenteritis for testing and also to detect the etiologic agent in both of them. in a school outbreak in louisiana, eighty-seven persons (sixty-seven students and twenty faculty members) experienced abdominal cramps after eating at the school's annual "turkey day" the day before. stool specimens and the turkey with the gravy were both positive for clostridium perfringens with the same pulse field gel electrophoresis (pfge) pattern (merlos ) . the inspection of the school cafeteria revealed several food handling violations such as storing, cooling and reheating of the food items served. other than illnesses among food handlers, these types of improper food handling or storage are the most common causes of foodborne outbreaks. a different type of outbreak is emerging as the world is getting smaller. in other words persons and food can travel more easily and faster from continent to continent and so do infectious diseases with them. foodborne outbreaks related to imported contaminated food items are normally widespread, involving many states and countries and therefore are frequently identified. in , a large outbreak of cyclospora cayetanensis occurred in us states and ontario, canada and was linked to contaminated raspberries imported from south america. several hundred laboratory confirmed cases were reported, most of them in immunocompetent persons (cdc ) . a very useful molecular tool to identify same isolates from different geographic areas is sub-typing enteric bacteria with pfge. in the us, the pulsenet database allows state health department to compare their isolates with other states and therefore increase the recognition of nationwide outbreaks linked to the same food item (swaminathan et al. ) . in a different scenario, a widely distributed food item with low-level contamination might result in an increase of cases within a large geographic area and therefore might be not get detected on a local level. this kind of outbreak might only be detected by chance if the number of cases increased in one location and the local health department alerts other states to be on the lookout for a certain isolate. another type of outbreak is the introduction of a new pathogen into a new geographic area as it happened in when vibrio cholerae was inadvertently introduced in the waters off the gulf coast of the united states. in the u.s., however, most cases are usually traced back to people who traveled to areas with a high cholera risk or to people who ate food imported from cholera-risk countries and only sporadic vibrio cholerae cases are associated with the consumption of raw or undercooked shellfish from the gulf of mexico (cdc b). food can not only be contaminated by the end of the food handling process i.e. by infected food handlers but also can be contaminated by any event earlier in the chain of food production. in , an ice cream outbreak of salmonella enteritidis in a national brand of ice cream resulted in , illnesses. the outbreak was detected by routine surveillance because of a dramatic increase of salmonella enteritidis in south minnesota. the cause of the outbreak was a basic failure on an industrial scale to separate raw products from cooked products. the ice cream premix was pasteurized and then transported to the ice cream factory in tanker trucks which had been used to haul raw eggs. this resulted in the contamination of the ice cream and subsequent salmonella cases (hennessey et al. ) . surveys are useful to provide information for which there is no data source or no reliable data source. surveys are time consuming and are often seen as a last choice to obtain information. however, too often unreliable information is used because it is easily available. for example, any assessment of the legionella problem using passive case detection will be unreliable due to under-diagnosis and under-reporting. most cases of legionellosis are treated empirically as community acquired pneumonias and are never formally diagnosed. in developing countries, surveys are often necessary to evaluate health problems since data collected routinely (disease surveillance, hospital records, case registers) are often incomplete and of poor quality. in industrialized nations, although many sources of data are available, there are some circumstances where surveys may be necessary. prior to carrying out surveys involving human subjects, special procedures need to be followed. in industrialized countries, a human subject investigation review board has to evaluate the project's value and ethics. in developing countries, however, such boards may not be formalized but it is important to obtain permission from medical, national and local political authorities before proceeding. surveys of human subjects are carried out by mail, telephone, personal interviews, and behavioral observations. in infectious diseases, the collection of biological specimens in humans (i.e. blood for serologic surveys) or the collection of environmental samples (food, water, environmental surfaces) is very common. personal interviews and specimen collection require face to face interaction with the individual surveyed. these are carried out in offices or by house to house surveys. non-respondents are an important problem for infectious disease surveys. those with an infection may be absent from school, may not answer the door or may be unwilling to donate blood for a serologic survey, thus introducing a systematic bias into the survey results. since surveys are expensive, they cannot be easily repeated. all field procedures, questionnaires, biological sample collection methods and laboratory tests should be tested prior to launching the survey itself. feasibility, acceptability and reliability can be tested in a small scale pilot study. more details on survey methods are to be found in chap. i. of this handbook. sampling . . since surveys are labor intensive, they are rarely carried out on an entire population but rather on a sample. to do a correct sampling, it is necessary to have a sampling base (data elements for the entire population) from which to draw the sample. examples of sampling bases are population census, telephone directory (for the phone subscriber population), school roster or a school list. in developing countries such lists are not often available and may have to be prepared before sampling can start. more information on sampling designs can be found in chap. iv. of this handbook. community surveys (house to house surveys) . . most community surveys are carried out in developing countries because reliable data sources are rare. the sampling base often ends up to the physical layout of the population. a trip and geographical reconnaissance of the area are necessary. the most common types of surveys undertaken in developing countries are done at the village level; they are based on maps and a census of the village. in small communities, it is important to obtain the participation of the population. villagers are often wary of government officials counting people and going from door to door. to avoid misinterpretations and rumors, influential people in the community should be told about the survey. their agreement is indispensable and their help is needed to explain the objectives of the survey and particularly its potential benefits. increasing the knowledge about disease, disease prevention and advancing science are abstract notions that are usually poorly understood or valued by villagers who are, in general, very practical people. if a more immediate benefit can be built into the survey, there will be an increase in cooperation of the population. incentives such as offering to diagnose and treat an infection or drugs for the treatment of common ailments such as headaches or malaria enhance the acceptance of the survey. in practically all societies the household is a primary economic and social unit. it can be defined as the smallest social unit of people who have the same residency and maintain a collective organization. the usual method for collecting data is to visit each household and collect samples or administer a questionnaire. medical staff may feel left out or even threatened whenever a medical intervention (such as a survey) is done in their area. a common concern is that people will go to their medical care provider and ask questions about the survey or about specimen collection and results. it is therefore important to involve and inform local medical providers as much as practical. a rare example of a house to house survey in an industrialized nation was carried out in slidell, louisiana for the primary purpose of determining the prevalence of west nile infection in a southern us focus. since the goal was to obtain a random sample of serum from humans living in the focus, the only method was a survey of this type. a cluster sampling design was used to obtain a representative number of households. the area was not stratified because of its homogeneity. census blocks were grouped so that each cluster contained a minimum of households. the probability of including an individual cluster was determined by the proportion of houses selected in that cluster and the number of persons participating given the number of adults in the household. a quota sampling technique was used, with a goal of enlisting participating households in each cluster. inclusion criteria included age (at least years of age) and length of residence (at least years). the household would be included only if an adult household resident was present. a standardized questionnaire was used to interview each participant. information was collected on demographics, any recent febrile illness, knowledge, attitudes, and behaviors to prevent wnv infection and potential exposures to mosquitoes. a serum sample for wnv antibody testing was drawn. in addition, a second questionnaire regarding selected household characteristics and peridomestic mosquito reduction measures was completed. informed consent was obtained from each participant, and all participants were advised that they could receive notification of their blood test results if they wished. institutional review board approvals were obtained. logistics for specimen collection, preservation and transportation to the laboratory were arranged. interpretation of serologic tests and necessary follow up were determined prior to the survey and incorporated in the methods submitted to the ethics committee. sampling weights, consisting of components for block selection, householdwithin-block selection, and individual-within-household participation, were used to estimate population parameters and % confidence intervals (ci). statistical tests were performed incorporating these weights and the stratified cluster sampling design. in this survey, households were surveyed (a % response rate), including participants. there were igm seropositive persons, for a weighted seroprevalence of . % (with a % confidence interval of . %- . %) (vicari et al. ). program evaluation is a systematic way to determine if prevention or intervention programs for the infectious disease of interest are effective and to see how they can be improved. it is beyond the scope of this chapter to explain program evaluation in detail however there is abundant information available i.e. the cdc's framework for program evaluation in public health (cdc a) as well as text books on program evaluation (fink ) . most importantly, evaluators have to understand the program such as the epidemiology of the disease of interest, the program's target population and their risk factors, program activities and resources. they have to identify the main objectives of the control actions and determine the most important steps. indicators define the program attributes and translate general concepts into measurable variables. data are then collected and analyzed so that conclusions and recommendations for the program are evidence based. evaluating an infectious disease control program requires a clear understanding of the microorganism, its mode of transmission, the susceptible population and the risk factors. the following example of evaluation of tuberculosis control shows the need to clearly understand the priorities. most of tuberculosis transmission comes from active pulmonary tuberculosis cases who have positive sputum smear (confirmed as tuberculosis mycobacteria on culture). to a lesser extent, smear negative culture positive pulmonary cases are also transmitting the infection. therefore priority must be given to find sputum positive pulmonary cases. the incidence of smear positive tuberculosis cases is the most important incidence indicator. incidences of active pulmonary cases and of all active cases (pulmonary and extra-pulmonary) are also calculated but are of lesser interest. the following proportions are used to detect anomalies in case finding or case ascertainment: all tuberculosis cases who are pulmonary versus extra-pulmonary, smear positive, culture positive, pulmonary cases versus smear negative, culture positive, pulmonary cases, culture positive, pulmonary cases versus culture negative, pulmonary cases. poor laboratory techniques or low interest in obtaining sputa for smears or cultures may result in underestimating bacteriological confirmed cases. excessive diagnosis of tuberculosis with reliance on chest x-rays on the other hand may overestimate unconfirmed tuberculosis cases. once identified, tuberculosis cases are placed under treatment. treatment of infectious cases is an important preventive measure. treatment efficacy is evaluated by sputum conversion (both on smear and culture) of the active pulmonary cases. after months of an effective regimen, % of active pulmonary cases should have converted their sputum from positive to negative. therefore the rate of sputum conversion at months becomes an important indicator of program effectiveness. this indicator must be calculated for those who are smear positive and with a lesser importance for the other active pulmonary cases. to ensure adequate treatment and prevent the development of acquired resistance, tuberculosis cases are placed under directly observed therapy (dot). this measure is quite labor intensive. priority must therefore be given to those at highest risk of relapse. these are the smear positive culture proven active pulmonary cases. dot on extra-pulmonary cases is much less important from a public health standpoint. the same considerations apply to contact investigation and preventive treatment in countries that can afford a tuberculosis contact program. a recently infected contact is at the highest risk of developing tuberculosis the first year after infection; hence the best preventive return is to identify contacts of infectious cases. those contacts are likely to have been recently infected. systematic screening of large population groups would also identify infected individuals but most would be 'old' infections at lower risk of developing disease. individuals infected with tuberculosis and hiv are at extremely high risk of developing active tuberculosis. therefore the tuberculosis control program should focus on the population at high risk of hiv infection. often, program evaluation is performed by epidemiologists who have not taken the time to understand the dynamics of a disease in the community. rates or proportions are calculated, no priorities are established and precious resources are wasted on activities with little preventive value. for example, attempting to treat all tuberculosis cases, whether pulmonary or not with dot, investigating all contacts regardless of the bacteriologic status of the index case, would be wasteful. today the world is smaller than ever before, international travel and a worldwide food market make us all potentially vulnerable to infectious diseases no matter where we live. new pathogens are emerging such as the sars or spreading through new territories such as wnv. wnv introduced in the us in , became endemic in the us over the next years. hospital-associated and community-associated methicillin resistant staphylococcus aureus (mrsa) and resistant tuberculosis cases and outbreaks are on the rise. public health professionals are concerned that a novel recombinant strain of influenza will cause a new pandemic. but not only the world and the etiologic agents are changing, the world population is changing as well. in industrialized countries, the life expectancy is increasing and the elderly are more likely to acquire a chronic disease, cancer or diabetes in their lifetime. because of underlying conditions or the treatment of these diseases, older populations also have an increased susceptibility for infectious diseases and are more likely to develop life-threatening complications. knowledge in the field of infectious disease epidemiology is expanding. while basic epidemiological methods and principles still apply today, improved laboratory diagnoses and techniques help to confirm cases faster, see how cases are related to each other and therefore can support the prevention of spread of the specific disease. better computers can improve the data analysis and internet allows access to in depth disease specific information. computer connectivity improves disease reporting for surveillance purposes and the epidemiologist can implement faster preventive measures if necessary and is also able to identify disease clusters and outbreaks on a timelier basis. the global threat of bioterrorism adds a new dimension. the intentional release of anthrax spores, and the infection and death of persons who contracted the disease created a scare of contaminated letters in the us population. with all these changes, there is renewed emphasis on infectious disease epidemiology and makes it a challenging field to work in. detection of bordetella pertussis and respiratory syncytial virus in air samples from hospital rooms transmission of tuberculosis in new york city. an analysis by dna fingerprinting and conventional epidemiologic methods report of the committee on infectious diseases in: chin j (ed) control of communicable diseases manual, th edn geographical information system (gis), gimmick or tool for health district management update: international outbreak of restaurant-associated botulism -vancouver epidemiologic notes and reports restaurant associated botulism from mushrooms bottled in-house -vancouver outbreaks of cyclospora cayetanensis infection -united states case definitions for infectious conditions under public health surveillance cdc ( a) framework for program evaluation in public health summary of infections reported to vibrio surveillance system (http:||www.cdc.gov|ncidod|dbmd|diseaseinfo|files|vibcste web.pdf) accessed norowalk-like viruses": public health consequences and outbreak management updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group outbreaks of gastroenteritis associated with noroviruses on cruise ships -united states diagnosis and management of foodborne illnesses: a primer for physicians and other health care professionals epiinfo (http:||www.cdc.gov|epiinfo|) accessed evaluation fundamentals: guiding health programs, research and policy yellow fever. in: cox cr (ed) the wellcome trust illustrated history of tropical diseases a national outbreak of salmonella enteritidis infections from ice cream encephalitis outbreak in louisiana in simple school questionnaires can map both schistosoma mansoni and schistosoma haematobium in the democratic republic of congo molecular and conventional epidemiology of mycobacterium tuberculosis in botswana: a population-based prospective study of pulmonary tuberculosis patients dolin r (eds) ( ) mandell, douglas, and bennett's principles and practice of infectious diseases epidemiology, principles and methods. little, brown and company an uninvited guest at "turkey day trends in hospitalizations associated with gastroenteritis among adults in the united states the next influenza pandemic: lessons from hong kong on the mode of communication of cholera pulsenet: the molecular subtyping network for foodborne bacterial disease surveillance, united states late-breaker report presented at the cdc "annual epidemic intelligence service conference http:||www.who.int|whr | | archives|index.htm) accessed statistical annex. (http:||www.who.int| whr | |archives| |en|pdf|statisticalannex.pdf) accessed cholera, the black one. in: yellow fever black goddess, the coevolution of people and plagues four tales from the new decameron. in: yellow fever black goddess, the coevolution of people and plagues key: cord- -la sum authors: feldblyum, tamara v.; segal, david m. title: seasonal and pandemic influenza surveillance and disease severity date: - - journal: global virology i - identifying and investigating viral diseases doi: . / - - - - _ sha: doc_id: cord_uid: la sum continuous investments in influenza research, surveillance, and prevention efforts are critical to mitigate the consequences of annual influenza epidemics and pandemics. new influenza viruses emerge due to antigenic drift and antigenic shift evading human immune system and causing annual epidemics and pandemics. three pandemics with varying disease severity occurred in the last years. the disease burden and determinants of influenza severity depend on circulating viral strains and individual demographic and clinical factors. surveillance is the most effective strategy for appropriate public health response. active and passive surveillance methods are utilized to monitor influenza epidemics and emergence of novel viruses. meaningful use of electronic health records could be a cost-effective approach to improved influenza surveillance the recent infl uenza pandemic in caused by infl uenza a/h n reassortant with high human-to-human transmissibility, demonstrated the unpredictable nature of emerging viruses and importance of continuous surveillance. during the - infl uenza season, the h n virus infected approximately million persons and caused an estimated , hospitalizations and , deaths in the usa [ ] . this novel virus caused severe morbidity and mortality in pregnant women [ - ] and younger adults with % of deaths occurring in persons younger than years of age [ ] . in addition to the human toll, annual infl uenza epidemics and pandemics carry substantial economic consequences in health-care utilization costs, intervention costs, and reduced productivity. the cost of annual infl uenza epidemics in the usa is estimated to range between $ and $ billion [ ] . individual risk factors for severe outcomes of infl uenza infection vary between seasons and are associated with circulating infl uenza virus types and subtypes, as well as with individual demographic characteristics, such as age, ethnicity, and clinical conditions, such as asthma, diabetes, cardiovascular, lung, and neurological diseases [ - ] . due to variations in infl uenza virus activity, the capacity to respond to seasonal epidemics and pandemics depends on the availability of accurate and timely information and swift and early identifi cation of pandemic and epidemic strains. the us national infl uenza surveillance systems include syndromic, clinical, and virologic monitoring. information on infl uenza-like illness (ili), infl uenza hospitalizations, infl uenza and pneumonia associated mortality, infl uenza-associated pediatric mortality, and laboratory testing of a subset of specimens from patients with ili to characterize the circulating viruses are reported. these surveillance systems are resource-intensive [ , ] and require sustained funding for epidemiologic and virologic information gathering at the national and local levels [ ] . enhanced and timely syndromic surveillance methods that use electronic health records (ehr) could improve the assessment of infl uenza medical and economic disease burden and associated risk factors leading to identifi cation of at risk population groups, targeted and appropriate public health interventions, and estimates of economic burden associated with the disease [ , - ] . ehrs capturing information using the international classifi cation of diseases, ninth revision, clinical modifi cation (icd- -cm) codes lend themselves to effi cient quantitative analyses and have been used in numerous epidemiologic studies and infl uenza surveillance [ , , - ] . with the growing focus of the us health care system on the meaningful use of electronic medical records, one of the practical applications is expanding biosurveillance and preparedness capabilities, such as surveillance of infl uenza severity and associated risk factors during seasonal epidemics and pandemics [ , ] . traditional infl uenza surveillance data are based on laboratory testing of a limited number of samples, case reporting by participating health care providers, hospitalbased primary data, and deaths reported by statistics offi ces [ ] . data extracted from electronic medical records can enrich reporting of risk factors for disease severity or clinical diagnoses, even in the absence of laboratory testing, and augment the traditional surveillance. in addition, monitoring patients ehrs may enable detection of disease outbreaks for which no laboratory diagnostics were requested including emerging pathogens and biothreat events [ ] . the timely reporting of information on circulating infl uenza viruses and the disease burden associated with seasonal and pandemic infl uenza is essential for optimal public health response, identifi cation of vulnerable populations, and for prevention and patient management strategies. large electronic datasets of hospital discharge records, such as the nationwide inpatient sample (nis), could provide information on risk factors for disease enhancing infl uenza surveillance methods [ , ] . the use of much larger more representative national population repositories from existing electronic medical records can potentially augment or replace small hospital case series studies often employed for assessment of infl uenza severity. every year, emerging and reemerging infl uenza viruses lead to tens of millions of respiratory infections and up to , fatalities worldwide. unpredictability of antigenic drift or antigenic shift leading to emergence of viral strains with limited or no immunity in human population results in variable disease spread and severity. a novel high pathogenicity virus adapted to human-to-human transmission could cause a global pandemic with millions of deaths [ ] . timely detection and reporting of disease in specifi c populations through an effective biosurveillance system is the most promising strategy for mitigating the impact from disease outbreaks caused by naturally occurring epidemics or bioterrorism events [ ] . infl uenza virus surveillance informs selection of the annual vaccine strains and guides antiviral therapy. monitoring infl uenza outbreaks is of particular interest because they represent a proxy for research of potential biothreat surveillance systems. early clinical symptoms of many biologic warfare agents such as aerosolized b. anthracis , tularemia, and smallpox resemble infl uenza like illness [ , ] . surveillance of infectious diseases can be conducted using passive or active approaches. active methods based on laboratory testing and case reporting are usually resource intensive and require ongoing reporting by participating physicians, hospitals, and laboratories [ ] . only a subset of specimens can be tested [ ] and cases are often underreported. passive syndromic surveillance methods may be less accurate but they are also less expansive and enable assessment of the disease spread and severity in the population. implementing syndromic surveillance based on signs and symptoms, diagnosis, and large volumes of other health related data for disease of interest can greatly improve the quality and timeliness of passive surveillance [ ] . information acquired integrating both methods can generate a more complete picture of an outbreak or an epidemic [ ] . in the usa, the national infl uenza surveillance is lead by the cdc as a collaborative effort of state and local health departments and laboratories, health-care providers, hospitals, and clinical laboratories. the data on circulating infl uenza viruses and the disease activity including incidence, morbidity, and mortality is collected year round, compiled, and published weekly with a - -week reporting delay [ ] . infl uenza virologic surveillance throughout the usa is conducted by approximately laboratories comprising the who and national respiratory and enteric virus surveillance system (nrevss) laboratory networks. they collect information on the proportion of infl uenza a and b positive respiratory specimens and determine infl uenza a subtypes. a subset of the infl uenza positive samples, especially if the subtypes cannot be determined by standard diagnostic tests, are sent to cdc for further characterization by gene sequencing to monitor emergence of novel viruses and antiviral resistance [ ] . the second component of the surveillance system is the illness surveillance network (ilinet) comprised of approximately , healthcare providers voluntarily reporting all outpatient visits and the number of visits due to infl uenza-like illness (ili) stratifi ed by age group. the percentage of weekly ili visits weighted to refl ect the population size of reporting states are compared to the national baseline of ili visits outside of infl uenza season to monitor ili activity levels in each state [ ] . vital statistics offi ces in participating us cities report the total number of deaths and the number of deaths caused by pneumonia or infl uenza (p&i) stratifi ed by age groups. statistical methods are used to calculate the weekly level of p&i mortality above the seasonal baseline or epidemic threshold. in , pediatric infl uenza-associated mortality for children - years of age became a nationally notifi able condition. infl uenza hospitalization network comprised of hospitals in over counties in states collects information from hospital records and reports on laboratory-confi rmed infl uenza hospitalizations for children and adults. the information on geographic spread of infl uenza activity is augmented by state and territorial health department epidemiologists' reports [ ] . in addition to the cdc surveillance systems, the armed forces operate the global emerging infections surveillance and response system (geis) to protect military personnel and their families [ ] . respiratory infections surveillance is one of the geis programs contributing to the global infl uenza surveillance network. the program leverages established laboratory and research facilities in host countries and collaborations with global partners. its activities are coordinated and information regarding circulating infl uenza viruses, disease burden, and epidemiology is shared with cdc, who, and host countries. the data is also used in research and for development of vaccines and diagnostics [ ] . international infl uenza surveillance is accomplished through the who global infl uenza surveillance network collaborating centers including the cdc. global infl uenza surveillance information is shared through the who flunet tool and it provides advance signals of infl uenza activity and trends, informs selection of annual vaccine strains, and enables member countries to better prepare for upcoming infl uenza season [ ] . in addition to the active surveillance efforts, alternative methods such as syndromic surveillance, electronic patient records from emergency room or ambulatory doctor visits, and hospital discharge records have been used for surveillance of infl uenza and other infectious diseases with growing frequency [ , , , ] . syndromic surveillance provides clues on disease patterns collected from multiple information sources such as emergency department visits, ambulatory health-care visits, calls to health information hotlines, internet health information seeking, and over-the-counter medication purchases. indication of potential disease outbreak from syndromic surveillance is usually available before laboratory test results are reported [ ] . essence, the electronic surveillance system for the early notifi cation of community-based epidemics is an example of syndromic surveillance system implemented by the department of defense (dod) to automatically download data from the electronic health records of military personnel and their families. the system captures information coded in accordance with the international classifi cation of diseases, ninth revision, clinical modifi cation (icd- -cm) standards from over , weekly outpatient visits to us military treatment facilities [ ] . it monitors disease outbreaks based on health care utilization patterns and uses icd- -cm codes to group diagnosis into one of the eight disease syndromes. another national electronic surveillance system, biosense, launched in and operated by the cdc collects and analyzes icd- -cm coded data from outpatient visits to health-care facilities and emergency departments, hospitalized patients, laboratory tests, and information on over-the-counter medications sold in pharmacies [ ] . although different studies reported variable utility of syndromic disease surveillance systems for local disease outbreaks, the majority of them indicated that it was useful for monitoring respiratory disease activity and the annual infl uenza seasons [ , , ] . sensitivity of icd- -cm based detectors of acute respiratory disease and infl uenza epidemics varied from to % for acute respiratory disease to % for infl uenza outbreak [ ] and specifi city ranged between and % [ , ] . sensitivity was found to be moderate and likely not suffi cient to detect a small disease outbreak, e.g., in the event of a local bioterrorism incidence. however, icd- -cm coded data can be useful for infl uenza surveillance when accuracy, completeness, and timeliness are carefully considered [ ] before using such data for decision making. for a comprehensive infl uenza surveillance system, it is critical to include hospitals that would collect epidemiological and virological information on severe cases. this data enables characterization of severe ili, identifi cation of at risk population groups, tracking of genetic changes in the circulating viruses, and serve as a monitoring tool for emerging pandemics [ ] . hospital based case series studies yield valuable information on risk factors for sever infl uenza during an ongoing or past infl uenza seasons. although these studies can inform vaccination and therapy decisions, majority of them have a limited sample size, are recourse intensive, and the results are not generalizable on the national level. the lack of this data became especially apparent during the h n pandemic when the disease incidence rate was very high resulting in declaration of phase pandemic while the disease severity on a national level was not ascertained [ ] . hospital-based electronic surveillance is a cost-effective approach to identify infl uenza season-specifi c populations at high risk for ili complications and fatal outcomes. detailed clinical information on each individual case is coded in patients' records and can be used to augment active surveillance in public health response planning and implementation [ ] . advances in information technologies enabled new global and national surveillance methods and real time information sharing among multiple stakeholders. monitoring indicators other than the traditional information captured by health-care providers can be a cost effective approach to augment respiratory disease surveillance. rise in purchases of over-the-counter cold medications, school absenteeism, internet health information searches, and utilization of health advice phone lines were shown to correlate with increased infl uenza activity [ ] . the rise in health information seeking preceded doctors' visits by about week and was also correlated with media coverage of the health concern [ ] . an approach to infl uenza surveillance monitoring ili health-seeking internet queries was launched by google and cdc during the - infl uenza season. the system analyzed logs of web searches related to ili information and reported data with only day lag instead of the usual delay of - weeks. the accuracy of the ili estimates was - % as compared to the actual disease incidence reported by cdc infl uenza surveillance [ ] . a health map web based data collection system was employed during the h n pandemic to monitor the internet, compile, and report infl uenza activity in geographically diverse locations through an interactive map. data was collected from news media, blogs, and other nontraditional sources as well as from the who, cdc, and the public health agency of canada. the median lag between reported and confi rmed cases ranged from to days with considerable variations between the countries infl uenced by public health infrastructure, political system restricting information, and media coverage. the nontraditional information sources may enable earlier detection of outbreaks and epidemics, expand population coverage, improve sensitivity of emerging diseases detection, and place the epidemic or pandemic in the context of the affected population [ ] . while electronic surveillance based on nonclinical data such as over-the-counter medication sales, school absenteeism, and health information seeking may provide preliminary signs of potential infection spread, prompt release of electronic health records (ehr) containing diagnosis and clinical outcomes can lead to a more informative and timely disease surveillance [ ] . increasing utilization of patient electronic records could play an important role in attaining public health objectives and complimenting other information sources. information from electronic medical records captured through surveillance platforms or stored in local or centralized databases has been used in numerous studies for monitoring disease incidence, prevalence, severity, risk factors, and medical care decisions. analyses of electronic medical records were employed to augment the traditional approaches [ , ] during respiratory seasons in the usa. standard surveillance was not suffi cient during the recent infl uenza h n pandemic when several states, including new york [ ] , wisconsin [ ] , and california [ ] implemented additional information gathering methods based on electronic medical records to gain a more complete understanding of the ongoing pandemic severity. ehr-based surveillance systems such as electronic medical record support for public health (esp) implemented in ohio and massachusetts and biosense were successfully used for analyzing icd- diagnosis codes, reporting notifi able disease cases, surveillance of ili, identifi cation of infl uenza or upper respiratory infection risk factors among hospitalized patients, and for monitoring diabetes prevalence, risk factors, and disease severity [ , ] . the results of infl uenza risk factor analyses based on icd- coded data overall agreed with earlier observations based on primary data collected through the emerging infections program during - infl uenza seasons [ ] and in manitoba, canada during h n pandemic [ ] as well as with laboratory confi rmed infl uenza hospitalizations reported to the cdc during the pandemic [ , , ] demonstrated that optimally selected icd- code groups can be used in an automated surveillance system drawing information from electronic medical records for accurate monitoring of infl uenza activity. in this study of the us air force personnel and their dependents outpatient visits the syndromic surveillance results correlated with the results of sentinel ili surveillance conducted by the cdc. placzek and madoff ( ) used administrative hospital discharge records to estimate the hospitalization rates and characterize patients hospitalized with ili during the seasonal fl u epidemics and the h n pandemic in massachusetts. they evaluated two sets ("maximum" and "minimum") of icd- diagnosis codes for their relevance and accuracy in identifying infl uenza-associated hospitalizations and disease severity and concluded the proposed minimum icd- criteria more accurately refl ected the actual infl uenza cases. icd- coded diagnosis alone or in conjunction with other electronic health data were used in monitoring of ili severity and risk factors [ , , ] , and for modeling early detection of local respiratory disease outbreak [ ] . this approach was adopted for other disease surveillance, such as sars [ ] , diabetes incidence and management [ ] , and pertussis [ ] . the study results suggest that timely ili surveillance is feasible using icd- -cm coded electronic medical records and emphasized the importance of the appropriate icd- -cm code selection for case defi nition for accurate assessment of disease activity and severity [ , ] . current infl uenza surveillance systems are resource intensive and provide limited information on patients at-risk for severe infl uenza. to date, no study has been conducted using a large sample of electronic health records (ehr) to examine the risk factors for infl uenza in hospitalized patients across the usa. larger data sets of ehrs will enable the creation of statistically signifi cant age-specifi c models of infl uenza severity and predict more representative infl uenza risk factors and vulnerable groups. a recent study utilized the nationwide inpatient sample (nis) which is a repository of eight million electronic hospital discharge records from , participating hospitals in over states representing approximately % of all us hospitalizations [ ] . this data source is maintained by the healthcare cost and utilization project (hcup) sponsored by the agency for healthcare research and quality (ahrq). results from the retrospective unmatched case-control study of nis patients hospitalized with infl uenza during the h n pandemic and severe a/h n - epidemic seasons confi rmed the utility of using an existing electronic resource to identify comorbidities and demographic risk factors for severity of clinical outcomes associated with pandemic and epidemic infl uenza viruses [ ] . the use of primary diagnosis icd- -cm codes .xx- .xx to correctly identify infl uenza hospitalizations from nis was verifi ed by comparing the temporal trends of monthly hospitalization counts identifi ed in nis records (table . findings from these studies demonstrate that large datasets of electronic medical records are an essential component of infl uenza epidemic surveillance. integration of icd- diagnosis codes into more complex disease detection algorithms can further improve the sensitivity and specifi city of surveillance systems based on electronic medical records [ ] . however, this approach is limited if electronic records are fragmented between different providers using different disease algorithms whereas the icd- codes even though potentially less specifi c are standardized among all users and may be more applicable to nationwide surveillance [ ] . further standardization of data coding and selection criteria, and interoperability among private and government surveillance efforts has the potential to enhance the electronic data quality and timeliness [ , ] . this methodology can be especially advantageous for public health applications as it uses routinely collected data and requires modest investments for maintenance and operation [ ] . infl uenza virus is a zoonotic pathogen causing annual epidemics and pandemics resulting in human toll and economic losses all over the world. infl uenza-associated morbidity and mortality are especially high among persons with chronic health conditions and usually among the very old or the very young [ ] . although the virus was identifi ed and isolated only years ago, infl uenza disease outbreaks can be traced back to middle ages and identifi ed by signs and symptoms, sudden start of the epidemic, and excess mortality in historical sources dating to [ ] . shope demonstrated in s that the infectious agent causing fl u in humans could adapt to other species and cause similar disease in swine. the infl uenza virus adaptability to the host immune system enables sustained human-to-human transmission and the emergence of novel viral strains [ ] . it also poses a challenge to the public health efforts to predict and control the annual infl uenza epidemics and pandemics. infl uenza viruses belong to the orthomyxoviridae family and are divided into three genera or types, infl uenza virus a, b, and c [ ] . infl uenza a viruses are further classifi ed into subtypes defi ned by one of the hemagglutinin and one of the ten neuraminidase subtypes present in the virus [ ] . infl uenza b viruses are not classifi ed into distinct subtypes but are divided into two genetic lineages, yamagata and victoria [ ] . it is an enveloped single stranded rna virus with a genome fragmented into eight segments encoding proteins. the surface glycoproteins hemagglutinin (ha) and neuraminidase (na) play the most important role in viral infection and transmission. ha attaches the virus to the target cell's sialic acids receptors facilitating the viral rna entry into the cell. the na enzymatic activity cleaves the sialic acid releasing the newly produced viral particles [ , ] . the annual epidemics are caused by infl uenza a and infl uenza b, but only infl uenza a can adapt to multiple hosts and emerge as a novel virus causing pandemics. antigenic drift due to mutations in ha and na genes allows the virus to evade preexisting antibodies in the human immune system conferring the pathogenicity and virulence. antigenic shift occurs when infl uenza viruses containing diverse ha and na subtypes coinfect the same host, triggering a reassortment event and producing progeny with genomic segments from both parental viral subtypes [ ] . wild birds are the natural reservoir for infl uenza viruses. sixteen hemagglutinin and nine neuraminidase infl uenza a subtypes were isolated from aquatic birds and only the most recent ha was isolated from fruit bats [ ] . according to the mixing vessel theory, pigs are considered the main mammalian host where the adaptation of an avian infl uenza viruses to human host and reassortment events occur [ ] . pigs' cell-receptors match both human and avian infl uenza, rendering them susceptible to infection with viruses from both hosts [ ] . infl uenza a viruses have been also isolated from other animals, including a horse, dog, cat, tiger, and leopard [ ] . infl uenza type b and c is rarely found in hosts other than humans, although infl uenza b has been found in seals and infl uenza c has been reported in swine and dogs [ ] . despite the investments in infl uenza research, surveillance, and prevention efforts, infl uenza virus remains a cause of respiratory infection in the usa and in the world. annual infl uenza-associated deaths in the usa range between , and , [ ] and, on the average, , are hospitalized due to severe disease [ ] . the variations in mortality can be attributed to difference in the circulating viral types and subtypes. the average mortality rates are . times higher during the seasons when infl uenza a(h n ) subtype is predominant as compared to seasons when infl uenza b or other infl uenza a subtypes are the predominantly circulating viruses. during a typical infl uenza season, severe illness and death occur most frequently among individuals years and older ( . %) or children younger than years of age [ ] . persons of any age with underlying health conditions are also at a greater risk for severe outcomes associated with infl uenza infections [ ] . infl uenza viruses are transmissible among humans via the respiratory rout. during seasonal epidemics and pandemics, each case transmits the virus at - day interval to . - . and . - . individuals respectively [ ] . the human-to-human transmission occurs in one of the three ways: direct contact with infected persons, touching object contaminated with the virus and then transferring it from hands to mucus surfaces of the nose or eyes, and inhaling virus-containing droplets produced by infected person when coughing or sneezing [ , ] . the effi ciency of infl uenza transmission aerosolized in droplets depends on the size of the droplet, viral concentration, and humidity. yang and marr ( ) demonstrated that the concentration of infectious infl uenza virus in cough droplets is inversely related to the relative humidity (rh) in indoor settings, while the droplet size is directly related to relative humidity. in a dryer environment, the smaller droplets tend to stay in the air longer, infecting larger number of sensitive hosts. in a humid environment, the virus in large droplets settles on objects (fomites) and can survive for several days. viable infl uenza viruses in mucus were detected on paper money bills after h and in some cases up to days [ ] . in temperate climates, infl uenza epidemics occur in a seasonal pattern during the colder months of the year, while in the tropical climates, infl uenza circulates all year round with patterns associated with rainy seasons. multiple reasons for this periodicity such as sunlight, temperature, humidity, human mobility, and contact rates, and functions of the immune system have been explored without arriving at a defi nitive conclusion [ , ] . yang and marr ( ) suggested that the winter seasonality can be partially explained by higher concentration of droplet-suspended infl uenza viruses in heated buildings due to lower humidity. other environmental factors, such as colder temperature and reduced ultraviolet radiation, are also independently associated with virus survival and seasonality. temperature and humidity also effect the human immune system, diminishing the blood fl ow and leukocyte supply in low temperatures while increasing viral shedding [ ] . lowen and palese ( ) confi rmed that cold and dry conditions facilitated viral transmission through aerosolized droplets, while warm or humid environment ( °c, % rh) prevented the viral spread. they proposed that seasonal pattern of infl uenza epidemics in temperate climate occurs due to viral transmission by aerosolized droplets, while year-round infections occur through fomites or direct contacts in tropical climate. the exception to this pattern was the spring outbreak of the swine-origin infl uenza a h n , which possibly could be explained by the increased frequency of transmission via direct contact due to the absence of human immunity to the novel antigenic strain. variations in temperature and humidity did not affect viral spread by direct contact [ ] . infl uenza pandemics are caused by novel viruses for which the world population has no immunity [ , ] . each of the six pandemics in the last years were caused by a different novel infl uenza a virus that has undergone antigenic shift, reassortment of gene segments encoding ha and/or na, and successfully adapted to the human host [ ] . however, of the multiple possible combinations between ha genes and na genes, infl uenza viruses with only three combinations (h n , h n , and h n ) have adapted to enable human-to-human transmission suggesting the presence of inherent limitations in viral ability to adapt [ , ] . of the documented pandemics, the most devastating occurred in - (the spanish infl uenza), causing more than , deaths in the usa and over million deaths in the world [ ] . the avian origin infl uenza a h n virus which caused the pandemic had a case-fatality rate of . %, with the majority of the deaths occurring among otherwise healthy young adults - years of age [ ] . the high mortality appeared to be associated with pneumonia caused by bacterial coinfection [ ] . world war i potentially contributed to the spread and severity of the pandemic. crowded conditions, increased stress, and malnutrition could have weakened the immune system of the troops while increased travel of the armed forces and civilians facilitated the spread of the virus throughout the world [ ] . the sequence data of the infl uenza a h n virus suggest that the virus was not a reassortant but rather all eight viral segments were novel with no prior immunity in the human population. in contrast, the viruses that caused the (h n ) and (h n ) pandemics were direct descendants of the infl uenza and evolved from the existing strains through reassortment events with genes from avian infl uenza viruses [ ] . the h n virus with two surface proteins new to humans caused the asian pandemic, resulting in approximately , deaths in the usa and two million deaths worldwide. the h n "hong kong'" virus was associated with , deaths in the usa and approximately , , excess deaths globally. the disease caused by the pandemic h n was relatively mild and the virus became seasonal and is circulating to date [ , ] . predictions that high pathogenicity avian infl uenza (hpai) h n would be the next pandemic strain were the subject of public health concern. the h n continues to spread, causing disease in poultry and occasional human infections through direct contacts with infected poultry. data pertaining to the h n iav strain adaptation to human host is limited, but it appears that human-to-human transmission has not occurred. meanwhile, a fourth generation swine origin descendant of the virus caused a pandemic in [ , ] . three strains of viruses, derived from birds, pigs, and humans, gave rise to the pandemic virus by antigenic shift, reassortment, and recombination in pigs [ ] . human infections with the novel triple reassortment swine origin virus pdm h n were fi rst detected in mexico and then in california in april of , followed by the declaration of public health emergency in the usa [ ] . due to the fast spread of the virus worldwide, the who declared infl uenza pandemic in june [ ] . despite the high transmissibility, the disease severity was moderate which is not typical of most pandemic strains [ ] . a distinguishing feature of the h n virus, also observed in previous pandemics, was the off-season timing for the start of the pandemic and young age prevalence among infl uenza cases, hospitalizations, and deaths. in mexico in the early stage of the pandemic, % of deaths were reported for patients -to -years-old [ ] . in the northern hemisphere, the majority of deaths, . - . %, occurred among adults - years and only . - . % of deaths were reported in adults older than years [ ] compared to a typical infl uenza season when estimated % of deaths occur in this age group [ ] . among hospitalized patients . % of fatalities occurred among adults - years of age during the pandemic while . % fatalities occurred among patients years and older during the preceding infl uenza season [ ] . underlying medical conditions contributed to disease severity in all age groups. cross-reactive immunity was found more frequently among persons older than years of age due to earlier exposure to infl uenza a/h n strains derived from the pandemic virus [ , ] . the impact of infl uenza epidemics or pandemics on the affected population has been associated with predominantly circulating viral types and subtypes and their relation to the preexisting immunity of the human host [ , ] . infl uenza infections may cause especially severe disease in populations already burdened with a high prevalence of chronic pulmonary conditions [ ] . galiano et al. ( ) suggested that the major determinant of infl uenza disease severity was host-related and included immune response, individual genetic background, and likely environmental factors surrounding human host and the virus. they based their hypothesis on the fact that a complete sequence of the a/fujian/ / -like h n virus isolates from cases that died and those who survived did not reveal any genetic differences that could be associated with disease severity or increased mortality [ ] . because the mechanisms by which viruses evolve and adapt to human hosts remain undetermined and the seasonal infl uenza disease continues to cause substantial public health threat, identifying the most vulnerable population groups in a timely manner remains a critical component of public health response. interventions to prevent or mitigate the impact of epidemics and pandemics include vaccination, antiviral drug therapies, and non-pharmaceutical methods. vaccination is considered the most effective prevention method because it creates herd immunity by protecting not only the vaccinated individual but also precluding the viral transmission to those who did not receive the vaccine. however, effective protection can be achieved only if the vaccine strains antigenically match the circulating viral strains [ ] . antiviral therapy is benefi cial, especially when a new viral strain emerges for which there is no vaccine. novel therapeutic technologies against infl uenza offer great promise such as the use of sirna and ribozymes delivered by intranasal spray or retroviral carriage [ ] . non-pharmaceutical methods include social distancing to reduce crowding and personal interactions and travel restrictions [ ] . infl uenza symptoms range from mild upper respiratory ailment to severe complications resulting in patient hospitalizations and in extreme cases, death [ ] . the symptoms of infl uenza-like-illness (ili) include fever, chills, sore throat, or cough [ , ] . depending on the circulating viral strains, diarrhea or vomiting may also be associated with infl uenza infection, especially in children [ ] . infl uenza may be diffi cult to diagnose based on clinical symptoms alone because the clinical presentation may be similar to other respiratory viral and some bacterial infections [ ] . presence of infl uenza virus can be confi rmed by laboratory testing. the disease severity can be characterized by outcome indicators such as hospitalizations, admissions to intensive care units, length of hospital stay (los), utilization of mechanical ventilators, and fl u-associated mortality [ , , , ] . on the average, the frequency of severe cases requiring hospitalization or resulting in death is higher during the seasons when a(h n ) viruses are predominant [ , ] . during the pandemic, an estimated . % of the pdmh n infl uenza cases required hospitalization and could be characterized as severe; approximately , of the cases or . % died [ ] . in a review of studies characterizing the disease severity in the beginning of the h n pandemic, found a wide range of hospitalization rates ( - . %), icu admission rates ( - . %), and fatality rates ( - . %) among infl uenza cases. the fatality rate was signifi cantly higher ( - . %) among patients admitted to the icu. a prospective study in canadian population measured the outcomes of severe infl uenza a (h n ) cases as mortality, length of stay (los) in an icu, and duration of mechanical ventilation. in this study of critically ill patients, % required mechanical ventilation, the median icu stay was days, and . % died within days [ ] . annual infl uenza vaccination is universally recommended in the usa as the most effective prevention method for children older than months of age and for all adults [ ] . vaccinating in advance % of the us population even with loweffi cacy vaccine in combination with school closure could be a cost-effective approach to reducing the disease burden [ ] . susceptibility to infl uenza and severity of the disease is affected by multiple factors including characteristics of the circulating virus strain, genetics of the host, prior infection history, comorbidities, age, and environmental factors [ , ] . higher proportion of younger adults aged - [ ] were more frequently infected during the h n pandemic than traditionally more vulnerable age group years or older during the seasonal infl uenza epidemics while pediatric mortality and morbidity was of a greater concern during the - season [ , ] . this unpredictability of the virus-host interactions and consequences to population's health underscores the need for continuous timely and informative infl uenza surveillance. multiple studies conducted during different infl uenza seasons demonstrated increased severity of infl uenza when chronic conditions such as asthma, diabetes, neurologic disorders, obesity, and cardiovascular disease are present in children and adults [ , , , ] . underlying health conditions, especially chronic lung and heart disease [ ] were more prevalent among the cases admitted to icu or those who died compared to other hospitalized patients diagnosed with infl uenza [ ] . in an international study of more than , hospitalized patients with laboratory confi rmed h n pdm infl uenza proportion of patients with underlying chronic conditions increased with disease severity and constituted . % of those admitted into icu and . % of those who died [ ] . during the pandemic, mortality was higher among individuals with underlying medical conditions regardless of their age [ ] . the presence of any chronic disease was also associated with infl uenza severity among hospitalized cases in the usa during the pandemic and preceding seasonal epidemics [ , ] . underlying health conditions including hiv, cancer, heart disease, lung and respiratory conditions, diabetes, neuromuscular and neurological disorders, obesity, and pregnancy were reported to be associated with increased risk for infl uenza infection or disease severity. however, results were often controversial or not confi rmed to be statistically signifi cant. slightly more than half of a sample from the nis hospitalization records ( . % in - and % in ) reported at least one underlying health condition assessed (fig. . ) [ ] . for both the h n pandemic and a/h n - epidemic seasons, the proportion of records with comorbidities among severe cases ( . % and . % respectively) and among those who died in the hospital ( and . %) was similar and signifi cantly higher than among the hospitalizations with moderate disease ( . and . % respectively). the hospitalized patients with any comorbidity had greater odds of severe seasonal and pandemic infl uenza (or = . and . respectively) and inpatient death (or = . and . respectively) [ ] . during the h n pandemic, a greater proportion of immunocompromised hiv-positive persons were hospitalized with infl uenza compared to hiv prevalence in general population but the h n pdm-associated disease severity and mortality were not substantially affected. in a us study of hospitalized patients with confi rmed pandemic infl uenza a h n , there was no statistically signifi cant difference between the proportion of immunosuppressed patients among those with pneumonia ( %) compared to patients without pneumonia ( %) [ ] . in low prevalence settings the severity of seasonal infl uenza does not appear to change signifi cantly in adults infected with hiv. however, in high hiv prevalence populations, infl uenza may pose a higher morbidity and mortality risk due to compromised immune functions and the presence of tuberculosis, hepatitis, and other comorbidities [ ] . in south african population with high prevalence of hiv among patients with confi rmed infl uenza a (h n ) infection referred to icu, . % were immunosuppressed due to either hiv or immunosuppressive therapy [ ] . cancer patients receiving chemotherapy or after hematopoietic cell transplant (hct) have suppressed immune functions and are susceptible to infections including seasonal or pandemic infl uenza viruses. infl uenza infection outcomes in hct recipients vary depending on the infl uenza virus type and subtype [ ] . studies comparing seasonal and pandemic infl uenza disease in children and adults undergoing cancer therapy found signifi cant differences in clinical symptoms at presentation and in clinical outcomes [ - ] . although children infected with h n were healthier at presentation and had fewer comorbidities they more frequently had pneumonia, stayed longer in the hospital, were more frequently admitted to icu [ ] , and experienced higher mortality ( % vs. %) due to complications compared to children with seasonal infl uenza infections. males were especially at high risk for developing pneumonia. timely antiviral therapy mitigated the infl uenza disease severity in children and adult recipients of hct [ , ] . chronic heart disease is a known risk factor for severe outcomes among persons with infl uenza-like illness. during the h n pandemic, heart disease was the second most prevalent medical comorbidity present in approximately % of reported deaths among adults and in almost % of fatalities among persons years or older [ ] . heart and lung disease were also frequent comorbidities with diabetes and kidney disease among the infl uenza case fatalities. in a dataset pooled from multiple countries in europe, asia, and america chronic heart disease was present in . % of all hospitalized patients with ph n infection, . % of icu admissions, and . % of deaths [ ] . lung diseases were the most frequently reported chronic conditions for the h n infl uenza case fatalities with the chronic obstructive pulmonary disease (copd) most prevalent in adults and asthma in children [ ] . regardless of asthma severity, its prevalence tends to grow with escalating infl uenza disease severity [ ] . infl uenza virus infection is known to exacerbate asthma and asthma is a known risk factor for infl uenza infection. it was the most frequently reported underlying medical condition in pediatric deaths associated with infl uenza a/ h n [ ] . the impact of asthma may also depend on the circulating infl uenza viruses. in a canadian studies of pediatric population hospitalized with infl uenza, children with pandemic h n infl uenza in were signifi cantly more likely to have asthma ( %) than those with seasonal infl uenza during the - seasons ( %) although there were no difference in severity or clinical presentation of asthma between the pandemic and seasonal pediatric infl uenza cases [ ] . asthma was also more prevalent among the children admitted to icu with ph n and developing ph n -associated pneumonia compared with seasonal infl uenza in - [ ] . patients with chronic lung and airways diseases such as copd are at a greater risk for severe morbidity and mortality associated with infl uenza infection. evidence suggests that bacterial coinfections in copd cases may further impact the disease severity. in a study of patients hospitalized with severe copd in italy, viral infection was detected in . % and viral-bacterial coinfection in % of patients hospitalized with copd exacerbation. infl uenza was one of the most frequently identifi ed infections adversely effecting lung function and extending hospital stay [ ] . although many national guidelines recommend infl uenza vaccination, there is only limited evidence that vaccine is effective in copd patients. however, some observational studies suggest that vaccine reduces both hospitalizations and mortality [ ] . the association between diabetes type and type and a greater risk for infl uenza associated complications may be explained by adverse impact of excessive blood glucose on immunity, as well as heart, kidney, and lung functions [ ] . infl uenza surveillance data in wisconsin and new mexico during the h n pandemic indicated that diabetes was the second most frequent comorbidity following asthma and was present in - % of hospitalized infl uenza cases [ , ] . van kerkhove et al. ( ) reported that diabetes was an underlying chronic condition in % of infl uenza-associated hospitalizations and . % of cases admitted to the icu in a sample representing countries with diverse populations and healthcare systems. diabetes was present in % of infl uenza a/h n associated fatalities in england [ ] , . % fatalities in a large international sample [ ] , and % of fatalities in new mexico [ ] . the higher proportion of diabetes in new mexico potentially could be due to a higher than % obesity among hospitalized patients older than years. diabetes prevalence is on the rise in the usa, especially among the aging population, reaching almost % prevalence among persons years of age or older [ ] . infl uenza surveillance and timely characterization of clinical disease course are important for potential prevention and treatment of diabetic infl uenza cases [ ] . neurological and neuromuscular disorders (nnmd) are risk factors for infl uenza infections possibly due to diffi culty clearing secretions from respiratory tract due to impaired or reduced muscle tone and lung function could lead to severe disease [ ] . persons with nnmd also may have an increased susceptibility to recurrent respiratory infection due to diminished ability to protect airways through cough [ ] and a higher risk (or, . ) of infl uenza-related neurologic complications such as seizures [ ] . in a study of infl uenza-associated pediatric deaths during the - infl uenza season, % of the children had neuromuscular or neurologic disorder [ ] . louie et al. ( ) further confi rmed that neurologic diseases with the potential to compromise respiratory function were present in more than % of severe infl uenza cases among children. nnmd were the most prevalent chronic diseases associated with respiratory failure in hospitalized children with laboratory-confi rmed infl uenza diagnosis followed by chronic lung and chronic heart conditions [ ] . a study of pediatric deaths reported to cdc during the h n pandemic showed that % of case fatalities had neurologic disorders. majority of the children also had additional comorbidities such as heart disease [ ] . adult patients who developed pneumonia as a consequence of infl uenza h n infection were more than twice as likely to have a neurological disease compared to patients who had no complications [ ] . neurological disorders found among patients hospitalized due to infl uenza included down syndrome, cerebral palsy, developmental delay, history of stroke [ ] , seizures, spinal cord injuries [ ] , neuromuscular disorders, hydrocephalus, and epilepsy [ ] . pediatric deaths due to pandemic infl uenza fi ve times exceeded the annual average number of deaths caused by seasonal infl uenza viruses during the fi ve proceeding seasons. neurologic disorders were the most frequent comorbidities found in infl uenza-associated pediatric deaths [ ] underscoring the importance of continues surveillance of disease severity and the need for timely characterization of risk factors during an ongoing infl uenza season. during the h n pandemic, obese individuals with body mass index (bmi) exceeding kg/m were at a higher risk for infl uenza infection; they were more likely to be hospitalized and were disproportionately represented among the patients in icus, those with longer duration of mechanical ventilation, longer hospital stay, and those who died compared with those who were not obese [ , , ] . in a study of california adults the prevalence of obesity and extreme obesity among infl uenza cases was . and . times higher respectively than the us population average. the odds ratio (or) for fatality among the extremely obese (bmi > ) patients was . - . [ ] . these fi ndings corroborated the results of kwong, campitelli, and rosella ( ) suggesting that obese individuals were at a greater risk for hospitalization than persons with normal weight during prepandemic infl uenza seasons with or = . and . , for individuals with bmi - . and ≥ respectively. the association between obesity and infection can be explained by impaired immune response or by strain of infection on respiratory system and reduced mechanical function of lungs and airways. obese persons consume high percentage of oxygen to maintain normal respiratory function; they have increased airway resistance and may suffer from hypoventilation and chronic infl ammation of the respiratory tract altering the immune function and the ability to respond to challenges to respiratory system [ , ] . the role of obesity as an independent risk factor may be diffi cult to ascertain, especially in studies with a limited sample size, as it is often directly correlated with other underlying health conditions (e.g., diabetes and heart disease) known to increase risk for infl uenza infections and severe outcomes [ ] . however, because more than % of adults in the usa [ ] and million worldwide [ ] are obese it may be a major contributor to excess morbidity and mortality associated with infl uenza and warrants further investigation. pregnancy has been reported as a risk factor for seasonal and pandemic infl uenza infections and severe disease outcomes using historical and current data. about % of pregnant women infected with infl uenza developed pneumonia during the and pandemics [ ] . pregnancy was reported to be a risk factor for infection with infl uenza and severe disease outcome during the infl uenza a/ h n pandemic as well. in a review of publications on h n pandemic epidemiology in the northern hemisphere, reported that . - . % of hospitalized cases were pregnant women and they comprised . - . % of icu admissions. compared to nonpregnant women diagnosed with infl uenza, they were seven times more likely to be hospitalized and twice more likely to have fatal outcomes [ ] . in a uk study of a population with an estimated % prevalence of pregnancy, % of patients hospitalized with laboratory confi rmed infl uenza h n were pregnant and the majority of them were in the second or third trimester. the case fatality rate ranged between and % [ ] . the rate of respiratory hospitalizations among pregnant women in nova scotia during non-pandemic infl uenza seasons between and was almost times higher for pregnant women than the year before they became pregnant [ ] . pregnant women with comorbidities such as asthma, anemia, and heart or renal disease were at the greatest risk for infl uenza-associated hospitalization. the fi ndings on infl uenza severity association with pregnancy were not consistent. in several countries as the level of disease severity increased the proportion of pregnant women diminished and the odds ratio for death among hospitalized pregnant women was < [ ] . interestingly, in a study of ili hospitalized patients during the - infl uenza seasons, pregnancy was protective against pneumonia (or . ), possibly due higher likelihood of hospitalizing pregnant women with severe respiratory infection [ ] . this observation was supported by a uk study reporting that maternal outcomes were no more severe that for nonpregnant women of similar age hospitalized for infl uenza [ ] . an increased susceptibility to infl uenza infection and severe disease among pregnant women could be partially explained by changes in immune response due to lower plasma levels of adiponectin regulating macrophage activity [ ] . an additional explanation could be psychosocial changes that may occur during pregnancy such as perceived increased stress, anxiety, and negative mood which also were shown to alter the immune functions and increase the risk for respiratory tract infections [ ] . in addition to clinical comorbidities demographic characteristics and socioeconomic conditions also can increase the risk for infl uenza infections. close human contacts in crowded housing during the infl uenza season, infl uenza vaccine uptake in a community, awareness of infl uenza transmission routs, and following the nonpharmaceutical prevention practices effect infl uenza virus spread and attack rates in population. the risk for infl uenza infection may also vary in individuals from different racial/ethnic backgrounds and age groups. historically, higher attack rates and more severe disease outcomes were observed among minorities since s including during the infl uenza pandemic [ , ] . in an analysis of infl uenza h n cases pooled from countries, van kerkhove et al. ( ) reported that indigenous populations and minority groups were disproportionately represented among hospitalized infl uenza cases and fatalities in canada, australia, and new zealand, while in mexico and thailand minority groups did not carry excess disease burden. in a canadian case-control study of laboratory-confi rmed ph n cases, % were represented by the first nation residents. the odds ratio was . for the first nation individuals being admitted to the icu compared to other ethnic groups even when controlling for socioeconomic status, age, residency settings, comorbidities, and time to treatment [ ] . similar results for infl uenza severity were observed in the usa where the risk for ph n infl uenza hospitalization in new mexico was . times higher among american indians, . times higher for blacks, and . times higher for hispanics compared to non-hispanic whites [ ] . surveillance data from states showed that the rate of mortality attributed to ph n was four times higher among the american indians and alaska natives (ai/an ) and they had the highest rate ( . %) of underlying health conditions than all other ethnic groups [ ] . higher proportion of pediatric hospitalizations among minorities was observed during the pre-pandemic seasons as well, including the - season [ ] and - when infl uenza a/ fujiian was the prevalent circulating virus [ ] . although the reasons for disparities in infl uenza susceptibility and severity among the racial and ethnic populations are not fully identifi ed several explanations have been proposed including socioeconomic status and resulting differences in living conditions, crowding, health behaviors, and access to medical care [ ] . cultural differences may affect utilization of available health care or vaccination uptake. difference in genetic susceptibility and higher prevalence of chronic conditions associated with increased risk for infl uenza disease severity may also impact the attack rates and the disease outcome in ethnic minority communities [ ] . traditionally populations at the extremes of the age spectrum, young children and older adults are the most vulnerable groups during seasonal infl uenza epidemics while pandemics exhibit a characteristic shift towards younger adults in infl uenzarelated deaths [ , , ] . persons younger than years of age accounted for a greater proportion of deaths during all three pandemics in the twentieth century as well as during the h n pandemic when young adults were at an increased risk for morbidity and mortality. age was an independent risk factor for severe disease outcomes and death. in a study of hospitalized infl uenza cases in washington state the odds of icu admission or death were . and . times greater among adults - years and - years of age respectively compared with children younger than years when controlling for other risk factors [ ] . the lower infl uenza incidence rate and mortality among adults over years observed during pandemics could be explained by antigen recycling mechanism, a partial protection due to earlier exposure to a similar virus [ ] . however, if infected, this age group had the highest mortality rate among the hospitalized patients [ ] potentially due to the presence of comorbidities, effect of medications, and bacterial coinfections. explanations for severe disease among young adults included antibody-dependent enhanced infection and strong infl ammatory response in the lungs leading to lung injury and ards [ ] . once infected with a novel infl uenza virus younger persons may retain long-lasting immunity better than older persons [ ] . during the seasonal infl uenza epidemics older adults and young children are usually at a higher risk for severe disease and death. the proportion of infl uenzaattributable deaths during the - infl uenza seasons in canada increased with age from % in - age group to % in persons years and older. the case fatality rate for infl uenza hospitalized patients increased from to % for population - years to years or older respectively and over % of deaths occurred in persons older than years of age [ ] . during the - season when infl uenza a fujian strain was predominantly circulating virus increased morbidity and mortality was observed among children younger than years of age [ , ] while children hospitalized due to severe infl uenza during the h n pandemic were signifi cantly older with a larger proportion older than years of age as compared to pediatric admissions during the pre-pandemic infl uenza seasons [ ] . developing immune system and absence of immunity to circulating viruses in young children and weakened immune response to vaccination among the older adults renders both groups especially susceptible to seasonal infl uenza infection [ , , ] . although the health conditions described in this chapter contribute to infl uenza virus susceptibility and severity of the disease, their prevalence and impact may vary during different infl uenza seasons. during the infl uenza pandemic, only one third of the , hospitalized cases representing countries had an identifi ed chronic clinical comorbidity while approximately two thirds of hospitalized cases and % of fatal cases did not have any identifi ed preexisting disease. for the infl uenza pandemic, the overall difference in demographic and clinical factors between the disease severity groups and moderate disease controls suggests that age, sex, race, and all clinical conditions of interest showed overall statistically signifi cant association with infl uenza severity. however, pregnancy was not associated with infl uenza severity for women of childbearing age [ ] . the differences of risk factors and clinical outcomes in different countries further highlighted the need for country-specifi c and global surveillance as well as data sharing internationally [ ] . timely information on circulating infl uenza viruses and the disease burden associated with seasonal and pandemic infl uenza is essential for optimal public health response, identifi cation of vulnerable populations, and for prevention and patient management strategies. susceptibility to infl uenza and severity of the disease is affected by multiple factors including characteristics of the circulating virus strain, genetics of the host, prior infection history, comorbidities, age, and environmental factors. the unpredictability of the virus-host interactions and consequences to population's health underscores the need for continuous timely and informative infl uenza surveillance. clinical surveillance is critical for identifi cation of at risk population groups which also may change depending on the circulating virus as well as for monitoring the disease spread in the population and severity. syndromic surveillance based on nonclinical indicators may contribute to a signal of epidemic spread and increase of cases. to better predict viral strains for effective vaccines and monitor novel emerging viral strains 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infl uenza exposure on rates of hospital admissions and physician visits because of respiratory illness among pregnant women pneumonia in us hospitalized patients with infl uenza-like illness: biosense psychosocial factors and susceptibility to or outcome of acute respiratory tract infections racial and ethnic disparities in hospitalizations and deaths associated with pandemic infl uenza a (h n ) virus infections in the united states deaths related to pandemic infl uenza a (h n ) among american indian/alaska natives- states population-based surveillance for hospitalizations associated with respiratory syncytial virus, infl uenza virus, and parainfl uenza viruses among young children pandemic versus epidemic infl uenza mortality: a pattern of changing age distribution lone simonsen, president of sage analytica, inc., graciously donated the nis dataset used in our research study for which we are currently preparing a manuscript of our detailed results [ ] . key: cord- -repm vw authors: ungchusak, kumnuan; heymann, david; pollack, marjorie title: public health surveillance: a vital alert and response function date: - - journal: the palgrave handbook of global health data methods for policy and practice doi: . / - - - - _ sha: doc_id: cord_uid: repm vw ungchusak, heymann and pollack address the critical global issue of public health surveillance. they describe how epidemiologists collect and use surveillance data to detect unusual events or outbreaks and to guide control programmes. drawing on their combined international experience, the authors explain the vital role that data play in alerting authorities to respond to outbreaks such as severe acute respiratory syndrome, ebola, zika virus and avian influenza. they point to the importance of sharing information globally while ensuring equal benefits to providers of data, coordinating surveillance activities across sectors, building capacity for surveillance and coordinating national surveillance activities. the authors emphasise the need for enhanced global cooperation to prepare for future public health emergencies of international concern. a three-month delay in identifying the outbreak of ebola virus in rural guinea in late resulted in its rapid spread to urban areas and to neighbouring liberia and sierra leone [ ] . once local and international responders identified the virus, they took a year to interrupt its widespread transmission. by april , ebola had accounted for more than , cases and over , deaths. people around the world watched with increasing alarm, as this tragic course of events played out, and with concern that air travel could enable the virus to spread across continents. this epidemic highlighted not only the inadequacy of local health systems to recognise and respond but also that international organisations were not ready to provide timely expertise and resources to control the situation and ameliorate the virus's spread through the region. had health officials identified ebola in west africa promptly, they could have minimised its impact on the lives and livelihoods of the populations of west africa by implementing appropriate control procedures. public health officials coined the term surveillance to describe systems they set up to watch out for and control occurrence of health threats. just as police, for example, set up closed-circuit television devices and community watch programmes to detect and prevent crime, public health surveillance systems engage all possible means to detect unwanted health events and prevent them from escalating and damaging population health. while public health surveillance originated to control spread of infectious diseases such as plague and cholera, it has evolved to include some non-communicable diseases, occupational health and injuries as well as surveillance of biological, behavioural and social determinants of these conditions. we start by reviewing the public health need for surveillance and the development by the international community of regulations to control infectious diseases and other public health emergencies of international concern (pheic). we describe how epidemiologists use surveillance data to detect unusual events or outbreaks and to guide control programmes, and we provide guidance about maintaining data quality. we examine networks that contribute to global surveillance systems and highlight the role of social media and information technology in providing data to monitor new events of international importance. we consider challenges facing epidemiologists responsible for surveillance and describe efforts to address them. public health surveillance is vital to the functioning of national and global health systems. policymakers and health administrators need surveillance information to set priorities to address population health problems, allocate resources and monitor progress of prevention and control programmes; they need surveillance systems to alert them immediately of public health threats. emerging infectious diseases, such as avian influenza of different subtypes, severe acute respiratory syndrome (sars) coronavirus, pandemic influenza h n and the zika virus (zikv) have the potential to spread rapidly causing severe loss of life and to impact socio-economic activity, especially trade and travel [ ] . the outbreak of sars in november highlighted the importance of every country having functioning and connected surveillance systems (see box . ). surveillance requires high-level government support, well-trained health workers, strong health information systems, well-functioning laboratories, effective communication systems and operational health facilities. to be effective, surveillance systems also require a strong legal framework to ensure that individual data can be shared while maintaining confidentiality as far as possible. global cooperation between countries, with up-to-date international health agreements to build and maintain these capacities, is essential to decrease risk of international spread of infectious diseases and contain the risk of bio-terrorism. sars originated in wildlife and spread silently among humans as atypical pneumonia in guangdong province, china, two months before officials became aware of it. authorities began surveillance to identify atypical pneumonia cases but this, and the containment response, were too late to stop sars spreading. a chinese urologist who was infected travelled to hong kong and spread sars to another persons. within weeks, sars spread to countries with more than , reported cases ( fig. . ) [ ] . by the end of the epidemic in july , sars had killed people [ ] . although unable to contain the outbreak of sars, the international community was able to bring the epidemic under control within six months-by collaborating across countries to identify and isolate all probable cases. nevertheless, the asian development bank estimated that the economic loss due to sars in affected countries was up to us $ billion with us$ . billion on mainland china (approximately . per cent of its annual gross domestic product (gdp)) and us$ . billion in hong kong (approximately per cent of its annual gdp) [ ] . plague ravaged europe during the fourteenth century and although authorities had no cure, they realised it was important to swiftly identify and isolate cases to prevent and control this lethal condition. authorities understood that international spread of such diseases followed cross-border trade, pilgrimage and war; and so prevention of disease was a national security issue. in the city-state of venice, authorities instigated quarantine measures-keeping arriving ships in the harbour for days before docking, and holding people in isolation for days at land borders to prevent entry of plague [ ] . in the mid-nineteenth century, recognising that quarantine measures were not enough, governments agreed international conventions aimed at stopping spread of plague and cholera-and two other infectious diseases, yellow fever and smallpox. the conventions required each country to report outbreaks of these diseases to all signatories of the convention, and permitted application of certain public health measures at international borders once a country reported of one of the diseases. in the early twentieth century, governments in the americas and in europe set up regional conventions called international sanitary bureaus. in , the newly formed world health organization (who) led establishment of the international sanitary regulations (isr) to foster global cooperation in reporting and acting at international borders to guard against spread of cholera, plague, yellow fever and smallpox. in , the who replaced the isr with the international health regulations (ihr) which required countries to report any cases of cholera, plague, yellow fever and smallpox to who [ ] . if a country reported one of these diseases, other countries could apply pre-established control measures at international borders-such as a requirement of proof of vaccination against yellow fever of any passenger arriving from a country that reported yellow fever to who. some countries reported to who late, or not at all, because of lack of capacity for public health surveillance, or because of fear of stigmatisation and economic repercussions. after hiv spread across international borders before being identified in , the international community realised that infectious diseases could not be stopped at borders. diseases often cross borders while still being incubated in humans, or in non-human hosts-insects, animals, and food and agricultural goods. in , after the sars outbreak, who updated and revised the ihr as a legal framework to include more diseases, and developed real-time evidence-based recommendations for prevention and control of outbreaks. who evaluates each newly identified outbreak for its potential to become a pheic by the country in which it is occurring. the ihr mandate who member countries to report immediately the occurrence of a single case of four diseases (smallpox, poliomyelitis due to wild type poliovirus, human influenza caused by a new subtype, and sars) [ ] . even though the world eradicated smallpox in , the ihr still maintain it on the list to cover the risk of the virus escaping from a laboratory. each country has an additional list of diseases that it requires its health workers to report by law. diseases of greatest public health threat are reportable, meaning that health workers or laboratory technicians must report individual cases as they occur. reportable diseases include those required by ihr and, for example, anthrax, cholera, ebola, legionellosis, plague and the zikv. other conditions are notifiable, meaning that health workers should report the number of cases that have occurred in a given time period. the number, frequency of reporting and breakdown of reportable and notifiable diseases varies by country. diarrheal cases, influenza cases, tuberculosis, aids and other significant endemic diseases are usually required to be notified to local health authorities. in some countries the notifiable list can include non-infectious conditions such as maternal or infant deaths. the ihr require countries to develop core capacities in public health, including surveillance systems and epidemiology services, that can analyse and act on surveillance information to detect and respond to diseases where and when they occur so that their potential to spread internationally is decreased. the purpose of surveillance activities is to: ( ) detect at an early stage, acute public health threats from all hazards-biological, chemical, radiation, natural disaster and deliberate acts-which require rapid investigation and response; and ( ) guide control programmes by measuring disease burden, monitoring trends, describing disease distribution and evaluating public health programme effectiveness (see table . ). the structure of government responsibilities for public health surveillance varies across countries. most often, countries set up dedicated early warning and rapid response surveillance teams that work with or complement surveillance activities of vertical control programmes such as malaria, hiv/aids or tuberculosis. surveillance and response teams detect early stage public health threats while control programmes gather disease (or condition) specific information to plan activities. control programmes share information with surveillance teams as required. a national network of public health laboratories, often linked to international reference laboratories, confirms etiologic agents, genetic strains and antibiotic resistance patterns. surveillance activities are said to be active when health workers pro-actively seek out cases and passive when the system relies on patients to report themselves to a clinic. using standard case definitions, health workers report individual cases of reportable and notifiable diseases to the local or national surveillance centre where staff aggregates reports, and clean and analyse the data. in cleaning the data, staff look for coding and classification errors, and for duplicate reports. epidemiologists analyse the data to determine how many new cases have occurred during the past day or week and their distribution in time, place and by person to see whether the magnitude and pattern of the disease under surveillance is changing. they note any changes in frequency, clustering or distribution and flag them for verification and explanation. box . illustrates how careful data analysis led to malaysia identifying nipah virus in [ ] . reporting of specific information about cases or patients or behaviour of populations under surveillance produces indicator-based data, that is individual or aggregated data derived from patients diagnosed-by syndrome description, clinical or laboratory confirmation-and identified through routine collection or active case search. the surveillance unit will also use eventbased data about outbreaks, unusual events or changes in human exposure [ ] . rather than wait for official reports, the surveillance team gathers information and rumours through the media, internet and unusual events reported by the community, and investigates these reports. the team captures abnormal health events in real-time and confirms potential outbreaks by triangulating these data with indicator-based data. epidemiologists responsible for surveillance use standard epidemiological methods to analyse trends, identify clusters and investigate suspected risk factors (see chap. for an overview of epidemiological methods). for example, high numbers of reported cases of kaposi sarcoma among young men in new york and california during the early s led to an investigation which showed a japanese encephalitis commonly occurs in school-age children of both sexes. there is a seasonal pattern of disease related to the rainy season when transmission and therefore disease occurrence, increases; there is no difference in occurrence between ethnic or religious groups. from september to april , surveillance teams sent reports of cases of febrile encephalitis ( per cent fatal) to the malaysian ministry of health [ ] . initially, the ministry considered japanese encephalitis virus to be the probable etiologic agent for this outbreak, and instituted conventional interventions of vaccination and insecticide to control mosquitoes. when they examined the surveillance data closely, the epidemiological pattern of encephalitis cases was different to what they expected-the disease occurred mostly among male adults of chinese ethnic origin whose occupations related to pig farming. the ministry sought a different cause and found the etiologic agent to be a new paramyxovirus, later named nipah virus. common risk factor of homosexual behaviour and its relationship with hiv/ aids [ ] . using increasingly sophisticated technologies for data capture and analysis, surveillance teams can monitor real-time occurrence, in time and place, of unusual events such as cholera or legionella, or seasonal outbreaks such as malaria (see chap. for an introduction to spatial and spatio-temporal techniques and to chap. which discusses predicting climate-related health outcomes such as malaria). once epidemiologists have concluded their analyses (sometimes in realtime), they prepare reports which can trigger immediate action by a rapid response team to visit the site of the events, investigate the situation and contain the outbreak. the team also sends reports to clinicians in hospitals and to local and national programme managers. many countries publish weekly disease surveillance reports that are also available to the general public: for example, the us centers for disease control and prevention (cdc) publish the morbidity and mortality weekly report (mmwr) [ ] , the european centre for disease control (ecdc) publishes eurosurveillance [ ] , and the who publishes the weekly epidemiological record [ ] . box . shows how epidemiologists associated microcephaly with zikv which led who to declare zikv a pheic [ ] . public health surveillance guides control programmes by undertaking the following functions: in late , zikv spread rapidly through latin america especially in brazil and el salvador. surveillance of birth defects in brazil identified a major increase in microcephaly during the period when zikv transmission increased. this alerted policymakers and epidemiologists to study whether the increase in birth defects was associated with zikv infection during pregnancy. who declared the suspected increase in microcephaly in association with zikv infection of pregnant women a pheic and recommended pregnant women to protect themselves from mosquito bites and to avoid travel to areas with known zikv transmission. the observation that men who travelled to areas with known zikv transmission could sexually transmit zikv to their partners led who to recommend practising safer sex or abstinence for a period of six months for men and women returning from areas of active transmission. its epidemiological patterns disease in humans results from interactions between the human host and causative agents or hazards of all types. the natural and socio-economic environment influences these interactions. diseases usually occur in the same pattern when there is no change in the causative agent (such as mutation), in the human host (such as vaccination) or in the environment (such as climate change). a surveillance system can closely monitor any changes in these dynamic factors and their consequences, as illustrated by the case of nipah virus in malaysia (box . ). public health surveillance must also address risk. for example, surveillance of annual per capita cigarette consumption in the us showed an increased trend from cigarettes in to , cigarettes in . researchers related this trend to advertising and an expansion in the number of cigarette companies. in , after the first studies suggesting cigarette consumption was related to lung cancer, and the us surgeon general issued a warning, the annual per capita consumption decreased to , [ ] . with surveillance information, epidemiologists can forecast an increase in lung cancer without intervention thereby providing evidence for policy to implement effective interventions such as taxation to prevent smoking. evaluating performance of control programmes after they have implemented interventions, health authorities use surveillance data to see if disease incidence declines. for example, when vaccine coverage increases, the number of cases of vaccine preventable diseases is expected to decrease. increasing taxes on cigarettes is one way to reduce consumption. surveillance data can document a correlation between increasing taxes and decreasing trends in cigarette consumption. to achieve these functions, programme managers collect data through patient records, surveys, programme records or informal sources. types of data include determinants of the condition, behaviours or risk factors associated with the condition, morbidity and mortality associated with the condition, programme responses, and abnormal or unusual events associated with the condition. table . provides examples of these types of data for surveillance of an hiv/aids control programme. to ensure surveillance programmes have adequate resources and produce useful information, public health authorities regularly review their surveillance activities. in , the us cdc issued guidelines to evaluate surveillance systems which, with some updating, are still widely used [ ] . these guidelines focus evaluation of public health surveillance on three areas: ( ) the surveillance system itself, describing the system, its structure, diseases under surveillance, sources of data, and how data are processed, analysed and disseminated; ( ) the resources used to operate the system, including funding sources, adequately trained staff and information technology; and ( ) the usefulness and quality of surveillance information, using the following indicators: usefulness of data do the data and information disseminated to data providers and users contain comprehensible facts and findings and useful recommendations to improve control measures and guide programme management? has the system detected outbreaks? how many of the detected outbreaks were investigated and controlled in a timely manner? timeliness of data and other information is data dissemination timely and regular? for example, epidemic prone diseases require weekly summary, while other diseases require only monthly or quarterly summaries. are these requirements met? validity and completeness of data much of the data come from clinical diagnoses that do not have laboratory confirmation. it is useful to conduct studies to determine the accuracy of diagnoses using standard laboratory confirmation testing. this helps in preparing estimates of the proportion of confirmed cases among all reported cases. when undertaking field investigations, investigators can compare the number of actual cases they find with the number of cases reported through the system. this provides an estimate of reporting completeness of the system. global public health surveillance is the collection, analysis and use of standardised information about health threats or their risk factors from more than one country, and usually worldwide. while surveillance mainly focuses on infectious diseases, global systems also seek to identify deliberate use of biological agents or toxins to cause harm. who leads the global public health surveillance system, gathering information from formal and informal sources working through its country and regional offices. who extends its reach through the global outbreak and response network (goarn) [ ] which comprises over national technical institutions that support who to detect public health threats and respond to outbreaks. who uses the information for risk assessment and analysis as part of its routine disease control and prevention programme activities. when requested by countries for support, who works with goarn institutions to recruit suitable experts. goarn includes regional networks of countries that cooperate independently to prevent and control infectious diseases occurring in their regions, for example, the east african integrated disease surveillance network (eaidsnet), [ ] and the mekong basin disease surveillance network (mbds) [ ] . who leads global networks that work to control specific diseases. these networks depend on cooperation of governments, public health workers and scientists to report cases, provide specimens and share information so that specific diseases can be controlled globally. these include: networks to support influenza control through vaccine development the global influenza surveillance and response system (gisrs) consists of national sentinel centres and national and regional laboratories which annually collect , - , nasal swabs from patients presenting with influenza-like illness. their analyses provide information about the distribution of strains circulating each year and enable scientists to recommend the influenza vaccine composition for the following year based on predominant sequences. gisrs also uses flunet, a public web-based data collection and reporting tool that tracks movement of influenza viruses globally and provides epidemiological data about influenza outbreaks [ ] . initiative. clinical health workers and epidemiologists report all cases of acute flaccid paralysis (afp) in children under years of age from whom they have collected stool specimens for isolation and identification of the poliovirus. through its network of national, regional and specialised laboratories, gpln determines whether polio was the cause of the afp, genetically sequences viruses and compares them to a global database to understand their geographic source. if a polio virus is found, gpln informs the national authority and who regional office for appropriate action. project on anti-tuberculosis drug resistance surveillance [ ] is a common surveillance platform to which countries can provide data that are then used to monitor the evolution and spread of multi-drug resistant tuberculosis (mdr-tb) and extensively drug-resistant tuberculosis (xdr-tb). national laboratories provide susceptibility testing of tuberculosis organisms collected from patients, supported by a supranational tuberculosis reference laboratory network. the global project provides understanding of the prevalence and distribution of tuberculosis resistance worldwide. [ ] . its goal is to develop a standardised strategy to collect, analyse and share clinical, laboratory and epidemiological data globally, assess the burden and support local, national and global strategies to control amr. until recently, surveillance systems depended on paper-based reporting, compilation and analysis of data. computers and electronic reporting have made compilation and analysis of data much easier, and the world wide web (www) and the internet improve the comprehensiveness of reporting. digital and internet-based technology can retrieve information from medical records on a daily basis-but this must be done without infringing personal privacy. hospitals, especially private ones, may refuse to provide patient information to the public health sector unless privacy issues are addressed. cell phone technology has extended the scope of informal and event-based surveillance while social media has transformed exploring rumours of new events. some ground-breaking examples of the use of information and communication technology include: electronic reporting of events the programme for monitoring emerging diseases (promed-mail) is a fully moderated internet-based listserv, that receives and publishes reports of public health events in humans, animals, wildlife and plants from its subscribers and other traditional and nontraditional information sources [ ] . promed-mail uses information available on the www and from voluntary listserv reporters who actively search for and report public health events in realtime from the media, internet blogs and other sites. promed-mail editors and expert moderators review, analyse, evaluate and where possible validate reports, and then disseminate them to listserv members and post them on its website. using big data to identify events the subscription-based application global public health intelligence network (gphin) continuously scans the www gathering information from multiple source news aggregators in real-time [ ] . gphin searches in nine languages for key words that could indicate infectious disease outbreaks, or environmental, radioactive and natural disasters. analysts identify new events and inform subscribers-who are governmental and non-governmental agencies with an established public health mandate. every hours, analysts communicate new information to who which validates reports through its network of regional and country offices. who discusses events that it validates in confidence with health departments in the countries involved. mapping events in real-time healthmap, a fully automated application, utilises online informal sources for disease outbreak monitoring and real-time surveillance of emerging public health threats [ ] . healthmap trawls www sources of information (in nine languages) including online eyewitness reports, expert-curated discussions such as promed-mail, validated official reports, for example from who, or the food and agriculture organization of the united nations, and news aggregation services such as google news. using open source software, healthmap displays the events by time, geographic location and aetiology. participatory flu tracking diseases and abnormal events happen all the time in the community. only some patients, especially those presenting with severe disease manifestations, seek medical care. flu near you invites anyone living north america, over years of age, to report if they have an influenza-like illness [ ] . once registered, participants are asked weekly by e-mail to complete a brief survey that seeks information on ten symptoms linked to influ-enza, and other information such as whether or not the registered participant has had an influenza vaccination. other countries, including the uk, have adopted similar participatory influenza surveillance systems, thereby adding a greater understanding of the epidemiology of influenza around the world. participatory onehealth disease detection (podd) chang mai university in thailand, with support from the skool foundation, developed this mobile application which connects volunteers in local governments. when volunteers notice an abnormal event such as poultry dying off or sickness in animals or humans, they use podd to notify local authorities who dispatch a surveillance and rapid response team to investigate and contain the event. after months of implementation, podd has enabled the detection of , abnormal events, including chicken high-mortality outbreaks, four cattle disease outbreaks, three pig disease outbreaks and three fish disease outbreaks, all of which were detected and controlled [ ] . since revision of the ihr in , outbreaks due to infections, including the middle east respiratory syndrome coronavirus and ebola virus, have highlighted continued weaknesses in public health surveillance and response capacities in most countries, with international spread causing disruptions in trade and travel, and negatively impacting economies. we present some challenges and suggest some solutions. most countries have established disease control programmes each with a surveillance component reporting from grassroots through provincial and national levels. national surveillance units may have sufficient staff for each disease control programme, but at lower levels of the health system, the same individuals often manage more than one programme and are heavily burdened by reporting requirements. there is also duplication of effort in reporting between programmes. who supports countries to coordinate surveillance activities across departments, programmes and administrative levels through integrated disease surveillance and response (idsr) [ ] . idsr links surveillance with other health information activities and strengthens overall capacity of countries to maintain public health surveillance. the ihr obligates countries to develop comprehensive disease surveillance, detection and response when and where infectious diseases and other acute public health threats occur. in reality, national surveillance capacity in many countries is still not at expected and necessary levels. this may be, as the ebola epidemic demonstrated in west africa, that health systems are weak and under-funded, or that the surveillance system itself does not function efficiently. regular evaluation of the system, as we describe in sect. . , can identify which components need to be strengthened. an over-riding issue is for the system to deploy and maintain enough professionals throughout the system with the required skills-understanding the nature and limitations of the data they are working with and able to interpret and draw important findings from the analyses of the surveillance data. approximately per cent of newly identified human diseases are zoonotic in origin [ ] and per cent of these diseases have their origins in wildlife [ ] . since the outbreak of h n avian influenza in hong kong, animal surveillance and human surveillance units have begun to share information and alert each other of unusual events. environmental factors are also crucial to disease occurrence, for example, paralytic shellfish poisoning among people who consume shellfish affected by harmful algae growth in the sea [ ] . the one health approach involves sharing information between multiple health sectors and working together to identify and resolve outbreaks [ ] . during the avian influenza outbreak, who requested all affected countries to share the virus isolated from humans for further study and vaccine development. some governments expressed concern about potential negative economic consequences of sharing information and about possible inequities in the benefits of sharing. this led to the jakarta declaration on responsible practices for sharing avian influenza viruses and resulting benefits [ ] . this declaration underlined need for continued open, timely and equitable sharing of information, data and biological specimens related to influenza; it also emphasised need for more equitable sharing of benefits for example in the generation of diagnostics, drugs and vaccines. the jakarta declaration led to the pandemic influenza preparedness framework (pip) under which manufacturers of influenza vaccines, diagnostics and pharmaceuticals that use gisrs information make annual financial contributions to who. who uses approximately per cent of these contributions for pandemic preparedness activities and surveillance, and per cent for pandemic response including purchase of vaccines and antivirals at the time of a pandemic for countries without access to these supplies. in may , the chatham house centre on global health security, after a series of roundtable consultation with experts in public health surveillance, produced a guide on strengthening data sharing for public health surveillance. this guide facilitates both informal and formal data sharing. the guide proposes seven principles: building trust; articulating the value; planning; using quality data; understanding the legal context; coming to agreement; and evaluating. the guidelines help create the right environment for data sharing and to facilitate good practice in addressing technical, political, ethical, economic and legal concerns that may arise. the guidelines aim to ensure, to the greatest extent possible, that any benefits arising from use of the data are shared equitably [ ] . similar to clinical or public health practice, institutions or agencies responsible for public health surveillance need a set of ethical principles to guide their operations. the who guidelines on ethical issues in public health surveillance proposed guidelines [ ] . these guidelines fall into three major groups: first, the mandate and broad responsibility of the agency to undertake surveillance and subject it to ethical scrutiny; second, the obligation to ensure appropriate protection and rights of individuals under surveillance; and third, considerations in making decisions about how to communicate and share surveillance data to pursue common good and equity of population without harm to individual. the west african ebola outbreak provided a costly lesson that policymakers must commit to establishing, maintaining and advancing public health surveillance systems to protect and promote population health. to prepare for the next major outbreak, the world needs to invest in a strong warning and response system led by a global institution with sufficient authority and funding to react swiftly [ ] . who serves this role but is chronically underfunded. similar investment is needed in countries where a fully supported, well-functioning surveillance office or programme must coordinate different components of the surveillance system. surveillance information should be disseminated widely to alert the public and health programmes of outbreaks so that they can contain the disease at source before it spreads internationally. because the world urgently needs reliable and timely surveillance information, public health surveillance should continue to make innovative use of new technology to gather and share information strategically and fairly. • the ebola outbreak highlighted inadequacies of national and global surveillance systems to detect and respond to public health threats. • surveillance provides critical data and information to guide, improve and protect public health. • more trained staff are needed for effective and efficient surveillance especially in low-and middle-income countries. • innovative use of information technology and social media can aid detection of public health threats. one year into the ebola epidemic: a deadly, tenacious and unforgiving virus: world health organization emerging infectious diseases in . years after the institute of medicine report. mbio sars molecular epidemiology: a chinese fairy tale of controlling an emerging zoonotic disease in the genomics era the legacies of sars -international preparedness and readiness to respond to future threats in the western pacific region. western pacific surveillance and response journal sars economic impacts and implications. economic and research department policy brief no . asian development bank lessons from the history of quarantine, from plague to influenza a. emerging infectious diseases world health organization. the international health regulations world health organization early detection, assessment and response to acute public health events: implementation of early warning and response with a focus on event-based surveillance: interim version who/hse/gcr/lyo/ . world health organization world health organization. who director-general summarizes the outcome of the emergency committee regarding clusters of microcephaly and guillain-barré syndrome achievements in public health, - : tobacco use-united states updated guidelines for evaluating public health surveillance systems the global outbreak alert and response network. global public health east african integrated disease surveillance network mekong basin disease surveillance network global polio laboratory network world health organization. surveillance of drug resistance in tuberculosis global anti-microbial resistance surveillance system (glass) epidemic intelligence -systematic event detection podd: an innovative one health surveillance system preventing pandemics with animal origins training programs in epidemiology and public health interventions network risk factors for human disease emergence global trends in emerging infectious diseases woods hole oceanographic institution. harmful algae one health initiative. one health initiative will unite human and veterinary medicine jakarta declaration on virus sharing: a strategic step to more equitable and affordable avian flu vaccines distribution a guide to sharing the data and benefits of public health surveillance. london (uk): the royal institute of international affairs chatham house world health organization. who guidelines on ethical issues in public health surveillance the next epidemic-lessons from ebola key: cord- - iwgebvp authors: siegel, frederic r. title: disease protection in sea coast (and inland) cities: problems in dense populations with shantytowns/slums date: - - journal: adaptations of coastal cities to global warming, sea level rise, climate change and endemic hazards doi: . / - - - - _ sha: doc_id: cord_uid: iwgebvp as discussed earlier in this book, there are sea coast cities worldwide that are at risk from floods, storm surges, and extreme weather conditions such as wind-driven high category hurricanes (typhoons, monsoons), or drought and heat waves, plus along pacific ocean coasts, earthquakes and tsunamis. a municipality and its public health services have to be prepared to adapt to their preparations to deal with what contemporary experiences and history reveal are the most likely physical hazards and diseases to impact it. the two main preparatives are first to be able to care for the injured during a hazard event at well-staffed and supplied hospitals and medical clinics or field hospitals. the second is to fill the basic needs of affected populations with clean water, food, shelter, toilets, waste collection, and if necessary power restoration. these primary responses will help to reduce the chance of an onset and spread of disease. it is important to activate search and rescue teams help citizens isolated or trapped by the event that did not or could not evacuate such as in the case of extreme weather and flooding. in addition, where there have been deaths, bodies should be recovered and interred as soon as possible in order to prevent sickness that might originate from them. for instances of collapsed structures with people trapped inside, equipment that can move debris and rescue people or recover the deceased is necessary as would be the case for less common strong earthquakes as well as some extreme storm events. what was just described may be a good template to follow, but in reality, many sea coast cities in developing and less developed countries do not have the resources to cope with severe hazards that might impact them. here, the world health organization and developed nations, large and small, have sent in personnel, supplies, and equipment to help an impacted sea coast city (or inland city) in the past and will surely continue to do so in the future. as discussed earlier in this book, there are sea coast cities worldwide that are at risk from floods, storm surges, and extreme weather conditions such as wind-driven high category hurricanes (typhoons, monsoons), or drought and heat waves, plus along pacific ocean coasts, earthquakes and tsunamis. a municipality and its public health services have to be prepared to adapt to their preparations to deal with what contemporary experiences and history reveal are the most likely physical hazards and diseases to impact it. the two main preparatives are first to be able to care for the injured during a hazard event at well-staffed and supplied hospitals and medical clinics or field hospitals. the second is to fill the basic needs of affected populations with clean water, food, shelter, toilets, waste collection, and if necessary power restoration. these primary responses will help to reduce the chance of an onset and spread of disease. it is important to activate search and rescue teams help citizens isolated or trapped by the event that did not or could not evacuate such as in the case of extreme weather and flooding. in addition, where there have been deaths, bodies should be recovered and interred as soon as possible in order to prevent sickness that might originate from them. for instances of collapsed structures with people trapped inside, equipment that can move debris and rescue people or recover the deceased is necessary as would be the case for less common strong earthquakes as well as some extreme storm events. what was just described may be a good template to follow, but in reality, many sea coast cities in developing and less developed countries do not have the resources to cope with severe hazards that might impact them. here, the world health organization and developed nations, large and small, have sent in personnel, supplies, and equipment to help an impacted sea coast city (or inland city) in the past and will surely continue to do so in the future. disease control after a natural disaster often means initially dealing with diarrheal diseases (dysentery) from the ingestion of contaminated water or food, and lack of sanitation facilities (toilets, hand-washing stations). it may mean dealing with cases of the infectious diseases like cholera (bacterium vibrio cholera) or typhoid (bacterium salmonella typhi) that originate from contaminated water or food or from contacts with cholera or typhoid carriers. to prevent a spreading epidemic status means making safe water and food, sanitation and hand-washing posts available. in the case of cholera, an epidemic may be avoided by isolating the cholera carriers and treating them (with hydration and antibiotics) or oral cholera vaccines where the disease is endemic until the threat of spread is under control. during march - , , the monsoon idai flood ravaged beira, mozambique, a sea coast city of , people, and tracked inland into areas of zimbabwe and malawi flooding them and killing a total of over people. in beira, the drinking water supply and the sewage infrastructure were damaged. cholera cases were diagnosed and the disease spread rapidly with over cases identified with deaths by the april . the who assembled more , doses of cholera vaccine (oral) and starting april delivered more than , initial doses to beira citizens and those in other affected populations, but only about weeks after the disease was first diagnosed. a second inoculation weeks later gives full protection from the disease. adaptation would require vaccine storage stations set up in regions where outbreaks of endemic and other infectious diseases occur so that those that can be controlled by vaccination could be serviced more rapidly than was the case in mozambique. in the above section, infectious diseases that could follow natural disasters that affect sea coast cities were discussed. in this section, infectious diseases that can affect sea coast cities by developing from within or those that are carried in from outside sources are considered. these would be water-and food-borne contaminants and vector-borne bacteria/viruses (by insects, animals, humans). important factors that have to be considered by public health personnel in sea coast cities in order to be prepared to deal with disease include a location's latitude and elevation as they influence climate (temperature and humidity). important sea coast cities are located in most geographic climatic zones. as such, and based on a history of urban health problems, their populations may be susceptible to one or more than one infectious disease that public health services should be prepared to treat. six principal climate zones (mediterranean, tropical, arid, temperate, polar, and mountainous) are classified on the basis of average monthly temperature and precipitation. temperature and precipitation are determined in grand part by latitude, altitude/landform, and the influence of surface and subsurface ocean currents flow paths, location of mountains and their elevations, and seasonal wind directions. the principal zones are a simplification of a zone classification, each with sub-zones (table . ) [ ] . missing from this classification is a mountainous one because with their masses and elevations they establish their own micro-zones and affect weather patterns of some of the other zones. sea coast cities in a similar stage of economic development and in the same climate zones are likely to have exposure to a similar group of endemic infectious diseases. for example, in southeast asia, jakarta, bangkok, and ho chi minh city, are hot ( °c) with higher humidity ( % rh) and have endemic infectious diseases that originate from contaminated water and food, and those that originate from vector-borne carriers. country reports show that in the endemic infectious group, all live with the threat of bacterial diarrheal diseases, and two cities live with the threat of hepatitis a and typhoid fever. in the second group, the three cities have exposure to dengue fever and malaria while two are threatened by japanese encephalitis, and one with hiv/aids and tuberculosis. in africa, mombasa, kenya in east africa, population , and luanda, angola in west africa, population . million, are located relatively close to the equator ( ° ′s and ° ′s, respectively) and have similar temperatures of - °c and - % relative humidity defining their mediterranean/sub-tropical to hot humid tropical climates. however, unlike the southeast asian sea coast cities with similar infectious disease burdens cited above, mombasa and luanda infectious disease burdens differ greatly. mombasa residents are at risk from malaria, tuberculosis, hiv/aids and recently from a resurgent occurrence of polio, measles, and kanazaar (parasitic disease leishmania donovani). luanda, angola residents are at risk from yellow fever, malaria, typhoid fever, and hepatitis a. mombasa, an important kenyan port city also serves uganda, rwanda, the eastern region of the dr congo. this put the mombasa population at risk of infectious diseases that may be carried from those countries by people or products. this possibility for infectious disease transfer was supported when public health personnel recently identified an ebola strain in kenyans who visited west africa. the west african region had suffered an ebola epidemic during - . the disease likely began in a village of southeast guinea as a young boy infected by bats (bushmeat) was carried by exposed villagers to its capital and port city conakry (population . + million). the ebola virus was spread by carriers to the liberian capital and port city monrovia (population million with doctors), and to the sierra leone capital and port city freetown (population + million). the world health organization, medical teams and ngos from other nations worked to stop the epidemic but only after more than , died and more than , were infected and saved. an as-yet unapproved ebola vaccine was used here and was instrumental in halting the spread of the ebola virus. major sea coast cities in south america and the indian sub-continent have similarities and differences of infectious disease risks as do the sea coast cities mombasa and luanda cited above. public health services staffed with appropriate personnel, supported financially, with medicine stocks plus who vaccination stores can be prepared to use them to halt the spread of an infectious disease to prevent an epidemic or to deal with an epidemic that may develop. on august , , there was an outbreak of zaire ebola virus in north kivu province of the dr congo that spread to ituri province. in march, , who reported that there were more than cases and deaths. the public health ministry medical experts identified the disease, did contact tracing, contained and treated infected people, and worked to educate the threatened populations of the risk of not reporting ebola cases, and unsafe burial practices (touching and washing the dead) that is meeting cultural resistance. as important in dealing with the epidemic was the acceptance of the use of the experimental ebola vaccine that helped stop the spread of the ebola disease in west africa a few years earlier. the vaccine was given to people who had direct or indirect contact with people with the disease. however, the application of the ebola vaccine and a halt in the rise of infections has been hampered by armed group violence and unsafe burial practices. the disease is recurrent in the dr congo with ten outbreaks since the discovery of the disease in [ ] . the hope is that it will not become entrenched. one adaptation to infectious disease prevention in this time of ready transnational transport of people globally via sea, air, and land is vaccination control. vaccinations against infectious diseases keep a nation healthy and productive. a low rate of vaccination in a country or regions for years on end can be damning to its citizens and to people where they travel within that country or internationally. the who warns that vaccine hesitancy is one of the top ten global threats of . vaccination hesitancy can be the result of personal philosophical or religious beliefs that allow parents to opt out of this protection for their children as in the united states where there have been major measles outbreaks in eight states and reported cases in other states. in many of these states legislatures are proposing laws to minimize exemptions to measles vaccination requirements for children of school age. a judge in brooklyn, new york, following a serious outbreak of measles in children of a religious sect during april, , mandated application of the mmr measles vaccine (including mumps and rubella) to all their children to protect the general public. this was done over the religion leaders objections to vaccinations. during , europe ( countries) reported that measles killed children and adults of the , people that contracted the measles virus due in part to a vaccination rate (< %) less than the % who deems necessary to prevent epidemics and in part to easily travel between countries. in a recent deadly outbreak, madagascar is working to contain a measles epidemic that began in september, and by february, had killed more than people ( % children to age ) and infected more than , persons. this epidemic is the result of a low immunization rate for many years and has spread throughout the country including madagascar's most important seaport, toamasina, and other ports of entry. this, in spite of the fact that a safe and effective vaccine, has been available for the past years but whose application has not been mandated by the national government. travelers from madagascar to other countries are potential carriers for about weeks ( days before a rash appears, plus days during the rash, and for days after the rash disappears). for this reason, the vaccination history of people entering a sea coast city or other ports of entry who come from or have passed through areas that are suffering or have suffered infectious disease events is important. this would require the collaboration of immigration officials who would be charged with checking the vaccination cards of people (ship personnel, tourists, immigrants, and asylum seekers) at sea coast city ports of entry. the same would be true for other ports of entry. it may require making spot checks of, for example, fever or an observational feature (sweating, paleness) of arriving passengers from specific locations suffering from outbreaks of infectious diseases. this was the case by fever checks in airports and other points of national entry after china revealed the spread of the sars (severe acute, respiratory syndrome) infectious disease during - . non-communicable diseases whether in highly and densely populated sea coast or inland cities can sicken and kill citizens. the focus on the causes of noncommunicable diseases (ncds) that are responsible for million of premature deaths annually and a far greater number of debilitating sicknesses has been on behavioral and metabolic causes and how to reduce their threats to societal health and its effect on city and national economies. these causes include tobacco use, alcohol abuse, poor nutrition, lack of exercise, obesity, and hypertension. of the million premature deaths cited above, ~ million are in low and middle income countries. seventy-nine percent of the ncds are attributed to cardiovascular disease ( . million), cancer ( million), respiratory disease ( . ) million, and diabetes ( . million). public health education programs have to be continually implemented to reduce harmful behavior and causes [ ] . to this point in time, not enough attention has been given to pollution as a principal factor in ncds illnesses and even premature deaths and their socio-economic consequences [ ] . these are often expressed by a calculation of dalys (disability adjusted life years), the total number of years lost to illnesses, disability, or premature deaths in a given population for a certain disease or disorder [ ] . this can be a serious problem in high population sea coast and inland cities with high densities of citizens especially when there are edge neighborhoods with poor and marginalized people living in shantytowns/slums with unsafe water, lack of toilets, and problems with garbage/waste collection. the loss of productivity because of dalys lowers gdp levels by up to % annually, especially in low middle income countries (incomes of us$ to us$ ), such as bangladesh, egypt, india, indonesia, pakistan, and vietnam. cities are most affected because they account for % of global economic business [ ] . pollution is a bane of humanity. air, water, soil, and food pollutants contribute to the premature deaths of millions citizens and sickens many millions more from ncds. pollution-caused diseases accounted for about million premature deaths in or % of all deaths globally (> million). of the million premature deaths, about % of these (e.g., cardiovascular disease, copd) were from pollutioncaused ncds, mainly in low income and low middle income countries [ ] . pollution is most damaging to people and economies where environmental laws protecting people and ecosystems from pollution have not been instituted or if laws have been passed but are not enforced. pollution does not distinguish between sea coast and inland cities but as noted above is most damaging where there are high and dense populations breathing, drinking, and eating pollutant bearing matter. the effect of ingestion of pollutants that cause ncds is not instantaneous but acts slowly over a period of time gradually attacking the normal functioning of body organs as pollutants access and bioaccumulate in them. pollution can be greatly mitigated in three ways: ( ) taxation has been effective in minimizing/eliminating the mass of pollutants emitted into the atmosphere or discharged into waterways or onto soils; ( ) investment in pollutant capture and control equipment and safe disposal of the captured pollutants; and ( ) legislation enforced by the threat and actions of closure of a pollutant-generating operation, by fines, or by incarceration of responsible individuals. of the ~ million premature deaths caused by pollution, ~ million are from indoor air pollution ( . million) and outdoor air pollution ( . million) [ ] . there are . million people that die annually from water pollution (bacterial, viral, heavy metals) and . million from soils polluted with heavy metals and toxic chemicals (e.g., pesticides) ingested through foods. from an economic perspective, pollution frequency costs in the range of - % of a nation's gdp and for some this exceeds what they receive in development aid [ ]. diseases that originate from indoor air pollution include stroke, coronary artery and ischemic heart diseases, chronic obstruction pulmonary disease (copd), acute lower respiratory infections in children, and lung cancer. outdoor air pollution diseases are much the same but with less coronary artery disease. sickness and deaths from indoor air pollution are the result of poor or no venting of gases and fine size particulates (< . - μm) from the fuel used (e.g., soft coal, charcoal, wood, dried animal waste) for cooking and/or heating. improved venting of indoor generated pollutants to the outside can reduce sickness and deaths especially for the more susceptible old people and small children but will add these toxins to outdoor air pollution. the effects of indoor air pollution can be eliminated by bringing natural gas to homes for cooking and heating and proper venting of emissions. this solution is lacking in some major sea coast and inland cities with highly populated shantytown/slum in inner city or edge neighborhoods. outdoor air pollution comes from multiple sources such as coal-burning power plants, various industries, vehicular traffic, and construction. these sources, individually or in consort, emit or raise fine size particulates (< . - μm), and other toxic emissions into the air that include sulfur dioxide, nitrous oxide, and heavy metals (e.g., lead [pb], mercury [hg], arsenic [as], cadmium [cd], and others). these toxic emissions may be inhaled or deposited onto soils or into waters where they can enter a food chain via agricultural crops (e.g., cd in rice) and food fish (e.g., hg in tuna, swordfish, king mackerel). under some meteorological conditions, toxic emissions from industries and vehicular emissions will contribute to a sickening or killer smog. air pollutant abetted diseases are most prevalent among major sea coast and inland cities with upwind nearby major industrial development and in shantytown/slum populations vs. economically more advantaged populations. the mandated use of pollutant capture and control technologies can mitigate the emissions of toxins from industrial sources cited and together with the inclusion of catalytic converters on vehicles can reduce the number of premature deaths abetted by outdoor air pollution. as cited previously, taxation of emission masses can mitigate the problem as can fines, plant closure until facilities becomes compliant with government legislative mandates, or the threat of imprisonment of plant owners [ ] . several major sea coast cities with high populations with very high and relatively high densities in developing countries suffer greatly from air pollution. these include dhaka ( , people/km ), mumbai ( , /km ), abidjan ( , /km ), kolkata ( , /km ), jakarta ( /km ), ho chi minh city ( /km ), bangkok ( /km ), guangzhou ( /km ), zhangjiang ( /km ), tianjin ( /km ), and ningbo ( /km ). in some of these cities, the air pollution threat is elevated by high numbers of poor populations living in shantytowns/slums where densities are higher than the average for a city and where the health status of a population is not as resistant to sickness and disease as it is in economically advantaged inner city populations. for example, in . because of the concern for citizens health and the economic losses from workdays lost or even years of an individual's contribution to a nation's development lost, municipal governments have been monitoring air pollution and identifying specific sources of air-borne toxins. in some cases, they are enforcing existing legislation or carefully planning legislation with enforcement powers that mandate the use of best available technologies at all toxic emissions sources and safe disposal of the captured pollutants. if enforcement is applied, it will greatly reduce toxin loading of pollutants into the atmosphere and hence reduce the risk to the health and premature deaths of great numbers of people. this will be effective only if enforcement of laws curbing air pollution is not just an empty threat because of a nation's drive for continued economic development or because of a culture of corruption. water pollution comes from the atmosphere as rain precipitates toxins from industrial emissions, especially coal-fired power plants and other sources into waterways or onto land where contaminants may run off into waterways. water pollution comes as well from toxins in industrial effluents discharged onto land that can runoff into streams and rivers. rainwater or streams that flow through talus piles of mine wastes can dissolve out heavy metals and carry them overland into waterways. acid mine drainage from abandoned and functioning operations contaminates streams/rivers into which it discharges. bacterial pollutants from human wastes where toilets are not used or where the wastes are collected but not treated can pollute waterways as do wastes and agricultural chemicals as runoff from farmland and commercial animal husbandry (e.g., cattle feed lots, chicken production). the toxin runoff, whatever the source(s), affects not only humans but ecosystems and their natural resources that are important to people. contaminated waters can be cleansed to drinking standards by passing them through a treatment plant before distribution to consumers. toxin-bearing surface runoff cited above can also seep through a surface cover into aquifers and contaminate groundwater. under some conditions, groundwater can dissolve heavy metals that may be in aquifer rocks thus polluting water downflow from where the toxic metals originate. as with surface sources of water, polluted groundwater can be treated to remove a heavy metal pollutant or more than one, or other chemicals before being released for human use (drinking, cooking, personal hygiene) or for irrigation of food crops. the sickening of citizens in well-populated sea coast cities from ingestion of pollutants in unsafe drinking (and cooking) water is a grave concern especially in poor neighborhoods that lack access to clean water vs. economically advantaged neighborhoods that receive treated clean water [ ] . sea coast and inland cities with large and growing populations (e.g., in the millions) obtain much of their foods from distant national and international sources. the reality is that some agricultural products (e.g., grains, vegetables, tobacco) may contain toxins taken up from soil or irrigation water polluted by human activities. these include heavy metals and pesticides/herbicides. if contaminated products are consumed over time, their toxin contents can build up in body organs (e.g., kidneys, lungs) and cause ncds (see sect. . ). toxin build up can also disrupt the normal functioning of nervous and cardiovascular systems, and renewal of skeletal matter. the ingestion of human generated soil/food pollutants can cause ncds [ ] [ ] [ ] . in addition to soils being polluted by industrial and agricultural sources, soils can have natural high concentrations of potentially toxic metals. if a rock contains heavy metals (e.g., black shale) and a soil forms from it as it degrades and decomposes, contaminant metal(s) may be retained in the soil. one pathway to humans is ingestion of one or more of the potentially toxic metals taken up by agricultural crops (food or a non-edible product such as tobacco) being grown in a heavy metal polluted soil. another pathway is from rainwater that seeps through a naturally contaminated soil that reacts with it to release a heavy metal that seeps through the soil into an underlying aquifer. here, the path is toxic metal from rock to soil to agricultural products or drinking/cooking water that can do the same harm to the human body as noted in the previous paragraph by bioaccumulation in and damage to vital organs and also cause medical symptoms and the onset of a ncd. examples of ncds caused by heavy metal poisoning abound in the literature [ , ] . whether a ncd will develop from ingestion of a potently toxic metal depends on several factors. these are the dose absorbed by a body, the route and duration of exposure to chemical species that can cause chronic or acute illness, age, gender, genetics, and health conditions of exposed individuals. perhaps most studied in recent years are cadmium (cd), lead (pb), arsenic (as), mercury (hg), and chromium (cr). there are natural sources of potentially toxic metals (e.g., rocks containing arsenic-bearing minerals, ores, volcanic gases, and particulates). however, great masses of the heavy metal pollutant load in the atmosphere, water bodies, and soils come from human sources. these include industrial operations emissions and effluents, especially from coal-fired power plants, mining and smelters, and factories that manufacture batteries, pharmaceuticals, fertilizer and biocides, paper, pulp, and wood preservatives, among others. sources for each metal or a group of metals that are thought to be linked to an epidemiology-defined disease node can be traced by following geological/geochemical principles used in the search for mineral deposits. governments can then take action to greatly reduce or eliminate the source(s). the fundamental question exists as to whether a government should make regular spot analyses to determine if there is a risk of heavy metal poisoning from agricultural food stuffs. in theory, the answer is yes. realistically, however, analyses would be done but only after a heavy metal was identified fortuitously or if public health professionals detected symptoms of heavy metal poisoning in a population. heavy metals are important to industrial development as well as to human health. there are heavy metals that are essential micronutrients for the optimal functioning of biological processes and organs in humans in a quantity of a few milligrams (mg) or micrograms (μg) daily. these are fe (iron), zn (zinc), co (cobalt), as (arsenic), cr (chromium), cu (copper), mn (manganese), mo (molybdenum), se (selenium), and v (vanadium). however, these may become toxic if ingested and bioaccumulated over time to higher concentrations than are necessary to satisfy nutritional needs. as noted in a previous paragraph, heavy metals that are most toxic to humans (+ animals and ecosystems) if ingested regularly in water and/or food are as, pb, hg, and cd. these metals can bioaccumulate in a body and have toxic effects on different vital organs. in addition, they can interfere in the functioning of essential elements and their metabolic processes. these four heavy metals have been well-studied by toxicologists and other scientists for sources, mobility in ecosystems, and toxic effects. their maximum allowable world health organization limits via oral intake in μg/day are pb ( ), hg-inorganic ( ) , as-inorganic ( ) , and cd ( ) [ ] . in the past and the present, old established sea coast cities have been centers of industrial development ofttimes within and/or upwind and upriver of cities. this, and increasing urbanization as populations grow, fueled increased utility and transportation needs. the release of heavy metals from industrialization, utility sources, and vehicles into a city environment and associated ecosystems on land and in the oceans can be an inherited legacy or existing danger to public health through bad air and contaminated water, soils, and foods. how sea coast cities (and inner ones) and national governments have adapted to mitigate toxic metal pollution is reflected in the health status of their populations. lead represents an inherited legacy that is an existing health threat in and surrounding old homes from pb in drinking water that passes through utility and home pipes containing pb or pipes joined with pb-containing solder. this problem is being addressed by replacing the pipes, by regularly flushing and cleansing city water pipe networks, and by meeting who standards at treatment facilities. lead bearing soil dust from vehicular sources has been basically eliminated since the phasing out of leaded gasoline that began in the s and s with the introduction of catalytic converters. however, inherited pb in soils can be a health hazard. a city may opt to replace a soil, cover it, or otherwise remediate it especially at playgrounds. cities were early in adapting to the pb-based paint problem by recommendations that the amount of pb in interior and exterior paint be reduced to < % and subsequently to < . % in . the un and world bank aim to eliminate its use in . a reasonable recommendation to deal with the millions of homes with pb-based paint is not to strip it off thus releasing toxic dust and chips to the air and surrounding soil but rather to repaint over it when the paint starts flaking. lead reduction programs are essential to protect babies and small children who are especially threatened by pb poisoning if they regularly ingest pb through drinking water, hand to mouth transfer of contaminated soils, or chewing on flaking paint. poisoning manifests itself by brain damage that can result in behavioral problems, learning deficits, and lowered iq. cadmium is an existential health danger, a known carcinogen. a principal source of the toxin is in rice grown in cd-rich soils or irrigated with cd-rich water. a public outcry arose in in guangzhou, china, a port city of . million people, when a report revealed that the rice tested in of samples from local markets had cd concentrations above china's national food safety standards. citizens were upset because of the knowledge that a long-term bioaccumulation of cd in the body had . heavy metal pollutants harmful to people and ecosystems been linked to chronic kidney disease/failure, the bone diseases osteoporosis and osteomalacia, diabetes, lung disease, impairment, cardiovascular disease, and cancer [ ] [ ] [ ] . rice is a staple food for close to half the world's population (~ . billion people) especially for poor populations in low and low middle income countries and its purity has to be assured. about % of the consumption is in asia, with a rising demand in africa and latin america, regions with a great number of commercially important sea coast cities. awareness of the potential problem has resulted in spot checks of rice crops to assure their purity. scientists are studying how to reduce the bioavailability of cd in a soil (paddy or not) to growing plants rice grains, by adjusting the ph of the growing medium [ , ] . geneticists that have improved micronutrient contents in rice crops and the plant tolerance to pests and herbicides are researching low cd-accumulating rice cultivars growing in cd-rich soils. these may reveal a gene that damps cd uptake. once identified, genetic engineers can modify receptive rice species that will greatly reduce uptake of soil cd yet maintain their nutritional values. cadmium can also access a body by inhaling cigarette smoke from tobacco grown in cd-rich soils. arsenic has been shown to be the cause of chronic and ncds in many countries with commercially important and high/densely populated port cities, especially in bangladesh, india, pakistan, thailand, vietnam, taiwan, and china. contaminated drinking water and cooking with untreated or insufficiency treated aquifer water is a principal source of the toxin. the as may develop within an aquifer as conditions change with water drawdown during growing season permitting oxidation and release from a mineral in aquifer rock into the water as either the oxidized chemical species arsenite (as + ) or arsenate (as + ). both species can be toxic if enough is ingested but the (as + ) form is times more toxic [ ] . the chronic response to as poisoning may be the appearance of lesions as pinkish to tan or skin colored papules and keratosis, an epidermal wart-like growth. acute afflictions caused by the longterm as bioaccumulation are cancer of the lungs, liver, bladder, or skin [ , ] . the world health organization warned that more than million people are at risk of as poisoning from aquifer water. water treatment facilities can eliminate this problem if installed, supplied with necessary cadres and chemical supplies, and maintained with a clean water distribution network that reaches all of a population. mercury in food fish is an existing public health threat. mercury emitted into the atmosphere from industrial sources, especially coal-fired power plants, rains into the oceans where microorganisms in the marine food chain convert it to the very toxic methyl mercury form (ch hg + ) [ ] . this chemical bioaccumulates in prey of large predator food fish (e.g., king mackerel, swordfish, tuna, tilefish, orange roughy, shark, marlin, and escolar) that further concentrate it to levels that can harm humans that regularly eat them. shellfish bioaccumulate the toxin as well. sea coast populations include much fish/shellfish in their diets, especially poorer citizens that count on fish/shellfish for % of their protein nourishment and % of their calorie intake. mercury intake can be especially dangerous to pregnant women or women that breast feed their infants because the ch hg + can pass to a growing fetus or an infant and cause neural damage. in adults, hg poisoning can manifest itself as tremors, memory problems, depression, or diminished hearing and vision. governments recommend how much hg accumulating fish meat is safe to eat. for example, canada recommends that pregnant women limit their intake to g/month ( / oz), youngsters - years old eat no more than g/month (~ / oz), and children - years old eat no more than g/month (~ / oz). these values vary with the food fish eaten [ ] . lastly, it should be noted that there is a possible legacy source of heavy metal toxins or other pollutants: buried/hidden toxic wastes. these are disposal sites left by long gone industries in or near long established sea coast (and inner) cities. as urban centers grow to accommodate expanding populations ( . billion more people in than in ), perhaps million added to port cities, areas to be urbanized should first be assessed to assure that no buried/hidden toxic waste disposal sites are present that could, over time, release toxins into an inhabited zone and pose a danger to people. if detected, such sites must be cleaned up before habitation is allowed. köppen climate classification ebola virus epidemic in war-torn estern dr congo pollution and non-communicable disease: time to end the neglect world bank group, independent evaluation group ( ) towards a clean world for all: an ieg evaluation of the world bank group's support for pollution management ambient air pollution: a global assessment of exposure and burden of disease. who, geneva, p. apps.who world urban areas (built up urban areas or world agglomerations) guidelines for drinking-water quality heavy metals, toxicity and the environment. a review toxicity, mechanism and health effects of some heavy metals cadmium stress in rice: toxic effects, tolerance mechanisms, and management: a critical review heavy metals causing toxicity in animals and fishes high cadmium levels found in guangzhou rice, south china strange osteomalacia by pollution from cadmium mining cadmium (cd) stress in rice; photo-availability, toxic effects and mitigation measures -a critical review effective method to reduce cadmium accumulation in rice grain worldwide occurrence of arsenic in groundwater global mercury emissions to the atmosphere from anthropogenic and natural sources health canada ( ) mercury in fish. consumption advice: making informed choices about fish key: cord- -cus sqka authors: nadal, david title: pediatric infectious diseases — quo vadis ? date: journal: pediatric infectious diseases revisited doi: . / - - - - _ sha: doc_id: cord_uid: cus sqka in modern medicine the discipline pediatric infectious diseases is an essential medical specialty. the challenging and complex tasks in the next years include meticulous consolidation and careful extension of existing activities aiming at conducting high level research, offering high standard teaching, and providing high quality patient management. this can only be accomplished by exquisitely dedicated individuals with extraordinary communication and integrative skills following painstaking continued training and formation. potential careers in the discipline can be envisioned not only in academics, but also in government, public health, and industry, whilst less likely in private practice. the discipline pediatric infectious diseases has evolved to an essential medical specialty and faces major challenges in the years to come. one of the most important tasks of pediatricians has always been the management of patients with communicable diseases. the main reason for this is the higher frequency of infectious diseases in infants and young children compared to older children and adults due to the limited adaptive immunity repertoire and thus increased susceptibility to common pathogens. therefore, pediatricians are considerably involved in the diagnosis, treatment and prevention of infectious diseases. in consequence, every pediatrician must be considered also an infectious disease specialist. this, in turn, has been a downside for the development of pediatric infectious diseases as a medical discipline recognized on its own in many countries. nevertheless, the multiple technical advances in the recent years have led to substantially improved prevention and treatment success rates in many pediatric disciplines, and a plethora of these success rates are linked to the integral role of pediatric infectious disease specialists providing profound knowledge, expertise and quality assurance. accordingly, pediatric infectious disease specialists nowadays play a pivotal role both for community pediatrics and for clinical pediatrics in highly specialized medical centers. this chapter attempts to summarize the current different activities of pediatric infectious disease specialists, to delineate their interactions with other medical disciplines and to speculate on the near future goals and development of this specialty with the widest scope compared to all the specialties in medicine. pediatric infectious disease specialists are based mainly in hospital settings and have very similar activities in clinics, teaching and research compared to their counterparts in adult medicine. the four disciplines microbiology, epidemiology, immunology, and pharmacology build up the essential basis for both pediatric and adult infectious disease specialists. nevertheless, despite several overlaps that are beneficial for constructive professional interactions, the position of the pediatric infectious disease specialists differ from those of specialists for adult infectious diseases. these differ not only in relation to the basic training in pediatrics and internal medicine, respectively, but also in relation to distinct focuses in the clinics obviously mandated by many age-related uniqueness of patients in the pediatric age (tab. ). etiology, epidemiology, pathogenesis, management and prevention of infectious diseases in children may substantially differ from those in adults. one important example of the uniqueness of pediatric infectious diseases is the need to deal with infections in newborns. newborns have distinct pathophysiological characteristics, which mainly relate to the immature immune system. another example of uniqueness comes from the age-related and more frequent contacts to potential infectious sources or index cases in nurseries, day-care centers or schools. these contacts lead to increased risks to preferentially acquire respiratory or gastrointestinal infections. similar reasons account for higher frequency of outbreaks of infectious diseases in children compared to adults. infants and toddlers are often the source of infections within a household, for health care workers or medical personnel as well as for nursery employees and teachers. infections represent the reason for up to % of the hospitalization of children. etiological diagnosis of these infections may be hampered by the limited volumes of biological samples including blood or cerebrospinal fluid available from young children, often affording rather judicious, and to this-age-group-adapted, diagnostic approaches. moreover, most of the hospitalized children are prescribed one or more antibiotics [ ] . in this context it needs to be underscored that the pharmacokinetics and pharmacodynamics of antimicrobial substances are rather different in children compared to adults. this may afford the use of distinct preparations or dosages in children. in addition, pharmacology and toxicology of antimicrobial drugs in newborns and specifically in preterm or small-for-date babies are rather special. accordingly, in pediatrics special knowledge in the distinct uniqueness of newborns and other age groups, other disciplines and on nosocomial infections in neonatal and pediatric nurseries and intensive care units is warranted. finally, vaccinations make up a larger proportion of the preventive measures in pediatrics than in adult medicine, and this is mirrored by the extraordinary success of general immunization campaigns in children [ ] . pediatricians in private practice and, in some countries or regions, also general practitioners, are in charge of the management of children with common and frequent infectious diseases [ ] . the quality of this management benefits highly from the continued access to and availability of a pediatric infectious disease specialist during the medical formation and training as well as throughout private practice activities. pediatric infectious disease specialists provide important recommendations on the use of microbiological and other diagnostic tests, application of antimicrobial drugs, and measures for infection control, which may substantially differ in children compared to in adults. furthermore, infectious disease specialists possess the required expertise for the establishment of standards of care for frequent communicable diseases and relevant guidelines for the community. pediatric infectious disease specialists are involved in the care of both outpatients and inpatients [ ] . the impact of the pediatric infectious disease specialist within a hospital can easily be deduced from the number of consultations related to infectious disease or infection control issues requested by both experienced table . specific clinical tasks of the pediatric infectious disease specialist [ ] -integrative discipline -provision of primary care and consultative services to patients from all pediatric disciplines -implementation of quality assurance programs in hospitals and other health care settings, e.g., infection control, hospital epidemiology, antimicrobial management programs -engagement in preventive efforts through implementation of vaccine strategies and other means; play a significant role in public health programs at all political levels -conduction of research seeking cures for new diseases as well as preventive measures, such as new vaccines -teaching and leadership in academic health institutions and non-experienced physicians within or outside the hospital. bedside consultations and phone consultations both play an important role [ ] . the multiple interactions result, e.g., in a more considerate selection of diagnostic measures and assays, more judicious and less costly use of antimicrobials, and reduction of formal consultations and hospitalizations [ ] ( fig. ) . much of the shared knowledge originates from pediatric infectious diseases research programs, as they substantially contribute to the development of improved diagnostic, treatment and prevention means as well as to the understanding of pathogenesis and epidemiology of infectious diseases. the multifaceted roles of the pediatric infectious disease specialist clearly improve the quality of patient care and teach physicians who are involved in primary health care [ ] . specialty in pediatric infectious diseases is the paradigm of an integral and integrative discipline providing paramount professional help, advice and support to other pediatric disciplines and to disciplines from adult medicine. obvious examples are consultations for patients with underlying conditions including congenital heart disease, cystic fibrosis, primary or secondary immunodeficiencies such as due to hiv infection or iatrogenic immunosuppression following allograft transplantation, or tumors. many of these patients nowadays survive beyond the pediatric age and need to undergo the difficult process of transition to medical care for adults [ ] . thus, close interactions with colleagues from adult medicine taking over the care of these patients before, during and after the transition process are indispensable to ensure satisfaction and compliance of the patients with often heavy burdens in addition to the burdens of adolescence. infectious disease specialists have a considerable number of skills at their disposal [ ] . experienced infectious disease specialists, for example, often reduce the use of expensive diagnostic measures even in the most complex patient situations, apply intravenous antimicrobial treatment also to outpatients and switch from intravenous to apt oral medication on time. hence, infectious disease specialists increase the satisfaction of patients while ensuring management quality at lowest possible expenses. an outlook into the future cannot be undertaken without careful consideration of the past and the current situation. thus, recent changes in the spectrum of infectious diseases, progress in the field of vaccinology, advances in microbiology, and quantum leap in communication technology are likely to determine new developments and areas of activity for the pediatric infectious disease specialist. the variety of topics covered in the chapters of this book nicely mirrors the wide spectrum of pediatric infectious diseases and the most recent novel developments in the field. several achievements including clean water, improved sanitation, vaccination and antimicrobial therapies have brought many important infectious diseases under control. nevertheless, we have had to face the emergence of pathogens that are resistant to antimicrobials and of new pathogens that have not been previously detected in humans. the principal diseases of the last decade can be segregated into three major groups: (i) infections against which significant progresses have been achieved; (ii) newly emerged infections; and (iii) infections on which we had no impact [ ] . in industrialized countries, infections with hiv or hepatitis c virus (hcv) have been transformed from diseases with no cure to manageable chronic infections due to newly available treatment or prevention modalities. most importantly, mother-to-child transmission rates in these countries have fallen from around - % to below %, and where preventive measures are strictly applied, vertical transmission of hiv has virtually vanished [ ] . this success story, however, evolved at the expense of intrauterine and neonatal exposure to drugs with a considerable toxic potential [ ] . thus, pediatric infectious disease specialists need to conduct long-term surveys on the evolution of these children following exposure to antiretrovi-ral drugs in a life period with highest vulnerability, especially of the central nervous system. testing of blood products has not only virtually abolished transfusion-related hiv infections but also hcv transmission [ ] . poliomyelitis vaccination campaigns have been extremely effective both in industrialized and in non-industrialized countries. globally, the number of poliomyelitis cases has been reduced by % from cases in to less than cases in [ ] . the goal to eradicate poliomyelitis, however, seems to be hurdled by unprecedented reemergence of poliomyelitis due to "escape" variants [ ] or due to outbreaks in communities reluctant to vaccination, mainly for religious reasons and in countries where there are governmental obstacles to vaccination campaigns [ ] . the tasks waiting the pediatric infectious disease specialists are to promote vaccination at the individual, at the community and at the country levels. this will demand persuasion activities focusing on individuals and on politicians. similarly, measles, rubella and mumps are three viruses against which we possess excellent vaccines, and thus could be eliminated given that the only host for these viruses is humans. we will eventually defeat theses viruses only if pediatric infectious disease specialists succeed in convincing parents of the necessity of vaccination. many parents are no longer familiar with the disastrous consequences of these viruses simply because of the decreased circulation of these viruses in the populations due to the fact that a large proportion has been previously vaccinated. but convincing just the parents will not be sufficient, physicians and politicians will need to be convinced too [ ] . the general introduction of the conjugate vaccine against haemophilus influenzae type b for infants and young children early in the s has resulted in a dramatic reduction of h. influenzae type b invasive infections including meningitis, epiglottitis, arthritis, and osteomyelitis [ ] . more recently, conjugate vaccines against streptococcus pneumoniae or neisseria meningitidis type c have also been introduced in general vaccination programs, and it appears that we will again witness a success. nevertheless, not all s. pneumoniae serotypes are represented in the vaccine and the serotypes against which the vaccine elicits immunity may be replaced by other serotypes. furthermore, a universal vaccine against n. meningitidis type b is still lacking. thus, the reduction of s. pneumoniae or n. meningitidis-induced disease will not be as impressive as for h. influenzae type b. in consequence, pediatric infectious disease specialists will have to explore modalities to improve surveillance and treatment of these prominent and potentially deadly bacterial infections. it goes without saying that more research on the elucidation of bacterial and host-related pathogenetic mechanisms is needed to cut the imminent danger from these pathogens [ ] [ ] [ ] . we have also witnessed the emergence of an unprecedented number of infections. most of these infections are of animal origin: avian influenza, severe acute respiratory syndrome (sars), west nile, ebola, and variant creutzfeldt-jacob disease. another unprecedented observation was the increase in the prevalence of antibiotic-resistant bacteria and the reemergence of previously eradicated pathogens as agents of bioterror. among the most feared and serious antibiotic-resistant bacteria are methicillin-resistant staphylococcus aureus. multiple antibiotic resistances are a problem also with s. pneumoniae, enterococcus faecalis, pseudomonas aeruginosa and mycobacterium tuberculosis [ ] . a main challenge for pediatric infectious disease specialists in addition to the challenge faced by their adult counterparts in this context will be the availability of apt antimicrobials in apt formulations. this in turn will demand that pharmacokinetic, efficacy and safety clinical trials for new drugs are conducted in parallel for adults and different age groups of children, to acquire the needed antimicrobial armamentarium on time. unfortunately, during the last decade we had no impact on tuberculosis, malaria and worldwide hiv, the three leading killer infectious diseases which contribute to half of the global burden of mortality from infectious diseases. in fact, the absolute number of the epidemics has steadily increased. this may cause repercussions in industrialized countries. thus, we pediatric infectious disease specialists who are in a privileged situation cannot neglect these unsolved medical problems, but rather need to increase our efforts to share our time, knowledge and expertise for the benefit of those who need it most. vaccines against these three pathogens are, without a doubt, of paramount priority. finally, another important issue has come up recently: pediatric infectious disease specialists have to deal with aspects of biological terrorism against children (see the chapter by kwang sik kim). the development of several vaccines has been hampered by technical difficulties. vaccines can be developed following the principles of pasteur, i.e., isolating, inactivating and injecting causative microorganisms. such development, however, is not apt for all pathogens, especially for those which cannot be grown in cultures, including hcv, papillomaviruses and and mycobacterium leprae or for antigenically hypervariable microorganisms such as n. meningitidis type b, n. gonorrhoea, malaria and hiv [ ] . in recent years, many obstacles in the engineering of vaccines have been overcome. using "reverse vaccinology" [ ] , a process in which computer analysis, microarrays, proteomics and other genome-based systematic approaches are used to select genomic sequences of microorganisms, antigens likely to confer protective immunity can be identified. candidate antigens can be expressed by recombinant dna and be tested in animal models. reverse vaccinology has enabled the production of vaccines against hcv, human papillomaviruses, and meningococci type b. these examples will be followed for other pathogens representing a threat to infants and children. the pediatric infectious disease specialist will have to define priorities, and will have to conceive plans to test the safety and efficacy of these future vaccines, as well as the surveillance of the epidemiology of the targeted pathogens following the introduction of the vaccines on a larger scale. a change in paradigm in vaccinology has come from the recognition that conquering the most difficult infections such as hiv and malaria may require the t cell arm of the immune system. most vaccines available today work by inducing antibodies, and quantification of these antibodies is often used as a parameter for immunogenicity of and protection by a given vaccine. unfortunately, protective antibody levels are not clearly defined for every available vaccine. moreover, as in the example of hbv vaccine, the levels of specific antibodies may not be indicative for the status of protection. the level of specific antibodies may be below the limit of detection but vaccinated individuals may be still protected against hbv infection by the cellular immune responses. the effective stimulation of cytotoxic t cells can be obtained using engineered non-replicating viral vectors, such as modified vaccinia virus, replication-incompetent adenoviruses and dna vaccines [ ] . another recent quantum jump has been that we -as other living organisms -possess a conserved "innate" immune defense against pathogens. the innate immune defense senses invading microorganisms or their components, and determines the type of adaptive immune response that will eventually result in protection. toll-like receptors and nod proteins are involved in this process. an improved knowledge of the pathways of innate immunity, their selectivity and their interactions is likely to improve the efficacy of vaccines, since certain compounds triggering innate immune defenses, e.g., unmethylated cpg, which mimics microbial dna or lipopolysaccharide as a bacterial cell wall component, could be used as novel vaccine adjuvants to enhance immunity. the field of innate immunity is certainly one of the most promising fields for laboratory and clinic-based research in pediatric infectious diseases [ ] . among the most important developments resulting in unprecedented insights into pathogenesis, susceptibility and diagnosis of infectious diseases are advances in microbiology, immunology and genetics. important changes, with introduction of molecular biology techniques and laboratory automation, have increased the accuracy and velocity of microbiological diagnosis (fig. ) , and new tools are still being developed [ ] . the pediatric infectious disease specialist will considerably benefit from close collaborations with microbiologists both at the research and at the routine level. an equally symbiotic relationship between pediatric immunologists and geneticists will help establish the reasons for increased susceptibility to distinct patho-gens as, for example, mycobacteria or salmonella [ ] and novel treatment modalities for immunocompromised children [ ] . the sick child has the right to receive the best possible medical attention. this includes the caring physicians calling in specialists for consultations, and interdisciplinary consultations can be predicted to become a pivotal component of standard care for patients in the future. who would dare to prevent a sick child from getting optimal remedial management? given the growing medical knowledge and the increasing complexity of modern medical care, pediatric infectious disease specialists can be anticipated to become highly solicited. thus, intra-and interdisciplinary interactions will be more than ever crucial for pediatric infectious disease specialists in the years to come. continued extensive communication, and close collaboration and partnership with other pediatric infectious disease specialists as well as with experts from pediatric immunology, clinical microbiology, pharmacy, epidemiology and all other pediatric subspecialties will build up the key for pediatric infectious disease specialists to ensure the indispensable optimal patient care, efficient teaching, and prosperous research. the most demanding challenge for pediatric infectious disease specialists will therefore be to comprehensively compile expertise, knowledge and cutting edge research for the ultimate benefit of the patient. whereas improved communication within the own hospital setting will help to cope with unqualified management of the sick child as much as pos- sible, installment of a regular and frequent dialog with other centers will not only provide helpful suggestions from peers for the management of patients, but also facilitate and improve continuous education in the field and ensure exchange of ideas for independent and collaborative patient-related or laboratory-based research. the rapidly evolving communication technology has established excellent and affordable tools to allow for quick and reliable data and digital picture transfer as well as for audiovisual conferences at the national, international and intercontinental levels. indeed, digital picture documentation of clinical and laboratory findings is advancing and will evolve. the improved communication at the national and international level should pave the way towards standardized training curricula and the development of training quality evaluation programs. in countries where medical specialty units specifically devoted to pediatric infectious diseases await establishment, support from national and international professional societies will be required to promote the specialty, and communication networks will certainly contribute to expediting this process. the goal to install a pediatric infectious disease service at least in every large medical center is justified. networking will become more and more important to conduct multicenter studies devoted to the pathogenesis, diagnosis or management of less common infectious diseases to enable inclusion of sufficient patients in an appropriate time frame or to adequately respond to emerging infectious diseases [ ] . further, networking that also included experts other than pediatric infectious disease specialists will become increasingly essential to collect and exchange data pertinent to interdisciplinary managed patients as, for example, neonates, cystic fibrosis patients or transplant recipients, to optimize clinical research and management as well as issuing guidelines. such guidelines will gain importance, e.g. in preventing misuse of highly expensive biologicals or drugs (http://www.swiss-paediatrics.org/paediatrica/vol /n /palivizumab -ge.htm). the pediatric infectious disease specialist faces many challenging and complex tasks in the next few years. these tasks will include meticulous consolidation and careful extension of existing activities aiming at conducting high-level research, offering high-standard teaching, and providing highquality patient management. these contributions to modern health care and medicine in general and pediatrics in particular can only be accomplished by dedicated individuals with extraordinary communication and integrative skills following painstaking continued training and formation. potential careers in the discipline can be envisioned not only in academics, but also in government, public health, and industry, although less likely in private practice. the diversity of issues and questions to be confronted makes the specialty of pediatric infectious diseases the specialty with the widest scope compared to all the specialties in medicine. accordingly, commitment to pediatric infectious diseases will be extremely demanding. since not all imposed tasks can be successfully completed by one person only, it will be of paramount importance to focus the activities and to carefully define priorities. nevertheless, such demanding commitment will be fully compensated by manifold societal and personal rewards. prospective survey of antibiotic utilization in pediatric hospitalized patients to identify targets for improvement of prescription the japanese experience with vaccinating schoolchildren against influenza trends in doctor consultations, antibiotic prescription, and specialist referrals for otitis media in children the pediatric infectious disease physician: current roles and importance of the specialty in the care of children. strategic development and long-range planning committee prospective study of telephone consultation and communication in pediatric infectious diseases medical errors detected and corrected by a pediatric infectious diseases consultation service strategies for improving transition to adult cystic fibrosis care, based on patient and parent views withdrawal of pneumocystis jirovecii prophylaxis in hiv-infected children under highly active antiretroviral therapy from pasteur to genomics: progress and challenges in infectious diseases prevention of vertical hiv transmission: additive protective effect of elective cesarean section and zidovudine prophylaxis persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues current and emerging infectious risks of blood transfusions polio endgame. polio: the final assault? poliomyelitis eradication -a dangerous endgame is the cultural context of mmr rejection a key to an effective public health discourse? worldwide haemophilus influenzae type b disease at the beginning of the st century: global analysis of the disease burden years after the use of the polysaccharide vaccine and a decade after the advent of conjugates invasive pneumococcal disease among infants before and after introduction of pneumococcal conjugate vaccine effect of introduction of the pneumococcal conjugate vaccine on drug-resistant streptococcus pneumoniae reverse vaccinology, a genome-based approach to vaccine development reverse vaccinology -in search of a genome-derived meningococcal vaccine therapeutic potential of toll-like receptor activation what does the future hold for clinical microbiology? human genetics of infectious diseases: fundamental insights from clinical studies treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the european experience i thank horst schroten, christoph berger, christian kahlert, and erika schläpfer for their most valuable comments on this manuscript. key: cord- -l d t wh authors: djukanoviĆ, ljubica; radovanoviĆ, zoran title: balkan nephropathy date: journal: clinical nephrotoxins doi: . / - - - - _ sha: doc_id: cord_uid: l d t wh balkan (or endemic) nephropathy is a chronic tubulointerstitial disease of unknown, presumably exotoxic etiology. it has been shown to exist only in some parts of the southeastern europe. while there have been many meetings and papers [ , ] concerning both cause and treatment of balkan nephropathy, sociopolitical turmoil, including wars, and economical hardship prevented any meaningful research on the problem during the ’s. thus, despite numerous proceedings and a large number of publications on the subject, many features of balkan nephropathy, its etiology and natural history in particular, remained nearly as mysterious as when described in the mid-fifties. meetings organized by international organizations [ - ] had a key role in informing the international scientific community on the disease. a recent source of information is a bilingual (in english and serbian) monograph published in [ ]. alkan (or endemic) nephropathy is a chronic tubulointerstitial disease of unknown, presumably exotoxic etiology. it has been shown to exist only in some parts of the southeastern europe. while there have been many meetings and papers [ , ] concerning both cause and treatment of balkan nephropathy, sociopolitical turmoil, including wars, and economical hardship prevented any meaningful research on the problem during the 's. thus, despite numerous proceedings and a large number of publications on the subject, many features of balkan nephropathy, its etiology and natural history in particular, remained nearly as mysterious as when described in the mid-fifties. meetings organized by international organizations [ ] [ ] [ ] [ ] [ ] had a key role in informing the international scientific community on the disease. a recent source of information is a bilingual (in english and serbian) monograph published in [ ] . though exclusive geographical restriction of the agent(s) of balkan nephropathy is not very likely, the disease has been diagnosed only among people living (or those who used to live) in more or less well defined areas of the balkans. along with bulgaria and romania , three republics of the former yugoslavia have been affected: bosnia , croatia and serbia , including kosovo ( figure ). as recently summarized [ ] , the affected territory has a shape of a rhomboid. its longer diameter spreads over km (from the vratza municipality in bulgaria to villages west of slavonski brod in croatia ), while its transversal diameter has about km (from endemic foci in eastern romania to vitina municipality in kosovo ). the disease affects individuals who live (or used to live) in rural environment. there are spared households even in the most affected areas, leading to frequently cited remarks on mosaic distribution of the disease. topography of the terrain differs between endemic regions. all endemic villages in croatia are located in a single lowland municipality, at an altitude of about m, while bosnian foci are found up to m. about % of all endemic settlements in serbia are also situated at a low altitude, below m. [ ] , either in large plains, river valleys or, much more seldom, in hilly regions. there have been no studies of medical geography of balkan nephropathy in romania for the last years and endemic localities are yet to be determined [ ] , but the original findings pointed out to hilly areas, with endemic villages laying at the bottom of valleys eroded by flooding, at an altitude of - m. the endemic region in bulgaria was described as mountainous or semi-mountainous, without any relationship between endemicity and altitude. hydrogeological features [ ] and lack of floods differentiate (at least some) bulgarian foci from other typical endemic regions. controversial data on the frequency of balkan nephropathy were mainly result of methodological shortcomings [ ] . a main obstacle was the operational definition of the disease, leading to huge differences in estimated prevalence rates. the highest ever recorded average annual incidence rate was . per in cakonica , bulgaria. the average cause-specific mortality rate from balkan nephropathy over years in one of the most affected serbian foci was . per [ ] . during initial balkan nephropathy research, patients were frequently in their thirties [ ] , and it was widely accepted that azotemia usually affects the age group - [ ] . later an apparent shift towards the older ages occurred, with most identified patients being above the age of [ ] . the diagnosis of clinical forms before the age of was rare and never independently confirmed. despite occasional statements on laboratory and bioptic abnormalities in the first decade of life among clinically healthy children from endemic areas, no follow up study ever showed that these children developed subsequently kidney disorder. both genders are similarly affected, especially considering mortality. as explained in details elsewhere [ ] , higher prevalence rates among women reported by some authors appears to be a consequence of unreliable diagnostic criteria. a vast majority of experts believe that a link exist between agricultural activity and exposure to the agent(s) of balkan nephropathy. there is also near consensus on the absence of ethnic and/or religious differences as a risk of developing the disease. the most convincing data come from croatia, where the large group of ukrainians who settled a century ago had the same odds of being affected as the indigenous population. the first generation immigrants developed balkan nephropathy usually a couple of decades after moving into an endemic region. initial studies of affected households showed a low standard of living, poor hygienic level, and insufficient nutrition. however, socio-economic factors, including living conditions and well water quality, did not differ between contiguous affected and non-affected households or between endemic and neighboring nonendemic villages. the initial description of balkan nephropathy emanated from bulgaria [ , ] and serbia [ ] [ ] [ ] . by , the disease was recognized in bosnia and croatia and by in romania [ ] . on retrospect, it was not a newly emerging condition but rather recognition of an already existing endemic process, a previous epidemic wave seems having occurred in the early forties. unfortunately, attempts to trace the disease prior to world war are speculative, due to the absence of reliable data and a high frequency of the competing causes of morbidity, notably tuberculosis and malaria . as for the secular trend , two facts, common to all endemic areas, are crucial in assessing any future dynamics of the disease. these are, an apparent shift of the age distribution of the incidence towards the older age groups, and a much longer natural history of the condition compared to previous data. consequently, it suggests that the intensity of exposure diminished (if still present at all). clustering of cases within a household is one of the most conspicuous features of the disease. it is generally agreed that the disease affects both blood related and non-blood related family members. the "phenomenon of simultaneous deaths" (dying of parents and their children within a short interval) was also observed. between / and / of patients with balkan nephropathy develop urothelial tumors [ ] . an exceptionally high frequency of these tumors was also observed in the general population of endemic regions [ ] . when initially studied, the attributive risk of developing upper urothelial tumors in inhabitants of endemic foci amounted to several dozen or even to as much as - . there is no evidence that domestic and/or wild animals in endemic regions develop a similar condition. there is general agreement on the following descriptive-epidemiological characteristics of balkan nephropathy [ ] : the disease is known to exist only in some parts of the southeastern europe, with central serbia as the most affected region. balkan nephropathy does not spread beyond its already defined foci; the disease is distributed mosaically : non-endemic villages exist in the most affected regions, and there are spared families and households in the most affected settlements. clustering of cases in families and households has been described. children and adolescents are spared of clinical disease. incidence is proportional to age, except for the oldest age groups. there are no major sex differences in the cause-specific mortality rates. the excess risk of developing transitional cell urothelial tumors was expressed by two-or even threedigit numbers. the large majority of researchers support the following statements [ ] : autochthonous urban population is spared; rural way of life, i.e., agricultural activity is needed for exposure to the agent(s). separation from an endemic focus early may prevent the disease, while immigration to an endemic area provides risk of disease development, providing that the exposure was sufficient. prevalence of the disease has been stable over many years, but now appears to decline in most affected settlements. incidence rates are shifting towards the older age groups, and the clinical course is much more protracted suggesting a less intensive contact with the agent(s) and, consequently, possible future spontaneous disappearance of balkan nephropathy. the most elaborate and, seemingly consistent, hypotheses regarding etiology initially came from proponents of heredity as an explanation of the disease occurrence. these authors assumed that the risk of developing the disease was restricted only to specific, ethnically distinct, population groups, irrespectively of their place of birth and residence history. wider acceptance of these hypotheses was hampered by the different perception of descriptive epidemiology of balkan nephropathy by a majority of researchers on the topic. a specific chromosome marker ( q ) in balkan nephropathy patients from bulgaria was identified, and this isolated finding was used to support arguments in favor of a crucial role of genetic factors [ ] [ ] [ ] . more recently, the same authors acknowledged that environment is also important [ ] . some aberrations of the x chromosome have been reported, but they resembled changes occurring after exposure to ochratoxin a [ ] . major anomalies of urinary organs allegedly occurred in a high percentage of otherwise healthy children from affected households. however, such a finding has never been replicated. genetic epidemiological approach suggest two possibilities, either polygenic type of inheritance with an insufficient expression of the main gene [ ] , or monofactorial model with a crucial role of a single gene of incomplete penetrance [ ] . in both cases, contributing environmental factor is needed. there is no evidence supporting an immunological mechanism in balkan nephropathy. unspecified viral particles [ ] , an unidentified cythopathogenic agent, serially propagated slow viruses [ ] , and an unknown virus associated with foci of natural infection [ ] have been mentioned in the context of balkan nephropathy etiology. several specific viruses, notably west nile [ ] , coronavirus [ ] , and papova virus [ ] , were also suggested as a causative agent. a common feature of all these hypotheses was unimpressive supporting evidence and ignorance of basic epidemiological features of the disease, in particular its absence of spreading [ ] . bacteria received particular attention in initial stages of the balkan nephropathy research but their possible etiological importance has been unanimously considered as ruled out [ ] . protozoa have never attracted any attention. toxic fungal products were until recently the principle and prime potential culprits. most efforts have concentrated on ochratoxin a , a mycotoxin responsible for porcine (swine) nephropathy [ ] . the substance is found in endemic foci but it is also present in neighboring non-endemic areas, and the differences are not statistically significant [ , ] . still, the consistent isolating of ochratoxin a in greater frequency and higher concentrations from food and sera samples obtained from endemic, compared to control villages, offered some arguments in favor of this hypothesis. association of ochratoxin a with chronic interstitial nephropathy in tunisia [ ] and its relation to renal tumors [ ] provides additional support for the idea of the etiological role of this mycotoxin. other fungal toxins, as zerealenone , citrinin [ ] and aflatoxin were also isolated in endemic foci. experimental models suggested that a combination of mycotoxins, rather than a single one, might be involved in the etiology of balkan nephropathy [ ] . aristolochic acid and its salts, originated from a weed, aristolochia clematitis, have toxic and carcinogenic effects to the kidneys and urothelium [ ] , respectively. ivic [ ] postulated that this plant may be a cause of balkan nephropathy, but failed to provide convincing evidence from field surveys. evidence that a. clematitis played a central role in the etiology of chinese herb nephropathy [ ] [ ] [ ] , a condition similar to balkan nephropathy, initiated a second look at this previously abandoned hypothesis and it gained a lot of weight by recent data on the association between dna adduct formation derived from aa, mutation pattern and tumour development in ben [ ] (see also chapter ) . no local practice in terms of the use of teas or folk medicine could have been implicated. no one has ever studied flora of the local wells. chronologically, lead poisoning was first offered as an explanation for the occurrence of balkan nephropathy [ ] [ ] [ ] . the idea on lead-contaminated flour led to abandonment of water mills in a part of central serbia . this energetic public health action had no impact on the disease frequency. effects of non-occupational exposure to cadmium [ ] , itai-itai disease in particular [ , ] , were occasionally compared with kidney damage seen in balkan nephropathy patients. in spite of some resembling features, the idea of a common etiology between cadmium nephropathy (including itai-itai disease) and balkan nephropathy was refuted [ , ] . many other metals, including radioactive ones such as uranium [ ] , were also suggested as possible causative agents of the disease. results were non-convincing and non-reproducible. inability to identify a single toxic effect of any metal or metalloid as a cause of balkan nephropathy led researchers to two alternative approaches. first, deficiency, rather than abundance of such a chemical element was proposed [ ] , with selenium as the most likely candidate [ ] . second, attention was paid to a combined adverse effect of several elements. synergism of uranium and some other elements, none of which exceeding maximal allowed levels, was proposed [ ] . it was also noted that criteria used in occupational medicine (exposure only during working hours) have been applied to an ecological problem (constant exposure) and that concentrations of lead or cadmium within formally acceptable level, combined with other factors, such as selenium deficiency, might lead to the disease [ ] . all these suggestions remained speculative. as for non-metals, there were attempts to relate balkan nephropathy to silicon [ ] [ ] [ ] [ ] . however, when affected and non-affected households were compared, there was even an inverse relationship between the silica content and endemicity. on one occasion, small differences in silica content happened to reach the level of statistical significance but the association was explained as a result of confounding variables [ ] . common hydrogeological characteristics of endemic foci [ ] and inverse relationship between altitude of wells and disease frequency in a longitudinal (cohort) study [ ] , pointed to potable water as a vehicle of the agent(s). however, none of the already mentioned or several dozen other non-organic substances were associated with the disease [ ] . organics in water have been investigated and pro-vided some interesting data [ ] . except for nitrites [ ] , chemically unstable substances have not been studied. wells associated with the disease were reported as situated on alkali soil [ ] , but the finding was restricted to a single endemic area and never reproduced. based on chronological data, it is clear that no pesticides , fertilizers or chemicals introduced during the last few decades may be blamed for the occurrence of balkan nephropathy. except for exposure to agricultural activities, no occupation , habit (e.g., smoking, alcohol consumption), or hobby (e.g., hunting, fishing) might have been shown to precede the disease onset. genetic factors may play a role in different individual risk of developing balkan nephropathy, upper urothelial tumors, both diseases or none of them [ ] . however, epidemiological data indicate that one or more external, environmental factors are crucial for the occurrence of both balkan nephropathy and excessive frequency of these tumors in endemic areas. among biological agents and their products, the candidates for etiological agents are mycotoxins and, much more probably, toxic plants, notably aristolochia clematitis. the possible role of viruses is very unlikely, indeed. as for inanimate environment, there is no chemical element that has been consistently detected in higher concentrations in biological material of balkan nephropathy patients and/or their environment, as compared to the controls. however, though unlikely, insufficiency of an essential element has not been completely ruled out. speculations on a combination of vaguely defined environmental factors have never been substantiated by facts. balkan nephropathy is non-destructive and noninflammatory tubulointerstitial renal disease [ ] . the changes are non-specific and in the chronic, sclerotic phase they may be quite similar to changes observed in other chronic interstitial diseases such as analgesic nephropathy [ ] , vascular nephrosclerosis [ ] cyclosporine-induced nephropathy [ ] , radiation nephritis [ , ] and aging [ ] , intoxication with silicate, cadmium , lead, uranium [ ] , mycotoxin ochratoxin (autopsy, a man aged ) . a [ ] , aristolochia clematitis [ ] , and recently with chinese herbs [ , ] . before introduction of hemodialysis in the treatment of chronic renal patients, the kidneys of patients who died of balkan nephropathy used to be the smallest seen at post mortem examinations, weighing . - g each ( figure a ) the difference between the left and right kidneys being small ( - g) [ , [ ] [ ] [ ] . surface of the kidneys is smooth, occasionally wavy but never granulated or roughly nodular. the section shows markedly narrowed cortex, pyramid and bertin's columns are fairly well preserved, and corticomedular border is well differentiated. papillary necrosis of the pyramids has not been found. small, papillary, usually multiple tumors of the renal pelvis and ureters are also one of the characteristic findings ( figure b ]. in post-mortem studies tumors were reported in - % of cases [ , ] . diffuse fibrosis of cortical interstitium and tubular atrophy may be observed along in the absence of sig-a b nificant cellular interstitial infiltration. in contrast to the cortex, bertin's columns are less markedly affected. even in severe tubulointerstitial cortical changes, glomeruli are well preserved, partially collapsed, and subsequently subjected to focal or generalized sclerosis mainly collapsing. glomeruli in bertin's column are occasionally compensatorily enlarged. pyramids are preserved or less markedly affected [ , , ] . blood vessels, arcuate or interlobular arteries, and arterioles are affected in the form of intimal sclerosis and thickening of lamina elastica interna. in addition, the blood vessels are compressed and torsioned [ ] . in cases of tumors of the renal pelvis and ureters morphological signs of pyelonephritis are often found [ ] . in oligosymptomatic clinical cases, rare disseminated foci of interstitial fibrosis and tubular atrophy with preserved glomeruli are seen. these changes have no special predilection of distribution and are not inflammatory. they tend to be triangular, with the base oriented toward the renal surface [ , ] . in cases with initial renal failure, the fields of acellular interstitial fibrosis are larger and even diffuse. the striking atrophic process observed in balkan nephropathy suggests that apoptosis may play a role in this disease. in this context it is of interest that savin et al. observed an increased apoptosis to proliferation ratio at the level of the tubuli [ ] . the glomeruli are usually affected by generalized [ %] or segmental sclerosis [ %]/ and only in % hyalinosis is recorded. double contour glomerular basement membrane was recorded in % of the cases. in . - % of cases fetal-like glomeruli can be seen in the kidneys, while glomerular hypercellularity was recorded in % [ , , ] . the most interesting changes are recognized in pre-and postglomerular blood vessels. in about % of cases pas positive proteins are deposited in vas afferens walls in a focal segmental or circumferent manner in the form of droplets, bands or granules [ , , ] . interlobular capillaries are filled with thick proteinaceous substances that are also deposited below the capillary endothelium and may even be found free in the interstitium. these changes are described as capil-lary sclerosis [ ] . although renal vascular changes in balkan nephropathy have been pointed out as very important, they are not specific and can be encountered in other renal diseases. ferluga et al. [ ] and sindjić [ ] commented on their similarities with cyclosporine induced nephrotoxic changes. immunofluorescence revealed irregular and scarce deposits of c , fibrin and igm, and occasionally iga, c q and c , mainly on the vascular walls, bowman's capsule and some sclerotic glomeruli [ , , ] . electron microscopic findings are either normal or correspond to degenerative and sclerotic changes. while some authors describe virus-like particles [ , , ] , others point out that such particles were not found [ , ] . despite these findings, some authors described balkan nephropathy as a form of glomerulonephritis [ , ] . however, the lack of reliable evidence supporting glomerulonephritis has lead to it being discarded [ , ] and abandoned even by its advocates [ ] . optic microscopic, immunofluorescent and electron microscopic studies of renal biopsies in children aged - from affected families in endemic regions failed to detect any balkan nephropathy related changes [ ] . it is generally agreed that the morphological changes of balkan nephropathy are not specific and correspond to non-destructive, non-inflammatory kidney disease accompanied by marked changes on the blood vessels in both early and late stages of the disease, interstitial, multifocal fibrous expansion and severe tubular atrophy mainly in the upper cortex [ , , , , ] . changes on kidneys arterioles have been described suggesting that the changes in early stage of the disease may be responsible for the development of multifocal, ischemic, vascular nephrosclerosis encountered in chronic stages of the disease [ , ] . on the other hand, close similarity of balkan nephropathy with analgesic and cyclosporin-induced nephropathy has been recognized [ , , ] . all this leads to a suggestion that balkan nephropathy develops following a model of toxic nephropathy, targeting primarily the vascular endothelium where the tubular epithelium is affected either directly or indirectly due to accompanying ischemia. balkan nephropathy is a chronic tubulointerstitial disease with occult, insidious onset, usually progressing slowly with no apparent signs of symptoms. after a long asymptomatic period, the disease is manifested as chronic renal failure. less commonly blunt lumbar pain or renal colic may develop or, occasionally, dysuric symptoms induced by urinary tract infection. if hematuria exists, urothelial tumor should be suspected. in an advanced case polyuria and nocturia are present due to impaired concentrating ability of the kidneys. the disease is tolerated well and the patients preserve their working ability until advanced stages of renal failure [ , , , ] . objective examination reveals characteristic skin tan of balkan nephropathy patients: a pale yellow with copperish glow on the cheeks has been recognized since the augural reports on the disease [ , ] . besides, xantochromia of the palms and soles is also frequently observed. in the advanced phase of the disease physical examination detects signs of chronic renal failure [ ] . patients with balkan nephropathy do not suffer from edema, and their blood pressure is usually described as normal [ , [ ] [ ] [ ] . recently, several studies reported a higher prevalence of hypertension even in offspring of balkan nephropathy families [ , , ] . as balkan nephropathy is characterized with slow asymptomatic course, most authors identify two main stages of the disease: the first, asymptomatic (latent, subclinical) and second, manifest (symptomatic). the latter is usually subdivided into the stage without renal failure (early, compensated balkan nephropathy, with no azotemia) and chronic renal failure (decompensated balkan nephropathy, uremia) [ , , ] . an important feature of balkan nephropathy is its association with a high incidence of tumors of the renal pelvis and ureters, but not urinary bladder tumors [ , , ] . however, the difference between the incidence of upper urothelial tumors in endemic and non-endemic regions diminished in the last decades. in the sixties and seventies the incidence of these tumors was reported to be several dozen times higher in endemic than in non-endemic regions, while in the last decades this difference almost disappeared [ , , , , ] . upper urothelial tumors of patients originating from the region with balkan nephropathy differ from tumors identified in patients from other regions in their similar incidence in both sexes, bilateral occurrence, and more common association with chronic renal failure [ ] . appearance and urine color are unchanged in most patients with balkan nephropathy. urine sediment is usually scarce, while microhematuria or leukocyturia are usually associated with the occurrence of tumors or urinary tract infection [ , ] . bacteriological studies usually reveal sterile urine, but in . - . % significant bacteriuria was confirmed and considered as superimposed urinary tract infection [ , ] . proteinuria is a common finding in patients with balkan nephropathy [ , ] . it is usually intermittent, less than g per day and it becomes permanent in advanced renal failure [ ] . although proteinuria is one of the criteria for diagnosis of balkan nephropathy, it has been reported in healthy members of endemic families [ , , ] . tubular proteinuria is the most common and increased excretion of low-molecular weight proteins such as β -microglobulins, lysozyme, ribonuclease, light chains of immunoglobulin, retinolbinding protein has been reported [ ] [ ] [ ] [ ] [ ] [ ] . beside tubular proteinuria, smaller numbers of patients manifest mixed proteinuria, while in patients with renal failure, glomerular proteinuria may be encountered [ , ] . anemia has been noted in patients with balkan nephropathy in early studies [ ] and described as normocytic and normochromic or mildly hypochromic [ ] . it has been suggested that anemia occurs earlier in the course of the disease progression than is the case in other renal diseases and that it precedes azotemia [ , ] . however, recent studies have failed to substantiate this claim [ , ] . also, there is no evidence that anemia in balkan nephropathy differs from anemia accompanying other renal diseases in either features [ ] or rate of deterioration in the progression of renal failure [ ] . nevertheless, anemia in balkan nephropathy patients treated with hemodialysis is more severe than in patients with other renal diseases [ ] . the leukocyte count in the peripheral blood of patients with balkan nephropathy is normal and without pathological changes in the differential count and bone marrow [ , ] . investigation of renal function in patients with endemic nephropathy has revealed tubular dysfunctions in the earliest stage of the disease: renal glycosuria , increased uric acid and amino acid excretion [ ] , as well as increased excretion of low molecular weight proteins [ ] . significantly higher activity of cellular enzymes in the urine and increased urinary excretion of tamm-horsfall protein was described in patients with balkan nephropathy, as well as in healthy members of endemic families [ ] . findings of a distal tubular disorders (impaired urinary acidification, impaired urine concentrating ability) were described in earlier studies [ , ] but could not be confirmed in studies conducted in larger groups of patients with normal or mildly impaired glomerular filtration rate [ , ] . the occurrence of certain disorders of the tubular function recorded in the course of chronic renal failure (increased natriuria, phosphaturia) can be considered as the result of kidney adaptation to the lost nephron mass, instead of balkan nephropathy properties [ ] . the immunological studies have failed to indicate that immune disorders participate in the pathogenesis of balkan nephropathy, with some of detected changes having been attributed to advanced renal failure [ ] . different methods of kidney imaging have shown that balkan nephropathy patients with chronic renal failure have symmetrically shrunken kidneys with smooth surface and no calcifications [ ] . the time at which the shrinking occurs remains to be determined. while some authors suggest that the size of the kidneys remains normal in patients in the latent phase of the disease and with normal renal function, others report cases of shrunken kidneys in patients in an early phase with normal glomerular filtration rate, and it was even proposed that the disease was characterized with primarily small kidneys [ , , ] . as ultrasound became a standard imaging method in the evaluation of kidney dimensions, several recent studies that used this method showed diminished kidney length and cortex width in members of balkan nephropathy families with normal kidney function [ , ] . besides, significantly shorter kidney length, as well as higher protein, albumin and b -microglobulin excretion was found among offspring with a maternal history of balkan endemic nephropathy (ben), not a paternal one [ ] . excretory urography does not reveal changes in the pyelocaliceal system, except in cases with secondary infection or urothelial tumors. radionuclide methods have shown that renal plasma flow impairment is the first sign of the early phase. glomerular and tubular functions correspond to the severity of the disease. the most commonly used criteria for the diagnosis of balkan nephropathy are still those proposed by danilović [ ] . they include: ) farmers living in the endemic villages, ( ) familial history positive for balkan nephropathy, ( ) mild proteinuria, ( ) low specific gravity of the urine, ( ) anemia, ( ) retention of nitrogen compounds in the blood (urea > mg/dl, creatinine > . mg/dl) and ( ) symmetrically shrunken kidneys . using these criteria, danilović suggested classification of patients in field studies into the following groups: . potential, a group with intermittent proteinuria, those that fulfill at least the first three criteria, . suspected patients, that in addition to the first three fulfill at least one of the remaining three criteria, . affected patients, that fulfill at least out of criteria, . decompensated patients that fulfill at least out of criteria and have urea values > mg% and manifested signs of uremia. analysis of these criteria leads to the conclusion that they enable detection only of patients with overt disease, and the criteria are not sufficiently specific to enable a reliable diagnosis. therefore, numerous studies have been focused on developing sufficiently sensitive and specific criteria to enable diagnosis in the early phase. although markers of tubular disorders, particularly tubular proteinuria , may be used as sufficiently specific diagnostic criteria, so far not a single clinical or laboratory finding is considered pathognomonic for balkan nephropathy when differentiate it from other, specially, tubulointerstitial diseases. the diagnosis of balkan nephropathy is now established according to the first two criteria (residence in endemic village and positive family history) suggested by danilović [ ] , presence of tubular proteinuria and ruling out other renal diseases. histopathological analysis makes the diagnosis of balkan nephropathy significantly easier [ , ] , and it is considered indispensable in classifying the following groups of patients with urinary abnormalities suggestive of endemic nephropathy: . patients from families that were not previously been affected with endemic nephropathy, but live in an endemic village, . in cases of nephropathy of unknown etiology in villages close to endemic foci, . in immigrants to endemic regions and in emigrants from these regions [ ] . differential diagnosis of balkan nephropathy should include all chronic, slowly progressive renal diseases, primarily chronic tubulointerstitial diseases. although no specific indicators of balkan nephropathy have been recognized, epidemiological data, familial history as well as clinical characteristics of the disease enable differential diagnosis. thus, shrunken kidneys with smooth surface are characteristic of balkan nephropathy and they differentiate it from analgesic nephropathy, pyelonephritis or reflux nephropathy that are characterized by shrunken kidneys with uneven surface. pyelocaliceal system of the kidneys remains unaffected in patients with balkan nephropathy, unlike the characteristic changes observed in pyelonephritis or obstructive nephropathy . absence of papillary necrosis/calcifications also enables differentiation of balkan nephropathy from analgesic, obstructive, reflux nephropathy [ , ] . recently similarity of balkan nephropathy and nephropathy induced by chinese herbs used in slimming diets have been suggested [ ] . nevertheless, chinese herb nephropathy is rapid progressive tubulointerstitial diseases with pronounced fibrosis and progression towards end-stage renal disease within few years, clearly different from the protracted clinical course of balkan nephropathy. balkan nephropathy is a disease of unknown etiopathogenesis, so that recommendations regarding effective prevention are not possible. efforts have been made to improve the living conditions, bring high quality drinking water to endemic villages and undertake other hygienic measures. treatment is planned according to the stage of the disease. in principle, the treatment involves the measures for slowing down deterioration of renal function and those applied in chronic renal failure [ ] . end-stage renal disease is treated with dialysis and kidney transplantation. hypertension and cardiovascular diseases affect the balkan nephropathy patients less frequently, so they tolerate hemodialysis rather well compared to patients with other renal diseases. the balkan nephropathy patients on long-term hemodialysis frequently develop upper urothelial or urinary bladder carcinoma. although the number of reported cases with kidney transplant is small, neither specific post-transplantation problems nor disease recurrency on the transplanted kidney have been described. however, recent studies indicated that patients with balkan nephropathy are at increased risk for the development of upper urothelial tumors in both native and transplanted kidneys [ ] . balkan nephropathy is a chronic tubulointerstitial disease with insidious occult onset progressing without symptoms. agreement as to how to define the early asymptomatic phase of the disease is lacking, since no specific indicators for the diagnosis have been recognized. the diagnosis is established according to epidemiological criteria (farmers in endemic villages, familial history positive for endemic nephropathy), presence of tubular proteinuria, findings of symmetrically shrunk kidneys with smooth surface, without calcifications and ruling out of other renal disease. renal biopsy may make the diagnosis easier, although the changes are non-specific. one of the important features of balkan nephropathy is its association to high incidence of tumors of the renal pelvic and ureters, comparable to analgesic nephropathy (se chapter ) and aristolochic acid nephropathy (see chapter ). so far, laboratory studies have failed to detect any disorder as a specific marker for early detection of the disease or a reliable indicator for differential diagnosis. laboratory studies have confirmed that balkan nephropathy is a tubulointerstitial disease so that tubular disorders precede impairment of glomerular filtration. although anemia is one of the criteria for the diagnosis of the disease, it has not been evidenced that pathogenesis and features of this anemia differ from that observed in other chronic renal diseases. it is only more severe in end-stage balkan nephropathy patients than in patients with other kidney diseases. photos of all tissue specimens were provided by professor jovan dimitrijević. endemic nephropathy -bibliography aetiology of balkan nephropathy: a reappraisal after years world health organization. the 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relatives at risk particules virales dans le rein de la nephropathie endemique balcanique endemic balkan nephropathy, a slow virus disease? endemska (balkanska) nefropatija kao prirodno-žarišna infekcija? studien über die endemische nephrozirrhose der balkanhalbinsel evidence of a viral etiology in endemic (balkan) nephropathy further data on the prevalence of serum antibodies to papova viruses (bk and sv ) in subjects from the romanian area with balkan endemic nephropathy epidemiological evidence on balkan nephropathy as a viral disease causal association of mycotoxic nephropathy ochratoxin a contamination of cereals in an area of high incidence of balkan endemic nephropathy in bulgaria ochratoxinogenicity of aspergillus ochraceus strains from nephropathic and non-nephropathic areas in yugoslavia foodstuffs and human blood contamination by the mycotoxin ochratoxin a: correlation with chronic interstitial nephropathy in tunisia ochratoxin a genotoxicity, relation to renal tumors nephrotoxicity of penicillium aurantiogriseum, a possible factor in the aetiology of balkan endemic nephropathy relevance of a rat model of papillary necrosis and upper urothelial carcinoma in understanding the role of ochratoxin a in balkan endemic nephropathy and its associated carcinoma the carcinogenic action of aristolochic acid in rats problem etiologije endemske nefropatije rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including chinese herbs chinese herbs nephropathy: a clue to balkan endemic nephropathy? kidny int chronic aristolochic acid toxicity in rabbits: a model of chinese herbs nephropathy? aristolochic acid and the etiology of endemic (balkan) nephropathy renal dysfunctions of inhabitants in a cadmium-polluted area cadmium and health: a toxicological and epidemiological appraisal urinary excretion of cadmium and zink among women with balkan endemic nephropathy prilog proučavanju etiologije hroničnog nefrita balkan endemic nephropathy in yugoslavia and geochemical studies selenium deficiency and balkan endemic nephropathy environmental renal disease: lead, cadmium and balkan endemic nephropathy essai d'explication de la manifestation de l'affection rénale de masse å slavonski kobas role étiopathogénique des silicates érosifs dans la néphrite endémique. presse méd experimental chronic interstitial nephritis compared with endemic human nephropathy fiberglass or silica exposure and increased nephritis or esrd (end-stage renal disease) well water characteristics and the balkan nephropathy groundwater chemistry and the incidence of balkan endemic nephropathy relationship between low-rank coal deposits and balkan endemic nephropathy different forms of mineral nitrogen in drinking water and the balkan nephropathy ispitivanje ovisnosti pojave endemske nefropatije i sastava zemljišta iz kojeg stanovništvo uzima vodu za piće u opštini modriča upper urothelial tumours and the balkan nephropathy -inference from the study of a family pedigree pathomorphology of the kidneys in endemic nephropathy nephropathy due to analgesics morphology of cyclosporin nephropathy renal function, protein excretion and pathology of balkan endemic nephropathy. iii light and electron micorscopy studies renal pathology with clinical and functional correlations resultats des recheches patho-anatomiques, faites jusqu'a present, concernant la nephropathie endemique enviromental ochratoxin a and balkan (endemic) nephropathy: evidence for support of a casual relationship endemic nephropathy in yugoslavia aspects anatomo-cliniques de la néphropathie endémique balkanique investigation of chronic endemic nephropathy in yugoslavia. ii renal pathology morphological changes in kidneys affected by endemic nephropathy the significance of apoptosis for early diagnosis of balkan nephropathy biopsy specimen examination during the early stage of endemic nephropathy endemska nefropatija -imunofluorescentna mikroskopska ispitivanja renal function, protein excretion and pathology of balkan endemic nephropathy. iv-immunohistology viruses in endemic (balkan) nephropathy isolation of a coronavirus from urinary tract tumours of endemic balkannephropathy patients ultrastructure of nephrons in kidney of endemic nephropathy patients immunohistochemical studies in balkan endemic nephropathy a clinical study of nephropathy of an endemic character in the people's republic of rumania clinical nephrotoxins. renal injury from drugs and chemicals. de broe me detection of renal dysfunctions in family members of patients with balkan endemic nephropathy clinical markers in adult offspring of families with and without balkan endemic nephropathy the prevalence of balkan endemic nephropathy has not changed since in the kolubara region in serbia epidemiology and treatment of renal pelvic and ureeral tumours tumors of the upper urothelium and endemic nephropathy decreasing incidence of urothelial cancer in a balkan endemic nephropathy region in serbia. a surgery based study from to survival of patients with transitional cell carcinoma of the ureter and renal pelvis in balkan endemic nephropathy and non-endemic areas of serbia renal function, protein excretion and pathology of balkan endemic nephropathy. i. renal function characteristics of proteinuria in endemic nephropathy ciba foundation study group no. renal function studies inindividuals with the tubular proteinuria of endemic balkan nephropathy etude elctrophoretique de la proteinurie de sujet crates vivant en pays de nephropathie endemique urinary proteins in renal diseases. ii. alpha -microglobulins in tubular protienuria with special reference to endemic nephropathy beta -microglobulin excretion as an index of renal tubular disorders with special reference to endemic balkan nephropathy renal function, protein excretion, and pathology of balkan endemic nephropathy. ii. protein excretion strahinji} s & stefanovi} v (editors) anaemia in balkan endemic nephropathy erythropoietin and anemia in the progression of balkan endemic nephropathy and other renal diseases relationship between tubular and tamm-horsfall proteinuria in balkan endemic nephropathy laboratory studies oxford textbook of clinical nephrology study of pathogenesis based on genetic factors clinical markers in adult offspring of families with and without balkan endemic nephropathy creatinine clearance and kidney size in balkan endemic nephropathy patients high diagnostic performance of ct scan for analgesic nephropathy in patients with incipient to severe renal failure clinical picture, diagnostics and treatment of endemic nephropathy renal transplantation in patients with balkan endemic nephropathy key: cord- -rt i wca authors: tosam, mbih jerome; ambe, j. radeino; chi, primus che title: global emerging pathogens, poverty and vulnerability: an ethical analysis date: - - journal: socio-cultural dimensions of emerging infectious diseases in africa doi: . / - - - - _ sha: doc_id: cord_uid: rt i wca in the last few decades, the world has witnessed the emergence and re-emergence of new and old infectious diseases. emerging infectious diseases (eids) have the capacity to spread rapidly from one region of the world to another, within a very short time, due to world travel and increased global interdependence. the impact of this varies from one region to another. resource poor countries suffer the most due to an already high disease burden, poor infrastructures, lack of clean, potable water and sanitation, as well as an acute shortage of qualified health personnel to manage, control and contain the crisis/spread. poor and marginalized communities are the most vulnerable because infectious diseases cause not only suffering and death, but also severe economic hardship. the outbreak of hiv/aids, tuberculosis and ebola virus disease (evd) in the developing world has shown the extent to which economic and social conditions can affect vulnerable populations. these socio-economic, cultural and environmental conditions accelerate the spread of, and exacerbate the negative impact of emerging pathogens. this chapter will undertake an analysis of the trend in global emerging pathogens, their economic impact, the global vulnerability status and ethical implications. better tackle these diseases and prevent them from assuming epidemic and pandemic levels. socio-economic, cultural and environmental conditions play a fundamental role in the emergence, spread and control/management of eids. in poor communities, a large part of the population live in overcrowded and squalid conditions. in these communities, mostly in the major cities of developing countries, there is a lack of clean drinking water, poor hygiene and sanitation. this environment creates opportunities for waterborne diseases and different forms of pollution. it is in the slums of the main cities of developing countries that most eids begin and spread and it is also in such areas of the cities that the greatest number of deaths are usually recorded. infectious diseases like ebola, hiv, tb, usually spread easily and widely from poor communities in emerging cities and through health workers who serve such communities. hence, poverty creates a favourable condition for the spread of infectious diseases and makes it difficult for affected people to get adequate access to prevention and care (who a, b) . the journal of infectious disease of poverty was launched in with the principal aim of fostering "interdisciplinary and transdisciplinary research that explicitly highlights the intersection of poverty and other ecological factors with disease" (xia et al. ) . in this chapter, we critically examine the socio-economic and environmental factors that influence the emergence and spread of eids and discuss the ethical issues that arise from the global response and management of eids. globally, the trend in the outbreak of eids has been increasing. jones et al. ( ) analysed the trend of eids events from to , identifying a total of eid events. they found that during this period, the number of eid events ranged from just over between and to close to events between and , with a peak of close to events between and as shown on fig. . . this peak was associated with the hiv/aids pandemic. this increasing trend in eid mirrors the overall global trend in all human infectious diseases. for instance, smith et al. ( ) found that within a -year dataset , , outbreaks of human infectious diseases were reported, with more than million cases occurring in nations. emerging infectious diseases are distributed all over the world, although their rate of emergence and spread varies from one setting to another. farmer ( ) observed that their emergence and spread appear to be high in areas with huge social inequalities. additionally, eids are more common in areas rich in wildlife and zoonotic pathogens from wildlife while vector-borne pathogens are more concentrated in lower latitudes, such as tropical africa, latin america and asia (jones et al. ). also, specific eids appear to be common within certain geographical regions. for example, evd has mainly been in sub-saharan africa while the zvd has been largely limited to the americas, situations which might be associated with the geographical location of the vectors that spread the disease. different authors have developed global maps highlighting the areas where eids have originated or are most likely to originate. these areas have been described as eid 'hotpots'. one of those maps has been developed by morse and colleagues, and illustrates the risk of emergence of infectious diseases originating from wildlife as shown in fig. . below (morse et al. ). the emergence of these diseases is driven by socio-economic, environmental and ecological factors (jones et al. ). the major factors include ecological changes (including those due to economic development and land use), human demographics, behaviour, international travel and commerce, technology and industry, microbial adaptation and change, and breakdown in public health or control measures (morse ) . examples of specific factors affecting infectious disease emergence is shown on table . . there is a direct correlation between poverty and the emergence of infectious diseases which can be seen through different factors. for instance, the ecological footprint left by humankind is evident by the direct impact on the land, air and water of a specific area or region, used to sustain the depletion of natural resources for an individual or a community. among the eids of zoonotic origin that make up more than % of all eids, evidence suggests that between and their emergence was largely attributed to changes in land use ( %), human susceptibility to infection ( %), intensification of agricultural practices ( %), and changes in the food industry ( %) (keesing et al. ) . a combination of 'other' factors (international travel and commerce, changes in human demographics and behaviour, changes in the medical industry, climate and weather, breakdown of public health measures, and unspecified causes) accounted for % of the drivers. figure . shows the global percentage of emergence events caused by each driver (a) and the countries in which the emergence events took place, and the drivers of emergence (b) from to (keesing et al. ). this study found that a decrease in the diversity ecosystem that is associated with changes in land use, changes in agricultural and other food production practices such as wildlife hunting, which has led to increasing contacts between humans and other animals has facilitated the emergence of infectious diseases of zoonotic origins (keesing et al. ). there is a close nexus between poverty and infectious diseases. poverty has provoked a wave of rural urban migration of people in search of new opportunities, which has led to population explosion in the major cities of most developing countries. the result of this has been the growth of slums and expanding cities with "new opportunities for infectious diseases to flourish and spread" (eisenstein ) . the concentration of people in squalid conditions leads to waterborne diseases and different forms of pollution. also, the magnitude and pace of the spread of infectious diseases is usually influenced by overcrowding in tandem with poor hygiene and examples of specific factors relationship to poverty ecological changes (including those due to economic development and land use) agriculture, dams, changes in water ecosystems, deforestation/reforestation, flood/drought, famine, climate change due to poverty, humans may move into new areas (eg. in search of food or shelter -forest clearing for agriculture, wildlife hunting) where there is a higher likelihood for infection. for example forest clearing and wildlife hunting exposes humans more to wildlife that may serve as reservoirs for these infections human demographics, behaviour societal events: population migration (movement from rural areas to cities), war or civil conflict, economic impoverishment, urban decay, factors in human behaviour such as the commercial sex trade, intravenous drug use, outdoor recreation, use of childcare facilities and other high-density settings growing urbanisation or conflicts, may force humans, especially the poor into behaviours (risky sexual habits) that may increase the likelihood of mergence and spread of infectious diseases. worldwide movement of goods and people, air travel increasing international travels to major international cities may increase the cost of living and force poorer individuals to areas where there is increased contact with eid vectors or engage in risky behaviours that may lead to infectious disease emergence/ re-emergence technology and industry food production and processing: globalisation of food supplies, changes in food processing and packaging poverty may cause poorer people to sell their organs which may decrease their immunecompetence and enhance the re-emergence of infectious disease health care: new medical devices, organ or tissue transplantation, drugs causing immunosuppression, widespread use of antibiotics microbial adaptation and change microbial evolution, response to selection in the environment poorer people are more likely to be engage in self-prescription of antibiotics due to the cost associated with seeing a qualified health professional. such practices might increase the re-emergence of eids associated with antibiotics resistance. (continued) sanitation. for example, it is reported that the recent ebola outbreak in west africa spread widely and rapidly in densely populated and highly mobile, urban slums in cities like monrovia and conakry (eisenstein ) . it was particularly among poor people in the slums, who lacked basic hygiene and sanitation infrastructure in the cities, that there was a serious outbreak. the un-habitat estimates that million people-one-third of the developing world's urban population live in slums. most slums do not have access to safe and clean drinking water and where there exist, it is usually easily open to contamination. these conditions are favourable for the spread of diseases such as cholera, a bacterial diarrhoea and typhoid infections which are transmitted through food and water. for instance, cholera outbreaks were almost an annual occurrence in the northern part of cameroon during the past years. what accounts for this is extreme water shortage and climate variability, poor sanitation, poor drainage systems with no toilet facilities, in some areas, so that during the rainy season, water carries all human and animal waste into the main sources of water for household use. in some parts of this region, humans and animals depend on the same source of water (nfor ) . this exposes the people to waterborne and food related diseases like cholera and typhoid. moreover, the "urban poor" usually travel far and wide in search of work, greatly increasing the areas that could be affected by the virus and making contact tracing very difficult (eisenstein ) . in slums, there are small ponds, abandoned vehicles, tyres and plastic waste which serve as ideal habitat for the insects that spread dengue and yellow fever as well as malaria. improvement in living conditions-less crowding, fewer animals and higher quality homes and education may help reduce the possibility of people contracting and spreading infectious diseases. in addition to the aforementioned factors, high burden of disease, fragile health systems and socio-economic disparity aids in the proliferation of disease vectors and increases vulnerability which can be seen in the patterns of global warming on the african continent. the earth has an ecological system that is comprised of biospheres that are interconnected and rely on each other. once that is interrupted, for instance, with migration, this disrupts the balance of the biospheres and adds to the burden of this disruption and environmental decay (abayomi and cowan ) . other factors are increased temperatures, rising sea levels, and increased air pollution. these adverse climatic conditions cause food and waterborne diseases as well as regular ruthless natural disasters (tosam and mbih ) . moreover, circumstantial evidence shows that deforestation may have played a role in the west african ebola epidemic of / . the index case for the west african ebola virus disease outbreak, lived in a district known as maliandou, in a village called guéckédou, in guinea-conakry (marí saéz ). this area is known as the forest region, however only approximately % of the trees are still standing. the area has lost most of the vegetation due to the mining of iron ore, bauxite, gold and aluminium (marí saéz ). wild animals have lost their ecosystems and many use the roofs of thatched huts to nest, living in closer proximity to the villagers. the few remaining trees, are colonized by bats and other animals. it is thought that the insectivorous bats in the hollowed-out tree, in the yard of the index case, may have been the reservoir for the ebola virus, zaire strain (ebov). research shows that this particular species (mops condylurus) are able to survive infection by ebov (marí saéz ) . amongst the first twelve evd cases, none were hunters and the index case, was a toddler which led epidemiologists to believe that domestic spaces were in danger for the spread of the disease. clearly, close living proximity between the bats and the people of the village provides circumstantial evidence of the mode of transmission of this disease, given the issues with deforestation (borchert et al. ) . while eids have contributed to exacerbating global health inequalities, inequalities in socio-economic conditions globally have arguably also contributed to the emergence and re-emergence of infectious diseases. as has been demonstrated in the earlier sections, developing regions (mostly low and middle-income countries) and impoverished communities and people tend to bear the brunt of eids and it negative impacts. considering the prevailing situation, it would be expected that any efforts in managing the emergence and re-emergence of infectious disease would place more attention and resources in addressing the root causes, especially in developing and low-resource countries. additionally, one will expect that countries and regions with relatively lower capacity to detect these diseases should receive more attention as the pace with which disease outbreaks are recognized is critical for establishing effective control efforts (kluberg et al. ) . however, existing evidence suggests that global resources devoted to countering the emergence of infectious diseases are poorly allocated, with the majority of the scientific and surveillance effort focused on countries from where the next important eid is least likely to originate (jones et al. ). this raises serious ethical issues as it would be expected that for a more effective global response to the prevention and control of eids, more resources should be channeled to regions and countries with the greatest risk of experiencing the emergence of these diseases. due to our shared elements of vulnerability, there is an urgent need for international cooperative endeavours to promote and preserve health since eids know no geographical and economic borders. in the past, vulnerability to eids and other health challenges was defined by geographic location and economic factors, but because of the high level of international interactions and movement of persons and goods across borders, this is no longer the case. and since those in affluent countries benefited from the accident of geography and climate as well as efficient health infrastructures which protects them from many threats to eids, it is difficult for many to identify with vulnerable people in poor countries. on the contrary, poor people, as a result of their geographical location (mostly in tropical regions) with harsh climatic and economic situations, weak and inefficient health infrastructures; lack access to the health goods needed to prevent them from eids (west-oram and bux , ). however, because of globalization and climate change, the tides are changing and vulnerability is being redefined. today, protection or exposure to eids and other tropical diseases is no longer determined by geographical location or economic situation; "all persons are increasingly united in their vulnerability to emerging threats" (west-oram and bux , ). there is need for global cooperation and solidarity between the affluent and poor nations. this can only be effective if both parties identify with each other and acknowledge their shared vulnerability. for example, with the ease in international air travels, an eid in sub-saharan africa (ssa) or asia can reach europe or the usa within a couple of hours. the recent evd and zika virus pandemics have firmly revealed the extent of global vulnerability and response to eids. while evd-infected expatriate health personnel were flown to their countries for treatment, local evd-infected health personnel were not accorded such treatment. this attitude was motivated by the failure to recognize the similarity between citizens in rich and poor countries which emerging health threats has exposed us to in our increasingly interconnected interdependent world. it further reflects the discrepancy that existed in international health in the past which does not longer hold today. this calls for a paradigm shift from the charity-based approach to a solidaristic one (west-oram and bux , ) . an approach that acknowledges our global interdependence and shared vulnerability to global health threats such as eids; and recognizes that if a neighbour's home is on fire, efforts must be made by all to put off the fire, otherwise it may spread and consume more homes (including ours) that may even be further away from the initial home on fire! in fact, the who has proposed solidarity as one of the key ethical principles in the management of infectious disease outbreaks globally (who ) . this principle justifies engagement in collective action in the face of common threats such as eids, while supporting efforts to overcome inequalities that undermine the well-being of minorities and groups suffering from discrimination. one potential application of this principle globally is the provision of financial, technical, and scientific assistance by high income and developed countries to low-income and impoverished countries to boost their capacities to prevent and manage ongoing and future eids. this is in fact one of the obligations of governments and the international community in the who 'guidance for managing ethical issues in infectious disease outbreaks' (who ) . therefore, ensuring support for low resource and poorer countries in the prevention and management of eids through global solidarity, global health will also be enhanced by reducing the risk of eids spreading to other countries. this can be achieved through strengthening lmics' capacities to adopt and effectively implement the international health regulations, a legally binding instrument of international law that aims among others to assist countries to work together to save lives and livelihoods endangered by the international spread of diseases and other health risks. it is expected that affluent countries have a moral responsibility to support or sponsor research for neglected tropical diseases as well as emerging infectious diseases, and the countries and regions most affected need to take the lead in responding to and in contributing resources to support affected persons, not only as a duty, but on the basis of rational self-interest. moreover, solidarity does not require that only the rich should identify with the poor; it involves identifying with all persons be they rich or poor. for example, during evd outbreak in west africa in - , most african countries and the african union were sluggish in taking the lead in the fight against the disease (metz ) . by the time western countries had pledged billion us dollars, african countries had barely managed to raise $ . .cuba, alone, had sent more than health workers whilst the au had just started deploying only medical personnel (metz ) . developing countries are usually ill-prepared to monitor, prevent and manage the outbreak of these diseases. in this chapter, we have shown that there is an inextricable link between socioeconomic, cultural and environmental conditions and the emergence or re-emergence of eids. eids have contributed in exacerbating global health inequalities as most areas where eids are common are also areas that experience lack of access to basic life-saving and preventive medicines. for any fight against these diseases to be successful, mechanisms must be put in place to redress these determinants as well as bring together the intellectual, financial and health resources of the world for all, especially people from low and middle-income countries where the local capacity to appropriately manage eids is relatively weak. this is because in our interconnected and interdependent world, no individual, group or nation is insulated from the threats of eids. it is important that rich countries play a fundamental role in dedicating resources and increasing funding for research in capacity-building and drugs for eids in developing countries, not only because their own populations are also vulnerable to eids, but also for the sake of global solidarity. also, the countries where eids are more likely to occur and those whose capacity to effectively manage eids is weak, must also play a leading role in addressing the socio-economic, cultural and environmental conditions which facilitate the emergence and spread of infectious diseases. open access this chapter is licensed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. the hiv/aids epidemic in south africa: convergence with tuberculosis, socioecological vulnerability, and climate change patterns a closer look at the ebola outbreak in west africa disease: poverty and pathogens social inequalities and emerging infectious diseases global trends in emerging infectious diseases impacts of biodiversity on the emergence and transmission of infectious diseases global capacity for emerging infectious disease detection identification and diagnosis of newly emerging pathogens how to deal with neglected tropical diseases in the light of an african ethic factors and determinants of disease emergence. revue scientifique et technique-office international des epizooties prediction and prevention of the next pandemic zoonosis recurrent cholera outbreak in far-north cameroon highlights development gaps investigating the zoonotic origin of the west african ebola epidemic climate change, health, and sustainable development in africa guidance for managing ethical issues in infectious disease outbreaks. geneva: world health organization social determinants of health: social exclusion combating infectious diseases of poverty: a year on key: cord- -pyn enz authors: zaras, nikolaos v. title: case study – greece date: - - journal: biopreparedness and public health doi: . / - - - - _ sha: doc_id: cord_uid: pyn enz biological terrorism and the need for biological defence is a relatively new concept for greece. although defence against weaponized pathogens was part of cbrn training in the military, it was the / massacre followed by the anthrax letters horror that triggered a more active involvement of the greek public health sector. in that historical moment a third bullet was added to the already existing disease outbreak classification – naturally, accidental and now deliberate. these incidents and the subsequent olympic games in athens drove the greek government to focus on biodefence and revise existing civil emergency planning by inclusion of new emerging threats. naturally occurring or accidental outbreaks of a disease usually take place in both urban and country environments. big cities are usually the targets of bioterrorism due to the high density of population resulting in both physical and psychological casualties. if the disease does not start from one's own country then early warning might be possible, leading to preventive measures all the way from the borders into the community. the h n virus pandemic is an example of this globalization of medical information that is useful to both countries and citizens. one important parameter of the epidemiology of infectious diseases is the movement of the populace either for professional, recreational or immigration reasons. in the past, moving from one location to another, even within the same country borders, took considerable time. in modern times, usually less than h is needed to cross the world. we witnessed the contribution of faster travel to the spread of disease recently during the fl u pandemic. apart from the legal movement of a population, mass illegal immigration also poses a signi fi cant problem in certain parts of the world -e.g. in greece -in relation to the spread of a disease or reemergence of old diseases like malaria or tuberculosis. the geographical location of greece and its porous borders due to the signi fi cant coastline make it an attractive destination for those seeking a better living environment or as a way to enter other eu countries as a fi nal destination. greece receives a considerable number of tourists annually that exceeds its own population. greeks also travel globally for the reasons mentioned above. this constant movement of a populace makes epidemiologic surveillance and disease prevention extremely dif fi cult. the reality of disease transmission as a result of immigration and travel is re fl ected below in the results from the hellenic centre for disease control and prevention (cdcp) [ ] and various relevant ngos addressing the health status of immigrants and transmission of old and new infectious diseases. the percentage of declared cases of speci fi c diseases attributed to greek citizens and immigrants is shown in table . . timely information is crucial when it comes to a natural or accidental outbreak of a disease. this information might be bene fi cial to laboratory or institution workers or the population that needs to be protected. of course, in most cases, basic hygiene measures (personal or collective, at home or in a wider infrastructure such as schools) can prevent these diseases. defence against a deliberate outbreak of a disease requires intelligence. this type of medical intelligence is attributed to national intelligence service both civilian and military. usually international collaboration is mandatory when weaponized pathogens are the problem. risk identi fi cation and assessment contribute to national defence as well. it is a continuous process dealing with both the deliberate and non-deliberate forms of disease outbreaks. internal (sanitary institutions, police reports, etc.) and external (neighboring countries, world health organization, eu public health surveillance systems, etc.) hints can assist experts to perform a risk assessment leading to an alert of the public health system. current geopolitical instability and turmoil in our own region combined with the existing direct and indirect, overt and covert threats against western societies make bioterrorism attacks a potential risk. production of biological weapons is both easy and cost effective. of course we must discriminate between the production and weaponization of pathogens that is not as easy and needs specialized equipment. pathogen production does not require large factories and existing facilities in commercial infrastructure (food industry, drug industry) can be used for this purpose. on a smaller scale, pathogens can be cultivated in small laboratories or mobile caravans similar to those used to produce illegal drugs. identi fi cation of such illegal laboratories is very dif fi cult. viral pathogens are more dif fi cult to produce as compared to bacteria and also need some extra precautions and equipment. large quantities of biological weapons can still be produced in a short period of time (days or weeks) in small laboratories. according to kathleen c. bailey, former assistant director, of fi ce for disarmament and armaments control, who visited many biotechnology and pharmacology companies, a complete biolab requires no more than a room of . m × . m and a budget of usd , for supplies [ ] . in such a room, trillions of bacteria can be quickly produced with low risk and with minimum personal protection equipment such as a gas mask and a plastic suit over clothing. dif fi culties relevant to the production of biological weapons include: dif fi culties in the protection of workers at all levels of production, transportation, • and fi nal dispersal of biological weapons; low level of training and expertise can lead to accidents and exposure to • pathogens; vaccination of those involved is not always protective/effective; • controlling the quality and quantity of produced material is dif fi cult; • dispersion is not without problems since dispersal device explosives, uv expo-• sure, or weather conditions such as rain or drying may have negative effects on pathogens or spores; storage of pathogens poses additional problems; speci fi c conditions are required • to maintain the ef fi cacy, and it is dif fi cult to maintain them in a form ready for dispersion over long periods of time. key stakeholders in public health preparedness and response systems are: epidemiologic monitoring is the systematic and continuous collection, analysis and interpretation of sanitary/medical information relevant to public health. the objectives of epidemiologic monitoring are: follow-up of tendencies (estimate the impact of a disease or health problem the system of mandatory reporting of diseases represents the basis of epidemiologic monitoring in most countries; usually it is supplemented by more specialized systems, networks or studies with speci fi c objectives. the objectives of this system are: speci fi c (for the system of mandatory reporting of diseases) -detection of spo-• radic cases; detection of epidemic cases generic (for every system of epidemiologic monitoring) -estimation of reper- the reporting process can start from the clinical or laboratory doctor or the hospital's infectious diseases nurse but has to be sent immediately (by fax) to the regional health directorates and cdcp. the reporting form includes the following data: after reporting, evaluation of the validity/completeness of the reported elements will follow along with a thorough investigation of the case that will lead to a systematic/ rapid analysis and interpretation/export of the conclusions. brie fi ng of public health/ sanitary/medical/nursing services will follow a complete evaluation of the system. : elementary mandatory reporting of diseases (newspaper of government, no. , / / ). - : legislation on systematic mandatory reporting of diseases (cholera , smallpox , plague ) . : "measures taken against infectious diseases justifying their reporting as mandatory", art. : mandatory reporting of diseases (rd / - - ) . : essential improvement of mandatory reporting system (national centre of epidemiologic surveillance and intervention). : "organization and modernization of public health services and other provisions", art. , law / - - : epidemiologic monitoring of pestiferous diseases is practiced and coordinated by cdcp. : "regulations applied for regional systems of health and providence", art. , law / - - : cdcp -each private or public medical institution or individual doctor, operating legally, is obliged to inform cdcp of each case of pestiferous disease that comes to his/her attention. hellenic personal data protection authority: : "protection of individuals from the manipulation/exposure of data of per-• sonal character", art. , law / : exceptionally, it is allowed: if it concerns subjects of health; -if it is executed by a health professional in duty of secrecy; -if it is essential for medical prevention. this system was set in operation in and revised in september . it deals with common diseases with minor indications (usually). its scope is to support the health system through data gathering and processing, to make a clear estimate of diachronic trends and detect a possible epidemic elevation in an area or region. a large number of selected primary care doctors participate in this system/ programme. these doctors are distributed all over the country in the following networks: private doctors network ( physicians); • regional health care centres/clinics ( physicians); • social security institute health units network ( physicians). • the diseases included in the system of illness observers at the fi rst degree health care centres are: whooping cough, measles, mumps, rubella, varicella, in fl uenza of infective etiology, respiratory infection with fever (> . °c). a weekly report is done of the number of cases and patients. the report is done according to the clinical fi ndings and de fi nitions. all public sector services, in the case of a suspected or con fi rmed biological incident -deliberate or not -that needs to be treated, alert the civil protection operations' centre of gscp. gscp then activates the crisis management team (cmt) which consists of representatives from police, fire service, first aid national center (fanc) [ ] , national defence general staff, centre for disease control and prevention (cdcp) and the gscp itself. gscp's representative coordinates the functions of the cmt through telephone or video conference. after the thorough evaluation of the severity of the incident and the classi fi cation with different color codes if necessary, cmt will conduct a meeting at the gscp building for better coordination of the operation. when an initial estimation has been made, medical directorates in various/all regions of the country are informed and guidelines are issued. medical directorates are obliged to report immediately to the gscp about any laboratory result following citizens' examinations and inform the public according to the guidelines of gscp. different missions are given to police and fire service depending on the incident's nature. if needed, national defence general staff contributes resources through its military hospitals, laboratories, mobile laboratories, medical personnel, services (mass vaccination) and equipment (direct supply of masks with fi lters against biological agents, personal protective suits, decontaminants, antidotes, drugs, mobile toilets, and decontamination facilities) or other supportive units (e.g. to clear or secure an area, for quick transportation or relocation of people, etc.). in case of a cbrn agent release, hellenic national defence general staff activates its special joint cbrn company which has the capability to be airborne and deploy anywhere in greece within h (maximum), to conduct a cbrn search, survey, identi fi cation, sampling, decontamination, and provide specialized fi rst aid. for bioterrorism agents, this company has the capability to operate portable biological detectors that can identify pathogens of special interest, such as those causing anthrax or plague, within min (up to biological samples can be processed simultaneously). the platoon was established after the olympic games by merging the two specialized units (one fi eld unit operating in both hot and warm zones and one hospital-based unit deployed at the army general hospital of athens) that were created and deployed during the games in support of fi rst responders. the optimal function of the system include: systematic weekly reporting • sharing small amounts of information with constant fl ow from the laboratories syndromic surveillance (special systems) this system is activated in special conditions or when there is a speci fi c objective. it applies to the reporting of predetermined clinical conditions ("syndromes") and not diagnosed diseases (e.g. "respiratory infection with fever" instead of syndromic surveillance applies to the system of illness observers, early detection of epidemic elevations or individual incidents with public health importance hepatitis a (syndrome compatible with acute hepatitis) • septic/unexplained shock monitoring of hospital infections) and studies with speci fi c objectives also exist. these are clinical-laboratory networks for special pathogens, such as hospital bio-pathology laboratories, reference laboratories, specialized laboratories, and special clinical units • contagious spongiform encephalopathy (centres for spongiform encephalopathy • reporting) poliomyelitis (centres for poliovirus reporting) • meningitis (centres for meningitis reporting) weapons of mass destruction: costs versus bene fi ts supplement to the general plan for civil protection "xenokrates" with the specialized plan for the management of human casualties" . ministry of citizen protection key: cord- -v ye authors: ganguly, nirmal kumar; saha, gautam kumar title: pharmacogenomics and personalized medicine for infectious diseases date: - - journal: omics for personalized medicine doi: . / - - - - _ sha: doc_id: cord_uid: v ye humans have been plagued by the scourge of invasion by pathogens leading to infectious diseases from the time in memoriam and are still the cause of morbidity and mortality among millions of individuals. trying to understand the disease mechanisms and finding the remedial measures have been the quest of humankind. the susceptibility to disease of an individual in a given population is determined by ones genetic buildup. response to treatment and the disease prognosis also depends upon individual’s genetic predisposition. the environmental stress induces mutations and is leading to the emergence of ever-increasing more dreaded infectious pathogens, and now we are in the era of increasing antibiotic resistance that has thrown up a challenge to find new treatment regimes. discoveries in the science of high-throughput sequencing and array technologies have shown new hope and are bringing a revolution in human health. the information gained from sequencing of both human and pathogen genomes is a way forward in deciphering host-pathogen interactions. deciphering the pathogen virulence factors, host susceptibility genes, and the molecular programs involved in the pathogenesis of disease has paved the way for discovery of new molecular targets for drugs, diagnostic markers, and vaccines. the genomic diversity in the human population leads to differences in host responses to drugs and vaccines and is the cause of poor response to treatment as well as adverse reactions. the study of pharmacogenomics of infectious diseases is still at an early stage of development, and many intricacies of the host-pathogen interaction are yet to be understood in full measure. however, progress has been made over the decades of research in some of the important infectious diseases revealing how the host genetic polymorphisms of drug-metabolizing enzymes and transporters affect the bioavailability of the drugs which further determine the efficacy and toxicology of the drugs used for treatment. further, the field of structural biology and chemistry has intertwined to give rise to medical structural genomics leading the way to the discovery of new drug targets against infectious diseases. this chapter explores how the advent of “omics” technologies is making a beginning in bringing about a change in the prevention, diagnosis, and treatments of the infectious diseases and hence paving way for personalized medicine. over the millennia with the progression of human civilization, the condition of human health has changed considerably. the lifespan of the human has increased considerably with the advent of vaccines against several diseases which has eradicated small pox, and now we are embarking on the global campaign to eradicate poliomyelitis and have controlled the disease in most parts of the world. the treatment of infectious diseases got a boost with the discovery of penicillin by alexander fleming in the earlier part of the twentieth century. as a range of antibiotics were later discovered, infectious diseases such as meningitis, bacterial pneumonia, sepsis, and other lifethreatening bacterial infection were treatable. also the survivability of patients undergoing operative procedures and aggressive chemotherapy was feasible and their recovery high. the last years of the twentieth century have been eventful with the discovery of antimicrobials that had given us the hope that we shall eradicate all infectious diseases. despite all the efforts and progress we have made in medical science, infectious disease remained a major health problem. but the golden era of the antibiotics will not be long if we go on unregulated administering and promoting rampant use of antibiotics, as is evident from the rise of antibiotic resistance (caramia and ruffi ni ) , and the new emerging diseases have posed major challenge (table . ). in the last few decades with the advent of fi eld of genomics, there is a new hope in prevention, diagnostics, and treatment of infectious diseases. we will explore in this chapter the table highlights the organisms that are of public health importance and their year of discovery source : world health organization: newly discovered organisms of public health importance: page . from who regional offi ce east-asia: combating emerging infectious diseases in south east asia region ( ) how the host genetic polymorphisms of drug-metabolizing enzymes and transporters affect the bioavailability of the drugs which further determine the effi cacy and toxicology of the drugs used for treatment. further, the fi eld of structural biology and chemistry has intertwined to give rise to medical structural genomics leading the way to the discovery of new drug targets against infectious diseases. this chapter explores how the advent of "omics" technologies is making a beginning in bringing about a change in the prevention, diagnosis, and treatments of the infectious diseases and hence paving way for personalized medicine. how the advent of new technologies is bringing about a change in medical treatment of the infectious diseases. for the use of "omics" technology to be successful requires considerable information of pathogen genome as well as genome information of the host. the pathogen genomic and proteomic information helps to identify antigens that can give us information necessary for making a diagnostic tool and vaccine design. the pathogen genome on one hand gives us the information about the important genes conferring disease pathogenesis as well as drug resistance, while the genome of the host on the other hand will reveal the susceptibility genes, and the further knowledge of polymorphisms in genes of the host metabolic and immune system will lead to the new vaccine strategies, drugs targets, and also their treatment outcomes. rapid advances of biotechnological and informatics tools in the past few decades mainly in fi elds of genetics, genomics, and proteomics are leading the way in identifying treating and thus improving the health of human beings. the effective treatment in a patient can only be achieved by fi rst rapid diagnosis of the disease and also identifying its causative agent that is particularly important in the cases of infectious diseases. new insights gained by the analysis of genome and structural feature of pathogen macromolecules have brought about new hope in the treatment of the dreaded diseases. the knowledge of system biology in respect to the microbial infections is still in development, and data available is mostly for few human infections. the rapid development of new generation sequencing technologies have led to generation of new knowledge base and with more advancement of such technologies in the coming years has brought in a hope that all diseases will conquered. in the near future, we will have complete sequences of the total transcriptomes, like genome sequences a decade earlier, and proteomic technologies will attain the throughput and sensitivity of microarrays. other technologies like metabolomics, glycomics, lipidomics, and phosphoproteomics when referred to in the context of infectious diseases are still in various stages of development, but we are taking the right steps in the direction of development of such technologies (antony et al. ). the technologies of transcriptomics and functional genomics are transforming our understanding of microbial infections and helping us decipher the reason of infections susceptibility in the humans. transcriptomics have been developed and used by scientists to broaden our understanding of infectious diseases. to elucidate the hostpathogen interaction, cdna microarrays have been widely used. the studies have focused on how the pathogens effect the host cell gene expression. the wild-type virulent strains and isogenic mutants have been used to gauge the responses of the host cell. the major fi ndings of these studies have shown how the pathogen virulence factors modify host cell factor expression (roy and mocarski ) . the role of pathogen recognition receptors (prr) affecting hostpathogen interaction has been studied. these studies have shown that the host cell responses have an alarm signal (jenner and young ) . studies have also shown that gene cluster signals are responsible for generating the alarm signals that are the target of attack by the invading pathogens (hamon and cossart ) . array technologies are being used with molecular probes of host human (also animals/plants) and microbial genes to monitor and at the same time point the expression of genes from host cells and those of the pathogens to better understand the full complexity of host-pathogen interaction. functional genomics have also led to the development of tools to decipher infectious diseases by manipulating the cellular mechanisms. the technology of the rna interference (rnai) has undergone tremendous development in the last decade which has led to the large-scale reverse genetic screens in human cells and model organisms (boutros and ahringer ) . rnai technology uses double-stranded ribonucleic acid (dsrna) with having complementary sequence to the target mrna sequence and is used to silence or downregulate the gene expression of its target. long dsrna induces interferons or other unspecifi c responses in mammalian cells which is avoided by the use of small interfering dsrna (sirna) directly or small hairpin rna (shrna). rnai screening using rna probes, which induces loss of function of host genes, leads to discovery of host resistance factors (hrf). it is made possible when silencing this restrictive factors leads to invading pathogen replication enhancement and may also identify host susceptibility factors (hsf) and also identify permissive factors, that when silenced will decrease the pathogen replication. the rnai screens still have some limitations due to offtarget sirna effect (echeverri et al. ) . thus, the primary screening validation is made by using additional sirna screens. from the several rnai screenings in human and in fruit fl y cells, only host factors were validated from about , and , targets identifi ed in the initial screen (agaisse et al. ; brass et al. ; konig et al. ; krishnan et al. ; zhou et al. ) . to make the system biological tools like rnai more effective in fi nding mechanisms in host-pathogen interaction and thus fi nding cure for the microbial infections, there is a need for integrations of all data obtained from the omics technologies. in addition several rounds of biological experimentations are required by using mutant pathogens, cellular rnai knockdowns, or humanized animal models using mice or primate infection model. the resulting inferences from the validated data would help us build predictive models which could lead us to the better understanding of pathogen interactions with the host. it is evident from human history of infectious diseases that not everybody in a given population is affected by an infective disease. for an infective organism to cause an infection, both the virulence of the pathogen and the host susceptibility are important. the identifi cation of genetic factors of host innate and adaptive immunity that determine the protection from pathogen is an important endeavor of scientists. animal models of infectious diseases especially mouse models have been used to fi nd the genetic factors and biochemical mechanism of disease susceptibility (marquet and schurr ) . the identifi cation of candidate genes responsible for disease susceptibility or resistance and the occurrence of genetic polymorphism in them give us the best possible biological scenario of the disease. researchers have found that in bacterial diseases, tuberculosis and leprosy seem to have similar genetic susceptibility determinants in the host as exemplifi ed from the fi nding that higher incidence of these diseases was found in the monozygotic twins than in dizygotic twins and siblings (abel et al. ; vidal et al. ) . mouse model of infections has revealed that gene encoding the natural resistance-associated macrophage protein ( nramp ) confers natural resistance to infections caused by mycobacterium , salmonella , and leishmania . nramp is found in the membrane of the phagosome of the macrophages where it seems to be probably affecting the replication of the infecting intracellular bacterium (gruenheid et al. ) . the genomic analysis in humans has found a similar gene to that of mice which is also having similar pattern of expression. hence, it has been inferred that humans too carry similar susceptibility gene. there have been further studies which have shown that polymorphisms found in the nramp gene are related to the infectivity of leprosy and tuberculosis (abel et al. ). in one of the studies, it was found that persons who carry an nramp heterozygous variant will be four times more likely to be infected by tuberculosis than persons who are carrying more common variants of the nramp (bellamy et al. ). cell-mediated immunity plays an important role in the context of tuberculosis and is well studied. research has further gone ahead to fi nd links between tuberculosis susceptibility and polymorphism in the gene coding for receptors of interferon-γ and interleukin- , which are cytokines belonging to t-helper cell type (th ). the absence of functional copies of either of these genes in families and of isolated patients leads to high susceptibility to m. tuberculosis infection (jouanguy et al. (jouanguy et al. , newport et al. ; altare et al. ) . the highly polymorphic human leukocyte antigen (hla) system is the name of the major histocompatibility complex (mhc) in humans. the hla class i glycoproteins are highly expressed on the surface of every nucleated human cell, and they present endogenous peptides derived from the cell to the cytotoxic t cells. hla class i glycoprotein plays a major role during the viral infection as it presents intracellular viral peptides on the surface which leads to cellmediated immune response, which further leads to the destruction of virus-infected cells. hla class ii glycoproteins which are present on the surface of antigen-presenting cells (apcs) on the other hand present - amino acids long peptides that are derived from the engulfed pathogen and displayed on the surface which then are recognized by t cell as foreign antigens, and it will elicit an immune response to the antigen. the length of antigen as well as composition are important in deciding if the antigenic peptide will bind to the antigenic peptide-binding cleft. polymorphisms occur almost solely in the peptide-binding cleft of hla class i and ii in the glycoproteins. the diversity of hla-binding region ensures that some pathogenic peptides will be preferentially presented compared to the others. thus, in a given population, the hla diversity ensures the advantage that some of the hla glycoprotein peptide-binding clefts will be able to bind and present the pathogenic antigen peptide which will lead to an immune response to any invading pathogen. this ensures the survivability of the species against an infection. thus, genomic studies have focused on the identifi cation of susceptibility genes which would lead to the better management of infectious diseases in the population. the tuberculosis susceptibility has been associated with hla class ii genetics. the association is evident from the studies that have been found between pulmonary tuberculosis and class ii hla antigens in several populations (marquet and schurr ) . as is now clear, the knowledge of the mechanism of action of the pathogen and the identifi cation of the susceptibility genes goes a long way in the management of the disease in context of a public health perspective to prevent, diagnose, identify, and target the vulnerable populations against a given infectious disease. the requirement of the genomic information of both the host and pathogen is important to fully carry out infectious disease management. the fi rst sequence map of human genome being completed in june (lander et al. ; venter et al. ) , followed by discovery of genome-wide single-nucleotide polymorphisms (sachidanandam et al. ) and further genomic sequencing of several pathogens by institutes like the j. craig venter institute ( www.jcvi.org ) gradually opened the pathway to create new treatment and disease management methods. the ultimate aim of genomic technologies to bring personalized medicine for every infectious disease scenario is still decades away, but here we will focus only on important breakthrough which has shown us the way forward in respect to infectious disease identifi cation and treatment. neisseria meningitidis is a bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a lifethreaten-ing sepsis ( www.cdc.gov/meningococcal/ ). n. meningitidis is a major cause of morbidity and mortality during childhood in industrialized countries and has been responsible for epidemics in africa and in asia . neisseria meningitidis serogroup b is responsible for causing about one third of infection. the genome sequence of n. meningitidis has opened a new way for disease management (pizza et al. ; tettelin et al. ) . the vaccine that was used only contained capsular polysaccharides from the serogroups a, c, y, and w only. serogroup b polysaccharide contained elements that resemble human polysialic acid and hence is poorly immunogenic and might generate autoantibodies (hayrinen et al. ) . in this scenario the n. meningitidis serogroup b was sequenced, and potential antigens from the serogroup b were expressed in escherichia coli to fi nd the potential vaccine candidate. the expressed proteins are injected into mice to fi nd immunogenic antigens that can developed into a vaccine. similar strategies are being used to fi nd vaccine candidates for other serotypes of neisseria species and for other pathogenic organisms. pathogen genomic information is also being used to fi nd the immunologically important peptides for cytotoxic t lymphocytes (ctls) epitopes. the response of ctls is to seek out virally infected cells by recognizing the peptides presented by human leukocyte antigen (hla) glycoproteins on the cell surface and killing the infected cells. ctl epitopes are the viral peptide that is presented by the hla and recognized by ctls. the peptides are of the length of amino acids, and genome sequence is used to fi nd out and synthesize these peptides for immunogenic evaluation. amino acids are divided into segments of peptide, measuring amino acids in length and overlapping the previous peptides amino acids, for example, west nile virus genome translates into , amino acids which can be segmented into , peptides that are amino acids in length. immunoinformatics, a fi eld of bioinformatics, is speeding up the fi nding of ctl epitopes for the scientist working in the fi eld. algorithms on computer softwares are being used to match the viral peptides with the hla glycoproteins in silico for binding based on previously known results and are being tested (de groot et al. ). informatics based algorithms help eliminate % of the peptides that would not be used in the experimental screens. thus, the time and effort to screen for the ctl epitopes have been reduced drastically. ctl epitopes may be used for making subunit-based vaccines and diagnostic tests. virusspecifi c antibody found using ctl epitopes can be used in enzyme-linked immunosorbent assay. it may be even possible to use ctl epitopes to test for the antigen itself. basically as of now only four types of molecular diagnostic tests are carried out to detect infection in laboratory setup. first is by direct detection where the pathogens can be detected directly by imaging technologies of microscopy and cell culture. second method of diagnosis is by the detection of proteins produced by pathogens by the use of specifi c antibodies, like that used in enzymelinked immunosorbent assay (elisa). third method is by detection of the specifi c antibodies iga, igm, and igg against the pathogens and the changes in their titers using antibody capture assay. fourth method uses detection of nucleic acid of the pathogens and amplifying their signal using techniques like polymerase chain reaction. latest diagnostic technologies have been developed on these basic four biotechnological technologies (speers ) . pathogen genome can also be used to identify the infecting organism itself. microbial dna in the clinical specimen can be used to identify the disease-causing pathogens. human immunodeficiency virus (hiv), hepatitis virus, borrelia burgdorferi (causative agent of lyme disease), and mycobacteria are few examples of pathogens that can be identifi ed by their genomic sequences. mycobacterium tuberculosis antimicrobial resistance strain-caused infections are becoming quite common, and genomic information has deciphered few potential candidates like katg gene mutations in resistant strains (siqueira et al. a , b ; marahatta et al. ) . the traditional culture test for mycobacterium which is time-consuming and less sensitive is giving way to restriction fragment length polymorphism (rflp), a specifi c technique used in dna fi ngerprinting (van soolingen ). the technology uses restriction enzymes that cut dna at the places having certain particular nucleotide sequences. the nucleotide pattern that is obtained is then compared to the previously identifi ed specifi c nucleotide pattern of the genome of the pathogen dna. dna patterns can be separated on the basis of length, and the pattern of dna fragments in the dna fi ngerprint is characteristic of particular isolate, and each particular pathogen has a unique pattern. dna fi ngerprint technology is faster and reliable than culturing of the mycobacteria, ideal for discovering new drug-resistant strains from unique genomic sequences of each mycobacterium. the technology is very useful for identification of strains during the time of outbreaks and further epidemiological studies. the knowledge of viral load in patients is also important for dosage determination in drug therapy; hence, detection of the viral pathogenic dna and rna in clinical specimens is of paramount importance. treatment of viral diseases like hiv, chronic hepatitis b, and hepatitis c often depends on the knowledge of viral load (revets et al. ) . for example, hiv viral loads are detected by enzymatic amplifi cation of the viral nucleic acid and detection of the signal from the labeled probes that hybridizes with them. the signal usually is either a color signal conjugated to the probe or a chemiluminescent probe, and the intensity of the signal corresponds to the number of copies of the nucleic acid rna. capillary electrophoresis detects hybridized probes at a very high sensitivity with detecting as low as , copies of hiv rna in milliliters of plasma (kolesar et al. ). the rampant uncontrolled use of antimicrobials has led to increased number of antibiotic bacterial strains. genomic mutations allow the certain bacterial strains to overcome the effects of antimicrobials and are able to propagate in spite of the presence of antibiotics. fluoroquinolones are drugs that act on the bacterial dna replication by binding to bacterial enzymes involved in bacterial dna replication, that is, dna gyrase and topoisomerase. the bacterial resistance to quinolone occurs due to mutation in the quinolone-binding site in the enzymes mentioned above. the mutation leads to change in the amino acid at the site of binding of fl uoroquinolones to the enzymes. if both the bacterial enzymes are mutated, then high-level resistance occurs to the quinolone drug affecting the treatment of infection as compared to when either of the enzymes is mutated (hooper ) . now genetic test is available to detect antimicrobial resistance in the infecting pathogens. the information is important because it would lead to a better treatment management of the infection. the methicillin-resistant staphylococcus aureus phenotype is detected when cultured in the presence of oxacillin after a period of h. before the era of omics technology, the only means of resistance detection was by culture test which is a very time-consuming test. methicillin resistance in s . aureus is controlled by alternations of penicillin-binding protein pbp a. gene meca controls the production of pbp a. polymerase chain reaction test is used to detect the presence of meca in reference laboratories, while commercially developed kit can detect the same using a fl uorescein-labeled meca probe. both dna probe and pcr technology when used for analysis can detect meca -resistant gene in a given sample in less than h. the rapid detection of antimicrobial resistance in pathogens helps patients in providing adequate treatment opportunities (louie et al. ) . the study of the host genome becomes important to fully understand the drug effects and as such design more effective methods of treatments. although the ultimate goal is to decipher the system biological effect, the trend of single gene effects is also very important. cytokines play a very important role in human immunity (paul and seder ) . in hepatitis c infection, interferon alpha is used to stimulate cell-mediated immunity against the viral infection and is the primary treatment. however, studies have shown that response to interferon-alpha treatment is only % in some cases even when combined with other antiviral treatment (manns et al. ) . further studies have shown that if chronic hepatitis c patients have il- polymorphism variant, it leads to the reduction in expression of il- itself, and they will have fi ve times more chance of effective treatment with interferon alpha than those who do not carry the polymorphism (edwards-smith et al. ) . interleukin- (il- ) is a polymorphic cytokine and is a t-helper cell type ii (th ) cytokine that is associated with the induction of the production of large amount of antibodies in body's immune response. th cytokines which promote cell-mediated immunity inhibit th response and vice versa. thus, people with high-expressing il- genotype if infected and suffering with chronic hepatitis c infection are less likely to respond to interferon-alpha treatment. new treatment regimes have to be developed for patients suffering from chronic hepatitis c infection and carrying il- polymorphism associated with high cytokine expression. vaccine responses can be used as a system of gauging the state of immune system ( poland a , b ) . vaccines are administered to large number of population as an integral part of public health system. vaccines are used to mimic the infective disease conditions that induce immunological memory to protect the individual against subsequent exposure to the pathogen and lead to prevention of disease. the phenomenon to gain protective immunity against a pathogen upon being vaccinated for the particular pathogen depends on individual genetic build. as studies have shown, not all healthy individuals are able to generate a protective immune response upon vaccination. it has been observed in the case of measles vaccination that only % of the population was seronegative and clustered in family (poland a , b ; poland et al. c ). both hla i and hla ii class alleles have been responsible for the measles vaccine response, while hla-b , hla-b , hla-drb * , and hla-dqa * are associated with positive measles vaccine response, and hla-b, hla-dr, and hla-dqa have been responsible for the vaccine being noneffective (hayney et al. (hayney et al. , poland et al. ). drugs used for targeting any pathogenic infection can only be successful if we are aware how it is affecting the host and pathogen at genomic level and hence are able to explain the host effi cacy and toxicity. we look at few important infectious diseases where pharmacogenomic research has been bringing a landscape change in the disease treatment. leishmaniasis is a very complex major tropical infection transmitted by the vector sand fl y is all right. the infection is caused by intracellular protozoan parasites of leishmania genus. there are more than species of leishmania . the type of infective species, virulence factors, and host immune responses and depending on the clinical symptoms, the disease is categorized into cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). vl is also known as kala-azar; the origin of the name is from the eastern and northeastern part of the indian subcontinent where the disease is endemic. depending upon the place where one has acquired the infection, cl is further categorized into "new world" from central america and south america and "old world" if from asia, middle east, africa, or southern europe. more than - . million cases of leishmaniasis occur worldwide (about countries are affected) with major countries being the developing nations of asia, africa, and latin america ( www.who.int/topics/leishmaniasis/en/ ) . species of leishmania are several causing different clinical manifestations of the infectious disease. l . donovani produces primary cutaneous disease as well as gives rise to visceral leishmaniasis (vl) and also post-kala-azar dermal leishmaniasis (pkdl) that is manifest after the treatment of the initial visceral disease. visceral leishmaniasis main causative pathogen is the l. donovani complex with old world vl disease being caused by the species l. donovani and l. infantum, and new world disease is mainly caused by different species of l. chagasi in the cases of infection caused by l. braziliensis complex, there is always a chance that the infection dissemination to mucosal region can occur to give rise to mucocutaneous leishmaniasis (mcl) (herwaldt ) . the complex disease is manifested due to multiple factors ranging from environmental factors such as time and number of exposure with infected vector sand fl ies, species of the infecting leishmania pathogen, to host genetic factors that include immune status of both innate and adoptive immune systems that determine the clinical outcome of the disease. other reasons for leishmania disease susceptibility are malnutrition, immunodefi ciency with hiv coinfection, and young age. the protection against invading pathogenic leishmania protozoa and even the curative resolution of the disease is provided by th cytokine response involving cytokine interferon gamma (ifn-γ), interleukin (il)- , and tumor necrosis factor alpha (tnf-α), whereas th response cytokines il- , transforming (tgf)-β, and il- have been implicated in increasing susceptibility to the disease in the experimental animals (reed and scott ; sacks and noben-trauth ) . nonhealing lesions and diffused lesions in cl have been implicated to th response, while self-healing lesion has been associated with th response (melby et al. ) . however, in some situation il- (a th cytokine) has been implicated to induce il- production and lead to th cytokine response, and it has also been found in some cases that th response occurs independent of il- (alexander and bryson ; mansueto et al. ). leishmania infection is a complex infection depending on host factors as well as strain polymorphism. leishmania mexicana cysteine proteases which target il- that prevents th protective response (buxbaum et al. ) , while the leishmania analogue of activated c kinase (lack) from the leishmania major induces th response that leads to host parasitization (kelly et al. ) . polymorphism of l. braziliensis also affects disease outcomes (cupolillo et al. ; schriefer et al. ). pkdl is a complication arising after treatment of vl, affecting % of vl patients in sudan (study carried out in united sudan) and also - % patients in india. pkdl has been found to be associated with increased levels of il- (zijlstra et al. ; ganguly et al. ). the major treatment regime of cl which has propensity of dissemination towards vl and mcl is with parenteral antimonials like sodium stibogluconate or meglumine antimoniate, pentamidine, and oral miltefosine (olliaro et al. ; ameen ; amato et al. ) , whereas cl with low risk of spread is treated with local and physical therapies such as intralesional antimonials, topical paromomycin, cryotherapy, and thermotherapy or by oral azoles. however, when the disease progresses to mcl, treatment is prolonged, and toxicity from such long-duration drug use is a common occurrence (marsden ; amato et al. ) . vaccine is still elusive in the case of leishmania. some trials with dna vaccines do have shown a way forward. these vaccines have shown the promise to be effective as they have been able to induce l- production, which was in response from the persistent antigen exposure from the dna vaccine (requena et al. ). in venezuela killed leishmania promastigotes along with bacillus calmette-guerin (bcg) used as immunotherapy have shown results with a high cure rate in clinical trials by inducing th response (convit et al. ) . l. major vaccine trial with bcg and parenteral antimony combined have been successfully used for treatment of pkdl (mansueto et al. ). the search for effective vaccine for leishmania had got a boost with knowledge from genome sequence data of several leishmania strains. more vaccine candidate genes will be evaluated in the future (stober et al. ). in the absence of an effective vaccine with recurring infection such as pkdl, dissemination infection to mucosa leads to aggravating of the disease. prolonged treatment with parenteral antimonials that give rise to high-level risk of toxicity with high morbidity and mortality from the disease is a problem of concern (convit et al. ; muse et al. (samaranayake et al. ) . l. donovani though normally associated with causing vl is shown in few places, kenya, yemen, cyprus, and the himalayan region of north india, and is the main causative pathogen of cl (mebrahtu et al. ; pratlong et al. ; sharma et al. ; antoniou et al. ) . to deduce the genetic susceptibility of the leishmania disease, experimental murine animal models along with clonal parasite line (to control environmental variable) have been used to fi nd the genes responsible for disease progression along with their human homologues of disease susceptibility (handman et al. ) . first genes that were used to deduce from such analysis in murine model were nramp and the h- locus had been implicated in l. donovani infection (blackwell et al. ) . hla class ii antigen hla-pq is found to be associated with cl in venezuela (lara et al. ) and mcl in brazil caused by l. braziliensis (petzl-erler et al. ) . pcr genotyping studies in mexico on leishmania patients has found an association with hla class ii genes with cutaneous leishmaniasis (cl) (olivo-diaz et al. ) . high blood tnf has been found to be associated with mcl (castes et al. ) and acute vl (barral-netto et al. a , b ) . a venezuelan study has implicated that allele of tnf-β polymorphism with high risk of developing mcl caused l. braziliensis and higher frequency of allele of tnf-α polymorphism was also associated with mcl (cabrera et al. ) . in brazil by using family-based disequilibrium test analysis (tdt), investigation has shown that tnf polymorphism has been linked to l. chagasi infection (karplus et al. ) . in asymptomatic patients having positive skin test, l. chagasi has been associated with tnf- allele of tnf-α gene, while in case of symptomatic l. chagasi vl patients, tnf- allele is implicated. due to parasite heterogeneity, this tnf polymorphism association has not been correlated to infection by other leishmania species such as in l. infantum vl (meddeb-garnaoui et al. ) and l. major cl (kamali-sarvestani et al. ) . variation in promoter of il- and ifn-γ gene polymorphism has been found to be linked to l. major cl disease susceptibility and progression respectively in an iranian study, while in a sudanese vl patient study, il- polymorphism has been shown to increase disease susceptibility (mohamed et al. ) . polymorphism in promoter region of il- gene leads to higher il- production which has been shown to increase the risk of having skin lesions during an infection of l. braziliensis (salhi et al. ). il- can diminish the high th proinfl ammatory response that occurs when l. braziliensis cl progresses to mcl (hatzigeorgiou et al. ; bacellar et al. ) . il- polymorphism plays an important role in the progression of l. braziliensis cl to mcl, and this fi nding is important since their genetic markers have high prognostic value (castellucci et al. ) . genome-wide linkage have been performed for l. donovani-infected vl patients in artinga ethnic group in sudan to help identify loci on chromosome q and is associated with disease susceptibility genes (bucheton et al. a , b ) . il- receptor β chain (il rb) gene is present in the highly susceptible loci on chromosome q that was identifi ed from this study. il- receptor has been detected in high levels during vl infection and plays a critical role in t cell genetic responses (barral-netto b ) . further studies have shown il rb polymorphism in association with l. donovani vl (bucheton et al. ) . another candidate gene found is slc a (formerly nrampi) on chromosome q , an innate resistance gene that regulates macrophage activation and contributes to increased vl risk in sudanese population (bucheton et al. a , b ; mohamed et al. ) as well as increased susceptibility to several intracellular pathogens (blackwell et al. ) . other studies have shown that genotypes having signifi cantly high level of mannan binding lecithin occur more prominently in patients with clinical vl. an opsonin, mannanbinding protein, is known to enhance pathogen infection. polymorphism in mannan-binding gene has been shown to increase risk of developing l. chagasi vl in brazilian study population (alonso et al. ) . in pkdl there is elevated level of ifn-γ. polymorphism of ifn-γ receptor from study in sudan has been implicated in pkdl (salih et al. ). the ifn receptor expression is important for the activation of macrophages via ifn-γ. drug treatments are not very effi cient in the treatment of leishmaniasis disease; more effective treatment regimes can be developed by thoroughly knowing the genetic factors that lead to disease progression. thus, unnecessary drug use and adverse reaction can be avoided. as various genetic susceptibility studies have shown, cytokine response determines the disease progression in leishmaniasis. role of il- in pathogenesis of leishmaniasis is known and is well established, and il- polymorphisms have shown to increase risk of lesions in l. braziliensis infection. in a study with l. guyanensis infected cl patients from french guiana, high level of mrna il- within lesions leads to poor chemotherapy response and treatment failure (bourreau et al. ) . it is hypothesized that il- might be regulating the response to chemotherapy by blocking the th response. the increased level of il- has been linked to the active vl (nylen and sacks ) and pkdl (saha et al. ) and also associated with persistent cl infection occurring from l. major (melby et al. ) and l. mexicana (louzir et al. ). success of vl treatment with amphotericin b and the complete elimination of il- are associated with one another (saha et al. ). on the other hand, mcl is associated with low il- receptor expression and low il- secretion that decrease the ability for modulation of proinfl ammatory response (faria et al. ) . progress has been made to fi nd the susceptibility genes and will provide further insight into disease pathogenesis and will lead to progress in the fi eld of diagnostic markers, drug targets, and vaccine development to control, treat, and eradicate leishmaniasis. improving the treatment of malaria by pharmacogenomics malaria is vector-borne (mosquito) disease that has been one of the top causes of mortality in the world for generations especially in tropical countries of asia and africa. even after renewed global efforts, still there is high infectivity and mortality. three billion people are at risk with - million deaths attributed to malaria each year ( www.who.int/topics/malaria/en/ ). four species of protozoan parasite are involved from genus plasmodium, i.e., p. falciparum , p. vivax , p. malariae, and p. ovale . these malaria-causing combination parasitic species occur in human population and occur in infected individuals (gurarie et al. ) . in respect to prevalence, virulence, and multidrug resistance, p. falciparum has been a major cause of mortality and morbidity. p. falciparum accounts for about % cases of malaria in africa (roca-feltrer et al. ). next to it is p. vivax which causes - million cases annually (price et al. ). the most commonly used drugs are chloroquine (cq) and sulfadoxine-pyrimethamine (s-p fansidar ® ) that are becoming less effective due to the development of resistance in malaria parasite by p. falciparum, and the species has become predominant and become a threat to travelers and people alike (schlagenhauf and petersen ) . in the absence of vaccine and in addition, development of resistance even in the mosquito vector control against chemical methods using insecticides has thrown new challenges for the researchers (greenwood et al. ) (fig. . a ). some of the recent developments in malarial treatment using pharmacogenomics are bringing about improvements in the effi cacy of treatment regime of malaria. current treatment regimes have recommended artemisinin combination treatments (acts) in cases of uncomplicated falciparum malaria in nonpregnant adults (lin et al. ) . the drug regime is highly effi cacious and has reduced development to resistance. in cases of uncomplicated malaria, the act is being used in countries by . in the coming years, a number of patients including women and children will be brought under act therapy regime as per world health organization. like the treatment of hiv and tuberculosis, combination therapy is now being used for malaria treatment too, which reduces resistance among the highly effi cacious drug the artemisinins which rapidly eliminate the parasite from blood and thus limit the number of parasites so that the other more bioavailable drugs given in combination act on the parasite. unrelated mode of action of two or more combination drugs also reduces the chances of resistance (yeung et al. ). among many other factors which contribute to drug effectiveness, malarial drug bioavailability and tolerability are depended upon the host metabolic mechanisms. the severe drug reaction to primaquine in the s used in antimalarial treatment was instrumental in the discovery of glucose- -phosphate dehydrogenase (g pd) defi ciency in ; thus, importance of the use of pharmacogenetics in malarial treatment was realized (alving et al. ). the polymorphism leading to variation g pd or even its defi ciency is a grave problem in designing the effective drugs. even now during malarial terminal prophylaxis to decrease transmission, primaquines are administered. thus, the g pd status of patient becomes quite important (luzzatto ) . knowledge of both the host and parasitic genetics is necessary to designing drugs and dosage for effective treatment regimes. parasitic genetics helps us in deciphering the modes of resistance, and host genetics help us in giving the information about host drug bioavailability and explain adverse reaction to the drugs. g pd polymorphisms and genetic variation in cyp c can play pivotal role in point of care diagnostics, but these genetic testings will have to be incorporated into the laboratories and national health programs. the knowledge of this important genetic variations in population would ultimately reduce cost and make the treatment regime more effective and with lesser adverse reaction and ultimately reduce the suffering of the patients. the pharmacogenetic drug policy in context of malaria is slowly becoming a reality as per efforts of the who and other agencies. genetics is becoming a guide to new drug policy. amodiaquine was generally known to be tolerated in malarial treatment, but later when it was found in the caucasian population during the decades of and to be responsible to cause agranulocytosis with fatalities and also cause hepatotoxicity (hatton et al. ; raymond et al. ; phillips-howard and west ) , the drug was fi rst removed from the list of essential drugs against malaria but then had to be added back to the list as alternate drugs started showing resistance. amodiaquine induced adverse reaction in individuals was attributed to genetic make up of the individual. the genotypes of individuals harboring cyp c , cypia , and cyp b have been reported in studies to show immunogenic adverse reaction to amodiaquine (li et al. ; kerb et al. ). some population in africa has shown hepatotoxicity and leucopenia with only two doses given weeks apart (orrell et al. ) . amodiaquine when administered to an individual with reduced cyp c activity impairs the metabolism of the drug and hence leads to the cause of hepatotoxicity and agranulocytosis. other common variants of the enzymes cyp c * and cyp c * have been associated with decrease in the metabolizing activity of cyp c enzyme as is evident from studies with anticancer drugs (dai et al. ) . individuals with cyp c * genotype have no cyp c enzymatic activity in vitro (parikh et al. ). in a study from burkina faso, patients carrying cyp c * genotype showed common adverse effects to amodiaquine and in addition patients have also reported to experience more abdominal pain when compared to healthy individuals. the study from burkina faso and ghana could not clearly establish the relation between drug efficacy and cyp c genotype (adjei et al. ) . though the inactivated gene of cyp c is not very high in population, estimates have shown that cyp c * and cyp c * occur in about . % of the population in zanzibar, united republic of tanzania, which was about , patients of the total malarial patients ~ , (cavaco et al. ) . in ghana it was found that . % of the population has been estimated to have metabolic variants of cyp c . hence, due to high disease burden, the study of pharmacogenomics for drug metabolism was carried out in large patient samples from the population to get a clear correlation between genotype and effi cacy of drug treatment as well as adverse reaction. major active antimalaria metabolite of artemisinin is dihydroartemisinin (dha) (ilett et al. ) . artesunate is rapidly converted to its active metabolite catalyzed via cyp a which is a major enzyme; conversion to dha also includes minor enzymes cyp b , cyp a , and cyp a (li et al. ) . cyp a has about variant forms of which at least have been implicated as slow metabolizing enzymes, and have been reported to show no activity in vitro (di et al. ) . hence, lower level of cypba enzymes in patients will have reduced bioavailability of dha the major antimalarial metabolite and hence have lower antimalarial activity. major endemic areas of malaria like sub-saharan africa, ghana, sabah region of malaysia have been evaluated for the presence of cyp a genotype. among these population of ghana has high wild-type cyp a along with % alleles being cyp a * a (gyamfi et al. ) , whereas malaysian population has an allele cyp a * a frequency of % with only % wild-type enzyme (yusof and gan ). other asian populations have been reported to carry several other alleles of cyp a with even alleles that do not show any cyp a enzyme activity at all. no activity variant of cyp a is found about . - . % in japanese, chinese, and thai populations (gyamfi et al. ) . hence, artesunate is expected to be more effective in population of ghana. in some parts of thailand, about % of patients have shown resistance to artemisinins (white ) . though it has been found by study that about % frequency of cypz alleles have no activity in the thai population and the antibiotic resistance is indicative to be related to cyp a activity and ability to convert artesunate to dha (noedl et al. ), more studies require to be done to clearly establish the relation between the genotype and resistance to artemisinin-based therapy. several mutations in gene targeted by antimalarial drug have been identifi ed which led to the resistance in vivo of act drug partners such as mefl oquine, lumefantrine, amodiaquine, and chlorproguanil (kerb et al. ; mehlotra et al. ). identifi cation of genes and mechanism is important for controlling the infection. research has yielded the information regarding the gene responsible and underlying mechanism of action resistance of some drugs against p. falciparum and p. vivax . chloroquine resistance (cqr) in p. falciparum has been linked to point mutation cq resistance transporter gene (pfcrt chromosome ). the mutation pfcrt -k t is a reliable marker for cqr. while cq-sensitive strain carries wild-type allele cvmnk , the variant cqr alleles are s agt vmnt (asia, south america, africa), s tct vmnt (south america), cvmnt (south america, philippines), cviet (southeast asia, africa), and cvmet (colombia). another multidrug resistance gene ( pfmdr chromosome ) is a parasite transporter gene. polymorphism, point mutation, and copy number variation have been implicated in multi drug resistance. in different geographic regions, the pfmdr two mutant alleles have been reported, namely, y_ y_ s_ n_ d found mostly in asia and africa and n_ f_ c_ d_ y predominantly from south america (valderramos and fidock ) . the pfcrt- and pfmdr - y mutations have been related jointly to contribute in giving rise to cqr phenotype in addition to other likely parasite genes (hayton and su ) . p . falciparum dhps enzyme ( pf-dhps , chromosome ) has been linked to resistance to the sulfa class of antimalarial drugs, while mutations in dhfr ( pf-dhfr , chromosome ) domain have been linked to high level of pyrimethamine resistance. combination of sulfadoxine-pyrimethamine (s-p) treatment failure has been found to be associated with pf-dhps double mutant ( g with either e or g), combined with the pf-dhfr triple mutant ( n_ _ r) (hayton and su ; hyde ) . point mutations in p. vivax ortholog of pfcrt (pcvg ) are associated with clinical cqr. pfmdr p. vivax ortholog that is pvmdr has been proven and has also been identifi ed. y cf point mutation of pvmdr has been linked to cqr. pv-dhs and pv-dhr gene point mutations have been identifi ed and are suspected to link to clinical resistance in antimalarial s-p treatment (hayton and su ) . more data is required for new mutations in the parasite genes, and in addition more data is needed for therapy of other act drug partners like sulfadoxine-pyrimethamine and lumefantrine. a new rejuvenation is taking place in pharmacology and pharmacokinetics development of databases of antimalarial pharmacogenetics. worldwide antimalarial resistance network ( http://www.wwarn.org/ ) has set up a module together with high-quality pharmacological research data for optimum drug dosage in light drug resistance information and adverse event reporting. the aim to achieve global cooperation will go a long way to personalized malarial treatment as per population needs. during the last half a century (for about years), the most effective treatment regimes have been the combination therapies of drugs that was because a single drug treatment was found to be in invariantly leading to resistance for the drug, leading to much more severity and complications (crofton ) . due to rampant and unregulated use of tuberculosis drugs, however, this has led to emergence of multidrug resistant tuberculosis (mdr) (fig. . b ) . now the treatment course is usually for months with the combination of isoniazid, rifampicin, pyrazinamide, and ethambutol for the fi rst months. this has to be followed up by the next months with isoniazid and rifampicin treatment. if the treatment is taken up with diligence and patient completes the whole drug course, then it has been reported that effi ciency of the treatment is very high with more than > % patients getting cured and relapse is in less than % of patients (menzies et al. ). another advantage of multidrug treatment is that the treatment regime helps in treating different population of tubercle bacilli. for the last years, knowledge from the fi eld of genetic molecular basis of drug treatment outcomes has helped us in the better management of and understanding of treatment effi ciency and of drug. the difference in drug response is found among different individuals of the population. the individual person tends to show similar type of response to tuberculosis drugs that do not change over time. thus, in light of above observations, we say that there is a huge variation in drug response among individuals due to variation in genes involved in drug metabolism, drug transporters, and drug targets compared to minimal within-subject variation as found from studies. further studies on drug response revealed that - % of variation in drug pharmacokinetics is due to genetic factors (kalow et al. ). the sequence variation in drug-metabolizing enzymes, drug transporters, or drug targets leads to the variation in drug response among individuals (evans and relling ; evans and johnson ) . some nongenetic factors such as nutrition organ function, age and other concomitant therapies, nature of disease, and drug interaction can also effect in drug response, but genetic determinant remains constant throughout the lifetime of the individual. pharmacogenomics have played an important role in deciphering therapeutic effi cacy of drug metabolism and occurrence of adverse events. though research is still being pursued to decipher the intricacies of how genetic differences play an important role in regard to clinical application of the drug however, through research we have gained information on the role of genetic polymorphism with respect to drug effi cacy for the treatment of tuberculosis. in this section we will discuss the knowledge we have gained through newer technologies in regard to different drugs being used for tuberculosis. since isoniazid has been in use for antituberculosis treatment since , it is the most well studied of the lot (ellard and gammon ) . this drug has been found to be tuberculosis specifi c in its action against tubercle bacilli and has relatively minimal toxicity. now pharmacogenomics is playing a very important role in making isoniazid the fi rst-line treatment drug. acetylation of isoniazid takes place mainly in the liver and gut mucosa. for any drug ingested in the body, it is absorbed and metabolized and then its soluble by-products are released or excreted out of the body. the drugs have specifi c retention and metabolizing rates depending upon their chemical composition and the genetic polymorphisms of the metabolizing enzymes. the activation of isoniazid is catalyzed by highly polymorphic enzyme n-acetyltransferase (nat ) and leads to formation of acetyl isoniazid. this is formed by the transfer of acetyl group from the acetyl coenzyme a to acceptor amine leading to formation of an amide. acetyl isoniazid combines with several other cellular compounds to give a variety of metabolites which do not have any antituberculosis activity. the level of acetylating isoniazid that will be subjected to during metabolism in the body determines the disease outcome. the level of bioavailability of the drugs determines whether the drug would be effective for elimination of the invading pathogen or toxic to the human body. acetylation of isoniazid varies from individual to individual depending as per his or her genetic predisposition. genetics determines the amount of active nat enzyme that an individual expresses. the metabolism of isoniazid is catalyzed by nat enzyme which takes place in liver or gut mucosa. thus, the level of nat gene expression is controlled by the type of polymorphism in nat that particular individual carries. thus, for the pharmacogenomic and personal medicine in effect to succeed, the dosage for the drugs that are metabolized by nat should be tailor made as per the enzymatic activity depending upon the polymorphic variant (roy et al. ) . the enzymatic activity being highly variable cascorbi and roots ( ) has been studied over the years in human subjects who have been categorized as slow or rapid inactivators ( http://www. brti.co.zw ). the categorization has been based on the measure of capacity of nat enzyme to acetylate isoniazid to acetyl isoniazid thus inactivating it. here the rapid inactivators are those who have more concentration of active nat enzymes than slow inactivators. based on the new technologies, genotypic studies have led to further classifi cation depending upon enzymatic activity nat variant as rapid acetylators, that is, the wild type gene which codes for the completely active enzyme. rapid acetylators are highly active forms of the enzyme denoted by nat * allele. patients harboring these alleles can tolerate conventional dosage of drug that is rapidly metabolized by nat enzyme. individuals who carry nat heterozygous alleles where only one of the allele is active/functional should be administered lower than average drug (those are nat metabolized) dosage to get an optimum effective drug response without adverse drug response. mutations in nat enzyme in human individuals designated as nat * a , nat * b , nat * a, and several others which lead to rendering the nat gene activity are termed as slow acetylators which can lead to diminished drug clearance and toxicity. the variation of frequency of slow acetylator gene is depended on the race, population type, and the ethnicity from one country to the next. it is found in a study that % of middle eastern, % in south indian population, caucasian and negroid, and % of the us population harbor slow acetylator gene. in mongoloid populations like the eskimos, japanese, and the chinese, slow acetylators are found in only % of study subjects. in another study carried out in a population of healthy caucasian, there is variability in isoniazid clearance. while isoniazid preparation is responsible for only % variation and body weight accounted for only % variation in isoniazid clearance, the majority variation of % in isoniazid clearance was due to nat genotypes (kinzig-schippers et al. ) . highactivity nat allele-carrying individuals have higher isoniazid clearance. other studies have shown that - times more isoniazid concentration in individual is carrying slow acetylator nat genotype (parkin et al. ) . a study estimating the comparison of urinary isoniazid excretion in japanese patients to normal, healthy individuals showed that persons with higher number of active nat alleles had higher level of isoniazid acetylation (kita et al. ) . the relation between isoniazid concentration in blood with drug effi cacy and toxicity knowledge is important. peak isoniazid concentration to minimum inhibitory concentration ratio has been proposed to serve as a means to outcome tuberculosis treatment (mitchison ) . mean early bactericidal activity of isoniazid depends on its level in the plasma which in turn depends upon the variant nat genotype carried by an individual. comparatively the mean bacterial activity is lower in rapid acetylators than in the slow acetylators (donald et al. ) . therapeutic failure or relapse of infection is thus attributed to the lower plasma level due to rapid metabolism of isoniazid in rapid acetylator genotypes, while on other hand high level of isoniazid in slow acetylators may lead to the high level of toxicity (weiner et al. ) . nat allele genotyping of tuberculosis patients prior to the treatment with isoniazid is the way forward. dosage adjustment of isoniazid could be carried out depending upon if the patient is harboring none, one, or two alleles nat rapid acetylators. thus, isoniazid would be more pharmaceutically viable for treatment of tuberculosis. in pulmonary tuberculosis patients with known acetylator state, the response to isoniazid treatment analysis was carried out when it is administered alone or in combination with p-aminosalicylic acid. the study compared isoniazid response between nat slow and fast acetylators, and the study revealed that there is association with treatment response and bacteriological negativity (selkon et al. ) . tuberculosis treatment trials used dosage regimes of daily, twice weekly (tuberculosis research centre madras, study , , or three times weekly drug regimes (ellard and gammon ) . by means of controlled clinical trials, it was observed that using once a week uptake of isoniazid showed better clinical response to treatment compared to rapid acetylators, with cure rate of - %. it was postulated that metabolic status of isoniazid may have lesser clinical signifi cance for daily isoniazid treatment regime as compared to thrice weekly or twice weekly treatments. in slow acetylator individuals, the peak concentration of isoniazid was higher than rapid acetylators, and the level of isoniazid decreased more gradually. the effectiveness of a drug in tuberculosis treatment is determined in terms of coverage and the exposure. coverage has been defi ned as the number of hours for which bacteriostatic concentration of isoniazid is ( . µg/ml) maintained in the blood, while exposure is defi ned as area under concentration time curve. both parameters have been found to be signifi cantly greater in slow acetylators. hence, in rapid acetylator individuals, there is a suboptimal concentration of isoniazid which leads to failure of once-weekly regime of isoniazid (sarma et al. ) . other studies using onceweekly isoniazid-rifapentine were compared with twice-weekly isoniazid-rifampicin; treatment also showed that in case of once-weekly treatment regimes, treatment outcome was poor and was associated with isoniazid acetylator status of the patients (weiner et al. ) . the clinical studies have shown that rapid acetylators having infected from combined tuberculosis and hiv infection are at a further disadvantage since it has been found that antituberculosis drug bioavailability becomes suboptimal in those individuals (gurumurthy et al. ) . tuberculosis patients having chronic renal failure are also at a risk from adverse drug reactions if they also happen to harbor slow acetylator genotypes of nat . studies have shown that slow acetylators have higher peak isoniazid concentration, exposure, and half-life compared to rapid acetylators and healthy subjects ( gurumurthy et al. ) . hence, in the case of pulmonary tuberculosis patient also suffering from chronic renal failure, the isoniazid dosage should be administered based on their nat genotypes status. in adult pulmonary patients, studies were carried out to determine correlation between isoniazid dosage and nat genotypic and phenotypic status. determination of isoniazid therapeutic dosage has shown that the fast acetylators need higher drug dosage to have an optimum positive treatment response. fast acetylators tuberculosis patients when administered with mg/kg isoniazid had similar exposure level as mg/kg isoniazid administered to slow acetylators does (donald et al. ). in a further study in a population of south african tuberculosis patients, it was found that current treatment regimes were causing suboptimal exposure of isoniazid in patients having rapid acetylator status (wilkins et al. ) . several fi eld studies have further suggested that there is a need for calibration of isoniazid dosage as per the individual tuberculosis patient's age, acetylator status, and disease process for an effective antimicrobial outcome of drug treatment (jeena et al. ) . in children affected with tuberculosis, it was shown through several studies that the exposure of isoniazid was reduced in the rapid acetylators when compared to the slow acetylators and thus likely to affect the outcome of the treatment of tuberculosis (cranswick and mulholland ; schaaf et al. ; mcilleron et al. ). though isoniazid has been found to be nontoxic during conventional regimes, two types of adverse reactions to isoniazid have been reported. the most common isoniazid toxicity reported is hepatotoxicity which affects - % of the patients (tostmann et al. ) . another isoniazid-associated adverse event is peripheral neuropathy. neuropathy usually occurs in slow acetylators due to administration of high doses of isoniazid (devadatta et al. ) . hepatotoxicity is the major adverse reaction of isoniazid, and the factors that are responsible are nat acetylation, oxidation by cytochrome p s oxidation (cyp) ei, and detoxifi cation by glutathione s-transferase (gst) enzyme activity (roy et al. ) . accumulation of acetyl hydrazine, a toxic metabolite of isoniazid, has been implicated in peripheral neuropathy, and the condition in humans is reversible by concomitant administration of pyridoxine (zilber et al. ) . further, it has also been deduced that hepatotoxicity occurs due to hydrazine metabolites of isoniazid. rifampicin also causes induction of isoniazid metabolism and inducing isoniazid hydrolase to produce isonicotinic acid and hydrazine. the rifampicin induction is more pronounced in slow acetylators compared to rapid acetylators (sarma et al. ) . in some populations studies have established association with nat acetylator status and isoniazid-induced hepatotoxicity, while in other studies it has not. studies in japanese and taiwanese populations have shown that the acetylator status of nat increased the risk factor for hepatotoxicity by -fold isoniazidinduced hepatotoxicity (ohno et al. ; huang et al. ) . in another study on the korean population, the nat slow acetylator status has been implicated to increase isoniazidinduced hepatotoxicity by two-to eightfold, and hence the nat acetylator genotype could serve as predictor of hepatotoxicity (cho et al. ) . nat * / b , nat * a/ a , it/t, and a/a diplotypes have been indicated and could be used as biomarkers for prediction of antituberculosis drug-induced toxicity (ben mahmoud et al. ) . slow acetylator nat alleles have been attributed to increase - -fold to -fold higher risk in isoniazid-induced hepatotoxicity. but other studies in tuberculosis patients have not been able to fi nd any association of nat acetylator status and the drug-induced hepatotoxicity. case studies of caucasian origin patients with tuberculosis (leiro-fernandez et al. ) , genotyping in an indian population (roy et al. ) , and study on heterogeneous population of hispanics, africans, caucasian, south american, and asian have not reported any linkage between nat acetylator status and isoniazid-induced hepatotoxicity polymorphisms (vuilleumier et al. ) . cytochrome p e is one of the enzymes of the hepatic microsomal enzyme system. cyp e gene encodes a member of the cytochrome p superfamily of enzymes. the cytochrome p proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids. cyp e is an enzyme which brings about conversion of acetyl hydrazine to hepatotoxins, such as acetyl diazone and ketene, and brings about conversion of acetylonium ion (nelson et al. ) . polymorphism in cyp e has been linked with increasing the risk factor associated with isoniazid-induced liver injury (lee et al. ) . the enzyme relocates to the endoplasmic reticulum and can be induced by isoniazid or its metabolite hydrazine. in animal model studies using rat, it has been found that cy pe activity is linked to blood isoniazid levels (yue et al. ). in the presence of variant genotype of cyp e , isoniazid could on the other hand inhibit the activity of the cytochrome p e enzyme. enhanced cytochrome p e activity leads to the increased production of hepatotoxins and hence causing hepatotoxicity. both nat and cyp e polymorphisms have been shown to be associated with susceptibility of fi rst-line druginduced hepatitis. cyp e polymorphisms have been related to increase in risk of antituberculosis drug-induced liver toxicity. the common * a/* a genotype of cyp e , in tuberculosis patients from taiwan, has been linked to increase in the liver damage risk by . times. presence of both slow nat acetylator status and the * a/* a genotype further increases risk of hepatotoxicity when compared to presence of either of the single polymorphism (huang et al. ) . the cyple * and * a* * d haplotypes in indian pediatric patients have been shown in sep-arate study and have shown to increase the liver toxicity. further, the common * a allele at cyp e has been implicated to hepatotoxicity in various heterogeneous population comprising of asians, africans, caucasians, hispanics, and south americans (leiro-fernandez et al. ), but study done on a korean population on the other hand could not fi nd any association between cyp e polymorphism and liver toxicity (huang et al. ) . the glutathione s-transferases are class of two distinct supergene families of proteins located in cytosolic and membrane-bound forms. glutathione s-transferases are a class of enzymes that are responsible for detoxifi cation of therapeutic medication, carcinogens, therapeutic medication, and toxic chemicals that are mostly electrophilic in nature. gsts are present both in eukaryotes and prokaryotes. at present, eight distinct classes of the soluble and cytoplasmic mammalian glutathione s-transferases have been identifi ed: alpha, kappa, mu, omega, pi, sigma, theta, and zeta. the cytosolic gst enzymes are encoded by at least fi ve different loci coding for gst enzymes, distantly related gene families (designated class alpha, mu, pi, sigma, and theta gst), whereas the membrane-bound enzymes, microsomal gst, and leukotriene c synthetase are encoded by single genes and both have arisen separately from the soluble gst (simon et al. ; strange et al. ) . glutathione s-transferase catalyzed elimination of toxic chemicals from the human body is carried out by making the toxic chemical soluble by conjugation with glutathione. in context of isoniazid-related hepatotoxicity, studies have indicated that deletions of gst mu ( gstm ) and gst theta ( gstt ) are associated with liver damage (cho et al. ; huang et al. ) . gst enzymes play an important role in removing the harmful metabolites of isoniazid from the body. the toxic metabolites generated by isoniazid metabolisms are from intracellular free radicals that are scavenged by conjugation with glutathione in reactions catalyzed by gst enzymes. now, studies in indian patients suffering from tuberculosis show that those harboring homozygous gstm mutations have higher risk of hepatotoxicity. it was also found in a study on taiwanese tuberculosis that patients have twice the risk of isoniazid-induced hepatotoxicity if they have homozygous gstm deletion. thus, it can be inferred from similar studies that identifi cation of gstm deletion in patients will lead to the better management of isoniazidinduced hepatotoxicity. reactive oxygen species as we have been aware is a causative agent for damage to hepatic tissue. it has been deduced that level of mitochondrial oxygen species is reduced by the action of manganese superoxide dismutase (msd). as the name suggests, msd catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide and is the fi rst line of defense against reactive oxygen species. polymorphism in the msd enzyme has been found in a study in the population in taiwan, where genotypes having t > c polymorphism in codon lead to variant amino acid valine in place of alanine which increases in risk associated with antituberculosis druginduced hepatotoxicity (huang et al. ) . the presence of valine at codon causes the increased activity in the enzyme manganese superoxide dismutase which leads to the accumulation of the toxic byproduct hydrogen peroxide which can cause hepatotoxicity. rifampicin has been proven to show concentration-dependent activity against m. tuberculosis and is a very important fi rst-line drug against tuberculosis (ji et al. ; jayaram et al. ) . drug transporters p-glycoprotein and oatp b transporters uptake rifampicin as a substrate and hence play an important role in distributing the drug throughout the body. the drug transporters are transcriptionally regulated by the nuclear receptors, i.e., pregnane x receptor and constitutive androstane receptor. the phenomenon variation in bioavailability of rifampicin among individuals in a population on administration of standard dosage has been subjected to investigation by the scientists. the pharmacokinetics of rifampicin depends upon the uptake of machinery of the cells in the body. it has been found that there is a relation between pharmacokinetics and polymorphisms of genes that is responsible for drug effl ux and infl ux. a study group of individuals suffering from tuberculosis who were categorize as per place of origin africans versus non-africans, it was observed that in drug transporter gene, slco b c > a polymorphism leads to reduced rifampicin exposure and bioavailability (weiner et al. ) . the people of african origin (black subjects) that carry slco b c > a gene polymorphisms have been associated with more pronounced reduced rifampicin exposure compared to people from other races. the study thus showed for the very fi rst time that marked interindividual variation in rifampicin exposure can be attributed to slco b polymorphism. another study in south africa has also highlighted that the variant allele of slco b rs polymorphism reduced the bioavailability of rifampicin in the body of the patients when present both in the homozygous and heterozygous states (chigutsa et al. ). this fi nding has been attributed to the observation that there is about % variability in drug clearance among the patients. polymorphisms in the abcb , pxr, and car genes have not been found to affect the pharmacokinetics of rifampicin in the patients in any signifi cant manner. researchers have further predicted by means of stimulation that increasing the dosage of rifampicin in patients carrying slco b rs would increase the plasma availability of the drug and thus would have a positive impact on the treatment outcome. however, more studies needed to be carried out to know the exact association of slco b gene polymorphisms between rifampicin bioavailability to provide an effective treatment regime. as has been already mentioned, the variation in human leukocyte antigens also is known cause of disease susceptibility and the response to treatment has also in indian patients the lack of human allele hla dqai* , while the presence of dqb * has been reported to be associated with antituberculosis-induced hepatotoxicity (sharma et al. ) . aminoglycosides are antibiotics which are molecules that consist of amino-modifi ed sugars, and some of the drugs of this class have been used for treatment of tuberculosis. aminoglycosides such as kanamycin, streptomycin, and amikacin have been used to treat tuberculosis. aminoglycosides have known to cause ototoxicity. ototoxicity is term used when there is damage to the ear (oto-), specifi cally the cochlea or auditory nerve and sometimes the vestibular system due to toxins. the association between aminoglycosideinduced ototoxicity and mitochondrial mutations has been found in a study in chinese family. the deafness phenotype was found to be associated with c > t s rrna gene polymorphisms which could be induced with the administration of aminoglycosides or even get more aggravated (zhao et al. ). there is still no clear relationship of ethnicity and genetic background and response to antituberculosis treatment, and no single variant of nat and cyp e genes is associated with signifi cant liver damage (yamada et al. ). more extensive pharmacogenomic research is still needed for realization of robust personalized medicine for tuberculosis. the antifungal medicine amphotericin b has been found to be effective and well quite toxic. further investigations revealed that the immunomodulatory role of amphotericin b also involves the induction of production of proinfl ammatory cytokine. in human cell the amphotericin-induced higher mrna expression and cytokine production have been detected in studies (rogers et al. ) . the discovery of induction of proinfl ammatory cytokine production was able to explain the infusion-related toxicity effect like nausea, fever, chills, and hypotension that are characteristics of this cytokine release. it was also able to explain the mechanism of action of amphotericin b since the proinfl ammatory cytokines are responsible for the activation of monocytes, macrophages, and promote chemotaxis that led to enhanced immune response to the infection. since the advent of the era of omics technology, the number of drugs that have been discovered have not delivered as was fi rst predicted especially in the case of infectious disease. some of the shortcoming and the remedial measures have already been discovered in the previous sections. it has been found that even with high-throughput screening of number of drug, candidates have not been successful always (payne et al. ). the fi eld of scientifi c research which has become all encompassing and interdisciplinary has added strength along the way and opened new avenues. the fi eld of biology is intertwined with structural biology and chemistry has given rise to the fi eld of medical structural genomics. the exact causes of failure of high-throughput screens have not been well defi ned. the fi eld of structure-based drug discovery has tried to overcome these limitations in the availability of chemical libraries and absence of structural information of many of the targets. the fi eld of structure-based drug discovery has its origin from the fi eld of x-ray crystallography and nuclear magnetic resonance (nmr) technology. with the availability of human genome sequences and pathogen genome sequence databases, the fi eld of structural genomics has gained importance, and hence over the past decade, more than such projects have been taken up. the fi eld of structural biology has got a boost with the coming together of robotics and informatics in the biological research sphere (haquin et al. ) . for the synthesis of an effective drug, by means of medical structural genomics, the protein which drug will affect should be well defi ned experimentally in both structure and functional aspects as the potential target. the protein should not only be well characterized structurally but also should be well defi ned as essential for the survival of the pathogen. once drug and its protein target in a microorganism is identifi ed, the fi eld of medical structural genomics provides rapid mechanisms using high-throughput x-ray crystallography and nmr assay system to fi nd the ligand-bound structures. to identify such drug targets, it is very essential to know the complex host and pathogen interaction. the mode the pathogen uses to cause the infections is very diffi cult to elucidate and is a long process. the technologies of rna interference and other gene knockout techniques should be complimented with experimental chemical biology approaches as microorganisms adopt multiple mechanisms for survival. this has been emphasized for the fact examples of efforts of scientist for targeting the fatty acid biosynthesis pathways of bacteria. at fi rst drugs were found to have high bioavailability and are potent against the bacterial replication in vitro. these compounds were subjected to be tested in animals and have been found to be not effective, the reason being that the bacterium utilizes the fatty acids present in their host vertebrates (brinster et al. ). hence, this study proves that there is need for more effective screening using the services of scientist from several spectra of biology like microbiologist, biochemists along with structural biologist, and chemical biologists to fi nd effective molecules and compounds which can eliminate the pathogen under proper infective conditions (hoon et al. ) . in pharmaceutical research scenario, it might also be possible that the drug target for a cell active compound is not known and then medical structural genomics provides a number of purifi ed protein targets which can be assayed for binding interaction with bioactive compound by means of number of biophysical techniques like thermal stability (ericsson et al. ). such efforts have already been carried out in the fi eld of protozoan pathogens. the program of medical structural genomics of protozoan pathogens ( http://msgpp.org/description.shtml ) has been initiated to screen for drugs for ten protozoan diseases. the initiative has screening of thousands of potential antimalarial drugs against about putative plasmodium falciparum protein targets by expressing them in bacterial expression system in the laboratory and deciphering their d structures. further, the com-pounds are assayed for their effectiveness in live organisms and further validated in appropriate disease model. the terms chemical validation and drugability are often used in conjunction in such cases. drugability is meant to be used how tractable a given drug target is for the development of a drug candidate, while chemical validation means that drugs have been found to be active in live organism. drugs which fulfi ll the abovementioned criteria are worth the effort, time, and resources. in the future more collaborative efforts between medical structural genomic centers and the chemical biology institutes would be possible with the availability of collection of phenotypically defi ned compound that would have proven anti-pathogen activity resulting in the synergistic target validation and hit to lead development using structure-based drug design. pharmaceutical industry has now taken fragment-based drug discovery methodology as an alternatively less expensive and at times more effective than high throughput screening. variety of methods like x-ray crystallography, nmr, surface fl uorescence polarization, plasmon resonance yield, and differential thermal denaturation have been used to obtain macromolecular structure to screen libraries of small fragment that are obtained from compounds that are building blocks of drugs and hence can be more drug like. the fragment-based drug discovery is based on the screening libraries of small molecules on the rule of three which has the molecular weight < da, the calculated log of octanol/water coeffi cient (clog p) < , and ≤ rotatable bonds and hydrogen bonds (rees et al. ; congreve et al. ) . protein-protein interactions are important for all biological processes. metabolic activities in the biological system are catalyzed by proteinbased enzyme where in certain cases their activities are regulated by modulation of an equilibrium of an alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) that have now been described as mode allostery. the oligomerization from the protein-protein interaction need not lead to gain in free energy, and it has been found that small molecules can block or disrupt any protein-protein interaction that is necessary for biological systems, for example, being in the development of potent peptidomimetic inhibitor of hsv ribonucleotide reductase with antiviral activity (liuzzi et al. ). the discoveries have opened avenues where structure-based information can be used to develop small novel antimicrobial molecules that can be made which can target protein-protein interfaces (wells and mcclendon ) . an example of this technology has been used to fi nd small-molecule species-specifi c allosteric drugs for porphobilinogen synthase (pbgs). the oligomeric equilibrium for porphobilinogen synthase (pbgs) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. in silico docking analysis from a small molecule library helped in selecting suitable compounds and molecules that had more affi nity for docking pbgs allosteric site and thus were subjected to testing in vitro . in one compound whose inhibition mechanism is species specifi c, conversion of pbgs octamers to hexamers was thus identifi ed (lawrence et al. ) . the above fi ndings have led the way of targeting of oligomeric enzymes in pathogenic organism bacteria. prime example is bacterial inorganic pyrophosphatases, which function as hexamer (kankare et al. ) . on the other hand, the eukaryotic, cytosolic, and mitochondrial pyrophosphatases function as homodimers (oksanen et al. ) and hence have different interfaces than its bacterial counterparts as evident from the study of evolutionary aspects of inorganic phosphatases. in this context the strategy has been to target the oligomeric state of the bacterial inorganic pyrophosphatase enzymes to inhibit their activity rather than their conserved active site (sivula et al. ) . the technology has opened a novel pathway where more antibiotics can be developed. the amount of knowledge of protein structures being generated is enormous; the need of the hour is dissemination of the knowledge databases among scientists and academic researchers on a worldwide scale. the protein structural know-how should be in the public domain without any constrains and copyright restrictions; also in addition the databases should be available free of any monetary charge. structural genomic projects the world over have solved the structures of many proteins and have made the knowledge available for world community by submitting the structures to protein data bank {pdb; http://www.wwpdb.org/ }. worldwide protein data bank is the site whose mission is to maintain a single protein structural public database which can be accessed by the global community (berman et al. ) . there is lot of structural data of protein-ligand complexes that is in private pharmaceutical industries not in the public domain. the economic incentives of drug discovery are driving force for this secrecy, but in this process there are a lot of valuable data that are duplicated and lots of valuable resources and energy efforts. the learning process from failures and successes in pharmaceutical corporate sectors is never known to the scientifi c community, and a major loss is of most valuable time. hence, as we can see, the drug discovery resources are not being adequately utilized across the academia and industry, so there is suggestion to have open-access industry-academia partnerships as possible mechanisms to overcome the problem. a frame work is need where both fi nancial and intellectual properties of the innovators are safeguarded when there structural data are deposited in the databases like pdb. a simple proposition would delay the release date of such structural data so that protection of intellectual property is feasible. policies which can bring into the public domain structural data from the corporate world could only be possible by the concerted efforts of all stakeholders from industry, national, and international research funding agencies from all nations (edwards et al. ). apart from easier dissemination of structural information related to infectious diseases and collaboration of structural biologist with medical chemist and molecular biologist, there is need for development of automation in several technologies to bring about unprecedented growth in the new drug discovery. fragment-based drug design needs the support of high throughput technologies such that along with structural genomics, there will be more success in the determining protein-ligand structure determination. decades of experience have shown that the infectious diseases would emerge with more vigor and virulence. when the diseases are not controlled, then it would take a considerable toll of human health both in terms of mortality and morbidity. the life would be affected by emerging microbial disease-causing pathogen whatever the region, ethnicity, lifestyle, socioeconomic status, and ethnic background. hence, the threat from infectious diseases is real and the situation is overtly challenging. the great advances in the genetics, genomics, and proteomics have the potential to take up the challenge in the coming decades. it is evident that these technologies have the potential to change the fi eld of diagnostics, treatment, and discovery of drugs and vaccines. the need of the hour is to strengthen the public health 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cord- -drjfwcdg authors: shephard, roy j. title: building the infrastructure and regulations needed for public health and fitness date: - - journal: a history of health & fitness: implications for policy today doi: . / - - - - _ sha: doc_id: cord_uid: drjfwcdg . to recognize the importance to the maintenance of good health of adequate public health regulations and an infrastructure that provides clean water and appropriate waste management. . to see the lack of such amenities over many centuries, but the progressive development of public health bureaucracies dedicated to provision of an appropriate infrastructure for healthy cities, beginning during the victorian era. . to observe how responsibility for the provision of adequate housing for poorer city dwellers has been shared between government, benevolent entrepreneurs and charities. . to note the new challenges to public health presented by such current issues as the abuse of tobacco and mood-altering drugs, continuing toxic auto-emissions, the epidemic of hiv/aids, a decreased acceptance of mmr vaccinations, and the ready spread of infectious diseases by air travel. opportunities for the spread of communicable diseases have increased with the growth in size of cities. the success of urban living has depended in great part on governmental ability to maintain population health through the building of an adequate infrastructure to provide clean water and to dispose of waste, as well as the enactment of appropriate regulations to control the prevent the spread of infectious diseases. in this chapter, we will look at success in meeting these objectives in various communities from early history through the classical era, the arab world, mediaeval europe, the renaissance, and the enlightenment to the victorian era, concluding with some comments on current challenges to public health. in the hey-day of the persian empire, heat, cold, dirt, stench, old age and anxiety were all thought to contribute to ill-health. cyrus the great ( - bce) thus taught his soldiers not to urinate or spit into running water. dead matter was also carefully removed from water-courses, and the clothing of dead people was systematically burnt. during the mediaeval era, interest in public health was much more advanced in the islamic world than in northern and western europe. ali ibn-rabban ( - ce), a well-respected physician living on the south coast of the caspian sea, wrote in his seven-part medical work paradise of wisdom that: "no one should live in any country which does not have four things: a just government, useful medicaments, flowing water and an educated physician." in the eleventh century, the arabic biographer al-mussawir emphasized that the main duty of a monarch was the preservation of health and well-being in his subjects. thus, islamic legislation required physicians to pay regular visits to army units, prisons and people living in outlying areas. the practice of medicine was regulated through a religious office, the hisba, headed by an official called the muhtasib with some of the powers of a modern ombudsperson. one function of the muhtasib was to act as the city medical officer of health. he prevented people with elephantiasis from using the public baths, regulated the cleanliness of public places such as markets, and ensured that garbage collectors did not handle food. one interesting example of applied hygiene was the method used to determine an appropriate location for construction of the main hospital in baghdad (chap. ). the merits of various sites were compared by hanging up pieces of meat, and noting the location where decomposition proceeded the most slowly. the city of córdoba under moorish rule further illustrates the infrastructure typical of the arab world during the tenth century ce. among other facilities, the city boasted public baths. in northern and western europe, public health infra-structure such as aqueducts and sewers fell into disrepair following departure of the roman garrisons, and during the mediaeval era the sanitary conditions in most cities were appalling. positive developments were the development of quarantine procedures and the re-emergence of a few public baths. food inspectors were appointed, and some cities also introduced zoning regulations, requiring malodorous trades such as tanning to be undertaken outside the city walls. water supply and sewage disposal untreated waste was thrown directly into the rivers of london and paris, and travellers were advised: "wise men go over bridges, and fools go under them." in ce, king edward iii ordered the mayor of london to: "cause the human faeces and other filth lying in the streets and lanes in the city top be removed with all speed to places far distant, so that no greater cause of mortality may arise…" a fourteenth century ordinance prohibiting the emptying of latrines into a creek near london's city wall remained largely ignored, so that in the fifteenth century the stream was buried underground. substantial populations of hogs and cattle roamed the streets of many large cities, adding to the urban stench. uncontaminated water was a rarity, and a lack of refuse disposal encouraged rat infestations. infrequent bathing and unwashed woolen clothing led to a proliferation of fleas and other insect vectors of infection. during the mid-fourteenth century, two thirds of the european population was killed by the flea-borne bubonic plague (the "black death", - ce). many doctors deserted their patients during this epidemic, and others proposed preposterous remedies. guy de chauliac wrote: "so contagious was the disease…. that no one could see or approach the patient without taking the disease…for self-preservation, there was nothing better than to flee the region… to purge oneself with pills of aloes, to diminish the blood by phlebotomy and to purify the air by fire and to comfort the heart with senna and things of good odor and to soothe the humours with armenian bole and resist putrefaction by means of acid things." chauliac unwittingly kept the rats and fleas away from pope clement vi, by surrounding his bedside with charcoal burners. a few years following the black death, observant physicians hypothesized that ships arriving from overseas were contributing to the recurring epidemics of plague. at first, hostels for sick townsfolk and newly arrived visitors were set up outside the city, but this was not entirely effective in containing infection. thus in ce, a trentino ( days) of isolation on an uninhabited island was required at many european ports of entry. subsequently, the isolation period was extended to days, perhaps because of an ancient greek doctrine that a contagious disease became manifest within days. in britain, recently arriving travellers were quarantined on guardships, anchored in the thames estuary ( fig. . ). most of the roman baths in northern europe had been abandoned by the mediaeval era, in part because of the high cost of heating the bath water, and in part because the church considered public bathing as a common prelude to venal sins. the church also had concerns about reinforcing belief in the supposed healing powers of celtic water deities (chap. ). nevertheless, as prosperity increased in the latter part of the middle ages, public baths were built or reopened in various parts of europe. in britain, the king's bath was built over the sulis minerva temple in the city of bath, and paris had established public baths by the thirteenth century. in germany, the tradition of river bathing had persisted from celtic times, and a growing number of new public bath-houses were constructed during the th and th centuries. admission to a bath-house was expensive, and poorer germans considered the payment of "bath money" a great blessing. the full luxury package of a spa treatment included washing, scouring and slapping of the body with a sheaf of twigs, a steam bath, rubbing to induce perspiration, swatting the skin with wet rags, scratching, hair washing, cutting and combing, lavendering, and blood letting. unfortunately, some of the baths subsequently became the scene of debauchery, prostitution and infection, and by the sixteenth century, many were closed for fears of spreading syphilis, leprosy and plague ( fig. . ). personal hygiene substantial quantities of soap were traded during the mediaeval era, but this was used more for the washing of wool than for cleansing of the skin. monasteries boasted laundry rooms, and many women listed their trade as "laundry woman." however, the laundering of clothes was an infrequent luxury for poorer people, and indeed many had no spare set of clothing, so that fleas flourished in the poorer households. food inspection basic foodstuffs such as wine, beer, bread, meat, fish and salt were frequently adulterated in mediaeval times. to counter such abuses, several european governments appointed food inspectors. in britain, in , the assize of bread and ale regulated the price of these staples in relation to the price of corn. occasional renaissance scholars expressed some interest in health promotion. the english diplomat and scholar thomas elyot (c. - ) wrote a book entitle the "castell of health," summarizing the latest medical knowledge for those unfamiliar with greek, and the venetian nobleman luigi cornaro ( - ) wrote a book on the art of living a long life. santo santorio (chap. ) also sought to put hygiene on a mathematical basis. most of renaissance society showed little interest in public health or hygiene, as shown by the outbreak and management of the great plague. however, boards of public health were set up in some cities. two small advances in personal hygiene were the introduction of cotton clothing and a growing use of toothbrushes. diligent housewives adopted a few other simple changes in household management to preserve the health of their families, and cambridge university insisted on a direct control of its food supply, the great plague the london "plague" of ce was one in a series of european epidemics of bubonic plague dating back to the "black death." the great plague claimed at least , lives in central london, this being about a half of the population who had not fled from the city. indeed, the death count was probably underestimated, since publically appointed street monitors were open to bribery by those who did not wish to disclose that their house had become infected. samuel pepys commented that the prevalence of the disease was such that corpses could not removed during the hours of darkness ( fig. . ). people were confined to their homes if one family member was infected, thus virtually ensured the death of the entire household. two watchmen were posted at the doors of infected homes for days, at a cost of d per house per day, and the victims received a public stipend of d per day to pay for food, fuel and medicaments. believing that the disease was conveyed by miasmata, the college of physicians recommended using bonfires to displace the infected air. there was probably some incidental benefit from these fires, since the smoke tended to drive away the flea-ridden rats that were vectors of the disease. the epidemic was eventually checked by the great fire, which consumed both the rats and the plagueinfested slum dwellings. in europe, local boards of public health were established; they adopted various measures for the containment of epidemics and the provision of social support to the community. in some cases, they designated specific physicians to attend plague victims, and in florence, local doctors prepared a public information booklet that summarized current knowledge on plague prevention. a further responsibility of these boards was to deal with doctors who failed to report communicable diseases in wealthy patients. one roman doctor who was arrested for this offence was ordered to serve as resident physician at the local pest-house. outbreaks of the plague placed a severe financial stress upon some municipalities. in milan, extra funding was needed to hire physicians and grave-diggers, to pay for operating a quarantine "pest-house," and to reimburse the infected for two-thirds of the estimated value of their possessions, which were summarily burned. some municipalities set up immigration offices on mountain passes to control the arrival of infected travelers, and others restricted imports, exports, market trading, travel and funerals, although it was unclear how far these costly measures were successful in reducing the toll of disease and mortality. personal hygiene and household management one positive development during the renaissance was the introduction of washable cotton clothing and sheets. this greatly curtailed the spread of insect-borne diseases, particularly among those with sufficient wealth to own several changes of clothing. another innovation was popularization of the bristle toothbrush. this device had been invented by the chinese in the thirteenth century, but did not become popular in england until the late seventeenth century, beginning with the aristocracy. without necessarily knowing why, tudor housewives achieved some sterilization of their dairy equipment by scouring with salt and hot water, and then exposing utensils to bright sunlight. infestation of houses by fleas was also countered by sprinkling appropriate herbs beneath the rush mats that covered their floors. the renaissance saw further occasional attempts to control the quality of food, particularly for the wealthy. cambridge university insisted that the direct supervision of their refectories was important to preserving the health and well-being of their students. one of those promoting hygiene during the enlightenment was the physician james mackenzie, who in wrote a text on "the history of health and the art of preserving it." the enlightenment saw some improvement of health infra-structure many dwellings for the poorer citizens of europe.were now constructed of brick and boasted glass windows. and samuel johnson ( - ) was urging a pro-active response to the prevention of disease:"we must consider how many diseases proceed from our own laziness, intemperance or negligence… and beware of imputing to god, the consequences of luxury, riot and debauchery." the diderot encyclopédie, first published in , included a section on hygiene, which wss defined as: "the things which mankind uses or handles… and their influence on our constitution and organs." gottfried wilhelm leibniz ( - ) was perhaps the greatest enthusiast for public health during this era. he strove to establish a pattern of medical training that was oriented towards public health and preventive medicine rather than the treatment of disease. he reminded his colleagues that hippocrates had registered every successful cure, and he urged a similar meticulous recording of outcomes in order to provide a modern preventive medicine data-base. he proposed that standardized questionnaires should be developed to examine eating habits, and that careful mor-the enlightenment tality statistics should be collected so that findings could be correlated with the local climate, air conditions and the nature of the soil. a few other scientists such as hales (who improved the water supply for his village of teddington), and bernouilli (with authored a probability study demonstrating the merits of vaccination) were also interested in public health. but concern about the provision of clean drinking water, adequate treatment of sewage and garbage, and protection against communicable diseases remained the exception rather than the rule, with most countries making a poor showing on indices of population health. francis bacon published studies on the percolation, filtration, distillation and coagulation of water as early as . anton van leeuenhoek described the microscopic animalicules that he had seen in dutch drinking water in , and the french scientist joseph amy patented a water filter in . however, the quality of water in most large cities left much to be desired. philippe de la hire ( - ) mapped the area around paris, seeking to improve the water supply to versailles, probably as much to service the palace ornamental fountains as to provide clean drinking water in the town, and he built a massive aqueduct for this purpose (fig. . ) . he further suggested that householders should install a sand filter to purify the water collected from the roofs of their dwellings, although he noted that one alternative source of water, from underground aquifers, was rarely polluted. in , paisley, scotland became the first british city to establish a municipal water treatment plant. it used a sand filter that had been developed by robert thom. in , paris also constructed a large water treatment facility on the seine; here, river water was allowed to settle for hours, and was then passed through sponge pre-filters and main filters that contained sand and charcoal. despite these advances, the residents of broad st., in central london, faced a massive outbreak of cholera as late as , because they were drawing water from a shallow well that was located close to a cholera-contaminated cess-pit. too often, the city dwellers of the enlightenment continued to pass sewage into open gullies or cess-pits that were fig. . ruins of an aqueduct, built by philippe de la hire to improve the water supply to the city of versailles and its royal palace (source: http://en. wikipedia.org/wiki/ canal_de_l'eure) close to wells, and garbage was thrown directly onto the street. however, in , the conseil supérieur of new france ruled that in order to reduce infection, the houses in quebec city must have latrines, and that garbage must be carried to the river st. lawrence, rather than simply thrown out of the door. populatiion health during the enlightenment vital statistics provide simple objective indices of overall population health during the enlightenment. at birth, the average european could expect to live no more than years. a third to a half of the population died before reaching the age of years. those who survived to their mid-teens lived into their s or even their early s, and at the age of the aristocracy could expect to live a further - years; this was an improvement over the years of adult survival typical of the fourteenth century. survival prospects were much worse in north america than in europe during the enlightenment. many of the population succumbed to fevers, intestinal diseases, and, in the case of the african slaves, to harsh working conditions. a quarter of european immigrant children did not survive until their first birthday, and half of all marriages ended in the death of one partner before their seventh wedding anniversary. epidemics of beri-beri, smallpox, malaria and yellow fever wreaked havoc among early colonists. two of every three deaths were attributed to typhoid, dysentery or salt poisoning. in an attempt to reduce this terrible toll, newly arrived immigrants were initially isolated in "guest houses." replacement of contaminated water by wine, beer or cider, a reduced consumption of infected clams, and a scattering of the population to areas where there were copious fresh water springs reduced deaths from typhoid and dysentery, but progress in reducing overall mortality was slow. in early canada, dispersal of the population along the major rivers made major epidemics less likely than in the urban settlements of the united states, but isolation, accidents and harsh winters made canadian life expectancy worse than those in either europe or the u.s. only a small fraction of the population lived beyond years, and many of the children suffered from rickets and anaemia. typhus and smallpox were also recurrent problems. the victorian era was marked by growing government responsibility for the health of the public in large european cities. there was a gradual improvement in the quality of housing, and demographics showed a burgeoning birth rate. social reformers also succeeded in abolishing child labour and slavery from western society (chap. ). in this section, we will discuss the role of boards of health, continued deaths from poisoning, and improvements in housing conditions. major epidemics of influenza, cholera, typhus, typhoid fever and scarlet fever sparked a deep concern about population health in victorian england. in london, england, cholera killed , people in - and , in (chap. ). however, leaders of a new sanitary movement such as edwin chadwick ( - ) and thomas southwood smith ( - ) began to recognize that ill-health of the individual soon became ill-health of the population. they thus made urgent calls for the provision of clean drinking water, proper removal of refuse and sewage treatment. chadwick and smith sat as commissioners on london's general board of health that regulated the water supply and sewer connections for all new housing in the city, and provided adequate burial grounds for those who died. the quality of london's drinking water was rapidly upgraded, and money was spent on methods of preventing death during childbirth. the public health acts of and also established public baths and wash-houses, and by the s, health-conscious municipalities were building public swimming pools. in lower canada (quebec), a physician was appointed as health officer in , with the primary responsibility of monitoring the sick and starving people who were arriving on immigrant ships from europe. by , a strengthened five-member board of health was supervising quarantine arrangements on grosse isle, in the st. lawrence river near to quebec city. nevertheless, the number of immigrants was such that this holding facility was at times overwhelmed, and cholera periodically reached quebec and montreal, killing between - % of the population. in , people also died of typhus while they were quarantined at grosse isle. a central board of health for both upper and lower canada was created in . compulsory vaccination against smallpox was introduced in the early s. in the united states, organization of sanitary reform began rather later than in canada. the city of new york enacted the metropolitan health bill in , creating a -person board of health. immigrants were processed on ellis island, just outside new york city. the original wooden structure was quickly destroyed by a catastrophic fire, but a stone replacement building opened in . many immigrants spent only a few hours in the facility, but those with contagious disease were summarily denied admission to the united states. continued deaths from poisoning many victorians died from eating adulterated or diseased food. one report to the british privy council ( ) estimated that % of meat came from diseased cattle. flour was expensive, and bakers frequently adulterated it with chalk (to whiten it) and alum; often, the bakers also kneaded the mixture with their bare feet. an act prohibiting the adulteration of food was passed in , but its enforcement was an option for local authorities, so that it was not very effective. cooking was typically done in tin-lined copper pans; wealthier citizens replaced the pots when the tin had worn away, but the poor could not afford to do this, and in consequence they sometimes developed copper poisoning. other sources of poisoning in the victorian home were leaking gas pipes, lead used in white paint, and arsenic used to colour wallpapers. in the early nineteenth century, the sudden influx of country folk into the major cities of europe created hideous slums: "in big, once handsome houses, thirty or more people of all ages may inhabit a single room." housing gradually improved over the victorian era, as many workers accumulated sufficient funds to purchase modest but well-built homes. enlightened industrialists also constructed model housing estates for their employees. robert owen ( - ) organized a model community for his workers at the new lanark mills, in scotland, complete with a nursery school. he envisaged an even more ambitious employee housing project in new harmony, in, but this project failed within two years. the quaker chocolate manufacturer george cadbury ( - ) built a model village for his employees around his factory at bournville, near birmingham, and in the u.s. george pullman, the railway carriage czar, built a model town at pullman, il, in . charitable foundations such as the peabody trust began to replace the worst of london's slums with solidly-built if spartan apartments (fig. . ). the first peabody block, at spitalfields, included dwellings for the poor, shops complete with accommodation for the shopkeepers, and on the top floor baths and laundry facilities for a total cost of £ , . in the united states, building codes were improved during the victorian era, and a national housing association was founded in , under the aegis of the commission on the congestion of population in new york. there were also attempts to persuade philanthropists to build model tenements at low rents; buildings were bought, renovated, and then rented to relocated slum dwellers who were given "friendly instruction" on management of their new households. despite substantial progress in the delivery of public health, there remain a number of continuing challenges in the twenty-first century. current issues include the definitive control of the sales of tobacco and mood-altering drugs, the regulation of automotive emissions and other source of urban air pollution, management of the hiv/aids epidemic, concern over a growing reluctance to accept childhood vaccinations, and the management of infections spread by international air travel. in the edwardian era, cigarette manufacturers had promoted their wares as the cure for various respiratory conditions such as asthma and hay fever. but in , the american physician isaac adler pointed to a growing incidence of lung cancer, and he speculated that the abuse of tobacco and alcohol might be responsible. anti-smoking groups developed in germany following world war i, and a magazine (german tobacco opponents) was published from to . the nazi regime was opposed to smoking, with hitler declaring it a waste of money. in particular, women who smoked were considered as unsuitable to be german wives and mothers. during world war ii, the axis powers made much propaganda from the fact that hitler, franco and mussolini were non-smokers, whereas churchill, roosevelt and stalin were all heavy users of tobacco. evidence of the toxicity of tobacco steadily accumulated during the modern era. in , fritz linkint dresden demonstrated an increased prevalence of lung cancers in smokers. his research was confirmed in , with a case-control study by franz hermann muller of cologne. during the s, ernst wynder at the sloan-kettering institute in new york and richard peto and bradford hill at oxford university advanced even more compelling evidence that cigarettes were carcinogenic. hill concluded that consuming cigarettes per day increased the odds of dying from lung cancer as much as forty-fold. other damning evidence came from cellular pathology, animal experimentation and the demonstration of toxic chemicals in cigarette smoke. however, for a substantial part of the post-modern era, public health workers had to combat a deliberate campaign by the cigarette manufacturers to confuse and deceive the general public. the manufacturers were well aware of the damning facts by the early s, but their misleading propaganda was able to increase u.s. cigarette sales to a peak of billion units in . as late as , only a third of u.s. doctors considered smoking as "a major cause of cancer," and % of physicians were still smoking on a regular basis. beginning in the mid s, there was a dramatic decrease in the social acceptability of cigarette smoking, and growing restrictions were placed on public areas where smoking was permitted. this resulted from demonstrations that passive exposure to cigarette smoke gave rise to small but significant increases in the risks of chronic respiratory disease and asthma in childhood, and carcinoma of the lungs and cardiovascular disease in adults. public polls showed a growing acceptance of public health measures to control smoking in public spaces. cigarette manufactur-ers went to particularly great pains to obfuscate the risks of passive exposure to cigarette smoke, but adverse effects were clearly demonstrated during the s, not only by epidemiological research, but also by the exposure of volunteers to machine-generated cigarette smoke while they exercised in closed chambers. public health workers continue to face many challenges in reducing the sales of tobacco products, as manufacturers doggedly resist measures to reduce consumption through increased taxation, prohibition of sponsorships, and plain packaging. they constantly seek methods of creating new addicts, both through extensive advertising in third world countries and through such tactics as the marketing of electronic cigarettes. as recently as , cigarette smoking still accounted for . % of deaths world-wide. the toll from cigarettes is now compounded by the effects of mood-altering drugs. several countries (including canada) have abandoned attempts to prohibit the marketing of marijuana, with as yet no clear standards of dosages compatible with worker and road safety, and an ever growing segment of the north american population is becoming addicted to powerful opiates, with a high risk of deaths from overdoses. british columbia alone had deaths from opiate overdoses in , despite providing emergency workers with supplies of the antidote naloxone. the modern era saw a dramatic drop in the sulphurdioxide/large particulate smog associated with coal fires in many developed societies, but air pollution problems have continued from coal-fired power station and sautomotive emissions, particularly during thermal inversions. the exposure of cyclists and pedestrians to carbon monoxide was studied during the s. substantial concentrations of carbon monoxide were recorded on congested city streets, particularly if air movement was impeded by tall buildings, but any build-up of carboxyhaemoglobin in the blood stream was reversed quite quickly when the individual moved to a less polluted area. the only adverse clinical effect from carbon monoxide exposure was a somewhat earlier onset of angina if a person with coronary atherosclerosis exercised on a heavily polluted street. chamber experiments by steve horvath in santa barbara, ca, and larry folinsbee in toronto documented acceptable ceilings of exposures to the ozone that was formed by the action of sunlight upon the nitrogen oxides from vehicle and aircraft exhaust. the threshold concentration causing a minor disturbance of respiratory function in healthy exercisers was around . p.p.m., a level that was exceeded in some north american cities on heavily polluted days. to date, in many cities improved automotive emission controls have done little more than match the increase in vehicle registrations, and places such as paris and beijing have needed to forbid the access of drivers to the centre of cities on alternating days in order to reduce pollution levels. since ozone levels show a marked diurnal cycle, one immediate remedy for the active individual is to exercise at less heavily polluted times of the day (early morning or late at night). the ultimate solution to the problem of automotive exhaust probably lies in the replacement of gasoline-driven by electric or hydrogen-powered vehicles. the hiv/aids epidemic officially began in the u.s. in , when the centers for disease control reported a clustering of cases of pneumocystis pneumonia among homosexual men in los angeles. it was quickly realized that the condition was not limited to homosexual individuals, but was seen also in intravenous drug users, haemophiliacs and others receiving blood transfusions. thus, in august , the cdc coined the new term aids. a year later, luc montagnier and his associates at the pasteur institute in paris discovered the virus responsible for this disease. much effort has since been devoted not only to finding highly effective antiretroviral agents, but also in devising measures to reduce transmission of the disease. particular emphasis has been placed upon the wearing of condoms during sexual intercourse, in providing sterile needles for intravenous drug users through programmes of needle exchange and supervised injection sites, in closer control of blood banks and in ensuring sterility during drug injection treatments of tropical diseases. nevertheless, success in controlling the epidemic has as yet been only partial. in the u.s. the disease had already claimed , lives by ; a further million were living with the disease, and , fresh cases were diagnosed in that year. in rural africa, the situation remains even worse, with as many as a third of young adults currently infected. during the early part of the post-modern era, successful childhood vaccination campaigns brought the incidence of mumps, measles and rubella to a very low level in most developed countries, and the who set the year ce for the total elimination of measles and rubella from the european region. however, the percentage of children receiving vaccination has decreased in recent years, with parents weighing the low current risk of infections relative to the supposed dangers of developing meningo-encephalitis and autism. fears that vaccination would cause autism stemmed from a paper published by the british physician andrew wakefield, in . extensive research found no evidence to support his claims, and the british medical journal recently declared that the original article was fraudulent. further, the british general medical council found wakefield had been guilty of serious professional misconduct, and he was struck from the medical register. there have since been small outbreaks of measles consequent upon the decreased proportion of vaccinations in britain and in canada, and unfortunately many of the general public remain convinced that vaccination can cause autism. infectious diseases can now spread very rapidly, due to the ever-growing number of people who engage in global air travel. this problem is well exemplified by an epidemic of sars (severe acute respiratory syndrome). this began in mainland china in november of , and due to delayed reporting by the chinese authorities it spread rapidly around the world. the who issued a global health alert on april th . fortunately, application of rigid quarantine measures contained the epidemic, with relatively few deaths in north america, and by july th , the who was able to declare that the sars epidemic was over. many of the major epidemics of earlier eras were due largely to poor hygiene-a lack of clean water, poor sewage treatment, and an inadequate control of people who were already infected. although we often assume that these issues have now been resolved, it is important to recognize that in many third world countries supplies of clean water and adequate supplies of food are still lacking, with shortages often exacerbated by ethnic conflicts. the same issues of clean water, waste disposal and burial of the dead could still arise in wealthier countries today if there were to be an earthquake, a typhoon or a tsunami, and emergency services must be prepared to give the highest priority to an early re-establishment of the basic health infrastructure following any natural disaster. issues in the adulteration of food have now been largely overcome in developed society, but the current obesity epidemic underlines that problems still have to be resolved in terms of persuading food processors to avoid tactics designed to persuade consumers to overeat. for those who can afford housing, the modern single-family home is generally well-equiped to optimize the health of those who are living in it. massive tower blocks are less suited to a healthy and active life-style, particularly for families with young children. moreover, ever-increasing minimum specifications for housing, a growing world population and a lack of land is presenting public health agencies with the issue of a growing proportion of homeless individuals in many large cities. globalization is presenting new challenges to public health, not only with the rapid spread of infections, but also with the international enforcement of regulations on issues ranging from emission controls on cars to the quality of foods and medications. the ideal forum for developing appropriate preventive measures would seem the world health organisation, but unfortunately (as with many international bodies) its effectiveness is often limited by political considerations, including threats from some nations to slash funding unless criticism of their practices is shelved. . are the infrastructure constraints of an earlier era still compromising public health in third world countries? questions for discussion child against measles, mumps and rubella? what will be the likely new challenges to public health agencies over the next years? public health foundations: concepts and practices cigarette smoking: health effects and challenges for tobacco control plague and the poor in renaissance florence principles of water resources bathing in public in the roman world mission and method: the early nineteenth century french public health movement history of hygiene asian medical systems: a comparative study hygiene in the early medical tradition public health victorian medicine and popular culture private choices and public health. the aids epidemic in an economic perspective health, civilization and the state; a history of public health from ancient to modern times the nazi war on cancer housing in urban britain environmental policy and public health: air pollution, global climate change and wilderness a history of public health shephard rj. the risks of passive smoking on the mode of communication of cholera the new public health the establishment of a board of health for new york city in further reading some early societies had an infrastructure that provided clean water and the removal of sewage, but since this was usually available only to wealthy citizens, its impact upon the course of epidemics was limited. major cities such as london did not build a comprehensive infrastructure until the middle of the victorian era, when appropriate initiatives were taken by newly formed boards of public health. although the traditional concerns of public health have now been largely met in developed societies, new challenges are constantly arising. these include the control of tobacco products and mood-altering drugs, the reduction of automotive emissions and other forms of urban pollution, management of the hiv/aids epidemic, overcoming a growing reluctance to vaccinate infants, and countering the rapid spread of infections by air travel. key: cord- -e phpqes authors: nan title: cis annual meeting: immune deficiency & dysregulation north american conference date: - - journal: j clin immunol doi: . /s - - -z sha: doc_id: cord_uid: e phpqes nan mutations in the genes encoding proteasome subunits (psmb , psmb , psma and psmb ) have been identified as the cause of candle syndrome. these mutations lead to malfunction of the proteasome, which results in buildup of cellular waste products. it is hypothesized that dysregulation in the interferon (ifn) signaling pathway in response to this waste is the driving mechanism of the inflammatory response, and may serve as a therapeutic target in these patients. objectives: to describe a case of suspected candle syndrome successfully treated with tofacitinib. methods: retrospective chart review was conducted with respect to diagnosis, treatment and response. results: a -month old caucasian male was admitted to the hospital for evaluation of profound anemia. his medical history was significant for extreme prematurity (born at weeks from premature labor), intraventricular hemorrhage grade iv that resulted in hydrocephalus needing ventriculoperitoneal shunting, and developmental delay. he was noted to have a hemoglobin of . g/dl during a neurosurgical evaluation for routine shunt revision. he developed hemodynamic decompensation and required hospital admission for packed red-blood cell transfusion. review of systems was remarkable for intermittent pruritic macular rash, daily temperature fluctuations (fever to hypothermia), joint pain/swelling/ stiffness of multiple sites, poor weight gain, irritability, irregular breathing, abdominal distention, and regression of gross motor milestones (no longer rolling over, sitting without support, or pulling up to stand). workup excluded infections and lymphoproliferative malignancies, and he met clinical criteria for systemic juvenile idiopathic arthritis. his initial laboratory studies showed systemic inflammation (wbc x ^ /ul, hgb . g/dl, platelets x ^ /uul, crp . mg/dl, sedimentation rate mm/h, ferritin ng/ml). imaging studies revealed serositis with right-sided pleural and pericardial effusions. he also had myositis supported by imaging and elevation of muscle enzymes (ast u/l, aldolase . u/l). the patient was started on pulse iv methylprednisolone mg/kg daily x days, followed by oral prednisolone mg/kg/day and anakinra mg/kg/day with partial improvement and he was discharged home. he was readmitted . weeks later due to concerns for macrophage activation syndrome (ferritin , ng/ml) in the setting of a gastrointestinal infection and anakinra was increased to . mg/kg/day. however, he continued to have persistently elevated inflammatory markers and so the dose was increased again to mg/kg/day. three months after initial presentation, he had an upper respiratory and ear infection and became ill with generalized rash, increased work of breathing, and poor perfusion. anakinra was considered a treatment failure at that time. he required several doses of pulse steroids and initiation of tocilizumab mg/kg iv every weeks with improvement on systemic symptoms. methotrexate mg/m² weekly was added soon after for persistent arthritis and inability to wean systemic steroids. he continued to have abnormal inflammatory indices, including ferritin ( , ng/ml) and il- levels ( , pg/ml, normal - ). proband only whole exome sequencing revealed a single heterozygous mutation in the psmb gene (c.- g>a), a published pathologic variant. based on this finding, the patient was started on tofacitinib . mg orally twice a day with a dramatic response. laboratory markers of inflammation normalized, and he was able to walk within the first month of treatment. further genetic testing to detect an additional proteasome subunit variant, as well as functional testing on a research basis to demonstrate an interferon signature are being pursued. conclusions: this case highlights the value of early genetic studies in patients with autoinflammation so that initiation of targeted therapy is not delayed in efforts to achieve control of symptoms and evade future complications. this case also illustrates the challenges in diagnosing monogenic autoinflammatory disorders in young patients that present with recurrent fevers, generalized rash, arthritis, and systemic inflammation that mimic systemic juvenile idiopathic arthritis. our experience contributes to the understanding of janus kinase inhibition in type i interferonopathies. of his recurrent infections and etiology of myasthenia gravis. results of the ct chest are notable for a thymoma. thymectomy with biopsy reveals benign pathology with a mixture of type a and b cells. he continues to have persistent fatigue, generalized weakness, diplopia, diarrhea and recurrent respiratory infections after thymectomy. immunoglobulin and lymphocyte subset panels reveal hypogammaglobulinemia with absent b cells. endoscopy reveals villous atrophy and blunting without evidence of celiac disease, inflammatory bowel disease or infection suggesting autoimmune enteropathy. the constellation of clinical and laboratory features are consistent with good syndrome with evans syndrome, seronegative myasthenia gravis and autoimmune enteropathy. the patient is started on immunoglobulin replacement therapy and pyridostigmine with resolution of recurrent infections and improvement of fatigue, generalized weakness and diplopia. three years later his fatigue and evans syndrome recur with new onset loss of appetite and a thirty pound weight loss. repeat immunologic labs were notable for elevated cd , borderline low cd and highly elevated cd cells with low absolute number and fraction naïve cd and cd cells suggesting worsening combined immunodeficiency with peripheral t cell expansion. a bone marrow biopsy reveals large granular lymphocytic (lgl) leukemia and he is started on methotrexate. serum antibodies targeting ifn, and il- are negative four years after removal of thymoma. conclusions: this case is consistent with a classic presentation of good syndrome represented by thymoma, t and b cell-mediated immunodeficiency, increased susceptibility to infections and autoimmune manifestations of evans syndrome, myasthenia gravis and autoimmune enteropathy. in this case the combination of evans syndrome, autoimmune enteropathy and lgl leukemia as malignancy further worsen prognosis and is typically not seen together in good syndrome. this case depicts well the crossroad of infection, autoimmunity and malignancy in late onset immunodeficiencies. introduction/background: dedicator of cytokinesis (dock ) deficiency is a known cause of autosomal recessive hyper-ige syndrome with a combined immunodeficiency. most of the mutations in dock are lossof-function homozygous or compound heterozygous point mutations or deletions. dock deficiency has been associated with low lymphocyte counts with impaired antibody responses, as well as eosinophilia, recurrent bacterial and cutaneous viral infections, malignancies, and severe atopy. we report the case of a year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock , who was noted to have two variants of unknown significance in the dock gene. objectives: we report the case of a year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock , who was noted to have two variants of unknown significance in the dock gene. methods: a year-old man presented to us for evaluation of known hyper-ige syndrome. he had a long history of elevated ige, peripheral eosinophilia, severe atopic dermatitis, food allergies, asthma, severe eczema since early childhood which failed to respond to methotrexate, mycophenolate, cyclosporine, and omalizmuab, but ultimately responded to intravenous immunoglobulin (ivig). his infectious history included mrsa skin infections and one episode of pneumonia, and he reported a history of fungal skin infections but the history was unclear. initial immune workup revealed eosinophilia of , ige level (as high as , ), igg level , igm level , and iga level . he had no random antibodies to streptococcus pneumonia serotypes, but he had protective antibodies to diphtheria and tetanus. lymphocyte subsets showed cd , cd , cd , cd . he had normal mitogen stimulation to pha but decreased mitogen stimulation to candida. dna testing for a stat mutation was negative. results: we found two missense variants of uncertain significance in the dock gene ( .p.v i, nm_ . :c. g>a and .p.l v,nm_ . :c. c>g ). the first variant had previously been reported in the clinvar database as a variant of uncertain significance, and the second variant had not been previously reported in the literature to our knowledge. our assay could not determine if the two dock variants were on the same allele or on different alleles. dock protein expression testing is currently pending. conclusions: our patient presented with history of elevated ige, eosinophilia, atopy, severe eczema, and cutaneous mrsa and fungal infections. he was noted to have variants of uncertain significance in the dock gene. homozygous or compound heterozygous pathogenic variants in dock are associated with an autosomal recessive hyper-ige syndrome and combined immunodeficiency with clinical features of recurrent bacterial infections, cutaneous viral infections, severe atopic disease, as well as susceptibility to malignancy. our patient does not have all the typical features of dock deficiency and he seems to have a less severe phenotype. notably, he does not have the cutaneous viral infections or malignancy often seen in dock mutation hyper-ige cases. our case demonstrates new missense mutations, which have not previously been described in the literature, possibly causing a milder phenotype of dock deficiency. a case of igm deficiency and adult-onset still's disease negative. previous biopsy of her cervical and thoracic lymphadenopathy was unremarkable for malignancy. during her hospitalization, serum immunoglobulins were performed, which demonstrated normal levels of igg and iga, with igm level of < mg/dl (reference range - mg/dl), consistent with selective igm deficiency. liver function tests revealed an elevated aspartate aminotransferase (ast) of u/l and an alanine aminotransferase (alt) of u/l with a total bilirubin of . mg/dl and an alkaline phosphatase of u/l. her ferritin was elevated at g/l. the patient fulfilled yamaguchi criteria for aosd with three major criteria of evanescent rash, intermittent fevers in a quotidian pattern, bilateral arthralgias in the hips, knees, and ankles. she also met two minor criteria of liver abnormalities and lymphadenopathy. conclusions: selective igm deficiency is an uncommon immunodeficiency disorder associated with increased risk for autoimmune disorders. the recognition of co-morbid autoimmune illnesses in an immunodeficient patient is often complicated by a paucity of examples in the literature and potential confounding of laboratory serology analysis. we report the first case of a patient with selective igm deficiency and aosd. introduction/background: anaphylaxis to protamine is an uncommon but life-threatening complication of cardiac surgery and insulin therapy. here we present a case of recurrent protamine hypersensitivity during vascular surgery. objectives . recognize clinical signs of protamine hypersensitivity . recognize recurrent hypersensitivity to protamine as a serious complication of anesthesia methods: a year old man with a history of diabetes, previously on nph insulin, hypertension, hyperlipidemia, chronic smoking, and peripheral artery disease with multiple vascular interventions was admitted to undergo a right lower extremity saphenous vein graft bypass. three years earlier during a similar intervention, the patient had developed intraoperative hypotension after protamine sulfate administration. protamine was subsequently held for additional surgeries, however the patient was able to tolerate protamine with slower infusion one year later. for the current vascular surgery, the patient was pretreated the day of surgery with diphenhydramine and dexamethasone, and a test dose of protamine was infused prior to full dosing. the patient initially appeared to tolerate the full protamine dose, but quickly developed facial erythema and angioedema. due to concern for laryngeal edema he remained intubated and was transferred to the surgical intensive care unit, where he received additional diphenhydramine and dexamethasone. his symptoms resolved and he was successfully extubated the next morning. results: anaphylaxis to protamine is an uncommon but lifethreatening complication of cardiac surgery and insulin therapy. protamine sulfate is a polypeptide used widely to neutralize heparin anticoagulation during cardiac and vascular surgeries, and in nph insulin. severe anaphylactic or anaphylactoid reactions caused by injection of protamine sulfate are well documented in literature, and the product contains a black box warning for such. the pathophysiologic mechanisms underlying these reactions are not clear, but ige-mediated hypersensitivity appears to play a role in many reactions, and prior sensitization or cross-sensitization (eg, to fish) have been suggested. type b adverse drug reactions are idiosyncratic drug reactions and are often unpredictable, as in our patient who previously tolerated protamine but subsequently developed an adverse reaction. hypersensitivity reactions during anesthesia should be thoroughly studied to identify the responsible drug and minimize exposure in recurrent surgeries. conclusions: this case illustrates the potential for severe reactions even with newer protamine formulations, and highlights the unpredictable nature of type b adverse drug reactions. it is important for clinicians to exhibit awareness of the potential adverse effects of protamine sulfate in such situations. introduction/background: x-linked lymphoproliferative syndrome type (xlp- ) is a rare primary immune deficiency caused by loss of function in the x-linked inhibitor of apoptosis protein (xiap). common reported manifestations include recurrent hemophagocytic lymphohistiocytosis, splenomegaly, crohns-like inflammatory bowel disease, and transient hypogammaglobulinemia without reductions in major t cell or b cell repertoires, with the exception of inkt cells and mait cells. however, with only~ known cases worldwide, we are likely only beginning to understand the phenotypic spectrum of this disease. objectives: to describe additional manifestations of xlp- that expand our current understanding of its phenotype. methods: a year-old male with adult-onset, treatment refractory ulcerative colitis was evaluated in the immunology clinic for a history of recurrent sinopulmonary infections, skin abscesses, and recurrent ebv and vzv infections. extensive laboratory testing was performed in the course of his evaluation, including lymphocyte immunophenotyping, lymphocyte proliferation and cytotoxicity studies, quantification of total immunoglobulin levels and specific antibody function, hiv testing, and genetic testing. results: laboratory testing was significant for persistent cd lymphocytopenia ranging from - cells/mcl (rr: cells/mcl). total b cell count was normal but b cell subsets showed an elevation in the percentage of naïve b cells (range: . . %), low non-switched memory b cells (range: . - . %, rr: . - . %), and low to low-normal switched memory b cells (range: . %- . %, rr: . - . %), a pattern that has been seen in some autoimmune diseases. genetic testing with a commercial immune deficiency panel (invitae corp) showed a pathogenic mutation in xiap [exon , c. c>t (p.arg *)]. this mutation has previously been reported to cause a premature stop codon and reduced xiap function. the patient was referred for hematopoietic stem cell transplant and is currently awaiting transplant with a matched unrelated donor. conclusions: xlp- is typically reported as having normal t cell, b cell, and nk cell counts, but the presence of persistent cd lymphocytopenia in this patient illustrates that this is not always the case. our patient also had abnormalities in his b cell repertoire that have not been previously reported in xlp- . additionally, xlp- has been associated with crohns disease and celiac-like bowel diseases, while our case indicates that the phenotype may also include ulcerative colitis. ( ) submission id# a case report: enteroviral encephalitis as a consequence of partial humoral immunodeficiency in a chronic lymphocytic leukaemia patient treated with rituximab hadeil morsi, st immunology st , oxford university hospitals introduction/background: enteroviral (ev) infections are prevalent and usually self limited or cause mild gastrointestinal manifestations. however , in the context of primary antibody deficiency , rare cases has been reported to develop meningoencephalitis and been linked to poor outcome with fatality or chronic course. ev meningoencephalitis is even far rare reported in the era of rising secondary humoral immunodeficiency as a consequence of b cell depleting therapy e.g. rituximab and lymphoproliferative malignancies. limited treatments for ev encephalitis are available to date, apart from intravenous immunoglobulin replacement which has variable efficiency. objectives: studying such rare cases of ev meningoencephalitis as a consequence of antibody deficiencies would help to develop guidelines for intravenous immunogobulin replacement for treating these infections to improve outcome as well as predicting patients at higher risk who should be considered for prophylactic immunoglobulin therapy. methods: herein, we report a rare case of proven enteroviral meningoencephalitis following rituximab based therapy for b-cell chronic lymphocytic leukaemia and an uneventful six months period of follow up. he was found to have persistent absent b cells six months after completing six cycles of fludarabine, cyclophosphamide and rituximab therapy. interestingly, he had partial pneumococcal igg serotypes deficiency, whilst his total igg, igm and iga were all within normal limits throughout the course of the disease. the patient was treated empirically with intravenous immunoglobulin when his subtle confusion progressed to overt behavioural changes. initially his level of consciousness continued to deteriorate and he was not communicating. results: fortunately enough, the patient did have a remarkable improvement of gcs within couple of days and a slower recovery of higher mental functions e.g. memory and calculations in the next couple of months. conclusions: early suspicion and detection of entervorial meningoencephalitis in patients at risk of secondary antibody deficiency is crucial for timely ivig replacement and better outcome. patients with haematological malignancies and those on b cell depleting immunotherapy should be screened for pneumococal igg serotypes as part of secondary immunodeficiency workup. further studies on enteroviral neurological meningo/encephalitis are required to optimise ivig therapy and prognostication. furthermore, such studies provide an important asset to reveal the underlying mechanisms for humoral/b-cell mediated protective response against ev compared to other t-cell mediated viral immunity, whilst highlighting the mechanisms of immunodeficiency in cll and immunotherapy. ( ) submission id# a comparison of immune reconstitution following human placenta-derived stem cells (hpdsc) with umbilical cord blood transplantation (ucbt) vs. ucbt alone in pediatric recipients with malignant and non-malignant diseases introduction/background: ucbt is a safe and effective treatment in children (geyer/cairo et. al bjh, ) . however, due to a limited concentration of hematopoietic progenitor cells (cd +) in ucb, ucbt has been associated with delayed hematopoietic reconstitution and a higher incidence of engraftment failure. hpdscs contain a rich population of hpcs, are low in hla class i/ii expression and t-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (cairo et al bmt, ) . objectives: to determine whether ucbt + hpdsc (vs. ucbt alone) is associated with enhanced hematopoietic and immune cell reconstitution in children with malignant and non-malignant diseases. methods: immune cell reconstitution at days + , , and was assessed in children who received ucbt with hpdscs at nymc (nct , ind# ). minimum tnc was x ^ /kg ( / hla match) or . x ^ /kg ( - / hla match). immune cell subset counts at these time points were compared to those from a historical population of pediatric recipients of ucbt alone (geyer/cairo et. al bjh, ) . results: twenty four patients years were enrolled. mean age was (range, . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] years. malignant diseases = , non-malignant diseases = . fourteen patients received myeloablative conditioning (mac) and ten patients received reduced toxicity conditioning (rtc). there were no severe adverse events associated with hpdsc infusion. two patients with non-malignant disease receiving rtc using alemtuzumab experienced primary graft failure. probability of neutrophil engraftment was . %, median day . of evaluable patients at day , the probability of platelet engraftment in neutrophil engrafted patients was %, median day . ( - ) . at days , , and , mean percent donor chimerism in whole blood was , , , and %, respectively. average percent of whole blood hpdsc chimerism was % at day and < % at beyond day . one patient with malignant disease relapsed. month overall survival was . %. there was no significant difference in cd , cd , cd , cd and cd immune cell reconstitution following ucbt + hpdsc vs. ucbt alone (image ). conclusions: these results suggest that ucbt ± hpdsc results in similar immune cell reconstitution. a larger cohort with extended follow-up would be required to confirm these preliminary findings. supported by a grant from celgene cellular therapeutics. a decade of disseminated abscesses due to mycoplasma faucium in a patient with activated pi k syndrome (apds ) introduction/background: pik r monoallelic mutations are known to be responsible for apds-like syndrome, a rare form of primary immunodeficiency presenting as combined immunodeficiency or hyper-igm like phenotype. this study reports a patient carrying heterozygous pik r mutation with early onset and long-term disseminated abscesses due to mycoplasma faucium in both peritoneal abscess and skin, with generalized involvement in neck and both upper extremities. objectives: we describe clinical management of retroperitoneal and skin abscesses before molecular diagnosis was available in a patient with a primary immunodeficiency. identification by rdna s in so-called sterile abscesses may confirm the clinical suspect of an oportunistic infection. furthermore, this study offers insight on the pik r suspicion even in the absence of higm-like phenotype. methods: a -year-old girl with -year history of recurrent peritoneal effusion, which had been drained repeatedly was admitted in our institution for a -year history of multiple supurative cutaneous and lymph node-abscesses (fig & a-c). she had prior diagnosis of agammaglobulinemia under standard subcutaneous immunoglobulin replacement therapy and subcutaneous interferon-gamma treatment. on physical examination at the age of years, she was stunted (weight and height below the rd percentile), with facial, arm skin abscesses and right fistulized axillary lymphadenopaties, - cm hepatomegaly and giant splenomegaly results: she had her first immunological work up at the age of years during one isolated episode of knee arthritis and first episode of skin abscesses. serum immunoglobulins revealed panhypogammaglobulinemia (igg< . mg/kg, iga < . mg/kg, igm < . mg/kg) with low b cell count. on her back, there was a x cm, elastic, neither painful nor tender mass. after proper assessment by ct scan and mri she had her retroperitoneal abscess drained percutaneously, and healed with sclerotherapy (percutaneous alcohol and polidocanol instilation) by the interventional radiologist ( fig b) . analysis of drained pus as well as pus of skin abscesses was made by s rdna pcr, having coincidence of . % with mycoplasma faucium . combination antibiotic therapy (doxycycline and ciprofloxacin) was started with favourable response. unfortunately skin abscesses then relapsed. t-cell phenotype only showed t-cell lymphopenia with senescent (tem & temra expansion) phenotype. whole exome sequencing revealed a heterozygous mutation, previously reported (c. + g>t) conclusions: in summary, this report emphasizes the suspicion of a combined immunodeficiency in the presence of multiple abscesses by mycoplasma, the usefulness of rdna s in order to achieve proper objectives: we describe a -year-old male patient with novel heterozygous mutation of ep gene; his first manifestations were initially characterized by infections, cytopenia and hypogammaglobulinemia suggesting a common variable immunodeficiency (cvid), but later on, persisting lymphopenia was suggestive of a combined immunodeficiency. methods: the patient was born to unrelated healthy italian parents at weeks gestation with adequate weight for gestational age. shortly after birth, he underwent several surgical procedures due to interventricular defect, aortic coarctation, double outlet right ventricle, open botallis duct, and gastroesophageal reflux. at the age of four, he came to our attention due to stomatitis. clinical examination revealed dysmorphisms (microcephaly, wide forehead, sparse eyebrows, high nasal root, low-hanging columella, thick lips, micrognathia), splenomegaly (spleen diameter . cm at abdominal ultrasound), and severe developmental delay. in the course of the infectious episode, blood tests showed leukopenia associated with neutropenia (white blood cells . /mm ; neutrophils / mm ) and thrombocytopenia (platelets . /mm ). analysis of bone marrow aspirate revealed normal differentiation of both myeloid and erythroid lineages. treatment with high doses immunoglobulin resulted in increase of platelet counts (up to /mm after month), while neutrophil counts spontaneously returned to normal when the infection resolved. however, thrombocytopenia relapsed ( /mm ) after months and intravenous high-doses of corticosteroids did not achieve normal platelets count. despite oral corticosteroid treatment started at the age of six, two episodes of autoimmune hemolytic anemia occurred. during the following six years of follow-up, the patient experienced recurrent infections (stomatitis, upper respiratory tract infections, and skin abscesses), but none of the episodes has required hospitalization. but, at the age of ten, he was admitted to the hospital because of severe cultures negative diarrhea. despite immunoglobulin replacement therapy was started at the age of fourteen, he was admitted twice due to bilateral pneumonia requiring continuous positive airway pressure and, a few months later, acute respiratory failure with evidence of mycoplasma pneumoniae and rhinovirus infections. immunological evaluation under chronic corticosteroid treatment at different time points showed persisting lymphopenia, with lymphocyte counts ranging from /mmc to /mm , thrombocytopenia (platelets ranging from /mm to /mm ), undetectable anti-diphteria and anti-tetanus toxoid antibodies, and splenomegaly. interestingly, analysis of isohemoagglutinins, revealed low titers of anti-a ( : ) at years of age, but normal immunoglobulins (igg mg/dl, iga mg/dl, and igm mg/dl). at the age of seven, reduced mitogen proliferation, hypogammaglobulinemia (igg mg/dl; iga mg/dl, igm mg/dl), increased cd +tcr+cd cd t-cell counts ( . / . %) and impaired fas mediated apoptosis as measured in two separate assays (table ) . at the age of fourteen, evaluation of b-cell subsets showed increase of cd locd lo cells and reduction of switched memory b-cells. analysis of t-cell compartment unveiled a decreased proportion of cd +ccr +cd ra+ recent thymic emigrants (rte) cells and ccr +cd ra+ naive cells, with prevalence of effector memory t-cells (ccr -cd ra-) ( table ) . interferon signature gene expression showed borderline levels of ifi (data not shown). because of the decrease of igg and the infectious episodes ivig treatment was started at age of fourteen. a molecular investigation performed by whole exome sequencing (wes) revealed a novel heterozygous missense mutation (nm_ . :c. t>c , p.met thr) in the exon of the gene ep encoding the histone acetyltransferase (hat) protein p . results: few immunological reports are available in rsts patients - . in keeping with previous data , , , our patient presented with progressive b-and t-cell lymphopenia, hypogammaglobulinemia with poor antibody response but also reduced naïve t cells, evans syndrome, splenomegaly, and defective lymphocyte apoptosis with increased dnt. at the age of seven, the patient presented the features of cvid. flow cytometry revealed expansion of cd hicd locd lo b cells, that is frequently associated with splenomegaly in cvid patients , and reduced switched memory b-cell, previously reported in a rsts patient with crebbp mutation (fig. s ). lougaris et al. reported expansion of cd locd lo b-cells in nf-kb haploinsufficiency , and this suggests in our opinion that alterations in the nf-kb pathway due to ep mutations may affect b-cell differentiation. compared to healthy controls and cvid patients with predominant infectious complications, upregulation of interferon responsive genes in cvid subgroup with noninfectious complications (i.e hematologic autoimmunity, lymphoproliferation) and lymphopenia with reduced total b cells and switched-memory b cells has been demonstrated . borderline levels of ifi expression under corticosteroid treatment represent a novel finding in rsts. interferon signature may identify and better characterize subgroup of rsts patients with autoimmune cytopenias and lymphopenia. at the age of fourteen, analysis of lymphocyte subsets revealed decreased total cd +, cd +, cd +, and of both naïve cd + and cd + cells. elevated and persisting igm levels were also observed (fig. s ). according to our data, increased igm levels may be related to high proportion of terminal differentiated igm+ cells. these data (infections requiring hospitalization, immune dysregulation, lymphopenia, reduced naïve t cells, and reduced proliferation to mitogen) together with clinical history (fisher evans syndrome and lymphoproliferation) and exclusion of known syndromic immunodeficiencies, suggested a diagnosis of combined immunodeficiency . conclusions: our case underlines the value of wes in patients with difficult phenotype-genotype correlation. no rsts typical traits were present and, prior to wes, several syndromes and immunodeficiencies were excluded. our report expands the phenotypic spectrum of ep mutations, thus in syndromic patients with clinical and immunological overlap between cvid and cid ruling out ep mutation should be advisable. furthermore, immunological work-up should be taken into consideration in rsts patients, in order to early identify immunological abnormalities that may lead to severe immune-hematological complications. introduction/background: interferon gamma receptor (ifngr )-related disorders are rare variants of mendelian susceptibility to mycobacterial diseases. although hematopoietic stem cell transplantation (hsct) is curative, it is complicated by high rates of delayed or failed engraftment thought to be due to high concentrations of interferon (ifn)-gamma. umbilical cord blood transplantation additionally increases risk of graft failure. objectives: describe a pediatric patient with non-functional ifngr who successfully underwent umbilical cord blood transplantation. methods: direct clinical care of described patient with additional electronic medical record chart review. results: the patient is a -month-old boy of yemeni descent who initially presented with significant hepatosplenomegaly and extensive lymphadenopathy, including a large mediastinal mass. he then developed salmonella enteritidis sepsis requiring numerous antimicrobials, vasopressor support, intubation and continuous renal replacement therapy. his evaluation showed a hyperinflammatory state with elevations in ferritin, ifn-gamma, scd , il- , il- , il- , il- and il- levels. maximal ferritin and ifn-gamma levels reached . ng/ml and pg/ml (normal < pg/ml), respectively. flow cytometry revealed normal expression of ifngr and il r but absent ifn-gammastimulated stat phosphorylation, suggesting defective ifngr signaling. genetic testing showed a previously unreported homozygous mutation in ifngr (c. + t>c) which affects a donor splice site in intron and is predicted to cause absent protein function. dexamethasone and a single dose of alemtuzumab ( . mg/kg) were given to decrease inflammation. he then underwent allogeneic hsct using a / human leukocyte antigen matched umbilical cord unit following a reduced-toxicity conditioning regimen of alemtuzumab ( . mg/kg), fludarabine ( mg/m ) and busulfan (auc mg/l*h). plasma ifn-gamma was undetectable prior to starting conditioning and on the day of transplant. neutrophil engraftment occurred on day + with day + posttransplant chimerism analysis of peripheral blood myeloid cells showing the presence of donor cells only. conclusions: these early results suggest that umbilical cord blood transplantation may be feasible in patients with ifngr -related disorders provided adequate control of inflammation is gained prior to transplant. introduction/background: introduction: btk is a cytoplasmic tyrosine kinase that activates phospholipase c (plc ) via phosphorylation, which ultimately leads to the activation of nfk, which is essential for b cell development and survival. mutations in btk lead to x-linked agammaglobulinemia (xla). in addition to the pleckstrin homology and tyrosine kinase domains, btk contains two src homology domains, sh and sh , which are essential for btk function. we describe a novel btk mutation (c. a>t) resulting in v a substitution in the sh domain that results in aberrant xla function with nearly normal btk protein expression. objectives: case report/results: the male proband presented with recurrent otitis media, persistent fevers and neutropenia beginning in the first year of life with an igg level of mg/dl and a lack of b cells ( cell/mm ), as demonstrated by flow cytometry. btk protein expression in monocytes, also determined by flow cytometry, was equivalent to controls. family history is significant for a maternal uncle with history of recurrent sinus infections and pneumonias with low iga and igm, low to normal ige, and an absent vaccine response. flow cytometry also showed an absence of b cells and essentially normal btk protein expression in his monocytes compared to controls. targeted high throughput sequencing of both proband and the uncle revealed a previously unreported missense mutation in exon , leading to the substitution of an aspartic acid residue for a valine (v d.) the mutation is in the highly conserved sh domain of btk (conservation phylop conservation score . .) the probands mother and his sister were shown to be carriers of the same mutation and had normal serum immunoglobulin levels and normal numbers of b cells. methods: we hypothesized that if the btk v d mutant protein was non-functional, the female carriers of the mutation would only express wild type (wt) btk in their b cells while their monocytes would express both wt and mutant btk. to test this hypothesis cd + b cells and cd + monocytes were purified from pbmc by fluorescence activated cell sorting (facs) from the sister, cdnas were generated from the respective populations of cells, and the btk cdna was sequenced using high-throughput sequencing. results: at a read-depth greater than , , cd + b cells demonstrated btk expression only from the wt allele (~ % wt btk) whereas both the wild type and mutant allele of btk were expressed at approximately equal levels in monocytes. conclusions: discussion: these results define a novel mutation in btk that nominally affects protein expression, but alters function. the v a substitution is found in the d structural element of the sh domain that is part of a hydrophobic phosphotyrosine binding pocket. a mutation in the adjacent residue, y s, has been shown to alter protein conformation and decrease binding affinity to plc by roughly -fold resulting in xla. our study, using a carrier harboring the c. a>t mutant btk, demonstrated that in contrast to mononuclear cells, b cells only expressed the wt allele. this is consistent with the loss of function of v a btk protein, thereby causing xla in both the proband and affected uncle. introduction/background: pyoderma gangrenosum (pg) is often associated with systemic autoimmune diseases but it has rarely been reported with common variable immune deficiency (cvid). while genetic analysis has been increasing in both disease domains, there has been little investigation into the genetic components associated with the cooccurrence of these entities. heterogeneous nfkb mutations have recently been identified in familial cases of cvid, though rarely have they been associated with pg. objectives: this case describes a novel nfkb mutation that may link both cvid and pg, and bolsters the recent identification of heterogeneous nfkb mutations in cvid. methods: a -year-old woman with a history of frequent skin infections in childhood presented with persistent, infected wounds following cholecystectomy. upon admission, she was started on broad spectrum antibiotics but continued to have fevers and leukocytosis. labs were also notable for elevated crp ( ; normal (n): . - ) and esr ( ; , with low c ( ; n: - ), c (< ; n: - ) , and ch (< ; n:> ). wound cultures grew multi-drug resistant coagulase negative staphylococcus. despite broad spectrum antibiotics, the wounds failed to heal. dermatology was consulted and punch biopsy revealed a dense neutrophilic infiltrate and no identifiable pathogens, which supports the diagnosis of pg. the patient was started on high dose steroids ( mg/kg/ day) and had a rapid response with decreased skin inflammation and lesion expansion. unfortunately, the patient developed posterior reversible encephalopathy syndrome (pres) on steroids and therefore pg treatment was changed to infliximab as recommended by dermatology. further laboratory testing found that the patient also had low igg ( ; n: - ) and iga ( ; , and a diagnosis of cvid was made given her clinical history of recurrent skin infections. genetic testing was pursued to evaluate additional pg therapy options and this revealed a heterozygous mutation in nfkb (c.a g; p.q q), located base pairs upstream of the splice donor site for exon twenty-one. while this mutation has not been previously identified as a pathogenic variant, similar mutations in this gene have been linked to autosomal dominant cvid. the patients father also carried a similar mutation but without any evident clinical phenotype. results: nfkb plays a crucial role in both immune and inflammatory responses. this case highlights a novel mutation in nfkb that has not been previously described as a disease-causing change. other mutations resulting in nfkb haploinsufficiency have been associated with cvid and rarely with concurrent pg, as in this case. based on the location of the mutation, it is expected that the variant causes obliteration of the normal splice site and therefore results in defective mrna that encodes p / p . interestingly, studies on p knockout mice show decreased levels of igg, iga, and ige but not igm and our patient similarly had low levels of igg and iga but normal igm. conclusions: further studies are needed to determine if there are other links to this novel nfkb mutation in patients with cvid and pg. ( ) submission id# a rapid flow cytometric analysis of dna repair proteins reveals a radiosensitive phenotype in bcl b deficiency associated with severe combined immunodeficiency (scid). introduction/background: we present the second report in the literature of a patient with immunodeficiency, dysmorphic features, growth retardation, and a homozygous variant in the dna ligase i (lig ) gene with associated absence of full-length lig protein. results: this is a now year old girl who was the fourth child of parents who are first cousins. she has one healthy older brother, a second older brother who died within hours of birth of meconium aspiration, and an older sister who died at six months of age of an upper respiratory illness / pneumonia after a history of congenital anemia, poor weight gain, cardiomegaly and hepatomegaly. she was born at weeks and spent the first five weeks of life in a neighboring hospital neonatal intensive care unit (nicu) for hepatomegaly, mild cardiomegaly (previously identified on fetal ultrasound) and congenital anemia (requiring transfusions). her exam was and remains notable for weight, height, and head circumference below rd percentile, prominent forehead, hypotelorism with epicanthal folds, downslanting palpebral fissures, and low set, posteriorly rotated and prominent ears. after discharge to home from the nicu, her course was subsequently complicated by poor weight gain, chronic diarrhea beginning after her first rotavirus vaccine, and multiple deep vein thromboses. she then became critically ill at months of age with respiratory failure, and was transferred to our institution for respiratory oscillator support. absolute lymphocyte count on admission to our institution was . k/μl (total wbc . k/μl, anc . k/μl) with agammaglobulinemia. she was diagnosed with and treated for pneumocystis jirovecii pneumonia, gradually weaned from oscillator to room air, and was discharged home five weeks later on . mg/kg every other week igg replacement. she has had no serious infections requiring hospitalization in the months since. alc has remained persistently below . k/μl with a corresponding uniform deficiency of t, b, and nk cells and no detectable trec positive t-cells. whole exome sequencing identified homozygosity for a c. g>a coding region variant in the lig gene not previously reported in the literature. a fibroblast cell line was successfully established and western blot shows an absence of full-length lig protein. further molecular characterization is in progress. conclusions: this second reported case provides further evidence for dna ligase i deficiency as a distinct clinical entity comprising immunodeficiency, dysmorphic features, and growth retardation. introduction/background: chronic mucocutaneous candidiasis (cmc) is associated with a heterogeneous group of primary immunodeficiencies. autosomal dominant stat gain-of-function (gof) mutations have been identified in up to % of patients with cmc. these mutations lead to impaired il a/f t cell immunity although the underlying mechanism is unclear. there seems to be no genotype-phenotype correlation. recently, jak inhibitor therapy has been reported to improve cmc and autoimmunity in patients with stat gof mutation. objectives: we describe an infant with cmc associated with a novel stat gof mutation. results: a -month-old girl was referred to our immunodeficiency clinic with chronic diaper rash since weeks of life, failure-to-thrive, and history of labial abscess complicated by rectolabial fistula. she was subsequently diagnosed with food protein-induced enterocolitis syndrome triggered by cows milk-based formula. laboratory evaluation revealed normal cbc with differential, lymphocyte subsets, mitogen response, immunoglobulin levels, antigen response to candida, and neutrophil oxidative burst assay. whole exome sequencing identified a de novo heterozygous variant in stat (c. t >c, p.cys arg) . further evaluation of this mutation revealed increased gas (gamma activation sequence) reporter activity in response to ifng stimulation suggesting that this is a gain-of-function mutation. the patient later developed significantly elevated liver enzymes while on fluconazole treatment, candida parapsilosis sepsis, granulomatous lesions in the liver, splenic lesions, intermittent thrombocytopenia and n o r m o c y t i c a n e m i a . s e p s i s a n d l i v e r l e s i o n s r e s o l v e d on amphotericin treatment but other findings, including tpn dependency persisted. we are planning to initiate a jak inhibitor therapy, ruxolitinib. conclusions: this case is a possible genotypic and phenotypic expansion of cmc due to stat gof. professor, the university of british columbia introduction/background: b cell cll/lymphoma b (bcl b) is a zinc finger protein transcription factor with a multitude of regulatory functions in the integumentary, central nervous, cardiac, and immune systems. it is critical for t cell lineage commitment, development, differentiation, survival, and function. in addition, it also specifies the identity and function of innate-like lymphocytes, including t cells, innate lymphoid cells (ilcs), and invariant natural killer t cells (inkt). however, little is known about its function in the human immune system, especially in the context of immune disorders. objectives: to understand the immunopathogenesis of a novel p.c y bcl b variant. methods: research study protocols were approved by our institutional research ethics board. two members of the family were enrolled (the index patient and her father). written informed consent for genetic testing and participation was provided by the parents for the child. genetic, bioinformatic, proteomic, and biochemical analyses were performed. results: we have identified the second described case of immune disease caused by a de novo heterozygous damaging variant of bcl b (p.c y). this young girl presented with intellectual disability, microcephaly, severe atopy, eczema, alopecia totalis, and brittle nails. extensive clinical immunophenotyping of patient blood showed initially unremarkable b and t cell populations. however, the patient possessed abnormal rare innate-like lymphocyte populations (inkt, dn t cells). using mass cytometry (cytof), a technique capable of concurrently analyzing parameters in a single cell, we were able to examine various innatelike lymphocyte populations, including t cells, ilc - , and nk cells. we found that the patient possessed severely compromised numbers of t cells, thus potentially implicating the p.c y variant in t cell development and function. conclusions: the identification of decreased t cells in a patient with a p.c y variant of bcl b suggests that bcl b is important for human t cell development and provides novel insights into the roles of both bcl b and t cells in regulating atopy and autoimmunity. introduction/background: adenosine deaminase deficiency caused by mutations in ada gene is a newly recognized disorder. it is associated with a spectrum of vascular and inflammatory phenotypes, ranging from early onset recurrent stroke to systemic vasculopathy or vasculitis. objectives: we describe a year old female patient with features of early onset immune thrombocytopenia (itp), autoimmune hemolytic anemia (aiha), chronic splenomegaly and variable abdominal lymphadenopathy. she was diagnosed with evans-syndrome and treated with rituximab at and month of age. from years of age she developed recurrent infections, hypogammaglobulinaemia with specific antibody deficiency, progressively decreasing class-switched memory b cells, and increased cd +cd -cd -//t cells ( %). differential diagnosis included common variable immunodeficiency (cvid) or autoimmune lymphoproliferative syndrome and therefore a broad search for causative genetic defect was initiated. the parents are first cousins of middle-eastern origin suggesting an autosomal recessive inheritance. patient was stable on long-term mycophenolate mofetil (mmf) and immunomodulatory dose ( g/kg/ month) ivig treatment. methods: genomic dna of the patient was sequenced with next generation sequencing technology. a panel of genes linked to primary immunodeficiency was analyzed. the identified variant was confirmed by sanger sequencing. results: genetic testing revealed a homozygous pathogenic mutation in the ada gene with one base pair duplication in exon (c. dup. p.arg alafs* ) that creates a frame shift starting at codon arg . the new reading frame ends at a stop codon positions downstream, likely resulting in a truncated protein. plasma ada activity of the patient was markedly reduced ( . mu/ml, normal . - . ) and confirmed the diagnosis of ada deficiency. the parents of the patient are heterozygous carriers of the same mutation. unlike most previously reported cases, this patient had an extended phenotype with no neurological evidence of vascular pathology, however brain mri revealed two silent lacunar infarct or vasculitis related changes. we speculate whether the long-term mmf or ivig therapy might be protective against vasculitis. conclusions: ada deficiency may present with a wide spectrum of clinical phenotypes beyond classical vasculopathy. the diagnosis should be considered in patients with hematological autoimmune disease, splenomegaly and/or cvid like presentation. better understanding of pathophysiology of ada deficiency may help diagnosis and targeted treatment. professor, university of california, los angeles ca introduction/background: adenosine deaminase (ada) deficiency as a cause of severe combined immunodeficiency (scid) is distinct from other forms of scid in several ways. historically, survival and clinical outcome of infants with ada scid have been inferior compared to infants with other scid genotypes. there are multiple treatment modalities available for ada-scid, including enzyme replacement therapy (ert), allogeneic hematopoietic cell transplant (hct) and experimental autologous transplant of gene corrected cells (in recent years preceded by low dose busulfan), designated as gene therapy (gt). in addition, there is a growing body of evidence for effects of ada deficiency on non-immunologic organ systems that may contribute to the historically poorer outcomes of these infants. therefore, it is important to evaluate the cohort of patients with ada scid separately from other scid cases. objectives: to capture incidence and treatment trends and to compare outcomes following available treatments for this rare inborn error of metabolism and other forms of scid, the primary immune deficiency treatment consortium (pidtc), a network of north american immunology and transplant centers, has collected standardized data for analysis. methods: ada scid patients, first treated between through , were enrolled from centers (range - subjects/site). ada accounted for % of the total pidtc scid patients treated during that time. patients were entered into either a retrospective protocol (pidtc , n= ) or a prospective protocol starting in (pidtc , n= ) . ada-scid patients who received an hct as first therapy entered either of two strata, as with other scid patients in pidtc studies, based on whether their initial presentation met definitions for typical (n= ) or leaky scid (n= ); in contrast, patients initially treated with either ert or gt were entered into a separate stratum (n= ). results: sixty-four patients ( % of all ada-scid enrollees) had ert as first therapy, but only in this cohort received ert as sole therapy; went on to have subsequent hct (n= ) or gt (n= ). there were various combinations of treatment cycles among these ada-scid patients. most received hct {+/-subsequent treatments} ( %), ert followed by hct {+/-subsequent treatments} ( %), or ert followed by gt {+/-subsequent treatment} ( %); several patients received multiple successive treatment modalities, representing either failure of initial treatment or planned progression from ert to cellular therapy. two-year survival has improved over time from % in ( - to % - (p= . ) (figure ). the survival for all other non-ada scid patients registered by pidtc over these two eras were: % ( - ) and % ( - ) . hct (either as sole therapy or after ert) accounted for > % of cellular therapies between and ; in contrast, since , gt was used as commonly as hct (n= vs. n= , respectively). there was a trend toward better two-year survival for patients receiving gt as first cellular therapy since ( %, n= , all after initial ert) compared to those receiving hct over the same time period ( %, n= , either as first therapy or after ert), although this did not achieve statistical significance (p= . ). conclusions: this study reveals the improved prognosis for patients with ada scid in recent years and the emergence of gt as a new treatment modality. further analyses are investigating the impacts of prior infection and treatment modality, including effects of conditioning, on outcomes (survival, event free survival, clinical outcomes and completeness of immune reconstitution) for ada-scid in successive eras. this study may identify optimal treatment approaches for future ada scid patients. sponsored by the pidtc, a member of the rare diseases clinical research network (rdcrn) and funded by u ai (niaid and ordr, ncats, nih). dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. jp discloses that her spouse is employed at invitae, a dna sequencing company. intern, imam abdulrahman bin faisal university introduction/background: patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. in fact, vaccines containing thymus-dependent antigens protect patients with diabetes as they produce massive and complex immune response and feature immunologic memory. the recommended vaccinations for patients with diabetes mellitus are influenza vaccination yearly and pneumococcal vaccination. in observational studies, influenza vaccine has been shown to be similarly effective in adults < years of age with diabetes as in older patients with or without diabetes [ ] . among immunocompetent elderly, vaccine efficacy of the -valent pneumococcal conjugate-vaccine (pcv ) was modified by dm with higher vaccine efficacy among subjects with dm [ ] . the hepatitis b vaccination should be given to unvaccinated adults with diabetes mellitus who are ages to years. for older patients administration only after assessment of benefits and risks of acquiring hepatitis b virus (hbv). in fact, one review suggests that dm is associated with the progression of severe liver outcomes in adults with hbv [ ] . on the other hand, tetanus and diphtheria vaccinations should be updated. in addition to, vaccinations such tick-borne encephalitis, meningococcal infections and other infections that put in risk diabetic patients travelling abroad. accordingly, theres a variability of vaccines that can offer a preventive method to reduce morbidity, mortality, and medical expense. in our multicenter study among eastern province saudi arabia evaluated the degree of adherence of the physicians to the immunization recommendations for adult patients with diabetes mellitus type and type to increased awareness of the immunization importance in diabetic patients. objectives: to increased awareness of the immunization importance in diabetic patients. in fact, patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. methods: this is a cross sectional study involving adult patients with type and type diabetes mellitus using a questionnaire. patients will be recruited from outpatient clinics including the primary care clinics and inpatient words of king fahd hospital-al khobar and other centers in the eastern province saudi arabia. after an informed consent, baseline data will be collected. patients will be then asked if they received the recommended vaccines and who was the provider. their knowledge regarding the needed immunization will be also tested. they will then be asked about frequency of upper respiratory tract infections and pneumonia they had over the last years. results: we are expecting to find low adherence to the recommended immunizations by the physician. we may also find that patients who received the vaccines has low incidence of related infections. conclusions: increased awareness of the immunization importance in diabetic patients and adherence to immunization as part of standard care of adult patients with diabetes mellitus that offer a preventive method to reduce hospitalizations, mortality, and medical expense. - , - , and - bases. repeatability and reproducibility of the assays were . and . , respectively. . % of the target regions were covered with over x sequencing depth. we showed that the assays had . sensitivity to detect single-exon deldups and . sensitivity to detect copy number aberrations covering two or more exons. using acmg guidelines for variant classification a diagnosis was established in % patients that were sent for comprehensive panel analysis. conclusions: conclusions: our results demonstrate the analytic validity of the developed tests and show that the technology is well-suited for clinical diagnostics of inherited eye disorders. it also demonstrated a cost-effective diagnostic tool to simultaneously diagnose various types of mutations from snvs to copy number variations. introduction/background: loss of function (lof) and null mutations in orai and stim cause a rare autosomal recessive immunodeficiency by abolishing calcium release-activated calcium (crac) channel function and store-operated ca + entry. the clinical presentation is characterized by scid-like disease, dental enamel defects, muscular hypotonia and anhidrotic ectodermal dysplasia. objectives: here we present the outcome of calcium assessments performed on lymphocytes from an adult patient with unusual infections and a purported novel single pathogenic variant in orai . methods: the ca + response in lymphocytes following activation by varying concentrations of non-cross-linked anti-cd was assessed by flow cytometry. results: the patient was a year old female with a history of seasonal and medication allergies and recurrent sinus infections, who had recently developed an acute infection of her right first metatarsal joint. cultures from the joint space grew neisseria gonorrhea and atypical mycobacteria at two different time points. hiv screening was negative. follow-up testing with the mantoux test and quantiferon gold suggested that she also had latent tuberculosis infection, for which she was started on rifampicin therapy. immunologic evaluation revealed normal complete blood count and differential, normal t, b and nk cell counts, normal immunoglobulin g, a, and m levels, as well as normal responses to polysaccharide vaccines and normal t cell proliferative responses to mitogen and antigen stimulation. however, given the identification of atypical organisms from joint fluid cultures as well as latent tuberculosis (despite the lack of significant risk factors), genetic testing was recommended by her local physicians to rule out an underlying primary immunodeficiency. an invitae primary immunodeficiency panel identified a single novel pathogenic variant in orai . she was then referred to our institution for further evaluation. so far, individuals identified as heterozygous for lof or null mutations in either orai or stim have lacked any phenotype associated with crac channelopathy. however, there have been reports of abnormalities in calcium response in parents of a number of these patients, who are heterozygous for the disease-causing mutation. to examine the functional effect of the observed mutation in our patient, her freshly isolated pbmcs were loaded with indo- and the ca + response of her lymphocytes were assessed by flow cytometry. this showed a dose-dependent decrease in the patients t cell response to non-cross-linked anti-cd in comparison to the normal control, i.e. there was a clear decrease in the patients ca + response at microgram/ml in comparison to the normal control, but the decrease was rectified upon stimulation with microgram/ml or more of anti-cd . conclusions: these findings provide functional support for the identification of a new pathogenic mutation in orai . nevertheless, it is not yet clear if the mutation has any mechanistic role in the patients recent clinical presentation. introduction/background: patient : years old boy, born to nonconsanguineous parents, with hypothesis of autoimmune encephalitis (vasculitis), which was not characterized. csf has already been routed to barcelona times and to vienna time. refractory epilepsy remains (uses drugs yet). he had continuous fever during the entire hospitalization. he had adhd (took ritalin for years) and central auditory processing deficit. one year ago he began to have fever for days, improving later but evolving with bilateral otitis, predominantly on the left ear, accompanied by sinusitis. soon after that he began presenting epilepsy that worsened severely. he was interned and took acyclovir ev, associated with hydantoinate (had stevens-johnson by the drug, suspending and improving quickly). in march had uti + amo, several infections requiring meropenem + vanco, among others. received flebogamma mg/ day/ days, twice; he also received weekly rituximab for a few months. in use of carbamazepine, clobazan, phenobarbital, levotiracetam and vigabatrin times a day. personal antecedents: allergic rhinitis, bronchial asthma, recurrent otitis media, iga deficiency. patient : male, years old, born to non-consanguineous parents, with history of repeated infections in the upper respiratory tract from one year of age with prolonged dry cough and sore throat in all episodes, treated with dexamethasone without improvement, followed by antibiotics with resolution of the condition. at age five, in february , he had a new episode of sore throat and cough that lasted three months, improving spontaneously thereafter. in june , new episode of sore throat with elevated fever and whitish plaques in the tonsils, being prescribed benzetacil, without improvement in three days. he returned to the same emergency room, the antibiotic was replaced by zinnat (axetil-cefuroxime), but within the hospital he began to convulsionate, entering into an epileptic crisis, being hospitalized for days in this service and being transferred to another hospital specialized in pediatrics, intensely investigated and treated with partial improvement of the condition. he remained in coma, not walking and talking for some months, recovering slowly with physical therapy and speech therapy. of relevant exams have: reduced iga, but before it was normal (probably induced by anticonvulsants); full-body magnetic resonance imaging demonstrates generalized lymphadenomegaly and hepatosplenomegaly; pet-ct showing signs of hypoperfusion in temporal (right), occipital and cerebellum regions (suggestive of hypoperfusion -vasculitis?) my first impression was of possible mevalonate kinase (mvk) or autoimmune lymphoproliferative syndrome (alps) deficiency. with these tests described above, i believe that the first diagnostic suspicion is that the epilepsy was triggered by hemophagocytosis in the central nervous system and consequent extremely severe epilepsy, triggered by ebv infection. objectives: to compare the clinical and genetic similarities and differences of both patients. methods: both patients wer submitted to whole exome sequencing looking for the genetic alterations associated to the disease of these patients. results: wes of patient showed an allelic variant in the gene of rai (c. g.a; p.arg gln) possibly pathogenic and that could be related to the clinical features of the patient. wes of patient showed an allelic variant in the gene of cd (c. g>a; p.arg his) heterozygous and of uncertain significance. another allelic variant was found in the gene of btk (c. g>a; p.arg gln) classified as of uncertain significance and hemizygous (as btk is in chromosome x). the expression of both proteins evaluated by flow cytometry is normal, decreasing but not abolishing the possibility of pathogenicity. conclusions: the similarity of the clinical presentations is striking, but the genetic alterations are totally different, leading to the presentation of this abstract. (inf-) have been associated with adult onset immunodeficiency in patients of asian origin. pathogens that cause infections in these patients include mycobacterium avium-intracellularae (mai), non-typhoidal salmonella, cytomegalovirus, penicillium marneffei, and varicella zoster virus. methods: chart review of one patient results: we present a thirty-two-year-old filipino female, with sjogrens syndrome, penicillin and vancomycin allergy, and shellfish allergy suffering from recurrent mai spinal osteomyelitis. after three months of conservative management of back pain, mri showed an abscess at l /l and l / s vertebrae, which was diagnosed as acid fast bacilli on biopsy. she was treated for mycobacterium tuberculosis with rifampin, isoniazid, pyrizinamide, and ethambutal with subsequent change in antibiotic therapy after six weeks once cultures grew mai. mri showed spread of abscess to l -s vertebrae. three months into treatment, she was found to have a new abscess at a different spinal site, and antibiotics were again changed. two months later, she had recurrence of disease with multiple large iliopsoas abscesses, cutaneous fistulas, insufficiency fractures of the sacrum bilaterally, and osteonecrosis of the l vertebra, requiring extensive surgical debridement. medications were adjusted and she was referred to immunology eight months after initial presentation to infectious disease. laboratories were notable for elevated igg ( mg/dl) and iga ( mg/dl) and decreased cd ( cell/ul) and cd / ( cell/ul) cells. serum electrophoresis showed low albumin, elevated gamma fraction, and polyclonal gammopathy. specific antibody titers, lymphocyte proliferation assay, ch , and immunofixation were within normal limits. cytokine panel was significant for elevated il- receptor cd ( pg/ml), il- ( pg/ml), and il- ( pg/ml), and normal tnf, inf, il- , il- , il- , il- , il- , il- , il- , and il- . further serologic testing was positive for autoantibodies to inf-. patient continues treatment with iv antibiotics and is awaiting enrollment in a rituximab trial. conclusions: autoantibodies to inf-should be considered in patients of asian origin presenting with adult onset immunodeficiency, particularly those with severe or recurrent infection with mai. a high level of suspicion is required to make this diagnosis: failure to consider this disease entity leads to delay in diagnosis with potentially significant consequences for the patient. allergy/immunology, university of south florida at johns hopkins all childrens hospital introduction/background: autoimmune and inflammatory conditions are common in cvid. these have been associated with increased morbidity and mortality. objectives: we sought to further understand and evaluate the prevalence of autoimmune and rheumatologic manifestations in patients with common variable immunodeficiency (cvid). methods: we performed a retrospective analysis of cvid patients with rheumatologic/autoimmune complications in the partners healthcare cvid cohort. we evaluated baseline patient characteristics as well as autoimmune and rheumatologic complications in this cohort of patients. results: in the partners cvid cohort, / ( %) had autoimmune or rheumatologic disease. autoimmune cytopenias were reported in / ( %) patients, including coombs positive autoimmune hemolytic anemia (n= ), idiopathic thrombocytopenic purpura (n= ), and autoimmune neutropenia (n= ). autoimmune thyroid disease was reported in / ( %) patients, including hypothyroidism (n= ) and hashimotos thyroiditis (n= ). inflammatory arthritis was present in / ( %), most commonly seronegative rheumatoid arthritis (ra) (n= ), followed by inflammatory arthritis (n= ), seropositive ra (+rf or +ccp antibody) (n = ), psoriatic arthritis (n= ), and juvenile idiopathic arthritis (n= ). systemic autoantibody disease was diagnosed in patients ( %), with diagnoses including vasculitis (n= ), systemic lupus erythematosus (n= ), polymyalgia rheumatica (n= ), antiphospholipid syndrome (n= ), mixed connective tissue disease (n= ), crest/ scleroderma (n= ), myositis (n= ), sjogrens syndrome (n= ), and discoid lupus erythematosus (n= ). inflammatory neuropathy was diagnosed in patients, with small fiber polyneuropathy (n= ), uveitis (n= ), myasthenia gravis (n= ), bells palsy (n= ), and multiple sclerosis (n= ). autoimmune skin conditions were diagnosed in patients with diagnoses including psoriasis (n= ), alopecia (n= ), and vitiligo (n= ). while the mean igm was higher in the patients with autoimmune/rheumatologic manifestations than in other cvid patients ( vs mg/dl), this difference did not reach statistical significance (p= . ). conclusions: autoimmune and rheumatologic complications are present in over half of patients with cvid. increased vigilance for autoimmune and rheumatologic complications is important as survival outcome are worse in cvid patients with non-infectious complications as previously described. further evaluation of these patients to understand the mechanism of immune dysregulation is essential, as this may promote targeted therapies and improve clinical outcomes. introduction/background: immunoglobulin concentrates have been successfully used for decades to treat patients with primary or secondary immunodeficiency disorders. this treatment has substantially decreased the frequency of life-threatening infections in these patients. octanorm is a newly developed maltose-formulated subcutaneous immune globulin (human) . % liquid for the treatment of patients with primary immune deficiency (pid) and secondary immune deficiency (sid). objectives: biochemical and physico-chemical properties were investigated. methods: molecular size distribution of monomers, dimers, polymers and fragments were determined (according to european pharmacopeia (ep) monograph . ) by size exclusion chromatography (sec). igg and igg subclass concentrations were quantified by respective nephelometric methods. functionality of the igg was demonstrated by measurement of fc function, opsonophagocytosis and fc gamma receptor binding assays. dynamic light scattering measurement and size exclusion chromatography were used to characterize the integrity of the igg molecule. measurement of potential procoagulant activity was done by natem and tga (fxia-like activity). the capacity of the octanorm manufacturing process to robustly inactivate/remove pathogens was investigated in spiking experiments with prions and viruses. results: octanorm contains more than % of human igg and is characterized by an especially low content of polymers and aggregates, low viscosity, low isoagglutinin titres, low iga and igm contents with a broad spectrum of antibodies against infectious agents. it has a distribution of immunoglobulin g subclasses closely proportional to that in native human plasma. in the final product, potential procoagulant activity is not detectable. functionality and physico-chemical properties of the igg molecules were demonstrated by state-of-the-art methods. virus safety of octanorm is obtained via a combination of three validated orthogonal methods as part of the manufacturing process: cold-ethanol fractionation, solvent/ detergent (s/d) and ph treatment. a substantial depletion of prions during the manufacturing process was demonstrated. conclusions: octanorm is a state-of-the-art subcutaneous immunoglobulin. based on the excellent stability the intended shelf life of octanorm is months stored at + °c to + °c protected from light. within its total shelf life the product can be stored at room temperature up to + °c for up to six months. efficacy and very good tolerability of this new subcutaneous normal immune globulin . % were shown in a clinical phase iii study performed in centers in north america and europe. professor, sapienza university of rome introduction/background: primary antibody deficiencies (pad) are characterized by defective ig production resulting in high susceptibility to bacterial infections, especially caused by s. pneumoniae and h. influenzae. there is a limited evidence on the rate of microbial airway epithelial colonization and on the role of bacterial carriage on the development of recurrent respiratory tracts infections in such populations. objectives: the aim of this study was to investigate the prevalence of s. pneumoniae and haemophilus influenzae colonization in pad adults in italy and its clinical and immunological correlates. methods: nasopharyngeal and oropharyngeal swabs were obtained from cvid and patients with idiopathic primary hypogammaglobulinemia (iph) over years of age and under ig replacement treatment during the period october -april . presence of s. pneumoniae and h. influenzae was investigated using conventional cultural methods and rt pcr. s. pneumoniae isolates were serotyped by the quellung reaction; capsular type of h. influenzae isolates was determined by pcr. the pattern of associations between the two species and potential risk factors were investigated. respiratory infections rate was recorded over months of follow up. results: among cvid prevalence of carriage assessed by traditional culture was % and % for s. pneumoniae and h. influenzae, respectively. rt pcr allowed to identify a higher rate of carriage of s. pneumoniae and h. influenzae compared to standard culture. cvid and iph had not different rate of pneumococcal colonization, whereas cvid had higher rate of h. influenzae carriage identified by a culture methods and rt pcr. no synergistic association between s. pneumoniae and h. influenzae colonization was observed. among cvid, s pneumonia and h. influenzae carriage were associated to low iga and igm levels. cvid under antibiotic prophylaxis did not have an increased prevalence of carriage. no association was found between carrier status detected by culture and having chronic lung disease, bronchiectasis or the rate of infections during the follow up. rt pcr identified merely the association between igm levels and h. influenzae carriage. antibiotic resistance from isolated stains was also assessed. conclusions: this is the first study assessing the prevalence of s. pneumonia and h. influenzae carriage in cvid. objectives: to discuss clinical challenges in a subject with congenital hair hypoplasia detected on newborn screening with preserved t-cell mitogen responses but declining naive t-cell counts. parents are jehovah's witnesses, which adds complexity to clinical decisionmaking. methods: immune evaluation included complete blood count, lymphocyte subsets, flow cytometry to define t-cell subsets, immune globulins, repeat trec assay from peripheral blood, human immunodeficiency virus (hiv) and cytomegalovirus (cmv) dna pcr, screen for maternal engraftment, chromosome microarray analysis, lymphocyte proliferation to mitogens, t-cell proliferation to interleukins, t-cell receptor v-beta diversity analysis by spectratyping, and thymic ultrasound. results: repeat trec assay from peripheral blood on day confirmed undetectable trecs. cd + thymic emigrants were low at (reference - cells/ul). naïve cd + and cd + t cell compartments declined as did naïve cd + t cells from to cells/ul and naïve cd + t cells from to cells/ul. lymphocyte proliferation to mitogens was preserved except for the cd + response to pokeweed mitogen. interleukin proliferation of cd + lymphocytes was slightly decreased after stimulation with anti-cd ( %, normal > %). however, t-cell proliferation with other interleukins (il- , anti-cd +il- , anticd as %cd ) was preserved. the t cell receptor repertoire had intermediate diversity. antibody replacement therapy was prescribed for declining igg with no history of severe infections except for rhinovirus prior to discharge from the nicu at weeks, during which she required continuous positive airway pressure (cpap) therapy. the viral load for cmv and hiv dna were undetectable. breastfeeding was discontinued early because the mothers cmv serology revealed positive cmv igg. the child has had intermittent anemia, which is common in patients with chh. [ ] tests for autoimmune hemolytic anemia were not performed because the patient required ivig from an early age. the parents of the child are jehovahs witnesses, complicating requests for blood transfusions. conclusions: this case highlights challenges in clinical decision making for a newborn with chh identified on nbs. t-cell function is preserved, but declining naïve t cell counts and absent trecs lead us to consider hematopoietic stem cell transplant (hsct). indication and timing of elective hsct is unclear and may depend on the natural progression of disease, including infections and anemia division of immunology and allergy, the hospital for sick children introduction/background: cartilage-hair hypoplasia (chh), caused by mutations in the ribonuclease mitochondrial rna-processing (rmrp) gene, is associated with diverse immune abnormalities including combined immune deficiency (cid). most patients with chh are managed with supportive measurements, while few have received allogeneic hematopoietic stem cell transplantations (hsct). the progression of the immune abnormalities and the impact of hsct in patients with chh and cid have not been well characterized. objectives: to characterize the progression of the immune abnormalities and the impact of hsct in patients with chh and cid methods: the clinical and laboratory findings of siblings diagnosed in infancy with chh and cid due to the common a>g mutation in rmrp, including the effects of hsct performed in of them, were compared. results: both patients suffered from recurrent respiratory infections at early age with reduced t cells numbers and responses. patient # immune function continued to deteriorate leading to hsct from an hla-matched sibling at . years of age. the patient suffered acute and chronic graft versus host disease of the skin with residual mild joint contractures and scleroderma-like skin changes. seven years after hsct patient # has normal immune function. immune evaluations of patient # in the first years of life indicated mild improvement. the patient did not have a suitable related hsct donor and the family elected to continue with supportive care. at years of age, patient # is clinically well and thriving with persistent t cell abnormalities. conclusions: close monitoring of immune function in early life for patients with chh and cid as well as the availability of suitable donors assists in determining management, including hsct introduction/background: leukocyte adhesion deficiency (lad) represents a group of distinct inherited disorders, which inhibit the normal extravasation of neutrophils and their recruitment to sites of infection or inflammation. objectives presentation of case: the patient is a girl of years old with no history of primary inmunological disease (pid) in family members, including a healthy brother (currently years old). she received all the immunization schedule until year old (according to peruvian schedule). she had several admission to the hospital since newborn; the st hospital admission was at days of life with diagnosis of: sepsis, pneumony, onphalitis (leukocytes count: ), she had more hospital admission and the leukocytes count were always above . in summary, she presented other infections besides the ones admitted at hospital like: episodes of sepsis, episode of pneumony, episodes of cellulitis (left eye, left elbow, right thigh and vaginal ( ), episodes of otitis, episodes of tonsillitis, episodes of diarrhea, episodes of rhinoadenoiditis, episodes of sinusitis, episodes of gingivitis and episode of whooping cough. she received different antibiotics for treatment, even broad-spectrum as vancomycin, meropenem and cefepime. the diagnosis of leukocyte adhesion deficiency (lad) was made at year months by clinical features like delaying in separation of umbilical cord, recurrent infections and persistent leukocytosis> . flow citometry was taken at years old, resulting cd b/cd : , %. results: discussionf: or the severe phenotype, in which leukocytes express < % of normal levels of cd , death occurs at an early age because of severe infection unless patients receive bone marrow transplant. however, it does not happened with her so we suspected in a possible reversion in lad so we took a second flow citometry at years old and the results were: total leukocyte: ; linfocyte: cd b+/cd +: , %; granulocyte: cd b+/cd +: , % and the conclusion was c receptor (cd b/cd ) absent in linfocyte, monocyte and granulocyte. molecular study was taken at years old, resulting a homozygous substitution c. c>t identified in exon , causing a nonsense mutation: p.arg , confirming lad diagnosis. she is, currently, receiving profilactic antibiotic and antimicotic which has reduced considerably recurrent infections. it seems that our patient may have a mixed phenotype due to a clinical expression, which may not require hematopoietic cell transplantation (hct) despite features of the severe type. conclusions conclusion: we conclude that the clinical evolution of this patient is unsual because she has severe lad , she has not transplanted yet, profilaxis treatment has improved to decrease frecuency of infections, she exceeded life expectancy and last flow citometry confirmed that lad was not reverted. , ph.d. , luigi d. notarangelo, md , troy r. torgerson, m.d. ph.d. , ph.d. , hans d. ochs, m.d. , m.d., ph.d. introduction/background: patients with x-linked hyper-igm syndrome (x-higm) due to cd ligand (cd l) deficiency often present with low blood neutrophil counts. however, even when not neutropenic and despite immunoglobulin (ig) replacement therapy, cd l-deficient patients are susceptible to life-threatening infections by opportunistic pathogens, suggesting impaired function of phagocytes, and requiring novel therapeutic approaches. objectives: to analyze whether peripheral neutrophils from cd l-deficient patients display functional defects and to explore the in vitro effects of recombinant human interferon (rhifn)-on such cells. methods: we investigated the microbicidal activity, respiratory burst and transcriptome profile of neutrophils from cd l-deficient patients. in addition, we evaluated whether the lack of cd l in mice also affects neutrophil responses. results: neutrophils from cd l-deficient patients exhibited defective respiratory burst and microbicidal activity which were significantly improved in vitro by rhifn-. similar to humans with cd l deficiency, cd l-deficient mice were found to have defective neutrophil responses. moreover, neutrophils from cd l-deficient patients showed reduced cd protein expression and a dysregulated transcriptome profile suggestive of impaired differentiation. conclusions: our data suggest a non-redundant role of cd l-cd interaction in neutrophil development and function that could be improved in vitro by rhifn-, indicating a potential novel therapeutic application for this cytokine. methods: in this study, through the use of healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from hcc patients. results: increased expression of cd on nk cells was observed in intratumoral but not peritumoral regions, along with increased expression of its ligand cd and a poor prognosis. human cd + nk cells exhibited functional exhaustion, showing decreased ifn-and tnf-productions, impaired cytolysis in response to in vitro stimulation, and high gene expression of il- and tgf- with low expression of t-bet, il- , perforin and granzyme b by global transcriptomic analysis of sorted cd + and cd -nk cells. blocking tgf- specifically inhibited cd expression and reversed the dysfunction of nk cells. in addition, we compared other two receptors, cd and tigit, which share common ligand cd with cd , and found cd plays a more important role in nk exhaustion. conclusions: these findings indicate that human cd + nk cells have features of functional exhaustion, suggesting that cd -cd blockade has the potential to restore immunity against liver tumors by reversing nk cell exhaustion. introduction/background: mutations in ncf (encoding protein p phox of the nadph oxidase) result in an autosomal recessive form of chronic granulomatous disease (cgd), a rare genetic disease with impaired phagocyte production of reactive oxygen species and recurrent infections. diagnosis of p phox cgd is based on abnormal dihydrorhodamine assay and absence of p phox protein by immunoblotting; however, these assays fail to diagnose carriers of ncf mutations. instead, carrier status is inferred after the birth of a child with p phox cgd. furthermore, identification of the specific genetic defect in patients with p phox cgd is complicated by two highly conserved (> %) pseudogenes. the ncf gene has a gtgt at the start of exon , while the pseudogenes (ncf b and ncf c) delete one gt (gt). in p phox cgd, the most common mutation in ncf is gt causing c. _ delgt; p.tyr fsx . sequence homology between the wild type gene and pseudogenes precludes using standard sanger sequencing to identify specific mutations in ncf . objectives: to identify phenotypic and genotypic differences that facilitate the diagnosis of patients and carriers with p phox cgd. methods: expression of p phox in neutrophils is determined by fixing and permeabilizing whole blood with intraprep, and then incubating with either anti-p phox antibody or its corresponding isotype. alexafluor -conjugated secondary antibody is used to detect the target antigen. expression of p phox is based on the mean fluorescence intensity of cells within the neutrophil population gated using forward and side light scatter. differential expression of p phox by flow cytometry is validated using quantitative immunoblotting. to screen for the gt mutation, a droplet digital polymerase chain reaction (ddpcr) with two distinct probes recognizing either the wild-type gtgt sequence or the gt sequence was used to quantitate the ratio of gtgt vs. gt copies. a second ddpcr reaction established copy number by comparing one probe for an invariant region of ncf /ncf b/ncf c and a second probe for the single-copy telomerase reverse transcriptase gene, tert. the results of these two assays were combined to determine the total number of gtgtcontaining and gt-containing ncf copies. results: analysis of p phox expression in permeabilized neutrophils determined that neutrophils from p phox cgd patients had negligible p phox expression; neutrophils from p phox cgd carriers exhibited~ % of p phox expression compared to healthy volunteers independent of the mutation in ncf . of all p phox cgd patients tested by ddpcr, . % ( / ) exhibited copies of gtgt, . % ( / ) exhibited copy of gtgt (compound heterozygotes with non-gt mutation), and . % ( / ) exhibited copies of gtgt (two non-gt mutations). moreover, ddpcr can identify the carriers among kindreds within the gt p phox cgd families. unexpectedly, among normal subjects tested, only . % exhibited the expected copies of gtgt per total ncf /ncf b/ncf c copies, designated / ; a significant number exhibited more than two copies of gtgt ( . % with / , . % with / ); others exhibited ncf /ncf b/ncf c copy number variation ( . % with / and . % with / ). conclusions: flow cytometric analysis of neutrophil intracellular p phox staining provides a quick method to identify patients and carriers of p phox cgd. droplet digital pcr can be used to identify patients and carriers of p phox gt, the most common mutation in p phox cgd copy number variation is observed at the ncf locus among normal subjects tested. introduction/background: defects in the cd subunits of the tcr/cd complex account for a small percentage of the scid presentations. cd deficiencies are characterized by profound and t-cells lymphocytopenia, and normal numbers of b-and natural killer (nk) cells in the peripheral blood (t-b+nk+ scid). thymocyte development from double negative stage to double positive stage results arrested. affected individuals typically present with severe and opportunistic infections in the early infancy. objectives: to evaluate possible underlying immunodeficiency disorder in the setting of chronic ebv infection. methods: here we report our experience of a patient with an atypical presentation of cd deficiency. results: a -year-old girl previously healthy, first generation americanborn of gambia immigrants was referred to our clinic for further evaluation of chronic ebv infection. one year before presentation, she developed bilateral parotid enlargement, cervical, axillar, and hilar lymphadenopathy, bronchiectasis, and pulmonary nodules. relevant previous studies included: ebv viremia (pcr quant , copies), ebv igg and ea positive whereas igm and ebna were negative. peripheral blood phenotyping was not suggestive for malignant process and cervical lymph node biopsy was consistent with a reactive process without evidence of a clonal lymphoproliferative disorder and bal studies positive for ebv only. hiv and tb both negative. she was otherwise thriving with no history of recurrent infections and family history was negative for consanguinity or immunodeficiency. our evaluation revealed: normal blood counts, ebv pcr quant , copies, normal immunoglobulin levels and vaccine titers. she had a normal lymphocyte subsets, but skewed cd ra/ro consistent with low thymic output (very low cd naïve t cells ( . %), low cd + naïve t cells ( . %), and elevated temra ( . %)), poor mitogen, and antigen responses. all these findings were suggestive of a scid like phenotype; therefore, scid next generation sequencing panel was pursued, and showed a novel homozygous splice site mutation (c. + g>t) in the cd gene. conclusions: a homozygous mutation in the cd gene might not necessarily imply profound t-cell lymphopenia. though this patient did not present with classic clinical course, and immune findings; her inability to clear ebv with persistent significant viremia does support a t-cell immune deficiency. she remains at risk for ebv-associated lymphoproliferative disorder, infections, and autoimmunity. bmt will be a curative treatment option. however, it is difficult to predict evolution of clinical phenotype given the atypical presentation. this case illustrates the importance of contextual interpretation of clinical findings, laboratory data, and genetic analysis for treatment approach. introduction/background: pol is multi-subunit polymerase that includes both pole with catalytic activity and additional pole , and . this holoenzyme plays a key role in proofreading damaged dna and is required for proper dna replication in proliferating cells, such as lymphocytes. germline mutations are linked to rare cause of primary immunodeficiencies whereas somatic mutations are described in colon cancer. primary immunodeficiencies are reported pole- deficiency in members of a large consanguineous french kindred and a palestinian female with fils (facial dysmorphism, immunodeficiency, livedo, and short stature). all reported cases with pole deficiency have homozygous intronic splice site variant (c. + a>g) that result in a deletion of exon which lead to subsequent frame shift (from p.s v onwards) and a premature stop codon at position ; this transcript results in a degraded product. the proportion of the pole transcript in t lymphoblasts is significantly lower ( %) in patients then carriers or healthy individuals. objectives: hereby we describe the clinical progression and treatment challeneges of the palestinian female with pole- deficiency secondary to homozygous g.g + a>g substitution. results: initially patient presented with viral and recurrent ear infections and cmv viremia. with age, patient had less episodes of infections even with intermittent pause in immunoglobulin replacement therapy (igrt), however had multiple admissions for fever of unknown origin with negative cultures but increased ferritin level ( ng/ml) and low platelet count ( , count/ml). autoimmune and inflammatory complications were not reported among the french kindred. her skin also worsened with poor wound healing and scarring throughout that hinders igrt via subcutaneous route. furthermore, igrt with intravenous administration has resulted in symptoms of aseptic meningitis likely related to the underlying inflammatory state. in addition, patient shows decline in immune dysfunction. initially, she had normal immunoglobulin g (igg) however by years of age, she developed hypogammaglobulinemia with low igm unlike in the french family with patients. also, pneumococcal, diptheria, tetanus titers were non-protective off of immunoglobulin replacement therapy (igrt). beyond low switched memory b cells, patient also developed low b cell by yo age. t cell dysfunction continued to decline from decreased lymphocyte proliferation to antigens at yo age to fully absent lymphocyte proliferation to antigens and mitogens. naïve cd and cd compartments continue to be preserved. currently management challenges include treatment strategies for thrombocytopenia, inflammation and progressive skin disease that complicates the proper selection of route for optimal igrt. conclusions: beyond progression of immunological decline, our patient developed inflammatory phenotype with age. the progression of her immunological decline may be related to further decrease in the proportion of the wild type pole transcript and yet to be examined. overall, clinical follow up is essential in patients with pole- deficiency as phenotype can change with age and may pose new challenges. longitudinal follow up studies are needed to uncover the potential role of germline pole and pole pathogenic variants in cancer susceptibility. introduction/background: x-linked hyper-igm syndrome (xhigm) is one type of primary immunodeficiency diseases, resulting from defects in the cd ligand/cd signaling pathways. objectives: here, we retrospectively reviewed clinical, laboratory and genetic characteristic of xhigm in chinese population, thus further improving diagnosis and treatment for xhigm. methods: we collected and analyzed chinese patients, who were diagnosed and followed up in hospitals affiliated to shanghai jiao tong university school of medicine from to . targeted gene capture combined with next-generation sequencing technology and sanger sequencing were used to find out related gene mutation. results: the median onset age of these patients was months (range: days months). thirty-six percent of them had positive family histories, with a shorter diagnosis lag. the most common symptoms were recurrent sinopulmonary infections ( patients, %), neutropenia ( patients, %), protracted diarrhea ( patients, %), and oral ulcer ( patients, %). ten patients had bcgitis. six patients received hematopoietic stem cell transplantations and four of them had immune reconstructions and clinical remissions. twenty-seven unique mutations in cd l gene were identified in these patients, with novel mutations. conclusions: to our knowledge, this report provides the largest cohort of patients with xhigm in china. mutation analysis is an important tool for xhigm diagnosis. infants with scid are asymptomatic at birth and unless prompt diagnosis of the disease is made they may horrifically be vaccinated. simultaneous appearance of two live vaccine associated infections in one person is rarely reported. objectives: in this study we present two infants with scid, who received bcg and oral polio vaccines early in life before the diagnosis of immune deficiency was made. both patients developed localized and disseminated infections originating from the bcg vaccine (bcgitis and bcgiosis, respectively) and in addition were diagnosed with chronic fecal secretion of vaccine-derived polio virus (vdpv); alarmingly, in both cases, the vdpv underwent reverse mutation of attenuated sites to the neurovirulent genotype. the rarity of concomitant infection from two live vaccines in one recipient, together with the multiple complexities originating from these infections in immunodeficient infants, led us to report these cases and to inquire the pathogenesis that underlies this unique condition. methods: immunological evaluation:: cell surface markers of peripheral blood mononuclear cells (pbmcs) were measured by immunofluorescent staining and flow cytometry serum concentrations of immunoglobulins were measured using nephelometry. quantitative analysis of the tcr v repertoire was performed by means of flow cytometry. quantification of t cell receptor excision circles (trecs) was determined by real-time quantitative (rq)-pcr. genetic analysis: genetic diagnosis of scid was made for patient by direct sanger sequencing of candidate genes and retrieval of mutation in rag , and for patient by wes (whole exome sequencing), followed by validation of the dna cross-link repair c (dclre c) mutation using sanger sequencing. poliovirus detection and characterization: stool samples were collected monthly and transported to the national poliovirus laboratory located at israel central virology laboratory (icvl) for polio detection and characterization. results: in both patients, immunological workup revealed undetectable serum iga and igm levels with normal igg levels (table ) , lymphocyte immune-phenotyping using flow cytometry revealed complete absence of t and b cells with presence of nk cells (table ) . trecs, a dna marker of naive t cells and thymic output, were absent in both patients. the diagnosis of scid was made. we initiated prophylactic antibiotic (trimethoprim-sulfamethoxazole) and anti-fungal (fluconazole) treatment, as well as monthly intravenous immunoglobulin (ivig) infusions. genetic workup: the t-b-nk+ scid phenotype in patient led us to search for a mutation in the rag complex genes. indeed, a sanger sequencing of the rag gene, revealed a g t homozygous mutation which predicts an amino acid substitution from glycine to valine in position (fig. ). for patient , who had a similar t-b-nk+ scid phenotype, we identified a homozygous mutation in the dclre c gene (del. bp, c. -cttt) (fig. ) , using wes. the genetic evaluation confirmed the diagnosis of scid due to rag deficiency in patient and artemis deficiency in patient . clinical course: during hospitalization patient developed disseminated bcg related disease with skeletal lesions involving the phalanx, tibia and maxillary bones, as well as involvement of the spleen, liver and pancreas. anti-tubercular therapy with isoniazid, rifampicin, ethambutol and ciprofloxacin was initiated. due to lack of response, empirical trial of g-csf (granulocyte colony-stimulating factor), in order to enhance macrophage activity . the patient showed good response to this combination therapy. patient developed a palpable rigid mass on her left shoulder with surrounded redness at the site of the bcg vaccine at the age of months. the clinical diagnosis of bcgitis was established and triple antitubercular therapy with isoniazid, rifampicin and ethambutol was initiated with good response. throughout their hospitalization, both infants suffered from intermittent diarrhea. pcr for enterovirus was performed and detected the presence of type and type vaccine-derived poliovirus (vdpv) in patient and , respectively. during the follow-up, stool samples collection revealed accumulation of several polio virus mutations and some of the neuro-virulence attenuation sites were reverted to the neuro-virulent genotype .fortunately, both patients did not show any signs of flaccid paralysis. precautionary measures of isolation were taken to prevent spread of the vdpv. eventually, both patients underwent allogeneic bone marrow transplantation (bmt): patient had bmt without pre-conditioning, from a matched sibling donor. due to engraftment failure, a second bmt was repeated, this time successfully. on follow up examination her t cell repertoire showed a normal tcr v polyclonality and trec was detected, indicating the emergence of new t cells. due to ongoing low immunoglobulin levels this patient is still on regular ivig-infusions and prophylactic antibiotic treatment, as well as isoniazid. currently, she is well, her bcgitis is not active and her stool specimens are negative for polio. patient underwent an urgent haplo-identical bmt with alpha-beta t cell depletion without pre-conditioning, due to her unstable medical condition. flow cytometry analysis six months post bmt revealed lymphopenia of . % ( /mm³) with low mature t cells (cd %) and absent b cells. trecs were barely detected. microsatellite analysis as a marker for engraftment revealed a stable donor chimerism of %. the patient is still on immunosuppressive therapy doing well, her bcgitis is not active and her stool specimens are negative for polio. conclusions: these cases highlight the importance of early recognition of scid by neonatal screening or thorough family anamnesis, and the need to further defer the timing of administration of live vaccines. introduction/background: measurement of b cells, b cell subsets and specific antibodies produced in response to vaccination are key tests used to investigate immune system function. specific antibody production may indicate b cell functionality. objectives: in this study the correlation between the different b cell subsets and antibody responses to pneumovax® in an immunocompromised population was investigated. methods: b cell subsets were assessed by flow cytometry and pneumococcal responses measured using the vacczyme pneumococcal capsular polysaccharide (pcp) igg, iga and igm elisas (the binding site group, birmingham, uk) in primary immunodeficiency patients (pid) vaccinated with pneumovax®. lower limits of normal were defined as follows: b cells: . %; naive b cells: . %; non-switched memory b cells: . %; switched memory b cells: . %; pcp igg: mg/l; pcp iga: u/ml; and pcp igm: u/ml. results: the correlation coefficients between percentage of b cells/b cell subsets and igg, iga or igm pneumovax® responses ranged from - . to . . the percentage of the cohort achieving a normal b cell and normal igg, iga or igm response to pneumovax® was %, % and % respectively. b-cell responses were measureable in the remaining patients but they did not produce normal concentrations of pcp igg, iga or igm. further stratification of patients who achieved normal percentage of switched or un-switched b cells but who failed to achieve normal igg, iga or igm responses to pneumovax® were %, % and %, respectively, for un-switched and %, % and %, respectively, for switched b cells. conclusions: the combined measurement of b cells and response to vaccination are required to provide a detailed insight into these disorders. introduction/background: this is a -year-old boy of african american heritage with multiple congenital malformations who was diagnosed with very early-onset inflammatory bowel disease, which proved to be resistant to treatment. subsequent testing revealed a heterozygous mutation in exon of ctla . this variant has not been previously reported in the literature in individuals with ctla -related disease. heterozygous mutations in ctla cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. early onset colitis has been reported with ctla -related disease and is unique to our patient's initial clinical presentation. objectives: this is a -year-old boy with cloacal exstrophy of the urinary bladder, omphalocele, imperforate anus, polydactyly, and sacral agenesis who was diagnosed with very early-onset inflammatory bowel disease at six months of age. he underwent cloacal exstrophy closure, omphalocele repair, and colostomy placement in the first week of life. at six months of age, he presented with dark tarry stools. upper endoscopy and colonoscopy revealed polyps, ileitis, and colitis. he was p-anca positive and started on sulfasalazine. unfortunately, he continued to have symptoms suggestive of active colitis, prompting a change to prednisone and azathioprine. despite therapy, his colitis persisted leading to chronic bloody diarrhea and growth failure. initial immune evaluation consisted of a normal complete blood count, serum immunoglobulin, lymphocyte subsets, and neutrophil oxidase burst assay. foxp analysis by flow cytometry showed a moderately elevated percentage of foxp +cd + cells in the cd + t cell population, but the regulatory t cell immunophenotype was normal. because suspicion was high for a monogenic immunologic disease to explain his symptoms, genetic sequencing was performed. a candidate gene panel was sequenced by next generation sequencing, and a heterozygous mutation in exon of ctla (c. g>a; p.arg gln) was found. this variant has not been previously reported but is predicted to be pathogenic in exac and polyphen databases. results: the patients diagnosis of ctla- haploinsufficiency associated with very early-onset inflammatory bowel disease has provided opportunity for targeted treatment of his specific molecular defect. given his poor response to treatment thus far, the patient will be started on abatacept. abatacept is fda approved for the treatment of rheumatoid arthritis but has been used successfully for the treatment of disease-related manifestations of ctla- haploinsufficiency. abatacept is a ctla- fusion protein formed by the igg fc region linked with the extracellular domain of ctla- ; it replaces the defective protein in ctla- haploinsufficiency. in addition, given other manifestations of ctla- haploinsufficiency including lymphoproliferative disease in non-lymphoid organs, particularly the brain and lung, we have initiated further evaluation of these organs to evaluate for disease-specific manifestations. conclusions: the protein cytotoxic t lymphocyte antigen- (ctla- ) is an essential negative regulator of t cells. heterozygous mutations in ctla cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. inflammatory bowel disease may be associated with certain variants in ctla , but the literature remains limited, both by number of papers published as well as by ethnic subsets studied. clinicians who are presented with children who have early-onset colitis, and particularly inflammatory bowel disease that is difficult to treat, should consider possible genetic abnormalities, such as ctla- haploinsufficiency, as these can impact therapeutic decision-making and outcomes. introduction/background: dock deficiency is an autosomal recessive combined immunodeficiency syndrome associated with recurrent infections, eczema and other atopic diseases. the infections are usually viral, bacterial and fungal resulting in predominantly cutaneous and sinopulmonary manifestations. homozygous or compound heterozygous deletions or mutations in the dock gene ( p ) lead to abnormal cytoskeletal organization and impaired function of dendritic cells and lymphocytes. an aniline derivative belonging to the group of synthetic sulfones, dapsone has been employed in the treatment of chronic skin diseases characterized by an accumulation of neutrophils and eosinophils. methods: chart review of one patient results: we present a four-year-old male with severe eczema, persistent asthma, allergic rhinitis, as well as peanut and egg allergy suffering from recurrent skin abscesses and prurigo nodularis. abscesses began at age months and required prolonged courses of antibiotics, eight in total prior to presentation. other infectious history included otitis media and lymphadenitis. there was no history of pneumonia or other severe infections. skin abscesses responded to oral antibiotics, but recurred shortly after completing extended courses of treatment. laboratory results including quantitative immunoglobulins, specific antibody titers, myeloperoxidase staining, neutrophil oxidative burst and complement were within normal limits. laboratories were notable for elevated ige ( iu/ml) and eosinophilia ( eosinophils/microl). lymphocyte immunophenotype was significant for mild elevations in cd and cd . dock genetic sequencing by genedx revealed a heterozygous missense mutation in exon (c. c>a, amino acid change p.ala asp). abscess cultures grew methicillin sensitive staphylococcus aureus (mssa) and enterococcus faecalis. mssa was sensitive to ampicillin/sulbactam, cefazolin, gentamycin, moxifloxacin, oxacillin, rifampin, tetracycline, and vancomycin. isolate was resistant to ciprofloxacin, clindamycin, erythromycin, levofloxacin, penicillin, and trimethoprim/ sulfamethoxazole. the patient was initially treated with emollients, mupirocin washes, topical steroids, anti-histamines, bleach baths, and cephalexin three times a day. he improved clinically but was unable to tolerate cephalexin for more than ten days secondary to abdominal pain. cephalexin, with addition of probiotic, was attempted several months later and again had to be discontinued because of abdominal pain and vomiting. due to limited antibiotic options, dapsone was started after ruling out glucose- -phosphate dehydrogenase deficiency. dapsone was initiated at a dose of . mg/kg, and the patient was monitored weekly for hemolytic anemia. after two weeks of treatment, anemia was noted and the dose was decreased to mg/kg. he has continued on dapsone mg/kg once a day, with significant improvement in abscess number and severity. he has not required other systemic antimicrobials since starting dapsone. conclusions: dapsone may be considered as a treatment option for children with heterozygous dock mutation and recurrent abscesses, particularly those requiring prophylaxis, long term treatment and lacking antibiotic options. introduction/background: autosomal dominant hyper-ige syndrome (ad-hies) is a rare complicated primary immunodeficiency disease (pid). signal transducer and activator of transcription (stat ) gene mutation is found to cause ad-hies. tlr / signaling plays multiple roles in b cell proliferation, activation, class-switch recombination, and cytokine and antibody production. however, little is known about b cell response to tlr / agonist in patients with ad-hies. objectives: here, we aim to study the response of b cells from ad-hies patients to the tlr / agonist. methods: pbmcs were isolated from peripheral blood of ad-hies patients and age matched healthy controls. pbmcs were stimulated with tlr and tlr agonist (r and cpg odn , respectively), then b cells were analyzed for proliferation, the expression of certain surface markers (cd , cd and cd ), intracellular immunoglobulin levels (igm and igg) and intracellular cytokine levels (il- and il- ) by flow cytometry. results: in response to tlr / agonist, proliferative capabilities of b cells were reduced in ad-hies patients compared with those in age-matched healthy controls. besides, defective costimulatory molecule cd expression was observed in b cells from ad-hies patients. furthermore, significantly lower igm and igg levels, and il- production was detected in b cells from ad-hies patients. however, there was no significant difference in b cell apoptosis between ad-hies patients and healthy controls. conclusions: these data demonstrated that stat gene mutations in ad-hies patients contributed to impaired b cell tlr / signaling, and further affected b cell proliferation, activation, cytokine secretion and antibody production. introduction/background: antibody function is most commonly measured by a rise in antibody titers in response to antigen introduced by vaccination or natural infection. specific antibody deficiency is defined as normal serum levels of immunoglobulins with reduced or absent antibody response to antigens, often after administration of the pneumococcal vaccine polyvalent (pneumovax® ). a paucity of information exists about measurement of antigen-antibody binding, or avidity, as a measure of antibody function, including persons with recurrent sinopulmonary infections who have normal response to immunization with pneumococcal vaccine polyvalent. objectives: the aims of this study are to identify and evaluate children with recurrent sinopulmonary infections who had appropriate rise in pneumococcal antibody titers following immunization with pneumococcal vaccine polyvalent but low response by avidity, and to assess response with igg replacement therapy in these patients. methods: a retrospective chart review involved eight children with recurrent sinopulmonary infections with discordant pneumococcal antibody and avidity results following vaccination with pneumococcal vaccine polyvalent. these eight children subsequently received igg replacement therapy. results: the mean age of subjects was . (range - ) years. the mean number of serotypes with a normal antibody response (> . ug/ml) among children following immunization with pneumococcal vaccine polyvalent was . (range - ) of serotypes while the mean number of serotypes with a normal avidity response ( . ) was . (range - ) of serotypes. igg replacement was administered subcutaneously in children. the mean igg level was mg/dl. local reactions were all mild and observed in / ( %) children. no serious adverse events were reported. all children experienced a marked reduction in respiratory illnesses while on igg replacement therapy. conclusions: discordance between pneumococcal antibody titers and pneumococcal avidity titers was identified in eight children with recurrent respiratory illnesses. in children with recurrent sinopulmonary infections despite normal antibody response to pneumococcal vaccine polyvalent, measurement of pneumococcal avidity may identify patients with poor pneumococcal antibody function. igg replacement in these children was well tolerated and associated with a decrease number of respiratory infections. introduction/background: exome sequencing (es) is a powerful genomic tool that can be used to identify novel molecular causes of disorders with multiple etiologies. immunologic disorders are clinically and genetically heterogeneous, and therefore present unique diagnostic challenges both in the clinic and in the laboratory. objectives: the objective of this study was to assess the utility of es for determining the genetic etiology of immunological disorders and describe diagnostic yield and outcomes of exome sequencing (es) for patients with immunologic disorders and immunologic phenotypes. methods: a retrospective review was performed of individuals referred for clinical es for primary abnormalities of the immune system and individuals with additional phenotypes where multiple immunologic features were reported as part of the clinical picture, as determined during internal curation. analysis of clinical es data was performed by boardcertified clinical geneticists and all variants reported were confirmed by a secondary methodology. positive es outcomes required a pathogenic or likely pathogenic variant in a gene with autosomal dominant or x-linked inheritance, or compound heterozygous or homozygous pathogenic or likely pathogenic variants in a gene with recessive inheritance. results: the most common clinical indications for es in this cohort were hypogammaglobulinemia ( %), neutropenia ( %), immune dysregulation ( %), lymphopenia ( %), and combined immunodeficiency ( %). the gender distribution was % male (n= ) and % female (n= ); % of cases were pediatric (< years, n= ) and % were adult (>= years, n= ). positive results were reported in cases ( %), comparable to the overall diagnostic yield of es at our laboratory (retterer et al., ) . this included / ( %) of cases submitted as proband-only, / ( %) submitted as a trio, and / ( %) submitted as duo, quad or alternative family structure. diagnostic results spanned different genes and recurrently reported genes with identified pathogenic variants included flg (n= ), rag (n= ), sbds (n= ), lrba (n= ), stat (n= ), and pik cd (n= ). variants possibly associated with the phenotype, but not considered diagnostic, were reported in % of cases (n= ), while % of cases (n= ) had reportable findings in a candidate gene. conclusions: these results support that exome sequencing for individuals with immunologic-based phenotypes has similar diagnostic utility as the overall rate for clinical es. immunologic es cases with trio family structures have a higher diagnostic yield than proband-only cases, as inheritance information improves confidence in classification of variants as pathogenic or likely pathogenic. genetic heterogeneity, as demonstrated by the large number of distinct genes represented in this cohort of diagnostic es cases and rapid candidate gene discovery make es a valuable tool for genetic diagnosis in patients with immunological disorders. introduction/background: vaccination response to the -valent polysaccharide vaccine (ppsv ) is often used in the diagnosis of common variable immunodeficiency (cvid). unfortunately, ppsv titers are often difficult to interpret and many cvid patients are started on igg replacement therapy (igrt) before adequate evaluation. unlike ppsv titers, the enzyme-linked immunosorbent spot assay (elispot) is independent of igrt and can provide an ex vivo functional measurement of specific antibody production on the b cell level. objectives: develop and test an elispot assay to better determine vaccination response to ppsv compared to ppsv titers in cvid patients on igrt, healthy controls, and igrt patients without immunodeficiency. methods: an elispot assay was successfully optimized and used to evaluate the ppsv -specific b cell response in healthy adult controls. elispots were performed on day , and day (when plasmablasts are best evaluated). ppsv titers were measured on day , day , and day . for igrt patients, flow cytometry for b cell subpopulations will be performed to further validate the assay. results: normal controls demonstrated a significant increase in ppsv antibody spot forming units (sfu) between day and after ppsv vaccination. ppsv titers showed generally robust initial titers at day , and no significant change at day , with day results pending. conclusions: here we optimized an elispot assay that functionally measures the specific antibody response to ppsv in normal controls with ppsv titer results pending. we are actively recruiting patients on igrt (both with cvid and without immunodeficiency) for comparison. we hope to validate our assay as a useful alternative to ppsv titers that may be particularly useful when patients are on igrt. previous studies have demonstrated bal may be a sensitive diagnostic method for treatment failures of clinically diagnosed pneumonias, even if performed under treatment with empiric antibiotics, and can lead to a culture-directed change in antimicrobial therapy in the majority of cases. however, it has also been reported that at least in one piddchronic granulomatous disease (cgd)the diagnostic yield of bal was inferior to that of other diagnostic methods (marciano et al., ) . further information on the diagnostic yield of bal and other invasive procedures to obtain a specific organism diagnosis in pidd patients with suspected pulmonary infection is needed. objectives: to characterize the yield of diagnostic procedures used in pidd patients with pneumonia or other suspected pulmonary infections at the ucsf benioff childrens hospital. methods: we screened our database of pidd patients (encompassing patients seen from september , to september , cared for by the pediatric immunology service at the university of california, san francisco to identify patients with history of at least bal or other invasive diagnostic procedure (fna or open lung biopsy) for etiologic diagnosis of suspected pulmonary infection. if multiple bals were performed during a single episode of illness, only the first was used for this analysis. results: we identified pidd patients with history of at least one bal or other invasive diagnostic procedure, for a total of events. most procedures (n= ) were performed at our institution, with documented in outside hospital records. patient diagnoses included cgd ( ), nemo deficiency ( ), cvid ( ), stat -deficient ad-hies ( ), dock deficiency ( ) , and mhc class ii deficiency ( ) . of bals, / ( %) grew a predominant organism, but only / ( %) were positive for an organism believed to be causative by providers and/or for which the overall result of the bronchoscopy affected antimicrobial treatment. bal yield was highest in patients with a clinical and/or radiologic diagnosis of pneumonia ( / , %). yield was poor ( / , %) in minimally or chronically symptomatic patients referred for bal for interval changes on chest ct (i.e. suspected fungal infection). our nemo deficiency cohort had the highest rate of positive organism isolation by bal ( / , %) for diagnosis of pulmonary infection. in our cgd cohort, / ( . %) bals grew a causative organism. lung biopsy yielded positive organism isolation in / cases ( / in cgd, burkholderia cepacia and nocardia cyriacigeorgica; / in cvid) and fna in / cases of cgd (aspergillus fumigatus). fna or biopsy was done concurrently or after bal in cases; in / ( %), fna or biopsy, but not bal, was positive for a causative organism. conclusions: at our institution, bal overall had a % rate of causative organism isolation in pidd patients, but had up to a % rate of organism isolation in those with clinical and/or features of pneumonia. our rate of causative organism isolation was slightly higher than in previous reports. however, in specific instances, biopsy was still required to make a definitive diagnosis. bal may have limitations in certain populations of pidd patients, such as in cgd, but it may be a reasonable starting point in the diagnosis of pneumonia or worsening pulmonary disease in pidd. prospective research is needed to evaluate whether fna or lung biopsy, though more invasive, could result in overall shorter time to institution of appropriate directed therapy and shorter hospitalizations for specific pidd patients. introduction/background: prior to the introduction of newborn screening, cases of severe combined immunodeficiency often presented with severe or disseminated infections. herein, we report an infant from india who presented for evaluation and treatment of a periorbital mass, presumed to be malignant. however, he was found to have disseminated bacille calmette guerin (bcg), as well as multiple other infections, and was eventually diagnosed with x-linked scid. results: a -month old boy was born to unrelated parents in india. he initially presented with growing periorbital mass and fever for two weeks. pet scan showed hypermetabolic areas in the bone marrow, spleen, mesenteric lymph nodes, and left shoulder, along with the periorbital mass. biopsy of the mass revealed numerous b lymphocytes without malignant transformation. there was no evidence for malignancy on bone marrow biopsy, though numerous granulomas and a significant decrease in t lymphocytes were seen. peripheral blood flow cytometry showed a complete lack of t cells. the bone marrow biopsy was reexamined, and innumerous acid-fast bacilli were found. cultures grew mycobacterium bovis, and he was diagnosed with disseminated bcg disease. genotyping revealed a novel splice site variant in il rg, consistent with x-linked scid. further evaluation revealed multiple other infections. these included extended-spectrum beta lactamase-produce e. coli bacteremia, human metapneumovirus, cytomegalovirus, and pneumocystic jiroveci. he was also tested for vaccine-associated poliovirus because he had received the oral polio vaccine, but this was negative. four-drug therapy was started for the bcg, and the periorbital lesion completely resolved within several weeks. additionally, he was treated with intravenous ribavirin for the human metapneumovirus, and sulfamethoxazole-trimethoprim for the p. jiroveci. although the cmv was initially treated with ganciclovir, the virus eventually developed resistance and required treatment with foscarnet. due to his many infections, he underwent haploidentical stem cell transplant plus donor lymphocyte infusion from mom. this transplant failed to engraft, but a matched unrelated donor was eventually found, and our patient received a second bone marrow transplant. he currently is showing signs of engraftment, and is continuing to be treated for his multiple infections. conclusions: disseminated mycobacterial disease due to the bacille calmette-guérin (bcg) vaccination has been noted in cases of xlinked scid previously, often consisting of lymphatic, skin, and pulmonary manifestations. however, there is a paucity of published cases presenting with multiple other co-infections. nor are there reports of disseminated bcg presenting as a localized mass. this case highlights the unique considerations when evaluating a patient with immunodeficiency from another country, where vaccination practices and epidemiology differ. specifically, unusual presentations of infections or masses may warrant investigation for severe immunodeficiency. prototypic t-b+nk+ immune phenotype is caused by mutations in the iil ra gene. the il signaling has an important role during t-cell development in the thymus, contributing to cell proliferation and survival. in addition, in mouse models, the rearrangement of the t cell receptor genes, specifically the gamma locus (trg), has been shown to be regulated by il signaling. similar to other scid phenotypes, patients with il ra deficiency are predisposed to acquire opportunistic infections early in life and to display poor outcome and death, unless their immune system is restored by mean of hematopoietic stem cell transplantation (hsct). while most patients with il ra mutations have full il ra deficiency resulted in a severe t cell depletion, some have a partial deficiency with residual cells or leaky phenotype, or even present symptoms later in life. objectives: here we report two non-related infants detected by the israeli national newborn screening program for scid. despite having similar il ra missense mutation (f l) they displayed distinct clinical and immunological course, resulted in a completely different treatment approaches; observation in one patient with an unusual recovery, and hsct in the other patient methods: patient lymphocytes were examined for subset counts, thymic output (via excision circles), t cell receptor repertoire diversity (tcrb) and il ra expression and function. the pathogenic il ra mutation was found by whole exome sequencing (wes). high throughput immunesequencing was performed to characterize the trg repertoire. results: we established patients' diagnosis by validating the pathogenic il ra mutation, and showing profoundly impaired t cell immune work up and abnormal il ra expression and function, determined by stat phosphorylation assay. all these measurements improved over time for the patient with less severe clinical presentation, while remained low in the patient with the severe phenotype. characterization of their trg immune repertoire using high throughput immune-sequencing revealed restriction of t cell receptor repertoires of both patients upon their initial diagnosis, compared to healthy controls. however, skewed usage of variable (v) gene segments and abnormalities of the cdr length distribution were more prominent in the patient with the severe phenotype. conclusions: these studies illustrate the gap that exists in our understanding of other non-genetic parameters that may influence disease course and severity in patients harboring a similar genetic defect. furthermore, the results reinforce the role of il- signaling not only in cell proliferation but also in trg rearrangements introduction/background: dock immunodeficiency syndrome is primary immunodeficiency disease caused by loos of function mutations in the dock gene, which was known to play a critical role in the survival, proliferation and function of several types of immune system, especially lymphocyte. dock immunodeficiency syndrome is the most common cause of autosomal recessive hyper-immunoglobulin e syndromes (hies) and mainly expressed as recurrent infections and severe allergic disease affecting the skin. in addition, autoimmune features including systemic lupus erythematosus, hemolytic anemia or idiopathic thrombocytopenic purpura may be presented in dock immunodeficiency syndrome. objectives: we report a case of -month-old boy diagnosed with dock immunodeficiency syndrome, which was initially expressed as sle without recurrent skin infections. methods: a child with atopic dermatitis was admitted to another hospital because of fever lasting more than days accompanied by swelling of the hands and foot. he developed whole body edema, perioral purpura and oliguria. complete blood count was normal and blood urea nitrogen , creatinine and albumin levels were normal on his laboratory findings. however, c-reactive protein level was high at . mg/dl, coagulation parameters were abnormal (prothrombin time . sec; activated partial thromboplastin time . sec, d-dimer . ug/ml). so he was transferred to our hospital for further examination and treatment on the th day of fever. additionally, ulceration of the tonsil and maculopapular rashes on the abdomen and both legs were observed in physical examination. we suspected a meningococcal infection and administered antibiotics. however, no bacterial isolates were identified in the blood and csf culture test. fever persisted despite the administration of antibiotics. we checked immunoglobulin level, complement level and autoantibodies based on fever of unknown origin. immunoglobulin g, m and a were normal, complement fractions c , c , and ch were low at . mg/ dl, . mg/dl and . u/ml, respectively. antinuclear antibodies were positive at : with homogenous fluorescence. anti-ds dna antibody was positive at . . the tests for anti-ssa, anti-ssb, anti-ribonucleoprotein, anti-scleroderma , and anti jo antibodies were all negative. he developed leukopenia and thrombocytopenia over time. results: he was treated with steroid satisfied diagnostic criteria for systemic lupus erythematosus (sle) and fever subsided. he was confirmed lupus nephritis by renal biopsy later. because of onset of sle at the young age, we performed diagnostic whole exome sequencing and multiplex ligation-dependent probe amplification assays. conclusions: he was confirmed dock gene deletion (a deletion on one allele and point mutation on the other allele). he is preparing for hematopoietic stem cell transplantation due to autoimmunity and nonreversible parenchymal organ damage form infections although he has not yet experienced a life-threatening infection. introduction/background: during immunological investigation, it is important to distinguish those individuals who may hav e hypogammaglobulinemia (hypo) without fulfilling the criteria for the severe antibody deficiency common variable immunodeficiency (cvid) e.g. unspecified hypogammaglobulinemia from those with cvid. objectives: since low igg concentrations may support a diagnosis of cvid, we sought to investigate whether the measurement of additional igg subclass antibodies (iggsc) may provide further discrimination between patients with cvid and those with hypo. methods: iggsc concentrations were measured in serum samples from cvid patients (n= , : . m:f, median age . years, range - ) and hypo patients (n= - , : . m:f, median age . years, range - ). results: cvid patients had lower median iggsc concentrations for all iggsc: igg mg/dl (range - ) vs mg/dl (range - ); igg - mg/dl (range - ) vs mg/dl (range - ); igg - mg/dl (range - ) vs mg/dl (range - ); igg mg/dl (range . - ) vs mg/dl (range . - ). this was significantly lower for igg and igg (p= . and . respectively). a higher percentage of cvid patients had iggsc below the lower limit of the normal range compared to hypo patients: igg ( vs . %); igg ( vs . %); igg ( vs . %); igg ( . vs . %). % of cvid patients had low concentrations of or more iggsc vs only . % of hypo patients (p= . ). . % and . % of hypo patients had low levels of or iggsc, respectively. conclusions: igg subclass measurements may have some utility in distinguishing cvid patients from hypogammaglobulinemia patients. introduction/background: the thymus is often removed during cardiac surgery for repair of congenital heart disease, but the extent of tissue removed varies between procedures and surgeons. previous studies have shown decreased t cell counts after thymectomy but there is limited data on the effect of thymectomy on t cell receptor excision circle (trec) levels and infection risk. objectives: to determine the effect of partial and complete thymectomy during cardiac repair surgery on trec levels and infection risk. methods: a retrospective study of electronic medical records was performed on children who received cardiac surgery before age one at new york presbyterian/morgan stanley childrens hospital between / / and / / . patients with heart transplant or primary immunodeficiency were excluded. data was recorded on trec levels (abnormal trec < copies/μl on new york state newborn screen), number of positive cultures, viral pcr panels, and infiltrates on chest x-ray. patients were followed for a minimum of six months after cardiac surgery. study was irb approved. results: cardiac surgery was performed on patients and data was available for patients. of patients included, had a partial and had a complete thymectomy. trec levels after surgery were recorded for patients. only % of patients had an abnormal trec level on newborn screen. there was no difference between partial and complete thymectomy on risk of abnormal trec (p = . ) and mean total number of infections at months (p = . ). conclusions: thymectomy rarely causes low trec levels in children undergoing cardiac surgery. complete thymectomy does not significantly increase infection rates in these children compared to partial thymectomy. these findings are possibly due to presence of ectopic thymic tissue or thymic regeneration and are reassuring for children undergoing complex cardiac surgeries. however, long-term follow-up of these children will be necessary to determine residual function of the thymus and clinical response. introduction/background: new sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (pid), not only by establishing a gene-based diagnosis, but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. objectives: we sought to determine whether blood collected by guthrie cards could be shipped across continents by regular airmail to a cliaapproved laboratory for confirmatory testing. methods: blood was collected and blotted onto the filter paper of guthrie cards by completely filling three circles. we enrolled male patients with presumptive x-linked agammaglobulinemia (xla) cared for at the vietnam national children's hospital, their mothers and several sisters for carrier analysis. dbs were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a cliacertified laboratory in the us for sanger sequencing. the protocol for sanger sequencing was modified to account for the reduced quantity of gdna extracted from dbs. results: high-quality gdna could be extracted from every specimen. btk mutations were identified in of patients studied, confirming the diagnosis of xla in % of the study cohort. type and location of the mutations were similar to those reported in previous reviews. the mean age when xla was suspected clinically was . years, similar to that reported by western countries. two of mothers, each with an affected boy, had a normal btk sequence, suggesting gonadal mosaicism. conclusions: dbs collected on guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gdna for sanger sequencing overseas. using this method of collecting gdna we were able to confirm the diagnosis of xla in of vietnamese patients with the clinical diagnosis of agammaglobulinemia. introduction/background: znf is a positive regulator of stat expression. it has recently been described that nonsense mutations in znf account for the stat -like phenotype in four autosomalrecessive kindred. patients presented with reduced stat expression and diminished th cell numbers, in absence of stat mutations. objectives: here, we decribed a turkish case having nonsense mutation in znf developed dual malignancy in years. results: a -year-old female patient presented with severe eczema, recurrent cold skin abscesses, herpetic skin lesions, sinopulmonary infection, otitis media and hearing loss. the parents are cousins. two younger sisters of the index case had eczema and recurrent skin infections since their infancy period. physical examination of the patient revealed severe eczema, high palate, micrognathia, maxillary hypoplasia and hearing loss. laboratory findings showed reversed cd /cd ratio, high serum ige level ( . u / l) and low igg level ( mg / dl). the patient was diagnosed as hyper ige syndrome and ivig therapy ( mg / kg, every weeks) was initiated because of igg subgroup deficiency and recurrent sinopulmonary infections. at the age of , a polypoid mass filling the left nasal cavity was detected in her examination. paranasal sinus ct revealed a mass obliterating the left nasal cavity, left ethmoid sinus and frontal sinus. immunohistochemical stains showed a small round cell malignant tumor in the nasal cavity. she was treated with chemoradiotherapy successfully. a homozygous nonsense mutation has been detected at exon in the znf gene (c. c> t) (kindly provided by grimbacher's lab) very recently. she developed papillary thyroid carcinoma two years after completing the cancer therapy. conclusions: the relationship between znf defect and cancer development is unknown. the development of a second malignancy in this patient for a short time of completing the therapy might imply us a tendency for malignancy in znf patients. additionally, the likelihood of increased radiosensitivity in these patients should be taken into consideration. introduction/background: prometic % igiv contains purified igg, % as monomer; with a distribution of igg subclasses proportional to that in native human plasma. we report the interim results from a phase trial in the usa of prometic % igiv in adults and children with pidd. objectives: this was a phase , single-arm, open-label, multicenter trial to evaluate the safety, tolerability, and efficacy of prometic % igiv in adults and children with pidd. methods: adults and pediatric subjects with pidd on a stable dose of igg replacement therapy ( - mg/kg) for at least months with serum igg trough levels > mg/dl were included. subjects received prometic % igiv every to weeks for approximately year at the same dose and schedule as their previous igg replacement therapy. results: an interim analysis was conducted when data were available on adult subjects who received at least one dose of prometic % igiv (total of infusions), with subjects receiving at least months of treatment (exposure = . subject years). at this time, pediatric exposure was only . subject years. there were no serious bacterial infections (sbis) reported, and rate/yr of infections other than sbis was . which was comparable to rate while on commercial product ( . ) all subjects achieved an igg trough level > mg/dl. there were no deaths, and no subject had a study drug-related serious adverse event or an adverse event that resulted in permanent discontinuation of study drug. a total of adverse reactions (ar) ( . /infusion) occurred in subjects ( . %), with infusions ( . %) associated with an ar. most infusions ( . %) were completed without a rate reduction. most ars were mild or moderate in severity, with severe ars ( . /infusion) occurring in subjects ( . %). the most frequent ars were headache ( . % subjects or . /infusion) and fatigue ( . % subjects or . /infusion). conclusions: in adults treated with prometic % igiv, there were no sbis and infusions were well tolerated. director, sean n. parker center for allergy and asthma research at stanford naddisy foundation, professor of medicine and pediatrics, stanford university introduction/background: desensitization to food allergies is being studied in clinical trials using oral immunotherapy (oit). there are limited data regarding the immune changes associated with successful oit. epigenetics involves heritable changes in gene function without modification of the underlying dna sequence. this is mediated by methylation, histone modification, or changes in microrna. objectives: to study methylation changes in the loci of four key genes of immune cells involved in allergy, interleukin (il- ), interferon gamma (ifn-g), forkhead box protein (foxp ), and interleukin (il- ), comparing baseline to post-oit. methods: we completed a phase , randomized, placebo-controlled, multi-food oit trial using omalizumab, an anti-ige biologic, to facilitate desensitization for multi-food allergic individuals. double-blind, placebo-controlled food challenges (dbpcfcs) to multiple foods were conducted at entry and after weeks of treatment, the primary endpoint. omalizumab (n= ) or placebo (n= ) was administered for weeks, with oit for - foods starting weeks after the beginning of omalizumab or placebo. after weeks ( weeks of oit), participants underwent dbpcfcs to their offending foods. treatment failures (n= ) were offered open-label omalizumab. pyrosequencing of bisulfite treated genomic dna purified from pbmcs from each participant at baseline and post-oit was undertaken to investigate changes in methylation. results: forty-four participants achieved successful desensitization, defined as passing dbpcfcs to or more foods following oit. we found that the - cpg site in the il- promoter region is hypermethylated over time during successful multi-food oit (fdr-adjusted p < . by wilcoxon signed-rank test). the median % of methylation at baseline was . (interquartile range . %) and was . post oit (interquartile range . %). there were no statistically significant (with a significance level of fdr adjusted p value of . ) changes in the il- , foxp , or ifn-g loci in the cpg sites we studied. conclusions: these preliminary results suggest that one immune mechanism involved in successful desensitization may involve suppression of th function by hypermethylation of il- in immune cells in the peripheral blood. introduction/background: atopic dermatitis (ad) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. objectives: we investigated the effects of esculetin from fraxinus rhynchophylla on atopic skin inflammation. methods: for induction of atopic skin inflammation, we exposed the ears of female balb/c mice with house dust mite (dermatophagoides farinae extract, dfe) and , -dinitrochlorobenzene (dncb) during weeks. results: oral administration of esculetin reduced dfe/dncbinduced atopic skin inflammation symptoms based on ears swelling and scratch numbers. the immunoglobulin (ig) e, igg a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. it suppressed th , th and th responses by inhibiting the production of inflammatory cytokines such as tumor necrosis factor (tnf)-, interferon (ifn)-, interleukin (il)- , il- , il- and il- in the ear tissue. further, we investigated the effects of escueltin on activated keratinocytes, one of the most representative cells for studying pathogenesis of acute and chronic atopic skin inflammation. as results, esculetin suppressed gene expression of th , th and th cytokines and activation of nuclear factor-b and signal transducer and activator of transcription in tnf-/ifn-stimulated keratinocytes. conclusions: taken together, the results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin might be a potential therapeutic candidate for the treatment of ad. introduction/background: autoimmunity is often seen in common variable immune deficiency (cvid) with immune thrombocytopenic purpura (itp) being the most frequent manifestation at a prevalence of - % in cvid patients. in such patients, itp is often recognized and treated long before cvid, the implications of which are unknown. primary itp is a clinicopathologic diagnosis which includes an evaluation of other conditions that may mimic it including cvid. currently, it is unknown how frequently cvid is evaluated during the diagnostic workup of itp and what percentage of those patient actually have cvid. objectives: the two main objectives of this study were to determine the number of itp patients that had an igg level checked during their clinical course and if the globulin fraction can be used as a marker for hypogammaglobulinemia in itp patients at the time of diagnosis. methods: a retrospective chart review was undertaken at a large academic medical center of patients with a new diagnosis of itp between january and january . igg levels were collected and globulin fractions were calculated as the difference between serum total protein and albumin within days of the initial itp diagnosis. results: six hundred and twenty-three patients were found to have a new diagnosis of itp in the given timeframe. of these, only ( . %) had igg levels checked at any point during their clinical course. twelve of the ( . %) had hypogammaglobulinemia with only of the ( %) having a formal immunologic follow-up evaluation. two were diagnosed with a primary immunodeficiency ( cvid and ctla deficiency). globulin fractions were calculated on patients at the time of itp diagnosis. mean calculated globulin fraction in hypogammaglobulinemic patients was . (range . . ), versus . (range . . ) in patients without hypogammaglobulinemia (p= . ). conclusions: the diagnosis of cvid is often delayed from the onset of symptoms which can include autoimmune conditions such as itp. our data indicate that clinicians do not routinely check igg levels at the time of itp diagnosis which should be considered standard of care based on the current guidelines. our data suggest that although calculated globulin fractions were significantly lower in hypogammaglobulinemic patients, the variability was substantial and hypogammaglobulinemic patients would be missed using this as an indicator of low immunoglobulins. future directives include a prospective study using igg levels checked at the time of itp diagnosis, with formal evaluation for cvid in any hypogammaglobulinemic patients to evaluate the true prevalence of cvid among itp patients. introduction/background: autoimmune lymphoproliferative syndrome (alps) is a disorder characterized by immune dysregulation due to the rupture of lymphocyte homeostasis, which occurs as a result of mutations in the apoptotic pathway mediated by fas. this disease is sometimes misdiagnosed due to its variable phenotypic expression and the overlapping of symptoms with many other hematological and immunological disorders. ( ) a patient diagnosed with evans-fisher syndrome (sef) was referred to the laboratory for immunity studies. this is a patient who from infancy had multiple admissions for severe sepsis, with severe anemia and severe thrombocytopenia. in the evaluation of the history of the disease by the work team, a series of clinical data was observed that suggested the possibility of the patient presenting an alps, so it was decided to incorporate as part of the study, the quantification of the cells t cd + cd -cd -(double negative t cells or dnt) that express tcr +. the differentiation pathways of dnt tcr + and the role of fas in this process are not clear, some authors hypothesize that they may be represented by direct descendants of chronically activated positive simple t cells, with deregulated co-receptors, in a state of differentiation in which they were destined to perish by fas-mediated apoptosis. ( ) ( ) ( ) ( ) the population of tcr + dnt cells required for diagnosis must be derived from the particular study of each laboratory in their populations, but several working groups agree that the pathological limit values are , % in total lymphocytes or , % in cd + lymphocytes. ( ) evidence shows that dnt alps cells are not simply accumulated in senescent withdrawal; they and their precursors remain active and proliferate under the influence of activation signals. ( ) although numerous genetic deficiencies lead to lymphoproliferation of t cells, only that caused by a defective fas pathway is dominated by negative double t cells. ( ) in clinical immunology, the distinction between cd ra + and cd ro + cells is particularly useful for determining the state of the "naive" cell compartment in relation to its thymic origin. primary immunodeficiency disorders are characterized by a decrease / absence of thymus performance, and often involve a decrease in cd ra + t lymphocytes. scientific evidence suggests the important role of the "naive" subpopulation in the origin and maintenance of self-reactive effectors in the periphery. ( ) regulatory t cells are able to effectively control autoreactive t cells, especially when negative thymic selection is defective. its differentiation and function is controlled by foxp . the decrease or absence of regulatory t cells leads to autoimmune diseases, specifically those mediated by cd + t cells, and to lymphoproliferation characterized by multiorgan inflammation and other autoimmune disorders. ( , ) objectives general: establish the diagnosis of a pediatric patient with suspected primary immunodeficiency due to dysregulation, an autoimmune lymphoproliferative syndrome (alps). specific: evaluate the tcr + dnt population, relevant in the diagnosis of alps. quantify cell populations that exhibit markers of activation, differentiation and regulation of the relevant t cells in the study of this disease. methods: monoclonal antibodies (mab) cd fitc / cd pe / cd pc triple labeled (beckman coulter), tcrapc, cd pe, cd rapercp, cd rofitc, cd fitc, cd rope, cd apc, cd f, cd pe, cd percp, cd pe, cd pe, cd percp, cd fitc, cd fitc were used , hla dr and the cd fitc / cd pe / foxp apc regulatory t cell kit, all from miltenyi biotec. the trial included the use of a healthy control. both samples were processed in unison to ensure reproducibility. the samples were acquired in a beckman coulter gallios cytometer, with the use of the kaluza program, version . . the analysis strategy included the formation of overlapping "gate" windows for the quantification of dnt tcr + and regulatory t cells. results: the patient studied showed . % of tcr + negative double t cells, in relation to . % of the healthy control. the presence of this population of t cells in patients diagnosed with evans syndrome, even in the absence of lymphoproliferation, is consistent with alps. ( ) (graph ) the presence of increased tcr + dnt and lymphoproliferationexpressed as lymphadenopathies and splenomegaly of noninfectious or malignant cause, of at least months of evolution; plus the typical immunohistological findings found in the patient's lymph node biopsy (paracortical hyperplasia), the presence of autoimmune cytopenias (hemolytic anemia and thrombocytopenia) and hypergammaglubulinemia, led to the "probable diagnosis" of alps in the patient studied. the accumulation of dnt in the lymph nodes and other peripheral organs is accompanied by qualitative changes in the composition of the t cell repertoire, so that the immunophenotype performed included markers of activation, differentiation and regulation. the evaluation of the activation on t lymphocytes and nk cells showed that there was a slight decrease in the expression of the receptor for il- , cd . the cd + cd + population was expressed in greater percentalthough discretely-in the patient than in the healthy control, which was directly related to the presence of hypergammaglobulinemia, elevated iga and the presence of autoantibodies. it was also highlighted by the high expression of the cd + cd + autoreactive population in the patient studied ( . % vs . %). in order to know the impact on cell differentiation, the subpopulations of effector and memory cells for cd + and cd + t lymphocytes were evaluated by combining the cd ra and cd ro isoforms. cd ra is expressed in naive t lymphocytes. particularly, the cd + cd ra + population has an essential function as a suppression inducer, and it is diminished in the patient studied, in relation to the control. ( . % vs. . %, respectively) the same behavior was shown by the cd + cd ro + memory and effector cells, . % vs . %. in the cd + population itself, it was possible to confirm the presence of a clone that expressed both receptors (cd ra and cd ro), probably a temra population (terminally differentiated effector memory cells). when comparing the results, it was unexpectedly found that there was a slight decrease between patient and control ( . % vs . %). as the characteristic phenotype of this cell population could not be corroborated, conclusive assessments could not be made. the combination of cd isoforms was also used to study cd + t lymphocytes, but similar behavior between patient and healthy control was observed. (the lab results are shown in table ) the analysis of the regulation of the immune response showed a decrease in the cd + cd + population and the expression of the foxp transcription factor in the patient with respect to the control. ( . % vs. . %, . % vs. . %, respectively) (graph ) conclusions . the markedly high quantification of cd -cd -tcr + t lymphocytes allowed to define the "probable diagnosis" of autoimmune lymphoproliferative syndrome in the patient under study. . the increased activation of cd + cells and the presence of the cd + cd + self-reactive population, largely responsible for autoimmunity and lymphoproliferation in the alps, could be confirmed. . the decrease in the cd + cd ra + t-cell suppressor-inducing population evidenced corroborated its involvement in this disorder by primary immunodeficiency, in relation to the thymus dysfunction that originates and maintains self-reactive effectors in the periphery. . the decrease in the expression of the foxp transcription factor observed, points towards a low regulation of the response that leads to autoimmunity and to lymphoproliferation, specifically mediated by cd + t cells. allergy and immunology arnp, nicklaus children's hospital allergy and immunology division director, nicklaus children's hospital introduction/background: specific antibody deficiency syndrome is characterized by a weak antibody response to bacterial polysaccharide antigens when no other immune system abnormalities can be found. low titers to pneumococcal vaccine have become one of the most frequently recognized immune abnormalities in pediatric patients with recurrent sinopulmonary infections. nonetheless, insufficient data and lack of consistent testing of the response to pneumococcal polysaccharides continues to affect the optimal diagnosis and management of this specific antibody deficiency. objectives: to characterize the pre-and post-immunization igg antibody trend for each specific serotype included in the pneumococcal -valent conjugate vaccine (pcv ), as well as others that are routinely tested, in a cohort of pediatric patients with recurrent sinopulmonary infections. secondarily, to understand differences in the immune response to the vaccine booster between age groups. methods: this retrospective review identified patients with recurrent sinopulmonary infections. in this cohort, required an immune workup, and were found to have low pneumococcal titers needing a pcv vaccine booster. baseline pneumococcal serotype-specific antibody titers at initial visit and weeks after the vaccine booster were obtained. patients were categorized by age: years, - , - , and - . an adequate response to the pneumococcal conjugate vaccine was deemed to be a -fold increase over baseline and/or a post-immunization titer of . μg/ml or greater. results: overall, pcv booster provided a significant improvement in the number of protective titers, increasing from . ( % ci: . - . ) serotypes at baseline to . ( % ci: . - . ) serotypes at weeks (p < . ). this increase correlated with improved clinical outcomes % showed no signs of recurrent infection after the first booster and % after a second dose. all those who did not improve clinically suffered from co-morbidities (genetic abnormalities and rheumatologic diseases). post-immunization antibody concentrations were significantly higher than at baseline for all serotypes (p < . ) and only , n, and f did not exhibit a greater than -fold increase (p > . ) weeks following the booster. across age groups, only , f, and v showed pre-immunization differences in titers. there were no differences between ages in post-immunization titer levels for all serotypes. similarly, all age groups had a comparable number of baseline titers (p = . ) and at follow-up (p = . ). conclusions: in pediatric patients with recurrent sinopulmonary infections, an additional pneumococcal booster proved to be effective in the protection of these children from further infections. the pcv booster substantially increased titer levels and concentrations, and significantly improved clinical outcomes, independent of age. this investigation has provided us with a better understanding of the response after booster vaccination, and its role in the protection of patients from recurrent sinopulmonary infections. further studies are needed to elucidate whether a fifth dose of pcv should be optional as part of the vaccination schedule of patients with recurrent sinopulmonary infections. introduction/background: many primary immunodeficiencies (pids) share overlapping presentations, complicating clinical diagnosis. due to the ability to include many genes in one assay and the rapid turnaround time that these panels allow, expanded next-generation sequencing (ngs) panels are valuable in facilitating the diagnosis of patients with pids. objectives: we aimed to determine the clinical utility of an expanded ngs panel for the genetic diagnosis of patients with suspected pids. methods: we performed a retrospective analysis of the clinical utility of a -gene pid ngs panel used in a clinical diagnostic laboratory. from april to october , panels were ordered for patients with suspected or known pids. results: seventy-four pathogenic or likely pathogenic (p/lp) variants were identified in ( . %) patients. eight ( . %) of the p/lp variants were copy number variations ( deletions, duplications). fifty-two patients ( %) had p/lp variant, and patients ( . %) had p/lp variants. of positive patients, . % (n = ) were heterozygous carriers for autosomal recessive conditions where a second variant was not identified. twenty-two patients had p/lp heterozygous variants in genes with autosomal recessive and autosomal dominant inheritance patterns, in which the positive findings may or may not explain the patient's phenotype. for example, variants in this category were heterozygous variants in tnfrsf b (taci). genetic diagnoses were established or likely in % of patients with p/lp variants ( . % of all patients). five patients were heterozygous for a single p/lp variant and a variant of unknown significance in the same autosomal recessive gene. four patients in whom a genetic diagnosis was determined were also heterozygous carriers for a second, unrelated condition. one patient was found to have two distinct genetic diagnoses. variants of uncertain significance (vus) were identified in most ( . %) patients. the average turnaround time from test requisition to return of results was days. in total, % percent of genetic diagnoses were for conditions that are treatable with hematopoietic cell transplantation. conclusions: these results illustrate the utility of broad ngs panels for the diagnosis of patients with pids. introduction/background: severe combined immunodeficiency (scid) is a life-threatening immune deficiency manifest by extreme susceptibility to infection. early diagnosis and definitive treatment with either hematopoietic cell transplant (hct) or, in select cases, gene therapy (gt) has been shown to significantly improve survival. newborn screening for scid has allowed for the opportunity to promptly identify these patients before significant infections occur. at ucsf, newly diagnosed infants with scid are admitted to the hospital for management and remain in isolation for definitive treatment and until adequate immune reconstitution occurs. previous work by our group demonstrated up to % of these parents experience psychosocial trauma manifested by depression and post-traumatic stress disorder (ptsd). the psychosocial challenges that contribute to depression and ptsd in these parents have not been qualitatively described. objectives: to understand the range of experiences and feelings of parents/caregivers of infants with scid diagnosed by newborn screening throughout their prolonged hospitalization and isolation in order to better support patients, parents, and their families. methods: voluntary participation was elicited from parents of children with scid who were status post hct/gt for one year or longer. semi-structured, in-person interviews lasting approximately - minutes were conducted with parents; interviews were recorded and transcribed. parents were asked to discuss their experiences from first notification of an abnormal screening result through discharge after hct or gt. emerging themes were identified from the transcribed interviews. results: we interviewed mothers and fathers of infants with scid. six infants received hct, while one underwent gt for ada-scid. all children were alive and well at the times when interviews were conducted. overall, once admitted, parents reported feeling well supported by the medical team and support staff. however parents identified a number of stressful events. uniformly reported key stressors included: receiving the first phone call regarding their childs abnormal newborn screening results, preparing for hct, coping with prolonged isolation, and transitioning from hospital isolation to care with ongoing isolation at home. other challenges described reflected the additional stressors of caring for a newborn, including coping with postpartum depression. overall, we identified three major themes encompassing the challenges faced by parents of hospitalized scid patients: (i) loss of normalcy and control over multiple aspects of life; (ii) prolonged waiting periods (especially the wait between diagnosis and hct and between hct and evidence of t cell engraftment); and (iii) perceived lack of guidance on realistic expectations during the hospital stay . parents sought and relied on peer support from other scid parents to learn about coping with the nuances of daily life as a parent of an infant with scid. conclusions: we identified multiple psychosocial stressors and challenges uniquely faced by parents and caregivers of infants diagnosed with scid by nbs. parents described barriers to caring for their own physical and mental health, which can be especially harmful to parents experiencing postpartum depression. recognizing these challenges allowed for the identification of opportunities to improve both healthcare delivery to their children and institutional support for future families affected by scid (table i) . emphasis should be placed on providing parents with scid-specific resources as early as the time of diagnosis, connecting parents with scid support networks, and facilitating access to psychosocial and mental health services for caregivers introduction/background: chronic granulomatous disease (cgd) is caused by a genetic defect that impairs phagocyte function. this disease results in recurrent infections and granuloma formation. rarely do patients develop cutaneous symptoms, unless associated with autoimmune disorders such as systemic erythematous lupus. previously described cutaneous findings include granulomas, abscesses, photosensitivity, malar rash, discoid lupus, vasculitis, and rarely vesicular rashes. here we describe two term infants diagnosed with x-linked cgd who present, in addition to frequent infection, with a unique papulopustular skin rash initially diagnosed as non-classic appearing eczema refractory to usual eczema treatment and antibiotics. objectives: to characterize cutaneous findings in x-linked cgd and emphasize the importance of considering further work in patients who present with similar rashes in conjunction with concerning features for primary immunodeficiency. methods: each infant was diagnosed with cgd based on abnormal dhr testing and/or genetic evaluation. after obtaining consent from both families, we have documented photographs of the development of rash in two newly diagnosed infants with cgd. one infant underwent cutaneous biopsy with resulting pathologic evaluation. results: our first patient presented at months of age with episodic fever with chronic leukocytosis, iron deficiency anemia, thrombocytosis, elevated inflammatory markers, proctocolitis with elevated calprotectin, and rash. egd with flexible sigmoidoscopy showed no signs of inflammatory bowel disease, the left colon had macroscopically raised erythematous lesions but was microscopically normal with no signs of active colitis. he was initially diagnosed with fpies and eczema in the setting of diagnosis of various infections (otitis, upper respiratory illness). the skin rash was described as non-pruritic, generalized pink-purple papulopustular lesions with a pink base, most prominent on upper and lower extremities, mostly sparing the trunk. skin biopsy histopathology revealed an essentially unremarkable appearing epidermis and within the dermis, there was a superficial perivascular lymphohistiocytic infiltrate. eosinophils and plasma cells were not abundant. the histologic changes were thought to be non-specific, but could be seen in a drug reaction or urticaria. a viral exanthem could also demonstrate these changes. giemsa stain and a stain for mast cell tryptase revealed normal numbers of mast cells in the biopsy specimen. he was not on any oral medications at the time and did not respond to treatment with topical moisturizers, oral antihistamines, topical steroids, or any standard for eczema care. he had a maternal uncle who passed away in infancy from infection. heightened clinical suspicion for primary immunodeficiency led to obtaining a neutrophil respiratory burst assay which was consistent with cgd and genetic testing was positive for x-linked cgd. pathogenic mutation nucleic acid change was c. c>t; hemizygous; amino acid alteration was p. arg>stp within the cybb gene locus. he had no other features of auto-immune disease including negative ana obtained later in his clinical course and his colitis diagnosed as cgd-associated colitis. his rash did not respond to systemic treatments for his colitis, oral antibiotics, and during hsct. our second patient presented at months of age with persistent fevers, inguinal lymphadenopathy, leukocytosis, elevated inflammatory markers, non-bloody diarrhea and rash. there was a family history of autoimmune gi disease. he presented to the hospital with fever of unknown origin with concern for infection, atypical kawasakis, and drug rash. his rash was described as a blotchy, pink, papular rash most prominent on the upper arms but also present on lower arms, legs, and chest, faint on face. there is some induration and thickness to the rash in some areas (confluent on the arms with more erythema and induration) and more faint pink papules (scattered on legs) elsewhere. a biopsy of his inguinal lymph node showed granulomatous lymphadenitis with neutrophilic abscess formation, and culture was positive for serratia marcescens. neutrophil respiratory burst assay was consistent with cgd and genetic testing was positive for x-linked cgd, mutation with the cybb gene. pathogenic nucleic acid change was c. + g>a; hemizygous; amino acid alteration was p. deletion of exon . the rash remained unchanged with treatment for infection, initiation of antifungal and bacterial prophylaxis, along with topical steroid therapy. conclusions: in patients who present with frequent infections and who have unusual cutaneous findings that do not fit with common infant rashes, consideration of work up for cgd should be pursued. specifically, generalized papulopustular rash with a negative skin biopsy can be misdiagnosed as atopic dermatitis, and in the right clinical context cgd should be considered. introduction/background: chronic lung disease in common variable immunodeficiency (cvid) is heterogeneous, and it is a leading cause of morbidity and mortality in this population. currently, the diagnosis and monitoring of chronic lung disease in cvid rely on radiographic findings, biopsy and/or pulmonary function tests. fractional exhaled nitric oxide (feno) is a noninvasive biomarker of airway inflammation, and it has been widely utilized to aid the in-office diagnosis, characterization, and management of inflammatory airway disease, such as asthma. however, exhaled nitric oxide in cvid patients with chronic pulmonary complications has not been examined. objectives: we aimed to determine fractional exhaled nitric oxide levels in cvid patients. methods: we measured exhaled nitric oxide in cvid patients with or without chronic lung disease. results: feno measurements were obtained in cvid patients (mean age: , range - ). five patients had no known lung disease; patients had chronic lung disease (bronchiectasis, n= ; lung granulomas, n= ; hepatopulmonary syndrome, n= ). four patients were on inhaled steroid (bronchiectasis, n= ; lung granulomas, n= ), patients were on systemic steroid (lung granulomas, n= ; hepatopulmonary syndrome, n= ), and patient was on oral budesonide (no known lung disease). feno was elevated (> parts per billion [ppb]) in of patients, of whom had known chronic lung disease. patients with granulomatous lung disease had higher feno (mean . ppb, range - ppb) compared to patients without known lung disease (mean ppb, range - ppb, p= . ), patients with bronchiectasis (mean . ppb, range - , p= . ), and the patient with hepatopulmonary syndrome ( ppb). feno levels remained elevated in granulomatous lung disease in patients with inhaled or systemic steroid use. conclusions: this is the first report of feno measurements in cvid patients. feno is elevated in a subgroup of cvid patients, and it may differ according to the underlying lung pathology. further investigation is warranted to determine the utility of feno in the diagnosis and management of chronic lung disease in cvid. professor of pediatrics, university of british columbia introduction/background: whole exome sequencing (wes) has revolutionized the discovery, diagnosis, and treatment of primary immune deficiency diseases (pids), a group of disorders with high genetic and phenotypic heterogeneity. current bioinformatic analysis approaches for wes rely heavily on population databases to exclude variants present in the populations represented by these databases. this approach may confound the ability to detect true disease causing variants, and conversely, flag variants that are absent from population databases only by virtue of originating from minority populations. ultimately, the pathogenicity of novel variants can only be mechanistically established through rigorous biochemical and functional validation. objectives: to evaluate the pathogenicity of a novel homozygous variant in caspase recruitment domain family member (card ) (c. g>t, p.c y), in a patient with features of combined immunodeficiency (cid). methods: research study protocols were approved by our institutional review board. six members of one family (the affected child, healthy siblings and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: targeted sanger sequencing confirmed the presence of the homozygous card variant c. g>t, p.c y in the index patient (and subsequently in one apparently healthy sister). each parent was found to be a heterozygous carrier of the variant. nfkb activation in vitro was found to be normal for both the index patient and sister, and was indistinguishable from unaffected family members. conclusions: although multiple in silico tools predicted the c. g>t, p.c y card variant to be pathogenic, the patients b and t cells did not have aberrant nfkb activation in vitro, as would be predicted given the central role of card in nfkb activation. this practical experience highlights how imperative it is to functionally characterize novel variants found by wes, even when variants are predicted to be damaging. chief, clinical immunology -faculdade de medicina do abc introduction/background: gata is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis. heterozygous mutations leading to gata deficiency were first described in . the age of clinical presentation ranges from early childhood to late adulthood, with most of them in adolescence to early adulthood. patients present clinical findings as monocytopenia, nontuberculous mycobacterial infections, myelodisplasia, viral (mainly hpv and ebv), fungal and bacterial infections. patiens may arise with many phenopytes, showing how complex is the effect of this transcription factor. objectives: we report a patient with gata mutation. methods: report: a year old female patient was referred due to a mycobacterial non-tuberculosis pneumonia. she was a healthy child until puberty. at the age of she presented genital herpes and recalcitrant vulvo-vaginal warts (hpv). at , she complained of lower back pain with no diagnosis for months, medication was unnefective, and she developed ischemic stroke. hemiparesia was mild and temporary. endocarditis with no agent identified was also diagnosed. therapy with acetilsalicilic acid was mantained until the age of . five years later, vaginal hpv spread through vulve and anal region evolving to neoplasia. she was submitted to surgery, radio and chemotherapy until october . one year later, profound cytopenias led to hospitalization. during that time, she had cough and fatigue and pulmonary tuberculosis was diagnosed. despite therapy with rifampin, isoniazid and pyrazinamid, there was no improvement. bronchoalveolar lavage was positive for mycobacterium avium. main immunological evaluation showed: monocytes ( %), t cells /mm ; cd +: ( , %), cd +: ( %); b cells /mm ( , %); nk: /mm ( , %); normal immunoglobulin levels; incomplete response to pneumococcus serotypes; igm and igg positive for cmv; negative serologies for hiv, ebv and htlv / . molecular analysis identified an heterozygous mutation c. c>tp.(thr met) in gene gata . results: we report a patient with gata mutation. the same gata mutation was previously described in national institute of health nih-usa (n= ) and in australia (n= ). conclusions: it took almost years for diagnosis suspicion. gynecologists should be warned about this diagnosis in order to improve patients prognosis. early diagnosis is crucial with adequate prophylaxis, prompt treatment of infection, surveillance of malignancy, and, moreover, family screening and genetic counseling. this is the first report of this mutation in latin america. introduction/background: severe combined immunodeficiency (scid) due to adenosine deaminase (ada) deficiency was the first human monogenic disease to be approached with gene therapy, and ongoing research advances over years led to the approval by the european medicines agency of a stem cell gene therapy product for its treatment using a gammaretroviral vector (gv), strimvelis®. despite the high success rate using gvs for ada gene transfer without vector-related complications, the development of leukoproliferative complications from the use of gvs in gene therapy of other disorders led us to develop lentiviral vectors (lvs) to deliver the corrective ada sequence/cdna (corrigan-curay et al, mol ther ; modlich et al, mol ther .). lvs, typically derived from hiv- and devoid of all viral genes, can be produced in a self-inactivating (sin) configuration, in which the viral long-terminal repeat enhancers are absent, eliminating the major identified cause of insertional oncogenesis due to gvs. we developed a lv (efs-ada) that carries a normal human ada cdna (codon-optimized) and demonstrated in pre-clinical studies its efficacy to transfer and express the ada protein, with evidence of significantly decreased potential for insertional mutagenesis compared to gammaretroviral vectors using the immortalization (ivim) assay and in murine bone marrow transplant models (carbonaro et al, mol ther, ) . this new lv was evaluated for safety and efficacy in parallel clinical trials of gene therapy for ada scid performed at sites in the u.s (university of california, los angeles and the national institutes of health {nih} clinical center) and u.k. (university college london/great ormond street hospital) enrolling subjects between and . we report here results from the patients treated in the u.s. between - . objectives: this is a prospective, non-randomized phase i/ii clinical study to assess the safety and efficacy of efs-ada lentiviral vector stem cell gene therapy in ada-scid subjects older than month. methods: subjects with ada-scid were enrolled, screened to document eligibility and underwent bone marrow harvest ( - cc/kg). bone marrow was processed to isolate cd + cells, which were pre-stimulated by overnight culture in serum-free medium containing c-kit ligand, flt- ligand, and tpo followed by culture with the efs-ada lentiviral vector overnight. subjects received a single dose of busulfan ( mg/kg) iv for reduced intensity conditioning. cells were removed from culture, washed, formulated and administered by iv infusion at least hours after busulfan administration. enzyme replacement therapy (ert) with pegylated bovine ada (peg-ada) was continued for one month post-transplant and then stopped. subjects were followed over months to assess safety and efficacy end-points. results: with - months follow-up, overall survival is %. eventfree survival has also been %, as all subjects are alive, remain off peg-ada ert, and none have required a second transplant. successful engraftment of gene-corrected cells was observed in all subjects at months, and persisted over the months of observation, based on vector gene marking in granulocytes and peripheral blood mononuclear cells (pbmcs), changes from baseline in rbc ada enzyme activity, and levels of metabolic detoxification of deoxyadenosine nucleotides. immune reconstitution was observed in all subjects and was sustained over the two years of observation, based on improvement of peripheral blood absolute lymphocyte counts and lymphocyte subsets (t, b and nk cells, and naïve cd + t cells). eighteen of patients have been able to stop receiving immunoglobulin replacement therapy. subjects who had routine infections all recovered with standard of care treatment, and there were no severe or opportunistic infections. conclusions: conclusions: gene therapy using the efs-ada lv has a favorable safety profile and was efficacious in this trial. a current followon trial at ucla is using a cryopreserved formulation of the cell product and pharmacokinetic-adjusted busulfan dosing, sponsored by the california institute for regenerative medicine (clin - ) and orchard therapeutics. acknowledgements: this study was supported by research grants from the national institutes of health (u ai ; p hl - ; nhlbi gtrp rsas # and ) and the california institute for regenerative medicine (cl - ; fa - ); support from the ucla david geffen school of medicine human gene and cell therapy program and the ucla eli & edythe broad center of regenerative medicine and stem cell research; and funding from the nhgri intramural program. dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and as an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. introduction/background: x-linked severe combined immune deficiency (scid-xl) is a rare monogenic primary immunodeficiency disorder (pid) where male infants are born without an adaptive and innate immune system. it is a life-threatening disease due to patients inability to fight viral and bacterial infections. scid-xl is driven by any of the two hundred known pathogenic mutations in the interleukin gamma receptor (il rg) gene, which function is required for proper development of t, b and nk cells. the most effective treatment for scid-xl, if performed in the first few months of life, is allogeneic hematopoietic stem cell transplantation (allo-hsct). this treatment is limited by absence of match donors, incomplete immune reconstitution, graft versus host disease and the need for long-term immunosuppression. an alternative curative treatment for scid-xl would be genome editing-based gene therapy by ex vivo genome correction of the patients long-term hematopoietic stem cells (lt-hscs) prior to autologous stem cell transplantation (auto-sct). objectives: here we report a proof-of-concept genome editing-based approach for correcting scid-xl disease. methods: using a crispr/cas -raav platform, we deliver a fulllength codon optimized il rg complementary dna (cdna) at the endogenous start site in cd + hematopoietic stem and progenitor cells (hspcs). results: using an optimized genome editing protocol we achieved > % genome editing as early as h and a median of % genome targeting while retaining > % viability and promoting greater than % ex vivo expansion of cd + hspcs from healthy donors. we demonstrate that our approach retains proper il rg signaling function in t-cells derived from healthy male donors and rescues the lymphopoietic defect from a patients derived mobilized cd + hspcs both in vitro and in vivo. we further show robust in vivo primary human engraftment potential and multi-lineage hematopoietic reconstitution of il rg gene targeted hspcs: a median of % (bone marrow), % (spleen) and % (liver) of il rg genome targeted engrafted cells was achieved at four months following primary transplantation into nsg mice and a median of . % - % was detected at months from secondary transplants of il rg targeted hspcs, thus achieving clinical levels of editing of lt-hscs. lastly, our observation of ( ) an intact hematopoiesis derived from il rg targeted cd + hspcs combined with ( ) a normal karyotype analysis and ( ) our deep analysis of potential off-target activity that showed only off-target sites of no known functional significant with < . % frequency of off-target activity presents strong evidence for the safety of our genome editing approach. conclusions: in sum, this pre-clinical study provides specificity, toxicity and efficacy data supportive of continued development of genome editing to treat scid-xl. objectives: to determine the risk of gvhd in msd hsct for scid patients compared to matched related donor (mrd). methods: retrospective cohort study comparing msd with mrd and the outcome of gvhd in all scid patients who underwent hsct between and . all statistical analysis was done using ibm spss statistic software. results: scid patients underwent hsct, ( %) received gvhd prophylaxis. gvhd occurred in ( . %); / ( %) had gvhd prophylaxis compared to / ( %) that did not, p value = . . acute gvhd occurred at a higher rate in msd / ( . %) compared to mrd / ( . %) p value = . . we analyzed outcome also according to period of hsct. first periods was to ; hsct, msd: , mrd: ; all had gvhd prophylaxis and there was no difference in gvhd. the second period was to : hsct, had msd and had mrd, gvhd prophylaxis was used in . % in msd and % in mrd, p value = . . gvhd was significantly higher in the msd ( . %) compared to mrd ( . %) odds ratio of . ( ci . to . ) p value = . . conclusions: gvhd prophylaxis in msd transplant may have a role to be considered in scid patients. introduction/background: xiap deficiency (also known as x-linked lymphoproliferative disease type xlp ; mim: ) is an x-linked primary immunodeficiency associated with mutations in the gene encoding the x-linked inhibitor of apoptosis (xiap; mim: ). the pathophysiology is characterized by immune dysregulation, usually triggered by epstein-barr virus (ebv) infection. primary ebv infection is followed by hemophagocytic lymphohistiocytosis (hlh) with high grade persistent fever, splenomegaly, hematologic cytopenias and hepatitis. most patients die during this acute phase, and those who survive usually evolve with hypogammaglobulinemia, recurrent infections, cytopenias, inflammatory bowel disease (ibd) and low counts of inkt cells. dysbiosis of intestinal microbiota is believed to fuel ibd and possibly contribute to the initiation and/or perpetuation of the disease. experimental studies have provided solid evidence to support a role for the indigenous gut microbiota in the pathogenesis of autoimmune diseases, thereby raising the possibility that an altered gut microbiota is an environmental risk factor for xiap disease. objectives: in this study, we sought to investigate the identity and abundance of the bacteria in gut microbial communities in a years old male patient with xiap deficiency. case presentation: at years of age, the patient presented with positive serology of active ebv infection, hlh, severe hepatitis, encephalitis and myocarditis. after recovery, the patient evolved well with few manifestations for several years. approximately years ago, the patient showed slow progression of hypogammaglobulinemia predisposing him to infections of the upper and lower respiratory system that required intravenous immunoglobulin replacement. immunological evaluation revealed reduction (but not absence) of inkt cells. at that time, the patient presented with intermittent diarrhea and abdominal pain that became more frequent and severe. after evaluation as ibd, the patients had been treated but without much improvement of diarrhea or resolution of his pain. after approximately months, the patient presented noted pain and fistulas lesions at the scrotal and gluteal regions. the exact causes of ibd in xiap deficiency are not known, but the abnormal activation of the mucosal immune system due to exaggerated response to the commensal bacteria associated to the dysregulation of nod and nod signaling might play an important role in the development and maintenance of the inflammatory status. methods: the gut bacterial composition was assessed by targeted metagenomic from the patients stool sample, collected before initiation of ibd antibiotics therapy. the s rrna amplified and its sequences were analyzed using a bioinformatic pipeline based on mothur software. we determined the bacterial community composition using , filtered reads using illumina miseq platform. results: according to the ezbiocloud database, the obtained dataset included operational taxonomic units at % dissimilarity, distributed among the following groups: bacteroidetes ( . %) with . % of b. dorei, , % b. vulgates, and . % b. fragilis, firmicutes ( . %), proteobacteria ( . %), bacteria_uc ( . %), actinobacteria ( . %). conclusions: increased abundance of bacteroides species including b. dorei and b. vulgatus have been implicated in inflammation in several gut diseases such as ulcerative colitis, irritable bowel disease, and celiac disease. although this experience is limited to a single patient, the results of the present study suggest an association between altered gut microbiota and the pathogenesis of ibd in xiap disease and may be of relevance to the future development of novel therapeutic strategies for xiap deficiency. ( ) submission id# hematopoietic stem cell transplantation in patients with primary immune regulatory disorders: a primary immune deficiency treatment consortium (pidtc) and inborn errors working party (iewp) study introduction/background: primary immune regulatory disorder (pird) is a newly recognized group of immune-mediated diseases with prominent features of autoimmunity, autoinflammation, and non-malignant lymphoproliferation in addition to immunodeficiency. the clinical manifestations of pirds are frequently difficult to manage and hematopoietic cell transplantation (hct) can be considered as a treatment option, often in those with the most severe disease. we sought to aggregate data from patients who have undergone hct for genetically defined pird or features of immune dysregulation. objectives: we sought to aggregate data from patients with pird who have undergone hct in order to determine the quantity of patients, clinical manifestations, indication and hct, and overall outcome. methods: a questionnaire based survey was sent to all primary immunodeficiency treatment consortium sites and hct referral centers in europe to determine the quantity and characteristics of patients with pirds who have undergone hct. the survey captured clinical manifestations, timing and indication of hct, strategy of hct, and outcomes from - . results: patients from centers ( in north america and in europe) were included with either known genetic defects or considered to have immune dysregulation regardless of gene defect. known genetic defects were identified in subjects, while had symptoms of immune dysregulation but lacked a genetic diagnosis. the mean age of onset of disease was years (range - years). clinical manifestations included gastrointestinal disorders ( %), failure to thrive ( %), dermatitis ( %), hematologic cytopenias ( %), and lymphoproliferative disease ( %). recurrent infections ( %), immunodeficiency ( %), autoimmunity ( %), and autoinflammation ( %) were also common. organ specific autoinflammation occurred most commonly in the lung ( %) and brain ( %). the median age of hct was years ( - years). graft sources included matched unrelated donors ( %), matched related donors ( %), mismatched unrelated donors ( %) and haploidentical donors ( %). reduced or minimal intensity conditioning was used in % of transplants. five-year overall survival was % and the majority of survivors had resolution of symptoms that led to transplantation. among those patients that died, infection was the most common cause. conclusions: based on our survey data, pird patients commonly develop clinical features of autoimmunity, autoinflammation, and susceptibility to infection at a young age. hct can be successful and lead to disease resolution. however, further studies to define the appropriate patient, timing of hct, donor selection and pre-hct conditioning regimen are necessary to improve outcomes of patients with pird. introduction/background: pathogenic variants in tnfrsf b (taci) are relatively common (found in about % of the population) but have been seen in about - % of cvid patients, interestingly in both homozygous and heterozygous states. recent articles suggest that the heterozygous state increases the risk of developing autoimmunity due to an effect on autoreactive b cell selection and activation. objectives ) illustrate the importance of genetic testing in patients with difficulty to treat inflammatory disease ) present a case report of inflammatory bowel disease associated with a pathogenic mutation in the tnfrsf b gene results: yo wf was diagnosed with crohns disease due to the chronic abdominal pain, vomiting, and bloody stools since years of age. intestinal biopsy revealed inflammatory changes in the entire intestine with active, submucosal lymphoid hyperplasia, neutrophilic cryptitis with focal areas of crypathic damage, and submucosal epithelioid granulomas more predominantly seen in the colon and rectum. she was started on immunosuppressant medications but developed anaphylactic reaction to both infliximab and adalimumab. she was then treated with azathioprine, mesalamine, methotrexate, and oral budesonide. despite these medications, she continued to have frequent relapses, - episodes a year and required periodic systemic corticosteroid bursts. other biologics, vedolizumab and ustekinumab, were also tried without success, and she subsequently underwent a colectomy. her postoperative course was complicated by ards, poor abdominal wound healing, and sepsis. due to her complicated clinical course, immune work up was performed which revealed a normal cbc and lymphocyte subpopulations, but hypogammaglobulinemia with low isohemagglutinin titers and specific antibody levels. comprehensive genetic testing ruled out chronic granulomatous disease and other known primary immunodeficiencies but revealed a rare missense mutation in tnfrsf b (taci). this variant, c t (p.cys arg) (rs , exac . %) is likely pathogenic. this heterozygous variant has been seen in both cvid cases and unaffected relatives but significantly more common among cvid patients. moreover, the studies on b cells of these relatives showed impaired function. increased number of autoreactive b cells were also found in the bone marrow of heterozygous individuals and these cells could give a risk of developing autoimmunity. conclusions: in difficult-to-treat autoimmune diseases, identifying the underlying immune defect may aid in the treatment decision. in this case, b cell targeted treatment such as anti-cd monoclonal antibody could be beneficial. introduction/background: defects in immunoproteasome caused by biallelic or digenic loss-of-function mutations in proteasome catalytic subunits cause an autoinflammatory disease identified as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (candle) associated to an increased interferon type i gene signature. the proteasome maturation protein (pomp) is a chaperone for both standard and immuno-proteasome assembly and is critical for the incorporation of catalytic subunits. here, we characterize and describe pomp-related autoinflammatory immunodeficiency disease (praid) in two unrelated patients and identify the underlying genetic mechanism of disease. objectives: determine the genetic cause and mechanism of disease in two patients with pomp variants . methods: whole-exome sequencing (wes) was performed to identify a genetic cause of our patients dysregulatory syndrome. proteasome assembly and catalytic function was assessed by sds-page and native gel respectively, using patient derived cell lines. expression of interferon type i-induced genes was measured by rt-qpcr. pomp protein was identified by western blot. results: we identified two unrelated individuals with a unique syndrome characterized by neonatal onset autoinflammation, neutrophilic dermatosis, autoimmunity, and combined immune deficiency with severe systemic viral and bacterial infections. immunologic evaluation for both individuals revealed elevated immunoglobulins, low cd + t cell numbers and extremely low b cell counts with persistently high titers of autoantibodies and increased expression of interferon type i-induced genes. in both individuals, truncating heterozygous de novo frameshift variants in pomp were identified by wes and confirmed by sanger sequencing. most mrna transcripts with premature termination codons should undergo nonsense-mediated decay (nmd), however in both of our patients, cdna sequencing revealed these transcripts escaped nmd. the expression wildtype and truncated versions of pomp protein was further confirmed by western blot. transfection of mutant constructs into an otherwise healthy cell line recapitulated an increased interferon signature suggesting a dominant negative mechanism. conclusions: we define praid in two unrelated individuals characterized by neonatal onset immune dysregulation and combined immunodeficiency caused by truncating variants in pomp in which transcripts that escape nmd result in a truncated protein that leads to a dominant negative (i.e antimorphic) allele. to our knowledge, praid is the first inherent defect of immunity mechanistically characterized by nmd escape. introduction/background: parvovirus viremia may occur in pediatric heart transplant patients who underwent thymectomy and developed secondary t cell lymphopenia. high dose intravenous immunoglobulin (hdivig) has been used to treat parvovirus infection in these cases. objectives: we aim to review different routes of immunoglobulin treatment in pediatric cardiac transplant patients with parvovirus viremia and compare the patients immunological phenotype. methods: data from three pediatric heart transplant patients with parvovirus viremia in a tertiary care center was reviewed including t cell counts, parvovirus viral load, route, dosage, and frequency of immunoglobulin treatment. results: all three patients received hdivig. patient and tolerated the treatment and viremia improved. patient developed recurrent aseptic meningitis from hdivig treatment and his viral load remained > million copies/ml. compared to the other two cases, patient had a much lower t cell count that likely contributed to the persistence of viremia. to improve his quality of life and reduce healthcare costs, a facilitated subcutaneous immunoglobulin (scig) treatment option was explored. scig treatment was well tolerated and led to a dramatic decrease in parvovirus viral loads in patient . conclusions: most pediatric cardiac transplant patients with persistent chronic parvovirus viremia respond well to hdivig, scig may serve as an alternative treatment option in refractory cases, especially in those with severe t cell lymphopenia. severe combined immunodeficiency (scid), by detecting tcell receptor excision circles (trecs) from dried blood spots (dbs) on routine newborn screening (nbs). this has lead to improved estimates of the incidence and prevalence of scid, decreased diagnostic delay, and improved patient outcomes. preliminary studies outside the us have demonstrated that nbs can be adapted to include screening for b-cell deficient infants before symptom onset by quantifying kappa-deleting recombination excision circles (krecs) on dbs. objectives: we report results of the initial characterization of a high throughput triplex trec/krec/rnasep assay run in , samples in new york state (nys). methods: dbs from anonymous, de-identified infants were included in the current study. dna from patients with confirmed primary immunodeficiencies (n= ), including, but not limited to, x-linked agammaglobulinemia (n= ) and scid(n= ) were obtained from the centers for disease control and prevention, and clinical immunologists working with the nbs programs in massachusetts, minnesota, wisconsin, and nys, were used as positive disease controls. all dbs were extracted and processed according to current nys nbs protocols. a trec/krec/rnasep triplex assay was designed and optimized to minimize reagent use, and maximize target amplification. cycle threshold (ct) was determined, and error detection cutoffs were identified to optimize sensitivity. results: pcr efficiency, assay quantification, intra-assay reproducibility, and error detection rates all met nys nbs standards. error detection rate for the triplex trec/krec/rnase p assay is %, comparable to the current error detection rate of % for the current duplex trec/rnase p assay. samples falling into the error detection range are repeated (analysis in process) to determine a receiver operating characteristic curve. conclusions: we show that the high-throughput trec/krec/rnase p triplex assay is feasible in a large, racially and ethnically diverse population in nys. compared to the current duplex assay, this assay has favorable performance characteristics and provides additional immunologic characterization. due to assay optimization, we were able to add the krec test at no additional cost. work is underway to further characterize other assay parameters such as sensitivity and specificity, in preparation for adoption of the triplex assay as part of routine nys nbs. highly accurate wiskott-aldrich syndrome diagnosis via rapid flowbased was protein staining samuel chiang , sue vergamini , ammar husami , marianne ifversen , kejian zhang , jack bleesing, md, phd , yenan bryceson , rebecca marsh, md introduction/background: wiskott aldrich syndrome (was) is a rare xlinked hemizygous disease commonly associated with symptoms of immune deficiency. diagnosis is based on clinical parameters including thrombocytopenia and reoccurring infections, but currently does not include any disease specific marker. as the only permanent treatment for was is hematopoietic stem cell transplantation, it is imperative that a swift and accurate diagnosis be made. absent or lowered was protein (wasp) levels have been reported in sporadic was cases. however, no systemic evaluation exists to date on the accuracy of wasp quantification for was diagnosis. objectives: to determine the accuracy of wasp staining in predicting was genetic abnormalities. methods: we retrospectively evaluated results from a rapid whole blood flow cytometry based assay on a cohort of suspected was patients and compared relative wasp staining levels to was genotype. roc curves as well as accuracy calculations were generated. results: a total of patients with normal and patients with a genetic abnormality in was were collected. missense mutations were most common but insertions, deletions, and gross mutations were also found ( fig a) . comparing was sequencing results to whole blood wasp expression levels provided an . % sensitivity and % specificity for a combined accuracy of . % when juxtaposed against genetic sequencing. when variants of unknown clinical significance (vucs) were removed, the sensitivity improved to . % (fig b) . conclusions: staining for wasp is a quick, simple, and accurate assay for the prediction of genetic was defects. introduction/background: cytotoxic t lymphocyte antigen (ctla ) is an inhibitory co-receptor essential for regulatory t cell (treg) function and a central regulator of t cell proliferation and expansion. ctla haploinsufficiency is a recently described autosomal dominant disease, in which heterozygous ctla mutations result in severe immune dysregulation with variable age of onset and a wide array of clinical manifestations. herein we describe atypical findings in a patient with a novel pathogenic variant in ctla . objectives ) to understand whether the novel ctla variant identified is pathogenic and ) to describe eosinophilic gastrointestinal inflammation and exocrine pancreatic insufficiency as possible manifestations of ctla haploinsufficiency. methods: next generation sequencing (blueprint genetics ©) was used to identify the ctla variant. polyphen and sift (blueprint genetics ©) were used for in silico analysis for the prediction of the effect of this genetic variant on protein structure/function. flow cytometry was used to evaluate ctla expression of regulatory t cells. results: a -year-old boy with type diabetes mellitus and autoimmune thyroiditis presented with abdominal pain, diarrhea, and weight loss. initial studies revealed markedly elevated peripheral blood eosinophils (> cells/μl) and exocrine pancreatic insufficiency (< μg elastase per gram of stool). prominent eosinophilic inflammation was appreciated in biopsies of the stomach, duodenum, jejunum, and terminal ileum. no parasitic infection or inciting drug/food trigger was identified. additional blood studies revealed normal total quantification of t cells but with increased memory t cells ( %, cd +cd +cd ro+) and decreased treg ( %, cd + cd +cd hifoxp hi). b cell quantification and serum immunoglobulin (ig) levels were unremarkable, save a modestly elevated ige level ( iu/ml). comprehensive next-generation sequencing of genes associated with primary immune deficiency revealed a novel heterozygous missense mutation (c. g>a, p.asp asn) affecting the last nucleotide in the ligand binding domain (exon ) of ctla . subsequent analyses revealed decreased ctla expression in the patients t cells compared to healthy controls as well as evolving hypogammaglobulinemia. treatment consisted of methylprednisolone and parenteral nutrition followed by sirolimus and abatacept to which the patient responded favorably. conclusions: we report a -year-old boy with a history of type diabetes mellitus and autoimmune thyroiditis presenting with hypereosinophilia, eosinophilic gastroenteritis, and exocrine pancreatic ins uff iciency as unique m anifestat ions o f ctla haploinsufficiency. although not previously reported in individuals with ctla haploinsufficiency, peripheral blood eosinophilia and eosinophilic inflammation of the gastrointestinal tract have been observed in patients receiving ipilimumab (ctla blocking antibody) suggesting a potential mechanism for the aforementioned findings. severe exocrine pancreatic insufficiency is a rare but observed manifestation in individuals with type diabetes mellitus. whether severe exocrine pancreatic insufficiency would be expected t o o c c u r m o r e f r e q u e n t l y i n i n d i v i d u a l s w i t h c t l a haploinsufficiency and type diabetes mellitus is unclear; however, our reported case and surveillance of others with ctla haploinsufficiency could elucidate incidence and prevalence of this manifestation. introduction/background: mild asymptomatic hypogammaglobulinemia during pregnancy is a well-described phenomenon due to hemodilution. in patients with known humoral primary immunodeficiency such as common variable immunodeficiency, women require an upwards titration of their immunoglobulin replacement dose. isolated symptomatic hypogammaglobulinemia during pregnancy in a patient is not well described in the literature. objectives . understand the physiology of igg during pregnancy. . define a rare entity with a likely genetic predisposition that manifests as hypogammaglobulinemia isolated in pregnancy. . management of this entity with defining goals of treatment. results: year old gravida para female presented with progressive hypogammaglobulinemia restricted to pregnancy starting with rd pregnancy, recurrent otitis media requiring sets of myringotomy tubes, recurrent sinusitis, and streptococcal pharyngitis. her son has common variable immunodeficiency (cvid) requiring immunoglobulin replacement (igrt). prior to conception, her igg was mg/dl. during th week of pregnancy, her igg level was mg/dl. during weeks of pregnancy, she complained of fatigue, and developed an episode of sinusitis that required different antibiotic treatments. at that time, her igg was mg/dl. she was started on subcutaneous igrt maintain igg troughs of > mg/dl. she remained infection-free during her pregnancy and igrt was stopped a few months after delivery with her serum igg level returning to pre-pregnancy levels. patient was initially evaluated during her rd pregnancy with recurrent streptococcal pharyngitis. at that time, her igg was mg/dl. diphtheria, haemophilus influenza, mumps, measles and rubella titers were protective, tetanus titer was non protective with / antipneumococcal titers protective. she was vaccinated with pneumovax and tdap with development of protective titers and remained infectionfree. igrt was not given and post-delivery, her igg levels improved to mg/dl. she was seen one and half years later, for prenatal counseling for her th pregnancy. her immune evaluation included an igg . she demonstrated protection to tetanus, diphtheria and streptococcal pneumoniae. at weeks of gestation, she developed recurrent upper respiratory infections requiring antibiotics. her igg was mg/dl. at weeks of gestation, her igg was mg/dl. igrt was recommended, but patient refused at that time. after delivery, igg improved to mg/dl. conclusions: decreased immunoglobulin levels during pregnancy are welldescribed phenomenon which can be attributed to hemodilution of pregnancy. igg transport to fetus generally begins in the second trimester and reaches its pinnacle in the third trimester. patients with known cvid require dose adjustments (higher) during pregnancy as they can be more symptomatic during this time. here we describe a patient who has an almost normal immune evaluation except for mildly low iga during absence of pregnancy, but during pregnancy, develops recurrent infections with significantly low igg level. her family history of a son with cvid, new daughter with low igg levels like to be transient hypogammaglobulinemia of infancy (thi), another daughter with thi suggests a genetic b-cell defect that manifests as cvid with mildly low iga and hypogammaglobinemia during metabolic stress such as pregnancy. there is only one similar report of a single pregnancy of transient symptomatic hypogammaglobulinemia during pregnancy. such patients should be adequately worked up and treated during pregnancy with igrt to decrease maternal and fetal mortality. introduction/background: card encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nf-b, jnk, and mtorc . germline mutations in card are known to give rise to distinct primary immune disorders in humans, including scid (null mutations), b cell expansion with nf-b and t cell anergy (benta; gain-of-function mutations), and severe atopic disease (loss-of-function, dominant interfering mutations). objectives: here we report our experience with an expanded cohort of patients harboring novel heterozygous card mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with card mutations. methods: cell transfections and primary t cell assays were utilized to evaluate signaling and function of card variants. results: we demonstrate that in addition to severe atopy, heterozygous missense mutations in card associated with dominant negative activity can present with immunologic phenotypes similar to those observed in stat lof, dock deficiency, common variable immune deficiency (cvid), congenital neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. evaluation of rare or novel card variants found in affected patients showed that dominant negative activity was largely confined to the card or coiled-coil domains, but did not always manifest in atopic disease. conclusions: these results illuminate a broader phenotypic spectrum associated with card mutations in humans, and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity. introduction/background: increasing number of states have been screening for severe combined immune deficiency (scid) as part of the expanded newborn screening program for nearly a decade. in the era of newborn screening, patients with scid present often asymptomatically and are prepared for early hematopoietic stem cell transplantation (hsct). with advances in genetic testing, mutations in over genes have been associated with development of the scid or leaky-scid phenotype. objectives: to present unique cases of hypomorphic x-linked scid where no known pathogenic mutations were identified on initial genetic testing, the il r gamma-chain as protein was expressed, but subsequent testing months later revealed pathogenic il rg mutation affecting either translation or protein function. to expedite earlier identification of pathogenic il rg mutation, we propose screening with x-inactivation studies in maternal t lymphocytes and assessing gamma-chain function by evaluating il r signaling in select male infants with abnormal newborn screen for scid, specifically those with scid phenotype but no identified pathogenic gene mutation on initial genetic testing, and the presence of gamma-chain expression. male patient a presented during the first week of life after his newborn screen was found to be abnormal with undetectable t cell receptor excision circle (trec) count. a phenotype of t-b+nk-scid was established and the patient was sent for bone marrow transplant evaluation. genetic testing revealed a novel hemizygous missense mutation in il rg (p.glu gln), which was a variant of unknown significance. the patient had common gamma-chain expression by flow cytometry on b and nk cells. he underwent haploidentical hsct with his father as donor. unfortunately, his transplant was complicated by prolonged neutropenia, slow t cell reconstitution, and eventual graft failure. to review his next treatment options, it was necessary to prove that his il rg mutation was pathogenic. male patient b presented with concern for an underlying immune deficiency after being hospitalized for peumocystis jirovecii pneumonia. his newborn screening for scid was inadequate at birth and follow up was delayed. retrospective analysis of the newborn screening card at birth confirmed absence of trecs. his phenotype was also t-b+nk-, consistent with x-linked scid. he also proceeded to hsct with a haploidentical parent donor. despite hhv- viremia pre-hsct which persisted posttransplant, he has had appropriate t cell engraftment. comprehensive genetic testing on whole exome level did not reveal any known mutations contributing to his phenotype. patient did have expression of gamma-chain by flow cytometry on t, b and nk cells. however, further testing revealed an il rg utr deletion of aa that, based on similar findings from a prior study can possibly lead to mrna abnormalities. to expedite the association of scid phenotype with x-linked disease, implying gamma-chain pathology, we obtained x-inactivation studies in maternal t-cells that showed severe skewing in both cases. furthermore, il r signaling was impaired on b-cells in each case. the combination of these two assays proved that both patients carry a pathogenic il rg mutation. conclusions: in the era of newborn screening for scid, we are discovering that phenotypic variability of scid patients can be very broad and caused by hypomorphic mutations in the common chain gene. exonbased genetic testing cannot exclude all variants and novel variants of unknown significance have to be evaluated by additional assays, including functional studies for causal effect. it is important to expedite early proof of association with gamma-chain pathology, especially in the era of gene therapy. we propose that in male infants with abnormal scid newborn screening and no known or previously described pathogenic mutation on genetic screen, evaluation continues for hypomorphic il rg mutations. the probability of this process can be increased by a simple screening test for x-inactivation of maternal t lymphocytes. allergy / immunology, allergy / immunology associates inc. / case western reserve university introduction/background: igg -related disease (igg -rd) is an immunologic disorder with multiple clinical presentations previously thought unrelated. it is characterized by the frequent presence of tumor-like swelling of the affected organs and several histopathological findings including tissue lymphoplasmacytic infiltrates with predominantly igg -positive plasma cells and lymphocytes, storiform fibrosis and obliterative phlebitis. humoral immunodeficiency is a term that encompasses several disease entities associated with impaired antibody production. it is suspected in patients who present with recurrent, frequently severe, sinopulmonary infections with encapsulated bacteria, which leads to evaluation of quantitative immunoglobulin levels and vaccine responsiveness. despite a few reports of primary immunodeficiency in patients with serum igg elevation, no adult case has been reported of igg -rd in a patient with concomitant humoral immunodeficiency. objectives: to present a unique case with the presence of concomitant igg -related disease and humoral immunodeficiency. methods: comprehensive chart review of our patient and all performed exams. literature review for igg -related disease, igg elevation in humoral immunodeficiency and concomitance of igg -related disease with humoral immunodeficiency. results: our patient is an -year-old caucasian male with relevant past medical history of chronic bronchitis who was referred to our practice after several episodes of pneumonia in the previous years, with six courses of antibiotics just in the year prior for recurrent sinopulmonary infections. blood tests revealed hypergammaglobulinemia and low level of vaccine responsiveness. chest ct showed multiple bilateral pulmonary nodules and hilar and mediastinal lymphadenopathy. sinus ct showed left maxillary sinus opacification. pulmonary function testing was normal. he later presented with left eye edema, proptosis, diplopia, and painless submandibular salivary gland enlargement. laboratory investigation showed an igg of mg/dl, igg of mg/dl. the patient denied any history of pancreatitis or abdominal pain and abdominal ultrasound was normal. biopsy of a salivary gland was normal. mri of the left orbit was obtained, showing lacrimal gland enlargement. based on the patients recurrent infections, lack of response to tetanus immunization, and limited, non-sustained response to pneumococcal immunization, the patient was started on ivig therapy. the patient was also diagnosed with possible igg -rd based on his salivary gland enlargement and orbital disease in association with hypereosinophilia and increased plasmablast levels. oral prednisone mg daily was started for four weeks, later followed by slow steroid taper (reducing mg every two weeks) with considerable improvement in left eye swelling and proptosis. a few months after discontinuation of the steroids, the orbital disease returned to its previous severity. left lacrimal gland biopsy confirmed igg -related disease, with many areas showing greater than one hundred igg -positive plasma cells per high-power field. after another course of steroids, oral prednisone was weaned to a maintenance dose of mg daily and the patient became asymptomatic from his ophthalmologic complaints with normalization of his ophthalmologic exam. his last checked igg was mg/dl. igg was still elevated at . mg/dl , but given controlled symptoms the patient was spaced to monthly ivig infusions and continued on that daily steroid dosage. conclusions: our patient, initially diagnosed with a humoral immunodeficiency, was later also diagnosed with biopsy-proven igg -related disease, which is a novel association of this two diseases in an adult patient. previous rare reports of association between elevated serum levels of igg and patients with concomitant humoral immunodeficiency were in the presence of isolated igg elevation and not in the presence of igg related disease. this novel association creates a therapeutic dilemma since the patient in question is hypergammaglobulinemic, yet needs ivig, which can lead to side effects such as thrombosis due to a hyperviscous state. the description of additional concomitant cases of both diseases and further understanding of their pathophysiology will be crucial to create awareness and obtain earlier diagnosis, to refine therapeutic options and design adequate treatment protocols. igm and iga anti-pneumococcal capsular polysaccharides as prognostic tool for common variable immunodeficiency: a longitudinal study. professor, sapienza university of rome introduction/background: the clinical spectrum of cvid ranges from a poorly symptomatic form to severe phenotypes characterized by high susceptibility to infections, autoimmunity, granulomatous inflammation, lymphoproliferative disorders, and malignancies. due to high prognosis heterogeneity, prognostic factors are required. objectives: with the aim to identify additional prognostic factors, we evaluated the anti-polysaccharide iga and igm responses by elisa assay in cvid in a longitudinal study over a -year period. methods: patients were immunized at baseline with the -valent pneumococcal polysaccharide vaccine (pneumovax®). twenty healthy donors (hd) were also included. results: as expected, cvid patient had lower igm/iga response than hd. for cvid, four immunological phenotypes were identified by postvaccination igm and iga levels: igm and iga responders ( %), igmhigh responders ( %), igm-low responders ( %) and non-responders ( %). to simplify, we analysed igm-high group with igm and iga responders and igm-low with non-responders. during the follow up, concomitant cvid-related conditions, immunoglobulin serum levels, respiratory infections and outcome were recorded by medical files. cvid igm-low/non-responders developed more frequently respiratory, gastro enteric and autoimmune manifestation and malignancies in comparison to igm-high/igm and iga responders (respectively, pneumonia: % vs % ; chronic diarrhea: % vs %; autoimmunity % vs %). autoimmune cytopenias were not found in the igm-high/igm and iga responders group. eleven ( %) patients died during the study time. survival analysis according to the igm/iga responder status showed that the -years estimated survival for igm-high/igm and iga responders vs igm-low/non-responders group was respectively: % vs %, % vs %, % vs %, % vs %, % vs %, % vs %, % vs %. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. conclusions: in conclusion, even if patients could not raise the protective humoral level, in cvid the anti-polysaccharide iga and igm responses could represent a prognostic factor, individuating groups of patients with less immunological impairment, lower risk of comorbidities and better survival. introduction/background: gaucher disease (gd) is a rare autosomal recessive disorder characterized by a defective function of the catabolic enzyme -glucocerebrosidase (gba) leading to a progressive accumulation of its substrate-glucocerebroside (gc) -in various organs in particular in mononuclear phagocite system. hepatosplenomegaly and cytopenia represent the most common features of the disease. moreover, gd patients also show hyperinflammatory features -secondary to machrophages engorgement and actviation-hypergammaglobulinemia, and a immune-dysregulation involving b , t and nk cells. since clinical phenotpye can be subdolous, symtoms can overlap with alps, however, few data are available on specific immunity pattern in these patients. objectives: to evaluate immune-phenotype and other alps parameters in a cohort of patients with gd methods: we evaluated lymphocytes subsets, immunophenotypic and serological features of alps (dnts, tcr alfa/beta b , b-memory cells, tregs/hla-dr ratio, il- , il- ), and test of apoptosis in a cohort of patients with gd followed-up at igg. results: patients ( in treatment, not) were studied. dnts and tcr alfa/beta b + resulted to be > . % of t-lymphocytes and > % in / ( %) and in / ( %), respectively. b-memory cells and t-regs/hla-dr ratio were < % and < in / patients ( %). / evaluable ( %) had all these parameters concomitantly alterated. / ( %) evaluable patients were resistant to apoposis. il- was pathological in / ( %) patients. all patients had normal levels of il- and sfas. conclusions: this study shows that some patients with gd may present an immune-dysregulation pattern that can overlap with alps features. therefore, the differential diagnosis of gd should be taken into consideration by clinicians during diagnostic work-up of patients with an alpslike phenotype. introduction/background: allogeneic hematopoietic stem cell transplantation (hsct) using unrelated and haploidentical donors is complicated by increased rates of graft-versus-host disease (gvhd) and slow immune reconstitution. selective depletion of alpha/beta t lymphocytes and b cells is a recently developed method of graft manipulation that retains mature natural killer (nk) and gamma/delta t lymphocytes, both of which may exert a graft-versus-leukemia effect and protection against life-threatening infections. objectives: to describe the rate and quality of immune reconstitution, incidence of transplant-related complications, including viral reactivation and gvhd, and overall outcomes following tcr-alpha/beta-and cd depleted hsct for hematologic malignancy in pediatric patients. methods: forty patients of median age . years ( . - . ) underwent hsct for acute myeloid leukemia (n= ), acute lymphoblastic leukemia (n= ), and myelodysplastic syndrome (n= ). grafts were from unrelated (n= ) and haploidentical (n= ) donors. tcr-alpha/beta and cd depletion was performed with the miltenyi clinicmacs plus system. median cd + cell dose was . x /kg ( . - ), and median cd + cell dose was . x /kg ( . - . ). conditioning was with myeloablative busulfan or total body irradiation, cyclophosphamide, and thiotepa. twenty of unrelated donor hscts and / haploidentical hscts also included antithymocyte globulin x . no patient received post-transplantation gvhd prophylaxis. all but patients received rituximab on day + per protocol for recipient positive epstein barr virus (ebv) serology. results: all patients engrafted. median time to neutrophil engraftment was ( - ) days, and median time to platelet engraftment was ( - ) days. one patient experienced graft rejection on day + , and twelve patients relapsed at a median of ( - ) days. overall survival was / ( %) at a median of . ( . - ) months follow-up. two ( %) patients developed grade iii or higher acute gvhd, and ( %) patients developed extensive chronic gvhd. cumulative incidence of cytomegalovirus (cmv) and adenovirus reactivation were / ( . %) and / ( . %), respectively. nine ( . %) patients developed bk hemorrhagic cystitis +/-viremia. ebv reactivation was not observed. median total, myeloid, t cell, and b cell donor chimerism were all % (ranges - %, - %, - %, and - %) at year post-hsct. immune reconstitution of all cells lines was rapid ( table ) . eighteen of ( %) patients had detectable t cell receptor excision circles (trecs) by months with a median trec count of ( - ) per ^ cd t cells, and recovery of the naïve t-cell compartment was observed by months in / ( %) of patients. t cell function as measured by response to pha was normal by months in / ( %) patients and continued to increase steadily with time. despite rituximab on day + for / ( . %) patients, there was rapid b cell reconstitution. nineteen of ( . %) patients had present switched memory b cells at months, and / ( . %) surviving patients are off immunoglobulin replacement at a median of ( - ) months. conclusions: selective tcr-alpha/beta and cd depletion of haploidentical and unrelated grafts results in high engraftment and rapid immune reconstitution with low incidence of gvhd in children with hematologic malignancy. cd depletion and routine post-hsct rituximab on day + are effective at preventing ebv reactivation. introduction/background: hyper-igd syndrome (hids; ) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal disturbance, and skin rash. the diagnostic hallmark of hids is a constitutively elevated level of serum immunoglobulin d (igd), although patients have been reported with normal igd levels. the disease is associated with mutations in the gene of mevalonate kinase. objectives: male patient, years old, born to non-consanguineous parents, with a history of severe diarrhea since childhood, followed by respiratory infections and pneumonias. moreover he presented several episodes of severe abdominal pain, with intestinal obstruction since seven months old, needing surgical intervention due to acute abdomen. this picture repeated several times until years of age, being submitted to new surgeries due to intestinal suboclusion. at and years he had gastroenteritis and then pancreatitis. years ago new severe acute gastroenterocolitis with suboclusion, submitted to laparotomy and resection of a little part of the gut. he has frequent diarrheas, triggered by coffee and tea. usually evacuates times a day. he was evaluated by several pediatric immunologists in childhood, who distrusted alpha heavy chain disease. he had an intense reaction to the bcg vaccine, and then did not make any more vaccines (only opv (sabin) in the campaigns). he was tonsillectomized at years of age. he had measles, chickenpox, and mumps (at years old). cellulitis at years, repeating several times since then. he presented improvement of pneumonias but still has sinusitis and otitis (approximately times a year introduction/background: pitthopkins syndrome is a rare neurological disorder caused by mutations the tcf gene on chromosome q . clinical features include severe intellectual disability, constipation, microcephaly, and seizures. features distinguishing it from other neurodevelopmental syndromes such as rett syndrome and angelman syndrome include breathing abnormalities (either apneic episodes or hyperventilation) and atypical facial features. typical facial dysmorphism includes bitemporal narrowing, deep-set eyes, an m shaped upper lip, and widely spaced teeth. although a very rare diagnosis (slightly over reported cases), it is not known to be associated with underlying humoral or cellular immunodeficiency. there is only one report of igm abnormalities described in a patient with pitt-hopkins syndrome. objectives: to present a case of pitthopkins syndrome with humoral immunodeficiency. methods: this is a case presentation of a patient with pitt-hopkins syndrome requiring immunoglobulin replacement therapy. results: year old female with genetically diagnosed pitt-hopkins syndrome who presented to our office for immunological evaluation in the setting of recurrent sinopulmonary infections. she was placed on chronic antibiotics by the department of otholaryngology for approximately two years prior to presentation. she was found to be hypogammaglobulinemic (igg: mg/dl, iga: mg/dl, igm: mg/dl), had non-protective titers to streptococcus igg antibody ( / titers protective greater than . mcg/ml) despite booster vaccination with pneumovax, and had borderline tetanus ( . iu/ml) and diphtheria titers ( . iu/ml). given this laboratory evaluation and her recurrent illness, immunoglobulin replacement therapy (igrt) was started. whole exome sequencing was completed to assess for any other genetic cause of her immunodeficiency. the only abnormality was her previous known pathologic variant p.q hfsx c. _ delga (gln his) in exon in the tcf gene. she continued to have infections despite therapeutic igrt. chronic antibiotic treatment was initially tapered, however needed to be reintroduced as ivig alone was not stopping her infections, despite a igg level over mg/dl. conclusions: humoral immunologic deficits are not known to be associated with pitt hopkins syndrome. there has been one case report of a patient with poliomyelitis-like syndrome following an asthma attack in a patient with pitt hopkins syndrome, which was treated with igrt and resulted in a nearly complete recovery. however, igrt was not used for reasons of underlying immunodeficiency. to our knowledge this is the first patient with pitt hopkins syndrome with persistent hypogammaglobulinemia and frequent infections requiring immunoglobulin replacement therapy. it remains unclear why the patient continued to have infections despite igg levels > mg/dl, yet with the combination of igrt and prophylactic antibiotics the patient remains healthy. introduction/background: gata deficiency is a rare disease that typically presents in late childhood or early adulthood with heterogeneous phenotypes including emberger syndrome. emberger syndrome is characterized by lymphedema and predisposition for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). over % of patients with gata mutations have immune deficiencies. definitive diagnosis of gata deficiency is made by gene sequencing, and treatment includes infection control and potentially hematopoietic stem cell transplant (hsct). objectives: the goal of this report is to contribute data to the small documented cohort of patients with gata deficiency to aid in diagnosis and management of this rare, heterogeneous disorder. methods: we present a case series of three siblings with identical gata mutations with variable phenotypes. a usidnet query resulted patients with gata mutations. results: three siblings ( year-old female (a), year-old male twin (b), and year-old female twin (c)) and their mother had congenital deafness. clinical symptoms include (a) h n influenza requiring mechanical ventilation, warts, and hypogammaglobulinemia, (b) streptococcus pyogenes neck abscess, warts, acne, and mds, and (c) lymphedema and acne. all three patients had absolute monocyte count (amc) of and lymphopenia without documented lymphoid cell dysfunction. a mutation on exon , c. delg, confirmed the diagnosis. all three patients were started on mycobacterial prophylaxis with azithromycin and recommended hpv vaccination. the usidnet query average age of symptom onset was years, and average age at diagnosis was . years. . % ( / ) of patients had a family history of gata deficiency, % ( / ) of patients had warts, . % ( / ) had lymphedema and only patient had sensorineural deafness. % ( / ) of patients had amc of . functional data was limited. conclusions: life threatening infections as well as hematologic malignancies have been reported in patients with gata deficiency, which can be successfully treated with hsct. to our knowledge, the gata mutation detected in this family has not been previously reported. the clinical presentation in these three patients was heterogeneous despite identical genotypes, and diagnosis occurred years after initial symptoms. variable phenotypes were found in the usidnet gata deficiency cohort as well. a high index of suspicion for the disorder and early recognition of clinical manifestations and laboratory abnormalities may aid in timely diagnosis of gata deficiency, with potential for improved outcomes. consulting medical advisor, immune deficiency foundation introduction/background: as individuals with antibody deficiencies age they are susceptible to developing shingles. patients with antibody deficiencies are advised not to receive live viral vaccines such as zostavax, the shingles vaccine. objectives: our survey aimed to determine the frequency of shingles and use of zostavax in common variable immunodeficiency (cvid) and hypogammaglobulinemia patients. methods: , email invitations delivered to members of the immune deficiency foundation database requesting participation in an online survey about zoster, influenza and varicella experiences. data from individuals age years old or older with cvid (n= ; mean age years old; % female; % white non-hispanic) or hypogammaglobulinemia (n= ; mean age years old; % female; % white non-hispanic) were analyzed. results: close to one fifth ( %, n= ) of adults age or older with cvid or hypogammaglobulinemia (n= ) had received shingles vaccination. the majority of those who were vaccinated reported receiving zostavax once ( %, n= ), while % (n= ) received a booster vaccination as well. mild side effects (e.g., skin rash and muscle pain) were only reported by % (n= ) of vaccine recipients after receiving their first vaccination. no side effects were reported after receiving the booster vaccination and no hospitalizations were reported as a result of receiving zostavax. when comparing shingles diagnosis and shingles vaccination, of the adults age years old and older, % (n= ) had been diagnosed with shingles, and of those diagnosed % (n= ) did not receive zostavax. of adults age years old or older, % (n= ) reported a shingles diagnosis. similarly, the cdc reports almost % people in the united states will develop shingles during their lifetime. more than half ( %, n= ) of those ever diagnosed with shingles reported experiencing shingles once, % (n= ) had shingles twice and % (n= ) had shingles three or more times. respondents with more than three shingles episodes were more likely to report their rash lasted more than two months and their blisters became infected. conclusions: almost % of adults with cvid or hypogammaglobulinemia reported receiving zostavax despite recommendations against vaccinations for immunodeficient individuals. however, side effects in those pi patients who received the shingles vaccine appears minimal. though it is possible these individuals were vaccinated prior to diagnosis of their pi; additional patient and physician education on live vaccines and immunodeficiency may be needed as well. the approval of the new non-live virus component varicella zoster vaccine may be of benefit to patients with pi. introduction/background: inclusion body myositis (ibm) is a rare disorder characterized as an inflammatory myopathy with endomysial inflammation and numerous red-rimmed vacuoles seen on biopsy. five cases of ibm have been described in the literature in patients with common variable immunodeficiency (cvid). objectives: to make immunologists aware of ibm as a complication of cvid, that may be incorrectly diagnosed as myositis or autoimmune neuropathy. methods: case description results: we report a -year-old man with common variable immunodeficiency on gammaglobulin replacement who presented complaining of progressively worsening lower extremity pain, weakness, and fatigue. he states that over the last couple of years, it has become difficult climbing and descending steps, arising from a seated position, and has begun experiencing frequent falls. his creatinine kinase level was found to be elevated at u/l and he continued to be lymphopenic with total lymphocyte counts ranging from to k/ul (cd + t-cells %, cd + t-cells %, cd + bcells %). his esr ranged from to mm/h. anti-glutamic acid decarboxy antibody (gad) was initially elevated at . u/ml and rose to . u/ml within months suggestive of a neuropathy. based on an electromyography (emg) and a muscle biopsy, he was diagnosed with polymyositis. he was treated with high dose steroids with no improvement. his intravenous gammaglobulin dose was then increased from mg every weeks to mg per day for straight days every weeks. months later, his creatinine kinase level dropped into the normal range (< u/l), however, he continued to complain of worsening weakness. physical exam showed decreased muscle bulk in forearms and quadriceps bilaterally, lack of a quadriceps tendon reflex, strength / in flexor digitorum profundus and / in hip flexors, and a broad-based gait. histology and electron microscopy of a repeat muscle biopsy identified rimmed muscle vacuoles as typically noted in inclusion body myositis. conclusions: inclusion body myositis is a potential rare complication of common variable immunodeficiency. it can mimic polymyositis and inflammatory demyelinating disorders. high dose steroids and ivig are of no clinical benefit in ibm, despite decreasing serum creatinine kinase levels, which may raise a false impression of a clinical benefit. objectives: in this abstract we present the case of the development of generalized cmv infection in a child with scid. girl n. at the age of months entered the children's infectious clinical hospital with complaints of cough, high febrile temperature for days, refusal to eat. from the anamnesis of life the girl from the st pregnancy, birth, was born full term in weeks gestation, birth weight g. for months of life, a bad increase in body weight was noted and at the time of admission, the weight in months was g. according to the parents, the child had atopic dermatitis. from the anamnesis of the disease on . . , the temperature rose to . °c, there was a cough and a mucous discharge from the nose. then the child refused to eat, the body temperature rose to . °c. at this time, the girl's mom was borne by the ari. january , patient was hospitalized in the hospital with a diagnosis: acute respiratory viral infection, acute rhinitis, pharyngitis, acute bronchitis, toxicosis of - degrees. acute pneumonia? atopic dermatitis, infant form. on january , , due to the worsening of the condition associated with the increase in oxygen (o ) -dependence, the child was transferred to the department of anesthesiology and resuscitation. methods: in the general analysis of blood upon admission, leukocytes are . x / l, hemoglobin is g / l, platelets are x / l, esr is mm / hour, stabs are % (abs. - . x / l), segmented - % (abs- . x / l), lymphocytes - % (abs . x / l), monocytes - % (abs - . x / l). in a biochemical study, the total protein is g / l, total bilirubin is . mol / l, urea is . mmol / l, creatinine is mol / l, lactate dehydrogenase is u / l, alt is u / l, asat - e / l, crp - . mg / l. radiography of the lung from / / -data in favor of interstitial pneumonia. the study of the acid-base ph state is . , pc is . mmhg, po is . mmhg, lactate is . mmol / l. a blood test was performed using the elisa and pcr method for markers of hsv, cmv, enterovirus and toxoplasmosis. ultrasound of the abdominal cavity revealed moderate hepatomegaly, signs of thickening of bile, splenomegaly. moderate diffuse changes in the renal parenchyma (toxic-inflammatory?). the minimum amount of free fluid in the abdominal cavity. ultrasound of the brain revealed signs of subependimal microcyst on the right. according to the immunogram, a sharp decrease in cd + % ( - %) was detected, activated t-lymphocytes (cd + hla-dr +) were . % ( - %), t helper / inducers (cd + cd - . % ( - %) and t suppressors / cytotoxic (cd + cd -) . % ( - %), a high ratio of tx / tc (cd + cd +) was detected . % ( . - . ), cytotoxic non-t cells (cd -cd +) - , , an increase in the number of b-lymphocytes (cd +) - . % ( - %), natural killers (cd + cd +) - . % ( - %), natural t-killers (cd + cd + cd +) - . ( - %), leukocyte gates (cd + cd -) - % ( - %). the absolute content of t-lymphocytes was . x / l, b -lymphocytes - . x / l. the number of thymic migrants (cd + cd ra + cd +) was not detected ( %). according to the results of the immunogram the diagnosis is made: severe combined immunodeficiency (t-b + nk +). / / ct scan of the chest was diagnosed ct signs of a polysergic two-sided inflammatory process in the lungs (figure ). when blood was sown for sterility on january , , staphylococcus epidermidis was isolated in an amount of , sensitive to linezolid, gentamicin resistant to amoxicillin, amoxicillin / clavulonic acid, and ciprofloxacin. on january , , cmv dna was detected in an amount of . × copies / ml. results: since the arrival clarithromycin was administered at a dose of mg / kg per day in divided doses from / / to / / . from / / to / / . change of antibacterial therapy for azithromycin intravenous at a dose of mg / kg per day once a day. . . - / / the state of the child is very severe with negative clinical and laboratory dynamics despite the ongoing therapy. antibacterial therapy was changed to meropenem in a dose of mg / kg intravenously every hours, linezolid at a dose of mg / kg per day and oseltamivir at a dose of mg / kg per day from / / to / / . . . - . . substitution therapy with an octagam in a dose of . g / kg was intravenously dripped. the patient's condition without significant dynamics. based on the results of pcr on cmv, ganciclovir was administered at a dose of mg / kg intravenously drip times a day. / / due to a decrease in platelet count, the platelet mass is transfused and there was a rash all over the body at night, which is associated with the development of the "graft versus host" reaction (gvhr). despite the ongoing therapy, a fatal outcome occurred. the main diagnosis: primary immunodeficiency (severe combined immunodeficiency, t b + nk +). complications: sepsis. septic shock. spon: ards, renal failure, dis, thrombocytopenia, anemia . two-sided lower-lobe pneumonia. generalized cmv infection. gvhd, acute dermal form. concomitant: atopic dermatitis, infant form. conclusions: the peculiarity of the described clinical case was that the patient's first symptoms of scid developed in the first months of life and were manifested by a bad weight gain, atopic dermatitis and the development of a life-threatening generalized cytomegalovirus infection with the development of bilateral low-grade pneumonia, respiratory insufficiency and acute cutaneous gvhd form, after transfusion of unirradiated platelet mass. an expanded immunological study confirmed the diagnosis of scid. methods: this study included patients ( boys and girls) aged months to years. the reasons for entering the hospital were manifestations of severe hepatitis (in children), acute respiratory infection ( children) and patient with symptoms of infectious mononucleosis. all patients were examined according to clinical protocols and given the severity and atypicality of the course of any infectious diseases, patients underwent immunological examination of the blood and they were consulted by an immunologist. all children were diagnosed with congenital immunodeficiency. results: in cases, the trigger for the realization of the immunodeficiency state was infection (e. meningoseptica + kl. pneumonia + b. pertussis; cmv; veb); in patients, giant cell hepatitis occurred. in patients, despite the ongoing therapy, the disease had an unfavorable (lethal) outcome ( patient with hepatitis and patient with generalized cmv infection). conclusions: thus, it should be noted that timely diagnosis of a congenital defect of the immune system and thus timely therapy will avoid adverse outcomes. interferon gamma (actimmune®) effects on severe burkholderia cepacia pneumonia in variant x-linked chronic granulomatous disease professor of pediatrics, university of utah associate professor of clinical pediatrics, keck school of medicine at the university of southern california professor of medicine, university of utah introduction/background: interferon gamma (ifnγ; actimmune®) has been proven to significantly decrease the overall number of infections in patients with chronic granulomatous disease (cgd) when given prophylactically (nejm : , ) . therapy with ifnγ has also been employed to treat severe overwhelming infections in some instances, such as severe aspergillosis with success (jid : , ) . we report here, two patients with very severe burkholderia cepacia (b. cepacia) infection, one of whom was placed on a respirator for approximately two weeks and another who was on extracorporeal membrane oxygenation for an extended period of time. both were treated with ifnγ (actimmune®) in addition to appropriate antimicrobial therapy in an attempt to affect these lifethreatening infections. objectives: the objective of this presentation is to describe two very severe variant x-linked cgd patients with b. cepacia pneumonia who were treated with ifnγ. in addition, we measured both super oxide production as well as nitric oxide production in the stimulated or unstimulated phagocytes from these patients in the presence or absence of interferon gamma. methods: case histories of both patients were reviewed in respect to the severity of their infection, the time spent on a respiratory or extracorporeal membrane oxygenation, the antimicrobial therapy administered, and the clinical results following the administration of interferon gamma as adjunctive immunomodulatory treatment. a standardized neutrophil oxidative burst assay was employed using cytochrome c reduction to measure super oxide production. in addition, nitric oxide was measured in the phagoctyes of the patients after stimulation with phorbol myristate acetate (pma) in the presence or absence of ifnγ using daf- fluorescence dye to detect the production of intracellular nitric oxide. results: a -year-old male developed a left lobar pneumonia and was admitted to primary children's medical center's intensive care unit and treated with iv cefotaxime, clindamycin, later, vancomycin and azithromycin were added. ct scan revealed a left-sided pneumonia and moderate parapneumonic effusion. subsequently, the patient decompensated, was intubated, and placed on respirator therapy. broncheoalveolar lavage and blood grew b. cepacia. neutrophil dihydrorhodamine fluorescence (dhr) demonstrated an intermediate broad peak of fluorescence with a small peak of unactivated cells, while the mother's dhr showed a broad intermediate peak suggesting the carrier state of variant x-linked cgd. both the patient, a . month old younger brother, and the carrier mother were found to have a g to a splice site mutation in exon of the gp -phox gene at position c. confirming the diagnosis of x-linked variant cgd. after approximately weeks on the respirator, ifnγ (actimmune®) therapy was instituted with significant improvement of the patient's lung function. he was taken off the respirator approximately days after the ifnγ therapy was instituted. following addition of ifnγ to his pma-stimulated neutrophil, there was a % increase in superoxide production and a fold increase in nitric oxide in his monocytes. the second patient was a -year-old male who presented with fever and cough and was diagnosed with right-sided middle and lower lobe pneumonia with cavitations. bronchoalveolar lavage grew b. cepacia and nocardia. following increasing ventilatory and circulatory collapse he was placed on extracorporeal membrane oxygenation and treated with - antimicrobial agents. after days of such therapy, ifnγ therapy was initiated and he was weaned from ecmo after days and has remained essentially healthy since then when he is on ifnγ prophylaxis. dhr revealed a broad intermediate peak in the patient and normal and intermediate peaks in the mother suggesting variant x-linked cgd in the patient and the carrier state of x-linked variant cgd in the mother. targeted sequencing revealed a g to a splice site mutation in exom of the cybb gene at position c. confirming the diagnosis of x-linked variant cgd. following addition of ifnγ to this patient's pma stimulated phagocytes, there was a % increase in superoxide production and a % increase in monocyte nitric oxide production. conclusions: two male variant x-linked cgd patients with splice site mutations in the cybb gene and severe life-threatening b. cepacia pneumonia, one on a respirator and one on ecmo were administered ifnγ (actimmune®) and each responded dramatically within - days recovering their respiratory capacity and coming off of assisted ventilation and ecmo, and have continued to do well when on ifnγ (actimmune®) therapy. introduction/background: hyqvia is a recombinant human hyaluronidase (rhuph )-facilitated subcutaneous immunoglobulin (ighy) % replacement therapy for patients with primary immunodeficiency diseases (pidd). objectives: to acquire long-term safety data on ighy, and assess prescribed treatment regimens and administration in routine clinical practice, a global postauthorization safety study (pass) is being conducted. methods: this is an ongoing prospective, non-interventional, open-label, uncontrolled, multicenter study initiated in the united states in november to assess local and systemic effects of ighy within a routine clinical setting. patients aged years with pidd who have been prescribed and/ or have started ighy are eligible for enrollment. patients are followed according to standard clinical practice and their treatment regimen is at the discretion of the treating physician. the presence of anti-rhuph antibody titers is evaluated on a voluntary basis. results: as of august , patients had been enrolled at us study sites. there were no serious aes which were deemed treatment related. sixteen patients experienced a causally related non-serious local ae ( . %; . events/patient-year, . events per infusion) and patients experienced a causally related non-serious systemic ae ( . %, . events/patient year, . events per infusion). of the patients with immunogenicity data, had positive binding antibody test to rhuph (titers : ); no neutralizing rhuph antibodies were detected. conclusions: this interim analysis of prospectively-collected data of ighy use in routine clinical practice indicates that ighy is well tolerated with no treatment-related saes and has not been associated with neutralizing anti-rhuph antibodies in patients with pidd. introduction/background: idiopathic thrombocytopenic purpura (itp) and/or hemolytic anemia accompanied by splenomegaly occurs in up to % of patients with common variable immunodeficiency (cvid). treatments include steroids, other immune suppressants and rituximab. however, in some that do not respond, splenectomy may be performed. while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. objectives: while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. methods: we report three cases of pah following splenectomy for cytopenias in patients with cvid. results: the first is a yo female, with long standing cvid complicated by interstitial lung disease, nodular regenerative liver disease and itp post splenectomy. the second is a yo man with severe bronchiectasis, cirrhosis/nodular regenerative liver disease and itp post splenectomy. the third is a yo woman with cvid (taci compound) complicated by cirrhosis/nodular regenerative liver disease, lung nodules and evans syndrome. all developed severe pah requiring chronic medications. pah in these patients is best classified as multifactorial, group v. conclusions: whether due to thrombus formation, continued cytopenias, and/or vascular changes, we suggest that pah may be a long-term complication of splenectomy in complex cvid. connective tissue, skeletal and vascular abnormalities. two isoforms exist, stat , a amino acid protein, and stat , a amino acid protein produced by alternative splicing of exon resulting in a frame shift and truncated protein at the c-terminus. objectives: we follow patients from families with stat mutations leading to altered c-terminal proteins. the patients have high ige but milder features of ad-hies. methods: clinical data were collected. stat sequencing, stat functional assays as well as lymphocyte phenotyping were performed. results: patient is a year old man diagnosed with hies as a child due to eczema, recurrent boils and high ige ( s iu/ml). he has tortuous and dilated coronary arteries, however denies lung infections, cmc, retained teeth, scoliosis, minimal trauma fractures, or hyperextensible joints. as an adult he developed avascular necrosis of both hips. whole exome sequencing revealed a novel splice mutation, c. + g>t at the end of exon causing skipping of the nucleotide exon as well as utilization of the stat alternative splice acceptor in exon , resulting in a nucleotide deletion. the mutant stat protein product has a amino acid, in-frame deletion encompassing both y and s phosphorylation sites. no stat is made from the mutant allele. lymphocyte phenotyping was unremarkable, however total stat protein levels were decreased in ebv transformed b cell lines and there was decreased y phosphorylation after stimulation. patient (family ) was healthy until diagnosed with severe, refractory coccidiodies pneumonia complicated by pneumothorax with prolonged bronchopleural fistulae at age years. this led to an immune evaluation in which he was found to have elevated ige ( iu/ml, ref . - . iu/ ml). he had one perianal abscess, primary teeth requiring extraction and mild scoliosis, but denies cmc, bacterial pneumonias, eczema, or minimal trauma fractures. stat sequencing revealed a single base insertion in the transactivation domain, c. _ insc causing a frameshift in the stat isoform, p.r pfsx , occurring immediately after the s phoshporylation site. the deletion occurs within the alternatively spliced region of exon , providing an intact, wild-type stat . stimulation with il- or il- showed reduced pstat at y , and elevated pstat which is often seen in other ad-hies patients. lymphocyte phenotyping was unremarkable and th cell analysis showed low-normal levels of th cells while ebv transformed b cells showed reduced total stat levels. his mother and infant sister also share this mutation -the month old infant had normal ige, and intermittent rashes; his mother has high ige ( iu/ml), recurrent sinopulmonary infections (complicated by tobacco use) but without bronchiectasis or pneumatocele, and denies cmc with the exception of pregnancy related vaginal candidiasis. conclusions: loss of function and gain of function mutations in stat lead to distinct syndromes, but it appears that stat mutations affecting the isoform expression, such as those reported here, can also lead to immune dysregulation with incomplete features of ad-hies. these stat mutations will allow us to better understand the relative roles of the isoforms stat and stat in somatic and immune cell signaling. physicians and is associated with immunological defects aswell. mutationsin the kmt d and kdm a genes are the most common genetic changes that lead to kabuki syndrome but for many cases the genetic basis remains unknown. objectives: recognize the varied presentation for a unique immunodeficiency syndrome methods: this is a case series. results: this is a case series describing three patients with ks and their clinical presentations which predominantly involve immunodeficiency and autoimmunity. our first patient is a -year-old male with autoimmune hemolytic anemia (aiha) at a young age, recurrent respiratory infections and hypogammaglobinemia which led to a diagnosis of cvid at the age of six. in addition, he has dysmorphic facial features, intellectual disabilities, and short stature but it was not until his late twenties where he was found to have a missense mutation in kmt d (p.arg cys) which has been described in patients with ks. the second patient is a -year-old female with hypogammaglobinemia, evan's syndrome, short stature, and severe complications which include granulomatous-lymphocytic interstitial lung disease, pulmonary hypertension, and chronic kidney disease. she was also found to have a missense mutation in kmt d (p.arg cys) in her early thirties and passed away from complications of her disease. the third patient is a -year-old female with a history of low iga/igg and poor vaccine titers, aiha, neutropenia, pulmonary nodules, and developmental delay who was diagnosed with cvid in her mid-twenties. for her immunodeficiency and autoimmunity, she was treated with immunoglobulin replacement, rituximab and cyclosporine and was found to have a missense mutation in kmt d (p.cys trp). this mutation was a de novo mutation in this patient and has also been reported in another patient with ks. conclusions: these cases highlight that the presentation of ks is varied and frequently includes immunodeficiency and autoimmunity in addition to the characteristic short stature and developmental delay. a diagnosis of ks remains challenging due the diversity of symptoms and disease severity, the need for genetic testing, and due to overlapping clinical presentations with other developmental conditions. thus, often times, like in these cases, the diagnosis of ks is delayed. chief medical officer, rocket pharma introduction/background: lad-i is a rare disorder of leukocyte adhesion, resulting from itgb gene mutations encoding for the beta- integrin component cd . cd deficiencies prevent integrin dimerization and endothelial leukocyte adhesion, essential for extravasation and antimicrobial activity. severe lad-i (< % of normal neutrophil [pmn] cd levels) is characterized by recurrent serious infections and early mortality unless treated by allogeneic hematopoietic stem cell transplant (hsct). mortality for severe lad-i was reported as % by age in an initial multicenter retrospective study. moderate lad-i ( - % of pmn cd levels) is more indolent; although most patients (pts) survive childhood with recurrent skin and mucosal surface infections; mortality by age can exceed %. lad-i is characterized by umbilical cord complications (delayed separation and omphalitis), poor wound healing and leukocytosis. objectives: reports regarding lad-i have been published in recent decades but no recent comprehensive prognostic assessments are available. we sought an updated understanding of severe lad-i with emphasis on prognosis in the absence of hsct, hsct outcomes and association of cd expression with clinical features. methods: we created a database of all published lad-i cases via pubmed searches and review of available references. results: three hundred twenty-three lad-i cases were reported between - in publications ( case-reports; largest series n= ). the nations reporting the most cases were iran (n= ), usa (n= ), and india (n= ); the highest number of publications were from us centers ( ). pts were considered to have severe lad-i, moderate and were not classified. pmn cd expression levels was reported for cases and was < % in patients ( %) and >= % in pts. four pts with cd > % were considered to have severe lad-i (cd % range . . ). gender was noted for pts; ( %) were male. age at presentation was reported for cases. for pts with cd < %, median presentation was age m (range . - m); for pts with cd >= %, median presentation was age m (range . - m). infection details and cd % were available for ( %) cases. the most frequent infections in pts with cd < % were respiratory tract ( %), sepsis ( %) and otitis media ( %) and for pts with cd >= % they were periodontal ( %), otitis media ( %) and sepsis ( %). perianal skin infections and necrotic skin ulcers were noted in > %. umbilical complications were more frequent in severe lad-i ( of pts with cd < % [ %] and of with cd >= % [ %; p = . ]). for severe lad-i pts with years of follow-up (or death prior to y), there was correlation between absence of umbilical complications and survival to m (p < . ). wbcs were reported in cases (median x /l; range x /l). there were limited correlations between cd expression and wbc (r < . ) and between cd and cd expression (r < . ). mutation analyses were reported in cases with > gene locations noted and mutations on exons , and accounting for % of specified cases. in cases, cd expression was > %; in of cases where cd expression was noted, at least one cd moiety was reported as < %. we sought to understand whether prognosis for severe lad-i in the absence of hsct is similar to the initially-reported % survival to age . there were severe lad-i cases (per investigator assessment or cd < %) for whom survival to years was reported, of whom died prior to age ( % mortality). mortality was similar for the subset of cases reported since ( %, deaths). early mortality was substantially lower in patients with cd >= % and the majority of pts with cd > % survived to adulthood. outcomes for pts who received hsct were consistent with recent series; phenotypic correction was reported in % of pts with hla-matched sibling donors. mortality was % overall ( % for hlamatched sibling recipients). for pts receiving haploidentical hsct there was % mortality and % received subsequent hsct. conclusions: severe lad-i remains a life-threatening condition with limited -year survival in the absence of allogeneic hsct. umbilical complications and granulocytosis are frequent early manifestations; respiratory tract, ear, sepsis, oral and skin infections are common. hsct is potentially curative; transplant-mortality and other complications are frequent, especially in haploidentical recipients. diverse itgb mutations result in lad-i, and genetic evaluation may be valuable for diagnosis and prognosis. rapid identification of pts with potential lad-i (unusual or severe infections in infancy, granulocytosis and umbilical complications) is essential to enable referral to centers with disease expertise. objectives: to determine the phosophorylation of stat in ad-hies patients with known stat mutations. methods: peripheral blood mononuclear cells (pbmcs) were collected from ad-hies patients under irb-approved institutional research protocols. pbmcs were then stimulated with il- or il- for , and minutes. cells were surface stained for cd , cd , cd , cd and cd . they were then fixed, permeabilized and stained with anti-y -stat to evaluate for phosphorylation of stat at position y . cells were then washed and data acquired using flow cytometry. results: pbmcs from one ad-hies patient with a stat sh domain mutation (p.y c) demonstrated normal y phosphorylation after il- stimulation. pbmcs from another ad-hies patient with a dbd mutation (p.h y) exhibited highly reduced stat phosphorylation after il- stimulation and absent phosphorylation after il- stimulation. conclusions: these findings highlight that, in the context of ad-hies, the domain location of the stat mutation does not predict stat phosphorylation potential following stimulation and challenges the current paradigm. ( ) submission id# cheng sun associate professor, institute of immunology introduction/background: as the predominant lymphocyte subset in the liver, natural killer (nk) cells have been shown to be highly correlated with the outcomes of patients with hepatocellular carcinoma (hcc). previously, we reported that nk cells were decreased and functional deficiency in hcc. however, the mechanism underline remains unknown. objectives: in this study, through the use of healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from hcc patients, methods: we have evaluated the expression of cd and its co-ligand receptor btla on hepatic cd + t cells and nk cells. results: decreased expression of cd on nk cells was observed in intratumoural but not pt regions, along with nk cell dysfunction, poor prognosis and tumour metastasis. human cd + nk cells exhibited functional activated, high capacity of ifn-secretion and nk mediated immunity by global transcriptomic analysis of sorted cd + and cd -hepatic nk cells. blocking tgf- specifically reversed the ifn-production of cd + nk cells. in addition, this decreased cd expression is predominantly on cd bright nk cells. conclusions: these findings indicate that cd expression reduction contributes to nk cell exhaustion and tumour immune escape, suggesting that cd has the therapeutic potential for fighting liver cancer. introduction/background: the low number of circulating lymphocytes in the blood is a marker for cellular immunodeficiency in young children. ethnicity also affects the lymphocyte count and ethnicity-specific lymphocyte norms have been used in many countries. this study analyzed the lymphocyte counts in a large cohort of infants and young children from the arabian peninsula. objectives -to define the normal lymphocyte counts in arab children -to define the possible cutoff lymphocyte count that define lymphopenia. methods: this is a cross-sectional analysis of the lymphocyte counts in , arab children. the age groups were: day, - months, - months, - years, - years, - years, - years, and - years, % females. we analyzed the first blood count performed during their visit to the abu dhabi seha ambulatory healthcare services between april and october . the median, th percentile and th percentile counts were calculated. the th percentile lymphocyte count was used to define lymphopenia. the kolmogorov-smirnov test, a non-parametric test, was used to compare lymphocyte counts between groups. statistical significance was defined by a two-tailed p< . . results: the median counts were higher during infancy. the variability (disparity) of the counts (reference intervals) progressively decreased from birth to years of life. the th percentile lymphocyte counts were relatively constant from birth to years ( . - . x /l) and from to years ( . - . x /l), table . the lymphocyte counts were similar in boys and girls. the lymphocyte counts were compared to those from five other studies. conclusions: arab children have lower lymphocyte counts ( th percentile) than children in the united states, brazil and south africa, but their counts are similar to children in china and uganda. our study results support the development and use of ethnicity-specific lymphocyte count standards. the implication of our results is that using these lower cutoff values for lymphopenia will prevent a large number of arab children from having unnecessarily investigated for immunodeficiency. introduction/background: chronic granulomatous disease (cgd) is a rare phagocytic defect caused by mutations in the nadph oxidase system leading to reduced or absent reactive oxygen species production. in addition to specific infectious susceptibility, patients with cgd are predisposed to hyperinflammation in response to infectious agents, autoimmunity, colitis, and other forms of autoinflammation. cgd patients with mutations in p phox are at increased risk of diabetes and cardiovascular disease. hyperinflammatory and auto-inflammation responses are often difficult to predict and manage. intracellular adhesion molecule- (icam- ) and e-selectin are endothelial adhesion markers that facilitate the adhesion and transendothelial migration of leukocytes and elevations in these markers have been associated with cardiovascular disease, glomerular injury, and thrombotic events. expression of adhesion molecules is induced by pro-inflammatory cytokines and are associated with a hyperinflammatory state. objectives: to determine if e-selectin and icam- are elevated in patients with cgd and to determine of endothelial adhesion markers could serve as a biomarker of inflammatory disease in cgd patients. methods: thirty-eight pediatric and adult subjects with cgd ( xlinked (xl-cgd), p phox deficient and p phox deficient, and x-linked cgd carriers were enrolled. e-selectin (pg/ml) and icam- (ng/ml) were measured from the plasma of patients via sandwich elisa. results: all cgd patients had histories of severe infection, active infection, chronic colitis, or other autoimmune disease at time of evaluation. nine p phox deficient cgd patients had history of diabetes and/or early onset cardiovascular disease. one subject with x-linked cgd had undergone hematopoietic stem cell transplantation (hsct). plasma levels of e-selectin were significantly elevated above healthy controls (median , pg/ml) in subjects with xl-cgd (median , pg/ ml, p< . ), p phox deficient cgd (median , pg/ml, p= . ) and p phox deficient cgd ( , pg/ml). plasma levels of icam- were also elevated above healthy controls in subjects with xl-cgd (median . ng/ml), p phox deficient cgd (median . ng/ml), and p phox deficient cgd ( . ng/ml), although none were statistically significant. plasma quantities of e-selectin and icam- increased further in those p phox deficient patients with diabetes and/or cardiovascular disease (median e-selectin , pg/ml, icam- . ng/ml) but neither reached statistical significance. e-selectin and icam- quantities in female carriers of xl-cgd and in xl-cgd cured by hsct were similar to values found in healthy controls. conclusions: immune dysregulatory features and hyperinflammation in cgd can be difficult to predict and manage. the endothelial adhesion markers e-selectin and icam- are elevated in patients with xl-cgd and p phox deficient cgd that worsens with presence of early onset cardiovascular diseases and resolves post-hsct. elevations in e-selectin and icam- in the serum of cgd patients may serve as surrogate markers of inflammation and suggest a chronic endotheliopathy in cgd patients. introduction/background: the phenotypic presentation of ctla haploinsufficiency was only recently described. the management of these patients in the medical literature is limited to anecdotal case reports. we aimed to detail our experience with short and long term immunomodulatory therapy in treating autoimmune cytopenias in the background of ctla impairment. objectives: we aimed to assess the efficacy of mtor inhibitors in treating autoimmune cytopenias in patients with ctla haploinsufficiency. methods: we retrospectively identified patients with proven ctla mutations and documented refractory autoimmune cytopenias while receiving care at nih clinical center (from july to august ). the complete (cr) and partial (pr) clinical response was assessed after six weeks of treatment and defined as hgb > g/dl, platelets > k/ul and hgb > g/ dl, platelets > k/ul, respectively without transfusion requirement. results: all analyzed patients failed or exhibited disease recurrence on at least one prior medical therapy, including: corticosteroids, rituximab, romiplostim and eltrombopag. the initial response rate to evrolimus and/or sirolimus was % ( cr and pr). one of the partial responders had recurrence of idiopathic thrombocytopenic purpura at four months while on rapalogs. overall, we used mtor inhibitors in patients for a total of patient years to treatm multiple modalities. the top three most common recorded adverse events were: clostridium difficile colitis (n= , in patients), lipid abnormalities (n = , patients required treatment) and bacterial pneumonia (n= , in patients). conclusions: our limited retrospective data suggests that mtor inhibitors might be efficacious in the treatment of autoimmune cytopenias in ctla haploinsufficent patients. further prospective studies are required to assess safety and efficacy of mtor inhibitors in this patient population. association with frequent upper respiratory tract infections and pharyngitis. from years of age she continued to have recurrent febrile episodes in the absence of infection, with fever of - degrees celsius on a monthly basis, normally lasting - days. she also developed episodes of urticaria, temporally unrelated to her febrile episodes. the rash was noted to be induced by exposure to the cold, and would generally last - days with some clinical response to antihistamines and naproxen. her clinical picture then progressed and she developed joint pain and swelling, particularly affecting her hands and feet. over the following years the patient continued to have recurrent episodes of urticaria as well as progressive joint involvement and limitation. there was some initial improvement with naproxen and prednisone. her symptoms were refractory to subsequent treatment with methotrexate, leflunomide, infliximab and tocilizumab and she remained corticodependent. immunological work up including lymphocyte phenotype, immunoglobulins, vaccine responses to both protein and live vaccines and ch were normal. there was no evidence of raised inflammatory markers with esr - mm/h, crp < . mg/l and ferritin ug/l. autoimmune workup including ana, ena, anti-dna, anca and c and c was normal. the initial differential diagnoses included systemic juvenile arthritis, periodic fever syndrome or cryopyrin associated periodic syndromes (caps). the patient therefore had a periodic fever gene panel that identified a heterozygous mutation in exon of nlrp , c. c>t (p.arg trp). whilst mutations in nlrp are known to be associated with familial cold autoinflammatory syndrome, this was reported as a variant of unknown significance. we therefore proceeded with functional in vitro testing to demonstrate pathogenicity. the patients monocytes showed increased secretion of il b upon stimulation with lps as compared to healthy donors. when tested in a luciferase reporter assay, the mutated nlrp partially lost the capacity to inhibit the nfkb pathway. overall these in vitro studies show that this nlrp mutation results in defective regulation of the inflammatory response. the patient was commenced on anti-il therapy with canakinumab, with no clinical improvement so was therefore discontinued. we report a case of familial cold autoinflammatory syndrome due to a mutation in exon of nlrp , that to this point has remained refractory to multiple treatment modalities. functional testing was able to demonstrate mutation causality and defective regulation of the inflammatory response. objectives: authors aim to evaluate the spectrum of clinical phenotypes associated with nemo hypomorphic mutations within the usidnet registry. methods: investigators obtained demographic, laboratory, and clinical data on patients with a defect in nemo within the usidnet registry. results: there were male patients within the usidnet registry with a diagnosis of eda-id attributed to nemo hypomorphic mutation. of these, were associated with a known variant in nemo (e x, f l, m v), and were associated with previously unreported variants (d v, e del, c. - g>c). for patients, a mutation was not specified. most reported having an affected family member (n= , %). median age of symptom onset and diagnosis were year (iqr . - y) and years (iqr - y), respectively. median age at most recent visit was years (iqr - y). infections, additional clinical features, treatments, and outcomes are summarized in table . skin manifestations (n= , %) and pulmonary complaints (n= , %) were common, with eczema (n= , %) and asthma being most prevalent (n= , %). gastrointestinal conditions (n= , %) were also frequently reported, and included non-specific diarrheal illness, enteropathy, colitis, enteritis, and inflammatory bowel disease. neurologic features (n= , %), including seizures, hearing defect, peripheral neuropathy, and encephalopathy, were unexpectedly common, suggesting a previously unrecognized disease association. conclusions: we observed that allergic diseases, including asthma and eczema, were common in patients with nemo mutation. notably, varied neurologic features were more prevalent than previously reported. this study highlights the potential of cross-institutional registry analysis to deepen our understanding of extremely rare genetic diseases. coordinated effort across institutions is required to better characterize the spectrum of clinical phenotypes associated with hypomorphic mutations in nemo. mycobacterial lysate (ml) and purified protein (ppd) in the diagnosis of patients with mendelian susceptibility to mycobacterial disease (msmd) elimination of this infection depends mainly on the success of the interaction between macrophages and infected t lymphocytes. patients with mendelian susceptibility to mycobacterial disease (msmd) present severe and recurrent infections due to impaired signaling of the ifn/il- axis. objectives: our aim was to evaluated the ifn/il- axis of patients with clinical history suggestive of msmd using mycobacterial lysate (ml) and purified protein (ppd) by elisa assay. methods: samples of patients (n= ) with a clinical history suggestive of msmd arrived in our laboratory. for the diagnosis, ml of blood diluted ( : ) in rpmi culture medium supplemented were used. the samples were distributed in two different plates, one used in the dosage of il ( h) and the other in the ifn dosage ( h). thus, they were stimulated with ml ( ug/ml for the h plate and ug/ml for the h plate) and with ppd ( ug/ml for the h plate and ug/ml for plate of h). at the same time, in half of the wells stimulated with lm and ppd, the cytokine ifn ( iu/ml) or il p ( ng/ml) were added in the plate. next, the plates were incubated at °c and % co² and the supernatant were collected and quantified by the elisa assay. the results were evaluated by the statistical mann whitney u test. results: six of the patients presented alterations in the evaluation of the ifn/il- axis. the age of the diagnosis of male patients ranged from to years. the only two female patients diagnosed were and years old. the clinical history was heterogeneous: had lymph node hyperplasia, pneumonia, colitis, herpes zozter, another had a urinary tract infection and bcgitis. the pathogens isolated were the m. tuberculosis, m. abscessus, m. gordonae, m. genavense and m. konsossi species found in different patients. statistical analysis of the ifn-/il- axis evaluation by elisa was performed.the results of the dosage of ifn were significant in samples with lm and lm plus il- (***). similar results were observed in samples treated with ppd and ppd plus il- (**) when compared with healthy controls. in the il- dosage, statistical difference was observed in the samples with lm (***) and with lm plus ifn (*). in samples stimulated with ppd, the results did not show statistical differences (ns.) but ppd + ifn (*) were significantly different. conclusions: six patients were diagnosed by the evaluation the il- / ifn axis. the use of micobacterial lysate (ml) showed reliable results to the diagnosis of patients with il /ifn pathway defects. the genetics diagnosis will be performed. introduction/background: invasive infections due to mycobacterial species are a feared complication in patients with t-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. cd + th t-cell immunity is known to be critical to antimycobacterial defense; accordingly, adoptive t-cell immunotherapy with mycobacteria-specific t-cells (mst) may be a beneficial therapy for combating these infections. objectives: to determine if ex vivo expansion of t-cells targeting common mycobacterial antigens is feasible from healthy donors, and whether the same antigens are recognized by patients with primary immunodeficiency (pid) and invasive mycobacterial infections. methods: peripheral blood mononuclear cells (pbmc) from healthy donors were pulsed with overlapping -mer peptide libraries encompassing five mycobacterial antigens (ag b, ppe , esat- , cpf , adk) and expanded for days with cytokines il- and il- . expanded msts were tested for specificity against the targeted antigens via ifn-g elispot, multiplex cytokine analysis, and flow cytometry. pbmcs from pid patients with invasive mycobacterial infections were similarly tested for presence of t-cells recognizing the tested mycobacterial antigens. a minimum of spots per x cells above negative control was considered specific on elispot. results: ten healthy donors and eight patients with pid were tested. specificity against - mycobacterial antigens (median ) was confirmed in all ten healthy donors, with a mean . -fold cellular expansion during the -day culture. msts were predominantly cd + t-cells (mean/sd: +/- %), with both central memory (mean/sd . +/ - . %) and effector memory (mean/sd . +/- . %) populations. there was no clear difference in antigen specificity between bcg immunized (n= ) and bcg naïve (n= ) healthy donors. six of pid patients had no detectable immunity to tested mycobacterial antigens. one patient with combined immunodeficiency has a low detectable specificity to ag b (mean spot forming colonies[sfc]), and a patient with nfkb haploinsufficiency mounted a response against ag b (mean sfc) and ppe (mean sfc). conclusions: mycobacteria-specific t-cells can be rapidly expanded from healthy donors utilizing a protocol that could easily be translated to a good manufacturing practices facility. the majority of tested pid patients lacked immunity to the targeted antigens. adoptive immunotherapy with msts derived from third-party healthy donors may be a beneficial adjunctive therapy for pid patients with invasive mycobacterial infections. is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. dihydrorhodamine (dhr) flow cytometry is the standard diagnostic test for cgd, and correlates with nadph oxidase activity. while there may be partial genotype correlation with the dhr flow pattern, in several patients, there is no correlation. objectives: in such patients, assessment by flow cytometric evaluation of nadph oxidase-specific (nox) proteins provides a convenient and rapid means of genetic triage (table) . methods: we performed dhr flow cytometry and nox flow cytometry on granulocytes and monocytes of cgd patients. results: phenotypic and laboratory patient data shown in table. *p had decreased p phox (% and mfi) monocytes, but not in granulocytes. all other siblings (p , p , and p ), and mother (p ) had relatively higher p phox (%) monocytes, but still lower than the healthy control. p had normal %p phox monocytes, comparable to control. however, p -p , and p had normal p phox (mfi) in monocytes and granulocytes. p - , and p have normal %gp phox+ granulocytes, while p and p have modestly decreased %gp phox in monocytes. p - , and p have all moderately decreased gp phox protein (mfi) in granulocytes and monocytes compared to healthy control, with a single population for protein expression. p , p and p have bimodal populations for gp phox in monocytes but not in granulocytes, with a larger positive population, and a much smaller negative population. the data from p - suggest that the amount of gp phox does not necessarily correlate with neutrophil oxidative burst, as measured by dhr. also, not all cybb variants affect p phox protein expression, though both proteins are membrane-bound. the cyba vus in p does not appear to have affected p phox protein expression in either monocytes or granulocytes. conclusions: the atypical clinical presentation of some cgd patients can make genotype-phenotype correlation with dhr flow data challenging. genetic testing, while necessary, can take several weeks. however, nadph-oxidase specific-protein flow assessment offers a rapid alternative to identification of the underlying genetic defect, and can be utilized as a reflex test to an abnormal dhr flow. further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes. head, division of immunology and allergy, the hospital for sick children introduction/background: adenosine deaminase (ada) is a ubiquitous enzyme important for purine metabolism. few studies have indicated that ada deficiency, in addition to causing profound lymphopenia and susceptibility to infections, is associated with neutrophils abnormalities. objectives: determine whether ada deficiency directly affects neutrophils and what are the mechanisms involved. methods: peripheral blood (pb) and bone marrow (bm) from . weeks old ada-deficient (ada-/-) mice that closely recapitulate the phenotype observed in ada-deficient patients, as well as ada+/-littermates were used to study neutrophils development and function. some experiments were supplemented with -week old ada-/-mice, maintained until weeks of age with ada enzyme replacement, and littermates. results: the number of neutrophils in pb of ada-/-mice at . and weeks of age was similar to ada+/-mice. the function of pb neutrophils from ada-/-mice, determined by oxidative burst, was also normal. the percentage of lin-/c-kit+/sca + hematopoietic progenitor cells in bm demonstrated significant reduction in ada-/-mice compared to littermates ( . ± . % and . ± . %, respectively, p= . ). moreover, expansion of bm isolated from ada-/-mice in methylcellulose resulted in significantly less cd b+/ly- g+ neutrophils compared to healthy controls ( . ± . % compared to . ± . %, respectively, p= . ). proliferation of bm cells, determined by brdu incorporation into cells dna, was higher in ada-/-mice than in littermates, possibly contributing to the normal neutrophil numbers in pb of ada-/-mice. conclusions: ada deficiency directly affects neutrophil development. further studies will help understand the significance of these effects and potential therapies for ada-deficient patients. objectives: we present here the clinico-pathologic features of a -yearold female with a heterozygous fancd gene deletion/mutation with evidence of cellular and humoral immune dysregulation. methods: we evaluated the patient using standard immunology anatomic, cellular, and biochemical functional assays. results: the patient has multiple dysmorphias including total anomalous venous return (repaired), mesomelia, absent ear canal, radial ray dysplasia, and short stature. her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. whole exome sequencing demonstrated deletion of exons - and a pathologic mutation (c. g>a, p.arg gln). repeated blood samples and immunophenotyping demonstrated severe lymphopenia. there were markedly low cd + t-cell counts with a low cd :cd ratio ( . ). changes in the composition of the b-cell population included: significantly diminished absolute total b-cells, elevated immature cells, low levels of transitional cells, and undetectable advanced b-cell populations. there was no immunogenic response to pcv- or varicella/tetanus/ diphtheria vaccination. the nk-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. bone marrow analysis demonstrated hypocellularity without dysplasia. conclusions: we report here a pediatric patient with a novel fancd deletion/mutation presenting with severe lymphopenia in two cell compartments (b and t cells) and susceptibility to invasive bacterial infection. the findings are suggestive of combined immune deficiency. the cellular immune profile suggests that fancd may be involved in the transition of immature b and t cells to mature cells, a process that requires substantial dna recombination. additional genetic and biochemical evaluation is needed to further characterize this rare clinical finding. introduction/background: familial hemophagocytic lymphohistiocytosis type (hlh) is a rare fatal condition due to a mutation in the pfr gene on chromosome q - inherited in an autosomal recessive pattern which results in overactivation of the immune system. symptoms usually manifest before year of age. a -year-old previously healthy male presented with acute onset of bilateral lower extremity pain and weakness, with subsequent inability to ambulate over a -day period. neurological exam demonstrated decreased lower extremity power, sensation, absent patellar and achilles reflexes, and a wide-based gait. objectives: not applicable methods: not applicable results: laboratory data was significant for neutropenia and thrombocytopenia, elevated levels of ferritin and serum cd , and a positive ebv pcr. patient was initially treated with ivig for possible ebv-driven guillain-barre syndrome with some improvement in neurologic symptoms as well as thrombocytopenia. a mass of retroperitoneal lymph nodes were noted on spinal mri. testing was negative for alps and bone marrow biopsy was negative for leukemia/lymphoma. further work-up revealed absent perforin expression in cytotoxic cells and normal sap protein expression on staining, with poor nk function. genetic testing revealed a pathogenic mutation in the pfr gene with additional variant of unknown significance. the patient was treated with rituximab for persistent ebvand returned with worsening lower extremity weakness. on mri, focal enhancements were found in the brain as well as worsening mass compression of the lumbosacral nerve roots. nerve root biopsy showed histiocytes with hemophagocytosis and a dense lymphohistiocytic infiltrate which stained positive for cd , cd , and granzyme b, with some loss of cd and no perforin. bone marrow biopsy was negative for hemophagocytes. conclusions: the patient was diagnosed with worsening familial hlh with cns involvement. hlh-directed chemotherapy (dexamethasone, cyclosporine, etoposide, intra-ommaya methotrexate/hydrocortisone) was started and hsct was performed. familial hlh is a rare and often lethal disorder that generally presents at a very young age. the acute onset and severity of presentation as occurred in this previously healthy adolescent is uncommon. familial hlh should be considered even in older patients with unexplained overactivation of the immune system. is the most profound form of primary immune deficiency, and is usually fatal in the first year of life without treatment. newborn screening for scid using quantitative analysis of t-cell receptor excision circles (trecs), has become the accepted method to facilitate early diagnosis and treatment in most of the united states, as scid babies typically do not make trecs. ikbkb deficiency is a rare form of autosomal recessive scid found in the northern cree first nations people of canada, where t cells develop normally but are non-functional. trec analysis is expected to be normal in ikbkb scid, and does not identify these cases. objectives: the objective of our study was to determine the feasibility of targeted genetic newborn testing for ikbkb deficiency. methods: we implemented a pilot project of prospective targeted genetic testing for the previously described homozygous ikbkb mutation (c. dupg) in newborns from small northern manitoba communities. between and , dna was extracted from dried blood spots of newborns, and targeted sanger sequencing of the mutationharbouring ikbkb exon was performed. results: all infants born in the selected communities underwent testing. fifty-five infants ( . %, or / . ) were found to be heterozygous carriers. one affected infant was identified, and underwent hematopoietic stem cell transplant before onset of infections. our findings are consistent with the predicted homozygosity for this mutation ( / . x / . x / = / births). conclusions: we demonstrated that targeted newborn testing for ikbkb deficiency was feasible, and provided the first prospective estimate of the ikbkb mutation carrier frequency in select manitoba northern cree first nations populations. we suggest that if we are to capture all babies with scid in manitoba, future newborn screening should be universal and include both trecs and direct mutation testing for population-specific mutations, including the first nations ikbkb mutation. high throughput analysis for trecs and targeted mutations will be introduced for universal newborn screening in manitoba. introduction/background: immunoglobulin class-switch recombination (csr) and somatic hypermutations (shm) are prerequisites of antibody and immunoglobulin receptor maturation and diversity within the adaptive immune system. the mismatch repair (mmr) machinery, consisting of homologues of mutsa, mutla, and mutsb (msh /msh , mlh /pms , and msh /msh , respectively) and other enzymes, is involved in csr, e.g. as backup of nonhomologous end-joining repair of activation-induced cytidine deaminaseinduced dna mismatches, and furthermore, in addition to errorprone polymerases, in the repair of shm-induced dna breaks. in line, a varying degree of antibody deficiency, from iga or selective igg subclass deficiency, to common variable immunodeficiency and hyper-igm syndrome have been shown in small numbers of patients with constitutional mmr deficiency (cmmrd) in addition to the known severe cancer predisposition due to genomic instability of patients with biallelic loss-of function mutations in one the mmr components. objectives: to elucidate the clinical relevance of primary immunodeficiency (pid) in cmmrd, we collected history and laboratory data of a novel cohort of consecutive patients from families with homozygous mutations in pms (n= ), msh (n= ), and mlh (n= ) reported to the consortium care for cmmrd (c cmmrd) between and , most of whom manifested with typical malignancies during childhood. methods: retrospective chart review according to a specific questionnaire and extended routine immunological analyses were performed with irb approval from the medical university of graz, austria. results: none of the presented patients fulfilled any classical or extended clinical warning signs of pid (infections, immune dysregulation, inflammation). furthermore, analyzing multiple specific laboratory parameters of the humoral and cellular immune system, we could not detect a uniform pattern of abnormalities. importantly, our data do not confirm previous suggestive evidence of iga or igg subclass deficiency, a specific antibody formation, or a b memory cell maturation defect. results of next generation sequencingbased detection of impaired class switch recombination and somatic hypermutations are pending. the t cell subsets and receptor repertoires were unaffected. together, neither clinical nor laboratory parameters were suggestive of pid in the present series of novel cmmrd patients. conclusions: we conclude that patients with cmmrd do not generally show a clinically relevant pid that could facilitate early diagnosis. on the contrary, these data support the prospect of potentially successful immune therapy of malignancies in the context of cmmrd. introduction/background: ada- deficiency is immune dysregulation diseases caused by an autosomal recessive mutation on cecr gene characterized by polyarteritis nodosa, childhood-onset, early-onset recurrent ischemic stroke and fever. objectives: report a new mutation on the cecr gene resulting in ada- deficient children. methods: female, -year-old, presented history of multiple ischemic strokes at one-year-old associated with recurrent fever and livedo racemosa. she has no siblings and parents are not consanguineous. results: the laboratory evaluation shows red cell= . x /ml, hemoglobin= . g/dl, leucocytes= . x /ml, neutrophils= . x / ml, lymphocytes= . x /ml and plateletes= x /ml. ige< ui/ml, igg= mg/dl, igm= . mg/dl and iga= . mg/dl. subsets lymphocytes shows cd += . %, cd /cd = . , cd + = % and cd / = . %. due the clinical history, we performed cecr gene sequence homozygous substitution located at position - of acceptor splice site intron , c. - g>a. this is a high conservative region with no alteration along phylogenetic studies and predicted to be pathogenic. to confirm the functional alteration, the ada- activity was tested in dried plasma spot showing . mu/g protein confirm the gene loss of function and the mutation pathogenicity. conclusions: the authors presented a novel mutation of cecr gene, the first one described in splice site causing gene loss of function and confirmed by extremely reduced ada activity. introduction/background: severe combined immunodeficiency (scid) is the most severe form of primary immunodeficiency characterized by severe, life threatening infections during early infancy. scid is a medical emergency associated with significant mortality if hematopoietic stem cell transplantations is not instituted early in the course of the disease. scid is a genetically heterogeneous disease caused by mutations in more than different genes. different genes are implicated in different ethnic populations and geographical locales depending on the rates of consanguinity and endogamy in these populations. however, following the institution of newborn screening of scid in almost all state of the us and the widespread use of next generation sequencing in primary immunodeficiency diseases ar-scid due to mutations in rag and rag genes are found to be more prevalent than reported earlier. objectives: we performed a retrospective analysis of scid cases diagnosed at our centre and referred to us from other centres to determine the clinical, immunological and genetic basis of the disease in these cases. genetic variants both recurrent and novel were analysed in detail. methods: fifty six ( ) of the suspected patients met the esid diagnostic criteria. the clinical features, immunological defects and the gene sequencing results of these patients were analysed. gene sequencing was performed at the our centre and other collaborative centres at dept of pediatrics and adolescent medicine, queen mary hospital, hong kong, national defense medical college, saitama japan, kazusa dna research centre, chiba, japan and duke medical university centre, usa. mutations were detected in of the patients. mutations were classified as recurrent or novel after checking different databases such as exome aggregation consortium (exac), human gene mutation database (hgmd) and other relevant scid databases. the effect of novel, previously unreported mutations was determined using in-silico prediction tools such as sift and polyphen . functional studies were also performed in few cases to determine the effect of novel mutations. results: mutations were detected in patients. mutations were more common in genes causing autosomal recessive form of scid than the x-linked variant. mutations were detected in il rg gene in patients followed by mutation in the rag gene in patients, dclre c in patients and rag in patients. mutations were also detected in ada gene ( patients, mutations), il r ( patients), stim , pnp and nhej ( patient each). nine novel mutations were detected. three in il rg gene, in rag , in ada and one each in the nhej and il r gene. conclusions: autosomal recessive form of scid was more common in our cohort compared to x-linked form of the disease. mutations in rag and rag genes were the commonest ( patients) followed by mutation in il rg gene ( patients). nine novel mutations in different pid genes were detected in our cohort of scid patients introduction/background: rasgrp is a guanine-nucleotide exchange factor which phosphorylates ras-gdp to the activated form ras-gtp in response to t-cell receptor stimulation, resulting in ras activation. mutations in the gene coding for rasgrp have been recently described in four patients with profound t-cell deficiency, resulting in recurrent bacterial and viral infections, autoimmunity and malignancy. here we describe a two-year-old male presenting with recurrent sino-pulmonary infections, found to have two variants in rasgrp , one not previously described. objectives . to describe a case of combined immunodeficiency with two pathogenic compound heterozygous rasgrp mutations. . to compare the clinical phenotype of rasgrp deficiency of our patient with that of previously described cases. . to argue for early hematopoietic stem cell transplantation in view of the increased susceptibility to epstein barr virus (ebv) induced lymphoma in patients with rasgrp deficiency. methods: a two-year-old male was referred to seattle children's immunology clinic for recurrent otitis media and two episodes of pneumonia. the diagnosis of combined immunodeficiency was considered based on a profound t-cell deficiency during immune evaluation and he was started on azithromycin and tmp/smx prophylaxis. at age ½ years he was hospitalized with a bladder outlet obstruction and found to have two abdominal masses. biopsies were obtained and he was diagnosed with an ebv driven b cell lymphoproliferative disorder. in addition, his csf and bone marrow were considered positive based on pcr and staining, respectively. treatment with cyclophosphamide, prednisone, rituximab, and intrathecal methotrexate was initiated. due to poor csf ebv clearance, intrathecal therapy was escalated to rituximab. results: initial laboratory evaluation showed elevated igg ( mg/dl), normal number of cd b-lymphocytes, and adequate response to tetanus, prevnar- and varicella vaccine. b-cell phenotyping showed elevated immature/transitional b-cells. a profound t-cell defect was identified with cd t-cell lymphopenia ( /mm ), elevated cd t-cells ( /mm ), inverted cd /cd ratio ( . ) and absent proliferation in response to mitogens (pha, anti-cd ) and antigen (tetanus). forty-three percent of peripheral blood t-cells were / positive. t-cell phenotyping revealed decreased cd and cd naïve t-cells with elevated proportion of cd + t-effector memory t-cells. cervical lymph node and retroperitoneal mass biopsies showed atypical lymphoproliferation without malignant transformation. exome sequencing revealed two variants in rasgrp . the first variant (c. + g>a) is located at a splice site predicting an unstable transcript targeted for degradation. the second variant (c. c>t) is a novel mutation resulting in a stop codon. conclusions: recurrent sino-pulmonary infections are often a presentation of antibody deficiency. in this case, further investigation showed a profound t-cell defect, resembling that reported in patients with homozygous nonsense mutations in the catalytic domain of rasgrp and patients with homozygous insertion mutations leading to a premature stop codon at the bzip domain. our patient had biallelic mutations in rasgrp downstream of the catalytic domain, both leading to unstable transcripts. he developed an ebv induced atypical lymphoproliferative disorder, a complication reported in one rasgrp deficient patient whose disease progressed to b cell lymphoma and unsuccessful hsct. another patient developed ebv induced lymphoproliferative disorder after hsct for ebv-positive hodgkin lymphoma. two additional patients presented with recurrent infections, developed b-cell lymphoma and one was successfully transplanted. we are preparing the patient for hsct after chemotherapy for his lymphoproliferative disease to correct the underlying immune defect given that these patients are at high risk of developing lymphoma following ebv infection. introduction/background: immunodeficiency-centromeric instabilityfacial anomaly is a group of rare genetic disorders typically involving agammaglobulinemia. type four is caused by variants in the hells gene. the five patients previously reported with icf have fit the phenotype of agammaglobulinemia. here we report a patient with novel phenotype including neutropenia and neuroblastoma. objectives: describe a unique presentation of immunodeficiencycentromeric-instability-facial anomaly syndrome to further expand our understanding of this disease. methods: retrospective chart review results: six-month old male was transferred to our tertiary care facility for ongoing chronic respiratory infection, chronic diarrhea, and failure to thrive. his past medical history was significant for -week prematurity due to rupture of membranes requiring a two month nicu stay for bronchopulmonary disease. upon discharge, he was bottle feeding and on room air. he had recurrent congestion for three months with two courses of antibiotics and one and a half weeks of diarrhea leading up to admission for difficulty breathing. he was found to have multiple infections including rhinovirus and parainfluenza virus on nasal wash, pjp pneumonia, norovirus, and pseudomonal cellulitis of his nose causing significant destruction. although previous laboratory studies revealed a normal absolute neutrophil count (anc), his anc quickly dropped to cells/ul. his igg, iga, and igm were undetectable. while his total b cell count ( cells/ul) was normal, he lacked any switched memory b cells. he had near normal total t cell count (cd cells/ul) and cd count ( cells/ul) and a markedly decreased cd count ( cells/ul) and poor proliferative response to low concentrations of phytohaemagglutinin and pokeweed mitogen. a . cm x . cm x . cm paraspinal mass was found on chest ct, which was subsequently characterized as mibg-avid with a curie score neuroblastoma. metastatic evaluation including bone marrow aspirate and biopsy was negative for malignancy. however, marked granulocytic hypoplasia and maturation arrest were present suggesting severe congenital neutropenia or, less likely, immune-mediated. whole exome sequencing detected homozygous variant of unknown significance in the hells gene (p.m t). he was treated with intravenous immunoglobulin and g-csf with clinical and laboratory improvement. his neuroblastoma was initially observed, then subsequently removed due to a > % increase in size. pathology confirmed mycn non-amplified favorable histology. he remains in remission months after resection. he is currently awaiting bone marrow transplant for his immunodeficiency. conclusions: the significance of this case report is the novel presentation of icf . neutropenia and malignancies have been reported in immunodeficiency-centromeric-instability-facial anomaly syndrome (icf ) but not icf . this case report thus expands upon the clinical picture of icf patients to include neutropenia and malignancies, and further describes the immunodeficiency. associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: identification of newborns with severe combined immunodeficiency using state wide newborn screening (nbs) began in florida in . abnormal results require extensive confirmatory diagnostic testing and prophylactic antimicrobial medications are needed to effectively evaluate and treat the infant. several barriers have been identified within government-sponsored health insurance programs that impede delivery of these evaluations and medications, often resulting in delays and/or inpatient hospitalization in order to provide timely and appropriate care. objectives: determine the cost differential between the initial evaluation and treatment of a scid patient detected by newborn screening in the inpatient versus outpatient setting. methods: the cost utilization of inpatient versus outpatient management of newly identified scid patients from nbs were analysed to include the cost of confirmatory testing and initiation of prophylaxis within the inpatient versus outpatient setting. laboratory tests included assessment of t cell immunity with quantitative and functional assessment, immunoglobulin measurement, genetic testing for scid variants, evaluation for maternal engraftment, and hla typing. medications included ig supplementation, pentamidine, fluconazole, and acyclovir. we compared the actual cost of inpatient stay and inpatient evaluation versus the approximate cost that would have been accrued if the patient were not admitted to the hospital. results: from - , infants with government-sponsored health insurance had abnormal nbs and were confirmed to have scid after evaluation at our institution. all infants were admitted into the hospital for initial evaluation and initiation of appropriate medications for an average of days. total average cost of medication administration for days was $ , , total cost of laboratory testing was $ , , and average inpatient stay averaged $ , per patient. conversely, the cost that would have been accrued in the outpatient setting for medication would have been $ , for days. laboratory testing costs would be no different as an outpatient. in total, the cost for inpatient evaluation was $ , versus $ , as an outpatient. conclusions: standard laboratory assessments and medications are necessary for infants identified with scid by population based nbs. despite government sponsoring of the florida nbs program, unnecessary barriers exist by government sponsored insurers that lead to a delay appropriate care. inpatient admission alleviates these barriers, but significantly increases cost. we advocate that standard ambulatory scid outpatient evaluation and initial treatment be authorized in children identified with scid through nbs without delay. patients with cd g mutations reveal a role for human cd g in treg diversity and suppressive function. introduction/background: integrity of the tcr/cd complex is crucial for positive and negative selection of t cells in the thymus, and for effector and regulatory functions of peripheral t lymphocytes. genetic defects that reduce, but do not abrogate tcr signaling, are associated with a variable degree of immune deficiency and immune dysregulation. in particular, while cd d, cd e, and cd z gene defects in humans present mainly with severe immune deficiency, cd g mutations lead to milder phenotypes, mainly characterized by autoimmunity. however, the role of cd , encoded by cd g, in establishing and maintaining immune tolerance has not been elucidated. objectives: we aimed to investigate abnormalities of treg cell repertoire and function in patients with genetic defects in cd g with evidence of clinical autoimmunity. methods: high throughput sequencing (hts) was used to study composition and diversity of the t cell receptor (trb) repertoire in treg, conventional cd + (tconv), and cd + cells from patients with cd g mutations and in healthy controls. treg function was assessed by studying their ability to suppress proliferation of tconv cells. results: treg cells of patients with cd g defects had reduced diversity, increased clonality, and reduced suppressive function. the trb repertoire of tconv cells from patients with cd g deficiency was enriched for hydrophobic amino acids at position and of the cdr , a biomarker of self-reactivity. overlap between treg and tconv cell repertoires was observed in cd g mutated patients. conclusions: the treg and tconv cell repertoire of patients with cd g mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. introduction/background: a common concern with b cell-depleting therapies is their potential effect on humoral immunity. although there have been reports of prolonged hypogammaglobulinemia in adult patients receiving rituximab, little is know about this phenomenon in children. objectives: we sought to assess humoral immunity in children receiving rituximab and determine risk factors leading to low immunoglobulin levels and infections. methods: we conducted a retrospective study on all pediatric patients ( years) who received rituximab for the first time between january to december in a single tertiary pediatric hospital. charts were reviewed and data was collected prior to rituximab treatment and at , , and > months after treatment. patients who received rituximab after hematopoietic cell transplantation (hct) or for a malignancy and those with an underlying primary immune deficiency (pid) at the time of treatment were excluded. results: in total, patients received rituximab during the study period. of those, were excluded (hct: n= , lymphoma: n= , pid: n= ). sixtyeight patients were eligible. indications for rituximab treatment were renal disease (n= ), neurologic disease (n= ), hematologic disease (n= ), rheumatologic disease (n= ), ebv control (n= ), other (n= ). one patient who died from autoimmune encephalitis days after rituximab was excluded from the follow-up study. at any time after rituximab treatment, low igg was present in / ( . %), low iga in / ( . %), and low igm in / ( . %) of patients. over a year after their last rituximab dose, / ( . %) of patients still had low b cell counts for age, and / ( %) had low memory b cell counts (cd + among cd + cells: mean value = . % +/- . % sd). hospitalisation for infection was required in / ( . %) patients in the year following rituximab treatment, which was associated with having either low igg ( . % vs . %, p= . ) or low iga ( . % vs . %, p= . ), but not with low igm levels ( . % vs . %, p= . ). also, receiving a treatment with more than one rituximab cycle was a risk factor for low igg ( . % vs . %, p= . ). conclusions: hypogammaglobulinemia following rituximab treatment was frequent, and the presence of low igg and iga were associated with a higher risk of serious infection in this context. introduction/background: subcutaneous immunoglobulin (scig) replacement therapy for patients with primary immunodeficiency (pid) is usually administered once a week. however, a variety of dosing regimens can be used to provide flexibility for patients. objectives: we used pharmacokinetic (pk) analysis to evaluate the pk characteristics of weekly and biweekly (once every weeks) scig administration in patients with pid. methods: this pk substudy was part of a prospective, open-label, phase study (nct ) in patients with pid treated with igpro (hizentra®, csl behring, bern, switzerland). a noncompartmental analysis of serum igg concentrations was used to calculate pk parameters and compare pk outcomes on weekly and biweekly dosing. results: of the patients included in the pk substudy, provided samples for both weekly and biweekly regimens. the dose-adjusted area under the concentration-time curve was comparable for both treatment regimens: . and . (h*g/l)/mg for the weekly and biweekly regimens, respectively. the igg clearance was also similar, being . for the weekly and . ml/h for the biweekly regimen. median peak igg concentrations occurred later with the biweekly regimen ( . days) compared to . days for the weekly regimen. igg trough levels were close for both treatment regimens, with arithmetic means slightly lower for biweekly than for weekly regimens, at . vs. . g/l respectively. the minimum igg concentrations within a dosing interval were also comparable, with arithmetic means of . and . g/l for the weekly and biweekly treatment regimens, respectively. conclusions: biweekly and weekly hizentra® administration at the same total monthly igg doses resulted in similar igg exposures. pharmacokinetics, efficacy, tolerability and safety of a new subcutaneous human immunoglobulin . % in primary immune deficiency introduction/background: patients with primary immune deficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors in patient acceptance. a new . % ig preparation (octapharma, lachen) was developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was to assess efficacy of a new . % subcutaneous human immunoglobulin preparation in preventing serious bacterial infections. secondary endpoints included evaluating tolerability and safety, determining the pk profile, the number and rate of other infections and changes in quality of life measurements. methods: a prospective, open-label, single-arm phase study involving patients was conducted at centers in north america and europe. pid patients who were stable on ivig treatment for at least months and with igg trough levels . g/l underwent a -week wash-in/wash-out period consisting of weekly scig doses . times the previous ivig dose (based on published conversion rates for marketed scig products), followed by a -week efficacy period ( scig infusions in total). of patients enrolled had complete pharmacokinetic assessments at different time points: before the switch from ivig to scig (pkiv), after the wash-in/wash-out phase (pksc ) and at week of the efficacy period (pksc ). results: patients (age: - years; mean age . years; . % female) receiving a total of , scig infusions ( . g/kg/week in young children ( years and < years of age) and . g/kg/week in adults; average overall: . g/kg) were included in the full analysis sets. no serious bacterial infections were recorded. among the other infections observed during the efficacy period only one infection was graded as severe (bronchiolitis due to rsv virus), which led to hospitalization ( days). all other infections were mild ( . %) or moderate ( . %) in intensity. infection rate per person-year was . . of the reported adverse events, only were assessed as being related to the study drug; all of these events were non-serious. five non-study drug related serious adverse events were reported in patients ( . %). serum igg trough levels were nearly constant during the study with a minimum trough level of . g/l and mean trough plasma concentrations of . ± . g/l and . ± . g/l for pksc and pksc . median igg trough levels after scig treatment were . to . g/l higher compared to ivig treatment prior to enrollment. a dosing conversion factor (dcf) of . was determined by auc (area under the curve) measurements, allowing dose adjustment to achieve bioequivalence between ivig and scig dosing. improved quality of life measurements, utilizing sf- v , were observed in both physical and mental health parameters when compared from the first to last scig infusion. conclusions: this study demonstrated that the new subcutaneous human normal immunoglobulin . % is well tolerated, safe and effective in patients with pid. introduction/background: atopic dermatitis (ad) is a chronic, relapsing, inflammatory skin disorder with associated pruritus that affects percent of children in the united states. severe atopic dermatitis refractory to conventional therapy can be concerning for an underlying immunodeficiency, especially in infants. increased incidence of hypogammaglobulinemia has been associated severe ad. a handful of cases describe a correlation with transient hypogammaglobulinemia of infancy (thi), but a thorough immunological evaluation is often missing to further understand this relationship between ad and characterization of thi. objectives: define various phenotypes of thi with their clinical presentation and laboratory findings. compare thi vs. thi associated with severe a.d. methods: a case series of six patients was conducted at a single academic center from / / to / / for patients with severe atopic dermatitis with low igg levels. all available immunological laboratory data were retrospectively collected during this time period. descriptive statistical analysis was utilized for data comparison. results: of the six patients, four had no infectious history. of the remaining two, one had recurrent skin abscesses associated with his poorly controlled atopic dermatitis requiring oral antibiotics and one patient had two episodes of staphylococcus aureus superinfection of the eczema. at the time of presentation, the mean age was months with mean igg of mg/dl and mean ige of , ku/l. iga and igm were within normal age cut offs. all patients had normal protein and polysaccharide specific antibody titers after completion of vaccination series. mean cd + count was , /ul with normal cd +, cd +, cd + and cd + cell counts. three patients were tested for lymphocyte mitogen proliferation and complement function which were normal. two patients, who were tested, had normal phagocyte work up. mean age of igg improvement to igg> mg/dl, was months. patients had total protein and albumin levels with mean . ku/l and . ku/l, respectively which eventually normalized. four patients improved with skin care and dietary modification to hypoallergenic formula. one patient improved with extensively hydrolyzed formula and three patients improved with amino acid formula. one patient improved with aggressive skin care. one patient, who was noncompliant with dietary recommendations and aggressive skin care, did not improve. conclusions: one prospective study described an increased incidence of hypogammaglobulinemia in patients with atopic dermatitis compared to controls regardless of the severity. no further prospective studies have characterized hypogammaglobulinemia in this population. according to the primary immunodeficiency practice parameters, children with thi often present with frequent viral and bacterial respiratory illnesses, low igg levels and normal vaccine responses. in one study, the period of hypogammaglobulinemia spontaneously corrects to normal by mean age of months with all patients reaching normal levels by months. these patients may have low igm or iga and decreased t or b cells, which eventually normalize. management is often with antibiotic prophylaxis and if refractory to prophylaxis or unable to tolerate, igg administration (igrt) based on severity of symptoms is recommended. we describe a less severe variant of thi associated with severe atopic dermatitis and characterized by very transiently decreased igg levels with earlier resolution than typical thi, normal igm and iga levels, normal specific antibody levels and normal t, b and nk cells. these patients typically do not manifest recurrent viral or bacterial respiratory illnesses. they also do not require antibiotic prophylaxis or igrt. severe atopic dermatitis maybe a positive prognostic indicator for patients with thi and is associated with an earlier self-resolution of hypogammaglobulinemia, lack of typical infections, normal iga, igm and normal t and b cell numbers. a possible mechanism for thi associated with severe ad may be transdermal loss of protein which was supported with mildly low total protein and albumin levels rather than immaturity of the immune system in patients with true thi. introduction/background: in recent years it was found that heterozygous mutation in pik cd gene produces an autosomal dominant primary immunodeficiency characterized by onset of recurrent sinopulmonary and other infections in early childhood with defects in both b-and t-cell populations and a special susceptibility to uncontrolled viral infections. many patients develop chronic lymphoproliferation and there is also an increased susceptibility to b-cell lymphomas. here we present a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik cd gene. objectives: to describe a case of a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik cd gene. results: year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis, chronic diarrhea without diagnosis, mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was following up in other center and in she was diagnosis with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but she referred low adherence to it. she did not referred history of lymphoproliferation nor significant viral infections. she has a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid, she deceased at years old because pulmonary infection. other daughter and sons referred healthy. in our first immunologic studies we found severe hypogammaglobulinemia (igg mg%, no dosable iga and igm) with absent of b cells in peripheral blood. we started with high doses of ivig ( mg/k/month) and antibiotic prophilaxis with improvement of the functional respiratory test and without new infections. we are planning colonoscopy to study her chronic diarrhea. thinking that her clinical picture could be other than cvid we order a genetic study. a nextera exome capture and next generation sequence with illumina hiseq was made and an heterozygous mutation in pik cd gene (chr : . . , p.pro ala) was found. family and functional studies are still pending. conclusions: due that clinical presentations of primary immunodeficiencies are becoming more complex, its diagnosis is a challenge for immunologist now a days. studies with next generation sequence is a very useful tool in indefinite cases, especially when more than one member in the family are involved. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: heterozygous gain-of-function mutations in pik cd as well as heterozygous pik r mutations that affect interaction of p with p lead to constitutive hyperactivation of the pi k pathway and cause activated pi k delta syndrome type or type (apds , apds ), respectively. we describe a female with apds with short stature, diffuse lymphadenopathy, recurrent upper respiratory tract infections, elevated igm, persistent ebvand cmv viremia and disseminated toxoplasmosis who gave birth to a genetically affected daughter with severe congenital toxoplasmosis. objectives: to characterize the molecular and cellular defects underlying severe toxoplasmosis in this family methods: investigation of the molecular basis of the disease was performed through whole exome sequencing, and results were validated by sanger sequencing. functionality of the pi k pathway was assessed by analyzing akt and s phosphorylation in freshly isolate b cells with and without stimulation with anti-igm. an excisional lymph node biopsy from the affected mother was stained with anti-pd antibody to detect t follicular helper cells, and with igm and igg specific antibodies to analyze the proportion of isotype-specific b and plasma cells results: whole exome sequencing of maternal dna with targeted analysis of pid genes identified a heterozygous mutation at an essential donor splice site of pik r (nm_ . :c. + g> a). sanger sequencing confirmed the presence of this mutation in both the mother and her child. functional studies on b cells freshly isolated from both patients confirmed an increase in baseline akt and s phosphorylation, suggesting constitutive activation of the pi k-mtor signaling pathway. a lymph node biopsied from the mother contained numerous pd- + tfh cells and igm+ plasma cells conclusions: toxoplasma gondii is an obligate intracellular parasite that is usually only symptomatic in immunocompromised hosts. severe toxoplasmosis has been reported in the following primary immunodeficiencies: cd ligand deficiency, tap deficiency, cvid, nfkb deficiency, and immunodeficiency due to anti-ifn-autoantibodies. importantly, toxoplasma infection was previously reported in a month-old infant with apds , and ocular involvement has been described in a -year-old patient with apds . this, however, is the first report of systemic and severe congenital toxoplamosis in a mother and child with apds. to evade innate host defenses, t. gondii induces the activation of the pi k/akt signaling pathway, reducing intracellular reactive oxygen species and creating an intracellular environment that is hospitable to parasite survival and proliferation. therefore, we postulate that the pik r mutation may lead to a hospitable cellular environment for toxoplasmosis replication. this work was partially supported by the division of intramural research, niaid, nih (protocol # -i- ). additional authors of this work are: ottavia delmonte and kerry dobbs, from the laboratory of clinical immunology and microbiology, niaid, nih. introduction/background: aplaid (autoinflammation and plc-gamma- -associated antibody deficiency and immune dysregulation) is a term that was proposed for the newly discovered autoinflammatory condition resulting from a pathogenic missense variant, ser tyr, in the cterminal sh (csh ) domain of plc-gamma- in order to distinguish it from plaid, a distinct clinical entity that results from intragenic deletions of portions of the csh domain of the same protein. plc-gamma- is a phosphodiesterase that is predominantly expressed in hematopoietic cell lines and acts on pip to produce ip and dag in the pkc and ras/raf/ erk pathways. the only formally published case of aplaid described a father-daughter pair with an autoinflammatory clinical syndrome affecting the skin, mucosa, eyes, pulmonary and gastrointestinal systems. objectives: to describe the aplaid phenotype resulting from a novel genetic variant of the phosphodiesterase plc-gamma- in two unrelated families methods: clinical review and case presentation results: we report a -year-old female with a long-standing history of recurrent pneumonia, cellulitis and cystitis with obstructive lung disease characterized as bronchiolitis and dynamic airway collapse, with negative alpha- -antitrypsin testing. she had a history of childhood onset granuloma annulare and pressure-induced urticaria, as well as episcleritis. immune testing revealed low igm ( mg/dl), elevated baff levels and a low percentage of cd + memory b-cells, prompting sequencing of plcg , which identified a c. t>a, p.met lys variant in the calcium binding c domain. her -month-old daughter, who has a history of bullous skin lesions, failure to thrive, febrile episodes and recurrent respiratory infections was likewise found to have this plcg variant. like her mother, the daughter also has low igm and elevated baff levels. similar symptoms of recurrent sinopulmonary infections and hypogammaglobulinemia, have been described in an unrelated family that shares this same plcg variant in a doctoral thesis by rozmus. this work also describes functional analysis, in which this particular variant of plc-gamma- was demonstrated to have dysregulated plcgamma- activity leading to aberrant intracellular calcium signaling and increased apoptosis of immature b-cell subsets. conclusions: we describe our experience in evaluation and treatment of this family with a previously undiagnosed disorder. together, these new cases add to the expanding body of knowledge regarding plc-gamma- and its importance for the development of autoinflammatory and primary immunodeficiency conditions. ( ) submission id# present a case.non-celiac gluten sensitivity is an emerging entity with symptoms similar to celiac disease, but without positivity in specific diagnostic tests. it is considered more common than celiac disease patients with sigad have a greater risk of concomitant autoimmune disorders than health individuals. sigad was previously to be associated with celiac disease, but not usually in non -celiac gluten sensitive. objectives: describir a case ataxia non celiac gluten sensitivity, methods: clinic case description. results: he patient has negative serology test for gluten, but clinically respond as celiac disease. conclusions: all patient has negative serology test for gluten, but clinically respond as celiac disease, could has non-celiac disease, this case described here ,it suggest than this entity should to thought before than the celiac disease, since not-celiac disease is it more common. associate professor, federal university of rio de janeiro introduction/background: primary cutaneous actinomycosis is a rare condition caused by gram-positive filamentous bacteria and generally occur after traumatic inoculation. objectives: to report an unusual etiology of skin lesions in a patient under anti-tnf-alpha therapy. methods: we report the case of a -years-old female receiving conventional doses of adalimumab for ankylosing spondylitis who presented, two weeks before referral for dermatological assessment, with an erythematous nodule with purulent discharge on right pretibial region and pruritic erythematous plaques with scaling and peripheral pustules in the trunk and nose tip. results: a fungal etiology was suspected and scraping specimens from several cutaneous lesions were submitted to direct microscopic examination and culture, which were negative for fungi. cutaneous biopsy of the pretibial lesion was sent for histopathology and microbial cultures. adalimumab was interrupted and empirical therapy with oral terbinafine ( mg/day) was started with close clinical follow-up. a folliculitis reaction pattern without granulomas was observed during histopathological examination and gram-positive cocci were isolated from biopsy sample and further identified as saccharopolyspora sp. by molecular typing. sulfamethoxazole+trimethoprim was added and discontinued after one week due to a cutaneous rash. terbinafine ( mg/day, p.o) was used for months with complete clearing of all skin lesions and very good tolerability. spondyloarthritis signs and symptoms were unremarkable and no anti-inflammatory or immunomodulating treatment was necessary. conclusions: increased risk of skin actinomycotic infections in patients under anti-tnf therapy is not consistently reported in the literature. nevertheless, they should be included as a differential diagnosis of atypical skin lesions in individuals under anti-tnf therapy. where we aim to determine the prevalence, incidence, characteristics, treatment and outcomes of primary immunodeficiency disease (pid) patients in qatar. pids are rare heterogeneous disorders of the immune system that result in an increased susceptibility to infection, immune dysregulation and occasionally, to cancer. objectives: determine the range of pids with important epidemiological data in qatar after analyzing the database and creating a registry. methods: this is a retrospective study of pid patients followed at hamad medical corporation from to using medical records. all patients who were diagnosed with a pid irrespective of age were included. patients were classified according to the international union of immunological societies expert committee on pid. the data is captured under sectionsa) patient demographics including age, gender, ethnicity b) clinical presentation, c) immunodeficiency profile including age at diagnosis, type of immunodeficiency, family history d) treatment modality and e) lab/genetic data. results: we registered patients ( females and males) over a span of four years. mean age at onset, diagnosis and diagnostic delay were . , . and years respectively. majority of the patients were arabs % followed by people from the asian subcontinent %. antibody deficiency was seen in %, immune dysregulation %, well defined immunodeficiency (at, hige, digeorge,wiskott aldrich) % and t/b cell cid %. rare diagnoses (ipex, msmd) were recorded whereas no cases of toll like receptor and complement deficiency were seen. consanguinity rate was (n = , % = ) and first degree cousin marriage (n = , % = ). family history was positive in % (n= ) of the patients. maximum diagnostic delay was seen in scid ( % > months) and agammaglobulinemia ( % > months).during the patients life, infection was the most common presenting complaint ( %) followed by sinopulmonary disease ( %) and gi-tract manifestations ( %). the most common infections were pneumonia ( %), otitis media and conjunctivitis ( % each) followed by failure to thrive ( %) and sepsis ( %).microbial isolates particularly seen as causative agents of infections were p.aeruginosa and salmonella ( %) each, mrsa and e.coli ( %) each. a genetic defect was confirmed in % of ataxic telangiectasia and % of scid patients. active infections were treated and prophylactic antibiotics were prescribed in cases ( %). prophylactic antibiotics were prescribed to % of patients with immune dysregulation and to % of well-defined syndromes. out of patients who had hsct, % had successful transplants. ivig was given to % of the total pid patients with humoral immunodeficiency patients receiving the most ( %). one patient had gene therapy and two required interferon gamma treatment for msmd. mortality rate at st year of life was % whereas total mortality rate was %, excluding cases that passed away before pid was diagnosed. conclusions: the estimated prevalence of pid in qatar is found to be per . over the years, physicians have become increasingly aware of pid and survival rate has improved. initiation of newborn screening for scid and agammaglobulinemia will lead to earlier diagnosis and initiation of therapy with better outcomes. introduction/background: agammaglobulinemia is typically associated with a near absence of b cells secondary to a developmental block in bone marrow, and, usually but not always, manifests in early life. most of such patients are males with x-linked agammaglobulinemia (btk deficiency). females with agammaglobulinemia, of either autosomal recessive or dominant inheritance, are rare. objectives: we present and discuss the differential diagnosis for the conflicting clinical and immunological phenotypes of two adult females who present with infections, cytopenias, and agammaglobulinemia with low to normal b cell count. methods: retrospective chart review of clinical and laboratory data results case : a -year-old female who, at age years, had evans syndrome (autoimmune thrombocytopenia and hemolytic anemia) that resolved after steroid and high-dose gammaglobulin treatment. at age years, immunologic workups revealed a complete absence of serum immunoglobulins (igg, iga, igm) and low b cells ( %) (normal range - ). however, three years prior, patient had detectable igm ( mg/ dl). b cell subset analysis showed an expansion of cd hi lo b cells ( %, normal . - . %), with a marked decrease in switched memory b cells ( . %, normal - %). additional immunophenotyping revealed a reduced frequency of naïve cd + ( . %, normal > %), cd +( %, normal > %) t cells, and normal lymphocyte proliferative responses to mitogens and anti-cd , anti-cd /anti-cd , and anti-cd /il- . case : a -year-old female with recurrent upper respiratory tract infection, undetectable igg, iga, igm and ige, and no response to vaccinations (tetanus and pneumococcal). thrombocytopenia ( x count/microliter) was noted once during her thirdtrimester pregnancy without evidence of pre-eclampsia. postpartum cd + t cell count was low ( cell/microliter, normal - cell//microliter). immunophenotyping revealed normal b cell count with reduced frequency of naïve cd + ( . %, normal > %) and cd + ( %, normal > %) t cells. conclusions: we report two adult females who present in early adulthood with recurrent infections and cytopenias. both had agammaglobulinemia and decreased naïve t cells suggestive of late-onset combined immunodeficiency (locid). the presence of peripheral b cells makes autosomal recessive defects in b cell receptor signaling (lamda , iga, igb, igm, blnk) less likely. differential diagnosis of locid with cytopenias includes ctla- haploinsufficiency, gain-of-function pik cd mutations, ikaros defects and autosomal recessive rag deficiency. both patients remain at risk for developing autoimmune complications. a molecular diagnosis will facilitate targeted therapy for the underlying defect. professor, director of the laboratory of childhood immunology, ku leuven introduction/background: complement factor properdin (cfp) is a soluble glycoprotein which has a unique known role as a positive regulator of the alternative complement pathway by binding and stabilizing the inherently labile c /c convertase enzymes. mutations in the cfp gene lead to aberrant protein expression or to expression of a dysfunctional protein which results in high susceptibility to pyogenic infections especially neisseria meningitidis. properdin-deficient individuals are at greater risk of fulminant meningococcal disease, with mortality rates as high as %. objectives: case report: a year old belgium boy from nonconsanguineous parents presented with achronic purulent cough and recurrent infections of upper and lower airways. he suffered from a severe pneumonia at the age of . ct thorax showed bronchiectasis of the right middle lobe and left lower lobe. in addition, he had recurrent acute otitis media since the age of year old. immunological work-up showed an absent ap activity, with normal ch , c and c . results: genetic and functional analysis: sanger sequencing of cfp gene identified a hemizygous c. t>g, p.y g (cadd score . , msc_cadd . ) mutation in the patient and his mother. properdin elisa (hycult biotech) showed absent and % of the normal healthy control value of serum properdin concentrations in patient and the healthy mother, respectively. conclusions: conclusion: we diagnosed properdin deficiency in a y old boy presenting recurrent lower and upper respiratory tract infections. ap testing, added to ch , should therefore be part of initial workup for patients with recurrent severe respiratory tract infections. indeed early diagnosis allows for appropriate prolonged antibiotic prophylaxis and immunization to reduce the risk of fatal meningococcal disease, to reduce or prevent organ damage and to allow for genetic counselling in the family. - , - , - and - ) and child depression inventory (cdi) were implemented to both children and parents in addition to sociodemographic data form; beck depression inventory (bdi), beck anxiety inventory and zarit caregiver burden scale were implemented only to parents. results: the depression inventory parent and child form values of the patient group with primary immunodeficiency were significantly higher than the control group (p= , and p= , ). according to beck depression and anxiety inventories, it was seen that the depression and anxiety inventory scores of the parents of the patient group were higher than the control group (p= , and p= , respectively). it was determined that the cdi parent form scores of the patients with hospitalization history were statistically higher than the patients without hospitalization history ( , ± , and , ± , ; p= , ). bdi scores were significantly higher in the group which received ivig (p= , ). while the quality of life of the patients compared to their parents was perceived as worse than the healthy children in all dimensions (p= , p< , and p< , ), the quality of life of the children was worse only in psychosocial and total quality of life fields (p< , p= , and p= , ). it was seen that quality of life of children physical health scores of only the patients with hospitalization history were statistically significantly lower ( , ± , and , ± , ; p= , ) . while no statistically significant difference was found in terms of quality of life of the child scores between the groups which received and did not receive ivig replacement treatment, psychosocial and total qualify of life scores of parents were statistically significantly lower in the group which received ivig replacement treatment (p= , p= , ). zarit care giver burden scale scores were similar in patient and control groups. although both groups were on the limits of mild to moderate caregiving burden, it was seen that the scores of the group which received ivig were significantly higher than the group which did not receive ivig (p= , ). conclusions: as a conclusion, we think that it will be appropriate to inform and monitor the entire family in relation to psychosocial difficulties and care giving burden they may experience in time as well as the medical aspects of the disease in order to develop a holistic approach to children with primary immunodeficiency. introduction/background: chronic lung disease is the most common complications of cvid, affecting - % of patients. it includes bronchiectasis affecting % of patients and granulomatous lymphocytic interstitial lung disease (glild) in to %. both are associated with an increased morbidity and mortality. pulmonary functional studies and ct scans have been proposed as screening procedures for lung involvement in cvid. the definitive diagnosis of glild is established histologically, but it is too invasive in patients with radiological abnormalities and no symptoms. objectives: we aim to describe the pulmonary complications of our cohort of patients with cvid comparing them with subjects affected with other types of hypogammaglobulinemia. methods: we reviewed all clinical records of the patients with a diagnosis of hypogammaglobulinemia of any cause until december . we looked at all pulmonary function tests (pft), -minute walk tests and ct scans performed. we classified patients according to the ct scan pulmonary disease pattern. we compared the demographic data and pulmonary characteristics of each group and intended to describe similarities and differences between them. results: we collected patients and ct scans from patients. patients were grouped for further analysis as follows: no ct performed: ; normal ct: ; bronchiectasis: ; glild: and bronchiectasis + glild: . eight patients ( %) had no ct including six with cvid, one with x linked lymphoprolipherative disease (xlp) and one had a syndromic deletion in chromosome . the median age of symptom onset was . yo, with a median delay to diagnosis of year. nine subjects ( %) had ct scans with no chronic disease, six had cvid, rituximab induced hypogammaglobulinemia (rih) and mgus with hypogammaglobulinemia (mguswh). the median age of symptom onset was years old with a median delay to diagnosis of years. nine patients ( %) presented persistent bronchiectasis, with cvid, with an igg deficiency (sigg d) and with xlp. median age at symptoms onset was yo and median delay to diagnosis years. seven patients ( %) had glild; all afected with cvid. median symptoms onset was years old, and median delay to diagnosis was years. one patient ( %) was included in the bronchiectasis and glild group. she was referred with a diagnosis of rih, in the context of a pulmonary malt lymphoma. however, her ct did not show a typical ct lung lymphoma pattern, lymphocyte monoclonality was never shown and she had a reduced pre-treatment low gamma globulin concentration and a history of respiratory infections since adolescence, we believed her diagnosis is cvid. pft with an obstructive pattern identified patients with bronchiectasis (p< . ) and patients with glild had a significantly lower basal and post minute walk o saturation (p< . , n= ) conclusions: using a systematic approach, we identified that roughly % of patients with hypogammaglobulinemia have chronic pulmonary ct abnormalities. half of them had bronchiectasis, that were associated to hypogammaglobulinemia of any cause, a longer disease course and a reduction in fvc and fev with a shorter distance reached in the minute walk test. the other half had glild, spirometries in this group were useless, but o saturation was significantly lower, basal and after the minute walk test. we found a high frequency of pulmonary disease in our cohort and a disease progression study is now in place. hies) is a primary immunodeficiency characterized by eczema, sinopulmonary infections, and musculoskeletal and vascular abnormalities. care of patients with this disease is largely supportive with the use of prophylactic antibiotics and topical eczema therapies. the use of replacement immunoglobulin is increasing. hematopoietic stem cell transplant (hsct) is being considered more frequently, but many questions remain regarding which patients should undergo transplant. as pulmonary complications are a leading cause of morbidity and mortality in this disease, and potentially improved with replacement immunoglobulin and hsct, we sought to examine more closely the patients with more frequent pulmonary hospitalizations and structural lung disease. objectives: to determine the rates of pulmonary complications in our large cohort of ad-hies patients, and examine the relationship between immunologic markers and pulmonary disease. methods: we retrospectively reviewed the records of ad-hies patients seen more than one time at nih between and . there were pediatric patients under the age of years. we reviewed the number of and cause for hospitalizations, and reviewed radiology reports for structural lung disease including bronchiectasis and pneumatoceles. we correlated these findings with specific antibody responses and lymphocyte phenotyping, such as the number of memory b lymphocytes. results: the patients range in age from years to years, with a median age of years. patients had chest cts, with percent having bronchiectasis, and percent having cavitary lesions. percent of patients had both. these abnormalities were more prevalent in patients with low memory b cells. percent of patients receive replacement immunoglobulin. in patients not receiving replacement ig, percent had appropriate response to pneumococcal vaccine. during this time period, there were hospitalizations, the majority of which were associated with pulmonary infections. the rate of overall hospitalization was higher in the group with low memory b cells (p= . ), but there was no difference associated with age. conclusions: immunologic abnormalities may assist in determining the long-term prognosis of patients with ad-hies, and can be considered in management plans such as the use of immune globulin and consideration for hsct. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is an autosomal recessive disorder affecting the purine salvage pathway causing a (severe) combined immunodeficiency disorder, autoimmunity, and neurological symptoms. patients typically present in the first few years of life with frequent infections and a failure to thrive. decreased plasma levels of uric acid are suggestive, while neutropenia is a rare complication. prognosis is generally poor with few patients making it to adulthood without treatment. treatment options are limited to general supportive care and hematopoietic cell transplantation. methods: we present the cases of three patients, one sister and two brothers, from a consanguineous french canadian family. they presented in early adulthood with nearly identical histories of repeated pneumonias and chronic rhinosinusitis. one patient had necessitated filgrastim to treat a parainfectious neutropenia. they did not report any neurological abnormalities or symptoms of autoimmunity. results: a primary immune deficiency was suspected. initial evaluation showed normal immunoglobulin levels, a lack of response to vaccination, positive ebv ebna iggs, and auto-antibodies. the patients were severely lymphopenic with reduced numbers of t, b and nk cells ( l: , cd + %, cd + %, cd + %, cd + %, cd -cd + %). in particular, they had a severe depletion of naive t-helper recent thymic emigrant cells (cd +cd ra+cd + %). additional studies revealed increased urinary inosine, guanosine, deoxyinosine, and deoxyguanosine, severely reduced erythrocyte pnp activity ( % residual activity), and a normal uric acid level. a homozygous, missense mutation, c. c>g (p.his asp) was found and described as pathogenic in silico. the diagnosis of pnp deficiency was made; tmp-smx prophylaxis and ivigs were started. conclusions: theses cases are atypical for a number of reasons. the first is the relatively benign immunological course, the lack of neurological symptoms, and the advanced age at the time of the diagnosis, possibly due to residual enzymatic activity. in addition, pnp deficiency has been classically described as causing a marked reduction of t cells with a relative sparing of b cells. however, b cell lymphopenia have been reported, particularly with latter presentations. while this specific mutation had been identified once previously in the literature, as a compound heterozygote, these are the first confirmed cases of homozygotes. the pathogenicity of the mutation was not only suggested in silico, it was confirmed biochemically. in addition, in vitro functional studies have demonstrated the importance of his for the binding of pnp to its substrate, particularly in the formation of the early transition state. indeed, pnp (p.his asp) has been shown to have a greatly reduced affinity for its substrates and catalytic activity. further study is needed to identify the optimal management for these patients. introduction/background: whole exome sequencing has become an integral part of diagnosis and treatment of rare immunodeficiency diseases. one of these diseases is tetratricopeptide repeat domain a(ttc a) mutations; a rare disorder associated with multiple intestinal atresias and severe combined immunodeficiency. histologic assessment of organ biopsies of patients with ttc a deficiency suggest it may play a role in multiple organs as it is expressed in the cytoplasma of intestinal, thymus and pancreatic cells. many patients with ttc a deficiency have moderate to severe combined immunodeficiency. nine patients described in the literature with ttc a mutations have been treated with hematopoietic stem cell transplant, mostly at a young age. objectives: this case report describes a rare presentation of an adolescent with ttc a deficiency, her clinical presentation, immune evaluation and treatment with eventual referral to bone marrow transplant at the age of . methods: retrospective chart review under irb approval was performed. whole exome sequencing performed at baylor texas childrens hospital and mutations were confirmed by sanger sequencing. extensive immune and nk cell phenotyping were performed by flow cytometry and nk cell function was tested using standard cr release cytotoxicity assays. results: a year old female was referred to immunology for severe refractory warts, history of severe diarrhea with epithelial dysplasia but no atresia, failure to thrive requiring parenteral nutrition until years old and multiple infections with staphylococcus aureus and candida. at years of age, she developed rapidly worsening pulmonary function that improved with monthly pulse methylprednisone. her immune phenotype demonstrated a combined immunodeficiency including severely low cd , cd , b and nk cells. she had no igd-cd + memory b cells and undetectable igg, iga and igm, isohemagglutinins and vaccine titers to diphtheria and tetanus. her mitogen proliferation response was low and she had abnormal tcr v-beta repertoire. she was referred to the texas childrens hospital center for human immunobiology for the nk cell evaluation and reasearch clinic (near) the patient was found to have impaired nk cell lytic function and terminal maturation. this was demonstrated by the decreased frequency of cells that matured to the cd dim subset and was accompanied by decreased expression of the lytic effector molecule perforin and the fc receptor cd . this phenotype was conserved when we isolated cd + hematopoietic precursors and performed nk cell differentiation in vitro. whole exome sequencing demonstrated a compound heterozygous mutation in the ttc a gene, including a c. + _ + aagt mutation, which has been previously described as a pathologic founder mutation in the french canadian population. the second mutation is a previously unreported c. g>a (p.e k), a variant of unknown significance. mutations were confirmed by sanger sequencing, and parents are carriers of the mutations. she was referred for hematopoietic stem cell transplantation with a / unrelated donor option. conclusions: ttc a deficiency is a genetic mutation associated with high morbidity and mortality leading to gut atresia and dysfunction in combination with severe immunodysfunction. we have described a unique case of ttc a deficiency with a novel mutation which is associated with intestinal epithelial dysplasia with no atresias and a late presentation and evolution of combined immune deficiency. nk cell assessments show significantly impaired terminal maturation suggesting a critical role for ttc a in human nk cell development. introduction/background: ras-associated autoimmune leukoproliferative disorder (rald) is a rare condition with significant overlap of clinical and laboratory findings with malignant disorders, notably juvenile myelomonocytic leukemia (jmml) and chronic myelomonocytic leukemia (cmml), but with much better prognosis without specific therapy. we report a case of rald in a month-old male infant originally suspected to have jmml. results a -month-old male infant, born to an unrelated ethiopian father and turkish mother, presented with splenomegaly and leukocytosis ( , /l), monocytosis ( , /l), and thrombocytopenia ( , /l); hemoglobin was gm/dl and fetal hemoglobin concentration . %. family history indicated early death of three paternal uncles, two at age - months and one at age years. jmml was suspected and bone m a r r o w a sp i r a t i o n w a s p e r fo r m e d ; h ow e v e r, r e s u l t s o f immunophenotypic testing with flow cytometry analysis and cytogenetic testing with fish did not support this diagnosis. patient had a normal b level. serum igg ( . mg/dl [nl . - . mg/dl]) and igm ( . mg/dl [nl . - . mg/dl] levels were elevated and iga was normal. lymphocyte subset analysis showed % cd [nl - %], % cd [nl - %], % cd [nl - %] positive cells with normal percentages of cd and cd /cd cells; there were no cd -/ cd -t cells detected. given the findings of leukocytosis, monocytosis, splenomegaly, hypergammaglobulinemia, and a dearth of cd -/cd -t cells, the patient appeared to meet criteria for rald. mutation testing with next generation sequencing showed an nras missense mutation [c. g>t; pgly cys] in % of t cells and % of myeloid cells. the presence of the nras mutation, in the absence of other typical jmml findings or rasopathy syndrome features, supported the diagnosis of rald. there was no therapeutic intervention and, at one year of age, the patient was reported to remain clinically well; he relocated to ethiopia with his parents. conclusions rald is a nonmalignant clinical syndrome originally classified as a subtype of autoimmune lymphoproliferative syndrome (alps) but subsequently distinguished to be a separate entity due to lack of double negative t-cells, a mutation in fas/fasl/caspase- , or consistently elevated serum b levels. though also present in % of jmml patients, a somatic mutation in ras signaling protein (kras or nras), which controls b-cell tolerance and production of autoantibodies, is present in all reported cases of rald. while all previously reported cases of rald had somatic kras or nras mutations, our patient's nras mutation was present in more than % of t cells and myeloid cells, suggesting a heterozygous germline mutation; this has not previously been reported in rald and needs to be further confirmed ( ) the primary objective of this study was to evaluate hct outcomes in patients with was who underwent hct since in north america. we hypothesized that survival after hct from alternative donors such as cord blood has improved compared to published results, but that overall survival would be superior in patients who undergoing hct at a young age. methods: patients were enrolled on pidtc protocol , a multicenter retrospective natural history study of patients treated for was in north america since . clinical features, disease status, hct type and post-hct outcomes were analyzed in patients who underwent hct at pidtc centers between - . descriptive statistics such as median and range for continuous variables and counts and percentages for categorical variables were used to summarize characteristics of the study population. in addition, kaplan-meier curves were used for estimating survival probabilities results: diagnosis of was was confirmed by an expert review panel for eligibility. mutation in the was gene was available for patients, including nonsense (n= , %), frameshift (n= , %), missense (n= , %), splicing (n= , %), gross deletion (n= , %), in-frame deletion (n= , %), pr complex (indel) (n= , %). donor types included matched sibling (n= , %), unrelated adult volunteer bone marrow or peripheral blood stem cells (n= , %), umbilical cord blood (n= , %) and other related donors ( each, phenotypically matched related, haploidentical) . median age at time of hct was . months (range, . . months). the vast majority of patients received busulfan containing conditioning regimens ( %), with some receiving other myeloablative ( %) or reduced intensity regimens ( %). with a median follow-up of . years, overall survival was excellent with -year and -year survival probabilities of % ( % ci, - %) and % ( % ci, - %), respectively. survival was similar at year for recipients of hct from matched sibling ( %, % ci, - %), matched unrelated donor ( %, % ci, - %) or umbilical cord blood ( %, % ci, - % see figure) . importantly we confirmed that survival at year was better in patients who were < years old (n= ) compared to those who were years old (n= ) at the time of hct ( % versus %, respectively p= . ). this difference persisted when only unrelated donor recipients were analyzed ( % vs %, p= . ). overall the percentage of patients in our study who underwent hct at a young age was high ( %) compared to the literature ( % in moratto et al, , blood) . the rate of second hct was only % (n= ). cumulative incidence of acute grade - at days, acute grade - at days and chronic graft-versushost disease at year were % ( % ci - %), % ( % ci - %) and % ( % ci - ), respectively. conclusions: outcome of hct for was since shows excellent overall survival for all donor types, including umbilical cord blood with very low rates of second hct. importantly, hct at a younger age (< years old) continued to be associated with superior survival supporting the provision of hct earlier in the course of the disease. further analysis of the complete cohort is planned to determine whether age at hct has decreased in the modern era compared to pre- and to analyze factors associated with platelet and immune reconstitution, donor chimerism and autoimmunity. introduction/background: comel-netherton syndrome is a rare disease hallmarked by congenital ichthyosis, atopy, trichorrhexis invaginata (bamboo hair) and, within the past years, has been defined as a primary immunodeficiency. specific, rare genetic polymorphisms (c. t>a, pl h) in the fc receptor iiia (fcgr a) on natural killer (nk) cells have been shown to decrease nk cell function. patients with these defects present with susceptibility to severe and recurrent viral infections, however, not all fcgr a mutations result in this clinical phenotype. here we report on a child with a mixed genotype, presenting with congenital ichthyosis, atopy and recurrent bacterial and viral infections. methods: immunophenotyping of lymphocyte subpopulations were evaluated by flow cytometry. genomic dna was sequenced using next generation sequencing. all detected variants were then sequenced using sanger sequencing. cd dual epitope assay and evaluation of nk cell maturation was also performed. intravenous immunoglobulin replacement therapy was initiated. results: our patient presented at months of age for evaluation of food allergy. she has congenital ichthyotic erythroderma with pruritic atopic dermatitis-like skin eruptions and a history of hypernatremic dehydration immediately following birth, thin and easily broken hair and a history of failure to gain weight with appropriate catch-up growth following formula fortification. she has had multiple episodes of acute otitis media and recurrent upper respiratory tract infections associated with wheezing. t-, b-and nk cell quantitation by flow cytometry was normal, including naïve and memory b cells. immunoglobulins and vaccine antibody levels to haemophilus influenza type b, tetanus and streptococcus pneumoniae were normal. lymphocyte proliferation to mitogens was normal. natural nk cell cytotoxicity was initially decreased, however, subsequent cytotoxicity testing performed at months of age was normal. whole exome sequencing revealed a homozygous mutation in spink (c. - a>g) and a homozygous missense mutation in the first immunoglobulin domain of fcgr a (c. t>a), both variants of unknown significance and under additional investigation. this homozygous mutation in spink was not detected in the patients healthy, unaffected sibling. conclusions: this is a case of an infant with a clinical phenotype consistent with comel-netherton syndrome, however genotyping has revealed homozygous mutations in spink and fcgr a, suggestive of a possible mixed genotype. a recent large study investigating the use of whole exome sequencing to identify variants implicated in primary immunodeficiency found that in % of families, more than gene contributed to the immunodeficiency phenotype. it should therefore be kept in mind that variability in an individuals clinical phenotype may be attributable to the presence of a mixed genotype. introduction/background: cartilage-hair hypoplasia (chh) is a rare autosomal recessive disease caused by mutations in the rmrp gene and can manifest with scid. allogeneic hct is a curative therapeutic option for scid associated with chh. bordon et al. reported an overall survival of % ( / patients) following hct with a predominantly myeloablative conditioning regimen with busulfan and cyclophosphamide. data on outcomes of hct using a ric regimen are limited. objectives: we herein report our experience with allogeneic ric hct for scid associated with chh. methods: we reviewed records of all patients who underwent allogeneic hct for scid associated with chh at our institution, with a ric regimen containing alemtuzumab, fludarabine and melphalan. results: five patients ( male, female) underwent allogeneic ric hct for chh at median age of months (range, months years). all patients had biallelic mutations in the rmrp gene, and met pidtc criteria for scid, prior to hct. two patients were diagnosed by newborn screening for scid. one patient received serotherapy only (rituximab mg/m daily x days, anti-thymocyte globulin . mg/kg daily x days) conditioning for initial hct but developed graft failure and subsequently received a ric regimen for second hct. all patients received a ric regimen consisting of alemtuzumab mg/kg over days(n= ) or mg/kg over days (n= ) and fludarabine mg/kg (weight < kg, n= ) or mg/m over days (n= ), and single dose of melphalan . mg/ kg(weight < kg, n= ) or mg/ (n= , dose reduced by % for preexisting sclerosing cholangitis with grade liver fibrosis). patients received matched (n= ) or - allele mismatched (n= ) bone marrow grafts and all but grafts were from unrelated donors. all patients received cyclosporine and steroids for gvh prophylaxis. all patients engrafted with full donor chimerism. three patients developed mixed chimerism, but continue to maintain donor t cell chimerism > %. two patients developed vod of the liver (one patient developed mild vod whereas the patient with pre-existing sclerosing cholangitis developed severe vod). one patient developed grade skin gvhd and one patient developed limited chronic skin gvhd. none of the patients developed liver or gi-gvhd. all patients remain alive at a median follow up of years (range months- years) with good t-cell and b-cell immune reconstitution. conclusions: our experience suggests that allogeneic ric hct with alemtuzumab, fludarabine and melphalan for scid associated with chh is curative, offers durable t-cell engraftment, low gvhd along with excellent survival and might be preferable over a myeloablative conditioning regimen, to further limit toxicity in young infants, especially in the era of newborn screening. refractory thrombocytopenia in a patient with wiskott-aldrich syndrome despite hematopoietic stem cell transplantation: eltrombopag as a therapeutic option. mauricio chaparro-alzogaray , marcela estupiñan-peñaloza , gisela barros-garcía , oscar correa-jimenez pediatric hematologist/oncologist, hsct unit, fundación homi hospital de la misericordia pediatric hematologist/oncologist, fundación homi hospital de la misericordia pediatrics resident, universidad nacional de colombia introduction/background: wiskott-aldrich syndrome (was) is an xlinked disorder characterized by: immunodeficiency, eczema and hemorrhage due to thrombocytopenia [ ] . hematopoietic stem cell transplantation is the treatment of choice, with an overall survival of approximately % regardless of the source of the stem cells [ ] . eltrombopag has been used as a pre-transplant stabilization therapy [ ] . in our knowledge, there are no reports of its use for the management of post-transplant autoimmune cytopenias in was. objectives: to present a clinical case in which the complexity of diagnosis and management of wiskott-aldrich syndrome is highlighted, as well as the usefulness of eltrombopag in post-transplant persistent thrombocytopenia. methods: clinical case presentation and review of literature. results: clinical case: -month-old infant with a history of thrombocytopenia identified at the third day of life (positive serology and viral load for cmv), bacteremia due to serratia marcescens at months, intermittent diarrhea from months, multiple platelet transfusions, ivig cycles, and ambulatory management with corticosteroids; who consulted the er for a -day of bloody diarrheic stools, generalized petechiae and fever. he was irritable with generalized petechiae in the lower extremities, diaper area dermatitis with signs of superinfection. admission cbc: wbc: /mm , neutrophils /mm , lymphocytes /mm , monocytes /mm , hb . g/dl, ht . % mcv . fl, mch . g/dl, mcmh . %, platelets /mm mpv fl. bone marrow aspiration was performed that ruled out proliferative syndrome. immunological profile with normal immunoglobulins and flow cytometry showed t lymphocytosis with cd / cd inversed ratio, direct coombs was positive. he progressed to refractory thrombocytopenia, with intracranial bleeding and transfusion platelet requirement every h. molecular diagnosis of wiskott-aldrich syndrome was made and it was decided to carry out an allogeneic transplant of unrelated umbilical cord. he showed adequate response, % chimerism; however, he persisted with important cytopenias, without response to management with ivig and corticosteroids, so that on day + he restarted eltrombopag that he received prior to transplantation. from day + he received rituximab for weeks. he continued with eltrombopag months post-transplant. currently without cytopenia and without complications, he completed the post-transplant year without additional complications. conclusions: was represents a great diagnostic challenge in pediatric clinical practice. despite the therapeutic option of transplantation of hematopoietic stem cells, patients may persist with different complications, within these; autoimmune cytopenias will require additional therapies. eltrombopag, in addition to being used as a pretransplant transient measure, is useful for the management of post-transplant persistent thrombocytopenia in these cases. references: . immunol allergy clin north am. ; ( ) methods: retrospective study of medical records in two centers of immunology results: in our center we have p with pid, with made retrospective study of medical records, up today we have registered p ( %). according to this record, these diseases are distributed in the following way: predominantly antibody disorders: p ( %), predominantly t cell deficiencies: p ( , %), phagocytic disorders: p ( . %), complement deficiencies: p( . ), other well pid: p( . ), autoimmune and immune dysregulation syndromes: p( . %), unclassified immunodeficien cies: p( . %). predominantly antibody deficiencies are the most common pid, which comprise more than half of our all p. these group is represented by: specific iga deficiency (sad): p, specific igg deficiency: p, transient hipogammaglobulinemia: p, common variable immunodeficiency (cvid): p, agammaglobulinemia linked x: p, agammaglobulinemia unknown causes: p, secondary hipogamma globulinemia: p, selective igm deficiency: p, subclasses deficiency: p, cd l deficiency: p, hyper igm unknown causes: p, other hyogammaglobulinemia: p. among them, selective iga is the most common pid.in our cohort of unclassified immunodeficiency, we have p with auto inflammatory syndrome, such as family mediterranean fever, hyper igd syndrome and candle like-syndrome and p with nk deficiency. in all our pid p, are under replacement gammaglobulin (gg)treatment, p use intravenous gg and p use subcutaneous gg conclusions: the lasid registry model represents a powerful tool to improve health policies, showing that are under diagnosed and should receive more attention. more data are needed to define the exact prevalence of pid to avoid underestimation of these diseases due to under reporting. as different reports in different countries, in our centers predominantly antibody deficiencies are the most prevalent. although the number of patient diagnosed with pid, is growing. many physicians still know little about these disorders. introduction/background: a number of anticonvulsant medications have been shown to cause hypogammaglobulinemia. lamotrigine is a phenyltriazine anticonvulsant medication that is approved to treat seizure disorders and bipolar disorder. objectives: we describe a patient who developed hypogammaglobulinemia secondary to lamotrigine use. methods: we performed a chart review and case-based literature review. results: a- -year old female presented to immunology clinic for evaluation of panhypogammglobulinemia. she was initially evaluated by general internal medicine for lightheadedness and fatigue and was found to have serum igg of mg/dl ( - mg/dl), igm mg/dl ( - mg/dl) and iga mg/dl ( - mg/dl). the patient reported a history of recurrent sinus infections occurring around twice per year. she reported resolution with oral antibiotics. she denied any history of pneumonia and was never hospitalized for treatment of infection. she did report increased number of recurrent upper respiratory infections; around - per year for the last several months. she denied any family history of primary immune deficiency. she was never prescribed corticosteroids or immuno-modulators. her past medical history was significant for bipolar disorder type for which she was prescribed lamotrigine mg oral twice daily seven months prior to her initial visit. the medication was prescribed prior to the onset of recurrent sinus infections. she had protective post-vaccination titers against tetanus, diphtheria, and acellular pertussis and non-protective pre-vaccination pneumococcal titers. post vaccination pneumococcal titers were not obtained due to the cost of the pneumonia vaccine. the patient was started on prophylactic azithromycin three times weekly. lamotrigine was discontinued and the patient switched to lurasidone due to concern for anticonvulsant-induced panhypogammaglobulinemia. her serum immunoglobulin levels increased after . months off of lamotrigine (igg mg/dl, igm mg/dl, iga mg/dl), and she is currently asymptomatic without further infections. conclusions: lamotrigine is likely responsible for the reversible hypogammaglobulinemia in this patient. serial immunoglobulin levels should be checked in all patients who experience recurrent sinopulmonary infections while on lamotrigine. in two separate reports, lamotrigine induced hypogammaglobulinemia began within months and months of starting therapy respectively. in another study, hypogammoglubinemia was reported in % of patients taking lamotrigine. further studies are needed to accurately describe onset and frequency of hypogammgeobulinemia in these patients. currently, it is unclear whether post vaccine titers are protective in patients with lamotrigine induced hypogammaglobulinemia. introduction/background: the association of vaccine strain rubella virus with cutaneous and sometimes visceral granulomatous disease has been reported previously in patients with various primary immunodeficiency disorders (pids). the majority ( / ) of these pid patients with rubella positive granulomas had dna repair disorders, namely ataxia telangiectasia (at) (n= ) or nijmegen breakage syndrome (nbs) (n= ) or rag (n= ) and rag (n= ) deficiency. objectives: to support this line of inquiry, we provide additional descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders as well as additional previously unreported pid patients with rubella virus induced cutaneous granulomas. methods: we provide in-depth descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders including at (n= ) and dna ligase deficiency (n= ). we also provide in-depth descriptive data on additional previously unreported pid patients with rubella virus induced cutaneous granulomas, including cartilage-hair hypoplasia (n= ), mhc class ii deficiency (n= ), whim syndrome (n= ), and coronin- a deficiency (n= ). results: the median age of the patients is . years (range - ). the majority are females ( %). cutaneous granulomas have been documented in all cases while visceral granulomas (spleen and liver) were observed in cases. t cell and b cell lymphopenia as well as hypogammaglobulinemia or impaired antibody formation were present in most patients. all patients had received rubella virus vaccine. the median age at presentation of cutaneous granulomas was months (range - ). the median duration of time elapsed from vaccination to the development of cutaneous granulomas was months (range - ). the diagnosis of rubella was made by pcr in % of patients and by immunohistochemistry in the remainder. one patient was confirmed to have vaccine strain rubella virus. hematopoietic cell transplantation was reported in three patients. rubella associated complications did not contribute to death among those patients who died ( %). conclusions: of the now cases, ( %) share the diagnosis of a dna repair disorder and confirm that chronic rubella virus infection is associated with cutaneous granuloma formation. analysis of patients with dna repair disorders and other pids with this complication will help clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection and may lead to defect-specific therapies. introduction/background: introduction/background: hizentra® is a % liquid igg product approved for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). limited information on use of hizentra® is available for children who have received hematopoietic stem cell transplantation (hsct). objectives: objectives: the aims of this study are to determine the safety and efficacy of hizentra® in pediatric patients post hsct, and to characterize reasons for switch from intravenous igg (ivig) to subcutaneous (scig) delivery following hsct. methods: methods: a retrospective chart review involved pidd infants and children (mean age . [range . to . ] months) status post hsct who received hizentra®. ten patients received hizentra® by pump administration, and patients by manual push. results: results: hizentra® administered weekly to children included an average of infusions (range - ). the mean dose was mg/kg/ weeks. the mean igg level was mg/dl while on hizentra® in patients compared to a mean trough igg level of mg/dl in patients during immunoglobulin administration prior to hizentra® of most children. four patients naïve to igg therapy were started on hizentra®. average infusion time was . (range - ) minutes for manual entry and (range - ) minutes for pump entry, and the average number of infusion sites was . (range to ). local reactions were mild and observed in / ( . %) children. ten patients had no local reactions. no serious adverse events were reported. the rate of serious bacterial infections (sbi) was . per patient-year while receiving hizentra®, similar to reported efficacy studies. the reasons for switch from ivig to scig in patients (some patients had multiple reasons) were improved igg serum levels and physician desire for steady state serum igg levels (n= ), loss/lack of venous access (n= ), patient/caregiver preference (n= ), home site of care preference (n= ), and physician preference for scig (n= ). conclusions: conclusions: hizentra® is a safe and effective option in children who have received hsct. reasons for switch from ivig to scig included improved serum igg levels, desire for steady state serum igg levels, and patient/caregiver preference. consulting medical advisor, immune deficiency foundation introduction/background: it is currently unknown how many persons with primary immunodeficiency (pi) receive the seasonal influenza vaccination, and what proportion becomes ill. additionally, the effect of immunoglobulin (ig) replacement therapy on the frequency of influenza diagnosis and severity of symptoms is not known. objectives: the current study sought to measure the prevalence of seasonal influenza vaccination and diagnosis among persons with pi, specifically those with antibody deficiencies: x-linked agammaglobulinemia (xla), common variable immunodeficiency (cvid) and hypogammaglobulinemia. methods: , email invitations were delivered to members of the immune deficiency foundation database requesting participation in an online survey regarding their zoster, influenza and varicella vaccination experiences. data from persons with xla (total n= ; children age< ; n= ; adults age> , n= ; % male; % white non-hispanic), cvid (total n= ; children age< , n= ; adults age> , n= ; % female; % white non-hispanic), and hypogammaglobulinemia (total n= ; children age< , n= ; adults age> , n= ; % female; % white non-hispanic) were analyzed for the - influenza season. results: overall, % (n= ) of the sample received a seasonal influenza vaccination during the - influenza season. persons with xla were less likely to receive an influenza vaccination ( %, n= ), than persons with cvid ( %, n= ) and hypogammaglobulinemia ( %, n= ). a fifth of respondents ( % overall, % of children and % of adults) who attend school and/or work stayed home at some time to avoid seasonal influenza. though the majority ( %, n= ) were not diagnosed with influenza, when stratified by age, children were twice as likely to be diagnosed than adults. of the children sampled, % (n= ) were diagnosed with seasonal flu compared to % (n= ) of the adults that were sampled. the role of replacement ig therapy in protection against flu can best be examined in the patients with xla since these patients cannot make specific antibodies to vaccine and only % (n= ) received the vaccine. although % (n= ) of the individuals with xla were exposed to flu, only % (n= ) were diagnosed with influenza; two of whom were not receiving ig therapy at time of diagnosis. concerning influenza severity, individuals receiving ig therapy at the time of flu diagnosis tended to have modestly milder symptoms than those not receiving ig treatment (e.g., less likely to report sore throat ( % versus %, p<. )), but the sample size is too low to draw firm conclusions. conclusions: a high proportion of antibody deficient persons received a seasonal influenza vaccination for the - influenza season. in addition to vaccination, many individuals attempted to avoid influenza infection by remaining home from school and/or work. there was a suggestion that ig replacement therapy may partially protect xla patients from symptomatic flu, although the role of cell mediated immunity in protection against flu is not clear. a prospective study could determine if ig replacement therapy partially protects against clinical flu symptoms in xla and cvid patients using two groups those that receive the current flu vaccine vs. those that do not receive the flu vaccine. introduction/background: fanconi anemia (fa) is an autosomal recesive or x-linked genetic disorder, characterized by cytopenia or bone marrow failure, this will lead to a severe anemia, neutropenia and thrombocytopenia, requiring frequent interventions with diversified therapies, including hematopoietic stem cell transplant (hsct). a complete immune reconstitution is requiere for a success transplant. one of the main upcoming events asociated after the hsct, is the secundary immunodeficiency, which is asociated with a significant morbility and mortality in the patients. to rich a successful alogenenic-hsct, is impending a complete reconstitution of the t cells immunity, for this it is crucial the presence of factors like; thymic activity of the hsct recipient, biological features of the allograft (eg, degree of histocompatibility, number and type of infused donor t cells) and preparative regimens. objectives: describe the kinetics of the immune reconstitution in fa patients after alogenic-hsct, as well as the infections associated during this process and other comorbilities. methods: we decribed the lympocyte population (t, b and nk) in pb measured by flow cytometry in fa patients on days + , + , + , + , + and + post alogenic hsct from a match related donor. the conditioning was base on fludarabine mg/m , cyclophosphamide mg/kg and antithymocyte globulin rabbit mg/kg. infectious-disease survillance for virus was determined by the quantification by dna pcr-rt for cmv, ebv, adenovirus, as well as the presence of galactomannan and candida sp antibodies, or isolation of fungus or bacterial culturing in the cases of infectious disease of known or uknown aetiology. results: the lymphocyte population mesurement was performed in a total of five fa patients who undergo to an allogenic hsct, all of them received stem cells from a match related donor and the source was bone marrow. successful engraftment was observed in all patients, there were no deaths reported. after the hsct was performed, the kinetics of recovering for the distinct lymphocytes subsets was the the following: nk cells (cd +cd +) were the first to recover, followed by cd+ t cells, b lymphocyte and finally cd+ t cells (figure ) all of them rich normal values and remain stable. three out of five patients presented infectious disease: two of them were cmv positive, one patient has a concurrent detection of adenovirus, and in the other was detected aspergillus, in both of them it was presented at day + . the third one developed acute gvhd which progressed to a chronic gvhd, at day + was diagnosed on him listeria monocytogenes meningitis, at that momento he has just cd+ +cd and cd + reconstituded. there is no new infectous disease detected in any of the patients after they reconstituted cd + t cells. conclusions: complete immune reconstitution is the decisive for the presence of several morbilities and mortalities, mainly because of oportunistic infecctions and gvhd. we show that the kinetics of recovery of the different populations of lymphocytes follows those patterns also described for patients with other hematological malignancies: early recovery of nk cells, followed by effector cytotoxic t cells and b cells, and finally, cd + t-helper cells. the utility of post-transplant monitoring of pb-lymphocyte subsets for improved follow-up of patients undergoing bmt and prevent opportunistic infections. introduction/background: early detection of primary immunodeficiency diseases (pids) before serious infection is of utmost importance and a question of patient survival. one of the main organs involved in pids is skin with mucocutaneous manifestations being one of the common complaints in pids. the presenting skin symptoms may serve as an essential element in early diagnosis of pids. objectives: this study aims to determine characteristics, frequency, nature and incidence of skin manifestation in pid patients seen in qatar. methods: this retrospective study was conducted at hamad medical corporation the only tertiary hospital in qatar from january to july .the subjects included were pid patients < years old, who had dermatological complaints. information collected included dermatological diagnosis, gender, age at onset of signs and symptoms, age at definite diagnosis, family history of related disease, any lab results such as pathology or viral studies obtained from the clinical medical record. patients were diagnosed and classified according to clinical and laboratory criteria by the international union of immunological societies primary immunodeficiency committee. results: a total of patients were studied and skin/mucocutaneous manifestations were found in patients ( %) with male to female ratio of . : . age at onset of skin manifestations ranged from to years. skin manifestations were divided in two categories according to presentation before pid diagnosis (primary manifestations n= , %) and during the disease period (secondary manifestations n= , %). the type of pids predominantly having primary manifestation were scid (n= , %) cvid and cgd (n= , %, each), xla, at and griscelli syndrome (n= , %, each).various manifestations which were atypical in their presentation and persistently found in patients who did not respond to any effective treatment (n= ) led to the basis of clinical immunological study and diagnosis including rare diagnosis such as ipex. whereas secondary manifestations were primarily reported in scid (n= , %) cgd, at and digeorge syndrome (n= , %, each). the nature of skin manifestations varied in both groups, primary manifestations notably had % cutaneous infections comprising of thrush ( %) stomatitis ( %) viral rashes ( %) bacterial rashes ( %) impetigo ( %) and fungal rash ( %). eczema/atopic dermatitis were % and other minor miscellaneous skin alteration cases found were apthous mouth ulcer ( ) erythroderma ( ) gvhd like rash ( ) alopecia ( ) psoriasis ( ) and scleroderma ( ) . secondary manifestations were identified as % cutaneous infections % eczema and infantile seborrheic dermatitis ( ) pruritus ( ) gvhd and alopecia ( ) each. the causative microorganisms were confirmed in % of the cases via lab cultures whereas common infections such as chicken pox and herpes were validated through clinical symptoms. none of the cases were biopsied. overall, highest skin infection was seen in ataxia telangiectasia ( ). other pids with prominent cutaneous manifestations included cgd ( ), digeorge syndrome ( ), hyper ige syndrome ( ) and scid (rag or , cases). more than two types of skin manifestations were found in % patients over the due course of their illness. conclusions: an awareness of various cutaneous and skin disorders associated with pid which are persistent and unresponsive to treatments among dermatologist and family physicians is crucial to raise suspicion for early detection, timely management and prevention of complications. introduction/background: ataxic telangiectasia (at) is a rare neurodegenerative autosomal recessive disease associated with immunodeficiency, poor coordination and disability. ataxia telangiectasia has a diverse clinical heterogeneity which may often lead to an incorrect diagnosis or late detection of the disorder resulting in exposing the patient to unnecessary radiation from various sources. objectives: we present a female child from the asian subcontinent that was seen for the first time with ulcerative skin lesions, failure to thrive and marked lymphopenia. methods: a search of the pubmed database was carried out, using different combination of the terms "ataxia", "telangiectasia", typical , atypical and "presentation" results: four-year-old pakistani girl, product of first degree nuptial presented with generalized vesicular rashes mainly on abdomen and scalp which turned into ulcers with residual hypo pigmented lesions and diarrhea. she had short stature, failure to thrive (weight and height < th percentile) and no previous infection or family history of primary immunodeficiency diseases (pid). mild delay in milestones was observed especially in speech. immunizations were up-to-date including bcg. on examination she had mild ocular telangiectasia but no cutaneous involvement. investigations revealed lymphopenia ( . ) and eosinophilia. workup for diarrhea including duodenoscopy revealing subtotal villous atrophy however the biomarkers for celiac disease were negative. immune system workup showed high igg levels, normal iga, ige and igm level. igg subclasses: low igg , and , antibody titers to h.influenza and pneumococcus vaccine were relatively low. lymphocyte subsets showed low cd ,cd and high nk cells ( %), low naïve t cells ( %) and inverted cd /cd ratio.t cell function with post pha was . % but had normal response to cd . alfa feto protein was requested due to continued low cd count and the level was found to be high: . iu/ml. next-generation sequencing (ngs) showed homozygous mutation for trp fs in chromosome of atm gene confirmed by whole exome sequencing. immediate treatment with ivig was started leading to mark improvement of skin lesions, diarrhea and weight gain. conclusions: the index of suspicion of at should be highlighted when deciphering lymphocyte subset. currently there is no neonatal screening for pid diseases and next generation sequences in qatar. once implemented it may prove beneficial in discovering cases from early infancy and reaching upon a definite diagnosis. introduction/background: chronic granulomatous disease (cgd) is a genetic disorder of the nadph oxidase complex in phagocytes which results in impaired production of microbicidal reactive oxygen species that can lead to recurrent life-threatening bacterial and fungal infections. the majority of affected patients in the united states have a x-linked defect in the cybb that encodes gp phox protein followed by an autosomal recessive defect in the ncf gene that encodes p phox . x-linked female carriers show populations of neutrophils and following lyonization, and severe skewing of x-chromosome inactivation can get cgd type infections. a recent study has shown that the lower dihydrorhodamine (dhr) percent in female carriers predicts a higher infection risk but carrier state on its own predicts autoimmunity results: a -year-old female presented for an evaluation of recurrent fevers. she had a history of several lobar pneumonias in childhood. at age , she developed erythematous bumps on her abdomen that were initially non-bothersome but later became painful. she then developed recurrent fevers, chills, and rapid weight gain. biopsy of the skin lesions showed subcutaneous panniculitis-like t cell lymphoma. she was treated with chemotherapy and found to be in complete remission. she had no previous family history of recurrent infections. at the completion of her chemotherapy, her -year-old son became septic in the hospital and was then diagnosed with cgd from mutated cybb gene. post chemotherapy, the patient had a prolonged course with an atypical lung infection that required bronchoscopy and video thorascopic surgery, however no bacteria was ever identified. she did improve after a long course of ivantifungals. evaluation for immunodeficiency was done after she had a recurrence of low grade fevers, cough, and fatigue. dhr flow cytometry with phorbol myristate acetate (pma) was done which showed . % neutrophil oxidative burst activity after stimulation, consistent with a highly skewed lyonization pattern in x-linked cgd. conclusions: this case demonstrates an interesting lesson in a woman with recurrent infections since childhood whose clinical picture was confounded by her history of malignancy. the abnormal lung infections prior to malignancy were alarming and gave cause to do additional immunology testing. however, it begs the questions, if she did not have a son, would she have been diagnosed with severe x-lined carrier disease. introduction/background: measurement of the specific antibody response following vaccine challenge provides clinicians with a better understanding of the adaptive immune response in individuals undergoing immunological evaluation. objectives: we hypothesised that after classification of primary immunodeficiency (pid) patients based on vaccine response (vr), further division using igg subclass (iggsc) - measurements may identify additional patients with abnormal b cell function and patients with different frequencies of infection at presentation. methods: vrs and serum iggsc concentrations were quantified using the vacczyme anti-pneumococcal polysaccharide (pcp) igg elisa and human iggsc liquid reagent kits (the binding site group limited, uk) in pid patients ( : . m:f, median age . years, range - ). all patients were immunised with pneumovax® (sanofi pasteur msd). the lower limits of published normal iggsc ranges were used as cut-off values (igg . g/l, igg . g/l and igg . g/l). pcp concentrations equal to or less than mg/l post-vaccination was considered an abnormal response. results: agreement between vr and iggsc measurements was % (p= . ). the frequency of respiratory tract infections at presentation among pid patients with a normal pcp igg vr was % and % among patients with an abnormal vr. subsequently, four separate groups could be identified by including iggsc measurements. the frequencies of infections at presentation were for the vr+/iggsc+ group (n= ) % vs. % for the vr+/iggsc-group (n= ); and % for the vr-/iggsc+ group (n= ) vs. % for vr-/iggsc-individuals (n= ). conclusions: these results confirm that vr and iggsc measurements are independent serum biomarkers of humoral immunity. taken together the vr and iggsc results provide more detailed information about the immune status that may influence diagnosis, treatment and monitoring decisions. introduction/background: hizentra® is a % liquid igg licensed for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). subcutaneous immunoglobulin (scig) use in autoimmune conditions is reported. stiff person syndrome (sps), a rare neurologic disorder characterized by fluctuating muscle spasms and rigidity, is mediated by autoantibodies to glutamic acid decarboxylase (gad). symptoms of sps have been shown to improve after administration of intravenous immunoglobulin (ivig) however, there is a paucity of information regarding use of scig in sps. objectives: the aim of this study is to describe the use of hizentra® in patients with sps, including indications for scig, clinical characteristics of patients, and clinical and laboratory response to scig. methods: a multicenter retrospective chart review examined patients with stiff person syndrome treated with hizentra®. results: sps was diagnosed in patients at and years. both patients were started on ivig, steroids, and rituximab prior to initiation of weekly hizentra® (ages and years). the average dose of hizentra was mg/ kg weekly. the average igg level while receiving ivig was , . mg/dl, similar to the average igg level while on hizentra was , mg/dl. the number of administration sites ranged from - , and duration of infusions ranged from - minutes. serum anti-gad antibody levels prior to hizentra® were u/ml and u/ml respectively. anti-gad antibody level during treatment with hizentra (available for one patient) was > , u/ml, and , u/ml following discontinuation of hizentra®. one of the reasons for switching to hizentra® was lack of response while on ivig. the average number of hizentra infusions were . patients reported improvement in spasticity related to sps while on hizentra®, and one patient had improvement in seizures. one patient discontinued hizentra® in favor of intravenous immunoglobulin (ivig) due to physician preference. the most common side effects were local reactions including pain, pruritus, and redness. no serious adverse events were reported. conclusions: hizentra® was associated with improved symptoms in sps in both patients including decreased spasticity, and improved seizure frequency in one patient. serum anti-gad levels did not decrease following administration of hizentra. hizentra® was well tolerated in patients with sps, with most side effects reported as mild. hizentra® may be considered as an alternative to ivig treatment in patients with sps. introduction/background: combined immunodeficiency (cid) has been associated with a spectrum of secondary gastrointestinal manifestations, including infectious and non-infectious causes. abatacept, a soluble ctla -igg fusion protein targeting t cell activation, has demonstrated benefit in the treatment of autoinflammatory manifestations in patients with cid due to underlying heterozygous germline mutations in ctla and lrba. objectives: herein, we report two patients with cid characterized by low immunoglobulin levels, low class-switched memory b cell counts, and low peripheral naïve cd + t cell counts. both patients suffered from severe and persistent diarrhea complicated by protein-wasting and malnutrition, ultimately diagnosed as biopsy-consistent autoimmune enteropathy. the clinical history of patient was also notable for granulomatous lung disease and autoimmune cytopenias. methods: a t-regulatory cell disorder was considered in both cases, however, ctla and lrba were found to be unaffected by whole exome sequencing (patient ) and/or flow cytometry (patient ). due to progressive worsening of the autoimmune enteropathy despite management with chronic steroids (both patients), combination rituximab/mycophenolate mofetil (patient ), and total parenteral nutrition (tpn) complicated by recurrent infections (patient ), abatacept was trialed at mg sc weekly. results: in both patients, the start of abatacept therapy produced a dramatic improvement as measured by decreased stool frequency, improved weight gain, and decreased protein-wasting. patient has been maintained on this monotherapy for over one year, with improvement in the gastrointestinal as well as pulmonary autoinflammatory complications. the only documented adverse event has been hepatitis b reactivation, managed with tenofovir and abatacept continuation. patient has required azathioprine/abatacept combination therapy for clinical stabilization and is without a significant adverse event to date. conclusions: we conclude that abatacept may be a relatively safe and effective therapeutic in the management of severe autoimmune enteropathy in the background of cid, even when used outside of the classical clinical context of ctla or lrba haploinsufficiency. objectives: as increasing the outbreak of allergic diseases, study for treatments comes to the force. in this research, we aimed to assess the effects of sg-sp , a derivative of gallic acid, on mast cell-mediated allergic inflammation using various animal and in vitro models. methods: ovalbumin-induced systemic anaphylaxis and immunoglobulin e (ige)-induced passive cutaneous anaphylaxis are standard animal models for immediate-type hypersensitivity. oral administration of sg-sp hindered the allergic symptoms in both animal models. these inhibitions were deeply related to the reductions of histamine and interleukin- . results: sg-sp reduced degranulation of mast cells and expression of inflammatory cytokines in a dose dependent manner. sg-sp showed better anti-allergic effects compared to gallic acid and dexamethasone. down regulations of intracellular calcium level and nuclear factor-b activation by sg-sp were causative of the reduction of allergic mediators. to anticipate the exact target of sg-sp , phosphorylation of proteins involved in mast cell signalling was assessed. sg-sp suppressed the activations from lyn and was aggregated with high affinity ige receptor (fcri). conclusions: from these results, we assured that sg-sp directly interact with fcri. all together, we propose that sg-sp might be a therapeutic candidate for allergic disorders. ( ) submission id# systematic assessment of pain in patients with primary immune deficiency using validated pain questionnaires: a prospective study. introduction/background: the number of primary immunodeficiency (pid) patients is rising dramatically because of good medical care as well as increased awareness. around in live births are affected. more than disorders have been discovered so far and this number is expected to rise in the coming years. as with any other chronic illness, pid patients are also prone to acute and chronic pains. chronic pain is a big challenge and lack of understanding of the etiology and underlying mechanisms limit our ability to diagnose or treat it effectively. objectives: to systematically assess chronic pain in patients with pid using validated questionnaires and to try to understand the underlying mechanisms of neuropathic versus non neuropathic pain. methods: short-form mcgill pain questionnaire (sf-mpq) is a recognized way to ascertain different pain characteristics as well as severity. a validated arabic version of the sf-mpq was used to prospectively assess chronic pain in patients with pid. furthermore, a validated arabic version of the neuropathic pain questionnaire-short form (npq-sf) was also used to assess neuropathic pain and to differentiate it from nonneuropathic pain. a total of patients with pid were included. results: males: females were . %: . % respectively. mean age was . years. commonest diagnosis was combined immune deficiency in % of the patients followed by common variable immune deficiency which was . %. chronic pain was found in % of the patients that participated in the study. % of the patients who complained of pain found it to be tiring and exhausting. % each had aching or heavy or sharp pain. % had cramping pain, % had tender pain and % characterized their pain as throbbing or burning. % felt shooting pain, % had splitting pain and % had gnawing pain. % found it to be frightening and % described it as being sickening in itself. the commonest pain complaint was abdominal pain in % of the patients followed by headache % and chest pain %. pain attributed to neuropathy was present in about . % of the study population. most patients described that they had been experiencing pain for at least - years. conclusions: this is the first international study to understand the prevalence, duration and severity of chronic pain among pid patients. a significant number of patients reported ongoing pain. this is the first time any kind of pain has been studied systematically in the patients with primary immune deficiency. treating pain should have a major impact on improving the patients quality of life. introduction/background: primary immunodeficiency (pidd) patients, particularly those with severe t cell defects, are at increased risk of infections. bone marrow transplant also contributes to significant t cell lymphocytopenia, which can be associated with similar risks. several prophylactic measures, which vary per institution, are placed on these patients in order to prevent infections. we report on a likely outbreak of rapid growing nontuberculous mycobacteria (ntm) at our institution, with a suspected association to tap water objectives: to recognize nontuberculous mycobacteria as a threat to patients with immunodeficiency disorders methods: case series of consecutive patients admitted to one institution with similar infections results: in a period of months, / to / , patients admitted to our institution were found to have rapid growing ntm species on central line culture or bronchoalveolar fluid. these cultures were obtained due to fever and symptoms of systemic infection. per institutional practices no peripheral blood samples were obtained at the time of initial culture. all had significant t cell dysfunction: wiskott-aldrich syndrome ( ), an undefined combined immunodeficiency ( ) , and three patients post bone marrow transplantation (bmt), one months ( days) post an unrelated cord blood transplant for farber syndrome, one days out of a matched related transplant for epstein barr virus (ebv)-associated lymphoproliferative disorder, and one patient with high risk neuroblastoma days out of his second tandem autologous transplant. both allogeneic transplant recipients had received serotherapy. all species were rapid growing, identified as mycobacterium mucogenicum (n= ), immunogenum (n= ), or abscessus-chelonae complex (n= ). the patients had not shared rooms, caregivers, invasive procedures, or medications from the same batch. three ( %) cases met cdc criteria for hospital acquired infections (hai). all patients were in general ward rooms, and were on standard precautions given diagnosis had not been established (n= ) or they were considered outside the typical window of strict bmt precautions (n= ). ntm species were subsequently isolated from hospital tap water. this has resulted in a significant increase in infection precautions, including the use of sterile water only for cares, as well as bottled water for drinking (with no use of ice machines), for all patients being evaluated for pidd or with significant lymphocytopenia. conclusions: ntm are a threat to patients with pidd. tap water is a potential source of mycobacterial infections in pidd patients. minimizing exposure risk to water sources containing ntm is very important in this population. patients with concern for pidd or significant t cell lymphocytopenia should take steps to avoid ntm exposures, including the use of sterile water for cares, and bottled water for drinking. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is a rare autosomal recessive condition leading to severe combined immunodeficiency and neurologic impairment, typically presenting in early childhood. the condition is progressive and typically fatal in the first or second decade of life without hematopoietic stem cell transplantation (hsct). objectives: to illustrate the clinical and laboratory presentation of pnp deficiency with a novel mutation in the pnp gene via a case study. results: case: a four year old female of hispanic descent, with a past medical history of spastic diplegia, initially presented with chronic nasal congestion, recurrent sinusitis, and cough. laboratory studies were significant for an alc cells/mcl ( - cells/mcl). she was lost to follow-up for two years, before returning to medical attention for pneumonia. evaluation of lymphocyte subsets at age years revealed cd + cells/mcl ( - cells/mcl), cd + cells/mcl ( - cells/ mcl), cd + cells/mcl ( - cells/mcl), and cd + cells/mcl ( - cells/mcl). t cell mitogen stimulation to pha measured by flow cytometry was % of control. pnp activity was nearly absent and urine guanosine and inosine were significantly elevated. gene sequencing revealed a homozygous c. a>g mutation in the pnp gene. prophylactic antimicrobials were started. despite strong recommendation for hsct, the patient was again lost to follow up until age years, at which time she was found to have progressive lymphopenia, bronchiectasis, and developmental delay. t cell mitogen stimulation to pha measured by thymidine uptake assay was % of control and pnp functional activity was undetectable. the patient underwent a / matched unrelated hsct six years after her initial presentation. one year post-transplant, the patient continues on immunoglobulin replacement therapy. her course was complicated by cutaneous gvhd, treated successfully with topical corticosteroids. conclusions: this case study illustrates the progressive nature of pnp deficiency in the first decade of life. our patient is notable in that she survived without significant medical intervention to the age of years. her presentation at age years was not unlike those previously reported in the literature, with muscle spasticity, ataxia, and recurring bacterial infections. to the authors knowledge, this case reports a novel mutation in the pnp gene. introduction/background: severe congenital neutropenia (scn) is a primary immunodeficiency disease characterized by early onset recurrent infections, persistent severe neutropenia and congenital genetic defect. severe idiopathic neutropenia (sin) is a rare disease defined by persistent severe neutropenia, in the absence of an identifiable etiology. objectives: here, we aim to find out clinical, laboratory, genetic characteristic and remission status in children with scn and sin in chinese population. methods: in this study, we enrolled chinese children who experienced severe neutropenia longer than months without any virus infection or auto-immune antibodies from june to july in hospitals affiliated to shanghai jiao tong university school of medicine. their clinical, laboratory and molecular characteristics were analyzed and the patients were followed up to observe their remission status. targeted gene capture combined with next-generation sequencing technology was used to find out related gene mutation. results: patients in this study had a mean age of . ± . months. molecular analysis revealed that patients had associated mutations of scn, including elane and g pc . among patients with continuous follow-up, one died for unknown reason. ten patients have recovered from sin (r-sin) with mean neutropenia duration of . ± . months. scn patients had more frequent infection ( . ± . times per year) than sin ( . ± . times per year, p= . ) and r-sin patients (p= . , . ± . times per year). scn patients had significantly higher count of anc and monocytes than sin (p= . ) and r-sin patients (p= . ). however, there was no difference in anc and monocytes counts between sin and r-sin patients. bone marrow examinations demonstrated a myeloid maturation arrest at the myelocyte-metamyelocyte stage in scn patients, while most of sin and r-sin patients were normal. conclusions: our study indicated that, patients with mild infection, lower anc, monocytes count and normal bone marrow are likely to be sin. whereas others with relatively more severe infection, higher anc, monocytes count and maturation arrest in bone marrow are inclined to be scn. introduction/background: patients with qualitative and/or quantitative defects of humoral immunity often require immunoglobulin (igg) replacement therapy (igrt). usual starting doses range between - mg/kg and the dose is adjusted as needed depending on the patient. there is a paucity of information about whether and how extreme bmi (obese or underweight) and route of administration may affect a patients rate of infections. objectives: the objective of the study is to determine whether rate of infection is associated with bmi, dose or route of administration in patients receiving igrt. methods: this is a retrospective chart review from december -october . we included patients between the age of month and years old who were evaluated initially and had at least one follow up evaluation. data reviewed included the route of administration, dose, infection history, igg serum levels, height and weight to calculate bmi, gender, age and diagnosis requiring immunoglobulin replacement therapy. participants were excluded if the type of immunodeficiency is unknown or if the participant had incomplete data for the requested data fields. the number of infections between visits was modeled using poisson regression as a function of dose, route of administration and the bmi with the log of the follow up interval as an exposure offset. results: eighty-five patients were eligible, and preliminary results for patients are presented here. the mean infection rate per weeks of follow up was . in obese patients, . in overweight patients and . in underweight/normal weight patients adjusted for route of administration; however these rates do not differ significantly from one another. the mean infection rate per weeks of follow up differed by administration route; . infections for ivig versus . infections for scig when adjusting for bmi (p< . ). the mean infection rate per weeks of follow up was not associated with dosage. conclusions: overweight patients may experience more infections than obese or underweight patients, regardless of administration route. ivig patients may have a lower rate of infections compared to scig patients regardless of bmi. work is ongoing to complete analyses for the remainder of the eligible patient population. ( ) submission id# introduction/background: primary immunodeficiencies (pids) are a rare group of genetic disorders associated with a tendency to infectious diseases and an increased incidence of cancer. there is no data regarding the prevalence of malignancies in patients with pid in turkey. objectives: in this study, we aimed to evaluate patients who diagnosed as pids and developed malignancies, and calculate the estimated frequency of malignancies associated with pids in turkey. methods: forty five patients who were diagnosed with malignancy in the follow-up period of pid at the four tertiary immunology clinics between and years were included in the study. the data were obtained retrospectively from the hospital records and the database of esid online patient registry. results: the prevalence of malignancies in patients with pid was found as . % ( / ). the male to female ratio of the patients was / , the median age was . years (minimum: . , maximum: ) and the median age at which patients get diagnosed with malignancy was years (minimum: . , maximum: ) . there was no cancer history in their family members. the most common type of pid which associated with malignancy was ataxia telangiectasia (n= , . %). non-hodgkin lymphoma was the most common malignancy (n = , . %) in our group (table ) . ebv quantitative pcr was positive in lymphoma cases ( . %). the median number of x-rays and ct scans in patients with at and bloom syndrome before malignancy developed were (minimum: , maximum: ) and (minimum: , maximum: ), respectively. two cases had dual malignancies (papillary thyroid cancer and anal adenocancer). twenty cases were treated with chemotherapy, cases with hematopoietic stem cell transplantation, cases with radiotherapy, and cases with surgical treatment, treatment information of patients was not reached. remission was detected in cases, while resistance to therapy in cases and recurrences in two patients were observed. four patients are still on chemotherapy. twenty cases died. conclusions: the tendency of malignancy in patients with pids is due to the deficiency in the immune response that lead to failed surveillance against oncogenic viruses, premalignant/malignant cells, or both. lymphoid malignancies are the most common malignancies associated with pids. pids-associated malignancy incidence has increased in recent years because of that improved survival of the patients. this study is the largest cohort investigating the association of malignancy in patients with pid in turkey. additionally, we first reported tendency to malignancy in a patient with znf deficiency. introduction/background: next-generation sequencing (ngs) has become an integral tool in the evaluation of primary immunodeficiency disorders (pid). we describe a patient with a previously described pathogenic foxp variant who met clinical and laboratory criteria for cvid. objectives: describe a patient with a previously described pathogenic foxp variant who met clinical and laboratory criteria for cvid. results: case description: an -year-old male born premature at weeks presented with a history of recurrent infections. family history was negative for immunodeficiency. the patient developed recurrent acute otitis media beginning at year of age, three episodes of pneumonia beginning at years of age, and recurrent sinus infections requiring treatment on average four times a year beginning at years of age. initial immunologic evaluation at age was notable for: igg mg/dl (reference - mg/dl), igm mg/dl (reference - mg/dl), iga mg/dl (reference - mg/dl), ige iu/ml (reference - iu/ml). lymphocyte subpopulations were normal. specific responses to vaccines showed: protective antibody titers to diphtheria, but not to tetanus or pneumococcal antigens. he did not respond to booster vaccination and was started on ivig with significantly reduced frequency of infections. at age , while on ivig, he developed oral ulcers (biopsy consistent with ulcerative eosinophilic granuloma), abdominal pain, and recurrent arthralgias involving ankles, elbows, hips and the sacroiliac joint. magnetic resonance imaging (mri) was consistent with sacroilitis. subsequent imaging was consistent with chronic relapsing osteomyelitis (crmo). gastrointestinal biopsies showed severe active chronic pangastritis with antralized oxyntic gastric mucosa with enterochromaffin cell hyperplasia; suggestive of autoimmune gastritis. plasma cells were present throughout the gastrointestinal tract. ngs (bcm-ngs, baylor miraca genetics laboratory, houston tx) identified a hemizygous pathogenic missense variant, c. g>a (p.r q) in the x-linked foxp gene, that has been reported previously in patients with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. flow cytometry studies showed a decreased percentage of treg cells of total cd expressing cells, . % (reference . - . %), but normal foxp expression within these cells, % of treg cells with foxp expression (reference - %). interestingly, treg cell subset phenotyping obtained at the same time showed a normal percentage of natural tregs, . % cd t cells (reference . - . %), as well as normal percentage of naive tregs, . % cd t cells (reference . - . %). up to this point, he had not had any signs of diabetes, thyroid disease or frank enteropathy involving the small or large intestine. conclusions: we report a pathogenic foxp variant, occurring in a patient with a cvid-like phenotype, autoimmune gastritis, and an association with crmo. this case demonstrates the increasing utility of ngs, which can profoundly impact prognostic and therapeutic considerations. ( ) submission id# the natural history of patients with profound combined immunodeficiency (pcid): interims analysis of an international prospective multicenter study. immunodeficiency in this patient group. so far, patients were transplanted after enrolment, overall patients died ( in the hsct group, in the non-hsct group). analysis of the hsct decisions revealed the divergent decisions in patients with similar disease burden, favoring an ongoing prospective matched pair analysis of patients with similar disease severity with or without transplantation. so far, neither the genetic diagnosis nor simple measurements of t-cell immunity emerged as good predictors of disease evolution. conclusions: the p-cid study for the first time defines and characterizes a group of patients with non-scid t-cell deficiencies from a therapeutic perspective. since genetic and simple t-cell parameters provide limited guidance, prospective data from this study will be an important resource for guiding the difficult hsct decisions in p-cid patients. ( ) submission id# the plasma contact system and its role in common variable immunodeficiency: an explorative study. introduction/background: a growing body of evidence suggests that the contact system is involved in the activation of various vascular and immunological pathways and acts as an interface to help regulate allergic reactions, coagulation, complement, innate immunity and inflammation. as demonstrated in mice experiments, contact activation and high molecular weight kininogen (hk)-derived peptides increased homing of t and b lymphocytes into lymph nodes, which suggests an important area of research for understanding the contact systems role, specifically fxii, in immune-mediated inflammation and immune dysregulation. this novel mechanism prompted further inquiry into its role of various human disease states characterized by inflammation. plasma hk cleavage has been proposed as a useful and minimally invasive biomarker in various inflammatory disease states. this pathway has not been explored in cvid, in which inflammatory complications are found in one-third of patients with an unidentified genetic cause. characterizing the contact system biomarkers in cvid patients could elucidate a role in pathogenesis. objectives: assess the presence of contact activation at baseline in sera from cvid patients with and without inflammatory complications compared to healthy controls. methods: cvid patients were recruited in the outpatient setting and the measurement of cleaved plasma hk (chk) levels was determined by western blot analysis, under reducing conditions, with quantitation of total and chk bands using an odyssey imaging system (licor). a one-way anova test for differences among the studied groups will be applied. c inhibitor levels, c and c levels and high-sensitivity crp were also measured as comparable biomarkers for inflammation. results: to date, cvid patients were studied, with and without inflammatory complications. repeated determinations of cleaved hk% (chk%) revealed an average of . % (range: . - . %) in cvid patients with inflammatory complications and those without complications averaged . % (range: . - . %). healthy controls had an average chk of . % (n= , range: . - . %). conclusions: cleaved kininogen detected in the sera of cvid patients was found at similar levels compared to healthy controls (chk< %). findings suggest that systemic activation of the contact system might be absent in cvid, however, future considerations include developing detection methods for local tissue activation. ( ) submission id# the underlying primary immunodeficiencies and lung diseases, and low cd and cd counts are associated with recurrent pneumonia in hiv negative lymphopenia patients. clinical fellow, yale university school of medicine introduction/background: lymphopenia can be considered as primary or acquired immunodeficiency. lymphopenia is associated with a considerable increase in susceptibility to infections and the treatment focus for lymphopenia is mainly consists of prophylaxis and treatment of opportunistic infections. aids is the most well known cause of acquired lymphopenia and the cd count serves as an effective surrogate marker for disease progression and guideline for prophylaxis for hiv positive lymphopenia patients (hplp). hiv negative lymphopenia patients (hnlp) have been following the same prophylaxis guideline for hplp patients. however, it is unclear whether same prophylaxis guideline will be appropriate for both groups since the underlying immune mechanisms are different between these two groups. objectives: we aimed to define the optimal treatment and prophylaxis guideline for hnlp. in this study, we compared the clinical phenotypes and absolute counts of lymphocyte subsets between hnlp with recurrent pneumonia (pna), recurrent upper respiratory infection (uri) and no pulmonary infection. methods: electronic medical records of hnlp (n= ) seen at an academic immunology clinic between the year of and were reviewed retrospectively. lymphopenia was defined as absolute cd count less than /μl. the age, absolute counts of cd , cd , cd , cd , nkt and nk cell counts, history of antibiotic or antiviral prophylaxis use, autoimmune disease, lung disease, immunosuppressive therapy use, hypogammaglobinemia and ivig therapy use were compared between patients with recurrent pna (n= ), recurrent uri (n= ) and no pulmonary infection (n= ). results: this study showed that patients with recurrent pna had significantly lower absolute cd , cd , nk cell counts and age compared to the patients with recurrent uri ( table ) . none of the clinical phenotype was significantly different between these two groups. when we compared the patients with recurrent pna vs no infection, all lymphocyte subset counts and age were similar between these two groups except the frequency of underlying lung disease which was significantly higher in the recurrent pna group (table ) . lastly, we grouped the recurrent pna and uri groups together as the pulmonary infection group. when we compared the pulmonary infection group with the no infection group, lymphocyte subset counts were not significantly different, however, infection group showed significantly higher incidence of pid and the trend of lower rate of is therapy use than the no infection group (table ) . conclusions: the initial study has several limitations. this was a retrospective study from a single clinic and patient population was limited to patients. despite these limitations, we believe that this study provides valuable messages regarding prophylaxis guideline for pulmonary infection in nhlp; the underlying pids including icl and cvid, lung diseases particularly bronchiectasis and the absolute cd and cd counts less than /μl and / μl, respectively, are associated with recurrent pna and the patients with these risk factors likely will benefit from antibiotic prophylaxis. in addition, patients with acquired lymphopenia due to chronic use of is therapies without underlying pid or lung disease less likely develop pulmonary infection despite of low cd and cd counts. further investigations are crucial to elucidate the clinical significance of our initial observation by increasing the patient population and analysis of detailed immunologic and genetic profile of these patients which will likely reveal immunological markers and genes that are involved in the pathogenesis of both primary and hiv negative acquired lymphopenia. director, neuro-oncology program, division of hematology/oncology, ucsf benioff children's hospital oakland, ca introduction/background: gata is a hematopoietic transcription factor, required for the development and maintenance of a healthy stem cell pool. heterozygous mutation of gata has been associated with different but overlapping syndromes affecting both myeloid and lymphoid cell lines including aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, pulmonary alveolar proteinosis and lymphedema. it is also associated with immunodeficiency and susceptibility to mycobacterial, fungal, and viral infections. hematopoietic stem cell transplantation (hsct) is the only available cure which should ideally occur before patients develop neoplasia, severe infections or lung disease. objectives -to describe the clinical presentation of a genetically confirmed case of gata mutation -to describe the characteristic hematological and immunological profile of patients with gata mutation -to emphasize the importance of high index of suspicion and early diagnosis in improving outcome with hsct methods: case: a -year-old female presented with prolonged fever, fatigue, nonspecific rash, and unremarkable clinical exam. laboratory evaluation was significant for mild pancytopenia with profound monocytopenia. bone marrow analysis was remarkable for hypocellularity without evidence of dysplasia or malignancy. peripheral blood flow cytometry showed decreased t-and b-cells and absent nk cells. results: the constellation of pancytopenia, marked monocytopenia and absent nk cells, were suggestive of gata deficiency. sanger gene sequencing of gata revealed a heterozygous nonsense mutation (p.arg *). conclusions: mutation of gata is the underlying defect in overlapping clinical syndromes and is associated with immunodeficiency and malignant predisposition. incidence of organ dysfunction, infections and neoplasia increases with age. confirming the diagnosis during the phase of marrow hypocellularity/monocytopenia and pursuing hsct prior to malignant transformation may improve patient outcome. introduction/background: reduction in child mortality has kept pace with improved immunization and nutritional status. however, there are children with severe infections who have no identifiable reason. the search for children with pid was initiated in a tertiary care referral hospital from a region with no documentation of the prevalence of this disorder, but with a high rate of consanguinity. financial constraints, poor availability of laboratory facilities or therapeutic options locally, limited awareness among pediatricians and vast distances between premier centres were major obstacles. objectives to identify children with primary immune deficiency and study the spectrum of pid in north kerala to prevent infectious and other complications in these children to provide curative therapy whenever feasible methods . acquisition of knowledge and skills to diagnose and manage pids . establishment of an immune deficiency clinic . liaison with centres across the country offering diagnostic tests and stem cell transplant . formation of project team and submission of a project to the central government . improvement of diagnostic facilities at home institution results: children with recurrent, severe or persistent infections or with two or more esid warning signs were screened for pid. severe combined immune deficiency was diagnosed in children, wiskott -aldrich syndrome in children, chronic granulomatous disease in children and x linked agammaglobulinemia in children and leucocyte adhesion deficiency in children. prophylaxis with ivig was initiated in children and stem cell transplant was done for children. conclusions: in a country with resource constraints, limited awareness among health care providers and vast distances, it is possible to make a difference to the lives of families of children with pid by networking across centers with expertise in immunological and molecular genetic diagnostic methods and life -saving therapeutic modalities like stem cell transplantation. introduction/background: complete thymic aplasia is a rare cause of scid, and requires thymic transplantation for curative treatment. because thymic transplantation is not widely available, there can be significant delay between diagnosis and curative therapy placing the infant at risk of invasive life threatening infections. objectives: describe modalities of therapy for adenoviremia in a patient with scid due to thymic aplasia. methods: chart review was performed. treatment including hlapartially matched third party cytotoxic t cell therapy and matched related hematopoietic cell transplant were assessed for treatment of life threatening adenoviremia in a scid patient with thymic aplasia. we identified an infant with a mutation in tbox transcription factor (tbx ) (c. _ dup ) by way of state newborn screening for scid. she had severe t cell lymphopenia and abnormal t cell function. at months age, she developed adenoviremia with associated fulminant hepatitis, and an initial viral load of million copies/ml. despite prolonged therapy with cidofovir, viral load increased to as high as million copies/ml. treatment with two infusions of partially hlamatched third-party cytotoxic t cells specific to adenovirus ( / and / hla matched, with hla class ii-mediated antiviral restrictions) led to partial clinical improvement without viral clearance. due to continued severe adenovirus-related hepatic dysfunction, unmanipulated bone marrow from an hla-identical sibling was infused without conditioning ( million cd + cells/kg and . x cd + cells/kg), at months of age. after an initial surge in adenoviral loads attributed to massive viral lysis, the degree of viremia progressively declined and was < , copies/ml within weeks of marrow infusion. antiviral tcell activity against adenovirus was detected at low level in peripheral blood via ifn elispot at weeks post-marrow infusion. she subsequently developed acute cutaneous and hepatic gvhd responsive to tacrolimus and steroids without recrudescence of viral illness. conclusions: delay in curative therapy in scid substantially increases risk of invasive life threatening infections. one strategy of allogeneic hct can be to eradicate severe infection in scid by providing the necessary t cell directed therapy against infectious agents. antigen-specific partial immune reconstitution can be achieved with hct in patients with thymic aplasia but concern regarding the development of full immunologic t cell diversity in athymic patients remains. senior technician, sanquin bloodsupply amsterdam introduction/background: chronic granulomatous disease (cgd), an inherited disorder of granulocyte function caused by a failure of intracellular superoxide production, normally presents as severe recurrent bacterial and fungal infections in the first years of life. the majority of affected individuals are diagnosed before the age of years, although patients may remain undiagnosed until adulthood despite the early onset of the symptoms objectives: investigation of fungal infection in adult cgd patients methods: nbt and dhr for detection of cgd and molecular analysis for detection of type of mutation in cgd and fungal characterization. results: we report here the detection of causative fungal infections in adult patients with cgd. in the first patients we found paecilomyces formosus infection in an adult patient ( years old female) with undiagnosed cgd who was referred to the shahid beheshti university hospital (tehran, iran) complaining of cough, dyspnea and fever for weeks prior to admission. microscopic analysis revealed branching solitary phialide with ellipsoidal conidia with long chain arrangement and many chlamydospores which mostly resemble botryotrichum species but during subcultures on potato dextrose agar (pda) and sabouraud dextrose agar (sda), phialides typical of paecilomyces species appeared. typically, paecilomyces spp. rarely cause infections in humans and if these fungi are detected in blood urine or cerebrospinal fluid cultures they are considered as contaminants. the second patient was a -year-old man was referred to the hospital with weight loss, fever, hepatosplenomegaly and coughing. he had previously been diagnosed with lymphoadenopathy in the neck at age and prescribed antituberculosis treatment. bal and serum galactomannan tests were negative. low, subnormal levels of ros were produced following stimulation of purified neutrophils with the phorbol ester pma. genomic dna was extracted and gene scan was used to determine the ratio between the number of exon sequences of neutrophil cytosolic factor (ncf ) gene, which encodes p phox, and the number of -ncf exon sequences. in addition, the fungal culture was disrupted with glass beads and dna was extracted. the dna sequence results were blasted using the ncbi genebank database, which showed % similarity to an aspergillus terreus isolate in the gene bank fungal library with accession no . conclusions: thus, despite its current relative rarity in older patients, the presence of fungal infection is changing our understanding of the diagnosis, management and outcome of cgd. greater appreciation of the potential of fungal infection in older cgd patients is important in regions, such as iran, where tuberculosis is endemic and that sarcoidosis and cgd are considered as differential diagnosis. the demonstration of the successful patient-orientated treatment after using sequencing to confirm cgd and to identify the presence of the specific infectious agent emphasises the importance of adopting this approach across the region. introduction/background: primary immunodeficiencies (pi) represent a heterogeneous group of over genetically distinct disorders which interrupt normal host-defense mechanisms and predispose to significant morbidity and mortality. presently, we are only able to screen for severe t-cell deficiencies at birth; however, the most common forms of pi often go undetected for years leading to adverse patient outcomes and excessive healthcare spending. given the relatively high incidence of pi in the general population, informatic measures could be useful for determining individual risk for pi and facilitating earlier, correct diagnosis and appropriate treatment across the spectrum of pi. objectives: the purpose of this study was to test the jeffrey modell foundations spirit (software for primary immunodeficiency recognition intervention and tracking) analyzer on the texas childrens health plan. major aims were to identify individuals with medium-high risk for pi, assess the clinical characteristics of at risk patients and determine if risk identification led to diagnosis of pi over a month period. methods: after removing all known pi diagnosed patients from the database, , individual texas childrens health plan enrollees were screened for risk of pi with the spirit analyzer using relevant, weighted icd and icd codes. patient characteristics are shown in table . following identification of medium-high risk (mhr) individuals, letters were sent to their primary care physicians to alert them of patient risk. a second analysis of the mhr individuals was performed months later. detailed chart reviews were conducted on mhr individuals to further assess clinical features of this group. the study was approved by the baylor college of medicine institutional review board (study h- ). results: of the original cohort, ( . %) were identified as mhr for pi. from that group, ( . %) were accessible for analysis and ( . %) had electronic health records for review. in the months following the first analysis, ( . %) were diagnosed with a pi. (figure ) another patients had concerning diagnoses coded warranting further investigation. (figure ) concerning diagnoses included: cellulitis( ), abscess ( ), recurrent otitis media( ), recurrent sinusitis ( ), osteomyelitis ( ), and mastoiditis ( ) among others. in total patients had a pi diagnosis or a history concerning for pi ( . % of main cohort; . % of mhr cohort). conclusions: the spirit analyzer is effective at identifying persons at risk for pi and facilitates diagnosis. potential mhr yield by the analyzer is over %. these patients are treatable and will benefit from targeted intervention once identified. the analyzer can also highlight concerning conditions worthy of additional assessment. future work should focus on longitudinal healthcare outcomes for patients diagnosed with pi via spirt and physician perspectives on the utility of the tool. introduction/background: genetic variants in card contribute to several diseases caused by dysregulation of the adaptive immune system. dominant-negative, loss-of-function and gain-of-function variants in card variants lead to primary immunodeficiency disease. however, primary immunodeficiency disease caused by card gain-of-function variants often progresses to b-cell malignancy. the clinical course and treatment options depend on the type of card mutation. unfortunately lymphocyte immunophenotyping and traditional proliferation assays can't distinguish the variant effect or predict the likelihood of malignancy. objectives: to use multiplexed functional assays for determining variant effect to distinguish between dominant-negative, loss-and gain-offunction effects in card . our ultimate goal is to generate a variant function map that can be used to guide diagnoses and treatment of immune dysregulation caused by mutations in card . methods: we co-delivered crispr/cas ribonucleoprotein complexes with libraries of single-stranded oligonucleotide repair templates thus generating lymphoma cell populations containing all possible singlenucleotide variants (~ different protein coding changes) in the nterminal amino acids of card . following culture with and without bcr pathway inhibitors, we used next generation sequencing to quantify variant abundance before and after culture, both with and without bcr pathway inhibitors. results: due a requirement for card in these lymphoma cells, those with dominant-negative and loss-of-function card variants grow more slowly, whereas those with gain-of-function variants grow faster in the presence b cell receptor pathway inhibitors. by tracking the relative abundance of each variant in the population by next generation sequencing over multiple conditions, we determined the functional effect of each. we assessed the functional effects of thousands of card variants in parallel. this enabled us to confirm several previously reported gain-offunction and dominant-negative variants in card , as well as identify several additional novel variants. finally, we evaluated previously undescribed dominant-negative, loss-of-function and gain-of-function variants during differentiation of primary human b cells and during nf-b signaling. conclusions: the results of our experiments demonstrate the utility of multiplexed functional assays for determining variant effect in proteins where distinguishing between dominant-negative, loss-and gain-offunction effects are required to guide diagnoses and treatment. introduction/background: primary immune-deficiencies (pids) are a heterogeneous group of nearly monogenic inborn errors of immunity. in recent years, whole exome sequencing (wes) became a valuable diagnostic approach for the identification of molecular defects in patients with clinical manifestations suggestive of pid. this approach provides a definitive diagnosis and may help in genetic counseling, prenatal diagnosis and pre-symptomatic identification of patients with a potentially lethal disease. the diagnostic yield using wes was found to be~ % in rare mendelian disorders and~ % in pids. we present here a markedly higher yield, of %, in pids with a high percentage of consanguinity ( % in this study). objectives: using the whole exome sequencing (wes) approach in israeli pid patients with high percentage of consanguinity to identify the genetic causes underlying their diseases for better diagnosis and clinical management. methods: wes was performed on genomic dna obtained from wbc of immune deficient patients with potential genetic causes. the sequencing data was analyzed bioinformatically. each of the discovered mutations was validated and the familial segregation was confirmed, using sanger sequencing. results: the probands ( males and females) ranged in age from weeks to years with a mean of . years. of them, patients are jewish ( %), palestinians ( %) and ( %) is from a greek ethnicity (cyprus). based on their clinical and immunologic phenotype at the time of initial evaluation, patients were assigned to one of seven pid groups: (i) humoral immunodeficiency patients ( %); (ii) combined immunodeficiency (cid) ( %); (iii) cid with syndromic features ( %); (iv) scid ( %); (v) congenital defects of phagocytes ( %); (vi) immune dysregulation ( %) and (vii) phenocopy of pid ( %). we identified mutated alleles, all in coding regions, that are highly likely to be causative in of the patients, achieving a % molecular diagnostic yield. among the patients, the mode of inheritance in patients ( %) is autosomal recessive and in is compound heterozygote ( %). four patients ( %) harbor a non-inherited mutation on one allele, either de novo or somatic. the inheritance of the mutation in one patient ( %) is x-linked. the high rate of bi-allelic inheritance ( % of the alleles) is mainly due to the high frequency ( %) of consanguinity among the studied cohort. twenty eight mutated genes were identified in this study. of them, were found to be novel in causing an inherited disease in man. interestingly, some genetic defects in known genes were found in patients with atypical phenotypes. in patients ( % of the total number of patients and % of the wes diagnosed) the discovery of the genetic cause led to a change of therapy, towards a more targeted and personalized one. the revised treatments included bone marrow transplantation, conditioning protocols, reduced intensity of immune suppression, and prevention of unnecessary treatments due to their possible deleterious outcome. conclusions: except of being a useful tool for diagnosing and deciphering novel or atypical forms of pids, our wes study demonstrates an immediate and powerful impact on patient therapy in pids. early intervention with bone marrow transplant or gene therapy is critical and best when the infant in uninfected. newborn screening for scid and t-cell lymphopenia has been implemented in states. the screening test is performed from dried blood spots collected at birth and involves pcr quantification of circular dna byproducts of t-cell receptor gene rearrangement, t-cell receptor excision circles (trec). trecs are generated during t-cell maturation in the thymus and are indicative of naïve t-cell output. assays and protocols to measure trecs vary by state and there is no standardized guideline at this time. washington state is unique in that it is one of few states where all newborns undergo two or more independent newborn screens, the first by which igrt treatment helps in these patients is unreported. here we present the first case of an mbl deficient patient with other complicating conditions (low igg and multiple cf polymorphisms, ciliary dyskinesia) who did not show improvement on igrt, thus refuting current literature. results: a -year-old caucasian male patient presented due to recalcitrant warts of the hands and feet. he had childhood otitis media status post tympanoplasty, mild molluscum contagiosum, and eczema. also, history of migraines with a negative brain mri. a trial of weekly pegylated interferon alpha mcg for his warts was discontinued due to significant neutropenia and depression. physical exam was notable for bulky skin colored warts on the lateral and dorsal fingers and dorsal hands including periungual skin. clusters of verrucae were noted on the feet. initial laboratory testing was notable for mild hypogammaglobulinemia with protective specific antibodies. lymphocyte subset analysis revealed a predominantly t cell lymphopenia with decreased naïve and memory cd and cd subsets, normal absolute numbers of b cells but with low memory subsets, and normal numbers and function of nk cells. whole exome sequencing ultimately revealed homozygous r q mutations in the cecr , a mutation previously described as causing ada deficiency. ada activity was about %. conclusions: ada deficiency is a relatively newly defined genetic defect, with a clinical phenotype that continues to evolve with newly diagnosed cases. our patient had not had evidence of vasculitis or stroke, but had recalcitrant warts with lymphopenia as his primary presentation. approach to therapy for those without vasculitis or significant cytopenias remains unknown. introduction/background: whole exome sequencing has added greatly to our library of primary immune deficiencies. however, interpretation of findings is not always straightforward. clinicians need to understand the limitations of this diagnostic tool and be familiar with the next steps in order to achieve a diagnosis. objectives: a year old male presents for evaluation of bronchiectasis of bilateral lower lobes and poor growth. past medical history was significant for gerd with poor weight gain during infancy and childhood, oral thrush, recurrent respiratory infections and bilateral partial hearing loss. the bal showed abundant neutrophils and was positive for strep pneumoniae and haemophilus influenza. methods: serum immunoglobulins were normal with an elevated iga ( ) and mildly elevated ige ( ). tetanus igg was protective ( . iu/ml) but post-vaccine responses to the pneumococcal polysaccharide vaccine were poor with protective titers achieved to / serotypes. lymphocyte phenotyping was remarkable for a complete absence of b and nk cells. cd :cd ratio was inverted ( . ) and the majority of the cd + t cells were memory phenotype. mitogen proliferation was essentially normal. due to the absence of b cells and despite normal total serum immunoglobulins, the patient was started on sc ig weekly and antibiotic prophylaxis with trimethoprim-sulfamethoxazole and fluconazole. clinically he demonstrated good weight gain and a reduction in cough and respiratory symptoms. results: whole exome sequencing was performed. a single base mutation in ada (c. t>c) was identified in one allele. this mutation (p.l p) is known to be deleterious and when homozygous is associated with typical scid. however, this mutation was not present on the other allele. secondary analysis looking for large deletions and duplications failed to identify any abnormality in sequence in the normal allele. but the expression of this allele was markedly diminished causing us to suspect that its function might be impaired. functional testing was performed and demonstrated that there was no ada activity in the patients red blood cells, thus confirming a diagnosis of combined immune deficiency due to ada deficiency. conclusions: null mutations in ada result in an absence of ada function with profound cd cell lymphopenia and dysfunction. clinically affected infants have typical scid. hypomorphic mutations may lead to partial function of ada with more variable immune defects. interestingly, most patients with ada deficiency have neurologic complications, frequently hearing loss. we believe that most likely this patient has had some degree of spontaneous reversion of the mutation in the normal allele leading to a less severe phenotype. as reported previously in a case involving a mutation in il rg chain, normal function was not restored and the patient remains with a combined immune deficiency. this case highlights an important limitation of whole exome sequencing and the need for confirming the impact of a genetic defect with a functional assay. introduction/background: the autosomal dominant hyper-ige-syndrome (ad-hies) is a rare primary immunodeficiency and multisystem disorder resulting from heterozygous loss-of-function mutations in the stat gene. ad-hies is characterized by skeletal dysplasia, recurrent pulmonary and skin infections (e.g. staphylococcal abscesses, eczematoid dermatitis) due to an increased susceptibility to bacteria and fungi. since many patients do rather well on anti-infective prophylaxis and supportive care, and early case reports suggested no benefit of allogeneic hematopoetic stem cell transplantation (hsct), ad-hies patients are rarely referred for hsct. the literature still contains only a handful of patients who underwent hsct. all these patients had experienced severe disease related complications before hsct and consequently the benefit of hsct was reported to be variable. objectives: currently, the general consensus is to only consider patients with severe pulmonary disease for hsct. it could however be postulated that transplanting patients earlier in their disease course and correcting their immunodeficiency before permanent organ damage due to infectious complications has occurred may extend life-expectancy and improve the quality of life of ad-hies patients. methods: we report on a year old female ad-hies-patient who presented with bronchiectases following recurrent pneumonias, one pneumatocele, and normal pulmonary function tests. her past medical history also included recurrent skin infections, serious haemoptysis, pathological fractures and atopic eczema. considering that lung infections are the major life-limiting complication in ad-hies and are potentially positively influenced by hsct, our patient had enquired about a transplant. after extensive discussion of the pros and cons and obtaining full informed consent from her and her family, she underwent an elective hsct. she received bone marrow from an hla-matched sibling donor after a reduced-intensity conditioning consisting of alemtuzumab ( x , mg/kg), treosulfan ( x g/ m ), fludarabine ( x mg/m ) and thiotepa ( x mg/kg), and graftversus-host disease (gvhd) prophylaxis with cyclosporine a (csa) and mycophenolate mofetil (mmf). results: the peri-transplant course was complicated by acute lower gastrointestinal tract bleeding and renal failure of unknown origin. continued kidney function impairment led to early tapering and discontinuation of csa on day + after hsct in the absence of any acute gvhd. neutrophils and platelets engrafted on days + and + respectively. she is currently on day + , free of gvhd or infection, exhibiting full donor chimerism and recovered kidney function. in view of the preexisting pulmonary damage she currently remains on antibiotic prophylaxis and inhalation therapy. conclusions: this ad-hies patient who underwent hsct with few pre-hsct disease complications and relatively little permanent organ damage may add to our understanding of whether early hsct will lead to improvement of quality of life and possibly increased life-expectancy in ad-hies patients. it remains to be elucidated whether her rather uncommon peri-hsct complications are connected to her underlying disease. future research should be directed at identifying ad-hies patients at high risk of severe pulmonary complications early, so these could be referred for timely hsct. objectives: this is a case study of a patient who had refractory ibd symptoms and recurrent infections who was found to have xiap deficiency and mefv variant mutations. methods: a year old male presented at age with recalcitrant ibd unresponsive to multiple medications including steroids, mesalamine, azathioprine, infliximab, adalimumab, methotrexate, and vedolibumab. in addition, he developed severe infections from a combination of immune dysregulation and immunosuppressant medications. currently he is on ustekinumab but still has severe abdominal symptoms. patient does not have a history of hemophagocytic lymphohistiocytosis (hlh). family history was significant for ibd in both his mother and sister. he has had persistent lymphopenia which ranged between to cells/ mm . t/b/nk panel showed decreased cd t cells ( cells/mm ) with normal cd , cd , and cd /cd ratios. b and nk cells were normal in quantity. t cell antigen/mitogen assays showed normal response to all mitogens (pha, pwm, con a) and most antigens (tetanus, candida, hsv, vzv, adv) but low response to cmv antigen. igg was elevated at , , but iga, igm, and ige were normal. his ebv dna pcr is negative. results: exome sequencing revealed a novel xiap hemizygous variant at position c. g>a (p.=?) of unknown significance and a mefv heterozygous variant. functional testing performed at medical college of wisconsin showed no expression of xiap on lymphocytes and a defect in nod pathway. given the xiap deficiency, bone marrow transplant was discussed as an option for refractory ibd and prevention of hlh. rituximab was also offered to decrease the possibility of hlh. currently the patient is in the decision making process of both treatment options. conclusions: genetic evaluation with clinical exome in early onset refractory ibd, family history of ibd, and recurrent infections demonstrated a novel mutation in the xiap gene with possible contribution of mefv variant as well. the absence of xiap protein expression and abnormal functional assay of the nod pathway confirm the pathogenicity of the mutation. identification of this genetic variant will help guide future therapeutic options and prognosis for this patient. introduction/background: x-linked agammaglobulinemia (xla) is a primary immunodeficiency disease caused by mutations of bruton tyrosine kinase (btk), which is essential in b cell maturation. xla is typically associated with bacterial infections of upper and lower respiratory systems, enteritis and increased risk for malignancies. objectives: to present the evolution and treatment of a complicated flexispira (helicobacter bilis) infection with delayed diagnosis in an xla patient. methods: clinical and laboratory features of a patient with xla evaluated at the national institutes of health. results: a -year-old male patient with xla presented for initial evaluation of indurated lower extremity and torso lesions. he was diagnosed with xla at age years secondary to bacterial sepsis pneumonia and empyema. after starting ivig at age , he was well without significant infections except for recurrent otitis media. at years of age, he developed right leg edema below the knee, which progressed to patchy skin thickening and discoloration. a tissue biopsy at years of age revealed marked fibrous thickening of the subcutaneous septum with diffuse infiltrate of eosinophils with negative cultures for bacterial, fungal, and mycobacterial infections and thought to be consistent with eosinophilic fasciitis. mri demonstrated infiltration in the superficial and deep muscle compartments with fibrosis. symptoms persisted despite empirical treatment with iv antibiotics and steroids. at years of age his left leg became involved with similar findings, while under treatment including multiple immunosuppressants (dapsone, methotrexate, tacrolimus, hydroxychloroquine, iv cyclophosphamide, remicade, cytoxan, and enbrel) targeting eosinophilic fasciitis. at age , he developed ulcerations over the left shin and ankle and was diagnosed with chronic multifocal osteomyelitis based on mri findings. physical exam was notable for bilateral leg swelling below the knees with woody appearance and induration, hyperpigmentation, and tenderness. indurated and nodular lesions without discoloration were noted above the waist line, on right forearm and above right nipple. skin biopsies of right lower extremity and right forearm were positive for numerous spirochetal-like organisms with warthin-starry stain. treatment was initiated with meropenem and gentamicin. gentamicin was discontinued due to vestibular ototoxicity six weeks later and doxycycline was added. initial response was followed by worsening of symptoms and intolerance to treatment, which led to the addition of tigecycline and azithromycin. with continued progression, seven months into initial evaluation, chloramphenicol and nitazoxanide were added to the regimen. due to the persistence of flexispira organisms on skin biopsy warthin-starry stains, fresh frozen plasma (ffp) was introduced four months later ( months after initial evaluation) as an adjunctive treatment. cultures performed at the cdc were negative; however, pcr and sequencing resulted in identification of helicobacter bilis. units of ffp was infused weekly for over three years, then reduced to every two weeks, with goal igm levels > mg/dl. subsequently, labs including cytopenias, inflammatory markers, and immunoglobulins improved as well as a negative warthin-starry stain. symptoms have improved with almost complete resolution of findings with only residual small areas of discoloration over both lower extremities. conclusions: xla immune deficiency is associated with flexispira (helicobacter bilis) infections, with typical appearance of discoloration and induration, which may evolve to osteomyelitis due to delayed treatment. although typically observed over the lower extremities, immunosuppressive treatment may lead to further expansion above the waist line. approach to therapy with weekly to bimonthly ffp infusions in addition to antibacterial treatment has proven to be beneficial in controlling the infection. higher igm levels resulting from the ffp may also provide antibacterial effects. introduction/background: x-linked severe combined immunodeficiency (scid) is a well described primary immunodeficiency associated with mutations in the common gamma chain. patients with x-linked scid classically present with profoundly low or absent t cells and nk cells with a variable number of b cells. the lymphocytes that are present typically have a proliferation index < % control when stimulated with mitogens and antigens. patients must undergo corrective therapy with bone marrow transplant (bmt) or gene therapy to avoid the life-threatening infections that are associated with the nearly absent adaptive immune system. objectives: a -week-old boy presented to childrens national immunology clinic for initial evaluation of critical result on newborn screen. methods: targeted partial exome sequencing was performed on a -dayold patient who was picked up via trec assay on the maryland newborn screen. flow cytometry was completed at childrens national and proliferation studies completed at cincinnati childrens hospital diagnostic immunology lab. results: flow cytometry revealed markedly decreased lymphocytes with nearly absent cd + t cells and low cd + t cells with a r e l a t i v e i n c r e a s e i n c d + c d r o t c e l l s ( r a t i o cd ra:cd ro: %: %). the b and nk cells were within the reference range for age. mitogen proliferation studies showed a mild decrease to pha and normal responses to pokeweed and cona ( cpm, cpm, and cpm, respectively). partial exome sequencing revealed a hemizygous nonsense substitution in il rg (c. c>t, p.r ) . maternal engraftment accounted for % of the t cells. the patient was started on prophylaxis with ivig, bactrim, and fluconazole with the plan to proceed with bone marrow transplant. as patient approached bmt maternal engraftment became absent in whole blood and repeat proliferation studies revealed normalization of the response to pha (stim index) with continued normal responses to cona and pokeweed. the patients flow cytometry values and ratios remained unchanged. patient completed a reduced intensity preparative regimen of busulfan, fludarabine and alemtuzumab prior to receiving his / matched unrelated donor bone marrow transplant. conclusions: it remains to be determined why initial proliferation studies showed > % function with improvement over time in a patient with a well-described genetic mutation causing scid ( ) director, division of intramural research, nih/niaid introduction/background: the advent of next-generation sequencing (ngs) has led to a proliferation of newly discovered genetic diseases and expanded phenotypes of known immunodeficiencies. availability of specific gene panels or whole exome sequencing (wes) with targeted analysis based on broad phenotypes, coupled with clinicians increasing awareness has led to higher utilization of ngs. published reports from high throughput sequencing labs indicate exome analysis identifies causative mutations in only - % of probands. results: we have seen several patients who underwent high quality ngs in whom causative mutations were not identified. two separate families with multigenerational histories of leukemia, aplastic anemia, myelodysplastic syndrome, and cytopenias suggestive of gata deficiency had myeloid gene panel screens at commercial labs without causative mutations identified. targeted gata sequencing in the first family identified a novel change in gata , c. g>t, p.l l. cdna analysis demonstrated this synonymous variant resulted in aberrant splicing leading to a frameshift and premature termination. the wes bioinformatics pipeline failed to recognize the splice mutation. in the second family, pcr amplification spanning the terminal exons revealed a shortened pcr product with a base deletion fully encompassing the penultimate exon and leading to a amino acid in-frame deletion. the deletion spanned all capture probes for the exon resulting in only the wild-type allele being captured and sequenced. additionally, capture kits targeting only coding regions of genes fail to capture deep intronic mutations such as those seen in gata (hsu, ) or in the untranslated region of ikbkg, encoding nemo, (mooster, ; hsu, submitted) . lastly, even with good capture and sequencing, the presence of pseudogenes may confound downstream sequence alignment as seen in ncf , encoding p phox preventing recognition of disease causing mutations. conclusions: with ngs becoming more widely available as a clinical diagnostic tool, it is important to remember that wes results, unlike many laboratory tests, are not binary. inadequate bioinformatics pipelines, deletions, intronic or untranslated mutations, and pseudogenes can all mask the presence of causative mutations. targeted panel captures or analysis will miss novel genes. astute clinicians need to recognize the limitations of the current technology and pursue alternate assays when suspicions warrant. a cell based assay for the detection of autoantibodies to il- in human serum. matt phillips, phd , vijaya knight, md, phd senior scientist, national jewish health director of immunology and complement adx labs, national jewish health introduction/background: patients with chronic candida infections are typically deficient in some aspect of il- signaling. one mechanism, which has recently come to light, is through the production of il- autoantibodies, particularly in patients who already suffer from specific autoimmune diseases. objectives: our objectives are to develop a diagnostic assay to accurately and easily detect il- autoantibodies in patient serum. methods: we developed a cell-based reporter assay using hek blue il- cells (invivogen) to detect the ability of patient serum to block il- receptor signaling. once stimulated with il- , the cells secrete alkaline phosphatase (ap) into the surrounding media which is detected by invivogen hek blue media and an absorbance reader. addition of serum containing blocking antibodies inhibits secretion of ap. results: we were able to demonstrate, using a single patients serum with known il- autoantibodies, the inhibition of il- signaling in hek blue il- reporter cells. we further characterized the sensitivity of our assay with a commercially available anti-il- monoclonal and found it to be sensitive to between . x - m and . x - m. conclusions: loss of il- signaling can lead to problematic immune deficiencies including difficulty in clearing extracellular pathogens such as candida. some people who have an immune deficiency in the il- pathway may have developed autoantibodies to il- and thus have difficulty generating an appropriate immune response. we have developed a relatively low maintenance, cost effective, and simple test for detecting il- autoantibodies in human serum. alternations in repertoire of t and b cell subsets in patients with partial recombination activating gene (rag) deficiency with autoimmunity and history of viral infections introduction/background: patients with partial deficiency of recombination-activating genes or (rag / ) can present with a wide spectrum of primary immunodeficiencies including combined immunodeficiency with granuloma and/or autoimmunity (cid-g/ai). prior case reports have highlighted alterations in b and t cell compartments; however comprehensive characterization of t and b cell receptor repertoires of lymphocyte subsets regarding diversity and autoreactivity has not been reported. objectives: defects in v(d)j recombination due to rag deficiency results in a skewed t and b cell repertoire that may be further modified by viral infections and promote inflammatory or autoimmune phenotype. methods: peripheral t and b cell compartments were sorted from two patients with combined immunodeficiency secondary to hypomorphic rag and rag mutations. b cells were stimulated with cd l, cpg and il- to transition to antibody secreting cells (ascs), mimicking viral infection. repertoire analysis and single cell cloning of bcr heavy and light chain variable regions from sorted b cell populations has been performed. repertoire of t cell subsets (treg and follicular helper) were also examined results: we noted skewing towards proximal j usage in all b cell compartments (mature naïve, marginal zone, cd -/low and memory) of two rag deficient patients compared to healthy controls. b cell clones with v - v genes with low rate of somatic hypermutation expanded during b cell development. after in vitro stimulation mature naïve b cells from rag deficient patient were capable of transitioning to antibody secreting cells and enriched for polyreactivity to dsdna, insulin, lps and ifn cytokine ( to %) compared to healthy control ( to %). in connection to altered b cell compartments, restricted repertoire of regulatory t cells and an expanded and skewed follicular helper t subset were detected. conclusions: our data indicate that patients with partial rag deficiency have skewed t and b cell subsets that can further be altered towards antibody secretion and polyreactivity after stimulation such as viral infections. chief of the division of allergy and immunology, division of allergy immunology, the childrens hospital of philadelphia, philadelphia, pa usa introduction/background: the clinical features of q . deletion syndrome include virtually every organ of the body. t cell lymphopenia, as a consequence of thymic hypoplasia, is the most commonly described immunologic feature and is most prominent in childhood. later in life, t cell exhaustion may be seen and secondary deficiencies of antibody function have been described in patients with q . deletion syndrome. objectives: the role of deletion breakpoints in determining q . deletion syndrome immunophenotype is unknown. in this study, we examined the effect of q . deletions with and without tbx on lymphocyte counts. methods: lymphocyte counts were compared between total q . patients with tbx -containing deletion (a-b, a-c, a-d deletions), and a total of patients with tbx -noncontaining deletion (b-d, c-d, d-e, d-f deletions). lymphocyte counts of patients with q . deletions were compared to a set of patients with a q . duplication including the tbx locus. lymphocyte subset counts for each group were analyzed by t-test. results: cd counts were significantly lower in the tbx -deleted cohort compared to the other two cohorts (mean cells/mm in tbx deleted cohort, cd cells/mm in the non-tbx deleted cohort, and in the duplication cohort, p< . for all). similarly, cd counts were lower in the tbx -deleted patients compared to the other two cohorts. there were no significant differences in cd , cd , and nk cell counts between the three cohorts. conclusions: these represent the first data to examine t cell counts in q . deletion syndrome patients with different breakpoints. our data highlights an important role for tbx or other genes in the a-b region in regulating t cell production. paracoccidioidomycosis associated with a heterozygous stat mutation and impaired ifn-immunity introduction/background: mutations in genes affecting ifn-immunity have contributed to understand the essential role of this cytokine in the protection against intracellular bacteria and fungi. however, inborn errors in stat , which controls il- responses, have not yet been reported. objectives: to determine the underlying genetic defect in a family with a history of paracoccidioidomycosis (pcm) disease. methods: genetic analysis was performed by whole-exome sequencing and sanger sequencing. stat phosphorylation and translocation from the cytosol to the nucleus, as well as ifnrelease by patient lymphocytes were assessed. the effect on stat function was evaluated by site-directed mutagenesis using a lymphoblastoid b cell line (b-lcl) and u a cells. microbicidal activity of patient monocytes/macrophages was also analyzed. results: a heterozygous missense mutation, c. a>t (p.e v) in stat was identified in the index patient and her father. patients and fathers lymphocytes showed reduced stat phosphorylation and nuclear translocation as well as impaired ifn-production. in accordance, b-lcl and u a cells carrying the stat mutant displayed reduced stat phosphorylation. patient's and father's pbmcs and macrophages (alone or in the presence of t cells) displayed impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human (rh) ifn-, but not rhil- . conclusions: our data suggest autosomal dominant stat deficiency as a novel inborn error of il- -dependent ifn-immunity associated with susceptibility to pcm disease. profound b cell lymphopenia in gof-stat that improves post ruxolitinib associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: subjects with gain of function signal transducer and activator of transcription (gof-stat ) mutations have a variable clinical phenotype including combined immunodeficiency (cid). ruxolitinib, a janus kinase / inhibitor has been successful at treating immune dysregulation in subjects with gof-stat . two subjects with profound b cell and/or t cell lymphopenia as a major manifestation are described, one of which was successfully treated with ruxolitinib. objectives: to discuss gof-stat mutations, their effect on the immune system, and the potential benefit of ruxolitinib in these subjects. methods: retrospective chart review was performed. results: subject (c. a>g) is a year old male with a history of recurrent shingles, chronic mucocutaneous candidiasis (cmc), pneumocysitis jiroveccii pneumonia, varicella zoster meningitis, severe enteropathy, cerebral aneurysm and lymphoproliferation, and autoimmune hypothyroidism. he has profound lymphopenia predominantly affecting t and b cells (cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul). subject (c. g>t) is a year old male with a history of severe cmc, recurrent pneumonia, enteropathy, and autoimmune thyroiditis. he had severe b cell lymphopenia (cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul, cd + cells/ul). treatment with ruxolitinib . mg bid led to clinical improvement of enteropathy and increased b cell counts in subject (cd + cells/ul). ruxolitinib has not yet been initiated in subject . both subjects were treated with anti-microbial prophylaxis and immunoglobulin supplementation. conclusions: combined immunodeficiency with variable degrees of b and t cell lymphopenia and hypogammaglobulinemia can be profound in subjects with gof-stat mutation. despite proper anti-microbial prophylaxis, this immunodeficiency can lead to severe infections. in addition to treating the autoimmune and immune dysregulatory features, treatment with ruxolitinib can improve the cid present and potentially reduce infectious susceptibility. igg -related disease (igg -rd), its common mimickers and response to anti-il -(reslizumab) treatment rachel eisenberg, md , arye rubinstein, md-phd fellow in allergy and immunology, montefiore medical center chief division of allergy and immunology, albert einstein college of medicine and montefiore hospital, bronx, ny, usa introduction/background: we describe a complicated case of igg related disease (igg -rd) both in its presentation and novel treatment objectives: to review the common mimickers of igg -related diseases which often lead to delayed diagnosis and treatment. to discuss novel therapeutic treatments for igg -related disease results: a -year-old woman with a history of thyroid disease, sicca symptoms, lipodystrophy, relapsing parotid enlargement, asthma and erdheim chester syndrome initially presented with recurrent bacterial and fungal sinusitis despite multiple sinus surgeries. immunologic workup was notable for lymphopenia of /ml, cd count of ( - cells/ul normal range) and elevated igg of ( . - . normal range). imaging was notable for nasal septal perforations and hypoplastic maxillary sinuses. there was high suspicion for igg disease however the patient was lost to follow up during which time she developed cachexia, eosinophilic pleural effusions ( % eosinophils), lung mass and a parotid mass with predominant t cell infiltrate misdiagnosed as follicular lymphoma. features consistent with igg -rd included > % ratio of igg /igg and a predominant t cell infiltrate a biopsied lung mass showed igg plasma cell > /hpf also consistent with igg -rd. bone marrow biopsy was within normal limits. the patient was treated with rituximab, an effective treatment for igg -rd. on treatment her igg levels normalized, however she developed recurrent large eosinophilic lung effusions requiring repeat drainage. fractional exhaled no (feno) was elevated to ppb. she was started on reslizumab at a dose of mg/ kg resulting in marked improvement in her respiratory status along with normalization of peripheral eosinophilia and reduction of feno to the normal level of ppb. conclusions: igg -rd is a fibro-inflammatory condition which can affect any organ system and is diagnosed via tissue histology showing igg positive plasma cells and a typical morphologic pattern. this case outlines the common mimickers of igg -rd often leading to a delayed diagnosis. before the final diagnosis of ig -rd was made by us, the patient carried multiple diagnoses including: thyroid disease, recurrent parotid enlargement and seronegative sjogrens. these diagnoses in hindsight may have been mikuliczs syndrome, kuttners tumor and/or riedels thyroiditis which are common manifestations of igg -rd. chronic sinusitis, atopic diseases, peripheral eosinophilia and destructive osseous lesions as noted in our patient are seen in up to % of patients with igg -rd. destructive bony lesions and eosinophilia can mimic granulomatous polyangiitis, which was ruled out in our patient. her cachectic appearance and diagnosis of lipodystrophy can be explained by destruction of osseous tissue in the craniofacial skeleton which was later confirmed on imaging. lymphoid inflammatory infiltrates are commonly seen in igg -rd and are can be misdiagnosed as a follicular lymphoma as in our case. a novelty in this case is the successful treatment with reslizumab targeted at the eosinophilic component of the disease. on reslizumab our patients asthma was for the first time controlled, pleuritis improved, fractional exhaled no (feno) normalized and her cachexia is improving. treatment with reslizumab should be considered in patients with igg -rd who manifest with eosinophilic respiratory disease. introduction/background: ikbkb deficiency (c. dupg in exon ) is a rare autosomal recessive form of severe combined immune deficiency (scid) originally described in canadian infants of northern cree descent. ikbkb scid is characterized by normal lymphocyte development, but impaired t-cell activation, along with innate immune defects. objectives: to report the clinical presentation, immunologic phenotype, and outcomes for patients with confirmed or suspected ikbkb scid due to this founder mutation. methods: we retrospectively reviewed hospital records dating back to of patients with confirmed homozygous ikbkb mutations, as well as patients suspected to be affected due to their clinical presentation and family relations to molecularly confirmed cases. results: fifteen patients were included. they presented early in life (average age months) with invasive and disseminated viral, bacterial, mycobacterial, and fungal infections. patients had concurrent and multiple infectious organisms, with a notable predilection for candida, gram negative organisms, and mycobacteria. four patients in our cohort received bcg vaccination at birth, resulting in fatal disseminated m. bovis infection in all, and additional patients succumbed to atypical mycobacterial infection following hematopoietic stem cell transplant (hsct). one newborn was identified in through new initiatives for targeted newborn screening for the mutation. immunologic features at presentation included normal to elevated lymphocyte counts with normal to elevated cd , cd , and cd t cells. when tested, response to pha varied from absent ( / ), to low ( / ), to normal ( / ). most patients had hypogammaglobulinemia, most often of the igg isotype ( / ). six had assessment of trec levels, and all had values above thresholds for screening programs. eight patients died before they could undergo hsct, and received transplants in the setting of ongoing severe, lifethreatening infections. only patient underwent hsct prior to the onset of infection. in our cohort, there are only long-term survivors. conclusions: ikbkb deficiency is a severe form of scid with early onset of invasive and disseminated multi-organism infection. the immunologic phenotype is characterized by normal to elevated lymphocyte numbers which do not meet pidtc criteria for scid, variable (and sometimes normal) mitogen response, normal trec levels, and low igg levels. the disease is universally fatal without hsct, however, conclusions regarding efficacy and long-term outcomes of hsct are uncertain given the small sample size. immune -dysregulation mimicking systemic lupus erythematosus in a patient with lysinuric protein intolerance introduction/background: lysinuric protein intolerance (lpi) is an inherited aminoaciduria caused by defective amino acid transport in epithelial cells of the intestine and kidney due to bi-allelic, pathogenic variants in slc a . the clinical phenotype of lpi includes failure to thrive and multi-system disease including hematologic, neurologic, pulmonary and renal manifestations. individual presentations are extremely variable, often leading to misdiagnosis or delayed diagnosis. here we describe a patient that presented with suspected immunodeficiency in the setting of early-onset systemic lupus erythematosus (sle), including renal involvement, who was subsequently diagnosed with lpi post-mortem. objectives: describe a clinical a patient with lysinuric protein intolerance that presented as early-onset systemic lupus erythematosus (sle), including renal involvement and primary immunodeficiency. methods: after informed consent was obtained, dna samples were obtained from the proband and his parents. trio whole exome sequencing was performed to identify a cause of early onset autoinflammation resembling systemic lupus erythematosus. results: the male proband had a history of failure to thrive starting at months of age, recurrent bacterial otitis media, and one episode of severe bacterial pneumonia requiring hospitalization. he presented at months of age with multifocal pneumonia, anemia (hgb . mg/dl) and mild thrombocytopenia. initial laboratory studies revealed low albumin ( . mg/dl), elevated ldh ( ), and mild hepatomegaly. renal and liver function testing was initially normal. immunologic evaluation for suspected primary immune deficiency showed normal immunoglobulin titers, low c ( ) and low c ( . ). lymphocyte phenotyping revealed low b cell counts ( . % of total lymphocytes) with t cells and nk cells within the normal range. despite antibiotic therapy, the patient worsened, developing fevers, a generalized erythematous rash, edema and nephrotic syndrome with oliguria. renal biopsy uncovered glomeruli with accentuated, global thickening and diffuse, peripheral capillary loops, as well as focal spiculated defects, there was endothelial swelling and other signs of acute damage including epithelial flattening, adluminal irregularity and extensive intraluminal proteinaceous detritus. endocapillary proliferative lesions, extracapillary crecents or tubular atrophy was not observed. immunofluorescence studies were positive for c , igg and c q granular deposits, mainly at the mesangium, interpreted as lupus nephropathy. endothelial swelling and massive, subepithelial electron-dense deposits with spike formation from the basement membrane were noted on electron microscopy, mimicking a stage ii membranous pattern of injury. autoantibodies included ana ( : ), anti-dsdna, smith, ssa and rnp were positive in agreement with the diagnosis of sle. immunosuppressive therapy with high dose iv corticosteroids and cyclophosphamide was initiated. despite this, the patient developed pancytopenia, elevated ferritin levels, increased triglycerides, and low fibrinogen. bone marrow biopsy displayed erythrocyte phagocytosis by macrophages, confirming a diagnosis of hemophagocytic lymphohistiocytosis (hlh). the patient subsequently died despite aggressive immunosuppression with high dose methylprednisolone and high dose iv immunoglobulin and dialysis. samples were collected from the deceased patient and his parents for research whole exome sequencing. trio analysis identified compound heterozygous missense variants in slc a . ammonia levels were not evaluated during the patients hospitalization. conclusions: lysinuric protein intolerance is a severe metabolic disorder that can present with protean systemic features including primary i m m u n o d e f i c i e n c y. i m p a i r e d l y m p h o c y t e f u n c t i o n , hypocomplementemia, immune-mediated glomerulonephritis, autoantibodies, and hlh are known complications of lpi. exactly how ineffective amino acid transport triggers these systemic inflammatory features is not yet understood. lpi should be considered in the differential diagnosis of early-onset sle, particularly in the absence of response to immunosuppressive therapy. director, national institute of immunohaematology introduction/background: chronic granulomatous disease (cgd) is a primary immunodeciency disorder with recurrent pyogenic infections and granulomatous inflammation resulting from loss of phagocyte superoxide production. mutations in any one of the five structural genes of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex viz. cybb and cyba encoding for membrane bound gp phox and p phox; and ncf , ncf , and ncf encoding for cytosolic components p phox, p phox, and p phox respectively, have been found to cause cgd. the relative incidence of these gene defects varies significantly depending on the ethnic background of the population. identification of molecular defect is important for patient management as well as for prenatal diagnosis in the affected families. the present study was aimed at studying the pattern of underlying genetic defects in a cohort of indian patients affected with cgd. objectives .to identify the underlying genetic defect in patients with chronic granulomatous disease in india . clinical,immunological and molecular characterisation . to utilise this information for genetic counselling and prenatal diagnosis of the affected families methods: eighty-seven (n= ) patients with abnormal nbt and dhr were included in this study. in case of male patients, mothers were first screened for carrier status to rule out x-linked cgd (xl-cgd). those patients where mother is not showing mosaic pattern were suspected for autosomal recessive cgd (ar-cgd) and were screened for the ratio of ncf gene to pseudo ncf gene by genescan analysis. additionally, evaluation of nadph oxidase components expression by flow cytometry also helped us to determine the underlined genetic defect and it is validated by dna sequencing of respective genes. results: eighty-seven patients were molecularly characterized to identify disease causing mutation which includes: novel mutations in cybb, in cyba, in ncf gene in our cohort. . % (n= ) of the patients belonged to xl-cgd. . % (n= ) of the patients are suspected to have ncf gene defect among which; homozygous delgt mutation was identified in patients. . % and . % patients showed abnormal p phox and p phox expression suggesting defect in cyba gene and ncf gene respectively. spectrum of mutations involve: % of delgt mutations, % nonsense, % missense, % deletion, % insertion, % other than homozygous delgt mutations. male to female ratio is . : . consanguinity is noted in % of the patients. conclusions: despite the male predominance ar-cgd is more common ( %) as compare to xl-cgd ( %) in this cohort of indian patients, which is distinct from the western data. % are the novel mutations suggesting, a wide heterogeneity in the nature of mutations in indian cgd patients. flow cytometric evaluation of nadph oxidase component is used as a secondary screening test to identify cgd sub-group. molecular characterisation of cgd genes was not only used in the confirmation of diagnosis but also in genetic counselling and pre-natal diagnosis in affected families. novel nlrc gain-of-function mutation presenting with neonatal enterocolitis and autoinflammation, with positive clinical response to rapamycin and anakinra. senior investigator, viral immunology section, national institute of neurological disorders and stroke senior investigator, translational neuroradiology section, national institute of neurological disorders and stroke staff clinician, neuroimmunology clinic, national institute of neurological disorders and stroke staff clinician, laboratory of clinical immunology and microbiolology, niaid, nih introduction/background: cytotoxic t-lymphocyte antigen- (ctla- ) is an essential negative regulator of the immune response and its function is critical for immune homeostasis. uni-allelic mutation in the ctla gene leading to reduced function or expression of ctla- , termed ctla- haploinsufficiency, can lead to systemic immune dysregulation with wide spread clinical disease, but with variable clinical penetrance. the neurological manifestations of ctla- haploinsufficiency are not known. objectives: to perform detailed phenotyping of the neurological manifestations of ctla- haploinsufficiency. methods: a retrospective review and prospective collection of clinical, imaging, cerebral spinal fluid, and pathological specimens was performed in a cohort of genetically confirmed patients (n= ) with ctla mutations who are followed at the national institutes of health. neurological symptoms and exams were collected on patient visits and from historical records. the data collected included brain mris and spinal cord mris that were visually inspected for evidence of inflammation. cerebral spinal fluid values were obtained from patients including flow cytometry in patients. pathological tissue from brain biopsies of inflammatory lesions was examined from patients. results: central nervous system (cns) inflammation was found in / ( %) of the cohort. common clinical manifestations from the patients with cns inflammation were headaches / and seizure / . focal deficits were rare. mri findings included contrast enhancing neuroinflammatory lesions in the brain / , brainstem/cerebellum, and spinal cord / . figure a -c show representative inflammatory lesions. lesions were multifocal in / patients and / had recurrent inflammatory lesions on longitudinal follow up. lesions were, at times, extremely large, / with a lesion > cm^ . leptomeningeal enhancement (lme) was seen in / patients and clearly preceded intraparenchymal lesion development in patients. figure d shows a site of lme (green chevron) that develops into an intraparenchymal lesion (yellow chevron), mris are separated by days. spinal fluid analysis showed a lymphocytic pleocytosis (mean cells/mm^ ) with the presence of oligoclonal bands in patients. pathological features included a mixed cellular infiltrate, predominantly lymphocytes or plasma cells, with little evidence of demyelination or necrosis (figure e). conclusions: the neurological manifestations of ctla- haploinsufficiency include recurrent and, at times, severe neuroinflammation. however, even large lesions and lesions in eloquent anatomical locations had little to no focal clinical defects resulting in a striking clinical-radiological dissociation. future studies into the mechanisms of cns-related disease may reveal important information related to peripheral and central immune system functioning. conditioning with anti-cd immunotoxin in a mouse model of hypomorphic rag deficiency allows complete reconstitution of the immune system with lack of toxicity enrica calzoni, md , cristina corsino, technician , marita bosticardo, phd , yasuhiro yamazaki, md phd , hsin-hui yu, md phd , lisa ott de bruin, md , john manis, md , rahul palchaudhuri, phd , david scadden, md , luigi d. notarangelo, md treated mice and % in cd -sap/ treated mice. at sacrifice, in the bm we observed strong selective advantage for donor b cells at all developmental stages, but in particular in the most mature subsets, in both cd -sap and cd -sap/ treated mice, rescuing the block in development at pre-b cell stage found in untreated f l mice. donor engraftment in bm hsc reached levels around % and % in cd -sap and cd -sap/ treated mice, respectively. donor chimerism in t and b cells in the spleen was also higher than % in both cd -sap and cd -sap/ groups. in the thymus, full donor chimerism was achieved in both cd -sap and cd -sap/ treated mice, starting at the dn stage and persisting at dp, sp and sp . importantly, in both treatment groups, t cell development was corrected both in terms of subset distribution and absolute numbers to levels comparable to those of wt mice. finally, the thymic epithelial cell compartment was also fully reconstituted, with a normal number, distribution and maturation of both cortical and medullary thymic epithelial cells. conclusions: in conclusion, we show here that conditioning with cd -sap immunotoxin, alone or in combination with rads tbi, is safe and leads to full reconstitution of the immune system in rag hypomorphic mice, suggesting that this conditioning regimen should be considered for testing in clinical setting. introduction/background: foxp is a key transcription factor for the maintenance of immune tolerance. foxp mutations result in dysfunction of foxp + regulatory t cells (tregs) causing immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome, a severe early onset autoimmune disease, which can be fatal if not promptly diagnosed and treated. our recent international study analyzing the longterm outcome in i.e. patients of the two currently available treatments, pharmacological immune suppression and allogeneic hematopoietic stem cell (hsc) transplantation, showed poor long-term disease-free survival or overall survival limitations, respectively (barzaghi f. et al, jaci, ) . ipex syndrome is a good candidate for gene therapy as it has been demonstrated that reconstitution of wild-type treg cells can control the disease. however, foxp expression is highly regulated, and its safe and physiological expression in treg and teffector (teff) cells is challenging. lentiviral-mediated (lv) foxp gene transfer successfully converts ipex patients-derived cd + t cells into treg-like cells (cd lv-foxp t cells) with stable suppressive capacity (passerini l. et al, sci transl med, ) . these ex vivo converted tregs are ideal as a short term cell-based therapy for ipex patients, but this approach does not reestablish regulated foxp expression in teff cells, that also likely contribute to the ipex pathology. thus, we are further characterizing cd lv-foxp t cells and, at the same time, developing gene editing strategies for ipex, whereby autologous t cells or hscs are genetically modified or corrected, respectively, and reinfused into the patients. objectives: to provide more effective treatments for ipex patients, we are i) optimizing lv-foxp gene transfer in t cells to be suitable for clinical use, and ii) establishing a novel foxp gene editing in hscs and testing both approaches in preclinical models. methods: lv-foxp gene transfer can be obtained in cd + t cells activated polyclonally or in an antigen-specific manner. the vector construct is bidirectional, foxp expression is under the ef a-promoter and the truncated form of ngfr, used as marker gene, is under cmv promoter. foxp gene editing is performed using a combination of crispr/cas , a chemically modified sgrna targeting foxp , and an aav packaged homologous donor dna template and the efficacy and safety of the resulting construct is tested in different cell types in vitro and in humanized mice. results: we demonstrate that cd lv-foxp t cells can successfully be generated specific to different antigens. this result opens to new potential clinical benefit of cd lv-foxp t cells with more safe and specific regulatory effect than polyclonal cd lv-foxp t cells. we are currently adapting the protocol to optimal in vitro production for clinical use and assessing dose, survival and efficacy of the cd lv-foxp t cells using different in vivo models. due to the wide distribution of identified mutations throughout the foxp gene, we have designed a gene editing strategy that uses homology directed repair to insert the coding sequence of the foxp gene at the start codon of the endogenous mutated foxp gene. this strategy permits regulated expression of the inserted wild-type, functional foxp protein in patient cells independent of the location of the downstream mutation. using this site-specific gene knock-in, we find that the system effectively targets expression of foxp in different cell types, namely tregs, teff cells and primary human cord blood-or bone marrow-derived hscs. gene editing of normal donor and ipex tregs and teff cells allowed us to test for regulated gene expression and for establishment of normal treg suppressor function and t cell proliferation upon activation. additionally, preliminary results demonstrate that gene edited hscs can be transplanted into nsg mice for long-term reconstitution. conclusions: our results show the feasibility of different gene therapy approaches for ipex syndrome. in addition, they suggest that cd lv-foxp t cells, either polyclonal or antigen-specific, could be applied not only in ipex but also in immune mediated diseases of different origins. the results from the foxp gene editing support the use of crispr/ cas to treat ipex syndrome patients with autologous edited hscs. this gene editing approach may also be applied to treat other pediatric monogenic blood and immune disorders. human pi kgamma deficiency with humoral defects and lymphocytic infiltration of barrier tissues andrew takeda, bs , william comrie, phd , yu zhang, phd , paul tyler, bs , koneti rao, md , carrie l. lucas, phd graduate student, yale university clinician, niaid, nih assistant professor of immunobiology, yale university introduction/background: the phosphatidylinositol -kinase (pi k) signaling pathways play a key role in transducing signals from a diverse array of stimuli by producing the pip second messenger. class ib pi k is primarily activated by g protein-coupled receptors (gpcrs), and this class is comprised of the p gamma catalytic subunit in complex with the p or p regulatory subunit. in contrast to the class ia pi k subunits, inherited mutations in the genes encoding the class ib subunits have not been described. objectives: given the leukocyte-restricted expression pattern of p gamma, we hypothesized that mutations affecting this kinase may be found in cohorts of patients with rare immunodeficiency disorders. our objective was to identify such mutations and determine molecular, biochemical, and cellular derangements in patients with mutated p gamma. methods: we used whole-exome sequencing of families to identify inherited gene mutations and determined the mechanistic basis of disease using biochemical assays to assess effects on protein function and cellbased assays to define functional defects with disease relevance. results: we identified a patient (here called a. ) harboring compound heterozygous mutations in pik cg, the gene encoding p gamma, who presented in early life with autoimmune cytopenias and eczema and, at the age of years, developed cryptogenic organizing pneumonia and prominent t cell infiltration of the lungs. she also has a history of skin infections, lymphadenopathy/splenomegaly, eosinophilia, defective antibody production, and more recently, lymphocytic colitis. she inherited a frameshift pik cg mutation from her mother and a missense mutation resulting in an r p amino acid substitution from her father. expression of p gamma protein was lost, and stability of its p binding partner was reduced. despite defective t cell signaling responses to chemokines (i.e., gpcr stimulation), chemotaxis of patient t cell blasts in vitro was normal. intriguingly, the frequency of peripheral blood treg cells was low in patient a. , and her cd t cells more frequently expressed the tissue-homing cxcr chemokine receptor. consistently, serum levels of cxcr ligands were elevated in patient a. . moreover, we found augmented inflammatory cytokine production from m -polarized macrophages differentiated from patient a. monocytes or from thp cells treated with p gamma inhibitor or stably expressing pik cg shrna. conclusions: we report the first human with loss of pi kgamma activity and present her clinical presentation with notable t cell infiltration of barrier tissues. based on our analyses, we propose that loss of p gamma activity in humans causes t cell-intrinsic effects of reduced tregs and increased tissue-homing propensity and the t cell-extrinsic effect of augmented inflammatory responses in macrophages. together, these consequences of p gamma deficiency drive aberrant accumulation of t cells in lung and gut. introduction/background: pulmonary disease is a frequent complication across many primary immunodeficiencies (pidds), however its impact on the quality of life (qol) in pidds is not well characterized. objectives: to ascertain the types of infectious and non-infectious pulmonary complications occurring in pidds and to determine how these complications affect qol. methods: we analyzed the pulmonary complications, disability descriptions, and clinical status of subjects with pidds in the usidnet registry using descriptive statistics. karnofsky or lansky performance indices (n= ) and promis qol data (n= ) were also analyzed. the t-test/mann-whitney test and chi square test were utilized to compare continuous and categorical variables, respectively. results: infectious pulmonary disease was reported in a majority of subjects ( . %), most commonly pneumonia ( . %) and bronchitis ( . %). non-infectious pulmonary disease was reported in . % of all subjects, most commonly asthma/reactive airway disease ( . %), bronchiectasis ( . %) and interstitial lung disease ( . %). pulmonary insufficiency was listed as a cause of disability in . % of all subjects with pidds, with highest rates of this disability in subjects with immune dysregulation ( . %). lower karnofsky/lansky performance scores were observed in subjects with pneumonia, lung abscess, bronchiectasis, interstitial lung disease, and emphysema/copd as compared to without these disorders (p< . ). promis qol metrics were largely similar among subjects with and without pulmonary disease, although physical function scores were significantly worse in those with copd/emphysema (mean= . +/- . ) as compared to without (mean= . +/- . , p = . ). promis physical function scores were also worse in subjects with non-infectious pulmonary disease (mean = . +/- . ) compared to those with infectious pulmonary disease only (mean = . +/- . , p= . ). a significantly greater percentage of patients with a history of copd/emphysema ( . % vs. . %) or interstitial lung disease ( . % vs. . %) were deceased as compared to those without a history of these disorders (p< . ). conclusions: both infectious and non-infectious pulmonary disorders cause significant morbidity in pidds and are associated with higher mortality in this population. infectious and non-infectious pulmonary complications were often associated with worse karnofsky/lansky scores while there was limited impact on promis qol measures. latin-american consensus on the management of patients with severe combined immunodeficiency, part : supportive measures during the time from diagnosis to definitive treatment. juan carlos bustamante ogando , armando partida-gaytán , francisco espinosa rosales , lasid "consensus on scid" study group pediatric allergy and clinical immunology specialist, clinical immunologist and researcher at primary immunodeficiency research unit, national institute of pediatrics pediatric allergy and clinical immunology specialist, researcher at primary immunodeficiency research unit, national institute of pediatrics pediatric allergy and clinical immunology specialist, president, fundación mexicana para niñas y niños con inmunodeficiencias primarias (fumeni) result in a majority of patients with late diagnosis, more comorbidities and reduced access to curative treatments. the interventions during such period are vital to keeping optimum health status to improve the probability of success of curative therapies. many interventions are not supported by clinical trials, are based mainly on clinical experience, and there are no clinical guidelines to standardize such treatments. objectives: to generate a consensus on the supportive care of patients with scid, from the diagnosis until a curative treatment is given, under a latin-american perspective taking into account particular challenges for our region. methods: in a first step, we gathered available information about scid diagnostic and therapeutic guidelines from two sources: a) literature search and b) personal communications with pid experts from europe and usa. next, we developed an expert consensus through a modified delphi technique (electronic and anonymous). we used google® forms® to gather the information and microsoft office excel® for the analysis of agreement through kappa coefficient and rounds concordance through repeated measures analysis of variance (anova). results: we gathered an expert panel of subjects from latin-american countries (argentina, brazil, chile, costa rica, mexico, and peru) including the primary centers caring for scid patients. we generated a document with agreed diagnostic and therapeutic interventions grouped in topic-domains (i.e. protective and isolation methods to decrease the risk of infections, antimicrobial prophylaxis, immunoglobulin treatment, immunizations, nutritional aspects, antimicrobial treatment, blood derivatives use, routine laboratory workup, imaging and other studies, conventional multidisciplinary approach). we also included nonagreed interventions, but where relevant arguments are shared, to allow for particular clinical scenario decisions. conclusions: this is the first document of its type, and it intends to standardize clinical care of latin-american patients with scid, reduce disease burden and ultimately improve health outcomes. we see this effort as a starting point for the continuous improvement of our professional care to such patients and is intended to help as a tool not only for immunologists but for primary care physicians and other specialists involved in scid patient's care. this work will hopefully be published during as a lasid collaborative work, and it will help as a guide for clinicians caring for scid patients not only in latin america but in other world regions. also in the future, this consensus may be improved by collaboration from immunologists worldwide. no significant difference in hospitalizations one year before vs. year after treatment for prophylactic antibiotics (p= . ) or igrt (p= . ). baseline igg was higher in prophylactic antibiotics vs. igrt ( . vs. . mg/dl, p= . ) . sex, severity of sad, igg subclasses deficiency, and lymphocyte counts were not significantly different between treatment groups. conclusions: prophylactic antibiotics are not inferior to igrt in preventing infections in some sad patients. while, clearly some patients with sad will need igrt, our date indicate that larger prospective studies are needed to identify patients who will benefit most from igrt vs prophylactic antibiotics alone. richard and barbara schiffrin presidents distinguished professor of microbiology and director, institute for immunology, university of pennsylvania introduction/background: t cell thymic development is dependent on signals received via the pre-tcr complex and we here report the first case of pre-tcr alpha (ptcra) autosomal recessive t cell immunodeficiency in an infant with a positive scid newborn screen (nbs). objectives: we sought to uncover the mechanistic links between ptcra mutations and immune dysfunction. methods: the patient was tracked clinically, with serial clinical immunophenotyping and t cell function testing. in addition, we performed deep immunophenotyping with mass cytometry and single cell rna sequencing to delineate the molecular circuitry underlying her immune phenotype. results: the patient presented with t cell lymphopenia and impaired response to mitogen stimulation. hsct was considered, but she did not meet clinical criteria and remained healthy, so she was watched closely on prophylaxis while awaiting genetic testing. response to serial mitogen stimulation remained between~ - %, response to serial cd /cd activation was normal and tcrv spectratyping was normal. whole exome sequencing revealed two mutations in ptcra. no prior human cases of ptcra deficiency have been published, but a mouse model bears a striking resemblance to this case (fehling et al, nature, ) , with elevated t cells and decreased t cells. her mitogen stimulation responses became persistently normal around years of age with stable t cell lymphopenia, elevated t cells and normal switched memory b cells. anti-fungal prophylaxis was halted, and she remained on atovaquone alone with persistent t lymphopenia. she was able to mount an antibody response to rabies vaccine at . years and was weaned off scig replacement and is planned to initiate vaccination. deep immunoprofiling with mass cytometry (cytof) demonstrated a unique immunophenotype. single cell rna sequencing confirmed normal cd and cd tcr clonotypic diversity but increased clonotype diversity in t cells and increased and transcript levels. in addition, cd naïve, cd memory and cd naive t cells demonstrated both increased numbers of expressed genes and transcriptomic diversity, with altered cytoskeletal and tcr proximal signaling pathways across t cell subsets versus control. this may reflect a peripheral role for ptcra, a durable imprint of thymic signaling events mediated by ptcra, evidence of homeostatic proliferation or a combination of the above. conclusions: scid nbs led to identification of homozygous variants in ptcra causing a novel t cell immunodeficiency characterized by t cell lymphopenia, altered proximal tcr and cytoskeletal signaling and increased number of altered t cells. we will continue to pursue the mechanism of these mutations by developing ipsc and studying their t cell differentiation capacity in vitro, as well as further defining her immunometabolic phenotype. rag hypomorphic mouse mutants show partial preservation of thymocyte development but peculiar abnormalities of thymic epithelial cell phenotype introduction/background: the recombination-activating gene (rag) and rag proteins are essential for v(d)j recombination. in the absence of these proteins, the development of b and t cells is blocked at early progenitor stages, resulting in severe combined immunodeficiency (scid). hypomorphic mutations in rag , allowing residual activity, result in delayed-onset combined immunodeficiency with residual development of t and b lymphocytes, associated with autoimmunity and/or granulomas (cid-g/ai). objectives: to study in details the effect of rag hypomorphic mutations at the early stages of t cell development, we have generated mouse models carrying mutations described in patients with cid-g/ai (r q, r w, f l) (niaid animal protocol: lcim e). methods: we performed an extensive evaluation of the thymic phenotype in the mouse models. results: the number of total thymocytes was found to be drastically reduced in all three models. however, two of these mouse models (r q and f l) retained a significant level of rag activity, and resulted in the development of mature t cells in the thymus, while the mouse model carrying the r w mutation had minimal rag activity and presented a phenotype more similar to that of complete rag knockout mice. in r w mice, almost all thymocytes were blocked at the double negative (dn) stage and there were virtually no mature t cells, as found in rag ko mice. on the other hand, r q and f l mice presented double positive (dp) and single positive (sp) and sp cells. the cross talk between t cells and thymic epithelial cells (tec) in the thymus is fundamental for the development and maturation of both types of cells. in rag -/-mice, and consequently in the absence of mature t cells, tecs cannot complete their maturation, and the mtec subset is virtually absent. these results were also observed in the r w mouse model. instead, in r q and f l mice, the residual rag gene activity allows development of a reduced number of mature t cells. although the number of tecs was markedly reduced in r q and f l mice, ctecs and mtecs were both present, but with an excess of ctecs. furthermore, mtecs were predominantly mhc-iihigh (mtechi), and only a minority of mtecs were mhc-iilow (mteclo) cells, the opposite of what found in adult wt mice. finally, mtechi cells from rag mutant mice were found to express aire to levels and frequencies comparable to those of wild-type (wt) mice. conclusions: our results show that tec in mouse models carrying rag hypomorphic mutations are affected both in terms of absolute numbers and in terms of subset distribution and maturation state. to further investigate the functional consequences of impaired cross-talk between thymocytes and tecs in rag mutant mice, we have performed rnaseq in sp and tecs sorted from r q, f l and wt mice. analysis of the gene expression profile in tec may thus provide novel insights in the mechanisms that govern normal and pathologic thymic t cell development. vedolizumab for autoimmune enteropathy in primary immunodeficiency: a case series of outcomes introduction/background: gastrointestinal complications are common in patients with primary immunodeficiency. infections are the leading cause, but autoimmune enteropathies including inflammatory bowel disease (ibd)-like colitis, sprue-like enteropathy, and nodular lymphoid hyperplasia (nlh) have been recognized in a subset of these patients. to date, there is no established treatment for these noninfectious disorders. vedolizumab is a humanized monoclonal antibody that binds to the alpha- beta- integrin, inhibiting the migration of memory tlymphocytes across the endothelium into inflamed gastrointestinal parenchymal tissue. it is fda approved as first-line therapy for inflammatory bowel disease. the safety and efficacy of treating autoimmune enteropathy with vedolizumab in patients with concurrent primary immunodeficiency (pid) has not previously been reviewed. objectives: to review the outcomes of a series of patients with hypogammaglobulinemia and autoimmune enteropathy following vedolizumab therapy methods: patients ( male, female) at mount sinai with enteric biopsies demonstrating inflammatory enteropathy with t cell infiltrates have been treated with vedolizumab. results: five of the seven patients completed induction therapy. one patient was recently started on therapy. therapy was aborted in one patient who developed acute hepatitis during induction. another developed severe cytomegalovirus enteropathy, prompting discontinuation. two patients discontinued therapy due to response failure. at present, two patients remain on therapy at months with symptomatic improvement. conclusions: vedolizumab was effective in cases, but had no benefit or deleterious side effects in subjects. its effectiveness in another patient is presently under investigation. introduction/background: ataxia telangiectasia (at) is an immunodeficiency most often associated with t cell abnormalities and abnormalities in serum immunoglobulin levels, primarily iga. there is a subset of patients with a hyper-igm phenotype, some with cutaneous granulomas, which may reflect a distinct clinical phenotype. a yearold female presented for evaluation of concern for immunodeficiency because of frequent illnesses, presumed to be viral. she was found to have an ataxic gait, some speech delay, mild ocular and ear pinna telangiectasia, and an ulcerative rash on the left upper and right lower extremity. initial blood work showed elevated -fetoprotein levels ( ng/ml), elevated serum igm ( mg/dl), low igg (< mg/dl), and iga ( . mg/dl). objectives: to determine if the atm mutations in this patient are associated with perturbations in the frequencies, distributions and functions of b and t cell subsets which account for the observed phenotype. methods: next generation sequencing was used to identify the mutations in the atm gene. b and t cells were purified from the patients peripheral blood by positive selection. intracellular staining for foxp and t-bet was performed. b cells were activated in the presence of polyclonal f(ab) rabbit anti-human igm, multimeric, soluble, recombinant-human cd l, gardiquimod (tlr agonist), or cpg (tlr agonist). the treg suppression assay was carried out by co-culturing cd +cd hicd lo tregs and cd +cd cd + responder t cells at a : ratio in the presence of beads loaded with anti-cd , anti-cd , and anti-cd for . days. results: next generation sequencing revealed two pathogenic mutations in the atm gene, a novel mutation creating a premature stop codon [c. dela,(p.lys asnfs )], and a nonsense mutation [c. c>g, (p.tyr ter)]. proliferative responses of pbmc to mitogens (pha, cona, pwm) were reduced to roughly half of the control responses; the response to tetanus was normal whereas the response to c. albicans was absent. serum cytokine analyses demonstrated elevations in levels of tnf ( . pg/ml) and il- ( pg/ml); levels of ifn, il- , il- , il- , and il- were below the limits of detection. b cell abnormalities included markedly increased percentages of cd locd lo cells ( %) expressing t-bet and fas. activation of these cd /low b cells through the b cell receptor, tlr and tlr , and cd was decreased in response to all of the stimuli as evidenced by a lower percentage of b cells expressing the activation markers cd and cd relative to healthy control samples. the frequency of unswitched cd +igd+ memory b cells was also increased ( %). among the naive b cells, the proportion of cd + cd cd cd +igmhi transitional b cells that newly emigrated from the bone marrow (bm) was found to be diminished to . % of the naive b cell compartment. in the t cell compartment, there was a decreased frequency of total cd + cells but normal absolute numbers of cd +cd + t cells. there was also a decreased proportion of naive cd +cd +cd rocd l+ t cells and a striking increase in the cd +cd +cd ro+ memory t cells ( %). this appeared to be largely attributed to the increased proportion of cd +cd +cd ro+cd l effector memory t cells ( %). the circulating t follicular receptor (ctfh) cell frequency in the patient was -fold higher ( %) than the average for healthy donors but icos expression levels were normal. treg frequency was decreased but suppressive capacity was not impaired. conclusions: the mutations in atm described here add to the growing understanding of the heterogeneity in degree and complex nature of the immunodeficiency seen in patients with at. these mutations resulted in perturbations in frequencies and distributions of normal and atypical b and t cell subsets, which can explain some immunologic aspects of the clinical phenotype in this patient. the immunophenotype seen here may also differentiate at patients with granulomas from those without cutaneous lesions. supported in part by grifols, the joanne siegel memorial fund, the dreizessen fund (to ewg), grants from niams t ar - (km) and niaid r ai (em). introduction/background: a -month-old male presented with pancytopenia, b cell deficiency and developmental delay. he was born at weeks with weight of . kg. he was severely anemic with a hb of . , and transfused on day of life. he received hep b and bcg vaccines without complications. a month later he had a hb of . with a febrile illness. a bone marrow aspiration performed at days, showed dyserythropoiesis without hemophagocytosis, and normal numbers of precursors. t and b cells were decreased. further evaluation with repeat bone marrow showed decrease in all cell lineages. exome sequencing of the family showed homozygous variant in mysm (c. _ delp. (lys arg/s* ) omim: * ) in the patient. both parents and hla-matched sister, were heterozygous for the same variant in mysm . treatment consistent of replacement immunoglobulin, packed rbcs, and g-csf. there was no history of recurrent viral or severe bacterial infections except for - episodes of urinary tract infections, which were treated with antibiotics. physical exam revealed low set ears, sunken and wide set eyes, depressed nasal bridge, mild micrognathia, frontal bossing, and cm x cm cafeau-lait spot noted behind left knee. he was pancytopenic with a wbc count ranging /mcl to /mcl, and anc ranging from /mcl to /mcl (on intermittent g-csf). hb = . gm/dl requiring transfusions every - weeks, and platelet count was , /mcl. b cell deficiency was confirmed with total b cell count of cells/mcl. b-cell maturation was essentially normal. t cell counts were normal with ageappropriate distribution of naïve and memory t cells, and t cell function. there was normal t cell receptor repertoire diversity. objectives: to assess defect in dna repair using a flow cytometry-based assay in a patient with mysm deficiency. methods: patients with mysm deficiency are reported to have increased genomic instability. deb testing, and telomere length analysis revealed normal results. defects in the dna repair pathway were assessed using a flow cytometry-based assay measuring phosphorylation of atm (patm), smc (psmc ) and h ax (gh ax) without irradiation, or h or h after low-dose ( gy) radiation using a cs source. the analysis was performed in t, b and nk cells. results: the patient had higher patm and psmc in t cells compared to the experimental controls (hc) at h post-irradiation. also, the amount of gh ax in nk cells was significantly higher than hc at h post-irradiation. interestingly, the patients b cells showed approximately % of b cells with constitutive gh ax even without irradiation, and this subset increased slightly to % at h after irradiation. the mfi (amount) of gh ax also increased at this time-point. at h post-irradiation, there was normal dephosphorylation in healthy control lymphocyte subsets. however, the patients t cells did not de-phosphorylate completely and showed higher residual patm, and psmc in both t and b cells. also, both t and b cells, at h, demonstrated a small subset of t cells ( %) with constitutive gh ax without irradiation, which increased to % after irradiation. there was also an increase in gh ax mfi in the irradiated sample. in b cells, % showed constitutive gh ax without irradiation at h, and this increased to % after irradiation, with a corresponding increase in mfi. conclusions: in summary, this rapid flow analysis revealed defects in the dna repair pathway, including higher patm, psmc and h ax phosphorylation in t, b and nk cells at h post-irradiation. at h, only t cells showed a residual subset with patm expression. but, psmc , a downstream target of atm, revealed higher levels in t, b and nk cells at h post-irradiation. this assay, which allows lineage-specific analysis, permitted dissection of dna repair defects, in individual lymphocyte subsets revealing heterogeneity within the cell subset to radiation susceptibility. the practical benefit of this rapid multi-parameter flow assay is selection of appropriate conditioning regimen for hematopoietic transplantation, as was the case with this patient. this has significant practical implications for treatment of patients with radiosensitive immunodeficiencies. ctla- haploinsufficiency-associated inflammation can occur independently of t-cell hyperproliferation introduction/background: cd and ctla provide opposing proliferative signals to t cells. we identified an -year-old female subject (s ) with heterozygous deletions of cd and ctla and multi-organ inflammatory disease characterized by a lack of t cell infiltrates in affected organs. inflammatory disease was remarkably responsive to s ctla -ig therapy. objectives: our goal was to characterize the immunologic consequences of combined deletion of cd and ctla , specifically assessing t cell proliferation and treg function in comparison to patients with alps associated ctla haploinsufficiency. we further sought to explain how this s s inflammatory diseases could occur without a pathologic t cell infiltrate and why they were amenable to ctla -ig therapy. methods: we performed phenotypic analyses of subject t cells and innate lymphoid cells (ilcs). we functionally characterized subject t cells. we created serum cytokine profiles. we stained and analyzed tissue biopsies. results: cd and ctla expression on s t cells were half that of control t cells. s t cells were hypoproliferative. s tregs were scarce and lacked suppressive function similarly to alps tregs. s tregs could suppress autologous t responder cells, likely due to their poor proliferative capacity. s colonic biopsies featured significantly fewer infiltrating intraepithelial lymphoid cells than biopsies from an alps patients. unlike alps patients whose colonic gland infiltrates were overwhelmingly t cells, s intraepithelial lymphoid cells were neither t cells nor b cells, suggesting the presence of ilcs. indeed, a greatly expanded population of type innate lymphoid cells (ilc ) and prototypical ilc cytokines were identified in s peripheral blood. ilc frequency and cytokine levels decreased in response to treatment with ctla -ig, corresponding with marked improvement in enterocolitis, hepatitis and pericarditis. conclusions: we report a novel genetic syndrome of combined cd /ctla deletion and describe the immunolopathologic correlates of this disease. dual ctla -and cd -haploinsufficiency results in a phenotype of multi-organ inflammatory disease characterized by ilc expansion in the setting of t-cell hypoproliferation and quantitative and qualitative treg defects. our patients clinical response to ctla -ig parallels published mouse studies and suggests the existence of additional stimulatory b receptor(s) preferentially expressed on ilc s over conventional t-cell populations. diagnosis of radiosensitivity and dna repair defect in dna ligase iv deficiency with a rapid flow cytometry assay. introduction/background: dna ligase deficiency (lig -scid) is one of several monogenic defects affecting dna repair, and causing lymphopenia (t-b-nk+) and a radiosensitive scid (rs-scid) phenotype. the assignment of a timely diagnosis is vital in the management of patients with rs-scid. laboratory assessment of radiosensitivity is laborious, and utilizes fibroblasts (non-hematopoietic) or lymphoblastoid cell lines, and can take several weeks to months for results. objectives: we demonstrate for the first time, the application of a flow cytometric-based kinetic analysis of phosphorylated h ax (h ax) in lymphocyte subsets, especially nk cells, for the diagnostic assessment of lig -scid. methods: simultaneous measurement of multiple dna repair markers phosphorylated (p) atm, smc and h ax (h ax) was performed by flow cytometry to assess dna repair defects in a -year-old korean female. the patient was evaluated for recurrent fevers, chronic respiratory tract infections, chronic diarrhea, and rash. genetic testing revealed compound heterozygous variants (nm_ , c. g>t, p.trp cys and nm_ , c. a>t, p.asp val) in lig . functional assessment (phosphorylation) was measured in t and nk cells (b cells were absent), before irradiation (background control), or after low-dose ( gy) irradiation ( and hours). results: we observed maximal h ax generation at hour post-irradiation, with progressive dephosphorylation at hours post-irradiation in healthy controls. the patient showed normal frequencies (%) of t cells and nk cells positive for h ax ( . % and . % respectively); (controls (n= ) t cells = . % and %; nk cells = . % and . %), but increased intracellular levels (mean fluorescence intensity, mfi) of h ax (t cells = . and nk cells = . ) compared to controls (t cells = . and . ; nk cells = . and . ) at hour post-irradiation. however, more importantly, at hours post irradiation there was a lack of dephosphorylation in a substantial proportion of lymphocytes ( % of t cells and % of nk cells) compared to healthy controls (t cells= . % and . %; nk cells = . % and . %). further, while there was dephosphorylation of h ax at h in patient lymphocytes as compared to h, the amount, as measured by mfi, remained elevated at h (t cells = . , nk cells = . ) compared to controls (t cells = . and . ; nk cells = . and . ). the data from patm and psmc were uninformative for the evaluation of lig -scid. conclusions: flow-based kinetic analysis of h ax is a useful marker for the diagnosis of lig -scid, and can be performed with a small amount ( cc) of blood, and provides a result in - days, facilitating rapid assessment of radiosensitivity in this condition. human plcg haploinsufficiency results in nk cell immunodeficiency and herpesvirus susceptibility objectives: we aimed to investigate the cause of disease in three patients from two kindreds with recurrent or severe herpesvirus infections and nk cell dysfunction. methods: we used exome sequencing and mass cytometry (cytof), as well as traditional immunologic techniques, to investigate the genetic causes, immune cell subpopulations/signaling, and nk cell function of these patients. we additionally used mouse models, crispr cell lines and in vitro assays to assess the role of plcg haploinsufficiency in disease. results: kindred a consisted of two patients presenting with hsv susceptibility and autoimmunity. kindred b consisted of one patient with severe cmv myocarditis and adenoviral hepatitis. both kindreds were evaluated for nk cell function and showed reductions in target killing in spite of normal cytotoxic granule degranulation against the same target. microscopy analysis suggested that granule mobility was reduced in at least one kindred. cytof revealed reductions in plcg phosphorylation after receptor crosslinking in the nk cells of both kindreds. kindred a also presented with a reduction in naïve b cells without perturbations in immunoglobulin output, b cell memory formation or class switching. trio whole exome sequencing was performed and revealed rare heterozygous plcg mutations in both kindreds. functional analysis, as well as mouse and crispr models, support a functional haploinsufficiency as a cause for nkd in these patients. conclusions: heterozygous loss-of-function point mutations in plcg have not been previously investigated as a cause of nk cell deficiency or recurrent herpesvirus infection. thus, these patients represent a novel immunodeficiency involving plcg haploinsufficiency, nk cell dysfunction, and herpesvirus susceptibility. hypomorphic rag mutations alter the pre-immune repertoire at early stages of lymphoid development introduction/background: human rag deficiency is associated with a spectrum of clinical phenotypes. while the most severe forms of rag deficiency manifest with severe combined immune deficiency or omenn syndrome since the first weeks of life, more recently patients have been identified who present to medical attention at a much older age predominantly with symptoms of autoimmunity and/or inflammation. many of the mutations associated with this atypical syndrome are found in the c-terminal domain (ctd) of the rag gene and allow residual development of t and b cells. these patients have an abnormal peripheral t and b cell repertoire, but how this is affected by abnormalities in the composition of the pre-immune repertoire vs. antigen-mediated selection and homeostatic proliferation in the periphery is unknown. objectives: in order to investigate whether mouse models with hypomorphic mutations in the rag ctd recapitulate the phenotype observed in patients with cid-g/ai, and to study how these mutations affect repertoire composition, cell selection and survival during t and b cell development, we generated three mouse models carrying homozygous rag mutations (f l, r q, and r w), corresponding to human mutations (f l, r q, r w) previously reported in patients with late-onset combined immune deficiency with granuloma and/ or autoimmunity (cid-g/ai). methods: mice were generated using crispr/cas mediated gene editing. t and b cell development, including apoptosis was studied by flow cytometry. immunoglobulins in naïve mice, baff levels and specific antibody responses were measured by elisa. serum igm autoantibodies were measured using a microarray (utsw). analysis of t cell receptor (trb) repertoire in several sorted t cell populations and immunoglobulin heavy chain (igh) repertoire in pre-b cells was performed by adaptive biotechnologies. in order to be able to detect both dj and vdj rearrangements, pro-b cells and spleen b cells were sequenced using high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (htgts-rep-seq). analysis of vk-jk rearrangements in pre-b cells was performed by pcr amplification. results: immunological characterization showed partial development of t and b lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with cid-g/ai. by using high throughput sequencing, we identified marked skewing of igh v and trb v gene usage in early progenitors, with a bias for productive rearrangements after selection occurred, and increased apoptosis of b cell progenitors. this suggested that more alleles remained in germline configuration. moreover, in the rearranged igh loci, the distal v gene segments were preferentially rearranged already at the earliest stages of b cell development, a finding that has not been previously reported. in addition, rearrangement at the igh locus was impaired, and polyreactive igm antibodies were detected. conclusions: in conclusion, this study demonstrates that hypomorphic rag mutations reported in cid-g/ai cause abnormalities of the primary b and t cell repertoire. these changes may affect survival and selection of t and b cells, and thereby contribute to the immune dysregulation often seen in patients with cid-g/ai. senior clinician, nih/niaid/lcim introduction/background: autosomal dominant hyper ige syndrome (ad-hies) is a primary immunodeficiency due to loss of function stat mutations. disease manifestations include recurrent skin and pulmonary infections, eczema, mucocutaneous candidiasis, as well as tion and in vitro studies demonstrated that the enhanced signaling could be controlled by ruxolitinib, an approved jak / inhibitor. informed by these experimental data, the patients were treated with ruxolitinib with remarkable improvement in a variety of clinical end-points, including hematological profiles and growth parameters. conclusions: this characterization of a human jak gain-of-function mutation expands our current understanding of the role of jak in eosinophil biology, hematopoiesis and immune function. chronic granulomatous disease, ornithine transcarbamylase deficiency and x-inactivation is a primary immune deficiency characterized by defects in the nadph oxidase enzyme complex resulting in a susceptibility to a narrow spectrum of bacteria and fungi. mutations in cybb encoding gp phox and located at xp . are associated with the most common form of cgd. deletions and rearrangements in this region are associated with other genetic diseases such as mcleod syndrome (xk), retinitis pigmentosa (rpgr), duchenes muscular dystrophy (dmd), ornithine transcarbamylase deficiency (otc), and x-linked mental retardation (tspan ). patient phenotype depends on the extent and position of the deletion, creating a "contiguous x-chromosome gene deletion syndrome. objectives: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. methods: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. the patient is the second born of a set of non-identical triplets from a clomiphene assisted pregnancy at weeks gestation. (weight at birth: lbs oz). after a day stay in the nicu, and cpap for one day she was discharged home. by the second month, she began having problems gaining weight and had persistent vomiting. at months, she was admitted with a pneumonia diagnosed as methicillin resistant staphylococcus aureus by lung biopsy. during that hospitalization, the mother noted an enlarging lesion on the infants left hand which was biopsy proven serratia marscecens osteomyelitis and was treated with intravenous cefepime for weeks. at this point a dihydrorhodamine assay (dhr) showed only . % positive cells (nl - %) and she was diagnosed as a carrier of x-linked cgd. bactrim was initiated for antibacterial prophylaxis but it was discontinued due to recurrent diarrhea; she was unable to tolerate antifungal prophylaxis as well due to liver function abnormalities. she continued having frequent vomiting episodes, irritability, failure to thrive and development delay. ulcerations in esophagus were confirmed by egd and colonoscopy, probably related to persistent emesis. diarrhea was unresolved. at months of life she was admitted with acute encephalitis, a serum ammonia level of and elevated urine orotic acid. results: given the demonstrated carrier status for cybb and apparent otc deficiency, comparative genomic hybridization was performed, revealing a deletion from xp . to xp . this . mb loss includes genes that are known to cause disease. since the diagnosis, the patient has been on reduced protein diet, resolving her diarrhea, she has subsequently grown and is on th percentile for weight and height. her dhr is now % positive. at years she was diagnosed with cone rod dystrophy. currently she is on bactrim for cgd prophylaxis and l-citrulline for the otc deficiency. she continues to gain weight, and has shown great improvement in her development delay and no new cgd-related infections have recurred. conclusions: this case reminds us that x-linked carriers with large deletions may be symptomatic and genetic analysis to determine other affected genes can be important for medical management. introduction/background: wiskott-aldrich syndrome (was) is a rare and severe x-linked disorder with variable clinical phenotypes correlating with the type of mutations in the was gene. the long-term prognosis of this syndrome is generally poor, with hematopoietic stem cell transplantation (hsct) remaining the only curative choice. the syndrome is poorly characterized in china. objectives: we retrospectively reviewed patients with was referred to our hospital from to , and summarize their clinical manifestations and genetic features. methods: sixty-four children suspected to be was from unrelated families were enrolled in this study. the clinical data of children were reviewed in the present study. distribution of lymphocyte subsets from peripheral blood and was protein (wasp) expression in peripheral blood mononuclear cells was examined by ow cytometry (fcm). wasp mutations were identified by direct sequencing of pcramplified genomic dna results: among patients with primary immunodeficiency diseases (pid), ( . %) were finally diagnosed as was with gene identified. the mean time of diagnosis was . months (range, . - . ). the common onset clinical manifestation was diarrhea, and most patients had recurrent upper respiratory tract infection, otitis media, pneumonia, and skin abscess. one patient had nephrotic syndrome and no patient with malignancy. all patients had classical was phenotype with was clinical scores - . total mutations in wasp were identified, including novel mutations. six patients received hsct, five survived, with one died because of gvhd. compared with the other patients without wasp mutations, was patients had lower numbers of cd + t cells and b cells, and higher eos and ige level. there was a negative association between the number of b cells and the was clinical scores. conclusions: in china, diagnosis of was has improved over the last decade, although a much higher number of cases had been expected. establishing more diagnostic centers dedicated to the care of pid will facilitate early, correct diagnosis and better care of was in china. regulatory cells (tregs) are precisely quantified by measuring demethylation of the treg-specific-region of foxp . more recently, we have identified highly cell type-specific dna regions of demethylation for further cell populations. objectives: this novel technology allows the implementation of differential immune phenotyping into the newborn screening procedure. here, we aimed at epigenetically quantify multiple immune cell types in different biological samples including dried blood spots and samples from patients with pid with immundysregulation, where currently no approach is available. methods: using cell type-specific demethylation sites, we developed epigenetic qpcrs for quantification of t-, treg, b-, nk-, monocyte and granulocyte cell population. epigenetic qpcr is applicable for relative and absolute quantification in whole blood and dried blood spots using isolated, bisulfite converted dna. results: we demonstrated > % concordance with flow cytometric analyses of the same fresh blood samples from healthy subjects. we have validated the method in children with symptoms of immundysregulation resulting from monogenic defects, including for example foxp , cd , stat , ctla , and leading to treg/teffector cell imbalance where treg deficiency has been difficult to assess by flow cytometry. furthermore, we tested dried blood spot (guthrie card) samples from healthy newborns and patients with diverse pids and correctly identified / pid patients, indicating that this method holds promise for newborn screening. conclusions: the method we established for immune cell quantification based on epigenetic cell type-specific markers, is feasible and reliable in biological samples either fresh, frozen or archived, including dried blood spot. the analysis of further immune cell types introduces an innovative opportunity to diagnose a variety of pids and immunodysregulatory disorders as early as newborn screening. pancytopenia and immunodeficiency with mds in an infant due to samd l mutation we present our data on behalf of the pcid study consortium of the inborn errors working party worth profound combined immunodeficiencies (p-cid) are inherited diseases with impaired t-cell function leading to infections, immune dysregulation or malignancies. genetic, immunologic and clinical heterogeneity make patient specific decisions on indication and timing of hematopoietic stem cell transplantation (hsct) difficult. objectives: since the pcid study recruits non-transplanted p-cid patients aged - years to prospectively compare natural histories of age and severity-matched patients with or without subsequent transplantation and to determine whether immunological and/or clinical parameters may be predictive for outcome methods: our prospective/retrospective international observational multicenter study recruits pediatric p-cid patients to identify biomarkers and clinical parameters that are predictive of outcome. results: so far > growth hormone deficiency comprised the majority of neuroendocrine cases ( %, / ). other notable neurologic diagnoses included headache ( %, / ), seizure ( %, / ), cerebrovascular accident ( %, / ), and neurologic tumors ( . %, / ). in addition to somatic neurologic conditions, many cvid patients ( . %, / ) had reported diagnoses of depression, anxiety, and post-traumatic stress disorder. conclusions: our findings suggest that neurologic diagnoses are more common in cvid patients than previously recognized. patients with neurologic autoimmune disease appear to have a more severe phenotype with earlier age at symptom onset. many cvid patients had depression, anxiety clinical outcomes of human herpesvirus reactivation after hematopoietic stem cell transplantation prognostic factors and outcome of epsteinbarr virus dnaemia in high-risk recipients of allogeneic stem cell transplantation treated with preemptive rituximab cytomegalovirus in hematopoietic stem cell transplant recipients daratumumab controls life-threatening post-hsct autoimmune haemolytic anaemia objectives: we present a month old pancytopenic male who was diagnosed with myelodysplastic syndrome (mds) with monosomy due to samd l mutation. methods: whole exome sequencing was performed on a male, who presented at months of age with findings concerning for a bone marrow failure (bmf) syndrome despite a normal bmf genetic panel. results: the patient presented at months of age, with severe pancytopenia, fevers, e. coli bacteremia, pancolitis, and echtyma gangrenosum. bone marrow showed severe aplasia with occasional macrophages.he was treated with antibiotics, as well as steroids, etoposide and cyclosporine for presumed hemophagocytic lymphohistiocytosis (hlh). a bone marrow failure and hlh genetic panels were normal conclusions: this is one of the first reported cases of samd l mutations causing mds since its initial discovery earlier this year. samd l mutation should be considered in patients who present with pancytopenia and monosomy mds. as new defects continue to be identified, further evaluation outside of typical bmf panels may be relevant primary immune deficiency disease in patients over age : an analysis from a proprietary immunology patient registry roger ucla school of medicine director objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd- codes d conclusions: our data suggest that pidd in patients over age may be more prevalent than previously reported introduction/background: the patient is a -month-old boy, born at + weeks gestation to non-consanguineous parents of italian origin. he was admitted to the intensive care unit at days of age with profuse bloody diarrhoea, weight loss, severe metabolic acidosis and acute renal failure. he had a rapid respiratory deterioration necessitating intubation, ventilation and inotropic support. the patient developed features of macrophage activation syndrome with: (i) prolonged fever > . °c, (ii) hepatosplenomegaly, (iii) bicytopenia (anaemia and thrombocytopaenia), (iv) hypertriglyceridemia, (v) high ferritin ( , ) and (vi) haemophagocytosis on bone marrow smear. he also presented with three interesting features (i) a macular erythematous rash that slowly resolved and was replaced by reticulo-livedoid rash, (ii) a marked hypereosinophilia and (iii) no significant elevation of hladr/cd + t cells on lymphocyte immunophenotyping ( - %). the patient underwent rectal biopsy which confirmed the presence of eosinophils, but without significant inflammation or architectural changes. stool microscopy showed presence of partially necrotic intestinal epithelial cells. immune work up demonstrated global t lymphopaenia without balanced subpopulation and eliminated a familial hemophagocytic lymphohistiocytosis (normal perforin and cd a expression on cd + t-cell and nk cells). circulating foxp + cd +cd lowcd + t cells were within normal range. a dihydrorhodamine reduction assay was normal. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: hematopoietic stem cell transplantation (hsct) has been used for the treatment of hematologic malignancies and primary immunodeficiencies (pid), for several decades with increasing efficacy. however, toxicity related to conditioning regimens based on the use of chemotherapy and/or irradiation to ensure engraftment of donor cells, remains a significant problem. recently, an alternative, potentially low-toxicity, approach has been proposed, which makes use of an immunotoxin targeting cd -expressing cells, which include hsc and more mature leukocytes. this approach is particularly attractive for leaky forms of severe combined immune deficiency (scid), with residual production of dysfunctional t and/or b cells, such as atypical forms of rag deficiency. objectives: we have developed a mouse model carrying a hypomorphic mutation in the rag gene (p.f l) resulting in a combined immunodeficiency with signs of autoimmunity, recapitulating the phenotype seen in patients. methods: using this model, we have tested the efficacy of conditioning with an anti-cd immunotoxin (cd -sap) alone or in combination with low irradiation ( rads; cd -sap/ ), and compared these regimens to a myeloablative dose of irradiation ( rads) [niaid protocol lcim e]. following conditioning, the mice were transplanted with wild-type (wt) bone marrow (bm) lineage-negative cells and followed over time to evaluate immune reconstitution. results: conditioning with cd -sap alone or with cd -sap/ led to a consistent engraftment of donor t and b cells in the peripheral blood (pb) of f l that increased overtime, reaching % donor chimerism at weeks. myeloid (cd b+) and nk cells in pb of f l mice also showed high level of donor engraftment that remained stable at around % in cd -sap around hours of life and the second after one week. there is no data on the utility of one versus two screens for scid screening. here we present our data evaluating whether patients with scid or t-cell lymphopenia were identified on the first or second trec screen. objectives . determine the benefit of a second trec screen in identifying scid and t-cell lymphopenia at birth. . examine outcomes of trec screening in washington state since implementation. methods: results of scid newborn screening performed in washington state between january and june were reviewed retrospectively with the staff of the washington department of health laboratory where the trec assay is performed. trec thresholds (copies/μl) were defined as follows: absent ( ), low ( - ), borderline ( - ) and normal (> ). all trec assays were run with a beta-actin control to assure sample adequacy. a screen is considered abnormal if there is one low/ absent trec or two borderline trec. newborns with abnormal trec screening have follow-up diagnostic testing consisting of lymphocyte flow cytometry to evaluate numbers of naïve and mature t cells, b cells, and nk cells, performed at seattle childrens hospital. results: a total of positive trec screens were found in washington state between january and june . five patients who did not have diagnostic flow cytometry testing were excluded from the analysis (one protocol deviation, one lost to follow-up and three who died before testing could be performed). the first screen was abnormal in forty three patients, while the second screen was abnormal in patients. five patients had an abnormal third or fourth trec drawn for other newborn screen follow-up. three patients with scid were identified, all with abnormal values on first screen. fortyfive patients with t-cell lymphopenia were identified; from the first screen and from the second screen. there was one patient with mhc ii deficiency was missed by both first and second screens because she did not have t-cell lymphopenia. the false positive rate with the first screen was % versus % with subsequent screens. the false positive rate dropped to % with two abnormal trec. the positive predictive value of scid or t-cell lymphopenia with the first abnormal trec was % versus % with two abnormal trec. average age of collection among infants with a positive screen was . hours for the st nbs and . days for the nd nbs. live viral vaccines were postponed in three patients who had an abnormal secondary screen (one with idiopathic t-cell lymphopenia, one with ectrodactyly-ectodermal dysplasia-clefting (eec) syndrome and one with q . deletion). one of these was started on pjp prophylaxis. conclusions: the practice of obtaining a second nbs from all newborns in washington state has led to increased identification of patients with tcell lymphopenia but did not result in identification of additional patients with scid. the false positive rate of the first and subsequent newborn screens was similar and decreased in patients with two abnormal trec. interventions including delaying live viral vaccines and pjp prophylaxis were instituted in patients who had a normal initial trec but abnormal secondary screen and documented t cell lymphopenia. two trec screens in all newborns can result in identification of additional patients with t-cell lymphopenia who may require intervention and additional follow-up. it is not yet clear whether the cost of a second mandatory scid newborn screen is balanced by the additional sensitivity gained by this approach. introduction/background: mannose-binding lectin (mbl) is a multimeric lectin that recognizes a wide array of pathogens independently of specific antibody, initiates the lectin pathway of the complement system, and acts as a proinflammatory mediator. mbl deficiency is reported to increase the frequency of infections in patients with impaired immune systems or cystic fibrosis (cf) patients. mbl replacement is experimental and unavailable. immunoglobulin replacement therapy (igrt) is controversial in the treatment of mbl deficiency; however, there are no reports of its efficacy or role in this condition. we describe a cf carrier patient with mbl deficiency, ciliary dyskinesia and mildly low igg who did not respond to igrt. objectives . understand when mbl deficiency can be symptomatic. . define the relationship between mbl deficiency and cf. . describe the treatment options of mbl deficiency. . define the efficacy of igrt and mbl deficiency. methods: a single case report. results: year old girl with mbl deficiency, cf carrier state with polymorphisms ( - a>g cf variant with t/ t and m v polymorphism) and ciliary dyskinesia (diagnosed by ciliary biopsy) who initially presented at eight years of age with recurrent sinusitis, recurrent otitis media status post tympanostomy tube placement, reactive airway disease and tracheomalacia. she had nine episodes of recurrent sinusitis with six negative sinus cultures and one positive for pseudomonas aeruginosa. she required a total of nine courses of antibiotics. labs showed several mbl levels < ng/ml on three occasions, normal ch , ah , igg - mg/dl (low normal for age), normal iga, igm, t cells, b cells, nk cells, and robust specific antibody titers. despite adequate pulmonary hygiene including nebulized levalbuterol, budesonide, dornase alfa, ipratropium, hypertonic saline and compression vest twice daily, she continued to have recurrent bronchitis and cough. in addition, even with sinus rinses and intranasal corticosteroid, she continued to have - episodes of sinusitis yearly. for this reason, she underwent bilateral total ethmoidectomies and maxillary antrostomies with modest reduction in frequency of sinus infections and symptoms. however after two years, her bacterial sinusitis recurred. four episodes were positive for methicillin staphylococcus aureus or pseudomonas aeruginosa. this did not improve significantly with a trial of intranasal mupirocin. due to increased frequency of bacterial sinusitis refractory to traditional therapy, she was started on subcutaneous igrt dosed approximately mg/kg/month dosing maintaining igg troughs around mg/dl. after a six month trial, she did not have improvement in the frequency of sinusitis, bronchitis and otitis media. she required - courses of antibiotics for bacterial upper respiratory tract infections and igrt was stopped. prophylactic antibiotics and repeat sinus surgery were instituted. conclusions: majority of the patients with low/deficient mbl levels do not manifest significant symptomology due to the redundancy of the innate immunity. the increased susceptibility to infections is thought to be due to additional factors that compromise other components of the immune system. specifically in cf patients, mbl deficiency is associated with earlier colonization with pseudomonas, more rapid decline in lung function and earlier death secondary to end-stage lung disease. there are no reports of combined mbl and cf carrier symptomatic patients similar to this patient. there are no validated age-corrected values for mbl levels in pediatric patients. the clinical relevance of these levels to infection frequency, severity, or treatment of mbl deficiency remains to be proven. it has been proposed that < ng/ml is considered deficient in children. mbl therapy is still experimental and not commercially available. management when provided for severe or frequent infections includes prompt treatment with antibiotics, prophylactic antibiotics, appropriate vaccinations, and a trial of igrt. there are no reported cases describing the efficacy of igrt in mbl deficiency, and the mechanism subsequent genetic analysis identified the presence of a de-novo heterozygous mutation in the nucleotide binding domain of nlrc (c. g>c, p.val leu). a mutation involving this amino-acid position has already been described but with a different substitution pattern in a boy and his father who presented with mas (p.val ala) ( ) . in order to prove the causality of this mutation, our team generated thp- cell lines expressing the two different mutations through gene-editing with the crispr system. in this system the mutation p.val leu, as well as the p.val ala mutation, were responsible for spontaneous activation of caspase , as evidenced by flica assay. the patient was treated with iv methylprednisone mg/kg/day. he continued to have progression of his inflammatory state, and was therefore commenced on anakinra. following confirmation of mutation, he was started on rapamycin, reasoning that (i) through autophagy induction, rapamycin could potentiate the action of anakinra ( , ) and (ii) through mtor inhibition counteract the effect of il- on t-cells ( ). with a combinatory therapy of anakinra up to mg/kg/day and ramapycin (with trough levels of - ng/l), the patient showed a marked clinical improvement, allowing weaning of steroids and establishment of enteral feeds. ferritin levels reduced to - ng/ml. we observed a significant decrease in il- plasmatic level following treatment initiation (pre-vs post-treatment levels of pg/ml and pg/ml, respectively). we report a novel nlrc gain-of-function mutation, presenting with neonatal enterocolitis and autoinflammation with improvement under combinatory therapy of anakinra and rapamycin. to our knowledge this is the first case to report the use of rapamycin in this disease, with what appears to be encouraging results. further studies are required to elucidate the potential role of rapamycin in the management other inflammasome disorders. introduction/background: allogeneic hematopoietic stem cell transplantation (hct) is currently standard treatment for patients with severe combined immunodeficiency (scid), with -year overall survival > % for typical scid (heimall blood ). previous studies revealed that poor clinical outcomes correlated with poor long-term t cell reconstitution, including low cd t cell counts and low naïve cd + t cell counts (pai nejm ) . we hypothesized that t cells developing in a poorly reconstituted immunologic environment would show features of chronically activated t cells with increased expression of inhibitory co-receptors. we further hypothesized that the intensity of the conditioning regimen would correlate with the expression of inhibitory co-receptors. objectives: to characterize the t cell phenotype of scid patients at > years post-hct and to investigate the impact of conditioning regimen on the quality of t cell reconstitution and the expression of inhibitory coreceptors methods: we analyzed scid patients - yrs (median yrs) after hct. we excluded from the analysis patients with chronic graft-versushost disease (gvhd), chronic dna viral infections or patients who had received donor lymphocyte infusion or boost in the months prior to study. scid genotypes (n) included il rg/jak ( ), rag /rag / dclre c ( ), ada ( ), il r ( ) and other/unidentified ( ). nine patients had received a reduced intensity (ric) or myeloablative (mac) conditioning regimen, while had received either no conditioning or immunosuppression only (none/is). poor t cell reconstitution was defined as cd t cell counts below cells/mm ( patients). t cell phenotype, including expression of inhibitory co-receptors, was assessed by flow cytometry. results: compared to patients with cd counts above cells/mm , patients with low cd counts had low naïve cd ra+ccr + t cells (p= . ) and high cd ra-ccr -t effector memory (tem) cells (p= . ), low numbers of naïve thymic cd ra+ cd + t cells, low numbers of trecs and a less diverse t cell repertoire (p< . ). additionally, they had an increased frequency of cd t cells expressing pd ( % vs %), ctla ( % vs . %), cd ( % vs %, p= . ), and b ( % vs %, p= . ) inhibitory co-receptors. increased inhibitory receptor expression was associated with a differentiation profile skewed toward a tem phenotype, reduced t cell diversity, increased markers of t cell activation, and the development of a highly exhausted cd high pd- high t cell population. more importantly, a fraction of ccr + cd ra+ cd t cells expressed pd ( % vs %, p= . ) and b ( % vs %, p= . ) in patients with low cd counts, suggesting that some naïve t cells of poorly reconstituted patients were chronically activated. inhibitory receptor expression did not increase with hla disparities between the donor and recipient, a history of gvhd after transplant or the infection status of the patient prior to transplant. however, inhibitory co-receptor expression was correlated with conditioning regimen, with increased frequency of b + cd t cells in unconditioned patients ( % vs % in none/is and ric/mac patients respectively, p= . ). conversely, ric/mac conditioning was associated with higher naïve cd t cell numbers (p= . ), higher naïve thymic cd ra+ cd + t cell numbers (p= . ), a more diverse t cell repertoire (p= . ) and low expression of inhibitory co-receptors. ric/mac conditioning was also associated with improved naïve t cell generation and limited expression of inhibitory receptors in il rg/jak patients ( % vs % for b , % vs % for cd in ric/mac versus none/is il rg/ jak patients respectively), a genotype permissive to t cell engraftment. conclusions: collectively, our results suggest that the expression of inhibitory co-receptors may be a biomarker of poor t cell reconstitution in transplanted scid patients. further, we propose that lack of conditioning limits t cell reconstitution, which correlates with increased expression of inhibitory receptors on circulating cd t cells. antibodies targeting inhibitory receptors are now available in clinical trials to treat cancer and viral infections. it will be necessary to evaluate the relationship between inhibitory receptor expression, t cell function and clinical outcome, to see if a selected group of scid patients could benefit from these immunotherapies. wallace chair, chief of allergy immunology, children's hospital of philadelphia introduction/background: prophylactic antibiotics (abx) and immunoglobulin replacement (igrt) are commonly used to treat specific antibody deficiency (sad), but the optimal therapy is not established. objectives: to compared outcomes (number of infections and hospitalizations) in sad treated with igrt vs. prophylactic antibiotics. methods: two-center, retrospective chart review of sad patients from jan -may . we excluded patients with hypogammaglobinemia and/or other immunodeficiency diagnosis. characteristics and treatment were reported, rates of infections/hospitalizations among treatment groups were compared using linear regression model. results: sad patients included. mean age was years, % were females. ( . %) received prophylactic antibiotics, ( . %) received igrt, ( . %) did not receive any specific treatment. number of infections decreased from . (year before treatment) to . (year after treatment) in prophylactic antibiotics group (p= . ), and from . to . in igrt group (p< . ). various musculoskeletal and vascular abnormalities. little is known of gynecologic-obstetric complications, but with improved therapies women are living longer making reproductive health more pertinent. objectives: to learn more about obstetric and gynecological health in women with stat loss of function. methods: we prospectively interviewed and retrospectively reviewed medical records of adult women with ad-hies evaluated at the nih between - . results: of patients aged - years (mean years), women were interviewed, and chart reviews were performed on . age of menarche in our cohort was consistent with the national average ( . vs. . years). five of patients ( . %) reported having worsening lung symptoms with menstruation, and of participants ( %) reported worsening eczema during menstruation. with regard to routine health maintenance, of women reported having regular cervical cytology testing; ( %) reported an abnormal result. of these , had hpv that responded to treatment or was hpv only not requiring treatment; had reactive changes due to yeast and ascus that was subsequently normal. mastitis and breast abscesses occurred in women. eleven women reported vulvar cysts/abscesses requiring drainage. nine women had progestin-releasing iuds placed, in some to suppress menses-associated vulvar eczema/abscess flares; no infectious complications were reported from progestin iud use. over % of women chose not to conceive given underlying disease. of women with pregnancies, women had live births, of ( %) had miscarriages, and of ( %) experienced recurrent pregnancy loss. three women whose pulmonary symptoms worsened during pregnancy were diagnosed with progression of parenchymal lung disease post-partum. one woman experienced worsening of skin manifestations. other reported postpartum complications included one wound infection (after caesarean) and one hemorrhage leading to hysterectomy. conclusions: as women with ad-hies are living longer, significant infectious and disease-related exacerbations related to both menstruation and pregnancy were observed in this patient population. it is important to focus on maintenance of their gynecologic and obstetric health and carefully monitor for these morbidities. finally, while these women may choose to attempt pregnancy, the risk of recurrent pregnancy loss and worsening disease warrants discussion. rna sequencing identifies aichi virus as the cause of chronic infection with lymphoproliferation in a patient with x-linked agammaglobulinemia objectives: we here present an xla patient with a complicated course in whom we detected aichi virus (aiv ). methods: case report: the patient was diagnosed with xla at age years, based on agammaglobulinemia with absent b cells and a known pathogenic mutation in btk (c c>t, p.r c). he had been suffering from recurrent respiratory and gastrointestinal infections since the age of months. he was started on immunoglobulin (ig) substitution, which resulted in complete control of infections with igg trough levels at g/l. however, at years of age he developed unexplained fever, refractory temporal epilepsy, hepatitis, progressive nephromegaly with chronic renal failure, splenomegaly, episodic diarrhea and growth failure. ultrasound identified multiple focal lesions in the liver, spleen and kidney. a liver biopsy showed severe chronic hepatitis with initial perisinusoidal fibrosis. serial kidney biopsies showed variable oligoclonal cytotoxic t cell infiltrates, suggestive of chronic viral infection. standard diagnostics failed to reveal a pathogen in blood or stool samples and on biopsies. results: we finally resorted to rna sequencing on a kidney biopsy sample. this technique identified aiv , a kobuvirus of the family picornaviridae, with a high read number. subsequently pcr confirmed the presence of aiv in both liver and spleen. results for cerebrospinal fluid, blood and feces are pending. conclusions: aiv is a picornavirus responsible for self-limiting gastroenteritis in humans. confirmatory pcrs on blood, csf and stool samples are ongoing. however, given the unequivocal result of the rna sequencing and confirmatory pcrs in other affected organs, we believe that the complications in this xla patient can be explained by aiv chronic infection. these findings confirm the potential of next generation sequencing techniques to identify infectious agents in patients with primary immunodeficiency. characterization and successful treatment of a novel autosomal dominant immune dysregulatory syndrome caused by a jak gain-offunction mutation. introduction/background: janus kinase (jak ) plays an essential, nonredundant role in the jak/stat signaling cascade, a key pathway in the control of hematopoiesis and immune function. significant progress has been made in elucidating the role of jak , but gaps in our knowledge still persist. to date, somatic gain-of-function mutations in jak have been linked to t-cell acute lymphoblastic leukemia. objectives: to understand and treat human jak gain-of-function mutations. methods: research study protocols were approved by our institutional review board. four members of the family (the affected children and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: we describe the first known patients carrying a germ-line gainof-function mutation in jak . the clinical phenotype includes severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, autoimmunity, and failure to thrive. functional analysis established the gain of function phenotype caused by the muta- introduction/background: herpesviridae infection after hsct for hematologic malignancies, specifically cytomegalovirus (cmv), epstein-barr virus (ebv) and human herpes virus- (hhv- ), have been associated with various outcomes including post transplant lymphoproliferative disorder (ptld), graft-versus-host disease (gvhd) and mortality ( ) ( ) ( ) . patients with primary immunodeficiency may have different incidence and outcomes with respect to herpesviridae given their differences in age at transplant, conditioning choices and underlying disease susceptibility to this viral family. objectives: the objective of this study was to describe the incidence and outcomes of cmv, ebv and hhv- post hsct for the primary immunodeficiency population. methods: a single center retrospective chart review of primary immunodeficiency registry patients (research ethics board protocol no. ) who received hsct from january december was undertaken. patients who received gene therapy were excluded. antiviral prophylaxis was given according to institutional protocol. demographic and clinical data were collated and analyzed with microsoft excel . the primary outcome was incidence and time to dnaemia for cmv, ebv and hhv- . results: sixty one patients who underwent hsct for primary immunodeficiency from january -december were reviewed. diagnoses are noted in table . the average age of transplant was . months (range . - . ). transplant recipients received busulfan and cyclophosphamide conditioning ( with anti-thymocyte globulin (atg), with alemtuzumab added), transplants received busulfan, fludarabine and either atg or alemtuzumab, and received atg alone. six transplants were unconditioned. . % ( / ) of patients developed gvhd requiring systemic immune suppression. the overall incidence of cmv, ebv and hhv- post hsct for primary immunodeficiency was . % ( / ), . % ( / ) and . % ( / ) respectively. in those with severe or profound combined immune deficiency the incidence of cmv was . % ( / ), ebv . % ( / ), and hhv- . % ( / ). in the patients with chronic granulomatous disease, cmv incidence was % ( / ), and ebv and hhv- were both % ( / ). in recipients with pre-transplant negative pcr for ebv, cmv and hhv- (r-), time to dnaemia post transplant is seen in figure . ptld was seen in patients with rag and il r, both of whom were d+r-for ebv status with ebv dnaemia, and one of whom died attributed to ptld. two year mortality for all patients was % ( / ), with mortality . % ( / ) for those with either cmv, ebv or hhv- dnaemia vs . % ( / ) in those without (p= . ). disseminated cmv prior to transplant was attributed to cause of death for one case. conclusions: cmv incidence was rare, likely from screening for cmv negative hsct donors. cmv, ebv and hhv- dnaemia was not associated with differences in mortality in this cohort. ebv incidence was common, and ptld incidence was similar to previously published outcomes for hsct for other disease ( ) . the advent of cytotoxic t cell therapy for ebv may help abrogate this risk. professor, senior physician, ulm university medical center, pediatrics, germany introduction/background: new-onset aiha occurs in - % of pediatric patients post-hsct. incomplete immune recovery may predispose to immune dysregulation following hsct including autoimmune cytopenias. although prednisolone or other immunosuppressive drugs control most episodes, some patients respond incompletely to first or second line therapies including rituximab. objectives: we describe an innovative therapy for post-bmt aiha refractory to proteasome inhibition. three patients responded to anti-cd antibody (daratumumab) therapy after failing treatment with bortezomib. methods: we retrospectively evaluated data from three patients treated with daratumumab for post-transplant aiha. patients and were treated according to the positive response reported for patient . results: aiha occurred between - months following hsct. daratumumab was curative in patients, the third one had only transient response and relapsed months after this treatment had been initiated. following daratumumab patients no longer required any prbc transfusions. conclusions: in potentially life-threatening aiha in the context of hsct daratumumab may be an effective rescue therapy in combination with rituximab. early post-natal thymus development is strictly dependent on the level of foxn expression in tec these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed groups of nu/+ and +/+ littermates, divided by age: day, - days, weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl , cxcl , dll , and scf, four key foxn target genes. the study was performed in accordance to niaid animal protocol lcim e. results: at day and - days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl , cxcl , dll , and scf at day . reduced expression of ccl persisted at day , but normalized at weeks. conclusions: these data indicate that foxn haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: foxn is the master regulator gene for the development and maturation of the thymic epithelial cells (tecs). by inducing the expression of chemokine receptors such as ccl and cxcl , foxn also allows the migration of early thymic progenitor (etp) cells from the bone marrow. lack of foxn leads to the nude (nu)/severe combined immunodeficiency (scid) phenotype in humans and mice. recently, a number of newborns have been identified with low t cell receptor excision circles (trecs) at birth, associated with heterozygous foxn mutations. these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed groups of nu/+ and +/+ littermates, divided by age: day, - days, weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl , cxcl , dll , and scf, four key foxn target genes. the study was performed in accordance to niaid animal protocol lcim e. results: at day and - days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl , cxcl , dll , and scf at day . reduced expression of ccl persisted at day , but normalized at weeks. conclusions: these data indicate that foxn haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: immunoglobulin g rd is an immunemediated disease most commonly seen in middle-aged and older men. clinical features include autoimmune pancreatitis, salivary gland disease, orbital disease and retroperitoneal fibrosis. pathologic features include a lymphoplasmacytic infiltrate enriched in igg -positive plasma cells and fibrosis in a storiform pattern. laboratory evaluation usually reveals an elevated serum igg concentration, and glucocorticoids are often used as treatment in early stages of disease. however, disease may recur off of steroids, and prolonged illness or steroid non-responsive disease is usually treated with rituximab. objectives: the objective of this case presentation is to discuss a presentation of a igg rd is a young adult male. results: year old male adopted from thailand, initially presented to hospital with five days of intermittent abdominal pain, night sweats and worsening fatigue. ct abdomen and pelvis revealed bronchiectasis in lung bases, hepatic masses and soft tissue infiltration in the porta hepatis extending into the liver, pulmonary nodules (read as possible metastases), bilateral renal masses, retroperitoneal nodes and ileocolic intussusception secondary to possible lymphoma. one year prior to presentation he developed new onset bilateral cervical lymphadenopathy. biopsy revealed a heterogenous lymphoid population and lymphoma was ruled out. he had a repeat lymph node biopsies during the admission, which was all negative for malignant cells, and was compatible with reactive lymphoid tissue with plasmacytosis. immunophenotype showed % lymphocytes with normal cd /cd ratio, and % cd -,cd -, tcr ++ t cells. immunohistochemical stain of a lymph node fine needle biopsy showed a mixture of cd + b and cd + t cells, abundant cd +, igg+ plasma cells(pcs) , with some igg+ pcs(ct of the neck and chest was completed for possible staging and revealed cervical lymphadenopathy, nasal polyposis, a soft tissue mass vs. enlarged lateral rectus muscle (left orbit), and mediastinal lymphadenopathy. a diagnosis of autoimmune lymphoproliferative syndrome (alps) was presumed by the hematologic/oncologic team given his elevated igg ( , mg/dl) and elevated vitamin b (> ) with multiorgan involvement. alps panel revealed a mutation in faslg (allele , c.- c>t) which is a variant of uncertain clinical significance. alps criteria/scorewas + for / criteria (cd +cd +/hla dr ratio < . ). he had been referred to the nih for further management of alps before initial presentation to our office. immunologic work up revealed immunoglobulins of igg: , mg/dl, iga: mg/dl, igm: mg/dl; / protective streptococcal titers (> . mcg/ml); hib: . mg/l; negative quantiferon gold, and hiv-serology. at that point igg rd vs. castleman disease was suspected instead of alps. further work up showed a normal il- level, and human herpes virus was also negative. igg subsets showed an elevated igg ( mg/dl), igg ( mg/dl), igg ( mg/dl) and normal igg ( . mg/dl). excisional lymph node biopsy was recommended to rule out castleman syndrome or igg rd. a left salivary gland was removed and showed mainly igg positive pcs with no multicentric lymphocytic infiltrates. dilution of the patients serum to determine if there was a prozone affect that minimized the igg level showed an elevated igg level of . mg/dl. this is in the st percentile of all cases of igg rd in terms of serum igg concentration. he was diagnosed with igg rd, a form previously called mikulicz disease, which is comprised of lacrimal and parotid gland enlargement. rituximab was given initially because of the severity of his disease. after two cycles he showed decreased lymphadenopathy, notable weight gain and marked decrease in fatigue. conclusions: igg rd is a rare and complex immunologically based disease process rarely seen in children or adolescents. patients with igg rd often undiagnosed at initial evaluation. normal serum igg levels are seen in % of patients with igg rd and thus a normal igg level should not be used as a biomarker to make the diagnosis of igg rd or in treating this disease. furthermore, the possibility of having a prozone affect in measuring igg needs to be considered and evaluated. meticulous correlation of clinical, pathologic, and imaging findings is required to make the diagnosis. modelling human immune deficiency from novel missense mutations with orthologous heterozygous mutations engineered in mice by crispr/cas introduction/background: next generation sequencing has resulted in substantial progress in identification of mendelian immune deficiency syndromes. in some cases, however, putative causal mutations occur in single kindreds, or even individual patients. under these circumstances, functional analysis of patient derived cells combined with in vitro analysis of genetically manipulated cell lines can provide additional evidence in support of genetic causation, but this might not be conclusive. objectives: understanding how genetic defects result in complex syndromes of immune deficiency and immune dysregulation can be impossible to achieve in vitro. one method for overcoming these obstacles is to generate accurate mouse models of human immune deficiency methods: mouse models of human immune deficiency are a valuable tool in which the murine genome is engineered to introduce a mutation orthologous to that discovered in the patient. we have applied this strategy to elucidate causation and mechanism of immunological defect in several mutations affecting the nf-kb pathway. results: so far, defects in both canonical and non-canonical pathways of nf-kb activation have been shown to cause immune deficiency, often associated with immune dysregulation. we describe a known defects and novel putative defect identified in the canonical nf-kb pathway conclusions: crispr-cas mouse models can be used to elucidate mechanism of disease and provide compelling evidence that mutations are causative. introduction/background: primary immune deficiencies (pid) with or without immundysregulation are rare diseases resulting from monogenetic aberrations leading to either infections or autoimmune manifestations or both. early diagnosis and treatment are crucial for reducing morbidity and mortality. beside genetic diagnosis not commonly yet performed, current standard methods for early diagnosis are dependent on either fresh samples or limited to certain cell types. to overcome those limitations, especially in newborn screening, a novel technology of methylation-based qpcr can be applied. among the known epigenetic modifications, dna demethylation is the most stable and genomic loci with highly cell type specific demethylation sites can be identified. differential methylation can be measured from blood samples of limited availability, or with suboptimal storage. we previously showed that thymic-derived t population, and determine whether there are unique diagnostic and treatment considerations within this demographic. whitney goulstone , elizabeth tough executive director, canadian immunodeficiencies patient organization lpn, alberta health services introduction/background: primary immunodeficiency diseases (pids) represent a significant collection of immune system disorders that increase susceptibility to infection, which in some cases are serious or life-threatening. patients with pid often require immunoglobulin g (igg, commonly referred to as ig) replacement therapy to prevent infections and associated comorbidities. pid treatment, in addition to symptoms and associated social and emotional impacts, has a significant impact on patients quality of life (qol). information available on the real-world diagnosis, management, and outcomes of the canadian pid patient population is limited. objectives: to better understand diagnosis and treatment of canadian patients with pid, we surveyed canadian patients with pid associated with the canadian immunodeficiencies patient organization (cipo) the primary goal of the survey was to gain insight into the canadian pid patient population with regards to demographics, diagnosis, treatment, including regimes, qol, and communication and support . methods: the authors conducted a cross-sectional survey to measure health-related qol in a cohort of patients with pid. eligible participants were identified through the canadian immunodeficiencies patient organization (cipo). the questionnaire consisted of questions that covered patient-reported outcomes including diagnosis, qol, treatment regimes, and communication. results: surveys were returned by patients with pid. participants conveyed significant impact on qol including personal, occupational, financial, emotional, and social impacts as a result of pid symptoms, risks, and treatment logistics, limitations, and side effects. the most common diagnoses were related to b-lymphocyte disorders ( . %). common treatments include intravenous immunoglobulin (ivig; in hospital) and subcutaneous immunoglobulin (scig; at home), in addition to antibiotics and antifungals as required. respondents reported feeling average before treatment on a scale of - (mean . ± . ) with increased health after treatment (mean . ± . ). respondents felt current treatment was convenient (mean . ± . ) and were comfortable with self-infusions (mean . ± . ). conclusions: patients with pid are not uncommon in the canadian community, and in these patients pid is associated with a significant impairment in qol. experiences range with regards to a particular treatments advantages and disadvantages, cost, travel, and convenience. respondents hope to achieve improved qol through the following solutions: better treatment, improved infusions, gene modification, more research and clinical trials, a cure, and education and outreach. improved financial, medical, and social supports were also requested. introduction/background: severe combined immunodeficiency (scid), the most severe form of t cell immunodeficiency, is detectable through quantification of t cell receptor excision circles (trecs) in dried blood spots (dbs) obtained at birth. for many professional, humanitarian and financial reasons, newborn screening (nbs) for scid is warranted. implementation of this screening test is highly important where high frequency of consanguinity is known to exist. objectives: since october , israel has conducted national scid nbs. this important, life-saving screening test is available at no cost for every newborn in israel. methods: herein, we describe two years results of the israeli scid newborn screening (nbs) program. validation includes cbc, lymphocyte subsets, trec (in a different method), t cell receptor (tcr) repertoire and response to mitogenic stimulation. whole exome sequence (wes) for genetic detection, and next generation sequencing (ngs) to demonstrate tcr clonality are used as well, in some unsolved cases. results: of , births screened, ( . %) had abnormal trec in their first screen ( terms, pre-terms), ( . %) had a repeated abnormal trec also in their second screen and were referred for a validation process. fourteen scid patients were diagnosed, so far, through the nbs program in its first years, revealing an incidence of : , births in the israeli population. consanguine marriages and muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both il r and dclre c deficiency scid. other diagnoses were made as follows: cases were found to have t cell lymphopenia; cases were diagnosed with syndromes; cases were found to have secondary t cell lymphopenia; cases were pre-term infants and cases were considered as false positive with normal evaluation. lymphocyte subset analysis and trec quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky scid and ruling out false positive results. detection of secondary targets (infants with non-scid lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. we could also report several perspectives regarding t cell development in non immunodeficient newborns that emerged from the accumulated data. conclusions: already in a short term, trec nbs in israel has achieved early diagnosis of scid and other conditions with t-cell lymphopenia, facilitating management and optimizing outcomes. this program has also enabled gaining insights on t cell development in health babies. (hct) . surprisingly, % of these infections were acquired during the interval between confirmation of the scid diagnosis and hct (heimall, blood ). to investigate further, in late we surveyed pre-hct management practices for scid patients at pidtc centers. physicians representing north american centers responded, including immunologists, transplant specialists and who identified as both. % of centers lacked a standard procedure for the management of infants with a positive nbs result. to confirm the scid diagnosis, basic t, b and nk cell flow cytometry was performed by most. testing for naïve t cells was generally included, but testing of lymphocyte mitogen proliferation was inconsistent. specialists were notified of a patients positive nbs test at median age . days ( - days) , and management of patients as scid was started at median age days ( - days). when unconditioned hct was anticipated, % of respondents began planning as soon as possible after diagnosis, while % awaited genetic testing results before hct. respondants consistently implemented pre-hct prophylaxis with trimethoprim/sulfamethoxazole ( %), fluconazole ( %) and immunoglobulin infusions ( %), although timing of initiation varied. palivizumab was used by %. there was little consensus regarding viral monitoring. although most physicians screened for cmv by blood pcr ( %), only about half routinely screened for ebvor adenovirus. while % of physicians started prophylaxis against double-stranded dna viruses in all patients, % did so only in selected situations, such as active genital hsv at the time of delivery, or when the mother was cmv-seropositive. although all centers used only acyclovir as antiviral prophylaxis, doses and timing varied widely: from - mg/kg/day, divided into or doses, continued in most centers until immune reconstitution. finally, % of physicians recommended that cmvseropositive mothers stop breast-feeding. there was no consensus on where patients should reside prior to hct. hospital or home were favored equally, although need for a reliable family was indicated by % as a criterion for home-based management. for hospitalized patients, % of centers required patients to be in a reverseisolation/positive-pressure room and over half required staff to wear gown, gloves, and mask. with regard to visitors, % required parents/ relatives to perform hand hygiene. approximately % did not require a gown, gloves, or mask for visitors. finally, there was no consensus on allowing siblings or friends to visit, but the majority permitted grandparents or other adult relatives. this survey revealed wide variability in the diagnostic pathway, viral surveillance, and isolation practices for scid patients, although pretransplant prophylaxis with immunoglobulin, fluconazole, and trimethoprim/sulfamethoxazole were utilized consistently. we conclude there is considerable opportunity to develop diagnostic and pre-hct management pathways for scid. prospective tracking of management practices could reveal which are important for avoiding pre-hct infections. evidence-based practice guidance is needed to maximize the potential to bring each scid patient identified via nbs to hct infection-free. ikaros/ikzf is an essential transcription factor expressed throughout hematopoiesis. in humans, somatic mutations in ikzf are linked to b-cell acute lymphoblastic leukemia (all), and germline heterozygous haploinsufficient mutations cause common variable immunodeficiency-like disorder with incomplete penetrance. herein, we report seven unrelated patients with an earlyonset novel combined immunodeficiency associated with de-novo, fully-penetrant, germline heterozygous dominant negative mutations affecting amino acid n in ikzf dna binding domain. patients presented with different infections, but pneumocystis jirovecii pneumonia was common to all. one patient developed a t-cell all. additional findings included decreased b-cells, neutrophils, eosinophils and myeloid dendritic cells as well as t-cell and monocyte dysfunction. t-cells exhibited a profound naïve/recent thymic emigrant/ t-helper phenotype and were unable to evolve into effector memory cells; monocytes failed to respond to different stimuli or facilitate t-cell activation. this new defect expands the spectrum of human ikzf -associated immunodeficiency diseases from haploinsufficient to dominant negative. the beta subunit of the il- receptor (il rb; cd ) is essential for il- and il- mediated signal transduction in a variety of hematopoietic cell types including t and nk cells. here we report the clinical and immunologic phenotypes of two siblings born to consanguineous parents harboring a variant in il rb, resulting in autoimmunity, with lymphoproliferation of cd + t and cd hi nk cells, and decreased regulatory t cell frequency. the proband presented with inflammatory enteropathy, failure to thrive, and disseminated cmv infection at months of age, and later developed atopy, lymphocytic interstitial pneumonitis, and red blood cell autoantibodies. his younger sister presented with severe autoimmune hemolytic anemia and cmv viremia at months of age, and later developed lymphocytic interstitial pneumonitis. whole exome sequencing and chromosomal microarray studies revealed a homozygous deletion within the highly conserved wsxws motif of the extracellular domain of il rb (c. _ delcctggagcc, p.pro _ser del). the deletion results in reduced il rb expression (cell surface and intracellular) in t and nk cells. the functional consequences of the defect include complete impairment of stat phosphorylation through the il- receptor but only partial impairment through the il- receptor, as well as a compensatory increase in serum il- and il- levels (> pg/ml). cd + t cell proliferation responses to in vitro t cell receptor (tcr) stimulation were reduced compared to an age-matched, healthy control, but partially rescued by supraphysiologic levels of il- and il- . despite reduced cd + t cell proliferation responses, the proband displayed a t cell population skewed toward cd + t cells, with an oligoclonal expansion of effector memory cells. arguing against effects of pervasive cmv infection alone, the sister displayed similar phenotypic and functional abnormalities at birth, prior to her cmv infection. our data suggest that the identified hypomorphic mutation in il rb results in a survival advantage for those cd + t and cd hi nk cells most sensitive to the exuberant serum il- and il- levels produced in response to the defect. these surviving cd + t and cd hi nk cells have great lymphoproliferative potential, leading to multisystem autoimmunity and inflammatory complications. therefore, we describe il rb deficiency as a novel primary immunodeficiency disease with prominent immune dysregulation and selective cd + t and cd hi nk cell lymphoproliferation. key: cord- -cbkrve h authors: nan title: defending against catastrophic terrorism date: journal: intelligence and security informatics for international security doi: . / - - - _ sha: doc_id: cord_uid: cbkrve h nan health information network messaging system (phin ms) messages to the portal at pre-specified time intervals. the connector at the portal side runs a data receiver daemon listening for incoming messages. after a message is received, the connector checks for data integrity syntactically and invokes the data normalization subroutine. then the connector stores the verified message in the portal's internal data store through its data ingest control module. other data sources (e.g., those from usgs) may have "pull"-type connectors which periodically download information from the source web sites and examine and store data in the portal's internal data store. the wnv-bot portal makes available the spatial temporal visualizer (stv) (buetow et al., ) to facilitate exploration of infectious disease case data and to summarize query results. the stv has three integrated and synchronized views: periodic, timeline, and gis. figure - illustrates how these three views can be used to explore an infectious disease dataset. the top-left panel shows the gis view. the user can select multiple datasets to be shown on the map in a layered manner using the checkboxes. the topright panel corresponds to the timeline view displaying the occurrences of various cases using a gantt chart-like display. the user can also access case details easily using the tree display located left of the timeline display. below the timeline view is the periodic view through which the user can identify periodic temporal patterns (e.g., which months have an unusually high number of cases). the bottom portion of the interface allows the user to specify subsets of data to be displayed and analyzed. our project has supported exploration of, and experimentation with, technological infrastructures needed for the full-fledged implementation of a national infectious disease information infrastructure and has helped foster information sharing and collaboration among related government agencies at state and federal levels. in addition, we have obtained important insights and hands-on experience with various important policy-related challenges faced by developing a national infrastructure. for example, a significant part of our project activity has been centered on developing privacy-sensitive data sharing agreements between project partners from different states. our ongoing technical research is focusing on two aspects of infectious disease informatics: hotspot analysis and efficient alerting and dissemination. for wnv, localized clusters of dead birds typically identify high-risk disease areas. automatic detection of dead bird clusters using hotspot analysis can help predict disease outbreaks and allocate prevention/control resources effectively. initial experimental results indicate that these techniques are promising for disease informatics analysis. we are planning to augment existing predictive models by considering additional environmental factors (e.g., weather information, bird migration patterns) and tailoring data mining techniques for infectious disease datasets that have prominent temporal features. in infectious disease informatics and bioterrorism studies, measurements of interest are often made at various locations and with timestamps. in the public health domain, the disease cases reported to the centers for disease control and prevention (cdc) through its national notifiable diseases surveillance system are collected with timestamps at various places across the entire nation. similar disease case reporting practices exist at state and local jurisdictions, typically with cases identified with specific geo-locations. recent years have seen increasing interest in answering the following central questions of great practical importance arising in spatio-temporal data analysis and related predictive modeling: (a) how can we identify areas having exceptionally high or low measures? (b) how can we determine whether the unusual measures can be attributed to known random variations or are statistically significant? in the latter case, how do we assess the explanatory factors? (c) how can we identify any statistically significant changes (e.g., in rates of health syndromes or crime occurrences) in a timely manner in a geographic area? two types of approaches have been developed in the literature to address some of these questions. the first type of approach is considered the retrospective model. it is aimed at testing statistically whether a disease is randomly distributed over space and time for a predefined geographical region during a predetermined time period. the second type of approach is prospective in nature, with repeated time periodic analyses targeted at identification of statistically significant changes in real time. both approaches have been applied in security informatics practice. for instance, the new york city department of health and mental hygiene collected geo-coded information concerning dead birds infected by west nile virus (wnv). applying retrospective methods to avian fatality data, they were able to detect possible wnv outbreaks before human case data became available (zeng et al., ). our study focuses on retrospective models. although a wide range of methods have been proposed for retrospective spatio-temporal data analysis, the space scan statistic, in particular, has become one of the most popular methods for detection of disease clusters and is being widely used by many public health departments and researchers. algorithmically, the space scan statistic method imposes a circular window on the map under study and moves the center of the circle over the area so that at different positions the window includes different sets of neighboring cases. over the course of data analysis, the method creates a large number of distinct circular windows (other shapes such as rectangle and ellipse have also been used), each with a different set of neighboring areas within it and each a possible candidate for containing a cluster of events. conditioning on the observed total number of cases, the spatial scan statistic is defined as the maximum likelihood ratio over all possible circles. the likelihood ratio for a circle indicates how likely the observed data are given a differential rate of events within and outside the zone. despite its success, there are a number of limitations associated with the scan statistic approach. first, its efficiency depends on the use of simple, fixed symmetrical shapes of regions. as a result, when the real underlying clusters do not conform to such shapes, the identified regions are often not well localized. second, it is difficult to customize and fine-tune the clustering results using the scan statistic approach. users often have different needs as to the level of granularity and number of the resulting clusters and they may have different degrees of tolerance regarding outliers. these limitations have motivated our research aimed at exploring and developing alternative and complementary modeling approaches for spatiotemporal data analysis in the context of security informatics. this case study reports our effort in studying two such approaches: risk-adjusted nearest neighbor hierarchical clustering (rnnh), which was initially developed for crime analysis; and risk-adjusted support vector clustering (rsvc), a new hotspot analysis approach we have recently developed. developed for crime hotspot analysis, rnnh is based on the well-known nearest neighbor hierarchical clustering (nnh) method, combining the hierarchical clustering capabilities with kernel density interpolation techniques. the standard nnh approach identifies clusters of data points that are close together (based on a threshold distance). many such clusters, however, are due to some background or baseline factors (e.g., the population which is not evenly distributed over the entire area of interest). rnnh is primarily motivated to identify clusters of data points relative to the baseline factor. algorithmically, it dynamically adjusts the threshold distance inversely proportional to some density measure of the baseline factor (e.g., the threshold should be shorter in regions where the population is high). such density measures are computed using kernel density based on the distances between the location under study and some or all other data points. rsvc is the result of our recent attempt to combine the risk adjustment idea of rnnh with a modern, robust clustering mechanism such as support vector machines (svm) to improve the quality of hotspot analysis. svms are the most well-known of a class of algorithms that use the idea of kernel substitution. svm-based data description and novelty detection (ddnd) is particularly relevant to our research. svm-based ddnd methods are aimed at identifying the support of a data distribution. they can single out data clusters in complex shapes and have been well-tested in complex, noisy domains (e.g., handwritten symbol recognition). the standard version of svm-based ddnd does not tale into consideration baseline data points and therefore cannot be directly used in spatio-temporal data analysis. as such, we have developed a risk-adjusted variation, called rsvc, based on ideas similar to those in rnnh. in this new approach we first compute the kernel density estimations using the baseline data points and then adjust width parameter in the gaussian kernel hnction based on such density estimations. we have conducted a series of computational studies to evaluate the effectiveness of the three hotspot analysis techniques (satscan, rnnh, rsvc) discussed above. in our experiment, we used artificially generated datasets with known underlying probability distributions to precisely and quantitatively evaluate the efficacy of these techniques. we use the wellknown measures from information retrieval to evaluate the performance of hotspot techniques: precision, recall, and f-measure. in the spatial data analysis context, we define these measures as follows. let a denote the size of the hotspot(s) identified by a given algorithm, b the size of the true hotspot(s), and c the size of the overlapped area between the algorithm-identified hotspot(s) and true hotspot(s). precision is defined as cia. recall is defined as c/b. f-measure is defined as the harmonic mean of precision and recall ( * precision * recall / (precision + recall)). high recall indicates low false negatives and high precision indicates low false positives. notice that achieving a high level of one measure often impacts negatively achieving a high level of the other measure. f-measure represents a balance and trade-off between precision and recall. we report three artificially-generated scenarios with which we have experimented. in the first scenario, as shown in figure - , we first randomly generated baseline points in one circle (whose center is around ( . , . ) on the graph). we then generated case points of interest in total. the first points were generated inside of the baseline circle. (as such, these data points do not represent a new unusual hotspot.) the next points were generated within another circle (whose center is around ( . , . ) ). it is clear that the only true hotspot is this second circle. to make the problem more interesting, we introduced some noise -- outlier baseline points and outlier case points over the entire map. for statistical testing purposes, we repeated the above data generation process times to produce instances of the template scenario by moving the centers of the two circles randomly across the map and also by varying the distances between them so long as they did not overlap. to collect the performance data, we ran all hotspot analysis methods under study on all the problem instances. table - summarizes these methods' average performance across all instances. overall rsvc achieves the best f-score at . %. two additional scenarios have been investigated in our study. in scenario , the true hotspots are the two pieces left of a rectangle when its middle section is removed by a circle. in scenario , the true spot is a square with its circular-shaped center removed. the purpose behind the use of these scenarios is to test how robust these hotspot methods are when dealing with hotspots of irregular shape. tables - and - summarize the average performance of these methods when applied to identify hotspots in randomly-generated instances of scenario and scenario , respectively. some observations are in order. in general, rsvc and the spatial scan method have similar levels of recall across different scenarios. however, rsvc has higher precision than the spatial scan method (confirmed by statistical tests). rnnh has the highest precision level but typically with the lowest recall. when considering the combined f-measure, rsvc consistently delivers the best results. for complex, irregular shapes, rsvc typically performs the best among the three techniques. we believe rsvc could be a strong candidate for hotspot identification in national security applications (e.g., disease surveillance, crime hotspot analysis). over the past ten years we have witnessed the many devastating global impacts of infectious diseases such as sars, foot-and-mouth disease, avian influenza, west nile virus, etc. many of these diseases have strong implications for national security (i.e., as bioagents). disease and bioagent surveillance is one of the most critical research topics of relevance to homeland security. traditionally, disease informatics research is conducted by epidemiologists in universities and local, state, and federal public health agencies. however, there are many opportunities for advanced information sharing, retrieval, and visualization research which could adapt some of the new digital library, search engine, and information visualization techniques. in addition, the discipline can benefit greatly from new temporal and spatial data mining techniques that could complement the prevailing statistical analysis techniques in epidemiology. standards and ontologies are also critically needed in disease informatics research. . how should the data confidentiality issue of animal and human disease information be addressed? how can social network analysis and hotspot analysis techniques be applied to other types of diseases? how can other predictive and environmental modeling techniques be incorporated? key: cord- -a t u authors: nan title: alphabetic listing of diseases and conditions date: - - journal: handbook of autopsy practice doi: . / - - - - _ sha: doc_id: cord_uid: a t u part ii begins with a list of special histologic stains, their for use and their corresponding references. at the end of this list is a procedure for removal of formalin precipitate from tissue sections. diseases. there may also be a list of possible associated conditions. these entities are generally linked pathogenetically to the main disease entry. any asterisk after a related disease indicates that that disorder is also listed as a disease entry. many disease entries will be followed by a three-column table that provides the reader with a listing of the pathologic findings to be expected with the disease as well as the prosection and dissection procedures necessary to demonstrate those findings. it is expected that routine hematoxylin-eosin stains will be done on all sections submitted for histologic examination. special stains will be recommended in the procedures column of the tables, when indicated. any table immediately following the two columns of disease entries always refers to the disease in the right column. prepare smears of undiluted blood. obtain blood for molecular studies for preservation of small intestinal mucosa and for preparation for study under dissecting microscope, see part i, chapter . submit sample for histologic study. submit stool for chemical analysis. record weight and submit sample for histologic study. freeze liver for molecular studies record appearance of spine (see also chest roentgenogram). for removal and specimen preparation, see chapter . request luxol fast blue stain. for removal and specimen preparation, see chapter . below-normal weight in infants. kyphoscoliosis. very low concentrations of cholesterol and decreased triglycerides; serum~-lipoprotein or absent; a.-lipoproteins present. acanthocytosis (spiny red cells). gene mutations ( ) . abnormal shape of villi; vacuolation of epithelial cells. fatty stools fatty changes. gene mutations ( ) systemic manifestations of malabsorption syndrome* and of vitamin a deficiency. * kyphoscoliosis. axonal degeneration of the spinocerebellar tracts; demyelination of the fasciculus cuneatus and gracilis ( ) . possible involvement of posterior columns, pyramidal tracts, and peripheral nerves. atypical retinitis pigmentosa ( ) with involvement of macula. angioid streaks ( ) . synonym: cerebral abscess. note: for microbiologic study of tissues and abscesses, see part i, chapter . include samples for anaerobic culture. it is best to study the brain after fixation but if specimen is examined fresh, aspirate and prepare smears of abscess content. photograph surface and coronal slices of brain. request giemsa stain, gram stain, pas stain, and grocott's methenamine silver stain for fungi. external examination if there is evidence of trauma, see also under "injury, head." prepare roentgenograms of chest and skull. submit for microbiologic study. for removal and specimen preparation, see chapter . for microbiologic study, photography, and special stains, see under "note." for exposure of venous sinuses, see chapter . sample walls of sinuses for histologic study. for exposure of paranasal sinuses, mastoid cells, and middle ears, see chapter . for removal and specimen preparation, see chapter . procedures depend on suspected lesions as listed in right-hand column. skin infections in upper half of face. edema of forehead, eyelids, and base of nose, proptosis, and chemosis indicate cerebral venous sinus thrombosis. * trauma; craniotomy wounds. skull fracture and other traumatic lesions. for possible intrathoracic lesions, see below under "other organs." the national transportation safety board (ntsb)* has authority over aircraft wreckage and the legal authority to investigate and to determine the cause of air crashes. ( ) the dead are the responsibility of the medical examiner or coroner. local police will seal off the area of the crash. other than for the purpose of determining that death has occurred, no one should be allowed to approach the bodies or any objects until the identification teams and the medical examiner or coroner have taken charge. the sudden influx of bodies after a commercial air carrier accident and the request for speedy identification of the victims would overburden almost any institution. managing such a disaster is eased by writing a contingency plan beforehand. temporary morgue facilities may have to be established near the scene of the crash. refrigerated trucks may serve as storage space. a practical approach is to deal first with those bodies that seem to be the easiest to identify, in order to narrow the field for the more difficult cases. if bodies are scattered, their locations can be referenced to stakes in the ground or spray paint on pavement; only then should these bodies (or parts) and personal effects be collected. for large-scale crashes a locations can be referenced to a string-line grid benchmarked to gps coordinates. records and diagrams of the relative positions of victims are prepared during this phase. if bodies are still within the airplane, their positions are recorded, and photographed. the personnel of the medical examiner or coroner can augmented by d-mort team staffed by forensic pathologists, anthropologists, dentists, morgue technicians, and investigators supplied by the national disaster medical system. ** the airline will provide a list of the passengers and the federal bureau of investigation (fbi) disaster team will make itself available to take and identify fingerprints and aid in the acquisition of other identifying data such as age, race, weight, height, and hair color and style. if dental records can be obtained, this provides one of the most certain methods of identification. a medical history indicating amputations, internal prostheses, or other characteristic surgical interventions or the presence of nephrolithiasis, gallstones, and the like will be helpful. fingerprints (and footprints of babies) should be taken in all instances. wallets with identification cards,jewelry, name tags in clothing, or other personal belongings may provide the fastest tentative identification. the medical examiner may elect to autopsy only the flight crew but not the passengers of an aircraft crash. however, the grossly identifiable fatal injuries should be described, photographed, and x-rayed. this may reveal identifying body changes. if comparison of somatic radiographs, dental records, fingerprints, or photographs do not identify the victim, dna comparison must be considered. burned or fragmented bodies of passengers and the bodies ofcrew members, and particularly the pilots, must have a complete autopsy, including roentgenographic and toxicologic examinations, which must always include alcohol and carbon monoxide determinations. internal examination might reveal a coronary occlusion, or roentgenograms may disclose a bullet as evidence that violence preceded the crash. in some airplane crashes, particularly in light airplane accidents, suicide must be considered. in such cases police investigation is required to determine if the pilot exhibited suicidal ideation in the recent past.. when resources permit, autopsies should be performed on all deceased occupants of aircraft crashes, including passengers, in order to distinguish among blunt impact trauma, smoke inhalation, and flash fires as causes ofdeath, and to answer future questions concerning pain and suffering, intoxication, and sequence of survivorship. after a crash victim has been identified, the coroner or medical examiner will issue a death certificate. if remains of a decedent cannot be found, a judge can, upon petition, declare a passenger dead and sign a death certificate prepared by a medical examiner. *phone # ofntsb command center: - - **phone # of dmort: - - . entry should be followed. usually, the circumstances that led to drowning are not apparent from the autopsy findings but can be reconstructed from reports of witnesses and the police. because the reflex drive to seek air is triggered by hypercarbia, not hypoxia, loss of consciousness and drowning can ensue after hyperventilation and breath-holding by experienced swimmers who then drown without a struggle. there are no specific autopsy findings. a search for trauma, including a posterior neck dissection, should be made in all instances. head and cervical injuries may be responsible for loss of consciousness and drowning, usually in individuals diving into shallow water. toxicologic examination as described below for scuba diving accidents is always indicated. with scuba diving fatalities, investigation of the equipment and circumstances is usually more important than the autopsy. scuba fatalities should be studied by or with the aid of diving experts-for instance, members of a diving club or shop (not the one providing the gear used by the decedent) or the u.s. navy. ( ) careful investigation of the scene and study of reports of witnesses and the police are essential. the investigation should ascertain the site of diving (currents and other underwater hazards), the estimated depth, the water temperature (exposure to cold), and a description of water clarity. electrocution should be considered if the site has electric underwater cables (see "injury, electric"). cerebral concussion should be considered if explosives were used in the vicinity. knowledge of the method of recovery of the body and the type of resuscitation efforts can aid in the interpretation of apparent wounds. the medical history of the diving victim should be sought, as it may lead to a diagnosis for which the autopsy is typically silent, such as seizure disorder, or may reveal asthma, emphysema, or chronic bronchitis, all of which increase the risk of air trapping and arterial air embolism. although drowning may be the terminal event in some scuba deaths, the investigation should be focused on the adverse environmental and equipment factors that place a capable swimmer at risk of drowning (see "embolism, air" and "sickness, decompression"). because scuba divers risk arterial air embolism if they ascend with a closed glottis, on can attempt to document gas bubbles at autopsy, but their interpretation is problematic: bodies recovered immediately are subjected to resuscitation efforts, which can by themselves produce extra-alveolar air artifacts, and bodies not recovered immediately tend to be found in a putrefied condition, full of postmortem gas. in the remaining cases, the pathologist must consider the potential of introducing artifactual gas bubbles by the forcible retraction of the chest plate and by sawing the calvarium. the following procedures apply primarily to scuba diving accidents. interrogation of witnesses is important; the behavior and complaints of the decedent, if any, might help distinguish between a natural death by heart disease and an unnatural death by air embolism. external examination eyes and ears head (skull and brain) chest blood (from heart and peripheral vessels) heart tracheobronchial tree and lungs a procedures photograph victim as recovered and after removal of wet suit and other diving gear. record condition of clothing and gear. impound all diving equipment for study by experts, particularly scuba tank, breathing hoses, and regulators. residual air in tank should be analyzed. record color of skin (including face, back, soles, palms, and scalp). palpate skin and record presence or absence of crepitation. record extent and character of wounds. prepare histologic specimens. record appearance of face (including oral and nasal cavities) and of ears. prepare roentgenograms. if air embolism must be expected, as in the presence of pneumomediastinum, follow procedures described under "embolism, air." for evaluation of findings, see also above under "note." if decompression sickness (caisson disease) is suspected, also prepare roentgenograms of the elbows, hips, and knees. otoscopic examination. funduscopic examination. save vitreous for possible toxicologic and other studies. for removal of brain, see chapter . record contents of arteries of the circle of willis and its major branches and basilar artery. strip dura from base of skull and from calvarium. for removal and specimen preparation, see chapter . for demonstration of pneumothorax, see under "pneumothorax". if gas is visible in coronary arteries, photograph. photograph and aspirate gas in heart chambers. submit samples of heart blood and peripheral blood for toxicologic study and drug screen. examine lungs in situ. save bronchial washings for analysis of debris. fresh dissection is recommended. if decompression sickness is suspected, prepare sudan stains from fresh-frozen lung sections. complete toxicologic sampling should be carried out (see chapter ). record nature of gastric contents. remove neck organs toward end of autopsy. for posterior neck dissection, see chapter . incise tongue. for removal, see chapter . for removal, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . consult roentgenograms. in decompression sickness, fatty change of liver, and ischemic infarctions of many organs. interstitial emphysema. aspiration (see above). trauma to cervical spine. mottled pallor of tongue after air embolism. contusion of tongue after convulsive chewing. nitrogen bubbles in spinal cord arteries may occur after rapid ascent. air embolism;' cerebral edema in decompression sickness. aseptic necroses (infarcts, "dysbaric osteonecrosis"), most often in head of femur, distal femur, and proximal tibia. infarcts indicate repeated hyperbaric exposures. nitrogen bubbles in and about joints and in periosteal vessels ("bends") occur during rapid ascent. related terms: automobile accident; motorcycle accident. note: a visit to the scene can make the interpretation of the autopsy findings easier. the vehicle can also be inspected in a more leisurely fashion at the impound lot. this is particularly useful for correlating patterned injuries with objects in the vehicle. most vehicular crashes occur as intersection crashes or because a vehicle with excessive speed left a curved road. the medical examiner or coroner should gain a basic understanding of the crash mechanism so that informed descriptions can be rendered, e.g., "impact to the b pillar of the decedent's automobile by the front of a pickup truck which failed to stop for a stop sign at an intersection, resulting in a -feet intrusion into the cabin; restraint belts not employed; air bag deployed; extrication required which took minutes." police are responsible for determining mechanical and environmental risk factors for the crash and for determining some human risk factors such as suicidal or homicidal intent. the pathologist determines other risk factors for crashes such as heart disease, a history of epilepsy, and intoxication by carbon monoxide, drugs, and alcohol. suicide as a manner of death should be considered when a single-occupant vehicle strikes a bridge abutment or a large tree head-on, with no evidence of evasive action or braking. in such a situation, the standard police traffic investigation should be supplemented of interviews of the victim's family and friends. the ambulance run sheet is an invaluable source of observations that often are not available from the police. this document should be acquired in all instances, even if the paramedics determined that death occurred and did not transport. the basic autopsy procedures are listed below. most traffic victims who die at the scene or who are dead on arrival at the hospital died from neurogenic shock caused by wounds of the head or vertebral column, or from exsanguination from a tom vessel or heart. as such, they have little lividity, and little blood is found in the vehicles. presence ofintense lividity may indicate suffocation or heart disease as a cause of death. if postural asphyxia is suspected, the first responders to the scene should be interviewed to determine the position of the decedent in the vehicle, and the vital signs, ifany, ofthe decedent from the time of the crash to the time of extrication. posterior neck dissection is indicated in these instances. if manifestations of heart disease, intense lividity, and absence oflethal wounds suggest that a crash occurred because the driver was dead, other drivers on the road may have observed that the victim was slumped at the wheel before the crash. the determination of heart attack at the wheel is usually simple, because most such victims realize that something is wrong, and bring the vehicle to a stop at the side of the road, or coast gently into a fixed object. in such instances, damage to the vehicle is minor, and wounds to the decedent are usually trivial. while pattemed wounds can often be matched to objects (see below), patternless wounds usually cannot be visually matched to specific objects, although an opinion can sometimes be given as to what object was struck, based on the direction of motion and position ofthe body with respect to the vehicle. impacts with the a-pillar produce narrow vertical zones of facial laceration and fractures extending from forehead to jaw. tempered glass shatters into small cubes on impact, and leaves so-called "dicing" wounds, which are abraded cuts arranged in a somewhat rectilinear pattern. windshield glass leaves shallow, abraded, vertically oriented cuts on the face or scalp. with pedestrians, the lower extremities are of particular forensic interest, to determine the height and direction of impact from vehicles that left the scene. scalp hair and blood should be collected from such "hit and run" victims and from occupants of a suspect car if police have a question as to which occupant was the driver; these exemplars can be compared to fibers and tissue recovered from the vehicle in question. likewise, foreign material in wounds can sometimes be matched to suspect vehicles, and should be sought and retained as evidence. for pedestrians, the distance between the impact point on the lower extremities and the soles of the feet should be recorded. the legs should be opened to inspect tibial fractures; cortical fractures initiate propagation opposite to the side of impact, where they usually have a pulled-apart appearance, and then splinter the cortex at the side of impact. abrasions are better impact markers than contusions, because subcutaneous blood extravasation can be caused not only by impact to the skin, but also from blood extravasating from underlying fractures. if no cutaneous abrasions or fractures of the leg bones are found, the skin of the legs should be incised to expose contusions. fracture descriptions should include location in the bone (e.g., proximal metaphysis or shaft), whether the fracture is complete or incomplete, and whether the fracture is displaced or distracted. lacerations of intervertebral disks, facet joint capsules, and ligamenta flava should not be loosely termed "fractures." the presence or absence of blood extravasation in soft tissue adjacent to the fractures should be recorded, and its volume estimated if it appears severe enough. venous air embolism from tom dural sinuses cannot be diagnosed without a pre-autopsy chest radiograph or an in situ bubble test. if an x-ray machine is readily available, an anterior-posterior chest radiograph should be obtained in every traffic victim who dies at the scene or after a failed resuscitation attempt. if a hemothorax is suspected, the rib cuts should be placed further lateral and the chest plate reflected so that the internal mammary vessels can be inspected before the chest plate is removed. after measuring and removing the bloody effusion, the underlying serosal surfaces should be inspected for defects. lacerations of the heart and aorta will be obvious. tamponaded lacerations of the aorta, around which the adventitia still holds, must be noted as such. if no lacerations are found at the usual sites, lacerations of the azygous veins must be considered, especially in association with fracture dislocations of the thoracic vertebral column; other sites are the internal mammary arteries, especially with fractures of ribs i and or of the sternum, and intercostal arteries with displaced rib fractures. only after the serosal defect is identified should the organs be removed, because that procedure creates many more holes in the serosa. for that reason, as much information as possible should be gained by in situ observation. the only evidence of concussion of the heart may be a cardiac contusion or a sternal fracture. the usual clinical history suggests cardiovascular instability that is not associated with craniocerebral trauma and which does not respond to the infusion of intravenous volume agents. the autopsy assistant may saw but should not retract the skull cap and remove the brain. the pathologist should observe in situ whether shallow lacerations of the pontomedullary junction with stretching of the midbrain are present. these lesions cannot be distinguished from artifact by examining the brain later. thus, only after appropriate in situ inspection should the pathologist remove the brain. a posterior neck dissection is required if no lethal craniocerebral or cardiovascular trauma is found, or if suffocation is suspected; neck trauma must be ruled out to diagnose suffocation in a traffic fatality. sudden death in a patient with seemingly trivial wounds may be caused by undiagnosed trauma of the craniocervical articulation. a posterior neck dissection is required in these instances. the diagnosis of diffuse axonal injury of the brain in victims with no appreciable survival interval requires that suffocation be ruled out and that no resuscitation from a cardiac arrest has been attempted. clinicians are quick to apply the label "closed head injury" when a victim of a traffic crash has cerebral edema on a computerized axial tomogram of the head, even if no cerebral contusions, scalp contusions, or skull fractures are evident. this may be a misinterpretation, because cerebral edema can be caused by hypoxic encephalopathy made evident after resuscitation from a cardiac arrest, or from hypoxia caused by suffocation. procedures possible or expected findings record presence of lividity. photograph all external wounds; measure all lacerations and any abrasions or contusions with a pattern. collect scalp hair and blood (see below) from victims of hit and run accidents. collect foreign material in wounds. intense lividity and absence of lethal wounds may indicate that the crash occurred because the driver was dead from heart disease or suffocation. wound documentation. patterned injuries often sometimes be matched to objects in or about the vehicle (the most common patterned wound is that from tempered glass; see above under "note"). impact patterns in pedestrians may help to reconstruct the accident. hair and blood of the victim may be matched to transfer evidence on a vehicle suspected of having left the scene. part ii / diseases and conditions internal examination of body cavities heart and great vessels abdomen skull and brain; neck soft tissue compartments at any location prepare roentgenograms of chest is cases with head impact and skull fractures. collect samples for toxicologic study from all victims, including passengers. create pleural window to detect pneumothorax. if blood is seen, examine internal mammary vessels (see under "note"). measure volume of blood in cavity bleeds, and note whether chambers of heart and great vessels are collapsed or filled. record evidence of cardiac contusion, sprain of intracardiac inferior vena cava, laceration of pericardial sac, and fracture of sternum. laceration of heart or great vessels (measure volume of blood). follow routine procedures for dissection of heart and great vessels (see chapter ) . in situ bubble test to confirm venous air embolism. record evidence of trauma and volume of blood in peritoneal cavity; estimated volume of blood in retroperitoneal soft tissues. autopsy assistant may saw the skull but pathologist should inspect brain in situ and remove it personally. for removal and specimen preparation of brain, see chapter . record brain weight. posterior neck dissection is indicated if there is no craniocerebral or cardio-vascular trauma, or if suffocation is suspected. record evidence of trauma and estimate volume of blood. venous air embolism.' evidence of alcohol or drug intoxication. pneumothorax, hemothorax, e.g., after laceration of internal mammary vessels. evidence of significant hemorrhage. indirect evidence of cardiac concussion. evidence of exsanguinating wounds. evidence of cardiovascular disease that may have felled the driver before the crash. in european countries, the concentration is expressed in promille (grams per liter). in the united states, it has become customary to refer to concentration by percentage (grams per deciliter), and values in these units have been written into legislation and included in the uniform vehicle codes. unless qualified, the use of promille or percentage does not indicate whether the result of the analysis is weight/weight, weight/ volume, orvolume/volume. another common way ofexpressing concentration, milligrams per deciliter, has also been used to indicate alcohol concentrations. the method ofexpressing concentration must be clearly specified whenever the alcohol level is mentioned. the desired expression canbe derived from the toxicologic report by using the following equation: i, ~g/ml = mg/dl = . g/dl = . mmolll = . promille = . % what is the legal interpretation of alcohol (ethanol) intoxication? objective impairment of driving ability is observed at threshold blood alcohol concentrations of . -. g/dl. as of august all states and the district of columbia have adopted laws that make it criminal offense for a driver to operate a motor vehicle with a blood alcohol concentration of . g/ dl or greater. many states have an enhanced penalty for high concentrations such as . g/dl or above. several states have zero tolerance laws, under which drivers who are minors are legally operating only if their blood alcohol concentration is . g/dl or less, and in some states, not detectable at all. blood alcohol concentrations obtained at autopsy are valid until putrefaction begins. specimen tubes with sodium fluoride should be used, and the specimen should be stored in the refrigerator. if the air space above the blood samples in the container is large, alcohol can evaporate and a falsely low blood alcohol level can result. putrefactive changes before autopsy or during storage may cause a falsely high blood alcohol concentration. ethanol can be produced in the specimen container; this is more likely in the absence of a preservative. because fluoride inhibits bacteria far more than fungi, higher fluoride concentrations are required for the inhibition of fungal growth ( ) . although there is no major difference in the alcohol concentrations ofblood samples from the intact heart chambers and the femoral vessels ( ), autopsy samples from pooled blood in the pericardial sac or pleural cavity are unsatisfactory. we therefore recommend that blood be withdrawn from peripheral vessels. is there normal "endogenous" blood alcohol (ethanol) in a living person? blood alcohol concentrations are generally believed to be negligible in the absence of ingested alcohol. "endogenous" ethanol in human blood exists at a concentration of about . g/dl, which is below the limit of detection for most methods ( ) . first in such a list would be postural asphyxia, for example, in drunks who fall asleep face down. also, depressant drugs in the tricyclic, analgesic, barbiturate, and benzodiazepine classes all potentiate the effect of alcohol ( ) . also included in such a list would be infancy and childhood; ischemic heart disease;' chronic bronchitis and emphysema;' other chronic debilitating diseases; poisoning with carbon tetrachloride' or carbon monoxide;' and other causes of hypoxia.' how can one estimate blood alcohol (ethanol) concentrations from vitreous, urine, or tissue alcohol levels and from alcohol in stomach contents? the ratio of serum, plasma, urine, vitreous, and various tissues has been compiled by garriot ( ) . the values may vary considerably. for vitreous, the ratios varied from . - . . these variations may depend on whether blood alcohol concentrations were increasing or decreasing at the time of death. most other body fluids and tissues showed ranges closer to . most urine values were above the blood alcohol concentrations. in another study ( ) , the blood/vitreous (bn) ratio in the early absorption phase was . (range, . - . ; sd . ) and in the late absorption and elimination phase, the bn ratio was . (range, . - . ; sd . ). blood ethanol concentrations probably can be estimated using b = . v for early absorption and b = . v for later phases. a urinelblood ethanol ratio of . or less indicates that the deceased was in the early absorption phase. how can one use alcohol (ethanol) concentrations in postmortem specimens to estimate the blood alcohol concentration at various times before death? with certain limitations, one can base calculations of this kind on the assumption that the blood alcohol level decreases from its peak at a fairly constant rate of . -q. g/dl/h until death ( ) . if blood is not available, conversion factors (see above) must be used. alcoholics have been reported to metabolize at a rate of up to . g/dl/h ( ) . example: the driver of an automobile drinks at a party until midnight. he leaves his host at about : a.m. and is involved in a head-on collision at : a.m. he dies in the emergency room at : a.m. there are multiple injuries and the patient exsanguinates. the autopsy is done at : p.m. although this appears quite unlikely, let us assume that no satisfactory blood sample was obtained before death and that no blood or plasma expanders were given. if under such circumstances the alcohol concentration in the vitreous was found to be . g/dl, what was the alcohol concentration in the blood at the time of the accident? vitreous and blood alcohol concentrations may be assumed to have remained unchanged after death. therefore, the blood alcohol level at the time of death must have been approx . (vitreous humor alcohol) x . (conversion factor, see above) = . g/dl. the time interval between the accident ( : a.m.) and death ( : a.m.) is hand min or / h. if we assume that the decedent was not an alcoholic and that the blood alcohol concentration was decreasing from its peak at a constant rate of . g/dl/h, then the concentration at the time ofthe accident is estimated to have been . (concentration at time of death) + ( / x . ) = . + . = . g/dl or . %. the blood alcohol concentration at the time of the accident could have been lower if the victim stopped drinking later than h or / h before the accident. in the latter case, the peak alcohol level would have occurred after the accident, reflecting the time to absorb the latest drink. the blood alcohol concentration at the time of the accident could have been lower or higher if the time when the patient stopped drinking, the time of the accident, or the time of the death is uncertain. the blood alcohol concentration at the time of the accident could have been higher if the victim was a chronic alcoholic. the elimination rate in such persons may be as high as . mg/dl, which would change the figures in our example above to . + ( / x . ) = . + . = . g/d or . %. only rough estimates are possible. first, the peak blood alcohol level must be determined or calculated, as described in the previous paragraphs. tables (see below) are available that relate blood alcohol level to the minimal amounts of whiskey, wine, or beer that must have been consumed ( ) . however, tables of this type are often based on the minimum amount of alcohol circulating in the body after specific numbers of drinks; such tables do not yield reliable results if used conversely. furthermore, inasmuch as drinking and elimination of alcohol may take place concomitantly, over a longer period the total amount of alcohol consumed may have been much greater than the tables would indicate. it cannot be lower. according to these tables, pints of ordinary beer or fl oz of whiskey would be the minimal amounts needed to produce a blood alcohol level of about mg/dl in a person weighing - pounds. the total body alcohol can be calculated from the blood alcohol level by using widmark's formula: average concentration of alcohol in entire body = . concentration of alcohol in the blood in a person weighing kg, the blood alcohol concentration would be increased mg/dl ( . %) by the absorption of oz of ethanol ( z of -proof whiskey). strength of alcohol is measured in "proof'; absolute alcohol is proof. therefore, in the united states, alcohol content as volume percent is half the proof (for example, -proof whiskey contains % alcohol by volume). the alcohol content of various beverages is shown in the following table. approximate alcohol content in various beverages t toata from glaister, rentoul e. medical jurisprudence and toxicology, th ed. e & s livingstone, edinburgh, with permission. twithin h after consumption of diluted alcohol (approx %) on an empty stomach, assuming body weight of - pounds ( . - . kg) reproduced from ( ) with permission. *one ounce (about ml) of whiskey or z (about ml) of beer. what is the toxicity of alcohol other than ethanol? in general, the toxicity increases as the number of carbon atoms in the alcohol increases. thus, butyl alcohol is two times as toxic as ethyl alcohol: but isopropyl alcohol is only twothirds as toxic as isobutyl alcohol and one-half as toxic as amyl alcohol. primary alcohols are more toxic than the corresponding secondary isomers ( ) . anemia, hemolytic synonyms and related terms: acquired hemolytic anemia; extracorpuscular hemolytic anemia; hereditary hemolytic anemia (hereditary elliptocytosis, pyropoikilocytosis, stomatocytosis. spherocytosis); immunohemolytic anemia; intracor-puscular hemolytic anemia; microangiopathic hemolytic anemia; spur cell anemia. possible associated conditions: disseminated intravascular coagulation;* eclampsia;* glucose- -phosphatase deficiency (g pd); hemolytic uremic syndrome;* malignant hypertension; lymphoma* and other malignancies; paroxysmal nocturnal hemo-globinuria; sickle cell disease;*thalassemia;* thrombotic thrombocytopenic purpura.* (see also below under "note.") note: hemolysis also may be caused by conditions such as poisoning with chemicals or drugs, heat injury, snake bite,* or infections or may develop as a transfusion reaction* or be secondary to adenocarcinoma, heart valve prostheses (see below), liver disease (see below), renal disease, or congenital erythropoietic porphyria. * procedures prepare skeletal roentgenograms. jaundice; skin ulcers over malleoli. in young patients: thickening of frontal and parietal bones with loss of outer table ("hairon-end" appearance); paravertebral masses caused by extramedullary hematopoiesis; deformities of metacarpals, metatarsals, and phalanges. osteonecrosis* of femoral heads. remove and place in fixative as early as possible in order to minimize autolysis (alternatively, formalin can be injected in situ; see below). samples should include oxyntic corpus and fundus mucosa. record weights. submit tissue samples for histologic study. record weight of thyroid gland. for removal and specimen preparation, see chapter . request luxol fast blue stain. for removal and specimen preparation, see chapter . if there is a clinical diagnosis of anemia-related amblyopia, follow procedures described under "amblyopia, nutritional." jaundice. manifestations of malnutrition. * stomatitis with cheilosis and perianal ulcerations due to folic acid deficiency. chronic exfoliative skin disorders. vitiligo. macrocytosis; poikilocytosis; macroovalocytes; hypersegmentation of leukocytes; abnormal platelets. atrophic glossitis with ulcers. pharyngoesophagitis (folic acid deficiency). previous total or subtotal gastrectomy. carcinoma of stomach. autoimmune gastritis (diffuse corporal atrophic gastritis) with intestinal metaplasia. crohn's disease;* sprue;* other chronic inflammatory disorders; jejunal diverticula; intestinal malignancies; fish tapeworm infestation; previous intestinal resection or blind intestinal loop; enteric fistulas. hepatosplenomegaly. alcoholic liver disease. * giant epithelial cells. hyperthyroid goiter; thyroiditis. demyelination of cerebral white matter (in advanced cases). demyelination in posterior and lateral columns of spinal cord, most frequently in thoracic and cervical segments. demyelination of peripheral nerves. retinal hemorrhages; demyelination of optic nerves. hypercellular; megaloblastic. myeloproliferative disorder. brain other organs if mycotic aneurysms are expected and microbiologic studies are intended, follow procedures described below under "aneurysm, mycotic aortic." request verhoeff-van gieson, gram, and grocott's methenamine silver stains. for cerebral arteriography, see chapter . if arteriography cannot be carried out, rinse fresh blood gently from base of brain until aneurysm can be identified. record site of rupture and estimated amount of extravascular blood. for paraffin embedding of aneurysms, careful positioning is required. expected findings depend on type of aneurysm. mycotic aneurysms are often multiple and deep in brain substance. berry aneurysms are the most frequent types and often are multiple. most frequent sites are the bifurcations and trifurcations of the circle of willis. saccular atherosclerotic aneurysms are more common than dissecting aneurysms, which are very rare. with congenital cerebral artery aneurysm: coarctation of aorta;* manifestations of hypertension;* and polycystic renal disease. with mycotic aneurysm: infective endocarditis;* pulmonary suppurative processes; and pyemia. aneurysm, dissecting aortic (see "dissection, aortic.") aneurysm, membranous septum of heart note: for general dissection techniques, see chapter . most aneurysms ofthe membranous septum probably repre-sent spontaneous closure of a membranous ventricular septal defect by the septalleafiet of the tricuspid valve. aneurysm, mycotic aortic note: (i) collect all tissues that appear to be infected. ( ) request aerobic, anaerobic, and fungal cultures. ( ) request gram and grocott methenamine silver stains. ( ) no special precautions are indicated. ( ) no serologic studies are available. ( ) this is not a reportable disease. chest and abdominal organs aorta other organs submit blood samples for bacterial culture. en masse removal of adjacent organs is recommended. photograph all grossly identifiable lesions. aspirate material from aneurysm or para-aortic abscess and submit for culture. prepare sections and smears of wall of aneurysm and of aorta distant from aneurysm. request verhoeffvan gieson and gram stains. septicemia and infective endocarditis. * streptococcus, staphylococcus, spirochetes, and salmonella can be found in mycotic aneurysm. para-aortic abscess. septic emboli with infarction or abscess formation. aneurysm, syphilitic aortic part ii / diseases and conditions heart and aorta other organs en masse removal of organs is recommended. for coronary arteriography, see chapter . request verhoeff-van gieson stain from sections at different levels of aorta, adjacent great vessels, and coronary arteries. see also under "syphilis." aneurysm usually in ascending aorta. may erode adjacent bone (sternum). syphilitic aortitis may cause intimal wrinkling, narrowing of coronary ostia, and shortening of aortic cusps. disruption of medial elastic fibrils. aortic valvulitis and insufficiency;* syphilitic coronary arteritis; syphilitic myocarditis. external examination aorta prepare chest and abdominal roentgenograms. open aorta along line of blood flow, or bisect into anterior and posterior halves. photograph tear(s). measure bloody effusions in body cavities. measure or estimate amount of blood in mediastinum. request verhoeff-van gieson stain. cutaneous impact trauma. mediastinum widened by hemorrhage in case of tarnponaded dissection. a bleed into a body cavity of less-thanexsanguinating volume should point to an alternate mechanism of death such as neurogenic shock or lethal concussion; a posterior neck dissection may be required in such instances. microscopy may show transmural rupture, false aneurysm, or localized dissection. angiitis (see "arteritis, all types or type unspecified.") angina pectoris note: see under "disease, ischemic heart" and chapter . angiokeratoma corporis dittusum (see "disease, fabry's.") angiomatosis, encephalotrigeminal (see "disease, sturge-weber-dimitri.") angiopathy, congophilic cerebral synonyms and related terms: beta amyloid angiopathy due to~-amyloid peptide deposition (~a ) (associated with alzheimer's disease; hereditary cerebral hemorrhage with amyloid angiopathy of dutch type; or sporadic beta amyloid angiopathy); hereditary cerebral amyloid angiopathy, due to deposition of other amyloidogenic proteins such as cystatin c (icelandic type) and others (e.g., transthyretin, gelsolin) ( ). procedures possible or expected findings request stains for amyloid, particularly congo red, and thioflavine s (examine with polarized and ultraviolet light, respectively). request immunostain for~a . some tissue should be kept frozen for biochemical studies. multiple recent cerebral cortical infarctions or small cortical hemorrhages, or both, or massive hemispheric hemorrhages, both recent and old. amyloid deposition in leptomeninges and cortical blood vessels. senile plaques are usually present. in some cases, angiopathy is part of alzheimer's disease. * other organs a prepare material for electron microscopy. electron microscopic study permits definite confirmation of diagnosis. organs and tissues may be minimally affected by amyloidosis. anomaly, coronary artery possible associated conditions: with double outlet right ventricle; persistent truncal artery; tetralogy of fallot;* and transposition of the great arteries.* note: coronary artery between aorta and pulmonary artery, often with flap-valve angulated coronary ostium. coronary artery may communicate with cardiac chamber, coronary sinus, or other cardiac veins, or with mediastinal vessel through pericardial vessel. saccular aneurysm of coronary artery with abnor-mal flow, infective endarteritis of arteriovenous fistula, and myocardial infarction may be present. ifone or both coronary arteries originate from pulmonary trunk, myocardial infarction may be present. heart perform coronary angiography. if infective endarteritis is suspected, submit blood sample for microbiologic study. ectopic origin of coronary arteries or single coronary artery. sudden death. for a detailed description of possible additional findings, see above under "note." anomaly, ebstein's (see "malformation, ebstein's") anorexia nervosa note: sudden death from tachyarrhythmias may occur in advanced cases and thus, autopsy findings may not reveal the immediate cause of death. external examination all organs record height and weight, and prepare photographs to show cachectic features. record abnormalities as listed in righthand column. follow procedures described under "starvation." record weight of endocrine organs and submit samples for histologic study. cachexia, often with preserved breast tissue; hirsutism; dry, scaly, and yellow skin (carotenemia). mild edema may be present. parotid glands may be enlarged. manifestations of starvation.* ovaries tend to be atrophic; other endocrine organs should not show abnormalities. synonyms: cutaneous anthrax; gastrointestinal anthrax; pulmonary (inhalational) anthrax. note: ( ) collect all tissues that appear to be infected. this is a reportable disease. bioterrorism must be considered in current cases. external examination and skin blood photograph cutaneous papules, vesicles, and pustules. prepare smears and histologic sections. submit samples for bacteriologic study. submit sample for serologic study. disseminated anthrax infection may occur without skin lesions. edema of neck and anterior chest in nasopharyngeal anthrax. anthrax septicemia. see above under "note." part ii i diseases and conditions lungs gastrointestinal tracts and mesentery neck organs record character and volume of effusions. after sampling for bacteriologic study (see above under "note") perfuse one or both lungs with formalin. extensive sampling for histologic study is indicated. extensive sampling for histologic study is indicated. photograph meningeal hemorrhage in situ. pleural effusions;* hemorrhagic mediastinitis; anthrax pneumonia (inhalational anthrax; woolsorter's disease). histologic sections reveal hemorrhagic necrosis, often with minimal inflammation and gram-positive, spore-forming, encapsulated bacilli. gastrointestinal anthrax with mucosal edema and ulcerations. hemorrhagic mesenteric lymphadenitis. tongue, nasopharynx, and tonsils may be involved. hemorrhagic meningitis (hemorrhage tends to predominate). external examination distal colon and rectum photograph perineum. measure depth of anal pit, if any. dissect distal colon, rectum, and perirectal pelvic organs in situ (as much as possible). search for opening of fistulous tracts from lumen. use roentgenologic study or dissection, or both, to determine course of tract. absence of normally located anus; anal dimple. abnormal termination of the bowel into the trigone of the urinary bladder, the urethra distal to the verumontanum, the posterior wall of the vagina, the vulva, or the perineum. aortitis note: see also under "arteritis" and "aneurysm, ascending aortic." heart and aorta other organs and tissues remove heart with whole length of aorta and adjacent major arteries. record width and circumference of aorta at different levels. describe and photograph appearance of intima and of orifices of coronary arteries and other aortic branches. submit multiple samples for histologic study and request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. secondary aortic atherosclerosis or intimal fibroplasia. widening of aorta; syphilitic aneurysm. * giant cell aortitis; rheumatoid aortitis; syphilitic aortitis; takayasu's arteritis.* manifestations of rheumatoid arthritis, * syphilis,* systemic sclerosis,* hodgkin's lymphoma, and many other diseases associated with vasculitis. external examination brain spine and spinal cord other organs prepare roentgenogram of spine. for removal and specimen preparation, see chapter . for removal of spinal cord and specimen preparation, see chapter . expose nerve roots. record appearance and photograph spinal cord in situ. submit samples of spinal cord and inflamed tissue for histologic study. request gram, gomori's iron, and grocott's methenamine silver stains. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. signs of previous spinal surgery or lumbar puncture (myelography). evidence of previous trauma or previous myelography. cerebral arachnoiditis. fibrous arachnoidal adhesions and loculated cysts. tuberculosis;* syphilis;* fungal or parasitic infection. systemic infection (see above). ascending urinary infection or other manifestations of paraplegia. arch, aortic, interrupted synonym: severe coarctation. note: the basic anomaly is a discrete imperforate region in the aortic arch, with a patent ductal artery joining the descending thoracic aorta. type a interruption is between the left subclavian and ductal arteries; type b between the left subclavian and left common carotid arteries; and type c (rare) between the left common carotid and brachiocephalic (innominate) arteries. for general dissection techniques, see part i, chapter . possible associated conditions: bicuspid aortic valve (with type a); di george syndrome* with thymic and parathyroid aplasia (with type b); hypoplasia of ascending aorta (with all types); persistent truncal artery (truncus arteriosus); ventricular septal defect. arrhythmia, cardiac note: see also under "death, sudden cardiac." toxicologic studies may be indicated, for instance, if digitalis toxicity (see "poisoning, digitalis") is suspected. if a cardiac pacemaker had been implanted, the instrument should be tested for malfunction. arteriosclerosis (see "atherosclerosis.") arteritis, all types or type unspecified synonyms and related terms: allergic angiitis and granulomatosis (churg-strauss);* allergic vasculitis; anaphylactoid purpura* and its synonyms; angiitis; buerger's disease;* cranial arteritis; giant cell arteritis;* granulomatous arteritis (angiitis); hypersensitivity angiitis; infectious angiitis; necrotizing arteritis; polyarteritis nodosa;* rheumatic arteritis; rheumatoid arteritis, syphilitic arteritis; takayasu's arteritis;* temporal arteritis; thromboangiitis obliterans; and others (see also below under "note"). note: autopsy procedures depend on ( ) the expected type of arteritis, such as giant cell arteritis,* polyarteritis nodosa,* or thromboangiitis obliterans (buerger's disease*); and ( ) the nature of suspected associated or underlying disease, such as aortic arch syndrome,* beh~et's syndrome,* cogan's syndrome, degos' disease,* dermatomyositis,* erythema nodosum and multiforme,* goodpasture's syndrome,* polymyositis, rheumatic fever, * rheumatoid arthritis,* syphilis,* and other nonspecific infectious diseases, systemic lupus erythematosus,* systemic sclerosis (scleroderma),* or takayasu's disease. for histologic study of blood vessels, verhoeff-van gieson stain or a similar stain is recommended. temporal and ophthalmic arteritis. arteritis of ciliary and retinal vessels. clinically, polymyalgia. anemia. arteritis, takayasu's synonyms: aortic arch syndrome; pulseless disease. external examination heart, aorta, and adjacent great vessels kidney eyes and optic nerve brain for in situ aortography, clamp distal descending thoracic aorta and neck vessels as distal as possible from takeoff at aortic arch. remove heart together with aorta and long sleeves of neck vessels. for coronary arteriography, see chapter (method designed to show coronary ostia). test competence of aortic valve. open aortic arch anteriorly and measure (with calipers) lumen at origin of great neck vessels. photograph aorta and neck vessels and submit samples for histologic study. request verhoeffvan gieson stain. submit tissue for histologic examination. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . facial muscular atrophy and pigmentation. narrowing at origin of brachiocephalic arteries. dilated ascending aorta. narrowing of coronary arteries at origins. myocardial infarction. aortic insufficiency. * aortic atherosclerosis. thromboses of brachiocephalic arteries. giant cell arteritis. * diffuse mesangial proliferative glomeulonephritis ( ) . atrophy of optic nerve, retina, and iris; cataracts; retinal pigmentation. ischemic lesions. artery, patent ductal synonym: patent ductus arteriosus. note: the basic anomaly is persistent postnatal patency of the ductal artery, usually as an isolated finding (in % of cases in infants, and in % in adults). it is more common in premature than full-term infants and at high altitudes than at sea level. possible complications in unoperated cases include congestive heart failure, * plexogenic pulmonary hypertension,* ductal artery aneurysm or rupture, fatal pulmonary embolism,* or sudden death. in some conditions, such as aortic atresia* or transposition with an intact ventricular septum,* ductal patency may be necessary for survival. possible associated conditions: atrial or ventricular septal defect;* coarctation ofthe aorta;* conotruncal anomalies; necrotizing enterocolitis in premature infants; postrubella syndrome; and valvular or vascular obstructions. artery, persistent truncal synonym and related terms: type i, pulmonary arteries arise from single pulmonary trunk (in %); type , pulmonary arteries arise separately but close-by (in %); type , pulmonary arteries arise separately but distal from one another (in %). note: the basic anomaly is a common truncal artery, with truncal valve, giving rise to aorta, pulmonary arteries, and coronary arteries, usually with a ventricular septal defect. interventions include complete rastelli-type repair, with closure of ventricular septal defect, and insertion of valved extracardiac conduit between right ventricle and detached pulmonary arteries. possible associated conditions: absent pulmonary artery (in %); atrial septal defect (in %); absent ductal artery (in %); coronary ostial anomalies (in %); di george syndrome;* double aortic arch; extracardiac anomalies (in %); interrupted aortic arch* (in %); right aortic arch (in %); truncal valve insufficiency (uncommon) or stenosis (rare); trun-cal valve with three (in %), four (in %), or two (in %) cusps. heart and great vessels if infective endocarditis is suspected, follow culture procedures for endocardial vegetation described in chapter . request verhoeff-van gieson stain. infective endocarditis,* usually of truncal valve. late postoperative conduit obstruction. postoperative late progressive truncal artery dilation with truncal valve insufficiency. hypertensive pulmonary vascular disease. cerebral abscess,* if right-to-ieft-shunt was present. arthritis, all types or type unspecified note: for extra-articular changes, see under the name of the suspected underlying conditions. infectious diseases that may be associated with arthritis include bacillary dysentery, * brucellosis, * gonorrhea, rubella,* syphilis, * tuberculosis, * typhoid fever, * and varicella. * noninfectious diseases in this category include acromegaly,* beh<;et's syndrome,* felty's syndrome,* gout,* rheumatoid arthritis,* and many others, too numerous to mention. remove synovial fluid and prepare smears. submit synovial fluid for microbiologic and chemical study. for removal of joints, prosthetic repair, and specimen preparation, see chapter . for removal and specimen preparation, see chapter . in the polyarticular variant, facial asymmetry may be noted. rheumatoid factor positive in some cases. pericarditis.* interstitial pneumonitis; pleuritis. (see also under "arthritis, rheumatoid.") lymphadenopathy. splenomegaly. monarthritis or severe, erosive polyarthritis; see also under "arthritis, rheumatoid" and above under "externalexamination and skin." ankylosing spondylitis* may be present. chronic iridocyclitis. see "arthritis, rheumatoid." arthritis, rheumatoid synonyms and related terms: ankylosing spondylitis;* felty's syndrome;* juvenile rheumatoid arthritis* (still's disease); rheumatoid disease; and others. possible associated conditions: amyloidosis;* polymyositis (dermatomyositis*); psoriasis;* sjogren's syndrome;* systemic lupus erythematosus;* systemic vasculitis, and others. subcutaneous rheumatoid nodules on elbows, back, areas overlying ischial and femoral tuberosities, heads of phalangeal and metacarpal bones, and occiput. deformities and subluxation of peripheral joints (see also below under "joints"). subaxial dislocation of cervical spine may be cause of sudden death. pneumothorax;* pleural empyema.* t-cell abnormalities ( ) . bacteremia. positive rheumatoid factor. rheumatoid granulomas in myocardium (septum), pericardium, and at base of aortic and mitral valves; constrictive pericarditis;* aortic stenosis;* coronary arteritis. systemic vasculitis (arteritis*). rheumatoid granulomas in pleura and lung (with pneumoconiosis*); bronchopleural fistula; rheumatoid pneumonia with interstitial pulmonary fibrosis and honeycombing; bronchiectasis;* bronchiolitis with cystic changes; pulmonary arteritis. pneumoconiosis* in caplan arthrogryposis ( ) may be a primary muscle disease, or it may involve abnormalities of the brain, spinal cord, and/or peripheral nerves. etiologies are numerous, as are the modes of inheritance. critical to making the appropriate diagnosis is the collection of muscles from various sites for routine histology, muscle histochemistry, and electron microscopy. portions of peripheral motor nerves must also be prepared for histology and electron microscopy. abdominal cavity intra-abdominal lymphatic system puncture abdominal cavity and submit fluid for microbiologic study. record volume of exudate or transudate and submit sample for determination of fat and cholesterol content. prior to routine dissection, lymphangiography (see below) may be indicated. possible associated conditions: with pulmonary aspergillosis-bronchiectasis; * bronchocentric granulomatosis;* sarcoidosis;* tuberculosis. * with systemic aspergillosisleukemia;* lymphoma;* and other conditions complicated by immunosuppression (l, ) . other organs a carefully make multiple parasagittal sections through the unperfused lungs. culture areas of consolidation. if diagnosis was confirmed, perfuse lungs with formalin. prepare histologic sections from walls of cavities, cavity contents, and pneumonic infiltrates. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. assault note: all procedures described under "homicide" must be followed. asthma note: spray death* may occur in asthma sufferers from pressurized aerosol bronchodilators. record thickness and position. perfuse one lung with formalin. because mucous plugs may block bronchial tree, attach perfusion apparatus to pulmonary artery or to bronchus and pulmonary artery. monitor perfusion to ensure proper inflation. prepare photograph of fixed cut section. submit samples of pulmonary parenchyma and bronchi for histologic study. request azure-eosin and verhoeff-van gieson stains. record weight and thickness of walls. leave attached to stomach. photograph and submit samples for histologic study. eczema. conjunctival hemorrhages and subcutaneous emphysema may be present after fatal attack. pneumothorax;* mediastinal emphysema. low diaphragm (see below). increased igeconcentrations in fatal asthma; postmortem tryptase determination is of doubtful value in this regard ( ) . hypertrophy. low position of diaphragm. hyperinflated lungs. thick-walled bronchi with prominent viscid mucous plugs. typical microscopic inflammatory changes ( ) . asthmatic bronchitis with eosinophilic infiltrates. bronchocentric granulomatosis.* pulmonary atherosclerosis with breakup of elastic fibers. paucity of ecosinophils in mucous ( ) . cor pulmonale. refl ux esophagitis ( ) . peptic ulcer. * pneumatosis of small intestine; emphysema of colon. centrilobular congestion and necrosis. petechial hemorrhages in hypothalamus; necrosis of cerebellar folia; anoxic changes in cortex, globus pallidus, thalamus, sommer's sector of hippocampus, and purkinje cells of cerebellum. suspected changes in anterior hom cells of spinal cord in patients with asthma-associated poliomyelitis-like illness (hopkins syndrome) ( ). allergic polyps and other allergic inflammatory changes ( ) . increased erythropoiesis. atresia, aortic valvular synonym: aortic atresia; aortic atresia with intact ventricular septum; hypoplastic left heart syndrome. note: the basic anomaly is an imperforate aortic valve, with secondary hypoplasia ofleft-sided chambers and ascending aorta. for possible surgical interventions, see two-stage norwood and modified fontan procedures in chapter . possible associated conditions: atrial septal defect* (or patent foramen ovale, usually restrictive); dilatation of myocardial sinusoids thatcommunicate with coronary vessels; dilatation of right atrium, right ventricle, and pulmonary trunk; fibroelastosis ofleft atrial and left ventricular endocardium; hypertrophy of ventricular and atrial walls; hypoplastic left atrium, mitral valve, left ventricle, and ascending aorta; mitral atresia* with minute left ventricle; patent ductal artery (ductus arteriosus); small left ventricle with hypertrophic wall; tubular hypoplasia of aortic arch, with or without discrete coarctation. synonyms and related terms: congenital biliary atresia; extrahepatic biliary atresia; infantile obstructive cholangio-pathy; syndromic (alagille's syndrome) or nonsyndromic paucity of intrahepatic bile ducts ("intrahepatic" biliary atresia). possible associated conditions: alpha]-antitrypsin deficiency;* choledochal cyst;* congenital rubella syndrome;* polysplenia syndrome* ( ); small bowel atresia; trisomy - ; trisomy ; turner's syndrome;* viral infections (cytomegalovirus infection;* rubella*). dissect extrahepatic bile ducts in situ or leave hepatoduodenalligament intact for later fixation and sectioning (see below). record appearance and contents of gallbladder and course of cystic duct. in postoperative cases, submit sample of anastomosed hepatic hilar tissue for demonstration of microscopic bile ducts. remove liver with hepatoduodenalligament. prepare horizontal sections through ligament and submit for histologic identification of ducts or duct remnants. prepare frontal slices of liver and sample for histologic study. request pas stain with diastase digestion. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. jaundice. congenital rubella and other viral infections. alpha]-antitrypsin deficiency;* defects in bile acid synthesis. chromosomal abnormalities. in atresia of the hepatic duct, the gallbladder will be empty. in isolated atresia of the common bile duct, the gallbladder contains bile but it cannot be squeezed into the duodenum. atresia or hypoplasia of bile duct(s); choledochal cyst(s). biliary drainage created by kasai operation. obliterative cholangiopathy ( ) . intrahepatic cholelithiasis; postoperative ascending cholangitis; secondary biliary cirrhosis; giant cell transformation; paucity of intrahepatic bile ducts. pas-positive inclusions in alphal-antitrypsin deficiency.* polysplenia syndrome* ( ) with malrotation, situs inversus, preduodenal portal vein, absent inferior vena cava, anomalous hepatic artery supply, and cardiac defects. for other abnormalities outside the biliary tree, see under "possible associated conditions"). nephromegaly ( ) . atresia, cardiac valves (see "atresia, aortic valvular," "atresia, mitral valvular," "atresia pulmonary valvular, with intact ventricular septum," "atresia, pulmonary valvular, with ventricular septal defect," and "atresia, tricuspid valvular.") atresia, duodenal possible associated conditions: with membranous obstruction of the duodenum-annular pancreas; atresia of esophagus* with tracheoesophageal fistula; congenital heart disease; cystic fibrosis;* down's syndrome;* hirschsprung's disease; imperforate anus* or other congenital obstructions of the intestinal tract ( ); intestinal malrotation; lumbosacral, rib-, and digitllimb anomalies; single umbilical artery; spinal defects; undescended testis ( ). see also under "atresia, small intestinal." the basic anomaly is an imperforate pulmonary valve, with a hypoplastic right ventricle. in unoperated cases, ductal closure is the most common cause of death. for possible surgical interventions, see modified blalock-taussig shunt, mod-ified fontan procedure, and pulmonary valvulotomy in chapter . for general dissection techniques, see chapter . possible associated conditions: dilated myocardial sinusoids that may communicate with epicardial coronary arteries or veins; patent ductal artery (ductus arteriosus); patent oval foramen (foramen orale); tricuspid atresia with minute right ven-tricle; tricuspid stenosis with hypoplastic right ventricle (in %); tricuspid insufficiency with dilated right ventricle (in %). synonym: tetralogy of fallot with pulmonary atresia. note: the basic anomaly is atresia of the pulmonary valve and ofvariable length ofpulmonary artery, and ventricular septal defect (membranous or outlet type), with overriding aorta, and with pulmonary blood supply from ductal or systemic collateral arteries. for possible surgical interventions, see rastelli-type repair and unifocalization of multiple collateral arteries in chapter . possible associated conditions: right ventricular outflow tract a short blind-ended pouch ( %) or absent ( %); atresia of pulmonary artery bifurcation, with nonconfluent pulmonary arteries; right aortic arch ( %); atrial septal defect ( %); persistent left superior vena cava; anomalous pulmonary venous connection; tricuspid stenosis or atresia; complete atrioventricular septal defect; transposed great arteries; double inlet left ventricle; asplenia, polysplenia, or velocardiofacial syndromes; dilated ascending aorta, with aortic insufficiency. related term: jejuno-ileal atresia. possible associated findings: esophageal atresia* with tracheoesophageal fistula; lumbosacral, rib-, or digit/limb anom -alies; undescended testes (l) . note: see also under "atresia, duodena ." fascia lata, blood, or liver these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter ) . intestinal tract for mesenteric angiography, see chapter . leave mesentery attached to small bowel, particularly to the atretic portion. trisomy . multiple atresias; proximal dilatation; volvulus; malrotation; meconium impaction; other evidence of cystic fibrosis. anorectal malformation (l) . annular pancreas ( ). atresia, tricuspid valvular note: the basic anomaly is an absent right atrioventricular connection ( %) or imperforate tricuspid valve ( %), with a hypoplastic right ventricle ( %), muscular ventricular septal defect ( %) that is restrictive ( %), and a patent oval atresia, urethral foramen ( %) or secundum atrial septal defect ( %). for possible surgical interventions, see modified fontan or glenn procedures in chapter . for general dissection techniques, see chapter . possible associated conditions: juxtaposed atrial appendages; large left ventricular valvular orifice; large left ventricular chamber; persistent left superior vena cava; pulmonary atresia; transposition of the great arteries ( %), with aortic co-arctation ( % of those); anomalies of musculoskeletal or digestive systems ( %); down's,* asplenia, or other syndromes. heart aorta and cervical arteries brain if infective endocarditis* is suspected, culture using the method described in chapter . for dissection of carotid and vertebral arteries, see chapter . for removal and specimen preparation, and cerebral anteriography, see chapter . if a foreign body is discovered during a medicolegal autopsy or if the discovery of a foreign body may have medicolegal impli-cations (e.g., presence of a surgical instrument in the abdominal cavity), the rules of the chain of custody apply. for the handling of bullets or bullet fragments, see "injury, firearm." if analysis offoreign material is required, commercial laboratories may be helpful. bolus (see "obstruction, acute airway!') burns note: fatal bums should be reported to the medical examiner's or coroner's office. the questions to be answered by the pathologist depend on whether the incident was accidental, sui-cidal, or homicidal, and whether the victim survivied to be treated in the hospital. a pending death certificate should be issued if the fire and police investigators are not sure of the circumstances at the time of the autopsy. for electrical bums, see under "injury, electrical." for victims who were treated at the hospital, autopsy procedures should be directed toward the discovery or confirmation of the mechanism of death, such as sepsis or pulmonary embolism.* death can be caused primarily by heart disease, with other-wise minor bums and smoke inhalation serving as the trigger that leads to lethal ventricular arrhythmia. because carbon monoxide concentrations are halved approx every min with % oxygen therapy, the pathologist must obtain the first clinical laboratory test results for co-hemoglobin. soot can be detected with the naked eye or d after inhalation of smoke. ambulance records should be examined to determine whether a persistent coma might have been caused by hypoxic encephalopathy following resuscitation from cardiac arrest at the scene. admission blood samples should be acquired to test for cohemoglobin and alcohol. this may not have been done in the emergency room. persons suffering from chronic alcoholism succumb to fire deaths more often than persons who do not drink. a very high initial serum alcohol concentration suggests a risk factor for the fire and presence of chronic alcoholism. patients with chronic alcoholism typically are deprived of alcohol when they are in the bum unit and this can cause sudden, presumably cardiac, death,just as it occurs under similarcircum-stances, not complicated by bums. under these circumstances, the heart fails to show major abnormalities. this mode of dying seems to have no relationship to the presence or absence of liver disease. if the body is found dead and charred at the scene, prepare whole body roentgenograms, before and after removal of remanants of clothing. see also under "identification of the body" and "external examination" in chapter ). one or two fingerpads may yield sufficient ridge detail for identification. if this is not possible, ante-and postmortem somatic and dental radiographs must be compared for identification, or dna comparison must be used. external examination, heart and lungs abdominal cavity and liver see below under "cardiomyopathy, dilated." record volume of ascites. record actual and expected weight of liver. request iron stain. see below under "cardiomyopathy, dilated." alcoholic cirrhosis and alcoholic cardiomyopathy rarely coexist. however, in genetic hemochromatosis,* cirrhosis and heart failure are common findings. cardiomyopathy, dilated (idiopathic, familial, and secondary types) note: for general dissection techniques, see chapter . external examination heart other organs and tissues record actual and expected weights. record ventricular thicknesses and valvular circumferences. evaluate relative atrial and ventricular chamber sizes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. note: huntington's disease maps to the short arm of chromosome . the gene is widely expressed but of unknown function; it contains a cag repeat sequence, which is expanded (range, to ) in patients with huntington's disease. a sensitive diag-nostic test is based on the determination of this cag sequence, which can be done on fresh-frozen tissue or blood ( ) . in the absence of genetic confirmation, sampling of organs and tissues cannot be excessive because a complex differential diagnosis must be resolved. note: disseminated intravascular coagulation (dic) often is a complication of obstetrical mishaps such as abruptio placentae or amniotic fluid embolism,* or it complicates malignancies (such as adenocarcinomas or leukemia*) or bacterial, viral, and other infections. other conditions such as aortic aneurysm* or hemolytic uremic syndrome* are known causes also. ifthe nature of the underlying disease is known, follow the procedures under the appropriate heading also. note: this is a cause of diarrhea. microscopic colitis is associated with older age; collagenous colitis is associated with female sex ( ). the colon is grossly normal but microscopically, increased lymphocytes in the lamina propria and a subepithelial band of collagen is found. if only the lymphocytic infiltrate is found, the term "lymphocytic colitis" or "microscopic colitis" should be applied. a trichrome stain should be ordered in all instances, because the collagen band may be difficult to see without the special stain. i death, anaphylactic synonym: generalized anaphylaxis. note: autopsy should be done as soon as possible after death. neck organs should be removed before embalming. if death is believed to be caused by drug anaphylaxis, inquire about type of drug(s), drug dose, and route of administration (intravenous, intramuscular, and oral or other). this will determine proper sampling procedures-for instance, after penicillin anaphylaxis. allergy to bee stings, wasp stings, fire ants, and certain plants may also be responsible for anaphylaxis. however, envenomation also can be fatal in the absence of anaphylaxis. external examination search for injection sites or sting marks. if such lesions are present, photograph and excise with -cm margin. freeze excised tissue at - °c for possible analysis. prepare chest roentgenogram. foam in front of mouth and nostrils. swelling of involved tissue. antigen-antibody reaction in involved tissues. antibodies against suspected antigen. laryngeal edema may recede soon after death. foamy edema in trachea and bronchi; diffuse or focal pulmonary distention ("acute emphysema") alternating with collapse; pulmonary edema and congestion; accumulation of eosinophilic leukocytes. eosinophilic leukocytes in red pulp. death, anesthesia-associated. note: there are many possible causes of anesthesiaassociated death that are not drug-related, such as acute airway obstruction* by external compression, aspiration, arrhythmia of a heart not previously known to be diseased, tumor, or an inflammatory process. some ofthe complications are characteristically linked to a specific phase of the anesthesia, and many are not revealed by customary morphologic techniques. the task for the pathologist charged with investigating an anesthesia-associated death is to reconstruct the chain of physiologic events culminating in cessation of vital signs. autopsy morphology plays a supporting role; the main investigations center around the record left by the anesthesiologist, testing of anesthesia equipment, and toxicological testing. a consulting anesthesiologist can divine much more information from the anesthesia and recovery room records than can the pathologist, and can suggest avenues of further investigation. therefore, the most important step in these autopsies is to obtain the anesthesiaassociated records and to secure the consulting services of an independent anesthesiologist. the changes in the vital signs during and after anesthesia will help to focus the investigation toward a cardiac mechanism ofdeath or depression ofbrainstem function as a terminal mechanism. when information is gathered about drugs and chemical agents that have been administered or to which the victim may have had access, the pathologist must keep in mind that some non-medical chemicals and many drugs are known to affect anesthesia. drugs and their metabolic products, additives, stabilizers, impurities, and deterioration products (one of which can be carbon monoxide) may be present and can be identified in postmortem tissues. therefore, all appropriate body fluids and solid tissue should be submitted for toxicological examination. if the anesthetic agent was injected into or near the spinal canal, spinal fluid should be withdrawn from above the injected site into a standard toxicologist's collection tube with fluoride preservative. if the anesthetic agent was injected locally, tissue should be excised around the needle puncture marks at a radius of - em. serial postmortem analysis of specimens may permit extrapolation to tissue concentration at the time of death. the time interval between drug administration and death sometimes can be calculated from the distribution and ratio ofadministered drugs and their metabolic products. for a review of anesthetic death investigation, see ref. ( ) . halothane anesthesia and some other anesthetic agents may cause fulminant hepatitis and hepatic failure. the autopsy procedures suggested under "hepatitis, viral" should be followed. note: for special autopsy procedures in postoperative deaths, see chapter . in some instances, procedures described under "death, anesthesia-associated" may be indicated. for a review of investigational procedures and autopsy techniques in operating-room-associated deaths, see ref. ( ) . if the autopsy will involve anatomy or dissection techniques that are unfamiliar, the pathologist should not hesitate to invite the surgeon to the autopsy. in patients who develop a cerebral infarction after open heart surgery, arterial air embolism should be considered as a possible cause. the diagnosis often must be based on excluding other causes because the air has been absorbed prior to death. if a patient dies rapidly, the hospital records may be incomplete or scanty. for example, if a patient bleeds to death despite attempted repair of hepatic lacerations, hospital records may not suffice to reach the correct cause-of-death opinion; personal accounts from the surgeon and anesthesiologist may be needed. autopsy data on patients dying following thoracic surgery may be found in ref ( ) . d death, restaurant (see "obstruction, acute airway.") death, sniffing and spray related terms: glue sniffing; sudden sniffing death syndrome. note: no anatomic abnormalities will be noted at autopsy. sudden death may occur after cardiac dysrhythmia or respiratory arrest. procedures possible or expected findings lungs brain if poison had been inhaled at the time when death occurred, tie main bronchi. submit lungs in glass container for gas analysis. submit samples of small bronchi for histologic study. for removal and specimen preparation, see chapter . submit samples of fresh or frozen brain for toxicologic study. submit samples in glass containers (not plastic) for toxicologic study. trichloroethane, fluorinated refrigerants, and other volatile hydrocarbons are most often involved in the "sudden sniffing death syndrome." spray death may occur in asthma sufferers using pressurized aerosol bronchodilators. freons and related propellants may also be responsible for sudden death. toxic components of glue-such as toluene-accumulate in the brain of glue sniffers. also present in various glues are acetone, aliphatic acetates, cyclohexane, hexane, isopropanol, methylethyl ketone, and methylisobutyl ketone. aerosols may occlude the airway by freezing the larynx. carbon tetrachloride sniffing may cause hepatorenal syndrome (see also under "poisoning, carbon tetrachloride"). death, sudden unexpected, of adult note: medicolegal autopsies are usually indicated, and appropriate procedures should be followed. ifanaphylactic death is suspected, see also under that heading. for all unexpected deaths, the pathologist should learn the circumstances of the death, in order to determine whether the mechanism of death was rapid or slow, and to guide the selection of ancillary tests. whenever paramedics attended a person, the run sheet should be obtained to look for a history of recent drinking or ofchronic alcoholism may be an important clue. the combination of a history ofalcoholism, a negative test for ethanol, and absence ofcardiovascular disease, should suggest alcohol withdrawal as the cause ofa sudden death. the list of"possible or expected findings" below is not complete. for general toxicologic sampling, see chapter . possible associated conditions: atrial septal defect;*bicuspid aortic valve;* coarctation,* hypoplasia, or interruption (type a) of aortic arch; coronary artery from main pulmonary artery; right atrial arch; patent ductal artery;* right pulmonary artery from ascending aorta; subaortic stenosis;* tetralogy of fallot;* ventricular septal defect. * (in approx % of the cases, one or more of these associated conditions are found.) defect, atrial septal note: the basic anomaly is a defect of the atrial septum, usually at the oval fossa (in %). possible complications in unoperated cases include atrial arrhythmias, congestive heart failure; paradoxic embolism; plexogenic pulmonary hypertension « %), and pulmonary artery aneurysm. possible surgical interventions include surgical and transcatheter closure of defect. for deficiency, vitamin c synonyms: hypovitaminosis c; scurvy. external examination and skin other organs bones, joints, and soft tissues record extent and character of skin lesions; prepare sections of skin. describe appearance of gums, and prepare sections. record evidence of bleeding. for removal, prosthetic repair, and specimen preparation of bones and joints, see chapter . hyperkeratotic hair follicles with perifollicular hemorrhages (posterior thighs, anterior forearms, abdomen); petechiae and ecchymoses (inner and posterior thighs); subcutaneous hemorrhages. gingivitis. in rare instances, gastrointestinal or genitourinary hemorrhages. hemorrhages into muscles and joints. subperiosteal hemorrhages occur primarily in distal femora, proximal humeri, tibiae, and costochondral junctions (scorbutic rosary). deficiency, vitamin d synonyms: hypovitaminosis d; rickets. note: features or rickets may be found in familial hypophosphatemia (vitamin d-resistent rickets; fanconi syndrome). vitreous or blood (serum) other organs prepare skeletal roentgenograms. in infants with suspected rickets, record size of anterior fontanelle and shape of head; state of dentition; and shape of costochondral junctions, wrists, long bones, and spine. submit samples for calcium, magnesium, and phosphate determination. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. weigh parathyroid glands and submit samples for histologic study. submit samples of intestine for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter . in infantile rickets, diagnostic sites for histologic sampling are costochondral junctions, distal ends of radius and ulna, and proximal ends of tibia and humerus. for adults, see under "osteomalacia." in infants, rachitic changes at costochondral junctions; in adults, osteoporosis* and osteomalacia*-with or without pseudofractures (milkman's syndrome ( ) . note: the term spinocerebellar degeneration encompasses a variety of lesions whose classification is controversial. a new approach has come from linkage analysis and molecular biology. for instance, friedreich's ataxia, the classic form of hereditary ataxia, is due to an intronic expansion of a gaa tri-nucleotide repeat. other forms are also identified by their specific gene loci. neuropathologic examination still is important and ample sampling is suggested, which should include cerebral cortex, basal ganglia (caudate nucleus, putamen, and globus pallidus), thalamus, subthalamic nucleus, midbrain (red nucleus and substantia nigra), pons (pontine nuclei), spinal cord (at cer-vical, thoracic, and lumbar levels), optic tract, optic nerves with lateral geniculate nucleus, and sensory and motor peripheral nerves. for removal and specimen preparation, see chapter . enlargement of head. poor demarcation between cortex and gelatinous white matter. extensive demyelination and vacuolation of white matter, particularly subcortically. optic atrophy. degeneration, striatonigral (see "atrophy, multiple system.") related term: thirst. note: possible underlying conditions not related to inaccessibility of water include bums, exposure to heat, gastrointestinal diseases, recent paracentesis, renal diseases, and use of diuretic drugs. see also under "disorder, electrolyte(s)." external examination vitreous urine prepare histologic sections of blisters, ulcers, or skin abrasions. submit sample for sodium, chloride, and urea nitrogen determination. skin turgor may be decreased and eyes may be sunken. microscopic changes help to decide whether skin lesions are antemortem or postmortem. sodium concentrations more than meqll, chloride concentrations more than meq/ and urea nitrogen concentrations between and meq/dl indicate dehydration. absence or minimal amount of urine. dementia (see "disease, alzheimer's.") drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." drug abuse, cocaine note: cocaine is spontaneously hydrolyzed by blood esterases, even after death. however, one of its major metabolite, benzoylecgonine, is routinely identifiable by immunoassay screening tests. when cocaine is abused concurrently with heroin or other depressant drugs, it may be difficult to ascribe deth to a single agent, unless circumstances clearly point to a rapid cardiac mechanism or a slow brainstem depression mechanism. note: if narcotic paraphernalia and samples of the drug itself are found at the scene of the death, they should be submitted for analysis. helpful information about the nature of a drug may be obtained from witnesses. state crime laboratories may provide much assistance. if name of drug is known, see also under "poisoning,..." the slang name of a drug may be insufficient for identification because these names often are used for different compounds at different times of places. opoid narcotics can be injected intravenously, or subcutaneously, or snorted. death may occur with such speed that the bodies may be found with needles and syringes in the veins or clenched in the hands. drug abuse may be associated with a multitude of local (see below) or systemic complications, including malaria* and tetanus. * as stated in chapter , for a growing number of analytes, most notably tricyclic antidepressants, peripheral blood is preferred over central blood. peripheral blood is aspirated by percutaneous puncture before autopsy, from the femoral vein or the subclavian vein. the authors prefer the femoral approach in order to avoid any question of artifact in the diagnosis of venous air embolism. it may be pru-dent to add naf to some of the samples. related term: childhood dermatomyositis (or polymyositis) associated with vasculitis; dermatomyositis (or polymyositis) associated with neoplasia or collagen vascular disease; primary idiopathic dermatomyositis; primary idiopathic polymyositis. possible associated conditions: carcinoma (lung, stomach, intestine, and prostate in males; breast, ovary, and uterus in females; miscellaneous sites in both sexes); lymphoma* (rare) and other malignancies ( ); lupus erythematosus;* mixed connective tissue disease; progressive systemic sclerosis;* rheumatoid arthritis;* sjogren's syndrome;* and others. vasculitis of childhood polymyositis (dermatomyositis). external examination and skin heart lungs esophagus and gastrointestinal tract photograph grossly involved skin. prepare sections of involved (anterior chest, knuckles, knees) and grossly uninvolved skin and subcutaneous tissue. prepare roentgenograms. submit samples from myocardium for histologic study. perfuse one lung with formalin. submit samples from all segments for histologic study. arteritis* and phlebitis* with thrombosis, fibrosis, and infarctions. steatohepatitis and manifestations of diabetes mellitus* may be found ( ) . myositis with muscular atrophy and fibrosis; vasculitis in childhood cases. polyneuropathy (rare) ( ). arthritis. diabetes mellitus synonyms: type i (insulin-dependent or juvenile-onset) diabetes mellitus; type ii (insulin-independent or adult onset) diabetes mellitus; secondary diabetes mellitus (e.g., due to drugs or pancreatic disease). note: in infants of diabetic mothers, macrosomia and congenital malformations must be expected. record size and weight of placenta and total weight and length, crown to rump length, and crown to heel length of infant. compare with expected measurements (see part iii). expected histologic finding in-clude hyperpla-sia with relative increase ofb cells of the islands of langerhans with interstitial and peri-insular eosinophilic infiltrates, decid-ual changes of the endometrium, enhanced follicle growth in the ovaries, and leydig cell hyperplasia. possible associated conditions: acanthosis nigricans; acro-megaly;* amyotrophic lateral sclerosis; * ataxia telangiectasia;* fanconi's anemia;* friedreich's ataxia;* gout;* hemochro-matosis; *hyperlipoproteinemia; * hyperthroidism;* obesity;* turner's syndrome;* and many others, too numerous to mention. note: the term "caroli's syndrome" often is used for cases that also show histologic features of congenital he-patic fibrosis or other manifestations of fibropolycystic liver disease,* whereas the name "caroli's disease" refers to idiopathic dilatation of intrahepatic bile ducts, without associated abnormalities. possible associated conditions: choledochal cyst* and related extrahepatic biliary abnormalities ( ); congenital hepatic fibrosis; * cysts of kidneys (renal tubular ectasia or medullary sponge kidney; autosomal-recessive polycystic kidney disease, and rarely, autosomal-dominant polycystic kidney disease [ ] )* and of pancreas. record volume of effusions. prepare smears of fresh blood or of buffy coat, or make thick-drop preparation. submit sample for xenodiagnosis or animal inoculation and for serologic study. record weight. in chronic chagas' disease, perfuse intact heart with formalin (chapter ) and slice fixed heart in a frontal plane so as to create anterior and posterior halves. prepare photographs. histologic samples should include conduction system. include several sections of atrial (auricular) walls for histologic study of autonomous ganglia. perfuse at least one lung with formalin. leave affected hollow viscera intact and fill with formalin. cut fixed organs in half, photograph, and cut histologic sections on edge. record liver weight and submit samples for histologic study. record weight. prepare photographs of abnormalities. weigh and examine. prepare histologic sections. for removal and specimen preparation, see chapter . autopsy is desirable in suspected cases because the diagnosis can only be firmly established after neuropathologic examination. serologic studies are not available. unfortunately, all tissues (not just the brain and spinal cord) may remain infectious even after prolonged fixation and histologic processing. thus, the autopsy recommendations for most other infectious diseases do not apply here. this is a reportable disease in some states. special precautions are indicated and therefore, the procedures described here should be followed strictly ( ) ( ) ( ) ( ) : all persons in the autopsy room must wear disposable long-sleeved gowns, gloves, and masks. contamination of the autopsy table should be prevented by covering it with a disposable, non-permeable plastic sheet. autopsy generally should be restricted to the brain. if organs in the chest or abdomen need to be examined, this is best done in situ. to prevent aerosolization of potentially infectious bone dust, a hood or other protective device should be used while opening the skull with a stryker saw. after completing the autopsy, instruments and other potentially contaminated objects should be autoclaved in a steam autoclave ( h at °c). porous load is considered more effective than gravity displacement autoclaves. immerse autopsy instruments in distilled water before and during autoclaving, in order to protect them from corrosion. ifno autoclave is available, chemical disinfection (see below) is a satisfactory alternative. disposable items should be put in a container for infectious hospital waste and ultimately incinerated. contaminated objects not suitable for autoclaving (such as the stryker saw) should be soaked with a nnaoh solution for h (alternatively, nnaoh may be used for h). contaminated surfaces should be thoroughly washed with the same solution. aluminum should be treated for h with a fresh % naoci (sodium hypochlorite) solution with at least , ppm free chloride. wash waters should be collected; if no autoclave is available, n naoh or > volumes of % sodium hypochlorite bleach should be added to the water and left for a minimum of h before being discarded. before removing the body from the autopsy room, it should be sponged with % sodium hypochlorite. to deactivate cjd infectivity, tissue blocks, mm or less in thickness, should be fixed in formalin in a formalin-totissue ratio of at least : for at least h and then soaked in concentrated formic acid ( - %) for i h, followed by another h of formalin fixation. the fixation fluid should be collected and decontaminated, as described earlier for wash water. glassware and tissue carriers should also be decontaminated as previously described. after this deactivation, the tissue blocks can be processed in a routine fashion. at any stage of these procedures, special care must be taken to avoid cuts with potentially contaminated glassware, blades, or other objects. parenteral exposure to potentially contaminated material also should be avoided. remains of patients who have died of the disease should not be accepted for anatomy teaching for students. if specimens are prepared for pathology collections, they should be handled with great caution. morticians and mortuary workers should be warned of possible hazards posed by tissues of patients with transmissible spongiforme encephalopathies; they should be advised about proper use of disinfectants. clinical laboratories that receive autopsy tissues or fluids must be warned about the infectious nature of the material. if possible, decontamination should be done at the site where the autopsy was done. for the shipping of potentially infected material, see chapter . increased concentrations of nse ( ). spongiforme changes, astrocytosis, neuronal loss, amyloid plaque formation, prp deposition, and proliferation of activated microglia ( ). cerebrospinal fluid brain submit sample for neuron-specific enolase (nse). for removal and specimen preparation, see chapter and above under "note." submit fresh-frozen material for confirmation of diagnosis by histoblot technique on protease k-digested frozen tissue or western blot preparations on brain homogenates. immunohistochemical localization ofprp and hla-dr protein on paraffin-embedded tissue is possible. disease, demyelinating (see "degeneration, spongy, of white matter," "encephalomyelitis, all types or type unspecified," "leukodystrophy, globoid cell," "leukodystrophy, sudanophilic," "sclerosis, multiple;' and "sclerosis, schilder's cerebral.") disease, diffuse alveolar synonym: diffuse pulmonary disease. note: autopsy procedures are listed under the more specific diagnoses, such as "hemosiderosis, idiopathic pulmonary," "lipoproteinosis, pulmonary alveolar," "microlithiasis, pulmonary alveolar," "pneumonia, lipoid," and "syndrome, goodpasture's." glycosphingolipid storage in cornea; lens opacities; dilated vessels in conjunctiva and lens; thrombi in blood vessels ( ). disease, fibropolycystic, of the liver and biliary tract note: "fibropolycystic disease of the liver and biliary tract" comprises a group of well defined conditions, which may occur together and hence need a collective designation. the conditions include autosomal-recessive (infantile) and auto-somal dominant (adult) polycystic disease of the liver; caroli's disease or syndrome;* choledochal cyst,* congenital hepatic fibro-sis,* multiple biliary microhamartomas, and related disorders. for autopsy procedures, see also under more specific designations. disease, glycogen storage synonyms: andersen's disease or brancher deficiency (glycogenosis, type iv); cori's or forbes' disease (glycogenosis, type ill); cyclic amp dependent kinase (type x); glycogen synthetase deficiency (type ); hers' disease (glycogenosis, type vi); mcardle's disease (glycogenosis type v); phosphorylase b kinase deficiency (types ixa, b, and c); pompe's disease (glycogenosis, type it); tarui disease (glycogenosis type vii); von gierke's disease (glycogenosis, type ia); x-linked glycogenosis (type vill). note: if the diagnosis had not been confirmed prior to death, samples of liver, skeletal muscle, blood, and fascia (for fibroblast culture, see below) should be snap-frozen for enzyme assay, which will determine the specific deficiency. types ia and b, iii, vi, and hepatic phosphorylase b kinase deficiency (types ixa, b and c) are hepatic-hypoglycemic disorders, whereas types v and vii affect muscle energy processes. type ii also affects the musculature, whereas type iv may cause cirrhosis and death in infancy from extreme hypotonia. determination of type of glycogenosis usually can be based on (i) pattern of glycogen storage in liver, ( ) presence or absence of nuclear hyperglycogenation in liver, ( ) cytoplasmic lipid in liver, ( ) presence or absence of liver cirrhosis, and ( ) presence or absence of glycogen and basophilic deposits in skeletal muscles. possible associated conditions: fanconi syndrome* or gout* with type ia glycogenosis; neutropenia, recurrent infections, and crohn's disease with types ib or ie. glycogen primarily in retinal ganglion cells and ciliary muscle. glycogen in sympathetic nerve ganglia and neurons of cranial nerves in type vii. gouty arthritis. disease, graft-versus-host note: this disease occurs most commonly after bone marrow transplantation. the disease has also occurred after transfusion of viable lymphocytes, for example, to patients with cancer or leukemia. * in patients with graft-versus-host disease (gvhd), autopsy also may reveal recurrence of the underlying disease such as leukemia. possible associated conditions: alphal-antitrypsin deficiency;* amyloidosis;* ankylosing spondylitis;* primary sclerosing cholangitis;* sjogren's syndrome. * see also below under "possible or expected findings." note: in many instances, either chronic ulcerative colitis or crohn's disease* had been diagnosed clinically, but sometimes, the distinction is difficult to make, even at autopsy. many features described below occur in chronic ulcerative colitis but some manifestations of crohn's disease or conditions that may occur in all types of inflammatory bowel disease also are listed so that both positive and negative findings can be recorded properly. osteoporosis;* ankylosing spondylitis;* arthritis of peripheral joints; periarthritis; hypertrophic osteoarthropathy;* tendinitis (particularly of ankle and achilles tendons). disease, iron storage (see "hemochromatosis.") related terms: atherosclerotic heart disease. note: the most common anatomic finding at autopsy in subjects older than yr is coronary atherosclerosis. unusual under-lying or associated conditions include chronic aortic stenosis or regurgitation; coronary artery anomalies; coronary artery dissection; coronary embolism; coronary ostial stenosis (due to calcification of aortic sinotubular junction or, rarely, to syphilitic aortitis); coronary vasculitis (for instance, in polyarteritis nodosa* or acute hypersensitivity arteritis); hyperthyroidism,* gastrointestinal hemorrhage; * hypothyroidism, * idiopathic arterial calcification of infancy; intramural coronary amyloidosis; pheochromocytoma, polycythemia vera; * pseudoxanthoma elasticum,* radiationinduced coronary stenosis; severe pulmonary hypertension (with right ventricular ischemia); sickle cell disease;* and others. if bypass surgery had been performed, see "surgery, coronary bypass." macular rash ( ). multifocal fibrinopurulent pneumonia with sparing of the bronchi and bronchioles. exudate is rich in phagocytes, fibrin, and karyorrhectic debris. synonym: lyme arthritis note: this infection is caused by the spirochete, borrelia burgdoiferi, which is transmitted from rodents to human by the hard deer ticks, ixodes dammini, . ricinus, and others. brain and spinal cord for removal and specimen preparation, see chapter . request luxol fast blue stain for myelin. symmetric and zonal demyelination in corpus callosum, anterior commissure, optic chiasm, optic tracts, and white matter of frontal lobes. external examination and skin; oral cavity lungs aorta record distribution of skin lesions and submit tissue samples for histologic study. for preparation of angiograms of the pulmonary arterial and venous vasculature, see chapter . if aneurysm or dissection is present, follow procedures described under those headings. telangiectatic (often papular) lesions most commonly found in cheeks, scalp, nasal orifices, oral cavity, ears, neck, shoulders, fingers, toes, and nail beds. cyanosis and clubbing may be prominent. arteriovenous malformations/fistulas. aneurysm; * aortic dissection. * if cirrhosis is present, prepare angiograms of hepatic arteries and veins (chapter ). photograph and prepare sections of angiomatous lesions. note: parkinson's syndrome is caused by conditions that may simulate parkinson's disease; these include carbon monoxide* and manganese poisoning, corticobasal degeneration, druginduced parkinsonism, huntington's disease, multiple system atrophy,* progressive supranuclear palsy* (steele-richardson-olszewski syndrome), space-occupying lesions (rare), trauma (dementia pugilistica), and causes related to tumors and vascular diseases. brain for removal and specimen preparation, see chapter . histologic sections should include midbrain (substantia nigra), upper pons (locus ceruleus), medulla, nucleus basalis (substantia innominata), and basal ganglia. if parkinsonian syndrome was diagnosed, follow procedures described under the name of the suspected underlying condition (see above under "note"). depigmentation of substantia nigra and locus coeruleus; neuronal loss and reactive gliosis; eosinophilic intracytoplasmic inclusion bodies (lewy bodies) in some of the surviving neurons; no significant changes in basal ganglia. disease, pelizaeus-merzbacher synonyms: sudanophilic (orthochromatic) leukodystrophy. brain and spinal cord for removal and specimen preparation, see chapter . request luxol fast blueipas stain for myelin and bielschowsky's stain for axons. prepare frozen sections for sudan stain. brain generally atrophic. myelin loss in centrum ovale, cerebellum, and part of brain stem, with a tigroid pattern of residual myelin near vessels. axons are preserved. diffuse gliosis with relatively few lipoid-containing macrophages, compared to the myelin loss. lipoid material stains with sudan. brain and spinal cord for removal and specimen preparation, see chapter . request silver stains (bielchowsky or bodian stain). histochemical stains in pick's cells and bodies reveal phosphorylated neurofilaments, ubiquitin, and tubulin. some tissue should be kept frozen for biochemical studies. severe cerebral atrophy, involving primarily frontal and anterior temporal lobes (knifeblade atrophy; walnut brain). microscopically, severe neuronal loss accompanied by astrocytosis. characteristic argyrophilic, intracytoplasmic inclusions (pick's bodies), particularly in hippocampus and swollen, distended "ballooned" neurons (pick's cells). these changes are not always present. external examination, skin, and adipose tissue blood cerebrospinal fluid heart liver and kidneys brain, spinal cord, and peripheral nerves eyes submit sample for determinaion of phytanic acid concentration and for molecular studies. for obtaining a sample, see chapter . sample for histologic study. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . ichthyosis. phytanic acid accumulation in adipose tissues. phytanic acidemia, mutation of phyh or pex ( ). increased protein concentrations. cardiomyopathy.* phytanic acid accumulation. axonal neuropathy. retinitis pigmentosa. hypoalphalipoproteinemia. lymphadenopathy with diffuse deposition of cholesterol esters. premature atherosclerotic cardiovascular disease ( ). hepatosplenomegaly with foam cells. enlarged tonsils with characteristic orange discoloration. polyneuropathy ( ) . in adults, corneal infiltrates. foam cells. request pas stain. in granulomas, bacilli are not always pas positive ( ) . section all grossly involved tissues for histologic examination. submit section for electron microscopy. emaciation. hyperpigmentation, particularly of exposed skin and in scars. hyperkeratosis. arthritis involving ankles, knees, shoulders, and wrists. ascites; fibrinous peritonitis. * nodules in peritoneum containing sickle-form particlecontaining cells (spc cells submit sample for determination of sodium, potassium, chloride, glucose, urea nitrogen, and creatinine concentrations. calcium and phosphate concentrations can also be tested. if sample is small, indicate priority for testing. if indicated, submit sample for chemical study. submit tissue samples for histologic study. considerably increased or decreased values for sodium (more than meqll or less than meqll) and chloride (more than meqll or less than meqll) indicate that changes were present before death. for further interpretation, see chapter . postmortem electrolyte concentrations are quite unreliable. may be useful for calcium determination. vacuolar nephropathy (vacuolar changes in proximal convoluted tubules) in potassium deficiency (may also occur after infusion of hypertonic solutions). disorder, hemorrhagic (see "coagulation, disseminated intravascular," ''disease, christmas:' ''disease, von willebrand's," "hemophilia," and "purpura,.••") disorder, inherited, of phagocyte function note: several conditions represent phagocyte function disorders. autopsy procedures for one of these disorders can be found under "disease, chronic granulomatous." consult this entry for other phagocyte function disorders. synonyms and related terms: fabry's disease* (angiokeratoma corporis diffusum); gangliosidosis;* gaucher's disease;* glycogenosis,* type ii; leukodystrophies (krabbe's or globoidcell,* metachromatic leukoencephalopathy*); mucopolysaccharidoses* (hunter, hurler, morquio, and sanfilippo disease); mucolipidosis; niemann pick disease* (type a, b, c, or sphingomyelinase deficiency); neuraminidase deficiency; neuronal ceroid lipofuscinosis (batten's disease or kufs' disease). hypopharyngeal pulsion diverticulum (zenker's diverticulum) at lower margin of inferior constrictor muscle of pharynx. traction diverticulum at midesophagus after an inflammatory process-for instance, tuberculous lymphadenitis. epiphrenic diverticulum may also occur. luxtacardiac or juxtapyloric diverticulum. heterotopic tissue in meckel's diverticulum, with or without peptic ulceration. colonic muscular hypertrophy and stenosis, usually in sigmoid colon. diverticulitis with perforation, fistulas, or peritonitis. * diving (see "accident, diving (skin or scuba).") related terms: dry drowning; fresh-water drowning; near-drowning; salt (sea)-water drowning (see the following table). primary drowning ("immediate drowning") deaths occurring within minutes after immersion, before or without resuscitative measures deaths from hypoxia and acidosis caused by glottal spasm on breath holding. there may be no evidence of water entering stomach or lungs and no appreciable morphologic changes at autopsy. note: the diagnosis is one of exclusion. the pathologist should help the police to determine: i) how did the person (or dead body) get in the water, and ) why could that person not get out of the water? it is not enough to ask if a person could swim but investigators should find out how well (what strokes did the victim know?) and how far he or she could swim. the inquiry must include the depth of the water and must address hazards such as undertow or underwater debris, and the behavior deaths occurring from within min to several weeks after resuscitation, because of metabolic acidosis, pulmonary edema, or infective or chemical pneumonitis deaths from hypoxia and acidosis caused by obstruction of airway by water related to: hypervolemia hemolysis hyponatremia hypochloremia hyperkalemia of the victim immediately before submerging. deaths of adults in bathtubs and swimming pools are usually from natural, cardiac causes, or they are suicides, unless the victim was drunk. diatom tests ( ) have not proven useful in the united states but there is enthusiasm for such tests among european pathologists. the distinction between hyponatremic deaths in fresh water and hypernatremic deaths in salt water derives from experimental studies; in practice, one cannot reliably predict the salinity of the immersion medium from autopsy studies. because many bodies of drowning victims are recovered only after the body floats to the surface, decomposition will often obscure even the nondiagnostic findings such as pleural effusions, which are often associated with drowning. external examination and skin (wounds) organ samples for diatom search serosal surfaces and cavities if identity of drowning victim is not known, record identifying features as described in chapter . prepare dental and whole-body roentgenograms. submit tissue samples for histologic study of wounds. inspect inside of hands. collect fingernail scrapings. record appearance and contents of body orifices. record features indicative of drowning. photograph face from front and in profile. take pictures of all injuries, with and without scale and autopsy number. remove vitreous for analysis. if diatom search is intended, clean body thoroughly before dissection to avoid contamination of organs and body fluids with algae and diatoms (see below). submit sample for toxicologic study. sample early during autopsy, before carrying out other dissections. use fresh instruments for removal of specimens to avoid contamination. submit subpleural portion of lung: subcapsular portions of liver, spleen, and kidneys; bone marrow; and brain. store samples in clean glass jars. for technique of diatom detection, see below. record volume of fluid in pleural spaces. photograph petechial hemorrhages. photograph layerwise neck dissection if strangulation* is suspected. open airways posteriorly, and photograph, remove and save mud, algae, and any other material in tracheobronchial tree. record size and weight of lungs. there may be wounds that were inflicted before drowning occurred-for instance, in shipwrecks or vehicular and diving accidents. other wounds may be inflicted after deathfor instance, from ship propellers or marine animals. sometimes, premortem and postmortem wounds can be distinguished histologically. object (hair?) held by hands in cadaveric spasm. cutis anserina and "washerwoman" changes of hands and feet are of no diagnostic help. foreign bodies; semen (see also under "rape"). foam cap over mouth and nose. in the autopsy room, water running from nose and mouth is usually pulmonary edema or water from the stomach. high concentrations of alcohol indicate intoxication (see under "alcoholism and alcohol intoxication"). evidence of alcohol intoxication may be found. diatoms may occur in the liver and in other organs of persons who have died from causes other than drowning. comparison with diatoms in water sample from area of drowning may be helpful. penny-sized or smaller hemorrhages may indicate violent respiratory efforts or merely intense lividity. presence of pleural fluid suggests drowning. for diatom detection (l) , boil - g oftissue for -- min in rnl of concentrated nitric acid and . rnl of concentrated sulfuric acid. then, add sodium nitrate in small quantities until the black color of the charred organic matter has been dispelled. it may be necessary to warm the acid-digested material with weak sodium hydroxide, but the material must soon be washed free from alkali to avoid dissolving the diatoms. the diatoms should be washed, concentrated, and stored in distilled water. for examination, allow a drop of the concentrate to evaporate on a slide, and then mount it in a resin of high refractive index. all equipment must be well-cleaned, and distilled water must be used for all solutions. there are several variations and adaptations of this method. drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." ductus arteriosus, patent (see "artery, patent ductal.") synonyms and related terms. achondroplastic dwarf; asexual dwarf; ateliotic dwarf; micromelic dwarf; normal dwarf; pituitary dwarf; true dwarf; and many other terms, too numerous to mention. external examination bones and joints record height and weight. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter . growth retardation. abnormal growth of epiphyseal cartilage with enlargement of metaphysis. long bones and pelvis most commonly affected. cavernous hemangiomas (maffucci's syndrome). see above under "external examination." chondrosarcoma. dyscrasia, plasma cell note: these conditions are characterized by abnormally proliferated b-immunocytes that produce a monoclonal immunoglobulin. multiple myeloma, * plasma cell leukemia, plasma-cytoma, and waldenstrom's macroglobulinemia* as well as heavy-chain diseases and monoclonal gammopathies of unknown type belong to this disease family. amyloidosis* is closely related to these conditions. for autopsy procedures, see under "amyloidosis," "macroglobulinemia," or "multiple myeloma" and under name of condition that may have caused the plasma cell dyscrasia. such conditions include carcinoma (colon, breast, or biliary tract), gaucher's disease,* hyperlipoproteinemia, * infectious or noninfectious chronic inflammatory diseases, and previous cardiac surgery. synonym: shigella dysentery. note: (i) collect all tissues that appear to be infected. blood bowel eyes joints submit sample for culture and for serologic study. submit sample of feces or preferably bloodtinged mucus for culture. if bacteriologic diagnosis has already been confirmed, pin colon on corkboard, photograph, and fix in formalin for histologic study. submit sample of vitreous for study of sodium, potassium, chloride, and urea nitrogen concentrations. for removal and specimen preparation of eyes, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . escherichia coli septicemia. colitis with microabscesses; transverse shallow ulcers and hemorrhages, most often in terminal ileum and colon. dehydration* pattern of electrolytes and urea nitrogen. serous arthritis* of knee joints is a late complication. external examination record extent of pigmentation, facial features, and primary and secondary sex characteristics. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter . record size of apertures of cranial nerves in base of skull. unilateral skin pigmentation and precocious puberty in females (albright's syndrome), less commonly in males. synonyms and related terms: becker's muscular dystrophy; congenital muscular dystrophy; duchenne's progressive muscular dystrophy; dystrophinopathy; em-ery-dreifuss mucular dystrophy; facioscapulohumeral dystrophy; limb girdle dystrophy; myotonic muscular dystrophy. external examination record pattern of scalp hair. record status of skeletal musculature. obtain sections for histologic examination. dystrophin staining of the sarcolemma is absent in duchenne's muscular dystrophy and patchy in becker's dystrophy. frontal baldness (in myotonic muscular dystrophy). atrophy and wasting of muscles (generalized or local: predominantly distal in myotonic muscular dystrophy). pseudohypertrophy of calf muscles in duchenne's muscular dystrophy. dystrophic changes include variations in fiber size, fiber degeneration and regeneration, peri-and endomysial fibrosis, and fatty replacement of muscle. the liver, especially the right lobe, is the most common site of involvement. secondary infection or calcification may be present. the lung is the second most common site of involvement. fluid and air may be visible on the roentgenogram. cysts may be present in the abdominal cavity, muscles, kidneys, spleen, bones, heart, and brain. eosinophilia. edema, angioneurotic synonym: angioedema. note: possible causes and suggested autopsy procedures are described under "death, anaphylactic." related term: silo-filler's disease. n<yfe: this condition is causedby inhalationoftoxic gases, such as oxides of nitrogen (silo-filler's disease) and phosgene (coci ). see also "bronchitis, acute chemical" and "poisoning, gas." ( ) gunshot and shotgun wounds of the head involving dural sinuses; ( ) injury to large veins, particularly cranial sinuses (during neurosurgical procedures) and veins ofthe neck (knife wounds, surgery) or uterus (criminal abortion); ( ) insufflation offallopian tubes (particularly in pregnancy or during menstrual period); ( ) infusion of blood components or crystalloid; ( ) malfunctioning of dialysis machines; ( ) subclavian vein catheterization in the semi-fowler position; and ( ) fracture in the hub of a central venous catheter used for parenteral nutrition. possible causes of arterial air embolism include: ( ) open heart surgery involving the aorta, left atrium, or left ventricle; ( ) positive-pressure ventilation in newborn infants; and ( ) underwater ascent with closed glottis (see accident, diving) ifair embolism is suspected, the autopsy should be performed as soon after death as possible. decomposition gases may be produced within a few hours. roentgenography of the whole body may detect large quantities of air, and the roentgenograms may serve as a guide to the most advantageous way of dissection. the postmortem diagnosis of arterial air embolism is made largely on the basis of medical history and circumstances. the autopsy serves to rule out competing conditions. the diagnosis of venous air embolism is made by chest roentgenography before the autopsy ( ). the air in the right chambers of the heart can be confirmed at autopsy by aspiration into a syringe. the formerly recommended procedure of clamping the internal mammary vessels below the sternoclavicular joints, and then cutting across the sternum distal to these clamped vessels so that the sternoclavicular joint area remains intact, is designed to prevent the production of a vacuum that pulls air into the veins. the procedure is not necessary if the air is welldocumented by roentgenography before autopsy. at autopsy, a large fatal pulmonary air embolism is readily apparent. the right atrium and ventricle are distended with fine, frothy, brightred blood, which also may distend the pulmonary arteries and superior vena cava. microbiologic examination ofblood and pericardial sac contents may help to rule out the presence of gas-forming bacteria that may simulate air embolism (fig. ii-i). however, the presence of putrefactive emphysema in any part of the body points toward a bacterial origin of the gas. differentiation of air and decomposition gases at the autopsy table with the pyrogallol test is described below. a % pyrogallol solution is prepared (it should be waterclear). two lo-ml syringes (syringe a and syringe b) are loaded with ml of the pyrogallol solution in each, without permitting any air to enter the system. immediately before the solution is used, drops of . n naoh is aspirated through the needle of syringe a to adjust the ph to about ( drop per ml of solution); the mixture will turn faint yellow. six ml of gas is then aspirated from the heart or blood vessels. the needle is immediately sealed with a cork or replaced by a cap, and the syringe is vigorously shaken for about min. in the presence of air, the pyrogallol solution will turn brown. if the solution remains clear, decomposition gases were present. in the latter instance, drops of . n naoh and ml of room air should be aspirated into syringe b, which is then also sealed and shaken for min. the mixture should turn brown, thus serving as a fig. · . gas-forming bacteria simulating air embolism. the pericardial sac is opened and filled with water. the heart is kept submerged with a pair of scissors. the coronary arteries have been incised with a scalpel. note gas bubbles and foam on the water surface. no discoloration of % pyrogallol was noted. blood cultures were positive for enterococcus organisms. microscopically, gram-negative rods were found in most tissues. control that the pyrogallol solution had been properly prepared. syringe b may also serve as a reserve. if only one syringe is used, the decomposition gas can be expel ed and room air can be aspirated for the control test. if only small amounts of gas can be aspirated, the volume of the pyrogallol solution should be decreased so that the gas-fluid volume ratio is at least : . if no bacterial gas formation is present, the edges of the pericardial incision are elevated and the pericardial sac is fil ed with water. clamping of the ascending aorta and venae cavae prevents the escape of gas into these vessels. the heart is held under water while the coronary arteries are incised, and the escape of bubbles is recorded. when the right coronary artery is opened, care must be taken that the right atrium is not incised. air in the coronary arteries indicates systemic embolism. the heart chambers are then incised. when there is gas in any of the arteries or heart chambers, gas bubbles rise to the surface of the water in the pericardial sac ("bubble test"). sometimes the vessels have to be somewhat compressed in order to cause the gas to escape. because large amounts of air or other gases cause the heart to float, it must be kept submerged before the vessels and chambers are incised. basically the same procedure is used for demonstrating the presence of gas in the superior or inferior vena cava and the pelvic veins (for example, in cases of criminal abortion). in this situation, the abdominal cavity is filled with water and the inferior vena cava and its tributaries are incised. in support of the diagnosis of systemic arterial air embolism, the skull vault may be removed without puncturing the meninges, so that the cerebral arteries can be inspected for gas bubbles. the demonstration of gas bubbles in the meningeal vessels and in the circle of willis is meaningful only when the neck vessels are still intact and the internal carotid artery and basilar artery have been clamped before the brain is removed. in acute cases, gas bubbles will be visible within the cerebral vessels. they are released under water when the clamps are removed and the vessels are slightly compressed. for the collection of gas from blood vessels or cavities, a system oflittle quantitative reliability is an air-tight, water-filled glass syringe with a needle. the needle is inserted into the vessel or cavity in question and gas is carefully aspirated. a combined qualitative and quantitative method has been described by kulka ( , ) (see fig. - ). he devised an apparatus for gas collection and described it as shown in fig. - and caption. the entire system is filled with mineral oil so that, when the funnel is level with the upright bottle, the oil fills only about half of the funnel. in operation, the funnel is first raised to a position - cm above the level of the upright bottle. all the cocks are opened and the position is held until every trace of gas has been driven from the system through the needle, which is thereby coated on the inside by a film of oil. after all air has been expelled, the cocks are closed and the funnel is lowered to its original position. as a precautionary measure and control, the air-tightness of the whole system should be tested before operation. this is done by inserting the needle into musculature or skin and attempting aspiration in the manner described in the next paragraph. to make the test, the bottle is inverted and the needle is inserted into the cavity in question. when the needle is in position, all cocks are opened. the funnel is lowered about - cm, or until adequate suction is created. this aspirates the contents of the cavity, which may consist of air or other gases, either pure or mixed with blood or other liquid. any gas or liquid entering this system may be observed through the wall of the short bent glass tubing. in a positive test, gas bubbles will collect in the bottle above the level of the oil. if desired, this gas can be saved for further examination by closing all the cocks and returning the bottle to its upright position ( ). the volume ofintravenous air needed to cause death in adults depends of the cross sectional area of the cardiac cavity or great vessel that is the site of the gas lock, which in tum depends on the position of the decedent at the time of the embolism ( ). ml of gas can pass through an intravenous hub in just a few seconds. small amounts of air entering the systemic circulation may cause death within minutes. delayed air embolism with fatal outcome may also occur. other organs if purpura is present, prepare photographs and record extent. submit sample (from right atrium) for microbiologic study. collect blood from right atrium and right ventricle. after the heart has been removed, allow blood in pulmonary vessels to pool in the pericardial sac. centrifuge this blood and submit sample of flocculent layer above buffy coat for microscopic study ( ) . submit a section of lung for bacteriologic study. dissect pulmonary arteries; prepare histologic sections of all lobes; request mucicarmine stain and the aldan blue and phloxinetartrazine stain of lendrum. also request sudan stain on frozen sections. skin purpura. vernix, lanugo hairs, and meconium can be found in pericardial blood pool. meconium-type material in blood vessels. in histologic sections, squamous epithelium, meconium, and fat from vernix caseosa. complete or incomplete lower uterine tear; chorioamnionitis. manifestations ofdisseminated intravascular coagulation* and fibrinolysis. intrauterine pneumonia. large amounts of debris in the blood vessels of all sections of the lungs may be considered to be lethal if there is no other cause of death. small amounts in one or more blocks of pulmonary tissue are more likely incidental ( ). a small uterine tear is more likely followed by a fatal amniotic fluid embolization than is a large tear, which may result in fatal hemorrhage or fibrinogen depletion. chorioamnionitis, intrauterine pneumonia, and positive lung cultures of the mother indicate infection of the amniotic fluid. record weight and sample for histologic study. for removal and specimen preparation, see chapter . photograph horizontal sections through brain, brain stem, and spinal cord. prepare frozen sections and request sudan stain. prepare frozen sections of one-half of gland and paraffin sections of the other half. petechial hemorrhages of skin (chest, neck, and face). wounds; other traumatic lesions. bone fractures. petechiae of conjunctivas and retinas. fat may accumulate on surface ofblood pool. no useful technique is available for estimating the amount of fat globules in the blood. fat emboli in lumen of small vessels and in pulmonary air spaces. severe fatty changes may be the cause offat embolism. fractures are the most common cause of fat embolism. petechial hemorrhages, fat emboli ( ). abdominal cavity submit sample of subphrenic exudate for gram stain and cultures. record location and volume of subphrenic exudate. possible causes of subphrenic empyema include appendicitis, cholecystitis, * diverticulitis, intrahepatic abscess, pancreatitis,* ruptured viscus; penetrating abdominal wound(s), perforated ulcer of stomach or duodenum,* and other conditions. pleural cavities and lungs e procedures record volume of effusion or exudate in pleural space. basal pleuritis and pneumonia, adjacent to empyema. encephalitis, au types or type unspecified synonyms and related terms: acute disseminated encephalomyelitis;* acute hemorrhagic encephalitis; acute infective encephalitis or encephalomyelitis; acute poliovirus encephalitis or encephalomyelitis; amoebic encephalitis; arbovirus encephalitis (japanese encephalitis; eastern encephalitis, western encephalitis, venezuelan equine encephalitis, st. louis encep-halitis); bulbar encephalitis;* brain stem encephalitis;* herpes encephalitis (cytomegalovirus encephalits, epstein-barr virus encephalitis, varicella-zoster encephalitis); herpes simplex ence-phalitis; hiv encephalitis; measles encephalitis; measles inclusionbody encephalitis; postinfectiousencephalitis; postvac-cinal encephalitis; progressive multifocal leukoencephalitis or leukoencephalopathy; rabies* encephalitis; subacute encephalitis; subacute sclerosing panencephalitis; viral encephalitis, west nile encephalitis and other terms ( ), too numerous to mention. see also under "note" and under "possible or expected findings." note: if the condition that caused the encephalitis is known, see also under that heading. if the cause of the encephalitis is unknown, submit samples of tissue for microbiologic and toxicologic study, particularly if there is a suspicion of lead poisoning.* see also under "encephalitis, brain stem," "encephalomyelitis,...," "encephalopathy" and "myelopathy, myelitis." encephalitis, brain stem synonyms and related terms: brain stem abscess; infectious brain stem encephalitis; listeria monocytogenes brain stem encephalitis; viral brain stem encephalitis. note: see also under "encephalitis, limbic." brain for removal and specimen preparation, see chapter . necrotizing encephalitis, with or without abscess formation ( enterocolitis, other types or type undetermined note: a multitude of infectious and noninfectious agents may cause inflammation of the small bowel, large bowel, or both. if the condition is not listed under "colitis," "enteritis," or "enterocolitis" or under another specific heading such as "dysentery, bacillary," obtain sufficient material for microbio-logic and histologic study to identify organisms such as clos-tridium, chlamydia, shigella, salmonella, yersinia, helicobacter, verotoxic e. coli, and others. if lymphogranuloma venereum,* or tuberculosis* are suspected, see also under these headings. see also under "disease, inflammatory bowel" and "disease, crohn's." synonyms and related terms: c. difficile colitis; darmbrand; hemorrhagic necrosis (gangrene; infarction) of intestine; ischemic enteritis or enterocolitis;* neutropenic enterocolitis;* pseudomembranous colitis. note: the name "pseudomembranous enterocolitis" is descriptive; the condition may be infectious, ischemic, or both. if the intestinal changes are clearly ischemic, see above under "enterocolitis, ischemic." if the cause is in doubt and if pseudomembranes can be identified, follow the procedures described here. (l) collect all tissues that appear to be infected. for other infectious intestinal diseases, see under specific names, such as "enterocolitis, neutropenic" or "enterocolitis, staphylococcal." intestinal tract and mesentery other organs collect material from pseudomembranes for culture and for c. difficile toxin assay. sample intestinal wall with pseudomembranes for histologic study. if the condition is suspected to be caused by ischemia, follow procedures described above under "enterocolitis, ischemic." note: myoclonic seizures also have been described in a number of progressive encephalopathies with complex neurological symptoms, such as gmi and gm gangliosidosis,* and niemann-pick* and krabbe's disease but also acquired disorders, including alzheimer's disease,* creutzfeldt-jakob epilepsy, myoclonus (continued) disease,* posthypoxic encephalopathy, and subacute sclerosing panencephalitis. mitochondrial encephalomyopathy also can present with myoclonus epilepsy and a mitochondrial myopathy with ragged red fibers (merrf syndrome) in skeletal muscles. lafora-body-type material in the heart, liver, retinas, peripheral nerves, skeletal muscles, and sweat gland ducts (especially axillary). ragged red fibers in mitochondrial myopathies. epilepsy, symptomatic note: possible causes or underlying conditions include cerebrovascular diseases, congenital malformations ofthe brain, degenerative and demyelinating diseases of the brain, head injury,* intracranial and cerebral infections, toxic or metabolic disorders (alcoholism,* barbiturate,* carbon monoxide,* and lead poisoning,* hemodilution, hypocalcemia, or hypoglycemia),* and tumors of the brain. * f failure, congestive heart note: coronary atherosclerosis and manifestations of ischemic heart disease, * valvular heart disease, congenital cardio-vascular diseases, and manifestations of systemic or pulmonary hypertension are the most frequent findings in patients dying of or with congestive cardiac failure. other causes include cardiomyopathies* and secondary myocardial disease (such as amyloid or pericardial constriction). if the cause of the congestive cardiac failure is unknown or not immediately evident after dissection of the heart and of the great vessels, myocardium and other appropriate tissues may be submitted for microbiologic study-including viral cultures-and for electron microscopy. specimens can also be snap-frozen for possible immunofluorescent, biochemical, or histochemical studies, particularly of the myocardium. possible causes of congestive cardiac failure are too numerous to mention. dilatation of heart, with or without mural thrombosis. myocardium may be soft, normal, or firm. pulmonary congestion, with or without hemosiderosis; congestion of viscera with organomegaly. other organ manifestations include bowel edema or hemorrhagic enteropathy (without mechanical vascular occlusion) and zonal hepatic steatosis, fibrosis, or necrosis, with or withoutevidence of liver failure. acute renal tubular necrosis may be present also. synonyms and related terms: acute kidney failure; chronic kidney failure; renal failure; uremia. note: if acute kidney failure had been diagnosed, the autopsy procedures will depend on the expected causes, such as poisoning with ethylene glycol,* lead,* mercury,* or methyl alcohol;* disseminated intravascular coagulation* and its various underlying conditions; glomerulonephritis* and its various underlying conditions; diabetes mellitus;* or multiple myeloma. * the procedures described below deal primarily with chronic renal failure. if the patient had had dialysis, see also under "dialysis (for chronic renal failure )." if transplantation had been carried out, see also under "transplantation, kidney." lassa fever is a highly communicable disease and autopsy studies are not recommended in the usually available surround-ings. if lassa fever is suspected, contact the state health department and the centers for disease control and prevention, atlanta, ga, for disposition and further studies ( ).ifan autopsy is done, disinfection can be accomplished by washing instruments with . % phenol in detergent (i.e., lysol), . % hypochlorite solution, formalin, or paracetic acid. for shipping procedures, see chapter . this is not a reportable disease. synonyms: borreliosis; louseborne (epidemic) relapsing fever; tickborne (endemic) relapsing fever. note: (i) collect all tissues that appear to be infected. ( ) rat/mouse inoculation with infected blood is the most sensitive method for the detection of the organism. consult the state health department. ( ) before the autopsy, consultation with the microbiology laboratory is advised. ( ) request direct dark-field examination and giemsa or wright stain. ( ) no special precautions are indicated. ( ) serologic studies are of questionable value due to the antigenic variability of the organism. ( ) fever, scarlet note: usually, this disease is a nonfatal group a streptococcal tonsillitis and pharyngitis with skin rash. potentially fatal complications include suppurative otitis media,* mastoiditis, and pharyngeal abscess ( ), with or without septicemia. ( ) collect all tissues that appear to be infected. ( ) atresia, but may also be seen with viral myocarditis, type ii glycogen storage disease (pompe disease) and carnitine deficiency. thus, a metabolic disorder must be considered. it is ideal to perform the autopsy as soon after death as possible ( ) . urine plasma for removal, prosthetic repair, and specimen preparation, see chapter . malnutrition ( ) muscle necrosis (clostridial myonecrosis) and accumulation of gas; little leukocytic infiltration. other organs g procedures depend on expected findings or grossly identified abnonnalities as listed in right-hand column. see also above under "note" and under "skeletal muscles." synonyms and related terms: acute postinfectious glomerulonephritis (nonstreptococcal postinfectious glomerulonephritis;*minimalchangedisease;mesangialproliferativeglomerulonephritis;* focal and segmental glomerulosclerosis with hyalinosis (focal sclerosis); poststreptococcal glomerulonephritis; idiopathic nephrotic syndrome; iga nephropathy (berger's disease); membranous glomerulonephritis; membranoproliferative glomerulonephritis; mesangial proliferative glomerulonephritis; rapidly progressive glomerulonephritis (associated with systemic infectious or immunologic multisystem diseases; drug idiosyncrasy; or as primary crescentic glomerulonephritis or superimposed on another primary glomerular disease). possible associated conditions: acquired immunodeficiency syndrome (aids);* alport's syndrome;* amyloidosis; anaphylactoid purpura; bee stings; chronic allograft rejection; dennatomyositis;* dennatitis herpetiformis; diabetes mellitus;* drug dependence;*fabry's disease;* goodpasture's syndrome;* guillain-barre syndrome;* henoch-schonlein purpura;* hemolytic uremic syndrome;* infective endocarditis;* leprosy;* malig-nancies;mixedconnectivetissuedisease;myxedema;polyarteritis nodosa;* rheumatoid arthritis;* preeclamptic toxemia; renovascular hypertension; sarcoidosis;* serum sickness;* sjogren's syndrome;*syphilis;*systemiclupuserythematosus;*systemic sclerosis;* thrombotic thrombocytopenic purpura;* thyroiditis;* vasculitis; viral hepatitis;* wegener's granulomatosis;* and many other conditions. urate deposits in scleras and corneas. granulocytopenia note: midline granulomas may belong to the angiocentric immunoproliferative lesions, which are related to lymphomatoid granulomatosis* and malignant lymphoma. the name "idiopathic midline granuloma" should be reserved for the few cases without evidence of malignant lymphoma or wegener's granulomatosis* ( ). the diagnosis of idiopathic midline granuloma also can be ruled out if studies reveal fungal organisms or features of leishmaniasis;* leprosy,* rhinoscleroma, pseudotumor of the orbit or tuberculosis. * complications of nasal cocaine abuse also may mimic midline granuloma ( ). eosinophilic pneumonitis (degenerating eosinophils with charcot-leyden crystals) and granulomas. angiitis (mostly arteritis), typically with giant cells in tunica media ( ) . findings resemble those in polyarteritis nodosa. * heart ( ), grastrointestinal tract ( ), skin, muscles, and joints are commonly involved. however, renal disease is often (but not always) mild or absent. necrotizing vasculitis of small arteries and veins is present, with extravascular granulomas and eosinophilic infiltration of vessels and perivascular tissues. optic neuritis may be found ( ). may be affected by the vasculitis ( ). pcr studies on paraffin sections via rna in situ hybridization may confirm presence of epstein-barr virus-positive b-cell proliferations combined with dense t-cell accumulations ( ) ( ) ( ) . the condition closely resembles angiocentric t-/nk cell lymphoma ( ). infiltration of lymphocytoid cells, plasma cells, and macrophages with necroses and granulomatous features, which are found primarily in the vicinity of blood vessels. special stains may reveal evidence of infection. lymphoreticular and granulomatous infiltrates (see "lung"). lymphoreticular and granulomatous infiltrates (see "lung"). characteristic infiltrates may be present in all organs and tissues. involvement of spleen, lymph nodes, and bone marrow is uncommon. in rare instances, the disease is confined to the abdomen. in most instances, characteristic infiltrates are present ( ). septicemia; circulating immunoglobulin complexes, and antiendothelial antibodies ( ). angiocentric granulomatosis ( ) hemorrhage, intracranial (see under "hematoma, subdural" and under name of suspected underlying condition, such as "aneurysm, cerebral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemorrhage, subarachnoid (see under name of suspected underlying condition, such as "aneurysm, cere-bral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemosiderosis, idiopathic pulmonary note: this diagnosis is made by exclusion; involvement of organs other than the lungs (see below under "kidneys") suggests another disease. hepatitis, chronic synonyms and related terms: autoimmune hepatitis; chronic viral hepatitis b (with or without d) and c. note: the term "chronic hepatitis" is not a complete etiologic diagnosis and related names such as chronic active (aggressive) hepatitis, chronic active liver disease, and chronic per-sistent hepatitis are obsolete. most cases in these categories represent autoimmune hepatitis or chronic viral hepatitis b or c. many other liver diseases, including drug-induced hepatitis, inborn errors ofmetabolism (e.g., alphal-antitrypsin deficiency* or wilson's disease*), developmental disorders, and chronic biliary diseases such as primary biliary cirrhosis or primary sclerosing cholangitis also may present as chronic hepatitis. ifchronic hepatitis was the cause ofdeath, submassive hepatic necrosis or cirrhosis* is usually present, often with manifestations ofportal hypertension,*hepatic encephalopathy, hepatorenal syndrome,* and with ascites or spontaneous bacterial peritonitis. possible associated conditions: see also below under "possible or expected findings." conditions that may be associated with hepatitis c include ( ): autoimmune hepatitis; behcet's disease; diabetes mellitus (type );* glomerulonephritis;* guillain-barre syndrome;* idiopathic pulmonary fibrosis; idiopathic thrombocytopenic purpura; iga deficiency; lichen planus; mixed essential cryoglobulinemia; mooren's corneal ulcers; polyarthritis; porphyria cutanea tarda;* thyroiditis. * note: coinfection with other hepatitis viruses (e.g., c and g) and/or systemic viruses such as the immunodeficiency virus are common, particularly in drug addicts ( , ) . in many cases of fulminant hepatitis, tests for known hepatitis viruses are negative ( , ) . homicide note: not all of the following procedures can be carried out or will be required in all cases, nor will this checklist be sufficient in all instances. consult also the entries indicating the cause of death, such as "injury, firearm" or "injury, stabbing." if the victim is an infant, see all under "infanticide." before the body arrives or before autopsy is begun: . investigate the scene where the body was found and where the crime may have been committed (these may be two separate locations). if this is not possible, study the report and photographs submitted by the investigator or the police. study all available information. request previous medical records and roentgenograms of the victim. . emphasize to first responders and autopsy personnel that the body of the victim should not be undressed, washed, or otherwise disturbed until it has been inspected by the pathologist. embalming is not permitted before completion of the autopsy. advise first responders or transporters to put paper bags over the hands of the victim. this will protect possible evidence, such as hair from the assailant. . prepare record sheets to document the time of arrival and release of the body; chain of custody for the body and specimens; the name, age, sex, and other information about the victim; the names ofthe technician, pathologist and guests; and the specimens taken. . prepare roentgenographic and photographic equipment. after body arrives but before autopsy is begun: . if the body is left unguarded-for instance, overnight-a lock should be placed on the refrigerator or cool room where the deceased is kept, and the key should be retained by the technician. . if the pathologist is not accustomed to a busy autopsy room, he or she should feel free to ask all persons other than the pathologist(s), technicians, and law enforcement personnel who are not immediately involved in the actual performance of the autopsy to leave the morgue. . the body temperature of the victim can be recorded at the autopsy facility, but this is rarely useful, particularly if the victim seems to have been deadfor longer than a day ortwo or ifthe body had previously been stored in arefrigeratoror cool room. insert thermometer deep into the anus (about - cm). record temperature and manner and time of procedure. . aspirate vitreous from one or both eyes and store the specimen in a refrigerator. . prepare roentgenograms. in each case, a decision must be made whether roentgenograms should be made of the entire body or only of parts of it-or not at all-and whether they should be made before the victim is undressed, after the victim is undressed, or at both times. . take overall survey photographs that depict the anterior surfaces of the body as it is on arrival to the facility, before any undressing, manipulation for roentgenographs, or cleaning. . take close-up photographs of any trace evidence such as fibers or flakes of gunpowder before undressing or cleaning. . photograph the face of the victim for identification purposes after it has been cleaned of any blood and foreign matter. to avoid perspective distortion from wide angle lenses, the lens should be about ft from the face. . photograph all injuries and other forensically significant findings after blood and foreign matter have been cleaned off the body. a moist towel is useful; brushes are too abrasive. two photographs should be taken of each finding. one should include the autopsy number and a scale. a second photograph should be taken without any extraneous objects. collection and documentation of evidence: . fingerprinting can be done before or after the autopsy but should be done in all homicide cases. in cases involving close contact between assailant and victim, fingernail scrapings or clippings (use a new clipper) and hair with roots should be collected, and its source should be identified (hair pulled from scalp, axillae, and pubis is identified as that of the victim; hair in hands or under fingernails or on clothing of victim may be from the assailant). hair exemplars can be collected in cases of homicide by firearm but are rarely of use. if the body is decomposed or mutilated or for any other reason has not been identified, prepare roentgenograms ofthe head, neck, and torso before the autopsy (the radiographic appearance is altered by the autopsy). these can be used for comparison purposes if antemortem films are located. if films of the extremities are needed they can be prepared after the autopsy. dental roentgeno-.grams are taken, usually after the autopsy, when investigators have located antemortem dental records. detailed descriptions and sizes of clothing, with photographs of clothing, can be useful then there is no putative identity. . in cases in which sexual assault may have been involved, collect pieces of clothing that may contain seminal fluid stains. follow procedures described under "rape." . preserve clothing or part of clothing as evidence. wet clothing should be dried before it is stored in labeled and sealed bags. the autopsy: . the measured length and weight, extent of rigor, and color and distribution of livor should be recorded, along with the state of preservation, nutrition, and hydration. do not omit examination of the hands, particularly of the volar surfaces. . if air embolism is suspected, see procedure described under part ii "embolism, air". if pneumothorax is suspected, see procedure described under part ii "pneumothorax". if there is evidence of strangulation or other neck injury, perform a layerwise dissection of the neck that includes the hyoid bone and tongue. . prepare diagrams of wounds and identify their location by anatomic region. for wounds of the torso, state the distance from the soles of a foot and the distance laterally from to the right or left of the midline. . submit samples of wounds for histologic study when there is any question of the age of the wounds. . the records should show when body fluids and tissues were collected for laboratory analysis, the volume and appearance of such specimens, and what was done with them. if most of the toxicology specimens are collected immediately after the internal examination has commenced, the time of the internal examination serves as the collection time. in all instances, the following items should be collected: blood ofvictim for dna comparison and toxicologic study (determination of alcohol, carbon monoxide, and drug concentrations will be requested most frequently); vitreous; urine; gastric contents; at least one solid organ specimen for toxicologic study (liver and brain have the most comparison data if an extracerebral congenital malformation is suspected, follow procedures described under "malformation, arnold-chiari." if cerebrospinal fluid is aspirated with a syringe, record volume. record weight of brain; record size of brain in relation to inner dimensions of skull. describe size of ventricles. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. if a surgical shunt is present, its location should be recorded and both ends of the implanted specimen that was used for shunting may be submitted for microbiologic study. if the shunt is not patent, record site and nature of obstruction. related terms: antibody-mediated hydrops fetalis; erythroblastosis fetalis;* nonimmune hydrops fetalis. note: the classic example of antibody mediated (rh incompatibility) hydrops fetalis is hemolytic disease of the newborn (erythroblastosis fetalis*). however, nonimmune hydrops fetalis also may be caused by hematologic disorders, e.g., alpha-thalassemia* ( ) or it may have no known cause. infec-tions, e.g., with human parvovirus bl ( ), cytomegalovirus, or syphilis;* heart and vascular diseases ( ) (cardiac tumors, cardi-omyopathy,* myocarditis,* arterial calcification, and others); storage disease ( ); tumors (including neonatal leukemia*); and many other fetal (e.g., congenital chylothorax* or lymphatic dysplasia; pulmonary sequestration and cystic adenomatoid malformation) or maternal conditions, e.g., maternal thyrotoxicosis, also may cause nonimmune hydrops fetalis. if coronary insufficiency or myocardial infarction is suspected, follow procedures described under "disease, ischemic heart." for coronary arteriography, see chapter to. record distribution of atherosclerotic lesions in aorta. samples for histologic study should include aorta, coronary arteries, and peripheral arteries. see also above under "heart." submit samples of head, corpus, and tail for histologic study. if applicable, see also under "diabetes mellitus." for special procedures and stains, see above under "heart." other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . obesity* is a common finding. eruptive xanthomas (palms, elbows, knees) in some but not all types of hyperlipoproteinemia. most prominent in apoprotein cii deficiency. xanthomas of tendons (knees, elbows, dorsum of hands), xanthelasmas, and arcus comeae in familial hypercholesterolemia. gangrene of lower extremities (see below under "arteries"). severe coronary atherosclerosis and myocardial infarcts in type hyperlipoproteinemia and multiple lipoprotein-type hyperlipidemia. atherosclerosis of abdominal aorta and its branches and of carotid arteries. coronary atherosclerosis (see above). pancreatitis* in familial apoprotein cii deficiency. foam cells with triglycerides in liver, spleen, and bone marrow in familial lipoprotein lipase deficiency. manifestations of diabetes mellitus* and hypothyroidism* in some cases of type hyperlipoproteinemia. cerebral infarct (stroke*) in type hyperlipoproteinemia. hypernatremia (see "disorder, electrolyte(s).") hyperoxaluria synonyms and related terms: oxalosis; primary hyperoxaluria type i (a anineglyoxylate aminotransferase deficiency); primary hyperoxaluria type ii (d-glyceric acid dehydrogenase deficiency); secondary hyperoxaluria (see under "note"). note: in ethylene glycol poisoning,*oxalatecrystals in media ofsmall arteries, with associated ischemic lesions. similardeposits may occur after long-term hemodialysis ( ). these conditions must be distinguished from the genetic disease. also, oxa-iate nephropathy may be a complication of short bowel syndrome. if patient with congenital hyperoxaluria underwent liver or combined liverlkidney transplantation ( ) , see also under these headings. remove vitreous for biochemical evalvation. submit tissue (i.e., fascia lata) for karyotype analysis. polycystic ovaries, testicular adrenal rests ( note: there are no diagnostic autopsy findings for hypoxia. possible causes of acute hypoxia include anesthesia-associated death,' compression of the torso by a vehicle, diving accident,' exposure to toxic gases-forinstance, carbon monoxide poisoning;' mechanical failure ofan oxygen supply system, as in an airplane; and sudden mechanical airway obstruction,' as in aspiration ofa foreign body orstrangulation. causes ofchronic asphyxia include prolonged exposure to high altitude and chronic pulmonary disease. profound medial hypertrophy of small pulmonary arteries and of pulmonary veins from chronic exposure to hypoxia. acute pulmonary edema may also occur in chronic hypoxia. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. malnutrition. empty and collapsed colon; small amounts of gray and dry meconium in terminal ileum; meconium masses in dilated and hypertrophied mid-ileum; liquid contents in proximal intestine ( ); appendicitis; bowel perforation. glandular inspissation and atrophy of intestinal mucosa. volvulus, infarction, necrosis, perforation, peritonitis, and acquired intestinal atresia may be present. absence of neural plexuses in congential megacolon* (hirschsprung's disease). manifestations of cystic fibrosis, with or without cirrhosis;* biliary atresia* ( ). immunodeficiency (see "syndrome, acquired immunodeficiency" and "syndrome, primary immunodeficiency.") impression, basilar note: basilar impression constitutes an upward bulging of the margins of the foramen magnum. when occipital condyles are displaced above the plane of the foramen magnum, basilar invagination is present. possible associated conditions: klippel-feil syndrome;* osteogenesis imperfecta;* osteomalacia;* paget's disease of bone;* rheumatoid arthritis; rickets; syringobulbia; syringomylia. * compression and secondary ischemic injury to lower brain stem and spinal cord. see also above under "skull and spine" and under "possible associated conditions." incompetence,••• (see "insufficiency,..•") related terms: battered-child syndrome; child-abuse or child-neglect death. note: vitreous should not be aspirated until the skull has been opened and trauma ruled out, because there is a risk of artifactual damage to the retina. in the presence of craniocerebral trauma the pathologist can opt to examine the eyes by removal, fixation, and histologic study; or retinal photography. follow general procedures described under "homicide" and, if necessary,. the procedures described under "rape." external examination see above under "note." prepare roentgenograms of entire body. see above under "note." in other situations, or after study of the fundus, submit sample of vitreous for chemical and possible toxicologic study. umbilical cord attachment prepare histologic sections of umbilicus. and end of umbilical cord blood submit sample for toxicologic study. consider retaining dried blood on filter paper for possible dna testing for paternity investigation. if there is a possibility that the cadaver represents a stillbirth, see part ii, "stillbirth". if infant is thought to have died shortly after birth, determine the location of air in the intestinal tract; roentgenograms may be helpful. save stomach contents and record amount. other organs see also under "homicide." in the abused child, bruises, hematomas, burn marks, or other patterned injuries. in any child, diaper rash, mongolian spots, self-inflicted fingernail scratches, skin infections, and emaciation. in the previously battered child, fractures in various states of healing. in hypertonic dehydration,' sodium concentrations more than meq/l. this may be caused by organic disease, improper medical treatment, or physical neglect. if infant was born alive, inflammatory changes may be found, depending on survival interval. for the hydrostatic lung test, see "stillbirth." the test is unreliable. extraneous material in air passages indicates that child was alive. air reaches the stomach after min, the small intestine after - h, the colon after - h, and the rectum after l h. there is no difference in the speed of gas propulsion between full-term and premature infants. bacterial gas production and previous resuscitation attempts are potential sources of errors. in questionable stillbirth, presence of milk proves that infant was alive and had been nursed. traumatic lesions and signs of neglect may be present. unilateral ulcers in visceral zoster. in rare instances, diffuse meningoencephalitis may occur. limited necrotic and inflammatory lesions in the cord or brain stem at the level of the affected ganglion are common. posterior hom myelitis ( ). ganglion cell necrosis; lymphocytic infiltration, hemorrhage, and, later, fibrosis. as a rule, only one ganglion is severely involved, but less severe lesions may occur in the ganglia that are immediately adjacent. diffuse lymphocytic infiltration; demyelination; axonal destruction; fibrosis. conjunctivitis; keratitis; iridocyclitis; retrobulbar neuritis; neuroretinitis; occlusion of retinal vessels. evidence of hematologic malignancies ( ) or other conditions, as listed above under "note." infection, middle ear (see "otitis media.") infection, pneumocystis carinii synonym: pneumocystosis. note: ( ) collect all tissues that appear to be infected. ( ) this organism cannot be cultured at present but is frequently associated with viral, bacterial, or fungal infections that can be diagnosed by culture. ( ) for rapid staining of aspirates, use gram-weigert stain, which will stain cysts of pneumocystis carinii and also fungi and bacteria. pneumocystis carinii is best demonstrated with grocott's methenamine silver stain. ( ) no special precautions are indicated. ( ) usually, no serologic studies are available. ( ) this is not a reportable disease. injury, fire (see "burns.") injury, firearm note: protect all drains of autopsy table, which will prevent accidental loss of bullets or bullet fragments. recovery of bullet fragments and pellets can also be improved by passing tissue fragments, blood, and other appropriate materials through a fine nonmetallic sieve. caution must be exercised because sharp edges andjagged projections ofbullets and bullet fragments may cause injury ( ). this applies particularly to the black talon bullet. bullets and bullet fragments should not be touched with forceps or other metal instruments that may produce artifactual markings; they must be placed in properly labeled evidence containers, and the chain of custody must be preserved. from a birdshot wound, at least pellets should be recovered. the firearms examiner will divide the total pellet weight by and derive median pellet weight. from a buckshot wound, all pellets should be recovered. in many of these cases, procedures described under "homicide" must also be followed. do not excise wounds before completion of autopsy (see below). as an academic exercise, excised firearm wounds may be used later for analysis of metal traces by neutron activation or for tests for carboxyhemoglobin. in practice, however, a high quality photograph is adequate to establish the presence or absence of gunpowder stippling or soot deposition toward the end of establishing the range of fire. the dimensions of the stippled areas and soot deposits should be recorded. after completion of external examination, dry the wet clothing and keep as evidence. clothing may also be used for possible test firing or for stain, powder, or particle analysis. external examination if identity of victim is unknown, follow procedures described in chapter . prepare initial roentgenograms of the body regions that have been shot, before disrobing of body, and then lateral films of body regions with bullets. follow up with films of other body regions as needed in the course of autopsy. if the body is decomposed to the extent that the external examination cannot be relied upon to determine cutaneous gunshot perforations, prepare roentgenograms of the entire body before autopsy. record location and number of bullet holes in all layers of garments, indicating whether the involved area is bloodstained or shows gunpowder or soot. if there are several cutaneous perforations, number or letter each consecutively and refer to these numbers in all records. location of bullet holes should be described by recording distance from soles of feet or top of head, and from midline of body. prepare diagrams and photograph holes, with and without labels. photographs should show surrounding garments and extent of blood stains. in hairy areas, shave around bullet wounds for better photographic documentation. for evaluation of distance from where a shot had been fired, see fig. additional roentgenograms during the autopsy may be needed to find bullets embolized to the femoral veins or down the spinal canal. systemic roentgenograms may reveal bullets from obscure entrance wounds, particularly in decomposed bodies, old bullets, bullets in the skin flaps reflected at autopsy, or bullets external to the body in clothing. bullet(s) may have been arrested in hollow viscus or blood vessel and may have been transported to distant sites by peristalsis or bloodstream ("bullet embolus"). bullets may be deflected in unexpected directions from bony surfaces. soot ("smudging") and gunpowder stippling may occur around entrance wound, depending on the muzzle-to-skin distance. there may also be an impression from a recoiling handgun or from a power piston. wounds may be stellate, round, jagged, or slit-like, with or without wide margins of abrasion. primer residues on hand of suicide victim after use of a handgun. fatal secondary injuries, particularly hemorrhages. alcohol intoxication is a frequent finding. fig. · . bullet wounds. differences in the appearance of cutaneous wounds of entrance and of exit and differences in wounds of entrance according to the distance between muzzle and skin at the moment of fire. entrance wounds often differ from exit wounds in that the former are usually surrounded by a narrow zone of abrasion. if the muzzle of a gun is in close contact with the skin at the moment of fire, the combustion products will be blown into the wound and will not be visible on the surface. for close-range wounds, the stippling of the skin around the wound, produced by particles of burned and unburned powder, becomes progressively more dispersed as the range of fire increases and is ordinarily not perceptible if the range has been greater than inches ( synonyms and related terms: acute radiation syndrome; chronic (delayed) radiation injury; radiation enterocolitis; radiation nephritis; radiation pneumonitis. note: procedures and expected findings depend on type of radiation damage, whether acute or chronic, localized or whole-body irradiation. if suspected radiation injury was associated with the administration of radionuclides such as p, , or au, follow procedures suggested in chapter . in fatal whole-body irradiation, findings are most likely related to bone marrow injury, and the suggested procedures and expected findings are those described under "pancytopenia." record extent of oropharyngeal and intestinal ulcerations and hemorrhages. late complications include malignant tumors (carcinoma, leukemia,* lymphoma*), manifestations of hypothyroidism,* and cataracts. organ changes in localized radiation injury depend on site of irradiation (lung, brain, kidney, intestine). the skin is involved in both acute (erythema) and chronic (atrophy, epilation) radiation injury. related terms: cutting injury; knife injury. note: in many of these cases, procedures described under "homicide" must also be followed. insuffiency, adrenal synonyms and related terms: adrenocortical insufficiency; polyglandular autoimmune syndrome (type i, usually in childhood, with parathyroid insufficiency and chronic mucocutaneous moniliasis; type ii, usually in adults, with two or more autoimmune endocrine disorders such as thyroiditis and diabetes mellitus*); primary adrenal insufficiency (addison's disease); secondary adrenal insufficiency (in hypothalamic-pituitary disease or steroid induced); x-linked adrenal hypoplasia. is caused by anatomic changes (see below under "adrenal glands"), drugs (enzyme inhibitors or cytotoxic drugs), or acth-blocking antibodies. possible associated conditions: aids with systemic infections ( ); antiphospholipidantibody syndrome ( ); autoimmune hepatitis; chronic lymphocytic thyroiditis; type i diabetes mellitus;* hypoparathyroidism;* hypothyroidism;* megaloblastic anemia;* surgical procedures, such as heart surgery or orthopedic procedures. large cell lymphoma ( ). should be removed as soon as possible (before embalming), either from cervical midline incision or from chest (neck of cadaver must be well-extended). photograph obstruction before larynx is opened, and inside of larynx and epiglottis after larynx is is opened in posterior midline. make gram-stained touch preparations. make histologic sections of larynx and epiglottis. hemophilus injluenzae (cannot always be isolated from larynx synonyms and related terms: acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, and hairy cell leukemia. multiple subtypes have been identified; they are characterized by cell surface markers, chromosomal abnormalities, staining reactions, and morphologic features. note: at the time of autopsy, most leukemias have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent of the disease and the presence of complications. if the leukemia had not been classified or if the features might have changed from the time of the last work-up (e.g., in suspected cases of superimposed diffuse large cell lymphoma [richter's syndrome]), material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation,* see also under that heading. possible associated conditions: agnogenic myeloid metaplasia with myelofibrosis; bloom's syndrome;* cutaneous mastocytosis; down's syndrome;* immunodeficiency syndromes* (bruton's disease; louis-bar syndrome; wiskott-aldrich syndrome); infantile genetic agranulocytosis; klinefelter's syndrome;* lymphoma;* multiple myeloma;* polycythemia;* and many others. for sampling and specimen preparation, see chapter . increased protein concentration. material in sediment stains red with toluidine blue. metachromatic material. arylsulfatase-a deficiency. excessive loss of myelin with large amounts of metachromatic material (see above under "urine") in white matter and also in some neurons. demyelination with metachromatic material (see above under "urine"). leukoencephalopathy, progressive multifocal possible associated conditions: aids* and other immunosuppressed states; carcinoma; malignant myeloproliferative or lymphoproliferative disorder; sarcoidosis;* tuberculosis. * procedures submit portion of fresh brain for viral culture. fix remainder of brain and spinal cord in formalin and submit samples for histologic study. in situ hybridization for ic virus is available on paraffin-embedded tissue. small patches of demyelination with tendency to form confluent areas in the cerebral white matter. white matter necrosis may be present. eosinophilic intranuclear inclusions occur in affected oligodendroglia cells. subsequently, large, bizarre astrocytes develop. no inflammatory (lymphocyte) cell reaction is present. lightning (see "injury, lightning.") lipoproteinemia (see "hyperlipoproteinemia.") lipoproteinosis, pulmonary alveolar synonym: idiopathic alveolar lipoproteinosis. note: the condition has been described in allografts ( ); in such cases, see also under "transplantation, lung." elephantiasis of penis, scrotum, or vulva (in chronic cases); skin rash (l) and conjunctivitis (in acute cases); genital ulcers. suppurative or fibrosing inguinal, iliac, or pelvic lymphadenitis, with or without sinus tracts. rectal strictures and fistulas in chronic cases ( ) . systemic involvement in the acute stage with pericarditis,* and arthritis,* and meningitis,* proctitis ( ) lymphoma synonyms and related terms: aids-related lymphoma; adult t-cellieukemiallymphoma; angioimmunoblastic lymphadenopathy; b-celllymphoma; burkitt's lymphoma; cutaneous t-cell lymphoma (includes mycosis fungoides and sezary syndrome); hodgkin's disease; non-hodgkin's lymphoma (low grade, intermediate grade, high grade, all with multiple subtypes too numerous to list, which vary in natural history); natural killer cell neoplasms; post-transplant lymphoproliferative disorders (ptld); t-cell lymphoma. note: at the time of autopsy, most lymphomas have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent ofthe disease and the presence of complications. if the lymphoma had not been classified or if the features might have changed from the time of the last work-up, material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation, * see also under that heading. for current terminologies of hodgkin's disease and non-hodgkin lymphomas as well as for staging criteria, appropriate hematologic textbooks should be consulted. possible associated conditions: acquired immunodeficiency diseases (e.g., after organ transplantation; human immunodeficiency virus-l infection); autoimmune disease (e.g., celiac sprue,*rheumatoid arthritis,* systemic lupus erythematosus,* or sjogren's syndrome*); chemotherapy with or without radiation treatment; epstein-barr virus infection; human t-cell leukemia virus infection; inherited diseases with immunodeficiency (e.g., klinefelter syndrome; * common variable immunodeficiency disease, wiskott-aldrich syndrome); radiation. the lesions are found most commonly in the urinary bladder and other parts of the urinary tract, but may also occur at many other sites, including skin ( ) and upper respiratory tract ( ). note: (i) collect all tissues that appear to be infected. ( ) usually, cultures are not indicated. ( ) request giernsa or wright stain. tissue blocks should be rinsed of blood and be as thin as possible before fixation in refrigerated buffered and neutral % formalin solution. this is to avoid precipitationofformalin pigment that may be confused with malaria pigment. the formalin solution should be used in a ratio of parts formalin to one part tissue and should be agitated every few hours. if one is dealing with tissues that contain formalin pigment, this pigment may be removed with a bleaching solution. ( ) usually, no special precautions are indicated. ( ) possible associated conditions: histoplasmosis* and other chronic fungal infections; immune connective tissue diseases such as rheumatoid arthritis* (l) ; malignancies (l); sarcoidosis;* silicosis;* tuberculosis* (l) . note record location and extent of skin changes or skin defects on back. prepare skeletal roentgenograms. if meningitis is suspected, submit sample of cerebrospinal fluid for culture. dse the posterior approach to remove the spinal cord. atrophic skin over meningocele, lacking rete pegs and skin appendages; skin defect in complete rachischisis. bony defects of spine. in meningocele and spina bifida occulta, arachnoid and dura herniate through the vertebral defect. spinal cord and roots are generally not involved. lumbosacral mass in meningomyelocele, with a highly vascular mass (area medullovasculosa) in spina bifida aperta. neural defects in complete rachischisis. diastematomyelia, hydrocephalus ( ) . pyelonephritis;* enlarged urinary bladder ("neurogenic bladder"). external examination heart lungs procedures prepare chest roentgenogram. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. record weights. prepare photographs and roentgenograms of fresh and fixed lung specimens. perfuse one lung with formalin. for preparation of paper-mounted sections, see chapter . submit one lobe for microbiologic and chemical study. decalcify tissue for histologic study. request van gieson's, hale's colloidal iron, pas, and sudan stains. miliary mottling in lower lung fields. mitral stenosis* may be a cause of microlithiasis (mostly intra-alveolar ossification). * cor pulmonale and heart failure ( ) (l) , see also under that heading. ( ) collect all tissues that appear to be infected. ( ) viral isolation, especially with ebv, is not diagnostically useful, due to long incubation periods. ( ) note: these diseases are characterized by a deficiency of a variety of hydrolases, resulting in accumulation of gly-cosaminoglycans (mucopolysaccharides) and glycolipids within lysosomes of fibroblasts, macrophages, white cells, and parenchymal cells of many organs ( , ) . mucopolysaccharides are also excreted in the urine. the general approach is similar in all types. formalin may dissolve all of the stored material and leave empty vacuoles in the involved cells. therefore, frozen sections should be utilized or tissues should be fixed in absolute alcohol. the accumulated material will show intense metachromasia if stained with toluidine blue. it will also stain with pas, alcian blue, and colloidal iron. oil red a will also stain the material from frozen sections. for specimen preparation for elec-tron microscopy, see chapter is. characteristic external features include dwarfism; thickened long bones; coarse facial features; coarse hair; macrocephaly; prognathism; hypertelorism; malformed teeth, and scaphocephalic skull with hyperostosis of sagittal suture; short neck; chest deformity; umbilical and inguinal hernias; lower thoracic and lumbar gibbus; genu valgum and coxa valga; pes planus and other joint deformities; wide hands (clawhands) and feet. coarse thickened skin, covered with lanugo-like hair. hyperlordosis; ovoid deformities of vertebrae; odontoid hypoplasia; large, shoe-shaped sella turcica; kyphosis; hypoplasia of femoral heads; osteoporosis.* if patient underwent bone marrow transplantation ( ), see also under that heading. chronic upper respiratory infections. large cytoplasmic granules in neutrophils. storage of mucopolysaccharides in osteocytes, chondrocytes, and fibroblasts of periosteum, tendons, fasciae, and other connective tissues; dysostosis multiplex. other tissues histologic sampling cannot be excessive. ( ) collect all tissues that appear to be infected. ( ), see also under that heading. blood; other organs and tissues record extent of skin lesions and prepare photographs; prepare sections of affected and unaffected skin. request gram stain of sections and smears. submit samples of blood and grossly affected organs or tissues for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. extensive epidermal necrosis. shedding of granular and horny layers of epidermis. septicemia; staphylococcal infection of nose, throat, ears, eyes, heart valves, urogenital tract, and other sites. bronchial epithelial detachment and bacterial pneumonia ( ). synonyms and related terms: acute kidney failure; *acute tubular necrosis; lower nephron nephrosis. note: the morphologic diagnosis of this condition may be difficult to discern from autolysis. the features that suggest tubular necrosis are: tubular dilation with epithelial flattening, intertubular edema and necrotic epithelial cells in the collecting ducts. the autopsy should be performed as soon as possible. needle specimens of the kidneys obtained in the immediate postmortem period may yield acceptable material. ifnephrotoxic drugs or chemicals are thought to be responsible for tubular necrosis, submit samples for toxicologic study (see also under synonyms and related terms: multiple endocrine neoplasia (men), type (parathyroid hyperplasia or adenoma; pancreatic islet cell hyperplasia, adenoma, or carcinoma; pituitary hyperplasia or adenoma); or type a (medullary thyroid carcinoma, parathyroid hyperplasia or adenoma; and pheochromocytoma); or type b (medullary thyroid carcinoma, pheochromocytoma, mucosal and gastrointestinal neuromas, and marfanoid features). note: in men, type , foregut carcinoids and subcutaneous and visceral lipomas also may be found. in type a, cutaneous lichen amyloidosis may be observed. mixed syndromes include (i) familial pheochromocytoma and islet cell tumor, ( ) von hippel-lindau syndrome, pheochromocytoma and islet cell tumor, ( ) neurofibromatosis* with features of men or , and myxomas, spotty skin pigmentation, and generalized endocrine overactivity (carney complex). in all instances, the autopsy should be done as soon as possible so that tissues for biochemical study can be frozen without delay. nephritis note: see under specific designation, such as "glomerulonephritis" and "pyelonephritis," or under name of suspected underlying condition, such as "gout" or "lupus erythematosus, systemic." nephroblastoma (see "tumor of the kidney(s).") synonyms and related terms: renal stones; urolithiasis. possible associated conditions: carcinomatosis; cushing's syndrome;* cystinuria;* fanconi syndrome;* hyperoxaluria;* hypervitaminosis d;* gout;* multiple myeloma;* osteoporosis;* polycystic renal disease;* primary hyperparathyroidism;* rheumatoid arthritis* ( ); sarcoidosis* ( ). kidneys ureters, urinary bladder, and urethra other organs remove kidneys, ureters, and urinary bladder en bloc. excise and bivalve kidneys to reveal renal pelves. open ureters. record size, number, and appearance of stones, and save for chemical analysis ( ). record heart weight. dissect and record weights of all parathyroid glands. other procedures depend on suspected underlying conditions, as listed above under "possible associated conditions." nephropathy note: see under name of suspected underlying condition, such as "diabetes mellitus," "disorder, electrolyte(s)" (hypercalcemia, potassium depletion), "gout," "hypertension (arterial), all types or type unspecified," or "poisoning,.. ." (heavy metal). if kidney failure was present, procedures under that heading should also be followed. renal tissue may need decalcification or fixation in water-free solution. ( ) note: in this condition, lesions in the brain and cranial nerves, spinal cord, and spinal roots may be schwannomas (including bilateral vestibular schwannomas); multiple meningiomas; gliomas (generally of spinal cord), mostly ependymomas ( %) and pilocytic astrocytomas; or schwannosis of spinal dorsal root entry zones. intracortical meningioangiomatosis; glial hamartia (intracortical, basal ganglia, thalamus, cerebellum, and dorsal horns of spinal cord) and cerebral calcifications also may be found. cutaneous abscess (j). acute necrotizing nocardial pneumonia with abscesses or sinus formation into surrounding tissues; empyema. pulmonary and mediastinallymph nodes other organs submit samples for culture and histologic study. submit samples from all organs and tissues with suspicious gross lesions for culture and histologic study. poorly encapsulated abscesses of any organ; endocarditis ( ) . related term: total parenteral nutrition. note: air embolism* or blood loss may have occurred if line became detached from the catheter hub. metabolic complications such as fluid overload or disturbances of acid-base and electrolyte balance often cannot be diagnosed reliably at autopsy. the disease(s) that may have necessitated parenteral nutrition therapy are not considered here; they include the acquired immunodeficiency syndrome (aids);* cancer cachexia; inflammatory bowel disease; liver or kidney failure;* severe pancreatitis;* short bowel syndrome, and others. obstruction, acute airway synonyms and related terms: aspiration; bolus death; "cafe coronary"; croup; restaurant death. note: ifpermission has been obtained, remove neck organs through straight incision from chest to chin to avoid dislodging a foreign body. ifdissection has to be accomplished from chest, neck of unembalmed cadaver should remain well extended during procedure. inspect larynx and trachea from above and below, respectively, before opening them carefully along the posterior midline. for removal, prosthetic repair, and specimen preparation, see chapter . submit samples of osteocartilaginous and synovial tissues for histologic study; sagittal or frontal saw section through spine provides for best routine evaluation. heberden's nodes at interphalangeal joints of fingers. degenerative changes, primarily of spine, hip joints, and knee joints. histologic and macroscopic degeneration of cartilage; exposure of subchrondral bone; formation of marginal osteophytes; bone cysts; synovial fibrosis; hypertrophic synovitis. o procedures if artificial joints (e.g., hip or knee) had been implanted, record state of implant site. loose joint prostheses. synonyms: hypertrophic pulmonary osteoarthropathy; pac-hydermoperiostosis (idiopathic hypertrophic osteoarthropathy [ j). note: in all instances, an underlying disease must be identified; they include cardiac disease (cyanotic congenital heart dis-ease with right-to-left shunt; infective endocarditis*); gastroeso-phageal reflux ( ); neoplasms of lungs, esophagus, intestine, and liver; chronic liver disease; inflammatory bowel disease;* pulmonary disease (e.g., abscess;* bronchiectasis;* cystic fibrosis;* emp-yema;* emphysema;* lipoid pneumonia* ( ); pneumocystis pneumonia; sarcoidosis;* tuberculosis*); hyperthyroidism. * external examination elastic arteries other organs prepare roentgenograms of extremities. for removal, prosthetic repair, and specimen preparation, see chapter . record presence of aneurysms (thoracic aorta, subclavian) or arteriovenous fistula of brachial vessels. if abdominal aortic aneurysm had been surgically repaired, search for evidence of graft infection ( ) . procedures depend on expected findings or grossly identified abnormalities as listed above under "note." clubbing of fingers or toes (see below under "elastic arteries"); swelling of extremities. periosteal new bone formation in distal shafts of bones of forearms and legs. all bones of extremities may be involved. see above under "external examination." unilateral clubbing. clubbing of toes but not of fingers. swelling; fistulas. bone defect(s) and focal osteosclerosis. bacterial or fungal infections, most common in metaphyseal region of bones; paravertebral or psoas abscess in osteomyelitis of spine. bacterial or fungal osteomyelitis, with or without cavitation and fistulas. trauma, surgery, insertion of prosthesis, generalized ( ) or contiguous infection, inflammatory bowel disease ( ), diabetes mellitus,* and peripheral vascular diseases, deep vein thrombosis ( ). synonyms and related terms: aseptic necrosis of bone; avascular necrosis of bone; idiopathic osteonecrosis; perthes' dis-ease; postfracture osteonecrosis; renal transplant associated osteonecrosis. note: possible underlying conditions include chronic alcoholism,* decompression sickness,* diseases that have been treated with high doses of corticosteroids ( ), gaucher's disease,* human immunodeficiency virus infection* ( ), tuberculosis* ( ), sickle cell disease, * systemic lupus erythematosus,* tuberculosis* ( ), and bisphosphonate therapy ( ). other organs submit sample for biochemical study. for removal, prosthetic repair, and specimen preparation, see chapter . if osteonecrosis is suspected because one of the possible underlying conditions (see above) is present, prepare saw sections through femoral heads, medial femoral condyles, and heads of humeri. procedures depend on suspected underlying conditions, as listed above under "note." osteopetrosis synonymsand related terms: albers-schonberg disease; autosomal-dominantosteopetrosis; carbonic anhydrase-it deficiency; malignant infantile osteopetrosis ( ); marble bone disease. note: manifestations of renal tubular acidosis may have been a clinical complication. possible associated conditions: bone marrow transplantation* (for infantile osteopetrosis). anemia, recurrent infections, bleeding, and bruises may have resulted from myelophthisis associated with osteopetrosis. upper airway obstruction in malignant infantile osteopetrosis may have necessitated tracheostomy ( ). in heritable osteoporotic disorders of connective tissue (osteogenesis imperfecta,* marfan' s syndrome,* and others) are pre-sented separately. for"osteomalacia," see above. for "osteodystrophy," see under "failure, kidney." possible associated conditions: acromegaly;* chronic alco-holism;* chronic obstructive pulmonary disease; chronic kidney failure* ( ); cushing's syndrome;* debilitating disease (various kinds, often with immobilization); diabetes mellitus* ( ); epilepsy;* hyperthyroidism ( );* hypogonadism; malabsorption syndrome;* malnutrition;* primary biliary cirrhosis;*rheu-matoid arthritis;* scurvy;* steroid therapy or anticonvulsant medication. normal parathyroid glands in uncomplicated cases. hyperparathyroidism* ( ) (without osteitis fibrosa) may be present, however. features of osteitis fibrosa (osteoclastic osteoporosis; see "hyperparathyroidism") or osteomalacia* exclude the diagnosis of uncomplicated osteoporosis. osteoporosis, which may be localized, occurs in various neoplastic (e.g., mastocytosis) and inflammatory diseases. external examination brain and base of skull with middle ears examine external ear canal. for removal and specimen preparation, see chapter . culture any lesion appearing to be infectious. draining, foul-smelling greasy material associated with acquired cholesteatoma. bacterial or viral infection, with or without mastoid osteitis. neck abscess, sinus thrombosis ( ) and brain abscess* and meningitis* may complicate otitis media. otosclerosis ( , ) note: otosclerosis may be a measles-virus-associated disease ( ) and therefore, studies to rule out a past infection may be indicated. for removal and specimen preparation, see chapter . for current methods of temporal bone studies, see ref. ( ) . spongy bone in the capsule of the labyrinth. trabeculae of woven bone show pagetoid changes. if stapedectomy had been done, a prosthesis may be in place. brain is externally normal or mildly atrophic. characteristic is midbrain atrophy with aqueduct dilatation and depigmentation of the substantia nigra. neuronal loss with reactive gliosis, associated with neurofibrillary tangles (globose tangles). primarily affected are globus pallidus, subthalamic nucleus, red nucleus, substantia nigra, tectum, periaqueductal gray matter, and dentate nucleus (grumose degeneration). palsy, pseudobulbar (see "disease, motor neuron.") related terms: acute pancreatitis; alcoholic pancreatitis; chronic (or chronic fibrosing) pancreatitis; interstitial pancreatitis. note: some causes of pancreatitis such as adverse drug reactions (e.g., due to azathioprine, furosemide, or estrogens) cannot be diagnosed at autopsy. possible associated conditions: acute fatty liver of pregnancy; apolipoprotein cli deficiency; cystic fibrosis; *hyperparathyroidism;* kidney transplantation. * status post endoscopic retrograde cholangiopancreatography. synonyms and related terms: calcifying panniculitis; histiocytic cytophagic panniculitis; mesenteric panniculitis (mes-enteric lipodystrophy); "sclerema neonatorum." note: the term weber-christian disease described systemic panniculitis with fever, bleeding, pulmonary and pancreatic lesions, and other abnormalities also involving lungs and pancreas, among others. however, this condition is not a specific entity and thus, the name has become obsolete. the term histiocytic cytophagic panniculitis is more descriptive and preferred. leukemia* and subcutaneous t-celllymphoma* may closely simulate panniculitis. possible associated conditions: alphaj-antitrypsin deficiency;* dermatomyositis;* rheumatoid arthritis;* scleroderma and morphea; sjogren's syndrome;* systemic lupus erythematosus.* external examination, skin, subcutaneous tissue, and breasts chest heart lungs liver and spleen record extent and character of skin lesions. record size, location, and gross appearance of subcutaneous nodules. submit tissue samples for histologic study of grossly unaffected skin, skin lesions, and subcutaneous nodules. request verhoeff-van gieson, gram, and grocott's methenamine silver stains. request s-l protein stain. ascertain that cell infiltrates are not leukemic or lymphomatous ( ) . dimpling of skin; necroses; fistulas. panniculitis with subcutaneous nodules in trunk, breasts, and thighs ( ) . calcification may occur in breast tissue but also at other sites (e.g., in kidney failure*). pancreatic enzymeinduced fat necroses associated with severe pancreatitis. widespread hardening of fat tissue with rupture of fat cells and crystal formation in "sclerema neonatorum." focal traumatic panniculitis also may occur in newborns. s-ioo stain negative in panniculitis but positive in rosai-dorfman disease (sinus histiocytosis with massive lymphadenopathy). mediastinal panniculitis. pericarditis;* interstitial myocarditis. * pneumonia;* pleuritis. fat embolism. * features of alphal-antitrypsin deficiency* ( ) . fatty changes of liver; splenitis. intestinal mucosal erosions and ulcers, with or without perforation. massive gastrointestinal hemorrhage in histiocytic cytophagic panniculitis. blind intestinal loop ( ). for sampling and specimen preparation, see chapter . prepare samples for electron microscopy. vacuolar myopathy. ( ) . external examination, skin, and mouth record extent of skin lesions; photograph; submit multiple samples for histologic study, preferably of areas that are free of secondary infection. prepare sections for immunofluorescent staining. bullous and other lesions of scalp, eyelids, nose, axillae, umbilicus, inframammary areas, back, hands, groins, genitalia, anus, knees, and feet. similar lesions may occur in the mouth. acantholysis is suprabasal or near granular layer. igg deposits on the surfaces of keratinocytes. note: (i) collect all tissues that appear to be infected. collect inguinal, popliteal, axillary, and supraclavicular lymph nodes for aerobic culture. photograph enlarged lymph nodes, and prepare smears of fresh cut surfaces. submit consolidated areas for microbiologic study (see above under "note"). perfuse both lungs with formalin. submit samples of pulmonary tissue and bronchial lymph nodes for histologic study. submit samples of grossly abnormal organs, exudates, or drainage fluids for culture and gram stain. hemorrhagic necrosis; variable suppuration. severe hemorrhagic edema; pneumonia with lobular to lobar features. large areas of necrosis teeming with organisms and minimal to suppurative inflammation. plasmacytoma (see "myeloma, multiple.") platybasia note: possible causes or associated conditions include arnold-chiari malformation;* basilar impression* orinvagination; fusion ofatlas to the foramen magnum; klippel-feil syndrome;* malpositioning of odontoid process; osteitis deformans (paget's disease of bone*); osteogenesis imperfecta;* osteomalacia;* rickets; syringobulbia, syringomyelia. * skull and spine prepare roentgenograms of skull and cervical spine. flattening of the base of the skull (angle formed bythe planeoftheclivus andtheplane of the anterior fossa exceeds °). pleuritis (see "effusion(s) and exudate(s), pleural.") pleurodynia, epidemic synonyms: bornholm disease; devil's grip; epidemic myalgia; epidemic myositis. blood heart and pericardium submit samples for viral culture. sample for histologic study and for viral cultures (coxsackievirus a and b; echoviruses). if pericardia! fluid can be obtained, submit for viral culture also. cultures may be negative and search for viral rna by in situ hybridization may be indicated. chest organs other organs dissect thoracic duct and its tributaries (see chapter ). perfuse one or both lungs with formalin. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit samples of cystic lesions for histologic study. submit samples of cystic lesions for histologic study. gas in thoracic duct. emphysema* of lungs. necrotizing enterocolitis in premature infants. pyloric stenosis, colitis ( ), redundant sigmoid colon, ischemic bowel disease. mucosal pseudolipomatosis ( ) . usually, cysts are subserosal in adults and located between muscularis mucosae and muscularis propria in infants and children. mucosa may be inflamed. extraintestinal cysts, as listed above under "abdomen." cysts may also occur in vaginal mucosa. include bright-field and polarized light microscopy, transmission electron microscopy, scanning or transmission electron microscopy with energy dispersive x-ray analysis ( ), ion beam instrumentation, and secondary ion mass spectrometry. the last three methods are time-consuming, require much skill and expensive equipment, and do not lend themselves well to quantitation. ideally, bulk analysis and in situ microanalysis should be used in combination. analyses can be conducted in specialized laboratories at the following centers (for charges and other information, consult the appropriate laboratories): prepare chest roentgenogram. submit sample for microbiologic study. refrigerate sample for possible immunologic study. submit one lobe or samples of all lobes for bulk analysis and for in situ microanalysis (see above under "note"). microincineration may permit preliminary dust analysis. for demonstration of asbestos fibers, see chapter and ref. ( ) . submit one lobe or segment for microbiologic study. for pulmonary arteriography and bronchography, see chapter . prepare roentgenograms of entire lungs and slices. perfuse one lung (or both, if only routine studies are intended) with formalin. submit samples for histologic study of nodular dust lesions, diffuse fibrotic lesions, grossly uninvolved areas, bronchi, and bronchopulmonary lymph nodes. for preparation of paper-mounted sections, see chapter . prepare samples for transmission and scanning electron microscopic study. for removal, prosthetic repair, and specimen preparation, see chapter . alveolar rupture with dissection of air into the mediastinum in neonates with respiratory distress (see "syndrome, respiratory distress, of infant") and in adult patients ventilated on volume respirators. external examination prepare chest roentgenogram. photograph mediastinum. explore major veins and record compression in cases of tension pneumomediatinum. roentgenograms provide the best permanent record of a pneumomediastinum. air bubbles in mediastinal soft tissues. part ii / diseases and conditions abdomen and neck organs perfuse one lung with formalin. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. search for evidence of trauma of other lesions that may have allowed air to enter soft tissues. intubation injury (j); perforation or rupture of major bronchus, trachea, or esophagus. alveolar rupture, e.g., in asthma* ( ), may not be discernible. bronchiolitis obliterans ( ), interstitial pneumonia* ( ) and other lung conditions also may lead to pneumomediastinum. dissection of air from lesions in abdomen (e.g., retroperitoneal colonic perforation [ ] ) or in neck area. pneumonia, all types or type unspecified note: if the type of underlying infection is known, follow procedures suggested under the name of the infectious disease. if the etiologic agent of the pneumonia is unknown, proceed as follows: (l) collect all tissues that appear to be infected. and grocott's methenamine silver stains. ( ) special precautions may be required (see chapter ). ( ) serologic studies may be helpful once a specific etiologic agent is suspected. thus, collect serum at the time of autopsy or procure serum that was collected prior to death. ( ) this may be a reportable disease. related terms: acute interstitial pneumonia (hamman-rich syndrome); desquamative interstitial pneumonia (dip); idiopathic organizing pneumonia (or "bronchiolitis obliterans with patchy organizing pneumonia [boop]" or "cryptogenic organizing pneumonitis"); idiopathic pulmonary fibrosis; lymphoid interstitial pneumonitis (lip); nonspecific interstitial pneumonia (nsip) (or "cellular interstitial pneumonia" [eip]); usual interstitial pneumonia (uip); fibrosing alveolitis; pulmonary alveolitis; and many others. note: the conditions listed under "related terms" are histologic variants of idiopathic interstitial pneumonia. vip is synonymous with idiopathic pulmonary fibrosis (ipf) ( ). diffuse alveolar damage is the histologic finding in patients with the adult respiratory distress syndrome* (ards) causing interstitial and intra-alveolar fibrosis. this is an acute condition, referred to as acute interstitial pneumonia when it occurs as an idiopathic form of rapidly progressive interstitial pneumonia. possible associated conditions: acquired immunodeficiency syndrome* (aids) in patients with with lip or nonspecific interstitial pneumonia ( , ); trauma and shock in ards. secondary pulmonary fibrosis from inhalants (extrinsic allergic alveolitis or pneumonia; pneumoconiosis*), in drug-induced pneumonia, hypersensitivity pneumonitis (microgranulomatous hypersensitivity reaction of lung), rheumatoid arthritis,* sjogren's syndrome,* and other collagen-vascular diseases; primary biliary cirrhosis in patients with nonspecific interstitial pneumonia ( ). external examination postmortem chest roentgenograms provide the only reliable and permanent record of a pneumothorax and its main complication, mediastinal shift due to a tension pneumothorax (fig. - ) . if roentgenograms cannot be prepared, a reasonably reliable diagnosis still can be made if the prosector inserts a needle through the lateral chest wall. the needle should be connected to a water-filled flask. ifa pneumothorax is present, gas bubbles appear in the flask as shown in fig. - . one can also expose the intact parietal pleura and observe if the lung tissue is separated from the parietal pleura by gas. incising the thorax at the base of a water-filled skin pocket is the least reliable method. infants can be totally submerged under water before the chest cavity is incised. however, care must be taken not to make an accidental incision into the underlying lung tissue. poisoning, all types or type unspecified note: if a specific substance is suspected-for instance, arsenic or ethylene glycol-follow procedures described under the appropriate entry. similar entries can be found for poisons whose general character or source is known-for instance, gas or mushroom poisoning. for some substances, the appropriate entry can be found under "abuse,...," "death,...," or "dependence, ..." in all instances, routine sampling of toxicologic material should be done as described in chapter . if no specific substances can be incriminated, the toxicologist must be provided with all available clinical information, as shown in chapter . in most cases of fatal poisoning, the coroner or medical examiner must be notified. poisoning, alkaloid note: see under specific name of alkaloid-for instance, "dependence, cocaine," "poisoning, atropine," "poisoning, digitalis," or "poisoning, strychnine." only a fraction of this large group of plant poisons has been listed. whether the specific name of the alkaloid is known or unknown, complete toxicologic sampling is recommended. poisoning, antimony ote: toxicologic material should be submitted for anal ysis, as suggested under "poisoning, arsenic." iatrogenic antimony toxicity may occur after treatment with antimony compounds for conditions such as filariasis, fungal infections, and schistosomiasis.* external examination and eyes pharynx and gastrointestinal tract heart, liver, and kidneys record skin changes and eye abnormalities. for toxicologic sampling of contents, see chapter . submit tissue samples for histologic study. request frozen sections for sudan stain. if exposure was from du t in smelting work, dermatitis and conjunctivitis may be present. severe gastroenteritis in acute poisoning. if victim drank antimony trichloride, ulcerative pharyngitis and gastritis may be present. fatty changes of myocardium and hepatic and renal parenchyma. poisoning, arsenic note: toxicologic material will be contaminated by bringing it in contact with fluids. keratinized tissues take up arsenic from solutions. put plastic bags over hands of victim. if exhumed bodies are investigated for arsenic poisoning, include material from surrounding soil and coffin along with tissues submitted for chemical analysis. interpretation of toxicologic findings: after fatal poisoning, arsenic concentrations in the liver tend to exceed . - . mg/ioo g wet tissue. in acute poisoning, arsenic concentrations in hair may reach ! g/g ( ) and in nails ! g/g. organs collect all contents, particularly in accidental poisoning in children. body dry and warm after death. mydriasis. iatrogenic atrioventricular block ( ) . fruits of atropa belladonna or seeds of datura stramonium may be found. no characteristic findings. poisoning, barbiturate(s) note: this type of poisoning has become uncommon. barbiturates may cause sudden death. in all instances, concomitant alcohol intoxication* must be ruled out. standard toxicologic sampling is sufficient. blood bile urine esophagus and stomach liver and brain procedures submit samples of blood from portal vein and peripheral veins or heart for toxicologic study. refrigerate for possible toxicologic study. record total volume and ph value. request tests for protein, glucose, and ketones; request drug screen. submit all contents and record their character. analyze for barbiturates and alcohol. submit samples for toxicologic study. evidence of alcohol intoxication.* poisoning by other addictive drugs. gritty residues of unabsorbed tablets, powder, or capsules. mucosal corrosion, ulceration, and discoloration from capsules may occur. concentration of barbiturate in parenchyma important for interpretation. poisoning, bismuth note: accidental poisoning is common (industrial exposure or drugs with soluble bismuth compounds). search also for other heavy metals. acute kidney failure* may be the cause of death. external examination and oral cavity stomatitis with bluish black discoloration of gums; loose teeth; sticky white membranous patches in mouth and throat. jaundice. protein casts and tubular epithelial cells in sediment. gray or black mucosal membranes; swelling of mucosa; intestinal ulcers that may be perforated. hemosiderosis. fatty changes; hemosiderosis. fatty changes; renal tubular degeneration with amorphic basophilic deposits in epithelium of convoluted tubules. hemosiderosis. see above under "external examination." for removal and specimen preparation, see chapter . peripheral neuritis. synonyms: bromine poisoning; bromism. note: the lethal dose is about . g in children and i g in adults (ingested). after fatal methyl bromide poisoning, headspace gas chromatography revealed a subclavian blood concentration of . microgram/ml whereas inorganic bromide concentrations were micrograms/ml in the blood ( ). tissue concentrations were lower than those in the blood. toxicologic samples should include liver and kidneys. chemical bums on face and conjunctivitis indicate direct exposure; skin pustules over body and nodose bromoderma of the legs indicate bromism. blood is best suited for bromide determination. after ingestion of bromide, necrosis with brown discoloration of mucosa of upper gastrointestinal tract may be present. after inhalation of bromide, swelling and inflammation ofmucous membranes in upper and lower respiratory tracts may be present. there may be pulmonary edema. pneumonia occurs in bromism. organs to be analyzed for cd should have no contact with water or be contaminated with blood; they should be sealed in polyethylene bags. cd leaks into fixation fluid. postmortem blood concentrations are very high and no indicator of the antemortem values ( ). external examination and oral cavity blood urine gastrointestinal tract other organs procedures submit sample for toxicologic study. submit sample for determination of cadmium concentration. submit sample for toxicologic study and a portion of one lobe for microbiologic study. perfuse one lung with formalin. fix bowel as soon as possible. collect renal tissue for light microscopic and electron microscopic study. sample for toxicologic study. submit samples for histologic study also. poisoning, carbon monoxide note: if the victim had been in a fire, see also under "bums." carbon monoxide poisoning may rarely be responsible for automobile accidents. relatively low carboxyhemoglobin concentrations may contribute to death if there is concomitant poisoning-forinstance, with alcohol or drugs, particularly sedatives. anemia, atherosclerotic heart disease, and chronic pulmonary disease also increase sensitivity to carbon monoxide. ifblood had been withdrawn at time of hospital admissionfor instance, for crossmatching-submit this for carboxyhemoglobin determination. if no blood can be obtained, see under "heart, kidneys, and other organs." for quick-orienting qualitative tests, for quantitative methods of carbon monoxide determi-nation, and for interpretation of toxicologic findings, see below. request also determination of hemoglobin concentrations and of blood alcohol. request drug screen. for shipping of blood and tissues for carbon monoxide determination, see chapter . it should be noted that losses of up to % of the original saturation occurred when blood was kept in uncapped container at room temperature for yz wk or at °c for wk. external examination blood heart, kidneys, and other organs brain record color of fingernails, particularly in heavily pigmented persons in whom lividity is difficult to discern. record appearance of blood and submit sample of postmortem blood for toxicologic study. see also above under "note." if no blood can be obtained, prepare water extract of spleen, kidneys, or other organs. request determination of carbon monoxide content and of carbon monoxide-binding capacity of this mixture. submit tissue samples for histologic study. for removal and specimen preparation, see chapter . pink skin and fingernails; bullous edema of skin; decubital ulcers. blood tends to be cherry red. for interpretation of toxicologic findings, see below. necrosis of papillary muscles in the heart or myocardial infarction may occur. renal tubular degeneration may also be found. acute kidney failure* has been observed after rhabdomyolysis complicated by compartment syndrome ( ) . hemorrhagic necrosis of basal ganglia (lenticular nucleus in globus pallidus); diffuse petechial hemorrhages in white matter; cerebral edema. acute hydrocephalus in infants ( ) . many methods of carbon monoxide determination have been described. currently, carboxyhemoglobin is detected in most medical examiner toxicology laboratories by visible spectrophotometry or gas chromatography. in hospitals, carboxyhemoglobin is frequently detected and reported in the course of routine arterial blood gas analysis. pink discoloration of skin and organs usually indicates the presence of more than % carboxyhemoglobin (but rule out cyanide poisoning* and exposure to cold*). in a healthy, middle-aged person, a carboxyhemoglobin concentration greater than - % is usually fatal. if the victim was anemic or suffered from chronic lung disease, particularly emphysema* or atherosclerotic heart disease, the concentration may be lower. in association with alcohol, sedatives, and other drugs, carboxyhemoglobin levels may also be much lower and yet fatal. a heavy cigarette smoker may have a carboxyhemoglobin concentration of - %, and higher levels may occur in police officers and other persons exposed to automobile exhaust in dense traffic. if the victim survived the carbon monoxide poisoning for several hours, postmortem blood samples usually will fail to show the presence of carboxyhemoglobin. in these instances, blood taken at the time of admission to the hospital may still be available and of particular value. if the victim had spent h in fresh air before death, - % of the carbon monoxide will have been removed, and - % will have been removed during each subsequent hour. even though clearance may be complete, death may still occur-primarily from brain damage and infectious complications in prolonged coma. or delayed by only a few hours, particularly after inhalation of carbon tetrachloride. sudden death probably is caused by cardiac dysrrhythmia. alcohol concentrations should be determined in all cases or, if death was delayed, evidence of drinking at the time of exposure should be sought. alcohol considerably increases the hazards of carbon tetrachloride. note: see also under "bronchitis, acute chemical" and under "poisoning, gas." hydrochloric acid is sold by plumbing supply houses and pool supply companies as muriatic acid. it is a liquid. chlorine is a water-soluble gas. as supplied for pool sanitation, liquid chlorine is usually acidic. for convenience, chlorine and hydrochloric acid are discussed here together. prepare photographs of face. conjunctivitis and cyanosis in chlorine gas poisoning; burns of lips from hydrochloric acid. see also above under "larynx and trachea." photograph opened esophagus and stomach. sample for histologic study, particularly if there is doubt whether a perforation was antemortem or postmortem. for removal and specimen preparation, see chapter . severe pulmonary edema, bronchopneumonia, and swelling of mucous membranes in chlorine poisoning. arterial thrombosis may occur. pulmonary fibrosis may develop after prolonged survival. swelling and ulceration of mucous membranes in chlorine poisoning; acute laryngotracheitis. tracheoesophageal perforation. corrosion of mucosa with thickening, hemorrhage, and blackish discoloration after ingestion of hydrochloric acid. antemortem and postmortem perforation may occur. glomerular capillary thromboses. hemorrhages in white matter ( ). poisoning, cyanide synonym: hydrocyanic acid (hydrogen cyanide) poisoning. note: hydrocyanic acid (hydrogen cyanide, hcn) is a water-soluble gas. its salts, sodium cyanide and potassium cyanide are sold as "eggs" to the jewelry industry. hydrocyanic acid is formed when cyanide salts are dissolved in acidic solutions. containers from which the poison might have been ingested or inhaled should also be submitted for toxicologic examination. for cyanide screening tests in the autopsy room and for interpretation offindings, see below. caution: stomach may still contain cyanide gas, formed by acidic reaction of cyanide salt. it may be best to open the stomach under a hood ( ). the odor is quite characteristic for cyanide poisoning but most persons are unable to smell this odor. it is helpful to know in advance if any person in an office or laboratory can smell cyanide. forensic pathologists who can smell the compound state that it has its own specific odor, which differs from the often quoted smell of bitter almonds. autopsies also can be done in a negatively pressured isolation room ( ). for odor, see above under "note." bright red skin color is not always present. corrosion around mouth and in oral cavity may be found after ingestion of potassium or sodium cyanide. blood is fluid and sometimes bright red. if potassium or sodium cyanide was ingested, brown-red mucosal corrosion may be present in stomach or in upper digestive tract. pulmonary edema. for odor, see above under "note." if death was not instantaneous, there may be hyaline thrombi in small blood vessels, minute hemorrhages, and necroses of lenticular nuclei. this test can be used for blood and gastric contents ( ) . dip squares of filter paper in a small amount ofsaturated picric acid. let these squares dry until barely moist. place a drop ofthe material to be tested-e.g., blood or gastric contents--on a piece of paper. let material dry for a moment, and then place one drop of % sodium carbonate in the center of the material to be tested. ifcyanide is present, a reddish purple color will chromatograph out from the material. the higher the concentration of cyanide the more blue the color will be. it is possible to recognize whole blood because the blood turns a rather dark brown and the reddish to purple color is clearly visible. high concentrations of sulfide interfere by giving a false-positive test. another screening test is done as follows ( ) . dip filter paper into normal blood. then treat the paper with potassium chlorate, whereupon brown methemoglobin forms. place this preparation into the fluid suspected of containing cyanide (e.g., blood, gastric contents, pulmonary edema fluid). if bright red cyanmethemoglobin forms, the reaction is positive. if the concentration of cyanide in the stomach is high and the concentration in the lungs is low, cyanide was ingested. alternatively, if the pulmonary cyanide concentration is high and the concentrtaion in the gastric contents is low, hydrogen cyanide most likely was inhaled. occasionally, minimal cyanide levels will be present in decomposed bodies. related term: digoxin toxicity. note: certain drugs may interfere with correct digitalis determination. blood heart vitreous procedures submit sample of peripheral blood for digoxin radioimmunoassay. freeze fresh myocardium for digitalis extraction. submit sample for digoxin radioimmunoassay. poisoning, drug(s) (see "dependence, drug(s), all types or type unspecified" or under "poisoning,•••" followed by specific name of drug.) poisoning, ethanol (ethyl alcohol) (see "alcoholism and alcohol intoxication," "cardiomyopathy, alcoholic," "disease, alcoholic liver," "syndrome, fetal alcoholic," and syndrome, wernicke-korsakorr.") poisoning, ethylene glycol related term: antifreeze poisoning. note: pulmonary and cerebral manifestations are the main findings in acute poisoning, and renal tubular necrosis is the primary finding in chronic poisoning. for general toxicologic sampling, see chapter . calcium oxalate crystals can be demonstrated in routine histologic sections but also in scanning electron micrographs of thick deparaffinized sections. eyes blood urine for removal and specimen preparation, see chapter . in acute cases, submit sample for ethylene glycol determination; in chronic cases, request determination of calcium concentrations. prepare sediment. papilledema; optic nerve atrophy. ethylene glycol in serum (i) . protein casts; calcium oxalate crystals ( ) . crystals are light yellow and birefringent, arranged as sheaves, rhomboids, or prisms. poisoning, food related terms: bacillary dysentery* (shigella food poisoning); botulism;* clostridium perfringens food poisoning; favism; mushroom poisoning;* salmonella food poisoning; staphylococcal food poisoning. note: if cause of food poisoning is unknown, submit suspected food for aerobic and anaerobic cultures, gram stain of smears, and routine toxicologic study. this should include tests for heavy metals (antimony, cadmium, and lead) that may have leaked from old cooking utensils. test for the presence of staphylococcal enterotoxin are done only in specialized laboratories. if botulism is suspected, follow procedures described under that heading. mushroom poisoning also is listed as a separate entity. if salmonella food poisoning is suspected, see under "fever, typhoid." see also under "enteritis" or "enterocolitis" or under another specific heading such as "dysentery, bacillary." obtain sufficient material for microbiologic and histologic study to identify organisms such as chlamydia, clostridium (type f strains), salmonella, shigella, verotoxic e. coli, yersinia, and others. external examination gastrointestinal tract submit contents for aerobic and anaerobic cultures (see above under "note"); prepare smears of contents for gram stain. submit samples for histologic study. debilitated states; patients in extremes of life. enteritis or enterocolitis. poisoning, gas note: anesthesia-associated death, * carbon monoxide poisoning,* and sniffing and spray death* are presented under the appropriate headings. procedures discussed here deal with other volatile substances, including chemical irritants such as ammonia (nh ), chlorine or hydrochloric acid poisoning (see also under that heading); methylene chloride, phosgene (coci ), sulfurous acid (h s ), or sulfur dioxide (s ) ' gases from body cavities, heart chambers, or blood vessels can be removed as described under "embolism, air." gases can also be trapped with a rubber dam after cutting organs under water. samples from various organs should be shipped in hermetically sealed nonplastic containers or in analyzing solutions. external examination, and oral cavity blood larynx and trachea record extent of chemical bums. submit sample for gas analysis. in many instances, inhaled gases can be demonstrated chromatographically in gas from head space above sealed blood specimen. chemical bums in and around mouth or conjunctivas. chemical bums. other organs if gas was inhaled and is to be analyzed, submit intact lungs with bronchi ligated in airtight, nonplastic container to laboratory that can conduct gas analysis. if survival was short, formalin perfusion of lungs is not recommended; it may cause artifactual ballooning and internal ruptures of organ. submit samples of tissue for routine histologic study. see above under "note." chemical pneumonia; pulmonary edema. after longer survival, obliterating fibrous bronchiolitis, chronic bronchitis, and saccular bronchiectasis* may occur. poisoning, glycol (see "poisoning, ethylene glycol.") poisoning, halogen (see "fluorosis," "poisoning, bromide," "poisoning, chlorine or hydrochloric acid," "poisoning, gas?, and "poisoning, iodine!') poisoning, heavy metal (see "poisoning, antimony," "poisoning, arsenic," "poisoning, cadmium," "poisoning, lead," poisoning, mercury," "poisoning, thallium!') poisoning, insecticide (see "poisoning, organophosphate(s)*) poisoning, iodine related terms: lugol's solution; tincture of iodine. for toxicologic sampling, see chapter . external examination and eyes urine, blood, and parenchymal organs lungs and upper respiratory tract stomach record color of skin and extent of corrosive lesions. submit samples for toxicologic study. submit samples for histologic study. submit contents for toxicologic study; photograph mucosa; prepare histologic sections. see above under "stomach." perioral corrosive lesions; yellow discoloration of skin; conjunctivitis after exposure to vapors. acute inflammation of respiratory tract after inhalation of vapors. corrosive gastritis; if starch was used as antidote, gastric lining will be bluish. histologically, well-preserved mucosa is present because of in vivo fixation. mucosa may show same changes as stomach. swelling of tubular epithelium. synonyms: propanol; rubbing alcohol. procedures see under "alcoholism and alcohol intoxication." nonspecific autopsy findings: visceral congestion; pulmonary and cerebral edema. poisoning, lead note: lead-free syringes and lead-free polyethylene containers should be used. blood lead concentrations can be determined by inductively coupled plasma mass spectrometry ( ). for screening methods, see ref. ( ) . this is a reportable disease in some states. external examination, oral cavity, and hair bluish lead line at gingival margin in victims with poor oral hygiene. external examination, oral cavity, and hair for diagnosis of chronic plumbism, analysis of scalp hair may be useful. analysis is done by neutron activation (see also under "poisoning, arsenic"). remove samples with lead-free syringe (see above under "note") or -ml vacutainer tubes (becton, dickinson and company). do not add anti-coagulant or preservative. for coliection procedures, see above under "blood" and under "note." request also determination of coproporphyrin concentrations. submit sample for histologic study; submit remaining tissue for toxicologic analysis. submit with contents for toxicologic study, particularly in acute poisoning. submit sample from each kidney for histologic study; submit remaining tissue of both kidneys separately for toxicologic study. submit at least g of fresh bone for toxicologic study. for removal and specimen preparation, see chapter . poisoning, lsd (d-lysergic acid diethylamide) (see "abuse, hallucinogen(s).") poisoning, lye related terms: ammonium hydroxide poisoning; calcium oxide or quicklime poisoning; poisoning by alkaline corrosives; potassium hydroxide poisoning; sodium hydroxide poisoning. external examination and oral cavity blood neck organs, esophagus, trachea, and lungs record extent of oral, perioral, and other facial corrosive injuries. photograph lesions. prepare histologic sections of tissue from inside of lips or mouth. submit sample for toxicologic study. after removal of heart, remove neck organs with hypopharynx, esophagus, larynx, and trachea. leave stomach attached to esophagus. open pharynx and esophagus along posterior midline. in acute cases, formalin perfusion of lungs is not recommended; it may cause artifactual baliooning and internal ruptures of organ. lye bums on face and chest in acute cases; scars and manifestations of malnutrition* in chronic cases. sweliing, edema, and necrosis of mucous membranes in acute poisoning. fibrosis and strictures in chronic cases. bronchitis* and bronchopneumonia. submit specimen from pharynx for histologic study. for removal and specimen preparation, see chapter . submit sample of brain for toxicologic study. exfoliative dermatitis. blue line at gingival margin; hypertrophy of gum; acute and chronic gingivitis; exfoliation and loss of teeth ( ) . degeneration of myocardium. increased concentrations of mercury ( ) . congestion. erosive gastritis and colitis. increased concentrations of mercury ( ) . degeneration of proximal tubules; calcifications. chronic kidney failure* may be the cause of death. induration of mucosa. increased concentrations of mercury ( ) . cortical hemorrhages. cholinesterase activity will be low. pulmonary edema if poison was inhaled. airways may contain aspirated material. cholinesterase activity can be determined reliably even after decomposition and embalming. clinically, guillain-barre syndrome has been observed after poisoning with organophosphate. in acute poisoning, the cholinesterase levels may be % of the normal values (see below). the cholinesterase levels in the blood are not affected by the duration of the postmortem interval; measurement may be attempted even on decomposed or exhumed bodies. severe fatty changes; periportal necroses. fatty changes in myocardium, skeletal muscles, and other organs. for toxicologic sampling (gastric contents, urine, blood, brain, and other organs), see chapter . rigor mortis after fatal strychnine poisoning may occur very soon after death and may be very severe (opisthotonos); it may persist until decomposition sets in. congestion of viscera; no characteristic morphologic autopsy findings. acute pancreatitis has been observed ( ). high strychnine concentrations (also demonstrable in exhumed bodies [ ] ). poisoning, thallium note: for toxicologic sampling, see below and chapter . thallium is used as a rodenticide and pesticide, and has some industrial applications. retrobulbar neuritis. chapter and . submit brain for toxicologic study. submit sections of brain and optic nerves for histologic study. submit specimens for toxicologic study (take from lower extremity). for removal, prosthetic repair, and specimen osteomalacia;* osteomyelofibrosis. preparation of bones, see chapter . for preparation of sections and smears of bone marrow, see chapter . submit samples for toxicologic study. synonym: acute anterior poliomyelitis. note: the disease has been nearly eliminated in the usa but not in many other countries. ( ) collect all tissues that appear to be infected. ( for removal and specimen preparation, see chapter . in acute cases, submit portions of brain and spinal cord for viral culture. neurogenic atrophy of skeletal muscles in areas of paralysis. electrolyte disorder. * hypertensive heart disease; myocarditis.* aspiration or bronchopneumonia (or both); edema; atelectasis; embolism;* alveolar wall necrosis (acute or organizing diffuse alveolar damage) after oxygen toxicity. acute ulcers. acute gastric dilatation; acute gastroduodenal ulcers; gastrointestinal erosions and hemorrhages. dilatation of colon; perforation of cecum. urolithiasis and nephrolithiasis,* pyonephrosis and pyelonephritis. * phlebothrombosis of legs, most commonly on left side. necrosis of anterior hom cells of spinal cord, with neuronophagia and perivascular inflammatory reaction. old lesions show neuronal loss and gliosis. medulla ("bulbar polio") and other areas of brain stem, cerebellum, and cerebrum, particularly the motor cortex, may be affected in various degrees. part ii / diseases and conditions pregnancy note: in some instances, procedures described under "death, abortion-associated," "embolism, amniotic fluid," or "toxemia of pregnancy" may be indicated. synonym: werner syndrome. external examination and skin abdomen procedures record volume of intraperitoneal fluid; prepare smears; submit samples of peritoneum for histologic study. colloid carcinoma of stomach or colon. well-differentated adenocarcinoma of ovary or, rarely, of appendix; ruptured appendiceal mucinous adenoma is the most common cause of peritoneal adenomucinosis. pseudotumor cerebri synonym: benign intracranial hypertension; meningeal hydrops. note: this condition, which generally affects young, obese females, is characterized by symptoms and signs of increased intracranial pressure without a demonstrable cause. hence, intra-cranial mass lesions, infections, and related conditions should be excluded. such conditions include adrenal insufficiency;* guillain-barre syndrome* (increased colloid-osmotic pressure); hyperadrenalism; hypervitaminosis a* (e.g., after treatment of acne); hypoparathyroidism;* hypothroidism;* infectious mono-nucleosis;* lyme disease; pregnancy; sydenham's chorea; throm-bus ofthe lateral or superior sagittal sinus (otitic hydrocephalus); and wiskott-aldrich syndrome. submit samples of all accessible organs and tissues, with or without gross lesions. submit material for electron microscopy. for removal and specimen preparation, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . heart small bowel liver procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. fix the bowel as soon as possible. record weight. submit samples for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter . aortitis involving ascending aorta and aortic valve with regurgitation; mitral valve and adjacent myocardium and conduction system also may be involved. sprue-like changes with loss of villi. fibrosis or cirrhosis and other abnormalities, particularly in patients who had been taking methotrexate. psoriatic arthritis and spondylitis. synonyms and related terms: allergic purpura; anaphylactoid purpura; hypersensitivity vasculitis. external examination and skin rabies note: (i) in most instances, an autopsy limited to the brain is sufficient to confirm the diagnosis. ( ) rabies virus is especially infectious and thus, universal precautions should be strictly followed (see chapter ) . avoid the use of scalpels whenever possible. the generation ofaerosols should be assidu- see above under "note." if a complete autopsy is done, sample heart, lungs, kidneys, pancreas, submaxillary salivary glands, and adrenal glands; freeze or refrigerate sampled tissues and submit for viral study (see below under "brain and spinal cord"). obtain serum for serologic studies. freeze or refrigerate cerebral tissue immediately after removal and submit frozen to special laboratory for fluorescent antibody staining. take these sections from hippocampus or brain stem. place the refrigerated tissues in % neutral glycerol saline solution for preservation. fix remaining brain and spinal cord tissue in % formalin and submit for histologic study. as an alternative to freezing of tissue, immunoperoxidase methods for detection of rabies viral antigens can now be applied to formalin-fixed tissue ( in almost all instances, the procedures described under "assault" and under "homicide" must also be followed. see also refs. ( ) and ( ) . for the evaluation of bite marks, photographs with and without scales and black and white film should be used. a forensic dentist is required to compare the victim's bite marks to the teeth of suspects. swabs of possible sperm or other fluids must be sent to the crime lab for proper evaluation. external examination other organs and tissues genital organs comb pubic hair over a towel and then pull sample; also, pull samples of hair from head. collect fingernail clippings and place in containers marked "right" and "left." collect blood, hair, fibers, and residues of urine or saliva and/or semen that may have stained the victim's clothing or that may be found on the skin of the victim. for study of stains and scrapings, see below. introduce sterile dry cotton swab into the posterior vault of the vagina or-preferablyinto the cervical canal. prepare smear on glass slide and send to crime laboratory for identification of sperm (see also above under "note" using a sterile cotton swab moistened with sterile saline, lift suspected dried spermatic fluid from hair or skin of external genitalia, perineum, buttocks, or other sites. stains on clothing will be extracted by forensic laboratory personnel. let specimens air dry in absence of sunlight and then place in paper bags or envelopes and seal. smears are made for the demonstration of spermatozoa, cytologic changes, bacteria, and other possible findings (some crime labs prefer to make their own smears from the swabs). other samples are used for the determination of acid phosphatase, as described below. usually, the survival time of morphologically recognizable spermatozoa in the vagina is less than h, but on occasion this may be much longer. thus, within the first h after coitus, % of cervical smears have been found to be positive for spermatozoa. at day , spermatozoa have been found in % of the smears. obviously, if the assailant had had a vasectomy, spermatozoa will not be found at any time. spermatozoa have been found in the vagina of some women several days or weeks after death. dried stains (see above) may give positive results after longer intervals. acid phosphatase activities greater than bodansky units (for method, see above) indicate that probably less than h have elapsed since the time of intercourse (i) . in another study, vaginal swab specimens showed acid phosphatase activities of more than , king if there is evidence of parotid involvement, other salivary glands should also be studied. parotid gland can be biopsied from scalp incision. submaxillary gland can be removed with floor of mouth. for sampling and specimen preparation, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . chronic meningitis; space-occupying lesions (submeningeal nodular granulomas). granulomatosis. iridocyclitis; chorioretinitis; papilledema; posterior uveitis; keratoconjunctivitis sicca; conjunctival follicles; cataracts. involvement of lacrimal glands and other orbital tissues ( ) . sarcoidosis of parotid gland is common. sarcoid neuropathy and sarcoid myopathy. cystic changes, primarily of small bones of hands and feet. sarcoidosis of joints ( ). synonyms and related terms: bilharziasis; schistosoma haematobium infection; schistosoma japonicum infection; schis-tosoma mansoni infection. note: unless specifically stated, the changes listed below refer to chronic schistosomiasis mansoni and japonica. ( ) collect all tissues that appear to be infected. ( ) request direct examination for schistosoma. the following procedures have been described and recommended ( ). for demonstration of schistosoma eggs, compress -mm tissue fragments-for instance, mucosa of the urinary bladder-between glass slides. if this gives negative results, digest a -g portion of tissue in potassium hydroxide. for the recovery of adult worms of schistosoma mansoni and schistosoma haematobium, remove the viscera en bloc and rinse with water. separate the intestines from the mesentery. subsequently, perfuse the portal vein system, the liver, and one lung with saline ( ) . then pass the perfusion fluid through a monofilament nylon cloth with an aperture size of m. submerge the cloth in water and examine with a dissecting microscope. fix worms in formalin solution. examine the intestinal mucosa directly. from the urinary bladder, ureters, and surrounding connective tissue, worms can be recovered as follows. inject water into the tissue until the tissue increases or times in thickness. then compress the tissue gently with a glass plate. cut slices . - . cm in thickness. compress these slices between the glass plate and the stage of a dissecting microscope and examine for the presence ofadult worms. many worms also will be present in the fluid expressed from the tissue as it is cut. for the counting of eggs in tissues, urine, and feces, see ref. ( ) . immunodiagnostic methods (elisa and imrnunoblot) also have been developed prepare skeletal roentgenograms. there are no diagnostic laboratory tests but it still is advisable to save a blood sample. record heart weight. test competence of valves (chapter ). open heart in line of blood flow. photograph and measure valvular lesions. prepare sections of valves, myocardium, conduction system (if there was a history of heart block), and ascending aorta. perfuse at least one lung with formalin. submit any consolidated area for microbiologic study. fix intestines as soon as possible. abnormalities as listed in right-hand column. sample for histologic study and request amyloid stains or renal parenchyma. prepare roentgenograms of spine, sacroiliac joints, symphysis ossium pubis, and manubriosternal, sternoclavicular, and humeroscapular joints. if spine cannot be removed in its entirety, it can be split in midline and one half can be removed, with costovertebral and costotransversal joints. hip joints should be exposed. histologic sections should include synovia and periarticular tissue. secondary and peripheral osteoarthritis* may be present. leukemia* developed in some patients who had had radiation treatment ( or earlier). acute anterior uveitis. ( ) request fungal culture. ( ) request grocott's methenamine silver stain. ( ) record weight of all organs (for expected weights, see part iii). submit samples of all major organs, including endocrine glands, lymphatic and fat tissue, bone, and bone marrow for histologic study. hunger edema; skin changes secondary to vitamin deficiencies. hypoproteinemia. atrophy; hemosiderosis of spleen. degree of atrophy varies from organ to organ; atrophy of fat tissue, lymphoid tissue, and gonads usually is most pronounced. severe infections, such as tuberculosis,* may be present without having been apparent clinically. steatohepatitis, nonalcoholic (nash) note: the morphologic findings in the liver are indistinguishable from those in alcoholic liver disease* ( ). follow autopsy procedures described under "disease, alcoholic liver." if the patient had received a liver transplant, procedures described under "transplantation, liver" should be followed also. possible associated conditions: celiac sprue (rare); diabetes mellitus* (type ); hyperlipidemia; malnutrition* from bulemia (rare); morbid obesity with liver failure particularly after episodes of rapid weight loss (e.g., after recent gastroplasty); lipodystrophy; mild obesity;* short bowel syndrome (rare); total parenteral nutrition. * stenosis, congenital valvular pulmonary related terms: isolated (pure, simple, or dome-shaped) pulmonary stenosis. note: the pulmonary valve is usually acommissural in isolated congenital pulmonary stenosis. with other coexistant congenital heart disease (such as tetralogy), the pulmonary valve is usually bicuspid and hypoplastic, but may be unicommissural or dys-plastic and tricuspid. heart and great vessels; peripheral pulmonary arteries brain procedures culture any grossly infected valve. for general dissection techniques, see chapter . record weight of heart, thickness of ventricles, and annular circumferences. for removal and specimen preparation, see chapter . infective endocarditis* of pulmonary valve and tricuspid valve; infective endarteritis at bifurcation of pulmonary trunk. it is preferable to have autopsies of fetuses and stillborns performed by pathologists experienced in perinatal pathology. if such personnel are not immediately available, the attending pathologist may nevertheless collect important information. if attempts to induce abortion appear to have caused the death of the mother, see "death, abortion-associated." the placenta should be immediately procured from the delivery room should it not arrive in the laboratory with the fetus. this will avoid the possibility of the placenta being discarded by the delivery room staff. no fetal autopsy is complete without a careful examination of the placenta (see part i, chapter ). pathologic changes of placenta that are causative of stillbirth are summarized in ref. . fascia lata or other aseptically obtained tissue should be collected for tissue culture for karyotype analysis. if the fetus is at all autolyzed, then the fascia lata cells may not grow in tissue culture. therefore, if the fetus shows any evidence of autolysis, tissue should be taken aseptically, from placenta immediately beneath the chorionic plate. a portion ofplacenta and liver should also be snap-frozen for possible molecular analysis. the initial stage of the autopsy should include photography and radiography, taking anterior-posterior and lateral views. the photographs will record the degree of maceration, which can be roughly correlated with the duration of fetal demise before delivery [ ] external measurements should include body weight, circumference ofhead, chest and abdomen, crown-rump length, crown-heel length, and foot length. these measurements are compared with tables of standards for normal fetuses (see part iii of this book). assessment of growth retardation may be based on these data. a careful external examination should search for abnormalities such as jaundice, bulging fontanel, cranial bone softening, hyper-or hypotelorism, choanal atresia, external ear anomalies, cleft lip, cleft palate, macroglossia, micrognathia, colobomata, cystic hygroma, shortened neck, contractures, omphalocele, gastroschisis, abnormal external genitalia, anal atresia, absent vagina, sacral pits, open neural tube defects, hemihypertrophy, syndactyly, clinodactyly, transverse palmar creases, or incomplete descent of testes. the organ bloc may be removed in the manner similar to the adult, using the technique of letulle (see chapter ) . if the thyroid gland is noted to be in its usual location and if it appears normal, then the tongue may be left in the body. in macerated fetuses, it is suggested that the organ bloc be fixed overnight in formalin solution prior to dissection. to aid adequate fixation, the following simple steps may be done: i) wash the organ bloc thoroughly prior to fixation; ) place multiple transverse cuts through the liver and lungs; ) dissect the posterior leaves of the diaphragm away from the adrenal glands and kidneys; ) bivalve the adrenal glands and kidneys in the coronal plane; ) instill formalin in the lumen of the intestine, using a syringe; and ) gently instill formalin into both ventricles of the heart, being careful to avoid the ventricular septum. the procedure for dissection of the organs is similar to that of the adult except: ) the venous and arterial connections of the heart, including the patency of the ductus arteriosus must be determined before the heart is removed; ) the esophagus must be opened posteriorly prior to its complete removal so that esophageal atresia or tracheo-esophageal fistula may be recognized and photographed prior to further dissection; ) the location of the appendix and of the testes should be recorded. until the intestine has been examined for stenosis or atresia, the mesentery should be left attached. the degree of autolysis as seen grossly and with histologic examination of both the fetus and the placenta can be used to estimate the duration of time between fetal death and delivery ( , , ) . trichrome stain is useful for better visualizing histologic features in severely autolyzed tissue. to remove the brain, benecke's technique of one of its modifications may be used. stillbirth vs livebirth. a decision has to be made whether the infant was born alive or was stillborn. the hydrostatic lung test, described in the previous edition, appears unreliable. the presence of gas in the lungs does not rule out stillbirth. after death, air can be introduced into the lungs, or putrefaction gases might be present. however, air artificially introduced after death will not distend the alveoli and can be squeezed out, whereas this does not seem to be the case after active ventilation. the distribution of fat in the fetal zone of the adrenal cortex may indicate whether intrauterine death was acute, more prolonged, or chronic ( ). this is particularly helpful if the stillborn baby is macerated. if the mother died also, see under "death, abortion-associated" strangulation note: in many instances, procedures described under "hom-icide" must also be followed. if a rope or some other material had been used (see also under "hanging"), leave ligature in place until autopsy can be done. see also under "hypoxia." toxicologic sampling, particularly for alcohol, should be done in all instances (chapter ). results than body fluids. ( ) universal precautions should be strictly followed. the generation of aerosols should be minimized. to sterilize tissue surfaces in preparation for culture, swab the surface with povidone iodine. avoid searing the tissue surface since this will produce an aerosol. keep no more than one scalpel in the dissecting area at anyone time. have an assistant available with clean, gloved hands to receive specimens in containers, so as to minimize the degree of contamination on the outside of the containers. for cleaning procedures and related information, see chapter . ( ) hiv-l in tissue may be demonstrated using polymerase chain reaction (pcr), immunohistochemistry, in situ hybridization, or immunofluorescence. ( ) for immunocytochemical and molecular studies, fix tissue in ethanol or carnoy's solution. the virus can be identified using pcr in most tissues, whether fresh, frozen, or fixed in ethanol. ( ) serologic tests as well as direct fluorescent antibody tests are avail-able for many of the expected infections. ( ) this is not a reportable disease. external examination and skin record body weight and evidence of lipodystrophy following aids medications. record and photograph any skin lesions. examine oral cavity. if the diagnosis is in doubt, samples can be submitted for enzyme immunoassay. cachexia. severe loss of subcutaneous fat in face and extremities, associated with large fat deposits on upper back and upper abdomen ("protease paunch"). cutaneous kaposi's sarcoma (l) . test may be positive for many weeks after death ( ) . syndrome, myelodysplastic synonyms and related terms: chronic myelomonocytic leukemia;* refractory anemia; refractory anemia with excess of blasts; refractory anemia with excess of blasts in transformation; refractory anemia with ringed sideroblasts; refractory dysmyelopoietic anemias. note: the myelodysplastic syndromes are represented by a heterogeneous group of normocytic anemias, often with neutropenia, thrombocytopenia, and monocytosis. for expected bone marrow changes, see above under "synonyms and related terms." autopsy procedures are similar to those recommended for most cases of leukemia, with particular attention paid to intercurrent infections and thrombocytopenic hemorrhages. in all instances, material should be collected using aseptic technique for tissue culture for chromosome analysis. common findings in these conditions are deletion of the long arm ofchromosome , deletion of chromosome or , or trisomy . syndrome, nephrotic note: see under name of suspected underlying condition, such as amyloidosis,* anaphylactoid purpura,*diabetes mellitus,* glomerulonephritis,* goodpasture's syndrome,* heavy metal poisoning, hemolytic uremic syndrome,* infective endo-carditis,* polyarteritis nodosa,* syphilis, * or systemic lupus erythematosus. * if accelerated hypertension or constrictive pericarditis is the suspected underlying condition, see under "hypertension (arterial), all types or type unspecified" or "pericarditis," respectively. in all instances, the renal veins and the inferior vena cava should be opened in situ. "en masse" removal of organs is recommended for this purpose. if thrombosis is found, record exact location and size of clot and submit sample of clot with wall of veins for histologic study. see also under "thrombosis, venous." coronary atherosclerosis and its complications seem to be increased in patients with the nephrotic syndrome. submit sample for bacteriologic, viral, and serologic study. record weight and submit samples for histologic study. if heart block was present, submit samples of conduction system for histologic study (chapter ). submit consolidated areas for microbiologic study. perfuse at least one lung with formalin. for removal of urethra, see chapter . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . consult roentgenograms (see above under "external examination, skin, and oral cavity"). keratoderma (keratosis blennorrhagica) with vasculitis ( ) of sole of feet, of palms, and of circumcised glans penis. arthritis* of knees, ankles, and metatarsal and midtarsal joints, with or without ankylosis. osteoporosis.* usually, sterile culture. pericarditis; * myocarditis.* aortic valve lesions. pleuritis and pneumonia.* pulmonary fibrosis involving upper lobes. erosions, papules, and plaques in urethral or bladder mucosa. enteritis. thrombophlebitis. conjunctivitis; multifocal choroiditis; acute anterior uveitis; iritis; keratitis. arthritis* (see above under "external examination, skin, and oral cavity") resembling rheumatoid arthritis. * nonspecific synovitis. for removal and specimen preparation, see chapter . syndrome, reye's note: hypoglycemia* may have been present, but that condition is difficult or impossible to confirm after death. an immediate autopsy is indicated (see below under "liver" and "pancreas"). vitreous blood urine heart lungs submit sample for sodium, chloride, and urea nitrogen determination. submit sample for microbiologic (viral) and serologic study, for ammonia and bilirubin determination, and for determination of salicylate levels if there is a history of treatment with this drug. obtain sample for biochemical and toxicologic analysis. record weight. request frozen sections for sudan stain. submit fresh tissue for bacterial and viral culture. request paraffin sections and frozen sections for fat stain (see above under "heart"). influenza;* varicella.* manifestations of liver failure. evidence of salicylate administration. assay for organic acids should rule out acylcoenzyme a dehydrogenase deficiency, and toxicity, e.g., of acetaminophen and valproic acid (l) . cardiomegaly; fatty changes of myocardium and patchy myocytolysis. viral pneumonia rarely present. acute interstitial pneumonia;* bronchitis;* hemorrhages. lipid-laden histiocytes in alveoli. record body weight and length, stature, and distribution of head, axillary, and pubic hair. record and prepare photographs of features of face and neck. prepare skeletal roentgenograms. submit samples of breast tissues for histologic study. these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter ) . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit for histologic study. submit samples of all portions for histologic study. if biliary atresia is suspected, follow procedures described under that heading. dissect kidneys and ureters in situ and record findings. submit samples of gonads or-if gonads cannot be identified-equivalent ridges on mesosalpinx for histologic study. also submit samples of endometrium, cervix, and vagina. record weight and submit sample for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for sampling and specimen preparation, see chapter . peroxidase-labeled gastrin antibodies seem to react with cells in all gastrinomas. aberrant gastrinoma may occur at hilus of spleen. metastases are found in regional lymph nodes and liver. there may be manifestations of multiple endocrine neoplasia. * secondary syphilis. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody techniques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. ( ) in adult autopsies, no special precautions are indicated (see below under "liver"). ( ) serologic studies are available from local and state health department laboratories. ( ) this is a reportable disease. external examination record and prepare photographs of all abnormalities listed in right-hand column. prepare smears and sections of acute lesions; prepare sections of older skin lesions or anogenital mucosal lesions. hunterian chancre in primary syphilis; condylomata lata. noduloulcerative gummas and scarring in later stages. cerebrospinal fluid lymph nodes heart and aorta other organs and tissues prior to wk gestation, the destructive effects of syphilis may not be seen. gummata are rare in neonates. tabes dorsalis is also uncommon. serologic diagnosis is difficult in the neonate because of transplacental transfer of maternal igg antibodies. acquired syphilis (syphilis in adulthood) is presented above under a separate heading. ( ) collect all tissues that appear to be infected. ( ) culture methods are not available, but animal inoculation can be performed. consultation with a microbiology laboratory is recommended. ( ) special stains for treponema pallidum rarely are positive except with material from fresh lesions of primary or secondary syphilis and of syphilitic hepatitis of the newborn. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody tech-niques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. ( ) in neonates, special precautions are indicated (see below under "liver") ( ) serologic studies are available from local and state health department laboratories. ( ) this is a report· able disease. (i) collect all tissues that appear infected. ( ) request aerobic and anaerobic cultures. however, the presence of tetanus bacilli established in culture is not diagnostic, since spores of c. tetani frequently contaminate wounds. record body weight and length and appearance of wound(s); photograph and excise wound(s) for histologic study. record evidence of parenteral drug abuse, especially subcutaneous injection (i.e., "skin popping"). prepare chest roentgenogram. evidence of weight loss; subcutaneous abscesses. tetanus may occur in drug addicts. tension pneumothorax* after mechanical ventilation. submit sample for microbiologic study. submit any consolidated area for microbiologic study. bacterial meningitis must be ruled out. aspiration and bronchopneumonia; embolism;* atelectasis. tetany note: see under name ofsuspected underlying conditions, such as hyperparathyroidism,* malabsorption syndrome,* or vitamin deficiency. * respiratory or metabolic alkalosis* cannot be confirmed after death; determination of blood ph is not helpful because acidity increases rapidly after death. the concentration of serum phosphates also increases after death. synonym: large ventricular septal defect with pulmonary stenosis* or atresia.* note: the basic anomaly consists of subpulmonary stenosis, ventricular septal defect, overriding aorta, and secondary right ventricular hypertrophy. for general dissection techniques, see chapter . surgical interventions include modified blalock-taus-sig subclavian-to-pulmonary arterial shunt; complete repair with patch closure of ventricular septal defect, and reconstruction of right ventricular outflow tract (with a patch or with an extracardial conduit). possible associated conditions: origin of left pulmonary artery from aorta; minor abnormalities of the tricuspid valve; absent ductal artery ( %); atrial septal defect* (in %; pentalogy of fallot); bicuspid pulmonary valve;* dextroposition of aorta; double aortic arch; hypoplastic pulmonary arteries; patent ductal artery;* patent oval foramen; complete atrioven-tricular septal defect* (usually with down's syndrome*); persis-tent left superior vena cava; pulmonary valve atresia (see "atresia, pulmonary valve, with ventricular septal defect"); right aortic arch ( %); second ventricular septal defect; origin ofleft anterior descending (lad) or right coronary artery (rca) from contralateral aortic sinus or coronary artery ( %); syndrome with absent pulmonary valve and massively dilated pulmonary arteries (rare). chest cavity heart lungs other organs record course of superior vena cava and of its tributaries. record course of thoracic aorta and of its main branches. record origin of pulmonary arteries. if infective endocarditis is suspected, follow procedures described under that heading (chapter ). for coronary arteriography, see chapter . perfuse both lungs with formalin. request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. persistent left superior vena cava. right aortic arch with or without right-sided ductal artery; double aortic arch; absent ductal artery. origin of the left pulmonary artery from descending aorta. for possible additional findings, see above under "note" and under "possible associated conditions." infective endocarditis* (usually of pulmonary or aortic valve). see also above under "possible associated conditions." marked right ventricular hypertrophy. right ventricular dilatation and fibrosis with late postoperative right-sided heart failure or sudden death due to arrhythmia. old in situ thrombosis of small pulmonary artery branches. paradoxic embolism; cerebral abscess. * thalassemia synonyms and related terms: congenital hemolytic anemia; alpha-thalassemia; beta-thalassemia major (cooley's anemia); beta thalassemia minor (beta-thalassemia trait). note: the changes described below are observed primarily in beta-thalassemia major. unless the cause of the renal vein thrombosis and the nature of the complicating or underlying renal disease are known, follow procedures described under "glomerulonephritis." procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. in infants, manifestations of dehydration. * pulmonary embolism. * tumor of the kidney* (renal cell carcinoma); aortic aneurysm; lymphadenopathy; other mass lesions. inferior vena cava thrombosis involving orifice or whole length of renal veins. tumor thrombus (e.g., from renal cell carcinoma). femoral vein thrombosis. rare venous malformations also may cause renal vein thrombosis (l) . hemorrhagic infartion. parenchymal renal disease with nephrotic syndrome,* including amyloidosis* and vasculitis* (these last conditions may be associated with renal vein thrombosis). acute gastroenteritis in infancy. hyperparathyroidism,* pregnancy,* trauma, and other conditions. thrombosis, venous note: for peripheral venous thrombosis, the autopsy procedures are essentially similar to those described under "phlebitis." see also under "hypertension, portal," "syndrome, budd-chiari," "thrombosis, cerebral venous sinus," "thrombosis, lateral sinus," and "thrombosis, renal vein." thymoma (see "'fumor of the thymus.") thyroiditis synonymsand related terms: chronic fibrosing thyroiditis (riedel's struma); chronic thyroiditis with transient thyrotoxicosis; hashimoto's thyroiditis; pyogenic thyroiditis; subacute (granulomatous, giant cell, de quervain's) thyroiditis. possibleassociatedconditions: withhashimoto's thyroiditisautoimmune (chronic) hepatitis,*megaloblastic anemia,*rheumatoid arthritis,*sjogren's syndrome,*and systemic lupus erythematosus.* with riedel's struma-retroperitoneal fibrosis,* sclerosing cholangitis,* sclerosing mediastinitis,*and other conditions with idiopathic fibrosis. with subacute thyroiditis-viral infection. submit sample for determination of protein concentration; record appearance of sediment. for removal and specimen preparation, see chapter . thromboses of small vessels. glomerulonephritis.* hemorrhagic necroses. proteinuria; abnormal sediment. abruptio placentae, small placenta with accelerated maturation of villi; infarcts; fibrinoid necrosis of decidual arterioles. thrombotic occlusion of small vessels with hemorrhagic necroses; anterior lobe of pituitary gland may contain necroses (see also "syndrome, sheehan's"). submit samples for histologic study. if infection is expected, submit material for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. transposition, complete, of the great arteries note: the basic anomaly is the origin of the aorta from the right ventricle, and of the pulmonary artery from the left ventricle, with a shunt, and usually with a right anterior aorta. there are four major types: ( ) with an intact ventricular septum ( %), ( ) with a ventricular septal defect* ( %), ( ) with a ventricular septal defect and subvalvular pulmonary stenosis* ( %), and ( ) with an intact ventricular septum and subvalvular pulmonary stenosis* ( %). for general dissection techniques, see chapter . interventions include atrial septostomy or septectomy; mustard or senning atrial switch procedure; rastelli-type repair with a valved extracardiac conduit; and jatene arterial switch procedure with lecompte maneuver. possible associated conditions: abnormal origin ofcoronary arteries ( %); atrial septal defect* ( %); coarctation of the aorta;* interrution of the aortic arch;* juxtaposition of atrial appendages ( %); overriding aorta ( %); overriding pulmonary artery ( %); patent ductal artery;* patent oval foramen; subvalvular pulmonary stenosis* ( %); tubular hypoplasia ofthe aortic arch;* ventricular septal defect* ( %), often mal-alignment type ( %). synonyms: atrioventricular and ventriculoarterial discordance; l-transposition. note: the basic anomaly is a mirror-image ventricular inversion, with a left anterior aorta, and with blood flow from right atrium to left ventricle to pulmonary artery, and from left atrium to right ventricle to aorta. for general dissection techniques, see chapter . interventions include patch closure of the ventricular septal defect; relief of pulmonary stenosis; relief of tricuspid insufficiency; and insertion of pacemakers. possible associated conditions: anterior and posterior av nodes ( %), prone to develop complete heart block; dysplasia or epstein's malformation* ofleft-sided tricuspid valve ( %); mirror-image epicardial coronary artery distribution ( %); right-sided mitral valve anomalies; subvalvular pulmonary stenosis* ( %); ventricular septal defect* ( %). kinyoun's, or other acid-fast stains. polymerase chain reaction for mycobacterial dna may be helpful in the differential diagnosis ofgranulomas ( ). ( ) universal precautions should be strictly followed and aerolization should be avoided. ( ) reliable serologic studies are not available. a definite diagnosis requires isolation of the organism. ( ) this is a reportable disease. cavitary, fibrocalcific, miliary, bronchial, and other types of pulmonary tuberculosis; granulomas in hilar lymph nodes. most organs and tissues may be involved, including liver, pancreas, spleen, kidneys, adrenal glands and other endocrine glands, gonads, and lymph nodes. tuberculous meningitis; tuberculoma. iridocyclitis or panophthalmitis. tuberculous arthritis and synovitis (hips, spine, knee); tuberculous osteomyelitis (anterior aspect of vertebrae; metaphysis of long bones). part ii / diseases and conditions procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. chronic ulcerative colitis may be associated with carcinoma of bile ducts, typically arising in psc.* metastases common in regional lymph nodes and lungs. i for removal, prosthetic repair, and specimen preparation and decalcification procedures, see chapter . if osteoblastic metastases appear to be present, search for primary tumor in prostate and breast, carcinoid tumors and hodgkin's lymphoma. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. cachexia. evidence of hypercalcemia (particularly in presence of osteolytic metastases). metastases to bone (e.g., from carcinoma of prostate, breast, bronchi, thyroid gland, kidney, or urinary bladder) usually involve red bone marrow. therefore, distal extremities are rarely involved by metastatic tumors in adults. metastases, commonly in lungs. metastatic calcification in patients with hypercalcemia. eosinophilia. myopathy.* metastases in liver and regional lymph nodes. see also above under "possible associated conditions." malakoplakia (rare) ( ) . part ii / diseases and conditions other organs and peripheral arteries tumor(s). if tumor is within a heart chamber (usually a myxoma), record extent of mobility and capability of tumor to obstruct a valvular orifice. submit samples of tumor(s) for histologic study and, particularly if the tumor is of unknown type, for immunohistochemical study and electron microscopy (chapter ). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. metastatic carcinoma or melanoma. secondary cardiac effects by tumors include carcinoid heart disease, amyloid heart disease in multiple myeloma, and lymphocytic myocarditis in pheochromocytoma. treatment effects such as adriamycin cardiotoxicity may be noted also. tumor (myxoma) emboli in pulmonary or peripheral vessels ( ) . multiple aneurysms* of cerebral and other arteries may rarely be associated with atrial myxomas. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. part ii / diseases and conditions if there was evidence of endocrine activity (see above), snap-freeze portion of fresh tumor for hormone assay. perfuse lungs with formalin. sample neoplastic and non-neoplastic tissue for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . see above under "possible associated conditions." note that hypoglycemia* generally cannot be confirmed at autopsy. nonbacterial thrombotic endocarditis.* carcinoma may be associated with asbestosis or other types of pneumoconiosis,* chronic bronchitis,* emphysema,* interstitial pneumonia,* and many other bronchopulmonary diseases. see above under "possible associated conditions." metastases (regional lymph nodes, liver, bones, brain, and many other sites) and metastatic calcification. migratory thrombophlebitis.* encephalomyelitis;* cerebellar corticoid degeneration;* subacute spinocerebellar degeneration. * crooke cell hyperplasia. myasthenic syndrome (eaton-lambert syndrome); myopathy;* dermatomyositis.* peripheral neuropathy. see above under "external examination and skin." bones (with red marrow) are common sites of metastases. record presence or absence of testes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. thmor of the soft tissues the possible sites and characteristics of soft tissue tumors vary so much that no universally applicable autopsy techniques can be presented. in all instances, the size, weight, and location of the tumor(s) must be recorded and tissue must be sampled for histologic study. if the tumor had not been classified prior to death, samples should be snap-frozen for immunohistochemical study. other samples should be prepared for electron microscopic study. evidence of paraneoplastic syndromes (see below) may require additional procedures. possible associated conditions: only a few paraneoplastic syndromes or systemic complications can be presented here. for the type of soft tissue tumor that was associated with each condition, see title of reference. kasabach-merritt syndrome ( ) (thrombocytopenia, microangiopathic hemolytic anemia, and acute or chronic coagulopathy associated with a rapidly enlarging hemangioma); liver function abnormalities ( ); neurofibromatosis ( ); osteomalacia ( ). heart esophagus, stomach, and duodenum other organs and tissues t in most instances, detennination of vitreous sugar concentration and of blood group is not indicated. inspect valves and prepare photographs and sections of vegetations. leave esophagus and part of duodenum attached to stomach. submit samples of tumor and of grossly uninvolved stomach for histologic study. request pas stain and warthin-starry stain for identification of h. pylori. portions of endocrine gastric tumor should be snap-frozen for immunohistochemical study and possible hormone assay. record location of tumor metastases. other organs procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for dissection of the thoracic duct (for search of tumor cell clusters), see chapter . tumor of the uterus (with cervix) possible associated conditions: acquired immunodeficiency syndrome* (aids) ( ) if peritonitis is present, submit exudate for bacteriologic study. record volume of exudate and location of perforation. see "disease, ischemic heart." other procedures depend on grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. perfuse at least one lung with formalin. for celiac arteriography, see chapter . open stomach and duodenum in situ and record site of perforation or penetration. record measured or estimated volume of blood in gastrointestinal tract. rinse ulcer with saline to locate eroded vessel(s). pin stomach and duodenum on corckboard (serosa toward board) and fix specimen in formalin before sectioning. prepare histologic sections of ulcer(s) and of remainder of stomach. request warthin-starry stain for h. pylori. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. free air in abdomen suggests perforation of ulcer. peritonitis. * perforation of ulcer. coronary atherosclerosis and manifestations of coronary insufficiency are commonly associated with peptic ulcer disease. in rare instances, pericardial fistula may be present from ulcer in hiatus hernia. peptic ulcers may be associated with emphysema,* tuberculosis,* and other chronic pulmonary diseases. vasculitis (see "aortitis," "arteritis, .•.," "phlebitis," and "purpura,•••") ventricle, double inlet left ( ) synonyms: single functional ventricle; univentricular heart; univentricular atrioventricular connection; holmes heart. note: the basic anomaly is the connection of both atrioventricular valves to the left ventricle, often with transposed great arteries and a restrictive ventricular septal defect. there are four major types, based on the ventriculoarterial connection: ( ) with congenitally corrected transposition ( %); ( ) with complete transposition ( %); ( ) with normally related great arteries (holmes heart; %); and ( ) double outlet, persistent truncal artery, or pulmonary atresia ( %). for general dissection techniques, see chapter . possible associated conditions: bicuspid pulmonary valve; bilateral mirror-image mitral valves (without tricuspid morphology); subvalvular aortic stenosis,* often with hypoplasia, coarctation, or interruption of the aortic arch; subvalvular pulmonary stenosis;* dual av nodes and progressive heart block in patients with congenitally corrected transposition. ventricle, double outlet right synonym: origin of both great arteries from right ventricle; taussig-bing anomaly. note: the basic anomaly is the origin of the aorta and pulmonary artery primarily from the right ventricle, usually with a ventricular septal defect, and often with subpulmonary stenosis. for general dissection techniques, see chapter . possible associated conditions: complete atrioventricular septal defect (often with asplenia syndrome); muscular discontinuity between aortic and mitral valves; right ventricular infundibular stenosis; ventricular septal defect* that may be subaortic, subpulmonary, doubly committed, or remote. examine histologically with h&e and oil red a stains. hepatomegaly and splenomegaly, jaundice. deficiency of acid lipase ( ). foam cells in the lamina propria. hepatomegaly, severe steatosis with cholesterol clefts in hepatocytes and kupffer cells. fibrosis. splenomegaly with lipid-laden foam cells. symmetric enlargement with dystrophic calcifications, giving the adrenals a gritty texture. vacuolated cells of the inner zona fasciculata and entire zona reticularis with cholesterol clefts. foam cells may be found in the lungs, bone marrow, lymph nodes, vessel walls, as well as schwann and ganglion cells. new insights into lipoprotein assembly and vitamin e metabolism from a rare genetic disease angioid streaks associated with abetalipoproteinemia familial hypobetalipoproteinemia: genetics and metabolism aviation pathology scuba diving accidents: decompression sickness, air embolism aetiology and occurrence of diving injuries. a review of diving safety scuba decompression illness and diving fatalities in an overseas military community diving-related emergencies bodies found in water achalasia and squamous cell carcinoma of the esophagus: analysis of patients esophageal achalasia and adenocarcinoma in barrett's esophagus: a report of two cases and a review of the literature a technique for necropsy evaluation of stenosis of the foramen magnum and rostral spinal canal in osteochondrodysplasia mutation analysis of the men i gene in multiple endocrine neoplasia type i, familial acromegaly and familial isolated hyperparathyroidism. i increased incidence of neoplasia in females with acromegaly benign and malignant tumors in patients with acromegaly pathology of acromegaly ethanol in sequestered hematomas stages of acute alcoholic influence/intoxication electrolyte imbalance in alcoholic liver disease collection and storage of specimens for alcohol analysis analysis for alcohol in postmortem specimens disposition of alcohol in man medicolegal aspects of alcohol. garriott jc blood, urine and other tissue specimens for alcohol analysis relationship between postmortem blood and vitreous humor ethanol levels larson cpo alcohol: fact and fallacy gradwohl's legal medicine left ventricular hypertrophy is more prominent in patients with primary aldosteronism than in patients with other types of secondary hypertension intracranial aneurysm and hemorrhagic stroke in glucocorticoid-remediable aldosteronism association of adrenal medullae and cortical nodular hyperplasia: a report of two cases with clinical and morpho-functional consideration amyloidosis: recognition, confirmation, prognosis, and therapy electron microscopy of primary and secondary cutaneous amyloidosis and systemic amyloidosis ocular amyloidosis current status review: cerebral amyloid spinal cord compression by amyloid deposits the burden of "sticky" amyloid: typing challenges quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with congo red histochemical stain kidney involvement in takayasu arteritis pathogenesis of rheumatoid arthritis splenic involvement in rheumatic diseases aspergillus sinusitis in patients with aids: report of three cases and review invasive aspergillosis in systemic lupus erythematosus aspiration (see "obstruction, acute airway aspergillus endocarditis, myocarditis and pericarditis complicating necrotizing fasciitis. case report and subject review aspergillus-related lung disease biliary atresia and the polysplenia syndrome: its impact on final outcome biliary atresia nephromegaly and elevated hepatocyte growth factor in children with biliary atresia coccidiomycosis pneumonia in a nonendemic area associated with inftiximab codeine (see "dependence, drug[s], all types or type unspecified all types or type unspecified (see "enterocolitis, other types or type undetermined chronic ulcerative (see "disease, inflammatory bowei cytologic diagnosis of cryp -tococcus neofonnans in hiv-positive patients cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis hypereosinophilia in disseminated cryptococcal disease cryptococcosis-a review of autopsy cases from a -year period in a large hospital cryptosporidiosis in persons with hiv infection broncho-pulmonary cryptosporidiosis in four hiv-infected patients cryptosporidiosis and inflammatory bowel disease sclerosing cholangitis associated with chronic cryptosporidiosis in a child with a congenital immunodeficiency disorder cyanide (see "poisoning, cyanide cyst(s), choledochal choledochal cyst-dinical features and classification cyst(s), liver (see "disease, fibropolycystic, of the liver and biliary tract pulmonary related terms: congenital cystic adenomatoid malformation; congenital pulmonary lymphangiectasis; intralobular bronchopulmonary sequestration. references infection or calcification of cysts; pyelonephritis;* perinephric abscess. obstructive uropathy in recessive polycycstic renal disease, congenital hepatic fibrosis is present and cystic bile ducts may be present in dominant cases ( ). see above under "possible associated conditions the pathology ofhuman renal cystic disease cystic kidney: genetics, pathologic anatomy, clinical picture, and prenatal diagnosis hepatic fibrosis associated with hereditary cystinosis: a novel form of noncirrhotic portal hypertension ophthalmic manifestations and histopathology of 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additional risk factor for cirrhosis and hepatocellular carcinoma? alpha i-antitrypsin deficiency and inflammatory bowel disease severe alphal-antitrypsin deficiency (piz homozygosity) with membranoproliferative glomerulonephritis and nephrotic syndrome, reversi-bleafterorthotopic livertransplantation klippel-feil syndrome associated with malfonned larynx. case report klippel-feil syndrome in association with a craniocervical dennoid cyst presenting as aseptic meningitis in an adult: case report klippel-feil syndrome accompanied by an aneurysm of the non-coronary sinus of valsalva the hereditary ataxias clinical and genetic characterizations of q-linked autosomal dominant spinocerebellar ataxia (ad-sca) and frequency analysis of ad-sca in the japanese population cocain-related medical problems. consecutive series of cases direct cocaine cardiotoxicity demonstrated by endomyocardial biopsy the pathology and etiology of cocaineinduced heart disease ischemic colitis related to cocaine abuse hepatic dysfunction accompanying acute cocaine intoxication high incidence of malignancies in patients with dennatomyositis and polymyositis: an li-year analysis partial lipodystrophy associated with juvenile dennatomyositis: report of two cases chronic pneumomediastinum and subcutaneous emphysema: association with dennatomyositis spontaneous esophageal rupture in adult dennatomyositis polyneuropathy in juvenile dermatomyositis combined glucose and lactate values in vitreous humor for postmortem diagnosis of diabetes mellitus diabetes is related to cerebral infarction but not to ad pathology in older persons hepatic lesions resembling alcoholic liver disease relationship between cardiomyopathy and liver disease in chronic alcoholism cyanamide-associated alcoholic liver disease: a sequential histologic evaluation caroli's disease: - experiences caroli 's disease and autosomal dominant polycystic kidney disease: a rare asso-ciation? spontaneous rupture of a bile duct and its endoscopic management in a patient with caroli's syndrome bartonella henselae endocarditis in an immunocompetent adult cat-scratch disease and bacillary angiomatosis attack, transient cerebral ischemic" and "infarction, cerebral!') cholesteryl ester storage disease: hepatopathology and effects of therapy with lovestatin clinical, biochemical and histological analysis of seven patients with cholesterol ester storage disease cutaneous manifestations of chronic granulomatous disease. a report of four cases and review of the literature aspergillus endocarditis in chronic granulomatous disease colitis complicating chronic granulomatous disease. a clinicopathological case report hypertrophic cranial pachymeningitis with spinal epidural granulomatous lesion ocular pathologic findings of chronic granulomatous disease of childhood review: creutzfeldt-jakob disease survival of creutzfeldt-jakob disease virus in formol-fixed brain tissue guidelines for high risk autopsy cases: special precautions for creutzfeldt-jakob disease tissue handling in suspected creutzfeldt-jakob disease and other human spongiforme encephalopathies (prion diseases) cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of creutzfeldt-jakob disease autopsy case of sporadic creutzfeldt-jakob disease presenting with signs suggestive of brainstem and spinal cord involvement metastatic crohn's disease: a rare cutaneous manifestation crohn's disease with pulmonary involvement in a year old boy mesenteric fibromatosis asso-ciated with crohn's disease cholangiocarcinoma and crohn's disease the pancreas as a site of granulomatous inflammation in crohn's disease dermatomyositis associated with crohn's disease cardiocyte storage and hypertrophy as a sole manifestation of fabry's disease. report on a case simulating hypertrophic non-obstructive cardiomyopathy an atypical variant of fabry's disease with manifestations confined to the myocardium pulmonary involvement in fabry disease cerebrovascular complications offabry's disease high incidence of thrombosis in fabry's disease oral involvement in chronic graft versus host disease after allogenic bone marrow transplantation massive pericardial effusion complicating the course of chronic graft-versus-host disease (cgvhd) in a child with acute lymphoblastic leukemia following allogeneic bone marrow transplantation the histological spectrum of pulmonary graft-versushost disease in bone marrow transplant recipients bronchiectasis in bone marow transplantation oncocytic metaplasia of bile duct epithelium in hepatic gvhd immune-mediated myelopathy after allogeneic marrow transplantation gunther's (see ''porphyria, congenital erythropoietic heavy-chain synonyms and related terms: gamma heavy-chain inflammatory bowel disease (first of two parts) takayasu's arteritis associated with idiopathic ulcerative colitis cerebrovascular complications ofinflammatory bowel disease pathology of inflammatory bowel disease legionnaires' disease source of infection for sporadic legionnaires' disease: a review legionnaire's disease associated with macular rash; two cases severe skin loss after meningococcal septicemia: complications in treatment osteonecrosis following meningococcemia and disseminated intravascular coagulation in an adult: case report and review rhabdomyolysis during the subacute stage of meningococcal sepsis primary meningococcal arthritis in a child: case report and literature review chondrosarcoma as a complicating factor in paget's disease of bone lymphoma arising in paget's disease osteoarthritis and paget's disease sickle cell disease sickle cell disease and sudden death: a retrospective/prospective study of autopsy cases and literature review vascular lesions of the liver in sickle cell disease. a clinicopathologic study in living patients whipple's disease and "tropheryma whippelii periodic acid-schiff-negative granulomatous lymphadenopathy in patient with whipple's disease. localization of the whipple bacillus to noncaseating granulomas by electron microscopy serial imaging studies of cerebral whipple's disease: from onset to end the wilson's disease gene is a putative copper transporting p-type atpase similar to menke's gene the liver biopsy diagnosis of wilson's disease. methods in pathology hepatolenticular degeneration (wilson's disease) venous air embolism in homicidal blunt impact head trauma: case reports a practical device for demonstrating air embolism amniotic fluid . kulka w. laboratory methods and technical notes: a practical device for demonstrating air embolism proof of fatal air embolism venous air embolism from head and neck wounds hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease liver involvement in hemorrhagic fever with renal syndrome hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hanta virus pulmonary syndrome lassa fever in the united states. investigation ofa case and new guidelines for management lassa fever: review of virology, immunopathogenesis, and algorithms for control and therapy early diagnosis of lassa fever by reverse transcription-pcr rheumatic pneumonia: reappearance of a previously recognized complication of rheumatic fever acute rheumatic fever and poststreptococcal acute glomerulonephritis caused by t serotype streptococcus scleritis, uveitis, and glaucoma in a patient with rheumatic fever comparison of clinical features and pathologic findings in fatal cases of typhoid fever during the initial and later stages of the disease typhoid fever complicated by acute renal failure and hepatitis: case reports and review hemophagocytosis and granulomas in the bone marrow of a child with down syndrome diseases involving intrahepatic bile ducts idiopathic eosinophilic esophagitis with stricture formation in a patient with longstanding eosinophilic gastroenteritis eosinophilic gastroenteritis presenting with colitis and cholangitis eosinophilic gastroenteritis presenting with acute pancreatitis idiopathic midline destructive disease: does it . mendenhallwmetai.lethalmidlinegranoloma-nasalnaturalkillerff-celi exist? nasal cocain abuse mimicking midline granuloma pulmonary lymphomatoid granulomatosis in acquired immunodeficiency syndrome: lesions with epstein-barr virus infection recurrent bilateral spontaneous pneumothoraces associated with pulmonary angiocentric immunoproliferative lesion lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications wegener's granulomatosis: histological documentation of common and uncommon manifestations in patients necrotizing granulomatosis of the breast wegener's granulomatosis with gangrene of toes pathology of pulmonary granulomatous vasculitis. sarcoidosis gunshot (see "injury, firearm splenic involvement in rheumatic diseases wegener's granulomatosis, acute laryngotracheal airway obstruction and death in a -year-old female: case report and review of the literature antiendothelial antibodies in patients with wegener's granulomatosis: prevalence and correlation with disease activity and manifestations. i rheumatoi transfusion-transmitted disease references flattening, broadening, and possible fusion of villi. phlegmonous inflammation and edema, mainly in ileocecal region encephalitis.* leukopenia; thrombocytopenia; aplastic anemia ( ) (pancytopenia*) synovitis (arthritis*) hepatitis g virus infection in hepatitis c virus-positive patients co-infected or not with hepatitis b virus and/or human immunodeficiency virus hepatitis g and c coinfection in children tumor of the liver diaphragmatic related terms: congenital diaphragmatic hernia fulminant hepatitis in patients undergoing liver transplantation: evidence for anon-a, non-b, non-c marrow transplantation for hepatitis-associated aplastic anemia: a follow up oflong-term survivors langerhans cell histiocytosis research. past, present, and future an update on c onality, cytokines, and viral etiology in langerhans cell histiocytosis langerhans cell histiocytosis in adults pulmonary langerhans cell granulomatosis central nervous system disease in langerhans cell histiocytosis immunohistochemical analysis oflangerin in langerhans cell histocytosis and pulmonary inflammatory and infectious diseases pathological approach to the diagnosis of hydrocephalus thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report infectious causes ofhydrops fetalis human parvovirus b infection associated with hydrops fetalis cardiac abnormalities associated with hydrops fetal is gmi-gangliosidosis presenting as nonimmune hydrops fetalis: a case report a case of recurrent infantile polycystic kidney associated with hydrops fetalis the pathologist and the hydropic placenta, fetus, or infant chronic diarrhea associated with thymoma and hypogammaglobulinemia (good's syndrome) systemic amyloidosis in a patient with hypogammaglobulinemia. i hcv infection in patients with primary defects of immunoglobulin production encepahomyelitis in primary hypogammaglobulinemia retinitis pigmentosaand hypogammaglobulinemia intestinal obstruction in the newborn with congenital syphilis meconium ileus and its equivalent as a risk factor for the development of cirrhosis: an autopsy study in cystic fibrosis biliary atresia and meconium ileus associated with nieman-pick disease postmortem cranial mri and autopsy correlation in suspected child abuse cytomegalovirus-associated pseudotumor simulating gastric malignancy in acquired immuno-deficiency syndrome: a case report with review of literature bone marrow fibrin ring granulomas and cytomegalovirus infection cutaneous reactions following herpes zoster infections: report of three cases and a review of the literature herpes zoster and internal malignancy posteriorhorn varicella-zoster virus myelitis death or injury caused by electrocution myocardial hemorrhagic necrosis in delayed death from electrocution safety in bullet recovery procedures: a study of the black talon bullet lightning injuries: prognostic signs of death lightning injuries adrenal insufficiency, recurrent bacteremia, and disseminated abscesses caused by nocardia asteroides in a patient with acquired immunodficiency syndrome ards and adrenal insufficiencyassociated with the antiphospholipid antibody syndrome adrenal insufficiency from bilateral adrenal hemorrhage after total knee replacement surgery non-hodgkin's lymphoma presenting with adrenal insufficiency and hypothyroidism: an autopsy case report a case ofprimary unilateral adrenal burkitt-like large cell lymphoma presenting as adrenal insufficiency effect of growth hormone on fatty liver in panhypopituitarism increased fracture frequency in adult patients with hypopituitarism and gh deficiency pituitary abscess the liver: an atlas and text of ultrastructural pathology intubation (see "injury, intubation iodine (see "poisoning, iodine attack, transient cerebral ischemic" and "infarction, cerebral heart (see "disease, ischemic heart isomorism (see "syndrome, polysplenia and asplenia a study on performance of two serological assays for diagnosis of leprosy detection of mycobacterium leprae infection by pcr pathology of eye in leprosy references intervillous abscesses containing grampositive, non-acid-fast bacilli bacteria are predominantly intracellular. enterocolitis. listeria monocytogenes can be cultured from meconium. generalized lymphadenitis. disseminated abscesses and/or granulomas, particularly after transplacental infection listeria monocytogenes empyema in an hiv infected patient fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis liver abscesses due to listeria monocytogenes yust . brainstem abscess and meningitis due to listeria monocytogenes in an adult with juvenile chronic arthritis pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions the liver in systemic lupus erythematosus: pathologic analysis of cases and review of japanese autopsy registry data chronic pancreatitis: a complication of systemic lupus erythematosus disseminated perivenous necrotizing encephalomyelitis in systemic lupus erythematosus: report of an autopsy case skeletal muscle lymphocytic vasculitis in systemic lupus erythematosus: relation to disease activity clinically occult avascular necrosis of the hip in systemic lupus erythematosus storage histiocytes and hemophagocytosis: a common finding in the bone marrow of patients with active systemic lupus erythematosus extensive medium vessel vasculitis with sle: an unsual association cutaneous manifestations of sexually transmitted ing cause of proctitis in men who have sex with men lymphogranuloma venereum as a cause cutaneous malakoplakia in a patient with acquired immunodeficiency syndrome (aids) extensive malakoplakia ofthe nasopharynx: management of a rare disease malakoplakia and colorectal adenocarcinoma coinfection is common in measles associated pneumonia giant cell pneumonia: light microscopy, immunohistochemical, and ultrastructural study of an autopsy case subacute sclerosing panencephalitis manifesting as viral retinitis: clinical and histopathologic findings intestinal neuronal dysplasia thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report disease, meningococcal encephalitis, all types or type unspecified" and "meningitis mercury (see "poisoning, mercury with myelofibrosis synonym: idiopathic myelofibrosis acute cardiac tamponade associated with pericardial extramedullary hematopoiesis in agnogenic myeloid metaplasia methyl alcohol) (see "poisoning, methanol (methyl alcohol) thrombotic thrombocytopenic (see "purpura, thrombotic thrombocytopenic epstein-barr virus in inflammatory diseases of the liver and liver allografts: an in situ hybridization study fulminant hepatitis due to epstein-barr virus infection the mucopolysaccharidoses: a clinical review and guide to management biochemical diagnosis of mucopolysaccharidoses: experience of diagnoses in a -year period ( - ) mucormycosis synonym: phycomycosis; zygomycosis. note: diseases that may be complicated by mucormycosis include bums,* diabetes mellitus,* leukemia,* lymphoma,* and tuberculosis cardiac involvement in mucopolysaccharidosis: effects of allogeneic bone marrow transplantation mitral and aortic regurgitation in patients with mucopolysaccharidosis toxic epidermal necrolysis possibly linked to hyperacute graft-versus-host disease after allogeneic bone marrow transplantation pulmonary complications in toxic epidermal necro-iysis: a prospective clinical study references possible or expected findings pyelonephritis;* nephrocalcinosis; granulomas; tumor infiltrates; manifestations of the conditions listed below under "other organs renal stones in patients with rheumatoid arthritis nephrolithiasis as a presenting feature ofchronic sarcoidosis: a prospective study nephrolithiasis and risk of hypertension posterior lens opacities; retinal hamartomas. peripheral neuropathy with focal schwannomatous changes or onion-bulb-like schwann cell or perineural cell proliferation neurofibromatosis type i. in: pathology and genetics of tumours of the nervous system neurofibromatosis type . in: pathology and genetics oftumours ofthe nervous system neurological complications involving the central nervous system in neurofibromatosis type i primary cutaneous nocardia asteroides infection after heart transplantation native valve endocarditis due to nocardia-like organisms hepatobiliary complications of total parenteral nutrition total parenteral nutrition: a histopathologic analysis of the liver changes in children obesity and breast cancer risk nonalcoholic steatohepatitis obesity cardiomyopathy: pathogenesis and pathophysiology bennett cpo pachydennoperiostitis in childhood unilateral hypertrophic osteoarthropathy associated with aortofemoral graft infection gastroesophageal reflux and esophagitis-associated hypertrophic osteoarthropathy hypertrophicosteoarthropathy caused by lipoid pneumonia achondroplasia" and dyschondroplasia, oilier's osteogenesis imperfecta synonyms and related terms: lobstein's syndrome osteogenesis imperfecta congenita; type i, ii, and iii; osteogenesis imperfecta cystica hypercalcemia in osteogenesis imperfecta treated with pamidronate gastrointestinal problems in patients who have type-iii osteogenesis imperfecta osteomalacia related terms: osteoporosis;* renal osteodystrophy; rickets. note: many possible causes of osteomalacia may not be apparent at autopsy, e.g., sodium fluoride or diphosphonate toxicity or use of anticonvulsant drugs osteomalacia associated with adult fanconi syndrome: clinical and diagnostic features epstein-barr virus in b-celllymphomas associated with chronic suppurative inflammation osteomyelitis and osteonecrosis in inflammatory bowel disease references hyperlipidemia; hyperuricemia. fracture or traumatic dislocation of hip see above under "note osteomyelitis and osteonecrosis in inflammatory bowel disease osteonecrosis and human immunodeficiency virus infection report of fifteen cases. therapeutic recommendations malignant infantile osteopetrosis: otolaryngological complications and management renal tubular acidosis and osteopetrosis with carbonic anhydrase ii deficiency: pathogenesis of impaired acidification spondylolysis in children who have osteopetrosis osteopetrosis with arnold chiari malformation type and brain stem compression osteoporosis and atherosclerosis in chronic renal failure endocrine disorders and osteoporosis otosclerosis: a measles virus associated inflammatory disease correlations between pathologic changes in the stapes and conductive hearing loss in otosclerosis the aetiology of otosclerosis: a review of the literature etiologic links between chronic pancreatitis and pancreatic cancer hyperparathyroidism and pancreatitis during pregnancy pancytopenia related terms: agranulocytosis;* aplastic anemia; fanconi's anemia.* note: some causes of pancytopenia such as adverse drug reactions (e.g., due to antimetabolites, sulfa drugs thrombotic thrombocytopenic purpura/hemolytic uremic syndrome secondary to pancreatitis retinopathy as asys-temic complication of acute pancreatitis references mesenteric panniculitis necrotizing pancreatitis. * vasculitis with thrombi; adrenocortical infarctions. vasculitis with thrombi and infarctions. relapsing polychondritis ( ). see also above under "possible associated conditions subcutaneous panniculitic t-cell lymphoma is a tumor of cytotoxic t lymphocytes abecassism. alpha i-antitrypsindefi-ciencyassociated panniculitis: resolution with intravenous alpha l-anti-trypsin administration and liver transplantation blind loop syndrome: an unusual cause of panniculitis. j am acad paracoccidioidomycosis synonyms: paracoccidioides brasiliensis infection cutaneous panniculitis and relapsing polychondritis: two cases normal subcutaneous fat, necrosis of adipocytes and classification of the panniculitides paraneoplastic pemphigus a case of pemphigus vulgaris with esophageal involvement rheumatoid constrictive pericarditis (clinical conference) benign paroxysmal (see "fever, familial mediterranean pneumatosis intestinalis in pediatric acquired immunodeficiency syndrome pneumatosis cystoides intestinalis: an unusual complication of systemic amyloidosis pneumatosis intestinalis in children with primary combined immunodeficiency pneumatosis cystoides intestinalis with abdominal free air in a -year-old girl after allogeneic bone marrow transplantation pneumatosis intestinalis: a review pneumatosis coli: a proposed pathogenesis based on study of cases and review of the literature collagenous inorganic dust pneumoconiosis, diffuse type: aluminosis; asbestosis; chronic pulmonary berylliosis; talcosis; collagenous inorganic dust pneumoconiosis, nodular type: silicosis; noncollagenous inorganic dust pneumoconiosis (includes mixed-dust fibrosis): baritosis; china clay pneumoconiosis; chromite pneumoconiosis; coal worker's pneumoconiosis; fuller's earth pneumoconiosis; hematite or magnetite miner's lung; siderosis or welder's lung; stannosis; organic dust pneumoconiosis: byssinosis. note: in addition to the aforementioned classic forms of pneumoconiosis, many other airborne substances have been impli-cated in recent years, for example, cerium uncommon causes ofoccupational interstitial lung diseases rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review diagnostic criteria for chronic beryl ium disease (cbd) based on the uk registry - pneumocystis carinii (see "infection, pneumocystis carinii this condition is diagnosed by inspection, palpation, and roentgenography. tension pneumomediastinum may autopsy evaluation of asbestos exposure: retrospective study of cases with quantitation of ferruginous bodies in digested lung tissue atypical mycobacteriosis as a complication of talc pneumoconiosis silicosis, mixed dust pneumoconiosis, and lung cancer complications associated with the use ofthe esophageal-tracheal combitube pneumomediastinum: an unusual complication of asthma in a young man spontaneous pneumomediastinum in a patient with bronchiolitis obliterans after bone marrow references i. katzenstein a-l, myers j. idiopathic pulmonary fibrosis: clinical relevance of pathologic classification update on lymphoid interstitial pneumonitis lymphocytic interstitial pneumonitis and nonspecific interstitial pneumonitis histologic diagnosis of extrinsic allergic alveolitis role of electron microscopy in interstitial lung disease detennination of arsenic in hair using neutron activation arsenic intoxication associated with tubulointerstitial nephritis atrioventricular block after administration of atropine in patients follow-ing cardiac transplantation death following intentional methyl bromide poisoning: toxicological data and literature review pulmonary edema, alveolar wall damage, and interstitial pneumonia after acute inhalation. severe pulmonary fibrosis may develop in chronic cases. gastroenteritis after nonlethal food poisoning. degeneration of proximal tubules and proteinuria in acute poisoning degenerative changes of liver and myocardium problems in the analysis of cadmium in autopsied tissues elevated urinary cadmium concentrations in a patient with acute cadmium pneumonitis cadmium-associated renal disease the effects of storage conditions on the stability of carbon monoxide in postmortem blood acute renal failure, poisoning, carbon tetrachloride synonym: tetrachloromethane poisoning. note: toxicologic sampling of body fluids and organs should be done routinely in all cases. in many instances, however, death occurs i wk to d after exposure, and by this time no carbon tetrachloride is demonstrable. death may be sudden compartment syndrome, and systemic capillary leak syndrome complicating carbon monoxide poisoning southern je acute hydrocephalus following carbon monoxide poisoning prevention of occupational cyanide exposure in autopsy prosectors gradwohl's legal medicine references possible or expected findings digoxin values in digitalis toxicity are greater than ng/ml ( ) digoxin concentration may be higher or lower than concentration in serum, depending on how long before death drug was taken (i) digoxin concentrations in postmortem specimens after overdose and therapeutic use a fluorimetric determination ofmyocardial digoxin at autopsy, with identification of digitalis leaf, digitoxin and gitonin ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis ethylene glycol poisoning: case report of a record-high level and a review lead concentrations in human plasma, urine and whole blood trace metals (lead and cadmium screening) an emg case report oflead neuropathy years after a gunshot injury aminoaciduria and glycosuria following severe childhood lead poisoning risk of nervous system cancer among workers exposed to lead demonstration of mercury in the human brain and other organs years after metallic mercury exposure spontaneous exfoliation of teeth following severe elemental mercury poisoning: case report and histological investigation for mechanism. oral surg oral med oral pathoi acute chemical pancreatitis associated with nonfatal strychnine poisoning homicide by strychnine poisoning coronary arteritis, with or without aneurysms and infarctions, primarily in childhood. myocardial hypertrophy secondary to hypertension. * minimal or no involvement by polyarteritis nodosa; considerable involvement in other types ofnecrotizing vasculitis (see "arteritis, all types or type unspecified") with or without formation of aneurysms and infarctions, may occur in all organs. the liver may show bile duct injury and rarely, nodular regenerative hyperplasia ( ) more frequently involved in giant cell elastic arteritis.* polyarteritis of small muscular arteries, including vasa nervorum. arthritis* may rarely be present with swollen joints. infarctions;* subarachnoid hemorrhage; arteritis of cerebral arteries. papillitis; retinal hemorrhages polyarteritis nodosa-like vasculitis in human immunodeficiency virus infection the coexistence of familial mediterranian fever and polyarteritis nodosa: report of a case microscopic polyarteritis during polymyalgia rheumatica remission development of polyarteritis nodosa in the course of inactive systemic lupus erythematosus bone marrow pathology in relapsing polychonditis: high frequency of myelodysplastic syndrome relapsing polychondritis associated with subclinical sjo-gren's syndrome and phlegmon of the neck giant cell arteritis and polymyalgia rheumatica posterior scleritis and polymyalgia rheumatica polymyalgia rheumatica in patients with ankylosing spondylitis: a report of cases inflammatory changes of hip synovial structures in polymyalgia rheumatica polymyositis (see "dermatomyositis polyneuritis (see "syndrome, guillain-barre familial, and related syndromes related terms: cowden's disease (multiple hamartoma syndrome); familial colonic polyposis gardner's syndrome; non-polyposis syndrome (hereditary nonpolyposis colorectal cancer syndrome) sampling for histologic study depends on expected findings or grossly identified abnormalities as listed in right-hand column. (see also below under "soft tissues syndrome; mucocutaneous pigmentations (buccal, perioral, priorbital, distal extremities) in peutz-jeghers syndrome;* papules in face and oral mucosa in cowden's disease; tumors of skin and subcutis (see below under "soft tissues gardner's syndrome. osteomas or exostoses (mandible, calvara desmoid in familial adenomatous polyposis malignant potential in intestinal juvenile polyposis syndromes ampullary carcinoma in familial adenomatous polyposis adenoma of the common bile duct in gardner's syndrome may cause relapsing acute pancreatitis orbital osteoma in gardner's syndrome myelopathylmyelitis," and "syndrome, guillain-barre.") references possible or expected findings hypertrichosis; erythrodontia; scarring and mutilation of hands and face. hemolytic anemia. erythrocytes contain large amounts of uroporphyrin i; nonnoblasts and reticulocytes exhibit intense red fluorescence uroporphyrin i and coproporphyrin i in high concentrations. splenomegaly (may have been treated by splenectomy) correction of congenital erythropoietic porphyria by bone marrow transplantation successful cord blood stem cell transplantation for congenital erythropoietic porphyria (gunther's disease) congenital erythropoietic porphyria associated with nephrotic syndrome atypical red cell inclusions in congenital erythropoietic porphyria possible associated conditions: adverse drug reaction; chronic alcoholism;* chronic hepatitis c ( ); human immunodeficiency virus infection porphyria cutanea tarda and human immunodeficiency virus: two cases associated with hepatitis c porphyria cutanea tarda and antibodies to hepatitis c virus an unhappy triad: hemochromatosis, porphyria cutanea tarda and hepatocel ularcarcinoma-a case report variegate porphyria possible associated conditions: ebstein's malformation of tricuspid valve. references possible or expected findings chronic eczematous skin lesions or superficial scarring; nail changes. perivascular pas-positive hyaline ( ) brown protoporphyrin deposits with red to yellow birefringence with a maltese cross configuration in hepatocytes, kupffer cells, and bile canaliculi erythropoietic protoporphyria cytofluorometry as a diagnosis of protoporphyria recessive inheritance of erythropoietic protoporphyria with liver failure pseudogout synonym: calcium pyrophosphate dihydrate (cppd) deposition disease puncture grossly affected joints and submit sample of synovial fluid for crystal analysis under compensated polarized light ( ) references possible or expected findings punctate calcium deposits in kneejoints and, less commonly, in joints of hips, ankles, shoulders, or wrists or in symphysis ossium pubis and intervertebral disks. arthritis* with synovitis. crystals of calcium pyrophosphate dihydrate in periarticular tissue ( ) and synovial fluid. cartilage contains calcium salts of pyrophosphate, hydroxyapatite, and orthophosphate. alkaptonuria;* gout calcium pyrophosphatedihydratedeposition disease presenting as tumoral calcinosis (periarticularpseudogout) calcifications in dermis; calcifications of blood vessels. diaphragmatic hernia.* hypertensive cardiomegaly. characteristic plaques in pericardium and endocardium, with or without mitral valve involvement. coronary atherosclerosis may have been a causeofangina( ).coronarythrombosisand myocardial infarction may be present also. accelerated atherosclerosis; calcification of peripheral vessels. gastrointestinal hemorrhage;* peptic ulcer degeneration of bruch's membrane with retinal hemorrhages; sclerosis of choroid vessels calcification of elastic fibers in pseudoxanthoma elasticum new report of severe coronary artery disease in an eighteen-year-old girl with pseudoxanthoma e asticum. case report and review of the literature hivrelated thrombotic thrombocytopenic purpura: report of cases and a review of the literature beham-pyelonephritis synonyms and related terms: acute ascending pyelone angiotropic large cell lymphoma presenting as thrombotic micro-angiopathy (thrombotic thrombocytopenic purpura) splenic pathology in thrombotic thrombocytopenic purpura emphysematous pyelonephritis in diabetic patients nephrobronchialfistulaandlungabscessresulting from nephrolithiasis and pyelonephritis a case ofan enterorenal pyothorax (see "empyema, pleurai.") fistula and pyelonephritis with airin renal pelvis systemic amyloidosis secondary to pyonephrosis. resolution after nephrectomy medicolegal investigation of death the laboratory's role in detecting sexual assault toluidine blue in the detection at autopsy of perineal and anal lacerations in victimes of sexual abuse sarcoidosis presenting as heart disease sarcoidosis and its ocular manifestations rheumatic features of sarcoidosis cutaneous sarcoidosis: differential diagnosis schistosoma mansoni and s. haematobium infections in egypt. i. evaluation of techniques for recovery of worms and eggs at necropsy. am i a quantitative post-mortem study of schistosomiasis mansoni in man immunodiagnosis of schistosomiasis. screen with fast-elisa and confinn with imrnunoblot hepatic connective tissue changes in hepatosplenic schistosomiasis schistosomiasis in women: manifestations in the upper reproductive tract sclerosis, systemic amyotrophic lateral (see "disease, motor neuron diffuse (see "sclerosis, schilder's cerebrai multiple synonyms and related terms: acute and subacute variants: acute multiple sclerosis (marburg type), acute necrotizing myelopathy; concentric sclerosis (ba type); concentric lacunar leukoencephalopathy; encephalitis periaxialis diffusa (schilder's type; see also next entry silicone ganuloma in acral skin in a patient with silicone-gel implants and systemic sclerosis organizing diffuse alveolar damage associated with progressive systemic sclerosis bayle , fiessinger in. brain involvement in scleroderma: two autopsy cases skeletal muscle pathology in systemic sclerosis mechanisms of scleroderma-induced lung disease thberous sclerosis complex and subependymal giant cell astrocytoma bilateral temporal arachnoid cysts associated with tuberous sclerosis poisoning, alkaloid accident, diving [skin or scuba scurvy (see "deficiency, vitamin c shigellosis (see "dysentery, bacillary shock related terms: anaphylactic shock; bacteremic shock; many others. note: see also under name of suspected underlying condition, such as "bums intestinal histological and ultrastructura inflammatorychanges in spondyloarthropathy and rheumatoid arthritis the sacroiliac joint in the spondyloarthropathies granulomatous ileitis in a patient with ankylosing spondylitis sporotrichosis synonym: sporothrix (sporotrichum) schenckii infection. note: ( ) mckiernan . partial lipodystrophy in coeliac disease coeliac disease and lymphoma: current status review: nonalcoholic steatohepatitis alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease non-alcoholic fatty liver disease in the metabolic syndrome the placenta in stillbirth estimating the time of death in stillborn fetuses: i. histologic examination of fetal organs: an autopsy study of stillborns estimating the time of death in stillborn fetuses: ii. histologic evaluation ofthe placenta; a study of stillborns estimating the time ofdeath in stillborn fetuses: iii. external fetal examination; a study of stillborns fat distribution in the adrenal cortex as an indication of the mode of intrauterine death stimulant(s) (see "dependence, drug(s), all types or type undetermined cutaneous manifestations of the acquired immunodeficiency syndrome aids: cardiac findings from autopsies the liver in hiv infection neuropathology of the spinal cord in the acquired immunodeficiency syndrome postmortem enzyme immunoassay for human immunodeficiency virus bk virus-associated renal failure among hiv patients - or write to american registry ofpathology sales office pathology of acquired immunodeficiency syndrome (aids) in children thymus involution in the acquired immunodeficiency syndrome micrencephaly in children congenitally infected with human immunodeficiency virus-a gross-anatomical morphometric study a rapid method for detecting the predominant ashkenazi jewish mutation in the bloom's syndrome gene bloom syndrome: multiple retinopathies in a chromosome breakage disorder severe eczema in a patient with di-george's syndrome digeorge's syndrome orthe iii -iv pharyngeal pouch syndrome: pathology and a theory of pathogenesis basal ganglia calcification and psychosis in q . deletion syndrome down's synonyms and related terms: mongolism possible associated conditions: acute lymphocytic, my references epicanthal folds; cleft lip/palate; high arched palate; furrowed tongue; rhagades. depressed nasal bridge small, broad or fiat nose; slanted palpebral fissures; fiat occiput; brachycephaly; palmar "simian crease"; short fifth middle finger ventricular septal defect;* complete atrioventricular septal defect* ( ). duodenal stenosis and atresia; imperforate anus. microcephaly; poorly developed secondary gyri hypoplastic brain stem, medulla and cerebellum; polymicrogyria; neurofibrillary tangles; meningomyelocoele. acute lymphocytic leukemia; acute myelocytic leukemia; acute megakaryocytic leukemia cardiovascular disease in down syndrome possible or expected findings hyperelasticity of skin, bruises or scars, hemorrhages, and hyperpigmentation. lipomatous pseudotumors that may be calcified or ossified. normal or abnormal amounts and fragmentation of elastic tissue. abnormalities of collagen. dislocation of hip, shoulder, patella, radius, or clavicle. loose-end clavicles; spondylolisthesis mitral valve prolapse; aortic insufficiency.* aortic dissection, aneurysm, ectasia, occlusion ( ). various congenital anomalies. congenital anomalies of gastrointestinal and genitourinary tracts. bleeding from various organ sites vascular ehlers-danlos syndrome: imaging findings note: this syndrome belongs to a large family (with more photograph abnormal features as listed in right-hand column. record appearance of external genitalia. prepare skeletal roentgenograms. procure long bones and vertebral bodies short-limb dwarfism* with narrowing of the rib cage; polydactyly; dysplasia of fingernails; thin and sparse hair; premature eruption of teeth; defective dentition; eye abnormalities; upper lip bound down by multiple frenula. cryptorchidism; epispadias; hypospadias. chondrodysplasia with acromelic micromelia (shortening of the distal segment of the limb); fusion of capitate and hamate bones of wrist; defects of lateral aspect of proximal tibia see lesions listed above under "external examination empty sella synonyms and related terms: primary empty sella syndrome; secondary empty sella syndrome (e.g., after surgical removal or spontaneous infarction of pituitary adenoma) chorea, philic pneumonia: histopathologic features in nine cases. am rev resp hereditary ascariasis and hookworm chronic eosinophilic pneupophosphatemic vitamin d-resistant rickets; galactosemia;transport defect; tyrosinemia. * excludes fan- weight and external measurements; record and photograph abnormalities as listed in right-hand column. prepare skeletal roentgenograms. radiographs of long bones. submit for tissue culture for possible enzyme analysis. possible or expected findings redlblond hair, fair skin (diminished pigmentation); dehydration;* stigmata of hypothyroidism;* delay of sexual maturation. rickets; osteomalacia.* positive rheumatoid factor. various types of pneumonia after splenectomy, presence of accessory spleen may account for treatment failure. manifestations of portal hypertension.* lymphadenopathy of mediastinal and paraaortic lymph nodes. anemia;* neutropenia sinusoidal lymphocytosis of the liver in felty's syndrome with a review of the liver involvement in felty's syndrome splenic involvement in rheumatic diseases felty's and pseudo-felty's syndrome nodular regenerative hyperplasia of the liver in rheu-matic diseases: report of seven cases and review of the literature fetal alcohol syndrome: craniofacial and central nervous system manifestations goodpasture's syndrome anti-glomerular basement membrane glomerulonephritis: a morphologic study of cases gronblad-strandberg (see "pseudoxanthoma elasticum acute inflammatory polyradiculoneuropathy; guillain-barre-strohl syndrome hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: pathophysiology and treatment cyclosporin-induced hemolytic uremic syndrome: factors that obscure its diagnosis enterohemorrhagic escherichia coli :h : an emerging pathogen hemolytic uremic syndrome as a presenting form of hiv infection hemolytic uremic syndrome in prostatic carcinoma diabetes secondary to genetic disorders. baillieres suprasellar tumors of maldevelopmental origin in klinefelter's syndrome. a report of two cases klippel-feu synonym: congenital fusion of cervical vertebrae primary yolk sac tumor of the prostate in a patient with klinefelter's syndrome extragonodal germ cell tumors are often associated with klinefelter syndrome organs and tissues procedures possible or expected findings external examination prepare roentgenograms of chest, neck, and head skull, spine, brain, and spinal cord myasthenia gravis disorders with dysraphia (see below), fusion of cervical vertebrae. congenital elevation of the scapula (sprengel's deformity) chiari malformation;* basilar impression;* meningomyelocele; platybasia;* spinal cord compression; weight and length; record and photograph abnormalities as listed in right-hand column. record weight and sample for histologic study. if renal transplantation ( ) had been carried out, see also under that heading. follow procedures described under "glomerulonephritis retinal dystrophy ( ) and other retinal changes the cardinal manifestations of bardet-biedl syndrome, a form of laurence-moon-biedl syndrome laurence-moon-biedl syndrome accompanied by congenital hepatic fibrosis pediatric renal transplantation in laurence-moon-biedl syn-drome possible or expected findings typical skeletal proportions. arachnodactyly; pectus excavatum genu recurvatum; dislocation of joints diaphragmatic hernia. * infective endocarditis. * atrial septal defect. * myxomatous transformation of mitral ring. mitral valve prolapse. aortic and pulmonary dilatation and valvular insufficiency.* aortic dissection* with dissection of adjacent vessels. ascending aortic aneurysm multiple cysts (see "cyst(s), pulmonary") aortopulmonary fistula: an uncommon complication in dystrophic aortic aneurysm peripartum acute myocardial infarction in marfan's syndrome aneurysm of the cervical internal carotid artery associated with marfan's syndrome--ease report noonan's syndrome in association with acute leukemia lymphatic dysplasia in a neonate with noonan's syndrome noonan syndrome: a clinical description emphasizing the cardiac findings cerebral arteriovenous malformation in noonan's syndrome clinical variability in a noonan syndrome family with a new ptpnii gene mutation peutz-jeghers syndrome bilateral large-cell calcifying sertoli cell tumor of the testes in peutz-jeghers syndrome: a case report ovarian tumors associated with the peutz-jeghers syndrome robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome g -gter congenital heart disease in the pierre robin syndrome glossoptosis-apnea syndrome chiari i malformation, caudal regression syndrome, and pierre robin syndrome: previously unreported combination risk factors for squamous cell carcinoma of the esophagus heterotaxy syndrome; ivemark's syndrome possible associated conditions: with asplenia: right isomerism (bilateral mirror-image right-sided symmetry) of heart, lungs, and abdominal viscera; common atrium; total anomalous pulmonary venous connection; absent coronary sinus; complete atrioventricular septal defect;* subpulmonary stenosis;* pulmonary valve atresia;* midline symmetric liver; malrotation of bowel; absent spleen. with polysplenia: left isomerism (bilateral mirror-image left-sided symmetry) ofheart and lungs, with variable sidedness ofabdominal viscera primary immunodeficiencies reiter's syndrome-like patternin aids-associated psoriasiformdermatitis reference pneumothorax;* pneumomediastinum;* pneumoperitoneum. septicemia. right ventricular hypertrophy and/or dilatation. intubation trauma and mural edema and hemorrhage in trachea; hyaline membrane disease intubation trauma. hemorrhages or other evidence of birth trauma; germinal matrix hemorrhages in premature infants; infarctions of subcortical white matter in term infants coalson . pathology of bronchopulmonary dysplasia pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia inborn errors of metabolism and reye's syndrome: differential diagnosis prevalence and non-specificity of microvesicular fatty changes liver histopathology in clinical reye syndrome ultrastructural study of intranuclear inclusions in the exo-crine pancreas in reye's syndrome neuropathologic findings in reye syndrome sanfilippo's (see "mucopolysaccharidosis scheie's (see "mucopolysaccharidosis segawa's (see "syndrome, dystonia severe acute respiratory (sars) note: this highly contagious illness, caused by a coronavirus (sars-cov)details, see biosafety level laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects pathology and pathogenesis of severe acute respiratory syndrome pulmonary involvement in primary sjogren's syndrome polymyositis and sjogren's syndrome associated with bronchiolitis obliterans organizing pneumonia the gastrointestinal manifestations of sjogren's syndrome clinicopathological factors relating malignant lymphoma with sjogren's syndrome acute transverse myelopathy and cutaneous vasculopathy in primary sjogren's syndrome primary localized cutaneous amyloidosis with unusual clinical features in a patient with sjogren's syndrome disease, parkinson's erythema multiforme man related terms: armadillo disease; continuous muscle fiber activity; neuromyotonia autoimmune central nervous system paraneoplastic disorders: mechanisms, diagnosis, and therapeutic options sudden infant death (sids) (see "death, sudden unexpected, of infant.") syndrome, superior vena cava related term: superior vena cava obstruction continue dissection of veins into neck and axillas. record and photograph site of thrombosis or of compression by surrounding pathologic conditions. submit samples for histologic study. benign or malignant tumors; fibrosing mediastinitis;* postradiation fibrosis; infectious disease (tuberculosis,* histoplasmosis*); thoracic aortic aneurysm;* chronic constrictive pericarditis;* chest trauma; arteriovenous fistula between ascending aorta and superior vena cava toxic shock synonyms and related terms: staphylococcal scarlet fever. note: ( ) collect all tissues this is a reportable disease. premature aging; increased number of pigmented nevi; infantile sex organs; webbed neck; broad chest with wide spacing of nipples. short fourth metacarpal; abnormal epiphyseal fusions; osteochondrosis-like changes of spine bicuspid aortic valve;* coarctation of aorta. * rarely other anomalies ( ) decreased/absent follicles. intestinal telangiectases. biliary atresia. * horseshoe kidney; double ureters. streak gonads without germ cells or follicles hashimoto's thyroiditis. * manifestations of diabetes mellitus,* hypertension,* or thyrotoxicosis. neuroblastoma and related tumors ( ). keratoconus acute aortic dissection, aortic insufficiency, and a single coronary artery in a patient with turner's syndrome neuroblastoma and related tumors in turner's syndrome turner's syndrome associated with bilateral retinal detachments weil's (see "leptospirosis related terms: alcoholic wernicke's encephalopathy; korsakoff's psychosis; nonalcoholic wernicke's encephalopathy ( , ); and specimen preparation, see chapter . for selection of histologic samples, see right-hand column melissas . wernicke's encephalopathy after vertical banded gastroplasty for morbid obesity syndrome, respiratory distress, of infant.") syndrome, wiskott-aldrich (see "syndrome, primary immunodeficiency malformation, ebstein's" and "preexcitation, ventricular alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease cerebro-hepato-renal syndrome; infantile refsum's disease. * note: this congenital familial cholestatic syndrome results from impaired assembly of peroxisomes and has its main manifestations in the brain, liver, and kidneys postmortem findings and prenatal diagnosis ofzellwegersyndrome. case report alcoholism and alcohol intoxication" and "disease, alcoholic liver possible associated condition: multiple endocrine neoplasia primary cardiac gastrinoma causing zollinger-ellison syndrome localization, syphilis, acquired synonym: treponema pallidum infection. note: congenital syphilis is presented below under a separate heading and surgical management of gastrinoma fundic argyrophil carcinoid tumor in a patient with sporadic-type zollinger-ellison syndrome nonimmune hydrops fetalis due to congenital syphilis associated with negative intrapartum mater-nal serology screening syringomyelia synonyms and related term: hydromyelia; idiopathic syringomyelia; secondary syringomyelia; syringobulbia. possible associated conditions: with idiopathic syringomyelia-arnold chiari malformation, type i;* basilar impression;* . oppenheimer eh, dahms bb. congenital syphilis in the fetus and neonate congenital syphilis: detection of treponema pallidum in stillborns with secondary syringomyelia-intramedullary gliomas (ependymoma, pilocytic astrocytoma) and vascular tumors; spinal arachnoiditis and pachymeningitislisted in right-hand column. prepare roentgenograms of spine and joints. for removal and specimen preparation, see chapter . for histologic sampling, see right-hand column. hypertrophy of body parts. muscle atrophy of upper extremities and hands cervical spinal cord is swollen and tense, with cavitation (syrinx) containing clear fluid. wall of cavity consists of degenerated glial and neural elements, with marked gliosis. spinal cord parenchyma is markedly compressed. see also above under hydrops fetalis caused by alpha-thalassemia: an emerging health care problem limb defects in homozygous alpha-thalassemia: report of three cases poisoning, thallium thirst (see "dehydration thromboangiitis obliterans (see "disease, buerger's purpura, thrombotic thrombocytopenic" and "syndrome, hemolytic uremic the pathological manifestations of pulmonary toxoplasmosis in the acquired immunodeficiency syndrome a spectrum in the pathology of toxoplasmosis in patients with acquired immu transfusion (see "reaction to transfusion bone marrow note: if the patient had symptoms of acute or chronic graft-versus-host disease (gvhd), see "disease, graftversus-host morphology and diagnostics of human toxoplasmosis fatal myocardial coinfection by toxoplasma gondii and parvovirus b in an hiv patient if there is evidence of infection, submit material for microbiologic studies. if there is evidence of recurrent leukemia or lymphoma, sample as described under those headings. if the patient had symptoms of thrombotic thrombocytopenic purpura (tip) or hemolytic uremic syndrome (hus), see these headings. record average size. fix specimens in b-plus®. make touch preparations. request giemsa or wright stain. for preparation of sections and smears (imprints), see chapter . manifestations of graft-versus-host disease (e.g., scleroderma-like changes).* septicemia. interstitial pneumonia* and cytomegalovirus infection* or human herpesvirus infection ( ). bacterial, fungal bacterial or fungal infections in gvhd.* other manifestations of gvhd. * recurrent leukemia,* lymphoma* (including posttransplant, epstein-barr virus associated lymphoproliferative disorder). recurrent solid tumor, e.g., carcinoma of breast, lung (small cell carcinoma), ovary lymphomatous or leukemic infiltrates myeloid cells may be absent in marrow graft rejection or nonimmunologic marrow graft failure. references hematomas; hemorrhagic necroses; infarcts; bacterial or fungal infections leukoencephal-opathy; vascular siderocalcinosis; neuro-axonal spheroids ( ) human herpesvirus infection and associated pathogenesis following bone marrow transplantation pulmonary veno-occlusive disease in an adult following bone marrow transplantation. case report and review of the literature acute renal failure following bone mar-row transplantation transplantation-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome thrombotic microangiopathies associated with drugs and bone marrow transplantation record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record weight, ventricular thickness, and valve circumferences. take multiple sections from all areas of myocardium. cut coronary arteries in cross sections; request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. left ventricular hypertrophy, from cyclosporine-related hypertension. acute transplant rejection (for grading, see ref. ). chronic transplant vasculopathy with coronary artery stenosis ( ) opportunistic infection ( ). pneumonia a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group autopsy findings in cardiac transplant patients: a lo-year experience fulminant toxoplasmosis following heart transplantation banff schema for grading liver allograft rejection: an international consensus document record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record lung weights separately. if lung infection is suspected, submit material for microbiologic possible or expected findings cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. opportunistic infection (in patients with a single lung transplant, infections may involve references the nontransplant lung, as well). chronic bronchitis* and bronchiectasis.* acute rejection (rare); chronic airway rejection (obliterative bronchiolitis); chronic vascular rejection. (for grading of rejection, see ref .) post-transplant lymphoprolijerative disorder a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group recurrent salmonella enteritidis and hepatic tuberculosis tularemia synonyms and related terms: francisella tularensis infection; pasteurella tularensis infection cerebral abscesses complicating tularemia meningitis endocrine (see "neoplasia, multiple endocrine any type note: ifthe tumor had been treated by surgery, irradiation, chemotherapy, or other means (the most common situation possible associated conditions: with adrenocortical adenoma-conn's syndrome with adrenocortical carci-noma-cushing's syndrome;* hypoglycemia;* virilization. with pheochromocytoma-cerebellarhemangioblastoma ery-throcytosis; hypercalcemia; hypertension,* multiple enarteries record body weight and abnormal features. photograph skin changes. record heart weight and thickness of ventricles. procure multiple sections of myocardium possible associated conditions.") focal myocarditis. * hypertensive cardiovascular disease (see "hypertension myocardial infarction without severe coronary artery disease (with pheochromocytoma) asystematicreviewoftheassociationbetween barrett's esophagus and colon neoplasm malakoplakia and colorectal adenocarcinoma an unusual case of colonic mixed adenoendocrine carcinoma: collision vs composite tumor. a case report and review of the literature submit lymph nodes for histologic study. remove neck organs with tongue together with esophagus. open pharynx and esophagus in posterior midline. if fistulas and abscesses are suspected, dissect esophagus but leave attached to mediastinum, stomach and diaphragm. record width of lumen of esophagus at various levels. photograph and record size and location of tumor; sample tumor and uninvolved esophagus for histologic study. request pas stain of uninvolved esophagus (sample from all levels). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. eczematoid dermatitis with adult renal cell carcinoma. cushing's syndrome,* galactorrhea or feminization or masculinization in some cases of renal cell carcinoma. evidence of hyperalcemia. pulmonary tumor embolism after invasion of renal vein and inferior vena cava. acquired renal cystic disease prolactin, renin, and prostaglandins in some renal cell carcinomas. see above under "possible associated conditions wilms tumor associated with polycythemia: case report and review of the literature renal medullary carcinoma: a potential sickle cell nephropathy of children and adolescents acquired cystic kidney disease byler's disease, carcinoid syndrome,*cirrhosis* of any type (mostly nonbiliary), congenital hepatic fibrosis, * cushing's syndrome,* erythrocytosis, genetic hemochromatosis, * glycogen storage disease (type ),* hepatitis b or c virus infection, hereditary tyrosinemia (type i),* hypercalcemia, hypercholesterolemia, hypoglycemia,* neurofibromatosis,* osler-rendu-weber disease* (hereditary hemorrhagic telangiectasia), polycythemia,* porphyria (acute intermittent and porphyria cutanea tarda), * pseudohyperparathyroidism,* and thorium (thorotrast) deposition. with bile duct carcinoma (cholangiocarcinoma}-clonorchis sinensis or opisthorchis viverrini infection, fibropolycystic liver and biliary tract disease,* hepatolithiasis, hypercalcemia, pri-mary sclerosing cholangitis,* and thorium (thorotrast) deposition. with hepatoblastoma-alpha-fetoproteinemia, cardiac and renal malformations, cleft palate, diaphragmatic hernia,* down's syndrome,* familial colonic polyposis,* hemihypertrophy, nephroblastoma. with angiosarcoma-thorium (thorotrast) deposition. see also below under "possible or expected findings describe and photograph cut surfaces. sample tumor and nonneoplastic liver for histologic study. if thorium deposition is suspected, prepare roentgenograms of liver slices and submit samples for energy-dispersive x-ray microanalysis of paraffin sections. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. cachexia. precocious puberty. feminization and gynecomastia in rare cases of hcc. spider angiomas; clubbing of fingers. see above under "possible associated conditions thorotrast storage may cause cirrhosis, hcc, bile duct carcinoma, or angiosarcoma. see above under "possible associated conditions tumor of the lung or bronchus possible associated conditions: abnormal concentrations of hormons or other metabolites in blood and tumor tissue (adrenocorticotropic, antidiuretic, growth hormone, parathyroid-like substances, or -hydroxyindolacetic acid); acanthosis nigricans s syndrome;*dermal hyperpigmentation; feminization; hyperglycemia; hypercalcemia; hypoglycemia;* hypokalemia; hyponatremia; precocious puberty. for syndromes affecting the brain, peripheral nerves or muscles, see below under "possible or expected findings organs and tissues procedures possible or expected findings external examination and skin record body weight. record abnormal features as listed in right-hand column; prepare photographs. prepare histologic sections of normal and grossly abnormal skin clubbing of fingers; spider angiomas. for other rare tumor-related changes, see above under "possible associated conditions thmor of the pancreas possible associated conditions: with carcinoma of the exocrine pancreas--cushing's syndrome;* diabetes mellitus* (rare); hypercalcemia; hyperglycemia with islet cell tumor-abnormal concentrations of hormons in blood and tumor tissue (see below under "pancreas"); features. dermal hyperpigmentation. for other rare tumor-related changes, see above under "possible associated conditions biliary obstruction caused by tumor in head of pancreas. islet cell tumor with adrenocorticotropic hormone, gastrin, glucagon, insulin, or other peptide hormones. see above under "possible associated conditions paraneoplastic pemphigus associated with pancreatic carcinoma pancreatic cystadenocarcinoma in peutz-jeghers syndrome testes may have been surgically removed to achieve androgen deprivation. urinary obstruction and hydronephrosis. * osteoblastic metastases hemolytic uremic syndrome in prostatic carcinoma thmor of the small intestine possible associated conditions: acquired immunode oncogenic osteomalacia associated with prostatic cancer gardner's syndrome) submit sample of non-neoplastic small bowel for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above. cachexia. mucocutaneous pigmentations associated with peutz-jeghers syndrome. * carcinoid tumor. lymphoma (see also above under "possible associated conditions"). familial polyposis or related syndrome. * celiac sprue. * crohn's disease. * see above under "possible associated conditions kasabach merritt syndrome in infants paraneoplastic hepatopathy associated with soft tissue sarcoma neurofibro-thmor of the spinal cord possible associated conditions: with angioma-cerebellar hemangioblastoma; segmental cutaneous vascular nevi; with matosis in children with soft tissue sarcoma tissues record abnormal features; photograph and submit nevi for histologic study. for removal and specimen preparation, see chapter . see also below under "vertebral column bone erosion and calcification of spinal canal. vertebral hemangiomas may be associated with arteriovenous malformation of spinal cord. manifestations of von hippel-lindau disease. * thmor of the stomach possible associated conditions: hypoglycemia;* megaloblastic anemia;* skin changes (as listed under "possible or expected findings)weight. record abnormal features; photograph and submit samples of normal and abnormal skin for histologic study. possible or expected findings cachexia; lower leg lymphoedema ( ) metastases common in liver, retroperitoneal and supraclavicular lymph nodes (virchow's node), ovaries (krukenberg tumor), and peritoneal cui de sac (blumer's shelf') carcinoma of the stomach with hyperkeratosis palmaris and plantaris and acanthosis of the esophagus gastric lymphoma ofmucosa-associated lymphoid tissue and helicobacter pylori gastric signet-ring cell carcinoma: unilateral lower extremity lymphoedema as the presenting feature thmor of the testis possible associated conditions: demyelinating neuropathy record location, size, and weight of both testes and of testicular tumor. submit samples of tumor and of ininvolved testis and epididymis for histologic study. snap-freeze tumor tissue for hormone assay. possible or expected findings cachexia. cryptorchism; hypospadia. gynecomastia. increased concentrations of alphafetoprotein and human chorionic gonadotropin testicular microlithiasis. secondary testicular tumors (metastases to the testes) are rare, except in association with leukemia* in children. references pulmonari embolism.* paraneoplastic diseases as listed above under "possible associated conditions chronic inflammatory demyelinating polyneuropathy (clop) associated with seminoma dermatomyositis associated with intratubular germ cell tumor and metastatic germ cell cancer testicular cancer in association with developmental renal anomalies and hypospadias submit samples of lymph nodes, spleen, peyer's plaques, and bone marrow for histologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. dermal hyperpigmentation. pemphigus foliaceus (rare). hypogammaglobulinemia and other immunoglobulin abnormalities. usually, thymoma presents as an infiltrating, anterior mediastinal mass that rarely metastasizes. carcinoid tumor, malignant lymphoma* (hodgkin's disease), and metastases from carcinomaofthe breast* and other tumors also may occur in this location herpes simplex*) and fungal infections (e.g., candidiasis*) due to thymoma-related immunodeficiency ( ). manifestations of paraneoplastic diseases and conditions as listed above under "possible associated conditions thrombotic thrombocytopenic purpura accompanied by transient pure red cell aplasia and thymoma glomerulonephritis associated with myasthenia gravis thymoma and cellular immune deficiency in an adolescent possible associated conditions: with papillary carcinoma-familial adenomatous polyposis thyroid carcinoma associated with familial adenomatous polyposis an association between acromegaly and thyroid carcinoma thyroid carcinoma causing fatal laryngeal obstruction prostate cancer metastasis to thyroid gland schistosomiasis and the risk of bladder cancer in human papilloma virus infection ( ) or herpesvirus infection ( ) with invasive cervical carcinoma. erythropoietin may be found in some tumors. thrombosis. * myopathy* may be associated with carcinoma of the cervix. cerebellar cortical degeneration* may be associated with carcinoma of the uterus. manifestations of uremia (see "failure, kidney") invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients association of risk factors for cervical cancer and human papilloma viruses in invasive cervical cancer association of herpesvirus infection with the development of genital cancer tyrosinemia type ii: a challenge for ophthalmologists and dermatologists hereditary tyrosinemia type i (chronic form): pathologic findings in the liver richner-hanhart syndrome (tyrosinemia ii): early diagnosis of an incomplete presentation with unusual findings acute pancreatitis-associated acute gastrointestinal mucosal lesions: incidence, characteristics, and clinical significance uncinariasis (see "ancylostomiasis uremia (see "failure, kidney from: handbook of autopsy practice urticaria pigmentosa of childhood (see ''mastocytosis, systemic acute aortic dissection;* aneurysma(s) of cerebral arteries; aortic insufficiency;* calcific aortic stenosis,* coarctation of the aorta;* infective endocarditis;* shone's syndrome; turner's syndrome. * organs and tissues procedures possible or expected findings heart if infective endocarditis is suspected, see chapter . open heart in cross-sections (see chapter ). photograph aortic valve and test valvular competence note: reye's syndrome* is a possible postviral complication of varicella that must be distinguished from varicella encephalitis. varicella may also cause exacerbation of tuberculosis.* (i) collect all tissues that appear infected bicuspid aortic valves are associated with aortic dilatation out of proportion to coexistent valvular lesions varicella-zoster virus infection in children with underlying immunodeficiency virus infection group a beta-hemolytic streptococcal epiglottitis as a complication of varicella infection optic neuritis: a rare complication of primary varicella infection hsv-l-induced acute retinal necrosis syndrome presenting with severe inflammatory orbitopathy, proptosis, and optic nerve involvement varicella with acute motor axonal neuropathy descending necrotizing mediastinitis in a child with chicken pox double outlet right ventricle with intact ventricular septum respiratory syncytial (see "pneumonia, all types or type unspecified vitamin a (see ''deficiency, vitamin a" and "hypervitaminosis a vitamin bt (thiamine) (see "syndrome, wernicke-korsakott vitamin bt (see "anemia, megaloblastic deficiency deficiency, vitamin d" and "hypervitaminosis d waldenstrom's macroglobulinemia (see ''macroglobulinemia, waldenstrom's.) waterhouse-friderichsen syndrome (see "disease, meningococcal wegener's granulomatosis (see "granulomatosis, wegener's werdnig-hottman disease (see "disease, motor neuron acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using -methylumbelliferyl palmitate which may be secondarily infected by bacteria. dark field microscopy will identify the organism expected findings mucosal ulcers, resembling those of typhoid fever, primarily of distal ileum and colon. villous shortening, crypt hyperplasia with mucosal microabscesses. microabscesses in regional mesenteric lymph nodes zellweger's syndrome (see "syndrome mucormycosis" and procedures under "diabetes mellitus abscesses and granulomas may occur in all organs and tissues. granulomatous lesions in central nervous system ( ) and eyes ( ). fungal osteomyelitis* that may be multifocal, including sites such as metacarpals and metatarsals. external examination other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. osteosclerosis of vertebrae, ribs, clavicles, pelvic bones, scapulae, skull, and metaphyseal ends of femur and humerus. osteomyelofibrosis or osteoreticulosis; rarely, panhyperplasia of bone marrow; increase of megakaryocytes.changes associated with chronic granulocytic leukemia* or with polycythemia vera* may imitate idiopathic myelofibrosis. widespread extramedullary hematopoiesis ( ) with splenomegaly. infectious diseases, including tuberculosis;* gouty arthritis; ascites and other manifestations of portal hypertension;* or hepatic vein thrombosis (budd-chiari syndrome*). cardiac tamponade ( ) . synonyms and related terms: hepatic porphyria; protoporphyrinogen oxidase deficiency ( ) .note: the manifestations of the disease closely resemble those in porphyria cutanea tarda and hereditary coproporphyria. measurements of porphyrins and porphyrin precursors are the only clearly distinguishing features. for autopsy procedures, see under "porphyria cutanea tarda." fibrin and platelet thrombi, with or without microaneurysms and purpura in kidneys, adrenal glands, pancreas, heart, and brain; lesions may also be present in liver; spleen ( ), lymph nodes, muscle, bone marrow, and synovium. fibrin thrombi can be demonstrated with labeled antihuman fibrin antibodies. lesions in precapillary arterioles. hypertrophic osteoarthropathy* (with pleural mesothelioma).pleural effusions. * asbestosis may be complicated by pleural mesothelioma. other organs and tissue sample bladder tumor and uninvolved urinary bladder for histologic study.cachexia. hydronephrosis* (obstructive uropathy) and hydroureter, usually caused by distal ureteral obstruction. recurrent nephrolithiasis* or pyelitis may have been present and is a risk factor for urinary bladder carcinoma.chronic urocystitis; infestation with schistosoma haematobium ( ) (uncommon in north america). manifestations of uremia (see "failure, kidney"). key: cord- - d eq y authors: nan title: espr date: - - journal: pediatr radiol doi: . /s - - - sha: doc_id: cord_uid: d eq y nan prof. michael riccabona undertook his medical school & university training at the university of innsbruck, tirol, austria, completing his md at karl franzens university in graz, austria. following an internship in neurology, surgery and internal medicine he then specialized in paediatrics at the dept. of paediatrics, university hospital in graz. there he took charge of the paediatric radiology and sonography sections at university hospital in graz, as associate professor of paediatrics. in he additionally started to specialise in radiology, becoming associate professor of radiology in -then taking charge of the subsection of paediatric sonography at the dept. of radiology, university hospital in graz, where in march he was appointed full univ. prof at the medical university graz, austria. he has a distinguished academic career and written over papers, more than chapters and several textbooks, and is a very popular international speaker, delivering numerous lectures at many high profile scientific meetings. he is an active member of several reputable international societies, has been chair of the paediatric ultrasound section of the austrian ultrasound society since , and president of the society of german speaking paediatric radiologists ( ) ( ) ( ) ( ) ( ) ( ) ( ) . he is a constant source of inspiration within his subspecialist areas of interest in ultrasound and abdominal radiology in children. he has been course director at several important meetings and served as president of espr in graz in and as lead of the paediatric subcommittee at ecr in . he has provided inspirational leadership as chair of the espr task force on uroradiology since writing state of the art guidelines and procedural recommendations to facilitate standardised best practice for imaging within paediatric uroradiology. he has been a reviewer for many international journals. he has on-going active roles in postgraduate education for medical colleagues from eastern europe, including basic ultrasound education and refresher courses, and workshops. michael riccabona is very deserving of honorary membership of espr, which reflects his seminal role within paediatric radiology in europe and his tireless dedication working for the good of children. & to discuss how to optimally adapt the various imaging techniques minimising radiation exposure and risks during diagnostic imaging in children. & to consider common restrictions, challenges, and possible solutions in paediatric radiology within the different settings in different countries, regions, continents and clinical scenarios -discussing all these aspects with colleagues, and to mingle with experts from all over the world learning from each other and fostering networking in paediatric radiology to try to grant optimal imaging for all children. an application for the rd annual meeting as well as for the th post graduate course has been submitted to the eaccme® for cme accreditation of this event. the eaccme is an institution of the uems (www.uems.net). the number of cme points will be announced at the espr congress website. each medical specialist should only claim those hours of credit that he/she actually spent in the educational activity. certificates of attendance will be available in the espr myuserarea after the meeting. the answers right away is: yes we can and we constantly have to. it is part of human nature to recognize problems and to find solutions. we define ideals but we also face reality. being aware of the gap in between we are constantly driven to improve. this overview will highlight some milestones and disputes throughout the years of development in the use of plain x-ray imaging. it will hint on scientific literature and sources of information. and it will hint on some swiss contributions as the espr meeting will be held in davos, switzerland. fighting the glow not the fire - years of x-ray imaging development and improvement: in the beginning of the clinical use of x-ray imaging, there was great enthusiasm in its potential without knowing the unfavorable dangers of uncontrolled use of x-ray. the dangers were recognized and the 'beast' was tamed and domesticated. in respect to radiation protection, the most significant achievements took place in the first half of that development. throughout the recent decades, some further considerable steps in dose reduction took place mainly by the improvement of film-screen systems and the recent introduction of computed (cr) and digitized radiography (dr). even if the early computerized x-ray imaging brought a slight increase in patient doses (which was overcompensated later on by direct digital radiography) a whole new world of further advantages launched the digital era in which we live today. as we all know new dangers arose with these techniques as the uncontrolled distribution of images and thereby confidential patient data over hospital departments and across borders throughout. also, the risk of an evitable overexposure in digital radiography is a significant issue. throughout the process of taming the radiation and controlling it, today's doses are attained within the lowest range of the danger scale. this range still is perceived as a black box within which we do not exactly know which concept reflects the potential harm best. the linear no-threshold model [lnt] is acknowledged as the concept which most reliably supports the idea of radiation protection. other concepts partly oppose the linear idea and question the relevance of that dose range because there lie so much greater health benefits in the appropriate use of diagnostic x-rays. several scientists even propagate the idea that very low levels are producing health benefits instead of physical harm [hormesis model]. nevertheless, the lnt model is widely accepted as the most helpful in the context of diagnostic radiology. some recent studies were able to support the idea of potential harm at very low dose levels as they were able to prove the induction of attributable cancers in the pediatric age group. so today we are fighting the glow, not the fire. as trained medical professionals we are fully aware of the fact that there is only little potential harm to the patient by using x-rays in the current state of the art. but on the other hand, we also have to be aware of the fact that our patients and their parents still fear the fire. one of our main tasks, therefore, is to explain the risks and benefits to the patients and their advocates and to educate the public. developed straight from the first radiographic technique's digital radiography today is state-of-the-art in plain d-imaging. throughout the last decade, it has replaced cr and conventional radiography in many institutions. in the united states of america, one of the most developed healthcare systems, healthcare authorities propagate incentives to abolish cr and older imaging systems by making them financially unattractive. the market is fully concentrated on the spread of dr systems. momentarily there are no real milestones but many refinements of existing systems such as tomosynthesis, dual energy subtraction and advanced auto-stitching, fluoroscopy capability, basic angiography applications and -d cone-beam ct images are made available. combinations of these features can be found in some recently designed x-ray machines. grid-less imaging software can reduce patient dose significantly. concerning detectors, there are cr retrofit systems which will support the easy upgrade of existing systems to dr capability. wireless detectors with large internal storage, different sizes and high resolutions of microns are available. dr is becoming a part of the system in the current era of full digitalization of our lives and big data, digital radiology is a cornerstone of our healthcare systems. ris and pacs as part of integrated healthcare (ihs) systems are widely disseminated. at the next step, all accessible data will be used for analyses. the major vendors of imaging systems, as well as pacs suppliers and independent companies, offer readymade software tools for reports and evaluations of all kind. the doses from different x-ray sources can be screened internally and be used for optimization purposes. they also can be sent to remote servers for dose monitoring, comparison and optimization in multi-hospital health care provider settings or to comprehensive databases like the american college of radiology dose index registry, cancer registries, or for central billing. has everything been invented? many technologies have been declared dead before a new transformation appeared. this was the case for example with single slice ct before the invention of spiral ct by willi kalender, germany and peter vock, switzerland in . often the plain x-ray image was meant to be needless or redundant as newer technologies like ct or mri approached. but it still is of value because of different reasons as the low dose, high availability, well known and easy interpretation to name a few. there are some new and sophisticated techniques on the way like the "smart x-ray source" which uses coherent beams of x-rays from an array of micronsized point sources, developed by scientists at the massachusetts institute of technology (mit). the developers promise less radiation, less weight of the equipment and a far better soft tissue resolution. another promising approach is phase contrast x-ray imaging which has the potential to reduce the dose up to / of the actual value. it also has its strengths in additional soft tissue information as recent experimental publications show (paul scherrer institute switzerland) e. g. in functional evaluation of lung fluid (munich, germany). functional imaging of the lungs can also be achieved without any radiation as the development of the known concept of electrical impedance tomography highlights. this functional imaging method usually is not within the modality spectrum of radiologists. dose control and reduction -local -regional -international the most effective measures to achieve significant dose reductions in your own department are still the same strategies which are based on the "eternal rules" as we know them from our teachers: avoiding unnecessary exposures by strictly controlling the appropriateness of a referral. justification is a shared responsibility between radiologists and clinicians. there are many tools available for justification like the appropriateness criteria, guidelines or rules (like wrist or ankle rules) of several national societies and different study groups. the process of optimization is mainly in the hands of the technicians. as many studies show, the proper collimation still has the greatest effect on dose reduction. other important factors are the positioning of the patient and the shielding of radiosensitive organs which are not relevant for image interpretation so that they may be covered by lead shields. the proper use of the grid in bigger children can now partially be replaced by software solutions. in digital radiography, a profound knowledge of postprocessing possibilities is mandatory as well as the active control of the exposure indices. dose limitation procedures should be regularly checked in a team-based approach to avoid overexposure by less experienced staff or "exposure creep". existing standards should be actively used to guarantee a constant satisfactory image quality. in , the image gently campaign released a safety checklist for performing digital radiography examinations on pediatric patients which is easily applicable to every radiology service. organizational improvements: at regional and national level, efforts should be made to check for best practice use in the departments and to compare and discuss imaging strategies. the establishment of national and international dose reference levels helps to keep the overall doses low and to protect the population from unnecessary overexposure. the pidrl project prepared the "european diagnostic reference levels for pediatric imaging" as part of the eurosafe project. momentarily the results of pidrl-workgroup are harmonized with international organizations. the european guidelines on drls for paediatric imaging can be accessed as a preliminary final for workshop drafts on the internet. on a worldwide basis, the world health organization has published a fundamental information brochure concerning radiation risks and the communication of health professionals and patients. health care professionals have a shared responsibility for communicating risks and benefits of imaging procedures to patients, especially in the case of pediatric patients. the document "communicating radiation risks in paediatric imaging-information to support health care discussions about benefit and risk" is intended to serve as a tool for health care providers, to communicate known or potential radiation risks associated with pediatric imaging procedures and to support risk-benefit dialogue in health care settings. as said before we are fighting the glow, not the fire. the paper of the swiss pediatric oncology group stirred a broad discussion. among other issues, there was a question if it shouldn't be a logical consequence to transfer kids from areas with higher background radiation to safer areas. the author's answers were clear: that swiss health authorities better concentrate their efforts more effectively and with greater benefit for more people by supporting prevention "toward modifiable environmental factors leading to larger numbers of deaths from several causes, such as exposure to radon, air pollution, and second-hand tobacco smoke". this leads to the conclusion that we as medical radiological professionals do have the obligation to make every effort to prevent our patients and personnel from harm of the usage or non-usage of radiation. as health specialists, we also should support the fields of prevention with broad mass effects as far as we have the opportunity. and as human beings, we are summoned to do so in respect to other beings, to our environment and to the resources we all share. radiation protection and quality improvement is just a small part of it all, but it is our field -and 'yes we can'. "communicating radiation risks in paediatric imaging. freely available at the who homepage." computed tomography: are we doing enough? e. sorantin; graz/at summary: already in the alara principle was publishedbut the implementation is still far from complete. according to the surveys of the ec tender project "pidrl -european diagnostic reference levels for paediatric imaging" the most frequent computed tomography (ct) examinations in children are, in descending order, head/neck, chest and abdomen thus counting for about % of all pediatric ct's. therefore it makes sense to optimize these examinations first. surveys of the "international atomic energy agency (iaea)" in countries have shown, there is considerable lack of organizationeg in about % of facilities protocols for children were missing, indication based protocols available only in %, ctdi values for head and chest two to five times of those for adults. all of these simple facts indicate we are not doing enough for radiation protection in pediatric ct. actions to lower dose in ct can be categorized in organisational, optimization and alternatives. the interdisciplinary implementation of international guidelines for ct in minor head trauma with trauma surgeons could serve as an example of organisational actions. for dose optimization knowledge about dose relevant factors according the "imaging chain" is mandatory as well as adjusting kv to pediatric needs. dose influence on image quality must be known, by exploiting the fact, that, if all ct parameters are kept constant but hte slice thickness is just halve there must be an increase in noisein particular about two times more. therefore if a standard examination is reconstructed at half slice thickness and image quality is still appropriate the amount of waste radiation is in the range of %. therefore if the next examination will be reduced with eg % mas setting less will be for sure in appropriate quality and the process can be started again. after a couple of examinations the optimal dose will be reached. thus the "half slice thickness" approach is easy to do, does not need special equipment or human resources and will help to find the appropriate dose. the third point is alternatives -ultrasound and mri being the candidates in the first row. new, radiation free, techniques like electrical impedance tomography and others are already developed and can be expected to be release soon. take home points: & we are not doing enough for ct dose savingeven more than years after release of alara principle & dose saving actions can be categorized inthe subtasks organisation, optimization and alternatives & "the half slice thickness approach" is an easy to do technique to elaborate the optimal dose on an particular ct machine. prenatal thoracic mr l. alamo; lausanne/ch the generalization of screening us has considerably increased the detection of congenital anomalies in utero. in the last years, important technological advances and especially, the development of fast heavily t -weighted sequences has led to an increasing use of prenatal mri as additional diagnostic imaging method. mri is increasingly used for evaluation of thoracic pathology, including tumours and vascular malformations as well as anomalies of the diaphragm, the lungs and more recently, even of the foetal heart: -thoracic tumours and vascular malformations: the diagnosis of a congenital tumor during pregnancy involves a tremendous emotional impact for a family. the most frequently observed thoracic tumours are teratoma, myocardial rhabdomyoma and exceptionally, pleuropulmonary blastomas. mri may provide relevant additional information concerning the origin of the lesion and its real anatomical extent. -diaphragmatic pathology: congenital hernia is by far the most commonly reported foetal diaphragmatic anomaly. the large field of view and the multiplanar possibilities of mri may help to clarify the position of the herniated organs and to evaluate the severity of lung hypoplasia, considered the most important parameter for predicting outcome. other rare pathologies include eventration, paralysis and diaphragmatic lung sequestrations. -lung anomalies: congenital lung abnormalities are a heterogeneous group of pathologies consisting of isolated bronchopulmonary or vascular anomalies or a combination of both of them. congenital pulmonary airway malformation, bronchopulmonary sequestration and bronchial atresia are the most often observed pathologies but they present significant overlap imaging findings. mri allows accurate information concerning the location and extension of the lesion and the volume of the normal and abnormal lung. -heart pathology: the evaluation of the foetal heart remains extremely difficult because of its small size and high rate of battements. the unpredictable foetal motions during data acquisition and the absence of a foetal ecg signal to synchronize data acquisition are additional problems. in the last years, different approaches have been made to overcome these challenges. radiologists should know the typical imaging findings of the thoracic pathology most often observed in foetuses. prenatal mri may provide additional relevant information in a wide spectrum of congenital thoracic anomalies, but in general, it should only be performed if it is considered that additional results might influence the management of the pregnancy and/or the therapeutic approach. therefore, it is important to know the right indications for mri and to recognize the limits of the method. interruptions during embryogenesis of the muellerian or wolffian ducts result in various, potentially complex genitourinary abnormalities of a wide spectrum or combinations. multiple imaging modalities are employed to evaluate patients with these abnormalities. ultrasound is the frontline imaging modality. mr imaging is mostly reserved for complex cases and may incorporate an mr urography, too. other imaging modalities are less frequently used or provide only ancillary information. this presentation will demonstrate the utility of ultrasound and mr imaging, in particular, in the routine diagnostic imaging of patients with the wide spectrum of muellerian and wolffian duct abnormalities. & mr imaging is reserved for more complex cases. neonatal hepatic tumors and vascular malformations d. pariente, s. franchi-abella; le kremlin bicêtre/fr neonatal hepatic tumours and vascular malformations are rare but imaging plays a key role in diagnosis and treatment. the most frequent hepatic tumour is haemangioma (fig ) which often is asymptomatic but may be complicated by cardiac failure, coagulopathy or compartment syndrome. the differential diagnosis mainly includes hepatoblastoma, hematoma (fig ), abscess, mesenchymal hamartoma, choriocarcinoma in the solitary form and metastatic neuroblastoma, cirrhosis, neonatal leukemia in the multifocal form. pertinent biological data are alpha-fetoprotein (but level may be normally high in neonate), betahcg, and urinary catechol amines. hepatic vascular malformations are rarer and include intra or extra porto-systemic shunts (pss), arterio-portal fistula or complex mixed forms. intrahepatic pss may be associated with haemangioma and regress in most cases rapidly (fig ). on the contrary the extrahepatic pss which are located below the portal vein, should be urgently closed to avoid occurrence of agenesis of the portal vein. the best imaging modality is us which must be performed with high frequency probes and colour doppler to identify hepatic vessels and assess patency, direction of flow, abnormal communication. mri and ct with contrast injection may also be useful. hepatic mass in a do neonate with increased crp and afp. us showing a hyperechoic mass with thrombosis of the left portal vein (black arrow) and a track (white) extending to the mass: hematoma due to malposition of an umbilical vein catheter. hemangioma of antenatal diagnosis on d . the mass is composed of a large anterior vascular lake corresponding to a porto-systemic shunt and a tissular hyperechoic part. the infant has remained asymptomatic. intrahepatic porto-systemic shunt between the left portal branch of segment (white arrow) and the left hepatic vein (black arrow) in a neonate. at months of age this shunt has completely resolved. haemangioma is the most frequent hepatic tumour in the neonate and is often asymptomatic with spontaneous resolution. levels of alphafetoprotein are physiologically high in the neonate, and can be misleading. hepatic hematoma can be secondary to traumatic delivery, to coagulation disorders or to umbilical vein catheterization. intrahepatic porto-hepatic shunts are the most frequent vascular malformations and regress in most of the cases in the first year of life. us with colour doppler remains the best imaging modality in the neonatal period. imaging in crohn disease: state of the art in diagnosis, prognosis and followup n. colavolpe, a. aschero, b. bourliere-najean, c. roman, f. khachab, h. pico, m. kheiri, g. gorincour, c. desvignes, p. petit; marseille/fr summary: during the past years the inflammatory bowed diseases (ibd) have increase in frequency ( ). less than twenty-five percent of them occur in children of less than years ( ) and crohn's disease (cd) is twice as frequent than ulcerative colitis (uc) in the pediatric age group. specific phenotypic and genotypic subtype of ibd occur in younger children. early onset (eo) pediatric ibd (before years of age) represent % of childhood ibd ( ) . uc and undetermined colitis are more frequent in this age group. eo cd showed a more frequent isolated colonic and upper gastrointestinal involvement than later-onset disease where locations are predominantly colic and terminal ileum later on childhood. some pediatric ibd specificities exist than can interfere with the imaging findings: -cd can be limited to the terminal ileum or to the colon in up to % of children ( ) . isolated jejunal involvement is reported to occur in - % of children. this location is more frequent in the youngest and is more at risk of complicated course of disease ( ) . for auvin et al. ( ) the small bowel is involved in % of cases with less involvement of the terminal ileum than in the adult population. -uc: the classical contiguous alteration of the bowel wall from the rectum to the caecum is inconstant. a macroscopic rectal sparing is reported from et % and the absence of continuous disease from rectum to caecum (caecal patch) described in % of children. transmural inflammation may be present in severe form as well as terminal ileitis without granulomata (backwash ileitis) ( ) . in order to assess these pathologies, and more specifically cd small bowel locations which are difficulty explored by others modalities, small bowel follow-through, barium enema, ultrasound, computed tomography and mr imaging have been widely used. among them, mr-enterography has gained worldwide acceptance due to multiple factors including: a high contrast resolution, a multiplanar ability, an absence of radiation, the possibility to explore in the same exploration the whole bowel and the extra-bowel diseases (perianal fistulae, sacroiliac joint, biliary tract), the ability to compare of side by side consecutive studies in a reproducible manner, a more easily understood exploration by the clinicians than ultrasound, and first of all for its performances. in order to technically harmonize this exploration a recent consensus statements on mre protocol has been published by the esgar and the espr societies ( ) . preparation: -depending on their age children must not have solid oral intake from to hours prior to the examination to reduce bowel wall motility. morning mr appointment is more favorable for this purpose. no gasless fluid restriction is recommended but is reabsorbed too quickly to distend enough the small bowel. none hyperosmolar non absorbable solution is superior to another. its ingestion must start to minutes prior to mre. the recommended volume is ml/kg with a maximum up to ml/kg. explanations long before the mre concerning the importance of such absorption and the use of a refreshed product mixed with aromatized flavors will facilitate the child's participation. -the use of spasmolytic agents is optional. however, there are recommended in adults by multiple societies including esgar ( ) , the society of abdominal radiology ( ) and the acr (www.acr.org/ quality-safety/standards-guidelines). but, mre without antiperistaltic agents result has reached a high diagnostic confidence and excellent agreement with ct enterography for the presence of cd ( ) . if used, they need to be administered immediately prior to motion sensitive sequence (t w dynamic enhanced sequences). if the pictures obtain with these medications are of better quality, there is no evidence that they change the final diagnosis and the children's therapeutic management ( ) . the use of these products increase the length of the exploration and their side effects are frequent (nausea > vomiting) which balance their visual benefice ( ) . if a spasmolytic agent is used, the recommended first line spasmolytic agent is i.v. hyoscine butylbromide ( . mg/kg i.v). the recommended second line agent is i.v. glucagon, . mg (< . kg) and mg (> . kg), given as a slow infusion with i.v. saline at an infusion rate at ml/s. -no rectal enema is needed. -exploration can be performed either at . tesla or testla. more chemical shift and susceptibility artifacts are present with the latter. prone position has been demonstrated to allow better small bowel distension than the supine one with reduction of the peristaltism but without better lesion detections ( , ) . large multi-elements coils are needed to cover with high resolution from the perineum up to the left colonic flexure. sequences: both morphologic steady state free precession gradient echo and d -t -weighted images are needed in the axial and coronal planes. fat saturation in one of this plane is recommended and maximal slice thickness of mm is required. nowadays, non-enhanced then enhanced d t -fat saturation weighted sequences are mandatory. slice thickness does not exceed mm. enhance sequence need to be acquired at the portal phase of injection. however, in recent studies the need for gadolinium has been questioned when dwi is added to the morphologic sequences. dwi sequences have been considered optional ( , ) but we consider their place essential in pediatric practice. they must be done with high b values, from up to in the coronal and axial planes with to mm contiguous cut in free breathing. axial plane is less prone to artefact than the coronal plane. interestingly enough shenoy et al ( ) report in pediatric patients that dwi does not perform as well as standard mre for detection of active crohn disease but the combination of dwi and mre increases imaging accuracy for determining disease activity compared with either technique alone. seo et al ( ) in young adults said that dwi mre was noninferior to contrast-enhanced mre for the evaluation of inflammation in cd. based on the exploration of cd adult and pediatric population, dwi proved to be efficient and would avoid gadolinium injection ( ) . sirin et al. ( ) report in children that dwi revealed lesions that were not detectable with mre done with gadolinium injection. finally, respectively dubron et al. ( ) in children and neubauer et al ( ) in children and young adults demonstrate better performance of dwi than gadolinium enhanced imaging. like the existing mr protocols for suspected appendicitis ( ) it will not be surprising to see fast mr ibd explorations becoming an alternative to emergency us as already proposed ( ) . this fast mr limited to a morphologic t sequence in two planes associated with dwi sequences will allow a positive diagnosis and the ibd work up. apart from bowel obstruction and its spontaneous bowel distension one of the limiting factors will be the need for an oral water agent uptake in a potential surgical patient. however, it has been published in the adult literature than an oral or rectal preparation was not necessary to rule out uc ( ) nor a cd ( ) . the other limiting factor is the length of exploration. mre can be shorten especially if the patient's positioning is easy to do (dorsal decubitus) ( ) and if there is no need for injection, either for spasmolotytic agent and for gadolinium chelates. the suppression of the iv line, the absence of potential side effects (nausea, vomiting) of paralytic agents and the decreased of repeated long apneas with no loss of significant information will be strong progresses toward the holy grail. -positive diagnosis, disease activity, prognosis and follow-up: mre has a better accuracy to detect inflammation for the small bowel than for the colon ( ) . one of its goal is to try to accurately identify features of active inflammation vs fibrotic disease. this is of paramount importance since the former may respond to medical treatment and the latter may need surgical resection. however, inflammation and fibrosis are associated within the same bowel segment and progress in a parallel way making the goal difficult to reach ( ) ( ) ( ) . in their study based on the analyze of children operated for cd strictures, barkmeier et al. ( ) report than strictures demonstrating > cm upstream dilatation with associated feces sign were highly associated with transmural fibrosis. the most severely fibrotic strictures were associated to the greatest amount of inflammation and there was no significant correlation between stricture length, mural thickness, degree of post-contrast enhancement (arterial and delayed venous phases), diffusion-weighted imaging apparent diffusion coefficient, pattern of post-contrast enhancement, or normalized t -weighted signal intensity and histological fibrosis or inflammation scores. however, correlation with histological specimens of cd done on a other series s ( ) (suppl ):s -s pediatr radiol demonstrated that the enhancement ratio of the wall is positively correlated with disease chronicity due to a possible increasing microvessels permeability and inversely correlated to acute disease ( ). on the other hand, several authors have tried to correlate the adc values to cd activity. fibrotic tissue does not restrict diffusion and presents a decrease of signal at high b values and high adc values whether acute inflammation shows decrease adc values. variable thresholds from . x - mm /s to . x - mm /s have been proposed to separate active vs non active disease ( ) . however, others authors have reported low adc value of fibrosis compare to histology ( ). even if promising results have been published with high correlation with the crohn disease endoscopic index of severity ( ) , adc measurements are associated with sever limitation factors including sample size overlap between the bowel wall and its atmosphere, lack of reproducibility between mri-units and mri-vendors, non-standardized sequence b-values parameters ( ) . two mre scores are available to quantify the activity of cd. one is using gadolinium injection ( ) and the other dwi ( ) . due to the complexity of the formula, both are difficult to use in daily practice and have not been evaluated in paediatric practice. interestingly enough if a simplify mre paediatric protocol appears to become a reality, us stays a good imaging challenger and ( ). in a recent meta-analysis, based on adult and pediatric series, calabrese et al ( ) reported that bowel us showed . % sensitivity and . % specificity for the diagnosis of suspected cd, and % sensitivity and . % specificity for initial assessment in established patients with cd. bowel us identified ileal cd with . % sensitivity, . % specificity, and colon cd with . % sensitivity, . % specificity, with lower accuracy for detecting proximal lesions. the absence of abnormal thickness wall would have a negative predictive value, high enough to exclude the need for further exploration, especially when cd is concerned ( , ). concordance between us and mre have been variably reported from excellent ( ) to just correct ( ). rosembaum and al ( ) report that the us findings present in children operated for cd include: bowel wall thickness above . mm (mean, . mm) and an increased frequency of loss of mural stratification and fibrofatty proliferation. others us technologies are used in children to better approach the disease activity. it includes, hydrosonograpy using specific oral agents (mannitol, sorbitol, polyethylene glycol, etc…), contrast-enhanced ultrasound and dynamic contrast-enhanced ultrasound (nowadays, contrast agent is offlabel in children) ( ) and elastography ( ). their enthusiastic results and their efficiency to assess disease activity need to be confirmed ( ). in conclusion, as we suspected years ago ( ), mre has dramatically modified our approach of pediatric ibd especially when considering its orientation toward a less invasive exploration and the increasing importance of dwi imaging. a cost benefice between mre and us remains to be done on this increasing disease. heterotaxy and isomerism c. lapierre; montreal/ca summary: objectives: to review the classification of visceroatrial situs to describe the associated cardiac and non-cardiac anomalies to illustrate typical findings in fetuses, neonates and children to discuss the surgical consideration and the long-term follow-up in these patients abstract: by definition, the type of situs is determined by the relationship between the atria and the adjacent organs. anatomically, the atrial chamber differentiation is based on the morphologic aspect of the atrial appendages, earlike extensions of the atria. three types of situs exist: solitus (normal), inversus (mirror image) and ambiguus. a single type of situs is present in a patient. when the situs is neither solitus nor inversus, it is referred to as situs ambiguus or heterotaxy. heterotaxy may manifest with various abnormal visceroatrial configurations that are associated with cardiac (in - % of cases) and extracardiac anomalies such as splenic abnormalities, biliary atresia and intestinal malrotation. two subsets of situs ambiguus are well-recognized: right isomerism (asplenia) and left isomerism (polysplenia). in heterotaxy, the venoatrial connections are frequently abnormal. left isomerism is usually indicated by bilateral bilobed lungs, interruption of the ivc and multiple spleens. the more likely found cardiac anomalies are: pulmonary or aortic stenosis, isolated atrial and ventricular septal defects, cardiac arrhythmia due to sinus node dysfunction as well as pulmonary veins that drain into both the right and the left atria. in the presence of right isomerism, bilateral trilobed lungs, a large symmetric liver, and absence of the spleen are frequently observed. at the cardiac level, patients are more likely to have a common atrioventricular defect, a double outlet right ventricle and pulmonary stenosis. total anomaly of the pulmonary venous return and absence of coronary sinus will always be present in right isomerism. heterotaxy can be diagnosed with high accuracy by prenatal echography. a diagnosis should be suggested in the presence of congenital heart disease, visceroatrial heterotaxy and interruption of inferior vena cava with azygos continuation for left isomerism or abnormally closed juxtaposition of inferior vena cava and descending aorta in right isomerism. the mortality in fetuses is high in the presence of heart block and hydrops whereas the cardiac anomalies influence the long-term outcome. as discussed in the literature, the clinical outcomes and long-term prognosis in these patients are relatively poor when compared with non-heterotaxy patients. the risk factors are cardiac (underlying anatomy and arrhythmia risk) and non-cardiac. based on the cardiac anatomy, one of the main determinants is left versus right isomerism. with right isomerism, the cardiac malformation is more severe and an univentricular correction is more frequent. another predictor of mortality is pulmonary vein stenosis/obstruction. whatever the severity of cardiac lesions, the postoperative or discharge mortality is higher in patients with heterotaxy. prenatal diagnosis seems not improve the survival. extracardiac anomalies also contribute to the increased morbidity and mortality. three of the more challenging entities are respiratory, immunologic and gastrointestinal. recurrent respiratory infections, failed extubation or chronic respiratory failure are frequently observed in patients with heterotaxy. recent studies revealed an association between heterotaxy and primary ciliary dyskinesia which can explain the increased postoperative respiratory complications. the spleen is important for the bacterial clearance. patients with asplenia or polysplenia are thought to have "functional asplenia". so, they are at risk for sepsis and severe bacterial infection. the incidence of intestinal malrotation is high, approximately % to %. observation versus prophylactic ladd procedure and screening for asymptomatic intestinal malrotation are a growing area of debate. the trend seems to go along conservative management and surveillance of malrotation. bronchopulmonary malformations, such as congenital pulmonary airway malformation (cpam), bronchopulmonary sequestration (bps), and congenital lobar emphysema (currently known as congenital lobar overinflation [clo] ), are common congenital lung diseases. these conditions are detected prenatally, usually in the second trimester, in countries where obstetric sonography is routinely performed. the malformations are seen as hyperechoic images with respect to normal fetal lung parenchyma, with a mass effect and homogenous appearance or with coexisting cysts. the lesions usually decrease in size along gestation. a residual mass is seen on postnatal chest radiography, the first imaging technique performed, in only % of cases. cpam and bps are predominantly located in the posterior lower chest and can be identified postnatally on ultrasound using a small vector probe and a subcostal and subxiphoid approach. potential feeding arteries can be visualized using color or power doppler. based on clinical and sonographic findings, the differential diagnosis between congenital lung malformations and tumors such as neuroblastoma, type i pleuropulmonary blastoma, and myofibroblastic tumor will be discussed. postnatal management and imaging of newborns with congenital lung malformations is controversial, particularly in asymptomatic patients (approximately % of cases). chest radiography is mandatory at birth and chest ultrasound is also recommended to confirm the prenatal diagnosis. computed tomography (ct) or magnetic resonance imaging (mri) using angiographic techniques should be performed some months ( months) after birth in asymptomatic patients. these techniques are also recommended in symptomatic newborns and before surgery to characterize the arterial supply and venous drainage in cpam and bps, as ultrasound is limited in this regard. in premature infants, sonography complements radiography in the study of prematurity-related lung diseases such as respiratory distress syndrome and its pulmonary complications (eg, pneumothorax), in predicting bronchopulmonary dysplasia, and in diagnosing transient tachypnea of the newborn when clinical and radiographic features are inconclusive. the main ultrasound finding in these conditions is visualization of numerous "b-lines", vertical narrow-based hyperechoic bands extending from the pleural surface to the end of the field of view, representing what is currently known as "sonographic interstitial syndrome". b-lines are artifacts originating from variations in the air-fluid relationship of the lung and are better seen using high-frequency linear probes . use of sonography for follow-up of these patients will reduce the number of the chest plain films performed, and therefore, the amount of radiation exposure in this vulnerable population. for proper interpretation of the sonographic findings in these conditions, the radiologist should be familiar with current related terms, such as lines a, lines b, comet tail artifact, interstitial-alveolar syndrome, septal syndrome, and white lung. trauma is the leading cause of mortality and morbidity in children after the first year of life. motor vehicle accidents are the leading cause of death from unintentional injury in children up to the age of . of these cases, the abdomen is the fourth most commonly injured area. in pediatric patients non-operative management of these injuries predominate, hence the importance of early radiologic assessment for appropriate clinical follow-up. anatomically, compared to adults, childrens' abdomens are more square, less muscular and with less intraperitoneal and subcutaneous fat to absorb impact. the diaphragm is more horizontal causing downward displacement of the liver and the spleen outside the protective casement of the ribs. the pelvis is smaller and hence the bladder is displaced upward, also resulting in more vulnerability to this organ. the organ surface area is larger in children and they have a smaller body mass-hence more force applied per-unit of body surface area. the ribs are flexible, and although we see fewer rib fractures, this results in more internal damage. physiologically, children maintain hemodynamic stability longer, often presenting with only mild tachycardia, even when in severe hemodynamic shock. decrease in blood pressure may not be evident before the loss of % blood volume. nevertheless, bleeding is less severe and operative intervention is rarely performed. mechanics of blunt abdominal trauma include organ compression from seat belt injury with the presence of erythema, ecchymossis or abrasion on the abdominal wall increasing the likelihood of internal organ injury ( % likelihood of injury). other common mechanisms include pedestrian-car collisions( % with intra-abdominal injuries), falls ( % with intra-abdominal injuries), or handle bar injuries ( % with intraabdominal injuries). after the child arrives in the hospital, a trauma algorithm is initiated. generally, for the unstable patient, algorithms are similar and require a rapid atls protocol, followed by a fast ultrasound to confirm free fluid prior to operation. in stable patients, institutional algorithms vary greatly between countries and in different centers. some rely solely on mechanism to determine the need for fast vs ct (not complete ultrasound), others will rely on clinical exam (in a conscious patient with reliable exam) and blood work to determine the need for imaging (ct or us) and others may chose to perform an initial us and complete the exam with a constrastenhanced us during work hours. in the literature many management prediction rules exist based on the history, physical examination, mechanism of injury and are supplemented by blood work and/or intial imaging. most are based on retrospective reviews, with only a few controlled clinical trials. however, the validity of these studies is limited because of different populations, institutional policies and variable radiological practices in terms of when imaging is performed, which modalities are most beneficial and which are less valuable, all the while, considering the utilization of the least irradiating techniques. a representative sample of such algorithms will be discussed. routine and extensive initial trauma panels are not required according to a number of studies. abdominal ultrasound and urinalysis together have been found to confirm % of all intra-abdominal injures, in some studies. serial haemoglobins/hematocrit is valuable for determining ongoing s ( ) (suppl ):s -s pediatr radiol blood loss and assists clinical surveillance. electrolyte abnormalities are uncommon in children unless severe shock is present (metabolic acidosis). liver function tests are elevated in most cases of blunt abdominal trauma, hence, are often performed for its high sensitivity, to avoid ct if the liver panel is negative. imaging, however, is needed for grading of the potential liver injury if the liver panel is positive. abdominal xray is not useful in blunt abdominal trauma, and is usually normal. ultrasound has an important role in the pediatric community, as a sensitive and non-irradiating modality. however, this sensitivity is dependent on the type of ultrasound performed (fast vs. complete abdominal ultrasound vs. contrast-enhanced ultrasound) but also on the qualifications and experience of the performing physician. a meta-analysis of fast in pediatrics demonstrates that it has a sensitivity of % (grade i-ii evidence) for identifying hemoperitoneum. a negative fast is not sufficient to rule out intra-abdominal trauma. one prospective observational trial demonstrasted that % of patients without free fluid on fast (performed by formally trained pediatric truama surgeons demonstrated at least grade iii liver or splenic injuries on ct). we know that pediatric ultrasound is operator-dependent, and generally an ultrasound performed by the skillful hand of a pediatric radiologist is more sensitive than that performed by surgeons or by adult radiologists. furthermore, we know that the benefits of contrast-enhanced ultrasound in pediatric trauma exist-highly accurate in visualising lesions, hence avoiding non-contributive ct imaging, however, the feasibility of providing -hr contrast-enhanced ultrasound by a qualified radiologist is resource intesive: both structurally and with respect to personnel. published indications for abdominal ct in stable pediatric patients included suspected mechanism of blunt abdominal trauma, significant fluid resusitation without apparant blood loss, hemoglobin < mg/l without obvious blood loss, multisystem trauma and unreliable abdominal exam. one series with children undergoing ct for blunt abdominal trauma demonstrate postive findings in ( %), of which all solid organ injuries and % of hollow viscus injuries were identified on ct. however, ct has its limitations: it was found to identify gastrointestinal perforation in only % of patients with known perforation, but with findings of free fluid, wall thickening and/or bowel dilatation. it is also less accurate in identifying pancreatic trauma, with normal scans in - % of children with pancreatic trauma. again, findings of pancreatic trauma can be non-specific: free fluid or, less commonly, thickening of the gerota's fascia, presence of mesenteric fluid or of fluid between the pancreas and the superior mesenteric vein. when and where to perform ct depends on the imaging algorithms established by individual centers. generally, unstable patients with very high grade visceral injuries are taken to surgery. the stable patients are treated with non-operative management. the literature on angiographic embolization in pediatric blunt trauma is limited to case series that demonstrate a limited utility in hemodynamically stable patients with ongoing blood loss or for the definitive treatment of traumatic pseudoanevryms. a dialogue with the interventional radiologist is imperative in such cases. common imaging findings and pitfalls will be illustrated with case examples. in conclusion, a child's anatomy and physiology must be taken into account when determing the level of urgency and appropriate imaging work-up in blunt abdominal trauma. imaging of these patients cannot follow a standard algorithm as institutions vary with respect to types of personel, training, frequency of trauma, emergency department trauma protocols and availability of an in-house pediatric radiologist. ultrasound and ct have their advantages and disadvantages with associated pitfalls that the pediatric radiologist must recognize to provide an optimal diagnostic workup with minium irradiation. take home points: a child's anatomy and physiology must be taken into account when determing the level of urgency and appropriate imaging work-up in blunt abdominal trauma. imaging of pediatric abdominal trauma cannot follow a standard algorithm as institutions vary with respect to types of personel, training, frequency of trauma, emergency department trauma protocols and availability of a pediatric radiologist. ultrasound and ct have their advantages and disadvantages with associated pitfalls that the pediatric radiologist must recognize to provide an optimal diagnostic workup with minium irradiation. sport injuries d. jaramillo; miami, fl/us the growing skeleton has unique vulnerabilities to acute and chronic injuries due to sports. the practice of intensive sports during puberty and adolescence has led to a great increase in the incidence of sportsrelated injuries. during the growth spurt of early adolescence, the physis becomes weak, and is the site of fractures and avulsions (particularly in the apophyses) and of physeal widening due to repeated stresses, such as the wrist in gymnasts or the proximal humerus of baseball pitchers. both lesions can result in growth arrest. the chondro-osseous junctions of the ossifying epiphyses and apophyses are also vulnerable to avulsions, and the avulsed fragment may be entirely cartilaginous and not visible radiographically (such as in the patellar sleeve fracture). repeated trauma to epiphyses or round bones can lead to osteonecrosis (panner's disease) but more often to osteochondritis dissecans (ocd). in adolescents, ocd occurs most frequently in the medial femoral condyle, the capitellum of the elbow and the talar dome. juvenile ocd has a better prognosis than the adult form. when the skeleton begins to mature, there are fractures unique to partially closing physes such as the triplane and tillaux fracture. some sturctures have propensity to unique injuries during adolescence. a stress on the anterior cruciate ligament (acl) can lead to a tibial eminence avulsion in puberty ( figure) , an incomplete acl tear in early adolescence or a complete acl tear later. meniscal tears are almost always vertical and often involve large meniscal fragments that can flip. patellar dislocations often result in osteochondral injuries. this review will cover the main types of sports-related injuries and the imaging modalities used to diagnose them. year-old with pain and popping sensation during a fall on a football game. ap radiograph is normal & it is important to take into account the specific sport in order to anticipate subtle injuries that may be difficult to detect. a. c. offiah; sheffield/uk the radiographs obtained when inflicted injury is suspected are collectively termed the "skeletal survey". a full skeletal survey should be performed in all children below years of age in whom abuse is suspected. the investigation is not complete until follow-up skeletal imaging has been performed in the to days following the initial survey. children below one year of age should also receive a ct brain. neurological imaging in older children will depend on the clinical scenario. ct chest/abdomen is indicated when visceral injury is suspected. in terms of imaging in suspected abuse, espr has adopted the rcr guidelines. in the absence of a history of significant trauma, fractures highly specific for abuse in pre-ambulatory children include rib, metaphyseal and diaphyseal fractures. simple linear skull fractures have a relatively low specificity for abuse. the combination of subdural haemorrhage, retinal haemorrhage and diffuse cerebral oedema/encephalopathy (the so-called, "triad") suggests shaking. whereas the presence of a skull fracture implies impact. visceral injury often results from direct blunt trauma and may therefore be accompanied by anterior and/or costochondral rib fractures. the posterior rib arcs are protected by soft tissue and posterior rib fractures result from compressive/squeezing forces rather than direct trauma. the dating of fractures has a subjective element and it is more important to recognise that fractures are in different stages of healing, rather than to assign a definite age/age range to the injuries. the major differential diagnoses are accidental trauma and osteogenesis imperfecta. if rickets is the cause of the fractures, then radiology and/or biochemistry will show evidence of rickets. a low vitamin d level, in the absence of rachitic features, is not the cause of fractures. close liaison between radiologists and paediatricians is vital and any siblings/children in the same household who are below years of age should also receive a skeletal survey. remember that the presence of injury does not always mean abuse and that the absence of injury does not always exclude abuse. scoliosis may be primitive, structural, particularly during adolescence; during this period, careful follow-up is mandatory, because worsening is frequent. clinical examination with evaluation of a hump (gibbosity) with a scoliometer is mandatory, with also neurological assessment. beside radiography, additional tools have been developed to avoid xray exposure: "spinal mouse", back surface topography systems, ultrasound and other computer-assisted systems. but scoliosis can also be secondary, and imaging is important to find a cause and adapt management. among the etiologies, radiologist must recognize spine malformations, dysplastic and neuromuscular scoliosis. in addition, scoliosis may also be in relation with a primitive lesion, tumor-related or not, whether the initial disease could be within the spinal canal, spinal or paravertebral. imaging studies lies first on pa and lateral full spine x-rays, if possible with a low dose device (flat panel, slot-scanning system), keeping in mind that follow-up with repetitive exposures may be necessary. reproducible measures of different curvatures help to assess the overall static spine and the importance of scoliosis with cobb angle. the assessment of axial rotation can be obtained through d simulations, with frontal and axial views (see figure) . morphologic evaluation of the s ( ) (suppl ):s -s pediatr radiol spine is mandatory: if a secondary scoliosis is suspected, the research to etiology needs to perform ct or mri, depending on the clinical signs and the results of plain x rays evaluation. similarly, these explorations are useful in the preoperative assessment when surgical treatment is necessary. girl scoliosis, pa and lateral views with eos®, d simulation, coronal and axial views take home points: clinical evaluation is always the first step in subject with suspected scoliosis radiation burning is quite low with new devices, but repetitive exposures for follow-up need to carefully respect justification for x-rays exposures new tools are available to appreciate d spinal deformation and evaluate prognosis and surgical procedures ct and/or mri are useful in presurgical assessment and to look for etiologies in suspected secondary scoliosis malformations of the spine and spinal cord a. rossi; genoa/it summary: embryology and classification: spinal cord development occurs through three consecutive periods: (i) gastrulation ( nd gestational week): the embryonic disk is converted from a bilaminar into a trilaminar arrangement, with formation of the intervening mesoderm; the notochord is laid down along the midline, identifying the craniocaudal embryonic axis; (ii) primary neurulation ( th - th day): under the induction of the notochord, the midline ectoderm specializes into neural ectoderm. the initially flat neural plate progressively bends and folds until it fuses in the midline to form the neural tube. the primary neural tube produces the uppermost / of spinal cord; (iii) secondary neurulation ( th - th day): a secondary neural tube is laid down caudad to the termination of the primary neural tube. retrogressive differentiation of the secondary neural tube results in the tip of the conus medullaris and filum terminale. defects in one of these three embryological steps produce spinal dysraphisms, characterized by anomalous differentiation and fusion of dorsal midline structures. spinal dysraphisms may be categorized clinically in two subsets: open and closed spinal dysraphisms. in open spinal dysraphisms (osd) the placode (non-neurulated neural tissue) is exposed to the environment through a cutaneous defect along the child's back. osd include myelomeningocele, myelocele, hemimyelomeningocele and hemimyelocele, and are associated with a chiari ii malformation. myelomeningocele is by far the most common of these forms; the placode protrudes through a posterior defect and is elevated above the skin surface due to concurrent dilatation of the subarachnoid spaces. closed spinal dysraphisms (csd) are covered by intact skin, although cutaneous stigmata usually indicate their presence. two subsets may be identified based on whether a subcutaneous mass is present. csd with tumefaction comprise lipomas with dural defect (lipomyelocele and lipomyelomeningocele), meningocele, and myelocystocele. lipomas with dural defect are more common; they are differentiated from one another based on the position of the cord-lipoma interface, that lies within the spinal canal in lipomyelocele, and outside the spinal canal (ie, into a meningocele) in lipomyelomeningocele. csd without tumefaction comprise complex dysraphic states (ranging from complete dorsal enteric fistula to neurenteric cysts, diastematomyelia, dermal sinuses, caudal agenesis, and spinal segmental dysgenesis), bony spina bifida, tight filum terminale, filar and intradural lipomas, and persisting terminal ventricle. the most complicated forms (complex dysraphic states), including diastematomyelia, caudal regression, and segmental spinal dysgenesis) are related to faulty gastrulation. diastematomyelia (literally, split cord) is caused by failure of midline notochordal integration, resulting into two separate hemineural plates. caudal agenesis and segmental spinal dysgenesis are related to defective notochordal formation, characterized by absence or hypoplasia of a segment of the notochord, in turn resulting into absence or hypoplasia of a corresponding segment of the spinal cord. functional neuroimaging of cns is a fast advancing field with frequent new developments in scanner's hardware, protocols, clinical indications, and post-processing techniques. for radiation safety reasons in the case of children, functional neuroimaging is mostly based on mr techniques especially designed to focus on the assessment of functional tissue characteristics, such as neuronal activity (fmri),, metabolism (mrs) and perfusion (dsc perfusion, asl). pediatric coils with multiple elements, multiple slice excitation, d spectroscopy, d asl, reduced fov (zoom) and improved motion compensation techniques are important tools available to meet the permanent challenges of pediatric mr functional imaging: fast motionless acquisitions and increased resolution. functional mri (fmri) reveals brain activation during performance of behavioral tasks, based on the blood oxygen level dependent (bold) mri signal, which is modulated by neural activity via a process of neurovascular coupling. for children, especially of younger age unable to follow a task, resting-state fmri (rfmri) can be performed and correlates brain areas with similar spontaneous fluctuations in the bold signalthereby enabling estimates of 'functional connectivity.' main clinical applications of fmri are the delineation of eloquent cortex near a space-occupying lesion and the determination of the "dominant hemisphere" for language. intense research is conducted in the areas of language organization and development, brain plasticity, and neurobehavioral disorders (e.g. adhd). magnetic resonance spectroscopy (mrs) is a noninvasive mr technique, that detects intracellular metabolites, and may provide neuroimaging biomarkers of normal biological and pathological processes or response to a therapeutic intervention. although the main field of application of mrs is the brain tumors, it has also been of particular ( ) (suppl ):s -s pediatr radiol usefulness in assessing ischemic or traumatic brain injury and neurometabolic disorders. perfusion mr imaging methods detect signal changes that accompany the passage of a tracer through the cerebrovascular system. a less invasive approach is arterial spin labeling (asl) that uses arterial water as an endogenous tracer to measure cbf and thus it is more suitable for pediatric studies. mr perfusion is applied in the evaluation of brain tumors, neurological diseases and developmental disorders. functional neuroimaging clinical applications are expected to expand greatly in the future due to the increasing availability of their techniques, as well as the continuous advancements in the field of pediatric research. good knowledge of these techniques will become more necessary for an effective clinical practice and will enhance the role of radiology in the healthcare system. functional neuroimaging advanced techniques based on mri allow us to study complex cns processes such as cerebral perfusion (dsc, asl), metabolic activity (mrs) and brain activation (fmri). functional neuroimaging techniques already have significant clinical pediatric applications and assisted by recent advances in mr technology are expected to become even more powerful in the near future. kidney: perfusion, excretion, obstruction k. darge; philadelphia/us the functional imaging of the urinary tract entails the evaluation of the renal perfusion and excretion. in this complex process the sites of the main abnormalities could be pre-renal, renal parenchymal, renal pelvicalyceal or post-renal or even a combination at different sites. functional mr urography (fmru) is an advanced tool that not only allows the exquisite morphological depiction of the urinary tract, but also makes it possible to generate comprehensive functional data. these provide information about the function of the kidney as well as the excretion of urine from the renal parenchyma into the pelvicalyces and ureter. the functional results are mainly divided into two groups: . transit timesthese are recorded in minutes and a side comparison gives idea how much time it takes for the contrast to go through the renal parenchymathe longer the more abnormal in general. . differential renal functionsthese can be based on the enhanced renal parenchymal volume or the patalk number generated from this area and provides in percentage the split renal function. this presentation will discuss in detail the functional aspect of mr urography and demonstrate its utility in routine pediatric uroradiologic imaging. in chronic childhood lung disease (e.g. cystic fibrosis) global pulmonary function tests (pft) can be normal although lung damage is already present. moreover, in comparison to imaging, pft is challenging in young children. thus, cross-sectional imaging became more important in the past two decades. regarding morphological evaluation, multidetector computed tomography (mdct) serves as the most sensitive and reproducible modality. for functional evaluation perfusion/ventilation scintigraphy remains the reference standard. although the individual radiation burden by a single chest ct has decreased significantly in the past, radiation doses can cumulate considerably when repeated examinations are performed in a longterm follow-up. pulmonary mri exists as an alternative method, especially for paediatric patients. however, standard h+mr sequences do not demonstrate small airway disease due to inherent limitations of low signal and rapid t * signal decay of lung tissue. for comprehensive diagnosis, functional mri offers the unique possibility to measure regional ventilation and perfusion, and mapping relaxation times and diffusion. focussing on research applications, a variety of methods are available for these purposes. in this context, ventilation imaging using inert fluorinated gas indicates to overcome the limitations of the expensive setting necessary for imaging with hyperpolarized noble gasses. regarding lung perfusion, dynamic contrast-enhanced mri (dce-mri) is the most established method in clinical practice. however, especially in children, techniques that are completely non-invasive and do not require i.v.-contrast agents administration or gas inhalation could be promising to achieve broad acceptance. concerning non-invasive methods, ventilation can be assessed by sequences with ultra-short echo times (ute), perfusion by arterial-spin-labeling (asl) and both by fourier decomposition mri (fd-mri). in conclusion, pulmonary mri offers both, the assessment of morphology and the unique possibility to measure regional ventilation and perfusion, and mapping relaxation times and diffusion. new mr techniques that are completely non-invasive are now available. however, further scientific evaluation is needed. ibd and related arthropaties d. jaramillo; miami, fl/us musculoskeletal diseases affect about % of patients with crohn's disease and are the most frequent extra-intestinal manifestation of inflammatory disease. the articular manifestations of inflammatory bowel disease (ibd) are one of the seronegative arthritides, although they have a lower incidence of hla -b than other seronegative arthritis such as ankylosing spondylitis. there are manifestations in the joints of the extremities, and findings in the pelvis, especially in the sacro-iliac joints, and spine. involvement of the extremities occurs in about % of patients with ibd related arthropathies, are more common with crohn's disease, and can have either manifestations related olygoarticular jia, or can have symmetrical involvement of smaller joints. the axial manifestations include ankylosing spondylitis and sacro-iliitis. sacroilliitis is typically bilateral (figure) and often has radiographic as well as mri abnormalities. enthesitis, tenosynovitis and dactylitis can occur with ibd just as they occur with other arthritides. it is important to differentiate ibd related arthritis from septic arthritis due to extension of an enteric fistula. deceased bone mineral density is a common finding in inflammatory disease. it occurs as a combination of malabsorption of vitamin d due to intestinal involvement and the effects of therapy, particularly corticosteroids. insufficiency fractures of the spine, sacrum and extremities can mimic the symptoms of arthritis. finally, ibd can be associated with chronic non-bacterial osteomyelitis, although this association is relatively rare. this review will illustrate several of the skeletal manifestations of ibd, focusing on the arthropathies. juvenile idiopathic arthritis (jia) can be defined as an arthritis of unknown cause occuring in children younger than years and of at least weeks duration. juvenile spondyloarthritis (jspa) is a subset of jia and is characterized by enthesitis (inflammation at the attachment of tendons, ligaments and the joint capsule), arthritis and an increased risk of axial disease. there is also a strong association with human leukocyte antigen b . jspa accounts for approximately - % of juveniles arthritis cases in europe and is the most common form of juvenile arthritis in asia. the condition is associated with significant long-term morbidity, high health-care costs and poorer outcomes compared with other forms of juvenile arthritis as well as its adult counterpart. up to % of patients continue to be at risk of developing ankylosing spondylitis (as) during the disease course. recognizing spondyloarthritis (spa) in children is challenging, particularly early in the course of disease, as the signs and symptoms at disease onset differ from those seen in adults. jspa typically presents with hip and lower limb arthritis in conjunction with enthesitis. inflammatory back pain as a presenting symptom is less common. as a consequence, jspa may be missed or confused with other juvenile arthritides and patients often experience prolonged delays in diagnosis. currently there is no single diagnostic or classification system that is representative of the jspa population. according to the international league of associations for rheumatology (ilar) classification system, most childhood spa's are classified as enthesitis-related arthritis (era), psoriatic arthritis or undifferentiated arthritis. recent studies indicate that there are two clinical phenotypes of era: those with early axial disease often associated with hip arthritis in addition to peripheral arthritis; and those who follow a more peripheral disease course with arthritis and enthesitis and do not develop axial disease. the ilar classification system places patients with both axial and peripheral involvement into the era subtype, and does not specifically address children who meet the criteria for as. the correct approach to the classification of era is uncertain, and this issue is confusing to both pediatric and adult rheumatologists. unlike other categories of juvenile arthritis, jspa affects boys more often than girls, and peak age of onset is early adolescence. enthesitis is a defining characteristic of jspa. it is more common and affects more sites in the paediatric population compared with the adult one. the most commonly tender entheses are the insertions of the patellar ligament at the inferior patella, plantar fascia at the calcaneus, and the achilles tendon. arthritis in jspa is typically asymmetrical, oligoarticular (< joints) and involves predominantely the weightbearing joints. isolated hip joint arthritis may be the presenting feature and predicts early axial disease. involvement of the small toe joints is common in jspa but rare in other forms of jia. midfoot arthritis and tarsitis (inflammation of the intertarsal bones, overlying tendons, entheses and soft tissue) is highly suggestive of spondyloarthritis. in adults, inflammatory back pain typically heralds the onset of sacroiliitis, whereas children seldom present with symptoms of axial disease. however, according to several studies, sacroilitis can be asymptomatic in jspa and only detectable by imaging. other axial manifestations in jspa are inflammation of the lumbar apophyseal joints and interspinous ligaments, corner lesions of the spine and other sites of axial enthesitis-osteitis including the various ligamentous and muscular attachments of the pelvis. extraarticular manifestations of jspa are highly associated with axial disease and include acute anterior uveitis, bowel inflammation, psoriasis, and cardiac disease. clinical diagnosis of jspa can be difficult and the role of imaging may be more critical than in adult disease. the major goal of imaging in jspa is to identify children with early signs of axial disease, as this group is at the greatest risk for progression to as. the presence of axial disease in spa has also major implications for treatment decisions, since traditional firstline therapies appear to have minimal effectiveness in the management of axial inflammation. in addition, recent studies in adults suggest that earlier initiation of biologic agents (anti-tnfs) may slow radiographic progression. x-rays are not sensitive to acute inflammatory changes and will only show advanced disease in the sacroiliac joints. for these reasons plain radiographs are not useful in children or adolescents. ultrasound is a non-invasive, non-ionizing and relatively inexpensive technique that can be performed in a clinical setting. it is emerging as a valid diagnostic tool in spa and can be used to visualize peripheral synovitis, tendonitis and enthesitis, but the method is heavily operator-dependent and there does not yet exist a clear definition for the diagnosis and grading of enthesitis in children. secondary changes (calcifications, enthesophytes) have been observed much less in children compared with adults. there is a need for better consensus on abnormal ultrasonographic findings that define enthesitis lesions and standardization of methods. magnetic resonance imaging (mri) is a radiation free and sensitive imaging modality for detection of synovitis as well as cartilage and bone destruction. mri of the sacroiliac joints is increasingly obtained for early detection of inflammatory changes, as it shows active inflammatory (bone marrow edema, osteitis, enthesitis and capsulitis) and structural (erosions, subchondral sclerosis, subchondral fatty change and bony ankylosis) lesions of sacroiliitis long before radiographic changes become evident. in adults, mri has become the gold standard imaging modality for detecting arthritis and enthesitis. consensus definitions of lesions indicating pathology on mri are now incorporated into diagnostic criteria for adult with spa. in children and adolescents there is no gold standard mri technique and it is therefore not clearly defined whether changes s ( ) (suppl ):s -s pediatr radiol seen in the sacroiliac joints are pathologic or part of normal maturation in the growing skeleton. the use of contrast enhanced imaging for the detection of active sacroiliitis on mri in jspa is a major controversy. synovial enhancement can be detected without accompanying bone marrow edema in children, and it can be argued that contrast should be administered in order not to miss the diagnosis. some authors argue that contrast administration does not change or add substantially to the mri findings made on non-enhanced scans. certainly, given the risks associated with gadolinium administration, contrast should be used with caution. perhaps the use of contrast agents should be limited to selected cases when high stir signal in the joint is the only finding in order to confirm the presence of synovitis, and when the differential diagnosis includes etiologies such as infection or tumor. the development of new mri techniques has made it possible to perform whole body mri scans (wbmri) that allow assessment of the full range of affected entheses and joints. there is limited data on the utility of wbmri in the pediatric population. it is worth noting that edema-like changes seen in the marrow of healthy children is an important potential pitfall to consider during interpretation and further studies are required in order to establish specific reference standards for mri of the pediatric skeleton. diffusion-weighted imaging (dwi) offers a new approach to detect inflammation. inflammation produces an increase in the apparent diffusion coefficient (adc) of water molecules in affected tissues. several studies in adults and a few recent studies in children have demonstrated that adc is elevated in sacroilitis versus controls and that diffusion scores correlates well with stir images. dwi is promising as a potential biomarker of disease activity in jia and presents a novel approach to contrast-free imaging of synovitis. however, further studies are needed before it can be implemented in clinical practice. jspa is distinct from adult spa and manifests more frequently as peripheral arthritis and enthesitis. symptoms involving the spine and sacroiliac joints often occures later in this population. clinical diagnosis of jspa can be difficult, and imaging therefore plays an important role in the diagnostic workup of disease. identifying early signs of axial disease has major implications for treatment decisions and mri of the sacroiliac joints is increasingly obtained for early detection of inflammatory changes. however, mri criteria for sacroilitis in children are lacking. a major controversy in imaging of sacroilitis in jspa is the use of contrast, as children can have sacroilitis without accompanying bone-marrow edema. dwi presents a novel approach to contrast-free imaging of synovitis but further studies are needed before it can be used in clinical practice.wbmri has been shown to be more sensitive than clinical examination in the assessment of both disease activity and extent, but there is limited data on wbmri in children. normal variants in the growing skeleton may mimic pathologic changes and potentially cause overdiagnosing and -staging of disease. hence, there is an urgent need to establish specific reference standards for mri of the pediatric skeleton and to develop a gold standard mri technique for the axial skeleton in children and adolescents. juvenile idiopathic arthritis o. olsen; london/uk summary: juvenile idiopathic arthritis (jia) is common (about : , children). diagnosis and classification are based on clinical criteria. these criteria are in flux depending on ) contemporaneous knowledge about aetiology and ) available treatment options. radiology has currently no role in establishing the diagnosis. the clinical classification rests on whether the child has few joints affected (oligo jia), many joints (poly jia), has a condition similar to adult spondyloarthritis (entesitis-related arthritis) or other clinical presentations (systemic-onset jia, psoriatic jia, etc). radiology can potentially assess expressions of jia, such as synovitis, tenosynovitis, systemic manifestations and permanent damage caused by inflammation. it is therefore thought to play a part in gauging the disease activity. the clinical care aims at optimising the child's everyday function, reducing acute symptoms (pain, swelling, joint restriction), allowing normal growth, minimising long-term sequelae (joint deformity) and minimising adverse effects of medical treatment. medical treatment in jia is systemic (immuno-modulation) and local (steroid injection to joints and tendon sheaths). both modes of therapy may to some degree be guided by imaging. however, there currently is no evidence that any form of whole-body imaging is efficacious for guiding treatment. this means that, in principle, indication for imaging should be ) specific clinical questions, e.g. uncertainty regarding active inflammation at specific sites, or ) a high pre-test likelihood of inflammation at a site which is difficult to assess clinically and where imaging offers reasonable accuracy. one example of the latter are the temporo-mandibular joints where destruction is frequently seen at an early stage, often without prior symptomatic warning. there is one fundamental challenge for imaging research in jia: what is the reference standard? for lack of anything better, a standardised clinical examination is often used as 'ground truth'. the dilemma is obvious. if clinical examination is reliable and accurate, then why bother with imaging? but we think imaging offers an improvement, then we cannot use an inferior method to set the standard. this problem is not unique to jia. as is often the case, radiology in jia is all about: knowing your clinicians (i.e. the pretest likelihood for disease) being technically eloquent (e.g. using high-resolution us probes, not delaying post-contrast mri acquisitions) knowing what is normal (e.g. normal undulations in the articular surface, focal bone marrow signal variation) not being dogmatic about individual observations or measurements interpreting your findings in a clinical context the lecture will demonstrate similarities and differences among joints and modalities in children with variable-severity jia. the following points will be made: focal areas in the bone marrow with high signal (t ) and corresponding enhancement are often seen in healthy children. in isolation, these do not signify active inflammation. active synovitis in children often is not associated with (much) effusion the combination of synovial thickening with hyperaemia (us)/abnormal contrast enhancement (mri) and surrounding softtissue swelling suggests active inflammation, however there is (yet) no established system for quantifying hyperaemia/enhancement focal pits in the carpal bones do not represent erosions unless there is an associated cartilage defect radiographs are useful for detection of destructive abnormality in mri, scan fairly soon after injecting contrast. gadolinium physiologically leaks into the synovial fluid making it difficult to delineate the synovium a few differential diagnoses to keep in mind when there is mass-like swelling within or adjacent to a joint: vascular and neoplastic lesion, pigmented villonodular synovitis, synovial chondromatosis, lipoma arborescens. synovial inflammation is not always primary. even when there is an established diagnosis of jia, do consider that it may be secondary to biomechanical abnormality (erosion, osteochondral lesion, deformity). focal areas in the bone marrow with high signal (t ) and corresponding enhancement are often seen in healthy children. in isolation, these do not signify active inflammation. active synovitis in children often is not associated with (much) effusion the combination of synovial thickening with hyperaemia (us)/abnormal contrast enhancement (mri) and surrounding soft-tissue swelling suggests active inflammation, however there is (yet) no established system for quantifying hyperaemia/enhancement focal pits in the carpal s ( ) (suppl ):s -s pediatr radiol bones do not represent erosions unless there is an associated cartilage defect radiographs are useful for detection of destructive abnormality in mri, scan fairly soon after injecting contrast. gadolinium physiologically leaks into the synovial fluid making it difficult to delineate the synovium pulmonary manifestation of connective tissue disorders c. m. owens; london/uk summary: connective tissue diseases are an important cause of morbidity and mortality in children with very varied presentations. nomenclature is confusing and a more appropriate descriptive term would be "multisystem inflammatory disorder +/-autoimmunity". it is important for the radiologist to be aware of the protean radiological appearances and clinical manifestations. take home points: different patterns of diffuse lung disease (eg, desquamative interstitial pneumonia, non specific interstitial pneumonia, lymphocytic interstitial pneumonia, organising pneumonia, diffuse alveolar damage) may be present in several forms of collagen vascular disease, (and indeed other rheumatological conditions such as jia) including scleroderma, systemic lupus erythematosis, juvenile dermato and polymyositis, sjogren's syndrome and mixed connective tissue disease. these will be discussed in detail with illustrations for thin section high resolution ct with histopathological correlation. the clinical presentation, prognosis and response to therapy vary depending on the histological pattern of diffuse lung disease, as well as on the underlying collagen vascular disease. whole body imaging in children: sonography, ct, mri, nuclear medicine -what and when? r. a. nievelstein; utrecht/nl there are several (benign and malignant) disease processes in children that frequently involve more than one organ system or body region. diagnostic imaging of children with such multifocal or multisystem diseases has been quite challenging, often requiring a combination of different imaging techniques for a whole body coverage. the recent technical developments in computed tomography (ct), magnetic resonance imaging (mri) and nuclear medicine (nm) have changed the role of imaging in these children revolutionary. in the past, imaging techniques have been mainly used as a tool to detect the cause of illness and to assess the extent of disease spread before, during and after therapy (i.e. structural imaging). but nowadays, it has also become possible to use imaging techniques to gain information on the biological behavior of diseases before and during therapy (i.e. functional imaging). plain radiography, ultrasonography (us) and computed tomography (ct) have been the structural imaging techniques of choice for many decades, more recently supplemented by functional imaging techniques like single-photon emission tomography (spect) and positron emission tomography (pet). a major disadvantage of most of these techniques is the use of ionizing radiation, which may be associated with induction of second cancers later during life. this small but not negligible health risk is of particular concern in children as their tissues are more radiosensitive than adults and they have more years ahead in which cancerous changes might occur. that is why there is an increasing interest in the use of alternative imaging techniques that do not use ionizing radiation. with mri it is nowadays possible to acquire images with a high spatial resolution and excellent soft tissue contrast throughout the body, which makes it an ideal radiation-free tool for the detection of pathology, especially in soft tissue, parenchymal and bone marrow locations. moreover, recent technological advances have resulted in fast diagnostic sequences for whole-body mr imaging (wb-mri), including functional techniques such as diffusion weighted imaging (dwi). as a result, wb-mri has become a clinically feasible imaging modality for diagnosis and follow-up of multifocal and multisystem diseases in children. in this scope, the recent development of integrated pet/mri systems is very interesting, combining the superior structural imaging of mri with the functional (molecular) information of both imaging techniques while decreasing the radiation dose. traditionally, whole body imaging techniques have been mainly used for oncological indications, such as staging of malignancies, and monitoring of the effectiveness of therapy. however, whole-body imaging techniques are increasingly used for the diagnostic imaging of other benign multisystem diseases and indications, including chronic recurrent multifocal osteomyelitis (crmo), rheumatological diseases, neuromuscular diseases, neurofibromatosis type , generalized vascular malformations, multifocal osteonecrosis after intensive chemotherapy, fever of unknown origin, and post-mortem imaging. finally, these imaging techniques may be used for the screening of children with a cancer predisposition syndrome. during this lecture, imaging protocols and indications of the different whole body imaging techniques will be discussed with a focus on their clinical application in children with benign and malignant multifocal or multisystem diseases. ( ) (suppl ):s -s pediatr radiol appearances is important for any radiologist involved in child imaging, because we have an important role in characterizing the lesions and guiding purposeful and minimally invasive but successful diagnostic procedures. most head and neck masses in children are benign and have an inflammatory, infective, vascular or congenital cause (cf. special presentation on vascular malformations). malignant lesions are less common, however, early diagnosis is paramount as many of these cancers are readily treatable and often curable. differential diagnosis is guided by patient age, clinical presentation, tumour localisation, and imaging characteristics. while some masses such as (epi-)dermoids, fibromatosis colli and swollen lymph nodes including atypical mycobacterial infections (mott) may be readily diagnosed by clinical inspection und ultrasound, others present special diagnostic challenges. fibromatosis, for example, is a benign lesion with an often complex and potentially destructive local spread. some malignant lesions tend to be localised such as the embryonal rhabdomyosarcoma, while others may be part of a systemic disease such as lymphoma and langerhans cell histiocytosis (lchc). in case of a suspected malignancy, patients should be referred to a specialized centre which will be able to provide the full spectrum of multidisciplinary evaluation and treatment according to the guidelines of an international oncology study group. this is also important for image guided or surgical biopsies as long term outcome and survival of many of the young patients are directly associated with these initial diagnostic and therapeutic strategies. with its excellent spatial resolution in the near field, ultrasound is the method of choice for all superficial masses. an experienced paediatric radiologist will be able to identify most of the benign lesions and in other cases will be able to guide further diagnostic decisions. tumours in the midline require thorough workup to exclude an encephalocele or a dermal sinus with connection to the intracranial space. high resolution mri is required if such an extension cannot be ruled out by ultrasound or if a tumour is larger than the transducer's scan area. soft tissue tumours in the deeper parts of face and neck as well as tumours of osseous origin are also best delineated by mri. in lesions adjacent to the skull base contrast enhanced and fat saturated mr images with high spatial resolution are of utmost importance to completely depict the tumour's extension through the foramina and along the meninges (fig. ) . ct can provide additional information on the involvement of osseous structures. embryonal rhabdomyosarcoma. high resolution mri with fat saturation after contrast injection depicts the tumour's extension through the foramen ovale (long arrow) and along the meninges (short arrows). skull base and face lesions are less frequent in children than in adults. symptoms may be subtle or unspecific. depending on their localization, clinical findings may be common (nasal obstruction, otitis…) or more disturbing (cranial nerves palsies, exophthalmos, vision loss …). clinical history and physical examination findings are important to reduce the spectrum of differential diagnosis, but imaging data are the key features to determine the nature of these lesions. ct and mri play an important role in diagnosis, treatment survey and surgery planning of skull base and face lesions. skull base and face bone lesions are either intrinsic lesions of the bone or secondary to soft-tissue tumors or pseudo tumors invasion. this lecture will focus on bone intrinsic lesions, and include soft-tissue and pseudo tumors only as differential diagnoses. computed tomography plays the role for skull base and face of plain radiograph for long bones. therefore, the same semiology may be used to determine if the lesion is slowly or rapidly growing, aggressive or looks benign. helical ct allows reconstructions with both soft-tissue and bone algorithms as well as multiplanar reformations. it gives a good visualization of the anatomy of the skull base and allows a good depiction of the bone architecture. ct is first used for the initial work up of the disease but also for surgery and therapeutic planning (endoscopic sinus surgery with navigation). however, ct analysis may be challenging in children due to growth changes: normal process of pneumatization according to age, sutures not yet fused has to be recognized. some variations in pneumatization must not be mistaken for pathology: asymmetrical pneumatization of the petrous apex and arrested pneumatization of the sphenoid mimicking intraosseous lesion are the most common. both ct and mr imaging are complementary: most preferably, contrast-enhanced mr is associated with non-contrast high resolution ct. mri allows a good delineation of bone involvement of skull base lesion due to bone marrow changes, whether ct can fail to detect subtle extension within the bone. in addition to t and t weighted sequences, the use of specific sequences and/or techniques such as fat-saturation, diffusion, dynamic-contrast-enhanced sequences, and mr angiography helps to characterize the lesions. t spin echo sequence is mandatory to appreciate bone marrow infiltration in adults and older children. but when red bone marrow has not yet be replaced by fatty bone marrow, in young children, this can be challenging. it is useful to know the bone marrow fatty conversion of the skull base chronology. cranial mr can also be associated to whole body mr to look for multifocal or metastatic disease. epidemiologic data concerning bone tumors of the skull base are scarce due the rarity of these lesions. they can be classified according to their location within anterior, middle or posterior cranial fossa or classified according to their origin: osteogenic (osteoblastoma, osteoma, osteosarcoma...), chondrogenic (chondroma, chondrosarcoma), fibrous ( fibrous dysplasia, fibro-s ( ) (suppl ):s -s pediatr radiol osseous lesions..), notochord (chordoma), hematopoietic (leukemia, histiocytosis ), vascular (hemangioma), neuro ectodermic ( ewing sarcoma) or unknown origin (aneurysmal cyst, giant cell tumor). the aim of this presentation is to draw attention to skull base growth changes that can mimic pathology and to describe the imaging specificities of the most common bone tumors of the skull base and face in children. because conflicted nomenclature can cause confusion, accurate diagnosis and classification of these anomalies is important for proper clinical evaluation and management. many of these patients require multidisciplinary care, consequently the usage of a correct nomenclature across all disciplines is a sine qua non. the international society for the study of vascular anomalies (issva) classification, updated in , offers a comprehensive classification accepted by many subspecialities. this approach/ classification has facilitated correct communication for all medical subspecialties involved in the care of these complex vascular anomalies. pediatric radiologists play a critical role in evaluating these patients since the majority present during childhood. in this presentation, we present a state of the art mri imaging protocol with exemplary cases of the most common types of vascular anomalies in the pediatric trunk and extremities, using the current issva classification. in addition, we discuss the common syndromes associated with vascular anomalies such as klippel-trenaunay and lumbar syndrome. genetic skeletal disorders (gsd's) are a heterogeneous group of syndromes characterized by an intrinsic abnormality in growth and (re-)modeling of cartilage and bone. a large sub-group of gsd's may have additional involvement of other structures/organs beside the skeleton, such as the central nervous system (cns). cns abnormalities have an important role in long-term prognosis of children with gsd's and should consequently not be missed. sensitive and specific identification of cns lesions while evaluating a child with a gsd requires a detailed knowledge of the possible associated cns abnormalities. here, we will present and discuss a pattern-recognition approach for identifying relevant neuroimaging findings in gsd's guided by the obvious skeletal manifestations of gsd. in particular, we will discuss which cns findings should be ruled out for the various gsd. to facilitate this diagnostic approach the multiple gsd are classified based on the pattern of skeletal involvement ( . abnormal metaphysis or epiphysis, ) abnormal size/number of bones, ) abnormal shape of bones and joints, and ) abnormal dynamic or structural changes). skeletal involvement is defined in accordance with online mendelian inheritance in man. the spectrum of co-existing cns involvement is extracted from an extensive literature search. selected examples will be shown based on prevalence of the diseases and significance of the cns involvement. cns involvement is common in gsd's. a wide spectrum of morphological abnormalities is associated with gsd's. early diagnosis of cns involvement is important in the management of children with gsd's. this pattern-recognition approach aims to assist and guide physicians in the diagnostic work-up of cns involvement in children with gsd's and their management. not infrequently the correct radiological differentiation of skeletal and/or central nervous system findings secondary to non-accidental injury versus inherited genetic and/or metabolic disorders may be challenging. imaging findings may be non-specific, can result in incorrect diagnosis and subsequently inadequate patient management or initiation of faulty treatment. the diagnostic work-up of children suspected of non-accidental injury or genetic/metabolic disorders requires a multi-disciplinary approach involving many key players including physicans of various disciplines, nurses, psychologists, social workers and many more. a proper and detailed medical history and physical examination of the patient, collection of the relevant family history, a metabolic and genetic work up, a detailed interview of care givers, friends and family are essential for the correct and comprehensive evaluation of imaging findings. in the current session, various exemplary and possibly confusing cases will be interactively discussed with the audience by a panel of experts (susan blaser, thierry a.g.m. huisman and andrea superti-furga). goal is to offer a case based approach to challenging patients with discussion of the best diagnostic approach including differential considerations. the zikv is transmitted mainly by the bite of female aedes aegypti and aedes albopictus mosquitoes. other forms of transmission, including through sexual intercourse, blood transfusion, and neonatal, are currently under evaluation, although more elements are still needed to assess the real importance of these transmission routes . the course of the zikv infection is self-limited. so far, no specific symptoms have been attributed to the disease, and a wide variety of manifestations ranging from absent to mild symptoms (in % of cases) have been described. when symptoms are present, they may lead to a misdiagnosis of other bacterial and viral infections, especially other arboviroses in endemic areas. the most frequently reported symptoms are mild fever, cutaneous rash, fatigue, arthralgia/myalgia, and conjunctivitis. dizziness, malaise, edema of the extremities, anorexia, retro orbital pain, photophobia, gastrointestinal disorders, sore throat, cough, sweating, and lymphadenopathy have also been reported. infection by the zikv in adults may be associated with autoimmune complications such as guillain-barré syndrome . the laboratory diagnosis of zikv infection is based on the demonstration of the virus in the urine and blood using real-time reverse transcription polymerase chain reaction (rt-pcr). the main limitation of this diagnostic method is a false-negative result after the viremia is resolved. the serological diagnosis of the disease is limited due to cross-reactivity of the zikv with other viruses of the flavivirus genus, especially those causing dengue and chikungunya. physicians should be aware of this fact when the diagnosis of zikv infection relies solely on serological results. the diagnosis is also possible by igm measurement in serum, urine, or cerebrospinal fluid using enzyme-linked immunosorbent assay (elisa) . the prevention against zikv infection is similar to that of other arboviroses, including vector control and mosquito bite prevention. the first major zikv epidemics were reported in the french polynesia in and . at that time, some neurological changes were observed in neonates of infected pregnant women but were not associated with a maternal-fetal transmission of the virus. the growing increase in the number of cases and the severity of the infection specific to this subpopulation then led to the evidence of a congenital disease . in brazil, the situation became alarming with the report of a high number of infected individuals in the second half of , . the brazilian ministry of health attributed to congenital zikv infection the -fold increase in cases of neonatal microcephaly in the northeastern part of the country, particularly in the state of pernambuco. this led the world health organization (who) to declare the zikv infection a "public health emergency of international concern" in february . the main challenge for radiologists practicing in regions of endemic zikv infection is to become familiarized with findings of congenital zikv infection in perinatal imaging studies; this is particularly important for the prenatal screening of pregnant women , . the diagnosis of zikv infection in the fetus by neuroimaging is based on prenatal ultrasound (us), especially in the third trimester, and complemented with magnetic resonance imaging (mri). postnatal imaging was obtained by transfontanellar us, ct or mri. the main imaging findings on ct are microcephaly, an exuberant external occipital protuberance, rectification of the frontonasal angle, and a redundant scalp skin. three-dimensional ( d) reconstruction of al skull permits a better evaluation of these findings and enhances the parents' understanding of the disease. moreover, ct scan data may yield a d virtual physical model that can maybe obtained from ct scan data and printed onto using thermoplastic acrylonitrile butadiene styrene . the aim of this study was to describe the perinatal imaging findings in cases of congenital zikv infection. we studied mothers diagnosed with zikv infection from october to november . they had all presented a maculopapular rash and fever during the first or second trimester of pregnancy, and their neonates presented neurological defects that were attributed to intrauterine transmission of the zikv. the maternal diagnosis of zikv infection was confirmed by serology (n= ) or rt-pcr (n= ). all patients were torch (toxoplasma, rubella, cytomegalovirus, herpes simplex) negative. prenatal us was performed every weeks after the first imaging findings, and fetal mri was obtained in all cases. microcephaly was considered present when the infant's head circumference was two standard deviations below the mean value for age and sex or below the second percentile. postnatal imaging follow-up was obtained in all cases by transfontanellar us, ct or mri. we found several cns malformations, including lissencephaly, pachygyria and/or polymicrogyria, cerebral atrophy (panel ), enlarged cisterna magna with abnormalities of the corpus callosum, ventriculomegaly, brainstem hypoplasia, malformation of the cortical development, and cortical and/or periventricular calcifications mainly in the junction between the cortical and subcortical white matter (panel ). the skull of the infants had a collapsed appearance, with overlapping sutures and redundant skinfolds (panel ). craniofacial disproportion was easily identifiable, and arthrogryposis was identified in one case. similar neurological findings were observed in the infected patients and seemed to differ from findings of other infectious diseases. the finding of microcephaly in neonates with congenital zikv infection seems to be only the tip of the iceberg, as several cns malformations have been identified in connection with the disease. in brazil, a spectrum of imaging findings associated with congenital zikv infection has been observed. such findings are useful in helping radiologists to identify suspected cases of the disease. panel : prenatal ultrasound ( weeks) shows calcifications (arrows) and microcephaly. axial and sagittal t shows relative smoothness of the brain surface (arrows) and assymmetric colpocephaly. panel :ax t -wi multiple cortical-subcortical fronto-parietal hyperintense foci (arrows) and markedly hypointense on swi. sagittal t : dysgenesis of the corpus callosum, with dilation of the posterior horns of the lateral ventricles (colpocephaly). pre-and postnatal imaging in zika virus: where are we? early insights into zika's microcephaly physiopathology, from the epicenter of the outbreak: a case for teratogenic apoptosis of central nervous system. p. jungmann; recife/br early insights into zika's microcephaly physiopathology, from the epicenter of the outbreak: a case for teratogenic apoptosis of central nervous system. in mid-october , intense interaction among surgical pathology and fetal medicine specialists from university of pernambuco was only focused on the dramatic and non explained ultrasonographic (us) findings and hopelessness due to lack of explanations on the odd us discoveries on the first gestational cases of zika's microcephaly. this is the field of our history of a physiopathological hypothesis on zika virus (zikv) related microcephaly when it first struck pernambuco state (pe), northeast brazil, the place that has been at the front line of the global response to the microcephaly and responsible for a large amount of data from affected children. the outbreak onset came with a sudden increase in microcephalic newborns being reported in pe state from august (panel, fig. ) . zikv was previously thought to cause a relatively mild disease, but was recently accepted to lead to severe and diverse neurologic conditions in s ( ) (suppl ):s -s pediatr radiol some children born from infected mothers and in adults . the scientific community is actively trying to uncover the extent of these disorders but little has been reported on the early days of the outbreak when doctors were approaching the unknown. while evidence that zikv is related to microcephaly in newborns is accumulating, the mechanisms of how the virus affects the fetus is still uncertain. in the outbreak onset we had to face daunting challenges to search the cause of microcephaly and the emotional toll on the families. we took a very early approach from microcephalic fetuses on gestation and microcephalic babies on clinical follow-up from different pe areas, evaluated between october and december in oswaldo cruz hospital, to propose the early physiopathologic hypothesis that, a viral-related brain developmental disruption could be the basic neuropathogenesis in zikv babies instead of a direct injurious process due to viral insult followed by active inflammation. the eight pregnant women were all in the rd gestational trimester and had had normal us follow-ups till week th . crucially, we were facing a temporal-geographic association of cases presenting an unanticipated pattern of us alterations. because of their late alarming findings they were re-examined and the us scans revealed sudden encephalic alterations after th gestational week. such devastating us clustering images were not seen here before, but are now considered as part of the "congenital zika syndrome". we observed late appearing severe dysmorphic encephalic changes in out of fetuses, including small skull, small brain, sub arachnoidal space enlargement, ventricular dilation, brain calcifications of varied shape and distribution, inclined frontal bone, progressive decline of head growth potential, early fontanels closure and redundant scalp (panel, figs. a, b). we had no clues on the causes and mechanisms responsible for this phenotype of severe alterations. thus, we had no explanation to offer to patients, in particular, or to the medical community. both as physicians and human beings, we were committed straightaway to continue the study of these victims of an unknown medical tragedy, engaging our expertise in fetal imaging and immunopathology. from beginning october, the first microcephalic babies were referred to the upe pediatric infectology service for initial investigation. strikingly, the newborns exhibited "healthy" appearance, excluded the microcephaly itself and motor sequels. we then looked for csf analysis of the microcephalic babies. for that, we obtained from dr. patricia travassos, a csf specialist at upe, a cohort of csf samples that have been studied for signs of meningitis or encephalitis. about % ( cases) of the csf analyzed looked normal for any signs of central nervous system ongoing inflammatory responses (panel, fig. ). the babies had been examined by outpatient clinic dr angela rocha, from the upe hospital infectology reference center that have stated that although small, the babies were near to full term gestation ( - weeks gestation), had good apgar scores and variable degrees of microcephaly and neurologic impairments, i.e. contractures, spasms, irritability and in some retinal macular atrophy. during the follow-up, the babies were cared at home, breastfeeding, gaining weight and having routine vaccines. none of them expressed signs of ongoing inflammatory reaction in the cns (panel, fig. ) or alterations on peripheral blood count and other routine laboratory tests up to months of age. despite the striking neurological phenotype, % of the babies were negative for torch agents, no deaths were recorded. furthermore only in january , the first evidence associating zikv to microcephaly from rt-pcr test on amniotic fluid was reported . astonishingly, a particular kind of physiopathological process linked to fetal brain development was arising without clinical manifestation of inflammatory reactions or necrotic processes in these babies. unfortunately, no necroscopic samples of affected brain tissues were available to us to monitor the presence of putative neural dysgenesis and the very nature of brain calcifications background offering histological support for our hypothesis. nevertheless, with this restricted dataset we hypothesized that whatever the etiologic agent involved in these cases, its physiopathologic mechanism must trigger the cellular death programthe apoptotic process -at a particular development window on the cns, assuming clinically that the agent was not encephaalitogenic but silently tertogenic. if not, the clinical outcome of affected babies would not be so mild as far as signs of inflammation on cns was concerned. consequently, the inflammation-free clinical status of patients suggested us that a massive enhanced apoptotic cell death during the window of telencephalic expansion was the most probable physiopathologic process operating this microcephaly phenotype, with no direct direct lytic brain lesion or significant necrosis due to usual injury. furthermore, knowles and penn stated that this window is very active to select the "fittest" neural cells by a constitutive apoptotic pathway. we so hypothetized that during this developmental time window, the "fit or not fit" status of the rapid, transient amplifying neural progenitors cells facing zikv, would heavily shift the selective process toward the self-elimination of virusbearing cells through apoptotic pathways. thus, zikv-enhanced constitutive apoptotic mechanisms would lead a massive loss of developing telencephalic neuronal precursors and, consequently, provoking losses of dividing cells and the arrest of further brain development. this could be particularly inferred by the absence of the characteristic morphology of late stages structures of neocortex, according with our us images of zikv microcephalics in gestation. similar processo could also be inferred to neurocrest derivatives as deformities in the viscercrany always accompany the cephalic malformation. our initial understandings based on clinical examination on the field, when no specific laboratory test, necroscopic data or experimental evidence on the disease causality were available, conducted our physiopathological approach to the "apoptosis hypothesis" for zika microcephaly that is now gaining strong support. in february, mlarkar et al showed clear connection between zikv and microcephaly, presenting cns histopathologic analyses, revealing remnants of neural germinative matrix, intense gliosis, alterations in cortical ribbon, calcifications in gray and white matters without associated necrosis, encephalitis or meningitis and the presence of the virus, further supporting neurodevelopmental arrest. similar results were showed by driggers et al . the ct scans from microcephalic babies from hazin at al , have added details of brain development arrest with no radiological signs of brain destruction or active inflammation. finally, experimental models have provided a body of evidence f or neuroprogenitors permissiveness to zikv and viralinduced apoptotic process. tang et al demonstrated by icq that the zikv infection of cortical neural progenitors attenuates their growth and increases caspase- activation, calling for an apoptotic process. this finding was corroborated by the up regulations of caspase- genes by rna sequencing. nowakowsky et al demonstrated that zikv may hijack axl protein as an entryway to infection. interestingly, axl is highly abundant on the surface of neural stem cells but not on differentiated neurons in the developing brain. recently, cugola et al demonstrated that zikv was able to cause cns congenital brain dysgenesis upon vertical transmission in mice. in parallel, human brain organoids infected by zikv show a reduction of proliferative zones and disrupted cortical layers, so targeting cortical progenitors and inducing apoptotic cell death with impaired development. for babies born with zikv-related microcephaly, the many expected consequences besides the evolving congenital neurosequels, are the unanticipated pattern of persistence of zikv in cns host cells, unsafe maintenance of neuron genome stability on remaining arrested populations, implying risks for brain tumors, risks for impaired adult type neuron wiring and neuron survival in an affected neuronal circuitry. in brief, evolve life with a wide vulnerable brain. the outbreak of zikv in the americas will eventually decline as herd immunity increases, but the world remains at risk of further waves of infection in affected countries and spread into new territories . while experimental studies will be carried out to fully understand the pathophysiology of zikv infection in the developing fetus, our findings provide a coherent and testable physiopathological hypothesis for cns teratogenic phenotype linked to zikv congenital infection, which may be critical for the clinical care of pregnant mothers and their babies before and after birth. take home points: fetal dysmorphisms detected by ultrassonographic and mri images in congenital zika syndrome are late findings, usually after the th gestational week and requires acurate analyses. clinically, zika's virus microcephaly is an infectiuos congenital condition that is not encephalitogenic but primarily teratogenic on the nervous system. the most important process leading to zika's virus microcephaly is pathologically induced apoptosis in telencephalic neuroprecursosrs cells and neurocrest precurssors cells. viral induced autophagy and low antiviral responses during the fetal period are linked to zika virus persistence in the central nervous system of affected new borns and babies a. vossough; philadelphia/us summary: susceptibility-weighted imaging (swi) has proven to be a valuable mr imaging sequence in a variety of applications. pediatric imaging has also immensely benefitted from this technique. in this presentation we will review pediatric neuroimaging applications in trauma, arterial and venous vascular disorders, hypoxic-anoxic injury, congenital malformations, congenital heart disorders, neoplasms, and pediatric degenerative disease. use of swi in pediatrics other than demonstrating hemorrhage and calcification will be reviewed. challenges in the clinical use of swi in pediatrics, interpretive pitfalls, and sources of clinical misinterpretation of swi will also be explored. we will also briefly present ongoing research and clinical use of swi in pediatrics and potentials for future collaborative investigations. & swi is highly sensitive in detection of susceptibility effects on mri. & in many cases, but not all, swi processing can differentiate between calclium and blood products. & quantitation information can also be obtained from swi with further processing. state of the art imaging of the single ventricle d.m. biko; philadelphia/us there are many congenital heart defects that result in a functional single ventricle. this may be functional or anatomical as a result of a dysfunctional valve or absent or ineffective pumping chamber. the repair of single ventricle physiology most often involves a staged reconstruction due to changing physiology ultimately resulting in a total cavopulomonary connection or fontan procedure. to appropriately image the single ventricle throughout its stages of palliation, familiarity with the physiology of the various steps in surgical palliation of the single ventricle is essential although echocardiography is a mainstay of cardiac imaging, cross sectional imaging has a vital role in the evaluation of the single ventricle. the role of ct angiography is mostly for anatomic evaluation. although it is fast and has high spatial resolution for evaluation of vasculature, ct has lower temporal resolution than mri and is unable to quantify flow. ventricular performance along with quantification of flow can be performed with mri. systemic to pulmonary collateral flow, which has been shown to result in adverse outcomes after fontan, can be quantified. valvular insufficiency and myocardial scarring can also be assessed. additionally, high anatomic vascular detail can be obtained with mri, particularly with the recent investigational use of the blood pool agent ferumoxytol. mri also has the ability to assess the lymphatics either through non-contrast t weighted imaging and/or dynamic contrast mr lymphangiography as lymphatic pathology may play a role in postsurgical hemodynamics in single ventricle patients. this lecture will focus on the use of ct and mri in the evaluation of the single ventricle particularly concentrating on the developing use of mri for anatomic and physiologic assessment. take home points: in single ventricle physiology, there is only one effective pumping chamber. familiarity with the physiology of the various steps in surgical palliation of the single ventricle is essential in imaging this disorder ct angiography provides high anatomic detail but limited in its assessment of physiology since it cannot quantify flow and has lower temporal resolution than mri. mri can evaluate ventricular performance, quantify flow and valvular insufficiency, and assess myocardial scarring. high anatomic vascular detail can also be obtained with mri particularly with the emerging investigational use of ferumoxytol. with non-contrast t weighted mri and/or dynamic contrast mr lymphangiography, lymphatic evaluation can be performed which may play a role in post-surgical hemodynamics in single ventricle patients. neuroimaging in head trauma m. argyropoulou, g. alexiou; ioannina/gr summary: objective: head trauma in children is one of the most common reasons for visiting emergency department. however, only a small portion of patients will have a traumatic brain injury. patients with moderate or severe head trauma should undergo ct scan, however, a debate exists for the indication and yield of neuroimaging for minor head trauma. we performed a systematic literature review on the accuracy of symptoms and signs in children with minor head trauma in order to identify those with severe intracranial injuries. materials: a systematic literature search of medline ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was performed to identify studies assessing the diagnosis of intracranial injuries in children. the authors independently performed critical appraisal and data extraction. results: we identified studies that evaluated the performance of findings for detecting intracranial injury using the reference standard of neuroimaging or follow-up examination. mechanism of injury, multiple vomiting episodes and decline in gcs score were more commonly associated with severe intracranial injury on ct. normal variations in the amount of joint fluid, ganglion cysts, bone marrow edema, and bony depressions that resemble erosions are frequent in the wrists of children. the results of a follow-up of a healthy cohort aged - will be presented. the cohort was examined twice with mr of the wrist, and the second time also with a cartilage sequence for better visualization of the bony depressions. knowledge of these normal variations is important because they can resemble disease. bone marrow edema, joint fluid more than mm, and bony depressions that can resemble erosion are frequent findings in the normal wrist. take home points: bone marrow edema, joint fluid more than mm, and bony depressions that can resemble erosion are frequent findings in the normal wrist. these findings can not be attributed to dissease without additional findings of synovitis. a cartilage sequence can be of use in the differentiation between true erosions and bony depressions. mri scoring of the wrist in patients with jia-current status and future perspectives c. nusman; amsterdam/nl the wrist is a frequently affected joint in patients with juvenile idiopathic arthritis. due to recent improvements in treatment strategies, permanent damage is not that common anymore. also, imaging has been playing a key role in monitoring the disease activity in the wrist of jia patients. the past years lots of efforts have been made to improve the assessment of acute and permanent changes of the jia wrist. requisites and recommendations for the mri protocol to use for of the jia wrist are available in literature. currently, the features of scoring the jia wrist are synovitis, tenosynovitis, bone marrow edema and bone erosions. the repeatability of the above-mentioned scoring features proved to be acceptable. recent studies showed that the appearance of the wrist in healthy children can mimic pathology. therefore, construct validity of the scoring features needs to be assessed by comparing wrists of healthy children with the wrists of jia patients. & construct validity of the scoring features needs to be assessed by comparing wrists of healthy children with the wrists of jia patients a novel radiographic scoring system for permanent hip involvement l. tanturri de horatio , p.l. di paolo , s.c. shelmerdine , p. toma , k. rosendahl ; rome/it, london/uk, approximately - % of children with jia, particularly those with systemic onset disease, will have hip-involvement within - years after disease onset. as scoring systems for radiographic changes in children with hip involvement are lacking, we aimed to examine the reliability of potential markers and suggest a novel scoring system. a set of hip-radiographs from children with jia and clinical hipinvolvement: seen at the outpatient clinic at great ormond street hospital (gosh), london, and seen at ospedale pediatrico bambino gesù, rome, was used. all hip radiographs were scored in a blinded fashion, once by an experienced paediatric radiologist and a paediatric radiologist with minor experience in musculo-skeletal imaging in rome, and twice by an experienced radiologist and a research fellow in bergen/ london. radiographic findings suggestive of ) destructive change (bone erosion, flattening of the femoral head, squaring of the femoral head contour, presence of sclerosis, joint space height, and ) growth abnormality (length and width of the femoral neck, varus/valgus deformity, the ccd angle and the trochanteric-femoral head height) were assessed. assessment of erosions of the femoral head, femoral neck and the acetabulum showed moderate to good agreement for the same reader. the inter-reader agreement was, however lower. there was a high to moderate ( ) (suppl ):s -s pediatr radiol agreement for the assessment of femoral head flattening using the mose' circle. the measurements of femoral neck length and width, the ccd and trochanteric-femoral head lengths were precise, with % limits of agreements within - % of the mean. we have identified a set of relative robust radiographic findings suggestive of growth abnormalities and destructive change in children with hip-jia, and suggested a novel scoring system. x-ray of a years-old jia patient with severe chronic hip involvement. x-ray of a years-old boy with growth abnormalities on hips (bilateral coxa magna). in jia hip involvement is often a predictor of a severe disease course. radiographic findings vary according to mode of onset and age: in younger children the initial findings may be developmental rather than destructive while children with later onset jia may have destruction/narrowed joint space as the first feature. several of the commonly used radiographic findings for chronic hipchange are inaccurate. we have identified a set of relative robust radiographic findings suggestive of growth abnormalities and destructive changes in children with hip-jia, and suggested a novel scoring system. bone age assessment -statement from the msk task force k. rosendahl; bergen/no summary: age assessment is an important, yet complex and challenging issue that authorities may need to perform to determine whether an individual is an adult or a child in circumstances where their age is unknown. there is currently no method which can identify the exact age of an individual and there are concerns about the invasiveness and accuracy of the methods in use, namely analysis of documentary evidence, interviews, physical or other form of medical examination such as imaging. the main imaging methods include carpal, collar bone and dental examinations. whilst many countries make use of these methods they do not apply them in the same way and often use different combinations and/or order. one of the main reasons for this is the fact that age assessment procedures remain to a large extent determined by national legislation, with procedures evolving through national jurisprudence (ref.: european asylum support office (easo age assessment practice in europe)). the ethical and legal aspects of using bone age to determine age will be addressed in a statement from the msk task force. the ethical and legal aspects of using bone age to determine age will be addressed in a statement from the msk task force. & the application of drls should be the responsibility of all providers of x-ray imaging. this means that drls should also be applied to imaging performed outside the radiology department. & the physical quantity used to establish drls should be an easily measurable quantity, usually directly obtainable from the x-ray equipment console, obtained either by manual recording or preferably by automatic recording and analysis. organ doses and effective dose are not considered feasible as a drl quantity because these cannot be easily determined. the ultimate mission of eurosafe imaging is to support and strengthen medical radiation protection across europe following a holistic, inclusive approach. most common imaging procedures in children and their contribution to collective dose e. sorantin , c. granata ; graz/at, genova/it summary: several countries have released "diagnostic reference levels (drl)" for imaging procedures using ionizing radiation. unfortunately those drl differ in types of procedures and granularity as well as information about the proportion of pediatric patients within the different examinations are sparse. therefore an more evidence based approach seems to be feasable -meaning releasing drl first for frequent and radiation burdened examinations. therefore a survey within europe was conducted and a questionnaire was sent to key persons of the european society of pediatric radiology (www.espr.org) as well to members of a large academic, interdisciplinary, international network within the ceepus programme (central european exchange programm for university studieswww.ceepus.info). alltogether centers were contacted and an response was received from ( . .%). from one center only frequencies for interventional radiology was sent. plain films: most frequent procedures are extremities ( . %), followed by chest films ( . %) -both account together for more than ¾. flouroscopy: voding cysto urethrography (vcu) . %, followed by upper gastro intestinal (gi) series with . % -again representing / of those examinations. computed tomography: head & neck . %, chest . %, abdomen . % -together almost %. interventional radiology and cardiac interventions: only limited data available and procedures quite hardly standardize and comparable. it seems adviseable, that only a few procedures are suitable for drl like peripheral insertion of vascular lines, occlusion of ductus arteriosus botalli or stent implantation for coarctation. in order to estimate the contribution to the relative collective dose all values were normalized to a chest xrays ( . ) and the following numbers could be calculated: abdominal plain film . , skull . , ct head . , ct chest . , ct abdomen . . the most frequent imaging procedures using on ionizing radiation are: in plain films extremities and chest xrays in flouroscopy vcu and upper gi series in ct head, chest and abdomen therefore eu wide drl should be released for those examinations. as it could be expected chest ct is the main contributor to the collective dose. since the espr abdominal (gi and gu) imaging task force has changed its name and agenda, extending from initially only genitourinary queries to also other abdominal imaging topics, new projects have been added such as for example imaging in anorectal and cloacal malformations, imaging in paediatric inflammatory bowel disease (ibd, a joint project with esgar), or paediatric abdominal ceus applications. results of these new projects will be presented in the upcoming talkshoping that again (as the last procedural recommendations and proposed imaging algorithms) our proposals and recommendations will help to standardise paediatric imaging, to reduce radiation burden, and to facilitate comparable imaging data for future research. other topics in this session are a proposal for a more standardised approach to gastrointestinal ultrasonography, and considerations on gadolinium applications in children in the light of new observations (i.e., gadolinium deposit in tissue even in children with normal renal function). the work goes ononly achievable with active participation of interested and competent members. many interesting topics for either recommendations or joint research are on the list such as addressing late decompensating pujo or specific imaging needs in ibd in early childhood; other new ones may be proposed by any task force member. thus all espr members are invited to join the group, work with us and share their expertise. ( ) (suppl ):s -s pediatr radiol contrast enhanced us in childhood -applications in children: literature review and results from the questionnaire c. bruno; verona/it in adults, following the characterization of focal liver lesions, several applications of contrast-enhanced ultrasound (ceus) have emerged in the last two decades, since second-generation contrast agents have been introduced and approved for use in most european countries. from many points of view, children represent an ideal population for ceus, because of the absence of radiation exposure and of need of sedation. moreover, due to the small body size many anatomical targets in children can be adequately explored with high-frequency ultrasound, obtaining images with higher spatial resolution than in adults. however, to date comparatively few data on pediatric ceus are available. although very rare and usually mild, possible adverse effects of contrast agents probably limit their use in many centers. in addition, the intravenous administration of ultrasound contrast agents in children is still off-label in europe, which makes informed consent necessary in every case. finally, for unclear reasons information on this topic does not flow easily. & from the comparison between the data available, similar or better results are likely to be obtained with ceus in children than in adults, and some specific pediatric indications might be proposed. imaging in ibd-joint recommendation statement with esgar f.e. avni , m. napolitano , p. petit ; brussels/be, milan/it, marseille/fr the first joint esgar/espr consensus statement on the technical performance of cross-sectional small bowel and colonic imaging ( ) objective: to develop guidelines describing a standardized approach to patient preparation and acquisition protocols for magnetic resonance imaging (mri), computed tomography (ct) and ultrasound (us) of the small bowel and colon, with an emphasis on imaging inflammatory bowel disease. methods: an expert consensus committee of members from the european society of gastrointestinal and abdominal radiology (esgar) and european society of paediatric radiology (espr) undertook a six-stage modified delphi process, including a detailed literature review, to create a series of consensus statements concerning patient preparation, imaging hardware and image acquisition protocols in pediatric and adult patients. the delphi process is constructed as follow: step questionnaire construction to includes all contents relevant to the guideline and set up of working groups; step questionnaire completed by all committee member, step literature search; step draft consensus produced by each wg based on the literature review and questionnaire responses; step committee members indicate agreement or otherwise for each individual draft consensus; step acceptance of agreed statements (more than % of members), face to face meeting to modify statements without agreement. committee members indicate agreement or otherwise for each modified consensus statement and final consensus statements. the questionnaire was split into four broad topics, each of them treated by a subgroup including in each of them a pediatric radiologist: ( ) patient preparation for mre/mr enteroclysis/cte/ct enteroclysis, ( ) mre/ mr enteroclysis technique and sequence selection, ( ) cte/ct enteroclysis technique, and ( ) enteric us patient preparation and technique. after an extensive literature research each member were instructed to always base their statements on the retrieved literature wherever possible, and to this end graded the strength of retrieved relevant publications from i (high) to v (low) using the criteria of the oxford centre for evidence based medicine ( ) during their review process. if no relevant literature was available for a particular item, members used expert opinion to construct the consensus statements. the pediatric guidelines were based on the opinion of pediatric radiologists and adult radiologists who have experience in pediatric practice. & it is recommended that children aged - should not eat any solid & it is recommended that the use of a spasmolytic agent is optional. unlike adult practice, the use of spasmolytic prior to mre is considered optional in paediatric patients and use is likely dependent on the age of the patient, with older children more likely to tolerate spasmolytic injection. there are data supporting the benefits of glucagon on image quality, at the expense of prolonged imaging time and precipitation of nausea in just under half of paediatric patients ( , ) . however, high diagnostic accuracy can also be achieved without spasmolytic ( ) . & it is recommended that children aged over years should be nil by mouth for carbonated and milk beverages for - h. ingestion of still water or non-carbonated fruit juice is recommended. & it is recommended that for dedicated colonic evaluation, a standard protocol without specific modification is used. & use of a spasmolytic agent is not recommended. & the use of i.v. us contrast is not recommended. & it is recommended that scan coverage should include an abdominal and pelvic examination, including the liver. there are no specific recommendations as to the use of hydro us in the paediatric patient as practice is not well developed. if oral contrast is given prior to us, it would seem sensible to follow the recommendations for mre in the paediatric population & it is recommended that if ct scanning is used in the paediatric population, no specific preparation is usually required although administration of positive oral contrast could be considered; for example, prior to percutaneous drainage of abscesses. limitations: there is little evidence in the literature to ascertain all these proposals. the recommendations were mainly based on expert opinion. no recommendations have been proposed for children before years of age. especially the benefice of mre under sedation ( ) in the younger compare to us doppler need to be explored. contrast media application is essential for a number of mri studies in children. there is some evidence that gadolinium-based contrast agents (gbca) are well tolerated in infants and children. the risk of adverse reaction is no higher in children than in adults. there are only few data available about pharamakokinetics in children, especially for the use of gbca in neonates. age-adapted reference values of the glomerular filtration rate (gfr) have to be used to identify children with a potential risk. in the past few years there was some attention toward the potential cellular toxicity of gadolinium and its role in the development of nephrogenic systemic fibrosis (nsf). there were only few children identified with proof of nsf. but, particulary renal insufficiency, poor hydration, acidosis and inflammation increase the risk for nsf. because the cases of nsf have been observed with linear componds the guidelines ( ) (suppl ):s -s pediatr radiol from the esur and the espr and others propose to avoid linear compounds and to prefer macrocyclic gbca. in the past year several studies have described observations about possible gadolinium retention in the brain; hyperintense brain structures in native t weighted sequences were verified -globus pallidum and dentate nucleaus -also in children undergoing multiple mri examinations with gbca application. so, repeated mr investigations within a short time should be avoided -the cumulative dose of gbca should be recorded. consider all these points, the benefit of a contrast-enhanced study should be weighted against the potential risks before administering a gbca for each child separately. but, never deny a child an indicated cemri study. use single dose application ( . - . ml/kg body weight), improve renal function and hydration, balance acidosis -and ask your pediatric nephrologist íf necessary. gadolinium-based contrast agents are safe. macrocyclic compounds should be used in children. avoid contrast media in neonates and be careful in infants. identify risk factors. avoid repetivite application. procedural recommendation: how to perform pediatric gastrointestinal us m.l. lobo , m. riccabona ; lisbon/pt, graz/at summary: ultrasound (us) is the first imaging modality applied in the investigation of abdominal complaints in children, and an increasingly valuable imaging tool in the assessment of the gastrointestinal (gi) tract in neonates, infants and children. a comprehensive us examination is a critical first-step to optimize the potential of us diagnostic yield in many paediatric gi conditions. using proper high resolution transducers and graded compression technique is an essential part of gi us examination. a methodical and systematic analysis is crucial to facilitate a thorough evaluation of the bowel segments as complete as possible: follow bowel in a cross section, complete by longitudinal and oblique views. for some bowel sections filling is essentialsuch as stomach for gastroesophageal reflux and pyloric function, and distensibility and size of the colon by enema (e.g. for query microcolon). modern us methods are valuable, but not a pre-requisite. proper documentation of abnormal size of the gi tract segments, their luminal content, peristalsis, bowel wall characteristics and its surroundings, as well as local tenderness should be noted. a proposal for recommendation on how to perform paediatric gastrointestinal us will be presented for public discussion. & careful and dedicated us examination is crucial to obtain maximum anatomic and functional information in many gastrointestinal disorders in children. & systematic and methodical analysis helps to assess the bowel as complete as possible. & satndardization of us technique is essential to optimize us diagnostic capabilities and to allow for comparable examinations wich is essential to improve future evidence-based knowledge. hominid evo-devo: reconstructing the evolution of human development c. zollikofer; zurich/ch from an evolutionary biologist's perspective, modern humans represent the only surviving species of a group of highly specialized "bipedal great apes". they evolved more than seven million years ago in africa and managed to spread over the entire globe. in this talk, i will trace the history of our species with an emphasis on key developmental innovations that underlie major evolutionary innovations. why are we born with brains that have the size of adult great ape brains? and why do we grow up so slowly and get so old? i will highlight how advanced biomedical imaging methods help addressing these questions, and show how combined fossil, clinical and great ape data yield surprising insights into the evolution of our development. to present our experience with innovative imaging in pediatric interventional radiology. imaging technologies presented will include: . use of bubble contrast (lumeson) for indicatons including; complex pleural effusion and abdominal collection assessment pre and post therapy, primary g tube placement, renal perfusion pre and post rena artery angioplasty, vascular patency during central venous line placement, vascular malformation therapy and biliary tube assessment. . intravascular us (ivus) in arterial intervention pre and post angioplasty and venous thrombolysis intervention. . optical coherence tomography pilot study assesssment for renal artery intervention -validation in normal subjects. currently this imagng which uses laser light technology to assess vascular mural detail at the micron level, is only validated in coronary artery intervention in adults. . mr overlay -a technology that fuses mr imaging with low dose fluoroscopy and can faciltate biopsy of mr positive/ct negative lesions in the ir suite. focus will be on bone lesion biopsy and vascular malformation therapy. critical structures to be avoided can be outlined on the mr and transposed onto the fluoroscopic image during biopsy. in our experience this technology has promise in the pediatric setting with significant dose reduction when compared to ct. . mr fusion and i guide fusion technology enables an mr positive/ct negative lesion that would require ct guided imaging to be biopsed, using low dose c -arm ct, with fusion of the ct and mri images performed using landmarks, facilitating fluoroscopically guided biopsy in the ir suite. critical landmarks/structures to be avoided can be outlined on the ct or mr and transposed onto the fluoroscopic image during biopsy path planning and orchestration. focus will be on bone lesion biopsy. . color parametric flow related imaging in vascular interventionthis software enables time to peak opacification of arterial or venous contrast to be color coded in time and can provide adjunctive information for assessent of perfusion change during vascular intervention such as renal artery angioplasty, dialysis access intervention and cerebral embolization. . mr guided intervention -this focus will be on the initial development of an mr interventional program and our initial experience with mr arthrography. discussion will also involve the use of this modality for vascular malformation sclerotherapy and other msk interventions such as biopsy and nerve injections. . high frequency us imaging-focus will be on the use of a mhz us probe in the ir suite for various indications including visualization of smaller targets such as neonatal central venous access, superficial vascular malformation therapy and thyroid fine needle biopsy. . participants will become more familiar with exisitng and emerging innovative imaging technologies for pediatric intervention. participants will learn about the various indications and limitations of these technologies. . participants will gain insight into the process of introducing new imaging modalities into their pediatric interventional practice. increasing evidence supports the notion that autism spectrum disorder is associated with anomalies of brain function and connectivity. it is also evident that there are atypicalities in development/maturation of brain systems. particular promise arises from findings of atypical electrophysiology -indexing brain neuronal activity in real time. in particular, this talk will address a characteristic electrophsyiologic signature of delayed auditory evoked response latency (at~ ms). this, and related timing anomalies, have been proposed as biomarkers for asd -with candidate use for diagnosis, prognosis, stratification and therapy monitoring. progress along each of these axes will be discussed. however, to justify the term "biomarker", we demonstrate converging evidence from spectrally-edited (megapress) mrs and diffusion-mri. mrs offers insights into neurotransmitter levels, especially gaba and glutamate, imbalance of which may be associated with anomalous electrophysiologic oscillations in the gamma band. diffusion offers insights into the white matter of the brain (auditory pathway will be illustrated) and an interpretation of diffusion parameters as an index of central conduction velocity will be offered. combining these mechanistic measures with the spectrospatio-temporal capabilities of magnetoencephalography (meg), this talk will present a state of the art review of multimodal biomarker development in asd. take home points: meg captures brain activity in space and time as well as showing sensitivity to activity at different frequencies (where, when and what) delays in cortical neuronal response latency are evidence in asd atypical coupling between diffusion evidence of conduction velocity and timing of cortical responses in shown in asd oscillatory activity is atypical in asd (elevated "noise", decreased "synchrony") diminished inhibitory neurotransmitter (gaba) levels are shown in asd disturbance of teh typical coupling between gaba and gamma-band oscillations in development leads to anomalous adult oscillatory activity (taken to index local circuit function). multimodal and longitudinal approaches may be required to tackjle the complex and heterogeneous landscape of asd the paediatric radiologist can play an important role in establishing vascular access in paediatric patients ranging from neonates to teenagers. a breadth of knowledge and skills are needed to deal with changing body morphology and varied pathology in this age range. some of the skills particular to performing and managing vascular access in children will be discussed. different devices which can be placed, their indications, advantages and disadvantages will be reviewed. choice of access vessel is important in children, because there are known long term complication such as central venous stenosis and thrombosis, which can have a huge impact for future venous procedures or potential creation of an arteriovenous fistula of the arm for dialysis. preserving venous access sites is a ( ) (suppl ):s -s pediatr radiol key responsibility especially in children with complex medical and surgical co-morbidities. because vascular access in children has associated morbidity it's important to manage and maintain devices that are placed. the risk of infection when repairing or exchanging a broken line will be highlighted. image guided biopsy is a very frequent procedure in pediatric patients. they range from random organ parenchyma for the diagnosis of medical disease up to tumor biopsies for histopathology analysis. different imaging modalities can be used for guidance as well as different biopsy devices and needles. ultrasound guidance is the most common modality used for this purpose in the pediatric population. the success of this procedure depends on multiple factors: from pain control up to choosing the correct device and area to sample. the radiologist performing the procedure also needs to be familiar with the potential complications of the intervention, how to prevent them and how to manage them. the intention is to perform the safer procedure as possible, obtaining the best quality of sample. the goal of this lecture is to present in a didactic way technical tips to perform safe and effective image guided pediatric biopsies, which may be applicable to different groups of operators, ranging from general pediatric radiologists performing occasional biopsies up to pediatric interventional radiologists. the objectives will be: to identify the safest approach to different types of biopsies; to describe ways to obtain the better quality of sample as possible; to demonstrate the use of different approaches in challenging clinical scenarios; to illustrate new devices currently used in specific applications; to discuss potential complications and its management and to show imaging modality integration applied to biopsy planning an performance. image guided biopsy is a frequent procedure in pediatric patients. a pre-procedure planning is fundamental in the success of the intervention. the operator must be aware of the aims of the biopsy and based on this choose the best approach, device and site for sampling. preparation and competency to manage complications is mandatory. pediatric interventional oncology: big cases in little people m. heran; vancouver/ca summary: the pediatric patient presents unique challenges in diagnosis and management of oncologic disorders. interventional radiology (ir) has a prominent role in the care of these children, with improvements in imaging and equipment offering better and safer options to traditional diagnostic and therapeutic procedures. as cancer can involve any organ system, consultations to the ir service can involve any part of the body, and can be non-vascular and vascular, simple and complex. the most common ir procedures in the pediatric oncology patient are enteric tube placement/change, vascular access, and percutaneous image-guided tissue/organ biopsy. however, with the explosion of interventional oncology in the adult setting, the variety and complexity of ir in pediatric oncology has begun to increase as well. ir techniques, such as thermal ablation, transarterial pharmacotherapy, and preoperative embolization, are now increasingly discussed in multi-disciplinary conferences as complementary or primary modes of treatment of oncologic disorders or related diseases/complications. however, although the principles of these diagnostic and therapeutic ir procedures remain essentially the same in their translation from adults to children, well recognized differences in pediatric physiology and metabolism, as well as the range in weight, size, and age of children, result in a practical question of "how do we do this?" the aim of this presentation is to provide an overview of the role of ir in the pediatric oncology patient, and to highlight areas of research and innovation. vascular anomalies encompass a spectrum of disorders including vascular tumours and vascular malformations. incorrect nomenclature and misdiagnoses resulting in inappropriate treatment are commonly experienced by patients with vascular anomalies. the currently accepted method for classification of vascular anomalies is straightforward and clinically relevant. vascular malformations can be divided into high flow lesions such as arteriovenous malformation or low flow lesions such as venous or lymphatic malformations. in children, a diagnosis can often be made with the history, examination and ultrasound. the classification of vascular anomalies will be briefly reviewed with examples of commonly encountered pathologies. a multidisciplinary team approach to the management of these conditions is vital. paediatric radiologists can play a key role not only in diagnosis but also in management, principally by injection sclerotherapy of low flow lesions and embolization of the much rarer arteriovenous malformation. many sclerotherapy agents are available with sodium tetradecyl sulphate the most commonly used for venous malformations and doxycycline for lymphatic malformations. different sclerotherapy agents have different characteristics and uses which will be covered. symptomatic relief is often achieved with treatment but multiple treatment episodes may be needed to achieve the desired outcome. ensuring the child and family understand this is vital to ensure they are satisfied with the management of the condition. contrast media is commonly used during imaging in children whatever their age and whatever the pathologic conditions. still, youngest patients are vulnerable and unstable. therefore, in neonates and infants the use of s ( ) (suppl ):s -s pediatr radiol contrast media should be carefully evaluated and customized putting in balance the risk versus the benefit of its use. when using contrast media in neonates and infants, several features should be highlighted: -prematures and neonates have rather immature kidneys and some contrast media might be difficult harmful -the thyroid gland in prematures may be (transitorily) depressed by iodinated contrast media -the use of high osmolar contrast may induce a fluid shift and dehydration especially in premature and neonates -most contrast media are used off label; almost none has obtained the authorization to be used in neonates. -there are very few studies evaluating the short and long term adverse reactions in neonates and infants below the age of two. fortunately these reactions seem very rare in these age groups. -using contrast extends the duration of the examination and the need for sedation different types of techniques will potentially need ingestion, instillation or injection of contrast media: ) opacification of the entire gi tract pre-and post-operatively ) retrograde uretro-cystography ) contrast enhanced ct ) contrast enhanced mr imaging ) contrast enhanced us ) angiography furthermore, different types of contrast media can be used to achieve these purposes ) barium (sulfate) ) iodinated water-soluble contrast media (hyper-, iso-or hypoosmolar) remarks regarding opacification of the upper gi tract: -the upper or lower gi tract should be opacified using water soluble contrast in the immediate postoperative period or whenever a bowel perforation is suspected. -air can be used to confirm esophageal atresia and duodenal atresia -barium should be preferred in case of t-e fistula -either barium or water soluble iodinate contrast can be used in order to opacify (sub)obstructed upper gi tract remarks regarding the opacification of the lower gi tract -iodinated iso/hypo osmolar contrast should be used to opacify the colon in case of obstruction -a higher osmolarity iodinated contrast can be used in case of suspected meconium ileus or plug; still this contrast should be used diluted and under close clinical surveillance and adequate hydration. -in some more specific cases, for instance whenever hirschprung disease or a stenosis post nectotizing enterocolitis are suspected, barium enema can be used remarks regarding ct scan -contrast enhancement may help for the global assessment of various pathologies especially in case of cardio-vascular malformations or for the evaluation of abdominal masses. any iodinate contrast among those available is acceptable in neonates. higher osmolality contrast allows to inject a lower volume -injected volumes of . ml/kg seem adequate using - gauge needles -power injectors are acceptable as long as adequate catheters can be used -allergic or side effects are very rare and should be managed similarly to adults. remarks regarding mr imaging -the use of gd chelates in neonates remains controversial as there is no data available on the long term effects of gd injected so early in life -gd should be used only when enhancement may provide additional information compared to the non-enhanced study (cns infections, tumors, cardiovascular imaging, abdominal tumors, uro-mr imaging...) -only gd with low nsf risk should be used -gd should not be used in children with renal failure remarks regarding contrast enhanced us -little is known about the use of ce-us in neonates -indications seem equal to older children -there are very few side or allergic effects -doses suggested are . ml/year of age children present varied histological types of brain tumours. it's now possible to combine different information and image techniques to improve the diagnosis of paediatric brain tumours. the multimodal approach has increased the diagnostic specificity and permits, in most cases, the pre-operative differentiation between low and grade tumours. children with low grade lesions, and in particular the less accessible tumours, would benefit the most from avoiding biopsy. in addition, preoperative spinal mri evaluation to rule out drop metastases should be performed in patients with suspected high grade tumours. in general paediatric brain tumours are less necrotic, i.e. aggressive tumours in paediatric patients tend to be more hypercellular and homogeneous. because of its ready availability and speed, computed tomography ( ) (suppl ):s -s pediatr radiol (ct) is the first investigation generally performed for a suspected brain tumour. ct can rule out haemorrhage or calcifications, but can also be used to evaluate tumour cellularity. a hyperdense tumour on ct reflects hypercellularity and is very often high grade. medulloblastoma are, for example, typically hyperdense on ct scans and paediatric low-grade astrocytomas are almost always hypodense. mri plays a major role in the evaluation of brain tumours. in conventional mri, the "general aspect" is the single most important parameter in predicting high-grade tumours in children. the same does not hold true for low-grade tumours, of which only % can be predicted using the general aspect. in our previous study, hyperintensity on t -w and the lack of diffusion changes were the most important single parameters with % positive prediction. embryonic tumours, such as medulloblastoma or pnet have high tumour cellularity with consequent very low adc and hypo/isointense t compared to the cortex. adc values derived from dwi have been shown to be decrease in highly cellular tumours. adc values cannot reliably be used in individual cases due to the substantial overlap between tumour types previously described in the literature. nevertheless, adc has a higher predictive value in children and increases the accuracy of preoperative differentiation between low grade and high grade paediatric tumours. the cut-off values for differentiating between low and high grade paediatric brain tumours are . x mm /s and . x mm /s for minimum adc and average adc values, respectively. perfusion with relative cerebral blood volume (rcbv) is considered a marker of angiogenesis and is helpful in distinguishing high and low grade tumours. however, perfusion can be difficult to perform in small children; small catheters with manual injection are therefore used in such cases (or, as an alternative, arterial labelling). it should however be taken into account that choroid plexus tumours can have high rcbvs resulting from highly leaky capillaries. mr spectroscopy (mrs) shows the metabolic profile of the tumour. high grade tumours show elevated choline (cho) -reflecting increase in cell membrane turnover -and decreased n-acetylaspartate (naa), which represents a neuronal marker. the absolute values of the mrs peaks are not used by us; we favour to normalize the signal intensities of metabolites to their values in contralateral brain tissue. mrs is helpful not only as guidance for stereotactic biopsy (cho hot spot) but also for determining whether the tumour is high or low grade. as a rule of thumb, a % increase of cho when compared to the contralateral brain tissue is highly suggestive of a high-grade tumour. however, in children, increased cho levels can also be found in pilocytic astrocytoma; in this case the typical aspect with cystic component and location can suggest the diagnosis, despite the mrs result. therefore, in children, high cho levels do not necessarily imply the presence of a malignant tumour. task based functional mri (fmri) can be used for pre-operative localization of the eloquent cortex together with the identification of the language and somatomotor function. in the future, small children who are unable to cooperate will probably profit from resting-state fmri. pet mri has the advantage of integrating structural mr imaging with physiologic pet. take home points: take home points although the histology of paediatric brain tumours is diverse, their general morphological aspect on mri has a very high diagnostic reliability. unlike adult grade iv brain tumours, malignant paediatric brain tumours are less necrotic, but are highly cellular with high nuclear-to-cytoplasmic ratios. adding information on signal intensities on t w and dwi further increases the diagnostic accuracy of conventional mri. the solid areas of high-grade tumours are iso-or hypointense on t w and hyperintense on dwi, whereas low-grade tumours show inverse signal characteristics. advanced mr techniques (perfusion and spectroscopy) provide important biological information which can be used to correctly identify grading (high vs. low) and to guide biopsy. in children high cho levels, although suggestive, do not necessarily mean a malignant tumour. experience with central review of paediatric renal tumours g. khanna; st louis/us summary: central imaging review of pediatric renal tumors has been performed in children's oncology group since . to date, more than cases of pediatric renal tumors have been centrally reviewed real time by the study radiologists. the mean time for central review was < days. discrepancies between local and central risk stratification were identified for detection of bilateral disease and pulmonary metastasis. in addition, central archiving of images has created a rich repository of cases for future research. the role of imaging in detection of key diagnostic features in pediatric renal tumors will be reviewed. the diagnostic performance of imaging for staging, detection of vascular invasion and tumor rupture will be discussed. real time central review of imaging is feasible in pediatric oncology wilms tumor remains the most common pediatric renal malignancy, followed by renal cell carcinoma cystic nephroma typically presents as a bosniak lesion, and has high association with dicer- mutations is there a role for dwi in nephroblastoma? a.s. littooij; utrecht/nl wilms tumour or nephroblastoma is the most common malignant renal tumour in children. ultrasound is usually the first line investigation. mri of the abdomen is often performed to further delineate the tumor and its surroundings. the addition of diffusion-weighted imaging (dwi) to the standard mri protocol may enable subtype characterisation and allows assessing treatment response beyond necrosis and volume change. overall, the survival rate in patients with nephroblastoma is relatively good and the current focus is on finding biomarkers to further improve outcomes while reducing therapy-related side effects in these children. therefore, identifying low-or high-risk type nephroblastoma might be relevant for treatment planning. diffuse anaplastic nephroblastoma and extensive blastema in residual tumour after preoperative chemotherapy may require more intensive treatment. the limited available literature suggest a linear relation between adc values and subtypes nephroblastoma at histopathology. furthermore, the addition of dwi to the standard mri protocol may detect lesions (e.g. nephrogenic rests of nephroblastomatosis) that remain undetected at post contrast t -weighted images. unfortunately, there is a considerable heterogeneity in acquisition techniques and methods of adc measurements. nephroblastoma often contains areas of necrosis and/or hemorrhage that can demonstrate very low adc values and consequently mimic highly cellular portions of tumours. therefore these areas should be excluded from further analysis. this lecture will highlight the potential additional benefit and limitations of dwi in children presenting with renal tumour. significantly lower radiation exposure even in comparison to low-dose pet/ct, (b) the higher diagnostic accuracy as compared to pet/ct even when using diagnostic contrast-enhanced ct, (c) the unique possibility to combine distinct mr-inherent contrasts (e.g. dwi) with specific pettracers (e.g. cu-labeled antibody imaging) for the evaluation of novel targeted therapies, and (d) the opportunity to stage local and systemic tumour burden within a single and highly resolved examination. on the other hand, many circumstances are challenging the extensive use of pet/mri in children. in general, the availability of pet/mri systems is low, particularly for children. thus, only a few sites in europe have experience with this technique in children, and therefore the generated scientific evidence is limited. moreover, whole-body-mri is still not a broadly adopted method for the combined assessment of local disease extent and whole-body staging, potentially replacing other whole-body modalities like the bone scan. in this context, especially the detection of pulmonary metastases is biased also against pet/mri. finally harmonized sequence protocols and specific recommendations for trace dosage are not available for pet/mri. in conclusion, further efforts are needed to keep the promises of pet-mri in the daily practice. common artefacts in paediatric mri-how to recognise, avoid or take advantage of them c. kellenberger; zurich/ch summary: while mri is a robust and radiation free imaging technique for assessing anatomy and pathology of most tissues and organs throughout the body, it is inherently prone to artefacts as no other imaging modality is. mri artefacts may impair image quality potentially leading to difficulties or errors in interpretation, but in some instances can contribute diagnostic information. main sources of image degradation are motion, disturbances of the local magnetic field and other factors inherent to image acquisition. strategies to reduce effects from various kinds of motion and adjustment of sequence parameters for eliminating artefacts will be discussed. & understanding the origin and effects of artefacts encountered in paediatric mri is essential for modification of mri protocols, so that artefacts and associated errors can be avoided. & for safely and successfully imaging children with implants and devices, the composition, location and functionality of the foreign body needs to be known. injuries to the central nervous system in abusive head trauma are responsible for the primary cause of morbidity and mortality in infants. neuroradiology has an important role in diagnosis but also in depicting injury and extent of brain damage of poor outcome. computerized tomography (ct) and magnetic resonance imaging (mri) are the primary imaging techniques. ct is usually performed in the acute phase while mri is performed the following days after injury. some injuries are better identified on mri such as diffuse axonal injury and cerebral edema with susceptibility and diffusion weighted images. abusive head trauma (aht) is the primary cause of morbidity and mortality in infancy, especially during the first year of life. aht is clinically characterized by a triad consisting of subdural hematoma, retinal hemorrhage and encephalopathy caused by brain swelling ( ). the most common mechanism responsible for brain damage is thought to be caused by whiplash shaking injury explaining that abusive head trauma is also referred as shaken baby syndrome. impaction, compression and penetrating injury are also possible mechanisms as well as strangulation. however because of the variability of types and severity of injury, clinical symptoms vary from subtle to severe such as alteration of consciousness or coma ( ) . the most common symptoms include vomiting, seizure, lethargy, poor feeding and apnea of which vomiting and respiratory pauses are non-specific ( ). poor feeding, irritability or lethargy is also nonspecific signs. however apnea and/or retinal hemorrhages seen in children with brain injury are strongly associated with inflicted trauma ( ) . in contrast to acute injury some children may manifest with increased head circumference related to chronic subdural hematomas. neuroimaging is therefore playing a crucial role to assess infants and children with a suspicion of abusive head trauma. computerized tomography (ct) and magnetic resonance imaging (mri) are the primary imaging techniques. ct is performed for the initial evaluation in cases with acute symptoms to look for hemorrhagic intracranial injury as subdural hematoma. mri is more often performed in the following days to further evaluate brain injury and to look for spine and spinal cord damage ( , ) or in the presence of normal or equivocal ct findings ( ) . however brain mri may be the first option in children presenting with increased head circumference. recently the study from flom et al showed the high sensitivity of mri for intracranial hemorrhage in well appearing infants at risk for abusive head trauma suggesting mri as a screening tool with pulse sequences (axial t , axial gradient recalled echo and coronal t weighted inversion recovery) ( ) . ct is generally performed without intravenous contrast injection with d volume rendering (vr) reconstructions for identification of fractures. in some cases postcontrast images are also obtained specially to rule out deep venous thrombosis especially when children present with nonspecific clinical symptoms. mri protocol should include axial t , t * or susceptibility weighted images, coronal t images, diffusion or diffusion tensor images, and postcontrast dt images including mip reconstructions to evaluate the venous structures. mr venography can also be performed. susceptibility-weighted images are usually preferred because they allow the depiction of smaller hemorrhagic dai lesions and greater number of lesions compared to gre t ( ) . it was also reported by colbert et al ( ) that the presence of micro-hemorrhages alone was useful for outcome prediction in abusive head trauma with significant poor long-term outcome. the sensitivity and specificity of microhemorrhages was also higher than the other clinical (such as retinal hemorrhages and glasgow coma scale score) and other imaging findings for prediction of outcome. diffusion tensor imaging (dti) measurements were reported in abusive head trauma by imagawa et al: decreased axial diffusivity related to axonal injury with consequent reduced mean diffusivity did correlate with poor outcomes ( ) . magnetic resonance spectroscopy (mrs) is usually not part of the standard protocol. however aaen et al ( ) showed that n-acetylaspartate/creatine and/or nacetylaspartate/choline ratios were decreased significantly in the corpus callosum, frontal white matter, parieto-occipital white matter, and parietooccipital gray matter in children with poor outcomes. this study mentioned above also reported that the prediction of outcome was accurate in % of patients by using a logistic regression model that include age, initial glasgow coma scale score, presence of retinal hemorrhage, lactate on mrs, and mean total n-acetylaspartate/creatine. functional mri, ( ) (suppl ):s -s pediatr radiol volumetry may be performed in long-term follow up of victims of child abuse. physical abuse is associated with altered emotion with greater activation in the salience network in response to negative stimuli, that includes amygdala, thalamus, putamen and anterior insula ( ) . increased responsiveness of the right amygdala to fearful and angry faces (negative stimuli) and structural changes as reduced hippocampal volume, are reported by dannlowski et al ( ) . impaired attention was also reported in patients with childhood abuse ( ) with reduced activation during attention tasks in the left hemispheric ventral and dorsolateral prefrontal regions. intracranial injuries include extracerebral hemorrhages and parenchymal damage as brain swelling and ischemia, venous infarction, diffuse axonal injury, contusions and intraparenchymal hematomas ( , ) . extracerebral hemorrhages subdural hematoma is a characteristic finding of inflicted traumatic brain injury, is generally multifocal and most commonly seen along the posterior interhemispheric scissure, over de convexities at the vertex level and/ or in the posterior fossa ( ) ( ) ( ) . subdural hematomas are most likely bilateral but may be unilateral. all locations are related to disruption of bridging veins. the identification of bridging vein rupture allows the diagnosis of traumatism in relation to acceleration/deceleration, rotational and shearing forces due to violent shaking ( ) . a mixed density appearance of subdural hematomas is frequent but is also seen in accidental traumatic brain injury ( ) ( ) ( ) . indeed this feature is often present in the very early hours following trauma and is thought to be secondary to early sedimentation of blood clots and supernatant serum. tubular high density is often seen on non-contrast ct over the convexities in abusive head trauma. this ct feature is related to a clot secondary to venous disruption ( , ) that can end up in thrombophlebitis. this tubular high density was reported more recently as tadpole sign ( ) and lollipop sign ( ) respectively seen in and % of abusive head trauma. this appearance is strongly associated to inflicted trauma and much less frequent in accidental trauma ( out of cases ( , %) of accidental trauma in our experience). associated venous infarction is reported in % of cases of abusive head trauma ( ) and often located in the parieto-occipital region, unilaterally at the site of venous disruption of bridging veins. subdural hemorrhages, when multiple, in the convexity and interhemispheric, or in the posterior fossa were found significantly associated with abusive head trauma in the meta-analysis reported by kemp et al ( ). in addition subdural hematoma, cerebral ischemia, skull fracture, retinal hemorrhage and intracranial injury were significantly associated with abusive head trauma in the review from piteau et al ( ). subarachnoid hemorrhages (sah) and epidural hematomas are also found in inflicted trauma and are not considered discriminant-imaging features. however epidural hemorrhages, isolated skull fracture and scalp swelling were reported as significantly associated with accidental traumatic brain injury ( ). sah in shaking injury is usually caused by tears of the vessels within the pia and arachnoid predominantly in the interhemispheric fissure and high convexity ( ). parenchymal injury parenchymal injury include brain swelling and ischemia, venous infarction (discussed above), diffuse axonal injury related to rotationallyinduced shear-strain injury with different inertia for grey and white matter due to their different specific gravities, contusions seen in deceleration trauma with friction between the skull and brain, and in blunt trauma and intraparenchymal hematomas related to lacerated vessels. brain swelling/ischemia may be related to increased blood volume (congestive swelling), increased presence of water in the nervous tissue, and the combination of both. increased water in the nervous tissue may manifest as vasogenic edema located in the white matter due to extravasation of plasma like fluid related to incompetent blood-brain-barrier and as cytotoxic edema located in the grey matter, related to ionic imbalance. cerebral edema can be recognize on ct within the hours following injury as loss of gray-white matter differentiation and decreased attenuation of grey and white matter. cytotoxic and vasogenic edema are better characterized on mri with diffusion-weighted imaging. brain swelling and edema occur early after trauma with consequent underestimation of subdural hematoma. therefore imaging should be repeated (ct or mri) especially when neurologic symptoms change rapidly. brain swelling/ edema may also involve the posterior fossa and is better identified on brain mri. two frequent patterns have been reported in abusive head trauma ( ). diffuse supratentorial brain swelling (infarction) involving the cortex and white matter was reported in % of cases and is considered as severe hypoxic-ischemic injury with poor outcome ( ). watershed infarction was reported in % of cases and considered a less severe form of hypoxia-ischemia. apparent diffusion coefficient (adc) values are strongly associated with poor neurodevelopmental outcomes in the acute phase (within days) especially basal ganglia, thalamus, brainstem, cerebral cortex, cerebellar vermis, cerebellar cortex and mean total brain ( ). during the early phase up to month adc values in fewer regions (basal ganglia, thalamus, brainstem and corpus callosum) were associated with poor outcome. when patients with and without parenchymal lesions are compared, the detection of diffuse lesions during the first months as well as beyond months is significantly associated with severe developmental outcome ( ). late mri (beyond months after injury) also showed that recovery depends on the extent of brain damage. patients with diffuse lesions show more severe motor and intellectual impairments and are more likely to have blindness and epilepsy than patients with focal or hemispheric lesions ( ). diffuse axonal injury (dai) is related to shear-strain injury of small medullary veins and was reported in % of cases of abusive head trauma ( ). it is encountered in trauma with sudden acceleration-deceleration associated with rotational angular forces and in shaking-impact trauma. the lesions may be hemorrhagic or non hemorrhagic (related to axonal swelling). dai is most often located in the subcortical white matter at the gray-white matter junction, corpus callosum, basal ganglia, brainstem and internal capsule. if the lesions are large enough and hemorrhagic dai may be seen on ct. however dai is usually better identified on mri with susceptibility and diffusion weighted images. the detection of changes in the basal ganglia or brainstem during the first days as well as during the first month after injury is significantly associated with poor long-term outcome in survivors ( ). the presence of intraparenchymal brain micro-haemorrhages detected on swi in children with abusive head trauma correlates with significantly poor long-term neurologic outcome ( ) contusion is also reported in abusive head trauma and is seen in blunt trauma with impact with or without contrecoup contusion. contusions are located at the surface of the brain (crest of gyri) and may be pial and haemorrhagic (disruption of cortical arteries). they are also found in the frontal and temporal regions related to impact of the brain on the roof of the orbit, middle cranial fossa and sphenoid wing. white matter tears are also seen in the frontal and temporal area related to the vulnerability of unmyelinated and soft white matter in infants. skull fractures are seen in blunt impact and are less frequent than long bones and rib fractures in non-accidental trauma. the most common site is the parietal bone (because of bulging of parietal bones below year of age). the fracture may be linear as in accidental trauma. radiologic features significant for inflicted trauma are multiple fractures, bilateral fractures and fractures that cross suture lines ( , ). focal underlying brain damage can be seen such as subdural hematoma and hemorrhagic contusion. hypoxic-ischemic encephalopathy is seen in strangulation injury with involvement of the territories of the internal carotid artery related to their anatomic vulnerability. neuroradiology (ct and mr) is crucial for the diagnosis of trauma, to predict outcome when showing edema and hypoxic-ischemic injury. this presentation will present an update on post mortem mri (pmmr) with relevance to clinical developments over the last years. in particular, reference will be made to diagnostic accuracy of pmmr across different body parts, the current limitations of post mortem mr, and protocol development at different field strengths. imaging correlates of post mortem interval are also being investigated. maceration (autolysis within intrauterine fluid) and perimortem hypoxic brain changes caused difficulties in image interpretation, which more advanced and quantitative techniques may be able to address. jawad take home points: below g, . -t pmmr shows a significant reduction in diagnostic yield, compared with conventional autopsy, and therefore its clinical usefulness in this setting will depend on individual circumstances. t pmmr performs better than . t particularly < weeks gestation, and particularly for the chest, heart and abdomen. diffusion characteristics in different fetal brain areas are multifactorial, with maceration the strongest predictor in most areas. international pm ct protocols c.y. gerrard , o.j. arthurs ; albuquerque, nm/us, london/uk the european society of pediatric radiology (espr) taskforce and the international society of forensic radiology and imaging (isfri) pediatric working group have combined efforts to establish best practice standards for performing perinatal and pediatric post mortem computed tomography (pmct) examinations. use of pmct in the investigation of pediatric death has increased significantly in the past decade. due to quick acquisition times and the ability to acquire thin slice, high detailed images of the whole body, ( ) (suppl ):s -s pediatr radiol many hospitals and forensic institutes have implemented pmct into daily practice. however, there lack an overall standardization of how cases are triaged and the acquisition methods when comparing institutes using pmct. in an effort to address inconsistencies in acquisition parameters, post processing, and case selection, pmct protocols were compiled from international institutes and centres currently performing pediatric imaging. this paper will describe both the uniform and divergent elements of image acquisition and procedural uses identified among the participating centres. the outcome is to provide a single source of information that can guide already established and new centres on the best practice standards for implementing pediatric pmct. take home points: describe how pediatric post mortem computed tomography (pmct) has increased in utility over the past decade. identify the differences in acquisition methods for clinical computed tomography versus post mortem computed tomography. discuss the overall consensus of case triage and scan acquisitions when comparing institutes in aggregate. provide comprehensive statement of best practice standards for pediatric pmct. post mortem imaging research: updates and future proposals o.j. arthurs; london/uk paediatric and perinatal post mortem imaging is a new and rapidly growing field, and the post mortem imaging taskforce was founded in graz at espr . the pmi taskforce aims to help reach consensus and guidance regarding imaging protocols and the potential yield of post mortem ultrasound, ct and mr. the key priorities are the themes of collaboration, image acquisition, best practice guidelines, training and education, raising awareness and access to imaging. this presentation will give updates on the latest developments in perinatal and paediatric imaging, with particular focus on where the pmi taskforce can help. in particular, protocol development is underway, and the espr meeting acts as an opportunity for collaborative working and network development, to facilitate best clinical practice and welcome new members. arthurs oj et al., espr post mortem imaging task force: where we begin. pediatr radiol ( ) ; : - take home points: post mortem imaging is an exciting sub-specialty which requires a combination of in depth fetal medicine, perinatal autopsy and pediatric imaging knowledge to help shape and grow the clinical and research arena. dedicated personnel have an opportunity to create the evidence-based behind a growing clinical service, with clear benefits to patients, families and referring clinicians. abstracts appear as submitted to the online submission system and have not been checked for correctness and completeness. sequences, are an emerging tool for evaluating intracranial vessel disease. improved survival due to emended treatment protocols results in an increasing number of long-term medulloblastoma survivors who experience delayed treatment effects. microbleedings, developement of cavernomas, vasculitis and atherosclerotic lesions are cerebrovascular structures affecting sequelae of the applied radiochemotherapy. this study evaluates radiation-induced intracranial vascular changes. twenty-two long-term pediatric medulloblastoma survivors (mean age . years, range - years; mean years after primary radiochemotherapy . years, range - years) underwent mri. the scan protocol included precontrast -dimensional time of flight (tof)magnetic resonance angiography (mra), precontrast d t -and d t -vwisequences and postcontrast d t -vwi-sequences of the medium and large intracranial arteries. vessel wall thickening, contrast enhancement and luminal narrowing were analyzed. additionally precontrast t -, t -swi and t -weighted images of the supra-and infratentorial brain were acquired. results: vwi-sequences: vessel wall changes could be found in ( %) and patients ( %) of the right and left ica, respectively. for the ba ( %) patients revealed vessel wall changes; for the left and right va ( %) patients were detected with vessel wall changes, respectively. in the tof angiography no alteration of the ica, ba or vas could be identified. in total vessel wall changes for the vertebrobasilar system and the icas could be found in ( %) patients. swi-sequences: all patients ( %) revealed swi lesions, the smallest lesion measuring less than mm, the biggest up to mm. sixteen patients ( %) were presented with lesions > mm, suspicious for cavernomas. to ensure quality of life in long term childhood medulloblastoma survivors, monitoring of long-term effects, like vascular changes after rct is gaining in importance. high resolution mri, including swi and vwisequences could be used here for. this study images, asymptomatic vessel wall alterations in former childhood medulloblastoma patients through vwi sequences and micro bleedings through swi sequences. vessel wall alterations, revealing rct induced arteriosclerosis, can lead to symptomatic intracranial stenosis which is associated with ischemia, furthermore micro bleedings and cavernomas can lead to intracranial hemorrhage. however further studies are needed to standardize mri sequence protocols to ensure a high standard follow up protocol, detecting clinically still asymptomatic vascular changes. fast "black-bone" mr imaging in evaluation of craniofacial abnormalities: comparison with high resolution ct z. habib, a. talib, c. parks, s. avula, l.j. abernethy; liverpool/uk to evaluate the feasibility and diagnostic value of a fast field echo, "black bone" mri sequence in children with craniofacial abnormalities. a fast "black bone" mri sequence has been used in addition to standard brain mri in children (mean age months, age range months to years and months) referred to the supra-regional craniofacial surgery unit at alder hey children's hospital, liverpool, uk. a subgroup of of these patients with complex craniofacial abnormalities additionally had high resolution volume ct performed at the same visit. "black bone" mr imaging was performed on philips ingenia t and . t scanners, using a d fast field echo sequence (tr= . ms, te= . ms, flip angle ). this sequence can be performed with an acquisition time of less than minutes. the "black bone" sequences were assessed for accuracy in evaluating the patency of the sagittal, coronal and lambdoid sutures, and, where applicable, were compared with high resolution ct. the fast "black bone" mri sequence was shown to be technically feasible in all cases. the resultant images successfully demonstrated both patent sutures, which were confidently seen, and prematurely fused sutures which were confidently not seen. visualisation of patent sutures was found to be further enhanced by the use of minimum intensity projection. in the subgroup of patients with complex craniofacial abnormalities, comparison with high resolution volume ct confirmed good sensitivity for patency of cranial sutures. there was complete agreement in out of sutures assessed. the "black bone" mr images were also found to produce good-quality surface-rendered images and were also suitable for -d printing of models for pre-operative planning. fast "black-bone" mri has proven to be technically feasible and to demonstrate cranial suture patency with good agreement with high resolution ct. additionally "black-bone" mri can be used to produce good quality surface-rendered images and -d printed models for surgical planning. main symptom of mucopolysaccharidosis type iva (mps iva) is progressive systemic skeletal dysplasia. this is routinely monitored by cerebral and spinal mri. the vascular system is generally not in the primary focus of interest. in our population of mps iva patients we observed vessel shape alterations of the vertebrobasilar arteries, which has not been described before materials: mri-datasets of patients with mps iva acquired between and were eligible for retrospective analysis of the vertebrobasilar arteries. the vessel length and angle of the basilar artery (ba) and both vertebral arteries (va) were analyzed. a deflection angle between °and °in the vessel course was defined as tortuosity, less than °as kinking. the results were compared to an matched control group of patients not suffering from mps. the deflection angle [°] of the va and ba was significantly decreased in the majority ( %) of mps iva patients (fig. ) mps iva is associated with significantly increased tortuosity of vertebrobasilar arteries. therefore the vascular system of mps iva patients should be monitored on routinely basis, as vessel shape alterations had been associated with dissections, leading to a higher risk of cerebrovascular events. in the pediatric population, intraspinal cysts (arachnoid or neurenteric cysts) are rare lesions mainly located in the thoracic region, whose acute onset is not well described in the literature. ( ) (suppl ):s -s pediatr radiol we present a series of four children seen in the last two years as spinal cord emergencies and discuss the clinical aspects, imaging diagnosis, and management approaches, particularly in the emergency setting. a comparison of our cases with those reported in the literature is also provided. as in other types of spinal cord lesions, mr imaging is the diagnostic procedure of choice, because of its potential to demonstrate the exact location and extent of the cyst and its relationship to the spinal cord, valuable information for planning surgical treatment. this is a retrospective review of cases of pediatric intraspinal cyst occurring in boys and girl, aged to years, treated at our institution between and . onset was sudden in all cases and mimicked transverse myelitis or infarction. all our affected patients had no preceding history of trauma and presented with signs of spinal cord compression-back pain and less commonly abdominal pain-followed by weakness. all patients underwent emergent mr imaging, including t , t , t *, d ciss, diffusion imaging and enhanced t sequences, mainly in the sagittal and axial planes. in each sequence, mr imaging showed a well-defined cystic lesion with signal intensity similar to cerebrospinal fluid, and secondary spinal cord compression that was severe in most cases. blood remnants were not visualized within or around the arachnoid cyst in any patient, which correlated with the absence of trauma antecedents. three of the four cysts were located in an anterior position relative to the spinal cord, and only one was located posteriorly; this latter had an associated subdural effusion. none of our patients had an associated neural tube defect. all patients were urgently treated with cyst wall fenestration or resection. the symptoms improved in all except one patient, whose symptoms did not abate, but ceased to progress. a prompt emergent diagnosis with mr imaging is important, as the symptoms can resolve if surgical treatment is performed before the spinal cord becomes irreversibly damaged. urgent surgery is essential in these cases, particularly if progressive neurological dysfunction develops over the course of spinal cord compression. the outcome following surgical fenestration or excision is excellent in most cases. nevertheless, a long-term imaging follow-up is recommended to detect possible recurrence. the objective of this study was to evaluate the usefulness of multiparametric quantitative mri model for myelination quantification in children. twenty-two children (age range: - , days) were scanned with multiparametric quantitative mri. total volume of myelin water fraction (mwf) (msum), the percentage of msum within the whole brain parenchyma (mbpv), and the percentage of msum within intracranial volume (micv) were obtained. mwf values of brain regions were acquired by drawing regions of interests. the values were fitted to representative models of myelin maturation. spatiotemporal pattern of mwf mapping was visually assessed. values of msum, mbpv, and micv well fitted to a developmental model of myelination. mwf of brain regions well fitted to a developmental model with high r values: pons (r = . ), middle cerebeller peduncle (r = . ), genu of corpus callosum (r = . ), splenium of corpus callosum (r = . ), thalamus (r = . ), frontal white matter (wm) (r = . ), parietal wm (r = . ), temporal wm (r = . ), occipital wm (r = . ), and centrum semiovale (r = . ). mwf mapping followed the known spatiotemporal pattern of myelination. multiparametric quantitative mri is a useful tool for mwf quantification in children. retinoblastoma is the most common intraocular tumour of childhood. it is a highly malignant. retinoblastoma is curable. if detected while still confined to the globe and if there are no metastatic risk factors, the child will nearly always survive following appropriate treatment. our aim is to assess diagnostic accuracy of preoperatively performed magnetic resonance (mr) imaging for detection of tumor extent in patients with histopathologically proved retinoblastoma. local ethics committee approval and informed consent were required for reviewing of patients' images and records. fifty-eight eyes in girls and boys with retinoblastoma (mean age at diagnosis was months ± . ) were reviewed on unenhanced t wi, t wi, and gadolinium-enhanced t -weighted mri with and without fat suppression. mri parameters such as anterior chamber hyperintensity, involvement of choroid, ciliary body, optic nerve, sclera, orbital fat, and pineal gland were determined. maximum tumor diameter was measured and correlated to metastatic risk factors. imaging and pathologic findings were compared. choroidal invasion was suspected with mr imaging in / eyes; findings were false-positive in eyes and false-negative in two (accuracy, . %; sensitivity, . %; specificity, %). mr imaging findings were true-positive in of eyes with proved prelaminar optic nerve invasion ( % sensitivity) and false-positive in ( . % specificity, . % accuracy). postlaminar optic nerve invasion was correctly detected in eyes; eyes were false positive, in other eyes, this metastatic risk factor was missed (accuracy, . %; sensitivity, . %; specificity, %). of nine eyes with histologically proven scleral invasion, eyes were true positive . in the other eyes, scleral involvement was missed on mri (accuracy, %; sensitivity, . %; specificity, %).extraocular fat invasion was suspected on mri in / eyes. of these, findings were truly positive in eyes ( %) and in eye ( %) was incorrect (false positive) (accuracy, . %; sensitivity, %; specificity, %).anterior chamber hyperintensity on t -weighted mr images obtained after contrast agent administration correlated well with main mri and histolopathology findings. tumor size (assessed in our study by the maximum diameter in mm) was statistically associated with postlaminar optic nerve invasion (ρ=. ) and choroidal invasion (ρ=. ). mr imaging shows promising role for tumor staging and detection of metastatic risk factors. tumor diameter, measured with mr imaging, is associated with postlaminar optic nerve and choroidal involvement. patterns of the cortical watershed continuum of term gestation hypoxic ischaemic injurythe "wish-bone sign" a. chacko , s. andronikou , s. vedajallam , j. thai ; east london/za, bristol/uk objective: background partial-prolonged term hypoxic ischaemic injury (hii) involves the cortical and subcortical watershed zones of the brain, which are visually difficult to conceive. new innovative methods of demonstrating watershed cortical atrophy using flattened maps of the brain surface gives added insight into distribution of the watershed zone by demonstrating the entire brain surface. aim determining and validating patterns of hii sustained at birth in term infants using cross-sectional mri and the innovative mercator and scroll map views of cortical surface anatomy, to define the distribution of the watershed zones in children with partial-prolonged injury. one hundred paediatric mri brain scans with an mri and clinical diagnosis of chronic term hypoxic injury were read by radiologists independently. all sites of abnormality were recorded and patterns classified. ( ) (suppl ):s -s pediatr radiol patients with partial-prolonged and combined patterns were evaluated using mercator and scroll map reconstructions, generating schematics of the watershed zone. predominant patterns of disease were partial-prolonged and acuteprofound types. the watershed zone was demonstrated, on the derived maps, representing a continuum of involvement in the shape of a 'wish-bone' extending bilateral from frontal lobes to posterior parietal lobes in band-like fashion along the para-falcine cortex and intersected by another band of atrophy in the peri-rolandic regions extending along peri-sylvian cortices. this is defined in schematics as a visual aid. predominant patterns of injury in term hypoxic ischaemic injury are described and quantified, with the 'wish-bone sign' introduced to describe the typical distribution pattern of partial-prolonged hii in the watershed zone. correlation of brain edema degree and biochemical parameters in pediatric posterior reversible encephalopathy syndrome with hematologic/oncologic diseases t. akbas , s. ulus , b. karagun , t. arpaci , c. kalayci , b. antmen ; adana/tr, istanbul/tr posterior reversible encephalopathy syndrome (pres) often associated with hypertension is characterized by typical transient parietooccipital predominantly brain edema on magnetic resonance imaging (mri) with neurological symptoms such as seizures, headache and visual disturbances. even if endothelial dysfunction, increased blood-brain barrier permeability and hyper-hypoperfusion remain as controversial mechanisms to explain, the pathophysiology of pres is unremain. the aim of our study was to investigate the correlation between brain edema degree on mri and serum biochemical parameters such as lactate dehydrogenase (ldh), albumin (alb), creatinine, uric acid (ua) and urea. a total of pediatric hematology and oncology patients ( male, female, aged - , mean age: years months) diagnosed with pres during treatment and after hematopoietic stem cell transplantation (hsct) were included in this retrospective study. underlying diseases were beta thalassemia (n: ), aplastic anemia (n: ), acute lymphoblastic leukemia (n: ), acute myeloid leukemia (n: ), lymphoid leukemia (n: ) and burkitt's lymphoma (n: ). pres was seen after undergoing hsct in patients. the brain edema degree according to specified anatomical regions on fluid attenuation inversion recovery (flair) mri sequence was scored by two radiologists blinded to patients' records. the levels of serum biochemical parameters at onset of symptoms were correlated with score of brain edema degree on mri. serum ldh concentration was statistically correlated with the score of brain edema degree (spearman's rho correlation, r= . , p= . ). no relationship was found between other biochemical parameters and the score of brain edema degree. our results suggest that increased serum ldh as a marker of endothelial dysfunction is the main biomarker for development of brain edema in pediatric pres patients under treatment and after hsct with underlying hematologic and oncologic diseases. objective: gadolinium based contrast agents (gbcas) have been associated with increasing signal intensities in deep brain nuclei on unenhanced t -weighted brain imaging. until now, most studies have been performed in adults, while results on pediatric patients are sparse. therefore, the aim of this study was to evaluate if there is any difference between signs of gadolinium retention in pediatric and adult patients. in this irb-approved, single center retrospective study, we extracted all patients with at least contrast-enhanced mris archived on pacs between - . all patients with gadobenate dimeglumine only enhanced mris were reviewed. seventy-six pediatric patients with the most injections and adult patients with the most injections were included in the final evaluation. therapies were documented. t signal intensity measurements for the initial and last unenhanced brain mris were performed for dentate nucleus, pons, globus pallidus and thalamus. signal intensity ratios for dentate-to-pons (dnp) and globus pallidus-to-thalamus (gpt) were calculated and correlated with number of injections and time interval as well as therapy. differences between adults and pediatrics were assessed. mean age for the pediatric group was . years compared to . years in the adults. no significant difference was found for gender distribution ( vs. % females) and follow up time ( . vs. years). there was no difference concerning the signal intensities on first and last mri in children and adults (p= . / . , respectively). for each additional year of follow-up the change in ratio increases by . for adults but only . for peds (p= . ). comparing therapies, in children a statistically significant difference between patients with and without former radiation was found (p< . ) while there was no difference in adult patients with and without therapy (p= . ). children and adults show a similar increase in t signal in deep brain nuclei ascribed to gadolinium deposition. in children, radiation and chemotherapy) seem to have a higher influence on gadolinium deposition. this correlation cannot be found in our adult cohort, indicating therapies have no (additional) influence. kearns-sayre syndrome (kss) is a rare mitochondrial dna-deletion syndrome characterized by early onset (< years), progressive external ophthalmoplegia and pigmentary retinopathy, often associated with cerebellar ataxia, muscle weakness, bilateral sensorineural hearing loss and cardiomyopathy. pyramidal symptoms may be present in kss, but they are poorly reported in the literature. through this case series, we aim to evaluate the concordance with the imaging patterns proposed by literature, correlating them with clinical and laboratory data, and to investigate possible microstructural damage with diffusion tensor imaging (dti) and magnetic resonance spectroscopy (mrs). we evaluated eight patients ( - years of age) with genetically confirmed diagnosis of kss. all pts. were studied with t/ . t mri. in / pts. the study was completed by mrs and in / by dti imaging with reconstruction of cortico-spinal tracts (cst) using a -rois approach. a t-test comparative study between mean fractional anisotropy (fa) of cst in the kss patients with dti and a group of healthy controls was performed. cst reconstruction in a patient suffering from kss (images a-c), compared to an healthy control (images d-f). the dti study showed significantly reduced fa values, pointing out a possible microstructural damage. the disease showed an mr pattern of mixed white and gray matter signal abnormality, with periventricular and/or subcortical white matter hyperintense lesions, which in / patient presented a "tigroid pattern" (fig. ) three patients displayed a disease extension to the cervical spinal cord. (fig. ) dwi images demonstrated restricted diffusivity in almost all lesions (fig. ) , with persistence of low adc values. mrs study documented a high lactate peak in / pts. and a naa reduction in / pts; an increment of gsh was noted in one patient (fig. ) . the t-test comparative study of cst showed a significant reduction of mean fa value in kss patients compared to healthy controls (p= , ). involvement of the spinal cord (a-c, yellow arrows). comorbidity was suspected in "a" (myelitis). below (d-f): pale nuclei (d, green arrows) and subcortical white matter (e) alterations. right image displays the "tigroid pattern" (purple arrow). mrs showing the presence of a gsh peak, which may suggest an augmented antioxidative activity within the encephalic tissue. below: dwi hyperintensity in many regions of the brain in patients suffering from kss, due to diffusion resctriction. the integration of neuroimaging with clinical data can implement the diagnosis of mitochondrial diseases such as kss. according to our experience, comorbidities can delay the achievement of a correct diagnosis. the finding of an altered signal in the spinal cord of / pts. may suggest a new possible localization of the disease, while in one patient was referable to myelitis (fig. , a) the evidence of a "tigroid patter" in should be taken in count in the differential diagnosis with lysosomal disorders. the presence of a prominent gsh peak may represent an augmented antioxidant activity, which may correlate with a more favorable outcome. an involvment of cst can be speculated even if pyramidal symptoms are poorly represented in kss. remotely distractible, magnetically controlled growing rod (mcgr, fig. ) system has been developed to allow for gradual lengthening on an outpatient basis. this allows for safe spinal lengthening with continuous neurologic monitoring and real-time feedback by the patient. this study aims to evaluate retrospectively our ultrasound (us) geometric method and his accuracy compared with the plain radiograph (gold standard) for assessing mcgr distractions. this is a retrospective study that includes patients with early-onset scoliosis undergoing multiple consecutive distractions after mcgr implant. the rods length was measured for with us, for each distraction ( -months interval), and compared with plain radiograph follow-up ( -year interval). all patients included were treated with dual-rod systems. distraction length was monitored by a senior radiologist with us at each visit, one rod at a ( ) (suppl ):s -s pediatr radiol time, before and after magnetic lengthening, with our geometric measurement method (fig. ) . low-dose upright two-projections radiograph were taken immediately after surgery and at -year intervals and measured by two radiologists ( and years of experience respectfully) (fig. ) . we compared measurements with the wilcoxon signed-rank test. from january to october , a total of patients ( females and male), which diagnoses included mitochondrial encephalopathy syndrome (n= ), spina bifida (n= ), ataxia of unknown cause (n= ), juvenile idiopathic scoliosis (n= ) and trisomy (n= ), with a mean of distractions per patient (standard deviation [sd] ± , ), were recruited. fifty distractions for each system ( measurements in total) were performed, targeting different lengths of distraction (from - . mm to + . mm) on each occasion. a total of sets of plain radiographs were taken. from these, sets of data points were used for correlation analysis. the mean distracted length per year on plain radiographs was , mm (sd ± , mm) and the mean distracted length on us per -months interval was , mm (sd ± , mm). excellent correlation was observed between radiographic and ultrasound measurements. in particular, correlation between rx measurements and ultrasound was excellent both for junior ( . <p< . , w= ) and senior radiologist (p< . , w= , ). there were no rod's breaches identified neither in ultrasound or radiographs in this series. the measured changes in rod length between the two imaging modalities were highly correlated. thus, ultrasonography is at least as accurate as radiographs in measuring changes in rod length. our geometric measurement method adds reproducibility to an effective, non-ionizing and lowcost procedure for monitoring distraction of mcgr in children population. refuting child abuse denialistshealing rickets is clearly different from child abuse metaphyseal fracture a.e. oestreich; cincinnati/us objective: child abuse denialists, who testify against the diagnosis of child abuse, often claim that radiographic findings generally recognized by pediatric radiologists as likely due to abusive trauma are instead a consequence of rickets, including healing rickets. in particular, bucket handle transverse bony density at metaphyses are declared by some denialists to be identical to the pattern of healing rickets. i demonstrate by reviewing the details of these different patterns that this is an incorrect declaration. additionally, concave distal ulna margins alone are not a sign of rickets. rickets is radiologically distinct from classic metaphyseal fractures. i reviewed medicolegal documents and published articles by several well known physicians who claim that certain children suspected of having suffered child abuse instead demonstrate rachitic bone disease. their statements and images in support of their opinions are critically reviewed and contrasted with known examples of rickets, including healing rickets. at the physeal region of long bones in rickets, the zone of provisional calcification (zpc) is not calcified and the normal metaphyseal collar shaftward from that zone is not maintained. in metaphyseal fractures from abuse, the zpc and collar are maintained. in early healing rickets, the zone of provisional calcification calcifies first, giving a transverse calcification separated from the already ossified metaphysis. this calcification is different from a bucket handle fracture pattern without signs of rickets. moreover, concave distal margin of the ulna without other signs of rickets is seen in about % of normal infants. repeatedly, denialists have testified against the diagnosis of child abuse, claiming rickets in the absence of radiographic evidence -i critique one well known child abuse is a serious diagnosisclaiming by denialists of an alternate diagnosis, such as rickets, when radiographically no rickets is present, should be refuted. a clear understanding of the differences between abuse fractures and rickets is obligatory. when no rickets is seen at other growth sites, including seeing the presence of zones of provisional calcification, rickets is not present. simultaneous multi-slice turbo spin echo: a -fold time-saving alternative to conventional tse in mr imaging of the pediatric knee s. benali, a. gholipour, p.r. johnston, s.d. bixby; boston/us objective: simultaneous multi-slice (sms) is a novel iteration of parallel imaging in mri that reduces acquisition time at least two-fold over conventional turbo spin echo (tse) acquisitions. the aim of this study is to determine whether -dimensional ( d) sms tse can replace conventional d tse in imaging of the knee in pediatric patients without compromising diagnostic quality. this was an irb-approved retrospective study. subjects included all pediatric patients referred for routine -tesla mri of the knee between october through december . mri examination consisted of institutional basic protocol with both traditional t tse and sms t tse in axial and sagittal planes with fat suppression. two anonymized study folders were created for each subject, one containing all basic sequences (basic exam), and one in which t tse sequences were substituted with t sms sequences (sms exam). two pediatric radiologists independently and blindly reviewed each study and noted presence or absence of pre-determined abnormalities (table ) . discrepancies between radiologists were settled by consensus. qualitative assessment was performed using a likert scale to evaluate edge blurring, contrast resolution, image noise, and flow artifact. thirty-five subjects comprised the study population (mean age . years, range - , female, male). diagnostic performance was assessed by comparing findings (table ) from basic exam to sms exam based on absolute agreement and cohen's kappa statistic. for all findings, the proportion of absolute agreements between basic and sms exam was > . for reader , > . for reader , and . for consensus between readers. kappas for consensus reads were . on all structures (p< . , lower % confidence limit > . ). for reader , kappas were . for / structures (p< . ) and . for pcl. for reader , kappas were . for / structures (p< . ) and . for cartilage defects. paired t-test was used to compare mean likert scores for image quality characteristics. for both readers, sms was preferred for flow artifacts whereas tse was preferred for the three remaining image quality characteristics (p< . ). our primary assessment suggests that sms t tse is comparable to standard tse in terms of diagnostic performance in the evaluation of the pediatric knee despite modest decrease in overall image quality. the -fold decreased acquisition time of sms is a significant advantage which is felt to offset the mild decrease in image quality, particularly as it increases the likelihood that children will tolerate the examination without motion. mri for sacroiliitis in children: panel findings and inter-observer evaluation using standardised reporting k.e. orr , m.j. bramham , s. andronikou ; plymouth/uk, bristol/uk there is little evidence regarding mri for diagnosing sacroiliitis in children with juvenile idiopathic arthritis (jia). the limited literature presents varied opinions but no published recommendations for standardisation of reporting. axial disease in jia responds poorly to conventional first-line treatments but identifying these children using history and examination findings is unreliable. standardised mri reporting ( ) (suppl ):s -s pediatr radiol may improve diagnosis and selection of patients in whom newer biologic treatments are indicated. the aim was to use a standardised reporting proforma based on published definitions for recording mri findings in suspected sacroiliitis to evaluate inter-observer agreement and determine the reliability of findings according to specific sequences. ninety-nine sacroiliac joint mris ( joints) were included, were initial examinations and were follow-up mris. the age range was between . and . years (mean age . years). three readers retrospectively reported all mris using the standardised proforma. 'reader ' was the study group panel while readers and were specialist paediatric radiology consultants working in the united kingdom. readers were blinded to additional clinical information and other imaging. inter-reader variation was evaluated for the presence of bone marrow oedema, erosions, effusions, ankylosis, sclerosis and enhancement, as well as the presence or absence of sacroiliitis. the quality of mri examinations was evaluated, including presence and adequacy of sequences performed and alignment of the coronal/oblique studies. mri findings were correlated with clinical details and final diagnosis. there is significant variability in sacroiliac joint mri protocols. refinement of these to include only necessary sequences based on inter-reader reliability and reinforcement of good positioning will improve reporting and result in universal standardisation. there is inconsistency in current reporting practice of sacroiliac joint mri in children but increasingly, clinicians rely on imaging to select patients with sacroiliitis and guide appropriate treatment. using a standardised reporting proforma may improve the quality and consistency of reporting. ultrasound-guided steroid tendon sheath injections in juvenile idiopathic arthritis s. peters, d.a. parra; toronto/ca objective: juvenile idiopathic arthritis (jia) is the most common chronic rheumatic disease in childhood. tenosynovitis is one of the manifestations of jia, which can explain the absence of response to treatment when adjacent joints are injected. steroid injection is one of the treatment options for tenosynovitis and it has been shown to be effective in the literature. utilizing ultrasound (us) guidance for injections into tendon sheaths has shown clinical advantage to conventional blind injections in the adult rheumatoid arthritis population. the aims of this study are to: (a) identify tendon sheaths most commonly treated in our patient population with jia referred for steroid injections; (b) describe technical aspects of the procedure; (c) characterize sonographic appearance of tenosynovitis in jia; (d) assess agreement between clinical request and sites injected. this was a year single-center retrospective study ( may -april in which we recruited patients with jia referred by rheumatology for us-guided tendon sheath injections. we collected patient demographics, clinical assessment information, sonographic appearance of the tendons and technical aspects of the intervention from the procedure records. we collected data from visits of patients ( % female, mean age years months) with a total of injections. the ankle region was most commonly injected ( %), specifically the tendon sheaths of tibialis posterior ( %), peroneus longus ( %) and brevis ( %). % of the procedures were performed under general anesthesia and triamcinolone hexacetonide was used in % of the injections. an "out of plane" approach was used in % of the interventions and the mhz "hockey stick" us probe was preferred for guidance ( %). we found minor intra-procedure complications without sequelae. the majority of treated sites ( %) showed peritendinous fluid and sheath thickening on us. other findings were increased color-doppler signal and echogenic peritendinous fluid. a strong agreement between clinical request and sites injected was observed and most patients required one visit ( %). us-guided tendon sheath injections are used frequently to treat patients with jia. it is a safe intervention with a high technical success rate. the ankle region, specifically the medial compartment, is the area most commonly injected in this cohort of patients. the most common sonographic finding is peritendinous fluid and sheath thickening. these findings might assist radiologists and rheumatologists to characterize and more effectively manage tenosynovitis in patients with jia. to evaluate the accuracy of the software for automatic bone age (ba) estimation based on deep learning technique, and to validate the feasibility of this system in clinical practice. the software for automatic ba estimation was developed based on deep learning technique using , left hand radiographs and estimated ba of each radiograph based on greulich-pyle method. ba estimation was done for left hand radiographs of consecutive patients ( months - years; boys and girls) in three methods: ( ) ai bone age (assessed by the software), ( ) ai-assisted ba (assessed by two radiologists with the assistance of the software), ( ) gp atlas-assisted ba (assessed by two radiologists with only gp atlas but the software). the reference ba was determined by two radiologists by consensus. the accuracy of the estimated ba by each method was assessed using concordance rate (%), pearson's correlation analysis, the root mean square error (rmse), and bland-altman plot. reading time for ba estimation by each method was evaluated. ai bone age showed % of concordance rate, and a significant correlation with reference ba (r = . , p< . ). the bland-altman plot of agreement between the reference ba and ai bone age showed the mean difference of - . years ( % limit of agreement, ± . years). rmse was . years. in reviewer , concordance rates were same between both gp atlasassisted ba and ai-assisted ba ( %), and rmse of ai-assisted ba ( . ) was slightly lower than that of gp atlas-assisted ba s ( ) (suppl ):s -s pediatr radiol ( . ). in reviewer , concordance rate was slightly higher in aiassisted ba ( %) than gp atlas-assisted ba ( %), and rmse was almost the same ( . in ai-assisted ba, . in gp atlasassisted ba). the reading time was reduced . % in reviewer and . % in reviewer . the software for automatic ba estimation based on deep learning technique showed high accuracy and may enhance work efficiency in ba estimation by allowing radiologists to save reading time and to improve accuracy. temporomandibular joint mri findings in adolescents with primary disk displacement in comparison to those in juvenile idiopathic arthritis j. bucheli, d. ettlin, c. kellenberger; zurich/ch to investigate potential differences of morphology and degree of inflammation in temporomandibular joints (tmjs) affected by primary anterior disk displacement (add) and juvenile idiopathic arthritis (jia). in adolescents ( female, age ± y), contrast enhanced magnetic resonance images (fig. a) of tmjs with add were retrospectively compared to those of age-and gender-matched controls with jia. morphology of articular disk and bony structures were described. osseous deformity and inflammation were qualitatively scored with progressive -grade scales and compared between groups with mann-whitney-u test. mandibular ramus length, measured on gradient echo minimum intensity projection images (fig. b) , was compared between groups and to normal values with independent samples t-test. in the add-group, / disks were dislocated anteriorly and showed thickening of the posterior band ( / ). in contrast, tmj disks of jia patients were mainly flattened (n= ) and/or centrally perforated (n= ) and rarely dislocated (n= ). tmjs with add showed similar overall grades of inflammation (p= . ) and osseous deformation (p= . ) as tmjs in the jia group. while erosions were frequent in both groups (add / ; jia / , p= . ), the mandibular condyle (p< . ) and glenoid fossa (p< . ) were less flattened in tmjs with add. in add tmjs, bone marrow oedema was less frequent (p= . ) and grades of joint enhancement slightly lower (p= . ), but presence of synovial thickening (p= . ) and degree of effusion (p= . ) were not significantly different between groups. mandibular ramus length was not significantly different (p= . ) between groups, but in both groups clearly decreased compared to mean normal values (p< . ). articular disks in tmjs affected by jia are rarely dislocated. surprisingly, tmjs with primary add show considerable inflammatory change including condylar erosions. still, chronic systemic inflammation in jia joints results in considerable higher deformity of the mandibular condyle and the temporal joint surface. observation of the mostly preserved normal shape of the temporal bone may help differentiating primary add from jia. retrospective magnetic resonance imaging (mri) study of consecutive jia patients ( female, median age y) with at least two consecutive tmj mri examinations ≥ y apart and no csi. degree of tmj inflammation was determined on t -weighted and contrast-enhanced t weighted fast spin echo images (fig. a) , and degree of osseous deformity on gradient echo images (fig. b) by progressive -grade scales ( - ). change of respective grades was assessed with wilcoxon test. mandibular growth was determined by ramus length change and compared to normal values. over a median period of . y (interquartile range, . - . y), degree of tmj inflammation improved (p< . ) with decrease in frequency of grade ( . % to %) and grade ( . % to . %). inflammatory grades improved both in patients with (n= , p= . ) and without (n= , p= . ) systemic disease modifying medication. the degree of osseous deformation slightly improved (p= . ), with decrease in frequency of grade ( . % to . %) and grade ( . % to . %), and increase of grade ( % to . %). overall growth rates of mandibular ramus (median, . mm/y) were not significantly different from normal growth rates (p= . ) (fig. c) . growth rates of tmjs from patients only receiving non-steroidal anti-inflammatory drugs (median, . mm/y) were not significantly different (p= . ) compared to patients treated with systemic disease modifying drugs (median, . mm/y). in patients with systemic treatment of jia, both the degree of tmj inflammation and osseous deformity as seen on mri improved at midterm follow-up. normal growth of the mandibular ramus was maintained. these results are in contrast to those from an earlier cohort treated with csi, in which on average deformities deteriorated and growth was impaired. objective: pediatric ileocolic intussusception, ici, is a common abdominal condition for which pediatric radiologists are asked to attempt emergency pneumatic reduction. because of the high success and low complication rates of pneumatic reductions, radiologists are able to make several attempts at reduction in stable patients if the initial enema attempt is unsuccessful. we have observed patients with successful reductions with rather long periods between initial symptoms of ici and performance of the air enema. we hypothesize that successful pneumatic reduction rates are independent of length of symptoms and in stable patients, repeated reduction attempts can be performed with the expectation of successful reduction. we performed an irb-approved retrospective review of all ici with a pneumatic reduction attempt between - at xxx. clinical, imaging and surgical data was reviewed. time to enema was defined as the time from first symptom to first air enema attempt. linear and second order polynomial statistical analysis was performed to assess the relationship between time to enema and enema outcome. results: ici were identified in patients. air enema was successful in ici, %. the mean time to enema was . hours, range - hours with sd of . hours for successfully reduced ici and . hours, range - hours with sd of . hours for unsuccessfully reduced ici. surgical resection was required in patients with ischemic bowel including one with an irreducible meckel's diverticulum as lead point. there was no correlation between time to enema and successful reduction, fig . no patient with a successful pneumatic reduction of a ici required subsequent bowel resection. conclusions: air enema for ici can be safely performed despite prolonged time to enema with the anticipation of a successful reduction. the lack of correlation of pneumatic reducibility and time to enema suggests that in surgically cleared patients with ici, the pneumatic reduction attempt may not be a true emergency and that repeated attempts at reduction are safe. additionally, though our numbers are small, they suggest that an ici is reducible or not from the beginning and do not "become irreducible" with prolongation of the time to enema. evaluation of splenic stiffness measurements for the diagnosis and the follow-up of portal stenosis after paediatric liver transplantation c. escalard , a. dabadie , s. chapeliere , d. pariente , c. adamsbaum , s. franchi ; le kremlin-bicêtre, paris/fr, la timone, marseille/fr to report our preliminary findings about the role of splenic and hepatic supersonic shear-wave elastography (sswe) in the diagnosis and followup after treatment of portal stenosis in paediatric liver graft recipients. all paediatric liver recipients with portal stenosis treated by the interventional radiology procedure, and who underwent splenic and hepatic sswe pre and post interventional procedures, were retrospectively reviewed. demographics, data about the portal stenosis (delay post transplantation, clinical presentation, initial radiological findings, hemoglobin and platelet counts), ir procedure performed, clinical and ultrasonographic follow-up and spleen stiffness pre and post ir procedure were collected. four patients were included, median age , years (range , months to years) and median delay post transplantation , years (range month to . years). two patients presented with anemia, associated in one case with progressive splenomegaly. one patient had liver test abnormalities, and one had decreased portal flow found on systematic doppler followup. spleen stiffness was elevated pre-procedure in all patients, from to kpa (normal < kpa), and liver stiffness was normal or mildly elevated in all. portal stenosis was successfully treated by ir in patients. spleen stiffness decreased rapidly, ranging from to % (figure ) . however, the size of the spleen remained unchanged. in the last patient, angioplasty of the portal stenosis failed leading to portal thrombosis. spleen stiffness increased on the subsequent ultrasound ( figure ). mr elastography (mre) is a novel imaging technique that provides a non-invasive evaluation of liver fibrosis. the standard sequence used for this purpose on a siemens scanner has been gradient echo (gre). we also implemented echo planar imaging (epi) available as a work-in-progress (wip). our aim is to compare the liver elastogram values between gre and epi in children. after consent from both research and referred clinical subjects, a dedicated mre of the liver was performed on a t mr scanner (magnetom® skyra, siemens) with a pediatric mechanical driver over the right upper quadrant. an axial t blade with fat saturation, coronal t vibe dixon and axial diffusion weighted imaging (dwi) were obtained. elastograms were obtained using both standard gre and epi, in the axial plane. for the gre sequence, different slices were selected and each scanned sequentially. the epi sequence incorporated different slices in just one series. images were post-processed placing regions-of-interest (roi) and measuring the stiffness in kilopascals (kpa). for each sequence and each slice the mean stiffness and then the average of the means was calculated. a spleen elastogram was simultaneously generated, without changing the mechanical driver location, and the mean stiffness was also calculated. increased stiffness was defined as > . kpa in the liver and > . kpa in the spleen. we focused on a technical comparison between the sequences without clinical or histological correlation of findings. we included subjects that had elastogram measurements of liver and of them spleen stiffness on both gre and epi sequences. mean liver stiffness on gre was . (sd+/- . ) and on epi was . (sd+/- . ), with a pearson's correlation of r= . (p< . ). increased liver stiffness was found in / ( . %) of the cases in gre and / ( %) of the cases in epi. mean spleen stiffness on gre was . (sd+/- . ) and on epi was . (sd +/- . ) with a pearson's correlation of r= . (p= . ). epi reported consistently higher values than gre in both liver and spleen stiffness. our preliminary data shows a moderate to high correlation between gre and epi sequences; however, the epi values were higher in both liver and spleen. in the future, larger studies are needed to validate these thresholds and patterns among different sequences. were also reviewed if done. patient's medical & surgical treatment, and clinical progress were also reviewed. active telephone follow-up days after cevus was performed. results: patients giving a total of pelviureteric units were referred for vus study during the study period, with age ranging from month to years old. no contrast-related complication was encountered. except cases with failed catheterization, were investigations of urinary tract infection (uti), antenatal hydronephrosis and congenital anomalies etc., and remaining were follow up studies of known reflux. of all cases of uti, refluxing units were picked up by vus, ranging from grade i to v. of the refluxing units diagnosed by cevus, were missed on mcu, among which were high grade refluxes (grade iii to v) requiring treatment; whereas cevus only missed one grade i refluxing unit detected by mcu. besides, one grade iv refluxing unit identified on vus was under graded by mcu to grade i. regarding patient outcomes, one patient with mcu-missed refluxing unit presented with breakthrough uti on follow up. two refluxing units that were missed on mcu but detected on cevus demonstrated scarring on dmsa. conclusion: cevus is shown to be more sensitive in detecting vesicoureteric reflux than mcu. the fact that mcu-missed refluxes detected by cevus were associated with breakthrough urinary tract infection and scarring on dmsa indicated that the extra sensitivity brought by cevus did translate to clinical significance. difficulty in visualizing low-grade reflux is a potential limitation of this technique. with favourable diagnostic performance and safety profile, cevus can be further applied in this community in the era of radiation reduction. percutaneous transbiliary needle or forceps biopsy in hepatic masses with biliary dilatation a. dabadie , s. franchi , d. pariente ; la timone, marseille/fr, le kremlin-bicêtre, paris/fr hepatic masses with biliary dilatation are rare in children and mainly include rhabdomyosarcoma of the biliary ducts, but also other masses or pseudo-masses compressing the hepatic hilum. in these patients histological diagnosis of the lesion as well as temporary biliary drainage are warranted. the objective of this study is to report our experience in percutaneous transbiliary biopsy performed simultaneously and using the same access as the percutaneous biliary drainage in children with hepatic mass obstructing the biliary ducts. children presenting with a hepatic mass causing biliary obstruction, with need for biliary drainage, were considered candidates for percutaneous transbiliary biopsy of the lesion performed at the same time. the biopsy was performed under ultrasound guidance, through a sheath introduced in the dilated biliary system, using a semi-automatic gauge needle or the transluminal biliary biopsy forceps set (cook medical, bloomington, usa). between and , four patients were included, three females and one male, median age . years (range . - . ). all presented with jaundice and were diagnosed with a hepatic mass with secondary biliary obstruction. percutaneous transbiliary biopsy was performed in all patients using the gauge needle. in one patient, the biopsy did not demonstrate any tumoral cells and a second biopsy was performed using the forceps device through the same biliary access. the samples deemed adequate for analysis by the pathology department in all patients, however the samples were larger when using the needle. a retrospective -prospective study included patients of both sexes ( , +/- , y), in a two-year span. patients were divided into two groups according to the used diagnostic method (positivegroup a on us and a on mri, with intestine mural thickness above mm, and negativegroup b on us and b on mri, with mural thickness below mm). overall sensitivity and specificity of us and mri in diagnosing ibd was calculated in comparison to pathohistological (ph) findings. us examination showed an average intestinal mural thickness of . ± . mm and . + . mm in group a ( patients) and group b ( patients) respectively. mri examination showed an average intestinal mural thickness of . ± . mm and , + , mm in group a ( patients) and group b ( patients) respectively. out of patients from group a, ( %) had irregular mural architecture, contrary to group b in which mural architecture irregularities have not been observed. in groups a and b ( . %) and ( . %) patients had irregular mural architecture respectively. average length of affected intestinal segment on us and mri was mm and mm respectively. five patients from group a and four from group a had signs of fibrosis. color doppler showed hyperemia in and patients of group a and a respectively. transmural signs of inflammation were found in % of patients on us, and . % of patients on mri. average longer diameter of mesentery lymph nodes measured by us and mri was . ± . mm and . ± . mm, respectively. overall sensitivity of us and mri was . % and . % respectively. both us and mri showed a specificity of %. us and mri are reliable and compatible methods in diagnosing ibd, with mri being slightly more accurate. us is an extremely valuable and widely available imaging modality in every-day clinical work, both in diagnosing and follow-up of therapy effects in children with ibd. findings in percutaneous transhepatic cholecysto-cholangiography in neonates and young infants presenting with conjugated hyperbilirubinemia d.a. parra, s. peters, j. amaral; toronto/ca objective: conjugated hyperbilirubinemia is a concerning finding in neonates and young infants, biliary atresia (ba) being one of the main diagnostic considerations. ba is a rare disease characterized by fibrosis of the biliary tree. the obliteration of the biliary system leads to cholestasis and ultimately liver parenchymal injury, cirrhosis and death. an early diagnosis of ba along with a kasai portoenterostomy operation significantly improves the long-term prognosis. percutaneous transhepatic cholecysto-cholangiography (ptcc) is one of the options described in the diagnostic algorithm of ba. the aims of this study are to: (a) describe ptcc findings in patients with conjugated hyperbilirubinemia; (b) identify the abnormal patterns encountered that justify further investigations; (c) analyze technical aspects of the procedure. this is a year single-center retrospective study ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in which we recruited patients with the diagnosis of cholestasis (less than months old) referred for ptcc. we collected patient demographics, clinical information, findings in ptcc, post-procedure management and long term clinical outcome. eigthy-nine patients were referred for ptcc in the study period. the procedure was technically feasible and successfully performed in patients ( % male, mean age . months). forty-one had a pre-procedure hida scan suggestive of ba. fifty-nine patients had an ultrasound-guided biopsy in conjunction with the ptcc and in all of them the cholangiography was performed through a needle placed using ultrasound guidance in the gallbladder. % ( ) of the patients had a normal ptcc. abnormal patterns encountered were: ) variable degrees of hypoplastic bile ducts seen in %; ) atretic gallbladder without demonstration of communication with bile ducts seen in %; and ) gallbladder communication with a cystic structure not communicated with the biliary ducts (cystic biliary atresia) seen in %. the most common diagnosis in the abnormal group was ba ( %). alagille's syndrome, alpha- antitrypsin deficiency and progressive familial intrahepatic cholestasis were other diagnoses in this group. no complications related to the procedure were observed. ptcc is a safe and effective option in the diagnostic algorithm of patients presenting with cholestasis early in life. visualization of the gallbladder is fundamental to perform the procedure. the majority of studies were normal in our patient population preventing further invasive investigations. three types of abnormal ptcc patters were encountered, with ba being the most common diagnosis in this group of patients. to evaluate the additive role of shear wave elastography in the sonographic distinction of biliary atresia from other causes of neonatal/ infantile cholestatic liver disease. neonates and infants with clinical and biochemical diagnosis of cholestatic jaundice were enrolled in our study after obtaining informed written consent from the parents. grey scale, doppler and shear wave elastographic findings were recorded after hours of fasting using aixplorer® ultrasound system (supersonic imagine, aix en provence, france). sedation was not needed during the study. for obtaining elastographic values, linear transducer ( - hz) was used and after image stabilization a q-box measuring mm was placed in the most homogenous vessel free area. the mean of three elastographic values were recorded. hida scan, liver biopsy, intra-operative cholangiogram and histopathological evaluation of resected specimens was done wherever feasible and clinically indicated. the prospectively obtained elastographic values were retrospectively evaluated. eleven of patients included in our study were proven to be biliary atresia (ba) by intra operative cholangiogram and histopathological reports. the diagnosis in the remaining patients included other causes of infantile cholestatic jaundice like infantile choledochal cyst, neonatal idiopathic hepatitis, progressive familial intrahepatic cholestasis, abernathy malformation, cmv hepatitis etc. the elastographic values of ba and non-ba patients were compared. six of infants were younger than days which included four patients with ba and their elastographic values ( . ± . kpa) were significantly different from that of non-biliary atresia ( ± kpa) in the same age group (p value < . ). similarly, for patients aged > days also we had a significant difference (p value < . ) in elastographic stiffness between ba ( . ± kpa; n= ) and non-ba ( . ± . kpa; n= ) groups. the mean echogenic area anterior to right portal vein (earpv) was . ± . mm in ba and . ± . mm in non-ba group (p value < . ). the mean gall bladder (gb) length was . ± . mm in biliary atresia group in contrast to . ± . mm in the rest (p value < . ). the roc plot for earpv and gb length gave a youden index cut off value of > . mm (sensitivity . & specificity . %) and < . cm (sensitivity & specificity . %) respectively. infants with biliary atresia have a significantly higher elastographic value when compared to age matched patients with other causes of neonatal cholestasis. we expect to validate the findings in our ongoing study with a larger sample size. to retrospectively define in a large pediatric population the association between testicular microlithiasis and testicular neoplasia. retrospective multicenter study of scrotal ultrasounds performed between january and april in subjects < years of age. all unique subject scrotal ultrasound reports from each institution were reviewed for mention of microlithiasis. for subjects with serial exams, the most recent exam performed was included in the analysis. all exams mentioning microlithiasis were reviewed by site-specific investigators to confirm the presence of ≥ punctate calcifications in the testicle on a single image. the presence of testicular germ cell and stromal tumors were determined for subjects with and without microlithiasis through review of institutional pathology and imaging databases. the risk of testicular neoplasia in the context of microlithiasis was expressed in terms of odds ratios with (a-or) and without adjustment (u-or) for fixed study site (institution) effects by logistic regression. the study population included , unique subjects with confirmed microlithiasis in , ( . %). mean subject age was . ± . years for subjects with microlithiasis and . ± . years for subjects without (p< . ). one hundred thirty-nine subjects ( . this large, multicenter study confirms that there is a significant, strong association between testicular microlithiasis and testicular neoplasia, particularly malignant germ cell tumors. children with microlithiasis have approximately x greater odds of having a malignant germ cell tumor than children without microlithiasis. this reinforces the need for a large prospective study assessing the risk of developing testicular neoplasia in children with incidentally identified diffuse microlithiasis. do adc-values reflect renal function or obstruction in children with uretero-pelvic-junction obstruction? p. grehten, a.c. eichenberger, c. kellenberger; zurich/ch the use of diffusion weighted imaging (dwi) in renal mri is increasing. in adults as well as in infants a positive linear correlation between adcvalues and glomerular filtration rate has been demonstrated. the aim of our study was to assess whether renal dwi can provide information on the grade of urinary tract obstruction or renal function in children with uretero-pelvic-junction (upj)-obstruction. retrospective analysis of children (age . +/- . y) with unilateral upj-obstruction who underwent pre-and postoperative mri at . t and normal controls (age . +/- . y). functional mr-urography and multiple b-value dwi were part of the mr-protocol. renal adc-values were correlated to measures of obstruction and function, and compared between obstructed and non-obstructed kidneys and between pre-and postoperative studies. no correlation was found between mean parenchymal, cortical or medullary adc-values and calyceal transit time (ctt), renal transit time (rtt) and measures of differential renal function (%parenchymal s ( ) (suppl ):s -s pediatr radiol volume, vdrf, pdrf). there was moderate correlation with absolute parenchymal volume and total kidney volume, and low correlation with pelvic volume. adc-values showed high correlation with age and patient's weight. adc-values normalized for age or weight showed low correlation with rtt and ctt, but no correlation with functional measures. adc-values were not significantly different between obstructed and contralateral normal kidneys (p= . - . ) or between pre-and postoperative studies (p= . - ). renal adc is dependent on age and weight in young children and does not correlate with differential renal function. for assessing urinary tract obstruction with adc normative values need to be established. to determine the level of knowledge and awareness of medical staff, medical students and parents concerning possible risks associated with ionizing radiation. a prospective study has been conducted at children's hospital, center for adult's radiology, and medical faculty, by filling out two anonymous questionnaires (questionnaire medical staff and medical students, questionnaire parents of the children exposed to x-ray based procedures), and it included participants. statistical analysis was performed using the spss . . the majority of examinees assessed their knowledge about ionizing radiation as moderate. knowledge level was statistically significantly higher only in the group of medical students who passed the course of radiology, in comparison to the group of those who have not attended the course yet. only % of radiologists and up to . % of pediatricians, pediatric surgeons and anesthesiologists are informed about "image gently" campaign. up to % of radiologists, and up to % of clinicians, both specialists and residents, are aware of alara principle. over % of medical doctors think that diagnostic radiology procedures are very often performed unnecessarily among children, while only . % of parents share this opinion. most of the radiologists and clinicians consider it necessary to inform parents about potentially harmful effects of ionizing radiation, but even though - % of clinicians claim they do inform parents in every-day clinical practice, over % of parents affirm that they had never been informed about effects of ionizing radiation before diagnostic procedures were performed on their children. only % of pediatric surgeons and pediatricians, but . % of radiologist and % of anesthesiologists are concerned that informing parents about ionizing radiation would cause problems in every-day work. nearly % of parents claimed that they would not refuse to expose their child to x-ray based diagnostic procedure, after the given information about potential harmful effects. over % of radiologists and less than % of pediatric surgeons and pediatricians support the initiative to calculate the total effective dose child was exposed to during hospitalization, and place it on the discharge list. between % and % of pediatricians and pediatric surgeons greatly underestimated the effective doses in ct and fluoroscopy procedures. there are - % of clinicians who are aware that ct increases the risk of carcinoma development. this study showed that general knowledge about ionizing radiation, potential risks and effective doses in pediatric population is poor, and that organized education is required. fluoroscopy in pediatric radiology -how important is an individual impact to radiation exposure of children? j. lovrenski, i. varga; novi sad/rs to determine whether there are differences between different pediatric radiologists and radiology residents in exposure of pediatric population to ionizing radiation during fluoroscopy procedures. a retrospective study has been conducted at the regional children's hospital, and included all the diagnostic fluoroscopy examinations performed within a one-year period. the fluoroscopic data along with the names of pediatric radiologists/radiology residents performing these examinations were retrieved from the evidentiary notebooks, and included: dose-area product (dap), skin dose, and fluoroscopy time. there were radiologists (r -r ), and radiology residents (r -r ) involved in fluoroscopic examinations. we found all the fluoroscopic findings in the hospital's data base, which enabled a differentiation between positive and negative findings. statistical analysis was performed using the spss . . a p-value less than . was considered statistically significant. a total of fluoroscopy procedures in children (mean age , y, males and females) have been performed within a one-year period, most of which were voiding cystourethrograms (vcug) - , and an upper gastrointestinal (gi) series - examinations. radiology residents and radiologists carried out and examinations respectively. duration of fluoroscopy procedures performed by residents (av. . s) was statistically significantly shorter in comparison with duration of fluoroscopy examinations performed by radiologists (av. s). dap and skin dose did not show statistically significant difference between these two groups, as well as the number of positive and negative fluoroscopic findings in groups of examinations performed by radiologists and radiology residents. mean dap value ranged from . μgym (r ) to . μgym (r ) when performing vcugs, and from . μgym (r ) to . μgym (r ) for upper gi series. mean skin dose ranged from . mgy (r ) to . mgy (r ) for vcugs, and from . mgy (r ) to . mgy (r ) for upper gi series. mean fluoroscopy time ranged from . s (r ) to . s (r ) for vcug, and from . s (r ) to . s (r ) for upper gi series. statistically significant difference was shown only between radiologists r and r for dap and skin dose values in performing vcug, and for fluoroscopy time in performing an upper gi series. for all examinations dap and skin dose were statistically significantly higher in the group of positive fluoroscopic findings. this study has shown that exposure of children to ionizing radiation during fluoroscopy procedures significantly depends on radiologist/ radiology resident and the nature of fluoroscopic finding. to evaluate image quality and radiation exposure of non-contrast pediatric chest ct with automated tube voltage selection (atvs), in combination with automated tube current modulation (atcm). non-contrast chest ct scans of children ( male and female; mean age, . ± . years) were analysed retrospectively with regard to radiation exposure and image quality before and after the implementation of an automated tube voltage selection. correlations of volume ct dose index (ctdi vol ) and the effective diameter (edm), before and after the implementation of atvs were compared, and confidence intervals related to the change in correlations with and without atvs were determined using fisher's z-transformation. image quality was assessed by mean signal-difference-tonoise ratios (snrs) in the aorta and in the left principal bronchus with the independent samples t-test. subjective image quality was rated by two pediatric radiologists and a general radiologist on a point scale. agreement between the readers was assessed using weighted kappa coefficients. a p< . were considered significant. automated tube voltage selection, in combination with an automated tube current modulation, resulted in optimization of scan protocols, homogeneity of image quality, and reduction of radiation exposure for pediatric patients. advantages and disadvantages of cone beam ct for pediatric interventions l. dance, r.b. towbin, d. aria, c. schaefer, r. kaye; phoenix/us objective: illustrate the advantages and disadvantages of cone beam ct (cbct) as an alternative to conventional ct guidance and an adjunct to angiography. there is a steep learning curve to optimize utilization of cbct. we found that cbct reliably identifies high-contrast lesions. however, the lower dose and decreased penetration of cbct resulted in poorer visualization of low-contrast lesions. also cbct can be degraded by streak artifact from hardware or dense contrast. the relatively narrow field of view can be restrictive for peripherally located lesions in larger patients. however, the anatomic display is adequate for guidance in most instances. these findings are illustrated in a series of cbct-guided cases including pulmonary nodule localization, osteoid osteoma ablation, abc sclerotherapy, renal av fistula embolization, and liver lesion biopsy. the advent of cbct as an adjunct modality in the ir suite has significantly decreased the use of conventional ct guidance and significantly decreased the radiation dose in children. we have found cbct to be a practice changer. the aim of this study is to review our local drl in pediatric fluoroscopy and to compare them to values proposed by pidrl guidelines and recent international litterature. data were prospectively collected on consecutive procedures ( total) performed from january to december on different fluoroscopy units (siemens iconos r , luminos drf). of each procedure patients data (name, weight and birth date), examination-data (kind of procedure, date, dap [cgy*cm ], total fluoroscopy time, number of images) were recorded. data from micturating-cystourethrography(mcu), barium meal/swallow(bs) and most commonly performed procedures were divided into weight-groups (< kg, - kg, - kg, - kg) and of each one th-percentile was calculated. data were compared to europeandrl and recent literature data (by age:newborn, -, -, years old). weight-groups are considered a representative sample if at least -patients per procedure-type and per patient-group are included. our local-drl for mcu are (< kg), ( - kg), ( - kg) and ( - kg). they results to be lower than pidrls values ( , , , ) but higher if compared to a previous local survey of ( , , , ) . bs data are (< kg), ( - kg), ( - kg); these data are lower than that of a previous local survey of ( , , ) . the update of local-drl is helpful in daily practice to identify (and solve) critical issues such as incorrect technique or poor practice with new flat-panel equipment. pidrl guidelines: a review of local drl for pediatric head, thorax and abdomen ct in a italian referral center a. magistrelli, v. cannatà, e. genovese, m. cirillo, r. lombardi, p. toma; rome/it the aim of this study is to review our local drl in pediatric ct and to compare them to values proposed by pidrl guidelines and recent international litterature. data were prospectively collected on consecutive procedures ( total) performed from january to june on a somatom definition flash siemens. of each procedure patients data (name, weight and birth date), examination-data (kind of procedure, clincal question, date, ctdivol / and dlp / ) were recorded. ctdivo/dlp from head ct were divided into age-groups (< weeks, weeks- y, - y,≥ y) and of each one th-percentile was calculated. ctdivol/dlp from thorax (chest, cardiovascular ct angiography) and abdomen+pelvis ct examination were divided into weightgroups (< kg, - kg, - kg, - kg,> kg) and of each one th-percentile was calculated. data were compared to europeandrl and recent literature data. weight-groups are considered a representative sample if at least patients per procedure-type and per patient-group are included. our local drl are substantially lower than that proposed by pidrl guidelines. specifically ctdivol/dlp for chest ct are / (< kg), , / ( - kg), , / ( - kg), , / ( - kg), , / (> kg) respectively. for cardiovascular ct angiography are , / (< kg), , / ( - kg), , / ( - kg), , / ( - kg), , / (> kg). while for abdomen+pelvis ct are , / (< kg), , / ( - kg), , / ( - kg), , / ( - kg), , / (> kg). data for trunk sere not collected. for head ct local drl are higher in age-group and but lower in other age-group if compared to routine head ct pidrl ones. the update of local-drl allowed us to identify (and solve) some critical issues such as incorrect technique. drl-curve in optimization of pediatric body ct r. seuri , p. laarne , a. nikkola-sihto , k. nygaard bolstad , m.s. perhomaa , a. thilander klang , k. rosendahl , j. ruohonen , e. tyrvainen ; helsinki/fi, tampere/fi, seinäjoki/fi, bergen/no, oulu/fi, gothenburg/se, kuopio/fi objective: diagnostic reference levels (drls) in medical imaging represent valuable tools to study dose optimization in clinical practice. this is particularly important in pediatric computed tomography (ct) as the number of the examinations in many institutions is low. drls are typically given as a percentile point, usually as % or rd quartile of the observed distribution of patient dose. in pediatric practice drls are often given for each age-or weight group separately. we present continuous drl-curve as a feasible way to compare dose levels in pediatric body ct. during - a selected group of nordic hospitals collected dose values (ct dose index by volume, ctdi vol , and dose-length product, dlp) from pediatric body ct examinations on children aged - years. the dose values were imported into a dynamic excel table, previously established by the radiation and nuclear safety authority in finland, stuk (fig ) . the stuk-table includes a graphic presentation of a continuous drl-curve presented as a function of body weight, and the program automatically calculates a dose curve and compares it to the established reference level (fig ) . the dose values were easily exported to the excel tables, and the graphic presentation and comparison with an established drl-curve was clear and readily understandable for both radiologists and radiographers. in some of the institutions included in the present study, the weight of the patient was not recorded routinely. this represents a challenge for the use of the drl-curves provided by stuk. the drl-curves provided by stuk were feasible for clinical practice. the automatic calculation of the dose curve and graphic presentation were helpful to interpret the results. the drl-curve also allows relevant comparison even with a smaller number of patients. fifty randomly selected ct chest studies performed over years to assess diffuse lung disease were included in the study sample ( females, males; mean age . years + . years), comprising disorders. two pediatric radiologists and a pediatric radiology fellow blinded to the results of the cts evaluated four subsets of complete chest cts ( slices, every third slice, every other slice, and all images below the thyroid) and compared the subsets with the entire chest ct, interpreted as the control. accuracy of evaluating the primary diagnosis and determination if significant diagnoses were missed in the reduced slice ct subsets were rendered. we assume linear distribution of dose across the anatomy to estimate dose reduction on reduced slice subsets. most significant findings were present on all reduced slice ct subsets. all relevant findings were present in % of subthyroid, % of every other slice, % of every rd slice, and % of regional slice subsets respectively. excluded findings included small foci of ground glass opacity, consolidation, focal mosaic attenuation, and linear parenchymal bands; peribronchial thickening, dextrocardia vs dextropositioning, tree-in-bud opacities, extent of mild bronchiectasis. with the exception of consolidation in of the studies, these findings were not thought to inhibit diagnostic assessment. the underlying diagnosis was correctly identified in most of the subsets: % subthyroid and every other slice, % every rd slice, and % of regional slice subsets. dose is significantly decreased by using any of these methods. while some findings are excluded with increasing gaps between slices, equivalent diagnostic information can be provided on reduced slice ct and can serve as a viable strategy to reduce lifetime radiation dose to children and young adults with diffuse lung disease imaged for routine follow-up. as findings are missed with larger gaps, this strategy should be used with caution in patients presenting with acute symptoms . to extrapolate the significance of early diagnosis which will compliment to treatment planning and management. case presentation: types a and b niemann-pick disease are lysosomal storage disorders that result from deficient acid sphingomyelinase activity and lead to the accumulation of sphingomyelin, primarily in tissues of the reticuloendothelial system. type b niemann-pick disease manifestations are hepatosplenomegaly, excess bleeding and bruising, growth retardation, and recurrent respiratory infections. features of hrct include thickened peribronchovascular and interlobular septal thickening, ground-glass opacities. the intermixed regions could be characterized as showing crazy paving, although this is not the predominant pattern. type b niemann-pick disease should be added to the list of clinical entities that can demonstrate crazy paving. our patient is a sevenyear old girl, presented with dry cough and fever. physical examination revealed hepato splenomegaly. radiological work up included abdominal ultrasound examination, which showed mild hepatosplenomegaly. chest radiography revealed diffuse reticulonodular infiltration in both lungs. chest hrct was done for more comprehensive evaluation which showed multilobar bilateral peribronchovascular interstitial thickening and interlobular septal thickening with ground-glass opacities and crazy paving appearance. no honeycomb pattern was seen. no sizable pulmonary nodule or sizable mediastinal lymphadenopathy was seen. no pleural effusion was seen. finding were indicating extensive pulmonary intestitial disease. a corroborative analysis along with lab tests and genetic studies revealed the diagnosis of type b niemann pick disease. . the spectra of hrct features including crazy paving pattern may be encountered; though not frequent. hence should be included in the differential diagnosis of crazy paving pattern. blast from the past: lemierre's syndrome in adolescents with sore throat o. kvist; stockholm/se a minor ailment such as a sore throat could prove to be a severe disorder known as lemierre's syndrome. this syndrome mostly affects previously healthy adolescents and young adults and in its classical form should meet four diagnostic criteria; primary infection of the oropharynx, septicemia, clinical-or radiographic evidence of thrombosis of the internal jugular vein (ijv) plus secondary metastatic abscesses. the infection is caused by fusobacterium necrophorum, a species of obligate anaerobe bacteria forming part of the normal human flora. the syndrome should be suspected in any patient with pharyngitis, cervicalgia and pulmonary symptoms. the incidence of lemierre's syndrome decreased dramatically after the introduction of antibiotics but has, of unknown reasons, increased over the past years. we will present four patients diagnosed with lemierre's syndrome in our department during the last years. the purpose of this case report is to raise awareness of this "forgotten disease". of the four patients diagnosed with lemierre's syndrome two fulfilled all criteria while two fulfilled out of . (table ). the first two presented at the emergency department with one week's history of a sore throat, left sided cervical lymphadenopathy, erythematous tonsils, leukocytosis and elevated crp. in both cases the clinical condition deteriorated and they were referred to the icu. one developed ards and required initiation of ecmo. in both patients, chest ct revealed multiple pulmonary consolidations with cavitations, findings consistent with septic emboli (image , and ). incidentally ct-neck revealed thrombosis in the left ejvand ijv (image ). ultrasound of the neck veins confirmed the finding (image and ). blood cultures taken on admission later proved positive to f. necrophorum. the third and fourth case, with similar clinical histories but with a less aggressive development, had positive blood cultures but no thrombosis and vice versa. (table and all four patients recovered and could be discharged with oral antibiotics and anticoagulants. unique teaching points: in conclusion, lemierre's syndrome is less common today thanks to antibiotics but may still occur in previously healthy adolescents and may lead to a fatal outcome. the pediatric radiologist should be aware of typical findings like septic emboli in the lungs and thrombosis in the ijv. unicameral bone cyst associated with secondary aneurysmal bone cyst of clavicle i. dasic, g.j. djuricic, s. ducic; belgrade/rs objective: aneurysmal bone cyst (abc) accounts for , % of all bone tumors. they are benign but locally destructive lesion of the bone characterized by presence of spongy or multiloculated cystic tissue filled with blood. abcs are metaphyseal, excentric, bulging, fluid-filled and multicameral, and may develop in all bones of the skeleton. most common locations include the proximal humerus, distal femur, proximal tibia, and spine. clavicle is a very rare site for aneurysmal bone cyst with only few cases reported in literature. a -year-old boy reported to the university children's hospital for detailed examination of swelling of right shoulder. - days before admission parents noticed tumefaction of right shoulder. there was no history of trauma or fever. physical examination revealed tumefaction of the right shoulder, in projection of acromial end of clavicle, measuring approximately x cm, which was tender and fixed. the swelling was not hot to the touch, and there was no skin discoloration over that area. regional lymph nodes were not palpable. (fig. a) x-ray revealed osteolytic, expansible lesion in the lateral end of clavicle and there was no pathological fracture. (fig. b) laboratory analyzes were within normal limits. blood cultures remained sterile. chest x ray and abdominal ultrasound were normal. computed tomography (ct) revealed a thinwalled multiloculate lesion in lateral end of right clavicle. (fig. a) there was no extension in the soft tissues on magnetic resonance imaging (mri). mri shows the multiloculate cavities and fluid levels. (fig. b) . the open biopsy was done. histopathological examination confirmed the secondary aneurysmal bone cyst on the field of simple bone cyst of clavicle. the clavicle is an uncommon site for bone tumors. review of literature shows clavicle accounts for less than % of all bone tumors. the patient with an aneurysmal bone cyst generally presents with pain and swelling, which may vary in duration from weeks to several years. up to % of bone tumors occur in less than years of age with peak incidence in second decade. radiologically, lesion is lytic and may have a soap-bubble appearance with ballooned distension of the periosteum. the differential diagnosis for aneurysmal bone cyst include giant cell tumor, chondromyxoid fibroma and telangiectatic osteosarcoma. distinction from telangiectatic osteosarcoma is difficult because the conditions have overlapping clinical and radiologic features. the differentiation is made from the histologic features. imaging of glomus tumor of liver in a child (case report) n. tewattanarat, j. srinakarind, j. wongwiwatchai, p. komvilaisak, s. areemit, p. ungarereevittaya, p. intarawichian; khonkaen/th objective: glomus tumors occur preferentially in subcutaneous tissue of fingers and toes, but extremely rare in visceral organs. most cases of the tumors are diagnosed in adults. several cases of glomus tumors in liver have been reported in adults. a literature review, no case of glomus tumor in liver in children was published. therefore, we present clinical, imaging findings of the first case of pediatric patient with glomus tumor in liver and also histopathological features. a previously healthy year-old-girl was admitted with a twoweek history of progressive dyspnea on exertion and vomiting. family history was unremarkable. physical examination revealed hypertension and smooth and firm mass at epigastrium. systolic apical murmur on heart examination was noted. liver function test ( ) (suppl ):s -s pediatr radiol showed elevated cholesterol ( mg/dl). other laboratory tests (complete blood count, blood chemistry, renal and liver function test, coagulation test, hepatitis profiles and alpha-fetoprotein) were within normal limits. echocardiogram found mitral and tricuspid regurgitation and poor left ventricular systolic dysfunction. abdominal mri demonstrated a -cm well-defined exophytic hypervascular mass with intratumoral hemorrhage at segment / b of the liver. there were no other suspicious lesions in other organs. the biopsy was done and revealed glomus tumor. patient underwent preoperative embolization and the liver mass revealed decreased size to -cm after -month follow up with ultrasound. after that, exploratory laparotomy with left lateral segmentectomy was performed. the pathological results showed dilated vascular channels surrounded by uniform neoplastic cells, uniform with round nuclei, fine chromatin, inconspicuous nucleoli, and pale eosinophilic cytoplasm, and well-defined cytoplasmic border. no mitotic figures and necrosis are identified. immunohistochemical (ihc) staining of tumor was positive for cd , smooth muscle actin (sma) and h-caldesmon. others ihc including ae /ae , heppar , cd , desmin and myogenin were negative. from these findings, the tumor was finally diagnosed as glomus tumor of uncertain malignant potential due to deep location and large size. primary glomus tumor is a rare entity of liver tumor diagnosed in children. however, it should be considered in the differential diagnosis of a hypervascular liver mass. most of these tumors are benign, however tumor in liver have malignant potential due to deep seated position. therefore, tumor removal with pre-operative embolization should be considered. brain mri in a pediatric patient with linear scleroderma en coup de sabre m. mortilla, a. rosati, e. canale, c. filippi; florence/it objective: linear scleroderma "en coup de sabre" (ecds) is a rare subset of localized scleroderma. affected individuals typically have a characteristic atrophic skin lesion involving the fronto-parietal scalp. the disease usually has a benign course but rare neurologic symptoms can be seen associated: the most common described is epilepsy. intracranial mri findings described in the literature include: focal brain atrophy, calcifications and t -hyperintense white matter lesions that may demonstrate contrast enhancement. white matter lesions and calcifications are found in the cerebral hemisphere ipsilateral to the skin abnormality. in the literature only a few pediatric cases have been described. a yrs. old girl was hospitalized at our institution for evaluation of a lesion of the frontal skin associated to a history of febrile seizures and mri alterations. she presented febrile seizures at the age of on april . on january parents noted a frontal cutaneous lesion that was defined as "linear scleroderma, port-wine stain type". on november she performed an mri at another institution showing a diffuse white matter alteration in the left emisphere with focal lesions with high susceptibility and mild contrast enhancement. she was addressed to immunosuppresive therapy with steroids and methotrexate, with steroids stopped after months. a clinical cutaneous improvement was noted. on july a second mri showed a worsening of the findings. we describe a case of a little girl with ecds with no neurologic deficits or symptoms that shows extensive and progressive neuroradiologic alterations. only a few pediatric cases have been described, but it has to be known that also in absence of symptoms, patients with linear scleroderma should be screened with mri to look for cns involvement in this immune disease. brain mri can also be used to monitor the progression of the disease and the response to therapy. mals is a vascular compression syndrome which symptoms can overlap chronic functional abdominal pain. in mals the proximal part of the celiac artery is compressed by the too low located median arcuate ligament during expiration resulting in hemodynamically significant symptoms. we report two cases with mals diagnosed primarily by ultrasonography. case -year-old girl was admitted to tartu university children's clinic (tucc) due to recurrent acute epigastric pain episodes with nausea and loss of appetite during years. previous analyses were normal, abdominal uss and gastroscopy did not show any abnormalities. she was referred to paediatric radiology department for doppler us (dus) which showed narrowed proximal celiac artery (ca) with turbulent flow, increased peak-systolic and end-diastolic velocities on deep inspiration and expiration, and positive ca deflexion angle on expiration. superior mesenteric artery (sma) was markedly widened, indicating possible collateral blood-supply due to severe ca stenosis. according us findings mals was suspected. abdominal mra showed proximal ca kinking, stenosis and poststenotic dilatation and confirmed diagnosis. during dsa collateral blood-supply from sma via pancreaticoduodenale arcade (pda) was seen. laparoscopic release of mal resulted in relief of patient's symptoms, she has been pain-free for two years. case -year-old girl applied to tucc due to recurrent abdominal pain episodes for - years. usually, pain occurred - times per week about minutes after the start of intense cycling training or competitions, and passed about minutes resting in squat position. mild mid-epigastric bruit was audible at physical examination. dus showed two-fold increase in expiratory peak-systolic and enddiastolic blood flow velocities compared to inspiratory velocities which indicated to the hemodynamically significant worsening of ca compression by mal during expiration. mra showed proximal ca compression, upward angulation and poststenotic dilatation. preoperative ct-angiography depicted collateral supply via pda. during laparoscopic surgery ca was released by transecting mal and surrounding fibrous tissue. after surgery the girl has been pain-free for one year except single pain episode during intense competition. the diagnosis of median arcuate ligament syndrome should be considered in patients with postprandial abdominal pain that does not have other clearly established etiology. colour doppler us should be the first choice imaging method. to confirm diagnosis in pediatric patients abdominal mra is preferred in our institution, but as mra may still have a tendency to movement artifacts and inadequate spatial resolution for smaller blood vessels, in these two cases mra was followed by cta or dsa. understand the unique predilection of infantile malignancies to metastasize and present as skin-based masses, most commonly lymphoma/leukemia. case presentation: an otherwise healthy day old male presented to dermatology with a pedunculated, friable red glabellar mass (centered between the eyes). first noticed as a flat, bluish lesion at days, its subsequent rapid growth led to an emergency department visit where dermatology diagnosed a hemangioma and initiated propranolol treatment. despite this, the mass continued to grow rapidly, encroaching upon the patient's right eye. the patient was admitted for further workup. an elevated beta hcg, anemia ( . mg/dl), and thrombocytopenia ( , ) suggested an alternate diagnosis. an mri and ultrasound led to a percutaneous biopsy; pathology was consistent with choriocarcinoma. pet ct found fdg-avid glabellar, liver and lung lesions. maternal and placental testing was negative for choriocarcinoma. ultrasound demonstrates a hypoechoic hypervascular mass. mri brain demonstrates cutaneous confinement of the solid avidly enhancing glabellar mass. ct shows a peripherally enhancing liver mass with a masslike area of consolidation in the right lung. initial pet/ct demonstrated fdg avid liver and lung metastases with a small focus of residual activity at the glabella consistent with incomplete resection. follow-up pet/ct showed astoundingly rapid re-growth of the glabellar mass and enlargement of the hepatic and pulmonary masses just days later demonstrating the extremely aggressive nature of this cancer. month follow-up pet/ct showed significantly decreased size and activity of the metastases consistent with a treatment response. in a series of infants with cutaneous metastases, the following diseases presented with cutaneous involvement (ordered most to least common): leukemia, langerhans cell histiocytosis, neuroblastoma, rhabdoid tumor, rhabdomyosarcoma, primitive neuroectodermal tumor, choriocarcinoma, and adrenocortical carcinoma. pathology slides ( ) (suppl ):s -s pediatr radiol unique teaching points: considered one of the fastest growing tumors, infantile choriocarcinoma classically presents with hepatomegaly, anemia, failure to thrive, and precocious puberty between days and months of life. left untreated, the disease is usually fatal within weeks of presentation. a solitary cutaneous metastasis can be mistaken for infantile hemangioma both clinically and radiographically. atypical mri appearance is one important clue that can suggest an alternative diagnosis. pet/ct may be useful for staging and follow-up. a rare case of ovarian juvenile granulosa cell tumor associated with ollier's disease -generalised mesodermal dysplasia p. joshi; pune/in to demonstrate a rare case of mesodermal dysplasia -association of ovarian granulosa cell tumour with enchondromatosis case presentation: two year month old girl presented with precocious puberty i.e thelarche. left hand radiograph showed the radiological age corresponding to chronological age, suggestive of peripheral precious puberty. the patient subsequently underwent a sonography which revealed a pelvic mass probably arising from the right ovary ? sex cord stromal tumour. a mri of the abdomen and pelvis confirmed the pelvic mass and revealed multiple bone lesions in the right hemipelvis -on the side of the tumour she was later operated. hpe of pelvis mass revealed juvenile granulosa cell tumour. ultrasound pelvis images reveal a solid pelvic mass, probably ovarian in etiology mri pelvis also reveals multiple bone lesions unique teaching points: the aim of the poster is to create awareness about this association. the bone lesions should not be mistaken for metastasis juvenile granulosa cell tumour of the ovary (jgct) is a well-known sexcord stromal ovarian neoplasm. ollier's disease is a rare, non hereditary mesosermal dysplasia consisting of multiple enchondromas. the association of granulosa call tumour with asymmetric ipsilateral hemiskeletal distribution may indicate generalised mesodermal dysplasia as there is also association of jgct with maffucci's syndrome, other dysplastic conditions such as microcephaly, facial asymmetry,' and potter's syndrome. review of literature showed previous cases of juvenile granulosa cell tumor associated with enchondromatosis, three associated with maffucci's syndrome, and the rest with ollier's disease goldbloom's syndrome is a paediatric idiopathic disease characterized by transient bone marrow oedema with recurrent crisis of bone pain, periosteal hyperostosis, fever, increased inflammatory markers and dysproteinaemia. a case series of wbmr studies in goldbloom's syndrome is reported and differential diagnosis discussed. case presentation: a -year-old female girl was admitted to our paediatric department because of daily crisis of bone pain of the lower limbs, associated with fever spikes, limping and nocturnal awakenings. no history of trauma was reported. laboratory tests showed mild anaemia (hb . g/dl), thrombocytosis (plt /mmc), increased inflammatory markers (ers mm/h, crp mg/dl), high streptolysine o and dnase-b antibody levels (aso iu/ml and adn-b ui/ml, respectively). throat swab was positive for group a β-haemolytic streptococcus (gas). unusual dysproteinaemia, characterized by hypoalbuminemia ( . g/dl) with increased a , a and g globulinaemia, was noted. x-ray examinations of both legs resulted normal. wbmri showed markedly delineated, high and homogeneous hyper-hypointensity respectively in stir/t of the distal tibialperoneal meta-diaphysis of both legs (fig a,b). distal metaphysis of femur, humerus, radius-ulna and proximal tibia were also homogeneously mildly hyperintense on stir sequences bilaterally (fig a). bone biopsy revealed signs of chronic inflammation. infectious and neoplastic diseases were ruled out and the diagnosis of gs with dysproteinaemia seemed conceivable. steroid treatment was started in association with indomethacin, leading to a prompt resolution of the clinical picture within a few days. the follow-up stir total body mri, performed after months, showed the complete resolution of bone oedema. (fig a,b) the sock sign is a pathognomonic whole-body magnetic resonance imaging (wbmri) feature of goldbloom's syndrome (gs).it is a well marked, symmetric, homogeneous and high bone marrow hyperintensity, localized both at the distal tibial and peroneal meta-diaphysis, which looks like a pair of socks. objective: left ventricle hypoplasia is generally thought as a part of hypoplastic left ventricle syndrome or aortic hypoplasia. it is estimated that about - ml/m left ventricle volume is needed in order to support systemic circulation. less than that volume generally precludes biventricular repair. however conditions associated with severe preload decrease such as total anomolous pulmonary venous return (tapvr) should be considered in the differential diagnosis. tapvr presenting as hypoplastic left ventricle syndrome is presented in this study. six month old female patient admitted to emergency service with symptoms of fever, dyspnea and coughing. emergency staff started intravenous antibiotic theraphy and from medical records learned that she has been followed for partial anomolous pulmonary venous return (papvr) and atrial septal defect (asd). lung x-rays revealed pulmonary edema. echocardiography was performed and revealed very small left ventricle, papvr and mm wide asd. ecg gated cardiac ct was requested with the prediagnosis of hypoplastic left ventricle syndrome. ct images revealed dilated right cavities, very small left ventricle, pulmonary edema, tapvd and peritoneal fluid plus hepatomegaly. we then retrospectively searched our archive and found she was diagnosed as papvr when she was days old. all the cavities that time, were normal sized. according to these we confirmed our diagnosis as tapvr and hypoplastic appearing cavities due to reduced preload and right chamber dilatation due pulmonary overcirculation. surgical team decided to perform corrective operation and they confirmed our diagnosis unique teaching points: small left ventricle cavity in an infant need not to be due to intrinsic hypoplasia. whenever we experience such a situtation we should search for other reasons of pseudohypoplasia in order to give a chance for corrective surgery instead of palliative procesures. we present a case report of kimura disease, a rare benign chronic inflammatory disease that involves the deep subcutaneous tissues and lymph nodes of the head and neck. we report the case of a thirteen year old male who presented with a right sided facial mass which had been present for two years but had enlarged rapidly in the preceding three months. us and mr were interpreted locally as an arteriovenous malformation. review of these examinations and catheter angiography performed at this quaternary referral centre favoured a vascular tumour. subsequent percutaneous biopsy demonstrated angiolymphoid hyperplasia with eosinophilia and blood tests showed a serum eosinophilia, consistent with kimura disease. us shows a mass consisting of scattered heterogenous foci within the fat with multiple large feeding vessels. contrast enhanced mri demonstrated a solid, homogenously enhancing, mass with multiple vascular flow voids from the right external carotid artery branches. catheter angiography showed tumour blood supply from branches of the right transverse facial artery and distal right ima. the dominant supply arose superficially from the transverse facial artery. kimura disease is a rare chronic inflammatory disorder of unknown aetiology that involves the deep subcutaneous tissues and lymph nodes of the head and neck region, most common in asian men in the third decade and sporadic in the non-asian population. the histopathological and biochemical characteristics are eosinophilic lymphfolliculoid granuloma, increased eosinophils in the peripheral blood and increased ige levels. whilst ultrasound and mri are effective imaging modalities, imaging alone does not allow confident differentiation from malignant lesions and biopsy is necessitated. kimura disease has a benign indolent course with an excellent prognosis following surgical excision although local recurrence has been reported. increased naa: is it surely canavan disease? e. varga, p. barsi, g. rudas; budapest/hu leukodystrophies are a group of rare genetic, metabolic diseases that affect the central nervous system, mainly the brain. each type of them is caused by a specific gene abnormality that leads to abnormal development or destruction of the white matter of the brain. the differential diagnosis are made on the basis of clinical and neuroradiological signs. there are some diseases which show typical changes on mr spectroscopy. we present a case of a year-old boy, who has been investigated due to somatomental retardation and muscle dystrophy since his six months of age. his perinatal period was normal except of a nystagmus visible from his birth. the child has muscle dystrophy, spastic quadriparesis, contractures, scoliosis, truncal hypotonia and ataxia and mental retardation. we started examinations to find out the background pathology of his idiopathic encephalo-myopathy. the brain mri showed a bilateral, symmetrical white matter signal alteration, which referred to some kind of metabolic ( ) (suppl ):s -s pediatr radiol disease. the mr spectroscopy revealed decreased cholin and increased naa levels, which are typical of canavan disease. despite of this, the clinical aspects and the location of the involved brain areas were more typical of pelizaeus-merzbacher disease (pmd). the pmd is a genetic disorder, which is originated of the mutation of the proteolipid protein gene (plp ) located on long arm of x-chromosome (xq - ) . this gene has an impact on growth of the myelin sheath. various types of mutations (deletion, duplication, point mutation, insertion) of plp gene lead to various severity of clinical picture. all form of mutations show decreased naa level on spectroscopy, except the duplication of plp gene. in connection with our case, we present briefly the clinical and neuroradiological differences between the two entities. magnetic resonance imaging findings in medium-chain acyl-coenzyme a dehydrogenase (mcad) deficiency l. talamanca, d. narese, m.c. rossi espagnet, l. pasquini, d. longo; rome/it we report serial brain magnetic resonance (mri) in a patient with medium-chain acyl-coenzyme a dehydrogenase (mcad) deficiency who developed acute encephalopathy. a -months-old girl was admitted in the emergency department of our hospital with sudden onset of acute encephalopathy with drowsiness. baseline laboratory investigations revealed severe hypoglycemia, hyperammonemia, hyperchloremic metabolic acidosis and hyperuricemia. the patient was treated with glucose solution infusion that resulted in a gradual resolution of symptoms. the first brain mri, performed within hours of onset of symptoms showed bilateral symmetric restricted diffusion on diffusion-weighted imaging (dwi) in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter; the adc map showed reduced diffusivity (fig ) . the second mri, at hours after the onset, revealed bilateral and symmetric hyperintensity on t -weighted images in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter. dwi showed restricted diffusion in both globus pallidus (fig ) . a single voxel h-mrs study performed by placing a roi in the right nucleus lenticularis revealed increased values of gaba and glutamine (fig ) . a further mri was performed weeks after the first neuroimaging and indicated widespread atrophy and the appearance of a hyperintense signal in t -wi in both globus pallidus while dwi did not reveal any remarkable signal abnormality. single-voxel mrs of the same region showed a normalization of gaba and glutamine values. brain mri showed bilateral symmetric restricted diffusion on diffusionweighted imaging (dwi) in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter; the adc map showed reduced diffusivity the second mri, at hours after the onset, revealed bilateral symmetric restricted diffusion on diffusion-weighted imaging (dwi) in both globus pallidus. a single voxel h-mrs study performed by placing a roi in the right nucleus lenticularis revealed increased values of gaba and glutamine. mcad is an enzyme of the mitochondrial b-oxidation of fatty acids, an essential source of energy for cells during stress. mcad deficiency is the most common genetic disorder of fatty acid oxidation. the clinical manifestation of the disorder is typically precipitated by stress due to fasting, vomiting, fever or muscular exertion and occurs in the majority of cases before the age of with the onset of acute hypoketotic hypoglycemia. clinical features of this decompensated state include seizures and lethargy proceeding to coma and death in the absence of prompt treatment with intravenous dextrose infusion. mcad deficiency usually appears in an acute form and has high morbidity and mortality rates; early diagnosis is therefore extremely important in order to promptly begin treatment and obtain a complete recovery from symptoms. mr can play a significant role in the early diagnosis of the decompensated state of the disease; in our case dwi revealed the presence of lesions with a bilateral symmetric topographic distribution that strongly suggested a metabolic disease leading to acute encephalopathy. a full-term male neonate ( days old) with external perineal anomalies was referred to our hospital. the physical perineal examination revealed a bifid scrotum containing palpable testis and a normal configured penis located at the bottom of the bifid scrotum. two soft masses of and cm respectively, divided from a cutaneous notch, were located below the bifid scrotum and on the right of the midline. the rear biggest mass was normal epithelized, instead the other one was a rugged pigmented mass, which resembled the scrotum (figure ). there were no additional abnormalities of the external genitalia. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content. a mild hydrocele in the right scrotum and a sliding testis on the left side were also revealed. us examination showed a hyperechoic solid tissue, corresponding to the rear biggest perineal mass. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content (figure ) . a mild hydrocele in the right scrotum and a sliding testis on the left side were also revealed. mri also confirmed two perineal peduncolar masses: the biggest and posterior one, was made up by homogeneous fatty matter without contrast-enhancement after intravenous gadolinium injection (figure ). the patient underwent excision of perineal masses and no complications occurred in the surgery. the histopathological examination of the perineal masses revealed two areas with different histological features: the first one was characterized by the presence of smooth muscle bundles dispersed in the dermal collagen, instead the other contiguous area showed abundant mature adipose tissue in the deep dermis and hypodermis ( figure ). at last the rugged swelling mass was definitively diagnosed as as without testis tissue inside, and the rear mass was diagnosed as lipoma. the physical perineal examination revealed a bifid scrotum containing palpable testis. two soft masses of and cm respectively, divided from a cutaneous notch, were located below the bifid scrotum. us examination showed a hyperechoic solid tissue, corresponding to the rear biggest perineal mass. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content mri confirmed the presence of two perineal peduncolar masses: the biggest and posterior one, was made up by homogeneous fatty matter without contrast-enhancement after intravenous gadolinium injection. neonates presenting with perineal masses are uncommon. these anomalies can occur isolated or more rarely in combination with other abnormalities such as uro-genital or ano-rectal anomalies or with contiguous subcutaneous tumors. when perineal masses are found, with prenatal diagnosis or during a newborn physical examination, it is important to look for any associated congenital anomalies or subcutaneous tumors by using imaging. to describe and emphasize the significance of the "half-moon" sign in pelvic mri. a -year-old adolescent, karate athlete, was submitted with left hip pain, decreased range of movement and asymmetry in thigh circumference. markers for infection or inflammation were negative. frog-leg radiograph was negative for hip effusion, slipped epiphysis and equivocal for a left trochanteric abnormality. mri demonstrated a half-moon pattern of bone marrow edema at the left intertrochanteric area and at the major trochanter, surrounding an apophyseal low-intensity lesion. ap radiograph and limited ct confirmed the presence of a lytic lesion with sclerotic margins, containing calcified chondroid matrix. chondroblastoma was histologically confirmed following excision. mri, coronal stir sequence, demonstrates semilunar-shaped hyperintense area abutting the growth plate and the cortex of the femoral neck, consistent with the half-moon sign. note edema surrounding an apophyseal low-intensity lesion and soft-tissue edema. ct confirms a typical apophyseal lesion with sclerotic margins containing chondroid matrix. unique teaching points: "half-moon" sign refers to a semilunar shape of bone marrow edema at the intertrochanteric area of the hip with its base located at the cortex of the femoral neck. this distribution differs from the distribution of edema in metaphyseal and metaphyseal-equivalent osteomyelitis. "half-moon" sign has been described in patients with stress fractures and osteoid osteomas. to our knowledge, this is the first case of chondroblastoma exhibiting this sign. whenever the "half-moon" pattern of edema is identified at pelvic mri scans, a thorough search for an occult fracture line or a nidus corresponding to an osteoid osteoma or a chondroblastoma is mandatory. mr elastography (mre) is a noninvasive imaging technique that quantitatively measures liver stiffness and provides an estimate of the degree of fibrosis. our aim was to evaluate the feasibility of performing mre using both gradient echo (gre) and echo planar (epi) sequences on siemens scanners. a dedicated mre of the liver was performed on a t mr scanner (magnetom® skyra, siemens) with a pediatric mechanical ( ) (suppl ):s -s pediatr radiol driver (courtesy of mayo clinic) over the right upper quadrant. an axial t blade with fat saturation, a coronal t vibe dixon and axial diffusion weighted imaging (dwi) were obtained. elastograms were obtained using both an axial standard gre and a works in-progress (wip) epi sequence. for the gre sequence, different slices were selected and each scanned sequentially. the epi sequence incorporated different slices in just one series. images were post-processed placing regions-of-interest (roi) and measuring the stiffness in kilopascals (kpa). for each sequence and each slice the stiffness mean was measured and then the average of the means was obtained. a spleen elastogram was simultaneously generated, without changing the mechanical driver location, and mean stiffness was also calculated. based on cutoffs in the literature, values were considered abnormal if liver stiffness > . kpa and spleen stiffness > . kpa. our initial experience shows that mre is feasible on siemens scanners using both gre and epi sequences. epi sequences are a promising addition to standard gre. prone versus supine ultrasound positioning for evaluation of urinary tract dilation (utd) in children c. maya , y. gorfu , e. dunn , k. darge , s. back ; philadelphia/us, addis ababa/et objective: ultrasound (us) is used in the initial evaluation and surveillance of utd in children. utd classification systems, including the multidisciplinary consensus, assess anterior-posterior renal pelvic diameter (aprpd) and calyceal dilation. there is currently no consensus regarding optimal patient positioning-prone versus supine-during us assessment of utd. this study was performed to determine if there is a significant difference in the measurement of the aprpd, presence of calyceal dilation, or resulting utd consensus score obtained between supine and prone positions. two raters retrospectively reviewed renal bladder ultrasounds of patients with utd of one or both kidneys. technically adequate ultrasound examinations of orthotopic kidneys that were imaged in both supine and prone positions were included. those with renal anomalies or prior surgery were excluded. aprpd measurements, as well as central and peripheral calyceal dilation, were documented in both prone and supine positions. a postnatal utd consensus score was assigned to each kidney based only on these features. kidneys ( left) in subjects had utd in either the supine or prone position. mean age was . years (range: . - . y). female to male ratio was : ( / ). the interclass correlation (icc) of the aprpd between raters was . and . in the supine and prone positions respectively (ps< . ). central calyceal dilation was found in / supine kidneys and / prone kidneys by rater and / supine and / prone kidneys by rater (kappa . ). peripheral calyceal dilation was found in / supine kidneys and / prone kidneys by rater and / supine kidneys and / prone kidneys by rater (kappa . ). as such the results are presented as one. the aprpd tended to be greater when prone with a strong correlation between prone and supine measurements ( . , p< . ). the mean difference between supine and prone aprpd was . mm (p< . ). in kidneys, calyceal dilation was seen in the prone position and not supine while kidney had central calyceal dilation only when supine. the utd score differed between supine and prone in / kidneys, with all but one higher when prone. in other kidneys, the aprpd differed between positions however concurrent calyceal dilation resulted in no change in utd class. as a screening tool, performing ultrasounds in the prone position may help identify more kidneys with utd. further research is needed to determine if these differences are clinically significant. during the evaluation of magnetic resonance enterography (mre), diffusion restriction (dr) has been utilized as a marker for bowel inflammation, but in our practice we commonly see dr in otherwise normal segments of jejunum. the purpose of this article is to assess the dr in normal loops of jejunum on mre and to determine if there is a correlation between dr and luminal distention, age, magnet field strength, and bowel segment location. a retrospective analysis of subjects with a normal mre and normal clinical work up (based on available clinical history, endoscopy reports, serum white blood cell count and inflammatory markers, and stool samples) was performed. the abdomen was divided into quadrants. if available, loops of jejunum were randomly chosen in each quadrant. two radiologists independently evaluated these same loops of jejunum for the following: luminal distension, wall thickness, and enhancement pattern. additionally, the loops were then evaluated for the presence or absence of dr. inter-rater reliability was determined. disagreement was resolved by consensus. presence or absence of dr was correlated with luminal distension, age, magnet field strength ( . versus tesla), and abdominal quadrant. one hundred ninety-seven loops of jejunum were evaluated in patients. not all subjects had jejunal loops in all quadrants. sixteen subjects ( %) had jejunal loops with dr for a total of loops. one loop had increased wall thickness and another increased enhancement but both did not demonstrate dr. no other loops demonstrate increased enhancement or wall thickening. for the presence or absence of dr, inter-rater reliability was fair (kappa= . ). there was no correlation between the presence/ absence of dr in relation to luminal distension, age, magnet field strength, or quadrant location. of the subjects who had a single loop with dr, a nd loop with dr was found in %. year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. a. coronal haste demonstrates the craniocaudal position of the axial diffusion sequence for reference (line). year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. b. axial diffusion weighted seqeunce (b= ) shows diffusion restriction within loops of jejunum (arrow) within the anterior abdomen. year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. c. corresponding adc map demonstrates low signal within the jejunal wall consistent with diffusion restriction (arrow). diffusion restriction in normal loops of jejunum on mre was present in % of patients. if dr is seen in an otherwise normal segment of jejunum, this can be considered non-pathologic. a patient with a loop of jejunum with dr is likely to have an additional loop of jejunum demonstrating dr. there is no correlation with dr of normal jejunum with luminal distension, magnet field strength, or patient age. our data may help reduce overestimation of disease burden when clinically applied. imaging findings in the newborn with meconium peritonitis that require surgery p. caro dominguez , a. zani , a. daneman ; cordoba/es, toronto/ca objective: meconium peritonitis is a rare condition caused by an in-utero bowel perforation resulting in spillage of meconium into the peritoneal cavity and subsequent calcification. the role of prenatal and postnatal imaging is to identify infants who require surgery. the aim of this study was to evaluate the role of postnatal imaging in meconium peritonitis and to correlate the radiologic and sonographic patterns with the need for surgery. imaging studies in infants with meconium peritonitis performed between and at our institution were reviewed separately by a pediatric radiologist, a pediatric radiology fellow and a pediatric surgeon. patients were divided in a surgical and a non-surgical group. clinical, surgical and pathology reports were reviewed to validate the diagnosis. statistical analysis: comparisons between sonographic and radiographic findings and patterns in the surgical and non-surgical groups were performed using unpaired t-test and chi-square. during the study period, there were infants with meconium peritonitis managed at our institution. in the ( %) who needed surgery, the most frequent surgical findings were idiopathic perforation, jejunal and ileal atresia. ultrasound identified more cases with hepatic calcifications, meconium pseudocyst, ascites and pneumoperitoneum than radiography and radiography more cases of small bowel obstruction. ascites identified with ultrasound (p= . ) [fig ] and bowel obstruction [fig ] diagnosed either with ultrasound (p= . ) or radiography (p= . ) were associated with the need for surgical intervention. one third of children with meconium pseudocysts ( / ) [fig ] , did not require surgery. diffuse peritoneal or hepatic calcifications as an isolated postnatal finding were not associated with the need for surgery. both radiography and ultrasonography give valuable information to the surgeon to take the decision for surgery. dilatation of bowel loops and ascites detected postnatally with radiography and/or ultrasound require surgical intervention in children with meconium peritonitis. interestingly, a large proportion of infants with meconium peritonitis can be managed conservatively. . - . ) . those included had complete fmru analysis, dti (b= and b= , directions), and upjo configuration in at least kidney. cases with motion artifact (n= ), post-pyeloplasty (n= ) or duplex collecting systems (n= ) were excluded. pelvicalyceal dilation grade (pcd), corticomedullary differentiation (cmd), and functional parameters were included. pyeloplasty following fmru was recorded. dti tractography was reconstructed using a fractional anisotropy (fa) and an angle threshold of . and °, respectively (figure ) . user-defined regions-of-interest (roi) of the renal parenchyma, excluding the collecting system, were drawn to quantify dti parameters: mean fa, apparent diffusion coefficient (adc), tract length and tract volume. the relationships between dti quantitative parameters and fmru parameters were analyzed. age and adc (roi) (p< . , r = . ), tract volume (p< . , r = . ) and tract length (p< . , r = . ) were positively correlated. age and fa (roi) (p< . , r = . ) were negatively correlated. there was a correlation between fmru parenchymal volume and tract volume (p< . , r = . ), but median volumes were higher on dti (tractography= . cm vs. fmru= . cm ; p< . ). of the children, had pyeloplasty, had nephrectomy, were managed conservatively and was lost to follow-up. fa was significantly lower in kidneys that went on to have pyeloplasty in comparison to those without pyeloplasty, but the %ci and the iqr overlapped (table ) . the adc, tract length and tract volume were similar between these groups (table ) . there was no difference between the adc of fa values in kidneys with and without pcd or cmd (p> . ). linear hierarchical regressions controlling the age did not show a significant relation between adc and cortical or renal transit times (p> . ), but lower fa values were related to a higher renal transit time (p< . , r = . ). table . quantitative dti parameters between kidneys with and without pyeloplasty following fmru. renal adc, fa, tract volume and tract length change with age but tractography overestimates renal parenchymal volume. there was a tendency towards a lower fa in kidneys that went on to pyeloplasty. otherwise, none of the quantitative parameters evaluated in this study differentiated degrees of upjo. echo-enhanced voiding urosonography (eevus) has become an important imaging tool in urodiagnostics; however, it has been observed that during eevus the premature destruction of ultrasound contrast agent microbubbles might occur. the purpose of this study was to evaluate the possible causes of contrast vanishing during investigations and propose the protocol to avoid false negative results. eevus was performed in children from april to december . sonovue mixed with saline solution in a plastic bottle is applied by continuous flow through the urine catheter. the collected data according to the protocol in this prospective study was completed in children, aged from weeks to . years. the protocol included general patient information, indication for eevus, duration of eevus in minutes, and the presence of vesicoureteric reflux. extensive data about sonovue were recorded: charge number, expiration date, time since opening, amount of initially administered contrast (ml sonovue/ml saline solution), grading of the initial contrast opacification of the bladder, the need for immediate readministration of contrast (dose), grading of contrast opacification during examination, and the need for readministration of contrast later in the course of the examination (dose). in addition, the data regarding bladder (ratio real/predicted bladder volume, wall thickness, ureter dilatation), saline solution, the size of urine catheter (french), and the type of antibiotic prophylaxis were collected. child observation included grading of crying and muscle stiffness. normal contrast opacification of urinary tract during examination was found in / children, while in / ( . %) the contrast opacification was insufficient. in / ( . %) microbubble destruction occurred during the first minute, in ( . %) in minutes, and in in minutes after the beginning of contrast administration. the reason for unsatisfactory contrast opacification at the beginning of the eevus is probably due to small urine catheter size ( % of children with fr catheter had insufficient opacification compared to . % with fr in whole cohort), time since the contrast is opened (more than hours in children), and insufficient bladder emptying at the beginning of the procedure. the reason for microbubble destruction later in the course of the examination is bladder overfilling in combination with increased muscle stiffness and strong crying, which led to increased bladder pressure. there was no correlation between the type of antibiotics and microbubble destruction. we should be aware of possible false negative vur results during eevus caused by premature microbubble destruction. patients with fontan-type palliation of univentricular congenital heart disease have elevated central venous pressure due to their passive pulmonary flow. the altered circulation has a negative impact on several visceral organs, and these patients have chronic liver congestion. they are at risk of developing hepatic fibrosis and cirrhosis with potential malignant transformation. these changes can occur from only a few years after fontan palliation, making early detection and grading of major importance. the patchy pattern of hepatic changes makes liver biopsy an unreliable diagnostic tool. magnetic resonance imaging (mri) t mapping has been suggested as a technique for non-invasive assessment and quantification of hepatic fibrosis/cirrhosis. the aim of this study was to compare two different t mapping sequences of the liver in adolescents with fontan palliation, and in healthy controls. materials: adolescents ( - y) with fontan circulation and young healthy adults ( - y) were included as a part of an ongoing national population-based study. all underwent mri ( . tesla) pre-and post-gadolinium contrast, including two types of t mapping of the liver. a d t volumetric interpolated breath-hold examination ( d vibe) sequence with dual flips with b correction and a modified look-locker inversion recovery (molli) sequence. t relaxation times (ms) were measured by placing five standardized circular regions of interest (roi) in the mid-section of the liver and one in the spleen (fig ) . statistical analysis was performed comparing measurements pre-and post-contrast, between sequences, and patients and controls. there was a significant difference in the measurements between molli and d vibe with increased values for the latter. within each sequence there were small, but significant regional differences in relaxation times (table ). the same pattern was seen in pre-and post-contrast images in both groups. there were significantly increased native t times on both sequences in all regions in the fontan group as compared to the controls, but not post contrast. t relaxation times differ between the t mapping sequences, molli and d vibe, pre-and post-contrast. t mapping of the liver revealed significantly increased native t times in adolescents with fontan palliation compared to healthy slightly older controls. these findings suggest hepatic fibrosis/cirrhosis, but may also represent a component of congestion. diagnostic accuracy of ultrasound, computed tomography and wedge portography in the work-up for mesenterico-rex bypass in children with extrahepatic portal hypertension s. toso, r. breguet, m. annoshiravani, s. terraz; geneva/ch to identify the diagnostic accuracy of ultrasound (us), computed tomography (ct) scan and portography (wedge hepatic vein portography or direct portography) in the pre-operative work-up of mesenterico-rex bypass performed for extrahepatic portal hypertension in children. we conducted a retrospective analysis of pre-operative imaging for mesenterico-rex bypass in our tertiary hospital over the last years. we analyzed all patients between the ages of - years, with extrahepatic portal hypertension necessitating surgical treatment that underwent us, ct and portography. three reviewers independently analysed the patency of the left portal vein, mesenteric vein, splenic vein and the presence of communication between the left and right portal vein on preoperative imaging with correlation to surgical findings. statistical analysis of diagnostic accuracy was performed. eleven patients underwent mesenterico-rex bypass for portal hypertension secondary to portal vein thrombosis. two patients had partial liver transplant. ct with ultrasound correlation was sufficient in responding to the preoperative criteria in % ( / ) cases. portography was useful in the % ( / ) cases where ct could not respond to preoperative criteria, in particular the presence of left-right communication. there was good inter-rater correlation for each modality and good correlation of findings between modalities. in the majority of cases the use of ultrasound and ct is sufficient for preoperative planning for mesentrico-rex bypass. portography is mandatory in cases with large intra-hepatic cavernoma, where the left-right communication could not be confirmed on ct. contemporaneous clinical data was reviewed where available, and a clinical decision on disease severity and activity on a likert scale made with and without imaging. fifty-three patients underwent mre and bowel us in the specified timeframe ( male; median age . years, range - years). twenty patients had sufficient contemporaneous clinical information to be analysed. inter-observer variability for the imaging scores was assessed using bland-altman plots. where variability was beyond pre-determined limits, the studies were consensus reviewed. mean scores were used for the studies within accepted limits of variability. there was no significant difference between total mre and us scores (wilcoxon signed-rank test z= . , p= . ). at the bowel segment level, there was no significant difference between the mre and us segment scores for the ileum and terminal ileum (wilcoxon-signed rank test, z= . , p= . ), but significant differences were present between the imaging scores for other bowel segments, with mre identifying more abnormalities. there is a significant positive correlation between mre and clinical consensus scores (spearman's rho= . , p= . ) and between us and clinical consensus scores (spearman's rho = . , p= . ). imaging caused a refinement to the original clinical assessment in of the cases, with jejunal and ileal disease the most common reason for 'upgrading' a score and absence of any detectable abnormality on us and mre the most common reason for 'downgrading' a score. we found good agreement between mre and us for total patient imaging scores, ileal and terminal ileal scores. both mre and us scores correlated well with the gold standard clinical consensus, with imaging altering the original clinical decision in % of cases. although us detected fewer abnormalities than mr, it correlates marginally better with the clinical consensus, suggesting it is at least equally clinically valuable. background: differentiating between acute osteomyelitis (om) and acute bone infarct (bi) in children with sickle cell disease (scd) is a challenge for clinicians and radiologists, particularly when blood cultures are negative. although bone aspiration is the gold standard test for om diagnosis, it is an invasive procedure and infrequently performed. magnetic resonance imaging (mri) has shown a potential role in differentiating between acute bi and acute om. the goal of this case series is to evaluate the utility of fluid signal on unenhanced fat-suppressed (fs) t -weighted mr sequence in distinguishing acute bi and om in children with scd. methods: we reviewed a total of children with scd admitted with long bone pain during the one -year study period - attributed to either an acute bi or an acute om. twelve of patients with available bone aspiration, blood culture, and mri data were evaluated for fluid signal, marrow signal and other criteria. of patients, nine patients were diagnosed as acute bi and two patients had acute om and one with coexisting bi and om. the diagnosis was based on the fluid signal on t unenhanced t fs mr images as compared to aspiration cytology in which eight of nine patients with bi had hyperintense fluid signal on non-contrast t fs mr images while one of two patients with om demonstrated hypointense fluid signal. the last patient was diagnosed as a probable coexisting lesion (om&bi) based on a giant well demarcated hypointense marrow signal with an extraosseous hyperintense fluid signal. in acute bi, an abnormal hyperintense subperiosteal or paraosteal fluid signal is frequently observed on unenhanced t -fs weighted images. this finding was present in the majority of cases in our study population regardless of age, sex or site in the appendicular skeleton. mri fluid signal characteristic on unenhanced t fs shows promise as a criterion to differentiate between acute bi and om. role of mri to assess skeletal age in pediatric celiac disease s. bernardo, m. martino, a. laghi, e. tomei; rome/it objective: coeliac children are often subject to weight loss and lower somatic growth rate, compared to healthy children of the same age. the purpose of this study was to asses the feasibility of magnetic resonance imaging (mri) of the hand and the wrist to assess skeletal age and growth delay. we enrolled in our study coeliac children ( males and females) affected by histological proven coeliac disease, with a chronological age ranged between years and month and years and months (mean age of years, +/ years and months standard deviation). a single mri sequence (t d se, acquisition time: minute seconds) of the hand and wrist in coronal plane was performed of each patient to estimate the skeletal age. patients' data were compared with a population of normal subjects. the preliminary results showed a delay in skeletal age in children affected by coeliac disease in , % of the simple study, with a delay of maturity of . years (+/- , years of sd). only children showed advance mri skeletal age when compared to normal subjects. mri of hand/wrist to assess skeletal age may be considered as a reliable indicator of somatic growth. mri, without radiation exposure, can be an used as a diagnostic tool in skeletal delay. it could play an important role in the follow up of coeliac children, after glutenfree diet. the prevalence of metaphyseal injury and its mimickers in otherwise healthy children under two years of age p. eide, Å. djuve, r.e. gjøsaeter, k.f. forseth, a. nøttveit, c. brudvik, k. rosendahl; bergen/no objective: metaphyseal lesions in infants and toddlers are believed to have a high specificity for inflicted injury, however, normal metaphyseal irregularities may mimic pathology and lead to overdiagnosis. during the period - all children between and years, seen at the a&e department in bergen (bergen legevakt) due to an injury, and who had radiographs taken, were included. data on previous injury, age, sex and injury mechanism were drawn from the medical notes and pacs archive. all radiographs were reviewed by two researchers and an experienced paediatric radiologist, registering the following: number, site and type of fractures, signs of healing (yes, no), bone structure (normal, pathological) and metaphyseal appearances (shape (normal, metaphyseal collar, metaphyseal irregularity), injury). the study was approved by the institutional review board. six hundred one children ( girls) between and months of age (mean . months) were included, of whom ( girls) had a total of fractures. one hundred eight of the fractures ( . %) involved the forearm, followed by leg-fractures ( / , . %) and fractures to the clavicle ( / , . %). one epiphyseal separation and one metaphyseal lesion (without a history of trauma) were seen. one thousand three hundred twenty metaphysis were analysed, of which ( . %) were defined as either irregular ( / , . %) or demonstrating a metaphyseal collar ( / , . %). metaphyseal lesions with a history of trauma did not occur in otherwise healthy neonates and infants under years of age, indicating that this type of fracture has a particular mechanism. metaphyseal irregularities are frequent, particularly around the knee, and should not be mistaken for clms to evaluate whether mri might be used for age estimation, based on greulich-pyle (gp) atlas criteria. . tesla mri of the left hand was conducted in adolescents, and subjectively evaluated by two blinded radiologists. for sequence optimization, coronal mri sequences (t tirm, t vibe- d-we, and t se) and a left hand x-ray were compared in ten patients (eight male, two female; mean age, . years). the ages of healthy volunteers ( s ( ) (suppl ):s -s pediatr radiol male, female; mean age, years) were assessed from coronal t vibe- d-we. bland-altman plots, intraclass correlation coefficients (icc), and logistic regression models were calculated. coronal t vibe- d-we achieved the best image quality. the correlation between estimated patients' ages on x-ray and mri was high. icc showed high inter-observer agreement ( . for x-ray, . for mri). the estimated age of the healthy volunteers tended to be older than their chronological age. the probability of overestimation was higher in girls than in boys. coronal t vibe- d-we of the left hand is feasible for skeletal age estimation by gp criteria with a high readers' agreement. the likelihood of overestimation of healthy children makes it necessary to develop a new hand atlas representing changes since the s. to assess the relationship between the radiographic findings of metabolic bone disease (mbd) and serum biochemical markers in preterm infants. preterm infants in our neonatal intensive care unit between january and september were included. two readers retrospectively reviewed the wrist radiography for grading according to mbd severity. we recorded the levels of alkaline phosphatase (alp) and phosphorous (p) immediately after birth, on the same day of the first wrist radiography (alp-s, p-s), the highest alp levels before the first wrist radiography (alp-hb) and during follow-up (alp-h), and the lowest p levels before the first wrist radiography (p-lb) and during follow-up (p-l). patients were subdivided into four groups according to mbd severity determined by wrist radiography for the first analysis, and were divided into two groups according to mbd presence for the second analysis. one-way analysis of variance with a tukey multiple comparison and the student's t-test were used for statistical comparisons in the two analyses, respectively. a receiver operator characteristic (roc) curve was constructed to determine the optimal cut-off values of the biochemical markers for the radiological prediction of mbd. of the patients, , , , and infants were classified into grades , , and , respectively. in the first analysis, alp-s, alp-hb, and alp-h were significantly different between grades - and - (all p< . ). plb was significantly different between grades and (p= . ) and p-l was significantly different between grades and or (p< . or p= . ). in the second analysis, alp-s, alp-hb, alp-h, p-s, p-lb, and p-l were all significantly different between the two groups (p< . ). the roc curve of alp-h showed the largest area under the curve values ( . , % confidence interval= . - . ; p= . ) for detection of a radiographic change. the optimal cut-off value of alp-h was . u/l, and the sensitivity and specificity were . % and . %, respectively. the first wrist radiography was obtained at . ± . weeks after birth, and alp-h was measured at . ± . weeks after birth. the cut-off value of alp for the prediction of abnormal radiological changes in wrist radiography was determined to be was . u/l. our findings indicate that the highest alp level at around . weeks after birth could be a valuable predictor of radiological mbd in preterm infants, including those with very low and extremely low birth weights. quantitative grading of tmj synovitis in children with jia-influence of mr-coil, timing after contrast-injection and location of measurements on joint-to-muscle enhancement ratio a. hamardzumyan schmid, c. kellenberger; zurich/ch objective: assessment of signal intensity ratio between joint space and longus capitis muscle on contrast-enhanced mri has been proposed as reliable method across different mr-scanners and protocols for grading temporomandibular joint (tmj) arthritis in juvenile idiopathic arthritis (jia) with a cut-off of . for diagnosing synovitis. the aim of this study was to investigate potential influences on such enhancement ratios (er). retrospective evaluation of contrast-enhanced mr-studies of tmjs in girls with jia (age . ± . y) obtained at two occasions with two different coils on a . t scanner. joint-to-muscle er were calculated from signal intensity measurements in different joint compartments, muscles and sequences obtained with varying delay after contrast-injection, and compared with paired sample t-test. er of tmjs without synovitis (n= ) and tmjs with synovitis (n= ), determined by qualitative criteria, were compared to er reported in the literature. superior and inferior joint space to longus capitis muscle er for normal tmjs ( . ± . ; . ± . respectively) exceeded . in all but one case (figure) and for tmjs with synovitis ( . ± . , . ± . ) were significantly higher than in cases with synovitis from the literature ( . ± . , p≤ ). the same er were higher when obtained with dual-ring coil ( . ± . ; . ± . ) than with multichannel surface coil ( . ± . ; . ± . ; p≤ . ). while er to longus capitis muscle were higher than those to pterygoideus muscle for both coils (p≤ . ), er to pterygoideus muscle did not differ between coils (p> . ). not considering the timing of the scan, er to pterygoideus muscle were highest in the inferior joint space ( . ± . ), followed by the anterior joint recess ( . ± . ) and superior joint space ( . ± . ). comparing images acquired immediately after contrast injection to later images (median delay min, range - min), pterygoideus muscle er in the superior ( . ± . to . ± . ) and inferior ( . ± . to . ± . ) joint space increased substantially (p< . ), while er in anterior recess showed no significant increase ( . ± . to . ± . , p= . ). conclusion: joint-to-muscle er are clearly dependent on ) the signal profile of the mr coil with muscles located further away from the coil providing higher er, ) the time of image acquisition after contrast-injection with later obtained images providing higher er, and ) where the joint signal intensity is measured. as these factors need to be accounted for, the described normal and pathologic ranges of joint to longus capitis muscle er cannot be generalised for every mr-system and imaging protocol. integration of d c-arm ct images with navigational software provides real-time fluoroscopic guidance during percutaneous interventions in the interventional radiology (ir) suite. a trajectory, drawn from skin entry point to the target lesion on the d c-arm ct data, is overlaid on intraprocedural fluoroscopy for real-time needle guidance. this study describes our experience with syngo iguide (siemens) needle guidance software in a range of clinical applications in the pediatric ir suite, including technical success, radiation dose and procedure time. in this irb approved study, all percutaneous interventions performed in the ir suite using syngo iguide over a -year period were included. cases were classified by procedure type; for each type, mean effective radiation dose (msv) was estimated using pcxmc program (v . . . , stuk) and procedure times were evaluated. forty-five patients ( male, female; mean age: ± years) underwent iguide-assisted interventions including: bone biopsies - / ( pelvic, lumbar, and lower extremity), intra-articular steroid injections - / ( sacroiliac, and temporomandibular joint), lumbar punctures - / , percutaneous catheter placements - / (cecostomy, and chest tube placement) and bone biopsy with radiofrequency (rf) ablation - / . iguide was used in particular for the cecostomy procedure due to high sub-hepatic cecal pole position, and in the chest tube procedure due to the presence of loculated pneumothoraces. all procedures were technically successful. the diagnostic bone biopsy rate was . %. the mean estimated dose and procedure times for each procedure type are listed in table . sonography of neonatal spine (sus) is a simple, non-invasive, quick, relatively inexpensive method to evaluate lumbar spine anomalies in infants less than months of age. unossified posterior neural arches allow beam penetration to obtain high-resolution images of the intra-spinal contents. sus is carried out at the bedside, does not utilize radiation & requires no sedation. linear array transducers with extended field-of-view permit diagnostic sensitivity equal to mri. factors affecting mri resolution like patient movement, pulsation & vascular flow do not affect sus. we use sus as first-line screening test in neonates with lumbosacral cutaneous stigmata & spinal dysraphism (sd) associated syndromes. this was a prospective study approved by the institutional ethics committee. thirty five children (age range of to years) with clinically suspected and complicated pulmonary tb were enrolled in the study. lung mri and ct scan was performed in all the patients. the sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of lung mri in detection of radiological findings that were considered highly suggestive or diagnostic for tb, were calculated, with ct as the standard of reference. lung mri performed equivalent to ct in detection of pleural effusion, mediastinal/hilar lymphadenopathy and lung cavitation with sensitivity and specificity of %. agreement between ct and mri in detection of each finding was almost perfect (k: . - ). lung mri was found to be comparable to ct scan for detecting various radiological abnormalities which were highly suggestive for tuberculosis. being a radiation free imaging modality, it has the potential, particularly in children, to replace chest radiographs and ct scan in the coming years. to evaluate differences of myocardial strain assessed by feature tracking using ssfp cardiac mri sequences between pectus excavatum (pe) patients and healthy volunteers. in this prospective study, cardiac mri was performed in pe patients (with a pathologic haller-index above . ) and healthy volunteers ( males and females, respectively; age range - years) including short-and long-axis cine-ssfp sequences on a t scanner. post-examination analysis included standard cardiac volumetry with measurements of the biventricular ejection fractions (ef). additionally, manual biventricular contouring by an experienced radiologist, and subsequent automated strain assessment using dedicated software (circle cvi ®) was performed. longitudinal, radial, and circumferential peak systolic strain and strain rates were analyzed for both ventricles. left-ventricular ef was normal in all patients. five pe patients had a normal right-ventricular ef, in pe patients rvef was slightly impaired ( - %), all healthy volunteers had a normal rvef. compared with healthy volunteers, pe patients showed a significantly higher apical left-ventricular strain (radial: ± . vs. ± %, p< . ; circumferential: - . ± . vs. - . ± %, p= . ) and strain rate (radial: . ± . vs . ± . s - , p< . ; circumferential: - . ± . vs. - . ± . s - , p= . ). mid right-ventricular strain (radial: . ± . vs. . ± . %, p= . ; circumferential: - . ± . vs. - . ± . %, p= . ) and strain rate (radial: . ± . vs. . ± . s - , p= . ; circumferential: - . ± . vs. - . ± . s - , p= . ), as well as apical right-ventricular strain (radial: . ± . vs. . ± . %, p= . ; circumferential: - . ± . vs. - . ± %, p= . ) and circumferential strain rate (- ± . vs. - . ± . s - , p= . ) were also significantly higher in pe patients than in healthy volunteers. left-and especially right-ventricular radial and circumferential strain and strain rate increased from the bases to apices in pe patients. longitudinal strain and strain rate did not differ significantly between pe patients and healthy volunteers. myocardial strain assessed by cardiac mri differs significantly between pe patients and healthy volunteers. as the chest wall deformity usually leads to a compression of the basal parts of the ventricles, higher values of myocardial strain in the mid and apical ventricles in pe patients might indicate a compensation mechanism to enhance especially right ventricular output against sternal compression. to determine the normal range of the haller index (hi) value, and its dependence on the age, sex, and respiratory phase. evaluate the possibility of reduction of the effective dose (ed) of ionizing radiation using a single-slice ct scan technique. the retrospective-prospective study included patients (av. y, sd y). it consisted of parts. the prospective study included evaluation of ct scans performed by single-slice technique in patients with pectus excavatum both in inspiratory and expiratory phase, without topogram. hi was measured in each patient in both respiratory phases. in retrospective study, ct scans of the chest in children without pectus excavatum were analyzed to determine normal range of hi values depending on the age ( - y, - y, - y, - y) and gender. the retrospective study also included the analysis of another ct scans in patients who were operated or diagnosed with pectus excavatum. in the latter group of patients the average value of ed of ionizing radiation was calculated, and the values were compared with the average ed obtained using low-dose ct examinations applied in the new protocol (single-slice technique). the normal value of hi was . ± . . a significant positive correlation between age and value of hi was found. older patients had higher hi ( - y: . ± . , - y: . ± . ). results of mann-whitney test did not demonstrate any difference between gender in the observed group, however girls had generally higher hi in all age groups. in the group of patients who were operated/diagnosed with pectus excavatum, hi was . ± . . the average value of hi in inspirium in children with diagnosed deformity was . ± . , while in expirium it was . ± . . only / ( %) patients had hi value over . (a boundary value for surgical treatment) during inspirium, while / ( %) patients had it in expirium, which showed statistically significant difference (p= . ). single-slice ct technique during the inspiratory and expiratory phase showed average ed of . msv, which is an equivalent of chest xray. it reduced ed more than times in comparison with low-dose whole chest ct. the value of haller index increases with the age and in expiratory phase. we propose the single-slice ct technique without topogram in expiratory phase, as a sufficient and reliable technique in evaluation of haller index and preoperative preparation. mps iva is a lysosomal storage disorder caused by a deficiency of nacetylgalactosamine-sulfatase. main symptom is a systemic skeletal dysplasia. affection of the vascular system has not been described yet. our goal is the analysis of the vascular system in patients with mps iva, based on the example of the aorta. in a retrospective study, patients with mps iva were included. the aorta in its course from th thoracic vertebrae to th was analyzed on the basis of craniospinal mr and ct examinations. to describe the course of the aorta, the area around the vertebral body was devided into equal parts (fig. ) . high buckled arteries in relation to the length of the affected aortal part were indicated as aortal kinking, and a moderate twist in relation to the length of the affected aortal part as aortal tortuosity. results: twelve of patients had an aortal kinking, of patients an aortal tortuosity, of these had moderate and strongly tortuous aortae. seven patients had a normal aortal course, couldn't be analyzed. one patient revealed both, aortal kinking and tortuosity. this study reveals the occurrence of aortic tortuosity in patients with mps iva. we suggest that this complication could be due to glycosaminoglycane deposition in the aortic intima, which may be s ( ) (suppl ):s -s pediatr radiol associated with an increased vulnerability of the vascular wall. we conclude that the examination of the vascular system should be included in regular follow-up protocols. lung ultrasound in the diagnosis and follow-up of pneumonia in children -is it really as reliable as chest x-ray? s. balj-barbir, j. lovrenski, s. petrović; novi sad/rs to investigate the role of lung ultrasound (lus) both in the diagnosis and follow-up of pneumonia in children. a prospective study was carried out in the regional children's hospital, and included children (av. . y, sd . y) with clinically suspected pneumonia, in whom initial lus and subsequent chest x-ray (cxr) were performed within h. the final diagnosis of pneumonia at discharge was used as a reference test to determine the reliability of lus, cxr, clinical and laboratory findings in the diagnosis of pneumonia. children with pneumonia formed a study group, while the control group consisted of children without diagnosed pneumonia. lus finding of subpleural lung consolidation was considered a diagnostic sign for pneumonia. the children with lus signs of pneumonia were followed-up until complete resolution of the lus findings. there were from one to five follow-up lus examinations performed. a final diagnosis of pneumonia was confirmed in / ( . %) patients, and / ( . %) were hospitalized (av. . , sd . hospital days). in diagnosis of pneumonia lus, cxr, auscultation, elevated crp, and tachypnea showed sensitivity of . %, . %, %, % and . %, and specificity of %, %, %, % and % respectively. lus detected lung consolidations in of children with final diagnosis of pneumonia, and in / patients lus showed air-bronchogram (figures , ) . lus was superior to cxr in the detection of lung consolidations smaller than mm. interstitial lung changes were detected by lus in / ( . %) patients, and by cxr in / ( %). lus and cxr detected pleural effusion in / ( . %) and / ( . %) patients respectively. mcnemar's test showed no statistically significant difference, and cohen's kappa coefficient showed almost perfect agreement ( . ) between us diagnosis of pneumonia and final diagnosis of pneumonia. during the follow-ups, moderate to substantial agreement between lus and clinical evaluation of the course of the disease was obtained (k= . - . ). in children with complete clinical and incomplete us regression of pneumonia, consolidations of less than mm were the most prevalent finding. the average time period until complete resolution of the lus findings was . ± . days. children with us detected pulmonary consolidations larger than mm were statistically significantly longer hospitalized than others. lung ultrasound in the diagnosis of pneumonia in children is just as reliable as radiography, and should be included in the standard diagnostic protocol. the latest uk nice guidelines for childhood tb contact screening require that a chest x-ray (cxr) be requested only when mantoux or igra testing is positive or if there is a documented reason e.g. clinical concern. nice clarifies the role of cxr in determining treatment choice. we aimed to review cxr referral and treatment in the current climate of european migrant screening. retrospective review of paediatric referrals to the infectious diseases clinic for tb contact screening of whom had cxrs, from october to august and correlation with the medical notes. a panel of paediatric radiologists independently interpreted radiographs in the clinical context of tb contact screening and a majority decision was reached. of patients referred to the infectious diseases unit, underwent cxr in addition to a mantoux and igra test. of those cxr's, were reported as having features of pulmonary tb but only / ( %) were treated as active tb. eighteen of the ( %) cxr's which were reported as having no features of pulmonary tb, were treated as active tb. of those , only / ( . %) had a clearly documented reason. review of the radiographs (mean age years) by the panel of radiologists noted that all were of readable quality, radiographs were in keeping with a diagnosis of tb, were inconclusive and were normal. the diagnosis of tb was based on lymphadenopathy in and ( ) (suppl ):s -s pediatr radiol milliary nodules in . parenchymal abnormality was seen in patients [one was the milliary] and effusion was seen in . this correlated well with the initial radiology reports of duty radiologists. of the who underwent cxr, referral information was available in . ( . %) of these had been appropriately referred because of a +ve mantoux/igra. only out of ( . %) of those who had cxr despite a -ve mantoux or igra, had a documented reason. according to nice guidelines, % of cxr reported positive for tb were not treated for active tb. this may represent a lack of clarity regarding the definition of 'latent tb'. furthermore, only a third of the % of patients who received treatment despite negative radiographs had a documented reason. the current migrant crisis requires clarity of x-ray definitions of latent tb to avoid the % under-treatment and % overtreatment identified in our population. is there really no cardiac problem for performing sports Ö.İ. koska, p. bayindir, h. alper; izmir/tr objective: sudden death in young is a rare condition excluding known anomalies and sudden infant death sydrome; but its consequences are devastating because they are so unexpected. % of them occur in a context of sports event. everyday parents of millions of children admit hospitals in order to get permission for participating in sports events. and after physical examination and ecg, physicians are expected to decide such an important issue. however there are a number of silent reasons that may lead to child to sudden death. altough we don't perform ct scans for such indications, we have encountered several cases with abnormalities that can lead to sudden child death and while reporting an examination, awareness of these devastating conditions may be usefull. we searched our database from . . to . . in order to see how often we diagnosed such a silent reason from the ct images that are performed for some reasons. as our center is a tertiary center we have performed cardiac ct examinations in that period that are mainly for excluding or defining complex cardiac anomalies. in order to prepare a pictorial review of unexpected but ct detectable sudden cardiac death reasons, we excluded congenital heart diseases (namely obstructive, shunting or complex anomalies) and ecg detectable arrhytmic diseases. the non arrhytmic, non traumatic reasons for sudden cardiac death excluding congenital heart diseases in the papers are: hypertrophic cardiomyopathy (cmp) (% ), some coronary artery path and origin anomalies (mainly abnormal left coronary artery from pulmonary artery (alcapa), and interarterial path)(% ), increased cardiac mass (% ), dilated cmp (% ), marfan disease (% ), myocarditis (% ), ischemic heart disease (% ). we detected examinations according to our inclusion criteria and selected one cases of each; rca path anomaly, alcapa, dilated cmp, hypertrophic cmp, subaortic discrete membrane and increased cardiac mass for presentation although sudden cardiac death is rare in young children it is a so devastating condition that it must be taken into account for every situation. awareness of silent conditions and active search of them may protect professionals from medicolegal issues and unpleasent results. to describe the spectrum of chest ct scan findings of pulmonary involvement in childhood langerhans cell histiocytosis (lch) and propose a simple scoring system to evaluate the profusion and distribution of the main lung lesions. one hundred forty-six chest ct scans of the pediatric patients with pulmonary lch enrolled in the french national database for lch until april , could be retrospectively and independently reviewed by pediatric radiologists. for each ct scan a semi-quantitative analysis was performed for nodular opacities and cystic abnormalities. the chest was divided in fields (upper, medium and lower field of the left and the right lung) and for each field, both for the nodules and the cysts the score was =no lesion, = lesions involving up to % of the parenchyma, = - %, = - % and =more than %. of patients evaluated at diagnosis, patients ( %) presented with nodules, patients ( %) presented with cysts and patients ( %) presented a combination of both nodular and cystic lesions. on the initial ct scan, median nodules total score was and median cysts total score was . during subsequent ct scans almost the same percentage of patients with nodules ( patients, %) was found but we observed an increase number of patients with cysts ( patients, %), median nodules total score was and median cysts total score was . the distribution of nodules and cysts was symmetric in the upper, medium and lower fields with an involvement of costo-phrenic angles in % of the cases. patients with pneumothorax ( patients, %) had a higher cysts median score ( ) than patients without pneumothorax ( ). we found alveolar condensation in patients ( %). none of them showed signs of infection at bal examination or any improvement after a treatment with a standard antibiotic therapy while they did show regression under the lch standard regimen of chemotherapy. we proposed a score for semiquantitative analysis of distribution and profusion of nodular and cystic lesions on chest ct scans that can be a useful tool in pediatric population to monitor lung involvement. we found a significant correlation between pneumothorax and a high cysts median score. alveolar condensation could be considered as a possible manifestation of plch in children. lung bases involvement was found in % of the cases, representing an important different imaging feature from adult plch. high resolution computed tomography for chronic small airway disease in hiv infected adolescents a.-m. du plessis , s. andronikou , h. zar ; cape town/za, bristol/uk early treatment with antiretroviral therapy (art) and decline in infected infants due to prevention of mother-to-child transmission has resulted in an increase in the population of hiv-infected adolescents. pulmonary disease is common among them. cxr is considered insensitive and terminology inconsistent. therefore, despite concerns related to radiation dose in paediatric patients, high resolution computed tomography (hrct) is the modality of choice for the evaluation of small and large airway pathology, prominent in chronic pulmonary disease. hrct findings are used for prognosis, treatment decisions and defining anatomic extent of bronchiectasis for surgical intervention. the aim of this paper is to demonstrate the spectrum, frequency and extent of airway pathology in hiv-infected adolescents using hrct. a nested sub study was undertaken within the cape town adolescent antiretroviral cohort (ctaac); a prospective, descriptive cohort study of hiv-infected adolescents on art and age matched hiv-s ( ) (suppl ):s -s pediatr radiol negative controls. hrct was performed on patients who demonstrated abnormal lung function (defined by forced expiratory volume in second (fev ) of < % and/or low lung diffusion capacity (dlco)). single phase, contrasted multi-detector volume acquisitions were performed from the thoracic inlet to the diaphragms at full inspiration and image data postprocessed to yield thin ( , mm) and thicker slice images ( mm). three mm slices at cm intervals were performed in full expiration. three radiologists interpreted the c t scans independently, with strict imaging definitions, and a majority decision was generated for each finding. ages of patients ranged from between to years with a mean of , . there were females and males with a ratio of : , . bronchiolitis obliterans was seen in % of patients and bronchiectasis was demonstrated in %, % of which was classified as severe (involving either an entire lobe or more than % of at least lobes). there was an absence of lymphadenopathy (a sign of primary tuberculosis (tb)), lymphocytic interstitial pneumonitis (lip) and post tuberculous apical architechtural distortion. miliary tb was identified in a single patient. ground glass was seen in % and consolidation in %. the majority of hiv infected adolescents with poor lung function demonstrated bronchiolitis obliterans strongly emphasizing the use of hrct for confirming small airways disease. hrct was also useful for demonstrating extent of associated bronchiectasis in %. hrct allows classification of patients into those with diffuse small airways disease requiring medical management and those with local disease requiring surgery. background: chronic recurrent multifocal osteomyelitis (crmo) is an autoinflammatory paediatric non-infectious bone disease. presenting symptoms are non-specific, prolonging diagnosis, and leading to deformity, morbidity and unnecessary procedures. imaging is critical to diagnosis, with whole-body mri (wbmri) commonly used in all stages of care. in our institution, a baseline whole-body coronal stir sequence is routinely obtained. aim: to determine lesion distribution and extent on baseline wbmri via retrospective panel review of all patients clinically diagnosed with crmo, and to determine any patterns of involvement that could facilitate earlier radiological diagnosis. method: all patients diagnosed with crmo since december using published bristol criteria were identified and baseline whole body mris reviewed. the reviewing radiologists were blinded to the original report and previous investigations. each mri was reviewed for focal lesions consistent with crmo. the extent of metaphyseal and epiphyseal lesions was categorized into involvement of thirds of the width of the structure. the wbmri of forty children ( girls, boys), averaging years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were reviewed by the panel using a majority decision rule. three hundred three lesions were recorded, averaging . lesions ( - ). the tibia was most affected ( lesions), most commonly the distal tibial metaphysis ( lesions in patients, bilateral). rib, metatarsals and distal femoral epiphyseals lesions were common. humeral, hand and skull lesions were few. complete metaphyseal involvement, the 'smouldering physis', was most prevalent within the proximal and distal tibial metaphyses. although ranked seventh, the reportedly more common clavicular lesions were the site of the most florid lesions, demonstrating bone expansion and periosteal reactions. two clear patterns of involvement emerged. in patients with clavicular lesions, fewer overall lesions were observed (average . ), mainly affecting the axial skeleton and feet. patients with tibial involvement had a higher number of overall lesions (average . ), but few lesions outside the lower limbs. only four patients had a both clavicular and tibial lesions. twelve vertebral lesions and four cases of spodylo-discitis were identified; two at t / level, one at t / level, and one involving both t / and t / . our series of cases of crmo with baseline wbmri, one of the largest in the published literature, identifies the common sites that should be interrogated for involvement. this study also demonstrates potential as-yet undescribed patterns of skeletal involvement that can be used to aid radiological diagnosis and highlights a non-infective cause for spondylo-discitis. hepatic hemangiomas (hh) are the most common benign vascular tumors encountered in the pediatric population. two main types have been described congenital and infantile, which both display distinct clinical courses and biological features. hemangioendothelioma in differential diagnosis of hh is controversy. recent literature suggests that congenital hepatic hemangiomas present in a focal form, whereas infantile hepatic hemangiomas present in either a multifocal or diffuse form. the goal of this study is to evaluate the features associated with focal and multifocal hh. the records of patients diagnosed with a hepatic hemangioma at a tertiary pediatric hospital from to were reviewed. we divided our series into groups: focal or multifocal including diffuse form. clinical endpoints are: age of diagnosis, presence of cutaneous hemangioma, alpha-fetoprotein, thrombocytopenia, cardiac insufficiency. imaging endpoints were echogenicity on us (hypoechoic, hyperechoic or mixed), vessels density on color doppler (< ; - , > cm ). presence of calcifications, venous lakes, visible vessels and aortic tapering were assessed on us, ct-scan, mr and angio. treatment and outcome were analyzed. univariate and bivariate analysis were done. this study included focal ( m, f) and multifocal ( m, f) hh. antenatal diagnosis was done in focal and multifocal hh. focal lesions were associated with the presence of cutaneous hemangiomas (p< . ) and calcifications (p< . ). no other variable was significant. conservative management was decided in focal and multifocal hh. steroids (focal: , multifocal: ), steroid-interferon (focal: , multifocal: ), propranolol-steroid (focal: , multifocal: ) and surgery in one focal form. complete regression was observed in most lesions (focal: n= , multifocal n= ), whereas incomplete regression < % was observed in patients (focal: n= , multifocal: n= ) and patients in the focal group with the pathology diagnosis of hemangioendothelioma. hepatic hemangiomas demonstrate a wide range of radiological features, with important overlaps in focal, multifocal or diffuse forms. focal and multifocal hh can be seen in congenital hemangioma, infantile hemangioma or hemangioendothelioma. the association of cutaneous infantile hemangioma in the focal group confirmed that the focal lesion can be seen in infantile hemangioma. however, calcifications are more frequent in focal hh which is described in congenital hemangioma or hemangioendothelioma. ( ) (suppl ):s -s pediatr radiol symptomatic and asymptomatic meckel's diverticulum in the pediatric population -a retrospective analysis of imaging findings with histopathologic correlation n. abu ata, r. cytter-kuint, j. bar-ziv, i. hadas-halpern; jerusalem/il objective: though meckel's diverticulum (md) is a relatively common gastrointestinal anomaly, many of the symptomatic and most of the asymptomatic md's are often missed on abdominal imaging. our purpose is to describe the radiologic appearance of symptomatic and asymptomatic md in the pediatric population and to correlate the radiologic findings with histopathology. a retrospective analysis of all children diagnosed with md between / - / and had relevant imaging (n= ) was done. imaging studies-ultrasound (us), computed tomography (ct) and magnetic resonance imaging (mri) were retrospectively reviewed and evaluated for visualization of md in symptomatic and asymptomatic patients. findings were compared with the preoperative radiology report and the pathology specimen. symptomatic group (n= ): mean age . ± years, nineteen males. md presented with abdominal pain in patients, small bowel obstruction (sbo) in patients, gastrointestinal bleeding or anemia in patients and intussusception in cases. md was identified in preoperative reports ( . %) and retrospectively identified in more cases (overall patients, . %). in cases, an inflamed or perforated md were found. in cases, mucosal lining resembling gastric folds was seen. inverted meckel and prominent tissue surrounding the diverticula were seen in patients. in a single case md was misdiagnosed as a duplication cyst. asymptomatic group (n= ): mean age . ± , eight males. md was not mentioned in any of the original reports and only md's were identified retrospectively ( . %). no mucosal abnormality or irregularity were noted. histopathology: ectopic gastric mucosa was found in / ( . %) of the symptomatic patients vs. / ( . %) in the asymptomatic group. all patients with sonographic appearance of gastric mucosa had gastric mucosa in pathology (specificity- %, positive predictive value- %). md has a variety of radiologic appearances. it can be detected in most of the symptomatic patients but is almost undetectable in asymptomatic patients. heterotopic gastric mucosa is more common in the symptomatic group. inflamed gastric mucosa may have a typical appearance resembling a small stomach, a sign that was not described before and has both high specificity and high positive predictive value for gastric mucosa within a meckel diverticulum. preliminary results on dna damage from ct irradiation in pediatric patients i. dilevska, e. nagy, w. schwinger, e. sorantin; graz/at the increased radiation sensitivity in children, compared to adults, is a well-recognised fact in the pediatric radiology community. the high-dose irradiation induced dna damage has been well established, however the dosages that are clinically used in everyday ct procedures are so low, that it remains unclear how this severely affects the dna and can induce cancer in the long run. the aim of this study is to assess the effects of lowdose ionizing radiation from ct in children by establishing a standardization curve ranging from the high to the low, medically significant ctdi values. this is done by measuring the phosphorylation of the h ax histone (γh ax), which is considered a biomarker for quantification of radiation induced dna double-strand breaks (ddsbs). the detection of the γh ax histone was done by two methods: immunofluorescence microscopy (im), which is an established method for detection and quantification of this histone and the new, promising flow cytometry technique (facs). for this study, leucocyte samples ("buffy coats") were provided by the local transfusion department and these samples were irradiated with a clinical ct scanner (aqilionone, tmse) with values ranging from , to , mgy ctdi. afterwards the samples were processed with both methods. for the immunofluorescence, twostep immunostaining was used with two different antibodies and the cell and foci counting were done on an olympus xc microscope, while the facs staining was done with a one-step antibody and the samples were measured on a navios flow cytometer (beckman coulter). comparable results were detected with both methods, with a good correlation between the facs and im, with a linear incline (r > . ) in the high and in the low dosages from , to . mgy ctdi. however, in the samples irradiated with doses below . mgy ctdi, there seems to be less phosphorylated h ax than in the native samples. two possible explanations arise: a) low dose irradiation initiates repair that extends to ddsbs occurring naturally or b) low dose irradiation doesn´t cause phosphorylation of this histone, but affects other dna damage and repair pathways. the preliminary findings show that the facs analysis can be used as a valid replacement method for the labor-intensive if method in the higher dosages. however more analysis should be done to establish its accuracy in the lower regions since underlying mechanisms are not clear yet. a. turkaj , g. cicero , e. sorantin , r. coroiu ; graz/at, mesina/it, cluj-napoca/ro there is only little information available regarding imaging procedures in the trauma setting of pediatric patients. such data can serve as a rational basis for running pediatric trauma units. the purpose of the paper is to investigate the number, types and distribution of imaging procedures in a tertiary pediatric trauma center serving children of about . million inhabitants with approximately . children. all trauma-caused admission to the emergency room and their imaging procedures were analyzed retrospectively occurring within a period of months. a cohort of patients (m:f= . : ) were analyzed. patients were grouped according to age into the following categories: neonates, infants, middle childhood, early adolescence, late adolescence. imaging procedures were classified into plain films, us, ct and mri. furthermore, the time of admission was noted and categorized in time slots : - : , : - : , : - : and : - : . referral cause was divided in domestic accidents, motor-scooter-bicycle accidents, car accidents, sport injuries, falls from height and others. the average age was . ± . years, aligned in the following age-groups neonates ( %), infants ( %), middle childhood ( %), early adolescence ( %), late adolescence ( %). a total of imaging procedures were performed, of which plain films ( %), us ( %), ct ( %) and mri ( %). there was a statistically significant difference of imaging procedures due to age in particular in us and ct. regarding the timeslots: : - : ; patients ( %), : - : ; patients ( %), : - : ; patients ( %), : - : ; patients ( %). domestic accidents were the leading referral cause with cases ( %) prevailed age groups were infants and middle childhoods corresponding for more than %. motorscooter/bicycle accidents accounted for cases ( %) of which most were early and late adolescence (more than %), s ( ) (suppl ):s -s pediatr radiol sport's accidents ( . %) equally shared among middle childhood, early and late adolescences. car accidents ( . %) cases and fall from height ( , %) did not show any prevalence according to the age groups. for the first time detailed data about imaging procedures at the emergency room for pediatric patients are now available. over half of the admissions ( %) occur outside regular work hours thus representing a challenge for the staff in duty and this fact should be considered in working schedules. due to strict interdisciplinary developed diagnostic pathways the number of ct examinations was reasonable low. head ct in a regional children's hospital without mri -effective doses and justification of clinical indications j. lovrenski, n. milenković; novi sad/rs to calculate effective doses (ed) for pediatric head computed tomography (ct), to determine the most common referral diagnoses, and the share of normal and pathological ct findings. a retrospectiveprospective study comprised all the children with performed ct examination ( -slice scanner) within a one-year period. pediatric ct protocols were used. the values of ed for head cts were calculated based on the two different models (shrimpton's and icrp publication ). average ed for different age groups was expressed as the number of chest x-rays (cxrs) ( cxr . msv). the most common non-traumatic referral diagnoses for head cts were determined, as well as percentage and type of pathological ct findings. a share of pathological ct findings was also determined for trauma as a referral diagnosis. head cts were represented with ( %) in total number of ct examinations within a one-year period. the different calculation models have shown the difference in ed values of up to . %. eds for head cts were equivalent of ( years of age and older) to (younger than months of age) cxrs for one sequence of scanning. the most common non-traumatic referral diagnoses for head cts were: loss of consciousness, epilepsy, headache, convulsions, and vertigo. in this group of patients, % of completely normal ct findings were found. pathological findings in this group consisted of the patients with the most common non-traumatic referral diagnoses were as follows: cortical atrophy ( patients), arachnoid cyst ( ), ischemia ( ), porencephalic cyst ( ), agenesis of the corpus callosum ( ), chiari malformation -type i ( ), open-lip schizencephaly ( ), and tumor of the posterior cranial fossa ( ). most common incidental, extracerebral pathology discovered included sinusitis and otomastoiditis. in patients with trauma as referral diagnosis, the share of pathological ct findings was . %. it is necessary to get clinicians familiar with the extent of ionizing radiation that children are exposed to during the head ct examinations. a more careful selection of children for head cts is necessary in an every-day clinical practice, especially for patients with non-traumatic referral diagnoses. diffuse and symmetric diffusion restriction involving the white matter of the brain in patients with neonatal seizures j.-y. hwang , y.j. lee , y.-w. kim ; yangsan-si, gyeongsangnam-do/ kr, yangsan-si/kr this study aimed to evaluate magnetic resonance (mr) imaging findings in patients with neonatal seizures focused on the diffuse white matter lesions on diffusion weighted image (dwi) in addition to clinical manifestations. a total of neonates aged less than -week old underwent brain mr imaging for evaluation of neonatal seizures between november and december . among them, patients showed diffuse and symmetric pattern of high signal intensity on dwi. clinical, laboratory, and mr images were analyzed retrospectively. nine patients were males and three patients were females. patient age was . ± . days (range, - days). all the patients were born at full term. the most frequent month of the hospital visit was march (n= ) and january (n= ). eight patients showed generalized clonic seizure and four patients showed partial clonic seizure. stool viral test was performed in nine patients. among them, five patients were positive for the rotavirus and one patient was positive for the astrovirus. nine patients underwent cerebrospinal fluid analysis, however, all showed negative results. mr imaging was performed at . ± . days after onset of seizures. diffuse and symmetric diffusion restriction were distributed along the cerebral white matter tracts and both thalami (fig ) accompanied with high signal intensity on either t -weighted images or on the fluid-attenuated inversion recovery (flair) sequence. multiple foci of high signal intensity on t -weighted images at the centrum semiovale that was affected on dwi were also observed. follow-up period was . ± . months (range, . - . months) and developmental delay was encountered in three patients. six patients underwent follow-up mr imaging at the age of . ± . months (median, . months; range, . - months). five patients showed volume loss in cerebral white matter on both sides of the brain and four patients showed high signal intensity of the periventricular white matter on either t weighted images or flair sequences (fig ) . myelination delay was not observed in follow-up mr images. diffuse and symmetric diffusion restriction involving the cerebral white matters can be seen in patients with neonatal seizures on mr imaging. our study shows that rotavirus is commonly encountered, but not exclusively detected in these patients. nevertheless, viral infection-associated encephalopathy should be considered when a patient is presented with characteristic clinical and mr findings. whole body mri on diagnosis and follow-up of neurofibromatosis type d. grassi, v. tostes, e. caran, h.m. lederman; sao paulo/br demonstrate that whole body mri is effective on showing neurofibromatosis type involvement of different regions of the body not known by the clinicians. review of patients with neurofibromatosis type (nf ) who underwent whole body mri throughout their follow-up with the majority of them had only brain and spine imaging studies. it was possible to demonstrate that whole body mri provides an overview of nf systemic manifestations and neurofibroma's extension beyond the clinic expectation. despite being rare, sarcomatous degeneration was suspected when there was any difference on the characteristics of the neurofibromas. whole body overview where its possible to see the neurofibroma's extension in right cervical region, scoliosis and multiple plexiform neurofibromas. only the biggest neurofibroma was detected by clinical exam. however it is possible to identify two others neurofibromas. whole body view of multiple plexiform neurofibromas. whole body mri is a radiation-free exam and it is useful on the diagnosis of nf and on patient's follow-up. it provides an overview of the systemic s ( ) (suppl ):s -s pediatr radiol involvement and neurofibroma's extension beyond the clinical expectation. during patients follow up, it could also show tumor's characteristics modification, which was considered as a possible sarcomatous degeneration. accuracy of non-radiologists and lay-persons for identifying children with cerebral cortical atrophy from 'mercator map' curved reconstructions of the brain s. vedajallam , a. chacko , s. andronikou , e. simpson , j. thai ; east london/za, bristol/uk objective: background: communication of cortical brain atrophy in children with term hypoxic ischaemic injury (hii) to parents and the legal fraternity contesting compensation rights can be very difficult using text and standard cross-sectional images. when demonstrating the cortex in hii, a single image of the brain surface, much like the way a map of the earth is derived from a globe, can be generated from curved reconstruction of coronal magnetic resonance imaging (mri) scans i.e. a mercator map. lay people's ability to identify abnormal scans from such maps without prior training requires evaluation before routine use. aim: to determine the sensitivity and specificity of lay people in detecting abnormal brain scans through review of mercator flat-earth maps of the brain, without prior training. ten mercator map images were provided to participants with a distribution of hii, cortical dysplasia and reported normal. participants were required to identify abnormal scans. sensitivity and specificity overall and for sub-groups were derived by averaging true positives and negatives; false positives and negatives. the results show a strong ability for lay-people to identify normal versus abnormal mri brain studies using mercator maps. the sensitivity and specificity in this group is % and % respectively. non-radiologist physicians and radiographers performed slightly better than lay people as expected. radiologists of course had very high sensitivity and specificity of % and %. the mercator map is therefore a viable tool in the communication of complex mr imaging to the lay-person. safety and efficacy of sphenopalatine ganglion blockade in childreninitial experience l. dance, c. schaefer, d. aria, r. kaye, r.b. towbin; phoenix/us objective: sphenopalatine ganglion (spg) blockade is known to be a safe and effective migraine headache treatment among adults. this paper will report the initial experience in the pediatric population with spg blockade. one hundred thirty-three procedures were performed in patients ages to from february through november . pre-intervention headache scores were recorded on a scale of to . the procedure was performed supine with neck in hyperextension. anesthesia of the bilateral nares was accomplished with lidocaine spray and gel. contrast was injected using a sphenocath confirming catheter position. % lidocaine was injected. patients remained supine with neck in hyperextension for minutes. post-intervention headache scores were recorded. mean pre-treatment score of . decreased to . post-treatment (Δ . , % ci . - . , p< . ). there were no complications. spg blockade is a safe and effective treatment for migraine headaches in children which results in decreased reliance on intravenous drug therapy. orbital masses represent a spectrum of benign and malignant lesions in children that can be challenging to diagnose and treat. imaging plays an important role in diagnosis, due to a potentially limited clinical examination and risks associated with biopsy. mr imaging is a powerful tool for imaging the orbit, due to the excellent tissue contrast it provides. yet conventional mri has a limitation in discriminate the benign from malignant lesions. diffusion-weighted imaging (dwi) is non-invasive rapid technique uses the water diffusibility to produce contrast among different kinds of tissues. our propose was to assess the role of dwi and calculated apparent diffusion coefficient (adc) values in characterization of the pediatric orbital masses regarding benignancy or malignancy. one hundred and thirty patients with recently diagnosed orbital masses and who underwent preoperative conventional mri and dwi were included in this study. the orbit was divided into six compartments: the eye globe, retroocular fat, optic nerve, lacrimal system, bony boundaries and extra-ocular muscles. the average adc obtained from each tumor was compared with the histopathological diagnosis determined from subsequent surgical sample. seventy girls and sixty boys with orbital masses were included in this study. their age was ranged from month to years. the globe is the seat of lesions in / cases, optic nerve in / case. seven cases have lesions in the lacrimal gland. forty-five of cases was diagnosed as having benign masses & of cases have malignant lesions. there is a statistically significant difference between the mean adc value of the benign lesions ( . ± . x - mm /s) and the mean adc value of the malignant lesions ( . ± . x - mm /s) (p< . ). the optimal adc cutoff value that was determined for discrimination between these lesions is: . x - mm /s), with sensitivity of . % and specificity of %. using conventional mri alone in predicting benign and malignant lesions has the sensitivity of % and specificity of % with % positive predictive value and % negative predicative value. combining dwi and conventional mri has increased accuracy, as the sensitivity and specificity were %, % respectively with % positive predictive value and % negative predicative value. ( ) (suppl ):s -s pediatr radiol adc values provide an accurate, sensitive, fast, and non-invasive mean of characterization of pediatric orbital tumors. a priori tumor characterization is useful in timing and treatment planning for orbital tumors. utiliy of resting state fmri in children for preoperative language mapping l.-m. leiber, m. delion, a. ter minassian; angers/fr to assess if resting state fmri is able to detect language eloquent areas in childrens. six children, from to years old suffering from brain lesions were enrolled in this study. they underwent mri with one dt morphology session and three minutes fmri sessions, including one resting state fmri and two language task induced activity fmri sessions. analysis was performed using a generalized linear model for the first one and a spatial independent component analysis approach for the two others. language maps were compared with cortical mapping obtained by intraoperative direct stimulation. language network was identified systematically by resting state session but not by task induced activity sessions. moreover, in two of the six patients, resting state fmri was able to detect eloquent areas found during intraoperative cortical mapping that were not present in task induced activity sessions. resting state fmri appears superior to task induced activity fmri in detecting language eloquent areas. is sclerotherapy an effective treatment option for ranulas or thyroglossal duct cysts in children? d. aria, l. dance, c. schaefer, r. kaye, r.b. towbin; phoenix/us to assess the utility of sclerotherapy in the treatment of ranulas and thyroglossal duct cysts materials: from - , patients varying in age from months to years were referred to the ir department for sclerotherapy. of the patients, had a diagnosis of ranula while had the diagnosis of thyroglossal duct cyst by either mr, ct, or us. sclerotherapy treatments were performed with standard sclerosing agents, i.e. sotradecol % foam, absolute ethanol, and bleomycin. in the subset of patients with ranulas, sclerotherapy was commonly performed in accordance with salivary (submandibular and/or sublingual) gland botox injection or ethanol ablation. -gauge or f sheathed needles were used for us-guided access to the lesions, with ranula sclerotherapy being performed after placement of side-hole drainage catheters ( - f) due to their increased viscosity. the preferred sclerosing agent was injected with dwell times ranging from mins to hours. salivary gland injection/ablation was performed under usguidance using a -gauge needle with volume injection targeted centrally within the gland or in the portion of the gland abutting the ranula. after treatment, all patients were scheduled for follow-up ultrasounds at a minimum of weeks to assess lesion response or residual disease. a total of sclerotherapy treatments were performed. of the patients, were lost to follow-up after single sessions for ranula and thyroglossal duct cyst. the other patients all had follow-up ultrasounds after each of the remaining sclerotherapy sessions. four of these patients showed initial improvement with either decreased size of lesion or lesion resolution while the other showed no improvement with either stable or increased size on initial follow-up. the patients who initially showed promising response unfortunately had recurrence on follow-up imaging and ultimately, demonstrated no favorable response to sclerotherapy after subsequent treatments regardless of whether treatment was combined with ethanol/botox salivary gland injection. in summary, all patients who were successfully followed show no appreciable response to treatment for ranula or thyroglossal duct cyst. despite the emergence of clinical requests for sclerotherapy of ranulas and thyroglossal duct cysts, in our case series, sclerotherapy has not proven to be an effective treatment option using our current drug regimen. role of the susceptibility-weighted imaging (swi) in the neuroimaging of term newborns g. rudas, e. varga, p. barsi, l. kozák, Ü. méder; budapest/hu objective: susceptibility-weighted imaging (swi) was introduced in the neonatal neuroimaging only a few years ago. we can find only a few publications about its advantages and disadvantages. according to our experience, swi is extremely useful not only for detecting bleedings but for the diagnosis of other diseases as well. during the last year we had mri examinations on term newborns ( - days of life) and the swi gave additional information in cases. we used a t philips insignia scanner. in the case of the questionable hypoxic-ischemic-encephalopathy ( cases) and the metabolic diseases ( cases) we could find increased signal intensity in the cortex; in the case of stroke we could find the thrombus itself in cases; the avm were much clearer using the swi in cases; at the pvl in cases we could visualize the cysts better using swi; in the case of congenital heart disease ( cases) and in the case of sinus thrombosis ( ) we could find microbleedings and/or dilated veins; in cases the position of the lateral ventricle drain or shunt was much clearer using the swi. the swi gave important additional information in / ( %). the swi is a strongly recommended new sequence at the mri examination of the term newborns' brain. a disadvantage of swi is that it requires ca. three minutes examination time (in contrast to t * which is only minute long). mechanical birth-related trauma: imaging of the "accidents of birth" a. chaturvedi, j.g. blickman; rochester/us objective: . to discuss definition, incidence and risk factors leading to "mechanical birth-related trauma" and compare these with existing literature. offer an organ-system based classification scheme encompassing the varied manifestations of birth-related trauma and describing the implications on care decisions. materials: the hospital imaging department database was searched for neonates who presented with history of difficult/traumatic birth at our obstetric center between january , -june , . search software used was primordial customised radiology solutions, san mateo, ca. the search terms used were "macrosomia", "shoulder dystocia", "instrumental delivery", "malpresentation", "cephalopelvic disproportion", "forceps" and "vacuum". initial and follow-up imaging and clinical data on these neonates was reviewed and compiled by two board-certified pediatric radiologists. the relevant literature was reviewed and findings compared. organ-system based classification scheme for birth-realted trauma. in our study, mechanical trauma of birth was seen to manifest within different organ systems, which have been listed below in the order of occurrence within our sample. injuries to the skull (sutural overlap, dents and fractures), scalp hemorrhages (subgaleal hematoma, cephalhematoma, caput). intracranial intraand extra-axial hemorrhages (subdural, subarachnoid, epidural, intraparenchymal). clavicle fractures neonatal brachial plexus injury. sternocleidomastoid hematomas. adrenal hemorrhages. cervical spinal cord contusions. schematic diagram depicting intra-and extracranial hemorrhages by location. -year-old with history of calvarial fracture at birth-fracture did not heal but enlarged secondary to leptomeningeal entrapment at the fracture sitean entity called "growing fracture" or "leptomeningeal cyst". multiple newborn organ systems can be injured from mechanical trauma of birth. our numbers compare favourably with the existing literature. mechanical birth-related trauma can occur simultaneously with hypoxic-ischemic birth injury. although most of these injuries spontaneously and completely resolve, long-term complications can be seen in some cases. few of these injuries are life-threatening. imaging plays a crucial role in diagnosis and follow-up, and can assist in decision making as well as in counselling the parents. ewing sarcoma of tibia in an infant girl a. seehofnerova, j. skotáková, i. Červinková; brno/cz objective: ewing sarcoma (es) is the second most common primary bone malignancy in children. it histologically originates from neuroectodermal tissue and consists of small round blue cells. ewing tumour family is very close to primitive neuroectoderm tumour (pnet) family with diverse stage of differentiation, ewing sarcoma being less differentiated. approximately % of the cases occur between ten and twenty years of age with slightly higher prevalence in male gender. nine-month-old caucasian girl presented to local surgery department after she had wedged her lower leg in a bed. the right lower leg was swollen and painful. she was initially diagnosed with a ligament injury and underwent standard treatment. oedema gradually disappeared, but swelling and pain increased after three weeks. she also suffered from a fever of . °c ( . o f). at that point x-ray of her right lower leg was performed with report describing pathologically changed structure of tibia and she was referred to our university centre. ( ) (suppl ):s -s pediatr radiol we made a second reading of the plain film, reporting sclerotic heterogeneous bone structure of the right tibial diaphysis and distal metaphysis, onion-like periosteal reaction with sunburst spiculation and cortical bone destruction. her laboratory results were: crp . mg/l, ld . μkat/l, nse . μg/l, ferritin μg/l. crp has been raising for a week to mg/ l, then decreased to normal level. differential diagnosis was established as a primary bone malignancy (especially es) or, less likely, an osteomyelitis. mri revealed pathological signal of bone marrow of diaphysis of the whole tibia with cortical scalloping and periosteal spiculated apposition. epiphyses were spared. dorsal cortical bone was interrupted with extraosseal spread of the process. intraosseal part enhanced heterogeneously, whereas extraosseal component enhanced almost homogeneously after contrast medium administration. total size of the tumour was assessed as x . x mm ( . ml) . adjacent muscles were oedematous with post-contrast enhancement. there were also few enlarged lymph nodes in popliteal region. results from the biopsy confirmed ews with positive ews/fli gene. tumour was assessed as a localized disease, enneking iib. patient underwent chemotherapy according to aews doc protocol and a knee-exarticulation with no traces of tumour in resection lines. nowadays she is in the first complete remission. x-ray: ap view mri: etw _tse postcontrast, sagittal view, pre-treatment mri: etw _tse postcontrast, sagittal view, after initial treatment unique teaching points: ewing sarcoma belongs to common primary bone tumours in children but is a very rare unit in infants. despite the age predilection it is necessary to consider this diagnosis even in children younger than one year of age. scimitar syndrome together with pulmonary sequestration and horseshoe lung: congenital pulmonary venolobar syndrome b.e. derinkuyu, h.n. Özcan, y. tasci-yildiz, h g. cınar, u.a. orun; ankara/tr objective: congenital pulmonary venolobar syndrome (cpvls) comprises of a spectrum of pulmonary developmental anomalies. the main components of cpvls are hypogenetic lung partial anomalous pulmonary venous return, absence of pulmonary artery, pulmonary sequestration, systemic arterialization of lung, absence of inferior vena cava. minor components of cpvls include tracheal trifurcation, eventration and partial absence of the diaphragm, phrenic cyst, horseshoe lung, esophageal and gastric lung, anomalous superior vena cava, and absence of the pericardium. in this case presentation, we present a baby with scimitar syndrome, pulmonary sequestration, horseshoe lung and right aberran subclavian artery. a month-old girl was admitted to our hospital with the suspicion of scimitar syndrome from a different hospital. she did not have any symptoms. the physical examination was unremarkable. on plain radiograph, the baby had dextrocardia. there was a doubtful tubular structure with the shape of scimitar and a nodular radioopacity behind the heart (figure ). transthoracic echocardiography demonstrated the dextrocardia, atrial septal defect and the right pulmonary artery hypoplasia. afterwards, the ct angiography was done for confirmation of scimitar syndrome and other accompanying abnormalities. on the ct angiography, there was a partial anomalous pulmonary venous return to the suprahepatic inferior vena cava known as scimitar syndrome. besides this, there was a right pulmonary extralobar sequestration in the lung base. the arterial supply was arising from the celiac trunk, while the venous drainage was going directly to the inferior vena cava. therefore, the right lung was hypoplastic of which the tongue of the right pulmonary parenchyma passing between the aorta and heart, appearing confluent with the left lung in a horseshoe configuration. there was dextrocardia and right aberran s ( ) (suppl ):s -s pediatr radiol subclavian artery. the patient was subjected to catheterization and angiography for treatment. on plain radiograph, the baby had dextrocardia. there was a doubtful tubular structure with the shape of scimitar and a nodular radioopacity behind the heart unique teaching points: the term cpvls is an umbrella to a group of pulmonary parenchymal and vascular anomalies that may present in combination. mdct is a helpful diagnostic tool in the preoperative evaluation for delineation of the components of this syndrome. congenital pulmonary venolobar syndrome refers to a wide spectrum of pulmonary developmental anomalies that may appear single or in combination. the main components of congenital pulmonary venolobar syndrome are hypogenetic lung (including lobar agenesis, aplasia, or hypoplasia), partial anomalous pulmonary venous return, absence of pulmonary artery, pulmonary sequestration, systemic arterialization of lung, absence of inferior vena cava, and accessory diaphragm. in this case presentation, we describe a child with scimitar syndrome, bilateral sequestration, hypogenetic lung (single lobed left lung) and right aberran subclavian artery. an year-old syrian girl was admitted to our hospital with the history of heart defect. she did not have syncope or ciyanosis whereas she has easy fatigue and palpitation. on plain radiograph the anomalous draining vein was seen as a tubular structure paralleling the right heart border in the shape of a turkish sword ("scimitar") ( figure ) . transthoracic echocardiography demonstrated the scimitar vein as well as large patent ductus arteriosus (pda), atrial septal defect and left pulmonary hypoplasia. afterwards, the ct angiography was done for confirmation of scimitar syndrome and other accompanying abnormalities. on the ct angiography, there was a partial anomalous pulmonary venous return to the suprahepatic inferior vena cava known as scimitar syndrome. besides this, there was a bilateral intralobar pulmonary sequestration in the lung bases. the arterial supply of the right side was arising from the celiac trunk, while the left side feeding artery was originating directly from the descending aorta. therefore, the left lung had a single lobe with single pulmonary vein draining to left atrium. there was a large pda and right aberran subclavian artery. the patient was subjected to catheterization and angiography for treatment. the right sided anomalous draining pulmonary vein and the feeding artery of the right sequestration were closed in the first session. the procedure was completed without any complication. afterwards, the closure of the feeding artery of the left pulmonary sequestration and the pda were planned in the next sessions. on plain radiograph the anomalous draining vein was seen as a tubular structure paralleling the right heart border in the shape of a turkish sword ("scimitar") unique teaching points: congenital pulmonary venolobar syndrome comprises a heterogeneous group of uncommon abnormalities that may occur in combination. diagnosis of congenital pulmonary venolobar syndrome can be confirmed by ct angiography that allows detailed evaluation of vascular, tracheobronchial, and pulmonary parenchymal abnormalities with a single short, noninvasive procedure. neck infection disclosing diagnosis of congenital fourth branchial arc anomaly in a girl h.n. Özcan, z. aycan, b. ardıclı, m. haliloglu; ankara/tr objective: congenital branchial arc anomalies are rare entities. herein, we describe the imaging findings of acute suppurative infection of the neck caused by fourth branchial fistula in a child. case presentation: an -year-old girl presented to our pediatric emergency department with fever, left sided neck swelling and redness. her complaints were started five days ago. on her physical examination, there was a x cm, stiff, painful mass lesion with redness on the left side of the neck. blood count and thyroid function tests were in normal range; however, c-reactive protein level and erythrocyte sedimentation rate were elevated. neck ultrasonography revealed diffuse soft tissue swelling, a hypoechoic mass consistent with abscess in the left thyroid lobe and perithyroid tissue. the left lobe of the thyroid gland had poorly defined margin and a focus of air. contrast-enhanced neck mr imaging demonstrated an abscess in the left thyroid and perithyroid tissue ( figure ) and enhancement of the soft tissue plane around the left pyriform fossa (figure ). barium swallow revealed the sinus tract originating from the left pyriform sinus apex. the patient was operated after antibiotic treatment and sinus tract was surgically excised. the aim of this report is to describe three cases of right kidney wilms' tumor with cavoatrial tumor extension, referred to our institution between january and september . case presentation: three children, two girls ( and years old) and one boy ( years old) were admitted at the emergency service with cardiac failure symptoms; the latter had also liver failure. echocardiography showed right atrial thrombus in all three patients, as an extension of massive obstructive thrombosis of the inferior vena cava (ivc). abdominal ultrasonography revealed in all patients a right renal mass, associated to right renal vein thrombosis that extended to the ivc and to the right hepatic vein. contrast enhenced computed tomography confirmed findings. patients were treated primarily with chemotherapy before surgery, with partial regression of the thrombus in two patients and no response in one. ( ) (suppl ):s -s pediatr radiol unique teaching points: wilms' tumor is the most common renal malignancy in children and its intravascular extension is a well-recognized event. incidence of tumor extension to inferior vena cava (ivc) is reported to be of - % and intra-atrial extension of , - , %. it occurs most commonly in tumors located in the right kidney (probably due to the shorter path of the right renal vein compared to the left one). this complication does not directly influence the prognosis of malignancy, but the degree of intravascular extension determines technical surgical strategy and increases difficulty of the surgical procedure, especially when there is intracardiac involvement, which increases morbidity. several classifications have been proposed in the adult age group, but due to the similarity of the degree of intraoperative difficulty, the same classifications are used in children. pritchett et al. ( ) described the relation between thrombus and hepatic vessels: level i -intrahepatic intravascular extension; level iiintrahepatic extension; and level iii -suprahepatic or atrial extension. staehler et al. ( ) proposed a different classification that was posteriorly modified and detailed by daum ( ) : stage i -small extension (thrombus size within ivc < cm); stage iilarge thrombus (> cm within the ivc), but still below the hepatic vessels; stage iii -thrombus extending to the level and above the hepatic vessels; stage iv -intra-atrial thrombus. a year old boy presented with a soft tissue mass in his forearm which appeared to have grown quickly in size over a period of three to four months. physical examination demonstrated a welldefined mass in the dorsal aspect of the forearm with no pulsatile bruit. intial differentials included a vascular anomaly or a sarcomatous lesion. the patient proceeded to have an ultrasound examination which revealed a very well-defined heterogenous subcutaneous mass, mostly solid in substance. the lesion measured . cm x . cm x . cm (transverse x length x depth). there was no evidence of muscle invasion. prominent internal arterial vascularisation was demonstrated and the mass was classed as inderminate in nature. subsequent mr findings demonstrated a mass with t signal isointense to muscle, hyperintense t signal and marked homogenous enhancement. small foci of intralesional t hyperintensity and larger areas of t * gradient hypointensity were noted, in keeping with small areas of intralesional blood. vessels were seen to extend from the subcutaneous fat into the lesion. the mass slightly distorted the underlying extensor muscles and tendons of the forearm but there was no deep extension across the fascia. findings deemed the lesion to be more malignant in nature. the patient underwent incisional biopsy and histological findings confirmed a diagnosis of angiomatous fibrous histiocytoma. these tumours are rare soft tissue tumours which most commonly occur in children, adolescents and young adults. while it is rare, there is a potential for local recurrence and metastasis. therefore, it is essential to identify these tumours where possible or at least consider them as a differential for a soft tissue mass in a child. the surgeon commented that the imaging findings and report were essential in making the initial decision about whether to perform an incisional or excisional biopsy as the best treatment for the tumour is wide surgical excision with clearance of margins. unique teaching points: angiomatous fibrous histiocytomas are rare lesions with potential for recurrence and metastasis and therefore should be identified and managed appropriately as a malignant tumour. they are often confused as soft tissue haemangiomas or complex haematomas. it is very important to be aware of the presentation and imaging findings, remembering this form of tumour as a key differential for a soft tissue mass. nasopharyngeal anlage tumor in a neonate with the initial presentation of respiratory difficulty: correlation between imaging and clinicopathologic findings p.-s. tsai, d.-c. lin, s.-l. shih; taipei/tw the etiologies of nasal or nasopharygeal obstruction are variable in neonates. the respiratory symptoms are varied in these cases. mass lesions in nasal cavity or nasopharynx are extremely rare during the neonatal period. however, we must keep it in mind when respiratory problems occur in the neonatal period. here, we report a case presenting with sleep apnea resulting from nasal obstruction by a rare benign salivary anlage tumor in nasopharynx and discuss the imaging findings as well as clinicopathologic characteristics. the -day-old female infant had loud breathing sound, slow feeding and sleep apnea since birth. nasal endoscope and laryngoscope disclosed a polypoid tumor in nasopharyngeal cavity with a stalk connecting with posterior nasal septum. further magnetic resonance imaging (mri) revealed a lobulated mass about . cm in greatest diameter occupying posterior nasal cavity to the nasopharynx that was intermediate signal intensity on t -weighted/t -weighted images and heterogeneous gadolinium enhancement. the patient then received endoscopic resection. the tumor was shown locating in nasopharyngeal cavity and having a stalk from posterior nasal septum, partially occluding the choanae as well. resected tissue fragments displayed tan and whitish in color grossly. microscopic examination demonstrated duct-like structures and mesenchymal elements in a nodular pattern which are typical features of salivary gland anlage tumor. until now, there is no tumor recurrence for four years. unique teaching points: "salivary gland anlage tumor (sgat)" was firstly introduced in a report by dehner et al in . the tumor that has histologic resemblance to the developing salivary gland, is believed to be a hamartoma originating from minor salivary gland rather than a true neoplasm. congenital sgat displays male predilection and is a rare cause of neonatal airway obstruction. the mass is usual in the midline and attached to the posterior nasal septum or posterior nasopharygeal wall by a delicate pedicle. favorable results with simple excision are obtained. once massrelated airway obstruction is established, further examination with computed tomography (ct) or mri is helpful in anatomic evaluation, size measurement, characteristics definition and intracranial involvement. if mass induced airway obstruction is suspected in a neonate and sgat is considered based on imaging studies, invasive procedure should be careful due to the potential of tumor dislodgement from its fine pedicle resulting in complete airway obstruction. the association of intussusception with malrotation is referred to as waugh syndrome. [ ] malrotation occurs in approximately in live births. [ ] the incidence of malrotation amongchildren with intussusception is %. we hereby present a case report of waugh's syndrome associated with midgut volvulus. case presentation: a month old male child reported to the emergency department with the clinical history of vomiting, abdominal distension, bloody mucoid stools and incessant cry. routine blood examination revealed hb: . gm%, tlc: /cu mm, plt- . lac/cu. mm. ultrasound (us) examination was performed and it revealed dilated fluid-filled small bowel loops with moderate amount of free fluid, right iliac fossa showed bowel within bowel appearance suggestive of target/pseudo kidney sign of bowel intussusception. no pathologic lead point was identified. transverse ultrasound image through the upper abdomen showed superior mesenteric vein noted to the left of the superior mesenteric artery hence malrotation should be considered. in view of surgical emergency non contrast enhanced ct was done and axial image showed target/sausage shaped soft tissue density mass it had alternating areas of low and high attenuation due to bowel wall and mesentry. on emergency laparotomy patient was found to have intestinal malrotation with duodenojejunal junction on the right of the midline and mid gut volvulus in clockwise direction. intussusception with terminal ileum (gangrenous), caecum, appendix, whole of ascending colon, transverse colon were telescoping into descending and sigmoid colon. the volvulus was derotated and the in tussusceptum was reduced. the gangrenous terminal ileum and appendix was resected and ladd's procedure was done, a diverting ileostomy was created. the patient recovered uneventfully after which an ileo-colonic anastomosis was created transverse ultrasound shows a mass with a swirled appearance of alternating hypoechoic and hyperechoic "bowel-within-bowel" appearance (target sign) unique teaching points: on ultrasonography multiple, concentric, target like appearance of wall layers of invaginated segments (target sign) on axial scan, as well as pseudokidney sign (sandwich sign) on longitudinal scans were accepted as diagnostic criteria for intussusception. [ ] it can assess the relative positions of the smv and sma which are mostly abnormal in malrotation. upper gastrointestinal contrast study is the imaging reference standard for diagnosis of malrotation with or without volvulus. abnormal position of the duodeno-jejunal junction. spiral, "corkscrew" or z-shaped course of the distal duodenum and proximal jejunum, and location of the proximal jejunum in the right abdomen. [ ] a high degree of clinical suspicion and radiologist's awareness of this entity is helpful in guiding the surgeons towards diagnosis and prevention of morbidity and mortality. a rare case of epidermal naevus syndrome p. joshi; pune/in to acquaint the radiologists with the entity of epidermal nevus syndromes (enss) which are a group of rare complex disorders characterized by the presence of skin lesions known as epidermal nevi associated with additional extra-cutaneous abnormalities, most often affecting the brain, eye and skeletal systems case presentation: this one and a half year old child was referred to us for neuroimaging. he had multiple hairy naevi over his face, limbs including the palms, since birth, associated with blackish discolouration of his entire trunk. unique teaching points: epidermal nevi are overgrowths of structures and tissue of the epidermis, the outermost layer of the skin. the different types of epidermal nevi can vary in size, number, location, distribution and appearance. neurological abnormalities that can be associated with enss can include seizures, cognitive impairment, developmental delays and paralysis of one side of the body (hemiparesis). skeletal abnormalities can include abnormal curvature of the spine, the term "epidermal nevus syndrome" has generated significant controversy and confusion in the medical literature. originally, the term was used to denote a disorder that was actually several different disorders erroneously grouped together. in the recent past, the term was used to denote a specific disorder now known as schimmelpenning syndrome. however, the term epidermal nevus syndrome could be correctly applied to several different disorders. therefore, the umbrella term "epidermal nevus syndromes" now represents a group of distinct disorders that have in common the presence of one of the various types of epidermal nevi. however, there is so far no general agreement how to classify the types of this diverse group of disorders, adding to the confusion within the medical literature. these disorders are quite different from one another and are not "variants" of each other as is sometimes mistakenly stated in the medical literature. in the future, as the genetic molecular basis of these disorders is better understood, the classification may change or expand. bilateral axillary lump in a newborn diagnosed as hematoma h.n. Özcan, u. aydingoz, m. haliloglu; ankara/tr objective: most birth traumas are self-limiting and have a favorable outcome. injuries to the infant that result from mechanical forces during the birth process are not uncommon. they occur most commonly on head and neck after vaginal breech delivery. however, soft tissue hematomas can be rarely seen after caesarian section (c/s). herein, we describe imaging findings of a newborn with bilateral axillary lump diagnosed as hematoma. case presentation: a -year-old woman was admitted to an outside hospital at weeks' gestation for c/s due to prior caesarean operation. it was her fourth pregnancy (g p ). the pregnancy was unremarkable and she had normal ultrasounds at gestation. there was no history of trauma or fall during antenatal period. according to the c/s reports, the process of operation was uneventful any undue prolongation and without having used any other instrumentation. the weight of the female baby was . kg at birth. on the rd postnatal day, her mother noticed a left axillary swelling, then admitted to a tertiary children's hospital. her physical examination revealed, bilateral axillary asymmetry with a fluctuant, nontender swellings. there was no redness or discoloration of the skin. there was no clinical feature suggestive of trauma or bleeding diathesis. a superficial ultrasonography showed solid heterogeneous, hyperechogenic masses x mm in the left axillary region and x mm in the right side. doppler study did not reveal any flow in the masses. contrast enhanced mr imaging demonstrated, bilateral axillary mass lesions with fluid levels and smooth contours (figure and ) . t w images demonstrated hyperintense component suggesting hemorrhage. after the administration of the gadolinium-based contrast material, lesions showed peripheral enhancement (figure ) . a diagnosis of hematoma was entertained. the child was managed non-operatively. she was monitored clinically and radiologically. follow-up ultrasounds scan revealed significant regression of the swellings. unique teaching points: soft tissue hematomas can be rarely seen in newborns. the formation of axillary hematoma on the background of c/s is a rare complication, which, to the best of our knowledge, has not been previously reported. ultrasonography and mr imaging readily depicts hematoma and aids in the differential diagnosis. colorectal carcinoma (crc) is extremely rare in pediatric age, with an estimated annual incidence of approximately case per million individuals. the majority of reported cases occur in adolescence, while the incidence is further lower for children under years. the distribution between males and females is not equal, with higher prevalence in males (ratio of : ). the etiology in children is unclear as these tumors are often sporadic and not linked to a preexisting adenomatous polyp, unlike adults. predisposing factors such as familial polyposis of the colon, other polyposis syndromes, ulcerative colitis and familial multiple cancer syndromes were reported in % of cases. advanced stage at diagnosis, aggressive histologic subtypes (poorly differentiated, signet ring and mucinous adenocarcinoma) and poor survival are the hallmarks of pediatric crc. case presentation: a -year-old male presented with a history of dyspeptic symptoms (recurrent epigastric-right flank colic pain and heartburn) for the last eight months, without evidence of irregular bowel function. after a prior diagnosis of esophagitis secondary to a gastroesophageal reflux disease, physical and laboratory examinations revealed anorexia, progressive body weight loss, microcytic iron deficiency anemia and positive fecal occult blood test. during an emergency access, abdominal ultrasound identified rounded target liver lesions and circumferential heterogeneous mural thickening of the ascending colon. contrast-enhanced computed tomography scan (cect) demonstrated a marked circumferential wall thickening of the ascending colon and cecum with a longitudinal extension of mm and thickness of mm; the mass contained lowdensity areas and calcifications. furthermore hypovascular hepatic lesions along with lymph node metastases containing calcifications were identified. no lung metastases were found. histopathological analysis confirmed the diagnosis of metastatic colon adenocarcinoma. after chemo-and radio-therapy, only the hepatic lesions showed reduction in size and number. the patient subsequently underwent right hemicolectomy. one month after surgery he is in a rigorous follow-up through ultrasonographic evaluation of pleural effusion and ascites and cect. unique teaching points: crc, although rare, should be suspected in children presenting with unexplained persistent abdominal pain, progressive body weight loss and positive fecal occult blood test. ultrasound imaging can be appropriate in the preliminary detection of abnormal bowel wall thickening, lymph node and liver metastases; cect is mandatory to confirm the radiological diagnosis and complete the staging. to increase awareness of this rare syndrome and its varied presentation in order to facilitate its early diagnosis and treatment to prevent poor prognostic outcomes. case presentation: lemierre syndrome is a rare disease characterized by an initial infection of the head and neck leading to the development of a septic thrombophlebitis which has a propensity to spread and involve the jugular and facial veins. this progressive infection then leads to the development of metastatic septic emboli to the respiratory tract. we present the case of a year old boy who attended with a week history of fever and a cough. initial imaging on admission demonstrated a large left sided hydropneumothorax with multiple cavitating lesions throughout the lung parenchyma in addition to thrombosis of some of the segmental pulmonary veins. the hydropneumothorax was surgically drained and the patient was transferred to the paediatric intensive care unit after further deterioration with the development of a broncho-pleural fistula. following a short course of antibiotics there was no clinical or radiological improvement and sputum cultures grew coliform organisms which raised suspicion for a more distant source. when pus was noted to be discharging from the left ear, a contrast enhanced ct of the head and neck revealed a left mastoiditis with multiple cerebral abscesses and occlusive thrombi in the left jugular vein, transverse venous sinus, sagittal and straight sinuses. following this diagnosis antibiotic therapy was modified and targeted at anaerobes, which was vital in assisting the patients recovery and successful discharge home. unique teaching points: clasically the majority of lemierres syndrome begins in the oropharynxinvolving the palantine tonsils and peritonsillar tissue often presenting with fever, sore throat and neck pain. our case demonstrates an atypical presentation with sepsis and respiratory symptoms as a result of the septic emboli which delayed diagnosis. we have learnt from this case the importance of considering lemierres syndrome in patients presenting with signs of a respiratory infectionin particular cavitating pulmonary lesions-that have not improved with conventional therapy and to have a low threshold to investigate the head and neck as a potential source of infection. when the working hypothesis of meningitis could not help e. kovacs , n. pinter , g. balázs , a. machovitsch , a. arany , z. liptai , l. fonyad , p. benke ; budapest/hu, amherst/us objective: neuroinfection still represents a diagnostic challenge in the everyday practice, where clinical evaluation, imaging, laboratory and pathological workup and treatment goes hand in hand under the pressure of time. we summarized a case in which, despite the extensive multilateral collaboration the battle was lost, to bring attention to the possible causes. a two year old, previously healthy female was taken to the emergency department for altered state of consciousness and fever. she also suffered from gingivitis. the unconscious child underwent an emergency ct scan: hydrocephalus with signs of raised intraventricular pressure was detected. subsequently mri of the head and spine was performed, and showed signs of diffuse leptomeningeal enhancement with basal predominance. multiple dwi restricted parenchymal lesions with basal predominance were also found. repeated csf and blood tests did not reveal any causative organism, although the gradually increasing crp suggested infection. two weeks after the onset of symptoms a follow up mri study showed extensive cerebral and spinal swelling with no focal lesion. the child passed away three days later due to cardiac failure. autopsy and neuropathological evaluation could not reveal the cause of the disease, which was identified only weeks after the child died, by culturing sputum and csf. unique teaching points: an overview of the clinical and radiological presentation of meningitis basilaris is carried out. attention is given to the circumstances, when tuberculotic infection should be suspected, and antituberculotic treatment should be started, even before the confirmation of the presence of mycobacteria can be obtained. to describe the clinical, laboratory and mri findings of chronic nonbacterial osteomyelitis(cno) in a patient with a negative radiograph and emphasize useful imaging findings, including an unusual radial pattern of edema in both femoral heads. case presentation: a -year-old adolescent, was referred with progressive debilitating hip pain and inability to walk since days, that was unsuccessfully treated with non-steroidal anti-inflammatory drugs. during hospitalization he developed fever up to ο with normal full blood count and smear, elevated esr ( mm/h) and crp ( . mg/dl), positive serologic markers for streptococcus (asto) and ebv and received antibiotics with relative good response. blood cultures did not grow any pathogens, the rest of serology was negative for acute infection, tuberculin skin test was negative and immunological investigation was unremarkable. pelvic radiographs were negative. mri showed a symmetric pattern of bone marrow involvement around both triradiate cartillages, at both femoral heads and ( ) (suppl ):s -s pediatr radiol major trochanters. complementary evaluation of tibial areas with a limited protocol disclosed asymptomatic involvement of tibial epiphyses and apophyses. a radial pattern of edema was seen at the femoral heads with alternating stripes of involved and uninvolved areas. clinical course and imaging appearances were highly suggestive of cno. rapid clinical improvement occurred during hospitalization while a repeat mri months later showed complete resolution of hip findings and the patient was free of any symptoms or signs. coronal stir sequence at presentation showing the radial pattern of bone marrow edema (arrowheads) alternating with stripes of normal marrow (*) at both femoral epiphyses. note hyperinense edema (arrows) around triradial cartillages. coronal stir sequence showing the predilection of bone marrow edema symmetrically around triradial cartillages (arrows) and at major and minor trochanters (arrowheads). coronal stir sequence at -months follow-up shows resolution of edema. unique teaching points: cno is a not well known chronic autoinflammatory bone disorder affecting primarily children and adolescents. positive serology for streptococcus or other infectious agents has been previously reported as in our case and may be a triggering factor. striking mri findings with a negative radiograph may occur at initial stages. symmetrical distribution of non-specific bone marrow edema around epiphyses and apophyses is highly suggestive of the diagnosis in the appropriate clinical setting and following exclusion of suppurative bone infections as well as bone or hematologic malignancies. the radial pattern of edema in our patient is unusual and considered to comply with the direction of main trabecular systems in femoral heads. in / chest cts, nodules (median size . mm) were detected. display mode a with mm mip yielded the best interreader variability (κ= . ) and the highest sensitivity ( . %) compared to mode b and c (κ= . , sensitivity . % and κ= . , sensitivity . %, respectively). perifissural nodules were detected in all subgroups. conclusion: mip improves the detection of pulmonary nodules in chest cts of young children, but overall interreader agreement is only fair. nodules, including perifissural nodules, occur in children with and without malignancy. images were subsequently read and interpreted by board-certified radiologists and nuclear medicine physicians in communal reading. in case of identifying suspicious lesions in cect additional imaging (mri) or biopsy was performed. compared to pet/ct employing only low dose ct (ldct), the use of cect resulted in the identification of additional suspicious lesions in patients. furthermore the use of cect allowed us to qualify lesions as benign/ physiologic which in pet/ldct were identified as suspicious and lesions suspect for metastases or tumor. in those patients who received combined integrated fdg pet/ct including both ldct and cect the ctdi ranged in between , - . mgy (n= . mgy) and the dose length product (dlp) ranged in between . - mgy *cm (n= . mgy *cm) specificity was significantly higher combining pet and ct compared to stand-alone ct and pet. our study showed that the acquisition of cect in combined integrated pet/ct leads to an increased specificity and thus represents an essential component of a good fdg pet/ct in pediatric oncology. in assessment of lymph nodes, inflammatory foci and liver lesions diagnostic contrast enhanced ct is essential. comparison of the detectability of ubos in neurofibromatosis type i patients with proton density-weighted and flair sequences in t mri l. porto, s. lescher, n. hillenbrand; frankfurt/de objective: neurofibromatosis type (nf ) is an autosomal-dominant congenital disease. in nf patients, significant numbers of so-called unidentified bright objects (ubos) can be found in brain imaging, with predilection sites at the basal ganglia and the dentate nucleus. ubos seem to develop at a very early age, contrary to other criteria leading to diagnosis. the detection of ubos might therefore prove helpful in the early diagnosis of nf , complementing the clinical diagnosis based on criteria of the "national institutes of health consensus development conference". the aim of the study was to investigate whether the detectability of ubos increases at t by comparing proton density-weighted images (pdw) with fluid-attenuated inversion recovery (flair) sequences. a total of nf patients ( male, female, between and years old, mean age . years) were examined by a t magnetic resonance scanner. the presence of ubos was evaluated on pd-w and flair images by evaluators ( experienced neuroradiologists, junior radiologist and student in his final year). detectability was rated by a three-point scoring system for dedicated regions: lesions which were "well defined/detectable", "suspicious" or "detected after a second look". the wilcoxon signed-rank test was used for comparisons between the raters. the level of significance was p< . . significantly more lesions were marked as "well defined/detectable" in the pd-w sequence compared to flair (p< , for all four evaluators together, as well as for each evaluator separately). in particular, pd-w proved to be superior for detecting ubos located in the medulla oblongata (p= , ) dentate nucleus (p= , ) and hippocampal region (p= , ), regardless of the level of the raters' experience. this is the first study that compares flair and pd-w at t for the diagnosis of ubos in nf . significantly more ubos are detected in the pd-w compared to flair sequences, especially for the infratentorial regions. as ubos occur at very early stages of the disease in patients with suspected nf , pd-w might aid an early diagnosis in these patients. assessment of radiation doses from diagnostic imaging in the followup of paediatric oncology patients p. logan , r. harbron , k. mchugh ; london/uk, newcastle-upon-tyne/uk objective: previous literature ( , ) has suggested paediatric oncology patients accumulate a large radiation burden as a consequence of routine diagnostic imaging examinations during therapy. we retrospectively looked at the effective doses from routine ct and nuclear medicine in three cohorts of children, namely patients with hepatoblastoma, wilms' tumours and rhabdomyosarcoma (rms). of note, in our centre we rely on repeated mris of the primary site for many tumours. effective doses (e), in millisieverts (msv), were estimated using the ncict dose estimation tool (lee et al ) , based on details specific to each procedure: patient age, scan region, scanner type and ct dose index (ctdi -an indicator of radiation exposure recorded at the time of each scan). doses for general radiography were estimated using pcxmc v . monte carlo simulations, assuming standard exposure factors and field size. there were patients in total ( hepatoblastoma, wilms', rms). there were boys. the mean age was years months (ranging from days - years months). the mean and median cumulative effective doses from ct for the whole cohort were . msv and . msv respectively. four patients in the wilms' cohort had a dmsa nuclear scintigram ( . - . msv), no hepatoblastoma patient had any nuclear medicine imaging, and patients with rms received a bone scan ( - . msv) or a pet scan (approximately msv). cumulative radiation doses from routine radiological investigations in paediatric oncology can be kept in a much lower range than reported in the literature ( , ). in our institution, the followup of solid intra-abdominal tumours with mri, with additional ct or nuclear medicine only when clinically justified, has resulted in a significantly low radiation exposure in these patient cohorts. mri of the primary tumour site should be implemented as a replacement for ct imaging when there is no significant detriment to the diagnostic information obtained. ( ) (suppl ):s -s pediatr radiol mri-based evaluation of multiorgan iron overload is a predictor of adverse outcomes in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation f. zennaro , d. zanon , r. simeone , g. boz , f. degrassi , m. gregori , g. schillani , c. boyer , n. maximova ; nice/fr, trieste/it objective: iron overload is associated with poor clinical outcomes in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). although the effects of hepatic and cardiac siderosis on patient outcomes have been extensively studied, less is known about the effects of siderosis in other organs. the medical records of consecutive pediatric patients who underwent allogeneic hsct in our institute from to were retrospectively reviewed. mri was used to measure iron concentrations in the liver, spleen, pancreas and bone. these patients were divided into two groups, with non-elevated (< μmol/g; group ) and with elevated (> μmol/g; group ) liver iron concentration (lic) at baseline. in group , only two patients had normal iron concentrations in all organs. none of the patients of group presented with pathological iron concentrations in only two organs. comparisons of baseline data with results of the first follow-up mri performed - months after hsct, showed a general worsening of iron accumulation. in group , none of the patients showed complete absence of iron overload in a single organ. in group , none of the patients showed a total absence of siderosis involving fewer than three organs. this study confirms the correlations between iron overload and the risks of transplant-related complications, such as transplant related mortality, sinusoidal obstruction syndrome, infections, pancreatic insufficiency, and metabolic syndrome, in transplant recipients with systemic siderosis. another important finding of this study was the close correlations between pre-transplant bic and times to neutrophil and platelet engraftment (p< . each). ( ), ganglioneuromas (gn, ) and ganglioneuroblastoma (gnb, ), examined by t mri were retrospectively grouped according to tumor entity, risk factors (bone marrow metastasis, mycn amplification or p deletion) and therapeutic regime (observation versus chemotherapy). dw (b values , and ) and conventional mri images (t , t pre and post contrast) were analyzed for tumor size, relative si-and absolute adc-values at baseline (base; no therapy), and after (fu ) and (fu ) months. adc values in nb were lower than in gnb and gn ( . * - mm /s versus . * - mm /s; p< . ). there was a tendency towards lower adc values in tumors with risk factors (n= ) versus no risk factors (n= ) at baseline, which did not reach statistical significance (p= . ). during follow-up shrinkage of tumor volume was noted (baseline ml, fu ml, fu ml; p< . baseline vs. fu ; p= . baseline vs. fu ). in the observation group, tumor adc values rose without relapse ( . * - to . * - mm /s). only in eventually relapsing tumors adc values tended to decrease further ( . * - to . * - mm /s, p= . ), despite initial reduction in tumor size. to establish inter and intra-observer variability in the radiological detection and assessment of pulmonary nodules at diagnosis in children with wilms tumours. a test set of ct thoraxes at diagnosis from patients enrolled in the multicentre 'improving population outcomes of renal tumours of childhood' (import) study in the uk were assessed. five radiologists ( chest, paediatric) from different centres ( uk, netherlands) completed a scoring sheet for nodule assessment on the same studies on two occasions, months apart. the readers were blinded to patient respiratory symptoms, the original radiology reports and also that they were scoring identical cases. descriptive statistics, modified bland altman graph and fleiss kappa scores were used for statistical assessment. in total, different pulmonary nodules across the ct thoraces at both rounds were scored by at least one reader. ( %) were seen by at least one reader in round and ( %) in round , ( . %) nodules were seen by at least one reader in both rounds. only ( %) nodules were scored by all readers in round , ( %) by all readers in round , and ( %) nodules by all readers in both rounds. of the nodules seen in the first round, were measured to be > mm in at least one dimension and of these, were classified as malignant by all readers. the limits of agreement for mean difference in nodule size in anterior-posterior, transverse and longitudinal measurements were ± . mm, ± . mm and ± . mm respectively. the fleiss kappa scores ranked from poor to fair agreement for nodule border smoothness ( . ), nodule shape ( . ), solidity ( . ) and impression of malignancy ( . ). within the same readers for both rounds, nodule detection rates of agreement were between . - . %. the average intra-reader percentage of observed agreements for nodule border smoothness, shape, solidity and impression of malignancy were . %, . %, . % and . % respectively. conclusion: detection and characterisation of pulmonary nodules on ct thorax shows both intra-and inter-observer variability. this has important implications for the interpretation of metastatic disease at presentation. fever without a focus is defined as febrile illness without an initial obvious cause or localizing signs. our aim is to assess the diagnostic value of whole-body mri (wb-mri) in the diagnostic work-up of children with fever without a focus. we retrospectively searched for subjects who underwent wb-mri for fever without a focus. a total of children (m= , f= ), mean age . years (range: . - . ) were included. / ( . %) subjects were immunosuppressed and / ( . %) subjects were hospitalized at onset of fever. the reference standard was based on positive cultures, biopsy or surgery. when this was not possible, a probable diagnosis was made based on clinical follow-up or serology. initially, the wb-mri images were reviewed independently by pediatric radiologists blinded to all clinical information. at the end of each case the final diagnosis and the diagnostic category ( categories: a. normal, b. infection, c. oncologic, d. rheumatologic, e. miscellaneous) was recorded. this was followed by a consensus read for comparison with the reference standard. for statistical analyses all subjects were treated as fever without a focus. results: reference standard: the diagnostic category of the reference standard was as follows: infectious / ( . %), oncologic / ( . %), rheumatologic / ( . %), miscellaneous. / ( . %). even after extensive work-up in / ( . %) no clear cause for the fever was found table . wb-mri: wb-mri diagnosed the cause of fever without a focus in / subjects ( . %) ( table ). in subjects ( . %) wb-mri results were falsely positive ( jia and myositis), and in the remaining subjects no imaging findings compatible a cause of febrile disease were found. interobserver agreement was fair (kappa . ). in children with fever without a focus wb-mri provided the diagnosis in in almost a quarter of the cases. given the multiplicity of causes of fever without a focus, some of them not possible to visualize on mr imaging, wb-mri may be considered in routine imaging practice when evaluating pediatric patients with fever without a focus. to compare linear measurement/volume to direct volumetric measurements using dimensional( d) post-processing software. for this irb approved study initial diagnostic ct or mr exams in patients( mo- yr) with solid tumors were reviewed by radiologists and technologists. radiologists recorded measurements in axes in their routine method, described tumor shape (sphere, ellipse, cone) and surface texture (smooth, almost smooth, or mildly, moderately, markedly irregular). three technologists individually, and radiologists by consensus, used d processing software (intellispace portal, philips, cleveland, oh) to directly measure tumor volume. tumor volume (v) was calculated from linear measuments using the following equations: sphere v= / πr , ellipsoid v= πr or πr , conicalv= πr or πr , and cuboid v=(xyz). inter-reader variability in tumor measurement in all tumors and for tumors divided by surface characteristics was assessed amongst radiologists and technologists, and radiologist consensus using coefficient of variation (cov). tumor shape analysis was reported as sphere, ellipse, cone, and surface texture smooth, almost smooth, mildly irregular, moderately irregular, markedly irregular. inter-reader variability of as much as , cc above to cc below the mean tumor volume was found when using radiologist determined linear measurements, with standard deviation (sd), range . - . inter-reader variability amongst technologist derived volumes was considerably less, range cc above to cc below the mean, with sd, range . - . cov analysis shows a greater degree of variation in tumor volume calculated from linear measurements [smooth( %), almost smooth( %), mildly( %), moderately( %), markedly( %) irregular] than direct volume determination [smooth( %), almost smooth( %), mildly( %), moderately( %), markedly( %) irregular]. variation was significant only for tumor with irregular surface texture [smooth (p= . ), almost smooth (p= . ), mildly (p= . ), moderately (p= . ), or markedly (p= . ) irregular]. variation in linear/volume measurements in very irregular tumors. light blue=middle % tumor volume measurements by pediatric radiologists. whiskers mark limits of range. ▲♦ • markers =measurements by technologists. note broad degree of variation. ( ) (suppl ):s -s pediatr radiol variation in linear/volume measurements in almost smooth tumors. light blue=middle % tumor volume measurements by pediatric radiologists. whiskers mark limits of range. ▲♦ • markers =measurements by technologists. note narrow degree of variation. both graphs show the same informationthe % relative variation in tumor volume measurements determined by dimensional linear measurements ( pediatric radiologists) v. volumetric processing (technologists & consensus group). radiologist generated measurements are subjective and unreliable. variation in measurement technique leads to differences in calculated tumor volume which significantly over or under estimate volume in tumors with irregular texture and is not significant in smooth tumors. quail-quantitative mri-based evaluation of pancreatic iron overload in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation f. zennaro , m. gregori , f. degrassi , e. cattaruzzi , y. diascorn , c. boyer , n. maximova ; trieste/it, muggia/it, nice/fr objective: iron overload (io) is a relatively common but often neglected transplantrelated complication and has been associated with poor prognosis in patients undergoing allogeneic hsct for hemato-oncological disease. pancreatic io is frequent among patients with transfusion-dependent anemias, but is uncommon among patients with hematologic malignancies. the causes of pancreatic io and the potential effects of pancreas iron deposits on transplant outcomes or on the risk of developing significant late effects in long-term hsct survivors have not been yet determined. our institute routinely uses magnetic resonance imaging (mri) with various gradient-recalled-echo (gre) sequences to quantitatively measure the iron concentration in abdominal parenchymal organs in all pediatric patients before and after allogeneic hsct. this study retrospectively analyzes the correlations of pancreas io with the type of conditioning regimen and pretransplant liver iron concentration (lic) in pediatric patients who underwent allogeneic hsct in our transplant unit over the last years. we enrolled patients, age - years. pre-transplant mean lic was , μmol/g (normal values μmol/g). ( %) patients had mild liver io and ( %) had moderate or severe io. pretransplant mean pancreatic iron concentration (pic) was , μmol/g, whose only ( %) had mild pancreatic io and none had severe io. post-transplant mean lic was , μmol/g, only one patient had mild liver io but patients ( %) had moderate or severe io. post-transplant mean pic was , μmol/g, ( %) patients had moderate or severe io. mean pre-transplant pancreatic volume was , cm , while mean post-transplant pancreatic volume (evaluated days after transplantation) was , cm . ( , %, p< , ) patients with post-transplant moderate or severe pancreatic io underwent tbi-based conditioning. mean reduction of pancreas volume in tbi group was , cm (p< , ). no pancreatic volume reduction was observed in chemotherapy-based group. all patients with pancreatic io have had exocrine pancreatic insufficiency and ( , %) patients have had metabolic syndrome. volume reduction well correlate (mean , %, p< , ) with pancreatic io. this study confirms that pancreatic iron overload is not so rare in patients with hematologic malignancy underwent allogeneic hsct, with increased risk of metabolic syndrome and total deficit of exocrine pancreatic activity, but not of endocrine activity. iron overload monitoring allows for chelation therapy optimization. mr is fast, reproducible and more reliable compared to serum ferritin and transfusional history and allows a multi organ evaluation. pulmonary tb is common in south africa, with many children affected. diagnosis can be challenging and chest x-ray remains fundamental for diagnosis. interpretation is difficult and shown to have wide inter-reader variability. no study however has compared cxr findings and interreader agreement between ambulatory and hospitalised patients. this study compares the frequency of cxr changes, as well as interreader agreement in ambulatory compared to hospitalised children with suspected tb. from nolungile clinic and red cross children's hospital respectively was done. each sample contained % proven tb and % negative controls. two paediatric radiologists and one paediatrician served as blinded, independent readers for the database using standardised ticksheets. our study demonstrated no significant difference in lymphadenopathy, but an increase in parenchymal change in the hospitalised group. we otherwise showed similar results to literature regarding finding frequency, but poor inter-observer agreement. if the least expert reader were removed, results were comparable with available literature. this highlights the need for development and study of explicit cxr criteria for lymphadenopathy to improve the value of cxr for paediatric tb in all settings. lung ultrasound in pediatric pneumonia -why is it necessary to use the additional trans-abdominal approach? j. lovrenski; novi sad/rs objective: to emphasize the need of lung ultrasound (lus) technique modification, which enables detection of pneumonia in children not visualized by using solely the standard trans-thoracic approach. a prospective study was carried out in the regional children's hospital, and comprised a -year period. the inclusion criterion was us finding of pneumonia detected by trans-abdominal, and not with trans-thoracic approach. lus examinations were performed using a combined, trans-abdominal and trans-thoracic approach. longitudinal, transversal (intercostal), and oblique sections were used. trans-abdominal examination included transhepatic and trans-splenic approach. the ultrasound probe was angulated from the most anterior to the most posterior sections while examining the lung bases by trans-abdominal approach. a pneumonia-positive lus finding included subpleural consolidation with air-bronchogram, or with an adjacent area of interstitial/ alveolar-interstitial edema. lus was always performed before the other diagnostic modalities (chest x-ray (cxr) and computed tomography (ct)), if they were indicated by pediatrician or radiologist. within a -year period in children (mean age . y, sd . y) the pneumonic focus was discovered using the trans-abdominal approach, while the trans-thoracic approach showed a normal lus pattern. all the children had the clinical symptoms of pneumonia (fever and cough, with or without dyspnea/tachypnea). the auscultatory finding was positive in children. cxr was performed in three children, showed a right-sided pneumonia in two, and was negative in one patient. one child had a contrastenhanced chest ct, which confirmed a left-sided pulmonary base abscess detected during lus examination by trans-splenic approach only (figures , ) . apart from pulmonary symptoms, there has not been any other associated diseases found, apart from otitis media in two children. each child responded to the antibiotics treatment with resolution of infection and us signs of pneumonia. in this oral presentation we will explain and give anatomical and technical reasons for pneumonia-positive us findings within lung bases, that remained undetected by the trans-thoracic approach. left-sided abscess abutted on the spleen (s), and was detected by trans-splenic us approach. it did not contact the pleural surface approachable by trans-thoracic ultrasound (black semi-lunar mark). l-liver. conclusion: trans-abdominal (trans-hepatic and trans-splenic) approach should become an inseparable part of each lus examination, along with a standard trans-thoracic approach. this modification of technique is expected to result in a further increase of lus sensitivity in diagnosing pneumonia. is thoracic ultrasound really competitive to computed tomography in children -a two-year retrospective study j. lovrenski, k. antolović; novi sad/rs to compare diagnostic accuracy of thoracic ultrasound (us) and computed tomography (ct) in children. a retrospective study was conducted in the regional children's hospital, and comprised a -year period. the inclusion criteria were: chest ct performed within h after the us examination of thorax, and us and ct examinations in the same patient performed by different pediatric radiologists. all us examinations were performed using a combined transabdominal-transthoracic approach. ct examinations were done ( ) (suppl ):s -s pediatr radiol according to the body mass based pediatric ct protocols. each hemithorax was analyzed separately in terms of comparison between us and ct findings. statistical analysis included the calculation of sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of ultrasound in diagnosis of pulmonary pathological entities. out of children with chest ct, of them (mean age , y, sd , y) fulfilled the criteria to enter the study group. lung us showed sensitivity, specificity, ppv and npv in diagnosis of pleural effusion: %, , %, %, %; lung consolidation: %, %, %, %; lung abscess: %, %, %, %; and interstitial lung disease: %, %, %, %, respectively. within hemithoraces multiseptation of pleural effusion was observed by us only. air bronchogram within lung consolidation was observed in hemithoraces both by us and ct examinations. necrotic areas within pulmonary consolidations were detected by us in hemithoraces, which was later confirmed by ct examination. lung abscesses were diagnosed in hemithoraces by both us and ct. two small lung abscesses filled with air ( hemithorax) and bronchiectasis ( hemithoraces) were detected only by ct examinations. other pathological findings detected both by us and ct examinations were: congenital pulmonary airway malformation (cpam) ( hemithorax), pulmonary sequestration ( hemithorax), partial pneumothorax ( hemithoraces), hidropneumotorax ( hemithorax), inflamed pneumatocele ( hemithorax), hydatid cyst ( hemithorax), pericardial effusion ( patients), soft tissue masses of thoracic wall with initial bone destruction ( patients), and lymphomas ( patients) (figures - ) . in one patient us and ct revealed cysts and an extremely dilated bronchus within lung consolidation (pathohistological finding: cpam type combined with subsegmental bronchial atresia, and extensive bronchopneumonia). us examination, unlike ct, could not differentiate between eventration of the left hemidiaphragm and diaphragmatic hernia in one patient. to determine and compare the accuracy of frontal cxrs alone and 'combination frontal-lateral' set of cxrs for diagnosing lymphadenopathy in children with tb using patients with confirmed tb and controls without tb, and to compare findings in hiv-infected and hiv-uninfected children. a total of children (ie: children with gene xpert confirmed tb and control patients admitted with lower respiratory tract infections), which were part of a larger south african study, who had both frontal and lateral cxrs, were included. three qualified radiologists read the cxrs in separate sittings one month apart (one for the frontal x-ray alone and one for the 'combination frontal-lateral' cxrs) for the presence of lymphadenopathy. odds ratios and % confidence intervals were calculated to determine the presence of lymphadenopathy using a consensus reading on the frontal cxr and frontal-lateral cxr combination according to the final diagnosis of tb. inter reader agreement was determined using the kappa statistic. lymphadenopathy was reported in ( %) patients on the frontal cxr alone and in ( %) patients on the frontal-lateral cxr combination. ( %) of the patients with lymphadenopathy on the frontal cxr alone were gene xpert positive versus ( %) of the patients with lymphadenopathy on the frontal-lateral cxr combination. in all patients, the consensus reading using a frontal-lateral cxr combination resulted in a -fold increase (or , ; % ci , ) in calling lymphadenopathy compared to using a frontal cxr only in the gene xpert positive group, the consensus reading using a frontal and lateral cxr combination resulted in a fold increase (or , ; % ci , - , ) in calling lymphadenopathy compared to a frontal cxr only. overall inter reader agreement for all readers when evaluating for lymphadenopathy was 'fair' on both the frontal cxr (k= , ) and the frontal-lateral cxr combination (k= , ). the addition of a lateral view to the standard frontal cxr increased the rate of calling lymphadenopathy. however, the accuracy of diagnosing lymphadenopathy on chest x-ray as a marker for tb was poor. this poor accuracy was further hampered by only 'fair' inter reader agreement for the presence of lymphadenopathy on chest x-ray. dynamic d ct imaging in children has significant advantages over routine ct scanning, bronchography and bronchoscopy for diagnosing trachebronchomalacia because it can be performed during free breathing without anaesthesia or invasive airwayaccess.itcanalsodemonstratevascularcausesoftracheo-bronchomalaciain the same sitting. the technique is currently performed in paediatric center in the uk. we aimed to report pitfalls encountered while setting up a dynamic d ct imaging service for children and report the findings of studies performed. materials: dynamic d ctscanning was introduced after installation of a large array ( slice) ct scanner, applications specialist training and review of the literature. imaging parameters in use by greenberg and colleagues (arkansas children's hospital, usa) were applied. referral indications, pitfalls encountered, quality of scanning and imaging findings/diagnoses were reviewed and enumerated. results: nineteen paediatric dynamic d ct scans ( females, males; days - years months; mean months) were performed over months. the first studies were performed without ivi contrast due to lack of experience and subsequent studies were performed with contrast ( figure major pitfalls included initial failure to perform contrasted studies for simultaneous evaluation of vessels, initial failure to withdraw the endotracheal tube, patient motion under care of nurses and clinicians, failure to appreciate the value of imaging the full lung volume while trying to keep dose length product to a minimum and failure to appreciate that collapse of the airway is often in the ap plane and not appreciated on coronal slab projections -rotating d volume rendered images is a requirement ( figure ). additional obstacles were initial clinician and radiologist lack of support after early failures and colleague concerns regarding the radiation dose. objective: diagnosis of pulmonary tuberculosis (ptb) in children relies on chest radiography, however there is wide inter-observer agreement in detecting lymphadenopathy, the hallmark of ptb. paediatric airways are pliable, thus detection of airway compression may be a more objective criterion for the presence of lymphadenopathy. thus the objctive was to assess the usefulness of airway compression on chest radiographs for diagnosis of ptb in children. chest radiographs of children admitted to red cross children's hospital with suspected ptb were read by two readers according to a standardised format and a rd when there was disagreement. radiographs of children with definite ptb were compared to those with lower respiratory tract infection (lrti) from another cause. the prevalence and location of airway compression was evaluated. findings were correlated with hiv status and age. inter-observer agreement was assessed using kappa statistic. . % in older children (or . ; %ci . - . ). no association with airway compression and hiv infection was found. inter-observer agreement ranged from . - . . eighteen-month-old male patient diagnosed with ptb; hiv negative. majority agreement of airway compression at lmb indicative of lymphadenopathy. left upper lobe opacity is in keeping with a ghon focus. there is a strong association between airway compression on chest radiographs and confirmed ptb, particularly in infants, irrespective of hiv status. however, clinical use is limited by poor inter-observer agreement. paediatric ultrasound-guided biopsies in a tertiary oncology centre: five years experience n. parvizi, m. smedley, s. chakraborty; oxford/uk objective: histological diagnosis is almost always essential to guide appropriate therapy for children diagnosed with cancer. tissue can either be obtained by surgical/open or image-guided percutaneous biopsy. the aim of this study is to assess the safety and diagnostic accuracy of ultrasound-guided biopsies in a tertiary oncology referral centre. a retrospective analysis of clinical data, imaging findings and histological diagnosis of patients aged to years between january and december was carried out. a total of ultrasound-guided biopsies were performed in our institution on patients. most of the biopsies were performed in theatre with the patient under general anesthetic and with an -gauge spring-loaded core biopsy needle with a minimum of two cores per patient. in % of lesions the needle biopsy was diagnostic. the single nondiagnostic case did not have sufficient material to make a full diagnosis and a surgical biopsy was required. eighty-two of the biopsied lesions were malignant and were benign. in no cases was a repeat biopsy required. the vast majority of the biopsies were performed within one week of request with over half performed within days. all biopsies were performed without complication and in the majority of cases the patients were discharged the same day or following an overnight stay. ultrasound-guided percutaneous biopsy is an accurate and safe technique in order to acquire tissue from suspected malignant lesions in children. these can be performed instead of or in addition to open biopsy and will often result in a shorter hospital admission and recovery time. the role of imaging in the diagnosis of thymoma in paediatric patients with myasthenia gravis j. adu, t. a. watson; london/uk thymomas are exceedingly rare tumours in the paediatric age group, with only very few cases having been reported in the literature. thymomas are commonly associated with myasthenia gravis (mg), with thymectomy being potentially curative. ct is the mainstay imaging modality for thymoma diagnosis in the adult population. while, chemical shift mr imaging can be helpful to distinguish thymoma from other anterior mediastinal abnormalities. currently, there is no consensus on the imaging pathway for children with mg with suspected thymoma. our aim is to review the imaging of patients who were referred to our institution for management of mg, and suggest an imaging pathway in cases where thymoma is suspected. we performed a retrospective search of the local pacs system of cases between and using the search terms "thymoma" and "myasthenia gravis" in the clinical indication for the study and the body of the final report. forty-three cases were identified using the search criteria. eight cases were excluded owing to an absence of cardiothoracic imaging. / of all cases ( %) had chest x-rays (cxr's), of these / ( %) were normal. the three remaining patients who had abnormal cxr's went on to have ct scans, which confirmed an anterior mediastinal mass (amm) in all three cases. / of all cases ( %) had cross-sectional imaging (mri / cases, ct / cases). of those, / of cases ( %) had normal studies. specifically, all mri studies ( % of cases) were normal, while only / ct scans ( %) demonstrated an anterior mediastinal abnormality. / of all cases ( %) had both cxr and cross sectional studies. / of these cases ( %) had a normal ct or mri. in the remaining three cases, the amm was clearly demonstrated on both cxr and the crosssectional imaging. in our series, radiography, ct and mri studies were normal in the vast majority of cases. however, given that thymectomy is potentially s ( ) (suppl ):s -s pediatr radiol curative, it is appreciated that clinicians may still be keen to radiologically investigate paediatric patients with myasthenia gravis. cxr is not an efficacious imaging modality in this context, as patients with a normal cxr may be falsely negative, and patients with an abnormal cxr may undergo cross-sectional imaging regardless. we propose that mri should be used as first line investigation for patients in this population. this approach will negate the need for ionizing radiation, maximize diagnostic yield, and facilitate surgical planning if deemed clinically appropriate. increased risk of venous thrombosis of the arm with multiple peripherally inserted central catheters insertion in paediatric patients r. gnannt , n. waespe , j. donnellan , k. liu , l. brandao , b. connolly ; zurich/ch, toronto/ca objective: peripherally inserted central catheters (piccs) are associated with superficial and deep venous thrombosis of the arm. the impact on the incidence of developing a thrombosis of the arm when inserting a subsequent picc remains unclear. the purpose of this study was to analyze the incidence of deep, upper limb thrombosis of repeated upper limb piccs in children. the study population included all patients who underwent their first successful arm picc insertion between january and december . subsequent ipsilateral arm piccs were included in the analysis. patients were followed until march or until any alternative central venous line insertion (jugular, femoral, saphenous or umbilical vein lines -because of their thrombogenic effect). for each picc insertion the following data were collected: date of insertion and removal, weight of the patient, type of picc ( . fr, . fr, fr, fr, fr), left or right arm, and vein cannulated (basilic, brachial, cephalic). all symptomatic deep and superficial thrombosis of the arm were correlated with the picc database. four thousand one hundred thirty-eight piccs were inserted. applying inclusion and exclusion criteria, piccs remained for analysis. first, nd , rd , and th picc insertions in the same arm were identified in , , and patients, respectively. in total there were upper body deep symptomatic thrombotic events diagnosed with ultrasound. a st , nd , rd , and th picc insertion was associated with / (incidence . %), / ( . %), / ( . %), and / ( . %) thrombotic events, respectively. an increasing hazard ratio was seen with higher numbers of picc insertions, which was significant when comparing the st with the rd picc insertion in the same arm (hr . , ci % . - . , p= . ). after excluding any confounder, double lumen piccs were associated with a significantly higher risk of thrombosis than single lumen (or . , ci . - . , p= . ). repetitive picc insertions in the same arm are associated with an increased risk of thrombosis. double lumen piccs are associated with a higher risk of thrombosis compared to single lumen lines. diagnostic performance of lung ultrasound for the detection of community acquired pneumonia in children j.a.m. stadler , s. androunikou , h. zar ; paarl/za, bristol/uk, cape town/za objective: chest radiographs (cxr) are considered the first line imaging modality when investigating cases of suspected community acquired pneumonia (cap) in children. however, cxr interpretation is limited by moderate sensitivity and specificity and poor inter-and intra-rater reliability and expose children to potentially harmful ionizing radiation. point-of-care lung ultrasound (lus) has been proposed as alternative to cxr for diagnosis of pneumonia in children and some published data suggest accuracy and reliability as good as or better than cxr. most of these studies however, were performed in in-hospital settings creating a bias for selceting more severe disease and consequently more overt radiological findings. the mean age of children in most of these studies were also well above one year, while the highest incidence and risk of complicated pneumonia occurs during the first year of life. the purpose of our study was to assess the diagnostic performance of lus for the diagnosis of pneumonia in both hospitalised and non-hospitalised children in an age group representative of the most at risk segment of the population. we performed a lus on children who presented with clinical signs consistent with the who case definition for childhood pneumonia. one hundred of these patients also had chest radiographs performed as part of routine clinical care. inter-rater reliability (irr) between a general practitioner and an expert paediatric radiologist were assessed for the interpretation of lus findings consistent with pneumonia. where radiographs were available concordance between lus and cxr findings of pneumonia were also assessed. results: seventy-four hospitalised and non-hospitalised clinically defined pneumonia cases were included with a median age of . years. our general practitioner reported lus findings consistent with pneumonia in / ( %) compared with / ( %) by the paediatric radiologist. substantial overall agreement between the reporters was found with an overall agreement proportion of . and kappa= . . agreement for the presence of lung consolidation or for a normal scan was also substantial with kappa of . and . respectively. agreement on the finding of interstitial syndrome was moderate with kappa= . . agreement was higher in hospitalised than in non-hospitalised cases with kappa of . and . for the respective categories. results showing concordance between lus and cxr findings are pending. conclusion: lus shows substantial irr for the diagnosis of pneumonia in children. irr are higher for the detection of consolidation or for no pathology than for interstitial syndrome. irr also appears to be lower in clinically less severe disease. 'white-out' on plain chest radiograph-a late presentation of congenital diaphragmatic hernia a. fagan , c. stewart , k. halliday , s. rao , d.t. chang kwok ; peterborough/uk, lincoln/uk, objective: awareness of the limitations of plain radiograph and computed tomography in diagnosis of late presentation of congenital diaphragmatic hernia. case presentation: a year old boy presented with a day history of pyrexia, vomiting and respiratory distress. he was haemodynamically stable, and had no audible air entry over his upper left thorax with occasional wheeze over the left base. he had bronchiolitis previously but did not require ventilatory support. he was otherwise well with unremarkable antenatal scans. initial chest x-ray showed a large air collection with fluid or soft tissue density within the left hemi-thorax and mediastinal shift to the right. repeat x-ray (figure ) demonstrated the nasogastric tube below the diaphragm. complicated pneumonia was suspected but as the findings were atypical a non-contrast ct was performed. this was interpreted as showing a large hydropneumothorax. (figure ) . a chest drain was inserted which drained only a small volume of fluid, and a repeat chest film showed no change. ct chest and abdomen with oral and intravenous contrast revealed a bochdalek diaphragmatic hernia (figure ) . fortunately the chest drain had not entered the herniated stomach. the hernia was surgically corrected and the child recovered well. ( ) (suppl ):s -s pediatr radiol bochdalek is the most common congenital diaphragmatic hernia (cdh). it is often diagnosed on prenatal ultrasound, with mri used for confirmation. cdh which is not diagnosed in the perinatal period may be asymptomatic and imaging findings can be confusing. postnatal x-ray typically shows an opacified hemi-thorax with or without gas bubbles. there can be mass effect with mediastinal shift. the position of an ng tube can be helpful in localising the stomach, but in this case the infradiaphragmatic position of the tube gave false reassurance. in neonates, the position of an umbilical venous catheter may demonstrate abnormal location of the liver. computed tomography generally demonstrates a posterolateral defect (foramen of bochdalek), which is located on the left in % of cases. ct is useful for excluding lung masses or bronchopulmonary foregut malformations, which may appear similar to cdh on x-ray. ct can also identify anatomical abnormalities associated with cdh. late presenting cdh is often misdiagnosed as pleural effusion or pneumothorax. there are other case reports published where chest drains were inserted before cdh was diagnosed. it is important to keep cdh in mind as a potential cause of unilateral hemithorax opacification, even in previously asymptomatic older children. ct with oral contrast can be useful in diagnosis. ovarian tuberculosis with peritoneal dissemination mimicking ovarian tumor with peritoneal seeding d. grassi, v. tostes, a. duarte, s. abib, h.m. lederman; sao paulo/br consider tuberculosis (tb) as a differential diagnosis whenever the case enrolls in an endemic region. case presentation: female, years old adolescent, who presents with abdominal pain and weight loss. abdominal sonography was performed in a public family practice location and bilateral ovarian masses were detected. she was referred to an oncology pediatric facility for further investigation. abdominal mri and chest ct were performed where dissemination through the peritoneal and mesenteric lymph nodes could be detected; chest ct was normal. the patient underwent surgical intervention for diagnosis and on pathology the findings in the bilateral ovarian masses were secondary to tb involvement. sonography showing bilateral pelvic masses. t -weighted coronal overview bilateral ovarian masses. unique teaching points: whenever a case enrolls in an endemic region of tuberculosis, it is important to consider it as a possible differential diagnosis. in this case, the initial presentation mimicked ovarian tumor with mesenteric seeding. however, only after surgical approach was possible to diagnose ovarian tuberculosis with mesenteric lymph nodes and peritoneal involvement. retrospectively, patient's uncle was discovered as having pulmonary tb. langerhans'-cell histiocytosis with thoracic involvement in infant and young child: ct findings s.-l. shih , k. tsai , w. huang , f.-s. yang ; taipei/tw, taitung/tw the purpose of the study was to evaluate the ct changes of thorax in the patients with langerhans'-cell histiocytosis. the -month-old female infant presented with generalized hemorrhagic macular rash over the skin for months. the laboratory findings showed hemoglobin . gm/dl (normal . ~ . gm/dl). the chest radiograph showed bilateral reticulonodular infiltration. high-resolution computed tomography (hrct) of chest showed multiple cystic-like lesions ( - mm) in the right middle and bilateral lower lobes. the pathological report was langerhans'-cell histiocytosis after skin biopsy from upper chest. then she was on scheduled chemotherapy. she was in remission after one-year treatment. the y m-old girl presented with fever for months. the physical examination revealed hemorrhagic-macular rash over the skin in the anterior chest wall and hepatosplenomegaly. the laboratory findings revealed albumin . g/dl (normal . - . g/dl) and hemoglobin . g/dl (normal . - . g/dl). hrct of chest showed multiple cystic-like lesions ( - mm) in the bilateral lower lobes with left pleural effusion as well as multiple osteolytic lesions in the vertebral bodies of t , t , t and t . the pathological report was langerhans'-cell histiocytosis after skin biopy from anterior chest wall. then she was on scheduled chemotherapy. she was doing well years after treatment. the y m-old girl presented with yellowish discoloration of skin for one month. the laboratory findings revealed direct/total bilirubin . / . mg/dl (normal . - . / . - . mg/dl), got iu/l ( - iu/l) and gpt iu/l ( - iu/l). the chest radiograph revealed enlargement of upper mediastinum. the ct scan of chest and upper abdomen showed punctuate calcification with heterogeneous enhancement in the upper mediastinum and several minute cysts in the lower lobes of lung (hrct) as well as dilatation of bilateral intrahepatic bile ducts in the liver. the pathological report was langerhans'-cell histiocytosis after biopsy from thymus and liver. then she was on scheduled chemotherapy and got initial response. unique teaching points: langerhans'-cell histiocytosis affecting the lungs and thymus may be in isolation or as part of a multiorgan disease. the pulmonary changes on ct scan may not have corresponding respiratory symptoms. ct scan of thorax may have multiple minute cysts ( - mm) in the lungs, pleural effusion, calcification in the thymus and osteolytic lesions in the thoracic spine. case of fungal infection of the soft tissue in a child with acute myeloid leukemia (ultrasound aspects of diagnosis) i. begun, s. kondaurova; minsk region/by objective: early diagnosis of fungal infections of the tissues is essential for a successful and complete recovery. we describe a clinical case of fungal infection of the soft tissue in a child with acute myeloid leukemia (aml). ultrasound were made for the characteristics of the structural changes in the area of interest to perform biopsies followed by bacteriological culture studies. case presentation: patient k., years old, diagnosed with aml, from which after a course of induction chemotherapy with neutropenia about weeks on the skin of the foreskin appeared removable hard white coating. cultures of plaque it possible to establish the presence of fungi of the genus trichosporon spp. after days, there were hyperemia, compaction and ulceration of the glans penis, which led to extensive tissue defects. with help ultrasound were determined the structural deformation of the glans penis with the pronounced around changed tissues vascularization. after days in the rear surface projection of the left thigh and the lateral surface of the left calf were defined erythematous papules which progressed to ulceration with central black scab. by standard ultrasound were visualized: subcutaneous nodal education oval , х , sm on hip and echogenic skin thickened portion having an average degree of severity of dorsal acoustic shadow on the lower leg (weakening of the signal behind scab). in cultures of biopsies of subcutaneous foci were revealed fungi of the genus trichosporon spp too. the patient received the combination treatment (intravenous liposomal amphotericin b and surgical rehabilitation of lesions of glans and corpus cavernosum of penis). after the stabilization of patient state the treatment of the underlying disease was continued. unique teaching points: for some patients, lesions of superficial tissues may be the only sign of systemic fungal infections, and rapid recognition of these lesions may contribute to early diagnosis and treatment. ultrasound examination in such a situation naturally becomes an main imaging tool and by choice method. the scanning high-resolution of foci of the thigh of the patient k. in grayscale made possibility to determine the configuration consisting of the central echogenic focus surrounded by a hypoechoic rim (fig. ) with peripheral changes in the type of "infiltrative" according by the active fungal infection at the exit of cytopenia. duplex and triplex ultrasound scanning were indicating to the perifocal vascularization with low level vascular resistance around of the affected area (see fig. - ) . to increase knowledge and awareness of rare cases and diseases in order to be able to better manage and treat patients in the future. case presentation: an -month-old female was presented to our hospital with abdominal distention that increased in the past months associated with low-grade fever, loss of weight and mild respiratory distress. abdominal ultrasonography revealed an enlarged liver with multifocal hypoechoic lesions scattered all over the liver (fig ) . a ct scan with iv contrast (mri was not available at that time in our district) revealed severe hepatomegaly with the presence of multiple, variable in size, hepatic hypodense lesions which had peripheral (ring) enhancement after contrast injection in the arterial phase (fig ) . progressive centripetal filling in portal phase is seen and in the delayed images many of the lesions were completely filled (fig ) . reduction in the aortic caliber (mid-aortic syndrome) below the level of celiac branch was noted. a diagnosis of hemangioendothelioma was made although liver biopsy was not done due to fear of hemorrhage. alternative diagnosis to infantile hemangioendothelioma in this age group is hepatoblastoma, mesenchymal hamartoma and metastatic neuroblastoma. the patient was transferred to another city to a hospital with pediatric oncology department for follow up and treatment. unfortunately the lack of experience and knowledge of such rare cases led to mismanagement and delayed treatment and after less than months the patient was brought back to our hospital to the pediatric icu due to deterioration of her status due to congestive heart failure. unfortunately the patient died shortly afterwards. hemangioendothelioma is twice as common in girls and can have complications due to high output chf secondary to arteriovenous shunting hemangioendotheliomas tend to involute spontaneously without therapy over a course of months to years. they are followed with sequential ultrasounds. medical therapy is reserved for severely symptomatic lesions (e.g. anemia, consumptive coagulopathy, high-output chf) and includes high-dose steroids or alpha-interferon. in cases of failed medical management, surgical resection should be performed. if partial hepatectomy is not technically achievable, transarterial embolization should be used either as definitive therapy or as a temporizing measure until liver transplantation is possible. the sad outcome of this case was mainly due to mismanagment due to lack of medical experience and knowledge of such rare cases so we suggest that such rare cases should be catalogued in a national data bank for future consultation and teaching purposes. fatal outcome of acute gastric dilatation causing acute abdomen compartment syndrome in a child: a case review c.s. yoon; seoul/kr to describe and review presumed acute abdominal compartment syndrome in a child. case presentation: a years and months old boy was admitted to emergency room due to abdominal distention. he suffered abdominal pain and vomited since yesterday after lunch. on physical examination, his abdomen was rigid and distended. body temperature is . °c. the white cell count was increased ( , /μl). esr is mm/hr and c-reactive protein was . mg/l. creatinine was increased ( . mg/dl). amylase and lipase were increased ( u/l and u/l respectively). prothrombin time was prolonged ( . sec). plain abdomen radiograph shows markedly distended stomach with air-fluid level (fig. ) . first trial of nasogastric tube insertion was failed due to kinking of tube at gastroesophageal junction. contrast-enhanced abdomen ctscan shows marked distensionofstomachwithlargeamountoffoodmaterialsandintraluminalairwith prominent external compression in the duodenal rd- th junction. esophageal air distention is also markedly noted with l-tube insertion. no opacification of large vessel with contrast media, without contrast enhancement of spleen, pancreas and left kidney is noted (fig. ) . prob. markedly compressed and poorly defined lower abdominal aorta with faintly visible both common iliac arteries and femoral arteries. after ctscan, nasogastric tube exchange was performed due to poor drainage of gastric fluid. about cc of gastric fluid was drained. however, sudden cardiac arrest of the patient was developed. although vigorous cardiopulmonary resuscitation was performed, the patient was died. ( ) (suppl ):s -s pediatr radiol unique teaching points: acute abdomen compartment syndrome is a very serious and lifethreatening disease. as soon as possible, rapid diagnosis and adequate treatment are necessary for good prognosis. delayed diagnosis and treatment may result in fatal outcome. pleuroperitoneal fistula in a pediatric patient with primary hyperoxaluria type w.p. chu; hang hau/hk to illustrate the imaging features of pleuroperitoneal fistula in a pediatric patient suffering from primary hyperoxaluria type case presentation: an -year-old girl with the history of primary hyperoxaluria type was repeatedly admitted to the hospital for recurrent right pleural effusion despite chest drain insertion. the right pleural fluid was transudative in nature and the microbiological cultures for bacteria and mycobacterial species were negative. the radiographic examination [ figure ] showed moderate right pleural effusion a n d f e a t u r e s o f o x a l o s i s i n c l u d i n g b i l a t e r a l c o r t i c a l nephrocalcinosis and generalized increased in bone sclerosis. delayed planar images of the peritoneal scinitigraphy [ figure ] obtained and hours after injection of technetium- m suphlur colloid found diffuse tracer activity at the right hemithorax, suggestive of pleuro-peritoneal fistula. the patient subsequently required thoracoscopy and surgical decortication at the right hemithorax and renal transplantation. primary hyperoxaluria is due to defective glyoxylate metabolism and results in increased synthesis of oxalic acid. cortical nephrocalcinosis and diffusely increased bone sclerosis are characteristic radiographic features. pleuroperitoneal fistula is unusual in patients without peritoneal dialysis. possible cause in this patient is increased intra-abdominal pressure related to portal hypertension and cirrhosis. osteosarcoma with pulmonary intra-arterial tumor embolism metastasis a. alzaher, f. alzaher; dammam/sa objective: osteosarcoma rarely invade the veins and small number of cases has been reported with venous invasion at the presentation. however, to our knowledge, no case has been reported with venous invasion and isolated distal metastasis as intra-arterial pulmonary embolisms. we are presenting a case of pediatric pelvic osteosarcoma with venous invasion and pulmonary arterial tumor embolisms as isolated distant metastasis at the presentation. the purpose of this case report is to describe the rare presentation of distant metastasis as isolated pulmonary arterial embolism that might be overlooked radiological. additionally, such tumor embolism might cause respiratory symptoms and differentiating tumor emblism from pulmonary thromboembolism is crucial to avoid the unnecessary anticoagulation. case presentation: fourteen year old boy who presented with months history of right hip and lower limb pain after trauma. this was associated with lower limb swelling. the plain radiography showed right pelvic iliac bone aggressive mass, along with lobulated, soft-tissue components, extensive areas of osseous matrix, and malignant periosteal reaction. the patient could not tolerate the mri and ct scan was performed and it showed that the mass was invading the right external and internal iliac vein with imaging appearance was most consistent with osteosarcoma. patient staging was then carried on with mri under anesthesia and chest, abdomen and pelvic ct scan. the unenhanced and iv contrast enhanced chest ct scan showed multiple beaded expansion of sub segmental pulmonary arteries with soft tissue destinies and calcification suggestive of intra-arterial pulmonary tumor embolisms. there was no isolated pulmonary nodule or any other site of distant metastasis. unique teaching points: we present this case to increase the awareness of isolated intra-arterial pulmonary tumor embolisms as osteosarcoma metastasis especially with the present of venous invasion. additionally, such condition might be with respiratory symptoms and differentiating the tumor embolism from pulmonary thromboembolism is crucial to avoid the unnecessary anticoagulation. case presentation: a -year old boy with acute myelodysplastic syndrome presented with recurrent, acute severe anemia (hemoglobin g/dl) and melena. his past history was significant for bone marrow transplant twice followed by graft-versus-host-disease of intestines, bilateral lung transplants for bronchiolitis obliterans, renal failure, scleroderma and acute pancreatitis. ct angiography performed previously did not identify active extravasation. several days before, upper gi endoscopy had demonstrated ulceration of the greater curvature of the gastric wall that was initially treated with epinephrine injection and surgical clip placement. at the time of referral, endoscopic interventions were unsuccessful leading to progressive clinical deterioration. a decision was taken to proceed to angiography to isolate the arterial source of hemorrhage, with an intention to embolize, if feasible. catheter angiography via transfemoral fr access revealed a left gastric artery pseudoaneurysm with active extravasation into the gastric lumen through the ulcer. after selecting the feeding pedicle of the left gastric artery with a microcatheter, the pseudoaneurysm was embolized using % nbca in lipiodol, resulting in complete angiographic obliteration of the bleeding source. on repeat cbc hours post-procedure, the hemoglobin had increased from to g/dl. the patient remained hemodynamically stable in the intensive care unit. there is no evidence of bleeding recurrence days later. unique teaching points: catheter angiography can define the bleeding source with greater accuracy than cta in children. there should be a low threshold to perform catheter angiography, with an intention to proceed to treatment. nbca embolization is a feasible and effective option for treatment of acute gi bleeding in children. case presentation: an infant born by cesarean section at weeks of gestation, after nonreassuring cardiotocoghraphy, with meconium aspiration at birth, severe hepatocellular failure with hyperbilirubinemia, signs of hemorrhage, edema, ascites, hypoglycemia, increased ferritin values, and lactic acidosis was referred for ultrasound and magnetic resonance. both examinations showed signs of liver cirrhosis with portal hypertension; in addition, on t -weighted images and gradient-echo images, the signal intensity of the liver and the pancreas was lower than that of the spleen and skeletal muscle, a finding consistent with abnormal iron deposition in those organs. a biopsy of the lower lip confirmed the diagnosis of neonatal hemochromatosis. unique teaching points: although the diagnosis may be suspected clinically, it must be confirmed by demonstrating the generalized iron overload affecting, among other organs, the salivary glands, liver and pancreas, with sparing of the reticuloendothelial system. the underlying cause may be associated with an an alloimmune mechanism; thus, intravenous immunoglobulin during gestation is administered in selected cases to prevent the severity of neonatal hemochromatosis. diagnosis is then crucial not only for management of the affected infant, but also for prevention in the future offspring. fishing for the answer -a rare case of paediatric exogenous lipoid pneumonia secondary to fish oil aspiration h. moodley, d. white, g.d. baker; johannesburg/za objective: lipoid pneumonia is a rare condition caused by the intrapulmonary accumulation of endogenous or exogenous fat containing substances. in the acute exogenous form secondary to aspiration of oil, it is important to make the diagnosis and remove the causative agent to prevent or arrest the progression of pulmonary fibrosis. radiopathological findings usually prompt the diagnosis, as aspiration of mineral oils is usually unnoticed due to the lack of reactive airway symptoms and patients present with vague chronic respiratory symptoms. case presentation: we present the clinical, radiological and pathological correlation of exogenous lipoid pneumonia in a -month-old male patient with recurrent respiratory tract infections. a ct chest demonstrated an extensive crazy paving pattern of the dependent lung segments bilaterally. the lung biopsy findings of occasional intra -alveolar macrophages with larger ( ) (suppl ):s -s pediatr radiol foamy cytoplasmic vacuoles, raised the possibility of an exogenous lipoid pneumonia secondary to aspiration. on further history, the patient was found to have been fed fish oil by his mother, confirming the diagnosis. unique teaching points: the rare diagnosis of exogenous lipoid pneumonia can be confirmed on ct chest by measuring the hounsfield units in the most hyperdense components of consolidation (typically - to - hu). histopathological confirmation can be obtained provided that the specimens are not embedded in paraffin. the possible role of visual evaluation of dwibs in childhood renal masses based on our five cases e. varga, g. rudas; budapest/hu objective: nowadays, the diffusion-weighted mri has a great importance not only in the differential diagnosis and follow-ups of childhood renal tumors, but in the early detection of recurrence of the disease as well. the dwibs with appropriate b-values and the adc calculation can be helpful in distinguishing between benign and malignant processes. however, the adc calculation is a time consuming method and in addition, there are cases when we cannot use this technique, but we can still apply the visual evaluation of diffusion. case presentation: between - , we had cases in which the visual assessment of dwibs was the best method which helped to make the appropriate therapeutic decisions. left kidney of an infant with nephroblastomatosis was removed because of an arising wilms tumor. , years later, in the contralateral kidney, a small area of diffusion restriction appeared on the dwibs in one of the cystic residual lesions, but the anatomic sequences haven't showed any changes comparing with the previous examinations. in another patient with beckwidt-wiedemann syndrome, the follow-up ultrasound examination showed a little bulging of the surface of the left kidney. accordingly, the mri showed a barely distinguishable nodule, but the dwibs referred to a wilms tumor. in a -month-old child, more nodules were visible in both kidney on the dwibs than on other sequences. with the help of visual evaluation of dwibs, we were able to detect the malignant lesion easily and quickly, among a lot of cystic and solid nodules of the kidneys in a seven years old patient with sclerosis tuberosa. an -month-old infant was followed with a benign cystic renal disease and a new small solid nodule was found on the last ultrasound examination. instead, the visual assessment of dwibs indicated a multilocular cystic wilms' tumor. unique teaching points: the diffusion-weighted mri is suitable for differentiate benign and malignant renal lesions in children. the dwibs (with appropriate b-values) and the adc calculation are very sensitive methods in pediatric oncoradiology. the adc calculation is a long process andas our cases demonstrated -we cannot apply in every cases. the visual evaluation of dwibs is a time saving method which is spared from limitations of adc histogram-based assessment, so it may become very useful in the everyday practice. we can use it in the differential diagnosis and follow-ups of childhood renal tumors and we can detect the recurrence of the malgnancy very early and easily. mr urography in a -years-old female with unusual urinary dribbling m.c. terranova, c. tudisca, d. narese, g. li voti, s. salerno; palermo/it objective: congenital anomalies of kidney and urinary tract (cakut) occurs in up to . % of infants, and clinically they can range from asymptomatic patients, in which anomaly is detected incidentally even in adulthood, to ante-natal or post-natal mortality due to bilateral kidney agenesis or acute renal failure. dmsa renal scintigraphy is considered gold standard, for evaluation of those cases electable for surgery, in order to assess renal function, depict and locate ectopic kidney and guide surgical management, but has the important limit of radiation exposure and may undetect poorly functional renal moieties. the advent of modern magnetic resonance technics proven to be able to assess anatomical malformations and renal function, overcoming the limits of dmsa scintigraphy, may be used as a valid alternative, especially in vulnerable pediatric population. we herein describe a case of a young girl with small left renal bud and ectopic ureter, draining in vagina, discovered by mr and undetected by previous dsma scintigraphy. case presentation: a years old girl was referred for continuous urinary dribbling, after starting toilet training, with normal bladder voiding pattern, unrelated to any physical and psychological events, and no history of urinary tract infections. physical examination revealed vaginal septa and micturition training was practiced, with no symptoms improvement. abdominal us study was performed, reporting empty left renal fossa and hypertrophic right kidney; no ectopic kidney nor sign of urine stasis or other urogenital anomalies were detected, and dmsa renal scintigraphy was planned. it depicted only normal right kidney radionuclide uptake but no evidence of left renal or ectopic renal tissues activity. patient then underwent mr evaluation for suspected genito urinary malformation, that revealed a small cystic formation, with a slight cortex, at the level of the iv lumbar vertebra -that represented the left immature renal bud -supplied by a short fluid-filled tubular structure, located postero-medially to the bladder -that configured the left ectopic ureter, draining in left vaginal wall. bladder was normal, and regularly connected with the right orthotopic ureter (fig ) . pre-surgical cystoscopy and vaginoscopy, followed by left ascending urethrogram were performed, confirmed previous mr findings, and patient underwent successfull laparoscopic left nephron-ureterectomy. unique teaching points: mr urography has proven to be a rapid, safe, radiation free, systematic diagnostic tool especially in the evaluation of poorly functioning renal systems, and of collecting system, bladder and ureteral abnormalities, overcoming the limits of conventional imaging technics agenesis of the dorsal pancreas: case report c. lanza, g. pieroni, l. amici, a. giovagnoni; ancona/it objective: agenesis of the dorsal pancreas (adp) is a rare malformation. since and until , cases have been reported. majority of the patients with this anomaly are asymptomatic or associated with abdominal pain, hyperglycemia, diabetes mellitus, and acute or chronic pancreatitis. case presentation: we present a case report of a -year-old girl with adp, diagnosed incidentally during radiological evaluation for abdominal pain. she was hospitalized in the pediatric department for recurrent abdominal pain for the past months. there was no history of nausea, vomiting or trauma. biochemical investigations showed a normal random serum glucose, serum amylase levels slightly increased ( u/l; reference value - u/l) and slightly elevated serum pancreatic lipase levels ( u/l; reference value - u/l). the day after serum amylase levels decresed up to u/l and lipase levels to u/l. us revealed increased -size pancreatic head with normal contour and echotexture with no parenchymal calcification or duct dilatation. the body and the tail of the pancreas were poorly visualized. mr imaging examinations revealed only a partial visualization of the pancreas: the pancreatic head and the uncinate process were visualized with defined margins with peripancreatic fat stranding, but the distal neck, body, and tail of the pancreas were absent. on mrcp, the dorsal pancreatic duct of santorini and the minor duodenal papilla could not be visualized. the ventral pancreatic duct of wirsung and the common bile duct were normal and clearly visualized. these findings were compatible with complete adp, eliminating the need for ercp. unique teaching points: the clinical presentation of dpa varies greatly ranging from incidental detection on x-ray, surgery or autopsy through to the development of a ductal adenocarcinoma of the pancreas. abdominal pain and diabetes are the most frequent clinical manifestations reflecting exocrine and endocrine insufficiency as most of the islands of langerhans are located in the tail of the pancreas. there have also been reports of an increase in the size of the remnant pancreas and recurrent acute pancreatitis as a form of presentation. diagnosis requires confirmation of the absence of the neck, body and tail of the pancreas and duct of wirsung using endoscopic retrograde cholangiopancreatography (ercp) or mrcp. one hundred four mr images of foetal cns with a us suspicion of acc were retrospectively reviewed. foetal mri was performed at . t magnetom avanto (siemens, erlangen, germany) without motherfoetal sedation. polymicrogyria, lissencephaly, schizencephaly, subependymal heterotopias and migration disorders were evaluated. cortical findings were compared to three types of acc (complete agenesis, partial agenesis and hypoplasia). genetic tests were collected. postnatal mri or foetopsy for diagnostic confirmation were collected. on foetuses, fetal mri was able to detect cortical malformations in cases even in early gestational ages (< gw). the mean gestational weeks (gw) at mr diagnosis was (range: - gw). mr imaging found / polymicrogyria, / lissencephaly, / schizencephaly, / subependymal heterotopias and / neuronal migration disorders. / had complete acc, / had partial acc and / had cc hypoplasia. statistically significant correlations (p< . ) between complete acc, focal polymicrogyria and cortical dysmorphism affecting frontal lobes were found. fetal cns mri can detect cortical development malformations in complex acc, providing further information for the clinician to assess the severity of perinatal outcome. mri is a useful tool in improving obstetrical genetic prenatal counselling to predict pregnancy and foetal prognosis. clinical signs of the neonatal lymphatic flow disorder (nlfd) are a combination of the congenital chylothorax, chylous ascites and body edema. it can present as neonatal chylothorax (nc), neonatal chylous ascites, or congenital lymphatic dysplasia (cld). the prenatal appearance of lymphangiectasia has been described as nutmeg lung. the purpose of this study is to describe prenatal and postnatal imaging features and outcomes of neonates with nlfd. materials: this is a retrospective case series of neonates in our institution that had pre-and postnatal lymphatic imaging and nlfd. all patients had prenatal imaging (fetal mri and us) and underwent postnatal dynamic contrast mr lymphangiography (dcmrl) with a three-dimensional ( d) t space. conventional lymphangiography (cl) when performed was also reviewed. six patients with nlfd were identified ( with nc and with cld). one patient had congenital heart disease. nutmeg lung was seen in all patients on fetal mri and patients on fetal us. / patients had pleural effusions, / had ascites and / had body wall edema prenatally. postnatal mri with d t space revealed soft tissue edema in the upper chest and neck ( / patients), mediastinal edema ( / patients), interstitial lung edema ( / patients), retroperitoneal edema ( / patients), and ascites ( / patients). dcmrl demonstrated lymphatic flow to the pleural space ( / patients) and to the abdominal cavity ( / patients) and dermal backflow ( / patients). cl was performed in patients, all of which had collateral lymphatic flow to the lung. lymphatic intervention was performed in patients, lipiodol injection for patients with nc and thoracic duct embolization (tde) for patient with cld. mean hospital duration in the first months of life was days (range - ) for nc and days (range - ) for cld. all patients with cld died after months of age due to respiratory distress including the patient that had tde and both with findings of dermal backflow. the pleural effusions in the patients with nc resolved post lipiodol injection and in the other patient with nc it resolved with conservative therapy. conclusion: nlfd is a disorder that can be recognized on prenatal and postnatal imaging. in this small series, nutmeg lung was present in all patients with nlfd and may be easier to recognize with fetal mr than us. dermal backflow on dcmrl suggests a poor prognosis. both prenatal and postnatal imaging may guide treatment and interventions in nlfd. fetal mri and postnatal ct scans of prenatally diagnosed bpms from patients with available histology were analyzed retrospectively. the fetal mri and ct images were reviewed by two radiologists blinded to histological findings. specific diagnosis was assigned based on predetermined criteria. the accuracy of fetal mri was evaluated. the agreement rate in fetal mri diagnosis between two radiologists was %. an overlap of % in fetal mri and histopathological diagnosis was reached. when comparing fetal mri and postnatal ct examinations, the agreement of the results was also %. the least matching histological diagnosis was bronchopulmonary sequestration (bps). fetal mri is very accurate in characterizing the bpm spectrum and provides important information on lesion type and structure when compared with histology. with relatively small number of patients high correlation between prenatal mri and postnatal ct was reached. therefore, further investigation with more patients is needed. we hypotethise that fetal mri in late pregnancy could in the future replace early (neonatal) ct examinations if fetal mri provides sufficient inforfmation for clinical management. real time virtual sonography: a new integrated approach for the evaluation of fetal cerebral pathologies? s. bernardo, a. antonelli, v. vinci, m. saldari, c. catalano, l. manganaro; rome/it objective: real-time virtual sonography (rvs) is a new technique that uses magnetic navigation and computer software for the synchronized display of real-time us and multiplanar reconstruction mri images. the purpose of this study was to evaluate the feasibility and ability of rvs to assess the main cerebral pathologies in fetuses with suspected us anomalies. materials: this is a prospective study. fusion imaging (hitachi hi vision ascendus) was offered to patients undergone fetal mri for a us suspicion of cerebral pathology. the mri image dataset acquired was loaded into the fusion system using a cd support and displayed together with the us image. both sets of images were then manually synchronized and images were registered. the possibility to record the images in a video format allowed, however, the possibility to re-evaluated the examination. results: rvs was technically possible in all cases. data registration, matching and fusion imaging were performed in minutes at the beginning and in less than - minutes after practice. the ability of rvs imaging to assess the main anatomical sites and fetal anomalies was evaluated and compared with standard us and mri images. the principal application of rvs was the study of midline, cerebral gyration and vascular malformations because it also allowed adding a real time doppler signal on mri images. fusion imaging helped the diagnosis in %. in the / cases of encephalic pathology, fusion imaging improved the diagnosis; in the other cases mri was superior to us even using the rvs. this is a preliminary study on the feasibility and practical use of a fetal mri-us real-time fusion imaging. both techniques are complementary but still independent and the retrospective synthesis of these exams allows optimal analysis of fetal cerebral anomalies. this technique has many advantages especially on the pedagogic plan. however, rvs is currently limited to the research area. role of foetal mri in the evaluation of ischaemic-haemorrhagic lesions of the foetal brain s. bernardo, a. antonelli, v. vinci, m. saldari, c. catalano, l. manganaro; rome/it the aim of this study was to define the role of fetal magnetic resonance imaging in the evaluation of cerebral ischaemic-haemorrhagic lesions and the extension of parenchymal injuries. from september to december we performed fetal mri of cerebral region in foetuses with suspected abnormalities on ultrasound or cmv infection and toxoplasma serum conversion. fetal mri was performed with a . -t magnet system without materno-fetal sedation. fetal mri detected ischaemic-haemorrhagic lesions in / fetuses, revealing a % pathology incidence. mri confirmed the diagnosis in / cases with us suspect of ischaemic-haemorrhagic lesions associated with ventriculomegaly. in / cases with us findings of cerebellar haemorrhage, mri confirmed and provided additional information regarding the parenchymal ischaemic injury. in / cases with us suspect of ventriculomegaly (n= ), corpus callosum agenesis ( ), cerebellar vermis hypoplasia ( ), holoprosencephaly ( ), spina bifida ( ) mri detected ischaemic and haemorrhagic lesions unidentified at us examination. in / fetuses with us suspicion of intracerebral tissue space-occupying lesion, mri modified the diagnosis to extra-axial haematoma associated with dural sinus malformation. results were compared to fetopsy or after-birth follow up. fetal mri is an additional imaging modality in the diagnosis of cerebral ischaemic-haemorrhagic lesions and it is useful in providing further information on the extension of parenchyma injury and associated abnormalities and in improving delivery management. the contribution of mid-trimester virtual autopsy with mr imaging a. d'hondt, n. d'haene, j. rommens, m. cassart, f.e. avni; brussels/be the aim of the study was to assess the potential contribution of fetal virtopsy (post-mortem mr imaging (pm-mri)) in the second trimester of pregnancy. during a one-year period, post-mortem mr imaging (pm-mri) was performed in all fetuses who died in utero or whose pregnancy was interrupted due to major malformations. the study was performed in agreement with the local ethical committee. fetuses of < weeks that underwent obstetrical ultrasound and pm-mr were included. mr imaging examination was performed on a . tesla magnet with a standardized protocol. the findings on pm-mri were compared to obstetrical sonographic findings (and to pathology when available). we have analyzed separately the findings in the central nervous system and those in the rest of the fetus (chest, abdomen and skeleton). the results were classified in three categories according to the diagnostic accuracy: ultrasound>pm-mri, ultrasound=pm-mri and pm-mri>ultrasound. the us and pm-mri data of ten fetuses were analyzed. their gestational age ranged from . - weeks and their bodyweight ranged from - g. for the cns malformation: pm-mri offered a better diagnostic accuracy than us in cases ( %) (e.g. agenesis of the corpus callosum and holoprosencephaly). in cases ( %) us offered the same information than pm-mri. there was no case where us was more accurate than pm-mri. for the rest of the body malformations: pm-mri offered a better diagnostic accuracy in cases ( %) (e.g. heterotaxy anomalies or vertebral segmentation anomalies). in cases ( %), us offered the same information as pm-mri. there were cases ( %) where us showed major malformations that were not diagnosed on the pm-mri (two cases of cardiac malformation). post mortem mr imaging is more accurate than obstetrical ultrasound in detecting major malformations in the cns as well as in the rest of the body. the present exceptions are cardiac malformations. the examination offers an easy evaluation of the deceased fetus. it provides, in most cases, important additional information. diffusion coefficient and perfusion fraction parameters correlate with gestational age in normal human in vivo placenta: a preliminary study a. antonelli, m. guerreri, s. bernardo, s. capuani, c. catalano, l. manganaro; rome/it to investigate the potential of diffusion parameters derived from a biexponential analysis as marker to evaluate the perfusion quality of normal in vivo placenta. eighteen normal pregnancies, fulfilling the study inclusion criteria, have been analysed at . t magnetom avanto (siemens, erlangen, germany) without mother-foetal sedation. dw imaging was collected using seven b values: , , , , , , (s/mm ). three regions of interest (rois) have been considered -central (c), peripheral (p) and umbilical (u) regions. a bi-exponential model was used to obtain perfusion fraction (f), pseudo-perfusion (d*) and apparent diffusion (d) coefficients. pearson test was performed to investigate correlation between diffusion parameters and gestation weeks (gw), body mass index (bmi) and basal glycaemia (bg). the average values on all rois were d= . ± . • - (mm /s), d*= . ± . • - (mm /s), f= . ± . • - , in good agreement with the literature. in the c roi, a positive correlation (p< . ) was observed between f and gw. after gw in the p roi a positive correlation between f and gw (p< . ) and a negative correlation between d and gw (p< . ) were found. no correlation was found between d, d*, f, bmi and bg. conclusion: the f increase reflects normal placenta perfusion physiology. on the other hand, the decrease of d highlights placental parenchyma maturation becoming more fibrotic during late gestational age. bi-exponential model provides more and useful information about placental morphological changes compared to mono-exponential diffusion model. to demonstrate the diagnostic value of fetal mri in the detection of fetal central nervous system (cns) impairment in prenatally echocardiographic diagnosed congenital heart diseases. we retrospectively examined fetuses between gestational weeks and gestational weeks who performed a fetal mri in our institution after a second-line ultrasonography, between april and october . fetal heart and cns studies were performed with a . tesla magnet (siemens magnetm avanto) without maternal sedation. prenatal findings were compared to fetopsy results, fetal mri after gw or postnatal mri. in our sample of cases, / had interatrial septal defect (iasd),intervertricular septal defect (ivsd), and atrioventricular canal defect (cavc), / had cardiac rhabdomyomas, / had hypoplastic left heart syndrome and hypoplastic aorta, / had transposition of the great vessels, / had fallot tetralogy, / had aorta coartation and / had intracardiac masses of uncertain significance. magnetic resonance imaging was able to detect cns impairment: we recognize / corpus callosum (cc) dysgenesis ( / cc hypoplasia, / complete cc agenesis, / partial cc agenesis), / ventriculomegalies or hydrocephalus, / subtentorial anomalies (dandy-walker, vermian hypoplasia and vermian malrotation) and / gyration anomalies. due to the high risk of cns involvement in prenatal congenital heart diseases, it is essential to suggest an mri study of the evolving fetal brain especially in complexes forms that suggest a syndromic background. fetal mri of the cns is mandatory in the study of congenital heart disease due to the high rate of encephalic anomalies associated, particularly in iasd, ivsd and cavc. first experiences and diagnostic utility of micro-ct for fetal autopsy j.c. hutchinson , x. kang , s.c. shelmerdine , m. cannie , v. segers , n. sebire , j. jani , o.j. arthurs ; newcastle upon tyne/uk, brussels/ be, london/uk perinatal autopsy remains poorly accepted by parents, despite yielding information that affects the management of future pregnancies in around % of cases. microcomputed tomography (micro-ct) has shown promising results in the examination of ex-vivo fetal organs, and may provide diagnostic imaging in cases where traditional autopsy is challenging, and s ( ) (suppl ):s -s pediatr radiol existing post mortem imaging techniques (ct and mri) provide insufficient diagnostic resolution. our objective was to examine whole fetuses non-invasively using micro-ct, and compare the findings with standard autopsy as the gold standard. in this ethically approved double blinded study, terminated fetuses or miscarriages underwent iodinated micro-ct examination followed by conventional autopsy. images were acquired using a nikon xth st microfocus-ct scanner with individual specimen image optimisation. forty indices normally assessed at perinatal autopsy were evaluated for each imaging dataset by two independent reporters and a consensus report produced. autopsies were performed blinded to the imaging findings by one of two perinatal pathologists. we examined fetuses, with a gestational age range of - gestational weeks. / indices were non-diagnostic ( %), but there was agreement for / diagnostic indices (overall concordance of . % ( % ci . , . %). in seven out of eight fetuses ( . %), the same final diagnosis was made following micro-ct examination and autopsy examination; in one case, micro-ct was non-diagnostic. ten false negatives indices included a vsd, laryngeal anomaly, ambiguous genitalia and incomplete bowel rotation, none of which changed the overall diagnosis. three apparent false positives on micro ct were a cloacal anomaly, incidental cystic neck lesion and thymic atrophy, which were not detected at autopsy. micro-ct of early gestation whole fetuses can provide highly accurate datasets with three-dimensional renderings of complex disease processes. this approach confirms the potential of this technology for non-invasive examination of small fetuses. investigation of perinatal body organ diffusion-weighted post mortem mri s.c. shelmerdine , m. cheryl , j.c. hutchinson , n. sebire , o.j. arthurs ; london/uk, southampton/uk objective: diffusion weighted magnetic resonance imaging (dwi) uses water molecule diffusion to generate mr contrast images, and can reveal microstructural or functional changes in tissues, quantified by measuring the apparent diffusion coefficient (adc). the application of dwi to the post mortem setting is appealing as it does not require the administration of an exogenous contrast agent. a recent pilot study of paediatric cases suggested that lung adc values at pm mri may be a useful marker of post mortem interval (time since death; pmi) which has forensic relevance, but other body organs have not been comprehensively evaluated. the aim of this study was therefore to evaluate the relationship between pmi and body organ adc values in a larger cohort of subjects across a wider gestational range in the setting of perinatal death. whole body perinatal postmortem mri with dwi sequences were performed at . t, with b values of , , mm /s. mean adc values were calculated from regions of interest (rois) placed in the lungs, myocardium, spleen, renal cortex, liver and psoas muscle. the values were measured by two independent readers, correlated against gestational age and post mortem interval (pmi) using the pearson product-moment correlation coefficient. bland-altman plots were created, and the limits of agreement used to assess the inter-observer agreement of mean adc values. results: eighty fetal deaths and stillbirths were imaged with mean gestational age . weeks (range: - weeks). the mean pmi was . days (range - days). there was a weakly positive correlation between pmi and mean lung adc (r = . ) and spleen adc (r = . ). no correlation was found with between adc and pmi for the other body organs. there was reasonable inter-observer agreement between the two readers, with mean adc difference . mm /s (+/- . mm /s). perinatal lung and splenic adc values show a mild increase with increasing pmi. together with other imaging parameters, this may be useful to evaluate organ-specific changes which occur in the post mortem period, particularly in a forensic setting. further research is needed to understand the organ-specific changes which occur in the post-mortem period. usefulness of combined grey-scale and color doppler ultrasonography(us) findings in the evaluation of acute pyelonephritis in children k. lee, j.h. lee; anyang/kr objective: us diagnosis of apn in children can give a valuable information to the clinicians for the early treatment. but the problem of us in the diagnosis of apn is wide range of sensitivity, which is - %. the purpose of this presentation is to evaluate the usefulness of grey-scale us and color doppler us in the diagnosis of acute pyelonephritis in children. from march to february , children( kidneys), boys and girls, aged weeks to years (mean age, . months) underwent kidney us as an initial diagnostic tool for acute pyelonephritis and follow up dmsa scintigraphy within a week. criteria for acute pyelonephritis on grey-scale image were focal/diffusely increased/decreased echogenicity or loss of corticomedullary differentiation. on color doppler sonography, the criterion was decreased color flow. we classified the us diagnosis of apn into categories. definite, suggestive, possible and normal. when above two grey-scale us criteria and color doppler us criterion are seen, we classified it as 'definite'. when one of greyscale us and color doppler us finding are seen, it was classified as 'suggestive' of apn. 'possible' apn was abnormal finding either on grey-scale or color doppler us. 'normal' was no abnormal findings on grey-scale and color doppler us. we compared above findings with dmsa scan, which is considered as gold standard for diagnosing apn. statistical analysis was performed on all kidneys. the overall sensitivity of our study was %( / ) and specificity was %( / ). the positive predictive value for each definite, suggestive, possible groups were %, %, and % respectively. the negative predictive value for normal group was %, which means the false ppv was %. the p-value of the definite and suggestive was statistically significant, but the possible was statistically insignificant. in the diagnosis of apn in children, abnormal us finding either on greyscale or color doppler us is not optimal. abnormal us findings both grey-scale us and color doppler us showed good association with dmsa scan and statistically significant. combined grey-scale and color doppler us findings can give a more reliable information in the diagnosis of apn in children. the greater degree of gastric and/or duodenal wall thickening and increased echogenicity are helpful sonographic features in differentiating congenital duodenal anomalies from malrotation. our findings confirm the superiority of us vs ugi for evaluation of duodenal obstruction in neonates and evaluation of gastric and duodenal wall must be added to the constellation of other features to be assessed on us examinations. a measure of renal morphology as an indicator for potential renal failure a.c. eichenberger, p. grehten, c. kellenberger; zurich/ch this study introduces a measure of renal morphology, herein labelled split renal volume (srv), that should be applied as an indicator for potential renal failure and eventual surgical treatment of obstructive uropathy in children. current practice applies dynamic contrast enhanced functional renal imaging (fri) with complex post-processing methods. fri generates a measure of split renal function (srf). reduced values of srf under % are currently considered to be an indicator for surgical treatment. this retrospective study compares the accuracy of srv with the accuracy of srf as methods for assessing potential renal failure. materials: srv is a quotient of volumetric measurements. total renal volume is described by the sum of parenchymal volume and intra-renal collecting system volume. srv is designated in this study as the quotient of two ratios: first, the ratio of total renal volume to parenchymal volume of the left kidney; and second, the ratio of total renal volume to parenchymal volume of the right kidney. twenty-two children were studied: (age . ± . y) with unilateral asymptomatic intrinsic uretero-pelvic-junction obstruction (upjo), and normal controls (age . ± . y). all subjects underwent mr urography at . t, which provided estimates of srf and srv for each of the examined kidneys. the sensitivity and specificity of both srf and srv for predicting surgical management were determined by comparing the indicators with an expert review panel's decision to operate. the panel was blinded to values of srv. results: when a cut-off value of % for srf was used, the resultant sensitivity and specificity of srf for the detection of kidneys at risk were found to be % and %. the values of srv ranged between . and . . it was found that a value greater than . indicated kidneys at risk. when the cut-off value of . for srv was used, the resultant sensitivity and specificity of srv for the detection of kidneys at risk were both %. in this small population, srv proved to be % accurate and is superior to srf for detecting kidneys at risk of failure due to obstruction. routine application of srv promises to simplify mr urography by obviating dynamic contrast enhanced imaging studies. further prospective studies are necessary in order to select an optimal cut-off value of srv. factors that can distort the dj flexure mimicking malrotation v. bhalla , s. mohan , k.a. bradshaw , m. thyagarajan ; stoke-on-trent/uk, birmingham/uk to highlight the varied radiological appearances and position of the duodenal-jejunal flexure in children and to discuss its importance in assessing for malrotation materials: retrospective analysis of the multiple fluoroscopic examinations performed in the assessment for malrotation over the past years in a busy tertiary centre results: the classic position of the dj flexure is to the left of left pedicle of l and at the level of the duodenal bulb on frontal views and posterior (retroperitoneal) on lateral views. however variations of the normal location can appear, particularly on frontal views, in the upper gi series that can mimic malrotation which has shown to be more common in neonates. we present cases with examples to illustrate the variability in position due to various causes and its implications in the diagnosis of malrotation and volvulus. our case mix includes patients with excessively distended stomachs, large bowel obstruction, renal pelvic dilatation, repeated naso-jejunal and gastro-jejunal tube insertion and in patients post liver transplantation. malrotation and its assessment have serious management and prognostic implications. this presentation demonstrates that the imaging features can be varied, and knowledge about factors distorting the position of the dj flexure is vital in the accurate management of neonates presenting with bilious vomits. retrospective study of prospectively collected data performed at a single tertiary paediatric institution over a . year period. a total of consecutive patients, aged < years, were reviewed who underwent native renal biopsy. all biopsies were performed within the interventional radiology department. all patients had renal disease requiring a renal biopsy for diagnosis. outcome measures include technical success, early and late complications and the adequacy of histological samples. in addition, age, body weight, glomeruli number, histological data, number of cores, size of the biopsy needle, use of co-axial needle and the rate of tract embolisation/plugging were recorded. results: from september to april , patients (mean age: . years +/- . ; range . - . years) underwent native renal biopsy. one hundred ninety-one patients were < years of age. nine hundred forty-six patients ( . %) had a biopsy of the right kidney, patients ( . %) had a biopsy of the left kidney and patient ( . %) had a biopsy of a horseshoe kidney. five hundred fifteen patients were female ( . %). seven hundred sixtynine patients ( . %) had the procedure performed under general anaes-thetic and of patients ( . %) had the procedure performed under local anaesthetic (+/-sedation/entonox). mean number of passes of the core biopsy needle through the renal capsule was . . a gauge core biopsy needle was used in % of the patients. . % of the patients had three or less passes of the biopsy needle though the renal capsule. the overall complication rate was . % (n= ). . % (n= ) of patients had a non-diagnostic biopsy. fifty-five patients underwent a post biopsy ultrasound due to clinical concerns. twenty patients developed perinephric haematoma ( were treated conservatively; one underwent embolisation and subsequent nephrectomy). four patients developed arteriovenous fistulas. two patients developed post procedure infections (one at the skin site and one a perinephric collection). histology results were reviewed in all patients. the mean number of glomeruli obtained was . (range - ). glomerulonephritis was the most common histological diagnosis (n= ; . %) conclusion: renal biopsy is an extremely useful diagnostic tool for renal disease. there is no published data of this size assessing the outcome of native renal biopsies in the paediatric population. jr usa a. lassrich germany j. sauvegrain france c. fauré france a. giedion switzerland e. willich germany r. astley united kingdom ringertz sweden d.g. shaw united kingdom r. lebowitz usa b. lombay hungary pena spain gold medallists london/united kingdom the dutch group of paediatric radiologists, the hague/the netherlands g. stake ringertz (espr) & d. kirks (spr) chicago/united states future espr meeting italy european courses of paediatric radiology (ecpr) genoa/italy (neuroradiology) r.fotter, graz/austria (abdomen) brussels/belgium (thorax) j-n. dacher paediatric musculoskeletal imaging) references: . -stellungnahme-lnt-modell.pdf [internet]. [zitiert an evaluation of paediatric projection radiography in ireland contrast imaging -> application -dectris background ionizing radiation and the risk of childhood cancer: a census-based nationwide cohort study best practices in digital radiography communicating radiation risks in paediatric imaging kinderradiologie-besonderheiten des strahlenschutzes diagnostic imaging and ionizing radiation -canadian nuclear safety commission epidemiology without biology: false paradigms, unfounded assumptions, and specious statistics in radiation science (with commentaries by inge schmitz-feuerhake and christopher busby and a reply by the authors) european guidelines for ap/pa chest x-rays: routinely satisfiable in a paediatric radiology division? eurosafe imaging together -for patient safety image gently campaign back to basics initiative: ten steps to help manage radiation dose in pediatric digital radiography hostens j, u. a. in-vivo dark-field and phase-contrast x-ray imaging safety commission cn. linear-non-threshold model optimisation of paediatric chest radiography optimizing digital radiography of children radiation exposure in diagnostic imaging: wisdom and prudence, but still a lot to understand radiation shielding for diagnostic radiology strahlenhygienische aspekte bei der röntgenuntersuchung des thorax the image gently pediatric digital radiography safety checklist: tools for improving pediatric radiography the standardized exposure index for digital radiography: an opportunity for optimization of radiation dose to the pediatric population gastroenterology and radiology records were searched to identify ibd patients with colonic strictures. all patients underwent an mre within months of colonoscopy. the following colonic parameters were evaluated: bowel wall thickening with luminal narrowing, pre-stenotic bowel dilatation, bowel wall enhancement, and diffusion restriction (if performed). colonoscopy and operative notes were correlated. results: fourteen patients met the inclusion criteria, one with colonic strictures. bowel wall thickening with luminal narrowing at the site of the reported stricture was present in all cases. pre-stenotic bowel dilatation (> . cm) proximal to the reported stricture was present in / cases. using luminal narrowing and prestenotic dilatation as criteria for diagnosis of a colonic stricture, / cases were therefore positive on mre. when comparing to colonoscopy, mre diagnosed colonic strictures in / cases ( %). in the six patients who had surgery, mre accurately diagnosed colonic strictures in / cases ( %). conclusion: mre is not the primary modality for colonic evaluation, yet diagnosing colonic pathology on mre, particularly strictures, may be beneficial for the referring gastroenterologist in the assessment of these patients. potential strictures on colonoscopy did not agree with mre in all cases, but when correlating with surgery % of colonic strictures were accurately diagnosed in a small subset. although mre is not optimized for the evaluation of the colon, colonic strictures can be recongnized in children with crohn's disease.disorders of sexual differentiations in neonates: standardized sonographic evaluation and proposal of a reading grid h. lerisson, e. amzallag -bellenger, f.e. avni, m. cartigny; lille/fr to propose a systematic and structured sonographic approach in neonates with disorders of sexual differentiation (dsd) materials: review of the us pelvic, external genital and adrenal findings in consecutive patients with clinical suspicion of dsd evaluated in the neonatal period. the us survey included: the uterus (absent or visible -with or without hormonal impregnation), the vagina (absent or present (complete or partial)), the gonads (ovaries, testis or unsetermineddysgenetic ) as well as the adrenals (normal, too small or enlarged). the us conclusions were correlated with the endocrinological and genetical work-up of each patient results: twenty cases of dsd have been included us had correctly identified the presence of a uterus in patients. there was one false positive case; among the patients did not show the physiological hormonal impregnation. the vaginal anomalies were correctly evaluated. the gonads were defined correctly as normal testis in patients, normal ovaries in and dysgenetic gonads in . they could not be visualized in patients. adrenals were considered normal in patients (one false negative), hypertrophied in and small in one patient. to compare hepatic d shear wave elastography ( d swe) in children between free-breathing and breath-hold conditions, in terms of measurement agreement and time expenditure. a cohort of children ( . ± . years) who underwent standardized d swe between may and october were retrospectively evaluated. liver elastograms were obtained under free-breathing and breath-hold conditions and time expenditure was measured. median stiffness, interquartile range (iqr), and iqr/median ratio were calculated based on , six, and three elastograms. results were compared using pearson correlation coefficient, intraclass correlation coefficient (icc), bland-altman analysis, and student's t. median liver stiffness under free-breathing and breath-hold conditions correlated strongly ( . ± . kpa vs. . ± . kpa; r= . , p< . ). time to acquire elastograms with free-breathing was lower than that with breath-holding ( . ± . sec vs. . ± . sec, p< . ). results for median liver stiffness based of , six, and three elastograms demonstrated very high agreement for free-breathing (icc . ) and for breath-hold conditions (icc . ). hepatic d swe performed with free-breathing yields results similar to the breath-hold condition. with a substantially lower time requirement, which can be further reduced by lowering the number of elastograms, the free-breathing technique may be suitable for infants and less cooperative children not capable of breath-holding. abstract: pelvi-ureteric junction obstruction (pujo), classified into intrinsic and estrinsic is one of the most frequent urological diseases affecting the pediatric population. extrinsic causes include the presence of crossing vessels, kinks or adhesions. in cases with extrinsic obstruction of puj, colour doppler ultrasound (cd-us) can detect the presence of crossing vessels. in presence of crossing vessels pyeloplasty or vascular hitch can be performed. the aim of the study is to analyze the sensitivity of cd-us and magnetic resonance urography (mru) in visualizing crossing vessels in extrinsic pediatric hydronephrosis in order to decide the correct diagnostic pathway and evaluate in the pre-operative phase which surgical technique and approach (open, laparoscopic or robotic) is the ideal to be performed. a retrospective review of medical records for patients who underwent surgical treatment for hydronephrosis from august to february was performed. a descriptive statistical analysis was performed. the presence of crossing vessels at surgery was considered the gold standard. the sensitivity was calculated for both the imaging techniques as a measure of accuracy, evaluating the ratio between the positive cases divided by the those with aberrant vessels identified at surgery. results clinical charts were reviewed. crossing vessels identified at surgery were ( , % of pujo). the median age was higher in the group with crossing vessels compared to the group without crossing vessels (p< , ). the sensitivity of cd-us was higher compared to mru ( , % vs , %). before the surgical time knowing which technique and approach have to be managed in hydronephrotic patients with crossing vessels could be very important. according to our preliminary datacollection cd-us has got a higher sensitivity and could be the gold standard technique. study limitations include the absence of specificity, positive and negative predictive values. in the future it could be useful to perform a double blind trial in which children with moderate-severe hydronephrosis will be subjected to both imaging techniques to evaluate not only the sensitivity, but also the specificity, the positive predictive value and the negative predictive value conclusion: conclusions in the pre-operative phase, cd-us could be sufficient for the surgeon to discern between pujo with the presence or the absence of crossing vessels, as it has a higher sensitivity and lower costs compared to mru.urosonography -nonradiant alternative for voiding cystourethrography o.m. fufezan, c.a. asavoaie, s. tatar; cluj-napoca/ro voiding cystourethrography (vcug) was considered the gold standard in the diagnosis and monitoring of vesicoureteric reflux (vur). this method is invasive due to the radiation exposure. in the present the diagnosis of vur can also be established by contrast ultrasound examination, also known as voiding urosnography (vus). the authors will present the role of vus in the diagnosis and grading of the vur and the role of patient position in the detection of vur. the infants and children with congenital anomalies of the urinary tract and/or urinary tract infection have been evaluated with vus. iatrogenic vur, neurogenic bladder and urogenital sinus anomalies were excluded. the presence and the degree of the vur were evaluated. vus has been performed using a protocol similar to the one used for vcug. in conditions of sterile urine, . ml sonovue and saline solution have been introduced into the bladder until voiding started. the patients were examined both in a supine and an upright position and the following structures have been scanned: urinary bladder, distal part of the ureters and both pelvicaliceal systems during bladder filling, during and after voiding. the visualisation of the ultrasound contrast agent in the upper urinary tract represented a positive vur diagnosis. the grading of the vur has been established based on the same criteria as in vcug. sixty five patients ( renal units), ages between weeks and years were evaluated (median age ± sd: years ± years and months) through vus. vcug was performed in patients in a maximum of hours after vus. vur has been identified in patients ( . % renal units). a high vur grade (iv-v) was identified in . % of renal units. for the patients investigated with both methods, the results were concordant in patients. in two patients vur has not been identified by vcug, but was detected during vus. the upright position (in addition to decubitus) revealed vur in renal units in which the reflux was not detected in decubitus. conclusion: vus is extremely useful and reliable in diagnosing and grading vur in pediatrics. the changing of the patient position during examination can improve vur detection.new sonographic features useful in differentiating congenital duodenal anomalies from malrotation: gastric and duodenal wall thickening and hyperechogenicity p. caro dominguez , s. hameed , a. zani , r. moineddin , o.m. navarro kunstmann , a. daneman ; cordoba/es, london/uk, toronto/ca the clinical and plain radiographic differentiation of congenital duodenal anomalies (atresia, web, stenosis) and intestinal malrotation is not always clear. although sonography has been documented as an important diagnostic tool to differentiate these two entities, its role is still not widely appreciated. the purpose of this study was to assess the sonographic features of the gastric and duodenal wall in a large series of neonates with congenital duodenal obstruction as these have not been reported previously. neonates who had surgically proven congenital duodenal anomalies or malrotation were identified from the surgical database in a tertiary pediatric hospital in a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . those with an ultrasound performed within hours of surgery were included in the study. imaging was retrospectively and independently reviewed by two readers in chronological order blinded to final diagnosis. a wall thickness of ≥ mm of a distended loop was considered abnormal. hyperechogenicity was recorded when the wall of the stomach or duodenum was brighter than liver or splenic parenchyma. imaging findings in the group with congenital duodenal anomalies was compared to the group with malrotation using fisher's exact test. one hundred eight neonates were included in the study, with a congenital duodenal anomaly, with malrotation ( with volvulus) and with both. ugi was performed in neonates who had us. the correct diagnosis was provided only by us in of these newborns ( %), only by ugi in ( %), by both in ( %) and by neither in ( %). ugi was performed in children with malrotation and volvulus, eight were diagnosed only by us, four only by ugi and nine by both. the gastric and/or duodenal wall was significantly thicker and more hyperechoic in neonates with congenital duodenal anomalies than those with malrotation (p< . ) [fig , table ]. conversely an abnormal relationship between the superior mesenteric artery and vein, abnormal position of the third part of the duodenum and the whirlpool sign were found more commonly in neonates with malrotation than those with congenital anomalies (p< . ). key: cord- -vm d mj authors: bradt, david a.; drummond, christina m. title: technical annexes date: - - journal: reference manual for humanitarian health professionals doi: . / - - - - _ sha: doc_id: cord_uid: vm d mj this chapter provides guidance on technical issues in the health sector. the annexes contain selective compilations of frequently used reference information. material ( ) financial f. identification of deliverables and timetables g. consolidated reporting and dissemination ( ) group terms of reference ( ) meeting minutes ( ) epidemiology updates ( ) health sitreps ( ) component analysis ( ) field documentation (toolkit) for new arrivals . capacity building of host authorities . civil society partnership and support a. organize community leaders b. encourage gender mainstreaming c. encourage privatization d. discourage entitlements . advocacy . transition to early recovery d. strategy for livelihood/economic relief . restore productive assets (supply side interventions) a. in-kind donations (e.g. food, seeds, tools, fishing nets, etc.) b. types of community projects in food-for-assets programs ( ) natural resources development (a) water harvesting (b) soil conservation ( ) restoration of agri(aqua)culture potential (a) irrigation systems (b) seed systems ( ) infrastructure rehabilitation (a) schools (b) market places (c) community granaries (d) warehouses (e) roads (f) bridges ( ) diversification of livelihoods (a) training and experience sharing . increase individual purchasing power a. cash distribution b. cash for work (cash for assets) c. vouchers d. micro-credit e. job fairs f. artisanal production g. livelihoods/income generation . support market resumption a. market rehabilitation b. infrastructure rehabilitation c. micro-finance institutions e. goals-protect what's left ( month), restore the system ( months), improve the system ( ( ) non-food items c. financial assistance ( ) cash grants ( ) cash for work ( ) microfinance (loans) ( ) livelihood/income generation . ensure responsible resource management a. human resources management ( ) incident management command and control ( ) team structure and function ( ) staff selection (a) internationals (b) homologues ( ) field activities (a) briefing (b) meetings and reports (c) debriefing ( ) operations support (a) comms (b) transport (c) office (d) food and lodging ( ) personal health maintenance and morale b. material resources management c. financial resources management d. supervision e. monitoring and evaluation . scale up coverage of priority health interventions . address bottlenecks of the disrupted health system (otherwise temporary solutions become permanent) . protect essential public health infrastructures . build capacity of local authorities with focus on sustainable systems a. technical oversight-hiring of local experts b. material assistance-production of key commodities c. financial assistance . provide incentives for host government . support host country non-beneficiary population . find new partners in the development community . use health sustainable development goals as targets for recovery activities . seek opportunities and develop mechanisms for transition and phase out f. . programmatic constraints a. staff ( ) western trained ( ) hospital-based ( ) resource intensive ( ) technology dependent ( ) procedurally oriented ( ) invasive ( ) monolingual ( ) hazard naïve b. supervision ( ) limited responsibility ( ) limited authority ( ) limited accountability c. projects ( ) acute ( ) curative ( ) short-term ( ) intermittent d. systems ( ) inadequate security ( ) weak rule of law ( ) limited accountability framework ( ) uncoordinated . international cooperation to protect lives and health . timely and sustained high-level political leadership to the disease . transparency in reporting of cases of disease in humans and in animals caused by strains that have pandemic potential to increase understanding, enhance preparedness, and ensure rapid and timely response to potential outbreaks . immediate sharing of epidemiological data and clinical samples with the world health organization (who) and the international community to characterize the nature and evolution of any outbreaks as quickly as possible . prevention and containment of an incipient epidemic through capacity building and in-country collaboration with international partners . rapid response to the first signs of accelerated disease transmission . work in a manner supportive of key multilateral organizations (who, fao, oie) . timely coordination of bilateral and multilateral resource allocations; dedication of domestic resources (human and financial); improvements in public awareness; and development of economic and trade contingency plans . increased coordination and harmonization of preparedness, prevention, response and containment activities among nations . actions based on the best available science d. program innovations at community level . genocide (article )-acts committed with intent to destroy, in whole or in part, a national, ethnic, racial, or religious group a. killing members of the group b. causing serious bodily or mental harm to members of the group c. inflicting on the group conditions of life calculated to bring about its physical destruction in whole or in part d. imposing measures intended to prevent births within the group e. forcibly transferring children of the group to another group . crimes against humanity (article )-acts committed as part of a widespread or systematic attack against any civilian population, with knowledge of the attack a. murder b. extermination c. enslavement d. deportation e. imprisonment in violation of international law f. torture g. rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence h. persecution on political, racial, national, ethnic, cultural, religious, gender, or other grounds universally recognized as impermissible under international law i. enforced disappearance j. apartheid k. other inhumane acts intentionally causing great suffering or serious injury to body or to mental or physical health . war crimes (article ) a. grave breaches of the geneva conventions of aug ( ) willful killing ( ) torture or inhumane treatment including biological experiments ( ) willfully causing great suffering ( ) extensive destruction and appropriation of property ( ) compelling a pow to serve in the armed forces of a hostile power ( ) willfully depriving a pow of the right to a fair trial ( ) unlawful deportation ( ) taking of hostages b. serious violations of laws and customs applicable in international armed conflict ( ) intentionally directing attacks against the civilian population or against civilians not taking direct part in hostilities ( ) intentionally directing attacks against civilian objects ( ) intentionally directing attacks against personnel, installations, material, units, or vehicles involved in humanitarian assistance or peacekeeping mission ( ) intentionally launching an attack in the knowledge that it will cause incidental civilian loss of life or severe damage to the natural environment ( ) attacking undefended towns, villages, dwellings, or buildings which are not military targets ( ) killing or wounding a combatant who has surrendered ( ) improper use of a flag of truce, flag or insignia or uniform of the enemy or of the un, or emblems of the geneva conventions resulting in death or serious personal injury ( ) transfer by the occupying power of parts of its own civilian population into the territory it occupies, or the deportation or transfer of all or parts of the population of the occupied territory within or outside the territory ( ) intentionally directing attacks against buildings dedicated to religion, education, art, science, charitable purposes, historic monuments, hospitals, and places where sick are collected, provided they are not military objectives ( ) subjecting persons to physical mutilation or to medical or scientific experiments which are not justified by the medical treatment nor carried out in his/her interest ( ) killing or wounding treacherously individuals belonging to the hostile nation or army ( ) declaring that no quarter will be given ( ) destroying or seizing the enemy's property unless such be imperatively demanded by the necessities of war ( ) declaring abolished, suspended, or inadmissible in a court of law the rights and actions of the nationals of the hostile party ( ) compelling the nationals of the hostile party to take part in the operations of war directed against their own country ( ) pillaging a town or place, even when taken by assault ( ) employing poison or poison weapons ( ) employing asphyxiating, poisonous or other gases, and all analogous liquids, materials, or devices ( ) employing bullets which expand or flatten easily in the human body ( ) employing weapons, projectiles, material and methods of warfare which cause superfluous injury or unnecessary suffering ( ) committing outrages upon personal dignity, in particular humiliating and degrading treatment ( ) committing rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence ( ) utilizing a civilian or other protected person to render certain areas or military forces immune from military operations ( ) intentionally directing attacks against buildings, material, medical units, transport, and personnel using the emblems of the geneva conventions in conformity with international law ( ) conscripting or enlisting children under the age of years c. serious violations of common article applicable in non-international armed conflict, i.e. acts vs. persons taking no active part in the hostilities, including armed forces placed hors de combat by sickness, wounds, detention, or other cause ( ) violence to life and person ( ) outrages upon personal dignity ( ) taking of hostages ( ) passing of sentences and carrying out of executions d. non-applicability of c (above) to internal disturbances (riots, sporadic violence, etc.) e. other serious violations of laws and customs applicable in non-international armed conflict c. degradation of health system a. ppm = mg/kg (solids) = mg/l (liquids) = μg/ml (liquids) = basic unit of measure for chloroscopes :. , ppm = % a range of generic prevention measures should be considered for its impact on diseases in a biological "all-hazards" environment. overall, excreta disposal, water quantity, personal hygiene, and food hygiene commonly contribute more to environmental health than do other listed measures. epidemic threats will oblige heightened consideration of disease-specific strategies for prevention and control. note: in u , length is the preferred term over height . . this last includes kwashiorkor and marasmatic kwashiorkor in the wellcome classification. • sam = severe wasting cases or bilateral pitting edema cases (where due to malnutrition) • sam = whz < − , muac < . cm, or bilateral pitting edema (who). whm not in definition. • sam prevalence worldwide ≈ , , . • sam mortality ≈ × mortality of normally nourished child and its cfr can be - %. • gam = mam + sam • gam = moderate wasting cases, severe wasting cases, or bilateral pitting edema cases (where due to malnutrition) underweight • underweight is not used for screening or surveys in nutritional emergencies. it reflects past (chronic) and present (acute) undernutrition and is unable to distinguish between them. it encompasses children who are wasted and/or stunted. however, weight gain over time can be a sensitive indicator of growth faltering which is easily tracked on road to health charts. • stunting generally occurs before age . it is irreversible. • stunting prevalence worldwide ≈ , , . • stunting is not a good predictor of mortality, but the cfr from ids in cases of severe stunting ≈ × the cfr from ids in cases without stunting. reference standards can be absolute muac, centile, % of median reference, or z scores: • muac • easy to understand. an excellent predictor of mortality. permits comparisons between age groups insofar as the low growth velocity of muac in the u age group makes data roughly comparable. may be used alone in "quick-and-dirty" convenience samples to estimate local prevalence of wasting. however, not used alone in authoritative anthropometric surveys, and is commonly part of a two stage screening process to determine eligibility for feeding programs. easy to understand. permits comparisons between age groups and outliers. however, data are not convenient to convert. e.g. z - . = . nd percentile • % of median of reference population whm is the preferred indicator to determine eligibility for feeding programs (sphere). calculations are easy and are used in the who road to health charts. however, median reference data are not comparable between ages. eg % wt-for-age = severe malnutrition in infants = moderate malnutrition in school age kids moreover, median reference data are not comparable between indicators. eg % wt-for-age = severe malnutrition in infants % wt-for-ht = death • z scores preferred indicator (sphere, who) for reporting anthropometry survey results because it permits comparisons between age groups and nutritional indices. however, data may be difficult to understand. eg z score wt-for-age for y/o: . . kg kg -= − sd below median for his age overall: whz gives higher prevalence of malnutrition than whm for the same population. this is most marked where there is low baseline prevalence of disease, and especially for adolescents (who get subsequently over-referred). whz is more statistically valid, but whm is better predictor of mortality and is used for admission to tfcs. weight-for-age is influenced by weight-for-height and height-for-age. it can be difficult to interpret. blanket-all hh in geographically targeted catchment area (e.g. where ipc + and gam > % or - % with aggravating factors) targeted-some hh in catchment area (e.g. where gam - % or - % with aggravating factors); u and pregnant or lactating women vs. u alone vs. u alone depending on resources available and challenges with case finding) overall programmatic target- % coverage for sam in rural areas (sphere); % coverage for mam in rural areas admission criteria: pedes: age - mo, muac < . cm, with appetite, discharged from otp, no severe medical complications pregnant & lactating: muac < . cm, and nd- rd trimester or with infant < mo treatment: rusf as dry rations e.g. plumpy'sup ® , csb, csb + (supercereal), csb ++ (supercereal +) nb csb may also be cooked on-site as in emergency school feeding. discharge criteria (pedes): weight gain, muac > . cm, time in program > months community outreach with mobile brigades admission criteria for u : sam (whz < − , muac < . cm, or bilateral pitting edema) discharge criteria for u : whz >− . , no edema, and clinically well (generally takes - weeks) treatment protocol (who, icddr) shock severe dehydration: rl + d , ½ strength darrow's + d , or ½ ns + d dose: cc/kg iv death (~ % sam will die with good care, and % will die with mediocre care) c. measures of association quantify the strength or magnitude of the association between the exposure and the health problem of interest. they are independent of the size of the study and may be thought of as best guess of the true degree of association in the source population. however, they give no indication of the association's reliability. • cohort study-relative risk (rr) = riskexposed/riskunexposed • in acute outbreaks, risk is represented by the attack rate (ar) • case-control study-odds ratio (or) • no denominator with which to calculate an attack rate • cross-sectional-prevalence ratio or prevalence odds ratio c. survey designs (see r magnani [ ] , and f checchi [ ] ) . census-complete enumeration of the entire population . sample a. probability sampling ( ) simple random sampling (srs) it requires a complete enumeration of population n-names and locations of all persons or households (hh)-and sample size n nb much effort is necessary to conform to requirements of random sampling. it is easier to sample less often but take more specimens as a cluster. unfortunately, it is recognized that individuals from a cluster often share characteristics which < the precision of the method. ( ) systematic random sampling it requires a complete enumeration of population n, and sample size n, to calculate the skip interval k = n/n. ( ) stratified random sampling it requires a population size n divided into groups or strata l, then srs within each stratum. the method ensures over-sampling in under-represented groups. it yields separate estimates for each stratum at less cost. however, it requires extra info and has complicated analysis. ( ) cluster sampling, cluster sample survey (css) it is used when you don't have a complete enumeration n of all people in the area, and thus can't do random sampling; or when the area is too big to cover, and thus can't do systematic random sampling. • what should be done to compensate for the bias induced when one samples clusters rather than individuals? use n. empiric data on association within clusters in smallpox immunization suggests doubling n. if n = , n = . • what is the minimum number of clusters that can be selected and still fulfill requirements of the theory on which binomial sampling is based? . statistical theory demonstrates that > clusters help ensure cluster means have a normal distribution. the larger the number of clusters, the smaller the design effect (i.e. study efficiency improves, and the total number of study subjects needed will decrease). e.g. × (n = ) will prove more accurate and efficient than × (n = ). clusters × households will be more precise, but clusters × households may be more logistically feasible. choice of cluster should be driven by what one team can complete in a day. × css leaves . min/hh/team, but × css leaves min/hh/team. if a team can only measure kids/day (which is common), then it's best to increase the number of smaller clusters. • to permit an equal number of children to be selected from each of clusters, children would not achieve the necessary n. therefore, children are selected per cluster ( × = ). b. non-probability sampling ( ) convenience ( ) purposeful/judgment (most affected area, hhs, etc.) ( ) quota bias (see r magnani [ ] , f checchi [ ] , and smart [ ] ) systematic, non-sampling error which lowers accuracy of findings. it is usually not appreciated by the survey team. it is usually not apparent from the survey results. it cannot be arithmetically calculated or corrected. its extent cannot be judged by readers of the report. methods and materials must be explicit. report authors must discuss possible sources of bias as limitations to their study. accuracy depends on validity of findings. it is more important than precision (section e), and bias should be prevented at all costs. awareness of sources of bias is the first step in minimizing its impact on any study. as sample size increases, it is more difficult to control quality. more teams to train and supervise create higher risk of bias. it is better to have smaller sample size with less attendant precision but much less risk of bias. . selection bias-respondents are not representative of the population a. project bias-assessors work where a project may be conceptually familiar to them b. spatial/access bias-assessors work where access is easiest (roadside or "windshield" bias) c. refusal or non-response bias (self-selection) bias-subject nonparticipation may undermine representativeness of the sample d. survivor bias-assessments are conducted where households have disappeared due to family death or migration. mortality rate is thereby underestimated. this bias is most likely where hh size is low, recall period is long, mortality is high, and clustering is present. e. class/ethnic bias-different social classes or ethnic groups are inadequately included if not excluded from the assessment. local assessors may have ethnic bias, or the key informants may be drawn from one particular social class or ethnic group. f. season bias-assessments are conducted during harvest season or periods of weather when segments of the population may be under-represented g. time of day/schedule bias-assessments are conducted at a time of day when segments of the population may be under-represented nb items - below may also be grouped as information/measurement bias. . interview bias a. interviewer bias ( ) cultural bias-assessors cultural norms lead to incorrect assumptions about the interview subjects ( ) mandate or specialty bias-assessors mandate or specialty blinds them to needs outside of that mandate or specialty. e.g., a shelter specialist may only assess shelter needs while neglecting livelihood or nutrition needs. gender bias-assessors interview only one gender ( ) language bias-assessors may have a limited spectrum of people with whom they can communicate ( ) key informant bias-assessors may be partial to key informants who appear credible in ways meaningful to the assessors ( ) information/political bias-assessors focus on information that confirms preconceived notions rather than pursue evidence of alternate beliefs ( ) mistranslation ( ) interviewer error-assessors write down answers incorrectly b. subject (response) bias ( ) event recall bias-retrospective surveys only, esp. with recall periods > yr (a) informants underreport remote events (e.g. neonatal deaths) (b) calendar bias-informants over report events within the recall period ( ) event reporting bias (a) taboos-informants underreport taboo subjects (e.g. neonatal deaths) (b) lies-informants misinterpret surveys as registration activities and over report family members or underreport deaths to maintain assistance (c) political bias-informants present information that conforms to their political agenda ( ) age heaping/digit preference-informants exhibit digit preference . instrument/measurement bias-errors in design or use of instrument (e.g. questionnaire, lab equipment, etc.) a. random errors in measurement random errors in weight measurement, even if yielding equal numbers of high and low measurements, widen the distribution curve without altering the mean. hence, the prevalence of malnutrition is overestimated. the effect is greater for severe malnutrition than for moderate malnutrition, and greater when prevalence is low than when it is high. the data distribution should be checked for normal distribution with an sd between . and . z scores. improving the data quality thus appears to reduce the prevalence of malnutrition. b. systematic errors in measurement systematic errors in weight measurement, even if small (e.g. g error in presence of clothing), may alter the mean, but also widen the distribution curve. hence, the prevalence of malnutrition is overestimated. systematic errors in height measurement, such as erroneous lengthboard, may alter the mean without altering the sd. if the measurement is too short, there will be > stunting, albeit < wasting. if the measurement is too long, there will be < stunting, albeit > wasting. a standardization test is routine before undertaking anthropometric surveys. nb some scholars prefers terms "counted" and "calculated" to "measured" and "derived" . data entry bias . analytic bias a. anchoring bias-focusing on one major piece of information b. confirmation bias-favoring data which confirm underlying beliefs c. familiarity bias-weighing familiar/understandable events and spokespersons more than unfamiliar ones d. recency bias-weighing recent events more than remote ones e. salience bias-weighing vivid events more than mundane ones f. "time will tell" bias-collecting more data or letting time pass instead of making a hard decision e. imprecision (see r magnani [ ] , f checchi [ ] , and smart [ ] ) sampling, non-systematic error which lowers precision of findings and affects the level of certainty in extrapolating sampling estimates to the population's true value. it is always present, unavoidable, and a function of chance. its magnitude depends on sample size, sampling statistics, prevalence of condition, and length of recall period. precision refers to consistency of results obtained from repeated measurements. what is the sample size n of a random sample of binomial variables needed to yield a result of specified accuracy and precision? n = [(z pq)/d ] × design effect e.g. n = first estimate of sample size z = confidence limits (accuracy), or normal deviate. usually set at % :. z "score" = . p = proportion of the target population with attribute p q = proportion of the population without attribute p = -p. usually set at . to maximize the n of a study having a result of specified accuracy and precision. if you knew p and q, you would not need to do a survey. d = confidence interval (precision). usually set at +/− % :. d = . design effect (see e below) . . . once n is calculated, compare it to the size of the target population (n). if n < % of n, then use n as final sample size. if n > % of n, then recalculate the final sample size (n f ) by the following correction (a smaller sample size may be used). n n n n f = + / n f = / . n f = nb n to calculate the mean weight may be much smaller than n to calculate the prevalence of malnourished outliers ( vs. ). . sampling statistics and error measurement a. malnutrition prevalence or death rate the higher the prevalence (or death rate), the lesser the precision (higher d) available through a fixed sample size. (this is a consequence of the formula.) % gam is a common trigger for intervention. but, smart discourages use of this because high survey precision is needed (narrow ci). :. choose highest expected prevalence or rate-tends to > n. nb at levels of malnutrition and mortality generally found in emergencies, precision has much greater effect on sample size than suspected prevalence of malnutrition or death rate. n is related to d . e.g., if the malnutrition rate estimate is %, and assuming a design effect of : • survey statistic with a ci of +/− % requires n = • survey statistic with a ci of +/− % requires n = as rule of thumb, prevalence (%)/ approximates the range of appropriate ci. e.g. malnutrition prevalence of % calls for a precision of +/− % (range of %). it's generally unfeasible to achieve precision greater than +/− %. b. standard deviation (sd, σ). the degree to which individuals within the sample differ from the sample mean (μ); unaffected by sample size c. standard error (se = sd/√n) standard deviation of the sampling distribution of a statistic; decreases with larger sample sizes as estimate of the population mean improves, thus a lower se is more precise ( ) standard error of the mean (sem) is standard deviation of a sample mean's estimate of a population's true mean; an estimate of how close to the population's true mean the sample mean appears to be. ( ) relative standard error (rse)-sem/μ expressed as % • se of g on weight mean of kg = rse of % • se of g on weight mean of kg = rse of % d. confidence interval (ci = μ + z (se)) the margin of error around a point estimate. for normally distributed data, the ci yields the range in which a parameter is % likely to be found. a convention for reporting such data would be: "the most probable estimate of the parameter is x, and we are % confident the parameter lies somewhere between y and z [bounds of the ci]" (paraphrased from checchi, ) . nb in general, the lower the prevalence (or death rate), the greater the precision (lower d) needed to detect it and any subsequent changes in it. (this is intuitive.) overall, there is no benchmark for precision. increasing precision (decreasing d) slightly can dramatically increase n. +/− . deaths/ , /d is a practical limit in precision of mortality surveys. :. choose widest acceptable ci-tends to < n. e. design effect (d eff = variance study design /variance simple random sample ) a measure of the (in)efficiency of a cluster sample survey compared to that of a simple random sample. if d eff > , but the analysis treats it as a srs, then the confidence interval is inappropriately narrowed, and a test for differences is more likely to produce a positive result (type error). • if each child in a cluster had an unrelated probability of immunization, the precision of the sample estimate would match that of a simple random sample in which children were chosen. d eff = . however, this is generally not the case. • if each child in a cluster had an identical probability of immunization, the precision of the sample estimate would match that of a simple random sample in which children were chosen. d eff = cluster size of . nb focal phenomena create clustering of findings which increase the d eff . :. choose largest d eff -tends to > n. . length of recall period a. the shorter the recall period, the more accurate the mortality estimate (more distant events are more likely to be forgotten). b. the longer the recall period, the more precise the mortality estimate for a fixed sample size. the "sample" is effectively the number of person-days. for a fixed level of precision, the length of the recall period is inversely related to number of study subjects needed. if you cannot increase the sample size, you must increase the recall period. confounders are extraneous variables that correlate with both dependent and independent variables of interest (e.g. both the exposure of interest and the outcome of interest), are unevenly distributed across the levels of exposure, but are not causally linked to exposure and outcome. age and sex are the most common confounders. hence, the importance of matching in intervention and control groups. g. validity . study validity a. internal-capacity of the study to yield sound conclusions for the study population after considering bias, imprecision, and confounding (see d-f above) b. external-generalizability beyond the study population (ill-advised) . measurement validity a. criterion validity ( ) concurrent-sensitivity/specificity or correlation with a gold standard ( ) predictive-ability to predict an event b. face validity-common sense c. content validity-all relevant elements of a composite variable are included d. construct validity (usually for a new measure)-extent to which the measure corresponds to theoretical concepts (constructs) e. consensual validity-extent to which experts agree the measure is valid :. strength of evidence: face validity, criterion validity > content, construct, consensual validity. in absence of validity, a measurement may be embraced for its reliability (below). . death rates-calculated incidence of death expressed per , p/d or per p/mo; data collected by retrospective surveys (e.g. month period) to gauge severity of public health emergency particularly where sudden events lead to spike in mortality a. cdr-crude death rate b. asdr-age-specific death rate (e.g. u dr or death rate of children - yr) during a studied time interval (written as m or - dr); age of study cohort, e.g. - yr, should not be confused with study time intervals . mortality rates-calculated probability of dying before a specified age expressed per live births; data collected by national health authorities in periodic (annual) demographic surveys to reflect ongoing health status a. cmr-calculated probability of mortality in given population for specific time b. imr-calculated probability of a live borne child dying before yr c. u mr-calculated probability of a live borne child dying before yr nb mr ≠ dr. e.g. cmr ≠ cdr, u mr ≠ u dr. different rates measure different things and are not directly comparable. however, mrs may be converted into drs by the following: cdr or u dr (deaths/ , /d) = -ln( -p/ ) × . where p = cmr or u mr (deaths/ live births). however, this has little field utility. nb mmr-maternal mortality ratio has different units in numerators (maternal deaths) and denominators (live births), thus is a ratio, not a rate i. . stability-inter/intra-observer variation a. discrete variables-kappa coefficient b. continuous variables-correlation coefficient . internal consistency-correlation among all items in the measure . tests of reliability-cronbach's alpha, kuder-richardson, split halves j. conclusions the application of study findings to an entire population from which the sample was drawn. if the survey was well-conducted, the results may be considered representative of the entire population. this is scientifically justified. however a ci should accompany any parameter estimate of that population. the extension of study findings to a population or period which was not represented in the sample. it works by association-if populations appear to be experiencing similar conditions, the morbidity/mortality experience of one may be imputed to the other. this is not scientifically justified, but is often done where data are insufficient or impossible to collect. .k. • holo-endemic areas (e.g. congo) have an intense level of malaria transmission year-round. epidemics don't occur unless displacement brings in nonimmune populations. infection may be asymptomatic. effective partial immunity develops in adults which enables clinical tolerance of infection and protects against serious episodes. mortality is highest in pedes u and pregnant women. • hyper-endemic areas (e.g. w. africa) have an intense but unstable level of transmission in seasonal peaks when the climatic conditions are favorable. epidemics occur. infection is generally symptomatic. partial immunity fails to develop. mortality occurs across all age groups. • hypo-endemic areas (e.g. thai-burmese border) have a low level of transmission year-round. epidemics occur. infection is generally symptomatic. partial immunity fails to develop. mortality occurs across all age groups. think differential diagnosis (below). know the golden rules of infectious diseases (abstracted from a yung [ ] and used with permission). rigors are always important-serious bacterial infections are the most likely cause. severe muscle pain may be a symptom of sepsis even without fever. elderly patients with sepsis may be afebrile. in elderly patients, fever is rarely caused by a viral infection. septic patients who are hypothermic have a worse prognosis than those with high fever. treat as a medical emergency. fever in a postoperative patient is usually related to the surgical procedure (e.g. pneumonia, uti, wound, or deep infection). fever with jaundice is rarely due to viral hepatitis. think liver abscess, cholangitis, etc. the rash of early meningococcal infection may resemble a viral rash. generalized rashes involving the palms and soles may be due to drugs, viral infections, rickettsial infections, or syphilis. all febrile travelers in or returned from a malaria infected area must have malaria excluded. . disseminated tb must be suspected in all elderly patients with fever and multisystem disease who have been in an area with endemic tb. . septic arthritis may be present even in a joint which is mobile. ddx failure to thrive without f in infants is worked up like f without localizing signs. watch for clinical mimics-malaria presenting as pneumonia or diarrhea in pedes; vl presenting as malaria in adults; lepto presenting as mild df (esp in df endemic areas where the pt has mild onset of illness, worsening course, and no rash but jaundice after a week). do basic things well, use equipment you understand, teach others, delegate. h. count the number of fresh graves or bodies at health facilities and inquire as to cause. . orient the descriptive data-person, place, and time. a. tabulate data on affected patients. b. make a spot map. ( ) when and where was/were the first reported case(s) seen indicating an outbreak? c. plot an epidemic curve. ( ) what is the present # of patients/day or week? ( ) what is the usual # of patients/day or week? ( ) is this an increase? ( ) what is the present # of deaths/week or month? ( ) what is the usual # of deaths/week or month? ( ) is this an increase? d. calculate attack rates and case fatality ratios for total patients, u , o , and gender. . develop hypothesis. a. postulate sources of disease and mechanism of spread. b. estimate the population at risk of contracting disease and of dying from it. consider especially: those with limited access to health services pregnant and lactating ( ) infants not breast fed, children unvaccinated ( ) elderly . initiate control measures considering agent, host, and environment. a. what action has the community taken? b. identify local response capacity. ( ) what number and type of staff are locally available? ( ) what drugs and supplies are locally available? c. determine immediate unmet needs. ( ) specimen collection and lab diagnosis ( ) logistics ( ) support for clinical care-staff, drugs, and supplies ( ) support for environmental health d. undertake further necessary actions. ( ) case management with secondary prevention ( ) patient isolation ( ) health education ( ) agent and reservoir identification ( ) environmental decontamination ( ) primary prevention ( ) public information . inform authorities with investigation report. . initiate ongoing disease surveillance. during epidemic there is no clinical difference between them. other serogroups may cause disease in individuals, but not epidemics. when a suspected cholera serotype (strain) is isolated in the lab, one of the first tests performed is bacterial agglutination with o and o antisera. strains are thereby identified as v. cholerae o , o , or non-o /non-o . • if (+) agglutination to o antisera, then the strain is further tested for agglutination to antiserum of ogawa and inaba serotypes. • if (+) agglutination to o antisera, then the strain is not further subdivided (except as producer or nonproducer of ct as noted below). • if (−) agglutination to o and o antisera, then the strain is known as non-o , non-o v. cholerae. a strain is further identified as a producer or non-producer of cholera toxin (ct). ct production is a major determinant of disease development. strains lacking ct do not produce epidemics even if from the o or o serogroup. • serogroup o exists as main biotypes-classical and el tor-though hybrids also exist. each biotype occurs as two serotypes-ogawa and inaba. classic biotype caused the th and th pandemics but little epidemic disease since the s though it still causes cases in india. el tor biotype caused the th (current) pandemic and almost all recent outbreaks. el tor was first isolated in in el tor, egypt after importation by indonesian pilgrims travelling to mecca. it survives longer in the environment and produces ct similar to the classical biotype. presumably because of ct pathogenicity, the % of cholera patients with severe disease has doubled over the past yrs. these patients tend to require iv fluid therapy. • serogroup o may have evolved from strains of o el tor as they share many properties though not agglutination. in spring of in dhaka, o cases exceeded o el tor cases for the first time, and it was postulated that o may become the cause of an th pandemic. however, since then, o has again become dominant. infective dose depends on individual susceptibility. relevant host factors include immunity produced by prior infection with serogroup o as well as stomach acidity. id may be , orgs, so personal hygiene plays a lesser role than in shigellosis where the id is much lower. shigella has species. • s. dysenteriae type (sd or shiga bacillus) causes the severest disease of all shigella sp because of its neurotoxin (shiga toxin), longer duration of illness, higher abx resistance, higher cfr thru invasive complications, and great epidemic potential. • s. flexneri is the most common, and is generally endemic, in developing countries • s. sonnei is the most common in industrial countries • s. boydii and s. sonnei give mild disease. id may be orgs, so personal hygiene plays a greater role than in cholera. some kinds of e. coli produce a shiga toxin. shiga toxin genes reside in bacteriophage genome integrated into the bacterial chromosome. some abx, e.g. fluoroquinolones, induce expression of phage genes. the bacteria that make these toxins are variously called "shiga toxin-producing e. coli" (stec), "enterohemorrhagic e. coli" (ehec), or "verocytotoxic e. coli" (vtec). all terms refer to the same group of bacteria. • e. coli o :h (often called "e. coli o " or "o ") is the most commonly identified stec in north america, and it causes most e. coli outbreaks. approximately - % of ehec infections result in hus. • non-o stec serogroups also cause disease. in the usa, serogroups o , o , and o are the most commonly identified e. coli pathogens overall. diarrhea epidemiology is seasonally dependent. environmental temperature directly influences biologic activity-∆ °c is proportional to × risk of disease • temperate climates: bacterial diarrhea in warmer, humid season; rotavirus diarrhea in cooler, dry season • tropical climates: bacterial diarrhea in rainy season; rotavirus diarrhea year round with increased incidence in cooler season • most common pathogens for watery diarrhea-rotavirus, etec, v. cholerae; most important pathogen for epidemic watery diarrhea-v. cholerae • most common pathogens for dysentery-shigella species, salmonella species, campylobacter jejuni, clostridium difficile, eiec, ehec, e. coli o :h , entamoeba histolytica, yersinia enterocolitica; most important pathogens for epidemic dysentery-s. dysenteriae serotype (developing countries), e. coli o :h (developed countries) bangladesh has two seasonal cholera peaks: pre-monsoon with hot, humid weather (esp weeks - in apr-may) creating increased biological activity; post-monsoon (esp weeks - in aug-sep) with contamination of water sources. premonsoon epidemics are generally worse than post-monsoon ones. dysentery has low level year-round incidence, but epidemics occur roughly each decade. epidemic strains display new, additive antibiotic resistance which probably triggers the epidemic. once resistant strains have become endemic, antibiotic susceptibility rarely reappears. sd acquires resistance quickly. sf acquires it more slowly, and that resistance may wane with decreasing abx pressure. at icddr, annual proportional incidence approximates the following: • e. coli overall = % of cases, but etec = %. • e. coli tends to dominate before monsoon season and flooding. • cholera tends to dominate after monsoon season and flooding. • overall, - % of diarrhea cases may be vaccine-preventable. • % of pts have no pathogen identified. clean water and waste management for cholera. personal hygiene (hand washing with soap and clean towels) for shigella. water safe drinking water (boiled, chlorinated) nb sphere standards are not enough-you need increased quantities of chlorinated water at household level. san clean latrines for safe disposal of excreta hand washing with soap food safe food (cooked, stored) breast feeding fomites safe disposal of dead bodies with disinfection of clothing nb after outbreak of a fecal-oral pathogen, food hygiene and funereal practices may influence human-tohuman transmission more then water quality. health education to affected population wash hands with soap: after using toilets/latrines. after disposing of children's feces. before preparing food. before eating. before feeding children. is identical for all patients, and thus can't be given to pedes < yr because of volume loading. dukoral has been the main vaccine considered for use in high-risk populations. • morc-vax and shanchol-similar to dukoral except they do not contain the rbs, hence do not require a buffer, and are / the cost to produce. morc-vax, produced in vietnam, is derived from a vaccine administered to millions of people since , but is not who pre-qualified, and is not expected to have international distribution. shanchol, produced in india, has international distribution (e.g. used in the haiti cholera vaccination campaign of ), and is now the agent of choice for who. it confers immunity d p nd dose, effectiveness > % at mo, and protection > % at yr. also confers short-term protection vs etec. dose: . cc vaccine followed by water ingestion but no fasting needed; doses, wks apart; cold chain required except for day of use. orochol-bivalent formulation as in dukoral without rbs of ct. dose: single dose. no longer manufactured. who recommendations: "vaccination should not disrupt the provision of other high-priority health interventions to control or prevent cholera outbreaks. vaccines provide a short-term effect that can be implemented to bring about an immediate response while the longer term interventions of improving water and sanitation, which involve large investments, are put into place" [ ] . icddr recommendations: "because of limitations in terms of transport, formulation, and cost of the current dukoral vaccine, the cots program does not require the utilization of the vaccine during an outbreak; it is not necessary to vaccinate to overcome an outbreak. however, if dukoral is readily available and staff are properly trained in its use according to the guidelines that come with the vaccine, the cots program permits dukoral's use (ideally before an outbreak) in the following high-risk populations: refugee populations in which cholera is present, health care workers managing cholera cases, and communities in which the incidence rate is greater than in annually" [ ] . if undertaken, the following will apply: vaccination campaign requires numerous staff. community mobilizers are key. clinical staff should not be poached from their clinical duties. supervisors must be free to move at will. logistics is key-if the st day goes bad, the campaign goes bad. mark the domiciles which are done. hold after-action meetings each day. last day, use mobilizers with mobile broadcasting to find those missed. second phase vaccination should include chws with multi-purpose messages on water and sanitation. key lessons in epidemic response avoid: press exaggeration abx prophylaxis reliance on ivf and insufficient ors lab investigation of cases once epidemic etiology is ascertained prolonged hospitalization hospital discharge criteria requiring multiple negative stool cultures enthusiasm for ocv during epidemic exaggerated water purification objectives concentration of technical competencies in moh at expense of districts failure to share information with district stakeholders influenza viruses comprise genera-influenza types a, b, and c-each with species. • influenza type a is divided into subtypes based upon serological response to hemagglutinin (ha) and neuraminidase (na) glycoproteins. there are different ha subtypes and different na subtypes. h n , h n , and h n are responsible for the major human pandemics in the last century. h n virus circulated between and but currently does not. only influenza a subtypes infect birds, and all subtypes can do so. bird flu viruses do not usually infect humans. but, in , an outbreak of h n avian influenza in poultry in hong kong marked the first known direct human transmission of avian influenza virus from birds to humans. since then, h , h , and h avian influenza subtypes have been shown to infect humans. • influenza type b is morphologically similar to a and also creates seasonal and epidemic disease. • influenza type c is rare but can cause local epidemics. seasonal human influenza vaccine currently has strains-h n /h n /b. influenza disease in humans has a short incubation period ( - d) . early symptoms are non-specific. it is highly infectious, especially early in the course of the disease, with a large # of asymptomatic carriers. transmission potential (r ) is a function of infectivity, period of contagiousness, daily contact rate, and host immunity. in general, the faster the transmission, the less feasible is interrupting transmission thru usual disease control tools of case finding, isolation, contact tracing, and ring vaccination. case definitions may change and become more specific as epidemic evolves case management guidelines for communicable diseases with epidemic potential outbreak management protocol rapid response teams to investigate case reports epidemic investigation kits to mobilize specimens to collect labs to verify diagnosis and share specimens with peer labs pts to identify, isolate, and treat (ipd and opd settings) contacts to trace and ? quarantine hotline use and rumor investigation secondary prevention specific groups of exposed or at risk in the community-most likely to work when there is limited disease transmission in the area, most cases can be traced to a specific contact or setting, and intervention is considered likely to slow the spread of disease eg quara ntine of groups of people at known common source exposure (e.g. airplane, school, workplace, hospital, public gathering; ensure delivery of medical care, food, and social services to persons in quarantine with special attention to vulnerable groups) (useless once there is community-based spread) eg containment measures at specific sites or buildings of disease exposure (focused measures to > social distance) cancel public events (concerts, sports, movies) close buildings (recreational facilities, youth clubs) restrict access to certain sites or buildings community-wide measures (affecting exposed and non-exposed)-most likely to work where there is moderate to extensive disease transmission in the area, many cases cannot be traced, cases are increasing, and there is delay between sx onset and case isolation. eg infection control measures ari etiquette-cover nose/mouth during cough or sneeze, use tissues, wash hands avoidance of public gatherings by persons at high risk of complications nb use of masks by well persons is not recommended eg "snow" (stay-at-home) days and self-shielding (reverse quarantine) for initial d period of community outbreak-may reduce transmission without explicit activity restrictions eg closure of schools, offices, large group gatherings, public transport (pedes more likely to transmit disease than adults) nb community quarantine (cordon sanitaire)-restriction of travel in and out of an area is unlikely to prevent introduction or spread of disease international travel nb travel advisories to restrict international travel are generally useless in slowing epidemic spread nb health screening for fever and respiratory sx at ports of entry is also generally useless in slowing epidemic spread meningitis is a disease with significant mortality. meningococcus (neisseria meningitides) is renown for its rapid onset, rapid progression (death sometimes within hours), and high mortality ( % untreated). there are serogroups of neisseria meningitides but only (a, b, c, w, x, y) are known to cause epidemics. the bacteria spread from person to person via respiratory and nasal secretions. kissing, sharing eating and drinking utensils, cigarettes, coughing, and sneezing are recognized methods of transmission. close contacts over a period of time, as between household or dormitory residents, are most commonly affected. population movements (e.g. pilgrimages, displacement, military recruitment), poor living conditions, and overcrowding are epidemic risk factors. large, recurring epidemics of meningitis occur in the "meningitis belt" of sub-saharan africa where over million people live. this belt encompasses countries from senegal in the west to ethiopia in the east and as far south as tanzania and the democratic republic of congo. sub-saharan arica has epidemic seasonality. dry seasons and droughts favor epidemics. rains stop them. large regional epidemics, as well as epidemics in displaced populations and refugee camps, have mainly been due to meningococcus serogroup a. since , extensive use of meningococcal type a conjugate vaccine in the meningitis belt has reduced the incidence and case load of type a epidemics by nearly %. in , the most common lab confirmed meningitis isolate was streptococcus pneumoniae. in non-epidemic settings, neisseria.meningitidis, streptococcus pneumoniae, and haemophilus influenzae account for % of all cases of bacterial meningitis. prior to the availability of conjugate vaccines, h. influenza type b (hib) was the most common cause of childhood bacterial meningitis outside of epidemics. where hib vaccines are in the routine infant immunization schedule, hib meningitis has nearly disappeared. polysaccharide vaccines are available with serotypes (a and c), serotypes (a, c and w) or serotypes (a,c, w, and y). duration of immunity is approximately years. meningococcal protein conjugate vaccines confer longer immunity but at higher cost than polysaccharide vaccines. monovalent conjugate vaccine against group c dates from , and tetravalent (a, c, w and y) conjugate vaccine dates from . a group b vaccine made from bacterial proteins has been licensed since but is not readily available. meningococcal vaccines have a very low incidence of side effects. regular disease surveillance is necessary to detect outbreaks. the epidemic threshold is suspected cases/ , population in any given week. two suspected cases of meningitis in the same settlement should trigger an outbreak investigation. nasopharyngeal carriage rates do not predict epidemics. - % of meningococcal disease presents with meningitis. % of cases occur in patients < y/o. peak incidence in meningitis belt is ages - yrs. diagnosis is straightforward when patient presents with signs of meningitis-fever, headache, vomiting, changes in mental status. however, most patients have non-specific illness - days before onset of meningitis. cfr of untreated meningococcal meningitis can be %. cfr of properly treated meningococcal meningitis is < %. - % of meningococcal disease presents with septicemia unaccompanied by meningitis or other focal features. it is a dramatic illness which affects previously healthy children and young adults. it presents with acute fever leading to purpura fulminans (hemorrhagic or purpuric rash), shock, and waterhouse-friderichsen syndrome (acute adrenal failure). etiologic diagnosis can be easily missed. cfr of meningococcal septicemia is % and may be % even with proper treatment. diagnosis may be confirmed by agglutination tests, polymerase chain reaction, culture and sensitivity testing of spinal fluid and blood. in many situations, these tests are not available. throat swabs may be helpful on occasions. do not delay treatment for tests or test results. minutes count. it is more important to have a live patient without a confirmed diagnosis than a dead one with a diagnosis. differential diagnosis in a tropical patient with fever and altered mental status, but without purpura or shock, includes cerebral malaria. co-infection may occur. standardized case management of bacterial meningitis in developed countries involves - days of parenteral antibiotic therapy. drug of choice in adults and older children is ceftriaxone which also rapidly eliminates the carrier state. alternate drugs include ampicillin and benzylpenicillin which do not eliminate the carrier state. in developing countries, days of parenteral antibiotic therapy are empirically shown to be effective. in large epidemics in resource-poor settings, a single im dose of chloramphenicol in oil is the drug of choice. for patients who do not improve in h, a repeat dose may be given. viral meningitis is rarely serious and requires only supportive care, recovery is usually complete. patient isolation and disinfection of the room, clothing, or bedding are not necessary. respiratory precautions are advised particularly early in the course of treatment. chemoprophylaxis of contacts is available in some settings but rarely in the disaster setting. vigilance and education of close contacts is mandatory. epidemic preparedness and early detection of outbreaks are key. vaccines against n. meningitides serogroups a, c, y and w are very effective in controlling epidemics. in epidemic settings, children - are the priority target with serogroups a and c typically the priority antigens. rapid mass vaccination campaigns can contain outbreaks in - weeks. for immunocompetent patients over years, vaccine efficacy rate is % one week after injection. however, duration of immunity may be as little as years in younger children. in some countries, vaccine may also be used with close contacts of sporadic disease cases to prevent secondary cases. chemoprophylaxis of contacts is not recommended in epidemics, but community education and ready access to health care are essential. source control/reduction/elimination avoid unnecessary contact with suspected reservoir animals and known disease carrier species (e.g. primates). avoid direct or close contact with symptomatic patients. undertake quarantine and culling of sick reservoir animals and known disease carrier species. avoid unnecessary contact with or consumption of dead reservoir animals or known disease carrier species. establish appropriate communicable disease controls for burial of the dead. administrative controls environmental and engineering controls avoid needle stick exposure to blood specimens thru automated machine handling ppe use standard precautions-gloves, masks, and protective clothing-if handling infected animals or patients. wash hands after visiting sick patients. active surveillance and contact tracing (enhanced surveillance) through community-based mobile teams active case finding (screening and triage) and contact tracing dedicated isolation facility food provision to isolated patients so they are not dependent on family case definition treatment protocols emphasizing supportive care and treatment of complications essential drugs referral guidelines secondary prevention barrier nursing strictly enforced family and community education ministerial task force to address policy local health authority task force to address procedures national level task forces to comprise guidance note on using the cluster approach to strengthen humanitarian response panel on humanitarian financing report to the united nations secretary-general. too important to fail-addressing the humanitarian financing gap belgian development corporation, government of bulgaria, government of canada, et al. the grand bargain-a shared commitment to better serve people in need available from usaid's development experience clearinghouse gender equality and female empowerment policy national strategy for pandemic influenza selective primary health care-an interim strategy for disease control in developing countries ten great public health achievements-united states ten great public health achievements-united states water and excreta-related diseases: unitary environmental classification infections related to water and excreta: the health dimension of the decade addendum to ipc technical manual version . . tools and procedures for classification of acute malnutrition. rome: ipc global partnership integrated food security phase classification technical manual version . . evidence and standards for better food security decisions nbc domestic preparedness training hospital provider course. undated. curriculum available from the center for domestic preparedness sampling guide interpreting and using mortality data in humanitarian emergencies-a primer for non-epidemiologists. humanitarian practice network, network paper no . london: overseas development institute measuring mortality, nutritional status, and food security in crisis situations: smart methodology. v infectious diseases-a clinical approach cholera vaccines: who position paper cholera outbreak training and shigellosis (cots) program [cd-rom version . , undated history and epidemiology of global smallpox eradication retrieved from us department of health and human services geneva: world health organization. laboratory available? . what tests does it perform? . is there transport to and from the laboratory? . who prepares transport media? . who provides specimen collection material and supplies? . how can these supplies be obtained? . who provides cool packs, transport boxes, car, driver …? . what forms/information must be sent with the specimens? how does the epidemiologist obtain results? if a lab is not available, then you need a sampling strategy that addresses specimen acquisition, preparation, and transportation in compliance with international regulations on the transport of infetious substances. reference . world health organization department of communicable disease surveillance and response. highlights of specimen collection in emergency situations. undated. available from who laboratory and epidemiology capacity strengthening office regulation ) . leak-proof specimen container wrapped with enough absorbent material to absorb the entire content of the st container . leak-proof secondary container usually plastic or metal . outer shipping container whose smallest dimension is mm diagnostic specimens use iata packing instruction without biohazard label. infectious materials use iata packing instruction with biohazard label. what to send with the sample? lab request form with: • sender's name and contact info • patient name, age, sex • sample date, time • suspected clinical diagnosis with main signs and symptoms • sample macroscopic description • context-outbreak confirmation, ongoing verification, outbreak end, etc. • epidemiological or demographic data where to send the sample? a. prior to seasonal epidemic . establish a national coordinating committee (ncc). . designate a lead agency and lead official in the ncc. . establish a local coordinating committee (lcc). . designate a lead official in the lcc. . anticipate roles for partner agencies (e.g. inter-agency and team coordination, disease surveillance, field epidemiological investigation, laboratory identification, case management guideline development, outbreak logistics, public information, and social mobilization). . identify sources of funds. . intensify disease surveillance. . identify reference lab(s) for communicable diseases of epidemic potential. . ensure mechanism for specimen transport. initial response to suspected outbreak . form an emergency team to investigate and manage the outbreak a. identify key roles on the outbreak investigation team(s) ( ) epidemiology and surveillance ( ) case management ( ) water and sanitation ( ) laboratory services ( ) communication b. staff those roles ( ) epidemiologist-to monitor proper data collection and surveillance procedures ( ) physician-to confirm clinical s/sx and train health workers in case management ( ) water and sanitation expert-to develop a plan for reducing sources of contamination ( ) microbiologist-to take environmental/biological samples for laboratory confirmation, train health workers in proper sampling techniques, and confirm use of appropriate methods in the diagnostic laboratory ( ) behavior change communication (bcc) specialist-to assess the population's reaction to the outbreak, create, and disseminate appropriate health messages outbreak investigation protocol in place rapid response teams to investigate case reports epidemic investigation kits to mobilize specimens to collect labs to confirm dx of v. cholerae, s. dysenteriae, other shigella, and e. coli o :h dipstick identification on representative sample of specs is useful for cholera, but c&s is essential because dipsticks are not available for shigella, etec. vibrio are hardy if kept moist and cool. they can survive a week in cary blair media. shigella are fragile and difficult to recover if transport time > d. - isolates initially to confirm outbreak - isolates initially to create abx use policy (bacterial resistance renders cotrimoxazole, amp/amox, nalidixic acid, and tetracycline unusable) - isolates monthly from ipd and opd before abx therapy to assess evolving abx resistance - isolates periodically to reference laboratory to confirm abx resistance patterns and undertake molecular studies isolates at end of the outbreak to confirm that new diarrheas are not epidemic pathogens nb systematic sampling is most representative-e.g. every th pt or all pts q weeks adjusted as needed to collect the necessary specs. sensitivity >> important than specificity in rdt screening during an epidemic. pts from one geographic area are more likely to constitute a cluster involving a new pathogen. an area may be considered cholera-free after incubation periods (total of d) have passed without cholera disease. however, hospital monitoring should continue for a year due to tendency of enteric pathogens to re-emerge long after they are declared gone. cholera may be viable but nonculturable from the environment; environmental monitoring has many false negatives. consider improvements to existing diagnostic labs hotlines set up for reporting of rumor health reference and educational materials in place case definitions case management and referral guidelines for communicable diseases with epidemic potential pt, provider, and community educational materials specimen handling protocols epidemic command & control center established under local health authorities using principles and practices of incident mgmt unified command of multi-disciplinary specialists information channel to government and stakeholders support by government for technical actions coordination with technical sectors-particularly wash (cfr is a function of case mgmt, but ar overall is a function of wash) water supply, purification, and distribution systems bucket chlorination is low tech but reasonable way to reach individual hh or small communities water treatment units need ca hypochlorite, chlorimetric, and colimetric monitoring devices chlorinators worth considering at water sources of high public demand and epidemic activity hygiene promoters with environmental health assessors to address hand and food hygiene in communities around the outbreak area (think ring vaccination with knowledge) safe disposal of medical waste and infectious sludge from treatment facilities medical logistics-resource prepositioning and stockpiles cots (take one and have carpenter make copies) plastic sheets with defecation hole or sleeve buckets ( white color for stool-enables recognition of diarrhea color; different color for emesis; different color for domestic waste) ivf, iv sets, iv poles or suspension cords (cholera kits) key: cord- -qyf vymf authors: sica, valentina; izzo, valentina title: pathophysiologic role of autophagy in human airways date: - - journal: autophagy networks in inflammation doi: . / - - - - _ sha: doc_id: cord_uid: qyf vymf lung diseases are among the most common and widespread disorders worldwide. they refer to many different pathological conditions affecting the pulmonary system in acute or chronic forms, such as asthma, chronic obstructive pulmonary disease, infections, cystic fibrosis, lung cancer and many other breath complications. environmental, epigenetic and genetic co-factors are responsible for these pathologies that can lead to respiratory failure, and, even, ultimately death. increasing evidences have highlighted the implication of the autophagic pathways in the pathogenesis of lung diseases and, in some cases, the deregulated molecular mechanisms underlying autophagy may be considered as potential new therapeutic targets. this chapter summarizes recent advances in understanding the pathophysiological functions of autophagy and its possible roles in the causation and/or prevention of human lung diseases. lung diseases are some of the most common medical conditions in the world. the lung has the principal aim to mediate gas exchange [ ] . for this reason, the lung can be subjected to several insults, belonging to the environment (inspiration of foreign matter, particles, smoke), reactive oxygen species (ros) production, biological origins (e.g., viruses, bacteria), changes in o tension, and mechanical stresses (e.g., mechanical ventilation). it is possible to discriminate between diseases affecting: (i) the airways (asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, acute bronchitis and cystic fi brosis); (ii) the interstitium (sarcoidosis, idiopathic pulmonary fi brosis, autoimmune diseases, pneumonias and pulmonary edemas); (iii) the blood vessels (pulmonary embolism and hypertension); the pleura (pleural effusion, pneumothorax and mesothelioma); (iv) the chest wall (obesity hypoventilation syndrome and neuromuscular disorders). the development of lung diseases can be associated to both acute and chronic exposure to such insults. however, in most conditions, a favouring genetic is necessary [ ] . yet, the lung has various inducible defence mechanisms to protect itself. first, constitutive and inducible stress protein and antioxidant defences; second, innate immune responses; third, pro-and anti-apoptotic mechanisms [ , , ] . several studies have recently pinpointed the emerging role of macroautophagy (more often and hereby referred to as autophagy) in lung homeostasis and diseases. autophagy is a catabolic process that involves the sequential sequestration of cytoplasmic material within double-membraned vesicles (autophagosomes), the fusion of autophagosomes with lysosomes, and the degradation of autophagosomal cargoes (as well as of structural autophagosomal components) by lysosomal hydrolases [ ] . autophagy is mediated by a genetically encoded, evolutionary conserved machinery that is connected to most, if not all, major biochemical processes of the cell, including core metabolic circuitries as well as signal transduction pathways initiated by plasma membrane receptors [ ] . basically, autophagy responds to three major organismal needs: ( ) it preserves cellular homeostasis in physiological conditions; ( ) it plays a key role in cellular adaptation to stressful stimuli; and ( ) it participates in the communication of states of the danger to the whole organism [ ] . indeed, autophagy continuously operates to mediate the disposal of potentially dangerous structures that may otherwise accumulate in the cytoplasm as a consequence of normal cellular activities, like old (and damaged) organelles or protein aggregates [ ] . moreover, the autophagic fl ux is highly responsive to situations in which intracellular or extracellular homeostasis is perturbed, which generally involves either an increased offer of autophagic substrates (as it occurs in the course of viral infection) or an increased need for autophagic functions or products (as it occurs in response to nutrient deprivation) [ ] . in both these settings, profi cient autophagic responses are required for the optimal adaptation of cells to stress, as demonstrated in experiments involving pharmacological inhibitors of autophagy or the depletion of essential components of the autophagic machinery [ ] . finally, autophagy is required for cells experiencing so-called "oncogenic stress" (i.e., the boost of cellular functions driven by activating mutations in one oncogene or loss-of-function mutation in one tumor suppressor gene) to become senescent (a cell-intrinsic oncosuppressive mechanism) while secreting immunostimulatory cytokines and expressing on their surface ligands for activatory natural killer (nk)-cell receptors (hence triggering a cell-extrinsic mechanism of tumor suppression) [ ] . along similar lines, cancer cells succumbing to a peculiar form of apoptosis known as "immunogenic cell death" are able to recruit antigen-presenting cells and hence trigger an adaptive immune response only if they secrete atp as they die, a process that requires profi cient autophagic responses [ , ] . it should be noted that autophagy has also been causally implicated in some instances of cell death, especially in lower organisms like drosophila melanogaster [ , ] . however, in mammals autophagy mainly mediates robust cytoprotective functions, and -when cellular homeostasis is irremediably compromised -contributes to the maintenance of organismal homeostasis by playing a role in danger signalling. in line with this notion, defects in the autophagic machinery have been associated with a wide panel of human pathologies, including (but not limited to) malignant diseases, neurodegenerative disorders, as well as cardiovascular, renal and pulmonary conditions [ ] . an accurate description of the autophagy pathway and its role in immunity and infl ammation has been provided in several previous chapters of this book; therefore, here we will focus on the impact of autophagic in the etiology and treatment of human pulmonary diseases. acute lung injury (ali) and the acute respiratory distress syndrome (ards) describe clinical syndromes of acute respiratory failure with substantial morbidity and mortality. ali is characterised by acute infl ammation that causes disruption of the lung endothelial and epithelial barriers. the ali cellular features include loss of alveolar-capillary membrane integrity, excessive transepithelial neutrophil migration, and release of pro-infl ammatory, cytotoxic mediators. the treatment of ali is predominantly based on ventilatory strategies [ ] . however, prolonged exposure to high oxygen therapy (hyperoxia) can result in lung injury [ ] . few studies are present in the literature concerning the role of autophagy in ali, even so these works support the hypothesis that activation of autophagy has a protective role in this disease. it has been demonstrated that prolonged hyperoxia, which causes characteristic lung injury in mice, induced the increase of lc ii expression. moreover, in pulmonary epithelial cells, the genetic depletion of lc sentitizes the cells to hyperoxia-induced cell death suggesting that lc activation confers cytoprotection in oxygen-dependent cytotoxicity [ ] . besides, the involvement of mitophagy has also been identifi ed. the ability to resist hyperoxia is proportional to pten-induced putative kinase (pink ) expression. in fact, the pink −/− mice were more susceptible to hyperoxia when compared to wild-type mice. furthermore, genetic deletion of pink or pink silencing in the lung endothelium cells increased susceptibility to hyperoxia via alterations in autophagy/mitophagy, proteasome activation, apoptosis and oxidant generation [ ] . chronic obstructive pulmonary disease (copd) is a chronic infl ammatory lung disease that causes breathing diffi culty, cough, sputum production and dyspnoea. emphysema and chronic bronchitis can contribute to copd development. emphysema is a condition resulting from a severe damage of air sacs (the alveoli). chronic bronchitis is due to infl ammation of the lining of the bronchial tubes. the lung damage that leads to copd is caused by long-term exposure to irritating gases or particulate matter, most often from cigarette smoke (cs), air pollution or workplace exposure to dust, smoke or fumes. however, a genetic susceptibility to the disease should be considered as an important cofactor. patients with copd present increased risk of developing other pathologies, such as heart disease or lung cancer [ ] . multiple molecular mechanisms, not fully understood, participate to the copd evolution and, among others, the involvement of the autophagic pathway has been pointed out [ , ] . in lung tissue from copd patients, an increase of autophagic vacuoles as well as several autophagy markers (lc , atg , atg / , atg ) expression has been detected [ ] . these evidences are perhaps a result of defective autophagic fl ux. to corroborate this hypothesis, an increased accumulation of p and ubiquitinated proteins and a decreased expression levels of sirtuin (sirt ) have been evaluated in lung homogenates from copd patients [ ] . kuwano and colleagues hypothesize that the insuffi cient autophagic clearance is involved in the accelerated cell senescence observed in copd [ , ] . the cs induces mitochondrial damage, accompanied by increased ros production in vitro . the cs-induced mitophagy was inhibited by pink and park knockdown, resulting in enhanced mitochondrial ros production. moreover, a decreased expression of park in copd lungs compared with non-copd lungs has been detected, suggesting that insuffi cient mitophagy is a part of the pathogenic sequence and cellular senescence of copd [ ] . in addition, a defective xenophagy has been observed in alveolar macrophages of smokers, suggesting that the deregulation of this selective process may contribute to recurrent infections [ ] . in contrast, other fi ndings indicate that autophagy has an opposite role in copd favouring the pathological environment. it has been shown that rtp (also known as redd ) expression is increased in human emphysematous lungs and in lungs of mice exposed to cs, whereas rtp knockout mice were protected against acute cs-induced lung injury. rtp inhibits mammalian target of rapamycin (mtor), by stabilizing the tsc -tsc inhibitory complex. the inhibition of mtor is linked to autophagy induction, but rtp expression enhances oxidative stress-dependent cell death, amplifying the development of cs-induced lung injury [ ] . furthermore, the higher expression of autophagy proteins has been linked to lung epithelial cell death, airway dysfunction and emphysema in response to cs. genetic depletion of lc b in vivo ( map lc b −/− mice) suppressed cell death and emphysematous airspace enlargement during chronic cs exposure compared to the wild type mice [ ] . more recently, the same group demonstrated that mitophagy regulates necroptosis, which contributes to the copd pathogenesis. mice defi cient for pink were protected against mitochondrial dysfunction, airspace enlargement and mucociliary clearance (mcc) disruption during cs exposure [ ] . interestingly, they identifi ed the contribution of a novel selective autophagy-dependent pathway that regulates cilia length, "ciliophagy", in the copd pathophysiological evolution. exposure to cs reduced cilia length and autophagy-impaired ( beclin +/− or map lc b −/− ) mice resisted to the cs-induced cilia shortening via a mechanism involving histone deacetylase (hdac ) [ ] . accordingly, it has been shown that autophagy negatively regulate ciliogenesis by the degradation of the essential ciliary protein ift [ ] . conversely, hedgehog (hh) signalling from primary cilia promotes autophagy [ ] and autophagy promotes ciliogenesis by degrading ofd (oral facial digital syndrome) at centriolar satellites [ ] . further studies are necessary to clarify the dual relationship between these processes [ ] . in conclusion, these studies illustrate that the contribution of autophagy in copd pathophysiology is complex and show a context-specifi c role depending on the cell type and tissue as well as on the different stimuli involved. interstitial lung disease (ild) is a general category that includes all lung diseases affecting the interstitium, the tissue and space that extends throughout both lungs. among them the most common are sarcoidosis and idiopathic pulmonary fi brosis (ipf). sarcoidosis is a systemic infl ammatory disease caused by persistent reaction toward a stimulus (virus or antigens) that continues even when it is physiologically cleared from the body. lung interstitium fi brosis is the fi rst symptom in patients with sarcoidosis. conversely, ipf is characterized by specifi c fi brosis at interstitial level due to the increased extracellular matrix (ecm) protein deposition and hyper activation of myofi broblasts [ ] . recently, reduced lc ii expression and p accumulation has been found in lung tissue from ipf patients [ ] . the reduced expression of the transcription factor foxo a in ipf fi broblasts could be the cause for the reduction in the levels of lc protein as the expression of this latter is positively stimulated by foxo a [ ] . furthermore, in fi broblast of ipf patients, decreased expression in beclin- protein and increased expression of the anti-apoptotic protein bcl- have been found, confi rming a defect in the autophagy pathway at different level [ ] . moreover, fi broblastic foci (ff), that are the starting point for fi brogenesis, are enriched in ubiquitinated proteins and p , confi rming the insuffi cient autophagy at the basis of ipf pathogenesis [ ] . autophagy inhibition is able to induce acceleration of epithelial cell senescence and fi broblast to myofi broblast differentiation (fmd), which have a critical role in ipf development [ ] . transforming growth factor-β (tgf-β ) is one of the essential mediators of fi brosis since it stimulates fi broblasts to produce fi bronectin and the smooth muscle-α actin (α-sma), which is a myofi broblast marker. autophagy has been associated to fi brosis through tgf-β . in fact, genetic deletion of lc or beclin increases tgf-β activity as well as in vivo treatment with rapamycin can protect from fi brosis [ ] . tgf-β expression seems to be dependent on il- a, a proinfl ammatory cytokine involved in chronic infl ammation and autoimmune disease. blocking il- a might reduce the progression of fi brosis promoting the autophagic degradation of collagen [ ] . recently, lacking of matrix metalloproteinases- (mmp- ) has been associated with exacerbated fi brosis in the hyperplastic alveolar epithelium of ipf lungs [ ] . additionally, mmp- -defi cient mice exhibit diminished atg c protein expression, demonstrating a direct correlation between these two pathways [ ] . similar evidences from an independent group corroborate the role of autophagy in promoting fmd. in fact, atg b-defi cient mice exhibited reduction in autophagic activity in lungs, collagen accumulation and increased protein levels of the myofibroblast biomarker α-sma [ ] . pharmacological treatment with the alkaloid barberine has been proposed for ipf monitoring because of its capacity to inhibit the activation of mtor and to increase the expression of lc and beclin in an bleomycin in vivo model of airway-fi brosis [ ] . furthermore, the multiple tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of ips for its anti-fi brotic effect. it has been shown that nintedanib is able to reduce the expression of ecm proteins, fi bronectin and collagen as well as to induce a beclin dependent, atg independent autophagy [ ] . asthma is a chronic respiratory disease affecting million people worldwide. asthma manifests through several symptoms including wheezing, breathlessness, and chest tightness. asthmatic airways are characterized by chronic infl ammation, eosinophil infi ltration, epithelial fi brosis, mucus hyperproduction, and goblet cell hyperplasia [ ] . it is considered as chronic allergic infl ammatory disease, mostly mediated by a th response, but an initial th -type immune response seems to be the trigger for the subsequent th -type response [ ] . thus, th hyperactivation leads to persistent airway infl ammation and the occurring of asthma phenotype [ ] . emerging evidences suggest that activation of autophagy is associated with reduced lung function in asthmatic patients. in particular electron microscopy analysis of fi broblast and epithelial cells from asthmatic patients showed increased autophagic hallmarks "such as double membrane autophagosomes" compared to healthy patients [ ] . unfortunately, at present, the role of autophagy in asthma is still unclear. a recent study demonstrated that two single nucleotide polymorphisms (snps), namely rs and rs were associated with childhood asthma. in particular rs localises at the promoter of atg gene and could increase its expression in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic patients [ ] . another intronic snp variant (rs ) in atg gene was also shown to be associated with pre-bronchodilator forced expiratory volume in second (fev ) in asthmatic patients [ ] . atg is an essential player in the initiation of autophagy, but its role in asthma pathogenesis is controversial. on one hand atg could help viral elimination through the activation of xenophagy, and on the other hand it negatively regulates the antiviral properties of type i interferon (ifn) inhibiting innate anti-virus immune responses [ , ] . together with these fi ndings, lungs from conditional atg knockout mice manifest hyper-responsiveness to cholinergic stimuli, which is a common sign of asthma and chronic infl ammatory diseases [ ] . asthma severity has been directly correlated with the level of autophagic response in the sputum granulocytes, peripheral blood cells and peripheral blood eosinophils of severe and non-severe asthmatic patients [ ] . autophagy is also involved in the maintenance of intracellular ros homeostasis, and it has been well established that oxidative stress is associated with asthma so that exhaled levels of hydrogen peroxide (h o ) and nitric oxide (no) are currently used as predictors of asthma severity [ ] . chronic asthma is characterized by excessive ecm deposition and proliferation of myofi broblasts, leading to fi brosis in the airway wall [ ] . the accumulation of fi brotic tissue is mostly due to the production of collagen a and fi bronectin by the primary human airway smooth muscle through a mechanism autophagy-dependent that involves the tgfβ . this response is reverted by the silencing of the major key autophagy-inducing gene atg and atg [ ] . as already mentioned, asthma is a pathology mostly driven by th -type cytokines. among them, il- is extensively produced in activated cd + th lymphocytes and is overexpressed in the airway epithelium of asthmatic patients [ ] . here, il- is thought to be responsible for epithelial hypertrophy, mucus hypersecretion, adventitial fi brosis and goblet cell hyperplasia [ ] . it directly induces hypersecretion of mucin ac, oligomeric mucus/gel forming (muc ac) in airway epithelial cell and oxidant stress through a mechanism that is autophagy-dependent, as demonstrated in vitro by depletion of atg or atg in primary human trachealbronchial epithelial cells [ ] . autophagy might be involved in the pathophysiology of alternaria (alt-e)associated asthma. alt-e is an outdoor allergen able to activate autophagy, which in turn stimulates epithelial cells to release il- [ ] . this latter when produced is able to stimulate th differentiation from naïve cd + t-cells and ifn-γ production by th cells. il- level in serum of asthmatic patients might refl ect the degree of disease exacerbation [ ] . cystic fibrosis (cf) is one of the most common lethal genetic diseases in caucasian population. it is an autosomal recessive disease caused by mutation in the cystic fibrosis transmembrane conductance regulator (cftr) gene. approximately out of caucasians are carriers for mutation in this gene. up to date over types of different mutations have been discovered and classifi ed according to the degree of functional cftr protein ( http://www.genet.sickkids.on.ca/statisticspage. html ; [ ] ). among these, the most common one is the f del-cftr. approximately % of cf patients have at least one f del-cftr allele, and about % are homozygous for it. the cftr channel is located at the apical surface of epithelial cells and it is deputized to move out cl − from the cell. na + passes through the membranes passively, increasing the movement of water by osmosis. loss of functional cftr expression is thought to alter this homeostatic balance through the epithelial layer, leading to net volume depletion of mucus, increased viscosity, and ineffective bacterial clearance [ , ] . recurrent pulmonary infections in turn induce an increased infl ammatory response and signalling, thus starting a vicious cycle of mucus retention, infection, and infl ammation. since the cftr is localized in many organs, cf symptoms could go from malabsorption at pancreatic level and gastrointestinal obstruction to male infertility and liver disease. nevertheless, the main cause of death remains persistent and untreatable pulmonary pseudomonas aeruginosa infection. several recent studies have demonstrated an impairment of autophagy in cf. in fact, in epithelial cells, mutated/unfunctional cftr causes increased ros production with consequent increase in tissue transglutaminase type (tg ) levels. tg , in turn, leads to crosslinking of several targets including beclin [ , ] . beclin interactome displaces from the endoplasmic reticulum (er) leading to the sequestration of class iii-phosphoinositide -kinase (pi k) complex, accumulation of p with consequent inhibition of autophagosomes formation. the resulting accumulation of aggresomes leads to proteasome overload and may promote the accumulation of mutated cftr in intracellular aggregates [ ] . restoration of beclin activity, depletion of p by genetic manipulation or treatment with autophagy-stimulatory proteostasis regulators, such as cystamine, functionally rescue the cftr mutated protein at the apical surface of epithelial cells both in vitro and in vivo [ ] . heme oxygenases are enzymes involved in the catabolism of the heme ring to generate carbon monoxide, biliverdin-ixα, and ferrous iron. the inducible isoform heme oxygenase- (ho- ) is activated in response to stress such as oxidative stress, hypoxia, heavy metals exposure and cytokines. ho- , together with its enzymatic products, is able to inhibit apoptosis and related cell death pathways, conferring tissue protection in case of lung or vascular injury [ ] . ho- could represent the link between cf and impaired autophagy since its expression is increased in human bronchial cf cells. this increase has been associated either to the reduction of apoptosis/injury during p. aeruginosa challenge either to the expression of infl ammatory mediators [ ] . other evidences suggesting the cytoprotective role of ho- in cf showed that lipopolysaccharide (lps)-challenged cf macrophages fail to compartmentalize ho- to the cell surface and this mechanism seems to be dependent on the reduction in caveolin- (cav- ) expression [ ] . in fact, when ho- localises at the plasma membrane, is able to form a complex with cav- , which in turn binds and detaches myd from its complex with tlr thus terminating the cell death signal [ ] . autophagic clearance of bacteria (so-called xenophagy) could also be impaired in case of disease, inducing increased bacterial infection that is one of the most frequent injuries in cf patients [ ] . in fact it has been demonstrated that burkholderia cenocepacia has the capacity to survive in f del-cftr macrophages since immediately after the engulfment, the bacteria resides on lc -positive vacuoles that appear as arrested autophagosomes [ ] . this capacity is directly correlated to the levels of p , so that its depletion leads not only to a decreased bacterial survival in macrophages but also to the release of beclin from aggresomes allowing its recruitment to the b. cenocepacia vacuole and bacterial clearance via autophagy [ ] . b. cenocepacia represents a serious threat for cf patients since the infection results in persistent lung infl ammation and the bacteria are resistant to most of all available antibiotics [ ] . similar fi ndings showed that pharmacological or molecular inhibition of autophagy reduces the clearance of intracellular pseudomonas aeruginosa in vitro [ ] . treatment of cf mice with the mtor inhibitor rapamycin decreases bacterial burden in the lungs and drastically reduces signs of lung infl ammation [ ] . in a normal situation, autophagy can help not only removing polyubiquitinated protein but also controlling bacteria clearance; for these reasons novel strategies aimed at restoring autophagy are emerging as promising therapeutic approaches for cf patients [ ] . aatd is a hereditary disorder characterized by a low serum level of alpha- -antitrypsin (aat), a kda serine protease inhibitor, member of the serpin family [ ] . aat is essentially synthetized in the liver and secreted into the bloodstream, where it controls tissue degradation by the enzyme neutrophil elastase. the defi ciency in aat is associated with liver and lung disease due to the loss of anti-infl ammatory and antiproteolytic functions. the majority of patients with aat defi ciency are homozygotes for a missense mutation ("piz mutation": lysine replaces glutamic acid at position ) that alters protein folding. mutant aat molecules polymerize and aggregate in the er of hepatocytes, forming large intrahepatocytic globules, the characteristic features of this disease. the proteasome is responsible for degrading the soluble form of att by means of er-associated degradation while autophagy is involved in disposal of insoluble att polymers and aggregates [ ] . in fact, a signifi cant accumulation of autophagic vacuoles was found in vitro and in vivo in liver cells from aatd patients as well as in piz mouse model [ , ] . whereas in absence of autophagy the degradation of aat was retarded [ ] . moreover, it has been demonstrated that the stimulation of autophagy by carbamazepine or rapamycin treatment or by liver-directed gene transfer of transcription factor eb (tfeb), a gene regulating lysosomal function and autophagy [ ] , reduce the hepatic amount of aat as well as the hepatic fi brosis in mice expressing mutant aat [ , , ] . although these results should be corroborated, altogether indicate that autophagy exerts a protective role in aatd and open a real possibility to treat aatd with pro-autophagic molecules. pulmonary hypertension (ph) was fi rst identifi ed in by ernst von romberg. ph is a severe and progressive disease that consists in increased blood pressure of lung vasculature and, often, can be a complication of chronic lung disease [ ] . since the pathology has been classifi ed, by the world health organization (who), in fi ve groups on the basis of mechanisms underlying the pathogenesis of the multiple types of ph. the role of autophagy in pulmonary hypertension has mainly been described in correlation with pulmonary arterial hypertension (pah), who group i. little is known about the aetiology of ph, one of the most frequent genetic mutations causing idiopathic inherited form of ph is found in the gene encoding bone morphogenetic protein (bmp) receptor type-ii (bmpr ). in pah, the pulmonary artery smooth muscle cells (pasmcs) proliferate excessively and are resistant to apoptosis. chloroquine, a known inhibitor of autophagy fl ux, has been described as a drug preventing experimental pah progression. the induction of pah, by monocrotaline, in rat is associated with increased autophagy and decreased bmpr protein expression. the inhibition of autophagy by chloroquine ameliorates the level of bmpr , inhibits the proliferation and stimulates apoptosis of rat pasmcs [ ] . a recent publication [ ] confi rms that the inhibition of autophagy, by overexpressing mtor, is a promising therapeutic strategy against pah. however, the role of autophagy in ph is still unclear and controversial, in fact, its protective role has been described in the initial phase of the pathogenesis of ph. histochemical analysis of samples obtained from human ph lungs and mouse exposed to chronic hypoxia, showed an increase in the lipidated form of lc and in egr- , which regulates lc expression. moreover, lc −/− or egr- −/− , but not beclin +/− mice are more susceptible to ph and in vitro lc knockdown cells showed an increase of hypoxic cell proliferation, suggesting a role for lc in the adaptation during vascular remodelling under hypoxia [ ] . in most organs, including the lung, autophagy robustly counteracts malignant transformation, i.e. , the conversion of a healthy cell into a (pre-)neoplastic cell, and several mechanisms related to the ability of autophagy to preserve cellular or organismal homeostasis account for such a pronounced oncosuppressive activity [ ] . indeed, besides being required for oncogene-induced senescence and anticancer immunosurveillance (see above) [ ] , autophagy promotes the maintenance of genomic integrity by multiple mechanisms [ ] . first, it mediates the degradation of damaged mitochondria, which are prone to overproduce genotoxic ros and other redox active entities of endogenous and exogenous origin [ ] . second, profi cient autophagic responses appear to be required for optimal dna damage responses [ ] . third, autophagy is involved in the disposal of potentially oncogenic retrotransposons and micronuclei [ ] . moreover, autophagy generally mediates anti-infl ammatory effects, and chronic infl ammation is known to accelerate oncogenesis (at least in some tissues, including the lung) [ ] . finally, it has been proposed that autophagy is required for the preservation of normal tissue architecture, in particular at the level of the stem-cell compartment [ ] . although little is known on the deregulation of stem cells in pulmonary carcinogenesis, it cannot be excluded that autophagic defects may promote malignant transformation in the lung also via this mechanism [ ] . conversely, the ability of autophagy to preserve genomic and redox homeostasis seems very relevant in the context of lung tumorigenesis, which in a signifi cant proportion of cases is associated with tobacco smoking or exposure to environmental nanoparticles like asbestos crystals [ ] . indeed, the oncogenic effects of both smoking and asbestos have been linked to their ability to cause ros overgeneration along with genetic/genomic defects and chronic infl ammatory responses [ ] . all these effects are limited, at least to some extent, by profi cient autophagic responses. irrespective of the precise mechanisms whereby autophagy counteracts malignant transformation in the lung, various genetic interventions aimed at specifi cally disabling autophagy in the lungs have been shown to promote malignant transformation driven by several oncogenes, including mutated b-raf proto-oncogene, serine/threonine kinase ( braf ) [ ] , epidermal growth factor receptor ( egfr ) [ ] , kirsten rat sarcoma viral oncogene homolog ( kras ) [ , ] . intriguingly enough, in one of these models, accelerated oncogenesis caused by the lung-specifi c inactivation of atg was linked to increased tumor-infi ltration by immunosuppressive cd + cd + foxp + regulatory t cells [ ] . moreover, the concomitant bi-allelic inactivation of serine/threonine kinase (stk , best known as lkb ) and phosphatase and tensin homolog (pten), two tumor suppressor genes that inhibit autophagy [ , ] , has been shown to cause the formation of pulmonary squamous cell carcinomas that express high levels of the immunosuppressive molecule cd (best known as pd-l ) [ ] . these latter observations strongly corroborate the notion that autophagy mediates not only cell-intrinsic, but also cell-extrinsic oncosuppression. the capacity of autophagy to preserve cellular homeostasis is benefi cial to healthy cells, but also benefi cial to transformed cells. this implies that autophagy often (but not always) promotes tumor progression, i.e., the growth and evolution of a transformed cells into an ever more malignant cancer [ ] . indeed, malignant cells are often exposed to relatively adverse microenvironmental conditions, including a shortage of nutrients and oxygen (especially in poorly vascularized tumor areas), and autophagy is instrumental for these cells (as it is for their non-transformed counterparts) to cope with stress and proliferate. along similar lines, the ability of autophagy to preserve stemness is benefi cial for the host when it preserves normal tissue architecture, but detrimental when it sustains the malignant stem-cell compartment. finally, autophagy supports the survival of malignant cells in key step of tumor progression, the so-called "epithelial-to-mesenchymal transition" (emt). in this context, epithelial cancer cells "initially growing in situ " physically detach from ecm and become able to colonize surrounding tissues as well as distant organs. the emt is required for all malignancies to become locally and distantly invasive, and critically relies on profi cient autophagic responses [ ] . in the presence of autophagic defects or pharmacological inhibitors of autophagy, indeed, malignant cells undergoing the emt and detaching from the ecm, succumb to a form of regulated cell death often referred to as "anoikis" [ ] . corroborating these observations, the genetic and/or pharmacological inhibition of the autophagic machinery in established tumors has been shown to accelerate disease progression in various models of pulmonary oncogenesis, including (but not limited to) braf -and kras -driven tumorigenesis [ , , ] . autophagy provides malignant cells with an increased resistance to various perturbations of homeostasis, including the lack of nutrient and oxygen that cancer cells normally experience in poorly vascularized tumor areas, as well as the presence of xenobiotics like chemotherapeutic agents and physical stress conditions like irradiation. an abundant amount of literature demonstrates indeed that chemical inhibitors of autophagy as well as genetic interventions that compromise autophagic responses accelerate (rather than inhibit) the demise of malignant cells exposed to a wide panel of chemotherapeutics or to irradiation, both in vitro and in vivo . these observations provided a strong rationale to the development of combinatorial therapeutic strategies involving chemo-or radiotherapy given in combination with an inhibitor of autophagy [ ] . clinical grade highly specifi c chemical inhibitors of autophagy, however, have not yet been developed, and currently available molecules that can be used in the clinic, like chloroquine (a widely employed antimalarial agent) often operate as lysosomal inhibitors, i.e., they target several processes other than autophagy [ ] . moreover, concerns have been raised that inhibiting autophagy at the whole-body level may de facto favor malignant transformation in healthy tissues, refl ecting the prominent oncosuppressive functions of autophagy in physiological conditions [ ] . finally, recent data highlight the differential role of autophagy in cancer therapy in immunocompromised versus immunocompetent hosts [ ] . in this setting, the response to radiotherapy of human non-small cell lung carcinoma (nsclc) or murine colorectal carcinoma (crc) cells xenografted in nude mice was significantly improved when cells were rendered autophagy-defi cient by the stable depletion of atg or beclin [ ] . however, when murine crc cells were implanted in immunocompetent syngeneic mice, the stable knockdown of atg compromised the therapeutic activity of irradiation, a defect that could be restored (at least in part) by the intratumoral administration of a chemical inhibitor of extracellular atpases [ ] . these fi ndings demonstrate that inhibiting autophagy in immunocompetent hosts may prevent the elicitation of a therapeutically relevant immune response against dying cancer cells. in summary, although autophagy generally (but not always) promote the progression of pulmonary malignancies and increases the resistance of lung cancer cells to chemo-and radiotherapeutic regimens, additional experiments are required to understand whether combinatorial treatments involving autophagy inhibitors constitute a clinically viable approach against pulmonary neoplasms. similarly, further work is needed to clarify whether biomarkers of autophagy such as the expres-sion levels of beclin or the lipidation of lc have a positive or negative prognostic/ predictive value in patients with lung cancer, as preliminary results are rather controversial [ , ] . abundant evidences indicate that autophagy actively participates in a wide range of cellular responses to both physiologic-and pathologic-related events in the diverse tissues and cell types that constitute the lung system. nevertheless, much is yet to be learnt about its biological relevance, functional targets, and role in development and disease. as described in this chapter, lungs are the fi rst line of defence against several insults and associated diseases are growing both in number and chronicisation. a clear deregulation of the autophagic machinery has been highlighted in most of the lung diseases, suggesting that this process mainly exerts a defensive role. however, in some pathological contexts, it has been reported that the activation of the autophagic process contributes to damage. as a consequence, a detailed knowledge of the molecular mechanisms at the basis of autophagy in lung pathologies is required for the development of novel diagnostic tools and promising therapeutic strategies. autophagy stimulation by rapamycin suppresses lung infl ammation and infection by burkholderia cenocepacia in a model of cystic fi brosis depletion of the ubiquitin-binding adaptor molecule sqstm /p from macrophages harboring cftr Δf mutation improves the delivery of burkholderia cenocepacia to the autophagic machinery autophagy in the pathogenesis of pulmonary disease regulation of autophagy during ecm detachment is linked to a selective inhibition of mtorc by perk autophagy mechanisms in sputum and peripheral blood cells of patients with severe asthma: a new therapeutic target essential role for the atg b protease and autophagy in bleomycin-induced pulmonary fi brosis oxygen toxicity and tolerance egr- regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease autophagy protein microtubule-associated protein light chain- b (lc b) activates extrinsic apoptosis during cigarette smoke-induced emphysema oxidative stress and pulmonary fi brosis berberine inhibits smad and non-smad signaling cascades and enhancesautophagy against pulmonary fi brosis neutralizing tumor-promoting chronic infl ammation: a magic bullet autophagy, not apoptosis, is essential for midgut cell death in drosophila autophagy in infection, infl ammation and immunity il activates autophagy to regulate secretion in airway epithelial cells insuffi cient autophagy promotes bronchial epithelial cell senescence in chronic obstructive pulmonary disease essential versus accessory aspects of cell death: recommendations of the nccd metabolic control of autophagy autophagy in malignant transformation and cancer progression asthma and chronic obstructive pulmonary disease to be or not to be? how selective autophagy and cell death govern cell fate mitochondria and the autophagy-infl ammationcell death axis in organismal aging the bone marrow niche, stem cells, and leukemia: impact of drugs, chemicals, and the environment autophagy suppresses progression of k-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis hallmarks of cancer: the next generation regulation mechanisms and signaling pathways of autophagy cystic fi brosis-associated liver disease an autophagy-enhancing drug promotes degradation of mutant alpha -antitrypsin z and reduces hepatic fi brosis alpha anti-trypsin: one protein, many functions reduced foxo a expression causes low autophagy in idiopathic pulmonary fi brosis fi broblasts on collagen matrices inducible disruption of autophagy in the lung causes airway hyper-responsiveness -mediated mitophagy is involved in regulation of hbec senescence in copd pathogenesis matrix metalloproteinase (mmp)- -defi cient fi broblasts display a profi brotic phenotype lkb modulates lung cancer differentiation and metastasis acute lung injury: epidemiology, pathogenesis, and treatment the atg atg conjugate associates with innate antiviral immune responses autophagy enhances bacterial clearance during p. aeruginosa lung infection autophagy and role in asthma intracellular inclusions containing mutant alpha -antitrypsin z are propagated in the absence of autophagic activity light-chain a autophagic activity and prognostic signifi cance in non-small cell lung carcinomas rapamycin reduces intrahepatic alpha- -antitrypsin mutant z protein polymers and liver injury in a mouse model consensus guidelines for the detection of immunogenic cell death the molecular basis for disease variability in cystic fi brosis autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to defi cient immunogenic signalling immunogenic cell death in cancer therapy autophagy and the integrated stress response direct effects of interleukin- on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma histone deacetylase -mediated selective autophagy regulates copd-associated cilia dysfunction autophagic protein lc b confers resistance against hypoxia-induced pulmonary hypertension mammalian target of rapamycin overexpression antagonizes chronic hypoxia-triggered pulmonary arterial hypertension via the autophagic pathway autophagic and tumour suppressor activity of a novel beclin -binding protein uvrag chloroquine prevents progression of experimental pulmonary hypertension via inhibition of autophagy and lysosomal bone morphogenetic protein type ii receptor degradation global burden of copd defective cftr induces aggresome formation and lung infl ammation in cystic fi brosis through ros-mediated autophagy inhibition autophagy and cellular immune responses the holy grail of cystic fi brosis research: pharmacological repair of the f del-cftr mutation tissue transglutaminase activation modulates infl ammation in cystic fi brosis via ppargamma down-regulation functional variant in the autophagy-related gene promotor is associated with childhood asthma autophagy suppresses tumor progression by limiting chromosomal instability casarett and doull's toxicology: the basic science of poisons blocking il- a promotes the resolution of pulmonary infl ammation and fi brosis via tgf-beta -dependent and -independent mechanisms the ampk signalling pathway coordinates cell growth, autophagy and metabolism mitophagydependent necroptosis contributes to the pathogenesis of copd autophagy: renovation of cells and tissues identifi cation of an autophagy defect in smokers' alveolar macrophages heme oxygenase- , a critical arbitrator of cell death pathways in lung injury and disease alternaria extract activates autophagy that induces il- release from airway epithelial cells hydrogen peroxide content and ph of expired breath condensate from patients with asthma and copd autophagy in cancer stem cells: a potential link between chemoresistance, recurrence, and metastasis functional interaction between autophagy and ciliogenesis gene transfer of master autophagy regulator tfeb results in clearance of toxic protein and correction of hepatic disease in alpha- -anti-trypsin defi ciency autophagy in idiopathic pulmonary fi brosis autophagy inhibition suppresses pulmonary metastasis of hcc in mice via impairing anoikis resistance and colonization of hcc cells autophagic disposal of the aggregation-prone protein that causes liver infl ammation and carcinogenesis in α - -antitrypsin defi ciency genetic and histologic evidence for autophagy in asthma pathogenesis novel mechanisms for the anti-fi brotic action of nintedanib a dual role for autophagy in a murine model of lung cancer cystic fi brosis airway fi brosis in asthma: mechanisms, consequences, and potential for therapeutic intervention autophagic removal of micronuclei decreased expression of autophagic beclin protein in idiopathic pulmonary fi brosisfi broblasts predominant th -like bronchoalveolar t-lymphocyte population in atopic asthma autophagy modulation as a potential therapeutic target for diverse diseases heme oxygenase- /carbon monoxide: from metabolism to molecular therapy autophagy in the lung autophagy in lung disease pathogenesis and therapeutics the biology and clinical relevance of the pten tumor suppressor pathway oxygen sensing, homeostasis, and disease tfeb links autophagy to lysosomal biogenesis organelle-specifi c initiation of autophagy autophagy sustains mitochondrial glutamine metabolism and growth of brafv e-driven lung tumors autophagy induction by sirt through attenuation of insulin-like growth factor signaling is involved in the regulation of human bronchial epithelial cell senescence hyperoxiainduced lc b interacts with the fas apoptotic pathway in epithelial cell death il- might refl ect disease activity in mild and moderate asthma exacerbation autophagy promotes primary ciliogenesis by removing ofd from centriolar satellites fasting in alpha -antitrypsin defi cient liver: constitutive activation of autophagy retention of mutant alpha -antitrypsin z in endoplasmic reticulum is associated with an autophagic response intracellular survival of burkholderia cepacia complex in phagocytic cells the heme oxygenase- /carbon monoxide pathway suppresses tlr signaling by regulating the interaction of tlr with caveolin- egfr-mediated beclin phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance cytoskeleton: autophagy and ciliogenesis come together loss of lkb and pten leads to lung squamous cell carcinoma with elevated pd-l expression current concepts on oxidative/carbonyl stress, infl ammation and epigenetics in pathogenesis of chronic obstructive pulmonary disease autophagy regulates tgf-beta induced fi brosis in human airway smooth muscle cells rtp , a suppressor of mtor signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema matrix metalloproteinase- is a key regulator of lung fi brosis in mice and humans reduced caveolin- promotes hyperinfl ammation due to abnormal heme oxygenase- localization in lipopolysaccharide-challenged macrophages with dysfunctional cystic fi brosis transmembrane conductance regulator endothelial pink mediates the protective effects of nlrp defi ciency during lethal oxidant injury heme oxygenase- expression in human lungs with cystic fi brosis and cytoprotective effects against pseudomonas aeruginosa in vitro autophagic protein beclin serves as an independent positive prognostic biomarker for non-small cell lung cancer pulmonary expression of interleukin- causes infl ammation, mucus hypersecretion, subepithelial fi brosis, physiologic abnormalities, and eotaxin production mechanism of action of conventional and targeted anticancer therapies: reinstating immunosurveillance the authors report no confl ict of interest. key: cord- -tpm i authors: goodin, douglas g.; jonsson, colleen b.; allen, linda j. s.; owen, robert d. title: integrating landscape hierarchies in the discovery and modeling of ecological drivers of zoonotically transmitted disease from wildlife date: - - journal: the connections between ecology and infectious disease doi: . / - - - - _ sha: doc_id: cord_uid: tpm i changes in landscape and land use can drive the emergence of zoonoses, and hence, there has been great interest in understanding how land cover change and the cascade of ecological effect associated with it are associated with emerging infectious diseases. in this chapter, we review how a spatially hierarchical approach can be used to guide research into the links between landscape properties and zoonotic diseases. methodological advances have played a role in the revival of landscape epidemiology and we introduce the role of methodologies such as geospatial analysis and mathematical modeling. importantly, we discuss cross-scale analysis and how this would provide a richer perspective of the ecology of zoonotic diseases. finally, we will provide an overview of how hierarchical research strategies and modeling might be generally used in analyses of infectious zoonoses originating in wildlife. zoonotic diseases, or zoonoses, are infectious diseases transmitted to humans from animals and may be bacterial, viral, or parasitic in origin. approximately % of the pathogens associated with infectious diseases in humans have originated through spillover from wildlife-e.g., ebolaviruses, hantaviruses, coronaviruses, henipaviruses (jones et al. ; lloyd-smith et al. ; smith et al. ; woolhouse and gowtage-sequeria ) . since , zoonotic pathogens represent the bulk of the outbreaks in human populations in both number ( % versus % by vector-borne pathogens) and diversity (smith et al. ) . the recently reported increase in zoonoses has been attributed to a variety of reasons, although a major driver is changes in land use resulting from the increased demands of human populations on the natural environment through agriculture intensification and deforestation (jones et al. ; woolhouse and gowtage-sequeria ) . land alteration for food production, broadly defined to include both agriculture and pastoral activities, has produced profound changes in the type and structure of the earth's vegetation cover. it has also altered the way humans interact with their environment, for example, in some geographical areas suppressing wildlife-human interaction (e.g., contact with large predators common among hunter-gathers) and instead favoring contact between humans and peridomestic species such as rodents, which are common reservoirs for many zoonotic pathogens. numerous examples of emergent zoonoses have often accompanied land clearance, and hence, there has been great interest in understanding how land cover change and the cascade of ecological effects associated with it are ecologically associated with emerging infectious diseases (mcfarlane et al. ; woolhouse and gowtage-sequeria ) . a persistent question in modeling of pathogen emergence is what is the spatial scale at which these processes occur? how do we measure and integrate the impact of processes that occur at varying and nested spatial scales that result in the observed disease distribution (watts et al. ) ? for example, climate (which can vary in scale from local to continental) and landscape are each associated with disease patterns and have been cited as factors in zoonotic disease outbreaks. land cover change and land-use intensification often occurs at finer scales than climate and can thus be thought of as nested within the climate system. at still finer scales, population dynamics and habitat interactions of the pathogen and its reservoir communities (and, indeed, with the human populations vulnerable to disease transmissions from these communities) occur within the climate and landscape scales and are influenced, but not necessarily determined, by them. this hierarchical nesting ( fig. . ) of these processes complicates the overall study and modeling of the spillover and transmission dynamics of wildlife pathogens to human populations. however, there are existing bodies of theory which can help shed light on these processes. hence, we review some of these conceptual ideas, focusing especially on landscape epidemiology, a body of theory first developed in the s and more recently updated to include modern tools and techniques for studying both disease and environmental process (ostfeld et al. ) , and hierarchy theory, a framework which incorporates the idea of multiple, nested spatial scales (allen and starr ) . in the following, we will review how a spatially hierarchical approach can be used to guide research into the links between landscape properties and zoonotic diseases. this will show how landscape concepts have been used to analyze the occurrence and spatial patterning of zoonotic diseases and how many of these studies are being conducted at particular spatial scales. this review will be followed by an illustration of how an integrated, cross-scale analysis might be employed to gain a deeper understanding of the ecology of zoonotic diseases, using an example from our own research. as earlier noted, methodological advances have played a role in the revival of landscape epidemiology. we will therefore discuss the role of methodologies such as geospatial analysis and mathematical modeling-again using examples drawn from our current research. we will show how cross-scale analysis might be employed to gain a deeper understanding of the ecology of zoonotic diseases. finally, we will provide an overview of how hierarchical research strategies and modeling might be generally used in analyses of infectious zoonoses originating in wildlife. landscape epidemiology is the study of spatial patterns of disease and disease risk arising from underlying environmental causes. the fundamental concepts of landscape epidemiology, first proposed by evegeniĭ pavlovskiĭ, stem from the idea that the spatial occurrence of disease could be understood by studying landscape and environmental factors associated with the disease (pavlovskiĭ ). pavlovksiĭ's ideas have undergone a revival, stimulated in part by widespread availability of geospatial data, analysis tools, and models. in particular, satellite remote sensing data analyzed within a geographic information systems (gis) framework has equipped landscape epidemiologists with a powerful suite of tools for analyzing environmental patterns associated with disease occurrence. landscape epidemiology has also benefited from the theoretical perspective of landscape ecology, another relatively new discipline that attempts to understand the relationship between spatial pattern and ecological process (meentemeyer et al. ) . for example, landscape fig. . illustration of hierarchical nesting of possible key components in pathogen emergence in wildlife. each process outlined with a "box" represents a distinct spatial scale to consider in developing models epidemiologists have made use of ecological concepts such as fragmentation to analyze disease vectors (brownstein et al. ; reisen ). one aspect of landscape ecology that remains relatively unexplored in infectious disease applications is the concept of spatial hierarchy. hierarchy in ecology is a multifaceted theory incorporating elements of nonlinear dynamics and complexity; for a comprehensive treatment of hierarchy theory, see allen and starr ( ) . the fundamental concept of hierarchy theory is that processes occurring at finer scales (i.e., "lower" in the spatial hierarchy) are constrained by processes at higher levels. hierarchical levels can also be distinguished by the rates at which ecological processes occur-faster at finer scales, slower at coarser ones. hierarchy theory in ecology arose as a response to the need for a rigorous method of handling middle-number systems, that is, systems whose components are too few to treat statistically but too many to address with classical newtonian mathematics. hierarchy provides a framework by which these middle number systems can be decomposed into a series of manageable units, whose environmental drivers can be characterized by the scale (and thus the rate) at which they occur. such a framework is amenable to the study of landscape epidemiology, since the linkages between environmental factors and disease are often multivariate, nonlinear, and not confined to a specific spatial scale. spatial scale is a crucial aspect of hierarchy theory. any discussion of the relationship between ecological process and scale therefore must first define how the term is used and to what characteristic dimensions these terms apply. these definitions are complicated by the number of ways that the term "scale" is used. scale is frequently referred to by descriptive adjectives such as "large" or "small," which meentemeyer ( ) noted can have opposing meaning depending on whether one is referring to cartographic scale (where small scale refers to less spatial detail) or ecological scale, where smaller scale equates to smaller spatial area and greater detail. ecologists generally distinguish between the grain and extent of a process, where extent refers to the area over which a process occurs, and grain denotes the smallest resolvable component of the process. typically, grain can be described as either "fine," indicating small resolvable elements, or "coarse," indicating larger elements. thus, we might characterize the scale process like tropical deforestation as occurring at relatively large extent, because a large area is affected, but at relatively fine grain, because the individual deforested units can be quite small. csillag et al. ( ) argue that for ecological uses of scale, it is preferable to adopt specific terminology to distinguish between extent and grain. in hierarchy theory, spatial extent is probably the more commonly used sense of scale (jenerette and wu ) . in describing spatial hierarchies, it is often useful to apply descriptive names to realms of scale. terms such as "global," "continental," "regional," and "local" are often used, although the precise areas referred to often vary. for our review of hierarchy in infectious disease analysis, we will operationally define continental scale as areas exceeding km , regional scales as ranging from km - km , local scale from - km , and microscale < km . the lower end of the microscale represents the general size range of a small rodent's world. landscape analyses across spatial scale frequently use remote sensing data (kitron et al. ; wu ) . currently, imagery is available from an array of orbital sensors with widely varying spatial, temporal, and spectral resolutions (table . ). grain size (or resolution, as it is more commonly termed in remote sensing) is an engineered property of these sensors, dependent on the optical characteristics of the sensor and orbital characteristics of the platform. theoretically, there are no limitations to the extent of any of these systems, although practical limitations (particularly cost of data acquisition) dictate a rough correspondence between extent and resolution. thus, the extents listed in table . are based on these practical limitations. the variety of remote sensor data available has certainly facilitated hierarchical landscape analysis, but in some sense, it has also imposed limitations. each remote sensor represents a "window" through which ecological process at some combination of grain size and extent can be observed. these limitations also extend to the range of spectral wavelengths each sensor can detect and the number and width of bands in which these wavelengths are detected. combining these discrete views into an integrated picture is a central challenge for hierarchical analysis of infectious disease processes. there is a large body of work relating infectious disease to environmental factors. most of these studies have concentrated on a single class of causative factor operating over a characteristic spatial scale. in the following, we will briefly review several examples which represent different classes of landscape factors that have been shown to impact zoonotic viral emergence. since our intent is to show how landscape spatial hierarchies influence disease processes, we will concentrate on examples that show the influence of landscape and land cover processes at various spatial scales, using the concept and terminology of scale developed in the previous section. for convenience, we will group the literature reviewed here into two categories. first, we will review the relationship between land cover disturbance (including anthropogenic and natural disturbance) and zoonotic disease emergence. included in this category are causes due to landscape structure, including natural landscape barriers, land cover change and disturbance, and fragmentation. this category also includes disturbance due to agricultural practices. the second class of landscape impacts that we will review are climate-driven landscape changes, which include climate-influenced changes in vegetation phenology patterns as well as persistent or transient superficial changes such as flooding. processes contained within these two categories are not exclusive; however we will review them based on their predominant process. landscape structure refers to the pattern and arrangement of habitats on the earth's surface. like all landscape variables, structure is complex. it varies with scale and per organism. the same landscape might be structurally very different for birds, small mammals such as rodents, and larger animals, and since zoonotic disease reservoirs vary in their body size and habitat, structural effects on disease also vary. structure can arise naturally due to topographic, edaphic meaning related to the soil, or climatic conditions, as well as through human landscape alteration. both types of structural changes can be relevant to disease processes. landscape structure has been related to zoonotic disease at a variety of spatial scales. at regional scale, russell et al. ( ) show how natural landscape barriers such as rivers and preferred habitat can limit rabies spread by raccoons and be used to manage wildlife through vaccination efforts. smith et al. ( ) used landscape heterogeneity as a predictor of the spread of rabies. langlois et al. ( ) showed that the distribution of hantavirusbearing rodents in north america was influenced by landscape fragmentation. their analysis was unusual in that the extent of the study was continental, but the grain size was local ( km). estrada-peña and oteo ( ) showed that landscape structure, particularly landscape connectivity, showed a strong influence on the abundance of lyme disease vectors in spain. land cover changes are known to affect zoonotic diseases through controls on the population dynamics of reservoir species (especially wild mammals) as well as disease vectors (patz et al. ) . giraudoux et al. ( ) used regional-scale land cover changes in france and china to show how host mammal communities affect transmission dynamics of the endoparasite echinococcus multilocularis. using the rompa (ratio of optimal to marginal patch area) hypothesis of lidicker and others, it has been shown that regional-scale landscape dynamics of intermediate host species can in turn affect parasite egg survival and transmission (lidicker ) . the giraudoux et al. ( ) study also considered the role of landscape change on establishing minimum thresholds, which they termed "filters" or "screens" of suitability for disease transfer. agriculture practices led to the nipah virus outbreak in malaysia (epstein et al. ; pulliam et al. ) . using a combination of field, laboratory, and modeling approaches, these efforts have supported the hypothesis that emergences of viruses such as nipah are due to ecological and not evolutionary drivers. these findings underscore the importance of having multidisciplinary teams work together to build predictive models for discovery of the relationships between anthropogenic environmental change and the transmission or spillover of infectious agents. climate-driven landscape change in this context refers to the effect of atmospheric processes (most notably precipitation) on the habitat of zoonotic host organisms. variations in rainfall magnitude and frequency have notable effects on vegetation phenology, causing variations in surface greenness that can be tracked using remotesensing instruments (de beurs and henebry ; reed et al. ). the emergence of several zoonotic diseases including hantavirus pulmonary syndrome (hps) , argentine hemorrhagic fever (simone et al. ) , and bolivian hemorrhagic fever (kilgore et al. ) can be clearly linked to landscape. several studies have linked climate-driven changes to patterns of disease occurrence at different spatial scales. two groups have evaluated the relationship between temporal patterns of normalized difference vegetation index (ndvi) and occurrence of ebola virus in west africa (pinzon et al. ; tucker et al. ) . they found that ndvi trajectories showed distinctive "trigger events" prior to occurrences of the disease in humans and apes, which they hypothesized might be used to forecast conditions conducive to outbreaks of ebola hemorrhagic fever. the remotely sensed data for this analysis came from the noaa-avhrr sensor, with continental spatial extent and observation grain size (pixel resolution) of km . the observed ndvi trajectories were related to precipitation patterns, reinforcing the link between climate and disease occurrence. estrada-peña and oteo ( ) and estrada-peña et al. ( ) used coarse resolution vegetation index data to model and predict the continental-scale relationship between climate-driven landscape change and lyme disease. again, the resolution and extent of this study were consistent with the idea that climate constrains disease processes at higher levels in the spatial hierarchy. at finer spatial scales, glass et al. ( ) used patterns of reflectance in landsat thematic mapper (tm) data to statistically model the presence of hps in the southwestern united states. at this spatial scale, the spectral response of the surface incorporates climatic factors (especially antecedent precipitation) but also integrates structural and compositional factors of the vegetation canopy itself. models based on these techniques have shown some utility for predicting hantavirus cases (glass et al. ) . one of the important advantages of a hierarchical approach is that it allows a multifactorial explanation for the occurrence of the infection and disease. the environmental, landscape, and climatic processes each contribute to processes that may alter species interactions within their habitat. these extrinsic factors can alter the reservoir population dynamics, drive extinction, and affect maintenance (persistence) of the microorganism. essentially, these factors can create constraints within their spatial scale and across scales. in this section, we explore an example of hierarchical observation that can be used in conjunction with modeling to test hypotheses regarding the effects of environment, landscape, and climate upon zoonotic pathogen distribution. for this we draw upon our work and others in the study of hantavirus with a focus on south america palma et al. ). hierarchy theory suggests that processes at coarser grain also occur over longer time frames and can often be assumed to be static with respect to finer-scale processes. the phylogeographical patterns of south american hantaviruses within the southern cone ("el cono sur," a subcontinental region roughly defined as consisting of the country argentina, often including chile, plus sometimes considered to include uruguay and paraguay) mapped with environment at the coarsest spatial scales . in other words, the phylogenetic clades of hantaviruses from the southern cone of south america appear tied to coherent spatial patterns consistent with subcontinental-scale biogeographic features such as the major biomes ). in fig. . , the locations of strains from three major subclades of south american hantaviruses are shown in the context of the major biomes based on the world wildlife fund terrestrial ecoregions data (olson et al. ) . for example, we find members from one subclade (i.e., laguna negra, rio mamoré, and alto paraná viruses) carried by rodent reservoirs that span the tropical grass and shrubland and dry broadleaf forest areas along the western, central regions of south america johnson et al. ; richter et al. ; yahnke et al. ) . in contrast, a second subclade of rodents that harbor jabora, maporal, and necocli hantaviruses inhabit mainly the moist broadleaf forest biome stretching from venezuela and colombia into paraguay de oliveira et al. ; fulhorst et al. ; londoño et al. ) . knowing the extent of prevalence of these closely related viruses over this vast region would certainly be fascinating from a phylogeographical point of view. it is interesting that these viruses are not yet associated with cases of hps. there are numerous strains of hantaviruses that have been identified in the atlantic forest (extends along the eastern coast of brazil and into eastern paraguay) and in the temperate grass and shrubland of argentina. the third clade of rodents, those that harbor the juquitiba, oran, and lechiguanas viruses, resides in the more humid lower chaco (a region that encompasses the flooded savannas of southern paraguay and forms a transitional environment between the arid gran chaco) and temperate coniferous forests in argentina, brazil, and uruguay (de araujo et al. ; delfraro et al. ) . andes, maciel, and pergamino viruses reside in rodents in the temperate grass and shrubland biome in argentina (bohlman et al. ; gonzález-ittig et al. ) . andes virus is also found in chile in mediterranean woodland and shrub biome (torres-perez et al. ) . araraquara viruses are associated with rodents that largely reside within agriculturally transformed areas of brazil (i.e., sugarcane production fields) that were formerly areas of moist broadleaf forest biome (de araujo et al. ; de sousa et al. ; suzuki et al. ). coarse-scale analysis has shown a relationship between hantavirus genetics and broad ecological pattern. narrowing this view to a more regional scale begins to reveal how land cover (and land cover disturbance) affects the spatial variability of hantaviruses. like all zoonotic disease, the ecology of each species of hantavirus is closely related to that of its host organism; thus, generalization of virus-landscape relationships cannot be made without considering the habitat characteristics of the reservoir host. we do not have abundant data on the microhabitat characteristics of many host species (lozada and guthmann ) . however, general habitat types defined at grains sizes of~ km might be useful to study preferences among various (olson et al. ) . selected strains of closely related hantaviruses are presented from three distinct subclades as indicated by the color of the yellow or pink dot at their location. the viruses represented by the pink dot and pink with yellow represent two distinct lineages from one subclade host species, and be relevant to the landscape epidemiology of hantaviruses, regardless of whether certain land cover types are more closely associated with the presence of the virus. a regional-scale analysis of rodent reservoirs of hantaviruses in paraguay showed that the host species do indeed show patterns of land cover preference, even when land cover is mapped into very general categories. the most common hantavirus host rodents in paraguay, akodon montensis and oligoryzomys spp., both showed disproportionately high probabilities of occurring in areas subjected to large-scale agricultural disturbance . even more significant, however, was the fact that rodents found to be antibody positive for hantavirus (indicating exposure to the virus at some point) were more likely to be associated with the human-disturbed land cover types. this relationship held even when the underlying differences in habitat preference were controlled. this finding suggests that some aspect of land cover disturbance or change increases the likelihood that a member of a host species will be infected with the virus. while analysis at the regional scale shows that human land cover alteration is associated with hantavirus presence, discovering the specific nature and causes for this observation cannot be addressed at this scale. questions of causation must be addressed at finer spatial scales, in large part because that is the scale to which most host populations and individuals are interacting with their environment (lozada and guthmann ; owen et al. ). simply presumed, because rodents species have specific requirements with respect to habitat, and because each hantavirus species seemingly persists only in certain rodents species, one might assume that a predictive map can be made for of the prevalence of a particular hantavirus based on knowledge of the associated rodent species. unfortunately, for most rodents, we know very little about their general life cycles and other important biological information that is critical for modeling (e.g., age at sexual maturity, birth rates, litter sizes). further, we have only a minimal amount of information on their habitat preferences, although some recent studies have indicated that fine-grained evaluations are necessary to understand rodent species distributions and community composition lozada and guthmann ; poindexter et al. ; schnell et al. ). at the microscale level, pathogen survival and reproduction depend on the dynamics within the reservoir host, which in turn depend on the habitat or the reservoir. a rodent may live within an approximate km range for most of its life barring fire, flooding, and other natural disasters. in the case of a viral pathogen, the virus must overcome physical barriers of the host and be compatible with cell receptors to gain entry into a target cell (allen et al. ). viral replication depends on host and viral genetics and other host factors such as prior pathogen or other immunogenic exposure, nutritional status, coinfection, age, sex, reproductive status, and the host immune response (allen et al. ). these and other factors determine outcome when a pathogen enters a host, with the possibilities including severity of any associated disease and whether the pathogen either is to be cleared by the host immune system, or the pathogen will persists in the host. for example, the survival of hantaviruses in nature depends on maintenance of persistent infections within their specific rodent reservoir. hantaviruses infect and persist only in the rodent reservoir with which the virus has coevolved, and the infection is believed to last the life of the animal (meyer and schmaljohn ) . notably, persistent infection of rodent reservoirs by hantaviruses shows continuous virus replication, without complete clearance by the immune system, and no pathological changes vaheri et al. ) . humans are not a natural reservoir for these viruses, and as such humans typically become infected only upon contact with aerosolized excreta from the rodent reservoir. in humans, hantavirus infection can result in severe disease although outcomes vary with different hantaviral species. the molecular basis for different disease outcomes in humans has been attributed to difference in receptor preferences of nonpathogenic and pathogenic hantaviruses. models that connect data on the outcomes associated with immune response in reservoir versus human hosts are just beginning to be developed. mathematical models are valuable tools for synthesizing information and testing hypotheses to provide insight into how and why disease outbreaks or spatial patterns of infections might arise in wildlife populations. several books and review articles summarize some of the modeling efforts on zoonotic infectious diseases (alexander et al. ; allen et al. ; grenfell and dobson ; heesterbeek et al. ; hudson et al. ; lloyd-smith et al. ) . a variety of modeling formats, deterministic and stochastic, have been applied to the study of zoonotic diseases. these models include compartmental, agent-based, individual-based, metapopulation, network, and ecological niche, but these classifications overlap. agent-based, individual-based, and metapopulation models may be classified under network models, where the nodes are infectious agents, individuals or populations, connected via an underlying network (riley et al. ) . simple compartment models (seir-susceptible, exposed, infectious, and recovered) are connected via a dispersal network in what is often called a patch model or metapopulation model (allen et al. ; arino et al. ; mccormack and allen b) . ecological niche models contain less detail about individual dynamics, and instead they are closely related to landscape, presence/absence data, and gis-based climatic and environmental data (alexander et al. ; peterson ) . mathematical tractability and the complexity of interactions among pathogens, reservoirs, and human hosts and the environment have often restricted the model formulation to the reservoir host and to a single spatial scale-landscape, population, or within-host. coupling temporal scales (hours to years or longer), biological complexity levels (genes to cells to ecosystems), and spatial scales (local to global) have been a continuous challenge to modelers . recent theoretical investigations on coupling within-host and between-host models are advancing (feng et al. ; gilchrist and coombs ; mideo et al. ) . hantaviruses and rabies are two examples where spatial patterns of infection involving multiple species have been observed haydon et al. ; rhodes et al. ; smith et al. ) . spatially explicit computer simulations that incorporate landscape heterogeneity and spatial genetic structure are being applied to study control of rabies and other zoonotic diseases (alexander et al. ; parratt et al. ; real and biek ; rees et al. ) . models with multi-host species and multi-pathogens are being investigated. for example, mathematical models for hantavirus infection in rodents have been studied in the context of multiple host species, spatial spread, and environmental variability (abramson and kenkre ; abramson et al. ; allen et al. a allen et al. , b, mccormack and allen a, b) . these models have shown that those random or seasonal variations which impact an ecosystems carrying capacity for a particular rodent species can trigger outbreaks when rodent densities and contacts rates are high (allen et al. b ). in addition, theoretical analyses have shown that when multiple species, as opposed to a single species, are involved in the transmission process, there may be a dilution or amplification effect that impacts disease persistence (dobson ; mccormack and allen a) . models for spatial spread among discrete patches have shown the importance of there being at least one patch where the disease persists (arino et al. ; allen et al. ; mccormack and allen b) . wu and loucks ( ) have suggested that the most significant contribution of hierarchy theory is as a framework for explicitly incorporating heterogeneity and scale into ecological analysis. in this chapter, we have tried to show some ways in which hierarchical consideration of spatial (and to some extent, temporal) scale can be incorporated into ecological analyses of zoonotic diseases. our review of the literature also suggests some of the opportunities in infectious disease research resulting from the hierarchical consideration of scale but also some of the challenges. spatial patterns of zoonotic hosts, the pathogens they harbor, and the host-zoonosis relationships may appear quite different depending on the scale at which we view them. although patterns derived from different size scales are often referred to as "emergent" patterns, it is unclear whether the newly recognized large-scale or the small-scale patterns might not be emergent from scale levels either above or below them. although we could view these scales to be nested subsets of one another, it may be more useful both conceptually and in practice, to view the patterns derived from different scales as complementary information requiring integration, rather than as contradictory results requiring amelioration. one area of opportunity for the application of hierarchical concepts in zoonotic disease ecology corresponds to scale-related questions facing the global change research community in general; how does global environmental change, especially global warming, manifest itself spatially? it has long been recognized that the spatial distribution of both individuals and communities of species is linked to climate and that these are two-way linkages; however much uncertainty remains about where, when, and how species will respond to climate change (potter et al. ) . fundamentally, the issue reduces to one of scale; the potential impacts of climate change are most often conceptualized at the macroscale (i.e., regional or global) but operate across a range of scales including those more proximate to the host or reservoir organisms (ashcroft et al. ; diffenbaugh et al. ; suggitt et al. ) . adoption of the complementary, hierarchical view of scale provides a framework to address these and similar questions in disease ecology. model selection among the wide array of potential formats depends on the virushost ecological system being investigated, data availability, and the questions to be addressed. many challenges remain in model formulation, analysis, and simulation of zoonotic disease dynamics that relate to landscape and climate and the wide range of temporal and spatial scales (allen et al. ; buhnerkempe et al. ; heesterbeek et al. ; lloyd-smith et al. ; 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spatial dynamics of rabies epidemics on heterogeneous landscapes global rise in human infectious disease outbreaks habitat microclimates drive fine-scale variation in extreme temperatures identifying rodent hantavirus reservoirs peridomestic small mammals associated with confirmed cases of human hantavirus disease in southcentral chile climatic and ecological context of the - ebola outbreaks uncovering the mysteries of hantavirus infections (disease/disorder overview) multiscale, resurgent epidemics in a hierarchical metapopulation model host range and emerging and reemerging pathogens hierarchy and scaling: extrapolating information along a scaling ladder from balance of nature to hierarchical patch dynamics: a paradigm shift in ecology patterns of infection with laguna negra virus in wild populations of calomys laucha in the central paraguayan chaco the ecology and evolutionary history of an emergent disease: hantavirus pulmonary syndrome acknowledgments cbj and ljsa acknowledge the support of the national science foundation grants (nsf) dms- and dms- . cbj and rdo acknowledge the support of the national institutes of health grant (nih) i . cbj, ljsa, rdo, and dg acknowledge support from nih r tw - through the nih-nsf ecology of infectious disease program. rdo was partially supported by the programa nacional de incentivo a los investigadores (conacyt, paraguay). funding this study was funded by national science foundation grants (dms- and dms- ) and national institutes of health grant (r ai ). this work was supported by a grant from the fogarty international center (r tw - ) under the nih-nsf ecology of infectious diseases initiative.conflict of interest douglas g. goodin declares that he has no conflict of interest. colleen b. jonsson declares that she has no conflict of interest. linda j. s. allen declares that she has no conflict of interest. robert d. owen declares that he has no conflict of interest.ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. key: cord- -q f e authors: ranjan, prabhat; dey, asmita; sharma, vinod praveen; tiwari, neeraj kumar title: importance of natural proteins in infectious diseases date: - - journal: biomedical applications of natural proteins doi: . / - - - - _ sha: doc_id: cord_uid: q f e proteins are important biomolecules, extensively involved in almost all biological processes. a number of proteins are also implicated in infectious diseases. bacterial proteins used in adhesion to host epithelium, bacterial toxins, and viral membrane glycoproteins are some of the proteins involved in infectious diseases. even components of the host innate immune system like toll-like receptors and nod-like receptors and adaptive immune components like immunoglobulins aiding in defense against pathogens are important biological proteins. chaperones like acid and heat shock proteins provide protection from high temperatures, metabolic poisons, and other stressful conditions. several natural and artificial proteins are components of vaccines, a key strategy to control fatal diseases, lacking empirical treatment. it is necessary to investigate these proteins, to develop new biomedical tools and technologies, aiding in eradication of various diseases. thus, further research should be carried out in this field, for saving and improving quality of human lives. an infectious disease is a disease that is caused by the invasion of a host by agents whose activities harm the host's tissues (that is, they cause disease) and can be transmitted to other individuals (that is, they are infectious). infectious diseases, also known as transmissible diseases or communicable diseases, comprise clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) resulting from the infection, presence, and growth of pathogenic biological agents in an individual host organism. infections are caused by infectious agents such as viruses, and prions, microorganisms such as bacteria. proteins might be involved in causing a disease as well as protecting the host from it. various membrane proteins of microorganisms (virus, bacteria, parasites, etc.), prions are responsible for causing different diseases. proteins like heat shock proteins/chaperons aid in causing as well as protecting from diseases. proteins might also be involved in playing solely protective roles against infections like various cell surface receptor proteins (toll-like receptors, nod-like receptors, etc.), which trigger protective immune responses. nowadays, various antimicrobial peptides or antibiotics are known to kill microorganisms or inhibit their growth, thus aiding in treatment of diseases. thus, proteins, one of the most incredible biomolecules, are extensively involved in almost all biological mechanisms, especially those implicated in pathophysiology of infectious diseases. the understanding of this topic is essential for developing different biomedical tools, for combating various diseases, thus, improving the quality of human life. a number of bacterial proteins are involved in its pathogenesis and virulence. bacterial infections are usually initiated by adherence of the microbe to a specific epithelial surface of the host, otherwise the organism will get removed: ( ) fimbrial adhesions involved in mediating attachment of some bacteria to mammalian cell surfaces, e.g., neisseria gonorrhoeae use pilli to adhere to the mucous membrane of urethra and can resist the flushing action of urine [ , ] . ( ) nonfimbrial adhesion including the filamentous hemagglutinin of bordetella pertussia helping in attachment to respiratory epithelium, a mannose-resistant hemagglutinin produced by salmonella typhimurium and a fibrillar hemagglutinin from helecobacter pylori [ , ] . other adhesions like protein f produced by streptococcus pyogenes, aiding in attachment to pharyngeal epithelium [ ] . some extracellular bacterial proteins considered invasins like hyaluronidase, produced by streptococci, staphylococci, and clostridia, which degrades hyaluronic acid of connective tissue [ , ] , collagenase produced by clostridium sp, which dissolves collagen framework of muscles [ ] , neuraminidase produced by vibrio cholerae and shigella dysenteriae, which degrades neuraminic acid of intestinal mucosa [ ] . other extracellular proteins like invasive enzymes, e.g., coagulase, contributes to the formation of fibrin walls around staphylococcal lesions [ ] ; exotoxins (proteins released extracellularly), like neurotoxin (tetanus toxin, by clostridium tetani, botulinum toxin by clostridium botulinum) [ ] and cytotoxins (diphtheria toxin produced by corynebacterium dipthereae) [ , ] , also known as a-b toxins (consisting of subunits: one binds to cell surface receptor and the other is transferred into the cell to damage the cell) [ ] , cytolytic toxins (attacking cell constituents causing lysis) like hemolysins produced by bordetella pertussis, inducing apoptosis of host cells, super antigen toxins (e.g., superantigen, sized kda produced by - % of staphylococcus aureus isolates, causing toxic shock syndrome (tss) by stimulating the release of large amounts of interleukin- , interleukin- and tumor necrosis factor, etc.) [ ] . enterotoxins (exotoxins that act on the small intestine, generally causing massive secretion of fluid into the intestinal lumen, leading to vomiting and diarrhea) produced by vibrio cholerae, e. coli o :h . endotoxins (generally cell-bound toxins released only when cells are lysed) produced by most gram-negative bacteria are generally nonproteinaceous, lipopolysaccharide in nature [ ] (fig. . ). viral glycoproteins, made up of carbohydrates and proteins mainly help in adherence of viruses to host cell surfaces and internalization of viral components. the addition of sugar chains or glycosylation of the protein surface-components, can happen either at asparagine, and is termed n-glycosylation, or at hydroxylysine, hydroxyproline, serine, or threonine, and is termed o-glycosylation. glycosylation is often present in proteins that are at least in part located in extracellular space. the sugar group can assist in protein folding or improve its stability. glycoproteins also aid in immune cell recognition. viral glycoproteins are composed of three parts: external/ecto-domain, which interacts with host; transmembrane segment, which spans the viral envelope membrane, typically α-helix; and endo-domain, the internal part. ecto-domain can be of two classes: class i, found in orthomyxoviruses, paramyxoviruses, retroviruses, filoviruses and coronaviruses; class ii, found in flaviviruses and alphaviruses. other types of ecto-domains that do not fit in class i and ii are glycoprotein b (gb) of herpes simplex virus (hsv) and glycoprotein g of vesicular stomatitis virus (vsv) [ ] [ ] [ ] [ ] . glycoproteins on the surface of viruses are anchored in the lipid bilayer of the envelope by means of hydrophobic bonds, and only about amino acids penetrate into the virus. these transmembrane proteins have large external domains and small cytoplasmic domains. viral glycoproteins are oligomers that are associated with each other to form tetramers, etc. there are often the spikes seen on the virus surface. some glycoproteins such as the influenza hemagglutinin are anchored at both ends, thus forming a loop. in such cases a signal sequence at the amino end has been removed. there are two types of glycoproteins: external glycoprotein anchored in the envelope by a single transmembrane domain, and a short internal tail. these proteins are usually the major antigens of the virus and involved in functions such as hemagglutination, receptor binding, and membrane fusion. the other classes are channel proteins, which are mostly hydrophobic proteins that form a protein lined channel through the envelope. this protein alters permeability of the membrane (e.g., ion channel). such proteins are important in modifying the internal environment of the virus [ ] [ ] [ ] [ ] (fig. . ). the first line of defense against pathogenic microorganisms is innate immunity, and its activation is initiated by the recognition of microbial structures by pattern recognition receptors (prrs). the first and most studied class of prrs is that of a proteinaceous receptor named toll-like receptors (tlrs), named after the toll receptor of drosophila melanogaster. ten human tlrs have been identified, from tlr through , with important roles in host defense against bacteria, viruses, and fungi [ , ] . they recognize a large array of pathogen-associated molecular patterns (pamps), including peptidoglycan, lipoproteins, lipopeptides, phenol-soluble modulin, lipoteichoic acid, lipoarabinomannan, atypical lipopolysaccharides (lpss), porins, flagellin, heat shock proteins (hsp), lycoinositol phospholipids, glycolipids, zymosan, also nucleic acids like double-stranded rna of viruses, etc. [ , ] . tlr family members are characterized structurally by the presence of a leucine-rich repeat (lrr) domain in their extracellular domain and a tir (toll/interleukin- receptor homology) domain in their intracellular region [ ] . expression of tlrs is modulated by a variety of factors such as microbial invasion, microbial components, and cytokines. there are two distinct signaling pathways of tlrs: myd -dependent and myd -independent pathways. the myd -dependent pathway signals via myd , irak, and traf and leads to nf-κb activation. the activity of nf-κb (nuclear factor kappa-light-chain-enhancer of activated b cells) is regulated by association with i-κb, which sequesters nf-κb in the cytoplasm until phosphorylated on serine residues by the i-κb kinase (ikk) complex. this phosphorylation leads to the dissociation and nuclear translocation of nf-κb. nf-κb is a transcription factor involved in upregulation of genes responsible for both the innate and adaptive immune responses, e.g., genes involved in t cell development, maturation, and proliferation, cell apoptosis, etc. similarly, in the myd independent pathway an adaptor molecule named tir domain-containing adaptor protein (tirap)/myd -adaptor-like (mal) is involved (fig. . ). nucleotide-binding and oligomerization domain (nod)-like receptors (nlrs) are a group of evolutionarily conserved intracellular proteinaceous prrs that play a vital role in innate immunity and host physiology, in both plants and animals [ , ] . in humans there are known nlrs, and the association of mutations and single nucleotide polymorphisms (snps) in their genes with human diseases reflect their vital role in host defense. nlrs also play important roles in reproduction and embryonic development. the characteristic feature of nlrs is a central nod (or nacht) domain, required for oligomerization, an n-terminal homotypic protein-protein interaction domain and a c-terminal series of leucine-rich repeats (lrrs) involved in agonist sensing or ligand binding [ ] . mammalian nlrs are subdivided into four subfamilies based on the variation in their n-terminal domain: nlra or class ii transactivator (ciita) contains an acid transactivation domain, nlrbs or neuronal apoptosis inhibitor proteins (naips) possess a baculovirus inhibitor of apoptosis protein repeat (bir), nlrcs have a caspase-recruitment domain (card), and nlrps a pyrin domain (pyd). nlrx contains a card-related x effector domain. among the nlrs, nlrp , nlrp , nlrp , nlrp , nlrp , nlrc , and naip have been reported to operate via inflammasomes. other nlrs such as nod , nod , nlrp , nlrx , nlrc , and ciita do not directly engage the inflammatory caspases, but instead activate nuclear factor-kb (nf-kb), mitogen-activated protein kinases (mapks), and interferon (ifn) regulatory factors (irfs) to stimulate innate immunity [ ] . nod-like receptors have been described as master regulators of innate immunity. nlrs are essential in recognition of microbial-and pathogen-associated molecular patterns (mamps and pamps), and have the ability to initiate and support robust immune responses through the formation of inflammasomes and the activation of nf-kb, irf, and mapk pathways. functions such as the enhancement of mhc transcription and presentation implicate nlrs in adaptive immunity, and their roles in reproduction indicate a broader responsibility of this gene family than previously suspected. the potency of nlrs in inducing immune defenses is vital for the host, but can also provide serious problems when dysregulation or malfunction occurs (fig. . ). the concept of "protein interaction" is generally used to describe the physical contact between proteins and their interacting partners. however, protein interactions do not always have to be physical [ ] . protein interaction networks are useful resources in the abstraction of basic science knowledge and in the development of biomedical applications. therefore, protein interaction networks can elucidate the molecular basis of disease, which in turn can inform methods for prevention, diagnosis, and treatment [ ] . microorganisms frequently change the ph of their own habitat by producing acidic or basic metabolic waste products. if the external ph decreases to . or lower, chaperones such as acid shock proteins and heat shock proteins are synthesized. chaperones were first discovered because they dramatically increased in concentration when cells were exposed to high temperatures, metabolic poisons, and other stressful conditions. thus many chaperones are often called heat shock proteins or stress proteins. heat shock proteins influence infectious disease processes in a number of diverse ways: they are involved in the propagation of prions, replication and morphogenesis of viruses, and resistance of parasites to chemotherapy. several heat shock proteins function as intracellular chaperones for other proteins. they play an important role in protein-protein interactions such as folding and assisting in establishment of proper protein conformation (shape) and prevention of unwanted protein aggregation. by helping to stabilize partially unfolded proteins, hsps aid in transporting proteins across membranes within the cell [ ] . these proteins also appear to be important mediators of bacteria-host interactions and inflammation, the latter via interactions with cell surface molecules and structures such as toll-like receptors and lipid rafts. heat shock proteins can be expressed on the surface of infected cells, and this is likely to provide a target for the innate immune response. elevated levels of circulating hsp are present in infectious diseases and these proteins might therefore regulate inflammatory responses to pathogenic challenge on a systemic basis [ ] . for many years it was believed that polypeptides would spontaneously fold into their final native shape, either as they were synthesized by ribosomes or shortly after completion of protein synthesis. although the amino acid sequence of a polypeptide does determine its final conformation, it is now clear that special helper proteins aid the newly formed or nascent polypeptide in folding to its proper shape [ ] . these proteins, called molecular chaperones, recognize only unfolded polypeptides or partly denatured proteins and do not bind to normal, functional proteins. their role is essential because the cytoplasmic matrix is filled with nascent polypeptide chains and proteins. under such conditions it is quite likely that new polypeptide chains often will fold improperly and aggregate to form nonfunctional complexes. molecular chaperones are key players of the homeostasis of the proteome network and suppress incorrect folding and may reverse any incorrect folding that has already taken place [ ] . therefore, their expression and the activity are tightly regulated at both the transcriptional and posttranslational level at various age-related diseases (e.g., degenerative diseases and cancer) [ ] . chaperones and atp-dependent proteases collectively engaged in the first line of defense of mitochondrial protein quality control (mtpqc) system, and this way it monitors the removal of oxidatively damaged polypeptides. for example, 'lon protease' governs the removal of oxidized proteins in yeast and mammalian mitochondria [ ] . many transmembrane proteins are also involved in the protection from infectious diseases. for example, interferon-induced transmembrane protein- (ifitm ) is a virus restriction factor mediating cellular resistance to influenza viruses and other viruses that enter cells via the acidic endosome [ ] . transmembrane mucins and their o-glycans on the glycocalyx provide the transcellular barrier, a second layer of protection. cell surface glycans bind carbohydrate-binding proteins that respond to extrinsic signals and modulates pathogenic responses. apart from maintaining the homeostasis, it also restricts drug targeting of epithelial cells [ ] . some of the members of gap junction (gj) family proteins like intercellular channels, which connect the cytoplasm of neighboring cells and hemichannels that connect the intra-and extracellular milieu, are also known to participate in physiologic and pathologic processes including electrical conduction, inflammation, immune system activation, tissue repair/remodeling, and response to bacterial and viral infections. however, little is known about the role of gj channels in parasite infection. a number of proteins take part in protection of human body from external harmful agents. keratin protein is a component of skin which is considered the first line of defense. this innate immunity component serves to prevent microorganisms from entering the body. lysozyme, an enzyme present in tears and saliva, and thus, another protein also confer protection to the body. likewise, almost all the elements of our immune system, e.g., immunoglobulins (antibodies), various cell surface proteins (t and b cell receptors, major histocompatibility complex, cd , cd , cd , etc.), the several adaptor proteins and enzymes, involved in signal transduction pathways, are all of protein origin and crucial for combating against external infections. since peptides play a crucial role in the fundamental physiological and biochemical functions of life, they have for decades now attracted much attention for their potential therapeutic use. compared with small chemical entity drugs, peptide-based drugs possess certain favorable characteristics, including: • higher potency: peptide-based drugs generally are very active on their target receptor, which translates into a high effect at a low dose; • higher selectivity: peptides can very tightly fit to their receptors, which make them much more selective than smaller molecules. this means that peptides tend to bind only to their target receptor and therefore are less likely to be associated with serious adverse side effects; • naturally occurring biologics-better safety: peptides are naturally degraded in the blood stream by circulating enzymes to their component amino acids. as these are natural biological products, peptide drugs are also associated with less accumulation in body tissue and fewer toxicity findings also owing to their low doses. there are a few challenges associated with the use of peptides like they are generally short-lived, cannot be administered orally and have low product stability. various peptide/glycopeptides drugs are administered: vancomycin, vasopressin, insulin (recombinant), nesiritide, ceruletide, bentiromide, exenatide, etc. vaccination, composed of several natural and artificial proteins, is a key strategy for the control of various infectious diseases. many pathogens, such as streptococcus pneumoniae, haemophilus influenzae type b (hib), and neisseria meningitidis produce on their surfaces dense and complex glycan structures, which represent an optimal target for eliciting carbohydrate specific antibodies able to confer protection against those bacteria. the traditional mechanism of action of glycoconjugates has considered peptides generated from the carrier protein to be responsible for t cell help recruitment. progress of synthetic and biosynthetic methods for the preparation of glycoconjugates gives new insights for the design of improved carbohydrate-peptide conjugate vaccines [ ] . similarly, staphylococcus aureus is a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. methicillin-resistant s. aureus (mrsa), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the united states. s. aureus produces numerous molecules that can potentially promote immune evasion, including protein a (spa), an immunoglobulin (ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. this finding provides the foundation to develop a vaccine that prevents severe s. aureus infections [ ] . stanley b. prusiner coined the term prion in . it is basically derived from the words protein and infectious [ ] . the protein that prions are made of (prp) is found throughout the body, even in healthy people and animals. however, prp found in infectious material has a different structure and is resistant to proteases, the enzymes in the body that can normally break down proteins. the normal form of the protein is called prp c , while the infectious form is called prp sc -the c refers to 'cellular' or 'common' prp, while the sc refers to 'scrapie', the prototypic prion disease, occurring in sheep [ ] . a prion in the scrapie form (prp sc ) is an infectious agent composed of protein in a misfolded form [ ] . so, they are not considered living organisms but may propagate by transmitting a misfolded protein state. if a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the disease-associated, prion form; the prion acts as a template to guide the misfolding of more proteins into prion form. these newly formed prions can then go on to convert more proteins themselves; this triggers a chain reaction that produces large amounts of the prion form [ ] . this altered structure is extremely stable and accumulates in infected tissue, causing tissue damage and cell death [ ] . all long-term hematopoietic stem cells express prp on their cell membrane and those hematopoietic tissues with prp-null stem cells exhibit increased sensitivity to cell depletion [ ] . a naturally occurring disinfectant exists within common lichens and might actually be able to stop prions in the wild. christopher johnson and his team describe experiments with lichens, symbiotic collections of algae, fungus, and bacteria that casual observers might mistake for moss. three common species of lichens, the team has found, exude an enzyme that breaks down the prion. they showed that these lichen extracts efficiently degrade disease-associated prion protein (prptse), the probable etiological agent of the transmissible spongiform encephalopathies (tses), and suggest that some lichens could have potential to inactivate tse infectivity on the landscape or be a source for agents to degrade prions [ ] . proteins are one of the most important biomolecules, involved in any biological process of all living beings, playing useful as well as harmful roles in their survival. it is necessary to investigate these proteins, and to develop new biomedical tools and technologies, which will play a key role in evasion of various diseases, thus, saving and improving quality of lives. further research is ongoing and should be carried out to explore this area of utmost concern. role of adhesin release for mucosal colonization by a bacterial pathogen bacterial adhesins: function and structure molecular aspects of bordetella pertussis pathogenesis the role of the bacterial flagellum in adhesion and virulence molecular basis of group a streptococcal virulence role of hyaluronidase in subcutaneous spread and growth of group a streptococcus acceleration of hyaluronidase production in the course of batch cultivation of clostridium perfringens can be achieved with bacteriolytic enzymes bacterial collagenases: a review neuraminidase production by vibrio cholerae o and other diarrheagenic bacteria mcgraw hill, new york . brook i ( ) current concepts in the management of clostridium tetani infection university of texas medical branch at galveston, galveston microbial toxins: current research and future trends bacterial toxins toxic shock syndrome: role of the environment, the host and the microorganism bacterial endotoxin and current concepts in the diagnosis and treatment of endotoxaemia glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation, and intracellular trafficking human cytomegalovirus phosphoproteins and glycoproteins and their coding regions the glycoproteins of the human herpesviruses viral glycoprotein heterogeneity-enhancement of functional diversity the role of human immunodeficiency virus type envelope glycoproteins in virus infection virus receptors: implications for pathogenesis and the design of antiviral agents the influenza virus-infected host cell, a target for the immune response influenza: pathogenesis and host defense pathogen recognition and innate immunity toll-like receptors and innate immunity the repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors discrimination of bacterial lipoproteins by toll like receptor toll-like receptors genomic insights into the immunesys-temoftheseaurchin conservation of nlr-triggered immunity across plant lineages functions of nod-like receptors in human diseases interactome data and databases: different types of protein interaction protein interactions and disease: computational approaches to uncover the etiology of diseases molecular chaperones-cellular machines for protein folding prokaryotic and eukaryotic heat shock proteins in infectious disease molecular chaperones in cellular protein folding heat shock proteins: molecular chaperones of protein biosynthesis molecular chaperones and proteostasis regulation during redox imbalance mitochondrial atp-dependent proteases in protection against accumulation of carbonylated proteins. mitochondrion a diverse range of gene products are effectors of the type i interferon antiviral response the ocular surface epithelial barrier and other mechanisms of mucosal protection: from allergy to infectious diseases recent mechanistic insights on glycoconjugate vaccines and future perspectives staphylococcus aureus protein a promotes immune suppression prusiner: autobiography. nobelprize.org. accessed biomedicine. a view from the top prion diseases from , feet sherris medical microbiology unraveling prion strains with cell biology and organic chemistry the structural basis of protein folding and its links with human disease prion protein is expressed on longterm repopulating hematopoietic stem cells and is important for their self-renewal degradation of the disease-associated prion protein by a serine protease from lichens key: cord- -zd v b authors: kawashima, kent; matsumoto, tomotaka; akashi, hiroshi title: disease outbreaks: critical biological factors and control strategies date: - - journal: urban resilience doi: . / - - - - _ sha: doc_id: cord_uid: zd v b disease outbreaks remain a major threat to human health and welfare especially in urban areas in both developed and developing countries. a large body of theoretical work has been devoted to modeling disease emergence, and critical factors that predict outbreak occurrence and severity have been proposed. in this chapter, we focus on biological factors that underlie both theoretical models and urban planning. we describe the sars – pandemic as a case study of epidemic control of a human infectious disease. we then describe theoretical analyses of disease dynamics and control strategies. an important conclusion is that epidemic control will be strongly dependent on particular aspects of pathogen biology including host breadth, virulence, incubation time, and/or mutation rate. the probability, and potential cost, of future outbreaks, may be high and lessons from both past cases and theoretical work should inform urban design and policy. interdisciplinary collaboration in planning, swiftness of information dissemination and response, and willingness to forgo personal liberties during a crisis may be key factors in resilience to infectious disease outbreaks. infectious diseases pose an ever-present danger to human societies. despite tremendous advances in medical care, roughly one quarter of worldwide human deaths are attributed to infectious and parasitic disease (mathers et al. ) . several seemingly unalterable aspects of urban life, including long-distance travel and dense human contact networks, facilitate outbreaks from both known and newly evolved pathogens. epidemics are defined as widespread occurrences of infectious disease in a community at a particular time, and the th century bubonic plague, or "black death", was the most devastating epidemic in human history (benedictow ) . death rates were as high as - % in europe, africa, and asia from a disease caused by a bacterial infection (yersinia pestis) that persists in rodent populations and is transmitted by fleas to humans. close contact between humans and rats and worldwide travel contributed to the global impact of bubonic plague which appears to have originated in asia and traveled to europe via trade routes (especially rat-infested ships). the most destructive modern pandemic was the influenza that infected one third of the world's population (about million) and killed - million between january and december (taubenberger and morens ) . "spanish influenza", as the disease was named, is caused by the h n virus which is endemic in pigs and birds and often transitions into human populations. the lethality of the strain was high and showed an unusual relationship between lethality and patients' age: % of deaths were in the - age group which is the opposite pattern for milder flu strains (higher mortality among the very young and the aged). this partly reflected the impact of wwi where contagion was passed among troops both in training facilities as well as during warfare. however, the strain also had an unusual and lethal property; virulence was enhanced by a human immune over-reaction called a "cytokine storm" which causes the lungs to fill with liquid. some important aspects of the epidemic were: a deadly pathogen arose from a jump from animal to human (close between-species interactions were important in the origin of the virus), a few mutations were sufficient to confer strong lethality for the virus, and human travel allowed rapid spread (close quarters and massive troop movements helped to spread the virus and allowed new mutations to spread quickly). this chapter will focus on biological factors that are relevant for understanding and controlling epidemics. we will briefly describe some pathogens that cause human disease and their transmission mechanisms before analyzing the sars - epidemic as a case study of a modern urban epidemic. disease models will be discussed with a goal of determining how human societies can prepare to minimize the impact of future disease outbreaks. infectious diseases can be classified into two broad categories based on their pattern of transmission (table ) . "long-range" infectious diseases are infections that do not require close contact for transmission. for example, water-borne diseases, such as cholera, can rapidly spread throughout the community when the supply of drinking water becomes contaminated with the pathogen vibrio cholerae through poor sanitation or hygiene practices. food-borne infections follow a similar transmission pattern to water-borne diseases. transmission through contaminated food and water is also known as "fecal-oral transmission" because fecal matter is often the source of contamination while oral ingestion is the primary route for infection (mount sinai hospital ) . diseases transmitted by an animal vector, such as bubonic plague, are also considered long-range infections because a vector facilitates the spreading of the pathogen and direct contact is not necessary. one interesting aspect of some vector-borne infections is that direct contact with an infected individual cannot transmit the infection without the help of the vector. for example, dengue fever, caused by a mosquito-borne virus, can only be transmitted through the bite of an infected mosquito (us centers for disease control and prevention ). in contrast, plague, caused by bacteria living in fleas of rodents, is primarily transmitted through flea bites, but contact with contaminated body fluids like blood can also lead to plague bacterial infection (us centers for disease control and prevention a). in general, fecal-oral and vector-borne diseases are infections transmitted through an environmental (water, food) or a biological (animal) carrier that extends transmission range to large distances, but other routes are also possible depending on the specific pathogen. compared to long-range diseases, "short-range" infectious diseases are infections that transmit over limited distances and may require close or direct physical contact with an infectious individual. examples of short-range infections are pathogens that infect via contaminated airborne particles or expectorated droplets, and diseases that require contact with skin or bodily fluids such as blood or semen. infections capable of airborne transmission have the widest range among short-range infections and are caused by pathogens that spread through minute solid or liquid particles suspended in the air for an extended period of time (mount sinai hospital ) . in addition, the pathogen must be resistant to desiccation to remain viable for long periods of time outside its host. respiratory diseases are commonly believed to spread via airborne transmission of contaminated particles expectorated from coughing and sneezing. however, many respiratory pathogens do not have the capacity to withstand dry environments. instead, these pathogens transmit via "droplets"-expectorated moisture particles that are too big to indefinitely remain suspended in the air-to ensure ample moisture while outside the host. transmission occurs when contaminated droplets from an infected individual come in contact with surfaces of the eye, nose, or mouth. this mode of transmission is called "droplet contact". although diseases spreading via droplet contact have a more limited range than truly airborne infections, in the later sections, we will show how environmental factors can extend the range of droplet transmission. finally, diseases that transmit via direct contact generally have the most limited transmission range and some have stringent requirements for transmission. in the case of ebola, the disease is transmissible only via direct exposure of broken skin or mucous membranes with contaminated body fluids like blood, urine and semen, and excretions such as vomit and feces (us centers for disease control and prevention c). sexually transmitted diseases like hiv/aids are a special form of direct contact infection that requires sexual intercourse or sharing contaminated needles for exposure (us centers for disease control and prevention b). thus, short-range infections are characterized by some dependence on distance for infection and can be transmitted directly without a carrier. distinguishing between these two classes is important because measures to alleviate and control the spread of long-range infections are not applicable for short-range cases and vice versa. for instance, targeting the carrier or vector of the disease to control the spread of long-range infections (e.g., decontaminating or blocking off access to contaminated water or food) and reducing exposure to vectors of the disease, are irrelevant for mitigating the spread of short-range infections. in contrast, measures to control short-range diseases such as limiting person-to-person contact and imposing quarantine procedures do little to help alleviate the spread of water-borne or vector-borne illnesses. thus, identifying the mode of transmission is crucial to controlling the spread of any contagious infection. however, we will show that the distinction between long-and short-range transmissions is not always clear-cut. in this chapter, we focus on the emergence and spreading of severe acute respiratory syndrome or sars; the first worldwide pandemic in the age of globalized air travel and telecommunications. through theoretical analyses and data gathered from the epidemic, we examine how globalization exacerbates the problem of containing epidemics and show how urban environments can be especially prone to epidemics. the emergence and control of the sars epidemic is extensively documented. research on both the origin and epidemiology of the outbreak as well as the biological underpinnings of the disease making them excellent cases to determine methods to enhance urban resilience to epidemics. the history of the - global outbreak of severe acute respiratory syndrome (sars) provides key lessons on biological and policy factors that should be of general importance in designing resilient cities. we will summarize the history of the epidemic, with a focus on biological factors, before our discussion of disease models. according to the world health organization (who), over worldwide sars cases and over deaths occurred in different countries, mostly over a period of about four months (kamps and hoffmann ) . the severe, "atypical" pneumonia originated in guangdong province in southern china in mid-november . most of the early cases appear to have occurred among those who kill and sell animals and meat as well as food preparers and servers (breiman et al. ) . by mid to late january , the disease began to spread rapidly within the province, but a combination of symptoms difficult to distinguish from pneumonia (fever, dizziness, muscle soreness, coughing) and government policy to discourage coverage delayed the reporting of the epidemic until february . the initial communication reported cases (including > healthcare workers) and mortalities, but claimed that the epidemic was under control (enserink ) . the role of "superspreaders" and amplification in hospitals remained characteristics of sars as it spread to a worldwide epidemic. the first of several superspreading (generally defined as ten or more transmissions from a single infected individual) events occurred in hong kong on february , (braden et al. ). the index case was a physician from guangdong who stayed at the hotel metropole. the physician had treated sars patients in guangdong (although the disease was still unrecognized) and showed symptoms before his trip. he stayed only one night at the hotel before being hospitalized with severe symptoms but the short stay was sufficient to spread the infection to or more of the guests from the same floor of the hotel as well as a hong kong resident who visited one of the guests. eventually, over (almost half) of the documented sars cases could be traced to this "index" case. remarkably, there was no known direct contact in most of the transmissions among the hotel guests and visitors. the hong kong resident who visited a friend in the hotel subsequently infected over others at the prince of wales hospital in hong kong. others were business/holiday travelers who spread the pathogen to canada, vietnam, and singapore. as we will discuss below, this high transmission rate with little close contact in the metropole hotel remains mysterious. rapid recognition of a new epidemic was aided by a who disease expert, carlo urbani, who was asked to examine patients in a hanoi hospital. the affected included one of the metropole guests and roughly hospital staff who became affected not long after his admission. urbani recognized a severe, and possibly new, disease and warned who headquarters as well as the hospital and vietnam government before contracting, and eventually dying from, the disease (bourouiba et al. ) . response time is a critical parameter in epidemic control and his efforts played a large role in the effort to subdue the epidemic. who designated a new disease, "severe acute respiratory syndrome" (sars), on march and issued a global health alert on march followed by an emergency travel advisory on march . the etiological agent of sars was later discovered to be a novel coronavirus and was named sars-associated coronavirus (sars-cov). this discovery, in late march , came as a surprise to disease experts as previous human coronaviruses were only known to cause mild illness. in animals, related viruses were known to cause fatal respiratory as well as neurological diseases but coronaviruses are usually highly species-specific (kamps and hoffmann ) . forensic analysis of the metropole hotel in late april revealed physical components of sars in the common areas of the th floor including the corridor and elevator hall. however, no bacteria were found inside the guest rooms of the infected guests (the ventilation systems employed positive pressure within the guest rooms so that air was not shared among rooms). respiratory droplets, or suspended small particle aerosols generated by the index case-patient, are the most likely transmission mechanism (braden et al. ) . sars and other respiratory infections are considered to undergo short-range (approximately m) transmission via pathogen-infected droplets from host coughing or sneezing. such transmission requires "close contact", physical proximity between infected and susceptible individuals who can be infected when large droplets spray enter their bodies via air or touch. however, minute droplets or even solid residues that can arise via evaporation (droplet nuclei) may allow potential indirect and/or long-range transmission (bourouiba et al. ) . for example, contaminated gas clouds that form during coughing/sneezing may have carried the pathogen and extended its transmission range, removing the distinction between droplet contact and airborne modes of transmission. aerosol transmission probably caused high infection rates in an airline flight (air china ) from hong kong to beijing in which a single -year old individual infected at least others (olsen et al. ) . this feature of the disease may be highly relevant for medical and urban policy. long-range aerosol/nuclei transmission does not require direct contact between infected and uninfected individuals and can greatly elevate the number of "contacts" for a given infected individual. interestingly, genetic analysis showed that several sars strains entered hong kong, but only the hotel metropole index case was associated with the subsequent global outbreak (guan et al. ) . a related superspreading event occurred at a crowded high-rise residence, the amoy gardens, in hong kong. many of the infected individuals inhabited vertically placed apartments (in contrast to transmissions on a common floor at the metro hotel case). sanitary drainage fixtures that were malfunctioning and allowing air and sars-contaminated aerosols to flow back into resident bathrooms may have been the main driver of infection spread in the condominium (stein ) . the superspreader was likely a medical patient undergoing treatment for a kidney problem including hemodialysis, a medical treatment that inhibits immune capacity (stein ) . the index case carried a high viral load and suffered from diarrhea. an important feature of this event was again, a lack of direct contact between the spreader and the individuals he infected, and the "opportunity" for the pathogen to be exposed to a large number of individuals through airborne transmission (yu et al. ) . at the amoy gardens, more than individuals showed symptoms of sars almost simultaneously. high rates of hospital (nosocomial) transmission were an important and disturbing characteristic of the sars outbreak. the large fraction of infections among healthcare workers probably reflects a combination of contact from respiratory secretions from patients who were at a highly contagious stage (critically ill individuals also were the most infectious) as well as from medical procedures that inadvertently generated aerosol contamination. a single patient appears to have transmitted infections to over hospital staff in a span of two weeks at the prince of wales hospital in hong kong (see below). two other superspreader events occurred in hospitals in other countries (braden et al. ) . one infected patient (the son of one of the hotel metropole guests) infected over cases among patients, visitors, and healthcare workers at the acute care hospital in toronto, canada. finally, although taiwan instituted strict port entry screening and isolation of potentially exposed travelers entering the country, there was an outbreak in the ho ping hospital which spread into the community. in spite of a lock-down quarantine of over people in the hospital (included a large fraction of uninfected individuals), over cases emerged before the outbreak was contained. the initial rapid spread of sars caused widespread concern and panic and the epidemic seemed unstoppable. however, the disease was eventually contained within several months through efforts coordinated by the who. although advances in biomedical science and cooperative efforts among laboratories played key roles in isolating the infectious agent, "classic" epidemiological practices of patient isolation (separation of infected individuals from the general population), contact tracing, and large-scale quarantine (isolation of non-symptomatic individuals who have had contact with the infectious agent) were the main elements that halted the epidemic ). the - sars pandemic was caused by a moderately transmissible viral infection that produced . new cases for every infection (riley et al. ) and yet it spread to over countries across three continents potentially exposing tens of thousands of people in the span of only a few months. several studies have shown that the vast majority of infected cases had very low infectivity and that a few outliers were responsible for a disproportionate number of new infections riley et al. ; lipsitch et al. ; wong et al. ). in fact, riley et al. ( ) and lipsitch et al. ( ) found that early in the epidemic, an infected individual would only produce approximately three new infections when outliers are excluded. in singapore, % of the first probable sars cases showed no evidence of transmitting the infection yet cases appeared to have transmitted the disease to or more individuals (lipsitch et al. ) . shen et al. ( ) found a similar pattern in beijing where out of the confirmed cases did not infect others whereas four cases were responsible for infecting eight or more. the rapid spreading of sars despite only moderate average infectiousness has revived interest in the concept of superspreading events and heterogeneity in pathogen transmission. the transmission potential of an infectious disease is often described by the parameter r, the average number of new infections that infected cases produce over the course of their infection. r is the transmission potential of an infected individual within an otherwise completely susceptible population (dietz ) . however, population-based summary statistics may obscure individual variation of infectiousness and other types of heterogeneities. woolhouse et al. ( ) have shown that heterogeneities in infectiousness exist such that only % of the host population contributes at least % of a pathogen's transmission potential. these individuals who significantly transmit more than the average are called superspreaders. in hong kong, apart from the incident at hotel metropole, at least two large clusters of infection were attributed to superspreading events (riley et al. ) . data from the sars pandemic showed the effect that superspreaders and superspreading events could have on the trajectory of the epidemic. given their crucial role in intensifying an outbreak, we review the risk factors that facilitate superspreading events. co-infection and the presence of a comorbid disease could be risk factors for turning infected individuals into superspreaders (stein ) . studies on hiv/aids transmission showed that co-infection with another sexually transmitted pathogen increased the urethral shedding of hiv in infected individuals. moss et al. ( ) demonstrated that urethral hiv infection is associated with gonococcal infection and treatment for urethritis may reduce the risk of hiv transmission. in the case of sars, peiris et al. ( ) reported that other viral respiratory pathogens such as human metapneumovirus were detected in confirmed sars cases. in addition, the index case in the prince of wales hospital superspreading event was described to have a "runny nose" , an uncommon symptom for a lower-respiratory tract infection such as sars. these observations have led to the hypothesis that co-infection or presence of a comorbid condition could endow an infected individual with characteristics or behaviors that increases their infectiousness (bassetti et al. ) . for example, rhinovirus, the major cause of common colds, can cause swelling of nasal tissues that can elevate airflow speed and contribute to aerosol production (sherertz et al. ) . rhinovirus co-infection with more serious, but less transmissible respiratory ailments, such as sars, could be an important factor contributing to high infectivity. environmental factors also play an important role in facilitating superspreading events (stein ) . in the sars superspreading event at the prince of wales hospital, the index case was placed on a nebulized bronchodilator four times daily for one week (kamps and hoffmann ) . nebulized bronchodilators are often used to deliver drugs to the lungs of respiratory patients but may have inadvertently aerosolized the virus and left infected droplets in the immediate surroundings leading to extensive dissemination of the pathogen (tomlinson and cockram ) . tracheal intubation, which involves placing a flexible tube into a patient's windpipe to maintain an airway to deliver drugs, may also have inadvertently spread sars within hospitals. patients often emit respiratory secretions during the procedure. an outdated ventilation system and overcrowding likely also contributed to the spreading of the virus at the prince of wales hospital (riley et al. ; tomlinson and cockram ) . through a case-control study of hospitals treating sars patients, yu et al. ( ) confirmed overcrowding as one of the general risk factors of hospital-based sars superspreading events. the case-control study performed included wards in hospitals in guangzhou and wards in five hospitals in hong kong and showed that the main risk factors included closely arranged beds (less than m apart), a workload of more than two patients per healthcare worker, hospital staff that continued working despite experiencing symptoms of the disease, and lack of washing or changing facilities for staff. despite the explosive growth and global distribution of the sars outbreak, the pandemic was largely contained through isolation and quarantine, increasing social distance, and social behavioral adjustments (bell and world health organization working group on prevention of international and community transmission of sars ). isolation and quarantine were shown to significantly interrupt transmission of sars in several countries including hong kong (riley et al. ) , china (pang et al. ) , singapore (lipsitch et al. ) , taiwan (twu et al. ) , and canada (svoboda et al. ). in general, symptomatic cases were immediately placed in isolation while contacts of confirmed infected cases were placed in some form of quarantine. in some cases, contacts were not immediately confined but instead were monitored for the disease and isolated only when symptoms emerged. confinement was usually at home but designated facilities were available in countries like taiwan (twu et al. ) . in some cases, individuals under quarantine were allowed to travel with the permission from the local health authorities provided they wore masks and refrained from using public transportation or visiting crowded places. to further reduce the chance of transmission, hong kong and singapore also closed schools and public facilities, and canceled mass gatherings to "increase social distance". people were also required to wear masks when using public transport, entering hospitals, or in jobs where interacting with numerous people is unavoidable such as in restaurants (bell and world health organization working group on prevention of international and community transmission of sars ). the concerted effort has been marginally associated with the rapid reduction of new sars cases in several countries. however, because of the simultaneous introduction of these measures, it is difficult to evaluate the effectiveness of each. several characteristics of the infectious agent were important factors in controlling the sars epidemic. the incubation period from contact with the infectious agent to onset of symptoms was, on average, . days. importantly, peak infectivity coincided with clinical symptoms and often required an additional days or more ). thus, infectious individuals tended to be hospitalized before peak transmissibility. in addition, the two-week interval from exposure to high infectivity gave epidemiologists critical time to perform contact tracing to identify and quarantine potentially infected individuals before they reached high infectivity. this feature, in combination with moderate transmission rates (except in special cases), contributed to making sars a relatively controllable outbreak. in the next section, we present current theories on the emergence and spreading of epidemics and review the theoretical underpinnings behind control measures used to contain outbreaks. we briefly highlight different mathematical models used to describe epidemic dynamics in populations. we explain the factors that govern the emergence and transmission of diseases as well as the evolution of pathogens that cause them. finally, we examine how control measures such as isolation, quarantine, and vaccination mitigate the spread of infections. mathematical models have played an important role in our understanding of disease propagation. if biological factors can be accurately incorporated, such models may have predictive power to evaluate control strategies and guide policy. a key parameter in epidemic models is the total number of new infections that arise from a single affected host, the reproduction number, r. this value determines the outbreak potential of the infection; if r = , the infection will be maintained at a constant level (if we ignore random effects). r > leads to disease spread and r < predicts eventual extinction. however, r is not an intrinsic property of the pathogen. variability of the reproductive number across pathogens, hosts, and environments over time must be understood to accurately model disease. in the following three subsections, we discuss theoretical results on three important aspect of disease outbreak: ( ) the effect of "superspreaders" on the probability of outbreak, ( ) the impact of control strategies such as isolation and quarantine, and ( ) factors that affect the evolution of pathogen virulence. the - sars epidemic was characterized by the large impact of "superspreaders" on disease propagation. in theoretical models, superspreaders can be treated as individuals with large number of connections to other individuals. individual-based simulations incorporating network structures can efficiently address this topic and, in this subsection, we introduce three theoretical studies focused on the effect of network structure on disease outbreak. lipsitch et al. ( ) studied the effect of superspreaders on outbreak probability using the estimated parameters from the sars outbreak in singapore. the authors first estimated the distribution of the parameter r, which expresses the number of new infections from an infected host. probabilities of outbreak (persistence of initially introduced pathogen lineages) were determined for r distributions with a fixed mean but differing in variance. the authors found that large variance in r distribution greatly decreased the probability of outbreak (fig. ) . contrary to the expectation of the importance of superspreaders, their result showed that distributions strongly clustered around the mean had higher probabilities of outbreak than distributions that included superspreaders (right-hand tail outliers). one reason of this apparent inconsistency might be the assumption of a fixed mean r. under this assumption, increased variance in the r distribution increases both the numbers of individuals with extremely high r and low r. individuals with low r are essentially "dead ends" in disease infection and high numbers of such individuals will decrease outbreak risk. a similar result was obtained in meyers et al. ( ) . this study also focused on the case of sars outbreak in asian countries and used parameters estimated from the case study in individual-based simulations. meyers and co-workers examined differences in the probability of outbreak among three different networks among individuals. in the first network, termed "urban", many individuals have numerous contacts at public places including schools, hospitals, shopping centers and workplaces, and have more limited numbers of contacts at their home. the second network was a power law network, in which the distribution of the number of connections has a long right-hand tail. in such a distribution, a small fraction of people have large numbers of connections but most people have only a few connections. the third network was a poisson network, in which the majority of the people have numbers of connections close to the mean number. if the existence of superspreaders increases the probability of outbreak, then power law networks should show the highest outbreak probability. however, similar to lipsitch et al. ( ) , power law networks showed the lowest probabilities of outbreak. the reason might be similar to what we discussed above; in a power law network, the numbers of individuals with extremely small numbers of connection are elevated compared with the other two networks. pathogens cannot spread if they infect such individuals and will go extinct before they have a chance to infect superspreaders. fig. theoretically estimated probability that a single introduced pathogen persists after infinite time under a markov process with different mean (e) and variance (v) in the r distribution. in lipsitch et al. ( ) this persistence probability is considered as probability of an outbreak. modified from lipsitch et al. ( , fig. a) the two studies above indicated reduced probabilities of outbreak for populations that include superspreaders, but this conclusion may be strongly sensitive to model assumptions. networks with more total connections (including superspreaders) may realistically model urban environments (this relaxes the assumption of constant mean connectedness). fujie and odagaki ( ) modeled superspreaders as individuals with higher infection rates (strong infectiousness model) or more connections including connections with distant individuals (hub model). they calculated the probability of outbreak under different fractions of superspreaders in a population and showed that, as the fraction of superspreaders increases, the probability of outbreak increases greatly (fig. ) . they also analyzed several features of outbreaks like speed of disease spread and infection path between the two models and suggested that the hub model is consistent with data from the sars outbreak in singapore. these contrasting results highlight the need to validate model assumptions for applications to human society. higher outbreak probabilities with larger numbers of connections may seem obvious but this may be a realistic scenario for human society. a key issue is whether the number of connections of one person statistically affects that of others in human society. if not, the comparison between different fraction of number of superspreaders like fujie and odagaki ( ) would fig. theoretically estimated "percolation" probability of a single introduced pathogen under different fraction of superspreaders and population density in a hub model. in fujie and odagaki ( ) this percolation probability of percolation theory, in which a pathogen that has infected an individual in the bottom of × grid finally reaches an individual in the top of the grid, is considered the probability of an outbreak. as density becomes lower, distance between individuals becomes longer. the results for different fraction of superspreaders (λ) are shown in different markers. modified from fujie and odagaki ( , fig. ) more realistically predict the effect of superspreaders on the probability of outbreak. however, if higher number of connections of one person necessitates reduced numbers for others, the results in lipsitch et al. ( ) and meyers et al. ( ) could be more applicable for human society. in either case, models should focus on both outbreak probability as well as the nature (explosiveness) of disease spread. lloyd-smith et al. ( ) demonstrated that many previous human epidemics appear to have spread through superspreaders (although not to the same extent as sars). they showed that, although pathogen extinction probability increases with variance in reproductive number, populations with superspreaders experienced more rapid infection spread in cases of pathogen survival. under their model, host populations may suffer greatly from improbable epidemics. the first step to control the rise of any infectious disease is to understand how it transmits between hosts. often, we imagine these infections as readily communicable illnesses that can be caught by even the most fleeting contact. but as we have shown, exposure and transmission depends on the route the infectious disease pathogen takes. this means that some diseases can be transmitted even without direct or close contact with an infected individual. we have also shown how particular conditions can make a close-range disease transmit over extended distances, as is the case with sars transmission in the amoy gardens condominium complex. aside from mode of transmission, the timing between infectiousness and showing symptoms of the disease is another crucial factor to consider. an infectious disease is an illness caused by the presence of a pathogen within the host as well as the host's response to the invading pathogen. upon entry into the host, the pathogen begins to increase its numbers by redirecting resources to itself. after a certain time, its presence and the damage it has done to the host raises an internal host response to thwart the infection. it is at this stage of the infection that overt symptoms appear and the infection can be observed. the time elapsed between exposure to the pathogen and observing the initial signs and symptoms of the disease is called as the "incubation period" of the disease. the length of the incubation period varies among diseases and is affected by several factors such as dose and route of infection, and host susceptibility and ability to respond to the pathogen. because of these considerations, incubation period is described as a range of values depicting how short or how long it takes before an infection would show symptoms. during this period, the infected individual may or may not be contagious depending on the type of disease and the individual's health state. the disparity between the time we observe the symptoms of the infection and consider an individual ill and the time the individual is contagious are important aspects to consider in modeling as well as in prescribing infection control measures. the timings vary widely depending on the infectious disease (fig. ) . in the simplest scenario, the entire time an infected individual is contagious occurs after the first symptoms of the disease and ends well before the symptoms disappear. a completely overlapping timing where all symptomatic individuals are infectious would simplify identification and make control measures more effective. this timing pattern can be easily modeled by assuming that newly infected individuals simultaneously start to cause new infections to other individuals. and because the disease spreads specifically through a single class of individuals, control measures can simply identify symptomatic individuals to prevent new infections. in the case of sars, peak infectiousness occurs - days following the onset of disease symptoms and correlates with viral load over the course of the infection ). many believe this pattern helped contain the sars pandemic (chau and yip ; diamond ; fraser et al. ) despite exponential growth of the epidemic that quickly spread to multiple continents. in contrast, diseases such as hiv/aids have completely different infectious and symptomatic periods. the first signs of aids do not appear until the infecting pathogen has significantly damaged the host yet the infected individual is contagious throughout the asymptomatic phase and peak infectivity occurs before the onset of symptoms ). modeling diseases with disconnected infectious and symptomatic periods requires splitting the "infectious" class into "asymptomatic infectious" and "symptomatic infectious" classes to more accurately reflect the clinical characteristics of the disease. though sars and hiv/aids have significantly different timing patterns, the relationship between peak infectivity and symptomatic period is clear. however, some diseases exhibit partially overlapping contagious and symptomatic periods that make their outbreaks more difficult to stop. identifying the precise period that infected individuals are contagious is difficult because the values are affected by anderson et al. ( ) numerous factors such as susceptibility of the host, mechanism of infection, and immune response (baron ) . individual variation in incubation periods further complicates the problem. in dealing with diseases that exhibit partially overlapping periods such as pandemic influenza, it is best to rely on conservative measures that consider both exposed and likely infected individuals as targets of containment measures. note that it is possible to harbor an infection yet not show any signs or symptoms of the disease. called a "subclinical infection", this asymptomatic state may be a result of the pathogen infection strategy and the host's ability to tolerate an infection instead of purging it (baron ) . asymptomatic cases that are infectious can help spread the contagion despite strict control measures by being misclassified as uninfected individuals. asymptomatic cases are usually discovered by chance or by reviewing epidemiological data after an epidemic (baron ) . modeling asymptomatic cases requires adding an "asymptomatic infectious" class that is capable of exposing and transmitting the disease. containing the spread of an infectious disease suspected to have a high proportion of asymptomatic infected individuals is difficult but procedures such as contact tracing may reveal some of these asymptomatic carriers and quarantining of exposed and high-risk individuals can minimize their impact. most emerging infections have no available vaccine or treatment. thus the only way to control the spread of these diseases is to prevent exposure and further transmission. isolation and quarantine are two control measures that help block transmission by isolating the individuals who have, or may have, the contagious disease. "isolation" describes separating sick individuals (symptomatic) from people who are not sick (naïve) while quarantine pertains to the practice of separating and restricting the movement of asymptomatic individuals who may have been exposed to the disease to see whether they become sick. these control measures aim to progressively reduce the number of new secondary infections until the disease is eradicated from the population. formally, we can measure the effect of isolation and quarantine by taking a survey of new infected cases and deriving the basic reproduction number r of the infectious disease for each step of the outbreak. without any intervention, r is expected to eventually decrease as the number of susceptible individuals decreases in a finite population without migration. however, by the time the rate decreases to r < , a large proportion of the population has already been infected with the disease. by "removing" potentially infected individuals from the population, isolation and quarantine can more rapidly decrease r below by reducing the incidence of the disease, leading to fewer new infected cases capable of transmitting the infection. isolating symptomatic individuals prevents new cases by separating individuals spreading the pathogen from the host population. given a clearly defined set of symptoms to diagnose the disease, this strategy is intuitive and straightforward to implement from a public health point of view. a precise case definition also reduces misdiagnoses and prevents unnecessary isolation of non-target cases. however, many diseases share symptoms and may occur in combination with other infections so case definitions are not always precise. in the sars epidemic, infected individuals showing atypical symptoms were a major source of transmission, partly because co-infection may have elevated transmission rates (kamps and hoffmann ) . modern biomedical research may serve to quickly identify new pathogens and providing diagnostic tests may be the most important function of initial research (vaccines and treatments generally require months or years and may not be helpful for new diseases). isolating symptomatic individuals is most effective if peak infectivity occurs after observing the first symptoms of the disease and transmission only occurs in symptomatic cases ). while diseases like sars have shown such properties, other infections such as influenza appear to be transmissible even prior to showing overt symptoms. when peak infectivity occurs before the onset of symptoms, quarantine for symptomatic individuals may have little impact on dampening the spread of the infection . even for infectious diseases that transmit only after symptoms emerge, infected individuals may not immediately practice self-isolation or report to a healthcare facility. during the lag time between diagnosis and isolation, the pathogen can still spread to susceptible hosts undermining isolation as a way to control the spread of the infection. on the other hand, quarantining individuals that have been exposed to the disease addresses the shortcomings of isolation as a control measure. identifying exposure is dependent on how the pathogen spreads from one host to another. if the pathogen transmits via airborne droplets, then people present in the same room with an infected individual are considered "exposed". however, if the pathogen spreads only through sexual contact, then only individuals who have had sexual relations with the infected case are considered exposed. when the transmission mechanism is unknown, scenarios such as airborne transmission or via physical contact that lead to the most conservative outcome may be used instead. because the criteria to select individuals are independent of disease status, this strategy sacrifices sensitivity but works regardless of timing of infectivity and does not suffer from the lag time problem. such a conservative strategy is well suited for emerging infections, especially when the mechanisms of transmission and pathogenesis have yet to be revealed. in a perfect quarantine, all exposed individuals are expected to undergo quarantine regardless of whether they develop the disease or not, and during the quarantine period, exposed individuals do not transmit the disease. however, tracing all contacts is often problematic especially when an infected individual has traveled to numerous locations and when exposure occurred in public spaces and mass transit. compared to isolation, quarantine sometimes faces more resistance from expected participants especially from those who have been exposed but appear to be in a healthy condition. during the sars epidemic, mass quarantines were implemented in many countries. over , potentially exposed individuals were quarantined in taiwan, but in retrospect, the action may have spread panic among uninfected individuals and may not have been an effective strategy (university of louisville school of medicine ). in reality, quarantines are never perfect. compliance to the procedures is often problematic: quarantined individuals do not reduce their geographical movement or they only abide by the procedure for a short period. formal quarantines have good compliance rates but are costly and difficult to manage for a large number of cases. therefore a majority of quarantines are made voluntarily or with less monitoring than formal quarantines, but these suffer from reduced compliance and are less effective overall. knowledge about potential superspreaders to identify candidates for isolation can greatly enhance the efficacy of quarantines with much lower numbers of required isolations (diamond ) . although such knowledge may be rare at the beginning of an epidemic, rapid epidemiological analyses may play a critical role in reducing the costs of epidemic control. a critical aspect of human pathogens is their virulence or extent of damage to host. high virulence infectious disease such as hiv, plague or smallpox can be a great threat to human society, and the number of cases of pathogens that have been reported to have evolved virulence and/or resistance against drugs is alarming (altizer et al. ; holden et al. ). understanding the factors which affect the evolution of virulence in human society is an important issue. if it is possible to control these factors in urban design, human society can be more resilient against serious disease outbreaks. several classic theoretical studies on the evolution of virulence concluded that reduced virulence is generally adaptive and should evolve among pathogens. low virulence allows infected host individuals to survive, and pathogens can have more chance to spread to other host individuals. if pathogens have high virulence, they can propagate within an infected host individual, but risk killing the infected host and limiting their spread to other hosts. trade-offs between reproduction within a host and transmission among hosts is a well-studied explanation for the evolution of reduced virulence (anderson and may ; alizon et al. ). however, the balance (or equilibrium) of this trade-off can differ depending on biological characters of pathogens. ewald ( ) discussed how transmission mechanisms of pathogens can alter the predicted trajectory of virulence evolution. highly virulent diseases tend to immobilize hosts in early stage of infection. therefore, if pathogens are mainly transmitted by contacts between hosts, higher virulence would greatly decrease chances of new transmission. however, if pathogens can survive outside of the host and can be transmitted by air, water or vectors in which they are not virulent, host immobility should have less effect on the chances of new transmission. ewald ( ) noted that such pathogens, such as smallpox, tuberculosis or diphtheria, are often more virulent than pathogens that depend more directly on hosts for transmission. other factors can affect the balance of the trade-off and allow evolution of high virulence (galvani ) . for example, if multiple pathogen strains infect simultaneously and compete within individual hosts, high reproduction rate within a host (leading to high virulence) may be favored. in sexually-transmitted diseases, frequent exchange of sexual partners makes transmission of pathogens between hosts easier and as a result cause high virulence. this may be the case of hiv in human society (lipsitch and nowak ) . host population structure also affects the transmission of pathogens and therefore, has a large impact on the evolution of virulence. because urban planning and design can create or alter population structure by its use of the the environment, in the following paragraphs, we introduce two studies focused on the effect of host population structure on evolution of virulence. these studies are based on relatively simple models that may yield general insights. boots and sasaki ( ) incorporated a grid-like spatial structure of "sites" at which individuals can exist. each site can have one of three states: empty, occupied by susceptible individual, or occupied by infected individual. in the spatial structure, connections between individuals were divided into two types, those between neighbors and those between randomly chosen individuals. randomly chosen individuals can be in distant sites, and in such cases, pathogens can be transferred to distant locations. they found that pathogen virulence is favored as contact between hosts living in distant places becomes more common. in this model, a site becomes empty after death of an occupant. therefore, higher virulence is more likely to create a situation in which pathogens kill all susceptible hosts around them and can no longer spread. however, long-distance transfer allows pathogens to spread to new locations where they are surrounded by susceptible hosts. long distance transportation in human society allows contact between distant individuals and may be an important factor that facilitates the spread of outbreaks and favors pathogen virulence. boots and sasaki ( ) did not consider host immunity in their model. immune (infection-resistant) hosts can block pathogen spread and may have a large impact on the evolution of virulence. this question was theoretically addressed by the same authors. boots et al. ( ) incorporated the immune state after the recovery assuming a negative correlation between recovery rate and virulence and found that evolutionary trajectories could lead to low, or even extremely high, virulence depending on host population density. in host populations with high density, pathogens can easily find susceptible hosts and therefore, low virulence which increases the opportunity of infection to a new host evolves. on the other hand, in host populations with low density, immune hosts around a newly infected host efficiently block pathogen spread. in this case, highly lethal pathogens which kill infected hosts and make open spaces can spread more efficiently compared with pathogens with low virulence which induce immunity in hosts. even after killing some hosts, pathogens still have a chance to spread by infecting new susceptible hosts that emigrate to the open spaces. in boots and sasaki ( ) , infected hosts are assumed to be susceptible just after they recover and therefore, lower virulence pathogens spread more efficiently. however, in boots et al. ( ) , immune hosts block pathogen spread and create scenarios where highly lethal pathogens evolve. the results in boots and sasaki ( ) and boots et al. ( ) reveal scenarios in which low virulence can evolve to higher virulence depending on the structure of host populations. a key point is that outcomes are sensitive to the scenarios of population structure, transmission mechanisms, and host immunity. because of their short generation times and high genetic mutation rates, pathogens like rna viruses may evolve rapidly, even over the course of an outbreak. influenza virus, norovirus or dengue virus are well known examples of rna viruses that infect humans. because these viruses cause epidemics every year, controlling their impact is a very important aspect of urban resilience. as mentioned above, the models in boots and sasaki ( ) and boots et al. ( ) may be too simple to directly apply for particular diseases. theoretical studies under more realistic conditions based on structures that closely resemble actual human society and biological characteristics relevant to particular pathogens will be valuable to prevent and control outbreaks of high virulent diseases. important points to consider include parameters and assumption sensitivity for aspects of both host populations and pathogens. in addition, the definition of "connection" differs depending on the transmission mechanism of the pathogen. the concept of "network" must take the view of the pathogen and different networks may need to be considered for different diseases in the same human populations. many of the major human infectious diseases are zoonotic infections that have crossed over from animals into humans (wolfe et al. ). bubonic plague (schmid et al. ) , influenza (palese ) , hiv (gao et al. ) , ebola (marí saéz et al. ) , sars (lau et al. ; li et al. b ) and mers (memish et al. ; wang et al. ) have all been shown to have originated from animals before infecting humans. wolfe et al. ( ) surveyed major infectious diseases ranked by highest mortality and/or morbidity to identify patterns in their animal origins and geographical spreading. all the diseases they surveyed appeared to have originated from the old world (africa, asia, europe) and a remarkable proportion of causative pathogens arose from warm-blooded vertebrates while the remaining were attributed to birds. interestingly, the purported geographical origin of the disease was correlated with the type of animal to which the pathogen originally infected. for example, many diseases that trace back to tropical regions have come from wild non-human primates whereas diseases attributed to temperate regions often emerged from domestic animals. although the exact reason for this pattern is unknown, wolfe et al. ( ) suggested that, because livestock and pets were domesticated in the old world, ancestral pathogens had more opportunity to infect humans compared to more recently domesticated new world animals. for the disparity between old world and new world monkeys, they believe that closer genetic relatedness between human and old world monkeys may have aided in cross-species transmission. these results stress the importance of considering both environmental and biological factors as key determinants of cross-species transmission of infectious diseases. recent spreading of human population by urbanization exposes us to novel pathogens that were previously isolated from human society. the risk of zoonotic infections may be increasing and it is notable that many novel pathogens appear to have high virulence in human (reads ; schrag and wiener ) . during their long evolutionary history, pathogens and their original hosts may have been recurrently co-evolving by which hosts evolve to be resistant against the pathogens, and pathogens evolve to evade the resistant system (little ; woolhouse et al. ) . this means if hosts are exposed to a novel pathogen, it is highly possible that the hosts do not yet have immune resistance against the pathogen and are affected by high virulence (longdon et al. ) . there are also cases in which infections of novel pathogens cause inappropriate immune response and as a result, increase their virulence (graham and baric ). as introduced above, these highly virulent pathogens can spread in a host population depending on host spatial structure. however, it is important to note that not all novel pathogens have high virulence for human. highly virulent pathogens are more likely to be detected and studied and therefore, the patterns may result from ascertainment bias (alizon et al. ; longdon et al. ) . in any case, careful surveillance of both human and animal populations in regions of high human-animal contact may be an important component to defending against novel disease (woolhouse et al. ) . finding the original animal host of a new human pathogen requires scientific rigor but also guesswork and luck. the search for the animal reservoir of the sars pathogen first identified the himalayan palm civet (paguma larvata) after sars-like coronaviruses (sl-covs) were isolated from civets in live-animal markets in guangdong, china (guan et al. ) . however, tu et al. ( ) showed that while civets in live-animal markets were infected with sl-covs, civets on farms did not possess antibodies against the virus, which indicated that they have never been exposed to the pathogen. moreover, palm civets infected with sars-cov showed signs of illness contrary to the expectation that animal reservoirs should be clinically asymptomatic (calisher et al. ) . this observation and that other animals in the same live-animal markets were also infected by the virus (guan et al. ) indicated that the palm civets were infected in live-animal markets rather than being the ultimate source of the pathogen. surveillance of wild animals in the region later lead to the serendipitous discovery that chinese horseshoe bats (rhinolophus sinicus) are the original animal host of the coronavirus that became sars-cov (lau et al. ; li et al. b) . the focus on bats may have been inspired by outbreaks of nipah and hendra virus a decade before that were also traced back to these mammals (normile ). in addition, li et al. ( b) stated that the use of bat products in food and traditional medicine in southern china led them to investigate bats as a potential reservoir. interestingly, bats appear to harbor many human pathogens and have been implicated as the animal reservoir of nipah virus, hendra virus, ebola virus, and sars-cov. even mers-cov, initially transmitted from camels, have been traced back to bat through phylogenetic analysis and biochemical studies (wang et al. ; yang et al. ) . while sars-cov infection primarily affected the respiratory system, high concentrations of coronavirus were observed in bat feces and recovery from the small and large intestines indicate that replication is primarily through the excretory system (drexler et al. ) . lau et al. ( ) speculated that the use of bats in traditional medicine, especially bat feces, may have played a crucial role in the cross-species transmission of the virus. bat meat is also considered a delicacy and many chinese believe it possess therapeutic activity, which led to bat trade in live-animal markets such as those in guangdong, china. exposure between the pathogen reservoir and the new potential host species is a key factor dictating the probability of successful cross-species transmission. for example, hiv- and - seem to have transferred multiple times to humans since based on phylogenetic analysis, but only after was there a significant spreading of the infection (heeney et al. ) . one explanation suggests that the limited interactions between humans and primates created a barrier for the transference of the virus and insufficient interhuman encounters of infected cases delayed the rise of the epidemic (parrish et al. ) . to describe this phenomenon, let us model the underlying host contact network as a network of nodes (individuals) and connections (exposure). assuming a heterogeneously connected network such as human social network and contact networks (eubank et al. ) , we find that the probability of a new infection becoming extinct by chance is very high both because the pathogen may be poorly adapted to transmit in the new host (parrish et al. ; daszak et al. ; dobson and foufopoulos ) and because cross-species transmission events tend to occur in sparsely connected rural areas (tibayrenc ) . the limited connections inhibit emergence of the disease and only the few that avoid stochastic extinction proceed to produce an epidemic in the host population (lloyd-smith et al. ; eubank et al. ). this may explain why spillover events of animal infections, such as h n avian influenza, fail to take hold in the human population despite the hundreds of human cases and deaths that have been reported (parrish et al. ) . while distribution is skewed towards fewer connections in these networks, it is still possible that the cross-species transmission event occurs at a highly connected portion of the network. such an outcome will only make it more likely that the infection will take hold to produce an epidemic due to the presence of highly connected hubs that can spread the disease to a disproportionate number of hosts (rock et al. ) . lloyd-smith et al. ( ) expand this concept to show that any type of individual variance, for example infectiousness, produces the same effect. high individual variance increases the probability of extinction of an invading disease regardless of the strength of mean infectiousness. when the host population has a highly heterogeneously connected network, emergence of disease may be rare, but infections that survive stochastic extinction produce "explosive" epidemics similar to the case of sars in . these findings show that host population structure and demography significantly affects the probability of cross-species transmission as well as the subsequent epidemic that may follow. host factors also play a significant role in determining the success of new infections in novel hosts, especially for viruses. to infect a host, a virus must be able to interact with the host's cellular receptors to gain entry into cells and hijack the cell's machinery to replicate itself. at the same time, the pathogen must survive against the host's defense mechanisms. the initial interaction between the virus and host receptors is a critical step that determines host specificity and host range. for example, in sars as well as in other coronaviruses, the viral structure responsible for viral entry is the spike glycoprotein, which also appears to be the key determinant of host specificity (graham and baric ) . in humans, the receptor-binding domain on the spike glycoprotein interacts with a cell surface metalloproteinase called human angiotensin-converting enzyme (ace) to gain entry and infect lung epithelial cells (li et al. ) . however, ren et al. ( ) showed that sl-covs found in bats do not interact with palm civet or human ace receptors implying that changes must have occurred to gain this new interaction. in fact, there appears to be a sizeable difference between coronaviruses isolated from the putative bat reservoir and sars. sl-covs from bats were found to be at most only % similar compared to sars-cov (li et al. b) . later, ge et al. ( ) were able to isolate and characterize a sl-cov that utilizes the ace for cell entry in bats, palm civets and humans. this finding argues that ace utilization may have evolved prior to any cross-species transmission event. while gaining the ability to bind to a novel receptor appears to be a complicated process, in some instances, even a few amino acid changes may confer the ability to recognize a new species. initial studies comparing sars-cov isolated at different time points in the pandemic revealed the spike protein of viruses taken from palm civets and early human cases bind less efficiently than those from later on in the epidemic (yang et al. ) . further genetic and biophysical studies demonstrated that two amino acid changes had an enormous effect on the binding affinity of the sars spike protein to human lung epithelial cells (li et al. a, c; qu et al. ) . in most palm civet samples, lysine at position and serine at position of the spike protein were observed whereas asparagine and threonine were present in human samples. li et al. ( a) found that replacing lysine with asparagine removed the electrostatic interference with the histidine residue on the receptor while replacing serine with threonine provided a methyl group capable of filling in a hydrophobic pocket at the interface of the human ace receptor. although the structural changes appear to be subtle, these substitutions caused a thousand-fold increase in binding affinity to the human ace and lead to enhanced human transmission. however, li et al. ( a) also found that some civet specimens have asparagine instead of lysine at position yet this did not affect binding to the civet ace receptor. changes that are neutral to the original host but advantageous in the new host may have played a critical role in facilitating cross-species transmission between palm civets and humans. once a pathogen has evolved to reliably infect the new host's cells, the innate immune response is the host's first line of defense against the infection. when a virus successfully infects a cell, cytoplasmic enzymes that detect the production of double-stranded rna, a hallmark of virus replication, activate the expression and release of interferons from the cell. interferons act as an early-warning signal to other cells nearby by activating their intracellular antiviral response to combat viral infection and replication (roy and mocarski ) . because of the importance of this immune response against viral infection, many viruses have evolved features to subvert interferon signaling. for example, the influenza virus prevents the infected cell from detecting viral replication by using its ns protein to sequester double-stranded viral rna (lu et al. ) . another method to interfere with the innate immune response is to prevent interferons from activating antiviral mechanisms. nipah virus produces two proteins that prevent stat from translocating into the nucleus as well as another protein that sequesters stat present in the nucleus, obstructing the activation of interferon-stimulated genes (shaw et al. ). in the case of sars, kopecky-bromberg et al. ( ) found that sars-cov nucleocapsid and accessory proteins inhibit both the expression of interferon and associated transcription factors, as well as inhibiting cellular response to interferon by subverting the jak/stat activation of intracellular antiviral mechanisms. while infection with sars-cov did not induce production of interferons, coinfection with another virus did produce interferons. it appears that sars-cov does not induce interferon expression, yet does not shut down the whole pathway as interferon signaling continues to work when other stimuli are present (frieman et al. ) . by antagonizing the induction and response to interferons, the pathogen blocks the activation of more than interferon-stimulated genes which prevents the cell from going into an "antiviral state" (de lang et al. ). under the antiviral state, inhibitors are activated to prevent cell division, enzymes that digest proteins initiate programmed cell death, and proteins that present viral particles to activate the adaptive immune response are upregulated. blocking interferon signaling causes a general decrease of both innate and adaptive immune system response, allowing sars-cov to infect cells unimpeded and potentially cause a more serious disease. the rate at which a pathogen evolves is another biological factor that may determine risk of cross-species transmission. most recent emerging infections have been caused by rna viruses such as hiv (gao et al. ) , ebola virus (gire et al. ) , dengue virus (gubler ) , sars-cov (lee et al. ) and mers-cov (de groot et al. ) . rna viruses have an extremely high mutation rate because their rna polymerase, the enzyme that copies their genome, lacks proofreading activity which leads to error-prone replication. mutation rates for rna viruses range from − to − substitutions per nucleotide per cell infection, two orders of magnitude higher, on average, than dna viruses (sanjuán et al. ). at those rates, about out of , nucleotide changes every time an rna virus replicates itself. this may not seem high but note that hundreds of millions of viral particles may be produced during a single infection (haase ) , which gives the virus numerous opportunities to explore potentially advantageous mutations. although high mutation rates helps rna viruses to rapidly adopt advantageous changes and alter phenotype, deleterious mutations are also produced at an elevated rate. lauring et al. ( ) have demonstrated that viruses mitigate the effects of deleterious phenotypes by outcompeting and quickly purging these low-fitness variants. the ability of viruses to incorporate functional components made by other functional viruses within the same cell appears to also mitigate the negative effects of high mutation rates (makino et al. ). indeed, studies have shown that raising the mutation rate through mutagens can be used to create large numbers of dysfunctional mutants that rapidly leads to the extinction of the viral population (pathak and temin ; loeb et al. ; domingo ) . interestingly, in the case of sars, the coronavirus that caused the disease did not have a very high mutation rate relative to other rna viruses. coronaviruses have the largest genomes (approximately , nucleotides) among rna viruses and genome size and mutation rate appear to be negatively correlated (sanjuán et al. ) . one reason behind the relative stability of coronavirus genomes could be the presence of proofreading enzymes that guard against mutagenesis. in the case of sars-cov, smith et al. ( ) showed that the exoribonuclease domain in non-structural protein had proofreading activity and was responsible for protecting the viral genome against mutagenesis. although high mutation rates appear to facilitate adaptation of rna viruses to new environments, diseases of animal origin are not always caused by the fastest evolving rna viruses. the discussions above have introduced how several aspects of urban life, including high connectedness of individuals (including connections among distant individuals) and regions of high human/animal contact, are likely to elevate the risk of future epidemics. because these properties may be intrinsic to urban life and difficult to alter or control, monitoring and preparedness are critical for urban resilience to disease outbreak. opportunities for the evolution of new or variant human pathogens are difficult to limit and may, in fact, be increasing in modern societies. each contact between microbe and host can be considered a "trial" for a potential pathogen with random mutations in their genomes that may confer new functions or specificities. thousands of such trials occur daily in many regions and are likely spawning candidate emerging pathogens with the ability to reproduce within humans and possibly also to transmit from person to person. the trajectory of pathogen evolution depends strongly on the numbers of contacts (potential transmissions) among individuals in the host population as well as on chance. the vast majority of potential new pathogens are likely to be lost early in their histories. however, given continuous opportunities, the chance event of pathogen emergence is simply a matter of time. in guangdong province, several recent outbreaks of bird influenza (h n ) have led to limits on live poultry markets, but consumer preference for freshly slaughtered poultry and wild animals remain an impediment to regulating the high-risk concentration of multiple species (pig, poultry, dog, cat, rabbit, as well as reptiles, fish and numerous wild game) in close contact with one another (and often in poor health) as well as with humans. regions of recent human expansion where wild animal populations are in close proximity to high density human settlements must also be monitored carefully for new zoonotic diseases. new strains of swine and bird influenza are currently monitored as candidates for outbreaks but pathogen emergence is unpredictable and may come from completely unexpected sources. regardless of the source of new infectious agents, a major concern is that future pathogens may have properties that will make control much more difficult than sars. shorter incubation times and pre-symptomatic transmission strongly limit the efficacy of isolation and quarantine and may allow rapid disease spread. in this chapter, we have focused on biological factors that are central to disease emergence and control. policy prescriptions have been discussed extensively (university of louisville school of medicine ; beaglehole et al. ) and we highlight selected topics below. one of the important lessons from the sars crisis was the need for a rapid and organized response, even in the case of a relatively controllable disease. recognition of new epidemics through surveillance and global warnings and travel advisories are obvious critical factors but the necessary infrastructure has been difficult to implement, especially in developing regions. given the likely lag-time between the start of an outbreak and pathogen isolation and development of diagnostics, well-trained physicians and epidemiologists at the frontlines of the epidemic play a critical role in initial response. for an infected individual, the numbers of contacts and possible and actual transmissions increase rapidly with time so diagnosis and contact tracing are time-critical events. finally, communicating with, and educating the public and controlling panic are major concerns especially in the context of false reports and rumors. establishing trusted sources of information prior to emergencies should be a major objective for cities/regional governments. issues with coordination among government agencies or between medical and government agencies were strong obstacles in the response to sars in most affected regions (university of louisville school of medicine ). high transmission in medical care settings was one of the prominent features of both the sars and mers outbreaks. because infected individuals with weakened immune systems or co-infections may be the most difficult to diagnose and may show high infectiousness, proper training in pathogen containment is a critical element of epidemic preparation. similar basic techniques (proper use of gloves, gowns, masks, and goggles) were successful for sars and ebola suggesting that many practices will be of general value but specifics for particular transmission mechanisms (e.g. airborne versus vector transmission) are also critical. intervals between outbreak occurrences may be large, so regular confirmation of preparedness is important. low margins in health care are strongly linked to overcrowding and government and private organization incentives (e.g., increased funding for hospitals that rate highly on infection control training and preparedness) can greatly enhance hospital safety (committee on the future of emergency care in the united states health system ). the importance of patient isolation in limiting disease spread is clear from recent sars, ebola and mers outbreaks; hospitals must have containment facilities and "surge capacity" to limit superspreading events. although public health measures were sufficient to eventually control sars, additional measures including antiviral drugs and rapid vaccine development and production may be necessary for stronger pathogens. the economic impact of epidemics, roughly billion usd ( % of regional gdp) for east asia from sars and potentially over billion usd for pandemic influenza (the economist ) should help to justify the costs of outbreak preparation. informed sanitation (water purification, sewage treatment) and building regulations/inspections (e.g. airflow control) policies can play a key role in preventing disease emergence and spread. the sars example illustrates the need for extensive interdisciplinary efforts, combining expertise from physics (fluid mechanics), biology (especially understanding mechanisms of disease transmission), and building design for resilience to future outbreaks. isolation and quarantine were critical to controlling sars in hong kong, singapore, taiwan, china, vietnam and canada. compliance rates appeared to be high in all regions (university of louisville school of medicine ) perhaps partly because the "cultural" value placed on solidarity and cohesion was relatively high in these regions. more severe movement restrictions may be necessary for more transmissible and/or virulent pathogens. it is unclear whether similar measures can be employed with success in other regions where personal liberties are emphasized and/or government is less trusted. biological studies can help to determine the necessity and guide planning for future epidemics, but social and economic issues may be the more critical limiting factors in developing preparedness for disease outbreaks. understanding the social, psychological, and economic costs of previous and potential disease outbreaks among both citizens and government officials will be central to planning for resilient communities. the ability to overcome economic and psychological barriers (e.g., normalcy bias) to implementing such plans may require a fundamental transformation in human society. virulence evolution and the trade-off hypothesis: history, current state of affairs and the future rapid evolutionary dynamics and disease threats to biodiversity epidemiology, transmission dynamics and control of sars: the - epidemic. philosophical transactions of the royal society of 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co-evolution of pathogens and their hosts receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses evidence of airborne transmission of the severe acute respiratory syndrome virus why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others key: cord- -u k pds authors: mason, jay w.; trehan, sanjeev; renlund, dale g. title: myocarditis date: journal: cardiovascular medicine doi: . / - - - - _ sha: doc_id: cord_uid: u k pds viruses are the most common cause of myocarditis in economically advanced countries. enteroviruses and adenoviruses are the most common etiologic agents. viral myocarditis is a triphasic process. phase is the period of active viral replication in the myocardium during which the symptoms of myocardial damage range from none to cardiogenic shock. if the disease process continues, it enters phase , which is characterized by autoimmunity triggered by viral and myocardial proteins. heart failure often appears for the first time in phase . phase , dilated cardiomyopathy, is the end result in some patients. diagnostic procedures and treatment should be tailored to the phase of disease. viral myocarditis is a significant cause of dilated cardiomyopathy, as proved by the frequent presence of viral genomic material in the myocardium, and by improvement in ventricular function by immunomodulatory therapy. myocarditis of any etiology usually presents with heart failure, but the second most common presentation is ventricular arrhythmia. as a result, myocarditis is one of the most common causes of sudden death in young people and others without preexisting structural heart disease. myocarditis can be definitively diagnosed by endomyocardial biopsy. however, it is clear that existing criteria for the histologic diagnosis need to be refined, and that a variety of molecular markers in the myocardium and the circulation can be used to establish the diagnosis. treatment of myocarditis has been generally disappointing. accurate staging of the disease will undoubtedly improve treatment in the future. it is clear that immunosuppression and immunomodulation are effective in some patients, especially during phase , but may not be as useful in phases and . since myocarditis is often selflimited, bridging and recovery therapy with circulatory assistance may be effective. prevention by immunization or receptor blocking strategies is under development. giant cell myocarditis is an unusually fulminant form of the disease that progresses rapidly to heart failure or sudden death. rapid onset of disease in young people, especially those with other autoimmune manifestations, accompanied by heart failure or ventricular arrhythmias, suggests giant cell myocarditis. peripartum cardiomyopathy in economically developed countries is usually the result of myocarditis. jay w. mason, sanjeev trehan, and dale g. renlund • viruses are the most common cause of myocarditis in economically advanced countries. • enteroviruses and adenoviruses are the most common etiologic agents. • viral myocarditis is a triphasic process. phase is the period of active viral replication in the myocardium during which the symptoms of myocardial damage range from none to cardiogenic shock. if the disease process continues, it enters phase , which is characterized by autoimmunity triggered by viral and myocardial proteins. heart failure often appears for the first time in phase . phase , dilated cardiomyopathy, is the end result in some patients. diagnostic procedures and treatment should be tailored to the phase of disease. • viral myocarditis is a significant cause of dilated cardiomyopathy, as proved by the frequent presence of viral genomic material in the myocardium, and by improvement in ventricular function by immunomodulatory therapy. • myocarditis of any etiology usually presents with heart failure, but the second most common presentation is ventricular arrhythmia. as a result, myocarditis is one of the most common causes of sudden death in young people and others without preexisting structural heart disease. • myocarditis can be definitively diagnosed by endomyocardial biopsy. however, it is clear that existing criteria for the histologic diagnosis need to be refined, and that a variety of molecular markers in the myocardium and the circulation can be used to establish the diagnosis. • treatment of myocarditis has been generally disappointing. accurate staging of the disease will undoubtedly improve treatment in the future. it is clear that immunosuppression and immunomodulation are effective in some patients, especially during phase , but may not be as useful in phases and . since myocarditis is often selflimited, bridging and recovery therapy with circulatory assistance may be effective. prevention by immunization or receptor blocking strategies is under development. • giant cell myocarditis is an unusually fulminant form of the disease that progresses rapidly to heart failure or sudden death. rapid onset of disease in young people, especially those with other autoimmune manifestations, accompanied by heart failure or ventricular arrhythmias, suggests giant cell myocarditis. • peripartum cardiomyopathy in economically developed countries is usually the result of myocarditis. the difficulty of diagnosing and treating myocarditis was recognized by senac in : "the inflammation of the heart is difficult to diagnose and when we have diagnosed it, can we then treat it better?" after sobernheim in defined myocarditis as any inflammation or degeneration of the heart, the term myocarditis was used for nonvalvular myocardial diseases, including ischemic and hypertensive cardiomyopathies. nearly a century later, white suggested that the term myocarditis be restricted to "true inflammation of the myocardium." the last half-century has seen the development of endomyocardial biopsy techniques, histologic criteria, and serologic methods to diagnose myocarditis. as our knowledge of the immunopathologic mechanisms evolves, new therapeutic strategies are developing. the world health organization/international society and federation of cardiology task force on cardiomyopathies classified cardiomyopathies whenever possible by etiologic/pathogenetic factors. this classification recognizes chronic viral, postinfectious autoimmune, and primary autoimmune forms of dilated cardiomyopathy (dcm). the classification states that "myocarditis is diagnosed by established histological, immunological and immunohistochemical criteria." the dallas criteria provide consensus-derived histologic criteria: "an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of ischemic damage associated with coronary artery disease." however, many have speculated that less pronounced histologic abnormalities may be present and that additional molecular, immunologic, and immunohistochemical diagnostic criteria can be used productively. [ ] [ ] [ ] [ ] [ ] [ ] myocarditis, irrespective of the etiopathologic factors, remains an inflammatory cardiomyopathy associated with cardiac dysfunction. a wide variety of infectious and noninfectious causes are associated with myocarditis (tables . to . ) . several epidemiologic observations linking these agents with myocarditis have been corroborated by serologic, polymerase chain reaction (pcr), or in situ hybridization methods. the incidence of infectious myocarditis in the general population is largely unknown. in a prospective study over several years, in a predefined subpopulation, an incidence of . % was found. these cases were confirmed by myocardial enzyme leak and characteristic electrocardiographic (ecg) changes. the ecg abnormalities suggesting asymptomatic myocardial involvement, in the absence of enzyme release, have been noted in . % of military conscripts during the course of other acute infectious diseases. during an epidemic of influenza a, the incidence rose to . %. in a prospective trial of consecutive patients admitted to a large infectious disease hospital in sweden, % showed ecg abnormalities suggestive of myocarditis. approximately % of a virus-infected population may experience symptoms or findings suggestive of cardiac involvement. the incidence of myocarditis associated with nonviral infections is even more difficult to estimate. although the list of possible etiologic agents is large, the enteroviruses, specifically coxsackievirus b, over decades have been the most commonly identified etiologic agents of inflammatory cardiomyopathy. among healthy active adults, at least % have detectable serum antibodies indicating prior infection with coxsackievirus b. , the world health organization has surveyed viral infections related to cardiovascular disease globally. in a year period from to , coxsackievirus b had the highest incidence of cardiovascular disease ( . cases per population), followed by influenza b ( . cases), influenza a ( . cases), coxsackievirus a ( . cases), and cytomegalovirus (cmv) ( . cases). the predominance of enteroviruses among myocarditisassociated agents has been substantiated by several laboratory and clinical studies. [ ] [ ] [ ] using serologic methods, vikerfors and associates reported that nearly % of consecutively studied myocarditis patients had enterovirus immunoglobulin igm. frisk and coworkers found a similar incidence of coxsackievirus b igm antibodies by reverse herpes simplex in two, and cmv in one patient. the control group did not demonstrate any viral genome sequences. just as the incidence of specific viral infections varies over time, so should the relative proportion of agents responsible for myocarditis. in a recent study, bowles and colleagues supported the observation by martin and coworkers that adenovirus is the most common agent associated with myocarditis in children, but they also found that adenoviruses predominated over enteroviruses in adults. figure . shows the dominant role of adenoviruses and enteroviruses in myocarditis. note that parvovirus was detected in young people. parvovirus b- has recently been identified as a cause of myocarditis and, in some regions, it has been found in adults as well as children. [ ] [ ] [ ] [ ] [ ] these differences between previous and newer studies are due, at least in part, to geographical and temporal variation in the incidence of specific viral infections. cytomegalovirus is a recognized cause of acute infectious myocarditis, although it is rare in healthy individuals. , maisch and associates demonstrated, using in situ hybridization techniques, cmv-specific nucleotide sequences in % of patients with acute myopericarditis. certainly in transplant recipients, cmv infection is fairly common and has been reported to affect the transplanted heart. , hepatitis c virus infection is frequently noted in patients with dcm, and hepatitis c virus rna has also been recovered from lymphocytes infiltrating the myocardium in chronic active myocarditis. matsumori and colleagues , found a high incidence of hepatitis c viral genomic material in a wide variety of cardiac disorders in japan. myocarditis is a well-recognized complication of corynebacterium diphtheriae infection, although this is now rare in the western world. myocardial dysfunction is also seen in association with salmonella septicemia, although it is rarely clinically severe. , myocardial dysfunction is primarily related to the toxemia of the severe infection, which is also observed in meningococcal and nonrheumatic streptococcal infections. perhaps the best-recognized bacterial agent thought to be responsible for myocarditis is the β-hemolytic streptococcus that causes rheumatic fever. fortunately, rheumatic fever is seen in the western world with only a low frequency of sporadic cases in regional clusters. the incidence in the united states is less than per , , but in the developing world, rheumatic heart disease continues to be the leading cause of cardiac hospitalization in the -to -year-old age group. although the inflammatory component of rheumatic carditis is largely restricted to the valves, it has been believed to cause myocardial dysfunction. myocarditis is a well-documented complication of borrelia burgdorferi infection (lyme disease) and is reported in up to % of cases. cardiac involvement is often characterized by the development of atrioventricular (av) block and rarely progresses to left ventricular dysfunction and cardiomegaly. mycoplasma pneumoniae infection has also been associated with myocarditis. lewes and coworkers demonstrated asymptomatic myocardial involvement as documented by ecg changes in a third of the cases with acute mycoplasma infection. six percent of military conscripts with clinical myocarditis were found to have active m. pneumoniae infection. chlamydia infections have also been associated with myocarditis, especially among small children, often having fatal outcomes. c. pneumoniae infection has also been noted in a few cases of mild myocarditis and has been found with respiratory infection associated with myocarditis, resulting in sudden death in a young athlete. chlamydia psittaci infection may be associated with myocarditis in % to % of those affected, usually with minimal clinical signs or symptoms. pericarditis is more frequent and likely to cause cardiac morbidity with ornithosis. other causative infectious agents rickettsial infections, like rocky mountain spotted fever and scrub typhus, are frequently accompanied by myocardial involvement, although vasculitis is more prominent with these infections. q fever may also be associated with myocarditis. trypanosoma cruzi is a well-recognized cause of myocarditis and cardiomyopathy in south america (chagas' disease). toxoplasma gondii poses a significant problem among cardiac transplant recipients because a large number of the recipients lack antibodies against this agent, which may cause myocarditis. toxoplasmosis also poses a major threat to patients with aids. myocarditis has frequently been seen in human immunodeficiency virus (hiv)-infected populations with or without concomitant toxoplasma infection. , in two autopsy studies of patients with aids, myocarditis was found in almost half of the cases; in another study, % of prospectively studied patients with aids had echocardiographic evidence of myocardial dysfunction. , myocarditis may also occur in patients with aids as a result of t-cell restitution after antiviral therapy. myocarditis can also be seen with parasitic infections such as trichinella spiralis, which has an affinity for striated muscle, including the heart. other noninfectious causes noninfectious causes of myocarditis include druginduced hypersensitivity, - direct toxicity of specific pharmaceutical agents, , [ ] [ ] [ ] and systemic collagen vascular disorders. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] eosinophilic myocarditis [ ] [ ] [ ] [ ] and giant cell myocarditis (gcm) [ ] [ ] [ ] [ ] [ ] [ ] [ ] are distinct forms of inflammatory myocarditis of uncertain etiology. microorganisms are rarely isolated or demonstrated in heart muscle; hence, identification of a specific infectious etiologic agent depends on recognition of its systemic manifestations. once specific noninfectious and nonviral infectious agents are excluded, myocarditis is often assumed to be of viral etiology. although definitive serologic evidence of viral infection can be obtained in many patients, it is absent in the majority of patients with presumed myocarditis. a significant number of cases of myocarditis is due to autoimmune phenomena either induced by a viral infection or resulting from systemic autoimmune disease. since the establishment of definitive etiologic diagnoses is ambiguous, the terms viral myocarditis, idiopathic myocarditis, lymphocytic myocarditis, autoimmune myocarditis, and interstitial myocarditis are frequently used interchangeably. the pathophysiologic mechanisms of myocarditis in humans are not fully understood. clearly, multiple mechanisms exist, including direct infection by viruses, bacteria, and other organisms; noninfectious causes, such as toxins and drug hypersensitivity; and parainfectious etiologies, resulting from the immune response to infection. most cases of overt heart failure due to myocarditis in north america, europe, and japan are thought to arise from the latter type of mechanism, during and after viral infection of the heart. a triphasic disease process is observed , (fig. . a ). in the first phase, active viral infection of the myocardium results in a variable extent of muscle damage, which often may not be clinically apparent. phase develops in an unknown proportion of infected individuals after partial or complete resolution of active infection and is characterized by further myocardial damage, both by ongoing immune activation and by newly developed autoimmune activity. a small proportion of patients develops dilated cardiomyopathy, the third phase of disease, resulting from the cumulative damage caused by infection, immunity, and autoimmunity. during this phase, a considerable body of evidence suggests that the immune and autoimmune processes persist, in part as a result of viral persistence. figure . b depicts the three roles virus may play in bringing about dilatation and chronic heart failure. after the initial injury that occurs during active viral replication, latent, nonreplicating viruses can still alter myocyte function through viral genomic expression. even if the virus is completely eliminated and the immune response ceases, through the various mechanisms of adverse remodeling, the cardiomyopathy may progress inexorably. the most widely accepted models for the study of human myocarditis are those of enteroviral myocarditis induced by coxsackievirus b (cvb ) and the encephalomyocarditis virus. induction of chronic murine myocarditis by cvb requires the virus to have a cardiovirulence capacity and murine strains of certain genetic background. , infection of syngeneic weanling mice with cvb results in brief cardiac infection lasting about a week, beyond which the virus cannot be cultured. however, viral rna persists for several months after the initial infection. , several mechanisms have been hypothesized to explain the initiation of chronic inflammatory response in myocytes by the viral infection: . although antibodies to these antigens are frequently identified in association with myocarditis, the clinical significance and causal relationship are yet unresolved. cytotoxic lymphocytes (ctls) from mice with cvb induced myocarditis possess the ability in vitro to recognize and kill neonatal myocytes, fibroblasts, and endothelial cells infected with the same strain of the virus, suggesting that the recognition of a novel tissue antigen is induced by the infection. cross-reactive, concurrent recognition of unrelated cardiac epitopes also occurs because ctls also lyse uninfected myocytes in vitro. the production of perforin, a pore-forming protein, has been proposed as one of the mechanisms for the cytolysis induced by lymphocytes. perforins, when inserted into myocyte membrane, induce a lethal augmentation in cell permeability that results in cellular edema and death. perforin-independent mechanisms have also been proposed, including a fas (cd /apol)-based inositol- , , -triphosphate-mediated cytolysis that can be demonstrated in perforin-deficient gene-knockout mice. coxsackievirus-infected mice also develop additional immune sensitization to cardiac heavy chain myosin, possibly owing to the release of the sequestered myosin antigens from the virus-damaged cells. immunization of mice with the heavy chain myosin and an adjuvant produces a histomorphologically similar picture to cvb -induced myocarditis. adoptive transfer of splenocytes can also produce experimental autoimmune myocarditis after myocardial infarction in syngeneic rats. the sensitized lymphocytes when transferred to normal rats cause cardiac-specific cellular infiltration with accompanying myocyte necrosis. the genetic susceptibility, kinetics, and cellular composition of the infiltrates in these models are similar and suggest the role of endogenous antigens as an epitope for the inflammatory response. the pathways and cellular participants in the immunopathogenesis of experimental viral myocarditis are well recognized. the replicating viral particles can be readily identified in cardiac myocytes within a few hours of inoculation of cvb into mice. , the viral particles reach a numerical peak in to days, and usually at to days, they are no longer detectable. the inflammatory infiltrate is detectable by day and reaches a plateau by days to . the early inflammatory infiltrate consists of lymphocytes, macrophages, neutrophils, natural killer cells, and the associated cytokines and humoral effectors. [ ] [ ] [ ] the natural killer cells are the first to appear and are detected in the activated state in to days. these cells are capable of lysing virusinfected cells in vitro. the t lymphocytes and macrophages follow the natural killer cells in the temporal sequence and become the predominant cells infiltrating the myocardium in to days. although cvb replicates readily in myocytes in vitro, the cells are resistant to lysis in comparison with other cultured cell lines. direct myocytolysis appears to play a minimal role in cell lines derived from normal mice. the immunodeficient severe combined immunodeficiency (scid) mouse model has provided valuable insight into the early immune activity in response to the viral infection. the scid mice lack mature t-and b-lymphocyte function and develop extensive myocardial necrosis with pleomorphic infiltrates, rapid viral proliferation, and profound virus-associated myocytolysis when inoculated with cvb . the macrophage and natural killer cell activity is unaffected in the scid mouse model and may participate in the myocytolytic activity, although direct viral myocytolysis predominates. pharmacologically immunosuppressed mice demonstrate similar characteristics, with higher viral loads, delayed clearance, and extensive myocyte necrosis, although direct viral myocytolysis is not frequent in immunocompetent mice. , , - even noncardiovirulent strains may have sufficient time to replicate and transform into quasicardiovirulent species in the absence of a functional antiviral immune response, which can then result in fatal myocarditis. this may also explain the clinical observation that many severe and fatal cases of myocarditis develop in young children with immature and incompletely developed immune systems. virus-specific ctls play a major role in the inflammatory response to viral infection of the myocytc. , the inflammatory response can be diminished significantly by t-lymphocyte depletion with either antithymocyte globulin or thymectomy and irradiation. , the ctls must recognize the foreign antigen in association with the syngeneic major histocompatibility complex (mhc) class i antigen that is found on immune-derived cells. the cvb -infected cells can readily express mhc class i antigens. the mhc class i molecules provide peptide-binding sites that evoke effector responses on recognition of the foreign peptide by the antigenspecific receptors of the t lymphocyte. however, tlymphocyte depletion and specific immunosuppression using cyclosporine have varying effects, depending on the murine model, the virus, and the time of therapy, and are not uniformly beneficial. [ ] [ ] [ ] the virus can no longer be cultured from cells after to days; however, areas of inflammatory infiltrate and myocyte necrosis do demonstrate persistence of viral rna, and the virus-specific ctls may continue to see these as immunologic targets and, hence, perpetuate the myocyte damage. the infected myocyte can still remain a target for the ctls, even if the viral antigens are cleared, owing to expression of "neoantigens" either induced by the virus or unsequestered due to the injury. , even nonviral antigens on infected myocytes can react with ctls, such as those induced by actinomycin d, and new glycoproteins have been identified on the surface of cvb -infected cells that can be recognized by ctls from other syngeneic-infected mice. recent observations suggest that co-stimulatory molecules b - , b - , and cd- may be expressed on myocytes in patients with myocarditis and may make the myocytes into antigen-presenting cells for ctls and natural killer cells, thereby playing an important role in the direct myocardial damage by these lytic cells. another mechanism for ongoing myocyte damage is the antibody-mediated autoimmune response. since the majority of the proteins identified as cardiac autoantigens are intracellular, it is unclear how these antibodies could harm normal intact myocytes. several mechanisms are proposed. one suggests that after the antibody response is initiated, the circulating antibodies to intracellular antigens crossreact with the native membrane cardiac tissue proteins. thus, after a small number of myocytes are damaged by the viral infection and release intracellular antigens, the resulting antibody response may affect normal myocytes, leading to global myocardial dysfunction. this hypothesis is supported by the demonstration of a number of cross-reacting antibodies. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] also, the antibodies against the intracellular mitochondrial adenine nucleotide transferase protein cross-react with the myocyte sarcolemmal calcium ion channel protein, and binding of these channels can physiologically alter the metabolism and contractile function of the myocyte. another theory holds that ctls and antibodies target uninfected myocytes by recognition of self-antigens that were previously sequestered from immune surveillance. the processing and presentation of the self-immunogenic peptides complexed with the mhc is a prerequisite for this hypothesis. normal human cardiac myocytes do not express detectable levels of mhc class ii antigens, and their constitutive expression of mhc class i molecules remains controversial. a significant increase in the expression of mhc class i and class ii antigens by the myocytes has been demonstrated in association with myocardial inflammation, such as that seen with viral myocarditis or transplant rejection. [ ] [ ] [ ] the increased mhc expression has also been demonstrated in endomyocardial biopsy specimens from patients with idiopathic dcm and myocarditis, [ ] [ ] [ ] and immune regulatory dysfunction may have a genetic predisposition. there is also evidence for aberrant expression of intracellular antigens, such as adenine nucleotide translocator (ant) and branched-chain α-keto acid dehydrogenase (bckd), on the surface of the myocytes. the formation of antiidiotypic antibodies is an additional mechanism of immune regulation in which an antibody is formed to the idiotypic determinants (antigen recognition site) of the primary antibody. the antiidiotypic antibody may cross-react with unoccupied viral receptor sites on uninfected myocytes. this phenomenon has been reported with the reovirus, polyomavirus, and coxsackievirus b models of myocarditis. [ ] [ ] [ ] the passive transfer of antiidiotypic b cells from a cvb myocarditic mouse to a syngeneic mouse can cause nonviral myocarditis. the presence of a complex, cytokine-rich microenvironment is suggested by the heterogeneous inflammatory cell populations in the hearts of infected mice. the cytokines perform myriad immunomodulatory functions, including regulation of antibody production, preservation of self-tolerance, , conscription of ancillary cells in the inflammatory milieu, , and maintenance of clonal expansion of ctls. , certain cytokines regulate the collagenogenic and collagenolytic activity of fibroblasts. although mounting evidence supports the negative inotropic effects or the blunting of catecholamine response in myocytes exposed to various cytokines, there is no direct evidence to suggest that the cytokines are directly responsible for myocytolysis. in an in vitro model, barry demonstrated that high concentrations of interleukin (il)- , tumor necrosis factor-α (tnf-α), interferon-γ (ifn-γ), and il- have no effect on myocyte survival over hours, whereas the ctls from a mixed lymphocyte reaction cause virtually % killing. gulick and colleagues demonstrated that cultured neonatal myocytes, when exposed to macrophage-derived il- and tnf-α, have reduced levels of cyclic adenosine monophosphate and have a reduced inotropic response to catecholamines. the mechanism for decreased responsiveness to catecholamines is believed to be modulated by increases in nitric oxide production mediated by increased inducible nitric oxide synthase (inos) activity, and the blunting of the catecholamine response can be inhibited by the l-arginine analogue n g -monomethyl-l-arginine (l-nmma). the decreased contractile response of cardiac myocytes to β-adrenergic agonists following induction of inos also requires the presence of insulin and the co-induction of enzymes responsible for the production of tetrahydrobiopterin, a cofactor for nitric oxide synthase. the role of inos remains controversial because increased expression of inos mrna and that of other proinflammatory cytokines is evident and is associated with contractile dysfunction. there is evidence to support the idea that inos induction is crucial for the host response to cvb infection, and inos-deficient mice have significantly increased viral loads with extensive myocardial damage. inhibition of inos through suppression of nuclear factor (nf)-κb induction has recently been shown to prevent encephalomyocarditis virus myocarditis. other investigators have suggested that inflammatory cytokines may have direct negative inotropic effects, independent of the responsiveness to the β-adrenergic agonists. high doses of il- during chemotherapy have been reported to result in depression of myocardial function. exposure of cardiac myocytes to endotoxin results in increased nitric oxide production and direct depression of contractility owing to increased levels of cyclic guanosine monophosphate. further, tnf-α may induce direct negative inotropic effects by decreasing the ca + transient, with no change in the l-type ca + current and independent of nitric oxide synthesis. although the extent to which cytokines cause direct negative inotropic effects or attenuation of endogenous β-adrenergic agonist activity remains unclear, they do produce myocyte dysfunction and cardiac decompensation. transgenic mice with overexpression of tnf-α develop biventricular dilatation and cardiac failure, resulting in premature death. pathologic specimens from these mice reveal globular dilated hearts and transmural myocarditis with myocyte apoptosis. increased levels of intracellular adhesion molecule (icam- ), il- α, il- β, tnf-α, and macrophage-stimulating factor have been demonstrated in patients with myocarditis and idiopathic dcm. , furthermore, the susceptibility of mice in the cvb myocarditis model can be increased by pretreatment with these cytokines. transforming growth factor-β is identifiable by immunohistochemistry in the prenecrotic regions of infiltrates in the murine myocardium and decreases when the macrophages and fibroblasts migrate to the necrotic foci. these growth factors may be responsible for recruitment of the immunologic effectors and may directly affect cardiac function. an intriguing feature of cytokine activity remains their possible role in the secondary development of myocyte hypertrophy and interstitial fibrosis, characteristic of dilated cardiomyopathy. among animals with different forms of viral myocarditis associated with similar intensity of initial myocyte necrosis, only those animals with persistent inflammation develop interstitial fibrosis, reflected by fibroblast proliferation and an increase in the extracellular matrix. myocardial fibrosis correlates well with the presence of t lymphocytes and macrophages, which in their activated state release fibrogenic cytokines such as fibroblast growth factor and transforming growth factor-β. matrix metalloproteinases (mmps), and their inhibitors, are thought to play a critical role in the process of myocardial remodeling. some of the cytokines elaborated during the course of viral myocarditis, such as tnf-α, disturb the balance between mmps and their inhibitors by increasing mmp, leading to failure of collagen cross-linking and worsened ventricular function ( fig. . ). this pathophysiology may present opportunities for prevention of the development of dilated cardiomyopathy resulting from myocarditis. extracardial reservoir secondary transfer to the target organ (e.g., heart) viral replication in the target organ viral protein expression lymphocytic myocarditis models in animals have conclusively demonstrated the association of viral infection and myocarditis. this association clearly exists in humans, but the proportion of cases that can be accounted for by viral infection is not known. the myocardial damage in murine models of viral myocarditis occurs in two distinct phases: an early phase of direct viral cytotoxicity in which virusspecific t-lymphocyte-and antibody-mediated cytotoxicity predominate; and a late or chronic phase in which the persistent viral genome, reactive ctls, autoantibodies, cytokines, and microvascular damage mediate myocyte damage and dysfunction. the hypothetical mechanisms of virusinduced autoimmune heart disease are presented in figures . to . . the recognition that immune responses to specific viruses are consequential in the development of myocyte injury has led to exhaustive research to exploit the possibility of designing immunomodulatory and antiviral therapies. the pretreatment of mice with inactivated virus vaccine prevents the manifestations of encephalomyocarditis virus myocarditis. the administration of antiviral therapies reduces the viral load and attenuates the histologic findings of myocarditis. , the antiviral response can be augmented by ifn-α or the exogenous administration of il- . , recombinant murine ifn-γ has also been demonstrated to improve the prognosis of acute murine myocarditis caused by encephalomyocarditis virus by suppressing replication. the murine model has also been the subject of intensive study with clinically applied immunosuppressants, such as corticosteroids, nonsteroidal antiinflammatory agents, , and cyclophosphamide, all of which have demonstrated deleterious effects when given in the acute viremic phase. cyclosporine, when administered in the early viremic phase, worsens myocardial injury but, in the late immune phase, has a beneficial effect. , , similar results have been reported with tacrolimus, and survival improves significantly when immunosuppressants such as cyclosporine, azathioprine, and -deoxyspergualin are used in adjunct to immunomodulators, such as ifn-α. antibodies to tnf-α have been demonstrated to improve survival and reduce myocardial injury. cytokine inhibitors have had promising results in animal models, but human clinical trials have been inconsistent. vesnarinone, a phosphodiesterase iii inhibitor, has demonstrated beneficial hemodynamic effects and inhibits the production of tnf-α and favorably modulates induction of inos. amlodipine has also been shown to increase survival of mice with viral myocarditis by inhibiting expression of inos and production of nitric oxide in vivo and in vitro. the diversity of immunopathogenetic mechanisms and variability in the severity of observed disease in the murine model are only a preview to the potpourri of clinical manifestations of myocarditis in humans. the presentation of unexplained progressive cardiac dysfunction or ventricular arrhythmias should lead to the suspicion of myocarditis, especially when routine cardiac diagnostic studies do not reveal an etiology. the history of an antecedent viral infection or prodrome is often sought but seldom reported and rarely confirmed by convalescent serologies. the presence of mild elevation of creatine kinase mb isoenzyme (ck-mb) or troponin, leukocytosis, or ecg changes may further underscore the possibility of myocarditis. most patients with myocarditis likely remain asymptomatic and never seek medical attention. the high frequency of exposure to cardiotropic viruses and the observation of a fairly high incidence of ecg abnormalities in apparently healthy individuals support this speculation. the incidence of myocarditis in an autopsy series following traumatic deaths in previously healthy individuals has been reported at . %. others have reported incidences ranging from . % to as high as % in unselected autopsy series. , these studies may suggest that at any given time, a significant percentage of the asymptomatic general population has myocarditis. the most common presentation of myocarditis is an acute febrile syndrome associated with pericardial and sys- temic complaints. cardiotropic viruses may cause pericardial inflammation, and patients often present with a syndrome of myopericarditis. chest pain is the most common symptom and is secondary to pericarditis or myocardial injury. a rather dramatic presentation of myocarditis is one indistinguishable from an acute myocardial infarction, complete with chest pain, ecg features suggesting acute ischemic injury, enzymatic evidence of myocardial damage, and echocardiographic or ventriculographic regional wall motion abnormalities, but on endomyocardial biopsy myocarditis is confirmed. [ ] [ ] [ ] most patients presenting with this acute syndrome completely recover, although there are isolated instances where progressive myocyte loss and cardiac failure or sudden arrhythmic death is reported. the segmental wall motion abnormalities result from virus-mediated injury, although local coronary arteritis and vasospasm have been suggested as possible culprits. , symptoms of right and left ventricular failure and even cardiogenic shock are frequently found in patients with biopsy-proven myocarditis, since it is these symptoms that lead to medical attention. however, the true incidence of heart failure in patients with myocarditis is probably much lower. in patients presenting with recent-onset heart failure and biopsy-proven myocarditis, % to % have had an antecedent flu-like illness. neonatal myocarditis is often a fulminant syndrome consisting of fever, tachycardia, tachypnea, cyanosis, and rapid progression to circulatory collapse. mortality rates are the highest in this subpopulation, approaching %. children are known to present with syncope due to heart block. other atrial arrhythmias described with myocarditis include sinoatrial block, atrial standstill, av block, intraatrial conduction abnormalities, atrial tachycardia, flutter, and fibrillation. [ ] [ ] [ ] [ ] [ ] [ ] histologic evidence of possible myocarditis has been described in up to two thirds of patients with lone atrial fibrillation. complete heart block has also been described in certain viral infections, such as epstein-barr virus or mumps, and also with rickettsiae. [ ] [ ] [ ] myocarditis may also manifest as myocardial thickening and fibrosis presenting as diastolic dysfunction or restrictive cardiomyopathy, and asymmetric septal thickening resembling hypertrophic cardiomyopathy. [ ] [ ] [ ] lieberman and coworkers proposed a clinicopathologic description of myocarditis based on the initial manifestations, endomyocardial biopsy, and recovery (fulminant, acute, chronic active, or chronic persistent myocarditis). ventricular arrhythmias are frequently encountered in patients with myocarditis, ranging from innocuous premature ventricular contractions to malignant and incessant ventricular tachycardia, and myocarditis is often incriminated in otherwise unexplained ventricular arrhythmias and sudden death. , myocarditis has been documented as a cause of ventricular repolarization abnormalities in athletes with or without arrhythmias. , ventricular arrhythmias may also be precursors to sudden cardiac death in young athletes with occult myocarditis. in autopsy series, myocarditis accounts for % to % of sudden deaths in young, healthy people. , , , in a population-based retrospective study from turin, italy, an incidence of only . % was reported among , autopsies performed over three decades, but the application of standardized systematic histologic examination and criteria tends to give a higher incidence, in the range of %, among autopsies performed at a general hospital. wesslen and associates reported signs of active, healing, or healed myocarditis in of cases of sudden death in young swedes. among high-performance athletes, sudden death due to undiagnosed myocarditis often stirs media attention. myocarditis has also been anecdotally implicated in sudden infant death syndrome. ventricular arrhythmias are frequently the initial and most prominent presentation of giant cell myocarditis. [ ] [ ] [ ] [ ] , ventricular arrhythmias and sudden death are common in all forms of myocardial failure, but specific immunemediator-induced effects on myocyte electrophysiology could also account for a portion of these arrhythmias. binah summarized a number of the mechanisms recognized by work in his laboratory and in others. as noted above, perforin elaborated by ctls is capable of forming membrane channels that pass charged ions, resulting in action potential shortening and diastolic oscillations. in addition, fas ligand can lengthen the action potential and induce afterpotentials, in part through inhibiting i to and augmenting i cal . the physical findings in acute myocarditis are dependent on the extent of myocardial or pericardial involvement, inciting agent (cardiotropic virus), and other factors. fever occurs occasionally, and in the myocarditis treatment trial (mtt), it was noted in % of patients with myocarditis. sinus tachycardia may frequently accompany the febrile state but is often out of proportion to the fever and is more likely adrenergically mediated, owing to the hemodynamic alterations of the failing heart. significant ventricular dysfunction may also be associated with hypotension, gallops, murmurs of regurgitation, rales, jugular venous distention, hepatomegaly, ascites, pleural effusions, and peripheral edema. pericardial involvement may result in a friction rub. the physical findings are not specific for myocarditis. patients with myocarditis frequently have serologic evidence of an inflammatory state with elevation of nonspecific markers of inflammation, such as erythrocyte sedimentation rate, c-reactive protein, and leukocyte counts. a fourfold increase in virus-specific igg titers in the convalescent period is considered reliable evidence of recent infection and is found in % of patients with myocarditis. , in the mtt, more than half of the patients with biopsy-proven myocarditis had an elevated sedimentation rate. other markers noted to be elevated in myocarditis include tnf-α, icam- , vascular cell adhesion molecule- , interleukins, and soluble fas. , , , unfortunately, these markers are not specific for myocarditis. myocarditis, although associated with myocyte damage and necrosis, results in ck-mb elevation in only % of patients with biopsy-proven myocarditis. more recently, lauer and colleagues reported on ck-mb elevation in only one of fi ve patients with histologic evidence of myocarditis, but cardiac troponin t (ctnt), which is extremely specific for myocardial damage, was elevated in all five. additionally, ctnt was elevated in patients, of whom had immunohistologic evidence of myocarditis. thus, ctnt elevation appears to be highly predictive for myocarditis. in an analysis of stored sera on patients from the mtt, cardiac troponin i (ctni) was elevated in % of patients ( of ) with myocarditis, compared with % ( of ) without myocarditis. in contrast, ck-mb values were elevated in only . % of patients ( of ) with myocarditis. further, the ctni elevations correlated with less than month's duration of heart failure symptoms. antibodies to cardiac antigens can be detected in the serum of patients with myocarditis. , , anti-α-myosin igg antibodies may have promise as a diagnostic tool, and, along with other antibodies, probably play a functional role. , the clinical efficacy of igg immunoadsorption , in dcm supports this notion (see also fig. . ). historically, acute myocarditis was diagnosed with the constellation of clinical symptoms, physical signs, and ecg abnormalities. although no particular feature on the electrocardiogram is pathognomonic of acute myocarditis, sinus tachycardia, repolarization abnormalities, conduction abnormalities, and arrhythmias are common findings. in a series of patients with biopsy-proven myocarditis, morgera and associates noted an abnormal qrs duration in %; abnormal q waves in %; left bundle branch block (lbbb) and right bundle branch block (rbbb) patterns in % and %, respectively; st elevation in %; t-wave inversions in %; and advanced av block in %. in patients presenting earlier in the course of the disease, with symptoms of less than month's duration, % had advanced av block and % had st elevation with t-wave inversions. the latter finding has been noted to portend a poorer prognosis. other predictors of poor outcome include lbbb, rbbb, and other conduction abnormalities, which seem to suggest active, severe, and extensive myocarditis. patients may present with sustained ventricular tachycardia, and continuous ecg monitoring of patients with myocarditis often reveals complex ventricular ectopy and nonsustained ventricular tachycardia. , echocardiography is useful in assessing the extent of left ventricular systolic dysfunction, which may range from mild segmental hypokinesis to severe global hypokinesis or akinesis associated with severe congestive heart failure (chf). patients presenting with chest pain or arrhythmias without chf often have normal echocardiograms. the ventricular dimensions may remain normal or may be only mildly enlarged. there may be an increase in left ventricular sphericity and right ventricular elongation and an increase in wall thickness and left ventricular mass with the interstitial edema and compensatory hypertrophy. , restrictive filling patterns in the left ventricle identifying diastolic dys-function have been reported consistently in biopsy-proven myocarditis. mural thrombi in diffusely hypokinetic ventricles have been reported frequently. hyperrefractile myocardium and other qualitative and quantitative analyses of myocardial texture have been described to assess the degree of active myocardial inflammation. pericardial effusion is a helpful echocardiographic finding, reported in % of patients with myocarditis, though hemodynamic compromise with cardiac tamponade is infrequent. urhausen and associates recently demonstrated that cardiac tissue velocity imaging by ultrasound is more sensitive than magnetic resonance imaging (mri) in some cases in detecting myocarditis with subtle ventricular functional impairment. imaging of leukocyte-mediated inflammation through ultrasound fracture of phagocytosed microbubbles shows promise as a means for detecting many forms of myocardial inflammation, although the method remains to be fully evaluated in humans. cardiac scintigraphy has been proposed as a convenient, noninvasive test with high sensitivity to diagnose active myocarditis. gallium- imaging, which identifies areas of increased inflammation, has been studied in clinical settings and noted to have sensitivity and specificity of % and %, respectively, with a negative predictive value of % for the diagnosis of myocarditis. indium- antimyosin monoclonal antibodies have been extensively studied to identify areas of myocyte damage in acute myocarditis. , this technique has extremely high sensitivity and often detects myocarditis that, on endomyocardial biopsy, is not seen by routine histologic assessment but is detected by immunohistochemistry. dec and coworkers studied patients with dcm with radiolabeled antimyosin antibody and endomyocardial biopsy. thirty-nine patients had abnormal antimyosin scans, but only of had evidence of myocarditis (predictive value of %). however, functional improvement was more likely in antimyosin scan-positive patients irrespective of the biopsy. the left ventricular ejection fraction (lvef) improved significantly in both concordant-positive (scan and biopsy both positive) and discordant-positive (scan positive, biopsy negative) patients, but it did not markedly improve in the negative scan and negative biopsy subset. the investigators proposed that discordant-positive scans represented patients with myocarditis in whom there may have been a sampling error on biopsy, hence the reason for missing the diagnosis. anastasiou-nana's group in athens reported that a combination of minimal or no left ventricular dilatation and a positive indium- antimyosin monoclonal antibody scan is highly specific for myocarditis. other nuclear techniques, such as technetium- m ( m tc)-mibi single photon emission computed tomography (spect) imaging, may also be useful in detecting myocarditis. contrast media-enhanced cardiovascular mri in patients with myocarditis has also been demonstrated to be an excellent tool in visualizing the location, activity, and extent of inflammation. early in myocarditis (day ), the enhancement on mri signals is accentuated and focal, whereas later (day ), this seems to be attenuated and more diffuse. furthermore, the severity of change correlates with prognosis. myocardial phosphorus- magnetic resonance spectroscopy has been utilized in assessing abnormalities in cardiac high-energy phosphate metabolism in patients with dcm and allograft rejection, but its role in the diagnosis of active myocarditis remains to be elucidated. the antemortem diagnosis of myocarditis was made feasible by the development of the endomyocardial biopsy technique. myocardial samples could be obtained via a transvascular approach with minimal discomfort to the patient and a low complication rate. whereas other approaches for acquiring myocardial tissue included percutaneous biopsy and mediastinotomy, , these were fraught with complications, precluding their acceptance into clinical practice. the safe and successful transvascular endomyocardial biopsy first described by sakakibara and konno was readily accepted for surveillance of cardiac allograft rejection in transplant recipients. the use of endomyocardial biopsy for the diagnosis and management of myocarditis was first reported in . subsequently, many reports , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] documented myocarditis in patients presenting with unexplained heart failure or ventricular arrhythmias (table . ). however, there was considerable incongruity in the diagnostic criteria used in these largely anecdotal reports. the dallas criteria were developed in preparation for a large, randomized, multicenter clinical trial of immunosuppressive therapy in myocarditis. these criteria define active myocarditis (see also fig. . a) as "an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of ischemic damage associated with coronary artery disease." furthermore, other causes of inflammation (e.g., connective tissue disorders, infection, drugs) should be excluded. , the dallas criteria also defined borderline myocarditis as an inflammatory infiltrate that is sparse and lacks myocyte injury, and often ( %) on repeat biopsy, borderline myocarditis will histologically progress to active myocarditis. a limitation of endomyocardial biopsy is possible sampling error. the inflammation in myocarditis may be patchy or focal, unlike allograft rejection, which is a relatively diffuse process. although obtaining four samples from the right ventricular septum provides a high sensitivity for detection of allograft rejection in transplant recipients, this may not hold true for myocarditis. in an autopsy study of the right ventricular biopsy technique ( samples taken from the apical septum), only six of patients dying of myocarditis were correctly identified. left ventricular biopsy missed the diagnosis in eight of . in another study using the standard four to six samples, the sensitivity of right ventricular endomyocardial biopsy was reported at %. dec and colleagues reported that employing repeat left and right ventricular biopsies in patients with suspected myocarditis with an initial negative biopsy increases the yield by %. because an ideal study to evaluate sampling error has not been done, the true yield is unknown, but clearly a negative biopsy does not exclude active myocarditis. in the mtt, only % of patients screened had histologic evidence of myocarditis. the european study of epidemiology and treatment of cardiac inflammatory disease (esetcid) demonstrated a % incidence of biopsy-proven myocarditis by expanding the dallas criteria with the use of newer techniques of pcr and in situ hybridization. as discussed earlier, [ ] [ ] [ ] [ ] [ ] [ ] there is a need for validation of new histologic and nonhistologic criteria for diagnosis of this disease to improve upon the dallas histologic criteria. coronary arteriography is usually normal, although in animal models, coronary vasculitis has been reported. the one major exception is kawasaki disease, in which coronary artery aneurysms are frequently seen in association with myocarditis. ventriculograms may demonstrate global or regional ventricular dysfunction, associated valvular regurgitation, and mural thrombi. localized ventricular aneurysms with normal global systolic function have also been reported. the hemodynamic profiles of patients with acute myocarditis are representative of the extent of myocardial and pericardial involvement. in patients with significant ventricular dysfunction, elevated filling pressures with depressed cardiac output and stroke work indices are seen. a restrictive hemodynamic profile can be seen and must be differentiated from that seen with postviral constrictive pericarditis. the true natural history of myocarditis is largely unknown because the great majority of cases is perhaps subclinical and resolves without any significant residual cardiac dysfunction. clinically apparent myocardial dysfunction as seen with acute coxsackievirus b infections also resolves without any adverse sequelae in most cases. it has been estimated that only % of patients with clinically suspected acute myocarditis will proceed to develop dcm, but the true incidence is unknown. the murine myocarditis models frequently develop a pathologic process indistinguishable from that of the human form of idiopathic dcm. the direct link among viral infection, myocarditis, and dcm has not been conclusively proven. isolation of infectious virus from the heart has been achieved in only a few cases of acute fulminant myocarditis in neonates and infants. , given the hypothesis that dcm may develop after viral infection has been eradicated, the presence of virus in the myocardium is neither sufficient nor necessary to link virally mediated myocarditis with dcm. the indirect evidence of viral etiology of dcm relies on ( ) progression of viral myocarditis to dcm in experimental animal models, ( ) apparent progression of myocarditis in some patients to dcm, ( ) increased enteroviral antibody titers in patients with dcm, ( ) presence of viral genomic material in the myocardial tissue of patients with dcm, and ( ) improvement of ventricular function in subjects with dcm receiving immunomodulatory treatments. the major limitations are as follows: the relevance of disease in mice to humans is suspect, most cases of dcm are not preceded by documented myocarditis, and epidemiologic serologic evidence is incomplete. whereas coxsackievirus b igm antibodies are detected with greater frequency in patients with dcm than in normal controls, the frequency is similar to matched community controls and household contacts. , enteroviral genomic sequences are detected in the myocardium of % to % of patients with active myocarditis and in % to % of patients with dcm, but in data derived from most published studies, the average detection frequencies are % for active myocarditis, % for dcm, and not significantly different from % among healthy controls. , in a metaanalysis of the association of enteroviruses with human heart disease, baboonian and treasure concluded that although the causative role of enteroviruses in acute myocarditis, particularly in children, was supported by an overall odds ratio of . [confidence interval (ci), . to . ], and the association of dcm was suggested by an overall odds ratio of . (ci, . to . ), six of studies did not demonstrate an increased presence of viral remnants. the same investigators demonstrated more recently that pcr positivity is not found in minimally affected first-degree relatives of patients with familial dcm, suggesting that in this group, genetic predisposition to viral myocarditis does not underlie the inherited predisposition to development of dcm. in recent studies, other investigators have found strong evidence for a viral link, while others have found no viral vestiges in the myocardium of patients with end-stage heart failure. , regional variation in the etiology of dcm may be responsible in part for the reported differences in pcr positivity. responsiveness of patients with dcm to immunomodulatory interventions provides an interesting line of evidence supporting a viral/immune etiology of dcm. one would expect immune suppression to be an effective treatment in dcm if postviral and other forms of autoimmunity play a causative role in the disease. efficacy of such interventions has been reported in carefully selected patients. , , [ ] [ ] [ ] although the link between myocarditis and dcm is unclear, certain prognostic factors are identifiable. the presence of an abnormal qrs complex on ecg correlates with more severe left ventricular damage and is an independent predictor of survival. left atrial enlargement, atrial fibrillation, and lbbb are also associated with increased mortality. higher baseline lvef is positively associated with survival, whereas intensity of conventional therapy at baseline is negatively associated with survival. the presence of right ventricular dysfunction, as evidenced by abnormal right ventricular systolic shortening on echocardiography, was shown to be the most important predictor of death or need for cardiac transplantation in a group of patients with biopsy-proven myocarditis who were followed longterm. in addition, a net increase in lvef (between initial and final ejection fraction) was associated with improved survival, whereas baseline ejection fraction was not predictive of outcome. the presence and degree of left ventricular regional wall motion abnormalities did not affect the clinical course. light microscopic findings on biopsy have not been found to predict outcome in myocarditis. however, the extent of myocardial inflammation was a predictor of outcome after surgical ventricular remodeling for heart failure. higher baseline serum antibodies to cardiac igg by indirect immunofluorescence was associated with a better lvef and a smaller left ventricular end-diastolic dimension. general supportive measures for patients with myocarditis include a low-sodium diet, discontinuation of ethanol, and fluid restriction, especially in the presence of heart failure. patients with myopericarditis may need analgesics for pain control. recommendations for the limitation of physical activity are based on the murine model of cvb myocarditis, in which forced exercise during the acute phase of illness was associated with higher titers of infectious virus, increased inflammatory and necrotic lesions, and mortality. , , ibuprofen, indomethacin, and salicylates administered to mice after inoculation with cvb also resulted in increased viral titers, increased histologic severity of myocarditis, and increased mortality. this led to the suggestion that even nonsteroidal antiinflammatory drugs should be avoided in patients with active acute myocarditis. the american college of cardiology task force on myopericardial diseases recommends a convalescent period of approximately months after the onset of clinical manifestations before a return to competitive sports. the management of patients with presumed or confirmed myocarditis is primarily directed toward treatment of chf, arrhythmias, and symptoms from pericardial disease. diuretics, vasodilators, and digoxin should be administered to patients with mild-to-moderate systolic dysfunction. inotropic therapy and mechanical support with intraaortic balloon pump or ventricular-assist devices may be required for patients in refractory cardiogenic shock. cardiac transplantation is reserved for those patients who do not improve despite the measures described previously. although there are multiple studies on the use of angiotensin-converting enzyme inhibitors (aceis) in heart failure, the utility of aceis in myocarditis has been studied only in the murine model. early treatment with captopril in a cvb myocarditis model resulted in less inflammatory infiltrate, myocardial necrosis, and calcification. heart weight, heart/body weight ratio, and liver congestion diminished. even with delayed therapy, a reduction in left ventricular mass and liver congestion was evident. the aceis exert a potent vasodilator response, improve pump function, prevent ventricular remodeling, and may have antiarrhythmic properties. hence, all patients with systolic dysfunction, including those with myocarditis, should be placed on maximally tolerated doses of aceis. the use of beta-blockers in patients with mild-tomoderate heart failure due to dcm has been reported to be beneficial, but once again, no trials in humans with myocarditis have been performed. metoprolol-treated mice in an acute cvb murine myocarditis model have increased viral replication, myocyte necrosis, and -day mortality rates. carteolol, a nonselective beta-blocker, has been studied in a chronic myocarditis model and found to have beneficial effects with improved histologic scores, reduced heart weight and volume, and liver congestion. it appears that in the acute setting, beta-blockers should be avoided, and in the chronic heart failure stage, the nonselective beta-blockers may be beneficial. antiarrhythmic therapy may be needed for control of ventricular and supraventricular dysrhythmias. although the data from clinical trials of antiarrhythmic therapy in heart failure have not shown a primary mortality benefit, patients with active myocarditis were excluded in these trials. since immunosuppression is probably not helpful in myocarditis and no other specific therapy is available, one might consider treating the arrhythmias in the usual fashion, but there appears to be a rationale for making the diagnosis of myocarditis in patients who do not have profound ventricular dysfunction along with their arrhythmia. first, the majority of patients with myocarditis have a spontaneous resolution. second, current antiarrhythmic therapy of ventricular tachyarrhythmias is exacting, involving electrophysiologic studies and use of potentially toxic drugs or implantable defibrillators. the benefit of making the diagno-sis of myocarditis is that the patient may require only shortterm protection while the underlying process resolves, which can be provided by using amiodarone or other antiarrhythmic drugs under inpatient monitoring. if myocarditis resolves, antiarrhythmic therapy can be withdrawn. patients whose arrhythmias fail to improve despite histologic resolution of myocarditis, or who survived cardiac arrest, may be candidates for aggressive electrophysiologic approaches and implantable defibrillators. temporary and permanent pacemakers may be required in patients presenting with conduction system abnormalities. clinical trials of immunosuppressive therapy were first reported in children with clinical evidence of myocarditis, prior to the introduction of endomyocardial biopsy. in two series, in a total of eight children presenting with acute onset of severe chf, rapid improvement and survival were noted with adrenocorticotropic hormone or hydrocortisone treatment. , mason and associates reported patients with biopsy-proven myocarditis, half of whom improved with azathioprine and prednisone. gagliardi and coworkers followed children with biopsy-proven myocarditis who were treated with cyclosporine and prednisone. at year, of patients still had histologic evidence of myocarditis. no patient died or required transplantation. however, there was no control group. the data supporting an immunologic basis of myocarditis resulted in multiple treatment trials using immunosuppressants (table . ). the average proportion of patients showing improvement with a variety of immunosuppressants was %. a large number of the trials predated the development of the dallas criteria; thus, the histologic definition of myocarditis was not uniform. immunosuppressive regimens were arbitrary, and the lack of control groups made interpretation of these trials arduous. it was unclear whether immunosuppression was beneficial in those patients with myocarditis, as they can improve spontaneously. further, the infectious complications of immunosuppression were frequently seen and occasionally reported. , the conflicting results from these nonrandomized observations led to the mtt. in a multicenter, prospective, randomized design, the mtt enrolled patients with heart failure of recent onset (< years), left ventricular dysfunction (lvef < %), and biopsy-proven myocarditis (per the dallas criteria). the study screened patients; ( %) had endomyocardial biopsy evidence of myocarditis, and patients had a qualifying lvef of less than % and agreed to enrollment. patients were randomized to three treatment arms: prednisone and cyclosporine, prednisone and azathioprine, and no immunosuppressant treatment. all patients received conventional therapy for heart failure. the prednisone and azathioprine group was subsequently eliminated owing to low patient recruitment in the trial. patients were treated for weeks, and the primary end point was comparison of the mean increase in lvef at weeks. secondary analysis of other markers of left ventricular function, survival, and several immune parameters was performed. at both and weeks, no difference in lvef was observed in immunosuppressive-treated patients compared with untreated patients. at and years, there was no difference in survival or need for cardiac transplantation between groups (fig. . ) . on multivariate analysis, better baseline lvef, less intensive conventional therapy, and shorter illness duration were independent predictors of improvement in lvef during follow-up. analysis of immunologic variables (cardiac igg, circulating igg, natural killer and macrophage activity, helper t-cell level) suggested an association between better outcome and a more robust immune response. a higher level of cardiac igg was associated with a higher lvef and a smaller left ventricular size. the mortality rate for the entire trial was % at year and % at . years. the results of the mtt were important for diagnostic management because the authors recommended that in patients with unexplained chf, the performance of endomyocardial biopsy for the sole purpose of instituting immunosuppressive therapy was not warranted. nonetheless, certain subgroups may benefit from immunosuppressant therapy, including those with gcm, hypersensitivity myocarditis, or cardiac sarcoidosis. using a multicenter database, cooper and colleagues reviewed patients with gcm. the rate of death or cardiac transplanta-tion was %. median survival was . months from symptom onset to death or transplantation. the median survival in patients treated with corticosteroids was . months versus . months in untreated patients. however, patients treated with corticosteroids and azathioprine had an average survival of . months. cyclosporine in combination with corticosteroids, corticosteroids and azathioprine, and corticosteroids, azathioprine, and orthoclone okt survived an average of . months. the uncontrolled nature of this report decreases the reliability of its conclusions. patients with myocarditis associated with a known immune-mediated disease, such as systemic lupus erythematosus, may benefit from immunosuppressive therapy. other potential indications for a trial of immunosuppressant therapy include failure of myocarditis to resolve, progressive left ventricular dysfunction despite conventional therapy, continued active myocarditis on biopsy, or fulminant myocarditis that does not improve within to hours of full hemodynamic support, including mechanical assistance, and persistent ventricular tachycardia or fibrillation. smaller studies have used differing immunosuppressant regimens. kühl and schultheiss treated patients with biopsies classified as immunohistologically positive (more then two cells per high-power field and expression of adhesion molecules), negative dallas criteria, and left ventricular dysfunction. patients were treated with conventional therapy for months, followed by gradual tapering of methylprednisolone doses over weeks (following biopsy and lvef response). therapy was associated with an improvement in ejection fraction in % and improved new york heart association (nyha) functional class in %. four patients ( %) had no change in ejection fraction despite improvement in inflammatory infiltrates. however, study conclusions are limited by the absence of a control group. drucker and coworkers retrospectively reviewed children with congestive cardiomyopathy and dallas criteria of borderline or definite myocarditis. twenty-one patients were treated with intravenous igg ( g/kg over hours) and were compared to historical controls. overall survival was not improved, although there was a trend toward improvement in -year survival rates in the treated group. in the intravenous igg group, the left ventricular function was improved and persisted after adjustment for age, biopsy status, and use of aceis and inotropes. in a comparative study of ifn-α, thymomodulin, and conventional therapy in patients with biopsy-proven myocarditis or idiopathic dcm, an improvement in the active treatment groups was reported for ejection fraction (at rest and during exercise), maximal exercise time, functional class, and ecg abnormalities. in patients with chf, nyha class iii or iv, with symptoms of less than months' duration, intravenous igg resulted in an improvement in lvef and a functional improvement to nyha class i or ii at year of follow-up, in all nine patients who survived, regardless of biopsy results. perhaps strategies with alternative immunosuppressive regimens and different diagnostic criteria will be more successful in demonstrating the utility of immunosuppressants. the esetcid , is a prospective multicenter, placebocontrolled, double-blind study intended to address the natural course of myocarditis, myopericarditis, pericarditis, and the treatment regimens include conventional therapy with diuretics, aceis, digoxin, and antiarrhythmics or defibrillators; specific therapy for cmv and enteroviral myocarditis; and prednisolone and azathioprine for myocarditis without detectable virus. the duration of blinded therapy is months, with follow-up for months. the possible utility of more flexible diagnostic criteria for identification of responders to immunosuppressive therapy was recently suggested in a retrospective analysis by frustaci and colleagues. they found that the patients who had improved with immunosuppression had detectible circulating cardiac-specific autoantibodies but no detectible viral genomic material in their myocardium, while nonresponders had the opposite findings. these observations could be explained by successful suppression of an autoimmune response without the liability of suppressing ongoing antiviral immune activity. immunomodulatory therapies should be considered in cases of myocarditis that display ongoing adverse immune and autoimmune activity. immunosuppressive drug therapy, intravenous igg, tnf-α, and immunoadsorption are the forms of immunomodulation discussed above that have been used in humans (fig. . ). immunoadsorption has been applied primarily in dilated cardiomyopathy, but may hold promise in myocarditis, perhaps especially in phase ( fig. . a) of lymphocytic myocarditis. manipulation of cytokines, chemokines, and other factors that regulate proinflammatory and antiinflammatory processes , , , [ ] [ ] [ ] [ ] should receive attention in the development and assessment of new immunomodulatory therapies. myocarditis has emerged as a special indication for device therapy in recent years. circulatory-assist devices are especially attractive in myocarditis because the disease is usually self-limited. as a result, a relatively short period of circulatory assistance may allow time for the myocardium to recover and the injurious infectious, immune, and autoimmune processes to dissipate. this concept has been successfully tested in patients with severe heart failure due to myocarditis. [ ] [ ] [ ] implanted cardioverter-defibrillator devices have been used to treat ventricular tachyarrhythmias commonly associated with myocarditis. while such therapy may be lifesaving, consideration should always be given to antiarrhythmic drug therapy with protracted monitoring so as to avoid device implantation if possible. those patients with myocarditis who have survived cardiac arrest are candidates for implantable defibrillator devices. in a small series (n = ) composed predominantly of female patients ( %), the outcome of patients with active lymphocytic myocarditis confirmed by histologic examination of the explained heart was significantly worse than in controls undergoing transplantation for other diagnoses. this concern has not been validated in the analysis of outcome of , cardiac transplant recipients in the registry of the international society for heart and lung transplantation. one-year actuarial survival in all groups transplanted (idiopathic dcm, myocarditis, peripartum cardiomyopathy, versus other diagnoses) was %. nonetheless, myocarditis may recur in the transplanted heart. prevention prevention of myocarditis is an important developing strategy given the likelihood that a substantial proportion of cases of dcm worldwide are the result of preceding or ongoing myocarditis. several strategies have been considered, including immunization against the most common cardiotropic viruses, , , [ ] [ ] [ ] [ ] functional disablement of the coxsackie-adenovirus sarcolemmal receptor, , and early induction of immune tolerance. while immunization seems to have the greatest potential, scientific, medical, geographic, and political impediments are formidable. the advances in treatment strategies for hiv-infected patients have successfully resulted in prolonged survival times, and noninfectious complications of aids, such as dementia and heart disease, have become increasingly prevalent. early in the history of the aids epidemic, reports emerged of a rapidly fatal dcm affecting hiv-infected patients. , since the early reports, several clinical and echocardiographic series , [ ] [ ] [ ] [ ] [ ] have suggested that a subgroup of hiv-infected patients are predisposed to the development of progressive heart disease. in a prospective echocardiographic survey of hiv-infected adults over a period of years, patients were found to have significant cardiac dysfunction. dilated cardiomyopathy occurred in of and was strongly associated with a cd count of less than /mm and poorer survival. it has been estimated that clinically significant cardiac disease occurs in % to % of hiv-seropositive individuals. an interesting hypothesis to explain the high frequency of dilated heart muscle disease is the presence of myocarditis in hiv-infected patients with left ventricular dysfunction. reilly and colleagues reported in an autopsy series of consecutive aids patients a significantly higher incidence of myocarditis in those with clinically apparent cardiac disease or dcm. there have been other reports of higher prevalence of myocarditis in an endomyocardial biopsy series of hiv-seropositive patients compared with those without risk factors for hiv who were biopsied for suspected myocarditis. human immunodeficiency virus-related myocarditis has unique and atypical immunopathogenic features. it is characterized by increased cd t lymphocytes and sole induction of mhc class i, perhaps as a part of the systemic depletion of cd t cells. the myocarditis may not be readily apparent on histology owing to the accompanying lymphopenia, and special immunohistology and histochemistry techniques may need to be employed. although in situ hybridization techniques have demonstrated hiv- transcripts in cardiac myocytes, interstitial dendritic cells, and endothelial cells, the pathologic significance of this finding is still unclear because patients with evident transcripts may or may not have clinical disease. also, it is not evident that myocyte injury is a result of direct cytotoxicity of the virus, transcripts, cytokines, or other cardiotropic viruses. a large number of hiv-seropositive patients with left ventricular dysfunction also manifest evidence of nonpermissive or latent infection of myocytes with cmv immediate-early (cmv ie- ) genes. although evidence for classic intranuclear inclusions of active lytic cmv infection is rarely found, there is increasing speculation that the latent viral infection may be responsible for enhanced mhc expression and provide a stimulus for ongoing immune injury, as seen with most models of myocarditis. a role for direct cytokine-mediated cardiac injury has also been proposed in hiv-infected populations with myocardial dysfunction. both tnf-α and il- , known to be elevated in hiv infection, directly inhibit cardiac contractility in vitro, and the former has been implicated in causing myocardial dysfunction. increased catecholamines may be responsible for microvascular spasm and chronic ischemic dysfunction. the clinical management of patients with hiv-related myocarditis and cardiomyopathy is targeted toward improving congestive symptoms, afterload reduction, and digitalis for improved neurohormonal axis. a specific role for antiviral therapies is controversial, since medications like zidovudine and ifn-α are themselves recognized as cardiotoxins. zidovudine has been known to result in premature termination of myocyte mitochondrial dna chain replication. despite worldwide eradication of smallpox, the bioterrorism threat arising from the existence of stored variola virus has prompted military and civilian smallpox vaccination programs in the united states. myocarditis emerged as a known, rare complication of smallpox vaccination during the eradication effort in the s and s. its incidence varied with the vaccinia strain used to produce the vaccine, and with the method used to detect myocarditis. the true incidence is not known. current vaccination programs use the original new york city board of health strain of vaccinia (dryvax) and new vaccines. while the occurrence of myocarditis in the united states's current military dryvax vaccination program appears to be higher ( . %, or about one in , ) than historical estimates, its incidence after new vaccines has not been determined. previously vaccinated individuals have a much lower risk of developing myocarditis. full functional and symptomatic recovery occurs in most patients. while involvement of eosinophils has been noted, the mechanisms responsible for postvaccination myocarditis are not known. bacterial infection of the myocardium occurs frequently in association with infective endocarditis, usually in the form of myocardial abscesses adjacent to the valve ring (see chapter ) . myocardial involvement has also been reported in association with a wide range of bacterial pathogens in the absence of endocarditis. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] with most of these agents, myocardial involvement is uncommon and occurs principally in the setting of overwhelming systemic infection. cardiac involvement after streptococcal infection is usually manifested as acute rheumatic fever, which develops to weeks after onset of pharyngitis and has a distinctive histologic appearance (see chapter ) . streptococcal pharyngitis may also be associated with a nonrheumatic form of myocarditis that occurs concurrently with the febrile illness. [ ] [ ] [ ] [ ] the most common clinical manifestations are chest pain and marked st-segment and t-wave abnormalities on the electrocardiogram, which correlate with segmental wall motion abnormalities observed with echocardiography. cardiomegaly and chf are uncommon. histologic examination reveals lymphocytic infiltrates and myocyte necrosis in the absence of aschoff bodies, similar to the findings in viral or idiopathic myocarditis. bacteria are not present in the myocardium, and it is hypothesized that inflammation is caused by streptococcal exotoxins in a manner similar to that in diphtheritic myocarditis. although vaccination has virtually eliminated diphtheria in most western nations, it remains an important public health problem in many underdeveloped countries, and may be the most common etiology of myocarditis worldwide. infection with c. diphtheriae is usually confined to the respiratory mucosa. systemic manifestations are due to secretion of a potent exotoxin. the ecg abnormalities suggesting myocardial involvement are present in a high proportion of patients, but clinical evidence of cardiac dysfunction occurs in only % to % of cases. nevertheless, cardiac involvement is the most common cause of death in fatal infections. disturbances of av conduction, including bundle branch blocks and complete av block, are observed frequently in affected patients and are associated with a mortality rate of % to %. patients may also present with progressive cardiac dilatation and chf. histologic study reveals diffuse mononuclear cell infiltrates associated with myocyte necrosis. corticosteroid therapy does not appear to be effective in the prevention or treatment of diphtheritic myocarditis, although only one prospective trial has been performed. one report suggested that administration of carnitine may decrease the incidence and severity of cardiac involvement. spirochetal myocardial disease lyme disease lyme disease is caused by the spirochetal organism b. burgdorferi, which is transmitted to humans by certain species of deer ticks in endemic areas of north america, europe, and asia. the acute phase of the illness is characterized by fever, myalgia, lymphadenopathy, and a characteristic rash known as erythema chronicum migrans. the organism persists in many tissues, and chronic manifestations include arthritis and a variety of neurologic syndromes. manifestations of cardiac involvement develop in % to % of patients at an average of . weeks (range, days to months) after the acute illness. , , disturbances of av conduction are the most common manifestations, occurring in % of cases, with complete or high-grade block in more than %. the av block is usually supra-hisian, with a narrow complex escape rhythm. temporary transvenous pacing is required frequently, but av block almost always resolves within to days. endomyocardial biopsy may reveal lymphocytic infiltrates with associated myocyte necrosis, and spirochetes may be identified in biopsy specimens. lyme carditis occasionally develops in patients without a preceding rash or other symptoms of acute lyme disease. therapy with a to week course of antibiotics (doxycycline, amoxicillin or cefuroxime) is recommended for patients with lyme carditis. , antibiotic therapy has proved effective in the prevention and treatment of chronic arthritic and neurologic syndromes, but its use in cardiac disease has not been tested prospectively. evidence of diffuse myocardial involvement is common, including evolving stsegment and t-wave abnormalities on the electrocardiogram, reversible abnormalities of left ventricular wall motion, and diffuse myocardial uptake on gallium scan. one fatal case of pancarditis has been reported, but frank heart failure is uncommon. a high incidence of positive serologies for b. burgdorferi was reported in european patients with chronic dcm, and in two patients the organism was cultured from myocardial biopsies. , it has been suggested that unrecognized lyme carditis may be responsible for a small but significant proportion of cases of idiopathic dcm. evidence of severe myocarditis is present at autopsy in a high proportion of fatal cases of leptospirosis and relapsing fever. , nonspecific ecg abnormalities are common in these diseases, but clinical evidence of left ventricular dysfunction is rare. although previously uncommon, the incidence of fungal infections of the heart has increased markedly since the early s. this increased incidence is due to several factors, including the increasing use of antibiotics, immunosuppressive agents for transplantation, and chemotherapy, as well as the increasing application of cardiac surgery and the increasing prevalence of intravenous drug abuse. candida infection the most common fungal organisms causing cardiac infection are candida species. candida endocarditis occurs most frequently after thoracic surgery and in intravenous drug abusers. immunocompromised patients, on the other hand, are more likely to develop candida myocarditis without involvement of the valves or endocardium, usually in the setting of disseminated systemic infection. [ ] [ ] [ ] autopsy studies reveal extensive myocardial involvement in % to % of patients who die of systemic candidiasis. histologically, candida myocarditis consists of focal abscesses (usually microscopic, although gross nodules may be present) interspersed with areas of normal myocardium. clinical manifestations typically include nonspecific ecg abnormalities, disturbances of av conduction, including complete heart block, and tachyarrhythmias. cardiomegaly and chf are rare. myocardial involvement is usually not recognized antemortem. myocardial involvement is present in % of patients with disseminated aspergillosis, and myocardial invasion is almost always present in patients with aspergillus endocarditis. as in other tissues, histology is characterized by microscopic and macroscopic abscess formation. , extensive vascular invasion by fungal hyphae results in thrombosis and coagulation necrosis. although aspergillus endocarditis has been treated successfully, myocarditis is uniformly fatal. cardiac involvement in actinomycosis occurs in only % of cases and usually develops by direct extension from a contiguous focus of pulmonary or mediastinal infection. [ ] [ ] [ ] hematogenous seeding of the myocardium occurs occasionally. myocardial involvement is characterized by necrotizing abscess formation with masses of mycelial bodies and characteristic sulfur granules. in many cases, cardiac symptoms are absent, but patients may present with chest pain characteristic of pericarditis, pericardial tamponade, or chf. myocardial involvement has rarely been reported in immunocompromised patients with disseminated coccidioidomycosis and cryptococcosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cardiac involvement is usually not clinically apparent antemortem, although death due to progressive chf has been reported. cardiac involvement with blastomycosis and histoplasmosis is extremely uncommon and usually results from direct extension from a contiguous intrathoracic focus. rocky mountain spotted fever caused by infection with rickettsia rickettsii is characterized by a diffuse vasculitis, and in fatal cases, death is usually due to vascular collapse. vasculitis of the coronary vessels may also be present, and lymphocytic infiltrates with myocyte necrosis are present in approximately % of fatal cases. , although cardiac dilatation and cardiogenic pulmonary edema occur infrequently, echocardiography demonstrates systolic left ventricular dysfunction in the majority of patients. , clinical evidence of myocarditis has been reported in association with scrub typhus due to rickettsia tsutsugamushi, whereas q fever (coxiella burnetii) usually causes endocarditis in its chronic form. it is estimated that to million people in south and central america are infected with t. cruzi, and chagas' cardiomyopathy resulting from this infection is the most common cause of chf and cardiac death in these endemic areas. , the parasite is transferred to humans by triatomine insects known as reduviid bugs. the clinical course of infection is characterized by an acute phase, an indeterminate or latent phase of variable duration, and a chronic phase. , after inoculation, parasites are disseminated throughout the body, with the highest concentrations appearing in striated and cardiac muscle and autonomic ganglia. a lesion may appear at the point of entry, and an acute illness develops, characterized by fever, myalgia, edema of the face and lower extremities, hepatomegaly, and generalized lymphadenopathy. because of the nonspecific nature of the symptoms, the acute phase of the disease is usually unrecognized. rarely, acute inflammatory myocarditis develops during the acute phase, with ecg abnormalities, cardiomegaly, and chf. histologic examination in these cases demonstrates inflammatory infiltrates adjacent to myocytes containing large numbers of intracellular parasites. these findings suggest that cardiac manifestations during the acute phase of the illness may be due to direct lysis of myocytes by parasites. , the acute illness resolves over a period of weeks to months, and patients enter the indeterminate phase. these patients are asymptomatic, with low-level parasitemia, and antibodies to t. cruzi are present. although the electrocardiogram is normal, echocardiography and left ventricular cineangiography demonstrate focal wall motion abnormalities in a high proportion of cases, most commonly involving the left ventricular apex and posterior wall. endomyocardial biopsy is frequently normal but may reveal hypertrophy, fibrosis, and inflammatory infiltrates in up to % of patients without clinical manifestations. manifestations of chronic chagas' disease develop in % to % of infected patients after a highly variable period, which may be as long as years. , involvement of autonomic ganglia may cause megacolon or megaesophagus, but the heart is the organ most commonly affected. histology is characterized by focal areas of inflammation or fibrosis interspersed with areas of normal myocardium. endomyo-cardial biopsy reveals myocarditis in approximately % of patients. , this process frequently involves the specialized conducting tissue, and therefore disturbances of av conduction, especially rbbb with or without associated left anterior fascicular block, are present in up to % of patients. complete heart block may require permanent transvenous pacing. ventricular arrhythmias are also frequent, and the initial manifestation of the disease may be sudden death due to ventricular tachyarrhythmia or complete heart block. decreased ventricular function is present in almost all patients with chronic chagas' disease, and in its most advanced form, chagas' disease presents as a congestive cardiomyopathy with four-chamber dilatation. a characteristic apical aneurysm is usually present. [ ] [ ] [ ] left ventricular thrombus is frequently observed, and systemic embolization is common. , this advanced form of the disease is usually fatal within a few years. diagnosis of chronic chagas' cardiomyopathy is dependent on detection of circulating antibodies to t. cruzi by one of several serologic methods. parasites are usually not detected in the myocardium, but low-level parasitemia can be demonstrated by hemoculture or xenodiagnosis, using uninfected reduviid bugs allowed to ingest the patient's blood. the pathogenic mechanisms leading to myocardial injury, in some patients occurring many years after the initial infection, are poorly understood. the presence of inflammatory infiltrates in the absence of detectable parasites suggests the possibility of autoimmune injury, as postulated for viral and idiopathic myocarditis. support for this hypothesis includes the demonstration of antibodies to t. cruzi as well as antiidiotypic antibodies that cross-react with myocyte antigens. , histologic studies demonstrate loss of autonomic ganglia, and physiologic studies are suggestive of marked autonomic dysfunction. [ ] [ ] [ ] withdrawal of parasympathetic tone may lead to excess sympathetic stimulation, which can cause cardiomyopathy. histologic studies also demonstrate abnormalities of the microvascular bed, , and in vitro experiments demonstrate altered endothelial cell function and increased platelet-endothelial cell adhesion. , all three reports suggest that progressive focal myocardial disease is the result of ischemia due to obstruction of the microvascular bed. treatment of chronic chagas' cardiomyopathy is supportive, with the use of standard therapy for chf. dynamic cardiomyoplasty has resulted in symptomatic improvement in some patients. the role for left ventricular reduction or the commonly known batista procedure is controversial. antiarrhythmic therapy may be indicated for sustained ventricular tachyarrhythmias, and a permanent pacemaker should be implanted in patients with high-degree av block. two antiparasitic drugs are available for the treatment of american trypanosomiasis. both nifurtimox and benznidazole decrease the level and duration of parasitemia and decrease mortality in patients with acute chagas' disease. low-level parasitemia persists in most treated patients, however, and it is unclear whether therapy in the acute phase decreases the incidence of subsequent progression to chronic chagas' disease. whereas earlier studies with these drugs have not been shown to decrease progression from latent phase to chronic disease or to decrease symptoms or improve cardiac function in patients with chronic disease, , the recent studies with itraconazole and allopurinol have shown partial success with parasitologic cure and normalization of ecg changes in nearly half the patients. in a randomized, placebo-controlled trial of benznidazole, there was successful negative seroconversion of % of patients with early chronic disease as manifested by seropositivity for t. cruzispecific antibodies after treatment for days. immunosuppressive therapy in patients with malignancies or after organ transplantation has been associated with reactivation causing acute chagas' disease. , reactivation of chagas' disease in this setting has usually responded promptly to therapy. [ ] [ ] [ ] african trypanosomiasis african trypanosomiasis is caused by trypanosoma gambiense or t. rhodanese and characteristically presents with progressive somnolence owing to central nervous system involvement. autopsy studies demonstrate a pancarditis involving the mural and valvular endocardium as well as the myocardium in up to % of fatal cases. [ ] [ ] [ ] [ ] the conduction system and autonomic ganglia may also be involved. nonspecific abnormalities are often present on the electrocardiogram, but other clinical manifestations of the frequent cardiac involvement are apparently uncommon. patients with acute infection by t. gondii are usually asymptomatic, but they may have a transient syndrome of fever and lymphadenopathy. the infection usually persists in a latent phase, with cysts deposited predominantly in the brain and myocardium. immunosuppression after chemotherapy, in transplant recipients, and in patients with aids may be associated with disseminated infection characterized by severe encephalitis and myocarditis. [ ] [ ] [ ] [ ] myocarditis after transplantation occurs frequently in seronegative recipients of hearts from seropositive donors. [ ] [ ] [ ] endomyocardial biopsy demonstrates intracellular toxoplasma pseudocysts and a mixed interstitial infiltrate, frequently including eosinophils ( fig. . d ). toxoplasma myocarditis can be successfully prevented by a -week course of pyrimethamine imitated after early transplantation or treated with pyrimethamine and sulfadiazine. cardiac involvement in metazoal infections is uncommon. up to % of patients with echinococcosis have cardiac cysts. [ ] [ ] [ ] these patients may present with pericardial or atypical chest pain, chf owing to inflow or outflow obstruction, ventricular arrhythmias, or pulmonary hypertension owing to diffuse pulmonary embolization of scolices. the diagnosis is usually documented by two-dimensional echocardiography, and surgical excision is indicated, when possible, even in asymptomatic patients. trichinosis, caused by the parasite t. spiralis, is usually a benign syndrome characterized by fever, myositis, and eosinophilia. mild, asymptomatic myocardial involvement is probably common, as suggested by frequent ecg abnormalities and pericardial effusion noted by echocardiography. rarely, a severe myocarditis develops, which is the apparent cause of death in most fatal cases. [ ] [ ] [ ] eosinophils are prominent in the interstitial infiltrate. t. spiralis does not become encysted in the heart, and larvae are seldom identified in the myocardium. myocardial injury is thought to be immune mediated, and therapy with corticosteroids is generally recommended, although prospective trials have not been performed owing to the infrequent occurrence of this syndrome. the mucocutaneous lymph node syndrome or kawasaki disease occurs predominantly in children under the age of years and is most prevalent in japan. , it has been recognized worldwide, and in the united states and the developed world, it has replaced rheumatic fever as the most common cause of acquired heart disease in children. it is widely believed to have an infectious etiology, but no agent has yet been identified. its diagnosis is based on recognition of clinical features of the illness, which include remittent high-spiking fever with distinctive conjunctival injection, anterior uveitis, strawberry tongue with erythema, dryness, fissuring and peeling of the lips and mouth, erythematous truncal rash, redness of palms and soles with periungual desquamation, and cervical lymphadenopathy. the principal cardiovascular manifestation of the disease is a multisystem arteritis with frequent involvement of the coronary arteries. coronary arteritis leads to aneurysm formation and thrombosis. the most common cause of death is myocardial infarction due to aneurysm rupture or coronary occlusion. myocardium obtained by endomyocardial biopsy or at autopsy reveals histologic evidence of myocarditis in a high proportion of patients. [ ] [ ] [ ] [ ] segmental wall motion abnormalities and nonspecific ecg changes are frequently present in the absence of coronary aneurysms. , these findings have been attributed to myocarditis, but they might also reflect ischemia due to small vessel arteritis. congestive heart failure in the absence of infarction is uncommon. intravenous gamma-globulin and high-dose aspirin are effective in the prevention of coronary aneurysms and thrombosis, but their effect on myocarditis is not known. giant cell myocarditis is a rare but frequently fatal disorder. it is defined histologically by extensive but patchy myocyte necrosis with areas of intense multicellular inflammatory infiltration that includes histiocytes, lymphocytes, and the characteristic multinucleated giant cells (fig. . b) . , [ ] [ ] [ ] there has been a great deal of controversy as to whether gcm and cardiac sarcoidosis are distinct pathologic entities because multinucleated giant cells in gcm seldom organize to form granulomas. , , litovsky and associates showed that gcm is characterized by myocytic destruction mediated by cytotoxic t cells, macrophagic giant cells, and eosinophils. in contrast, cardiac sarcoid is an interstitial granulomatous disease without myocytic necrosis. although the etiology of gcm is unknown, it has been associated with a medley of autoimmune disorders and perhaps is immunologically mediated. thymomas, systemic lupus, rheumatoid arthritis, wegener's granulomatosis, ulcerative colitis, chronic hepatitis, myasthenia gravis, myositis, pernicious anemia, takayasu's arteritis, and lymphomas have been associated with gcm. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the clinical presentation of gcm may mimic lymphocytic myocarditis, although arrhythmias and heart failure are usually more severe and rapidly progressive. , frequently, patients with gcm present with conduction system abnormalities, ventricular tachycardia, or even sudden cardiac death. [ ] [ ] [ ] [ ] , , , , giant cell myocarditis has also been reported to present as asymmetric septal hypertrophy. the natural history of gcm is obscure owing to its rare occurrence, but the isolated reports in the literature suggest that it carries a poor prognosis. davidoff and coworkers reported that % of patients with gcm required cardiac transplantation or died during a -year follow-up period compared with the % of patients with lymphocytic myocarditis. cooper and colleagues reported on patients with gcm collected in a worldwide registry. the registry patients had an % rate of death or need for transplantation, which was significantly worse than that for the patients with lymphocytic myocarditis seen in the mtt. the median survival with gcm was . months. the patients treated with immunosuppressive regimens including cyclosporine, azathioprine, and prednisone had an average cardiac survival of . months compared with . months for the untreated patients. the rate of recurrent gcm in the transplanted patients was % (nine of ). the role of immunosuppressive therapy for gcm is unknown, but at least anecdotal and registry reports suggest possible benefit of cyclosporine and prednisone with or without azathioprine. cardiac transplantation remains the last therapeutic resort for these patients, although there is risk of recurrent disease, , , which seems to be associated with abatement of immunosuppressive therapy after transplantation and may represent atypical rejection in the allograft. it usually resolves with intensification of the immunosuppressive regimen. eosinophilic myocarditis loffler first described the association of eosinophils with cardiac disease, and reported "endocarditis parietalis fibroplastica" in association with eosinophilia. the endocardial disease with eosinophilia is well recognized and extensively reviewed elsewhere. , myocardial involvement is rare and frequently fatal; hence, diagnosis is often made postmortem. endomyocardial biopsy is essential to the antemortem diagnosis of eosinophilic myocarditis (fig. . c ). myocarditis is believed to represent a more fulminant and necrotic form of the endocardial disease. eosinophils have the ability to secrete highly toxic cationic proteins into areas of inflammation and to produce harmful oxygen radicals and potent lipid mediators, leading to myocyte necrosis as seen in proximity to degranulating cosinophils. animal experiments have confirmed that exposure of myocytes to eosinophil granule proteins is lethal, and ventricular function in the intact heart is reduced in hypereosinophilic states. eosinophilic myocardial infiltrates have been reported in association with profound eosinophilia caused by an allergic diathesis, parasitic infection, drug hypersensitivity, vasculitis, or churg-strauss syndrome, , , but eosinophilic myocarditis can occur in the absence of profound eosinophilia. further, eosinophilic myocarditis may present as acute myocardial infarction, sudden death, cardiogenic shock, or nonspecific chest pain and dyspnea. the natural history of eosinophilic myocarditis is usually swift and ominous with rapid evolution to refractory heart failure or intractable arrhythmias, leading to death. early biopsy-aided histologic confirmation is fundamental to antemortem diagnosis. clinical improvement may occur with corticosteroid therapy. cardiac sarcoidosis sarcoidosis is a multiorgan, noncaseating granulomatous disorder of unknown etiology. histologically, it may involve the lung, lymph nodes, skin, liver, spleen, parotid glands, and heart. right heart failure owing to pulmonary manifestations of pulmonary hypertension and pulmonary fibrosis is the predominant cardiac finding. asymptomatic cardiac involvement is common, with a quarter of the patients having sarcoid granulomas in the heart at autopsy. characteristically, the noncaseating granulomas infiltrate the ventricular walls and become fibrotic. they may involve the conduction system, although there is no definite predilection for specialized tissues. there may be transmural involvement with fibrous replacement of portions of the myocardium and aneurysm formation. the fibrous transition of granulomas may result in early diastolic dysfunction, but as the disease progresses and with extensive involvement, systolic impairment occurs. whereas cardiac involvement in sarcoidosis commonly occurs as part of the systemic affliction, isolated cardiac sarcoidosis in the absence of systemic disease has been described. the clinical presentation of cardiac sarcoidosis is variable and may depend on the amount of myocardium replaced with granulomas and the amount and location of scar tissue. rhythm abnormalities and conduction disorders predominate, although asymptomatic patients with mildly restrictive filling patterns may elude medical attention. patients with chf may show clinical features of restrictive cardiomyopathy or dcm. papillary dysfunction with mitral regurgitation and pericardial involvement with effusiveconstrictive disease have also been described. radionuclide myocardial imaging with thallium and gallium is helpful in identifying patients with myocardial involvement. magnetic resonance imaging has also been proposed as a diagnostic modality. , histologic diagnosis with endomyocardial biopsy is corroborative, but a negative biopsy does not rule out the possibility, owing to sampling error. the combination of bilateral hilar adenopathy and myocardial disease suggests cardiac sarcoidosis in a young person. corticosteroids are indicated when myocardial involvement, conduction abnormalities, and ventricular arrhythmias are present. patients with scintigraphic uptake of gallium may be more responsive to corticosteroid therapy. perma-nent pacemakers may be needed to treat the conduction abnormalities. implantable defibrillators may be utilized in the prevention of sudden death. heart failure is treated in the conventional manner, whereas heart transplantation is reserved for intractable heart failure. heart-lung transplants are performed infrequently for patients with pulmonary involvement, but there is a significant risk of recurrent disease. peripartum myocarditis/cardiomyopathy virchow and porak first reported the association of pregnancy with dcm in in an autopsy series. peripartum myocarditis/cardiomyopathy occurs in one of every , to , pregnancies. the incidence is higher in africa, and it increases with older age, multiparity, multiple gestations, and prior history of peripartum myocarditis/cardiomyopathy. peripartum cardiomyopathy is currently believed to be a myocarditis of unknown etiology, perhaps an infectious, autoimmune, or idiopathic process. the viral myocarditis hypothesis stems from the observations that pregnant mice are more susceptible to cardiotropic viruses, with increased viral replication, and with the increased hemodynamic burden of pregnancy, the myocardial lesions worsen. recently, it has been postulated that after delivery, the rapid degeneration of the uterus results in fragmentation of tropocollagen by enzymatic degradation. this releases actin, myosin, and their metabolites, and antibodies are formed that then cross-react with the myocardium. an association between tocolytic therapy and cardiomyopathy has also been reported. while the diagnosis of peripartum myocarditis/cardiomyopathy is traditionally made during the last trimester or during the first months postpartum, earlier occurrence has been reported. the presentation is usually of decompensated ventricular systolic failure in the absence of any identifiable cardiac pathology. the lvef normalizes in approximately % of women and is more likely to normalize if the initial lvef is > %. therapy is tailored to the decompensated state with diuretics, digoxin, and vasodilators (aceis are contraindicated in pregnancy). inotropic therapy may be needed for supporting those in cardiogenic shock, along with the use of mechanical circulatory-assist devices. although there are anecdotal reports of benefit of immunosuppressive therapy, the routine use of these agents cannot be recommended; in fact, the only indication would be biopsy-proven fulminant myocarditis. cardiac transplantation is an alternative therapeutic option and may be offered to those with intractable heart failure, but it is preferred that transplantation be delayed. the early outcome after transplantation in these patients is often unfavorable, with increased allograft rejection, and the natural history of peripartum myocarditis/cardiomyopathy suggests that more than half of the patients have spontaneous resolution. there are perhaps two different subgroups. one presents with a rapidly progressive, fulminant course with often nearcomplete resolution of myocardial dysfunction within days and excellent long-term prognosis. the other group has late, insidious onset and presents with progressively worsening heart failure with poor prognosis. it is often difficult to differentiate this from the common variety of dcm. myocarditis is a focal or diffuse inflammation of the myocardium, which has multiple infectious and noninfectious etiologies. autoimmunity, triggered most often by viral infections, is a prominent pathophysiologic mechanism of myocarditis. overt and clinically inapparent myocarditis is an important cause of dilated cardiomyopathy. virus-induced lymphocytic myocarditis progresses through three stages: active viral infection, autoimmunity, and dilated cardiomyopathy. myocarditis is no longer a diagnosis of exclusion; histology, histochemistry, dna and rna detection, tissue and circulating antibody detection, and a variety of imaging techniques can be used together or, in some cases, independently to make the diagnosis and to establish the disease stage. treatment of myocarditis must be tailored to the phase of disease. many new therapies based on knowledge of the molecular pathophysiology of 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transplantation peripartum cardiomyopathy: a comprehensive review viral myocarditis during pregnancy: encephalomyocarditis virus infection in mice viral myocarditis and cardiomyopathy peripartum heart failure associated with prolonged tocolytic therapy pregnancy-associated cardiomyopathy: clinical characteristics and a comparison between early and late presentation peripartum myocarditis and cardiomyopathy peripartum cardiomyopathy: clinical, hemodynamic, histologic and prognostic characteristics key: cord- - z ykb authors: healing, tim title: surveillance and control of communicable disease in conflicts and disasters date: journal: conflict and catastrophe medicine doi: . / - - - - _ sha: doc_id: cord_uid: z ykb nan tim healing • to describe the principles of health surveillance in conflict and disaster situations • to assist in organizing a health surveillance system in conflict and disaster situations • to describe the principles of control of communicable diseases in conflict and disaster situations • to assist in organizing a response to outbreaks and epidemics • to introduce the challenges associated with health surveillance and communicable diseases in conflict and disaster situations there are five fundamental principles for the control of communicable disease in emergencies: • rapid assessment -identify and quantify the main disease threats to the population and determine the population's health status • prevention -provision of basic health care, shelter, food, water, and sanitation • surveillance -monitor disease trends and detect outbreaks • outbreak control -control outbreaks of disease. involves proper preparedness and rapid response (confirmation, investigation, implementation of controls) • disease management -prompt diagnosis and effective treatment rapid assessment has been dealt with elsewhere in this book as have the prevention aspects of disease control (adequate shelter, clean water, sanitation, and food, together with basic individual health care). this chapter therefore covers surveillance, outbreak/epidemic control, and public health aspects of disease management. the topics are dealt with in general terms. more details can be found in references. disasters, particularly conflicts, by damaging or destroying the infrastructures of societies (health, sanitation, food supply) and by causing displacement of populations, generally lead to increased rates of disease. outbreaks and epidemics are not inevitable in these situations and are relatively rare after rapid-onset natural disasters, but there is a severe increase in the risk of epidemics during and after complex emergencies involving conflict, large-scale population displacement with many persons in camps and food shortages. in most wars more people die from illness than from trauma. preventing such problems, or at least limiting their effects, falls on those responsible for the health care of the population affected by the emergency. they must be able to • assess the health status of the population affected and identify the main health priorities • monitor the development and determine the severity of any health emergency that develops (including monitoring the incidence of and case fatality rates from diseases, receiving early warning of epidemics and monitoring responses) at first sight, undertaking public health activities in emergencies, especially in conflicts, may seem to be difficult or impossible. the destructive nature of warfare may prevent or inhibit the provision of adequate food and shelter, of clean water and sanitation and vaccination programs. despite the difficulties that warfare imposes, it is generally possible to undertake at least limited public health programs, including disease surveillance and control activities. in other types of disaster public health activities may be expected to be less affected by the security situation than in a war (although aid workers may be at risk if populations are severely deprived of resources such as food, shelter, water, or cash), and with limited access and damage to communication systems and other parts of the infrastructure assessment, surveillance and control activities can be severely restricted. for example, following the pakistan earthquake late in access was severely restricted for some time and the urgent need to treat the injured and provide food and shelter meant that the limited transport available was heavily committed. the surveillance and control of communicable disease require data which can be collected in one of three ways: . surveillance systems -covering all or at least a significant proportion of the population . surveys -in which data are collected from a small sample of the affected population considered to be representative of the whole . outbreak investigations -in-depth investigations designed to identify the cause of deaths or diseases and identify control measures although the latter two can provide valuable information for disease control and form part of the surveillance process, proper control of disease requires regular monitoring of the overall disease situation, which in turn requires the establishment of a properly designed health surveillance system. it is important therefore that responsibility for surveillance activities is defined at the beginning of planning for an aid mission. generally speaking, a team will be required, including a team leader (often an aid agency health coordinator), who should ideally have surveillance experience, clinical workers, a water and sanitation specialist, and representatives of the local health services and communities. the team may also need clerical, logistic, information technology and communications specialists. the world health organization defines health surveillance as "the ongoing systematic collection, analysis and interpretation of data in order to plan, implement and evaluate public health interventions." data for surveillance must be accurate, timely, relevant, representative, and easily analyzed, and the results must be disseminated in a timely manner to all who need to receive them. in addition the data collected, the methods used for collection and the output must be acceptable to those surveyed (health-care professionals and the population). in emergencies the time that can be given to surveillance by medical personnel is likely to be limited and surveillance activities will be far from the minds of most of those involved. therefore the methods used need to be rapid, practical, and consistent, and while the greatest possible accuracy must be achieved, "the best must not be the enemy of the good." it is necessary to strike a balance between collecting large amounts of information ("what we would like to know") and collecting too little which can lead to an ineffective response. those responsible for establishing surveillance programs must therefore try to determine what is really needed ("what we need to know"). it is better to err on the side of too much than of too little. ideally any existing surveillance system should be used. there is no point in establishing a system if one already exists, unless the existing one is inadequate or inappropriate or has broken down irretrievably. surveillance systems for use in conflict and disaster situations should therefore adhere as far as possible to the criteria given in table . . notes on these criteria: complexity and inflexibility are incompatible with surveillance systems generally and particularly when operating in emergencies where collection of data may be difficult and where situations can change very fast. defining what you "need to know" will allow you to set up the appropriate data collection methods (questionnaires, sites, etc.) and to design the system so that it can obtain and handle the information required. information that is accurate but out of date is useless for immediate disease control purposes and of little value for forward planning. communications therefore form an integral part of any surveillance system. do not try to overreach when setting up a system. for example, expatriate staff may best be used to recruit local staff for the system and in supervisory activities rather than in collecting data. this criterion is certainly a goal to aim for as sustainability must be the target for all aid work. however, there may be situations where an emergency system is needed rapidly and where it cannot readily be integrated into existing systems or be developed as a new long-term system. . based on standardized sampling methods the sampling system must use the same data collection methods throughout if data are to be comparable. ideally this should be methods that are internationally agreed and approved. agreement should be sought for the methods from the other agencies on the ground to ensure consistency. without case definitions that are agreed by all parties the likelihood of success of a surveillance system is very low. this is especially so when laboratory support is minimal or absent since clinical case definitions have to be drawn very tightly if different diseases are not to be confused. routine surveillance requires more than material from ad hoc sources. sites such as medical centers (in towns, villages, or refugee camps), hospitals, and/or public health units should be recruited. the more comprehensive the coverage of the system, the more likely is it that the data will be accurate and complete and that problems will not be missed. such coverage can be problematic. the coverage of the different systems that can be used is discussed below. the data collected and the methods used should ideally fit in with systems that are operating or have previously operated in the area. following from criterion , if systems are already in existence or in abeyance but revivable then this should be done so as to ensure compliance by local health-care services and continuity of data collection and analysis. existing records are of considerable value for predictive purposes. knowledge of past problems makes it possible to anticipate future trends and problems and allows for early planning decisions. if several health agencies are operating it is essential to ensure collaboration among them in surveillance activities to avoid confusion and duplication of effort. . involve collaboration with local services so as to avoid duplication as above, early involvement of local health and surveillance services will reduce workloads and avoid duplication of effort. if those from whom the data are collected, those who are collecting the data, and those who will receive the results are unhappy with the system, the system is unlikely to operate effectively. these criteria can be used to evaluate a plan for a surveillance system and also, with some additions, to evaluate an existing system. however, failure to fulfil all these criteria need not rule out a system. in many emergencies it can be difficult to meet such a wide range of "best case" criteria, and the question that must be asked is whether the proposed system is capable of fulfilling its purposecan it provide sufficiently accurate essential information to those who need it when they need it? the emphasis of an emergency surveillance program may need to be altered as the situation changes especially if a particular item emerges as being of key importance. those running the surveillance program should use the data gathered and a continuous assessment of the general running of the system, to alter the program as required (preferably after consultation with relevant stakeholders). when designing health surveillance systems, it is essential to do the following: the population under surveillance may be relatively small and well defined (such as the population of a refugee camp) or a much less defined group such as mobile groups of refugees or idps or the population of a village, town, or region, the size of whose population may be unknown or may be fluctuating because of a disaster. establishment of denominators may therefore be difficult. even refugees or idp camps may present a challenge as, while the size of the population may appear to be (or actually be) stable, its makeup may vary over time because of movements in and out. if the age or sex makeup of the camp alters, the pattern of disease may also alter. both the number of cases detected and the rate of factors such as morbidity or mortality per unit of population are important values needed to inform emergency programs. those responsible for all aspects of health care need to know what numbers of cases are involved so as to ensure adequate provision of services (amounts of medicines, numbers of hospital beds, etc.). however, simple numbers are of little value in assessing trends and patterns since increases or decreases in numbers of cases (or numbers of deaths) may reflect changes in population size (resulting, for example, from population displacement) rather than a trend due to (for example) a particular disease. in addition, several rates (such as the crude mortality rate) are key indicators in defining health emergencies (see below). knowing the demography of the affected population is therefore important and all agencies working in an emergency should agree on and use the same population figures. the essential demographic data needed include the following: • total population size • population structure -overall sex ratio and the sex ratio in defined age groups -population under years old, with age breakdown ( - years) -this group has special needs and is usually a key factor in planning the emergency response -age pyramid -ethnic composition and place of origin -number of vulnerable persons (e.g., pregnant and lactating women, members of female-headed households, unaccompanied children, destitute elderly, disabled and wounded persons) at the outset it is therefore important to establish methods to obtain demographic data. often the best that can be managed initially is a rough estimate, but this can usually be refined later. it is helpful to use several methods and cross-check the figures to obtain the best estimate. surrogates of the whole population (such as those attending a clinic) may be the best that can be achieved early on. the ease with which such data can be obtained usually depends on the size and scale of the population under consideration. the demography of a well-run refugee camp is quite easy to obtain but that of a larger area may be much more difficult. a lack of knowledge of the size of a displaced group can be confounded by a lack of knowledge of the size of the resident population. in many countries with poor infrastructures, accurate census data are not available. in some instances tax records may be helpful if these can be obtained. it should be noted that demographic data, especially if they involve refugees and idps, can be politically sensitive and interested parties may place undue weight on any figures that are given. ideally, communicable disease surveillance should be nationwide (or at least "affected area wide"), drawing information from a range of health-care centers that cover a sufficient proportion of the population to ensure that the great majority of cases (preferably all) of the relevant conditions are reported. a surveillance system in a refugee or idp camp is effectively a miniature comprehensive system as it is possible to cover the whole population. there are situations where comprehensive surveillance is not possible and these often arise in disasters. damaged access and communications and staff shortages frequently mean that only limited numbers of reporting sites (sentinel sites) can be used. as far as possible these should be chosen to ensure a wide coverage of the area and also to maximize the proportion of the population that is covered. sentinel surveillance systems are inherently less satisfactory than comprehensive systems largely because they provide a much less complete coverage. the calculation of rates can sometimes be difficult or impossible; such systems can be very labour intensive, and important events may be missed. both types of system may rely on notification of cases based solely on clinical evidence (and this is the most likely situation in conflicts and disasters at least in the early stages), or may include laboratory verification of some or (preferably) all diagnoses. if there is more than one center involved in establishing the diagnosis (for example, a clinical department, a hospital laboratory, and a reference laboratory) the channels of reporting must be very carefully set up so as to avoid duplicate reporting. surveillance must provide information on key health indicators, which should include the following: the selection of information sought in these categories must be done carefully. it is neither possible nor desirable to monitor everything, especially in the early stages of a disaster response. at that stage (the acute phase) the priority of surveillance is the detection of factors that can have the greatest and most rapid effect on the population. in terms of communicable disease this means diseases that affect large numbers of people and have epidemic potential. in most instances this also means diseases for which effective rapid control measures exist. while gathering data on other largescale disease problems should not be excluded, the main surveillance and control efforts should be aimed where they can do the most immediate good. in the very early stages, only clinical information may be available since laboratory diagnostic services will probably be damaged or simply unavailable. however, this need not be a problem if the medical response is also geared to a syndromic approach. as the situation stabilizes, laboratory support becomes available, and longer term control measures can be supported, the surveillance can become more refined and additional diseases (for example, those which can cause severe morbidity and mortality in the longer term -such as tuberculosis, hiv or aids, and stds) can be added to the list. the main morbidity figures that are routinely sought are as follows: • incidence -the number of new cases of a particular disease reported over a defined period • attack rate (used in outbreaks -usually expressed as percentage) (also called incidence proportion or cumulative incidence) -number of new cases within a specified time period/size of the population initially at risk (× ). (e.g., if per , persons develop a condition over weeks, the ar/ip/ci is / , [ . %]) • incidence rate -number of new cases per unit of person-time at risk. in the above example, the ir is / , person-weeks. (this statistic is useful where the amount of observation time differs between people, or when the population at risk varies with time) • prevalence -the total number of cases of a particular disease recorded in a population at a given time (also called "point prevalence") (nb: prevalence "rate" is the number of cases of a disease at a particular time/population at risk) there are a number of ways of estimating morbidity. health information systems based on health center attendance are the most common but are passive and rely on who presents to the services. other ways of gathering morbidity data include the following: • surveys -in which data are collected from a small sample of the emergencyaffected population deemed to be representative of the whole (or from a particular group for a specific purpose) • outbreak investigations -which entail in-depth investigations designed to identify the cause of deaths or diseases and identify control measures as with disease, changes in numbers of deaths may reflect changes in population size. determination of rates is needed because mortality rate is an important surveillance indicator in an emergency. often the first indication that a problem is developing is an increase in death rate, especially in particular vulnerable groups. all deaths occurring in the community must therefore be recorded. the following indicators can provide the essential information to define the health situation in a population: • crude mortality rate (cmr) is the most important indicator as it indicates the severity of the problem, and changes in cmr show how a medical emergency is developing. cmr is usually expressed as number of deaths per , persons per day. if the cmr rises above / , per day (> / , per day for young children) an acute emergency is developing and the emergency phase lasts until the daily cmr falls to / , per day or below. • age-specific mortality rate (number of deaths in individuals of a specific age due to a specific cause/defined number of individuals of that age/day). in children this is usually given as the number of deaths in children younger and older than years/ , children of each age/day). nb: if population data for the under s are not available, an estimate of % of the total population may be used. • maternal mortality rate. maternal mortality is a sensitive indicator of the effectiveness of health-care systems. a maternal death is usually defined as the death of a woman while pregnant or within days of the termination of the pregnancy (for whatever cause) from any cause related to or aggravated by the pregnancy or its management. the -day cut-off is recommended by who but some authorities use a time of up to a year. maternal mortality rate = (number of deaths from puerperal causes in a specified area in a year/number of live births in the area during the same year) × , (or × , ) • cause-specific death rates (case fatality rates -usually given as a percentage). proportion of cases of a specified condition which are fatal within a specified time. case fatality rate = (no. of deaths from given disease in a given period/no. of diagnosed cases of that disease in the same period) × the following indicators must be measured: • prevalence of global acute malnutrition (includes moderate and severe malnutrition) in children - months of age (or - cm in height) (percentage of children with weight for height under two standard deviations below the median value in a reference population and/or edema) • prevalence of severe acute malnutrition in children - months of age (or - cm in height) (percentage of children with weight for height under three standard deviations below the median value in a reference population and/or edema) • • estimate number of children needing to be cared for in selective feeding programs • estimate number of additional calories per day provided by selective feeding programs immunization programs are a vital part of the public health measures undertaken following disasters. for example, measles vaccination is one of the most important health activities in such situations. the need for campaigns may be assessed on the basis of national vaccination records if they exist. in the absence of such records questioning of mothers may provide the information required, or children or their parents may have written vaccination histories with them (rare). the effectiveness of the programs undertaken can be assessed in defined populations by recording the percentage of children vaccinated. in less well defined populations an assessment of coverage may be made using the numbers of children attending clinics as a surrogate for the population as a whole. items such as water, sanitation, food, and shelter are essential to maintain a healthy population and prevent communicable diseases. depending on the circumstances it may be necessary to monitor these elements in the affected population. indicators such as number of consultations per day, number of vaccinations, number of admissions to hospitals, number of children in feeding programs are typically reported. other factors such as effectiveness of the supply chain, maintenance of the cold chain, and laboratory activities may also be surveyed. activities in related sectors such as water and sanitation, shelter and security may also be included. the major sources of health data will be hospitals and clinics (both national and those established by aid agencies), individual medical practitioners, and other health-care workers. specialized agencies should be able to provide data on particular needs (e.g., food, water, sanitation, and shelter). case definitions are an essential part of surveillance. if the diseases (or syndromes) that are to be covered by the system are not clearly defined, and if the definitions are not adhered to, the results become meaningless -changes from week to week are as likely to be due to changes of definition as to real changes in numbers of cases. this is especially important when laboratory confirmation is not possible. it is therefore important that all agencies working in an emergency agree to and use the same case definitions so that there is consistency in reporting. case definitions must be prepared for each health event or disease or syndrome. if available, the case definitions used by the host country's moh should be used to ensure continuity of data. several different sets of case definitions already exist, either in generalized form (for example, those produced by the centers for disease control in atlanta) or sets prepared for specific emergencies (e.g., the who communicable disease toolkit for the iraq crisis in ). standard case definitions may have to be adapted according to the local situation. it should be noted that such case definitions are designed for the purposes of surveillance, not for use in the management of patients, nor are they an indication of intention to treat the patients. when case definitions based purely on clinical observations are used, each case can only be reported as suspected, not confirmed (see table . ). although lacking precision, such definitions can make it possible to establish the occurrence of an outbreak. samples can subsequently be sent to a referral laboratory for confirmation. once samples have been examined and the causative organism has been identified, a more specific case definition can be developed to detect further cases. visits to surveillance sites and discussions with staff involved will help define the recording and data transmission systems required. the great advances in information technology that have been made in recent years have greatly facilitated the collection, recording, transmission, and analysis of surveillance data, but care must be taken that the systems put in place are appropriate. in areas where electricity supplies are problematical and communications poor it may be better to use a paper recording system and verbal data transmission by radio than a computerized system. data verification is essential for the credibility of a surveillance system. those responsible for surveillance systems must ensure good adherence to case definitions if a symptom-based system is in operation and that laboratory quality control systems operate where appropriate. regular assessments of record keeping and the accuracy of data transfer are required. triangulation of results from several sources can sometimes help to detect anomalies. frequency of reporting will usually depend on the severity of the health situation. in general, daily reporting during the acute phase of an emergency will be needed, although in an acute medical emergency (such as a severe cholera outbreak) even more frequent reporting may be necessary, especially if the situation is fluctuating rapidly. the frequency may reduce to (say) weekly as the situation resolves. who is to analyze the data and how it is to be analyzed must be established at the outset. in a relatively defined area such as a camp, a data analysis session may be the last of the daily activities of the person responsible for surveillance. if record keeping and analysis protocols have been carefully worked out initially this task is not necessarily a large additional burden. surveillance systems that cover larger areas and bigger and more diffuse populations usually rely on a central data collection point where designated staff analyze the data. use of such a system requires good data transmission systems. output is as important as input. collecting data without dissemination of results is a sterile exercise and tends rapidly to demotivate those who are collecting the data. there are some important points to consider: • the results of surveillance must be presented in a readily comprehensible form. • surveillance reports should be produced regularly and widely distributed to aid agencies, and to national and international governments and organizations. this will help those involved to understand the overall picture, rather than just that in the area where they are working, and will allow them to take informed decisions about future actions. surveillance systems should be evaluated constantly to ensure that they are working properly, that the data are representative, analysis is appropriate and accurate, and that results are being disseminated to where they are needed. the public health aspects of communicable disease control can be broadly divided into preventive activities (such as vector control and vaccination programs) and the investigation and control of outbreaks and epidemics. experience from many emergencies and disasters has made it possible to identify a number of syndromes or diseases that are most likely to occur in such situations (table . ). this makes it possible to plan activities and interventions on the basis of likely occurrences, even before those involved are present at the scene of the disaster, and to make initial purchases and establish stockpiles of appropriate medicines and equipment. "prevention is better than cure" and proper attention to preventive measures from the earliest stage of the response to the disaster will greatly reduce the risks to the health of the population from infectious disease. a key method of preventing communicable disease is the provision of shelter, adequate amounts of clean water, sufficient safe food, and proper sanitation (latrines and facilities for personal hygiene, clothes washing, and drying). arthropod vectors (mosquitoes, ticks) can be controlled by appropriate spraying programs and also by habitat management (e.g., the removal of places where water can accumulate and mosquitoes breed). provision of bed nets, particularly nets impregnated with insecticide, is effective for reducing infection with agents such as malaria and leishmania. control of rodents, by proper control of rubbish, by rodent proofing food stores, by attention to domestic hygiene and by use of rodenticides, will reduce the risks of transmission of rodent-borne diseases such as plague and lassa fever. medical waste includes laboratory samples, needles and syringes, body tissues, and materials stained with body fluids. this requires careful handling, especially the sharps, as infectious agents such as those causing hepatitis b and c, hiv and aids, and viral hemorrhagic fevers can be transmitted by these materials. used sharps should be disposed of into suitable containers (proper sharps boxes are ideal but old metal containers such as coffee or milk powder tins are adequate). medical waste should ideally be burned in an incinerator. this should be close to the clinic or hospital but downwind of the prevailing wind. a -l oil drum can be used for this purpose with a metal grate half way up and a hole at the bottom to allow in air and for the removal of ash. larger-scale and more permanent incinerators can be constructed if necessary. burning pits can be used in emergency. if burning is not possible items should be buried at least . m deep. this is more suitable than burning for large items of human tissue such as amputated legs. ensure there is no risk of groundwater contamination. a few others, such as malaria and other vector-borne diseases (e.g., typhus and leishmaniasis), are also likely to occur but are region specific. tb and hiv or aids can also cause major problems in the longer term this is a complex process involving not just considerations of infection risk but also legal, sociocultural, and psychological factors. there are a number of specialist publications which can be of help. after almost every natural disaster, fear of disease has encouraged authorities to dispose rapidly of the bodies of the dead, often without identifying them, and this sometimes seems almost to take precedence over dealing with the living. however, in sudden impact disasters (such as the indian ocean tsunami in ), the pattern and incidence of disease found in the dead will generally reflect those in the living. the situation is much the same in wars and other long drawn out disasters, although these may affect disease patterns and create vulnerable groups. in fact dead bodies pose little risk to health (with some exceptions listed below) since few pathogenic microorganisms survive long after the death of their host. the diseased living are far more dangerous. the decay of cadavers is due mainly to organisms they already contain and these are not pathogenic. those most at risk are those handling the deceased, not the community. the most likely risks to them are as follows: mortuary facilities may need to be provided where the dead can be preserved until appropriate legal proceedings have been undertaken and where relatives, etc., may easily attend to identify and claim the deceased. cold stores and refrigerated vehicles can be used as temporary mass mortuary facilities. alternatively such facilities can be provided in buildings, huts, or tented structures, but refrigeration will be needed. the dead must always be treated with dignity and respect. as far as possible the appropriate customs of the local population or the group to which the deceased belonged should be observed. if the dead have to be buried in mass graves then the layout of the cemetery must be carefully mapped to facilitate exhumation if needed. when an individual may have died of a particularly dangerous infection, then body bags should be used (and also for damaged cadavers). in general, bodies should be buried rather than cremated (as exhumation for purposes of identification may be needed). bodies should be buried at least . m deep or, if more shallowly, should have earth piled at least m above the ground level and . m to each side of the grave (to prevent access by scavengers and burrowing insects). disinfectants such as chloride of lime should not be used. new burial sites should be at least m from drinking water sources and at least . m above the saturated zone. vaccination programs are an essential part of disease prevention. information about existing vaccination programs must be obtained during the assessment process and this should include information from external assessors (e.g., who, unicef, ngos) as to the effectiveness of the vaccination programs that have been undertaken in the past. it cannot be assumed that simply because children have received vaccines that these vaccines were effective. measles kills large number of children in developing countries and is one of the greatest causes of morbidity and mortality in children in refugee and idp camps. mass vaccination of children between the ages of months and years should be an absolute priority during the first week of activity in humanitarian situations and can be conducted with the distribution of vitamin a. a system for maintaining measles immunization must be established once the target population has been covered adequately in the initial campaign. this is necessary to ensure that children who may have been missed in the original campaign, children reaching the age of months, and children first vaccinated at the age of - months who must receive a second dose at months of age are all covered. some of the children vaccinated during such a mass campaign may have been vaccinated before. this does not matter and a second dose will have no adverse effect. it is essential to ensure full coverage against measles in the population. other epi vaccinations for children are not generally included in the emergency phase because they can only prevent a minor proportion of the overall morbidity and mortality at that stage. however, should specific outbreaks occur then the appropriate vaccine should be considered as a control measure. vaccination programs require the following: • appropriate types of vaccines. • appropriate amounts of these vaccines. • equipment (needles, syringes, sterilization equipment, sharps disposal). emergency immunization kits, including cold chain equipment, are available from a number of sources, including unicef and some ngos (e.g. msf). • logistics (transport, cold chain). • staff: a vaccination team may be quite large. it must include the following personnel: -a supervisor. -logistics staff. -staff to prepare and administer vaccines. -record keepers. -security staff (to maintain order and control crowds) may also be needed. maintenance of the cold chain is particularly important. this is the system of transporting and storing vaccines within a suitable temperature range from the point of manufacture to the point of administration. the effectiveness of vaccines can be reduced or lost if they are allowed to get too cold, too hot, or are exposed to direct sunlight or fluorescent light. careful note should be taken of the conditions needed to transport different vaccines because these can vary. the essential cold chain equipment needed to transport and store vaccines within a consistent safe temperature range includes the following: • dedicated refrigerators for storing vaccines and freezers for ice packs (fridges and freezers powered by gas or kerosene are available as alternatives to electric machines, and solar-powered fridge/freezer combinations specially designed for vaccine storage are also available) • a suitable thermometer and a chart for recording daily temperature readings if possible, vaccines should be stored in their original packaging because removing the packaging exposes them to room temperature and light. check the temperature to ensure the vaccines have not been exposed to temperatures outside the normal storage ranges for those vaccines (see table . ). max. storage time at the different levels: primary, months; region, months; district, month; health center, month; health post, daily usemax. month diluents must never be frozen. freeze-dried vaccines supplied packed with diluent must be stored between + and + °c. diluents supplied separately should be kept between + and + °c vaccines must be kept at the correct temperature since all are sensitive to heat and cold to some extent. all freeze-dried vaccines become much more heat-sensitive after they have been reconstituted. vaccines sensitive to cold will lose potency if exposed to temperatures lower than optimal for their storage, particularly if they are frozen. some vaccines (bcg, measles, mr, mmr, and rubella vaccines) are also sensitive to strong light and must always be protected against sunlight or fluorescent (neon) light. these vaccines are usually supplied in dark brown glass vials, which give them some protection against light damage, but they must still be covered and protected from strong light at all times. only vaccine stocks that are fit for use should be kept in the vaccine cold chain. expired or heat-damaged vials should be removed from cold storage. if unusable vaccines need to be kept for a period before disposal (e.g., until completion of accounting or auditing procedures) they should be kept outside the cold chain, separated from all usable stocks and carefully labelled to avoid mistaken use. diluents for vaccines are less sensitive to storage temperatures than are the vaccines with which they are used (although they must be kept cool), but may be kept in the cold chain between + and + °c if space permits. however, diluent vials must never be frozen (kept in a freezer or in contact with any frozen surface) as the vial may crack and become contaminated. when vaccines are reconstituted, the diluent should be at same temperature as the vaccine, so sufficient diluent for daily needs should be kept in the cold chain at the point of vaccine use (health center or vaccination post). at other levels of the cold chain (central, provincial, or district stores) it is only necessary to keep any diluent in the cold chain if it is planned to use it within the next h. freeze-dried vaccines and their diluents should always be distributed together in matching quantities. although the diluents do not need to be kept in the cold chain (unless needed for reconstituting vaccines within the next h), they must travel with the vaccine at all times, and must always be of the correct type, and from the same manufacturer as the vaccine that they are accompanying. each vaccine requires a specific diluent, and therefore, diluents are not interchangeable (for example, diluent made for measles vaccine must not be used for reconstituting bcg, yellow fever, or any other type of vaccine). likewise, diluent made by one manufacturer for use with a certain vaccine cannot be used for reconstituting the same type of vaccine produced by another manufacturer. some combination vaccines comprise a freeze-dried component (such as hib) which is designed to be reconstituted by a liquid vaccine (such as dtp or dtp-hepb liquid vaccine) instead of a normal diluent. for such combination vaccines, it is again vital that only vaccines manufactured and licensed for this purpose are combined. note also that for combination vaccines where the diluent is itself a vaccine, all components must now be kept in the cold chain between + and + °c at all times. as for all other freeze-dried vaccines, it is also essential that the "diluent" travels with the vaccine at all times. the effectiveness of a vaccination program will need to be assessed. the program can be evaluated both by routinely collected data and, if necessary, by a survey of vaccination coverage. routine data on coverage is obtained by comparing the numbers vaccinated with the estimated size of the target population (and clearly depends on accurate assessment of the latter). a coverage survey requires the use of a statistical technique called a two-stage cluster survey details of which can be found in the appropriate who/epi documents. information about the effectiveness of the campaign should be obtained from routine surveillance of communicable disease. if, for example, large number of measles cases continue to occur, or there is an outbreak, then data on coverage should be reexamined. if this is shown to be good (over %) then the efficacy of the vaccine must be suspected. if the field efficacy is below the theoretical value % (for measles vaccine -data on efficacy of other vaccines can be obtained online) then possible causes of a breakdown in the vaccination program must be investigated (failure of the cold chain, poorly respected vaccination schedule). methods for measuring vaccine efficacy can be found in the who/epi literature. mass chemoprophylaxis for bacterial infections such as cholera and meningitis is not usually recommended except on a small scale (for example, the use of rifampicin may be considered to prevent the spread of meningococcal meningitis among immediate contacts of a case), but the difficulties of overseeing such activities and the risks of the development of antibiotic resistance outweigh any benefits that might be gained. the use of chemoprophylaxis for malaria must be undertaken with care. it may be indicated for vulnerable groups of refugees/idps (for example, children and pregnant women) arriving in an endemic area, particularly if they come from a nonmalarious area, but care must be taken to provide drugs to which the local strains of malaria are sensitive. the spread of resistance means that many of the standard drugs are ineffective and the replacements are both costly and may have unwanted side effects. public health education and information activities play a vital role in disease prevention. vaccination programs will not work unless there is acceptance by the public of the necessity for such programs. individuals must be informed as to why these programs are necessary and also where and when they need to take their children for vaccination. such activities are also essential to inform people about particular health programs (for example, feeding programs or vector control programs) and about the steps they can take to protect their health and that of their families (e.g., good hygiene). information can be propagated in many ways: staff who are trained in this type of activity therefore play a key role in disease prevention. heath education also requires transport and equipment (such as video or film projectors, screens, generators, blackboards, etc.). details of the treatment of individuals for various infectious diseases and the facilities needed are covered elsewhere in this book and in many textbooks covering disasters and disease response. in terms of the population aspects of the treatment of disease, important requirements are to ensure that there are • appropriate laboratories (microbiological, parasitological, hematological, biochemical) available to confirm diagnoses and monitor treatment. • adequate supplies of appropriate antimicrobial agents available and the facilities to transport these, store, and distribute them under appropriate conditions (e.g., controlled temperature), together with relevant instruction for use. the provision of laboratory facilities in emergencies is usually limited to basic tests such as those for malaria. more advanced tests, including identification of microorganisms and the determination of antimicrobial sensitivities, require more sophisticated facilities. these may be available in the affected country but are unlikely to be operating in the disaster-affected area. it is more likely that specimens will have to be transported to laboratories abroad. collection of specimens requires appropriate equipment. this will include items such as swabs, transport media, needles, syringes, or vacum sampling systems for blood sampling, different blood collection bottles (with and without anticoagulants) and other sterile specimen tubes, and containers for faeces and urine. transporting specimens must be done safely, and packing specimens for shipment requiring specially trained personnel. treatment of disease requires good supplies of appropriate antimicrobial agents. it is important to ensure that the agents chosen are suitable for use in the area. it is common for doctors in affected areas to ask for the latest therapeutic agents. however, these agents, although effective, are often expensive and not part of the normal treatment programs in the region. the local doctors may not therefore be familiar with the use of these agents, nor may laboratories be capable of monitoring their use. it is better to use funds, which are often limited, to supply larger amounts of older (generic) agents. one caveat is the possibility that regular use may have allowed resistance to certain agents to develop in a country. data on this may be available from local surveillance records. antimicrobials should always be supplied with relevant guidelines in a language that can be understood locally. if local laboratories are unable to test microbes for resistance to antimicrobials, isolates or specimens should be sent as soon as possible to appropriate reference laboratories for testing. outbreaks of communicable disease may occur before preventive measures can take effect or because the measures are in some way inadequate or fail. an epidemic is generally defined as the occurrence in a population or region of a number of cases of a given disease in excess of normal expectancy. an outbreak is an epidemic limited to a small area (a town, village, or camp). the term alert threshold is used to define the point at which the possibility of an epidemic or outbreak needs to be considered and preparedness checked. the areas where vaccination campaigns are a priority need to be identified and campaigns started. the term epidemic (outbreak) threshold is used to define the point at which an urgent response is required. this will vary depending upon the disease involved (infectiousness, local endemicity, transmission mechanisms) and can be as low as a single case. infections where a single case represents a potential outbreak include the following: infections where the threshold is set higher, usually based on long-term collection of data, and will vary from location to location, include the following: • human african trypanosomiasis • visceral leishmaniasis a surveillance system that is functioning well should pick up the signs that an outbreak or epidemic is developing and should therefore allow time for measures to be introduced that will prevent or limit the scale of the event. however, this may not always work and it is essential therefore that plans are made to combat outbreaks or epidemics. in addition to the establishment of surveillance, outbreak preparation involves the following: • preparing an epidemic/outbreak response plan for different diseases covering the resources needed, the types of staff and their skills that may be needed and defining specific control measures. • ensuring that standard treatment protocols are available to all health facilities and health workers and that staff are properly trained. • stockpiling essential supplies. this includes supplies for treatment, for taking and shipping samples, other items to restock existing health facilities and the means to provide emergency health facilities if required. • identifying appropriate laboratories to confirm cases and support patient management, make arrangements for these laboratories to accept and test specimens in an emergency, and set up a system to ship specimens to the laboratory. • identifying emergency sources of vaccines for vaccine-preventable diseases and make arrangements for emergency purchase and shipment. ensure that vaccination supplies (needles, syringes, etc.) are adequate. make sure the cold chain can be maintained. • identifying sources for other supplies, including antimicrobials, and make arrangements for emergency purchase and shipment. if the number of reported cases is rising, is this in excess of the expected number? ideally work with rates rather than numbers (see above) because (for example) the number of cases in a refugee camp could increase if the number of people in the camp increases without an outbreak occurring. verify the diagnosis (laboratory confirmation) and search for links between cases (time and place). laboratory confirmation requires the collection of appropriate specimens and their transport to an appropriate laboratory. in the case of a limited outbreak this team should be set up by the lead agency with membership from other relevant organizations, including moh, who, other un organizations, ngos, etc. in the case of an epidemic the moh will probably take the lead or may ask who or another un agency to do so. the team will need to include a coordinator, and specialists from the various disciplines needed to control the outbreak. this may include health workers, laboratory staff, water and sanitation, vector control, and health education specialists, representatives of the moh or other local health authorities, representatives of local utilities (e.g., water supply), representatives of the police and/or military, and representatives of the local community. this team should meet at least once a day to review the situation and define the necessary responses. it has additional responsibilities, including implementing the response plan, overseeing the daily activities of the responders, ensuring that treatment protocols are followed, identifying resources (both material and human) to manage the outbreak and obtaining these as necessary, and coordinating with local, national, and international authorities as required. the team should also act as the point of contact for the media. a media liaison officer should be appointed and all media contact should be through this individual. this will allow team members to refer media representatives to a central point and reduce interference with their activities. it will also ensure that a consistent message based on the most complete data is given to the media. the appropriate national authorities should be informed of the outbreak. in addition to their responsibilities to their own population and to any refugees within their borders, they have a responsibility under the revised international health regulations ( ) to report outbreaks of certain diseases. these include four diseases regarded as public-health emergencies of international concern: • smallpox • polio (wild-type) in some cases, member states must report outbreaks of additional diseases: cholera, pneumonic plague, yellow fever, viral hemorrhagic fever, and west nile fever, and other diseases that are of special national or regional concern (e.g., dengue fever, rift valley fever, and meningococcal disease). once the diagnosis has been confirmed and the causative organism identified, then there are a number of steps that must be taken in addition to continuing to treat those affected: • produce a case definition for the outbreak. this is primarily a surveillance tool that will reduce the inclusion of cases that are not part of the outbreak and prevent dilution of the focus and activities of the main control effort. • collect and analyze descriptive data by time, person, and place (time and date of onset, individual characteristics of those affected -age, sex, occupation, etc., location of cases). plot the distribution of the cases on a map (can help locate source(s) of an outbreak and determine spread) and plot outbreak curves (which will help estimates of how the outbreak is evolving). • determine the population that is at risk. • determine the number of cases and the size of the affected population. calculate the attack rate. • formulate hypotheses for the pathogen about the possible source and routes of transmission. • conduct detailed epidemiological investigations to identify modes of transmission, vectors/carriers, risk factors). • report results and make recommendations for action. the two main statistical tools used to investigate outbreaks are as follows: • case-control studies in which the frequency of an attribute of the disease in individuals with the disease is compared to the same attribute in individuals without the disease matched in terms of age, sex, and location (the control group) • cohort studies in which the frequency of attributes of a disease is compared in members of a group (for example, those using a particular feeding center) who do or do not show symptoms however the design and methods involved in such studies are often too complex for the austere environment of conflict and disaster. • implement prevention and control measures specific to the disease organism (e.g., clean water, personal hygiene for diarrheal disease) • prevent infection (e.g., by vaccination programs) • prevent exposure (e.g., isolate cases or at the least provide a special treatment ward or wards) • evaluate the outbreak detection and response -were they appropriate, timely, and effective? • change/modify policies and preparedness to deal with outbreaks if required • what activities are needed to prevent similar outbreaks in the future (e.g., improved vaccination programs, new water treatment facilities, public health education, etc.)? • produce and disseminate an outbreak report. the report should include details of the outbreak, including the following: -cause -duration, location, and persons involved -cumulative attack rate (number of cases/exposed population) -incidence rate -case fatality rate -vaccine efficacy (if relevant) (no. of unvaccinated ill − no. of vaccinated ill/no. of unvaccinated ill) -proportion of vaccine-preventable cases (no. of vaccine-preventable cases/no. of cases) -recommendations this is an easy-to-use tool which is of great value for handling epidemiological data and for organizing study designs and results, which can be downloaded free of charge from the internet. it is produced by the centers for disease control (atlanta) and is a series of microcomputer programs which can be used both for surveillance and for outbreak investigation and includes features used by epidemiologists in statistical programs, such as sas or spss, and database programs such as dbase. public health action in emergencies caused by epidemics. geneva: who, . cdc atlanta. case definitions for infectious conditions under public health surveillance updated guidelines for evaluating public health surveillance systems epidemiology for the uninitiated communicable disease control in emergencies -a field manual last jm (ed). dictionary of epidemiology medicins sans frontieres. refugee health -an approach to emergency situations geneva: international committee of the red cross sphere project. humanitarian charter and minimum standards in disaster response. geneva: the sphere project key: cord- -s knxdne authors: perra, nicola; gonçalves, bruno title: modeling and predicting human infectious diseases date: - - journal: social phenomena doi: . / - - - - _ sha: doc_id: cord_uid: s knxdne the spreading of infectious diseases has dramatically shaped our history and society. the quest to understand and prevent their spreading dates more than two centuries. over the years, advances in medicine, biology, mathematics, physics, network science, computer science, and technology in general contributed to the development of modern epidemiology. in this chapter, we present a summary of different mathematical and computational approaches aimed at describing, modeling, and forecasting the diffusion of viruses. we start from the basic concepts and models in an unstructured population and gradually increase the realism by adding the effects of realistic contact structures within a population as well as the effects of human mobility coupling different subpopulations. building on these concepts we present two realistic data-driven epidemiological models able to forecast the spreading of infectious diseases at different geographical granularities. we conclude by introducing some recent developments in diseases modeling rooted in the big-data revolution. historically, the first quantitative attempt to understand and prevent infectious diseases dates back to when bernoulli studied the effectiveness of inoculation against smallpox [ ] . since then, and despite some initial lulls [ ] , an intense research activity has developed a rigorous formulation of pathogens' spreading. in this chapter, we present different approaches to model and predict the spreading of infectious diseases at different geographical resolutions and levels of detail. we focus on airborne illnesses transmitted from human to human. we are the carriers of such diseases. our contacts and mobility are the crucial ingredients to understand and model their spreading. interestingly, the access to large-scale data describing these human dynamics is a recent development in epidemiology. indeed, for many years only the biological roots of transmission were clearly understood, so it is not surprising that classical models in epidemiology neglect realistic human contact structures or mobility in favor of more mathematically tractable and simplified descriptions of unstructured populations. we start our chapter with these modeling approaches that offer us an intuitive way of introducing the basic quantities and concepts in epidemiology. advances in technology are resulting in increased data on human dynamics and behavior. consequently, modeling approaches in epidemiology are gradually becoming more detailed and starting to include realistic contact and mobility patterns. in sects. . and . we describe such developments and analyze the effects of heterogeneities in contact structures between individuals and between cities/subpopulations. with these ingredients in hand we then introduce state-of-the-art data-driven epidemiological models as examples of the modern capabilities in disease modeling and predictions. in particular, we consider gleam [ , ] , episims [ ] , and flute [ ] . the first model is based on the metapopulation framework, a paradigm where the inter-population dynamics is modeled using detailed mobility patterns, while the intra-population dynamics is described by coarse-grained techniques. the other tools are, instead, agent-based model (abm). this class of tools guarantees a very precise description of the unfolding of diseases, but need to be fed with extremely detailed data and are not computationally scalable. for these reasons their use so far has been limited to the study of disease spread within a limited numbers of countries. in comparison, metapopulation models include a reduced amount of data, while the approximated description of internal dynamics allows scaling the simulations to global scenarios. interestingly, the access to large-scale data on human activities has also started a new era in epidemiology. indeed, the big-data revolution naturally results in real time data on the health related behavior of individuals across the globe. such information can be obtained with tools that either require the active participation of individuals willing to share their health status or that is mined silently from individuals' health related data. epidemiology is becoming digital [ , ] . in sect. . we introduce the basic concepts, approaches, and results in this new field of epidemiology. in particular, we describe tools that, using search queries, microblogging, or other web-based data, are able to predict the incidence of a wide range of diseases two weeks ahead respect to traditional surveillance. epidemic models divide the progression of the disease into several states or compartments, with individuals transitioning compartments depending on their health status. the natural history of the disease is represented by the type of compartments and the transitions from one to another, and naturally varies from disease to disease. in some illnesses, susceptible individuals (s) become infected and infectious when coming in contact with one or more infectious (i) persons and remain so until their death. in this case the disease is described by the so-called si (susceptible-infected) model. in other diseases, as is the case for some sexual transmitted diseases, infected individuals recover becoming again susceptible to the disease. these diseases are described by the sis (susceptible-infected-susceptible) model. in the case of influenza like illnesses (ili), on the other hand, infected individuals recover becoming immune to future infections from the same pathogen. ilis are described by the sir (susceptible-infected-recovered) model. these basic compartments provide us with the fundamental description of the progression of an idealized infection in several general circumstances. further compartments can be added to accurately describe more realistic illnesses such as smallpox, chlamydia, meningitis, and ebola [ , , ] . keeping this important observation in mind, here we focus on the sir model. epidemic models are often represented using chart such as the one seen in fig. . . such illustrations are able to accurately represent the number of compartments and the disease's behavior in a concise and easily interpretable form. mathematically, models can also be accurately represented as reaction equations as we will see below. in general, epidemic models include two type of transitions, "interactive" and "spontaneous." interactive transitions require the contact between individuals in two different compartments, while spontaneous transitions occur naturally at a fixed rate per unit time. for example, in the transition between s to i, susceptible individuals become infected due to the interaction with infected individuals, i.e. sci ! i. the transition is mediated by individuals in the compartment i, see fig. but how can we model the infection process? intuitively we expect that the probability of single individual becoming infected must depend on ( ) the number of infected individuals in the population, ( ) the probability of infection given a contact with an infectious agent and, ( ) the number of such contacts. in this section we neglect the details of who is in contact with whom and consider instead individuals to be part of a homogeneously mixed population where everyone is assumed to be in contact with everyone else (we tackle heterogeneous contacts in sect. . ). in this limit, the per capita rate at which susceptible contract the disease, the force of infection , can be expressed in two forms depending on the type of population. in the first, often called mass-action law, the number of contacts per individual is independent of the total population size, and determined by the transmission rateǎ nd the probability of randomly contacting an infected individual, i.e. dˇi=n (where n is the population size). in the second case, often called pseudo massaction law, the number of contacts is assumed to scale with the population size, and the transmission rateˇ, i.e. dˇi. without loss of generality, in the following we focus on the first kind of contact. the sir framework is the crucial pillar to model ilis. think, for example, at the h n pandemic in , or the seasonal flu that every year spread across the globe. the progression of such diseases, from the first encounter to the recovery, happens in matters of days. for this reason, birth and death rates in the populations can be generally neglected, i.e. d t n Á for all times t. let us define the fraction of individuals in the susceptible, infected, and recovered compartments as s; i, and r. the sir model is then described by the following set of differential equations: where dˇi Áˇi n is the force of infection, and d t Á d dt . the first equation describes the infection process in a homogeneous mixed population. susceptible individuals become infected through random encounters with infected individuals. the second equation describes the balance between the in-flow (infection process, first term), and the out-flow (recovery process, second term) in compartment i. finally, the third equation accounts for the increase of the recovered population due to the recovery process. interestingly, the sir dynamical equations, although apparently very simple, due to their intrinsic non-linearity cannot be solved analytically. the description of the evolution of the disease can be obtained only through numerical integration of the system of differential equations. however, crucial analytic insight on the process can be obtained for early t t and late times t ! . under which conditions a disease starting from a small number, i , of individuals at time t is able to spread in the population? to answer this question let us consider the early stages of the spreading, i.e. t t . the equation for the infected compartment can be written as d t i d i.ˇs /, indicating an exponential behavior for early times. it then follows that if the initial fraction of susceptible individuals, s d s =n, is smaller than =ˇ, the exponent becomes negative and the disease dies out. we call this value the epidemic threshold [ ] of the sir model. the fraction of susceptibles in the population has to be larger than a certain value, that depends on the disease details, in order to observe an outbreak. typically, the initial cluster of infected individuals is small in comparison with the population size, i.e. s i , or s . in this case, the threshold condition can be re-written asˇ= > . the quantity: is called the basic reproductive number, and is a crucial quantity in epidemiology and provides a very simple interpretation of the epidemic threshold. indeed, the disease is able to spread if and only if each infected individual is able to infect, on average, more than one person before recovering. the meaning of r is then clear: it is simply the average number of infections generated by an initial infectious seed in a fully susceptible population [ ] . for any value of > , the sir dynamics will eventually reach a stationary, disease-free, state characterized by i d d t i d . indeed, infected individuals will keep recovering until they all reach the r compartment. what is the final number of recovered individuals? answering this apparently simple question is crucial to quantify the impact of the disease. we can tackle such conundrum dividing the first equation with the third equation in the system . . we obtain d r s d r s which in turn implies s t d s e r r t . unfortunately, this transcendent equation cannot be solved analytically. however, we can use it to gain some important insights on the sir dynamics. we note that for any r > , in the limit t ! , we must have s > . in other words, despite r , the disease-free equilibrium of an sir model is always characterized by some finite fraction of the population in the susceptible compartment, or, in other words, some individuals will always be able to avoid the infection. in the limit where r we can obtain an approximate solution for r (or equivalently for s d r ) by expanding s d s e r s at the second order around r . after a few basic algebraic manipulations we obtain in the previous sections we presented the basic concepts and models in epidemiology by considering a simple view of a population where individuals mix homogeneously. although such approximation allows a simple mathematical formulation, it is far from reality. individuals do not all have the same number of contacts, and more importantly, encounters are not completely random [ ] [ ] [ ] [ ] . some persons are more prone to social interactions than others, and contacts with family members, friends, and co-workers are much more likely than interactions with any other person in the population. over the last decade the network framework has been particularly effective in capturing the complex features and the heterogeneous nature of our contacts [ ] [ ] [ ] [ ] [ ] . in this approach, individuals are represented by nodes while links represent their interactions. as described in different chapters of the book (see chaps. , , and ), human contacts are not heterogeneous in both number and intensity [ ] [ ] [ ] [ ] ] but also change over time [ ] . this framework naturally introduces two timescales, the timescale at which the network connections evolve, g and the inherent timescale, p , of the process taking place over the network. although the dynamical nature of interactions might have crucial consequences on the disease spreading [ ] [ ] [ ] [ ] [ ] [ ] , the large majority of results in the literature deal with one of two limiting regimens [ , ] . when g p , the evolution of the network of contacts is much slower than the spreading of the disease and the network can be considered as static. on the other hand, when p g , the links are said to be annealed and changes in networks structure are much faster than the spreading of the pathogen. in both cases the two time-scales are well separated allowing for a simpler mathematical description. here we focus on the annealed approximation ( p g ) that provides a simple stage to model and understand the dynamical properties of epidemic processes. we refer the reader to chap. face-to-face interactions for recent approaches that relax this time-scale separation assumption. let us consider a network g .n; e/ characterized by n nodes connected by e edges. the number of contacts of each node is described by the degree k. the degree distribution p .k/ characterizes the probability of finding a node of degree k. empirical observations in many different domains show heavy-tailed degree distributions usually approximated as power-laws, i.e. p .k/ k ˛ [ , ] . furthermore, human contact networks are characterized by so-called assortative mixing, meaning a positive correlation between the degree of connected individuals. correlations are encoded in the conditional probability p .k jk/ that a node of degree k is connected with a node of degree k [ , ] . while including realistic correlations in epidemic models is crucial [ ] [ ] [ ] they introduce a wide set of mathematical challenges that are behind the scope of this chapter. in the following, we consider the simple case of uncorrelated networks in which the interdependence among degree classes is removed. how can we extend the sir model to include heterogeneous contact structures? here we must take a step further than simply treating all individuals the same. we start distinguishing nodes by degree while considering all vertices with the same degree as statistically equivalent. this is known as the degree block approximation and is exact for annealed networks. the quantities under study are now i k d i k n k ; s k d s k n k , and r k d r k n k , where the i k ; s k , and r k are the number of infected, susceptible, recovered individuals in the degree class k. n k instead describes the total number of nodes in the degree class k. the global averages are given by i d using this notation and heterogeneous mean field (hmf) theory [ ] , the system of differential equations ( . ) can now be written as: the contact structure introduces a force of infection function of the degree. in particular, k d k k where is the rate of infection per contact, i.e.ˇd k, and k describes the density of infected neighbors of nodes in the degree class k. intuitively, this density is a function of the conditional probability that a node k is connected to any node k and proportional to the number of infected nodes in each class in the simple case of uncorrelated networks the probability of finding a node of degree k in the neighborhood of a node in degree class k is independent of k. in this case k d d p k .k / p .k / i k =hki where the term k is due to the fact that at least one link of each infected node points to another infected vertex [ ] . in order to derive the epidemic threshold let us consider the early time limit of the epidemic process. as done in sect. . . . let us consider that at t t the population is formed mostly by susceptible individuals. in the present scenario this implies s k i k and r k k. the equation for the infected compartment then becomes d t i k d k i k . multiplying both sides for p .k/ and summing over all values of k we obtain d t i d hki i. in order to understand the behavior of i around t let us consider an equation built by multiplying both sides of the last equation by .k / p .k/ =hki and summing over all degree classes. we obtain d t d . hk i hki hki / . the fraction of infected individuals in each value of k will increase if and only if d t > . this condition is verified when [ ] : giving us the epidemic threshold of an sir process unfolding on an uncorrelated network. remarkably, due to their broad-tailed nature, real contact networks display fluctuations in the number of contacts (large hk i) that are significantly larger than the average degree hki resulting in very small thresholds. large degree nodes (hubs) facilitate an extremely efficient spreading of the infection by directly connecting many otherwise distant nodes. as soon as the hubs become infected diseases are able to reach a large fraction of the nodes in the network. real interaction networks are extremely fragile to disease spreading. while this finding is somehow worrisome, it suggests very efficient strategies to control and mitigate the outbreaks. indeed, hubs are central nodes and play a crucial role in the network connectivity [ ] and by vaccinating a small fraction of them one is able to quickly stop the spread of the disease and protect the rest of the population. it is important to mention that in realistic settings the knowledge of the networks' structure is often limited. hubs might not be easy to easily known and other indirect means must be employed. interestingly, the same feature of hubs that facilitates the spread of the disease also allows for their easy detection. since high degree nodes are connected to a large number of smaller degree nodes, one may simply randomly select a node, a, from the network and follow one of its links to reach another node, b. with high probability, node b has higher degree than a and is likely a hub. this effect became popularized as the friend paradox: on average your friends have more friends than you do [ ] . immunizing node b is then much more effective than immunizing node a. remarkably, as counter-intuitive as this methodology might seem, it works extremely well even in the case of quickly changing networks [ ] [ ] [ ] . the next step in the progression towards more realistic modeling approaches is to consider the internal structure of the nodes. if each node in the network represents a homogeneously mixed sub-population instead of a single individual and we consider the edges to represent interactions or mobility between the different subpopulations, then we are in the presence of what is known as meta-population. this concept was originally introduced by r. levins in [ ] for the study of geographically extended ecological populations where each node represents one of the ecological niches where a given population resides. the metapopulation framework was later extended for use in epidemic modeling by sattenspiel in . in a landmark paper [ ] sattenspiel considered two different types of interactions between individuals, local ones occurring within a given node, and social ones connecting individuals originating from different locations on the network. this idea was later expanded by sattenspiel and dietz to include the effects of mobility [ ] and thus laying the foundations for the development of epidemic models at the global scale. metapopulation epidemic models are extremely useful to describe particle reaction-diffusion models [ ] . in this type of model each node is allowed to have zero or more individuals that are free to diffuse among the nodes constituting the network. in our analysis, as done in the previous section, we follow the hmf approach and consider all nodes of degree k to be statistically equivalent and write all quantities in terms of the degree k. to start, let us define the average number of individuals in a node of degree k to be w k d where n k is the number of nodes with degree k and the sum is taken over all nodes i. the mean field dynamical equation describing the variation of the average number of individuals in a node of degree k is then: where p k and p kk represent, respectively, the rate at which particles diffuse out of a node of degree k and diffuse from a node of degree k to one of degree k . with these definitions, the meaning of each term of this equation becomes intuitively clear: the negative term represents individuals leaving the node, while the positive term accounts for individuals originating from other nodes arriving at this particular class of node. the conditional probability p .k jk/ encodes all the topological correlations of the network. by imposing that the total number of particles in the system remains constant, we obtain: that simply states that the number of particles arriving at nodes of degree k coming from nodes of degree k must be the same as the number of particles leaving nodes of degree k. the probabilities p k and p kk encode the details of the diffusion process [ ] . in the simplest case, the rate of movement of individuals is independent of the degree of their origin p k d p for all values of the degree. furthermore, if individuals that are moving simply select homogeneously among all of their connections, then we have p kk d p=k. in this case, the diffusion process will reach a stationary state when: where w d w=n, w is the total number of walkers in the system, and n the total number of nodes. the simple linear relation between w k and k serves as a strong reminder of the importance of network topology. nodes with higher degree will acquire larger populations of particles while nodes with smaller degrees will have proportionally smaller populations. however, even in the steady state, the diffusion process is ongoing, so individuals are continuously arriving and leaving any given node but are doing so in a way that maintains the total number of particles in each node constant. in more realistic settings, the traffic of individuals between two nodes is function of their degree [ ] : in this expression  modulates the strength of the diffusion flow between degree classes (empirical values are in the range : Ä Â Ä : [ ] ), where w is a constant and t k d w hk c i=hki is the proper normalization ensured by the condition in eq. ( . ). in these settings, the diffusion process reaches a stationary state when: note that for  d this solution coincides with the case of homogeneous diffusion [eq. ( . )]. combining this diffusion process with the (epidemic) reaction processes described above we finally obtain the full reaction-diffusion process. to do so we must simply write eq. ( . ) for each state of the disease (e.g., susceptible, infectious, and recovered for a simple sir model) and couple the resulting equations using the already familiar epidemic equations. the full significance of eq. ( . ) now becomes clear: nodes with higher degree have higher populations and are visited by more travelers, making them significantly more likely to also receive an infected individual that can act as the seed of a local epidemic. in a metapopulation epidemic context we must then consider two separate thresholds, the basic reproductive ratio, r , that determines whether or not a disease can spread within one population (node) and a critical diffusion rate, p c , that determines if individual mobility is sufficiently large to allow the disease to spread from one population to another. it is clear that if p d particles are completely unable to move from one population to another so the epidemic cannot spread across subpopulations and that if p d all individuals are in constant motion and the disease will inevitably spread to every subpopulation on the network with a transition occurring at some critical value p c . in general, the critical value p c cannot be calculated analytically using our approach as it depends non-trivially on the detailed structure of the network and the fluctuations of the diffusion rate of single individuals. however, in the case of uncorrelated networks a closed solution can be easily found for different mobility patterns. indeed, in the case where the mobility is regulated by eq. ( . ) we obtain: interestingly, the critical value of p is inversely proportional to the degree heterogeneity in the network, so that broad tailed networks have very low critical values. this simple fact explains why simply restricting travel between populations is a highly ineffective way to prevent the global spread of an epidemic. the mobility patterns considered so far are so-called markovian: individuals move without remembering where they have been nor they have a home where they return to after each trip. although this is a rough approximation of individuals behavior, markovian diffusion patterns are allowed to analytically describe the fundamental dynamical properties of many systems. recently, new analytic results have been proposed for non-markovian dynamics that include origin-destination matrices and realistic travel routes that follow shortest paths [ ] . in particular, the threshold within such mobility schemes reads as: the exponent Á, typically close to : in heterogeneous networks, emerges from the shortest paths routing patterns [ ] . interestingly, for values of Â Ä : , fixing Á d : , p c in the case of markovian mobility patterns is larger than the critical value in a system subject to non-markovian diffusion. the presence of origindestination matrices and shortest paths mobility lower the threshold facilitating the global spreading of the disease. instead, for values of  > : the contrary is true. in these models the internal contacts rate is considered constant across each subpopulation. interestingly, recent longitudinal studies on phone networks [ ] and twitter mention networks [ ] point to the evidence that contacts instead scale super-linearly with the subpopulation sizes. considering the heterogeneity in population sizes observed in real metapopulation networks, the scaling behavior entails deep consequence in the spreading dynamics. a recent study generalized the metapopulation framework considering such observations. interestingly, the critical mobility thresholds, in the case of mobility patterns described by eq. ( . ), changes significantly being lowered by such scaling features of human contacts [ ] . despite their simplicity, metapopulation models are extremely powerful tools in large scale study of epidemics. they easily lend themselves to large scale numerical stochastic simulations where the population and state of each node can be tracked and analyzed in great detail and multiple scenarios as well as interventions can be tested. the state of the art in the class of metapopulation approaches is currently defined by the global epidemic and mobility model (gleam) [ , ] . gleam integrates worldwide population estimates [ , ] with complete airline transportation and commuting databases to create a world wide description of mobility around the world that can then be used as the substrate on which the epidemic can spread. gleam divides the globe into transportation basins. each basin is defined empirically around an airport and the area of the basin is determined to be the region within which residents would likely use that airport for long distance travel. each basin represents a major metropolitan area such as new york, london, or paris. information about all civilian flights can be obtained from the international air transportation association (iata) [ ] and the official airline guide (oag) [ ] that are responsible for compiling up-to-date databases of flight information that airlines use to plan their operations. by connecting the population basins with the direct flight information from these databases we obtain the network that acts as a substrate for the reaction diffusion process. while most human mobility does not take place in the form of flights, the flight network provides the fundamental structure for long range travel that explains how diseases such as sars [ ] , smallpox [ ] , or ebola [ ] spread from country to country. to capture the finer details of within country mobility further information must be considered. gleam uses census information to create a commuting network at the basin level that connects neighboring metropolitan areas proportionally to the number of people who live in one are but work in the other. short-term short-distance mobility such as commuting is fundamentally different from medium-term long-distance airline travel. in one case, the typical timescale is work-day ( h) while in the other it is day. this timescale difference is taken into account in gleam in an effective, mean-field, manner instead of explicitly through a reaction process such as the one described above. this added layer is the final piece of the puzzle that brings the whole together and allows gleam to describe accurately the spread from one country to the next but also the spread happening within a given country [ ] . in fig. . we illustrate the progression in terms of detail that we have undergone since our initial description of simple homogeneously mixed epidemic models in a single population. with all these ingredients in place we have a fine grained description of mobility on a world wide scale on top of which we can finally build an epidemic model. within each basin, gleam still uses the homogeneous mixing approximation. this assumption is particularly suited for diseases that spread easily from person to person through airborne means such as ili. gleam describes influenza through an seir model as illustrated in fig. . . seir models are a modification of the sir model described above that includes a further compartment, exposed, to represent of the remaining symptomatic individuals, one half is sick enough to decide to not travel or commute while the remaining half continue to travel normally. despite their apparent complexity, large scale models such as gleam are controlled by just a small number of parameters and ultimately, it's the proper setting of these few parameters that is responsible for the proper calibration of the model and validity of the results obtained. most of the disease and mobility parameters are set directly from the literature or careful testing so that as little as possible remains unknown when it is time to apply it to a new outbreak. gleam was put to the test during the h n pandemic with great success. during the course of the epidemic, researchers were able to use official data as it was released by health authorities around the world. in the early days of the outbreak there was a great uncertainty about the correct value of the r for the /h n pdm strain in circulation so a methodology to determine it had to be conceived. one of the main advantages of epidemic metapopulation models is their computational tractability. it was this feature what proved invaluable when it came to determine the proper value of r . by plugging in a given set of parameters one is able to generate several hundreds or thousands of in silico outbreaks. each outbreak contains information not only about the number of cases in each city or country as a function of time but also information about the time when the first case occurs within a given country. in general, each outbreak will be different due to stochasticity and by combining all outbreaks generated for a certain parameter set we can calculate the probability distribution of the arrival times. the number of times that an outbreak generated the seeding of a country, say the uk, in the same day as it occurred in reality provides us with a measure of how likely the parameter values used are. by multiplying this probability for all countries with a known arrival time we can determine the overall likelihood of the simulation: where the product is taken over all countries c with known arrival time t c and the probability distribution of arrival times, p c .t/ is determined numerically for each set of input values. the set of parameters that maximizes this quantity is then the one whose values are the most likely to be correct. using this procedure the team behind gleam determined that the mostly likely value of the basic reproductive ratio was r d : [ ] , a value that was later confirmed by independent studies [ , ] . armed with an empirical estimate of the basic reproductive ratio for an ongoing pandemic, they then proceeded to use this value to estimate the future progression of the pandemic. their results predicting that the full peak of the pandemic would hit in october and november were published in early september [ ] . a comparison between these predictions and the official data published by the health authorities in each country would be published several years later [ ] clearly confirming the validity of gleam for epidemic forecasting in real time. indeed, the model predicted, months in advance, the correct peak week in % of countries in the north hemisphere for which real data was accessible. in the rest of cases the maximum error reported has been weeks. gleam can also be further extended to include age-structure [ ] , interventions and travel reductions. the next logical step in the hierarchy of large scale epidemic models is to take the description of the underlying population all the way down to the individual level with what are known as abm. the fundamental idea behind this class of model is a deceptively simple one: treat each individual in the population separately, assigning it properties such as age, gender, workplace, residence, family structure, etc: : : these added details give them a clear edge in terms of detail over metapopulation models but do so at the cost of much higher computational cost. the first step in building a model of this type is to generate a synthetic population that is statistically equivalent to the population we are interested in studying. typically this is in a hierarchical way, first generating individual households, aggregating households into neighborhoods, neighborhoods into communities, and communities into the census tracts that constitute the country. generating synthetic households in a way that reproduces the census data is far from a trivial task. the exact details vary depending on the end goal of the model and the level of details desired but the household size, age, and gender of household members are determined stochastically from the empirically observed distributions and conditional probabilities. one might start by determining the size of the household by extracting from the distribution of household size of the country of interest and selecting the age and gender of the head of the household proportionally to the number of heads of households for that household size that are in each age group. conditional on this synthetic individual we can then generate the remaining members, if any, of the household. the required conditional probability distributions and correlation tables can be easily generated [ ] from high quality census data that can be found for most countries in the world. this process is repeated until enough synthetic households have been generated. households are then aggregated into neighborhoods by selecting from the households according to the distribution of households in a specific neighborhood. neighborhoods are similarly aggregated into communities and communities into census tracts. each increasing level of aggregation (from household to country) represents a decrease in the level of social contact, with the most intimate contacts occurring at the household level and least intimate ones at the census tract or country level. the next step is to assign to each individual a profession and work place. workplaces are generated following a procedure similar to the generation of households and each employed individual is assigned a specific household. school age children are assigned a school. working individuals are assigned to work places in a different community or census tract in a way that reflects empirical commuting patterns. at this point, we have a fairly accurate description of where the entire population of a city or country lives and works. it is then not entirely surprising that this approach was first used to study in detail the demands imposed on the transportation system of a large metropolitan city. transims, the transportation analysis and simulation system [ ] , used an approach similar to the one described above to generate a synthetic population for the city of portland, in oregon (or) and coupled it with a route planner that would determine the actual route taken by each individual on her way to work or school as a way of modeling the daily toll on portland's transportation infrastructure and the effect that disruptions or modification might have in the daily lives of its population. episims [ ] was the logical extension of transims to the epidemic world. episims used the transims infrastructure to generate the contact network between individuals in portland, or. susceptible individuals are able to acquire the infection whenever they are in a location along with one or more infectious individuals. in this way the researchers are capable of observing as the disease spreads through the population and evaluate the effect that measures such as contact tracing and mass vaccination. more recent approaches have significantly simplified the mobility aspect of this kind of models and simply divide each h period into day time and nighttime. individuals are considered to be in contact with other members of their workplace during the day and with other household members during the night. in recent years, modelers have successfully expanded the large scale agent based approach to the country [ ] and even continent level [ ] . as the spatial scale of the models increased further modes of long-range transportation such as flights had to be considered. these are important to determine not only the seeding of the country under consideration through importation of cases from another country but also to connect distant regions in a more realistic way. flute [ ] is currently the most realistic large scale agent-based epidemic model of the continental united states. it considers that international seeding occurs at random in the locations that host the largest international airports in the us by, each day, randomly infecting in each location a number of individuals that is proportional to the international traffic of those airports. flute is a refinement of a previous model [ ] and it further refines the modeling of the infectious process by varying the infectiousness of an individual over time in the sir model that they consider. at the time of infection each individual is assigned one of six experimentally obtained viral load histories. each history prescribes the individuals viral load for each day of the infectious period and the infectiousness is considered to be proportional to the viral load. individuals may remain asymptotic for up to days after infection during which their infectiousness is reduced by % with respect to the symptomatic period. the total infectious period is set to days regardless of the length of the symptomatic period. given the complexity of the model the calibration of the disease parameters in order to obtain a given value of the basic reproductive ratio, r requires some finesse. chao et al. [ ] uses the definition of r to determine "experimentally" its value from the input parameters. it numerically simulates instances of the epidemic caused by a single individual within a person fully susceptible community for each possible age group of the seeding individual and use it to calculate the r a of each age group a. the final r is defined to the average of the various r a weighted by age dependent attack rate [ ] . the final result of this procedure is that the value of r is given by: where is the infection probability per unit contact and is given as input. flute was a pioneer in the way it completely released its source code, opening the doors of a new level of verifiability in this area. it has successfully used to study the spread of influenza viruses and analyze the effect of various interventions in the los angeles county [ ] and united states country level [ ] . the unprecedented amount of data on human dynamics made available by recent advances technology has allowed the development of realistic epidemic models able to capture and predict the unfolding of infectious disease at different geographical scales [ ] . in the previous sections, we described briefly some successful examples that have been made possible thanks to high resolution data on where we live, how we live, and how we move. data availability has started a second golden age in epidemic modeling [ ] . all models are judged against surveillance data collected by health departments. unfortunately, due to excessive costs, and other constraints their quality is far from ideal. for example, the influenza surveillance network in the usa, one of the most efficient systems in the world, is constituted of just providers that operate voluntarily. surveillance data is imprecise, incomplete, characterized by large backlogs, delays in reporting times, and the result of very small sample sizes. furthermore, the geographical coverage is not homogeneous across different regions, even within the same country. for these reasons the calibration and test of epidemic models with surveillance data induce strong limitations in the predictive capabilities of such tools. one of the most limiting issues is the geographical granularity of the data. in general, information are aggregated at the country or regional level. the lack of ground truth data at smaller scales does not allow a more precise selection and training of realistic epidemic models. how can we lift such limitations? data, data and more data is again the answer. at the end of almost billion of people had access to the internet while almost billion are phone subscribers, around % of which are actively using smartphones. the explosion of mobile usage boosted also the activity of social media platforms such as facebook, twitter, google+ etc. that now count several hundred million active users that are happy to share not just their thoughts, but also their gps coordinates. the incredible amount of information we create and access contain important epidemiologically relevant indicators. users complaining about catching a cold before the weekend on facebook or twitter, searching for symptoms of particular diseases on search engines, or wikipedia, canceling their dinner reservations on online platforms like opentable are just few examples. an intense research activity, across different disciplines, is clearly showing the potential, as well as the challenges and risks, of such digital traces for epidemiology [ ] . we are at the dawn of the digital revolution in epidemiology [ , ] . the new approach allows for the early detection of disease outbreaks [ ] , the real time monitoring of the evolution of a disease with an incredible geographical granularity [ ] [ ] [ ] , the access to health related behaviors, practices and sentiments at large scales [ , ] , inform data-driven epidemic models [ , ] , and development of statistical based models with prediction power [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the search for epidemiological indicators in digital traces follows two methodologies: active and passive. in active data collection users are asked to share their health status using apps and web-based platforms [ ] . examples are influenzanet that is available in different european countries [ ] , and flu near you in the usa [ ] that engage tens of thousands of users that together provide the information necessary for the creation of interactive maps of ili in almost real time. in passive data collection, instead, information about individuals health status is mined from other available sources that do not require the active participation of users. news articles [ ] , queries on search engines [ ] , posts on online social networks [ , [ ] [ ] [ ] [ ] , page view counts on wikipedia [ , ] or other online/offline behaviors [ , ] are typical examples. in the following, we focus on the prototypical, and most famous, method of digital epidemiology, google flu trends (gft) [ ] , while considering also other approaches based on twitter and wikipedia data. gft is by far the most famous model in digital epidemiology. launched in november together with a nature paper [ ] describing its methodology, it has continuously made predictions on the course of seasonal influenza in countries around the world. the method used by gft is extremely simple. the percentage of ili visits, a typical indicator used by surveillance systems to monitor the unfolding of the seasonal flu, is estimated with a linear model based on search engine queries. this approach is general, and used in many different fields of science. a quantity of interest, in this case the percentage of ili visits p, is estimated using a correlated signal, in this case the ili related queries fraction q, that acts as surrogate. the fit allows the estimate of p as a function of the value of q: logit .p/ dˇ cˇ logit .q/ c ; ( . ) where logit .x/ d ln x x ,ˇ andˇ are fitting parameters, and is an error term. as clear from the expression, the gft is a simple linear fit, where the unknown parameters are determined considering historical data. the innovation of the system lies on the definition of q that is evaluated using hundreds of billions of searches on google. indeed, gft scans all the queries we submit to google, without using information about users' identity, in search of those that ili related. this is the paradigm of passive data collection in digital epidemiology. in the original model the authors measured the correlation of millions search queries with historic cdc data, finding that of them were enough to ensure the best correlation between the number of searches and the number of ili cases. the identity of such terms has been kept secret in order to avoid changes in users' behavior. however, the authors provided a list of topics associated with each one of them: were associated with influenza complications, to cold/flu remedies, to general terms for influenza, etc. although the search for the terms has been performed without prior information, none of the most representative terms were unrelated to the disease. in these settings gft showed a mean correlation of : with real data and was able to predict the surveillance value with - weeks ahead. gft is based on proprietary data that for many different constraints cannot be shared with the research community. other data sources, different in nature, are instead easily accessible. twitter and wikipedia are the two examples. indeed, both systems are available for download, with some limitations, through their respective apis. the models based on twitter are built within the same paradigm of gft [ , [ ] [ ] [ ] ] . tweets are mined in search of ili-related tweets, or other health conditions such as insomnia, obesity, and other chronic diseases [ , ] , that are used to inform regression models. such tweets are determined either as done in gft, or through more involved methods based on support vector machine (svm) or other machine learning methods that, provided an annotated corpus, find disease related tweets beyond simple keywords matches [ , [ ] [ ] [ ] ] . the presence of gps information or other self-reported geographical data allows the models to probe different granularities ranging from countries [ , , , ] to cities [ ] . while models based on twitter analyze users' posts, those based on wikipedia focus on pages views [ , ] . the basic intuition is that wikipedia is used to learn more about a diseases or a medication. plus, the website is so popular that is most likely one of the first results of search queries on most search engines. the methods proposed so far monitor a set of pages related to the disease under study. examples are influenza, cold, fever, dengue, etc. page views at the daily or weekly basis are then used a surrogates in linear fitting models. interestingly, the correlation with surveillance data ranges from : in the case of ebola to : in for ilis [ , ] , and allows accurate predictions up to weeks ahead. one important limitation of wikipedia based methods is the lack of geographical granularity. indeed, the view counts are reported irrespective of readers' location but the language of the page can be used as a rough proxy for location. such approximation might be extremely good for localized languages like italian but it poses strong limitations in the case of global languages like english. indeed, it is reported that % of pages views for english pages are done in the usa, % in the uk, and the rest in australia, canada and other countries [ ] . besides, without making further approximation such methods cannot provide indications at scales smaller than the country level. despite these impressive correlations, especially in the case of ilis, much still remains to be done. gft offers a particular clear example of the possible limitations of such tools. indeed, despite the initial success, it completely failed to forecast the h n pandemic [ , ] . the model was updated in september to increase the number of terms to , including the terms present in the original version. nevertheless, gft missed high out of weeks in the season - . in gft predicted a peak height more than double the actual value causing the underlying model to be modified again later that year. what are the reasons underlying the limitations of gft and other similar tools? by construction, gft relies just on simple correlations causing it to detect not only the flu but also things that correlate strongly with the flu such as winter patterns. this is likely one of the reasons why the model was not able to capture the unfolding of an off-season pandemic such as the h n pandemic. also, changes in the google search engine, that can inadvertently modify users' behavior, were not taken into account in gft. this factor alone possibly explains the large overestimation of the peak height in . plus, simple auto-regressive models using just cdc data can perform as well or better than gft [ ] . the parable of gft clearly shows both the potential and the risks of digital tools for epidemic predictions. the limitations of gft can possibly affect all similar approaches based on digital passive data collection. in particular, the use of simple correlations measures does not guarantee the ability of capturing the phenomena across different scales in space and time with respect to those used in the training. not to mention that correlations might be completely spurious. in a recent study for example, a linear model based on twitter simply informed with the timeline of the term zombie was shown to be a good predictor of the seasonal flu [ ] . despite such observations the potential of these models is invaluable to probe data that cannot be predicted by simple auto-regressive models. for example, flu activity at high geographical granularities, although very important, is measured with great difficulties by the surveillance systems. gft and other spatially resolved tools can effectively access to these local indicators, and provide precious estimates that can be used a complement for the surveillance and as input for generating epidemic models [ , ] . the field of epidemiology is currently undergoing a digital revolution due to the seemingly endless availability of data and computational power. data on human behavior is allowing for the development of new tools and models while the commoditization of computer resources once available only for world leading research institutions is making highly detailed large scale numerical approaches feasible at last. in this chapter, we present a brief review not only of the fundamental mathematical tools and concepts of epidemiology but also of some of the state-of-the-art and computational approaches aimed at describing, modeling, and forecasting the diffusion of viruses. our focus was on the developments occurring over the past decade that are sure to form the foundation for developments in decades to come. essai dune nouvelle analyse de la mortalité causée par la petite vérole et des advantages de l'inocoulation pur la prévenir. mémoires de mathematique physique de l infectious diseases in humans the role of the airline transportation network in the prediction and predictability of global epidemics modeling the spatial spread of infectious diseases: the global epidemic and mobility computational model modelling disease outbreaks in realistic urban social networks flute, a publicly available stochastic influenza epidemic simulation model digital disease detection: harnessing the web for public health surveillance digital epidemiology the mathematical theory of infectious diseases modeling infectious diseases in humans and animals a contribution to the mathematical theory of epidemics networks: an introduction scale-free networks complex networks: structure, robustness and function dynamical processes on complex networks computational social science linked: how everything is connected to everything else and what it means temporal networks telling tails explain the discrepancy in sexual partner reports simulated epidemics in an empirical spatiotemporal network of , sexual contacts what's in a crowd? analysis of face-to-face behavioral networks activity driven modeling of dynamic networks time varying networks and the weakness of strong ties epidemic spreading in non-markovian timevarying networks thresholds for epidemic spreading in networks modeling dynamical processes in complex socio-technical systems epidemic spreading in correlated complex networks spread of epidemic disease on networks correlations in weighted networks efficient immunization strategies for computer networks and populations using friends as sensors to detect global-scale contagious outbreaks controlling contagion processes in activity driven networks some demographic and genetic consequences of environmental heterogeneity for biological control population structure and the spread of disease a structured epidemic model incorporating geographic mobility among regions reaction-diffusion equations and their applications to biology epidemic modeling in metapopulation systems with heterogeneous coupling pattern: theory and simulations modeling human mobility responses to the large-scale spreading of infectious diseases the scaling of human interactions with city size the scaling of human contacts in reaction-diffusion processes on heterogeneous metapopulation networks the gridded population of the world version (gpwv ): population grids. palisades global rural-urban mapping project (grump), alpha version: population grids. palisades predictability and epidemic pathways in global outbreaks of infectious diseases: the sars case study human mobility and the worldwide impact of intentional localized highly pathogenic virus release assessing the international spreading risk associated with the west african ebola outbreak multiscale mobility networks and the spatial spreading of infectious diseases seasonal 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analysis global capacity for emerging infectious disease detection web-based participatory surveillance of infectious diseases: the influenzanet participatory surveillance experience assessing vaccination sentiments with online social media: implications for infectious disease dynamics and control you are what you tweet: analyzing twitter for public health forecasting seasonal outbreaks of influenza forecasting seasonal influenza with stochastic microsimulations models assimilating digital surveillance data the use of twitter to track levels of disease activity and public concern in the us during the influenza a h n pandemic validating models for disease detection using twitter national and local influenza surveillance through twitter: an analysis of the - influenza epidemic towards detecting influenza epidemics by analyzing twitter messages detecting influenza epidemics using search engine query data detecting epidemics using wikipedia article views: a demonstration of feasibility with language as location proxy wikipedia usage estimates prevalence of influenzalike illness in the united states in near real-time guess who is not coming to dinner? evaluating online restaurant reservations for disease surveillance satellite imagery analysis: what can hospital parking lots tell us about a disease outbreak? public health for the people: participatory infectious disease surveillance in the digital age google flu trends using twitter to estimate h n influenza activity a content analysis of chronic diseases social groups on facebook and twitter. telemedicine and e-health reassessing google flu trends data for detection of seasonal and pandemic influenza: a comparative epidemiological study at three geographic scales predicting consumer behavior with web search acknowledgements bg was partially supported by the french anr project harms-flu (anr- -monu- ). key: cord- -gykw nvt authors: yadav, mahendra pal; singh, raj kumar; malik, yashpal singh title: emerging and transboundary animal viral diseases: perspectives and preparedness date: - - journal: emerging and transboundary animal viruses doi: . / - - - - _ sha: doc_id: cord_uid: gykw nvt the epidemics and pandemics of a few infectious diseases during the past couple of decades have accentuated the significance of emerging infectious diseases (eids) due to their influence on public health. although asia region has been identified as the epicentre of many eids and upcoming infections, several new pathogens have also emerged in the past in other parts of the world. furthermore, the emergence of new viral diseases/infections, such as rift valley fever, west nile fever, sars coronavirus, hendra virus, avian influenza a (h n ), nipah virus, zika virus and swine influenza a (h n ) virus, from time to time is a glaring example threatening adversely both animal and public health globally. infectious diseases are dynamic and concerning due to their epidemiology and aetiological agents, which is manifested within a host, pathogen and environment continuum involving domestic animals, wildlife and human populations. the complex relationship among host populations and other environmental factors creates conditions for the emergence of diseases. the factors driving the emergence of different emerging infectious disease (eid) interfaces include global travel, urbanisation and biomedical manipulations for human eids; agricultural intensification for domestic animal eids; translocation for wildlife eids; human encroachment, ex situ contact and ecological manipulation for wildlife–human eids; encroachment, new introductions and ‘spill-over’ and ‘spill-back’; and technology and industry for domestic animal–human eids. the concepts of sanitary and phytosanitary (sps) measures and biosecurity have gained recognition globally in almost all the realms of human activities, including livestock health and production management. this chapter provides the experience gained in the control and management of a few important tads and eids along with the successes, constraints, limitations and future research needs for developing better control approaches. for wildlife-human eids; encroachment, new introductions and 'spill-over ' and 'spill-back'; and technology and industry for domestic animal-human eids. the term 'emerging disease' is used to refer to changes in the disease dynamics in the population. emerging infectious diseases (eids) are those which have moved recently into a new host or have enhanced incidences or geographic range or are caused by evolving pathogens (lederberg et al. ; daszak et al. ) . this general definition covers a range of infectious diseases of man and animals which pose a significant threat to both medical and veterinary public health. among the oie-listed diseases of viral aetiology, major changes have been experienced in the occurrence of rinderpest, peste-des-petits ruminants (ppr), foot-and-mouth disease (fmd), african swine fever (asf), lumpy skin disease and rift valley fever (rvf). of these, rinderpest presents a success story from the s to as a result of fao, oie, eu and iaea (international atomic energy agency) guided and coordinated programmes including the pan african rinderpest campaign (parc), npre and nrep in india (yadav ) , global rinderpest eradication program (grep) of the fao and other national governments where the disease was endemic. these exemplary efforts led to the historic declaration of global rinderpest eradication by the fao on june , . the terms 'exotic disease' and transboundary animal diseases (tads) are often used interchangeably. though all transboundary diseases are of exotic origin, all exotic diseases are not included in tad listing. many eids are also transboundary diseases. the tads are defined as highly contagious and transmissible epidemic diseases of livestock which have the capability for rapid spread to new areas and regions regardless of national borders and have serious socio-economic and public health consequences. nearly all diseases affect livestock, poultry, fishes and other animals and adversely impact the quality and quantity of food and other products, such as hides and skins, bones, fibres, wool and animal draft power for tilling, transport and traction. the reduction in animal production, productivity and profitability due to tads affect the human livelihood. in the present scenario of fast-increasing globalisation, tads represent a serious threat to the economy and welfare of the public and affected nations as they drastically reduce production and productivity; disrupt trade and travel and local and national economies; and also threaten human health through inferior food quality and zoonotic diseases/infections. as such, consequences of tads could have a significant detrimental effect on the economy and public health of not only the affected nations but also the whole of the world. possibly the infectious agents which cause emerging and transboundary diseases are already present in the environment and get the opportunity to cause disease under certain altered circumstances. the transmission of the infectious agent could occur between animal and human; between wildlife, human and domestic animals; or between wildlife, domestic animal(s) and human. however, the main source for maintenance and transmission of the infectious agents in nature is determined by the zoonotic pool and spill-over and spill-back mechanisms. tads have become of great concern due to the risk for national security on account of their economic significance, zoonotic nature and ever-growing threat of newer tads in future. among the tads having zoonotic manifestations, a number of infectious diseases, such as highly pathogenic avian influenza (hpai), bse (mad cow disease caused by prion), west nile fever, rift valley fever, sars coronavirus, hendra virus, nipah virus, ebola virus, zika virus and cchf, to name a few, adversely affecting animal and human health have been in the news in recent times (malik and dhama ; munjal et al. ; singh et al. singh et al. , . the direct and indirect costs due to the fmd outbreak in the uk in were assessed to be over us$ billion. over million chicken died or were destroyed in southeast asia in to control hpai (h n ). the netherlands suffered an economic loss of $ . billion due to classical swine fever in [ ] [ ] . as per the estimates of fao nearly one-third of the world meat trade was facing import bans on account of bse, hpai and other animal diseases. there is evidence to suggest that threats from tads have increased over the years. the risk of animal disease outbreaks is likely to further grow in future as the higher incomes of people in developing countries will generate more demand for animal protein and products (milk, meat, egg, chicken and fish). the number of animals raised for meat is growing rapidly. during s poultry production in east asia has increased by about % per year to double every - years. similar to tads, new human viral diseases have emerged like ebola, sars, zika, cchf, nipah and bse as well as there is the emergence of new antigenic forms or new biotypes of the existing infectious diseases, such as a hypervirulent strain of ibd in poultry in europe and highly virulent strain of newcastle disease in the usa (riemenschneider ; singh et al. ) . vector-borne pathogens, namely, bluetongue, african horse sickness, rift valley fever and west nile fever, have the potential to spread in epidemic forms. riemenschneider ( ) has deliberated over several issues relevant in the control of tads as proposed in the institute of medicine (iom) report (anonymous ) . some of the points which could be responsible for the increased threat of tads are briefly discussed below. in the present-day world, higher quantitative levels of animal origin foods, as well as faster trade, new trade routes and air travel, have led to higher risks for contracting new infections and diseases. as it is now possible to reach any part of the world within h which is less than the incubation period of most of the infectious diseases, animals or people carrying the infectious agents go undetected in want of clinical disease/symptoms. fresh commodities vis-à-vis processed foods that have witnessed an increased trade are more likely to carry the pathogens to distant parts of the world-countries and continents. recent decades are witnessing higher demands for animal protein and other nutrients through meat and meat products, milk and milk products, eggs, and fish and fish products as a result of rising incomes in the developing countries and elsewhere which leads to the intensification of production systems and overcrowding of animals. this increased production is often required in peri-urban areas, having large human populations, under suboptimal husbandry practices. in such high-production areas, disease outbreaks affect a greater number of animals at a faster rate and speed, leading to heavy economic losses. drastic control measures are taken, such as the slaughter of infected and in-contact animals followed by burning or burial is not acceptable to the society at large. for example, the mass slaughter of pigs in the netherlands in - for the control of csf virus led to objection from the non-farm population which might influence the application of the stamping-out policy as a disease control approach in future. exposure of the domestic animals to forest niches due to deforestation and transformation of tropical rainforests for livestock grazing exposes the domestic livestock to a completely new range of pathogens and vectors which previously circulated in wildlife reservoir niches only. with the domestic livestock being fully susceptible and naïve to these infectious agents, the disease spreads more rapidly and severely in want of lack of diagnostic tests and vaccines against these new pathogens resulting in heavy morbidity, mortality, trade restrictions and economic losses. nowadays many countries face prolonged civil unrests besides inter-and intracountry conflicts, which may lead to enhanced threat of tads. civil disorders are known to disrupt enforcement of quarantine and other control measures due to refugee and army deployments/movements. breakdown in the institutional support for quarantine and difficulty in gaining access to border area due to landmines make disease surveillance more difficult. inflows of more food aids for such areas also pose additional risks as the food items may have contaminants. climate change and global warming seem to be altering rainfall and weather patterns. rising temperatures in the northern hemisphere are likely to shift the distribution of insect vectors of bluetongue, african horse sickness, rift valley fever and similar vector-borne diseases. the bluetongue virus (btv) having serotypes occurs in many parts of the world. however, until recently it was never reported from europe. the sudden incursions of some serotypes into spain, italy, greece, portugal and the balkan countries since , followed by germany, and the recent incursion of btv serotype in several farms in the netherlands, germany and belgium since august as well as serotype are also believed to be due to climate change as european weather has become hotter in recent decades. the btv serotype revealed that this serotype is closest to the nigerian strain. the incursion is believed to have been caused by the importation of an infected zoo animal or an infected midge. an upsurge of rift valley fever was observed in east and west africa due to climatic changes. many factors discussed above make the tads as a serious threat to national and international security. the developing countries are usually the worst sufferers. among other factors, veterinary public health services in developing countries are usually much behind than the medical public health services. moreover, unlike human disease reporting, animal disease reporting systems are usually based on passive reporting rather than active disease surveillance. a few other factors are also responsible for greater threat due to tads, namely ( ) lack of awareness of the farmers about the high-threat epizootic animal diseases; ( ) lack of diagnostic facilities for exotic diseases, and under-reporting of animal diseases like hpai due to the fear of loss of internal and export market till the country gets infection-free status as per oie-laid-down criteria; and ( ) poor and faulty compensation schemes. in the technological advances made in today's world, there is always a real risk of deliberate misuse of certain infectious agents/pathogens by terrorists as a means of biowarfare between nations to harm the people and/or livestock, poultry and other animals. potential for pathogenic disease agents not reported previously in a country and being misused or mishandled for bioterrorism is likely to threaten the ecosystem on a large scale. even new pathogens can be engineered as novel infectious agents. the animal diseases could even be a greater threat than human diseases as these may result in significant economic disruptions, besides causing food poisoning and deterioration, and zoonotic diseases in human beings. as animal diseases get less priority than human infections/diseases in undertaking immediate disease control measures, the threat scenario with the use of animal pathogens for bioterrorism or biowarfare will have many serious consequences. some of the viruses having significant bioterror potential for humans and or animals include hpai (h n ), when an exotic viral disease strikes a country for the first time, it may initially affect one animal, few animals or a large number of animals. the strategy to be adopted for containing the outbreak will depend on the nature of the virus, speed of its spread, role of vectors, risk assessment, communication and management, response time and country legislation on disease control and prevention. thus, there is a need to develop strategic plans for the prevention and control of exotic and tad on a case-to-case basis. examples of such viral diseases from indian perspective include african swine fever (asf)‚ transmissible gastroenteritis (tge)‚ and swine vesicular disease in pigs, rift valley fever, african horse sickness (ahs), west nile fever, eastern equine encephalomyelitis (eee), western equine encephalomyelitis (wee), and venezuelan equine encephalomyelitis (vee), fmd virus types 'c', 'sat i', 'sat ii' and 'sat iii', nipah virus, hendra virus, sars coronavirus, and prion diseases-bovine spongiform encephalopathy (bse), and scrapie. institution of appropriate and timely biosecurity measures is an important instrument for the protection and improvement of animal health. breach in biosecurity due to ignorance and avoidable lapses in the adoption of timely biosecurity and biosafety measures in the management of livestock, poultry and fish minimise the risks from infectious diseases including eids and tads. breach in biosecurity in livestock management is often an important reason for the high incidence of zoonotic and other infectious diseases of animals. this is more so in case of the viral diseases of livestock and poultry. closer contact between wildlife, animals and humans and rearing of livestock and poultry in close association with people promote spread of viral and other infectious diseases which have the potential for threatening health, economies and food security around the world. the emergence of new viral diseases/infections, such as rift valley fever, west nile fever, sars coronavirus, hendra virus, avian influenza a (h n ), nipah virus, zika virus and swine influenza a (h n ) virus, from time to time is a glaring example of zoonotic disease threats adversely affecting both animal health and public health, national economies and food and nutrition security globally. due to a lacuna in the biosecurity, viral diseases like the fmd had reoccurred in countries where these had not been reported for many decades, including the uk, a developed country. biosafety and biosecurity are interrelated terms but used in different contexts. the guidelines are developed by who, fao and oie. biosafety aims at the protection of person(s) at work and the facilities which are dealing with the biological agents, against their exposure to a disease agent, and prevents unintentional exposure to pathogens/toxins or their accidental release. thus, biosafety is the application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards. biosecurity, unlike biosafety, has divergent meanings in different contexts in which it is used. it deals with the protection of microbiological assets from spill-over, theft, loss, diversion or intentional release from laboratories, preventing the import of certain organisms/ toxins. biosecurity is a set of preventive measures designed to reduce the risk of intentional transmission of infectious diseases to safeguard the facilities containing sensitive biological materials with the potential of a biological weapon. in brief, biosecurity means bio-risk management. once a disease is eradicated globally, the policy for keeping the wild and vaccine strains of the virus along with vaccine stocks for emergency use and their subsequent destruction is decided by international agencies like fao, who and oie based on the recommendations of experts in the area. these include risk assessment; communication and management; quarantine of imported animals at seaports, dry ports and farm; establishment of check posts and vaccination stations at international and interstate borders for clinical surveillance; creation of immune belts at international borders; and planning and conducting structured disease surveillance including clinical surveillance and serosurveillance. biosafety and biosecurity need to be observed at all levels beginning from farm to national and international levels. for handling the most dangerous transboundary disease pathogens, bsl iii and bsl iv laboratories are required to ensure biosafety, biosecurity and biocontainment. proper zoo sanitary measures, such as quarantine; rodent and vector control; disinfection of animal sheds and premises; proper disposal of dung, urine, feed and fodder wastes; and proper carcass disposal, need to be adopted religiously for effective management of eids and tads. every country needs strict and foolproof biosecurity mechanism at its international borders as a safeguard against the entry of exotic infectious agents/diseases from abroad along with the import of livestock and other animals and their products. for example, india has contiguous and porous borders with countries like nepal, bhutan, pakistan and bangladesh, besides free trade with nepal and bhutan. since all these countries are vulnerable to tads, there is a need for regional biosecurity plan to ensure a biosecure region. it would never be possible to have a biosecure country if the bordering countries do not have effective biosecurity in place. different countries are at risk for a number of tads like anthrax, plague, glanders, lyme disease, contagious equine metritis, salmonella abortus equi, hpai virus, fmd virus (sat - ), lyssavirus, rabies, hendra and nipah viruses, west nile virus, highly pathogenic nd virus, rabbit haemorrhagic disease virus, bovine spongiform encephalopathy (bse), african horse sickness (ahs), equine encephalomyelitis (eee, vee, wee), equine infectious anaemia, chicken infectious anaemia, equine influenza, vesicular stomatitis, rift valley fever, malignant catarrhal fever (mcf) and other tses of sheep, goat and deer. biosecurity measures are required for preventing and containing the ingress of these diseases through international trade. the oie has facilitated safe trade in animals and animal products by developing effective standards to prevent the spread of animal diseases across the globe. prevention of transmission of pathogens across intra-and inter-country borders warrants devising of biosecurity measures at par with international standards. adequate infrastructure comprising check posts and quarantine facilities at seaports, airports and porous international land border are must to check the ingress of viral and other pathogens from across the borders. diagnostic facilities with trained human resource, and well equipped with instruments and pen-side diagnostic tests/kits, should be in place for ensuring the pathogen-free status of imported livestock and livestock products. biosecurity measures at national level incorporate the components of 'external biosecurity' preventing the ingress of exotic and transboundary animal diseases and 'internal biosecurity' within the country encompassing zonal, compartmental and farm-level biosecurity. regulations for animal movement through interstate borders in india are in place but need strict implementation. modern detection systems can be used for identification and tracking of animals and animal products to provide information regarding the origin of the animal, and environmental practices used in production and food safety. for effective disease prevention and control, integration of biosecurity into every operation at the farm is essential. farm biosecurity should be inclusive of both 'bioexclusion' (measures for preventing a pathogen from being introduced to a herd/ flock) and 'biocontainment'. the latter addresses the events after the introduction of the pathogen and its ability to spread among susceptible groups of animals at the farm or further spill-over to other farms. strict implementation of biosecurity at farm level has played a crucial role in preventing the spread of diseases. a suitable plan addressing important issues, such as location and layout of the farm, animal health practices in place and general management on the farm, needs to be chalked out. it should be flexible to include new knowledge, concepts and technology. a wide range of biosecurity practices have been recommended for different livestock species and production systems, both for specific infection risks or for disease prevention in general. biosecurity practices have been recommended for cattle, sheep, pig, poultry and fish production systems. general biosecurity practices and interventions that can be applicable across species and farms include: . maintaining a closed herd procurement/purchase of animals from known sources . minimising the number of animals purchased/transferred/exchanged and the number of herds from which the animals are introduced . avoiding purchases from markets or dealers . appropriate quarantine and testing of animals upon introduction or reintroduction in farm premises . discouraging farming practices such as hiring a bull or stallion and returning it after the breeding season . avoiding the introduction of biological material of uncertain health status . health and vaccination records should be obtained for all the newly introduced animals isolation/quarantine of such animals for - weeks in a separate quarantine facility should be practiced and the animals during this period should be observed for illness/symptoms and screened for important diseases before mixing with other stock at the farm. laboratory testing of appropriate samples collected during quarantine against important infectious diseases is recommended. the incoming stock can also be given vaccine against the endemic disease prevalent in the area at least weeks before release from quarantine to boost their protective immunity. animal diseases can spread from farm to farm resulting in animal sickness, death and economic losses. visitors to the livestock farm, disease laboratory, birds, rodents, vehicles, feed and fodder and other inanimate objects are often a source of infection. in addition to adverse effects on the economy, there can be negative effects on the environment and human health. the best designs are to implement effective biosecurity practices. baths by the laboratory workers after and before visiting animal farm or laboratory and putting on gum boots, disposable overall, head gear and gloves should be a mandatory requirement. all effluent from the laboratory should be pre-treated to ensure freedom from pathogens before their disposal to the environment. disinfectant foot bath for the workers and vehicles entering the farm at the gate, exclusive separate dress and shoes for laboratory and farm workers, minimum movement of the people and animals within the farm during the outbreak period, and personal health and hygiene of the staff are some of the minimum guidelines to strengthen farm biosecurity. timely, rapid and accurate disease reporting based on oie-approved diagnostic tests is a must for effective detection of the pathogen and instituting early response without giving much time for the disease to spread further. to face the new exotic diseases, it is recommended to have a standard sop in place along with technical guidelines, decision-taking levels along with adequate provision for funds and legal backup. it has been observed that lack or inadequate compensation for culling the diseased and in-contact animals and poultry and negative effect on the sale, sale price and exports deter the farmers from reporting animal diseases in time which eventually leads to the spread of tads. for example, due to bse cattle producers in the usa lost over $ billion in exports to japan. similarly, hpai resulted in over $ billion loss in poultry exports for thailand. trade concerns also discourage the use of preventive vaccinations for some diseases such as hpai and fmd. disease-free countries are generally reluctant to import animals or animal products from the countries practicing preventive vaccinations. with these adverse trade considerations, stamping-out policy was adopted by the uk over the vaccination for fmd in outbreak. from a public health point of view, a vaccination programme might reduce the viral load circulating in a country and thus reduce the risk of hpai spreading to humans. however, stamping out rather than vaccination is preferred by most of the countries to declare themselves free from disease/infection at the earliest possible to regain access to exports. to ensure the cooperation of livestock farmers, it is essential to provide adequate and timely compensations to reduce the losses suffered by them on account of culling and closing the units for a few months. the failure of timely disease reporting hastens the spread of tads within the country as well as between countries. it is believed that the hpai (h n ) avian influenza virus might have been circulating in the poultry for months in the affected region before it was reported to the international authorities in leading to the wide spread of the disease/infection. an ex ante study of the fmd outbreak of the uk suggested that the fmd virus was probably introduced - weeks before it was reported and followed by a ban on livestock movements. earlier reporting and ban on animal movements would have cut the spread of the disease by about %. it is believed that the eids and tads will continue to remain an ever-growing threat to animal and human health, economic sustenance of the world and global environment well-being. however, it is difficult to predict the number of these diseases which could rapidly escalate in a country or region threatening the animal and human life as well as the economy of that region or nation. the rising global demands for meat, particularly in east and south asia, have put humans and animals together in numbers never seen before in the world. the fact is that the farm biosecurity in these countries where meat production is growing most rapidly is often poor. this scenario creates a great scope for animal diseases to jump species to create human health problems. some of these issues were thoroughly described in the iom report, , and further debated and discussed by riemenschneider ( ) . the steps suggested include early detection and early response, preparedness plans, decentralisation of government structure, international coordination, understanding of ecology, microbial evolution and viral traffic, expanded surveillance system, disease intelligence, preparedness, collaboration and cooperation among government agencies and cross-field partnerships. in developing countries, the preparedness plans for animal diseases are often unsatisfactory. incentives such as adequate compensation should be provided to the affected farmers as an impetus for reporting animal diseases. the level of preparedness should be assessed by conducting mock drills. this will help in confidence building for rapid detection and response to both eids and tads that appear suddenly and are capable of spreading to large areas in a short time. import bans in response to an animal disease outbreak must be based on sound scientific evidence to ensure that the concerned countries also have the incentive to report the disease to international agencies, namely oie, fao and who. deficiencies in national veterinary services have been attributed for inability in early detection of the disease and response as investigation, and diagnostic services have deteriorated in many regions. a continuing structural upgradation programme for national veterinary services will have to be taken into account for their transformation from providers of services, such as diagnosis, vaccinations and treatment of sick animals to inspection and quality assurance services. disease surveillance, early warning and emergency preparedness need to be pursued more vigorously in africa, the middle east and southeast asia as vital components of national veterinary services. though public health and national security are under the perspective of national governments, the decentralised government structure and improved international coordination are essential to address the threat of tads effectively as they do not respect local, regional or national boundaries. nevertheless, government support at the administrative level is essential to assure sufficient and timely response to avoid the spread of disease through livestock movement controls, closing of live markets, sharing of diagnostic services, expertise, funding, etc. technical support and guidance of international agencies, such as fao, who and oie, are key in the formulation and timely implementation of the plans and modalities for the control and management of eids and tads. the fao in established the global framework for the control of tads (gf-tad) through the emergency centre for transboundary animal disease (ectad) operations and emergency prevention system (empres) for transboundary animal and plant pests and disease initiatives for early warning and response to disease threats, following a collaborative approach to investigation at animal-human-ecosystem interface. these mechanisms have proved to be of immense help and use in the control, prevention and eradication of disease(s). microbial evolution, particularly viral evolution, is a continuous process. it is, therefore, necessary to conduct basic research on emerging infectious diseases, both viral and another microbial origin, for providing new insights about the factors responsible for the emergence of new microbes. for understanding the ecology of disease, social factors, viral and microbial traffic and spread, ecological and demographic changes in human and animal populations due to migration and other factors work in tandem leading to precipitation of emerging infections. these signals for viral and microbial traffic should be seen as warning signs. biodiversity should include microbes and viruses, and environmental impact assessment should include health aspects into account in development planning. enhancing surveillance systems by establishing laboratory response network at national, regional and international level is important for which adequate funds should be provided. by linking the laboratories in public and private domains, such networks are expected to enhance the capabilities at all levels to detect and prevent the spread of eids, transmitted naturally or intentionally (anonymous ) . a network of more than laboratories world over by who for a constant survey of influenza viruses is one of the best examples of networking of laboratories for eids and tads. these laboratories should have multidisciplinary teams involving veterinarians, physicians, ecologists, entomologists, vaccinologists, epidemiologists, molecular biologists, immunologists and possibly other specialists. state-of-the art disease surveillance is required having the capability to forecast when and where a particular disease is likely to occur for more targeted surveillance. such actionable intelligence may derive from the analysis of changes in climatic conditions, vegetation, wildlife demographics, trade pattern or vector demographics and distribution (anonymous ). the disease-producing microbes, particularly viruses and bacterial agents, often change their antigenic make-up as a result of spontaneous mutations, and immune pressure when the wild strains of the infectious agent persist in the host in the presence of vaccinal antibodies. rna viruses having segmented genome are more prone to such antigenic changes as a result of recombination, gene deletion, etc. influenza a viruses of human and animals continuously evolve new virulent variants by exchanging haemagglutinin (h) and neuraminidase (n) genes of various h and n types circulating in human, birds, pigs and other species including equines. with the change in antigenic make-up, the current vaccine strains do not provide protection against the new types of the virus. similar situations occur in fmd virus having seven types and further subtypes, clades and genotypes: ppr virus and newcastle disease virus, to name a few. new antigenic types of a virus or pathogen may also be introduced from abroad through imported livestock and poultry. hence, there is a need to have a plan in place to upgrade the vaccine by incorporating the current strains of the pathogen which induce strong and lasting immunity. this will require the setting of repositories of field strains isolated from disease outbreaks, particularly the ones from vaccine failure cases. such updates of vaccines are routinely followed for influenza vaccines for poultry, equines and human, and fmd and csf vaccine for livestock. vaccination is a valuable and well-tested method in preventive veterinary medicine for promoting animal health and welfare and reducing the risk of human exposure to several zoonotic pathogens. prophylactic immunisation practices, principles and vaccination protocols have helped in significantly reducing the prevalence of many life-threatening viral and bacterial diseases. the risks of not vaccinating their stock on account of lack of awareness among the stakeholders, non-availability of costeffective diagnostics and vaccines, and poor delivery of veterinary services to the livestock farmers can have serious consequences on livelihoods of rural livestock producers. effective vaccination programmes if implemented properly with a broader perspective are likely to reduce the need for antimicrobials, which in turn can help reduce the risk of emergent antimicrobial resistance. the world veterinary association (wva) and health for animals believe that it is essential for the global veterinary profession to educate the public, particularly animal keepers and producers, about the benefits of vaccination for animals and humans. the major objectives and motive of veterinary vaccines are to protect, improve and promote the health and welfare of companion and food animals; increase the production of livestock in a cost-effective manner; and prevent animal-to-human transmission of infectious diseases from domestic animals and wildlife to humans through animal-origin food, close contact and other mechanisms. these diverse aims have led to different approaches to the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations and naked dna injections for immunisation of animals. the final successful outcome of vaccine research and development is the generation of a product that will be available in the marketplace on demand and suitable to be used in the field to achieve desired outcomes. successful veterinary vaccines have been produced against major bacterial, viral, protozoan and multicellular pathogens, which led to successful field application and adaptation of novel technologies. these veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health through increasing safe food supplies, namely milk, meat, eggs and fish, and preventing animal-to-human transmission of infectious diseases. the continued interaction between the researchers from veterinary and medical streams and health professionals will be a major impetus for adapting new technologies, providing animal models of human diseases and confronting new and emerging infectious diseases. over different veterinary vaccines are currently commercially available (meeusen et al. ). multivalent (bivalent, trivalent and polyvalent) vaccines should be given preference over monovalent vaccines to cover more than one disease prevalent during control programmes to save money, time and other expenses and also to reduce the burden on implementing agencies, such as veterinarians and para-health livestock workers. there should be a system in place to conduct post-vaccination sero-monitoring in the field by appropriate agencies for finding evidence for adequate seroconversion in the randomly collected samples as per standard procedure preferably using diva tests to differentiate between vaccine-induced immune response and the one induced by the virulent virus. the application of risk analysis concerning the spread of disease on account of international trade in live animals and their products, namely, import risk analysis (ira), has been largely driven by the sanitary and phytosanitary (sps) agreement of the world trade organization (wto). the ira standard established by the world organisation for animal health (oie), and associated guidance, meets the needs of the sps agreement. the use of scenario trees is the core modelling approach adopted to represent the steps necessary for the hazard to occur. there is scope to elaborate scenario trees for commodity ira so that the quantity of hazard at each step is assessed (peeler et al. ) . the dependence between exposure and establishment of the hazard suggests that they should fall within the same subcomponent. ira undertaken for trade reasons must include an assessment of consequences to meet sps criteria. the integration of epidemiological and economic modelling may open a path for better methods. matrices have been used in qualitative ira to combine estimates of entry and exposure, and consequences with likelihood, but this approach has flaws, and better methods are needed. ira standards and guidance provided by oie indicate that the volume of trade should be taken into account. some published qualitative iras have assumed current levels and patterns of trade without specifying the volume of trade, which constrains the use of ira to determine mitigation measures (to reduce risk to an acceptable level) and whether the principle of equivalence, fundamental to the sps agreement, has been observed. it is questionable whether qualitative ira can meet all the criteria set out in the sps agreement. nevertheless, scope exists to elaborate the current standards and guidance, so that they better serve the principle of science-based decision-making. options for trade from disease-free zones and disease-free compartments and trading in safe commodities are now available to have a positive mechanism for facilitating international trade. in india, fmd-control program (fmd-cp) is already in operation intending to create fmd-free zones. similar zones can be created for other diseases like hs, bluetongue, sheep pox, goat pox, ppr and other important diseases. compartmental biosecurity is the new concept for the management of biosecurity in a compartment through a single set of biosecurity measures. creation of zones/compartments will ensure a boost in international trade of livestock and poultry products. in india, legislation regarding the movement of animals across these zones and compartments are required by the central and state governments. the tads are a threat to animal health and production and cause huge losses to the economy of nations. recent outbreaks of bovine spongiform encephalopathy (bse), foot-and-mouth disease (fmd) and highly pathogenic avian influenza (hpai) have unfolded the real and growing global threat that animal diseases pose to livestock systems and to human health and welfare. the tads adversely affect the trade in live animals and their products. the detection of one bse-positive animal in in the usa led to an % drop in beef exports during [ ] [ ] . similarly, the losses in the uk were estimated to be over us$ billion during the ill-fated fmd outbreak. the economic losses due to hpai (h n ) avian influenza have been estimated from . % to . % of gdp in thailand and vietnam by rushton et al. ( ) . the outbreak of avian influenza due to h n strain in the netherlands destroyed as many as million birds. direct losses due to fmd in india have been estimated to the extent of inr , million per annum (anonymous - ) . ppr has been estimated to cause global losses between us$ . billion and $ . availability of adequate financial support for animal health r&d, especially in developing countries, is not always readily ensured. as the livestock keepers in these countries are mostly socio-economically poor, the local and national governments should come forward to support these programmes, particularly for the landless and marginal farmers keeping pigs, sheep, goats, backyard poultry and low-producing bovine stocks by providing incentives or subsidies for diagnostics and vaccines. raising venture fund for emergency disease control through public and private partnership could be considered to meet the urgent requirements, besides farmer-friendly insurance policies for livestock health protection. for important tads, such as avian influenza, ppr and fmd, multinational, regional or global programmes under the supervision of fao, who and oie under 'one health' concept are suggested for better coordination and results. the 'one world-one health' (owoh) concept steered by fao, who and oie has its roots in the interaction between living beings including humans, animals and pathogens, and the environment is considered as a unique dynamic system in which the health of each component is interconnected and dependent with other components. nowadays, a newly integrated 'one health one medicine' approach reflects this interdependence with a holistic view of the ecological system. the owoh can be defined as a collaborative and a multidisciplinary effort at the local, national and global level to guarantee an optimal healthy status for humans, animals and environment. the control of infectious diseases, which have influenced the course of human history, is to be considered strictly related to the one health concept. after its first occurrence in in china, the highly pathogenic avian influenza (hpai) a virus (h n ) has affected more than countries in asia, europe, africa and north america. the virus affected wild birds as well as domestic poultry. sporadic cases of transmission to humans in close contact of infected birds with sizeable mortality raised the pandemic concern of 'bird flu'. after the first report of the h n virus from india and bangladesh in and , respectively, both these countries are experiencing outbreaks almost every year. between february-march and february , india incurred an expenditure of more than inr . million, including inr . million for compensation and inr million on the culling of . million birds (anonymous - ) . avian influenza viruses (aivs) have become a continued threat to global health and economy. after its first outbreak in , the h n hpai serotype disseminated very fast from korea to other parts of asia, europe and north america, a feature not observed in case of other highly pathogenic aivs. however, the pathobiological features of the virus that favoured its global translocation are not known. results of simulation studies undertaken in migratory birds to identify pathobiological features supporting aiv intercontinental dissemination risk suggest that characteristic differences exist among h n and other aiv subtypes, e.g. h n and h n that have not spread as rapidly. lower infection recovery and mortality rates and migration recovery rates also favour translocation in migratory bird populations. although india has been reporting h n aiv since , the h n virus was first time reported in from migratory birds and poultry in the states of delhi, madhya pradesh, kerala, karnataka, punjab and haryana. studies undertaken on comparative epidemiology of influenza viruses h n and h n among human and bird populations to find out similarities and differences between the two viruses in their genetic characteristics, distribution patterns in human and bird populations and postulated mechanisms of global spread (bui et al. ) indicated that h n viruses are diversifying at a much greater rate than h n viruses. analyses of certain h n strains demonstrated similarities with engineered transmissible h n viruses, which make it more adaptable to the human respiratory tract. these differences in the epidemiology of h n and h n viruses in human and birds raise further questions as to how h n has spread at a greater rate than the h n virus. african swine fever (asf) is a highly contagious, deadly emerging disease of pigs in many countries. although first described in and it affected more than countries in africa, europe and south america, several key issues about its pathogenesis, immune evasion and epidemiology remain uncertain (arias et al. ). in the absence of a vaccine, the disease causes greater sanitary, social and economic impacts on swine herds compared to many other swine diseases. currently, asf is present in sub-saharan africa, sardinia, the trans-caucasus, the russian federation and central and eastern states of the european union. the disease continues to spread, with first reports in china (august ), bulgaria (august ), belgium (september ) and vietnam (february ) highlighting the increasing threat of asf to the global pig industry (netherton et al. ) . ongoing outbreaks have also been reported in hungary, latvia, moldova, poland, romania, russia, south africa, ukraine, cambodia, north korea, vietnam and laos. the disease was rampant in china during , and about half of china's breeding pigs died or were slaughtered. the threat of asf looms large as presently no licensed vaccine is available against this disease, and further research is desired in this area for the development of live attenuated vaccines for asfv. it has been possible to generate pigs resistant to classical swine fever virus and prrs virus (burkard et al. ) by using genetic modification of the host species. genetic modification can be attempted as a viable solution to increase the host resistance to asfv. wild suids, namely warthog or bush pig, sequences could be engineered into the domestic pig genome to produce animals in which replication of asf virus and/or disease burden after asfv infection is reduced. however, to generate pigs fully resistant to asfv infection, a more effective strategy such as targeting the virus receptors on the host cell to block the entry of virus and viral replication may be attempted. different clinical courses of asfv infection in pigs have been described based on the virulence of the virus isolates, and sequencing the genomes of isolates of reduced virulence has identified virus genes associated with this phenotype. targeted gene modifications and deletions and testing of the genetically modified viruses in macrophages and pigs have contributed to an understanding of virulence factors and how the virus modulates host responses. in the absence of a vaccine and rapid spread of asf in europe and asia, the main emphasis should be on strict customs and border protection to keep the negative countries free from asf virus infection/ disease. research is required on priority to explore the virulence genes and genes related to host protection and immune evasion, role of multigene families in antigenic variability, mechanism of evasion of the immune response, factors determining viral persistence and infection outcomes, and interactions between asfv and wild african suids, which are tolerant to asfv infection. such studies will provide a complete understanding of the pathogenesis of asf. the specific role of different hosts including wild suids, vectors and environmental factors in disease propagation needs to be elucidated for understanding different epidemiological scenarios. in this regard, the northern european scenario in which infected wild boars drive disease transmission and maintenance needs to be investigated further. presently, asf has become of great significance in china and a real threat to the pig and pork production. the affected countries are planning to compensate for the losses in pork production by increasing broiler poultry production. gaps in sanitary control of wild boar populations make asf control difficult. raising awareness among veterinarians, hunters and farmers should be the priorities for asf control. advances in non-invasive sampling are required to facilitate surveillance in affected areas. current and future tests need to be optimised for noninvasive matrices. the availability of a confirmatory serological test and cell lines for replacing primary cell cultures should be the priorities for future work. availability of safe and potent vaccine against asf could benefit disease control and prevention substantially, but despite some advances such vaccine is still lacking (arias et al. ). after the successful eradication of rinderpest from the globe in , foot-andmouth disease (fmd) of cloven-footed animals is another oie-listed important viral disease inflicting heavy economic losses and adversely affecting the trade of livestock and livestock products from endemic countries to fmd-free nations/ regions. knight-jones et al. ( ) have given a detailed account of global fmd research update along with gaps and an overview of global status and research needs. the conclusions are drawn to highlight that currently available vaccines and control tools have enabled fmd eradication from many countries of the developed world. however, in many developing countries, fmd remains uncontrolled. the main reason given is that biosecurity measures that have been fundamental to successful fmd control in the developed country are difficult to be implemented effectively in developing countries due to obvious reasons. in the present scenario, improved vaccines, with longer lasting protection against a wider range of fmdv strains and lower production costs, could be the single most important development to enhance our ability to control fmd. although encouraging progress has been made with several novel vaccine candidates, addressing key limitations of the current inactivated vaccines, a commercial vaccine is yet awaited. while new discoveries are crucial, current vaccines have been used to effectively control fmd on numerous occasions. however, for imparting better immunity, fmd vaccines should be subjected to adequate quality assurance and be made available in sufficient quantity to provide desired coverage following appropriate strategy. there is also a need for better training and support in the design and execution of vaccine-based fmd control programmes. another area of research is genetic and molecular studies on the virus to elucidate host-virus interactions. more powerful tools and analyses are increasing our understanding of various aspects of fmdv evolution, ecology and epidemiology. this, in turn, should benefit many areas of fmd research, from basic virology to the vaccine and diagnostic development. furthermore, improved genetic technologies have the potential to reveal information crucial for control, such as transmission chains, vaccine match and level of virus circulation. fmd control has been prioritised by many governments around the world. besides traditional bastions of established research institutes in europe and north and south america, notable work is being conducted in china, india and africa. experiences in south america and europe have shown that through decades of sustained investment fmd can be controlled, even in regions where once it was rampant and control was seemingly impossible. however, if improved and more widespread fmd control is to be achieved, continued investment in fmd research at the local and international level is a must. improved diva diagnostics increase our ability to detect infected animals in vaccinated populations. greater confidence in the ascertainment of fmd status of animals and products has, in turn, opened the way for international standards for trade and disease control that are more efficient and less restrictive. rigorous licensing procedures increase the time taken for new technologies for diagnostic kits and vaccines to reach the market. however, if authorisation is less rigorous, substandard products may be released onto the market. hence, there is a need to balance these two requirements. relaxation should be provided for necessary changes to the existing technologies, such as changing vaccine strains, particularly when the need is urgent (knight-jones et al. ). rinderpest, also known as 'cattle plague', was once a deadly serious threat to the livestock industry and agriculture economy in several regions of the globe, particularly in asia, africa, europe and the americas. it periodically swept through old world, resulting in devastating epizootics and huge economic losses. the disease could be successfully eliminated from the globe with mass vaccination programmes, zoo sanitary measures, policy support, international cooperation and political will. the morbidity and mortality rates in newly exposed naïve populations could be as high as - % leading to enormous economic losses. in india mortality rate of about , animals were recorded among the affected bovine population of , per annum during the first half of the s, indicating average mortality of %. throughout the history of humankind, the social, economic and ecological consequences due to rinderpest had been more catastrophic, even changing the history of nations and empires. in india, the presence of rinderpest was confirmed by the cattle plague commission (hallen et al. ) . this disease has been conquered successfully by following mass vaccination along with zoo sanitary measures. the fao declared the global eradication of rinderpest on june , marking it the first ever viral disease of animals eradicated globally about three decades after the eradication of smallpox, a viral disease of humans in (yadav et al. ) . constraints of availability of quality vaccine in sufficient quality, freeze-drying of vaccines and maintenance of cold chain for a vaccine in tropical countries, lack of infrastructure for structured clinical surveillance and sero-surveillance were some of the limitations in executing the mass vaccination programmes. in india, dividing the country into four zones based on the epidemiological picture of the disease and adopting strategic and focused vaccinations at interstate and international borders and migration routes of bovines and caprine for creating immune belts, coupled with rigorous clinical and sero-surveillance, were of great help in achieving freedom from the infection. the financial support and/or technical guidance from fao, oie, eu and iaea were the driving forces in achieving infection-free status for india in the year . with the successful eradication of rinderpest, the livestock sector across the globe became safer, and consequently the living standard of livestock farmers improved. the success of rinderpest control and eradication proved a rewarding experience and landmark for the veterinary services in india, providing capacity building and confidence among field veterinarians, researchers, policy planners and donor agencies and other stakeholders to undertake a successful control programme of livestock diseases at the national level. the freedom of the country from rinderpest not only enabled the growth of the dairy industry in india but has also boosted the export of meat and other dairy products in the recent decade. today india tops not only in milk production in the world but is also the largest exporter of buffalo meat. cost-benefit analyses indicated that every dollar spent on rinderpest control programme gained about $ to the indian dairy industry through more milk, meat and draft power for better agricultural productivity (uppal ). in the fate of rampant threat due to eids and tads, a diverse, dynamic and wellplanned structured disease surveillance and monitoring approach would be the key for the sustainability and welfare of healthy livestock production systems of any country. preparedness for combating the prevailing, emerging, re-emerging eids and tads requires robust monitoring and precision detection systems that are flexible, feasible and adaptable under field conditions. in this regard, pen-side diagnostic tests/lab-on-the chip tools are the need of the hour. the hurdles of sampling need to be curtailed opting non-invasive methods for sample collection from different animal species and wildlife. transparency in disease reporting needs to be adhered to and reported to oie. because of trading in animals and animal products, the international obligation for oie reportable diseases of high importance must be followed by all member countries of wto. it is high time to apply developed diagnostics and molecular detection tools in the field to ensure fast detection and confirmation of pathogens capable of causing diseases in humans and animals. this must be accompanied by national-level disease surveillance, monitoring and networking to enable an early warning system for infectious diseases based on forecasting (saminathan et al. ) . due priority is also required for development and application of new potent, safe and affordable vaccines and vaccine delivery systems and adopting innovative vaccination programmes and immunomodulatory and effective therapeutic modalities, which would help in devising timely prevention and control strategies against viral and other infectious diseases. besides these, good manage-ment and standard biosecurity and biosafety measures/practices and appropriate hygienic and zoo sanitary and quarantine measures should be observed. moreover, on-the-spot control and checking of the spread of pathogens and adequate trade restrictions as envisaged under the sps agreement of wto also need to be followed. a holistic vision and approaches are required for timely implementation of these concepts and strategies along with the strengthening of various multidimensional research and development programmes supported by appropriate funding resources. these measures will greatly help to minimise disease incidences and outbreaks, and lessen economic burdens due to infectious animal diseases and boost livestock and poultry health, reproduction and production to strengthen sustainable growth of livestock and poultry industry. reduction in pandemic threats and public health concerns eventually lead to an improvement in the socioeconomic status and welfare of the society at large under 'one health' umbrella. application of artificial intelligence (ai), gps, remote sensing and traceability in disease detection and management needs priority attention in developing countries. similarly, the latest techniques of gene editing, base editing, nanotechnologies, electronic nose, etc. should be applied for efficient disease diagnosis and drug delivery. while planning the breeding policies for livestock and poultry, both higher production performance and health of the progeny should be given equal weightage. modern techniques should be used for developing disease resistance (absolute or partial) in livestock and poultry using indigenous germplasm. institute of medicine (iom) report - ) annual report, icar-directorate of foot-and-mouth disease - ) annual report, department of animal husbandry, dairying and fisheries, ministry of agriculture and farmers welfare, govt. of india gaps in african swine fever: analysis and priorities a systematic review of the comparative epidemiology of avian and human influenza a h n and h n -lessons and unanswered questions pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection emerging infectious diseases of wildlife-threats to biodiversity and human health allijan mm ( ) the cattle plague commission report to government of india global foot-and-mouth disease research update and gap analysis: -overview of global status and research needs emerging infections: microbial threats to health in the united states zika virus-an imminent risk to the world current status of veterinary vaccines advances in developing therapies to combat zika virus: current knowledge and future perspectives the genetics of life and death: virus-host interactions underpinning resistance to african swine fever, a viral hemorrhagic disease animal disease import risk analysis-a review of current methods and practice: open access article avian influenza and other transboundary animal diseases, director, liaison office for north america, food and agriculture organization of the united nations. presentation at "health in foreign policy forum impact of avian influenza outbreaks in the poultry sectors of five south east asian countries prevalence, diagnosis, management and control of important diseases of ruminants with special reference to indian scenario ebola virus-epidemiology, diagnosis and control: threat to humans, lessons learnt and preparedness plans-an update on its year's journey nipah virus: epidemiology, pathology, immunobiology and advances in diagnosis, vaccine designing and control strategies-a comprehensive review peste des petits ruminants: sheep and goat plague. today and tomorrow's printers and publishers fao sponsored final project report "national testimonies" under the global rinderpest eradication programme (grep)-(gcp/glo/ /ec) fao sponsored final project report on "laboratory contributions for rinderpest eradication in india" under the global rinderpest eradication programme (grep)-(gcp/ glo/ /ec) animal sciences. in: singh rb (ed) years of agricultural sciences in india acknowledgements all the authors of the manuscript thank and acknowledge their respective universities and institutes. there is no conflict of interest. key: cord- -aknktp d authors: bello-orgaz, gema; hernandez-castro, julio; camacho, david title: a survey of social web mining applications for disease outbreak detection date: journal: intelligent distributed computing viii doi: . / - - - - _ sha: doc_id: cord_uid: aknktp d social web media is one of the most important sources of big data to extract and acquire new knowledge. social networks have become an important environment where users provide information of their preferences and relationships. this information can be used to measure the influence of ideas and the society opinions in real time, being very useful on several fields and research areas such as marketing campaigns, financial prediction or public healthcare among others. recently, the research on artificial intelligence techniques applied to develop technologies allowing monitoring web data sources for detecting public health events has emerged as a new relevant discipline called epidemic intelligence. epidemic intelligence systems are nowadays widely used by public health organizations like monitoring mechanisms for early detection of disease outbreaks to reduce the impact of epidemics. this paper presents a survey on current data mining applications and web systems based on web data for public healthcare over the last years. it tries to take special attention to machine learning and data mining techniques and how they have been applied to these web data to extract collective knowledge from twitter. web is one of the most important sources of data in the world producing amounts of public information. the exponentially increasing of websites and online web services in the last years has allowed new interdisciplinary challenges for several fields and computer science, such as marketing campaigns [ ] [ ] , financial prediction [ ] or public healthcare [ ] [ ] [ ] , among others. recently, the research on artificial intelligence techniques applied to develop technologies allowing monitoring web data sources for detecting public health events has been emerged as a new relevant discipline called epidemic intelligence (ei). ei can be defined as the early identification, assessment and verification of potential public health risks [ ] , and the timely dissemination of the appropriate alerts. this discipline includes surveillance techniques such as automated and continuous analysis of unstructured free text information available on web from social networks, blogs, digital news media or official sources. surveillance systems are nowadays widely used by public health organizations such as world health organization (who) or the european centre for disease prevention and control (ecdc) [ ] . tracking and monitoring mechanisms for early detection are critical in reducing the impact of epidemics giving a rapid response. for instance, several of these systems can be able to discover early events of the disease breakout during the a(h n ) influenza pandemic in [ ] . traditional epidemic surveillance systems are implemented from virology and clinical data, which is manually collected, and often these traditional systems have a delay reporting the emerging diseases. but in situations like epidemic outbreaks, real-time feedback and a rapid response is critical. social web media is a profitable medium to extract the society opinion in real time. blogs, micro-blogs (twitter), and social networks (facebook) enable people to publish their personal opinions in real time, including geo-information about their current locations. these big data with situation and context aware information about the users provide a useful source for public healthcare. however, the extraction of information from web is a difficult task due to its unstructured definition, high heterogeneity, and dynamically changing nature. because of this diversity in the data format, several computational methods are required for its processing and analysing [ ] (data mining, natural language processes (nlp), knowledge extraction, context awareness, etc...). this paper presents a survey on current data mining applications and web systems based on web data for public healthcare over the last years. it tries to take special attention to machine learning and data mining techniques, and how they have been applied to these web data to extract collective knowledge from social networks like twitter. the rest of the paper has been structured as follows: section shows the state of the art of the existing epidemic intelligence systems. section describes the different web mining techniques used to detect disease outbreaks. section provides an overview of twitter applications for monitoring and predicting epidemic and their experimental results. finally, the last section presents a discussion of the main features extracted from this survey. nowadays, large amounts of emergency and health data are increasingly coming from a large range of web and social media sources. this information can be very useful for disease surveillance and early outbreak detection, and several public web surveillance projects in this field have emerged over the recent years. one of the earliest surveillance systems is the global public health intelligence network (gphin) [ ] developed by public health agency of canada in collaboration with who. it is a secure web-based multilingual warning tool that is continuously monitoring and analysing global media data sources to identify information about disease outbreaks and other events related to public healthcare. the information is filtered for relevancy by an automated process, and categorized based on a specific taxonomy of categories. then this information is analysed by public health agency of canada gphin officials. from to years, this surveillance system was able to detect the outbreak of severe acute respiratory syndrome (sars). since , biocaster [ ] is an operational ontology-based system for monitoring online media data. this system is based on text mining techniques for detecting and tracking the infectious disease outbreaks through the search of linguistic signals. the system continuously analyses documents reported from over rss feeds, google news, who, promed-mail, and the european media monitor, among others providers. the extracted text are classified for topical relevance and plots them onto a google map using geo-information. the system consists of four main stages: topic classification, named entity recognition(ner), disease/location detection and event recognition. in the first stage, the texts are classified into relevant or non-relevant categories using to train a naive bayes classifier. then, for relevant document corpus are search entities of interest from concept types based on the biocaster ontology [ ] related to diseases, viruses, bacteria, locations and symptoms, see figure . healthmap project [ ] is a global disease alert map which uses data from different sources such as google news, expert-curated discussion such as promed-mail, and official organization reports such as world health organization (who) or euro surveillance. this is an automated real-time system that monitors, organizes, integrates, filters, visualizes, and disseminates online information about emerging diseases as can be seen in figure . other system which collects news from the web, related to human and animal health, and plot the data on a google maps mashupare is epispider [ ] . this tool automatically extracts infectious disease outbreak information from several sources including promed-mail and medical web sites, and it is used as surveillance system by public healthcare organizations, several universities and health research organization. additonally, this system automatically converted the topic and location information of the reports into rss feeds. other public health surveillance system used by a public health organization (the european centre of disease prevention and control) is medisys [ ] monitoring human and animal infectious diseases, as well as chemical, biological, radiological and nuclear (cbrn) threats in open-source media. medisys automatically collects articles concerning public health in various languages from news, which are classified according to pre-defined categories as can be seen in figure . users can display world maps in which event locations are highlighted as well as statistics on the reporting about diseases, countries and combinations of them, also can apply filters for language, disease or location. a specific and extensive application of predictive analytic techniques to public health approach are the monitoring systems of influenza through web and social media. google flu trends [ ] uses google search data to estimate flu activity during two weeks giving an early detection of disease activity, see figure . this web service correlates search term frequency with influenza statistics reported by the centers for disease control and prevention (cdc), and it enables a quicker response in a potential pandemic of influenza, thus reducing its impact. internet users perform search queries [ ] and post entries in blogs using terms related to influenza illness as its diagnosis and symptoms. an increase or decrease in the number of illness searches and posts in blogs, reflects a higher or lower potential outbreak focus for influenza illness and can therefore be used to monitor it. finally all the systems mentioned together with their main characteristics are listed in table . the problem of detecting and tracking epidemic outbreaks through social media can be defined as the task of extracting relevant knowledge about the epidemics in the real world given a stream of textual or multimedia data from social media. web mining is the application of data mining techniques to discover and retrieve useful knowledge from the web documents and services. therefore, the application of these techniques to knowledge extraction provides a better using and understanding of the data space on biomedical and health care domain [ ] . there are several health data sources very useful to detect and prevent new outbreaks of different diseases. social web media and web sites give a large amount of useful data for this purpose. other important data sources are search engines such as google and yahoo! [ ] [ ] . in this case, the objective is to detect specific searches that involve terms that indicate influenza-like-illness (ili) through the keywords of the queries performed. the complexity is to interpret the search context of the query, as the user may query about a particular drug, symptom or illness for a variety of reasons. finally, promed-mail [ ] is also a widely data source used for disease outbreaks detection. it is a human network of expert volunteers operating / as an official program of the international society for infectious diseases. their volunteers monitor global media reports and in many cases have a reporting time of outbreak disease alerts better than who reports. text mining techniques have been applied on biomedical text corpus to named entity recognition, text classification, terminology extraction, or relationship extraction [ ] . these methods are a human language processing algorithms that aim to convert unstructured textual data from large-scale collections to a specific format filtering them according to the needs. once the data have been extracted from the social media sites (rss feeds, www, social networks, promed-mail, search engines, etc...), the next stage is to perform the text analysis methods for the trend detection, identifying potential sources of disease outbreaks. these methods can be used to detect words related to diseases or their symptoms in published texts [ ] . but this goal can be difficult because the same word can refer to a different thing depending upon context. furthermore, a specific disease can have multiple names and symptoms associated which increases the complexity of the problem. ontologies can help to automate human understanding of key concepts and relations between them and allow that a level of filtering accuracy can be achieved. biomedical ontologies contain lists of terms and their human definitions, which are then given unique identifiers and classified into classes with common properties according to the specific domain treated. in the domain of ei it is necessary to identify and link term classes such as disease, symptom and species in order to detect potential focus diseases. currently there are various available ontologies that contain all the biomedical terms necessary. for example, biocaster ontology (bco) [ ] is in the owl semantic web language to support automated reasoning across terms in languages. a new unsupervised machine learning approach to detect public health events is proposed in fisichella et al. work [ ] which can complement existing systems since it allows to identify public health events (phe) even if no matching keywords or linguistic patterns can be found. this new approach defines a generative model for predictive event detection from document by modeling the features based on trajectory distributions. discovering time and location of the text is the value added by ei systems for high quality. in practice location names are often highly ambiguous because geotemporal disambiguation is so difficult, and because of the variety of ways in which cases are described across different texts. keller et al. [ ] work provides a review of the issues for epidemic surveillance and present a new method for tackling the identification of a disease outbreak location based on neural networks trained on surface feature patterns in a window around geo-entity expressions. finally, a different solution for outbreak detection is shown in leskovec et al paper [ ] , where the problem is modelled as a network in order to detect the spreading of the virus or disease as quickly as possible. they present a new methodology for selecting nodes to detect outbreaks of dynamic processes spreading over a graph. this work shows that many objective functions for detecting outbreaks in networks, such as detection time, likelihood, and population affected, are submodular. this means that, for instance, reading only a few blogs provides more new information than reading it after we have read many ones. they use this characteristic to develop an efficient approximation algorithm (celf) which achieves near-optimal solutions and it is times faster than a simple greedy algorithm. the increasing popularity and use of micro-blogging services such as twitter are recently a new valuable data source for web-based surveillance because of its message volume and frequency. twitter users may post about an illness, and their relationships in the network can give us information about which people could be in contact with. furthermore, user posts retrieved from the public twitter api can come with gps-based location tags, which can be used to detect potential disease outbreaks for a health surveillance system. recently, several works have already appeared shown the potential of twitter messages to track and predict disease outbreaks. ritterman et al. [ ] work is focused on using prediction market to model public belief about the possibility that h n virus will become a pandemic. in order to forecast the future prices of the prediction market, they decided to use the support vector machine algorithm to carry out regression. a document classifier to identify relevant messages is presented in culotta et al. paper [ ] . in this work, twitter messages related to flu were recollected during weeks using keywords such as flu, cough, sore throat or headache. then, several classification systems based on different regression models to correlate these messages with cdc statistics were compared, finding that the best model achieves a correlation of . (simple model regression). aramaki et al. [ ] presents a comparative study of various machine-learning methods to classify tweets related to influenza into two categories: positive or negative. their experimental results show that svm model using a polynomial kernel achieves the highest accuracy (fmeasure of . ) and the lowest training time. a novel real-time surveillance system to detect cancer and flu is described in paper [ ] . the proposed system continuously extracts text related the two specific diseases from twitter using twitter streaming api and applies spatial, temporal, and text mining to discover disease-related activities. the output of the three models is summarized as pie charts, time-series graphs, and us disease activity maps on the project website as can be seen in figure . this system can be useful not only for early prediction of disease outbreaks, but also for monitoring distribution of different cancer types and the effectiveness of the treatments used. well known regression models are evaluated on their ability to assess disease outbreaks from tweets in bodnar et al. [ ] . regression methods such as linear, multivariable an svm, are applied to the raw count of tweets that contain at least one of the keywords related to a specific disease, in this case "flu". the results confirmed that even using irrelevant tweets and randomly generated datasets, regression methods were able to assess disease levels comparatively well. finally a summary of all the systems mentioned and the machine learning techniques used is listed in table . it can be noticed that most of the works use regression models, and are usually focused on detecting influenza outbreaks. all the systems and solutions presented have demonstrated the successful and beneficial use of artificial intelligence techniques when applied to extract and acquire new knowledge for public healthcare purposes. the main challenge of these systems is to interpret the search context of a particular query or document, because an user can query about a particular drug, symptom or illness for a variety of reasons. this goal can be difficult because the same word can refer to a different thing depending upon context. furthermore, a specific disease can have multiple names and symptoms related to it, which increases the complexity of the problem. therefore, to develop strategies for reducing false alarms and decreasing percentage of irrelevant events detected by the epidemic systems can be an important issue for future works and researches on the field. additionally, to identify the time and location of messages is a value added for increasing the quality of detecting possible new diseases outbreaks. but in practice location names are often highly ambiguous because geo-temporal disambiguation is so difficult, and because of the variety of ways in which cases are described across different texts. there are several recent works show the potential of twitter to track and detect disease outbreaks. these works demonstrate that there are health evidences in social media which can be detected. but, there can be complications regarding the possible incorrect predictions because of the huge amount of social data existing compared with the small amount of relevant data related to potential diseases outbreaks. therefore, it is necessary to test and validate carefully all the models and methods used. twitter catches the flu: detecting influenza epidemics using twitter predicting the future with social media extracting collective trends from twitter using social-based data mining validating models for disease detection using twitter surveillance sans frontieres: internet-based emerging infectious disease intelligence and the healthmap project google trends: a web-based tool for real-time surveillance of disease outbreaks ai for global disease surveillance scalable influence maximization for prevalent viral marketing in large-scale social networks a survey of current work in biomedical text mining uncovering text mining: a survey of current work on web-based epidemic intelligence biocaster: detecting public health rumors with a web-based text mining system an ontology-driven system for detecting global health events towards detecting influenza epidemics by analyzing twitter messages detecting health events on the social web to enable epidemic intelligence detecting influenza epidemics using search engine query data the landscape of international eventbased biosurveillance social web mining and exploitation for serious applications: technosocial predictive analytics and related technologies for public health, environmental and national security surveillance use of unstructured event-based reports for global infectious disease surveillance automated vocabulary discovery for geo-parsing online epidemic intelligence nowcasting events from the social web with statistical learning real-time disease surveillance using twitter data: demonstration on flu and cancer costeffective outbreak detection in networks medisys: medical information system the global public health intelligence network and early warning outbreak detection epidemic intelligence: a new framework for strengthening disease surveillance in europe using internet searches for influenza surveillance using prediction markets and twitter to predict a swine flu pandemic promed-mail: an early warning system for emerging diseases detecting and tracking disease outbreaks by mining social media data key: cord- - su uan authors: lynteris, christos title: introduction: infectious animals and epidemic blame date: - - journal: framing animals as epidemic villains doi: . / - - - - _ sha: doc_id: cord_uid: su uan the introduction to the edited volume summarises the chapters of the volume and discusses their contribution in the context of current historical and anthropological studies of zoonotic and vector-borne disease, with a particular focus on how epidemic blame is articulated in different historical, social and political contexts. of 'emerging infectious diseases' (eid), which configures the rise of new diseases as carrying with it a potential for human extinction. this volume examines the history of the emergence and transformation of epidemiological and public health framings of non-human disease vectors and hosts across the globe. providing original studies of rats, mosquitoes, marmots, dogs and 'bushmeat', which at different points in the history of modern medicine and public health have come to embody social and scientific concerns about infection, this volume aims to elucidate the impact of framing non-human animals as epidemic villains. underlining the ethical, aesthetic, epistemological and political entanglement of non-human animals with shifting medical perspectives and agendas, ranging from tropical medicine to global health, the chapters in this volume come to remind us that, in spite of the rhetoric of one health and academic evocations of multispecies intimacies, the image and social life of non-human animals as epidemic villains is a constitutive part of modern epidemiology and public health as apparatuses of state and capitalist management. whereas the above approaches (including microbiome studies, and 'entanglement' frameworks in medical anthropology) do contribute to a much-needed shift in the intellectual landscape as regards the impact of animals on human health, their practical and political limitations are revealed each time there is an actual epidemic crisis. then, all talk of one health, multispecies relationships and partnerships melts into thin air, and what is swiftly put in place, to protect humanity from zoonotic or vector-borne diseases, is an apparatus of culling, stamping out, disinfection, disinfestation, separation and eradication; what we may call the sovereign heart of public health in relation to animal-borne diseases. for the maintenance and operation of this militarised apparatus, the framing of specific animals as epidemic villains is ideologically and biopolitically indispensable, even when blame of the 'villain' in question lacks conclusive scientific evidence (see thys, this volume). going against the grain of scholarship that in recent years has sought to portray the vilification of animals as hosts and spreaders of disease as a thing of the past, histories of non-human disease hosts and vectors aims to illuminate the continuous importance of this ideological and biopolitical cornerstone of modern epidemiology and public health. representations of animals as enemies, antagonists or sources of danger have, in different forms, shapes and degrees, been part and parcel of human interactions with the non-human world across history. it is, however, only at the turn of the nineteenth century that, as a result of bacteriological breakthroughs, non-human animals began to be systematically identified and framed as reservoirs and spreaders of diseases affecting humans. to take one famous example, before the end of the nineteenth century, rats were not believed to be carriers or spreaders of plague or any other infectious disease. whereas rats had long been considered to be damaging to human livelihood, due to consuming and spoiling food resources, their only redeeming characteristic was, erroneously, widely believed to be their supposed disease-free nature. hence while mid-seventeenth-century plague treatises noted the rat's destructive impact on fabrics and food, no mention of its connection with the disease was made. equally, two centuries later, when in - british colonial officers in india observed that, at the first sight of rat epizootics, garhwali villagers fled to the himalayan foothills in fear of the 'mahamari' disease, they dismissed this behaviour as merely superstitious. however, the bacteriological identification of rats as carriers of plague or mosquitoes as carriers and spreaders of yellow fever and malaria, at the end of the nineteenth century, was itself enabled and indeed complicated by an already-existing stratum of signification which, by the mid-seventeenth century, had led to the introduction of new symbolic, ontological and legal frameworks of thinking about animals as 'vermin'. vermin, in mary fissell's definition, 'are animals whom it is largely acceptable to kill', not because of some inherent characteristic they possess, but because, in specific historical contexts, 'they called into question some of the social relations which humans had built around themselves and animals'. paraphrasing fissell, we may say that, arising in early modern europe, the category 'vermin' problematised animals which devoured or destroyed the products of human labour and the means of human subsistence in terms of an agency or intentionality that confounded human efforts to control them. departing from the structuralist influences of mary douglas, which dominated animal studies in the s (see, for example, robert danton's work on the great cat massacre in france), and from keith thomas' 'modernisation' reading of vermin as simply animals that were of no use in an increasingly utilitarian world, fissell's discourse analysis of popular texts on vermin from seventeenth-century england was the first to dwell in the social historical reality of the emergence of this notion. however, more recent studies have opposed fissell's idea that what made vermin a threat to 'human civility' was their perceived 'greed and cunning', or their overall 'trickster' character. lucinda cole's recent monograph imperfect creatures argues that, 'what made vermin dangerous was less their breedspecific cleverness or greed than their prodigious powers of reproduction through which individual appetites took on new, collective power, especially in relation to uncertain food supplies'. the two approaches are not mutually exclusive. indeed, if approached anthropologically, they point to an entanglement between symbolic and economic aspects of vermin as threats to 'social integrity', something that is further supported by the association of vermin at the time with vagrancy and the poor. medical historians have in turn noted the association of vermin with miasma in disease aetiologies and public health practices of early modern europe, especially in times of epidemics when extensive legislation against them and prescriptions for their destruction are recorded. this was particularly the case in the context of plague outbreaks that had long been associated with 'putrid' and 'corrupt' vapours, which certain animals, like dogs, pigs, cats and poultry (and their excrements and carcasses), were believed to emanate. as in the late middle ages, the fear of pestilential miasmata emanating from offal and other meat products had led to the spatial regulation of butchery in england and other parts of europe (cf concerns with 'bushmeat' in relation to ebola; thys, this volume), william riguelle has shown that, in the course of the seventeenth century, concerns with 'noxious' animals played an important role in instituting limits of where these could be kept and where they could be allowed to roam in urban environments. the idea of miasma would continue to impact medical thinking into the nineteenth century. as a part of ontologies that escape both the straightjacket of recent anthropological classifications and classical medicalhistorical dichotomies of contagionism/anti-contagionism, the idea of miasma was malleable, adaptable and ambiguous enough to be compatible with, rather than antagonistic to, that of infection and contagion. however, as new medical and biopolitical challenges arose in the context of colonial conquest, the problematisation of animal-derived miasma or 'febrile poison' gave way to concerns about the climate as the driving force of epidemic disease. thus while the dawn of bacteriology, by the s, did not introduce understandings of animals as sources of disease ex nihilo, it did mark a drastic return to this idea, and, at the same time, led to a significant conceptual shift as regards the ontology of the diseases transmitted, and the mechanism involved in this transmission. this transformation was catalysed by an intense medical, economic and political interest and concern over cattle epizootics, which, as historians have shown, catalysed both the emergence of veterinary medicine and the medicalisation of animals across the globe in the second half of the century. as regards infectious diseases affecting humans, the medicalisation of non-human animals and their transformation into 'epidemic villains' involved an interlinked, two-part framing of their epidemiological significance: on the one hand, as spreaders and, on the other hand, as reservoirs of diseases. the historiography of the identification and study of non-human animals as spreaders of infectious diseases has for some time now stopped being the foray of heroic biographies of men like ronald ross, paul-louis simond or carlos chagas. focused on the social, political and epistemological histories of scientific studies of zoonosis and vector-borne diseases, historians, anthropologists and sts scholars have underlined the ways in which, within epidemiology, bacteriology and parasitology, non-human animals constituted active agents in complex networks of power and knowledge, and how they assumed different epistemic value in diverse colonial and metropolitan contexts. framed as spreaders of infectious diseases, animals also came to play an important role in what charles rosenberg has famously described as the dramaturgy of epidemics. assuming a protagonistic role in a series of epidemic and public health dramas, animals came to be seen as the ultimate source of disease outbreaks. no longer simply a nuisance or 'pests', the transformed image of a series of animals (mosquitos, rats, ticks, lice and flies in particular) as enemies of humanity was invested with militaristic tropes and colonial moralities. these animals formed as it were a global repertoire of disease spreaders, while at the same time assuming importantly diverse local forms, often in interaction with concerns and social imaginaries about other, regionally specific, disease hosts and vectors (beetles, bats, sandflies, etc.). while it is not in the scope of this introduction to map these 'glocal' interactions, deborah nadal's chapter in this volume provides a detailed picture of the longue durée of dogs as spreaders of rabies in india. nadal's chapter underlines the complex and important semiotic and ontological workings and re-workings on dogs as spreaders of rabies from colonial india to our times. with dog-borne rabies being recognised as an important public health problem across the globe since the s, in india, where rabies is endemic, human understandings of the particular zoonosis were linked to practices of classifying dogs. for british colonials, distinguishing between rabies-prone and rabies-impervious dogs was key to the imperial project of mastery over both indian society and 'nature'. within the confines of tropical medicine and its biopolitical imperatives, the management of rabies made crucial the definition of dog-human relations in terms of ownership. believed to be able to spontaneously develop rabies, for the british, 'ownerless' dogs presented a distinct danger for the colony. seen as the source of infection amongst owned dogs (which were considered unable to develop spontaneous rabies), these animals, nadal argues, challenged victorian morality and were associated with two key notions: on the one hand the notion of 'stray', with its overtones of vagrancy, and, on the other hand, the notion of the 'pariah'-an anglicised caste term used by british colonials to refer to outcaste or untouchable communities. at the heart of these classifications lied ideas about domesticity and wildness, as well as a pervasive social hierarchical mentality. perceiving street life in general as a threat to colonial rule grouped dogs of distinct social status and social life under one, infectious category. transforming 'strays' from 'vermin' and 'nuisance' into epidemic villains that should be sacrificed in the name of human health was not, however, a frictionless process but, as nadal shows us, one that embroiled indian society in debates about the value of life and compassion (led by both anti-vivisectionists and mahatma gandhi). after , 'catch-and-kill' of dogs for the control of rabies continued unabated but also involved indian society in renewed debate involving civil society activists, animal welfarists and political parties. in nadal's reading, these dog-related conflicts underlined a lingering problem pertaining to the classification of dogs vis-à-vis rabies: the persistence of the term 'stray' (inclusive of its 'pariah' associations). the solution since , nadal argues, has been the emergence of a discourse around 'street dogs', which has marked a shift towards an accommodation between different attitudes towards the particular animals, allowing for the concept that they can be both masterless and hygienic. nadal's chapter thus points out that, at the same time as what we may call high-epidemiology redefined experiences of non-human animals as spreaders of disease, it also instituted regimes of hygienic hope. envisioning and putting in place programs of increasing separation between humans and non-human disease vectors became the hallmark of public health from onwards. whether this involved rat-proofing, ddt spraying, mosquito nets, the cleaning of streets from stray dogs or the drying of swamps, this sanitary-utopian aspiration to liberate humanity of zoonotic and vectorborne diseases was based on a vision of universal breaking of the 'chains of infection'; a separation and, at the same time, unshackling of humans from disease vectors that was aimed at confining pathogens in the animal realm. in this way, whereas separation from animals was seen as a sufficient means of protection of humans from zoonotic and vector-borne diseases, animals themselves were defined as ultimately hygienically unredeemable-they were, in other words, rendered indistinct from disease. hence, the naturalist ontology of the enlightenment, which in philippe descola's anthropological model defines humans and animals as unified under the rubric of nature, was unsettled by a radical divide that saw disease as a mode of being which was only inherently proper to non-human animals, and only tentatively, or, as sanitary utopians would have it, temporarily, part of the human species. sayer's chapter in this volume focuses on the - plague outbreak in freston (suffolk, uk)-the last outbreak of plague in the history of england-and excavates the epistemological, political, class and colonial history of such a regime of prevention and hope. analysing what she calls 'the vermin landscape' of the outbreak, sayer focuses on non-human animal actors so as to show that, in spite of the widespread epidemiological acceptance of the rat flea (xenopsylla cheopis ) as the true spreader of plague, ideas about locality and class created a medico-juridical matrix where it was the rat that constituted the main object of scientific investigation and public health intervention. situating the suffolk outbreak both within the third plague pandemic and within british imperial science politics, sayer stresses the ways in which suffolk was connected to india, as the prime locus of the pandemic and of plague science in the empire. as the outbreak in suffolk was experienced as an echo of the ongoing devastating epidemic in india, the rat became an object of epidemiological concern and fear. what if infected rats moved from the rural hotspots of the epidemic into urban areas, transforming them into the equivalents of plague-ravaged bombay on english soil? such fears were fostered not just by the perceived natural traits of rats (as invasive of migratory animals), but also through their association with the rural poor. tapping into complex imaginary registers involving victorian systems of class-related disgust, the english rural idyll, and the image of 'the labourer's country cottage […] as literal and figurative representation of the state of the nation', sayer argues that, 'because this rested in turn on the state of the rural labouring class, and that class were said here to be unsanitary and their cottages invaded by rat and plague, the indian racial other therefore ghosted a new category of (dead) undeserving poor'. as epidemic villains, in the eyes of epidemiologists and public health authorities, rats indianised the dwellings of rural labourers in suffolk. as 'plague was equated with "rat plague"', plague also became indian plague, and in turn necessitated control measures and legislation aimed at 'codif [ying] the rat in law and normalis[ing] its destruction'. formulated around an entanglement of class and interspecies relations, the suffolk plague crisis led, on the one hand, to an increasing medico-juridical investment of the rat in england, while, on the other hand, to a systematic neglect of 'the hares, cats, dogs that featured in gamekeepers' and labourers' narratives of the disease'. identifying and investing on a non-human epidemic protagonist (the rat) led to, and indeed required, a disinvestment and neglect of other species involved in the spread of the disease, and-perhaps most crucially-to overlooking the ecological complexity of disease persistence and transmission between different species in any given ecosystem. the rats and mice (destruction) act , 'which tasked every british citizen with a legal obligation to remove rats from their property', was the pinnacle of the configuration of the rat as an epidemic villain in england and of the institutionalisation of sanitary regimes of hope as regards the prevention of animal-borne infection. having conquered the globe by the mid- s, this regime of prevention and hope came to an end with the dawn of the emerging infectious diseases framework in the early s, when scientists began to focus on processes leading to new diseases, hitherto of non-human animals, infecting humans and to the 'specie-jump' processes (so-called spillover) leading to this phenomenon: 'rather than revolving around already-existing pathogens and how they circulate in specific ecological contexts, the focus on emergence required a shift of attention to what we may call "viral ontogenesis"'. over the past years, the rise of 'emergence' as the central framework of studying and understanding infectious diseases has led to a radical shift of scales and a reinvestment on zoonotic diseases that has been tied to a shift away from prevention towards preparedness. this is a regime of biosecurity that, as anthropologists like andrew lakoff, frédéric keck and carlo caduff have shown, is based on the anticipation of an unavoidable pandemic catastrophe, and which sets in place technologies of biosecurity that have come to increasingly dominate the realm of global health. envisioned as inevitable and catastrophic, 'emergence' has thus radically transformed the status of animals as epidemic villains. on the one hand, whereas in the sanitary-utopian framework of highepidemiology, animals were considered to be isolatable carriers of disease, in the eid framework infection is rendered inevitable. and, on the other hand, whereas for the sanitary-utopian framework, animal-human infection posed a limited threat to humanity, for eid it poses an unlimited one, or to be precise one associated with existential risk. it is telling that the mytho-historical event defining the conceptual horizon of the sanitaryutopian framework was the black death. believed by to have been rat-borne bubonic plague, the fourteenth-century pandemic was used by moderns as a key cautionary tale, and at the same time as a potent medical metaphor: black death was something that could 'return' (as hundreds of reports and news items made clear during the third plague pandemic) but whose impact would be effectively limited by grace of modern medicine and sanitation. on the other hand, as caduff has shown, the mytho-historical event defining the conceptual horizon of eid is the flu pandemic. the political ontology of this event for our contemporary pandemic imaginary is distinctly different from that of the black death for the early-tomid-twentieth-century public. for, as every contemporary epidemiological report and news broadcast makes clear, were an event like 'the spanish flu' to occur again today, globalisation and modern transport would transform it to an event of human extinction proportions; something not only nonpreventable, but whose control, once it has begun, is not guaranteed. both of these mytho-historical events have non-human animals at the heart of their causation narrative: the black death (at least so scientists believed at the time) rats, while the flu birds, probably chicken. however, while the sanitary myth of origin of the black death portrayed the rat as an ancient enemy of humanity whose days were numbered due to the advancement of science, the eid myth of origin frames chicken as just one example of a host of unknown species from which the 'killer virus' may emerge and against which the only action we can take is being prepared. séverine thys' chapter in this volume explores the consequences of the eid approach to non-human animals, as it applied to 'bushmeat' in the context of the recent ebola epidemic in west africa ( - ), with a focus on the impact of epidemiological and public health framings of 'bushmeat' hunting, butchering and consumption. especially affecting 'forest people' in macenta, guinea-conakry, the framing of a fluid host of animals as the source of epidemiologically illicit meat relies on persistent colonial tropes that imagine the 'tropical jungle' as an originally natural realm whose disturbance by human activity leads to the emergence of killer viruses. rehearsed time and again in films like outbreak ( ), this mortal link between nature and culture, thys reminds us, is currently being mediated by the figure of the bat-the in-between figure of a 'rogue' animal, which, james fairhead has shown, is being increasingly deployed as an epidemiological bridge in several zoonotic scenarios (ebola, mers, sars). thys follows other anthropologists in pointing out that this insistence on 'bushmeat' and contact with fruit-bats frames local cultures as pathogenic, in line with paul ewald's notion of 'culture vectors', and thus 'obscure[s] the actual, political, economic, and political-economic drivers of infectious disease patterns'. framed in terms of a 'transgression of species boundaries', ebola spillover events are thus pictured as resulting from a life led according to 'traditional' (and the implication is irrational) classificatory systems that fail to maintain 'us vs. them' boundaries. replete with visual and affective structures of disgust, this view, thys argues, is not challenged by the one health framework, which 'should provide a more nuanced and expanded account of the fluidity of bodies, categories and boundaries' so as to 'generate novel ways of addressing zoonotic diseases, which have closer integration with people's own cultural norms and understandings of human-animal dynamics'. key to this, according to thys, is to recognise and examine the historically dynamic nature of these classificatory and more broadly ontological systems (a view shared by nadal, this volume), and the explanatory models with which they are entangled. thys outlines the complex matrix of uses of non-farmed meat in the region (for nourishment, medicaments, trophies, etc.) and their transformation under the weight of regional and global commodity market networks. one may add that what is often neglected is the fact that 'bushmeat' was used by colonial authorities as a reward to local communities; in angola, for example, the portuguese rewarded local communities with 'bushmeat' for rat-catching in the colonial power's effort to contain plague during the s. the political investments of non-human animals as disease spreaders are further explored in gabriel lopes' and luísa reis-castro's chapter in this volume on the history of the aedes aegypti mosquito in modern brazil. following the social life of the particular mosquito species from the s until today, lopes and reis-castro stress that, while recognising that it has always constituted an 'epidemic villain', we need to pay closer attention to the particular diseases to which this villainous character has been linked to, and to the corresponding political system under which this identification has been undertaken, over the course of modern brazilian history. at the beginning of the twentieth century, aedes aegypti was associated with 'underdevelopment' as a key overarching ailment of brazil, with 'the image of a plagued country swarming with mosquitoes' filled with yellow fever playing an important role in bringing health under the rubric of the state and its modernising agenda. lopes and reis-castro follow gilberto hochman's classic work on the linkage between sanitation and nationbuilding in brazil in stressing that what began as a project of 'civilizing the tropics' by eliminating yellow fever across the country transformed by the early s into a more modest programme of preventing outbreaks in urban centres. by contrast to the liberal nation-building sanitary-utopian visions of oswaldo cruz and his collaborators in the first decades of the twentieth century, in the second half of the s a renewed focus on aedes aegypti was underscored by the politics of democratisation, following the end of the -year-long military dictatorship in . as by april it had become identified with dengue fever, as a new disease to plague urban 'areas marked by racialised histories of state abandonment and violence', the aedes aegypti became associated with a disease that was not as lethal as yellow fever, and which bore with it the sign of social, political and economic restitution. as public health had been the pejorative of left-wing and other democratic forces during the last decade of the dictatorship, calls to control dengue-carrying aedes aegypti as an embodiment of state violence and neglect contributed to the success of the 'sanitary reform movement' and the establishment, in , of brazil's sistema Único de saúde. lopes and reis-castro then turn their attention to the latest incarnation of aedes aegypti as a spreader of the zika virus. unfolding during the years of the impeachment (or judicial coup, depending on one's point of view) against dilma rousseff, the appearance of zika in brazil involved aedes aegypti in an international emergency. lopes and reis-castro examine the political struggles around zika-related mosquito control and argue that, focused on social inequality and the 'uneven effects of climate change', this new framing of the aedes aegypti on the one hand continues a longestablished practice of problematising it as a disease vector with specific political and political-economic parameters, while, on the other hand, introducing important gender-related critiques of public health. hence, while the authors claim that, 'the specific kind of virus in mosquitoes' bodies shaped what kind of epidemic villain the mosquito became', they also stress that, 'the mosquito as a vector carried not only three epidemiologically distinct viruses but very different political desires, struggles, and debates'. focusing on the recent zika crisis, in their chapter to this volume gustavo corrêa matta, lenir nascimento da silva, elaine teixeira rabello and carolina de oliveira nogueira in turn argue that the focus on mosquitoes' guilt and on the technological strategies developed to control these vectors unfolded within a context of profound political instability, and at the same time of epistemic uncertainty regarding key epidemiological traits of the disease. framing aedes aegypti as epidemic villains in this context, diverted attention from issues of social, economic and environmental injustice and inequality that were driving determinants of the outbreak, and legitimised the absence of governmental measures regarding the latter in response to the epidemic. the 'enactment of a global enemy, aedes aegypti, as the villain of the epidemic' thus allowed the brazilian government to paint an all-too-familiar and deceptive picture of a promethean struggle of the country as a unified whole (notwithstanding its enormous and often violent class, race, gender and ideological discrepancies and antagonisms) against a vile creature, which was solely held responsible for the disease. drawing on critical medical anthropological perspectives, matta et al. thus underline the structural violence inherent in both the discourse of epidemic villains and in the policies built and legitimated by this discourse. brazil's mosquitocentred policy in the face of zika, financially, politically and morally boosted by the declaration of public health emergency of international concern (pheic) by the who, relied on a securitisation framework that rhymed well with the broader neoliberal turn of the country and mobilised the image of the mosquito as a public enemy to create a spectacle of national unity that obscured 'iniquities, poverty, the skin colour of those bitten by mosquitoes, the house and streets where these fly, and the environment where they lay their eggs'. as mark honigsbaum has shown, disease ecology frameworks, arising in the usa in the s, framed non-human animals not simply as spreaders of infectious diseases but also as their 'reservoirs'. the 'great parrot fever epidemic' of - involved pet parrots in an epidemic panic across the globe, with a particular focus in the usa. as readers of the colonialist bande dessiné exemplar, tintin in the congo (published in the shadow of the epidemic in ), may remember, psittacosis (caused by chlamydia psittaci) is a zoonotic disease carried by parrots and parakeets that can infect humans. however, for karl f. meyer, a key contributor to the development of disease ecology, the ability of parrots and parakeets (popular pets at the time in the usa) to be asymptomatic carriers of the disease posed a more important problem that the immediate epidemic crisis; especially, honigsbaum explains, as '[t]hese latent infections were a particular problem in california where during the depression many people supplemented their incomes by breeding parakeets in backyard aviaries'. the discovery that psittacosis was not simply an 'exotic' disease imported to the usa by parrot traders, but one that had established itself endemically in american aviaries transformed the structure of epidemic blame from one focused on an outbreak to one focused on an endemic and, at the same time, from one revolving around an exotic invasion to one regarding unhygienic infrastructures at home. more profoundly, it also contributed to a shift towards a reframing of animal-borne disease in terms of disease ecology, a process which involved several decades of studies and interdisciplinary exchanges, but was ultimately triggered by an integration of charles elton's pathbreaking understanding of animal zoology in the realm of epidemiology. what is less well recognised historically is that the notion of the reservoir had a long history in epidemiological reasoning predating disease ecology. rats in particular were suspected, from as early as , as not only spreading plague (via their flea, xenopsylla cheopis ) but as also contributing to the maintenance of persistence of the disease in given urban settings. indeed, elton's interest in the role of disease in the regulation of animal populations was itself stimulated by earlier russian and chinese studies of the siberian marmot as a host of plague in the inner asian steppes. in chapter of this volume, christos lynteris returns to these studies to examine how the so-called tarbagan became the subject of investigations regarding plague's ability to survive the harsh winters of the region. the question was related to ideas about 'chronic plague', which in the case of the siberian marmot were linked to its hibernation between october and april. using an abundance of visual material, lynteris argues that, on the one hand, tarbagan burrows, which had been epistemic objects ever since the discovery of the species in , and, on the other hand, marmot hibernation, which had been the focus of scientific investigation in relation to host immunity already by , were tied together into an epidemiological duet as a result of the emergency of the manchurian plague epidemic of - . there is indeed a crucial metonymic work involved in this tying together the 'mystery of the survival of plague' over winter to marmot hibernation, and marmot underground dwellings. for the three actants in this network of what following genese sodikoff, we may call 'zoonotic semiotics'-latent plague, hibernating marmots, underground burrows-shared and maintained between them an image of 'mystery' and occultation which has been key both to epidemiological reasoning regarding infectious diseases and to the 'pandemic imaginary' underlying understandings of zoonosis. this image of plague taking advantage of unseen biological processes, materialities or infrastructures so it can assume an imperceptible form that would allow it to persevere over either human action against it or environmentally adverse conditions is of course reliant on pasteurian notions virulence, latency and attenuation. yet, more than simply illuminating a reiteration of bacteriological doctrine, what the tarbagan example points out to is a pervasive aspect of epidemiological reasoning; for the assumption that, when plague (or indeed any other disease) is not seen, this is because it is 'hiding', is part of what we may call a cynegetic complex in epidemiology. as john berger once noted, admittedly in a very different context, a key principle (and, one may add, a mythic structure) of cynegetic worlds is that, 'what has vanished has gone into hiding'. in the case of epidemiology, as with other cynegetic cosmologies, this implies an ambivalent relation. on the one hand, microbes are seen as predators of humanity, who lurk and hide so as to better ambush their prey. and on the other hand, as the enduring metaphor of 'virus hunters' amply illustrates, microbes are also seen as humanity's pray-which thus 'hide' to escape being caught and vanquished by us. as frédéric keck has stressed (following chamayou), '[w]hereas pastoral techniques are asymmetrical, relying on the pastor's superior gaze over the flock manifested by sacrifice, cynegetic techniques are symmetrical, as hunters and prey constantly change perspectives when displayed in rituals'. maurits meerwijk's chapter in the present volume shows that this is indeed a historically pervasive framework, which in the case of mosquitoes is carried over from tropical medicine into global health. comparing the discourses of ronald ross and bill gates, meerwijk shows how the cynegetic metaphor comes to encompass not only the pathogens in question but also their vectors. this points out at a transformative ontology underlying epidemiological reasoning, and its obsession with the 'invisibility' of disease, insofar as pathogens are seen, on the one hand, as able to persist by transforming themselves inside non-human animal hosts (by means of attenuation or mutation) and, on the other hand, as able to spread by transforming their hosts into bestial man-hunters. more than simply blaming non-human animals, in epidemiological reasoning, this double transformative ability configures the former into the loci par excellence of pathogenesis and, at the same time, necessitates techniques of rendering host-pathogen relations visible. visual images of non-human animals have played a historically important role in their configuration as epidemic villains. since the dawn of bacteriology, the scientific identification and examination of non-human hosts and vectors of infectious diseases have heavily relied on photographic technologies (including microphotography), diagrams and epidemic cartography. following sayer (this volume), animals have been 'fed into a data-focused visual regime', combining photography, mapping, diagrams and statistical graphs, that seeks to establish points of contact, habitats, interspecies boundaries and other forms of what hannah brown and ann h. kelly have called human/non-human 'material proximities'. in the context of high-modern epidemiology as well as in today's eid framework, these visualisations are part of a project of mastery aimed not so much at the subjugation of nature, as to the control of humanity's relations with nature. diagrammatic images of dissected mosquitoes played a key role in ronald ross' examination of the insects as malaria vectors, as, in later years, the microphotography of anopheles gambiae dissected ovaries would prove an indispensable, soviet-led method for identifying the capacity of a given mosquito to transmit the malaria plasmodium to humans. similarly, nicholas evans has shown, in the course of the third plague pandemic, comparative images between healthy and plague-infected rats became standard visual objects in epidemiological investigations and their published reports. but the visualisation of 'epidemic villains' did not always necessitate their direct representation. in her chapter for this volume, sayer draws an insightful comparison between two sets of visualising rat control, the first in the english port of liverpool and the second in british india. in both cases, the actual rats are imperceptible, with the photographic focus being on humans undertaking carefully orchestrated epidemiological work (rat dissection, flea collection); a fact which, in the case of liverpool, is underlined by the staged poses of the sanitary officers in questions, and, in the case of india, was permeated by colonial racial hierarchies in the representation of lab work. as representations of the relation between pandemic plague, medical science and empire, these images provide reassuring portraits of control in direct dialogue with the image of objectified rats, described by evans, thus 'making rats an integral part of plague'. similarly, with a focus on this relational aspect of human/non-human mastery and its visual regimes, in the second chapter of this volume lynteris illustrates how the epidemic framing of siberian marmots as reservoirs of plague in inner asia relied on photography and the diagrammatic cartography of their burrows. comprising in survey photographs of excavated marmot burrows and diagrammatic depictions of burrow systems, the visual regime constructed around this suspected host of plague following the manchurian plague outbreak of - comes to show, on the one hand, that intrusive practices of epidemiological visualisation were not limited to human dwellings, but also included those of non-human animals (photographing the marmot burrows required their prior excavation), and, on the other hand, that the visual framing of 'epidemic villains' is not limited to the representation of their role as spreaders of diseases. at the same time, the popularisation of the identity of specific mammals, birds and insects as disease spreaders has and continues to be mediated by their visual representation through photography, film and illustration. photographs of 'wet markets' in south china during and in the aftermath of the sars pandemic have been shown to incorporate a key principle of 'epidemic photography': the depiction of animal-related spaces as potential ground zeros of the 'next pandemic'. the practice of the public vilification of non-human animals and the framing of contact spaces between them and humans as infection hotspots was established for the first time in the course of anti-malarial and anti-yellow fever campaigns in the first decades of the twentieth century, but also during complex public health operations against plague in the context of the third plague pandemic ( - ) when the dreaded disease was often visually personified as the rat. indeed, quite often, the image of animals as enemies of humanity assumed anthropomorphic aspects, which under a colonialist gaze, involved racist inflections. in australian newspaper illustrations, for example, plague-carrying rats were depicted having chinese faces, thus both making an aetiological connection between plague and china (plague as an 'oriental disease' arriving from china, by chinese migrants) and fostering broader sinophobic bigotry at the time. in his examination of the framing of 'tiger mosquitoes' (aedes aegypti and aedes albopictus ) in this volume, meerwijk explores the rich visual culture supporting progressive framings of the specific mosquito species as infectious enemies of humanity. in a striking example, meerwijk shows how the diagrammatic juxtaposition of a mosquito and a tiger was used in a public health poster, meant to underline the predatory, man-eating qualities of aedes mosquitos. pointing at a pervasive tendency to talk about and visualise mosquitoes in terms of great predators (tigers, sharks) or 'enemies of humanity' (terrorists, vampires, prostitutes), meerwijk elucidates the work of the fusion between military, cynegetic and sexual metaphors and visual tropes employed in the depiction of mosquitoes across epidemiological paradigms. this is all the more important as the visualisation of animals as 'epidemic villains' was a trope that found application and success beyond epidemiology and public health. non-human animals were charismatic protagonists of political caricatures since the turn of the eighteenth century. in particular, lukas englemann notes, 'the "political bestiary", as gombrich calls the long tradition of depicting political issues through animal characters, acquired widespread popularity in the nineteenth century. the meaning many animals inhabited could be easily exploited to convey strong messages and almost always suggested degradation'. what changed at the turn of the nineteenth century was the introduction of a new aspect in the use of animals in caricature: their infectious nature. with political discourse utilising more and more medical terms at the time, the use of the visual form of the infectious animal to portray one's political enemies became an exemplary field of vilification. to mention only one example, in the course of the moscow trials, soon after the soviet state prosecutor, andrey vyshinsky, publicly pledged 'to stamp out the accursed vermin' who 'should be shot down like rabid dogs', the prolific cartoonist of the pravda, boris efimov (who was present at the trial), produced a striking caricature of leon trotsky and nikolai bukharin as a two-headed rabid dog held on the leash by the hand of the gestapo. however, as engelmann has shown in his examination of caricatures in the course of the plague outbreak in san francisco, the aim of depicting animals in the context of epidemic crises has not been limited to practices of blaming the former as spreaders or reservoirs of disease. in fact, animals were also used to critique and ridicule bacteriology itself. for example, in the case of san francisco, newspaper caricatures used animals to portray bacteriology 'as a science that formulated its judgments through experiments with animals, not in the treatment of people'. by visualising laboratory animals as 'vermin and pest', englemann argues, bacteriology was portrayed as 'a wasteful expenditure of public funds' and 'the medical laboratory was stripped of its progressive potential and instead appeared as an infliction of damage on the public good'. at the same time, as dawn day biehler has shown in her monograph on pests in twentieth-century us history, images of disease hosts, like rats, have also been used for subaltern purposes, such as the campaigns by the black panther party in the s- s against slumlords and the living conditions in african american neighbourhoods. for example, biehler argues, the well-known illustration by emory douglas, 'black misery! ain't we got right to the tree of life?', 'constrast[ed] with images of women afraid of rats; the woman's grip on the rat suggests determination, courage and fury'. here, the rat represented the unhygienic, exploitative and pestilential conditions imposed by white capital on working-class african americans, and the latter's determination to face up to this social injustice. the prolific use of images of non-human animals as 'epidemic villains' in diverse fields of social practice as public health campaigns, political propaganda, the critique of bacteriology and subaltern critiques of power and domination, points at the importance placed on the infectious nature or potential of animals both as a reality and as a metaphor in the modern world. however, whether it is to convey a threat to the national body, or to mock science, the use of these images also points at the fascination and discomfort of moderns towards non-human agency. underlining how epidemiology and public health emerged in relation to, and continue to be informed by framings of non-human animals as epidemic villains, the chapters in this volume explore the layered political, symbolic and epistemic investments of non-human animals, as these have become rhetorically and visually enabled in distinct ways over the past years. whether it is stray dogs as spreaders of rabies in colonial and contemporary india, bushmeat as the source of ebola in west africa, mosquitoes as vectors of malaria, dengue, zika and yellow fever in the global south, or rats and marmots as hosts of plague during the third pandemic, this volume shows framings of non-human animals to be entangled in local webs of signification and, at the same time, to be global agents of modern epidemic imaginaries. civet cats, fried grasshoppers, and david beckham's pajamas: unruly bodies after sars' the pandemic perhaps: dramatic events in a public culture of danger the scale politics of emerging diseases more than one world more than one health: reconfiguring inter-species health i am using animal-borne diseases here as a term inclusive of zoonotic and vector-borne diseases the rat-catcher's prank: interspecies cunningness and scavenging in henry mayhew's for an influential example of the rat being described as disease-free, see cristofano and the plague: a study in the history of public health in the age of galileo the rat would become suspect of carrying plague for the first time during the inaugural outbreak of the third plague pandemic, in hong kong, with another decade elapsing before the universal acceptance of the link between the animal and human plague. the first scientific study showing the role of the rat and its flea in the propagation of plague was: p. l. simond, 'la propagation de la peste imagining vermin in early modern england the great cat massacre and other episodes in french cultural history religion and the decline of magic: studies in popular beliefs in sixteenth and seventeenth century england imagining vermin in early modern england imperfect creatures: vermin, literature, and the sciences of life on vermin and the poor, see p. camporesi, bread of dreams: food and fantasy in early modern europe animal bodies, renaissance culture filth is the mother of corruption". plague, the poor and the environment in early modern florence que la peste soit de l'animal! la législation à l'encontre des animaux en période d'épidémies dans les villes des pays-bas méridionaux et de la principauté de liège ( - ) que la peste soit de l'animal!'; on ideas of miasma emanating from butchered meat see d. r. carr, 'controlling the butchers in late medieval english towns great stenches, horrible sights and deadly abominations": butchery and the battle against plague in late medieval english towns infection," and the logic of quarantine in the nineteenth century fractured states: smallpox, public health and vaccination policy in british india toxic histories: poison and pollution in modern india as kathleen kete has shown, the modern transformation of this connection, before the dawn of bacteriology, was fostered by a sexualisation of the disease, which rendered it comparable to uncontrollable impulses or lust. commenting on kete's work, linda kalof writes: 'since nymphomania and uncontrollable sexual desire in men were considered the result of prolonged sexual abstinence the beast in the boudoir: petkeeping in nineteenth-century paris looking at animals in human history healing the herds: disease, livestock economies, and the globalization of veterinary medicine veterinary research and the african rinderpest epizootic: the cape colony the great epizootic of - : networks of animal disease in north american urban environments' beastly encounters of the raj: livelihoods, livestock and veterinary health in india animals and disease: an introduction to the history of comparative medicine from coordinated campaigns to watertight compartments: diseased sheep and their investigation in britain, c. - unpacking the politics of zoonosis research and policy catching the rat: understanding multiple and contradictory human-rat relations as situated practices blaming the rat? accounting for plague in colonial indian medicine the bacteriological city and its discontents' malarial subjects: empire, medicine and nonhumans in british india what is an epidemic? aids in historical perspective wartime rat control, rodent ecology, and the rise and fall of chemical rodenticides urban mosquitoes, situational publics, and the pursuit of interspecies separation in dar es salaam the colonial disease: a social history of sleeping sickness in northern zaire the mobile workshop: the tsetse fly and african knowledge production cat and mouse: animal technologies, trans-imperial networks and public health from below building out the rat: animal intimacies and prophylactic settlement in s south africa'. american anthropological association (engagement modern" management of rats: british agricultural science in farm and field during the twentieth century of rats, rice, and race: the great hanoi rat massacre, an episode in french colonial history for a more detailed discussion of this process, see c. lynteris, 'zoonotic diagrams: mastering and unsettling human-animal relations' curing their ills: colonial power and african illness rethinking human-nonhuman primate contact and pathogenic disease spillover the scale politics of emerging diseases the pandemic perhaps avian preparedness: simulations of bird diseases and reverse scenarios of extinction in hong kong unprepared: global health in a time of emergency the pandemic perhaps great anticipations human extinction and the pandemic imaginary for discussion, see k. ostherr, cinematic prophylaxis: globalization and contagion in the discourse of world health inclusivity and the rogue bats and the war against "the invisible enemy as fairhead argues, this entanglement of 'native culture' with 'rogue animals' has the effect of transferring the status of the 'rogue' to the 'culture' in question; fairhead, 'technology, inclusivity and the rogue bats and the war against "the invisible enemy"'. see also m. leach and i. scoones, 'the social and political lives of zoonotic disease models: narratives for a discussion of disgust and animal disease, see a. l. olmstead, arresting contagion. science, policy and conflicts over animal disease control it needs to be noted here that, following fissell, the emergence of the early modern notion of 'vermin' was not associated with disgust-something that points to the introduction of this affective and sensory structure in the nineteenth century imagining vermin in early modern england serviço permanente de prevenção e combate à peste bubónica no sul de angola: relatório (lisboa: agência geral das colónias the sanitation of brazil: nation, state, and public health latent infections, and the birth of modern ideas of disease ecology' tipping the balance blaming the rat? plague and the regulation of numbers in wild mammals Évolution de la peste chez la marmotte pendant l'hibernation'. comptes rendus hebdomadaires des séances de l'académie des sciences for a more detailed examination of zoosemiotics in the case of marmots, see c. lynteris, 'speaking marmots, deaf hunters: animal-human semiotic breakdown as the cause of the manchurian pneumonic plague of - on the ambivalence as applies to hunters and gatherers, see r. willerslev lessons in medical nihilism. virus hunters, neoliberalism and the aids pandemic in cameroon on chamayou's theory, see grégoire chamayou, manhunts: a philosophical history for a discussion of the mythic ability of pathogens to transform their hosts into man-hunters, see c. lynteris, 'the epidemiologist as culture hero: visualizing humanity in the age of "the next pandemic on diagrams and the configuration of zoonosis, see lynteris the evolution of ebola zoonotic cycles'. contagion material proximities and hotspots: toward an anthropology of viral hemorrhagic fevers' human extinction and the pandemic imaginary seeing cellular debris, remembering a soviet method' blaming the rat? on the practice of intrusive epidemic photography as regards human dwellings, see r. peckham, 'plague views. epidemic, photography and the ruined city the prophetic faculty of epidemic photography: chinese wet markets and the imagination of the next pandemic this 'global visual economy' was so pervasive in fact so as to lead to a retrospective diagnosis of the presence of rats in paintings such as nicholas poussin's the plague of ashdod as evidence of a pre-bacteriological knowledge of this zoonotic connection; for a critique, see s. barker yellow peril epidemics: the political ontology of degeneration and emergence a plague of kinyounism: the caricatures of bacteriology in san francisco the sharp weapon of soviet laughter: boris efimov and visual humor a plague of kinyounism', p. . . ibid pests in the city: flies, bedbugs, cockroaches, and rats (washington key: cord- -b r authors: labrunda, michelle; amin, naushad title: the emerging threat of ebola date: - - journal: global health security doi: . / - - - - _ sha: doc_id: cord_uid: b r ebola is one of the deadliest infectious disease of the modern era. over % of those infected die. prior to , the disease was unknown. no one knows exactly where it came from, but it is postulated that a mutation in an animal virus allowed it to jump species and infect humans. in simultaneous outbreaks of ebola occurred in what is now south sudan and the democratic republic of the congo (drc). for years, only sporadic cases were seen, but in a new outbreak occurred killing hundreds in the drc. since that time the frequency of these outbreaks has been increasing. it is uncertain why this is occurring, but many associate it with increasing human encroachment into forested areas bringing people and animals into more intimate contact and increased mobility of previously remote population. this chapter will navigate ebola in the context of global health and security. there are multiple objectives of this chapter. first is to provide a basic understanding of ebola disease processes and outbreak patterns. second, is to explore the interplay between social determinants of health and ebola. the role of technology in spreading ebola outbreaks will be explained as will ebola’s potential as a bioweapon. readers will gain understanding of the link between environmental degradation and ebola outbreaks. this chapter will be divided into five main sections. these are ( ) a case study; ( ) ebola disease process; ( ) social determinants of health and ebola; ( ) ebola in the modern era, and ( ) the link between ebola and environmental degradation. who contracts ebola. the story will be told from her perspective. she will describe from her why she thinks the outbreak has occurred. her husband has died of ebola despite efforts of traditional healers. she will discuss burial rites in the context of her religious beliefs. the next section looks at the disease itself. the history, epidemiology, transmission, and signs/symptoms will be described. prevention measures including the use of personal protective equipment and vaccination strategies will be discussed. the basics of diagnosis and treatment will be covered. the section will end with a discussion of ebola epidemics. social determents of health play an important role in the epidemiology and transmission of ebola. factors impacting spread include, high population mobility, porous international borders, and ongoing conflict resulting in displaced populations. poverty, cultural beliefs and practices and prior ineffective public health messages have all played a role in the emergence of ebola. the following section will explore ebola in the era of technology. the role of air travel in disease spread and the effectiveness of airport screening measures will be discussed. ebola's potential for use in bioterrorism will also be discussed in this section. the relationship between environmental encroachment and disease emergence will be explored. global warming, and the impact of a growing population in ebola outbreaks will be explored. the chapter will end with a discussion of future directions. in this last section the important of international collaborations for disease prevention and public education programs will be discussed. sia waited nervously in the small one room house where she lives. she was waiting for her brother-in-law to return with the body of her dead husband, saa. he had died yesterday of the bush illness that was killing so many in her community, ebola the outsiders called it. just weeks ago, the world had seemed a different place. sia had sat with the other women of the kissi tribe at church joking and planning for the upcoming rice harvest. yes, they practiced christianity, but also followed the traditions of their ancestors. women in her village prayed to jesus and god, but also to their ancestors. outsiders sometimes questioned how the kissi could follow both christianity and their old traditions, but sia had never seen a problem. ancestors after all, were the ones who communicated with god. when someone in the family died, they were escorted to the realm of the ancestors where they were able to protect the living family and speak to god on their behalf. ancestors continued to live in the village, but in their new form. sia shivered thinking of what happened to those who died and were not escorted to the realm of the ancestors. ceremonies were usually performed by the brother of the deceased. if the ceremonies were not done properly, a loved one would become a wandering ghost instead of an ancestor. wandering ghosts torment the living bringing misfortune to everyone in the village, especially to the family that failed to perform the proper rights. sia did not like to think of such things, but there had been several deaths in a nearby village and she could not help but to wonder if it was the work of a wandering ghost. that was the day it started. saa was fine when he woke up, but while they were at the church, he started to get sick. he got sick so quickly that sia suggested that they return home early so he could rest. it wasn't a far walk, but by the time they arrived home, saa was having chills, headache, nausea, and said his joints hurt. while saa rested, sia prepared a tonic to ease the pain and ward off evil spirits. saa's eyes were red and he felt hot to touch. "a powerful spirit must be involved", sia thought to herself. she couldn't imagine who would have cursed her husband this way. he hadn't argued with anyone that she knew. for days sia cared for her husband with special food, potions, and prayers. she had even sacrificed a chicken, but instead of getting better he started vomiting and having diarrhea. obviously, she needed assistance from someone with greater influence in the spirit realm. kai, a local medicine-man of conservable powerful agreed to help but needed time to make the necessary preparations. by that evening saa had stopped eating altogether and his gums started to bleed. kai belonged to a secret society that added to his powers. sia was not allowed to attend kai's ceremony but was told that saa had cried blood and started to hiccough uncontrollably. kai was notable to defeat the evil spirits even with his most powerful incantations. some of the villagers wanted to take saa to a treatment center set up by some foreigners to see if they could help him. sia was hesitant, but by the next morning saa had developed a yellow color to his skin and was having black diarrhea, so she agreed. after a bunch of questions saa was taken into the camp that the foreigners set up, but they would not let sia or anyone else in the family enter. that was the last time she had seen saa alive. two days later sia was informed that saa had died. he was to be buried in a mass grave and no one was allowed to see his body. saa's brother said that he thinks the foreigners killed him. they weren't really there to help but part of a government plan to destroy the kissi. workers in the camp were removing the internal organs of the sick while they were still alive and selling them. that is why no one was allowed into the camp or to bury the bodies properly. they weren't just attacking the living, but also trying to destroy the ancestors by preventing the death ceremony from happening. luckily saa's brother knew people. it had cost everything that the family owned, but the man driving the truck full of bodies agreed to meet a short distance from the foreigners' camp. he would give them the body there, but there were not to tell anyone. as saa's brother walked into the house carrying saa's body, sia felt an overwhelming sense of relief. all the worry gave her a headache and made her feel weak. now that they had saa's body it will be better. they will do the rituals this evening and burry saa in the morning. he will be able to walk with the ancestors. one of the world's deadliest pathogen, the ebola virus made its first appearance in in not just one but two simultaneous outbreaks. the first of its deadly attacks were in what is now known as nzara, south sudan while the second occurred in a small village community near the ebola river bank in yambuku, democratic republic of congo (drc) [ ] . of the known to be infected, lost their lives. since that time, we have learned much about the ebola virus and the disease it causes. ebola virus is an uncommon virus which infects both human and non-human primates. it belongs to the family filoviridae, a negative stranded rna virus. when magnified, it appears as a filamentous structure fig. . the ebolavirus genus has six known species, zaire, sudan, tai forest (formerly côte d'ivoire ebolavirus), bundibugyo, reston, and the recently described bombali [ ] . reston is highly pathogenic for non-human primates and pigs, and bombali has been discovered in free-tailed bats as part of ongoing research to discover the ebola reservoir. the zaire species was responsible for the first ebola virus outbreak in and is considered to be the deadliest of the six [ ] . initially the disease caused by ebola virus was called ebola hemorrhagic fever, but later studies showed that the hemorrhagic manifestations were less common than initially thought and subsequently the name was changed to ebola virus disease (ebd). until , the ebola virus isolated sporadic outbreaks occurred only in central africa with counts numbering in the hundreds or less, and only lasting days to weeks. however, in march the who confirmed an epidemic of the zaire species of ebola virus emerging in west africa. this outbreak lasted years and grew to be one of the world's deadliest epidemics. there were , case and , fatalities documented by the world health organization (who). the index case of this epidemic is thought to be a -year-old child who became ill in late . the child eventually succumbed to the illness with symptoms of fever, chills, vomiting, and black-tarry stool [ ] . this was in guinea, west africa a country where ebola supposedly did not exist. from here it spread to liberia, sierra leone, nigeria, and mali. the natural reservoir of ebola virus is not known with certainty, although research has suggested that it may be bats. human infection may occur through direct contact with the mystery reservoir or through contact with infected primates. this can occur when hunting and preparing bush-meat or via contact with body fluids from an infected person. ebola is highly transmissible. the disease pattern of evd has shifted over the last years. currently, ebola has been found across central and west africa, with occasional exported cases to other regions. for obscure reasons, outbreaks seem to be occurring with increasing frequency. this may be linked to environmental degradation and increasing mobility of local populations. ebola spread is through contaminated body fluids. unfortunatly, traditional funerary practices across africa put funeral attendees in contact with body fluids from those who have died of ebola. initial international efforts to control ebola spread during outbreaks have often resulted in clashes and conflict as control measures confront tradition. inadequate public health messages, distrust of those providing the health messages, political instability, and regional conflict have allowed ebola to spread and kill thousands when early containment could have been within reach. ebola is one of the most fatal infectious diseases humans have encountered. even with the best medical care the disease is deadly. unfortunatly, the developing countries where evd occurs are not equipped with optimal medical or public health facilities. to complicate the situation further, survivors of evd are not hailed as heroes, but instead may be left with chronic illness and stigmatized in their communities. transmission of ebola disease is still being studied, but it is known that person-toperson contact is the most common form of spread. infection occurs primarily through direct contact with body fluids from infected people or animals (fig. ). viral antigens have been isolated from the skin of those infected suggesting that skin contact alone may be sufficient to spread disease [ ] . it has also been shown that, at least in primates, ebola can be spread through intramuscular injection, and inoculation can occur through contact of the conjunctiva or oral mucosa with infected body fluids [ ] . blood, vomitus, and feces are the body fluid most likely to spread infection because of the frequency with which they are encountered during the course of the illness, but other fluid such as urine, semen, vaginal fluid, tears, sweat, and breast milk also have potential for viral transmission [ , , , , , ] . caring for an infected person with ebola, whether at home or in the hospital has been identified as a high-risk activity for acquiring ebola. household members who provide direct care to an ebola victim are - times more likely to contract ebola than household members who share a residence but do not participate in patient care [ , ] . healthcare workers are also at high risk for acquiring ebola. one study found the risk of developing evd for healthcare workers to be times that of the general community during an outbreak of ebola in sierra leone [ ] . there are many factors contributing to the spread of ebola amongst healthcare workers. the presentation of ebola is non-specific so early on in the disease process it may be diagnosed as malaria, influenza, or other non-specific viral illness. if a patient is initially misdiagnosed, then proper protective measures to limit the spread of ebola will not be initiated. also, the use of personal protective equipment (ppe) including gloves and gowns for routine patient care is less common in developing countries than in more developed countries due to financial restriction. there is a risk of iatrogenic spread of ebola. in the initial outbreak of , health care workers reusing glass syringes and needles in a community clinic may have inadvertently caused spread of infection. the facility consisted of a -bed hospital and a busy outpatient center which treated between and , people per month. at the beginning of each day, nurses were given five syringes each which were reused after a warm water rinse. unfortunatly, this is where ebola made its first appearance. potentially hundreds were exposed from this clinic alone [ , ] . there have been many other instances where hospitals have turned into epicenters for ebola outbreaks [ , ] . early detection and isolation is key to preventing similar incidents in the future. the greatest risk of transmission of evd from human to human occurs when a patient is acutely ill. risk also corelates with severity of illness. the sicker a patient is the more infective she is. in early phase of acute illness, the viral load is relatively low, however it increases exponentially during the latter part of the acute illness, and high viral loads are associated with high mortality rates and infectivity [ ] . those who handle corpses of ebola victims after death also run considerable risk of acquiring the disease. many funerary customs in ebola-prone regions involve extensive physical contact with the dead body. despite the risk of transmission, many still engage in these traditional practices. without these preparations, some local traditions hold that misfortune will plague the living and the dead will not be able to pass into the spirit realm. family who do not engage in expected funerary practice may be viewed negatively in the communities where they live. one funeral ceremony alone has been linked to additional cases of ebola [ ] . transmissibility of ebola virus depends on the phase of infection of the ill-person. the viral load corresponds to the severity of illness [ ] . in other words, the sicker a person is, the higher concentration the concentration viral particles in the blood stream. as an ill person succumbs to ebola, they become more debilitated and require more care. at the same time, the viral load increases as the victim declines. because of this, family caring for the ill are more likely to be infected in the later stages and corpses of those killed by ebola are highly infectious [ ] . even after a person has recovered from ebola and no virus can be isolated from blood, it may still be found in other tissues and able to transmit disease. live virus has been isolated from breastmilk after recovery raising the issue of transmission to mother to infant [ ] . ebola has been isolated from semen up to months after onset of symptoms, in urine for days, sweat for days, aqueous humor of the eye for weeks, and in cerebral spinal fluid for months [ , , , , ] . there has been at least one case where a man who recovered from ebola transmitted the infection to a sexual partner days after his initial illness [ ] . to prevent sexual transmission of ebola, the who recommends systematic testing for ebola virus in semen. for the first months after infection, the semen of male ebola survivors should be assumed to be infectious. three months after the day symptoms started semen testing for ebola should be initiated. if the result is negative, then it should be repeated in week. if the test is positive, then it should be repeated monthly until a negative result is obtained. once two consecutive negative results have been obtained sexual activities can be resumed [ ] . vaginal secretions have been found to contain virus up to days after the initiation of symptoms, but no official testing recommendations exist for vaginal secretions [ , ] . other methods of ebola spread have been postulated, but do not appear to be significant sources of transmission. surfaces contaminated with body fluids produce a theoretical risk of transmission, but no confirmed documented cases of fomite transmission of ebola exist. ebola virus has been shown to persist in the environment supporting the need for close attention to decontamination of surfaces [ , ] . medical procedures can augment disease spread if proper precautions are not taken [ ] . hunting and capturing infected animals for bush meat or for trading in black market as exotic pets can result in exposure and transmission of ebola. there have been numerous instances of human infection resulting from contact with dead primates [ , ] . contact with wild primates, especially those found dead should be avoided to curb the risk of contracting ebola. there is another step in ebola transmission that continues to be elusive. humans and other primates can catch ebola from each other, but they are not the reservoir. the reservoir is not known with certainty, but there is some evidence linking bats to ebola [ ] . the evidence for bats as the ebola reservoir is suggestive but not compelling. antibodies against ebola have been found in bat species, but the significance of this is unclear. antibodies are formed when an organism has been exposed to an infectious organism. this is evidence of exposure and immune response, but not of long-term infection or viral shedding [ ] . only one small study has ever isolated ebola rna from bats [ ] . attempts to infect bats then isolate viral rna or shedding have not met with success [ , ] . as the systematic search for the reservoir continues, negative findings are as important as positive one. plants and arthropods have not been shown to harbor ebola [ , ] . ebola virus disease is an acute febrile illness that has been associated with hemorrhagic manifestations. it has an incubation period of - days, but presentation of symptoms is most common between day and after exposure [ ] . it is unclear whether or not infected people can transmit disease prior to developing symptoms, but those with symptoms should be assumed to be contagious. evd typically begins with abrupt onset of malaise, fever, and chills. it is also common to experience vomiting, headache, diarrhea, and loss of appetite early in the disease course. the diarrhea can be profuse and water losses of up to l per day have been reported [ ] . dehydration and hypovolemic can result. relative bradycardia can also be seen in ebola [ ] . a maculopapular rash commonly develops - days after onset of illness. the rash is not a consistent finding and seems to vary from region to region [ ] . hemorrhage is the most dramatic symptom associated with evd but is not as common as first feared. usually it manifests as gastrointestinal bleeding, but petechia, ecchymosis, bleeding oral mucosa can also be seen [ ] . bleeding is multifactorial and likely due to a combination of thrombocytopenia, coagulopathy from liver involvement, and in some instance disseminated intravascular coagulation (dic). evd can cause involve a number of different organ systems. neurologically, it can cause meningoencephalitis, confusion, chronic cognitive decline, and seizures. neurological symptoms typically occur - days after onset of illness [ , ] . cardiomyopathy and respiratory muscle fatigue have been described [ ] . eye involvement is also common early in the disease course and may persist. patients frequently report blurred vision, photophobia and blindness [ ] . laboratory findings during the course of the infection can include leukopenia, elevated renal profile, abnormal coagulation panel, thrombocytopenia, anemia, and elevated liver function tests [ ] . hiccoughs are common late in the acute phase of illness. symptoms typically abate after weeks of illness. even after the acute illness has resolved, ebola victims can have long term symptoms. these include fatigue, insomnia, headaches, myalgias, arthralgias, cognitive decline, and hair loss. uveitis and hearing loss are both common after recovery from evd [ , ] . even after the resolution of acute evd, new symptoms can develop. in a study looking at early clinical sequela, % of ebola survivors developed arthralgias, % ocular symptoms, % auditory symptoms, and % uveitis [ ] . studies evaluating the long -erm sequela of evd are ongoing. prevention strategies for ebola are numerous, but essentially boils down to avoiding all contact with skin and body fluids that could potentially harbor the ebola virus. of course, this is more easily said than done especially in health care settings, and for families of those infected. health care providers deal with rapidly changing conditions often in limited resource settings and are at high risk for contracting ebola if prevention protocols are not followed. families of ebola victims face similar, but even more daunting challenges. ebola may be found in secretions of those who have recovered for months or even years after the acute illness has resolved. while not common, cases of transmission have occurred months after a person has recovered. active ebola virus can persist in urine, vaginal secretions, breast milk, semen, ocular fluid, and cerebrospinal fluid even after recovery making prevention more challenging. while not heavily researched as an effective prevention strategy, people who eat bushmeat should be encouraged to take precautions to prevent ebola infection. this means avoiding contact with fluids from slaughtered animals as much as possible. ebola virus is inactivated by thorough cooking, so through cooking of bush meat should be encouraged [ ]. ebola is highly pathogenic and easily transmitted. both the who and the center for disease control (cdc) have published detailed guidelines on prevention which are freely available online [ , , ] . the who recommends the following key elements to prevent transmission of ebola virus in the hospital setting: • hand hygiene • gloves • facial protection (covering eyes, nose and mouth) • gowns (or overalls) • sharps safety • respiratory hygiene for both health care providers and patients • environmental cleaning • safe linen transport and cleaning • proper waste disposal • proper sanitation of patient care equipment ebola prevention requires attention to and special training in donning and removing personal protective equipment (ppe). specific instructions and videos for use of this equipment is available at the who prevention cdc websites. health care workers who use ppe equipment properly are safe from ebola infection, but can develop other health issues from the ppe itself. the ppe suits are hot, uncomfortable, and require constant surveillance to ensure that all the equipment remains in place and undamaged. areas prone to ebola outbreaks tend to be hot, humid, and lack resources for air conditioning, wearing ebola suits creates a risk for development of heat related illness and dehydration. the cdc has published guidelines for preventing heat related illness for those providing care to ebola patients in hot african climates [ ] . as previously mentioned, people have survived initial ebola infection may still be able to transmit the disease to others. with proper preventive measures the risk of transmission can be ameliorated. as with other aspects of ebola, both the cdc and who have published extensive guidelines available on their websites. for healthcare workers, no special precautions are needed for basic patient care. the cdc does recommend that additional ppe be used when caring for ebola survivors if contact with testes, urine, breast, breast milk, spinal fluid, or intraocular fluid is anticipated during patient care [ ] . in the home, additional precautions may be needed. cases of transmission through sexual contact and breast milk have been describe in the literature [ , ] . cdc guidelines recommend abstinence from sexual activity of all types including oral, anal, and vaginal. if abstinence is not possible then condoms and avoidance of contact with semen is recommended. the who has recommended that semen be tested months after the onset of disease in men. if the test is negative, then it should be repeated in week. after two negative test sexual activity can be resumed. if the test is positive, it should be repeated every month until a negative test is obtained. once a negative test occurs, it should be repeated in week, and after two negatives sexual activity can be resumed [ ] . maternity issues around ebola are complex. it is unclear when it is safe for a woman to become pregnant after recovering from ebola. some organizations have suggested that a woman wait a few months prior to becoming pregnant, but so far this recommendation has not been supported by clinical data. breastmilk can transmit ebola virus from a mother who has recovered from evd to her child. if feasible, breastfeeding should be avoided. the data on ebola transmission through breasting is limited, and resources in ebola-prone areas make repeat testing of breastmilk impractical. suggested strategies have recommended avoiding breasting feeding for months after recovery [ ] . travel restrictions may occur during ebola outbreaks. it is generally accepted practice that those who have potentially been exposed to ebola virus not travel for days after the last possible day of their exposure. as an alternative for those at low risk, close monitoring with no restrictions on travel may be done. balancing individual rights with community safety creates ethical and regulatory challenges in cases of potential exposure. additional information on monitoring and travel restriction can be found at both the cdc and who websites. vaccination development is in place, but there is currently no federal drug administration (fda) approved vaccination for ebola. currently, there are different clinical trials running with the goal of developing a safe and effective ebola vaccine [ ] . an investigational vaccine called rvsv-zebov is presently being used in drc under "compassionate use". this vaccine is specific for the zaire strain of ebolavirus. this same vaccine was previously administered to , volunteers during an outbreak in . so far, the vaccine appears safe with few side effects, but insufficient data is available for licensing [ ] . preliminary reports suggest an efficacy of %, but duration of protection is currently not known [ ] . even though there are no specific therapies to treat ebola, diagnosis is important to prevent spread and to ensure administration of appropriate supportive care and monitoring. anyone who has had any potential exposure to ebola in the last days should be evaluated if symptoms of ebola develop. while awaiting the result of ebola testing, appropriate infection control practices should be implemented. diagnosis is done by reverse-transcription polymerase chain reaction (rt-pcr). the test should be done days after the onset of symptoms [ ] . false negatives can occur if the lab is collected before h of symptom onset. a positive test confirms ebola virus disease and that the patient is infective. considering repeat testing in patients whose clinical picture is highly suspicious of ebd and have a negative initial test. ebola virus disease has a broad differential, and simultaneous testing for other illnesses should be undertaken as clinically warranted. this differential includes, malaria, lassa fever, typhoid fever, influenza, meningococcal meningitis (neisseria meningitidis), measles, crimean-congo hemorrhagic fever, yellow fever, marburg, and the familiar travelers' diarrhea among many others [ ] . supportive care is the only treatment for ebola. there are no antimicrobial agents proven to be effective in ebd. when possible, care should be provided at a facility familiar with the clinical progression of ebola. supportive care in ebola is no different than for any other critically ill patient. give intravenous fluids to prevent dehydration and shock. patients with ebola suffer from vomiting and diarrhea and may easily dehydrate. if intravenous fluids are unavailable or prohibitively expensive, oral hydration should be undertaken. ebola can lead not only to hypovolemic shock, but also septic shock [ ] so close patient monitoring is warranted. electrolytes will require close monitoring and should be repleted as needed. vasopressors may be required if blood pressure cannot be maintained. ebola can result in significant hematological abnormalities [ , ] . it can also lead to liver failure followed by coagulopathy [ ] . thrombocytopenia, leukopenia, and anemia are all common and treatment should be based on the specific abnormality encountered. other management may include antipyretics, respiratory support, analgesics, antimotility agents for diarrhea, antiemetics for nausea and vomiting, antibiotics, nutritional support and renal replacement therapy. these and other supportive measures must be tailored to the individual patient need. the first reported outbreak of ebola-like illness occurred in in sudan and zaire [ ] [now south sudan and the democratic republic of congo (drc)]. it is probable that sporadic outbreaks happened earlier but were not identified. outbreaks appear to be occurring more frequently than before. this is not only due to improved detection techniques, but also due to environmental encroachment, increasing population mobility, and changing weather patterns. the following section will summarize data on known ebola social determinants of health are the conditions in which a person lives and grows. there is no one list of these factors, but they are generally considered to include influences such as school, (un)employment, the community where one resides, food, and transportation. the factors are driven by forces outside of one's sphere of control such as poverty and war as well as some potentially self-directed choices such as belief system and friend circle. for example, social determinants of health are a way of describing why when a . magnitude earthquake hits haiti buildings collapse and people die and when a . magnitude earthquake and the same earthquake on guam causes no damage. social determinants of health significantly affected how ebola has impacted affected countries. poverty affects every aspect of life for most. according to world bank data, the rate of poverty in sub-sahara africa is trending downwards but is still over % of the population. poverty leads to lack of education, limited medical resources, poor nutrition, and crowded living conditions. people in poverty will eat a dead animal if they find one because it may be all they have to eat. they are unlikely to seek medical care outside of traditional healers because it is all they know and can afford. they may insist on washing the bodies of the dead because their only knowledge of science are traditions passed from generation to generation. all of which contributes to the spread of ebola. anyone who reads the history the countries that make up the peri-equatorial regional of africa will quickly notice that the region has suffered from nearly continuous war since even before the european occupation. there are pockets of stability in the region, but conflict is a way of life for many. conflict leads to destruction of infrastructure, fear, stress, distrust, and population displacement. currently, an ebola outbreak is occurring in drc. refugees from drc continually flee into neighboring countries, especially uganda. conflict driven human movement is a means by which ebola can be spread. no widespread outbreak of ebola has occurred in a refugee camp, but these types of settlements are fertile soil where an outbreak could start and flourish before an alarm is raised. the ugandan government is working with the international federation of the red cross and red crescent societies (ifrc), unicef, and the who collaborating to develop an ebola emergency preparedness plan [ ] . political and economic instability across have resulted in a debilitated medical and public health infrastructure. official data is limited, but media sources have reported that liberia has experienced a severe shortage of trained health workers within the country. media sources list general practitioners, public health specialists, pediatricians, surgeons, obstetrician-gynecologists, ophthalmologists, internists, dentists, psychiatrists, family medicine specialists, orthopedic surgeons, radiologists, pathologist, ear-nose-throat specialist, veterinarian, and dermatologist as comprising the entire formally trained health community (excluding nursing professionals) [ ] . the cia world factbook lists the number of physicians per people to be . for liberia, . for sierra leona, . for guinea, . in drc, and . in uganda [ ] . even some of these numbers are almost years-old making it difficult to assess the actual situation in the region. regardless, it is a safe conclusion that none of these countries are even close to having the recommended physician per residents recommended by the who. each of these countries is unique in the health care challenges it faces, and only are mentioned here because they have all been touched by ebola. infrastructure development is generally associated with improved health and decreased disease burden, but this is not always the case. while lack of infrastructure such as water and sanitation is thought to lead to increased transmission. increased connectivity via road and boat is thought to increase the risk of transmission through increased number of contacts [ ] . one of the most fascinating aspects of ebola occurs at the intersection of culture and public health. for generations, a mixture of traditional beliefs and mainstream religion has served as a cultural foundation in many tribal areas across central and western africa. funerary practices in these tribes are some of the most important in their belief system. it is these practices that have been exploited by the ebola virus allowing it to spread. exposure has been associated with attendance of funerals and contact with dead bodies in multiple countries [ , , ] . as public health and medical personnel tried to curb ebola spread, conflict has occurred. those most at risk for ebola suddenly felt threatened not only by the disease itself, but also by those where were trying to help as their core beliefs were suddenly targeted. from the perspective of the health care workers trying to save lives, the cultural beliefs were generally considered as just another barrier to be surmounted. this lack of understanding between those at risk and the health care workers lead to conflict, distrust, which at times drove ebola victims into hiding rather than seeking care. bribes were made, bodies were stolen, aid workers were attacked, and ebola spread. some of the cultural beliefs common in central and western africa will be discussed here with the goal of fostering cultural understanding of disease. given the diversity of human beliefs, it is likely that future events will again put disease control against traditional beliefs. a good starting point in cultural sensitivity is viewing an idea from the point-ofview of the other party. in the case of ebola, it is important to understand what different groups of people believe to be the etiology of disease. most educated health professionals view disease as an understandable biological process. infections are caused by microbes. in the case of ebola, it is a filovirus. in many traditional african cultures, disease is believed to be due to witchcraft [ ] . consultation with traditional healers is a common practice across africa. in many regions traditional healers are the only locally available medical provider. even if modern medical facilities exist, many will turn to the traditional healers first because they are more trusted, and their beliefs tend to align more closely with those of the community. there are many different traditional healing practices, sometimes traditions are passed down through generations in specific families. one description of a traditional medical ceremony in sudan describes a medicine man and his assistants. first, ritualistic dance and chants are performed. next the medicine man shows his spiritual power by having a large rock placed on his abdomen and broken by an ax while he remains still. once his strength has been established, his attention can be turned to his patient. the medicine man's diagnosis is mental illness caused by evil ancestors who have returned with the purpose of tormenting the patient. incantations are the treatment [ ] . beliefs and practices such as this are common in rural central africa. in these societies, illness is viewed as a disruption in the relationship between god, ancestors, and the person affected. witchcraft, sorcery, angry ancestors, and evil spirits may all be at the root of disease and a powerful medicine man can restore the proper balance in these relationships thus curing disease [ , ] . the individual customs and beliefs associated with the cause and treatment of disease is too long to be included here, but those interested in additional information should read the articles cited in this section for additional details. traditional healers can be a great asset to a community, but there have been unfortunate instances where they actually promoted the spread of ebola. some traditional healers claimed to be able to cure ebola. unfortunatly, their attempts at cure have been known to spread the disease to those in attendance of curative ceremonies as well as to themselves [ ] . traditional healers can also charge a significant amount of money putting a family who is already dealing with the loss of a loved one in additional financial stress [ ] . not all traditional healers seek the good of the community but instead are motivated by personal gain. many societies in central africa practice religious beliefs based on a combination of mainstream religion and ancestor worship. occult ceremonies, secret societies, and rituals are common, and the details of these practices are often covert, only known to a small subpopulation. the ceremonies may be benign such as the one described in the preceding paragraph or may involve animal or human sacrifice [ , ] . while many of these practices involve sacrifice and exposure to blood no studies have been published linking these activities to ebola transmission. it is the traditional funerary practices that have been most closely associated with the spread of ebola. many central and western african cultures view the death ceremony as one of the most important. when people die, they must be guided to the realm of the ancestors. from this realm, ancestors are able to hear the requests and see the needs of the living family and communicate these needs to god. the living family prays directly to the ancestors. if death rights are not done correctly then instead of becoming an ancestor, the deceased may become an angry ghost which torments the family [ ] . a common funerary practice in liberia is for an elder family member to bathe the body of the deceased. it is common for mourners to touch the face and kiss the forehead of the deceased. in some traditions the spouse of the deceased continues to share a bed with the corpse until the time of burial. another tradition involves dance. on the night prior to the funeral, men dance with the dead body while women wail. several traditions involve sacrifice and exposure to the blood of a bull as part of their ceremony [ ] . to prevent the spread of disease the governments in liberia and guinea passed laws requiring safe burial teams or cremation when the number of grave sites was insufficient for the number of bodies. numerous reports of bribing health workers responsible for collecting and properly disposing of the bodies allowed ebola to persist in this region [ ] . people stopped going to the health care facilities, and families would try to hide the cause of death from officials. at the height of the epidemic in sierra leone, the number of ebola care beds was insufficient for the number of patients. many were transferred from facility to facility and their families were not notified. rumors began to spread that the ebola facilities were harvesting organs and killing people [ ] . poor communication resulted in suspicion and distrust. it took thousands of deaths, but finally both sides began to compromise. the government and health care workers started to work with local religious leaders and traditional healers to find solutions that would let the people honor the dead without exposing themselves. many muslim leaders told their followers to abstain from washing bodies until the outbreak ended. bodies were buried with families nearby and although the could not touch the bodies prayers could be said. burial teams started to dress corpses in clothing requested by the family and often placed requested jewelry. once all sides compromised and started working together the epidemic was able to be contained [ ] . even if someone survives ebola the battle is not over. there is poor understanding of disease and disease transmission. survivors may be ostracized and shunned by their communities because there is fear that they can spread disease. survivors have had their houses burned, families attacked, and lost their jobs due to irrational community fear. during the west african ebola outbreak survivors were issued certificates stating that they were no longer contagious in an attempt to combat social stigma. this is not to say that it is all gloom-and-doom in countries that have experienced ebola outbreaks. social determinants of health are not isolated static elements. technology and globalization are bringing health improvements at an unprecedented rate. if one reviews data for the countries where significant ebola outbreaks have occurred, guinea, uganda, drc, south sudan, and liberia. all of these countries have had a decrease in infant mortality rates, decrease in maternal mortality rates, and extreme poverty rate have been steadily dropping over the last years despite the presence of ebola [ ] . anyone interested in additional information on measurable global trends, whether they be economic, or health based is encouraged to visit gapminder (www.gapminder.org). not every country that faces ebola descends into a public health crisis. in july multiple cases of evd were diagnosed in lagos, nigeria. lagos is a densely populated city and the capital of nigeria. the nigerian ministry of health was able to rapidly contain the situation before a full-scale epidemic began. the nigerian government had access to trained health care providers able to do contact tracing, able to mobilize a rapid efficient response, and worked closely in cooperation with the who to implement standardized epidemiologic practices. the epidemic in nigeria was halted before it was able to start [ ] . ebola in the technology era the concept of quarantine was first developed in the fourteenth century to control the spread of plague [ ] . quarantine is a required separation of incoming people or animals prior to mixing with the local population with the goal of preventing the spread of disease. it is one of the oldest and most effective public health measures, but very unpopular with those whose movements are restricted by quarantine. recently, kaci hickcox, a nurse volunteering in sierra leone returned to the us. she possibly had been exposed to the ebola virus. ms. hickcox was placed on a mandatory home quarantine of days, but she defied the quarantine order and proceeded with her day-to-day activities [ ] . in reality, she was at very low risk for developing the disease, and there was essentially no risk for widespread ebola transmission in the us, but her unwillingness to comply with the quarantine brought attention to many public issues surrounding quarantine. specifically, the conflict between individual civil liberty and the well-being of the general public [ ] . since when quarantine laws were first written technology has expanded drastically. surely there exists a technology that allows us to abolish the antiquated quarantine system. whether an intentional act of terrorism or through accidental contagion spread, travelers pose a significant threat to homeland security. various measures have been attempted to try and identify sick travelers with the goal of limiting epidemic spread. the following is a discussion of currently available boarder control measures aimed at preventing the spread of disease, and evaluation of the effectiveness of these measures, and a discussion of technologies that may be of utility in the future in preventing cross-border ebola spread. two-point-five million people fly in or out of the united states every day [ ] and an estimated one-million more per day cross via land and sea [ ] . with millions of border crossings daily, transmission of communicable disease between remote locations is inevitable. the vast majority of communicable diseases spread by travelers are upper respiratory viruses such as the common cold or influenza. generally, these are self-limited illnesses with few long-term consequences. every few years though, something new with greater lethality emerges and threatens the security of the us travelers, their contacts, and the broader population at home. ebola, severe acute respiratory syndrome (sars), and even the relatively benign zika virus have made media headlines with travelers seen as potential harbingers of disease. another factor that must be taken into account is the increasing population density and urbanization. the united nations (un) predicts that % of all people will live in cities by the year [ ] . a megacity is defined as an urban population of over ten million people. the first to reach megacity status was new york city in the 's [ ] . by , the megacity count rose to [ ] . large numbers of people in a small area constitute a vulnerability when looking at epidemic risk assessment. a single ill traveler arriving to a megacity has the potential to start a local chain of infection that could rapidly spread to millions. with the widespread availability and affordability of trains, planes, automobiles, buses, and boats it is easy for microbes as well as humans to travel rapidly across the globe. travel provides individual freedom for pleasure and commerce but, at the expense of national security. small disease outbreaks are continually occurring across the globe. multiple international monitoring systems are in effect and the center for disease control (cdc) has issued official recommendations for travel restrictions for persons with higher-risk exposure to communicable diseases of public health concern [ ] . briefly, these guidelines state that a person who meets the following criteria will have their travel restricted [ ] : be known or likely infectious with, or exposed to, a communicable disease that poses a public health threat and meet one of the following three criteria: . be unaware of diagnosis, noncompliant with public health recommendations, or unable to be located. or . be at risk for traveling on a commercial flight, or internationally by any means. or . travel restrictions are warranted to respond effectively to a communicable disease outbreak or to enforce a federal or local public health order. while the above criteria may be the best legally available option, it leaves a multitude of holes by which a person with a communicable illness could slip into a us city and start a new epidemic. ideally, additional layers of protection would allow potentially ill travelers to be identified and detained prior to entry to the united states. an infectious agent can travel across the globe in h if spread via airplanes [ ] . this has important implications for those trying to prevent disease from spreading. land and boat entry into the united states present other challenges. the sheer number of people crossing by land on a daily basis makes any screening difficult. boat traffic can also present unique screening challenges. a cruise boat, for example, may arrive with thousands of people who all debark within a short period of time. though screens are impractical in these situations. even if screening technology was employed allowing security agents to detect fever there are so many causes of fever that timely interpretation of the data would be difficult. with so much international travel occurring, there is a continual search for ways to improve screening for ill travelers with the goal of preventing importation of disease. many different methods have been tried, most centered around a specific pandemic rather than continual monitoring. none have had great success. these methods have included entry-screens, exit-screens, and post-entry monitoring. the us division of quarantine is not only authorized, but required to identify and detain anyone entering the country with actual or suspected diphtheria, any viral hemorrhagic fever including ebola, cholera, tuberculosis, small pox, plague, novel influenza strains or yellow fever [ , , ] . in theory, this is an excellent regulation, but how can millions of travelers be efficiently screened and detained if needed? after the outbreak of sars in many countries starting using boarder screening to try to identify possibly ill people in hopes of limiting spread of infectious disease, others jumped on board after the h n influenza pandemic. the issue then resurged in the wake of the ebola outbreak in west africa. as with many things, there must be an understanding of the costs, potential benefits and effectiveness of programs aimed at preventing a possible public health disaster. an article by the cdc, published around the same time as the article recommending travel restriction for high-risk individuals, concludes that border screens are expensive and not effective in preventing the spread of disease [ ] . while point-of-care screens are not yet considered an effective means of controlling certain biosecurity threats, progress is being made. temperature screens have been developed with the goal of identifying people with fever. what happens when a fever is detected depends on where a person is traveling to and from, and the current state of outbreaks occurring in the world. there are several types of temperature readers including ear gun thermometers, full body infrared scanners, and hand-held infrared thermometers [ ] . none of these methods is highly effective and most screening devices can be fooled with minimal training and effort. once study found that thermal screens were only about % effective in detecting fever. the authors of this study concluded that temperature screens were ineffective in identifying ill travelers [ ] . the european center for disease control (ecdc) has also investigated the feasibility of using temperature screens to identify ill travelers and came to similar conclusions. this report was done during the ebola of and geared towards diagnosing travelers potentially infected with ebola. they estimate that even under ideal conditions % of symptomatic illness would be missed due to low sensitive of temperature devices [ ] . additionally, it was concluded that those intentionally trying to mask their temperature could easily do so and that those who had not developed symptoms would be missed by the screen. even if fevers screens were accurate and difficult to manipulate that would still be a poor screening measure. first of all, with many illnesses including chicken pox, flu, the common cold and countless others, people can be contagious before a fever starts. it is not yet known if an infected person can spread ebola before symptoms begin. secondly, not all fevers indicate an infectious disease. fevers can be due to drug reactions, blood clots, and even cancer. third, not everyone reacts to an infection the same way. some people naturally tend to have fever and others tend not to. one expression commonly taught in medical schools across the us is, "the older the colder". this is a reminder to students that elderly patients may never have a fever even if they are extremely ill with an infectious disease. lastly, what determines what constitutes a fever? the medical field defines fever as a temperature of degrees celsius ( . f) or higher. are these same numbers valid for travelers or should different cut offs be used? while temperature screens may have their place in emergency settings, they are far from an ideal way of detecting an ill passenger and the day to day use of temperature screens is not generally considered an effective means of identifying ill travelers. when foreign agencies are cooperative screening may be done prior to departure. exit screening was done during the ebola outbreak of for travels from west africa to the united states. the goal of exit screening is to identify those potentially infected with a specific disease and prevent them from departing for the united states until they can be medically cleared. the cdc considers this to be one of the more effective forms of preventing disease importation to the united states [ ] . departure screens are not routinely used except during times of known outbreaks. during the west african ebola outbreak exit screening measures were implemented. the general process used for screening during the outbreak was as follows. travelers were instructed to arrive earlier than they normally would for their travel due to increased processing times. general instructions to travelers instructed them to postpone travel if they were ill. in addition to the regular airport screening, all travelers were required to have their temperature taken and fill out a "traveler public health declaration". travelers who were febrile or considered at risk based on the answers to their health declaration forms were detained and their travel delayed [ ] . during the ebola outbreak the who provided resources for predeparture screening that were detailed yet used easy-to-follow language and including flow charts for those performing the screen. basic information on ebola and its symptoms so that the illness was more well understood and the disease symptoms familiar. directions for using personal protective equipment for those performing the screening. written tools and the public health declaration form were provided. additional resources included a data collection log and a traveler information card that could be distributed to travelers [ ] . the ebola screening was done in two steps, a primary screen and a secondary screen. the primary screen included three questions: ( ) is the traveler febrile?; ( ) is the traveler demonstrating symptoms of ebola?; and ( ) has the traveler marked "yes" to any questions on the health declaration form? an affirmative response to any of these questions resulted in secondary screening. secondary screening involved a public health interview and filling of the secondary health screen form, repeat temperature measurement preferably with an accurate thermometer, and focused medical exam. if the secondary screen found a temperature < . , no risk factors for ebola in the public health interview, and no symptoms of ebola on the public health interview they were allowed to proceed to check-in. if the above criteria were not met, check-in was denied until health clearance could be obtained [ ] . this strategy was considered effective. the limitations include the time and money required to implement the program, frustrating travel delays for travelers, and the inability to identify illnesses other than ebola or similar diseases. its usefulness is limited to known and identified epidemics. this strategy will likely continue to be used in future outbreaks to prevent exportation of disease [ ] . temperature screens have been used during five epidemics to date, dengue, sars, ebola, and influenza during both the entry and exit process. screening for fever in taiwan entry points during a dengue outbreak was reported to be effective. one research study reports that % of imported dengue cases were able to be identified through airport screening [ ] . during the sars outbreak, singapore entry points screened , people and identified no cases, canada entry points screened . million people and identified no cases, and hong kong entry points screened . million people identifying only two cases of sars [ ] . fever screening was used during the - influenza pandemic and even with a low threshold for defining fever was found to have a sensitivity in the . % range. exit screening done in west africa during the ebola outbreak identified fever in out of , travels screened. of these, none had ebola [ ] ). active monitoring is another technique that can be used in preventing disease spread within ebola naive countries such as the united states. it involves allowing a traveler freedom to come into the us, freedom from quarantine, but also allows health authorities to monitor the health status of potentially infected people. if someone begins to develop symptoms then measures can be taken to isolate, diagnose, and treat the ill person. this method is best applied to those who are reliable and at low risk for developing illness. there has not been much experience with widespread use of active monitoring systems with the exception of the western africa ebola outbreak. during this outbreak, travelers from liberia, sierra leone, and guinea to the us were given care (check and report ebola) kits upon arrival to the us [ ] . care kits provided resources to travelers from ebola affected countries. travelers were given information on the signs and symptoms of ebola, educated on the basic pathophysiology of ebola, provided a thermometer with detailed use instructions and given a cell phone to ease the communication process. travelers were allowed to travel freely but were required to check in with public health officials daily. during these check-ins, Àhealth reports were given including the development of any new symptoms, and daily temperature readings for days. ebola has a highly variable incubation period. twenty-one days was the longest interval between exposure and disease presentation to have been reported accounting for its use in both care packages and quarantine [ ] . while the cdc coordinated active monitoring programs, the programs were managed at the state level. all states eventually participated, but with varying start dates. new york, pennsylvania, maryland, virginia, new jersey, and georgia were those to first initiate the program. seventy percent of travelers from west africa enter through these states making them logical starting points for the program [ ] . after much legal debate and unwanted publicity, ms. hickcox mentioned in the introduction, eventually went into active monitoring program which restored most of her personal freedoms while at the same time protecting public interests. currently available technology is considered insufficient to prevent entry of ill individual into ebola naive countries. the general public continues to demand protection of civil liberties that include the freedom to travel and protection of privacy. despite recommendations by the cdc, it is difficult to identify an ill traveler either before a person embarks for the us or at the point-of-entry. post entry monitoring of reliably low risk travels is a socially acceptable alternative to quarantine and considered reliable although not widely tested. screening technologies such as infrared screens may not be considered useful on a daily use basis but may prove of utility under certain circumstances such as an active ebola outbreak. as research continues, technology advances, and better models to study patterns of disease spread are developed, new methods of pointof-entry biosecurity are sure to emerge. bioterrorism is the intentional spread of disease with the goal of destabilizing an opposing group. it is thought to have roots extending back to at least bce when the hittites used infected sheep to spread infection and destabilize their opponents [ ] . since that time, technology has improved and along with it the threat of bioterrorism has augmented. the center for disease control (cdc) divides bioterrorism agents into three separate categories a, b, and c. category a agents are those which are considered to be of highest risk. characteristics group a pathogens are, easy transmission, high mortality rate, protentional for social disruption, and require special action. category b agents are of concern, but considered to have a lower potential for disease than those in group a. this category is comprised of pathogens that are moderately easy to spread, have moderate morbidity, low mortality and require specific diagnostic and surveillance tools. group c are agents of some concern. this group is made of pathogens that are easily available, easy to produce and disseminate, and potentially have significant medical and public health implications. emerging infections also fall within group c pathogens. ebola is considered to be a high threat level a biothreat [ ] . bioweapons are at least as large a threat to homeland security as are traditional weapons. biological weapons are attractive to potential terrorists because they are relatively inexpensive to manufacture, easy to encounter, and easy to distribute [ ] . in the biological weapons convention went into effect. it has been signed by countries and prohibits the development of biological agents for the purpose of warfare. unfortunatly, terrorists fail to abide by this convention, and it is rumored that even some of the countries that signed the convention document continue to engage in clandestine research into biological agents for warfare. characteristics of a pathogen with bioterrorism potential are those with consistent disease induction and progression, high infectivity, are easily transmissible between people, are difficult to diagnose, and have a high mortality rate [ ] . it is also important that the pathogen be stable during production, storage, and distribution [ ] . lack of immunity in the targeted population and diseases that are difficult to diagnoses are also attractive to would-be terrorists. ebola possesses many of these characterizes. ebola possesses many features of an ideal bioterrorism weapon. in the early stages, ebola presents as an acute viral illness. by the time clinical features unique to ebola infection have developed, it is likely that the illness will already have be transmitted to others. particularly vulnerable are those caring for infected patients including family members and health care workers. despite being limited to transmission through body fluids, ebola is highly contagious. ebola has a high mortally rate and is attractive to terrorists because there is already widespread fear associated with ebola infection. reston virus, a non-human pathogen in the ebola family, can be transmitted. there is concern that with genetic manipulation evd could be transformed into an airborne illness and distributed as a bioterrorism weapon [ ] . ebola is one of the many pathogens that could potentially be converted into a biological weapon. preparedness plans at the local, state, and national level all include sections applicable to ebola. all hospitals in the nation have received training on ebola identification and response. continued vigilance and repetitive training sessions are required to ensure that should ebola be used as a biological weapon, it will be rapidly identified and contained. ebola virus is an agent that could be used as a bioterrorism agent. it is deadly, can result in long term infection in survivors, and non-specific clinical presentation make it an attractive choice for would be terrorists. also, for many people, the word ebola creates fear out of proportion to the actual risk of disease. this visceral reaction and exaggerated fear make ebola a tempting agent. on the other hand, the lack of airborne spread and existence of effective vaccine (even if not licensed) are deterrents to its use. it is impossible to know with certainty when the first ebola infection occurred. most likely it was in a remote african jungle and those infected died without a diagnosis other than that provided by the local traditional healer. what can be said with certainty is that the outbreaks are occurring with more frequency. no one knows with certainty why this is. hypothesis tend to center around issues of environmental degradation in association with increased population mobility. increasing population, global warming, and continued human encroachment into forested areas have been put forth as potential contributing factors. increasing population is theorized to be contributing to the increasing frequency of ebola outbreaks. increasing populations, particularly in developing countries, tend to lead to congesting living conditions and rapid disease spread, but this would not explain how the index case in an outbreak becomes infected. experts opinion often lists expanding population as contributing to the ebola outbreak, and intuitively it is credible, but there is little in the way of direct evidence to support this theory. literally hundreds of studies have been conducted on ebola since the outbreak, but none directly addresses the relationship between population growth in africa and increasing frequency of ebola outbreaks. it is likely that the impact of increasing human populations in endemic areas will not be fully understood until the reservoir of ebola has been determined. what we can say with certainty is that once started, ebola spreads more quickly than it did in the past and is killing more people. population level research on ebola has yielded interesting results. for a start, risk of ebola infection has been associated with a higher level of education [ , ] . lower risk for acquisition of ebola at the population level has been associated with urban residence, households with no or low-quality sanitary system, and married men in blue-collar professions in the outbreak in west africa [ ] . other studies have found different results when examining the interplay between population dynamics and the emergence of ebola. for example, in contrast to the study by levy & odoi, ebola transmission has been positively correlated with population density, and proximity to ebola treatment centers in other investigations [ ] . another study found that . % of people who tested positive for ebola cases lived within a -km of roads connecting rural towns and densely populated cities [ ] . basic public health principles hold that increasing population density allows infectious disease to spread more quickly, but it is unclear what the impact is on the emergence of ebola. it is safe that there is a relationship between population density, population distribution, and ebola but the exact nature of that relationship remains elusive. climate change has been cited by mass media sources as the source of emerging disease such as ebola. elevated atmospheric temperature have been associated with the development of evd, but then so have low temperatures [ ] . there does appear to be a relationship between ebola and temperature, but the character of that relationship is not clear. ebola virus is sensitive to high temperatures so intuitively, higher temperatures would not create a more active form of the virus. what may change is the human response to higher temperatures. when it is hot, people sweat more, drink more, and may wear different clothing. it may be that the human response to hot weather is responsible for the noted difference rather than changes in viral activity. it is also possible that temperature changes correlate with other phenomena such as rain storms and that rain, or the response of vegetation to rain somehow impacts the emergence of ebola. climate change, whether due to human activities or natural climatic cycles will change patterns of disease across the globe. how changing weather patterns may affect the distribution and frequency of ebola cases remains to be seen. possibly once the reservoir of ebola virus has been discovered scientists can predict with greater certainty how climate change will impact the emergence of ebola. it is also postulated that ebola is occurring with greater frequency due to increasing human activities within previously untouched natural areas. at least one study has linked deforestation to evd outbreaks [ ] . again, there are limited studies confirming this idea, but logic does suggest that it would be true. expert opinion, and the mass media purport that the increasing frequent outbreaks of ebola are due to environmental encroachment [ ] . as roads are build, forests are cut, and mineral resources exploited humans are in more intimate contact with the forest and its inhabitants including the reservoir for ebola. the reservoir is unknown, but it is probably found in african jungles. a study looking at vegetation cover, population density and incidence of ebola found that vegetation was protective until the population reached people per square km. at this population density vegetation became associated with and increase incidence of evd [ ] . there is a relationship between environmental encroachment and the emergence of ebola, but until the reservoir is found it will be difficult to determine the exact nature of this relationship. the frequency of ebola outbreaks has been increasing. international collaboration is essential to better understand how and why this is occurring. traditional tribal regions do not always follow country lines and both official and unofficial border crossing are common. contact tracing is essential for containment of ebola outbreaks requires countries to coordinate as people cross borders. epidemiological evaluation and experience in treating the disease also require a global rather than country approach. the study of ebola requires systematic evaluation and intercountry coordination to most effectively predict outbreaks and limit their spread once they do occur. the global community would also benefit from international standards for diagnosis, prevention, and treatment. luckily, framework already exists for this collaboration, at least in times of epidemics with pandemic potential. the international health regulations (ihr) agreement is legally binding accord signed by countries. it stipulated that these countries must act to contain the threat if a public health emergency of international concern (pheic) is declared by the who director general. a pheic was declared in august in response to the ebola outbreak in west africa [ ] . the ihr helps to ensure that an appropriate global health response will be made once a public health disaster is well underway. intervention at this level will help curb progression of the disaster. along this same line of thinking, mitigation and preparedness efforts are needed prior to development of a public health disaster. if a pheic is declared, then local measures have failed. improved regional collaboration is needed to help minimize the impact of ebola in the region. many countries at risk for outbreaks of evd would benefit from bolstering of their public health and medical programs. outside assistance is a starting point, but capacity building is required for long term solutions. in countries with weak public health infrastructure international efforts need to focus on programs to develop a sustainable public health system. the challenges are considerable particularly in areas of chronic conflict, but progress has already been made and with continued support will continue into the future. a basic public health infrastructure will help contain ebola as well as whatever threat comes next. when an ebola outbreak hits the general public needs to be educated on how to respond. if ebola preparedness is part of the local education, then lives can be saved. the public can help with surveillance efforts. this would require the population to trust the public health community, believe that their input is useful, and that they be trained to recognize potential ebola in the community. public health education can also assist with limiting spread if an outbreak does occur. this education can be provided through schools, community outreach campaigns, or religious institutions. the education does not need to be complex, just consistent, concise, true, and culturally appropriate. outbreaks of evd have been occurring with increasing frequency. thousands have died and thousands more have been lives have suffered because of the disease. the disease is highly fatal, but even more insipid, it exploits traditional ceremonies and death-rights as a means of spread. poverty, both at personal and national level has resulted in an infrastructure ill-equipped to deal with events such as ebola. overcrowding promotes transmission and lack of financial incentives have delayed vaccine development. despite the barriers, evd is slowing being more well understood, thousands of research articles have been published, and guidelines for every aspect of the disease have been published by the who, cdc, or other government level organizations. progress is being made. esposure patterns driving ebola transmission in west africa: a retrospective observational study assessment of the risk of ebola virus 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interim guidance ebola virus disease: faq: compassionate use of ebola vaccine in the context of the ebola outbreak in north kivu, democratic republic of the congo who: international commission ( ) ebola haemorrhagic fever in zaire who: international study team ( ) ebola haemorrhagic fever in sudan who interim guidance for ebola: exit screening at airports, ports and land crossings november ) who interim guidance for ebola: exit screening at airports, ports and land crossings ebola fact sheet the cdc's new quarantine rule could violate civil liberties. the atlantic a novel immunohistochemical assay for the detection of ebola virus in skin: implications for diagnosis, spread, and surveillance of ebola hemorrhagic fever key: cord- -blwguyl authors: guleria, randeep; mathur, vartika; dhanuka, ashutosh title: health effects of changing environment date: - - journal: natural resource management: ecological perspectives doi: . / - - - - _ sha: doc_id: cord_uid: blwguyl environment plays a crucial role in our economic, social and cultural behaviour as well as on health. however, since the beginning of industrialization era, focus on economic development has caused detrimental effects on the environment. last two centuries have witnessed changes in global environmental factors such as rise in temperature leading to global warming, depletion of stratospheric ozone layer, loss of biodiversity and marked degradation in air and water quality due to atmospheric pollution, thereby causing upsurge in infectious and non-infectious diseases. environmental health has emerged as an important part of medicine. the world health organization (who) estimates that % of global disease burden and % of all deaths can be attributed to environmental factors. deaths from heart disease, cancer, respiratory disorders and many vector-borne diseases such as malaria, dengue, chikungunya and cholera have increased due to changes in climate, especially in developing countries. besides limited attention to sanitation, hygiene, as well as quality of food and drinking water, factors such as deforestation, increasing vehicular traffic, migration from rural to urban areas, decreasing water resources and inadequate drainage systems contribute to increase incidence of diseases. the need of the hour is to sensitize ourselves about the way our ecology is being degraded and the health effects it is causing. a holistic view is needed to address the problem of environmental health where agriculture, animal husbandry, public health, water safety and air pollution need to be looked at in a combined manner for education, planning and resource allocation. therefore, a close association between scientists, public health professionals and administrators is needed for integrated design and development of framework to attain harmony between man and nature. an ecosystem is defined as a community of living beings surviving and interacting in mutual and interdependent relationship with their physical environment. for thousands of years, man has lived in harmony with their natural surroundings. environment has played a crucial role in his economic, social and cultural behaviour as well as on his health. the role of environment in various diseases has been well documented, both in communicable and non-communicable diseases. since the dawn of industrialization era in europe years ago and its subsequent spread to the rest of world, economic development and physical comfort for mankind have increased at a tremendous pace. this increase is perhaps the most rapid over the last three decades. often this has been done, knowingly or unknowingly, at the cost of our environment. the last years have witnessed a sharp increase in the global temperature from its levels around years ago, owing to industrialization (mann et al. ; marcott et al. ) . moreover, other concerns such as depletion of carbon fuels at alarming rates, damage to the ozone layer and rise in seawater levels, combined with global warming, have damaged our environment extensively, leading to changes in the aquatic biodiversity and to the extinction of many species of plants and animals (thomas et al. ) . increase in urbanization has led to loss of dense forests. air pollution has risen to the extent that many big cities in the world have a highly toxic air quality. however, very little has been done by various governmental and non-governmental agencies with almost no visible results. the need of the hour is to sensitize the scientific community, as well as the common man, about the way our ecology is being degraded and the health effects it is causing and to suggest ways to get remedies for this situation. environmental health has emerged as an important part of medicine, due to the rapid environmental changes linked to industrialization and urbanization. it is being increasingly recognized that environmental factors play a key role in human health and are linked to many chronic and infectious diseases. deaths from heart disease and respiratory illness are increasing, and many diseases such as malaria, dengue, chikungunya and cholera are sensitive to changes in the climate (mcmichael et al. ; patz et al. ) . according to the world health organization (who), 'in its broadest sense, environmental health comprises those aspects of human health, disease and injuries that are determined or influenced by factors in the environment. this includes the study of both direct pathological effects of various chemical, physical and biological agents, as well as effects on health of the broad physical and social environment, which include housing, urban development, land use and transportation, industry and agriculture'. the who estimates that % of the global disease burden and % of all deaths can be attributed to environmental factors. moreover, environmental factors have a much bigger impact in developing countries than developed ones, and this effect is seen much more in the vulnerable population such as children and elderly. in a developing country like india, the burden of various diseases is increasing due to environmental factors and the changes in our environment. it is estimated that % diarrhoeal disease burden may be attributed to environmental factors such as unsafe food and drinking water, as well as poor sanitation and hygiene. similarly, in india there is strong evidence linking lower respiratory tract infection to indoor air pollution caused by the use of solid fuels in household. almost % of acute lower respiratory tract infections in developing countries are attributable to environmental factors. besides this, a close association of vector-borne diseases and environmental conditions has been established. furthermore, factors such as deforestation, increasing vehicular traffic, migration from rural to urban areas, decreasing water resources and inadequate drainage system are important environmental and ecological factors that contribute to infectious diseases. the temperature of the earth has increased by about . °c over the past years (griggs and noguer ; mccarthy ) . winters are shortening and average temperature is rising. intergovernmental panel on climate change (ipcc) of united nations predicts that the global temperature will rise by . - . °c by the turn of this century, if no remediable actions are taken (houghton et al. ). this will lead to rise in sea level by - cm and drowning of coastal cities, which comprise % of world's major cities (crutzen ; fitzgerald et al. ; nicholls and cazenave ) . higher temperatures will lead to melting of polar ice, melting of glaciers, floods and droughts (patz et al. ) . average temperature shall rise during both summers and winters. heat waves will increase, and average annual precipitation will also increase correspondingly. heat waves, floods and droughts lead to natural calamities, shortage of food supplies, increased risk of infectious diseases and increased human mortality (haines et al. ). clean water is essential for the survival of humans. water pollution due to environmental changes therefore constitutes another serious risk to the health of our planet. water pollution occurs when energy and substances are released and degrades the quality of water for other users. anything that is added to water, which is more than its capacity to break it down, constitutes water pollution. anthropogenic activities such as industrial waste effluents, sewage disposal and agricultural activities are some of the major causes of water pollution (manivasakam ; tilman et al. ) . chemical pollution of surface water causes major health problems as it can be used directly for drinking or it may contaminate shallow wells, used for drinking. ground water, which is much deeper, has very few pathogens as it gets filtered when it passes through many underground layers. it can be polluted by toxic chemicals such as fluoride and arsenic which may be present in the soil or the rock layers. similarly, pollution of coastal water can cause contamination of sea food (guleria ) . changing environment has a serious effect on safe water, affecting not only human health but also changing the ecology of plants. the global effect of water pollution has not been studied in detail and is limited to mainly outbreaks of waterborne infections or certain chemical toxins in limited areas, such as arsenic in drinking water in bangladesh, 'minamata' disease in japan, etc. (argos et al. ; harada ) . the burden of waterborne diseases is grossly underreported in india due to lack of data, poor surveillance and reporting. according to a report from the ministry of health and family welfare, nearly million people are affected by waterborne diseases such as diarrhoea, enteric fever, amoebiasis and helminthic infestations, every year. who estimates > , deaths annually, in india alone, due to contaminated water consumption. moreover, floods and droughts also affect human health. floods lead to physical injury as well as spread of waterborne diseases such as diarrhoea, enteric fever and viral hepatitis. overcrowding occurs and sanitation is affected, leading to respiratory infections. diseases such as malaria and dengue may turn into epidemics. on the other hand, drought leads to lack of sanitation, decreased food production and ultimately malnutrition. another aspect of waterborne diseases is chemical contamination leading to diseases such as fluorosis and methemoglobinemia, due to contamination of soil water owing to fluoride and fertilizers. chronic exposure to contaminated water can cause significant health effects and can lead to liver and kidney damages. this occurs due to chronic exposure to copper, cadmium, arsenic, mercury, chromium and chlorobenzene. endocrine effects have been reported, and problems relating to reproduction, development and behaviour have also been observed. enso is a cycle of seawater temperature and pressure changes occurring over the southern pacific ocean at an interval of - years and lasting for - months. this leads to episodes of floods in the southwest united states, mexico and western coast of latin america and droughts in southeast asia and the pacific islands (kovats et al. ). this may be followed by cold waves called la niña. higher global temperatures are predicted to lead to more frequent and severe ensos, and this will lead to significant effects on human health, in the coming years (bouma et al. ). change in the global climate has led to higher temperatures, humidity and floods, which has made the environment more conducive for parasites such as mosquitoes and fleas (patz et al. ) . malaria, dengue and other vector-borne diseases are expected to increase both in magnitude and their geographical reach (haines et al. ) . people living at higher altitudes may also likely experience resurgence in vector-borne diseases, due to a rise of average temperatures in these regions. moreover, these diseases can spread to any part of the world in a very short time (at times during the incubation period) and cause an outbreak in a community where these diseases do not usually occur, resulting in diagnostic difficulties. this has recently been seen during the ebola and the mers coronavirus outbreaks. other factors such as breakdown of public health infrastructure, shortage of medical supplies and changes in land use also contribute to adversities in health, due to water pollution. air pollutants affect the human body through the inhalational route. environmental changes due to industrialization have drastically altered the quality of the air we breathe. there are hundred substances that pollute the air and may harm human health. pollutants are generally classified as primary or secondary pollutants. chemicals that are directly emitted from a source are known as primary pollutants. these include sulphur dioxide, nitrogen oxides, carbon monoxide, volatile organic compounds, etc. moreover, particulate matter emitted due to combustion from automobile exhaust, heating, cooking and industrial sources are also primary pollutants. secondary pollutants, such as formaldehyde, nitric acid and different aldehydes, on the other hand, are formed from chemical or photochemical reaction in the atmosphere. on exposure to sunlight, volatile organic compounds and nitrogen oxides react photochemically, producing pollutants such as ozone. air pollution and occupational exposure may cause a variety of negative health outcomes, including reduced lung function in children as well as increased susceptibility to infections, airway inflammation and cardiovascular diseases. respiratory disorders due to air pollution are emerging to be a major contributor to mortality, according to recent epidemiologic studies. moreover, low-level air pollution is recently being recognized as a risk factor for lung diseases and death from copd (bosson and blomberg ) . with newer insights into the immunopathogenesis of asthma, the contribution of air pollution to allergen sensitization and airway hyperresponsiveness are being established. for example, increased exposure to nitrogen dioxide during infancy correlates with increased risk for asthma in later childhood. ozone can produce significant adverse effects on human health (gryparis et al. ; teague and bayer ; uysal and schapira ) . moreover, recent research is now linking air pollution to increased risk of respiratory symptoms and duration of respiratory tract symptoms. international agency for research on cancer recently designated diesel exhaust as a human carcinogen. sulphur oxides are produced mainly from industrial activities processing materials that contain sulphur, such as generation of electricity from coal, oil or gas, as well as by combustion of fossil fuels. sulphur dioxide is also present in motor vehicle emission. together with ozone, it is known to cause foliar injury and reduction in plant growth (smith ; tingey and reinert ) . it is mainly absorbed in the upper airways as it is water soluble. its exposure is known to cause symptoms such as nose and throat irritation. it may travel to the lower airways and cause bronchoconstriction and dyspnoea, especially in asthmatic individuals, thus worsening their condition (balmes et al. ; ierodiakonou et al. ) . nitrogen oxides are emitted primarily from motor vehicle exhausts, as well as from stationary sources such as electric utilities and industrial boilers. compounds such as sulphur and nitrogen oxides cause chemical reactions in air and acid rains. although acid rains do not affect humans radically, they may indirectly cause health problems, particularly difficulty in breathing and, in extreme cases, lung problems such as asthma or chronic bronchitis. moreover, nitrogen oxides are the main precursors in the formation of tropospheric ozone. they also form nitrate particles and acid aerosols. exposure to nitrogen dioxide for a short term leads to changes in airway responsiveness and deterioration in pulmonary function in individuals with underlying lung disease. long-term exposure may lead to increased chances of recurrent respiratory tract infections and alter lung mechanics (berglund et al. ) . carbon monoxide is produced mainly due to motor vehicle emission. in urban areas more than % of the carbon monoxide emission may be due to motor vehicles. besides this, the combustion of coal, oil and gas also leads to carbon monoxide production. moreover, tobacco smoke is one of the main sources of indoor pollution of carbon monoxide. high levels of carbon monoxide are extremely dangerous to humans, more so because it is colourless, tasteless and odourless and therefore cannot be detected by humans. early symptoms of carbon monoxide include weakness, headache, nausea, dizziness, confusion, disorientation and visual instability. carbon monoxide quickly enters the blood stream and forms carboxyhaemoglobin which causes more systemic effects. it reduces oxygen delivery to the tissues and may have a serious health threat to those with underlying heart disease (badman and jaffé ) . prolonged or severe exposure may result in lethal arrhythmias, electrocardiographic changes, pulmonary oedema, various neurological symptoms as well as death, most likely due to cardiac failure. carbon monoxide is known to cause foetal development disorders, brain lesions and, in extreme cases, even mortality (raub et al. ) . the atmospheric levels of lead have decreased due to the use of unleaded fuel. however, lead toxicity continues to be a problem, due to the exposure occurring in drinking water. lead exposure leads to adverse effects on the central nervous system, causing neurological symptoms such as sleep disorders, fatigue, trembles in limbs, blurred vision and slurred speech, as well as kidney and liver disorders (kampa and castanas ) . lead toxicity can lead to lower intelligence, learning deficits and behavioural disturbances. ozone is an important secondary pollutant and is a component of photochemical smog. it is a pulmonary irritant and an oxidant. it may produce significant adverse effects on human health. exposure to ozone causes airway inflammation, airway hyperreactivity and a decline in lung functions. ozone exposure causes cough, chest tightness and wheezing. the increase in the levels of tropospheric ozone is associated with reduced baseline lung functional as well as structural abnormalities, exacerbation of asthma and premature mortality. recent studies have shown increased admissions for chest complaints and worsening of asthma on exposure to even low levels of ozone. studies looking at long-term exposure to ozone suggest that a cumulative long-term exposure in childhood may affect lung function, especially that of the small airways of the lung, in adult life (künzli et al. ) . ozone also affects mucous membrane and causes pulmonary inflammation and has both a local and systemic effect on the immune system. patients with underlying respiratory illness such as asthma and chronic obstructive airway disease are more prone to the harmful effect of ozone. high ozone concentrations have been linked with increased hospital admissions for pneumonia, copd and asthma (gryparis et al. ; teague and bayer ; uysal and schapira ) . particulate matter consists of liquid or solid mass contained in an aerosol. it is a mixture of numerous different chemicals, with varying properties. major sources of particulate matter are factories, power plants, incinerators, motor vehicles, construction activities, fire and dust. broadly particulate matters from . to μm in diameter are coarse particulate matter. coarse particulate matter consists mainly of airborne soil dust and elements such as silicon and aluminium. fine particles of less than . μm are composed mainly of sulphate and organic material. particulate matter in air is associated with allergic rhinitis, lung inflammation, pulmonary disorders, cardiac arrhythmia, ischemic cardiovascular events, higher incidences of cancer and shortening of life (carlsten and georas ; dockery et al. ; kampa and castanas ; pope iii et al. ; raaschou-nielsen et al. ) . only recently we began to understand the cardiovascular effects of air pollution. high levels of air pollution worsen underlying heart disease. but now it is becoming clear that persistent exposure to high levels of air pollution may also lead to heart disease. this is especially true for particulate matter. inflammation in lungs also causes inflammation in the blood, leading to atherosclerosis and an increase incidence of coronary artery disease that may be fatal (fig. . ). many well-conducted studies have demonstrated a - % higher risk of coronary artery disease in individuals exposed to high levels of air pollution, for many years (cesaroni et al. ; miller et al. ). this increased risk has been linked to higher levels of pm . in the ambient air. studies have also looked at subclinical atherosclerosis, which is the pathological process associated with coronary artery disease. a positive association between subclinical atherosclerosis in the carotid and the coronary arteries has been observed with long-term exposure to high levels of air pollution (künzli et al. ; künzli et al. ) . there is therefore now a significant body of evidence linking air pollution to cardiovascular diseases and increased mortality. many investigators argue that air pollution should now be considered as a preventable risk factor like smoking and dyslipidemia for the development of coronary artery disease, and steps should be taken to bring down the exposure to air pollution. indoor air pollution and its effect on human health are important as individuals spend more than % of their time indoors. cooking is an integral part of indoor human activity. the who has estimated that about % of the world's population, or about billion people, still uses solid fuel for their household energy needs. of these, about . billion people use biological material (wood, charcoal, crop waste and dung), and the remaining use coal. in india, about % of the population has been estimated to depend upon wood, and about % depend upon dung for energy. although this number is slowly decreasing and moving towards the use of other fuels such as liquefied petroleum gas (lpg) and kerosene, it is still very significant. in india in , of the . billion people, about million still used solid fuel for cooking or heating. many studies over the last three decades have documented the link between solid fuel exposure and different respiratory diseases. lim et al. ( ) estimated more than million premature deaths per year due to indoor air pollution, because of solid fuel used for cooking purposes. exposure to high concentrations of harmful substances in smoke during use of biomass fuel causes significant illness amongst homemakers and young children. it has been shown that biomass fuel is a less efficient means of energy production and a number of carcinogenic constituents are released during biomass combustion (chafe et al. ; smith and sagar ) . inhalation of these particles in high concentration leads to 'lung overloading' and sustained inflammation. this results in the release of reactive oxygen that causes deoxyribonucleic acid (dna) damage. indoor smoke produced due to burning of solid fuel contains many pollutants. particulate matter, nitrogen oxides, carbon monoxide, benzene, . butadiene, polycyclic aromatic hydrocarbons, free radicals and volatile organic compounds are many of the toxic substances that have been found in smoke produced by burning solid fuels. chronic exposure to these harmful substances leads to lung fibrosis and subsequently the development of lung cancer. the evidence for the development of lung cancer due to biomass exposure has been shown in experimental animals, but the evidence in humans is not that strong. indoor air pollution thus accounts for a significant proportion of the global burden of disease in developing countries. the link between solid fuel exposure and chronic obstructive lung disease in women and acute respiratory tract infection in children is strong. the commendable initiative by the government of india called 'give it up' is a step in trying to decrease the effects of indoor air pollution on human health. also steps to improve ventilation in kitchens or use smokeless stoves chulla may also help in reducing the exposure to indoor air pollution (reddy et al. ). waste generated from used electronic devices and household appliances constitutes e-waste. it comprises of a wide range of equipments and devices falling under 'hazardous' and 'non-hazardous' categories such as computers, mobile phones, refrigerators, washing machines, air conditioners, personal stereos, consumer electronics, etc., that are discarded by users (puckett et al. ) . pollution due to electronic and electrical waste has rapidly grown over the last decade due to progressive increase in production of electronics, lack of proper disposal facilities in india and dumping of e-waste from developed countries. in alone, india generated about . million tons of e-waste (double the amount as compared to ), which is progressing rapidly. e-waste may contain many toxic substances which may be harmful to the environment and human health. this can have a significant economic and social impact on society. iron and steel constitute about % of the e-waste followed by plastics ( %), nonferrous metals ( %) and other constituents ( %). others include nonferrous metals like copper, aluminium, silver, gold, platinum, palladium, etc. the presence of elements such as lead, mercury, arsenic, cadmium, selenium and hexavalent chromium, with flame retardants beyond threshold quantities of e-waste, classifies them as hazardous waste. manual recycling of e-waste is done predominantly via the unorganized sector, and the work force involved consists predominantly of individuals with low literacy and hardly any training to protect themselves from ill effects and to identify warning signals of toxicity. accordingly, a significant percentage of health problems due to e-waste results from direct contact with harmful materials and inhalation of toxic fumes. moreover, these materials may get accumulated in the food and water and are consumed. heavy metals such as lead can cause kidney failure, neurologic manifestations and hypertension. mercury toxicity can lead to central and peripheral nervous system damage and hepatic and renal toxicity (guleria ) . furthermore, uncontrolled burning, disposal and dismantling of e-waste can cause a number of problems including air pollution and water pollution. there is a lack of an environmentally effective recycling infrastructure for e-waste, and this leads to pollution of the environment. this is gradually changing our ecology. there is, therefore, a need to increase public awareness about the harmful effects of e-waste and develop an effective recycling and disposal plan, to prevent or minimize air and water pollution. there should be general awareness of how changes in climate and environment lead to significant acute and chronic effects on human health. these effects can be both for infectious and non-infectious illnesses. a holistic view is needed to address the problem of environmental health where agriculture, animal husbandry, public health, water safety and air pollution need to be looked at in a combined manner for education, planning and resource allocation. general population should also be made aware about the ways to reduce harm to our environment. intergovernmental efforts should be made to check climate change, avoid deforestation and use alternative sources of energy like solar energy instead of petroleum products. ultimately, as embedded in its definition, ecosystem is a community, and unless all people in community put efforts to conserve it, no amount of individual effort can suffice. therefore, a close teamwork between scientists, public health professionals and administrators is needed for integrated vertical and horizontal planning. arsenic exposure from drinking water, and all-cause and chronic-disease mortalities in bangladesh (heals): a prospective cohort study blood and air pollution; state of knowledge and research needs symptomatic bronchoconstriction after shortterm inhalation of sulfur dioxide health risk evaluation of nitrogen oxides update in environmental and occupational medicine global assessment of el niño's disaster burden update in environmental and occupational lung diseases long term exposure to ambient air pollution and incidence of acute coronary events: prospective cohort study and meta-analysis in european cohorts from the escape project household cooking with solid fuels contributes to ambient pm . air pollution and the burden of disease an association between air pollution and mortality in six us cities coastal impacts due to sea-level rise climate change : the scientific basis. contribution of working group i to the third assessment report of the intergovernmental panel on climate change acute effects of ozone on mortality from the "air pollution and health: a european approach" project basic considerations of environmental and occupational diseases climate change and human health: impacts, vulnerability and public health minamata disease: methylmercury poisoning in japan caused by environmental pollution climate change : the scientific basis ambient air pollution, lung function, and airway responsiveness in asthmatic children human health effects of air pollution el niño and health association between lifetime ambient ozone exposure and pulmonary function in college freshmen-results of a pilot study ambient air pollution and atherosclerosis in los angeles ambient air pollution and the progression of atherosclerosis in adults a comparative risk assessment of burden of disease and injury attributable to risk factors in regions - : a systematic analysis for the global burden of disease study physico-chemical examination of water sewage and industrial effluents. physico-chemical examination of water sewage and industrial effluents proxy-based reconstructions of hemispheric and global surface temperature variations over the past two millennia a reconstruction of regional and global temperature for the past , years climate change : impacts, adaptation, and vulnerability: contribution of working group ii to the third assessment report of the intergovernmental panel on climate change climate change and human health: present and future risks long-term exposure to air pollution and incidence of cardiovascular events in women sea-level rise and its impact on coastal zones effects of environmental change on emerging parasitic diseases the potential health impacts of climate variability and change for the united states: executive summary of the report of the health sector of the us national assessment impact of regional climate change on human health particulate air pollution as a predictor of mortality in a prospective study of us adults exporting harm: the high-tech trashing of asia. basel action network. e-waste/technotrashfinalcom air pollution and lung cancer incidence in european cohorts: prospective analyses from the european study of cohorts for air pollution effects (escape) carbon monoxide poisoning-a public health perspective domestic cooking fuel and lung functions in healthy non-smoking women air pollution and forests: interactions between air contaminants and forest ecosystems making the clean available: escaping india's chulha trap outdoor air pollution: asthma and other concerns extinction risk from climate change forecasting agriculturally driven global environmental change the effect of ozone and sulphur dioxide singly and in combination on plant growth effects of ozone on lung function and lung diseases key: cord- -of vx og authors: saghazadeh, amene; rezaei, nima title: the physical burden of immunoperception date: - - journal: biophysics and neurophysiology of the sixth sense doi: . / - - - - _ sha: doc_id: cord_uid: of vx og the previous chapter introduced the immunoemotional regulatory system (immers). also, there was a brief discussion about psychological states/psychiatric disorders that so far have been linked to the immers. the present chapter considers another aspect of the immers in which physiological states/physical diseases can be fit to the immers. such as pemphigus [ , ] . further, human studies provided evidence pointing to the increased development of emotional problems and edr-related disorders in patients with various types of aids, such as sle and multiple sclerosis (ms), in a disease state/severity-dependent manner [ ] [ ] [ ] [ ] [ ] [ ] . for example, among patients with childhood-onset sle, % manifest neuropsychiatric sle (nsle). mood and anxiety disorders were the most common psychiatric conditions with the prevalence rate of % and % [ ] . even about % of patients with sle without cns manifestations suffer from psychological distress compared with % in controls. it is, thus, not surprising that both emotional coping and depressive symptoms were correlated with non-nsle [ , ] . interestingly, there was an increased activation of the brain regions related to emotion regulation/processing (e.g., the amygdala and superior temporal) in sle patients. however further analyses led to identifying this increased activity of emotional circuit as a consequence of cns involvement by sle [ ] . among patients with ms, emotional troubles were more than twofold more likely to occur in patients who had an exacerbation or progressive nonremitting ms compared to stable patients. this was reflected by an increased rate of using emotion-focused coping styles in patients with relapsingremitting multiple sclerosis (rrms) compared to stable patients [ , ] . mood disturbance was correlated negatively with sil- r levels and positively with joint pain in patients with ra [ ] . consistent with data from human studies, animal experiments have also supported the link between emotionality-related behaviors and aids. clearly, aids result from immdr. interestingly, the aids-related immdr has been observed in the specific brain regions associated with emotional behaviors, particularly anxiety-and depressive-like behaviors [ ] . in this manner, the link between aids and immers is strengthened. a high rate of increased emotionality and emotional-like behaviors in aids led to propose the term autoimmune-associated behavioral syndrome (aabs). studies emphasize the pivotal role of cytokines and neuroendocrine factors in the pathogenesis of aabs [ ] . b-cell-activating factor (baff) transgenic mice model, which is used as an experimental model of systemic lupus erythematosus (sle), rheumatoid arthritis (ra), and sjögren syndrome, exhibited an anxious phenotype along with the following changes in immune brain signaling, such as increased igg titers in the hippocampus, hypothalamus, and cortex and increased cd (as a maker of activated microglia/macrophages) and gfap (as a maker of activated astrocytes) immunoreactivity in the hippocampus in mice at . - months of age, but not in young ( months of age) mice [ ] . eae models of ms showed an increase in levels of il- β and tnf-α in the hypothalamus. this indicates an inflammatory central basis behind anxiety-and depressive-like behaviors [ ] . these emotional deficits were shown to display before the onset of ms [ , ] . consistently, behavioral problems usually manifest before symptoms of impaired cognitive and motor performance in dementia. moreover, this model showed an early (at day ) and a meaningful increase in circulating cytokine levels and cd + t cell counts. of note, these inflammatory markers began to decrease in the periphery (at day ) almost when their infiltration in the cns (at day ) started [ ] . in the mrl-lpr model of aid, which is a well-documented model of emotional deficits [ , ] , a reduced preference to glucose, and as an index of emotionality, was detected in -to -week-old mice [ ] . this deficit could be diminished by immunosuppressive treatment with cyclophosphamide and was pronounced by means of chronic administration of il- [ ] . along with psychosocial stressors, either chronic and acute, and social networkrelated factors (i.e., social ties, social conflict, and social support), the experience of unpleasant emotions, including anger, depression, sadness, and stress, or, in general, extremely exciting emotions, often promptly, pulls susceptible individuals into a steep road leading to cardiovascular events, particularly acs (for review see references [ ] [ ] [ ] ). for example, emotional stress is ranked as the second most common neuropsychological cause of acute myocardial infarction (ami) owing to its record in approximately - % of these patients [ , ] . at the molecular levels, these patients have shown increased levels of the proinflammatory cytokines (e.g., tnf-α, il- , il- , il- , and il- ) and decreased levels of the anti-inflammatory cytokine (il- ). thus, it is not surprising that the inflammatory response and respective cytokines are supposed as one of the possible mechanisms linking the experience of negative emotions or er-related disorders and the progression of cardiovascular diseases, of course along with the neuroendocrine system and apoptosis signaling pathways [ , , [ ] [ ] [ ] [ ] . it is to be noted that when patients with cardiovascular diseases are stratified according to their emotional background, some cytokines are more highlighted than others, for example, tnf-α and il- , but not il- , considering depressive symptoms in chf patients [ ] . cognitive reappraisal is found to correlate positively with the engagement of the lateral and prefrontal regions and inversely with the engagement of the amygdala and medial orbitofrontal cortex [ ] . the role of the inflammatory cytokine il- in the progression of cardiovascular diseases is widely appreciated [ ] . studies show that il- is significantly involved in efforts to arbitrate between the sides of the relationship between the reappraisal-related activation of the dorsal anterior cingulate cortex and preclinical atherosclerosis (evaluated by carotid artery intima-media thickness and inter-adventitial diameter) in healthy individuals [ ] . the normative aging study carried out a -year follow-up study in older men (mean . ± . years). there was a dose-response relationship between negative emotions, evaluated by the minnesota multiphasic personality inventory (mmpi), and incidence of coronary heart disease (chd) within the duration of the study (p = . ) [ ] . meanwhile, the circulating levels of il- were positively associated with the reappraisal-related activation of the dorsal anterior cingulate cortex in healthy subjects [ ] . however, higher reappraisals and suppressions were positively and inversely associated with the serum levels of crp, respectively [ ] . on the other hand, the reflection of watching the ice hockey match, as a real-life emotional excitement, on serum levels of endothelin- (et- ) and il- was more pronounced in spectators with coronary artery disease compared with healthy spectators [ ] . patients with type d personality display concurrently two absolute opposite, positive and negative, tendencies towards the experience and the expression of negative emotions by themselves and in front of other people, correspondingly. meta-analysis studies and comprehensive literature reviews have revealed that this type of personality is positively associated with contracting cardiovascular conditions and their consequent mortality and morbidity, as well as with a constellation of non-cardiovascular complaints (for details, see [ ] [ ] [ ] ). also, individual studies, either cross-sectional or follow-up, have providence evidence of increased levels of proinflammatory cytokine tnf-α and its receptors, stnfr and stnfr [ ] [ ] [ ] [ ] , an enhanced il- /il- ratio, and decreased levels of anti-inflammatory cytokine, il- , in chf patients with type d personality compared to those without type d personality [ ] . interestingly, in chf patients, the inflammatory effect of type d personality appears to resemble closely the effect of aging. there was a similar increased pattern of stnfr and stnfr in younger chf patients with type d personality and older patients without this personality trait [ ] . plasminogen activator inhibitor- (pai- ) is a factor contributing to thrombosisrelated cardiovascular diseases in elderly people. both cytokines and hormones take part in the regulation of the gene expression of pai- (for review, see reference [ ] ). in a model of premature immunosenescence, mice were assigned to either fast or slow group if the amount of time taken to explore the first arm of the maze was ≤ s or > s, correspondingly [ ] . when compared to fast mice, slow mice expressed high emotional response to stress and had lower life span [ ] . at the immunological levels, slow mice showed a reduction in proliferative response to concanavalin a (con a) and related release of il- and il- β and nk cell activity, while increasing the production of tnf-α [ ] . an investigation on women who had to undergo breast biopsy indicated that this procedure should be considered as an emotional stressor if the final diagnosis is determined benign. in parallel with this emotional stress, the immune system prepares itself before the procedure and seeks for ways to prolong this preparation even months after the procedure. this is a reflection of the joint regulation of our body by both the immune system and the emotional brain [ ] . the immune system responds to this challenge by decreasing nk cell activity, decreasing production of ifn-γ, and increasing production of il- , il- , and il- [ ] . further, there was a significantly positive relationship between mothers with breast cancer and their adult daughters on distress levels. this persuaded scientists to investigate the immune profile and its association with distress in daughters' group. daughters' distress levels were inversely associated with il- , il- , and ifn-γ production and also with il- -induced natural cytotoxic activity (nca) [ , ] . further, nca activity and the production of th cytokines were both negatively related to the emotional distress degree [ ] . antoni and his colleagues accomplished a genome-wide transcriptional analysis on leukocyte samples taken from women subject to the treatment of stage -iii breast cancer and demonstrated that the negative affect, evaluated by the affects balance scale (abs), was significantly associated with greater than % increased expression of leukocyte transcripts such as proinflammatory marker-related genes [ ] . another multiplex analysis on circulating concentration of cytokines identified the il- profile as the predictor of physical and cognitive functioning and also the vascular endothelial growth factor (vegf) profile as the predictor of emotional functioning [ ] . further, the experience of childhood emotional neglect/abuse was associated with lower levels of nca at the first evaluation after breast cancer surgery [ ] . emotional processing and expression (evaluated by emotional approach coping scale), respectively, tended to be inversely and positively correlated with plasma levels of il- , soluble tnf-receptor type (stnf-rii), and crp in male patients with prostate cancer [ ] . however, among those correlations, two of them, the correlations of emotional expression with il- and crp, were not found significant (p < . ) [ ] . in vivo model of ultraviolet-b light-induced squamous cell carcinoma concluded that the high stress and anxiety levels can leave mice prone to the more considerably progression of the tumor through increasing the expression of immunosuppressive (ccl and t regulatory cells) and angiogenic (vegf: vascular endothelial growth factor) markers and decreasing the expression of antitumor immune markers (ctack/ccl , il- , and ifn-γ) [ ] . on the other side, a peripheral tumor, by itself, could lead to a reduction in the hippocampal function, as reflected in increased depressive-like behaviors and memory impairment. this was, at least in part, underpinned by triggering an inflammatory process both in the hippocampus (↑il- β, ↑il- , ↑il- , and ↑tnf-α) and in the circulation (↑il- , ↑il- β, and ↑il- ) [ , ] . this process was found to be significantly strengthened in infection models compared to peripheral tumor models, explaining the presence and absence of the sickness state in these models, respectively [ ] . using hospital anxiety and depression scale (hads) test, it was estimated that nearly half of patients with hemodialysis (hd) were in a depressive mood, which was significantly higher than what was reported for control group [ ] . also, there was a higher production of il- in hd patients with anxiety (hads≥ ) than those without anxiety (hads≤ ) [ ] . at least one major psychiatric illness, particularly depression, affects greater than % of hiv patients [ ] . by virtue of the fact that specific substances of abuse aggregate further the situation of hiv patients at the neuropathological level [ ] , the inverse correlation between affect regulation and regular substance motivates us to utilize er as a therapeutic intervention in this population [ ] . hiv patients encounter commonly with situations where the social self is threatened. this threat causes shame feelings, which have been associated with increased proinflammatory cytokines [ ] . both emotional and environmental factors affect the gut [ ] . similar to that mentioned for asthma, this affect is mediated by crf and similar neuropeptides and also by mucosal mast cells [ ] . immunoregulatory factors along with genetic and environmental factors contribute to the pathogenesis of inflammatory bowel disease (ibd). patients with ibd confront various er-related problems in their social life in a disease severity-dependent manner, such as higher sensitivity to negative emotions, fewer dropping into bar/disco and delayed falling in love, and experiencing more depressive and anxious symptoms, not only compared to controls, but also compared to patients with other chronic conditions [ ] [ ] [ ] [ ] [ ] . neuroimaging studies have indicated a reduction in both the volume and the activation of brain regions related to emotional processing [ , ] . the gray matter (gm) volume of both the frontal cortex and the anterior midcingulate cortex was reduced in patients with crohn's disease (a type of ibd) compared to controls. more interestingly, disease duration was found to correlate with the gm volumes of some brain regions, importantly, limbic areas [ ] . also, patients with ulcerative colitis (another type of ibd) showed reduced activity, evaluated by bold signal, within the amygdala, thalamic regions, and cerebellar areas during the emotional visual task, compared to the control group [ ] . for the first time in , cohen and his colleagues demonstrated that higher psychological stress is associated with lower resistance to respiratory viruses (rhinovirus type , , or , respiratory syncytial virus, or coronavirus type e) in a dose-response manner [ ] , while positive emotional style (pes), but not negative emotional style (nes), was found to correlate inversely with susceptibility to common cold and upper respiratory infections following exposure to rhinoviruses and influenza a virus in a dose-response manner [ , ] . various regression analyses showed that this correlation is independent of prechallenge virus-specific antibody, virus type, age, sex, education, race, body mass, season, and nes, optimism, extraversion, mastery, self-esteem, purpose, and selfreported health [ , ] . by contrast, childhood socioeconomic status, as assessed by "the number of childhood years during which their parents owned their home," was found to correspond negatively with both the risk of illness and infection and, in a word, with vulnerability to common colds [ ] . this finding along with approximately the similar increased risk of common colds in "those whose parents did not own their home during their early life but did during adolescence" in "those whose parents never owned their home" [ ] indicate that (a) the childhood period takes more impression of socioeconomic status of their family than other lifetime periods (e.g., adolescence) such that (b) it would influence the mind-body background of future life. meanwhile, pes and nes were negatively and positively related to the subjective report of unfounded symptoms of common cold, respectively [ , ] . however, the basal protein levels of all the investigated proinflammatory cytokines, e.g., il- β, il- , and il- , were associated with illness symptoms/ signs after exposure to rhinoviruses. however, il- was the best cytokine which could predict nasal symptoms/signs [ ] . further, daily evaluation of emotional style and cytokine production in infected individuals on each one of days after exposure to rhinoviruses and influenza virus showed that the production of inflammatory cytokines including il- , il- β, and tnf-α was negatively related to positive affect (pa) on that day or on the next day [ ] . neurological diseases including parkinson's disease (pd) and alzheimer's disease (ad) are accompanied by serious shortfalls in emotional processing in a severity-dependent manner. for example, patients with frontotemporal dementia (ftd) represent a poor recognition of several basic emotions, e.g., anger, sadness, disgust, fear, and contempt. also, patients with the probable ad are more likely to fail to recognize fear and contempt compared with controls [ ] . in patients with mild ad, the recognition of more basic emotions are missed, and they are less able to differentiate between some emotions, e.g., happiness and sadness [ ] . apathy in patients with ad was found to correlate positively with dysfunction in the prefrontal and anterior temporal regions [ ] . regarding memory recall, individuals with ad presented no preference to recall better emotional memories other than nonemotional ones, standing in stark contrast to healthy subjects, either young or older [ ] . experimental models provided evidence that there are deficits in the emotional memory performance in ad, which can be diminished by treatment with cytotoxic necrotizing factor (cnf ) [ ] . this pleasant effect of cnf was accompanied by a reduced il- β expression in the hippocampus, along with other encouraging events, especially enhancing the energy amount evaluated by the atp (adenosine triphosphate) levels [ ] . there is a spectrum of behavioral problems in patients with ad [ ] . agitation is the second most common behavior in ad, after apathy. it has been associated with cognitive impairment [ ] . inflammatory changes appear to pave the way for agitation. there were higher il- β levels and decreased nk cell activity in both the morning and evening periods corresponding with preagitation and agitation phases of ad [ ] . even, esterling and his colleagues demonstrated changes in the immune profile of ad patients' spousal caregivers, either former or current. there was a reduced response of enriched nk cells to either ril- or rifn-γ cytokines in patients' caregivers than controls [ ] . interestingly, this response was related positively to the emotional and tangible social support levels [ ] . women with severe or morbid obesity had significantly increased levels of proinflammatory markers il- and hscrp in a bmi-dependent manner, which were closely related to anxiety and depression subscales of neuroticism, even after the bmi adjustment [ ] . since these patients had to undergo gastric surgery, these markers were measured again after surgery. interestingly, decreased levels of il- and hscrp were correlated with lower anxiety and depressive behaviors postoperation [ ] . the long-term maternal exposure ( weeks before mating and during pregnancy and lactation) to a high-fat diet (hfd) led to a decrease in the basal serum levels of cort in offspring [ ] . in addition, the steps toward normalizing the stress-induced cort levels were made with the lower speed in long-term hfd-exposed offspring than standard chow diet (sd)-exposed offspring at the end of stress challenge [ ] . regarding inflammation-related markers, the increased expression of il- and il- ra in long-term hfd-exposed offspring than chow-exposed ones in the amygdala was found in both females and males [ ] , while changes in the expression of nf-kb and i-kappa-b-alpha (ikba) were observed only in female, but not in male, offspring [ ] . mice subjected to short-term ( - weeks) hfd also exhibited anxiety-like behaviors in addition to learning and memory impairments and had significantly higher levels of homovanillic acid-a metabolite of dopamine-in their hippocampus and cortex but without any alteration in the gene expression of inflammatory markers [ ] . further, chronic western diet (wd) intake led to the increased responsiveness to lps, which was represented in higher and more prolonged protein/mrna measures of il- in both plasma and hypothalamus, while there was no significant difference in the plasma levels of other proinflammatory cytokines such as tnf-α, il- β, and ifn-γ between wd and sd groups [ ] . in parallel with the increased expression of il- , there was significantly increased mrna expression of socs- , which belonged to the suppressors-of-cytokinesignaling (socs) family of proteins, in the hypothalamus in wd than sd mice [ ] . however, the lps-induced mrna expressions of tnf-α and ifn-γ in the hippocampus were significantly higher in wd than sd mice. also, lps augmented the levels of adipokines, e.g., cst, leptin, and resistin, more significantly in wd mice when compared with mice exposed to sd [ ] . altogether, both short-term and long-term obesity in either young adult or maternally can lead to display disturbed anxiety-like behaviors and impaired learning/memory, and brain inflammation might be one of the reasons behind these hfd-related events [ ] [ ] [ ] [ ] . chronologically, at first, learning/memory and then anxiety-like behaviors are impaired, and disturbance in depressive-like behaviors is subject to exposure to an immune challenge, such as lps [ ] . a high age-adjusted prevalence rate of ~ % is estimated for metabolic syndrome (visceral obesity, dyslipidemia, hyperglycemia, and hypertension) in the united states [ ] . both er-and edr-related subscales have been associated with the metabolic syndrome factor [ ] . even, a disease pathway involving edr can be proposed, which is triggered by low socioeconomic status (ses), followed with low reserve capacity for high negative emotions and eventuated in the metabolic syndrome factor [ ] . inflammation plays a major role in metabolic syndrome [ ] . it has also been indicated that this role is not performed in the periphery merely, but a mice model of metabolic syndrome proved the presence of central inflammation (↑tnf-α, ↑il- β, and ↑il- ) in the hippocampus, explaining the anxiety-like behavior in this model [ ] . a possible pathogenic pathway for metabolic syndrome is initiated by emotional stress and ensuing enhancement in the levels of proinflammatory cytokines, e.g., il- , il- , and tnf-α. then, these cytokines lead to increased levels of ngf, which in turn stimulates a series of cascades toward insulin resistance and finally resulting in diabetes mellitus (for review, see [ ] ). skin diseases are frequently associated with troubled er, reflecting in problematic emotional expression. for instance, patients with psoriasis, who are more likely to be alexithymic, employ more control over negative emotions and more avoidance of emotional closeness and intimacy compared with controls [ ] . it may explain the negative relationship between psoriasis symptoms and affective expression in a severity-dependent manner [ ] . when patients with atopic dermatitis were compared to healthy controls, the effect of psychological stress on the various immune parameters, such as ↑ eosinophil count, ↑ cd + /cd + and cla + t cells, and ↑ cytokines (il- and ifn-γ), was significantly strengthened [ ] . the route from edr to immdr in acnegenesis has been explained elsewhere [ ] . in summary, emotional distress would make sebocytes prone to the increased expression of receptors for crh, melanocortins, b-endorphin, vasoactive intestinal polypeptide, neuropeptide y, and calcitonin gene-related peptide, and then the production of proinflammatory cytokines are stimulated by means of these receptors and binding to their ligands. this notion that sleep disturbances are, irrespective of their cause, seen as chronic stressors is supported by evidence at different levels, e.g., immunological, neuropathological, and neuroimaging studies (for review, see [ ] ). to assess the effects of sleep and its efficiency on susceptibility to respiratory infections, cohen and his colleagues conducted an investigation on healthy individuals and recorded at first their sleep duration and its efficiency within consecutive days and then made them exposed to rhinoviruses, and after days postchallenge, calculated the rate of clinical cold development [ ] . this investigation indicated that those who had average sleep duration (asd) ≤ hours were three times more susceptible to clinical cold than those with asd≥ hours. further, there was a . -fold increased risk of clinical cold in individuals with sleep efficiency < % compared to those with an efficiency of ≥ %. therefore, it is well expected that circadian arrhythmia has been associated with edr-related parameters, e.g., decreased social motivation/functioning, decreased exploratory anxiety, and decreased emotional functioning [ , ] . in a cancer population, the presence of circadian arrhythmia was associated with decreased levels of all the investigated cytokines, e.g., tnf-α, tgf-α, and il- [ ] . alexithymic patients were more likely to suffer from severe forms of stroke when compared with non-alexithymic patients. this alexithymia trait appears to contain an inflammatory component either as a cause or an effect [ ] . study of patients with a first-ever symptomatic ischemic stroke revealed that (a) the circulating levels of il- were correlated positively with the severity of alexithymia, (b) stratification of patients made this correlation more statistically significant in those who had right hemisphere lesions, and (c) these increased il- levels were pronounced in alexithymic (tas- score ) than non-alexithymic patients [ ] . in patients hospitalized for orthopedic injuries, the use of emotion-focused coping was found to correlate positively with the levels of proinflammatory cytokines, il- and il- , whereas it was negatively correlated with tgf-β levels [ ] . compared with controls who received placebo, circulating levels of cytokines, in particular, il- , were increased at hours after the first-ever typhoid vaccination. it coincided with significant mood impairment [ , ] . it has been elucidated that when subjects perform psychological tasks (i.e., stress condition) after injection, the il- response is inversely related to optimism in either the typhim vi typhoid vaccine or saline placebo group [ ] . in response to the implicit emotional face perception task, there was an increased activity in the subgenual anterior cingulate cortex (sacc) along with reduced functional connectivity between sacc and reduced activity within the anterior rostral medial prefrontal cortex (armpfc), mni coordinates, nucleus accumbens, right amygdala, sts, and ffas. these changes were observed in inflammation-associated mood change compared to the placebo group [ ] . chapter presented evidence supporting the notion that there are a variety of psychological states/psychiatric diseases where the immune responses, as well as the emotion regulation, are impaired. this chapter provided evidence linking physiological states/physical diseases to the impairment of both the immune system and emotion regulation. altogether, the immunoemotional regulatory system (immers) covers both psychological states/psychiatric diseases and physiological states/physical diseases. inevitably, such a system must comprise both the immune mediators and the neuroendocrine messengers, which will be discussed in the next chapter. 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and susceptibility to the common cold circadian arrhythmia dysregulates emotional behaviors in aged siberian hamsters elevated serum cytokines correlated with altered behavior, serum cortisol rhythm, and dampened -hour rest-activity patterns in patients with metastatic colorectal cancer disease outcome, alexithymia and depression are differently associated with serum il- levels in acute stroke cytokine levels as potential biomarkers for predicting the development of posttraumatic stress symptoms in casualties of accidents inflammation causes mood changes through alterations in subgenual cingulate activity and mesolimbic connectivity neural origins of human sickness in interoceptive responses to inflammation dispositional optimism and stressinduced changes in immunity and negative mood key: cord- - px s c authors: hopkins, richard s.; magnuson, j. a. title: informatics in disease prevention and epidemiology date: - - journal: public health informatics and information systems doi: . / - - - - _ sha: doc_id: cord_uid: px s c this chapter provides a description of the components of disease prevention and control programs, and then focuses on information systems designed to support public health surveillance, epidemiologic investigation of cases and outbreaks, and case management. for each such system, we describe sources used to acquire necessary data for use by public health agencies, and the technology used to clean, manage, organize, and display the information. we discuss challenges and successes in sharing information among these various systems, and opportunities presented by emerging technologies. systems to support public health surveillance may support traditional passive case-reporting, as enhanced by electronic laboratory reporting and (emerging) direct reporting from electronic health records, and also a wide variety of different surveillance systems. we address syndromic surveillance and other novel approaches including registries for reporting and follow-up of cases of cancer, birth defects, lead poisoning, hepatitis b, etc., and population-based surveys (such as brfss or prams). systems to support epidemiologic investigation of outbreaks and clusters include generic tools such as excel, sas, spss, and r, and specialized tool-kits for epidemiologic analysis such as epi-info. in addition to supporting outbreak investigation, agencies also need systems to collect and manage summary information about outbreaks, investigations, and responses. systems to support case management, contact tracing, and case-based disease control interventions are often integrated to some degree with surveillance systems. we focus on opportunities and choices in the design and implementation of these systems. systems to support case management, contact tracing, and case-based disease control interventions are often integrated to some degree with surveillance systems. we focus on opportunities and choices in the design and implementation of these systems. public health programs to prevent disease typically have been designed and implemented one disease at a time. each disease has its own patterns of distribution in populations, risk factors, and optimal and practical intervention strategies that are effective in controlling, preventing, or even eliminating cases of the disease. for example, an important strategy to prevent measles is vaccination, the main strategy to prevent gonorrhea is antibiotic treatment of case contacts before they become ill themselves, an important strategy to prevent cervical cancer is screening with pap smears and treatment of preclinical disease, and the main strategy for prevention of neural tube defects is folic acid supplementation of selected foods. still, each disease prevention program's components are drawn from a relatively short list: • planning and evaluation • public health surveillance • outbreak or cluster recognition and response • policy and guidance development • clinical services -screening -immunization -prophylaxis -treatment • laboratory services • case-contact identifi cation and interventions • education and training for clinicians • public education • regulation (for example, of food services, drinking water, child-care centers, hospitals, etc.) • administration and fi nancial management ideally, program managers choose the most effective combination of these program components to prevent or control the disease or diseases they are charged with addressing. however, as this must be done within the constraints imposed by the available funds, cost-effectiveness is the usual criterion for choosing the preferred combination of program components. public health agencies typically are organized both by disease and by function. for example, each disease-specifi c program usually does not have its own laboratory, and a single public health clinical facility and its staff may provide varied services such as immunizations for well children, treatment of people with tuberculosis (tb) and their contacts, and pap smear services. to variable degrees, they may even combine activities in a single patient encounter, for example, testing women for gonorrhea and chlamydia trachomatis infections at the same visit where they get a pap smear, or offering hepatitis b vaccination during a visit for sexually transmitted diseases (std) treatment. as information technology has become more widely used in public health and replaced paper-based systems, it has typically been implemented program area by program area, as resources became available. this has led to the creation of information 'silos.' for example, laboratory information systems usually have developed in isolation from those to support clinical care or public health surveillance. information systems to support clinical operations of public health departments (for example, clinical services for stds, childhood immunizations, hiv/aids, tb, or family planning services) have characteristics similar to those of other electronic health record systems in ambulatory care. however, in some health departments, clinical information systems have been separated by disease or clinic. if one were to design information systems from scratch for a set of disease prevention programs, there would be potential savings and effi ciencies from identifying the ways that one program component depends on information from another, or can serve multiple programs, and then designing the system to provide that information seamlessly. one can identify potential effi ciencies from two perspectives: in reality, it is rare to have an opportunity to design such extensive information systems as a single project. one is dealing with numerous legacy systems that were designed to support program-specifi c workfl ows. so a key challenge for the public health informaticist is to help their agency make decisions about where information system 'integration' will yield substantial benefi ts and where it will not. for example, if it is desired to know (one time) how many people in the jurisdiction have been reported during a particular time interval with both syphilis and hepatitis b, one could do an ad hoc match of information in two independent surveillance information systems. this task might take an analyst a few days or weeks to accomplish -which is almost certainly inexpensive compared to the cost of building a new information system that could do this task almost immediately. for many purposes, it may be useful and suffi cient to be able to display multiple streams of surveillance or programmatic data in the same environment, on the same screen or even in the same chart. in florida, de-identifi ed reportable disease case information and death certifi cate information are imported into the essence analytic environment that was originally designed for syndromic surveillance [ ] , so that trends for similar conditions by age, sex, and geographic area in the two data streams can be easily compared. on the other hand, if it is desired to have real-time information available to the std clinic staff about past diagnoses of hepatitis b, or about past receipt of hepatitis b vaccine, then information systems need to be designed to support this kind of look-up; the usual solution is a shared person index between the two systems. alternatively, a common data repository can be designed in which all information about each person is permanently linked. as mentioned earlier, there are a number of components common to disease control and prevention programs. in this chapter, we will address information systems designed to support the following: • public health surveillance • outbreak or cluster recognition and response • acquisition of laboratory information • case-contact identifi cation and intervention cdc defi nes public health surveillance as "the ongoing, systematic collection, analysis, and interpretation of health data, essential to the planning, implementation, and evaluation of public health practice, closely integrated with the dissemination of these data to those who need to know and linked to prevention and control" [ ] . each word of this defi nition is carefully chosen, and has implications for the design of surveillance information systems. a one-time data collection activity is not surveillance. data collection for research purposes is not surveillance. surveillance data are collected to support public health action, and analyses and recommendations based on these data must be shared with those who provided the data and with others who need to know. objectives of surveillance systems differ at the local, state, and federal levels [ ] . at the local level, immediate response to individual cases is relatively more important, while at the federal level the analysis of larger-scale patterns is the most important function of surveillance. for state health departments, both uses of surveillance data may be important, depending on the disease and the size of the state. public health surveillance systems may be based on data capture from a variety of sources, including case reports, population-based surveys, sentinel providers, electronic health records (including laboratory information management systems for elr and emergency department records for syndromic surveillance), or administrative data (like hospital or physician claims for reimbursement). for some noninfectious diseases, surveillance is carried out through registries (see below). information systems to support reportable disease surveillance contain records representing case reports that currently are, for the most part, entered manually into an application by public health staff, based on information received from doctors, infection control practitioners, hospitals, and laboratories. increasingly, the laboratory information in these records comes from electronic records transmitted by the public health laboratory, hospital laboratories, and commercial laboratories, when there is a positive result meeting certain reporting criteria (like a positive igm antibody test for hepatitis a). these records typically contain a combination of clinical, laboratory, and epidemiologic information about each case. in future, increasing proportions of these case reports will be entered directly into a website by the practitioner creating the case report, or be transmitted electronically from the practitioner's electronic health record (ehr) system. currently almost half the states in the us use the cdc-provided nedss base system (nbs) as their platform for managing case reports. the remainder use either a system developed in-house or one of several commercially-available solutions [ ] . in case-based surveillance practice, there is usually a relatively short list of required elements in the initial case report. for some diseases this is the only information received on all cases. for other diseases, usually of more importance and with lower case numbers, an additional data collection form is initiated by the receiving health department, which gathers information as appropriate from the ill person, the treating physician, and health records. the optimum amount of information to collect in the initial case report, as opposed to the disease-specifi c case report form, is a matter of judgment and may change as technology changes. in a largely manual system, health departments typically desire to minimize barriers to reporting of cases, so the incentive is to keep the initial case report form short. if much of the information desired for the disease-specifi c case report form can in fact be extracted from an electronic medical record with no additional effort by the person making an electronic case report, then the balance changes. careful decisions are needed: for which cases of which diseases are follow-up interviews necessary [ ] ? until very recently, virtually all of the case-based surveillance information used at the federal level was collected initially at the local (or sometimes state) level, where it was used in the fi rst instance for local response. as the case report information passes from the local to the state to the federal level, it is subjected to validation and cleaning: cases not meeting the surveillance case defi nition have been removed from the data submitted to the federal level, missing data have been fi lled in to the extent possible, and cases have been classifi ed as to whether they are confi rmed, probable, or suspected using standard national surveillance case defi nitions (these case defi nitions are developed by the council of state and territorial epidemiologists in consultation with cdc) [ ] . more recently, advances in technology have allowed case reports, and the information on which they are based, to move almost instantaneously from electronic health record systems, maintained by doctors, hospitals, and laboratories, to public health authorities. there are no technical barriers to these data being available at the federal level essentially as early as they are at the local and state levels. this ready availability of unfi ltered clinical information may allow more rapid awareness by public health offi cials at all levels of individual cases of high-priority diseases (like botulism or hemorrhagic fevers like ebola virus infection), and thus lead to more rapid detection and characterization of likely outbreaks. the simultaneous availability of raw data to multiple agencies at different levels of government also presents certain challenges. the user at the local level will have ready access to information from many sources about local conditions and events, and can use this information to interpret local observations. they will be in a position to understand when an apparent anomaly in their surveillance data is due to an artifact or to local conditions that are not a cause for alarm. they will also know whether a problem is already under investigation. a user at a state or federal level will be able to see patterns over a larger area, and thus may be able to identify multijurisdictional outbreaks, patterns, or trends that are not evident at a local level. the fact that several users may be examining the same raw data at the same time requires that these multiple users be in frequent communication about what they are seeing in their data and which apparent anomalies are already explained or need further investigation. there is a danger that users at a higher level may prematurely disseminate or act on information that, while based on facts, is incomplete or misleading. similarly, users at a local level may not realize that what they are seeing is part of a larger phenomenon. in the syndromic surveillance domain, the biosense . governance group [ ] has adopted a set of etiquette principles which participating jurisdictions will be required to agree to, that spell out the mutual obligations of analysts at each level of the system (scott gordon , association of state and territorial health offi cials, , personal communication). from an information management perspective, an important question is where to put human review of case reports in this information fl ow. for example, it is becoming technically possible for likely cases of reportable diseases to be recognized automatically in health care electronic record systems. some of these could be passed on to public health authorities without human review, in the same way that reportable laboratory results are already passed on in electronic laboratory reporting (elr). for which constellations of fi ndings in the electronic health record would this be appropriate? should some electronic case reports generated by electronic health record systems be passed to state or even federal public health offi cials before they are reviewed and validated at the local or state levels? if so, which ones? as always, there is a tension between the speed of information fl ow and its quality and completeness. there is a need for research to determine which constellations of fi ndings in electronic health records have adequate specifi city and sensitivity to warrant automated identifi cation of a person as being likely to have a case of a reportable disease. the acceptable sensitivity and specifi city will vary by disease. in , cdc published the updated guidelines for evaluating public health surveillance systems [ ] . this document identifi es a set of key attributes of surveillance systems to be assessed during a surveillance system evaluation, including simplicity, fl exibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness, and stability. these are also useful attributes to consider when designing a surveillance information system [ ] . the relative importance of these attributes will vary depending on the condition under surveillance and the main purposes for surveillance. for example, a surveillance system to detect cases of botulism for immediate public health response puts a high premium on timeliness, and its operators are likely to be willing to accept a modest number of false-positive reports (a lower positive predictive value ) in order to assure that reports are received very quickly. on the other hand, surveillance to support planning of cancer prevention programs and treatment services is less time-sensitive, given the quite long incubation periods for most cancers, and therefore more concerned with diagnostic accuracy of every case report than with speed of reporting. timeliness, positive predictive value, and sensitivity of a public health surveillance system are always in tension with each other; increasing two of these always compromises the third. in systems based on case-reporting from doctors, hospitals, and laboratories, and receipt of electronic health records from these same organizations, records for an individual can in principle be linked with records for that same individual in numerous public health information systems, including those supporting clinical service, immunization registries, case investigation, partner or contact identifi cation, partner or contact notifi cation, and provision of interventions to partners or contacts. sometimes this will be done best by automated messaging of structured data from one system to another, sometimes by supporting real-time look-up capabilities, and sometimes by development of a master person index to underlie some or all of these applications. one key decision is which application to consider as the hub for this information sharing, for example, the surveillance application itself or a clinical application. surveillance systems that are based on sample surveys (such as the behavioral risk factor surveillance system, brfss [ ] ), on sentinel practices (such as ili-net for surveillance of infl uenza-like illness [ ] ) or on syndromic surveillance do not have individual patient identifi ers, and so intrinsically cannot be linked at the individual level to information systems supporting other disease control program components. their data are typically managed in systems built on standard statistical software packages, or other independent systems. syndromic surveillance systems are based on rapid acquisition of unfi ltered, real-time, electronic records without individual identifi ers from hospital emergency rooms [ ] and urgent care centers, and also, increasingly, from outpatient physicians' offi ces and from hospital admissions [ ] . the primary purpose of these systems is to support detection and characterization of community disease outbreaks, as they are refl ected in care received at emergency departments, physicians' offi ces, or hospitals. each visit to an emergency department is assigned to a category or syndrome , based on words and strings contained in the patient's chief complaint and/or the triage nurse's notes. as the records received by the health department do not have individual identifi ers, they cannot be linked to records in other information systems. however, records received by the syndromic surveillance system should contain unique identifi ers that could allow the epidemiologist analyzing the data to work back through the sending facility to an identifi ed clinical record. this traceback might become necessary if the person appeared to have a case of a reportable disease or to be part of a signifi cant outbreak. adding outpatient visits and hospital admissions to the scope of syndromic surveillance is opening up additional uses for this technology, especially in the areas of real-time non-infectious disease surveillance. surveillance for cancers [ ] , stroke [ ] , birth defects [ ] , and some other chronic diseases like amyotrophic lateral sclerosis (als) is carried out through registries. registries are usually established by specifi c legislation, and typically relate to a single topic -for example a registry of records for a disease, or of immunization records. registries may be restricted to a geographic region. a distinctive feature of registries is that individual case reports are kept open for long periods of time, up to several or many years, allowing additional information about treatment, hospitalization, and death or other outcomes to be added. registries thus serve as systems to monitor type, duration, and outcome of treatment for these diseases, in addition to the occurrence of new cases of disease (disease incidence ). they may also support outreach efforts to patients or their families, as a way to document that appropriate steps have been taken to link patients to needed types and sources of care. most cases recorded in state-level cancer registries are acquired from hospitallevel registries, using an electronic case report in a standardized format [ ] . some case abstracts are obtained directly by registry personnel or contractors, when hospitals do not have suitable registries of their own. case reports require extensive review and abstraction of medical records by trained workers. birth defect registries may also be built by active search for cases in hospital and other medical records, and abstraction of those records to make case reports. they also may be built by electronically linking records from vital statistics (birth and death records), centralized hospital discharge record systems, and clinical service providers for children with birth defects (such as state programs for children with special medical needs) [ ] . the latter are much less expensive to develop but cannot be assumed to have captured all cases of the disease under surveillance, or captured them correctly [ ] . a disease outbreak is defi ned as a number of cases greater than the number expected during a particular time interval in a geographic area or population. this term usually is used for events due to infectious diseases, and sometimes for those of toxic origin. a similar increase above expected numbers for a non-infectious disease, such as birth defects or cancer, is usually called a cluster . outbreaks and clusters may be due to diseases for which individual cases are reportable (like shigellosis or breast cancer), or diseases for which they are not (like food poisoning due to staphylococcal or clostridium perfringens toxins in most states, sars when it was new, or multiple sclerosis). surveillance systems are designed to facilitate recognition of outbreaks or clusters by frequent examination of the most current information available. the design of the user interface is particularly important. the interface should allow users to: fl exibly display line lists, bar charts by date of event (epidemic curves), and maps of location of cases; fl exibly select subsets of cases for display; apply appropriate statistical tests to detect improbable increases in case counts; and display multiple streams of data on the same chart. for example, users may want to display the epidemic curve of an infl uenza outbreak for several different regions of a state or for several different age groups, or to display counts of positive infl uenza tests and emergency department visits for infl uenza-like illness on the same graph with different scales for each. syndromic surveillance systems have been leaders in developing and evaluating statistical algorithms for automated detection of anomalies which may, on investigation, turn out to be outbreaks. such algorithms have less frequently been applied for automated detection of possible outbreaks or clusters in reportable disease data streams. most outbreaks and clusters are in fact not recognized by examination of regularly-collected surveillance system data. instead, they are recognized by private citizens (such as the organizer of a social event, a teacher or school nurse, the manager of a child care center, the manager of a food service facility, an employer, or the ill people themselves) or by practicing doctors, and brought to public health attention via a phone call or e-mail or entry on a web site established for the purpose [ ] . public health workers assess the information and make the decision whether or not to do a formal investigation of the outbreak. one part of such an assessment is to look at available streams of surveillance data and determine whether there is information supporting the occurrence of an outbreak. for example, a report of a possible infl uenza outbreak in a high school might prompt closer examination of syndromic surveillance data from nearby hospital emergency departments to determine whether there is a more general increase in visits for infl uenza-like illness. a report of a neighborhood cluster of brain cancers would prompt closer examination of available cancer registry information, which might or might not support an interim conclusion that such a cluster is real and statistically signifi cant. in order to be accountable for the effectiveness of their work, local and state health departments need to track the occurrence of outbreaks and the public health response to those outbreaks. since outbreaks can be due to reportable or nonreportable diseases, this cannot be done only by actions such as identifying some cases in the reportable disease data system as being part of an outbreak. systems to track the occurrence of outbreaks need to document the following: • time and date the fi rst and last cases occurred • total (estimated or counted) number of cases • population group most affected (by age, sex, location) • setting of the outbreak (school, workplace, restaurant, wedding, etc.) • suspected or confi rmed agent • most common clinical presentation • suspected or confi rmed source and mode of spread • methods used to investigate agent, source and mode of spread • control measures recommended • control measures implemented • lessons learned for prevention of future outbreaks and improved investigation and response in future events this information about outbreaks should be stored for ready retrieval, and to serve as a basis for quality improvement efforts. for quality improvement purposes, it is also helpful to document the content of the summary report written about each outbreak. when the outbreak is due to a reportable disease, individual cases in the reportable disease surveillance information system can be linked to the outbreak, for example by having an outbreak identifi er attached to their records. if preliminary information about outbreaks in a jurisdiction is entered into the outbreak information system in real time, as the investigation is proceeding, and if the outbreak database is readily searchable by all communicable disease investigators in the jurisdiction, then local investigators can use the outbreak database to help them with investigations of new illness or outbreak complaints [ ] . for example, if they receive a complaint that illness has occurred in people who consumed a particular food product, they can look in the database and determine whether other recent or current complaints or outbreaks mention the same food product. if they receive a report about a gastroenteritis outbreak in a childcare center, they can determine what agents have been found to be responsible for recent or current similar outbreaks in nearby communities; this can help focus their laboratory testing and initial control strategies. some us states have had long-standing systems to document all outbreaks investigated by local or state personnel, but others have not. a major variable in the design of such systems is the state-local division of responsibilities in each state, including the degree of state oversight of 'routine' local outbreak investigations. the actual investigation of an outbreak or cluster may involve enhanced "active" case-fi nding, use of case-report forms, group surveys, and formal epidemiologic studies. active case-fi nding involves regular solicitation of case reports from doctors, hospitals, and laboratories. managing the reports of possible, probable, and confi rmed cases that are part of the outbreak is an important task. for a reportable disease, the jurisdiction's reportable disease surveillance system may be adequate to manage reported cases. it may be necessary, however, to create a continuouslyupdated line list of possible cases and their current status, which is outside the scope of the standard reportable disease application. outbreak investigation surveys will typically involve interviewing everyone with a possible exposure (like all attendees of a wedding reception), whether they were ill or not. formal studies may involve interviewing selected non-ill people, for example, as part of a case-control study. the investigation may also involve obtaining and sending to a laboratory a large number of specimens from ill persons, and sometimes from exposed non-ill persons and from environmental sources (food, water, air, soil, etc.). managing these disparate types of information is a challenge, especially in a large outbreak or one involving multiple jurisdictions. there is currently no one widely-accepted and satisfactory way to manage data in such settings. each investigation team typically uses the tools it is most familiar with, including some combination of data management tools like ms excel, ms access, or epiinfo [ ] , and standard statistical packages. many health departments maintain libraries of standard questionnaires with associated empty data bases, for use during outbreak investigations. when cdc is involved in a multistate outbreak, the investigation team at the local or state level needs to be able to produce and transmit timely case report and other information in the format desired by cdc. the services of an experienced public health informaticist can be extremely helpful to the investigation team when outbreaks are large and multifocal. an ongoing challenge for cdc and the states is how to make the transition from specialized case reporting during an outbreak of a new disease, such as west nile virus encephalitis or sars, to routine case-based surveillance. if this transition is not well-managed, it is likely to result in the creation of a permanent stand-alone surveillance information system (or silo) for that disease. if the new disease is of national importance, cases should be made nationally notifi able and its surveillance should be incorporated into existing systems. laboratory information is a critical component of disease surveillance and prevention. laboratory data form the foundation of many surveillance systems. there are different types of laboratories involved in the public health data stream. laboratories providing data to public health fall into the general categories of commercial or private industry, hospital or clinical, and public health laboratories. public health laboratory information systems (lis) contain information about test results on specimens submitted for primary diagnosis, for confi rmation of a commercial or hospital laboratory's results, for identifi cation of unusual organisms, or for further characterization of organisms into subgroupings (like serotypes) that are of epidemiologic importance. in some states, all clinical laboratories must submit all isolates of certain organisms to the public health laboratory. many of the results obtained in a public health laboratory turn out to be for diseases that are not reportable and not targets of specifi c prevention programs. some of those results may, however, be for cases of non-reportable diseases that are historically rare in the jurisdiction but of great public health importance, or are new or newly-recognized. the main business of clinical laboratories (located both inside and outside hospitals) is to test specimens for pathogens or groups of pathogens specifi ed by the ordering physician, and return the results to the person who ordered the test. public health agencies have, since the early s, asked or required such laboratories to also identify results meeting certain criteria (indicating the presence of a case of a reportable disease) and send a copy of the results to the public health agency for public health surveillance. initially, case reporting by laboratories was accomplished on paper forms, which were mailed or faxed to public health departments. some laboratories very soon moved to mailing printouts of relevant laboratory results, then to sending diskettes, then to transferring computerized fi les containing laboratory results by direct modem-to-modem transfer, and eventually to transferring such fi les via the internet using standard formats and vocabularies. in some states, public clinics (for example, std clinics) have used contract laboratories for their testing needs. in this situation, the outside laboratory supplies both positive and negative results to the public health agency, increasingly by transfer of electronic results in standard formats. laboratories provide data on reportable conditions to their local or state public health authority. reportable diseases are determined by each state; clinicians, hospitals, and/or laboratories must report to public health when these conditions are identifi ed. some reportable conditions are also nationally notifi able. deidentifi ed cases of these are voluntarily notifi ed by states and territories to cdc, which, in collaboration with the council of state and territorial epidemiologists, maintains a listing of nationally notifi able conditions that includes both infectious (e.g., rabies, tb) and non-infectious (e.g., blood lead, cancer) conditions [ ] . the public health partnership with laboratories has led to the very successful and still increasing implementation of electronic laboratory reporting (elr) in the us. elr refers to the secure, electronic, standards-based reporting of laboratory data to public health. elr implementation has been steadily escalating since its inception around the year , replacing previous reporting systems that relied on slower, more labor-intensive paper reporting. the elr national working group conducted annual surveys from to [ ] which gathered data from all states as well as from several territories and large metropolitan areas. these data were supplemented with data for years - , retroactively gathered in the survey. the tracked growth of elr (fig. . ) illustrates its rapid rise in the us, from the start of early stage planning to fully operational elr [ ] . the expected benefi ts of elr include more rapid reporting of reportable cases to public health departments, allowing faster recognition of priority cases and outbreaks for investigation and response, and thus more effective prevention and control [ ] . elr also is expected to reduce the number of missed cases, as automated systems do not require laboratory staff to actively remember to make case reports, and to improve the item-level completeness and quality of case reports. although experience shows that the expected improvements in timeliness, sensitivity, completeness, and accuracy are generally being realized [ ] , timeliness may not be improved substantially for those diseases where clinicians routinely report based on clinical suspicion without waiting for laboratory confi rmation (for example, meningococcal disease) [ ] . in addition, laboratories (especially referral laboratories) often do not have access in their own information systems to home addresses for people whose specimens they are testing, and have struggled with providing complete demographic information to public health agencies. implementation of an operational elr system is not a trivial undertaking. laboratories must confi gure data into an acceptable message format, most commonly health level seven (hl ® ) [ ] . laboratory tests and results should be reported with correlated vocabulary or content codes. two of the most common code systems used for laboratory tests and their associated results are logical observations identifi ers names and codes (loinc ® ) [ ] and systematized nomenclature of medicine (snomed ct ® ) [ ] . neither of these systems is suffi cient by itself to encode all the information needed for public health surveillance. public health jurisdictions have introduced elr to their partner laboratories using one or more of the following approaches: • the "charm" approach -relies on establishing goodwill and collaboration with laboratory partners. while this collegial approach is very appealing, it may be unable to overcome signifi cant barriers such as lack of laboratory funding or resources, and some facilities will supply data only in methods specifi cally required by law. • the incentive approach -involves offering either fi nancial or technical assistance to laboratory partners, assisting them in the startup process of elr. while this approach may be preferred by many laboratories, relatively few jurisdictions have the discretionary funds (or are able to receive federal assistance funds) to implement the approach. • the enforcement or legislative approach -requires reporting rules or legislation that requires laboratories to participate in elr. the most successful enforcement approach will include low-cost options for smaller laboratories, such as web data entry, so that they may benefi t from an elr -"lite" implementation [ ] . the mainstreaming of elr systems in the us has pioneered a clear path forward for public health to begin maximizing its presence in the domain of electronic data interchange. at a local level, case reports for communicable diseases prompt action. although the specifi c action varies by disease, the general approach is the same. it starts with an interview of the ill person (or that person's parents or other surrogates) to determine who or what the person was in contact with in ways that facilitate transmission, both to determine a likely source of infection and to identify other people who may be at risk from exposure to this person. information systems to support contact tracing, partner notifi cation, and postexposure prophylaxis (for stds or tb, for example) contain records about all elicited contacts (exposed persons) for each reported case of the disease in question. these records contain information about each contact, such as whether they were located, whether they received post-exposure prophylaxis, and the results of any additional partner-elicitation interviews or clinical testing that were completed. information systems to support surveillance for other reportable diseases also increasingly contain information about what disease-appropriate action was taken in response to each case; such actions may include identifi cation of contacts, education of household members, vaccination or antibiotic prophylaxis of contacts, isolation of the case (including staying home from work or school), or quarantine of exposed people. std and tb information systems typically capture full locating information for contacts, and can be used both to support fi eld work and to generate statistics on effectiveness of partner notifi cation activities worker by worker and in the aggregate. systems for other reportable diseases may capture only the fact that various interventions were done, and the date that these were initiated. information about the timeliness of initiation of recommended control measures is now required as a performance measure for selected diseases by cdc's public health emergency preparedness cooperative agreement [ ] . in the investigation of a case of meningococcal disease, contacts are people who had very close contact with the original person, for example a household member, boyfriend, or regular playmate. health department staff determines who the close contacts are. each will then be offered specifi c antibiotic treatment to prevent illness. for syphilis, contacts are people who have had sex with the original case. contacts will be examined by a clinician and assessed serologically to see if they are already infected, and offered appropriate prophylactic or curative antibiotic treatment. for measles, contacts may include anyone who spent even a few minutes in the same room as a case. contacts whose exposure was recent enough, and who are not fully immunized already, will receive a dose of measles-containing vaccine, and all contacts will be asked to self-isolate immediately if they develop symptoms of measles. in investigating a common-source outbreak of legionellosis, histoplasmosis, or anthrax, the local health department may want to locate everyone who had a specifi ed exposure to the apparent source of the infection. these exposed people may need antibiotic prophylaxis or may be advised to seek medical care promptly if they become ill. information systems to support this type of work typically have three purposes: . serve as a place for workers to record and look up information about people who are or may be contacts, and to track which contacts have and have not yet received needed interventions. . serve as a source of information for calculating indices of program or worker timeliness and performance, such as the average number of sexual contacts elicited per syphilis patient interviewed, or the percentage of measles contacts who were identifi ed in a timely way and who received post-exposure measles vaccine prophylaxis. . document the workload and effort put in by epidemiology and disease control fi eld staff it seems logical that the surveillance information system should serve as the basis for a system to support fi eld investigation, and this is often the case. the fact that the recommended interventions vary by disease makes designing a single system more complex. existing systems that track fi eld worker activities in detail are much more common for std and tb programs than for others. for general communicable disease fi eldwork, it is currently more common that the system simply documents which interventions were done and when, rather than use the application to track specifi c named contacts or exposed people. the public health informatics institute has published a detailed analysis [ ] of the typical workfl ow involved in surveillance, investigation, and intervention for reportable diseases, and the corresponding information system requirements. the work group that phii convened had representatives of nine different state and local health departments, who were able to identify a large number of processes that were common to all nine jurisdictions, such as case-fi nding, case investigation, data analysis and visualization, monitoring and reporting, case/contact specifi c intervention, and others. these common processes can then serve as a basis for designing information systems to support case-reporting, surveillance, and case-based intervention work that are useable in multiple jurisdictions. consider existing or planned surveillance systems for multiple diseases and conditions. broadly, there are three functions in each of these systems -acquiring the raw data, cleaning and managing the data, and making the data available to users. each of these functions potentially can be integrated, to varying degrees. for example, multiple surveillance systems may benefi t from receiving electronic laboratory reports with a result indicating the presence of a case of a reportable disease. laboratories appreciate having a single set of instructions and a single destination for all their required reports, as this simplifi es their work. the laboratories then benefi t from the ability of the recipient health department to route the reports internally to the right surveillance information system. at the other end of the data pathway, users appreciate having a single interface with which to examine data about multiple conditions or diseases, using the same commands and defi nitions. the users do not have to understand how different surveillance information systems may internally code the same concept in different ways. they also appreciate being able to directly compare information that originally was submitted for the use of different program areas -for example, hepatitis b and gonorrhea in the same chart or table. in the short to medium term, it is not necessary to build a single integrated data repository or a master person index to achieve these goals, even if that is what one would have designed if one were starting from the beginning. however, if one wants to be able to see information about the same person that originates and is stored in multiple systems -for example, so that tb clinicians can see hiv data on their patients and vice versa -then an integrated data repository, or a master person index, or a query system that is extremely accurate in fi nding data on the right person, is needed. modifying existing systems to be able to carry out these functions is time consuming and expensive, so the business case and requirements need to be especially clear. florida's essence system: from syndromic surveillance to routine epidemiologic analysis across syndromic and nonsyndromic data sources (abstract) history of public health surveillance blueprint for a national public health surveillance system for the st century status of state electronic disease surveillance systems -united states prioritizing investigations of reported cases of selected enteric infections. paper presented at council of state and territorial epidemiologists nationally notifi able disease surveillance system case defi nitions association of state and territorial health offi cers. biosense . governance updated guidelines for evaluating public health surveillance systems, recommendations from the guidelines working group design and operation of local and state infectious disease surveillance systems oxford handbook of public health practice behavioral risk factor surveillance system overview of infl uenza surveillance in the united states international society for disease surveillance meaningful use workgroup. final recommendation: core processes and ehr requirements for public health syndromic surveillance electronic syndromic surveillance using hospital in patient and ambulatory clinical care electronic health record data national program of cancer registries coverdell national acute stroke registry surveillance -four states atlanta congenital defects program (macdp) north american association of central cancer registries, inc. (naaccr). implementation guidelines and recommendations report on birth defects in florida a comparison of two surveillance strategies for selected birth defects in florida online food and waterborne illness complaint form biosurveillance plan for human health, version . . atlanta national notifi able diseases surveillance system (nndss), cdc. accessed at http:// wwwn.cdc.gov/nndss/ on available from www.coast coastin-formatics.com national electronic laboratory reporting (elr) snapshot survey. available from www.coast coastinformatics.com . cited statewide system of electronic notifi able disease reporting from clinical laboratories: comparing automated reporting with conventional methods a comparison of the completeness and timeliness of automated electronic laboratory reporting and spontaneous reporting of notifi able conditions potential effects of electronic laboratory reporting on improving timeliness of infectious disease notifi cation -florida health level seven (hl ® ) homepage. available at logical observation identifi ers names and codes (loinc ® ) systematized nomenclature of medicine-clinical terms (snomed ct ® ) see for example section d- . ( ) of the florida administrative code: notifi cation by laboratories public health emergency preparedness cooperative agreement, budget period , performance measures specifi cation and implementation guidance, at-a-glance summary redesigning public health surveillance in an ehealth world on . what are some of the methods for surveillance besides case-reporting? . how are registries different from other surveillance information systems? . what are the advantages and disadvantages of building a master person index across surveillance information systems for multiple diseases? . what are the expected benefi ts of electronic laboratory reporting as a method to enhance surveillance? . what are the advantages and disadvantages of building a system to manage information about case contacts as part of the surveillance information system? . who determines for which diseases cases are nationally notifi able? key: cord- -sy cpff authors: mitrovic, stéphane; feist, eugen; fautrel, bruno title: adult-onset still’s disease date: - - journal: periodic and non-periodic fevers doi: . / - - - - _ sha: doc_id: cord_uid: sy cpff adult-onset still’s disease (aosd) is a rare but clinically well-known, polygenic, systemic autoinflammatory disease. it is typically characterized by four main (cardinal) symptoms: spiking fever ≥ °c, arthralgia or arthritis, skin rash, and hyperleukocytosis (≥ , cells/mm( )). however, many other clinical features are possible, and it can appear in all age groups with potentially severe inflammatory onset accompanied by a broad spectrum of disease manifestation and complications. hence, it remains a diagnostic challenge, and the clinician should first rule out infectious, tumoral, or inflammatory differential diagnoses. determination of the total and glycosylated ferritin levels, although not pathognomonic, can help in diagnosis. new biomarkers have recently been described, but they need to be validated. the disease evolution of aosd can be monocyclic, polycyclic, or chronic. in chronic disease, a joint involvement is often predominant, and erosions are noted in one-third of patients. many progresses have been made in the understanding of the pathogenesis over the last decades. this chapter provides a comprehensive insight into the complex and heterogeneous nature of aosd describing the identified cytokine signaling pathways and biomarkers. it also discusses the current evidence for the usage of biologics in aosd to provide guidance for treatment decisions, taking into account both the efficacy and the safety of the different therapeutic options. adult-onset still's disease (aosd was first described in the early s, by bywaters, as an inflammatory condition occurring in young adults. the disease was very similar to childhood-onset still's disease, today called systemic-onset juvenile idiopathic arthritis (sojia), described a century ago by sir still [ ] . although the exact pathogenic mechanisms of the disease remain unknown, substantial advances have been made first to confirm the homology between aosd and sojia, and then the disease became the archetype of nonfamilial, or sporadic, systemic autoinflammatory disorders (said) [ ] [ ] [ ] . by definition, aosd affects people older than , either de novo or those with a history of systemic jia [ , , ] . in the latter, a disease-free interval between the childhood episode and the adulthood recurrence differentiates aosd from persistent sojia. in most patients, aosd is characterized by four cardinal symptoms: spiking fever, an evanescent salmon-pink maculopapular rash, arthritis, and a white blood cell count (wbc) ≥ , /mm , mainly neutrophilic polymorphonuclear cells (pmns). other features are sore throat or pharyngitis, myalgia, lymph node or spleen enlargement, pleuritis or pericarditis, multivisceral involvement, elevated levels of liver enzymes, and other hematologic abnormalities [ , , ] . none of these symptoms is disease specific, so diagnosis of aosd is difficult, and clinicians must first rule out neoplastic, infectious, or inflammatory conditions. to date, epidemiologic data about aosd have been scarce and imprecise, because of the heterogeneous clinical presentation of the disease and the complex assessment of diagnosis. the disease occurs worldwide, and no specific familial aggregation has been reported. aosd satisfies the definition of an orphan disease because historically its prevalence was estimated between one case per million in europe [ ] and ten cases per million in japan [ ] . in the japanese study, the incidence was estimated at about one case per million in the mid- s. however, because of the substantial advances in aosd diagnosis as well as differential diagnoses during the last two decades, these prevalence and incidence estimates lack robustness, and new studies are clearly needed to update these figures. initially, the disease was characterized as affecting exclusively young adults (i.e., - years of age [ ] ); however, more recent series identified cases in adults older than and even elderly people [ , [ ] [ ] [ ] [ ] . the sex ratio is almost balanced, with only a slight female predominance [ , , , [ ] [ ] [ ] ]. clinical expression aosd is traditionally characterized by four "cardinal" symptoms (three are clinical, one is biological), with many other manifestations possibly associated [ , , ]. fever is constant when the disease is active, except in patients with established disease, for whom residual inflammation could remain without it. typically, fever starts suddenly, and temperature rapidly reaches °c or more, associated with shivers. it evolves with daily evening spikes for more than a week ( fig. . ). patients usually experience rapid deterioration of general health as well as significant weight loss. of importance, fever may be the only clinical symptom of aosd as a potential diagnosis in patients with fever of unknown origin [ ] . joint pain or arthritis is the second most common symptom, with synovitis, usually with concomitant fever spikes, occurring in more than two-thirds of patients. all joints can be involved, including sacroiliac and distal interphalangeal joints. in some patients, the presentation is that of a bilateral symmetrical rheumatoid arthritis (ra)-like polyarthritis [ , ] . synovial fluid analysis displays high cellularity, > cells/mm , which confirms joint inflammation. when performed, synovium biopsy reveals only nonspecific synovitis. during the evolution, arthritis becomes erosive in one-third of patients; in these patients, isolated bilateral carpal ankylosis (i.e., without structural damage of metacarpophalangeal or proximal interphalangeal joints, in contrast to ra) is very suggestive of aosd ( fig. . ) [ , , ]. skin rash is typically salmon pink-colored macular or maculopapular (figs. . and . ). the rash is transient, mainly visible during fever spikes on the proximal limbs or trunk, and rarely involves the face, palms, or soles of the feet [ , , ] . no specific histological feature has been described. misdiagnosis with drug allergy is frequent and usually attributed to nonsteroidal anti-inflammatory drugs (nsaids) prescribed for joint symptoms or fever. complete regression without scars is the rule. atypical patterns also reported are urticarial or pruritic with dermographism [ , , ] . presence of purpuric lesions should lead to urgent coagulation workup because these lesions are suggestive of the aosd hematologic complications of hemophagocytic syndrome or reactive hemophagocytic lymphoproliferation (rhl), disseminated intravascular coagulation (dic), or thrombotic microangiopathy (tma), also called thrombocytic thrombocytopenic purpura or moschcowitz syndrome [ , ] . sore throat (odynophagia) and sometimes pharyngitis are classical symptoms, concomitant with fever (and not just a few weeks previous, e.g., in post-streptococcal arthritis) [ ] . all microbiological test results are negative. the presence of this symptom in such a systemic context seems to exclude lymphoma or another neoplastic disease [ ] . diffuse and symmetrical lymphadenopathy is possible, eventually associated with splenomegaly or even hepatomegaly. however, large and nonsymmetrical lymphadenopathy should lead to the exclusion of malignant lymphoma by biopsy: reactive polyclonal lymphoid hyperplasia is the usual pattern in aosd [ , ] . kikuchi necrotizing lymphadenitis has been described in a few cases [ ] . myalgia is frequent, but myositis or polymyositis is exceptional [ , , , ] . finally, as in many other inflammatory disorders, nonspecific manifestations have been reported: abdominal pain related to deep lymphadenitis, aseptic peritonitis or rarely acute pancreatitis, pleural effusion or pericarditis, or interstitial lung infiltrates [ , ] . high serum ferritin level, an indicator of macrophage activation, has been frequently reported [ , , [ ] [ ] [ ] [ ] . although hyperferritinemia was first considered suggestive of aosd, several studies showed that hyperferritinemia, whatever the threshold used, has poor positive predictive value in isolation without a suggestive context [ , ] . besides total ferritin level, the diagnostic performance of glycosylated ferritin (gf) level has been investigated [ ] . the gf level normally represents more than half of the total ferritin level. in inflammatory conditions, the concentration of gf decreases and usually ranges from % to %; this decrease has been related to the saturation of glycosylation mechanisms due to hyperferritinemia. however, in aosd, gf level is typically markedly decreased (below %), which suggests a more specific phenomenon. more extensive data revealed a sensitivity and specificity of gf ≤ % for aosd diagnosis of % and %, respectively [ ] . the combination of both hyperferritinemia and gf level ≤ % yielded a sensitivity and specificity of % and %, respectively. of note, although serum ferritin level fluctuates according to systemic inflammation, gf level remains low several weeks to several months after disease remission [ ] . however, as mentioned previously, low gf level is not completely specific to aosd and is observed in other inflammatory processes, such as severe systemic infections [ ] . moreover, the gf level is usually low, ≤ %, in hemophagocytic syndromes, regardless of the cause, such as infection, neoplasm, or inflammation [ ] [ ] [ ] . results of immunological workups performed to exclude other connective tissue diseases or inflammatory joint diseases must be negative. approximately ten years ago, mcgonagle and mcdermott formulated the hypothesis of two main pathogenic mechanisms underlying immune-mediated inflammation against the self and proposed a new classification for immunological diseases contrasting autoimmunity and autoinflammation [ , ] . autoimmunity referred to adaptive immunity and was defined as aberrant dendritic, b-and t-cell responses in primary and secondary lymphoid organs leading to tolerance break and development of immune reactivity toward native antigens (with autoantibodies in most cases), whereas autoinflammation referred to innate immunity and was defined as dysregulated activation of macrophages and neutrophils in response to a danger signal leading to tissue damage. these categories represent a continuum, which allowed for a new classification of immune-mediated inflammatory disorders that was refined in the following years [ , ] . although autoimmune diseases were rather easily diagnosed based on autoantibody or autoantigen-specific t and b cells, no specific biomarker exists for said. the definition mainly relies on similarities with monogenic, hereditary periodic fever syndromes, which were at the origin of the concept. they include several key features: intense inflammation with periodic fever, tissue inflammation depending on the disease, increased leukocyte and neutrophil count, elevated erythrocyte sedimentation rate (esr) and c-reactive protein (crp) level, and, more recently, a pathogenic role of inflammasome and response to interleukin- (il- ) blockade [ ] . besides monogenic familial syndromes, crohn's disease was the first nonfamilial polygenic aid, mainly tissue specific, with predominant gut involvement [ , ] . a few years later, still's disease was described as another nonfamilial aid, becoming one of the most characteristic polygenic systemic said [ ] . the mechanisms underlying aosd pathogenesis are largely hypothetical, but, from the evident pathogenic pathway seen in sojia patients and the growing understanding of said, a pro-inflammatory cascade can be proposed (fig. . ) [ , , , , ] . the starting point is likely specific danger signals, such as pathogen-or damageassociated molecular patterns (i.e., pamps or damps). danger signals are transmitted to macrophages and neutrophils via specific toll-like receptors that activate specific inflammasomes, likely nod-like receptor family pyrin domain containing (nlrp ), leading to caspase activation and overproduction of active il- [ ] [ ] [ ] [ ] . this step seems to be central to the aosd pathogenesis and leads to intense innate immune cell activation and overproduction of several pro-inflammatory cytokines including il- , il- , tumor necrosis factor (tnf), as well as il- and il- . several factors actively contribute to an amplified inflammatory response, often referred to as the cytokine "burst" or "storm" [ , ] . in addition to il- itself, conferring retrograde activation of macrophages and neutrophils; different alarmins, such as the s protein-s a protein seeming to be more specific to still's disease in children-and advanced glycosylated end (age) products are involved in these processes [ , [ ] [ ] [ ] [ ] [ ] . besides amplification mechanisms, said pathogenesis has been suggested to involve deficiency or failure in regulatory or antiinflammatory mechanisms: deficiency in t regulator or natural killer cells, insufficient il- production, and deficiency in resolution lipid mediators, soluble receptors of ages (rages), or other resolution-associated molecular patterns [ ] [ ] [ ] [ ] . [ , , ] . in addition, patients often experience odynophagia or pharyngitis just before the start of the disease or the relapse, which could correspond to the infectious danger signal that will trigger toll-like receptors and start the intense inflammatory process. in contrast to monogenic, hereditary, periodic fever syndromes, the underlying genetic background of aosd is largely unknown. the disease is present in different geographic regions and different ethnic groups [ , , , , ] . association studies have suggested a potential predisposing genetic background. hla-bw was the first identified as a susceptibility antigen and associated with a mild self-limiting pattern of the disease [ ] . hla-dr was found more prevalent in aosd cases versus healthy controls, and hla-drw was associated with the occurrence of proximal arthralgia [ ] . however, no functional analysis has confirmed these hypotheses [ , , ] . recently, two major findings have been reported in sojia patients. firstly, a homoallelic missense mutation in the enzyme laccase (multicopper oxidoreductase) domain-containing -lacc -has been identified in sojia patients from five saudi consanguineous families [ ] . although familial sojia is not the rule, this study raises the potential role of this laccase in the pathogenesis of sojia. secondly, a large association study performed in sojia children and healthy controls identified a strong association between sojia and different hla-drb * haplotypes which all contain glutamate [ ] . this finding is more challenging since it would involve adaptive immunity in the aosd pathogenesis which was not expected [ ] . finally, substantial advances have been made in exploring the human genome, which will facilitate the identification of potential mutations in genes involved in autoinflammatory pathways, including de novo germinal mutations or somatic mutations occurring during fetus development or after birth [ , ] . several severe manifestations have been described in aosd, which explains the potentially pejorative prognosis of the disease [ , ] . the disease can be lifethreatening because of the predominant involvement of one organ or of systemic complications with multiple organ failure from the outset, while the diagnosis isn't clearly confirmed. the articular signs may often fall by the wayside because of the gravity of the complications of such clinical forms, which can contribute to misdirect the clinicians who are not familiar with aosd. these complications are given in table . , and the most frequent and relevant ones are discussed in more detail. this is a common complication of aosd at the time of diagnosis, right after treatment introduction or during the course of the disease. in systemic-onset jia, it is also called macrophage activation syndrome. this serious complication should be suspected in a patient with persisting fever (in contrast to evening spiking fever) and decrease in initially elevated leukocyte and neutrophil counts [ , , ] . several other manifestations can be associated. the key issue with rhl is to determine whether its occurrence is related to aosd intense inflammation or to concomitant infection, potentially favored by immunomodulators introduced because of aosd. the list of possible infections is quite long, with viral reactivation at first place. aosd can be complicated by two serious coagulation disorders, mainly at the acute phase of the disease. the first disorder, disseminated intravascular coagulation (dic), is not rare and may occur at a frequency of - % [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this diagnosis should be suspected with the combination of thrombotic events and cutaneous or mucosal bleeding and sometimes specific organ involvements, such as fulminant hepatitis, cardiac or respiratory failure, or stroke. hemostasis workups reveal platelet and coagulation factor consumption, increased thromboplastin time, and high d-dimer levels. the other rare but quite severe coagulation disorder is thrombotic microangiopathy (tma). it must be suspected with unexplained multiorgan failure or stroke, related to multiple small thrombi leading to tissue ischemia and mechanical hemolytic anemia [ , , ] . acute vision impairment, such as blurred vision related to purtscher-like retinopathy, is frequently the first symptom [ ] . key diagnostic tests display thrombocytopenia by platelet consumption, anemia, and schistocytes (fragmented red blood cells), which are specific to this diagnosis. organ imaging may reveal multiple infarctions. in addition, adamts enzymatic activity needs to be tested because acquired deficiency has been found to predispose to tma [ , [ ] [ ] [ ] . tma has been mainly described during aosd flare, related to intense inflammation or to concomitant infection with shiga toxin-producing microorganisms [ ] . although pleural effusion or pericarditis is frequent, other serious cardiac or pulmonary manifestations have been described. specific attention should be dedicated to a rare and very severe aosd manifestation, pulmonary arterial hypertension (pah), with several cases recently reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pah, either idiopathic or occurring with aosd or other connective tissue diseases, is thought to be related at least in part to immune-related alteration of the endothelium with overproduction of il- , il- , il- , and tnf [ , , , , ] . pah may occur at aosd onset or later and seems to predominate in women. the diagnosis must be suspected with unexplained and often rapidly progressing dyspnea [ , , , , ] . electrocardiography is rarely contributive, eventually disclosing signs of right atrium hypertrophy. echocardiography is more useful for detection by measuring systolic pulmonary artery pressure, which is > mmhg, and the exclusion of left ventricle dysfunction. the formal pah diagnosis remains the right heart catheterization [ , ]. while liver abnormalities are most often limited to mild to moderate increases in aminotransferase activity which is frequent (up to % of cases), fulminant and fatal hepatitis have been reported [ , ] . the potential role of aspirin, nonsteroidal anti-inflammatory drugs (nsaids), or methotrexate has been mentioned by some authors [ ] . thus, clinicians should closely monitor liver function from the onset of the disease and particularly after prescription of potentially hepatotoxic drugs. self-medication should be avoided. liver biopsy, if performed, reveals nonspecific portal infiltrates of lymphocytes, plasma cells, and polymorphonuclears [ , , ] . hepatocytic lesions or massive necrosis has been described in fulminant hepatitis with rapidly progressive hepatic insufficiency. liver transplantation was necessary in a few exceptional cases [ , ] . amyloid a amyloidosis is becoming extremely rare owing to the better ability of aosd treatments to control inflammation [ ] [ ] [ ] [ ] [ ] . it could be observed in refractory patients or in patients who did not have access to adequate treatments for long periods of time. multiple organs can be involved. many other symptoms attributed to aosd have appeared in the literature. as these are almost exclusively in case reports, drawing conclusions is difficult: -ophthalmologic involvement related to sicca syndrome, conjunctivitis, uveitis, or episcleritis [ , , ] . -neurological manifestations, such as ischemic stroke, aseptic meningitis, or encephalitis [ ] . such symptoms might reveal hepatic failure or coagulation dysfunction, as in dic, rhl, or tma. -renal involvement limited to isolated proteinuria or related to glomerular or interstitial nephritis [ , ] . acute renal failure is possible in the context of severe myositis, hepatic insufficiency, or hematological complications [ , , [ ] [ ] [ ] . -secondary amyloid a amyloidosis (aa amyloidosis) is nowadays exceptional and is related to sustained uncontrolled inflammation [ ] [ ] [ ] [ ] [ ] . aosd is an exclusion diagnosis. because of presentation heterogeneity and of the lack of specificity of the clinical and biological manifestations, many other diagnoses might be evoked and afterward ruled out, before reasonably considering aosd (table . ) [ , , , ] . before finally diagnosing aosd, it is thus essential to first rule out neoplastic, infectious, or inflammatory conditions that can mimic the disease. infectious diseases represent the most complex issue in differential diagnoses because aosd therapies can be highly deleterious. the most misleading diagnoses are probably deep occult infections (e.g., biliary tract abscess or bone marrow infestation) or subacute endocarditis [ ] . whole blood culture, urine testing, and synovial fluid culture seem the minimal workup, while other tests might be considered depending on the context. among malignancies, malignant lymphomas-either hodgkin's disease or non-hodgkin's lymphomas-are probably the most misleading and the first to exclude if nonsymmetrical, fixed, and indurated lymph node enlargement is present [ , ] . less frequently, systemic inflammatory conditions have been described in the course of lymphoid leukemia or in paraneoplastic syndromes associated with solid cancers of either the lung or the breast [ , ] . autoimmune diseases that can mimic aosd include vasculitis, especially polyarteritis nodosa, and polymyositis [ ] . for patients with mainly chronic arthritis, the distinction between seronegative ra and aosd can be difficult. besides autoimmune diseases, hereditary or nonfamilial autoinflammatory syndrome (ais) can present with symptoms close to aosd because of the combination of fever, skin lesions, and arthritis in addition to other symptoms that differ among the ais [ ] [ ] [ ] . although the hereditary ais often begins during childhood, the diagnosis tends to be made when the individual is an adult; examples are hyperimmunoglobulin d (igd) syndrome or tnf receptor-associated periodic syndrome (traps). different mutations have been associated with different hereditary ais, but, to date, none has been associated with aosd. however, the similarities between these diseases and aosd might suggest common pathogenic pathways and place aosd as a nonfamilial, sporadic form of ais. finally, in rare cases, the severe and systemic drug hypersensitivity reaction can mimic aosd. the presence of urticarial skin lesions and increased eosinophilic pmn count are suggestive features, as is exposure to a potential agent within the previous days or weeks. after comprehensive investigations, no clinical sign or biological abnormality can be considered aosd specific enough to ascertain the diagnosis. thus, classification criteria may be helpful, although they have been developed more for clinical research than diagnosis. two sets of criteria have been validated (table . ). the yamaguchi criteria set, published in , is the most widely used [ ] ; however, it contains exclusion criteria that are problematic in clinical practice. the fautrel criteria set has the advantage of including ferritin and gf levels as diagnostic biomarkers and does not require exclusion criteria [ ] . in a recent validation study, both sets showed high sensitivity and specificity [ ] . the most relevant serum biomarkers identified to date are listed in table . . ferritins are the major biomarkers for aosd. serum ferritin level is a key biomarker to assess disease activity, but probably less relevant for diagnosis since its specificity is limited and no clear threshold has been identified so far [ , ] . the diagnostic value of glycosylated ferritin level, with the threshold of gf≤ %, is much more interesting [ , ] . (continued) identifying the disease subset might orientate the therapeutic strategy c serum ferritin levels are significantly higher in the systemic subtype [ ] , but high ferritin levels after adequate treatment may predict chronic articular course [ ] d calprotectin levels help rule out rheumatoid arthritis, but further studies are needed to validate it as a diagnostic biomarker because of no statistical difference between aosd and septic patients, although the populations were small [ ] e elevated plasma levels of il- β, il- , and tnfα have been found during aosd, but the cytokine profile is not specific and cannot differentiate aosd patients from those with sepsis f s a was found an efficient diagnostic and monitoring biomarker in systemic juvenile arthritis, but further studies are needed for validation in aosd procalcitonin, a marker of severe systemic infection, was also found elevated in patients with active aosd and does not appear relevant to distinguish acute infection from aosd flare [ , ] . serum level of calprotectin (i.e., s a /s a protein) could be an additional disease activity biomarker because alarmins seem to play a key role in aosd pathogenesis. however, it is not specific to aosd and may be elevated in many other inflammatory conditions [ , , , ] . finally, serum amyloid a protein (saa) is an inflammatory biomarker found predictive of amyloidosis [ ] . several research works assessed serum cytokine levels. high serum levels of il- , il- , and il- have been found in systemic forms of aosd and can be considered activity biomarkers. however, their additional value on top of crp level and other inflammatory biomarkers is unclear, and these cytokines are clearly not specific to aosd. thus, they are not recommended in routine investigation. of importance, il- seems to play a key role in rhl. age products and rages, involved in aosd pathogenesis, may be elevated in other inflammatory disorders. high serum levels have been associated with polycyclic or chronic/progressive evolution patterns [ ] . serum level of soluble cd , a macrophage activation biomarker, is elevated in aosd but is not specific to the disease. finally, several chemokines (c-x-c motif chemokine ligands and , macrophage inhibitory factor) have been found to be diagnostic biomarkers for aosd, which needs to be confirmed in larger patient samples [ ] . aosd evolution can be quite diverse and currently is impossible to predict. several patterns of evolution have been described, mainly based on case series and not robust epidemiological studies [ , , , , ]: it combines systemic manifestations and joint involvement: the initial flare evolves over a few weeks; remission is achieved with steroids or other immunomodulatory agents after a few days or weeks; and treatments can be progressively tapered and then stopped without relapse after a few months. the pattern may represent - % of cases. it is characterized by aosd relapses after a few months or years, under immunomodulatory treatment or after its discontinuation. in this pattern, one classical presentation is a first flare during childhood-diagnosed sojia, followed by sustained drug-free remission for several years, and then a relapse in adulthood. in most of these cases, recurrences combine systemic and joint involvement. the pattern represents - % of cases. in this form, a continuous inflammation is responsible for chronic and frequently erosive joint involvement with regular systemic flares. this pattern was the most frequent, estimated at - % of cases, in old series [ , , ] . although no new estimates are available, this pattern should be less likely with the most recent targeted therapies. the prognosis of aosd is dominated by potential life-threatening events as well as progressive functional impairment. the life prognosis is dominated by the severity of the following visceral involvements, in isolation or combination: hematological complications, such as dic, tma, or severe rhl; fulminant hepatitis with rapid liver failure; and acute respiratory distress syndrome, extensive myocarditis, or multiorgan failure [ , ] . importantly, exacerbation of these serious symptoms has been reported within the first days of treatment initiation, especially of biological therapies. this necessitates a close and very careful monitoring of such patients at the start of treatment. in addition to organ involvements, the development of secondary amyloidosis of the aa type remains possible in long-standing refractory chronic articular aosd [ ] . functional prognosis depends mainly on erosion and joint destruction in chronic articular aosd, which affects approximately one-third of patients [ , , ] . no clear predictor of erosive joint disease has been identified, but, as joint involvement is often limited, functional prognosis in aosd seems to be less severe than in ra or other inflammatory arthritides [ ] . corticosteroid side effects are also common because steroids are usually prescribed in high doses and the long term, which might contribute to the overall prognosis in aosd. aosd is rare, and no randomized controlled trial has been performed. thus, the only information on therapy is from observational studies and retrospective case series. recently, the management of aosd benefited from the proofs of the efficacy of targeted biotherapies. although nsaids, including aspirin, have been effective in systemic jia, they are rarely effective in aosd; only % of patients have achieved control with this therapy [ , , ] . of the nsaids, indometacin - mg/day seems to be the most effective [ , ] . liver enzymes must be monitored at the initial stages of the disease because severe hepatitis has been suggested to be related to treatment with nsaids [ , , ] . acetaminophen may help to reduce fever, although it is often not enough to suspend it. other analgesics might be necessary to relieve intense myalgia and joint paint. once the diagnosis is established, corticosteroids are usually required to induce symptom remission. optimal dosing relies on medium to high doses (i.e., . - mg/ kg/day of prednisone equivalent) [ , , , , ] . patients with serious visceral involvement might achieve a quick response with intravenous infusion of high-dose methylprednisolone [ , , ] . response to corticosteroids is often dramaticwithin a couple of hours or a few days [ , ] . the consensus is lacking on a therapeutic tapering scheme once clinical remission is achieved. however, owing to the potentially serious side effects of cumulative corticosteroid therapy, many others currently recommend to achieve the dose of . mg/kg/day after weeks of therapy and to stop completely after three months. if this is not possible, the response should be considered inadequate, and a diseasemodifying treatment, especially a targeted therapy, should be started (fig. . ) [ ]. methotrexate is used as an immunomodulatory agent in rheumatoid arthritis. it is efficient in controlling aosd disease activity and allowing for steroid dose sparing [ , [ ] [ ] [ ] . however, whether methotrexate can prevent or limit joint structural damage in the erosive form of aosd, as has been shown for ra, is unclear. the presence of liver enzyme abnormalities does not contraindicate methotrexate prescription, but close biological monitoring is necessary [ , , , ] . of importance, methotrexate can be associated with anti-il- -or anti-il- targeted therapies. different targeted therapies have been used to treat refractory aosd (table . ). the medical need is high, keeping in mind the unsatisfactory rates provided by classical therapies with nsaids or conventional disease-modifying antirheumatic drugs (dmards). the use of glucocorticoids is also limited by the known longterm safety issues, especially with higher doses. in fact, for at least - % of patients, the disease cannot be controlled by glucocorticoids even when combined with conventional dmards, such as methotrexate. the use of il- inhibitors for aosd contributed to the revival of considering this mode of action in rheumatology and now represents the primary choice for treating autoinflammatory diseases in general. however, it took surprisingly long until clinical development moved forward from cohort studies to randomized, placebocontrolled trials. currently, two il- blocking compounds are the focus of phase / phase studies with different designs. anakinra, a recombinant il- receptor antagonist, was the first biologic showing beneficial effects in treating the systemic and articular features of aosd in many case series, uncontrolled trials, and different national surveys. although the evidence for its efficacy has not been proven by controlled trials, the overall number of more than published cases provides convincing results. in fact, a clear and sustained improvement was described for systemic and also arthritic symptoms in most treated cases [ , [ ] [ ] [ ] [ ] [ ] . systemic features seem to show a more rapid response, and longer exposure seems usually required for improvement of arthritis. of major importance for long-term safety were consistent reports of a reduction or even discontinuation of glucocorticoids use as well as nsaids. in this context, meta-analyses from eight case series and three national surveys meeting predefined quality standards and including more than anakinra-treated aosd patients revealed a remission rate of approximately % and a reduced use of steroids in approximately % of patients [ ] . recently, a large observational retrospective multicenter study from italy added evidence for a strong impact on disease activity with the pouchot score as well as clinical and serological features regardless of sex, age, disease pattern, or co-medication [ ] . since september , a randomized, double-blind, placebo-controlled, multicenter, phase study in the united states has recruited patients for investigating two different doses of anakinra ( mg/kg/day [max mg/day] or mg/kg/day [max mg/day]) to evaluate its efficacy and safety in patients newly diagnosed with sojia and aosd. the primary end point, american college of rheumatology (acr ) response, has already been evaluated at week ; however, the overall treatment period is short, with only weeks of exposure followed by a -week safety follow-up [ ] . of note, anakinra is the only il- blocker for which we have substantial long-term results in terms of efficacy and safety in aosd [ ] , and it has now been approved in this indication. in contrast to monogenic autoinflammatory diseases, in aosd, remission can continue in some cases even after treatment is stopped. however, a relatively high withdrawal rate of % has been reported owing to loss of response over time and also to frequent injection site reactions to the required daily administrations. the other approved biologic for aosd is canakinumab, a fully human antibody against il- β [ ] . furthermore, canakinumab is also approved for treating other autoinflammatory diseases including cryopyrin-associated periodic syndromes, familial mediterranean fever, tnf receptor-associated periodic syndrome, and hyper-igd syndrome as well as sojia. although the overall experience with canakinumab in aosd is still limited, the reported response of systemic features and arthritis was rapid and sustained over many months to years in most patients, frequently allowing for tapering of steroids [ ] [ ] [ ] . of note, canakinumab was found highly effective for patients with aosd who were difficult to treat, including those showing failure of nsaids, glucocorticoids, and other il- inhibitors. with these promising results, a placebo-controlled, randomized, multicenter, phase study of canakinumab in aosd is ongoing [ ] . the dosage of canakinumab with monthly injections of up to mg/kg body weight (maximum dos of mg) is based on the pharmacokinetic profile in adolescent patients with sojia. the primary aim is to investigate the efficacy of canakinumab in patients with aosd and active joint involvement in terms of the proportion of patients with a clinically significant reduction in disease activity (disease activity score in joints [das ] > . ) following a treatment period of weeks. the overall placebocontrolled period is weeks, but nonresponders can be unblinded and show treatment rescue at week . the core study is followed by an optional long-term extension over years to provide additional safety results in the adult population. in summary, the published data for anti-il- agents in refractory aosd clearly show a consistently high rate of full or partial remission with the additional achievement of lowering or stopping glucocorticoid medication [ ] . inhibition of il- signaling is possible by two different approaches, the direct neutralization of the cytokine or the blockade of the respective receptor. none of these mechanisms has been investigated in aosd in a controlled setting of clinical trials in detail. for the two different il- receptor antagonists currently available in daily practice for treating rheumatic diseases, only the tocilizumab aosd case series has been published and reported at conferences [ ] [ ] [ ] [ ] . to summarize, the observed anti-inflammatory effects were strong, rapid, and sustained for most of the cases. usually, the systemic features disappeared entirely, but also the arthritic manifestations improved, and the serologic activity decreased [ , ] . however, because of limited data, the likelihood of response cannot be estimated as clearly as for il- inhibitors and seems to be between % to % in terms of full remission. a recently published meta-analysis of ten original studies ( individuals) on the efficacy of tocilizumab and aosd revealed overall high partial and complete remission rates of - %, respectively. tocilizumab prevented new flares, was well tolerated, and could significantly reduce the need for corticosteroids [ , ] . whether other il- inhibitors have similar effects remains unknown. as shown for ra treatment, with aosd, differences could exist with respect to safety, especially in comparison to the direct cytokine inhibitors. also, the advantages or disadvantages that distinguish il- and il- inhibition are unclear. although clearly il- as well as il- inhibitors have high response rates and represent alternative approaches in aosd, we have no way to predict the individual effects and define the ideal treatment algorithm. a novel compound is tadekinig alfa, a recombinant human il- binding protein [ ] . this drug was tested in healthy volunteers and patients with psoriasis and ra in phase i studies and demonstrated a good safety and tolerability profile with only mild adverse events at the injection site. because of the postulated role of il- in the pathogenesis of aosd, it was a logical step to investigate the effects of tadekinig alfa in this condition. a first open-label, dose-finding phase study involving multiple centers in europe was designed to capture safety information as the primary outcome. two doses ( and mg) were administered during weeks, and patients were followed up for more weeks. as a secondary outcome measurement, the efficacious dose at weeks was defined as normalization of body temperature and decrease of crp level by % or more of baseline values. although limited by the low patient number and short observation period in the study, the safety profile of tadekinig alfa was overall acceptable, with injection site reactions and infections being the most frequent adverse events. in terms of efficacy, reduced level of crp as well as other inflammatory markers (including il- , ferritin) was detected in some patients. furthermore, an improvement in skin rash and a slight but significant reduction in the prednisolone doses were reported. in summary, inhibition of il- by tadekinig alfa could be a promising new approach in asod that needs to be investigated in a controlled setting. many other treatments have been tried in aosd, with varying degrees of success. stimulated by encouraging results from other chronic inflammatory joint diseases, especially ra, tnf inhibitors have been the first biologics used for aosd [ ] [ ] [ ] [ ] [ ] [ ] . however, driven by the deceptive impression that aosd, with a predominant arthritic phenotype, is a subgroup of seronegative ra, the results from uncontrolled trials involving usually small cohorts of patients were inconsistent. since favorable outcomes have been seen in only a few patients without any specific characteristics, tnf inhibitors can only be considered third-line drugs, preferentially for patients with chronic arthritis. cyclosporine a has been proposed before the era of biotherapies, in patients with systemic features of aosd or with rhl, with an interesting efficacy. however, the tolerance of this drug limits its use, but it certainly can be of interest in complex or refractory situations. there is henceforth no place for intravenous immunoglobulins in aosd treatment, owing to the efficacy of the other therapies available. the strategy we propose for aosd patients is summarized in fig. . . management should include, firstly, symptomatic care in a medicine department or intensive care unit (icu); secondly, active and rapid search for an infection; and, thirdly, introduction of high-dose steroids. since rhl has described under il- therapy, it appears prudent to introduce this treatment only after rhl control by a few days of high-dose steroids. dic is a medical emergency whose care combines: . supportive measures, which could include platelet transfusion, coagulation factors (or fresh frozen plasma), and fibrinogen (in cryoprecipitate) infusion to control severe bleeding or sometimes heparin if no active bleeding is present [ ] . . specific anti-inflammatory agents often decided in multidisciplinary rounds, with mainly high-dose corticosteroids, either intravenous (methylprednisolone mg/kg/day) or oral (prednisone mg/kg/day) [ ] . to limit steroid exposure, the addition of other immunomodulatory agents, such as il- or il- blockers or cyclosporine, is recommended [ , , [ ] [ ] [ ] . tma treatment is complex and mandatorily includes [ ]: . supportive care in an icu, with plasma exchange being central [ ] . high-dose steroids intravenously and then orally other immunomodulatory agents proposed for refractory cases include anakinra [ ] , tocilizumab [ ] , intravenous immunoglobulins, cyclophosphamide, azathioprine, cyclosporine a, and rituximab [ , ] . despite this treatment, mortality associated with tma remains high, up to % [ ]. their treatment combines supportive care, high-dose steroids, and frequently il- or il- inhibitors. regarding pah, early diagnosis is central for the prognosis of this complication, with mortality remaining as high as % [ ] . the diagnosis includes [ , , , , , , ]: . hospitalization in an icu and rapid referral to a specific reference center for multidisciplinary round discussions. . vasodilating therapies frequently combining endothelin receptor antagonists (bosentan, ambrisentan), phosphodiesterase- inhibitors and other guanylate cyclase stimulators (sildenafil or others), and prostacyclin analogues. . high-dose steroids, initially intravenously and then orally. . rapid introduction of biologic immunomodulatory agents targeting il- or il- or other immunosuppressive agents, such as methotrexate, azathioprine, cyclophosphamide, cyclosporine, or rituximab. however, these treatments must be carefully monitored because a few cases of pah worsening have been described after their introduction. pah deserves to be well known by physicians caring for aosd patients because it was not described in most of the initial aosd case series for several reasons: now better diagnostic tools for pah; differential efficacy of large-spectrum immunomodulatory agents, such as steroids, as compared with more targeted therapies, such as il- or il- blockers; and exposure to 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infliximab: first experiences infliximab in the treatment of adult still's disease refractory to conventional therapy club rhumatismes et inflammation. tumour necrosis factor alpha blocking agents in refractory adult still's disease: an observational study of cases tocilizumab in refractory adult still's disease: refractory asd and tocilizumab tocilizumab in adultonset still's disease: the israeli experience tocilizumab for the treatment of adult-onset still's disease: results from a case series treatment of refractory adult-onset still's disease with tocilizumab: report of two cases and review of the literature a pilot study on tocilizumab for treating refractory adult-onset still's disease. sci rep efficacy of tocilizumab therapy in korean patients with adult-onset still's disease: a multicentre retrospective study of cases efficacy and safety of tocilizumab with inhibition of interleukin- in adult-onset still's disease: a meta-analysis the effect of tocilizumab on preventing relapses in adult-onset still's disease: a retrospective, single-center study open-label, multicentre, doseescalating phase ii clinical trial on the safety and efficacy of tadekinig alfa (il- bp) in adult-onset still's disease successful treatment of refractory adult onset still's disease with rituximab refractory adult-onset still disease successfully treated with abatacept efficacy of abatacept in a refractory case of adult-onset still's disease adult onset still's disease-the evidence that anti-interleukin- treatment is effective and well-tolerated (a comprehensive literature review) s.m. has received consultant fees from bms and pfizer. e.f. has received consultant fees from abbvie, biogen, bms, celgene, janssen, lilly, medac, msd, nordic pharma, novartis, pfizer, roche, sanofi-aventis, swedish orphan biovitrum, and ucb, as well as a research grant from bms, lilly, novartis, pfizer, and roche.b.f. has received consultant fees from abbvie, biogen, bms, celgene, janssen, lilly, medac, msd, nordic pharma, novartis, pfizer, roche, sanofi-aventis, swedish orphan biovitrum, and ucb, as well as research grants from abbvie, lilly, msd, and pfizer. key: cord- -tbfg vmd authors: brauer, fred; castillo-chavez, carlos title: epidemic models date: - - journal: mathematical models in population biology and epidemiology doi: . / - - - - _ sha: doc_id: cord_uid: tbfg vmd communicable diseases such as measles, influenza, and tuberculosis are a fact of life. we will be concerned with both epidemics, which are sudden outbreaks of a disease, and endemic situations, in which a disease is always present. the aids epidemic, the recent sars epidemic, recurring influenza pandemics, and outbursts of diseases such as the ebola virus are events of concern and interest to many people. the prevalence and effects of many diseases in less-developed countries are probably not as well known but may be of even more importance. every year millions, of people die of measles, respiratory infections, diarrhea, and other diseases that are easily treated and not considered dangerous in the western world. diseases such as malaria, typhus, cholera, schistosomiasis, and sleeping sickness are endemic in many parts of the world. the effects of high disease mortality on mean life span and of disease debilitation and mortality on the economy in afflicted countries are considerable. (german measles), and chicken pox, confer immunity against reinfection, while diseases transmitted by bacteria , such as tuberculosis, meningitis, and gonorrhea, confer no immunity against reinfection. other human diseases, such as malaria, are transmitted not directly from human to human but by vectors, agents (usually insects) that are infected by humans and who then transmit the disease to humans. there are also diseases such as west nile virus, that are transmitted back and forth between animals and vectors. heterosexual transmission of hiv/aids is also a vector process in which transmission goes back and forth between males and females. we will focus on the transmission dynamics of an infection from individual to individual in a population, but many of the same ideas arise in transmission of a genetic characteristic, such as gender, race, genetic diseases, a cultural "characteristic," such as language or religion, an addictive activity, such as drug use, and the gain or loss of information communicated through gossip, rumors, and so on. similarly, many of the ideas arise also with different characterizations of what is meant by an individual, including the types of cells in the study of disease dynamics of the immune system. in the study of chagas disease, a "house" (infested houses may correspond to "infected" individuals) may be chosen as an epidemiological unit; in tuberculosis, a household or community or a group of strongly linked individuals ("cluster") may be the chosen unit. an epidemic, which acts on a short temporal scale, may be described as a sudden outbreak of a disease that infects a substantial portion of the population in a region before it disappears. epidemics usually leave many members untouched. often these attacks recur with intervals of several years between outbreaks, possibly diminishing in severity as populations develop some immunity. this is an important aspect of the connection between epidemics and disease evolution. the historian w.h. mcneill argues, especially in his book plagues and peoples ( ) , that the spread of communicable diseases frequently has been an important influence in history. for example, there was a sharp population increase throughout the world in the eighteenth century; the population of china increased from million in to million in , and the population of europe increased from million in to million in . there were many factors involved in this increase, including changes in marriage age and technological improvements leading to increased food supplies, but these factors are not sufficient to explain the increase. demographic studies indicate that a satisfactory explanation requires recognition of a decrease in the mortality caused by periodic epidemic infections. this decrease came about partly through improvements in medicine, but a more important influence was probably the fact that more people developed immunities against infection as increased travel intensified the circulation and cocirculation of diseases. there are many biblical references to diseases as historical influences. the book of exodus describes the plagues that were brought down upon egypt in the time of moses. another example is the decision of sennacherib, the king of assyria, to abandon his attempt to capture jerusalem about bc because of the illness of his soldiers (isaiah , - ), and there are several other biblical descriptions of epidemic outbreaks. the fall of empires has been attributed directly or indirectly to epidemic diseases. in the second century ad, the so-called antonine plagues (possibly measles nomic hardships leading to disintegration of the empire because of disorganization, which facilitated invasions of barbarians. the han empire in china collapsed in the third century ad after a very similar sequence of events. the defeat of a population of millions of aztecs by cortez and his followers can be explained, in part, by a smallpox epidemic that devastated the aztecs but had almost no effect on the invading spaniards, thanks to their built-in immunities. the aztecs were not only weakened by disease but also confounded by what they interpreted as a divine force favoring the invaders. smallpox then spread southward to the incas in peru and was an important factor in the success of pizarro's invasion a few years later. smallpox was followed by other diseases such as measles and diphtheria imported from europe to north america. in some regions, the indigenous populations were reduced to one-tenth of their previous levels by these diseases: between and the indian population of mexico was reduced from million to million. the black death (probably bubonic plague) spread from asia throughout europe in several waves during the fourteenth century, beginning in , and is estimated to have caused the death of as much as one-third of the population of europe between and . the disease recurred regularly in various parts of europe for more than years, notably as the great plague of london of - . it then gradually withdrew from europe. since the plague struck some regions harshly while avoiding others, it had a profound effect on political and economic developments in medieval times. in the last bubonic plague epidemic in france ( - ), half the population of marseilles, percent of the population in nearby toulon, per cent of the population of arles, and percent of the population of aix and avignon died, but the epidemic did not spread beyond provence. expansions and interpretations of these historical remarks may be found in mcneill ( ) , which was our primary source on the history of the spread and effects of diseases. the above examples depict the sudden dramatic impact that diseases have had on the demography of human populations via disease-induced mortality. in considering the combined role of diseases, war, and natural disasters on mortality rates, one may conclude that historically humans who are more likely to survive and reproduce have either a good immune system, a propensity to avoid war and disasters, or, nowadays, excellent medical care and/or health insurance. descriptions of epidemics in ancient and medieval times frequently used the term "plague" because of a general belief that epidemics represented divine retribution for sinful living. more recently, some have described aids as punishment for sinful activities. such views have often hampered or delayed attempts to control this modern epidemic. there are many questions of interest to public health physicians confronted with a possible epidemic. for example, how severe will an epidemic be? this question may be interpreted in a variety of ways. for example, how many individuals will be affected and require treatment? what is the maximum number of people needing and) invaded the roman empire, causing drastic population reductions and eco-care at any particular time? how long will the epidemic last? how much good would quarantine of victims do in reducing the severity of the epidemic? scientific experiments usually are designed to obtain information and to test hypotheses. experiments in epidemiology with controls are often difficult or impossible to design, and even if it is possible to arrange an experiment, there are serious ethical questions involved in withholding treatment from a control group. sometimes data may be obtained after the fact from reports of epidemics or of endemic disease levels, but the data may be incomplete or inaccurate. in addition, data may contain enough irregularities to raise serious questions of interpretation, such as whether there is evidence of chaotic behavior [ellner, gallant, and theiler ( ) ]. hence, parameter estimation and model fitting are very difficult. these issues raise the question whether mathematical modeling in epidemiology is of value. mathematical modeling in epidemiology provides understanding of the underlying mechanisms that influence the spread of disease, and in the process, it suggests control strategies. in fact, models often identify behaviors that are unclear in experimental data-often because data are nonreproducible and the number of data points is limited and subject to errors in measurement. for example, one of the fundamental results in mathematical epidemiology is that most mathematical epidemic models, including those that include a high degree of heterogeneity, usually exhibit "threshold" behavior, which in epidemiological terms can be stated as follows: if the average number of secondary infections caused by an average infective is less than one, a disease will die out, while if it exceeds one there will be an epidemic. this broad principle, consistent with observations and quantified via epidemiological models, has been used routinely to estimate the effectiveness of vaccination policies and the likelihood that a disease may be eliminated or eradicated. hence, even if it is not possible to verify hypotheses accurately, agreement with hypotheses of a qualitative nature is often valuable. expressions for the basic reproductive number for hiv in various populations is being used to test the possible effectiveness of vaccines that may provide temporary protection by reducing either hiv-infectiousness or susceptibility to hiv. models are used to estimate how widespread a vaccination plan must be to prevent or reduce the spread of hiv. in the mathematical modeling of disease transmission, as in most other areas of mathematical modeling, there is always a trade-off between simple models, which omit most details and are designed only to highlight general qualitative behavior, and detailed models, usually designed for specific situations including short-term quantitative predictions. detailed models are generally difficult or impossible to solve analytically and hence their usefulness for theoretical purposes is limited, although their strategic value may be high. for public health professionals, who are faced with the need to make recommendations for strategies to deal with a specific situation, simple models are inadequate and numerical simulation of detailed models is necessary. in this chapter, we concentrate on simple models in order to establish broad principles. furthermore, these simple models have additional value since they are the building blocks of models that include detailed structure. a specific goal is to compare the dynamics of simple and slightly more detailed models primarily to see whether slightly different assumptions can lead to significant differences in qualitative behavior. many of the early developments in the mathematical modeling of communicable diseases are due to public health physicians. the first known result in mathematical epidemiology is a defense of the practice of inoculation against smallpox in by over three generations) who had been trained as a physician. the first contributions to modern mathematical epidemiology are due to p.d. en'ko between and [dietz ( ) ], and the foundations of the entire approach to epidemiology based on compartmental models were laid by public health physicians such as sir r.a. ross, w.h. hamer, a.g. mckendrick, and w.o. kermack between and , along with important contributions from a statistical perspective by j. brownlee. a particularly instructive example is the work of ross on malaria. dr. ross was awarded the second nobel prize in medicine for his demonstration of the dynamics of the transmission of malaria between mosquitoes and humans. although his work received immediate acceptance in the medical community, his conclusion that malaria could be controlled by controlling mosquitoes was dismissed on the grounds that it would be impossible to rid a region of mosquitoes completely and that in any case, mosquitoes would soon reinvade the region. after ross formulated a mathematical model that predicted that malaria outbreaks could be avoided if the mosquito population could be reduced below a critical threshold level, field trials supported his conclusions and led to sometimes brilliant successes in malaria control. unfortunately, the garki project provides a dramatic counterexample. this project worked to eradicate malaria from a region temporarily. however, people who have recovered from an attack of malaria have a temporary immunity against reinfection. thus elimination of malaria from a region leaves the inhabitants of this region without immunity when the campaign ends, and the result can be a serious outbreak of malaria. we formulate our descriptions as compartmental models, with the population under study being divided into compartments and with assumptions about the nature and time rate of transfer from one compartment to another. diseases that confer immunity have a different compartmental structure from diseases without immunity and from diseases transmitted by vectors. the rates of transfer between compartments are expressed mathematically as derivatives with respect to time of the sizes of the compartments, and as a result our models are formulated initially as differential equations. models in which the rates of transfer depend on the sizes of compartments over the past as well as at the instant of transfer lead to more general types of functional equations, such as differential-difference equations and integral equations. in this chapter we describe models for epidemics, acting on a sufficiently rapid time scale that demographic effects, such as births, natural deaths, immigration into and emigration out of a population may be ignored. in the next chapter we will describe models in which demographic effects are included. daniel bernouilli, a member of a famous family of mathematicians (eight spread throughout history, epidemics have had major effects on the course of events. for example, the black death, now identified as probably having been the bubonic plague which had actually invaded europe as early as the sixth century, spread from asia throughout europe in several waves during the fourteenth century, beginning in , and is estimated to have caused the death of as much as one third of the population of europe between and . the disease recurred regularly in various parts of europe for more than years, notably as the great plague of london of - . it then gradually withdrew from europe. more than % of the population of london died in the great plague ( - ). it appeared quite suddenly, grew in intensity, and then disappeared, leaving part of the population untouched. one of the early triumphs of mathematical epidemiology was the formulation of a simple model by kermack and mckendrick ( ) whose predictions are very similar to this behavior, observed in countless epidemics. the kermack-mckendrick model is a compartmental model based on relatively simple assumptions on the rates of flow between different classes of members of the population. in order to model such an epidemic we divide the population being studied into three classes labeled s, i, and r. we let s(t) denote the number of individuals who are susceptible to the disease, that is, who are not (yet) infected at time t. i(t) denotes the number of infected individuals, assumed infectious and able to spread the disease by contact with susceptibles. r(t) denotes the number of individuals who have been infected and then removed from the possibility of being infected again or of spreading infection. removal is carried out through isolation from the rest of the population, through immunization against infection, through recovery from the disease with full immunity against reinfection, or through death caused by the disease. these characterizations of removed members are different from an epidemiological perspective but are often equivalent from a modeling point of view that takes into account only the state of an individual with respect to the disease. we will use the terminology sir to describe a disease that confers immunity against reinfection, to indicate that the passage of individuals is from the susceptible class s to the infective class i to the removed class r. epidemics are usually diseases of this type. we would use the terminology sis to describe a disease with no immunity against re-infection, to indicate that the passage of individuals is from the susceptible class to the infective class and then back to the susceptible class. usually, diseases caused by a virus are of sir type, while diseases caused by bacteria are of sis type. in addition to the basic distinction between diseases for which recovery confers immunity against reinfection and diseases for which recovered members are susceptible to reinfection, and the intermediate possibility of temporary immunity signified by a model of sirs type, more complicated compartmental structure is possible. for example, there are seir and seis models, with an exposed period between being infected and becoming infective. when there are only a few infected members, the start of a disease outbreak depends on random contacts between small numbers of individuals. in the next section we will use this to describe an approach to the study of the beginning of a disease outbreak by means of branching processes, but we begin with a description of deterministic compartmental models. the independent variable in our compartmental models is the time t, and the rates of transfer between compartments are expressed mathematically as derivatives with respect to time of the sizes of the compartments, and as a result our models are formulated initially as differential equations. we are assuming that the epidemic process is deterministic, that is, that the behavior of a population is determined completely by its history and by the rules that describe the model. in formulating models in terms of the derivatives of the sizes of each compartment we are also assuming that the number of members in a compartment is a differentiable function of time. this assumption is plausible once a disease outbreak has become established but is not valid at the beginning of a disease outbreak when there are only a few infectives. in the next section we will describe a different approach for the initial stage of a disease outbreak. the basic compartmental models to describe the transmission of communicable diseases are contained in a sequence of three papers by w.o. kermack and a.g. mckendrick in mckendrick in , mckendrick in , and . the first of these papers described epidemic models. what is often called the kermack-mckendrick epidemic model is actually a special case of the general model introduced in this paper. the general model included dependence on age of infection, that is, the time since becoming infected, and can be used to provide a unified approach to compartmental epidemic models. the special case of the model proposed by kermack and mckendrick in , which is the starting point for our study of epidemic models, is a flow chart is shown in figure . . it is based on the following assumptions: (i) an average member of the population makes contact sufficient to transmit infection with β n others per unit time, where n represents total population size (mass action incidence). (ii) infectives leave the infective class at rate αi per unit time. (iii) there is no entry into or departure from the population, except possibly through death from the disease. (iv) there are no disease deaths, and the total population size is a constant n. according to (i), since the probability that a random contact by an infective is with a susceptible, who can then transmit infection, is s/n, the number of new infections in unit time per infective is (β n)(s/n), giving a rate of new infections (β n)(s/n)i = β si. alternatively, we may argue that for a contact by a susceptible the probability that this contact is with an infective is i/n and thus the rate of new infections per susceptible is (β n)(i/n), giving a rate of new infections (β n)(i/n)s = β si. note that both approaches give the same rate of new infections; in models with more complicated compartmental structure one may be more appropriate than the other. we need not give an algebraic expression for n, since it cancels out of the final model, but we should note that for an sir disease model, n = s + i + r. later, we will allow the possibility that some infectives recover while others die of the disease. the hypothesis (iii) really says that the time scale of the disease is much faster than the time scale of births and deaths, so that demographic effects on the population may be ignored. an alternative view is that we are interested only in studying the dynamics of a single epidemic outbreak. the assumption (ii) requires a fuller mathematical explanation, since the assumption of a recovery rate proportional to the number of infectives has no clear epidemiological meaning. we consider the "cohort" of members who were all infected at one time and let u(s) denote the number of these who are still infective s time units after having been infected. if a fraction α of these leave the infective class in unit time, then u = −αu , and the solution of this elementary differential equation is thus, the fraction of infectives remaining infective s time units after having become infective is e −αs , so that the length of the infective period is distributed exponentially with mean ∞ e −αs ds = /α, and this is what (ii) really assumes. if we assume, instead of (ii), that the fraction of infectives remaining infective a time τ after having become infective is p(τ), the second equation of ( . ) would be replaced by the integral equation where i (t) represents the members of the population who were infective at time t = and are still infective at time t. the assumptions of a rate of contacts proportional to population size n with constant of proportionality β and of an exponentially distributed recovery rate are unrealistically simple. more general models can be constructed and analyzed, but our goal here is to show what may be deduced from extremely simple models. it will turn out that that many more realistic models exhibit very similar qualitative behaviors. in our model r is determined once s and i are known, and we can drop the r equation from our model, leaving the system of two equations together with initial conditions we think of introducing a small number of infectives into a population of susceptibles and ask whether there will be an epidemic. we remark that the model makes sense only so long as s(t) and i(t) remain nonnegative. thus if either s(t) or i(t) reaches zero, we consider the system to have terminated. we observe that s < for all t and i > if and only if s > α/β . thus i increases so long as s > α/β , but since s decreases for all t, i ultimately decreases and approaches zero. if s < α/β , i decreases to zero (no epidemic), while if s > α/β , i first increases to a maximum attained when s = α/β and then decreases to zero (epidemic). the quantity β s /α is a threshold quantity, called the basic reproduction number [heesterbeek ( ) ] and denoted by r , which determines whether there is an epidemict. if r < , the infection dies out, while if r > , there is an epidemic. the definition of the basic reproduction number r is that it is the number of secondary infections caused by a single infective introduced into a wholly susceptible population of size n ≈ s over the course of the infection of this single infective. in this situation, an infective makes β n contacts in unit time, all of which are with susceptibles and thus produce new infections, and the mean infective period is /α; thus the basic reproduction number is actually β n/α rather than β s /α. another way to view this apparent discrepancy is to consider two ways in which an epidemic may begin. one way is an epidemic started by a member of the population being studied, for example by returning from travel with an infection acquired away from home. in this case we would have i > , s + i = n. a second way is for an epidemic to be started by a visitor from outside the population. in this case, we would have s = n. since ( . ) is a two-dimensional autonomous system of differential equations, the natural approach would be to find equilibria and linearize about each equilibrium to determine its stability. however, since every point with i = is an equilibrium, the system ( . ) has a line of equilibria, and this approach is not applicable (the linearization matrix at each equilibrium has a zero eigenvalue). fortunately, there is an alternative approach that enables us to analyze the system ( . ). the sum of the two equations of ( . ) is thus s + i is a nonnegative smooth decreasing function and therefore tends to a limit as t → ∞. also, it is not difficult to prove that the derivative of a nonnegative smooth decreasing function must tend to zero, and this shows that integration of the sum of the two equations of ( . ) from to ∞ gives division of the first equation of ( . ) by s and integration from to ∞ gives ) is called the final size relation. it gives a relationship between the basic reproduction number and the size of the epidemic. note that the final size of the epidemic, the number of members of the population who are infected over the course of the epidemic, is n − s ∞ . this is often described in terms of the attack rate [technically, the attack rate should be called an attack ratio, since it is dimensionless and is not a rate.] the final size relation ( . ) can be generalized to epidemic models with more complicated compartmental structure than the simple sir model ( . ), including models with exposed periods, treatment models, and models including quarantine of suspected individuals and isolation of diagnosed infectives. the original kermack-mckendrick model ( ) included dependence on the time since becoming infected (age of infection), and this includes such models. integration of the first equation of ( . ) from to t gives and this leads to the form this implicit relation between s and i describes the orbits of solutions of ( . ) in the (s, i) plane. in addition, since the right side of ( . ) is finite, the left side is also finite, and this shows that s ∞ > . the final size relation ( . ) is valid for a large variety of epidemic models, as we shall see in later sections. it is not difficult to prove that there is a unique solution of the final size relation ( . ). to see this, we define the function then, as shown in figure . , is monotone decreasing from a positive value at x = + to a negative value at x = n. thus there is a unique zero s ∞ of g(x) with s ∞ < n. is monotone decreasing from a positive value at x = + to a minimum at x = n/r and then increases to a negative value at x = n . thus there is a unique zero s ∞ of g(x) with in fact, it is generally difficult to estimate the contact rate β , which depends on the particular disease being studied but may also depend on social and behavioral factors. the quantities s and s ∞ may be estimated by serological studies (measurements of immune responses in blood samples) before and after an epidemic, and from these data the basic reproduction number r may be estimated using ( . ). this estimate, however, is a retrospective one, which can be derived only after the epidemic has run its course. the maximum number of infectives at any time is the number of infectives when the derivative of i is zero, that is, when s = α/β . this maximum is given by since detailed records were preserved and the community was persuaded to quarantine itself to try to prevent the spread of disease to other communities, the disease in eyam has been used as a case study for modeling [raggett ( ) ]. detailed examination of the data indicates that there were actually two outbreaks, of which the first was relatively mild. thus we shall try to fit the model ( . ) over the period from mid-may to mid-october , measuring time in months with an initial population of infectives and susceptibles, and a final population of . raggett ( ) gives values of susceptibles and infectives in eyam on various dates, beginning with s( ) = , i( ) = , shown in table . . the final size relation with s = , i = , s ∞ = gives β /α = . × − , α/β = . the infective period was days, or . month, so that α = . . then β = . . the relation ( . ) gives an estimate of . for the maximum number of infectives. we use the values obtained here for the parameters β and τ in the model ( . ) for simulations of both the phase plane, here the (s, i)-plane, and for graphs of s and i as functions of t (figures . , . , . ). figure . plots these data points together with the phase portrait given in figure . for the model ( . ). the actual data for the eyam epidemic are remarkably close to the predictions of this very simple model. however, the model is really too good to be true. our model assumes that infection is transmitted directly between people. while this is possible, bubonic plague is transmitted mainly by rat fleas. when an infected rat is bitten by a flea, the flea becomes extremely hungry and bites the host rat repeatedly, spreading the infection in the rat. when the host rat dies, its fleas move on to other rats, spreading the disease further. as the number of available rats decreases, the fleas move to human hosts, and this is how plague starts in a human population (although the second phase of the epidemic may have been the pneumonic form of bubonic plague, which can be spread from person to person). one of the main reasons for the spread of plague from asia into europe was the passage of many trading ships; in medieval times ships were invariably infested with rats. an accurate model of plague transmission would have to include flea and rat populations, as well as movement in space. such a model would be extremely complicated, and its predictions might well not be any closer to observations than our simple unrealistic model. very recent study of the data from eyam suggests that the rat population may not have been large enough to support the epidemic and human to human transmission was also a factor. raggett ( ) also used a stochastic model to fit the data, but the fit was rather poorer than the fit for the simple deterministic model( . ). in the village of eyam the rector persuaded the entire community to quarantine itself to prevent the spread of disease to other communities. one effect of this policy was to increase the infection rate in the village by keeping fleas, rats, and people in close contact with one another, and the mortality rate from bubonic plague was much higher in eyam than in london. further, the quarantine could do nothing to prevent the travel of rats and thus did little to prevent the spread of disease to other communities. one message this suggests to mathematical modelers is that control strategies based on false models may be harmful, and it is essential to distinguish between assumptions that simplify but do not alter the predicted effects substantially, and wrong assumptions that make an important difference. in order to prevent the occurrence of an epidemic if infectives are introduced into a population, it is necessary to reduce the basic reproductive number r below one. this may sometimes be achieved by immunization, which has the effect of transferring members of the population from the susceptible class to the removed class and thus of reducing s( ). if a fraction p of the population is successfully immunized, the effect is to replace s( ) by s( )( − p), and thus to reduce the basic reproductive number to β s( ) a large basic reproductive number means that the fraction that must be immunized to prevent the spread of infection is large. this relation is connected to the idea of herd immunity, which we shall introduce in the next chapter. initially, the number of infectives grows exponentially because the equation for i may be approximated by i = (β n − α)i and the initial growth rate is this initial growth rate r may be determined experimentally when an epidemic begins. then since n and α may be measured, β may be calculated as however, because of incomplete data and underreporting of cases, this estimate may not be very accurate. this inaccuracy is even more pronounced for an outbreak of a previously unknown disease, where early cases are likely to be misdiagnosed. because of the final size relation, estimation of β or r is an important question that has been studied by a variety of approaches. there are serious shortcomings in the simple kermack-mckendrick model as a description of the beginning of a disease outbreak, and a very different kind of model is required. exercises . the same survey of yale students described in example reported that . percent were susceptible to influenza at the beginning of the year and . percent were susceptible at the end of the year. estimate the basic reproductive number β /α and decide whether there was an epidemic. . what fraction of yale students in exercise would have had to be immunized to prevent an epidemic? . what was the maximum number of yale students in exercises and suffering from influenza at any time? . an influenza epidemic was reported at an english boarding school in that spread to of the students. estimate the basic reproductive number β /α. . what fraction of the boarding school students in exercise would have had to be immunized to prevent an epidemic? . what was the maximum number of boarding school students in exercises and suffering from influenza at any time? . a disease is introduced by two visitors into a town with inhabitants. an average infective is in contact with . inhabitants per day. the average duration of the infective period is days, and recovered infectives are immune against reinfection. how many inhabitants would have to be immunized to avoid an epidemic? . consider a disease with β = / , /α = days in a population of members. suppose the disease conferred immunity on recovered infectives. how many members would have to be immunized to avoid an epidemic? . a disease begins to spread in a population of . the infective period has an average duration of days and the average infective is in contact with . persons per day. what is the basic reproductive number? to what level must the average rate of contact be reduced so that the disease will die out? . european fox rabies is estimated to have a transmission coefficient β of km years/fox and an average infective period of days. there is a critical carrying capacity k c measured in foxes per km , such that in regions with fox density less than k c , rabies tends to die out, while in regions with fox density greater than k c , rabies tends to persist. estimate k c . [remark: it has been suggested in great britain that hunting to reduce the density of foxes below the critical carrying capacity would be a way to control the spread of rabies.] . a large english estate has a population of foxes with a density of . foxes/km . a large fox hunt is planned to reduce the fox population enough to prevent an outbreak of rabies. assuming that the contact number β /α is km /fox, find what fraction of the fox population must be caught. . following a complaint from the spca, organizers decide to replace the fox hunt of exercise by a mass inoculation of foxes for rabies. what fraction of the fox population must be inoculated to prevent a rabies outbreak? . what actually occurs on the estate of these exercises is that percent of the foxes are killed and percent are inoculated. is there danger of a rabies outbreak. . here is another approach to the analysis of the sir model ( . ). (i) divide the two equations of the model to give (ii) integrate to find the orbits in the (s, i)-plane, (iv) show that no orbit reaches the i-axis and deduce that s ∞ = lim t→∞ s(t) > , which implies that part of the population escapes infection. the kermack-mckendrick compartmental epidemic model assumes that the sizes of the compartments are large enough that the mixing of members is homogeneous, or at least that there is homogeneous mixing in each subgroup if the population is stratified by activity levels. however, at the beginning of a disease outbreak, there is a very small number of infective individuals, and the transmission of infection is a stochastic event depending on the pattern of contacts between members of the population; a description should take this pattern into account. our approach will be to give a stochastic-branching process description of the beginning of a disease outbreak to be applied as long as the number of infectives remains small, distinguishing a (minor) disease outbreak confined to this stage from a (major) epidemic, which occurs if the number of infectives begins to grow at an exponential rate. once an epidemic has started, we may switch to a deterministic compartmental model, arguing that in a major epidemic, contacts would tend to be more homogeneously distributed. implicitly, we are thinking of an infinite population, and by a major epidemic we mean a situation in which a nonzero fraction of the population is infected, and by a minor outbreak we mean a situation in which the infected population may grow but remains a negligible fraction of the population. there is an important difference between the behavior of branching process models and the behavior of models of kermack-mckendrick type, namely, as we shall see in this section that for a stochastic disease outbreak model if r < , the probability that the infection will die out is , but if r > , there is a positive probability that the infection will increase initially but will produce only a minor outbreak and will die out before triggering a major epidemic. we describe the network of contacts between individuals by a graph with members of the population represented by vertices and with contacts between individuals represented by edges. the study of graphs originated with the abstract theory of erdős and rényi of the s and s [erdős and rényi ( rényi ( , rényi ( , ]. it has become important in many areas of application, including social contacts and computer networks, as well as the spread of communicable diseases. we will think of networks as bidirectional, with disease transmission possible in either direction along an edge. an edge is a contact between vertices that can transmit infection. the number of edges of a graph at a vertex is called the degree of the vertex. the degree distribution of a graph is {p k }, where p k is the fraction of vertices having degree k. the degree distribution is fundamental in the description of the spread of disease. we think of a small number of infectives in a population of susceptibles large enough that in the initial stage, we may neglect the decrease in the size of the susceptible population. our development begins along the lines of that of [diekmann and heesterbeek ( ) ] and then develops along the lines of [callaway, newman, strogatz, and watts ( ), newman ( ), newman, strogatz, and watts ( )]. we assume that the infectives make contacts independently of one another and let p k denote the probability that the number of contacts by a randomly chosen individual is exactly k, with ∑ ∞ k= p k = . in other words, {p k } is the degree distribution of the vertices of the graph corresponding to the population network. for the moment, we assume that every contact leads to an infection, but we will relax this assumption later. it is convenient to define the probability generating function since ∑ ∞ k= p k = , this power series converges for ≤ z ≤ , and may be differentiated term by term. thus it is easy to verify that the generating function has the properties the mean degree, which we denote by k or z , is more generally, we define the moments when a disease is introduced into a network, we think of it as starting at a vertex (patient zero) that transmits infection to every individual to whom this individual is connected, that is, along every edge of the graph from the vertex corresponding to this individual. we may think of this individual as being inside the population, as when a member of a population returns from travel after being infected, or as being outside the population, as when someone visits a population and brings an infection. for transmission of disease after this initial contact we need to use the excess degree of a vertex. if we follow an edge to a vertex, the excess degree of this vertex is one less than the degree. we use the excess degree because infection cannot be transmitted back along the edge whence it came. the probability of reaching a vertex of degree k, or excess degree (k − ), by following a random edge is proportional to k, and thus the probability that a vertex at the end of a random edge has excess degree (k − ) is a constant multiple of kp k with the constant chosen to make the sum over k of the probabilities equal to . then the probability that a vertex has excess degree (k − ) is this leads to a generating function g (z) for the excess degree, and the mean excess degree, which we denote by k e , is we let r = g ( ), the mean excess degree. this is the mean number of secondary cases infected by patient zero and is the basic reproduction number as usually defined; the threshold for an epidemic is determined by r . the quantity k e = g ( ) is sometimes written in the form our next goal is to calculate the probability that the infection will die out and will not develop into a major epidemic, proceeding in two steps. first we find the probability that a secondary infected vertex (a vertex that has been infected by another vertex in the population) will not spark a major epidemic. suppose that the secondary infected vertex has excess degree j. we let z n denote the probability that this infection dies out within the next n generations. for the infection to die out in n generations, each of the j secondary infections coming from the initial secondary infected vertex must die out in (n − ) generations. the probability of this is z n− for each secondary infection, and the probability that all secondary infections will die out in (n − ) generations is z j n− . now z n is the sum over j of these probabilities, weighted by the probability q j of j secondary infections. thus since g (z) is an increasing function, the sequence z n is an increasing sequence and has a limit z ∞ , which is the probability that this infection will die out eventually. then z ∞ is the limit as n → ∞ of the solution of the difference equation thus z ∞ must be an equilibrium of this difference equation, that is, a solution of z = g (z). let w be the smallest positive solution of z = g (z). then, because it follows by induction that from this we deduce that z ∞ = w. the equation g (z) = z has a root z = , since g ( ) = . because the function g (z) − z has a positive second derivative, its derivative g (z) − is increasing and can have at most one zero. this implies that the equation g (z) = z has at most two and the equation g (z) = z has only one root, namely z = . on the other hand, if r > , the function g (z) − z is positive for z = and negative near z = since it is zero at z = , and its derivative is positive for z < and z near . thus in this case the equation g (z) = z has a second root z ∞ < . this root z ∞ is the probability that an infection transmitted along one of the edges at the initial secondary vertex will die out, and this probability is independent of the excess degree of the initial secondary vertex. it is also the probability that an infection originating outside the population, such as an infection brought from outside into the population under study, will die out. next, we calculate the probability that an infection originating at a primary infected vertex, such as an infection introduced by a visitor from outside the population under study, will die out. the probability that the disease outbreak will die out eventually is the sum over k of the probabilities that the initial infection in a vertex of degree k will die out, weighted by the degree distribution {p k } of the original infection, and this is to summarize this analysis, we see that if r < , the probability that the infection will die out is . on the other hand, and there is a probability − g (z ∞ ) > that the infection will persist, and will lead to an epidemic. however, there is a positive probability g (z ∞ ) that the infection will increase initially but will produce only a minor outbreak and will die out before triggering a major epidemic. this distinction between a minor outbreak and a major epidemic, and the result that if r > there may be only a minor outbreak and not a major epidemic, are aspects of stochastic models not reflected in deterministic models. if contacts between members of the population are random, corresponding to the assumption of mass action in the transmission of disease, then the probabilities p k are given by the poisson distribution the commonly observed situation that most infectives do not pass on infection but there are a few "superspreading events" [riley et al. ( ) ] corresponds to a probability distribution that is quite different from a poisson distribution, and could give a quite different probability that an epidemic will occur. for example, if r = . , the assumption of a poisson distribution gives z ∞ = . and g (z ∞ ) = . , so that the probability of an epidemic is . . the assumption that nine out of ten infectives do not transmit infection while the tenth transmits infections gives from which we see that the probability of an epidemic is . . another example, possibly more realistic, is to assume that a fraction ( − p) of the population follows a poisson distribution with constant r, while the remaining fraction p consists of superspreaders each of whom makes l contacts. this would give a generating function for example, if r = . , l = , p = . , numerical simulation gives so that the probability of an epidemic is . . these examples demonstrate that the probability of a major epidemic depends strongly on the nature of the contact network. simulations suggest that for a given value of the basic reproduction number, the poisson distribution is the one with the maximum probability of a major epidemic. it has been observed that in many situations there is a small number of long-range connections in the graph, allowing rapid spread of infection. there is a high degree of clustering (some vertices with many edges), and there are short path lengths. such a situation may arise if a disease is spread to a distant location by an air traveler. this type of network is called a small-world network. long range connections in a network can increase the likelihood of an epidemic dramatically. these examples indicate that the probability of an epidemic depends strongly on the contact network at the beginning of a disease outbreak. we will not explore network models further here, but we point out that this is an actively developing field of science. some basic references are [newman ( ( , strogatz ( ]. contacts do not necessarily transmit infection. for each contact between individuals of whom one has been infected and the other is susceptible, there is a probability that infection will actually be transmitted. this probability depends on such factors as the closeness of the contact, the infectivity of the member who has been infected, and the susceptibility of the susceptible member. we assume that there is a mean probability t , called the transmissibility, of transmission of infection. the transmissibility depends on the rate of contacts, the probability that a contact will transmit infection, the duration time of the infection, and the susceptibility. the development in section . assumed that all contacts transmit infection, that is, that t = . in this section, we will continue to assume that there is a network describing the contacts between members of the population whose degree distribution is given by the generating function g (z), but we will assume in addition that there is a mean transmissibility t . when disease begins in a network, it spreads to some of the vertices of the network. edges that are infected during a disease outbreak are called occupied, and the size of the disease outbreak is the cluster of vertices connected to the initial vertex by a continuous chain of occupied edges. the probability that exactly m infections are transmitted by an infective vertex of degree k is we define Γ (z, t )to be the generating function for the distribution of the number of occupied edges attached to a randomly chosen vertex, which is the same as the distribution of the infections transmitted by a randomly chosen individual for any (fixed) transmissibility t . then in this calculation we have used the binomial theorem to see that note that for secondary infections we need the generating function Γ (z, t ) for the distribution of occupied edges leaving a vertex reached by following a randomly chosen edge. this is obtained from the excess degree distribution in the same way, the basic reproduction number is now the calculation of the probability that the infection will die out and will not develop into a major epidemic follows the same lines as the argument for t = . the result is that if r = t g ( ) < , the probability that the infection will die out is . and a probability −Γ (z ∞ (t ), t ) > that the infection will persist, and will lead to an epidemic. however, there is a positive probability Γ (z ∞ (t ), t ) that the infection will increase initially but will produce only a minor outbreak and will die out before triggering a major epidemic. another interpretation of the basic reproduction number is that there is a critical transmissibility t c defined by in other words, the critical transmissibility is the transmissibility that makes the basic reproduction number equal to . if the mean transmissibility can be decreased below the critical transmissibility, then an epidemic can be prevented. the measures used to try to control an epidemic may include contact interventions, that is, measures affecting the network such as avoidance of public gatherings and rearrangement of the patterns of interaction between caregivers and patients in a hospital, and transmission interventions such as careful hand washing or face masks to decrease the probability that a contact will lead to disease transmission. in each exercise, assume that the transmissibility is . . show that it is not possible for a major epidemic to develop unless at least one member of the contact network has degree at least . . what is the probability of a major epidemic if every member of the contact network has degree . estimate (numerically) the probability of a major epidemic if c = . . . show that the probability generating function for an exponential distribution, given by for what values of α is it possible to normalize this (i.e., choose c to make ∑ p k = ? compartmental models for epidemics are not suitable for describing the beginning of a disease outbreak because they assume that all members of a population are equally likely to make contact with a very small number of infectives. thus, as we have seen in the preceding section, stochastic branching process models are better descriptions of the beginning of an epidemic. they allow the possibility that even if a disease outbreak has a reproduction number greater than , it may be only a minor outbreak and may not develop into a major epidemic. one possible approach to a more realistic description of an epidemic would be to use a branching process model initially and then make a transition to a compartmental model when the epidemic has become established and there are enough infectives that mass action mixing in the population is a reasonable approximation. another approach would be to continue to use a network model throughout the course of the epidemic. in this section we shall indicate how a compartmental approach and a network approach are related. we assume that there is a known static configuration model (cm) network in which the probability that a node u has degree k u is p(k u )). we let g (z) denote the probability generating function of the degree distribution, the per-edge from an infected node is assumed to be β , and it is assumed that infected nodes recover at a rate α. we use an edge-based compartmental model because the probability that a random neighbor is infected is not necessarily the same as the probability that a random individual is infected. we let s(t) denote the fraction of nodes that are susceptible at time t, i(t) the fraction of nodes that are infective at time t, and r(t) the fraction of nodes that are recovered at time t. it is easy to write an equation for r , the rate at which infectives recover. if we know s(t), we can find i(t), because a decrease in s gives a corresponding increase in i. since we need only find the probability that a randomly selected node is susceptible. we assume that the hazard of infection for a susceptible node u is proportional to the degree k u of the node. each contact is represented by an edge of the network joining u to a neighboring node. we let ϕ i denote the probability that this neighbor is infective. then the per-edge hazard of infection is assuming that edges are independent, u's hazard of infection at time t is consider a randomly selected node u and let θ (t) be the probability that a random neighbor has not transmitted infection to u. then the probability that u is susceptible is θ k u . averaging over all nodes, we see that the probability that a random node u is susceptible is ( . ) we break θ into three parts, with ϕ s the probability that a random neighbor v of u is susceptible, ϕ i the probability that a random neighbor v of u is infective but has not transmitted infection to u, and ϕ r the probability that a random neighbor v has recovered without transmitting infection to u. then the probability that v has transmitted infection to u is − θ . since infected neighbors recover at rate α, the flux from ϕ i to ϕ r is αϕ i . thus it is easy to see from this that r = αi. ( . ) since edges from infected neighbors transmit infection at rate β , the flux from to obtain ϕ i we need the flux into and out of the ϕ i compartment. the incoming flux from ϕ s results from infection of the neighbor. the outgoing flux to ϕ r corresponds to recovery of the neighbor without having transmitted infection, and the outgoing flux to ( −θ ) corresponds to transmission without recovery. the total outgoing flux is (α + β )ϕ i . to determine the flux from ϕ s to ϕ i , we need the rate at which a neighbor changes from susceptible to infective. consider a random neighbor v of u; the probability that v has degree k iskp(k)/ k . since there are (k − ) neighbors of v that could have infected v, the probability that v is susceptible is θ k− . averaging over all k, we see that the probability that a random neighbor v of u is susceptible is to calculate ϕ r , we note that the flux from ϕ i to ϕ r and the flux from ϕ i to ( −θ ) are proportional with proportionality constant α/β . since both ϕ r and ( − θ ) start at zero, now, using ( . ), ( . ), ( . ), and we now have a dynamic model consisting of equations ( . ), ( . ), ( . ), and s + i + r = . we wish to show a relationship between this set of equations and the simple kermack-mckendrick compartmental model ( . ). in order to accomplish this, we need only show under what conditions we would have s = −β si. differentiating ( . ) and using ( . ), we obtain consider a large population with n members, each making c ≤ n − contacts, so that we now let c → ∞ (which implies n → ∞) in such a way that we will now show that ϕ i θ ≈ , and this will yield the desired approximation the probability that an edge to a randomly chosen node has not transmitted infection is θ (assuming that the given target node cannot transmit infection), and the probability that in addition it is connected to an infected node is ϕ i . becausê β = βc is constant and therefore bounded as c grows, only a fraction no greater than a constant multiple of i/c of edges to the target node may have transmitted infection from a node that is still infected. for large values of c, ϕ i is approximately i. similarly, θ is approximately as c → ∞. thus ϕ i /θ ≈ i as c → ∞. this gives the desired approximate equation for s. the result remains valid if all degrees are close to the average degree as the average degree grows. the edge-based compartmental modeling approach that we have used can be generalized in several ways. for example, heterogeneity of mixing can be included. in general, one would expect that early infections would be in individuals having more contacts, and thus that an epidemic would develop more rapidly than a mass action compartmental model would predict. when contact duration is significant, as would be the case in sexually transmitted diseases, an individual with a contact would play no further role in disease transmission until a new contact is made, and this can be incorporated in a network model. the network approach to disease modeling is a rapidly developing field of study, and there will undoubtedly be fundamental developments in our understanding of the modeling of disease transmission. in the remainder of this chapter, we assume that we are in an epidemic situation following a disease outbreak that has been modeled initially by a branching process. thus we return to the study of compartmental models. we have established that the simple kermack-mckendrick epidemic model ( . ) has the following basic properties: . there is a basic reproduction number r such that if r < , the disease dies out while if r > , there is an epidemic. . the number of infectives always approaches zero and the number of susceptibles always approaches a positive limit as t → ∞. . there is a relationship between the reproduction number and the final size of the epidemic, which is an equality if there are no disease deaths. in fact, these properties hold for epidemic models with more complicated compartmental structure. we will describe some common epidemic models as examples. in many infectious diseases there is an exposed period after the transmission of infection from susceptibles to potentially infective members but before these potential infectives develop symptoms and can transmit infection. to incorporate an exposed period with mean exposed period /κ, we add an exposed class e and use compartments s, e, i, r and total population size n = s + e + i + r to give a generalization of the epidemic model ( . ) ( . ) a flow chart is shown in figure . . the analysis of this model is the same as the analysis of ( . ), but with i replaced by e + i. that is, instead of using the number of infectives as one of the variables, we use the total number of infected members, whether or not they are capable of transmitting infection. in some diseases there is some infectivity during the exposed period. this may be modeled by assuming infectivity reduced by a factor ε during the exposed period. a calculation of the rate of new infections per susceptible leads to a model we take initial conditions for this model, integration of the sum of the equations of ( . ) from to ∞ gives integration of the third equation of ( . ) gives and division of the first equation of ( . ) by s followed by integration from to ∞ gives in this final size relation there is an initial term β i /α, caused by the assumption that there are individuals infected originally who are beyond the exposed stage in which they would have had some infectivity. in order to obtain a final size relation without such an initial term it is necessary to assume i( ) = , that initial infectives are in the first stage in which they can transmit infection. if i( ) = , the final size relation has the form ( . ). one form of treatment that is possible for some diseases is vaccination to protect against infection before the beginning of an epidemic. for example, this approach is commonly used for protection against annual influenza outbreaks. a simple way to model this would be to reduce the total population size by the fraction of the population protected against infection. in reality, such inoculations are only partly effective, decreasing the rate of infection and also decreasing infectivity if a vaccinated person does become infected. this may be modeled by dividing the population into two groups with different model parameters, which would require some assumptions about the mixing between the two groups. this is not difficult, but we will not explore this direction here. if there is a treatment for infection once a person has been infected, this may be modeled by supposing that a fraction γ per unit time of infectives is selected for treatment, and that treatment reduces infectivity by a fraction δ . suppose that the rate of removal from the treated class is η. this leads to the sitr model, where t is the treatment class, given by a flow chart is shown in figure . . it is not difficult to prove, much as was done for the model ( . ), that in order to calculate the basic reproduction number, we may argue that an infective in a totally susceptible population causes β n new infections in unit time, and the mean time spent in the infective compartment is /(α + γ). in addition, a fraction γ/(α + γ) of infectives are treated. while in the treatment stage the number of new infections caused in unit time is δ β n, and the mean time in the treatment class is /η. thus r is it is also possible to establish the final size relation ( . ) by means very similar to those used for the simple model ( . ). we integrate the first equation of ( . ) to obtain integration of the third equation of ( . ) gives integration of the sum of the first two equations of ( . ) gives combination of these three equations and ( . ) gives ( . ). in some diseases, such as influenza, at the end of a stage individuals may proceed to one of two stages. there is a latent period after which a fraction p of latent individuals l proceeds to an infective stage i, while the remaining fraction ( − p) proceeds to an asymptomatic stage a, with infectivity reduced by a factor δ and a different period /η. a flow chart is shown in figure . . the model ( . ) is an example of a differential infectivity model. in such models, also used in the study of hiv/aids [hyman, li and stanley ( )], individuals enter a specific group when they become infected and stay in that group over the course of the infection. different groups may have different parameter values. for example, for influenza infective and asymptomatic members may have different infectivities and different periods of stay in the respective stages. . exposed members may be infective with infectivity reduced by a factor ε e , ≤ ε e < . . exposed members who are not isolated become infective at rate κ e . . we introduce a class q of quarantined members and a class j of isolated (hospitalized) members, and exposed members are quarantined at a proportional rate γ q in unit time (in practice, a quarantine will also be applied to many susceptibles, but we ignore this in the model). quarantine is not perfect, but it reduces the contact rate by a factor ε q . the effect of this assumption is that some susceptibles make fewer contacts than the model assumes. . infectives are diagnosed at a proportional rate γ j per unit time and isolated. isolation is imperfect, and there may be transmission of disease by isolated members, with an infectivity factor of ε j . . quarantined members are monitored, and when they develop symptoms at rate κ q they are isolated immediately. . infectives leave the infective class at rate α i and isolated members leave the isolated class at rate α j . these assumptions lead to the seqijr model [gumel et al. ( )]: the model before control measures are begun is the special case of ( . ). it is the same as ( . ). a flow chart is shown in figure . . we define the control reproduction number r c to be the number of secondary infections caused by a single infective in a population consisting essentially only of susceptibles with the control measures in place. it is analogous to the basic reproduction number, but instead of describing the very beginning of the disease outbreak it describes the beginning of the recognition of the epidemic. the basic reproduction number is the value of the control reproduction number with we have already calculated r for ( . ), and we may calculate r c in the same way but using the full model with quarantined and isolated classes. we obtain each term of r c has an epidemiological interpretation. the mean duration in e is /d with contact rate ε e β , giving a contribution to r c of ε e β n/d . a fraction κ e /d goes from e to i, with contact rate β and mean duration /d , giving a contribution of β nκ e /d d . a fraction γ q /d goes from e to q, with contact rate ε e ε q β and mean duration /κ q , giving a contribution of ε e ε q β nγ q /d κ q . a fraction κ e γ j /d d goes from e to i to j, with a contact rate of ε j β and a mean duration of /α j , giving a contribution of ε j β nκ e γ j /α j d d . finally, a fraction γ q /d goes from e to q to j with a contact rate of ε j β and a mean duration of /α j , giving a contribution of ε j β nγ q /d α j . the sum of these individual contributions gives r c . in the model ( . ) the parameters γ q and γ j are control parameters, which may be chosen in the attempt to manage the epidemic. the parameters ε q and ε j depend on the strictness of the quarantine and isolation processes and are thus also control measures in a sense. the other parameters of the model are specific to the disease being studied. while they are not variable, their measurements are subject to experimental error. the linearization of ( . ) at the disease-free equilibrium (n, , , , ) has matrix ⎡ the corresponding characteristic equation is a fourth-degree polynomial equation whose leading coefficient is and whose constant term is a positive constant multiple of − r c , thus positive if r c < and negative if r c > . if r c > , there is a positive eigenvalue, corresponding to an initial exponential growth rate of solutions of ( . ). if r c < , it is possible to show that all eigenvalues of the coefficient matrix have negative real part, and thus solutions of ( . ) die out exponentially [van den driessche and watmough ( )]. in order to show that analogues of the relation ( . ) and s ∞ > derived for the model ( . ) are valid for the management model ( . ), we begin by integrating the equations for s + e, q, i, j, of ( . ) with respect to t from t = to t = ∞, using the initial conditions we continue by integrating the equation for s, and then an argument similar to the one used for ( . ) but technically more complicated may be used to show that s ∞ > for the treatment model ( . ) and also to establish the final size relation thus the asymptotic behavior of the management model ( . ) is the same as that of the simpler model ( . ). in the various compartmental models that we have studied, there are significant common features. this suggests that compartmental models can be put into a more general framework. in fact, this general framework is the age of infection epidemic model originally introduced by kermack and mckendrick in . however, we will not explore this generalization here. these models represent an sir epidemic model and an seir epidemic model respectively with a mean infective period of days and a mean exposed period of days. do numerical simulations to decide whether the exposed period affects the behavior of the model noticeably. use the parameter values if you feel really ambitious, formulate and analyze an seir model with infectivity in the exposed period and treatment. . consider an sir model in which a fraction θ of infectives is isolated in a perfectly quarantined class q with standard incidence (meaning that individuals make a contacts in unit time of which a fraction i/(n − q) are infective), given by the system . isolation/quarantine is a complicated process because we don't live in a perfect world. in hospitals, patients may inadvertently or deliberately break from isolation and in the process have casual contacts with others including medical personnel and visitors. taking this into account, we are led to the model (i) determine all the parameters in the system and define each parameter. (ii) show that the population is constant. (iii) find all equilibria. (iv) find the reproductive number r . (v) describe the asymptotic behavior of the model, including its dependence on the basic reproduction number. . formulate a model analogous to ( . ) for which treatment is not started immediately, but begins at time τ > . can you say anything about the dependence of the reproduction number on τ? in the simple model ( . ) studied in section . we have assumed that the infective period is exponentially distributed. now let us consider an sir epidemic model in a population of constant size n with mass action incidence in which p(τ) is the fraction of individuals who are still infective a time τ after having become infected. the model is ( . ) here, i (t) is the number of individuals who were infective initially at t = who are still infective at time t. then because if all initial infectives were newly infected we would have equality in this relation, and if some initial infectives had been infected before the starting time t = , they would recover earlier. we assume that p(τ) is a nonnegative, nonincreasing function with p( ) = . we assume also that the mean infective period ∞ p(τ)dτ is finite. since a single infective causes β n new infections in unit time and ∞ p(τ)dτ is the mean infective period, it is easy to calculate since s is a nonnegative decreasing function, it follows as for ( . ) that s(t) deceases to a limit s ∞ as t → ∞, but we must proceed differently to show that i(t) → . this will follow if we can prove that t i(s) ds is bounded as t → ∞. we have since ∞ p(τ)dτ is assumed to be finite, it follows that t i(s) ds is bounded, and thence that i(t) → . now integration of the first equation in ( . ) from to ∞ gives and this shows that s ∞ > . if all initially infected individuals are newly infected, so that i (t) = (n −s )p(t), integration of the second equation of ( . ) gives and this is the final size relation, identical to ( . ). if there are individuals who were infected before time t = , a positive term the general epidemic model described by kermack and mckendrick ( ) included a dependence of infectivity on the time since becoming infected (age of infection). we let s(t) denote the number of susceptibles at time t and let ϕ(t) be the total infectivity at time t, defined as the sum of products of the number of infected members with each infection age and the mean infectivity for that infection age. we assume that on average, members of the population make a constant number a of contacts in unit time. we let b(τ) be the fraction of infected members remaining infected at infection age τ and let π(τ) with ≤ π(τ) ≤ be the mean infectivity at infection age τ. then we let the mean infectivity of members of the population with infection age τ. we assume that there are no disease deaths, so that the total population size is a constant n. the basic reproduction number is integration with respect to t from to ∞ gives here, ϕ (t) is the total infectivity of the initial infectives when they reach age of infection t. if all initial infectives have infection age zero at t = , then ϕ then ( . ) takes the form and this is the general final size relation. if there are initial infectives with infection age greater than zero, let u(τ) be the fraction of these individuals with infection age τ, ∞ u(τ)dτ = . at time t these individuals have infection age t + τ and mean infectivity a(t + τ). thus thus, the initial term satisfies the final size relation is sometimes presented in the form example . the seir model ( . ) can be viewed as an age of infection model with ϕ = εe + i. to use the age of infection interpretation, we need to determine the kernel a(τ) in order to calculate its integral. we let u(τ) be the fraction of infected members with infection age τ who are not yet infective and v(τ) the fraction of infected members who are infective. then the rate at which members become infective at infection age τ is κu(τ), and we have the solution of this system is and it is easy to calculate this gives the same value for r as was calculated directly. the age of infection model also includes the possibility of disease stages with distributions that are not exponential [feng ( ), feng, xu, and zhao ( )]. example . consider an seir model in which the exposed stage has an exponential distribution but the infective stage has a period distribution given by a function p, ( . ) with initial conditions if we define u(τ), v(τ) as in example , we again obtain u(τ) = e −κτ , and v satisfies for period distributions that are not exponential, it is possible to calculate ∞ a(τ)dτ without having to calculate the function a(τ) explicitly. example . consider an seir model in which the exposed period has a distribution given by a function q and the infective period has a distribution given by a function p. then in order to obtain an equation for i, we differentiate the equation for e, obtaining thus the input to i at time t is and the first term in this expression may be written as i (t), and the second term may be simplified, using interchange of the order of integration in the iterated integral, to yield we obtain then the model is which is in age of infection form with ϕ = i, and we have an explicit expression for a(τ). . interpret the models ( . ), ( . ), and ( . ) introduced earlier as age of infection models and use this interpretation to calculate their reproduction numbers. . calculate the basic reproduction number for the model ( . ) but with infectivity in the exposed class having a reduction factor ε. the assumption in the model ( . ) of a rate of contacts per infective that is proportional to population size n, called mass action incidence or bilinear incidence, was used in all the early epidemic models. however, it is quite unrealistic, except possibly in the early stages of an epidemic in a population of moderate size. it is more realistic to assume a contact rate that is a nonincreasing function of total population size. for example, a situation in which the number of contacts per infective in unit time is constant, called standard incidence, is a more accurate description for sexu-ally transmitted diseases. if there are no disease deaths, so that the total population size remains constant, such a distinction is unnecessary. we generalize the model ( . ) by dropping assumption (iv) and replacing assumption (i) by the assumption that an average member of the population makes c(n) contacts in unit time with c (n) ≥ [castillo-chavez, cooke, huang, and levin ( a), dietz ( )], and we define it is reasonable to assume β (n) ≤ to express the idea of saturation in the number of contacts. then mass action incidence because the total population size is now present in the model, we must include an equation for total population size in the model. this forces us to make a distinction between members of the population who die of the disease and members of the population who recover with immunity against reinfection. we assume that a fraction f of the αi members leaving the infective class at time t recover and the remaining fraction ( − f ) die of disease. we use s, i, and n as variables, with n = s + i + r. we now obtain a three-dimensional model ( . ) since n is now a decreasing function, we define n( ) = n = s + i . we also have the equation r = − f αi, but we need not include it in the model, since r is determined when s, i, and n are known. we should note that if f = , the total population size remains equal to the constant n, and the model ( . ) reduces to the simpler model ( . ) with β replaced by the constant β (n ). we wish to show that the model ( . ) has the same qualitative behavior as the model ( . ), namely that there is a basic reproduction number that distinguishes between disappearance of the disease and an epidemic outbreak, and that some members of the population are left untouched when the epidemic passes. these two properties are the central features of all epidemic models. for the model ( . ) the basic reproduction number is given by because a single infective introduced into a wholly susceptible population makes c(n ) = n β (n ) contacts in unit time, all of which are with susceptibles and thus produce new infections, and the mean infective period is /α. we assume that β ( ) is finite, thus ruling out standard incidence (standard incidence does not appear to be realistic if the total population n approaches zero, and it would be more natural to assume that c(n) grows linearly with n for small n). if we let t → ∞ in the sum of the first two equations of ( . ), we obtain the first equation of ( . ) may be written as − s (t) s(t) = β (n(t))i(t). since we now obtain a final size inequality if the disease death rate is small, the final size inequality is an approximate equality. it is not difficult to show that n(t) ≥ f n , and then a similar calculation using the inequality β (n) ≤ β ( f n ) < ∞ shows that from which we may deduce that s ∞ > . . for the model ( . ) show that the final total population size is given by to cope with annual seasonal influenza epidemics there is a program of vaccination before the "flu" season begins. each year, a vaccine is produced aimed at protecting against the three influenza strains considered most dangerous for the coming season. we formulate a model to add vaccination to the simple sir model ( . ) under the assumption that vaccination reduces susceptibility (the probability of infection if a contact with an infected member of the population is made). we consider a population of total size n and assume that a fraction γ of this population is vaccinated prior to a disease outbreak. thus we have a subpopulation of size n u = ( − γ)n of unvaccinated members and a subpopulation of size n v = γn of vaccinated members. we assume that vaccinated members have susceptibility to infection reduced by a factor σ , ≤ σ ≤ , with σ = describing a perfectly effective vaccine and σ = describing a vaccine that has no effect. we assume also that vaccinated individuals who are infected have infectivity reduced by a factor δ and may also have a recovery rate α v that is different from the recovery rate of infected unvaccinated individuals α u . we let s u , s v , i u , i v denote the number of unvaccinated susceptibles, the number of vaccinated susceptibles, the number of unvaccinated infectives, and the number of vaccianted infectives respectively. the resulting model is the initial conditions prescribe s u ( ), s v ( ), i u ( ), i v ( ), with since the infection now is beginning in a population that is not fully susceptible, we speak of the control reproduction number r c rather than the basic reproduction number. however, as we will soon see, calculation of the control reproduction number will require a more general definition and a considerable amount of technical computation. the computation method is applicable to both basic and control reproduction numbers. we will use the term reproduction number to denote either a basic reproduction number or a control reproduction number. we are able to obtain final size relations without knowledge of the reproduction number, but these final size relations do contain information about the reproduction number, and more. since s u and s v are decreasing nonnegative functions they have limits s u (∞) and s v (∞) respectively as t → ∞. the sum of the equations for s u and i u in ( . ) is from which we conclude, just as in the analysis of ( . ), that i u (t) → as t → ∞, and that α similarly, using the sum of the equations for s v and i v , we see that integration of the equation for s u in ( . ) and use of (??) gives a similar calculation using the equation for s v gives this pair of equations ( . ), ( . ) are the final size relations. they make it possible to calculate s u (∞), s v (∞) if the parameters of the model are known. it is convenient to define the matrix then the final size relations ( . ), ( . ) may be written the matrix k is closely related to the reproduction number. in the next section we describe a general method for calculating reproduction numbers that will involve this matrix. . suppose we want to model the spread of influenza in a city using an sliar model (susceptible-latent-infectious-asympotmatic-recovered, respectively). then our system of equation would be where β is the transmission coefficient, δ is the reduced transmissibility factor from asymptomatic contacts, κ is the rate of disease progression from the latent class, p is the proportion of individuals that are clinically diagnosed, η is the recovery rate from the asymptomatic class, γ is the recovery rate from the infectious (clinically diagnosed) class, and n is the total population size. (i) add a vaccination class to the model. assume that the vaccine imparts partial protection until it becomes fully effective. is the population of the new system constant? are there any endemic equilibria? (ii) vary the vaccination rate from . to . and determine how the number of infected individuals changes compared with the model without vaccination. does vaccination prevent the outbreak? up to this point, we have calculated reproduction numbers by following the secondary cases caused by a single infective introduced into a population. however, if there are subpopulations with different susceptibilities to infection, as in the vaccination model introduced in section . , it is necessary to follow the secondary infections in the subpopulations separately, and this approach will not yield the re-production number. it is necessary to give a more general approach to the meaning of the reproduction number, and this is done through the next generation matrix [diekmann and heesterbeek ( ) , diekmann, heesterbeek, and metz ( ), van den driessche and watmough ( )]. the underlying idea is that we must calculate the matrix whose (i, j) entry is the number of secondary infections caused in compartment i by an infected individual in compartment j. the procedure applies both to epidemic models, as studied in this chapter, and to models with demographics for endemic diseases, to be studied in the next chapter. in a compartmental disease transmission model we sort individuals into compartments based on a single, discrete state variable. a compartment is called a disease compartment if the individuals therein are infected. note that this use of the term disease is broader than the clinical definition and includes stages of infection such as exposed stages in which infected individuals are not necessarily infective. suppose there are n disease compartments and m nondisease compartments, and let x ∈ r n and y ∈ r m be the subpopulations in each of these compartments. further, we denote by f i the rate at which secondary infections increase the i − th disease compartment and by v i the rate at which disease progression, death, and recovery decrease the i − th compartment. the compartmental model can then be written in the form note that the decomposition of the dynamics into f and v and the designation of compartments as infected or uninfected may not be unique; different decompositions correspond to different epidemiological interpretations of the model. the definitions of f and v used here differ slightly from those in [van den driessche and watmough ( )]. the derivation of the basic reproduction number is based on the linearization of the ode model about a disease-free equilibrium. for an epidemic model with a line of equilibria, it is customary to use the equilibrium with all members of the population susceptible. we assume: • f i ( , y) = and v i ( , y) = for all y ≥ and i = ,..., n. • the disease-free system y = g( , y) has a unique equilibrium that is asymptotically stable, that is, all solutions with initial conditions of the form ( , y) approach a point ( , y o ) as t → ∞. we refer to this point as the disease-free equilibrium. the first assumption says that all new infections are secondary infections arising from infected hosts; there is no immigration of individuals into the disease compartments. it ensures that the disease-free set, which consists of all points of the form ( , y), is invariant. that is, any solution with no infected individuals at some point in time will be free of infection for all time. the second assumption ensures that the disease-free equilibrium is also an equilibrium of the full system. the uniqueness of the disease-free equilibrium in the second assumption is required for models with demographics, to be studied in the next chapter. although it is not satisfied in epidemic models, the specification of a specific disease-free equilibrium with all memebers of the population susceptible is sufficient to validate the results. next, we assume: • f i (x, y) ≥ for all nonnegative x and y and i = ,..., n. y) ≥ for all nonnegative x and y. the reasons for these assumptions are that the function f represents new infections and cannot be negative, each component v i represents a net outflow from compartment i and must be negative (inflow only) whenever the compartment is empty, and the sum ∑ n i= v i (x, y) represents the total outflow from all infected compartments. terms in the model leading to increases in ∑ n i= x i are assumed to represent secondary infections and therefore belong in f . suppose that a single infected person is introduced into a population originally free of disease. the initial ability of the disease to spread through the population is determined by an examination of the linearization of ( . ) about the disease-free equilibrium ( , y ). it is easy to see that the assumption for every pair (i, j). this implies that the linearized equations for the disease compartments x are decoupled from the remaining equations and can be written as where f and v are the n × n matrices with entries because of the assumption that the disease-free system y = g( , y) has a unique asymptotically stable equilibrium, the linear stability of the system ( . ) is completely determined by the linear stability of the matrix (f −v ) in ( . ). the number of secondary infections produced by a single infected individual can be expressed as the product of the expected duration of the infectious period and the rate at which secondary infections occur. for the general model with n disease compartments, these are computed for each compartment for a hypothetical index case. the expected time the index case spends in each compartment is given by is the solution of ( . ) with f = (no secondary infections) and nonnegative initial conditions x representing an infected index case: in effect, this solution shows the path of the index case through the disease compartments from the initial exposure through to death or recovery with the i − th component of ϕ(t, x ) interpreted as the probability that the index case (introduced at time t = ) is in disease state i at time t. the solution of ( . ) is φ (t, x ) = e −v t x , where the exponential of a matrix is defined by the taylor series this series converges for all t (see, for example, [hirsch and smale ( ) ]. thus ∞ ϕ(t, x ) dt = v − x , and the (i, j) entry of the matrix v − can be interpreted as the expected time an individual initially introduced into disease compartment j spends in disease compartment i. the (i, j) entry of the matrix f is the rate at which secondary infections are produced in compartment i by an index case in compartment j. hence, the expected number of secondary infections produced by the index case is given by following diekmann and heesterbeek ( ), the matrix k = fv − is referred to as the next generation matrix for the system at the disease-free equilibrium. the (i, j) entry of k is the expected number of secondary infections in compartment i produced by individuals initially in compartment j, assuming, of course, that the environment experienced by the individual remains homogeneous for the duration of its infection. shortly, we will describe some results from matrix theory that imply that the matrix k l = fv − , called the next generation matrix with small domain, is nonnegative and therefore has a nonnegative eigenvalue, r = ρ(fv − ), such that there are no other eigenvalues of k with modulus greater than r and there is a nonnegative eigenvector ω associated with r [berman and plemmons ( ), theorem . . ]. this eigenvector is in a sense the distribution of infected individuals that produces the greatest number r of secondary infections per generation. thus, r and the associated eigenvector ω suitably define a "typical" infective, and the basic reproduction number can be rigorously defined as the spectral radius of the matrix k l . the spectral radius of a matrix k l , denoted by ρ(k l ), is the maximum of the moduli of the eigenvalues of k l . if k l is irreducible, then r is a simple eigenvalue of k l and is strictly larger in modulus than all other eigenvalues of k l . however, if k l is reducible, which is often the case for diseases with multiple strains, then k l may have several positive real eigenvectors corresponding to reproduction numbers for each competing strain of the disease. we have interpreted the reproduction number for a disease as the number of secondary infections produced by an infected individual in a population of susceptible individuals. if the reproduction number r = ρ(fv − ) is consistent with the differential equation model, then it should follow that the disease-free equilibrium is asymptotically stable if r < and unstable if r > . this is shown through a sequence of lemmas. the spectral bound (or abscissa) of a matrix a is the maximum real part of all eigenvalues of a. if each entry of a matrix t is nonnegative, we write t ≥ and refer to t as a nonnegative matrix. a matrix of the form a = si − b, with b ≥ , is said to have the z sign pattern. these are matrices whose off-diagonal entries are negative or zero. if in addition, s ≥ ρ(b), then a is called an m-matrix. note that in this section, i denotes an identity matrix, not a population of infectious individuals. the following lemma is a standard result from [berman and plemmons ( )]. lemma . . if a has the z sign pattern, then a − ≥ if and only if a is a nonsingular m-matrix. the assumptions we have made imply that each entry of f is nonnegative and that the off-diagonal entries of v are negative or zero. thus v has the z sign pattern. also, the column sums of v are positive or zero, which, together with the z sign pattern, implies that v is a (possibly singular) m-matrix [berman and plemmons ( ), condition m of theorem . . ]. in what follows, it is assumed that v is nonsingular. in this case, v − ≥ , by lemma . . hence, k l = fv − is also nonnegative. theorem . . consider the disease transmission model given by ( . ). the diseasefree equilibrium of ( . ) is locally asymptotically stable if r < , but unstable if r > . proof. let f and v be as defined as above, and let j and j be the matrices of partial derivatives of g with respect to x and y evaluated at the disease-free equilibrium. the jacobian matrix for the linearization of the system about the disease-free equilibrium has the block structure the disease-free equilibrium is locally asymptotically stable if the eigenvalues of the jacobian matrix all have negative real parts. since the eigenvalues of j are those of (f −v ) and j , and the latter all have negative real parts by assumption, the diseasefree equilibrium is locally asymptotically stable if all eigenvalues of (f − v ) have negative real parts. by the assumptions on f and v , f is nonnegative and v is a nonsingular m-matrix. hence, by lemma all eigenvalues of (f −v ) have negative real parts if and only if ρ(fv − ) < . it follows that the disease-free equilibrium is locally asymptotically stable if r = ρ(fv − ) < . instability for r > can be established by a continuity argument. if r ≤ , then for any ε > , (( + ε)i − fv − ) is a nonsingular m-matrix and by lemma . , (( + ε)i − fv − ) − ≥ . by lemma . , all eigenvalues of (( + ε)v − f) have positive real parts. since ε > is arbitrary, and eigenvalues are continuous functions of the entries of the matrix, it follows that all eigenvalues of (v − f) have nonnegative real parts. to reverse the argument, suppose all the eigenvalues of (v − f) have nonnegative real parts. for any positive ε, (v + εi − f) is a nonsingular m-matrix, and by lemma . , ρ(f(v + εi) − ) < . again, since ε > is arbitrary, it follows that ρ(fv − ) ≤ . thus, (f −v ) has at least one eigenvalue with positive real part if and only if ρ(fv − ) > , and the disease-free equilibrium is unstable whenever r > . these results validate the extension of the definition of the reproduction number to more general situations. in the vaccination model ( . ) of the previous section we calculated a pair of final size relations that contained the elements of a matrix k. this matrix is precisely the next generation matrix with large domain k l = fv − that has been introduced in this section. example . consider the seir model with infectivity in the exposed stage, ( . ) here the disease states are e and i, then we may calculate since fv − has rank , it has only one nonzero eigenvalue, and since the trace of the matrix is equal to the sum of the eigenvalues, it is easy to see that the element in the first row and first column of fv − . if all new infections are in a single compartment, as is the case here, the basic reproduction number is the trace of the matrix fv − . in general, it is possible to reduce the size of the next generation matrix to the number of states at infection [diekmann and heesterbeek ( )]. the states at infection are those disease states in which there can be new infections. suppose that there are n disease states and k states at infection with k < n. then we may define an auxiliary n×k matrix e in which each column corresponds to a state at infection and has in the corresponding row and elsewhere. then the next generation matrix is it is easy to show, using the fact that ee t k l = k l , that the n × n matrix k l and the k × k matrix k have the same nonzero eigenvalues and therefore the same spectral radius. construction of the next generation matrix that has lower dimension than the next generation matrix with large domain may simplify the calculation of the basic reproduction number. in example above, the only disease state at infection is e, the matrix a is , and the next generation matrix k is the × matrix example . consider the vaccination model ( . ). the disease states are i u and i v . then it is easy to see that the next generation matrix with large domain is the matrix k calculated in section . . since each disease state is a disease state at infection, the next generation matrix is k, the same as the next generation matrix with large domain. as in example , the determinant of k is zero and k has rank . thus the control reproduction number is the trace of k, example . (a multi-strain model of gonorrhea) the following example comes from lima and torres ( ). the system of equations is where s is the susceptible class, i is the class infected with strain , and i is the class of individuals infected with a mutated strain. the "birth" rate of the population is ρ, μ is the natural mortality rate, c is the probability of successful contact, λ i is the of strain i, γ i is the recovery rate of strain i, and p is the proportion of the original infected population that become infected by the mutated strain. the disease-free equilibrium for this model is [s = n, i = , i = ]. next we will reorder our variables: di dt di dt t and note that we need only the infected classes to calculate r . then the new infection terms are cλ s(t)i (t) in the di dt equation and in the di dt equation, pγ i (t) enter the i class, but only after they have been infected with strain . then and v = (μ + γ )i (t) (μ + γ )i (t) − pγ i (t) . since we have only two infected classes, n = , and our jacobian matrices are then we can calculate the inverse of v, to calculate the spectral radius of fv − we find the eigenvalues of the matrix: we often call r = cλ μ+γ the reproductive number for strain and r = cλ μ+γ the reproductive number for strain . then the basic reproductive number is where s i refers to the susceptible class of species i, and i i refers to the infected class of species i for i = m, b, h, mosquitoes, birds, and humans, respectively. then p i is the mosquito biting preference for species i, μ i is the natural mortality rate of species i, b is the number of bites per mosquito per unit time, θ is the human recovery rate, β m is the transmission probability from mosquito to host per bite, β b is the transmission probability from birds to mosquito, and β h is the transmission probability from humans to mosquito. from which we calculate the eigenvalues and determine that the spectral radius is we have described the next generation matrix method for continuous models. there is an analogous theory for discrete systems, described in [allen and van den driessche ( )]. there are some situations in which r < in which it is possible to show that the asymptotic stability of the disease -free equilibrium is global, that is, all solutions approach the disease -free equilibrium, not only those with initial values sufficiently close to this equilibrium. we will say that a vector is nonnegative if each of its components is nonnegative, and that a matrix is nonnegative if each of its entries is non -negative. we rewrite the system ( . ) as ( . ) y j = g j (x, y) , j = ,..., m. if r < , we have shown that the disease -free equilibrium is asymptotically stable, and that −a = −(f −v ) is a non -singular m -matrix. theorem . (castillo-chavez, feng, and huang ( )). if −a is a nonsingular m-matrix andf ≥ , if the assumptions on the model ( . ) made earlier in this section are satisfied, and if r < , then the disease-free equilibrium of ( . ) is globally asymptotically stable. proof. the variation of constants formula for the first equation of ( . ) gives there are examples to show that the disease-free equilibrium may not be globally asymptotically stable if the conditionf ≥ is not satisfied. exercises . use the next generation approach to calculate the basic reproduction number for the model ( . ) but with infectivity in the exposed class having a reduction factor ε. . formulate an seitr model and calculate its reproduction number. . for each of the examples in this section determine whether the disease-free equilibrium is globally asymptotically stable when r < . a fundamental assumption in the model ( . ) is homogeneous mixing, that all individuals are equivalent in contacts. a more realistic approach would include separation of the population into subgroups with differences in behavior. for example, in many childhood diseases the contacts that transmit infection depend on the ages of the individuals, and a model should include a description of the rate of contact between individuals of different ages. other heterogeneities that may be important include activity levels of different groups and spatial distribution of populations. network models may be formulated to include heterogeneity of mixing, or more complicated compartmental models can be developed. an important question that should be kept in mind in the formulation of epidemic models is the extent to which the fundamental properties of the simple model ( . ) carry over to more elaborate models. an epidemic model for a disease in which recovery from infection brings only temporary immunity cannot be described by the models of this chapter because of the flow of new susceptibles into the population. this effectively includes demographics in the model, and such models will be described in the next chapter. many of the important underlying ideas of mathematical epidemiology arose in the study of malaria begun by sir r.a. ross ( ). malaria is one example of a disease with vector transmission, the infection being transmitted back and forth between vectors (mosquitoes) and hosts (humans). other vector diseases include west nile virus and hiv with heterosexual transmission. vector transmitted diseases require models that include both vectors and hosts. an actual epidemic differs considerably from the idealized models ( . ) and ( . ). some notable differences are these: . when it is realized that an epidemic has begun, individuals are likely to modify their behavior by avoiding crowds to reduce their contacts and by being more careful about hygiene to reduce the risk that a contact will produce infection. . if a vaccine is available for the disease that has broken out, public health measures will include vaccination of part of the population. various vaccination strategies are possible, including vaccination of health care workers and other first line responders to the epidemic, vaccination of members of the population who have been in contact with diagnosed infectives, and vaccination of members of the population who live in close proximity to diagnosed infectives. . isolation may be imperfect; in-hospital transmission of infection was a major problem in the sars epidemic. . in the sars epidemic of - , in-hospital transmission of disease from patients to health care workers or visitors because of imperfect isolation accounted for many of the cases. this points to an essential heterogeneity in disease transmission that must be included whenever there is any risk of such transmission. the discrete analogue of the continuous-time epidemic model ( . ) is s j+ = s j g j , where s j and i j denote the numbers of susceptible and infective individuals at time j, respectively, g j is the probability that a susceptible individual at time j will remain susceptible to time j + , and σ = e −α is the probability that an infected individual at time j will remain infected to time j + . assume that the initial conditions are s( ) = s > , i( ) = i > , and s + i = n. exercise . consider the system ( . ). (a) show that the sequence {s j + i j } has a limit s ∞ + i ∞ = lim j→∞ (s j + i j ). with r = β −σ . next, consider the case that there are k infected stages and there is treatment in some stages, with treatment rates that can be different in different stages. assume that selection of members for treatment occurs only at the beginning of a stage. let i (i) j and t (i) j denote the numbers of infected and treated individuals respectively in stage i (i = , ,..., k) at time j. let σ i i denote the probability that an infected individual in the i (i) stage continues on to the next stage, either treated or untreated, and let σ t i denote the probability that an individual in the t (i) stage continues on to the next treated stage. in addition, of the members leaving an infected stage i (i) , a fraction p i enters treatment in t (i+ ) , while the remaining fraction q i continues to i (i+ ) . let m i denote the fraction of infected members who go through the stage i (i) , and n i the fraction of infected members who go through the stage t (i) . then, ..., m k = q q · · ·q k , n = p , n = p + q p , ..., n k = p + q p + . . . + q q · · ·q k− p k . the discrete system with treatment is s j+ = s j g j , [i = ,..., k, j ≥ ], with where ε i is the relative infectivity of untreated individuals at stage i and δ i is the relative infectivity of treated individuals at stage i. consider the initial conditions exercise . consider the system ( . ). show that hint. equation ( . ) can be proved by showing the following equalities first: . project: fitting data for an influenza model consider an sir model ( . ) with basic reproduction number . . ), and ( . ) use the next generation approach to calculate their reproduction numbers describe the qualitative changes in (s, i, r) as a function of time for different values of β and α with β ∈ { discuss the result of part (a) in terms of the basic reproductive number (what is β /γ?). use a specific disease such as influenza to provide simple interpretations for the different time courses of the disease for the different choices of β and γ , , . }, and for each value of r , choose the best pair of values (β , α) that fits the slope before the first peak in the data found in the table for reported h n influenza cases in méxico below reluctant" means the class of mbts that come into the door as new hires without a disposition to learn new stuff from ℜ , discuss what would be the impact of changing parameters q, γ, and δ what are your conclusions from this model? cases assume that n(t) = r(t) + p(t) + m(t) +u(t) + i(t) and that the total number of mbts is constant, that is, n(t) = k μ for all t, where k is a constant. the model iswhere q, β , δ , μ, γ, and α are constants and ≤ q ≤ . . interpret the parameters. key: cord- - pyjhpbk authors: pilania, rakesh kumar; singh, surjit title: kawasaki disease date: - - journal: periodic and non-periodic fevers doi: . / - - - - _ sha: doc_id: cord_uid: pyjhpbk kawasaki disease (kd) is the commonest cause of acquired heart disease in children in the developed world and is increasingly being reported from developing countries. kd has a predilection for the coronary arteries. etiology of this disorder is remains an enigma. diagnosis of kd is essentially clinical with the help of set of clinical criteria. incomplete kd is said to occur when these criteria are not fulfilled. however, incomplete kd should not be considered as a milder form of the disease. d-echocardiography remains the imaging modality of choice for evaluation and monitoring of cardiac complications but often needs to be supplemented by ct coronary angiography. intravenous immunoglobulin along with aspirin is the gold standard therapy of treatment for kd. however, there is no consensus on treatment of resistant forms of kd. patients with kd should be on long-term follow-up especially if they have developed coronary artery abnormalities during the acute stage. kd is a complex disorder in which an infectious/environmental agent is believed to trigger the onset of disease in a genetically susceptible host [ ] . various susceptibility genes have been identified to have association with kd. these includes inositol , , -trisphosphate -kinase c (itpkc), caspase- calcium release-activated calcium modulator (orai ), and cd [ ] [ ] [ ] . knowledge about these susceptibility genes may provide new insights in etiopathogenesis of kd. reports have suggested association of transforming growth factor (tgf)-β variants with development of caas in patients with kd [ ] . although etiopathogenesis of kd is not clear, various epidemiological and laboratory studies have shown that an infectious agent triggers a cascade that causes the illness ( fig. . ). histopathologic studies have emanated from children with kd who have died from unrelated causes [ ] . the vasculitic process shows three distinct phases [ ] : (a) acute necrotizing arteritis phase: in which there is neutrophilic infiltration of intima and media. (b) subacute/chronic arteritis phase: in which the neutrophilic infiltrate gets replaced by lymphocytes, plasma cells, and macrophages. (c) luminal myofibroblastic phase: in which there is proliferation of smooth muscle cells in the media that can result in luminal narrowing [ , ] . japan, korea, taiwan: incidence rates of kd- , . , . / , children below . incidence of kd is still increasing in these countries europe, north america, australia, new zealand: incidence rates of kd-vary from - / , children below . incidence of kd in these regions has pleatued china, india: nationwide data are not available in these countries. city specific incidence rates that have been reported: sichuan (china) . ; shenghai (china) . ; beijing (china) . ; hong kong sar and chandigarh (india) . / , children below . latin america: chile has a reported incidence of . / , children below . putative genes associated with pathogenesis of kd the etiopathogenesis of kd is closely linked to an infectious process. evidence for this comes from both seasonability and clustering of cases of kd [ , ] . the presence of febrile exanthemata and cervical lymphadenopathy, uncommon occurrence in babies below months, and the rarity of cases in adults, further lends support to this hypothesis. some of the infectious agents that have been linked to the etiology of kd are parvovirus, epstein-barr virus, staphylococcus aureus, chlamydia, and mycobacteria. it has been hypothesized that the putative infectious trigger sets up a cytokine storm that manifests as kd [ ] . some investigators have also proposed a super-antigen theory that triggers an immune response against vascular endothelium [ ] . diagnosis of kd is essentially clinical. criteria for diagnosis of kd have been updated from time to time. currently there are two sets of guidelines-american heart association (aha) guidelines ( [ ] and [ ] ) and kawasaki disease research committee guidelines (japanese guidelines), (table . ) [ ] . aha guidelines (clinical criteria) for diagnosis of kd are given in table . . patients who fulfil the criteria are classified as having complete kd (also known as classical or typical kd), while those who do not fulfil criteria are classified as incomplete kd [ ] . principal clinical features in kd are reviewed in table . . the clinical course of kd has three distinct phases: (a) acute febrile phase, (b) subacute phase, and (c) convalescent phase [ ] . however in clinical practice, these features often overlap. acute phase: this phase starts with abrupt onset of high grade fever that is characteristically accompanied by significant irritability. it usually lasts for - days. fever with marked irritability may be the initial clinical presentation of kd, especially in young infants [ ] . presence of intermittent or remittent fever is not characteristic of kd. cough can be present in a small subset of patients but nasal catarrh is [ ] five of the following six criteria (at least five items of - should be satisfied for diagnosis of kd. however, patients with items of the principal symptoms can be diagnosed with kd when coronary aneurysm or dilatation is recognized by -d echocardiography or coronary angiography) . fever persisting ≥ days (inclusive of cases in whom the fever has subsided before the fifth day in response to therapy) . bilateral conjunctival congestion . changes of lips and oral cavity . polymorphous exanthema . changes of peripheral extremities . acute non-purulent cervical lymphadenopathy table . aha diagnostic criteria for kd [ ] diagnosis of classic kd can be proffered in the presence of fever for at least days associated with at least of the following principal clinical features. in the presence of ≥ principal clinical features, particularly when redness and swelling of the hands and feet are present, the diagnosis of kd can be made on day of fever also. principal clinical features: . changes in lips and oral cavity: erythema, lips cracking, strawberry tongue, diffuse injection of oral and pharyngeal mucosae . changes in extremities acute: erythema of palms, soles; edema of hands, feet subacute: periungual peeling of fingers and toes in weeks and . polymorphous exanthema (diffuse maculopapular, urticarial, erythroderma, erythemamultiforme like, not vesicular or bullous) . bilateral bulbar conjunctival injection without exudates . cervical lymphadenopathy (> . cm diameter), usually unilateral a careful history may reveal that ≥ principal clinical features were present during the illness but resolved by the time of presentation exclusion of other diseases with similar findings (e.g., scarlet fever, viral infections like measles, adenovirus, enterovirus, stevens-johnson syndrome, toxic shock syndrome, drug hypersensitivity reactions, systemic juvenile idiopathic arthritis) unusual for kd. rash in kd is usually generalized, erythematous, polymorphic ( fig. . b ), but is never vesicular or bullous. cervical lymphadenopathy (≥ . cm size) is usually unilateral and is often more commonly seen in the anterior cervical triangle. cervical lymphadenopathy may be mistaken for suppurative lymphadenitis. [ ] clinical manifestation characteristics fever is typically high grade, acute onset, unremitting, without any response to antimicrobials and can associated with extreme irritability. extremity changes (acute) in acute phase include erythema of palms and soles that is usually associated with edema over dorsum of hands and feet. diagnosis of kd can be made on day of illness if one of the features is redness and dorsal edema of extremities. extremity changes (subacute) periungual sheet like peeling of skin is a pathognomonic sign of kd that usually appears in the second to third week of illness. diffuse erythematous polymorphous rash usually appears in the first few days of illness and may be seen in > % patients. bullous, vesicular and petechial lesions are not seen in kd. bilateral nonexudative conjunctival injection with characteristic sparing of limbus is an important and specific clinical sign and often helps in making a clinical diagnosis. it may be seen in > % patients. changes in lips and oral cavity erythema of lips and oral cavity with vertical lip cracking in a febrile child is an important diagnostic clue and is seen in > % patients. in kd, there is enlargement of multiple lymph nodes along with retropharyngeal edema while bacterial lymphadenitis is mostly associated with a single lymph node with a central hypoechoic region [ ] . however, such differentiation on basis of ultrasound examination requires lot of expertise and that may not be readily available in the usual clinical setting. oral cavity and lip changes include redness of lips with bleeding and vertical cracking, oral mucosa redness and a strawberry tongue ( fig. . d ). oral ulcers are distinctly unusual in kd. conjunctival injection is characteristically nonexudative and with typical sparing of limbus ( fig. . a). conjunctivitis with discharge is a strong pointer towards an alternative diagnosis. edema of the dorsum of extremities is an early sign and is usually transient (fig. . e). perianal desquamation is virtually pathognomonic of kd and is a useful clinical sign for diagnosis of the disease during the acute phase ( fig. . c). arthritis in kd is typically oligoarthritis, involving large joints and resolves without sequelae [ ] . erythema at bcg injection site is an important clinical sign during acute stage of kd and more common in infants [ ] . hydrops of gall bladder is also an important finding during acute stage. mild pericardial effusion is a common finding on dechocardiography ( de). caas are usually not seen during the first week of illness. subacute phase: this stage usually lasts for another - weeks during which fever usually subsides. periungual peeling is characteristically seen during this stage ( fig. . f). irritability that is prominent during acute phase subsides completely in this phase. arthritis in kd can also develop in subacute phase. caas most commonly become apparent during this time. an important laboratory manifestation that is seen in the subacute phase is development of thrombocytosis and this in conjunction with periungual desquamation is very suggestive of kd. chronic phase: it lasts for few weeks to months; there are no symptoms during this phase and the inflammation tends to subside. beau's lines presents as horizontal ridging over nails and first manifest at the subsidence of the subacute phase ( fig. . g). this is the only clinical sign of kd that can be seen for several weeks. measles can present with similar clinical features; however, the presence of a viral prodrome, exudative conjunctivitis and koplik spots will help to differentiate measles from kd. in measles, lymphocytic leucocytosis is usually prominent (unless there is secondary infection) while children with kd have polymorphonuclear leucocytosis. procalcitonin levels are normal in measles but may be elevated in kd. other infectious causes that can mimic kd include viral infections (cytomegalovirus, adenovirus, epstein barr virus, and enterovirus), bacterial infections (bacterial cervical lymphadenitis, scarlet fever, toxic shock syndrome, staphylococcal scalded skin syndrome, and leptospira), and systemic juvenile idiopathic arthritis. absence of eye changes and lip changes, presence of sand paper rash, elevated antistreptolysin o titres, and a brisk response to antimicrobials are pointers towards scarlet fever. presence of exudative conjunctivitis, ulcerative lesions in oral cavity, exudative pharyngitis, generalized lymphadenopathy and significant running nose are certain clinical features that make the diagnosis of kd less likely [ ] . the diagnosis of kd is challenging and requires experience. clinical features of kd are nonspecific and overlap with various common childhood disorders especially infections [ ] . patients who are not fulfilling clinical criteria completely are labelled as incomplete kd. in these situations, one has to rely largely on clinical assessment supplemented by laboratory parameters. incomplete form of kd is more commonly seen in infants (especially in babies below months). approach for diagnosis of incomplete kd has been simplified in new aha guidelines [ ] . incomplete kd should, by no means, be considered a milder form of kd. a patient can be said to have atypical kd if the clinical manifestations are unusual. these atypical manifestations may include arthritis [ ] , nephritis [ ] , pneumonia [ ] , myositis, central nervous system involvement, uveitis [ ] , and retinal vasculitis [ ] . common neurological findings include extreme irritability and aseptic meningitis. rare neurological manifestations include transient peripheral facial nerve palsy (mostly unilateral) and profound sensorineural hearing loss. vomiting, diarrhea, and transient hepatitis are common gastrointestinal manifestations [ ] . other relatively uncommon findings include jaundice, gall bladder hydrops, and pancreatitis. genitourinary findings include urethritis which usually presents as sterile pyuria [ ] . atypical presentations of kd can pose difficult clinical problems for the attending physician. in infancy, and especially in babies below months, kd is often a diagnostic challenge because it may present with incomplete manifestations. kd in this age group may often remain undiagnosed for several days, leading to increased incidence of development of caas. this group of patients also has higher intravenous immunoglobulin (ivig) resistance [ , , , ] . sterile pyuria is another clinical presentation of kd in this age group and may mistakenly get treated as a urinary tract infection. the consequent delay in diagnosis can result in serious clinical sequelae [ ] . due to these reasons, kd in infants has been given special consideration in new aha guidelines. it has been highlighted that if an infant has fever for more than days without explanation, kd should always be in list of differential diagnosis. according to aha guidelines kd should be considered as a clinical possibility in the following situations: • babies < months who present with fever and extensive irritability. • infants having prolonged fever and unexplained aseptic meningitis. • children presenting with longer duration of fever and -culture-negative hypotensive shock. -cervical adenopathy that is unresponsive to antibiotic therapy. -parapharyngeal or retropharyngeal phlegmon that is unresponsive to antibiotic therapy. kd is uncommon in older children and may often go unrecognized. due to missed or delayed diagnosis, there is higher risk of caas in this age group. it may be difficult to assess caas by de in this age group of patients because of limited acoustic window and thick chest wall [ , ] . myocarditis in kd is said to be very common and may even be universal. it is not often recognized and can, at times, be severe and symptomatic. myocarditis can develop during acute stage and manifest with unexplained tachycardia, hemodynamic compromise, or cardiovascular collapse [ ] . approximately % patients with kd can present with cardiovascular collapse. this entity is known as kd shock syndrome (kdss). the shock in these patients is multifactorial and may have both cardiogenic and distributive components. the distributive shock may result from cytokine storm leading to uncontrolled inflammation [ ] . patients with kdss are usually present to emergency room and intensive care units with shock and may be inappropriately treated for bacterial sepsis and presumed septic shock. diagnosis of kd gets often delayed in these cases and can have devastating consequences. differentiation between kdss and septic shock in the early phase of clinical diagnosis is often challenging. however, de should always be performed in patients with fever and shock as presence of caas will suggest kdss. differentiation of kdss and septic shock is critical as management of both diseases is entirely different. presence of conjunctival injection, dorsal edema, perineal desquamation, incomplete response to antimicrobials, and no microbiological evidence for infection are indicators towards kd in these patients. these patients are reported to have increased risk of having ivig resistance, caas, and myocardial dysfunction [ , , ] . it is said that kdss should always be considered in children presenting with fever, cardiovascular collapse and myocardial dysfunction. kdss has been discussed at length in the aha revised guidelines because of these highlighted facts [ ] . infections have been commonly considered as triggers for kd. at the bedside, it is often difficult to differentiate the clinical features of kd from that of viral exanthemata. however, if a child is having typical clinical features of the kd, the diagnosis cannot be excluded even in the presence of a documented infection. adenovirus, coronavirus, dengue virus, enteroviruses, measles virus, respiratory syncytial virus have been reported to trigger the kd in children [ ] . toxin-mediated diseases (e.g., staphylococcal and streptococcal infection) have also been also closely associated with the pathogenesis of kd [ ] . candida infection has been linked to the causation of kd in mice models and as well as in epidemiological studies carried out on tropospheric wind patterns in japan and hawaii [ ] . the acute phase is characterized by a mild normocytic normochromic anemia, polymorphonuclear leucocytosis, raised erythrocyte sedimentation rate (esr), c-reactive protein (crp), and usually a normal platelet count. serum procalcitonin, that is usually thought to be a sensitive and specific marker for bacterial infection, may get elevated in kd as well. thrombocytosis develops during the end of second week or in the third week; however it may even develop earlier [ ] . presence of thrombocytopenia during acute stage suggests either macrophage activation syndrome or thrombotic microangiopathy-this is associated with poor prognosis [ ] . progressive thrombocytosis has been correlated with development of caas. urine may show sterile pyuria, secondary to urethritis and easily mistaken as urinary tract infection especially during infancy, thereby resulting in delay in diagnosis of kd [ ] . it is said that kd is the commonest cause of sterile pyuria in children. diagnosis of kd is essentially clinical and there is no gold standard for confirmation of diagnosis. it is no surprise, therefore, that several biomarkers have been extensively evaluated for their role in diagnosis of this condition. various cytokines (e.g., tumour necrosis factor-α (tnf-α), interleukin ) have been found to be raised during the acute phase of kd and decrease promptly following ivig administration. in patients with refractory kd or caas levels of tnf-α continue to remain elevated [ ] . various microarray based studies have been carried out to identify the genes associated with kd. expression of these genes may be used as a novel diagnostic and prognostic biomarker for kd [ ] . n terminal pro-b-type natriuretic peptide (nt-probnp) is a cardiac biomarker that has recently been found to be raised in children with kd during the acute phase. age-based nomograms for pro-bnp are available. these are helpful in differentiation of kd from other febrile illnesses [ ] . the values for pro-bnp are comparatively higher in patients who develop caas as compared to patients with normal coronaries [ ] . probnp levels also correlate with myocardial dysfunction [ ] . cardiac evaluation is an essential component in patients with kd. de is an important tool for coronary artery assessment and, evaluation of cardiac structures during acute phase as well as on follow-up. however, diagnosis of kd should never be ruled out on basis of normal de examination. the quality of scan obtained by de is operator dependent [ ] . criteria for definition of caas have been given by aha as well as by the japanese ministry of health. according to the latter coronary involvement in kd is categorized on basis of absolute internal diameter of coronary artery ( [ , ] . it is mandatory to use body surface area-adjusted 'z' scores for during this period may be mistakenly interpreted as having excluded coronary involvement. . while normative data are available only for proximal segment of coronary arteries, there is paucity of literature on data for middle and distal segments of coronary arteries. isolated involvement of distal segments of coronary arteries is uncommon but has been described [ ] . . it has been suggested that an aneurysm may be better defined as a dilatation that is . times or more than an adjacent segment. this circumvents the problems associated with z scores. . detailed and better evaluation of coronary arteries requires multiple transducer positions, imaging in multiple planes and high frequency transducers. coronary artery diameters refer to the maximal internal luminal diameter. measurements should not be taken at branching points. . limitations of de include difficulty in visualization of distal coronaries, frequent non-visualization of left circumflex coronary artery, difficulty in commenting on stenosis or thrombosis and limited field of vision in older children because of thick chest walls. another problem is due to artifacts especially while scanning the right coronary artery or left circumflex coronary artery. . several nomograms on z scores are available and there may be variations in the measurements. body surface area calculations have to be carried out meticulously and there is no consensus on the ideal method. it cannot be overemphasized that a trivial difference in measurement of weight and height (especially in infants and young children) can significantly impact the calculations of z scores. . there are no normative data for "z" scores for left circumflex coronary artery. although de has hitherto been considered the imaging modality of choice for coronary artery evaluation, ctca is now increasingly being performed for better delineations of coronary arteries. ctca is a useful modality for better characterization and delineation of coronary arteries dilatations, ectasia and aneurysms especially in the mid-and distal segments. it also provides precise details of aneurysm size, morphology, and thrombus. in the last decade, due to advancements in ct technology and the development of dual-source ct scanners (dsct), it is now possible to obtain high resolution motion-free images at acceptable radiation dose. in the convalescent phase, ctca can be used for delineation of complications such as intra-aneurysmal thrombus, segmental stenosis, and mural calcifications [ ] . main aim of therapy in kd is halting of acute phase inflammation and arterial damage. management includes use of ivig, aspirin with or without anticoagulant therapy. ivig is the standard of care for the patients with kd. it should ideally be administered in first days of illness. in the acute stage, ivig ( g/kg) is given over - h as a single infusion along with oral aspirin [ , ] . if diagnosis is made after days, ivig should still be given if fever is persistent, inflammatory parameters are raised or if caas are present. administration of ivig during acute stage has reduced the rate of coronary artery complications to less than %. approximately - % patients with kd can have recurrences [ ] . aspirin has anti-inflammatory and antiplatelet activity depending on the dose being used. it remains an essential component of management in kd. however, effect of aspirin on development of caas is inconclusive. during the acute phase of illness, aspirin should be administered at high ( - mg/kg/day) or moderate ( - mg/kg/day) doses - hourly [ , ] . anti-inflammatory dose of aspirin is usually continued until - h after the patient becomes afebrile. after discontinuation of anti-inflammatory dose of aspirin, low-dose aspirin ( - mg/kg/day) is started and continued for - weeks. at this time if there are no caas on de, aspirin can be discontinued. for children who develop caas, aspirin may need to be given indefinitely [ ] . complications related to caas during the acute phase of kd include thrombotic occlusion of a coronary artery aneurysm and rarely coronary artery rupture leading to sudden cardiac death. coronary artery thrombosis in patients with kd is contributed by acute inflammation, high platelet counts, endothelial dysfunction and stasis of blood flow due to abnormal dilatation. in patients with caas assessment with de is mandatory to monitor size of aneurysm and presence of thrombus. patients with large coronary artery aneurysms are at high risk of myocardial infarction especially in the first year after illness. they continue to be at increased life-time risk of developing coronary artery events [ ] . risk stratification of coronary artery aneurysm should take into account both current and maximal z score. along with a z score classification the shape, location, number of aneurysms and coronary artery wall abnormalities should also be considered for prognostication and risk stratification [ ] . in patients with small aneurysms, low-dose aspirin is adequate for thromboprophylaxis. however, treating physicians can consider addition of another antiplatelet agent (e.g., clopidogrel) in patients with moderate size aneurysms. patients having large or giant aneurysms are at very high risk for coronary artery complications including thrombosis and rupture. these patients should be treated with a combination of antiplatelet and therapeutic anticoagulation therapy. this includes low-dose aspirin along with either low-molecular-weight heparin (lmwh) or oral warfarin. lmwh should be given as mg/kg/dose subcutaneously every h. lmwh may be preferred to warfarin in acute phase because of added effects of anti-inflammatory and remodeling action. transition from lmwh to oral warfarin may be considered in situations where the aneurysm has stopped progressing [ ] . majority of patients will respond to first dose of ivig with rapid defervescence of fever and improvement in general well-being. however, approximately - % of patients with kd may go on to develop recrudescent or persistent fever. children who have axillary temperature ≥ . °c at h after completion of therapy are said to have resistant or refractory kd [ ] . there are multiple factors that have been associated with development of ivig resistance. these include severity of disease and host genetic factors (e.g., polymorphisms in the fcγ receptors). patients having refractory kd are at increased risk of developing caas. various scoring systems have been developed in japan to predict ivig resistance, but the validity of these scores in other countries remains a contentious issue. there are no clear guidelines on management of patients with refractory kd [ ] . the aha guidelines recommended use of a repeat dose of ivig ( g/kg) in these patients. alternatively, the guidelines emphasize the role of intravenous pulse methylprednisolone ( mg/kg/day, three doses) with tapering oral prednisolone. administration of tapering course of prednisolone for - weeks with second dose of ivig ( g/kg) may also be considered in the retreatment of refractory kd patients. infliximab (tnf-α blocker), given as a single dose ( - mg/kg) intravenously, is also an important choice in treatment of refractory kd and appears to decrease the chances of developing caas [ ] . use of infliximab is associated with prompt reduction of fever. at our centre we prefer to use infliximab as second line therapy [ ] . cyclosporine may also be considered as an option in these circumstances. in highly refractory cases, plasma exchange, cytotoxic agents, or other biologicals have been used [ ] . there are a few recent reports of successful use of the il- receptor antagonist, anakinra, for the treatment of highly refractory kd [ ] . long-term follow-up and management of patients with kd is based on risk stratification. children with large caas may need lifelong follow-up. long-term management includes counselling about a healthy lifestyle with balanced diet and exercise to avoid obesity and hypertension. because these patients have already developed a cardiac risk factor (even if there are no obvious caas during acute stage), a second risk factor like obesity, hyperlipidemia, or hypertension must be avoided [ ] . uses of statins have also been advocated in these situations. patients having large and giant aneurysms should undergo frequent de examinations for monitoring of size and associated complications. these patients may also require ctca at periodic (every - years) intervals [ ] . children with missed and untreated kd can have significant clinical consequences later in life. even though coronary arteries with remodeled aneurysms may become anatomically normal, they continue to remain functionally abnormal. an increasing number of young adults are presenting to cardiologists with myocardial ischemia and infarction-it is not often realized that these could well be late manifestations of kd that has been missed in childhood. these consequences of kd in adulthood are grossly under recognized [ ] . there is very little awareness amongst adult cardiologists regarding cardiovascular sequelae of kd and their management. clearly there is a need for an improved evidence base to guide the care and management of adults with coronary artery damage following kd in childhood. whenever a young adult is presenting with myocardial ischemia and there is marked ectasia or proximal coronary artery involvement, possibility of untreated kd in past should be entertained [ ] . the aha has published an update on criteria for diagnosis of kd in . while the presence of fever remains an essential criterion and there is no change in the five principal clinical manifestations of the disease, the scientific committee has suggested several amendments. the new guidelines emphasize that some clinical features of kd may subside by the time the patient reaches a clinician. in such situations the clinical findings that have disappeared can also be taken into account for fulfilment of criteria. similarly, the issue of diagnosis in young infants has been addressed at length. the guidelines mention that any infant with unexplained fever lasting for a week or more should be evaluated for underlying kd. this cannot be overemphasized. in the guidelines, the terms incomplete kd and atypical kd have been used interchangeably. however, these are distinct clinical entities and this fact needs to be kept in mind while evaluating a patient. desquamating groin rash has been included as one of the other clinical findings. it is known that erythema or desquamation of perineal/perianal region occurs earlier than classic periungual peeling and thus may facilitate early consideration of the diagnosis. the aha guidelines emphasize on the use of z score for assessment of coronary artery size. however, the difficulties involved in calculation of an accurate 'z' score that is reproducible across centres have not been detailed at length. and aha guidelines [ , , ] . in the japanese criteria fever is not taken as an essential prerequisite for diagnosis. in other words, a diagnosis of kd can be offered even in absence of fever while using the japanese criteria. . definition of coronary artery lesion in japanese ministry of health guidelines is based on absolute dimensions, while aha guidelines have classified the caas on basis of "z" scores (table . ). . refractory kd has been defined as persistence of fever more than h despite treatment as per aha guidelines. however, the cut-off for duration of fever is h in japanese guidelines. diagnosis of kawasaki disease diagnosis, treatment, and long-term management of kawasaki disease: a statement for health professionals from the committee on rheumatic fever, endocarditis and kawasaki disease, council on cardiovascular disease in the young acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children epidemiology of kawasaki disease in asia, europe, and the united states kawasaki disease: epidemiology and the lessons from it the epidemiology of kawasaki disease: a global update epidemiological observations of kawasaki disease in japan cumulative incidence of kawasaki disease in japan epidemiology and clinical features of kawasaki disease in south korea the global epidemiology of kawasaki disease: review and future perspectives epidemiologic features of kawasaki disease in shanghai from epidemiologic survey of kawasaki disease in jilin from through epidemiological and clinical characteristics of kawasaki disease and factors associated with coronary artery abnormalities in east china: nine years experience epidemiologic study on kawasaki disease in beijing from is kawasaki disease incidence rising in chandigarh descriptive epidemiology of kawasaki disease in japan genetics of kawasaki disease: what we know and don't know the genetics of kawasaki disease dissecting kawasaki disease: a state-of-the-art review variations in orai gene associated with kawasaki disease transforming growth factor-beta signaling pathway in patients with kawasaki disease is kawasaki disease an infectious disorder? three linked vasculopathic processes characterize kawasaki disease: a light and transmission electron microscopic study histopathological aspects of cardiovascular lesions in kawasaki disease spatial and temporal clustering of kawasaki syndrome cases the role of superantigens of group a streptococcus and staphylococcus aureus in kawasaki disease diagnosis, treatment, and long-term management of kawasaki disease: a scientific statement for health professionals from the american heart association revision of diagnostic guidelines for kawasaki disease (the th revised edition) kawasaki disease in infants below months: a clinical conundrum arthritis presenting during the acute phase of kawasaki disease bcg site reactivation in kawasaki disease controversies in diagnosis and management of kawasaki disease acute kidney injury in patients with kawasaki disease pulmonary presentation of kawasaki disease-a diagnostic challenge acute anterior uveitis as the presenting feature of kawasaki disease kawasaki disease as a cause of encephalitis kawasaki disease and the pediatric gastroenterologist: a diagnostic challenge pyuria in patients with kawasaki disease high risk of coronary artery aneurysms in infants younger than months of age with kawasaki disease mortality in children with kawasaki disease: years of experience from a tertiary care centre in north india kawasaki disease in older children and adolescents clinical assessment of coronary arteries in kawasaki disease: focus on echocardiographic assessment not just coronary arteritis, kawasaki disease is a myocarditis, too myocarditis and kawasaki disease early differentiation of kawasaki disease shock syndrome and toxic shock syndrome in a pediatric intensive care unit kawasaki disease in a pediatric intensive care unit: a case-control study tropospheric winds from northeastern china carry the etiologic agent of kawasaki disease from its source to japan kawasaki disease in india: increasing awareness or increased incidence? biomarkers for diagnosis of kawasaki disease screening of differentially expressed genes associated with kawasaki disease by microarray analysis pro-brain natriuretic peptide (probnp) levels in north indian children with kawasaki disease usefulness of natriuretic peptide for the diagnosis of kawasaki disease: a systematic review and meta-analysis guidelines for diagnosis and management of cardiovascular sequelae in kawasaki disease (jcs ). digest version improved classification of coronary artery abnormalities based only on coronary artery z-scores after kawasaki disease computed tomography coronary angiography for evaluation of children with kawasaki disease computed tomography coronary angiography for evaluation of children with kawasaki disease recurrent kawasaki disease: usa and japan aspirin dose and prevention of coronary abnormalities in kawasaki disease what dose of aspirin should be used in the initial treatment of kawasaki disease? the role of echocardiography in kawasaki disease adjunctive therapies in kawasaki disease infliximab is the new kid on the block in kawasaki disease: a single-centre study over years from north india the use of interleukin receptor antagonist (anakinra) in kawasaki disease: a retrospective cases series presentation of missed childhood kawasaki disease in adults: experience from a tertiary care center in north india management of sequelae of kawasaki disease in adults key: cord- -budyph u authors: bonnaud, laure; fortané, nicolas title: serge morand and muriel figuié (eds), , emergence de maladies infectieuses. risques et enjeux de société (the emergence of infectious diseases. societal risks and stakes): paris, quae, p date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: budyph u nan international organisations (world health organization (who), office international des epizooties or world organization for animal health (oie), food and agriculture organization of the united nations (fao)) play a leading role in coordinating the exchange of surveillance information and in facilitating the implementation of tools to fight disease. twenty-five years after stephen morse's seminal work which helped put emerging infectious diseases onto the political and scientific agenda (morse ), contemporary debates were very much influenced by an article published in nature in (jones et al. ). jones and his colleagues presented three major results: the regular and significant increase in the number of infectious diseases that had emerged since the s, the role played by viruses and bacteria (with parasites playing a secondary role), and finally, the animal origin of this phenomenon. in order to understand what might seem to be an inevitable biological evolution, the book takes a multidisciplinary approach: whilst the origin of these infections is definitely animal, it is human societies, through their lifestyles, which are responsible for their propagation-as s. morand immediately points out in the first chapter devoted to a presentation of definitions, the state of scientific debate, and the main available epidemiological data. the following three chapters set out to deconstruct the apparent evidence of international mobilisation against these emerging diseases. claude gilbert and nathalie bender show how the influenza pandemic constituted an interesting opportunity for who, an international organisation in crisis since the s. at the beginning of the s, who decided to reposition itself as a reference centre for biomedical expertise. its management of the sars epidemic, through its network of experts and its alert and response system, enabled it to gradually recover an authority that was both moral and technical. in the case of influenza, national experts-virologists in particular-also became lobbyists for this public issue, linking it to other problems (by presenting it as a model for preparations against bioterrorism) and suggesting solutions, such as mass vaccination. in the french case, the authors analyse the mechanism for countering h n flu and look at different ways of shaping the public problem, depending on whether the pandemic is deemed to be a public health issue, a stake of collective security, or a global problem affecting contemporary societies. these different appropriations nevertheless coexist without really impacting one another. they do not lead to any major redefinition of solutions, and mass vaccination remains the solution that health authorities recommend. in a chapter devoted to the cbrn risks (chemical, biological, radiological, and nuclear) in the usa, patrick zylberman retraces the origin of scenario planning, used by public authorities to prepare against threats. he shows that since the end of the cold war, health issues have been taken on board by national security actors whose tools and concepts have gradually taken over. among the norms, plans, and procedures that constitute the tools with which to prepare for biological, chemical, radiological, and pandemic threats, scenario planning takes pride of place. it is a case of preparing bin order to dissuade^, with the idea of a terrorist enemy being implicit. scenario planning allows one to set probabilities aside and thus bring public authorities into a worst-case logic. p. zylberman is sceptical about the fictional aspect they contain, which serves just as much the political and ideological motives of domestic policy as truly helping institutions to prepare against threats. muriel figuié takes a critical state-of-the-art look at the implementation of international public action in relation to animal health and more particularly at epidemiological surveillance and vaccination. she notes recurring observations by international experts of the difficulties involved in coordinating actors and discusses the literature that attempts to explain these issues. two types of explanations are generally put forward: on the one hand, those which relate to individual determinants and on the other, those which focus on cultural aspects. in the first group, we find cognitive and psychological factors, along with those relating to individual economic calculation. the second group concerns everything relating to inherited beliefs and know-how which prevent any proper understanding of the solutions suggested by experts from international institutions. m. figuié confronts these analyses with a reflection on conditions for collective action, talking for example about her works on the informal epidemiological surveillance networks which were developed in vietnam when avian influenza emerged. she reveals the many ambiguities surrounding the mechanisms proposed by southern countries and the controversies and conflicts between actors who are supposed to work together to protect animal health, now regarded as a global public good. we are thus moving away from the vision of a coherent project that local actors cannot or do not want to support, in favour of putting more nuance into the definition of what constitutes the implementation of global health policy instruments. in vietnam, poultry culling is a tool aiming at preventing the spread of the flu as well as a mean to modernise agricultural system by fostering the development of agri-food industrial actors. this viewpoint is examined in greater depth in the final chapter by françois roger, who focuses on the north/south inequalities that are embedded in plans to monitor and fight infectious diseases. all of these chapters form a coherent book which offers a very solid introduction to the examination of public action against emerging infectious diseases. they open the road to new research, be it disciplinary or interdisciplinary, which might throw further light on certain aspects currently left in the shadows; they will also be of use to experts and decision-makers. the analysis of this new global public health model is therefore a convincing one, even though its circulation and eventual local variants remain unknown. the priority given to international institutions might therefore be bolstered by an analysis of regional evolutions which at the very least would appear to have laid the ground for this new model: the european union's animal health strategy was thus perfectly adapted to a reclassification of animal diseases-but for internal budgetary reasons rather than to fight emerging diseases! (ollivier ) . the book also opens the way for in-depth studies of the roles played by the market and by private actors. it very rapidly sets out the foundations, the pharmaceutical laboratories, and the agri-food industry which are vital stakeholders in the fight against infectious diseases-or in their emergence (such as the development and spreading of antimicrobial-resistant bacteria, for example!). their role could in itself be a research object. finally, epidemiological surveillance networks aside, the book pays little attention to the implementation of other devices or policies to fight against emerging infectious diseases. in the northern countries, changes to healthcare systems, the medical and paramedical professions, the ngo relating to public health, and more broadly, migratory policies might also throw light on how certain infectious diseases are managed (aids is one well-documented example: gelly et al. ) . similarly, countries in the southern countries use a variety of devices to fight infectious diseases-such as triage centres or clinical trials set up as part of the fight against the ebola virus in africa (lakoff et al. )-which clearly demonstrate the social, political, technical, and biological complexity surrounding contemporary emergences. the next step towards understanding emerging diseases might therefore be to compare this new global health model with existing devices, in both the northern and southern countries. it nevertheless remains that this book, both through the concepts and policy instruments it presents and the perspectives it offers, marks an important step forward in the analysis of emerging infectious diseases. how did international agencies perceive the avian influenza problem? the adoption and manufacture of the bone world la fin du sida ? global trends in emerging infectious diseases ebola's ecologies, limn quand les vétérinaires et les animaux font l'europe: l'action publique européenne en santé animale key: cord- -spteznv authors: heikkilä, jaakko; niemi, jarkko k.; heinola, katriina; liski, eero; myyrä, sami title: anything left for animal disease insurance? a choice experiment approach date: - - journal: nan doi: . /s - - - sha: doc_id: cord_uid: spteznv animal disease insurance plays only a minor role in public activities related to animal diseases in animal production in europe, and the current situation is likely to persist as long as producers place strong faith on public compensation schemes. in this study, we undertook a farm survey in finland employing a choice experiment to study the willingness to pay for animal disease insurance products. we found that producers’ willingness to pay for animal disease insurance is relatively low, even if consequential losses are covered. however, attributes of the insurance products which increased the likelihood of the producer wishing to purchase the product in a statistically significant manner were identified. the most important attribute was a low deductible. using latent class analysis, four classes of producers were identified, those who were ( ) not interested, ( ) weakly interested or ( ) strongly interested in insurance, and additionally, ( ) a group who emphasised biosecurity measures but was not willing to purchase insurance. those primarily interested in insurance were typically young, well-educated producers from large farms, and they already had a good level of biosecurity on their farms. however, the majority of the respondents preferred not to purchase insurance. the analysis suggests that commercial production animal disease insurance may need to be subsidised or otherwise made more attractive to producers, and even so, many producers might consider it unnecessary. animal disease outbreaks can be costly to the producers as well as the society in general. the european union (eu) cofinances activities of the member states on selected zoonotic or otherwise societally important animal diseases. eradication, control and monitoring are co-financed if they are in accordance with the rules laid out in council decision / /eec. based on these regulations, the eu and the member states have taken an important role in covering costs of animal disease expenses. however, in recent years, there have been increasing calls, both in the eu as well as in individual member states, for cost sharing of publicly funded animal disease expenses. one potential alternative for cost sharing is animal disease insurance. various animal disease finance schemes, including insurances and funds, have been recently reviewed (european commission ; heikkilä and niemi ; oecd ). there appears to be considerable variation in compensation and finance schemes for animal disease losses around the world as the schemes are often tailored to meet specific needs (oecd ) . in the eu, insurance products in general seem to cover either fairly low (< %) or fairly high (> %) proportion of farms. high coverage has often been achieved either through compulsory nature of the insurance or through group insurance (heikkilä and niemi ) . in addition, other policy instruments such as legal facilitation, taxation and institutional arrangements are used to support the schemes (oecd ). the price of insurance products might be high due to underwriting losses that emerge because disease risk exposure is heterogeneous across farms. producers, who have better knowledge of their farm disease risks than the insurer, may use this knowledge to their private benefit (just et al. ) . besides adverse selection, moral hazard can also play an important role in animal disease insurance. in practice, if there is a risk of adverse selection and moral hazard, animal disease insurance requires producers to co-finance the losses (gramig et al. ), for instance through a deductible or restricted maximum compensation. insurance companies may also need to invest in verifying what measures their customers take to reduce the risk. in finland, the target country of our case study, many contagious diseases which cause problems in pig and poultry production elsewhere are either absent or have a low incidence. in addition, highly contagious notifiable animal diseases listed by the world organisation for animal health are not present in finland (finnish food safety authority evira ). all parts of the food chain are committed to the policy of preventive biosecurity. for instance, the occurrence of salmonella in pigs and poultry in finland is usually limited to few farms, if any, being infected each year. most producers have opted for a salmonella group insurance via their egg packaging company or slaughterhouse. according to our survey, about two thirds of the pig and poultry farms indicated that they have salmonella group insurance, and some % had additional animal disease insurance. unique social security, which is mandatory to all producers in finland (http://www.mela.fi/en/ about-mela), covers small minimum income in case of sickness or injury. at the time of data collection, producers also had basic cover for their yield losses through crop damage compensation scheme (myyrä and pietola ) . furthermore, almost all the farms in finland are insured against idiosyncratic risks like fire and theft. in general, finnish producers can be considered to be risk averse. the common agricultural policy (cap) proposal of the european commission introduced the possibility to use public funds to provide producers also with ex ante safety nets against animal disease outbreaks (council regulation / ). hence, eu agricultural policy has moved a step from ex post measures towards ex ante support for agricultural risk management. this structural change from ex post to ex ante measures might be challenging for private insurance markets. studies focusing on how public policies could promote risk management in agriculture (e.g. cafiero et al. ) suggest that public support for insurance premiums is justified only if market-based demand for insurance exists and the markets are unable to provide producers with affordable insurance products. otherwise, for instance if public catastrophic assistance is provided on an ex post and ad hoc basis, the markets may be crowded out and commercial products are eliminated by the public intervention (van asseldonk et al. ; botzen and van den bergh ) . despite the fact that information on the demand for animal disease insurance is important for developing insurance products that are attractive to the producers, empirical studies on this topic are hardly available. our contribution is to fill up a part of this gap. the main motivation for this paper is understanding whether there are sufficient incentives for producers to purchase commercial insurance against animal diseases by analysing the perceptions and producers' a priori willingness to pay (hereafter wtp) for an animal disease insurance. we do not consider the insurance companies' ability to provide affordable insurance products, but instead concentrate on revealing if market-based demand for insurance exists. the above topics were studied through a choice experiment, where finnish pig and poultry producers indicated their willingness to buy different kinds of insurance products. choice experiments have recently been applied widely to study consumer demand in different circumstances, including health insurance (bergrath et al. ) , crop insurance (mercadé et al. ; nganje et al. ) , price insurance (ranganathan et al. ) and flood insurance (botzen and van den bergh ), but to our knowledge not in relation to animal disease insurance. our study contributes to the literature by addressing wtp for animal disease insurance. the specific questions we aim to answer are as follows: ( ) how much demand is there for animal disease insurance, ( ) what are the preferred characteristics of insurance and what is the producers' wtp for them, and ( ) are there specific characteristics of the farms or producers that can be used to explain their wtp for animal disease insurance? the study analyses challenges related to cost sharing in a situation where government-financed schemes are already running, and producers are adapted to these schemes. the methods in the study are set to measure producers' wtp on top of the already existing public schemes. hence, our focus is on wtp for commercial animal disease insurance which is complementary to the existing schemes, and our estimates take into account also that producers evaluated implicitly reliability and adequacy of the current schemes when choosing among options presented to them in the questionnaire. our study examines commercial animal disease insurance products which are not available in the finnish markets but which could be introduced to the markets in the future if sufficient demand for them exists. in the next section, the estimation method and data are described. results of the study are presented in the third section, and the final section provides a discussion over study findings. to assess demand for animal disease insurance as well as to identify what kind of insurance characteristics the producers would prefer, we undertook a survey among the finnish pig and poultry producers. the pig and poultry farms were selected because these production lines operate in an intensive manner and the risk of contagious animal diseases is important and constantly present in pigs and poultry. pig and poultry producers are likely to be more aware of the role of insurance than cattle producers. in both pigs and poultry, there have been cases where an animal disease has caused losses, and compensations have been argued to be insufficient. these production lines were hit by a large feed-related salmonella outbreak in . most pig and poultry farms in finland are covered by salmonella group insurance. in pig production, there have been attempts to introduce also other disease insurance products. pig and poultry production in finland is concentrated in south-western and western parts of the country. in , the market value of pig production in finland was million euro ( % of the total market value of finnish agriculture, excluding subsidies) and the value of poultry production was million euro ( % of the total market value) (niemi and ahlstedt ). according to our sample, in , the average number of animals per specialised farm was , broilers (for % of the farms the number being between , and , ), laying hens ) , sows ( - ) or other pigs than sows . the broiler production in finland is characterised by high vertical integration (all stages from feed and chick production until meat processing) and fairly intensive production which is practiced in controlled housing conditions. egg production is characterised by a large proportion of small producers and a number of very large farms, which collaborate closely with egg packers. egg production is more heterogeneous than other sectors included in the survey. the pig sector is also fairly intensive, and production occurs under well-managed facilities, but the heterogeneity of farm types and sizes is larger than in the broiler sector. larger farms, which produce majority of the pig meat, are specialised, very professionally run and wellmanaged enterprises typically having - sows or - finishing pigs. piglet trade is usually coordinated by the slaughterhouses and animal auctions are uncommon. a questionnaire was sent to all commercial pig and poultry producers in finland, enquiring for instance their disease history (information on previous disease outbreaks at the farm), current insurance cover (all types of insurance) and the biosecurity measures used on their farm. more specifically, the questionnaire included a list of different biosecurity measures, such as whether the production facilities are compartmentalised, whether the farm employs all-in-all-outprinciple, whether there is protective clothing available for visitors, whether the producers participate in disease-related training and so on. the respondents chose the measures that they use on their farm. the final section in the questionnaire was the choice experiment, where different hypothetical insurance products were offered to the producers. choice experiment is an application of the characteristics theory of value (lancaster ) , combined with random utility theory. based on random utility theory, we assumed that the surveyed producers would be able to choose the best alternative from different insurance product choices in the choice set. the overall utility from a good can be divided into attributes: where u in is the utility of alternative i for individual n, v in is the explained part of the utility, z i denotes product-specific attributes and ε in is a random error, which is independent of other terms and independently and identically distributed (iid) with an identical type i extreme value distribution, representing the unobserved part of utility. the explained part of utility therefore specifies the attributes of a product that can be directly measured as well as the functional form v in , through which it explains the overall utility. discrete choice models describe individual choices among alternatives. the probability p in of individual n choosing alternative i is equal to the probability that the utility of alternative i is greater than, or equal to, the utility associated with alternative j for every alternative in the choice set (j = , …, j). formally: the multinomial logit model was derived under the assumption that the error term is iid for all i. the logit probability is (mcfadden ) : where v in = β ′ z i and β is a vector of parameters. in the multinomial logit model (eq. ), the choice probabilities of the different insurance options are modelled. several attributes (see table ) were used as explanatory variables, and their functional forms were specified by v in . it was assumed that this function is separable, additive and linear. discrete choice models measure the utility of respondents. thus, the estimated model coefficients are not interpretable in economic terms. therefore, to reveal the overall wtp for an insurance product, implicit price (ip) estimates of insurance attributes were calculated as: where β k is the coefficient of the kth attribute, and β p is the price coefficient. note that parameters β k and β p constitute vector β in eq. . to calculate the wtp for the product, the estimated ips for individual characteristics (summarised in table ) were summed up and compared to the wtp of the baseline case (status quo): where β base,k denotes the coefficient associated with the baseline case (status quo). typically, decision-makers feel that the greater their assets are, the less they should pay for insurance to cover a given risk (pratt ) . thus, producers' wtp for insurance against animal diseases might vary largely. as we were also interested in potential respondent segments, a latent class model was employed. it assumes that the respondents belong to heterogeneous latent classes based on their differing attitudes and perceptions of product attributes and other phenomena (swait ) . these differences are reflected in their segmentspecific choice behaviour. the latent class model reveals both the segments and the relative preferences prevailing in each segment (hu et al. ; vermunt and magidson ) . the best model having the optimal number of classes was selected using bayesian and akaike information criteria (bic and aic). insurance premiums are typically differentiated according to the location of a farm and other farm-or producer-related characteristics. these socioeconomic factors were also accounted for in the choice experiment. in the latent class model, they were included as covariates to explain class characteristics. however, they were set to be inactive, meaning that only the product characteristics and choice behaviour determined class membership, but socio-demographic and farmspecific characteristics were used to explain class membership. they therefore provide an opportunity to seek for recognisable groups among the potential buyers of insurance. alternative specific constants were excluded from the model table an example of a choice set offered to a pig farm (alternative , alternative , and the no buy-option) and the set of all attribute levels used in the choice experiment (right hand side column) i would not buy either product □ levels for alternatives and shown to each respondent were selected from the sets of all attribute levels, resulting in different choice sets because of multicollinearity with some of the product characteristics, primarily with the compensation level. because of this, the compensation level coefficients are negative, as they indicate deviation from the status quo and dislike towards buying the insurance. the data were analysed using latent gold statistical software (from statistical innovations, inc.). a pilot questionnaire was sent to farms in july . accounting for the responses to this questionnaire, the final form of the questionnaire was mailed to all finnish poultry and pig producers, altogether approximately farms, in august . the addresses of the producers were obtained from the information centre of ministry of agriculture and forestry. the choice experiment consisted of choice sets, each choice set included two insurance products, and each respondent was presented four of these sets. within each choice set, the respondent had three options: choose insurance product a, choose insurance product b or choose 'i would not buy either product'. the sets were formed using the ngene software (from choicemetrics pty ltd). in the final construction, the d-error was . and the a-error . . the lower these measures are, the more efficient the choice set design is (see, e.g. rose and bliemer ) . in the design, priors for the alternatives were based on the results of the pilot questionnaire. each choice alternative consisted of five attributes: insurance provider, biosecurity requirement, damages compensated by the insurance, deductible and the price. the levels of these attributes are presented in table . we briefly discuss these attributes below, based on heikkilä and niemi ( ) , european commission ( ), van asseldonk et al. ( ) and shaik et al. ( ) . the attribute combinations (and therefore the products offered) are hypothetical. however, the attributes and their levels were all set at realistic ranges, drawing on the above literature reviews in european insurance systems as well as our own experience on the insurance market in finland. the attributes can be found in products existing in different real-life contexts, and the specific attribute levels were tested in the pilot questionnaire. a private insurance company and producers' mutual fund were presented as possible insurance providers. we did not have any a priori information on whether the producers have preferences towards the provider of insurance, and therefore, this was included as an attribute. biosecurity requirement helps the insurance system to encourage the producer to reduce the disease risk. requirements for a specific level of biosecurity to be maintained at the farm will also help to manage asymmetric information and moral hazard. biosecurity management is, however, costly to the producer, as disease prevention measures incur costs (protective clothing, animal disposal, disinfection, and so forth). some producers may wish to have a lower insurance premium in return for adopting enhanced biosecurity measures compared to the national standard. the insurance scheme should provide producers with incentives to purchase insurance and to take disease prevention measures thereafter. increasing the level of compensation may raise the insurance premiums, but it may also provide a better safety net for the producers. however, when indemnities are based on losses, the risk of moral hazard needs to be taken into consideration. previous studies have suggested that producers are not very willing to purchase insurance that covers an extended amount of losses, for instance indirect losses (van asseldonk et al. ) . we tested this by including three levels of compensation in the choice sets as a scale attribute. the basic level covers only material damages and animals, and additional levels increase the cover to income loss, as well as protection against potential price fluctuations associated with the disease outbreak. the deductible is a necessarily part of a functioning insurance scheme, as it decreases moral hazard and creates incentives for producers to maintain sufficient disease prevention. however, if the deductible is set too high, it may undermine the incentives to detect disease at an early stage. we tested a range of deductibles, varying from to %. the price of the insurance defines the annual amount that the producer pays to the insurance provider for carrying the disease risk and compensating the damages in case of a disease outbreak. when the insurance is fairly priced, risk averse producers should, according to theory, insure, because they would not be worse off by purchasing than not purchasing the insurance (see e.g. mas-colell et al. ) . the questionnaire was sent to producers of different pig production types (finishing, farrowing, and farrowing-tofinishing pig farms) as well as to two types of poultry producers (broiler and laying hen farms). where 'other pig farm' is used, it refers to farrowing and farrowing-tofinishing farms collectively. each producer was quoted a price that was adjusted for their type of production. the prices were formed such that they represent a given percentage of market revenue generated by the animals. using this type of relative prices, we gave each production line a comparative set of prices. the prices that were included in the choice sets were . - . % of the market revenue. for instance, for the finishing pig producers, the annual insurance price varied from euro to euro per animal places (see table , where the prices denoted are for pig producers in finishing farms). in the regressions, each production line was analysed separately in relation to the price coefficient. an example of a choice set is provided in table . the respondents were also provided additional information regarding the biosecurity requirements, as well as an explanation to what the different levels of compensated damages would cover. additionally, a split sample feature was introduced to study whether the demand for insurance depends on the context in which it is embedded (jacobsen et al. ) . for instance, diseases included in the insurance scheme have to be clearly defined to limit the liability of the insurer. insurance cover would typically be introduced as an attribute, but we already had two attributes representing the cover (compensated damages and the deductible). therefore, we divided the sample into two sub-samples depending on which diseases the insurance would cover. half of the respondents were told that the insurance would cover a set of diseases which are present in many countries, although not necessarily in finland (hereafter called 'common diseases'), whereas the other half dealt with a set of highly contagious notifiable diseases which are not present in finland (hereafter called 'notifiable diseases') ( table ) . the term used here should not be confused with the official definition of a 'notifiable disease' in finland. a total of questionnaire responses were received, the response rate being . %. the main data characteristics are described in table . of all the insurance choice sets altogether % of the insurance products were not purchased-in other words, the 'i would not buy either product' option was chosen. thus, in % of the choice situations one of the offered hypothetical insurance products was purchased. some prejudice votes may also be included, as % of the respondents ( respondents) answered 'i would not buy either product' to all four choice sets they were presented. the result is overall very similar across the production types, although there are some minor differences between the groups. the pig producers in the common disease group purchased % of the insurance products compared to % in the notifiable disease group. the poultry producers' interest to buy insurance was similar in both groups, approximately - %. pig producers are therefore somewhat more willing to buy the insurance when it covers the common animal diseases, whereas the disease coverage of the insurance does not affect poultry producers. to study how the insurance purchase likelihood varies with the different attributes, logistic regression models were applied. the dependent variable in each regression model indicates whether the insurance product was chosen. a positive coefficient associated with a particular attribute (explanatory variable) implies a higher probability that the product is chosen and therefore a higher level of utility associated with the attribute when the value of the explanatory variable increases. likewise, a negative coefficient infers decreasing probability that the product is chosen. in addition to the generic case, a regression was run for both disease types separately. overall, the basic model explains the choices relatively well (table , 'all diseases'). although the class-specific r statistic is modest ( . ), the overall r ( ) statistic shows a larger value ( . ). the large difference is due to strong preference for the 'no buy' option. most of the explanatory variables are statistically significant and the signs of the coefficients are logical. moreover, in logistic models r statistic is often low even if the model classifies the observations correctly, in other words correctly predicts individuals or observations that would entail the purchase of insurance. the most significant variables are the compensated damages (low, medium, high). however, these capture also the constant associated with the 'no buy' option. the compensated damages are naturally zero if no product is chosen, and as this is the level to which the level of compensated damages is compared, also the 'no buy' constant is reflected here. the negative and highly significant coefficients of compensated damages suggest that regardless of the attribute levels, many respondents preferred to choose the no buy option, i.e. not to purchase insurance. the coefficients indicated that the respondents on average preferred more compensation to less compensation (compensated damages 'high' had a less negative coefficient than compensated damages 'medium', which in turn was less negative than compensated damages 'low'). however, the difference in magnitude between the medium and high levels of compensated damages is fairly small, indicating that increasing the coverage of compensated damages increased purchase intentions only very little, if at all. also, the price and the deductible were statistically significant attributes, the deductible being the second most important variable. the price had the expected negative sign: as the price increases, demand for insurance decreases. the price coefficients for different production lines are all negative and highly significant, but of different magnitudes, reflecting different values of the animal place as well as potentially different choice behaviour of the respondents in each line of production. the biosecurity requirement and the provider of the insurance were not statistically significant at % confidence level. these attributes have little impact on respondents' choice of insurance. when the two disease types were analysed separately ( table , 'common diseases' and 'notifiable diseases'), the results were qualitatively similar to the base case. the compensation level and the deductible were significant in both disease types. the impact of the deductible is somewhat larger in the common than in the notifiable diseases group. interestingly, the price is not statistically significant variable in the common diseases group (except for poultry producers), although it is statistically significant in the notifiable group (again, except for the poultry producers). the insurance provider on the other hand is significant in the common diseases group, lending some support for the slight preference for the insurance company over a mutual fund. altogether, there are no major differences between the disease types, a finding that was not anticipated beforehand. after having an overview of the demand for insurance, the next question was whether there are respondent or farmrelated characteristics that affect the demand for insurance. we therefore analysed the latent classes with the regression model. based on the bic and aic values, a four-class model was chosen (table ). the overall statistics were significantly improved from the one-class base model, r statistic being . and r ( ) statistic . . the parameters in table describe how different characteristics are related to the four classes. the wald p values indicate that the attributes were jointly significant, while the wald() p values. the statistic labeled wald() tests whether regression coefficients are equal between classes show that only the price, biosecurity requirement and some levels of compensated damages were class dependent. class includes % of the respondents. they are not interested in purchasing insurance ('non-buyers'). compared to the whole sample, the class is characterised by a somewhat lower share of young and somewhat higher share of older producers, and the respondents are more likely to have only primary education. the farms have a lower likelihood of having encountered a disease in the past years, and they are somewhat more likely to be poultry farms than pig farms. their farms are typically smaller and they have adopted fewer biosecurity measures than the other farms on average. in this class, coefficients for insurance price, provider and biosecurity requirement were statistically insignificant. all three compensation levels and the deductible had a large negative impact on demand for insurance in this class. hence, having insurance could reduce their utility and they had no willingness to pay for any kind of insurance. class includes % of the respondents. it is characterised by strongly interested buyers, who chose one of the insurance products in most choice situations ('strong buyers'). they are typically young and operate larger-than-average farms, on which they have a higher than average biosecurity. they have a positive wtp for insurance. demand for insurance in this class responds more sluggishly to changes in price, but more heavily on changes in the deductible. class includes % of the respondents. it is characterised by weakly interested buyers of insurance ('weak buyers'). additional biosecurity requirement has a negative impact on their demand for insurance, and also the impact of the deductible is fairly strong. the class includes a smaller share of the low-biosecurity farms as well of the high-biosecurity farms than the sample on average. the group is characterised by a somewhat higher share of those with agricultural education. also, female respondents are more presented in this group. all the attributes are vital for the demand for insurance in this group. hence, their demand for insurance responds strongly to changes in the type of insurance provider, price, biosecurity requirement, losses covered and the deductible. finally, class includes % of the respondents. it is characterised by producers who prefer additional biosecurity measures to be included in the insurance but who are not willing to purchase insurance themselves ('concerned non-buyers'). a much larger share of them has encountered an animal disease in the past than the sample on average. they are more likely than the average farm to have high level of biosecurity, but there were also several respondents with lower than average biosecurity in this class. the respondents are more often young, from large farms and with university education. as they are not willing to purchase insurance, their responses may indicate preference that insurance should include requirements for stronger biosecurity measures for other farms, such that the overall risk of disease is reduced. the results suggest that young (less than years) and highly educated (university education) respondents are more positive towards purchasing animal disease insurance than middle-aged or low-educated respondents. gender did not play a substantial role in the purchase behaviour, although female respondents were somewhat reluctant towards buying insurance. as for the farmlevel characteristics, there is no significant difference between pig and poultry producers in their willingness to buy insurance. however, there are signs that producers who have experienced an animal disease outbreak in the past years are more positive towards buying insurance (class ) as well as perhaps demanding others to take better precautions (class ) than producers without previous experience on disease outbreaks. finally, large farms (belonging to the largest % of the farms as measured by the number of animals) are more willing to purchase insurance. farms that have adopted only few biosecurity measures (belonging to the lowest % of the farms with respect to the adoption of biosecurity) are less keen on insurance (class and class ). the wtp estimates were calculated as described in the 'material and methods' section, comparing the utility the dependent variable is the stated purchase intention. the independent variables are mostly categorical, with the exceptions of price and the deductible. the covariates are inactive and describe the farm having the characteristic in question. wald () statistic tests whether coefficients are class independent a the farm indicated they have suffered from a disease in the past years b less than years old c more than years old d belonging to the largest quartile e belonging to the smallest quartile associated with given insurance products to the utility without the product. the wtp estimates are expressed in euro and can be interpreted as the annual price the producers are willing to pay per animal place. the wtps for some exemplary insurance products are reported in table . for class , it was not possible to calculate wtps due to statistically insignificant price coefficient, but this class includes those who would not buy the insurance in any case and to whom even a zero-price did not make a difference. positive wtps are found for classes and . the level of the deductible affects the wtp significantly. in class , each percentage point increase in the deductible decreases wtp by . euro (finishing pig producers), . euro (other pig producers) and . euro (poultry producers) per animal place. in class , the corresponding figures are . euro, . euro and . euro. in the model for all diseases, the wtp for the wider coverage of compensated damages increases moderately as the compensation level rises. thus, implicit price for the highest level of compensated damages is higher than for the second highest level, which in turn is higher than for the lowest level. although the relationships are statistically significant, the actual wtp is very low and in many cases negative. however, in classes and (table ), the wtp is highest at the medium level of damages (although in class it is still negative). the immediate reason for this is the lower negative coefficient for medium compensation than for high compensation. the result indicates that there does not seem to be much demand for the highest compensation level of the insurance. the wtps were also calculated separately for the two disease types, and it turned out that wtp was somewhat greater in the common diseases group than in the notifiable diseases group. however, as the impact of the deductible also differed in the two models, at higher levels of the deductible ( - %), the wtp was actually greater for the notifiable diseases than the common diseases. the wtps were translated to farm-level figures using the average number of animal places as the multiplier (table ) . for class , the wtps are the largest, varying from euro to euro for the low compensation level, euro to euro for the medium compensation level and euro to euro for the high compensation level. for class , the annual total wtps are approximately euro to euro for the low compensation level, euro to euro for the medium compensation level and euro to euro for the high compensation level. the wtps are highest for the broiler farms, followed by the finishing pig farms. the laying hen farms and other pig farms have a lower overall wtp. the farm-level wtps can be compared to the average annual insurance payments by the farms. this information was obtained in the questionnaire, by asking how much the farms currently pay annually for animal disease insurance. the average figures are euro for the pig farms and euro for the poultry farms. based on farm accountancy data, the animal disease insurance payments were euro for the pig farms and euro for the poultry farms in . these figures are in the same ballpark as the estimated wtps indicating that on top of the existing schemes, not much additional wtp exists. in this paper, we have analysed demand for animal disease insurance in pigs and poultry in finland. our results suggest that overall, there is currently limited demand for a new animal disease insurance as a commercial product. this concerns a product which could be brought to the market to complement currently existing insurance and public compensation policies. although wtp for such insurance products is low, in some cases even negative, it also varies by market segment, and there are important product attributes that increase the likelihood of the producer wishing to purchase insurance policy. the attributes include, perhaps most importantly, a low deductible. this result combined with % deductible required for a public insurance intervention (in connection to the cap) to be introduced to the markets reveals the possibilities for parallel existence of private and public insurance. public intervention does not crowd out all (shallow loss type) private market insurances for animal diseases. even though a low deductible was seen as an attractive characteristic of insurance, even a % deductible did not attract all the respondents to purchase insurance. compensated damages were offered at three levels (low, medium and high), and it was assumed that the larger levels were more desirable. however, this was hardly reflected in the choices made by the respondents. a higher coverage of damages compensated may increase purchase intentions, but the increase is relatively small. pearson x -tests of independence between the variables showed that the difference between compensation levels regarding the purchase intensity was not significant at the % level of confidence. a similar result has been found by van asseldonk et al. ( ) and nganje et al. ( ) . in flood insurance, botzen and van den bergh ( ) found that lowering the coverage from to % reduced the wtp by only about %. it is perhaps not worth supplying insurance products that cover all kinds of risks, for instance income risks, because different risk management tools can be used to cover different types of risks. a similar argument applies to different types of animal diseases. co-financing by the eu and by the member states for the prevention of officially controlled animal diseases decrease animal disease risks faced by producers. these policies cover only the most harmful diseases well beyond the % deductible at the farm level. because of these policies and other reasons, the remaining risks are so small that producers' incentives to participate in an animal disease insurance against these diseases are compromised. lower wtp for notifiable diseases suggests that most producers prefer not to purchase insurance and rather rely on public support or other means of managing the disease risk. also, time preference of the producers might weaken the demand for diseases that have a low probability of occurrence. for common diseases, the risks can be perceived to be too low and other management patterns to be efficient enough to induce substantial demand for animal disease insurance. the price of the insurance was found to have a negative and statistically significant although a fairly small impact on stated insurance purchases. the price was not as important a factor as anticipated. this result provokes reflection on whether the insurance support recently introduced by the cap will be based on farmers' mutual funds. these are article 'crop, animal, and plant insurance', article bmutual funds for adverse climatic events, animal and plant diseases, pest infestations and environmental incidents^and article bincome stabilisation tool^in (eu) no / . under these articles, support shall only be granted for insurance contracts (between producer and producers mutual fund) which cover for loss caused by an adverse climatic event, or by an animal or plant disease, or a pest infestation, or an environmental incident or a measure adopted in accordance with directive / /ec to eradicate or contain a plant disease, or pest which destroys more than % of the average annual production of the farmer in the preceding -year period or a -year average based on the preceding -year period, excluding the highest and lowest entry. respondents were overwhelmed by all the attributes, whether they had too many and too complicated options to address, or whether the impact of the price was overshadowed by the other attributes. in addition, the producers may have perceived difficulties in attaching a price to animal disease insurance as it is not widely available in the markets. where found positive, wtps were relatively similar compared to the insurance fees that the farms currently pay (on average about euro per farm per year). latent class analysis suggests that the type of supplier of the insurance does not play any significant role in determining the demand for animal disease insurance, although there are minor signs that a private insurance company may be somewhat preferred over producers' mutual fund. the lessons learned from eu member states show that a mutual fund covering animal disease expenses is the most commonly used form for cost sharing of direct costs in the netherlands (van asseldonk et al. ; meuwissen et al. ; oecd ) , germany (oecd ; british embassy berlin ) and france (cassagne ) . within the cap, mutual funds could be reported only if dealing with risks beyond farmers % deductible. hence, there seems to be room for marketorientated coordination on animal disease insurance. private companies handle shallow losses which is the area where cap-supported mutual funds are not allowed to operate. furthermore, private insurance company has the advantage of having experience from other fields of insurance and a functioning infrastructure for operating the insurance scheme. the producers' mutual fund pursues to promote the interests of the industry, but it may lack the necessary infrastructure which may reduce its attractiveness. whether the producer buys animal disease insurance is part of the wider risk management plan of the farm. in finland, most livestock farms are covered by property insurance, which covers for instance losses due to fire, and by salmonella group insurance. according to pukara ( , using data based on our questionnaire), % of the surveyed pig and poultry farms had a business interruption insurance and % had other production animal insurance. farm insurance products in finland are often sold as packages such that one product is purchased on top of another. some of these packages may cover animal disease losses as well. for instance, a farm can purchase an insurance to cover business interruption losses or generic production animal insurance, and in some cases (such as upon high mortality) these may cover animal disease losses. insurance products frequently include conditions to prevent the damage from occurring. many of the conditions in the salmonella group insurance are included in the production contracts that producers sign with slaughterhouses or egg packers. similar conditions may occur in other existing insurance products as well. hence, group insurance and production contracts constitute a set of rules and guidelines which encourage producers to apply proper management methods and to take care of biosecurity and other preventive measures. they are thus also setting a biosecurity standard in the field. in our analysis, the biosecurity requirements attached to the insurance did not affect the overall choices. however, the latent class analysis identified a group of respondents who preferred additional biosecurity measures related to the insurance product, but who were not willing to purchase any additional insurance themselves, to some extent because they were already better insured against animal diseases than the other respondents. our results provide some support for the notion that biosecurity and insurance are seen as complements by the producers. this follows from the fact that in classes where wtp was positive, the biosecurity levels were higher (and more importantly, were not lower) than average. a similar finding has been provided by ranganathan et al. ( ) who found that those who use alternative risk management measures also have a positive wtp for price insurance, and by botzen and van den bergh ( ) who found that probability for flood insurance is higher for those who already have other insurance products. this also suggests that adverse selection may not be an overwhelmingly large issue (see also botzen and van den bergh ) . a challenge for the insurance providers is to find incentives for those smaller farms that have a lower level of biosecurity to insure themselves against disease risks. if producers see biosecurity and insurance as substitutes to each other, any insurance scheme will face challenges. the producer and farm characteristics suggest that those primarily interested in insurance include young, educated producers from large farms, who mostly already have a good level of biosecurity on their farms. this may be related to producers' risk perceptions due to education, their debt obligations and the magnitude of risks involved in livestock farming at different stages of farm life cycle. the result is in line with the current situation in finland: the study by pukara ( ) on pig and poultry farms' current insurance coverage revealed that producer's high education, low age and large farm size were associated with increased probability to have their farm covered by different insurance policies. also more generally, the adoption of risk management tools can be linked to factors such as producer's age, education or farm size (e.g. velandia et al. ). producers who have faced an animal disease outbreak in the past are more willing to purchase insurance, suggesting that perhaps their preferences may have changed or that there are possible misconceptions regarding the extent of public support. having said that, the overall wtp for the insurance is mostly low, especially if the deductible is at the currently typical level of - %. the results also indicate that wtp for insurance varies according to the farm and producer characteristics. given the structural change towards fewer and larger livestock farms, producers' attitudes towards insurance may become more positive on average. our analysis suggests that currently, there are limited opportunities to increase animal disease insurance cover of pig and poultry farms. we identified two subgroups of respondents who would be willing to purchase animal disease insurance and one group who mostly already have one. those interested in insurance are typically young producers operating larger-than-average farm. the subgroups of producers could be analysed in greater detail in further studies. moreover, insurance characteristics such as price and the deductible matter, but their impact seems to be typically quite small. incentives for private insurance should be taken into account when designing potential ways for the government to support animal disease risk management. attracting health insurance buyers through selective contracting: results from a discrete-choice experiment among users of hospital services in the netherlands monetary valuation of insurance against flood risk under climate change livestock diseases compensation-systems available in germany to limit economic damage to producers/ farmers risk and crisis management in the reformed european agricultural policy managing compensation for economic losses in areas surrounding foot and mouth disease outbreaks: the response of france evaluation of the community animal health policy (cahp) - and alternatives for the future livestock disease indemnity design when moral hazard is followed by adverse selection eläintautivahinkojen rahoitusvaihtoehdot: käytännöt, kannustimet ja kustannukset (in finnish), helsinki, mtt taloustutkimus. mtt:n selvityksiä trading off health, environmental and genetic modification attributes in food embedding effects in choice experiment valuations of environmental preservation projects adverse selection in crop insurance: actuarial and asymmetric information incentives a new approach to consumer theory microeconomic theory conditional logit analysis of qualitative choice behaviour. in: zabrenka p (ed) frontiers in econometrics a choice experiment method to assess vegetable producers' preferences for crop insurance, paper prepared for presentation at the th eaae seminar ba resilient european food industry and food chain in a challenging world^ testing for moral hazard and ranking farms by their inclination to collect crop damage compensations, in: european association of agricultural economists livestock diseases: prevention, control and compensation schemes risk aversion in the small and in the large kotieläintuotannon vakuutusturvan laajuus ja siihen vaikuttavat tekijät sika-ja siipikarjatiloilla demand for price insurance among farmers in india: a choice experiment-based approach in: hensher d (ed) transport economics: critical concepts in economics the economics of livestock insurance: concepts, issues and international case studies a structural equation model of latent segmentation and product choice for cross-sectional revealed preference choice data trade-offs between catastrophic assistance and subsidised insurance in european agriculture factors affecting farmers' utilization of agricultural risk management tools: the case of crop insurance, forward contracting, and spreading sales latent gold® choice . user's manual. statistical innovations key: cord- -kc thr authors: bradt, david a.; drummond, christina m. title: technical annexes date: - - journal: pocket field guide for disaster health professionals doi: . / - - - - _ sha: doc_id: cord_uid: kc thr . humanitarian programs ; . security sector ; . health sector : core disciplines in disaster health . primary health care programs . disease prevention . clinical facilities . reproductive health . water and sanitation . food and nutrition . chemical weapons . epi methods ; . tropical medicine : tropical infectious diseases—vector-borne and zoonotic . tropical infectious diseases—non-vector-borne ; . epidemic preparedness and response ; . communicable disease control : diarrhea . influenza . malaria . measles . meningitis . viral hemorrhagic fever ; . diagnostic laboratory : indications, laboratory tests, and expected availability . specimen handling ; . acronyms ; this section provides guidance on technical issues in the health sector. the annexes contain compilations of frequently used reference information. • humanitarian programs-contains conceptual frameworks on global clusters, relief programs, humanitarian financing, and early recovery. • security sector-contains key definitions from the rome statute of the international criminal court • health sector-contains a broad range of core health technical information including environmental classification of water and excreta-related diseases, disease prevention measures, water treatment end points, anthropometric classifications, micronutrient deficiency states, management of chemical weapon exposures, and epi methods. • tropical medicine-contains clinical summaries of tropical infectious diseases with details on disease vector and host, clinical presentation, diagnostic lab tests, clinical epidemiology, and therapy. • epidemic preparedness and response-contains core principles of epidemic preparedness and response. • communicable disease control-contains an overview of selected communicable diseases of epidemic potential including diarrhea, influenza, malaria, measles, meningitis, and viral hemorrhagic fever. • diagnostic laboratory-contains guidance on lab specimen handling and testing. • acronyms-contains acronyms commonly used in disaster management and humanitarian assistance. a. in-kind donations (eg food, seeds, tools, fishing nets, etc) b. types of community projects in food-for-assets programs ( ) natural resources development (a) water harvesting (b) soil conservation ( ) restoration of agri(aqua)culture potential (a) irrigation systems (b) seed systems ( ) infrastructure rehabilitation (a) schools (b) market places (c) community granaries (d) warehouses (e) roads (f) bridges ( ) diversification of livelihoods (a) training and experience sharing . increase individual purchasing power a. cash distribution b. cash for work (cash for assets) c. vouchers d. micro-credit e. job fairs f . artisanal production g. livelihoods/income generation . support market resumption a. market rehabilitation b. infrastructure rehabilitation c. micro-finance institutions goals-protect what's left ( month), restore the system ( months), improve the system ( . promote transformational development support far-reaching, fundamental changes in relatively stable developing countries, with emphasis on improvements in governance and institutions, human capacity, and economic structure, so that countries can sustain further economic and social progress without depending on foreign aid. focus on those countries with significant need for assistance and with adequate (or better) commitment to ruling justly, promoting economic freedom, and investing in people. reduce fragility and establish the foundation for development progress by supporting stabilization, reform, and capacity development in fragile states when and where u.s. assistance can make a significant difference. . support strategic states help achieve major u.s. foreign policy goals in specific countries of high priority from a strategic standpoint. . international cooperation to protect lives and health . timely and sustained high-level political leadership to the disease . transparency in reporting of cases of disease in humans and in animals caused by strains that have pandemic potential to increase understanding, enhance preparedness, and ensure rapid and timely response to potential outbreaks . immediate sharing of epidemiological data and clinical samples with the world health organization (who) and the international community to characterize the nature and evolution of any outbreaks as quickly as possible . prevention and containment of an incipient epidemic through capacity building and in-country collaboration with international partners . rapid response to the first signs of accelerated disease transmission . work in a manner supportive of key multilateral organizations (who, fao, oie) . timely coordination of bilateral and multilateral resource allocations; dedication of domestic resources (human and financial); improvements in public awareness; and development of economic and trade contingency plans . increased coordination and harmonization of preparedness, prevention, response and containment activities among nations . actions based on the best available science . genocide (article )-acts committed with intent to destroy, in whole or in part, a national, ethnic, racial, or religious group a. killing members of the group b. causing serious bodily or mental harm to members of the group c. inflicting on the group conditions of life calculated to bring about its physical destruction in whole or in part d. imposing measures intended to prevent births within the group e. forcibly transferring children of the group to another group . crimes against humanity (article )-acts committed as part of a widespread or systematic attack against any civilian population, with knowledge of the attack a. murder b. extermination c. enslavement d. deportation e. imprisonment in violation of international law f. torture g. rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence h. persecution on political, racial, national, ethnic, cultural, religious, gender, or other grounds universally recognized as impermissible under international law i. enforced disappearance j. apartheid k. other inhumane acts intentionally causing great suffering or serious injury to body or to mental or physical health . war crimes (article ) a. grave breaches of the geneva conventions of aug ( ) willful killing ( ) torture or inhumane treatment including biological experiments ( ) willfully causing great suffering ( ) extensive destruction and appropriation of property ( ) compelling a pow to serve in the armed forces of a hostile power ( ) willfully depriving a pow of the right to a fair trial ( ) unlawful deportation ( ) taking of hostages b. serious violations of laws and customs applicable in international armed conflict ( ) intentionally directing attacks against the civilian population or against civilians not taking direct part in hostilities ( ) intentionally directing attacks against civilian objects ( ) intentionally directing attacks against personnel, installations, material, units, or vehicles involved in humanitarian assistance or peacekeeping mission ( ) intentionally launching an attack in the knowledge that it will cause incidental civilian loss of life or severe damage to the natural environment ( ) attacking undefended towns, villages, dwellings, or buildings which are not military targets ( ) killing or wounding a combatant who has surrendered ( ) improper use of a flag of truce, flag or insignia or uniform of the enemy or of the un, or emblems of the geneva conventions resulting in death or serious personal injury ( ) transfer by the occupying power of parts of its own civilian population into the territory it occupies, or the deportation or transfer of all or parts of the population of the occupied territory within or outside the territory ( ) intentionally directing attacks against buildings dedicated to religion, education, art, science, charitable purposes, historic monuments, hospitals, and places where sick are collected, provided they are not military objectives ( ) subjecting persons to physical mutilation or to medical or scientific experiments which are not justified by the medical treatment nor carried out in his/her interest ( ) killing or wounding treacherously individuals belonging to the hostile nation or army ( ) declaring that no quarter will be given ( ) destroying or seizing the enemy's property unless such be imperatively demanded by the necessities of war ( ) declaring abolished, suspended, or inadmissible in a court of law the rights and actions of the nationals of the hostile party ( ) compelling the nationals of the hostile party to take part in the operations of war directed against their own country ( ) pillaging a town or place, even when taken by assault ( ) a range of generic prevention measures should be considered for its impact on diseases in a biological "all-hazards" environment. overall, excreta disposal, water quantity, personal hygiene, and food hygiene commonly contribute more to environmental health than do other listed measures. epidemic threats will oblige heightened consideration of disease-specific strategies for prevention and control. c. water treatment (bold text of particular relevance in clinical facilities) ppm = mg/kg (solids) = mg/l (liquids) = ug/ml (liquids) = basic unit of measure for chloroscopes : , ppm = % • sam = whz < − , muac < . cm, or bilateral pitting edema (who). whm not in definition. • sam prevalence worldwide ≈ , , . • sam mortality ≈ x mortality of normally nourished child and its cfr can be - %. • gam = mam + sam • gam = moderate wasting cases, severe wasting cases, or bilateral pitting edema cases (where due to malnutrition) • underweight is not used for screening or surveys in nutritional emergencies. it reflects past (chronic) and present (acute) undernutrition and is unable to distinguish between them. it encompasses children who are wasted and/or stunted. however, weight gain over time can be a sensitive indicator of growth faltering which is easily tracked on road to health charts. • stunting generally occurs before age . it is irreversible. • stunting prevalence worldwide ≈ , , . • stunting is not a good predictor of mortality, but the cfr from ids in cases of severe stunting ≈ x the cfr from ids in cases without stunting. reference standards can be absolute muac, centile, % of median reference, or z scores: • muac easy to understand. an excellent predictor of mortality. permits comparisons between age groups insofar as the low growth velocity of muac in the u age group makes data roughly comparable. may be used alone in "quick-and-dirty" convenience samples to estimate local prevalence of wasting. however, not used alone in authoritative anthropometric surveys, and is commonly part of a two stage screening process to determine eligibility for feeding programs. • overall whz gives higher prevalence of malnutrition than whm for the same population. this is most marked where there is low baseline prevalence of disease, and especially for adolescents (who get subsequently over-referred). whz is more statistically valid, but whm is better predictor of mortality and is used for admission to tfcs. weight-for-age is influenced by weight-for-height and height-for-age. it can be difficult to interpret. b. adults and adolescents (o ) anthropometrics: bmi = weight (kg) / height (m) . death rates-calculated incidence of death expressed per , p/d or per p/mo; data collected by retrospective surveys (eg month period) to gauge severity of public health emergency particularly where sudden events lead to spike in mortality a. cdr-crude death rate b. asdr-age-specific death rate (eg u dr or death rate of children - yr) during a studied time interval (written as . mortality rates-calculated probability of dying before a specified age expressed per live births; data collected by national health authorities in periodic (annual) demographic surveys to reflect ongoing health status a. cmr-calculated probability of mortality in given population for specific time b. imr-calculated probability of a live borne child dying before yr c. u mr-calculated probability of a live borne child dying before yr nb mr ≠ dr. eg cmr ≠ cdr, u mr ≠ u dr. different rates measure different things and are not directly comparable. however, mrs may be converted into drs by the following: cdr or u dr (deaths/ , /d) = − ln( −p/ ) × . where p = cmr or u mr (deaths/ live births). however, this has little field utility. nb mmr-maternal mortality ratio has different units in numerators (maternal deaths) and denominators (live births), thus is a ratio, not a rate the application of study findings to an entire population from which the sample was drawn. if the survey was well-conducted, the results may be considered representative of the entire population. this is scientifically justified. however a confidence interval should accompany any parameter estimate of that population. extrapolation the extension of study findings to a population or period which was not represented in the sample. it works by association-if populations appear to be experiencing similar conditions, the morbidity/mortality experience of one may be imputed to the other. this is not scientifically justified, but is often done where data are insufficient or impossible to collect. s/sx think differential diagnosis (below). . severe muscle pain may be a symptom of sepsis even without fever. . elderly patients with sepsis may be afebrile. in elderly patients, fever is rarely caused by a viral infection. . septic patients who are hypothermic have a worse prognosis than those with high fever. treat as a medical emergency. . fever in a postoperative patient is usually related to the surgical procedure (eg pneumonia, uti, wound, or deep infection). . fever with jaundice is rarely due to viral hepatitis. think liver abscess, cholangitis, etc. . the rash of early meningococcal infection may resemble a viral rash. . generalized rashes involving the palms and soles may be due to drugs, viral infections, rickettsial infections, or syphilis. . all febrile travelers in or returned from a malaria infected area must have malaria excluded. . disseminated tb must be suspected in all elderly patients with fever and multisystem disease who have been in an area with endemic tb. . septic arthritis may be present even in a joint which is mobile. . back pain with fever may be caused by vertebral osteomyelitis or an epidural abscess. . a patient may have more than one infection requiring treatment (eg malaria and typhoid), especially if they are elderly, immunosuppressed, or have travelled. . always remember common infections, not just opportunistic infections, in aids patients with a fever. understand morbidity multipliers-measles, malnutrition, and tb/hiv. understand occult co-morbidities. for any undifferentiated illness, even in infants, think of hiv, tb, syphilis, and sarcoid. for any child, think of malaria, hookworm, and anemia. malarial anemia usually in pedes < year-old; hookworm anemia usually in pedes > year-old. for any icp, think of tb, vl, histoplasmosis, and strongyloides. must treat early. watch for clinical mimics-malaria presenting as pneumonia or diarrhea in pedes; vl presenting as malaria in adults; lepto presenting as mild df (esp in df endemic areas where the pt has mild onset of illness, worsening course, and no rash but jaundice). tx do basic things well, use equipment you understand, teach others, delegate. this annex profiles selected communicable diseases of epidemic potential whose incidence, management complexity, or mortality obliges particular attention. • if (+) agglutination to o antisera, then the strain is further tested for agglutination to antiserum of ogawa and inaba serotypes. • if (+) agglutination to o antisera, then the strain is not further subdivided (except as producer or non-producer of ct as noted below). • if (−) agglutination to o and o antisera, then the strain is known as non-o , non-o v. cholerae. a strain is further identified as a producer or non-producer of cholera toxin (ct). ct production is a major determinant of disease development. strains lacking ct do not produce epidemics even if from the o or o serogroup. • serogroup o exists as main biotypes-classical and el tor-though hybrids also exist. each biotype occurs as two serotypes-ogawa and inaba. classic biotype caused the th and th pandemics but little epidemic disease since the s though it still causes cases in india. el tor biotype caused the th (current) pandemic and almost all recent outbreaks. el tor was first isolated in in el tor, egypt after importation by indonesian pilgrims travelling to mecca. it survives longer in the environment and produces ct similar to the classical biotype. presumably because of ct pathogenicity, the % of cholera patients with severe disease has doubled over the past yrs. these patients tend to require iv fluid therapy. • serogroup o may have evolved from strains of o el tor as they share many properties though not agglutination. in spring of in dhaka, o cases exceeded o el tor cases for the first time, and it was postulated that o may become the cause of an th pandemic. however, since then, o has again become dominant. infective dose depends on individual susceptibility. relevant host factors include immunity produced by prior infection with serogroup o as well as stomach acidity. id may be , orgs, so personal hygiene plays a lesser role than in shigellosis where the id is much lower. shigella has species. • s. dysenteriae type (sd or shiga bacillus) causes the severest disease of all shigella sp because of its neurotoxin (shiga toxin), longer duration of illness, higher abx resistance, higher cfr thru invasive complications, and great epidemic potential. • s. flexneri is the most common, and is generally endemic, in developing countries • s. sonnei is the most common in industrial countries • s. boydii and s. sonnei give mild disease. some kinds of e. coli produce a shiga toxin. shiga toxin genes reside in a bacteriophage genome integrated into the bacterial chromosome. some abx, eg fluoroquinolones, induce expression of phage genes. the bacteria that make these toxins are variously called "shiga toxin-producing e. coli" (stec), "enterohemorrhagic e. coli" (ehec), or "verocytotoxic e. coli" (vtec). all terms refer to the same group of bacteria. • e. coli o :h (often called "e. coli o " or "o ") is the most commonly identified stec in north america, and it causes most e. coli outbreaks. approximately - % of ehec infections result in hus. • non-o stec serogroups also cause disease. in the usa, serogroups o , o , and o are the most commonly identified e. coli pathogens overall. weather (esp weeks - in apr-may) creating increased biological activity; post-monsoon (esp weeks - in aug-sep) with contamination of water sources. pre-monsoon epidemics are generally worse than postmonsoon ones. dysentery has low level year-round incidence, but epidemics occur roughly each decade. epidemic strains display new, additive antibiotic resistance which probably triggers the epidemic. once resistant strains have become endemic, antibiotic susceptibility rarely reappears. sd acquires resistance quickly. sf acquires it more slowly, and that resistance may wane with decreasing abx pressure. at icddr, annual proportional incidence approximates the following: clean water and waste management especially for cholera. personal hygiene (hand washing with soap and clean towels) especially for shigella. water safe drinking water (boiled, chlorinated) nb sphere standards are not enough-you need increased quantities of chlorinated water at household level. san clean latrines for safe disposal of excreta hand washing with soap food safe food (cooked, stored) breast feeding fomites safe disposal of dead bodies with disinfection of clothing nb after outbreak of a fecal-oral pathogen, food hygiene and funereal practices may influence human-to-human transmission more than water quality. health education to affected population wash hands with soap: after using toilets/latrines. after disposing of children's feces. before preparing food. before eating. before feeding children. dukoral has been the main vaccine considered for use in high-risk populations. • morc-vax and shanchol-similar to dukoral except they do not contain the rbs, hence do not require a buffer, and are / the cost to produce. morc-vax, produced in vietnam, is derived from a vaccine administered to millions of people since , but is not who pre-qualified, and is not expected to have international distribution. • shanchol, produced in india, has international distribution (eg used in the haiti cholera vaccination campaign of ), and is now the agent of choice for who. it confers immunity d p nd dose, effectiveness > % at mo, and protection > % at yr. also confers short-term protection vs etec. dose: . cc vaccine followed by water ingestion but no fasting needed; doses, wks apart; cold chain required except for day of use. • orochol-bivalent formulation as in dukoral without rbs of ct. dose: single dose. no longer manufactured. who recommendations: "vaccination should not disrupt the provision of other high-priority health interventions to control or prevent cholera outbreaks. vaccines provide a short-term effect that can be implemented to bring about an immediate response while the longer term interventions of improving water and sanitation, which involve large investments, are put into place." [ ] icddr recommendations: "because of limitations in terms of transport, formulation, and cost of the current dukoral vaccine, the cots program does not require the utilization of the vaccine during an outbreak; it is not necessary to vaccinate to overcome an outbreak. however, if dukoral is readily available and staff are properly trained in its use according to the guidelines that come with the vaccine, the cots program permits dukoral's use (ideally before an outbreak) in the following high-risk populations: refugee populations in which cholera is present, health care workers managing cholera cases, and communities in which the incidence rate is greater than in annually." [ ] epidemiological surveillance (specific to cholera) epidemiological assumptions (who, cots): estimated attack rates: - % extremely vulnerable hosts and poor environmental health (who) % (refugee camps with malnutrition) (cots) % (rural communities of < p) (cots) % (severe epidemic-good estimate of ultimate disease burden) (who) . % (endemic areas with bad sanitation) (cots) . % (endemic areas in open settings-suitable for initial calculations of early resource requirements) nb overall, % of cases are mild and difficult to distinguish from other types of d. nb asymptomatic carriers are very common ( x # of cases). referral rates for ivs % of cases (much higher- % at icddr as it shortens recovery time) case fatality ratios % (with good care) the following catchment populations will yield acute pts of whom will be severely dehydrated: refugee camp of people (ar of % = pts) open settings in endemic area with , people (ar . % = pts) a population of , infected individuals in an epidemic area will yield the following (who): population infected , clinical cases , ( % of infected population) cases needing early resources ( % of cases) cases needing iv therapy ( % of cases) anticipated deaths ( % cfr) nb in non-endemic areas, ar adults > ar pedes because adults have higher exposure risks. in endemic areas, ar pedes > ar adults because adults have been exposed since childhood delivery of health services shigella are fragile and difficult to recover if transport time > d. - isolates initially to confirm outbreak - isolates initially to create abx use policy (bacterial resistance renders cotrimoxazole, amp/amox, nalidixic acid, and tetracycline unusable) - isolates monthly from ipd and opd before abx therapy to assess evolving abx resistance - isolates periodically to reference laboratory to confirm abx resistance patterns and undertake molecular studies isolates at end of the outbreak to confirm that new diarrheas are not epidemic pathogens nb systematic sampling is most representative-eg every th pt or all pts q weeks adjusted as needed to collect the necessary specs. sensitivity > > important than specificity in rdt screening during an epidemic. pts from one geographic area are more likely to constitute a cluster involving a new pathogen. an area may be considered cholera-free after incubation periods (total of d) have passed without cholera disease. however, hospital monitoring should continue for a year due to tendency of enteric pathogens to re-emerge long after they are declared gone. cholera may be viable but nonculturable from the environment; environmental monitoring has many false negatives. consider improvements to existing diagnostic labs • hotline set up for reporting of rumor this often translates into a hastily conceived vaccination campaign that distracts from core principles of cholera management. for every symptomatic pt, there may be asymptomatic carriers. in an established epidemic, the affected community is already extensively infected. cholera vaccination, under these circumstances, has little public health benefit for the resource investment. if undertaken, the following will apply: • vaccination campaign requires numerous staff. community mobilizers are key. clinical staff should not be poached from their clinical duties. supervisors must be free to move at will. • logistics is key-if the st day goes badly, the campaign goes badly. • mark the domiciles which are done. • hold after-action meetings each day. • last day, use mobilizers with mobile broadcasting to attract those who missed out. • second phase vaccination should include chws with multi-purpose messages on water and sanitation. avoid: press exaggeration abx prophylaxis reliance on ivf and insufficient ors lab investigation of cases once epidemic etiology is ascertained prolonged hospitalization hospital discharge criteria requiring multiple negative stool cultures enthusiasm for ocv during epidemic exaggerated water purification objectives concentration of technical competencies in moh at expense of districts failure to share information with stakeholders influenza viruses comprise genera-influenza types a, b, and c-each with species. • influenza type a is divided into subtypes based upon serological response to hemagglutinin (ha) and neuraminidase (na) glycoproteins. there are different ha subtypes and different na subtypes. h n , h n , and h n are responsible for the major human pandemics in the last century. h n virus circulated between and but currently does not. only influenza a subtypes infect birds, and all subtypes can do so. bird flu viruses do not usually infect humans. but, in , an outbreak of h n avian influenza in poultry in hong kong marked the first known direct human transmission of avian influenza virus from birds to humans. since then, h , h , and h avian influenza subtypes have been shown to infect humans. • influenza type b is morphologically similar to a and also creates seasonal and epidemic disease. • influenza type c is rare but can cause local epidemics. seasonal human influenza vaccine currently has strains-h n /h n /b. influenza disease in humans has a short incubation period ( - d). early symptoms are non-specific. it is highly infectious, especially early in the course of the disease, with a large # of asymptomatic carriers. transmission potential (r ) is a function of infectivity, period of contagiousness, daily contact rate, and host immunity. in general, the faster the transmission, the less feasible is interrupting transmission thru usual disease control tools of case finding, isolation, contact tracing, and ring vaccination. • specific groups of exposed or at risk in the community-most likely to work when there is limited disease transmission in the area, most cases can be traced to a specific contact or setting, and intervention is considered likely to slow the spread of disease eg quarantine of groups of people at known common source exposure (airplane, school, workplace, hospital, public gathering; ensure delivery of medical care, food, and social services to persons in quarantine with special attention to vulnerable groups) (useless once there is community-based spread) eg containment measures at specific sites or buildings of disease exposure (focused measures to > social distance) cancel public events (concerts, sports, movies) close buildings (recreational facilities, youth clubs) restrict access to certain sites or buildings • community-wide measures (affecting exposed and non-exposed)most likely to work where there is moderate to extensive disease transmission in the area, many cases cannot be traced, cases are increasing, and there is delay between sx onset and case isolation. infection control measures ari etiquette-cover nose/mouth during cough or sneeze, use tissues, wash hands avoidance of public gatherings by persons at high risk of complications nb use of masks by well persons is not recommended "snow" (stay-at-home) days and self-shielding (reverse quarantine) for initial d period of community outbreak-may reduce transmission without explicit activity restrictions closure of schools, offices, large group gatherings, public transport (pedes more likely to transmit disease than adults) nb community quarantine (cordon sanitaire)-restriction of travel in and out of an area is unlikely to prevent introduction or spread of disease anopheles vector biology egg becomes adult mosquito in d adult mosquito becomes infective in d after bite on infected host susceptible human host becomes infective in d after bite from infected mosquito :. earliest human clinical disease in d after eggs are laid follow the -d rule: dusk and dawn stay indoors as much as possible with window screens in good repair dress in light colored long sleeve shirts and long pants when outside identify cause of the outbreak undertake vaccination campaign strengthen routine immunization and surveillance meningitis is a disease with significant mortality. meningococcus (neisseria meningitides) is renown for its rapid onset, rapid progression (death sometimes within hours), and high mortality ( % untreated). there are serogroups of neisseria meningitides but only (a, b, c, w, x, y) are known to cause epidemics. the bacteria spread from person to person via respiratory and nasal secretions. polysaccharide vaccines are available with serotypes (a and c), serotypes (a, c, and w) or serotypes (a, c, w, and y). duration of immunity is approximately years. meningococcal protein conjugate vaccines confer longer immunity but at higher cost than polysaccharide vaccines. monovalent conjugate vaccine against group c dates from , and tetravalent (a, c, w, and y) conjugate vaccine dates from . group b vaccine made from bacterial proteins has been licensed since but is not readily available. meningococcal vaccines have a very low incidence of side effects. regular disease surveillance is necessary to detect outbreaks. the epidemic threshold is suspected cases/ , population in any given week. two suspected cases of meningitis in the same settlement should trigger an outbreak investigation. nasopharyngeal carriage rates do not predict epidemics. - % of meningococcal disease presents with meningitis. % of cases occur in patients < y/o. peak incidence in meningitis belt is ages - yrs. diagnosis is straightforward when patient presents with signs of meningitis-fever, headache, vomiting, changes in mental status. however, most patients have non-specific illness - days before onset of meningitis. cfr of untreated meningococcal meningitis can be %. cfr of properly treated meningococcal meningitis is < %. - % of meningococcal disease presents with septicemia unaccompanied by meningitis or other focal features. it is a dramatic illness which affects previously healthy children and young adults. it presents with acute fever leading to purpura fulminans (hemorrhagic or purpuric rash), shock, and waterhouse-friderichsen syndrome (acute adrenal failure). etiologic diagnosis can be easily missed. cfr of meningococcal septicemia is % and may be % even with proper treatment. diagnosis may be confirmed by agglutination tests, polymerase chain reaction, culture and sensitivity testing of spinal fluid and blood. in many situations, these tests are not available. throat swabs may be helpful on occasions. do not delay treatment for tests or test results. minutes count. it is more important to have a live patient without a confirmed diagnosis than a dead one with a diagnosis. differential diagnosis in a tropical patient with fever and altered mental status, but without purpura or shock, includes cerebral malaria. co-infection may occur. standardized case management of bacterial meningitis in developed countries involves - days of parenteral antibiotic therapy. drug of choice in adults and older children is ceftriaxone which also rapidly eliminates the carrier state. alternate drugs include ampicillin and benzylpenicillin which do not eliminate the carrier state. in developing countries, days of parenteral antibiotic therapy are empirically shown to be effective. in large epidemics in resource-poor settings, a single im dose of chloramphenicol in oil is the drug of choice. for patients who do not improve in hours, a repeat dose may be given. viral meningitis is rarely serious and requires only supportive care, recovery is usually complete. patient isolation and disinfection of the room, clothing, or bedding are not necessary. respiratory precautions are advised particularly early in the course of treatment. chemoprophylaxis of contacts is available in some settings but rarely in the disaster setting. vigilance and education of close contacts is mandatory. epidemic preparedness and early detection of outbreaks are key. vaccines against n. meningitides serogroups a, c, y and w are very effective in controlling epidemics. in epidemic settings, children - are the priority target with serogroups a and c typically the priority antigens. rapid mass vaccination campaigns can contain outbreaks in - weeks. for immunocompetent patients over years, vaccine efficacy rate is % one week after injection. however, duration of immunity may be as little as years in younger children. in some countries, vaccine may also be used with close contacts of sporadic disease cases to prevent secondary cases. chemoprophylaxis of contacts is not recommended in epidemics, but community education and ready access to health care are essential. preventive medicine [ ] source control/reduction/elimination undertake quarantine and culling of sick reservoir animals and known disease carrier species. avoid unnecessary contact with or consumption of dead reservoir animals or known disease carrier species. avoid unnecessary contact with suspected reservoir animals and known disease carrier species (eg primates). avoid direct or close contact with symptomatic patients. establish appropriate communicable disease controls for burial of the dead. administrative controls (improve people's work practices) environmental and engineering controls (isolate people from the hazard) avoid needle stick exposure to blood specimens thru automated machine handling. ppe (protect people with ppe) use standard precautions-gloves, masks, and protective clothing-if handling infected animals or patients. wash hands after visiting sick patients. active surveillance and contact tracing (enhanced surveillance) through community-based mobile teams active case finding (screening and triage) and contact tracing dedicated isolation facility food provision to isolated patients so they are not dependent on family case definition treatment protocols emphasizing supportive care and treatment of complications essential drugs referral guidelines secondary prevention barrier nursing strictly enforced family and community education ministerial task force to address policy local health authority task force to address procedures national level task forces to comprise if a lab is not available, then you need a sampling strategy that addresses specimen acquisition, preparation, and transportation in compliance with international regulations on the transport of infectious substances. guidance note on using the cluster approach to strengthen humanitarian response international conference on primary health care selective primary health care-an interim strategy for disease control in developing countries water and excreta-related diseases: unitary environmental classification infections related to water and excreta: the health dimension of the decade world health organization. cholera vaccines: who position paper available from: international centre for diarrhoeal disease research history and epidemiology of global smallpox eradication available from: us department of health and human services communicable disease control in emergencies-a field manual. geneva: world health organization ebola: technical guidance documents for medical staff world health organization. manual for the care and management of patients in ebola care units/community care centers-interim emergency guidance. who/ evd/manual/ecu/ . . geneva: world health organization what tests does it perform? is there transport to and from the laboratory? who prepares transport media? who provides specimen collection material and supplies? how can these supplies be obtained? who provides cool packs, transport boxes, car, driver …? • refrigerate other vials for cytology, chemistry ( °c) leak-proof specimen container wrapped with enough absorbent material to absorb the entire content of the st container . leak-proof secondary container usually plastic or metal . outer shipping container whose smallest dimension is mm diagnostic specimens use iata packing instruction without biohazard label. infectious materials use iata packing instruction with biohazard label. what to send with the sample? lab request form with: • sender's name and contact info • patient name, age, sex • sample date, time • suspected clinical diagnosis with main signs and symptoms • sample macroscopic description • context-outbreak confirmation, ongoing verification, outbreak end, etc • epidemiological or demographic data where to send the sample? • reference lab • contact person what and when to expect results? source: world health organization world health organization department of communicable disease surveillance and response. highlights of specimen collection in emergency situations. undated . designate a lead official in the lcc. . anticipate roles for partner agencies (eg inter-agency and team coordination, disease surveillance, field epidemiological investigation, laboratory identification, case management guideline development, outbreak logistics, public information, and social mobilization). . identify sources of funds. . intensify disease surveillance. . identify reference lab(s) for communicable diseases of epidemic potential. . ensure mechanism for specimen transport. a. initial response to suspected outbreak . form an emergency team to investigate and manage the outbreak a. identify key roles on the outbreak investigation team(s) ( ) epidemiology and surveillance ( ) case management ( ) water and sanitation ( ) laboratory services ( ) communication b. staff those roles ( ) epidemiologist-to monitor proper data collection and surveillance procedures ( ) physician-to confirm clinical s/sx and train health workers in case management ( ) water and sanitation expert-to develop a plan for reducing sources of contamination ( ) microbiologist-to take environmental/biological samples for laboratory confirmation, train health workers in proper sampling techniques, and confirm use of appropriate methods in the diagnostic laboratory ( ) key: cord- -k wcibdk authors: pacheco, jorge m.; van segbroeck, sven; santos, francisco c. title: disease spreading in time-evolving networked communities date: - - journal: temporal network epidemiology doi: . / - - - - _ sha: doc_id: cord_uid: k wcibdk human communities are organized in complex webs of contacts that may be represented by a graph or network. in this graph, vertices identify individuals and edges establish the existence of some type of relations between them. in real communities, the possible edges may be active or not for variable periods of time. these so-called temporal networks typically result from an endogenous social dynamics, usually coupled to the process under study taking place in the community. for instance, disease spreading may be affected by local information that makes individuals aware of the health status of their social contacts, allowing them to reconsider maintaining or not their social contacts. here we investigate the impact of such a dynamical network structure on disease dynamics, where infection occurs along the edges of the network. to this end, we define an endogenous network dynamics coupled with disease spreading. we show that the effective infectiousness of a disease taking place along the edges of this temporal network depends on the population size, the number of infected individuals in the population and the capacity of healthy individuals to sever contacts with the infected, ultimately dictated by availability of information regarding each individual’s health status. importantly, we also show how dynamical networks strongly decrease the average time required to eradicate a disease. understanding disease spreading and evolution involves overcoming a multitude of complex, multi-scale challenges of mathematical and biological nature [ , ] . traditionally, the contact process between an infected individual and the susceptible ones was assumed to affect equally any susceptible in a population (mean-field approximation, well-mixed population approximation) or, alternatively, all those susceptible living in the physical neighborhood of the infected individual (spatial transmission). during recent years, however, it has become clear that disease spreading [ ] [ ] [ ] [ ] transcends geography: the contact process is no longer restricted to the immediate geographical neighbors, but exhibits the stereotypical small-world phenomenon [ ] [ ] [ ] [ ] , as testified by recent global pandemics (together with the impressive amount of research that has been carried out to investigate them) or, equally revealing, the dynamics associated with the spreading of computer viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recent advances in the science of networks [ , , , , ] also provided compelling evidence of the role that the networks of contacts between individuals or computers play in the dynamics of infectious diseases [ , ] . in the majority of cases in which complex networks of disease spreading have been considered [ ] , they were taken to be a single, static entity. however, contact networks are intrinsically temporal entities and, in general, one expects the contact process to proceed along the lines of several networks simultaneously [ , - , , , , - ] . in fact, modern societies have developed rapid means of information dissemination, both at local and at centralized levels, which one naturally expects to alter individuals' response to vaccination policies, their behavior with respect to other individuals and their perception of likelihood and risk of infection [ ] . in some cases one may even witness the adoption of centralized measures, such as travel restrictions [ , ] or the imposition of quarantine spanning parts of the population [ ] , which may induce abrupt dynamical features onto the structure of the contact networks. in other cases, social media can play a determinant role in defining the contact network, providing crucial information on the dynamical patterns of disease spreading [ ] . furthermore, the knowledge an individual has (based on local and/or social media information) about the health status of acquaintances, partners, relatives, etc., combined with individual preventive strategies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (such as condoms, vaccination, the use of face masks or prophylactic drugs, avoidance of visiting specific web-pages, staying away from public places, etc.), also leads to changes in the structure and shape of the contact networks that naturally acquire a temporal dimension that one should not overlook. naturally, the temporal dimension and multitude of contact networks involved in the process of disease spreading render this problem intractable from an analytic standpoint. recently, sophisticated computational platforms have been developed to deal with disease prevention and forecast [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the computational complexity of these models reflects the intrinsic complexity of the problem at stake, and their success relies on careful calibration and validation procedures requiring biological and socio-geographic knowledge of the process at stake. our goal here, instead, will be to answer the following question: what is the impact of a temporal contact network structure in the overall dynamics of disease progression? does one expect that it will lead to a rigid shift of the critical parameters driving disease evolution, as one witnesses whenever one includes spatial transmission patterns? or even to an evanescence of their values whenever one models the contact network as a (static and infinite) scale-free network, such that the variance of the network degree distribution becomes arbitrarily large? or will the temporal nature of the contact network lead to new dynamical features? and, if so, which features will emerge from the inclusion of this temporal dimension? to answer this question computationally constitutes, in general, a formidable challenge. we shall attempt to address the problem analytically, and to this end some simplifications will be required. however, the simplifications we shall introduce become plausible taking into consideration recent results (i) in the evolutionary dynamics of social dilemmas of cooperation, (ii) in the dynamics of peer-influence, and even (iii) in the investigation of how individual behavior determines and is determined by the global, population wide behavior. all these recent studies point out to the fact that the impact of temporal networks in the population dynamics stems mostly from the temporal part itself, and not so much from the detailed shape and structure of the network [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, we now know that (i) different models of adaptive network dynamics lead to similar qualitative features regarding their impact in what concerns the evolution of cooperation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , (ii) the degree of peer-influence is robust to the structural patterns associated with the underlying social networks [ ] , and (iii) the impact of temporal networks in connecting individual to collective behavior in the evolution of cooperation is very robust and related to a problem of n-body coordination [ , ] . altogether, these features justify that we model the temporal nature of the contact network in terms of a simple, adaptive network, the dynamics of which can be approximately described in terms a coupled system of odes. this "adaptive-linking" dynamics, as it was coined [ , [ ] [ ] [ ] , leads to network snapshot structures that do not replicate what one observes in real-life, in the same sense that the small-world model of watts and strogatz does not lead to the heterogeneous and diverse patterns observed in data snapshots of social networks. notwithstanding, the active-linking dynamics allows us to include, analytically, the temporal dimension into the problem of disease dynamics. the results [ ] , as we elaborate in sects. and , prove rewarding, showing that the temporal dimension of a contact network leads to a shift of the critical parameters (defined below) which is no longer rigid but, instead, becomes dependent on the frequency of infected individuals in the population. this, we believe, constitutes a very strong message with a profound impact whenever one tries to incorporate the temporal dimension into computational models of disease forecast. this chapter is organized as follows. in the following sect. , we introduce the standard disease models we shall employ, as well as the details of the temporal contact network model. section is devoted to present and discuss the results, and sect. contains a summary of the main conclusions of this work. in this section, we introduce the disease models we shall employ which, although well-known and widely studied already, are here introduced in the context of stochastic dynamics in finite populations, a formulation that has received less attention than the standard continuous model formulation in terms of coupled ordinary differential equations (odes). furthermore, we introduce and discuss in detail the temporal contact network model. here we introduce three standard models of disease transmission that we shall employ throughout the manuscript, using this section at profit to introduce also the appropriate notation associated with stochastic dynamics of finite populations and the markov chain techniques that we shall also employ in the remainder of this chapter. we shall start by discussing the models in the context of well-mixed populations, which will serve as a reference scenario for the disease dynamics, leaving for the next section the coupling of these disease models with the temporal network model described below. we investigate the popular susceptible-infected-susceptible (sis) model [ , ] , the susceptible-infected (si) model [ ] used to study, e.g., aids [ , ] , and the susceptible-infected-recovered (sir) model [ , ] , more appropriate to model, for instance, single season flu outbreaks [ ] or computer virus spreading [ ] . it is also worth pointing out that variations of these models have been used to successfully model virus dynamics and the interplay between virus dynamics and the response of the immune system [ ] . in the sis model individuals can be in one of two epidemiological states: infected (i) or susceptible (s). each disease is characterized by a recovery rate (•) and an infection rate (oe). in an infinite, well-mixed population, the fraction of infected individuals (x) changes in time according to the following differential equation where y d x is the fraction of susceptible individuals and hki the average number of contacts of each individual [ ] . there are two possible equilibria (p x d ): x d and x d r , where r d hki/ı denotes the basic reproductive ratio. the value of r determines the stability of these two equilibria: x d r is stable when r > and unstable when r < . let us now move to finite populations, and consider the well-mixed case where the population size is fixed and equal to n. we define a discrete stochastic markov process describing the disease dynamics associated with the sis model. each configuration of the population, which is defined by the number of infected individuals i, corresponds to one state of the markov chain. time evolves in discrete steps and two types of events may occur which change the composition of the population: infection events and recovery events. this means that, similar to computer simulations of the sis model on networked populations, at most one infection or recovery event will take place in each (discrete) time step. thus, the dynamics can be represented as a markov chain m with nc states [ , ] -as many as the number of possible configurations -illustrated in the following fig. . . in a finite, well-mixed population, the number i of infected will decrease at a rate given by where denotes the recovery time scale, i n the probability that a randomly selected individual is infected and ı the probability that this individual recovers. adopting as a reference, we assume that the higher the average number of contacts hki, the smaller the time scale inf at which infection update events occur ( inf d /hki) [ ] . consequently, the number of infected will also increase at a rate given by equations ( . ) and ( . ) define the transitions between different states. this way, we obtain the following transition matrix for m: where each element p kj of p represents the probability of moving from state k to state j during one time step. the state without any infected individual (id ) is an absorbing state of m. in other words, the disease always dies out and will never re-appear, once this happens. at this level of approximation, it is possible to derive an analytical expression for the average time t i it takes to reach the single absorbing state of the sis markov chain (i.e., the average time to absorption) starting from a configuration in which there are i infected individuals. denoting by p i (t) the probability that the disease disappears at time t when starting with i infected individuals at time , we may write [ ] using the properties of p i (t) we obtain the following recurrence relation for t i whereas for t n we may write defining the auxiliary variables i d l , a little algebra allows us to write, for t such that t i can be written as a function of t as follows the intrinsic stochasticity of the model, resulting from the finiteness of the population, makes the disease disappear from the population after a certain amount of time. as such, the population size plays an important role in the average time to absorption associated with a certain disease, a feature we shall return to below. equations ( . ) and ( . ) define the markov chain m just characterized. the fraction of time the population spends in each state is given by the stationary distribution of m, which is defined as the eigenvector associated with eigenvalue of the transition matrix of m [ , ] . the fact that in the sis model the state without infected (id ) is an absorbing state of the markov chain, implies that the standard stationary distribution will be completely dominated by this absorbing state, which precludes one to gather information on the relative importance of other configurations. this makes the so-called quasi-stationary distribution of m [ ] the quantity of interest. this quantity allows us to estimate the relative prevalence of the population in configurations other than the absorbing state, by computing the stationary distribution of the markov chain obtained from m by excluding the absorbing state id [ ] . it provides information on the fraction of time the population spends in each state, assuming the disease does not go extinct. the markov process m defined before provides a finite population analogue of the well-known mean-field equations written at the beginning of sect. . . . indeed, in the limit of large populations, g.i/ d t c .i/ t .i/ provides the rate of change of infected individuals. for large n, replacing i n by x and n i n by y, the gradients of infection which characterize the rate at which the number of infected are changing in the population, are given by again, we obtain two roots: g(i) d for i d and i r d n .n / ı hki . moreover, i r becomes the finite population equivalent of an interior equilibrium for r Á ı hki n n > (note that, for large n we have that n n ). the disease will most likely expand whenever i < i r , the opposite happening otherwise. the si model is mathematically equivalent to the sis model with ı d , and has been employed to study for instance the dynamics of aids. the markov chain representing the disease dynamics is therefore defined by transition matrix eq. ( . ), with t i d for all i. the remaining transition probabilities t c i ( < i < n) are exactly the same as for the sis model. since all t i equal zero, the markov chain has two absorbing states: the canonical one without any infected (id ) and the one without any susceptible (idn). the disease will expand monotonically as soon as one individual in the population gets infected, ultimately leading to a fully infected population. the average amount of time after which this happens, which we refer to as the average infection time, constitutes the main quantity of interest. this quantity can be calculated analytically [ ] : the average number of time steps needed to reach % infection, starting from i infected individuals is given by ( . ) with sir one models diseases in which individuals acquire immunity after recovering from infection. we distinguish three epidemiological states to model the dynamics of such diseases: susceptible (s), infected (i) and recovered (r), indicating those who have become immune to further infection. the sir model in infinite, well-mixed populations is defined by a recovery rate ı and an infection rate . the fraction of infected individuals x changes in time according to the following differential equation where y denotes the fraction of susceptible individuals, which in turn changes according to p y d hki xy: ( . ) finally, the fraction of individuals z in the recovered class changes according to p z d ı x: ( . ) to address the sir model in finite, well-mixed populations, we proceed in a way similar to what we have done so far with sis and si models. the markov chain describing the disease dynamics becomes slightly more complicated and has states (i, r), where i is the number of infected individuals in the population and r the number of recovered (and immune) individuals (i c r Ä n). a schematic representation of the markov chain is given in fig. . . note that the states ( , r), with Ä r Ä n, are absorbing states. each of these states corresponds to the number of individuals that are (or have become) immune at the time the disease goes extinct. consider a population of size n with average degree hki. the number of infected will increase with a rate where denotes the recovery time scale. as before, the gradient of infection g(i), such that g.i/ d t c .i/ t .i/, measures the likelihood for the disease to either expand or shrink in a given state, and is given by note that we recover eq. ( . ) in the limit n ! . for a fixed number of recovered individuals r , we have that g(i, r ) d for i d and for i r d n becomes the finite population analogue of an interior equilibrium. furthermore, one can show that the partial derivative @g.i;r/ @i has at most one single root in ( , ), possibly located at i r d i r Ä i r . hence, g(i, r ) reaches a local maximum at i r (given that at that point @ g.i;r/ @i ˇi r d hki n.n / < ). the number of infected will therefore most likely increase for i < i r (assuming r immune individuals), and most likely decrease otherwise. the gradient of infection also determines the probability to end up in each of the different absorbing states of the markov chain. these probabilities can be calculated analytically [ ] . to this end, let us use y a i;r to denote the probability that the population ends up in the absorbing state with a recovered individuals, starting from a state with i infected and r recovered. we obtain the following recurrence relationship for y a i;r y a i;r d t .i; r/ y a i ;rc c t c .i; r/ y a ic ;r c t .i; r/ t c .i; r/ y a i;r ; ( . ) which reduces to the following boundary conditions ( . ) allow us to compute y a i;r for every a, i and r. our network model explicitly considers a finite and constant population of n individuals. its temporal contact structure allows, however, for a variable number of overall links between individuals, which in turn will depend on the incidence of disease in the population. this way, infection proceeds along the links of a contact network whose structure may change based on each individual's health status and the availability of information regarding the health status of others. we shall assume the existence of some form of local information about the health status of social contacts. information is local, in the sense that individual behavior will rely on the nature of their links in the contact network. moreover, this will influence the way in which individuals may be more or less effective in avoiding contact with those infected while remaining in touch with the healthy. suppose all individuals seek to establish links at the same rate c. for simplicity, we assume that new links are established and removed randomly, a feature which usually does not always apply in real cases, where the limited social horizon of individuals or the nature of their social ties may constrain part of their neighborhood structure (see below). let us further assume that links may be broken off at different rates, based on the nature of the links and the information available about the individuals they connect: let us denote these rates by b pq for links of type pq (p , q fs, i, rg. we assume that links are bidirectional, which means that we have links of pq types si, sr, and ir. let l pq denote the number of links of type pq and l m pq the maximum possible number of links of that type, given the number of individuals of type s, i and r in the population. this allows us to write down (at a mean-field level) a system of odes [ , ] for the time evolution of the number of links of pq-type (l pq ) [ , ] which depends on the number of individuals in states p and q (l m pp d p .p / = and l m pq d pq for p ¤ q) and thereby couples the network dynamics to the disease dynamics. in the steady state of the linking dynamics ( p l pq d ), the number of links of each type is given by l pq d ' pq l m pq , with ' pq d c/(c c b pq ) the fractions of active pq-links, compared to the maximum possible number of links l m pq , for a given number of s, i and r. in the absence of disease only ss links exist, and hence ss determines the average connectivity of the network under disease free conditions, which one can use to characterize the type of the population under study. in the presence of i individuals, to the extent that s individuals manage to avoid contact with i, they succeed in escaping infection. thus, to the extent that individuals are capable of reshaping the contact network based on available information of the health status of other individuals, disease progression will be inhibited. in the extreme limit of perfect information and individual capacity to immediately break up contacts with infected, we are isolating all infected, and as such containing disease progression. our goal here, however, is to understand how and in which way local information, leading to a temporal reshaping of the network structure, affects overall disease dynamics. we investigate the validity of the approximations made to derive analytical results as well as their robustness by means of computer simulations. all individual-based simulations start from a complete network of size nd . disease spreading and network evolution proceed together under asynchronous updating. disease update events take place with probability ( c ) , where d net / dis . we define dis as the time-scale of disease progression, whereas net is the time scale of network change. the parameter d net / dis provides the relative time scale in terms of which we may interpolate between the limits when network adaptation is much slower than disease progression ( ! ) and the opposite limit when network adaptation is much faster than disease progression ( ! ). since d net / dis is the only relevant parameter, we can make, without loss of generality, dis d . for network update events, we randomly draw two nodes from the population. if connected, then the link disappears with probability given by the respective b pq . otherwise, a new link appears with probability c. when a disease update event occurs, a recovery event takes place with probability ( c hki) , an infection event otherwise. in both cases, an individual j is drawn randomly from the population. if j is infected and a recovery event has been selected then j will become susceptible (or recovered, model dependent) with probability •. if j is susceptible and an infection event occurs, then j will get infected with probability oe if a randomly chosen neighbor of j is infected. the quasi-stationary distributions are computed (in the case of the sis model) as the fraction of time the population spends in each configuration (i.e., number of infected individuals) during disease event updates ( generations; under asynchronous updating, one generation corresponds to n update events, where n is the population size; this means that in one generation, every individual has one chance, on average, to update her epidemic state). the average number of infected hii and the mean average degree of the network hki observed during these generations are kept for further plotting. we have checked that the results reported are independent of the initial number of infected in the network. finally, for the sir and si models, the disease progression in time, shown in the following sections, is calculated from independent simulations, each simulation starting with infected individual. the reported results correspond to the average amount of time at which i individuals become infected. in this section we start by (i) showing that a quickly adapting community induces profound changes in the dynamics of disease spreading, irrespective of the underlying epidemic model; then, (ii) we resort to computer simulations to study the robustness of these results for intermediate time-scales of network adaptation; finally, (iii) we profit from the framework introduced above to analyze the impact of information on average time for absorption and disease progression in adaptive networks. empirically, it is well-known that often individuals prevent infection by avoiding contact with infected once they know the state of their contacts or are aware of the potential risks of such infection [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] : such is the case of many sexually transmitted diseases [ , [ ] [ ] [ ] , for example, and, more recently, the voluntary use of face masks and the associated campaigns adopted by local authorities in response to the sars outbreak [ , [ ] [ ] [ ] or even the choice of contacting or not other individuals based on information on their health status gathered from social media [ , , ] . in the present study, individual decision is based on available local information about the health state of one's contacts. thus, we can study analytically the limit in which the network dynamics -resulting from adaptation to the flow of local information -is much faster than disease dynamics, as in this case, one may separate the time scales between network adaptation and contact (disease) dynamics: the network has time to reach a steady state before the next contact takes place. consequently, the probability of having an infected neighbor is modified by a neighborhood structure which will change in time depending on the impact of the disease in the population and the overall rates of severing links with infected. let us start with the sir model. the amount of information available translates into differences mostly between the break-up rates of links that may involve a potential risk for further infection (b si , b ir , b ii ), and those that do not (b ss , b sr , b rr ). therefore, we consider one particular rate b i for links involving infected individuals (b i Á b si d b ir d b ii ), and another one, b h , for links connecting healthy . in general, one expects b i to be maximal when each individual has perfect information about the state of her neighbors and to be (minimal and) equal to b h when no information is available, turning the ratio between these two rates into a quantitative measure of the efficiency with which links to infected are severed compared to other links. note that we reduce the model to two break-up rates in order to facilitate the discussion of the results. numerical simulations show that the general principles and conclusions remain valid when all break-up rates are incorporated explicitly. it is worth noticing that three out of these six rates are of particular importance for the overall disease dynamics: b ss , b sr and b si . these three rates, combined with the rate c of creating new links, define the fraction of active ss, sr and si links, and subsequent correlations between individuals [ ] , and therefore determine the probability for a susceptible to become infected (see models and methods). this probability will increase when considering higher values of c (assuming b i > b h ). in other words, when individuals create new links more often, therefore increasing the likelihood of establishing connections to infected individuals (when present), they need to be better informed about the health state of their contacts in order to escape infection. in the fast linking limit, the other three break-up rates (b ii , b ir and b rr ) will also influence disease progression since they contribute to changing the average degree of the network. when the time scale for network update ( net ) is much smaller than the one for disease spreading ( dis ), we can proceed analytically using at profit the separation of times scales. in practice, this means that the network has time to reach a steady state before the next disease event takes place. consequently, the probability of having an infected neighbor is modified by a neighborhood structure which will change in time depending on the impact of the disease in the population and the overall rates of severing links with infected individuals. for a given configuration (i,r) of the population, the stationary state of the network is characterized by the parameters ' ss , ' si and ' sr . consequently, the number of infected increases at a rate [ ] where we made d . the effect of the network dynamics becomes apparent in the third factor, which represents the probability that a randomly selected neighbor of a susceptible is infected. in addition, eq. ( . ) remains valid, as the linking dynamics does not affect the rate at which the number of infected decreases. it is noteworthy that we can write eq. ( . ) in the form which is formally equivalent to eq. ( . ) and shows that disease spreading in a temporal adaptive network is equivalent to that in a well-mixed population with (i) a frequency dependent average degree hki and (ii) a transmission probability that is rescaled compared to the original according to note that this expression remains valid for both sir, sis (r d ) and si (ı d , r d ) models. since the lifetime of a link depends on its type, the average degree hki of the network depends on the number of infected in the population, and hence becomes frequency (and time) dependent, as hki depends on the number of infected (through l m pq ) and changes in time. note that Á scales linearly with the frequency of infected in the population, decreasing as the number of infected increases (assuming ss ı si > ); moreover, it depends implicitly (via the ratio ss ı si ) on the amount of information available. it is important to stress the distinction between the description of the disease dynamics at the local level (in the vicinity of an infected individual) and that at the population wide level. strictly speaking, a dynamical network does not change the disease dynamics at the local level, meaning that infected individuals pass the disease to their neighbors with probability intrinsic to the disease itself. at the population level, on the other hand, disease progression proceeds as if the infectiousness of the disease effectively changes, as a result of the network dynamics. consequently, analyzing a temporal network scenario at a population level can be achieved via a renormalization of the transmission probability, keeping the (mathematically more attractive) well-mixed scenario. in this sense, from a well-mixed perspective, dynamical networks contribute to changing the effective infectiousness of the disease, which becomes frequency and information dependent. note further that this information dependence is a consequence of using a single temporal network for spreading the disease and information. interestingly, adaptive networks have been shown to have a similar impact in social dilemmas [ ] . from a global, population-wide perspective, it is as if the social dilemma at stake differs from the one every individual actually plays. as in sect. , one can define a gradient of infection g, which measures the tendency of the disease to either expand or shrink in a population with given configuration (defined by the number of individuals in each of the states s, i and r). to do so, we study the partial derivative @g.i;r/ @i at i d this quantity exceeds zero whenever note that taking r d yields the basic reproductive ratio r a for both sir and sis: on the other hand, whenever r a < , eradication of the disease is favored in the sis model (g(i)< ), irrespective of the fraction of infected, indicating how the presence of information (b h < b i ) changes the basic reproductive ratio. in fig. . we illustrate the role of information in the sis model by plotting g for different values of b i (assuming b h < b i ) and a fixed transmission probability . the corresponding quasi-stationary distributions are shown in the right panel and clearly reflect the sign of g. whenever g(i) is positive (negative), the dynamics will act to increase (decrease), on average, the number of infected. figure population and, once again, allows us to identify when disease expansion will be favored or not. figure . gives a complete picture of the gradient of infection, using the appropriate simplex structure in which all points satisfy the relation icrcsdn. the dashed line indicates the boundary g(i, r) d in case individuals do not have any information about the health status of their contacts, i.e., links that involve infected individuals disappear at the same rate as those that do not (b i d b h ). disease expansion is more likely than disease contraction (g(i, r) > ) when the population is in a configuration above the line, and less likely otherwise. similarly, the solid line indicates the boundary g(i, r) d when individuals share information about their health status, and use it to avoid contact with infected. once again, the availability of information modifies the disease dynamics, inhibiting disease progression for a broad range of configurations. up to now we have assumed that the network dynamics proceeds much faster than disease spreading (the limit ! ). this may not always be the case, and hence it is important to assess the domain of validity of this limit. in the following, we use computer simulations to verify to which extent these results, obtained analytically via time scale separation, remain valid for intermediate values of the relative timescale for the linking dynamics. we start with a complete network of size n, in which initially one individual is infected, the rest being susceptible. as stated before, disease spreading and network evolution proceed simultaneously under asynchronous updating. network update events take place with probability ( c ) , whereas a disease model (si, sis or sir) state update event occurs otherwise. for each value of , we run simulations. for the si model, the quantity of interest to calculate is the average number of generations after which the population becomes completely infected. these values are depicted in fig. . . the lower dashed line indicates the analytical prediction of the infection time in the limit ! (the limit when networks remain static), which we already recover in the simulations for > . when is smaller than , the average infection time significantly increases, and already reaches the analytical prediction for the limit ! (indicated by the upper dashed line) when < . hence, the validity of the time scale separation does again extend well beyond the limits one might expect. for the sir model, we let the simulations run until the disease goes extinct, and computed the average final fraction of individuals that have been affected by is given by eqs. ( . ) and ( . ) . one observes that linking dynamics does not affect disease dynamics for > . once drops below ten, a significantly smaller fraction of individuals is affected by the disease. this fraction reaches the analytical prediction for ! as soon as < . . hence, and again, results obtained via separation of time scales remain valid for a wide range of intermediate time scales. we finally investigate the role of intermediate time scales in the sis model. we performed computer simulations in the conditions discussed already, and computed several quantities that we plot in fig. . . figure . shows the average hii of the quasi-stationary distributions obtained via computer simulations (circles) as a function of the relative time scale of network update. whenever ! , we can characterize the disease dynamics analytically, assuming a well-mixed population (complete graph), whereas for ! we recover the analytical results obtained in the fast linking limit. at intermediate time scales, fig. . shows that as long as is smaller than ten, network dynamics contributes to inhibit disease spreading by effectively increasing the critical infection rate. overall, the validity of the time scale separation extends well beyond the limits one might anticipate based solely on the time separation ansatz. as long as the time scale for network update is smaller than the one for disease spreading ( < ), the analytical prediction for the limit ! , indicated by the lower dashed line in fig. . , remains valid. the analytical result in the extreme opposite limit ( ! ), indicated by the upper dashed line in fig. . , holds as long as > . moreover, it is noteworthy that the network dynamics influences the disease dynamics both by reducing the frequency of interactions between susceptible and infected, and by reducing the average degree of the network. these complementary effects are disentangled in intermediate regimes, in which the network dynamics is too slow to warrant sustained protection of susceptible individuals from contacts with infected, despite managing to reduce the average degree (not shown). in fact, for > the disease dynamics is mostly controlled by the average degree, as shown by the solid lines in fig. . . here, the average stationary distribution was determined by replacing, in the analytic expression for static networks, hki by the time-dependent average connectivity hki computed numerically. this, in turn, results from the frequency dependence of hki. when b i > b h , the network will reshape into a configuration with smaller hki as soon as the disease expansion occurs. for < , hki reflects the lifetime of ss links, as there are hardly any infected in the population. for < < , the network dynamics proceeds fast enough to reduce hki, but too slowly to reach its full potential in hindering disease progression. given the higher fraction of infected, and the fact that si and ii links have a shorter lifetime than ss links, the average degree drops when increasing from to . any further increase in leads to a higher average degree, as the network approaches its static limit. contrary to the deterministic sis model, the stochastic nature of disease spreading in finite populations ensures that the disease disappears after some time. however, this result is of little relevance given the times required to reach the absorbing state (except, possibly, in very small communities). indeed, the characteristic time scale of the dynamics plays a determinant role in the overall epidemiological process and constitutes a central issue in disease spreading. figure . shows the average time to absorption t in adaptive networks for different levels of information, illustrating the spectacular effect brought about by the network dynamics on t . while on networks without information (b i d b h ) t rapidly increases with the rate of infection oe, adding information moves the fraction of infected individuals rapidly to the absorbing state, and, therefore, to the disappearance of the disease. moreover, the size of the population can have a profound effect on t . with increasing population size, the population spends most of the time in the vicinity of the state associated with the interior root of g(i). for large populations, this acts to reduce the intrinsic stochasticity of the dynamics, dictating a very slow extinction of the disease, as shown in fig. . . when recovery from the disease is impossible, a situation captured by the si model, the population will never become disease-free again once it acquires at least one infected individual. the time to reach absorbing state in which all individuals are infected, again depends on the presence of information. when information prevails, susceptible individuals manage to resist infection for a long time, thereby delaying the rapid progression of the disease, as shown in the inset of fig. . . naturally, the average number of generations needed to reach a fully infected population increases with the availability of information, as illustrated in the main panel of fig. . . making use of three standard models of epidemics involving a finite population in which infection takes place along the links of a temporal graph, the nodes of which are occupied by individuals, we have shown analytically that the bias introduced into the graph dynamics resulting from the availability of information about the health status of others in the population induces fundamental changes in the overall dynamics of disease progression. the network dynamics employed here differs from those used in most other studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we argue, however, that the differences obtained stem mostly from the temporal aspect of the network, and not so much from the detailed dynamics that is implemented. importantly, temporal network dynamics leads to additional changes in r compared to those already obtained when moving from the well-mixed assumption to static networks [ ] . an important ingredient of our model, however, is that the average degree of the network results from the selforganization of the network structure, and co-evolves with the disease dynamics. a population suffering from high disease prevalence where individuals avoid contact in order to escape infection will therefore exhibit a lower average degree than a population with hardly any infected individuals. such a frequency-dependent average degree further prevents that containment of infected individuals would result in the formation of cliques of susceptible individuals, which are extremely vulnerable to future infection, as reported before [ , , ] . the description of disease spreading as a stochastic contact process embedded in a markov chain constitutes a second important ingredient of the present model. this approach allows for a direct comparison between analytical predictions and individual-based computer simulations, and for a detailed analysis of finite-size effects and convergence times, whose exponential growth will signal possible bistable disease scenarios. in such a framework, we were able to show that temporal adaptive networks in which individuals may be informed about the health status of others lead to a disease whose effective infectiousness depends on the overall number of infected in the population. in other words, disease propagation on temporal adaptive networks can be seen as mathematically equivalent to disease spreading on a well-mixed population, but with a rescaled effective infectiousness. in accord with the intuition advanced in the introduction, as long as individuals react promptly and consistently to accurate available information on whether their acquaintances are infected or not, network dynamics effectively weakens the disease burden the population suffers. last but not least, if recovery from the disease is possible, the time for disease eradication drastically reduces whenever individuals have access to accurate information about the health state of their acquaintances and use it to avoid contact with those infected. if recovery or immunity is impossible, the average time needed for a disease to spread increases significantly when such information is being used. in both cases, our model clearly shows how availability of information hinders disease progression (by means of quick action on infected, e.g., their containment via link removal), which constitutes a crucial factor to control the development of global pandemics. finally, it is also worth mentioning that knowledge about the health state of others may not always be accurate or available in time. this is for instance the case for diseases where recently infected individuals remain asymptomatic for a substantial period. the longer the incubation period associated with the disease, the less successful individuals will be in escaping infection, which in our model translates into a lower effective rate of breaking si links, with the above mentioned consequences. moreover, different (social) networks through which awareness of the health status of others proceeds may lead to different rates of information spread. one may take these features into account by modeling explicitly the spread of information through a coupled dynamics between disease expansion and individuals' awareness of the disease [ , ] . creation and destruction of links may for instance not always occur randomly, as we assumed here, but in a way that is biased by a variety of factors such as social and genetic distance, geographical proximity, family ties, etc. the resulting contact network may therefore become organized in a specific way, promoting the formation of particular structures, such as networks characterized by long-tailed degree distributions or with strong topological correlations among nodes [ , [ ] [ ] [ ] which, in turn, may influence the disease dynamics. the impact of combining such effects, resulting from specific disease scenarios, with those reported here will depend on the prevalence of such additional effects when compared to linkrewiring dynamics. a small fraction of non-random links, or of ties which cannot be broken, will likely induce small modifications on the average connectivity of the contact network, which can be incorporated in our analytic expressions without compromising their validity regarding population wide dynamics. on the other hand, when the contact network is highly heterogeneous (e.g., exhibiting pervasive long-tail degree distributions), non-random events may have very distinct effects, from being almost irrelevant (and hence can be ignored) to inducing hierarchical cascades of infection [ ] , in which case our results will not apply. modeling infectious diseases in humans and animals infectious diseases in humans evolution of networks: from biological nets to the internet and www dynamical processes in complex networks epidemic processes in complex networks small worlds: the dynamics of 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in evolutionary games repeated games and direct reciprocity under active linking reacting differently to adverse ties promotes cooperation in social networks selection pressure transforms the nature of social dilemmas in adaptive networks origin of peer influence in social networks linking individual and collective behavior in adaptive social networks uses and abuses of mathematics in biology a contribution to the mathematical theory of epidemics production of resistant hiv mutants during antiretroviral therapy a first course in stochastic processes stochastic processes in physics and chemistry fixation of strategies for an evolutionary game in finite populations on the quasi-stationary distribution of the stochastic logistic epidemic men's behavior change following infection with a sexually transmitted disease an examination of the social networks and social isolation in older and younger adults living with hiv/aids social stigmatization and hepatitis c virus infection assessing vaccination sentiments with online social media: implications for infectious disease dynamics and control social and news media enable estimation of epidemiological patterns early in the haitian cholera outbreak the effects of local spatial structure on epidemiological invasions epidemic processes over adaptive state-dependent networks classes of small-world networks statistical mechanics of complex networks the structure and function of complex networks velocity and hierarchical spread of epidemic outbreaks in scale-free networks key: cord- -mcxbl mv authors: vijayan, vannan k. title: diagnosis of pulmonary parasitic diseases date: - - journal: parasitic diseases of the lungs doi: . / - - - - _ sha: doc_id: cord_uid: mcxbl mv the protozoal and helminthic parasites that traverse the respiratory tract during their life cycles can cause lung diseases, though the most common habitats of these parasites are the gastrointestinal tract and the blood or lymphatic circulations. these diseases are commonly encountered in the tropical regions of the world. however, parasitic lung diseases are increasingly being reported from other parts of the world due to an increase in the occurrence of immunosuppression (acquired immunodeficiency syndrome, organ transplantations, the use of immunosuppressive drugs) and transcontinental travel. the lung diseases that may result from these infections range from asymptomatic phase to life-threatening acute respiratory distress syndrome. these diseases can also mimic common respiratory diseases such as bacterial pneumonias, pulmonary tuberculosis, lung cancer, bronchial asthma, interstitial lung disease, and pulmonary hypertension. the diagnosis of parasitic lung diseases is a challenge to physicians, if they are not aware of the entity or these diseases are not investigated properly. the diagnosis of these diseases is based on the identification of the causative organism in the stool, sputum, other body fluids, or tissue specimens. radiological imaging studies of the thorax including chest radiographs, high-resolution computerized tomograms, and ultrasonograms may aid in the diagnosis. in certain situations, invasive investigations such as fiberoptic bronchoscopic evaluation (transbronchial lung biopsies and bronchoalveolar lavage studies) and thoracic surgical procedures (thoracoscopy and open lung biopsy) may be required for a diagnosis and also to exclude other lung diseases. serologic and molecular diagnostic methods are being developed for accurate diagnosis of the parasitic diseases. pulmonary parasitic lung diseases are commonly diagnosed in countries where the prevalence of parasitic infection is high. however, there is an increase in the number of patients diagnosed as parasitic lung diseases recently, even in countries of low prevalence of parasitic infection which demands an awareness of such diseases in these countries. this increase in diagnosis in countries with low prevalence with parasitic infections has been attributed to an increase in the numbers of immunosuppressed individuals due to various reasons, organ transplantations and global travel [ ] . the parasites can cause a wide spectrum of lung diseases varying from mild self-limiting bronchitis to life-threatening acute respiratory distress syndrome [ ] . in addition, parasitic lung diseases may mimic diseases such as bacterial pneumonias, pulmonary tuberculosis, bronchial asthma, lung cancer, interstitial lung disease, and pulmonary hypertension. both protozoal and helminthic parasites can cause lung diseases and helminthic lung infections are important causes of eosinophilic lung diseases [ ] . though the treatment of parasitic lung diseases is with specifi c antiparasitic drugs, the physicians treating such diseases should be competent in tackling the specifi c issues related to lung injury and sequel that may follow such infections. the lung diseases that may result from infections with parasites are listed in table . [ ] . the important protozoal parasites that cause pulmonary diseases are entamoeba histolytica , leishmania donovani , malarial parasites ( plasmodium vivax , p . falciparum , p . malaria , p . ovale , and p . knowlesi ), babesia spp. ( babesia microti and babesia divergens ), and toxoplasma gondii . amebiasis results from ingestion of mature entamoeba histolytica cysts in fecally contaminated food, water or from hands. infection with entamoeba histolytica can lead to intestinal colonization, colitis or extraintestinal manifestations resulting from the hematogenous spread of infection from the intestine. about % of intestinal colonization is with nonpathogenic species, entamoeba dispar and entamoeba moshkovskii . invasive amebiasis occurs in % of persons colonized with e . histolytica . patients with amebic colitis present with several-week history of cramping abdominal pain, weight loss, and watery or bloody diarrhea. extraintestinal amebic infection can manifest as amebic liver abscess, splenic abscess, brain abscess, empyema, and pericarditis. nearly % of cases of amebic liver abscesses occur in the right lobe. the most common complication of amebic liver abscess is rupture into the pleural space resulting in pleuropulmonary amebiasis [ ] . the main symptoms in pleuropulmonary amebiasis are fever, cough, hemoptysis, right upper quadrant abdominal pain, and chest pain. some patients may present with respiratory distress and shock. lung abscess, hepatobronchial fi stula, and bronchopleural fi stula with pyopneumothorax have also been reported. expectoration of anchovy saucelike pus indicates amebiasis [ ] . the fi ndings of elevated hemidiaphragm, tender hepatomegaly, pleural effusion, and basal pulmonary involvement are suggestive of pleuropulmonary amebiasis. amebiasis can be suspected in patients with a history of immigration from or travel to developing countries and many patients give history of dysentery and alcoholism. amebiasis is commonly diagnosed by microscopy and cysts or motile trophozoites can be identifi ed on a saline wet mount of a stool specimen [ ] . microscopic examination of fresh stools, sputum or pleural pus, rectal smears or rectosigmoidoscopy materials, pus from liver abscesses, and colonic biopsy samples may reveal motile trophozoites, even though it is a relatively specifi c test but is not sensitive for the identifi cation of e . histolytica . the presence of ameba in the stool does not indicate that the disease is due to pathogenic e . histolytica as two other nonpathogenic species found in humans ( e . dispar and e . moshkovskii ) are indistinguishable morphologically [ , ] . this method can, therefore, give false-positive results if entamoeba dispar or entamoeba moshkovskii infection is present. however, it has been reported recently that several different genotypes of e . dispar can be potentially responsible for tissue damage similar to that observed with e . histolytica [ ] . a nonpathogenic entamoeba gingivalis which is present in the oral cavity has to be differentiated from entamoeba histolytica in sputum samples. a combination of serological tests with identification of the parasite by antigen detection by pcr is the best approach to diagnosis. in vitro culture by inoculation of portions of stool, liver abscess, or empyema fl uid into sterile culture media and incubating it at °c is also useful in the diagnosis of amebiasis. the culture media is examined for the growth of amebic trophozoites, which, if present, can be seen on the walls of test tubes or in debris [ ] . antibody detection and antigen detection are other important immunodiagnostic methods. indirect hemagglutination (iha) test is used for routine serodiagnosis of amebiasis. antigen consists of a crude soluble extract of axenically cultured organisms. the enzyme immunoassay (eia) test detects antibody specifi c for e . histolytica in approximately % of patients with extraintestinal amebiasis, % of patients with active intestinal infection, and % of asymptomatic persons who are passing cysts of e . histolytica . detectable e . histolytica -specifi c antibodies may persist for years after successful treatment, so the presence of antibodies does not necessarily indicate acute or current infection. specifi city is % or higher and false-positive reactions rarely occur. e . histolytica -specifi c antigen detection may be useful as an adjunct to microscopic diagnosis in detecting parasites and to distinguish between pathogenic and nonpathogenic infections. detection of circulating antigens in the serum has been found to be an important advancement in the diagnosis [ ] . polymerase chain reaction (pcr) assays are useful for the differentiation of e . histolytica , e . dispar , and e . moshkovskii and for genetic typing of isolates [ , ] . however, these tests are time-consuming and expensive and, hence, are not practical in areas endemic for amebiasis. infection with leishmania donovani causes visceral leishmaniasis and is transmitted by various species of phlebotomus , the sand fl y [ ] . there are no clinical symptoms and signs that are pathognomonic of visceral leishmaniasis (vl) or kala-azar. the symptoms and signs suggestive of visceral leishmaniasis are irregular fever, weight loss, enlargement of liver and spleen, and anemia. pneumonitis, septal fi brosis, pleural effusion, and mediastinal adenopathy are reported in patients coinfected with human immunodeficiency virus (hiv) [ ] . leishmaniasis has also been reported in lung transplant patients [ ] . leishmania amastigotes can be found in the alveoli, pulmonary septa, and bronchoalveolar lavage (bal) fl uid. diagnosis of leishmaniasis is by the demonstration of the parasites in bone marrow aspirates and by the identifi cation of specifi c dna sequences in tissues by molecular biology techniques [ ] . the differential diagnosis of leishmaniasis includes malaria, cirrhosis with portal hypertension, miliary tuberculosis, brucellosis, histoplasmosis, lymphoma, and leukemia [ ] . the diagnosis of leishmaniasis is based on the microscopical demonstration of leishmania amastigotes in the relevant tissue aspirates or biopsies such as bone marrow, spleen, lymph nodes, or liver, skin slit smears, or in the peripheral blood buffy coat [ ] . the smears can be stained with romnowsky's, hematoxylin-eosin, or immunoperoxidase stains. the amastigote stage seen in clinical samples is known as leishman-donovan (ld) bodies. the amastigotes observed in the smears have to be differentiated from "dot"-like structures (e.g., histoplasma spp., platelets) by looking for the characteristic size ( - mm in diameter), shape (round to oval), and the internal organelles (the nucleus and kinetoplast). culture of these specimens on solid nnn medium will demonstrate promastigotes. immunological methods of diagnosis include indirect fl uorescent test (ifa), direct agglutination test (dat), and enzymelinked immunosorbent assay (elisa) to detect antibodies against leishmania [ ] . antigen detection tests are better means of diagnosis of active leishmaniasis [ ] . the antigen detection is the ideal test in immunocompromised patients, where antibody response is very poor. pcr is found to be the most sensitive and specifi c molecular test and is useful in molecular epidemiological studies besides diagnosis [ ] . malaria is caused by the obligate intraerythrocytic protozoa of the genus plasmodium and is primarily transmitted by the bite of an infected female anopheles mosquito. five species of malarial parasites ( plasmodium vivax , p . falciparum , p . malariae , p . ovale , and p . knowlesi ) infect man [ ] . the main symptoms of malaria are periodic fever, chills, malaise, headache, abdominal pain, and vomiting, usually manifesting - days after mosquito bite. anemia and splenomegaly are other important fi ndings in malaria. falciparum malaria is the most deadly type. the pulmonary manifestations range from cough to severe and rapidly fatal non-cardiogenic pulmonary edema and acute respiratory distress syndrome (ards) ( fig. . ) [ ] . acute lung injury and ards have also been reported to occur in infection with p . vivax and p . ovale [ , ] . there has been no convincing evidence for the existence of true malarial pneumonitis, and if it occurs, it may be due to viral and secondary bacterial infections. diffusing capacity was signifi cantly impaired in patients with severe malaria. in addition to ards, falciparum malaria can cause many other severe complications such as cerebral malaria, acute renal failure, severe anemia, thrombocytopenia, bleeding, and gastrointestinal, hepatic, and metabolic complications [ ] . microscopic examination of the giemsa-stained blood smears is the gold standard for the diagnosis of malaria. microscopic diagnosis is based on staining thick and thin blood fi lms on a glass slide to visualize the malaria parasite [ ] . it is inexpensive, able to differentiate malaria species and quantify parasites. the detection threshold in giemsa-stained thick blood fi lm has been estimated to be - parasites/μl. plasmodium species can be correctly recognized in thin blood fi lm. sometimes malarial parasites cannot be detected in peripheral blood smear, but malaria pigments may be seen in circulating phagocytic leukocytes. this is a pathognomonic sign of recent infection. the parasite count, number of circulating pigment-containing phagocytes, and the presence of late asexual stages of the parasite observed in the blood smear are all positively correlated with a fatal outcome. bone marrow aspirate can also demonstrate malarial parasites, if thin smears of the peripheral blood do not show the parasites. quantitative buffy coat (qbc) method involves staining parasite deoxyribonucleic acid (dna) in micro-hematocrit tubes with fl uorescent dyes (e.g., acridine orange) and its subsequent detection by epifl uorescent microscopy. the parasite nuclei fl uoresces bright green and the cytoplasm appears yellow-orange [ ] . rapid diagnostic test (rdt) is a device that detects malaria antigen in a small amount of blood, usually - μl, by immunochromatographic assay with monoclonal antibodies directed against the target parasite antigen and impregnated on a test strip [ , ] . the result, usually a colored test line, is obtained in - min. histidine-rich protein (hrp- ) which is specifi c for p . falciparum is the most common malaria antigen targeted [ ] . plasmodium lactate dehydrogenase (pldh) enzyme is the other group of targeted antigens. monoclonal antibodies against pldh and aldolase enzymes are available for the detection of plasmodium spp. (pan malaria). hrp- often persists in the patient's blood for weeks after successful treatment. molecular methods such as pcr allow the specifi c amplifi cation of a selected region of the malarial genome. this is a specifi c and sensitive method and permits genotyping. drug-resistant parasites and mixed infections can be detected by pcr using single nucleotide polymorphism. a pcr-based detection of plasmodium falciparum in human urine and saliva samples has been described. the antibodies against asexual blood stages of malaria parasite can be detected by the immunofl uorescence assay (ifa). serological tests are useful in epidemiology surveys and are not suitable for the acute diagnosis of malaria [ ] . babesiosis is caused by hemoprotozoan parasites, babesia microti and babesia divergens [ ] . man gets the infection by the bite of an infected tick, ixodes scapularis , and can also be infected from a contaminated blood transfusion [ ] . the parasites attack the red blood cells and can be misdiagnosed as plasmodium . the symptoms are fever, drenching sweats, tiredness, loss of appetite, myalgia, and headache. acute respiratory distress syndrome occurring a few days after initiation of medical therapy is the important pulmonary manifestation [ ] . chest radiological features include bilateral infi ltrates with an alveolar pattern and thickening of the septa. the peripheral blood smears may show, in addition to ring forms, tetrads inside the red blood cells. these tetrads, known as maltese cross formations, are pathognomonic of babesiosis because they are not seen in malaria [ ] . specifi c diagnosis is made by examination of a giemsastained thin blood smear, dna amplifi cation using pcr, or detection of specifi c antibody [ ] . toxoplasmosis is caused by one-celled protozoan parasite, toxoplasma gondii . cats are the primary carriers of the organism [ ] . man gets the infection by eating parasitic cyst-contaminated raw or undercooked meat, vegetables, or milk products. the symptoms of toxoplasmosis are fl u-like syndrome, enlarged lymph nodes, or myalgia. chronic toxoplasmosis can cause chorioretinitis, jaundice, encephalitis, and convulsions. pulmonary toxoplasmosis has been reported with increasing frequency in patients with hiv infection. toxoplasma pneumonia can manifest as interstitial pneumonia/ diffuse alveolar damage or necrotizing pneumonia [ ] . diagnosis of toxoplasmosis is based on the detection of the protozoa in body tissues. antibody levels can be increased without active disease. a real-time pcr-based assay in bal fl uid has been reported in immunocompromised hiv-positive patients [ ] . the important helminthic parasites that cause lung diseases include cestodes ( echinococcus granulosus and echinococcus multilocularis ), trematodes ( schistosoma haematobium , schistosoma mansoni , schistosoma japonicum , and paragonimus westermani ), and nematodes ( ascaris lumbricoides , ancylostoma duodenale , necator americanus , strongyloides stercoralis , wuchereria bancrofti , brugia malayi , brugia timori , dirofi laria immitis , dirofi laria repens , toxocara canis or cati , and trichinella spiralis ). the parasite species that cause hydatid disease in man are echinococcus granulosus and echinococcus multilocularis . the adult e . granulosus resides in the small intestine of the defi nitive hosts, mainly dogs. the intermediate hosts including man are infected by ingestion of eggs excreted in the feces of the dogs. primary pulmonary cystic hydatid disease is usually single. multiple cysts may be seen in % of cases and may be unilateral or bilateral [ ] . secondary metastatic pulmonary cystic hydatid disease may occur by the rupture of a liver cyst in vena caval circulation or a heart cyst in the right ventricular cavity. patients are asymptomatic in the initial stages of infection. pulmonary symptoms include cough, fever, dyspnea, and chest pain [ ] . signs and symptoms can occur due to compression of adjacent tissue by the cysts. rupture of the cysts into a bronchus may result in hemoptysis and expectoration of cystic fl uid containing parasite membrane and can cause anaphylactic shock, respiratory distress, asthma-like symptoms, persistent pneumonia, and sepsis [ , ] . rupture of the cyst into the pleural space can result in hydropneumothorax, pleural effusion, and empyema. diagnosis of pulmonary hydatid cyst is based on thoracic imaging studies (chest radiography, thoracic computerized tomography (ct), and thoracic ultrasonography) [ ] and an uncomplicated cyst presents as a well-defi ned homogenous round (cannonball) opacity that may be lobulated by contiguous bronchovascular axes ( fig. . ) [ ] . chest radiographs show solitary or multiple round opacities that may mimic lung tumors. ct is helpful in doubtful cases, because the internal structure of the cyst can be analyzed and its density measured. ct is also useful to assess the state of the neighboring parenchyma and to evaluate the whole thorax and abdomen for associated cystic lesions or anomalies. ultrasonography using a portable ultrasound scanner has been found as reliable, inexpensive, and rapid technique in community-based screening surveys for cystic hydatid disease. the crescent sign, cumbo's sign (onion peel sign), water lily sign, and air-fl uid level are seen on chest radiography and computed tomography (ct) [ ] . inverse crescent sign, signet ring sign, and serpent sign are recognized as features of pulmonary hydatid cysts in computerized tomogram. laboratory tests are complementary to clinical and imaging investigations. eosinophilia and elevated immunoglobulin e (ige) levels are seen when the hydatid cyst ruptures [ ] . serologic tests are less sensitive in patients with lung hydatid disease than in those localized in liver. falsepositive tests may be observed in patients suffering from other helminthic infections. immunologic tests may be helpful to confi rm the hydatid origin of a cystic lesion and permit the serologic monitoring of medically or surgically treated patients [ , ] . pulmonary alveolar echinococcosis (ae) is due to hematogenous dissemination from hepatic lesions. the liver is the fi rst target of the parasite, with a silent and long incubation period ( - years). exogenous proliferation causes infi ltration of adjacent tissues and pressure necrosis. it can metastasize to distant organs mainly to lungs, brain, and bones [ ] . lung involvement results from metastatic dissemination or direct extension through the diaphragm of hepatic echinococcosis with intrathoracic rupture into the bronchial tree, pleural cavity, or mediastinum. direct extension to the right atrium through the inferior vena cava with recurrent episodes of pulmonary embolism has also been reported. imaging studies with radiography, ultrasonography, ct, and magnetic resonance imaging (mri) may help in the diagnosis of metastatic lung disease [ ] . biopsy may be needed to confi rm the diagnosis [ ] . serologic the schistosomes that cause human disease are schistosoma haematobium , schistosoma mansoni , and schistosoma japonicum . the fi nal habitat of s . haematobium is urinary bladder vesicle beds and of s . mansoni and s . japonicum is the mesenteric beds. the schistosome eggs are passed in urine ( s . haematobium ) or in feces ( s . mansoni and s . japonicum ) by the infected humans. the parasites can cause schistosoma dermatitis at the site of skin penetration. pulmonary schistosomiasis can manifest clinically as an acute form and a chronic form [ ] . acute symptoms can develop - weeks after skin penetration [ ] . the acute form, also known as katayama syndrome, presents with fever, chills, weight loss, diarrhea, abdominal pain, myalgia, and urticaria and is seen in nonimmune patients [ ] . pulmonary manifestations include shortness of breath, wheezing and dry cough. patients with chronic schistosomiasis present with features of pulmonary hypertension and cor pulmonale [ ] . massive hemoptysis and lobar consolidation and collapse have been reported in schistosomiasis [ ] . hepatosplenomegaly due to portal hypertension has been reported in patients infected with s . mansoni or s . japonicum [ ] . chest radiographic abnormalities range from multiple nodules to diffuse interstitial infi ltrates. small pulmonary nodules in ct have been described in acute schistosomiasis [ ] . diagnosis of chronic schistosomiasis is based on the demonstration of eggs in stool or urine by direct microscopy or rectal/bladder biopsy [ ] . multiple examinations of specimens are required in mild and chronic infections. in active infections, eggs contain live and mature miracidia. the incubation period of the infection is usually months and hence eggs can be detected after months of last known contact with fresh water. peripheral blood eosinophilia with mild leukocytosis, abnormal liver function test results and elevated ige levels are reported in acute schistosomiasis. hyperglobulinemia is observed in chronic schistosomiasis. serological tests with elisa are available, but cannot differentiate active from past infections [ ] . bronchoscopy and transbronchial biopsy may reveal eosinophilic pneumonitis. paragonimiasis is a food-borne zoonoses and is caused by infection with paragonimus species and manifests as subacute or chronic infl ammation of the lung. though more than species are known to cause paragonimiasis in man, the main species that cause paragonimiasis is paragonimus westermani . adult worms live in the lungs and the eggs are voided in sputum or feces. the man gets infection, when raw or undercooked crabs or crayfi shes infected with infective metacercariae are ingested. the parasite from the human gut passes through several organs and tissues to reach the lung. pulmonary paragonimiasis manifests as fever, chest pain, chronic cough, and bloodtinged sputum [ ] . the cough is dry at fi rst and later productive with blood-stained, rusty-brown tenacious sputum. occasionally, there is profuse hemoptysis. pulmonary paragonimiasis is confused with tuberculosis as the symptoms in both diseases are similar. chest radiographs may show infi ltrative, nodular, and cavitating shadows. pleural effusion or pneumothorax is an important fi nding in paragonimiasis [ , ] . ct scan may show single or multiple nodules in the lung parenchyma, calcifi ed spots and pleural thickening with interlobar pleuritis, and pleural effusion. mri may show conglomerated lesions with hemorrhage or tunnel signs. defi nitive diagnosis is based on the demonstration of eggs in sputum samples, bal fl uid, or lung biopsy specimens. ascaris lumbricoides is the most common intestinal helminthic infection. respiratory symptoms in ascariasis are due to larval pulmonary migration, airway hyper-reactivity, and bronchospasm. symptomatic pulmonary disease may range from mild cough to loffl er's syndrome [ ] . loffl er's syndrome is a self-limiting infl ammation of the lungs and is associated with blood and lung eosinophilia. this syndrome can occur as a result of parasitic infestations (especially ascariasis in children) and exposure to various drugs. patients may present with general symptoms of malaise, loss of appetite, fever lasting - days, headache, and myalgia. the respiratory symptoms include chest pain, cough with mucoid sputum, hemoptysis, shortness of breath, and wheezing [ ] . there may be rapid respiratory rate and rales can be heard on auscultation. leukocytosis particularly eosinophilia is an important laboratory fi nding. chest radiographs demonstrate unilateral or bilateral, transient, migratory, and non-segmental opacities of various sizes. these opacities are often peripherally situated and appear to be pleural based [ ] . the severity of symptoms will depend upon the larval burden. rarely chronic eosinophilic pneumonia or symptoms of upper airway obstruction can occur. a diagnosis of pulmonary disease due to ascariasis can be made in an endemic region in a patient who presents with dyspnea, dry cough, fever, and eosinophilia. sputum may show charcot-leyden crystals and the chest radiograph may reveal fl eeting pulmonary infi ltrates. because of the occurrence of respiratory symptoms during larval pulmonary migration, stool examination usually does not show ascaris eggs and stool samples may be negative until - months after respiratory symptoms occur, unless the patient was previously infected. however, larvae can sometimes be demonstrated in respiratory or gastric secretions [ ] . it has been suggested that measurement of ascaris -specifi c igg by elisa may be useful in the serodiagnosis of ascariasis [ ] . hookworm disease in humans results from infections with two species, ancylostoma duodenale and necator americanus . during pulmonary larval migration, patients may present with fever, cough, wheezing, and transient pulmonary infi ltrates in chest radiographs. this is associated with blood and pulmonary eosinophilia [ ] . the other characteristic feature is iron defi ciency anemia due to chronic blood loss [ ] . in severe hookworm anemia, patients may present with fatigue, exertional dyspnea, poor concentration, and cardiac murmurs. during massive infection from oral ingestion of hookworm larvae, patients can present with nausea, vomiting, cough, dyspnea, and eosinophilia, and this condition is termed as wakana disease. prominent gastrointestinal symptoms in hookworm disease are abdominal pain, nausea, anorexia, and diarrhea. a direct microscopical examination of stool demonstrates the presence of characteristic hookworm eggs. concentration method may be used when the infection is light. eosinophilia in the peripheral blood is a prominent fi nding. a peripheral blood smear examination will reveal microcytic hypochromic anemia. a polymerase chain reaction (pcr) to differentiate between a . duodenale and n . americanus has been developed [ ]. strongyloides stercoralis is seen worldwide and the unique feature of the life cycle of s . stercoralis is that it can complete its life cycle either in the human host or in the soil. it has been observed that - % of chronically infected people may be asymptomatic. although symptoms in individuals with chronic strongyloides stercoralis infection are usually mild, it can persist for many years due to autoinfection. this may occasionally progress to the hyperinfection syndrome with high mortality especially in immunosuppressed individuals [ , ] . the relative risk of s . stercoralis infection is increased in elderly men and patients who had recently used corticosteroids, had a hematologic malignancy, and had prior gastric surgery. other risk factors include chronic lung disease, use of histamine blockers, or chronic debilitating illness. strongyloidiasis is a chronic relapsing illness of mild to moderate severity characterized by gastrointestinal complaints (diarrhea, pain, tenderness, nausea, vomiting), peripheral blood eosinophilia, and hypoalbuminemia. pulmonary signs and symptoms include cough, shortness of breath, wheezing, and hemoptysis. in patients at high risk for strongyloidiasis, adult respiratory distress syndrome and septicemia due to intestinal transmural migration of bacteria can occur as a result of hyperinfection or disseminated strongyloidiasis [ , ] . in addition, acute anemia, acute renal failure, and systemic infl ammatory response syndrome are also reported in hyperinfection. strongyloidiasis can manifest as eosinophilic pleural effusion in both immunocompetent and immunocompromised individuals. rare pulmonary manifestations include acute respiratory failure due to respiratory muscle paralysis, granulomatous reaction in the lung with interlobular septal fi brosis, and pulmonary microcalcifi cations. a paradoxic therapeutic response of asthma to glucocorticosteroids, in which bronchial asthma symptoms worsened after treatment with parenteral corticosteroids, has been described in patients with strongyloides superinfections [ ] . exacerbations of chronic obstructive pulmonary disease and worsening of symptoms in idiopathic pulmonary fi brosis have also been reported in strongyloides stercoralis infection. in immunocompetent patients with strongyloidiasis, the parasite load is usually low and the larval output is irregular. as a result, the diagnosis of strongyloidiasis by examination of a single stool specimen using conventional techniques usually fails to detect larvae in up to % of cases [ ] . the diagnostic yield can be increased by examination of several stool specimens on consecutive days. examination of stool by agar plate culture method was found to be superior to direct smear and modifi ed baermann technique [ , ] . strongyloides stercoralis larvae can be demonstrated in duodenal aspirate. in disseminated disease, larvae and adult parasites can be seen in sputum, urine, bronchoalveolar lavage fl uid, and other body fl uids [ ] . a serological test using centers for disease control (cdc) enzyme immunoassay (eia) for detection of antibodies to strongyloidiasis was found to have a sensitivity of . % in patients with proven infection [ ] . tropical pulmonary eosinophilia (tpe) results from immunologic hyper-responsiveness to human fi larial parasites, wuchereria bancrofti and brugia malayi [ - ] . tpe is a systemic disease involving mainly the lungs, but other organs such as liver, spleen, lymph nodes, brain, and gastrointestinal tract may also be involved. the disease occurs predominantly in males, with a male to female ratio of : , and is mainly seen in older children and young adults between the ages and years. the systemic symptoms include fever, weight loss, and fatigue. patients with tpe usually present with respiratory symptoms that include paroxysmal cough, breathlessness, and wheeze and chest pain [ , ] . the symptoms occur predominantly at night, but can persist during the day. severe cough can lead to fractured ribs. sputum is usually scanty, viscous, and mucoid. the sputum often shows clumps of eosinophils, and rarely charcot-leyden crystals are observed. on examination, patients are often breathless. bilateral scattered rhonchi and rales may be heard on auscultation [ , ] . leukocytosis with an absolute increase in eosinophils in the peripheral blood is the hallmark of tpe. spontaneous fl uctuations in the eosinophil count can occur. absolute eosinophil counts are usually more than , cells/mm and may range from , to , [ ] . microfi lariae are rarely seen in the peripheral blood. as patients with tpe especially from endemic areas can be simultaneously infected with other helminthic parasites, stool examination may reveal ova or larvae of other helminthes ( ascaris , ancylostoma , whipworm, and strongyloides ) in % of patients with tpe. this observation does not deter the physician from making a diagnosis of tpe, if other conditions for diagnosis are fulfi lled. the chest radiological features of tpe include reticulonodular shadows predominantly seen in mid and lower zones and miliary mottling of - mm in diameter often indistinguishable from miliary tuberculosis (fig. . ) . twenty percent of patients have a normal chest radiograph. in patients with a long-standing history, a few patients have honeycomb lungs. radiological improvement occurs on specifi c therapy with dec, but some degree of radiological abnormality persists in some patients. lung function tests reveal mainly a restrictive ventilation defect with superimposed airway obstruction [ , ] . single breath carbon monoxide transfer factor (tlco) is reduced in % of untreated patients with tpe. the reduction in tlco is due to reduced pulmonary membrane diffusing capacity (dm) [ ] . the criteria suggested for the diagnosis of tpe are (a) appropriate exposure history (mosquito bite) in an endemic area of fi lariasis, (b) a history of paroxysmal nocturnal cough and breathlessness, (c) chest radiographic evidence of pulmonary infi ltrations, (d) leukocytosis in blood, (e) peripheral blood eosinophils more than , cells per cu mm, (f) elevated serum ige levels, (g) elevated serum antifi larial antibodies (igg and/or ige), and (h) a clinical response to diethylcarbamazine citrate [ , ] . pulmonary dirofi lariasis is a zoonotic infection caused by fi larial nematodes, dirofi laria immitis and dirofi laria repens . humans are accidental hosts of this parasite which is transmitted to man by the mosquito. the parasites are usually seen in the pulmonary artery where they produce an embolism ultimately leading to the formation of a pulmonary nodule or "coin lesion" [ ] . nearly % of subjects infected with dirofi lariasis are asymptomatic. clinical symptoms are chest pain, cough, fever, hemoptysis, and dyspnea. ct scan may show a well-defi ned nodule with smooth margin connected to an arterial branch [ ] . positron emission tomography scan can demonstrate hypermetabolic activity in a pulmonary infarct secondary to dirofi lariasis [ ] . a pcr-based diagnosis of d . repens in human pulmonary dirofi lariasis has been reported [ ] . a defi nitive histopathological diagnosis of pulmonary dirofi lariasis can be made tissue specimens obtained by wedge biopsy, by video-assisted thoracoscopy, or rarely by fi ne needle biopsy. toxocara larva migrans syndromes are important zoonotic infections. certain nematode parasites entering into an unnatural host (e.g., man) may not be able to complete their life cycle and their progress is arrested in the "unnatural host." the common parasites that cause visceral larva migrans (vlm) and eosinophilic lung disease in man are a dog ascarid ( toxocara canis ) and less commonly a cat ascarid ( toxocara cati ) [ ] . human toxocariasis occurs in all parts of the world wherever there is a large pool of infected dogs. visceral larva migrans (vlm) is characterized by leukocytosis and eosinophilia. the larva induces a granulomatous reaction in the tissues containing eosinophils and multinucleated giant cells. larvae can get encapsulated within the granuloma where they are either destroyed or persist for many years in a viable state. granulomata are found in the lungs, liver, central nervous system, and eyes. later fi brosis and calcifi cation occur. larval antigens can cross-react with human a and b blood group antigens. visceral larva migrans is usually reported in young children with a history of pica. a history of exposure to puppies or dogs supports the diagnosis of vlm. these children usually present with fever, cough, wheezing, eosinophilia, and hepatomegaly. however, most of the children infected with toxocara spp. are asymptomatic. the main symptoms in patients with visceral larva migrans are fever, cough, wheezing, seizures, anemia, and fatigue. pulmonary manifestations are reported in % of cases and patients may present with severe asthma [ ] . scattered rales and rhonchi are heard on auscultation. there will be intense blood eosinophilia. skiagram chest may reveal focal patchy infi ltrates. in some cases, severe eosinophilic pneumonia may lead to respiratory distress [ ] . other clinical features include generalized lymph node enlargement, hepatomegaly, and splenomegaly. skiagram chest may show patchy infi ltrates. nonspecifi c changes include hypergammaglobulinemia and elevated isohemagglutinin titers to a and b blood group antigens. serological tests by elisa method using excretory-secretary proteins obtained from cultured t . canis may be useful in the diagnosis. cross reactivity with other helminths limits the usefulness of this test in endemic areas. detection of ige antibodies by elisa and toxocara excretory-secretary antigens by western blotting procedure have also been reported for diagnosis [ , ] . however, serodiagnostic procedures cannot distinguish between past and present infections. histopathological examination of lung or liver biopsy specimens may demonstrate granulomas with eosinophils, multinucleated giant cells, and fi brosis. since man is not the defi nitive host of toxocara sp., eggs or larvae cannot be demonstrated in the feces. human trichinellosis is an important food-borne zoonosis. the most important species that infect man is trichinella spiralis . the parasite has a direct life cycle with complete development in one host (pig, rat, or man). man gets infection from raw and partially cooked pork, when infected pig's muscle containing larval trichinellae is eaten by man. the common symptoms of trichinellosis are muscle pain, periorbital edema, fever, and diarrhea [ ] . pulmonary symptoms include dyspnea, cough, and pulmonary infi ltrates. dyspnea may be due to the involvement of diaphragm [ ] . leukocytosis, eosinophilia, and elevated levels of serum muscle enzymes (creatine phosphokinase, lactate dehydrogenase, aldolase, and amino transferase) are important laboratory fi ndings. an enzyme-linked immunosorbent assay (elisa) for detection of anti-trichinella antibodies using excretory-secretary antigens may be useful in the diagnosis. a defi nitive diagnosis can be made by muscle biopsy (usually deltoid muscle) that may demonstrate larvae of t . spiralis [ ] . is the incidence of parasitic lung diseases increasing, and how may this effect modern respiratory medicine? emerging and established parasitic lung infestations immunopathogenesis and treatment of eosinophilic lung diseases in the tropics parasitic lung infections progress in research on entamoeba histolytica pathogenesis thoracic amoebiasis value of microscopy in the diagnosis of dysentery associated with invasive entamoeba histolytica diagnosis of amebiasis in bangladesh comparison of pcr, isoenzyme analysis and antigen detection for diagnosis of entamoeba histolytica infection human amebiasis: breaking the paradigm? a new liquid medium for xenic cultivation of entamoeba histolytica and other lumendwelling protozoa laboratory diagnosis of amebiasis genomic dna differences between pathogenic and non pathogenic entamoeba histolytica development of multiplex real-time polymerase chain reaction for detection of entamoeba histolytica, entamoeba dispar, and entamoeba moshkovskii in clinical specimens bronchopulmonary and mediastinal leishmaniasis: an unusual clinical presentation of leishmania donovani infection leishmaniasis among organ transplant recipients detection of leishmania infantum kinetoplast dna in laryngeal tissue from an immunocompetent patient new developments in diagnosis of leishmaniasis comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis latex agglutination test for the detection of urinary antigens in visceral leishmaniasis molecular diagnosis of leishmaniasis malaria prevention in short-term travelers lung biology in health and disease: tropical lung diseases acute lung injury and other serious complications of plasmodium vivax malaria acute respiratory distress syndrome in a case of plasmodium ovale world health organization. guidelines for the treatment of malaria. geneva: world health organization laboratory diagnosis of malaria infection-a short review of methods malaria diagnosis: a brief review rapid diagnostic tests for malaria parasites rapid diagnostic testing for malaria a review of malaria diagnostic tools: microscopy and rapid diagnostic test (rdt) co-infections acquired from ixode ticks acute respiratory failure in patients treated for babesiosis diagnosis of pulmonary infection with toxoplasma gondii in immunocompromised hiv-positive patients by real time pcr clinical and diagnostic management of toxoplasmosis in the immunocompromised patient eosinophilic lung disease in the tropics parasitic diseases of the respiratory tract spontaneous rupture of lung echinococcal cyst causing anaphylactic shock and respiratory distress syndrome expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans usefulness of four different echinococcus granulosus recombinant antigens for serodiagnosis of unilocular hydatid disease (uhd) and postsurgical follow-up of patients treated for uhd pulmonary cystic echinococcosis echinococcus multilocularis disseminated alveolar echinococcosis mimicking a metastatic malignancy the immunodiagnosis of echinococcus multilocularis infection pulmonary schistosomiasis pulmonary manifestations of early schistosoma infection in nonimmune travelers katayama fever; an acute manifestation of schistosomiasis schistosomiasis associated pulmonary hypertension a diagnostic dilemma of right lower lobe collapse caused by pulmonary bilharsiasis acute schistosomiasis in nonimmune travelers: chest ct fi ndings in patients pathophysiology, diagnosis, and treatment detection of circulating antigens in patients with active schistosoma haematobium infection studies on clinical manifestations, diagnosis and control of paragonimiasis in china pulmonary paragonimiasis: an evaluation of roentgen fi ndings in positive sputum patients in an endemic area in thailand clinicoradiologic features of pleuropulmonary paragonimus westermani on kyushu island evaluation of human igg subclass antibodies in the serodiagnosis of paragonimus heterotremus tropical parasitic lung diseases strongyloidiasis and other intestinal nematode infections eosinophilic lung disease (or how to slice pie) ascariasis and hookworm possible approach for serodiagnosis of ascariasis by evaluation of immunoglobulin g response using ascaris lumbricoides somatic antigen nature and causes of hookworm anemia differentiation between the human hookworm ancylostoma duodenale and necator americanus using pcr-rflp biology and immunology of human strongyloidiasis disseminated strongyloides stercoralis in human immunodefi ciency virus-infected patients: treatment failure and review of literature pulmonary manifestations of strongyloidiasis strongyloides stercoralis infestation associated with septicemia due to intestinal transmural migration of bacteria corticosteroidinduced asthma: a manifestation of limited hyperinfection syndrome due to strongyloides stercoralis diagnosis of strongyloides stercoralis infection a simple modification of the baermann method for diagnosis of strongyloidiasis evaluation of three methods for laboratory diagnosis of strongyloides stercoralis infection diagnosis of pulmonary strongyloidiasis by bronchoalveolar lavage serology and eosinophil count in the diagnosis and management of strongyloidiasis in a non-endemic area tropical pulmonary eosinophilia acute tropical pulmonary eosinophilia: characterization of the lower respiratory tract infl ammation and its response to therapy tropical pulmonary eosinophilia: analysis of antifi larial antibody localized to the lung pulmonary eosinophilia: progress in respiration research tropical pulmonary eosinophilia re-examination of the diagnostic criteria of tropical pulmonary eosinophilia the course of lung function in treated tropical eosinophilia diffusing capacity in acute untreated tropical eosinophilia pulmonary membrane diffusing capacity and capillary blood volume in tropical eosinophilia persistent lower respiratory tract infl ammation associated with interstitial lung disease in patients with tropical pulmonary eosinophilia following treatment with diethylcarbamazine how to diagnose and manage common parasitic pneumonias? public health aspects of dirofi lariasis in the united states pulmonary dirofi lariasis: computed tomographic fi ndings and correlation with pathologic features pet fi ndings in pulmonary dirofi lariasis detection of dirofi laria (nochtiella) repens dna by polymerase chain reaction in embedded paraffi n tissues from two human pulmonary locations highlights of human toxocariasis visceral larva migrans associated with the hypereosinophilic syndrome and the onset of severe asthma acute respiratory failure due to toxocara infections evaluation of an immunenzymatic assay detecting specifi c anti-toxocara immunoglobulin e for diagnosis and post treatment follow up of human toxocariasis application of the western-blotting procedure for the immunodiagnosis of human toxocariasis clinical aspects of infections with trichinella spp tropical infectious diseases: principles, pathogens and practice key: cord- - ndb rm authors: iwasa, yoh; sato, kazunori; takeuchi, yasuhiro title: mathematical studies of dynamics and evolution of infectious diseases date: journal: mathematics for life science and medicine doi: . / - - - - _ sha: doc_id: cord_uid: ndb rm nan the practical importance of understanding the dynamics and evolution of infectious diseases is steadily increasing in the contemporary world. one of the most important mortality factors for the human population is malaria. every year, hundreds of millions of people suffer from malaria, and more than a million children die. one of the obstacles of controlling malaria is the emergence of drug-resistant strains. pathogen strains resistant to antibiotics pose an important threat in developing countries. in addition, we observe new infectious diseases, such as hiv, ebora, and sars. the mathematical study of infectious disease dynamics has a long history. the classic work by kermack and mckendrick ( ) established the basis of modeling infectious disease dynamics. the variables indicate the numbers of host individuals in several different states -susceptive, infective and removed. this formalism is the basis of all current modeling of the dynamics and evolution of infectious diseases. since then, the number of theoretical papers on infectious diseases has increased steadily. especially influential was a series of papers by roy anderson and robert may, summarized in their book (anderson and may ) . anderson and may have developed population dynamic models of the host engaged in reproduction and migration. in a sense, they treated epidemic dynamics as a variant of ecological population dynamics of multiple species community. combining the increase of our knowledge of nonlinear dynamical systems (e. g. chaos), anderson and may also demonstrated the usefulness of simple models in understanding the basic principles of the system, and sometimes even in choosing a proper policy of infectious disease control. the dynamical systems for epidemics are characterized by nonlinearity. the systems include many processes at very different scales, from the population on earth to the individual level, and further to the immune system within a patient. hence, mathematical studies of epidemics need to face this dynamical diversity of phenomena. tools of modeling and analysis for situations including time delay and spatial heterogeneity are very important. as a consequence, there is no universal mathematical model that holds for all problems in epidemics. when we are given a set of epidemiological phenomena and questions to answer, we must "construct" mathematical models that can describe the phenomena and answer our questions. this is quite different from studies in "pure" mathematics, in which usually the models are given beforehand. one of the most important questions in mathematical studies of epidemics is the possibility of the eradication of disease. the standard local stability analysis of the endemic equilibrium and disease-free equilibrium is often not enough to answer the question, because it gives us information only on the local behavior, or the solution in the neighborhood of those equilibria. on the other hand, it is known that global stability analysis of the models is often very difficult, and even impossible in general cases, because the dynamics are highly nonlinear. even if the endemic equilibrium were unstable and the disease-free equilibrium were locally stable, the diseases can remain endemic and be sustained forever. sometimes, rather simple models show periodic or chaotic behavior. recently, the concept of "permanence" was introduced in population biology and has been studied extensively. this concept is very important in mathematical epidemiology as well. permanence implies that the disease will be maintained globally, irrespective of the initial composition. even if the endemic equilibrium were unstable, the disease will last forever, possibly with perpetual oscillation or chaotic fluctuation. since the epidemiological data supplied by medical and public health sectors are abundant, epidemiological models are in general much better tested than similar population models in ecology developed for wild animals and plants. the diversity of models is also extensive, including all the different levels of complexity. rather simple and abstract models are suitable to discuss general properties of the system, while more complex and realistic computerbased simulators are adopted for policy decision making incorporating details of the structure closely corresponding to available data. mathematical modeling of infectious diseases is the most advanced subfield of theoretical studies in biology and the life sciences. what is notable in this development is that, even if many computer-based detailed simulators become available, the rigorous mathematical analysis of simple models remains very useful, medically and biologically, in giving a clear understanding of the behavior of the system. recently, the evolutionary change of infectious agents in the host population or within a patient has attracted an increasing attention. mutations during genome replication would create pathogens that may differ slightly from the original types. this gives an opportunity for a novel strain to emerge and spread. as noted before, emergence of resistant strains is a major obstacle of infectious disease control. essentially the same evolutionary process occurs within the body of a single patient. a famous example is hiv, in which viral particles change and diversify their nucleotide sequences after they infect a patient. this supposedly reflects the selection by the immune system of the host working on the virus genome. a similar process of escape is involved in carcinogenesis -a process in which normal stem cells of the host become cancerous. the papers included in this volume are for mathematical studies of models on infectious diseases and cancer. most of them are based on presentations in the first international symposium on dynamical systems theory and its applications to biology and environmental sciences, held in hamamatsu, japan, on - march . this introductory chapter is followed by four papers on infectious disease dynamics, in which the roles of time delay (chaps. and ) and spatial structures (chaps. and ) are explored. then, there are two chapters that discuss competition between strains and evolution occurring in the host population (chap. ) and within a single patient (chap. ). finally, there are papers on models of the immune system and cancer (chaps. and ). below, we briefly summarize the contents of each chapter. in chap. , zhien ma and jianquan li give an introduction to the mathematical modeling of disease dynamics. then, they summarize a project of modeling the spread of sars in china by the authors and their colleagues. in chap. , yasuhiro takeuchi and wanbiao ma introduce mathematical studies of models with time delay. they first review past mathematical studies on this theme during the last few decades, and then introduce their own work on the stability of the equilibrium and the permanence of epidemiological dynamics. in chaps and , wendi wang and shigui ruan discuss the spatial aspect of epidemiology. the spread of a disease in a population previously not infected may appear as "wave of advance". this is often modeled as a reaction diffusion system, or by other models handling spatial aspects of population dynamics. the speed of disease propagation is analogous to the spread of invaders in a novel habitat in spatial ecology (shigesada and kawasaki ) . since microbes have a shorter generation time and huge numbers of individuals, they have much faster evolutionary changes, causing drug resistance and immune escape, among the most common problems in epidemiology. by considering the appearance of novel strains with different properties from those of the resident population of pathogens, and tracing their abundance, we can discuss the evolutionary dynamics of infectious diseases. in chap. , horst thieme summarized the work on the competition between different and competing strains, and the possibility of their coexistence and replacement. an important concept is the "maximal basic replacement ratio". if a host once infected and then recovered from a single strain is perfectly immune to all the other strains (i. e. cross immunity is perfect), then the one with the largest basic replacement ratio will win the competition among the strains. the author explores the extent to which this result can be generalized. he also discusses the coexistence of strains considering the aspect of maternal transmission as well. in chap. , yoh iwasa and his colleagues analyze the result of evolutionary change occurring within the body of a single patient. some of the pathogens, especially rna viruses have high mutation rates, due to an unreliable replication mechanism, and hence show rapid genetic change in a host. the nucleotide sequences just after infection by hiv will be quite different from those hiv occurring after several years. by mutation and natural selection under the control of the immune system, they become diversified and constantly evolve. iwasa and his colleagues derive a result that, without cross-immunity among strains, the pathogenicity of the disease tends to increase by any evolutionary changes. they explore several different forms of cross-immunity for which the result still seems to hold. in chap. , edoardo beretta and his colleagues discuss immune response based on mathematical models including time delay. the immune system has evolved to cope with infectious diseases and cancers. they have properties of immune memory and, once attached and recovered, they will no longer be susceptive to infection by the same strain. to achieve this, the body has a complicated network of diverse immune cells. beretta and his colleagues summarize their study of modeling of an immune system dynamics in which time delay is incorporated. in the last chapter, h.i. freedman studies cancer, which originates from the self-cells of the patient, but which then become hostile by mutations. there is much in common between cancer cells and pathogens originated from outside of the host body. freedman discusses the optimal chemotherapy, considering the cost and benefit of chemotherapy. this collection of papers gives an overview of theoretical studies of infectious disease dynamics and evolution, and hopefully will serve as a source in future studies of different aspects of infectious disease dynamics. here, the key words are time delay, spatial dynamics, and evolution. toward the end of this introductory chapter, we would like to note one limitation -all of the papers in this volume discuss deterministic models, which are accurate when the population size is very large. since the number of microparasites, such as bacteria, or viruses, or cancer cells, is often very large, the neglect of stochasticity due to the finiteness of individuals seems to be acceptable. however, when we consider the speed of the appearance of novel mutants, we do need stochastic models, because mutants always start from a small number. according to studies on the timing of cancer initiation, which starts from rare mutations followed by population growth of cancer cells, the predictions of deterministic models differ by several orders of magnitude from those of stochastic models and direct computer simulations. infectious diseases of humans a contribution to the mathematical theory of epidemics biological invasions: theory and practice key: cord- -a jsbunw authors: parthasarathy, a. title: training in pediatric infectious disease: need of the time date: - - journal: nan doi: . /j.pid. . . sha: doc_id: cord_uid: a jsbunw nan training in pediatric infectious disease: need of the time in recent years, there has been increased coverage by the media on infectious diseases thereby making it one of the most debated topics amongst scientific and public forums. public health officials, pharmaceutical professionals and policy makers need to understand the transmission patterns of infectious diseases, in order to be able to interpret and evaluate the epidemiological data and the findings of such studies. most recently, mathematical models and newer techniques for measurement and analysis have been applied to outbreaks and emerging epidemics, such as influenza a (h n ) and severe acute respiratory syndrome (sars). many such infectious diseases remain a leading cause of morbidity and mortality worldwide, with hiv, tuberculosis and malaria estimated to cause % of all deaths each year. worldwide, two thirds of all deaths in children under years of age are caused by infectious diseases. the world health organization (who) estimated that % of total mortality in children less than years of age is due to vaccine preventable diseases. this represents approximately . million of deaths. infections like diphtheria ( cases in ), malaria (yearly . million infections), tuberculosis ( . million cases and . million people dying every year) , have a high negative impact on society, and there is a great deal to be done in terms of awareness, treatment and control strategies. thus, the prevention and treatment of these infections remains a top priority to achieve global health. in india, clinical superspecialties such as neurology, cardiology, gastroenterology and nephrology are often preferred by medical students and hospitals, while infectious diseases as a specialty is frequently overlooked and thought to be less important. is it time to revisit this concept? a course in infectious diseases will educate and train healthcare professionals and will help promote high quality advanced care and research by strengthening their knowledge. this could facilitate clinicians, researchers and academicians to intensely understand and be involved in surveillance, policy making, program implementation on infections that directly contribute to morbidity and mortality in children, including new and emerging infections like sars, h n influenza, dengue, and infectious diarrhea. these trained and specialized health professionals will be involved in testing and implementing vaccination strategies against common infections such as measles, rubella and polio. such a training program would facilitate the formation of a 'think tank' of professionals who can be key opinion leaders guiding policy decisions. professionally-oriented training and education in pediatric infectious diseases is the 'need of the hour' and should be implemented on a "war footing" to achieve global health. i therefore, strongly feel that such a course will be useful not only for individuals interested in expanding their knowledge of the techniques available for analyzing and interpreting epidemiological data on infectious diseases, but also for the world and for india to achieve the goal of "health for all". the indian academy of pediatrics should come forward to establish a fellowship course in pediatric infectious diseases. the course and content can be worked out, and may be achieved through institutional participation, distant education, contact sessions, seminars, written and practical examinations. importance can be given to infection control both in the hospital as well as the community, with a strong focus on public health. the existing infectious diseases chapter should be upgraded into a full fledged 'infectious diseases academy', which could develop a suitable syllabus for the fellowship course. in time, the course could be expanded to include nursing and paramedical professionals as well. it is time that the indian academy of pediatrics and its infectious diseases academy take a step forward and act fast to expand our pool of professionally trained and committed infectious disease experts who will play an active role in our children's health in the near future. r e f e r e n c e s immunization surveillance, assessment and monitoring. vaccine-preventable diseases who vaccine preventable diseases monitoring system . immunization profile india new delhi: national rural health mission, department of health and family welfare rntcp status report, i am stopping tb, ministry of health and family welfare key: cord- - zfio u authors: tyring, stephen k. title: syndromal tropical dermatology date: - - journal: tropical dermatology doi: . /b - - - - . - sha: doc_id: cord_uid: zfio u nan stephen k. tyring with increasing numbers of persons from industrialized, temperate countries traveling and/or working in tropical lands, there is a marked need for physicians to be able to diagnose accurately and treat tropical diseases with mucocutaneous manifestations. while some studies demonstrate that approximately one-third to two-thirds of travelers returning from tropical countries experience some health problem, diarrhea is the most prevalent complaint. mucocutaneous problems, however, are among the top five health complaints of the returned traveler, and comprise - % of health concerns of persons returning from the tropics. during international conflicts, soldiers from north america, europe, and australia are often required to serve in tropical lands and sometimes develop diseases not familiar to physicians of their home countries. this was the case for french soldiers serving in vietnam in the s and american soldiers serving there in the s and s. recently hundreds of american and allied troops serving in iraq and afghanistan have developed "baghdad boils," i.e., leishmaniasis, transmitted by sand fly bites ( fig. . ) . likewise, millions of persons from tropical countries now live and work in temperate lands and may present with medical problems with which the physician is not familiar. whereas the cutaneous problems in the returned traveler are frequently the result of infectious diseases, skin diseases of non-infectious etiologies usually predominate. such non-infectious sources of skin problems include excessive sun exposure, cutaneous reactions to medications taken for prophylaxis (including phototoxic reactions) or exposures to marine, freshwater or other irritants. furthermore, whether it is the traveler or the immigrant presenting to the physician, many cutaneous complaints are unrelated to the person's travel or national origin, but are the same conditions seen daily in every physician's office. therefore, the physician should not ignore the common sources of dermatological problems while searching for an exotic etiology. another, somewhat recent source of patients with tropical skin diseases are adoptees who frequently originate in central america or southeast asia. these children could be infected with organisms having a long incubation period that may not have been detected by physical examinations and not preventable by available vaccines. tropical infections in temperate lands, however, are not totally unique to travelers. for example, the outbreak of monkeypox in wisconsin, usa, in was a result of prairie dogs acquiring the virus from gambian rats housed in adjacent cages in pet stores. the prairie dogs then transmitted the infection to humans who had never been near the usual range of monkeypox, i.e., central africa. occasionally, the patient with a tropical disease is neither the traveler nor exposed to an animal carrying an infectious agent. the carrier may be a friend or relative who is a returned traveler who has acquired a tropical infection and who has not yet developed signs or symptoms. this possibility has recently been given much attention due to the potential spread of severe acute respiratory syndrome (sars) or avian influenza virus. on the other hand, the contaminated food may have originated in a tropical or subtropic area, such as when oysters from the gulf of mexico are shipped to the midwest usa and are consumed raw. the resulting vibrio vulnificus or hepatitis a infection thus produces gastrointestinal and cutaneous manifestations in persons who may not have visited the source of the shellfish. therefore, it is always important to ask about new pets, changes in diet, or any other change in persons with a suspected tropical disease. on the other hand, the traveler may have purchased non-consumable items which are the source of their dermatoses. for example, animal skins used for rugs or blankets may be the source of anthrax. a non-infectious cause may include nickel-containing jewelry, to which the patient has developed contact dermatitis. whereas travelers naturally fear large carnivores while on camera safari or sharks and a variety of other aquatic animals while swimming or diving, it must be remembered that the animal (indirectly) responsible for most morbidity and mortality is the mosquito (i.e., malaria, dengue, etc.) ( fig. . ). an example of a mosquito-borne disease that was considered primarily "tropical" in the recent past but is now relatively common in much of north america is infection with the west nile virus ( fig. . ). sometimes the skin findings on physical examination are not the reason for the physician visit or even the patient's complaint. such skin findings may be cultural, such as tattoos, scarification, or the result of the use of kava or of chewing betel nuts. some cultural practices, however, would be considered abuse in industrialized countries, but are widely accepted religious/cultural practices in certain lands. an example of such practice is female circumcision, which is practiced in many countries in sub-saharan africa. on the other hand, the skin changes may be much more benign, transient, and may even be the result of previous therapies, such as cupping and coining, widely practiced by immigrants from southeast asia. considerations for deciding the differential diagnosis of cutaneous manifestations of tropical diseases and/or of diseases acquired while traveling must be based not only on the type of lesions and systemic symptoms but also on the patient's history of travel. because the incubation period of various infectious diseases differs widely, it is important to know when the patient traveled. for frequent travelers, the history may become complex if they report having visited many destinations within the past few months. because vectors differ with the climate, the season of travel is also noteworthy. even in a tropical country where the temperature is always hot or warm, there may be a dry season and a rainy season. because seasons are reversed north and south of the equator, it is important to know the season at the destination. the duration of the stay is significant, not only because it increases the chance of acquiring an infectious disease but also because it tells the physician if the person was in the tropics during the incubation period of the suspected disease. whether the visitor was only in an urban environment or also in a rural area is relevant. whereas a sexually transmitted disease could be acquired in either location, an arbovirus or a zoonosis might be more likely in a rural situation. the altitude of the destination could provide a clue to the etiology of the skin condition, as could the type of sleeping condition. for example, a sexually transmitted disease could easily be acquired in a five-star hotel, but an infection transmitted by a flea, louse, or mite would be more likely in someone who slept on the ground and/or in a tent. the type and preparation of food and drink consumed by the traveler would not only help explain gastrointestinal symptoms; it could also be a clue to cutaneous signs, i.e., unsafe drinking water or milk or raw or undercooked meat, fish, or shellfish. a list of the patient's current and recent medications can be very useful and should include prescription drugs, illicit drugs, and herbal remedies, because the source of the cutaneous problem may not be directly related to the travel destination, but rather may be due to medications taken to prevent travel-related illnesses. for example, many antimalarials, such as chloroquine, mefloquine, proguanil, quinine, and halofantrine, can cause cutaneous reactions, and chloroquine, doxycycline, and quinine can cause photosensitivity. interestingly, chloroquine can worsen psoriasis. a number of agents taken to treat or prevent diarrhea can also cause cutaneous reactions, such as quinolones (ciprofloxacin, ofloxacin, sparfloxacin, levofloxacin), furazolidone, metronidazole, trimethoprim-sulfamethoxazole and bismuth sulfate; quinolones are particularly likely to produce photosensitivity. antihelmintic medications, such as ivermectin, albendazole, and diethylcarbamazine, can also produce pruritus and rash. even diethyltoluamide (deet), used to prevent arthropod bites, can cause an irritant dermatitis when used in high concentrations. because many medications in tropical countries are sold over the counter and/or have different trade names than in industrialized lands, patients are not always certain what they received if treated during their travel. likewise, an injection or transfusion given in a tropical country might also carry an increased risk of contamination. a similar risk might be taken by having acupuncture, tattoos, or body piercing in tropical lands, but these procedures can be hazardous even in industrialized countries, because the first intervention is occasionally done by non-medical personnel and the other two are almost never done by medically trained persons. a history of pre-travel vaccinations and/or immunoglobulins would be useful for possible exclusion of certain suspected etiologies. for example, if the yellow fever vaccine and the hepatitis a and/or b vaccine series were administered in sufficient time before the travel, it is less likely that these viruses were the source of the medical complaint. the traveler's occupational or recreational exposure to dirt, water, or animals can be an important component of the history. an animal bite or scratch should be easy to remember, but the bite of many arthropods may not even be noticed until after a cutaneous reaction has appeared and the responsible fly, mite, or flea has moved on to the next victim. exposure to some animals may be more indirect. for example, the spelunker (cave-explorer) may inhale aerosolized bat guano and develop rabies without ever touching a bat. a history of swimming, boating, or surfing can be a clue to an aquatic/marine etiology. such fresh-or brackish-water activities may increase the risk of infection with schistosomiasis or with free-living ameba, while marine activities may be associated with jellyfish stings, contact with the venomous spines of certain fish, or irritant dermatitis from fire coral. a preexisting skin abrasion or laceration or a puncture wound from a sea urchin or sting ray may result in a secondary bacterial infection. thus, a complete medical and travel history and physical examination is imperative in helping to narrow the differential diagnoses in the returned traveler, the immigrant, or the adoptee with a tropical origin. the qualitative and quantitative nature of the skin lesions are very important and are discussed in detail later in this chapter. specific attention must be given to the age of the patient as well as to the person who is immunocompromised due to human immunodeficiency virus (hiv), internal malignancy, organ transplantation, or other iatrogenic source of immunocompromise. blood tests, e.g., liver/kidney function tests, complete blood counts with differentials, urinalysis, skin scraping, biopsy, and/or culture are often necessary to confirm the diagnosis. a recent example of the importance of knowing both the patient's national origin and their immune status was seen when an hiv-seropositive man from myanmar presented with the first case of penicillium marneffei reported from houston, tx ( fig. . ) . many viral diseases that were not considered "tropical" years ago are now much more frequently seen in immigrants from tropical countries or in travelers who did not receive their recommended childhood vaccines. three common examples are measles, rubella, and hepatitis b. until the s measles and rubella were very common sources of infection in temperate countries, but in the twenty-first century they have become rare in industrialized countries, except for imported cases. worldwide, however, almost one million children die of measles annually ( fig. . ) and rubella still causes many congenital abnormalities. measles is still the number one vaccine-preventable killer of children in the world. morbidity and mortality are often the result of secondary bacterial infections developing in malnourished infants with measles ( fig. . ). in east asia, sub-saharan africa, and many other parts of the tropical world, hepatitis b is very common and a major source of morbidity and mortality. although measles, rubella, and hepatitis b should not be a problem in the immunized traveler, many travelers have not received the proper immunizations because they or their parents had unfounded concerns about the safety of the vaccines. this problem continues to grow as more people reach child-bearing age without ever knowing anyone who has suffered from the childhood diseases common in the first half of the th century. therefore, they do not understand that the approved vaccines are a million-fold safer than the diseases they are designed to prevent. sexually transmitted diseases (stds) should be considered at the top of the differential diagnoses when a patient presents with syndromal tropical dermatology genital lesions and/or urogenital discharge. [ ] [ ] [ ] although many of the same considerations would be true whether the patient was a recent traveler or not, certain factors should be given attention in travelers: ■ was the person traveling without his/her spouse/family and therefore outside his/her usual social structure? ■ did the person travel to countries where sex workers are readily available? while sex workers are available in most parts of the world, legally or illegally, the traveler might be less likely to acquire an std in mecca during a haj than in amsterdam, bangkok, or nairobi, where sex workers are very prevalent. ■ did the person attend parties where large amounts or alcohol and/or drugs were consumed (e.g., "spring break" in the usa)? ■ if the traveler is strongly suspected of having an std, did he/she visit a destination where chancroid, granuloma inguinale (gi), or lymphogranuloma venereum (lgv) (l serovars of chlamydia trachomatis) is prevalent? if so, the diagnostic tests and therapy might need to be expanded beyond those under consideration for stds acquired in temperate lands. when one std is confirmed, there is an increased possibility of acquisition of additional stds. not only is this the case because the source partner(s) may have had multiple stds, but also because having certain stds makes a person more susceptible to other stds. the best example of this phenomenon is the twoto fivefold greater risk of acquiring hiv if the person with a genital ulcer disease (gud) has sex with an hiv-positive individual. the reasons for this increased risk include the reduced epithelial barrier in all guds as well as the infiltrate of cd + cells in certain guds such as genital herpes. these cd + cells are the targets for hiv infection. genital herpes is the most prevalent gud in industrialized countries. in fact, the centers for disease control and prevention estimate that there are million herpes simplex virus type (hsv- )-seropositive persons in the usa. in the tropics, chancroid has been the most frequently diagnosed gud, followed by syphilis and genital herpes, but the last two diseases are becoming more prevalent in certain tropical countries. depending on the travel destination, lgv and gi must also be considered. the dates and duration of travel are important components of the history because the primary clinical presentation of all these guds ranges between - days (genital herpes and chancroid) and weeks (syphilis and gi). currently, the world health organization (who) estimates that there are million hiv-seropositive persons in the world. many of these people have gud, which may be changed both qualitatively and quantitatively by hiv. therefore the traveler may have a "non-classical" presentation of gud. in addition, it should be remembered that the signs and symptoms of gud can also appear on the perianal area/buttock or in or around the mouth. other locations are possible, but less likely. in general, however, multiple, painful, usually bilateral vesicles which progress to ulcers on skin or start as ulcers on mucous membranes, then heal over after - without therapy or within - weeks with antiviral therapy, are consistent with genital herpes. because most true primary cases of genital herpes recur, a history of multiple recurrences of the vesicles or ulcers is highly consistent with genital herpes. this diagnosis can be confirmed by viral culture or serology. in the absence of these tools, a useful test is the tzanck smear, which usually demonstrates multinucleated giant cells in herpetic lesions, but is of low sensitivity and specificity. genital herpes, however, can present many diagnostic dilemmas because the first recognized clinical occurrence is often not the result of a recent infection but rather represents a first-episode, non-primary outbreak. whereas a true primary outbreak of genital herpes is usually consistent with acquisition of the virus days to weeks previously, a first-episode, non-primary outbreak may be consistent with an infection any time in the past. in this case, the patient's recent travel history may be of less importance than his/her sexual encounters of the most distant past. although syphilis is much more common in many developing countries than in the usa, western europe or australia, the lack of travel certainly does not exclude syphilis. this diagnosis should be suspected when the patient presents with a single, non-tender, genital, perianal, or lip ulcer associated with non-tender lymphadenopathy. while chancroid is uncommonly reported in industrialized countries, it is very common in the tropics. it is usually characterized by one or more painful genital ulcers and painful syndromal tropical dermatology lymphadenopathy. in lgv the primary lesion is usually very transient and is often not seen. the clinical presentation is usually that of tender inguinal lymphadenopathy, sometimes with a suppurating bubo. the diagnosis of gi is very rarely made outside the tropics. the presentation is usually that of one or more non-tender genital ulcers with inguinal swelling. if any of these bacterial guds is suspected, the appropriate diagnostic tests must be initiated, i.e., serology for syphilis and lgv, culture for chancroid and lgv, tissue examination for gi, and the appropriate antibiotic started. whereas a history of multiple recurrences of genital vesicles or ulcers would be consistent with genital herpes, a more difficult scenario is represented by the patient who reports a single outbreak of non-specific genital signs and symptoms that are resolved by the clinic visit. a western blot or type-specific serological test for hsv- would determine whether the person was infected with this virus, but it would not be definitive proof that that hsv- was responsible for the resolved outbreak. for example, a hsv- serologically positive person may acquire syphilis, but the genital ulcer may resolve without therapy, or with inadequate treatment, before the clinic visit at home. thus, a careful history may reveal the need for serology for hsv- as well as for syphilis. because hiv can be acquired concomitantly with or subsequently to these guds, but not produce genital manifestations, hiv testing should be conducted as well. although many patients may be hesitant to admit sexual activity that puts them at risk for stds, others will worry about these activities following travel (or any time) and ask to be tested for "everything." if the sexual encounter with a new partner has been very recent, the serological test may be falsenegative because serology for syphilis, hiv, or hsv- may require weeks to become positive in the majority of persons after initial infection. patients who ignore their primary genital lesions because of denial or difficulty finding medical care during their travels may believe that the problem is gone because the lesion has resolved. if syphilis is the cause of the gud, it may reappear weeks or months later as non-genital cutaneous manifestations in the form of secondary (or tertiary) syphilis ( fig. . ) . a careful history regarding the primary lesion may lead to the appropriate diagnostic tests and therapy. some stds may not produce any genital signs or symptoms and the disease may be diagnosed long after the travel (or the non-travel acquisition), making it more difficult to find the source of the infection. although over % of hiv-seropositive persons eventually develop indirect mucocutaneous manifestations of infection, the primary rash of seroconversion (if present) is not noticed by most patients. therefore, the diagnosis is usually made when the patient develops systemic signs and symptoms (fever, chills, diarrhea, weight loss, lymphadenopathy) and/or develops one or more of the opportunistic infections, neoplasms, or inflammatory skin problems frequently seen in hiv patients. similar to hiv, primary infection with hepatitis b rarely produces genital lesions. diagnosis is usually made long after infection due to systemic symptoms or non-specific skin changes such as jaundice. it is noteworthy that hepatitis b is the only std for which a prophylactic vaccine is available. therefore, a history of successful hepatitis b vaccination makes this diagnosis less likely. although the pustules of disseminated gonococcemia are distinctive, the consequences of untreated neisseria gonorrhoeae (gonococcus) are usually pelvic inflammatory disease, epididymitis, proctitis, pharyngitis, or conjunctivitis. pelvic inflammatory disease can also be caused by chlamydia trachomatis (non-l serovars), mycoplasma hominis, or various anaerobic bacteria. the non-l serovars of c. trachomatis can also cause epididymitis, proctitis, and conjunctivitis. pharyngitis can also be due to hsv- or entamoeba histolytica. the initial presentation of gc, c. trachomatis (non-l serovars), ureaplasma urealyticum, mycoplasma genitalium, trichomonas vaginalis, or even hsv- may be urethritis. vaginal discharge can be caused by any of these organisms as well as candida albicans, gardnerella vaginalis, peptostreptococci, bacteroides spp. or mobiluncus spp. these organisms can usually be diagnosed by smear, wet-mount, dna detection, serology, or culture. antimicrobial therapy is usually initiated based on the physical examination and smear or wet-mount and modified, as needed, when other laboratory studies are completed. infection with human papillomaviruses (hpv) is one of the most common stds in the world, but the clinical implications of the infection vary widely. there are over hpv types that can cause genital lesions, but most infections do not result in any visible lesions. because the incubation period of hpvs can be months, or even years, if and when genital lesions do develop, it is often very difficult for the patient to determine the source partner. therefore, it is usually a challenge for the physician to relate hpv lesions to travel, especially recent travel. non-oncogenic genital hpv, such as types and , result in condyloma acuminatum, which can be treated with cytodestructive therapy, surgery, or with the immune response modifier, imiquimod. oncogenic genital hpv, such as types and , can result in anogenital cancer, the most prevalent of which is cervical cancer. cervical cancer is the second most prevalent cancer killer of women in the world and sexually transmitted diseases over % of all cervical cancer is caused by hpv. most cervical cancer deaths are in tropical countries, making hpv one of the world's deadliest tropical diseases (although rarely listed with the other major tropical diseases). there are many reasons why more cervical cancer deaths occur in tropical countries. first, in industrialized countries most women receive regular pap smears, which result in early detection and subsequent therapy of cervical abnormalities, thus reducing progression to cervical cancer. if cancer is detected, surgery, radiation therapy, or chemotherapy is available. in most tropical countries, regular pap smears are not the standard of care. therefore, cervical cancer is often detected too late for successful intervention, even if it is available. second, there appears to be a genetic susceptibility that allows oncogenic hpv to progress to malignancy. this genetic susceptibility appears to be more prevalent in certain tropical countries. the rarity of male circumcision in many tropical countries appears to be a risk factor for the development of cervical cancer in these males' partners. third, most of the world's estimated million hiv-seropositive individuals live in tropical countries where no antiretroviral therapy is available. not only is cervical cancer an acquired immunodeficiency syndrome (aids)-defining illness in hiv seropositive women, the same hpv can cause anal cancer, which is a major problem in homosexual men with hiv. molluscum contagiosum (mc) is a poxvirus that can be sexually transmitted, resulting in wart-like lesions on the genitalia. in contrast to hpv, however, mc does not progress to malignancy. like condyloma acuminatum, however, mc can be treated with cytodestructive therapy, surgery, or imiquimod. ectoparasites such as scabies, sarcoptes scabiei, and pubic lice, phthirus pubis, can be sexually transmitted. in contrast to many stds, however, these ectoparasites can be easily treated with topical medications such as lindane or permethrin. like all stds, if the sexual partner is not treated concomitantly, reinfection is common. the most common cause of fever after tropical travel is malaria, which usually does not have specific cutaneous manifestations. dengue fever is the second most common cause of fever in the traveler and does have somewhat specific cutaneous manifestations, making dengue fever the leading cause of fever with rash in the traveler returning from a tropical destination ( fig. . ) . other common causes of fever and rash include hepatitis viruses, rickettsia, and some enteric fevers. it should always be kept in mind, however, that fever in the returned traveler may not be due to exposure during travel. for example, the fatigue of travel, i.e., jet lag, may make one more susceptible to influenza or other common infections in temperate lands. when both fever and rash are seen, the time between travel and onset of signs and symptoms becomes increasingly important. if travel preceded fever and rash by less than - weeks, considerations should include anthrax, dengue fever, diphtheria, ehrlichiosis, hemorrhagic fever viruses, leptospirosis, lyme disease, measles, meningococcal infections, plague, rickettsia, toxoplasmosis, trichinosis, tularemia, typhoid fever, and yellow fever. if the period between travel and fever/rash is up to a month, the list should be expanded to include hepatitis viruses (a, c, and e), hiv, rubella, schistosomiasis and trypanosomiasis. if at least months separate travel from fever/rash, the following infections should be considered: bartonellosis, filariasis, gnathostomiasis, hepatitis viruses (b and c), histoplasmosis, hiv, leishmaniasis, lyme disease, melioidosis, penicilliosis, syphilis, trypanosomiasis, and tuberculosis. in each case, however, the nature of the fever, the type of rash, the destination of the travel, and any other symptoms must be considered. because many infections producing fever with rash can be rapidly fatal and/or easily spread, it is imperative immediately to initiate diagnostic tests and antimicrobial therapy for the presumed cause of the infection. such infections include anthrax, bartonellosis, candida (macronodules), diphtheria, disseminated gonorrhoeae (papules and pustules over joints), hepatitis viruses, leptospirosis, meningitis (asymmetrical, scattered, petechiae, and purpura), plague, pseudomonas (ecthyma gangrenosum), relapsing fevers, rickettsia (scattered petechiae and purpura), staphylococcus (osler's nodes, diffuse toxic erythema), streptococcus (janeway lesions, diffuse toxic erythema), strongyloides (migratory petechiae and purpura), syphilis, tuberculosis, typhoid fever (rose spots) ( fig. . ), various gram-negative bacteria (i.e., peripheral gangrene), vibrio (especially v. vulnificus), and viral hemorrhagic fevers (petechiae, purpura, hemorrhage). eosinophilia may be due to diverse processes, such as allergic, neoplastic, and infectious diseases. although an allergic reaction could easily result from an exposure during travel, eosinophilia in the returned traveler may have nothing directly to do with the travel. on the other hand, it may be due to an infectious process or to a drug taken for prophylaxis or therapy during travel. if an infection is the cause of the eosinophilia, it is usually a parasitic disease, especially due to a helminth. only a few viral, bacterial, or fungal diseases are associated with both rash and eosinophilia, e.g., streptococcal (i.e., scarlet fever), tuberculosis, hiv, and coccidioidomycosis. protozoa only rarely provoke eosinophilia. the principal helminth that causes eosinophilia is strongyloides. when strongyloides is disseminated such as in the hyperinfection syndrome, skin lesions such as urticaria, papules, vesicles, petechiae, and migratory serpiginous lesions become common, especially if the patient is given systemic corticosteroids (because strongyloides was not considered). pruritic, erythematous papules can be seen as a result of schistosomal cercariae, as in swimmer's itch. eosinophils may be seen in the skin biopsy as well as in the blood. pruritic lesions of the skin and subcutaneous tissues are commonly associated with eosinophilia in onchocerciasis. lymphangitis, orchitis, and epididymitis are also commonly observed. in loiasis, fever and eosinophilia are typically seen. migratory lesions, especially angioedema, are usually erythematous and pruritic. likewise, gnathostomiasis produces recurrent edema after ingestion of raw fish. the skin lesions are usually erythematous, pruritic, and/or painful. drug hypersensitivity is a relatively common cause of eosinophilia and may be associated with non-specific skin changes such as urticaria and/or phototoxic reactions. although most drugs that cause eosinophilia may not be taken for purposes related to traveling, increased sun exposure during travel may make the problem clinically apparent. because antibiotics may be taken for prophylaxis or therapy more frequently during traveling, they should be given careful consideration when eosinophilia is detected. such antibiotics include penicillins, cephalosporins, quinolones, isoniazid, rifampin, and trimethoprim-sulfamethoxazole. non-specific cutaneous manifestations of tropical diseases may include pruritus and urticaria. frequently, more specific signs may accompany pruritus and urticaria, which are useful in narrowing the differential diagnoses. if eosinophilia is found with the pruritus and urticaria, helminthic infections should be considered. therefore, consideration should be given to trichinellosis, strongyloidiasis, schistosomiasis, onchocerciasis, loiasis, hookworms, gnathostomiasis, dracunculiasis, and cutaneous larva migrans. pinworms, as well as protozoa such as amebiasis, giardiasis, and trypanosomiasis, are less likely to produce eosinophilia. pruritus and urticaria are possible with spirochetes such as borrelia (e.g., relapsing fevers), spirillum (e.g., rat-bite fever) and treponema (i.e., syphilis and pinta). yersinia (e.g., plague) is another bacteria that produces pruritus and urticaria, which can be present before buboes form. the hepatitis viruses (e.g., a, b, and c) can produce pruritus and urticaria, as can a number of ectoparasites and biting arthropods, e.g., ticks, scabies, bedbugs, lice, fleas, mites, and flies. [ ] [ ] [ ] [ ] [ ] jaundice although hepatitis viruses can produce pruritus and urticaria, jaundice is a more specific indication that the problem has a hepatitic etiology. not only can all the hepatitis viruses (a-e) produce jaundice, other tropical viruses also do so commonly, e.g., yellow fever and rift valley fever. less frequently, dengue and epstein-barr viruses can cause jaundice, as can such bacteria as leptospira (i.e., leptospirosis), coxiella (i.e., q fever) and treponema (i.e., syphilis). protozoa, such as malaria, and drug reactions can also be responsible. although vesicles and bullae can appear as a result of contact dermatitis or drug eruption, including photodermatitis and photoexacerbated drug eruptions as well as toxic epidermal necrolysis, many cases represent the early stages of a viral or bacterial infection. the most common viral etiology in the traveler or non-traveler includes the herpesviruses, especially herpes simplex virus and , as well as varicella-zoster virus, both primary varicella and herpes zoster. measles and many enteroviruses (e.g., hand, foot, and mouth disease) can present with vesicles, as can certain alphaviruses. a number of poxviruses, such as vaccinia, variola, orf, tanapox, and monkeypox, can produce vesicles. less commonly, vesicles comprise an early stage of certain bacterial diseases such as vibrio vulnificus, bacillus anthracis, brucella spp., mycobacteria tuberculosis, mycoplasma spp., rickettsia akaru and staphylococcus (bullous impetigo). other organisms such as fungi that cause tinea pedis, protozoa (e.g., leishmania brasiliensis), and helminths (e.g., necator americanus) can occasionally cause vesicles. a wide variety of infectious and non-infectious etiologies are related to both macules and to papules. almost any of the vesicular diseases listed above may initiate first as a macule, then a papule, before becoming a vesicle. a number of drugs, arthropod bites (e.g., mosquito or flea) and infestations (e.g., scabies and other mites) commonly cause macules and/or papules. the range of terrestrial, freshwater, and marine contactants can elicit these cutaneous reactions, as can a spectrum of drugs. viral etiologies include hiv, as in the hiv seroconversion syndrome, epstein-barr virus (infectious mononucleosis), human herpesvirus (roseola), parvovirus b- (fifth disease), measles, rubella, and various hemorrhagic fever viruses. many bacteria can be responsible, such as rickettsia, bacillus anthracis, spirochetes (spirillum, leptospira, borrelia, treponema), coxiella burnetii, yersinia pestis, salmonella typhi, bartonella bacilliformis, and brucella. histoplasmosis and coccidioidomycosis are fungal diseases commonly associated with ulcers and other specific skin lesions use in bioterrorism. the best-recognized etiology of a noninfectious eschar is a bite from a brown recluse spider. petechiae and purpura can result from adverse reactions to a number of drugs. the most important infectious cause of petechiae and/or ecchymoses with fever is meningococcemia, which has a high rate of morbidity and mortality and is widespread throughout the tropical world and found sporadically in industrialized countries. other bacterial causes include borrelia, burkholderia, enterococcus, haemophilus, leptospira, pseudomonas, rickettsia, streptobacillis, treponema, vibrio, and yersinia. a number of hemorrhagic fever viruses can cause petechial or purpuric lesions, but the most prevalent viral causes are enteroviruses, cytomegalovirus, dengue, and yellow fever. protozoal diseases (e.g., malaria and toxoplasmosis) and helminths (e.g., trichinellosis) can also induce this clinical presentation. changes in pigmentation can be seen after a variety of medications, many of which are taken for prophylaxis or therapy related to travel. these agents include a spectrum of drugs such as antibiotics, antidiarrheals, anthelmintics, and antimalarials, many of which can also elicit photosensitization. a number of infectious agents can also alter pigmentation. leishmaniasis, pinta, and tinea versicolor may be associated with hypopigmentation or hyperpigmentation. leprosy, onchocerciasis, syphilis, and yaws are more often associated with hypopigmentation. erythrasma, hiv, and loiasis are more frequently causes of hyperpigmentation. with the exception of the movements of larvae of myiasis, mucocutaneous migratory lesions are usually due to infections with helminths. the best-recognized example is cutaneous larval migrans, but migratory lesions can also be due to dracunculiasis, fascioliasis, gnathostomiasis ( fig. . ) , hookworms, loiasis, paragonamiasis, sparganosis, or strongyloidiasis. in conclusion, the differential diagnoses of mucocutaneous lesions in the returned traveler, immigrant, or adoptee should be based on the morphology of the lesions, but the patient's symptoms, general medical, and exposure history must all be considered. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the physical examination and laboratory results must be integrated with the patient's vaccination and medication record. the travel destination(s), travel duration, living, work/recreation conditions, food and drink ingestion, and activities while traveling must all be taken into consideration. it must not be forgotten, however, that many mucocutaneous problems in the returned traveler or the immigrant/adoptee are not related to the travel or the country of origin but can be the same disorders seen daily in patients who have never left their local communities. health problems after travel to developing countries an overview of sexually transmitted diseases. part i (bacterial stds) an overview of sexually transmitted diseases. part ii (viral stds) an overview of sexually transmitted diseases. part iii. sexually transmitted diseases in hiv infected patients ulcers and other specific skin lesions dermatoses associated with travel to tropical countries: a prospective study of the diagnosis and management of patients presenting to a tropical disease unit cutaneous manifestations of intestinal helminthic infections parasitic infections of the skin skin problems in returning travelers dermatologic manifestations of parasitic disease the travel and tropical medicine manual kaposi's sarcoma and other manifestations of human herpesvirus mucocutaneous manifestations of viral diseases tropical dermatology: viral tropical diseases lyme borreliosis dermatologic infectious diseases in international travelers macules and/or papules. certain protozoa such as toxoplasma gondii and leishmania can also induce these types of lesions. among helminths, hookworm disease, strongyloidiasis, and onchocerciasis can be associated with macules and/or papules. although otherwise similar, papules are usually less than . - . cm, while nodules are larger than . - . cm in diameter. except for certain poxviruses which cause orf and milker's nodules, as well as warts and malignancies induced by hpv, viruses rarely form nodules. on the other hand, all subcutaneous and systemic mycoses can induce nodules. bacterial causes of nodules include bartonella (verruga peruana and cat-scratch disease), buckholderia mallei (glanders), calymmatobacterium granulomatis (gi), chlamydia trachomatis (lgv), klebsiella rhinoscleromatis (rhinoscleroma), leptospira autumnalis (leptospirosis), mycobacteria spp. (atypical mycobacteria, cutaneous tuberculosis, leprosy, etc.), nocardia brasiliensis (and other bacterial causes of mycetoma) and treponema pallidum (bejel, yaws). protozoan causes of nodules include amebiasis, leishmaniasis, and trypanosomiasis. almost all helminthic infections that have mucocutaneous manifestations can induce nodules, e.g., coenurosis, cysticercosis, dirofilariasis, dracunculiasis, echinococcosis, filariasis, gnathostomiasis, loiasis, onchocerciasis, paragonimiasis, schistosomiasis, sparganosis, and visceral larval migrans. if the helminthic nodule contains sufficient fluid, it will produce a cyst. cysts can be seen in helminthic infections such as coenurosis, echinococcosis, filariasis, gnathostomiasis, loiasis, and onchocerciasis. there are also arthropod causes of nodules such as myiasis, scabies, tick granulomas, and tungiasis. whereas ulcers can form as a result of breakdown of previously normal skin, they frequently develop from nodules after inflammation destroys the epidermis and papillary layer of the dermis. herpes simplex virus is a very common cause of ulcers in both tropical and temperate regions of the world. other causes of gud are bacterial, e.g., chancroid, gi, lgv, and primary syphilis. other bacterial diseases that commonly cause ulcers include anthrax, bacterial mycetomas, diphtheria, glanders, melioidosis, mycobacterial diseases (e.g., buruli ulcer, leprosy, tuberculosis), plague, rickettsia, tropical ulcers, tularemia, and yaws. a number of fungi can form nodules that break down into ulcers, or they can induce ulcers from systemic spread: blastomycosis, chromomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, lobomycosis, mycetomas, paracoccidioidomycosis, penicilliosis, and sporotrichosis. the most common helminthic cause of cutaneous ulcers is dracunculiasis, when the worm erupts from the skin. two protozoan diseases cause ulcers -amebiasis and leishmaniasis. arthropod causes of ulcers include myiasis and tungiasis. many bites (e.g., brown recluse spiders and various snakes), stings (e.g., insect, jellyfish, and scorpion) or venomous spines of various fish can also induce ulcers. an eschar can be seen in both temperate and tropical lands due to pseudomonas aeruginosa (i.e., ecthyma gangrenosum), but the most common causes of eschars are rickettsia. eschars due to anthrax can be seen in persons who work with animal skins, but anthrax has received much attention recently due to its potential key: cord- -v nw xh authors: willard, michael d.; twedt, david c. title: gastrointestinal, pancreatic, and hepatic disorders date: - - journal: small animal clinical diagnosis by laboratory methods doi: . /b - - - / - sha: doc_id: cord_uid: v nw xh nan expectoration is the coughing up of material from the lungs or major airways. the material typically is frothy mucus or red blood; bile is absent. the characteristic sequence of coughing followed by oral expulsion must be determined from the history. regurgitation and vomiting typically occur without simultaneous coughing, although regurgitation is often accompanied by tracheitis and aspiration pneumonia. of the three, expectoration should be the easiest to identify. regurgitation is due to oral, pharyngeal, or esophageal dysfunction and is typically characterized as a relatively passive expulsion of esophageal contents. gagging is the expulsion of oral or pharyngeal material and may be associated with disorders causing dysphagia (i.e., difficult swallowing) or regurgitation. the relatively minor abdominal contractions associated with gagging can often be differentiated from the vigorous abdominal contractions that occur with vomiting. regurgitation may follow seconds to hours after eating or drinking. if only saliva is regurgitated, eating may not have occurred for hours or even days before the act. regurgitated food material is undigested and sometimes has a tubular form conforming to the shape of the esophageal lumen. most clients cannot reliably distinguish undigested from digested food. regurgitated material that has remained in the esophagus for long time periods often becomes macerated and odoriferous and is mixed with saliva and mucus. if blood is present it is usually undigested (i.e., bright red), whereas blood originating from the stomach is usually partially digested by gastric acid and has a "coffee ground" appearance readily distinguishing it from the undigested form. it is sometimes difficult to differentiate vomiting from regurgitation via history, and in some patients the processes are concurrent. vomiting may cause secondary esophagitis and subsequent regurgitation, or a patient with longstanding esophageal disease may develop another concurrent disorder causing vomiting. it is therefore important to clarify the chronologic order of specific signs. finally, some patients with signs suggesting regurgitation are vomiting. to aid in differentiation, one may attempt to observe the act of expulsion by feeding the patient, although this is very unreliable ("watched" regurgitating patients often do not regurgitate). watching the patient eat may be helpful, because pharyngeal dysphagia is usually obvious and suggests oropharyngeal disease. some patients with pharyngeal dysphagia also have concurrent esophageal dysfunction. contrast radiographs of the pharynx and esophagus usually differentiate the two disorders. regurgitation is usually best evaluated by history, physical examination, plain and contrast radiographs, or esophagoscopy ( figure - ). contrast radiographs should use barium instead of iodide contrast agents unless esophageal rupture is strongly suspected. the main purpose of a contrast esophagram is to distinguish esophageal motility abnormalities from lesions such as an obstruction, diverticulum, or fistula. some drugs (e.g., xylazine) commonly used for restraint cause esophageal paralysis, making the radiographs potentially misleading. esophagoscopy may not diagnose esophageal muscular weakness but is effective for sampling mass lesions, differentiating intramural from extramural obstruction, identifying esophagitis, detecting diverticula, and removing foreign objects. patients with acquired esophageal weakness (i.e., megaesophagus) should be evaluated for myopathies, neuropathies, and myasthenia gravis (generalized or localized to the esophagus). occasionally, hypoadrenocorticism, hyperkalemia, lead poisoning, spirocerca lupi, and selected central nervous system (cns) disorders (e.g., distemper, hydrocephalus) may be responsible. generalized or localized myopathies and neuropathies have several causes, such as trauma, dermatomyositis, thymoma, botulism, tick paralysis, hypothyroidism, hyperadrenocorticism, systemic lupus erythematosus, nutritional factors, toxoplasmosis, and trypanosomiasis. dysautonomia has been recognized in dogs and cats and causes generalized dysfunction of the autonomic nervous system, including regulation of esophageal motility. hypothyroidism and systemic lupus erythematosus may exist without obvious clinical signs. it is important to detect these underlying disorders so that one may treat the cause rather than just the symptoms. it is also wise to evaluate patients with unexpected esophageal foreign objects (e.g., a relatively small bolus of food) for partial obstructions (e.g., subclinical vascular ring anomaly, stricture). vomiting is a reflex act originating in the cns that can be stimulated by various conditions. one must consider primary gi disease and non-gi disorders as causes of vomiting. examples of non-gi disorders include metabolic, inflammatory, and toxic conditions. many vomiting patients are probably vomiting from non-gi instead of primary gi problems. vomiting is classically characterized by prodromal nausea (i.e., salivation, licking of lips) followed by retching or forceful abdominal contractions. vomiting may occur any time after eating or drinking (seconds to hours). a patient may vomit food, water, fresh blood, or mucus that is indistinguishable from regurgitated material. bile, partially digested blood (i.e., "coffee grounds"), or expelled material with a ph of five or less confirms that vomiting is occurring. vomited duodenal contents may have a ph greater than or equal to six and are usually positive for bile. a urine dipstick with a ph indicator is useful in making ph determinations. clinically, vomiting patients are best divided into those with acute (< weeks) versus those with chronic (> weeks) vomiting. the most common categories of causes for each are listed in tables - and - . acute vomiting often spontaneously resolves if the patient is supported by fluid, electrolyte, and acid-base therapy. a thorough history and physical examination are indicated first. laboratory evaluation (including electrolytes and acid-base evaluations) or imaging should be considered next if the disease is severe. if vomiting persists, is progressive, or is attended by other clinical signs (e.g., polyuria-polydipsia [pupd] , weight loss, icterus, painful abdomen, ascites, weakness, hematemesis), additional testing is also indicated (figure - ). diet and parasites commonly cause acute and chronic vomiting; hence, dietary change (to a bland or hypoallergenic diet), fecal examination, and broad-spectrum anthelmintic therapy (e.g., fenbendazole, pyrantel) are reasonable initial choices in nonobstructed patients. continued vomiting is an indication for laboratory tests or imaging. gastric or intestinal obstruction does not require clinicopathologic testing for diagnosis. a complete blood count (cbc) may suggest sepsis, disseminated intravascular coagulation (dic), or severe blood loss. renal function, electrolyte, and acid-base evaluations are recommended before anesthesia. one cannot reliably predict changes in these parameters even when the site of obstruction is known. gastric vomiting sometimes causes hypokalemic, hypochloremic metabolic alkalosis with aciduria. these changes generally occur secondary to persistent and profuse vomiting, gastric outflow obstruction, or high duodenal obstruction. most patients with gastric vomiting are not alkalotic. insignificant acid-base changes or metabolic acidosis due to dehydration with resultant lactic acidosis is perhaps more common. intestinal obstruction may cause acidosis as the result of loss of pancreatic bicarbonate, although some patients have a normal blood ph or a metabolic alkalosis if the obstruction is high. abdominal palpation and imaging are the best initial diagnostic tests. in otherwise occult cases, contrast radiographs may be necessary. barium is preferred over iodide compounds unless intestinal rupture is strongly suspected. barium leakage causes peritonitis and requires vigorous abdominal lavage at the time of surgery (see chapter ). a serum chemistry profile should be obtained to help rule out hepatic disease (alanine aminotransferase [alt] , serum alkaline phosphatase [sap] , blood urea nitrogen [bun] , and albumin), hypoadrenocorticism (sodium and potassium), hypercalcemia (calcium and albumin), uremia (creatinine, bun, and urinalysis), and diabetic ketoacidosis (glucose and urinalysis). very young patients (those < to weeks of age) should undergo blood glucose monitoring to avoid secondary hypoglycemia. more precise testing is occasionally required to diagnose these disorders (e.g., serum bile acids for hepatic insufficiency, adrenocorticotropic hormone [acth]stimulation test for hypocortisolemia). other tests to consider are serum gastrin for gastrinoma, and serum thyroxine for feline hyperthyroidism. acute pancreatitis is recognized commonly. predisposing causes in dogs include hyperlipidemia, fatty meals, or obesity. pancreatitis can occur in any dog, but middle-aged obese female dogs, schnauzers, and yorkshire terriers seem to be most commonly affected. vomiting may or may not be associated with eating, abdominal pain, fasting hyperlipidemia, bloody diarrhea, and rarely, diffuse subcutaneous fat necrosis. on radiographic examination, a mass or indistinctness (as the result of localized peritonitis) may be visible in the cranial right abdominal quadrant. serum amylase and lipase activities can be measured, but falsenegative and false-positive results are common, necessitating reliance on other findings. leukocytosis with or without a left shift and with or without wbc toxicity (as the result of the sterile inflammation) and increased alt and sap concentrations (as the result of the proximity of the pancreas to the liver and obstruction of the biliary duct) are common. the latter occasionally causes extrahepatic biliary tract obstruction and subsequent icterus. mild to moderate hypocalcemia may occur. abdominal ultrasonography seems to be an excellent test for canine pancreatitis, and it usually reveals abnormalities in the recently an assay for immunoreactive canine pancreatic lipase has been developed and validated (gi laboratory at texas a&m university); this test seems to hold promise for being a sensitive, specific test for acute pancreatitis. once considered rare in cats, pancreatitis is being recognized with increasing frequency in cats. feline pancreatitis is more difficult to diagnose. chronic pancreatitis is not uncommon in older cats, occurring in conjunction with cholangiohepatitis and sometimes with inflammatory bowel disease. the presence of the three diseases together has been referred to as "triaditis syndrome." vomiting is not as prominent as in dogs. amylase and lipase values are usually in the normal reference range; feline trypsin-like immunoreactivity (ftli) concentrations are increased in some patients. abdominal ultrasonography may be useful if an obvious abnormality is found, but the sensitivity of ultrasonography is uncertain. a pancreatic biopsy may be required for a definitive diagnosis. feline pancreatitis occasionally is due to toxoplasmosis or to feline infectious peritonitis (fip) (see chapter ). chronic enteritis, colitis, or gastritis can cause various degrees of vomiting and may require mucosal biopsy for diagnosis. abdominal imaging may delineate infiltrative or inflammatory intestinal patterns. if gastritis or enteritis is suspected or if the other major causes of chronic vomiting have been ruled out, gastric and intestinal mucosal biopsies via endoscopy or laparotomy are indicated. inflammatory bowel disease is a significant cause of feline chronic vomiting. duodenitis is also a significant cause of vomiting without diarrhea in dogs; therefore, both gastric and intestinal biopsies should be performed. finally, because % to % of patients with colitis vomit, it is useful to perform endoscopy routinely on both the upper and lower intestinal tracts in patients (especially cats) with chronic vomiting. it is critical that mucosal tissue samples be taken and handled properly to avoid artifacts, which can render them nondiagnostic. hematemesis is the vomiting of blood. it suggests gastric ulceration. the character of the vomitus may be either bright-red blood or digested blood that resembles coffee grounds. administration of nonsteroidal anti-inflammatory drugs (especially concurrently with corticosteroids) is a major reason for canine ulceration. renal and hepatic failure, mast cell tumor, shock with poor mucosal perfusion, and coagulopathy must be considered. after these have been ruled out, endoscopy is indicated and allows diagnosis of ulceration (especially because of a foreign object, inflammatory disease, or neoplasia). alternatively, one may treat symptomatically for ulceration; however, such treatment may allow progression of underlying disease. septic or nonseptic peritonitis (or inflammation of any abdominal organ) may cause vomiting. abdominocentesis or abdominal lavage (see chapter ) may be needed, especially if physical examination or abdominal imaging suggests abdominal fluid. occult cases may require exploratory surgery for diagnosis. gastrinoma (e.g., zollinger-ellison syndrome) is a gastrin-secreting tumor of the pancreatic islet cells; it increases gastric acid production and produces duodenal ulceration. gastrinoma is rare but has been diagnosed more commonly since the advent of reliable serum gastrin assays. no other typical, unique clinicopathologic tests exist that suggest this disease. any chronically vomiting middle-aged or older dog with weight loss or diarrhea is a reasonable suspect. duodenal ulceration and reflux esophagitis are common. resting gastrin concentrations are usually increased, but in rare cases one must measure gastrin concentrations after administering food or secretin. causes of hyperamylasemia • decreased glomerular filtration (i.e., azotemia) and pancreatitis are two causes. hyperamylasemia as the result of renal dysfunction usually is less than two to three times the upper limit of normal. patients with pancreatitis may have normal to markedly increased values. intestinal disease, ruptured intestines, and hepatic disease have been suspected of causing increased serum amylase because of amylase present in these tissues. serum amylase level is an unreliable indicator of pancreatitis in cats. hyperamylasemia in a vomiting or anorexic animal is an indication to search for pancreatitis by cbc, serum chemistry profile (including alt and sap), abdominal imaging, serum trypsin-like immunoreactivity (tli), or a combination thereof. when abdominal fluid amylase is greater than serum amylase, a nonseptic exudate caused by pancreatic disease is possible. bowel rupture may also be possible. table - ) plus heparin. corticosteroids (dexamethasone) may increase serum lipase activity up to fivefold over baseline without potassium bromide (this is a reported but unproven association) salicylazosulfapyridine (azulfidine) sulfonamides tetracycline thiazide diuretics note: these drugs do not reliably cause pancreatitis, and a history of administration of one of these drugs plus signs of pancreatitis cannot be assumed to be cause and effect. a patient with acute pancreatitis that is receiving one of these drugs, however, should undergo drug withdrawal, if possible. note: different methods can give substantially different values. some saccharogenic methods are affected by normal canine serum maltase concentrations and should not be used in dogs. serum amylase activity is stable at room temperature for up to days and at °c for as long as month. histologic evidence of acute pancreatitis; however, the lipase activity is usually only slightly greater than the reference range. causes of hypolipasemia • not significant. this finding does not support a diagnosis of pancreatic insufficiency. causes of hyperlipasemia • these are similar to the causes of hyperamylasemia. renal dysfunction increases serum lipase, usually less than two to three times normal, although it may rarely be more than four times normal. not all patients with acute pancreatitis have increased serum lipase, and the increase in serum lipase activity is not proportional to the severity of the pancreatitis. extremely increased lipase values have been associated with pancreatic carcinomas. abdominal ultrasonography and increases in serum ftli appear to be more useful than serum amylase or lipase in the diagnosis of feline pancreatitis. in addition to serum tli, trypsin-activating peptide (tap) and phospholipase a have been investigated as tests for pancreatitis in dogs; however, none have become accepted as valued clinical tests. conversion of pg/ml to ng/l: multiply pg/ml × . = ng/l. artifacts • falsely decreased: hormone degradation as the result of storage for several days at temperatures above freezing. drug therapy that may increase gastrin • antacids including h receptor antagonist drugs, and proton pump inhibitors. causes of hypergastrinemia • atrophic gastritis (uncommon), antral g-cell hyperplasia (rare), short bowel syndrome, hyperparathyroidism, ulcers, gastric outlet obstruction, renal failure, and gastrinoma are the main causes. the last four are the most common. hepatic insufficiency does not appear to directly increase serum gastrin concentrations. if gastrinoma is suspected in a patient that has a normal or equivocal serum gastrin concentration, secretin or calcium stimulation tests may be performed. a rise in the serum gastrin concentration after giving either of these drugs suggests a gastrinoma. patients with diarrhea are best classified into those with acute (< to weeks) versus those with chronic (> to weeks) diarrhea. acute diarrhea (table - ) is usually selflimiting, although some conditions may be severe and cause mortality, such as acute hemorrhagic gastroenteritis, parvoviral disease, parasites (e.g., hookworms), or intoxication. history should explore the possibility of recent dietary change or exposure to infectious agents. diet, bacteria, viruses, and parasites are the major identifiable causes of acute diarrhea in dogs and cats. because intestinal parasites may contribute to any diarrheic state, multiple fecal examinations (direct and flotation) are warranted in all diarrheic patients. giardiasis may be particularly occult and require special diagnostic techniques (see fecal giardia detection). feeding with bland or hypoallergenic diets may be diagnostic and therapeutic. depressed, weak, and dehydrated patients should undergo electrolyte and acid-base evaluations to aid in selecting fluid replacement therapy. all patients less than to weeks of age and those that are emaciated or weighing less than pounds should undergo blood glucose monitoring to detect secondary hypoglycemia. febrile or depressed patients should undergo cbc analysis so that sepsis or transmural inflammation can be detected. to identify the cause of acute diarrhea that is not the result of diet or parasites (such as that occurring in kennels, pet stores, shelters, and households where more than one member has diarrhea), fecal cultures for salmonella spp., campylobacter jejuni, yersinia enterocolitica, verotoxin-positive escherichia coli, and other pathogens plus viral identification methods (i.e., elisa, electron microscopy) or toxin identification methods (i.e., elisa for clostridium perfringens or clostridium difficile toxins) or both may be used. not all patients with canine parvoviral diarrhea are severely ill, have identifiable leukopenia, have diarrhea, or have a fever. leukopenia may persist as briefly as to hours and can easily be missed if a cbc is not performed during that period. other diseases causing severe sepsis (i.e., perforating linear foreign body with peritonitis or overwhelming salmonellosis) can cause leukopenia indistinguishable from that of canine parvoviral diarrhea. routinely used vaccination schedules do not necessarily guarantee protection against canine parvovirus. finally, fecal shedding of viral particles may not occur for - days after signs begin and decreases rapidly with time. in-house elisa tests for parvovirus are performed on the feces and appear to be accurate in identifying the parvoviral antigen, but testing may be negative if done too early or too late. the test result should be strongly positive within days of the onset of clinical signs and remain positive for several days. a recent vaccination may result in a weakly positive fecal elisa. chronic diarrhea should first be defined as either small intestinal or large intestinal in origin, preferably by using the history and physical examination (table - ). occasionally, large and small intestines are concurrently involved. patients with chronic diarrhea in which clinical disease is not severe are often treated with therapeutic trials before aggressive diagnostics are instituted. all patients should once parasitism, dietary-responsive disease, and clostridial colitis are eliminated, additional simple diagnostic steps, such as rectal mucosal scrapings (not swabs) with cytologic examination (see color plate b) or fecal culture for c. jejuni, salmonella spp., y. enterocolitica, verotoxin-positive e. coli, or a combination of these might be appropriate. persistent large intestinal disease is usually an indication for colonoscopy plus biopsy, especially if the animal has hypoalbuminemia or has lost weight. rigid colonoscopy of the descending colon is adequate for diagnosis in most cases. flexible endoscopy allows access to the descending, transverse, and ascending colon; ileocolic valve; cecum; and ileum. if flexible endoscopy is unavailable, abdominal ultrasonography or a barium enema may reveal lesions in areas not accessible with rigid endoscopy. chronic and severe small intestinal diarrhea necessitates differentiation of maldigestion, protein-losing enteropathy (ple), and malabsorptive disease without protein loss ( figure - ). weight loss and diarrhea are usually present, but some patients only have weight loss. maldigestion resulting from bile acid insufficiency as the result of biliary obstruction is rare. intestinal lactase deficiency is uncommon, but a lactose-free diet may be tried in selected patients (especially cats). epi is the most common cause of canine maldigestion but is rare in cats. differentiation of epi from malabsorptive disease is important. the diagnosis is often overlooked in afflicted dogs or may inappropriately be made in patients without the malady. clinical trials using pancreatic enzyme preparations are commonly used to diagnose epi. unfortunately this method is unreliable. powdered enzyme is often superior to tablet formulations, and some enzyme preparations are clearly superior to others. even when appropriate enzymes are administered, some dogs with epi also require a low-fat diet, antacid therapy (rare), or treatment for concurrent antibiotic-responsive enteropathy (are) (common) before the enzyme replacement therapy becomes effective. too often, failure of the initial enzyme replacement therapy leads to unnecessary tests (i.e., exploratory laparotomy), because the correct diagnosis of epi was incorrectly eliminated. no consistent hematologic or serum chemistry profile changes are seen, and the serum amylase and lipase values are usually normal. undigested fats are often found in the feces; however, this is inconsistent. the fat absorption test is inexpensive but can yield false results. the tli assay is the best test for canine epi, and the ftli assay is the best test for feline epi. it is important to note that the tests are species specific. measurement of trypsin proteolytic activity also is an accurate means of diagnosing feline epi; however, it is more cumbersome and has limited availability. once maldigestion has been accurately ruled out, malabsorption becomes the most likely diagnosis in diarrheic animals with weight loss. one must then decide whether to perform diagnostic therapeutic trials or diagnostic tests. a definitive diagnosis usually necessitates intestinal biopsy. patients that are critically ill (i.e., are emaciated or have serum albumin < . g/dl) usually should next undergo abdominal ultrasonography and intestinal biopsies (preferably via endoscopy). patients that are not critically ill may be managed first using therapeutic trials. therapeutic trials may be chosen more rationally with the aid of minimal laboratory data such as a cbc, biochemical profile, and fecal examinations. the two major therapeutic trials are ( ) food trials for dietary intolerance and ( ) antibiotic trials for are. are (previously called "small intestinal bacterial overgrowth") may exist by itself or it may coexist with another gi malady. are may prevent therapy aimed at the underlying problem from resolving clinical signs. no consistent cbc or serum chemistry profile changes are seen in this syndrome. fecal culture is not informative, and intestinal biopsy is seldom diagnostic. a barium contrast study may identify a segmental lesion or partial obstruction responsible for secondary are. quantitated culture of duodenal or proximal jejunal fluid for aerobes and anaerobes is difficult to interpret, because clinically normal dogs may have as many or more bacteria than clinically affected dogs. finding an increased concentration of unconjugated serum bile acids is believed to be supportive of are. bacteria can deconjugate serum bile acids in the intestinal lumen, and these bile acids can be absorbed by the jejunum. however, the test is limited in its availability. serum vitamin b and folate concentrations have been used as screening procedures for are once epi has been ruled out. some patients with are have normal serum vitamin b and folate concentrations, however. response to empiric antibiotic therapy supports the diagnosis. signs secondary to are usually respond to appropriate antibiotic therapy (e.g., tetracycline, tylosin, ampicillin, metronidazole) unless irreversible mucosal changes or a primary underlying intestinal disease are seen. dietary intolerance is relatively common, and hypoallergenic diets (e.g., fish and potato, turkey and potato, tofu and beans) are reasonable trials. at least weeks (and preferably to weeks) should be allotted for such a dietary trial, during which time absolutely nothing else should be fed (including flavored treats or medications). if dietary, antibiotic, and repeated anthelmintic and antiprotozoal therapies are ineffective, small intestinal biopsy is probably necessary. laparotomy, laparoscopy, or endoscopy may be used. in most patients, the stomach, duodenum, ileum, and colon may be endoscopically sampled. duodenal cytology is helpful in some disorders (e.g., eosinophilic enteritis, purulent enteritis, giardiasis, lymphoma). if laparotomy is performed, multiple representative full-thickness specimens (e.g., stomach, duodenum, jejunum, ileum, mesenteric lymph node) are indicated, because lesions can be sporadic. if endoscopy is performed, multiple specimens (e.g., ≥ ) from each site are obtained. it is critical that the endoscopist be accomplished and trained in obtaining high-quality tissue samples. many endoscopic biopsies obtain nondiagnostic samples because of the operator's lack of training in this area. ples are often characterized by a decrease in serum concentrations of both serum albumin and globulin, which are lost through the gi tract. ple is uncommon in cats but seen with some regularity in dogs. dogs with inflammatory diseases causing hyperglobulinemia and some breeds (e.g., basenji dogs) may have only hypoalbuminemia. this occurs because the serum globulin concentration is greatly increased, and even though much of this fraction is lost into the intestines, the amount remaining keeps concentrations in the normal range. if red blood cells (rbcs) are also being lost, iron-deficiency anemia may occur (see chapter ). ple may be the result of various gi diseases (e.g., hookworms, chronic intussusception, fungal infections, ulcers and erosions), but inflammatory bowel disease, alimentary lymphosarcoma, and lymphangiectasia are the most common causes in adult dogs. intestinal lymphangiectasia causes severe ple in dogs (it is not reported in cats) and can produce some of the lowest serum protein levels (serum albumin < . g/dl) that occur in alimentary disease. because of the loss of lymph into the intestines, peripheral lymphocyte counts may be decreased; hypocholesterolemia and steatorrhea are common. if hepatic insufficiency and loss from the kidneys and skin have been eliminated in a hypoalbuminemic patient, ple becomes the major differential diagnosis by process of elimination. if ple is suspected in a patient that has another potential explanation for its hypoalbuminemia (e.g., renal protein loss or substantive hepatic insufficiency), then detecting abnormally high concentrations of alpha- protease inhibitor in the feces supports a diagnosis of gi protein loss. the relatively stable alpha- proteinase is resistant to gi degradation and consequently can be measured in the feces. intestinal biopsy is usually the definitive test. full-thickness biopsy may risk dehiscence if the serum albumin level is less than . g/dl; however, serosal patch graft techniques decrease the risk of dehiscence. gastroduodenoscopy-ileoscopy plus biopsy is safe and often (but not invariably) diagnostic. occasionally the intestinal lesion is inaccessible via endoscopy. although not recommended, dietary trial may be used in patients with ple. an ultra-low-fat diet is reasonable if lymphangiectasia is suspected; however, therapeutic trials with steroids are potentially dangerous and are not recommended without a definitive diagnosis. mucoid feces should be approached as a large intestinal or a distal small intestinal problem. in dogs and cats that have large bowel disease but no weight loss or hypoalbuminemia, multiple fecal examinations, digital rectal examination, and therapeutic trials (i.e., dietary, antibacterial or anthelmintic [or both]) are often the best initial steps. if these are unsuccessful, then colonileoscopy plus biopsy generally becomes the most useful diagnostic tool. hematochezia should also be considered as a large bowel problem. melena signifies swallowed blood from any source, coagulopathy, or gastric and upper intestinal bleeding. therefore before performing an exploratory laparotomy, one should consider all the possible causes of oral bleeding (e.g., coughing up blood from the respiratory tract, posterior nasal bleeding). ingestion of bismuth subsalicylate (pepto-bismol) or liver can cause feces to appear melenic. diet and changes in intestinal bacterial flora influence fecal color but do not generally signify disease. interpretation • finding clostridium perfringens enterotoxin in feces plus clinical signs of clostridial diarrhea has been considered diagnostic of clostridial colitis. results from elisa methodology appear to correlate better with disease than do results from rpla methodology. however, production of enterotoxin does not appear to be a consistent event (i.e., found in every bowel movement), especially in the later course of disease. in suspected cases with a negative toxin assay, it might be useful to wait and repeat the test again at the onset of recurrence of clinical signs or perform a therapeutic trial with amoxicillin or tylosin. fecal spore counts do not correlate well with clostridium perfringens enterotoxin production or with the presence of diarrhea. examining fecal smears (see fecal microscopic cytology) to look for the presence of spores no longer seems to be an acceptable screening procedure (e.g., clinically normal dogs may have spores in their feces and be positive for enterotoxin by rpla). finding evidence of clostridium difficile toxin a in feces of diarrheic patients seems suggestive of a cause-and-effect relationship, but this is currently being investigated. occasional indications • dogs and cats with persistent diarrhea (especially large bowel) of unknown origin, suspected contagious diarrhea, or a suspected infectious cause (e.g., diarrhea with concurrent fever, leukocytosis, neutrophilic fecal cytology, bloody diarrhea). enteric pathogens include c. perfringens, salmonella spp., c. jejuni, verotoxin-positive e. coli, clostridium perfringens, clostridium difficile, and y. enterocolitica. disadvantages • must specify which pathogens to culture, must provide the laboratory with fresh feces or feces submitted in appropriate transport media, and requires a microbiology laboratory familiar with the specific enrichment and isolation techniques for each pathogen for which a culture is attempted. using culture swabs is not adequate for isolation of most enteric pathogens. finally, growing a "pathogen" does not mean that it is responsible for clinical signs. analysis • fresh feces must be promptly submitted to the laboratory, and the laboratory must know the specific pathogen(s) sought. to submit old feces or feces that have not been collected or handled properly or to request a "general culture for pathogens" is generally a waste of time and money. it requires laboratories that are properly equipped to culture for enteric pathogens. culture for c. perfringens and clostridium difficile in particular is not usually diagnostically useful. interpretation • small numbers of any of the pathogens listed earlier might be found in normal pets, although y. enterocolitica is particularly uncommon in the united states. interpretation of the fecal culture must consider the history, physical examination, laboratory data, and sometimes numbers of pathologic organisms (i.e., number of bacterial colony-forming units per gram of feces) found. with diarrhea from any cause, the gi flora may change from a predominately anaerobic to a gram-negative aerobic population. rare indications • to detect malabsorption or maldigestion in animals with diarrhea or unexplained weight loss. minimal expense, availability, and reasonable accuracy as a screening test. advantages (quantitative analysis) • very sensitive. • expense, difficulty in collecting and storing feces and in differentiating between the causes of steatorrhea. analysis • the clinician performs a semiqualitative analysis for undigested fats by mixing a drop of fresh feces with a drop of sudan iii, heating the slide to a boil, and examining the smear microscopically. the clinician performs analysis for digested fats by mixing drop of fresh feces, drop of % acetic acid, and drop of sudan iii. this is put on a microscope slide, heated to boiling, and examined while still warm. in both cases, identifying orange droplets is a positive finding. it is important that the patient has been eating a moderate-to high-fat diet. feeding low-fat diets to malabsorptive dogs may cause the test result to be negative. quantitative analysis is rarely indicated or performed. the technique is described in prior editions. normal values • semiqualitative: few or no undigested and digested fat globules per high-power field (hpf). artifacts • the semiqualitative analysis may have unexplained false-negative and false-positive reactions. administration of barium sulfate, bismuth, psyllium fiber, mineral oil, or castor oil or feeding a low-fat diet may also confuse semiqualitative analysis. • decreased excretion may be caused by medium-chain triglyceride oil supplements (titrimetric analysis). increased quantitated fat excretion may be caused by azathioprine, orally administered aminoglycosides, and cholestyramine. • finding several orange globules/hpf, if repeatable on several examinations, is principally caused by malabsorption or maldigestion. it is a reasonable screening test and helps distinguish maldigestion because of epi (positive for undigested fats) from malabsorption (positive for digested fats). despite occasional false-positive reactions, strongly positive results for undigested fecal fat in a dog with signs consistent with maldigestion are an indication for more specific tests (e.g., tli). questionable results on the semiqualitative test should always be followed by more specific tests. with sudan staining, fecal fat may not be detectable in some dogs with epi. rare indications • chronic diarrhea or weight loss. disadvantages • negative and positive results that do not correlate with malabsorption and maldigestion. this test is not recommended. analysis • fecal smears are stained with % lugol's iodine. starch granules show up as dark blue-black granules when viewed microscopically. normal values • rare ( to ) granules/hpf, although this may vary with the diet. artifacts • falsely increased: contamination of feces with food. unexplained false-positive and false-negative results may also occur. starch • some diets may have more fecal starch excretion than others. • epi is most likely, but high-starch diets or conditions causing increased intestinal transit may cause amylorrhea. amylorrhea in a patient with weight loss or diarrhea is an indication for serum tli. rare indications • chronic, small bowel diarrhea or weight loss that is difficult to diagnose. rare indications • to detect maldigestion in animals with chronic diarrhea or weight loss of unknown cause. advantages • theoretically this test may diagnose epi in rare patients that have epi secondary to obstruction of the pancreatic duct or ducts. disadvantages • the radiograph film digestion test is useless and should never be used. the most reliable procedure for measuring fecal proteolytic activity is difficult to perform and requires special handling of the feces; it is described in prior editions. analysis • described in prior editions. the ftli test has replaced measurement of fecal proteolytic activity as the diagnostic test of choice for epi in cats. infrequent indications • hypoalbuminemia of uncertain cause or suspected ple. advantages • specifically indicates the gi tract as the source of protein loss. alpha- protease inhibitor is a plasma protein, which, if leaked into the intestinal lumen, is resistant to gi degradation and hence can be measured in the feces. the amount of alpha- protease inhibitor reflects the approximate loss of plasma proteins into the gi tract. disadvantages • limited availability of the test. the magnitude of alpha- protease inhibitor in the feces is variable and may not reflect the severity of the disease. analysis • three g fecal samples from three different bowel movements are submitted in tubes provided by the laboratory. it is critically important that three samples (preferably from different days or at least different bowel movements) be submitted, that the feces be collected promptly after defecation, and that the feces not be collected by digitally removing them from the rectum. samples must be frozen while one awaits shipping and must be shipped on a cold pack. currently the only laboratory offering this test for dogs is gi laboratory, college of veterinary medicine, texas a&m university, college station, tx - . normal values • . to . µg/g feces. abnormally high values in the feces indicate loss of serum proteins into the alimentary tract and might indicate that ple is the cause of hypoalbuminemia. interpretation of the magnitude of the loss is as per the laboratory. drug therapy • administration of barium and psyllium fiber may make interpretation difficult, and antibiotics change bacterial flora composition. causes of abnormalities • fecal wbcs (specifically neutrophils) are observed with bacterial (e.g., salmonellosis, campylobacteriosis) and inflammatory mucosal disease. transmural colitides occasionally have increased fecal wbcs. fecal wbcs are an indication to culture for specific bacterial pathogens or to biopsy colonic mucosa. eosinophils may be visible with allergic or parasitic colitis. increased numbers of yeast, fungal organisms, or a uniform population of bacteria may help identify the cause of diarrhea in a patient. rare indications • to detect gi bleeding that is not apparent grossly (i.e., melena, hematochezia). analysis • fresh feces are smeared on a test pad. the patient must have been on a meatfree diet for at least days before the feces are obtained. sensitivity varies markedly between assays. artifacts • falsely decreased: sampling unmixed feces (blood may not be distributed homogeneously throughout the feces) and vitamin c supplementation. falsely increased: diets containing fresh meats (i.e., hemoglobin) or fresh uncooked vegetables (i.e., peroxidases), which cause a positive reaction. • bleeding into the gi tract at any level and as the result of any cause may result in fecal occult blood. gi blood loss of volumes of ml blood/ kg body weight will give positive results. rare indications • to detect and distinguish maldigestion from malabsorption in chronic small intestinal diarrhea or unexplained weight loss. despite low cost and availability, this test has many false-negative and falsepositive results and is not recommended. analysis • the test is described in prior editions. rare indications • because of the accuracy and the availability of the tli test, the bentiromide (bt-paba) test is rarely used and is not recommended. artifacts • theoretically, epi caused by an obstructed pancreatic duct instead of acinar cell atrophy would yield a normal or even increased serum tli value. drug therapy that may alter tli • drugs causing acute pancreatitis (see table - ) might increase serum tli. oral pancreatic enzyme supplementation does not affect serum tli concentrations. causes of decreased tli • a serum tli concentration less than . µg/l (dog) or µg/l (cat) is generally considered diagnostic for epi, and tli is considered to be the test of choice for epi (see figure - ). subclinical canine epi may be suspected by finding intermediate values (> . µg/l and < . µg/l). in such cases repeated testing should be performed. some dogs will later develop epi, whereas others will not. • values greater than µg/l in dogs and greater than µg/l in cats may occur with pancreatitis, renal failure, prerenal azotemia (may increase two times), and malnutrition. the ftli test seems to be more specific for the diagnosis of pancreatitis in cats than amylase or lipase; however, the ftli test is not clearly as sensitive or specific for feline pancreatitis as pancreatic biopsy. in dogs, tli seems to increase early in pancreatitis but then returns to reference ranges. rare indications • it is not recommended for evaluating the gi tract. see prior editions for description. rare indications • same as for oral glucose absorption test. rare indications • it is not recommended for evaluating the gi tract. it is described in prior editions. analysis • a solution of to sugars obtained from the laboratory is administered orally. six hours later a spot urine sample is obtained and placed in a container with sodium azide to prevent bacterial degradation of the sugars. the concentration of the sugars is determined with high-pressure liquid chromatography, and the ratio of specific sugars is calculated. results are interpreted as per the laboratory. occasional indications • chronic small bowel diarrhea, unexplained weight loss, or uncertain but suspected small intestinal disease. advantages • need only one serum sample. disadvantages • uncertain sensitivity and specificity for small intestinal disease, are, or epi. this test should be used as an adjunct to other tests for maldigestion and malabsorption syndromes. the test is specific for cobalamin deficiency. analysis • measured in serum by bioassay or immunoassay. "no boil" methods are unreliable in dogs. serum should be transported in a covered tube. normal values • depend on the laboratory. normal ranges vary widely between laboratories. the particular laboratory must validate the assay for dogs or cats. danger values • none. artifacts • falsely decreased vitamin b : degradation caused by exposure of serum to sunlight. concentrations • the major recognized reasons for decreased serum b concentrations in dogs and cats are ileal disease or resection (rare), epi, intestinal mucosal disease, are, and in cats, hepatic disease. the major differentiation to be made is among epi, mucosal disease, and are; therefore, decreased serum b is an indication for serum tli. not all dogs with epi, mucosal disease, or are have decreased serum vitamin b . cats with epi, severe small intestinal disease (e.g., lymphoma, inflammatory bowel disease), and some hepatic diseases (e.g., idiopathic hepatic lipidosis) can have very low b concentrations. finding a significantly decreased serum b concentration can be an indication of small intestinal disease in animals that were previously not suspected to have such disease. severe mucosal disease of the proximal small intestine decreases serum folate. not all patients with such disease have decreased folate levels. • are, epi, and dietary supplementation are probably the major causes. many patients with these diseases do not have increased folate levels. the combination of low vitamin b plus increased folate is an indication to treat for are. rare indications • chronic small bowel diarrhea or unexplained weight loss. the test detects hydrogen production as a by-product of bacterial fermentation of carbohydrates. increase in hydrogen production indicates are or carbohydrate malabsorption. disadvantages • need for special equipment, unknown sensitivity and specificity, multiple confounding factors. this is not a routine diagnostic test, because it requires special hydrogen analysis equipment. analysis • the concentration of hydrogen in expired air is measured after ingestion of food or a carbohydrate such as d-xylose or lactulose. normal values • dogs, varies with laboratory analysis and the carbohydrate administered. artifacts • abnormal intestinal motility may delay or hasten expiration of hydrogen owing to colonic bacterial fermentation of carbohydrates. breath test • antibiotics and drugs that delay intestinal transit may cause decreased or delayed expiration of hydrogen. carbohydrate malabsorption and are may increase expired hydrogen. the only source of hydrogen is bacterial fermentation of carbohydrates. the sensitivity and specificity of this test for are in dogs are unknown. common indications • a screen for parasites and parasitic ova; any patient with diarrhea, melena, hematochezia, fecal mucus, weight loss, or vomiting. advantages • availability, ease of performing the test, and low cost. disadvantages • need for fresh feces and the frequency with which parasites and their ova or cysts are not detected. analysis • a thin smear is made of very fresh (< minutes old) feces, usually mixed with a drop of saline solution and coverslipped to prevent dehydration. it should be examined immediately. if protozoa are visible and better cytologic detail is desired, a drop of lugol's iodine or dobell and o'connor's iodine may be placed at the corner of the coverslip. artifacts • cooling of the slide or dehydration inhibits the motility of some protozoa and bacteria. • orally administered compounds containing kaolin, pectin, barium sulfate, bismuth, and other intestinally active compounds (e.g., cathartics, enemas) may make it difficult to find and identify parasites, ova, and cysts. be identified • this test is most useful in identifying giardia spp., tritrichomonas spp., entamoeba histolytica, balantidium coli, strongyloides stercoralis, and aleurostrongylus abstrusus. any ova may be found, but this test may be useful for detecting spirocerca lupi and trichuris vulpis ova. with oil immersion, small motile bacterial spirochetes in conjunction with fecal wbcs suggest campylobacter spp. as a possible cause. advantages • sensitivity, availability, and low cost. analysis • feces are well mixed with either a saturated sugar solution or a zinc sulfate solution (leib, ) . (zinc sulfate solution is made by mixing g znso • h o in l water to attain a specific gravity of . to . [as determined with a hydrometer]. this is supposedly the best fecal flotation technique for giardia spp. because it does not distort the cysts.) ova and cysts are allowed to rise to the surface and are retrieved with a coverslip. samples for giardia detection should be examined within minutes to avoid distortion and lysis of cysts. centrifugation of the sample increases the sensitivity of the procedure. samples that will be sent to an outside laboratory for analysis may be refrigerated (not frozen) for to days or preserved by mixing part feces with parts sodium acetate-acetic acid-formalin. this is prepared by mixing . g sodium acetate + ml glacial acetic acid + ml % formaldehyde solution + . ml water (kirkpatrick, ) . artifacts • falsely decreased: diarrhea may decrease ova concentration within a sample. identified • ancylostoma spp., toxocara spp., toxascaris leonina, t. vulpis, s. lupi, physaloptera rara (using dichromate solution), capillaria aerophilia, capillaria plica, onciolo canis, dioctophyme renale, isospora spp., giardia spp., toxoplasma gondii, cryptosporidium spp., paragonimus kellicotti, and some tapeworms. rare indications • same as for fecal smear and flotation, especially if flukes are being considered. if feces contain excessive fat, formalin and ethyl acetate is probably better than water sedimentation. disadvantages • requires more time than direct fecal smear or fecal flotation. analysis • feces are mixed with the sedimentation solution (e.g., water), usually strained once or twice to remove large debris, and note: iodine kills protozoa, thus stopping motility. allowed to settle for minutes to hours. the sediment is then examined microscopically. when formalin and ethyl acetate is used, the strained feces are centrifuged, the pellet is resuspended in ml of % formalin solution, ml ethyl acetate is added, and the mixture is shaken vigorously. this is recentrifuged, the debris at the formalin and ethyl acetate interface is discarded, and the sediment is then examined (kirkpatrick, hepatic disease may be heralded by relatively specific signs (e.g., hepatomegaly, microhepatia, icterus, ascites, hepatic encephalopathy associated with meals) or may be associated with nonspecific signs (e.g., depression, weight loss, anorexia, vomiting). the latter are common presenting complaints of many diseases, which is why serum biochemistry profiling is indicated in patients with chronic signs or evidence of systemic disease. it is important to note that no consistent signs or laboratory abnormalities are found in all patients with hepatic disease. when screening for hepatic disease, one should request at least cbc, serum alt, sap, gamma-glutamyl transpeptidase (ggt), total bilirubin, albumin, cholesterol, bun, glucose, urinalysis, and abdominal imaging. hepatic function tests (bile acids, ammonia tolerance, clotting times, and others) ultrasonographic examination, hepatic biopsy, or contrast angiography and portography is usually necessary for definitive diagnosis. abnormalities in hepatic-specific enzymes may result from primary hepatic disease but also occur because of secondary hepatic involvement from a primary nonhepatic disease (e.g., glucocorticoid hepatopathy, inflammatory bowel disease, pancreatitis). after identifying abnormalities in alt, aspartate transferase (ast), sap, or ggt, one should investigate first for a primary nonhepatic disease, because nonhepatic disease is the most common cause of increased values. in such cases the liver usually has reactive but reversible degenerative changes. laboratory tests should be used for two main purposes: ( ) to identify the presence of hepatic disease and ( ) to determine if a biopsy or radiographic contrast procedure is indicated. a small liver suggests atrophy (due to portosystemic shunts, hepatic arteriovenous [av] fistulas), fibrosis and cirrhosis, or diffuse massive hepatic necrosis (figure - ) . radiographically, hepatic atrophy tends to be characterized by sharp borders as opposed to the rounded or blunted hepatic margins typically associated with fibrosis and cirrhosis. some patients with primary hepatic fibrosis severe enough to cause portal hypertension also have sharp hepatic margins, however. many patients with marked hepatic atrophy due to portosystemic shunting are relatively young (< to years) and have had signs of hepatic disease since (or before) weaning, whereas most patients with cirrhosis are middle-aged or older and clearly have late onset of clinical signs. hepatic av fistula is an uncommon cause of microhepatia, but it is usually diagnosed in dogs less than years of age. however, some dogs are first diagnosed as having a single congenital portosystemic shunt when they are more than years old. likewise, although acquired portosystemic shunts are classically thought of as occurring in older dogs, they can be diagnosed in dogs less than months old. hepatic atrophy causes abnormalities in hepatic function tests (e.g., bile acids, ammonia, or ammonium chloride tolerance test) but may yield normal or abnormal alt, sap, bun, and serum albumin. a single normal or abnormal hepatic function test result does not mean that other hepatic function tests will have similar results. preprandial and postprandial serum bile acid concentrations are sensitive function tests. (note: cholestatic diseases also increase bile acids; therefore, bile acids are not a "pure" test of hepatic function.) however, if hepatic disease is strongly suspected and the serum bile acid concentrations are not as high as anticipated, one should not hesitate to perform other tests to characterize the liver. if hepatic atrophy is likely, abdominal ultrasonography, contrast portography, hepatic biopsy, or a combination of these might be considered. small livers with clearly rounded or blunt hepatic margins are usually cirrhotic. significant increases in serum alt and sap are often present, but some dogs with marked hepatic cirrhosis have normal hepatic enzymes. serum albumin and bun are more variable. if cirrhosis appears likely, a biopsy is indicated. most cirrhotic livers can be identified grossly by their nodular or "cobblestone" appearance. however, significant fibrosis can be present without major gross changes, and severe nodular hyperplasia may resemble a cirrhotic liver. acquired multiple shunts visible at laparoscopy or laparotomy are usually due to cirrhosis but can be secondary to congenital hepatic av fistula, venoocclusive disease, or portal vein obstruction. if the liver is not clearly cirrhotic or fibrotic, a mesenteric venoportogram may be indicated in patients with acquired shunting. focal or asymmetric hepatic enlargement generally necessitates further laboratory investigation, imaging, and possibly biopsy. neoplasia is a prominent but not invariable cause of focal hepatomegaly. the magnitude of the enlargement is not prognostic. generalized hepatomegaly necessitates careful clinicopathologic evaluation. hepatomegaly may be the result of primary or secondary hepatic disease. diagnosis may be confirmed with a history of exposure to certain toxins (tables - and - ) or diagnosis of a systemic disease (e.g., hyperadrenocorticism) known to affect the liver. changes in alt, sap, hepatic function tests, and hepatic size, although suggestive of hepatic disease, are not diagnostic of specific entities. this is true even in breeds with specific predispositions (e.g., doberman pinschers, bedlington terriers). changes in the sap or serum alt may also be the result of primary nonhepatic disease (e.g., hyperadrenocorticism, inflammatory bowel disease, diabetes mellitus, heart failure). a definitive diagnosis usually requires hepatic biopsy. the clinician should first seek to rule out nonhepatic causes of secondary hepatic dysfunction. hepatic biopsy should be considered in patients with obviously significant hepatic disease, those that do not have hyperadrenocorticism, and those that have persistent (more than month) changes in serum alt or sap consistent with chronic or progressive hepatic disease or abnormal hepatic function tests (see figure - ). it is not always possible to make these distinctions accurately; therefore, whenever hepatic biopsy is performed via laparotomy or laparoscopy, the rest of the abdomen should be explored and other organs sampled if the clinician figure - . diagnostic approach to altered hepatic shape or size in dogs and cats. doubts their involvement. fine-needle aspirates with cytology are sometimes useful in detecting diffuse hepatic infiltrative disease and hepatic lipidosis (see color plate e); however, fine-needle aspirates (even when guided by ultrasound) often miss infiltrative processes, and a negative cytologic finding never excludes an infiltrative disease in the liver. in general, laparoscopy allows for much superior hepatic biopsies compared with ultrasound-guided biopsies. abnormal behavior, sometimes associated with eating, may be caused by hepatic encephalopathy, although hypoglycemia, primary cns disease, and epilepsy must also be considered. whenever possible, glucose should be measured on blood obtained during an episode. evaluation of hepatic function is indicated in patients with behavioral changes, transient blindness, seizures, coma, or vague cns abnormalities. congenital (e.g., portosystemic shunt) and severe acquired hepatic disease (e.g., cirrhosis) may cause encephalopathy. routine biochemical profiling may be suggestive, but hepatic function testing is mandatory, because these diseases may not significantly change serum alt, sap, albumin, bun, glucose, or bilirubin determinations. resting plasma ammonia concentrations are meaningful only if they are increased. a patient in an episode of hepatic encephalopathy may have increased or normal resting plasma ammonia concentrations. ammonia tolerance testing (att) and pre-and postprandial serum bile acid concentrations appear to be the most sensitive note: these drugs do not reliably cause hepatic disease. in a patient with an increased alt that is receiving one of these drugs, the medication probably should be stopped, if possible, and the alt rechecked to weeks later. those drugs that most reliably increase alt are marked (important). the other drugs are less consistent but may still cause severe hepatic disease. almost any drug could cause an increased alt in a particular patient. icterus is detected at physical examination or when serum or plasma is inspected at the laboratory. hyperbilirubinemia always denotes hepatobiliary or hematopoietic disease (figure - ). hepatic and hematopoietic diseases are not always associated with icterus, and disease in either system may be secondary to other disorders. the presence or absence of icterus is not diagnostic or prognostic. sepsis, pancreatitis, and inflammatory bowel disease sometimes cause secondary hepatic dysfunction that may include icterus. occasional indications • icterus (on either physical examination or inspection of nonhemolyzed serum or plasma), bilirubinuria (any amount in a cat or significant amounts in a dog), or suspected hepatic disease that is not apparent on other tests. the sclera have detectable icterus when the serum bilirubin is greater than to mg/dl, and the plasma is icteric when the serum bilirubin is greater than . to mg/dl. measurement of direct (conjugated) and indirect (unconjugated) bilirubin fractions is not useful and should not be done. hemolytic, hepatic, and biliary tract diseases have unpredictable variation in the amount of each fraction. other tests are required to identify the cause of hyperbilirubinemia. analysis • measured in serum or heparinized plasma by spectrophotometric and dry reagent methods. the latter require dilutions if the bilirubin is greater than . mg/dl. bilirubin is stable at °c for days if not exposed to bright light. measurement of urine bilirubin is discussed in chapter . normal values • dogs, less than . mg/dl; cats, less than . mg/dl. danger values • dogs, uncertain, but values greater than mg/dl cause concern (i.e., kernicterus); cats, unknown. artifacts • exposure to bright sunlight or fluorescent lighting can decrease bilirubin by % per hour. see introduction to serum chemistries. bilirubin • decreased bilirubin may be caused by drugs that cause hepatic enzyme induction (e.g., phenobarbital). increased bilirubin may be the result of drugs causing hemolytic anemia (table - ) or acute hepatic necrosis (see table - ). causes of hyperbilirubinemia • hemolytic disease and hepatobiliary disease are the two main causes (see figure - ). a cbc should be determined in every icteric patient to help rule out hemolytic disease. rbc numbers must decrease rapidly and significantly to cause clinical icterus. very regenerative anemias may suggest that icterus is due to immune-mediated hemolytic anemia (imha). reticulocytosis, hemoglobinemia, hemoglobinuria, erythrocytic autoagglutination, spherocytosis, positive coombs' test results, splenomegaly, or hepatomegaly are often present. see chapter for further discussion of iha and other regenerative anemias (e.g., heinz body, zinc intoxication, haemobartonella). bilirubinuria theoretically should be absent in hemolytic disease but is often present in iha because canine kidneys conjugate bilirubin. the clinician must not be misled by increases in alt because severe, acute hemolytic anemia may cause increased alt (ostensibly caused by acute hepatic hypoxia). severe hepatic disease (especially acute necrosis) is sometimes accompanied by dic and subsequent hemolytic anemia. these cases may be difficult to distinguish from iha. however, anemia caused by dic is usually not as regenerative as in iha; in addition, the presence of rbc fragments, thrombocytopenia, increased fibrin degradation products (fdp), decreased antithrombin iii, prolonged clotting time, and abnormal hepatic function note: icterus is absent in many animals (especially dogs) with hepatic disease. serum bilirubin is not a sensitive test for hepatic disease. icterus due to hemolytic anemia autoimmune hemolytic anemia zinc toxicity blood parasites (see table - ) tests usually allows differentiation, as do vomiting, abdominal pain, and encephalopathy when present. dogs and cats often have relatively severe hepatic disease before icterus is observed; however, the magnitude of the total serum bilirubin is not prognostic or diagnostic. secondary hepatic disease (reactive disease or so-called bystander phenomenon as the result of septicemia, toxemia, or inflammation) may have icterus similar to that occurring in primary hepatic disease. certain bacterial endotoxins and acute-phase inflammatory mediators are thought to alter normal bilirubin metabolism and cause increases in total bilirubin concentrations. most feline hepatic diseases cause icterus; the most common conditions include hepatic lipidosis, cholangitis and cholangiohepatitis, hepatic lymphoma, and fip. icterus in cats is an indication for cbc and a serum biochemistry panel. icterus in cats that is not caused by hemolysis usually indicates a hepatic biopsy, because most of these cats have primary hepatic disease. biopsy is necessary to differentiate causes and institute specific treatment. common causes of nonhemolytic icterus in dogs include pancreatitis obstructing the bile duct, cholecystitis, chronic hepatitis, hepatic lymphoma, acute hepatic necrosis, hepatic cirrhosis, and intrahepatic cholestasis. icterus in dogs is an indication for cbc and a serum biochemistry panel (to include at least alt, sap, bun, cholesterol, and albumin). imaging (radiographs, ultrasonography, or both) is indicated to help determine if primary hepatic disease or biliary tract obstruction exists. if primary hepatic disease is diagnosed, hepatic biopsy is usually indicated. if pancreatitis is present, surgery is not indicated unless a chronic bile duct obstruction necessitates bypassing the common bile duct with a cholecystoduodenostomy (rarely needed) or a pancreatic abscess is present. if coexisting extrahepatic disease is found, it should be investigated. alt was formerly known as serum glutamicpyruvic transaminase (sgpt). common indications • systemic disease including weight loss, hepatomegaly, vomiting, diarrhea, icterus, ascites, depression, and anorexia; also, as a screening procedure for hepatic disease in any patient with undiagnosed illness. most patients with known chronic hepatitis should undergo periodic alt determinations to monitor the problem. disadvantages • lack of sensitivity (i.e., patients with significant hepatic disease such as cirrhosis or hepatic neoplasia may have normal alt) and inability to distinguish among different hepatic diseases or when there is secondary nonhepatic disease involvement. analysis • measured in serum (heparinized plasma in selected assays) by spectrophotometric and dry reagent methods. alt is stable in separated serum for approximately (at °c) to (at °c) days. normal values • serum enzyme activity may vary markedly among laboratories, depending on the technique and the units used. danger values • despite correlation between alt and active hepatic damage, no correlation exists between alt and hepatic function; hence, no danger values exist. drug therapy that may alter serum alt • any drug causing hepatocellular damage (i.e., drug-induced hepatopathy) may cause increased alt. the list of all drugs suspected to cause increased alt is extensive and includes many that are safe in the majority of patients. a list of selected drugs documented to cause increased alt in human beings, dogs, and cats is given in table - . administration of one of these drugs does not automatically explain an increased alt, however. causes of increased alt • increase in alt is principally caused by hepatocellular damage from any cause (table - ). rbcs and striated muscle cells contain small amounts of alt, and damage to these may cause relatively minor increases (i.e., less than two to three times normal) in serum alt, as may exercise. dogs with muscular dystrophy may have major increases in alt, but should also have increases in ast and creatine kinase (ck) values. hepatocytes contain substantial amounts of alt in the cytosol, and major increases in serum alt (i.e., three or more times normal) indicate hepatocellular leakage of the enzyme but do not always signify primary or irreversible hepatic disease. hepatic disease may have normal to significantly increased serum alt activity. the magnitude of the increase in alt does not correlate with the seriousness of the hepatic disease and is not a prognostic indicator unless a specific disease is being considered. the serum alt half-life is approximately to days or less, and serum alt is expected to decrease over to weeks once active hepatic damage ceases. it is thought that alt remains elevated during hepatic regeneration. after increased serum alt is identified, many factors must be considered (figure - ) . if no other evidence of disease is found, the increased alt indicates the need for periodic monitoring because it may be the first detectable sign of significant hepatic disease. if other abnormalities consistent with hepatic disease are found, the approach is like that in any other patient with hepatic disease. common causes of serum alt more than three times normal include hepatic anoxia, poor hepatic perfusion, spontaneous and surgical trauma (e.g., hit by a car, surgery), chronic hepatitis, cirrhosis, cholangitis and cholangiohepatitis, acute biliary obstruction, hepatic necrosis as the result of any cause, acute pancreatitis, hepatic neoplasia, sepsis, and certain drugs. sepsis, especially septicemia and toxemia, may secondarily damage hepatocytes. abdominal inflammation may do the same. the pancreas is close to the liver, and inflammation in the pancreas may cause mechanical damage to the liver. in doberman pinschers, bedlington terriers, dalmatians, and west highland white terriers, a persistently increased serum alt suggests chronic hepatitis that may or may not be associated with increased hepatic copper concentrations. ast was formerly known as serum glutamicoxaloacetic transaminase (sgot). other disorders anoxia because of anemia/shock anoxia because of anemia/shock iatrogenic (see table - ) iatrogenic (see table - ) note: almost any disease affecting the liver can cause increased alt levels. the disorders listed are those that may be more likely to cause a significant increase. however, any of these diseases can exist with minor or no increase in alt values. a patient can have an idiosyncratic reaction to almost any drug, causing an increased alt. occasional indications • same as for alt. disadvantages • not as specific for the liver as alt. analysis • same as for alt. decreased ast may be caused by metronidazole therapy. hepatotoxic drugs may cause increased ast (see table - ). causes of increased ast • like alt, ast is present in significant quantities in hepatocytes. although alt is present in the cytosol, ast is present in the mitochondria. increased serum alt reflects cell membrane damage and leakage; significant ast increases tend to reflect more serious hepatic damage because the mitochondria are not damaged as readily as is the cell membrane. ast is, however, present in significant quantities in many other tissues, including muscle and rbcs; therefore, increased ast is not as specific for hepatic injury as is increased alt. exercise and intramuscular (im) injections may increase serum ast. the most common causes of increased ast include hepatic disease, muscle disease (inflammation or necrosis), or hemolysis (spontaneous or artifactual). increased ast is an indication to check for ongoing hemolysis by measuring the hematocrit and observing the color of the plasma and serum on a centrifuged blood sample. if no hemolysis is found, the next step is to measure serum alt to determine whether the increased ast is from the liver (significant increases in both alt and ast suggest that ast increases are of hepatic origin). anorexia; also as a screen for hepatic disease and hyperadrenocorticism. advantages • useful in evaluating the liver for subtle cholestatic disease. disadvantages • affected by corticosteroids, bone lesions, and osteoblastic activity in young growing dogs. analysis • measured in serum or heparinized plasma by spectrophotometric methods. stability in heat ( °c) has been used to attempt to differentiate sap of bone origin (i.e., heat sensitive) from sap of hepatic origin (i.e., heat stable). it is difficult to obtain reproducible results with the heat-inactivation test; however, l-phenylalanine inhibits steroidinduced sap and can be used to help determine if increased sap is due to corticosteroids. alternatively, cellulose acetate electrophoresis can separate the isoenzymes more definitively. the diagnostic usefulness of determining the percentage of steroid fraction is questionable, because dogs with various types of hepatic disease often have considerable steroid involvement. normal values • may vary markedly from laboratory to laboratory. immature dogs characteristically have sap (bone origin) activities up to twice that of sexually mature dogs. danger values • because of the lack of correlation with hepatic function, no danger values exist for sap. drug therapy that may increase sap • any drug that causes hepatic enzyme induction or cholestasis (see table - ) may increase sap. glucocorticoids, primidone, and barbiturates typically increase sap in dogs, but other drugs are less consistent. although glucocorticoids can cause marked sap increases in dogs, cats are almost never affected. causes of increased sap • sap of bone origin is commonly increased (sap less than three times normal) in dogs less than to months old. bone disease (e.g., osteosarcoma, osteomyelitis) may increase sap (usually a minor increase) and generally denotes a guarded prognosis from presumed metastatic disease in the bones. increased sap is interpreted differently in dogs and cats (table - ). cats have less hepatocellular sap, which is readily excreted by their kidneys. therefore, any increase in feline sap is considered significant, indicating further tests. not all cats with hepatic disease have increased sap. the major causes of increased sap in cats are hepatic lipidosis, cholangitis and cholangiohepatitis, hyperthyroidism, and diabetes mellitus. sap increases are generally more specific than ggt in cats with hepatic lipidosis (cats with lipidosis classically have very high sap with little to no increase in ggt; however, this finding is not consistent enough to make a diagnosis). hyperadrenocorticism (spontaneous and iatrogenic) very, very rarely increases sap in cats. increased sap in a cat is an indication for serum thyroid hormone determination, urinalysis, blood glucose and serum alt measurement, and perhaps a hepatic function test (e.g., bile acid). if hepatic disease is the apparent cause of the increased sap, one must determine if hepatic biopsy is indicated (see the later discussion under bile acids). the major causes of sap values more than three times normal in dogs are hepatobiliary disease, hyperadrenocorticism, and therapy with glucocorticoids or anticonvulsants. hepatic disease with increased sap usually has a cholestatic component; however, this does not imply icterus or gross obstruction of the biliary tract. intrahepatic cholestasis caused by diffuse or focal compression of bile canaliculi may occur in various hepatopathies, even those secondary to septicemia, toxemia, and chronic stress-induced vacuolar (i.e., hydropic change) hepatopathy. acute hepatocellular necrosis can transiently increase sap (usually less than five times normal). extrahepatic biliary tract obstruction and enzyme induction caused by endogenous or exogenous glucocorticoids or drug administration may increase sap more than times normal. as with alt, the magnitude of the increase in sap does not correlate with the seriousness or prognosis of the disease. in dogs it is important first to rule out young age, drug therapy, and hyperadrenocorticism to avoid performing an unnecessary hepatic biopsy (figure - ) . hyperadrenocorticism can easily be confused with primary hepatic disease because it typically causes hepatomegaly, pu-pd, increased alt, and sometimes increased serum bile acids. unless a patient has signs of hepatic failure (i.e., icterus, hepatic encephalopathy, hypoglycemia, weight loss, vomiting, hypoalbuminemia, ascites, microhepatia), hyperadrenocorticism must be precluded by adrenal gland function testing. if a hepatic biopsy specimen is obtained from a patient with hyperadrenocorticism, vacuolar hepatopathy is documented. occasional indications • same as for sap. sap appears to be more sensitive for hepatobiliary disease in dogs; however, in cats, ggt has slightly greater sensitivity and perhaps greater specificity for hepatic disease except hepatic lipidosis. therefore, it is more frequently indicated in cats than in dogs. ggt is less influenced than sap by secondary hepatic disease conditions or enzyme-inducing drugs. the use of sap and ggt together has a higher predictive value of hepatic disease. analysis • measured in serum, urine, and body fluids by spectrophotometric methods. ggt is stable in serum at °c for at least days and at °c for up to year. lipidosis (where classically the sap is usually quite high, but ggt values show only a mild [or no] increase). ggt does not tend to increase after acute hepatic necrosis, as does sap. increased ggt should be pursued as for increased sap (see figure - ). increased ggt may suggest pancreatitis obstructing the bile duct, as for sap. increased -hour urinary excretion of ggt can be caused by various nephrotoxins (e.g., gentamicin). disadvantages • lack of specificity. the lactic dehydrogenase (ldh) test is not recommended. analysis • measured in serum, heparinized plasma, or cerebrospinal fluid (csf) by spectrophotometric methods. causes of increased ldh • ldh is found in so many body tissues that serum ldh is of questionable diagnostic value. inexplicable increases of small to great magnitude are not uncommon. rare indications • in general, sulfobromophthalein (bsp) dye retention is rarely used artifacts • very increased serum dehydrogenase activities may require modification of the spectrophotometric technique. severe lipemia (i.e., chylomicronemia) and hemolysis may falsely decrease bile acid measurements, and hypertriglyceridemia may falsely increase concentrations when spectrophotometric techniques are used, but they do not affect ria. this test is not indicated in icteric patients. that may alter serum bile acid concentration • cholestyramine lowers serum concentrations by binding to bile acids in the intestinal lumen, preventing their reabsorption. ursodeoxycholic acid (a synthetic bile acid) therapy may increase total serum bile acid concentrations. resection of the ileum (the principal site of bile acid reabsorption), severe ileal disease, or cholecystectomy may also cause serum bile acids to inaccurately reflect hepatic function. prolonged anorexia (> to days) may cause fasting serum bile acid concentrations to be less than would be found if the patient were eating normally. intestinal hypomotility may cause the -hour postprandial sample to be a less sensitive indicator of hepatic disease because of failure to deliver the bile acids to the ileum in a timely fashion. are may increase total serum bile acid concentrations because of bacterial deconjugation of bile acids with subsequent increased small intestinal uptake. hepatic insufficiency does not decrease serum bile acid concentrations. concentration • delayed gastric emptying, rapid intestinal transit, malabsorption disorders, and ileal resection may cause subnormal values. with are, total measurable moieties may or may not decrease, but it is expected that unconjugated serum bile acid concentrations may increase. concentration • serum bile acid concentrations are increased because of hepatocellular disease, cholestatic disease, or portosystemic shunting. when both fasting and -hour postprandial serum bile acid levels are determined, the sensitivity of these tests becomes greater than with other hepatic function tests. because of the ease of performing and wide availability of the test, it has replaced other clinical hepatic function tests. serum bile acids offer no additional information in icteric patients with hepatic or extrahepatic biliary tract disease. in nonicteric patients suspected of having hepatic disease, serum bile acids are a good screening test to support further diagnostic evaluations. not all patients with hepatic disease have increased serum bile acid concentrations, and the relative increase in bile acids is not diagnostic for the type of disease or the prognosis. reported fasting serum bile acids that are increased greater than µmol/l or postprandial values of greater than µmol/l suggest significant hepatic disease or portosystemic shunting and dictate further hepatic evaluation and possibly hepatic biopsy. generally, preprandial and postprandial bile acids are determined simultaneously; however, if only fasted values are determined and found to be normal, postprandial measurements are required. the magnitude of the rise or the percent increase from preprandial to postprandial values does not imply a specific diagnosis or prognosis. most animals with chronic hepatitis, marked hepatic necrosis, cholestasis, and hepatic neoplasia have abnormal values. bile acids are usually not markedly altered by secondary hepatic disease from nonhepatic disorders or with glucocorticoid or anticonvulsant therapy; however, in rare cases they may be. increased serum bile acids are possibly the most sensitive biochemical indicator of congenital portosystemic shunts. almost all animals with congenital portal vascular anomalies have increased postprandial bile acids; some of the highest concentrations occur in these cases. diuretics causing hypokalemia or alkalosis, hyperalimentation, ammonium salts, and high-protein meals (including blood from spontaneous gi bleeding). causes of hyperammonemia • urea cycle disorders (extremely rare) and hepatic insufficiency (especially congenital or acquired portosystemic shunting). resting blood ammonia concentrations are probably less sensitive than fasting serum bile acids in detecting hepatic dysfunction, whereas the att is possibly as sensitive as preprandial and postprandial bile acids in detecting portosystemic shunting. a significantly increased fasting blood ammonia concentration renders an att unnecessary. clinical signs are not well correlated with blood ammonia concentrations. an abnormal att result or resting ammonia value in a patient with hepatic disease is generally an indication for hepatic biopsy or a portogram. rarely, plasma ammonia is increased because of urinary tract obstruction, especially if complicated by infection with urease-producing bacteria. some young dogs (notably scottish deerhounds in great britain) have been found to have elevated resting blood ammonia values that spontaneously return to normal as the dog ages. therefore, caution must be used when diagnosing congenital portosystemic shunting in at least some breeds solely by evaluating the resting serum ammonia concentration. see chapter . weight loss has many causes (table - ). concurrent problems with fewer potential causes (e.g., regurgitation, vomiting, diarrhea, icterus) should be considered first. if a patient had a reasonable appetite when weight loss began, major differential diagnoses are intestinal disease, maldigestion, increased use of calories (e.g., hyperthyroidism, lactation), or increased loss of calories (e.g., diabetes mellitus). if no other identifiable problems (other than weight loss or anorexia) can be pursued, a systematic search is indicated (figure - ). one should first preclude as many causes as possible with the history and physical examination (i.e., lack of food, calorie-deficient food, inability to eat, regurgitation, vomiting and diarrhea). next, extensive clinicopathologic screening is indicated. imaging is considered an extension of the physical examination, and abdominal and thoracic radiographs are appropriate. thoracic radiographs may be very revealing, even if a patient does not have coughing or abnormal lung sounds. abdominal ultrasonography is particularly desirable and often more useful than radiographs if the operator is accomplished. if laboratory or radiographic abnormalities are not present or are unconvincing, one may repeat the tests at to week intervals, depending on the clinical condition of the patient, or immediately proceed to function tests, biopsies, or both. certain hepatic and adrenal gland diseases may require such function tests. it is noteworthy that severe gastric or intestinal disease may cause anorexia or severe weight loss without vomiting or diarrhea. gastroduodenoscopy and ileoscopy plus biopsy are reasonable in patients with severe weight loss of unknown cause. some cases with gastric neoplasia may present only for anorexia and weight loss. clinicians without access to endoscopic equipment may consider exploratory laparotomy. if surgery is performed, gastric, duodenal, jejunal, ileal, mesenteric lymph node, and hepatic biopsies are usually appropriate, regardless of a normal gross appearance of the organs. in cats, the pancreas should also be biopsied. cancer cachexia can be particularly difficult to diagnose. it is a poorly defined, multifaceted syndrome that may involve loss of taste, malabsorption, increased metabolism with energy wasting, and other mechanisms. almost any tumor can cause cancer cachexia, and no consistent laboratory findings exist. the causative cancer may be large or small, focal or diffuse; lymphomas and carcinomas are probably the most common causes. anorexia of unknown cause is similar to weight loss in being difficult to evaluate if no other identifiable abnormalities are seen. the diagnostic approach is similar to that for chronic weight loss (see figure - ; table - ). figure - . diagnostic approach to chronic weight loss in dogs and cats when no other abnormalities are found on history or physical examination and the animal is not ingesting adequate calories (see table - ). acth, adrenocorticotropic hormone; ana, antinuclear antibodies; cbc, complete blood count; csf, cerebrospinal fluid; emg, electromyogram; felv, feline leukemia virus; fiv, feline immunodeficiency virus; tli, trypsin-like immunore-activity; wbc, white blood cell. anorexia can be divided into three categories: ( ) pseudoanorexia associated with inability to eat (oral, pharyngeal, or esophageal disease), ( ) primary anorexia (rare) associated with a primary cns disorder, and ( ) secondary anorexia (the most common), which is the result of systemic or metabolic disease. if necessary, one may elect a therapeutic trial to treat for a suspected problem in a patient in whom a diagnosis cannot be made. it is vital that one design such therapy so that it is safe and extremely likely to succeed if the presumptive disease is present. then, if the trial fails, one may rule out that disease and go on to treat for something else. to do this, the clinician must be sure that the dose and duration of the treatment is sufficient. history, physical examination, radiographs, and ultrasonography are the initial tools in diagnosing the cause of abdominal pain (figure - ). extra-abdominal diseases such as spinal problems and patients predisposed to nonsurgical diseases (e.g., pancreatitis) must be identified early. in patients with severe, progressive, acute abdomen (severe unrelenting pain or shock or stupor in a deteriorating patient), surgery is often indicated as soon as fluid, electrolyte, and acid-base status are acceptable for anesthesia. imaging is desirable, but extensive laboratory testing is unlikely to identify the more common causes of acute abdomen (e.g., intestinal obstruction, gastric dilation and volvulus, peritonitis, organ ischemia, tumor, sepsis, or bleeding) and usually only delays surgical resolution of disease. abdominal exploration offers a good chance for definitive diagnosis plus resolution of the disease process. if a patient is not in severe pain and the disease is not progressing rapidly, one must differentiate between problems that ultimately necessitate surgery and those that usually do not (e.g., pancreatitis, hepatitis, cholecystitis, upper urinary tract infection, prostatitis, pansteatitis, heavy metal intoxication, rocky mountain spotted fever [rmsf] ). abdominal ultrasonography is useful to examine the liver, spleen, pancreas, kidneys, and prostate, as well as to detect peritoneal fluid. if abdominal fluid is present, abdominocentesis or abdominal lavage with cytologic analysis is indicated. if these procedures are not revealing and the problem continues, exploratory surgery may be necessary. contrast radiographs are rarely useful because thorough abdominal exploration should diagnose almost anything they reveal; finding an abnormality on radiographs simply is an indication for surgery. in rare situations the exhibited abdominal pain may be referred from other causes such as pulmonary disease or disk disease. clinicopathologic features of dogs with hepatic microvascular dysplasia with and without portosystemic shunts evaluation of a routine diagnostic fecal panel for dogs with diarrhea diagnostic procedures for evaluation of hepatic disease diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats comparison of the sensitivity of different diagnostic tests for pancreatitis in cats interferographs: user's guide to interferences in clinical chemistry instruments procedures for the evaluation of pancreatic and gastrointestinal diseases diseases of the small intestine diagnostic approach to hepatobiliary disease incidence and prognostic value of low plasma ionized calcium concentration in cats with acute pancreatitis: cases endoscopic aspiration of intestinal contents in dogs and cats: cases small intestinal bacterial overgrowth genotypic and phenotypic characterization of clostridium perfringens and clostridium difficile in diarrheic and healthy dogs subclinical exocrine pancreatic insufficiency in dogs artifacts • see introduction to serum chemistries.drug therapy that may affect ggt • same as for sap. causes of increased ggt • causes are similar to increased sap and tend to parallel the magnitude of the rise in sap, but bone lesions are not recognized to increase ggt. it is induced by glucocorticoid therapy and certain drugs, as is sap. in cats, ggt may increase more than sap, except in hepatic table - )iatrogenic (see table - ) * note: almost any disease affecting the liver can cause increased sap levels. the disorders listed are those that may be more likely to cause a significant increase. however, any of these can exist with minor or no increase in sap values. * rarely of importance.rare indications • same as for bile acids (i.e., when a sensitive function test is needed to prove hepatic disease in an animal in which easier tests do not allow diagnosis).advantages • good sensitivity and specificity.disadvantages • procedural requirements for submitting the samples, and the likelihood of vomiting or cns signs with att.analysis • measured in blood, serum, plasma (heparinized is recommended), csf, or urine by enzymatic, selective electrode, dry reagent, and resin absorption methods. there does not appear to be any advantage of arterial over venous blood. blood must be drawn into an ice-chilled tube, which is stoppered tightly after filling, immediately put back on ice, and promptly taken to the laboratory. a control sample should be taken at the same time using the same technique. the test must be performed within minutes, or the plasma must be frozen at − º c, which stabilizes the ammonia concentration for at least days. if an att is to be performed, samples for ammonia determination should be taken before and or minutes after administration of nh cl, mg/kg of body weight. the nh cl may be administered orally as a solution in to ml of water, as a % solution, as a dry powder in gelatin capsules, or rectally as a % solution. key: cord- -ralxw ad authors: oishi, peter; fineman, jeffrey r. title: diseases of the pulmonary vascular system date: - - journal: the respiratory tract in pediatric critical illness and injury doi: . / - - - - _ sha: doc_id: cord_uid: ralxw ad nan although historically considered the lesser circulation, pathology of the pulmonary circulation is a great source of pediatric morbidity and mortality. this is most commonly displayed in neonates with persistent pulmonary hypertension; neonates, infants, and children with congenital heart disease; and adolescents and young adults with primary pulmonary hypertension. recent evidence indicates that normal pulmonary vascular tone is regulated by a complex interaction of vasoactive substances that are locally produced by the vascular endothelium [ ] [ ] [ ] [ ] [ ] [ ] . these substances, such as nitric oxide (no) and endothelin- (et- ), are capable of producing vascular relaxation and/or constriction, modulating the propensity of blood to clot, and inducing and/or inhibiting smooth muscle cell migration and replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in fact, mounting data implicate endothelial injury and the subsequent aberration in the endogenous production of these substances in the pathophysiology of pulmonary hypertensive disorders [ ] [ ] [ ] [ ] [ ] . this chapter discusses the normal regulation of the fetal, transitional, and postnatal pulmonary circulations, the pathophysiology of pediatric pulmonary hypertensive disorders, and new therapeutic and preventative strategies for pulmonary hypertension. particular emphasis is placed on the role of the pulmonary vascular endothelium in these processes and treatment modalities. fl ow and decreases pulmonary vascular resistance but not to newborn values [ ] . a proportion of this decrease relates to alterations in the physical architecture of the alveoli and small pulmonary vessels that occur with mechanical distention [ ] . in addition, physical expansion of the lung results in the release of vasoactive substances, such as pgi , which increases pulmonary blood fl ow and decreases pulmonary vascular resistance in the fetal goat and lamb independent of the changes in oxygen tension [ ] [ ] [ ] [ ] [ ] [ ] . when ventilation is accompanied by changes in oxygen tension (i.e., ventilation with ambient air or supplemental oxygen), fetal pulmonary blood fl ow increases and pulmonary vascular resistance falls to newborn values. the exact mechanisms of this oxygen-induced pulmonary vasodilation remain unclear. alveolar and/or arterial oxygen may directly dilate pulmonary resistance vessels or may trigger the release of vasoactive substances, such as pgi or no. in fact, data indicate that no, in particular, participates in the decrease in pulmonary vascular resistance that accompanies increases in alveolar and arterial oxygen tension [ , ] however, despite its important role, inhibition of no does not impair the immediate fall in pulmonary vascular resistance seen after birth, further suggesting that multiple mechanisms are involved in this transitional physiology. in fact, recent data implicate fl uid shear forces across endothelial cells, which result in the production of both no and pgi , as an additional mechanism by which vasodilation occurs after birth [ ] . it is possible that this particular mechanism acts to maintain pulmonary vasodilation once it has been established by the mechanisms described earlier. in general, the dramatic increase in pulmonary blood fl ow with the initiation of ventilation and oxygenation at birth refl ects a shift from active pulmonary vasoconstriction in the fetus to active pulmonary vasodilatation in the newborn. failure to undergo this normal transition contributes substantially to the pathophysiology of many neonatal pulmonary hypertensive disorders, including bronchopulmonary dysplasia, persistent pulmonary hypertension of the newborn, chronic lung disease, and congenital heart disease [ , . the successful transition from the fetal to the postnatal pulmonary circulation is marked by the maintenance of the pulmonary vasculature in a dilated, low-resistance state [ ] . recent evidence suggests that basal no release, and the subsequent increase in smooth muscle cell cyclic guanosine monophosphate (cgmp) concentrations, in part mediate the low resting pulmonary vascular resistance of the newborn [ ] . other vasoactive substances, including histamine, -hydroxytryptamine, bradykinin, and metabolites of arachidonic acid by the cyclooxygenase and lipoxygenase pathways, have also been implicated in mediating postnatal pulmonary vascular tone; however, their roles are not well elucidated. two of the most important factors affecting pulmonary vascular resistance in the postnatal period are oxygen concentration and ph. decreasing oxygen tension and decreases in ph elicit pulmonary vasoconstriction [ ] . alveolar hypoxia constricts pulmonary arterioles, diverting blood fl ow away from hypoxic lung segments, toward well-oxygenated segments, thus enhancing ventilationperfusion matching [ ] . this response to hypoxia, unique to the pulmonary vasculature, is greater in the younger animal than in the adult [ ] . indeed, in most vascular beds (e.g., cerebral vasculature), hypoxia is a potent vasodilator. the exact mechanism of hypoxic pulmonary vasoconstriction remains incompletely under-stood but likely involves changes in the local concentration of reactive oxygen species that in turn regulate voltage-gated potassium channels and calcium channels [ , ] . acidosis potentiates hypoxic pulmonary vasoconstriction, whereas alkalosis reduces it [ ] . the exact mechanism of ph-mediated pulmonary vascular reactivity also remains incompletely understood but appears to be independent of paco [ ] . recent data suggest that potassium channels play an important role in mediating these responses as well [ ] . manipulating alveolar oxygen tension and systemic arterial ph are fundamental approaches to changing pulmonary vascular tone in the critical care setting. alveolar hyperoxia and alkalosis are often used to decrease pulmonary vascular tone because they generally relieve pulmonary vasoconstriction with little effect on the systemic circulation as a whole. however, severe alkalosis is generally avoided because of the detrimental effects of severe hypocarbia or alkalosis on cerebral and myocardial blood fl ow (see general treatment approach, later) [ , ] . despite extensive innervation of the lung, neural input is not a major determinant of basal pulmonary vascular tone. however, pulmonary neurohumoral receptors are sensitive to α-adrenergic, β-adrenergic, and dopaminergic agonists [ , ] . therefore, vasoactive agents that stimulate these receptors will affect the vascular tone of both the pulmonary and systemic circulations. alterations in vascular tone, in response to a given agent, are dependent on the relative tone of the vascular bed at a given time. therefore, the response of these agents is diffi cult to predict in an individual critically ill patient. pulmonary vascular resistance changes throughout gestation and after birth. the resistance of the pulmonary circulation at any one time is related to several factors and can be estimated by applying the resistance equation and the poiseuille-hagen relationship [ ] . the resistance equation (the hydraulic equivalent of ohm's law) states that the resistance to fl ow between two points along a tube equals the decrease in pressure between the two points divided by the fl ow [ , ] . for the pulmonary vascular bed, where rp is pulmonary vascular resistance and qp is pulmonary blood fl ow, the decrease in mean pressure is from the pulmonary artery (ppa) to the pulmonary vein (ppv) or left atrium, where la is mean left atrial pressure: rp = [ppa − ppv or la (mean)]/qp therefore, the calculated pulmonary vascular resistance increases when pulmonary arterial pressure increases or when pulmonary blood fl ow decreases. changes in pulmonary venous pressure or mean left atrial pressure are somewhat more complicated. in isolation, increases in pulmonary venous pressure and left atrial pressure would decrease the calculated pulmonary vascular resistance. however, increases in pulmonary venous pressure are generally accompanied by a greater increase in pulmonary arterial pressure (which maintains driving pressure), resulting in an increase in the calculated resistance across the pulmonary vascular bed. furthermore, changes in left atrial pressure, which occur independent of alterations in pulmonary vascular resistance, must be considered. for example, large intracardiac shunts (e.g., ventricular septal defect) may result in congestive heart failure with an elevation in left atrial pressure. closure of the ventricular septal defect may acutely decrease left atrial pressure, resulting in an elevation in the calculated pulmonary vascular resistance (provided that pulmonary arterial pressure does not decrease to the same extent), when in fact no change in pulmonary vascular tone has occurred [ ] . other factors that affect pulmonary vascular resistance can be defi ned by applying a modifi cation of the poiseuille-hagen relationship, which describes the resistance (r) to fl ow of a newtonian fl uid through a system of round, straight glass tubes of constant cross sectional area: where l is length of the system of vessels, n is vessel number, r is the internal radius of the system of vessels, and η is the viscosity of the fl uid. according to this relationship, increasing the viscosity of blood perfusing the lungs or decreasing the radius or crosssectional area (πr ) of the pulmonary vascular bed increases pulmonary vascular resistance. because the above equations describe steady, laminar fl ow of a newtonian fl uid in rigid, glass tubes, differences between physical and biologic systems should be considered. first, blood is not a newtonian fl uid. however, this is probably of little importance at normal hematocrit levels [ ] . the viscosity of blood is related to red cell number, fi brinogen concentration, and red cell deformability. an increased hematocrit (secondary to fetal hypoxemia, twin-to-twin transfusion, maternal-to-fetal transfusion, or delayed clamping of the umbilical cord) will increase viscosity [ , ] as pulmonary vascular resistance increases logarithmically when the hematocrit increases. second, pulmonary vessels are not rigid tubes. their walls are deformable, and their size and shape are infl uenced by transmural pressure. for example, as pulmonary blood fl ow or left atrial pressure increases, vessel diameter may change, and/or the recruitment of additional pulmonary vessels may occur. therefore, the fall in calculated pulmonary vascular resistance with increases in pulmonary blood fl ow is nonlinear [ , , ] . third, blood fl ow through the pulmonary circulation is pulsatile, not laminar, and the small pulmonary arteries are branched, curved, and tapered, not smooth [ ] . in addition, the small pulmonary arteries are in parallel, and the radii of these arteries may differ in different lung zones. despite these differences from physical models, the general effects of changes in physical factors, such as viscosity and radius, do apply [ ] [ ] [ ] . in fact, a change in luminal radius is the major factor responsible for maintaining a high pulmonary vascular resistance in the fetus. consideration of these factors, particularly viscosity and cross-sectional area of the vascular bed, is important in evaluating the pathophysiology of pulmonary hypertensive disorders. finally, it is important to note the overall relationship between lung volume and pulmonary vascular resistance, which has been described by several investigators [ , ] . these studies have shown that this relationship to be u-shaped (figure . ) with minimal pulmonary vascular resistance noted at functional residual capacity. using an open-chest model, pulmonary vascular resistance decreased as lungs were infl ated from a collapsed state and then progressively increased at higher lung volumes, which was thought to be related to infl ation pressure on the alveolar vessels. these observations support the concept that lung infl ation may have a variable effect on the distribution of pulmonary blood fl ow. when pressure is expressed in mm of hg and fl ow in l/min, units of resistance are derived as mm of hg/l/min (wood unit, u). however, comparisons among patients of differing weight and age are problematic. therefore, resistance is more commonly expressed in relation to body surface area, as u·m . multiplying u by converts to dynes/sec/cm − , a common form utilized to express resistance in other settings. pulmonary vascular resistance may be as high as - u·m immediately after birth and then, under normal conditions, falls as previously described to adult levels of - u·m by to weeks of life [ , ] . pulmonary hypertensive disorders are a signifi cant source of morbidity and mortality in the pediatric population. pulmonary hypertension is defi ned as a mean pulmonary artery pressure of greater than mm of hg at rest or greater than mm of hg during exercise. in addition, a calculated pulmonary vascular resistance of greater than u is generally considered abnormal. in neonates, the most common etiology results from a failure to undergo the normal fall in pulmonary vascular resistance at birth termed persistent pulmonary hypertension of the newborn (pphn) that has an incidence of ∼ per , live births. however, other pulmonary abnormalities, such as congenital diaphragmatic hernia, respiratory distress syndrome, and bronchopulmonary dysplasia, may also result in neonatal pulmonary hypertension. beyond the neonatal period, the majority of pediatric pulmonary hypertensive disorders are associated with congenital heart defects. other, less common causes of pediatric pulmonary vascular disease include primary (idiopathic) pulmonary hypertension, hypoxiainduced pulmonary vascular disease, rheumatologic disorders, sickle cell disease, portal hypertension, chronic thromboembolic disease, human immunodefi ciency virus disease, and drug-toxin induced disease. a number of clinical classifi cation systems for vascular endothelial cells are capable of producing a variety of vasoactive substances that participate in the regulation of normal vascular tone. a schematic of some of these endothelial factors is shown in figure . . these substances, such as no, et- , and prostacyclin are capable of producing vascular relaxation and/or constriction, modulating the propensity of the blood to clot, and inducing and/or inhibiting smooth muscle cell migration and replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nitric oxide is a labile humoral factor produced by nitric oxide synthase (nos) from l-arginine in the vascular endothelial cell [ ] [ ] [ ] . nitric oxide diffuses into the smooth muscle cell and produces vascular relaxation by increasing concentrations of guanosine ′ ′-monophosphate (cgmp) via the activation of soluble guanylate cyclase [ , ] . nitric oxide is released in response to a variety of factors, including shear stress (fl ow) and the binding of certain endothelium-dependent vasodilators (such as acetylcholine, adenosine triphosphate [atp], and bradykinin) to receptors on the endothelial cell [ , ] . basal no release is an important mediator of both resting pulmonary and systemic vascular tone in the fetus, newborn, and adult, as well as a mediator of the normal fall in pulmonary vascular resistance that occurs immediately after birth , , ] . in addition, aberrant no-cgmp signaling is integral to the pathophysiology of pulmonary hypertension, as well as a number of other biologic vascular disorders [ , , , , , , , ] . endothelin- is a amino acid polypeptide also produced by vascular endothelial cells [ ] . the vasoactive properties of et- are complex, and studies have shown varying hemodynamic effects on different vascular beds [ ] [ ] [ ] [ ] [ ] . however, its most striking property is its sustained hypertensive action. in fact, et- is the most potent vasoconstricting agent discovered, with a potency times that of angiotensin ii. the hemodynamic effects of et- are mediated by at least two distinct receptor populations, et a and et b [ , ] . the et a receptors are located on vascular smooth muscle cells and mediate vasoconstriction, whereas the et b receptors are located on endothelial cells and smooth muscle cells and thus may mediate both vasodilation and vasoconstriction, respectively. individual endothelins occur in low levels in the plasma, generally below their vasoactive thresholds. this suggests that they are primarily effective at the local site of release. even at these levels, they may potentiate the effects of other vasoconstrictors, such as norepinephrine and serotonin [ ] . the role of endogenous et- in the regulation of normal vascular tone is unclear at present [ ] . nevertheless, alterations in et- have been implicated in the pathophysiology of a number of disease states, including pulmonary hypertensive disorders, and has been implicated in the so-called rebound effect of inhaled no [ , , , , ] . endothelial-derived hyperpolarizing factor (edhf), a diffusible substance that causes vascular relaxation by hyperpolarizing the smooth muscle cell, is another important endothelial factor. endothelial-derived hyperpolarizing factor has not yet been identifi ed, but current evidence suggests that its action is dependent on k + channels [ ] . activation of potassium channels in the vascular smooth muscle results in cell membrane hyperpolarization, closure of voltage-dependent calcium channels, and ultimately vasodilation. potassium channels are also present in endothelial cells. activation within the endothelium results in changes in calcium fl ux and may be important in the release of no, prostacyclin, and edhf. potassium channel subtypes include atp-sensitive k + channels, ca + -dependent k + channels, voltage-dependent k + channels, and inward-rectifi er k + channels [ ] . the breakdown of phospholipids within vascular endothelial cells results in the production of the important byproducts of arachidonic acid, including prostacyclin (pgi ) and thromboxane (txa ). prostacyclin activates adenylate cyclase, resulting in increased camp production and subsequent vasodilation, whereas txa results in vasoconstriction via phospholipase c signaling. other prostaglandins and leukotrienes also have potent vasoactive properties. increasing evidence suggests that endothelial injury and the resulting alteration in the balance of these and other vasoactive substances has a signifi cant role in the development of pulmonary hypertension and increased vascular reactivity [ , , ] . support for this hypothesis is strengthened by observations that endothelial injury precedes pulmonary hypertension and its associated vascular remodeling in several animal models of pulmonary hypertension [ , ] . in humans, endothelial dysfunction, including histologic abnormalities of the endothelium, impairment of endothelium-dependent pulmonary vasodilation, and increased plasma et- concentrations have been described in children with congenital heart defects and pulmonary hypertension before the development of signifi cant vascular remodeling [ , , ] . in addition, neonates with pphn and adults with advanced pulmonary vascular disease have evidence of endothelial dysfunction, impairment of endothelium-dependent pulmonary vasodilation, increased plasma et- concentrations, and decreased prostacyclin production [ , , , ] . the mechanism of injury to the vascular endothelium is unclear but is likely multifactorial and in part dependent on the etiology of the pulmonary hypertension. for example, in children with congenital heart disease and increased pulmonary blood fl ow, the initiating endothelial injury is likely mediated by increased shear stress. however, once pulmonary arterial pressure is elevated, shear stress-mediated endothelial injury appears to promote the progression of the disease, independent of the underlying etiology. finally, a genetic disposition appears to be important in some subtypes of pulmonary vascular disease and remains an area of active research. for example, up to % of patients with familial idiopathic pulmonary hypertension have mutations resulting in the loss of function of bone morphogenetic protein receptor ii [ ] [ ] [ ] [ ] . following an initial endothelial injury, smooth muscle proliferation and progressive structural remodeling occurs. the progression of anatomic changes is best characterized in congenital heart disease (see later discussion) [ ] [ ] [ ] [ ] . however, regardless of the etiology, advanced disease is characterized by medial hypertrophy, intimal hyperplasia, angiomatoid formation, in situ thrombi, and eventual vascular obliteration. if the underlying stress remains untreated (e.g., delayed repair of cardiac shunt), these structural changes can progress to the point of becoming functionally "fi xed" or irreversible. an important goal of therapy is to halt this progression and reverse the early vascular remodeling if possible. regardless of the underlying etiology, the general treatment approach is similar and can be subdivided into four major goals: ( ) prevent and acutely treat active pulmonary vasoconstriction, ( ) support the failing right ventricle, ( ) treat the underlying etiology, and ( ) chronically promote, if possible, the regression of pulmonary vascular remodeling. in the intensive care setting, the prevention and treatment of active pulmonary vasoconstriction is a primary focus for the care of patients with underlying pulmonary vascular disease. it is well appreciated that these patients have augmented pulmonary vasoconstriction in response to such stimuli as hypoxia, acidosis, the catecholamine-mediated α -adrenergic stimulation associated with pain and agitation, and increases in intrathoracic pressure [ ] [ ] [ ] . in fact, acute increases in pulmonary vascular resistance can lead to signifi cant cardiopulmonary compromise (i.e., a pulmonary hypertensive crisis). the pathophysiology of such a crisis in outlined in figure . . following an acute increase in pulmonary arterial pressure, there is an acute increase in right ventricular afterload, producing right ventricular ischemia and, ultimately, failure [ , ] . the resulting increase in right ventricular end diastolic volume shifts the intraventricular septum to the left, decreasing left ventricular volume and cardiac output. decreased cardiac output results in decreased systemic perfusion and metabolic acidosis. increased pulmonary vascular resistance and right ventricular failure also decrease pulmonary blood fl ow, increasing dead space ventilation. distention of the pulmonary arteries and perivascular edema produce large and small airways obstruction, respectively, which impairs ventilation-perfusion matching and decreases lung compliance. in fact, the decrease in lung compliance can be so dramatic that chest wall movement is impaired, even with manual ventilation. the ensuing hypoxemia, hypercapnia, and acidosis (metabolic and/or respiratory) further increase pulmonary vascular resistance and perpetuate this cascade. prevention of pulmonary hypertensive crises may be accomplished by avoiding stimuli known to increase pulmonary vascular resistance, including hypoxia, acidosis, agitation, pulmonary overdistention, and polycythemia [ ] . various regimens have been utilized for this purpose, including the judicious use of supplemental oxygen, analgesics, sedatives, and muscle relaxants (especially before noxious stimuli, such as suctioning); the maintenance of an alkalotic ph with the use of controlled ventilation and buffer; aggressive evacuation of pneumothoraces and pleural effusions; the utilization of low lung volume ventilator strategies; the minimization of positive end-expiratory pressures; and the maintenance of the hematocrit below % [ , ] . in addition, data suggest that the use of pulmonary vasodilator therapy may decrease the incidence of pulmonary hypertensive crises [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . treatment of active pulmonary vasoconstriction is accomplished with the use of pulmonary vasodilator therapy. the mainstay of acute pulmonary vasodilator therapy remains supplemental oxygen and moderate alkalosis, as these therapies have minimal effects on the systemic vasculature. interestingly, the dose-dependent response of the pulmonary vasculature to these agents has not been well established. studies in newborn lambs demonstrate dosedependent pulmonary vasodilation in response to increasing ph from . to . , and a dose-dependent response to increasing inspired oxygen concentrations from . to . , with minimal effects at higher concentrations [ ] . several intravenous agents have been utilized to promote pulmonary vasodilation, including tolazoline, sodium nitroprusside, nitroglycerin, prostacyclin, prostaglandin e , nifedipine, and α-adrenergic antagonists, such as phenoxybenzamine [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the effi cacies of these agents are variable, at least in part because of their effects on the systemic vasculature. systemic afterload reduction can be advantageous in the setting of left ventricular dysfunction; however, signifi cant reductions in pulmonary arterial pressure without unacceptable systemic hypotension are often not possible [ ] [ ] [ ] . in addition, intravenous vasodilators can override intrinsic hypoxic pulmonary vasoconstriction, resulting in an increase in dead space ventilation, which may not be tolerated in some critically ill patients [ ] [ ] [ ] [ ] [ ] . more recent treatment modalities, most notably inhaled no, deliver short-acting vasodilators to the pulmonary vasculature via an inhalational route [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . when administered to the lung in its natural gaseous form, no diffuses through the alveolar wall to reach small pulmonary arteries. it then enters vascular smooth muscle cells, initiating a cascade that results in pulmonary vasodilation via increases in cgmp. after entering the blood vessel lumen, no is rapidly inactivated by hemoglobin, which confi nes its effects to the pulmonary vasculature. because of these properties, inhaled no has several advantages over other vasodilators, including ( ) selective pulmonary vasodilation caused by rapid inactivation by hemoglobin, ( ) rapid onset and elimination, and ( ) an improvement in ventilation-perfusion matching because of the limitation of delivery to well-ventilated lung regions. accordingly, inhaled no has become a mainstay of treatment for acute pulmonary hypertensive disorders and the assessment of pulmonary vascular reactivity. inhaled prostacyclin has similar pulmonary selectivity, secondary to rapid inactivation by hemoglobin. its vasodilating effects are secondary to increasing camp concentrations. currently, studies on the use of inhaled prostacyclin for pediatric pulmonary hypertension are sparse, and comparison studies between inhaled no and inhaled prostacyclin are lacking [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . inhibitors of phosphodiesterases (pdes), a family of enzymes that hydrolyze the cyclic nucleotides camp and cgmp, are a relatively new class of agents that have potent vasodilating and inotropic effects [ ] . milrinone is a pde inhibitor that increases camp concentrations. animal and human data demonstrate pulmonary vasodilation in response to milrinone that can be in excess of its systemic effects if the pulmonary vasculature is constricted [ ] [ ] [ ] [ ] . in addition, a large, randomized study demonstrates that its use decreases the incidence of low cardiac output syndrome following surgery for congenital heart disease [ ] . given these properties, milrinone is increasingly utilized in the postoperative management of patients with congenital heart disease and pulmonary hypertension. sildenafi l, a pde inhibitor, which increases cgmp concentrations, also has potent pulmonary vasodilating effects [ ] . the oral formulation is currently being investigated for chronic pulmonary hypertensive therapy, and recent short-term studies demonstrate benefi cial effects in children with advanced pulmonary vascular disease [ ] . the intravenous formulation is currently being investigated for acute pediatric pulmonary hypertensive disorders (pphn and perioperative pulmonary hypertension) [ , ] . increasing data implicate alterations in et- in the pathophysiology of pulmonary hypertension (see earlier) and suggest that et-receptor antagonism may be a useful therapeutic strategy [ , , , , ] . in fact, bosentan, an oral combined et a and et b receptor antagonist, has demonstrated effi cacy as a chronic therapy for advanced pulmonary vascular disease [ , ] . to date, there have been no large studies on the use of et receptor antagonists for acute pulmonary hypertensive disorders. in addition, the use of selective et receptor antagonism is under investigation but has not yet reached clinical trials. a signifi cant component of the pathophysiology of both acute and chronic pulmonary hypertension is the development of right ventricular dysfunction, which often requires pharmacologic support. maintenance of adequate preload is necessary to optimize cardiac output in patients with pulmonary hypertension. continuous central venous pressure monitoring may be helpful to guide volume therapy, keeping in mind that patients with a poorly compliant right ventricle or increased right ventricular afterload may require elevated central venous pressures to maintain an adequate cardiac output. frequent clinical assessment of liver size can be helpful, particularly in infants. despite adequate preload, cardiac output may be compromised secondary to elevated right ventricular afterload and/or biventricular myocardial dysfunction after cardiac surgery and cardiopulmonary bypass, necessitating the use of inotropic agents [ , ] . these agents increase stroke volume at a given preload and afterload by stimulating ß -adrenergic receptors [ , ] . however, some of these agents also stimulate ß -or α -adrenergic receptors, which are found on the smooth muscle cells of both the pulmonary and systemic arteries. agents that stimulate ß -adrenergic receptors decrease both pulmonary and systemic vascular resistance and improve right and left ventricular function [ , ] . agents that stimulate α -adrenergic receptors may increase both systemic and pulmonary vascular resistance. therefore, a rational approach to using inotropic agents in the setting of pulmonary hypertension is to utilize agents with ß -receptor selectivity and minimal α -adrenergic stimulation (i.e., dobutamine). although animal studies have shown that high doses of dopamine increase pulmonary vascular resistance, human studies have shown increased cardiac output with minimal effects on pulmonary vascular resistance [ , ] . milrinone is also a useful therapy for patients with pulmonary hypertension and myocardial dysfunction, given its vasodilatory and inotropic properties [ ] . in the setting of pulmonary hypertension secondary to congenital heart defects, an atrial communication can be benefi cial in that it allows the failing right ventricle to decompress when right atrial pressure rises [ ] . accordingly, atrial septal defects can be left unclosed (i.e., patent foramen ovale) or created at the time of surgery. the existence of an atrial level communication decreases the risk of right ventricular failure and maintains left-sided cardiac output. the resulting right-to-left shunt is generally well tolerated, particularly if high hemoglobin concentrations are maintained. as right ventricular function improves, right-to-left shunting decreases and oxygenation improves. atrial septostomy as a part of management for chronic pulmonary hypertension (e.g., primary pulmonary hypertension) has been advocated but must be considered carefully on an individual basis [ ] [ ] [ ] [ ] [ ] . in patients with refractory pulmonary hypertension, short-term postoperative extracorporeal support has been useful during the postoperative period of extreme vasoreactivity. however, its use should be limited to support those patients in which the underlying pulmonary vascular disease is deemed reversible. whenever possible, treatment of the underlying disorder must coincide with symptomatic treatment for pulmonary hypertension if attenuation and/ reversal of the disease are to be successful. for example, in neonates, this may involve correction of underlying metabolic disturbances, antibiotics for infectious etiologies, and exchange transfusions for polycythemia. for patients with congenital heart disease, repair of the underlying defect, after determining that the pulmonary vascular disease is reversible (see later), is mandatory. for hypoxia-induced disease, tonsillectomy and adenoidectomy may be required for sleep apnea, and a descent to sea level may be needed for high-altitude-related disease. finally, for rheumatologic disease, immunosuppression may be required. the mainstay of chronic therapy has been aimed at decreasing pulmonary vascular resistance, thereby assisting right ventricular function and perhaps attenuating the progression of vascular remodeling by decreasing the pressure to which the vasculature is exposed. the continuous infusion of prostacyclin (epoprostin) has been the most successful therapy to date in this regard [ ] [ ] [ ] [ ] [ ] . in fact, several studies in humans with advanced pulmonary vascular disease demonstrate improved -year survival and improved exercise tolerance. interestingly, even those patients without an initial vasodilating response to the infusion show signifi cant longterm benefi t, suggesting that effects beyond vasodilation, such as antiplatelet effects, camp-mediated inhibition of smooth muscle cell growth, or other unknown mechanisms may be responsible for the treatment effect [ ] . despite the impressive results, several factors limit its utilization, including the need for chronic intravascular assess with the associated infectious and thrombotic risks, and many other untoward effects, including headache, fl ushing, and acute cardiopulmonary compromise with disruption of the infusion [ ] . with the increasing appreciation for the role of et- in the pathophysiology of pulmonary vascular disease, et receptor antagonists have been developed as a potential treatment modality. to date, bosentan, a combined et a and et b receptor antagonist, is the most widely studied agent and is the only receptor antagonist approved for the treatment of pulmonary hypertension [ , ] . recent studies in adults with primary pulmonary hypertension demonstrate similar improvements in survival and exercise tolerance as those demonstrated with epoprostenol [ ] . the use of et receptor antagonists for pediatric pulmonary hypertensive disorders is currently under investigation. defi ciencies in the no-cgmp cascade in pulmonary vascular disease are well documented. in addition, the vasodilating effects, antiplatelet effects, and antiproliferative effects of augmenting this cascade are well appreciated. therefore, new chronic therapies that augment no-cgmp signaling, which include chronic inhaled no delivered by nasal cannula and sildenafi l, are currently under investigation [ ] . in fact, the short-term benefi t of sildenafi l in children with advanced pulmonary hypertension has recently been reported [ ] . data indicate that several of these new oral therapies, such as bosentan and sildenafi l, may offer additional benefi t by virtue of their ability to inhibit vascular smooth muscle growth and fi brosis [ ] . a number of other treatment strategies, including combination drug therapies, are currently under investigation. to date they have been used predominantly in advanced pulmonary vascular disease, but, due to these favorable characteristics, several potential applications warrant investigation. this includes their use in lung hypoplastic syndromes, in hypoxia-associated disease, and in congenital heart disease in order to improve the operability of patients with modest vascular changes [ ] . in a number of clinical conditions, pulmonary vascular resistance does not decrease normally at birth. as a result, pulmonary blood fl ow remains reduced and pulmonary arterial pressure remains high. the pathophysiologic effects are hypoxemia, myocardial dysfunction, and a resulting reduction in systemic oxygen delivery. the hypoxemia is most often secondary to extrapulmonary rightto-left shunting of blood at the atrial and/or ductal levels but may also be secondary to intrapulmonary right-to-left shunting of blood when associated with parenchymal lung disease. the pathophysiologic mechanisms preventing the normal pulmonary vasodilatation at birth remain unclear and are most likely multifactorial in etiology. within this defi nition of pphn, three major subgroups are often characterized: those with underdevelopment of the lung, those with maladaptation of the lung, and those with maldevelopment of the lung [ ] . these subgroups represent a spectrum of etiologies and pathophysiology. for example, underdevelopment of the lung represents disorders of vascular hypoplasia, which are usually associated with varying degrees of lung hypoplasia. within this subgroup, patients with congenital diaphragmatic hernia have been most thoroughly investigated. although the structural abnormalities are greatest on the side of the hernia, both of the lungs of these patients are smaller and have fewer alveoli than do lungs from a normal control population [ ] [ ] [ ] . their lungs also have fewer vessels per unit of lung [ ] . thus, the total cross-sectional area of the vascular bed is markedly decreased. furthermore, the existing pulmonary arteries have increased muscle mass with medial hypertrophy in normally muscularized arteries and an abnormal extension of muscle into the intra-acinar arteries. the increased muscularization may explain the labile, right-to-left extrapulmonary shunting of blood seen in such patients [ , ] . the response to therapy and long-term outcome is dictated by the degree of hypoplasia of the underlying vasculature. following acute therapies, which often include surgical repair, mechanical ventilation with inhaled no, and extracorporeal support, subacute and chronic pulmonary hypertension has been increasingly recognized as a major outcome variable in these patients. because ultimately lung and vascular growth are necessary to reverse the disease process, aggressive long-term support with agents that inhibit vascular remodeling (i.e., et receptor antagonists and pde inhibitors) is an emerging treatment approach to support these infants as they grow. maladaptation of the lung represents a stress event at the time of delivery that does not allow the normal dilating stimuli, such as increases in systemic arterial ph and oxygen tension, to occur. this may occur in the setting of apnea, pneumonia, sepsis, and aspiration of meconium or amniotic fl uid [ ] [ ] [ ] . the underlying pulmonary vasculature is often normal, and, thus, these neonates are likely to respond to vasodilator therapy and the correction of contributory metabolic abnormalities. maldevelopment of the lung represents a group of conditions in which the vasculature is thickened and abnormally distributed. for example, some newborns who die from persistent pulmonary hypertension have abnormally muscular pulmonary vascular beds, even when they die on the fi rst day of life. in particular, they have thickened muscular coats in the normally muscular preacinar arteries, and extension of muscle into the normally nonmuscular intra-acinar arteries [ , ] . because vascular remodeling takes time to develop, it has been hypothesized that this increased muscularization is caused by a chronic intrauterine stress. in animal models, this pathophysiology can be mimicked by chronic placental insuffi ciency, fetal hypoxemia, chronic constriction of the ductus arteriosus, and chronic no inhibition [ , , [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, pphn has been associated with maternal indomethacin use, which causes constriction of the ductus arteriosus [ , ] . the response to therapy in neonates with maldevelopment of the lung is variable and may be related to the extent and type of underlying structural vascular pathology. the primary therapeutic approach is to decrease pulmonary vascular resistance and support myocardial function. the specifi c treatment modality depends on the underlying etiology. if the cause is perinatal asphyxia, correcting alveolar hypoxia, hypercarbia, and metabolic acidosis by ventilation with % oxygen, and by administration of buffer, should decrease pulmonary vascular tone toward normal levels. if parenchymal disease (i.e., respiratory distress syndrome, meconium aspiration, or pneumonia) is causing pulmonary vasospasm due to alveolar hypoxia and hypercarbia, then infl ation of the alveoli with positive end-expiratory pressure, surfactant administration, and mechanical ventilation may reverse the pulmonary hypertension [ ] [ ] [ ] . the near-term child can exert substantial intrathoracic pressure opposing mechanical ventilation; thus, sedation and occasionally muscle paralysis may be necessary to obtain stable mechanical ventilation [ ] . when treatment of the underlying pulmonary parenchymal, infectious, or infl ammatory disease is ineffective, or if there is no such underlying disease, therapy is directed at reversing abnormal pulmonary vasoconstriction. this is generally accomplished with sedation, mechanical ventilation with % oxygen, and alkalinization. when further pulmonary vasodilation is needed, inhaled no is utilized with or without high-frequency ventilation. in fact, several multicentered, randomized trials have demonstrated that inhaled no improves oxygenation and decreases the need for extracorporeal life support in newborns with persistent pulmonary hypertension [ , , ) , although no differences in mortality were noted. the use of extracorporeal membrane oxygenation has substantially decreased overall mortality for most subsets of pphn. however, overall mortality rates remain substantial at %- % [ ] [ ] [ ] [ ] . the development of pulmonary hypertension and increased pulmonary vascular reactivity is associated with two major types of congenital heart disease: ( ) those with increased pulmonary blood fl ow and pulmonary arterial pressure and ( ) those with increased pulmonary venous pressure [ ] [ ] [ ] ] . after birth, large communications at the level of the ventricles or great vessels result in increased pulmonary blood fl ow and pulmonary arterial pressure, which produces progressive structural and functional abnormalities of the pulmonary vasculature [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . similarly, elevated pulmonary venous pressure results in progressive increases in pulmonary venous and arterial pressure, which produces structural abnormalities of the pulmonary vasculature. heath and edwards fi rst described the progression of these pulmonary vascular changes in [ ] . in their classifi cation, changes progress from medial hypertrophy (grade i) to intimal hyperplasia (grade ii), lumen occlusion (grade iii), arterial dilatation (grade iv), angiomatoid formation (grade v) and fi brinoid necrosis (grade vi). in addition, morphometric analysis shows progression of disturbed arterial growth and remodeling of the pulmonary vascular bed, which correlates with the aberrant hemodynamic state of the pulmonary circulation [ , , [ ] [ ] [ ] . these changes are characterized by ( ) abnormal extension of vascular smooth muscle into small peripheral pulmonary arteries and mild medial hypertrophy of normally muscular arteries (grade a), ( ) severe medial hypertrophy of normally muscular arteries (grade b), and ( ) decreased pulmonary arterial number (grade c). uncorrected, these vascular changes result in decreased cross-sectional area and obliteration of the pulmonary vascular bed and death secondary to severe cyanosis or myocardial failure. different congenital heart defects vary considerably in the frequency and severity of pulmonary hypertension. the risks and frequencies of developing advanced pulmonary vascular disease (pvd) for particular heart defects are summarized in table . . importantly, children with trisomy and congenital heart defects often have an accelerated development of advanced pulmonary vascular disease [ ] . this may be secondary to confounding factors such as airway obstruction or another unidentifi ed predisposition. after surgical correction, early vascular changes (grades i-iii, grades a, b) are reversible; however, more severe changes are irreversible and progressive. therefore, the pathophysiologic state of the pulmonary circulation is the main determinant of the clinical course and the success of surgical treatment, and it explains the trend toward early repair of congenital heart defects [ ] . although early surgical repair of congenital heart defects has decreased the incidence of irreversible pulmonary vascular disease, even those children with reversible vascular changes suffer morbidity and mortality in the perioperative period secondary to chronic and/or acute elevations in pulmonary vascular resistance ] . chronic elevations are related to the structural changes that decrease the cross-sectional area of the pulmonary vascular bed. these alterations may take several months to normalize following surgical repair. acute elevations in pulmonary vascular resistance are often seen immediately following surgery with cardiopulmonary bypass, when there is often a period of enhanced pulmonary vascular reactivity [ , , ] . this period may last up to - days and is most likely a manifestation of preexisting aberrant endothelial cell-smooth muscle cell interactions that are exacerbated at the time of surgery. during cardiopulmonary bypass, several factors including the disruption of pulmonary blood fl ow, complement activation, and neutrophil activation induce pulmonary vascular endothelial dysfunction. this results in an increase in the production and/or release of endothelial factors that promote vasoconstriction, such et- and txa , and a decrease in endothelial relaxing factors, most importantly no [ ] . this period of extreme reactivity may produce severe hypoxemia, acidosis, low cardiac output, and death if not treated immediately. classically, a preoperative determination of pulmonary vascular reactivity is made in the cardiac catheterization laboratory in order to assess the operability of a given patient, that is, the degree to which the pulmonary vascular disease is reversible, as well as the postoperative risk. this testing involves measuring pulmonary arterial pressure and calculating pulmonary vascular resistance under varying conditions. the vascular resistance following acute maximal vasodilator therapy (e.g., oxygen and no) represents the degree of structural pulmonary vascular disease that is present. despite the frequent utilization of such testing, there is no absolute pulmonary vascular resistance that is universally considered inoperable. in general, a larger reduction in resistance in response to vasodilator therapy correlates with an increased chance of reversibility and a lower risk of perioperative morbidity from pulmonary hypertension. recent studies suggest that the combination of % oxygen and inhaled no produces maximal pulmonary vasodilation and has some perioperative predictive value [ , ] . in fact, a % decrease in the ratio of the pulmonary-to-systemic vascular resistance with vasodilator therapy, and a nadir in this ratio of less than %, was % sensitive and % accurate in predicting a good surgical outcome. therefore, the combination of oxygen and inhaled no is now most commonly used for pulmonary vascular reactivity testing. reactivity testing may also be helpful in the intensive care unit in the setting of a persistent postoperative elevation of pulmonary arterial pressure in order to differentiate between residual anatomic defects and prolonged periods of increased tone [ ] . the optimal treatment for perioperative pulmonary hypertensive morbidity is prevention with early surgical repair. it is increasingly clear that the longer the pulmonary vasculature is exposed to the abnormal forces associated with increased blood fl ow and/or pressure, the greater the risk of perioperative pulmonary vascular reactivity. following surgery, the goal of perioperative management is to minimize active pulmonary vasoconstriction during the period of exaggerated reactivity and support the right ventricle. to this end, avoidance of those stimuli that increase pulmonary vascular resistance (hypoxia, acidosis, pain, agitation, increased intrathoracic pressure) is critical. continuous pulmonary arterial and right atrial pressure monitoring is often helpful by allowing prompt recognition of pulmonary hypertensive crises and evaluation of the response to therapeutic maneuvers. monitoring systemic arterial pressure and systemic arterial oxygen saturation is essential in that it allows changes in pulmonary arterial pressure to be interpreted in the context of the total cardiopulmonary response. for example, if systemic and pulmonary arterial pressures increase in response to pain and agitation, but right atrial pressure does not increase, and systemic perfusion and oxygen saturation remain adequate, then specifi c treatment directed at the pulmonary vasculature is not necessary. conversely, increases in pulmonary arterial pressure that are associated with increased right atrial pressure, decreased systemic pressure, and/or decreased systemic saturation might herald imminent collapse. the objective of vasodilator therapy is to decrease right ventricular afterload and prevent acute increases in pulmonary arterial pressure. inhaled no, in combination with oxygen, has been increasingly utilized because of its potent vasodilating effects, pulmonary selectivity, and rapid onset and elimination (see earlier). several studies demonstrate its potent vasodilating effects in this population [ ] [ ] [ ] [ ] ; however, large, randomized trails are lacking. one randomized trial did demonstrate that inhaled no decreased postoperative pulmonary vascular resistance, the incidence of pulmonary hypertensive crises, and the days of mechanical ventilation compared with placebo [ ] . in patients with a history of pulmonary venous hypertension (total anomalous pulmonary venous return, mitral valve disease), aggressive diuresis may be helpful because interstitial pulmonary edema may contribute signifi cantly to elevations in pulmonary vascular resistance. therapies that maintain an adequate cardiac output in this patient population are not dissimilar to therapies utilized in other patient populations, with the exception of the particular emphasis placed on right ventricular afterload reduction and support. it is noteworthy that patients with a poorly compliant right ventricle or with increased right ventricular afterload may require elevated central venous pressures to maintain an adequate preload. in addition, the use of inotropic agents with signifi cant α -adrenergic effect should be minimized to avoid the associated pulmonary vasoconstriction. agents such as dobutamine, milrinone, and dopamine are routinely utilized. the use of high levels of positive end-expiratory pressure (peep) is somewhat controversial. mechanical hyperventilation with high peep increases intrathoracic pressure and pulmonary vascular resistance [ , ] . this therapy should be avoided if adequate systemic arterial saturation can be achieved by other means. however, at low lung volumes, the use of peep may increase lung volume until very recently, pulmonary arterial hypertension of unknown etiology was termed primary pulmonary hypertension. however, recent evidence indicates a genetic disposition in a subset of patients with primary pulmonary hypertension, and a number of diseases that lead to pulmonary arterial hypertension with similar histological and pathophysiologic features have been uncovered [ ] [ ] [ ] [ ] . thus, at the third world symposium on pulmonary arterial hypertension, a new classifi cation was proposed to further classify primary pulmonary hypertension into the following subgroups: ( ) idiopathic pulmonary arterial hypertension (ipah), ( ) familial pulmonary arterial hypertension (fpah), and ( ) pulmonary arterial hypertension related to risk factors or associated conditions (apah) [ ] . unfortunately, mortality from primary pulmonary hypertension remains high and may be higher for children than adults. in fact, the primary pulmonary hypertension national institutes of health registry reports a median survival of only months for pediatric patients [ ] . however, recent data suggest that pediatric patients may respond differently than adults to new therapies and that these differences may portend a better outcome in younger patients [ , ] . the frequency of primary pulmonary hypertension in pediatric patients is not known, but it appears that the number of confi rmed cases is increasing. the incidence is slightly increased in females [ ] . the most common causes of death in children with primary pulmonary hypertension are right ventricular failure and sudden death, which may be related to malignant cardiac arrhythmias, pulmonary emboli, or acute right ventricular ischemia [ ] . physicians caring for children in an intensive care unit setting must be cognizant of this disorder, albeit rare, because relatively benign disease processes, such as pneumonia, can be life threatening for children with primary pulmonary hypertension, which may not have been previously identifi ed. as opposed to adults with primary pulmonary hypertension, who often have severe plexiform lesions resulting in relatively fi xed vascular changes, children display greater medial hypertrophy with less intimal fi brosis and fewer plexiform lesions [ , ] . in addition, pediatric patients have a decreased pulmonary arterial number and increased pulmonary vascular reactivity compared with adult patients. the molecular mechanisms underlying primary pulmonary hypertension remain speculative; however, studies suggest an integral role for endothelial dysfunction, resulting in an increase in factors that favor both vasoconstriction and mitogenesis, such as et- and txa , and a decrease in factors that promote vasodilation and smooth muscle antiproliferation, such as no and prostacyclin [ , , , [ ] [ ] [ ] [ ] . other mechanisms have been investigated including, altered gene expression, coagulation abnormalities (resulting in intravascular thrombosis), and defects of pulmonary vascular smooth muscle cell potassium channels [ ] [ ] [ ] . recent advances in the understanding of pulmonary hypertension have established an association with a number of disease processes and toxins. thus, it is now known that pulmonary hypertension can be related to collagen vascular disease, portal hypertension, human immunodefi ciency virus infection, chronic obstructive pulmonary disease, interstitial lung disease, sleepdisordered breathing, alveolar hypoventilation disorders, chronic exposure to high altitudes, thromboembolic disease, sickle cell disease, schistosomiasis, sarcoidosis, thyroid disorders, glycogen storage disease, gaucher disease, hereditary hemorrhagic telangiectasia, myeloproliferative disorders, and pulmonary capillary hemangiomatosis. in addition, drugs or toxins, most notably anorexigens, have been associated with the development of pulmonary hypertension [ , ] . in general the diagnostic work-up includes history and physical examination, electrocardiography, chest radiography, echocardiography, and cardiac catheterization. serologic evaluation in order to exclude secondary causes is required, and v/q scanning to evaluate for pulmonary emboli may be necessary. treatment strategies for pediatric pulmonary arterial hypertension are evolving. when the disease is associated with a known disorder, treatment must include specifi c therapy aimed at the underlying condition. however, general treatments include the approach reviewed above, with oxygen, calcium channel blockers, anticoagulation, et receptor antagonists, prostacyclin analogues, acute and chronic inhaled no, pde type inhibitors, atrial septostomy, and lung or heart-lung transplant considerations as indicated [ ] . patients with pulmonary arterial hypertension have histologic evidence of in situ pulmonary vascular thrombosis, which is the rationale for anticoagulation therapy. although several adult studies have demonstrated its effi cacy, pediatric studies are lacking [ , ] . currently, warfarin is the treatment of choice for adult patients and in large pediatric centers with signifi cant experience with pediatric pulmonary arterial hypertension. low-molecularweight heparin is another alternative [ ] ; aspirin does not have any demonstrated effi cacy. chronic calcium channel blockade is effi cacious for a subset of adults and children with pulmonary arterial hypertension. in fact, whereas less than % of adults respond to calcium channel blockers, up to % of children are positive responders [ , ] . it is worth noting that calcium channel blockers are not utilized in the management of other common causes of pediatric pulmonary hypertension, such as pphn and congenital heart disease. indeed, calcium channel blockade should be avoided in neonates and after congenital heart surgery. however, studies indicate that long-acting calcium channel blockers, such as nifedipine and amlodipine, are well tolerated in children with primary pulmonary hypertension. an important caveat is that a positive response to calcium channel blockers (i.e., an acute reduction in pulmonary arterial pressure) must be demonstrated as a part of acute vasodilator reactivity testing. children without a positive acute response do not benefi t from chronic treatment. prostaglandins are a mainstay of therapy for patients with pulmonary arterial hypertension. in general, prostacyclin (epoprostenol) is administered as a continuous infusion, necessitating a permanent indwelling central catheter, with its associated risks [ ] [ ] [ ] [ ] [ ] . however, various other formulations including oral, inhaled, and subcutaneous prostacyclin analogues have been developed and are in various stages of clinical investigation [ ] [ ] [ ] [ ] . supplemental oxygen, cardiac glycosides, antiarrhythmic therapy, and inotropic agents are also variably utilized in certain patients [ , ] . diuretic therapy is also often benefi cial, keeping in mind that these patients may require elevated right ventricular preload. based on an expanding understanding of the disease process, future therapies might include elastase inhibitors and gene therapy [ , ] . as noted previously, atrial septostomy may have a role in the management of a select group of patients [ ] . however, atrial septostomy in the setting of an acute exacerbation of chronic pulmonary hypertension may lead to unacceptable hypoxemia because of excess right-to-left atrial shunting. finally, heart-lung, single-lung, or bilateral lung transplantation has been successful in pediatric patients with terminal pulmonary hypertension [ , ] . the international society for heart and lung transplantation reports survival of approximately % at years in pediatric patients [ ] . consensus is lacking as to the best type of transplant. increases in pulmonary arterial pressure in response to hypoxia are well described. clinical and experimental evidence suggests that prolonged exposure or chronic intermittent exposure to hypoxia can result in functional and structural derangements of the pulmonary vasculature, leading to pulmonary hypertension [ ] [ ] [ ] . fortunately, elevations in pulmonary arterial pressure that occur in response to acute hypoxia (such as an acute ascent in altitude) are rapidly reversible. interestingly, there is great clinical variability in the response to hypoxia. for example, increased susceptibility to high-altitude pulmonary edema, which is associated with increased pulmonary arterial pressure, has been linked to certain major histocompatibility complexes [ , ] . the mechanisms of hypoxia-induced pulmonary hypertension continue to be an area of intense investigation. to date the precise mechanisms remain unclear, but it is known that a number of endothelial derived factors, such as no, et- , leukotrienes, and potassium channels, participate [ , [ ] [ ] [ ] [ ] . furthermore, additional genetic polymorphisms are also under investigation. pediatricians must consider this physiology in patients with conditions such as upper airway obstruction, central hypoventilation, and neuromuscular disorders that affect ventilation. in fact, many of these patients do develop evidence of pulmonary hypertension, with right ventricular enlargement. in most cases, addressing the underlying pathology is curative, but it can take some time to fully reverse the structural changes that have occurred. the pathophysiology of acute lung injury involves damage to both the alveolar epithelium and pulmonary vascular endothelium. vascular endothelial injury accounts for key features of acute lung injury, including intravascular thrombosis and capillary permeability that increases alveolar fl uid [ ] . in fact, pulmonary vascular injury, in the setting of acute lung injury, can lead to pulmonary arterial hypertension, resulting in increased intrapulmonary shunting, hypoxia, pulmonary edema, and right ventricular dysfunction [ ] [ ] [ ] [ ] . in children with acute lung injury, persistently elevated pulmonary arterial pressures have been associated with worse outcomes [ ] ; therefore, vasodilators have been utilized in the management of these patients. however, intravenous vasodila-tors that dilate both the systemic and pulmonary vasculature have signifi cant problems, including systemic hypotension, right ventricular ischemia, increased intrapulmonary shunting (i.e., increased v/q mismatch), and increased hypoxemia [ ] [ ] [ ] [ ] [ ] . consequently, selective pulmonary vasodilation with inhaled no has been utilized, as it improves v/q matching and oxygenation without untoward systemic effects [ , ] . unfortunately, improvements in oxygenation associated with inhaled no are transient, and large randomized trials have failed to demonstrate an improvement in mortality with its use [ , , ] . the routine use of inhaled no in patients with acute lung injury, therefore, cannot be justifi ed; however, it may be indicated in individual patients, particularly those with an acute hemodynamic compromise and refractory hypoxemia caused by elevated pulmonary arterial pressures. clearly, physicians caring for pediatric patients with acute lung injury must include an awareness of the pulmonary vascular aberrations associated with the disease in their management considerations. historically, diseases of the pulmonary vasculature, although not uncommon, have been underrecognized. this was caused, in part, by the paucity of effective treatments as well as an incomplete understanding of the vascular biologic mechanisms. in fact, over the past decade, the therapeutic gold standard has been the continuous infusion of prostacyclin. although certainly extending and improving the lives of many patients, intravenous prostacyclin administration has been predominantly limited to patients with irreversible disease, given the inconvenience and morbidity associated with its delivery. fortunately, an expanded understanding of the vascular endothelium, vascular smooth muscle cells, and the role of their interactions in the pathophysiology of pulmonary vascular disease have resulted in new effective treatments, with additional potential therapies evolving rapidly. oral agents such as bosentan and sildenafi l are two examples with great promise. in addition, accumulated experience and focused research have uncovered a multitude of disease processes that contribute directly or indirectly to the development of pulmonary hypertension. physicians caring for critically ill children must be aware of these illnesses, the pathophysiology of pulmonary hypertension, and the available treatment options in order to translate these advances into improved outcomes for patients. cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate a novel potent vasoconstrictor peptide produced by vascular endothelial cells vascular endothelial cells synthesize nitric oxide from l-arginine flow-induced release of endothelium-derived relaxing factor molecular mechanisms of endothelium-mediated vasodilation the l-arginine-nitric oxide pathway n omega-nitro-l-arginine attenuates endothelium-dependent pulmonary vasodilation in lambs endothelial modulation of pulmonary vascular tone nitric oxide-generating vasodilators and -bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells edrf inhibition augments pulmonary hypertension in intact newborn lambs in vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue m&b , a cgmp phosphodiesterase inhibitor, is a pulmonary vasodilator in lambs nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia endothelial dysfunction in pulmonary hypertension vascular endothelial growth factor increases endothelinconverting enzyme expression in vascular endothelial cells tone-dependent responses to endothelin in the isolated perfused fetal sheep pulmonary circulation in situ endothelin- produces pulmonary vasodilation in the intact newborn lamb developmental effects of endothelin- on the pulmonary circulation in sheep circulatory effects of endothelin in newborn piglets maturational changes in endothelium-derived relaxations in newborn piglet pulmonary circulation circulatory changes during growth in the fetal lamb impairment of endotheliumdependent pulmonary artery relaxation in children with congenital heart disease and abnormal pulmonary hemodynamics reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension expression of endothelin- in the lungs of patients with pulmonary hypertension altered endothelium-dependent responses in lambs with pulmonary hypertension and increased pulmonary blood fl ow morphological development of the pulmonary vascular bed in fetal lambs effects of birth-related events on blood fl ow distribution fetal and neonatal pulmonary circulation changes in the lungs of the new-born lamb control of the fetal and neonatal pulmonary circulation distribution and regulation of blood fl ow in the fetal and neonatal lamb regulation of pulmonary vascular tone in the perinatal period effect of lung expansion on the fetal lamb circulation the onset of breathing at birth stimulates pulmonary vascular prostacyclin synthesis increasing oxygen tension dilates fetal pulmonary circulation via endothelium-derived relaxing factor oxygen modulates endotheliumderived relaxing factor production in fetal pulmonary arteries pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn effects of birth-related stimuli on l-arginine-dependent pulmonary vasodilation in ovine fetus ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs endothelin- in congenital heart disease increased pulmonary blood fl ow alters the molecular regulation of vascular reactivity in the lamb altered regulation of the et- cascade in lambs with increased pulmonary blood fl ow and pulmonary hypertension alterations in nitric oxide production in -week-old lambs with increased pulmonary blood fl ow increased endothelial nos in lambs with increased pulmonary blood fl ow and pulmonary hypertension emergence of smooth muscle cell endothelin bmediated vasoconstriction in lambs with experimental congenital heart disease and increased pulmonary blood fl ow sgc and pde are elevated in lambs with increased pulmonary blood fl ow and pulmonary hypertension altered endothelial function in lambs with pulmonary hypertension and acute lung injury alterations in tgf-beta expression in lambs with increased pulmonary blood fl ow and pulmonary hypertension alterations in et- , not nitric oxide, in -week-old lambs with increased pulmonary blood fl ow in utero placement of aortopulmonary shunts. a model of postnatal pulmonary hypertension with increased pulmonary blood fl ow in lambs the pathophysiology of pulmonary hypertension in congenital heart disease altered endothelium-dependent relaxations in lambs with high pulmonary blood fl ow and pulmonary hypertension endothelin- vasoactive responses in lambs with pulmonary hypertension and increased pulmonary blood fl ow failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs persistent fetal circulation: newly recognized structural features the structural basis of persistent pulmonary hypertension of the newborn infant pharmacologic therapy for persistent pulmonary hypertension of the newborn: as "poly" as the disease itself structure and function in pulmonary hypertension. new perceptions elevated immunoreactive endothelin- levels in newborn infants with persistent pulmonary hypertension loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia an imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension pulmonary pkg- is upregulated following chronic hypoxia models of persistent pulmonary hypertension of the newborn (pphn) and the role of cyclic guanosine monophosphate (gmp) in pulmonary vasorelaxation physical factors affecting normal and serotonin-constricted pulmonary vessels response of the pulmonary vasculature to hypoxia and h+ ion concentration changes site and sensitivity for stimulation of hypoxic pulmonary vasoconstriction infl uence of alveolar oxygen on pulmonary vasoconstriction in newborn lambs versus sheep hypoxic pulmonary vasoconstriction. physiologic signifi cance, mechanism, and clinical relevance hypoxic pulmonary vasoconstriction increased arterial ph, not decreased paco , attenuates hypoxia-induced pulmonary vasoconstriction in newborn lambs the effect of hypocarbia on the cardiovascular system of puppies pulmonary vascular k+ channel expression and vasoreactivity in a model of congenital heart disease the nervous control of the circulation in the foetal and newly expanded lungs of the lamb physiology and pharmacology of the pulmonary circulation in the fetus and newborn poiseuille's law and its limitations in vascular systems mechanics of the pulmonary circulation applied hydro-and aeromechanics congenital diseases of the heart: clinical-physiological considerations relative viscosity of blood at varying hematocrits in pulmonary circulation effect of hematocrit and inertial losses on pressure-fl ow relations in the isolated hindpaw o the dog mechanical infl uences on the pulmonary microcirculation recruitment vs. distensibility in the pulmonary vascular bed relationship between regional lung volume and regional pulmonary vascular resistance infl uence of state of infl ation of the lung on pulmonary vascular resistance biosynthesis of nitric oxide and citrulline from l-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical pharmacology of endotheliumderived nitric oxide and nitrovasodilators activation of purifi ed soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid cyclic guanosine monophosphate as a mediator of vasodilation nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation cloning and expression of a cdna encoding an endothelin receptor cloning of a cdna encoding a non-isopeptide-selective subtype of the endothelin receptor regulatory functions of the vascular endothelium regulation of the cerebral circulation: role of endothelium and potassium channels pulmonary artery endothelial abnormalities in patients with congenital heart defects and pulmonary hypertension. a correlation of light with scanning electron microscopy and transmission electron microscopy impairment of pulmonary endothelium-dependent relaxation in patients with eisenmenger's syndrome development of crotalaria pulmonary hypertension: hemodynamic and structural study plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. evidence for increased production of endothelin in pulmonary circulation heterozygous germline mutations in bmpr , encoding a tgf-beta receptor, cause familial primary pulmonary hypertension. the international pph consortium sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding bmpr-ii, a receptor member of the tgf-beta family mutation in the gene for bone morphogenetic protein receptor ii as a cause of primary pulmonary hypertension in a large kindred bmpr germline mutations in pulmonary hypertension associated with fenfl uramine derivatives lung biopsy in congenital heart disease: a morphometric approach to pulmonary vascular disease ultrastructural fi ndings in lung biopsy material from children with congenital heart defects quantitative structural analysis of pulmonary vessels in isolated ventricular septal defect in infancy pulmonary vascular disease in different types of congenital heart disease. implications for interpretation of lung biopsy fi ndings in early childhood pulmonary vascular disease with congenital heart lesions: pathologic features and causes pulmonary hypertension in children: perioperative management diagnosis and management of postoperative pulmonary hypertensive crisis transient myocardial ischemia of the newborn infant: a form of severe cardiorespiratory distress in full-term infants persistence of atrioventricular valve regurgitation and electrocardiographic abnormalities following transient myocardial ischemia of the newborn lung mechanics in congenital heart disease with increased and decreased pulmonary blood fl ow a randomized, controlled study of the -hour and -hour effects of inhaled nitric oxide therapy in children with acute hypoxemic respiratory failure inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. the neonatal inhaled nitric oxide study group inhaled nitric oxide in the neonate with cardiac disease low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. clinical inhaled nitric oxide research group effects of inhaled nitric oxide in patients with acute respiratory distress syndrome: results of a randomized phase ii trial. inhaled nitric oxide in ards study group multicenter randomized controlled trial of the effects of inhaled nitric oxide therapy on gas exchange in children with acute hypoxemic respiratory failure inhaled nitric oxide therapy for persistent pulmonary hypertension of the newborn inhaled nitric oxide in congenital hypoplasia of the lungs due to diaphragmatic hernia or oligohydramnios randomized, multicenter trial of inhaled nitric oxide and high-frequency oscillatory ventilation in severe, persistent pulmonary hypertension of the newborn inhaled nitric oxide therapy inhaled nitric oxide and persistent pulmonary hypertension of the newborn. the inhaled nitric oxide study group the effects of inhaled nitric oxide on postoperative pulmonary hypertension in infants and children undergoing surgical repair of congenital heart disease inhaled nitric oxide in premature infants with the respiratory distress syndrome inhaled nitric oxide, oxygen, and alkalosis: dose-response interactions in a lamb model of pulmonary hypertension effects of nitroprusside and dopamine on pulmonary arterial vasculature in children after cardiac surgery comparison of effects of prostaglandin e and nitroprusside on pulmonary vascular resistance in children after open-heart surgery hemodynamic effects of nifedipine and oxygen in children with pulmonary hypertension. cardiovasc does prostacyclin enhance the selective pulmonary vasodilator effect of oxygen in children with congenital heart disease? hemodynamic effects of prostaglandin e in patients with congenital heart disease and pulmonary hypertension comparison between prostaglandin e and epoprostenol (prostacyclin) in infants after heart surgery use of atp-mgcl in the evaluation and treatment of children with pulmonary hypertension secondary to congenital heart defects refractory hypoxemia associated with neonatal pulmonary disease: the use and limitations of tolazoline hemodynamic effects of pulmonary arterial infusion of vasodilators in newborn lambs comparative studies on the hemodynamic effects of prostaglandin e prostacyclin, and tolazoline upon elevated pulmonary vascular resistance in neonatal swine comparison of ketanserin and sodium nitroprusside in patients with severe ards prostaglandin e and nitroglycerin reduce pulmonary capillary pressure but worsen ventilation-perfusion distributions in patients with adult respiratory distress syndrome the pathophysiology of failure in acute right ventricular hypertension: hemodynamic and biochemical correlations prostaglandin e in the adult respiratory distress syndrome. benefi t for pulmonary hypertension and cost for pulmonary gas exchange prostacyclin for the treatment of pulmonary hypertension in the adult respiratory distress syndrome: effects on pulmonary capillary pressure and ventilation-perfusion distributions inhaled prostacyclin is safe, effective, and affordable in patients with pulmonary hypertension, right heart dysfunction, and refractory hypoxemia after cardiothoracic surgery inhaled epoprostenol (prostacyclin) and pulmonary hypertension before cardiac surgery inhaled prostacyclin for term infants with persistent pulmonary hypertension refractory to inhaled nitric oxide the use of inhaled prostacyclin in nitroprusside-resistant pulmonary artery hypertension inhaled prostacyclin (pgi ) is an effective addition to the treatment of pulmonary hypertension and hypoxia in the operating room and intensive care unit aerosolized prostacyclin (epoprostenol) as an alternative to inhaled nitric oxide for patients with reperfusion injury after lung transplantation inhaled aerosolized prostacyclin and pulmonary hypertension during anesthesia for lung transplantation effects of inhaled prostacyclin analogue on chronic hypoxic pulmonary hypertension dose-response to inhaled aerosolized prostacyclin for hypoxemia due to ards inhaled prostacyclin in the treatment of pulmonary hypertension inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fi brosis cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms milrinone decreases both pulmonary arterial and venous resistances in the hypoxic dog van trigt p rd. milrinone improves pulmonary hemodynamics and right ventricular function in chronic pulmonary hypertension hemodynamic and inotropic effects of milrinone after heart transplantation in the setting of recipient pulmonary hypertension milrinone: systemic and pulmonary hemodynamic effects in neonates after cardiac surgery effi cacy and safety of milrinone in preventing low cardiac output syndrome in infants and children after corrective surgery for congenital heart disease sildenafi l for primary and secondary pulmonary hypertension benefi cial effect of oral sildenafi l therapy on childhood pulmonary arterial hypertension: twelve-month clinical trial of a single-drug, open-label, pilot study interaction between inhaled nitric oxide and intravenous sildenafi l in a porcine model of meconium aspiration syndrome intravenous sildenafi l and inhaled nitric oxide: a randomised trial in infants after cardiac surgery effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebocontrolled study bosentan therapy for pulmonary arterial hypertension surgical implications of pulmonary hypertension in congenital heart disease the effects of dopamine and isoproterenol on the pulmonary circulation acute circulatory effects of dopamine in patients with pulmonary hypertension dobutamine pharmacokinetics and cardiovascular responses in critically ill neonates dobutamine: a hemodynamic evaluation in children with shock effects of vasoactive drugs on thromboxane a mimetic-induced pulmonary hypertension in newborn lambs factors that infl uence the outcome of primary pulmonary hypertension atrial septostomy as palliative therapy for refractory primary pulmonary hypertension double balloon technique for dilation of valvular or vessel stenosis in congenital and acquired heart disease successful palliation of primary pulmonary hypertension by atrial septostomy blade balloon atrial septostomy in patients with severe primary pulmonary hypertension graded balloon dilation atrial septostomy in severe primary pulmonary hypertension. a therapeutic alternative for patients nonresponsive to vasodilator treatment long-term treatment of primary pulmonary hypertension with continuous intravenous epoprostenol (prostacyclin) survival in primary pulmonary hypertension with long-term continuous intravenous prostacyclin a comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. the primary pulmonary hypertension study group primary pulmonary hypertension: improved long-term effects and survival with continuous intravenous epoprostenol infusion reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension vasodilator therapy for primary pulmonary hypertension in children pulmonary arterial hypertension in children survival with fi rst-line bosentan in patients with primary pulmonary hypertension treatment of rebound and chronic pulmonary hypertension with oral sildenafi l in an infant with congenital diaphragmatic hernia ventilatory predictors of pulmonary hypoplasia in congenital diaphragmatic hernia, confi rmed by morphologic assessment unsuspected pulmonary vascular abnormalities associated with diaphragmatic hernia morphologic analysis of the pulmonary vascular bed in congenital left-sided diaphragmatic hernia 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periodontal disease. most ferrets, even on a dry diet, develop tartar that progresses with age. tartar tends to accumulate most heavily on the second and third upper premolars. biting and gnawing habits often result in discoloration, wearing, and breaking of the tips of the canine teeth ( fig. - ) . broken canine teeth do not usually result in obvious discomfort or pain unless the dental pulp is exposed. root canal restoration or surgical removal of the affected teeth may be necessary in some ferrets. tooth root abscesses are not common but can occur at any age. although dysphagia and drooling are sometimes seen, dental disease is often an incidental finding during physical examination. dental extractions and scaling can be performed with the animal under anesthesia. follow the basic principles for dental disease management that apply in the care of the dog or cat. ferrets cele formation. although this lesion is uncommon in ferrets, mucocele diagnosis and treatment have been described. , diagnosis of a mucocele is relatively straightforward. facial swellings are often seen in the commissures of the mouth or in the orbital area in the case of a zygomatic mucocele. other locations also are possible. aspirate the mass to obtain samples for cytologic analysis. the fluid is viscous or mucinous and clear or blood-tinged. cytologic examination reveals amorphous debris and occasional red blood cells. treatment for salivary mucoceles is usually surgery. in one reported case, scalpel blade lancing of the medial wall of the mucocele resulted in drainage and no recurrence. marsupialization into the mouth with the use of a wide circular incision in the medial wall of the mucocele may be effective for mucoceles that bulge into the oral cavity ( fig. - ). surgical excision of the affected salivary gland is ideal for avoiding recurrence (see chapter ) . it may be possible to inject contrast medium into the mucocele in an effort to trace the origin of the saliva. review the superficial anatomy of the head and neck region of the ferret before attempting surgical excision of a salivary gland. recurrence is possible. diseases of the esophagus are rare in ferrets. acquired megaesophagus has been reported in ferrets, and i have seen the condition several times in my practice. , megaesophagus describes an esophagus that is enlarged (dilated) on radiographic examination and that lacks normal motility. recognizing this disease is important because the prognosis in ferrets with megaesophagus is poor. clinical signs include lethargy, inappetence or anorexia, dysphagia, and weight loss. regurgitation is common. coughing or choking motions are sometimes described, and some ferrets have labored breathing. differential diagnosis includes the presence of an esophageal or gi foreign body, gastritis, influenza, and respiratory diseases. diagnosis is based on clinical signs and radiographic evidence. on radiographs, the esophagus is often dilated in both the cervical and thoracic segments ( fig. - ). food may be visualized in the esophagus. aspiration pneumonia and gastric gas are sometimes evident in addition to esophageal dilation. always take radiographs of the abdomen to exclude lower gi disease. administer barium ( ml/kg po) to delineate the esophagus and to evaluate mural lesions, strictures, or obstructions ( fig. - ). an endoscope can also be used to evaluate the esophagus. use fluoroscopy, if available, to determine the motility of the esophagus after a barium swallow. the cause of megaesophagus in ferrets is unknown. consider possibilities in the differential diagnosis as for dogs, and tailor the diagnostic workup accordingly. the management of ferrets with megaesophagus is similar to that of canine patients but is usually less successful. supportive care and antibiotics are palliative at best. administration of a gi motility enhancer such as metoclopramide ( . - mg/kg q - h po or sc) (reglan, ah robins company, inc., richmond, va) may be helpful. cisapride, which until recently was marketed for gastroesophageal reflux and gastroparesis in humans, reduces the frequency of regurgitation in dogs with megaesophagus when it is given at . mg/kg q - h po. however, this drug is no longer available commercially in the united states because of adverse cardiac effects in humans. its use in ferrets has not been evaluated. if esophagitis is suspected, add an h -receptor blocker, such as cimetidine, ranitidine (zantac, glaxo pharmaceuticals, research triangle park, nc), or famotidine (pepcid ac, johnson and johnson, fort washington, pa). the prognosis for ferrets with megaesophagus is poor; generally, they die or are euthanatized within days of diagnosis. affected ferrets are debilitated and suffer from malnutrition, hepatic lipidosis, and aspiration pneumonia. other causes of esophageal disease in the ferret are rare. esophageal foreign body has been reported in a ferret and was successfully managed surgically. gastric and duodenal ulceration has been reported in laboratory ferrets and is seen sporadically in pet ferrets. causes of gi ulceration include foreign body or toxin ingestion, helicobacter mustelae infection, treatment with ulcerogenic drugs, gi neoplasia, and azotemia caused by renal disease. the laboratory ferret is used as an animal model for the study of h. pylori infection in humans. h. mustelae isolated from the gastric mucosa of ferrets shares many molecular and biochemical features of h. pylori. h. mustelae infection in ferrets is associated with varying degrees of gastritis, with or without duodenitis, and it can result in ulcer formation. (see part ii for a discussion of h. mustelae infection.) ulcerogenic drugs such as nonsteroidal and steroidal antiinflammatory agents can be associated with ulcer formation. it is rare for ferrets to have gi bleeding when they are treated with corticosteroids at appropriate dosages; however, ulceration is possible with the prolonged use or overdose of other antiinflammatory agents such as ibuprofen (see chapter ) . severe uremia and associated melena can occur in ferrets with primary renal disease, but this is uncommon. gastritis in ferrets may be acute or chronic. clinical signs may include weight loss and vomiting. affected ferrets may hypersalivate and display tooth-grinding, which are indicative of nausea and abdominal pain. clinical signs of gastric or duodenal ulceration include melena, anorexia, lethargy, and weight loss. basic diagnostic testing includes whole-body radiography and screening blood tests. fast the ferret for a short time ( - hours) to facilitate visualization of a gastric foreign body or hairball. the diagnosis of h. mustelae gastritis may be a diagnosis of exclusion of other common disorders, such as the presence of a gi foreign body; treatment for h. mustelae gastritis is often based on a presumptive diagnosis. establish definitive diagnosis of helicobacter infection by histopathologic study of a gastric mucosal sample obtained by endoscopic or surgical biopsy. specialized techniques are necessary for culturing the organism, which is not shed consistently in feces of infected ferrets. treat gastritis and gastric ulceration with both specific therapy (according to the diagnosis) and supportive care. hospitalize sick and anorexic ferrets for fluid therapy and parenteral treatment. a broad-spectrum antibiotic, administered parenterally, is indicated for sick ferrets. for ferrets that are not vomiting, offer multiple small feedings of a bland, moist diet; avoid dry, ferrets high-fiber foods. for vomiting animals, withhold food for to hours while closely monitoring for any sign of hypoglycemia (older ferrets often have subclinical insulinomas); then, if vomiting has resolved, introduce small, frequent feedings. bismuth compounds have action against pepsin, a proteolytic enzyme believed to be an important factor in the development of peptic ulcers. administer bismuth subsalicylate at a dose of ml/kg q h po. sucralfate (carafate, marion merrell dow, inc., kansas city, mo) is a cytoprotective agent that binds to the erosion site and helps to form a protective barrier. it is a safe and useful adjunct to ulcer treatment and can be given orally in tablet ( / - / of a -g tablet) or suspension ( mg/kg) form every hours. systemic h -receptor antagonists, such as cimetidine and famotidine, are often used to treat gastric ulcers because they block the histamine receptor on the gastric parietal cell and reduce gastric acid secretion. the proton pump inhibitors, such as omeprazole (prilosec, astra merck, inc., wayne, pa), are occasionally used in ferrets. one quarter of the contents of a -mg capsule can be mixed with soft food and given orally. antacid therapy may not be helpful in the early treatment of helicobacter infection because affected ferrets usually develop hypochlorhydria. a standard treatment for helicobacter infection in humans is "triple therapy" with amoxicillin, metronidazole, and bismuth (see part ii). bismuth interferes with the colonization of h. pylori in humans and suppresses colonization of h. mustelae in ferrets. surgical removal is the treatment of choice for gi foreign bodies. two ferrets with gi polyps have been seen at the animal medical center (new york, ny). both ferrets showed lethargy, inappetence, melena, and weakness from anemia. abdominal radiographs suggested gi abnormalities. on surgical abdominal exploration, one ferret had a gastric polyp and the other had a small intestinal polyp. both ferrets did well after surgical resection of the polyps, which were histologically benign. gastric bloat is rarely seen in pet ferrets, but it has been reported on domestic ferret farms and in black-footed ferrets (mustela nigripes). , clinical signs are usually observed in weanling ferrets and include acute gastric distention, dyspnea, and cyanosis. sudden death can occur. the cause of gastric bloat is unknown but is thought to be related to an overgrowth of clostridium perfringens (previously called c. welchii). certain conditions may predispose to clostridial overgrowth, including increased concentration of carbohydrates in the gi tract from overeating, dietary changes, and intestinal hypomotility. c. perfringens multiplies rapidly, producing enterotoxins that attack the villous epithelial cells of the gut. gas production by the bacteria results in abdominal distention. prevention and treatment of the disease are difficult because of the ubiquitous nature of the organism and the short course of disease. these animals are in shock and need immediate aggressive therapy. relieve gastric pressure by trocharization or placement of an orogastric tube. follow therapeutic protocols as for bloat in canine patients. gi foreign bodies are very common in ferrets. ferrets are naturally very inquisitive and like to chew on miscellaneous environmental objects, particularly rubber or sponge products. rubber foreign bodies are most commonly ingested by young ferrets (younger than years of age); in contrast, trichobezoars (hair balls) are more common in older ferrets. linear foreign bodies, commonly ingested by cats, are rare in ferrets. the most common clinical signs of a gi foreign body in ferrets are lethargy, inappetence or anorexia, and diarrhea. vomiting is often not reported by the owner. however, if vomiting is observed, consider a gi foreign body (table - ). some ferrets display signs of nausea, including bruxism, ptyalism, and face rubbing. weakness can be profound in acutely obstructed animals; some of these ferrets are recumbent and reluctant to ambulate. if a gi foreign body is suspected, palpate the abdomen carefully. foreign bodies in the small intestine often are associated with very localized discomfort or pain. gastric foreign bodies are more difficult to palpate. take whole-body survey radiographs in these cases. although uncommon, esophageal disease is an important differential diagnosis in the anorexic, "vomiting" ferret. abnormal abdominal radiographic findings include segmental ileus, gaseous distention of the stomach, and, occasionally, a visible foreign object or trichobezoar ( fig. - ). contrast (barium) studies can be done but are rarely needed. base the diagnosis on history, clinical signs, palpation, and the results of radiography. at a minimum, submit a blood sample for a complete blood count (cbc) and plasma biochemical analysis in ferrets that are sick for longer than or days. ferrets rarely pass gi foreign bodies unassisted. occasionally, a small, partially obstructing object may pass with the administration of intestinal lubricants (cat laxatives) q h and replacement fluids. however, most gi foreign bodies must be removed surgically. stabilize debilitated ferrets before surgery. parenteral fluids are usually essential because these ferrets often have varying degrees of dehydration. perform an exploratory laparotomy as soon as possible. collect biopsy specimens from the liver, the spleen (if enlarged), and the stomach or intestines (if ulcerated or abnormal in appearance). some of these ferrets may also have h. mustelae-associated gastritis or gi lymphoma. check the adrenal glands and the pancreas in older ferrets; discovery of concurrent abdominal disease is not unusual during surgery. (see chapter for a description of the surgical procedure.) in most instances, recovery is rapid after gi foreign body removal, and ferrets are able ferrets to eat soft foods hours after surgery. most ferrets can be discharged within to hours after surgery. prevention of foreign body obstructions includes recommending the regular use of a cat laxative preparation during active shedding seasons and "ferret-proofing" the household. ferrets should not be left uncaged or unsupervised. advise owners to avoid giving small rubber "squeak" toys to pet ferrets. gi parasites are uncommon in ferrets. however, any ferret with diarrhea should have a complete fecal parasite check, including a direct fresh wet mount and fecal flotation. in juvenile ferrets, nematodiasis is rare, but coccidiosis and giardiasis are occasionally seen. coccidiosis can be subclinical in ferrets or it may be associated with diarrhea, lethargy, and dehydration. rectal prolapse is possible. base the diagnosis of coccidiosis on fecal testing, either by direct wet mount and microscopic examination or by fecal flotation. follow the same treatment protocols as for canine and feline patients with coccidiosis. cryptosporidiosis is described in ferrets but may not result in clinical disease. , young ferrets can have a subclinical infection that can persist for several weeks. the oocysts can be shed in the feces of clinically normal ferrets. histologically, the organism may be associated with an eosinophilic infiltrate in the lamina propria of the small intestine. it is not known whether zoonotic transmission of ferret cryptosporidia is possible; however, warn immunocompromised owners if the oocysts are detected in their ferrets. salmonellosis is a contagious disease characterized by fever, bloody diarrhea, and lethargy. conjunctivitis and anemia also may be present. salmonella newport, s. typhimurium, and s. choleraesuis may be involved. the incidence of salmonellosis in pet ferrets is very low and the infection may be associated with the feeding of uncooked meat, poultry, and meat by-products. isolation of salmonella organisms usually requires the collection of multiple fecal samples and the use of selective media. treatment consists of aggressive supportive care, use of antimicrobials, and shock therapy as needed. ferrets may be naturally or experimentally infected by the bovine, avian, and human tubercle bacilli. , , mycobacterium bovis and m. avium infections have been recognized in research and farm ferrets in england, europe, and new zealand. these infections may have been associated with the feeding of raw meat and poultry and unpasteurized dairy products. more recently, a pet ferret was reported to have a visceral infection caused by m. avium. the ferret had a long-term history of weight loss, diarrhea, and vomiting that was unresponsive to treatment. intestinal biopsy results revealed granulomatous inflammation and acid-fast bacteria. base the diagnosis of mycobacteriosis on the findings of tissue biopsy, including histopathologic examination with acid-fast staining, polymerase chain reaction (pcr) testing, and culture. m. avium can be detected in the small intestine, liver, spleen, and lymph nodes of affected ferrets. because of the zoonotic potential, treatment is not recommended. campylobacter jejuni is a bacterial enteric pathogen that is associated with diarrhea and enterocolitis in human beings and several animal species, including dogs, cats, calves, and sheep. c. jejuni can be isolated from the feces of normal ferrets. during the s, it was suspected to be the cause of proliferative colitis, which has since been renamed "proliferative bowel disease (pbd)" in ferrets. , however, inoculation of c. jejuni in conventionally reared and two gnotobiotic ferrets caused diarrhea but not the full spectrum of clinical signs and histopathologic lesions seen in pbd (see part ii). the agent of porcine proliferative enteropathy, which is identical to that causing pbd in ferrets, has now been assigned the new genus and species name lawsonia intracellularis. the importance of c. jejuni as a primary pathogen in pet ferrets is not known. infection with rotavirus causes diarrhea in young ferrets. farm outbreaks of diarrhea are associated with high morbidity and mortality rates in neonatal kits from to weeks of age. , the morbidity is low in adult ferrets, but infection may result in a transient green mucoid diarrhea. no antemortem testing is available for the diagnosis of rotaviral infection in ferrets. treatment is supportive; administer fluids and antibiotics to affected ferrets. canine distemper virus is a highly contagious paramyxovirus that causes fatal disease in unvaccinated ferrets. clinical signs are variable but often include diarrhea in conjunction with nasal and ocular discharges and a generalized orange-tinged dermatitis (see chapter ) . diarrhea may be acute or intermittent. fortunately, the widespread practice of vaccinating ferrets against canine distemper virus has greatly limited the occurrence of distemper and it has now has become an uncommon disease in ferrets. coldlike symptoms and diarrhea in newly purchased, unvaccinated ferrets should arouse suspicion. there is no treatment for distemper. ferrets affected with human influenza virus (an orthomyxovirus) sometimes have transient diarrhea. the virus also causes upper respiratory disease associated with coughing, sneezing, inappetence, and lethargy. affected ferrets are often febrile (see chapter ) . epizootic catarrhal enteritis (ece) is a highly transmissible diarrheal disease of ferrets that first appeared in in several rescue and breeder operations in the eastern united states. the causative agent is thought to be a coronavirus. in intestinal biopsy samples from affected ferrets, histologic findings include lymphocytic enteritis, villous atrophy, and blunting or degeneration of apical epithelium. ferrets with ece initially develop a profuse, green mucoid diarrhea that may progress to a loose, grainy stool resembling birdseed. adult ferrets are most susceptible to ece, and the typical history includes recent exposure to a new, young ferret that acts as an asymptomatic carrier. the incubation period is to hours, and affected ferrets are anorexic and lethargic. treat sick ferrets with signs of ece with aggressive fluid therapy, antibi-otics, and supportive care, and isolate these ferrets from asymptomatic or unexposed ferrets. although the morbidity rate can be high, the mortality rate is low in ferrets that are treated appropriately. after recovering from ece, some adult ferrets develop persistent, intermittent malabsorption with diarrhea. the clinical course can be prolonged in these ferrets, lasting weeks to months. treatment with a short course of steroids (prednisone mg/kg q h for days) and changing the diet to an easily absorbed food may speed recovery. inflammatory bowel disease is a relatively common cause of gastroenteritis in ferrets. the cause is unknown but may be related to dietary intolerance, hypersensitivity reaction, or another aberrant immune response. the inflammation typically is lymphoplasmacytic and should be distinguished from eosinophilic gastroenteritis, which often involves multiple tissues. this condition is easily overlooked in ferrets because it resembles viral diarrhea (ece), eosinophilic gastroenteritis, and helicobacter-associated gastroenteritis. affected ferrets can have loose stools, intermittent nausea, occasional vomiting, and weight loss. clinical signs can be subtle or, in rare cases, can resemble those of a gi foreign body in severity. these ferrets are usually young or middle-aged adults and in multiple-ferret households; typically only one ferret in the household is affected. results of blood tests may reveal an increase in concentrations of liver enzymes and serum globulins, and lymphocytosis is occasionally present. in some ferrets, laboratory results are unremarkable. diagnosis is based on clinical signs, a detailed clinical history that eliminates the possibility of exposure to ece, and results of diagnostic tests such as radiographs and routine blood tests. diseases such as helicobacter gastroenteritis and aleutian mink disease should be ruled out. definitive diagnosis can only be made by histologic examination of full-thickness gastric and intestinal biopsy samples. treatment is aimed at suppressing the immune response and dietary management. corticosteroids such as prednisone ( mg/kg po q - h) can be used, but some ferrets with inflammatory bowel disease respond poorly to long-term treatment with steroids. azathioprine (imuran, prometheus laboratories, san diego, ca) ( . mg/kg po q - h) is another treatment option and seems to be well tolerated in ferrets. hypoallergenic diets made for cats (z/d feline; hill's pet nutrition, topeka, ks) can be tried. lymphoma is the most common hepatic neoplasm seen in ferrets. other reported hepatic neoplasms include hemangiosarcoma, adenocarcinoma, and hepatocellular adenoma. pancreatic islet cell tumors can eventually metastasize to the liver. prognosis is guarded regardless of tumor type. other than neoplastic diseases, primary hepatopathies are uncommon in ferrets. vascular shunts have not been reported. hepatic lipidosis can be found in association with long-term anorexia. chronic gi diseases (e.g., trichobezoar formation) can lead to hepatic lipidosis. steroid hepatopathy is rare in ferrets, even with long-term steroid administration or hyperadrenocor-ticism. chronic-lymphocytic portal hepatitis has been found on histologic examination of hepatic biopsy samples. the cause of these entities is unknown but may be related to chronic visceral inflammation such as inflammatory bowel disease. chronic cholangiohepatitis with biliary hyperplasia of variable intensity was reported in of cohabitating ferrets. three ferrets had neoplastic lesions in the liver. spiral-shaped bacteria were identified in the livers of three ferrets, and bacteria with % similarity to helicobacter species were identified by pcr in the feces of one ferret. because of the clustering of cases and the pathologic findings, a possible infectious cause was suggested. copper toxicosis was diagnosed in two sibling ferrets on the basis of high hepatic copper concentrations and histologic changes in hepatic tissue. clinical signs in these two ferrets were mostly nonspecific and included severe central nervous system depression with hypothermia and hyperthermia, respectively. one ferret was icteric. both ferrets died within a few days of clinical evaluation despite supportive care. a genetic predisposition to copper toxicosis in these two ferrets was proposed because they were siblings with the same phenotypic coat color and because no environmental source of copper could be identified. a high concentration of alanine aminotransferase (> iu/l) is present on biochemical analysis in most ferrets with liver disease. alkaline phosphatase concentration is sometimes elevated. high total bilirubin levels are uncommon, and ferrets are rarely icteric. base the diagnosis of liver disease on observation of persistently high concentrations of liver enzymes, radiographic and ultrasound findings, and for definitive diagnosis, analysis of liver biopsy samples. ultrasound-guided needle biopsy of the liver is possible, but full abdominal exploration is often recommended because of the likelihood of concomitant disease in ferrets. the gi tract is not a common site of primary neoplasia in ferrets. the oral cavity is a rare location for neoplastic lesions. pyloric adenocarcinoma has been reported, and i have seen intestinal adenocarcinoma in one ferret. lymphoma frequently affects the gi tract of ferrets (see also part ii). visceral and mesenteric lymph nodes and the liver are common sites for lymphoma; intestinal lymphoma is uncommon. treatment involves surgical resection and debulking whenever possible. chemotherapy for lymphoma is described in chapter . rectal prolapse can occur in ferrets. it is most often associated with diarrhea and is usually a disease of young ferrets. possible causes include coccidiosis, pbd, colitis, campylobacteriosis, and neoplasia (see part ii). diagnostic tests should include a fecal wet mount and flotation to check for parasites. gi parasitism other than coccidiosis is uncommon. treat with antibiotics and antiparasitics, as indicated. prolapses often resolve with treatment of the causative condition. although rarely needed, rectal purse-string sutures can be placed if the prolapse is extensive; these sutures can be left in place for to days. include a careful rectal examination (visualization and palpation) in all ferret examinations. undescented ferrets may develop anal gland disease, including impactions and abscessation. palpation of the anal area may reveal either unilateral or -degree perianal swelling. manage anal gland disease as in the dog. be forewarned: anal gland odor is quite noxious. anal gland removal is described in chapter . neoplasia is rare in the rectal area, although i have seen one descented ferret with leiomyosarcoma that surrounded the rectal opening ( fig. - ). the ferret presented for a rectal prolapse, and a tumor was found on palpation. treatment involves surgical debulking and, possibly, a rectoplasty. prognosis is poor. owners may describe "vomiting" in their ferrets, but some of these animals may actually be regurgitating. in light of this, the differential diagnoses for emesis in ferrets include both esophageal diseases and gastroenteric disorders. in the clinical history, vomiting is not as frequently described in ferrets as it is in canine or feline patients. for example, ferrets rarely vomit hairballs, and often vomiting is not part of the history associated with foreign body ingestion. the reason for this is unclear. no anatomic feature prevents emesis in ferrets; in fact, ferrets have long been laboratory animal models for human emesis studies because vomiting can readily be induced in them in a laboratory setting. the major differential diagnoses for vomiting or regurgitation in ferrets include the presence of a gi foreign body, h. mustelae gastritis, gastroenteritis, and, rarely, megaesophagus. it is uncommon for ferrets with metabolic problems such as azotemia or hepatic disease to vomit. although definitive diagnosis is not always possible, it is important to recognize whether medical or surgical treatment is required. for example, most obstructions caused by a foreign body require surgery, whereas gastroenteritis is a medical disease. however, differentiating these two diagnoses is often quite challenging (see table - ). diagnosis begins with the history. pointedly question an owner regarding the chewing habits of the ferret: does the ferret have a squeak toy? is it unsupervised in the household or usually caged? has vomiting been observed? the description of any vomiting behavior is significant. also question the owner regarding the animal's appetite and obtain a description of the feces. on physical examination, some foreign bodies in the small intestine can be distinctly palpated. however, enlarged mesenteric lymph nodes can sometimes feel like foreign objects. also remember that foreign bodies in the stomach are difficult to detect on palpation. pbd may result in palpably thickened intestines in the ferret; however, vomiting is not usually a feature of this disease. radiography is the most important diagnostic test in the workup of a vomiting ferret. survey radiographs should include the whole body. radiographic signs of megaesophagus can be subtle. the heart may appear small because of hypovolemia from dehydration. varying amounts of gas can be seen with foreign body-related obstruction, and sometimes the incriminating object is visible. segmental ileus or a dilated and gas-or fluidfilled stomach is a typical radiographic sign of obstruction (see fig. - ) . not all cases of gi foreign body are radiographically obvious. if evidence of foreign body obstruction is not well defined, consider medical therapy and perform repeat radiography in hours. alternatively, give barium contrast for a series of contrast-enhanced films. if there is a strong indication of the presence of a foreign body, perform abdominal exploratory surgery the same day, preferably after parenteral fluid therapy has been started (see chapter ) . obtain tissue for biopsy as needed (e.g., the liver or spleen), and save any foreign object to show to the owner. always check the entire gut for lesions and examine the pancreas and adrenal glands, especially in older ferrets. if a foreign body is not found, collect gastric and duodenal biopsy samples and request special staining for helicobacter species. helicobacter infection is associated with gastritis, especially in the antral region and the proximal duodenum (see part ii). although results of exploratory surgery may be negative for a foreign object, histologic examination of biopsy samples may or may not reveal a diagnosis. the possibility of negative findings should be discussed with the owner before surgery. if surgery is not an option or is not recommended, consider treatment for h. mustelae-associated gastritis (see part ii). if obstruction is still a possibility, administer a cat hairball preparation (laxatone [evsco pharmaceuticals, buena, nj] or petromalt [vrx products, harbor city, ca]) at ml q - h. carefully examine all feces passed in the hospital; foreign objects or matter may sometimes be found in the stool. normal ferrets nibble on food all day. their gi transit time is short ( hours), so defecation is frequent in the healthy state. the normal stool is slightly soft and formed. diarrhea can range from mucoid and green to hemorrhagic. some owners describe a "birdseed" type of diarrhea that may be caused by malabsorption. anorexic ferrets may produce a very dark green (bile) stool that can resemble melena. unlike canine patients, diarrhea in ferrets is difficult to classify as originating in the small intestine or the large intestine. more important are the onset, duration, and severity of the diarrhea as well as concurrent clinical signs. several causes of diarrhea in ferrets are recognized. these can be separated into diseases of young or older ferrets as well as infectious or noninfectious causes. the most common noninfectious causes of diarrhea include dietary indiscretion, foreign body ingestion, trichobezoar, and inflammatory bowel disease. occasionally, severe metabolic disease can result in a green (biletinged) mucoid diarrhea. eosinophilic gastroenteritis typically affects mature ferrets but is uncommon (see table - and part ii). infectious agents are rare causes of diarrhea in closed groups or isolated ferrets, such as those kept as individual household pets. ferrets do not usually have gi parasites, but coccidia can be present in young, newly purchased ferrets. rotavirus can cause outbreaks of severe diarrhea, but most reports of this are in very young, unweaned ferrets. ferrets that have been exposed to unfamiliar ferrets, such as show ferrets, may be susceptible to ece. newly acquired young ferrets can also act as asymptomatic carriers of ece and expose naïve, older ferrets in a household group. in contrast, pbd usually affects young ferrets. canine distemper virus in the epitheliotropic form causes diarrhea in conjunction with respiratory and integumentary disease in unvaccinated ferrets. the clinical approach to the diagnosis of diarrhea depends on the severity and duration of clinical signs. obtain a vaccination and dietary history and perform a direct fecal wet mount and flotation to check for gi parasites. treat ferrets with mild diarrhea, without anorexia or vomiting, on an outpatient basis with an antibiotic such as chloramphenicol or sulfadimethoxine or with a cat hairball preparation if a trichobezoar is a possibility. sick ferrets need a more comprehensive workup that includes radiographs to check for obstructive lesions and a cbc and a plasma biochemical analysis to assess metabolic conditions. if simple diagnostic tests do not reveal a cause and therapy is unsuccessful, consider exploratory surgery to evaluate the gi tract and obtain biopsy samples. endoscopy can be difficult in ferrets because of their small size but may be an alternative to surgery. perform colonoscopy in ferrets with chronic colitis. culture the feces for c. jejuni or salmonella species, especially if the ferret is febrile or the feces are hemorrhagic. hospitalize sick or dehydrated ferrets for supportive care and a diagnostic workup. give fluids subcutaneously if a ferret is stable or intravenously if it is weak and dehydrated. administer antibiotics parenterally if possible. metronidazole, chloramphenicol, and amoxicillin are good choices for gi disease in the ferret. oral chloramphenicol does not usually cause the nausea and anorexia that it produces in some cats. a short course of a kaolin/pectin suspension ( - ml/kg po q - h prn) can be administered as a gi protectant until a more definitive diagnosis is established. drugs that affect the motility of the gi tract should never be administered without an initial diagnosis. motility-enhancing drugs such as metoclopramide and cisapride are contraindicated if a gi obstruction is present, and anti-cholinergic drugs can produce an ileus that may be difficult to interpret radiographically. judith a. bell, dvm, msc, phd h. mustelae gastritis, pbd, and eosinophilic gastroenteritis all cause diarrhea and wasting in ferrets. eosinophilic gastroenteritis has been infrequently diagnosed, and no specific causative agent has been identified. virtually all north american ferrets are likely to be exposed to h. mustelae as kits, becoming persistently infected at weaning and developing some degree of gastritis. pbd, also known as proliferative colitis or ileitis, is caused by an intracellular bacterium and is associated with chronic diarrhea and marked weight loss in ferrets. clinical or subclinical gastritis may develop concurrently with pbd and other gi or systemic diseases. other entities to be ruled out include partial gi obstruction by a foreign body, lymphosarcoma or other gastric neoplasia, aleutian mink disease, and persistent ece. h. mustelae is a gram-negative rod morphologically similar to campylobacter species that requires a microaerophilic environment for growth on artificial media. it is antigenically related and biochemically similar to h. pylori, a human pathogen associated with gastritis and ulcers. colonization of the antral area of the stomach and pyloric area of the duodenum with h. mustelae is very common in domestic ferrets, unless they are specifically treated or hand reared in isolation. , colonization is accompanied by a specific immune response, but infection persists despite high serum antibody titers. although infection is common, clinical gastritis and ulcers occur relatively infrequently. severe gastritis may be evident in gastric biopsy samples from ferrets showing no signs of clinical disease. the histopathologic lesions of h. mustelae-associated gastritis in human beings and ferrets consist of mucus depletion, gland loss and regeneration, and leukocyte infiltration. the organism can be observed in silver-stained histologic sections of gastric mucosa. affected ferrets that die usually have a single large pyloric ulcer or many small ones (fig. - ) , and the stomach and intestinal tract contain digested blood and mucus, causing the ingesta to appear very dark. cultures of h. mustelae from fecal samples are usually difficult to obtain, even when the organism is readily identified histologically in gastric biopsy samples. in humans, chronic infection with h. pylori leads to different clinical and pathologic outcomes, including chronic gastritis, peptic ulcer disease, and gastric neoplasia. the severity and distribution of the h. pylori-induced inflammation are key deter-ferrets minants of these outcomes. gastritis involving the antrum is associated with excessive acid secretion and a high risk of duodenal ulcer. gastritis involving the acid-secreting corpus region of the stomach is associated with hypochlorhydria, gastric atrophy, and increased risk of gastric cancer. as in humans infected with h. pylori, transient hypochlorhydria develops in ferrets approximately weeks after experimental infection. this condition probably facilitates fecal-oral transmission as well as recovery of h. mustelae from feces. hypochlorhydria is associated with urease production, which is detectable in gastric biopsy samples. it also correlates with the degree of colonization and the occurrence of gastritis in biopsy results. urease production is associated with the ability of h. mustelae to colonize the stomach, with non-urease-producing strains being nonpathogenic. a urease breath test is available for humans to aid in diagnosis, and a similar test has been used in ferrets under research conditions but is not practical for clinical use. h. mustelae inhibits secretion of acid by parietal cells in vitro, and this mechanism may also contribute to hypochlorhydria. in humans, chronic infection with h. pylori is associated with release of cytokines that impair function of enterochromaffin cells, which are neuroendocrine cells in the gastric mucosa that control acid secretion by releasing histamine. the impaired secretory function of these cells may predispose to hypochlorhydria and gastric carcinogenesis. gastrin is a hormone that stimulates gastric acid secretion and is secreted by the g cells of the gastric antrum. in humans and probably in ferrets, high levels of gastrin may initiate gi mucosal damage and ulceration. hypergastrinemia is probably a response to the presence of h. mustelae in the antrum or to its associated inflammation. hypergastrinemia is abolished after antibiotic therapy eradicates the helicobacter infection. in humans, helicobacter-associated gastritis has been implicated as a risk factor for gastric adenocarcinoma and gastric lymphoma. there is some evidence that this is also the case in ferrets. as h. pylori does in humans, infection with h. mustelae in ferrets apparently stimulates cell proliferation in the gastric mucosa. under research conditions, gastric adenocar-cinoma eventually developed in ferrets that were naturally infected with h. mustelae and treated with a known gastric carcinogen. spontaneously occurring gastric adenocarcinoma has been reported in pet ferrets. although h. mustelae was neither cultured from the lesions nor identified histologically in some cases, , , in others, silver-stained organisms that were morphologically compatible with h. mustelae were present in the neoplastic tissues. lymphoid follicles (mucosa-associated lymphoid tissue [malt]) are observed in the gastric mucosa of humans colonized with h. pylori and in that of ferrets colonized with h. mustelae but not in uninfected individuals. in humans this condition may progress to malt lymphoma. eradicating h. pylori usually causes early tumors to regress, implicating the infection as the cause of neoplasia. , gastric malt lymphoma associated with h. mustelae infection has also been reported in four ferrets to years of age. none of the affected ferrets were treated with antibiotics to eradicate h. mustelae either before or during the illness associated with neoplasia. illness may develop in ferrets to weeks of age under conditions of stress caused by a combination of factors, such as rapid growth, dietary changes or inadequacy, and concurrent diseases. infection is lifelong in untreated ferrets, and the severity of chronic gastritis increases with age. in mature ferrets, the disease may become clinically apparent in animals that are stressed by concurrent disease or by surgery for other conditions such as adrenal disease or insulinoma. ferrets with severe h. mustelae gastritis and ulcers are lethargic and anorexic, and they rapidly become emaciated. chronic vomiting may occur. excessive salivation and pawing at the mouth, which are signs of nausea in ferrets, may be evident. affected ferrets are often moderately to severely dehydrated and may have mild anemia. black, tarry fecal material often stains the fur of the tail and perineal region. pbd has been recognized for decades in pigs, hamsters, and ferrets. the cause in swine is a bacterium classified in a new genus and species, lawsonia intracellularis. the same agent causes pbd in hamsters and in ferrets and has more recently been implicated in proliferative enteropathies of other species, including white-tailed deer, ratite birds, and domestic horse foals. l. intracellularis is an obligate intracellular organism that cannot be propagated on artificial media. two tests that detect this organism have been developed and are used in ferret tissues under research conditions: a pcr test specific for the swine isolate, and an indirect fluorescent antibody test that identifies the omega antigen common to organisms found in pbd lesions of swine, hamsters, and ferrets. however, diagnosis of clinical cases usually depends on observing clinical signs and gross or histopathologic lesions. areas of intestine affected by pbd feel firm, appear grossly thickened, and are often discolored on the serosal surface. the colon, small intestine, or both may be involved. ridges of proliferative tissue, distinct from adjacent normal tissue, are obvious on the mucosal surface ( fig. - ) . occasionally the affected bowel perforates and causes fatal peritonitis. on histologic examination, epithelial proliferation with hypertrophy of the muscularis and infiltration of the bowel wall with either monocytic or granulocytic inflammatory cells, or both, are present. in silver-stained sections, comma-shaped organisms can be found inside enterocytes lining crypts or glands. the normal architectural pattern of the mucosa is lost. normally, straight tubular glands are covered evenly with enterocytes and numerous goblet cells. in pbd the irregular, branching, proliferative glands lack goblet cells, and necrotic debris accumulates in the crypts. severe glandular hyperplasia resembles neoplasia and may metastasize to extraintestinal sites. pbd occurs most frequently in rapidly growing juveniles, to weeks of age. environmental and nutritional stress factors appear to play a role in resistance of infected animals to clinical disease. l. intracellularis is probably transmitted by the oral-fecal route, and it may be assumed that all ferrets that are housed in groups will be equally exposed to the agent. however, clinical disease develops in only a small percentage (usually %- %) of group-housed juvenile ferrets. improvements in the quality of care and nutrition of pet ferrets may be responsible for the apparently decreasing incidence of pbd in recent years. affected ferrets have chronic diarrhea that may vary from dark, liquid feces streaked with bright red blood to scant, mucoid stool, often with bright green mucus. the fur of the tail and perineal area may be stained and wet with fecal material, and the preputial area of males is often wet with urine. rectal tissue may continuously or intermittently prolapse (fig. - ). affected animals moan or cry while straining. some continue to eat but lose weight at an alarming rate. if not appropriately treated, a ferret weighing g may lose g in less than weeks. these animals are moderately to severely dehydrated and may be hypoalbuminemic. they are weak and sleep most of the time. because of their general debility, ferrets with pbd are more susceptible to other infectious diseases. they may have upper respiratory tract infections that do not affect other healthy ferrets housed with them and often develop clinical gastritis or ulcers. severely affected animals will die if not treated appropriately, and most of those that die despite treatment have proliferative ileitis alone or in combination with colitis. eosinophilic gastroenteritis is a rare type of inflammatory bowel disease that occurs in ferrets as in other animals. in all reported cases, the ferrets were older than months of age; however, because of the small number of reports available, finding this disease in a younger animal may be possible. no specific causative agent has been found in ferrets, , dogs, or humans, but food allergy is implicated in most humans and in some dogs. in specific cases in humans and in other species, clinical signs were relieved when appropriate treatment for food allergies or parasitism was instituted. peripheral eosinophilia is a common but not a constant finding in affected dogs and humans but has been reported in most of the relatively few ferrets diagnosed with this disease. no reports of food elimination tests in affected ferrets have been published. the lesion of eosinophilic gastritis in ferrets, as in other animals and humans, is a mild to extensive infiltration of the mucosa, submucosa, and muscularis of the stomach and small intestine with eosinophils. focal eosinophilic granulomas may be found in the mesenteric lymph nodes of affected ferrets. no pathogens have been observed in or isolated from the lesions of affected ferrets. in humans and other affected species, granulomas may cause partial bowel obstruction. affected animals typically have chronic diarrhea, with or without mucus and blood, and severe weight loss. granulomas may be palpable. vomiting, anorexia, and dehydration are variable signs. signs may be clinically indistinguishable from those of gastritis, persistent ece, or gi obstruction by a foreign body. h. mustelae-associated gastritis and pbd may occur independently, sequentially, or concurrently in the same animal. pbd is a sufficient stressor to induce clinical gastritis in a ferret colonized with h. mustelae. although these two diseases are most common in ferrets to weeks of age, sufficiently stressed mature ferrets may also be affected. however, clinical disease in adult animals is more often associated with helicobacter-associated gastritis than with pbd. although eosinophilic gastroenteritis has been confirmed only in adults, it also may be occurring undiagnosed in younger animals. any of the wasting diseases can be diagnosed by gastric and intestinal biopsy. however, often a presumptive diagnosis may be based on clinical examination, an accurate history, and results of a cbc, without results from a complex or expensive laboratory workup. diagnosis may be "confirmed" by the response to appropriate treatment. characteristics of gi diseases that cause diarrhea and weight loss are summarized in table - . other important differential diagnoses for diarrhea and weight loss in domestic ferrets include lymphoma and aleutian disease. history question the owner of a lethargic, anorexic ferret with diarrhea and sudden weight loss about changes in the ferret's diet, feeding schedule, and access to water. the stress factor most commonly associated with wasting diseases is restriction of food for any reason, including the following: self-denial of food ferrets resist changing to a food that differs in flavor and texture from the one to which they are accustomed and may fast for several days rather than eat the new food. fasting depletes fat stores, which should not be confused with the loss of muscle mass associated with wasting diseases. ferrets consume about three times as much water as dry food pellets and cannot meet their nutritional requirements if water is restricted. restriction of access to food food hoppers used with some types of ferret cages may be easily blocked by large food pellets or pellets with unusual shapes, and the owner may not realize that the ferret is unable to get its food. children caring for ferrets are less likely than adults to understand the significance of an unchanging level of food in the hopper for several days. in addition, some ferrets habitually dig their food out of the container and refuse to eat food that becomes wet or contaminated on the cage floor. inappropriate, nutritionally deficient diet occasionally, new owners provide ferrets with inappropriate foods, such as dog food or poor-quality cat food, or offer them excessive amounts of treats, especially raisins, which are palatable but contain almost % sugar and no protein. rapidly growing young animals with nutritional deficiencies are much more susceptible to all infectious diseases. environmental stress exposure to extremes of temperature, particularly heat, is very stressful to ferrets. animals may be stressed during inclement weather if they are housed outdoors without adequate protection from wind and rain, especially if their food is of poor quality or subject to wetting, caking, and molding. ask the owner if the affected pet is their first ferret. you may identify stressors that the owner would not have taken into consideration. physical examination palpate the abdomen of an emaciated ferret. grossly thickened areas of gut in ferrets with pbd and eosinophilic gastroenteritis are usually palpable. a focal area of pain in the abdomen is more typical of the presence of a gi foreign body. splenomegaly is common in ferrets in association with many diseases, and mesenteric lymph nodes are likely to be enlarged in ferrets with any of the wasting diseases. projectile vomiting has been reported in one ferret with eosinophilic gastroenteritis. although rectal prolapse is not pathognomonic for pbd, it is safe to assume this diagnosis in a ferret with prolapse associated with diarrhea and weight loss. in young ferrets (younger than weeks of age and usually younger than weeks), coccidiosis may be associated with diarrhea and rectal prolapse, but coccidiosis is rarely associated with significant weight loss. both aleutian mink disease and lymphosarcoma are insidious; thin ferrets will have lost condition over a period of weeks or months and may not have diarrhea. radiography is the most useful tool for detecting a gi foreign body (see part i). contrast radiographs are sometimes helpful in identifying obstruction with a radiolucent foreign body, but radiographs may also suggest areas of gastric ulceration or intestinal mucosal proliferation in ferrets with either pbd or eosinophilic gastroenteritis. obtain a blood sample for a cbc to help eliminate the possibility of lymphosarcoma and eosinophilic gastroenteritis. ferrets with eosinophilic gastroenteritis usually have dramatic eosinophilia ( %- % eosinophils compared with %- % in normal ferrets). ferrets with lymphosarcoma may not be leukemic, and further tests, such as peripheral lymph node biopsy or a splenic aspirate, are necessary for diagnosis. inflammation associated with pbd often causes leukocytosis with neutrophilia and a left shift. ferrets with bleeding ulcers are usually anemic (normal hematocrit is %- % in spayed or neutered pets, lower in jills in estrus). dehydration may mask mild anemia and hypoproteinemia in emaciated animals; therefore repeat a cbc after rehydration. aleutian disease may cause diarrhea, anemia, leukocytosis, and wasting. serologic tests for aleutian disease virus are available (see chapter ), but many ferrets with positive test results show no clinical signs of aleutian mink disease. clinicians should not assume that any illness in ferrets serologically positive for aleutian disease virus is caused by the virus. l. intracellularis is sensitive to chloramphenicol. no other antibiotic consistently resolves pbd in ferrets. chloramphenicol is administered at a dose of mg/kg q h im or sc (chloramphenicol sodium succinate) or orally (chloramphenicol palmitate oral suspension) for at least days. a ferret with colitis of recent onset improves quickly with this treatment and gains to g/day within a few days of the first dose. l. intracellularis is also sensitive to tylosin, tetracyclines, tiamulin, and several other antimicrobials that are used to treat pbd in pigs and to erythromycin, which is commonly used in affected foals, but treatment of ferrets with any of these drugs is disappointing. treatment of infected but clinically normal -to week-old ferrets with oral tylosin ( mg/kg mixed in soft food once daily) appears to reduce the incidence of clinical pbd in a colony, but only chloramphenicol produces a dramatic improvement in sick ferrets. repair of rectal prolapse with a purse-string suture is rarely necessary because, as the colon heals, the prolapse usually disappears spontaneously. it may appear intermittently for weeks but causes no apparent distress. if a purse-string suture is used, the owner must closely monitor the ferret to make sure that it can defecate, especially when the stool regains its normal consistency. sutures should be removed in to days. chloramphenicol has no effect on h. mustelae. the original treatment for helicobacter in humans and ferrets was "triple therapy," a combination of amoxicillin, metronidazole, and bismuth subsalicylate, administered q h for at least weeks (see table - for dosages). colloidal bismuth subcitrate ( mg/kg po q h) may be substituted for bismuth subsalicylate. although h. mustelae is sensitive to either amoxicillin or metronidazole, drug resistance quickly develops unless both are given simultaneously. cimetidine ( mg/kg po q h), other h -receptor blockers, or sucralfate suspension ( - mg/kg po q h) may be helpful in very sick animals that are bleeding from extensive gastric ulcers. compounding pharmacists can prepare palat-able suspensions of many drugs available in tablet form. avoid fish-flavored suspensions when having drugs compounded because ferrets do not like this taste. when administering oral medications, scruff the ferret firmly and use a plastic dropper or dosing syringe. oral veterinary or pediatric amoxicillin suspensions are palatable and well accepted by most ferrets. other drug combinations have been used in ferrets to eradicate h. mustelae, with advantages of improved palatability and convenience of dosing (see table - ). a combination of ranitidine bismuth citrate and clarithromycin, recommended for treatment of h. pylori in humans, has been effective in treating ferrets under research conditions and has been used clinically. ranitidine bismuth citrate tablets may be crushed and mixed with a palatable liquid, and clarithromycin is available as a pediatric suspension (biaxin, abbott laboratories, north chicago, il). both drugs are administered for days (dosages given in table - ). a combination of clarithromycin, metronidazole, and omeprazole has proved more effective than the original "triple therapy" in eradicating h. mustelae in research ferrets. resistance to clarithromycin has not yet been reported in ferrets but does occur in humans. to prevent development of macrolide-resistant strains, clarithromycin should be combined with a second antibiotic not in the macrolide class. although eradication of h. mustelae is accompanied by decreasing antibody titers, lesions may take longer to resolve. ferrets from which the infection has been eradicated by antibiotic therapy may be reinfected with h. mustelae through contact with infected ferrets. for treated ferrets to remain free of h. mustelae, they should not be exposed to ferrets of unknown helicobacter status until the new ferrets have also been treated. humans, dogs, and cats with eosinophilic gastroenteritis usually respond to steroid treatment. because the disease in ferrets resembles that in other species, prednisone administration has been the treatment of choice. remission has occurred in ferrets treated with prednisone ( . - . mg/kg po q h for days and q h thereafter) until the ferret is clinically normal. immediate recovery also followed removal of an enlarged mesenteric lymph node in one ferret and treatment with ivermectin ( . mg/kg sc) in another. when eosinophilic gastroenteritis is a response to the presence of a parasite, eliminating the parasite is preferable to prolonged treatment with corticosteroids to relieve clinical signs. rehydrate sick ferrets with either intravenously (preferred) or subcutaneously administered balanced electrolyte solutions. these animals are often very weak and may allow a saphenous or cephalic catheter to be inserted with little resistance. among the advantages of giving intravenous fluids is that glucose can be administered by this route. however, if a catheter cannot be placed, fluids administered subcutaneously are well and rapidly absorbed. hospitalize an emaciated, dehydrated ferret until fluid and electrolyte balances are reestablished. when the life-threatening episode is over, the owner will probably be willing and able to give the supportive care needed to restore the ferret to health. while waiting for results of diagnostic tests, one can assume that most cachectic ferrets with diarrhea that do not have a gi foreign body or eosinophilia have both ileitis/colitis and h. mustelae gastritis with ulcers. if the ferret is treated for only one disease when it has both, the time required for recognizing treatment failure may be the factor that ultimately decides if the ferret will survive. unfortunately, the effective drugs used for treatment of the two diseases are different, and therapy necessitates multiple daily doses of several drugs for at least weeks. because the gut flora of ferrets is very simple and plays no vital role in digestion, long-term administration of broadspectrum antibiotics does not cause diarrhea in ferrets as it does in most animals. emaciated animals that die despite proper treatment usually have either very extensive gastric ulcers or severe ileitis, each of which drastically reduces the absorption of essential nutrients. age of the animal may help in the differential diagnosis; young ferrets are more likely to develop pbd, whereas older ferrets can develop severe, chronic gastritis associated with h. mustelae infection. also, h. mustelae-associated disease is more often associated with stress factors common in mature pet ferrets, such as concurrent disease or surgery (see table - ). emaciated animals have no energy reserve and should receive intensive care. offer a smorgasbord of premium cat foods and ferret diets. some animals refuse to eat their regular diet of dry pellets but do accept the same food mixed with water and heated in a microwave until it develops a porridge-like consistency. some ferrets eat meat baby foods, especially liver and chicken, and most drink milk. milk causes loose stool in normal ferrets and in those with colitis, but it is very palatable. if milk fat is increased to % to % with cream, it appears to be absorbed well and contributes to weight gain even though diarrhea continues. most sick ferrets accept nutri-cal (tomlyn, buena, nj) at numerous times during the day. many ferrets like the human food supplements such as ensure plus (abbott laboratories, ross products division, columbus, oh), boost plus (formerly sustacal; bristol myers, evansville, in), or isocal hn (mead johnston, evansville, in), and they sometimes eat softened pellets mixed with one of these products when they refuse pellets alone. alternatively, offer nutritional recovery foods such as maximum-calorie (the iams company, dayton, oh) or canine a/d (hill's pet nutrition); most sick ferrets accept these foods readily. nutritional recovery diets such as max-cal can be used as the sole source of nutrition for weeks at a time if necessary. when using these diets, calculate a minimum daily intake of kcal per kilogram of body weight. sick ferrets may not make the effort to get up and drink from a water bottle but do usually drink from a dish. offer fresh food and water by hand several times daily during hospitalization and home care; ferrets often take a few mouthfuls of every new offering but never go back for more. the first days are critical for an animal that has lost % to % of its body weight, and intensive supportive care is essential. when ferrets with pbd or ulcers regain their appetites, often within hours of the first doses of medication, and diarrhea has resolved significantly, owners may be tempted to stop treatment. however, ferrets treated for less than weeks often relapse, and some ferrets need antibiotic therapy for an additional to weeks if they are to recover completely. ferrets with eosinophilic gastroenteritis that do not respond to ivermectin require longer periods of monitoring and prednisone therapy. most ferrets with these wasting diseases can be saved with aggressive and persistent treatment. eosinophilic gastroenteritis prednisone . - . mg/kg q h po ivermectin . mg/kg sc, po once ranitidine bismuth citrate, azathioprine, and metronidazole can be prepared as suspensions by compounding pharmacists. *treat for a minimum of days relevance of the ferret model of helicobacter-induced gastritis to evaluation of antibacterial therapies natural and experimental helicobacter mustelae reinfection following successful antimicrobial eradication ferrets salivary mucocele in ferrets evaluation of campylobacter jejuni colonization of the domestic ferret intestine as a model of proliferative colitis biology and diseases of ferrets megaesophagus in nine ferrets enteric coccidiosis in a ferret a chronic granulomatous intestinal disease in ferrets caused by an acid-fast organism morphologically similar to mycobacterium paratuberculosis clinical aspects of inflammatory bowel disease in ferrets medical and surgical management of esophageal foreign body in a ferret diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a lawsonia intracellularis-specific multiplex pcr assay helicobacter mustelae-associated gastric malt lymphoma in ferrets eosinophilic gastroenteritis in a domestic ferret cisplatin-induced emesis in the ferret: a new animal model bacterial and mycoplasmal diseases systemic diseases. in fox jg, ed. biology and diseases of the ferret. philadelphia, lea & febiger bacterial and mycoplasmal diseases: helicobacter mustelae bacterial and mycoplasmal diseases: proliferative bowel disease-desulfovibrio spp. (lawsonia intracellularis) ferret as a potential reservoir for human campylobacteriosis campylobacter jejuni infection in the ferret: an animal model of human campylobacteriosis helicobacter mustelaeassociated gastritis in ferrets. an animal model of helicobacter pylori gastritis in humans helicobacter mustelaeassociated gastric adenocarcinoma in ferrets (mustela putorius furo) intracellular campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a desulfovibrio sp helicobacter mustelae infection in ferrets: pathogenesis, epizootiology, diagnosis, and treatment proliferative colitis in ferrets helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric helicobacter copper toxicosis in sibling ferrets hepatobiliary inflammation, neoplasia, and argyrophilic bacteria in a ferret colony neoplasia in ferrets measurement of dietary and dentifrice effects upon calculus accumulation rates in the domestic ferret megaesophagus in a domestic ferret increased gastric epithelial cell proliferation in helicobacter pylori associated follicular gastritis a rapid procedure for filling fractured canine teeth of ferrets equine proliferative enteropathy: a cause of weight loss, colic, diarrhea and hypoproteinaemia in foals on three breeding farms in canada proven or potential zoonotic diseases of ferrets ranitidine bismuth citrate and clarithromycin, alone or in combination, for eradication of helicobacter mustelae in ferrets development of a curea breath test in ferrets colonised with helicobacter mustelae: effects of treatment with bismuth, antibiotics, and urease inhibitors antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy zygomatic salivary gland mucocele in a ferret gastrointestinal foreign body in ferrets: cases molecular and cellular mechanisms involved in helicobacter pylori-induced inflammation and oxidative stress eradication of helicobacter mustelae from the ferret stomach: an animal model of helicobacter (campylobacter) pylori chemotherapy kirk's current veterinary therapy xi: small animal practice gross and microscopic anatomy of the major salivary glands of the ferret physiology of gastric enterochromaffin-like cells cryptosporidiosis in ferrets pyloric adenocarcinoma in a ferret gastroenteritis associated with clostridium perfringens type a in black-footed ferrets (mustela nigripes) granulomatous enteritis caused by mycobacterium avium in a ferret diseases of the intestines: eosinophilic gastroenteritis two cases of pyloric adenocarcinoma in the ferret (mustela putorius furo) gastric anti-secretory, mucosal protective, anti-pepsin and anti-helicobacter properties of ranitidine bismuth citrate multiple tumors in a ferret eosinophilic gastroenteritis: a clinicopathological study of patients with disease of the mucosa, muscle layer, and subserosal tissues cisapride: clinical experience with the newest gi prokinetic drug a: isolation of an atypical rotavirus causing diarrhea in neonatal ferrets coronavirus-associated epizootic catarrhal enteritis in ferrets a critical review of the effect of helicobacter pylori eradication on gastric malt lymphoma gastric lymphoma of mucosa-associated lymphoid tissue and helicobacter pylori key: cord- - pj fmv authors: perdue, michael l.; seal, bruce s. title: impact of avian viruses date: - - journal: viral ecology doi: . /b - - / - sha: doc_id: cord_uid: pj fmv nan in considering the ecology of the avian viruses and their impact on life on earth, it may be useful to first consider the host itself. the class aves first diverged from the reptiles between and million years ago, depending on which current paleontological interpretations one accepts. of the vertebrate classes, they are most often compared with the reptiles from which they evolved and with mammals because of their common warm-blooded nature. this shared feature with mammals is probably the most influential with respect to ecology, since a virus adapted to warm-blooded physiology would not fare well in the cold-blooded world, and vice versa. along these lines, our recent experience is that some virologists think that a bird is a bird, and that if a given virus replicates in one, it will replicate in them all. this is, of course, far from the truth and perhaps should be a starting point for discussing avian virus ecology. according to fossil records, the class aves emerged from the extinctions of the late cretaceous period , , years ago, somewhat bottlenecked, as did the class mammalia, but since that time they have undergone parallel evolution with mammals and are equally diverse in their own right. certainly some viruses, such as avian paramyxovirus (of which newcastle disease virus is the prototype), are infectious for numerous orders of birds. there are also other important avian virus strains such as the gallid herpesvirus (infectious laryngotracheitis) and the avihepadnaviridae, which appear to be exclusively confined to a single bird family or even genus. it is clear that birds, because of their close association with powered flight, do present a more homogeneous anatomy and physiology than do the class mammalia (feduccia, ) . whether this affects or specifies the molecular nature of viruses that infect birds is not really known. this feature, however, almost certainly uniquely impacts the natural distribution and ecology of the viruses that inhabit the flying birds. a virus infecting or persisting in an arctic tern could potentially be translocated up to , miles in a few weeks. viruses such as some avian orthomyxovirus or avian paramyxovirus strains, which may exhibit subclinical infections, might be shared among a migrating flock of waterfowl and persist for indefinite periods as the flocks move from lake to lake. along the way the virus might be shared with other birds crossing flight paths. so birds do present unique environments for transmission of viruses. the commercial practices of humans have further provided unique opportunities for transmission not normally seen for birds. the order galliformes in particular, which includes domestic and wild fowl; pheasants, quail, and turkeys, has been unquestionably affected. it would be quite safe to say that humans have both determined and upset the ecological balance among members of this order and the viruses that affect them. due to continuous breeding practices, live-virus vaccination regimes, and by housing tens of thousands of birds in a single enclosure, situations never encountered in natural settings are created. whether this has affected the virus-host ecological balance in other orders of undomesticated birds as well is not known; but it would seem highly likely. one of the most important features of a host-parasite relationship, of course, is the host's immune defense. while there are similarities shared with respect to the immune system, particularly the dual (humoral and cell-mediated) nature, there are marked differences between cold-and warm-blooded vertebrates and additional significant differences between birds and mammals (eerola et al., ) . the discovery of processing and maturation of immunoglobulin-producing lymphocytes was made as a result of characterization of an avian-specific organ, the bursa of fabricius (ratcliffe, ) . although functional equivalents exist in mammals, the bursa is a distinct and wholly avian-specific organ. since several viruses are known to affect this organ specifically, it should be considered a unique ecological niche. a second significant difference in the immune system of birds is in the apparent genetic content responsible for specifying the avian major histocompatibility complex (mhc). the chicken mhc appears considerably more simple (providing the oxymoron: a simpler complex) than the mammalian mhc (kaufman and wallny, ) . several alleles have arisen and considerable recombination documented in the mammalian mhcs that thus far have been studied. these are responsible for producing a great variety of class i-, ii-, and iii-type proteins used in recognition and presentation of antigen. in the chicken, some mhc haplotypes produce only one type of class i protein and there is no evidence for recombination at all (kaufman and wallny, ) . this has led to speculation that the relationship with the avian pathogens has evolved significantly differently from the mammals. one result of this difference may be the occurrence and frequency of either resistance or sensitivity to specific viral infections encountered in chickens (see below). thus, the class ayes presents several unique features that might ultimately affect the ecology of viruses that infect them. the classification of birds has presented a significant challenge to systematists. the number of species has actually decreased over the years because of the clearer genetic relationships that have emerged. conversely, additional species have been defined as recognition of convergent evolution has become clearer. currently, new species are identified at a rate of about two per year. while there remains some disagreement among specialists, most accept the current classification of orders, families, genera, and + species (gill, ). molecular analysis of avian genes for phylogenetic studies is still in its early stages. restriction fragment length polymorphism (rflp) analysis and sequence analysis of s mitochondria dna has yielded some molecular phylogenetic information but not enough to gain any insight regarding the viruses of birds (hedges et al., ; cooper and penny, ; mindell et al., ) . roughly speaking, the flightless orders represented by ostriches, rheas, cassowaries, and kiwis are thought to be the most ancient, while the bewildering array of members of the order passeriformes (representing % of known species and % of known families) are thought to be the most recent (gill, ) . trying to determine how long viruses have been associated with various avian groups is of course as impossible as it is with virus-host relationships in any other classes. orthomyxoviruses, paramyxoviruses, and coronaviruses have recently been isolated from ostriches, rheas, and emus, indicating no absolute barriers in these more ancient birds. the class aves is of course distributed worldwide. in addition, there are several hundred species that migrate, sometimes in spectacular fashion. biogeographers have divided the earth into six distinct faunal regions, which correspond somewhat with the major continental areas (welty, ) . these include the nearctic (north america and greenland), palearctic (asia, europe, and north africa), ethiopian (central and southern africa), oriental (india, southeast asia), australasian (australia, new guinea), and neotropical (south and central america). each area contains its own characteristic birds. in the northern hemisphere (nearctic and palearctic), most species are migratory, which is not the case in the other areas. the neotropical has the richest and most abundant bird life, and the southern hemisphere has by far the most families represented, as well as the most families peculiar to a given region. each year, billions of land birds and waterfowl in north america and asia head south to south america and africa, respectively, carrying their viruses with them. the size and scale of these geographic relocations are unmatched by any other land vertebrates. the sea mammals are the only other comparable migrating vertebrates, and they surely cross paths with the birds. in the most interesting putative contacts, purely avian-origin type a orthomyxovirus of at least two different subtypes were isolated from dead and dying seals off the coast of new england in (webster et al., b hinshaw et al., ) . this represents viral ecology at its most forceful, being effected between two warm-blooded vertebrate orders during their natural migration and geographic interaction. if one takes a broad look at the virus families associated with avian hosts (table i) , it is actually easier to list the families of viruses that infect vertebrates but that do not yet have a clear avian member. these are the iridoviridae, arenoviridae, african swine fever-like viruses, and filoviridae. a similar comparison of families that do not contain a mammalian member yields only the birnaviridae. so, in one sense, the mammalian host might be considered more virus-friendly, rather than the alternative. still, many investigators think of bird tissues, in particular the embryo, as being an ideal medium for identifying new viruses. if just from an experimental and practical, rather than a natural, point of view, the avian host has played a tremendous role in our understanding of viral ecology. many of the most important findings in virology have been made utilizing the chicken embryo and in chicken cell cultures. the egg also continues to provide an abundant and important substrate for the production of veterinary and human vaccines. if we think of ecology as the study of organisms and their relationship with their environment to include all other organisms, we most certainly encounter a unique relationship with viruses. unlike higher pathogens, viruses really do not mate, communicate, or colonize (except in the very broadest stretches of the imagination); they simply parasitize and replicate. "we try and make them more familiar by defining higher organism-type genetic alterations as "evolution," when in truth the viruses may simply be adapting to the evolutionary pressures encountered by, or within, its host. obviously, the host is central to the viruses' lifestyle and must be considered a major part of virus ecology. in this sense, information gathered on various viruses as they replicate in embryos or embryo cells, in the absence of immune pressure, should only be considered "natural" for those viruses that are transmitted vertically. the embryo or cell culture then can only provide a window on the natural ecology of the organism in which the immune system is not a factor. in ecological terms, only the natural state of the virus-host relationship becomes important. viruses being parasites, these relationships more often than not eventually result in a disease state. it is not possible to distinguish the molecular biology of avian viruses from that of non-avian viruses on the basis of features unique to the avian system. however, there are some important distinctions with which viruses must deal. one dramatic difference between birds and mammals is body temperature. on average, birds operate at -- ~ higher than mammals, and most can readily regulate their temperature, some dropping body temperature as much as ~ during the night (gill, ) . while this certainly could influence such things as rates of viral enzyme activity, polymerase fidelity, or protein stability, there are no situations documented in which a particular virus group is restricted to class aves solely on the basis of body temperature. it is possible to distinguish some important receptor-specific distinctions unique to avian systems. one example is found in the influenza type a viruses. the glycosidic linkages associated with sialic acid residues on the avian versus mammalian cell surface serves to restrict the strains of viruses able to replicate. influenza virus hemagglutinin (ha) proteins bind to sialic acid residues on the surface of the host cell. although there is no absolute barrier, those viruses that replicate well in avian cells have a receptor binding pocket on the surface of the ha that has a preference for the o~- , siallyl-sugar linkages abundant in bird tissues. those viruses that replicate well in mammalian cells have a preference for ~- , linkages more prevalent in mammalian cells (rogers and paulson, ; murphy and webster, ) . there are probably many more receptor specificities associated with other avian virus infections, particularly among the herpesviruses, where host range is narrowly dictated. another example of virus-specified tissue tropism unique to birds would be infectious bursal disease virus (ibdv; genus avibirnavirus), which has "evolved" an affinity for the bursa, a bird-specific organ. the other two known genera of birnaviridae infect fish and invertebrates. since there are as yet no mammalian members, it is tempting to speculate that ibdv is bird specific because of its evolved affinity for the bursa. air sacs are also uniquely avian structures. the bird lung is a fixed tissue incapable of expansion like mammalian lungs. the air sacs are a series of extensions of the respiratory system that expand with the musculature of the body cavity and allow the large-scale rapid oxygen transfer needed for powered flight. the air sac system is extensive throughout the bird, even encroaching into bone tissue; some birds have as many as nine distinct air sacs. many viruses replicate and cause disease in this unique organ system, and there are examples of apparent preferences by some viruses for air sac tissue over other respiratory tissue. finally, feathers, the most notable and prominent distinguishing feature of the class aves, provide a unique niche for at least two three viruses-avipox, marek's disease, and psittacine beak-and-feather disease virus --which can replicate in and are spread from feather follicles (biggs, ; tripathy and reed, ) . as mentioned previously, the immune system of the intact bird is a critical feature in the establishment of ecological relationships between many viruses and hosts. as the immune system plays a major role in the relationships, they will be covered, but there are some extraordinary examples that deserve their own mention. marek's disease, caused by an alphaherpesvirus, is most commonly associated with lymphomas developing relatively early in the life of a chicken. the initial infection in md is a respiratory infection probably initiated in macrophages. a typical field infection would then progress through the following phases in a bird: latency is established primarily in lymphoid cells, and mostly in activated t lymphocytes. in susceptible birds, the latent state progresses to a second round of lytic infections at multiple sites in the bird. interestingly, at this point the feather follicle epithelium is the only site where complete virus replication occurs and becomes a significant source of environmental infectious virus (calnek and witter, ) . concomitant and permanent immunosuppression occurs in the affected bird. the disease then progresses into a lymphoproliferative phase in susceptible birds that can range in severity depending on the virus strain and breed of chickens. some breeds have shown natural resistance to this progression, and, although the mechanism of resistance is not completely understood, it involves primarily lymphoid tissue and is dictated mostly by genes involved in the immune response (venugopal and payne, ) . it should be clear from this scenario that the immune system in this host-parasite relationship plays a critical role in the ecology of marek's disease virus. infectious bursal disease virus, as mentioned earlier, is a virus that replicates exclusively in lymphoid tissue. the virus can be detected replicating in the bursa of fabricius and within circulating lymphocytes as early as hours after infection of a chicken. it causes acute degeneration of various lymphoid tissues within the first day of infection and results in a severe, albeit age-dependent, depression in the humoral immune response, being most dramatic in very young birds. interestingly, the infection does not suppress b-cell responses to the viral antigens themselves; in fact, there is stimulation of proliferation of b cells committed to anti-ibdv antibody production (mcferran, ) . one may only speculate as to what role, if any, this may play in the viral ecology or replicative cycle, but the immune system is once again a major participant in this host-parasite relationship. finally, the effects of vaccination programs on the ecology of avian viruses cannot be overemphasized. in one sense, we have an ongoing experiment where humans control the type of viruses to which certain species of birds are exposed. since live viruses generally yield much better immune responses, they are employed most often. in the case of the single-stranded rna viruses, noted for their ability to rapidly mutate and avoid the immune system, this has the effect of artificially challenging the immune system, creating selection pressure between host and parasite that would not normally occur. this may be effective in the short run, protecting against disease, but the long-term effects are unknown. table i are some of the most important features of relationships of various viruses with their avian hosts as well as the wide variety of relationships that exist. it is not feasible to list all the interesting attributes for each virus-host relationship, but the table presents a variety of relationships that will be covered in more detail in the following sections. as such, the table should not be taken as the final word on each member virus. for example, in the case of turkey meningoencephalitis virus, a flavivirus, reduced egg production is listed under pathogenesis. reduction in egg production is a common feature in many virus infections of poultry, and while listing it under pathogenesis somewhat stretches the meaning of that word, this clinical sign is one of the most important aspects of that disease in turkeys. although there is variation in the economic or ecological impact of various viral groups from year to year and among geographic sites, the "top ten" list of virus groups exhibiting routine significant impact on commercial poultry worldwide (not necessarily in order of impact) are paramyxoviruses (newcastle disease); coronaviruses (infectious bronchitis); herpesviruses (infectious laryngotracheitis; marek's disease; duck enteritis); reoviruses (viral arthritis); picornaviruses (avian encephalomyelitis); adenoviruses (egg drop syndrome); retroviruses (lymphoid leukosis); orthomyxoviruses (avian influenza); poxviruses (fowlpox); and birnaviruses (infectious bursal disease). the circoviruses (chick anemia) could likely be included in the above list, except it is not yet known to what extent the viral infection alone influences morbidity and mortality (see below). what may not be obvious from table i is that if one searches for viruses in a given avian species one will likely find them. in some of the virus families listed, investigators were forced to clearly separate the disease-causing virus in question from accompanying "contaminant" viruses, which may or may not have influenced the original disease manifestation. there appear to be many viruses of birds that in certain ecological conditions and in certain species may be considered "normal flora" and are not associated with disease. these, obviously, are less interesting to any funding agencies and consequently do not receive much research attention. no one really knows to what extent their transmission and persistence in avian populations affect their own ecology or that of their hosts. the office international des epizooties (oie), the principal world organization for animal health, provides listings of the most serious infectious diseases of animals (oie, ) and divides them into two groups: list a diseases, which "includes those diseases that spread rapidly, the scope of which extends beyond national borders" and "have particularly serious socioeconomic or public health consequences"; and list b diseases, which include those "that are considered to be of socioeconomic and/or public health importance within countries." of the avian viral diseases listed above, only highly pathogenic avian influenza and velogenic newcastle disease are in list a; marek's disease, infectious bursal disease, infectious bronchitis, duck enteritis, and infectious laryngotracheitis are in list b. the only avian disease in oie's list and not on our top list is duck hepatitis, which is a complex of diseases caused by at least three virus families, and generally limited to country-specific origins. the ecological impact of viruses of birds ultimately interests us as homo sapiens, perhaps only to the extent that we are affected. in this respect, there is no question that the major impact thus far has been on raising birds as a food source for our species. ecologically speaking, this impact could have very significant consequences when one considers that poultry provide the most widely used protein source in the world. thus, we will consider for the most part how viruses affect this food source. the most significant and widespread infections of wild birds will be discussed as they are encountered relative to commercial and domestic birds. the most imminent and significant human public health concerns with regard to bird viruses appear to be twofold: the potential relationships with type a orthomyxoviruses that have become at least partially adapted in a totally nonpathogenic state to some avian orders. there is compelling evidence that these viruses may also replicate in pigs and re-assort with pig and/or human strains of influ-enza, yielding new variants capable of replicating and causing disease in humans. they also find their way into commercial poultry, sometimes with devastating consequences. with the recent documented transmission of a lethal avian influenza virus from commercial poultry to humans, these ecological relationships take on new significance. . the presence of a large reservoir of arboviruses in wild birds, some of which, when transmitted by invertebrate vectors to mammals, cause disease. beyond these two examples, other relationships are less directly important to human public health. other avian-origin viruses are capable of replicating in and sometimes causing mild disease in humans, but there is obviously not room in one chapter to cover each in detail. infectious laryngotracheitis (ilt) is a respiratory disease almost exclusively of chickens. infections in turkeys and pheasants have been reported, but surveys have yielded no wild bird reservoir or other domestic poultry reservoir (cranshaw and boycott, ) . based on this and the knowledge that ilt apparently exhibits little antigenic heterogeneity, it has been proposed that through proper husbandry practices and appropriate vaccination techniques the disease could be eliminated from commercial poultry (bagust and johnson, ) . the virus is a member of the alphaherpesvirinae subfamily and is identified taxonomically as gallid herpesvirus i. the disease is almost exclusively respiratory, with no systemic involvement. the severity of disease can vary from significant mortality ( %) in young birds to an inapparent infection of adult birds. there are age-dependent effects on the pathognomonic signs, and there do appear to be strain-specific virulence differences. however, different isolates do not exhibit sufficient genetic heterogeneity thus far to identify specific virulence factors (bagust and guy, ) . the most interesting aspect of ilt is its capacity for persistence in infected birds and flocks showing no disease signs. this persistence is most likely due to establishment of the latent state and recrudescence. numerous studies have demonstrated re-isolation of virus many months after initial infection, and one more recent study demonstrated reactivation of latent virus due to stress factors (hughes et al., ) . this latency achieved by herpesviruses could certainly be considered a unique ecological state, and most herpesvirus infections in birds are associated with its establishment. duck plague, also known as duck viral enteritis (dve), is caused by an alphaherpesvirus classified as anatid herpesvirus- , which infects free-living and domestic ducks, geese, and swans (sandhu and leibovitz, ) . the disease is acute and often associated with high morbidity and mortality. the virus has a worldwide distribution and has caused numerous documented outbreaks in free-living anatids. the first documented north american outbreak was in commercial ducks on long island, new york in (leibovitz and hwang, ) , and since that time sporadic reappearance of the virus in commercial and wild populations has occurred. major outbreaks in free-living birds along the mississippi flyway and a large epornitic in south dakota in killed tens of thousands of wild ducks and geese (brand, ) . vertical transmission and recrudescence of latent virus has been established experimentally in mallard ducks but has not been demonstrated in wild waterfowl. species susceptibility may vary among various waterfowl, although more than species have been shown to be naturally or experimentally infected and virulence differences among dve strains have been demonstrated. the other notable avian herpesvirus infection occurs in pigeons. the virus is taxonomically designated as columbid herpesvirus i and is antigenically indistinguishable from natural isolates taken from wild falcons and owls (vindevogel and duchatel, ) . the causative virus is antigenically distinct from ilt, mdv, herpesvirus of turkeys (hvt), and the anatid herpesvirus i. the disease associated with infection by columbid herpesvirus i is a major cause of growth retardation and bad performance in homing pigeons, though mortality is generally low. this virus, like ilt, becomes latent, reappears, and is shed in asymptomatic birds and flocks. other antigenically distinct herpesviruses have also been isolated from cormorants, quail, and storks. kaleta ( ) has proposed the division of these various herpesviruses into eight antigenic groups. the host specificity for each group varies, but in general they appear to be strictly adapted to the host of origin (as exemplified by the gallid herpesvirus i). gallid herpesvirus ii and hvt will be discussed in the following section. the impact of transmissible neoplastic diseases of poultry has been quite variable over the years. prior to vaccination, losses to marek's disease were often devastating, and even in marketable flocks condemnations due to lymphomas at processing plants could reach %. lymphomas caused by mdv and retroviruses are still the most common viral neoplastic diseases of poultry, and a recent increase in mortality and evolution of more virulent mdv strains indicates that the impact of these viruses will continue to be felt (witter, ) . marek's disease is caused by a herpesvirus that has two very similar relatives: a second nononcogenic serotype and the herpesvirus of turkeys (hvt). these are sometimes classified as serotypes - , respectively, of the gallid herpesvirus ii strain. they share common antigens that distinguish them from the nononcogenic herpesviruses but can be distinguished on the basis of antigenic differences (calnek and witter, ) . only the oncogenic mdvs (serotype ) cause significant problems in commercial poultry. the herpesvirus of turkeys is ubiquitous among commercial flocks and quite prevalent in wild turkeys but has not been directly associated with disease. the serotype strains were originally thought to be nononcogenic apathogenic mdv isolates until they were serologically distinguished from the original isolates. the type serotypes are associated with subclinical infections in chickens, although not as prevalent as turkey strains. the hvt isolate is a very interesting and important isolate, as it was used (and is still used) in vaccine formulations against marek's disease in chicken flocks. it has been very effective at protecting flocks against lymphomas and thus correctly billed as the first anticancer vaccine. marek's disease is relatively well controlled by using mixtures of primarily serotype and isolates in vaccine formulations. however, this vaccination program may ultimately extract a price. shown in figure is a diagrammatic representation of the evolution of virulence in mdv strains associated with changing vaccine formulations. the extent to which these formulations have influenced the evolution of virulence is not proven, but certainly the association is undeniable. the s have brought an increase in incidence of marek's disease cases, and the presence of these acutely virulent strains raises concerns for the future. what is clear is the role of humans in the generation of these strains. there is evidence that the commercial housing practices developed in the s and s resulted in generation of strains that were oncogenic and that this rapid evolution of virulence in the last years is probably due to human control of commercial bird populations. the avian retroviruses have one of the most interesting histories of all of the avian viruses. the first transmissible lymphomas were demonstrated in by ellermann and bang ( ) and the first cell-free transmissible solid tumor by peyton rous three years later (rous, ) . the etiologic agents of both of these diseases were later shown to be members of what is now known as the avian leukosis virus-avian sarcoma virus complex (alv-asv) of related retroviruses. scientists investigating this interesting group of viruses have garnered more nobel prizes (six) than with any other group. because of their relative simplicity in genetic content and their close association with the genetic character of their host, they have provided a bountiful model for the study of oncogenesis. these are rna-containing viruses that replicate via an intermediate dna stage that is most often incorporated into the host genome. the integrated viral genomes serve as templates for production of new progeny genomic rna molecules and the mrnas needed to make new viral proteins (coffin, ) . in the process, these viruses transform the host cell into a tumor cell. there is a significant array of viral subtypes and relationships that exist between the host and avian retroviruses and numerous reviews that may be consulted (crittenden, ; swanstrom and vogt, ) . there are several viral subgroups, based on antigenic differences alone, in the surface envelope glycoproteins. viral subtypes have also been grouped on the basis of whether the viruses rapidly induce neoplasia because of the presence of a viral oncogene, or whether they induce slow development of tumors due to their integration into the host genome and subsequent activation of cellular oncogenes. in commercial poultry, the predominant problems are caused by the latter, the slow-inducing lymphoid leukosis viruses (llvs). the sarcoma-, myeloblastosis-, and erythroblastosis-inducing strains cause only sporadic problems. additionally, it has been calculated that losses due to llv because of poultry performance decreases are actually greater than those due to the lymphomas. still, these viruses continue to cause condemnations at slaughter and must be distinguished from the more rapidly forming mdv-induced lymphomas. two other oncogenic avian retroviruses pose significant problems and possess much wider host ranges than the llv-sarcoma complex. the relatively new subgroup "j" has been characterized in europe as causing significant myelocytoma and endothelioma. in contrast to the llvs, subgroup j viruses have a high tropism for cells of the myelomonocytic series but a very low tropism for bursal cells (arshad et al., ) . a related subgroup j virus has been identified in broiler-breeder flocks in the united states as well . additionally, of note are those retroviruses of the reticuloendotheliosis virus (rev) group. infection with members of this group only rarely results in disease, and then disease most commonly occurs only in the hosts from which rev was first isolated: turkeys. still the rev group is predicted to have significant potential for causing problems in poultry (witter and johnson, ) , and the wide host range of the virus group has prompted speculation regarding potential as a public health risk. there are, in fact, data demonstrating antibodies to the virus in human and other mammalian sera . coronaviruses contain a large positive-sense rna genome of approximately kb. members of this virus family infect both mammals and birds (cavanagh et al., ) . infectious bronchitis, caused by a coronavirus, infectious bronchitis virus (ibv), is one of the major poultry viral diseases of worldwide economic importance. it is also one of the most rapidly spreading avian respiratory diseases known (mcmartin, ) . the virus is antigenically variable owing to a high mutation rate in the surface glycoprotein s gene. new antigenic variants of ibv continue to be isolated from various geographic regions. thus far, it has not been possible to determine the number of distinct ibv subtypes, but there are at least six (siddell, ) . strains can differ in their virulence and tissue tropism, but in general the disease is a rapid-onset respiratory distress that can cause significant mortality in young birds. in established flocks, the infection is often associated with growth retardation and reduction in egg production that may be exacerbated by other respiratory pathogens. estimates of a to % loss in market value have been made for a typical outbreak in a flock. vaccination is employed to control the disease, and an interesting related feature is the demonstration that vaccine strains can undergo recombination with wild-type strains. consequently, new ibv antigenic variants of the s gene emerge with characteristics of both viruses (cavanagh et al., ; wang et al., ) . ibv is also an excellent example of a virus exquisitely adapted to its host, as it is not found naturally in any other reservoir. chickens seem to be the only reservoir, and the virus is capable of persisting in some manner within populations and later being transmitted to naive flocks. evidence suggests the mode is primarily airborne transmission. in controlled studies, birds in houses feet away from an experimentally "seeded" house were infected via aerosol transmission (mcmartin, ) , and circumstantial data from natural outbreaks suggest transmission over distances of several hundred yards (cumming, ) . coronaviral enteritis of turkeys, also known as bluecomb disease, may also be species specific since chickens, pheasants, and quail do not exhibit any disease following inoculation with a strain of virus that is virulent for turkeys (hofstad et al., ; larsen, ) . coronaviruses have been isolated from ratites with enteric disease in zoological collections, but the relationship of these isolates with the turkey coronavirus (tcv) has not been investigated (frank and carpenter, ; kennedy and brenneman, ) . transmission of tcv appears to be restricted to the fecal-oral route, although cell-free lysates of the bursa from infected birds can be used to transmit the agent orally. among turkey flocks, the primary mode of transmission of coronaviral enteritis is via contaminated personnel and equipment. however, since tcv is excreted in fecal material and is very stable, it is conceivable that wild birds may serve as vectors for the agent. morbidity is usually as high as %, with mortality becoming as high as % in an infected turkey flock. mortality among poults may be much higher with increased numbers of secondary bacterial gastrointestinal infections contributing to severity of disease. turkey coronavirus also shares features reported for mammalian hemagglutinating coronaviruses and is closely related by sequence and antigenic crossreactivity to bovine coronavirus and a human coronavirus isolate (verbeek and tijssen, ) . this close relationship suggests recent interspecies transmission of the coronaviruses. while no etiologic agent has been pinpointed, a bovine origin coronavirus has been implicated in an emerging enteric disease of turkeys called poult enteritis mortality syndrome (pems) (barnes and guy, ) . the arthropod-bome viruses (arboviruses) present a unique challenge to evaluating ecological virus-host relationships. this grouping includes members from several virus families, six of which have members isolated from birds: togaviridae, flaviviridae, reoviridae, arenoviridae, bunyaviridae, and rhabdoviridae. of these, only the togaviridae and flaviviridae have strains that have caused documented disease in commercial poultry and game birds. although difficult to assess in feral populations, isolates from the remaining virus families as well as several isolates from the togaviridae and flaviviridae are not associated with any pathology in birds. of the togaviridae, the genus alphavirus contains the encephalitic strains causing eastern equine encephalitis (eee), western equine encephalitis (wee), and a closely related wild bird isolate --the highlands j virus (hjv). pheasants have been the primary targets of significant outbreaks of eee. signs of infection are primarily but not exclusively neural, and mortality rates have reached % in some natural outbreaks eleazer and hill, ) . economically significant outbreaks of disease due to eee virus in turkeys have occurred in wisconsin, with severity of the disease decreasing with increasing bird age. significant outbreaks have also been recorded in chukar partridges, ducks, and chickens. wee has been only rarely associated with disease in avian species, and the closely related hj virus appears to be the eastern united states equivalent to wee. hjv has caused severe neuropathogenic outbreaks in chukar partridges and has been associated with infections in turkey flocks resulting in acute reduction in egg production . of course, these encephalitic viruses also cause disease in humans and horses. another interesting alphavirus, ockelbo virus, related to sindbis virus, has been implicated in causing arthralgia and rash in humans following its isolation from mosquitos collected during the outbreak. these viruses are transmitted by mosquitos among bird populations, which may act as the vector for transmission between humans and avian species. ockelbo virus, therefore, is apparently maintained in an enzootic cycle involving birds and mosquitos with transmission to other hosts such as humans. antibodies to ockelbo virus, either experimentally or naturally infected, have been detected in passeriformes, galliformes, and anseriformes (lundstrom et al., ; lundstrom and niklasson, ) . viremia in the absence of disease resulting from infection with ockelbo has also been demonstrated in these bird groups. given the widespread occurrence of antibodies to these encephalitic alphaviruses, it seems logical to conclude that birds in many cases act as "natural" and reservoir hosts. the only flavivirus thus far associated with disease in birds is the israel turkey meningoencephalitis virus. infected birds exhibit neurological dysfunction and occasional significant mortality. this virus has also been identified in turkeys in south africa. double-stranded segmented rna viruses are unique and intriguing. the distinguished virologist dr. wolfgang k. joklik, when asked what in the world he thought they meant in the grand scheme of biology, replied (and i paraphrase), "they represent an evolutionary step forward in the establishment of the ideal genetic material." the reoviruses have fascinated virologists for some time. they have an unusual and complex replication strategy, are able to undergo reassortment of their genes, and are curious in that they retain their infecting subviral cores as their rna replication template. reoviruses are also quite stable outside the host, remaining viable for up to a year at room temperature (nibert et al., ) . the mammalian strains have a wide host range but are found without any associated clinical signs, the origin of the name reo (respiratory and enteric orphan) telling most of the story. in poultry, several antigenic subtypes have been identified, and the virus can be classified based on serotyping and virulence, but there is no unified typing scheme as yet (kawamura and tsubahara, ; robertson and wilcox, ) . the most severe and common pathology associated with reoviruses is arthritis-tenosynovitis, although associations with other clinical syndromes including respiratory and enteric disorders have been described (rosenberger and olson, ) . often, the clinical states are influenced by the presence of other pathogens. arthritis is a significant problem in birds but primarily only in young chicks and turkey poults. other reoviridae in birds include several members of the genera orbivirus (arboviruses; see below) and rotavirus, which have been associated with a runting-stunting syndrome in chickens. the birnaviridae are a relatively recently characterized family of viruses that contain two double-stranded segments of rna and have no mammalian members thus far (ictv, ) . they were difficult to classify for many years because of their cell-associated nature and slow replication in cell culture systems. currently, two serotypes ( and ) are accepted, with significant antigenic variation and numerous proposed subtypes within each serotype (mcferran et al., ; jackwood and saif, ) . chickens are the only animals known to develop disease and lesions when naturally infected by avibirnaviruses. both serotypes are distributed worldwide, but serotype strains are the only ones associated with pathogenicity and immunosuppression. the bursa is the primary target organ, and strains of differing virulence have been identified. the disease is most significantly manifested when young birds are infected and permanent immunosuppression results. this sets the stage for subsequent severe viral and bacterial infections later in life. interestingly, ibd is an example where maternal antibodies derived by either vaccinations or natural infections provide significant immune protection. highly virulent strains of these viruses exist in various countries worldwide, and there are indications that new virulent variants do arise in the face of immune pressure (chettle et al., b) . avian adenoviruses are double-stranded dna viruses containing - genes. these viruses can be loosely divided into three groups based on antigenic relationships of internal proteins (mcferran, ) , although the three groups have not been officially recognized by the ictv. the avian group i adenoviruses include at least serologically distinct types, all of which have been isolated from mildly or asymptomatic poultry. the type species is known as the celo (chick embryo lethal orphan), or phelps strain, or f (fowl) strain. there are also numerous isolates from other avian species plus electron microscopy and immunological evidence that the type i aviadenoviruses are widely distributed in birds. by themselves, these viruses present no particular clinical problems in commercial poultry, but they are thought to cause significant problems in mixed infections with immunosuppressive viruses such as ibdv and chick infectious anemia virus (ciav). a virus virtually indistinguishable from the celo strain known as quail bronchitis virus (qbv) can be devastating in commercial quail operations, causing as high as % mortality (dubose and grumbles, ; montreal, ) . virus isolations and significant antibody levels in wild quail suggest that the virus may present disease problems in nature (king et al., ) , although there are no documented cases or epizootics. the group ii aviadenoviruses include turkey hemorrhagic enteritis virus (hev), pheasant marble spleen disease virus (msdv), and the avian adenovirus splenomegaly (aas) of chickens. these three agents are serologically indistinguishable but induce differing clinical manifestations in the different species. infections caused by this virus group appear to target lymphoid tissue, often resulting in immunosuppression, and there are strain-specific differences in virulence among the various group ii isolates (pierson and domermuth, ) . hev reached epidemic proportions in the s and still causes significant problems in turkeyproducing states in the united states and elsewhere. msdv is a significant pathogen in confinement pheasant operations, causing significant economic losses. aas is virtually ubiquitous among chicken flocks in the united states, in which case mild respiratory signs and splenomegaly are occasionally associated. the syndrome is similar to the disease state observed in pheasants, but in general aas virus causes no significant problems among chickens. evidence indicates that these group ii avian adenoviruses are limited to the order galliformes, and that wild bird populations (even wild turkeys) are unaffected. the more interesting group iii adenoviruses first appeared in associated with a distinct clinical manifestation called egg drop syndrome (eds), in which egg production decreases and thin-shelled or shell-less eggs are produced. the disease has caused significant egg production losses mostly in the eurasian and australian-pacific poultry markets. a virus named eds , which has been found associated with this syndrome, may have been originally introduced via a contaminated vaccine, but it seems clear now that sporadic cases are initiated by introduction of the causative virus from domestic and wild waterfowl, mostly ducks and geese. when endemic in flocks, the virus is transmitted vertically and exhibits a latent phase, which is reactivated in laying hens, usually after egg production begins. the virus initially appears to replicate in lymphoid tissues but rapidly moves to the oviduct, where replication causes inflammation and production of aberrant eggs. unlike other avian adenoviruses, the group iii strains do not replicate in the intestinal mucosa. it is likely that eds virus or very similar strains are present ubiquitously in wild ducks and geese, but rarely appear to be associated with disease (mcferran, ) . the avipoxviruses are widely distributed throughout the class aves, having been isolated from some species representing avian taxonomic families. the avipoxviruses are responsible for economically important disease problems in commercial poultry and aviaries (tripathy, ) . they may cause a slowly developing cutaneous disease with low mortality or, conversely, significant mortality, and generalized infections when in the diphtheritic form on mucosal surfaces of the respiratory tract and associated areas. these large dna viruses replicate in the cytoplasm of the cell, where they form characteristic inclusion bodies within rapidly proliferating nodular lesions. the poxviruses may be transmitted by mechanical means such as introduction from poultry workers into abrasions in the skin of uninfected poultry. there is also real evidence for transmission of the disease by mosquitos and other vectors such as mites in close conditions, where the number of diseased birds is high. the avipoxviruses can also apparently establish a latent state and be naturally or chemically induced to reactivate (kirmse, b) . cutaneous lesions containing infectious virus persistent for more than a year have also been documented in wild birds (kirmse, a) . variant strains of avipoxviruses have since been isolated from previously vaccinated flocks that were experiencing significant mortality from the diphtheritic form of the disease (tripathy and reed, ) . this suggests that, similar to marek's disease, vaccination against poxviruses may ultimately lead to escape of more virulent forms of the viruses. the avipoxviruses are also considered one of the most promising dna virus vectors for delivery of recombinant poultry vaccines, and poxvirus-vectored vaccines against newcastle disease and avian influenza have been licensed. avian encephalomyelitis is primarily a disease of young chicks caused by a picornavirus. the disease was quite economically important prior to initiation of live-virus vaccination. the host range is very limited (order galliformes), and there is only one virus serotype. the natural isolate is enterotropic and is transmitted horizontally (orally) and vertically. there is a gradient of pathology dependent on the age of infection of young chicks. pre-immune chicks from non-immune parents will generally die if infected within to days of hatch. if infected between and days of age, they may live but exhibit significant nervous involvement (encephalomyelitis). if infected beyond that age, they may exhibit enteric pathology but not neural signs. at adulthood or full immunocompetence, they may be infected but refractory to any clinical signs. so the immune status of the host in this case is very important in affecting the course of the viral replication (calnek, ) . another avian picornavirus, distinct from aev, is the avian nephritis virus (shirai et al., ) . this virus is similar to aev in that it is primarily a problem in very young birds and has a very limited host range. as the name implies, a distinct clinical syndrome is associated with the agents, but it is has not yet been determined how extensive infections are in commercial poultry. finally, another picornavirus, duck hepatitis (dhv- ), has caused significant problems in com-mercial duck operations. there are still unknowns with regard to the overall importance of dhv- , and there are additional virus-induced types of hepatitis disease with at least two others often encountered. the most significant cost involved in raising birds for food sources is feeding them. consequently, diseases that affect the feed-protein conversion ratio will directly affect the cost of marketable birds. as such, enteric disease, even when nonlethal or mild in nature, can affect the performance of food-source birds. in recent years, investigators have identified rotaviruses in chickens, astroviruses in turkeys, and enterovirus-like particles in several avian species associated with enteric diseases. many of these have been associated with significant pathogenesis, particularly in mixed infections where immunosuppression occurs, with subsequent decrease in marketability due to weight loss (barnes, ) . the virus designated chicken infectious anemia virus (ciav) is a singlestranded circular dna-containing virus, tentatively classified with two similar agents in a new family: circoviridae. the agent has only recently been recognized, purified, and characterized, and its unequivocal role in economically important disease is not yet fully established. purified virus inoculated into young chicks results in severe anemia and immunosuppression, but inoculation of -to -week old birds results in little if any pathology (yuasa and imai, ) . the virus has been associated with adult anemic conditions, however, in conjunction with other viral and bacterial infections and may thus play a significant role. the agent is widespread in commercial poultry flocks worldwide, and infection with the virus has been statistically related to a decrease in overall growth and performance (chettle et al., a) . the extent to which two other tentative members of the family circoviridae --psittacine beak-and-feather disease virus (bfdv) and porcine circovirus-are related to ciav is under question, and these stated taxonomic relationships may differ in the future. bfdv has proven to be a significant pathogen in aviaries and commercial in psittacine birds to the extent that vaccines are now routinely administered. recent evidence also suggests that circovirus particles can be detected by em and isolated from several other wild and captive bird species. consequently, the circoviridae may emerge as a more significant disease-causing agent in the near future. the caliciviridae is a family of small single-strand positive-sense rna viruses with a polyadenylated genome. virus-like particles resembling caliciviruses have been isolated from a variety of wild-bird and captive-raised species. a chicken calicivirus has been replicated in cell culture and caused apparent gastrointestinal disease in specific pathogen-free day-old chicks (cubitt and barrett, ) . intestinal contents of goldfinches with hemorrhagic enteritis have been found to contain calicivirus-like particles and gastrointestinal disease associated with caliciviruses has been reported in guinea fowl and pheasants (gough et al., ) . caliciviruses were originally isolated from marine mammals and have caused disease in both domestic and feral swine due to consumption of uncooked garbage containing seafood. interestingly, caliciviruses have been detected in pelagic birds, such as the white tern (poet et al., ) . calicivirus isolations such as these lead to the speculation that wild sea birds may be important in the transmission of these agents across large areas or to other animals. parvoviruses are the smallest of the dna-containing viruses, carrying a singlestranded genome of about nucleotides. they cause or are associated with three infections in birds (kisary, ) . in wild and domestic geese and muscovy ducks, derzy's disease refers to a syndrome that had been variously called goose influenza, goose hepatitis, gosling enteritis, and infectious myocarditis in different countries. this collection of names gives some indication of the variety of signs associated with derzy's disease. the virus, which can be transmitted horizontally and vertically, induces differing pathologies depending on the age of the bird and, in the case of hatchlings, the level of maternal antibodies. an interesting pathology often associated with infection of older birds is the virtually complete loss of feathers. the extreme stability of the parvoviruses makes control of this disease difficult in commercial operations worldwide, and the disease can result in % mortality in young hatchlings. in chickens and young turkeys, parvoviruses have been associated with runting stunting syndrome (rss), as have other agents. experimental inoculations have indicated that the associated viruses, which are distinct from the goose viruses, can cause significant pathology, but the extent to which these viruses participate in the rss is not yet established (trampel et al., ; decaesstecker et al., ) . another parvovirus, the avian adeno-associated virus is almost always found associated with adenovirus infections in poultry. the avian adeno-associated viruses are grouped with their mammalian counterparts in the dependovirus genus, although they are serologically unrelated. the avian dependovirus appears to contribute to disease only in the sense that it can affect multiplication of the associated adenoviruses (yates and piela, ) . newcastle disease virus (ndv), classified as avian paramyxovirus- , contains a single-strand negative-sense rna genome of kb containing coding sequences for six genes. the virus infects all bird species tested to date, including flee-living and domestic species. one panzootic outbreak of the disease is thought to have originated in asia, with subsequent spread to europe, with outbreaks of disease first reported in poultry during the s in java, indonesia, and newcastle-upon-tyne, england. worldwide dissemination of the disease, particularly during the s and s, has been attributed to increased international trade of both commercial poultry and pet birds. this led to development of inactivated and live-virus vaccines for commercial poultry. the transmission of infectious ndv may occur by either ingestion or inhalation, and this knowledge is the basis for mass-application vaccination procedures during poultry production. isolates of ndv are grouped into three main pathotypes depending on the severity of disease that they cause (alexander and parsons, ) . mildly virulent "lentogenic" viruses may cause unnoticeable infections in adult chickens or a mild respiratory distress and are used extensively as live-virus vaccines. "mesogenic" ndvs are of intermediate virulence and cause respiratory distress, with mild infections of various organs detectable only by histopathology. highly virulent viruses that cause severe morbidity and mortality are termed "velogenic." velogenic viruses can manifest themselves as neurotropic or viscerotropic forms of newcastle disease with extensive systemic replication throughout a bird. virulent forms of ndv can replicate within cultures of most avian and mammalian cell types without the addition of trypsin, while lentogens require added proteases for replication in cell culture. the presence of dibasic amino acids at the proteolytic cleavage site (pcs) of the viral fusion protein and the ability of cellular proteases to cleave the fusion protein of various pathotypes specify the molecular basis for ndv virulence. fewer basic amino acids are present in the fusion protein cleavage site of lentogenic ndv than is the case for mesogenic or velogenic isolates. the presence of the increased number of dibasic amino acids in the fusion protein pcs sequence of ndv allows for systemic replication of these more pathogenic viruses in the host (nagai et al., ) . although the principal concern with ndv is its effect on poultry production, it may have severe consequences in other flee-living avian species. major outbreaks of newcastle disease have occurred in north american cormorants during the summers of and , and again in . outbreaks during and occurred in the north central united states and south central canada, while in newcastle disease occurred among cormorants of the western united states and canada. mortality in young nestlings in some areas was as high as to %, and the affected birds had characteristic neurotropic lesions (heckert et al., ) . during the outbreak in cormorants, an unvaccinated north dakota turkey flock became infected with ndv, resulting in high mortality. using nucleotide sequence analysis, the virus causing disease in turkeys was proven to be the same virus isolated from afflicted cormorants in the central united states and canada. the cormorant virus was also phylogenetically related to other known ndv isolates of psittacine origin (seal et al., ) that had caused a major outbreak in southern california poultry during the early s that resulted in a depopulation of two million chickens (utterback and schwartz, ) . illegal importation of pet bird species into the united states continues to be a source of highly virulent ndv and certainly may play a role in spread of viruses that threaten commercial poultry worldwide (panigrahy et al., ) . what role free-living birds play in spread of ndv is unclear. although persistent infections of chickens by ndv do not appear to occur, the virus' persistence in poultry flocks may result from virus reintroduced from wild populations. also, different bird species vary in their level of susceptibility to ndv. ducks, geese, and certain psittacine birds do not exhibit signs of disease when infected with highly virulent ndv, while other psittacine species may have high mortality. persistent infections have been demonstrated in various psittacine birds, with virus isolations noted up to a year following experimental infection of parrots with velogenic ndv chicken isolates (erickson et al., ) . although psittacine birds have been directly linked as a source of ndv highly virulent for gallinaceous birds, such as chickens and pheasants, no studies have shown direct isolation of ndv from feral psittacines. newcastle disease occurred in racing and show pigeons during the s in both western europe and north america. the outbreaks in western europe were linked to contaminated feed, and disease was controlled in both areas via vaccination with mildly virulent ndv commonly used for poultry. the virus isolated from these birds varied somewhat from traditionally virulent ndv in having only one set of dibasic amino acids in the fusion protein cleavage site. increased virulence for poultry subsequently occurred only after passage in chickens (collins et al.. ) . these examples demonstrate the fact that variant forms of ndv may arise in different bird species and that free-living birds may be a constant source ot viruses that affect both domestic and wild birds. vaccination programs against ndv for the most part have been effective al controlling the virus in commercial poultry. however, all of the vaccine strains available today, while effective against lethal disease, share less sequence identit with currently circulating disease strains. this is effectively illustrated in figure , which may also be considered a useful example for other rna-containin~ viruses. the phylogenetic tree demonstrates the relationship between currenl . phylogenetic relationships among newcastle disease virus isolates. nucleotide sequences from the fusion protein gene coding for the cleavage site from several newcastle disease virus isolates were aligned and analyzed by parsimony analysis. all the viruses listed are intermediately virulent mesogens or highly virulent velogenic isolates when inoculated into chickens, with the exception of the two vaccine viruses and the chicken/ field isolate. the data demonstrate that field isolates from chickens related to vaccine virus are isolated from poultry. the chicken/ virus caused the last major outbreak of newcastle disease in u.s. poultry and was epidemiologically linked to a pet bird. it is genetically related to a parrot isolate and viruses that caused disease in pigeons during the s. viruses isolated from cormorants in infected an unvaccinated turkey flock. these viruses appear to be circulating among cormorants, since closely related viruses were isolated again in . no viruses related to the virulent chicken viruses from have been isolated since that time, indicating these virus types may no longer be circulating among birds. all the recent virulent newcastle disease viruses were isolated from pet or exotic birds and chickens infected by free-living birds. these viruses are related to virulent viruses originally isolated during the s. avian influenza (ai) presents one of the most interesting ecological relationships between birds and their viruses. its ecology has been reviewed in depth hinshaw et al., ) , but ai presents such a complete picture of a virus with multiple impacts on several species that it must be included in this chapter. the type a orthomyxoviruses are essentially bird viruses. the infections in birds can range from clinically inapparent with minimal serologic response, to a devastating systemic disease that can result in % mortality within a matter of days. there are identified subtypes of ai viruses based on the hemagglutinin (ha) protein antigenic structure and nine subtypes based on the neuraminidase (na) structure. these two genes code for the predominant surface glycoproteins, which are embedded in the lipid bilayer of the viral envelope. these are only of genes coded for by the virus. the virus' genetic material is contained on eight separate negative-sensed single-stranded rna segments ranging from to nucleotides in length. all of the ha and na viral subtypes have been identified in feral waterbirds, and it has been proposed that these birds act as a "natural" reservoir for the virus slemons and easterday, ) . determination of the phylogenetic relationships of several genes from several subtypes of aiv collected from wild birds indicated that the general rate of evolution in the avian reservoir is low compared to the rate of evolution observed in human and other mammalian strains of type a orthomyxoviruses . other recent studies, however, have shown that the mutation rate of the ha gene and the ns gene even in wild birds approaches that seen in human strains . measurement of clinical signs among infected migrating waterbirds is, of course, a difficult task. thus far, only a single instance of severe clinical signs has been associated with free-living birds --a lethal outbreak in terns in south africa in caused by an h n strain (becker, ) . given the recently measured mutation rates, and the appearance of this severe lethality in a wild bird population, it must be considered that aivs do continue to evolve in feral birds (indeed subtypes have arisen already!). there is no reason to believe that the present count of subtypes will be the final tally. while some have proposed that the aiv strain in wild birds causes no disease problems as a result of "evolutionary stasis," without question the virus has dramatic effects when it leaves this proposed reservoir. in addition to the fixed influenza populations in pigs and horses, avian strains can directly infect mammals and have been identified in whales, seals, and mink (lang et al., ; berg et al., ) . the dramatic association with seals in and mentioned earlier clearly points out the potential impact of aivs. this avian reservoir then serves as a sort of base for the viral "biological invasions" that are a part of interspecies transmission. ecologically and economically, the transmission of these aiv subtypes to commercial poultry has perhaps yielded the most important impact. the situation is most clearly played out in minnesota each year. due to the large number of lakes in the state, migratory waterfowl, primarily anseriformes (ducks, geese), frequent the area in large numbers seasonably. nearby commercial turkey operations suffer infections nearly every year, particularly in the cooler fall months, caused by different influenza subtypes of different virulence (halvorson et al., ) . in severe years, the associated costs due to turkey mortality, as well as performance and egg production losses, can reach several millions of dollars (poss and halvorson, ) . in chickens, introduction of influenza viruses from waterfowl has been more devastating. in the northeastern united states, the h subtype caused the first large-scale outbreak of highly pathogenic ai in numerous flocks in . twentythree million birds were destroyed at a cost of more than $ , , in order to contain the infection. clinically, the disease was indistinguishable from the disease originally described as "fowl plague" in in europe (eckroade and silverman, ) . today we know that all of the classical "fowl plague"-type outbreaks have been associated exclusively with only two subtypes: h and h . evolution to virulence in these subtypes has been closely associated with accumulation of basic amino acids at the proteolytic cleavage site of the hemagglutinin protein (bosch et al., ; webster and rott, ) . in order for aiv strains to be infectious, the ha protein must be cleaved into ha and ha subunits, which subsequently allows structural rearrangement and exposure of a protein sequence needed to fuse the viral envelope with the plasma membrane of the target cell (klenk et al., ; skehel et al., ) . isolates of low pathogenicity lack multiple basic amino acids and are only cleaved by trypsin-like extracellular proteases. these proteases are abundant on the mucosal surfaces of the respiratory tract and gut, so the viruses replicate unrestricted at these sites. the highly pathogenic (hp) forms, however, have additional basic amino acids, which are recognized by the intracellular furin-like proteases ubiquitous in bird tissues (rott et al., ) . thus, in birds, these viruses are able to escape the respiratory tract and infect a wider variety of internal tissues and organs. this is one of the most important of the virulence factors (webster and rott, ) . only two subtypes have been shown to accumulate these basic amino acids: h and h . these subtypes appear to do so by either base substitution or by insertion events (wood et al., ; perdue et al., ) . nucleotide insertion events appear to be the most common mechanism, and table ii and figure illustrate what is presently known about occurrence of these hp virus isolates in commercial poultry. the hp isolates, with only two exceptions, have additional inserted basic amino acids within the well-conserved region surrounding the cleavage site. from the data in table ii and figure , it may be easily surmised that both the number of hp isolates and the number of basic amino acids at the cleavage site are increasing. athe amino acids surrounding the hemagglutinin protein cleavage sites of a representative member of each of the subtypes of avian influenza viruses are shown above. subtypes , , and , which cluster together in a phylogenetic tree, all have five amino acids between the conserved proline and arginine; the remaining subtypes have four. highly pathogenic isolates (subtypes h and h ) more often than not, have insertions of arginines and lysines, increasing the length of the cleavage site and making the hemagglutinin accessible to ubiquitous proteases. this has the effect of greatly increasing the tissue distribution and virulence of the virus. table ii for further explanation. the avian influenza viruses are distributed worldwide. in addition to the united states, outbreaks of highly pathogenic ai have occurred in mexico, canada, europe, asia, and australia. in most cases, there is some sort of connection between the affected flocks and nearby waterfowl. the extent to which the reservoir of viruses exists in waterfowl is not really known. surveys have shown that, in addition to the viruses being widespread, the various subtypes increase and decrease in prevalence over successive years. the order charadriiformes (shorebirds and allies), in addition to anseriiformes, has been shown to carry aiv strains (kawaoka et al., ) , and the extent to which aivs are carried in other orders has really only been superficially explored. molecular and phylogenetic evidence have clearly shown that these segmented viruses share various genes back and forth within the populations of circulating virus strains . they are also capable of donating genes through genetic reassortment to viruses in swine and those genes ultimately end up in humans (webster et al., ) . add this capacity to the known (albeit rare) transmission of purely avian influenza strains directly into mammals and one creates a potential ecological bonanza for the virus, and potential nightmares for new hosts. as advanced as our study of viruses has become, there are still a variety of unknowns with regard to the extent to which avian viruses infect other vertebrates. it would not be unreasonable to suggest that, because of their mobility and longevity of existence, that the class aves may have played a critical role in distribution of viruses to other classes. in the case of the avian orthomyxoviruses, phylogenetic evidence points to recent introduction of these viruses from birds to mammals. following introduction, the natural mutation and evolution of the virus produced strains that now appear to be adapted to their new hosts. there are swine, equine, and human strains of type a influenza viruses that do not replicate well outside their respective host species. if birds are indeed responsible for us coming down with the flu today, we know of no reason why they might not have participated over the eons in the establishment of other purely mammalian diseases. of course, one could easily argue the opposite relationship, but, intuitively, birds do present an ideal mobile vector, particularly for the enterically transmitted viruses. in addition to the diseases discussed here, there is a growing group of emerging diseases or diseases of as yet unknown etiology in commercial poultry (saif, ) . many of them have virus particles clearly associated with the syndrome, but only limited research and characterization has been carried out thus far. it is probably safe to assume that the future will continue to bring new avian diseases and bring to light new or undiscovered avian viruses. the relationships existing among birds, insects, and viruses is one area that should be explored in more detail by increased research efforts. in free-living birds, the large number of apparently innocuous infections with bunyaviridae, togaviridae, and flaviviridae suggest that birds may act as an ecological reservoir for maintenance of these virus populations. from a virocentric point of view (if there is such a thing), having a potentially highly mobile population of susceptible hosts on which transmission vectors may feed would be advantageous. it would provide a mechanism by which to transfer viruses over large geographical distances where they could then be transmitted by new related or unrelated vectors, thus increasing host range. of course, one runs the risk here of assuming that the virus would be ecologically improved by extending its replicative capacity in new hosts. this may not be a valid assumption. for example, if variola virus or human poliovirus type had been exploiting this strategy, they made a bad choice in extending their host range into humans! from one human vantage point, birds provide a useful monitor for detection of arboviral diseases in a given area. sentinel chickens are actually in use today along the east coast of the united states to gauge the extent to which some mosquito populations are carrying eastern equine encephalitis and st. louis encephalitis viruses. the future is perhaps the most important aspect to consider. what viruses other than the orthomyxoviruses may someday establish a similar relationship where transmission to a mammalian intermediate or even direct transmission may allow introduction into the human population? one fundamental unanswered question is whether birds, acting as reservoirs for arboviruses, should be considered an important ecological niche for those viruses. there is no evidence of which we are aware to indicate either yes or no. there have been no documented transmissions of arboviruses from birds to mammals without the insect vector in nature, however, so at this point wild birds could be considered dead-end hosts for the arboviruses. in commercial poultry during outbreaks of the alphavirus eeev, there is no evidence of infection of workers in close contact with infected birds. if, however, a bird provides the necessary reservoir of eeev that infected the human, then this bird becomes very important. but there are no data that as yet identify the bird as the required reservoir for disease transmission to human. newcastle disease virus provides an interesting new disease problem in humans that has only arisen as a result of increased vaccination of birds. a live-virus vaccine is available for control of ndv that is administered by aerosol spray. in a small number of cases where precautions have not been taken, vaccinators can contract a conjunctivitis caused by the vaccine. this outcome has been seen in the heavy poultry-producing areas of northern georgia, on a common enough basis now, to be easily recognized by ophthalmologists. there is usually no seroconversion to the virus in these cases, and they are not particularly serious infections. there is also suggestive evidence in the form of measured seroconversion that poultry workers occasionally may be subclinically infected with avian coronaviruses and avian retroviruses. the evidence for direct infection of humans with avian influenza viruses is growing. human strains have classically included only three subtypes (h - ). until recently, the only other strains shown to cause disease in humans were ot the h subtype. conjunctivitis caused by purely avian h subtype viruses has been reported on two occasions (kurtz et al., ; webster et al., a) . the most disturbing set of events, however, was the more recent highly publicized influenza outbreak in hong kong. a purely avian h subtype virus was isolated from some patients, of whom died, from may through december of . the index case, a -year-old child, in may followed a highly pathogenic outbreak of avian influenza that had just previously occurred in chickens in hong kong and its environs. the child had been exposed to ill birds at a day care center, and it was clear that the recovered virus was essentially the same as that recovered during the chicken outbreak. considerable efforts were made to determine whether the virus was a contaminant and whether it was replicating in the child. the evidence strongly suggested that it was indeed replicating (subbarao et al., ) . subsequent to this event, in november-december the remaining confirmed cases were reported and an intense effort was mounted to determine exactly how this virus was transmitted. it is clear now that the h n viruses infecting humans in hong kong were all of avian origin . there is limited serologic suggestion of human-to-human transmission but no genetic evidence of adaptation to humans. this case was touted as a premier test run for the next predicted influenza pandemic. whether this outbreak represents a simple dead-end zoonotic transmission or whether these purely avian influenza viruses can become fixed in the human population is a matter for conjecture. the avian h and h influenza strains have been the only natural influenza strains thus far to unequivocally cause systemic lethal infection in any species owing to the unique structure of their ha protein (see fig. ). a major fear is that by adding such ha subtypes to a population of mammalian viruses a whole new class of virulent strains might be created. our experience in working with numerous highly pathogenic and nonpathogenic strains has been one of very little or no evidence for human infection in the years that scientists and technicians have spent working with hundreds of different stains. but our experience has also been that, when one attempts to confine influenza viruses by setting biological rules for them, they usually find a way to break them. if ecology is the study of the relationship of organisms to their environment, for bird viruses, the active, pertinent, environment is always within the bird and its tissues. while one cannot discount the effects of the environment outside the host, there are no positive effects on an avian virus life cycle outside the host of which we are aware. that is, there are no activation events outside the avian host of which we are aware; only events of inactivation. certainly, avian viruses, as all viruses, have evolved survival strategies to allow passage from host to host, and some are much more refractory to environmental inactivation than others. but without really knowing which, if any, "evolutionary direction" viruses are taking, it is impossible to determine whether a large herpesvirus of genes is ecologically more fit than the enterovirus with five genes! the relationship of birds and insects in the transmission and life cycle of the arboviruses is poorly understood. until this recent transmission of h n influenza strains to humans, this relationship appeared to be the only significant one in which there is interaction among a genetically unaltered virus, an avian host, and a non-avian host. as mentioned earlier, other documented cases of avian-to-mammalian viral transmission are newcastle disease infections of humans and influenza a infections of mammals and humans. there is also serological evidence for avian retrovirus and avian coronavirus infections. in the case of newcastle disease, the documented infections are mostly minor conjunctivitis. in the case of influenza, discussed earlier, the avian viruses most often acquire rna segments from other sources before they are established in a new host. thus, in general, the impact of avian viruses on public health might currently be considered small, but clearly the potential exists for significant future impact. a major interesting question arises when evaluating virulence of avian-origin viruses. conventional wisdom seems to be suggesting that virus entry into naive host populations is more likely to result in a general decrease in virulence as the virus adapts to the new host (morse, ) . this may be true under truly "natural" conditions; but what happens under human-dictated conditions such as vaccination and by defining the host population through breeding and housing? in the case of marek's disease, we see an example of virulence increasing in the face of continued vaccination against the virus (fig. ) . whether this is occurring as a result of the vaccination or a combination of factors, the fact is that the population of marek's disease herpesviruses in nature is becoming more virulent. the vaccination programs for newcastle disease utilize vaccines whose genetic compositions are becoming farther and farther removed from the virulent strains circulating in wild fowl (fig. ) . this may not bode well for future control of virulent strains in commercial poultry. in the case of avian influenza viruses, we have seen the number of highly virulent outbreaks increase in recent years, often following prior circulation of a nonpathogenic precursor, and we have seen concomitant changes in genetic structure related to virulence (fig. ) . again there are unknowns in this system, but the fact remains that we are seeing more virulence as the nonpathogenic aiv subtypes replicate in commercial poultry settings. thus, this naive commercial population, unlike the waterfowl hosts to which the virus has adapted, appears to allow generation of heretofore unencountered virulence phenotypes. similar scenarios appear to be playing out as more virulent infectious bronchitis strains and infectious bursal disease strains appear worldwide. one must be cautious, then, in working strictly under the previously mentioned virulence-decrease paradigms for mammalian viruses. these have been suggested mostly by the experiences with artificial introduction of rabbit papilloma viruses in australia or where new introductions occur as human encroachment ensues (such as in the recent cases of ebola virus or monkey poxvirus infections). in these 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infections of birds diseases of poultry epizootology of velogenic viscerotropic newcastle disease in southern california molecular pathogenesis of marek's disease --recent developments sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronaviruses miscellaneous herpesvirus infections egg production drop in turkeys associated with alphaviruses: eastern equine encephalitis virus and highlands j virus evidence of natural recombination within the s gene of infectious bronchitis virus influenza virus a pathogenicity: the pivotal role of hemagglutinin conjunctivitis in human beings caused by influenza a virus of seals b). characterization of an influenza a virus from seals evolution and ecology of influenza a viruses influenza: a model of an emerging virus disease increased virulence of marek's disease virus field isolates epidemiology of reticuloendotheliosis virus in broiler breeder flocks deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza a viruses of h and h subtypes avian adenovirus-associated virus pathogenicity and antigenicity of eleven isolates of chicken anemia agent (caa) key: cord- -hsw dk d authors: thys, séverine title: contesting the (super)natural origins of ebola in macenta, guinea: biomedical and popular approaches date: - - journal: framing animals as epidemic villains doi: . / - - - - _ sha: doc_id: cord_uid: hsw dk d in december , a two-year-old child died from viral haemorrhagic fever in méliandou village in the south-east of guinea, and constituted the likely index case of a major epidemic. when the virus was formally identified as ebola, epidemiologists started to investigate the chains of transmission, while local people were trying to make sense out of these deaths. the epidemic control measures taken by national and international health agencies were soon faced by strong reluctance and a sometimes aggressive attitude of the affected communities. based on ethnographic work in macenta (forest region) in the autumn of for the global outbreak and alert response network (goarn) of the world health organization, this chapter shows that while epidemiologists involved in the outbreak response attributed the first ebola deaths in the forest region to the transmission of a virus from an unknown animal reservoir, local citizens believed these deaths were caused by the breach of a taboo. epidemiological and popular explanations, mainly evolving in parallel, but sometimes overlapping, were driven by different explanatory models: a biomedical model embodying nature in the guise of an animal disease reservoir, which in turn poses as threat to humanity, and a traditional-religious model wherein nature and culture are not dichotomized. the chapter will argue that epidemic responses must be flexible and need to systematically document popular discourse(s), rumours, codes, practices, knowledge and opinions related to the outbreak event. this precious information must be used not only to shape and adapt control interventions and health promotion messages, but also to trace the complex biosocial dynamics of such zoonotic disease beyond the usual narrow focus on wild animals as the sources of infection. at the end of december with the death of a two-year-old child in the village of méliandou in guéckédou prefecture, four days after the onset of symptoms (fever, black stools and vomiting). this patient would be considered from now on as the 'case zero', the index case stemming the severe ebola virus disease (evd) epidemic of west africa from apparently a single zoonotic transmission event. but then, with the idea of the spillover taking central stage the question arises: which animal species, the mythic 'animal zero', came to bear the burden of epidemic blame this time? while this retrospective epidemiological study was perceived as essential for limiting high-risk exposures and for quickly implementing the most appropriate control interventions, these investigations (biomedical experts deployed from the rich north) were tempted to mimic and fulfil the 'outbreak narrative' imposed by the global health governance. in this endeavour, rather than discovering the epidemiological origin, what becomes crucial is to quickly identify the carriers-'these vehicles necessary to drive forward the plot', which often function as the outbreak narrative's scapegoats. historically always located at the boundary of the human social body, the ideal candidate to carry this role in the evd epidemic of - was once again the wild and villainous non-human animal. because the pathways for emergence are in any way 'natural' or 'sylvatic', according to the dominant western biomedical model, the inclusion of wildlife in the epidemiology and the evolution of emerging infectious diseases is justified, yet its role is often misrepresented. although the probability of a humans contracting the disease from an infected animal still remains very low, certain cultural practices sometimes linked with poverty, especially 'bushmeat' hunting, continue to be seen as the main source of transgression of species boundaries. in the african context, research into emerging infections from animal sources implicates nonhuman primate 'bushmeat' hunting as the primary catalyst of new diseases. since the virus of ebola was identified for the first time in zaïre in and qualified as the first 'emerging' virus according to the new world clinic called 'global health', the link between animal and human health appears based on an 'us vs. them'. after the formal confirmation of the aetiological agent in march , the epidemic quickly took on an unprecedented scale and severity in several respects. it was declared by the who as an 'extraordinary event' because of its duration, the number of people infected, and its geographical extent which made it the largest ebola epidemic recorded in history until then. to these quantifiable impact measures were added sociological, ecological, political and economic phenomena that are much more complex to decrypt. these have had a profound impact on society, well beyond the remote rural environment that was typically affected by preceding epidemics. by threatening major urban areas, these 'geographies of blame' or 'hotspots' (usually at the margin of modern civilisation and configuring specific areas of the world or the environment into the breeding grounds of viral ontogenesis) have been mapped by 'virus-hunters' to update 'predictions about where in africa wild animals may harbour the virus and where the transmission of the virus from these animals to humans is possible'. in addition to this epidemic's extraordinary character, by spreading beyond the capacities of humanitarian aid, this new biomedically unsolved complexity conferred upon it a status of 'exceptionality' also by 'proclaiming the danger of putting the past in (geographical) proximity with the present'. this status had the effect, among others, of the most intense involvement, perhaps more visibly than before, of different disciplines, from human and animal health to the social sciences, in the international response. anthropology's response in particular was 'one of the most rapid and expansive anthropological interventions to a global health emergency in the discipline's history'. yet it is very critical that the collective social science experiences acquired during this west african ebola epidemic remained engaged to addressing future outbreaks and beyond. they translated and shared anthropological knowledge between scholars by including translation for public health specialists, transmitting that knowledge to junior scientists, and engaging in ongoing work to develop relevant methodology and theory. among the three west african countries most affected by the epidemic, guinea-conakry has been more marked by this dual 'exceptionality', that is to say, both epidemiological and social. beside the exceptionalism described by the senegalese anthropologist faye on the strong and sometimes violent demonstrations of popular reticence with regard to the activities of the 'riposte', guinea was also marked by a higher case fatality rate, as shown in the who report of march . globally raised up to more than % (while knowing that the number of cases and deaths was probably underreported), this case fatality rate confirmed the seriousness of the disease in a guinean context where the ebola virus had never hit before. neither the medical community, nor the population, nor the authorities had so far experienced it. despite all the measures implemented, to the question, why did we observe a higher case fatality rate in guinea compared to that of other countries, a multitude of factors can be advanced. the latter deserve to be the subject of multidimensional analyses, especially as this global lethality has manifested itself differently according to the geographical region of the country. the highest fatality rate was observed in forest guinea ( . %, / ), the region of origin of the index case and main epicentre of the epidemic. was this due to exclusively biomedical factors, such as a lower level of immunity among the guinean population? or was it because of late care that would have given patients less chance of surviving and fighting the virus? but then, why did people infected with the virus later arrive at ebola treatment centres (etc) in guinea? was it due to a poorer and more limited health system and frailer medical and health infrastructure than liberia and sierra leone at the time of the epidemic? or was it due to less effective coordination work by international and national teams in responding to the epidemic? or simply because in guinea the local communities were much more reluctant and intentionally opposed to the deployment of humanitarian and health assistance? although sharing broadly similar cultural worlds, what can therefore explain this notable difference of social resistance between the affected countries? combined with a divergent political practice and lived experiences of the state, especially between sierra leone and guinea, the working hypothesis drawn from my ethnographic observations in macenta and related literature review is that part of the continuing episodes of hostility and social resistance manifested by guinean communities regarding the adoption of the proposed control measures against the scourge of ebola has its origins in the divergence between explanatory systems of the disease; on the one hand, biomedical explanatory systems, and, on the other hand, popular explanatory systems. in march , when ebola hemorrhagic fever was formally identified a few months after the first death, epidemiologists and local populations each actively began to trace and understand this first human-to-human transmission chain of the disease, as well as its triggering event. evolving most often in parallel, and overlapping at times, these epidemiological and popular investigations generally refer to different explanatory models, some more biomedical ('natural') and others more mysticoreligious ('supernatural'). the purpose of this chapter is to trace and reflect on the interpretations of the origin and transmission of the ebola disease, as perceived and explained by the population, and to contrast them with the explanatory model of epidemiologists. in order to interrupt the two routes of evd transmission, namely from animal reservoirs to humans and between human infection, humanitarian responses followed the following public health logic: 'bushmeat' hunting, butchering and consumption should be banned and the ill should be isolated within etcs and burials should be made safe. yet, the interventions related to this reasoning had unattended consequences and, together with the ebola disease itself, they 'disrupted several intersecting but precarious social accommodations that had hitherto enabled radically different and massively unequal worlds to coexist'. carriers, in the case of human-to-human transmission, are generally perceived as the ones promulgating the epidemics and are marked with transgressive attributes intrinsic to their 'contagiousness' (e.g. wanton or deviant sexuality for the hiv epidemic, uncleanliness for the cholera epidemic, immigration for typhoid). however, in zoonosis-related diagnostic discourses, pathogens have the potential to reverse relations between humans and animals in such a way that the carrier becomes the victim. located at the 'interface' between humans, animals and the (natural) environmentalready proved to be a virtual place where deadly pandemic risks lie waiting for humanity-'forest people' from guinea were rendered both carriers of the disease and victims of the villainous role of nonhuman animals. the response to the fear of pandemics has been made unmistakable: we have to shield off humanity from nature. this mindset strongly adheres to the prevailing 'culture-nature divide' which is also depicted through zoonotic cycles diagrams further operating both as pilots of human mastery over human-animal relations and as crucial sites of unsettlement for the latter. wild animals became public enemy number one, together with those who were supposedly facilitating the transgression of the boundaries between the cultural and natural world with (or because of) their culturally 'primitive' or 'underdeveloped' practices. by framing 'bushmeat' hunting, as well as local burials, as the main persisting cultural practices among the 'forest people' to explain (or to justify) the maintenance of the evd transmission during the west african epidemic, the notion of culture that fuelled sensational news coverage has strongly stigmatised this 'patient zero' community both globally and within guinea, and has been employed to obscure the actual, political, economic and political-economic drivers of infectious disease patterns. appointed by my former institute, the institute of tropical medicine of antwerp, belgium (itm), to the who, i was sent to guinea-conakry from the end of october to the end of november for a four-week mission by the global outbreak alert and response network (goarn). since august , the country had been in the largest and longest phase of the epidemic, the second recrudescence which would also be the most intense one up until january . i first spent a week in conakry to follow the implementation of a social mobilisation project (project of monitoring committees at the level of each commune in the urban area). then, following an evaluation of the situation qualified as catastrophic by the national coordinator of the who, it was in macenta, forest guinea, where i was deployed. macenta, located east of guéckédou, was the prefecture considered to be the epicentre of this new outbreak of ebola and where transmission was the most intense. this district would remain one of guinea's most affected regions. by october , macenta, where catastrophic scenarios seemed possible, had already a cumulative number of almost cases since the beginning of the epidemic. the epidemiological situation was out of control, with a lack of material, human and financial resources. on arrival, there was still only one transit center (cdt). a new etc was being finalised by msf belgium. its management would be taken over a few weeks later by the french red cross. due to the long rainy season, the road used for bringing confirmed cases from macenta to the guéckédou treatment centre was in a deplorable state, slowing down the start of treatment and increasing the risk of transmission during transportation. it is as a medical anthropologist that i have been involved in guinea's national coordination platform for the fight against ebola and this within the commission of 'social mobilization and communities engagement', also named locally the 'communication' unit, in order to document, better understand and help to address the reluctance manifested by the local community. without going into the debate about the instrumentalisation of anthropologists as simple 'cultural mediators' at the service of humanitarians, i will simply recall here the specific objectives assigned for the mission. they consisted, on the one hand, in an analysis of rumours and crisis situations in order to propose responsive actions and, on the other hand, in adapting the responses and protocols of the various national and international institutions to local conditions, giving priority to comprehensive and participatory approaches. by integrating the 'communication' unit, i tried to support and animate the meticulous and sensitive work of a whole team working to rebuild trust with communities and to 'open' villages reluctant to receive care interventions. under the authority of unicef guinea, this communication team also hosted many local associations previously working for the prevention of infectious diseases, such as hiv/aids, in the region. the latter had already been mobilised to serve as a relay and to mitigate the unpredictable consequences of the epidemic not foreseen by the riposte, such as, among other things, sensitisation and reception of healed people and orphans of ebola, food distribution, and support for people and villages stigmatised by the disease for whom access to the market-purchase and sale of products-was forbidden. religious representatives of protestant and muslim communities also voluntarily joined this platform to learn and then preach preventive behaviour, to comfort the population, as well as to deconstruct and addressed rumours. their main message was to convince the public that ebola did indeed exist and 'was a real disease'. subsequently, the communication unit was finally able to associate the prefectural direction of traditional medicine of macenta counting traditional healers and distributed in the subprefectures of macenta. the main objective of this new activity was to engage all traditional healers in the fight against evd by raising the awareness of their patients and their entourage thanks to their high level of credibility in their respective communities. they also undertook to refer their patients directly to the tc if they came to present even one of the symptoms of evd (fever, diarrhoea [with blood], vomiting [with blood], loss of appetite). a 'health promotion' team managed and financed by msf belgium also acted on the ground. each morning, the different commissions and stakeholders of the riposte present in macenta were meeting at the prefectural health directorate (dps) to discuss and coordinate their activities in the field. alongside a guinean sociologist, consultant for the who and the assistant coordinator of the mission philafricaine, i was quickly immersed in the realities of the field and in the local strategies elaborated with respect of traditional hierarchies, despite the emergencies. their goal was to restore dialogue with the various village representatives who, since the officialisation of the epidemic, had decided to resist ebola interventions. this was, for instance, the case of the village of dandano, where deaths had risen to ; a village whose access was authorised the day after my arrival in macenta. although tragic, this coincidence made me earn some legitimacy from the other national and international 'fighters'. it is in this intense and difficult context that the ethnographic observations and their preliminary analysis, presented in this chapter, were collected. the methods employed are based on participant observation, including many informal discussions during meetings with villagers (representatives of youth/notables/sages/women), with religious representatives (protestant pastors, and imams), with drivers and partners of the coordination community (e.g. doctors without borders, guinean red cross, unicef among others). some formal interviews were also conducted with key informants such as healed individuals (ebola survivors), traditional healers, pastoralists and local actors in the fight. biomedical scientific literature and reports on epidemiological data, as well as observational notes, photographs and audio recordings collected in the field, allowed me to trace the interpretations of the origin and transmission of ebola in a dual perspective: that of epidemiologists, on the one hand, and that of the population on the other. it is through the concept of explanatory models or 'cultural models of the disease' developed by arthur kleinman that i attempted to interpret the observations ( fig. . ) . this is a conceptual framework that has already been used by barry and bonnie hewlett, alain epelboin and pierre formenty in their respective interventions during the previous outbreak of ebola haemorrhagic fever in the congo in . to be able to adapt the response and interrupt transmission, it is essential to know and understand how the population perceives the introduction of a disease, especially when it is such a deadly one. explanatory or cultural models refer to the explanations of an individual or a culture and to predictions about a particular disease. these are social and cultural systems that construct the clinical reality of the disease. culture is not the only factor that shapes their forms: political, economic, social, historical and environmental factors also play an important role in disease knowledge construction. in kleinmann's model, care systems are composed of three sectors (popular, professional, and traditional) that overlap. in each healthcare system, the disease is perceived, named and interpreted, and a specific type of care is applied. the sick subject encounters different discourses about the illness as she or he moves from one sector to another. kleinman defines the existence, in each sector, of explanatory models of the disease for the sick individual, for his/her family and for the practitioner, whether professional or not. in general, only one part of an explanatory model is conscious, the other is not. although the explanatory models seek from the health district mbomo in congo in , they identified five different cultural models including a sorcery model (sorcerer sending spiritual objects into victims), a religious sect (la rose croix, a christian sect devoted to study of mystical aspects of life), an illness model (fever, vomiting, diarrhoea with blood), an epidemic model (illness that comes rapidly with the air/wind and effects many people) and a biomedical model (ebola haemorrhagic fever). interestingly, none of the integrated non-biomedical models identified a specific non-human animal as potential source and/or carrier of evd or hunting and butchering as specific health risk activities for such illness. this further supports the epistemic dissonance observed during many epidemics (including the west african evd epidemic in this case), between the public health framing of wild meat as hazardous and the practical and social significance of the activities that occasion contact with that hazard. in the case of evd, it is the biomedical cultural model that prevails among western health workers. when the alert was launched by the local health authorities on march , two and a half months after the beginning of the disease of the index case, it was virologic investigations that were conducted at first, following the many deaths that occurred during this socalled silent phase. when the zaïre ebolavirus was identified as the causative agent, retrospective epidemiological investigations of the cases took place, which are crucial during the outbreak of an infectious disease responsible for such high mortality rate. the first chains of transmission of evd are presented in the below graph adapted from baize et al. ( ) (fig. . ). these investigations are mainly based on the identification of patients and the analysis of hospital documents and reports (results of blood tests carried out in the laboratory), as well as on testimonies and interviews with the affected families, the inhabitants of the villages where the cases occurred, suspected patients and their contacts, funeral participants, public health authorities and hospital staff members. virologic analyses suggest a single introduction of the virus into the human population. but the exact origin of the infection of this two-year-old child has not yet been definitively identified, even though the role of bats as natural hosts of the ebola virus, including this time also the insectivorous species, remains one of the most probable scientific hypotheses. up to now, the precise nature of the initial zoonotic event in guinea remains undetermined and the natural reservoir of the ebola virus more generally is not yet certain, beside for three species of fruit bat and other insectivorous african bat species known to carry ebola antibodies and rna. therefore, this informational gap was from the start filled with assumptions during the west african outbreak. among these assumptions, the elusive link between bats, wild animals and humans triggered high concerns over handling, butchering and consuming wild animals, commonly referred to as 'bushmeat'. consequently, these concerns were integrated into public health messages on disease prevention and were translated into a 'bushmeat ban' by governments across the region and enforced during the entire outbreak. this raises the question of the value of focusing on zoonotic transmission, in particular by fruit bats and non-human primates, which was quickly (s ) child, years-old onset dec. dotted arrows: the epidemiological links have not been well established deemed to be of minimal risk, when the biggest threat of infection was from other humans. furthermore, it raises the question of whether there is evidence to indicate and confirm that 'bushmeat'-related information included in public health campaigns in the region actually reduced ebola transmission. first, hunting and consuming 'bushmeat' for food have long been a part of human history occurring worldwide, serving as an important source of protein, and household income, especially where the ability to raise domestic animals is limited. the term itself encompasses an extensive list of taxa that are harvested in the wild (ranging from cane rats to elephants and including duiker, squirrels, porcupine, monkeys, non-human primates, bats and hogs) for food, medicine, trophies and other traditional, cultural uses. yet, designating the consumption of wild animal meat through the use of the term 'bushmeat' for west africans instead of 'game', as is the case for europeans and americans, by the media, scientific literature and public health campaigns that prohibit this practice, participates in 'semiotics of denigration' and has the effect of perpetuating 'exotic' and 'primitive' stereotypes of africa. although involuntary, the immediate and visceral effect produced in western minds by the thought of someone eating a chimpanzee, a dog or a bat, for instance, creates a feeling of disgust which downgrades this person, his/her needs and his/her claims on us. this issue has led to calls to replace the term with 'wild meat' or 'meat from wild animals'. secondly, while the term 'bushmeat' typically refers to the practice in the forests of africa, the trade of 'bushmeat', which has expanded over the past two decades, is considered as an example of an anthropogenic factor that provides opportunities for the transmission of diseases from wildlife to humans. the unsolved reconciliation between present policies and practices and the different values at stake (ecological, nutritional, economic and intrinsic values of wildlife hunted for food) in the actual 'bushmeat crisis', have accentuated the national and global conservation, development and health (infectious disease transmission related) concerns over hunting, eating and trading wild meat. thirdly, because of the many competing interests and realities involved, the proscription of hunting and consuming certain species of wild animals-in particular fruit bats and nonhuman primates during the west africa ebolavirus outbreak-has resulted in several unintended consequences, has incurred great cost and has had only a limited effect. in addition to being vague, inconsistent with scientific research and targeted to the wrong audience, messaging that unilaterally stressed the health risk posed by wild meat and fomite consumption contradicted the experiences of target publics, who consume wild meat without incident. consequently, in addition to having a negative impact on the livelihoods of people living at the frontlines of animal contact, the ban ran the risk of eroding public confidence in the response efforts and fuelling rumours as to the cause of evd (e.g. that the government was attempting to weaken villages in areas supporting the opposition party, as wild meat is considered an important source of physical 'strength' and energy). by focusing exclusively on the risk of spillover, we are distorting and concealing aspects of the dynamics at play. what if species boundaries are not perceived in the same way by everyone? what if the transgression of this 'invisible enemy' is spotted at a different intersection, beyond the nature/society binary? the first chains of human-to-human transmission led to the conclusion that the main vector of contamination was a health professional (s ) who spread the ebola virus in macenta, nzérékoré and kissidougou in february . the fifteenth patient, a doctor (s ), would have also contaminated his relatives in the same areas. the aetiological agent of this deadly disease (the ebola virus for some, the transgression of a taboo for others) remained hidden until then and finally became apparent because of clusters of cases in the hospitals of guéckédou and macenta. indeed, even though the high risk of exposures was elucidated, the problem remained hidden for a number of months, mainly because no doctor or health official had previously witnessed a case of ebola and because its clinical presentation was similar to many other endemic diseases experienced in guinea, such as cholera, which affects the region regularly. but these signals could also have been blurred by another narrative of the causative agent of these same symptoms. this is very similar to what genese marie sodikoff has identified during the recent bubonic plague epidemic in madagascar, when scientists elicited survivors' memories of dead rats in the vicinity to reconstruct the transmission chain. not only were these clues imperceptible to most, but residents had also constructed an alternative outbreak narrative based on different evidence. indeed, the mystico-religious beliefs deeply rooted in this region, even within the medical profession, have offered a different interpretation of causality according to a cultural model other than the biomedical model used by epidemiologists. following james fairhead, it is important to note that 'cultural' model does not tend here to slip into more totalising ideas of 'culture', such as a model being a 'kissi culture' (see below) nor its strict symmetrical opposite (e.g. a model of the 'humanitarian culture' or of a 'western culture'). origin and transmission chain according to an 'animist' model at the beginning of the epidemic, for some, the first deaths in forest guinea were due to the transmission of the filovirus through contact with animals' and/or patients' body fluids; while for others, these deaths originated from the transgression of a taboo related to the touch of a fetish belonging to a sick person, a member of a secret society belonging to one of the ethnic groups of the region. as a result, susceptibility to ebola was initially perceived to be restricted to this particular ethnic group, labelling ebola as an 'ethnic disease'. i decided to name this explanatory model of evd in forest guinea, the 'animist' model, not to further racialise this epidemic, but because it refers to the genies and fetishes that constitute principal aspects of the ancient religions of west africa and also because it describes a belief in a dual existence for all things-a physical, visible body and a psychic, invisible soul. according to a young pastor from macenta who i interviewed, and as confirmed by several other sources of key informants, the population of macenta initially attributed the origin of the disease (in this region at least) to a curse that was only affecting the kissi ethnic group because the first deaths solely affected people belonging to this ethnic group. here is what was stated: … on arrival with all the rumours we heard in conakry, i really did not believe in the beginning that it [the ebola virus disease] must be true because i thought it was an issue of the kissi (…) because it had started in macenta with the kissi, the first deaths were almost only kissi. so we thought it was something related to it … and so we, as toma, it was not going to touch us, it is like that at the beginning we perceived things (…) not something genetic, we thought about the fetishism and idolatry activities that people exercised and that can influence them in one way or another … the first rumour that was there, in macenta, the first death was the doctor who was dead in front of everyone's views. people said they have an idol called 'doma' and so when a person dies of that according to the tradition and according to what is done. and those who are on the thing [those who belong to the secret society of 'doma'] have no right to touch, to manipulate the corpse, or to see it otherwise they may die (…) and that, it existed before. it is a kind of secret society, so they have told us that it can certainly be that, that it is why they [the kissi] are just dying successively. according to these discourses, a health worker from guéckédou hospital (s ), who had gone to seek treatment at his friend's house at macenta hospital (s ), belonged, like him, to a secret initiation society called 'doma' which is also the name of a very powerful fetish; so powerful that it can cause a very fast death for its owner if it has been touched by someone else belonging to the same secret society. when the guéckédou health worker's body was moved, the doctor from macenta would have touched this fetish, idol, sacred object, often hidden in the owner's boubou (traditional clothing). by touching the sacred, the fetish got upset causing the brutal death of the director of macenta's hospital very soon after this event. at that point, in order to repair this transgression and calm the anger of the fetish, six more deaths must succeed each other to reach the symbolic number of seven. if the number of sudden and rapid deaths reaches eight, it means that the fetish is very powerful, and, as a result, seven additional deaths must occur to reach deaths to restore harmony and repair sacrilege. if we reach deaths, we must go to deaths before the disturbed order is restored and moreover that the stain is 'washed', and so on. since the first deaths of this second chain were indeed members of this kissi ethnic group (fig. . ), the 'animist' explanatory model of the disease was quite consistent with people's observations and gained legitimacy among the population at the expense of the biomedical discourse of the existence of evd. as the susceptibility of dying from ebola was initially and predominantly perceived as restricted to this particular ethnic group, no preventive measures were adopted by the non-kissi population of the region. among the kissi, the consequent epistemic dissonance between the public health logic and the transgression to be restored led between june and july twenty-six kissi-speaking villages in guéckedou prefecture to isolate themselves from ebola response, cutting bridges and felling trees to prevent vehicle access, and stoning intruding vehicles. because it is a disease of the social-of those who look after and visit others, and of those who attend funerals-there are of course many reasons why the ebola phenomenon was likely to be associated with sorcery. it is also not a coincidence that the triggering event, the transgression, in this explanatory model was attributed to medical doctors. as elite africans generally educated in european ways and relatively wealthy, this social group displays many characteristics of sorcerers (they lead a secluded life, do not share their gains, exchange abrupt greetings, eat large quantities of meat and eat alone). moreover, the intense preoccupation throughout this region with 'hidden evil in the world around you that finds dramatic expression in the clandestine activities of witches and the conspiracies of enemies' is exacerbated by tiny pathogens remaining largely invisible to our routine social practices, hence attracting suspicions of sorcery (fig. . ) . following the investigation of this 'animist' model in relation to the strong community resistance manifested in forest guinea, i interviewed a member of the riposte communication unit originating from macenta about the dandano case : yes, there is the specificity of dandano. (…) [in] dandano there was a great witch doctor who had gone to greet his counterpart witch doctor where there were a lot of cases. and that is where he got infected. he returned to dandano. three days later he developed the disease and died. afterwards, as he is a great, recognised witch doctor, people said to themselves, because he died, it was not ebola that killed him but his fetish that is taking revenge on him because it is a betrayal to leave one's domain to greet one's friend. maybe he went to spy on his friend and his friend hit him … well, there have been many versions. (…) among the old people who knew the drug he had, euh… his fetish, the grigri that he had, and that if it was his grigri who killed him, it means that all those who saw him, who saw his body, must also suffer. (…) [we could] see his dead body because he was not protected, because we had to wash him and there were medicines that had to be poured to annihilate his fetishes' power before burying him. so there must have been deaths, hence it was already premeditated. then there were deaths, as it was said, and they were successive deaths. that means there were deaths, two days, three days, so people put more anathema on what happened. and that is how dandano lived things. so there were deaths, we said it is the fetish that woke up because dandano is known as a village of powerful fetishes, that is known. (…) even all the sensitisation we do, we never stop in dandano on a manager, a notable, otherwise they can do something to you … so it is well recognised (…) dandano, is not where you have to go joking. (…) at the end, with a lot of deaths, a lot of funerals, they saw that no, it is not that [the fetish anger] anymore, and with the information here and there, it is ebola. and it is like that with all the negotiations (…). notably, these explanatory models are distinct from general beliefs about diseases and care techniques in the region. we cannot argue then that 'biomedicine' and 'kissi culture' are somehow distinct and opposed. chain of transmission according to the 'animist' cultural model. s and s are the two suspect cases as presented in the 'biomedical' chain of transmission (see fig. . ); the grey blocks are the kissi people of the 'animist' transmission chain these beliefs belong to the ideology of different sectors of the care system and exist independently of the illness of a subject. explanatory models are collected in response to a particular episode of illness in a given subject in a given sector and can evolve over time, depending on how the experience, knowledge and risk exposure of the concerned individual develop. this is precisely what has been reported to us and what has been observed in forest guinea. as the number of deceased progressed, and according to the religious and/or ethnic affiliation of the deceased, a new explanatory model was put in place as stated in this conversation: yes, at first it was said, when i was in conakry, since our country is predominantly muslim, it was said that it is a matter for christians since muslims do not eat apes. muslims do not eat the bat. it's only the foresters who eat that. and that's why this disease hits only the kissi and toma who are from the forest. so it's a kaf disease. -kaf? (séverine thys) -from unbelievers, pagans who do not know god. we call kaf, all those who do not believe in the god of muslims. this last extract particularly highlights the fact that these explanatory models are not fixed in time and space and are not impervious to each other either. indeed, the first health messages communicated to the population and built on the biomedical model were intensely focused on the need to avoid the consumption of 'bushmeat', especially wild animals identified as potential primary sources of contamination, namely monkeys and bats. the content of these messages gave birth to another popular model, in which the food taboos or eating habits observed by members affiliated to a certain religion allowed them to explain why this disease was affecting certain groups and not others. this quote also perfectly illustrates how popular discourses have integrated medical interpretations or public health messages. in the study conducted by bonwitt et al. about the local impact of the wild meat ban during the outbreak, all participants, irrespective of age or gender, were aware of wild mammals acting as a source of transmission for ebola. yet a confusion remained about which species in particular could transmit the ebola virus, which may be due to the content of public health messages that were inconsistent as regards the species shown to be potentially hazardous. messages are being absorbed, but in such chaos and fear, people process information according to their own worldview, according to the sources available to them, and following their personal experiences and instincts. furthermore, the criminalisation of wild meat consumption, which fuelled fears and rumours within communities, did entrench distrust towards outbreak responders and also exacerbated pre-existing tensions within villages, ethnicities and religions. following the kissi, it seemed that it was the muslim community that was hit by sudden and numerous deaths. to cope with this new upheaval, this new incomprehension, the operated explanatory model of these deaths' origin was, consequently, first that of a 'maraboutage': it started like that until a certain moment. and then it turned upside down. there have always been upheavals. it turned upside down, and instead of being weighed at a certain moment on the toma and the kissi, it was rather on the manyas, who are entirely, %, % even muslims. and so people started saying 'ha! that only attacks muslims, why not christians?'. so there has always been upheaval in all the procedures of this disease evolution. as noted by hewlett et al., 'patients, physicists, caregivers and local people in different parts of the world have cultural patterns for different diseases. providing care and appropriate treatment for a particular disease is often based on negotiation between these different models'. to be able to negotiate, it is necessary that each one, doctor and patient, partakes in the knowledge of the explanatory model of the other. while most health professionals rarely assume that people have and construct their own interpretation of the causal chain, my ethnographic observations presented in this chapter demonstrate that the a priori on which all interventions of sensitisation are based is not only incorrect, but also a source of blockages for the adoption of prescribed behaviours. this is because, to return to hewlett et al., 'people do not just follow the continuous thread of learning; they also develop an ability to articulate adherence to prescribed behaviours with the refusal of others, to cooperate at certain times and to show reluctance to others, inviting the analysis to move towards a sociology of compromise'. through the example of funerals, wilkinson and leach have also cast light on the presumption that the knowledge needed to stop the epidemic is held by public health experts and scientists, and not by local people. this very often leads to the development of protocols and procedures that completely negate the contribution of communities. this asymmetrical reflection between caregivers and care receivers, the structural violence that has cultivated inequalities in this region, the heterogeneity of experiences seen by the populations as fundamental contradictions between words and facts, the confidence and trust crisis since the 'demystification' programme initiated during sékou touré's time, and the traumas inflicted by a transgression of usages in the name of urgency and the exceptional nature of the ebola epidemic, are all realities that have fueled community reluctance and resistance. the late involvement of traditional healers, primarily consulted by guineans when experiencing illness, in the activities of the response in macenta, is another example of this asymmetry, which too often omits to acknowledge and relate to these other categories that support the social fabric, even if since alma ata in these stakeholders should no longer be on the margins of the health system. although the concept of explanatory models is not sufficient to explain all the failures of response in the context of guinea, or the bordering regions with sierra leone and liberia, nevertheless it allows to move past linear technical discussions of 'weak health systems' as the main reason for the scale of the disaster. the use of this conceptual framework for understanding popular interpretations of the origin of the disease and its transmission reveals the complex, historically rooted and multidimensional picture of the ebola crisis. several authors agree that, 'in any case, it is not a question of archaic beliefs or outlier depictions, but good answers -which can be called rational in this context -to a vital emergency situation, interpreted in the light of past and present experiences'. a better knowledge and comparison of these discourses and different cultural models of the disease, sometimes incorporated, sometimes hermetic, could nevertheless contribute considerably to the success of the fight against the epidemic, especially when it concerns the improvement of knowledge of the chains of disease transmission, the identification and understanding of the behaviours of local populations, and of the sources of denials and rumours. explanatory models proposed by the biomedical sciences are very often in competition and in contradiction with diagnoses made by traditional healers and especially with rumours involving divine punishments, breaches of prohibitions, the misdeeds of wizards or genies, or virologic warfare. if this 'animist' model is not identified nor recognised as making sense for others at the key moment, there will also be no negotiation and no understanding of the distances and proximities existing between the thought systems present in the concerned ecosystems. an anthropological approach remains essential to adapting this response to local realities. epelboin further argues that 'local models of causation regarding misfortune, often the most predominant, involve not only the virulence of the virus and human behaviour, but the evil actions of human and non-human individuals. the virologic model is then only one explanatory model among others, leaving the field open to all social, economic and political uses of misfortune'. following the re-emergence of this infectious disease of zoonotic origin in a whole new social ecosystem, a cross-sectoral research agenda, the so-called one health integrated approach, has finally emerged in the field of viral haemorrhagic fevers, also enabling the role of anthropology to be expanded to times of epidemic outbreak. until then, anthropologists were mandated to contribute to the adaptation and improvement of immediate public health interventions in relation to human-to-human transmission. yet, the growing interest of anthropologists in the interaction between humans and non-humans has made it possible to extend their research topic to the complex dynamics of the primary and secondary transmission of the virus. in addition, this anthropological interest has provided a new cross-cultural perspective on the movement of pathogens and has therefore improved knowledge about the mechanisms of emergence, propagation and amplification of a disease located at the interface between humans and wildlife. such was the role of almudena marí saéz and colleagues who, in a multidisciplinary team, conducted an ethnographic study in the village of the ebola epidemic's origin, the index case village, to better understand local social hunting practices and the relationships between bats and humans. however, the realm of the human-animal-disease interaction has been limited to 'natural versus cultural' domains and frequently conceived as a biological phenomenon in one health studies instead of a biocultural one integrating the social and cultural dimensions generated by human-animal relations. incorporating anthropology into one health approaches should provide a more nuanced and expanded account of the fluidity of bodies, categories and boundaries as drawn up by existing ethnographies on cattle in east and southern africa for example. epelboin et al. have stressed that, 'the anthropological approach in previous epidemics has confirmed that the urgency and severity of an epidemic must not prevent people from listening to them and thinking throughout the epidemic of taking into account indigenous codes, customs, knowledge, skills and beliefs'. by taking seriously the possibility that affected people in the places where we do research or implement control measures might not see things in the same way, we have to be willing to have our categories (such as culture/nature, human/animal, mind/body, male/female, caregivers/care receivers) unsettled, and to grapple with the practical implications of this for engagement in field sites, for knowledge-sharing and for the design of interventions, in the hope that such improvements might contribute to a future prevention of ebola and to public health policies more suitable to respond to people's basic needs. it also allows the affected people themselves to have a say in the matter. as philippe descola and other anthropologists have argued, on the basis of a comparative analysis of a wide range of ethnographic work across the continents, native classificatory systems usually offer a continuum, rather than sharp divisions, among humans and other animal species. indeed, human dispositions and behaviours are attributed not only to animals but also to spirits, monsters and artefacts, contrasting to modern western models, which generally see the categories of human and non-human as clearly defined and mutually exclusive. the ability to sense and avoid harmful environmental conditions is necessary for the survival of all living organisms and, as paul slovic has argued, 'humans have an additional capability that allows them to alter their environment as well as respond to it'. as regards the emerging violence in conservation as either against nature (e.g. culling bats) or in defence of it (e.g. rearranging landscapes within an inclusive 'one health' approach), james fairhead proposes that such violence is increasingly between 'the included' and 'rogues' in ways that transcend the nature/society binary. while the 'white', and african elites were seen by the affected population as 'antisocial' intruders or rogues, suspected of sorcery and using ebola as a tool for political manipulation, those involved in the struggle to address the ebola epidemic were not fighting just against the virus but also against the natural world that harboured it: the rogues which included villainous bats but moreover habitat destroyers, namely hunters, bushmeat traders and deforesters. these were the humans casted as the ones invading the habitat of the virus. since evd will be constantly reconceptualised, and because of new scientific discoveries (e.g. on natural reservoir, or vaccine development), control interventions must listen to and take into account popular perceptions as well as the socio-cultural and political context and their respective evolution. rumours must be identified and managed on a case-by-case basis without global generalisation that could reinforce misinterpretations on the assumption that ignorance alone generates these rumours, con-flicts, lack of trust and resistance. moreover, zoonotic epidemic fighters should follow macgregor's and waldman' recommendations by starting to think differently with and about animals and about species boundaries in order to generate novel ways of addressing zoonotic diseases, allowing for closer integration with people's own cultural norms and understandings of human-animal dynamics. and medicine ( ) ebola virus disease in guinea-update (situation as of aspects épidémiologiques de la maladie à virus ebola en guinée (décembre -avril emergence of zaire ebola virus disease in guinea investigating the zoonotic origin of the west african ebola epidemic zoonosis: prospects and challenges for in this outbreak story, a disease emerges in a remote location and spreads across a world highly connected by globalisation and air travel to threaten 'us all'-read the globally powerful north: see a entre science et fiction' contagious: cultures, carriers, and the outbreak narrative: wald priscilla the global focus on wildlife as a major contributor to emerging pathogens and infectious diseases in humans and domestic animals is due to reports which are not based on field, experimental or dedicated research but rather on surveys of literature and research regarding human immunodeficiency virus (hiv) and aids, severe acute respiratory syndrome (sars) and highly pathogenic avian influenza (hpai), all of which have an indirect wildlife link: r. kock, 'drivers of disease emergence and spread: is wildlife to blame on how and why 'bushmeat' hunting leads to the emergence of novel zoonotic pathogens see bushmeat hunting, deforestation, and prediction of zoonoses emergence uncovering zoonoses awareness in an emerging disease "hotspot"'. social science attempt of the zoonotic niche of evd, see contagious: cultures, carriers, and the outbreak narrative the term 'exceptionality' is borrowed from s. l. faye, 'l' "exceptionnalité" d'ebola et les "réticences" populaires en guinée-conakry. réflexions à partir d'une approche d'anthropologie symétrique to-plague-and-beyond-how-can-anthropologistsbest-engage-past-experience-to-prepare-for-new-epidemics. for the policy relevance of anthropological expertise and a (self-)critical reflection on ebola and on anthropological (and more broadly social scientific) engagements with humanitarian response, see a. menzel and a. schroven the term 'riposte' is the french name used to designate the official national mobilisation settled to respond to the evd crisis, structured into two poles, an inter-ministerial committee and a national coordination committee grouping together the international actors and the national non-governmental organisations; see m. fribault heterogeneities in the case fatality ratio in the west african ebola outbreak challenges in controlling the ebola outbreak in two prefectures in guinea: why did communities continue to resist? comparison of social resistance to ebola response in sierra leone and guinea suggests explanations lie in political configurations not culture understanding social resistance to the ebola response in the forest region of the republic of guinea: an anthropological perspective contagious: cultures, carriers, and the outbreak narrative zoonosis: prospects and challenges for medical anthropology the good, the bad and the ugly: framing debates on nature in a one health community understanding social resistance to the ebola response in the forest region of the republic of guinea: an anthropological perspective sustainability and contemporary man-nature divide: aspects of conflict and alienation on the visual ethnographic examination of the ebola zoonotic cycle transformed into tools of public health communication by the us cdc during the outbreak of medical anthropology and ebola in congo unintended consequences of the "bushmeat ban emergence of zaire ebola virus disease in guinea maladie à virus ebola: une zoonose orpheline?'. bulletin de l'académie vétérinaire de france inclusivity and the rogue bats and the war against "the invisible enemy about the natural reservoir for ebola virus see the evolution of ebola virus: insights from the - epidemic' a review of the role of food and the food system in the transmission and spread of ebolavirus mammalian biogeography and the ebola virus in africa for information on the 'bushmeat ban', see bonwitt et al., 'unintended consequences of the "bushmeat ban ebola virus disease epidemic emergence of zaire ebola virus disease in guinea'; world health organization, 'one year into the ebola epidemic: a deadly, tenacious and unforgiving virus caring for critically ill patients with ebola virus disease ebola-myths, realities, and structural violence'; and olival and hayman the threat to primates and other mammals from the bushmeat trade in africa, and how this threat could be diminished origins of major human infectious diseases'; centers for disease control and prevention take-a-semiotician-or-what-we-talk-aboutwhen-we-talk-about-bush-meat-by-adia-benton/. the kellogg institute on the feeling of disgust as a sentiment with powerful political valences, see also j. livingston, 'disgust, bodily aesthetics and the ethic of being human in botswana the anatomy of disgust world organisation for animal health the bushmeat trade: increased opportunities for transmission of zoonotic disease bushmeat crisis' is caused by the dual threats of wildlife extinctions and declining food and livelihood security of some of the poorest people on earth and whether the hunting of bushmeat is primarily an issue of biodiversity conservation or human livelihood, or both, varies according to perspective, place and over time; see unintended consequences of the "bushmeat ban impact of the ebola virus disease outbreak on market chains and trade of agricultural products in west africa'. food and agriculture organization of the united nations sending the right message: wild game and the west africa ebola outbreak bushmeat ban" in west africa during the - ebola virus disease epidemic'; p. richards, ebola: how a people's science helped end an epidemic les errances de la communication sur la maladie à virus ebola zoonotic semiotics: plague narratives and vanishing signs in madagascar' understanding social resistance to the ebola response in the forest region of the republic of guinea: an anthropological perspective one year on: why ebola is not yet over in guinea encyclopedia of medical anthropology : health and illness in the world's cultures extracts of the individual interview conducted with the pastor on the cultural and political role of initiation societies in the forest region and the related experiences of local citizens in relation to both the manding (often islamic) world to the north, and to the 'white' (often christian) colonial and neo-colonial order, see fairhead purity and danger, an analysis of concepts of pollution and taboo communication with rebellious communities during an outbreak of ebola virus disease in guinea: an anthropological approach understanding social resistance to the ebola response in the forest region of the republic of guinea: an anthropological perspective memories of the slave trade: ritual and historical imagination in sierra leone lifeworlds: essays in existential anthropology for more information about dandano village 'surrendering their sick and dead after being battered by the virus', see a. nossiter extracts of the individual interview conducted with a voluntary of the communication unit of macenta new therapeutic landscapes in africa: parental categories and practices in seeking infant health in republic of guinea extracts of the individual interview conducted with the pastor for similar narrative about muslim communities and food taboos regarding bats, see f. batty, 'reinventing "others" in a time of ebola unintended consequences of the "bushmeat ban extracts of the individual interview conducted with the pastor medical anthropology and ebola in congo: cultural models and humanistic care ebola en guinée: violences historiques et régimes de doute briefing: ebola-myths, realities, and structural violence l' "exceptionnalité" d'ebola et les "réticences" populaires en guinée-conakry ebola en guinée: violences historiques et régimes de doute'; wilkinson and leach traiter les corps comme des fagots' production sociale de l'indifférence en contexte ebola (guinée approche anthropologique de l'épidémie de fhv ebola en guinée conakry zoonosis: prospects and challenges for medical anthropology extending the "social": anthropological contributions to the study of viral haemorrhagic fevers investigating the zoonotic origin of the west african ebola epidemic views from many worlds: unsettling categories in interdisciplinary research on endemic zoonotic diseases animal spirits and mimetic affinities: the semiotics of intimacy in african human/animal identities annexe . contribution de l'anthropologie médicale à la lutte contre les épidémies de fièvres hémorragiques à virus ebola et marburg'. in world health organisation, Épidémies de fièvres hémorragiques à virus ebola et marburg: préparation, alerte, lutte et évaluation the morning after: anthropology and the ebola hangover beyond nature and culture biosecurity and the topologies of infected life: from borderlines to borderlands nature and society: anthropological perspectives by perception of risk inclusivity and the rogue bats and the war against "the invisible enemy views from many worlds acknowledgements i would like to thank tenin traoré, a guinean sociologist and consultant to who, and joseph kovoïgui, assistant coordinator of the philafrican mission and then consultant to who, for their commitment and engagement in the fight against ebola, their generosity, their knowledge, their experience and our fruitful collaboration in many respects. i would also like to thank the coordination team and the dps (prefectural health direction) of macenta for their welcome and sincere attention; goarn/who, antwerp institute of tropical medicine, and in particular prof. marleen boelaert for emotional, financial and logistical support; dr. alain epelboin for field preparation and numerous sharing with the francophone anthropological platform; and christos lynteris for his invitation to connect and exchange with the anglophone 'anthro-zoonoses' network and contribute to this timely collection. key: cord- -mfh ope authors: zlabinger, markus; hofstätter, sebastian; rekabsaz, navid; hanbury, allan title: dsr: a collection for the evaluation of graded disease-symptom relations date: - - journal: advances in information retrieval doi: . / - - - - _ sha: doc_id: cord_uid: mfh ope the effective extraction of ranked disease-symptom relationships is a critical component in various medical tasks, including computer-assisted medical diagnosis or the discovery of unexpected associations between diseases. while existing disease-symptom relationship extraction methods are used as the foundation in the various medical tasks, no collection is available to systematically evaluate the performance of such methods. in this paper, we introduce the disease-symptom relation collection (dsr-collection), created by five physicians as expert annotators. we provide graded symptom judgments for diseases by differentiating between relevant symptoms and primary symptoms. further, we provide several strong baselines, based on the methods used in previous studies. the first method is based on word embeddings, and the second on co-occurrences of mesh-keywords of medical articles. for the co-occurrence method, we propose an adaption in which not only keywords are considered, but also the full text of medical articles. the evaluation on the dsr-collection shows the effectiveness of the proposed adaption in terms of ndcg, precision, and recall. disease-symptom knowledge bases are the foundation for many medical tasks -including medical diagnosis [ ] or the discovery of unexpected associations between diseases [ , ] . most knowledge bases only capture a binary relationship between diseases and symptoms, neglecting the degree of the importance between a symptoms and a disease. for example, abdominal pain and nausea are both symptoms of an appendicitis, but while abdominal pain is a key differentiating factor, nausea does only little to distinguish appendicitis from other diseases of the digestive system. while several disease-symptom extraction methods have been proposed that retrieve a ranked list of symptoms for a disease [ , , , ] , no collection is available to systematically evaluate the performance of such methods [ ] . while these method are extensively used in downstream tasks, e.g., to increase the accuracy of computer-assisted medical diagnosis [ ] , their effectiveness for disease-symptom extraction remains unclear. in this paper, we introduce the disease-s ymptom relation collection (dsrcollection) for the evaluation of graded disease-symptom relations. the collection is annotated by five physicians and contains symptoms for diseases. we label the symptoms using graded judgments [ ] , where we differentiate between: relevant symptoms (graded as ) and primary symptoms (graded as ). primary symptoms-also called cardinal symptoms-are the leading symptoms that guide physicians in the process of disease diagnosis. the graded judgments allow us for the first time to measure the importance of different symptoms with grade-based metrics, such as ndcg [ ] . as baselines, we implement two methods from previous studies to compute graded disease-symptom relations: in the first method [ ] , the relation is the cosine similarity between the word vectors of a disease and a symptom, taken from a word embedding model. in the second method [ ] , the relation between a disease and symptom is calculated based on their co-occurrence in the meshkeywords of medical articles. we describe limitations of the keyword-based method [ ] and propose an adaption in which we calculate the relations not only on keywords of medical articles, but also on the full text and the title. we evaluate the baselines on the dsr-collection to compare their effectiveness in the extraction of graded disease-symptom relations. as evaluation metrics, we consider precision, recall, and ndcg. for all three metrics, our proposed adapted version of the keyword-based method outperforms the other methods, providing a strong baseline for the dsr-collection. the contributions of this paper are the following: -we introduce the dsr-collection for the evaluation of graded disease-symptom relations. we make the collection freely available to the research community. -we compare various baselines on the dsr-collection to give insights on their effectiveness in the extraction of disease-symptom relations. in this section, we describe the new disease-s ymptom relation collection (dsr-collection) for the evaluation of disease-symptom relations. we create the collection in two steps: in the first step, relevant disease-symptom pairs (e.g. appendicitis-nausea) are collected by two physicians. they collect the pairs in a collaborative effort from high-quality sources, including medical textbooks and an online information service that is curated by medical experts. in the second step, the primary symptoms of the collected disease-symptom pairs are annotated. the annotation of primary symptoms is conducted to incorporate a graded relevance information into the collection. for the annotation procedure, we develop guidelines that briefly describe the task and an online annotation tool. then, the annotation of primary symptoms is conducted by three physicians. the final label is obtained by a majority voting. based on the labels obtained from the majority voting, we assign the relevance score to primary symptoms and to the other symptoms, which we call relevant symptoms. in total, the dsr-collection contains relevant symptoms and primary symptoms for diseases. we give an overview of the collection in table . for the diseases, the collection contains a total of symptoms, of which are labeled as primary symptom (about %). the top- most occurring symptoms are: fatigue which appears for of the diseases, fever which appears for , and coughing which appears for . notice that the diseases are selected from different medical disciplines: mental (e.g. depression), dental (e.g. periodontitis), digestive (e.g. appendicitis), and respiration (e.g. asthma). we calculate the inter-annotator agreement using fleiss' kappa [ ] , a statistical measure to compute the agreement for three or more annotators. for the annotation of the primary symptoms, we measure a kappa value of κ = . , which indicates a substantial agreement between the three annotators [ ] . individual κ-values per disease are reported in table . by analyzing the disagreements, we found that the annotators labeled primary symptoms with varying frequencies: the first annotator annotated on average . primary symptoms per disease, the second . , and the third . . vocabulary compatibility: we map each disease and symptom of the collection to the unified medical language system (umls) vocabulary. the umls is a compendium of over vocabularies (e.g. icd- , mesh, snomed-ct) that are cross-linked with each other. this makes the collection compatible with the umls vocabulary and also with each of the over cross-linked vocabularies. although the different vocabularies are compatible with the collection, a fair comparison of methods is only possible when the methods utilize the same vocabulary since the vocabulary impacts the evaluation outcome. for instance, the symptom loss of appetite is categorized as a symptom in mesh; whereas, in the cross-linked umls vocabulary, it is categorized as a disease. therefore, the symptom loss of appetite can be identified when using the mesh vocabulary, but it cannot be identified when using the umls vocabulary. evaluation: we consider following evaluation metrics for the collection: recall@k, precision@k, and ndcg@k at the cutoff k = and k = . recall measures how many of the relevant symptoms are retrieved, precision measures how many of the retrieved symptoms are relevant, and finally, ndcg is a standard metric to evaluate graded relevance [ ] . in this section, we discuss disease-symptom extraction methods used in previous studies. a commonly used resource for the extraction of disease-symptom relations are the articles of the pubmed database. pubmed contains more than million biomedical articles, including the abstract, title, and various metadata. previous work [ , ] uses the abstracts of the pubmed articles together with rule-based approaches. in particular, hassan et al. [ ] derive patterns of disease-symptom relations from dependency graphs, followed by the automatic selection of the best patterns based on proposed selection criteria. martin et al. [ ] generate extraction rules automatically, which are then inspected for their viability by medical experts. xia et al. [ ] design special queries that include the name and synonyms of each disease and symptom. they use these queries to return the relevant articles, and use the number of retrieved results to perform a ranking via pointwise mutual information (pmi). the mentioned studies use resources that are not publicly available, i.e., rules in [ , ] and special queries in [ ] . to enable reproducibility in future studies, we define our baselines based on the methods that only utilize publicly available resources, described in the next section. here, we first describe two recently proposed methods [ , ] for the extraction of disease-symptom relations as our baselines. afterwards, we describe limitations of the method described in [ ] and propose an adapted version in which the limitations are addressed. we apply the methods on the open-access subset of the pubmed central (pmc) database, containing , , medical articles. to have a common representation for diseases/symptoms across methods (including an unique name and identifier), we consider the symptoms and , diseases from the medical subject headings (mesh) vocabulary [ ] . given the set of diseases (x) and symptoms (s), each method aims to compute a relation scoring function λ(x, s) ∈ r between a disease x ∈ x and a symptom s ∈ s. in the following, we explain each method in detail. embedding: proposed by shah et al. [ ] , the method is based on the cosine similarity of the vector representations of a disease and a symptom. we first apply metamap [ ] , a tool for the identification of medical concepts within a given text, to the full text of all pmc articles to substitute the identified diseases/symptoms by their unique names. then, we train a word vec model [ ] with dimensions and a window size of , following the parameter setting in [ ] . using the word embedding, the disease-symptom relation is defined as λ(x, s) = cos(e x , e s ), where e refers to the vector representation of a word. cooccur: this method, proposed by zhou et al. [ ] , calculates the relation of a disease and a symptom, by measuring the degree of their co-occurrences in the mesh-keywords of medical articles. the raw co-occurrence of the disease x and symptom s, is denoted by co(x, s). the raw co-occurrence does not consider the overall appearance of each symptom across diseases. for instance, symptoms like pain or obesity tend to co-occur with many diseases, and are therefore less informative. hence, the raw co-occurrence is normalized by an inverse symptom frequency (isf) measure, defined as isf(s) = |x| ns , where |x| is the total number of diseases and n s is the number of diseases that co-occur with s at least in one of the articles. finally, the disease-symptom relation is defined as λ(x, s) = co(x, s) × isf(s). we compute three variants of the cooccur method: -kwd: the disease-symptom relations are computed using the mesh-keywords of the ≈ . million pmc articles. -kwdlarge: while kwd uses the . million pmc articles, zhou et al. [ ] apply the exact same method on the ≈ million articles of the pubmed database. while they did not evaluate the effectiveness of their diseasesymptom relation extraction method, they published their relation scores which we will evaluate in this paper. -fulltext: applying the cooccur method only on mesh-keywords has two disadvantages: first, keywords are not available for all articles (e.g. only % of the ≈ . million pmc articles have keywords) and second, usually only the core topics of an article occur as keywords. we address these limitations by proposing an adaption of the cooccur method, in which we use the full text, the title, and the keywords of the ≈ . million pmc articles. specifically, we adapt the computation of the co-occurrence co(x, s), as follows: we first retrieve a set of relevant articles to a disease x, where an article is relevant if the disease exists in either the keyword, or the title section of the article. given these relevant articles and a symptom s, we compute the adapted co-occurrence co(x, s), which is the number of relevant articles in that the symptom occurs in the full text. the identification of the diseases in the title and symptoms in the full text is done using the metamap tool [ ] . we now compare the disease-symptom extraction baselines on the proposed dsrcollection. the results for various evaluation metrics are shown in table . the fulltext-variant of the cooccur method outperforms the other baselines on all evaluation metrics. this demonstrates the high effectiveness of our proposed adaption to the cooccur method. further, we see a clear advantage of the cooccur-method with meshkeywords from ≈ million pubmed articles as the resource (kwdlarge) -in comparison to the same method with keywords from approximately . million pmc articles (kwd). this highlights the importance of the number of input samples to the method. error analysis: a common error source is a result of the fine granularity of the symptoms in the medical vocabularies. for example, the utilized mesh vocabulary contains the symptoms abdominal pain and abdomen, acute . both symptoms can be found in the top ranks of the evaluated methods for the disease appendicitis (see table ). however, since the corpus is not labeled on such a fine-grained level, the symptom abdomen, acute is counted as a false positive. another error source is a result of the bias in medical articles towards specific disease-symptom relationships. for instance, between the symptom obesity and periodontitis a special relationship exists, which is the topic of various publications. despite obesity not being a characteristic symptom of a periodontitis, all methods return the symptom in the top- ranks. a promising research direction is the selective extraction of symptoms from biomedical literature by also considering the context (e.g. in a sentence) in that a disease/symptom appears. effective mapping of biomedical text to the umls metathesaurus: the metamap program measuring nominal scale agreement among many raters extracting disease-symptom relationships by learning syntactic patterns from dependency graphs cumulated gain-based evaluation of ir techniques binary and graded relevance in ir evaluations -comparison of the effects on ranking of ir systems the measurement of observer agreement for categorical data symptom extraction issue distributed representations of words and phrases and their compositionality automated medical diagnosis by ranking clusters across the symptom-disease network neural networks for mining the associations between diseases and symptoms in clinical notes enhancing ontology-driven diagnostic reasoning with a symptom-dependency-aware naïve bayes classifier evaluating wikipedia as a source of information for disease understanding mining disease-symptom relation from massive biomedical literature and its application in severe disease diagnosis human symptoms-disease network we introduced the disease-s ymptom relation collection (dsr-collection) for the evaluation of graded disease-symptom relations. we provided baseline results for two recent methods, one based on word embeddings and the second on the cooccurrence of mesh-keywords of medical articles. we proposed an adaption to the co-occurrence method to make it applicable to the full text of medical articles and showed significant improvement of effectiveness over the other methods. key: cord- -elzkwyz authors: day, brennan; burnice mckay, ruth; ishman, michael; chung, ed title: the new normal: lessons learned from sars for corporations operating in emerging markets date: - - journal: nan doi: . / sha: doc_id: cord_uid: elzkwyz the modern industrialized world was completely caught off guard by the recent sars outbreak. fortunately, for most organizations, the impact has been short lived, but management has been provided with a reminder of the impact of the external environment in a world of ever increasing globalization. as seen with the sars outbreak, a lack of preparedness can have devastating effects on business and warrant inclusion in a business definition of a crisis. this paper uses the recent sars epidemic as a background to highlight the importance of crisis planning, particularly in emerging economies, and suggests how organizations can address these concerns. the implementation of environmental scanning in strategic planning is critical to the survival of an organization. that businesses operate in the context of some larger system, not a vacuum, is today part of the accepted business perspective. however, we ought to recognize that scanning and the related processes of contingency planning presume our ability to discern future patterns. many well-known business mistakes, such as the big three's disastrous handling of the energy crisis in the s, can be attributed as much to inadequate managerial skills as ignorance about environmental trends. after all, it was not any existing predators that eradicated the dinosaurs, but instead it was something never before seen and hence could not have been anticipated. the fact that the meteor was not in the dinosaur's vernacular did not prevent a meteor from changing the course of earth's history. the scope of our environmental scanning activities expands as our knowledge increases. such scanning is informed by new developments that have often been prospering just below the corporate radar screen. the recent (and arguably not over) sever acute respiratory syndrome (sars) outbreak is a case in point. even though contagious diseases are well-known, and their fatal impact acknowledged, e.g. pneumonic plague, spanish-flu and bubonic plague, the world was caught completely off-guard by the sars outbreak. despite superhuman efforts by the many health-care workers around the world, it was months (and many deaths) before the world health organization pronounced the epidemic temporarily halted. the health-care establishment could not have predicted the outbreak. as devastating as it was for many businesses, strategic planners would have to be psychic to have anticipated the disease and to incorporate it in the planning process. however, it would be useful to delineate lessons that can be learned from the recent sars (and other) outbreaks, and discuss how strategic management may benefit from an appreciation of these lessons. in preparation for unforeseen external events organizations should have a plan in place to reduce the influence on the organization and provide an operation manual in the event of a crisis. however, many fortune , firms do not have a crisis-management plan at all (hickman and crandall, ) . as seen with the sars outbreak this lack of preparedness can have devastating effects on business and warrant inclusion in our business definition of a crisis. a crisis is defined as a low probability/high consequence event, caused by human actions, that threatens the most fundamental goals of an organization (weick, ) . mitroff and pearson ( ) categorized crises according to two dimensions: normal to sever and technical/economic to human/social. however, their crisis analysis included only industrial health problems, not epidemics. there is a need to broaden the scope of crisis to include epidemics. our paper aims to provide this addition. this paper will start by presenting background information on the sars epidemic and the impact on organizations, especially those operating in emerging markets. next, characteristics of countries termed emerging markets will be identified and the inherent risks and challenges organizations face operating in these markets will be discussed. third, we offer recommendations for minimizing the impact of operating in an emerging market where contagious diseases are a reality. this represents an important first step towards developing (and implementing) a robust contingency plan for purposes of business continuity. on the basis of purchasing power parity, emerging and developing economies account for percent of the world's economy (gingrich, ) . conservative predictions conclude that by the year the developing world will account for over percent of the total world output (belous et al., ) . given the substantial growth in these economies, it is not surprising that the us department of commerce's international trade administration recently predicted that the greatest commercial opportunities will be found in the big emerging markets such as the people's republic of china (china), india, singapore, mexico, vietnam, or south africa (qureshi, ) . china provides perhaps the clearest illustration. for example, in china received $ billion in direct foreign investment, overtaking the united states to make it the world's biggest recipient of direct foreign investment (buckman, ) . according to the us bureau of economic analysis in , china and hong kong accounted for nearly a quarter of the income of us overseas subsidiaries (karmin, ) . however, the recent sars outbreak has shown how quickly commerce can be impacted in the global market. according to richard miller, an economist and vice president of the world travel & tourism council: "never before has there been a massive impact of this nature other than a war shutting down an economy totally" (prystay, ) . as of june , , outbreaks of sars have occurred in countries, resulting in , individuals being infected and subsequent deaths (world health organization, ) . economists are predicting that china, as a result of sars may see gdp growth drop below percent to its lowest level in nearly a decade (leggett, ) . some areas have been hit more harshly than others. in the chinese province of guangdong, the place where sars is believed to have started, the country's largest export-trade fair saw orders drop in april nearly percent (leggett, ) . in beijing, draconian measures were imposed to try to stem the spread of sars. public schools and universities were closed, migrant workers were told to stay in the capital and make-shift roadblocks were set up in villages to prevent city dwellers from entering (kaufman and chen, ) . in may, hong kong retail sales were down to percent, restaurants and their suppliers were going broke and hotel occupancy rates were in the single digits. airlines were also severely impacted. cathay pacific airway during this time dealt with an percent decrease in air traffic from a year earlier (meredith, ) . such a decrease in travel is not surprising given the overall drop in business air travel globally. in a survey conducted by the society for human resource management in the usa, based on human resource professionals, percent reported cuts in employee travel as a result of sars (usa today, ) . kingsway international holdings limited, a hong kong investment holding company that engages in securities brokerage, corporate finance advisory services, fund and asset management, securities investments, and strategic investment in technology-related businesses has seen earning per share drop from $ . in june to $ . in june . the company attributes the results to the difficult operating conditions including the influence of sars (infomart, ) . march sales of desktop computers in hong kong plunged percent, laptop sales dropped percent and mobile-phone sales fell percent (leggett, ) . cell phone sales dropped percent in guangzhou, the shanghai auto show only drew one quarter of its expected visitors and motorola shut its -story asia pacific headquarters building in beijing (engardio et al., ) . businesses located in developed nations have also been impacted. in australia, some small-to-medium sized businesses could face losses of up to $ million due to sars. westpac bank has predicted that sars could reduce australia's economic growth by . percent (chong, ) . multinational corporations are also being impacted. for example m, the makers of the n face mask, one of the few face masks that are percent effective at filtering out airborne diseases like sars, does percent of its $ billion in annual sales in the asia pacific region. j.p. morgan lists m as well as intel and united technologies as american companies that could be hurt by sars (freedman, ) . other companies suggesting revenues will be down as a result of sars include american international group inc., yum brand inc., goodrich corp. (karmin, ) and cara operations ltd. (flavelle, ) . another industry feeling the impact of sars is the phone makers. motorola, a company holding percent of the chinese mobile phone market, has warned that it may fall short of sales and profit forecasts for the second quarter and year end citing weakening demand in asia because of sars (timmons, ) . four season's hotel inc. has a net loss in the first quarter of cents per share compared to net income of cents per share a year earlier (infomart, ) . sars is seen as a key contributor to this loss. the city of toronto also provides an indication of the economic impact of sars claiming the cost to the government for health care and income compensation for those quarantined for the first six months of totaled us$ million (chowdhury, ) . while the above figures are indicators of the effect of sars, the true impact may never be known. it is believed that china, in order to maintain a semblance of stability and economic prosperity at the time of changing government leaders, covered up the md , truth about the extent of sars. it went so far as to remove sars patients from hospitals to deceive inspectors from the world health organization (jue, ) . in april, jiang yanyong blew the whistle on chinese authorities' deceit regarding the truth about sars in beijing. in a letter to journalists, when beijing was saying that only people in the city were infected with sars, dr jiang disclosed that the true number of sars patients was at least , including ten who had died. in response to dr jiang's revelation the health minister and beijing mayor were fired. for a regime with a long history of deception and denial, this event has been viewed as a revolution in official openness (york, ) . in a step to reinforce openness the chinese government has threatened to execute anyone who knowingly spreads the sars virus (dean, ) . however, this may have the reverse impact of making people hesitant to admit they have sars for fear of punishment. by lying about the true number of sars patients, beijing damaged what little faith international economist has in its numbers. for example, repeated yearly claims of percent annual gdp may be questionable. for the new chinese leadership the incentive to cheat is even greater than usual. strong gdp growth has been the key to the communist party's hold on power. the new government would not want to be associated with instant economic failure (august, ) . in addition to the sars outbreak there is news that another deadly virus has emerged in southern china through the winter. the associated press ( ) reported an outbreak of encephalitis in southern china that has (at time of writing) affected several hundred people and resulted in more than a dozen deaths. at the same time as this new disease is spreading in china and hong kong, the government in hong kong is pushing ahead with tough new security laws which journalists fear could inhibit future reporting of crisis. under the new legislation reporters could well be jailed for publishing details of state secrets and confidential communication between hong kong and beijing. thomas crampton, president of the foreign correspondents club of hong kong stated that in a country where health statistics are a state secret the legislation "puts a pall of uncertainty over reporting in hong kong. it encourages self-censorship" (korporaal, ) . for businesses operating in this and related regions, another outbreak and the possibility of another chinese cover up could not be good news. while it is hard to beat the wage rates of to cents an hour in china, it is a constant corporate challenge to operate in an environment where propaganda and obfuscation are used as government marketing tools. given the rapid onset of sars, the presence of other contagious diseases in the world and the government and infrastructure challenges for corporations operating in emerging markets, it is essential that corporations are prepared for future contagious disease outbreaks. it is not clear if sars can be eradicated. the disease could gain a toehold in the southern hemisphere, or it could lie low until cold weather returns (regalado, ) . while sars is taking the center stage, areas in europe are dealing with another disturbing virus that is spreading among chickens and in some cases humans. the avian flu has been found in the netherlands, belgium and germany. the virus, which rarely causes serious illness in humans, infected people who came in contact with infected birds (fuhrmans, ) . it is also conceivable that corporations may be operating in regions of the world where they will face bio-terrorism. as an indication of the possibility of such sabotage the us department of justice and the federal emergency management agency staged a mock terrorist attack on chicago airports in may . this is the second such exercise, the first being staged at a denver concert hall in may . in this year's exercise, terrorists sprayed unseen germs over chicago airports, infecting thousands with a lethal pneumonia. the exercise was undertaken to determined level of preparedness in the event of bio-terrorism (chase, ) . according to the australian state chamber of commerce chief executive officer, margy osmond, "(t)he lack of preparation by many businesses in terms of handling workplace issues and risks arising from the (sars) virus is . . . a concern". she also notes that percent of corporate respondents had a risk management plan to deal with potential disruptions created by sars but only percent had made this plan known to their staff. benefits of a fast response can be seen in the fast reaction of the canadian health authorities dealing with sars preventing the outbreak from reaching unmanageable numbers. according to castillo-chavez, a mathematical epidemiologist at cornell university, if the canadian outbreak had been left to run unchecked it could easily have infected as many as , in the toronto area (brown, ) . the toronto sars experience is a warning that even a giant thriving city can be laid low by a nasty and highly contagious disease (vogel, ) . since emerging markets are increasingly important to the world economy and are at the same time susceptible to outbreaks of infectious diseases, we need to understand how we are linked together on an interdependent global level. that interdependency is highlighted in the following section. the days of national economies existing as relatively self-contained entities are rapidly disappearing. individual economies are no longer isolated from each other by barriers to cross-border trade and investment -by distance, time zones, and language -and by national differences in government regulation, culture, and business systems. national economies are merging into an interdependent global economic system and, consequently, the rich industrial countries are dominating the world economy relatively less than they used to. at the beginning of its fiscal year (ending on june ), countries had been identified by the world bank as developing economies (international bank for reconstruction and development /the world bank, , p. ). the developed world, however, currently represents more than percent of total world output, while the developing nations represent less than percent. this proportion is poised to change in the near future though, with conservative projections indicating that by the situation could be reversed, with the developing world representing well over percent of total world output (belous et al., , p. ). the developing countries that have been the frontrunners in economic advancement have been designated as emerging economies by the economist. this classification is based on both the gross domestic product of each nation and the capitalization of their stock markets. these countries have historically played an unparalleled role in the global economy -a role that is well positioned to steadily expand in international importance. the emerging economies are home to approximately percent of the world's population and the population growth rates of these economies are the highest of all countries. the five largest developing nations, home to . billion people, have a combined population that is four times that of the g- countries. the populations of china and india alone, . billion and more than billion respectively, easily outnumber those of many developed countries combined (cavusgil et al., , pp. , ) . many of the world's developed countries now have birth rates below those that are required to maintain their present population levels. furthermore, individuals in the developed world are living progressively longer and leaving the work force earlier. with fewer workers generating output, and with more of the population dependent on those who do, income growth, savings levels, and economic growth will all be lower. in contrast, the populations of emerging countries are comparatively young. the working-age population in these markets -which will be the portion of the population driving economic and consumption growth -will grow at rates three to four times that of the developed world. the emerging markets also offer a large pool of low-cost, unskilled labor and it has become increasingly commonplace for large, multinational corporations to transfer their assembly and production plants from developed locations to these areas (gingrich, ) . the size of the consumer markets in the emerging countries now rivals those of the developed markets in many cases. for instance, the combined gross domestic product of the ten largest emerging markets is now nearly two-thirds the size of that of the g- counties in purchasing power parity terms. five emerging economies -china, india, brazil, mexico, and indonesia -are among the largest economies in the world and have a combined purchasing power already half of that of the g- . according to the international monetary fund (imf), developing countries will achieve a growth rate of more than percent per year over the next two decades, whereas industrialized countries are likely to average . percent (cavusgil et al., , p. ) . if just three of the asian emerging economies -china, india, and indonesia -are able to maintain this growth rate of percent per year, the organisation for economic co-operation and development (oecd) has estimated that by approximately million people in those countries will have an average income equivalent to that of spain today. as a group, they are roughly equivalent in population to the usa, japan, and europe combined (us department of commerce, , p. ). given these trends, multinational corporations (mncs) face profound changes in the economic landscape. over the next to years, most of the total world growth in consumption of consumer goods will likely be concentrated within the emerging economies. the emerging markets have also become major suppliers of many of the natural resources that the industrialized world relies on. for example, mexico, venezuela, and indonesia, in addition to several middle eastern countries, are major sources of oilstill the most vital energy source for developed countries. similarly, brazil, russia, and china, along with developing countries papua new guinea and jamaica, supply half of the world's bauxite (aluminum ore) (day, , p. ) . looking to the future, the supplier role of the emerging countries will expand since the exploitation of natural resources in many of the wealthy countries has reached its limit. either the oil fields, mines, or forests have been tapped out, or environmental regulations reduce new exploration and development. industrialized nations are therefore becoming increasingly dependent on emerging markets to ensure that sufficient levels of natural resources are available. overall, development in the emerging markets has been steady, but none of these countries have been fortunate enough to avoid certain growing pains along the way. one historical trend of many emerging markets is the inability of their currencies to hold value. rather than attack the principal causes of currency depreciation, many governments opt instead for a short term, cosmetic solution. after several years of declining currency value, they might print a new currency that "lops off two zeros" from the denomination of the older currency (hooke, , p. ) . in , for example, mexico initiated the new peso that was worth , old pesos. these superficial actions diminish investor confidence. additionally, widespread corruption exists in virtually all emerging markets, with hong kong and singapore being the most notable exceptions. many politicians view a government career as similar to a private sector job, with one primary goal in mind: making money. with many top officials setting a poor example, low-level bureaucrats are active in demanding payments for business licenses, permits, and concessions. since public sector jobs, as compared to a private sector position, have low salaries and no stock options, the difference is often made up through bribes and insider deals. public employees and elected officials accept these prerequisites in exchange for government contracts, licenses, and privileges (hooke, , p. ) . environmental issues in the emerging world often run unchecked. due to their size, their growing industrialization, and the urgency they attach to business expansion and job creation, many emerging markets have barely begun to put in place effective environmental programs. they often lack, or claim to lack, the financing capability to address the environment at this stage of their development, especially in light of competing priorities such as infrastructure. despite these ever-present risks and challenges that exist in the emerging world, the staggering economic impact of many of these countries coupled with their substantial population growth has created a growth potential too significant to be ignored by the developed world. accordingly, many private sector and government analysts continue to point to emerging markets as a potential major source of western economic growth and profit in the coming years (day, , p. ) . over the past three decades emerging markets have consistently shown growth rates well above those of more mature economies and this trend is expected to continue. the economies that make up the emerging world will almost undoubtedly have an even greater impact on the overall global economy throughout the foreseeable future. emerging markets can present tremendous opportunities as well as unforeseen challenges for global organizations. the recent sars epidemic highlights how a disease centered in countries identified as emerging can create formidable obstacles for managers. in order to examine the potential long-term effect of sars on emerging economies, an examination of infectious disease is required. the next section provides a brief historical look at the impact of epidemics on communities and organizations. despite the advents of modern medical science, infectious diseases such as malaria continue to wreak havoc. "if seven boeing s, filled mostly with children, were crashing into mount kilimanjaro each day, something might be done about it. that, in the memorable phrase of wen kilama, a tanzanian researcher, is roughly the scale of the carnage wrought by one disease, malaria, mainly in africa" (the economist, ) . more recently, the spread of sars captured center stage, due as much to the disease's proclivity as to media's desire for the next major headline. augmented or not, that sars has been (and may continues to be) destructive is unequivocal. indeed, because md , most of the inflicted jurisdictions are important developing economies, sars poses serious implications for businesses around the world. as surowiecki ( ) noted in the new yorker magazine, "before sars, china was going to be the factory floor to the world. now it has to worry about becoming the sick ward of the east". it is important to note, however, that menacing as sars is, it is but one epidemic in a long series of infectious outbreaks that have plagued human history. and in spite of its highly vilified image, sars has caused relatively minor damage compared to past epidemics. as one commentator puts it, "sars has not yet reached the scope or virulence to be described as a pancosmic. so far it is a fleabite compared to previous plagues. you are more likely to be struck by lightning than sars" (howard, ) . a history of plagues is beyond the scope of this paper. however, it is important to offer some background on infectious diseases and their impact on humanity over the centuries. we also wish to point out that, quite aside from sars, infectious diseases (some with much more deadly and rapid effects) continue to threaten the world. while most have heard of the black death and yellow fever, perhaps the most famous plague of all happened centuries ago in athens ( - bc). though it is impossible to know with certitude what the disease actually was, it was of such devastation that even vultures refused to touch the corpses of the dead (howard, ) . fast forward to the s, black death swept across europe. as "recently" as the seventeenth century, the great plague of london killed , people, followed by the plague that killed up to , people in china and hong kong. indeed, within the next years, plagues had killed more than million people worldwide. lest we thought the plague is a thing of the past, outbreaks of bubonic and pneumonic plagues in killed more than in india (king, ) . recent reports by the washington post suggest that rotaviral infections, a virus that causes acute gastroenteritis, are fast becoming an emerging problem in the former soviet union. north america has never been exempt from the devastations of infectious diseases. the advance of european cultures over first people in north america, such as the mohawks and algonquin nations, was due to the importing of diseases such as tuberculosis (tb) that devastated the established cultures killing millions of people. yellow fever ravaged philadelphia in , killing thousands in the then capital of the usa (dalrymple, ) . in and , spanish influenza killed more than half a million people in the usa alone (king, ) , and many insurance companies saw their profits wiped out and had to reduce dividend payments because of the claims made from the illness (bell, ) . today, tb is perhaps one of the most lethal infectious diseases. even though was the tenth consecutive year of declining tb cases in the usa, each year some two million people die from tb and an estimated one-third of the world's population is infected with the bacteria (center for disease control, ) . other diseases such as malaria and ebola continue to claim the lives of thousands, and since they occur predominantly in under-developed economies with poor infrastructure and health services, their effects are staggering. a disease like ebola, for example, can have mortality rates as high as percent (washington post, ) . it is informative to note the difference between epidemics and endemic diseases, though with the ease of modern travel and human contacts, the lines are somewhat blurred. endemic diseases are those that are localized in certain regions. unlike epidemics which really have no "home base," so to speak, endemic diseases are associated with particular geographic locations (surowiecki, ) . usually because of inadequate infrastructure, these jurisdictions are ill-equipped to stem the disease. the disease not only negates current economic growth but also acts as a barrier to future economic growth since investment flows tend to avoid these regions where companies remain concerned about the health of their employees. as business becomes increasingly globalized, companies large and small will encounter infectious diseases of both the epidemic and endemic varieties. in the latter case, it may well be in business's interest to work with local and global health officials to improve local infrastructures, such as hospitals and health services, not only as a means to generate goodwill and promote good corporate citizenship, but also as a way to help create a more hospitable operational environment. for example, the state-run china national offshore oil corporation donated million renminbi (just over us$ million) to aid research on sars. one of the most widespread and publicized epidemics today is hiv/aids. while the disease may be well publicized, its connection with the business community is not often elaborated upon. in fact, otherwise innocuous actions on the part of businesses have a hand in spreading the disease. for instance, truckers working for long-distance transportation companies have been identified as a major transmitter of the disease throughout africa and asia. hiv/aids also has devastating effects on businesses themselves. barclay's bank of zambia, for example, reported losing more than a quarter of its senior managers to the disease. a mattress company in zimbabwe hires and trains three people for every position due to attrition by the disease (forstater et al., ) . aids has become south africa's biggest single killer. in a south africa's medical research council calculated that percent of the deaths of those aged between and in the previous year were due to the aids pandemic. in recognition of the impact hiv/aids is having on its work force, daimlerchrysler south africa has, since , provided funding for standardized treatment for hiv-related diseases and anti-retroviral medication for its employees (daimlerchrysler, ) . according to the african vice president of the world bank, callisto madavo, hiv is the single greatest threat to future economic development in africa. of equal concern though is the growth rate of aids in asia. aids is growing fastest in asia leading many health experts to believe that it may overtake africa in the number of people infected with hiv. this will create a vexing problem for investors in europe and north america as their economies peak and they increasingly turn to the third world for profits. inexpensive labor is appealing but what if you cannot find employees to complete an order (jeter, ) ? the above discussion seeks not to provide a chronological listing of infectious diseases, but to highlight the dangers the world continues to face. businesses, indeed the world community, cannot afford to be complacent in the efficacy of modern medicine and assume that sars is an anomaly. infectious diseases are a fact of life and will continue to impact human society in the future. it is imperative that businesses develop coherent strategies to deal with such diseases when necessary. there should be no excuse for being "caught off-guard". the following section will look at specific impacts of the sars epidemic on commerce and present suggestions for organizations to protect themselves against similar future spread epidemics. in this section a distinction is made between the impact on large and small organizations. suggestions for contingency planning in face of epidemics so far we have attempted to provide a comprehensive discussion of how infectious diseases can gravely impact a firm's ability to function smoothly, unless adequate contingency planning is incorporated into its strategic management function and crisis management plan. in this section, we attempt to provide suggestions to minimize the impact of operating in an emerging market where contagious diseases are a reality. there are several areas that a contingency plan should address, in order to deal with the kind of epidemics that we have recently witnessed. further work will be needed to more fully develop the precise parameters of these dimensions, but for our purposes, we offer below some of the key areas that we feel a contingency plan should seek to address. businesses will need to re-examine the level of inventory, so as to provide some level of buffer in case of emergency. certainly we recognize that this adds to the cost of doing business, but we suggest that operationally this merely represents an additional variable in modeling the optimal level of inventory necessary. furthermore, businesses will need to assess whether it is practicable to secure their supplies from more than one source. diversification, as we know, spreads the risk. as much as possible, we need to incorporate technology into our operations, and re-evaluate location attractiveness with an eye towards the ease of technology replication as well as emergency backups. when opportunities arise move employees around global locations. these moves can be for short stints. the objective is to share understanding of competitive advantage. this may help upper management in reestablishing their competitive advantage in an alternative location. it is also important that values central to the business be ingrained in the workers to the extent possible. in this instance, continual efforts of re-education may be a useful step to take. we posit that companies would be concerned about how its workers are affected by the epidemic in terms of health, safety, and even emotion. as major epidemics such as sars could not be foreseen, it is imperative that managers take an emergent approach to plowing through the problem. companies in the many emerging markets where government regulations and transparency are lacking may particularly need to do more than simply adopt the official government line regarding the effects of the epidemic. to the extent possible, businesses should consider hiring local management talents as well as rotating key personnel to multiple locations of operations so as to build a solid base within the company from which local knowledge and expertise can be tapped. it is critical also that this knowledge is not only the exclusive domain of senior management, but that open communication be instituted throughout the company to enable workers at all level to know what is going on and what to expect. one of the unfortunate results of secrecy is that it often leads to unfounded rumors that only serve to aggravate a dangerous situation. corporations need to revisit their health plan to consider the need to establish health policies for suppliers. the "we didn't know" defense does not work too well any more in today's world of instant communication. businesses need to not only have in place their own health policies related to epidemics, but should determine whether their suppliers have adopted effective health policies to deal with such crises. regardless of all that we know, we acknowledge the impossibility of predicting future events. consequently, companies should not waste time and resources attempting to plan for patterns that are simply unpredictable. rather, it is critical that companies pay appropriate attention to contingency planning (such as crisis management plans); the basis of which are already well understood. what we have done is to tease out the important lessons from the sars epidemic. we have looked at its impact on organizations operating in emerging markets and drawn suggestions from this event. crisis management plans must be robust enough to handle all forms of the unexpected. as events arise that give us insight into the unforeseen, it is essential that organizations reexamine their crisis management plans to see if they were designed effectively enough to handle the unique features of our evolving environment. for those organizations that have operated without a crisis management plan, sars is a reminder of the importance of designing such a plan. the sars epidemic also shows how a crisis management plan must be integrally connected with an organization's strategic planning, thus being emergent rather than static in structure. it would be informative to take stock of what has transpired since the outbreak of the sars epidemic. a year after the disease first broke, it would be of intellectual (and practical) interest to assess what, if anything, the business community has put in place to combat such medical challenges. furthermore, inasmuch as we have offered some useful suggestions in this paper to deal with similar crises, we believe the exercise would be well served by couching our recommendations in a framework based in theory. such a future study should reveal whether the business community has adopted any important lessons that might be learned from the sars outbreak, and render our proposed contingency planning recommendations more practical in light of actual corporate actions. encephalitis outbreak in mainland china statistics are first casualty flu pandemic hit insurers hard emerging markets and international development: options for us foreign policy china isn't the only alluring partner for investors: despite worries, southeast asia still draws firms doing business in emerging markets: entry and negotiation strategies exercise in terror municipal affairs minister david young says he hopes ottawa will 'rethink' its compensation offer for the province's sars outbreak how congress coped in the age of yellow fever emerging markets of the global marketplace the sars outbreak: cases flare in taiwan; china gets draconian sales take a holiday airline woes: sars take toll on cara masked peril europe struggles to eradicate avian flu outbreak five rules for winning emerging market consumers before disaster hits: a multifaceted approach to crisis management emerging markets: a practical guide for corporations, lenders, and investors infomart ( ), available at: www.infomart.ca/doss/doss_db_display.php?key ¼ corp/cfps international bank for reconstruction and development/the world bank aids plants crop of death in africa many us firms fear sars will hurt profits this quarter beijing imposes new sars curbs: city's theaters and cafés are shut down as china lists another cases epidemic disease since the black death hong kong worries about bill to curb sars reporting china's growth may fall below percent: sars causes drop in sales of electronic products, closing of stock exchanges crisis management: a diagnostic guide for improving your organization's crisis preparedness sars squeezes asia's travel sector: industry study says virus may cost economies billions of dollars, millions of jobs emerging markets and international development: options for us foreign policy infection rates might drop as spring temperatures rise the high cost of illness nokia warns of lower sales, blaming economy and sars the big emerging markets: outlook and sourcebook local health official continue to plan for crisis cumulative number of reported probable cases of sars china cool toward md who told sars truth: whistleblower treated both as a hero and political threat, writes geoffrey york in beijing key: cord- - etvgoxc authors: ellis, christine title: ferrets date: - - journal: saunders manual of small animal practice doi: . /b - - - / - sha: doc_id: cord_uid: etvgoxc nan m clinical techniques restraint • most pet ferrets are gentle, tractable, and are easy to restrain without assistance. often only minimal restraint is needed when performing a physical examination. some ferrets may be lightly restrained on the examination table. others will need to be restrained in a firmer manner. • tractable ferrets can be lightly restrained on an examination or treatment table by placing one hand under the chest and lifting slightly. • energetic ferrets may be restrained by scruffing ( fig. - ). use one hand to grasp the skin over the back of the neck and lift the ferret up, suspending all the limbs. stroke the abdomen with a downward motion to relax the ferret. the ferret's back may be supported with the other hand, or the ferret may then be reclined along the forearm of the arm used to scruff the ferret. most ferrets will become very relaxed, although some young ferrets and some females may resist. • firm restraint is often required when administering vaccinations or when performing treatment procedures. control the head by scruffing, or by cupping the back of the ferret's neck and placing the thumb and fingers along the caudal border of the mandibles. place the other hand over the pelvis to restrain the hindquarters on the table top with the hind legs underneath the body; do not pull the legs back. • aggressive ferrets, such as nursing females, kits, or ferrets raised with little human contact, are uncommon. restrain these ferrets by the scruff of the neck, using the techniques previously described. avoid using leather gloves, which are awkward. use sedation if necessary. there are several suitable sites for blood collection in ferrets: • • cephalic or lateral saphenous venipuncture may be used to obtain small amounts of blood (< . ml) for a packed cell volume (pcv), blood glucose, complete blood cell count (cbc), or serum biochemistry analysis. • the jugular vein, cranial vena cava, cephalic vein, or ventral tail artery may be used to collect larger volumes of blood. • use the jugular vein to collect blood for transfusion. christine ellis figure - . "scruffing" a ferret for restraint. • sedation: collection of blood from the jugular veins, cranial vena cava, or ventral tail artery may require sedation and/or the assistance of two people for restraint. sedation is rarely required for venipuncture of cephalic, lateral saphenous, or jugular veins. • if necessary, clip the hair over the venipuncture site to see the vein. • the normal hematocrit of ferrets is high; draw three times as much blood as the volume of plasma or serum required. (see tables - and - for blood values reported in normal ferrets.) • the pcv, red blood cell count (rbc), hemoglobin, white blood cell count (wbc), and plasma proteins often rapidly decrease after induction of isoflurane anesthesia. cephalic vein • collect blood from the cephalic vein in ferrets using the same restraint technique described for dogs and cats. • use an insulin syringe with a -gauge needle or a cc tuberculin syringe with a -gauge needle to collect volumes of blood up to . ml. larger volumes of blood may be collected with a cc syringe and a -gauge needle. • alternatively, place a -gauge needle in the vein and collect blood directly from the hub into small blood collection tubes. ‡these white blood cell counts are higher than those currently seen in clinical practice. at our laboratories, the normal white blood cell count is - ¥ /ml, and most are - ¥ /ml. adapted with permission from lee ej, moore we, fryer hc, minocha hc: hematological and serum chemistry profiles of ferrets (mustela putorius furo). lab anim : - , ; and thornton pc, wright pa, sacra pj, goodier tew: the ferret, mustela putorius furo, as a new species in toxicology. lab anim : - , . copyrights and lateral saphenous vein • the lateral saphenous vein runs diagonally across the lateral surface of the hindleg, just proximal to the hock. • use an insulin syringe with a -gauge needle or a -ml syringe with a -gauge needle to collect small blood samples (< . ml). • several methods are described for jugular venipuncture: • the ferret may be placed in sternal recumbency at the edge of the table. extend the head dorsally with the front legs held down, out of the path of the venipuncturist. • alternatively, wrap the ferret in a towel with the front legs drawn back along the thorax, leaving the head and neck extended from the towel. position the ferret in dorsal recumbency, and extend the head and neck by scruffing. • restrain the ferret in the same manner described for the cranial vena cava (see below). • ferrets that struggle should be sedated for this procedure. • use a -ml or -ml syringe with a -to -gauge needle for sample collection. • this procedure is referred to as cranial vena cava venipuncture, but, in reality, blood is collected from the jugular vein as it passes into the thoracic cavity at the thoracic inlet. m key point blood collection from the cranial vena cava requires complete immobilization of the ferret; otherwise, do not attempt the procedure. use sedation or the help of two assistants for restraint. • do not use this site if intrathoracic disease (e.g., mediastinal mass, mega-esophagus) or coagulopathy is suspected. . place the ferret in dorsal recumbency. one assistant restrains the head and neck in extension while holding the forelegs alongside the thorax. a second assistant restrains the hindquarters without pulling the rear legs back. precise positioning facilitates the procedure. . palpate the manubrium and locate the "notch" on either side where the manubrium and the first rib meet. . insert a -ml syringe with a -gauge, / -inch needle at either notch and direct the needle at a shallow angle (< degrees) along an imaginary line running from the notch toward the opposite rear leg. . insert the needle to the hub and gently aspirate while withdrawing the needle. . if the ferret struggles, abort the procedure, and do not make a second attempt until the ferret is quiet. ferrets that struggle persistently should be sedated for this procedure. • venipuncture at this site may be painful. . scruff the ferret and place it in dorsal recumbency (wrapping it in a towel may help with restraint), or anesthetize the ferret and place it in dorsal recumbency. . prepare the site aseptically. . use a -or -cc syringe and a -to -gauge needle for sample collection. . insert the needle on the ventral midline of the tail at -degree angle toward the body approximately to cm from the anus. . advance the needle to the bone; withdraw it slowly while applying a slight vacuum to the syringe. . apply direct pressure to the site for several minutes after the needle is withdrawn. a detailed discussion of the techniques used for blood collection for transfusion and blood transfusion is presented in the hematopoietic system section in this chapter. • use standard radiographic techniques (see chapter ), including sedation for correct positioning and to limit exposure of the technician, as well as high detail radiographic film and cassettes. • when interpreting films, it helps to think of the ferret as an elongated cat. • the kidneys are relatively short (about two lumbar vertebrae in length). • splenomegaly is a common radiographic finding. • for barium-contrast radiography of the gastrointestinal (gi) tract, give ml/kg of % barium solution po via syringe feeding or lavage tube. most ferrets will accept syringe feeding of barium. normal gi transit time is about to hours. • echocardiography may be used to evaluate the heart in ferrets with suspected cardiac disease (see "cardiovascular disease"). • other uses of ultrasound in ferrets include investigation of intra-abdominal or intra-thoracic masses, organomegaly, paraurethral cysts, or prostatic cysts. • the indications, guidelines, and techniques for bone marrow sampling are the same as those described for dogs and cats. (see chapter ) • preferred sites include the proximal femur and humerus. the iliac crest may be used for sample collection as well, but can be a difficult site to access. • sedation is required for bone marrow aspiration. use a -or -gauge spinal needle with stylet for sample collection. fine-needle aspiration of the spleen has been performed successfully in ferrets and is a rapid means of evaluating splenic cytology. in ferrets, the only contraindication is suspected hemangiosarcoma of the spleen. sedation is rarely necessary, but is recommended if the ferret persistently struggles. . two assistants are recommended for restraint. place the ferret in dorsal recumbency. one assistant should restrain the head and neck by scruffing the ferret with one hand. the other hand is used to restrain the forelimbs. a second assistant restrains the hind limbs by placing one hand around the pelvis. . palpate the spleen and position it against the left lateral or ventral body wall. . clip and prepare the site aseptically. . insert a -gauge needle attached to a -ml syringe to the hub at a perpendicular angle to the skin and aspirate from the spleen. . when a small amount of bloody fluid is visualized in the needle hub, withdraw the needle and prepare slides routinely for cytology. • urine may be collected by cystocentesis using the same technique described for the cat. • a -or -gauge needle on a -or -cc syringe may be used for sample collection. • anesthesia is recommended if the ferret is difficult to restrain. m key point sedation is often required for placement of a butterfly or indwelling intravenous (iv) catheter, or for small-volume iv therapy. • for small-volume iv therapy ( . - . ml), use an insulin syringe. the cephalic or lateral saphenous veins are the preferred sites for injection. sedation may not always be required for a single injection. • a -gauge butterfly catheter may be used to administer larger, single-dose volumes into the cephalic vein. • an indwelling catheter can be placed in the cephalic, lateral saphenous, or jugular vein. sedation is usually required. • flush indwelling catheters with small volumes of heparinized saline solution to maintain catheter patency. • peripheral indwelling catheters can be placed rapidly and are useful in emergency situations and for surgery. • restrain the ferret by scruffing. some ferrets may require two people for restraint. • sc injections may be given in the loose skin over the shoulders. • im injections may be given in the quadriceps, the semimembranosus-semitendonosus muscles of the hind limbs, or in the expaxial muscles of the lower back. • limit the volume of the im injections, due to the small muscle mass of the ferret. • fluids may be administered sc, iv, or io, depending on the needs of the patient. • the daily fluid requirement for ferrets has not been reported but can be estimated at to ml/kg/ day. adjust for dehydration and fluid loss. • administer sc fluids over the dorsal shoulder and thoracic region. • iv or io fluid therapy is used for a wide range of medical and surgical situations, and is recommended for ferrets that are > % dehydrated. • iv or io fluids must be administered with an infusion pump. fluids may be given as a continuous infusion, may be administered by continuous infusion, or may be given in to bolus doses over a -hour period. • ferrets may require the addition of dextrose to fluids because hypoglycemia is common. • oral medications are most easily given to ferrets in liquid form. • if possible, compound medications formulated in tablet or capsule form into liquid suspensions, or crush and mix them with a sweet-tasting substance such as nutri-cal (evsco pharmaceuticals) feline hairball laxative, or fruit-flavored syrup, and administer by syringe. • ferrets suffering from insulinoma should not be given sugar-based treats or medications if at all possible. hide medications in fatty acid supplements, vegetable oil, whipping cream, or meat baby food. • supplemental feeding is important in the management of anorectic or critically ill ferrets, and in the treatment and prevention of hypoglycemic episodes associated with insulinoma. • most ferrets can be force-fed dietary supplements by syringe. once they acquire a taste for a given supplement, it may be possible to offer it in a bowl. • feed ferrets as much food as they will take comfortably ( - ml) to times daily. • foods useful for force-feeding include the following: meat baby foods, slurried cat or ferret food, and science diet a/dliquid soy-based formulas (e.g. deliver . , mead johnson nutritionals) may be added to the mixture to increase the calorie content and improve palatability. • use of all medications is considered off-label for the ferret; there are no approved drugs available for ferrets in the united states. • several exotic animal formularies are commercially available that include drug dosage information on ferrets. • when dosing information is not available, use feline dosages with the following exceptions: • chloramphenicol: ( mg/kg) bid, iv, sc, im, or po. • aspirin: ( - mg/kg) bid-tid po (canine dosage). • many ferrets become lethargic when placed on enalapril (enacard, merck agvet) for cardiac disease. start with a very low dose ( . - . mg/kg) q h po. some ferrets cannot tolerate more than every-otherday therapy. • ivermectin ( . mg/kg) q d po for heartworm prevention; ( . mg/kg) once to weeks after adulticide treatment as a heartworm microfilaricide; ( . - . mg/kg) po, sc, repeat in days for sarcoptic mites; ( mg/kg) instill half the calculated dose into each ear and repeat in days for ear mites. a detailed discussion of the techniques used for blood collection for transfusion and blood transfusionis presented in the hematopoietic system section in this chapter. • sedation or isoflurane anesthesia facilitates urinary catheterization. the procedure may be difficult in ferrets with urethral disease or urethral calculi. . position in ventral recumbency with the hindquarters elevated and use a tomcat catheter or a . -fr. feeding tube with or without a stylet. . the urethral orifice is located on the ventral floor of the vaginal vault. catheterize the urethra blindly or after identification using a vaginal speculum. • the catheter may only pass part way into the urethra (often to the pelvic flexure). this may be sufficient to allow retrograde flushing of urethral calculi into the bladder, or to empty the bladder of a ferret with urethral obstruction secondary to prostatic enlargement. m key point isoflurane administered by face mask is the most convenient method to immobilize a ferret for procedures such as venipuncture and radiography. induction and recovery are rapid. doses for parenteral agents used in ferrets are listed in table - . • acepromazine is useful for sedation. • butorphanol tartrate has been used at sc, im, iv, but can cause very profound sedation in some ferrets. • ketamine alone does not produce effective muscle relaxation. use in combination with acepromazine for minor surgical procedures, or with diazepam for more complicated procedures. • medetomidine is not analgesic; animals will respond to painful stimuli. use with an analgesic agent. this agent is reversible (atapamazol). • tiletamine-zolazepam (telazol, fort dodge) gives variable muscle relaxation but is useful for immobilization for procedures such as venipuncture, radiography, and electrocardiography. recovery may be prolonged. • xylazine may cause bradycardia or vomiting, and is not recommended for use. • premedicate with parenteral agents followed by facemask induction. alternatively ferrets may be given • isoflurane is the inhalant anesthetic most commonly used in small mammal practice. sevoflurane is used in some practices as well. pharmacokinetic and pharmacologic isoflurane and sevoflurane are very similar; sevoflurane smells better and is better tolerated during face-mask induction. • isoflurane does not provide analgesia. administer an analgesic pre-or intra-anesthesia if a painful procedure is to be performed. analgesic agents commonly used in ferrets include buprenorphene ( . - . mg/kg q - h sc, im, iv), butorphanol ( . - . mg/kg q - h sc, im), carprofen ( mg/kg q - po), and flunixin meglumine ( . - . mg/kg q - h im, iv). • intubate ferrets to facilitate intermittent positive pressure ventilation (ippv). a . to . mm endotracheal tube usually is suitable. • the same planes of anesthetic depth reported for dogs and cats occur in the ferret. • follow basic principles of anesthesia for small animals, (see chapter ) and provide supplemental heat during surgery; administer iv fluids (isotonic electrolyte solutions supplemented with . - % dextrose) during long procedures and for insulinoma surgery. canine distemper and influenza are the two most common viral diseases of the ferret. influenza is zoonotic between humans and ferrets, and is typically passed from humans to ferrets. canine distemper is % fatal in the ferret, making distemper vaccination imperative. • the canine distemper virus (cdv) is a paramyxovirus. transmission occurs through direct contact with infected animals of any species, and through contact with fomites such as in shoes or clothing. • the incubation period for cdv in the ferret is typically to days; however, incubation for some strains of cdv may take up to days. (for discussion of cdv in dogs, see chapter ). • early in the disease the only clinical sign may be a mild unilateral or bilateral conjunctivitis. • pyrexia (> °c), anorexia, and profuse mucopurulent naso-ocular discharge develop as the disease progresses. • vaccinate all ferrets in the household or facility. currently only two vaccines are approved for use in ferrets: purevax (merial, athens, ga), and fervac-d (united vaccines, inc., madison, wi). give ml sc, using the following schedule: • if the dam is vaccinated: vaccinate kits at weeks of age and repeat vaccination every to weeks until the kits are weeks of age. • if the dam is unvaccinated: vaccinate the kits at weeks of age and repeat every to weeks until the kits are weeks of age. • revaccinate annually. sources claim that immunity lasts for years; however, outbreaks have been known to occur months after vaccination. • use of serum titers as a method to evaluate an animal's current immunological status is unsubstantiated. • quarantine new ferrets and canines for weeks before exposure to other resident animals. vaccinate new animals immediately after acquisition at the beginning of the quarantine period. • use of galaxy d (schering-plough animal health co., omaha, ne) for distemper vaccination has been described. use of this product in ferrets is extra-label. this product has proved effective in preventing canine distemper in young ferrets; however, duration of immunity is unknown. • do not use cdv vaccines that contain canine parvovirus, adenovirus, or other viruses. it is not necessary to vaccinate for leptospirosis unless there is exposure to wild rodents. the influenza virus is an orthomyxovirus. ferrets are susceptible to influenza a and b; this is the only documented zoonotic disease of the ferret. human-to-ferret transmission is more common than ferret-to-human transmission. transmission occurs by direct contact with naso-ocular discharges, and via inhalation of aerosolized droplets. • the incubation period is typically to days postexposure. • the clinical course of the disease is typically to days. m key point influenza typically causes only mild illness and discomfort, and is usually self-limiting in an otherwise healthy animal. • clinical signs may include any combination of the following: • sneezing with a clear, serous nasal discharge. • mild conjunctivitis with serous ocular discharge. crusting around the eyes may occur rarely. • a nonproductive cough that may be loud and paroxysmal, and often occurs more frequently at night. • diarrhea. vomiting may occur in rare cases. • partial to total anorexia, listlessness, and fever. • pneumonia, severe illness, or death may occur in neonates, geriatric patients, and in ferrets with concurrent diseases such as lymphosarcoma or insulinoma. • ferrets with underlying immunosuppressive disorders, especially lymphosarcoma, may develop repeated or cyclic episodes of influenza. rule out lymphosarcoma by performing a complete blood cell count (cbc), bone marrow biopsy/cytology, or a peripheral lymph node biopsy (see lymphoma in this chapter). diagnosis is based primarily on the clinical signs, history, and physical examination. • the history often indicates recent exposure to a human or another ferret with influenza or signs of upper respiratory tract disease. • the overall physical condition often remains good, although slight or moderate dehydration may be present if the animal is not eating or drinking normal amounts. • differential diagnoses include the very early stages of canine distemper, gi rotavirus infection, and lymphosarcoma. if mucopurulent nasal or ocular discharge is noted, consider early cdv or a secondary bacterial infection. • supportive care generally is sufficient. • encourage the ferret to eat and drink. offer to tablespoonfuls of hill's science diet a/d or strained meat baby food bid-qid if the animal refuses the regular diet. • if indicated, give an oral electrolyte solution that is palatable to ferrets. • if sneezing or coughing is excessive and interferes with eating or sleeping, give an antihistamine such as chlorpheniramine ( . - . mg/kg) bid-tid po, or diphenhydramine ( . - . mg/kg) bid-tid po. nasal solutions containing phenylephrine may be used to relieve nasal congestion. • antibiotics are not necessary unless secondary bacterial infection is present. • antiviral medications such as amantadine ( mg/kg) bid po (symmetrel, endo pharmaceuticals, chadds ford, pa) may be useful in the treatment of ferrets with influenza. zanamivir ( . mg/kg) once intranasally (relenza, glaxosmithkline, research triangle park, nc) has been shown experimentally to prevent influenza infection. good hygiene is the key to prevention. • discuss the zoonotic potential with the client. advise clients to wash their hands frequently, and to avoid holding the ferret near the face. • in the veterinary hospital, do not allow influenzainfected personnel to handle ferrets, especially if the animal is a neonate, a geriatric patient, or a patient debilitated by serious disease. • vaccination is not recommended; only short-term immunity results, and the wide variation of the influenza virus makes appropriate vaccination difficult. rabies is caused by a rhabdovirus that results in fatal disease in ferrets. it is transmitted via contact with an infected animal's saliva (see chapter ). this is a zoonotic disease; however, there has never been a report of ferret-to-human rabies transmission. experimentally, the incubation period is to days. m key point very little is known about rabies in naturally infected ferrets. • it is known that ferrets may become naturally infected; however, there is some question as to how easily they can contract the disease and the length of the incubation period. information about clinical signs is derived primarily from literature associated with laboratory-infected ferrets. signs are variable and include: • behavioral abnormalities that range from anxiety and hyperactivity to lethargy. • neurologic signs such as ascending paralysis, ataxia, hyperparesthesia, and posterior paresis. diagnosis is based on clinical signs, and/or a history of known or potential exposure to rabies. • history may include a recent bite wound or exposure to a rabid animal. • the ferret may be unvaccinated; however, development of rabies in vaccinated individuals has occurred in other animal species. • differential diagnoses include aleutian disease, botulism, brain hypoxia from severe seizures, cns neoplasia, insulinoma, intervertebral disc disease, and viral or bacterial encephalitis. • postmortem laboratory testing of brain tissue (fluorescent antibody staining [fas] or virus isolation) confirms the diagnosis (see chapter ). • there is no treatment for rabies. • euthanize the suspect animal to protect humans and other animals in its environment. submit animal for postmortem fas testing of brain tissue. m key point it has not been demonstrated that ferrets are carriers of rabies. many public health facilities now recognize and accept the -day quarantine period for ferrets; however, in some states, unvaccinated ferrets involved in biting incidents will be euthanized and submitted for rabies testing. it is important to be familiar with state and local laws regarding vaccination requirements and the laws following a biting incident. m key point the compendium of animal rabies prevention and control recommends that ferrets be confined and observed for days following human exposure. if signs compatible with rabies develop, the animal should be euthanized and protocols for rabies testing should be followed. vaccinated ferrets exposed to a potentially rabid animal should be revaccinated and quarantined for days. euthanize any unvaccinated ferret exposed to a rabid animal. • vaccination is the only prevention, and is mandatory in some states. • imrab (rhone merieux) is an inactivated rabies vaccine that is currently the only rabies vaccine approved for use in ferrets. administer at a dose of ml sc. • vaccinate initially at months of age. revaccinate annually. aleutian disease (adv) is caused by a parvovirus that affects both mink and ferrets. transmission occurs by direct contact or via contact with fomites contaminated with any infected body fluid, including blood. adv produces a progressive immune-mediated disease accompanied by the deposition of antigen-antibody complexes in multiple organs of the body. the virus is prevalent in the ferret population, but the percentage of ferrets that develop clinical illness is low. in one survey of ferrets, % were serologically positive, but only two animals developed clinically active disease. some ferrets may be asymptomatic carriers, while others may have natural immunity to the disease. m key point the clinical signs of aleutian disease are extremely variable, and the incubation period can be as short as day or as long as to days. • ataxia, mild incoordination, posterior paresis, or tremors may be the initial presenting signs. initially ferrets often continue to eat, and appear bright and alert. as the disease progresses, paresis progresses to the forelimbs, and wasting develops that may continue for weeks or months. • anorexia, lethargy, melena, and urinary incontinence are seen in later stages of the disease. • a slow wasting disease existing without neurologic signs may also occur. • diagnosis may based on the history, clinical signs, physical examination findings, the presence of high serum total protein and hypergammaglobulinemia, and a positive adv test. diagnosis is confirmed with histopathology. • differential diagnoses include bacterial or viral encephalitis, cns neoplasia, canine distemper, lymphosarcoma, gastric foreign body, tuberculosis, intervertebral disc disease, systemic mycoses, and rabies (in cases with behavioral changes and sudden paralysis). • exposure history is often not helpful because of the prevalence of asymptomatic carriers. • high serum total protein may be present. serum protein electrophoresis may demonstrate hypergammaglobulinemia (> % of the total serum protein). • blood samples may be submitted for counterimmunoelectrophoresis testing (united vaccines, inc., madison, we) or enzyme-linked immunosorbent assay (elisa) testing (avecon diagnostics, bath, pa). an in-house saliva sample kit is available as well (avecon diagnostics, bath, pa). • histopathology demonstrates lymphocytic plasmacytic infiltration and perivascular cuffing in many organ systems. the kidneys, liver, lymph nodes, and spleen are often affected. m key point there is no effective treatment for aleutian disease. provide supportive care and do not allow contact between clinically ill animals and healthy ferrets. euthanasia is usually indicated only for clinically affected animals. should not be euthanized because they may never become clinically ill. infected ferrets may remain asymptomatic for life, but can remain persistently infected. other ferrets may develop nonpersistent, self-limiting disease and fully recover. • administration of corticosteroid therapy and supportive care may prolong the life of some ferrets with clinically active disease. breeding colonies • breeding colonies should be closed. new animals should be adv tested and quarantined prior to introduction. • test all resident ferrets and remove serologically positive animals from the population. • adv-negative animals should be retested in months, before adding them to the colony, due to the potentially long incubation period. • it is not necessary to test a pet ferret unless it has been exposed to a clinically ill animal. • it is not necessary to euthanize a clinically normal, non-breeding adv-positive ferret or remove it from contact with other pet ferrets. advise the client, however, that there is a slight possibility that the pet may develop clinical illness. • do not house ferrets in close proximity to mink. • retest adv-positive animals in months since some animals may eventually eliminate the virus and become negative. rotavirus causes gastrointestinal infection and a bright green or yellowish-green diarrhea. rotavirus is described in the "gastrointestinal system" section in this chapter. lymphosarcoma is common in the ferret, and is discussed in the "neoplasia" section in this chapter. staphylococcus, streptococcus, escherichia coli, and other common bacteria from the environment can be introduced through penetrating wounds, punctures, abrasions, contact with mucous membranes, and by inhalation or ingestion. • abscessation is an uncommon form of bacterial infection in ferrets. • an abscess may occur in any part of the body, including the anal glands, mammary tissue, mouth, mucous membranes, reproductive tract, respiratory tract, subcutis, and prostatic tissue. • body temperature may be £ °c if bacterial sepsis is present. • bacterial dermatitis causes thickened, irritated areas of skin. affected ferrets may lick and chew these areas until they become denuded and ulcerated. • bacterial conjunctivitis causes a thick mucopurulent ocular discharge and swelling of the conjunctiva; corneal ulcerations may be present. • bacterial pneumonia causes lethargy, fever, anorexia, and dyspnea, and is often accompanied by mucopurulent nasal discharge and coughing. • bacterial mastitis occurs primarily in the lactating jill, and is accompanied by depression, fever, and anorexia. one or more mammary glands are swollen, discolored, and warm to the touch. • bacterial metritis may or may not cause a vaginal discharge; depression, fever, and partial or total anorexia are often present. • bacterial vaginitis causes a thick mucopurulent yellow-to-green vaginal discharge with little odor. fever is usually absent, and the animal does not appear clinically ill. • presumptive diagnosis is based on clinical signs, physical examination, demonstration of bacteria on routine cytology, results of bacterial culture, and sensitivity of the affected sites. the total wbc may demonstrate a marked leukocytosis (> , ). • treatment should consist of appropriate antibiotic therapy based on culture and sensitivity results, and surgical drainage or excision of the affected tissue when appropriate. • begin treatment with a broad spectrum antibiotic pending the results of culture and sensitivity testing, or when obtaining a culture is not feasible. • provide supportive treatment as needed, such as fluid therapy and nutritional support. • lance and thoroughly flush with an antiseptic solution. keep the area open and flush twice daily until healing occurs by second intention. • administer oral antibiotics until signs of infection are gone and a healthy bed of granulation tissue is present. see mastitis in the reproductive disease section in this chapter. see uterine infection in the reproductive disease section in this chapter. • if possible, perform a tracheal wash and submit samples for cytology, bacterial culture, and sensitivity testing. • if pleural effusion is evident on radiography, perform thoracocentesis. submit samples for cytology, bacterial culture, and sensitivity testing. • start oral broad-spectrum antibiotic therapy immediately, pending culture and sensitivity results. if pleural effusion is present, consider treating with a combination of clindamycin and cephalosporins (use cat dosages). • use of bronchodilating agents and/or nebulization therapy may be beneficial treatment modalities as well. • treat conjunctivitis with a broad-spectrum ophthalmic ointment. • perform a fluorescent corneal staining test to rule out corneal ulcers. (see chapter ) • submit samples for bacterial culture and sensitivity testing. • begin treatment with broad-spectrum oral antibiotics. modify treatment based on culture sensitivity results. • instruct owners to hot pack the affected area to minutes bid-tid. • treat until infection and swelling resolve, then perform anal sacculectomy. • continue antibiotic treatment to days postoperatively. • campylobacter spp. typically causes gi disease (see "gastrointestinal system"). • salmonella spp. may rarely cause gastroenteritis in ferrets (see "salmonella" within "gastrointestinal system"). • botulism is a rarely encountered disease in the domestic ferret caused by the ingestion of food contaminated with the clostridium botulinum toxin. c. botulinum is commonly found in the soil. • uncooked food or food contaminated with soil can be the source of the infection. • clinical cases of tuberculosis in the ferret are reported infrequently; however, ferrets are susceptible to bovine, avian, and human mycobacterium spp. infections. • the disease can be transmitted by ingestion of contaminated meat (poultry or meat), unpasteurized milk, or food contaminated by the droppings of infected wild or pet birds (see chapter for information about tuberculosis in dogs and cats). • clinical signs include chronic weight loss, and diarrhea that is unresponsive to treatment. vomiting may occur as well in some cases. • diagnosis is based on history, clinical signs, and the exclusion of other diseases; it is confirmed by intestinal biopsy. histopathologically granulomatous inflammation and acid-fast bacteria are identified. infection may also be confirmed by culturing the organism, and with polymerase chain reaction (pcr) testing. • because of the zoonotic potential of this disease, treatment is not recommended. affected animals should be euthanized. dermatophytosis is rare in the ferret and is typically caused by microsporum canis and trichophyton mentagrophytes. dermatophytes are transmitted by direct contact with infected animals or contaminated bedding, caging, and fomites. m key point ferrets are usually not carriers of these organisms. clinical disease is typically selflimiting. the most common source of infection of the pet ferret is the household cat. • young, debilitated, or geriatric ferrets are the most commonly affected. • lesions are consistent with those described in other species (see chapter ). alopecia with erythema, inflammation, hyperkeratosis, superficial crusting, lichenification, and erythema are present. pruritis is common and may lead to self-trauma and secondary pyoderma. • diagnosis is based on the identification of the fungal agent on skin scrapings, fungal culture, or a positive wood's light examination (m. canis). (see chapter ). • dermatophytosis is often self-limiting and may resolve without therapy. however, due to the zoonotic potential, treatment is recommended. • topical treatment includes the use of keratolytic shampoos, and/or lime sulfur dips. (see chapter ). • oral therapy consists of the administration of grisofulvin ( mg/kg) po sid for to days. perform a cbc every days while the ferret is receiving treatment. • disinfect the home by steam cleaning, the application of dilute ( : ) bleach or chlorhexidine solutions, and vacuuming thoroughly to remove infectious spores. dispose of the vacuum cleaner bag after vacuuming is complete. a thorough cleaning of the heat ducts and air conditioner/heater filters is also recommended. systemic mycoses are rare in the ferret; however blastomycosis, histoplasmosis, cryptococcosis, coccidioidomycosis, and aspergillosis have been reported. • consider these infections in the differential diagnosis of any systemic disease that is refractory to treatment and involves wasting, granulomatous lesions, persistent or recurring draining wound tracts, and respiratory tract disease. • diagnosis is based on the histopathological or cytological demonstration of the fungal organism in biopsies or aspirates (see chapter ). • complement fixation and precipitation tests have been used with variable success. • treatment is the same as described for the dog and cat (see chapter ). primary disease of the spleen is uncommon. splenomegaly is often a common incidental finding in a healthy adult ferret, or it may occur in association with a wide variety of disease conditions. perform a complete medical evaluation in all splenomegaly cases. • splenomegaly may be a normal or incidental finding in some patients. • pathological causes of splenomegaly may include chronic immune stimulation, erythroid bone marrow insufficiency, extra-medullary hematopoiesis (emh), hypersplenism, heart disease, and neoplasia. • splenomegaly can occur concurrently with adrenal gland disease and insulinoma, but it is usually an incidental finding. • extramedulllary hematopoiesis (emh) may cause enlargement of the spleen. the etiology of emh is unclear; compensation for myeloid insufficiency and chronic immune stimulation have been suggested as causes. ferrets with emh typically do not show evidence of hematological abnormalities. grossly the spleen has a normal shape and color, but it appears enlarged. • hypersplenism may cause enlargement of the spleen, but is rare in the ferret. • destruction of one or more blood cell lines by the splenic reticuloendothelial system occurs; affected ferrets will have anemia, leukopenia, thrombocytopenia, or pancytopenia. • bone marrow may be normal or hyperplastic in affected patients. • lymphoma is the most common neoplasia of the ferret spleen. hemangioma or hemangiosarcoma may occur as well. • when splenic lymphoma is present the spleen typically has irregular borders and a nodular texture. white or tan nodules may be noted grossly on the surface of the spleen and in the parenchyma. metastasis may be present. • splenic torsion and abscessation are rare in the ferret. • the normal ferret spleen measures approximately cm ¥ cm ¥ cm, and may be palpated in the left cranial abdominal quadrant. the texture of the spleen should be slightly firm, smooth, and the edges should be sharp. • an enlarged spleen is often noted on abdominal palpation as a firm, elongated smooth mass extending down the left side of the ferret abdomen, or crossing diagonally across the ventral abdomen from the left cranial abdominal quadrant to the caudal right abdominal quadrant. • abdominal distention may occur. • occasionally the spleen is so large and pendulous that the ferret can barely lift its abdomen off the ground. • abdominal discomfort due to splenomegaly appears to be uncommon in ferrets. • perform a cbc, platelet count, serum biochemical analysis, bone marrow cytology, and whole body radiography. • diagnosis of hypersplenism is based on the presence of one or more cytopenias, normal to hypercellular bone marrow cytology/biopsy, and the absence of blood loss, infection, or neoplasia. • obtain whole body radiographs to delineate the borders of the spleen and to rule out other abnormalities such as cardiomegaly or hepatomegaly that may contribute to splenomegaly. • splenic aspiration or biopsy may be performed. perform fine-needle aspiration of the spleen using a -gauge needle (see "clinical techniques"). do not perform splenic aspiration if hemangiosarcoma is suspected. • perform an abdominal ultrasound to evaluate the spleen. when the splenic parenchyma appears irregular, an ultrasound-guided biopsy or fine-needle aspiration may be performed. • perform a splenic biopsy during abdominal exploratory surgery, particularly if the spleen is irregular or discolored. treatment depends on the primary disease condition. usually splenectomy is not necessary. m key point indications for splenectomy are the same as for other species and include hypersplenism, splenitis, splenic abscess, torsion, rupture, neoplasia, and discomfort caused by excessive splenomegaly. • to perform a splenectomy, follow the surgical guidelines for splenectomy in dogs and cats (see chapter ). • anemia may result after splenectomy; the decision to perform splenectomy should be made cautiously, and with consideration to the health of the ferret as a whole. • administer antibiotics and fluid therapy pre-and postoperatively. • monitor asymptomatic ferrets with splenomegaly with periodic physical examination, cbc evaluation, imaging, and splenic aspiration. the clinical approach to anemia in ferrets is the same as for other species. anemias are classified as regenera-tive or nonregenerative; treatment is directed at the specific cause. there are many causes of anemia in ferrets; decreased erythropoiesis, destruction of red blood cells, and blood loss contribute to anemia. • nonregenerative anemia (normocytic, normochromic, nonregenerative anemia) occurs when bone marrow hematopoiesis is disrupted. bone marrow cytology of affected ferrets may appear normal. • decreased erythropoiesis may be caused by chronic metabolic disease (renal, hepatic), chronic inflammation, hyperestrogenism, bone marrow suppression, and neoplasia. • anemia of chronic disease can occur whenever long-term illness is present and is caused by decreased erythrocyte survival, decreased availability of iron, or a decreased response to the anemia treatment. • anemia associated with chronic inflammation is mediated by sustained inflammatory cytokine release. • hyperestrogenism may cause nonregenerative anemia due to estrogen-induced bone marrow suppression. unspayed female ferrets, female ferrets with ovarian remnants, or hyperestrogenism associated with chronic adrenal disease may contribute to this syndrome. • myeloid and leukemic neoplasias can cause suppression of bone marrow erythropoiesis due to replacement of normal bone marrow by neoplastic or fibrotic changes. • erythrocyte destruction may cause anemia; causes include immune-mediated disease, toxins, parasitism, or septicemia. • idiopathic immune-mediated hemolytic anemia, and immune-mediated hemolysis secondary to viral disease or blood parasites have not been reported in the ferret. • drug-induced hemolysis and heavy metal toxicosis (including zinc) are potential causes of hemolytic anemia. • anemia secondary to blood loss may be secondary to trauma, hemostatic disorders, bleeding lesions, and parasitism. • bleeding lesions may be internal or external. • bleeding ulcers may lead to anemia, and may be associated with h. mustelae gastritis, gastrointestinal foreign body, or chronic use of ulcerogenic drugs. • parasitism is uncommon in the ferret. coccidiosis in the young ferret or severe flea infestation may cause anemia. • hemostatic disorders include thrombocytopenia associated with estrogen toxicity, rodenticide poisoning, and liver disease. • clinical signs include weakness, pallor, lethargy, and inappetence. jaundice may be seen if hemolysis is present. • a soft systolic murmur is common in anemic ferrets. • a swollen vulva is present in ferrets with persistent estrus, an ovarian remnant, and in some cases of adrenal gland disease. hair loss may also be present on the shoulders and flanks. • ferrets with estrogen toxicity may have signs of thrombocytopenia such as petechiae, ecchymoses, and melena. • melena may be noted if gi bleeding is present. • palpate the spleen. splenomegaly may be caused by hypersplenism and subsequent anemia. • check carefully for fleas. perform a fecal examination. • diagnosis is based on the medical history, the physical examination findings, and a complete diagnostic work-up that includes a cbc, reticulocyte count, serum biochemical analysis, whole-body radiographs, and bone marrow cytology if indicated. • obtain a careful history regarding possible blood loss, toxicity, and foreign body ingestion. determine the duration of vulvar swelling (if present). • characterize the anemia based on rbc parameters and hemoglobin concentration. • the normal hematocrit for the ferret is % to %, higher than that of other animals. the erythrocyte count is higher as well; erythrocyte counts as high as . ¥ cells/µl have been reported. • the normal reticulocyte count may be as high as %. reticulocyte counts greater than % are indicative of a regenerative bone marrow response. • regenerative anemia is often the result of blood loss or hemolysis. • perform bone marrow aspiration (see clinical techniques), particularly if the anemia is nonregenerative, to identify infiltrative processes and assess the morphology of rbc precursors. bone marrow cytology from animals affected with anemia of chronic disease may be normal. bone marrow cytology is also indicated in ferrets with nonregenerative anemia that is unresponsive to treatment after to days. • obtain blood for blood lead concentration if lead poisoning is suspected. • whole-body radiographs are indicated to rule out abdominal neoplasia, gi foreign body, and thoracic neoplasia. a gi contrast study may be helpful to rule out the presence of gi ulcers. ultrasound may be helpful based on the rule outs established. m key point anemia in an intact female ferret with a swollen vulva for more than weeks most likely is due to estrogen toxicity. the objectives of treatment are to treat both the anemia and the underlying cause. • general supportive care includes oxygen therapy, subcutaneous fluids, and nutritional supplementation. • specific supportive care includes whole blood transfusion, iron dextran therapy, and the administration of erythropoietin. • oral iron supplements may be administered to replenish whole-body iron stores. • erythropoietin may be used to treat ferrets with nonregenerative anemia. administer u/kg three times per week until the pcv is stable, then administer to times a week. continue to monitor the pcv, and titrate the dose as needed. • indications for blood transfusion include the clinical status of the patient, a low packed-cell volume (pcv) of < %, the specific cause of the anemia, and the potential for continued blood loss. • ferrets lack specific blood types; transfusion reactions are rare in the ferret; up to three transfusions from the same donor and transfusions from multiple donors are considered safe. • the normal blood volume of an adult ferret may be calculated as % of the body weight. • the ideal value of the pcv post-transfusion would be within the normal reference range; a more likely goal is % to % higher than the pre-transfusion pcv. for dosage guidelines, see chapter . • before transfusion, administer a rapid-acting corticosteroid, such as dexamethasone sodium phosphate ( - mg/kg once iv) or prednisolone sodium succinate ( mg/kg once iv), as a slow bolus infusion, and administer an antihistamine such as diphenhydramine ( . - mg/kg iv, im, sc) to the recipient ferret. • the normal blood volume of ferrets has not been reported, but is estimated to be % to % of the total body weight (approximately ml/kg). twenty percent of the estimated blood volume (approximately ml) may be collected from healthy ferrets. • the jugular vein is the preferred site for the collection of large volumes of blood for transfusion. • sedate the donor ferret and place it in dorsal recumbency. • use a butterfly catheter to collect the blood into a syringe containing an anticoagulant such as sodium citrate ( . ml citrate per . ml blood) or acid-citratedextran (acd) ( ml per ml blood collected). • transfer the blood immediately to the recipient. blood should be filtered as it is transfused to the recipient. • administer fresh blood transfusions through an indwelling catheter or via a butterfly catheter into the cephalic or jugular vein. if a vein is inaccessible, administer into the peritoneal cavity or via the intraosseous route into the proximal femur. • whole blood is commercially available from marshall farms (marshall pet products inc., wolcott, ny). • hemoglobin substitutes such as oxyglobin (bioprure corporation, cambridge, ma) ( - ml/kg) iv or io over hours may be used if whole blood is unavailable. • administer oxyglobin slowly in normovolemic patients and in patients with renal disease, heart disease, or when the risk of pulmonary edema is present. • stop bleeding (internal or external). • correct the underlying cause of gi bleeding, including medical therapy for gi ulceration (see "gastrointestinal system"), surgery to remove gi foreign body, and antibiotics and supportive care for enteritis/colitis. • for anemia of chronic disease, treat the underlying primary disease process. • address metabolic disease (renal, hepatic) if present. • to correct estrogen toxicity in the intact female, terminate estrus (see below) and provide supportive care until the bone marrow is functional. broad-spectrum antibiotic therapy is important for the control of sepsis in leukopenic patients. estrogen toxicity associated with ovarian remnants is treated by surgical removal of the remnants when the ferret is stable enough for surgery. estrogen toxicity associated with adrenal gland disease is treated by adrenalectomy. preoperative care is the same as that described for the intact female (see below). (for diagnosis and treatment of adrenal tumors see "adrenal gland disease.") • treat fleas with any product that is safe for use in cats (see chapter ). • treat lead poisoning following the same protocols recommended for cats (see chapter ). • anemia secondary to neoplasia is associated with a poor prognosis. some cases of lymphoma may respond to treatment (see "neoplasia"). • administer human chorionic gonadotropin (hcg) in a single injection of iu (or usp) im. repeat this dose in to weeks if vulvar swelling has not diminished. • alternatively, give gonadotropin-releasing hormone (gnrh) at a dose of mg im or sc; repeat in weeks if necessary. • gnrh and hcg are effective only after the th day of estrus. bone marrow toxicity is not immediately reversible with termination of estrus; the pcv continues to fall for days to weeks. • monitor the pcv as a useful guide to therapy and prognosis: • pcv > %-the prognosis is good and termination of estrus is the only therapy required. • pcv % to %-the prognosis is guarded because the pcv level can decrease further after termination of estrus. • pcv < %-the prognosis is poor and aggressive supportive care is indicated, including multiple blood transfusions until bone marrow function is restored. • some causes of anemia in ferrets can be prevented. educate owners about proper husbandry techniques to avoid flea infestation, foreign body ingestion, and trauma. m key point to prevent estrogen toxicity, spay all female ferrets not used for breeding. insulinoma (pancreatic beta-cell tumor) is one of the most common neoplasias of the ferret. disease is most common in ferrets over years of age, and results in progressive, cyclic, or persistent hypoglycemia. • the incidence of insulinoma is typically higher in ferrets in the united states than in ferrets in other countries. the cause(s) are unknown. • possible etiologies include a limited genetic pool and diet. ferrets in the united states are typically fed processed foods containing large amounts of cereal grains. ferrets in other countries are typically fed a more natural diet consisting of meats and whole prey items. • early signs may be subtle and transient. cyclic or progressive episodes of profuse hypersalivation and pawing at the mouth (which is indicative of nausea), lethargy, depression, "stargazing," and posterior paresis may be seen during periods of hypoglycemia. • as the disease progresses, or during periods of inadequate food intake, symptoms become more pronounced and may progress to stupor or coma. seizures may occur. • splenomegaly is a common, unassociated finding on physical examination (see "splenomegaly" under "hematopoietic system" in this chapter). • adrenal disease is often identified concurrently in many ferrets with insulinoma (see "adrenal neoplasia" in this chapter). m key point base a presumptive diagnosis of insulinoma on the history, clinical signs, and repeated evidence of hypoglycemia in the presence of normal or elevated blood insulin levels. make a definitive diagnosis via surgical removal and histopathology of a pancreatic tumor or biopsy. • differential diagnoses include hepatic disease, sepsis, starvation, and laboratory error. • a carefully monitored fast of to hours is sufficient. • if necessary, obtain several samples over a period of several days. • normal fasting serum glucose concentration is to mg/dl. ferrets with insulinoma often have a fasting serum glucose of to mg/dl. ferrets with fasting serum glucose between to mg/dl are considered suspect and should be monitored. • do not fast the animal for more than hours. discontinue fast if signs of hypoglycemia occur. prolonged fasting may lead to collapse, coma, or seizures. feed the ferret a high-protein and high-fat meal as soon as possible after collection of blood. • measurement of blood insulin concentration is not consistently reliable in the ferret. • false positive results may occur if liver disease, nonislet cell tumors, or sepsis are present. • blood insulin concentrations greater than pmol/ liter (or mu/ml) are considered elevated in ferrets. however, if the ferret is severely hypoglycemic at the time of sample collection, the blood insulin value may be normal because insulin and glucose are in a constant dynamic state. • the serum biochemical profile is typically normal except for the presence of hypoglycemia. • the cbc is typically normal. • a slight elevation in alanine aminotransferase (alt) and aspartate aminotransferase (ast) may be noted. the cause is unknown and may incidental, or may indicate hepatic lipidosis due to chronic hypoglycemia or some hepatic pathology. m key point insulinoma is a progressive disease in ferrets. educate owners how to recognize the signs of hypoglycemia and how to manage hypoglycemia at home (see "medical therapy" and "hypoglycemia episodes"). • treatment options include medial therapy and/or surgical therapy. • medical therapy will need to be adjusted as the disease progresses. • surgery is used as a management tool and is not curative. insulinoma is a progressive disease, even after surgical intervention. medial therapy is often effective in controlling symptoms associated with insulinoma for to months. frequent feeding is the first step in treatment. add prednisone and diazoxide as clinical signs and hypoglycemia worsens. • feed high-quality protein and high-fat meals frequently, especially after exercise or a long sleep. avoid foods containing sugar or excessive carbohydrates (except to treat hypoglycemic episodes); these foods cause short term hyperglycemia followed by a period of hypoglycemia to hours later. • to tablespoons of hill's science diet a/d or meatbased baby foods may be given twice daily and as needed. • chromium has been anecdotally reported to stabilize blood glucose and insulin levels in humans. brewer's yeast, which is a rich source of chromium, has been beneficial in some ferrets with insulinoma. surgical therapy is the treatment of choice even though surgical removal or debulking of pancreatic tumors or partial pancreatectomy is palliative and provides only temporary remission of signs ( to months). • follow canine preoperative protocols (see chapter ). fast the ferret for only to hours preoperatively to avoid hypoglycemia. • administer iv or io isotonic fluids containing dextrose ( . - . %) to hours preoperatively if possible, during surgery, and continue postoperatively until the ferret is stable and is eating and drinking. • evaluate the blood glucose concentration pre-, intra-, and postoperatively if possible. • see chapter for information about the surgical removal of insulinoma. m key point perform a complete abdominal exploratory; insulinomas can metastasize to the regional lymph nodes, liver, and spleen (uncommon). concurrent adrenal tumors (see adrenal gland disease) are common. • if the spleen is enlarged and appears irregular or mottled, consider performing a complete or partial splenectomy and submit for histopathology. • postoperatively monitor the blood glucose concentration bid-tid until the ferret is discharged from the hospital. many ferrets become euglycemic immediately after surgery. some ferrets may remain hypoglycemic. rarely, ferrets may become transiently hyperglycemic after surgery. • most ferrets will require resumption of medical therapy to months after surgery. some ferrets will need medical therapy immediately postoperatively. • monitor blood glucose levels for to days after surgery and at to day intervals. • iatrogenic pancreatitis is rarely a problem in ferrets; however, as a precaution, withhold food and water for hours postoperatively; give . % to . % dextrose iv during this period. monitor blood glucose to times daily. • transient diabetes mellitus may occur postoperatively. hyperglycemia and glucosuria may be present for to days postoperatively; generally no treatment is required. • histopathologic examination of the pancreatic mass may reveal hyperplasia, adenoma, and/or adenocarcinoma of the pancreatic beta cells, even within a single tissue specimen. • the prognosis is guarded, but with surgery and medical treatment, ferrets have had a good quality of life for more than year after diagnosis of insulinoma. the median survival time was months (range, . - months) in one study of ferrets treated with surgery, medical management, or both. adrenal tumors are common in ferrets, and occur with approximately the same frequency as insulinomas. adrenal neoplasia and insulinomas often appear concurrently. adrenal tumors have been identified in ferrets as young as year of age, although they typically occur in ferrets > years of age. • adrenal gland disease in ferrets is not cushing's disease. excessive sex steroids, not corticosteroids, are produced by a hyperplastic or neoplastic adrenal gland. • the etiology is unknown. possible causes of adrenal disease in ferrets include early neutering, genetic factors, and lack of exposure to normal seasonal photoperiods. the incidence of adrenal neoplasia is higher in ferrets in the united states. in the u.s., ferrets are typically neutered at weeks of age, and are housed indoors under artificial light cycles. ferrets in europe and australia are typically housed outside, and are not neutered until months of age. • adrenal neoplasia in ferrets causes a variety of clinical signs, and appears to be the result of excessive secretion of estrogens and androgens, not cortisol. pituitary-dependent hyperadrenocorticism has not been documented in ferrets. • adrenal tumors most commonly arise from the adrenocortical tissue. common histopathological findings include hyperplasia, adenoma, and adenocarcinoma. • signs include progressive, bilaterally symmetric alopecia, usually starting at the tail base and progressing cranially. hair loss often starts in the early spring or fall. there may be a history of alopecia and spontaneous hair regrowth as well. • pruritis often is reported, along with excessive dryness of the skin and small excoriations. thinning of the skin is common. • an enlarged vulva, mimicking estrus, may be the only clinical sign in spayed females. mucoid or mucopurulent vulvar discharge may be noted. castrated males may exhibit territorial marking and sexual behaviors, and may develop the strong body odor and oily hair coats of intact males. mammary hyperplasia can occur in either sex. • male ferrets may present with partial to complete urinary obstruction. persistent elevation of adrenalderived androgenic hormones may cause development of prostatic hypertrophy, prostatic cysts, or periurethral cysts, which cause narrowing of the urethra. affected ferrets may present with stranguria, dysuria, azotemia, and severe metabolic derangement. male ferrets that are described as straining to urinate should be treated as an emergency (see "urinary system"). • atrophy of abdominal musculature and mobilization of fat to the ventral abdomen, leading to a pendulous appearance, may be seen. • atrophy of hind limb musculature and rear limb paresis can occur. • polyuria/polydipsia is uncommon but has been reported. • collapse, anemia, and petechiation resembling estrogen toxicity have been described in male and female ferrets with chronic or advanced adrenal disease (see hematopoietic system). • enlarged adrenal glands may occasionally be noted on the physical examination. the left adrenal gland is easier to palpate than the right. • radiographs are not typically helpful in confirming this disease. ultrasonography may be useful for identification of adrenalmegaly. • cbc is typically unremarkable unless estrogen toxicity-like anemia is present. the serum chemistry profile is typically within normal limits unless insulinoma is present. m key point a history of symmetric truncal hair loss suggests the diagnosis. differential diagnosis includes seasonal alopecia, which typically appears in the spring or fall, affects only the tail, and resolves after several weeks. • female ferrets often present with a swollen vulva. differential diagnoses include an intact female ferret, a female ferret with an ovarian remnant, and seasonal alopecia. perform a serum steroid panel or administer human chorionic gonadotropin ( iu) im to determine if a female ferret is unspayed or has an ovarian remnant. • a plasma steroid hormone assay may be used to support the diagnosis. elevated plasma concentration of estradiol, androstenedione, and/or -hydroxyprogesterone is a reliable indicator of adrenal gland disease (see table - ). a hormone panel is commercially available through the clinical endocrinology laboratory of the department of comparative medicine at the university of tennessee. • the adrenocorticotropic hormone (acth) stimulation test and the low-dose dexa-methasone suppression test are not useful in ferrets. ferrets with adrenal gland disease do not produce abnormal concentrations of cortisol, and adrenal gland disease in the ferret appears to be independent of acth. urine cortisol/creatinine ratio does not appear to be a specific indicator of adrenal gland disease. • perform exploratory surgery to confirm the diagnosis. • adrenal tumors can be managed medically or surgically. surgical management is preferred and recommended. • medical treatment may cause clinical signs to regress, but will not stop growth of the adrenal tumor. the goal of medical treatment is to decrease or eliminate the clinical signs of adrenal gland disease. medical therapy will not stop or prevent the growth of an existing tumor, and should be reserved for ferrets that are poor surgical candidates, ferrets with inoperable bilateral adrenal tumors, or ferrets with recurrent adrenal gland disease. • medical treatments described in the literature include mitotane, ketoconazole, androgen receptor blockers, aromatase inhibitors, and gonadotropinreleasing hormone analogs. • gonadotropin-releasing hormone analogs. there are two general types of gnrh analogs: gnrh agonists and gnrh antagonists. to date only gnrh agonists such as leuprolide acetate (lupron depot, tap pharmaceuticals inc., lake forest, il) have been used to control the signs of adrenal disease in the ferret. of the medical treatments described, anecdotal reports suggest that leuprolide acetate has been most effective in alleviating dermal and urogenital signs of adrenal disease. administer the month depot formulation of leuprolide acetate at a dose of ( µg/kg) im every days. • androgen receptor blockers theoretically block the actions of androgens at the receptor site, and decrease or reverse the signs of adrenal gland disease. in human medicine these drugs are used to treat men with prostatic carcinoma or prostatic hyperplasia. flutamide (eulexin, schering corporation, kenilworth, nj) and bicalutamide (casodex, astrazeneca pharmaceuticals lp, wilmington, de) have been used, primarily in male ferrets. results are variable. • aromatase inhibitors such as anastraozole (arimidex, astrazeneca pharmaceuticals lp) inhibit aromatase, an enzyme involved in estrogen production. some ferrets show decreased evidence of adrenal gland disease symptoms when treated with this drug. • mitotane ( ,p'-ddd) (lysodren, bristol-myers squibb oncology, princeton, nj) is rarely effective in ferrets with adrenal gland disease, presumably because ferrets do not develop pituitary-dependent hyperadrenocorticism. if clinical signs do resolve, they will often recur as soon as the mitotane therapy is withdrawn. m key point perform a fasting blood glucose test before starting mitotane therapy. do not use mitotane if blood glucose is low (indicative of concomitant insulinoma). mitotane causes a decrease in endogenous cortisol production; if insulinoma is present, serum glucose levels also may fall, causing a hypoglycemic crisis. • give mitotane ( mg/kg) po q h for days, then q h until clinical signs start to resolve. at that time decrease to q h until signs are fully resolved, then maintain the ferret on mg/kg once q - d as necessary. • mitotane must be compounded in -mg aliquots in # capsules. capsules must be administered intact. have owners coat the capsules with vegetable oil, push into the back of the throat, and follow with a palatable liquid or blenderized cat food to promote swallowing. • the most common side effect of mitotane is hypoglycemia. teach owners to recognize the signs of hypoglycemia, and have prednisone available at home. if side effects occur, discontinue mitotane and administer prednisone ( . - . mg) po. • if continuation of mitotane therapy is desired after a hypoglycemic crisis, administer concomitantly with prednisone (see insulinoma). • ketoconazole is not effective in the treatment of adrenal disease in the ferret. follow the adrenalectomy preoperative protocol described for dogs (see chapter ). • fast the ferret to hours preoperatively. place an indwelling iv or io catheter preoperatively, and administer fluids pre-, intra-, and postoperatively. if insulinoma is present concurrently treat and monitor appropriately. • perform a ventral midline laparotomy. palpate and visualize both adrenal glands carefully. normal adrenal glands are to mm ¥ - mm in size, are pale pink in color, and are typically surrounded by fat. • the left adrenal gland lies in a fat pad cranial to the left kidney. the right adrenal gland is located cranio-medial to the right kidney under the caudate liver lobe adjacent to the vena cava. it may be necessary to transect the hepatorenal ligament to fully visualize and palpate this gland. • adrenal changes may be subtle, especially in younger ferrets and because the adrenal glands are surrounded by fat. visual changes, such as dark circular lesions and small raised cysts, may be present instead of gross enlargement. • one or both adrenal glands may be affected. if only the left adrenal gland is affected, removal is relatively straightforward. if the right adrenal gland is affected, removal can be difficult because of the gland's proximity to the vena cava and liver (see chapter ). • if both adrenal glands are affected, remove the left adrenal gland and debulk the right adrenal gland. bilateral adrenalectomy has been described in the ferret, but should be done with caution. monitor postoperatively for development of acute adrenal hypocorticism. if acute ahc develops, treat as described for dogs (see chapter ). m key point always perform a complete abdominal exploratory. observe and palpate the pancreas at surgery for insulinomas, which often are found concurrently with adrenal neoplasia. • monitor fasting serum glucose levels every to days during mitotane therapy and after adrenalectomy, even if no pancreatic nodules were evident during surgery. • the prognosis following successful surgery is good. a full resolution of clinical signs can be expected in many cases. • recurrent or continued symptoms of adrenal gland disease may be associated with development of a tumor on the remaining adrenal gland, or recurrence of an adrenal tumor due to metastasis (which is rare). • even without treatment, ferrets may survive up to years or longer after diagnosis, although the hair loss is generally progressive. • potential sequelae to chronic adrenal gland disease include prostatic disease, bone marrow suppression, or mechanical interference with the vena cava (right adrenal gland). pheochromocytomas are adrenal tumors that arise from the adrenal medulla and produce excessive amounts of catecholamines. pheochromocytomas have been reported in ferrets, but are rare. treatment of choice is surgical removal of the affected gland. lymphosarcoma (lymphoma) is common in ferrets of all ages, and is similar in presentation to the disease in cats and dogs (see chapter ). three presentations may occur in the ferret and include lymphosarcoma, lymphocytic, and lymphoblastic forms. • a viral etiology has been hypothesized. clinical signs are variable, depending on the form of lymphoma present and the organ system involved. • lymphosarcoma: solid tissue tumors are present in the organs or lymph nodes. • lymphocytic lymphoma: adult ferrets are most commonly affected. the course and survival time can be long. peripheral lymphadenopathy is typically present and metastasis to visceral organs may occur. the neoplastic cell identified on cytology or histopathology is a mature, well-differentiated lymphocyte. • lymphoblastic lymphoma: young ferrets are most commonly affected. leukemia and neoplasia in visceral organs occur early in the course of this form of the disease. large immature lymphocytes are noted on cytology or histopathology. • other forms: cutaneous lymphoma may occur in the ferret. • clinical signs that may accompany any form of lymphoma include: • inappetence, lethargy, splenomegaly, and weight loss despite a normal appetite • dyspnea, tachypnea, and exercise intolerance • peripheral lymphadenopathy and/or abnormal cbc • acute collapse, often with pyrexia • fever of unknown origin • cutaneous masses • chronic diarrhea and/or rectal prolapse • some ferrets are asymptomatic; lymphoma may be an incidental finding during evaluation for another medical problem. • lymphoma tends to be a more acute, fulminant disease in younger animals. the method of diagnosis depends on the organ system involved. • obtain a thorough history and physical examination. • perform a cbc, platelet count, and a serum biochemistry profile. if the ferret is anemic, perform a reticulocyte count. • often the cbc and differential wbc counts are not diagnostic for lymphoma. the cbc may be normal or may reveal an absolute or relative lymphocytosis. anemia, leukopenia, and thrombocytopenia may be seen. abnormal lymphocytes may occasionally appear in the differential count. • persistent absolute lymphocyte counts greater than or a relative lymphocytosis (> %) are considered suspicious; repeat the cbc in to weeks and perform a bone marrow biopsy and/or lymph node biopsy if the cbc results are repeatable or if lymphadenopathy is present. • the serum chemistry profile may disclose elevated liver enzymes if the liver is involved; paraneoplastic syndromes are uncommon in the ferret. • perform thoracic and abdominal radiography and ultrasonography to evaluate for intra-thoracic and intra-abdominal masses. • perform fine-needle aspiration or biopsy of affected tissues for histological and cytological examination. fine-needle aspiration of the spleen is usually inconclusive. • lymph node biopsy is often the most helpful diagnostic tool for diagnosis of lymphoma. if possible, biopsy an enlarged lymph node. when lymphadenopathy is not present, biopsy the popliteal lymphnode. the popliteal lymph node is the most accessible peripheral node for biopsy. avoid biopsy of intra-abdominal lymph nodes if possible. • perform bone marrow aspiration to identify infiltration by neoplastic cells and the disease (see "clinical techniques"). • if the spleen is involved, perform a splenectomy (see chapter ) to reduce the overall tumor load. chemotherapy for lymphoma may be successful (approximately % remission rate). in general, protocols have been adapted from feline medicine (see chapters and ). • success of chemotherapy may be affected by the age of the ferret, concurrent disease (e.g., adrenal gland disease, insulinoma), concurrent medication, inappropriate use of and resistance to chemotherapeutic agents (ferrets treated with prednisone prior to chemotherapy), and the type of lymphoma present. • ferrets with bone marrow involvement or with solid tumors involving organs typically have a poor prognosis. • longer periods of remission tend to occur in individuals with adult onset or lymphocytic lymphoma. • iv chemotherapeutic agents are given via butterfly catheter or small-gauge needle with the ferret under sedation; face-mask administration of isoflurane is the most convenient and rapid method of sedation. • one chemotherapy protocol is outlined in table - . m key point monitor the cbc weekly. if the wbc falls below wbc/ml, or the rbc falls below ¥ /ml discontinue vincristine for week or more until the wbc count increases to at least wbc/ml. • palliative therapy may be attempted by administering oral prednisone ( . mg/kg) po q h. • supportive care is important (see "nutritional support for insulinoma"). • consider referral to an oncologist if experience with chemotherapeutic agents is limited. • chordoma: chordomas are tumors that arise from notochord remnants. tumors occur most often at the tip of the tail, but may occur in the cervical region as well. • git: gastric adenocarcinoma. • other tumors reported in ferrets include chondroma, chondrosarcoma, fibroma, fibrosarcoma, hepatic adenocarcinoma, hemangioma, hemangiosarcoma, mast cell tumor, mesothelioma, osteoma, osteosarcoma, schwannoma, squamous cell carcinoma, thymoma, and renal and pancreatic carcinomas. ferrets may experience dramatic seasonal changes in the haircoat triggered by photoperiod changes. this change is most apparent in the intact animal. if one is unfamiliar with these changes, normal coat changes may be interpreted incorrectly as a medical problem. m key point individual animals may exhibit different patterns of coat change each successive year. • a normal, diffuse, gradual thinning of the coat typically occurs in the spring when the photoperiod is increasing and continues through the summer. the coat typically becomes shorter and darker at this time, and the face mask may appear or disappear. focal alopecia should not be present. • some ferrets may experience a dramatic -day loss of the undercoat. • a normal, but dramatic, loss of body weight (up to %) may occur at this time as well. • hair growth will reverse in the fall and winter. coats typically become longer, thicker, and lighter. body weight may change (up to %) as well. • females in estrus and males "in season" may show an even more marked hair loss but should not have areas of alopecia. • males typically lose hair in the inguinal area because of constant rubbing to mark territory; the mid-and caudal abdomen is often wet with urine. • neutered ferrets or ferrets kept under artificial lighting conditions often experience no coat changes. • neutering or spaying may cause temporary, diffuse alopecia hair thinning postoperatively, particularly if the animal was reproductively active at the time of surgery. the preoperative color pattern may not return. • at any time of the year, regrowth of hair that has been shaved for medical procedures is slow. this is particularly true in the winter and summer when no active hair growth is occurring. • hair regrowth (regardless of the cause of alopecia) is often preceded by a blue to purple discoloration of the skin that can alarm the owner. this discoloration is caused by new hairs growing through the dermis, and is most noticeable on the abdomen and face. • intact jills may exhibit a bluish discoloration of the skin during estrus. if ovariohysterectomy is performed while a jill is in estrus, this discoloration may occur approximately days postoperatively. • pseudonails associated with hyperkeratosis of the footpads may occur in ferrets older than years of age that are housed on carpet or linoleum surfaces. trim pseudonails as necessary. rub a small amount of petroleum jelly or vitamin e oil into the pads daily to help prevent lesions. cutaneous bacterial infections in ferrets are typically manifested as abscesses or as a diffuse, ulcerative pyoderma. • abscesses may develop secondary to puncture wounds, bites, or may develop in the inguinal fat after traumatic injury (e.g., being stepped on). for diagnosis and treatment of abscesses, see "infectious diseases." ulcerative pyoderma is the second most commonly encountered form of bacterial dermatitis in the ferret. • various bacteria can cause ulcerative pyoderma. the most common agents are staphylococcus and streptococcus spp. • focal alopecia with diffusely hyperemic, thickened, ulcerated skin may occur over any area of the body. • perform a cutaneous punch biopsy (see chapter ) to rule out diffuse cutaneous mast cell tumor, which may have a similar gross appearance. • perform bacterial culture and sensitivity testing. • administer systemic antibiotics based on culture and sensitivity testing. antibiotics effective in the treatment of pyoderma in ferrets often include amoxicillin-clavulanate (clavamox, smithkline) ( - mg/kg) q h po and cephalosporins (use feline dosages). • topical treatments include twice-weekly cleansing with an antibacterial shampoo containing chlorhexidine or benzoyl peroxide. daily application of an antibacterial cream may be beneficial if the lesion is small and localized. dermatologic lesions are quite prominent with cdv infection in ferrets. • dermatologic signs typically begin with hyperemia around the lips, chin, eyes, and sometimes the inguinal area. with time, crusts and skin thickening may appear. • hyperkeratosis of the foot pads occurs as the disease progresses. • see infectious diseases in this chapter for a detailed discussion of cdv in ferrets; also see chapter for a discussion of cdv in dogs. • microsporum canis and trichophyton mentagrophytes are the most common causes of superficial mycotic infections in the ferret. • see "infectious diseases" in this chapter for diagnosis and treatment. • flea infestation may be encountered in pet ferrets. clinical signs are similar to those seen in cats (see chapter ). • flea shampoos, dips, or powders containing pyrethrin may be used. products containing lindane or organophosphates are not recommended for use in the ferret. • imidacloprid (advantage, bayer corporation, shawnee mission, ks) ( . ml) topically every weeks has been reported to be effective. no adverse effects have been noted. this drug may be used in conjunction with lufenuron. • lufenuron (program, norvartis animal health, greensboro, nc) ( mg) po every weeks has been anecdotally reported to be effective. advise clients that there is a -to -week period before flea numbers are observed to decline. • fipronil (frontline, merial ltd., iselin, nj). half the cat dose has been anecdotally reported to be effective. this drug may be used in conjunction with lufenuron. • selamectin (revolution, pfizer, new york, ny). administration of the cat dosage has been anecdotally reported to be effective. • flea collars are not recommended because they come off easily and small pieces can be ingested. • treat the environment for fleas. ear mite infection in ferrets is caused by otodectes cyanotis, the same parasite that infects cats and dogs. • ferrets rarely exhibit pruritis, even with heavy mite infestation. • • when chronic ear mite infestation is present, lichenification and a bluish pigment may appear on the inner surface of the pinnae. these changes are caused by a response to chronic irritation, and usually regress after treatment. • rarely, o. cyanotis may colonize other parts of the body. • examine all ferrets for ear mites; the incidence of infestation is high in some populations. • mites in the ear canal can often be visualized using an otoscope; however, otoscopic examination is often difficult because of the uncooperative nature of the patient and small size of the ear canal. • tresaderm (merck agvet, rahway, nj) may be used to treat ear mites in ferrets. administer drops in each ear q h for days, stop for days, then repeat. this medication has been reported to be effective in treatment of ear mites in the ferret. • selamectin may be used for treatment of ear mites in the ferret. use at the dose described for treatment of fleas. • bathe the ferret within to hours after treatment. wash all bedding and treat all other potential hosts in the household (see chapter ). • topical treatments may not be effective due to the narrow size of the ear canal, and patient resistance to treatment. • persistent infections may be due to the presence of ear mites on the body, or failure to deliver the topical agent effectively. in such cases, parenteral administration of ivermectin ( . mg/kg) sc every to days for treatments may be necessary. do not use topical and parenteral ivermectin together. • lesions are typically confined to the feet, which become hyperemic, swollen, and intensely pruritic. crusting often occurs around the nails, and in severe cases the nails may slough. • generalized alopecia, accompanied by intense pruritis, occurs rarely. • a positive diagnosis is based on clinical signs, exclusion of differential diagnoses, and positive skin scrapings obtained from several sites (false negative results do occur). • a common differential diagnosis is contact allergy. similar lesions have been observed in ferrets housed on plastic-floor cages. these lesions resolve when the cage bottom is changed to wire or wood. • advise clients of the zoonotic potential of this parasite. • treatment may need to be based on differential diagnoses; mites may be difficult to identify on skin scrapings. • administer ivermectin ( . mg/kg) sc every weeks for three treatments. • lime sulfur dips may be used instead of ivermectin. dip ferrets in % lime sulfur every days until signs have resolved for weeks. • wash all bedding and treat all potential contact hosts in the household. demodicosis is rare in the ferret. • otitis externa has been associated with demodicosis. this may be the only presenting sign. • localized alopecia accompanied by pruritis may occur. • mites may be identified on routine skin scrapings and examination of ear canal debris. • treatment can be difficult. use ivermectin at the daily dose described for dogs (see chapter ). • do not use mitotane. the etiology of tail alopecia in the ferret is unknown but is suspected to be caused by hormonal fluctuations because the disease responds to changes in the photoperiod. hair loss occurs most commonly at the time of the fall molt, when the photoperiod is becoming shorter, but may be seen any time of year under artificial lighting conditions. hair regrowth usually occurs in - weeks. the same pattern of alopecia is not always repeated annually. • hair loss, ranging from diffuse hair thinning to complete alopecia, occurs from the base of the tail to the tip. m key point alopecia occurs only on the tail. if alopecia extends to the body, suspect another form of endocrine disease, such as adrenal gland disease. • comedones and a brown, waxy scale may accompany the alopecia. • diagnosis is based on clinical signs. • differential diagnoses include the early stages of adrenal gland disease; however, hair loss on the body typically occurs as well when this condition is present. • no treatment is necessary. hair regrowth will occur when the photoperiod changes. • artificially lengthening the photoperiod may speed hair regrowth, although not reliably. • if the tail exhibits excessive amounts of waxy scaling or comedones, clean the tail weekly with a mild shampoo. alopecia may be seen in intact females that have been in estrus for month or longer. • bilaterally symmetrical hair loss over the shoulders and flanks, which eventually progresses to involve the entire body. hairs epilate easily, and the underlying skin appears normal. • a grossly enlarged vulva indicates a state of estrus. be aware that the ferret also may be anemic and thrombocytopenic (see "anemia"). • diagnosis is based on clinical signs in an intact female. • perform an ovariohysterectomy (see chapter ) if the ferret is stable enough for the procedure, or induce ovulation with hcg (see "termination of estrus"; "anemia"). • hair regrowth will recur rapidly after surgery or ovulation; however, changes in hair length, color, or thickness are common. • bilateral, symmetrical alopecia is a common sign of adrenal disease in the ferret (see "adrenal gland disease"). • hypothyroidism has not been documented in ferrets. neoplasia of the skin is the third most common neoplasia reported in the ferret and commonly occurs in ferrets year of age and older. complete removal of skin masses using wide surgical excision followed by histopathology is recommended. mast cell tumors are the most common skin masses encountered and are typically benign in the ferret. • individual tumors typically appear as slightly raised, flat, button-like cutaneous masses ranging in size from to mm. the tumors are often tan in color or may be hyperemic with a dark flaky crust. tumors may also appear as raised, ulcerated areas, or as diffuse areas of erythema and crusting. pruritis may be present at the site. • mast cell tumors have occasionally been associated with diffuse or generalized areas of alopecia that resolve with surgical removal of the tumor. • metastasis is rare but has been reported in the lung and gallbladder (see chapter for information about mast cell tumors in dogs and cats). • these tumors may also be referred to as haral cell tumors or sebaceous adenomas and are common in the ferret. • tumors may appear as wart-like, ulcerated, or cystic masses ranging in size from . to cm. • excision is usually curative. recurrence is rare, and metastasis is not reported. • other, less common neoplasms of the skin and subcutaneous tissues include: basal cell carcinoma, basi-squamo-sebaceous carcinoma, hemangioma, histiocytoma, leiomyosarcoma, lymphoma, myxosarcoma, neurofibrosarcoma, perianal gland adenocarcinoma, sebaceous gland adenocarcinoma, and squamous cell carcinoma. • adenocarcinomas often metastasize to regional lymph nodes, liver, and lungs. • diagnosis, treatment, and prognosis for these tumors in ferrets are the same as for dogs and cats (see chapter ). • cardiac auscultation is centered more caudally in the thorax than are auscultations in cats. • the heart extends from the sixth rib to the caudal border of the seventh or eighth rib (compared with cats, where it extends from the second to the sixth rib). • the heart rate averages to beats per minute. • a pronounced sinus arrhythmia and pronounced bradycardia are common during auscultation. • cardiac disease is relatively common in the ferret. quality of life and long-term prognosis for ferrets with cardiac disease depends on the type and severity of cardiac disease present, and the initial response to treatment. many ferrets do well for months on the appropriate medications. • ferrets appear to compensate well for early cardiac insufficiency, perhaps because a slight decrease in activity is not readily apparent to owners. • ferrets with congestive heart failure (chf) may present with clinical signs that resemble symptoms associated with other disease entities, such as anorexia, ascites, coughing, dehydration, dyspnea, exercise intolerance, generalized weakness, hindlimb weakness, hypothermia, lethargy, tachypnea, and weight loss. • pale or cyanotic mucus membranes and a prolonged capillary refill time (crt) may be noted on physical examination. • jugular pulses may be present when right-sided chf is present. • femoral pulses may be weak, irregular, or normal. • ascites, hepatomegaly, or splenomegaly may be noted on abdominal palpation. • murmurs may be noted on auscultation, and are typically associated with valvular insufficiency. • history and physical examination findings are important in the diagnosis of heart disease. • perform a complete physical examination, including auscultation of the heart, and evaluation of the capillary refill time. observe for tachypnea or dyspnea and auscult the lungs. palpate the abdomen and examine for ascites. m key point proceed with further testing only if the ferret is stable. otherwise, administer furosemide and oxygen therapy. • diagnosis requires information obtained by radiography, ecg, and echocardiography. • obtain whole-body radiographs. the cardiac silhouette typically appears enlarged and globoid in shape with rounded right and left ventricles. ascites, hepatomegaly, pleural effusion, and pulmonary edema may be present as well. • evaluate a cbc, serum biochemical profile, and urinalysis to determine if azotemia, electrolyte abnormalities, or other systemic diseases are present. perform a heartworm test if the history is supportive for potential exposure. • if thoracic or abdominal effusion is present, perform thoraco-or abdominocentesis and submit fluid for cytologic examination. perform centesis as described for cats; take into consideration the relatively caudal position of the heart in ferrets. sedation is usually necessary. a modified transudate is typically associated with chf. • perform standard six-lead electrocardiography (ecg) if possible (table - lists normal ferret ecg parameters). sedation may be necessary. electrocardiography may reveal atrial premature contractions, atrial tachycardia, atrial fibrillation, ventricular premature contractions, and ventricular tachycardia. m key point sedation with isoflurane is recommended when necessary. sedation with ketamine or a ketamine-diazepam combination raises the heart rate. the heart rate tends to decrease with ketamine-xylazine sedation; therefore, avoid using xylazine in ferrets with suspected cardiac disease. • echocardiography is the most useful diagnostic tool in the ferret. the same echocardiographic changes observed in the dog and cat are seen in the ferret. (table - ) . m key point treatment of acute chf should focus on improving oxygenation and reducing cardiac preload and afterload. • place the ferret in an oxygen-rich environment. administer supportive care such as subcutaneous fluids (e.g. . % saline and . % dextrose), and provide nutritional support for ferrets that are anorexic. • administer diuretics such as furosemide ( - mg/kg) im or iv bid-tid. • nitroglycerin % ointment may be applied to the skin in the axilla, inguinal area, or on a hairless body surface. • angiotensin-converting enzyme (ace) inhibitors may be given to reduce afterload and preload. give enalapril (enacard, merck agvet division) ( . mg/kg) po q h, then titrate up to q h if possible. ace inhibitors may cause hypotension in ferrets, titrate to effect. • when diuretics and ace inhibitors are used together it is important to monitor for azotemia. • perform thoracocentesis or abdominocentesis when indicated. • monitor body weight, crt, heart rate and rhythm, hydration status, mucous membrane color, respiratory rate, respiratory effort, bun, creatinine, and serum electrolytes. m key point chronic therapy typically includes the use of ace inhibitors, and diuretics with the addition of digoxin in ferrets with dilated cardiomyopathy. whenever possible, try to titrate the diuretic dose to the lowest possible dose without recurrence of pleural effusion or pulmonary edema. • administer digoxin elixir ( . mg/kg) po sid-bid to ferrets with dilated cardiomyopathy. • side effects associated with digoxin include anorexia, arrhythmias, diarrhea, lethargy, and vomiting. • serum digoxin levels should be monitored every to weeks. normal values have not been published for the ferret; reference values for dogs and cats are used for interpretation. • use of antiarrhythmic drugs such as atenolol or diltiazem is not well documented in the ferrets, but may be useful in the treatment of ferrets with hypertrophic cardiomyopathy. • salt-free diets may be beneficial; however, they are often unpalatable to ferrets. instruct the owner to avoid feeding snacks, treats, or food items with a highsalt content. • management includes periodic reevaluation of heart rate and rhythm, serum electrolytes, and renal values. radiographs should be used to monitor for the development of pulmonary edema or changes in the cardiac silhouette. ecg and echocardiography should be repeated periodically as well. cardiomyopathy may occur in ferrets years of age or older. dilated (congestive) and hypertrophic forms can occur; the dilated form is more common. the cause of dilated cardiomyopathy (dcm) in the ferret is unknown. • abdominal enlargement secondary to ascites, anorexia, dyspnea, lethargy, and weight loss are often noted. • ascites, heart murmur, pale mucous membranes, tachycardia, and weakness may be noted on physical examination. • moist rales and increased respiratory sounds may be noted when pulmonary edema is present. • pleural effusion may be present, and may cause an increased inspiratory effort. the heart may sound muffled on auscultation. • coughing generally is not noted. • see the chf section in this chapter. • treatment is the same as that described for chf. • taurine supplementation does not appear to have any effect on dcm in the ferret. • see the chf section in this chapter. the cause of hypertrophic cardiomyopathy (hcm) is unknown. • clinical signs may be compatible with those described for chf or dcm (see above). • other clinical signs are similar to those described for the cat, and include acute onset of congestive heart failure and/or sudden death. • follow the same guidelines described for dcm. • include hcm on the rule-out list when evidence of cardiac disease is noted on the physical examination or diagnostic evaluation. • radiographs may not be beneficial in the diagnosis of hcm. • echocardiography should be used for definitive diagnosis. • treatment should be aimed toward alleviating signs of chf and improving the diastolic efficiency of the left ventricle. • administer beta-adrenergic blocking drugs such as atenolol ( . - . mg) po sid. titrate to effect. • administer calcium channel blockers such as diltiazem ( . - . mg) po bid. titrate to effect. • diuretics are indicated if symptoms of chf are present (see above). • follow-up is the same as that described for chf and dcm. natural and experimental heartworm infections have been reported in ferrets (see chapter ). the clinical presentation of heartworm disease typically resembles that of cats; however, the life cycle of dirofilaria immitis in ferrets is similar to the life cycle present in the dog. reported adult worm burdens range from to . the presence of only one adult worm in the heart can be lethal. • heartworm disease is caused by the canine heartworm dirofilaria immitis, a filarial nematode that is transmitted via mosquitoes. • ferrets that are housed outdoors in endemic areas are at greatest risk of infection; however, ferrets kept indoors also can become infected. • clinical signs include coughing, dyspnea, hepatomegaly, inappetence, lethargy, melena, weakness, and symptoms associated with right-sided chf (pulmonary edema, pleural effusion, ascites). sudden death due to pulmonary artery obstruction may also occur. • microfilaria may be present in the blood of approximately % of infected ferrets. • diagnosis is based on the history, clinical signs, heartworm test results, radiographs, and echocardiography. • if the history is compatible with cardiac failure, inquire about possible mosquito exposure. • physical examination findings resemble those of heart failure (see above). m key point minimize stress in ferrets suspected of heartworm disease. if symptoms of congestive heart failure are present, delay further diagnostic evaluation until the patient is stabilized (see "treatment of congestive heart failure"). • obtain whole body radiographs. thoracic changes may include cardiomegaly with enlargement of the right atrium, caudal vena cava, and right ventricle. pleural edema and pleural effusion may be present as well. radiographic changes in the peripheral pulmonary arteries are not typically noted because the worms tend to reside in the right side of the heart and in the main pulmonary artery. abdominal changes often include hepatomegaly, splenomegaly, and ascites. • if possible, draw blood for the modified knott's test for microfilaria. microfilaria are identified in approximately % of infected ferrets. • submit blood for an enzyme-linked immunosorbent assay (elisa) for dirofilaria antigen. antigen is produced by adult female heartworms; there is a potential for false negative test results in ferrets with low worm burdens. a commercial assay (snap heartworm antigen test kit; idexx laboratories inc., portland, me) has been used to detect heartworm infection in the ferret. • perform a cbc, serum biochemical profile, and urinalysis to rule out the presence of other systemic diseases. • if pleural or abdominal effusion is present, submit fluid for cytology. a modified transudate is typically noted when chf is present. • echocardiography may be used to visualize heartworm(s) in the pulmonary artery, right ventricle, and right atrium; dilation of the right ventricle and right atrium may be assessed as well. doppler echocardiography may be used to evaluate the patient for the presence of pulmonary hypertension. • treatment of heartworm disease in ferrets is difficult. success is dependent on early diagnosis, diligent supportive care, and long-term antithrombotic therapy in conjunction with adulticide therapy. • if signs of chf are present, treat this first, and stabilize the patient (see the chf section). • if the patient is symptomatic and microfilaremia positive: • administer microfilaricidal therapy: ivermectin ( µg/kg) sc every days until clinical signs and microfilaremia resolve. • follow with adulticide therapy: melarsomine (immiticide, rhone merieux, athens, ga) using a two-stage protocol: stage : administer a single dose of melarsomine ( . mg/kg) im. stage : month later, administer two injections of melarsomine ( . mg/kg) im given hours apart. • transient swelling at the site of injection is common. • administer prednisone ( . mg/kg) po sid-tid during adulticide treatment and for as long as clinical signs persist. • if pleural effusion is present administer diuretics (see the chf section). • cage confinement is important for to weeks after treatment. • perform a post-treatment elisa for heartworm antigen months after adulticide therapy. repeat every days if results are positive. most ferrets become seronegative months after treatment. • begin heartworm prevention month after adulticide treatment. • if ferrets are microfilaria negative, administer adulticide therapy as described above. m key point because of the high mortality associated with heartworm disease, recommend preventive therapy for all ferrets in heartworm-endemic areas. • ivermectin may be given as preventive therapy beginning month before and continuing months after mosquito season. liquid ivermectin % may be diluted in propylene glycol ( . ml ivermectin in ml propylene glycol) and administered at a dose of ( . ml/kg) po every month. this solution must be stored in an amber glass bottle out of sunlight. feline heartguard (merck agvet) may be administered using the dose appropriate for a -to -lb cat. • if possible, house all ferrets in endemic areas within structures with mosquito-proof screening. • follow the same recommended guidelines for heartworm prevention in dogs and cats. valvular heart disease may occur in ferrets > years of age. • clinical signs depend on the severity of the underlying disease process. • mitral regurgitation may be ausculted as a systolic murmur in the left apical region. • tricuspid regurgitation is ausculted in the right parasternal region. • dyspnea and moist rales may be noted on auscultation of the lungs if chf is present. • obtain thoracic radiographs to evaluate the size of the heart and to determine if chf is present. pulmonary edema typically appears as a mixed alveolar and interstitial pattern in the caudodorsal lung lobes. • electrocardiography (ecg) may be normal or may demonstrate evidence of atrial arrhythmias. • echocardiography typically demonstrates thickening of affected valves and atrial enlargement. • doppler echocardiography may be used to identify and quantify the degree of regurgitation present. aortic regurgitation is often noted in ferrets and is considered an incidental finding. • treatment is recommended if chf is present, or if cardiac enlargement is significant (see the chf section). myocarditis occurs when the myocardium is infiltrated with inflammatory cells, resulting in the development of reduced myocardial function, arrhythmias, and replacement of the normal myocardial tissue with fibrous tissue. • causes include sepsis, systemic vasculitis, parasitic, bacterial or viral infection, and autoimmune disorders. • aleutian disease can cause fibrinoid necrosis and mononuclear cell infiltration of the arterioles of the heart. • antemortem diagnosis is difficult. • suspect myocarditis if arrhythmia and/or acute myocardial dysfunction is noted in association with multisystemic illness. • definitive diagnosis is made by histopathological evaluation of affected myocardial tissue. • treatment should be directed at identifying and treating the underlying systemic disease. • cardiovascular support should be provided and may include the use of diuretics or antiarrhythmic drugs (see the chf section). as clinical experience with pet ferrets increases, other types of cardiac disease are likely to be recognized. third-degree heart block (of unknown etiology) and various forms of valvular disease, including mitral and tricuspid insufficiency and endocarditis, have been seen in ferrets. • the approach to these conditions in ferrets is the same as for other companion animals; use the drug dosages given previously for cardiac myopathies. • the permanent teeth erupt between and days of age. • the dental formula is (i / , c / , pm / , m / ). • the third upper premolar (carnassial tooth) has three roots. the second lower molar has one root. all other premolars and molars have two roots. • the ferret is an obligate carnivore with a simple stomach, short intestinal tract, no cecum or ileocolic valve, and a short colon. • the duodenum terminates at the jejunoileum; there is no gross anatomic distinction between the jejunum and the ileum. • the junction of the jejunoileum and the colon is determined by evaluating the pattern of anastomosis between the jejunal artery and the ileocolic artery. • gi transit time is approximately to hours. • the anal sacs are located between the external and internal anal sphincter muscles at and o'clock. the ducts are located near the mucocutaneous junction. • the exact nutritional requirements of the ferret have not been determined. • the diet of the ferret must contain predominantly animal protein and fat. • due to the short digestive tract and rapid gi transit time, the ferret requires a concentrated maintenance diet high in protein ( - %) and fat ( - %), and low in fiber. the protein quality should be % to % digestible. • breeding ferrets and kits may require diets higher in protein and fat. • meat, poultry, meat and poultry meals, and other animal-based proteins should appear first, then several more times on the food ingredient list. • complex carbohydrates (starch, fiber) are not readily digested by the ferret. high-fiber diets can induce a relative protein-calorie deficiency; the ferret cannot eat enough of a low-density food to meet its high maintenance requirements. • premium cat foods and ferret diets typically meet the ferret's nutritional requirements for growth and reproduction. • treats and supplements should not exceed more than % of the daily diet. acceptable treats include meat baby foods, and moist cat or ferret diets. high-sugar or carbohydrate treats should be limited, especially if insulinoma is present. • fatty acid supplements should be given in measured amounts (a few drops per day). administration of large quantities of fatty acid supplements may reduce the intake of the balanced diet. • canine diets should not be fed to ferrets; the protein, fat, and carbohydrate content is not appropriate, and the diets often contain high percentages of grain and vegetable matter. • the long-term effect of formulated dry and canned diets on the long-term health of ferrets is controversial among some practitioners. • some practitioners feel that feeding commercial diets containing large quantities of plant-based ingredients contributes to the development of eosinophilic gastroenteritis, inflammatory bowel disease, insulinoma, urolithiasis, and general untriftiness. for example, most ferrets in the united states are fed dry kibbled diets, and the incidence of insulinoma is high. many ferrets in europe and australia are fed whole prey items (e.g., a "natural diet), and the incidence of insulinoma is low. • a correlation between diet and the development of certain diseases in ferrets is hypothetical at this time; however, this controversy demonstrates the need for longer-term diet studies in the ferret. • the canine teeth are often worn or broken at the tips due to biting and gnawing. • broken canine teeth typically are not painful unless the dental pulp is exposed. • dental tartar and periodontal disease are common in ferrets over years of age. • soft, moist diets may predispose ferrets to the development of dental disease. • tartar typically accumulates first on the second and third upper premolars. • dental abscesses are not common, but may be noted, even in young ferrets. • follow the basic medical and surgical treatment principles described for dental diseases in dogs and cats (see chapter ). • ferrets have five major pairs of salivary glands: the parotid, submandibular, sublingual, molar, and zygomatic. • salivary mucocele occurs secondary to trauma or infection of a salivary gland. • salivary mucocele typically presents as a soft to firm swelling in the region of the orbit, oral commissure, or mandibular lymph node. aspiration of the swelling often yields a clear to serosanguinous or mucinous fluid; microscopic examination demonstrates amorphous debris and occasional rbcs. • treatment of choice is surgical excision of the affected gland (see chapter ). • advise clients that recurrence is possible. megaesophagus is rare in ferrets. • the etiology of megaesophagus in ferrets is unknown (see list of possible causes in dogs in chapter ). • clinical signs resemble those described for the dog and include: lethargy, anorexia, dysphagia, coughing, choking, dyspnea, weight loss, and regurgitation. • clients may indicate that the ferret vomits up large boluses of food. • diagnosis may be based on clinical signs and radiographic evidence of megaesophagus. • obtain thoracic radiographs. the esophagus is often dilated and filled with air in the cervical and thoracic regions. food may be present within the lumen of the esophagus. • perform a barium contrast study to delineate the esophageal mucosa and to identify potential mural lesions, strictures, or obstructions. • aspiration pneumonia may be visible radiographically. • follow canine treatment protocols. the prognosis is poor; response to therapy is usually not successful. • gi promotility agents such as metoclopramide • administer antibiotics if indicated for aspiration pneumonia. • supportive care includes feeding high-calorie, highprotein slurried diets to times per day, and elevating the ferret for to minutes immediately after feeding. • ferrets, like other carnivores, are able to vomit. • differential diagnoses to consider for vomiting include esophageal and gastroenteric disorders (see below). • ferrets often demonstrate symptoms associated with nausea or vomiting when gastroenteritis, gi disease, gastric ulcers, helicobacter mustelae gastritis, or gi foreign bodies are present. hypoglycemia may cause signs of nausea as well (see discussion of insulinoma in this chapter). • signs of nausea include hypersalivation and pawing at the mouth. • ferrets may demonstrate bruxism (grinding of the teeth) when abdominal discomfort is present. • gi parasites are uncommon in the ferret. coccidiosis and giardiasis are occasionally seen. nematodiasis is rare. • routine fecal testing is still recommended, especially in young animals and ferrets with diarrhea or rectal prolapse. • young ferrets with coccidiosis may have diarrhea and may be severely dehydrated. • cryptosporidiosis may occur in ferrets, but typically does not result in clinical disease. the zoonotic potential is unknown; however, it may be prudent to warn immunosuppressed owners of the potential for zoonosis. • treat with appropriate anthelmentics following the protocols and dosages used for cats (see chapter ). • obtain fasting whole-body radiographs to help rule out the presence of a gi foreign body or trichobezoar. • a barium study may be used to demonstrate gi ulceration. • exploratory laparotomy/gastrotomy is often required for a definitive diagnosis. • diagnosis of h. mustelae gastritis is often a diagnosis of exclusion. definitive diagnosis requires the finding of organisms along typical histological lesions on gastric biopsy specimens. • debilitated, anorexic ferrets may require hospitalization for supportive care. • if the patient is vomiting, withhold food for to hours. administer iv fluids containing dextrose, and monitor for signs of hypoglycemia. when vomiting has resolved begin to offer small, bland meals. • feed small meals of a bland, moist diet tid-qid (see diet recommendations in "insulinoma" section). avoid feeding high-fiber dry foods. • administer broad spectrum antibiotics if the ferret is debilitated. • administer a gastric protectant. options include: • gi obstruction caused by foreign body ingestion or hairballs is one of the most common problems in pet ferrets. • foreign bodies typically occur in ferrets younger than year of age; trichobezoars (hairballs) are common in ferrets older than years of age. m key point suspect the presence of a gi foreign body in any young ferret presented for anorexia, even if no vomiting is reported. • rubber and foam objects are the most common foreign bodies. obstruction with a hairball (older ferrets), cloth, or plant material also may occur. • lethargy, partial or total anorexia, hypersalivation, bruxism, pawing at the mouth, weight loss, and diarrhea are the most common clinical signs of gi foreign body. hindlimb weakness, dehydration, and melena may be noted as well. • vomiting is uncommon; however, if the ferret is vomiting, be suspicious that a gi foreign body may be present. • diagnosis is based on the history, physical examination findings, radiographs, or exploratory laparotomy. • history: identify possible types or causes of foreign body ingestion. ask the owners if hairball preventative is used routinely. • physical examination: large gastric foreign bodies are often palpable. small foreign bodies in the small intestine may be associated with localized pain. • obtain fasting ( - hours) plain whole body radiographs. radiographs may reveal segmental ileus, and marked gaseous distention of the stomach and/or bowel. occasionally a foreign body or trichobezoar can be identified. • obtain a gi barium contrast study to identify small foreign bodies and to rule out gi ulceration. • perform a cbc and serum biochemical panel to rule out hepatic lipidosis and other systemic diseases. surgical removal is the treatment of choice. if the ferret is debilitated, begin supportive therapy, and perform surgery as soon as possible. • surgery: follow routine preoperative, operative, and postoperative procedures for gastrotomy or enterotomy (see chapters and ). ferret tissues are more delicate than those of a puppy or kitten of equivalent weight. use - or - suture material to close the gi tract. • perform gastric biopsy to rule-out underlying h. mustelae infection, and other gi diseases. perform biopsy of the liver. • evaluate the entire abdominal cavity prior to closure. older ferrets often have concurrent diseases such as insulinoma or adrenal gland disease. • the prognosis following gastrotomy is good with prompt therapy. m key point instruct owners to "ferret proof" the house if ferrets are allowed to roam. in particular, restrict access to rubber toys and rubber objects. • to prevent trichobezoars, administer a feline hairball laxative product ( - cm) po to times per week. epizootic catarrhal enteritis (ece, "green slime disease") is a highly infectious diarrheal disease that first appeared in . the etiological agent is thought to be a coronavirus. ece can spread rapidly through a ferret population, often affecting % of ferrets within hours. histological examination of intestinal biopsy samples reveals lymphocytic enteritis with villous atrophy and blunting, and degeneration of the apical epithelium. • the history often includes recent exposure of an older ferret to a new or young ferret that appears healthy. often within hours the older ferret becomes anorexic and lethargic. • four clinical syndromes are typically seen: . ece may cause relatively mild diarrhea that lasts several days in young ferrets with no underlying disease. . ece may cause severe diarrhea lasting for several days that may be followed by an acute onset of severe bloody diarrhea in older ferrets or ferrets with concomitant disease. anemia may develop as a sequelae. . a wasting disease with abnormal stools that have the appearance of bird-seed or of being grainy. these stools may develop in ferrets that initially appear to have recovered from the diarrheal phase. . voluminous green, watery diarrhea and occasional vomiting followed by chronic wasting may occur in some ferrets. • the clinical course of disease can be prolonged in some ferrets, and may last weeks to months. affected ferrets typically appear to recover, but continue to have persistent, intermittent diarrhea. • the disease may recur in previously affected ferrets; an asymptomatic carrier state appears to be possible. • do not house ferrets that have had ece with ferrets that have not had the disease. • rotavirus has been associated with several outbreaks of diarrhea and high mortality in ferret kits to weeks of age; it is often referred to as "ferret kit disease." • rotavirus also causes diarrhea in the young of several other species, including humans, cattle, swine, sheep, and rats. • in adult ferrets, rotavirus infection is rarely fatal, but may cause bright green mucoid diarrhea that lasts for several days. • there is no readily available antemortem test for the rotavirus infection; rotavirus particles can be identified in feces by electron microscopy. • treatment consists of supportive care. administer fluids, antibiotics, and nutritional support. salmonellosis is rare in the ferret, and is typically associated with exposure to contaminated raw meat and meat by-products. salmonella typhimurium, s. newport, and s. choleraesuis may be associated with clinical disease. • clinical signs include anorexia, lethargy, fever, and diarrhea (usually bloody). conjunctivitis and anemia have also been reported. • diagnosis is based on clinical signs and a positive fecal culture. multiple fecal samples must be collected, and selective media is used for culture. • treatment includes aggressive supportive care and antibiotic therapy. • ferrets may be presented in shock. iv fluids and administration of rapidly acting intravenous corticosteroids may be necessary for treatment of these patients. • other details of salmonellosis, including its public health significance, are discussed elsewhere in this text. eosinophilic gastroenteritis (ege) is an inflammatory bowel disease that occurs in ferrets and other animals. no specific etiological agent has been identified in the ferret, but food allergy is implicated in humans and other animals. • chronic diarrhea with or without mucus or blood, and weight loss are the most common signs. inappetence, intermittent vomiting, and skin lesions may be seen as well. • on physical examination, the mesenteric lymph nodes may be enlarged and the intestines may feel thickened. • a marked peripheral eosinophilia is often present on the cbc differential. • presumptive diagnosis is based on history, clinical signs, physical examination findings, the presence of a peripheral eosinophilia, and/or the presence of eosinophils on fecal cytology. • definitive diagnosis is based on histological examination of intestinal biopsy specimens. mild to extensive eosinophilic infiltration of the mucosa, submucosa, and muscularis of the stomach and small intestines are noted. focal eosinophilic granulomas maybe identified in the mesenteric lymph nodes. • treatment is similar to that described for treatment of dogs and cats. • begin corticosteroid therapy with prednisone ( . - . mg/kg) po sid every days. perform a recheck examination and cbc weeks after the last dose. if the ferret has improved clinically and the peripheral eosinophilia is resolving, decrease the prednisone dose by % every days, and recheck again. continue the prednisone taper at -week intervals until the ferret is tapered to the lowest possible dose, or withdrawn from the steroids altogether. • although food allergy has not been identified as a definitive etiological cause of ege, changing the ferret to a hypoallergenic diet, such as a feline lamb and rice-based diet may be helpful in resolution of signs. • there have been reports of ferrets with peripheral eosinophilia (up to %), and erythema and crusting of the feet, ears, and face. histological lesions in biopsy specimens from affected skin were consistent with allergic dermatitis. these ferrets were treated with corticosteroids, and did respond to treatment. one also responded to diet change. inflammatory bowel disease (ibd) can occur in the ferret. the etiology is unknown; dietary factors, hypersensitivity reactions, or an immune-mediated cause have been considered. • clinical signs can be subtle and include diarrhea; nausea; occasional vomiting; soft, malformed stools that resemble bird seed; and weight loss. these signs often resemble ece, ege, and helicobacter gastroenteritis. • affected ferrets are often young or middle-aged adults. • elevation of liver enzymes and serum globulins may be noted on serum biochemistry analysis. • lymphocytosis may be noted on the cbc. • diagnosis is based on the history, clinical signs, and diagnostic work-up. • definitive diagnosis is made by histological examination of gastric and intestinal biopsy samples. mild to severe lymphoplasmacytic gastritis and enteritis are noted on histopathology. • administer corticosteroids such as prednisone ( . - . mg/kg) po sid every days initially, and taper in a manner similar to that described for ege. some ferrets respond poorly to steroid therapy. • azathioprine (imuran, prometheus laboratories, san diego, ca) ( . mg/kg) po q - h may be used as an alternative to steroid treatment. • hypoallergenic diets may offer some benefit. proliferative bowel disease (pbd) in ferrets was first reported in , and is similar to the pbd that occurs in swine and hamsters. pbd was a commonly encountered disease in the late s and early s, but is relatively uncommon now. pbd is caused by lawsonia intracellularis, an intracellular bacteria that cannot be propagated by routine culture methods. • this disease affects primarily young ferrets to months of age. • acute and chronic forms of the disease can occur. • diarrhea is present and often contains mucus and blood. defecations are frequent and small; ferrets often cry out when they defecate. the rectum may be partially prolapsed. • other signs include lethargy, depression, inappetence, weight loss, dehydration, and pyrexia. • neurologic signs such as ataxia and muscle tremors may be present. • the intestines may feel firm or thickened on abdominal palpation. • a tentative diagnosis of proliferative bowel disease is based on clinical signs and physical examination. definitive diagnosis requires intestinal or colonic biopsy, but this rarely is warranted because response to therapy usually is good if initiated early. • a polymerase chain reaction (pcr) assay specific for the swine isolate, and an indirect fluorescent antibody test (ifa) are available. • necropsy lesions include gross thickening and discoloration of the small intestine and/or colon. ridges of proliferative tissue that are distinct from normal adjacent tissues are present on the mucosal surface. • histological examination of biopsy samples or necropsy specimens typically demonstrate epithelial proliferation, hypertrophy of the muscularis, and infiltration of the bowel wall with monocytic or granulocytic inflammatory cells. silver-stained tissues reveal intracellular, comma-shaped organisms in crypt epithelial cells. glandular hyperplasia consisting of irregular, branching proliferative glands that lack goblet cells, and necrotic debris may be identified in the crypts. severe glandular hyperplasia may resemble neoplasia and can metastasize. • treat mild cases on an outpatient basis. • hospitalization for supportive care (fluid therapy, nutritional support) may be necessary when severe disease is present. • administer chloramphenicol ( mg/kg) q h, po, iv, im, or sc as the drug of choice. treat for at least weeks; longer therapy often is necessary to prevent relapse. • metronidazole ( mg/kg) q h, po may be effective. • the prognosis is good with timely therapy. • some ferrets improve temporarily and then relapse at the end of the treatment period. use a long-term course of antibiotic therapy in these animals. • rectal prolapse is usually a disease of young ferrets, and is often associated with diarrhea. • possible causes of rectal prolapse include colitis, diarrhea, gi parasitism (e.g., coccidiosis), pbd, and other diseases that may cause straining or diarrhea. • other differentials include gi lymphoma, benign intestinal polyps, and postoperative complications of anal gland removal. • perform direct fecal and fecal flotation tests to screen for parasites. • medical treatment is similar to that described for other species. administer anthelmentics and antibiotics when indicated. • the prolapse may resolve without surgical intervention when the underlying disease process is resolved. • surgical correction is usually unnecessary (see below). • if indicated, perform a biopsy of the prolapsed tissue to rule out lymphoma. • flush the prolapsed tissues with sterile saline and replace them into the rectum. • place a purse-string suture in the anus with a small opening to allow passage of feces. keep the pursestring suture in place for to days. • in ferrets with chronic prolapse, surgery may be necessary to reduce the size of the anal opening. excise a small triangular wedge of anal mucosa and routinely close the defect by suturing. alternatively, consider abdominal exploratory surgery and colopexy (see chapter ) • clinical signs and physical examination findings in ferrets with an abscessed anal sac are the same as those described in dogs and cats. • the recommended treatments include antibiotic therapy, lancing and drainage of the abscess, or surgical removal of both anal sacs (see chapter ). anal sacculectomy is performed as a treatment for anal sac abscesses, or to decrease the musky "ferret" odor. for odor reduction, neutering should be performed simultaneously because the apocrine, perianal, sebaceous, and scent glands in the skin are under hormonal control and contribute to the overall musky odor. some clinicians believe that neutering is sufficient to decrease odor and that routine anal sacculectomy should be discouraged. . grasp the anal sac duct and hold it closed with mosquito forceps. make a circumferential skin incision around the duct opening. . apply gentle caudal traction to the anal sac, and use a scalpel blade or gauze to tease away the surrounding fascia. . leave the surgical sites open, and allow to heal by second intention. . make small, arc-like incisions just lateral to the duct openings. . dissect the subcutaneous tissues bluntly to reveal the neck of the anal sac; grasp the opening and hold it closed with mosquito forceps. . dissect the sac free of surrounding tissues, using gentle traction. . do not suture the incisions. ferrets reach sexual maturity during the first breeding season after birth. the breeding season runs from march to august under natural lighting conditions. • the opening of the prepuce is located just caudal to the umbilical area. • males (hobs) have a j-shaped os penis. • during the breeding season (march-august), testicle size is twice that noted in the fall and winter months. • prostatic tissue is located at the base of the urinary bladder and surrounds the urethra. prostatic disease associated with adrenal gland disease may occur in middle-aged and geriatric male ferrets (see "adrenal gland disease" and "prostatic disease"). • female ferrets (jills) are seasonally polyestrous and are induced ovulators. ovulation typically occurs to hours after mating. • the vulva is located in the perineal region ventral to the anus. in non-estrous females the vulva is small, and looks like a slit; during estrus (or when adrenal gland disease is present), the vulva becomes swollen and is easily visualized. • if mating is unsuccessful, pseudopregnancy results and lasts to days. • approximately % of females remain in estrus if they are not bred. the resultant prolonged elevation of serum estrogens can cause bone marrow toxicity and pancytopenia (see the discussion of anemia under "hematopoietic system" in this chapter). • submit blood for a cbc and platelet count if the ferret has been in estrus for more than days. • termination of estrus is recommended (see "termination of estrus in the hematopoietic".) • most pet male ferrets in the united states have already been neutered prior to weeks of age. • castrate intact male ferrets at to months of age in order to reduce aggressive behavior and odor. • castration is performed using techniques similar to those used in cats (see chapter ). • make an incision in the scrotum over each testicle. • most pet female ferrets in the united states have already been spayed prior to weeks of age. • spaying intact female ferrets is recommended to prevent estrogen-induced bone marrow hypoplasia. • ovariohysterectomy is similar to the procedure performed in cats (see chapter ). • the ventral midline incision is made approximately cm caudal to the umbilicus, and may be extended as necessary. • the uterus is bicornuate, and is located dorsal to the bladder. • ovarian vasculature may be difficult to locate due to the large amount of body fat typically present in this region. • pyometra and metritis are uncommon in pet ferrets in the united states because they are usually spayed prior to being sold as pets. • clinical signs may include anorexia, lethargy, pyrexia, and vulvar discharge. polyuria and polydipsia are not usually noted. • persistent estrus may predispose ferrets to pyometra. • preoperatively perform a cbc and a serum biochemical analysis to rule out estrogen-induced bone marrow hypoplasia (see "anemia" in "hematopoietic system"). • provide appropriate supportive care pre-and postoperatively. • perform ovariohysterectomy when the patient is stable (see chapter ). • start the ferret on broad-spectrum antibiotic therapy preoperatively, and continue postoperatively for to days. use broad-spectrum antibiotics. organisms commonly associated with pyometra include staphylococcus spp, streptococcus spp, corynebacterium spp, and e. coli. • vulvar swelling is an external sign of estrus in female ferrets. • in a spayed female, a swollen vulva indicates a remnant of ovarian tissue, or another source of estrogens and estrogen precursors such as adrenal gland disease (see "adrenal gland disease"). • ovarian remnants typically induce signs of estrus in ferrets younger than years of age. • administer hcg ( iu) im. vulvar swelling should subside if an ovarian remnant is present. if no changes occur, adrenal gland disease is probably the cause of the clinical signs. • perform exploratory laparotomy to remove the ovarian remnant (see chapter ). evaluate for uterine remnants and adrenal gland disease as well. • preoperatively evaluate a cbc to rule out estrogeninduced bone marrow hypoplasia. • pregnancy toxemia is a potentially life-threatening condition that occurs in late pregnancy. primiparous females are most commonly affected. • the disease results in high mortality of jills and kits. • toxemia can be induced if an accidental fast occurs in the last week of gestation. • pregnancy toxemia may also develop in primiparous jills that are carrying large litters due to nutritional compromise induced by the size of the gravid uterus and the resultant reduced capacity of the stomach. • advise owners that pregnant jills must have access to food and water al lib during pregnancy. • suspect pregnancy toxemia if acute lethargy develops in the last week of gestation. other clinical sings include dehydration, melena, hypoglycemia, ketonuria, and azotemia. • affected ferrets usually are presented in an acute state of shock. • treatment includes aggressive supportive care including iv or io fluids containing dextrose. perform an immediate cesarean section (see chapter ). • postoperative care includes continued supportive care, including frequent feedings of high-calorie critical care diets. • jills that survive pregnancy toxemia often do not produce milk. kits can be difficult to hand rear; if a foster jill is not available, attempts may be made to hand rear the kits using a kitten milk replacer (see below). kits born before days of gestation often do not survive. • the prognosis is usually poor, even with aggressive treatment. • do not breed females with a history of mastitis. • abrasions to the mammary tissue and nipples can cause mastitis. prevent trauma from occurring by providing a large nest box opening with smooth edges that allows the jill to pass through easily. • mastitis may be acute or chronic. • acute mastitis typically occurs immediately after whelping or during the third week of lactation. • affected glands appear swollen, firm, red to purple in color, and are painful. gangrene can develop within hours of clinical signs. • treatment must be aggressive. administer broadspectrum antibiotics, and apply hot packs to the affected area to times per day for days. debride necrotic tissue if present. provide supportive care and analgesic therapy. • submit a sample for bacterial culture and sensitivity testing. modify antibiotic therapy based on test results. • if there is no clinical response to medical therapy in days, or if gangrene rapidly develops, consider surgical removal of the affected mammary tissue. because of the potential for severe toxicity and life-threatening disease, do not delay surgery if gangrene is already present, or if there is no improvement with medical therapy. • if the jill continues to lactate, leave the kits with her. supplement feed the kits with a kitten milk replacer if necessary. do not foster the kits with another jill because this may result in mastitis in the foster jill. • ingestion of infected milk may cause gastroenteritis in the kits; kits may need to be treated with antibiotics as well. • chronic mastitis is often difficult to diagnose. the affected jill often appears normal, while the kits lose weight or fail to thrive. • mammary glands appear firm but are not painful or discolored; often the glands are presumed to be full of milk. • affected mammary glands become scarred and are no longer functional. affected jills should be culled from the breeding program. • hand-rearing kits from birth is difficult. prognosis is poor for survival. • it may be necessary to provide supplemental feeding for kits if the jill's milk production is reduced, or if the litter size is large. • whenever possible, foster kits with another lactating jill. most jills will accept kits of any size or age. • kits require a milk supplement that contains a high fat content ( %). kitten milk replacers mixed with cream may be used. • feed kits as much as they will eat times per day with a dropper or small pet nurser. • begin to mix solid food with the enriched milk replacer when kits are weeks of age. this mixture may be offered in a shallow dish or bowl. • kits may be weaned onto a solid diet at to weeks of age. feline or ferret growth diets are recommended. • the right kidney lies cranial to the left kidney. the cranial end of the right kidney often lies under the caudate lobe of the liver. • the bladder is small, and can hold up to ml of urine. • male ferrets have a small prostate gland that surrounds the urethra at the base of the bladder. • urinalysis: • the normal urine ph is . for ferrets on a meatbased diet. • normal values for urine-specific gravity have not been reported. • there is evidence that proteinuria may be normal in ferrets ( - mg/dl in males; - mg/dl in females) and that bilirubinuria can occur in the absence of liver disease. renal disease in not common in ferrets, but may occur. • clinical signs are similar to those described in other animals, and include ataxia, bruxism, halitosis, hindlimb weakness, inappetence, melena, mucus membrane ulceration, polyuria/polydipsia, vomiting, and weight loss. • physical examination findings may include cachexia, dehydration, irregularity in the shape and size of the kidneys, pale mucous membranes, and oral ulceration. • diagnosis is based on clinical signs, physical examination, and cbc, serum biochemical analysis, and urinalysis results. m key point hyperphosphatemia, hypocalcemia, and high bun may be noted on serum biochemical analysis. serum creatinine concentration is often normal or only moderately elevated. • treatment should address the underlying cause, if possible. • nonspecific treatment includes fluid therapy, nutritional supportive care, and antibiotic therapy based on culture and sensitivity when indicated. • prognosis is guarded, depending on laboratory findings and response to treatment. unilateral or bilateral renal cysts are relatively common in ferrets (see chapter for a description of this disease in dogs and cats). the condition is usually an incidental finding in middle-aged and older ferrets, although clinical signs associated with this condition can occur at any age. • the cause of renal cysts in the ferret is unknown. • heredity does not appear to be a factor. renal cysts are not associated with hepatic or biliary cysts. • renal cysts typically present as one or more smooth masses on the surface of the kidney. on abdominal palpation affected kidneys feel smoothly enlarged or irregular. • polycystic disease is unusual in the ferret. when present, affected kidneys appear rough and irregular; multiple cysts are often distributed throughout the renal tissue. cysts may be present in other organs as well. • usually there are no clinical signs associated with renal cysts. • rarely, there may be enough disruption of normal renal parenchyma to lead to renal failure, and subsequent clinical signs. • palpate the kidneys for irregular shape. • perform a cbc, serum biochemical profile, and urinalysis. • abdominal radiography usually is not helpful unless the kidneys are very irregular. intraosseous fluid therapy in small exotic animals exotic animal formulary biology and diseases of the ferret. philadelphia: lea & febiger ferret husbandry, medicine and surgery essentials of pet ferrets: a guide for practitioners ferrets, rabbits, and rodents: clinical medicine and surgery ferrets for dummies. indianapolis • perform an abdominal ultrasound to detect renal cysts, to evaluate renal architecture, and to rule out other conditions such as renal neoplasia.• intravenous pyelography or nuclear scintigraphy may be used to evaluate renal function.• renal cysts may be an incidental finding during abdominal surgery. • there is no specific treatment for renal cysts. no treatment is necessary in asymptomatic animals.• monitor affected ferrets by periodic abdominal palpation, serum biochemical profile, urinalysis, and ultrasound, if indicated.• if an affected kidney becomes very large, consider unilateral nephrectomy (if the opposite kidney is functional) (see chapter ).• symptomatic ferrets may be managed using the same supportive care methods used in dogs and cats with chronic renal failure.• the prognosis is grave for ferrets in renal failure. • hydronephrosis is uncommon in ferrets. iatrogenic hydronephrosis may occur as the result of inadvertent ligation of a ureter during ovariohysterectomy (see chapter for information about hydronephrosis in dogs and cats). • bacterial cystitis without urinary calculi is rare in pet ferrets. follow treatment protocols for cystitis in dogs (see chapter ). urinary calculi was a common cause of stranguria in ferrets at one time; improvement in the quality of ferret diets has decreased the incidence of calculi. calculi are usually composed of calcium oxalate or struvite (magnesium ammonium phosphate hexahydrate). cysteine calculi also have been reported. • the cause of urinary calculi is unknown; however, diet is believed to be a factor.• diets containing plant proteins or poor quality meatbased proteins may be associated with the development of urinary calculi. urolithiasis is uncommon in ferrets maintained on a high-quality feline or ferret diet containing high-quality animal-based proteins.• other factors may include urinary tract infection, metabolic, genetic, and congenital factors. clinical signs depend on the location of the urolith(s) and may include dysuria, stranguria, hematuria, persis-tent wetness in the perineal region, and frequent licking of the perineum.• urethral calculi may cause obstruction in both male and female ferrets. • ferrets with urethral obstruction often strain and cry as they attempt to urinate. • if complete obstruction is present ferrets often appear lethargic and anorexic, and may not demonstrate obvious signs of dysuria. • palpate the bladder to identify cystic calculi. the urinary bladder wall may be thickened; in ferrets with urethral obstruction, the bladder is distended and firm.• obtain abdominal radiographs to confirm the presence of radiopaque urinary calculi. cysteine calculi are not radiopaque and require contrast radiography or ultrasonography for diagnosis.m key point small stones located at the base of the os penis can be very hard to identify.• renal calculi may be an incidental finding on whole body radiographs, or may be associated with renal failure. m key point urethral obstruction is an emergency.severe metabolic derangement, coma, and death may occur if urethral obstruction is not diagnosed and treated quickly.• stabilize non-obstructed ferrets by providing supportive care, fluids, analgesics, and antibiotics (if indicated) prior to performing cystotomy to remove the urolith(s).• cystic calculi may be removed surgically via cystotomy; the procedure is similar to that used in cats and dogs (see chapter ). close the bladder wall with - or - absorbable sutures.• submit a urolith sample for analysis and bacterial culture/sensitivity testing.• administer antibiotics for a minimum of to days. use results of follow-up urinalysis, and urine culture/sensitivity testing to determine when to discontinue antibiotic therapy.• begin conversion to a high-quality-animal, proteinbased feline or ferret diet. urinary acidifies are not usually necessary once the ferret is on a high-quality animal, protein-based diet, since this diet alone will cause the urine to be acidic.• feline calculi-dissolving diets and preventative diets may be offered to ferrets; however, many ferrets do not find these diets palatable. • renal calculi can often be managed medically by administering antibiotic therapy and changing the diet. m key point the bladder is very fragile in ferrets.handle ferrets with obstruction gently to avoid bladder rupture.• urinary obstruction in the male ferret can be difficult to manage. catheter placement is challenging due to the small size of the urethra and the j-shaped os penis. (see urinary catheterization in the techniques section of this chapter.)• to facilitate placement of the urinary catheter, empty the bladder via cystocentesis prior to catheterization. submit urine samples for urinalysis and bacterial culture/sensitivity testing.• use either a ferret urinary catheter (slippery samferret urinary catheter, cook veterinary products), a standard tom cat catheter, or a . -fr red rubber catheter for catheterization.• inhalant anesthesia with isoflurane or sevoflurane is strongly recommended to facilitate catheter placement.• if the urinary catheter placement is not successful, consider emergency cystotomy, and attempt to perform anterograde flushing of the urethra via the cystotomy site.• perineal urethrostomy may be considered if cystotomy is unsuccessful (see chapter ). • feed a high-quality, animal protein-based feline or ferret diet. prostatic disease and subsequent urethral obstruction is a potentially life-threatening condition of middle-aged and geriatric male ferrets. this condition typically occurs in association with adrenal gland disease. • prostatic disease and prostatic cyst formation are presumed to be the effect of excessive androgens on the prostate. excessive androgen production occurs with adrenal gland disease.• squamous metaplasia of prostatic glandular epithelium occurs and may subsequently lead to the development of cysts ranging in size from to cm or larger. secondary bacterial infection and abscessation may occur.• prostatic abscesses associated with transitional cell tumor of the bladder, prostatic seminoma, and pro-static carcinoma have also been reported in the ferret, but are rare. • clinical signs associated with prostatic disease may include symptoms associated with a urinary tract infection, urethral obstruction, or urinary incontinence.• signs of adrenal gland disease are often present (see "adrenal gland disease"). • on physical examination, a large, firm, often painful caudal abdominal mass is usually palpable. with careful palpation, this mass is found to be bilobed, representing the urinary bladder and a cystic structure. ferrets with mild to moderate prostatic disease may appear to have a normal-sized prostate on abdominal palpation, yet are still symptomatic. it is important to remember that adrenal gland disease is usually the cause of prostatic disease. perform a complete diagnostic work-up that includes whole-body radiography, cbc, serum biochemistry analysis, and urinalysis. a plasma steroid hormone assay, and abdominal ultrasound may be indicated as well.• obtain abdominal radiographs; prostatic enlargement or prostatic cysts appear as mass lesions dorsal to the bladder.• perform abdominal exploratory surgery for a definitive diagnosis. • address urethral obstruction if present (see "urolithiasis").• manage medically until the ferret is stable for adrenalectomy and surgical drainage of the cysts.• medical management includes maintenance of urinary catheterization for several days, administration of fluids, antibiotic therapy, anti-inflammatory and analgesic therapy, and nutritional support as needed.• consider administration of an androgen receptor blocker (see "adrenal gland disease").• consider administration of leuprolide acetate -day depot formulation (lupron depot, bristol-myers-squibb oncology, princeton, nj) ( ug/kg) im; prostatic tissue shrinkage may occur within hours in some individuals. some ferrets have been maintained successfully on monthly injections of this drug, although results are highly variable.• perform adrenalectomy and drainage of the cysts.large cysts may require debulking. m key point omental pull-through procedures and marsupialization have been described as means of prostatic abscess management in the ferret. these procedures should be used with some caution. prostatic abscesses and prostatic cysts can be difficult to differentiate from paraurethral cysts. paraurethral cysts communicate with the urethra or bladder neck. consider performing contrast radiography to determine if there is communication between the cyst/abscess and the bladder prior to performing these procedures.• administer postoperative antibiotic therapy for a minimum of to days, along with androgen receptor blockers or leuprolide acetate.• base the decision to discontinue antibiotic therapy and androgen receptor blocker/leuprolide acetate therapy by monitoring changes on physical examination, follow-up radiography, and follow-up urinalysis. • the long-term prognosis is good if prostatic changes regress, and if subsequent adrenal gland disease does not occur in the remaining adrenal gland.• some ferrets may need to be maintained on androgen receptor blockers or leuprolide acetate indefinitely. paraurethral cysts are thin-walled single or multiple cysts present on the dorsal aspect of the bladder and proximal urethra. these cysts appear to also be associated with adrenal gland disease and can cause urethral obstruction.it is important to differentiate between prostatic cysts and paraurethral cysts when planning the surgical protocol. • paraurethral cysts have been reported in male and female ferrets.• clinical signs are similar to those described for prostatic disease, and include symptoms associated with a urinary tract infection, urethral obstruction, urinary incontinence.• clinical signs of adrenal gland disease are usually present (see "adrenal gland disease"). • a large, firm caudal abdominal mass is often palpable dorsal to the bladder, just cranial to the pelvic inlet. • perform a complete diagnostic work-up that includes whole-body radiography, cbc, serum biochemical analysis, and urinalysis.• because adrenal gland disease is usually the underlying etiology, consider performing a plasma steroid hormone assay.• radiographically, paraurethral cysts appear as mass lesions dorsal to the bladder.• ultrasonography may be useful in evaluation of the paraurethral cysts and adrenal glands. • surgical drainage and debulking of the cysts is the treatment of choice.• marsupialization is an alternative, but may lead to a formation of a permanent cystotomy.• do not perform an omental pull-through procedure. key: cord- - kqx arn authors: rueda, mario; lipsett, pamela a. title: hepatic failure date: - - journal: principles of adult surgical critical care doi: . / - - - - _ sha: doc_id: cord_uid: kqx arn the progression of liver disease can cause several physiologic derangements that may precipitate hepatic failure and require admission to an intensive care unit. the underlying pathology may be acute, acute-on chronic, or chronic in nature. liver failure may manifest with a variety of clinical signs and symptoms that need prompt attention. the compromised synthetic and metabolic activity of the failing liver affects all organ systems, from neurologic to integumentary. supportive care and specific therapies should be instituted in order to improve outcome and minimize time of recovery. in this chapter we will discuss the definition, clinical manifestations, workup, and management of acute and chronic liver failure and the general principles of treatment of these patients. management of liver failure secondary to certain common etiologies will also be presented. finally, liver transplantation and alternative therapies will also be discussed. accepted theories that revolve around impaired detoxification of substances normally cleared by the liver. • ammonia • the metabolism of nitrogen-containing compounds in the gastrointestinal system results in the production of ammonia. in its normal state, the liver converts this neurotoxic product into glutamine and urea. impaired liver function results in elevated blood ammonia. astrocytes contain the enzyme glutamine synthetase in their endoplasmic reticulum as a means of handling excessive ammonia. accumulation of glutamine within the astrocytes results in cell swelling which leads to a series of events that result in a neuroinhibitory state [ ] . • false neurotransmitters • the failing liver results in the production of false neurotransmitters. these molecules may interfere with normal brain functioning and have a net inhibitory effect [ ] . • amino acid imbalance • patients with hepatic failure have decreased plasma levels of the branched-chain amino acids (bcaa) valine, leucine, and isoleucine while experiencing increased levels of aromatic amino acids (aaa) phenylalanine, tryptophan, and tyrosine. this is thought to be related to increased muscle catabolism and therefore increased bcaa metabolism as well as decreased breakdown of aaa by the compromised liver. the end result is an imbalance that leads to an increased influx of aaa in the brain which has an inhibitory effect in the nervous system [ ] . • gaba receptor • thought to be mediated by inflammatory cells, neurosteroids are produced by myelinated glial cells. this results in positive modulation of gaba receptors that in turn enhance the inhibitory tone [ ] . besides the astrocyte swelling that is seen with the accumulation of glutamine explained above, overall neurologic dysfunction results in loss of autoregulation of intracranial pressure as well as reduced cerebral blood flow. the result of these changes may result in further neurologic derangement and compromise [ ] . besides hepatic encephalopathy, patients with alf can also present with cerebral edema. there is an overlap with the clinical features that are seen with encephalopathy and include nausea, vomiting, headache, and agitation. in advanced cases which can progress to brain herniation, hypertension, bradycardia, changes in pupillary exam or reflexes, as well as respiratory depression can be seen [ ] . patients with alf may present with nonspecific respiratory symptoms including dyspnea on exertion, orthopnea, anxiety, and air hunger. the affecting processes involved are very broad and can range from a simple pleural effusion to acute respiratory distress syndrome (ards) [ ] . the spectrum of respiratory pathology that is seen can be grouped in to two major categories: infectious and noninfectious (table . ) . pulmonary edema can be of cardiogenic or noncardiogenic etiology. the prevalence of pulmonary edema appears to be higher in those patients with cerebral edema, suggesting the accumulation of osmotic substances within the lung parenchyma and outside the vasculature [ ] . molecular imbalance and injury to endothelial cells, accompanied by a decrease in oncotic pressure, may play a role in the development of this disease. hepatopulmonary syndrome can be seen in both alf and chronic liver failure. it is thought to arise from microscopic shunting from arteriovenous dilations that occur in the pulmonary vasculature [ ] . the precise mechanism is unknown; however, it is thought that the elevated levels of nitric oxide seen in patients with liver failure may mediate the abnormal vasodilation that occurs in the pulmonary parenchyma. the result is an overperfusion with maintenance of ventilation; a vq mismatch occurs that ultimately leads to hypoxemia [ ] . as part of the pathophysiology associated with alf, there is low systemic vascular resistance and a hyperdynamic circulation with elevated cardiac output. the pathophysiology is multifactorial but vasoactive substances are thought to mediate the process [ ] . while the underlying pathophysiology may differ, hemodynamic variables appear very similar to those seen in sepsis and septic shock. in the failing liver, there is an increase in splanchnic blood pooling that is associated with the increased resistance of flow through the liver. this results in increased shear stress in the splanchnic circulation that causes upregulation of endothelial nitric oxide synthase (enos) and ultimately nitric oxide (no) production [ , ] . there is further systemic vasodilation causing a low effective circulating volume and relative hypotension despite an overall elevated intravascular volume. the systemic baroreceptors are unloaded and there is a compensatory increase in cardiac output as well as activation of the renin-angiotensinaldosterone system (raas) that may ultimately affect the renal system ( fig. . ) [ ] . patients with alf usually present with varying degrees of coagulopathy. as the liver fails, there is decrease in the synthesis of factors involved in both coagulation and anticoagulation, specifically fibrinogen, prothrombin, protein c, protein s, and factors v, vii, viii, ix, x, and xi. the end result is an increased in prothrombin and activated partial thromboplastin times as well as elevation of inr [ ] . overt bleeding is not typically seen, as there is a decrease in both coagulation and anticoagulation factors. however, mucosal bleeding from the oropharynx or the gastrointestinal mucosa can be frequently seen. this is compounded by the underlying platelet dysfunction that can occur in patients with liver failure. right upper quadrant pain, gastrointestinal bleeding, ascites, nausea, and vomiting can be seen in patients with alf. these symptoms are nonspecific and can be multifactorial. patients with acute viral or autoimmune hepatitis may experience liver parenchyma inflammation as part of the normal response to infection. this leads to an increase in the overall volume of the liver. the liver capsule may be unable to accommodate acute volume changes, and stretching of it results in activation of pain receptors and right upper quadrant pain. discomfort in this area can also be related to direct trauma causing bleeding. abdominal distention may be associated with ascites. the neurohumoral alterations are seen with alf leading to excessive sodium retention and ultimately plasma volume expansion. this, combined with a decrease in the overall circulating proteins due to compromised liver function, leads to overflow of fluid into the peritoneal cavity [ ] . tense ascites can result in compromise of respiratory, renal, and cardiovascular function due to direct compression of the diaphragm and vasculature. as part of its normal physiologic function, the liver is responsible for gluconeogenesis as well as glycogen storage. as liver function worsens, these two key metabolic functions are compromised. in up to % of patients, hypoglycemia is seen and treatment is warranted [ ] . compromised flow through the liver secondary to fibrosis or intrinsic disease acute kidney injury can be present in - % of patients with alf [ , ] . the etiology can be variable: prerenal azotemia, drug toxicity, and acute tubular necrosis have all been implicated. hepatorenal syndrome, especially type , has also been associated with the progression of this disease. acute kidney injury can be divided into oliguric vs. anuric failure, with the latter making fluid management difficult in the critical care setting [ ] . accompanying this derangement we can also see electrolyte disturbances: hyperkalemia, hyperphosphatemia, hypophosphatemia, hypercalcemia, and hypomagnesaemia that can lead to secondary arrhythmias and mental status changes [ ] . lactic acidosis can be seen in patients with alf. the accumulation of tissue lactate is multifactorial. the effective blood pressure is usually lower in those patients with liver failure. this causes a generalized tissue hypoxia that leads to the production of lactate. the compromised liver is unable to uptake and process the lactate, leading to its accumulation [ ] [ ] [ ] . in addition, acute kidney injury can further contribute to the underlying acidosis due to failure of fixed acid clearance [ ] . kupffer cells can be found around the hepatic sinusoids. because of their location, they are constantly exposed to gut bacteria and endotoxins. they play a key role in clearing these pathogens and in maintaining normal homeostasis. in patients with liver failure, their function is impaired, and there is an increased susceptibility to develop gram-positive and gram-negative bacterial infections as well as possible fungal and viral infections [ ] . hepatic encephalopathy has been linked to an increased incidence of infection [ ] . although the mechanism behind this has not been clearly elucidated, it is thought that cns depression alters the immune system modulation. in alf, there is also a change in the production as well as clearance of different cytokines in patients with liver failure and compromised neutrophil function. these problems will lead to decreased bacterial opsonization and clearance. these alterations ultimately contribute to the immunologic impairment [ ] [ ] [ ] . up to three quarters of patients with alf will develop a bacterial infection. the organisms that are most commonly seen include gram-negative-bacteria, streptococcus, staphylococcus, and candida. they may develop a systemic inflammatory response syndrome (sirs) that will be undistinguishable from noninfectious conditions including necrotic hepatocytes from the failing liver [ ] [ ] [ ] . jaundice and pruritus are common complaints of patients with alf. although not specific to liver failure, the presence of both symptoms should raise suspicion of compromised excretion of bilirubin by hepatocyte failure. a normal by-product of the metabolism of heme, bilirubin is usually excreted in bile and urine. the liver is responsible for conjugating glucuronic acid with bilirubin in order to make a soluble compound. as a result, conjugated bilirubin passes into the colon and is eventually eliminated. in the failing liver, there is a severe compromise of the ability to metabolize and excrete bilirubin secondary to the undergoing cell necrosis. there is buildup of unconjugated bilirubin in the blood resulting in eventual deposition of these molecules in mucous membranes, skin, and conjunctiva, what is known as jaundice [ ] . because of the yellow color of the pigment, the physical appearance of the patient changes, directly correlating with bilirubin levels. besides bilirubin, there is also accumulation and deposition of bile acids in the skin. this has been associated with pruritus. other mechanisms that may explain this symptom include the endogenous opioids theory which proposes that the liver failure patient has elevated opioid levels secondary to decrease clearance and metabolism. these molecules activate the mu opioid receptor which may produce pruritus [ ] [ ] [ ] . as explained throughout this chapter, the management strategies for patients with alf are different from those of patients that have chronic liver failure with an acute decompensation. it is imperative to determine what form of failure the patient is experiencing. for those with alf, early recognition and transfer to a transplant center will improve outcomes and mortality. on initial presentation, a patient's mental status will be affected to different degrees; however it may deteriorate further. getting a thorough history during the first encounter is therefore important as it can elucidate the possible cause of the acute failure. the intensivist should review all medications that the patient ingested in the last days. specific questions about ingestion of acetaminophen should be asked. dietary intake should also be explored, playing close attention to any exposure to mushrooms. exact time of ingestion is key in order to determine treatment and further steps in management. social history should also be reviewed in detail. recent travel to viral hepatitis endemic areas as well as contact with other patients that have required hospital visits should be evaluated. focus on alcohol and drug use, sexual behaviors, and vaccination status can help determine the causative mechanism for the liver failure. past medical history plays a key role in determining if the patient has chronic liver disease or if they are experiencing an acute failure. a history of hepatitis, ascites, jaundice, asterixis, and gynecomastia and family history of a metabolic disorder favor chronic liver disease with an acute exacerbation. history of malignancy and lack of screening for colorectal cancer should also make the intensivist suspicious for metastatic malignancy. physical exam may disclose important findings that can elicit cause. an effort to identify the clinical manifestations described previously should be done. laboratory values that should be routinely obtained are listed in table . . when testing for hepatitis b, it is important to evaluate for immunity (hepatitis b surface antibody), infectivity (hepatitis b e antigen), and the presence of an acute infection (hepatitis b core antibody igm). although hepatitis c can cause alf, it is usually associated with chronic liver failure [ ] . bun and co can usually be lower than reference values in patients with alf. this is secondary to poor muscle mass as well as a respiratory alkalosis experienced by these patients. presentation with concomitant renal failure will alter most serum electrolytes. elevation of liver enzymes can be indicative of acute hepatitis and alf. however, values that are within reference range may be markers of poor prognosis as it may be reflective of decreased effective liver mass [ , ] . workup should be started on presentation, even if patient is going to be transferred to a liver center. early identification of the etiology and early treatment can significantly improve outcome. it can also identify those patients that will need liver transplantation in order to treat their disorder. if during the history and physical assessment a cause can be clearly identified, treatment should be started empirically. waiting for laboratory values can be detrimental and result in further deterioration of the patient. consultation with hepatology/gastroenterology, transplant surgery, and the intensivist should be done upon determination of liver failure of any cause. the development of alf has very different etiologies as well as presentations. as such, the management may differ from patient to patient. identification of the causative agent and treatment of it is important. however, supportive care in the intensive care unit is critical for ensuring a positive outcome. patients that have evidence of encephalopathy will require intensive care unit (icu) admission and management while those with no neurologic derangement can be followed on a regular ward with close monitoring. patients should have frequent checks of their coagulation parameters, arterial blood gases, complete blood counts, metabolic panels, serum aminotransferases, alkaline phosphatase, and bilirubin levels. derangements warrant further investigation. hemodynamic monitoring, precise fluid management, and monitoring for infections are all essential. the grade of hepatic encephalopathy guides the management and treatment of the neurologic system in alf. this is because intracranial hypertension (ich) and cerebral edema characterize the severity of patient presentation. those with mild forms (grades i and ii) very rarely develop these devastating complications while - % of patients with grade iii and - % of those with grade iv present with ich [ ] . for those patients with grades i and ii, frequent neurologic assessments should be performed to follow possible neurological progression. maintaining the patient in a quiet environment helps minimize agitation. sedation should be minimized; however, if needed minimal doses of short-acting benzodiazepines should be used [ ] . for patients who present with or develop grade iii and iv neurological symptoms, securing an airway should be the first treatment strategy followed by mechanical ventilation. for sedation, propofol should be used since there is evidence that it decreases cerebral blood flow and allows for frequent ongoing neurological assessment [ ] . intracranial pressure (icp) monitoring devices are used in some icus in patients with alf and grade iii or grade iv encephalopathy [ ] . the main reason for its use is the early identification of ich and subsequent treatment. also, not all patients present with cushing's trial of systemic hypertension, bradycardia, and irregular respirations. several trials have shown that icp monitoring can be performed safely and successfully be used to manage ich [ ] [ ] [ ] . however, no trial has demonstrated a survival benefit. bleeding has been associated with the placement of monitors; however, recent literature reports that there is a decrease prevalence of this particular complication. the incidence of bleeding after placement of icp monitor device has been less than % [ ] . ct scan of the brain should be considered in those patients with an acute mental status change and those with coagulopathy in order to rule out intracranial bleed. this imaging modality does not diagnose cerebral edema or ich in all patients, and therefore, it is not needed in every case of encephalopathy. patients at risk of encephalopathy should also have the head of their bed elevated at ° [ ] , minimize et suctioning, and minimize pain as these factors can lead to ich [ ] . for those patients with elevated ammonia levels (greater than ug/dl) and alf, administration of lactulose can lower the incidence of cerebral edema and decrease mortality [ ] . prior to prescribing this drug, the route of drug administration must be considered as the patient's ability to tolerate po intake may be compromised. other compounds studied include l-ornithine l-aspartate but have failed to demonstrate any survival improvement [ ] . phenytoin has been proposed as a possible prophylactic measure to prevent cerebral edema. an initial study that involved evaluation of brain at autopsy showed that patients who were treated with prophylactic phenytoin had a decrease in cerebral edema [ ] . follow-up trials were unable to replicate these results and more importantly, there was no survival improvement when this agent was used prophylactically [ ] . the administration of intravenous mannitol has been shown to transiently decrease cerebral edema and may be helpful in cases in which ich is < mmhg [ ] . a dose of . - g/kg may be beneficial and it may be repeated if serum osmolality is below mosm/l. the use of hypertonic saline has also been suggested. there is a lower incidence of ich in patients with alf that are treated with hypertonic if it is used to achieve a serum sodium level between and meq/l [ ] . use of hypertonic saline can be limited by renal failure. a newer treatment technique that has been proposed to prevent ich is hypothermia. it is thought to mediate this benefit by preventing hyperemia [ ] . concerns regarding the use of hypothermia in the treatment of alf include worsening coagulopathy and compromise of hepatocyte recovery [ ] . hyperventilation and use of corticosteroids have been proposed as a management option to reduce icp. the former may achieve this goal via vasoconstriction. however, trials suggest that although there is a delay in the onset of cerebral herniation, there is no reduction in the incidence of cerebral edema and no survival benefit [ ] . hyperventilation should only be used after all other resources have failed. while hypoxemia in patients with alf arises from many causes, it is treated with supplemental oxygen. if the patient has grade iii or iv hepatic encephalopathy, a definite airway should be established. during intubation, cis-atracurium is the agent of choice since it does not increase icp [ ] . pleural effusions can be observed and may or may not be contributing to hypoxemia or other respiratory problems. the use of diuretics should be carefully considered as these patients are usually in a very delicate hemodynamic state. overuse of diuretics can precipitate renal failure [ ] . hepatopulmonary syndrome (hps) has been traditionally resistant to medical therapies [ ] . oxygen supplementation for hypoxemia is recommended. transjugular intrahepatic portosystemic shunt (tips) has been reported to improve hps; however, it is not currently recommended as its outcomes are variable [ , ] . liver transplantation is the only therapy that has been shown to improve oxygenation and decrease oxygen requirement [ ] . the diagnosis of hps should prompt immediate referral to a transplant center. decreases in blood pressure lead to compromised renal and brain perfusion. it is imperative to be attentive to blood pressure and heart rate values in order to ensure adequate hemodynamics and, most importantly, adequate perfusion. patients with alf should be resuscitated initially with crystalloid before considering vasoactive agents. the generally accepted goal mean arterial pressure is mmhg [ ] . if after adequate volume resuscitation the patient is still hypotensive and not meeting blood pressure goals, vasopressors should be considered. norepinephrine should be initiated and titrated to effect [ ] . for resistant hypotension consideration to vasopressin should be given, although it should be used with caution as it has been associated with cerebral vasodilation and increased ich [ , ] . terlipressin has also been suggested as adjuvant treatment but it is currently not available in the united states [ ] . other causes of hypotension resistant to vasopressor therapy should also be entertained including adrenal failure and severe acidosis. during liver transplantation, ich and hemodynamics improve immediately after hepatectomy, probably secondary to removal of vasoactive cytokines. hepatectomy can improve these derangements for up to h [ ] . hepatectomy is currently recommended only as a last resort and when a liver graft in the process of being delivered to the transplant institution [ ] . despite the derangements of coagulation laboratories in patients with alf, their coagulation status remains in equilibrium and overall hemostasis. in the absence of bleeding, no correction of laboratory parameters should be performed [ ] . transfusion should be discouraged because treatment with ffp may precipitate pulmonary problems including hypoxia, and transfusion also prevents the use of inr as a marker of hepatocyte recovery [ ] . if an invasive procedure is planned or if there is evidence of significant bleeding, correction of coagulopathy should be done. ffp can be used for this purpose; however, careful volume management should also be achieved. the use of plasmapheresis and recombinant activated factor vii (rfviia) can help in the correction of coagulopathy. rfviia has been proposed as it effectively corrects derangements without volume overload [ ] . however, administration does carry the risk of myocardial infarction and portal vein thrombosis [ ] . alf has also been associated with vitamin k deficiency and it should be administered routinely in these patients [ ] . thrombocytopenia has also been reported in patients with alf. platelets should not be administered in the absence of bleeding. if the patient has platelet counts that are greater than , /mm , no prophylactic transfusion should be given [ ] . if an invasive procedure is planned, platelets between , /mm and , /mm have been proposed, and in those bleeding, the intensivist should consider transfusion if platelets drop below , /mm [ , ] . bleeding from intestinal mucosa is rare but has been reported in patients with alf. histamine- receptor blockers have been used in critically ill patients as prophylaxis of gastrointestinal (gi) bleeding with great success [ ] . also, proton pump inhibitors (ppi) have contributed to the reduced incidence of upper gi bleeding in patients with liver dysfunction [ ] . it is therefore recommended that alf patients are started on prophylaxis while in the icu. nutrition can be compromised in patients with alf; therefore, enteral feedings should be started early unless there are contraindications. there is no evidence that using branched-chain amino acid formulas has benefits over other enteral tube feeds [ ] . protein supplementation should not be restricted but rather limited to g per day in most patients. if gastrointestinal feeding is contraindicated, parenteral nutrition may be considered. there is also evidence that the risk of gi bleeding is reduced in patients that are on enteral feeding [ ] . hypoglycemia should be actively treated in patients with alf. the intensivist should consider adding dextrose to crystalloids in the form of d . if hypoglycemia is severe, central replacement with d concentration should be used. frequent glucose checks should be performed in order to assess the response to glucose administration. improvement and eventually weaning can be achieved in those patients that experience hepatocyte recovery. right upper quadrant pain can be treated with narcotics. judicious doses should be used as metabolism of medications can be compromised with the failing liver [ ] . the management of ascites will be discussed with chronic liver failure. close urine output monitoring is paramount in patients with alf. hemodynamic changes and alterations in the cardiovascular system make the kidneys susceptible to injury. insertion of a urinary catheter should be performed upon determination of hepatic failure. besides serum electrolytes, measurement of urinary sodium and creatinine is necessary. high or normal urine sodium may indicate the presence of acute tubular necrosis, while a low urine sodium may indicate prerenal azotemia or hepatorenal syndrome. several electrolyte derangements may occur and correction should be attempted. accumulation of lactate may result from tissue hypoxia and combined with renal failure may cause life-threatening acidosis. renal replacement therapy may be necessary in these patients. when indicated, continuous dialysis should be used as studies have shown that it provides cardiovascular as well as intracranial pressure stability when compared to intermittent dialysis [ ] . the development of an infection in a patient with alf has been associated with worsening encephalopathy and cerebral edema. also, the presence of bacterial or fungal infections may compromise any attempts at performing a liver transplantation. because of the impact that it has, prophylactic antimicrobials have been proposed as a prevention strategy for these patients [ ] . prophylactic antibiotics have been used and shown to decrease the incidence of infections in patients with alf. in a prospective control trial by rolando n et al., patients with fulminant liver failure were randomized to receive either selective parenteral and enteral antimicrobials vs. no treatment until clinically indicated. patients were included in this study. thirty-four percent of those patients randomized to receive prophylactic antibiotics developed an infection compared to % of those that were treated when clinically indicated (p < . ). however, this did not translate into a survival benefit [ ] . it is currently recommended that if no prophylactic antibiotics are used, periodic sputum, urine, and blood cultures are performed to determine if there are bacterial infections [ ] . the use of antifungals has also been studied [ ] . it is routine practice of the authors to use prophylactic enteric fluconazole in patients that are expected to be in the icu for more than days, given that there is a decrease in fungal infections in high-risk critically ill surgical patients [ ] . it is paramount to perform an infectious workup to any patient with liver failure that develops a change in mental status as it may be a change precipitated by infection. the most common cause of alf in the united states is acetaminophen (paracetamol) toxicity [ ] . over-the-counter availability and the fact that it can be found in combination with other medications make it the cause of voluntary or involuntary overdoses that compromise liver function and may result in fulminant liver failure. acetaminophen is usually taken orally and absorbed via the gastrointestinal system. its half-life is usually - h with one exception being extended release preparations in which it is increased to more than h. total doses should not exceed g per day. ingesting doses less than . g per day is unlikely to result in acute toxicity; however, it can vary depending on underlying liver function [ ] . the metabolism of acetaminophen is performed in the liver. most of the compound, approximately %, is conjugated with sulfate or glucuronide and excreted in the urine. five percent of the remaining medication is excreted unchanged in the urine. the remaining acetaminophen is subject to metabolism by the cytochrome p pathway. it is converted into n-acetyl-p-benzoquinoneimine (napqi), a highly reactive and toxic compound that is immediately conjugated with hepatic glutathione and excreted in the urine. when glutathione levels drop below % physiologic levels, napqi forms covalent bonds via cysteine groups with hepatic molecules and proteins, leading to irreversible hepatocyte damage. a decrease in glutathione levels, enhanced cytochrome p activity secondary to medication use, acetaminophen overdose, or decreased liver function from chronic disease make patients more susceptible to developing toxicity. the clinical presentation of acetaminophen toxicity can be divided into four different stages (table . ). stage i includes a series of nonspecific gi symptoms that start shortly after ingestion. no liver abnormality can be seen. during stage ii, there is usually transaminitis with a high ast/alt ratio. stage iii is characterized by the clinical evidence of liver failure and, in some patients, renal failure. mortality is higher at this stage. those patients that survive this stage progress to stage iv in which there is normalization of most of their lab derangements. because patients may not show symptoms up to h after ingestion, it is very important to obtain a detailed history. standard workup should be initiated as discussed previously. contacting poison control will help coordinate efforts to treat and eventually transfer patient to a liver center [ ] . in order to determine the severity of the poisoning, a serum acetaminophen concentration ( h post ingestion or later) should be plotted against time on the modified rumack-matthew nomogram (fig. . ) [ , ] . patients with acetaminophen levels below the treatment line can be discharged home after psychiatric and social evaluation. all other patients should be admitted to the intensive care unit [ ] . for those patients that ingested a single dose of acetaminophen of more than . g less than h prior to presentation, administration of activated charcoal should be considered. review of several small studies demonstrated that activated charcoal was the best available option to reduce absorption [ ] [ ] [ ] . also, there is a decreased risk of developing liver injury if charcoal is given prior to other forms of treatment [ ] . if patient has an unstable airway, charcoal should not be administered until the airway is controlled. the antidote of choice for acetaminophen toxicity is n-acetylcysteine (nac). the exact mechanism of action is unclear; however, it appears to restore glutathione levels [ , ] . indications for administration include a serum acetaminophen level above the treatment line, ingestion of more than . g, serum acetaminophen level > mcg/ml if time of ingestion is unknown, evidence of liver injury, and a history of acetaminophen ingestion regardless of time of ingestion [ ] [ ] [ ] . oral and iv administration of nac have been studied and both appear effective [ ] . the main factor determining the mode of treatment should be the mental status of the patient. if the patient is confused or has evidence of encephalopathy, oral administration should be avoided. if the oral protocol is used, a loading dose of mg/kg should be given followed by doses of mg/kg given every h. if iv nac is used, a loading dose of mg/kg is given over h. a second dose of mg/kg is then given over h and finally a third dose of mg/kg is given over h. an alternative to nac is hemodialysis. this method effectively removes acetaminophen [ ] . however, because of the effectiveness of nac, it should be reserved for cases in which the antidote is not available. acetaminophen toxicity is best managed in a multidisciplinary setting with assistance from hepatology and surgery teams. ingestion of poisonous mushrooms can lead to lethal emergencies including alf. amanita phalloides, amanita bisporigera, amanita verna, and other mushroom species may cause alf. these mushrooms do not express repulsive smells or tastes, and they can be found throughout midsummer in moist oak forests. alpha-amanitin is the amatoxin responsible for liver failure. after gastrointestinal absorption, enterohepatic circulation is responsible for transportation into the liver, where via active transport it concentrates in hepatocytes. the toxin will bind to rna polymerase and inhibit protein synthesis, ultimately leading to apoptosis [ ] . the clinical presentation of patients that ingest amatoxin includes an initial asymptomatic period of a few hours. this is followed by gastrointestinal symptoms that include abdominal pain, nausea, vomiting, and diarrhea that can be bloody. liver enzymes will be elevated and will continue to increase. one to two days after ingestion, the second phase of the presentation begins with an apparent recovery with continuing elevation of ast and alt. in severe poisonings, coagulopathy and possible dic and renal failure may ensue. the last phase includes alf and typically starts days after ingestion. hypoglycemia and multi-organ failure can be seen. workup of a patient with suspected amanita ingestion should proceed as indicated earlier in this chapter. detection of amatoxin can be performed in urine samples using enzyme-linked immunoassay (elisa); this test is not readily available in all institutions and awaiting results should not preclude supportive treatment [ ] . supportive treatment should be started immediately after presentation. in addition, an effort to minimize toxin absorption should be attempted. activated charcoal can bind amatoxin, and if given in repeated doses, it can reduce mortality significantly by increasing elimination via gastrointestinal tract [ ] . medications that can inhibit uptake of this toxin have also been described. these include penicillin g and silymarin. the former is given as a continuous infusion and has been show to decrease mortality [ , ] . the latter is a more potent inhibitor and is available in iv and po formats. silymarin has been shown to minimize damage to hepatocytes [ , , ] . nac has also been used in the treatment of amatoxin intoxication. mortality appears to improve with implementation of protocols very similar to those of acetaminophen toxicity [ , ] . wilson's disease poses a different presentation from frank alf. it normally occurs in the background of chronic liver disease that has been unrecognized. treatment varies when presentation of this disease is acute, and this will be the focus of this section. a genetically recessive disease, it is estimated that - % of alf cases are related to wilson's disease [ ] . the majority of copper that is ingested is transported into the liver where it is incorporated into enzymes and copper-binding proteins (ceruloplasmin). excess copper is combined with apometallothionein and excreted into bile. in wilson's disease, the incorporation into ceruloplasmin is compromised and copper is accumulated in the liver. as the disease progresses, other organs are affected. besides parkinsonian movements and tremors, kayser-fleischer rings, psychiatric alterations, and renal problems, wilson's disease will present with liver disease: cirrhosis, chronic failure without cirrhosis, and acute liver failure. laboratory workup should include serum ceruloplasmin, which is usually low, as well as serum copper level (above mcg/dl) [ ] . in patients with evidence of alf, low transaminases, low alkaline phosphatase, hypokalemia, glycosuria, hypophosphatemia, and renal tubular acidosis, the diagnosis of wilson's disease should be considered. in patients with acute failure, the aim should be to remove copper. hemodialysis and peritoneal dialysis can successfully achieve this goal [ ] . albumin dialysis and the molecular absorbent recirculating system (mars) device have also been used with promising results [ , ] . penicillamine, zinc, and other medications used for treatment of wilson's disease do not play a role in alf. the development of alf from viral hepatitis may occur after acute infection; ostapowicz et al. estimated that the etiology of % of those patients that were diagnosed with alf was viral hepatitis [ ] . most of the clinical deteriorations that are seen in patients with this etiology of disease are related to chronic liver infection. alf is more common with hepatitis b but it can also present in patients with hepatitis a, c, and e [ ] . presentation of viral hepatitis is described in four phases. phase is characterized by lack of symptoms but changes in laboratory studies that may be suggestive of viral hepatitis. phase marks the development of symptoms that include nausea, vomiting, abdominal pain, arthralgias, and possible fevers. the next phase includes clinical characteristics of alf including right upper quadrant pain, becoming icteric, and possible coagulopathy. the last phase, , leads to the normalization of laboratory values and resolution of symptoms. diagnosis of viral hepatitis relies on serum laboratories. hepatitis b has several important antigens and antibodies. hepatitis b surface antigen (hbsag) is usually found in patients with acute infection. a second antigen, associated with infectivity, is hepatitis b e antigen. the first antibody that can be detected in patients acutely infected and that indicates acute presentation of disease is igm anti-hbcag. resolution of acute infection and recovery results in igg antibodies against this antigen. finally, anti-hbsag appears in the serum several months after infection, indicating resolution. they will also be found in patients with hepatitis b vaccine. igg anti-hepatitis c virus has been used to diagnose exposure to this viral infection. it can usually be found in the serum several months after an acute infection and contrary to anti-hbsag, it does not confer immunity to hepatitis c. use of elisa and riba testing for diagnosis has fallen out of favor. hcv rna pcr assays were developed in order to detect the presence of the virus. it has been successful in not only establishing the diagnosis but also the presence of an acute infection. treatment of acute hepatitis a is limited to supportive care as there are no medications that improve outcome. hepatitis b treatment usually follows the same principles as most antiviral therapy is directed toward treatment of chronic disease. however, recent studies have suggested that acute hepatitis b may benefit from administration of lamivudine [ ] . finally, acute hepatitis c has been treated with ifn therapy with resolution of hcv rna after several months of treatment [ ] . low perfusion pressure to the liver may result in clinical manifestations of alf known as ischemic or hypoxic hepatitis. it is an uncommon cause of liver failure, with a prevalence of per , hospital admissions [ ] . this can be a direct consequence of global hypoperfusion, hemodynamic instability, direct vascular occlusion during surgical procedures, hepatic artery disease (occlusion, dissection, thrombosis) in patients with portal vein thrombosis, or hepatic sickle cell crisis [ ] . hepatocytes in zone become ischemic and eventually necrotic leading to liver insufficiency. prognosis of ischemic hepatitis is poor. raurich et al. described an in-hospital mortality of . % in all patients that were diagnosed with this disease process. in those patients with concomitant septic shock and those that experienced cardiac arrest, mortality rates were higher, at . % and . %, respectively. risk factors for mortality included an elevated inr, need for renal replacement therapy, and diagnosis of septic shock. non-survivors were more likely to be on vasopressors and to require mechanical ventilation [ ] . patients with hepatitis secondary to shock present with several symptoms related to their hemodynamic instability including altered mental status, respiratory distress, severe hypotension, and renal failure. patients with a history of cardiac compromise may present with nausea, vomiting, right upper quadrant pain, and malaise. up to % of patients with septic shock will also have ischemic hepatitis, presenting with fevers and severe hypotension [ ] . laboratory examination reveals elevated aminotransferase levels, usually above , iu/l. the ratio of serum alanine aminotransferase to ldh less than . suggests ischemic hepatitis [ ] . if hypoperfusion is chronic in nature, synthetic function may be preserved and coagulation studies may be normal; however, in acute cases, there is severe derangements that continue to progress with time. if ischemic hepatitis is suspected, a right upper quadrant ultrasound with doppler should be immediately performed as it may reveal the etiology of the insufficiency. there is no specific treatment for ischemic hepatitis. management is centered around restoring cardiac output and reestablishing hepatic perfusion. appropriate resuscitation is necessary. excessive fluid administration may lead to vascular congestion which can in turn compromise perfusion of hepatocytes and aggravate the presentation. judicious use of diuretics should be exercised as diuresis may exacerbate hypoperfusion and therefore liver failure. intensivists should rule out ischemic hepatitis in any patient that presents with septic shock and has elevated aminotransferases [ ] . prompt recognition of hypoperfusion state may lead to early intervention and possible better outcomes. continuous hepatic injury that persists for more than months is considered chronic liver disease (cld). the liver parenchyma suffers continuous inflammation and potential destruction. the hepatic insult does not only result in damage but also in attempts of repair. ultimately this leads to a broad spectrum of clinical manifestations including fibrosis, cirrhosis, and hepatocellular carcinoma. these changes are accompanied by alterations in serum liver function tests and can include physical exam finding suggestive of physiologic alterations. in the united states, the most common causes of cirrhosis leading to liver transplantation are alcoholic liver disease, chronic viral hepatitis, and nonalcoholic liver disease (table . ) [ ] . this last etiology has increased significantly in incidence. most patients are generally asymptomatic until decompensation occurs, making the calculation of prevalence difficult. approximately , deaths in where associated with cld [ ] . patients with cld may present with compensated or uncompensated hepatic failure. the former may be asymptomatic prior to evaluation, but patients usually report nonspecific symptoms such as weight change, fatigue, and lack of appetite. those patients with an acute decompensation may show signs of active bleeding, confusion, and skin changes. because of the broad spectrum of the disease, presentation will vary between different patients. due to similar underlying pathophysiology, symptoms and findings may be similar to those described previously during the acute liver failure presentation. patients with cld may present with varying degrees of hepatic encephalopathy. classification and underlying pathophysiology are similar to those described previously in the alf section. an acute exacerbation with an underlying chronic liver dysfunction can cause rapid progression from confusion to coma. shortness of breath, dyspnea, and other nonspecific respiratory symptoms may also be reported. as with acute dysfunction, the etiology may be of infectious, metabolic, or of cardiac etiology. hepatopulmonary syndrome can also play a role in underlying hypoxemia [ ] . the mechanisms that lead to the respiratory derangements in cld are similar to those described in acute liver compromise. figure . explains the molecular mechanism behind the underlying decreased effective perfusion pressure seen in patients with liver failure. as a result, patients will have a lower than baseline blood pressure, with some of them transitioning from hypertensive to normotensive. the cardiac output in patients with liver disease is usually high; however it is important to understand that myocardial cells are actually depressed from exposure to the changes in cytokines and other molecules. there is a slightly elevated heart rate that compensates for the depression and overall results in increase cardiac output, in a normal-sized man, often in the range of - l/min [ ] . patients with cld may present with anemia, leukopenia, thrombocytopenia, and coagulopathy [ ] . the pathophysiology behind anemia is multifactorial, and it may include episodes of gastrointestinal bleeding associated with portal hypertension and coagulopathy. there may also be nutritional deficiencies such as folate deficiency that can lead to compromised production of red cells and vitamin k deficiency that can lead to decreased production of coagulation factors [ ] . aplastic anemia, hypersplenism, and hemolysis may contribute to the anemia experienced by patients with chronic failure [ ] . thrombocytopenia is associated with portal hypertension: an enlarged spleen can sequester the majority of the circulating platelet mass and lead to a decrease platelet count. it has also been described that patients with liver disease have decreased levels of thrombopoietin that will also lead to thrombocytopenia [ ] . patients experiencing cld can present with abdominal distention and pain, anorexia, nausea, and vomiting. physical exam may also show ascites, hypogonadism, hypersplenism, and evidence of gastrointestinal (gi) bleeding such as hematemesis, hematochezia, and melena. gi bleeding can be the result of mucosal injury and thrombocytopenia or a more severe and life-threatening event such as variceal hemorrhage. an umbilical hernia may be seen when ascites becomes prominent. for those patients with cld, there are significant changes in the hemodynamics of the portal vein. the hepatic microcirculation, sinusoids, undergoes constriction secondary to architectural changes that compromise the lumen of these systems. furthermore, there is active contraction of myofibroblasts and active smooth muscle secondary to cytokine changes (increased levels of intrahepatic et- ) that cause even more restriction in the radius of these sinusoids [ , ] . these changes lead to an increase in portal pressure. a second factor that impacts the pressure of the portal vein is the increased in blood flow in the portal vein. as shown in fig. . , there is a splanchnic arteriolar vasodilation that leads to increase venous outflow and, therefore, increased flow that results in further increases of portal pressure and eventually portal hypertension (pht) [ ] . the elevated blood pressure and flow are partially relieved by decompressing the inflow into the portal vein into systemic collaterals. the esophageal submucosal veins are a preferred method of decompression and may result in esophageal varices. as flow increases so does the vessel radius [ ] . this ultimately leads to an increase in wall tension that may end up in rupture and variceal bleeding [ , ] . ascites is also closely related to pht. in fact, patients without evidence of pht do not develop ascites even in the presence of cirrhosis. the threshold for formation of ascites appears to be mmhg at the level of the portal vein [ ] . as a response to this increase in pressure, there is splanchnic vasodilation leading to a decrease in effective arterial blood volume that is mediated by several molecules including nitric oxide (no). there is subsequently an activation of the renin-angiotensin-aldosterone system that increases renal sodium retention and plasma expansion that ultimately leads to accumulation of fluid in the peritoneal cavity [ ] . the low levels of circulating protein secondary to liver compromise may also favor the formation of ascites. on physical exam, we can find evidence of pht by placing a stethoscope over the epigastrium. if there are collateral connections between the portal system and the umbilical vein, a murmur can be auscultated. this finding is known as cruveilhier-baumgarten murmur. dizziness, diaphoresis, and overall malaise may be reflective of underlying hypoglycemia. patients with cld undergoing an acute exacerbation may see decreased levels of circulating glucose with corresponding changes in neurologic exam. male and female patients with cld can report abnormalities related to infertility, impotence, and in the case of women chronic anovulation. physical exam may show evidence of testicular atrophy in men, while ultrasound and other imaging may show atrophic ovaries and uterus. there are several possible mechanisms that explain these findings. the increased levels of follicle-stimulating hormone (fsh) and luteinizing hormone (lh) observed in some patients suggest the primary dysfunction of the testicles or ovaries. an alternative mechanism suggests suppression of the hypothalamicpituitary function. the dysfunction may be secondary to decreased clearance of estrogen, testosterone, prolactin, and other substances [ , ] . male patients with cld may complain of loss of male pattern pubic hair, chest and axillary hair loss, and gynecomastia. this finding is thought to be related to an overall increase in estradiol: the adrenal glands produce and increase quantities of androstenedione that undergoes aromatization into estrone and eventually to estradiol [ ] . similar to patients with alf, patients with cld can present with renal pathology. these may manifest as decreased urine output, arrhythmias, generalized body edema, and overall malaise. most of the changes are associated with the underlying liver dysfunction. in hospitalized patients with cld, it is estimated that approximately % of them will develop hepatorenal syndrome (hrs). the pathophysiology of hrs follows the development of pht. as explained in fig. . , there is dilation of the splanchnic circulation, leading to a decrease in perfusion pressure. the response is cardiac compensation as well as activation of the renin-angiotensin-aldosterone system. there is also vasoconstriction mediated by the sympathetic nervous system. these changes ultimately lead to low renal perfusion and a significant decrease of the glomerular filtration rate [ ] . electrolyte abnormalities can accompany the changes that are seen on the renal system. hyperkalemia, hyperphosphatemia, and hyponatremia can be detected in serum electrolytes. symptoms may be variable and depend not only on severity of derangement but acuity. dizziness, weakness, and palpitations may be reflections of these abnormalities. cld leads to acquired immune deficiency and makes these patients prone to developing infections. the mechanism by which the immune response is compromised includes the deficiency of serum complement [ ] as well as the compromised activity and function of phagocytes such as macrophages, pmns, and kupffer cells [ , ] . certainly, the presence of fevers should make the intensivist suspicious for an infectious process and further investigation is warranted in order to determine additional symptoms that may guide further treatment. however, patients who present with decompensated liver failure may have an infection causing the decompensation. thus, suspicion for the presence of infection should be high, and the threshold for obtaining cultures is low in any patient with liver failure who is acutely ill. abdominal pain that worsens and fevers should raise the suspicion for spontaneous bacterial peritonitis (sbp) in those patients with evidence of ascites. up to % of these patients may develop sbp [ ] . patients with cirrhosis have an increased intestinal permeability as well as altered intestinal motility. this may lead to the bacterial overgrowth and infection of ascites [ ] . the most common organism seen is escherichia coli; however, other organisms have also been described [ ] . typically sbp is monomicrobial and a polymicrobial infection should prompt consideration of a perforated viscous. similar to alf, skin and urine color can change in patients with cld. the increase in bilirubin secondary to compromised liver function leads to the accumulation in the skin leading to jaundice as well as dark appearance of urine. these changes are usually undetectable if the serum bilirubin is less than mg/dl. another change that can be appreciated in the skin of patients with cld includes palmar erythema. it is thought to be the consequence of altered sex hormone metabolism which may lead to capillary vasodilation [ ] . careful examination of the skin can also reveal vascular lesions characterized by the presence of a central arteriole with surrounding smaller vessels. these are called spider angiomata and their appearance is related to an increase in estradiol levels. the number as well as size of these lesions is related to the severity of liver disease although they are not specific for it [ ] . as an additional route to decompress the portal vein during pht, the umbilical vein may open leading to shunting into abdominal wall veins. these vessels engorge significantly making them very easy to identify during physical exam. this finding is known as caput medusa. initial workup and management of patients with cld should begin with a thorough history. onset of symptoms and identification of disease progression helps determine the pathophysiologic manifestations of the disease. previous medical diagnosis including viral hepatitis should be assessed. a thorough review of all medications that the patient takes can help identify potential additional mechanisms of liver injury. hospitalizations and transfusions should be reviewed. social history including exposure to high-risk behaviors such as intravenous drug use and alcohol abuse should be performed. family history of liver disease and personal history of malignancy (including oncologic treatment and surveillance studies) also play a key role in the development of disease and should be explored. a complete physical exam should be performed and an attempt to determine if any of the clinical manifestation discussed previously are present. the exam should include neurologic, rectal, and skin exam. assessment of vital signs in order to identify possible hypotension, hypoxemia, as well as end-organ perfusion should be performed. there is no serologic test that can diagnose cld accurately. laboratory abnormalities that are identified could be related to alf or another etiology with some degree of liver dysfunction. besides serologic tests, evaluation of the degree of liver fibrosis and additional characteristics of cld can be investigated with radiologic studies. the initial serologic studies that are performed as well as initial management are similar to those described in table . in the alf section. in addition, studies from ascitic fluid should also be performed when it is desired to identify etiology of fluid and possibility of infection. after paracentesis with removal of ml of ascites in a sterile fashion, the intensivist should send the fluid for cell count, cytology, albumin, total protein, triglycerides, amylase, adenosine deaminase, as well as culture [ ] . this should be accompanied by a serum albumin in order to calculate the serum-ascites albumin gradient (saag). this is done by subtracting the albumin in the ascitic fluid from the serum value. based on such studies, the etiology of ascites can be determined (table . ) . imaging studies that are routinely used include ultrasonography (us), ct scan, and magnetic resonance imaging (mri). us can help identify morphologic changes such as nodularity. with doppler us, patterns of flow as well as possible occlusions can be identified. ct and mri are able to identify nodularity and changes in volume of liver mass (hypertrophy or atrophy) as well as assess the portal vasculature [ ] . evaluation of collateral circulation, varices, and tumors can also be performed. since us does not use contrast, this can be very helpful in those patients with renal compromise [ , ] . if after a thorough workup, the diagnosis of cld cannot safely be established, liver biopsy should be considered. identifying changes consistent with cld may be very beneficial as it may prevent delays in therapy and potential worsening of the patient [ ] [ ] [ ] . surgery and interventional radiology teams should be involved in order to determine the safest and least invasive method that can render a diagnosis. suspicious findings for cld should prompt consultation with hepatology/gastroenterology and transplant surgery in order to determine if the patient will benefit from additional therapies and workup including possible transplantation. evidence of encephalopathy, compromised ventilation, hypotension, hypoperfusion, active bleeding, sepsis, and sbp should prompt admission to the icu. consideration of additional hemodynamic monitors such as an arterial line and central access may be considered in every patient. a foley catheter should be placed in all patients with hemodynamic instability or with poor renal function but avoided in those with anuria to prevent a urinary tract infection. it is also helpful to classify the severity of liver disease. the child-turcotte pugh (ctp) classification divides patients into three groups based on serum labs and clinical presentation. it can help in determining possible surgical treatments or additional therapies [ , ] . this specific scoring system is presented in table . . another classification system that is used for the allocation of organs in the unites states is the model for end-stage liver disease (meld). it consists of a formula that will assign a score to a patient and that accurately predicts mortality within months. the formula is based on three laboratory values (bilirubin, inr, and creatinine) and it is modified by etiology. the formula is shown below [ ] : if the disease process is alcohol, is assigned to etiology. if the liver failure is secondary to a cholestatic process, is assigned instead. several factors can modify the calculated meld score for allocation purposes, and these include dialysis and the presence of hepatocellular carcinoma. the ctp and meld system have been compared in several studies in order to determine which provides a better answer to prognosis for patients. although some studies show superiorities of meld, others show no difference and good predictions with both systems [ ] [ ] [ ] [ ] . a systematic review, suggested that the meld was better for predicting -month mortality but otherwise the systems were similar [ ] . because of its use with united network for organ sharing (unos) lists for allocation of organs, meld has become more popular. hepatic encephalopathy (he) is a diagnosis of exclusion, and therefore, an effort to identify other etiologies of altered mental status should be performed. it is also necessary to determine the precipitating event leading to the neurologic derangement which includes bleeding, renal failure, electrolyte abnormalities, changes in diet, and changes in medication [ ] . treatment principles are similar to those described in the alf section. they should be based on supportive care, attempts to correct precipitating factors, minimizing gi nitrogen intake, and establishment of therapy. admission to an icu is important as patients with he need constant neurologic assessments for progression or resolution. for grade iii and grade iv he, establishment of definite airway should be the first step in management. laboratory studies are key in order to identify possible precipitating events. a decrease in nitrogen production as well as nitrogen delivery should be attempted with medication. the most common therapy used is lactulose, which reduces the absorption of ammonia. twenty-five milliliter should be given twice a day and should be titrated to achieve two soft bowel movements [ ] . rifaximin has also been used as an add-on therapy to lactulose. it is an antibiotic with activity against grampositive and gram-negative aerobes and anaerobes. the usual dose is mg three times a day. trials have shown benefit in the treatment of he when rifaximin is used in addition to lactulose [ ] . another antibiotic that has been use is neomycin. this alternative treatment has been used for the treatment of overt hepatic encephalopathy [ ] . however, because it has been associated with complications such as ototoxicity and nephrotoxicity, neomycin is used less commonly today [ ] . an assessment of nitrogen intake by assessing a patient's diet is also very important. if a patient's he is unresponsive to the therapies described above, oral branched-chain amino acids (bcaa) should be considered in an attempt to reduce the hepatically metabolized nitrogen load. a recent metaanalysis showed that bcaa-enriched formulations may be beneficial in some patients with he and cld [ ] . the daily protein intake should be . - . g/kg/day as severe restriction may be detrimental in the catabolic state of cld [ ] . the first step in management of a patient with cld and ascites should be sodium restriction to no more than , mg per day [ ] . this should also be accompanied by oral spironolactone and possibly furosemide in order to perform natriuresis while maintaining normokalemia. spironolactone inhibits sodium reabsorption in the distal tubule and collecting ducts but it can lead to gynecomastia and hyperkalemia. furosemide is a loop diuretic and inhibits the luminal na-k- cl symporter causing natriuresis and also hypokalemia when used alone. combination therapy has been used more effectively in achieving sustained results. if the serum sodium is less than mmol/l, fluid restriction to no more than . l per day should also be done [ ] . for those patients that are not responsive to diuretic therapy, serial paracenteses can be performed in order to relieve symptoms [ ] . in carefully selected patients, transjugular intrahepatic portosystemic shunt (tips) should be considered. trials have demonstrated that there is better control of ascites and overall survival with this procedure; however, there is worsening hepatic encephalopathy [ ] . referral to a transplant center should be done for patients with refractory ascites. tense ascites with respiratory compromise and abdominal discomfort can also be the initial presentation of patients with cld. prior to sodium restriction, paracentesis should be performed. for large volume (> l) removal, albumin replacement should be done [ ] . replacement of - g of albumin per l of fluid removed has been shown to improve survival [ ] . replacement after paracentesis has remained a controversial topic. in one study performed by gines et al., patients with tense ascites were randomized to receive albumin or no replacement. those that did not receive albumin had more changes in serum electrolytes, plasma renin, and creatinine but had no survival advantage [ ] . there has been no study up to date demonstrating decreased survival in patients without replacement when compared to albumin [ ] . in a meta-analysis by bernardi et al., , patients from trials were analyzed. albumin was shown to be superior to other plasma expanders, with an infusion between and g of albumin per liter removed [ ] . angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aspirin, and nonsteroidal anti-inflammatory agents should be avoided in patients with cld and ascites: prostaglandin inhibition can severely affect renal hemodynamics as well as natriuresis. it is important to evaluate patients with ascites for ventral and umbilical hernias. for those patients with ascites, hernia repair should only be attempted after medical treatment of ascites. for those with refractory ascites, repair should be deferred until after liver transplantation. if the patient has an incarcerated or strangulated hernia, emergency repair is warranted, but special attention to the ascites postoperatively must be made. the diagnosis of spontaneous bacterial peritonitis (sbp) is established with studies sent from ascitic fluid revealing one of the following three findings: . leukocyte count of more than per mm . polymorphonuclear count of more than per mm the causative organism is usually a gram-negative enteric bacteria; if more than one organism is identified, secondary peritonitis should be considered. escherichia coli and klebsiella are responsible for more than % of the cases [ ] . therapy is tailored based on the most likely causative agent. if the patient has not been on empiric antibiotics prior to presentation, an intravenous third-generation cephalosporin should be started, preferably cefotaxime g every h. if the patient has been exposed prior to this medication, coverage should be based on hospital antibiogram [ ] . therapy should be started if there is a high suspicion for infection while cultures are pending. the recurrence rate of sbp can be as high as % and therefore prophylaxis is advocated. long-term antibiotic therapy, norfloxacin mg daily, is recommended [ ] . trimethoprim/sulfamethoxazole can be used as a secondline agent for those patients with sensitivities [ ] . the presence of esophageal varices in patients with cld warrants prophylactic therapy. the most effective medication has been propranolol that inhibits stimulation of the beta- venodilator receptors seen in varices. it should be started at low doses, mg orally twice a day, and titrated to reduction of pulse rate by %. if patients cannot take propranolol, isosorbide mononitrate can be used. if the patient is unable to tolerate medical therapy, esophagogastroduodenoscopy (egd) and variceal banding should be performed [ ] . three principles govern the management of an acute variceal bleed: stabilization and resuscitation, identification and treatment of bleeding, and prevention of recurrence. if a patient presents with evidence of gi bleeding, immediate type and cross should be performed, and if needed, transfusion of untyped and uncrossed blood should begin. waiting for laboratory values to show anemia may worsen the overall clinical condition of the patient. upper gi bleeding in a patient with presumed cld prompts urgent endoscopy to identify possible bleeding esophageal or gastric varices. if during endoscopy, no varices are seen, repeat evaluation should be done in years. if varices are identified but not bleeding, follow-up endoscopy should be done after year. if active bleeding is encountered and it appears to involve esophageal varices, an attempt at controlling the bleeding varices should be done. banding followed by sclerotherapy are the two most common methods of achieving control. if after appropriate attempts bleeding does not stop, a sengstaken-blakemore tube should be inserted. tips and surgical shunts should be considered if all previous methods fail. tips has shown improved outcomes [ ] ; however, it is associated with he [ ] . surgical shunts carry a high morbidity and should be considered a last resort. cld patients with gi bleeding are at risk of developing bacterial infections. some advocate the use of ceftriaxone for days while patients are gi bleeding [ , ] . if the patient stabilizes and tolerates oral intake, changing to norfloxacin is reasonable. the diagnostic criteria for hepatorenal syndrome (hrs) are shown in table . . hrs is a diagnosis of exclusion and it is important to rule out other etiologies including prerenal azotemia, intrinsic renal disease, and post renal failure. in order to diagnose hrs, all major criteria in table . must be met. minor criteria are not required; however, they provide supportive evidence that the pathophysiology is consistent with hrs. identification of precipitating event is also instrumental in the management of hrs as additional therapy can be instituted. when performing large volume (> l) paracentesis, it is recommended to replace volume with albumin (see ascites section above) as this procedure may lead to hrs. evaluation for possible sbp as well as workup for gi bleeding should be considered as they are well-established risk factors for the development of this syndrome. there are two manifestations of hrs: type i and type ii. the former shows a rapid decline in renal function with either an initial creatinine of greater than . mg/dl or a % reduction in the creatinine clearance. type ii usually leads to moderate renal failure that progresses slowly and is manifested as diuretic-resistant ascites [ ] . liver transplantation is the preferred treatment for patients with hrs. any patient with evidence of this syndrome should be referred to a liver transplantation center in order to be listed for transplantation [ ] . bridging with pharmacotherapy is necessary in most patients as there is rapid decompensation, especially in those with type i hrs. the basic principle behind the management of hrs is reversal of renal vasoconstriction and splanchnic vasodilation. dopamine, fenoldopam, and prostaglandins have been used in an attempt to cause direct renal vasodilation [ ] . results of several trials have not favored any of these agents as none have improved outcome [ ] [ ] [ ] . splanchnic vasoconstriction, in an attempt to reduce portal blood flow and decrease pressure, has been attempted with vasopressin, ornipressin, terlipressin, norepinephrine, and midodrine [ ] . ornipressin, with some promising results, resulted in an increase rate of ischemic events [ ] . terlipressin in combination with albumin has shown the most promising results, with improvements in renal function although its use has not been approved in the united states [ ] . norepinephrine and vasopressin have been used with improvement of renal function and successful bridging to transplantation [ ] . hemodialysis may be required in the treatment of these patients, especially those with type disease. those patients that are hospitalized in an icu should receive continuous dialysis rather than intermittent as it minimizes changes of abrupt hemodynamic changes and further compromise of these frail patients [ ] . patients with alf and cld may benefit from liver transplantation. this therapeutic option should be considered when medical therapy has failed and when there is progression of disease. referral to transplant center should occur once the patient has experienced ascites, variceal hemorrhage, hrs, and he. consultation with hepatology and transplant surgery teams ensures early consideration for transplantation. table . presents poor prognostic factors from the king's college criteria that may suggest that the need for transplantation is increased. prior to transplantation, a thorough evaluation is performed on patients regardless of etiology. this includes assessment of cardiac function, possible occult malignancy, identification of infection, contraindications to chronic steroid therapy, and appropriate social support. the rapidly progressive nature of alf designates that these patients are currently listed as status by the united network for organ sharing (unos) [ ] . approximately table . criteria for diagnosis of hepatorenal syndrome chronic or acute liver disease with advanced hepatic failure and portal hypertension low glomerular filtration rate serum creatinine > . mg/dl or h creatinine clearance < ml/min absence of shock, ongoing bacterial infection, and current or recent treatment with nephrotoxic drugs absence of gi fluid losses absence of renal fluid losses in response to diuretic therapy no sustained improvement in renal function after diuretic withdrawal and expansion of plasma volume with . l of plasma expander proteinuria < mg/day no obstructive uropathy, parenchymal renal disease, microhematuria minor criteria urine volume < ml/day urine sodium < meq/l urine osmolality greater than plasma osmolality urine rbcs < /high-power field serum sodium concentration < meq/l non-acetaminophen inr greater than . or three of the following five criteria: patient age of less than or greater than serum bilirubin of greater than μmol per liter time from onset of jaundice to the development of coma of greater than days inr greater than . drug toxicity, regardless of etiology of alf acetaminophen arterial ph < . inr greater than . creatinine greater than μmol per liter encephalopathy (grade iii or iv) % of patients with alf will undergo liver transplantation, % of them will improve with supportive care, and % will not survive their presentation; of those that have a liver transplant performed, the -year survival is approximately % [ ] . patients with failure secondary to viral hepatitis usually have better outcomes than those with drug reactions or metabolic causes. also, patients with alf have worst outcomes when compared with patients with cld. the -year survival for patients with cld that undergo liver transplantation is % [ ] . timing is not standard and is usually dependent on severity of meld. living donors have been used secondary to decrease in organ availability and it has been successful. this therapy has not been studied in patients with alf. liver replacement therapies (lrt), also known as liver dialysis, have been studied and used as a bridging therapy to transplant [ ] [ ] [ ] [ ] . several methods have been developed and they can be grouped into artificial and bioartificial devices. regardless of the mode of action, they attempt to clear toxins that are free and protein bound, as well as to regenerate or replace proteins that are affected by the liver failure process. among the artificial methods, the most studied is the molecular adsorbent recirculation system (mars). it effectively clears several toxic compounds and causes a dramatic improvement in serum laboratories and in some symptoms such as pruritus [ ] . unfortunately, this has not translated into clinical benefits [ ] . biologic methods include devices with porcine hepatocytes and with human hepatoblastoma cells [ , [ ] [ ] [ ] . their theoretical advantage is the production of proteins and compounds produced by a normal liver as well as detoxification functions. as opposed to artificial systems, technology is not readily available. the results from different trials have been promising, showing improvement in survival to transplantation and normalization of serum laboratories [ ] . an alternative to liver transplantation is hepatocyte transplantation. this consists of injecting human hepatocytes into the portal vein with an attempt to restore hepatic function [ ] . it has been principally used to correct errors of metabolism, and trials have shown improvement in encephalopathy and ammonia and serum laboratories in patients with alf that undergo this therapy [ ] . more trials are needed in order to establish the role of this treatment option. epidemiology of acute liver failure east meets west: acute liver failure in the global village the electroencephalograph in liver disease low myo-inositol and high glutamine levels in brain are associated with neuropsychological deterioration after induced hyperammonemia interorgan ammonia, glutamate, and glutamine trafficking in pigs with acute liver failure branched-chain amino acids in liver diseases an interaction between benzodiazepines and neuroactive steroids at gaba a receptors in cultured hippocampal neurons liver function, cerebral blood flow autoregulation, and hepatic encephalopathy 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liver failure moderate hypothermia in patients with acute liver failure and uncontrolled intracranial hypertension a double-blinded, randomized trial of hydrocortisone in acute hepatic failure. the acute hepatic failure study group atracurium infusions in patients with fulminant hepatic failure awaiting liver transplantation effect of transjugular intrahepatic portosystemic shunt on pulmonary gas exchange in patients with portal hypertension and hepatopulmonary syndrome transjugular intrahepatic portosystemic shunt: a successful treatment for hepatopulmonary syndrome improved survival after liver transplantation in patients with hepatopulmonary syndrome acute liver failure in adults: an evidence-based management protocol for clinicians management of acute liver failure comparison of terlipressin and noradrenalin on cerebral perfusion, intracranial pressure and cerebral extracellular concentrations of lactate and pyruvate in patients with acute liver failure in need of inotropic support worsening of cerebral hyperemia by the administration of terlipressin in acute liver failure with severe encephalopathy two-stage total hepatectomy and liver transplantation for acute deterioration of chronic liver disease: a new bridge to transplantation minimal effects of acute liver injury/acute liver failure on hemostasis as assessed by thromboelastography recombinant activated factor vii for coagulopathy in fulminant hepatic failure compared with conventional therapy fviia corrects the coagulopathy of fulminant hepatic failure but may be associated with thrombosis: a report of four cases pharmacokinetics and efficacy of oral versus intravenous mixed-micellar phylloquinone (vitamin k ) in severe acute liver disease the expert panel on preventive services: continuing the work of the uspstf coagulopathy of acute liver failure oral ranitidine as prophylaxis for gastric stress ulcers in intensive care unit patients: serum concentrations and cost comparisons proton pump inhibitors versus histamine receptor antagonists for stress ulcer prophylaxis in critically ill patients: a systematic review and meta-analysis parenteral nutrition with branched-chain amino acids in hepatic encephalopathy. a meta-analysis the value of early enteral nutrition in the prophylaxis of stress ulceration in the severely burned patient improved cardiovascular stability during continuous modes of renal replacement therapy in critically ill patients with acute hepatic and renal failure prospective study of bacterial infection in acute liver failure: an analysis of fifty patients prospective controlled trial of selective parenteral and enteral antimicrobial regimen in fulminant liver failure fungal infection: a common, unrecognised complication of acute liver failure double-blind placebo-controlled trial of fluconazole to prevent candidal infections in critically ill surgical patients annual report of the american association of poison control centers toxic exposure surveillance system assessment and treatment of acetaminophen overdose acetaminophen poisoning and toxicity acetaminophen hepatotoxicity: the first years a review of acetaminophen poisoning simulated acetaminophen overdose: pharmacokinetics and effectiveness of activated charcoal impact of activated charcoal after acute acetaminophen overdoses treated with n-acetylcysteine a prospective evaluation of the effect of activated charcoal before oral n-acetylcysteine in acetaminophen overdose comparison of oral and i.v. acetylcysteine in the treatment of acetaminophen poisoning treatment of paracetamol (acetaminophen) poisoning with n-acetylcysteine acetylcysteine for acetaminophen poisoning hemodialysis as adjunctive therapy for severe acetaminophen poisoning: a case report amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous amanita mushrooms phalloidin syndrome: role of elisa-based assay for the detection of alpha-and gamma-amanitins in urine. preliminary results treatment of amatoxin poisoning: -year retrospective analysis a rare case of amatoxin poisoning in the state of texas the use of silymarin in the treatment of liver diseases effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial amatoxin poisoning treatment decision-making: pharmaco-therapeutic clinical strategy assessment using multidimensional multivariate statistic analysis aasld practice guidelines: wilson disease fulminant wilson's disease with haemolysis and renal failure: copper studies and assessment of dialysis regimens molecular adsorbent recycling system (mars): clinical results of a new membrane-based blood purification system for bioartificial liver support albumin dialysis: effective removal of copper in a patient with fulminant wilson disease and successful bridging to liver transplantation: a new possibility for the elimination of protein-bound toxins results of a prospective study of acute liver failure at tertiary care centers in the united states lamivudine treatment for acute severe hepatitis b: a pilot study treatment of acute hepatitis c with interferon alfa- b hypoxic hepatitis hypoxic liver injury hypoxic hepatitis in critically ill patients: incidence, etiology and risk factors for mortality serum lactic dehydrogenase in the differential diagnosis of acute hepatocellular injury nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the united states us burden of disease collaborators. the state of us health, - : burden of diseases, injuries, and risk factors incidence, prevalence, and clinical significance of abnormal hematologic indices in compensated cirrhosis splenomegaly, hypersplenism and coagulation abnormalities in liver disease thrombopoietin in acute liver failure endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats hemodynamic mechanism of esophageal varices pathophysiology of portal hypertension and esophageal varices portal hypertension and its complications is sinusoidal portal hypertension a necessary factor for the development of hepatic ascites? pathogenesis of ascites in cirrhosis amenorrhoea in women with non-alcoholic chronic liver disease patterns of hypothalamic-pituitary-gonadal dysfunction in men with liver disease due to differing etiologies low-protein-concentration ascitic fluid is predisposed to spontaneous bacterial peritonitis role of kupffer cells in host defense and liver disease role of kupffer cells in the pathogenesis of liver disease spontaneous bacterial peritonitis: recent data on incidence and treatment intestinal permeability in cirrhotic patients with and without spontaneous bacterial peritonitis: is the ring closed? spontaneous bacterial peritonitis: pathogenesis, diagnosis, treatment clinical anatomy of the umbilicus aasld practice guideline management of adult patients with ascites due to cirrhosis: update ct and mri findings of cirrhosis-related benign nodules with ischaemia or infarction after variceal bleeding ultrasound of the liver the real capabilities of contrast-enhanced ultrasound in the characterization of solid focal liver lesions noninvasive tests for liver disease, fibrosis, and cirrhosis: is liver biopsy obsolete? sampling variability on percutaneous liver biopsy sampling variability of liver biopsy in nonalcoholic fatty liver disease resection for hepatocellular carcinoma is a good option in child-turcotte-pugh class a patients with cirrhosis who are eligible for liver transplantation evaluation of hernia repair operation in child-turcotte-pugh class c cirrhosis and refractory ascites the model for end-stage liver disease (meld) meld vs child-pugh and creatinine-modified child-pugh score for predicting survival in patients with decompensated cirrhosis meld score is better than child-pugh score in predicting -month survival of patients undergoing transjugular intrahepatic portosystemic shunt comparison and improvement of meld and child-pugh score accuracies for the prediction of -month mortality in cirrhotic patients comparison of meld, child-pugh, and emory model for the prediction of survival in patients undergoing transjugular intrahepatic portosystemic shunting systematic review: the model for end-stage liver disease -should it replace child-pugh's classification for assessing prognosis in cirrhosis? hepatic encephalopathy: from pathophysiology to therapeutic management hepatic encephalopathy in chronic liver disease: practice guideline by aasld and easl rifaximin treatment in hepatic encephalopathy neomycin reduces the intestinal production of ammonia from glutamine peritoneovenous shunting as compared with medical treatment in patients with alcoholic cirrhosis and massive ascites comparison of paracentesis and diuretics in the treatment of cirrhotics with tense ascites. results of a randomized study randomized controlled study of tips versus paracentesis plus albumin in cirrhosis with severe ascites recommendations for the use of albumin and immunoglobulins randomized study of therapeutic paracentesis with and without intravenous albumin in cirrhosis randomized trial comparing albumin, dextran , and polygeline in cirrhotic patients with ascites treated by paracentesis albumin infusion in patients undergoing large-volume paracentesis: a metaanalysis of randomized trials spontaneous bacterial peritonitis norfloxacin prevents spontaneous bacterial peritonitis recurrence in cirrhosis: results of a double-blind, placebo-controlled trial propranolol plus placebo versus propranolol plus isosorbide- -mononitrate in the prevention of a first variceal bleed: a double-blind rct improved survival after variceal bleeding in patients with cirrhosis over the past two decades antibiotic prophylaxis for the prevention of bacterial infections in cirrhotic patients with gastrointestinal bleeding: a metaanalysis definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis hepatorenal syndrome oral misoprostol or intravenous prostaglandin e do not improve renal function in patients with cirrhosis and ascites with hyponatremia or renal failure reversibility of hepatorenal syndrome by prolonged administration of ornipressin and plasma volume expansion terlipressin improves renal function in patients with cirrhosis and ascites without hepatorenal syndrome etiology and outcome for patients with acute liver failure in the united states analysis of long-term outcomes of liver prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure the treatment of acute liver failure with fractionated plasma separation and adsorption system: experience in applications in vitro comparison of the molecular adsorbent recirculation system (mars) and single-pass albumin dialysis (spad) extracorporeal liver support-albumin dialysis with the molecular adsorbent recirculating system (mars) treatment of resistant pruritus from cholestasis with albumin dialysis: combined analysis of patients from three centers extracorporeal albumin dialysis with the molecular adsorbent recirculating system in acute-on-chronic liver failure: the relief trial pilot-controlled trial of the extracorporeal liver assist device in acute liver failure foreword: prospects of liver cell transplantation and liver-directed gene therapy treatment of the crigler-najjar syndrome type i with hepatocyte transplantation key: cord- -z wpjr d authors: stephens, r. scott; wiener, charles m.; rubinson, lewis title: bioterrorism and the intensive care unit date: - - journal: clinical critical care medicine doi: . /b - - - - . -x sha: doc_id: cord_uid: z wpjr d nan • no country is fully prepared to avert illness when large portions of the population are covertly exposed to a serious bioweapons agent; a moderate or large-scale intentional release of a serious pathogen will likely cause lifethreatening illness in a large portion of exposed people. • intensivists will play a key role in the medical response to a bioterrorism event due to the clinical conditions caused by serious bioweapons pathogens, such as severe sepsis, septic shock, hypoxemic respiratory failure, and ventilatory failure. • compared with conventional disasters, bioterrorist attacks may not be readily recognized; thus, accurate clinical diagnoses and management on the basis of clinical suspicion are critical not only for appropriate care of individual patients but also for instituting an epidemiological investigation. • victims of a bioterrorist attack who require intensive care unit-level care may be more contagious than those who are less sick. • health care workers, accustomed to putting the welfare of patients ahead of their own in emergency situations, must be prepared for the proper use of personal protective equipment and trained in specific plans for the response to an infective or bioterrorism event. tive prophylactic countermeasures exist, a large portion of exposed people will likely develop life-threatening illness. although intensivists working in developed countries generally have little experience treating specific illnesses caused by serious bioweapon pathogens, these diseases result in clinical conditions that commonly require treatment in intensive care units (icus) (e.g., severe sepsis and septic shock, hypoxemic respiratory failure, and ventilatory failure). therefore, intensivists will play a key role in the medical response to a bioterrorism event. usual critical care practices will likely require modification for any event resulting in more than a few critically ill victims, and critical care specialists should participate in planning for such situations. capabilities to provide medical care, especially critical care, services to large numbers of contagious patients are very limited in most countries. local hospitals will be expected to care for seriously ill victims of a bioterrorist (bt) attack, and the ability to care for large numbers of critically ill patients will likely be a major determinant of the medical impact of such events. although the current risk of a large-scale bt event is uncertain, a number of groups throughout the world during past decades have deliberately exposed civilians to biologic agents. fortunately, none of these prior events produced a large number of casualties because of the nonlethal nature of the pathogens released (e.g., salmonella typhimurium in oregon in ), the lack of technical expertise to successfully disseminate lethal pathogens (aum shinrikyo in japan in ), or relatively limited exposure (the anthrax cases of in the united states). these limited exposures should not result in predictions of the numbers of potential casualties being reduced; rather, they simply reveal that an increasing number of groups throughout the world are willing to use biologic agents. the scope and effect of prior events could have been much greater if a contagious agent were used or if a lethal pathogen were widely disseminated. despite the increased attention to biodefense, the risk of subsequent bt events may paradoxically be increasing. rapid advancements in science are making synthetic and novel biologic agents more accessible and technologies to disseminate agents may no longer be restricted to only a few nations. to reduce the medical effect of a bt event, the major determinants of morbidity and mortality must be understood ( fig. . ). the number of deaths from a bt event depends in part on the lethality and infectivity of the released agent, in addition to how effectively and widespread it is delivered. many biologic agents could theoretically be used as weapons, but some are an intentional release of a biologic agent within a civilian population, exposing hundreds or thousands of people to a serious pathogen, is increasingly recognized as a plausible terrorism event. unlike most mass casualty incidents, releases of bioweapons agents may be covert, thus providing additional security and public health challenges to the medical response beyond the generic burden of scores of casualties. an optimal medical response to a bioweapon attack will require all or most of the following: early diagnosis, rapid case finding, large-scale distribution of countermeasures for postexposure prophylaxis or early treatment, immediate isolation of contagious victims, and enhanced capacity for providing medical care to seriously and critically ill victims. no country is fully prepared to avert illness when thousands of people are covertly exposed to a serious bioweapons agent. hence, after a moderate or large-scale intentional release of a serious pathogen, even one for which effec-more lethal, more available, and more easily disseminated (table . ). the agent's characteristics alone will not, however, determine the overall impact of the bt event. population characteristics (i.e., vulnerabilities) will also affect the impact of a bt event ( fig. . ). many agencies, professions, and community members must be involved in preparing and responding to a bt event. to optimally respond, hospital and public health cooperation, planning, and preparedness need to occur before the disaster. the integration and coordination of all these responders is very important, and detailed operational descriptions are beyond the scope of this chapter (see box . ). instead, this chapter is intended to be an introduction to the medical response issues for a bt event, specifically the critical care medical response. the centers for disease control and prevention (cdc) has compiled a list of potential agents of bioterrorism and divided these into three categories, a-c (see table . ). category a agents are those believed to pose the greatest threat in terms of potential lethality, ability for widespread dissemination, ability for subsequent human-to-human transmission, and disruptive impact on the community and the public health system. these agents include variola major (smallpox), bacillus anthracis (anthrax), yersinia pestis (plague), clostridium botulinum (botulism), francisella tularensis (tularemia), and viral hemorrhagic fevers (vhfs). this section provides a brief summary of the pathogenesis and diagnosis of each category a agent and also reviews current recommendations for treatment. bacillus anthracis is acquired from contact with infected animals or animal products and causes three forms of disease: cutaneous, inhalational, and gastrointestinal. approximately cases of cutaneous anthrax occur annually worldwide. inhalational anthrax has not occurred naturally within the united states since ; any case must therefore be considered a possible sentinel case of a bioterrorist event. gastrointestinal anthrax is uncommon in developed countries and is not discussed here. cutaneous and inhalational anthrax are the forms expected following an aerosol release of spores, with the latter being the most lethal. a estimate by the u.s. congressional office of technology assessment predicted that an aerosolized release of kg of "weaponized" anthrax over a populated city would cause , to million deaths, similar to the mortality of a thermonuclear detonation. inhalational anthrax results from spore particles to mm in diameter entering the alveolar spaces and being transported by macrophages to mediastinal lymph nodes. after an incubation period that ranges from days to www.acponline.org/bioterro www.upmc-biosecurity.org www.shea-online.org www.cdc.gov weeks (median of days during the cases and up to days after the release of spores in sverdlovsk in ), germination occurs, with the vegetative bacilli producing two toxinslethal toxin and edema toxin. initial symptoms of inhalational anthrax are nonspecific: fevers, chills, drenching sweats, nonproductive cough, dyspnea, nausea, vomiting, and fatigue. hemorrhagic thoracic lymphadenitis and mediastinitis develop, and hemorrhagic pleural effusions with compressive atelactasis are common. hemorrhagic meningitis may also occur. patients may rapidly develop hemodynamic collapse, which typically has been refractory to treatment if it develops prior to antimicrobial administration. diagnosis is predicated on a high index of suspicion. in the attacks, all patients with inhalational anthrax had an abnormal chest radiograph or thoracic computed tomography scan. mediastinal widening due to lymphadenopathy and large bilateral pleural effusions were the most common features (figs. . and . ). these findings in the setting of a previously healthy patient with the abrupt development of sepsis should raise suspicion of anthrax infection. sputum gram stain and culture are rarely positive. blood cultures may yield a diagnosis but require hours to days to grow the organism. as in many infections, blood cultures lose diagnostic utility if obtained after antibiotic administration. hemorrhagic meningitis was common ( % of patients) during the sverdlovsk incident but was only confirmed in of patients in . patients suffering from inhalational anthrax are likely to require icu care but do not require respiratory isolation. antibiotics must be started as soon as possible without waiting for diagnostic confirmation. treatment recommendations are summarized in table . . for adults, combination antimicrobial therapy with intravenous ciprofloxacin and one or two other agents is recommended. given the potential for meningitis, agents with good central nervous system penetration, such as rifampin, penicillin, or chloramphenicol, are recommended. clindamycin has been administered for the theoretical benefit of reducing toxin production by the vegetative bacilli, although this has been done in too few instances to critically evaluate its effectiveness. a change to oral therapy is acceptable once the patient is stable. therapy should continue for days. the concomitant use of an anthrax vaccine in a modified dosing regimen (three doses within the first month) may be limited by availability. there is anecdotal evidence that drainage of pleural effusions carries some benefit. whether the benefit is simply reduction of pleural fluid volume to improve oxygenation or actually helps to reduce toxin burden remains uncertain. during the outbreak, pleural drainage was accomplished via serial thoracenteses and tube thoracostomy. optimal management may require tube thoracostomy due to the hemorrhagic nature of the fluid. historically, inhalational anthrax had a mortality rate of approximately %. in the attacks, modern critical care interventions and use of multiple antimicrobial agents reduced mortality to %. cutaneous anthrax results after the inoculation of skin with anthrax spores. these patients are unlikely to require icu-level care if they are treated promptly. two exceptions are the possibilities of airway compromise due to a neck lesion with resulting edema or postoperative management of a compartment syndrome. local edema is the first feature of the condition, with the subsequent appearance of a macule or papule that rapidly ulcerates and develops into a painless black eschar ( fig. . ). systemic disease, including lymphadenopathy and lymphangitis, can follow. in the absence of antibiotic therapy, mortality has been reported to be as high as %; death is rare if adequate treatment is instituted. between and the early s, y. pestis, the causative agent of plague, swept through europe, eventually killing to million people-one-third of the population. plague continues to occur naturally as an insect-borne illness, infecting approxi-bioterrorism and the intensive care unit mately people annually worldwide. in the united states, most cases occur in the rural states of the southwest. plague occurs in three forms: bubonic, septicemic, and pneumonic. naturally occurring bubonic plague occurs when infected fleas bite a human and typically results in enlarged lymph nodes (bubo) and severe sepsis. a smaller percentage of patients may develop sepsis without bubo formation, and this is termed primary septicemic plague. rarely, patients with bubonic or septicemic plague develop pneumonia, and this is termed secondary pneumonic plague. patients with pneumonic plague can transmit disease through respiratory droplets. those who contract pneumonic plague from person-to-person transmission are considered to have primary pneumonic plague and do not develop buboes. both primary and secondary pneumonic plague are transmissible from person to person. the intentional release of aerosolized plague would result in primary pneumonic plague, a condition that is rare in naturally occurring plague. world health organization (who) estimates from predict that kg of y. pestis released over an urban area with million inhabitants would cause pneumonic plague in , , with , fatalities. exposure is followed to days later by fever, dyspnea, and cough with bloody, watery, or purulent sputum. gastrointestinal symptoms also occur. cervical buboes are rare. pneumonia progresses rapidly with unilateral or bilateral infiltrates or consolidation. severe sepsis and septic shock develop with leukocytosis, multisystem organ failure, and disseminated intravascular coagulation. in the absence of therapy, irreversible shock and death occur to days after exposure. a bioterrorist attack with aerosolized plague would likely present as an outbreak of severe pneumonia and sepsis. diagnosis depends on standard microbiologic studies, with confirmatory tests available only at select laboratories. hence, unless epidemiologic clues alert health care workers that these patients do not have community-acquired pneumonia, the first group of patients will likely be cared for with the hospital's usual infection control policies for this condition. unless droplet precautions are commonly used and strictly adhered to, a number of health care workers and addi- tional patients may be exposed to plague early in the outbreak. therapeutic recommendations for pneumonic plague appear in table . . streptomycin and gentamicin are the first-line agents. doxycycline, ciprofloxacin, and chloramphenicol are alternative choices. in the event of a mass casualty situation, or for postexposure prophylaxis, doxycycline or ciprofloxacin are the preferred agents for adults. francisella tularensis is an extremely infectious pathogen; exposure to as few as organisms can cause tularemia. naturally occurring throughout north america, europe, and asia, tularemia is transmitted to humans via arthropod bites, contact with small mammals or contaminated food, and inhalation. tularemia can take many clinical forms (box . ). disease manifestations depend on virulence, dose, and site of infection. the disease can begin in the skin, starting as a papule and resulting in an ulcer, and also involve regional lymph nodes (ulceroglandular). if contaminated water is ingested or contaminated droplets are inhaled, pharyngeal ulcers with cervical lymphadenitis can occur (oropharyngeal). the eyes can be the initial portal of entry leading to chemosis and lymphadenitis (oculoglandular). sometimes, lymphadenitis may occur without ulceration (glandular). inhalational tularemia may also occur naturally due to aerosolization of contaminated materials. this clinical scenario is the most likely after an intentional release, since aerosol release would be the most likely method of dissemination. who estimated that kg of aerosolized f. tularensis in a city of million would affect , people and cause , deaths. after an incubation period of to days, abrupt fever develops, accompanied by influenza-like symptoms (headache, chills, rigors, myalgias, coryza, and pharyngitis). bronchiolitis, pleuropneumonitis, and hilar lymphadenitis would be expected, although inhalational tularemia can often present as a systemic disease without respiratory features. progressive weakness, fever, chills, malaise, and anorexia rapidly incapacitate victims. hematogenous spread can lead to pleuropneumonia, sepsis, and meningitis. sepsis due to tularemia may manifest as severe sepsis or septic shock. mortality without antibiotic therapy can be as high as % to % for pneumonic and septic tularemia. current antimicrobial therapy results in a mortality rate of less than %. rapid diagnostic testing for tularemia is not widely available. the constellation of atypical pneumonia, pleuritis, and hilar lymphadenopathy in association with the previously described symptoms should raise suspicion for tularemia. in the wake of a bioterrorist attack, until a number of patients present, initial diagnosis may be delayed. most diagnoses are made serologically with a fourfold rise between acute and convalescent antibody titers. antibodies are slow to develop: titers will usually be negative at week, positive at weeks, and peak in to weeks. laboratories need to be specifically notified if tularemia is suspected, both to improve diagnostic accuracy and to protect laboratory workers. polymerase chain reaction (pcr) and antigen detection are rapid and available at reference laboratories in the united states through the laboratory response network. if the reference lab is alerted when specimens are sent, an answer can be given within hours. treatment recommendations for tularemia are presented in table . . in the event of a contained casualty situation, streptomycin is the preferred drug, with gentamicin as a first-line alternate. doxycycline, chloramphenicol, and ciprofloxacin are acceptable alternates. for mass casualties and for postexposure prophylaxis, oral doxycycline and ciprofloxacin are the recommended agents. treatment with aminoglycosides or fluoroquinolones should last days; tetracyclines and chloramphenicol require a -day course. botulinum toxin, produced by the bacteria c. botulinum and a few other clostridium species, is the most potent known neurotoxin. botulinum toxin inactivates proteins necessary for the release of acetylcholine into the neuromuscular junction. the toxin could be disseminated as an aerosolized agent or as a food contaminant. fewer than naturally occurring cases of botulism occur annually in the united states. the use of botulinum toxin by terrorists would result in inhalational botulism or foodborne botulism, depending on the mode of dispersal. the neurologic signs are identical regardless of whether the toxin enters the body via the lungs or the digestive tract. intestinal botulism may be preceded by gastrointestinal complaints. symptoms appear approximately to hours after exposure. botulism presents as an acute, symmetric, descending flaccid paralysis. there is no associated fever, and the bulbar musculature is always affected first. presenting complaints and findings are related to cranial nerve palsies and include diplopia, dysarthria, dysphonia, and dysphagia. hypotonia and generalized weakness ensue. loss of airway protection may necessitate intubation, and respiratory muscle paralysis may require mechanical ventilation. the course is variable and may require months of mechanical ventilation. during small outbreaks with sufficient medical resources, serial measurement of vital capacity can help identify patients with respiratory muscle weakness. elevation of paco is a late finding, and positive pressure ventilation should be instituted prior to frank ventilatory failure. notably, cognitive function is not affected; patients are completely awake and alert. a fever should raise suspicion of secondary infection or an alternative diagnosis. the diagnosis of botulinum intoxication is clinical and is classically described as the triad of symmetric cranial neuropathies with descending paralysis, clear sensorium, and lack of fever. other diagnoses to consider are listed in box . . in developed nations, the occurrence of a number of temporally related cases of acute paralysis points to botulinum intoxication. the edrophonium or "tensilon" test may be transiently positive in botulism, although it still may be helpful in distinguishing it from myasthenia gravis. csf is generally normal in botulism. an electromyogram demonstrating an incremental glandular ulceroglandular oculoglandular oropharyngeal pneumonic septic response with repetitive stimulation at hz may suggest botulism when the conduction velocity and sensory nerves are normal. culture of stool or gastric contents (for foodborne exposure) may yield clostridium. confirmation usually requires the mouse bioassay (mice are exposed to samples and those given polyvalent and specific antitoxin survive) but takes several days. samples for the mouse bioassay must be collected prior to the patient's receiving antitoxin. a clinician who suspects botulism must immediately notify local public health authorities to aid with epidemiologic and diagnostic investigations. most laboratory testing cannot be performed at hospitals. laboratories must be notified of suspicion regarding botulism, since samples can be potentially harmful to laboratory personnel. specific therapy consists of treatment with equine antitoxin (see table . ), which will not reverse extant paralysis but may prevent progression. it must therefore be administered as early as possible. in mass casualty situations, when the antitoxin supply may be limited, patients with weakness but not yet requiring mechanical ventilation may be the most appropriate for antitoxin therapy. supportive therapy is essential, with a specific focus on mechanical ventilation and efforts to prevent complicating events (e.g., ventilator-associated pneumonia, venous thromboembolism, and decubitus ulcers). nonventilated patients should be placed in the reverse trendelenberg position at to degrees to optimize respiratory muscle function and minimize the possibility of aspiration aminoglycosides and clindamycin should be avoided because of the potential for exacerbating the neuromuscular blockade. mortality for foodborne botulism averages %. the last naturally occurring case of smallpox was identified in , and the last case (due to a laboratory accident) was in . despite worldwide eradication, smallpox continues to concern biodefense experts due to uncertainties about available stocks of the virus. despite the mortality rate of smallpox ( %) being considerably lower than those of other bioweapons agents, its potential for harm is still very high because those who survive may be severely deformed or blinded and no proven specific therapy exists once exposed people become symptomatic. in addition, with cessation of worldwide vaccination, entire populations and especially younger persons are susceptible to infection. the agent of smallpox, the variola virus, belongs to the orthopoxvirus family. these viruses are quite stable in the environment, and hence an aerosol may be widely dispersed. any case of smallpox would be an emergency and must be considered to be the result of a deliberate act. the typical incubation period for smallpox is to days, with an average of to days for the majority of patients. initial symptoms include fever, rigors, backache, and headache. vomiting and delirium may develop in this prodromal phase. two or days later, a nonspecific erythematous rash begins. the rash first appears in the mouth and throat, with red spots appearing on the buccal and pharyngeal surfaces. the usual dictum is that a person is not contagious until the rash begins. in most patients, the macular lesions become papular followed by vesicles. the lesions then become pustular, which umbilicate and are deeply seated in the dermis. the crops of lesions appear at the same time and are all at the same stage on the affected part of the body (fig. . ). they usually begin and are more concentrated on the face and limbs rather than the trunk. this is in contrast to primary varicella, in which lesions on any given part of the body are in different stages of development (some macular, some vesicular, and some crusting) and in which the rash begins on the trunk and moves outward. after to days, scabs form at the sites of the pustules. in survivors, these become depressed depigmented scars. smallpox lesions also occur on the palms and soles, which rarely occurs with chickenpox. the previous description is seen in more than % of smallpox cases, but there are less common forms of smallpox as well. hemorrhagic smallpox is uniformly fatal (it tends to affect pregnant women more frequently), and it typically has a shorter incubation period and does not lead to the classic rash. instead, death follows development of a hemorrhagic rash. in the malignant or flat form of smallpox, the disease begins classically but does not progress to pustules. instead, the rash is confluent and may desquamate. also, variola minor is a less severe form of smallpox. suspicion of smallpox must initially be based on clinical findings; the possibility of this disease must be considered in any patient displaying fever and a characteristic centrifugal and uniform rash. definitive diagnosis requires specialized diagnostic techniques. electron microscopy can determine whether the virus is an orthopox, and confirmatory pcr techniques require primers specific to variola. laboratory confirmation will likely be required for the sentinel cases of an outbreak. after initial cases are confirmed, additional case identification can be based on clinically consistent criteria. miller-fisher variant of guillan-barré syndrome myasthenia gravis tick paralysis atropine poisoning paralytic shellfish/puffer fish poisoning if an exposure to smallpox is suspected but the patient is asymptomatic, administration of the vaccinia virus within a few days from exposure can prevent or greatly diminish the severity of the illness. once the disease develops, however, specific therapeutic options are limited (see table . ). there is evidence that cidofovir may have activity against the variola virus, although the evidence is based on alternative orthopox disease models and in vitro assays. supportive care for critically ill patients may limit mortality, but since the last case occurred more than years ago, it is uncertain what effect modern critical care will have on outcomes. mortality is expected to be approximately %, with far greater rates of disfigurement or disability. secondary transmission is most likely to occur through close contact with symptomatic patients (e.g., droplet and contact transmission), although fomite and airborne transmission have been documented. patients with a cough may be more likely to transmit droplet nuclei (i.e., airborne transmission), and those with atypical disease courses (e.g., hemorrhagic and malignant) may be more difficult to identify, so unprotected exposure of health care workers may be more likely. the viral hemorrhagic fevers believed to be possible agents of bt are listed in box . . the filoviruses and arenaviruses are transmissible from person to person. although the limited information available suggests that transmission is primarily via infected body fluids, mucosal transmission has been documented in experimental animals and airborne or droplet transmission has been suggested in several outbreaks. clinical manifestations will vary with the particular virus. in general, the vhfs have an incubation period ranging from to days (commonly, - days). initial symptoms are nonspecific and may last up to week. fever, malaise, headache, myal-gias, arthralgias, nausea, and gastrointestinal complaints are prominent. a rash may be present. on exam, patients are typically febrile, relatively bradycardic, hypotensive, and tachypnic. as the disease progresses, hemorrhagic manifestations, such as petechiae, mucosal bleeding, hematuria, hematemesis, and melena, may appear. eventually, disseminated intravascular coagulation, multiorgan system failure, and shock may develop. mortality rates vary greatly, but in the case of ebola virus they may be as high as %. confirmatory diagnosis of vhfs must be made at specialized laboratories. the diagnosis must be suspected in any patient presenting with acute fever, severe illness, and hemorrhagic manifestations. any patient who presents with a vhf who does not have a travel, contact, or exposure history consistent with the known natural occurrence of these illnesses must be considered as the possible victim of a bt attack. unfortunately, vhfs have a high lethality and supportive therapy is the only treatment (see table . ). there is evidence that ribavirin may have some effect against arenaviridae and bunyaviridae. no therapies have been shown to be effective against the filoviruses or flaviviruses. additionally, there is no recommended agent or vaccine for postexposure prophylaxis to any of the vhfs. many of the operational functions for the response to bioterrorism events are similar to those for other disasters (intentional or natural), such as requiring coordination and communication among a number of government agencies, professions, citizens, and community stakeholders for the response. however, bioterrorism events pose specific challenges for the medical response that may be serious enough that if not addressed may shut down hospitals and leave many victims without adequate options for health care (table . ). recognizing that a conventional disaster has occurred is usually immediate, and these events are limited both geographically and temporally. casualties have traumatic injuries, and a large portion of the survivors are taken to the nearest health care facility. within hours to a few days, the number of expected casualties is usually known. immediate death rates may be high, especially with structural collapse, but typically only a small bioterrorism and the intensive care unit fraction of survivors are critically ill (injury severity score > ). enclosed space explosions may lead to higher proportions of survivors with critical injuries, but the absolute number of critically ill patients is usually less than . after the initial chaotic response period, medical staff and equipment are usually not in short supply. if local hospitals are overwhelmed, additional staff and resources can be transported to the disaster area, or patients can be evacuated to unaffected areas. the recovery plans for the affected health care facilities are usually initiated within the same day or a few days following a conventional disaster. unlike conventional disasters, a release of a bioweapons agent may go undetected. in such situations, exposed people would present for medical care after the incubation period has passed. since people travel extensively in developed countries, and most incubation periods are days to weeks, patients are likely to present to a number of hospitals rather than to the facility located closest to the release. having patients distributed to a number of hospitals may lead to delayed recognition that a bt event has occurred. in addition, most diseases resulting from serious bioweapons agents initially cause symptoms and signs that are commonly seen every day in hospital emergency departments and outpatient clinics. there may be no pathognomonic signs that a bioterrorist event occurred in the sentinel patients initially presenting with respiratory failure or hemodynamic collapse. no diagnostic tests are available to help clinicians rapidly diagnose most diseases, so a bt event may go unnoticed until scores of ill victims arrive at hospitals. the presentation of multiple previously healthy patients with unusual and severe symptoms should prompt suspicion. because of the specialized diagnostic techniques required for these organisms, and the biosafety precautions that are frequently beyond the capabilities of most hospital-based clinical laboratories, confirmatory diagnostic testing for the category a agents in the united states is handled at laboratories of the national laboratory response network, which includes local and state labs as well as federal facilities, such as the u.s. army medical research institute of infectious diseases and the cdc. there will necessarily be a delay in final diagnosis because samples for confirmatory testing must be sent to off-site laboratories. this increases the importance of accurate clinical diagnoses and proceeding with management on the basis of clinical suspicion. prompt diagnosis is critical not only for appropriate care of individual patients but also for instituting an epidemiological investigation. the community or nationwide response hinges on the results of this rapid investigation. once the source, agent, and location of a bt attack have been deduced, other clinicians can be notified, resources can be mobilized, and the at-risk population can receive postexposure prophylaxis. the difficulties of recognizing initial exposures to a bt attack have profound implications for hospital functionality, particularly if the pathogen is contagious. health care workers (hcws) and other patients without adequate infection control protections may be exposed to contagious patients. during an outbreak of severe acute respiratory syndrome (sars), unprotected exposure of hcws and hospitalized patients to patients with sars was thought to be the major risk to a hospital remaining open. most victims of serious bioweapons attacks (e.g., anthrax, plague, smallpox, botulism toxin, tularemia, and vhfs) will develop illness that is rapidly progressive (ultimately requiring mechanical ventilation, hemodynamic support, or other aggressive therapeutic interventions) if they do not receive early medical intervention or if no disease-specific medical countermeasures exist. these critically ill patients will also likely require extended critical care for survival. few hospitals can provide even usual critical care services for an additional critically ill bt victims, especially if the pathogen is contagious. in the aftermath of a bt event, it may be very difficult to ascertain the extent of the exposure. incubation periods have a range, so the first cases may simply represent the tails of the gaussian distribution, and many more patients may require care in the following days. some ill patients may go unrecognized as cases, and patients may arrive at hospitals in a larger geographical area than is typical after a conventional disaster. the initially affected area may be quickly overwhelmed because of shortages of critical care resources. the unaffected regions may choose to wait to offer help until the size of the event becomes better delineated so that they do not send staff and resources away until they are certain they were unaffected. furthermore, if the pathogen is contagious, resources in affected areas may be more rapidly overwhelmed and unaffected regions may be even less likely to provide help. bt attacks resulting in a disproportion of critically ill victims to available icu beds are plausible. if such an event occurred today, many critically ill patients would have to forgo potentially life-sustaining critical care interventions. hospitals can plan to give traditional standards of critical care to the few who are fortunate to arrive early during the event, or they can modify critical care so that more patients have access to some of the most important critical care interventions (e.g., mechanical ventilation). methods to decide who should get critical care (e.g., triage algorithms), what critical care interventions should be provided, who should provide critical care, and where critical care should be provided need to be addressed before a bt event. through such planning, hospitals may "gracefully degrade" services rather than ceasing to function when overwhelmed. all efforts must be made to provide disease-specific therapies to victims of bioterrorism. unfortunately, not all of the serious bioweapons agents have effective treatments, and for those with treatments there is concern about development of antimicrobialresistant strains. systems must be in place for testing new treatments during an outbreak so that effective treatments can be rapidly communicated to other clinicians and ineffective or harmful treatments can be rapidly withdrawn. methodological issues, ethical concerns, skeleton protocol development, and information technology systems capable of making data rapidly available for analysis should all be developed and made functional before a disaster. patients seriously ill due to a bioweapons agent, regardless of whether a specific therapy exists, will likely require extensive supportive care, including interventions traditionally provided in icus. for small-scale events with few critically ill patients, traditional icu care will likely be provided. for larger events, deci- sions regarding which supportive care practices to continue and which to forgo will depend on the number of patients relative to the available resources. supportive care that is deemed most important can be better maintained if advanced planning and preparedness are undertaken. icu physicians should alert their hospitals to the potential need for rapid acquisition of additional mechanical ventilators, noninvasive respiratory aids, oxygen, palliative medications, and specialized staff in the event of a bt attack. perhaps the most critical aspect of caring for victims of a biologic attack or an emerging infective disease in an icu is the prevention of secondary transmission. in the sars outbreak of , % of cases in canada resulted from in-hospital exposure. in taiwan, the percentage of hospital-acquired cases was %. these data include other patients in the hospital who contracted the disease as well as health care workers who suffered occupational exposures. category a biologic agents that are transmissible from person to person are listed in box . . effective infection control measures are paramount in preventing the spread of disease through the hospital and, by extension, into the community. infection control is particularly important in the icu, in which a "perfect storm" for the rapid spread of an infection exists. victims of a bioterrorist attack who require icu-level care may be more contagious than those who are less sick due to higher levels of viremia or bacteremia. invasive procedures with their attendant risks of splashing or aerosolization of blood, respiratory secretions, or other bodily fluids are more commonly performed on critically ill patients. staff members in an icu are often called on to rapidly complete a number of tasks in stressful conditions, a situation conducive to errors in infection-control practices. since critically ill patients require a high level of frequent care, cumulative exposure to staff may be higher than in other areas of the hospital. finally, other patients in the icu are immunocompromised by virtue of their own critical illnesses, notwithstanding the disproportionate number of icu patients who are immunosuppressed secondary to organ transplantation, oncologic conditions, or infection with the human immunodeficiency virus (hiv). one of the mainstays of management of a chemical incident is rapid and effective decontamination of victims. decontamination serves both to limit the total dose of chemical agent received by the victims and to protect health care workers from remnant chemicals on patient skin or clothing. patients will not present for medical care after release of a biologic agent until the incubation period passes. decontamination is not necessary for these patients, since they are not likely to be grossly conta-minated at the time of presentation. for an overt attack, if the patient is grossly contaminated and there is concern about secondary aerosolization, it becomes reasonable to decontaminate the patient. since t mycotoxin can be transdermally absorbed, decontamination of patients grossly contaminated with this agent is also warranted. if possible, symptomatic victims of a communicable bioterrorism agent should be placed in a private room to prevent exposure to other patients. in the case of smallpox or a viral hemorrhagic fever, rooms should be under negative pressure and equipped with high-efficiency particulate air (hepa) filtration. the exhaust air from these rooms should be expelled directly to the outside, and the ventilation system should not be shared with other areas of the hospital. documented cases of smallpox transmission have occurred through ventilation systems. although the number of airborne infection isolation (aii) rooms in most hospitals is few, there are engineering modifications to increase modified aii capacity during an outbreak. planning for these modifications before an event is critical. assuming a large outbreak of disease, patients should be grouped together not only with respect to location but also with respect to nursing staff, physicians, and equipment. if no diagnostic test exists, care must be taken to minimize exposure of uninfected patients with similar signs or symptoms who may be inadvertently housed in the same location. although friends and family undoubtedly bring much comfort and support to critically ill patients, in the face of an infectious disease they become both potential victims and potential vectors. visitors of victims of bioterrorism with contagious agents, or victims of an emerging infectious disease, must be kept to an absolute minimum. they must be instructed and supervised in the use of proper protective equipment and notified that they must seek treatment immediately if they develop symptoms. in extreme circumstances, it may be necessary to completely preclude friends and family from visiting patients. health care providers are accustomed to putting the welfare of the patient ahead of their own; patient care, particularly in emergency situations, is often carried out without adequate protective equipment. this cannot be allowed in the case of extremely contagious agents, even in an emergency or "code" situation. a health care provider who has contact with a patient without suitable protective gear risks not only his or her own health but also the health of other patients, coworkers, visitors, and their own families. individual patient care issues must be secondary to adequate infection control practices, lest an epidemic of smallpox, sars, plague, or viral hemorrhagic fever spread unchecked. the cdc has developed categories of precaution that are to be applied to patients with potentially communicable diseases (box . ). these categories have been described at length elsewhere but are summarized in the following sections, along with their applicability to the category a biological agents. standard precautions should be applied to all patients and include measures designed to prevent transmission of blood- smallpox viral hemorrhagic fevers pneumonic plague cutaneous anthrax borne illnesses such as hiv and hepatitis b/c. most interactions with patients do not require any protective equipment, but gloves, gown, and face shield should be used for any activity that could potentially result in an exposure to blood or bodily fluids. of the category a agents, anthrax, tularemia, and botulinum toxin require only standard precautions because these diseases are not transmissible from person to person. cutaneous anthrax should perhaps be treated with contact precautions because transmission has been suggested following contact with the lesions of this type of anthrax. contact precautions are applicable to diseases that can be spread by touching the patient directly or indirectly by coming into contact with contaminated objects. common examples include scabies, herpes, clostridium difficile, and methicillin-resistant staphylococcus aureus. contact precautions must be used, if applicable, during all patient interactions, regardless of whether body fluid contact is expected. protective equipment consists of gloves and gown, and a face shield is mandatory if splashing or spraying of body fluids is possible. patients with smallpox and vhfs must be placed in contact precautions. patient care equipment must also be dedicated to these patients and not used on patients not suffering from these diseases. droplet precautions apply to diseases that are transmissible by large-particle droplets, defined as those greater than mm. due to the size of the droplets, transmission is highest over short distances (< m) and does not occur through ventilation systems. necessary equipment includes a face shield or surgical mask with eye protection, gown, and gloves. pneumonic plague requires droplet precautions. airborne precautions are required for diseases that are spread via droplet nuclei, which are less than mm. tuberculosis is the most familiar of airborne infectious agents, but of the category a agents, both smallpox and vhfs fit into this category. droplet nuclei may travel through ventilation systems, underscoring the importance of placing patients with these diseases in negativepressure rooms with hepa filters and exhaust of air to the outside. required equipment includes gown, gloves, and adequate respiratory protection. either an n respirator (specifications are described elsewhere) with eye protection or a powered air purifying respirator (papr) is acceptable. a misconception exists that once a patient is intubated and mechanically ventilated, both large droplets and droplet nuclei are no longer expelled into the air. this is true only if the ven-tilator expiratory circuit is fitted with a filter that meets hepa guidelines. unfortunately, many of the filters and heat/moisture exchanging filters commonly used do not meet hepa criteria. this poses dangers to health care personnel, visitors, and other patients not only for bioterrorist agents but also for tuberculosis, sars, and other emerging infections. hospitals would be well advised to stockpile hepa-grade filters for ventilator expiratory circuits. correct hand washing is an essential component of hospital infection control in all circumstances. this is perhaps even more true in the circumstances of an outbreak of an emerging infectious disease or possible agent of bioterrorism. hands must be washed after each patient contact even when protective gloves are used because a surprisingly high percentage of protective gloves contain microscopic holes, and holes may develop during the activities of routine patient care. failure to thoroughly wash hands following patient contact places other patients and health care workers at risk. there has been an increase in the use of waterless alcohol rubs in icus rather than soap and water. although these gels are generally effective against bacteria and viruses, they have been shown to be ineffective against bacterial spores such as anthrax. soap and water, antimicrobial or not, are effective at removing anthrax spores from hands. accordingly, we recommend the use of antimicrobial soap and water for the washing of hands after patient contact. complete recommendations for postexposure prophylaxis are given in table . . in the case of anthrax and tularemia, postexposure prophylaxis with vaccination has not been proven effective. there is no need for prophylactic antibiotic therapy for health care workers unless they were potentially exposed in the initial attack. vaccination does not confer protection against pneumonic plague. health care workers caring for patients with pneumonic plague should, however, receive prophylaxis with days of oral doxycycline. if symptoms such as fever develop, they should be aggressively treated with parenteral antibiotics. health care workers caring for patients with smallpox should be vaccinated as soon as possible because vaccination within days of exposure can prevent or limit the severity of subsequent illness. immediate isolation must follow the development of fever after exposure to smallpox. no postexposure prophylaxis exists for vhfs. although decontamination of victims of a biological attack is rarely necessary, the rooms in which they are treated can become contaminated with infectious organisms, particularly if sprays of bodily fluids or respiratory aerosols are produced. virions of smallpox, in particular, can persist in linens for extended periods of time; documented cases exist in which laundry workers contracted smallpox from handling contaminated bedding and clothing. the causal agents of vhfs may also be transmitted via contaminated linens. commercial hospital disinfectants and household bleach at a : dilution are effective at eliminating surface contamination with anthrax, smallpox, ebola and marburg viruses, tularemia, and plague. linens from infected patients should be incinerated, autoclaved, or the bodies of deceased patients with smallpox, plague, or vhfs continue to pose an infectious risk. autopsies or postmortem examinations should be avoided if possible. the bodies of victims of smallpox should be cremated. people who have died of a vhf should preferably be cremated, although prompt burial without embalmment is a secondary option. proper use of personal protective equipment is essential in order to protect staff from infectious disease. the equipment required depends on the particular disease, as described previously. effort must be made to ensure that adequate supplies of equipment exist, and requirements will likely be far greater than expected. calculations estimating the amount of equipment necessary for one nurse caring for four patients with a communicable disease are striking and sobering. during an -hour shift, one nurse would likely require sets of personal protective equipment: pairs of gloves; gowns; surgical masks, n masks, or papr hoods; and face shields. providing for the needs of physicians, respiratory therapists, and other ancillary personnel increases this equipment need markedly. beyond the availability of adequate stocks of equipment, health care workers must be adequately trained in their uses. n respirators require fit testing annually. the equipment must be used as designed, including donning and removing it correctly. removing equipment in the proper order is particularly important: the gloves must be removed first to avoid contamination of the face or clothing when removing the gown, mask, and eye shield. unfortunately, this correct sequence is not widely appreciated by health care workers. given the complexity of caring for victims of a bt attack or an emerging infectious disease, early planning for such an incident must be carried out at the institutional and icu levels. how will a hospital, or an icu, care for a potentially massive influx of patients with communicable disease or specific requirements? plans will differ in their specifics depending on each hospital and each icu's architecture and capabilities. general principles include the following: patients should be grouped according to infection, not necessarily by need. these patients should then be isolated from the remaining hospital population and staff. dedicated physicians, nurses, ancillary personnel, and equipment should be used so as to prevent exposure to other patients and keep the numbers of exposed health care workers to a minimum. the scale of a bioterrorist attack could potentially be enormous. as described previously, many of the category a agents could produce the same lethality as a nuclear explosion. the number of casualties could rapidly overwhelm any one hospital or even all of the hospitals in a community. staff safety must be paramount; if staff members believe themselves to be at risk, large numbers of nurses, physicians, and others may not show up to work, crippling or even forcing the closure of a hospital. although all risk cannot be avoided, all possible provisions must be made. all staff members must be trained in the proper use of personal protective equipment. beyond that, training should be provided in specific plans for the response to an infective or bt incident. physicians and nurses in particular should be educated with regard to possible agents of bt and presented with disease-specific issues. contingency plans must be made in advance for postexposure prophylaxis, either with antibiotics or vaccinations, as indicated. given the critical and limited time windows to initiate effective prophylaxis, plans for distribution of medication or vaccine must be thought through in advance and not made in an ad hoc manner. a large-scale bioterrorist incident could rapidly exhaust the resources of individual hospitals or even whole communities in a number of respects. the demand for personal protective equipment will be enormous. beyond that, there will be a need for pharmaceuticals of all types. antibiotics will be essential, but so will medications regularly employed in the icu setting: vasopressors, sedatives, narcotics, and others. mechanical ventilators may be at a premium for patients with acute respiratory distress syndrome, pneumonia, or ventilatory failure secondary to botulism. although in the united states, and likely in other countries, the federal government has assembled stockpiles of antibiotics, smallpox vaccine, and mechanical ventilators, it will take time for these to be deployed, and they may not contain all essentials. the prospect of bioterrorism is not an abstract one. it has been attempted before, and it will certainly be attempted again. that it has not happened is not reason for complacency. adequate response to a bioterrorist incident is possible, but it requires careful and thoughtful preparation. also, the preparation is hardly specific to the potential agents of bioterrorism. the principles of providing safe and effective care to victims of a bt incident are wholly applicable to the management of patients suffering from naturally acquired emerging infectious diseases. the age of bioterrorism is also that of sars, coronavirus, avian influenza, ebola virus, and, significantly, global travel. the potential for patients to present acutely ill with rare, unknown, and infectious diseases, whether naturally acquired or unleashed by criminals, has never been higher. planning and preparation are essential. botulinum toxin as a biological weapon. medical and public health management hemorrhagic fever viruses as biological weapons. medical and public health management tularemia as a biological weapon. medical and public health management the challenge of hospital infection control during a response to bioterrorist attacks smallpox as a biological weapon. medical and public health management plague as a biological weapon. medical and public health management updated recommendations for management key: cord- -vf xbaug authors: dysko, robert c.; nemzek, jean a.; levin, stephen i.; demarco, george j.; moalli, maria r. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - / - sha: doc_id: cord_uid: vf xbaug nan status as a cooperative companion animal of reasonable size. dogs were used in the mid- s by william harvey to study cardiac movement, by marcello malpighi to understand basic lung anatomy and function, and by sir christopher wren to demonstrate the feasibility of intravenous delivery of medications (gay, ) . the use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by pavlov to observe and document the conditioned reflex response and by banting and best to identify the role of insulin in diabetes mellitus. for a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for beagle for the years - , approximately % of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. the next most common areas of research using beagles were dental and periodontal disease and surgery ( % of publications), orthopedic surgery and skeletal physiology ( %), and radiation oncology ( %). other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies is maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. these dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions. although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. according to the u.s. department of agriculture ( ) , the number of dogs used in research has declined from a high use of , in , in to only , in . this decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola and icterohemorrhagiae, and bordetella bronchiseptica. rabies virus vaccination may also be included. purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (r. scipioni and j. ball, personal communication, ) . random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. options for procurement of dogs for biomedical research typically include purchase from a u.s. department of agriculturedesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . , definitions, and . , requirements and application. briefly, class a licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically class a dealers). class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. regulations regarding the sale of pound dogs to research facilities or class b dealers vary from state to state and include some bans on this practice. the best resource for identification of possible vendors is the "buyer's guide" issue of the periodical lab animal. typically the last issue of each year, the "buyer's guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within that issue of the journal. welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare. regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats, of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures--additional requirements for dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute of laboratory animal research (ilar) has written the "guide for the care and use of laboratory animals" (seventh edition, ) . the "guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the association for assessment and accreditation of laboratory animal care international (aaalac international) and other animal care evaluation groups to facilitate site visits and inspections. the ilar committee on dogs has also written "dogs: laboratory animal management " ( ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. growth data for beagles from a purpose-bred dog breeding facility are provided in table i . table ii features hematology data from beagles from the same commercial facility. table iii lists serum and urine chemical data for beagles. normal physiologic data for dogs (no breed specified) are provided in table iv . the information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. the basic nutrient requirements for dogs have been compiled by the nrc and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (national research council, ) . the reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. these diets do not necessarily provide the identical composition of ingredients from batch to batch. ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. an "openformula" (or "fixed-formula") diet provides more precise dietary control. in these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are generally safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the basal metabolic rate, or basal energy requirement (ber), refers to the amount of energy expended following sleep, - hours after food consumption, and during thermoneutral conditions (kleiber, ; lewis et al., ) . the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the ber (lewis et al., ) . for dogs weighing greater than kg, the mer may be calculated using this simplified linear equation: mer (metabolizable kcal/day) = ( weightkg + ) (national research council, ; lewis et al., ) . the quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the mer by the energy density of the diet. fat provides three major dietary functions, including absorption of fat-soluble vitamins (a, d, e, and k), enhancement of palatability, and provision of essential (unsaturated) fatty acids. dietary fat is an excellent, highly digestible energy source, providing . times more energy on a per weight basis than either soluble carbohydrates or proteins (lewis et al., ) . however, fats are not needed for this purpose when adequate carbohydrate and protein are present. consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. however, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." dogs require linoleic (f - ) acid, an essential fatty acid (national research council, ) , and more recently it has been demonstrated that the f - fatty acids may play a role in maintaining healthy skin (logas and kunkle, ) . supplementation with a balanced essential fatty acid product (e.g., derm caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (logas and kunkle, ; miller, ) . essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (lewis et al., ) . there are a-amino acids, of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. the essential amino acids and the minimal requirements for growth are listed elsewhere (lewis et al., ) . chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (lewis et al., ) . conversely, inadequate protein intake results in retardation of growth and adata graciously provided by r. scipioni and j. ball of marshall farms usa, inc., north rose, new york. beagles tested for period / / - / / . b s.d., standard deviation; wbc, white blood cells; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; rdw, red cell distribution width; hdw, hemoglobin distribution width; plt, platelets; mpv, mean platelet volume; neut, neutrophils; lymp, lymphocytes; mono, monocytes; eos, eosinophils; baso, basophils; luc, large unstained cells; li, lobularity index; mpxi, mean peroxidase activity index reduction in production and/or performance. protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. as a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. thus, caloric needs must be met before protein needs (lewis et al., ) , an important concept to bear in mind in the event of research experiments that may predispose to anorexia. in general, providing a good quality commercial diet that supplies the required amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. appropriate mineral balance in the diet is very important. the best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. the recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (lewis, ) . determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/ . (basal) . (anestrus) . c < . c - c - r (continues) deficiencies are similar and nonspecific. a definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. vitamins function as enzymes that regulate a wide variety of physiologic processes. they are divided into two groups based on their solubility. the fat-soluble vitamins include a, d, e, and k, whereas the rest are water-soluble. a list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (lewis et al., ) . cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (b complex and c) (lewis et al., ) . however, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. this section is largely based on information assimilated from texts such as "miller's anatomy of the dog" (evans and christensen, ) , "veterinary reproduction and obstetrics" (arthur et al., ) , and an issue of veterinary clinics of north america: small animal practice devoted to pediatrics of puppies and kittens (hoskins, ) . the ovaries of the bitch are attached to the dorsolateral walls of the abdominal cavity caudal to the kidneys by the broad ligaments and are not palpable abdominally. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the birchard and sherding ( ) . ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. thus, semen is deposited at the external cervical os during natural or artificial insemination. the vagina is a long musculomembranous canal that extends from the uterus to the vulva. when the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. examination should proceed at an angle of approximately ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the bitch has a monoestrous cycle, with clinical estrus occurring predominantly in january or february and again in july or august (although it can occur at any time of year). the estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. the average duration of proestrus is days. during this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. estrus generally lasts days, and the vulva is softer and smaller than in proestrus. a vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. the endocrine feature of estrus is the luteinizing hormone (lh) surge, followed by ovulation within - hours. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. serum progesterone levels peak during diestrus. the duration of anestrus is approximately months. anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. the cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at ~ lower than basal body temperature. sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. the penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. it is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. the accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. the dog does not have seminal vesicles or bulbourethral glands. the onset of puberty ranges from to months of age and is affected by breed, season, and nutritional and disease status. testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. the sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or leydig's, cells. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. there is a positive correlation with the size of the testicles as measured by scrotal circumference and the number of sperm produced. finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. in general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. on mounting, the initial thrusting and ejaculation of semen last about minute. the bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. the male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." ejaculation of the accessory gland fluid continues for - minutes. the continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. fertilization occurs in the oviduct and may occur as late as days after coitus, because of the long life span of sperm in the dog. however, once ovulated, oocytes generally remain viable for only - hours. therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear "anuclear" and are classified as "cornified" or "anuclear squames." cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. the percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear-cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. during proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. the behavioral hallmark of estrus is standing receptivity toward the male. during this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days , , and of the standing heat. fertilization is completed in the mid-to distal oviduct. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. pregnancy detection can be performed by abdominal palpation of the uterus days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings in the early gravid uterus. they are approximately inches in length at - days, the time at which pregnancy is most easily and accurately diagnosed. by day the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. fetal skeletons become calcified and are radiographically evident by day . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. real-time ultrasound can be utilized for pregnancy detection of vesicles as early as - days. an abrupt drop in body temperature to less than ~ indicates impending parturition within - hours. the process of parturition has been divided into three stages, stage of labor lasts - hours and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include hard panting and increased pulse and respiration rates. fetal expulsion occurs during stage , which lasts approximately - hours. as the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the ferguson reflex. oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. the bitch is usually recumbent during stage but is able to inhibit this stage if labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between delivery of each pup is irregular, but the average time lapse is less than hour between pups until parturition is complete. veterinary assistance is necessary if the bitch remains in stage for more than hours without delivering the first pup, or for more than hours before delivering subsequent pups. the placentas are expelled during stage of labor, immediately following delivery of a pup, or up to minutes thereafter. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. heat lamps may be placed hours prior to parturition and remain until all neonates dem-onstrate vigorous and successful suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. the postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. in these cases, - units of oxytocin may be administered intramuscularly. uterine involution occurs during anestrus within - weeks of parturition. during this time a greenish to red-brown vaginal discharge, or lochia, may be noted. although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the sixth postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. semen for ai is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the bitch's scent and manually stimulated. after collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to - ml. the semen is then drawn into a sterile or ml syringe attached to a sterile disposable insemination pipette. the bitch is inseminated either standing or with raised hindquarters. a gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. the tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. the semen is injected, and - ml of air are then flushed through the syringe and pipette. the pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. the bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. as with natural breeding, ai should be performed on days , , and of standing heat, or on the days of maximal vaginal cornification. the bitch should be palpated for pregnancy approximately weeks after the first insemination. false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. however, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (cheque drops) at an oral dose of ~tg/kg q hr for - days (brown, ) . reproductive performance in the bitch is optimal prior to years of age. although normal cycle lengths are reported to occur up to the ages of - years, the interestrous interval tends to increase by years of age. cycling does not completely cease; however, after years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common. beagles have been a popular animal model because of their docile nature. they are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ecg) recordings, oral gavage, and venipuncture. dogs are sexually mature by - months of age, but they are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from - weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. the extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. in general, breed-specific patterns do tend to emerge. for example, it appears that beagle pups are very motivated by food reward (overall, ). this is not surprising, because the breed was selected to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "clinical behavioral medicine for small animals" (overall, ). by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "current veterinary therapy" series (j. d. bonagura and r. w. kirk, eds.), "veterinary internal medicine" (s. j. ettinger and e. c. feldman, eds.), and "infectious diseases of the dog and cat" (c. e. greene, ed.) . full citations of some chapters from these texts are listed in the references (w. b. saunders co. of philadelphia publishes all three texts.) canine infectious tracheobronchitis (kennel cough complex) etiology. infectious tracheobronchitis (itb) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. several organisms have been incriminated as causative for this condition: bordetella bronchiseptica; canine parainfluenza virus (cpiv); canine adenovirus types and (cav- , cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasms and ureaplasms. clinical signs. clinical infectious tracheobronchitis can be subdivided into mild or severe forms. the mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. mild tracheobronchitis usually lasts - days, even if left untreated. the severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and may be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. these cases require more aggressive treatment and may be fatal. bordetella bronchiseptica is considered to be the respiratory tract of infected animals (bemis, ) . this bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (bemis, ) . transmission is favored by confined housing of multiple animals. in experimental studies, b. bronchiseptica transmission to susceptible individuals was % (thompson et al., ; mccandlish et al., ) . the incubation period is - days. cpiv and cav- are also spread by aerosols. of these two viruses, cav- is the most persistent, lasting for up to several months in the environment, whereas cpiv is fairly labile (hoskins, a) . both viruses can be destroyed by quaternary ammonium disenfectants. pathogenesis. the most common clinical isolates are cpiv and bordetella bronchiseptica. however, b. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. in cases of clinical infection, b. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv or cav- alone are usually subclinical; coinfections with b. bronchiseptica or other microbes may result in clinical itb (keil and fenwick, ; wagener et al., ) . the characteristic lesion from cpiv or cav- infection is necrotizing tracheobronchiolitis (dungworth, ) . pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction. diagnosis and differential diagnosis. diagnosis of infectious tracheobronchitis is often based on clinical signs. isolation of bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. viral isolation or paired serology can be done but is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (johnson, ) and should be considered as a differential for itb. prevention. prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. intranasal vaccine combinations for bordetella bronchiseptica and cpiv are preferred. intranasal vaccines protect against both infection and disease, can be given to dogs as young as weeks of age, and can produce immunity within days. control. sanitation and ventilation are critical for control. the animal care staff must practice proper hygiene to prevent fomite transmission. symptomatic animals should be isolated, and animal-to-animal contact avoided. kennels should be disinfected with agents such as bleach, chlorhexidine (nolvasan) or quaternary ammonium chloride (roccal-d). proper ventilation and humidity are important in controlling spread of these infectious agents; - air changes per hour at % relative humidity are recommended (sherding, ) . no specific treatment is available for viral infections. bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for days. use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. for severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. tis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. animals with clinical disease would also be poor surgical candidates. etiology. [ -hemolytic lancefield's group c streptococcus (streptococcus zooepidemicus) is a gram-positive non-spore-forming coccus and an etiologic agent for pneumonia and septicemia in dogs. clinical signs. clinical signs vary based on the organ system affected. pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. peracute death without clinical signs has been reported in a previously healthy research dog (bergdall et al., ) , and conjunctivitis can also be caused by this organism (murphy et al., ) . epizootiology and transmission. lancefield's group c streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (olson et al., ) . epizootics have been reported in both racing greyhounds and research colonies (sundberg et al., ; garnett et al., ) . in these epizootics, and in the reported case of peracute death (bergdall et al., ) , recent transportation (within days) was associated with the disease. as such, lancefield's group c streptococcus may be an opportunistic pathogen in dogs. pathologic findings. in the peracute case reported (bergdall et al., ) , hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. ecchymotic and petechial hemorrhages were seen on other organ surfaces. the lungs were heavy and wet, and blood oozed from cut surfaces. "bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (fig. ) . histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. gram-positive cocci were seen in both the lung and the tonsils. pathogenesis. the pathogenesis for disease caused by lancefield's group c streptococcus is unclear. strain variation with respect to virulence and host immune factors is probably significant. diagnosis and differential diagnosis. definitive diagnosis is made based on bacterial culture and identification. any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. bacterial pneumonias or septicemias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and bordetella bronchiseptica. nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control. too little is known about the pathogenesis of lancefield's group c streptococcus to make any recommendations about prevention and control. treatment. antibiotic therapy should be provided, based on culture and sensitivity. intravenous fluids are indicated for febrile or systemically ill patients. for dyspneic patients, oxygen therapy and strict activity restriction are required. research complications. clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. the association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. etiology. serovars of the spirochete leptospira interrogans sensu lato cause canine leptospirosis. disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. clinical signs. leptospirosis may present as either an acute or a chronic problem. clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. icterus, congested mucous membranes, or signs referable to disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. uveitis, abortions, and stillbirths have also been associated with leptospirosis. epizootiology and transmission. vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past years. wild animals, cattle, and rodents are reservoirs for leptospira. the epidemiology of the disease is not static, and recent changes have been observed. serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. this may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (rentko et al., ; brown et al., ; kalin et al., ) . transmission occurs primarily by environmental contact, and not directly from animal to animal. infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. recovered animals may shed organisms in their urine for months to years. the organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. leptospirosis is a zoonotic disease. pathologic findings. the kidneys consistently have gross and microscopic lesions. in the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (searcy, ) . in chronic stages of leptospirosis the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly. several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. the hematogenous phase lasts - days. acute renal failure or progressive renal failure leading to oliguria or anuria may occur. the most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (greene, ) . the nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. icterus, if it develops, is most common in the acute phase. the combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. disseminated intravascular coagulation is often a secondary complication. the severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient. diagnosis and differential diagnosis. zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection. prevention and control. vaccination for leptospirosis is standard veterinary practice. bivalent inactivated bacterins for serovars of l. interrogans canicola and serovars of l. interrogans icterohemorrhagiae are commercially available. however, immunization does not prevent development of the carrier state or protect against other serovars. for outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. control requires identification and either treatment or elimination of carrier animals. treatment. penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. aggressive fluid therapy and supportive care may also be needed. elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. should not be used in research studies because of the effects of the disease on renal and hepatic function. etiology. campylobacteriosis in dogs is caused by campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. clinical signs. most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. these signs are usually mild, may be intermittent, and typically last - days. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. epizootiology and transmission. the role of c. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . clinical signs of disease most often occur in dogs less than months of age, although any age may be affected. stress or immunosuppression may make animals more susceptible to clinical disease. pound and shelter populations have the highest rates of fecal excretion of c. jejuni (sherding and johnson, ) . transmission is via the fecal-oral route, mostly through fecally contaminated food or water. unpasteurized milk, poultry, and meat are other sources of infection. campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk. pathologic findings. the actual lesions observed depend upon the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. in cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. on histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. when translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. with warthin-starry silver staining, c. jejuni may be seen between enterocytes but only rarely inside them. pathogenesis. clinical disease may be produced by several different mechanisms after the campylobacter has populated the intestinal tract (van kruiningen, ) . after colonization of the enterocyte surface, c. jejuni can produce an enterotoxin that causes a secretory diarrhea. campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. in addition, c. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. this causes mesenteric lymphadenitis. diagnosis and differential diagnosis. fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of ~ any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, giardia, and salmonella infections; helminth infestations; and hemorrhagic gastroenteritis. clinical signs. based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (c. g. couto, personal communication, ; waddle and littman, ; woody and mcdonald, ) . a history of tick exposure may be noted prior to onset of signs. in the acute phase, clinical signs range from mild to severe and may last - weeks. they include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. central nervous system (cns) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (kuehn and gaunt, ) . clinical signs are generally absent during the subclinical phase. mild thrombocytopenia, anemia, or leukopenia may be seen. the chronic phase develops - months after the initial infection, and signs may be subclinical to severe. an extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. the following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. on bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in - % of e. canis seropositive or infected dogs (kuehn and gaunt, ; breitschwerdt et al., ; shimon et al., ) . proteinuria and/or hypoalbuminemia have also been seen. epizootiology and transmission. ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. the definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. the primary vector and reservoir is the brown dog tick, rhipicephalus sanguineus. ehrlichia canis is found worldwide and follows the distribution of the vector. infection in dogs is most prevalent in tropical and subtropical areas (greene, ) . in the united states, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (breitschwerdt, ) . transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as years. ticks become infected by feeding on an infected dog that is in the first - days of an acute infection (lewis et al., ) , and ticks can shed the organisms for up to months. within the tick population, e. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (groves et al., ) . pathogenesis. in experimental infections, the incubation period prior to the onset of the acute phase is - days. during the acute phase, which can last from - weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. the thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. the subclinical phase of the disease occurs - weeks after initial infection. during this stage, dogs that can mount an effective immune response clear the infection. those that cannot mount such a response progress to the chronic stage. infection does not confer protective immunity in dogs that recover. german shepherds and doberman pinschers seem to be more severely affected than other breeds. pathologic findings. gross lesions are varied and change, depending on the phase of the disease. the most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (woody and hoskins, ) . the most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. diagnosis and differential diagnosis. the most sensitive, specific, and commonly employed method for diagnosing e. canis infections is the indirect fluorescent antibody (ifa) test. antibodies can be detected as early as days postinfection, although some dogs may not seroconvert until days postinfection (buhles et al., ) . cross-reaction may occur between e. canis, e. chaffeensis, and e. ewingii. titers greater than : are considered positive and indicative of infection and may persist for up to year. effective treatment typically produces seronegative results in - months. in some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (bartsch and greene, ) . the exact mechanism for this finding has not been elucidated. ehrlichia canis morulae can be demonstrated in circulating monocytes of giemsa-stained blood smears. however, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. using buffy coat smears from capillary blood may increase the diagnostic yield. polymerase chain reaction (pcr) assays are also available to identify e. canis. differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. prevention. preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. avoid exercising dogs in areas infested with ticks. use topical acaricides to prevent tick infestations. keep kennel areas tick-free. dogs used as blood donors and dogs from unproven sources should be tested for e. canis. treatment. doxycycline is the drug of choice for treating monocytic ehrlichiosis. oral doses of either . - mg/kg q hr or mg/kg q hr for days are very effective at eliminating the organism. tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. in chronic cases, antibiotic treatment should be extended for an additional - weeks. research complications. the most significant research complication is the thrombocytopenia that persists for all stages of the disease. additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. for these reasons, dogs positive for antibodies to e. canis should not be used in research. etiology. this disease, caused by ehrlichia platys, was first described as cyclic thrombocytopenia by harvey et al. in . clinical signs. in most cases, infection with e. platys results in subclinical disease. a generalized lymphadenopathy may be noted. epizootiology and transmission. the vector for e. platys is assumed to be a tick; however, this mode of transmission has not been established. experimental studies by simpson et al. ( ) failed to demonstrate rhipicephalus sanguineus as a vector for e. platys. coinfection with e. canis has been reported, which suggests a common vector for both organisms (french and harvey, ; kordick et al., ) . dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (harvey et al., ; gaunt et al., ) . the geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other ehrlichia organisms. the highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as michigan and as far west as oklahoma (wilson, ; mathew et al, ) . the prevalence of seropositive dogs can be high in some parts of the country. a study by bradfield et al. ( ) reported that % of the dogs entering a research institute's quarantine facility from sources in eastern north carolina were seropositive for e. platys. hoskins et al. ( ) reported a . % seropositive prevalence in healthy dogs from kennels in louisiana. pathologic findings. gross and histopathologic findings during experimental e. platys infection in dogs have been described by baker et al. ( ) . generalized lymphadenopathy was the only gross lesion noted. follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. all dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. multifocal kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. pathogenesis. the pathogenesis of e. platys in dogs has primarily been determined through experimental infection (harvey et al., ) . after inoculation the organism directly infects platelets. thrombocytopenia occurs by day - and fluctu-ates, along with parasitemia, at to day intervals. in some cases the rebound may be within the normal range for thrombocyte counts. the nadir can be lower than , platelets/~d. concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of e. platys infection. the mechanism responsible for the cyclic nature of the infection has not been elucidated. diagnosis and differential diagnosis. ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. however, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. available ifa assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other ehrlichia species. dogs usually develop detectable titers - weeks postinfection. pcr assays for e. platys have now been developed as well (chang and pan, ; mathew et al., ) . differential diagnoses for thrombocytic ehrlichiosis include e. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (dic). platys is the same as described for e. canis, above. research complications. ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. coinfection with e. platys may potentiate the pathogenicity of other infectious agents, in particular e. canis (breitschwerdt, ) . etiology. lyme disease is caused by borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. clinical signs. clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. the onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. over the course of the disease, signs may wax and wane over a period of weeks to months. dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (greene, ; manley, ) . hematologic and biochemical profiles are generally unremarkable. lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the united states. it affects humans and dogs worldwide. the geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. three major endemic foci that have been identified in the united states account for % of reported human cases (appel and jacobson, ). the distribution of these cases is as follows: northeast/mid-atlantic focus, %; midwestern focus (michigan, wisconsin, minnesota, iowa, illinois, and missouri), %; and california and oregon, %. for the most part, dogs in the remainder of the country are not at risk for contracting lyme disease. borrelia burgdorferi is transmitted exclusively by ixodes ticks. other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. ixodes scapularis, a three-host tick with a to year life cycle, is the prototypical vector for north america. the spirochetes are spread by tick bites from both nymphs and adults. ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). in endemic areas, - % of adult ticks may be infected (appel and jacobson, ) . the primary reservoir for the organism is the whitefooted deer mouse, peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. evidence also indicates that the eastern chipmunk, tamias striatus, is an important reservoir (slajchert et al., ) , and birds may also be a significant reservoir. deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. pathogenesis. the pathogenesis of lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. infection can be induced experimentally by the bite of a single infected tick. clinical signs develop - days postinfection. some evidence points to the host's inflammatory response to the organism as etiologic for disease (pershing et al, ; greene, ) . seroconversion in dogs occurs - weeks after infection with b. burgdorferi. antibody titers may remain extremely elevated for at least months. igm titers also remain elevated for several months and are indicative of neither acute nor active infection (appel and jacobson, ) . because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for days with antibiotics) can be reactivated by steroid treatment up to days postinfection (straubinger et al., ) . diagnosis and differential diagnosis. appel and jacobson ( ) recommend that three of the following four criteria be met to establish a diagnosis of lyme disease in dogs: ( ) history of exposure to ixodes ticks in an endemic area, ( ) characteristic clinical signs, ( ) positive serology, and ( ) rapid resolution of clinical signs with antibiotic therapy. ifa or elisa tests for borrelia antibodies are the assays of choice. it should be re-membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. responses to vaccine versus infection may be distinguished by western blot. culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents. prevention and control. prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, section iii,a,l,e above). a vaccine against b. burgdorferi is available but should not be necessary in a research setting. treatment. doxycycline is the drug of choice for treating lyme borelliosis. a typical dosing regimen is mg/kg q hr for - weeks. amoxicillin, tetracycline, and the quinolones are also effective. of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. (fox and lee, ) . "helicobacter heilmannii" and h. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. this species, as well as h. rappini and h. canis, is considered to be zoonotic (fox and lee, ) . clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. epizootiology and transmission. the epizootiology and transmission of helicobacter spp. in the dog remains to be elucidated. the prevalence of canine helicobacter infections in colony or shelter situations has been reported to range from % to almost % (fox, ; hermanns et al., ) . both oral-oral and fecal-oral routes for transmission have been suggested. pathologic findings. no gross lesions are noted; the primary lesion is that of histologic gastritis. this is typically characterized by reduced mucus content of the surface epithelium; vacu-olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (hermanns et al., ) . focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. pathogenesis. some helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. helicobacter felis and "h. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (fox and lee, ) . the mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the helicobacter's ability to produce urease. urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (marshall et al., ; shimoyama and crabtree, ). diagnosis and differential diagnosis. any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. definitive diagnosis for dogs requires either endoscopic or surgical biopsy. confirmation of infection with helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by pcr. a positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. the use of warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. prevention and control. until more is known about the epizootiology and transmission of helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. treatment. combination therapy has proven to be the most effective method for treating helicobacter spp. infections in dogs. combination therapy of amoxicillin ( mg/kg q hr), metronidazole ( mg/kg q hr), and sucralfate ( . - . mg/kg q hr) for days has been suggested for dogs (hall and simpson, ) . replacing the sucralfate with famotidine ( . mg/kg q hr), omeprazole ( . mg/kg q hr), or bismuth subsalicylate ( . ml/kg q - hr) may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . the benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. research complications. helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis. clinical signs. clinical signs of canine parvovirus usually appear days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. profuse, intractable diarrhea ensues, which may become hemorrhagic. approximately % of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from - wbc/~d or less. repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. parvovirus can infect dogs of any age, but puppies between and weeks of age appear to be particularly susceptible. puppies less than weeks of age are generally protected from infection by passive maternal antibody. adult dogs probably incur mild or inapparent infections that result in seroconversion. pathogenesis. canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis. parvovirus can be detected in fecal samples with a commercially available elisa from cite. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. other lesions include myeloid degeneration and widespread lymphoid depletion. parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. prevention and control. prevention of transmission begins with isolation of affected animals and quarantine for week after full recovery. disinfection of potentially infected kennel and diagnostic areas with diluted bleach ( : ) or commercially prepared disinfectant (such as kennesol, available from alphatech, lexington, massachusetts) is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a commercially available modified live vaccine until - weeks of age. young rottweilers and doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every weeks ( times) from - weeks of age. treatment. treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance. research complications. infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. canine coronavirus infection is usually inapparent or causes minimal illness. this epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at - , - , and - weeks of age and then annually thereafter. the role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (hoskins, ) . etiology. canine distemper virus (cdv) belongs to the family paramyxoviridae, within the genus morbillivirus, which includes human measles virus and rinderpest virus of ruminants. although there is only one serotype of cdv, there is a wide difference in strain virulence and tissue tropism. some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. a transient subclinical fever and leukopenia occur - days after exposure, with a subsequent fever spike - days later, accompanied by conjunctivitis and rhinitis. other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. an immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (greene and appel, ) , because dogs that develop skin lesions often recover. neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete elimination by the immune system, possibly because of protective effects by the blood-brain barrier. classic neurologic signs that may occur in acute or chronic cdv infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. canine distemper is the most common cause of seizures in dogs less than months of age. dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. cdv has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis." epizootiology and transmission. the virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of - months, coincident with the waning of maternal antibody. transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. pathologic findings. the predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may .. be accompanied by gliosis, necrosis, edema, and macrophage infiltration. acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. diagnosis and differential diagnosis. diagnosis of cdv is based on history of exposure and clinical signs. young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have cdv. ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. attenuated strains of cdv, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. because of the variety of clinical signs, there are many differential diagnoses for canine distemper. an important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. prevention and treatment. a series of three immunizations from to weeks of age, followed by yearly boosters, is a recommended preventative. treatment is largely supportive, but because of the profound immunologic effects and significant morbidity of cdv, humane euthanasia is usually undertaken in the research setting. etiology. canine herpesvirus (chv) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than weeks of age. in adult dogs, chv causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress. clinical signs. clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. older puppies, aged - weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. infection in adult dogs may result in stillbirths, abortions, and infertility. lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. passage of puppies through the birth canal or venereally in adult dogs. puppies can also be horizontally infected by littermates. entire primiparous litters may be lost, with subsequent litters protected by colostral antibody. pathologic findings. pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings. diagnosis and differential diagnosis. diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. the diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (carmichael and greene, ) . differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia. there is no effective curative treatment. supportive therapy is unrewarding, and death usually ensues within hours in in-fected neonates. in general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled. adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. because of the severity of clinical illness in puppies, such animals should be humanely euthanatized. etiology. rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals. clinical signs. clinical progression of neurologic disease occurs in three stages. the first, or prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. the third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death usually occurs within - days of the onset of clinical signs, due to respiratory failure. epizootiology and transmission. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds. pathogenesis. the incubation period for rabies is generally - weeks from the time of exposure to the onset of clinical signs but can range from week to year. bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis. diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. definitive diagnosis is based on fluorescent antibody demonstration of the virus in negri bodies of hippocampal cells. prevention and treatment. puppies should be vaccinated at - months of age, "boostered" in year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. treatment of rabies is not recommended, because of the risk of human exposure. research complications. in a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. a healthy, purpose-bred dog that bites a human in a research facility should be quarantined for days and observed for signs of rabies. this quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. a random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. the brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. a rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. a. protozoa i. giardiasis etiology. giardiasis is a small-intestinal disease of the dog caused by giardia duodenalis (lamblia), a binucleate flagellate protozoan. clinical signs. most giardia infections are subclinical. when dogs are clinically affected, diarrhea is the most prominent sign. the diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. weight loss has also been associated with clinical infection. clinical illness is more often seen in young animals. epizootiology and transmission. giardia has a direct life cycle. dogs (and people) typically become infected when they consume water (or food) contaminated with giardia cysts. the ph change from the stomach (acid) to duodenum (neutral) causes excystation. trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle. pathologic findings. giardiasis is rarely fatal. on histopathology of duodenal or jejunal specimens, giardia trophozoites can be seen attached to enterocytes. mucosal inflammation and ulceration, and villous atrophy, have been observed. pathogenesis. the exact pathogenesis of giardia-induced illness is unknown. it is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. it is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. diagnosis and differential diagnosis. definitive diagnosis requires observation of the organism in fecal or intestinal samples. direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. commercial elisa kits and direct immunofluorescent tests are available to detect fecal giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (barr, ) . differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. prevention. high-quality water sources will eliminate the possibility of infection developing within an animal research facility. use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. control. once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of giardia cysts. treatment. the most common treatment for giardiasis is metronidazole (flagyl) at - mg/kg per os twice per day for - days. quinacrine hydrochloride (atabrine) at mg/kg per os once per day for days, furazolidone (furoxone) at mg/kg per os twice per day for - days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of giardia. a bendazole is recommended at mg/kg per os q hr for days, and fenbendazole at mg/kg per os q hr for days. fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (barr, ) . research complications. typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies. clinical diarrhea would clearly need to be treated before a dog could be used as a research subject. ii. coccidiosis etiology. intestinal coccidia that have been associated with enteropathy in dogs include cystoisospora canis, c. ohioensis, c. burrowsi, and c. neorivolta. clinical signs. dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. epizootiology and transmission. cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. an indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. the gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. pathologic findings. dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. the mucosa may appear normal, raised, or ulcerated. histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. the oocysts are usually readily apparent within the epithelial cells (van kruiningen, ) . pathogenesis. intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. such factors include immunodeficiency, malnutrition, and/or concurrent disease. overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. diagnosis and differential diagnosis. diagnosis is somewhat difficult, as coccidian oocysts (of both cystoisospora and non-cystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. other causes for diarrhea (e.g., parvovirus, roundworms, giardia spp., campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. prevention. clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-crowding, and providing as stress-free an environment as possible. treatment. treatment for the presence of coccidial oocysts may often not be necessary, because cystoisospora infections are typically self-limiting and clinically insignificant. treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/lb per os for days), trimethoprim sulfa ( mg/lb per os for days), or quinacrine ( mg/lb per os for days). amprolium, which is not labeled for dogs, can also be used as a coccidiostat. it can be given in gelatin capsules for - days at a daily dose of mg for small-breed pups and mg for larger breeds. research complications. as with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. dogs used in intestinal pharmacokinetic studies should be confirmed to be free of cystoisospora infections. b. nematodes i. ascarids etiology. the most common ascarid of dogs is toxocara canis. toxascaris leonina can also infect both dogs and cats. clinical signs. ascarid infestations are most commonly subclinical. however, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. puppies may have a classical "potbellied" appearance and dull hair coat. heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. visceral larval migrans caused by toxocara canis can cause pneumonia. epizootiology and transmission. toxocara canis typically infects puppies. in fact, a unique characteristic of t. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. pathologic findings. puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia. pathogenesis. the infective stage of t. canis is the third-stage larva (l ). infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. these larval migrations are often asymptomatic, and progression of the l larvae is arrested in the tissues. it is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. if the source of infection is transplacental, puppies may be born with l larvae in their lungs, because larval migration is already in progress (sherding, ). diagnosis and differential diagnosis. the characteristic large ( - ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. prevention and control. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. most anthelmintics are effective for treatment of ascariasis. pyrantel pamoate (nemex) and fenbendazole (panacur) are commonly used. treatment should be started early in puppies ( , , , and weeks) because of the possibility of prenatal or neonatal infection. pyrantel pamoate, dosed at mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see section iii,a, ,b,ii). in breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. extended fenbendazole therapy ( mg/kg per os twice per day for days or once per day from day of gestation through day of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. research complications. puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. hookworms etiology. the most common and most pathogenic hookworm of dogs is ancylostoma caninum. other, less pathogenic canine hookworms found in north america are a. braziliense, which can be found in the american tropics and southern united states, and uncinaria stenocephala, which is distributed in the northern united states and canada. clinical signs. only a. caninum infestation typically results in clinical illness, because of the amount of blood that it con-sumes. puppies with a. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. epizootiology and transmission. infective larvae (l ) are typically ingested by puppies and develop directly in the intestinal tract. ingestion can be from the bitch's milk (transmammary migration occurs with a. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. transplacental migration does occur with a. caninum, but to a much lesser extent than is seen with toxocara canis. larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. the prepatent period is weeks. pathologic findings. infected puppies often have severe anemia and eosinophilia. the anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. on gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (fig. a) . ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (fig. b) . pathogenesis. the severe pathogenicity of a. caninum is a direct result of its voracious consumption of blood and body fluids. each adult hookworm can consume . - . ml of blood; thus an extensive infection could deplete a puppy of ml of blood per day, which is approximately % of the blood volume of a . kg animal. in contrast, a. braziliense and u. stenocephala consume . and . ml per worm, respectively. diagnosis and differential diagnosis. diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. prevention and control. purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. pyrantel pamoate (nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. because of the possibility of transplacental or milk-borne infection, puppies should be treated every weeks from weeks - . a follow-up treatment at weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation. research complications. anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. iii. strongyloides etiology. strongyloides stercoralis is a small strongyle that can cause hemorrhagic enteritis in puppies. it is found in warm, humid climates such as the southeastern united states. fects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. the eggs of s. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. diagnosis and differential diagnosis. the baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva ( - ~tm x - ~tm). the larvae must be distinguished from larva of filaroides hirthi and hatched ancylostoma caninum. treatment. the usual treatment for s. stercoralis is fenbendazole (panacur) at mg/kg per day for days. iv. whipworms etiology. trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. the adult whipworm typically resides in the cecum or ascending colon. clinical signs. most whipworm infections are subclinical. in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss are also seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode. life cycle. adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. the eggs are very hardy and can persist for years. in optimal conditions, the eggs develop into an infective embryo within days. after ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. the prepatent period is typically - months long. pathologic findings. dogs do not typically die from whipworm infestations. lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. pathogenesis. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. diagnosis and differential diagnosis. whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. these eggs are barrel-shaped, with thick walls and bipolar plugs. because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. adult worms can be seen on colonoscopy (jergens and willard, ) . differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. prevention and control. trichuris eggs are resistant to disinfection, making control difficult. dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. treatment. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. research complications. whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis. etiology. heartworm disease of dogs is caused by the filarial worm, dirofilaria immitis. adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them. clinical signs. most heartworm infestations are asymptomatic. the most common clinical signs observed are coughing and dyspnea. clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. epizootiology and transmission. successful heartworm transmission requires the presence of mosquitoes. for this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. the microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. this stage is then returned to the canine bloodstream during feeding. this stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. in the united states, transmission of heartworm by mosquitoes occurs over a month or shorter period, except for the southeastern and gulf coast states. here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (knight, ) . pathologic findings. on necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (fig. a ). there may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (fig. b) . endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis. pathogenesis. the physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. however, the host immunologic response to this infestation, coupled with secretion by the heart-worms of physiomodulative factors, contributes significantly to the complications seen with this disease. endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. these changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. the heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (dillon, ) . these factors are discussed in more detail below, under "research complications." diagnosis and differential diagnosis. for dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. a diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. false-negative results can occur during the prepatent period after initial infection (first - months), and when the adult worm burden is light or predominantly male. infections consisting of more than three mature female worms are usually detected by antigenic serology (knight, ) . a significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). if a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test months later. examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified knott test and filter tests. tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of dipetalonema reconditum, a nonpathogenic filarial worm. other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. these same techniques can be used to diagnose clinical heartworm disease. additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis (complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure. prevention and control. for dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. purpose-bred dogs reared in such an environment are usually free from dirofilariasis. however, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. there are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). these could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. if a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. treatment. treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (hoskins, ) and immunologic reactions to dead worms lodged in the pulmonary vasculature. for this reason, medical treatment of heartworm disease is not usually attempted in research dogs. in a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (caparsolate) and ivermectin (ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). alternative choices include melarsomine (immiticide) as an adulticide and milbemycin (interceptor), levamisole (levasol), or fenthion (spotton) as microfilaricidal agents. dosing regimens for these agents are detailed in dillon ( ) . research complications. the physiomodulative properties of heartworm infection have been studied. such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (maksimowich et al., ; matsukura et al., ; mupanomunda et al., ) . the extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (kaiser et al., ) but refuted in others (tithof et al., ) . it is thought that the endothelium is perturbed by a factor released from the adult dirofilaria, possibly a cyclooxygenase product such as prostaglandin d (kaiser et al., (kaiser et al., , . these products have also been demonstrated to cause constriction in in vitro rat tracheal ring preparations (collins et al., ) , suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. platelet reactivity was also been found to be enhanced in dogs naturally infected with dirofilaria, when compared with uninfected controis (boudreaux and dillon, ) . based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements. etiology. several species of cestodes (tapeworms) parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. clinical signs. most cestode infestations are subclinical. severe infestations with dipylidium can be associated with diarrhea, weight loss, and poor growth. epizootiology and transmission. the cestode life cycle requires an intermediate host. for dipylidium caninum, the intermediate hosts are fleas and lice. thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. pathologic findings. adult cestodes in the small intestine are usually an incidental finding at necropsy. diagnosis and differential diagnosis. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. dipylidium egg packets are large ( x bm) and contain - eggs per packet (hall and simpson, ) . prevention and control. the most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. see the sections on these ectoparasites for effective means to treat infested dogs and kennels. treatment. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against dipylidium caninum (hall and simpson, ) . clinical signs. most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. epizootiology and transmission. the lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. on ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. pathologic findings. grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. pleural hemorrhages may also be caused by the migrating metacercariae (lopez, ) . pathogenesis. clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. diagnosis and differential diagnosis. definitive diagnosis of paragonimus infestation requires identification of the characteristic ovoid eggs ( - ~tm long) with a single operculum in either the feces or a transtracheal wash. identification from fecal samples requires sedimentation techniques. other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. prevention. use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal. treatment. praziquantel (at mg/kg q hr x days) or fenbendazole ( - mg/kg q hr x - days) are recommended for treatment of canine paragonimus infestation (hawkins, ) . effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. research complications. experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e. mites i. demodicosis etiology. canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles. it is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. clinical signs. demodex canis infestation is typically asymptomatic. clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and the face and around the ears (demanuelle, a). secondary bacterial pyoderma is a common complication. epizootiology and transmission. demodex canis mites pass to nursing pups from the dam. they live their entire lives on one dog and are not considered contagious to other dogs or humans. certain breeds are predisposed to the generalized form of demodex dermatitis (see "pathogenesis," below). beagles are among the predisposed breeds, as are german shepherds, doberman pinschers, old english sheepdogs, collies, boxers, and shorthair brachycephalic breeds (muller et al., ) . pathologic findings. histologically, demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. cases with generalized demodicosis (see "pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression . pathogenesis. when clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one foot affected, or five or more small areas, or one large body area). localized demodicosis is typically seen in juvenile dogs (< months) and usually resolves without treatment as natural immunological control develops. generalized demodicosis can develop in juvenile or adult populations. juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited t-lymphocyte dysfunction. adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration). diagnosis and differential diagnosis. demodex is readily identified from deep skin scrapings of lesioned areas (campbell, ; noli, ) . demodex canis has a characteristic "cigar shape," with short, stubby legs on a body - ~tm long. differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. prevention and control. dogs with generalized demodicosis should not be maintained in a breeding colony. treatment: ivermectin (ivomec) at - ~tg/kg and oral milbemycin (interceptor) at - mg/kg/day have been found to be effective treatments. these parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of demodex canis. amitraz (mitaban) dips ( ppm every days) can be used for more problematic cases. treatment duration can be extensive and must be accompanied by repeated skin scrapings. research complications. dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. sarcoptic mange etiology. canine sarcoptic mange is caused by sarcoptes scabiei var. canis. clinical signs. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. epizootiology and transmission. sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for - weeks away from the host, and it is this ability that enables them to spread from dog to dog. sarcoptes scabiei var. canis can also infect cats and humans. pathologic findings. histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis . pathogenesis. lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. the typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. diagnosis and differential diagnosis. sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige in either the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ). an important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. prevention and control. use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. for random-source dogs, an ectoparasite control program should be in place to limit possible infestations. many institutions use ivermectin as a means to control both endoparasites and ectoparasites. treatment. unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. in research colonies, the usual means of treatment is either ivermectin (ivomec) at - ~tg/kg q days or milbemycin (interceptor) at oral doses of mg/kg q days . neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. research complications. the local skin inflammation and systemic immune response to sarcoptic mange probably make infected dogs poor subjects for dermatologic and immunologic studies. f lice and ticks i. lice etiology. dogs can be infested by one species of sucking louse (linognathus setosus) and two species of biting lice (trichodectes canis and heterodoxus spiniger). clinical signs. mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. more severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. these lesions lead to excoriation and secondary bacterial dermatitis. severe linognathus infestations could cause anemia, because this species feeds on blood. epizootiology and transmission. louse infestations are uncommon in both pet animal practice and the research setting. they would most likely be seen in random-source dogs that were obtained from a pound or shelter. transmission is usually by direct contact, for lice spend their entire lives on the host species. lice are host-specific and not zoonotic. pathogenesis. the biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. trichodectes canis can serve as vector for the canine tapeworm dipylidium caninum. the most severe complication of infestations by the sucking louse is the potential anemia. diagnosis and differential diagnosis. pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (muller et al., ) . differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea. prevention. use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony. treatment. most commercially available insecticide shampoos and dips readily treat louse infestations. treatment should be repeated in - days, because any nits that were not killed would have hatched by that time (muller et al., ) . there is probably minimal interference with research, unless severe linognathus infestations cause anemia. ii. ticks etiology. ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. except for the brown dog tick (rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, and ixodes. the primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including rocky mountain spotted fever (caused by rickettsia rickettsii), lyme disease (borrelia burgdorferi), and the canine forms of ehrlichiosis (ehrlichia canis and e. platys), babesiosis (babesia canis), haemobartonellosis (haemobartonella canis), and hepatozoonosis (hepatozoon canis). clinical signs. as an entity unto itself, tick infestation causes minimal clinical signs. most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (merchant and taboada, ) . some species of ticks (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (malik and farrow, ) . the paralysis develops within - days of tick attachment and can result from a single tick. this paralysis is fatal once the respiratory musculature is affected. epizootiology and transmission. in dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. the brown dog tick may reside within kennels (attics, bedding, wall insulation) (garris, ) . pathologic findings. under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death). pathogenesis. tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., dermacentor) while consuming a blood meal (malik and farrow, ) . interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. the primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including lyme borreliosis, ehrlichiosis, babesiosis, and rocky mountain spotted fever. diagnosis and differential diagnosis. for uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . prevention. purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites. treatment. removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. dogs with tick-bite paralysis usually show improvement within hr, with complete recovery within hr (malik and farrow, ) to remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. the tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. the tick should be pulled away from the dog with steady pressure. many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. probably have minimal impact on research variables. the significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see sections iii,a,l,e-g). g. other i. flea infestation etiology. fleas are laterally flattened wingless insects that feed on animal blood. the most common flea to infest dogs is ctenocephalides felis, the cat flea. other fleas that can affect dogs are ctenocephalides canis, pulex irritans, and echidnophaga gallinacea. the fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (campbell, ) . clinical signs. flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as well as the flanks, thighs, and abdomen (muller et al., ) . the lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages. epizootiology and transmission. fleas are readily transmitted between animals and even between host species. they move readily between the host and the environment, making transmission easy and control difficult. because fleas require host blood for food, they can survive off of a host for only - months (muller et al., ) . pathologic findings. biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (muller et al., ) . pathogenesis. fleas are parasites that require animal blood for their meals. when they bite host animals, they inject some saliva into the host's skin. if the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. fleas can also transmit or serve as vectors for other pathogens (e.g., dipylidium tapeworms). flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (demanuelle, b) . circulating eosinophilia is seen in some dogs with flea allergy dermatitis. differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity). prevention. most dogs obtained from high-quality purposebred facilities should be free from flea infestations. dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility. control. thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. treatment. treatment for fleas needs to address treatment of both the dog and the environment. many insecticide formulations such as shampoos, sprays, dips, powders, and oral systemics can be used for initial treatment of the individual dog. the active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. flea control in the kennel may need to include outdoor areas in warm climates. typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (demanuelle, b) . in addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. oral prednisone or prednisolone at . mg/kg q hr for - days has been proposed as a starting therapy (muller et al., ) . the use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting. research complications. mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. in a research setting, the residual effects of insecticides may preclude their use in experimental animals. such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. the ability of fleas to transmit other parasitic diseases must also be considered. etiology. dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the united states are usually caused by either microsporum canis, m. gypseum, or trichophyton mentagrophytes (muller et al., ) . clinical signs. uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. these skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (muller et al., ) . ep&ootiology and transmission. the fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil. pathologic findings. on close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (muller et al., ) . stains that facilitate visualization of fungal elements include periodic acid-schiff (pas) or gomori methenamine-silver. the pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. pathogenesis. the dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. the hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. this causes short stubbly hair to be seen within the lesion. as the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. it is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (muller et al., ) . diagnosis and differential diagnosis. diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. these scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. fungal elements can also be seen on skin biopsy samples. for speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as sabouraud's medium or dermatophyte test medium (dtm). incubation should be at ~ with % humidity for - days. lesions caused by m. canis may fluoresce when inspected using a wood's ( . nm ultraviolet) light. unfortunately, some strains of m. canis do not fluoresce, and neither does m. gypseum or t. mentagrophytes. differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (muller et al., ) . prevention. purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals. control. in cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. treatment, if acceptable, should be started immediately. treatment. topical antifungal therapy is most commonly used. shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (stannard et al., ) . severe cases may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. however, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). many of the newer agents are also expensive and not labeled for use in dogs. impact on most research applications for dogs. unfortunately, the zoonotic implications of dermatophytoses force the issue of aggressive treatment, and many antifungal agents may not be compatible with biomedical research studies. systemic fungal infections disseminate to multiple organ systems from a single mode of entry (usually through the respiratory tract). dogs are susceptible to several fungi that characteristically cause systemic mycosis, including blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans var. neoformans. these diseases are not typically seen in the research setting, because of the low overall incidence and noncontagious nature of these disorders and because of the use of purpose-bred animals. these conditions could, however, present in the rare random-source dog that was subclinical at its point of origin, especially if the animal becomes immunosuppressed (either naturally or by virtue of experimental manipulation). typical clinical signs include weight loss, fever, lymphadenopathy, and cough and dyspnea (if the lungs are affected). the reader is advised to read veterinary medical text chapters (e.g., taboada, ) for more complete information on these disorders and their possible treatments. although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology. the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. both of these causes result in a gradual loss of functional thyroid tissue (kemppainen and clark, ) . lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs. because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic normochromic nonregenerative anemia and increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bischel et al., ; panciera, ) , and so the relationship between hypothyroidism and these problems has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause bradycardia as a result of decreased myocardial conductivity. abnormalities that may be detected by ecg include a decrease in p and r wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these electrocardiographic abnormalities are caused by lowered activity of atpases and calcium channel function. several reports have suggested that hypothyroidism is associated with von willebrand's disease and bleeding abnormalities. however, the relationship is probably one of shared breed predilection and not a true correlation. it has been demonstrated that dogs with hypothyroidism are not deficient in von willebrand's factor when compared with other dogs. in addition, the replacement of thyroid hormone in dogs did not increase the levels of vwf:ag in naturally occurring (panciera and johnson, ) or experimentally induced (panciera and johnson, ) hypothyroidism. epizootiology. the prevalence of hypothyroidism in the general canine population has been reported to be less than % (panciera, ) . the disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . in general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (panciera, ; peterson and ferguson, ). diagnosis and differential diagnosis. because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. the tests currently available and in popular use will be discussed further. however, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t in the body and is heavily proteinbound. free t represents the unbound fraction that is available to the tissues (peterson and ferguson, ) . using the measurement of serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (peterson and ferguson, ) . however, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. if the clinical signs are equivocal or if only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone in the body, the measurement of serum t levels is an unreliable indicator of hypothy-roidism (peterson and ferguson, ; ferguson, ) . like t , serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released, and conversion of t to t may be enhanced in the hypothyroid dog (peterson and ferguson, ; ferguson, ) . t was within normal limits in % of the hypothyroid dogs in one study (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous tsh (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous tsh is now available in dogs. however, tsh levels can be normal in some dogs with hypothyroidism, and high tsh levels have been noted in normal dogs. therefore, it is recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and hours after. a normal response to the administration of tsh should create an increase of t levels at least ktg/dl above the baseline levels or an absolute level that exceeds ~tg/dl (peterson and ferguson, ; wheeler et al., ) . treatment. the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . mg/kg once a day or . mg/m (body surface area)/day for very small or very large dogs. if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - hours after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are also frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research. etiology. obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy. excessive caloric intake resuits from overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (butterwick and hawthorne,. ). as in humans, genetics plays an important role in the development of obesity in dogs. it has been established that certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . in addition to genetics, several metabolic or hormonal changes are associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese when compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ) . in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . epizootiology. ewen, ) . obesity affects up to % of pet dogs (mac-diagnosis and differential diagnosis. the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see section iii,b,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment. restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. there has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick and thorne, ; butterwick and thorne, ) . it is important to control weight gain in research animals, because of the association of obesity and several metabolic changes. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . obesity in dogs over years of age appears to be related to an increase in cardiovascular problems (edney and smith, ) , and obesity has been linked to hypertension. joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (macewen, ) . in the laboratory setting, the majority of traumatic wounds will be small in size. in facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. to aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see table v ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. dirty wounds can occur as postsurgical infections or complications of initial wound therapy. when one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of the wound's classification. when first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. bleeding should be controlled with direct waldron and trevor ( ) . pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . when the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. then a water-soluble lubricant gel may be applied directly to the wound. a wide margin of hair should then be clipped from around the wound, using a # blade. after the clipping, a surgical scrub is performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a,b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. two solutions, . % chlorhexidine diacetate in water (lozier et al., ) and % povidone-iodine in saline, are most often recommended for wound lavage (waldron and trevor, ) . the chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (sanchez et al., ) . actually, the type of solution chosen may not be as important to wound care as the volume and pressure at which the solution is delivered. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a ml syringe and an -or gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be place d. subcutaneous closure should be performed with absorbable suture such as polydioxanone (pds), polyglactin (vicryl), or polyglycolic acid (dexon). it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. when the sponges are later pulled from the wound, dried exudates will also be removed. when the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . secondintention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. in skin wounds, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . etiology. pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. the compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. these factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (swaim and angarano, ) . clinical signs. at first, the skin at the developing site will appear red and irritated. over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. the severity of the sores may be graded from i to iv, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. epizootiology. the problem usually occurs in large-breed dogs, but any type of dog can be affected. prevention and control. minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. if the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain good flesh and adequate healing is also important (swaim and angarano, ) . treatment. the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. the affected area should be radiographed to assess bone involvement, and the wound should be cultured. all of the damaged tissue should be debrided, and wound management guidelines should be followed (see section iii,c, ). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary. bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. this will displace the forces acting on the wound over a larger area and over healthier tissue. then the doughnut is incorporated into the bandage. if a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ). bandages should be removed at least once or twice a day to allow wound care. after wound care has been initiated the causative factors for the pressure sore must be addressed (see "prevention and control," above). recumbent animals should be moved frequently to prevent continuous compression on the wound. if the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. etiology. acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. in a few cases, self-trauma begins because of identifiable neurologic or orthopedic causes (tarvin and prata, ) . however, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma. epizootiology. the lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (walton, ) . clinical signs. at first, lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ). the predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis. acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of these problems can be ruled out by the history of the animal. when in doubt, a biopsy should be taken. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control. behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. the environment of a dog with this problem can be enriched with exercise and the introduction of toys. in addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. these drugs may produce side effects, however (swaim and angarano, ) , and may interfere with experimental results. treatment. several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in ah cases. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. in one study, either naltrexone ( mg/kg sq) or nalmefene ( - mg/kg sq) successfully reduced the excessive licking behavior in of dogs; however, lesions returned after the drug was discontinued (dodman et al., ) . the use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide ( ml of banamine [schering] mixed with ml of synotic [diamond laboratories]) applied topically twice daily has also been shown to be effective (walton, ) . the prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. etiology. hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology. elbow hygromas are most frequently reported in large and giant breeds of dogs around - months of age (johnston, ; bellah, ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs. a dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. the animals are not usually lame. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma is secondarily infected, the animal may exhibit pain and fever (johnston, ; bellah, ) . pathology. the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is yellow or red and is a serous transudate. this fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (johnston, ) . treatment. the treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see section iii,c, ). more aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this treatment (johnston, ) . likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (johnston, ) . a technique that has been used successfully involves placement of multiple penrose drains. the drains are kept in place for - weeks, and the limb remains bandaged for weeks with this technique (bellah, ) . another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. the remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. the stents are removed in - days, and the wound is bandaged until suture removal in - days (newton et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology. in the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting. clinical signs. the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia. treatment. the treatment of corneal ulcers will depend on the depth and size of the affected area. deep ulcers may require debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given times a day for - days usually provides adequate treatment. ointments are preferred over drops, because use of the former requires less frequent. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. the catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. it is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. when appropriately maintained, indwelling catheters may remain functional for months without serious complication. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. this is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact vascular locations not specified). the lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. the traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. the visceral infarcts were noted in the spleen, kidney (fig. ) , and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. fatal hemorrhages were most often found in animals with experimentally induced hypertension. these animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm. etiology. the leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. clinical signs. the signs and treatment of systemic infection are covered in section iii,d, . problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. the signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. prevention. because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. the catheter should be made of nonthrombogenic material. in addition, it is recommended that catheters be as simple as possible. a catheter with extra ports or multiple lumens requires addi- tional management and supplies more routes for infection. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. the placement of the catheter should be verified by radiography. catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. after catheter placement, the animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "treatment"). throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. treatment. the treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ) . superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. aerobic and anaerobic cultures of blood and locally infected sites should be performed (ringler and peter, ) . localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. again, the catheter should be removed. if retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. it has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used in the catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . when placed and managed correctly, these ports may remain in place for months without complications. the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., , kwei et al., . these infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. there have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. this complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (meunier et al., ) . the chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (figs. a,b) as complications of intestinal access ports. the procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. it is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. an omental patch placed over the site of entry may help form a firm adhesion. in addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. the placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (meunier et al., ) . etiology. sepsis is defined as the systemic response to infection. most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. in laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs. the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis. the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~ as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis. in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (kirby, ) . treatment. the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is associated with the source of infection, the implant should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no "magic bullet" for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology. in research animals, aspiration into the lungs may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. clinical signs. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on a history consistent with aspiration and the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. pathogenesis. aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the ph, osmolality, and volume of the substance. the compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - hr. treatment. the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. in cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. when severe inflammation is present, systemic fluid therapy should be instituted. support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. the use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (nader-djahal et al., ) . several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. in humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be treated with antibiotics immediately when the aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. etiology. in laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs. the clinical signs vary with the type and degree of burn injury. initially, the injury may not be noticed. the first signs may be oozing from the skin and matting of the overlying hair. within a couple of days, progressive hair and skin loss may be observed (johnston, ) . the wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. superficial wounds appear as red, inflamed skin similar to sunburn in humans. the pain associated with these injuries usually subsides in - days, and the wound reepithelializes without complications in - days. deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. the wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. within - days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. several days later, gram-negative organisms can appear in the burn wound (johnston, ) . at this point, signs of wound infection and sepsis may occur (see section iii,d, ). treatment. appropriate and timely treatment of a burn wound will reduce the extent of the injury. thermal injuries should be immediately cooled to reduce edema and pain (demling and lalonde, ) . chemical burns should be thoroughly lavaged for min after wounding. the damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. after the initial treatment, all burn wounds should be gently cleansed - times a day (demling and lalonde, ) . burns involving the epidermis and part of the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, topical wound dressings are recommended in the early stages of treatment. a thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (demling and lalonde, ) . when signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. after the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. when the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected. prevention. obviously, prevention of burn wounds is preferable to a long course of treatment. care should be taken to prevent direct exposure to harsh chemicals. tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. etiology. research and/or anesthetic protocols may require the intravenous injection of various solutions. when these substances have a ph or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (swaim and angarano, ; waldron and trevor, ) . clinical signs. the immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. later, the area may appear red, swollen, and painful as inflammation progresses. often there will be eventual necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a to month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . prevention. because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. if a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. whenever possible, insertion of an indwelling catheter is extremely important. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. treatment. the treatment of perivascular injections will depend on the amount and type of substance injected. in most cases, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (swaim and angarano, ). the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) , and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (waldron and trevor, ) . despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful, and analgesia should be addressed. etiology. hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. this condition may be seen in young dogs with congenital portosystemic shunting of blood flow. however, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. a well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy. pathogenesis. when the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (hardy, ; center, ) . these compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance -aminobutyric acid (gaba). clinical signs. the signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. diagnosis and differential diagnosis. a presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. serum glucose and protein levels may be low if hepatic function is severely impaired. a low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (hardy, ) . measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal. treatment. because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. during initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. the majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either . % sodium chloride or . % sodium chloride with . % dextrose, supplemented with potassium chloride, is recommended (hardy, ) . the type of drug to be used for seizure control is controversial. the short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (maddison, ) . however, endogenous benzodiazepines mediate some of the cns signs seen with hepatic encephalopathy. therefore, the use of diazepam has been discouraged in favor of phenobarbital (johnson, ) . the drug selected for seizure control should be titrated carefully, given the altered liver metabolism. most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of h blockers such as cimetidine or ranitidine is suggested (swalec, ) . in addition, lactulose retention enemas should be performed ( - ml/lb of a % solution in water, retained for - min) (hardy, ) . lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. the decrease in colonic ph reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. lactulose will also cause an osmotic diarrhea. antibiotics such as neomycin ( mg/lb, - times/ day) or metronidazole ( mg/lb, times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (hardy, ) . when the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. diets suitable for dogs with renal insufficiency are recommended initially. this type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. these requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. to maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (center, ) . in addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels. the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had either mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. an instrument such as a tru-cut needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a mm x to . cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor, and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology. lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid ( , -d) as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . clinical signs. multicentric and alimentary lymphomas account for most cases of canine lymphoma. in multicentric lymphoma, animals usually present with enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. the tumors may occur as nodules, plaques, ulcers, or dermatitis. approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement. epizootiology. the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . pathologic findings. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. pathogenesis. all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. diagnosis and differential diagnosis. differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed. treatment. therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. macewen and young ( ) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. research complications. given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. etiology. the fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (pulley and stannard, ) . frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas. clinical signs. although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. fibrosarcomas are extremely variable in size and can grow to be quite large. in general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. epizootiology. most fibrosarcomas develop in adult and aged animals but can affect dogs as young as months or less. pathogenesis. fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. less frequently, spread to local lymph nodes is observed. diagnosis and differential diagnosis. differential diagnoses for fibrosarcomas vary with the location of the tumor. histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms. treatment. treatment of any soft-tissue sarcoma would begin with wide surgical excision. if the tissue margins indicate incomplete resection, radiotherapy could be used. for any highgrade tumors, adjuvant chemotherapy would be recommended (see macewen and withrow, a , for a complete discussion). research complications. because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies. etiology. neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas. clinical signs. these growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. generally they are well circumscribed, encapsulated, and soft on palpation. further, the skin is freely movable over the tumor. epizootiology. lipomas occur principally in aged animals (average years), and the incidence increases with age (pulley and stannard, ) . the tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed. pathologic findings. histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present. pathogenesis: lipomas are typically slow-growing and do not recur after complete surgical excision. diagnosis and differential diagnosis. lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. generally, the distinction can be made from the clinical history. treatment. treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. in such cases, surgical excision is usually curative. research complications. lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation. etiology. histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (pulley and stannard, ) . clinical signs. the most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. the masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure - cm in diameter. epizootiology. histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than years of age. pathologic findings. histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. the cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. despite being benign lesions, histiocytomas characteristically have a high mitotic index. pathogenesis. this tumor typically exhibits rapid growth ( - weeks) but does not spread. most histiocytomas will spontaneously regress in less than months. diagnosis and differential diagnosis. histiocytomas must be distinguished from potentially metastatic mast cell tumors. this is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple. treatment. although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. research complications. histiocytomas should not interfere with most studies. etiology. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (bostock, ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. clinical signs. well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, to cm nodules in the skin. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-guinal and scrotal areas. mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. epizootiology. these tumors tend to affect middle-aged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ). pathologic findings. because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis, and grade iii the worst prognosis. grade i tumors are well differentiated, with round to ovoid uniform cells. the nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. the cytoplasmic granules are few, but mitotic figures are much more frequent. in addition to skin lesions, mast cell tumors have been associated with gastric ulcers. these lesions are most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . the ulcers can be found in the fundus, pylorus, and/or proximal duodenum. although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis. mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple. treatment. initial treatment for mast cell tumors is generally wide surgical excision ( to cm margins). even with wide surgical margins, approximately % of mast cell tumors may recur. if the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. other alternatives include amputation (if on a limb) or radiation therapy alone. as an adjunct to surgery, grier et al. ( grier et al. ( , found that deionized water injected into surgical margins reduced tumor recurrence by hypo-osmotically lysing any mast cells left behind. this technique has recently been refuted by jaffe et al. ( ) . for systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (graham and o'keefe, ) . research complications. because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology. hemangiosarcomas are malignant tumors that originate from endothelial cells. clinical signs. these tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. clinical signs are associated with the site of involvement. vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. heart failure can be observed secondary to tumor burden or hemopericardium. when found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from to cm in diameter. the most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. epizootiology: hemangiosarcomas occur most frequently in -to -year-old dogs. the german shepherd dog is most commonly affected. pathologic findings. grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (fig. ) . the masses are spherical and reddish black and can range in size up to - cm in diameter. on cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. when the masses are found in the heart, the endocardium may be covered by a thrombus, giving the to cm tumors a reddish gray or yellow appearance. histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. these spaces may be filled with blood or thrombi. the neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures. pathogenesis. hemangiosarcomas can be found in one or many sites. in cases where multiple sites are involved, it may be impossible to identify the primary tumor. this neoplasia is highly malignant and spreads easily. metastasis occurs most frequently to the lungs but can be found in any tissue. diagnosis and differential diagnosis. splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. when the heart is affected, other causes of heart failure must be ruled out. echocardiography is a valuable tool for identifying the primary lesion. histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors. treatment. surgery is generally the first choice of treatment for hemangiosarcoma. dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. research complications. dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. the other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting. etiology. also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (nielsen and kennedy, ) . the origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (macewen, ) . clinical signs. the tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (fig. ) . less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission. transmissible venereal tumors are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions are believed to be a result of trauma prior to exposure to the tumor. pathogenesis. tumor growth is rapid after implantation but later slows. metastasis is rare (< % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. diagnosis and differential diagnosis. transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology. prevention. thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control. removing affected individuals from a breeding program should stop further spread through the colony. treatment. surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for - treatments will induce remission and cure in greater than % of the cases (macewen, ). research complications. experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology. dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs. single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. epizootiology. mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. median age at diagnosis is - years. mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . extensive discussions of classification, staging, and histopathologic correlations can be found in macewen and withrow ( lb) and moulton ( ) . pathogenesis. mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. futher, schneider et al. ( ) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs. diagnosis and differential diagnosis. both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis. prevention. mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. this is commonly done in the general pet population at months of age. recently, the topic of spaying sexually immature dogs ( - weeks of age) has received much attention for the control of the pet population. kustritz ( ) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age. treatment. surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (macewen and withrow, lb). research complications. because % of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-tinue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. etiology. beagles are among the breeds with the highest prevalence of thyroid carcinomas. benjamin et al. ( ) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle. clinical signs. thyroid carcinomas generally present as palpable cervical masses. affected animals may experience dysphagia, dyspnea, and vocalization changes. precaval syndrome resulting in facial edema is also observed in some cases. epizootiology and transmission. the mean age of dogs presented with thyroid carcinomas is years, with equal distribution of cases between the sexes. pathologic findings. grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. they tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. the masses are unilateral twice as often as bilateral (capen, ) . histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see capen, , for complete discussion). pathogenesis. thyroid carcinomas tend to grow rapidly and invade local structures. early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein. diagnosis and differential diagnosis. nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. often a preliminary diagnosis can be made by fineneedle aspirate. treatment. surgery is the treatment of choice for thyroid carcinomas that have not metastasized. when the tumor is freely movable, surgery may be curative. surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. when bilateral tumors are observed, preservation of the parathyroid glands may not be possible. in such cases, treatment for hypoparathyroidism will be necessary. both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (ogilvie, ) . in the research setting, treatment of this tumor may not be rewarding. only freely movable tumors can be practically treated without seriously affecting research efforts. euthanasia is warranted in the more advanced cases when clinical illness is apparent. beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, b) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). in addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor viii deficiency (von willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. at a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia ( . % of births) and open fontanelle ( . % of births) (r. scipioni and j. ball, personal communication, ) . other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology. cataract is an opacification of the lens or the lens capsule. it is the pathologic response of the lens to illness or injury, because the lens has no blood supply. cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. the ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract. clinical signs. the first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (helper, ) . moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. epizootiology and transmission. certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. dogs in studies for diabetes mellitus should be observed regularly for cataract development. toxicological studies may also induce formation of cataracts. pathogenesis. lens fibers respond to all biological or chemical insults by necrosis and liquefaction (render and carlton, ) , because they have no blood supply with which to recruit an inflammatory and repair process. disruption of these fibers by any means, therefore, leads to opacification. the exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. in diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. as these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (capen, ) . diagnosis and differential diagnosis. the ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. prevention. most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. treatment. because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. many dogs adjust quite well to the visual impairment caused by persistent cataracts. lens removal can be performed for dogs seriously affected by cataracts, but this would not be anticipated for dogs in the research setting. information on surgical lens extraction procedures can be found in helper ( ) or other veterinary ophthalmology textbooks. research complications. research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. etiology. hip dysplasia is a degenerative disease of the coxofemoral joint. a specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (pedersen et al., ) , modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (smith et al., ) . clinical signs. the initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. this may present as a gait abnormality without any indication of lameness or stiffness. eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. epizootiology and transmission. hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. in the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. diagnosis and differential diagnosis. hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (pedersen et al., ) . hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. this may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. radiographic calculation of the distraction index (di) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (smith et al., ) . prevention. because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. dogs should be provided a good plane of nutrition but not be allowed to become overweight. dogs that were limit-fed at % of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (kealy et al, ) . treatment. in the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. more advanced cases may require continuous analgesia. sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (pedersen et al., ) . surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. neither surgical treatment is likely in a research setting. research complications. long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. in studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. etiology. benign prostatic hyperplasia (bph) is an agerelated condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs. bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for bph. epizootiology and transmission. bph typically affects older dogs (> years), although it has been seen as early as years of age. pathologic findings. in its early stages, canine b ph is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis. bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to a-dihydrotestosterone (kustritz and klausner, ) . diagnosis and differential diagnosis. bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement. prevention. castration is the primary means for prevention of benign prostatic hyperplasia. treatment. the first and foremost treatment for b ph is castration. in pure cases of b ph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. newer drugs marketed for human males have also shown promise in treating canine bph. finasteride (proscar) is a a-reductase inhibitor that limits metabolism of testosterone to a-dihydrotestosterone. treatment at daily doses of - mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (kustritz and klausner, ) . dogs given . mg/kg were proven to still be fertile. there are also indications that lower doses may be effective in relieving b ph. androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of bph while maintaining libido and fertility (kustritz and klausner, ) . unfortunately, both the areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the united states. research complications. bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. it is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. etiology. juvenile polyarteritis syndrome (jps) is a painful disorder seen in young beagles (occasionally reported in other breeds). the lesion consistent with the syndrome is systemic necrotizing vasculitis. the cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs. clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to extend the neck ventrally. most dogs seem to be in pain when touched, especially in the neck region. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission. jps typically affects young beagles ( - months), with no sex predilection. pathologic findings. on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. these lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (fig. a) . the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen (fig. b) . a subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis. the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in / affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis. differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). are known at this time. no prevention and control measures brane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs. the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (fig. ) . excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. treatment. clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q hr, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. pathologic findings. typically the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis. prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . research complications. because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology. "cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-prevention. hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment. corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications. in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. etiology. interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (fig. ). the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). clinical signs. dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission. interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . pathologic findings. histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis. initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis. bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment. if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg ql h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require fig. . interdigital cyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on patients treated primarily with tetracycline and/or doxycycline dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog saunders manual of small animal practice neurologic manifestations associated with hypothyroidism in four dogs neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats platelet function, antithrombin-iii activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis ehrlichia platys infection in dogs the rickettsioses monoclonal gammopathy associated with naturally occurring canine ehrlichiosis variation in age at death of dogs of different sexes and breeds leptospira interrogans serovar grippotyphosa infection in dogs efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis infection, tetracycline therapy, and challenge inoculation comparison of campylobacter carriage rates in diarrheic and healthy pet animals. zentralbl advances in dietary management of obesity in dogs and cats effect of level and source of dietary fiber on food intake in the dog effect of amount and type of dietary fiber on food intake in energy-restricted dogs external parasites: identification and control tumors of the endocrine glands thomson's special veterinary pathology infectious diseases of the dog and cat nutritional support for dogs and cats with hepatobiliary disease specific amplification of ehrlichia platys dna from blood specimens by two step pcr detection of humoral antibody to the transmissible venereal tumor of the dog dirofilaria immitis: heartworm products contract rat trachea in vitro dogs: laboratory animal management management of septicemia in rhesus monkeys with chronic indwelling catheters client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis dirofilariasis in dogs and cats use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma study of obesity in dogs visiting veterinary practices in the united kingdom miller's anatomy of the dog update on diagnosis of canine hypothyroidism helicobacter-associated gastric disease in ferrets, dogs, and cats the role of helicobacter species in newly recognized gastrointestinal tract disease of animals serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test hemorrhagic streptococcal pneumonia in newly procured research dogs control of ticks platelet aggregation studies in dogs with acute ehrlichia platys infection health benefits of animal research: the dog as a research subject soft tissue sarcomas and mast cell tumors textbook of veterinary internal medicine infectious diseases of the dog and cat canine lyme borreliosis mast cell tumor destruction by deionized water mast cell tumour destruction in dogs by hypotonic solution transmission of ehrlichia canis to dogs by ticks (rhipicephalus sanguineus) textbook of veterinary internal medicine diseases of the liver and their treatment cyclic thrombocytopenia induced by a rickettsia-like agent in dogs shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of veterinary internal medicine canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats thiacetarsamide and its adverse effects infectious diseases of the dog and cat pediatrics: puppies and kittens canine viral diseases textbook of veterinary internal medicine antibodies to ehrlichia canis, ehrlichia platys, and spotted fever group rickettsia in louisiana dogs mastocytoma and gastroduodenal ulceration complications in the use of indwelling vascular catheters in laboratory animals deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours helicobacter infection textbook of veterinary internal medicine textbook of veterinary internal medicine hygroma of the elbow in dogs thermal injuries dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? depression of endotheliumdependent relaxation by filarial parasite products three cases of canine leptospirosis in quebec cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs role of bordetella bronchiseptica in infectious tracheobronchitis in dogs kirk's current veterinary therapy : small animal practice the fire of life kirk's current veterinary therapy : small animal practice coinfection with multiple tick-borne pathogens in a walker hound kennel in north carolina tarsal joint contracture in dogs with golden retriever muscular dystrophy clinical and hematological findings in canine ehrlichiosis early spay-neuter in the dog and cat textbook of veterinary internal medicine chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs the brown dog tick rhipicephalus sanguineus and the dog as experimental hosts of ehrlicha canis the clinical chemistry of laboratory animals double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease thomson's special veterinary pathology effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs transmissible venereal tumors kirk's current veterinary therapy : small animal practice soft tissue sarcomas tumors of the mammary gland canine lymphoma and lymphoid leukemias kirk's current veterinary therapy : small animal practice effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein tick paralysis in north america and australia saunders manual of small animal practice thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think urea protects helicobacter (campylobacter) pylori from the bactericidal effect of acid characterization of a new isolate of ehrlichia platys using electron microscopy and polymerase chain reaction decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension vaccination against canine bordetellosis: protection from contact challenge dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs clinical trial of dvm derm caps in the treatment of allergic diseases in dogs: a nonblinded study diagnosis of neoplasia tumors of the mammary gland dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior survey of conjunctival flora in dogs with clinical signs of external eye disease hyperoxia exacerbates microvascular injury following acid aspiration nutrient requirements of dogs surgical closure of elbow hygroma in the dog tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases walker's mammals of the world tumors of the endocrine system beta hemolytic streptococcus isolated from the canine vagina comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs clinical behavioral medicine for small animals hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs joint diseases of dogs and cats target imbalance: disparity of borrelia burgdorferi genetic material in synovial fluid from lyme arthritis patients textbook of veterinary internal medicine tumors of the skin and soft tissue thomson's special veterinary pathology canine leptospirosis: a retrospective study of cases dogs and cats as laboratory animals effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs epidemiology of thyroid diseases of dogs and cats factors influencing canine mammary cancer development and postsurgical survival muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology textbook of veterinary internal medicine canine infectious tracheobronchitis (kennel cough complex) saunders manual of small animal practice serum protein alterations in canine erhlichiosis bacterial factors and immune pathogenesis in helicobacter pylori evaluation of rhipicephalus sanguineus as a potential biologic vector of ehrlichia platys role of the eastern chipmunk (tamias striatus) in the epizootiology of lyme borreliosis in northwestern illinois evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs pathologic features of naturally occurring juvenile polyarteritis in beagle dogs textbook of veterinary internal medicine clinical manifestations, pathogenesis, and effect of antibiotic treatment on lyme borreliosis in dogs streptococcus zooepidemicus as the cause of septicemia in racing greyhounds trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs portosystemic shunts textbook of veterinary internal medicine lumbosacral stenosis in dogs experimental respiratory disease in dogs due to bordetella bronchiseptica dirofilaria immitis: depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro thyroiditis in a group of laboratory dogs: a study of beagles of agriculture, animal and plant health inspection service thomson's special veterinary pathology a retrospective study of cases of naturally occurring canine ehrlichiosis role of canine parainfluenza virus and bortedella bronchiseptica in kennel cough management of superficial skin wounds serum concentrations of thyroxine and , , '-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs ehrlichia platys in a michigan dog ehrlichial diseases of dogs canine ehrlichiosis. miss albert, r. e., benjamin, s. a., and shukla, r. ( ). life span and cancer mortality in the beagle dog and human. key: cord- - edzmj n authors: mitruka, kiren; wheeler, robert e. title: cruise ship travel date: - - journal: travel medicine doi: . /b - - - - . - sha: doc_id: cord_uid: edzmj n nan cruise ship travel has gained tremendous popularity over the last three decades. in , . million passengers sailed on north american cruises, representing a , % percent increase since . , a fleet of cruise ships, with , beds, sailed at % capacity that same year. surveys show that the top three reasons for passenger satisfaction with cruise vacations are being pampered, having the chance to visit several locations and fine dining. in fact, the modern-day cruise ship has evolved since the early s, when passenger ships were virtually the only means of crossing the ocean and when the focus was on efficient transatlantic transportation. the speed of jet air travel changed the focus of cruise ship travel to the pleasure of the voyage itself. cruise destinations such as the caribbean and mediterranean gained popularity due to climate as well as accessibility to many ports. over time cruise ships have come to resemble floating luxury resorts. the ever-expanding cruise itineraries, which include diverse ports of call, along with a growth in the number of embarkation ports and onboard amenities, provide travelers with convenient and comfortable means to sample different parts of the world in a short amount of time. with the growing popularity of recreational cruises, gastrointestinal (gi) and respiratory disease outbreaks have posed challenges for public health officials and the cruise industry to ensure a healthy cruise environment. many communicable diseases seen on board cruise ships are also on land. however, disease exposure and transmission aboard ships may be exacerbated by the densely populated, semiclosed cruise environment, which compels international passengers and crew to share many activities and resources. , , moreover, a ship can acquire new infectious disease reservoirs while in port through contaminated food and water or infected people. environmental contamination on cruise ships and infected crew and passengers who remain on board during successive voyages may result in protracted outbreaks on cruises. , an infectious disease acquired during the voyage may incubate in people disembarking from cruise ships and result in outbreaks in the travelers' home communities, especially in closed settings (such as nursing homes). therefore, the public health significance of illness aboard cruises lies not only in possible widespread illness onboard ships, but also the spread of diseases into communities all over the world. sanitation and disease surveillance programs developed through the cooperation of cruise industry and public health agencies have led to improved detection and control of communicable diseases. due to the potential impact on health, having knowledge about shipboard morbidity and illness prevention measures as well as cruise ship sanitation practices is important for embarking passengers and their health-care providers. understanding the most frequently reported diseases on cruise ships, their source and mode of transmission, prevention measures, and available ship medical care facilities can lead to better preparedness for a healthy cruise vacation. the north american cruise industry ships carrying or more passengers are considered to be passenger ships. they include sailboats, yachts, river cruise ships, and ocean cruise ships. sailboats and yachts are best known for niche travel, such as "eco-touring." river cruises are popular for providing an informal, intimate atmosphere while traveling to places such as the nile and the amazon. ocean cruises make up the greatest portion of ship-based leisure travel, with the north american cruise industry accounting for the major part of the global ocean cruise market. the north american cruise industry consists of cruise lines that primarily market cruises to north americans, but have embarkation ports all over the world. nevertheless, north american ports, particularly those in the united states, handle the vast majority ( % and %, respectively) of all global embarkations. the north american cruise industry provides significant benefits to the u.s. economy, creating , jobs and generating $ billion in . , during the same year, . million cruise passengers embarked on cruises from u.s. ports, with % of all cruise ship passengers sailing out of ports in florida. seaports in puerto rico and canada handled % each, and those in rest of the world (primarily europe and the mediterranean) had % of global embarkations in . , the caribbean remained the top cruise destination, followed by the mediterranean, other parts of europe, alaska, and mexico. depending on the type of cruise, the duration can range from a day (e.g. gambling cruises) to several months (e.g. round-the-world cruises). the average length of a cruise is days, and approximately % of cruising passengers choose -to -day cruises. the typical -day cruise allows passengers ample time to visit - ports and explore different locales and cultures. for many travelers the ship is a destination in itself, offering luxurious accommodation, a variety of food, exciting activities, and relaxation. over the last few years, the number of u.s. embarkation ports has steadily increased, allowing for lower costs and more convenience for u.s. residents wishing to take a cruise. data reported in by the cruise line international association (clia), the north american cruise industry's main marketing organization, indicate that up to million north americans might take a cruise within years. clia also notes that % of the u.s. population has already cruised once in their lifetime, with more than % of that number having been on a cruise more than once. in fact, % of all cruise passengers are u.s. residents. compared with u.s. resident noncruise vacationers (defined by those spending nights or more away from home for leisure trips), cruisers tend to be older ( % are over the age of years), have higher income levels, and be likely to plan a vacation - months in advance, allowing time for pretravel health preparation. a typical cruise ship will have a passenger-to-crew ratio of around : . , cruise ships employ crew members from all over the world; on average, nationalities will be represented in a crew of , . the origin of crew members will depend on the cruise line and the designated occupation on the ship. crew members may stay for months on successive voyages aboard a cruise ship, carrying out specialized tasks with the aim of achieving higher quality service. the international character of today's cruise industry is manifested not only by the variety of worldwide ports and the nationalities of passengers, crew members, and company ownership, but also by the flag status, or maritime registry, of the individual cruise ships. the flag or maritime registry administration is a regulatory agency that oversees the operational procedures, ship specifications, crew qualifications, practices, and conformity to laws of commercial vessels registered in a particular country. , , cruise lines may register their ships with maritime agencies in countries such as the netherlands, norway, the united kingdom, and the united states. they often choose to register their ships in the country of ownership of the cruise line or the site of the ship's production. some cruise lines opt to register their ships in countries that provide open maritime registration or "flags of convenience." panama, liberia, and the bahamas are three such countries that allow foreign vessel owners to register their ships through their respective flag administrations. together they account for more than % of the world's merchant fleet maritime registrations, including many cruise ships. the "flag of convenience" concept originated in panama just after world war i, when u.s. merchant and passenger vessel registrations began to be transferred to panama to take advantage of favorable tax incentives and to avoid restrictive prohibition laws. , the open maritime registrations continue to provide the respective countries with a substantial revenue flow and the registrants with significant financial advantages pertaining to tax, labor, and liability costs. each flag state, whether it provides open maritime registrations or the more traditional maritime state flag status, is required to be an international maritime organization (imo) member nation and abide by its maritime safety resolutions, conventions, and codes. cruise health, sanitation, and safety regulations the concept of monitoring health aboard ships emerged during the th century plague epidemic, when "black death," arrived from the east and swept across europe. to prevent the entry and spread of disease and protect commerce, venice enforced a mandatory -day anchor and observation of ships arriving from affected regions. this practice came to be known as "quarantine," derived from the italian word for (quaranta). thereafter, other global infectious disease outbreaks, such as the cholera epidemic during the mid- s, provoked multiple international treaties and conventions to develop world standards to prevent the spread of disease across borders. however, it was not until the world health organization (who) promulgated the international sanitary regulations (isr) in that such international standards were developed. the isr, renamed the international health regulations, were revised and updated in may to address emerging public health threats. , the overall goal of the revised ihr is to provide maximum security against the international spread of disease while avoiding unnecessary interference with international travel and trade. among other things, the ihr provide world standards for ship and port sanitation, surveillance, and response for infectious diseases that can have a serious public health impact globally. under the ihr, international ports and conveyance operators are required to take measures to ensure delivery of safe food or water, appropriate waste disposal, and vector control. in addition, the who guide to ship sanitation (referenced in the ihr) provides specific health requirements for ship construction and operation, and for sanitary measures on ships. it highlights the importance of applying control measures to reduce public health risks on ships. recently, these guidelines have been revised with a systematic review of documented outbreaks and diseases of public health significance aboard ships during the last three decades. the safety of cruise ship passengers and crew members is of paramount importance to cruise lines. safety regulations for international seagoing vessels, including cruise ships, are promulgated by the imo in its international convention for safety of life at sea (solas) and international safety management (ism) code. these regulations address a variety of issues pertaining to passenger and crew safety, including fire protection, lifesaving equipment and procedures, and radio communications. the primary responsibility for monitoring for compliance with the solas standards and other internationally recognized imo conventions lies with the ship's country of maritime registry or flag state. federal and state maritime agencies at ports of call, such as the united states coast guard for ships sailing in u.s. territorial waters, are provided with the legal authority to inspect vessels to ensure compliance with imo conventions. the centers for disease control and prevention (cdc) has responsibility for ensuring appropriate levels of sanitation and health aboard cruise ships arriving at u.s. ports. in , as a result of gastrointestinal outbreaks on cruise ships, cdc established the vessel sanitation program (vsp), in cooperation with the cruise industry, to minimize the risk of gastrointestinal diseases on cruise ships and establish comprehensive sanitation programs. in accordance with u.s. federal quarantine regulations, vsp requires all ships carrying or more passengers to report, at least hours before arrival at a u.s. port, the number of cases (including zero) of acute gastroenteritis (age). cdc defines age as three or more episodes of loose stool within chapter : cruise ship travel a -hour period, or vomiting plus one or more episodes of loose stool, fever, muscle aches, bloody stool, or headache. an epidemiologic and environmental investigation may be prompted if the cruise ship reports unusual cases, or at least % of the passenger or crew report symptoms of age. vsp posts summaries of recent and previous (since ) gastrointestinal outbreaks on its web site. those include the names of the associated cruise lines and cruise ships, sailing dates, illness symptoms, the percentage of passenger and crew affected, control measures, and causative agent, if known. in addition to monitoring and investigating gastrointestinal disease aboard cruise ships, vsp develops guidelines and provides consultation to help shipbuilders and renovators meet construction standards that protect public health. that guidance extends to facilities that could affect public health, such as food storage, ventilation systems, and pools or spas. vsp also conducts unannounced, biannual sanitation inspections of u.s.-bound cruise ships that have international itineraries. the cruise line pays the fee for this inspection, based on tonnage. ships are inspected for food and water sanitation, disinfection of spas and pools, personal hygiene and practices of employees, pest control, general cleanliness, the physical condition of the ship, surveillance for acute gastrointestinal disease, and other factors. a score of at least (out of ) is necessary for a ship to pass inspection. up-to-date sanitation inspection scores of cruise ships are available on the vsp web site and are published monthly in the "summary of inspections of international cruise ships" (green sheet). in general, lower inspection scores correlate with lower sanitation standards, but cannot provide an estimate for risk of acquiring gastrointestinal disease on the ships. other than age requirements, all international passenger conveyances bound for the united states are legally required to report, at least hours before arrival, onboard cases with certain febrile syndromes suggestive of a communicable disease, as well as any deaths. these reports are received and investigated by cdc's quarantine stations, located at major u.s. ports of entry or land border crossings, and operated by the cdc division of global migration and quarantine in atlanta, georgia. under federal quarantine regulations, the cdc quarantine stations have the authority to isolate, quarantine, and conduct medical surveillance of person(s) who are infected with, or exposed to, any of the communicable diseases listed in an executive order signed by the president of the united states. this list can be updated as new public health threats emerge. diseases on the list include cholera, diphtheria, infectious tuberculosis, plague, suspected smallpox, yellow fever, and viral hemorrhagic fever. recent additions include severe acute respiratory syndrome (sars) and influenza due to novel viruses with pandemic potential. although regulations provide an important framework for cruise health and sanitation, the cooperation of the cruise industry is crucial in establishing a safe and healthy environment on cruise ships. with this goal, clia, which recently merged with the international council of cruise lines, monitors and participates in domestic and international maritime policy development, and accordingly sets compliance standards among its member cruise lines (which represent the majority of the north america cruise industry). some of the clia's requirements of its member cruise lines include measures to ensure prevention of marine pollution, appropriate work environment for the crew, waste management, and ship safety and security. clia member cruise lines follow and exceed the "health care guidelines for cruise ship medical facilities," developed by the american college of emergency physicians (acep) section on cruise ship and maritime medicine. this acep section is composed of physicians actively involved in cruise ship medicine. their objective is to advance the capabilities of cruise ship medical facilities and the quality of medical care provided aboard cruise ships. the guidelines address standards for medical facility design, medical staff qualifications, diagnostic equipment, and formulary selection, with a goal of providing general and emergency medical services to passengers and crew. medical care aboard cruise ship is designed to provide cruise line passengers and crew members with timely access to comprehensive services for minor to severe illness and injury. the international maritime requirement to have a doctor aboard ship is actually determined by the size of the crew. only vessels with or more crew members on an international voyage of days or more are required to sail with a physician. no international maritime regulations require a doctor to be aboard ship specifically for passenger medical care. the availability of doctors and nurses aboard cruise ships for passenger medical care are a result of the maritime requirements for crew medical services. in reality, the provision of services to both crew members and passengers by medical staff has become the standard within the cruise industry to meet the needs and expectations of all people onboard the vessel. , cruise ship physicians are recruited from countries worldwide. employment by a particular cruise line may be dependent upon the doctor's nationality, as per the ship's flag state regulations, or the company's own hiring preferences. all physicians must meet the qualifications for appointment as a ship's medical officer. this typically includes an unrestricted medical license, medical board certification, years of clinical experience post medical school, competence in advanced life support and minor surgical skills, and fluency in the official language of the cruise line. cruise ship nurses have comparable requirements for employment. the number of medical staff aboard any particular ship is dictated primarily by the total number of people aboard ship. this can range from a lone doctor for a small ship of people to a medical staff of two physicians and five nurses for a mega-ship carrying more than , passengers and crew members. the medical facility aboard a modern cruise ship is designed to provide essential medical services within the space constraints of an ocean-going vessel (see infirmary photo). most of the medical conditions that arise aboard ship can be treated as they would be at a doctor's office or ambulatory care center at home. the infirmary is staffed several hours a day for routine medical evaluations. the medical staff is also available hours per day to respond to medical emergencies. more serious problems (such as myocardial infarction, respiratory distress, or cerebrovascular accidents) may require emergency evacuation to a fully staffed and equipped shore-side hospital after the patient is stabilized in the ship's medical facility. accordingly, one should view a ship's medical facility as an infirmary or sickbay and not a full-service hospital. basic specifications for medical equipment and drugs from the imo and the international labour organization require a "medicine chest" that includes the "international medical guide for ships" and the "list of essential drugs" published by who. , these regulations are aimed more toward the ship's master or other "medical person in charge" on smaller vessels without a doctor onboard. in addition, flag states may regulate supplemental equipment and supplies. however, the equipment and formulary found on a modern cruise ship are much more complex and comprehensive than the basic medicine chest, the result of an evolutionary process to optimize passenger and crew medical care. the specific needs of any particular ship are based on several factors, including the ship's size, total passenger and crew count, mean age, and baseline health status of those on board, and the destination and duration of the cruise. , the cruise lines have organized their medical departments on the basis of these factors and acep and other cruise industry guidelines. most modern cruise ships are equipped to perform a variety of laboratory tests (which may include complete blood count, blood sugar, electrolytes, chemistries, cardiac enzymes, pregnancy testing, and urinalysis), radiography, cardiac monitoring, and advanced life-support procedures. the ship's formulary includes medications for treating common medical problems and a variety of more serious conditions, including infections, injuries, respiratory distress, and cardiac disorders. the spectrum of illnesses occurring aboard cruise ships is broad and can vary depending on the demographics of passengers and crew on board. two studies involving retrospective reviews of cruise ship medical logs showed that about half of all passengers seeking care aboard cruise ships were older than age years. respiratory tract infection was the most common diagnosis, followed by injuries, nervous system problems (e.g. seasickness), and gastrointestinal illness. , about % of illnesses on cruise ships were not considered serious or life threatening, but of those which were, asthma, arrhythmia, angina, and congestive heart failure were among the most common. more than % of ill persons seen by the medical clinic were treated on board. the rest required either temporary or permanent disembarkation (before completion of the cruise) for shore-side medical care. , one study estimated that on an average cruise ship voyage, a medical facility can expect to encounter a potentially serious illness or injury four times and have a patient disembark for medical reasons once. antibiotics were the most frequently prescribed prescription medication on cruise ships. the risk of exposure to an infectious disease on a cruise is hard to quantify due to limited epidemiologic data and the wide range of environmental exposures that occur during cruise travel. documented outbreaks of infectious diseases abroad cruise ships have been most commonly related to gastrointestinal (norovirus) and respiratory infections (influenza, legionella). , clusters of illnesses related to vaccine-preventable diseases (other than influenza) such as rubella and varicella have also been reported in recent years. , upper respiratory tract infections are the most frequent diagnosis in cruise ship infirmaries, accounting for approximately % of passenger visits. the semiclosed and crowded environment of cruise ships may allow for increased person-to-person transmission of respiratory viruses. in addition, ship resources such as contaminated whirlpools or water supply, and even infected crew or passengers remaining on board for multiple voyages, may serve as reservoirs for respiratory pathogens, causing continuous transmission of illness on consecutive cruises. , the two most frequently documented etiologic agents of cruise ship-related pneumonia outbreaks are legionella and influenza viruses. , legionnaires' disease cruise travel-related legionnaires' disease may occur for several reasons. first, a large percentage of cruising passengers are of advanced age and may have underlying illnesses, placing them at a higher risk for this disease than the general population. second, cruise travel may involve high risk environmental exposures, such as potable water systems or cooling towers at ports of call in tropical climates. third, the ship's complex water system may fail in design, maintenance, or disinfection, leading to bacterial growth in the water supply, air conditioning, or whirlpools. , public health investigations often have difficulty linking a case of legionnaires' disease to a particular cruise ship due to delayed case detection and multiple exposures encountered during a cruise vacation. as a result of the long incubation period ( - days) of legionnaires' disease, cruise ship-associated clusters may go undetected because passengers may not develop symptoms until they have returned home. once symptoms do develop, physicians may not suspect or confirm the diagnosis of legionnaires' disease nor associate it with cruise travel. furthermore, even if a diagnosis is made, fewer than an estimated % of legionnaires' disease cases in the united states are reported to local or state public health departments, although reporting is legally required. upon case detection, public health investigators face several challenges in pinpointing the origin of travel-related legionnaires' disease. passengers on a particular cruise ship may have been exposed to multiple pathogen reservoirs, such as hotel showers, during port stops, as well as before and after cruise travel. moreover, firm linkage to a source requires isolation of the pathogen both from the suspected environment and from clinical specimens, which may no longer be available at the time of the public health investigation. the first passenger ship-associated case of legionnaires' disease was reported in . since then approximately incidents involving about cases were documented as of . in about % of these outbreaks, the ship's infectious source was not determined or reported. the most commonly established causes of the outbreaks included contamination of ship's water supply, air conditioning system, or spa pool. , the largest documented culture-confirmed cruise ship outbreak of legionnaires' disease occurred in . it involved a total of passengers during nine separate sailings of the same ship. illness due to infection through bacteria-laden aerosols generated by the spas was associated with immersion in, and spending time around, the whirlpool. the risk increased by % for every hour spent in the spa water and % for every hour spent around the spa. the same strain of legionella pneumophila serogroup was isolated from the sand filters of the whirlpool spas and a clinical respiratory specimen, pointing to inadequate bromination of the ships' three whirlpool spas as the cause for the outbreak. no cases were discovered in the crew, most likely due to their lack of exposure to the spas and younger age. this outbreak was detected months after it began, when a new jersey physician notified the state health department that three hospitalized patients with atypical pneumonia had been on the same cruise ship, which highlights the delay in detection of cruise-associated legionnaires' disease. in another, smaller, outbreak, three cases of legionnaires' disease were detected on two separate voyages of one ship in the autumn of and . a hot water supply contaminated with l. pneumophila serogroup was found to be the outbreak source. past cruise ship outbreaks of legionnaires' disease and better knowledge about legionella ecology have led to improvements in cruise ship prevention measures: design optimization of water-containing equipment, and standardization of disinfection, as well as maintenance, of spas and water supplies reduce the risk of bacterial growth and colonization. clinicians can also play an important role in the control of cruise travel-related legionnaires' infection through rapid case detection among pneumonia patients by ▪ inquiring about travel history, including cruise travel; ▪ performing appropriate diagnostic tests (both rapid and culturebased tests); and ▪ promptly reporting cases to state and local health departments. influenza influenza a and b outbreaks among cruise ships crew and passengers can occur throughout the year, even when seasonal influenza activity is absent in the region of the cruise. , the convergence and intermingling of international crew and passengers from parts of the world where influenza is in circulation, can lead to the introduction and rapid spread of influenza aboard ships. substantial morbidity may result from cruise influenza outbreaks due to the presence of a large percentage of elderly and chronically ill passengers, both of whom are at higher risk for complications and death due to influenza infection. , in september , an influenza outbreak occurred among passengers and crew aboard a cruise ship during several sailings between new york and montreal. the associated acute respiratory illness (ari: cough and/or sore throat) affected % of the crew ( of ) and % of , surveyed passengers, % ( ) of whom were age years or older and % ( ) had chronic health conditions. investigators found that the number of persons who sought treatment for respiratory illnesses from the ship's infirmary greatly underestimated the true magnitude of the outbreak. although this cruise was in the northern hemisphere, the isolated influenza a (h n ) strain was thought to have been introduced by travelers from the southern hemisphere, where influenza was in season and the implicated strain was circulating. this outbreak was contained through surveillance, isolation of cases, vaccination of crew member, antiviral prophylaxis and treatment of both crew and passengers, and other public health control measures. a more sustained outbreak occurred among land-and sea-based tourists to alaska and the yukon during the summer of , when % of an estimated . million tourists entered the region by cruise ship. some of these passengers also participated in overland tours. tour operators and returning travelers alerted public health officials about the increased risks of acquiring ari and pneumonia. reviews of cruise ship passenger medical logs revealed ari rates two to three times the rate of . visits per , passengers. this led to intense public health surveillance at multiple sites, including local hospitals, clinics, and cruise ship infirmaries. the surveillance ultimately revealed , cases of ari, including , influenza-like illness (ari and fever of ° f or . ° c or self-reported fever) and cases of pneumonia. most cases were among tourists and tourism workers (representing and countries, respectively), and only a small percentage were among alaska residents. the isolated influenza a (h n ) virus was the predominant circulating strain during the previous influenza season in the united states and canada. it was unclear if the virus was still circulating among alaskan residents and infected susceptible tourists or if infected tourists from the southern hemisphere (where the influenza season was ongoing) introduced this outbreak. despite control measures, the outbreak lasted for months due to a large weekly influx of susceptible overland and cruise ship tourists, ending only after the completion of tourism season. during the summer of , an increase in ari again occurred among tourists to the same region, prompting year-long surveillance. it also resulted in cdc's releasing the "preliminary guidelines on prevention, surveillance and control of influenza-like illness (ili) aboard cruise ships," compliance with which is voluntary for cruise lines. influenza b outbreaks have occurred more recently. in one outbreak during the summer of , a baltic cruise from the united kingdom carrying passengers primarily from the united states had a much greater ari attack rate among the crew ( % of ) than the passengers ( % of , ) . , confirmed cases initially tested negative by rapid influenza testing (designed to detect a and b strains). the final diagnosis of influenza b was made by viral culture and immunofluorescence antibody of nasopharyngeal specimens. the index case was found to be a passenger who boarded the ship ill and remained in her cabin except for meals, passing on the infection to both her cabin and dining room stewards. other crew members became infected by socializing or sharing cabins with the ill crew members, leading to the higher attack rate among crew than among passengers. this outbreak demonstrates the potential for person-to-person transmission of the influenza virus between passenger and crew. it also highlights the importance of appropriate isolation of ill persons and use of infection control precautions in preventing and controlling outbreaks, especially in crowded and confined settings such as cruise ships. prevention of influenza outbreaks aboard cruise ships involves joint efforts from the cruise industry, public health agencies, and the traveling public. cdc guidelines suggest that cruise lines should ensure yearly influenza vaccination of at least % of crew per ship. in addition, to ensure early recognition of an outbreak, ships should conduct surveillance for ari and ili, perform appropriate rapid influenza testing, expeditiously report suspected outbreaks to public health authorities, and implement control measures such as isolation, infection control, and treatment with antiviral medications, if warranted. clinicians can also play an important role in preventing influenza and other respiratory disease outbreaks aboard ships by ▪ asking travelers to refrain from traveling while ill and if illness develops during the trip, to practice respiratory hygiene and minimize contact with other people, including the cruise staff; and ▪ providing vaccination (or rarely, antiviral medications) as prevention, especially to high-risk populations as well as their close contacts, and those traveling in large tour groups, even if travel occurs during summer. the estimated likelihood of contracting gastroenteritis aboard a -day cruise is less than %. , gastrointestinal (gi) disease accounts for fewer than % of passenger visits to the ship's infirmary. cruise outbreaks of gastroenteritis due to bacterial and viral pathogens, particularly noroviruses (nov), are well recognized. , as a matter of fact, the number of outbreaks of gastroenteritis on cruise ships has increased significantly in recent years due to nov. acute gastroenteritis (age) outbreaks per cruises increased almost tenfold from to ( . to . ). a recent who review documented shipassociated food and waterborne outbreaks from to . data from outbreaks revealed , ill persons, hospitalizations, and deaths. however, these reported outbreaks were thought to underestimate the true incidence of gastrointestinal illness aboard cruises due to underdetection of cases. ill passengers may not seek care in the ship's infirmary for fear of ruining a vacation or other impositions, and ill crew may not present themselves due to concerns of losing income during isolation or premature disembarkation. a review of data from cruise ship waterborne diseases outbreaks during the period - showed that enterotoxigenic escherichia coli (etec) and nov are the two pathogens most frequently linked to cruise ship waterborne gi outbreaks. consumption of ice from contaminated water bunkered by cruise ships at non-u.s. ports, with subsequent onboard water treatment failure have resulted in number of etec outbreaks. in addition, factors such as crosscontamination of potable water with bilge water and ice machine contamination from failure of backflow prevention devices have resulted in nov gi outbreaks. more commonly, however, nov may not be implicated in waterborne disease outbreaks because of the difficulty in detecting nov in environmental samples. moreover, nov are more resistant to chlorination than enteric bacteria and require higher levels of residual chlorine levels to prevent outbreaks. other organisms that may cause waterborne gi outbreaks on cruise ships include salmonella spp, shigella sp, cryptoporidium sp, and giardia intestinalis. however, the specific cause cannot be identified in about one quarter of outbreaks. an outbreak of brainerd diarrhea was associated with consumption of unbottled water, ice, and raw sliced fruit and vegetables aboard a cruise to the galapagos islands in . deficiencies in the ship's water handling and chlorination may have contributed to this outbreak of chronic diarrhea among passengers. cruise ships can prevent waterborne disease outbreaks by ensuring that water is obtained from safe and reliable sources at ports and stored safely; loading water properly at ports to avoid cross contamination; conducting extra disinfection treatments of water if it is suspected to be contaminated; monitoring residual disinfectants in distribution systems; and ensuring performance of regular inspections and maintenance of the ship's potable water systems. an epidemiologic review of cruise ship foodborne disease outbreaks that occurred from through showed that % were caused by bacterial pathogens and the rest by viruses, parasites, or unknown sources. salmonella spp was the most common etiologic agent of cruise ship foodborne outbreaks, causing more than one quarter of the outbreaks. other common pathogens included enterotoxigenic e. coli (etec) ( % of outbreaks), shigella spp ( %), vibrio parahaemolyticus spp ( %), and nov ( %). outbreaks have resulted more rarely from staphylococcus aureus, vibrio cholerae, clostridium perfringens, trichinella spiralis, and cyclospora spp. seafood (causing % of outbreaks) has been the most commonly implicated food vehicle, followed by salads, poultry, eggs, and red meat. however, a contributing factor could be identified in fewer than half of all outbreaks. those identified factors most frequently included inadequate temperature control, infected food handlers, and contaminated raw ingredients. cruise dining typically offers elaborate meals consisting of a large assortment of foods, which usually involve preparation by multiple food handlers and in many steps, resulting in an increased chance of food mishandling and contamination. furthermore, meals are served to a large population in a short time, likely leading to a time gap between the preparation and serving. inadequate temperature control of foods during this time can allow bacteria to grow. for example, in a s. aureus gastrointestinal illness outbreak aboard a caribbean cruise ship, the suspected food was pastries, which were believed to have been contaminated during preparation. the same strain of s. aureus was detected from several food handlers and cases. additionally, possible subsequent storage of the pastries at warm temperatures led to the production of enterotoxin, which caused illness among passengers. to reduce the risk of foodborne outbreaks cruise ships should ensure that food (especially shellfish) is thoroughly cooked, use pasteurized eggs, cater shore-side meals rather than using local sources, and exclude ill food handlers from food preparation. norovirus from to , the incidence of outbreak-related diarrheal disease cases on cruise ships decreased from . cases per , passenger days to . cases per , . this decline was associated with improved sanitation aboard ships, as vsp inspection failure rates dropped from almost % in to about % in . however, during the period to , despite good environmental health standards on cruise ships, the incidence of outbreak-related diarrheal disease rose to cases per , passenger days. in fact, the total number of reported gi outbreaks on cruise ships increased from in to in . this increase in gi outbreaks as well as the frequency of age cases on cruise ships was attributed to noroviruses (nov). outbreaks of nov typically occur during the fall and winter months. however, during the summer of , the incidence of nov outbreaks peaked with the emergence of a previously unidentified strain called "farmington hills" (genogroup ii cluster nov). this was also the predominant circulating strain on land, causing % of the outbreaks. , this strain caused gi outbreaks on as many as six consecutive cruises, despite sanitation measures. it even resulted in a nursing home outbreak linked to an infected resident who returned from cruise travel. nov are the most common cause of viral gastroenteritis on cruise ships and age in the united states, with million cases yearly. nov are transmitted by fecal-oral route, directly person to person, from contaminated food and water, or by contact with contaminated surfaces or objects. aerosolized vomit has also been suspected as a mode of transmission. during cruise nov outbreaks, the original source of infection may be an infected person or food. further spread, resulting in large numbers of illnesses, mainly occurs by person-toperson transmission of the virus. most nov outbreaks can be characterized by having high attack rates, high prevalence of vomiting, short duration of illness, and an absence of identifiable pathogen on culture. recurrences of nov cases on consecutive cruises are common due to the virus' low infective dose, combined with its persistent reservoir; cruise staff remaining onboard during multiple voyages; and a contaminated cruise environment, which is difficult to disinfect with routine sanitary measures. , , furthermore, studies to identify effective disinfectants are difficult due to the lack of a nov culture system, making efficient decontamination even more challenging for the cruise industry. the key to controlling the spread of nov on cruise ships is the rapid implementation of control measures at the first signs of an age outbreak. in fact, each cruise line has detailed operational directives, based on cdc-recommended control measures, on how to deal with a pending or actual gastrointestinal outbreak. for example, routine cleaning is conducted when age affects fewer than % of passengers or crew. if the percentage affected reaches % or more (outbreak likely or in progress) or there are six or more cases in one day, the captain is notified immediately and a decision may be made to escalate sanitation measures. some examples of cdc-recommended nov control measures include strict application of food and water sanitation measures; prompt disinfection with suitable disinfectants (e.g. chlorine solutions at concentrations of at least , ppm, phenol-based compounds, and accelerated hydrogen peroxide products); and isolation of ill passengers and crew for - hours after clinical recovery. typically, the captain gives printed instructions to the passengers about illness prevention and control measures to be followed. these may include hand washing tips and a recommendation to seek medical care in the ship's infirmary as soon as gi symptoms develop. this letter also lists sanitation measures being undertaken by the ship, such as frequent cleaning of common areas, staterooms, elevator buttons, and hand rails. in addition to influenza, cruise ships have had outbreaks of other routine vaccine-preventable diseases (vpd), such as rubella and varicella. most often, such illnesses are traced to crew originating from countries in which immunity to routine vpd may be low. for example, susceptibility rates to rubella range from % to % in countries without a routine rubella vaccination program. the densely populated environment of cruise ships and the social interactions among crew and passengers allow for person-to-person spread of vpd among susceptible persons. in the united states an estimated % of women of childbearing age are susceptible to rubella. of the pregnant women infected during the first weeks of gestation, up to % of the infants will be born with congenital rubella syndrome (crs); the rate of crs for infants born to women infected during the first weeks of pregnancy is %. hence, women of childbearing age (i.e. - years), particularly pregnant women, along with other potentially susceptible groups of cruise travelers, such as the elderly, immunosuppressed, and children, need to check their immune status to routine vpd before travel. in , the cdc investigated two outbreaks of rubella among crew members on two cruise ships sailing from florida to the bahamas. in one outbreak, ( %) of crew members tested were found to have a positive igm for rubella. of these sixteen crew members with an acute rubella infection, % were asymptomatic. an additional % of tested crew members proved to be susceptible to infection. approximately % of crew members were born outside the united states (representing more than different countries), and % had no known immunity to rubella or had a negative antibody result on testing. although no cases appeared among passengers in either outbreak, a survey of , passengers on one of the cruise ships found % to be u.s.-born, % to be of childbearing age, and . % to be pregnant (half being in the first trimester), demonstrating the susceptibility of passengers to rubella. this outbreak was controlled by administration of measles-mumps-rubella (mmr) vaccine to crew members without documented immunity to rubella, active surveillance aboard the cruise ship for new rubella cases, and notification of all passengers at risk for exposure to rubella on the cruise ship. another outbreak of varicella aboard a cruise ship in revealed similar trends in vpd risk among the crew, with % being infected or susceptible, most being foreign-born (especially from tropical countries). persons are immune once they develop varicella, so the risk of infection decreases with age. in tropical areas, however, varicella typically occurs among adults, who are at risk of developing more severe disease. special attention should be given to immunocompromised persons among susceptibles, and they who should receive specific varicella-zoster immune globulin (or intravenous immune globulin if specific ig is not available) within hours of exposure. the cdc quarantine stations receive case reports from u.s.-bound ships of other communicable diseases (mostly among crew) such as measles, mumps, tuberculosis, typhoid, and hepatitis a and b. after respiratory infection, injuries are the second most common reason for passengers to seek medical care aboard cruise ships, accounting for % of infirmary visits. the most common injuries seen are sprains, contusions, and superficial wounds. reported cruise-related injuries most frequently occur on decks and stairs, in passengers' own cabins, or ashore during port calls. most cruise itineraries tend to be in the calm waters of the caribbean or the mediterranean. modern cruise ships are also constructed with roll stabilizers, which minimize turbulence. even so, sea sickness is a common concern of many cruise travelers and is among the top four reasons for infirmary visits. some passengers are sensitive to motion and require pharmacologic prevention and treatment with antihistamines, antimuscarinic, or antidopaminergic agents. alternative medicines may also help some individuals (for more information on sea sickness, see chapter ). the association between passenger cabin location and risk of motion sickness is controversial, but common perception persists that central cabins are the least sea sickness-inducing. a recent study found that cabin location is only associated with the risk of sea sickness when the passenger is seated or standing. passengers who are able to readily lie down can reduce their risk for motion sickness irrespective of their cabin location. pre-travel health preparation for cruise travel can be challenging because of planned visits to multiple countries and participation in a variety of shipboard and shore-side activities. in making health risk assessments, health-care providers should consider a broad range of issues. these include the travelers' health condition and immunity to routine vpd including influenza, the need for special immunizations and chemoprophylaxis based on the cruise ship's itinerary, and health-related risk behaviors during cruise travel. , , (see table during pre-travel counseling, clinicians should carefully review the traveler's medical conditions to assess if the person can endure the stress of travel and whether the person has any special health needs, for example, wheelchair access, oxygen, or dialysis. , cruise travel can expose travelers to infectious agents, pollutants, changes in diet, physical exertion, extremes of weather, and other conditions that can exacerbate chronic medical conditions. however, cruise tours are available that provide care by onboard specialists, such as pulmonologists and nephrologists, for persons with certain physical impairments, such as chronic obstructive pulmonary disease (copd) and renal failure requiring hemodialysis. travelers with medical conditions should be advised to contact and make arrangements with the cruise line about their specific medical needs before departure. , cruise ships built during the past years generally have cabins designed to accommodate wheelchairs. information regarding wheelchair access can be obtained from the individual cruise lines or the clia web site. , depending on the medical condition, some cruise lines may require that the traveler have a travel companion. cruise travelers can also verify a cruise ship's sanitation level and learn about ongoing or recent gi outbreaks through the cdc's vsp web site. , medical facilities on most modern cruise ships are comparable to those of a community urgent care center. however, limitations and variability exist between cruise lines and individual ships -and at shore-side hospitals during port stops -in the level of care available. , for this reason and to protect the health of others on board, passengers with acute medical complaints or those who acquire an infectious disease before travel should be encouraged to postpone travel and call the cruise lines to discuss alternatives. regardless of age and underlying medical conditions, all passengers should be up-to-date for routine age-appropriate vaccinations (see table . ). , given previous cruise ship outbreaks of vpd such as rubella, chickenpox, and influenza, immunity to these diseases should be ensured, especially in high-risk populations (e.g. elderly, immunosuppressed, or pregnant women). , , consideration for influenza vaccination should be given, especially to travelers at high risk for complications from influenza infections and their close contacts, or those who will be traveling with a large group (any time of the year) or visiting the tropics or the southern hemisphere during april to september. however, influenza vaccine may be largely unavailable in north america during the summer months. therefore, clinicians may consider prescribing a recommended antiviral medication for treatment or prophylaxis of influenza for high-risk patients. the traveler's planned itinerary -which countries will be visited, duration of stay, and shore-side excursions and activities -provides crucial insights for determining the need for special immunizations (such as typhoid, rabies, yellow fever, japanese encephalitis, and meningococcal) and chemoprophylaxis (e.g. for malaria and influenza). if warranted by location of shore-side stays and outdoor activities, travelers should be advised to include mosquito and sun protection in their travel kit (for more information on travel kits, see chapter ). travelers, especially those with known health conditions, should carry a written summary of essential health information which would facilitate their care on board or at a shore-side hospital during a medical emergency. this personal medical information sheet (sample available in reference ) should include information on the traveler's demographics, allergies, chronic conditions, blood type, medication list, contact information of the physician and next of kin, and medical and travel insurance information. , important laboratory information, such as an ecg or chest radiograph, if abnormal, should also be attached to the medical information sheet. , all prospective cruise travelers should be strongly advised to contact their health insurance carriers in advance of travel and to consider purchase of additional insurance to cover reimbursement for medical evacuation and health services in foreign countries. , , often, gaps in regular coverage require the additional travel insurance, which can often be found in a travel package offered by cruise lines, usually costing %- % of the total package price. however, if only supplemental medical evacuation insurance is needed, the cost can be as low as $ per person for year of coverage. clinicians should remind cruise travelers to exercise health-conscious behavior during their journey. travelers should use caution in selecting the food and water they consume and should practice good hygiene (wash hands, cover coughs and sneezes, etc.) to reduce their risk of getting ill from an infectious disease (see table . ) . , passengers should ensure that food they consume is thoroughly cooked, inquire if pasteurized eggs were used for foods requiring a large number of eggs as ingredients (e.g. custards or flan), and evaluate the risks of eating food off the ship at ports. they should be encouraged to follow the rule of "boil it, peel it, cook it, or forget it." during shore-side excursions, pre-packaged foods should not be kept for long hours at unsuitable temperatures, and passengers should drink bottled water. , practicing good hand and respiratory hygiene is important in preventing illnesses that are transmitted from person to person, either by direct contact, by respiratory routes, or through contaminated environments. , after travel passengers should be urged to follow up with their health-care provider for any fever or flu-like illness that develops up to a year after travel. clinicians should inquire about cruise travel in all cases of pneumonia, other respiratory illnesses, gastrointestinal illnesses, or suspected communicable disease. appropriate diagnostic testingboth rapid and culture-based-better enables public health investigations to link an illness to a source. viral isolation (via nasopharyngeal specimens) is essential to identify new and unusual imported strains of influenza and other respiratory pathogens. , clinicians can help enhance surveillance and characterization of cruise ship-associated illnesses by identifying and reporting notifiable diseases or conditions and possible clusters of diseases to public health agencies. insect repellent (based on itinerary and shore-side activities) (same resources as above) adequate supply of all medication written personal medical information (include patient demographics, health and travel insurance, contact information of health-care provider and next of kin, medical history, current medications and pertinent lab data (ekg) routine immunizations, if not up-to-date other immunizations if indicated (based on itinerary) malaria prophylaxis if indicated (based on itinerary) influenza antiviral medication (based on risk assessment) guidance about mosquito prevention guidance about sun protection travel advice -pre-travel health preparation, during travel healthy habits, and after travel follow-up (table . ) conclusion since , the north american cruise industry has seen an average annual passenger growth rate of . %. and the popularity of cruise travel is expected to continue to grow, reaching an estimated . million cruising passengers in . the occurrence of shipboard illnesses and outbreaks related to gastrointestinal, respiratory, and vaccine-preventable diseases has led to improved infectious diseases surveillance and control strategies by the cruise industry and public health agencies. , , pre-travel health preparations and knowledge of available medical care aboard cruise ships are important for cruise travelers, especially given that approximately one third are senior citizens who may have chronic illnesses or who may be at greater risk for some infectious diseases. because medical facilities aboard cruise ships are designed to provide basic emergency medical care, travelers should be encouraged to consult their health insurance provider regarding extra coverage while away from their home country and for medical evacuation. during travel (while on cruise and on-shore): to prevent getting food-and water-borne diseases ensure all food consumed is thoroughly cooked inquire about use of pasteurized eggs for foods with eggs as main ingredient (e.g. flan, omelets) evaluate the risks of eating any food, especially off the ship ensure correct temperature of cold and hot foods ensure pre-packaged food for shore-side excursions is stored at appropriate temperature to prevent spread of germs follow good hand hygiene: wash hands frequently with soap and water if soap and water not available, alcohol-based disposable wipes or gel sanitizers containing at least % alcohol may be used follow good respiratory hygiene: cover mouth and nose with tissue when coughing or sneezing if tissue not available, cough or sneeze into upper sleeve, not hands put used tissue in waste basket avoid close contact with people who are sick be sure to report illness to cruise staff if they are unaware stay well hydrated by drinking water get plenty of rest avoid excessive alcohol intake report to doctor for illnesses especially with fever or respiratory or symptoms new york: cruise lines international association (us) london: passenger shipping association brief history of the passenger ship history travelers' health: yellow book. health information for international travelers available from: www .ncid.cdc.gov/travel/yb/utils/ybget. asp?section=transportation&obj=cruise.htm&cssnav=browsecyb infectious diseases on cruise ships cruise ships: high-risk passengers and the global spread of new influenza viruses outbreaks of acute gastroenteritis on cruise ships and on land: identification of a predominant circulating strain of norovirus-united states norovirus transmission on cruise ship outbreak of legionnaires' disease among cruise ship passengers exposed to a contaminated whirlpool spa outbreaks of gastrointestinal diseases on cruise ships: lessons from three decades of progress international convention for the safety of life at sea (solas) available from: www.imo.org./home.asp?flash=false the contribution of the north american cruise industry to the us economy in business research and economic advisors for international council of cruise lines (us) the cruise industry economic summary cruise lines international association (former international council of cruise lines) (us); [date unknown travel health at sea: cruise ship medicine cdc vessel sanitation program regulation, representation and the flag market top merchant fleets of the world, flag registry united states department of transportation maritime administration investigation and management of infectious diseases on international conveyances (airplanes and cruise ships) international infectious disease law-revision of the world health organization's international health regulations sanitation on ships: compendium of outbreaks of foodborne and waterborne disease and legionnaire's disease associated with ships cruise industry source book- edition virginia): cruise lines international association national center for environmental health. vessel sanitation program atlanta: centers for disease control and prevention, department of health and human services (us) health care guidelines for cruise ship medical facilities american college of emergency physicians, section on cruise ship and maritime medicine ilolex: database of international labour standards ilo convention (no. ): health protection and medical care for seafarers evaluation of emergency air evacuation of critically ill patients from cruise ships descriptive epidemiology of injury and illness among cruise ship passengers anatomy of a world cruise sea, wind and pneumonia legionellosis associated with ships: to . comm dis public health legionnaire's disease on a cruise ship linked to the water supply system: clinical and public health implications a large outbreak of influenza a and b on a cruise ship causing widespread morbidity large summertime influenza a outbreak among tourists in alaska and the yukon territory preliminary guidelines for the prevention and control of influenza-like illness among passengers and crew members on cruise ships influenza b virus outbreak on a cruise ship-northern europe epidemiology of gastroenteritis on cruise ships epidemiology of diarrheal disease outbreaks on cruise ships a review of outbreaks of waterborne disease associated with ships: evidence for risk management traveler's diarrhea at sea: three outbreaks of waterborne enterotoxigenic escherichia coli on cruise ships an outbreak of brainerd diarrhea among travelers to the galapagos islands a review of outbreaks of foodborne disease associated with passenger ships: evidence for risk management staphylococcal food poisoning on a cruise ship the vessel sanitation program inspection team. diarrheal disease on cruise ships current trends vessel sanitation scores. mmwr outbreaks of gastroenteritis associated with noroviruses on cruise ships-united states epidemiologic notes and reports gastroenteritis outbreaks on two cruise ships. mmwr dealing with gastrointestinal illness on a cruise ship. part : description of sanitation measures. part : an isolation study rubella among crew members of commercial cruise ships-florida, . mmwr control and prevention of rubella: evaluation and management of suspected outbreaks, rubella in pregnant women, and surveillance for congenital rubella infecton effect of climatic factors and population density on varicella zoster virus epidemiology within a tropical country division of global migration and quarantine, national center for preparedness, detection, and control of infectious disease cabin location and the likelihood of motion sickness in cruise ship passengers atlanta: centers for disease control and prevention cruise ship accommodations for passengers with physical limitations due to disability or age new york: cruise lines international association (us) prevention and control of influenza. recommendations of the advisory committee on immunization practices. mmwr keep health needs in mind atlanta: centers for disease control and prevention, department of health and human services stopping the spread of germs at home, work & school [online]. atlanta: centers for disease control and prevention, department of health and human services key: cord- -hprwqi n authors: löscher, thomas; prüfer-krämer, luise title: emerging and re-emerging infectious diseases date: - - journal: modern infectious disease epidemiology doi: . / - - - - _ sha: doc_id: cord_uid: hprwqi n emerging infectious diseases (eids) are characterized by a new or an increased occurrence within the last few decades. they include the following categories emerging diagnosis of infectious diseases: old diseases that are newly classified as infectious diseases because of the discovery of a responsible infectious agent. europe including great britain as well as in india, china, and japan. emerging vector-borne disease events concentrated in densely populated subtropical and tropical regions mostly in india, indonesia, china, sub-saharan africa, and central america (see figs. . , . , and . ). the identification of new infectious agents in old diseases with unknown etiology is still the basis in many epidemiological studies. such newly detected bacteria and viruses in the last few decades are listed in table . . since the detection of helicobacter pylori in , this infection has been identified as the causative agent in % of b-gastritis cases. the risk of duodenal ulcer is increased by - -fold in patients with helicobacter-associated gastritis. who declared h. pylori as a carcinogen of first order because of its potential to enhance the risk of stomach carcinoma and malt lymphoma in long-term infection. in highprevalence regions for h. pylori, the frequency of stomach carcinoma is significantly higher compared to low-endemic areas (correa et al. ). the identification of h. pylori facilitates curative treatment of most associated diseases in individuals. but the most important epidemiological effect on associated diseases is attributed to increased hygienic standards in industrialized countries with a substantial reduction of h. pylori prevalences in younger age cohorts. transmission of h. pylori occurs mainly in childhood. in western developed countries the overall prevalence is around %, higher in older age groups due to a cohort effect, and this increases with low socioeconomic status (rothenbacher et al. ). in countries with low hygienic standards the prevalences are still high in younger age groups and reach % in developing countries. in developed countries, migrant subpopulations from less-developed regions show significantly higher prevalences in comparison to the nonmigrant population (mégraud ). since the early th century, a characteristic expanding skin lesion, erythema migrans (em), and an arthritis associated with previous tick bites were known. borrelia for many decades. increased outdoor activities facilitated contacts between humans and ticks in the s and the s and increased transmission of borrelia to humans at the northeastern coast of north america, leading to the discovery of borrelia burgdorferi in by willy burgdorfer. three different stages of the disease that describe the stage of infection and the involvement of different organ systems are known: stage , early localized infection; stage , early but disseminated infection; and stage , late stage with persistent infection. lyme disease is endemic at the east coast and in minnesota in the united states, in eastern and central europe, and russia. seroprevalence rates that reflect about % of nonclinical infections vary between and % in the general population in germany (hassler et al. ; weiland et al. ) . in high-risk groups like forest workers in germany the prevalences reach - % (robert koch institute a). in ticks (ixodes) the prevalences are between and % depending on the geographical area and the testing method used [immunofluorescence test, ift and polymerase chain reaction (pcr)]. in most studies the main risk factors of infection are age (children: - years, adults - years), outdoor activities, skin contacts with bushes and grass, and the presence of ticks in domestic animals (robert koch institute b) . the probability of infection (seroconversion) after a tick bite in germany is - % and the probability of a clinical disease is . - . %. the probability that the bite of an infectious tick leads to infection in the host is - %. this depends on the time duration that the tick is feeding on the human body. since the detection of the etiologic infectious agent and the subsequent development of laboratory diagnostic tests in the s, the number of reported cases of lyme disease has increased from to , per year, indicating that it is an "emerging diagnosis." the reported numbers vary depending on the reproduction of the hosting rodents for ticks as well as the contacts between humans and nature (spach et al. ) . ticks may live for several years and their survival, reproduction rate, and activity are directly affected by changes in seasonal climate through induced changes in vegetation zones and biodiversity, hence causing local alterations of the tick's habitat and in the occurrence of animals that are carriers of different pathogens (like small rodents). several studies in europe have shown that in recent decades the tick ixodes ricinus, transmitting lyme borreliosis and tick-borne encephalitis (tbe), has spread into higher latitudes (e.g., sweden) and altitudes (e.g., czech republic, austria), and has become more abundant in many places. such variations have been shown to be associated with recent variations in climate. as a result, new risk areas of both diseases have recently been reported from the czech republic. climate change in europe seems likely to facilitate the spread of lyme borreliosis and tbe into higher latitudes and altitudes, and to contribute to extended and more intense transmission seasons. currently, the most effective adaptive strategies available are tbe vaccination of risk populations and preventive information to the general public (danielova et al. ; lindgren et al. ; materna et al. ). an effective vaccine was licensed for b. burgdorferi in . in europe, where different variants of borrelia are present (mostly b. afzelii and b. garinii), this vaccine is not protective. trivalent vaccines for europe are in clinical trials. in recent years, norovirus infections are increasingly recognized as the cause of large outbreaks of diarrheal diseases in the general population, school classes, nursing homes, hospitals, and cruise ships in western countries with peaks in colder seasons (winter epidemics) (centers of disease control ; verhoef et al. ; robert koch institute a) . this is a typical example for emerging diagnosis due to increasing availability of routine pcr testing for these viruses in stool samples. noroviruses (family caliciviridae) are a group of related, single-stranded rna viruses first described in an outbreak of gastroenteritis in a school at norwalk, ohio, in . five genogroups are known. immunity seems to be strain specific and lasts only for limited periods, so individuals are likely to get the infection repeatedly throughout their life. it is estimated that noroviruses are the cause of about % of all food-borne outbreaks of gastroenteritis. for several years there has been an ongoing epidemic in several european countries due to drift variants of a new genotype (gg ii. jamboree) previously unknown to this nonimmune population (robert koch institute a). as a result of an analysis of outbreaks in the united states between and , direct contamination of food by a food handler was the most common cause ( %), person-to-person transmission was less prevalent ( %), and even less frequently waterborne transmission could be proved ( %) (centers for disease control ). vomiting is a frequent symptom of norovirus enteritis and may result in infectious droplets or aerosols causing airborne or contact transmission. this may explain the difficulty to stop outbreaks in hospitals, nursing homes, and similar settings despite precautions to prevent fecal-oral transmission. also on cruise ships, person-to-person transmission is most likely in those closed settings, and drinking tap water is a risk factor as well (verhoef et al. ). searching for an agent which causes large outbreaks of enterically transmitted non-a hepatitis in asia and other parts of the world, the hepatitis e virus (hev) was first described in and cloned and sequenced in (reyes et al. ). meanwhile, hev has been shown to be a zoonotic virus circulating in pigs and other animals. it is implicated in about % of sporadic cases of acute hepatitis in developing countries and associated with a high case fatality rate in the third trimester of pregnancy ( - %). hev is a major cause of large epidemics in asia, and to a lesser extent in africa and latin america, typically promoted through postmonsoon flooding with contamination of drinking water by human and animal feces. recent data show hev also to circulate in european countries and to be associated with severe and fatal disease not only during pregnancy but also in the elderly and in patients with chronic liver conditions. in patients with solid organ transplants, hev may even cause chronic hepatitis and liver cirrhosis (kamar et al. ) . a recombinant hev vaccine candidate has demonstrated a high protection rate of approximately % during clinical trials in nepal (shrestha et al. ). for years, specific human papillomaviruses have been linked to certain human cancers and have been identified as causative agents of malignant proliferations. in the s the detection of papillomavirus dna from cervical carcinoma biopsies were published, showing that hpv types and are the most frequent (dürst et al. ; boshart et al. ) . the relation of hpv infections and cancer is further discussed in chapter . definition: only infections that are newly discovered in humans are listed in this chapter: hiv, new variant of creutzfeldt-jakob disease (vcjd), hemorrhagic uremic syndrome (hus) caused by enterohemorrhagic escherichia coli, viral hemorrhagic fevers like hanta, lassa, ebola, and marburg fever, nipah virus encephalitis, monkeypox, human ehrlichiosis, severe acute respiratory syndrome (coronavirus infection, sars), and avian influenza (h n ) (see fig. . and table . ). these infections mostly have their origin in zoonotic wildlife (e.g., avian influenza, monkeypox, hantavirus, nipah virus, and filoviruses) or livestock (e.g., vcjd). factors promoting the spread of these infections in humans are contacts with wildlife, mass food production of animal origin, and globalization (migration, transportation of goods and vectors) (see fig. . ). in addition, new strains or variants of well-known pathogens have emerged showing increased or altered virulence such as clostridium difficile ribotype or staphylococcus aureus strains expressing the panton-valentine leukocidin (see also chapter ). the epidemiology of hiv is treated in chapter and that of avian influenza and new influenza h n in chapter . in the year , years after the peak of the bse epidemic in the united kingdom, with an annual incidence rate in cows of . per million bovines aged over months, the first mortalities in humans with a new variant of creutzfeldt-jakob disease were observed in the united kingdom. until , smaller incidence rates of bse cases had been reported by other european countries in indigenous bovines and up to more than , per million in in ireland. from , bse started to increase in switzerland and portugal, from in spain and in recent years has spread to eastern european countries (organisation mondiale de la santé animale ). the infectious agent is a self-replicating protein, a "prion." the source of infection for cows is infectious animal flour. the transmission to humans occurs through oral intake of cow products, most likely undercooked meat and nerval tissues as well as transplants of cornea, dura mater, contaminated surgical instruments, or the treatment with hypophyseal hormones extracted from animal tissues. after a statuary ban on the feeding of protein derived from ruminants to any ruminant and the export ban of all cow products from england, the epidemic of bse in cows and the occurrence of human infections decreased in the united kingdom since . by june the total number of deaths in definite/probable cases of vcjd in the united kingdom was (the national creutzfeldt-jakob disease surveillance unit ). only a few numbers of vcjd were reported from other european countries and the united states (who ). nipah virus encephalitis was first observed in / in malaysia. the disease was transmitted by pigs to laborers in slaughterhouses and showed a lethality of %. the infectious agent was detected in (chua et al. ; lam and chua ) . since then, several outbreaks of nipah virus infections have been observed in asian countries: singapore in , india , and bangladesh since (who a harit et al. ) . the virus has been isolated repeatedly from various species of fruit bats, which seem to be the natural reservoir (yob et al. ). west nile is a mosquito-borne flavivirus that was first isolated from a woman with a febrile illness in uganda in . from the s, west nile fever endemicity and epidemics started being reported from africa and the middle east. severe neurological symptoms were thought to be rare. more recent epidemics in northern africa, eastern europe, and russia suggested a higher prevalence of meningoencephalitis with case fatality rates of - %. in , west nile virus was identified as the cause of an epidemic of encephalitis at the east coast of the united states (nash et al. ) . a seroepidemiological household-based survey showed that the first outbreak consisted of about , infections of which about , developed fever and less than % experienced neurological disease ). since then, epidemics occur during summer months in north america each year, with an estimated , febrile illnesses and over , encephalitis or meningitis cases in the united states in (centers for disease control ). age above years is the main risk factor for developing severe disease. the virus is transmitted mainly by culex mosquitoes, but also by sandflies, ceratopogonids, and ticks, with birds as reservoir hosts and incidental hosts such as cats, dogs, and horses. efforts are made to reduce the transmitting mosquito population and to prevent mosquito bites through personal protection as well as to prevent transmission through blood donations by screening (centers of disease control ). the first case of sars occurred in guangdong (china) in november of , leading to an outbreak with cases in china and hong kong ( cases worldwide) until july . the case fatality rate was . %. a new coronavirus (sars-cov) was identified as the causative agent (drosten et al. ) , being transmitted first by infected semidomesticated animals such as the palm civet and subsequently from human to human. some cases were exported to other countries, causing smaller outbreaks there, canada being the most affected country outside asia with cases, before control of transmission was effective. eight thousand and ninety-six cases were reported worldwide, until july , then further transmission stopped (besides one more case of laboratory transmission in ), indicating an efficient international cooperation in disease control (who b) . recently, sars-cov has been found in horseshoe bats, which seem to be the natural reservoir of the virus. about , - , cases of hemorrhagic fever with renal syndrome (hfrs) caused by hantaviruses are reported annually worldwide, with more than half in china, many from russia and korea, and numerous cases from japan, finland, sweden, bulgaria, greece, hungary, france, and the balkan with different death rates depending on the responsible virus, ranging from . % in puumala to - % in hantaan infections (schmaljohn and hjelle ) . hantaviruses are transmitted from rodent to rodent through body fluids and excreta. only occasionally do humans get infected. different types of hantaviruses are circulating in europe and the eastern hemisphere, predominantly puumala virus, dobrava virus, and tula virus, adapted to different mouse species. depending on the virus type the case fatality rate is between and %. as an example, the annual rate of reported cases in germany was about cases per year from onward. this started to change in with reported cases and rose dramatically to cases in . that year, hantavirus infections were among the five most reported viral infections in germany. reasons for the rise in human infections were an increase in the hosting rodent population due to a very mild winter / and an early start of warm temperatures in spring which led to favorable nutritional situations for the mice influencing their population dynamics. in addition, favorable climatic conditions enhanced the outdoor behaviors of humans facilitating transmission in rural areas (robert koch institute b; hofmann et al. ) . since , a previously unknown group of hantaviruses (sin nombre, new york, black creek canal, bayou-in the united states and canada; andes, in south america) emerged in the americas as a cause of hantavirus pulmonary syndrome (hps), an acute respiratory disease with high case fatality rates (approx. %), causing a new, significant public health concern. a total of cases had been reported until march in states, most of them in the western part of the united states (centers for disease control ). lassa virus was detected for the first time in during an outbreak affecting nurses in a missionary hospital in lassa, nigeria. however, the disease had previously been described in the s. lassa virus is enzootic in a common peridomestic rodent in west africa, the multimammate rat mastomys natalensis, which is chronically infected and sheds the virus in urine and saliva. human infection through direct or indirect contact with rats or their excretions is rather common in some west african countries and estimates from seroepidemiological and clinical studies suggest that there are several hundred thousand cases annually. however, only a minority of infections seems to progress to severe hemorrhagic disease with a case fatality rate of - % in hospitalized cases. the virus can be transmitted by close person-to-person contact and nosocomial spread has been observed under poor hygienic conditions. marburg and ebola viruses, which were first detected during outbreaks in and , respectively, have so far been observed only during several limited outbreaks and a few isolated cases in certain countries of sub-saharan africa. however, very high case fatality rates ( - %), the occurrence of outbreaks that were difficult to control in resource-poor settings, and the obscure origin of these viruses have attracted considerable public interest worldwide. recently, evidence was found for both marburg and ebola viruses to occur in certain species of bats that probably constitute the natural reservoir of these filoviruses (towner et al. ). although the disease burden of these viral hemorrhagic fevers is low, they gained considerable international attention due to -their high case fatality rates, -the risk of person-to-person transmission, -several imported cases to industrialized countries, and -fears of abuse of these agents for bioterrorism. as a consequence, considerable resources have been invested, even in nonendemic countries, in the setting up of task forces and high containment facilities for both laboratory diagnostic services and treatment of patients using barrier nursing. this highly virulent strain of c. difficile expresses both cytotoxins a and b and, in addition, the binary toxin cdt, an adp-ribosyltransferase. due to a deletion in the regulatory tcdc gene, the synthesis rates of toxin a and b are increased by -and -fold, respectively. this strain was detected in for the first time in pittsburgh, usa. since then it has spread to canada, and in it reached europe causing multiple outbreaks in hospitals and nursing homes (warny et al. ) . c. difficile -associated colitis has shown high case fatality rates ( - %) and an increased relapse rate. containment of outbreaks in hospitals and other institutions necessitates isolation of patients or cohorts and strict hygienic measures. during recent decades, a large variety of well known infectious diseases has shown regional or global re-emergence with considerable public health relevance (table . ). globally, tuberculosis is probably the most important re-emerging infectious disease. in developing countries, tb infection still is extremely common and, in the wake of the hiv pandemic, the percentage of those developing overt disease has increased dramatically. worldwide, tb is the most common opportunistic infection in patients with aids. the significance of tb and hiv/tb coinfection is reviewed in chapters and . the re-emergence of some infectious diseases is closely related to the lack or the breakdown of basic infrastructures as seen in periurban slums and in refugee camps in developing countries, or as a consequence of war, breakdown of the civil society, or natural or man-made disasters. cholera is a formidable example for both re-emergence and epidemic spread under those conditions. another important group of re-emerging infectious diseases is caused by various vector-borne infections, such as malaria, dengue fever, and yellow fever. these major vector-borne diseases are treated in more detail in chapter . in addition, there are a variety of re-emerging infections transmitted by arthropod vectors such as various arboviral diseases and some protozoal diseases other than malaria (i.e., leishmaniasis, human african trypanosomiasis). the reasons for the emergence of several vector-borne diseases are rather variable and may range from climatic factors (e.g., global warming, rainfall), lack or breakdown of control, to changes in agriculture and farming and in human behavior (e.g., outdoor activities). these factors are usually quite specific for each of these diseases and largely depend on the specific ecology of the agent, its vectors, and reservoirs. cholera, an acute diarrheal infection transmitted by fecally contaminated water and food, had been endemic for centuries in the ganges and brahmaputra deltas in the th century before it started to spread to the rest of the world. since , six pandemics caused by the classical biotype of vibrio cholerae were recorded that killed millions of people across europe, africa, and the americas. it has been a major driving force for the improvement of sanitation and safe water supply. the seventh pandemic was caused by the el tor biotype, first isolated from pilgrims at the el tor quarantine station in sinai in . it started in in south asia, reached africa in , and is still ongoing. after more than hundred years, cholera spread to the americas in , and beginning in peru, a large epidemic hit numerous latin american countries with . million cases and more than , fatalities reported within years. out of the serogroups of v. cholerae, only o and o can cause epidemics. the serogroup o , first identified in bangladesh in , possesses the same virulence factors as o and creates a similar clinical picture. currently, the presence of o has been detected only in southeast and east asia, but it is still unclear whether v. cholerae o will extend to other regions. since , the re-emergence of cholera has been noted in parallel with the everincreasing size of vulnerable populations living in unsanitary conditions. cholera remains a global threat to public health and one of the key indicators of social development. while the disease is no longer an issue in countries where minimum hygiene standards are met, it remains a threat in almost every developing country. the number of cholera cases reported to the who during rose dramatically, reaching the level of the late s. a total of , cases were notified from countries, including , deaths, an overall increase of % compared with the number of cases reported in . this increased number of cases is the result of several major outbreaks that occurred in countries where cases had not been reported for several years such as sudan and angola. it is estimated that only a small proportion of cases -less than % -are reported. the true burden of disease is therefore grossly underestimated. the absence or the shortage of safe water and sufficient sanitation combined with a generally poor environmental status are the main causes of spread of the disease. typical at-risk areas include periurban slums where basic infrastructure is not available, as well as camps for internally displaced people or refugees where minimum requirements of clean water and sanitation are not met. however, it is important to stress that the belief that cholera epidemics are caused by dead bodies after disasters, whether natural or manmade, is false. on the other hand, the consequences of a disaster-such as disruption of water and sanitation systems or massive displacement of population to inadequate and overcrowded camps-will increase the risk of transmission. chikungunya virus, an arbovirus belonging to the alphavirus group, is transmitted by various mosquitoes. the virus was first isolated in tanzania in and since then has caused smaller epidemics in sub-saharan africa and parts of asia with low public health impact. in , the largest epidemic ever recorded started in east africa, spread to réunion and some other islands of the indian ocean, and then spread further to asia, with more than . million cases in india alone so far. characteristics of the disease are high fever and a debilitating polyarthritis, mainly of the small joints that can persist for months in some patients. now, for the first time, severe and fatal cases have been observed that may be due to certain mutations of the epidemic strain (parola et al. ) . the asian tiger mosquito aedes albopictus has proved to be an extremely effective vector in recent epidemics causing high transmission rates in big cities and leading to epidemics with high public health impact. this southeast asian mosquito species has been shipped by transport of used tires and plants harboring water contaminated with larvae to other continents and, since , ae. albopictus has successfully spread in italy and other parts of southern europe. in august , an outbreak of chikungunya fever occurred in northern italy with more than confirmed cases. the index case was a visitor from india who fell ill while visiting relatives in one of the villages and further transmission was facilitated by an abundant mosquito population during that time, as a consequence of seasonal synchronicity (rezza et al. ). ross river virus (rrv) is another arbovirus of the alphavirus group that causes an acute disease with or without fever and/or rash. most patients experience arthritis or arthralgia primarily affecting the wrist, knee, ankle, and small joints of the extremities (epidemic polyarthritis). about one-quarter of patients have rheumatic symptoms that persist for up to a year. the disease can cause incapacity and inability to work for months. it is the most common arboviral disease in australia with an average of almost , notified cases per year. rrv is transmitted by various mosquito species and circulates in a primary mosquito-mammal cycle involving kangaroos, wallabies, bats, and rodents. a human-mosquito cycle may be present in explosive outbreaks which occur irregularly during the summer months in australia and parts of oceania. heavy rainfalls as well as increasing travel and outdoor activities are considered as important factors contributing to the emergence of rrv epidemics. this flavivirus is transmitted by certain culex mosquitoes and is a leading cause of viral encephalitis in asia with , - , clinical cases reported annually. it occurs from the islands of the western pacific in the east to the pakistani border in the west, and from korea in the north to papua new guinea in the south. only in - infections will lead to encephalitis, which is, however, often severe with fatality rates of - % and with a high incidence of neurological sequelae. despite the availability of effective vaccines, je causes large epidemics and has spread to new areas during recent decades (e.g., india, sri lanka, pakistan, torres strait islands, and isolated cases in northern australia). je is particularly common in areas where flooded rice fields attract water fowl and other birds as the natural reservoir and provide abundant breeding sites for mosquitoes such as culex tritaeniorhynchus, which transmit the virus to humans. pigs act as important amplifying hosts, and therefore je distribution is very significantly linked to irrigated rice production combined with pig rearing. because of the critical role of pigs, je presence in muslim countries is low. crimean-congo virus is a bunyavirus causing an acute febrile disease often with extensive hepatitis resulting in jaundice in some cases. about one-quarter of patients present hemorrhages that can be severe. fatality rates of . - % have been reported in hospitalized patients. cchf is transmitted by hyalomma ticks to a wide range of domestic and wild animals including birds. human infection is acquired by tick bites or crushing infected ticks, and also by contact with blood or tissue from infected animals that usually do not become ill but do develop viremia. in addition, nosocomial transmission is possible and is usually related to extensive blood exposure or needle sticks. human cases have been reported from more than countries in africa, asia, southeastern europe, and the middle east. in recent years, an increase in the number of cases during tick seasons has been observed in several countries such as russia, south africa, kosovo, and greece. in turkey, where before no human cchf cases had been observed, a total of , confirmed cases, including deaths, were reported between and june . the emergence of cchf has been associated with factors such as climatic features (temperature, humidity, etc.), changes of vector population, geographical conditions, flora, wildlife, and the animal husbandry sector. rvf is a mosquito-borne bunyavirus infection occurring in many parts of sub-saharan africa. it infects primarily sheep, cattle, and goats, and is maintained in nature by transovarial transmission in floodwater aedes mosquitoes. it has been shown that infected eggs remain dormant in the dambos (i.e., depressions) of east africa and hatch after heavy rains and initiate mosquito-livestock-mosquito transmission giving rise to large epizootics. remote sensing via satellite can predict the likelihood of rvf transmission by detecting both the ecological changes associated with heavy rainfall and the depressions from which the floodwater mosquitoes emerge. transmission to humans is also possible from direct and aerosol exposure to blood and amniotic fluids of livestock. most human infections manifest themselves as uncomplicated febrile illness, but severe hemorrhagic disease, encephalitis, or retinal vasculitis is possible. in , rvf has been transported, probably by infected camels to egypt, where it caused major epidemics with several hundred thousand infections of humans. it has been suggested that introduction of rvf may be a risk to other potentially receptive areas such as parts of asia and the americas. floods occurring during the el niño phenomenon of in east africa subsequently gave rise to large epidemics and further spread to the arabian peninsula. most recent epidemics occurred in and following heavy rainfalls in kenya, somalia, and sudan, causing several hundred deaths. besides mosquito control, epidemics are best prevented by vaccination of livestock. leishmaniasis, a protozoal transmitted by sandflies, has shown a sharp increase in the number of recorded cases and spread to new endemic regions over the last decade. presently, countries are affected with an estimated million cases worldwide. there are about . million new cases of cutaneous and mucocutaneous leishmaniasis, a nonfatal but debilitating disease with % of cases occurring in afghanistan, brazil, bolivia, iran, peru, saudi arabia, and syria. the incidence of visceral leishmaniasis (vl), a disease with a high fatality rate when untreated, is estimated at around , per year. the situation is further aggravated by emerging drug resistance (table . ) and the deadly synergy of vl/hiv coinfection. epidemics usually affect the poorest part of the population and have occurred recently in bangladesh, brazil, india, nepal, and sudan. for many years, the public health impact of the leishmaniases has been grossly underestimated. they seriously hamper socioeconomic progress and epidemics have significantly delayed the implementation of numerous development programs. the spread of leishmaniasis is associated with factors favoring the vector such as deforestation, building of dams, new irrigation schemes, and climate changes, but also with urbanization, migration of nonimmune people to endemic areas, poverty, malnutrition, and the breakdown of public health. antimicrobial resistance of epidemiological relevance has emerged as a major problem in the treatment of many infectious diseases (table . ). resistance is no longer a problem that predominantly affects the chemotherapy of bacterial infections. it became increasingly important in parasitic and fungal diseases, and despite the short history of antiviral chemotherapy, it already plays a prominent role in the treatment of hiv infection and other viral diseases. resistance is also a problem in some of the emerging infections and will further complicate their treatment and control. resistance of bacterial pathogens has become a common feature in nosocomial infections, especially in the icu and in surgical wards. currently, the number one problem in most hospitals is s. aureus resistant to methicillin (mrsa, see chapter ). however, common problems of resistance also extend to other major bacterial pathogens such as enterococci, various gram-negative enteric bacilli, and pseudomonas species. resistance has developed not only to standard antibiotics (e.g., penicillins, cephalosporins, aminoglycosides, macrolides, or quinolones) but also to second-line antibiotics including carbapenems, glycopeptides, and newer quinolones. however, there is considerable geographic variation. in , the european antimicrobial resistance surveillance system (earss), a network of national surveillance systems, reported vancomycin-resistant rates among enterococci ranging from none in iceland, norway, romania, bulgaria, denmark, and hungary to % of enterococcus faecium strains in greece (earss ) . a surveillance study conducted in the united states hospitals from to showed that % of nosocomial bloodstream infections were caused by enterococci and that % of e. faecalis isolates and % of e. faecium isolates were vancomycin resistant (wisplinghof et al. ) . rates and spectrum of antibacterial resistance of e. coli and other gram-negative enteric bacilli may differ considerably from one hospital to the other. in some important pathogens of hospital-related infections such as klebsiella, enterobacter, and pseudomonas species, resistance to almost all available antimicrobials has been observed. this may complicate the choice of an effective initial chemotherapy considerably. therefore, each hospital has to monitor the epidemiological situation of resistance regularly, at least for the most important bacteria causing nosocomial infections, such as staphylococci, enterococci, gram-negative enteric bacilli, and pseudomonas. even in community-acquired infections, there has been a considerable increase in resistance problems. at present, approximately % of pneumococcal isolates in the united states are resistant to penicillin, and % exhibit intermediate resistance. the rate of resistance is lower in countries that, by tradition, are conservative in their antibiotic use (e.g., netherlands, germany) and higher in countries where use is more liberal (e.g., france). in hong kong and korea, resistance rates approach %. in addition, about one-quarter of all pneumococcal isolates in the united states are resistant to macrolides. this rate is even higher in strains highly resistant to penicillin, and increasingly there is multiresistance against other antibiotics such as cephalosporins. the prevalence of meningococci with reduced susceptibility to penicillin has been increasing, and high-level resistance has been reported in some countries (e.g., spain, united kingdom). although high-dose penicillin is effective in infections with strains of intermediate resistance, most national and international guidelines recommend broad-spectrum cephalosporins such as ceftriaxone as first-line drugs. however, in most developing countries, penicillin and chloramphenicol are the only affordable drugs. in recent years, certain strains of community-acquired s. aureus with resistance to methicillin (cmrsa) have been observed which produce a toxin (panton-valentine leukocidin) that is cytolytic to pmns, macrophages, and monocytes, and which are an emerging cause of community-acquired cases and outbreaks of necrotic lesions involving the skin or the mucosa, and in some patients also of necrotic hemorrhagic pneumonia with a high case fatality (vandenesch et al. ) . development of resistance is mainly determined by two factors: -the genetic potential of a certain pathogen, i.e., mobile elements such as plasmids, transposons, or bacteriophages, genes coding for resistance, and mutation rate. -the selection pressure caused by the therapeutic or the para-therapeutic application of antimicrobial drugs. in the hospital these factors are supported by -microbial strains that are highly adapted to this environment (e.g., rapid colonization of patients, resistance to disinfectants), -an increasing percentage of patients who are highly susceptible to infections due to old age, multimorbidity, immunosuppression, extended surgery, and invasive procedures, and -the frequent use of broad-spectrum antibiotics or combinations of antimicrobial drugs. another source of resistant bacteria has been identified in mass animal production and the use of antimicrobials as growth promoters (e.g., the glycopeptide avoparcin, the streptogramin virginiamycin) or as mass treatment in the therapy or the prevention of infections. the inadequate use of antimicrobial drugs is also an important factor responsible for the development of resistance in community-acquired infections. this is especially true in developing countries where only a limited spectrum of antibiotics is available, where shortage of drugs often leads to treatment that is underdosed or too short, and where uncontrolled sale and use of antibiotics is commonplace. as a consequence, resistance of gonococci is extremely frequent in southeast asia, and resistance of salmonella typhi, shigella, and campylobacter to standard antibiotics is common. some of the still effective second-line antibiotics have to be given parenterally or are not available because they are too costly. a typical example of the consequences of insufficient chemotherapy due to lack of compliance and/or unavailability of drugs is the alarming increase in multiresistance and extreme resistance in tb (see chapter ). resistance is also a problem in parasitic diseases such as malaria (see chapter ), leishmaniasis, or african trypanosomiasis. plasmodium falciparum developed resistance against all major antimalarial drugs as soon as they were used on a broad scale. resistance had contributed significantly to the increase in malaria-associated morbidity and mortality observed in many endemic areas (wongsichranalai et al. ) . a recent report on failures of the new artemisinin combination treatment for p. falciparum malaria at the thai-cambodian border supports fears of the development of resistance to this most promising class of drug at present (dondrop et al. ). resistance against antiviral drugs has developed almost from the beginning of antiviral chemotherapy (table . ). in the treatment of hiv infection, the risk of development of resistance has been drastically reduced by the combination of several drugs with different mechanisms of action (see chapter ). however, drug resistance remains the achilles' heel of the highly active antiretroviral therapy (haart) and may be at a considerable risk of expanding haart to the developing world. today, we have to realize that as we develop antimicrobial drugs, microbes will develop strategies of counterattack. antimicrobial resistance occurs at an alarming rate among all classes of pathogens. even in rich countries it causes real clinical problems in managing infections that were easily treatable just a few years ago. in life-threatening infections such as sepsis, nosocomial infections, or falciparum malaria, there is a substantial risk that the initial chemotherapy might not be effective. in addition, the delay caused by inadequate treatment might favor transmission to other people and support the spread of resistant pathogens (e.g., multiresistant tb). last but not the least, surveillance and control and the necessity to use expensive second-line drugs or combinations of antimicrobials are enormous cost factors. for developing countries this is a major limitation in the treatment and control of infections caused by resistant agents. so, in many ways, emerging resistance contributes to the emergence of infectious diseases. despite the availability of effective strategies for treatment and prevention, infectious diseases have remained a major cause of morbidity and mortality worldwide. however, the problems associated with infections are due to considerable changes. in industrialized countries the mortality caused by infectious diseases has decreased tremendously during more than years. however, during recent years, both mortality and morbidity associated with infections are increasing again. ironically, this is closely associated with the advances in medicine which have contributed to profound changes in the spectrum of both patients and their infections. advanced age, underlying conditions, and an altered immune response are common features in the seriously infected hospital patient today. immunosuppressive therapy is frequently used to treat neoplastic and inflammatory diseases or to prevent the rejection of transplants. some infections, most notably hiv/aids, cause immunosuppression by itself. in the compromised patient, infections are generally more severe or may be caused by opportunistic pathogens that will not harm the immunocompetent host. antimicrobial treatment is often less effective in these patients and tends to be further complicated by antimicrobial resistance which may manifest itself or develop at a higher frequency in the immunocompromised patient. an increasing percentage of infections are hospital acquired or otherwise health care associated. it is estimated that nosocomial infections affect . million patients and contribute to approximately , deaths in us hospitals annually (klevens et al. ). considering the rising number of elderly and immunocompromised patients, a further increase in severe infections can be predicted. in developing countries, the significance of infectious diseases has remained high for ages and despite the advances in medicine. until now, infections are by far the leading cause of both disability-adjusted life years and life years lost. the reasons are obvious and mostly related to poverty and lack of development causing poor and unhealthy living conditions, inadequate health systems, and lack of resources for prevention and treatment. this is, of course, just an integral part of the general socioeconomic problems of developing countries. however, poor health conditions per se are an important obstacle to development, and infections such as hiv/aids in sub-saharan africa can be a major cause of lack of development, increasing poverty, and political instability. generally, the situation of many developing countries has not improved during the last two decades, and the gap between the first and the third world has increased. however, most of the mortality and morbidity associated with infectious diseases is avoidable. as laid down in the millennium goals, a major task of the world community will be to counteract the imbalance between the industrialized and the developing countries and to find strategies to ensure participation in the progress of modern medicine for all. developing countries also carry the main burden of diseases caused by newly emerging and re-emerging infections (table . and . ) . however, the consequences of economical and political crises on emerging infectious diseases are obvious in industrialized countries also-such as the return of diphtheria or the increase in tb and multiresistant tb after the breakdown of the former soviet union. today, all countries worldwide are affected by emerging infections as well as by emerging antimicrobial resistance. in the age of globalization, travel and transport of people, animals, and goods of all kinds have increased tremendously. as a consequence, infectious agents may travel over long distances and at high speed. this is clearly evident with influenza pandemics or outbreaks such as the sars epidemic or with imported cases of viral hemorrhagic fever transmissible from person to person. the spread of antimicrobial resistance or the re-emergence of tb seems to be less spectacular, but the consequences may be at least as important in the long run. management and control of emerging and re-emerging infectious diseases can be very different from disease to disease and has to allow for all relevant factors of the populations at risk and of the specific disease including the ecology of the agent, its vectors, and reservoirs. however, some basic principles apply to all situations: -surveillance -information and communication -preemptive planning and preparedness -provision and implementation of • adequate treatment • adequate control and prevention -international cooperation active and passive surveillance systems with rapid reporting and analysis of data are essential for the early detection of outbreaks, changes in epidemiology, and other events of public health concern (see chapters and ). however, many resourcepoor countries do not have functional surveillance systems. in addition, reporting of infectious diseases may be neglected or delayed because of fears of stigma, international sanctions including trade and travel restrictions, or interference with tourism. classical examples are plague and cholera, but also recent examples such as the bse/vcjd crisis in the united kingdom or sars originating from china showed undue delays between first occurrence of cases and information to the public. although, in outbreaks of new and unknown diseases it may be difficult, or even impossible, to predict or assess the magnitude of the problem and the potential consequences, timely and adequate information and communication is not only obligatory, according to international regulations, but also the best strategy to avoid rumors, misbeliefs, panic, or disregard. in recent years, many countries have installed national plans of action for important epidemiological scenarios and outbreaks such as pandemic influenza, bioterrorism, import of viral hemorrhagic fevers transmissible from person to person, sars, and comparable diseases or outbreaks. all member states of the world health assembly that have so far not been able to install functional surveillance and/or pre-emptive planning are obliged to do so within a maximum of years after their ratification of the new international health regulations (who ) . preparedness not only means surveillance and planning but also has to include the provision of facilities to adequately treat and, if necessary, to isolate patients with infectious diseases of public health importance and relevant epidemic potential and/or at risk of transmission to other persons including health-care workers. task forces and high containment facilities for both laboratory diagnostic services and treatment of patients using barrier nursing have been set up in several countries. however, all health facilities of a certain level such as general hospitals should be prepared by their organization and structure to treat patients with infections of public health relevance such as multiresistant tb under appropriate isolation and barrier nursing conditions. this also applies to hospitals in resource-poor countries. adequate training of health-care workers and strict management have been effective to control outbreaks of highly contagious infections within rural african hospitals lacking sophisticated technical equipments (cdc ) . strategies for control and prevention may be quite different for various emerging infections. effective vaccinations are available only for some infections and are usually lacking for newly emerging infections (table . ). for the majority of emerging infections, control and prevention have to rely on information, education and exposure prophylaxis, interruption of transmission by vector control and control of reservoir hosts (e.g., rodents), and case finding with early diagnosis and treatment. for diseases and outbreaks caused by infections of public health relevance that are transmissible from person to person, containment procedures including isolation and treatment of patients under condition of barrier nursing as well as tracking and surveillance of contacts are warranted by national and international health regulations. here, international cooperation is essential to successfully contain outbreaks and epidemics such as the sars epidemic in . despite dramatic progress in their treatment and prevention, infectious diseases are still of enormous global significance with tremendous economic and political implications. emerging and re-emerging infectious diseases as well as emerging antimicrobial resistance are major challenges to all countries worldwide. for the management of current and future problems, it will be most important to counteract the imbalance between the industrialized world, new economies, and developing countries, and to adequately and timely react to new threats on a global scale. a new type of papillomavirus dna, its presence in genital cancer and in cell lines derived from genital cancer world health organization: infection control for viral haemorrhagic fevers in the african health care setting nipah virus: a recently emergent deadly paramyxovirus helicobacter and gastric carcinoma. serum antibody prevalence in populations with contrasting cancer risks effects of climate change on the incidence of tick-borne encephalitis in the czech republic in the past two decades artemisinin resistance in plasmodium falciparum malaria identification of a novel coronavirus in patients with severe acute respiratory syndrome a papillomavirus dann from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions susceptibility results for e. faecium isolates lyme-borreliose in einem europäischen endemiegebiet: antikörperprävalenz und klinisches spektrum hantavirus outbreak global trends in emerging infectious diseases hepatitis e virus and chronic hepatitis in organtransplant recipients estimating health care-associated infections and deaths in u.s. hospitals nipah virus encephalitis outbreak in malaysia lyme borreliosis in europe: influences of climate and climate change, epidemiology, ecology and adaptation measures. who regional office for europe altitudinal distribution limit of the tick ixodes ricinus shifted considerably towards higher altitudes in central europe: results of three years monitoring in the krkonose mts epidemiology of helicobacter pylori infection : results of a household-based seroepidemiological survey outbreak of west nile virus infection novel chikungunya virus variant in travelers returning from indian ocean islands isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis infection with chikungunya virus in italy: an outbreak in a temperate region waldarbeiter-studie berlin-brandenburg zu zeckenübertragenen und andere zoonosen risikofaktoren für lyme-borreliose: ergebnisse einer studie in einem brandenburger landkreis übertrifft die infektionszahlen der vorjahre zahl der hantavirus-erkrankungen erreichte in deutschland einen neuen höchststand prevalence and determinants of helicobacter pylori infection in preschool children: a population-based study from germany hantaviruses: a global disease problem safety and efficacy of a recombinant hepatitis e vaccine tick-borne diseases in the united states the national creutzfeld-jakob disease surveillance unit (ncjdsu) marburg virus infection detected in a common african bat community-acquired methicillin-resistant staphylococcus aureus carrying panton-valentine leukocidin genes: worldwide emergence multiple exposures during a norovirus outbreak on a river-cruise sailing through europe toxin production by an emerging strain of clostridium difficile associated with outbreaks of severe disease in north america and europe prevalence of borrelia burgdorferi antibodies in hamburg blood donors nipah virus outbreaks in bangladesh revision of the international health regulations nosocomial bloodstream infections in us hospitals: analysis of cases from a prospective nationwide surveillance study large outbreak of norovirus: the baker who should have known better epidemiology of drugresistant malaria nipah virus infection in bats (order chiroptera) in peninsular malaysia key: cord- - glqvzfx authors: funkhouser, william k. title: pathology: the clinical description of human disease date: - - journal: essential concepts in molecular pathology doi: . /b - - - - . - sha: doc_id: cord_uid: glqvzfx nan radiographic examination, fluid tests (blood, urine, sputum, stool), and possibly tissue biopsy. radiographically, abnormalities in abundance, density or chemical microenvironment of tissues allows distinction from surrounding normal tissues. traditionally, the absorption of electromagnetic waves by tissues led to summation differences in exposure of silver salt photographic film. tomographic approaches such as ct ( ) and nmr ( ) complemented summation radiology, allowing finely detailed visualization of internal anatomy in any plane of section. in the same era, ultrasound allowed visualization of tissue with density differences, such as a developing fetus or gallbladder stones. more recently, physiology of neoplasms can be screened with positron emission tomography (pet, ) for decay of short half-life isotopes such as fluorodeoxyglucose. neoplasms with high metabolism can be distinguished physiologically from adjacent low-metabolism tissues, and can be localized with respect to normal tissues by pairing pet with standard ct. the result is an astonishingly useful means of identifying and localizing new space-occupying masses, assigning a risk for malignant behavior and, if malignant, screening for metastases in distant sites. this technique is revolutionizing the preoperative decisionmaking of clinical teams, and improves the likelihood that patients undergo resections of new mass lesions only when at risk for morbidity from malignant behavior or interference with normal function. pathologically, disease is diagnosed by determining whether the morphologic features match the set of diagnostic criteria previously described for each disease. multivolume texts are devoted to the gross and microscopic diagnostic criteria used for diagnosis, prognosis, and prediction of response to therapy. pathologists diagnose disease by generating a differential diagnosis, then finding the best fit for the clinical presentation, the radiographic appearance, and the pathologic (both clinical lab and morphologic) findings. logically, the venn diagram of the clinical, radiologic, and pathologic differential diagnoses should overlap. unexpected features expand the differential diagnosis and may raise the possibility of previously undescribed diseases. for example, legionnaire's disease, human immunodeficiency virus (hiv), hantavirus pneumonia, and severe acute respiratory syndrome (sars) are examples of newly described diagnoses during the last years. the mental construct of etiology (cause), pathogenesis (progression), natural history (clinical outcome), and response to therapy is the standard approach for pathologists thinking about a disease. a disease may have one or more etiologies (initial causes, including toxins, mutagens, drugs, allergens, trauma, or genetic mutations). a disease is expected to follow a particular series of events in its development (pathogenesis), and to follow a particular clinical course (natural history). disease can result in a temporary or lasting change in normal function, including patient death. multiple diseases of different etiologies can affect a single organ, for example, infectious and neoplastic diseases involving the lung. different diseases can derive from a single etiology, for example, emphysema, chronic bronchitis, and small cell lung carcinoma in long-term smokers. the same disease (for instance, emphysema of the lung) can derive from different etiologies (emphysema from a- -antitrypsin deficiency or cigarette smoke). modern diagnostic pathology practice hinges on morphologic diagnosis, supplemented by histochemical stains, immunohistochemical stains, cytogenetics, and clinical laboratory findings, as well as the clinical and radiographic findings. sections that meet all of these criteria are diagnostic for the disease. if some, but not all, of the criteria are present to make a definitive diagnosis, the pathologist must either equivocate or make an alternate diagnosis. thus, a firm grasp of the diagnostic criteria and the instincts to rapidly create and sort through the differential diagnosis must be possessed by the diagnostic pathologist. the pathologic diagnosis has to make sense, not only from the morphologic perspective, but from the clinical and radiographic vantage points as well. it is both legally risky and professionally erosive to make a clinically and pathologically impossible diagnosis. in the recent past, limited computer networking meant numerous phone calls to gather the relevant clinical and radiographic information to make an informed morphologic diagnosis. for example, certain diseases such as squamous and small cell carcinomas of the lung are extremely rare in nonsmokers. thus, a small cell carcinoma in the lung of a nonsmoker merits screening for a nonpulmonary primary site. fortunately for pathologists, computing and networking technologies now allow us access to preoperative clinical workups, radiographs/reports, clinical laboratory data, and prior pathology reports. all of these data protect pathologists by providing them with the relevant clinical and radiographic information, and protect patients by improving diagnostic accuracy. just as research scientists ". . . ignore the literature at their peril. . .", diagnostic pathologists ". . . ignore the presentation, past history, workup, prior biopsies, and radiographs at their peril. . . ." there are limitations to morphologic diagnosis by h&e stains. first, lineage of certain classes of neoplasms (including small round blue cell tumors, clear cell neoplasms, spindle cell neoplasms, and undifferentiated malignant neoplasms) is usually clarified by immunohistochemistry, frequently by cytogenetics (when performed), and sometimes by electron microscopy. second, there are limitations inherent in a snapshot biopsy or resection. thus, the etiology and pathogenesis can be obscure or indeterminate, and rates of growth, invasion, or timing of metastasis cannot be inferred. third, the morphologic changes may not be specific for the underlying molecular abnormalities, particularly the rate-limiting (therapeutic target) step in the pathogenesis of a neoplasm. for example, ret gain of function mutations in a medullary thyroid carcinoma will require dna level screening to determine germline involvement, familial risk, and presence or absence of a therapeutic target. fourth, the same morphologic appearance may be identical for two different diseases, each of which would be treated differently. for example, there is no morphologic evidence by h&e stain alone to distinguish host lymphoid response to hepatitis c viral (hcv) antigens from host lymphoid response to allo-hla antigens in a liver allograft. this is obviously a major diagnostic challenge when the transplant was done for hcv-related cirrhosis, and the probability of recurrent hcv infection in the liver allograft is high. paraffin section immunohistochemistry has proven invaluable in neoplasm diagnosis for clarifying lineage, improving diagnostic accuracy, and guiding customized therapy. if neoplasms are poorly differentiated or undifferentiated, the lineage of the neoplasm may not be clear. for example, sheets of undifferentiated malignant neoplasm with prominent nucleoli could represent carcinoma, lymphoma, or melanoma. to clarify lineage, a panel of immunostains is performed for proteins that are expressed in some of the neoplasms, but not in others. relative probabilities are then used to lend support (rule in) or exclude (rule out) particular diagnoses in the differential diagnosis of these several morphologically similar undifferentiated neoplasms. the second role is to make critical distinctions in diagnosis that cannot be accurately made by h&e alone. examples of this would include demonstration of myoepithelial cell loss in invasive breast carcinoma but not in its mimic, sclerosing adenosis ( figure . ), or loss of basal cells in invasive prostate carcinoma (figure . ). the third role of immunohistochemistry is to identify particular proteins, such as nuclear estrogen receptor (er) (figure . ) or the plasma membrane her proteins (figure . ) , both of which can be targeted with inhibitors rather than generalized systemic chemotherapy. morphology remains the gold standard in this diagnostic process, such that immunohistochemical data support or fail to support the h&e findings, not vice versa. probability and statistics are regular considerations in immunohistochemical interpretation, since very few antigens are tissue-specific or lineage-specific. cytokeratin is positive in carcinomas, but also in synovial and epithelioid sarcomas. this example may imply aspects of the lineage of these two sarcomas that may be helpful in our categorization of these neoplasms. another example would be the diagnosis of small cell carcinoma in the lung of a nonsmoker. because lung primary small cell carcinoma is extremely uncommon, in non-smokers, this diagnosis would prompt the pathologist to inquire about screening results for other, nonpulmonary, primary sites. likewise, immunohistochemistry results are always put into the context of the morphologic, clinical, and radiographic findings. for example, an undifferentiated cd (þ) neoplasm of the testis supports embryonal carcinoma primary in the testis, whereas a lymph node effaced by sclerotic bands with admixed cd (þ) reed-sternberg cells supports nodular sclerosing hodgkin's disease. a wealth of information is conveyed to a service pathologist in a tried-and-true h&e section. analogous to the fact that a plain chest x-ray is the sum total of all densities in the beam path, the morphologic changes in diseased cells and tissues are the morphologic sum total of all of the disequilibria in the abnormal cells. for most neoplastic diseases, morphologic criteria are sufficient to predict the risk of invasion and metastasis (the malignant potential), the pattern of metastases, and the likely clinical outcomes. for example, the etiology and pathogenesis in small cell lung carcinoma can be inferred (cigarette smoking, with carcinogeninduced genetic mutations) and the outcome predicted (early metastasis to regional nodes and distant organs, with high probability of death within years of diagnosis). new molecular data for both neoplastic and non-neoplastic diseases will most likely benefit unaffected individuals by estimating disease risk, and will most likely benefit patients by defining the molecular subset for morphologically defined diagnostic entities, thus guiding individualized therapy. diagnostic pathology will continue to use morphology and complementary data from protein (immunohistochemical) and nucleic acid (cytogenetics, in situ hybridization, dna sequence, and rna abundance) screening assays. new data will be integrated into the diagnostic process by reducing the cost and turnaround time of current technologies, and by development of new technologies, some of which are described. for transplant candidates, major histocompatibility complex (mhc, hla in human) screening is evolving from cellular assays and serology toward sequencing of the alleles of the class i and ii hla loci. rapid sequencing of these alleles in newborn cord blood would allow databasing of the population's haplotypes, facilitating perfect matches for required bone marrow or solid organ transplants. current uses of in situ hybridization to screen for viruses (such as ebv), light chain restriction (in b lymphomas), and copy number variation (for instance, her gene amplification) demonstrate the benefit of in situ nucleic acid hybridization assays. it is possible that interphase fish/cish will become rapid enough to be used in the initial diagnostic workup of certain patients, including for sarcomaspecific translocations, ploidy analysis in hydatidiform moles, and gene amplification of receptor tyrosine kinase genes. current uses of nucleic acid screening for bcr-abl translocation, donor:recipient ratios after bone marrow transplant, microsatellite instability, quantitative viral load (for ebv, bk, cmv, and others), and single gene mutations (for cftr, factor , a- -antitrypsin) demonstrate the benefit of nucleic acid screening in diagnosis and management. it is possible that each new neoplasm will be promptly defined as to ploidy, translocations, gene copy number differences, dna mutations, and rna expression cluster subset, allowing residual disease screening as well as individualized therapy. current uses of morphology, immunohistochemistry, and molecular pathology demonstrate their benefit through improved diagnostic accuracy. however, diagnosis, extent of disease, and molecular subsets are currently imperfect estimators of prognosis and response to therapy. relational databases which correlate an individual's demographic data, family history, concurrent diseases, morphologic features, immunophenotype, and molecular subset, and which integrate disease prevalence by age, sex, and ethnicity using bayesian probabilities, should improve accuracy of prognosis and prediction of response to therapy. as risk correlates are developed, it is possible that healthy individuals will be screened and given risk estimates for development of different diseases. current uses of normal ranges for serum chemistry assumes a similar bell-curve distribution across ages, sexes, and races. this may be true for most but not all analytes. computer reference databases will likely generate normal ranges specific for the particular age/sex/ethnicity of individual patients. similarly, familial risk for an inherited disease may vary by ethnic group, and this variation should be used in bayesian calculations to define risk for unaffected at-risk family members. current uses of liver and renal impairment to guide drug dosage demonstrate the benefit of using patient physiology to customize therapy. it is likely that individual differences in enzymatic metabolism of particular drugs (for instance, warfarin or tamoxifen) will be defined at the enzyme sequence level, and that gene haplotype data will be determined for new patients prior to receipt of these drugs. current uses of prostate-specific antigen (psa) to screen for prostate carcinoma and its recurrence demonstrates the benefit of serum biomarkers in common neoplasms. it is likely that high-sensitivity screening of single and clustered serum analytes will lead to improved methods for early detection and persistence of neoplasms, autoimmune diseases, and infections. pathologists consider each disease to have a natural, mechanical, physicochemical basis. each disease has an etiology (initial cause), a pathogenesis (stepwise progression), and a natural history with effects on normal function (clinical outcome). pathologists collect the data needed to answer patients' and clinicians' questions, simply phrased as "what is it?" (diagnosis), "how it going to behave?" (prognosis), and "how do i treat it?" (prediction of response to therapy). instincts and diagnostic criteria, as well as the optical, mechanical, chemical, and computing technologies described previously, are the basis for modern service pathology. as the human genome is deciphered, and as the complex interactions of cellular biochemistry are refined, risk of disease in unaffected individuals will be calculable, disease diagnosis will be increasingly accurate and prognostic, and molecular subsets of morphologically defined disease entities will be used to guide customized therapy for individual patients. it is a great time in history to be a pathologist. n clinically, diseases present to front-line physicians as patients with sets of signs and symptoms. symptoms are the patient's complaints of perceived abnormalities. signs are detected by examination of the patient. the clinical team (including the pathologist) evaluate the patient based on the possible causes of the signs and symptoms (the differential diagnosis). n pathologically, disease is diagnosed by determining whether the morphologic features match the set of diagnostic criteria previously described for each disease. pathologists diagnose disease by generating a differential diagnosis, then finding the best fit for the clinical presentation, the radiographic appearance, and the pathologic (both clinical lab and morphologic) findings. n etiology describes the causes of a disease. one disease entity can have more than one etiology, and a single etiology can lead to more than one disease. for example, emphysema, chronic bronchitis, and small cell lung carcinoma can all occur in longterm smokers (different diseases derived from a single etiology). likewise, the same disease (for instance, emphysema of the lung) can derive from different etiologies (emphysema from a- -antitrypsin deficiency or cigarette smoke). n the pathogenesis of a disease describes its stepwise progression after initiation in response to a specific etiologic factor (or factors). pathogenesis can refer to the changes in the structure or function of an organism at the gross/clinical level, and it can refer to the stepwise molecular abnormalities leading to changes in cellular and tissue function. n the natural history of a disease describes the expected course of disease, including chronicity, functional impairment, and survival. however, not all patients with a given disease will naturally follow the same disease course, so differences in patient outcome do not necessarily correspond to incorrect diagnosis. variables that independently correlate with clinical outcome differences are called independent prognostic variables, and are assessed routinely in an effort to predict the natural history of the disease in the patient. n likewise, variables that independently correlate with response to therapy are called independent predictive variables, and are assessed routinely in an effort to optimize therapeutic response for each patient. localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody general nature of the genetic code for proteins an immunoglobulin heavy chain variable region gene is generated from three segments of dna: vh, d and jh in search of the origins of modern surgical pathology formation and detection of rna-dna hybrid molecules in cytological preparations expression in escherichia coli of chemically synthesized genes for human insulin contagiousness of puerperal fever berliner klinische wochenschrift continuous cultures of fused cells secreting antibody of predefined specificity initial sequencing and analysis of the human genome the immunology of transplantation specific enzymatic amplification of dna in vitro: the polymerase chain reaction the rna code and protein synthesis ignac semmelweis and the etiology of fetal and neonatal sepsis product differentiation by analysis of dna melting curves during the polymerase chain reaction the continuing role of morphology in the molecular age dna sequencing with chainterminating inhibitors fluorescence detection in automated dna sequence analysis rudolf virchow-father of cellular pathology the sequence of the human genome molecular structure of nucleic acids; a structure for deoxyribose nucleic acid key: cord- -pz htccp authors: kohn, dennis f.; barthold, stephen w. title: biology and diseases of rats date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: pz htccp nan the diversity of research for which the laboratory rat is used is probably greater than that associated with any other animal. the laboratory rat is a descendent of the wild rat, rattus norvégiens, which originated in asia and reached europe in the early s. wild and albino mutants were first used for ex perimental purposes in europe in the mid- s and in the united states shortly before . the wistar institute in phil adelphia was prominent in the development of the rat as a labo ratory animal, for here originated many of the rat strains now used worldwide. henry donaldson and his colleagues at the wistar institute used these early rat strains for a variety of stud ies dealing with neuroanatomy, nutrition, endocrinology, ge netics, and behavior. the history and evolution of the many rat strains used today have been recently summarized (lindsey, ) . the most commonly used outbred rat stocks in north amer ica are the wistar, sprague-dawley, long-evans, and holtzman. all are albino except the long-evans stock, which is usually marked with a black or gray hair coat over the shoul ders and is sometimes referred to as a "hooded rat." there are numerous inbred and mutant rat strains, although the number is less than that in the mouse. table i lists the more commonly used strains. there are a rather large number of commercial vendors of laboratory rats in the united states. most of the stocks and strains mentioned above can be obtained from more than one source. although the origin of an outbred stock, such as the sprague-dawley, may have been the same for a number of vendors, in many cases it has been to years since such a stock has been removed from its original breeding colony. ac cordingly, the genotype of outbred stocks and inbred strains may vary among sources and be reflected by differences in data when multiple sources of rats are used. a standardized scheme of identifying stocks and strains of rats has been devel oped and is now used by nearly all commercial vendors. more over, it is important that authors correctly identify stocks and strains that are used in their research since the success in re peating the work in another laboratory may be dependent upon the genotype (source of the rat). table ii summarizes the stan dardized nomenclature for outbred stocks as developed by the table i commonly used strains "national institutes of health ( ) . . letters preceding the colon designate the supplier/breeder code consist ing of a capital and two or three lowercase letters . capital letters following the colon are used by a breeder to identify his stock . letters in parentheses denote origin of stock . subscript symbols indicate rearing by means other than natural mother (f, fostered; fh, fostered by hand) international committee on laboratory animals (icla). table iii contains the scheme for designating inbred strains of rats (national institutes of health, ) . "animals for re search" (national academy of sciences, ), a directory of sources for laboratory animals sold in the united states and canada, lists all rodents according to standard nomenclature, and is a valuable aid in purchasing laboratory animals. commercial production of rats has markedly changed since the s due to the development of hysterectomy-derived and barrier-maintained breeding colonies. prior to the application of this technology to production colonies, infectious diseases were ubiquitous in rats from most sources. today, vendors can be selected who offer pathogen-defined animals for most stocks and strains. concomitant with changes in commercial sources of rats are the major advances made in the design and construction of institutional animal resources and husbandry practices within them. optimum housing of rats today includes provisions for quarantining and isolation of animals according to vendor subpopulations that have a similar microbial flora. there are various levels of sophistication to provide barriers to the spread of infections in rat colonies. since many rat pathogens are spread by aerosol, ventilation control is very important. nonrecirculating room air or high-efficiency panic ulate air (hepa)-filtered air has become a design standard in modern animal facilities. as discussed in chapter , clean/contaminated corridor-designed facilities aid in contain ment against the spread of pathogens by aerosol, personnel, table iii nomenclature for inbred rats . the strain designation is given in capital letters followed by a slash . the substrain designation follows the slash and is given as numbers or as individual or company codes. numbers are used to denote substrains that were derived from a common strain but separated before the completion of inbreeding . subscript symbols indicate rearing by means other than natural mother and contaminated equipment. a more complete barrier system may include an entry area in which incoming supplies and equipment are sterilized and in which personnel shower and don sterile clothing and filter masks before entering animal rooms. more recently, laminar-flow (mass air displacement) rooms and mobile units have become popular because they can be incorporated in existing buildings that lack design charac teristics mentioned above. environmental control in rat rooms is important to the com fort and health of the animals, as well as to the consistency of data derived from the rats. room temperatures between and °f are desirable, and the relative humidity should range be tween and %. daily fluctuations in temperature and hu midity act as significant stressors. these fluctuations may be associated with the environmental control system of a building or may be induced by procedures such as cleaning floors with a water hose or high pressure sprayer. twenty-four-hour tem perature/humidity recorders are useful in detecting changes in environmental conditions. light intensity should be evenly distributed to all animals within a room. seventy-five to fc have often been suggested as an optimal range for light inten sity. however, recent evidence indicates that this intensity can induce retinal degeneration in albino rats (anver and cohen, ) . light-timing devices are a convenient means to provide desired day/night cycles such as - or - hr. caution should be exercised in the use of insecticides and air-deodorizing chemicals, since some have been shown to in duce hepatic microsomal enzymes in rats. accordingly, their use in animal rooms is not usually recommended (baker et al., a) . rats can be housed in either wire-or solid-bottom cages. wire-bottom cages are more frequently used since they are less labor-intensive. frequency of cage and litter pan changing is a function of animal density. solid-bottom cages should be sani tized two to three times per week, while wire-bottom cages should be sanitized on a weekly or biweekly schedule with litter pans changed two or three times per week. feed should be contained in hoppers. either automatic systems or bottles are satisfactory for providing water to rats. some caution is necessary when using automatic systems, since weanling and newly arrived rats may not drink initially from such devices. to avoid undesirable microbial contamination, water bottles should be sanitized before they are refilled and automatic sys tems should be drained and flushed when racks are sanitized. acidification of water to a ph of . to . or chlorination at to ppm will control pseudomonas aeruginosa contamina tion of water (weisbroth, ) . however, this treatment is not necessary for immunocompetent animals. wood shavings or chips are the most commonly used contact bedding mate rials. hardwoods are preferred to softwoods, since the latter are capable of inducing hepatic microsomal enzymes (baker et al, a) . this section summarizes some of the anatomical characteris tics of the rat with emphasis on characteristics that are unique. the reader is advised to refer elsewhere in the literature for comprehensive descriptions (bivin et al., ; caster et al., ; hebel and stromberg, ; smith and calhoun, ; zeman and innés, ) . the rat dental formula is ( / , c / , pm / , m / ) = . the incisors are well developed and grow continuously. the rat lacks tonsils and water taste receptors. the major pairs of salivary glands are the parotid, submandibular (submaxillary), and sublingual. the parotid gland is a serous gland consisting of three to four lobes and is located ventrolaterally from the caudal border of the mandible to the clavicle. the submandibular glands are mixed glands located ventrally between the caudal border of the mandibles and the thoracic inlet. the sublingual glands are mucous glands and are much smaller than the parotid and submandibular glands. they are located at the rostral pole of the submandibular glands to which they are closely associated. brown fat deposits are present in the ventral cervical region. these multilocular deposits are well demarcated and can be confused with salivary glands or lymph nodes. the stomach of the rat is divided into two parts; the forestomach (nonglandular) and the corpus (glandular). the two portions are separated by a limiting ridge. the esophagus en ters at the lesser curvature of the stomach through a fold of the limiting ridge. this fold is responsible for the inability of the rat to vomit. the forestomach, which is thinner than the cor pus, is linked with an epithelium similar to that of the esopha gus and extends from the cardia to a narrow band of cardiac glands at the junction of the glandular portion. the small intestine is composed of the duodenum ( cm), jejunum ( cm), and ileum ( cm). the cecum is a thinwalled, comma-shaped pouch that has a prominent lymphoid mass in its apical portion. the colon is composed of the as cending colon, with prominent oblique mucosal ridges, trans verse and descending colons, with longitudinal mucosal folds; followed by a short rectum that is confined to the pelvic canal. the liver has four major lobes (median, right lateral, left, and caudate) and is capable of regeneration subsequent to par tial hepatectomy. the rat has no gallbladder. the bile ducts from each lobe form the common bile duct that enters the duo denum mm from the pyloric sphincter. the pancreas is a lobulated, diffuse organ that extends from the duodenal loop to the gastrosplenic omentum. it can be dif ferentiated from adjacent adipose tissue by its darker color and firmer consistency. up to excretory ducts fuse into - large ducts, which empty into the common bile duct. the nasal cavity is not markedly different from that of other mammals. the rat has a maxillary recess (sinus) located be tween the maxillary bone and the lateral lamina of the ethmoid bone. the recess contains the lateral iiasal gland (steno's gland) that secretes a watery product that is discharged at the rostral end of the nasal turbinate. it has been postulated that the nonviscous secretion contributes to the humidification of in spired air and acts to regulate the viscosity of the mucous layer overlying the nasal epithelium. the left lung has one large lobe, and the right lung is divided into four lobes (cranial, middle, accessory, and caudal). the pulmonary vein in the rat has cardiac striated muscle fibers within its wall that are contiguous with those in the heart. the rat does not have an adrenergic nerve supply to the bronchial musculature, and bronchoconstriction is controlled by vagai tone. unlike the guinea pig, the rat lung has a low concentra tion of histamine (bivin et al., ) . the heart and peripheral circulation in the rat differ little from that of other mammals. the blood supply to the heart is derived from both coronary and extracoronary arteries. the latter arise from the internal and subclavian arteries. the right kidney, which is more craniad than the left, has its cranial pole at the l, vertebra and its caudal pole at the level of l . the rat kidney is unipapillate as are kidneys of other ro dents, lagomorphs, and insectivores. having only one papillus and calyx makes the rat useful for studies in which cannulization of the kidney is done. the presence of superficial nephrons in the renal cortex has made the rat widely used as a model for studying nephron transport in an in vivo micropuncture system. the male reproductive system has a number of highly devel oped accessory sex glands. these include large seminal vesi cles, a bulbourethral gland, and a prostate gland composed of the coagulation gland (dorsocranial lobe) and ventral and dorsolateral lobes. the inguinal canal remains open throughout the life of a rat and testes descend initially by days of age. the female rat has a bicornate uterus that is classified as the duplex-type because the lumina of the uterine horns are com pletely separate with paired ossa uteri and cervices. the female urethra does not communicate with the vagina or vulva, but rather exits at the base of the clitoris. the brain of the rat has very large olfactory bulbs and a nonconvoluted cerebrum. the hypophysis is behind the optic chiasma and is attached to the base of the brain by a thin hol low stalk, the infundibulum. the blood supply to the brain is from the internal carotid and vertebral arteries. blood leaves the brain via a system of sinuses that are enclosed in the dura mater. the ventricular system is similar to that of other ani mals, but the rat lacks a foramen of magendie. it must be recognized that many of the normal values deter mined for a specific group of rats may be accurate for only that rat stock/strain, source, and conditions under which they are held. selected physiological, hematological, and clinical bio chemical parameters are listed in tables iv-vii. more com plete information on biological values is available (mitruka and rawnsley, ; ringler and dabich, ) . nutritionally adequate diets are readily available from com mercial sources. these standard rations are quite satisfactory for most applications. however, for some types of experimen tation there are factors, other than nutritional adequacy, which must be considered. the nutrient composition of diets and the contamination of feed components by mycotoxins, antibiotics, synthetic estrogens, heavy metals, and insecticides may have a profound impact on many studies. for instance, caloric intake and the percent of fat and protein in the diet of rats influence the incidence of neoplasia (altman and goodman, ) . sim ilarly, various contaminants have an adverse effect on data from toxicologie, gerontological, and reproductive studies. standard commercial diets are formulated from natural ingre dients and will vary in nutrient composition on a batch-tobatch basis due to differences in type and quality of ingredients used. commercial makers of rodent feeds take precautions to preclude the presence of contaminants in feeds, but only a few products have a defined profile of maximal levels of heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. for some investigative purposes, feeds formulated with re fined ingredients (purified diets) or with chemically defined compounds are useful when control of nutrient concentrations is essential (national research council, ) . these diets are, however, too expensive for general use. baker et al. ( b) and bivin et al. ( ). rats are commonly fed ad libitum, and food intake will vary according to requirements for growth, gestation, and lactation. the nutritive requirements for the rat are listed in table viii. the duration of storage and the temperature at which feeds are stored prior to use effect the nutritive quality of diets. com mercial diets are formulated to have a shelf life of up to months. however, storage in a hot or damp environment will reduce this shelf-life. to help assure that only fresh diets are used, products should be used which have milling dates identi fied on their containers (see chapter ). sexual maturity occurs between and weeks for both sexes, although the onset of first estrus in females occurs at about weeks. the vagina opens between and days, and the testes descend between and days, although they remain fully retractable in adults. rats ovulate spontaneously, but ovulation can also be induced by forced coitus during nonestrous intervals. vaginal stimulation during mating is impor tant in rat reproductive physiology. the more often a male inserts his penis into the vagina prior to ejaculation, the greater the probability of a resulting pregnancy. however, natural or artificial stimulation of the vagina within min of a first mat ing will abrogate pregnancy from the first mating by inhibition of sperm transport. a -hr estrous period recurs every to days and after parturition, without seasonal variation. estrus can be suppressed when females are housed in groups and syn chronized in the presence of a male or its excreta (whitten effect), but this effect is not as pronounced as in the mouse. female fertility wanes at to days, but estrous cycles may continue through months. male fertility is lost between and months. fertility of both sexes is generally regarded as maximal between and days of age (adler and zoluth, ; baker, ; farris, ; lane-petter, ; leathern, ) . males will mount estrous females numerous times with one or two rapid ejaculations in the course of to minutes. ejaculated semen coagulates, forming a copulatory plug that remains in the distal vagina for a few hours, after which time it dissolves or is extruded. copulation is usually nocturnal. du ration of gestation varies with strain, age, litter size, and other variables, and ranges from to days, with an average of or days. primiparous females tend to have a slightly longer gestation than multiparous females (farris, ) . estrus can be detected in a number of ways. females in es trus are hyperactive and brace themselves when touched. their ears quiver when they are stroked on the head or back, and stimulation of the pelvic region induces lordosis (farris, ) . the vulva becomes swollen, and the vagina becomes dry in contrast to the moist pink wall during metestrus or diestrus. as proestrus occurs (approximately hr), smears of va ginal cells contain nucleated epithelium, leukocytes, and occa sional cornified cells. estrus (approximately hr) begins with about % nucleated and % cornified cells, with cornified cells predominating as estrus continues. metestrus follows (ap proximately hr) with large numbers of leukocytes and corn ified cells, which form abundant caseous vaginal detritus. metestrus is characterized by the presence of large flat nucle ated (pavement) cells. diestrus persists for approximately hr (baker, ; farris, ) . breeding dates can be established by examination of vaginal swabs for spermatozoa or examining the distal vagina or cage pan for copulatory plugs. timed pregnancies are best achieved by placing the female in the male's cage in the afternoon and examining her for a plug or spermatozoa the following morning. abdominal enlargement becomes evident at about weeks. pseudopregnancy is rare (lane-petter, ) . rats reproduce successfully under a variety of conditions, but husbandry practices can significantly influence fecundity. rats can be bred as monogamous pairs, taking advantage of postpartem estrus for maximal breeding efficiency. polyg amous breeding is more economical, since only one male can be kept with to females. females are often removed to a separate cage prior to whelping, since they may not tolerate other females in the cage while nursing. they will tolerate their mates, however. females with litters do best on clean dust-free wood shavings in solid-bottom cages. due to heat regulation, pups neither thrive in overly spacious cages with wide flutuations in ambient temperature, nor in overly crowded cages where they cannot dissipate heat. the recom mended cage floor area for a female and her litter is in. . ambient room temperature and humidity should be within the acceptable range with minimal fluctuation. high ambient tem perature can cause male infertility (baker, ; baker et al., a; lane-petter, ) . the rat estrous cycle is particularly sensitive to variations in light. daily lighting at an average of fc with a spectrum approximating natural light for to hr is best for breeding. constant light for as few as days may induce persistent es trus, hyperestrogenism, polycystic ovaries and endometrial hy pertrophy or metaplasia (baker et ai, a; gralla, ) . nutrition may also affect reproductive performance. re quirements for certain components are increased during preg nancy and growth, but overfeeding is deleterious. caloric re striction may actually improve fertility and possibly reproduc tive life of the female (leathern, ) . excess dietary protein can adversely affect female sexual development. vitamin defi ciencies can cause infertility, particularly those vitamins (a, e, riboflavin, and thiamin) that are most labile to autoclaving or deterioration (baker, ) . it is not necessary to add nesting material to bedding for successful breeding, but rats will utilize it if offered. shredded paper or cotton nesting material will be readily accepted and used by prepartem and nursing dams. parturition is heralded by pronounced postural stretching and rear leg extension. a vagi nal discharge may be noted li- hr prepartum. parturition is usually complete in or hr, but can range from a few minutes to several hours depending on litter size. dystocia is exceed ingly rare. litters average between and pups, with highest fecundity through the sixth litter. inbred rats tend to produce smaller litters. although infrequent, cannibalism is most apt to occur with nervous or primiparous females subjected to stress (farris, ; lane-petter, ; leathern, ) . the neonate weighs about i gm, depending on litter size, sex, strain, and physical condition of the dam. pups are born hairless, blind, with closed ears, undeveloped limbs, and short they are fully haired between and days (baker, ; farris, ; lane-petter, ) . maternal antibody is trans ferred in utero, via the yolk sac and by intestinal absorption of colostrum by the neonate for up to days after birth (chev ille, ) . optimal weaning age is - days, although pups can be weaned as early as days. differentiation of sex in adult rats is relatively easy after the testes descend. the adult testes can be readily retracted through large inguinal canals. male neonates have a larger genital papillus and the anogenital space is greater in males than females. from national research council ( ) . h adequate to support growth, gestation, and lactation; based on % dry matter. ( linoleic acid, . %, is required. ^one-third to one-half can be supplied by l-cystine. ^one-third to one-half can be supplied by l-tyrosine. ^mixture of glycine, l-alanine, and l-serine. ^vitamin a, iu = . ìg retinol, . ìg retinyl acetate, . ìg retinyl palmitate. vitamin d, iu = . ì£ ergocalciferol. vitamin e, iu = mg dl-a-tocopheryl acetate. artificial insemination can be achieved in rats, but the major obstacle is the coagulative properties of their semen. sperm can be obtained by maceration of the epididymis and vasa or by electroejaculation, although the latter method is unreliable and the semen often rapidly coagulates. coagulation can be eliminated by prior surgical extirpation of the seminal vesicles and coagulating glands without significant effect on fertility. semen can be diluted with a number of media but frozen stor age of rodent semen has met with little success. insemination can be achieved surgically by direct injection of seminal fluid into the uterus and by nonsurgical means. successful concep tion seems to require not only insemination during estrus but also induction of pseudopregnancy by mating with a vasectomized male or mechanical stimulation within a few hours (before or after) insemination. egg harvest for transfer can be accomplished by excision of the preovulatory ovaries and teas ing from gravid follicles or recovery from the oviduct or uterus by flushing with transfer medium. superovulation by injection of gonadotropisms may enhance yield, but is usually not nec essary. eggs are generally injected directly into the uterus but the recipient uterus must be at the same stage of the uterine cycle (bennet and vickery, ) . synchronization of estrus can be achieved by vaginal inser tion of polyurethane sponges containing . mg medroxyprogesterone for days. females are then put in a cage previously occupied by male rats, sponges are removed, and the rats are injected with iu of pregnant mare's serum. within hr, % will be in estrus. this can also be attained by administer ing mg medroxyprogesterone in ml ethanol/liter drink ing water, prepared fresh daily for days, then intramuscular injection of iu of pregnant mare's serum (bennet and vick ery, ). the rat has been utilized extensively in a variety of research fields, including behavioral science. rats are docile, adapt to new surroundings, tend to explore, and are easily trained to a variety of sensory cues by positive or negative reinforcement. rats sleep during daylight hours and activity, including feed ing, is greater during the night and early morning. laboratory rats are easily handled, but strain differences exist. sprague-dawley and lew rats tend to be less fractious than long evans or f rats. docility is improved with routine and proper handling. rats become nervous and refractory to han dling when they hear others squeal. nutritional deficiency, particularly hypovitaminosis a, and mishandling can make rats vicious. rats seek entry into small openings, a trait that is utilized for coaxing them into restrait apparatus. like other rodents, rats are coprophagic, which must be taken into con sideration when administering drugs, measuring fecal output, or performing nutritional studies. unlike mice, rats are less apt to fight, and males can be housed together. in addition, rats are not gregarious like mice, and seem to tolerate single caging well. experimental studies indicate significant changes in plasma corticosteroid levels, depending on cage cohort size. levels tend to be least in rats housed singly, to increase in groups up to , to decrease in larger groups up to - , then rise again in groups up to (lane-petter, ). infectious agents constitute a significant environmental vari able that impacts on research data derived from laboratory rats. as is the case with other species, infectious agents induce a wide range of diseases in the rat that vary from inapparent to overt clinical disease. most investigations use large numbers of rats in which a specific group or colony consists of several to hundreds of rats. accordingly, emphasis on disease is one of prevention and placed at the colony level rather than on a sin gle or a few animals. curative use of antibiotics, which is important in the treatment of bacterial diseases of nonrodent species, is rarely useful in the laboratory rat. administration of drugs to obtain therapeutic blood levels is difficult to achieve in a colony; also some animals may improve clinically but re main colonized by the pathogen and serve as carriers, reinfecting other animals. rats seldom show clinical signs of disease upon arrival to the laboratory from commercial sources. however, these rats may harbor pathogens that are of low to moderate virulence and that are capable of severely compromising the health of animals when the rats are exposed to various types of experimental stress. moreover, some of these pathogens may never cause clinical disease, yet induce microscopic lesions or biochemical aberrations that can have profound effects on research data. for these reasons, investigators and clinicians should be aware of the pathogen status of the animals used in studies, both ini tially and throughout the course of the studies. this section on infectious diseases contains those agents that are of principal importance to the investigative use of the rat. a. streptococcosis. the causative organism, streptococcus pneumoniae, is a gram-positive coccus that is rather ubiq uitous among humans and animals. streptococcus pneumoniae is frequently recovered from respiratory tract lesions in guinea pigs, nonhuman primates, and some domestic animals. in hu mans, it is often present in the nasopharynx in the absence of clinical symptoms of infection. upper respiratory tract infec tion of conventionally raised rats has been reported to be com mon. however, it is seldom present in barrier-maintained, commercial rat sources. as in pneumococcal disease in hu mans, a number of serological types have been associated with respiratory disease in rats. streptococcus pneumoniae infection in rats often remains lo calized in the nasopharynx without the development of overt disease. a shift in the host-parasite balance due to stress or concurrent infection with another pathogen may result in bronchopneumonia and bacteremia. the most common signs of respiratory disease are serous to mucopurulent nasal discharge and "red tears" due to porphyrin pigments secreted from the harderian glands, dyspnea, rales, and depressed activity. ani mals will often die within a few days after the onset of pneu monic signs. the severity and prevalence of clinical disease within an infected colony are associated with environmental conditions that induce stress (e.g., experimental manipulation, overcrowding, fluctuations in ambient temperature and humid ity, and copathogens). although all age groups are susceptible to infection and clinical disease, young animals are more apt to be clinically affected. transmission between rats is by aerosol droplet. although both humans and rats can carry the same serotypes of s. pneumoniae, the authors are unaware of evi dence indicating zoonotic or human-to-animal transmission. the most characteristic gross lesions are pulmonary consol idation and fibrinopurulent pleuritis and pericarditis ( fig. ). an extensive fibrinopurulent peritonitis, orchitis, or meningitis may occur as well. if a bacteremia occurs early, the disease may be acute with few gross lesions. streptococcus pneu moniae induces an outpouring of exudate rich in fibrin, neutrophilic leukocytes, and erythrocytes into the alveoli. bron chioles are filled with neutrophilic leukocytes. embolie lesions may occur in multiple tissues which include the spleen, liver, kidneys, joints, and brain. streptococcosis is diagnosed by clinical signs, characteristic lesions, and isolation of s. pneumoniae from lesions. the per icarditis, pleuritis, and pleural effusion noted above differenti ate pneumococcal disease from pneumonia due to mycoplasma, although the two pathogens often are superimposed. this organism produces an á-hemolysis on blood agar plates similar to that of the streptococcus viridans group. streptococ cus pneumoniae isolates are most commonly differentiated from nonpathogenic s. viridans by the sensitivity of the former organism to optochin (hydrocuprein hydrochloride). optochin-impregnated discs are placed on a blood agar plate which has been inoculated with a pure culture of the clinical isolate. if the isolate is s. pneumoniae, a distinct zone of growth inhibition will be present around the disc. although typing of s. pneumoniae isolates is seldom done today, one can type an isolate by reacting known specific s. pneumoniae antisera with s. pneumoniae isolates. this serological test is the neufeld-quellung reaction and is based on the capsular swelling that is induced by specific antiserum. there is no effective means to control s. pneumoniae infec tion once it is enzootic in a colony. benzathine penicillin ( , units/ gm body weight) may be helpful in reducing the severity of the disease and as an aid in limiting infections to a subclinical mode in some animals. however, antibiotics will not eliminate the organism from rat colonies. hysterectomy rederivation of breeding stock from infected colonies is an ef fective method of initiating new stock free from pneumococcal infection (weisbroth, ) . b. pseudotuberculosis (corynebacteriosis). the causative agent of pseudotuberculosis is the gram-positive bacillus, corynebacterium kutscheri. on occasion, other corynebacterium species can cause similar syndromes in rats. typically, the or ganism causes inapparent infections in rats, with exacerbation of respiratory disease under conditions of stress. when clinically ill, the most commonly seen signs include serous oculonasal discharge, dyspnea, anorexia, and loss of weight or retarded growth. animals with severe pulmonary signs usually succumb within several weeks, while rats with less severe signs often survive much longer. most rats will have inapparent infections in which c. kutscheri cannot be isolated from internal organs. little is known concerning how c. kutscheri is carried or transmitted within a colony. it has been suggested that the organism is transmitted via aerosol droplet or direct contact. once rats are infected, a hematogenous spread may be involved, since lung lesions are initially interstitial and not bronchial. gross lesions are characterized by a variable number of grayish-yellow foci surrounded by red zones, particularly in the lung (fig. ) . in longer-standing cases, individual foci co alesce into raised lesions cm or larger in diameter. occasion ally, fibrous adhesions occur between the lungs and thoracic walls. similar lesions may be seen in other organs, including the liver, brain, and kidneys. the hepatic lesions resemble tu bercles and have caseous centers and fibrous capsules. prepucial adenitis, arthritis, and otitis media may also be caused by c. kutscheri. the lesions in various target organs appear to be due to septic emboli. pulmonary lesions initially consist of a polymorphonuclear cell and macrophage infiltrate of the bronchioles and interstitial tissue with a round cell infiltrate occurring later. bronchi become impacted with polymorphonuclear cells and necrotic leukocytes. giemsa or gram staining of infected tissues will reveal the rod-shaped c. kutscheri organisms. diagnosis of c. kutscheri infection is made on clinical signs, gross and microscopic lesions, and isolation of the bacterium from infected tissues. although the respiratory signs are simi lar to those present with mycoplasmosis, the rapidity with which c. kutscheri clinically affected rats succumb helps dif ferentiate it from mycoplasma pulmonis-'mduced disease. un like streptococcosis, fibrinopurulent pericarditis, peritonitis, and pleural effusion are not seen. whereas peribronchial lymphoid hyperplasia is a dominant lesion in mycoplasmosis, it is unremarkable in c. kutscheri infections. corynebacterium kutscheri is easily recovered from lesions and upper respiratory tract exudates by culturing on blood agar plates incubated aerobically at °c. epizootics of pseudotuberculosis may occur in conven tionally raised breeding colonies, but rarely occur in barrierraised colonies. epizootics often can be retrospectively associ ated with an environmental stress (e.g., fluctuation in ambient temperature or ventilation). culling of ill animals will not eliminate c. kutscheri from animals remaining in a colony. isolation of the organism from animals with subclinical infec tions is not usually successful. for this reason, cortisone ad ministration has been advocated as a means for surveillance of infection in colonies prior to necropsy and culturing for c. kutscheri. in the past, most serological methods have been un satisfactory in detecting antibody in animals with inapparent infections (weisbroth, ) . recently, however, enzymelinked immunoabsorbant assay (elisa) has been shown to be capable of detecting antibody in animals without clinical signs of infection (ackerman et al., ) . hysterectomy derivation is an effective means to establish a c. kutscheri-free colony. antibiotic therapy will not eliminate c. kutscheri from a colo ny, but a -day regimen of penicillin has been reported to be effective in curtailing an epidemic of c. kutscheri-'mductd pneumonia (fox et al., ) . since c. kutscheri infection is, in most cases, inapparent and manifests itself whenever the host is sufficiently stressed, it can be a significant problem in experimentally stressed rats. c. tyzzer's disease. tyzzer's disease is caused by the gram-negative, spore-forming rod, bacillus piliformis. this organism, which is not a true bacillus, is an intracellular pathogen that has not been cultivated on artificial media, and is, as yet, taxonomically undefined. in the laboratory, b. piliformis is propagated in the yolk sac of embryonated chick eggs. this disease occurs in other rodent species and appears to be widely distributed in many nonrodent species, but there ap pears to be a degree of species specificity among b. piliformis strains. it occurs occasionally in conventionally raised rat colo nies. the vegetative form of b. piliformis is unstable in the environment. however, spores of the organism are relatively stable and are believed to be the source of transmission among animals. clinical signs associated with tyzzer's disease are not partic ularly distinctive and, accordingly, only suggestive in making a diagnosis. typically, affected rats are apt to be adolescents with signs such as lethargy, weight loss, and distended abdo mens. diarrhea is not a common sign in rats with b. piliformis infection. animals displaying clinical signs generally die with in several weeks. clinically inapparent infections occur and are most probably responsible for transmission of the organism within a colony. clinically evident tyzzer's disease is usually associated with experimentation that compromises the immunocompetence of rats. the most remarkable gross lesions involve the liver, ileum, and myocardium. hepatic lesions consist of numerous small, pale foci on the surface and within the parenchyma. the intes tinal lesion has been termed "megaloileitis" due to a segmen tai dilatation and inflammation of the ileum (fig. ) (jonas et ai, ) . heal distension is not always present. in some rats, circumscribed gray foci also occur in the myocardium. the pathogenesis of the disease is believed to involve a pri mary intestinal infection with spread to the liver via the portal circulation. bacillus piliformis invades enterocytes, resulting in villus shortening, inflammation, necrosis, and hemorrhage. intracellular organisms are demonstratable in epithelium of crypts and villi. the necrotic foci in the liver are most often present near vessels. surrounding these foci are varying num bers of leukocytes, macrophages, and fibroblasts. intracytoplasmic bacteria may be seen in hepatocytes at the periph ery of the lesions, but may be present in very small numbers and thus be hard to find. organisms are also found in myocar dium around foci of necrosis (weisbroth, ) . a presumptive diagnosis can be made by the gross lesions, but a definitive diagnosis is dependent upon observation of the organism within hepatocytes, intestinal epithelium, or myocar dium. impression smears of liver taken at necropsy and stained with gram, giemsa, or méthylène blue stains may be useful for a rapid diagnosis. however, formalin-fixed specimens stained by giemsa or warthin-starry methods are usually per formed to confirm a diagnosis. the ileal distension seen in rat tyzzer's disease must be differentrated from other causes of adynamic ileus, particularly chloral hydrate-induced lesions. prevention of tyzzer's disease in a colony is dependent upon a barrier that excludes entry of the agent by contaminated cages, equipment, and infected animals. routine cage sanita tion probably is ineffective in killing the spores of b. piliformis, but exposure of spores to °c for min has been shown to inactivate them. sodium hypochlorite ( . %) is an effective disinfectant (ganaway, ) . although antibiotics have been shown to be effective under experimental conditions in mice, there is no evidence to indicate that antibiotic therapy can be of value under natural conditions within a colony of rats (weisbroth, ) . d. pasteur elio sis. pasteur ella pneumotropica frequently infects conventionally raised rats and has been recovered occa sionally in rats from barrier-and axenic-maintained colonies. it is a pathogen of very low virulence, and most infections remain clinically inapparent. only a relatively few reports doc ument p. pneumotropica as a primary pathogen in cases of penumonia, otitis media, and conjunctivitis. as a copathogen with either m'ycoplasma pulmonis or sendai virus, it has a con tributory role in the resultant respiratory lesions and otitis. its localization is not limited to the respiratory tract, since it is frequently isolated from the oral cavity, intestinal tract, and uterus. it also has been associated with mastitis and furunculosis in rats. it has been suggested that p. pneumotropica is essentially an enterotropic rather than a pneumotropic orga nism. the intestinal tract is probably the primary site for local ization of the organism in subclinical infections. horizontal transmission is by the oral-fecal route and direct contact. since p. pneumotropica is frequently carried in the uterus, vertical transmission can occur, and, accordingly, this can compromise the microbial status of axenic and gnotobiotic colonies. distinctive clinical signs and lesions do not occur with p. pneumotropica-induoed disease. accordingly, a diagnosis must be based upon its isolation as the sole pathogen or, as in many cases, as a copathogen within lesions. blood agar medi um is satisfactory for primary isolation from nonenteric sites. however, for recovery from the intestinal tract, enrichment in a medium such as gn broth is recommended before isolation is attempted on blood agar plates (weisbroth, ) . hysterectomy derivation and barrier maintenance are the only means to control infection. however, particular attention must be made to ensure that hysterectomy-derived young came from dams that had culturally negative uteri. antibiotic thera py is not effective in eliminating the organism from a colony. e. salmonellosis. salmonella species that infect rats in clude salmonella enteritidis, s. typhimurium, s. dublin, and s. meleagridis. salmonellosis, which was once a major cause of disease in laboratory rat and mouse colonies, is rarely reported in either species today. however, it still exists in wild popula tions of rodents and, therefore, remains a potential threat to laboratory rodents. infection in an immunologically naive colony typically re sults in an epizootic of clinically affected rats and a varying proportion of animals with inapparent infection. these latter animals act as subclinical carriers to render the infection as enzootic in a colony. acute outbreaks will occur intermittently whenever immunological and other host defense mechanisms are altered. signs associated with salmonellosis in the rat are anorexia, depressed activity, starry hair coats, and soft to formless feces. affected animals die in to weeks. lesions that occur in salmonellosis differ depending on the stage of the disease. salmonellae penetrate the intestinal mucosa at the level of the ileum and cecum. the earliest le sions occur in this locale and consist of a mild dilatation, thick ened intestinal walls, and a granular mucosal surface. involve ment of the reticuloendothelial system is reflected by enlarged peyer's patches, mesenteric lymph nodes, and spleen. in some infected animals, a bacteremic state occurs that results in the demise of the host before the development of further lesions. however, in animals not succumbing to septicemia, ulcération of the ileal, colonie, and cecal mucosa occurs. histologically, the villus epithelium of the ileum is markedly degenerated, and the lamina propria is infiltrated with neutrophils and mac rophages. concomitant with intestinal lesions is the develop ment of focal necrosis and granulomas in the spleen and liver due to hematogenous spread of the organism (buchbinder et al, ; maenzae/fl/., ) . in rats who are intermittent or chronic shedders of salmonel la, the most remarkable lesions are lymphadenitis of the mes enteric lymph nodes and ulcération of the cecal mucosa. rats from which salmonella is chronically shed have more ad vanced lesions than do intermittent shedders of the organism. a diagnosis of salmonellosis relies upon identification of an isolate as a salmonella sp. recovery of salmonella from the intestines, spleen, and liver is readily accomplished in rats clinically affected during an epizootic. however, this is not true for asymptomatic carriers, since some will shed the orga nism intermittently in the feces, and recovery from tissues is difficult. recovery in carrier animals is best accomplished by initial incubation of fecal pellets in an enrichment broth, such as selenite f plus cystine broth, followed by streaking onto brilliant green agar (weisbroth, ) . from this medium, possible salmonella colonies are inoculated into triple-sugariron slants. final identification is then made by biochemical tests and serotyping. prevention of this disease is based upon the exclusion of wild rodents from laboratory animal facilities and the use of only feed and bedding that has been properly processed and pack aged to ensure against salmonella contamination. /. pseudomoniasis. pseudomonas aeruginosa, a ubiq uitous gram-negative bacterium found in soil and water, colo nizes plants, insects, animals, and humans. it often colonizes the oropharynx and can be isolated from the intestinal tract of rodents. infection with this organism in immunocompetent rats is nearly always inapparent. however, when rats are immunosuppressed, p. aeruginosa invades the upper respiratory mucosa and cervical lymph nodes, becomes bacteremic and induces an acute, lethal disease. in some cases, rats develop facial edema, conjunctivitis, and nasal discharge. in genet ically thymic-deficient rats (nude), retro-orbital abscesses may occur prior to bacteremia. transmission in laboratory rodents occurs primarily by direct contact and contaminated water bottles and automatic watering systems. phenolics are usually effective disinfectants, but quaternary ammonium compounds may actually support its growth. diagnosis of pseudomoniasis is based upon a history of immunosuppression associated with an epizootic of acute disease and isolation of p. aeruginosa from the blood and organs of affected rats. facial edema in affected rats must be differenti ated from viral sialodacryoadenitis. pseudomonas aeruginosa grows well on blood agar and most other standard laboratory media. most strains are ß-hemolytic and produce a bluish-green pigment, pyocyanin, as well as fluorescein. the use of specialized media (pseudomonas p agar) enhances pigment production. the organism derives energy from carbohydrates via oxidation rather than fermentative me tabolism. identification of isolates as p. aeruginosa is easily made by the above characteristics and appropriate biochemical reactions (weisbroth, ) . in most research applications, p. aeruginosa-free rats are not necessary for the conduct of the work. it is a major problem, however, in rats used for burn research and in studies in which drugs or radiation induce immunosuppression. infection can be relatively well controlled in a colony by hyperchlorinating drinking water at ppm or by acidification of water to a ph of . - . . in a closed colony, it is also advisable to remove rats that remain culturally positive after water treatment has been instituted. in studies requiring pseudomonas-free rats, isolators are useful in which a gnotobiotic environment can be achieved. alternatively, laminar flow units may suffice if supplies and equipment are sterilized and personnel wear sterile garments. g. streptobacillosis. streptobacillus moniliformis is a commensal bacterium often present in the nasopharynx of con ventionally raised rats. although it may be involved occasion ally as a secondary invader within inflammatory lesions of the rat, the chief importance of s. moniliformis is that it is the principal agent causing rat-bite fever in humans (anderson et ai, ) . the other bacterium associated with this clinical syndrome is spirillum minus. clinical signs in humans usually occur within days of a rat bite and consist of headache, weakness, fever, a generalized rash, and arthritis. often clini cal signs subside in several days but then recur at irregular intervals for weeks or months (see chapter ). a. murine respiratory mycoplasmosis. murine respirato ry mycoplasmosis (mrm) is the term now accepted for a dis ease which, for many years, had an undefined etiology and a number of synonyms [i.e., infectious catarrh, enzootic bronchiectasis, chronic respiratory disease (crd), and chronic murine pneumonia]. since , the causal relationship of mycoplasma pulmonis with this disease has become well estab lished (kohn and kirk, ; lindsey et al., ; whittlestone et al., ) . of all the pathogens occurring in laboratory rats, m. pulmonis has had the greatest negative im pact on studies. this has been primarily due to the chronicity of the disease, which often manifests itself only after months of infection. long-term studies in areas of toxicology, carcinogenesis, nutrition, and gerontology, in particular, have been affected. prior to the use of gnotobiotic techniques and barrier maintenance in rat production colonies, m. pulmonis was enzootic in nearly all commercial and institutional colo nies. today, vendors can be selected who offer mycoplasmafree rats. my coplasma pulmonis is highly contagious and in duces a disease that frequently results in debilitation or demise of the host after a long period of time. the clinical signs associated with mrm range from negligi ble upper respiratory tract signs to systemic signs associated with pneumonia. the earliest and most common signs include snuffling and serous or mucopurulent oculonasal discharge. extension of m. pulmonis infection from the nasopharynx via the eustachian tubes to the middle ears is common. however, torticollis and circling due to involvement of the inner ear are infrequently observed, even though one or both middle ear bullae may be impacted with exudate. the onset of upper res piratory signs is variable, but often occurs within several weeks postinfection. signs of penumonia include dyspnea, rales, and systemic effects such as weight loss, starry hair coat, and hunched posture. characteristically, signs of pneumonia occur - months postinfection, but this is quite variable and is a function of environmental influences, such as intracage ammonia levels and the immune competence of the host. in a small percentage of cases, the disease will be nearly subclinical even in the presence of extensive pulmonary lesions. mycoplasma pulmonis is transmitted both horizontally and vertically from dams to their litters. in most instances, trans mission from the female occurs postpartum by direct contact, but if the genital tract of the dam is infected, antenatal infec tion can occur. horizontal transmission between postweanling rats of any age readily occurs, and there appears to be no sig nificant age-related resistance to either infection or disease. although little is known about differences in resistance among rat stocks and strains, the lew rat has been shown to be more susceptible to mrm than the f rat. there is little evidence available to indicate that transmission occurs through fomites such as caging equipment and garments worn by personnel. since aerosol droplet and direct contact appear to be the prima ry modes by which m. pulmonis infections are spread, the rapidity with which the organism is transmitted is dependent upon environmental factors, such as ventilation rates, degree of recirculation of air, and animal density within rooms. the basis for the pathogenicity of m. pulmonis is not well understood. mycoplasma pulmonis adsorbs to the cell mem brane of the ciliated, columnar or cuboidal epithelia in the res piratory tract (fig. ) . it has been suggested that adsorption is a means by which mycoplasmas damage host cells by uptake of essential cellular metabolites; release of cytotoxic products, such as h ; or cross reaction of antibody with cell mem brane components that are antigenically similar to or altered by mycoplasmas. infection severely distorts or ablates ciliary structures (fig. ) , interfering with mucociliary clearance mechanisms. the gross lesions in the upper respiratory tract include mucopurulent exudate in the nasal cavity, sinuses, and middle ear bullae. later, the exudate becomes caseous within the bul lae. lesions in the lower respiratory tract reflect those of a bronchopneumonia. the earliest lesion is a mucopurulent exu date within the trachea, bronchi, and bronchioles. this pre cedes grossly evident lesions of the lung parenchyma that ini tially consist of atelectasis due to bronchial occlusion. later, bronchiectatic lesions appear as numerous cream-colored nod ular abscesses on the surface of the lung. these lesions may be restricted to only a portion of a lobe or may involve nearly all of the parenchyma (fig. ) . microscopically, the inflammatory response is characterized by a lymphocyte and plasma cell infiltrate in the submucosa and neutrophilic leukocyte response within the lumina of the epithelium. nasal cavity, eustachian tubes, middle ears, and tracheobronchial tree. a consistent and prominent lesion in the lung is the peribronchial lymphoid hyperplasia that often be comes quite massive. within the lumina of the bronchi and bronchioles, mucin and neutrophil exudation increases during the course of the disease to the point of bronchiectasis. con comitant with the impaction of bronchi is a change in the epithelia from a ciliated, columnar type to a squamoid type. this change in epithelial architecture is likely associated with cytotoxic enzymes from autolyzed neutrophils, although a di rect cytotoxic effect from mycoplasmas could be involved. a tentative diagnosis of mrm can usually be made by obser vance of the clinical signs and gross lesions described above. clinical signs alone are not particularly helpful, since nasal exudates are present in bacterial infections such as s. pneumoniae. in addition, the reddish porphyrin deposition seen in the nares and periorbitally in sialodacryoadenitis virus infec tion and water deprivation may be confused with exudation. the gross lesions of otitis media and bronchiectasis are rather distinct. however, c. kutschen lung lesions may grossly mim ic those of mrm. histopathology and serological evidence will differentiate mrm from sendai virus infection, although the two infections are often superimposed. recently a filamen tous bacterium has been associated with bronchiectasis in wild and laboratory rats (mackenzie et al., ) . however, the causal relationship of this organism with lesions is undefined since the rats were also infected with m. pulmonis. this fila mentous bacterium has not been successfully grown on artifi cial media, and its presence is best verified by either histology, using the warthin-starry stain, or electron microscopy (fig. ) . although a definitive diagnosis of mrm is made by isola tion of m. pulmonis from involved tissues, it is evident that the existence of other agents must be evaluated to determine if copathogens are contributory to lesions. prevention of mrm in either breeding or experimental colo-nies is dependent upon barrier systems that preclude the entry of m. pulmonis into the facility. hysterectomy derivation is the only means of establishing an m. pulmonis-frtt breeding colo ny from a previously infected stock. due to the frequent local ization of this microorganism in the uterus, it is necessary to ensure that neonates taken by hysterectomy have not been in fected in utero. rats used in research animal facilities are ob tained from various commercial and institutional sources. ac cordingly, it is essential that the mycoplasma status of these sources is known and that the rats are housed by vendor or in groups with a similar microbial status. for assessment of whether a group of rats is m. pulmonisfree, the best sites for isolation in animals without gross lesions are the nasal cavity, middle ear, trachea, and uterus-oviduct. mycoplasma pulmonis is not particularly fastidious and grows well in several types of mycoplasma media (cassell et al., ; lentsch et al., ) . most formulations have a ph indi cator that is useful since m. pulmonis ferments glucose. in broth media, moderate to heavy growth is reflected by ph and color of the broth. in broth cultures in which the titer is low, a perceptable ph change may not occur. tissue and washing samples should be placed in broth rather than agar media, since recovery of the organism is more likely in those samples con taining few mycoplasmas. samples from broth cultures are transferred to agar media when a ph change is readily evident or at - days if no ph change occurs. mycoplasma colonies are evident in - days by observation with x stereoscopic microscopy. although culturing and histopathology have been the usual means to survey rat colonies, elisa testing has recently been shown to be a very sensitive serological assay and one that can be performed quickly in most clinical laboratories (cassell et al., a) . in vitro sensitivity tests show m. pulmonis to be susceptible to tetracycline and tylosin. tetracyline, given at mg/ml drinking water, may be useful in some situations (lindsey et al., ) . however, treatment with antibiotics seldom influences the disease course of mrm in a colony situation. b. murine genital mycoplasmosis. mycoplasma pulmonis recently has become recognized as an important pathogen in the female genital tract of rats, and thus is being treated here as a distinct disease rather than as a sequella to mrm. infection of the genital tract is usually inapparent. however, reduced fertility and fetal deaths can occur. infection of the oviduct and uterus occurs frequently in rats who have respiratory my coplasmosis. it is unknown whether localization in the genital tract occurs due to a hematogenous spread or to an ascending infection of the genital tract. it has been shown that subsequent to intravenous inoculation, m. pulmonis almost invariably lo calizes in the female oviduct-uterus. gross lesions, when present, consist of a purulent oophoritis, salpingitis (fig. ) , and pyometra. the lew strain is particu-Ä^sfcvvv ( Ä ß f/g. . electron micrograph of filamentous bacterium (large arrow) and m. pulmonis (small arrow) attached to epithelium of respiratory mucosa. the morphology of size of the filamentous bacterium are similar to that of the cilia. (courtesy of dr. w. f. mackenzie.) larly prone to develop gross lesions. mycoplasmapulmonis ad sorbs to the epithelial cells in the genital tract in a manner similar to that seen in the respiratory tract. salpingitis occurs most frequently and is characterized by exudation of neutrophils into the lumen, hyperplasia of oviductal epithelium, and a lymphoid response in the submucosa. the lesions in the ovarian bursa include edema and inflammation. uterine le sions can vary from a mild inflammatory change to pyometra (casselle/fl/., b). genital mycoplasmosis in the male rat has not been well doc umented. however, it is known that experimental inoculation can include an inflammatory response in the ductus efferens and epididymis. moreover, it is known that m. pulmonis is capable of adherence to spermatozoa in an in vitro system. since pasteur ella pneumotropica can also induce similar le sions in the female rat, a diagnosis of mycoplasmosis is depen dent upon isolation of m. pulmonis from the lesions. methods for culturing and identification are similar to those used for respiratory mycoplasmosis. because the rat is widely used in various types of reproduc tive biology research, m. pulmonis colonization, even without gross lesions, would probably impact on the validity of data. the grossly evident caseous lesions in the ovary and oviduct can be mistaken for neoplasia if microscopy is not done. c. mycoplasmal arthritis. the etiological agent of this disease is mycoplasma arthritidis. this mycoplasma species colonizes the pharynx, middle ears, and lungs of rats, although few studies have been done to document the relative frequency of this mycoplasma in rat sources. within the respiratory tract, m. arthritidis colonization is thought to induce negligible le sions, and it has been shown to coexist with m. pulmonis. although it is often considered to be the principal agent in volved in arthritis in rats, the disease has been rarely reported. nearly all reports of its involvement in clinically apparent ar thritis have been made prior to . it has been suggested that poor cage sanitation and abrasions of the extremities are in volved in entry of the organism to the joints by hematogenous spread or extension from surrounding tissues (ward and cole, ) . since the organism appears to be of low virulence, the immunocompetence of the host may be a major factor in the outcome of infection. arthritic animals limp and move with difficulty due to pain associated with the polyarthritis. any of the joints in the limbs and vertebrae can be affected, but the tibiotarsal and radiocarpal joints are most often involved. affected joints are hyperemic and swollen. incised joints reveal a purulent exudate in both articular and periarticular tissues. microscopically, there is exudation of neutrophils into the synovial spaces, and a lym phocyte and plasma cell infiltration in the synovial mem branes. destruction of the articular cartilage occurs subsequent to the inflammatory response. since polyarthritis can occur subsequent to septicemias asso ciated with other bacteria, particularly c. kutschen, a diag nosis of m. arthritidis-'mduced arthritis is contingent upon the demonstration of m. arthritidis by isolation or immunofluorescence techniques. this mycoplasma species grows well in me dia used to isolate m. pulmonis if arginine is added to the for mulation (cassell et al., ) . tetracyclines have been used to prevent the onset of arthritis when the organism has been inoculated intravenously, but there are no reports of its efficacy in spontaneous cases. mycoplasma arthritidis, like m. pulmonis, may contaminate transmissible tumors and caution should be exercised to ensure transplanted tissues are not contaminated. hemobartonellosis. the causative agent of this rickettsial disease is hemobartonella muris. this organism is an extra cellular parasite of erythrocytes and induces inapparent infec tions that may persist for long periods. the ability of the host to restrict the infection to a subclinical mode rests with the integrity of the reticuloendothelial system. evidence of infec tion is usually limited to splenomegaly and laboratory findings of mild parasitemia and reticulocytosis. transmission of h. muris involves the blood-sucking louse, polyplax spinulosa. transmission can occur during a blood meal or when rats crush infected lice and are inoculated via pruritis-induced abrasions. the organism can also be transmit ted inadvertantly with transplantable tumors and other biolog ical products. diagnosis of hemobartonellosis is dependent upon identifica tion of the organism in the peripheral blood of infected ani mals. the usual method of detection is by splenectomizing rats suspected of harboring the organism. in these rats, severe para sitemia and hemolytic anemia occur within weeks after sur gery. hemobartonella muris can be visualized on the surface of erythrocytes in romanowsky-stained blood smears as coc- coid bodies arranged singly, in clusters, or chains (cassell et al., ) . the rarity of reported cases would indicate h. mûris is no longer a significant problem in barrier-maintained colonies. however, conventionally maintained colonies may be exposed to infected wild rats and p. spinulosa and, accordingly, the disease still is of importance in the laboratory rat. the disease has had a negative impact on investigations of various types, but principally with those in which the host's immune compe tence has been impaired. a. parvoviral syndromes. parvoviruses that can infect rats include rat virus (rv), toolan h-l (h-l) virus, and minute virus of mice (mvm). parvoviruses are small nonenveloped viruses that resist extremes in temperature, ph, and drying. rat virus, or kilham rat virus (krv), has several antigenically related strains (rv, h- , x- , l , hb, sprv, her, hhp, kirk), all of which have been isolated as inadvertant contami nants of rat tissue or rat-passaged biological material. toolan h-l related serotypes (h-l and h-t) are antigenically distinct from rv serotypes. both rv and h-l are experimentally pathogenic, producing similar lesions, but only rv has been associated with natural disease. neonatal rats can be experi mentally infected with mvm, but the virus does not seem to cause natural infection. minute virus of mice antibody reac tivity can be present in rat serum, but this is probably non specific, since it can be found in germfree rat serum and is reduced or eliminated by receptor destroying enzyme. rat virus infection is usually subclinical or latent, but a num ber of clinical syndromes have been associated with it. infec tion of pregnant females can cause fetal résorption and birth of small litters. pups are runted, atactic, or jaundiced. neonates develop similar signs following postpartem exposure. rats in troduced to an infected colony can develop ruffled fur, de hydration, and sudden high mortality. a similar syndrome oc curs in latently infected adults subjected to immunosuppressive regimens. the rat is the only natural host for rv and h- , although experimental infection can be established in a number of other species. seroconversion to both rv and h-l virus is common, with a high prevalence of infection within an enzootically in fected colony. horizontal transmission is achieved by the oral and probably respiratory routes, with virus excretion primarily in the feces. some strains of rv can be excreted in the milk or in utero. clinical signs are manifest transiently upon introduc tion of rv into a previously uninfected population, but, there after, the virus spreads rapidly to produce subclinical or inapparent enzootic infection. rat virus can persist as a true latent infection in the presence of high circulating antibody, but dis ease can be activated by immunosuppression. it must, there- fore, be assumed that seropositive rats are persistently infected and can serve as a source of infection to other rats. pups infected in utero or as neonates develop intranuclear inclusions and necrosis in the outer germinal cell layer of the cerebellum. the recovered animal has severe depletion of the internal granular layer and disorganized purkinje cells. intra nuclear inclusions are also in hepatocytes, kupffer cells, endothelial cells, and biliary epithelial cells, resulting in necrotizing hepatitis and the sequellae thereof (bile retention, jaundice, peleosis, bile ductal hyperplasia, parenchymal collapse, nodu lar hyperplasia). in adults, infection is usually inapparent, but when acute disease is precipitated, rv injures vascular walls and hematopoietic elements, causing coagulative disorders, thrombosis, hemorrhage, and infarction within the central ner vous system (hemorrhagic encephalomyelopathy). hemorrhagic and necrotic lesions have also been noted in the per itoneum, testis, and epididymis. rat virus has broad tissue tropism and lesions or clinical signs may potentially be varied, depending on virus and host factors (coleman et al., ; jacoby et al, ) . infertility and unthrifty pups caused by rv must be differenti ated from environmental and husbandry factors or infectious agents such as mycoplasma or sendai virus. adult disease must be differentiated from toxicity, nutritional deficiency, and trau ma. diagnosis is made by the typical lesions, if present, virus isolation, and serology. seroconversion to each virus (rv or h-l) can be detected by serum neutralization, hemagglutination inhibition, complement fixation, and immunofluorescence. hemagglutination inhibition is currently the most commonly used means of antibody determination (jacoby et ai, ) . since rv infection is usually silent and persistent and can be transmitted either vertically or horizontally, effective control is best achieved by destroying the entire population, decon taminating, and repopulating with clean stock. virus-free rats can be obtained from selected commercial vendors or by caesarean rederivation. rederived progeny must be tested for vertically transmitted strains of virus. colonies can be kept virus-free by limiting entry to seronegative, virus-free rats (as well as transplantable rat neoplasms or tissues), periodic serological testing, and adequate physical containment. although parvovirus infection of rats is usually inapparent, there can be adverse effects on the research usefulness of in fected rats. immunosuppression may exacerbate illness and mortality in latent carriers. the viruses often contaminate transplantable tumors and cell lines, can modify immune re sponsiveness or cause teratological effects. a decision to work with infected animals should be made carefully. b. other dna virus infections. rats are susceptible to rat cytomegalovirus, which has a predilection for the salivary and lacrimai glands. infection is widespread among wild, but not laboratory rats (jacoby et al., ) . rats also seroconvert to mouse adenovirus, but it is not known if infection is due to a mouse or rat strain of virus. adenovirus-like inclusions have been reported in the intestine of rats treated with cancer chemotherapeutic agents (ward and young, ) . c. siaiodacryoadenitis virus and related coronaviral infections. two strains of coronavirus have been identified as pathogens of laboratory rats: siaiodacryoadenitis virus (sdav) and rat coronavirus (rcv). furthermore, rats are experimen tally susceptible to the coronavirus of mice, mouse hepatitis virus (mhv). coronaviruses are large, pleomorphic enveloped rna viruses with surface peplomers or spikes that confer a corona-like appearance to the virion. viruses of this group have complex antigenic interrelationships and cross-react ex tensively. common antigens are shared by sdav, rcv, and mhv, particularly by complement fixation, but antibody reac tivity is highest with homologous virus. siaiodacryoadenitis virus and rcv represent different strains of the same virus, but whether different strains of the same virus or separate viruses, they are both important natural pathogens in rats. the signifi cance of mhv for rats is not known, but the virus can replicate in the respiratory tract of intranasally inoculated rats (taguchi et al., ) . natural antibodies to mhv can occur in rats, but this is probably due to the closely related antigenicity of mhv to sdav and rcv rather than natural mhv infection of rats (barthold, ) . clinical signs of sdav infection vary widely in severity, but include blepharospasm, sneezing, porphyrin-pigmented nasal and ocular discharge, and cervical edema (fig. ) . some rats develop keratoconjunctivitis and other ocular lesions. signs persist approximately one week, but ocular sequellae can be permanent. acutely infected rats become anorectic, and estrus can cease temporarily. infection is subclinical in weanling or older rats, but intranasally inoculated neonates die and suck lings develop lower respiratory disease. siaiodacryoadenitis virus is highly contagious and spreads rapidly among susceptible rats by contact, aerosol, or fomite. susceptible rats of any age can be infected. when enzootic within a colony, clinical disease occurs only in sucklings, since adults are immune. infection is acute, lasting only about week, at which time rats seroconvert with no carrier state. maintenance of sdav in a colony requires continuous intro duction of susceptible stock as weanlings or newly introduced rats. the epizootiology of rcv is presumed to be similar to sdav. within days of intranasal inoculation, sdav causes rhi nitis followed by necrosis of the ductular and acinar epithelium of salivary and lacrimai glands, accompanied by intense in flammation and edema. tracheitis and peribronchial lymphoid hyperplasia can also be found. salivary glands appear swollen, pale, with interlobular and periglandular edema. harderian glands are flecked with yellow-gray foci. one, some, or all of the salivary or lacrimai glands can be affected, with the excep tion of the sublingual glands, which are spared. cervical lymph nodes become enlarged. glandular repair ensues within week, with squamous metaplasia of ductular epithelium and hyperplasia of acinar epithelium. the repair phase subsides within days with minimal residual lesions. interstitial pneu monia can occur in suckling, but not adult rats. conjunctivitis, keratitis, corneal ulcers, synechia, hypopyon, and hyphema can arise due to lacrimai dysfunction. eye lesions usually re solve, but can proceed to chronic keratitis, megaloglobus (fig. ) , and retinal degeneration. rat coronavirus infection causes rhinotracheitis and focal interstitial pneumonia. salivary but not lacrimai gland infection is rare, but when present, resem bles wild sdav lesions. infection with rcv also lasts approx imately week (barthold, ; jacoby et al., ) . nasal and ocular signs must be differentiated from those caused by mycoplasma, sendai virus, pathogenic bacteria, ex cess ammonia, or hypovitaminosis a. cervical swelling may fig. . epiphora and swelling of the ventral neck in a rat naturally infected with sdav. (from barthold, ; courtesy of hemisphere publishing corp.) fig. . megaloglobus and hyphema in a young rat naturally infected with sdav. (from barthoid, ; courtesy of hemisphere publishing corp.) also occur in immunosuppressed rats infected with p. aeruginosa. microscopic sdav lesions are characteristic. mild lower respiratory tract lesions associated with rcv must be differentiated from those of sendai virus or pneumonea virus of mice (pvm). seroconversion or rising complement fixing antibody titers following acute disease is confirmatory. how ever, antibody may be low or undetectable with this method. serum neutralization is another test that can be used, but the most sensitive antibody tests are immunofluorescence or elisa. either mouse or rat coronaviruses are used as antigen in these latter tests (smith, ) . rats can be kept free of sdav and rcv if they are isolated and if newly introduced rats are immune or unexposed. intro duction of a single subclinically infected rat can precipitate epizootic disease among naive rats. if an outbreak occurs, the infection will run its course and die out within - weeks if new rats are not introduced into the room and if breeding is temporarily ceased. routine disinfection of rooms and equip ment is sufficient to destroy environmental sources of virus. sialodacryoadenitis virus lesions can be confused with or contribute to changes induced by test compounds or nutritional deficiencies, particularly vitamin a. sialodoacyoadenitis virus disease can predispose to anesthetic death due to airway hypersecretion. eye lesions resulting from sdav infection can in terfere with eye research. both sdav and rcv can potentiate other respiratory infections. d. sendai viral infection. sendai virus commonly infects laboratory rats, but its clinical significance is less than in mice. sendai virus is a parainfluenza virus of the paramyxovirus family. paramyxoviruses are pleomorphic, enveloped, labile rna viruses. sendai virus infection in rats is usually subclinical, but can be manifested as ruffled fur, dyspnea, or anorexia. a decrease in average litter size and runted pups is common during outbreaks in breeding colonies. sendai virus is highly contagious and disseminates rapidly. outbreaks subside following development of an immune popu lation, with the potential of recurrence several months later as the susceptible population enlarges. sendai virus induces an acute respiratory infection with no natural carrier state. excre tion and transmission of virus occurs via the respiratory tract (jacoby <?r Ö/., ) . sendai viral lesions in the rat are similar to those in genet ically resistant strains of mice. following an initial necrotizing rhinitis, tracheobronchitis ensues within week of infection. lungs become consolidated and plum-colored in a patchy, pri marily anteroventral, distribution. necrotizing bronchiolitis, focal nonsuppurative interstitial pneumonia, and perivascular and peribronchial lymphoplasmacytic infiltrates are the hall marks of the lower respiratory component. in the rat, acute lower respiratory lesions last only a few days and coincide with clinical disease. repair of bronchial mucosa is effected through hyperplasia and squamous metaplasia. residual le sions can last for months in the mouse, but probably not in rats. during the acute stage of sendai virus infection, mucosal necrosis and inflammation can extend up the eustachian tubes to the middle ear. bacterial otitis media is often preceded by sendai viral infection in rodents (brownstein et al., ; d. g. brownstein, unpublished observations, ) . mild nonsuppurative pulmonary lesions can be seen with sendai, pvm, rcv and to a lesser extent, sdav. sendai virus can be superimposed upon and contribute to respiratory mycoplasmosis. squamous metaplasia of respiratory mucosa can mimic hypovitaminosis a and also occurs in my coplasmosis. virus isolation and serocon version or rising titers to sendai virus confirm a diagnosis based upon characteristic lesions and clinical signs. sendai virus antibody can be accu rately detected by complement fixation and hemagglutination inhibition, but newly developed immunofluorescence and elisa are replacing these methods (smith, ) . rats can be kept free of sendai virus in a similar manner as with sdav and rcv. recovered sendai virus antibody-posi tive stock can be utilized as breeders to reestablish a virus-free population. pups born of these parents will be virus-free and antibody negative, following decay of their maternal antibody. mice and hamsters can serve as sources of infection with paramyxoviruses. sendai virus infection, although usually subclinical, can cause significant pulmonary changes and act as a copathogen with mycoplasma or bacteria. squamous metaplasia and hy perplasia induced by these agents influence respiratory carcinogenesis and interpretation. sendai virus can cause immunosuppression for prolonged periods in rats (garlinghouse and vanhoosier, ) . e. pneumonia virus of mice infection. pneumonia virus of mice is a pneumovirus of the paramyxovirus family and thus shares many features in common with sendai virus. it has not been associated with clinical disease in the rat or mouse under natural conditions. several rodent species, including hamsters, guinea pigs, and gerbils, seroconvert to pvm under natural conditions. the virus seems to spread rapidly among suscepti ble rodents, based on serocon version. it is presumed to be transmitted in a manner analogous to sendai virus. the pathogenesis of pvm has been investigated in mice, but has not been thoroughly examined in rats, even though it pro duces more significant pulmonary lesions in rats than mice un der natural conditions. in the mouse, pvm replicates in the upper respiratory mucosa without obvious cytolysis following low-dose exposure (d. g. brownstein, unpublished observa tions, ) . viral antigen and cytolytic lesions are seen in the bronchial and bronchiolar epithelium as well as alveolar walls following apparently higher doses of virus in a pattern reminis cent of sendai virus. the ensuring lesion, like that of sendai virus, is necrotizing bronchiolitis and interstitial pneumonia (carthew and sparrow, ) . the active infection lasts about week, after which time lymphocytic infiltration of peribronchial and perivascular tissues and focal interstitial pneumonia can persist for several weeks. rat lungs develop prominent nonsup purative perivascular and mild interstitial inflammation in re sponse to both experimental and natural pvm infections (d. g. brownstein, unpublished observations, ; vogtsberger et al., ) . infection is usually diagnosed retrospectively in rats, where pulmonary lesions are observed following seroconversion to pvm in the absence of other respiratory pathogens. pulmonary lesions mimic those of sendai virus or possibly coronaviruses. antibody to pvm has usually been detected by hemagglutination inhibition or serum neutralization, but these tests are being replaced by immunofluorescence and elisa. hemagglutination inhibition is least expensive and highly accurate, however (smith, ) . pulmonary lesions induced by pvm can inter fere with the interpretation of experimental studies, or possibly pulmonary function, although this has not been established. hemorrhagic fever with renal syndrome, korean hemorrhagic fever, or muroid virus nephropathy is a human disease com plex caused by one or more serologically related bunyaviruses, including hantaan virus. this syndrome is mentioned here be cause of its emerging eminence as a major zoonotic disease in which several species of the suprafamily muroidae, including laboratory rats, can serve as carriers. the virus is transmitted to man by excretions and aerosols from the lungs, saliva, and urine of healthy rodent carriers. pathology, if any, in rats has not been described. the disease in humans includes proteinuria, azotemia, and, less often, petechiae, hemoconcentration, hypotension, and renal failure. human mortality can oc cur, but recovery with immunity to reinfection is usual. hemorrhagic fever with renal syndrome viruses cause human disease throughout much of eurasia, but on the basis of serological evidence, hfrs viruses exist world-wide in feral rodents and humans, including the united states. transmis sion of hfrs to laboratory personnel by silently infected al bino laboratory rats (rattus norvegicus) has been documented in japan and belgium. virus-infected lung and kidney cell lines provide excellent sources of antigen for sensitive immu nofluorescence tests, but serological monitoring of laboratory rats for hfrs virus antibody is not currently a common prac tice (gajdusek et al, ; anonymous, ) . g. other virus infections. rats harbor endogenous retroviruses or genomic retrovirus sequences that (in contrast to the mouse) are of minimal significance in terms of natural disease. a number of endogenous rat sequences have been artificially / / / recombined with mouse leukemia virus sequences or naturally recombined with other rat retrovirus sequences to form defec tive rat sarcoma viruses, for example kirsten and harvey rat sarcoma viruses, which are experimentally oncogenic to rats, mice, and hamsters (shih and scolnick, ) . natural seroconversion to reovirus- and mouse encephalomyelitis virus have been noted with no apparent disease. an enterovirus with neurotropic properties has been isolated from naturally infected, asymptomatic rats. although rats can be experimen tally infected with ectromelia and lymphocytic choriomeningitis virus, these agents are not of practical significance as natu ral infections of rats. testing for antibody to these agents is therefore a vacuous exercise. a number of other viruses or viruslike agents have been isolated or incriminated as causes of disease in rats, including rat submaxillary gland virus, novy virus, enzootic bronchiectasis agent, gray lung virus, and wild rat pneumonia agent. these agents, if extant at all, are of du bious importance in contemporary laboratory rat populations (jacoby et ai, ) . recently, clinically silent infection with an unidentified pox virus has been found in soviet rats. micro scopic necrotizing and inflammatory changes were restricted to the nose (kraft et ai, ) . the agent is believed to be cow pox virus. laboratory rats are host to far fewer parasites than wild rats, since husbandry practices interrupt complex life cycles and caesarean rederivation has simply eliminated many agents al together. control of these agents in infected colonies is also best achieved by these means. this section will mention only agents that have been reported in laboratory rats, although most are unlikely or rare. table ix outlines selected treatment regimens for control of common metazoan parasites, but the reader must beware that these drugs may render the treated rat useless for experimental work. decisions to treat rather than eliminate infected rodents must be made with care, in concert with the needs of the scientific investigator. a. protozoal infections. several flagellates can parasitize, rats but few are truly pathogenic. hemoflagellates (tripanosoma cruzi, t. lewisi) occur in wild rats, but these agents require anthropod vectors. nevertheless, t. lewisi can be en countered if a colony is infested with rat fleas. trypansoma lewisi is only mildly pathogenic even in heavy infections, and infection is short term. rats harboring this organism become ill when stressed or immunosuppressed. enteric flagellates (tritrichomonas sp., tetratrichomonas sp., pentatrichomas sp., trichomitis sp., hexamastix sp., enteromonas sp., retortamonas sp., chilomastix sp., monocercomonoides sp., and octomitus sp.) are very common but nonpathogenic in labora tory rats. giardia muris and spironucleus (hexamita) muris application - gm/liter drinking water for days, stop days, re peat days . gm/liter drinking water for days mg/liter drinking water or . mg/kg feed for weeks . mg/gm feed for day mg/g sc, single dose mg/kg po, single dose . - . % dust with or without pyrethrin hr/week on cage top for several weeks - gm of pellets in bedding for days, change bedding and cage, repeat at days, change and repeat at month . % dip - % dust, up to . % spray or up to % dip, repeat at days up to . % dip % spray "information kindly supplied by dr. j. w. streett, yale university. ; 'a number of these compounds have adverse effects on the experimental usefulness of treated rats and therefore should be used with discretion and full knowledge of the investigator. are also common and considered potentially pathogenic, caus ing unthriftiness, diarrhea, and intestinal lesions in severely affected animals. however, these are generalizations made from other species, since natural clinical disease has not been reported among rats. sporozoan parasites, common in wild rats, are encountered only on occasion in laboratory rats. hepatozoon muris infec tion is usually subclinical, but severely affected rats can have leucocytosis, splenomegaly, anemia, hepatic degeneration, emaciation, or death. infection requires ingestion of the vec tor, laelaps echidninus. toxoplasmosis (toxoplasma gondii) is subclinical in rats, requiring the ingestion of cat feces or contaminated tissues containing asexual stages, but can also be transmitted transplacentally. intracellular t. gondii organisms occur in several tissues, particularly lung and reticuloendothelial organs, and cysts are found in the central nervous system. sarcocystis muris, once common but now rare in labo ratory rats, has a life cycle similar to Ã. gondii. cysts are found in the muscle. frenkelia sp. has been described in a colony of laboratory rats, causing large thin-walled cysts and inflamma tion in the central nervous system, but no clinical signs. encephalitozoon cuniculi is apparently now uncommon in labora tory rats and seldom induces signs or lesions. its prevalence and significance are far greater in the laboratory rabbit. single or clusters of small spores form within cysts in a number of tissues. nonsuppurative inflammation can be found in the brain and kidneys. unlike t. gondii, e. cuniculi organisms are gram-positive. encephalitozoon cuniculi can be transmitted via contaminated transplantable tumors, tissue, or tissue fluids from infected rats. intestinal sporozoans include eimeria nieschulzi and e. miyairii, which infect the small intestine, and e. separata, which infect the large intestine of wild rats. they are exceedingly rare or nonexistent in laboratory rats. pneumocystis carinii, once considered to be a yeast but now considered a sporozoan, is an ubiquitous organism that infects the lung of a variety of species, including laboratory rats. prev alence of infection is high within infected colonies, but preva lence among various geographic areas in unknown. it seems to be very common among both conventional as well as barrierreared rats. normally p. carinii is not pathogenic, but infected rats can develop disease after several weeks of repeated steroid injections and starvation, particularly in younger rats. remark ably, rats from many commercial sources can be so treated and shown to be infected, even if free of other pathogens. under these circumstances, the lungs are expanded and rubbery. al veolar walls are thickened and hypercellular, and alveoli are distended with proteinaceous, eosinophilic foamy material containing macrophages and organisms in different stages of development. tissues can be stained with special stains, such as methanamine silver or periodic acid-schiff, to visualize the organisms, which are not visible with hematoxylin and eosin. other protozoa, including entamoeba muris and balantidium coli, are found in the large bowel of rats, but are not pathogenic. the reader is referred to more exhaustive coverage of protozoa for detailed means of diagnosis (flynn, ; hsu, ) . b. nematodiasis. syphacia muris is a common oxyurid (pinworm) of the cecum and colon. embryonated eggs are passed in the feces or deposited on the anus. rats are infected by ingestion of eggs or by larval migration into the colon from the anus. syphacia muris closely resembles s. obvelata, the mouse pinworm. both species of syphacia can patently infect rats or mice, but preference seems to be shown for the homolo gous host. another common oxyurid of rats and mice is aspicularis tetraptera, which is also found in the cecum and colon. aspicularis eggs are nonembryonated, and are not de posited near the anus. depending on parasite species, the pré paient period is - days. diagnosis is achieved by exam ination of large bowel contents (particularly the cecum and proximal colon) for nematodes, feces for ova, and, in the case of syphacia, cellophane tape impressions of the anus for ova. speciation is easily achieved by the differential morphology of ova. aspicularis ova are symmetrically ellipsoidal, while syphacia ova are flattened on one side. since eggs are ex tremely resistant to environmental factors and disinfectants, and autoinfection can occur with s. mûris, infection is difficult to eradicate. anthelminthic treatment ameliorates infection (table ix) , but all rats must be treated simultaneously and the room must be thoroughly cleaned. despite these actions, rein fection frequently occurs. caesarean rederivation and subse quent strict sanitation are the most effective means of control. pinworms are generally considered nonpathogenic, but heavy parasite loads can be deleterious to the host. concern has been raised regarding the effects of these agents on research but no effects have been documented in the rat. trichosomoides crassicauda is common in some rat colonies but is not generally encountered in commercially raised ani mals. it inhabits the transitional epithelium and lumen of the urinary tract. the small male resides within the reproductive tract of the female, whose anterior end embeds within the mucosa. embryonated ova with bipolar opercula are shed in the urine. following ingestion, eggs hatch in the stomach, lar vae penetrate the gastic wall, then migrate via the lungs, kidney, and ureter to the urinary bladder. migrating larvae can incite eosinophilia and formation of granulomata, particularly in the lung. drug treatment (table ix) is effective, as is cesarean rederivation. parasites do not incite an inflammatory response in the bladder, but can serve as a nidus for calculi or enhance natural and experimental bladder carcinogenesis. other nematodes are common in wild but rare in laboratory rats. trichinella spiralis adults occur in the intestine, and lar vae migrate extensively, encysting in muscle. transmission is effected by ingestion of contaminated meat. capii i aria hepatica eggs are ingested, hatch, and migrate through the cecum to the liver through the portal veins. adults mature and lay characteristic bipolar operculate ova in the liver, where they lie dormant until ingested by a carnivore. gongylonema neoplastium burrows in the squamous epithelium of the tongue and anterior stomach, where it incites a proliferative response or ulcération. the life cycle involves an intermediate insect host. angiostrongylus cantonesis, which requires the ingestion of a mollusk intermediate host, infects the brain and lung. the intestinal nematodes heterakis spumosa, nippostrongylus brasiliensis, strongyloides ratti, and trichuris muris lack in termediate hosts, but rarely occur naturally in laboratory rats. none is known to be very pathogenic. further details on nematode diagnosis and ova morphology are available (flynn, ; hsu, ; oldstone, ) . taenia taeniaformis, the cat tapeworm. following ingestion of ova, oncospheres migrate to the liver and form strobilocerci (cysticercus fasiolaris). host connective tissue capsules can give rise to sarcomas. this parasite has been used as a model of parasite-induced oncogenesis in the rat. cysticercus fasciolaris is on occasion encountered in laboratory rodents with contaminated food or bedding. hymenolepis nana and diminuta infect the small intestine of several species, including the rat. hymenolepis nana, the dwarf tapeworm, has a -mm wide segmented body ranging from to mm in length. the life cycle may be either direct or indirect. following ingestion of ova, oncospheres penetrate the intestinal mucosa, form cysticercoid larva, then emerge into the lumen and mature into adults that shed eggs in the feces. nonimmune hosts can be autoinfected; eggs are pro duced, hatched, and complete their life cycle within the intes tine of a single host. the indirect cycle involves intermediate hosts, such as grain beetles or fleas, in which cysticercoid lar vae form and await ingestion by the definitive host. hymenolepis diminuta is - mm wide and - mm long. the eggs are passed in the feces but must be ingested by an intermediate host (flour beetle, flea, moth), which in turn must be ingested by the definitive host to complete the life cycle. enteritis can occur in heavy infestations, which are most likely to be encountered with h. nana. both can infect primates, in cluding humans. hymenolepis nana, which does not need an intermediate host and can cause autoinfection, is a particular public health hazard. anthelmintic treatment is effective (table ix) . diagnosis of hymenolepis is made by finding ova containing hexacanth embryos in the feces or adults in the lumen of the bowel. differential features are detailed else where (hsu, ) . d. insect infestation. two anopluran (sucking) lice infest wild rats, polyplax spinulosa (the spined rat louse) and hoplopleura pacifica (the tropical rat louse). the latter has not been reported among laboratory rats. polyplax spinulosa is now rare in most laboratory rat populations. it completes its entire life cycle within the fur of the host and is transmitted by direct contact. infested rats are unthrifty, irritable, pruritic, restless, and anemic. polyplax spinulosa can transmit a num ber of infectious agents, which include hemobartonella muris and t. lewisi. diagnosis is made by identifying organisms in the fur. rat fleas (xenopsylla sp., leptopsylla sp., and nosopsyllus sp.) are rare in laboratory rats, since their life cycles are usu ally interrupted by proper sanitation. fleas can transmit other agents and can serve as the intermediate host of h. nana and diminuta (flynn, ; hsu, ; oldstone, ) . see sec tion iii,a, ,e for methods of control. e. arachnid infestation. mesostigmate mites can be en countered on occasion in laboratory rats. they are rapid blood suckers and have a nonselective host range. they are on the host only during feeding, spending much of their life cycle secreted in the environment. diagnosis must be attained by finding engorged mites on bedding, cages, and crevices. other hosts, including humans, fall prey to their painful and irritating bites. the most important mesostigmate is ornithonyssus bacon (tropical rat mite). heavily infested colonies contain de bilitated, anemic rats with reduced reproductive efficiency and occasional deaths. liponyssoides sanguineus has not been re ported in laboratory rats, but may be unrecognized because of its similarity to ornithonyssus sp. laelaps echidninus (spiny rat mite) does not adapt well to the laboratory rat environment unless husbandry is poor. it is a vector of hepatozoon. prostigmate mites have a more selective host range and spend their life cycle in the fur (or follicles) of the host. radfor dia ensifera, the rat fur mite, can be common in some rat colonies. this mite produces few ill effects, but heavy infesta tions can induce self-inflicted trauma (flynn, ; hsu, ; oldstone, ) . demodex sp. is also encountered, but its prevalence is unknown, since it lives deep within hair follicles and sebaceous glands where it produces minimal lesions (walbtxgetal., ) . astigmate mites include the mange mites, which have a se lective host range and complete their life cycle on the host. notoedres muris, the ear mange mite, infests the skin of the ear and to a lesser extent, the nonglabrous regions of the body. this mite burrows in the cornified epithelium, and elicit a pruritic dermatitis (flynn, ; hsu, ) . it is seldom seen in contemporary rat colonies. control of ectoparasites is gained by preventing the introduc tion of infected animals (including wild rodents), sanitation, treatment of rats, and, in the case of mesostigmates and fleas, treatment of the environment (table ix) . treatment seldom completely eliminates infestation and can have an adverse ef fect on the usefulness of the treated rats for research. repopulation or caesarean rederivation and subsequent preven tion by proper management is the best approach. deep mycoses are generally not considered to be significant natural diseases in laboratory rats. pulmonary aspergillosis is occasionally observed in rats and mice, particularly when immunocompromised. aspergillus sp. can be readily isolated or observed in affected lung tissue with selected histochemical techniques. pulmonary lesions consist of miliary granulomata containing langhans giant cells in areas with characteristic uniformly branched, septate hyphae. phycomycotic encepha litis has been reported as a natural disease in -to -week-old rats. phycomycotic fungi have thick, irregularly branched, nonseptate hyphae in tissue. dermatomycosis is probably more likely to be encountered than deep mycosis, but is also rare in laboratory rats. ring worm in rats is restricted largely to infection by trichophyton mentagrophytes. infected rats can be lesionless carriers or dis play patchy alopecia and erythema with scale, papule, or pus tule formation. fungal elements are visible within the stratum corneum and hair follicles. ecothrix spore formation in hair shafts can be present. the disease can be diagnosed by exam ination of skin scrapings treated with % potassium hydrox ide, histological sections of skin prepared with fungal stains, or isolation (weisbroth, ) . a. genetic anomalies. genetic anomalies in coat color and character, organ development, immune responsiveness, obesity, hypertension, metabolism, and others have been iden tified and in many cases developed as specific characteristics of various stocks and strains. the reader is referred tö several more reviews for further information (altman and katz, ; hansen et al., ; robinson, robinson, , . some strains nat urally develop disease syndromes, such as brattleboro rats with diabetes insipidus, while other strains have less obvious characteristics but react uniquely to different research vari ables. the researcher must be aware of these variations and judiciously select the appropriate stock or strain to accomplish the goals of the experiment. each stock, strain, substrain, and even group of rats that has drifted from its parental stock can develop its own unique dis eases, manifest its own incidence and rate of development of various spontaneous lesions, and react in different ways to in fectious and research variables. expression of genetic traits is further influenced by extraneous factors such as husbandry and diet. b. nutritional deficiencies. more is known about the nu tritional requirements and deficiencies of rats than perhaps any other species. natural deficiencies of single dietary compo nents are rare and seldom recognized. this is because rats ef fectively store fat-soluble vitamins (and b ), manufacture vi tamin c, and fulfill much of their requirement for b vitamins by coprophagy. furthermore, some deficiencies are influenced or compensated for by other dietary elements. the most com mon signs of nutritional deficiency are vague, such as poor hair coat, reduced weight gain or growth, diminished fertility, and susceptibility to infectious disease. commercially avail able rodent diets effectively supply balanced diets to rodents, but diets should not be utilized beyond their expiration date and ideally should be kept in cold storage. several components deteriorate with prolonged storage, heating, or sterilization, in cluding lysine, vitamins a and e, and, to a lesser extent, riboflavin and thiamin. nutritional requirements vary with the genetic strain, stage of life, and microbiological association. axenic or specific pathogen-free (spf) animals can lack gut microflora necessary for supplying certain vitamins, particu larly vitamin k. they must receive supplemented diets both because they need more and because autoclaving or pasteuriza tion reduce the levels of certain essential nutrients. antibiotic treatment, which affects intestinal microflora, can reduce the bacterial source of vitamins by coprophagy. nutritional ade quacy goes far beyond the needs of the rat, since the composi tion of nutritionally adequate diets is known to profoundly in fluence the biological responsiveness to research manipula tion, infectious disease, and expression of spontaneous lesions (national research council, ; rogers, ) . the signs of nutritional deficiency are often vague and must be differentiated from other syndromes. hemorrhage due to hypovitaminosis k can be confused with rv disease (which in itself can be precipitated by nutritional deficiency). infertility or poor production can be caused by rv, sdav, sendai virus, m. pulmonis, or nuturitional deficiency. squamous metaplasia in the salivary and lacrimai glands of rats recovering from sdav or in the respiratory tract of rats infected with sendai virus or m. pulmonis must be differentiated from hypo vitaminosis a, which in turn predisposes to respiratory infec tions. environmental factors, such as excess heat or low hu midity, contribute to infertility or skin disease which mimic hypovitaminosis e or other deficiencies. ordinarily mild infec tious diseases can become severe, such as pseudotuberculosis, due to nutritional deficiency. the diagnosis of many rat dis eases therefore requires review of nutritional practices. practices other than nutrition can also influence or cause dis ease in rats. sanitation is of obvious import. cage design, pop ulation density, and temperature influences have been men tioned in sections ii,d and e. outbreaks of opportunistic in fectious disease, such as pseudotuberculosis, can be precipi tated by sudden fluctuations in temperature and humidity. pseudomonas aeruginosa can be introduced and spread within a colony through inappropriate sanitation, particularly of water bottles. refilling water bottles at a faucet in the animal room is not advised for this reason. bottles and sipper tubes should be sanitized and water should be acidified or chlorinated. exces sive ammonia from dirty bedding, overcrowded cages, or poor ventilation predisposes to respiratory infections, particularly mycoplasmosis. a number of management-related syndromes can occur that are not related to sanitation or infectious disease. rats adapt well to automatic watering systems, but animals unfamiliar with these devices can become dehydrated. similarly, mal function of these systems and blockage of water bottle sippe tubes with metal filings or other detritus can have a similar effect. rats maintained for long periods on wire-bottom cages develop foot sores, which can result in severe lesions, discom fort, hemorrhage, and anemia. aging rats must be examined periodically for overgrowth of their incisors, a very common problem in rats held long term. teeth must be carefully clip ped, avoiding splitting or shattering. dusty bedding and food can result in foreign body pneumonia. plant, mineral, and even bone fragments can be found in sections of lung obtained from rats under these circumstances. softwood shavings used as bedding can cause intestinal impaction, particularly in young rats. high ambient light, or even light levels within the recom mended range, can cause retinal degeneration and cataracts in albino rats. rats maintained near ceiling light fixtures develop retinal degeneration, while those near the floor are spared. low relative humidity (< %) in concert with high tempera ture and other factors can cause one or more annular constric tions of the tail skin (ringtail) or toes. the segments distal to the constrictions swell or undergo dry gangrene (fig. ) . this is most apt to occur in suckling or preweanling rats and in rats kept in wire-bottom cages. increasing humidity, reducing air turnover rate, placing rats in solid-bottom cages, and providing nesting material for nursing dams and their litters are all ame liorative for this condition. axenic rats normally have enlarged ceca, which become ap proximately five times normal size. there is net efflux of fluid into the lumen in response to increased osmotic pressure of luminal content, reduced availability of ions needed for mucosal solute-coupled water résorption and vasoactive com pounds which exert their effect on smooth muscle tone. cecal enlargement can interfere with reproduction. dietary manip ulation can reduce the enlargement substantially (foster, ) . the effects on smooth muscle tone become pronounced in aging axenic rats, which can develop progressive cecal en largement due to atony. volvulus and torsion can result. the reader must be cognizant of the much larger list of en- vironmental variables that significantly alter physiological re sponsiveness of test animals (baker et al., a; gralla, ) . unlike the mouse, traumatic skin disease in the rat is more likely to be due to self-inflicted trauma than fight wounds. rats infested with ectoparasites or dermatophytes can develop excoriative dermatitis due to self-inflicted trauma. roughly sym metrical ulcerative dermatitis over the dorsal and lateral neck and shoulders has been reported (fig. ) . the etiology re mains to be determined, but coagulase-positive staphylococcus aureus is consistently isolated from the skin lesions, and colonies of gram-positive cocci are microscopically visible on the surface of the lesion. attempts to induce the disease in other rats with this agent have yielded inconsistent results (fox et al, ) . the rat is often considered "resistant" to infection and ac cordingly submitted to a variety of experimental procedures without regard to aseptic techniques. peritonitis and wound in fections are often found under such conditions. inappropriate handling of large rats by the tips of their tails will cause the skin to tear and slip off when they struggle. adynamic ileus occurs in rats given intraperitoneal injections of anesthetic preparations containing chloral hydrate or related compounds (fig. ) . for variable periods up to weeks after treatment, rats become lethargic, anorectic, and constipated with distended abdomens that may lead to death. gross nec ropsy findings reveal segmental atony and distension of the bowel, usually jejunum, ileum, and cecum. focal serosal in flammatory changes and fibrosis are seen microscopically (fleischman et al., ) . this syndrome mimics megaloileitis (tyzzer's disease), but can be differentiated on the basis of history, lack of characteristic bacteria in tissues, and lack of liver and heart lesions. in.addition, it should not be confused with the bowel dilatation observed in axenic rats. space exists in this chapter to only summarize the more fre quently reported tumors in the rat, and the topic will not in clude experimentally induced tumors. the reader is encouraged to refer to the articles and monographs referenced in the text for more complete information. there have been numerous studies done to assess the inci dence of neoplasia in various stocks and strains of rats (burek, ; coleman et al., ; international agency for research on cancer, mackenzie and garner, ; ringler and dabich, ; sher, ) . some of these have led to the development of animal models and provided baseline data for experimental carcinogenesis work for which the rat is regularly used. there is a wide range of values reported in regard to the prevalence and type of tumors found in a particular rat stock or strain. these differences are a function of genetic and environ mental influences and differences in sample selection. nutri tion is well known as a factor that influences the development of neoplastic disease. for instance, it has been shown that a high fat diet enhances tumorigenesis, that the protein-calorie ratio influences the occurrence of some tumors, and that re stricted food intake of post weanlings for or more weeks de creases tumor risk (ross and bras, ) . most of the surveys on tumor occurrence in rats either state nothing in regard to the presence of enzootic infectious disease or acknowledge that infectious diseases were present in the population. in many instances, infectious diseases can signifi cantly influence data on tumor risk because of their effect on longevity, preneoplastic changes, and masking of small tumors. with the exception of mammary fibroadenomas in many stocks and testicular tumors in f rats, the prevalence of tumors is quite low in rats under months of age. according ly, the age at which rats are surveyed is very important in de fining the incidence of neoplasia in a particular stock or strain. the f rat is used in the bioassay program of the national cancer institute and is widely used in toxicology studies. it has been selected for these types of studies because of its small size, longevity, and low incidence of most tumors. however, this strain has a moderate to high incidence for some tumors, such as those of the testis, mammary gland, uterus, and hematopoietic and endocrine systems (sher, ) . a sprague-dawley-derived stock that is commonly used in carcinogenesis studies is the crl:cd(sd)br rat. this stock has a high inci dence of mammary tumors and pituitary adenomas. papillomas and squamous cell carcinomas occur infre quently, but have been reported in many stocks of rats. squam ous cell carcinomas are usually located on the face and head, and tend to be highly invasive but not metastatic. most of these arise as sebaceosquamous tumors from the glands of zymbal in the ear. similar tumors occur on the prepuce and clitoris. in females from most stocks and strains, the mammary gland is the most frequent site of neoplasia, with incidences of - % being common. these tumors occur in males but much less frequently. benign fibroadenomas are the most common type, but adenocarcinomas may also occur. fibroadenomas have ductal epithelium and periductular connective tissue compo nents and tend to be less vascular than carcinomas. adenocar cinomas can metastasize to regional lymph nodes and the.lung. tumors of the mammary gland, which may arise at any site from the neck to the inguinal region, often attain a very large size (fig. ) . the prevalence of tumors in the alimentary tract is extremely low, with most cases involving the colon. although hepatic tumors are readily induced by chemical carcinogens, very few spontaneously occurring malignant neoplasms have been re ported. more commonly occurring are neoplastic nodular le sions. the term ò 'neoplastic nodule" is used to describe cir cumscribed nodules of proliferating hepatocytes that compress the parenchyma (altman and goodman, ) . there is some fig. . mammary fibroadenoma in an aged rat. controversy as to whether these nodules are in all cases neo plastic. however, there is evidence that some develop into carcinomas. leukemia is rare in many stocks and strains of rats. howev er, mononuclear cell leukemia is very frequently seen in f and wf rats. in one survey of male f rats, an incidence of % proved leukemia second in occurrence only to the testic ular interstitial cell tumors. the leukocyte count in leukemic rats ranged from , to , cells/ìÀ with an average of , (coleman et al., ) . other studies indicate a simi lar incidence for the wf rat. in the bn/bi rat, myelomonocytic leukemia has been reported to be the most common lymphoreticular tumor, with female and male incidences of and %, respectively (burek, ) . both types of leukemia oc cur in rats over months of age. neoplasms of the pituitary occur frequently with a higher proportion in females. surveys have shown an incidence of - % in f rats, % in om (osborne-mendel) rats, and - % in sprague-dawley rats. the most common pituitary tumor is the chromophobe adenoma. these are benign tumors that frequently become quite large, and, due to compression of the brain by the tumor, hydrocephalus and neurological signs may occur. they appear dark due to hemorrhagic areas arising from a vascular sinusoid component of the tumor. clinical manifestations may include neurological signs or cachexia, but these are often absent. pancreatic islet cell tumors are common in some stocks and strains. surveys have reported a % incidence in f rats, . % in holtzman rats, and a - . % in sprague-dawley stocks. tumors of the thyroid are usually parafollicular cell ade nomas. these benign tumors occur in many stocks with an incidence varying between and %. follicular cell car cinomas, which occur infrequently, can metastasize to the lung. adenomas of the adrenal cortex are very common in several strains. in at least three strains, buf/n, m /n, and om/n, over % of the females and % of the males have cortical tumors. pheochromocytomas are the most common medullary tumors. they are particularly common in the buf/n, f /n, m /m, and wn/n strains, and, unlike cortical tu mors, they appear more often in males. tumors of the brain have been found to occur rarely in those studies that included examination of the cns. less is known about the prevalence of tumors in the cns than in other sys tems because many studies have not included brain and spinal cord examination. the astrocytoma is the most commonly re ported tumor of the cns. less often reported tumors include oligodendroglioma, meningioma, ependymoma, and granular cell tumors. primary tumors of the lung are rare in the rat. the most fre quently reported types are bronchogenic carcinomas, car cinomas, sarcomas, and adenomas. tumors of the bladder are rare in most stocks and strains of rats. however, the bn/bi strain has an unusually high inci dence of carcinomas of the bladder and ureter. males of this strain have been reported to have a % incidence of bladder carcinoma, while % of females have carcinoma of the ureter (burek, ) . nephroblastomas, renal tubular adenomas, and adenocarcinomas have been reported in the rat kidney. tumors of the prostate are common in the axc rat. in one report, adenocarcinoma of the ventral prostate was histologically detected in % of -to -month-old axc rats. prostatic tumors are reportedly uncommon in most stocks and strains of rats. reportedly, however, some of these surveys did not include adequate sectioning to locate small adenomas and carcinomas. interstitial cell tumors are the most common testicular tumor type. nearly all aged f rats and the majority of aci/n rats develop these tumors, but the tumor is rare in most other strains. tumors of the vagina and cervix are rare, except in aged bn/bi rats. tumors of the uterus and ovary are common in several strains of rats. one om strain was reported to have a % incidence of granulosa cell tumors, and a high incidence of endometrial tumors has been reported in the f and m strains. congenital disorders in the rat are infrequently reported. this is probably a reflection of two factors; the first being that evidence indicates the incidence is extremely low in outbred stocks, and the second being that commercial/institutional sup pliers select against breeders who have produced abnormal young. the genitourinary system and the eye have been most associated with congenital disorders. one substrain of the axc rat has a greater than % inci dence of unilateral renal agenesis and hydronephrosis (fujikura, ) . a high incidence of unilateral and bilateral hy dronephrosis has been found in bn/bi rats (cohen et ai, ) . pseudohermaphroditism has been reported in rats that are phenotypically female but who have an xy karyotype. testicles are present either in the abdomen or inguinal canal. this condition is due to a sex-linked recessive gene that is expressed by an insensitivity of tissues to androgens (bardin et al., ) . a number of ocular disorders have been reported in the rat. retinal dystrophy is inherited as a single autosomal recessive gene (rdy). homozygotes have a progressive loss of the retinal photoreceptor cells and an overproduction of rhodopsin early in the disease process (lavail et al., ) . a retinal degener ation reported in the wag/rij rat appears to be an expression of an autosomal dominant gene. end-stage lesions include disap pearance of photoreceptor cells, migration of the pigment epi thelium, and disorganization of remaining retinal layers. this retinal disease appears to mimic the pathogenesis of retinitis pigmentosa in humans (lai et al., ) . other reported devel opmental disorders of the eye include colobomas and cataracts. reports of congenital heart disease are rare. among the few reports is one involving an inbred strain of long-evans rats (fox, ) . in this strain, approximately % of the neonates have interventricular septal defects. this malformation is be lieved to be of polygenic origin. unlike the mouse, there are few reported mutants that have disorders of the central nervous system. recently, a partially inbred strain has been developed in which one subline has nearly a % incidence of hydrocephalus (kohn et al., ) . the mode of inheritance appears to be polygenic. the hydrocephalus is classified as communicating and the pathogenesis may be due to poorly developed veins in the periosteal and durai layers, and to underdeveloped pia-arachnoid cells. neoplastic and nonneoplastic lesions vary in type, onset, and prevalence due to the hereditary and environmental factors as sociated with a specific colony of laboratory rat. the rat is widely used in gerontological research. those who use the rat must be clearly aware of the factors that can influence lesions and death in aged rats (anver and cohen, ; burek, ) . neoplastic disease, a cause of death in many aged rats, has been discussed previously, and this section will summarize the more commonly seen nonneoplastic lesions in aging rats. a. chronic progressive nephropathy (cpn). chronic pro gressive nephropathy is among the most commonly encoun tered lesions in the rat, particularly in the aged animal where it is a major life-limiting disease. because of its complex nature, cpn has a large number of synonyms, often with inaccurate descriptive titles. sequential development of lesions and fac tors that modify the course of cpn have been thoroughly stud ied, but the actual pathogenesis remains undetermined. chron ic progressive nephropathy occurs in most rats, but its rate of development is significantly influenced by numerous factors, including sex, genetics, age, diet, association with microflora, hormone treatment (particularly testosterone), and others. male rats have an earlier onset and more rapid progression of cpn than females, and albino strains and stocks seem to be more predisposed. diet is an extremely important predisposing factor, particularly the quality and quantity of dietary protein. significant differences in severity of cpn can be observed among rats of the same sex, strain, age, and source when fed different diets for their lifetime. axenic rats do not seem to develop significant cpn. rats with cpn develop progressively severe proteinuria, first with the selective excretion of only small molecules such as albumin. in late cpn there is nonselective excretion, and the electrophoretic pattern of urinary protein resembles that of serum. proteinuria related to cpn should not be confused with the normal urinary excretion of a-globulin in male rats, proba bly of tubular origin. the histopathology of cpn is charac terized as progressive basement membrane thickening of the glomerulus, bowman's capsule, and proximal tubule. base ment membranes split and wrinkle as tubules become atrophie and collapse. glomeruli enlarge, with mesangial deposition of protein and lipid, adhesion to bowman's capsule, and segmen tai sclerosis. tubular epithelium may contain granules of resorbed protein, which later is mixed with lipofuscin and hemosiderin. as glomerular protein leakage becomes more severe, tubules dilate and accumulate hyaline eosinophilic castes of protein within their lumina, resembling colloid. tubular epi thelium may undergo atrophy with collapse or dilatation. in terstitial fibrosis and leukocytic infiltration is frequently ob served. grossly, the kidneys become enlarged, pale with irregular surfaces and pigmented (fig. ) . cystic tubules can be observed. hypoproteinemia, parathyroid hyperplasia with serum chemical changes indicative of hyperparathyroidism, and nephrotic syndrome are observed in rats with severe cpn (barthold, ) . b. nephrocalcinosis. this disease, which occurs more fre quently in females, has been reported in numerous strains of rats. in most cases, affected rats have been fed purified diets (e.g., . renal lesions usually involve the corticomedullary junction and consist of mineral deposition in the lumina of the proximal tubules (nguyen and woodward, ) . e. polyarteritis nodosa. this disease is of unknown etiol ogy and affects muscular arteries, most commonly the mesenteric, pancreatic, and spermatic. the lesions may be either acute or chronic. in the acute stage, there is intimai and medial fibrinoid necrosis, focal thrombosis, disruption of the elastic lamellae, and an infiltration of polymorphonuclear leukocytes and mononuclear cells. in the chronic stage, the arteries are nodular, tortuous, and thick-walled. affected vessels fre quently have aneurysms and thrombi. both acute and chronic phases may be observed in the same artery in an animal. d. myocardial degeneration. degeneration is a commonly observed lesion that occurs in most stocks and strains, with an onset at - months of age. it occurs more frequently in males. the lesions are usually microscopic but, in advanced stages, can appear grossly as grayish foci. the papillary mus cles and their attachment sites in the wall of the left ventricle are the most frequent sites of the lesion (anver and cohen, ) . e. radiculoneuropathy. spinal nerve root degeneration has been reported in a number of rat stocks and strains. lesion distribution may vary among strains; however, the cauda equi na and ventral spinal nerve roots are commonly involved. pos terior paresis and paralysis have been associated with these lesions, but some suggest that these signs are associated with a separate degenerative process of the skeletal muscle (anver and cohen, ) . an enzyme linked immunosorbent assay for detection of antibodies to corynehacterium kutschen in experimentally infected rats copulatory behavior can inhibit preg nancy in female rats neoplastic diseases inbred and genetically defined strains of laboratory animals. part . mouse and rat rat-bite fever in animal research laboratory personnel muroid virus nephropathies lesions associated with aging reproduction and breeding housing to contro! research variables selected norma tive data. appendix i pseudohermaphroditic rat: end organ insensitivity to testoster one chronic progressive nephropathy in aging rats research complications and state of knowledge of rodent coronaviruses reproduction and breeding techniques for laboratory animals morphophysiology sendai virus infection in genetically resistant and susceptible mice observa tions on enzootic paratyphoid infection in a rat colony age-associated pathology a comparison in germ-free mice of the pathogenesis of sendai virus and pneumonia virus infection my coplasmal and rickettsial diseases detection of natural mycoplasma pulmonis infection in rats and mice by an enzyme-linked immunosorbent assay (elisa) adherence and colonization of mycoplasma pulmonis to genital epi thelium and spermatozoa in rats tissue weights of the rat. i. normal values determined by dissection and chemical methods immunopathology. in "cell pathology heritable hydronephrosis in a mutant strain of brown norway rats pathological changes during aging in barrier-reared fischer male rats naturally occurring lethal parvovirus infection of juvenile and young-adult rats breeding of the rat adynamic ileus in the rat induced by chloral hydrate parasites of laboratory animals gnotobiology ulcera tive dermatitis in the rat corynebacterium kutschen pneumonia in rats on long term tobacco inhalation studies evidence suggesting a multigenic origin of membranous septal defect in rats kidney malformations in fetuses of axc line rats bibli ography of hemorrhagic fever with renal syndrome (muroid virus nephropathies) effect of heat and selected chemical disinfectants upon infectivity of spores of bacillus piliformis (tyzzers disease) studies on adjuvantinduced arthritis, tumor transplantability and serologie response to bovine serum albumin in sendai virus-infected rats scientific considerations in monitoring and evaluating toxicological research nih rodents catalogue anatomy of the laboratory rat parasitic diseases viral diseases. /// "the laboratory rat tyzzer's disease in the rat pathogenicity of mycoplasma pulmonis in laboratory rats a new model of congenital hydrocephalus in the rat morphological evi dence for natural poxvirus infection in rats animal model: hereditary retinal degeneration in wag/rij rats the ufaw handbook on the care and management of laboratory animals photoreceptor pig ment epithelial cell relationships in rats with inherited retinal degenera tion comparison of techniques for primary isolation of respiratory mycoplasma pulmonis from rats historical foundations murine chronic respiratory disease. significance as a research complication and experimental production with mycoplasma pulmonis comparison of neoplasms in six sources of rats a filamentous bac terium associated with respiratory disease in wild rats salmonella enterocolitis in the rat clinical biochemical and hematological reference values in normal experimental animals intranephronic calculosis in rats helminths, ectoparasites and protozoa in rats and mice hematology and clinical biochemistry genetics of the norway rat nutrition lasting influence of early caloric re striction on prevalence of neoplasms in the rat tumors in control hamsters, rats, and mice: literature tabulation molecular biology of mammalian sarcoma viruses state of the art detection methods for rodent viruses the microscopic anatomy of the white rat asymptomatic infection of mouse hepatitis in the rat histological and serological response of b c f, mice and f rats to experimental pneumonia virus of mice infection demodicidosis in laboratory rats (rattus norvegicus) latent adenoviral infection of rats: intranuclear inclusions induced by treatment with a cancer chemotherapeutic agent the role of mycoplasmas and l forms of bacteria in disease bacterial and mycotic diseases respiratory disease in a colony of rats. ii. isolation of mycoplasma pulmonis from the natural disease, and the experimental disease induced with a cloned culture of this organism craigie's neuroanatomy of the rat key: cord- - tkc yq authors: hooks, john j.; detrick, barbara; nussenblatt, robert title: infections associated with retinal autoimmunity date: - - journal: infection and autoimmunity doi: . /b - - . - sha: doc_id: cord_uid: tkc yq nan the retina the topic of immune-mediated vision loss, with an emphasis on autoimmune reactivity and autoimmune disease in the eye, is a rapidly expanding area of research and therapy. numerous studies in other body sites have clearly identified links between infections and autoimmunity and autoimmune disease [ , ] . only a limited number of studies have been reported in which retinal disorders have been evaluated to study this relationship. we will begin this chapter with a brief overview of infection and autoimmunity in the eye. this will be followed by specific examples of infections and autoimmunity in the retina. we will highlight two human diseases triggered by onchocerca volvulus or toxoplasma gondii and an experimental model referred to as experimental coronavirus retinopathy (ecor), triggered by the murine coronavims, mouse hepatitis virus (mhv). the visual axis is a precious sense. the eye is an organ that is known to have immunologic processes that are both infectious and non-infectiously driven. the eye is unique in that it lacks lymphatics and still enjoys an intimate relationship with the immune system. an inflammatory process in the eye is termed an uveitis, and it in no way reflects the origin of the inflammatory process nor where it is located in the eye. while there are many descrip-tions of inflammatory processes in the eye, there are three major presentations of these conditions. if the inflammatory condition is centered in the front of the eye, the process is termed an anterior uveitis. if upon examination the dominant part of the inflammation is centered in the vitreous of the eye, it is termed an intermediate uveitis. finally, if inflammation occurs in the back of the eye, that is centered in the retina or the choroid of the eye, it is termed a posterior uveitis. clearly inflammatory conditions may involve several parts of the eye, and if all anatomic of the eye are involved it is termed a panuveitis. most of the cormqaents of this chapter will address disorders of the back of the eye, i.e., those involving the retina. eye specialists have the great advantage of being able to visualize directly the parts of the eye that can be involved in an inflammatory process. in addition to simple visualization, many additional tools can be readily applied. electrophysiologic testing is easily and frequently performed, this is an excellent way to evaluate the retina's ability to react to a light stimulus. fluorescein angiography, the use of dye injected into an arm vein and then rapid photographs are taken of the back of the eye. this approach helps to visualize the vascular system and the integrity of the retina. the severity of the inflammatory response can be graded by direct visualization of the inflammatory response in the eye. most inflammatory process that we recognize will have a cellular response associated with it. we also know that antibody mediated pathology, as seen in such entities as cancer associated retinopathy, can occur, but appears to be the distinct minority of cases. the eye is a complex organ from the point of view of the immune system. it is known that antigen placed into the anterior chamber of the eye will induce a deviated immune response, with a marked decrease in cell mediated responses, but an intact cytotoxic and b-cell response is seen [ ] . addtionally, the retina is a complex structure with several layers needed to turn the light stimulus into a chemical signal ultimately sent to the brain. at the photoreceptor level and the single layer just below it, the retinal pigment epithelium, a number of uveitogenic antigens have been identified and characterized [ ] . two antigens in particular, the retinal s-antigen and the interphotoreceptor retinal binding protein (irbp), have been used to develop a model of autoimmune ocular disease which is termed experimental autoimmune uveoretinitis (eau) [ ] . this model has many qualities of the disease seen in humans and has helped to better understand the underlying mechanisms that lead to disease. one major difference between this model and the human disease is of course that it is not spontaneous. it is not clear what triggers the human disease. this chapter will explore one such trigger, that of ocular infection. several entities, some based on animal models, other seen in the clinic, will be discussed to elucidate the possible role between infection and autoimmunity. experimental coronavirus retinopathy (ecor) is an animal model system that we generated in the 's to demonstrate that a virus can trigger a progressive retinal degenerative disease [ ] . studies during the past years have identified that this degenerative eye disease is composed of three basic components; a virus component, a genetic component and an immunologic component [ , ] . in our system, we selected a naturally occurring neurotrophic strain (jhm) of a mouse hepatitis virus that infects and persists within the retina. the virus causes an acute infection, marked by virus replication in distinct retinal cells, neutralizing antibody and the production of cytokines, namely ifn-gamma. this disease also has a genetic component. that is, different strains of mice behave differently after virus infection. two strains of mice, babl/c and cd- , were extensively studied after coronavirus infection. during the early phase of the disease (day - ) the virus infects and replicates within the retina of both balb/c and cd- mouse stains [ ] . however, on days to , only the balb/c mice experience a late phase of the disease which is marked by a retinal degeneration. the cd- mouse does not undergo the retinal degenerative phase but rather, the retina returns to a normal architecture within days. finally, the immune component of this disease is characterized by the presence of autoantibodies, specifically, anti-retinal and anti-rpe auto-antibodies. the presence of these antibodies are observed only in the retinal degenerative susceptible balb/c mice. these auto-antibodies are absent in the retinal degeneration resistant cd- mice. in summary, ecor is a virus triggered retinal degenerative disease that is influenced by both genetics and the immune response. in this session we will discuss in detail the virologic, pathologic, immunologic, genetic and autoimmune factors involved in this model system. coronaviruses are large, enveloped, positive strand rna viruses that cause significant diseases in a number of animal species and humans. in animals, coronaviruses are responsible for important diseases of livestock, poultry and laboratory rodents. until recently, man was known to be infected with two strains of coronavirus. either of these strains are responsible for approximately % of the common colds. a new human coronavirus has been identified as the causative agent for severe acute respiratory syndrome (sars) [ ] . one of the closest relatives to the human s ars-coronavirus is the murine coronavirus, mouse hepatitis virus (mhv). the jhm strain of mhv is the most thoroughly studied neurotropic coronavirus. it causes both acute and chronic central nervous system effects in mice and rats. acute encephalomyelitis and chronic cns disease have been observed in mice. in rats an autoimmune disease known as subacute demyelinating encephalomyelitis, has been described. initial studies in the ecor system, showed that inoculation of this jhm strain into the vitreous or anterior chamber of balb/c mice resulted in retinal tissue damage [ , ] . infectious virus could be detected within the retina between and days post inoculation (pi), reaching a peak level of . pfu/ ml at day [ ] . virus antigen was also identified within the retina between day and pi [ ] . virus antigen was first detected within the rpe cell and the ciliary body epithelial cell at day and this virus replication intensified at day and . between day and virus antigen was also detected in mullerlike cells that span the multiple layers of the neural retina. occasionly, virus antigen was also observed within the ganglion cells. after day , infectious virus and viral antigen could not be detected within the retina. however, in situ hybridization studies identified that the viral rna persisted within the retina until days pi [ ] . anti-virus neutralizing antibodies were first noted at day pi [ ] and coincided with the disappearance of infectious virus and viral antigen. after inoculation with jhm virus, two distinct patterns of retinal pathology were noted in the balb/c mice [ ] . the early phase of the disease, day to , was characterized by retinal vasculitis and perivasculitis. the late phase of the disease, after day , was characterized by retinal degenerative changes. the retinal layers revealed disorganization with large areas of outer and inner segment loss. in addition, the rpe cells were morphologically abnormal with focal rpe cell swelling or proliferation, or with focal rpe cell atrophy or loss. analysis of retinal cell function also revealed dramatic changes [ , ] . there was a significant decrease of complete loss of electroretinogram (erg) patterns and the disappearance of an important transport protein in the retina, the interphotoreceptor retinoid-binding protein (irbp). the host immune response to this virus infection was evaluated by tracking the cellular infiltrate and identifying the cytokine profile within the retina [ ] . the most prominent infiltrating cell was the macrophage. mac- staining was detected in % of the eyes at day and pi, and was occasionally seen on day and beyond. the second most prominent cell was the t cell. cd t cells were present in the retina at days and . this was followed by a shift to cd t cells which were observed at day and pi. a low number of cd t cells were still noted at day pi. b cells and nk cells were not detected. during the course of the disease, cytokine profiles were studied by evaluating retina tissue and sera [ ] . analysis of pooled retinal mrnas from untreated, mock-injected and virus infected balb/ c mice revealed the presence of il- , ifn-y and tnf-~ mrnas in virus infected retinas isolated during the acute disease, day and day pi. gene expression for these cytokines was not detected in retinas from untreated or mock-injected mice. eia analysis of sera identified the presence of these same cytokine proteins in virus infected mice and not in untreated or mock injected mice. the presence of retinal mrna for ifn-y was also associated with the upregulation of mhc class i and ii molecules within the retina. mhc class i and ii molecules were not identified within the normal or mock injected retinas. it was noted that the first cell to express these mhc molecules was the rpe cell. this cell is also the first cell to express new viral antigens during the infection in vivo and is persistently infected in vitro [ ] . it is critically important to point out that this rpe cell has been shown to process and present retinal and non-retinal antigens to sensitized t cells and is upregulated to express mhc class ii molecules during retinal autoimmune and degenerative processes [ , ] . the genetic constitution of the host can be a critical factor in determining the outcome of a virus infection [ ] . we therefore evaluated the possible role of host genetics in ecor. we inoculated selected strains of mice and evaluated the retinal disease. babl/c, c b , a/j and cd- mice were studied. when c b and ajj mice were evaluated, we observed a disease pattern similar to that seen in balb/c mice. however, retinal changes were less severe than those seen in balb/c mice. retinal tissue damage induced by jhm virus in cd- mice was very different (table ) . only the early phase of the disease, consisting of retinal vasculitis, was observed. these cd- mice did not develop the retinal degenerative disease. in fact, by day pi, the retina had a normal appearance. these studies underscored the role of genetics in ecor and showed that the genetics of the host profoundly affected the nature of retinal tissue damage. since the cd- mice did not exhibit the late retinal degenerative phase of the disease, we evaluated a variety of parameters and compared the findings with the data obtained in balb/c mice. for example, during the acute phase of the disease (days - ), virus load in the retina, production of anti-virus antibody, blood-retina barrier breakdown, lymphoid trafficking and mhc class i and ii staining were similar in both mouse strains. moreover, gene expression for ifn-~, in pooled retinas from cd- mice was positive at pi day and . again, this is similar to the pattern observed in balb/c mice. the disease pattern in the late phase was clearly different in the two mouse strains. balb/c mice displayed a retinal degeneration with blood-retina barrier breakdown, and cd- mice showed a normal retinal architecture. immuno-staining observed at day in cd- mice was clearly different from that observed in balb/c mice, in that cd- mice were negative for mhc class i and ii expression and cd t cells were absent from the retina. in ecor, the late phase of the disease was associated with the lack of direct evidence for viral replication within the retina. this observation suggested that the continued degenerative process may be associated with alterations directly induced by virus replication during the first few days after infection or it may be associated with additional factors. inasmuch as viruses are know to trigger an autoimmune phenomena and some human retinopathies may be associated with autoantibody formation, we studied the possible production of antiretinal autoantibodies [ ] . we found that the retinal degenerative process in balb/c mice was associated with the presence of antiretinal autoantibodies (table ). these autoantibodies were not found in sera from normal or mock-injected mice. the presence of antibodies to retinal tissue was evaluated by immunoperoxidase staining on frozen sections of normal rat eyes. two patterns of staining were observed, reactivity in the neural retinal and reactivity in the retinal pigment epithelium (rpe) (fig. ) . the antiretinal autoantibodies first appeared as igm class antibodies. this was later replaced by igg class autoantibodies. the anti-rpe cell autoantiboedies were predominantly of the igg class. as stated above, jhm virus infected cd- mice developed a retinal disease that is different than the retinal disease observed in balb/c mice. in cd- mice, only the early stage, consisting of retinal vasculitis was seen. the cd- mice recovered and were not susceptible to the later phase of the disease, the retinal degenerative disease. we therefore, evaluated the development of a retinal degenerative disease and the development of antiretinal autoantibodies in these two strains of mice after inoculation with jhm virus [ ] . the data summarized in table shows that all of the babl/c mice developed antiretinal antibodies and developed pathologic changes consistent with retinal degenerative processes. in contrast, none of the cd- mice developed antiretinal autoantibodies. that is, in those mice that failed to develop antiretinal autoantibodies, they also failed to develop a retinal degeneration. these findings suggest a role for autoimmunity in the pathogenesis of ecor. toxoplasmosis is a disorder that has a worldwide distribution. it is caused by the obligate intracellular parasite, toxoplasma gondii. over million people are believed to have the disease. in , the organism was first described in the brain of the north african rodent, the gondii, by nicolle and manceaux [ ] and by splendore in a rabbit [ ] . the first connection between this organism and human disease was made by janku [ ] who described the presence of the organism in a child who died of disseminated toxoplasmosis. while suspected for a long period, it was not till the early 's that the parasite was shown to cause ocular disease. helenor campbell wilder, working at the armed forces institute of pathology in washington, dc, identified the organism in eyes that were believed to have other types of inflammatory processes, particularly tuberculosis [ ] . it is interesting to note that a similar observation has been made more recently in nepal, where many cases of ocular tuberculosis have now been rediagnosed as toxoplasmosis of the eye. the cat (and perhaps related species) appears to be the definitive host. the sexual cycle is one of schizogony and gametogony leading to the the development of toxoplasma oocysts, which are - ~m in size and are found uniquely in the intestinal mucosa of cats. two forms of the organism can be found in man, cysts and tachyzoites. the tachyzoites (the proliferative intracellar form) are believed to be the cause of most of the tissue damage in human, though often it is very difficult to demonstrate the presence of the stage of the organism. the bradyzoites (the latent form of the organism found in cysts) are found in host cells. hundreds of bradyzoites (with very slow metabolic rates) have a propensity towards neural tissue such as the eye and brain, but are also found in skeletal muscle and heart. it is assumed that attacks occur with rupture of the cyst, leading to a pouring out of bradyzoites and then the conversion of bradyzoites to tachyzoites. the mechanisms that lead to cyst rupture are still unknown. while the hallmark of the disease is changes in the posterior portion of the eye, changes in the front of the eye are also noted. an anterior uveitis can be seen in many patients with this disorder. this is an interesting finding since the organism is not seen in the anterior segment of the eye except possibly in immunocompromised individuals. additionally, there is a loss of pigment in the iris that can be seen and is associated with changes in the back of the eye [ ] . this finding, termed fuch's heterochromia, is thought to be an autoimmune phenomenon. the classic finding in ocular toxoplasmosis is that of a retinal lesion, which is destructive. it is typically an oval lesion where all layers of the retina and frequently many layers of the choroid have been infected. it is the result of an immune response believed to have occurred against the toxoplasma organism. while there may be only one lesion, often there are multiple lesions surrounding an old large scar, and these are called satellite lesions. in addition to the lesion itself, during the active stage of the disease, evidence of retinal vascular leakage is seen. it has been hypothesized that this vasculitis is due to an immune complex related phenomenon. typically while stigmata of the disease may be present in both eyes, recurrences of the disorder occur only in one eye. additionally, while reactivation of the disease is believed due to the breakage of cysts and the presence of tachyzoites, it is rare to see this stage of the organism in the retina. patients who are immunocompromised, such as those with aids, will often have bilateral disease and multiple lesions, suggesting a different mechanism in these patients as compared to the immunocompetant patient. abrahams and gregerson [ ] evaluated five patients with ocular toxoplasmosis. this longitudinal study measured serum antibody responses to the retinal s-antigen, a "p" antigen (thought to contain rhodopsin), and new antigen designated p ag, all isolated from bovine retina. they reported that all the patients initially tested showed antibody responses to all three antigens. the anti-s-antigen responses tended to decrease with clinical improvement, while the anti-p antibodies remained high even after the acute attack was over. a more recent report by whittle and colleagues [ ] looked at a larger number of toxoplasmosis patients. in this study a total of patients with toxoplasma retinochoroiditis were evaluated for evidence of anti-retinal antibodies. thirty-four of these sera showed antibodies directed against the photoreceptor layer of the retina when tested with indirect immunofluorescence. six of controls showed a similar staining pattern with the p value as reported as < . . interestingly, using an eia to measure the presence of anti-s-antigen antibodies, the researchers observed that of sera from toxoplasmosis retinochoroiditis patients were positive, but so were of normals, with a calculated p value of greater than . . the antibodies seen in the two assays did not appear to run in parallel. the author's interpretation of their data was that the extent of anti-retinal antibodies could not be explained by anti-s-antigen findings alone. they argued that these findings probably did not reflect an epiphenomenon since patients with idiopathic retinal vasculitis were also evaluated. in that study the number of sera positive from patients with idiopathic retinal vasculitis was considerably lower than that found in the toxoplasmosis group. our group has had the chance to evaluate cell mediated responses of lymphocytes from patients with ocular toxoplasmosis. in a very early study [ ] in which we looked at proliferative responses from patients with all kinds of uveitic conditions, we reported that a small number of ocular toxoplasmosis patients' lymphocytes did respond to the uveitogenic retinal s-antigen. in a later study, we evaluated the proliferative cell mediated responses in patients with ocular toxoplasmosis. in addition to the retinal s-antigen, we also evaluated the response to crude toxoplasma antigen and to puri-fled antigens from the parasite [ ] . in addition, we performed an eia to look for anti-s-antigen antibodies and hla phenotyping to see if a specific hla type was associated with s-antigen responsiveness. of the patient's lymphocytes tested, ( %) had proliferative responses with a stimulation index above . (see fig. ). there appeared to be no correlation with this responsiveness and any hla phenotype. additionally, we were unable to demonstrate anti-s-antigen antibodies using eia. the ocular toxoplasmosis patients could be divided by their lymphocytes responsiveness to the various toxoplasma antigens tested. however, no correlation was seen in s-antigen responsiveness and the stimulation index to toxoplasmosis antigens. infection with the nematode parasite onchocerca volvulus can result in severe eye disease, often referred to as fiver blindness. it is estimated that approximately million people in tropical africa, the arabian peninsula and latin america are infected with the organism and of these, approximately one to two million are blind or have severe visual impairment. humans are infected with the helminth larvae by the bite of a black fly of the simulium genus and approximately one year after infection, the adult female worms produce microfilariae. in fact, the adult worm can live for up to years, producing to microfilariae per day. it is the microfilariae that are able to move through subcutaneous and ocular tissues. when these microfilariae die, they incite an immune response that is associated with clinical symptoms. onchocerciasis is one of the leading causes of blindness in the developing world. ocular disease occurring in the anterior segment of the eye consists of corneal opacification and sclerosing keratitis, whereas, ocular disease occurring in the posterior pole is characterized by retinal degeneration [ ] . clinical disease activity in the anterior segment is associated with microfilarial load and it is generally believed that ocular pathology is a result of host directed inflammatory responses to the nematode. sixteen of these had stimulation indices above . and were designated as "high responders". this responsiveness was not correlated to either a specific hla phenotype nor the vigor of the cell mediated response to toxoplasma antigens. (reprinted with permission.) in contrast, pathology associated with the retina and optic nerve has not been directly linked to microfilarial load. posterior ocular onchocerciasis is characterized by atrophy of the retinal pigment epithelium and as lesions advance, subretinal fibrosis occurs [ ] . a number of studies indicate that this retinal disease process may involve autoimmune responses. in , chan and associates identified that a majority of onchocerciasis patients had anti-retinal antibodies in their sera and vitreous [ ] . using fa assays on human retina tissue, they observed reactivity in the inner retina and photoreceptor layers. during the 's, braun, mckechnie and associates performed a number of studies to elucidate the nature of the autoimmune reactivity [ ] [ ] [ ] [ ] . they identified a recombinant antigen in o. volvulus that showed immunologic cross-reactivity with a component of the rpe [ , ] . by western blot analysis, an antibody to a , mw antigen (ov ) of o. volvulus recognized a , mw component of the rpe cell. subsequent studies have shown that hr ag is present in the optic nerve, epithelial layers of iris, ciliary body and rpe. although ov and the hr proteins are not homologus, they did show limited amino acid sequence identity [ ] . immunization of lewis rats with either ov from o. volvulus or hr from human retinal tissue, induced ocular pathology [ ] . the retinal disease in the rat was characterized by extensive breakdown of the posterior blood-ocular barrier, iridocyclitis and retinitis and the activation of retinal microglia. these studies indicate that, molecular mimicry between o. volvulus and human rpe protein may contribute to the retinopathy found in patients with onchoceriacis. a large group of clinical entities have been grouped under the title, white dot syndromes. as the name infers, they are all characterized by whitish lesions of varying sizes that are found strewn throughout the fundus. some have a significant inflammatory reaction associated with them while others do not. the natural history of some may lead to significant visual handicap while others may not. some of these disorders seem to progress while others fade away. the disorders that are included in this list include such entities as acute multifocal placoid posterior pigment epitheliopathy (amppe), serpiginous choroiditis, the multifocal evanescent white dot syndrome (mewds), and multifocal choroiditis. the underlying cause of these diseases is unknown. many of these disorders seem to be preceded by a viral illness, and one disorder, amppe, was hypothesized to be due to the an epstein-barr infection [ ] . this concept is no longer thought to be the case [ ] . however, a few patients have been treated with anti-viral medications, with unclear responses. the most common therapy for all of these conditions is that of immunosuppression and therapy is directed against what is believed to be an autoimmune, or least non-infectious, process in the back of the eye. in summary, we have reviewed the evidence that three distinct classes of infectious agents have been implicated in the development of autoimmune processes within the retina. these data also indicate that distinct pathogenic mechanisms are involved in the induction of autoimmunity triggered by these three organisms. in t. gondii infections, the persistence and chronic reactivation of the organism is probably responsible for introduction and presentation of sequestered retinal epitopes to the immune system. in o. volvulus infections, molecular mimicry between the organism and human rpe protein may contribute to the retinal pathology. in ecor, similar processes are induced in coronavirus-infected mice displaying either retinal degeneration susceptibility or retina degeneration resistance. however, recent evidence indicates that differences in time of induction, in duration and intensity of immune reactivity may contribute to autoimmune reactivity in balb/c mice. anterior chamber associated immune deviation (acaid): regulation, biological relevance, and implications for therapy experimental autoimmune uveoretinitis-rat and mouse amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity the role of infection in the pathogenesis of autoimmune disease retinopathies associated with antiretinal antibodies ocular tropisms of murine coronavirus (strain jhm) after inoculation by various routes murine coronavirus induces an acute and long-lasting disease of the retina genetic predisposition to coronavirus-induced retinal disease the critical role of ifngamma in experimental coronavirus retinopathy retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy sars-associated coronavirus the role of apoptosis within the retina of coronavirus-infected mice virus rna persists within the retina in coronavirus-induced retinopathy redistribution and reduction of interphotoreceptor retinoid-binding protein during ocular coronavirus infection blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and vegf in sensitive and resistant strains coronavirus (jhm) replication within the retina: analysis of cell tropism in mouse retinal cell cultures expression of hla-dr antigen on retinal pigment epithelial cells in retinitis pigmentosa cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation sur une infection a corps de leishman (ou organismes voisins) due gondii un nuovo protozoa parassita dei conigli: incontrato nell lesioni anatomiche d'une malattia che ricorda in molti punti kala-azar dell'uomo pathogenesis and pathologic anatomy of coloboma of macula lutea in eye of normal dimensions, and in microphthalmic eye, with parasites in the retina ocular toxoplasmosis: a th anniversary tribute to the contribution of helenor campbell wilder foerster fuchs' heterochromic cyclitis and ocular toxoplasmosis longitudinal study of serum antibody reponses to retinal antigens in acute ocular toxoplasmosis human antiretinal antibodies in toxoplasma retinochroiditis cellular immune responsiveness of uveitis patients to retinal santigen lymphocyte proliferative responses of pateints with ocular toxoplasmosis to parasite and retinal antigens pathogenesis of onchocercal keratitis (river blindness) onchocercal eye disease and the impact of mectizan treatment immunopathology of ocular onchocerciasis. . anti-retinal autoantibodies in serum and ocular fluids immunological crossreactivity between a cloned antigen of onchocerca volvulus and a component of the retinal pigment epithelium cross-reactive antigens in the pathogenesis of onchocerciasis immunologic cross-reactivity in the pathogenesis of ocular onchocerciasis immunization with the cross-reactive antigens ov from onchocerca volvulus and hr from human retinal tissue induces ocular pathology and activates retinal microglia molecular and immunological characterization of hr , a human ocular component immunologically cross-reactive with antigen ov of onchocerca volvulus epstein-barr viral antibodies in multifocal choroiditis and panuveitis epstein-barr virus antibodies in multifocal choroiditis and panuveitis key: cord- -yxttl gh authors: siegel, frederic r. title: progressive adaptation: the key to sustaining a growing global population date: - - journal: countering st century social-environmental threats to growing global populations doi: . / - - - - _ sha: doc_id: cord_uid: yxttl gh adaptation is an evolving long-term process during which a population of life forms adjusts to changes in its habitat and surrounding environments. adaptation by the global community as a unit is vital to cope with the effects of increasing populations, global warming/climate change, the chemical, biological, and physical impacts on life-sustaining ecosystems, and competition for life sustaining and economically important natural resources. the latter include water, food, energy, metal ores, industrial minerals, and wood. within this framework, it is necessary to adapt as well to changes in local and regional physical conditions brought on by natural and anthropogenic hazards, by health threats of epidemic or pandemic reach, by social conditions such as conflicts driven by religious and ethnic fanaticism, and by tribalism and clan ties. the principal problems with growing populations do not involve space although population density is a problem unto itself for reasons discussed in previous chapters. the main problems are how to nourish people with food and water. the chronic malnutrition that about billion people suffered from in is likely to grow in number in some regions due to global warming/climate change because humans cannot adapt to less food if they are already at subsistence rations. for example, the population in sub-saharan africa is million people. the population is projected to increase to about . billion in , an increase of about %. within the same time frame, the united nations estimates that acreage under maize cultivation in the region will decline by % because of heat and drought brought on by climate change. the loss of arable land for food production can be countered in sub-saharan africa if marker assisted hybridization of maize or maize genetically modified to withstand heat and drought come onto the seed market together with modified seeds for other food staples and if african nations that do not now accept gmo seeds do so in the future. if not, nations favored for food production by climate change will have a moral obligation to provide food staples to people in nations with declining food production at accessible costs based on their economies. it is clear that what happens in sub-saharan africa and other regions with declining cultivation acreage or that will bear other effects of climate change (e.g., drought, shifting rain patterns) will affect the rest of the worldwide community politically, economically, and socially. the earth's problems that associate with global warming/climate change will be further discussed in the last section of this chapter. water is the staff of life. it keeps the body hydrated and is necessary to grow food crops, hydrate food animals, and grow feed grains. chemically or biologically polluted water does not serve these ends. if ingested, contaminated water can result in sickness as discussed in chap. . water stokes industry and manufacturing as well, thus keeping economies in many countries contributing to a population's wellbeing by providing employment, goods, and services. ideally, these businesses contribute their fair share to a tax base that supports social needs (e.g., education, healthcare, maintenance of infrastructure). factory owners adapt and plan against water shortages by having a water recycling system in place but may also slow or stop production until operational water conditions return. citizens with a reliable water supply can adapt to periods of water shortage by limiting use according to mandates by government officials but still have water for basic daily needs. however, persons in nations with a chronic per capita water shortage may not have this option to serve their daily needs unless water is imported or new water sources are found (see chap. ). if imported water is not an option to meet immediate essential needs, an alternative adaptation for people (and animals) is to try to reach a location where water would be available to them. with growing populations, per capita water availability is greatly diminished (table . ), water shortages become endemic, and people are at risk of existing at subsistence levels or dying. most at risk from the lack of a basic water ration are pregnant women, infants, young children, and old people. water wars are a future possibility as nations battle for their peoples' survival unless political differences are set aside and projects are supported to develop and share water sources. in a welcome effort, jordan, israel, and the palestinian authority signed a memorandum of understanding in the world bank, december , with specific aims: ( ) produce millions of cubic meters of drinking water for a water-deficient region; ( ) pipe million cubic meters of water annually * km ( mi) from the red sea to the dead sea; ( ) build a desalination plant at aqaba that would supply water to aqaba and eilat; ( ) the israeli water utility would supply - million cubic meters of drinking water to the palestinian authority for the west bank population at a reduced cost; and ( ) there would be an inflow of water to slow and in the future perhaps abate and reverse the shrinking of the dead sea. funding for the estimated $ million, year project will come from the world bank, donor nations, and philanthropic groups. as the global population increases and more people in developing and less developed nations have more disposable income, there will be a growing draw on natural resources other than water and food to service their industrial, agricultural, and manufacturing needs and wants. competition can force economic wars among national and multinational corporations for the resources necessary to provide goods and services and thus drive up prices for resources. industries and manufacturing units that cannot compete economically for natural resources will shut down, thus contributing to unemployment and downturns in economies because of falling domestic demand. to keep order in the increasingly interdependent world economy, accommodation for shared natural resources (or substitutes for them) at affordable prices is the adaptationnecessary. this can be mandated by the world trade organization backed by other practical-minded international groups. another adverse effect of growing populations that is a national resource that can be lost at the expense of some countries to the benefit of others is brain power. this brain power has been cultivated at excellent universities in developing countries, often times at little or no cost to students (e.g., in medicine, science, engineering, economics, the arts) who attend and graduate in increasing numbers. where there are too many well-educated professionals but lack of employment opportunities for them in their fields of expertise, educated people have the option of relocating to another country that can nurture and use the expertise. many adapt to the employment problem by taking up this option. this may mean moving from a developing country to a developed country or from a less developed country to a developing or developed country. ultimately, this loss of citizens with special skills can hurt a country. to counter this brain drain or reverse it, a country can adapt by investing in its future to create programs and conditions that keep talented professionals home, or if they have emigrated, entice them to return. china and india are examples of countries that have successfully taken this tact. when there are increases in a population because of immigration, problems can ensue between immigrants and a general population. adaptation to diversity and the multicultural experiences it brings to a community is often not a comfortable change. the antipathy of some in a host country is based on slowness of the immigrants to learn the language and inability of host country citizens to understand what immigrants are saying among themselves. this makes citizens feel uneasy. some view immigrants as a threat to their own or a family member's employment or advancement. race difference is a factor that some cannot readily adapt to as is ethnicity with its traditions and customs unfamiliar to the general public. religion can be divisive if adherents to its beliefs engage in acts of hatred detrimental to the host country fueled by fundamentalists and zealots who interpret religious writings as giving them license to commit crimes or absolving them of the crimes. sadly, many citizens paint an entire religious community with the taint of the relatively few evildoers. adaptation to diversity is essential for our earth's citizenry with joint efforts by all to resolve worldwide issues (e.g., global warming/climate change) so as to become the keys to providing a sound future for coming generations. there has to be a shared attack on global threats, no matter what the language, race, ethnicity, or religious beliefs are, no matter social or economic status, no matter whether a threat affects less developed, developing, or developed countries. adaptation is a progressive process when dealing with natural hazards because as each type of natural hazard impacts global communities over time, lessons are learned from each one that give direction to the methods of adjustment. adaptation to living where hazards can be expected to strike and where populations continue to increase is dependent on what we learn from the study of past hazards. we can use this evaluation of measured and observed data to minimize the immediate effects and aftermaths of hazards and protect citizens from injury, death, and from damage or destruction of property or infrastructure when hazards strike in the future. in areas prone to earthquakes, we know that earthquakes do not kill and injure people but that collapsing buildings and infrastructure do. earthquakes are not predictable so that there is no adaptation by a timely evacuation to minimize deaths and injury. however, building structures to make them more earthquake resistant can save lives, reduce injuries, and protect property. thus, after a high-magnitude earthquake, forensic engineering teams come to assess the damage and determine where and why damage and destruction took place within the context of the magnitude of an earthquake, the type of motion it originated (shaking, jarring, rolling), its duration, the area it affected, and the geologic properties of rocks underlying structures' foundations. hazard assessment teams also evaluate other factors that contributed to additional damage such as ruptured gas lines that feed fires and ruptured water lines that inhibit fire control. the engineers establish how construction can be improved in the future in terms of construction techniques and materials to prevent the types of collapses and utility failures they investigated. municipalities revise building codes accordingly to direct reconstruction and future building projects. where possible, structures that withstood an earthquake with minor or no visible damage should be retrofitted to improve their resistance to the next "big one." with each event, we gain more data on how to better construct earthquake-resistant structures and alter building codes to more stringent specifications. in theory, this adaptationto an irregularly recurring global event is good, but in practice it is most applicable to nations with the economic resources for reconstruction according to revised building codes and where there is no corruption to allow a bypass of the code. the same can be stated for retrofitting to give more resistance to earthquakes to existing structures. many developed nations and nations rich in commodity exports (e.g., oil) have a moral obligation to donate funds, material, and expertise to help citizens in economically disadvantaged nations recover from a destructive earthquake. some commodity-rich and economically sound nations do not do so directly, whereas others, big and small, rally to help disaster victims. for example, immediately after megatyphoon haiyan devastated many regions in the central philippines in , israel sent medical doctors and nurses and field hospitals to help philippine citizens recover from the impacts of the typhoon. as discussed in an earlier chapter, volcanoes are predictable in terms of becoming active by emitting wisps of smoke, bulging on a slope, warming of the soil or nearby pond or lake waters, emitting increasing concentrations of gases, and showing increased low-frequency seismicity. however, this activity does not always result in an eruption. a marked increase in measurements and observations, especially the low-frequency seismic activity, suggests that an eruption is imminent. adaptation to living and working on or near a volcano means investing in equipment to monitor volcanic activity and listening to alerts from scientists monitoring its activity and being ready to evacuate by gathering important papers and precious mementos and prepared to load into transportation for evacuation to safe locations. governments adapt by charging geologists to map out areas considered as high-, moderate-, and low-hazard zones in the volcano environs. geologists do this by studying rocks deposited from past eruptions and assessments of the topography. municipalities then pass zoning regulations applicable to the hazard level. governments have adapted to repeated periodic flooding in areas by creating flood control systems described in chap. . dams hold water during times of heavy and/ or extended rainfall and release any overflow into channels that move water away from urban or rural population centers. levees increase the volume of water that can move through a channel, thereby keeping it from spreading into populated areas and cultivated farmland. for smaller waterways that flow through cities, municipalities may invest in deepening, widening, and straightening channels as well as erecting walls so that more water can flow through the area more rapidly without coming out of a channel. governments define zones on flood plains according to a recurrence interval of damaging floods (e.g., years) as being off limits for residential and factory/plant construction. as much as we plan to adjust to living in an area prone to flooding, there is always the possibility of a megaevent that can overcome in situ control systems. therefore, as described chap. , governments adapt to this possibility by installing flood prediction equipment in drainage basins to provide warning to those at risk from rising and sometimes raging waters. the warning gives people time to gather important documents and personal treasures and evacuate to safe areas. the apparent increase in the frequency and magnitude of storms and resulting flooding in recent years is thought by many weather scientists to be related to global warming and the increased amount of moisture in the atmosphere from warmer oceans that gathers in clouds and precipitates during storms. this will be discussed further in this chapter. adaptation to extreme weather events such as an extended period of drought, heat waves, and frigid weather means preparation to wait them out. some municipalities adapt to repeated, sometimes seasonal, times of short-term drought by storing a - month water supply in surface or underground reservoirs during periods of normal precipitation that can be tapped (conservatively) as needed. others may plan to move water via pipes or water tankers from where it is plentiful to where drought conditions exist. otherwise, to survive, people move as best they can to where they have access to water. in instances of years long drought, crops and livestock and other life forms may be lost. heat waves can kill. adaptation to heat wave conditions means that water has to be available to people to avoid dehydration. where possible, homes should have air-conditioning or fans to keep people comfortable and municipalities should have cooling centers to which people can go. personnel should check on senior citizens and escort them to cooling centers if necessary. clearly, economically advantaged nations have the resources to give support to citizens during natural hazards such as these. these nations, international organizations, and ngos have a moral obligation to help economically disadvantaged nations as is possible when hazard conditions such as these threaten populations. the most extreme of weather conditions that can injure and kill people and destroy housing and infrastructure are tropical storms that evolve into violent hurricanes (typhoons, monsoons) by increasing wind speeds and sucking up moisture (water) as they track across oceans toward land. when these storms make landfall, they drive storm surges that can wreak havoc onshore communities, and as they move inland precipitate heavy rains that cause life-threatening and destructive flooding. these violent storms are destructive to coastal populations and island nations and have regional reach inland as they move along paths until they finally spend their energy or move out to sea. on november , , the typhoon named haiyan, the strongest recorded typhoon ever to make landfall smashed into the central philippines killing more than , people, injuring about , , and displacing almost , people. there was a -m (* ft) storm surge driven by winds measured at over km/h ( mi/h) with gusts reaching km/h ( mi/h). the typhoon flattened the city of tacloban that was home to , residents, and there was major flooding inland. the weather alerts led to a government call for evacuation away from the predicted path of the storm, and about million people followed the evacuation warning, surely saving many lives. access to aid typhoon-ravaged areas was difficult, and there were shortages of water, food, and medical care for many evacuees for several days. the philippine central government and local officials were not prepared to deal with a storm of this magnitude but help started arriving from many nations worldwide. there was a post-event concern of attending to sanitation needs of survivors to prevent outbreaks of diseases such as cholera, typhoid fever, hepatitis, and dysentery. if the philippine government had adapted by adopting better policies with respect to response to high-category typhoons in addition to the call for evacuation, the impact of haiyan would have been ameliorated. one would hope that this deficiency would be dealt with to limit the effects of future like disasters. evacuation to prevent injury and death in coastal zones that could be struck by high winds, heavy sustained rains, and storm surges is dependent on weather bureau forecasts and warnings from police, firefighters, or other government-authorized personnel. homeowners adapt to hurricanes by securing roofing with additional nails or special fasteners as a retrofit precaution and by boarding up windows on structures before an incoming storm hits. governments have adapted to the onslaught of violent high-energy storms by constructing seawalls of varying designs and heights to protect population centers by damping the force of storm surges. in china, for example, a seawall . m (* ft) in height and that has been heightened in the past protects shanghai from the full damaging effects of high-category typhoons. as a result of rising sea level, the shanghai seawall and other that protect coastal cities from being flooded by surges from high-energy tropical storms will have to be heightened to afford a greater degree of protection to people and property. wildfires can be a natural hazard when ignited by a lightening strike. however, most wildfires are started by human carelessness such as tossing a lit cigarette on a forest floor or failing to completely extinguish a campfire, or by arsonists. one may adapt to living in an area with a history of wildfires in two ways, neither of which is practical or promises % protection. first would be to clear an area of vegetation in a -m ( ft) swath around a dwelling or site for building. second would be to build with nonflammable materials so that embers propelled during a wildfire could not ignite a structure. adaptation to the advance of a wildfire would be to heed warnings to evacuate carrying a prepared case with important documents and other items of personal value. to delay evacuation by going back to retrieve something from then home can be fatal as it was for two people in a recent (june, ) wildfire that destroyed almost homes in colorado springs, colorado, usa. when there is a hazard event coming that calls for evacuation, responsible and often economically advantaged governments have adapted to the threat by designating evacuation routes, by providing transportation for people who need it, by having evacuation centers stocked with water and food, cots and blankets, basic medical supplies and medical personnel, and by having phone service available for people that need it. in the case of a primary or triggered hazard that happens with little or no warning (e.g., an earthquake, a tsunami, a volcanic mud flow), search and rescue teams should be ready to move in soon after dangerous conditions ease and they can move with safety. there should be medical attention to treat injured survivors, and stations set up as soon as possible to provide water, food, and other essentials available to those that survived with little or no physical hurt. these first steps at adaptation are the keys to survival. recovery after a shock phase can be long and drawn out, depending in grand part on a nation's social and economic resources and physical and economic assistance from other nations, international institutions, and ngos. change on our earth's inhabitants global warming is a fact attested to by an overwhelming majority of the scientific community and unwaveringly supported by a february joint publication of the us national academy of sciences and the royal academy in the uk on the causes and evidence for global warming [ ] . as noted in earlier chapters, during the past century, measurements show that the earth has warmed by * . °c (* . °f). global warming is an ongoing process that is attributed in grand part to a slow but continuous and increasing buildup of greenhouse gases in the atmosphere. the greenhouse gas most associated with global warming is carbon dioxide (co ). a plot of the increase of co content in the atmosphere with time against the increase in global temperature shows an excellent correlation of one with the other. additional lesser contributors include methane (ch ), nitrous oxide (no ), and chlorofluorocarbons (cfcs). with the beginning of the industrial revolution and the increased use of coal as the principal energy source, the content of co in the atmosphere was parts per million ( . %). the combustion of coal and later oil (petroleum) and natural gas emits co to the atmosphere. initially, and for many years thereafter, the added greenhouse gases were taken up by vegetation for photosynthesis and was also absorbed by the oceans and other water bodies. this kept the atmosphere co close to the ppm pre-industrial level. however, with increased industrialization, the need for electrical power, and the use of internal combustion engines, the amount of co generated was greater than what could be absorbed by nature and the content of co in the atmosphere increased. during june , its concentration reached more than ppm, an increase of over % over the pre-industrial concentration (scripps institute of oceanography mauna loa measurement). the increasing co content, other greenhouse gases, aerosols, and particles acted as a media that admitted sunlight (heat energy) to the earth's surface but did not let all of the heat escape back into the atmosphere. this abets global warming. in the past two to three decades, the rush to industrialization in developing countries (e.g., china, india, and brazil) and their growing power needs and vehicular use has thwarted the implementation of international agreements to reduce emissions from coal-fired power plants, other industrial and manufacturing operations, and the transportation sector. a direct consequence of global warming is sea level rise (slr) caused by the progressive melting of icecaps and ice sheets in greenland, the arctic, and antarctica, and of mountain glaciers in the himalayas, the alps, the rocky mountains, and the andes. the * -cm (* in) sea level rise during the past century may see a rise of another * cm (* in)- m (* in) during this twenty-first century. one-third of the rise would be from the expansion of warmer sea water, one-third from icecap and ice sheet melt, and one-third from mountain glacier melt [ ] . in , other researchers used computer models on existing data and proposed that % of sea level rise between and was from glacial melt [ ] . following the same line of investigation, other scientists studied satellite data and ground measurements from alaska, the canadian arctic, greenland, the southern andes, the himalayas, and other high mountains of asia and estimated that glacier contributions to sea level rise from to was % and together with ice sheet melt explained % of slr [ ] . a publication in estimated that ocean thermal expansion - m deep and - m deep contributed up to % to sea level rise [ ] . these latter two estimations are in line with the ipcc prediction for melting ice and ocean thermal expansion contribution to the estimated rise of sea level by the end of the century [ ] . with a rise in sea level, marine waters encroach on land. as the rise continues, possibly at an increasing rate, it threatens habitation in lowlying islands, coastal villages and farmland in lowlying zones, and heavily populated cities worldwide settled on inshore terrain close to sea level (e.g., bangkok, ho chi minh city, jakarta, manila, miami, new york, boston, buenos aires, london, rotterdam). rising sea level and warming of ocean waters have other ramifications that affect coastal communities as well as inland areas. as explained in chap. , the warmer surface water releases more water vapor with heat energy into the atmosphere. when the water vapor molecules condense in clouds, heat energy is released. this energy gives more force to tropical storms as they form, track to shore, and move inland, or storms that move close to and along a coast. these storms may transition to hurricanes (typhoons, monsoons) with the violent winds that cause destruction, and heavy rainfall that triggers flooding if they move onto land. we recognize that rising sea level means that tropical storms that impact a coast with storm surges have a farther reach inland with their destructive energy that is more pronounced when the surge occurs at high tide. the surges also saturate farmland they reach with salt water that harms crops. they also carry salt water into fresh water marshes and ponds, thus disrupting ecosystems there. the increase in the number of these extreme weather events and the increase in violence and destruction they wreak on land compared with like weather events in the recent past (e.g., during the past years) strongly suggest that they are fueled to a significant degree by global warming. there are two possibilities for adapting to the effects of rising sea level on coastal urban centers, one impractical, the other very costly but doable. the impractical adaptation possibility is to move at-risk population centers inland, out of the reach of the destructive tropical storms. this does not lessen the threat of flooding. the move is possible in some cases where land is available, but such a move is not economically feasible. one practical but costly adaptation to mitigate encroachment from sea level rise and the effects of tropical storm surges is to surround cities at risk within place seawalls - m higher than recorded high tides or higher depending on historical records and contemporary published data. the walls can have a concave configuration so that surging waves lose energy when their lower parts hit and are curled back on themselves damping some wave energy or there can be a different configuration best for the site(s) to be protected. similarly, gates buried at strategic locations where there is ship access to consider can be built to be hydraulically driven so that they can rise from a near shore seabed site to mitigate the effects of storm surges. both techniques have been used at different global locations. we have read that climate change affects land-based agricultural production, both for crops and animal husbandry. the warming climate at higher mid-hemispheric latitudes and at higher altitudes does not favor the growth and normal yield and/or quality of many crops. depending upon the degree of climate change and the linked change(s) that may follow it, farmers can adapt in several ways to maintain or increase crop yield and nutrition value. for example, when warming starts diminishing the productivity of a traditional crop, farmers can sow crops that are known to grow well in warmer temperature and give a satisfactory economic benefit. however, new groups of weeds, pests, and diseases will migrate to the warmer growth environment and will have to be dealt with in order to protect the new crops. where the effect of global warming reduces water supply for rain-fed agriculture, for crops irrigated with surface waters, and for groundwater-irrigated crops when aquifer recharge does not balances discharge, agriculturalists can adapt in two ways. first is the use of a more efficient irrigation method that delivers water directly to a growing plant (e.g., drip or focused irrigation). this minimizes runoff and loss to evaporation. second and similar to what was mentioned earlier is to sow a crop that needs less water to thrive and that delivers a good yield, good-quality product. another result of global warming for some farmlands is a longer growing season. in this situation, growers can adapt by planting earlier and have the possibility of double cropping. they can also grow a cultivar that is later maturing and that gives a product that brings a good market price. however, switching to new crops in a warmer growth environment means that there will be an invasion of a new set of weeds, pests, and diseases to ward off. in any efficient operation, and as emphasized in earlier chapters, farmers adjust to a changing growth environment for a given cultivar by applying the optimum amounts of fertilizer and other agricultural chemicals as might be needed that nurture and protect it most effectively. this reduces agricultural costs and lessens runoff of these chemicals to ecosystems where they can be harmful. global warming can bring on abnormal weather extremes that affect agricultural productivity. in these cases, farmers have to plan ahead based on recent history of these conditions in their regions. drought, heat waves, and long-term rain or heavy rain in a short time present problems for both cultivars and food animals. periods of less than average precipitation may last months or years. depending on the amount of the deficit precipitation, adaptation can include storing water in reservoirs and cisterns during times of rainfall to be tapped during a drought to sustain food animals and crops during a short-term, not too severe drought. there is also the option of trucking in water to sustain livestock. long-term droughts when precipitation deficits are high take their toll on plants and animals to the detriment of agriculture in a region especially when accompanied by heat waves. they have caused recent disasters for crops and food animals on all continents less antarctica. farmers either wait out the "bad times," change the type of cropping they do, the livestock they tend to, or change careers. the adaptation from crops that have been grown successfully before the effects of global warming reduced yields and quality of a harvest, to those "same" crops that can grow successfully under the advancing warming changes just described generally means that hybridized species have to be developed and used as warming increases at a location and slowly tracks to higher latitudes and higher altitudes. thus, growers turn to plants that are created by hybridization as described in chap. : traditional methods and marker-assisted selection methods within the same species, and genetically engineered (-modified, -manipulated) methods using different species. hybridization is a slow process, sped up markedly by genetic engineering, a method that yields foodstuff not accepted by the european union and many nations outside the union, especially in africa. bred species are developed to carry one or more characteristics that favor crop resilience against the effects of climate change. these include resistance to disease, weeds, and pests, and tolerant of drought (water stress), heat, short-term inundation, and short-term saline exposure (see chap. ). hybrids have also been developed to give higher yields and more nutritious crops. thus far, research has been focused mainly on improving seed for world staples such as rice, maize (corn), wheat, sorghum, and soybean. there have been great successes where hybrid crops were agriculturalists' adaptation so that the possibility exists that we can feed the earth's growing populations and reduce chronic malnutrition. when this is coupled with the opening of additional arable acreage and the use of improved farming methods for seeding, watering, and harvesting, global food security can be strengthened for the existing world population and the future generations on earth. however, this will require economic and technical input by developed nations and international groups. without basic sustenance, people will have less resistance to diseases and there may be local or regional population crashes if diseases evolve into epidemics or pandemics that invade susceptible populations. warming of the open ocean water, enclosed aquaculture operations in ocean waters and on land water bodies has affected marine fisheries and marine and estuarine aquaculture that grow food fish and shellfish, and lakes that sustain fisheries. in marine fisheries worldwide (e.g., in the north atlantic, off the coast of peru, off the coast of the philippines), some food fish or fish captured for other purposes (e.g., to use in pet food, to use to make fertilizer) have migrated to cooler water in ecosystems with conditions conducive to their spawning and growth. in some cases, predators follow fish they prey upon that have migrated to cooler waters, but in other cases they find new prey to sustain them. in other situations, they may become prey for larger fish in an ecosystem. fishing fleets adapt by following the fish they hunt into cooler waters where ideally they capture the hunted species in quantities allotted them by national and international fishery governing body regulations. if the quota system is followed, this will allow recovery of fish populations and sustainable harvesting. aquaculture operations that provide important supplies of food fish worldwide can adapt to warming waters by raising food fish or shellfish that will grow and multiply under the changed range of day/night temperature conditions if the fish they are farming cannot survive in the warmer waters. aquaculturalists also have the option to move their facilities to cooler-temperature waters, but the economic feasibility of doing this has to be evaluated by a benefit to cost analysis. this analysis has to be for the time frame during which the cooler-ecosystem waters are estimated to remain stable within the framework of a time range against global warming/climate change. another adaptation is that food fish currently being raised can be genetically engineered to be resistant to a warmer growth environment without changing their nutrition yield, growth rate, and ability to reproduce. there are diseases that are global threats, others that put regions at risk, and yet others that menace smaller political divisions. humans adapt to the threat of sickness in a population or a sickness itself in several ways. scientists develop methods to eradicate a virus or bacterium health threat, or a chemical/radioactivity threat. failing this, health professionals act to control a disease, to slow or minimize its transmission, and to apply approved therapies and support research to find therapies to treat an illness if one is transmitted. the following discussion draws strongly on the disease fact sheets put out by the world health organization. vaccines provide a main line of defense against many diseases. smallpox has been eradicated on earth by vaccination. polio has all but been eradicated globally except for a few pockets of the disease in pakistan, afghanistan, and nigeria where, in some cases, religious fundamentalists have beaten and killed health workers tasked with giving the vaccine to children, and in other cases where parents have been warned by the zealots against allowing their children to be vaccinated. recently, polio cases were diagnosed mainly in somalia but also in kenya. this is attributed to the fact that by , , children in somalia have not received the vaccine and are at risk from this highly contagious disease. it is also attributed to crossborder migration of infected persons into kenya. both governments are stepping up their vaccination programs. there were cases of polio diagnosed in the rest of the world in . measles is a global disease that can be prevented by a vaccine that is safe and cost-effective. measles may soon reach the near-eradication stage. in and subsequent years, . million people, mainly children under years of age, died from measles. since , billion children were vaccinated, million in . by , % of the world's children received the measles vaccine, up from % in . from to , deaths from measles dropped to %, from , to , . when the vaccination rate reaches %, mainly in low-income countries, the world will have brought another disease close to elimination [ ] . seasonal influenza is a global viral illness that afflicts - million people. the sickness kills , - , people with severe symptoms annually. transmission of the virus takes place when an infected individual coughs or sneezes without covering his/her mouth and releases droplets that can be inhaled by someone up to a meter away. transmission can also be from hands carrying the virus. seasonal influenza affects all age groups, but children less than years old, people over , and those with complicating medical problems are most at risk. influenza is a disease to be controlled. the principal control is by safe and effective vaccines that can prevent - % of influenza cases in healthy adults. secondary controls are obvious for infected persons: cover the mouth when sneezing or coughing, and wash the hands frequently. the influenza vaccine is taken once annually. because strains of the influenza virus change from year to year, adaptation is needed. the adaptation is via a vaccine that is prepared with or strains that scientists determine will be most common during a coming season [ ] . other types of influenza and respiratory illnesses have the potential to cause an epidemic or pandemic. they include avian flu and its strains and swine flu if the strains develop the ability for person-to-person transmission after infection, and sars (severe acute respiratory syndrome) and middle east respiratory syndrome (mers) because there is human-to-human transmission of the sicknesses. to the present, the outbreaks of the animal influenza diseases have been contained by quarantining infected people during treatment and by culling flocks and herds, or if available, vaccination of healthy animals. the latter two respiratory illnesses are caused by the coronavirus, and infected people have been in isolation wards. for sars, an illness that broke out in and spread to countries, isolation of victims and treatment with antiviral drugs and steroids stopped the disease during . mers is a recent ( / ) illness that has been confined to jordan, saudi arabia, qatar, and the united arab emirates. the mers virus has been found in camels. infected persons are quarantined in hospitals, but an effective drug treatment is still being sought to complement the normal hospital care-afforded patients. hiv/aids is a global epidemic that killed million people in three decades since . worldwide, in , there were million people with hiv, mainly ( million or %) in sub-saharan africa and south/southeast asia. the illness is caused by the exchange of body fluids (semen, vaginal excretions, blood, breast milk) from an infected individual with an uninfected person. more than % of the cases of hiv are from heterosexual activity. there is no vaccine against hiv/aids, no cure for it, but there is a cocktail of medicines (antiretroviral treatment) that control viral replication and allow an infected person's immune system to strengthen. this keeps the illness at bay and afflicted people in general good health and productive in their communities. in , only . million (less than %) of those with hiv in low and middle economies received the antiretroviral treatment. this is changing as more hiv carriers have access to antiretroviral therapy and there are more donations from economically advantaged countries to support hiv stabilization and reduction programs. the number of new cases of hiv is not exploding because more than % of those infected are following protocols that reduce the transmission of the disease. the prevention of transmission methods include access to male and female condoms, blood screening before transfusions, and needle and syringe exchange programs for sterile injections by drug users. hiv testing and education programs and hiv treatment help prevent transmission because individuals in continuous treatment have a very low probability of passing on the disease. male circumcision reduces the infection in men by about %. there is still much progress to be made because there were . million new cases of hiv in , with . million of that total in sub-saharan africa. the hiv/aids is a global sickness that is slowly coming under control because of generous donations from governments and foundations in developed countries added to what low-and middle-income countries themselves provide to lower the prevalence and incidence of hiv in their populations [ ] . tuberculosis (tb) infected . million people globally in , killing . million persons. it is a bacterial disease that spreads among people when infected individuals cough, sneeze, or spit, releasing bacteria into the air where they can be inhaled by others a meter away. although tb occurs worldwide, developing countries carry the largest burden of cases and deaths ( %). the bulk of new cases are regional in asia ( %) with sub-saharan africa reporting a large share as well with , new cases per million inhabitants. there is no vaccination for tb, but the disease can be treated and cured. the treatment is a half-year course of four antimicrobial drugs that must be taken without fail and thus requires continual supervision by healthcare personnel. more than million people have been treated and cured of tb since and perhaps million lives saved by following the who stop tb strategy protocols including securing adequate, sustained financing, ensuring early reliable detection and diagnosis, and providing approved treatment with a secure effective drug supply. the number of people infected with tb is declining, and from to , the tb death rate dropped more than %. the success in dealing with tb is muted somewhat because a strain of the bacterium that causes tb has evolved to be multidrug resistant (mdr-tb). in , , cases of this variant were reported (of the . million cases worldwide), mainly from india, china, and the russian federation. these are treated with, but do not always respond to, the most effective anti-tb drugs. research into new drugs to deal with this problem is ongoing [ ] . there is the question of whether people visiting or immigrating from these countries should be screened before a host country issues them entry visas. regional illnesses threaten the health of s of millions of people mainly in tropical and subtropical areas and often affecting children. one of these, the guinea worm disease, is trending toward elimination, if not eradication. this is a parasitic disease caused when people swallow water contaminated with infected water fleas (microscopic copepods) carrying worm larva. the worms release, penetrate the intestines, and move through the body migrating under the skin until they emerge causing swelling and blistering. people infected with guinea worm disease cannot contribute to their communities for months. during the mid- s, there were . million cases mainly in african nations. but attention to where the sources were so that they could be avoided and treated, and assistance in generating clean water, were adaptations that brought the number of cases down to less than , in . the number of cases continued to decline and was reduced to in in four african countries: south sudan, chad, ethiopia, and mali. there is no vaccine against guinea worm disease. health officials adapt to counter this sickness in several ways. as noted above, access to clean drinking water is the best way to prevent infection. the prevention or transmission of the worms from infected individuals to healthy persons by proper treatment and hygiene and the use of the larvacide temephos to eliminate the parasite-infected water flea vector and other prevention protocols are important in the control and effort to eliminate/eradicate the disease [ ]. the (jimmy) carter institute, atlanta, georgia, usa, has been a principle force since in the fight to rid the world of guinea worm disease. in tropical and subtropical regions, there are three mosquito-vectored diseases that put millions of people at risk: yellow fever, malaria, and dengue fever. yellow fever is an endemic viral disease in tropical regions of africa and latin america with , cases reported annually that cause , deaths. there is no set treatment for afflicted people, but there is an adaptive preventive measure. a vaccine against yellow fever is available that is safe, affordable, and that gives lifelong immunity to the disease with one dose after - days for % of the people vaccinated. when there is the onset of a yellow fever outbreak where the population lacks vaccination protection, mosquito control is an essential first step in adaptation to prevent or slowdown transmission of the yellow fever virus. spraying insecticides to eliminate breeding sites and kill adult mosquitos is the control used during epidemics to make time for vaccination campaigns in a population and for immunity to take hold. there are limitations to the application of the yellow fever vaccine. first is that babies less than months of age should not be vaccinated or, during an epidemic babies less than - months of age should not receive the vaccine. second, pregnant women should not be vaccinated except when there is an outbreak of the disease. third, people with a strong allergy to egg protein or those with a marked immunodeficiency or with a thymus problem should not receive the vaccine [ ] . malaria is a parasitic disease caused by the bite of an infected mosquito. there is no vaccine against malaria, but one is undergoing a clinical trial in seven african nations with results expected in . a use or no use decision as a control method for malaria will be made in . promising results from an early-stage clinical trial of an unconventional vaccine prepared with live, weakened sporozoites of the malaria parasite were published in . plasmodium falciparum was given to healthy - year-old volunteers intravenously. the volunteers were grouped to receive - doses and subsequently exposed to bite by five mosquitoes carrying the parasite. none of the six that received five doses were infected with malaria. three of the that received four doses became infected, whereas of the that received lower doses became infected. of that received no vaccine, became infected. those that became infected were treated with malarial drugs and cured. clearly, higher dosages give protection against infection by malaria [ ] . more research and extensive clinical trials are necessary to determine how children respond to the vaccine with adjusted dosages and whether the results from earlystage trial are reproducible in larger volunteer populations. if the results of additional clinical trials go well, the hurdle of producing enough vaccine and adapting it to injection has to be faced. forty percent of the deaths from malaria are of african children in the democratic republic of congo and nigeria. in addition to sub-saharan africa, populations in asia (especially india and the greater mekong region) and latin america suffer from the disease. the effort to deal with the disease that is preventable and curable now centers on control and treatment to reduce the number of cases. in , the who reported that there were million cases and , deaths (with an uncertainty range of , - , ). in a report, researchers suggested that the number of deaths was understated and that their computer model gave a figure for almost double, , , deaths ( % uncertainty interval of , - , , ) [ ] . the who stood by its figure stating that much of the data in the cited study were based on verbal testimony of how people had died, not on laboratory diagnosis of samples. either figure represents too many deaths from the disease and have to be reduced. mosquito control is the adaptation that can reduce the transmission of the disease greatly. this includes personal protection by use of proper clothing and/or the application of mosquito repellent, the use of longlasting insecticidal (pyrethroids treated) nets to kill mosquitos and prevent nighttime bites, and indoor residual spraying (remains effective for months). those people infected can be treated with oral artemisinin monotherapy followed by a second drug. failure to complete the treatment as prescribed leaves parasites in a person's blood. no other antimalarial treatment is available so that parasite resistance could become a serious problem. for visitors to a malaria region, antimalarial drugs taken before, during, and after a trip can protect them from the disease. many countries in tropical and subtropical areas have used the above-cited strategies and others to work toward the elimination of malaria. malaria eradication is the goal of the who [ ] . dengue fever is a female mosquito-borne virus that infects people with an influenza-like disease in tropical and subtropical regions worldwide. the disease can kill if it evolves to severe dengue. it is endemic in latin america and asia where most cases now occur. since the s, the sickness has spread to more than countries putting about % of the world's population ( . billion people) at risk. dengue fever is especially endemic to urban/semi-urban environments. humans are the main carrier of the virus. after a mosquito bites an infected person, each subsequent bite by the infected mosquito creates another carrier. a mosquito can bite many people each time it feeds. in the americas alone, there were . billion cases of dengue fever reported in with , being severe dengue. there is no vaccination against dengue fever, but research continues to develop one. the main treatment for afflicted persons is to keep them hydrated. adaptation to deal with slowing or stopping the spread of dengue fever involves three main tracks in addition to spraying insecticide to kill mosquitos. the best control method to prevent the transmission of the virus is to deprive mosquitos of sites with shallow, standing water where they can lay eggs and multiply. control can be improved if communities cover and clean water storage containers regularly, and use proven insecticides on them as necessary. finally, individual protection such as the use of mosquito repellants and insecticide-impregnated bed nets can help reduce the incidence of dengue fever as it has with malaria [ ] . although controls are known, they are not always applied because of economics and other factors that prevent access to protection methods. the result is that the number of cases of dengue fever reported continues to grow globally. as populations increase in urban locations, the incidence of dengue fever can be expected to increase as well unless strict controls are enforced until a safe and cost-effective vaccine is developed. a positive aspect of the dengue fever problem is that recovery from one serotype of the virus gives immunity for life. however, there are four serotypes of the infectious virus so that recovery from one leaves a person susceptible to the others [ ] . chagas is another regional disease. it infects - million people annually, mostly in latin american countries. it is a parasitic illness that evolves after the bite of a blood-feeding triatomine bug, often on the face, where it defecates close by leaving parasite-bearing feces. parasites access the body when the feces are inadvertently smeared into the bite, the eyes, the mouth, or any skin lesion. the parasites circulate in the blood expressing their presence as a purplish swelling of one eyelid or as a skin lesion. there are several other symptoms as well in this acute stage of the illness, but these may be absent or mild. if diagnosed early during this stage, chagas disease is treatable. the parasite is killed with the medicines benznodazole and nifurtimox taken for months. there are limitations as to who can take these medicines (e.g., not by pregnant women or people with kidney or liver problems). the untreated sickness can cause cardiac alterations and digestive problems that show up - years after an untreated infection. chagas disease can be spread by blood transfusion and by organ transplant, making blood screening for the parasite essential before a procedure. it can also pass to a fetus from an infected woman. there is no vaccination against the illness so that control of the vector (triatomine sp.) is necessary. the controls adapted by many municipalities include insecticide spraying inside a home, the use of treated bed nets, and hygiene practices that protect food, its preparation, and its storage before eating it [ ] . the sickness may recur if control practices become lax. chagas disease is spreading as populations emigrate from latin america to northern countries. blood screening of visitors or immigrants from the countries where chagas is endemic may be necessary, and treatment followed by an infected individual before a host country issues an entrance visa. this would prevent the ingress and possible spread of chagas. outbreaks of diseases in town and cities is most often caused by bacterium-contaminated water or food and poor sanitation. sicknesses such as cholera, typhoid, and various other diarrhea types are examples of such diseases. they are all highly infectious if good hygiene practices are not followed. these diseases are endemic in many countries where populations do not have access to safe water and adequate sanitation. there are vaccinations for some of these sicknesses that may require more than one dose, but they may not be completely effective or long lasting and require revaccination at times specified by medical personnel (e.g., after - years). otherwise, infected persons can be treated with medicines such as oral rehydration pills or antibiotics. adaptation for prevention is easier called for than realistically available: washing hands with soap and clean water after visiting the toilet, and as noted above, access to safe water and good sanitation. given the millions of people infected by these bacterial diseases and the hundreds of thousand that die from them annually, generally in economically disadvantages countries, there should be an expanding global priority to eliminate the disease-causing conditions, and preparedness to combat an outbreak when it is reported. there are important factors to consider when adopting plans to halt or meliorate the effects of health threats to people in the near and extended future. one is the climate change-driven spread of tropical and subtropical diseases discussed earlier to newly warmer and moister higher-latitude and higher-altitude zones. another is the growth of populations mainly in tropical and subtropical regions in africa, asia, and latin america. together with this latter factor are the increasing populations and population densities in urban centers especially in the regions just cited. an additional factor to consider is whether there is accessibility to populations by healthcare workers or by people to healthcare clinics or hospitals, well-staffed and well-stocked with necessary pharmaceuticals. certainly, future planning has to include funding to support research to develop vaccines for diseases that do not have vaccination as an option against an illness (e. g., malaria, dengue fever) . in addition, improvement of vaccines that are available but that are not completely effective in terms of protection or the length of time they are effective should be a priority in pharmaceutical and biotechnology laboratories. scientists presented a fine review of the status of vaccine research from the design and development of vaccines to discussion of vaccines and infectious diseases (e.g., hiv, malaria, tuberculosis, pneumococcal disease, and influenza) [ ] . they also discuss vaccines against enteric infections and viral diseases of livestock as well as vaccines against non-infectious diseases (e.g., cancer) and against chronic noninfectious diseases. continued and repeated education classes on how to prevent the transmission of diseases and free supplies of materials that work to this end (e.g., insecticide-treated bed netting, male and female condoms) are essential to reducing the prevalence and incidence of diseases as are safe water and uncontaminated food. as new medicines or combinations of medicines are developed, tested, and found to be effective in controlling diseases, they become part of the protocol for either curing disease or controlling disease to reduce transmission while allowing persons to carry on with their lives. in these times of easy and rapid migration, one wonders whether screening of visitors or immigrants for diseases known to be endemic or active in the countries or regions from which they come should be required so as to prevent a carrier from infecting others and spreading a disease (e.g., chagas disease, cholera, tuberculosis). this was done at airports during the sars scare for people leaving or entering a country (e.g., china) and likely limited the transmission of the sars virus and spread of the disease. preparedness for a disease outbreak, response to an outbreak, and management of resources during and post-outbreak are the keys to adapting to health threats that could affect future generations. this means developing the capability to extend the reach of health services to regions where climate change brings warmer, moister conditions to higher-latitude and higher-altitude ecosystems that are now reached by disease vectors that have expanded into these formerly cooler and drier environments as a result of global warming. adapting to this reality and planning ahead makes it possible to deal with and stem an incipient outbreak of disease before it is transmitted and spread to the general population. this becomes essential when there is a future disease outbreak in large, dense populations in tropical and subtropical urban centers as well as those in regions warmed and humidified by climate change to subtropical and tropical settings. remember that urban populations worldwide, especially in africa, asia, and latin america, are where much of the global population growth will take place during the next few generations. under these conditions, diseases can spread rapidly in many ways. these include from bites of vectors, by respired droplets after an infected person coughs or sneezes, and by touching surfaces bearing viruses, bacteria, or parasites. diseases are also spread by ingestion of contaminated water and/or tainted food, and by other methods of infection transmission. disease transmission can be checked by rapid response teams with appropriate and sufficient supplies to treat (and perhaps places to quarantine) those in the infected population. lastly, it must be noted that there are many other diseases in addition to those cited previously for which prevention, treatment, and cures are research priorities in laboratories worldwide. in addition, there are addiction diseases that can trigger health problems in important segments of society. these include smoking (e.g., emphysema, lung cancer), alcoholism (e.g., cirrhosis of the liver), drugs (e.g., various psychological and physical ills), and overeating (obesity, diabetes, high blood pressure). adaptation to these health threats involves public education forums through various media outlets, counseling, and sponsored groups with their individual group meeting, and programs are assisting many in breaking from an addiction to the benefit of a healthier life. adaptation to meet the health challenges in the past, and in contemporary times has been a slow, progressive adventure with many successes but with much yet to be done. this is the planned path for the future: meet the challenges of societal health threats, resolve many, and keep researching to resolve others. a special ipcc report in examines in a general way adaptation to a changing climate as a risk management approach [ ] . it uses pre-planning to reduce exposure and vulnerability to extreme hazard events by preparing for them beforehand, responding to their impacts on people, structures, and infrastructure, and having in place recovery systems that can act when a danger condition eases. in this way, there will be an ability of populations to cope with future risks brought on by a changing force with which a hazard impacts a community, changes in the frequency of an occurrence, and extension of the spatial reach of its destructive power. much of this has been discussed in the chapters of the book you are reading. an understanding of what is being done now to adapt to the various problems society faces during the second decade of the twenty-first stimulates proposals of how to adapt to them as global conditions change in the future. to this end, the world bank commissioned a study on the effects global warming as it increased from . °c that exists on our planet now to what can be expected if the warming reached °c, a change that many scientists believe we can adapt to, and then reached °c as warming continues [ ] . the study centered on regions with high population growth and great susceptibility to be negatively impacted by climate changes: ( ) sub-saharan africa where food production is at risk: ( ) southeast asia where coastal zones and productivity are at risk; and ( ) south asia where there could be extremes of water scarcity and excess. the effects of higher temperatures from global warming and climate change included what has been discussed in previous chapters of this book: heat, drought, sea level rise, coastal zones, typhoons, flooding, river runoff, water availability, ecosystem shifts, crop yields, fishing, aquaculture, livestock, health and poverty, and tourism. projections such as those published in the world bank study give impetus to governments, international institutions, multinational companies, private foundations, and ngos to think now, to invest now, and to research now for adaptations that can be realized in good time and that will provide global citizenry with a good quality of life where needed. in this book, we have examined existing human populations and the problems they are experiencing in the second decade of the twenty-first century and have also considered growing populations globally and additional problems future generations will experience. we have discussed strategies on how to cope with manyfaceted threats to citizens. these include how to nourish those who need food and water, how to shelter people safely from natural and anthropogenic hazards, how to provide them with healthcare, education, and employment, and how to prepare them for the evolving global warming and the physical and biological dangers that ensue from climate change. given the present global conditions with about % of our earth's population suffering from malnutrition and more than % not having access to safe water, our capability of nourishing a billion and a half more people by is in question. also problematical is our capability to provide for an additional billion people years later, or a total of at least . billion people by the turn of the century, that is, if we reach those population figures or have population crashes such as from pandemics that can kill scores of millions if a disease is not immediately treatable, or an unlikely but possible nuclear conflagration that could do the same. less likely yet is an explosion of a small asteroid or comet in the atmosphere such as happened in a poorly inhabited area of siberia in . here, an exploding mass more than m in size knocked down millions of trees in an area greater than , km (close to mi ) with energy thought to be , times greater than the hiroshima atomic bomb. clearly, such an event could kill the population of a megacity if it were to occur. another question is whether national governments are economically strong enough and have the will to set priorities that adopt strategies to protect citizens from natural (e.g., earthquakes) and anthropogenic (e.g., pollution) hazards as well as from extreme weather conditions that are supported by global warming (pollution of the atmosphere) but are naturally occurring. countries can also improve social and economic conditions by investing in health care and education for their citizens in order to form a sound and knowledgeable cadre that would be attractive to investors interested in locating a development project that would provide employment. again, this is in question given limited national economic capabilities and the increasing numbers of people to be accommodated, especially in several developing and less developed countries in africa, asia, latin america, and the middle east. at this point, we must ask, "what is the carrying capacity of the earth?" have we reached it at billion given the billions who are today under served in developing and less developed countries? some scientists will answer yes, whereas others believe that advances in agriculture and technology can allow population expansion although to what point is not defined. can countries that are poisoning their environments do a turn around to save their citizens from grief? can they exert controls on operations that create unhealthy conditions that sicken people, lessen agricultural production, and otherwise disrupt local, regional, and global climate change: evidence and causes ( p) intergovernmental panel on climate change ( ) climate change (as four part report). part . the physical science basis mitigation of climate change past and future sea level change from the surface mass balance of glaciers a reconciled estimate of glacier contributions to sea level rise ocean thermal expansion and its contribution to sea level rise tuberculosis. fact sheet no. p . world health organization ( ) dracuncukiasis (guinea-worm disease) protection against malaria by intravenous immunization with a non-replicating sporozoite vaccine global mortality between and : a systematic analysis world health organization ( ) malaria. fact sheet no. p . world health organization ( ) dengue and severe dengue chagas disease (american trypanosomiasis). fact sheet no vaccines and global health ) ipcc special report summary for policy makers. managing the risks of extreme events and disasters to advance climate change adaptation turn down the heat: climate extremes regional impacts and the case for resilience. a report for the world bank prepared by potsdam institute for climate impact research and climate analytics key: cord- -a ku rke authors: tyring, stephen k. title: syndromal tropical dermatology date: - - journal: tropical dermatology doi: . /b - - - - . - sha: doc_id: cord_uid: a ku rke nan with increasing numbers of persons from industrialized, temperate countries traveling and / or working in tropical lands, there is a marked need for physicians to be able to diagnose accurately and treat tropical diseases with mucocutaneous manifestations. while some studies demonstrate that approximately one-third to two-thirds of travelers returning from tropical countries experience some health problem, diarrhea is the most prevalent complaint. mucocutaneous problems, however, are among the top five health complaints of the returned traveler, and comprise - % of health concerns of persons returning from the tropics. during international conflicts, soldiers from north america, europe, and australia are often required to serve in tropical lands and sometimes develop diseases not familiar to physicians of their home countries. this was the case for french soldiers serving in vietnam in the s and american soldiers serving there in the s and s. recently hundreds of american and allied troops serving in iraq and afghanistan have developed "baghdad boils" (i.e., leishmaniasis), transmitted by sand fly bites ( fig. - ) . likewise, millions of persons from tropical countries now live and work in temperate lands and may present with medical problems with which the physician is not familiar. whereas the cutaneous problems in the returned traveler are frequently the the person traveled. for frequent travelers, the history may become complex if patients report having visited many destinations within the past few months. because vectors differ with the climate, the season of travel is also noteworthy. even in a tropical country where the temperature is always hot or warm, there may be a dry season and a rainy season. because seasons are reversed north and south of the equator, it is important to know the season at the destination. the duration of the stay is significant, not only because a longer stay increases the chance of acquiring an infectious disease, but also because it tells the physician whether the person was in the tropics during the incubation period of the suspected disease. whether the visitor was only in an urban environment or also in a rural area is relevant. whereas a sexually transmitted disease (std) could be acquired in either location, an arbovirus or a zoonosis might be more likely in a rural situation. the altitude of the destination could provide a clue to the etiology of the skin condition, as could the type of sleeping condition. for example, a sexually transmitted disease could easily be acquired in a five-star hotel, but an infection transmitted by a flea, louse, or mite would be more likely in someone who had slept on the ground and / or in a tent. the type and preparation of food and drink consumed by the traveler would not only help explain gastrointestinal symptoms, but could also be a clue to cutaneous signs (i.e., unsafe drinking water or milk or raw or undercooked meat, fish, or shellfish). a list of the patient's current and recent medications can be very useful and should include prescription drugs, illicit drugs, and herbal remedies, because the source of the cutaneous problem may not be directly related to the travel destination, but rather may be due to medications taken to prevent travelrelated illnesses. for example, many antimalarials, such as chloroquine, mefloquine, proguanil, quinine, and halofantrine, can cause cutaneous reactions, and chloroquine, doxycycline, and quinine can cause photosensitivity. interestingly, chloroquine can worsen psoriasis. a number of agents taken to treat or prevent diarrhea can also cause cutaneous reactions, such as quinolones (ciprofloxacin, ofloxacin, sparfloxacin, levofloxacin), furazolidone, metronidazole, trimethoprim-sulfamethoxazole and bismuth sulfate; quinolones are particularly likely to produce photosensitivity. anthelmintic medications, such as on the other hand, contaminated food may have originated in a tropical or subtropical area, such as when oysters from the gulf of mexico are shipped to the midwest usa and are consumed raw. the resulting vibrio vulnificus or hepatitis a infection thus produces gastrointestinal and cutaneous manifestations in individuals who may not have visited the source of the shellfish. therefore, it is always important to ask about new pets, changes in diet, or any other change in persons with a suspected tropical disease. on the other hand, travelers may have purchased non-consumable items that are the source of their dermatoses. for example, animal skins used for rugs or blankets may be the source of anthrax. a non-infectious cause may include nickel-containing jewelry to which the patient has developed contact dermatitis. whereas travelers naturally fear large carnivores while on camera safari, or sharks and a variety of other aquatic animals while swimming or diving, it must be remembered that the animal (indirectly) responsible for most morbidity and mortality is the mosquito (i.e., malaria, dengue, etc.) ( fig. - ). an example of a mosquito-borne disease that was considered primarily "tropical" in the recent past but is now relatively common in much of north america is infection with the west nile virus (fig. - ) . sometimes the skin findings on physical examination are not the reason for the visit to a physician or even the patient's complaint. such skin findings may be cultural, such as tattoos or scarification, or the result of the use of kava or of chewing betel nuts. some cultural practices, however, would be considered abuse in industrialized countries, but are widely accepted religious / cultural practices in certain lands. an example of such practice is female circumcision, which is practiced in many countries in sub-saharan africa. on the other hand, the skin changes may be much more benign, transient, and may even be the result of previous therapies, such as cupping and coining, widely practiced by immigrants from southeast asia. considerations for deciding the differential diagnosis of cutaneous manifestations of tropical diseases and / or of diseases acquired while traveling must be based not only on the type of lesions and systemic symptoms but also on the patient's history of travel. because the incubation period of various infectious diseases differs widely, it is important to know when temperate countries, but in the st century they have become rare in industrialized countries, except for imported cases. due to non-compliance with recommended vaccinations, however, measles cases were reported in the us in , the highest number in the st century. a single outbreak in early , resulting from an infected person visiting disneyland, california, resulted in cases. worldwide, however, almost one million children die of measles annually ( fig. - ) and rubella still causes many congenital abnormalities. measles is still the number one vaccine-preventable killer of children in the world. morbidity and mortality are often the result of secondary bacterial infections developing in malnourished infants with measles ( fig. - ). in east asia, sub-saharan africa, and many other parts of the tropical world, hepatitis b is very common and a major source of morbidity and mortality. although measles, rubella, and hepatitis b should not be a problem in the immunized traveler, many travelers have not received the proper immunizations because they or their parents had unfounded concerns about the safety of the vaccines. this problem continues to grow as more people reach child-bearing age without ivermectin, albendazole, and diethylcarbamazine, can also produce pruritus and rash. even diethyltoluamide (deet), used to prevent arthropod bites, can cause an irritant dermatitis when used in high concentrations. because many medications in tropical countries are sold over the counter and / or have different trade names to those in industrialized lands, patients are not always certain what they have received if treated during their travel. likewise, an injection or transfusion given in a tropical country might also carry an increased risk of contamination. a similar risk might be taken by having acupuncture, tattoos, or body piercing in tropical lands, but these procedures can be hazardous even in industrialized countries because the first intervention is occasionally done by non-medical personnel and the other two are almost never done by medically trained persons. a history of pretravel vaccinations and / or immunoglobulins would be useful for possible exclusion of certain suspected etiologies. for example, if the yellow fever vaccine and the hepatitis a and / or b vaccine series were administered in sufficient time before the travel, it is less likely that these viruses were the source of the medical complaint. the traveler's occupational or recreational exposure to dirt, water, or animals can be an important component of the history. an animal bite or scratch should be easy to remember, but the bite of many arthropods may not even be noticed until after a cutaneous reaction has appeared and the fly, mite, or flea that is responsible has moved on to the next victim. exposure to some animals may be more indirect. for example, the spelunker (cave explorer) may inhale aerosolized bat guano and develop rabies without ever touching a bat. a history of swimming, boating, or surfing can be a clue to an aquatic / marine etiology. such fresh-or brackish-water activities may increase the risk of infection with schistosomiasis or with free-living ameba, whereas marine activities may be associated with jellyfish stings, contact with the venomous spines of certain fish, or irritant dermatitis from fire coral. a preexisting skin abrasion or laceration, or a puncture wound from a sea urchin or sting ray, may result in a secondary bacterial infection. thus, a complete medical and travel history and physical examination are imperative in helping to narrow the differential diagnoses in the returned traveler, the immigrant, or the adoptee with a tropical origin. the qualitative and quantitative nature of the skin lesions is very important and is discussed in detail later in this chapter. specific attention must be given to the age of the patient as well as to the person who is immunocompromised due to human immunodeficiency virus (hiv), internal malignancy, organ transplantation, or another iatrogenic source of immunocompromise. blood tests (e.g., liver / kidney function tests, complete blood counts [cbc] with differentials, urinalysis, skin scraping, biopsy, and / or culture) are often necessary to confirm the diagnosis. a recent example of the importance of knowing both the patient's national origin and their immune status was seen when an hiv-seropositive man from myanmar presented with the first case of penicillium marneffei, recently renamed talaromyces marneffei, reported from houston, tx ( fig. - ) . many viral diseases that were not considered "tropical" years ago are now much more frequently seen in immigrants from tropical countries, or in travelers who did not receive their recommended childhood vaccines. three common examples are measles, rubella, and hepatitis b. until the s, measles and rubella were very common sources of infection in • if the traveler is strongly suspected of having an std, did he / she visit a destination where chancroid, granuloma inguinale (gi), or lymphogranuloma venereum (lgv) (l serovars of chlamydia trachomatis) is prevalent? if so, the diagnostic tests and therapy might need to be expanded beyond those under consideration for stds acquired in temperate lands. when one std is confirmed, there is an increased possibility of acquisition of additional stds. not only is this the case because the source partner(s) may have had multiple stds, but also because having certain stds makes a person more susceptible to other stds. the best example of this phenomenon is the two-to fivefold greater risk of acquiring hiv if the person with a genital ulcer disease (gud) has sex with an hiv-positive individual. the reasons for this increased risk include the reduced epithelial barrier in all guds, as well as the infiltrate of cd + cells in certain guds such as genital herpes. these cd + cells are the targets for hiv infection. genital herpes is the most prevalent gud in industrialized countries. in fact, the centers for disease control and prevention (cdc) estimate that there are million herpes simplex virus type (hsv- )seropositive persons in the usa. in the tropics, chancroid has been the most frequently diagnosed gud, followed by syphilis and genital herpes, but the last two diseases are becoming more prevalent in certain tropical countries. depending on the travel destination, lgv and gi must also be considered. the dates and duration of travel are important components of the history because the primary clinical presentation of all these guds ranges between and days (genital herpes and chancroid) and weeks (syphilis and gi). currently, the world health organization (who) estimates that there are million hiv-seropositive persons in the world. many of these people have gud, which may be changed both qualitatively and quantitatively by hiv. therefore the traveler may have a "non-classical" presentation of gud. in addition, it should be remembered that the signs and symptoms of gud can also appear on the perianal area / buttock or in or around the mouth. other locations are possible, but less likely. in general, however, multiple painful, usually bilateral, vesicles that progress to ulcers on skin or start as ulcers on mucous membranes, then heal over after - weeks without therapy or within - weeks with antiviral therapy, are consistent with genital herpes. because most true primary cases of genital herpes recur, a history of multiple recurrences of the vesicles or ulcers is highly consistent with genital herpes. this diagnosis can be confirmed by viral culture or serology. in the absence of these tools, a useful test is the tzanck smear, which usually demonstrates multinucleated giant cells in herpetic lesions, but is of low sensitivity and specificity. genital herpes, however, can present many diagnostic dilemmas because the first recognized clinical occurrence is often not the result of a recent infection but rather represents a first-episode, non-primary outbreak. whereas a true primary outbreak of genital herpes is usually consistent with acquisition of the virus days to weeks previously, a first-episode, non-primary outbreak may be consistent with an infection at any time in the past. in this case, the patient's recent travel history may be of less importance than his / her sexual encounters of the more-distant past. although syphilis is much more common in many developing countries than in the usa, western europe, or australia, a lack of travel certainly does not exclude syphilis. this diagnosis should be suspected when the patient presents with a single, ever knowing anyone who has suffered from the childhood diseases common in the first half of the th century. therefore, they do not understand that the approved vaccines are a millionfold safer than the diseases they are designed to prevent. stds should be considered at the top of the differential diagnoses when a patient presents with genital lesions and / or urogenital discharge. [ ] [ ] [ ] although many of the same considerations would be true whether or not the patient was a recent traveler, certain factors should be given attention in travelers: • was the person traveling without his / her spouse / family and therefore outside his / her usual social structure? • did the person travel to countries where sex workers are readily available? although sex workers are available in most parts of the world, legally or illegally, the traveler might be less likely to acquire an std in mecca during a haj than in amsterdam, bangkok, or nairobi, where sex workers are very prevalent. • did the person attend parties where large amounts of alcohol and / or drugs were consumed (e.g., "spring break" in the usa)? fortunately, human papillomavirus (hpv) vaccines are now widely available, which can prevent up to nine of these sexually transmitted viruses. although the pustules of disseminated gonococcemia are distinctive, the consequences of untreated neisseria gonorrhoeae (gonococcus) are usually pelvic inflammatory disease, epididymitis, proctitis, pharyngitis, or conjunctivitis. pelvic inflammatory disease can also be caused by chlamydia trachomatis (non-l serovars), mycoplasma hominis, or various anaerobic bacteria. the non-l serovars of c. trachomatis can also cause epididymitis, proctitis, and conjunctivitis. pharyngitis can also be due to hsv- or entamoeba histolytica. the initial presentation of gc, c. trachomatis (non-l serovars), ureaplasma urealyticum, mycoplasma genitalium, trichomonas vaginalis, or even hsv- may be urethritis. vaginal discharge can be caused by any of these organisms as well as by candida albicans, gardnerella vaginalis, peptostreptococci, bacteroides spp. or mobiluncus spp. these organisms can usually be diagnosed by smear, wet-mount, dna detection, serology, or culture. antimicrobial therapy is usually initiated based on the physical examination and smear or wet-mount and modified, as needed, when other laboratory studies are completed. infection with hpv is one of the most common stds in the world, but the clinical implications of the infection vary widely. there are over hpv types that can cause genital lesions, but most infections do not result in any visible lesions. because the incubation period of hpvs can be months, or even years, if and when genital lesions do develop, it is often very difficult for the patient to determine the source partner. therefore, it is usually a challenge for the physician to relate hpv lesions to travel, especially recent travel. non-oncogenic genital hpv, such as types and , result in condyloma acuminatum, which can be treated with cytodestructive therapy, surgery, or with the non-tender, genital, perianal, or lip ulcer associated with nontender lymphadenopathy. whereas chancroid is uncommonly reported in industrialized countries, it is very common in the tropics. it is usually characterized by one or more painful genital ulcers and painful lymphadenopathy. in lgv the primary lesion is usually very transient and is often not seen. the clinical presentation is usually that of tender inguinal lymphadenopathy, sometimes with a suppurating bubo. the diagnosis of gi is very rarely made outside the tropics. the presentation is usually that of one or more non-tender genital ulcers with inguinal swelling. if any of these bacterial guds is suspected, the appropriate diagnostic tests must be initiated (i.e., serology for syphilis and lgv, culture for chancroid and lgv, or tissue examination for gi) and the appropriate antibiotic started. whereas a history of multiple recurrences of genital vesicles or ulcers would be consistent with genital herpes, a more difficult scenario is represented by the patient who reports a single outbreak of non-specific genital signs and symptoms that are resolved by the clinic visit. a western blot or type-specific serologic test for hsv- would determine whether the person was infected with this virus, but it would not be definitive proof that that hsv- was responsible for the resolved outbreak. for example, a hsv- serologically positive person may acquire syphilis, but the genital ulcer may resolve without therapy, or with inadequate treatment, before the clinic visit at home. thus, a careful history may reveal the need for serology for hsv- as well as for syphilis. because hiv can be acquired concomitantly with or subsequently to these guds, but not produce genital manifestations, hiv testing should be conducted as well. although many patients may be hesitant to admit sexual activity that puts them at risk for stds, others will worry about these activities following travel (or any time) and ask to be tested for "everything." if the sexual encounter with a new partner has been very recent, the serologic test may be false negative because serology for syphilis, hiv, or hsv- may require weeks to become positive in the majority of persons after initial infection. patients who ignore their primary genital lesions because of denial or difficulty finding medical care during their travels may believe that the problem is gone because the lesion has resolved. if syphilis is the cause of the gud, it may reappear weeks or months later as non-genital cutaneous manifestations in the form of secondary (or tertiary) syphilis ( fig. - ) . a careful history regarding the primary lesion may lead to the appropriate diagnostic tests and therapy. some stds may not produce any genital signs or symptoms and the disease may be diagnosed long after the travel (or the non-travel acquisition), making it more difficult to find the source of the infection. although over % of hiv-seropositive persons eventually develop indirect mucocutaneous manifestations of infection, the primary rash of seroconversion (if present) is not noticed by most patients. therefore, the diagnosis is usually made when the patient develops systemic signs and symptoms (e.g., fever, chills, diarrhea, weight loss, lymphadenopathy) and / or develops one or more of the opportunistic infections, neoplasms, or inflammatory skin problems frequently seen in hiv patients. similar to hiv, primary infection with hepatitis b rarely produces genital lesions. diagnosis is usually made long after infection due to systemic symptoms or non-specific skin changes such as jaundice. hepatitis b was the first std for which a prophylactic vaccine was available. therefore, a history of successful hepatitis b vaccination makes this diagnosis less likely. toxoplasmosis, trichinosis, tularemia, typhoid fever, and yellow fever. if the period between travel and fever / rash is up to a month, the list should be expanded to include hepatitis viruses (a, c, and e), hiv, rubella, schistosomiasis, and trypanosomiasis. if at least months separate travel from fever / rash, the following infections should be considered: bartonellosis, filariasis, gnathostomiasis, hepatitis viruses (b and c), histoplasmosis, hiv, leishmaniasis, lyme disease, melioidosis, penicilliosis, syphilis, trypanosomiasis, and tuberculosis. in each case, however, the nature of the fever, the type of rash, the destination of the travel, and any other symptoms must be considered. because many infections producing fever with rash can be rapidly fatal and / or easily spread, it is imperative to initiate immediately diagnostic tests and antimicrobial therapy for the presumed cause of the infection. such infections include anthrax, bartonellosis, candida (macronodules), diphtheria, disseminated gonorrhoeae (papules and pustules over joints), hepatitis viruses, leptospirosis, meningitis (asymmetrical, scattered, petechiae, and purpura), plague, pseudomonas (ecthyma gangrenosum), relapsing fevers, rickettsia (scattered petechiae and purpura), staphylococcus (osler's nodes, diffuse toxic erythema), streptococcus (janeway lesions, diffuse toxic erythema), strongyloides (migratory petechiae and purpura), syphilis, tuberculosis, typhoid fever (rose spots) ( fig. - ) , various gram-negative bacteria (i.e., peripheral gangrene), vibrio (especially v. vulnificus), and viral hemorrhagic fevers (petechiae, purpura, hemorrhage). eosinophilia may be due to diverse processes, such as allergic, neoplastic, and infectious diseases. although an allergic reaction could easily result from an exposure during travel, eosinophilia in the returned traveler may have nothing directly to do with the travel. on the other hand, it may be due to an infectious process or to a drug taken for prophylaxis or therapy during travel. if an infection is the cause of the eosinophilia, it is usually a parasitic disease, especially that due to a helminth. only a few viral, bacterial, or fungal diseases are associated with both rash and eosinophilia, for example, streptococcal fever (i.e., scarlet fever), tuberculosis, hiv, and coccidioidomycosis. protozoa only rarely provoke eosinophilia. immune response modifier imiquimod. oncogenic genital hpv, such as types and , can result in anogenital cancer, the most prevalent of which is cervical cancer. cervical cancer is the second most prevalent cancer killer of women in the world and over % of all cervical cancer is caused by hpv. most cervical cancer deaths are in tropical countries, making hpv one of the world's deadliest tropical diseases (although rarely listed with the other major tropical diseases). there are many reasons why more cervical cancer deaths occur in tropical countries. first, in industrialized countries most women receive regular pap smears, which result in early detection and subsequent therapy of cervical abnormalities, thus reducing progression to cervical cancer. if cancer is detected, surgery, radiation therapy, or chemotherapy is available. in addition hpv vaccines are now widely available in most industrialized countries. in most tropical countries, regular pap smears are not the standard of care. therefore, cervical cancer is often detected too late for successful intervention, even if this is available. second, there appears to be a genetic susceptibility that allows oncogenic hpv to progress to malignancy. this genetic susceptibility appears to be more prevalent in certain tropical countries. the rarity of male circumcision in many tropical countries appears to be a risk factor for the development of cervical cancer in these males' partners. third, most of the world's estimated million hiv-seropositive individuals live in tropical countries where no antiretroviral therapy is available. not only is cervical cancer an acquired immunodeficiency syndrome (aids)-defining illness in hiv-seropositive women, but the same hpv can also cause anal cancer, which is a major problem in homosexual men with hiv. molluscum contagiosum (mc) is a poxvirus that can be sexually transmitted, resulting in wart-like lesions on the genitalia. in contrast to hpv, however, mc does not progress to malignancy. like condyloma acuminatum, however, mc can be treated with cytodestructive therapy, surgery, or imiquimod. ectoparasites such as scabies, sarcoptes scabiei, and pubic lice, phthirus pubis, can be sexually transmitted. in contrast to many stds, however, these ectoparasites can be easily treated with topical medications such as lindane or permethrin. like all stds, if the sexual partner is not treated concomitantly then reinfection is common. the most common cause of fever after tropical travel is malaria, which usually does not have specific cutaneous manifestations. dengue fever is the second most common cause of fever in the traveler and does have somewhat specific cutaneous manifestations, making dengue fever the leading cause of fever with rash in the traveler returning from a tropical destination ( fig. - ) . other common causes of fever and rash include hepatitis viruses, rickettsia, and some enteric fevers. it should always be kept in mind, however, that fever in the returned traveler may not be due to exposure during travel. for example, the fatigue of travel (i.e., jet lag) may make one more susceptible to influenza or other common infections in temperate lands. when both fever and rash are seen, the time between travel and onset of signs and symptoms becomes increasingly important. if travel preceded fever and rash by less than - weeks, considerations should include anthrax, dengue fever, diphtheria, ehrlichiosis, hemorrhagic fever viruses, leptospirosis, lyme disease, measles, meningococcal infections, plague, rickettsia, be considered. therefore, consideration should be given to trichinellosis, strongyloidiasis, schistosomiasis, onchocerciasis, loiasis, hookworms, gnathostomiasis, dracunculiasis, and cutaneous larva migrans. pinworms, as well as protozoan infections such as amebiasis, giardiasis, and trypanosomiasis, are less likely to produce eosinophilia. pruritus and urticaria are possible with spirochetes such as borrelia (e.g., relapsing fevers), spirillum (e.g., rat-bite fever) and treponema (i.e., syphilis and pinta). yersinia (e.g., plague) is another bacteria that produces pruritus and urticaria, which can be present before buboes form. the hepatitis viruses (e.g., a, b, and c) can produce pruritus and urticaria, as can a number of ectoparasites and biting arthropods (e.g., ticks, scabies, bedbugs, lice, fleas, mites, and flies). [ ] [ ] [ ] [ ] [ ] jaundice although hepatitis viruses can produce pruritus and urticaria, jaundice is a more specific indication that the problem has a hepatitic etiology. not only can all the hepatitis viruses (a-e) produce jaundice, other tropical viruses also do so commonly, e.g., yellow fever and rift valley fever. less frequently, dengue and epstein-barr viruses can cause jaundice, as can bacteria such as leptospira (i.e., leptospirosis), coxiella (i.e., q fever) and treponema (i.e., syphilis). protozoa, such as malaria, and drug reactions can also be responsible. although vesicles and bullae can appear as a result of contact dermatitis or drug eruption, including photodermatitis and photo-exacerbated drug eruptions as well as toxic epidermal necrolysis, many cases represent the early stages of a viral or bacterial infection. the most common viral etiology in the traveler or non-traveler includes the herpesviruses, especially herpes simplex virus and , as well as varicella-zoster virus, both primary varicella and herpes zoster. measles and many enteroviruses (e.g., hand, foot, and mouth disease) can present with vesicles, as can certain alphaviruses. a number of poxviruses, such as vaccinia, variola, orf, tanapox, and monkeypox, can produce vesicles. less commonly, vesicles comprise an early stage of certain bacterial diseases such as those caused by vibrio vulnificus, bacillus anthracis, brucella spp., mycobacteria tuberculosis, mycoplasma spp., rickettsia akaru, and staphylococcus (bullous impetigo). other organisms such as fungi that cause tinea pedis, protozoa (e.g., leishmania brasiliensis), and helminths (e.g., necator americanus) can occasionally cause vesicles. a wide variety of infectious and non-infectious etiologies are related to both macules and papules. almost any of the vesicular diseases listed above may initiate first as a macule, then as a papule, before becoming a vesicle. a number of drugs, arthropod bites (e.g., mosquito or flea) and infestations (e.g., scabies and other mites) commonly cause macules and / or papules. a variety of terrestrial, freshwater, and marine contactants can elicit these cutaneous reactions, as can a spectrum of drugs. viral etiologies include hiv, as in the hiv seroconversion syndrome, epstein-barr virus (infectious mononucleosis), human herpesvirus (roseola), parvovirus b- (fifth disease), measles, the principal helminth that causes eosinophilia is strongyloides. when strongyloides is disseminated, such as in the hyperinfection syndrome, skin lesions such as urticaria, papules, vesicles, petechiae, and migratory serpiginous lesions become common, especially if the patient is given systemic corticosteroids (because strongyloides was not considered). pruritic, erythematous papules can be seen as a result of schistosomal cercariae, as in swimmer's itch. eosinophils may be seen in the skin biopsy as well as in the blood. pruritic lesions of the skin and subcutaneous tissues are commonly associated with eosinophilia in onchocerciasis. lymphangitis, orchitis, and epididymitis are also commonly observed. in loiasis, fever and eosinophilia are typically seen. migratory lesions, especially angioedema, are usually erythematous and pruritic. likewise, gnathostomiasis produces recurrent edema after ingestion of raw fish. the skin lesions are usually erythematous, pruritic, and / or painful. drug hypersensitivity is a relatively common cause of eosinophilia and may be associated with non-specific skin changes, such as urticaria and / or phototoxic reactions. although most drugs that cause eosinophilia may not be taken for purposes related to traveling, increased sun exposure during travel may make the problem clinically apparent. because antibiotics may be taken for prophylaxis or therapy more frequently during traveling, they should be given careful consideration when eosinophilia is detected. such antibiotics include penicillins, cephalosporins, quinolones, isoniazid, rifampin, and trimethoprim-sulfamethoxazole. non-specific cutaneous manifestations of tropical diseases may include pruritus and urticaria. frequently, more specific signs may accompany pruritus and urticaria, which are useful in narrowing the differential diagnoses. if eosinophilia is found with the pruritus and urticaria, helminthic infections should an eschar can be seen in both temperate and tropical lands due to pseudomonas aeruginosa (i.e., ecthyma gangrenosum), but the most common infectious causes of eschars are rickettsia. eschars due to anthrax can be seen in persons who work with animal skins, but anthrax has received much attention recently owing to its potential use in bioterrorism. the best-recognized etiology of a non-infectious eschar is a bite from a brown recluse spider. petechiae and purpura can result from adverse reactions to a number of drugs. the most important infectious cause of petechiae and / or ecchymoses with fever is meningococcemia, which has a high rate of morbidity and mortality, is widespread throughout the tropical world, and is found sporadically in industrialized countries. other bacterial causes include borrelia, burkholderia, enterococcus, haemophilus, leptospira, pseudomonas, rickettsia, streptobacillis, treponema, vibrio, and yersinia. a number of hemorrhagic fever viruses can cause petechial or purpuric lesions, but the most prevalent viral causes are enteroviruses, cytomegalovirus, dengue, and yellow fever. protozoal diseases (e.g., malaria and toxoplasmosis) and helminths (e.g., trichinellosis) can also induce this clinical presentation. changes in pigmentation can be seen after a variety of medications, many of which are taken for prophylaxis or therapy related to travel. these agents include a spectrum of drugs such as antibiotics, antidiarrheals, anthelmintics, and antimalarials, many of which can also elicit photosensitization. a number of infectious agents can also alter pigmentation. leishmaniasis, pinta, and tinea versicolor may be associated with hypopigmentation or hyperpigmentation. leprosy, onchocerciasis, syphilis, and yaws are more often associated with hypopigmentation. erythrasma, hiv, chikungunya and loiasis are more frequently causes of hyperpigmentation. with the exception of the movements of scabies and the larvae of myiasis, mucocutaneous migratory lesions are usually due to infections with helminths. the best-recognized example is cutaneous larval migrans, but migratory lesions can also be due to dracunculiasis, fascioliasis, gnathostomiasis ( fig. - ) , hookworms, loiasis, paragonamiasis, sparganosis, or strongyloidiasis. since the first publication of tropical dermatology in , many tropical diseases previously unknown in temperate countries have been reported to have been transmitted outside the tropics (e.g., ebola, chikungunya, and zika viruses), or have markedly increased their endemic areas (e.g., chagas disease). in addition, certain non-infectious diseases such as podoconiosis have become more widely recognized. rubella, and various hemorrhagic fever viruses. many bacteria can be responsible, such as rickettsia, bacillus anthracis, spirochetes (spirillum, leptospira, borrelia, treponema), coxiella burnetii, yersinia pestis, salmonella typhi, bartonella bacilliformis, and brucella. histoplasmosis and coccidioidomycosis are fungal diseases commonly associated with macules and / or papules. certain protozoa such as toxoplasma gondii and leishmania can also induce these types of lesions. among helminth diseases, hookworm disease, strongyloidiasis, and onchocerciasis can be associated with macules and / or papules. although otherwise similar, papules are usually less than . - . cm in diameter, whereas nodules are larger than . - . cm. except for certain poxviruses that cause orf and milker's nodules, as well as warts and malignancies induced by hpv, viruses rarely form nodules. on the other hand, all subcutaneous and systemic mycoses can induce nodules. bacterial causes of nodules include bartonella (verruga peruana and cat-scratch disease), buckholderia mallei (glanders), calymmatobacterium granulomatis (gi), chlamydia trachomatis (lgv), klebsiella rhinoscleromatis (rhinoscleroma), leptospira autumnalis (leptospirosis), mycobacteria spp. (atypical mycobacteria, cutaneous tuberculosis, leprosy, etc.), nocardia brasiliensis (and other bacterial causes of mycetoma), and treponema pallidum (bejel, yaws). protozoan causes of nodules include amebiasis, leishmaniasis, and trypanosomiasis. almost all helminthic infections that have mucocutaneous manifestations can induce nodules (e.g., coenurosis, cysticercosis, dirofilariasis, dracunculiasis, echinococcosis, filariasis, gnathostomiasis, loiasis, onchocerciasis, paragonimiasis, schistosomiasis, sparganosis, and visceral larval migrans). if the helminthic nodule contains sufficient fluid, it will produce a cyst. cysts can be seen in helminthic infections such as coenurosis, echinococcosis, filariasis, gnathostomiasis, loiasis, and onchocerciasis. there are also arthropod causes of nodules such as myiasis, scabies, tick granulomas, and tungiasis. although ulcers can form as a result of breakdown of previously normal skin, they frequently develop from nodules after inflammation destroys the epidermis and papillary layer of the dermis. herpes simplex virus is a very common cause of ulcers in both tropical and temperate regions of the world. other causes of gud are bacterial (e.g., chancroid, gi, lgv, and primary syphilis). other bacterial diseases that commonly cause ulcers include anthrax, bacterial mycetomas, diphtheria, glanders, melioidosis, mycobacterial diseases (e.g., buruli ulcer, leprosy, tuberculosis), plague, rickettsia, tropical ulcers, tularemia, and yaws. a number of fungi can form nodules that break down into ulcers, or they can induce ulcers from systemic spread (e.g., blastomycosis, chromomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, lobomycosis, mycetomas, paracoccidioidomycosis, penicilliosis, and sporotrichosis). the most common helminthic cause of cutaneous ulcers is dracunculiasis, when the worm erupts from the skin. two protozoan diseases cause ulcers -amebiasis and leishmaniasis. arthropod causes of ulcers include myiasis and tungiasis. many bites (e.g., those of brown recluse spiders and various snakes), stings (e.g., insect, jellyfish, and scorpion), or venomous spines of various fish can also induce ulcers. control, globalization, and emergence of another vector, a. albopictus, in addition to the main vector, a. aegypti. travelassociated cases have been reported in the usa since , with a significant increase in following the first case in the western hemisphere in december and the start of domestic transmission in . zika virus is a member of the family flaviviridae related to west nile, dengue, and yellow fever viruses ( fig. - ). from through , serologic evidence of human infection was reported from african countries and in parts of asia. zika virus is transmitted to humans primarily through aedes mosquitoes. in , an outbreak of cases was reported in yap island, micronesia. since then, outbreaks have been reported in french polynesia, and most recently in brazil and other countries in tropical south america. zika virus transmission has not yet been documented in the usa, but cases have been reported in returning travelers. these imported cases, and the fact that the vector is the same as for dengue and chikungunya, may result in local spread of the virus in some areas of the usa. , [ ] [ ] [ ] american trypanosomiais, also known as chagas disease (cd), is caused by trypanosoma cruzi. the disease is transmitted to humans by triatomine insects (blood-sucking bugs of the reduviidae family). these insects deposit their feces, which are laden with t. cruzi, at the time of biting. however, cd can also be transmitted via mother-to-child transmission, food-borne transmission, and blood transfusion. for this reason, screening of the us blood supply for cd began in early . cd infects ebola is a hemorrhagic fever disease caused by ebola virus, a member of the filoviridae family. ebola was first discovered in near the ebola river in congo. since then, outbreaks have appeared sporadically in central africa. the largest epidemic in history occurred in , affecting multiple countries in west africa. five different ebola viruses are known, with zaire ebola virus being the most virulent and causative agent in the most recent epidemic. with a fatality rate of up to %, the world health organization (who) declared ebola a 'public health emergency of international concern' . in the united states (usa) two imported cases, including one death, and two locally acquired cases in health-care workers were reported. ebola can be transmitted via direct contact with body fluids. dermatologists should be cautious of the high risk of contamination through skin biopsies and dermatologic examination. chikungunya is an acute febrile illness caused by the chikungunya virus, of the togaviridae family, and transmitted by aedes mosquitoes (fig. - ) . the spread of chikungunya worldwide has been attributed to a multitude of factors including mutation of the virus, absence of herd immunity, lack of efficient vector furthermore, cd is a leading cause of heart disease among people living in extreme poverty in the western hemisphere, especially in latin america ( fig. - ) . podoconiosis, a non-communicable, non-infectious, tropical disease, is a common cause of lower-leg lymphedema in tropical volcanic highland areas above meters with high annual rainfall. clinical characteristics include below-theknee bilateral lower-limb elephantiasis. it is found in barefoot subsistence farmers in fields with red soils formed from alkaline volcanic rock (see fig. - ) . although it was only recently designated a "neglected tropical disease" by who, it is responsible for a significant public health burden in ethiopia and nine other countries in the horn of africa, northern india, and latin america. the finding of an association of certain hla class ii loci with susceptibility to podoconiosis suggests that it is a genetically predisposed, t-cell-mediated inflammatory disease. in conclusion, the differential diagnoses of mucocutaneous lesions in the returned traveler, immigrant, or adoptee should be based on the morphology of the lesions, but the patient's symptoms, general medical, and exposure history must all be considered. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the physical examination and laboratory results must be integrated with the patient's vaccination and medication record. the travel destination(s), travel duration, living, work / recreation conditions, food and drink ingestion, and activities while traveling must all be taken into consideration. it must not be forgotten, however, that many mucocutaneous problems in the returned traveler or the immigrant / adoptee are not related to the travel or the country of origin, but can be the same disorders seen daily in patients who have never left their local communities. approximately million people and kills about yearly. in some preliminary estimates, mexico ranks number three, and the usa number seven, in terms of the number of infected individuals with cd in the world. [ ] [ ] in the usa, approximately cases are believed to be present, although one alternative estimate reports more than cases in texas alone, [ ] [ ] with up to one million or more cases nationwide. health problems after travel to developing countries disseminated talaromyces marneffei and mycobacterium intracellulare coinfection in an hiv-infected patient for healthcare professionals. clinical features morbid mortal wkly rep (mmwr) an overview of sexually transmitted diseases. part i (bacterial stds) an overview of sexually transmitted diseases. part ii (viral stds) an overview of sexually transmitted diseases. part iii. sexually transmitted diseases in hiv infected patients eosinophilia in travelers dermatoses associated with travel to tropical countries: a prospective study of the diagnosis and management of patients presenting to a tropical disease unit cutaneous manifestations of intestinal helminthic infections parasitic infections of the skin skin problems in returning travelers dermatologic manifestations of parasitic disease ebola (ebola virus disease) cutaneous manifestations of the ebola virus world health organization. ebola virus disease mucocutaneous manifestations of chikungunya fever centers for disease control and prevention. chikungunya virus in the united states zika virus outside africa zika virus outbreak on yap island, federated states of micronesia clinical evaluation & disease zika virus zika virus outbreak centers for disease control and prevention. zika virus. areas with zika zika: the new arbovirus threat for latin america detection of zika virus in urine recent developments in dermatological syndromes in returning travelers an unfolding tragedy of chagas disease in north america trypanosoma cruzi and chagas disease in the united states historical perspectives on the epidemiology of human chagas disease in texas and recommendations for enhanced understanding of clinical chagas disease in the southern united states podoconiosis: endemic nonfilarial elephantiasis hla class ii locus and susceptibility to podoconiosis the travel and tropical medicine manual kaposi's sarcoma and other manifestations of human herpesvirus human papillomaviruses mucocutaneous manifestations of viral diseases. london: informa healthcare tropical dermatology: viral tropical diseases lyme borreliosis dermatologic infectious diseases in international travelers mucocutaneous manifestations of helminth infections: trematodes and cestodes mucocutaneous manifestations of helminth infections: nematodes tropical dermatology: venomous arthropods and human skin: part ii. diplopoda, chilopoda, and arachnida tropical dermatology: venomous arthropods and human skin. part i. insecta tropical dermatology: marine and aquatic dermatology tropical dermatology: tropical diseases caused by protozoa tropical dermatology: bacterial tropical diseases tropical dermatology: fungal tropical diseases key: cord- - yiuuye authors: mims, cedric a.; dimmock, nigel j.; nash, anthony; stephen, john title: mechanisms of cell and tissue damage date: - - journal: mims' pathogenesis of infectious disease doi: . /b - - - - . - sha: doc_id: cord_uid: yiuuye nan the impact on the host of microbial damage depends very much on the tissue involved. damage to muscle in the shoulder or stomach wall, for instance, may not be serious, but in the heart the very existence of the host depends on a strong muscle contraction continuing to occur every second or so, and here the effect of minor functional changes may be catastrophic. the central nervous system is particularly vulnerable to slight damage. the passage of nerve impulses requires normal function in the neuronal cell membrane, and viruses especially have important effects on cell membranes. also a degree of cellular or tissue oedema that is tolerable in most tissues may have serious consequences if it occurs in the brain, enclosed in that more or less rigid box, the skull. therefore, encephalitis and meningitis tend to cause more severe illness than might be expected from the histological changes themselves. oedema is a serious matter also in the lung. oedema fluid or inflammatory cell exudates appear first in the space between the alveolar capillary and the alveolar wall, decreasing the efficiency of gaseous exchanges. respiratory function is more drastically impaired when fluid or cells accumulate in the alveolar air space.* the effect of tissue damage is much less in the case of organs such as the liver, pancreas or kidney, which have considerable functional reserves. more than two-thirds of the liver must be removed before there are signs of liver dysfunction. cell damage has profound effects if it is the endothelial cells of small blood vessels that are involved. the resulting circulatory changes may lead to anoxia or necrosis in the tissues supplied by these vessels. here too, the site of vascular lesions may be critical, effects on organs such as the brain or heart having a greater impact on the host, as discussed above. rickettsiae characteristically grow in vascular endothelium, and this is an important mechanism of disease production. by a combination of direct and immunopathological factors there is endothelial swelling, thrombosis, infarcts, haemorrhage and tissue anoxia. this is especially notable in the skin, and forms the basis for the striking rashes in typhus and the spotted fevers. these skin rashes, although important for the physician are less important for the patient than similar lesions in the central nervous system or heart. it is damage to cerebral vessels that accounts for the cerebral disturbances in typhus; involvement of pulmonary vessels causes pneumonitis, and involvement of myocardial vessels causes myocardial oedema. in q fever, rickettsiae sometimes localise in the endocardium, and this causes serious complications. sometimes an infectious agent damages an organ, and loss of function in this organ leads to a series of secondary disease features. the signs of liver dysfunction are an accepted result of infections of the liver, just as paralysis or coma is an accepted result of infection of the central nervous system. diabetes may turn out to be caused by infection of the islets of langerhans in the pancreas. coxsackie and other virus infections of the islets of langerhans can certainly cause diabetes in experimental animals, and coxsackieviruses have been associated with juvenile diabetes in man. there are many diseases of unknown aetiology for which an infectious origin has been suggested. sometimes it is fairly well established that an infectious agent can at least be one of the causes of the disease, but in most instances it is no more than a hypothesis, with little or no good evidence. for conditions as common and as serious as multiple sclerosis, cancer and rheumatoid arthritis it would be of immense importance if a microorganism were incriminated, since this would give the opportunity to prevent the disease by vaccination. accordingly, there is a temptation to accept or publicise new reports even though the evidence is weak or the observations poorly controlled. as if to warn us about this and remind us of possibilities from environmental toxins, parkinson's disease, a chronic neurological condition in which there is a loss of neurons in a sharply defined region of the brain (substantia nigra), can be caused by exposure to the chemical mptp. one example which raises the possibility that subtle cns disturbances may be caused by viruses is experimental infection with borna disease virus. this virus was used to infect tree shrews (tupaia glis) which are primitive primates. there is little overt disease, but afterwards the male is no longer able to enact the ritual courtship behaviour which (as students well know), is an essential preliminary to mating in all primates, and the frustrated male usually ends up bitten by the female. thus it can be said that infection with borna disease virus renders the male psychologically sterile. presumably the virus in some way alters the functioning of neurons concerned in this particular pathway. all other behavioural and physiological aspects appear normal. borna disease virus is not known to occur in man, but speculation about an analogous human situation is fuelled by the finding of borna disease virus-specific antibodies in patients with psychiatric/ behavioural disorders. in an entirely different clinical context, infection of a particular strain of rats with borna disease virus causes immense obesity, the underlying physiological basis of which is not understood. since the aetiology of such diseases raises interesting problems in pathogenesis, the present state of affairs is summarised in table . , which includes some of the human diseases whose infectious origin is probable, possible, conceivable, or inconceivable. causal connections between infection and disease states are particularly difficult to establish when the disease appears a long time after infection. it was not too difficult to prove and accept that the encephalitis that occasionally occurs during or immediately after measles was due to measles virus. but it was hard to accept that a very rare type of encephalitis (subacute sclerosing panencephalitis or sspe), occurring up to years after apparently complete recovery from measles, was also due to measles virus and this was only established after careful studies and the eventual difficult isolation of a mutant form of measles virus from brain cells. 'slow' infections, in which the first signs of disease appear a long time after infection are now an accepted part of our outlook. the disease kuru occurred in new guinea and was transmitted from person to person by cannibalism. the incubation period of the disease in man appears to be - years, and was caused by a nonisolatable infectious agent that grew in the brain. this was established when the same disease appeared in monkeys several years after the injection of material from the brain of kuru patients. a similar agent called scrapie (see ch. ) infects sheep, mice and other animals and also has an incubation period representing a large portion of the life span of the host. in both kuru and sspe the agent was eventually shown to be present in the brains of patients. so far this has only been demonstrated indirectly as, despite strenuous efforts, the causative agent has yet to be isolated. if in a slow infection the microorganism that initiated the pathological process is no longer present by the time the disease becomes manifest, then the problem of establishing a causal relationship will be much greater. this may possibly turn out to be true for diseases like multiple sclerosis and rheumatoid arthritis. liver cancer in humans and leukaemia in mice, cats, humans and cattle can be caused by slow type virus infections. cancer or leukaemia appears as a late and occasional sequel to infection. the virus, its antigens or fragments of its nucleic acid are detectable in malignant cells. one important factor that often controls the speed of an infectious process and the type of host response, is the rate of multiplication of a microorganism.* different infectious agents show doubling times * every infection is a race between the spread and multiplication of the microbe and the generation of an antimicrobial response by the host. a day or two's delay in this response may let the microbe reach the critical levels of growth that give tissue damage and disease. varying from min to weeks, and some of these are listed in table . . often the rate of multiplication in the infected host, in the presence of antimicrobial and other limiting factors and when many bacteria are obliged to multipy inside phagocytic cells, is much less than the optimal rate in artificial culture. clearly a microorganism with a doubling time of a day or two will tend to cause a more slowly evolving infection and disease than one that doubles in an hour or less. it is uncommon for an infectious agent to cause exactly the same disease in all those infected. its nature and severity will depend on infecting dose and route, and on the host's age, sex, nutritional status, genetic background, and so on. many infections are asymptomatic in more than % of individuals, clinically characterised disease occurring in only an occasional unfortunate host, as 'the tip of the iceberg'.* asymptomatically infected individuals are important because they are not identified, move normally in the community, and play an important part in transmission. this chapter deals with demonstrable cell and tissue damage or dysfunction in infectious diseases. but one of the earliest indications of illness is malaise, 'not feeling very well'. this is distinct from fever or a specific complaint such as a sore throat and although it is difficult to define and impossible to measure, we all know the feeling. it can precede the onset of more specific signs and symptoms, or accompany them. sometimes it is the only indication that an infection is taking place. almost nothing is known of the basis for this feeling. toxins', of course, have been invoked and the earliest response to pyrogens (see pp. - ) before body temperature has actually risen, may play a part. interferons may have something to do with it because pure preparations of human a or ß interferons cause malaise and often headaches and muscle aches after injection into normal individuals. if interferon is eventually recognised as an important antimicrobial force we may have to regard these side effects as unfortunate but acceptable. soluble mediators of immune and inflammatory responses, such as interleukin- (see glossary) or other cytokines may also play a part. in some infectious diseases weakness and debility are prominent during convalescence. this can be especially notable following influenza and hepatitis, but its basis is as mysterious as in the case of malaise. the infections that matter are those causing pathological changes and disease. before giving an account of the mechanisms by which these changes are produced, it is important to remember that many infectious agents cause little or no damage in the host. indeed, it is of some advantage to the microorganism to cause minimal host damage, as discussed in ch. . virus infections as often as not fall into this category. thus, although infection with rabies or measles viruses nearly always causes disease, there are many enterovirus, reovirus and myxovirus infections that are regularly asymptomatic. even viruses that are named for their association with disease (poliomyelitis, influenza, japanese b encephalitis) often give an antibody response as the only sign of infection in the host. tissue damage is too slight to cause detectable illness. there is also a tendency for persistent viruses to cause no more than minor or delayed cellular damage during their persistence in the body, even if the same virus has a more cytopathic effect during an acute infection, e.g. adenoviruses, herpes simplex (see ch. ). a few viruses are remarkable because they cause no pathological changes at all in the cell, even during a productive infection in which infectious virus particles are produced. for instance, mouse cells infected with lcm or leukaemia virus show no pathological changes. a mouse congenially infected with lcm virus shows a high degree of immune tolerance, and all tissues in the body are infected. throughout the life of the animal, virus and viral antigens are produced in the cerebellum, liver, retina etc. without discernible effect on cell function. but sometimes there are important functional changes in infected cells which lead to a pathological result. for example, the virus infects growth hormoneproducing cells in the anterior pituitary. although the cells appear perfectly healthy, the output of growth hormone is reduced, and as a result of this, suckling mice fail to gain weight normally and are runted. when bacteria invade tissues, they almost inevitably cause some damage, and this is also true for fungi and protozoa. the extent of direct damage, however, is sometimes slight. this is true for treponema pallidum, perhaps because the lipopolysaccharide-protein components that might have induced inflammatory responses, are not exposed on the surface of the bacteria. it produces no toxins, does not cause fever, and attaches to cells in vitro without harmful effects. leprosy and tubercle bacilli eventually damage and kill the macrophages in which they replicate, but pathological changes are to a large extent caused by indirect mechanisms (see below). in patients with untreated lepromatous leprosy, the bacteria in the skin invade blood vessels, and large numbers of bacteria, many of them free, may be found in the blood. in spite of the continued presence of up to bacteria m l - of blood there are no signs or symptoms of septicaemia or toxaemia. mycobacterium leprae can be regarded as a very successful parasite that induces very little host response in these patients, even when the bloodstream is invaded. the resident bacteria inhabiting the skin and intestines of man and animals do not invade tissues and are normally harmless; indeed, as discussed in ch. , they may benefit the host. bacteria such as meningococci and pneumococci, whose names imply pathogenicity, spend most of their time as harmless inhabitants of the normal human nasopharynx: only occasionally do they have the opportunity to invade tissues and give rise to meningitis or pneumonia. cell and tissue damage are sometimes due to the direct local action of the microorganism. however, it is not at all clear how viruses cause the death of cells. many virus infections result in a shutdown of rna synthesis (transcription), protein synthesis (translation) and dna synthesis in the host cell, but usually these are too slow to account for the death of the cell. after all, cells like neurons never synthesise dna, and the half life of most proteins and even rnas is at least several hours. a possible alternative mechanism is the alteration of the differential permeability of the plasma membrane. this is important as the cell has a high internal k + concentration and low n a + concentration, while the reverse is true of body fluids. viruses do alter membrane permeability, but the unresolved question is whether or not this is responsible for the death of the cell or is merely an after-effect. it now appears that at least some virus infections (hiv, adenovirus, influenza virus, sindbis virus) cause the cells to commit suicide by a mechanism called 'programmed cell death' or 'apoptosis'. this is the natural process by which the body controls cell numbers and rids itself of superfluous or redundant cells during development. a familiar example is a tadpole 'losing' its tail. cells do not disintegrate but round up, and are then removed by phagocytes. a possible example of apoptosis is seen in the common cold when caused by the rhino virus group of picorna viruses. rhinoviruses infect nasal epithelial cells, and at an early stage the cells round up, fall off the mucosal surface and are carried away, often with their cilia still beating, in a stream of fluid induced by the infection.* this leaves areas of raw mucosa, with the exposed underlying tissues inflamed, oedematous, and susceptible to infection by the normally harmless resident bacteria. the gross pathology of cells infected with viruses appears generally nonspecific, very like that induced in cells by the toxins of diphtheria bacilli and streptococci, or by physical and chemical agents. the most common and potentially reversible change, the oedema seen as 'cloudy swelling' by routine histology, is associated with membrane permeability changes. changes in the endoplasmic reticulum, mitochondria and polyribosomes are seen by electron microscopy at this stage. later the nuclear chromatin moves to the edge of the nucleus ('margination' of chromatin) and becomes condensed (pycnosis), but the cell has already died by this time. there are two more characteristic types of morphological change produced by certain viruses, and these were recognised by histologists more than years ago. the first are inclusion bodies, parts of the cell with altered staining behaviour which develop during infection. they often represent either cell organelles or virus factories in which viral proteins and/or nucleic acids are being synthesised and assembled. herpes group viruses form intranuclear inclusions, rabies and poxviruses intracytoplasmic inclusions, and measles virus both intranuclear and intracytoplasmic inclusions. the second characteristic morphological change caused by viruses is the formation of multinucleate giant cells. this occurs, for instance when hiv 'fusion' proteins (gpl -gp ) present in nascent virus particles budding from an infected cell attach to cd receptors in the plasma membranes of neighbouring cells; membranes then fuse and multinucleate cells are formed. it also happens in measles and certain herpes virus infections. before leaving the subject of direct damage by viruses, one supreme example will be given. here the direct damage is of such a magnitude that the susceptible host dies a mere six hours after infection. if rift valley fever virus, an arthropod-borne virus infecting cattle, sheep and man in africa, is injected in very large doses intravenously into mice, the injected virus passes straight through the kupffer cells and endothelial cells lining liver sinusoids (see ch. ) and infects nearly all hepatic cells. hepatic cells show nuclear inclusions within an hour, and necrosis by four hours. as the single cycle of growth in hepatic cells is completed, massive liver necrosis takes place, and mice die only six hours after initial infection. the host defences in the form of local lymph nodes, local tissue phagocytes etc. are completely overcome by the intravenous route of injection, and by the inability of kupffer cells to prevent infection of hepatic cells. direct damage by the replicating virus destroys hepatic cells long before immune or interferon responses have an opportunity to control the infection. this is the summit of virulence. the experimental situation is artificial but it illustrates direct and lethal damage to host tissues after all host defence mechanisms have been overwhelmed. most viruses, rickettsiae and chlamydia damage the cells in which they replicate, and it is possible that some of this damage is due to the action of toxic microbial products. this action, however, is confined to the infected cell, and toxic microbial products are not liberated to damage other cells. mycoplasma (see table a . ) can grow in special cell-free media, but in the infected individual they generally multiply while attached to the surface of host cells. as studied in culture and on the respiratory epithelium they 'burrow' down between cells, inhibit the beat of cilia and cause cell necrosis and detachment. the mechanism is not clear. if a complete lawn of mycoplasma covers the surface of the host cell, some effect on the health of the cell is to be expected, but it is possible that toxic materials are produced or are present on the surface of the mycoplasma. dental caries provides an interesting example of direct pathological action. colonisation of the tooth surface by streptococcus mutans leads to plaque formation, and the bacteria held in the plaque utilise dietary sugar and produce acid (see p. ). locally produced acid decalcifies the tooth to give caries. caries, arguably the commonest infectious disease of western man, might logically be controlled by removing plaque, withholding dietary sugar, or vaccinating against streptococcus mutans. however, fluoride in the water supply or in toothpaste has been the method of choice, and has been very successful. it acts by making teeth more resistant to acid. bacteria generally damage the cells in which they replicate, and these are mostly phagocytic cells (see ch. ). listeria, brucella and mycobacteria are specialists at intracellular growth, and the infected phagocyte is slowly destroyed as increasing numbers of bacteria are produced in it. bacteria such as staphylococci and streptococci grow primarily in extracellular fluids, but they are ingested by phagocytic cells, and virulent strains of bacteria in particular have the ability to destroy the phagocyte in which they find themselves, even growing in the phagocytes, as described in ch. . many bacteria cause extensive tissue damage by the liberation of toxins into extracellular fluids. various toxins have been identified and characterised. most act locally, but a few cause pathological changes after spreading systemically through the body. this is a huge and growing part of our subject and we need to define the term toxin, a task which is more difficult than one might think. an attempt was made by bonventre who in defined toxins as a 'special class' of poisons which differ from, for example, cyanide or mercury by virtue of their microbial origin, protein structure, high molecular weight, and antigenicity. this view is too embracing, because it includes proteins of doubtful significance in disease, and also too restrictive, because it excludes nonprotein toxic complexes such as endotoxin. another suggestion is that toxin must include all naturally occurring substances (of plant, animal, bacterial or whatever origin) which when introduced into a foreign host are adverse to the well-being or life of the victim. this, too, is unsatisfactory because some substances-potent toxins within the scope of this definition-are being used in some contexts as therapeutic agents! perhaps it is pointless to strive for an all-embracing definition, although the obvious differences between bacterial and fungal toxins warrant the continued use of the appropriate prefix. for example, bacterial toxins are usually of high molecular weight and hence antigenic, whereas fungal toxins tend to be low molecular weight and not antigenic. the problem of definition is compounded because there are substances (aggressins) which help to establish an infective focus as well as those whose action is uniquely or largely responsible for the disease syndrome. also there are substances known to be produced by bacteria in vitro, whose properties on a priori grounds make them potential determinants of disease, but which have not been shown to play a role in vivo. we will concentrate on those toxins known to be, or likely to be, involved in some aspect of disease causation. it is not difficult to demonstrate the production of'toxins' by bacteria in culture, as judged by some bioassays. primary consideration will be given to those substances which are produced under ecologically significant conditions (i.e. in the natural host or relevant animal model) and cause (also in biologically relevant systems) damage to cells or tissues thereby contributing to disease. exotoxins are produced and then either secreted by, or released upon lysis from, both gram-positive and gram-negative bacteria. they are proteins, some of which are enzymes. when liberated locally they can cause local cell and tissue damage. those that damage phagocytic cells and are therefore particularly useful to the microorganism have been described in ch. . those that promote the spread of bacteria in tissues have been referred to in ch. . a description of some of the more interesting exotoxins follows. proteases and hyaluronidases, which help the spread of bacteria through tissues have already been mentioned in ch. . here we consider toxins which act on extracellular substances and are responsible for many of the main features of the diseases caused by the infecting organism. pseudomonas aerwgmosa-elastase, and one of at least six proteases of legionella pneumophila, both induce fibrinopurulent exudation in the rat lung (a model for p. aeragmosa-induced pneumonia in human cystic fibrosis) and the guinea-pig lung (a model for legionnaires' disease) respectively. these characteristics almost certainly arise from the release of oligopeptides from extracellular matrix components of the host which are chemotactic for leucocytes and fibroblasts. the l. pneumophila protease is the same major secretory protein (the m r zinc metalloprotease) already considered in ch. in relation to survival within macrophages. staphylococcal exfoliatin is important in staphylococcal 'scalded skin syndrome' (ssss), a disease of newborn babies. the disease is characterised by a region of erythema which usually begins around the mouth and, in - days, extends over the whole body. during this period, small yellowish exudative lesions often appear. the most striking feature of the disease, however, is that the epidermis, although apparently healthy, can be displaced and wrinkled like the skin of a ripe peach by the slightest pressure. soon large areas of the epidermis become lifted by a layer of serous fluid and peel at the slightest touch. large areas of the body rapidly become denuded in this way and the symptoms resemble those of massive scalding. while the toxin causes cleavage of desmosomes (specialised cell membrane thickenings through which cells are attached to each other) in the stratum granulosum, it may also act intracellularly. despite numerous attempts to characterise the biological activity of exfoliatin, the genetically predicted serine protease and/or lipase activity has never been demonstrated. some enterotoxigenic e. coli elaborate families of low molecular weight heat stable (st) peptides as well as heat labile (lt; cholera-like) toxin. sts bind to a receptor which then activates a tightly coupled membranebound guanylate cyclase in gut cells, resulting in the transmission of a signal to the inside of the cell, thereby elevating cgmp, or some other second message. as described later in the section on diarrhoea this gives rise to efflux of ions, and hence water, from enterocytes. we know that these toxins are able to traverse membranes since their targets are intracellular; we still know very little about the details of how they achieve this. these proteins have common features: one component (usually described as the b fragment or subunit) binds to and interacts with a cell receptor and promotes the uptake of an active component (a fragment or subunit) in which resides the biological activity which confers toxicity. there are several ways in which one can classify these toxins. ( ) biologically-some kill cells (cytotoxic toxins) whereas others (sometimes called cytotonic toxins) 'deregulate' cells. ( ) biochemically-some belong to a well defined biochemical group recognised by their ability to cleave nad + into nicotinamide and adp-ribose moieties. they are designated adp-ribosyl (adpr) transferases since they transfer adpr to different target proteins; this group straddles groups and . ( ) genetically-the third possibility reflects the genetic origin of these toxins: (i) production from a single gene of a single peptide which undergoes post-translational modification into a and b fragments which are covalently linked; (ii) production from separate genes of a and b subunits which noncovalently associate into stable complexes; (iii) production from separate genes of different proteins which do not associate into stable complexes but which must act in concert to express toxicity. these are known as binary toxins; again, group straddles group . examples of each of these groups will be given illustrating how they work, their importance in disease, and surprisingly (!) how some of the most toxic substances known to man are being used or may be used for therapeutic purposes. diphtheria toxin (dt). corynebacterium diphtheriae organisms multiply on the epithelial surfaces of the body (nose, throat, skin) but do not penetrate deeply into underlying tissues. the infection on the body surface causes necrosis of mucosal cells with an inflammatory exudate and the formation of a thick 'membrane' (hence the name c. diphtheriae: gr., diphthera = membrane) and if the infection spreads into the larynx there may be respiratory obstruction. the toxin probably assists colonisation of the throat or skin by killing epithelial cells and polymorphs. active immunisation with diphtheria toxoid has made diphtheria a clinical rarity in developed countries. dt is disseminated from the infection site and has important actions, especially on the heart and nervous system. dt is encoded by lysogenic phage ß but its expression is controlled by the host bacterium under conditions of iron stress. it is synthesised and processed as shown in fig. . . there is intense interest in seeking to understand the mechanisms of uptake of the active moieties of diphtheria and other toxins whose target is intracellular. this is driven by the desire to understand fundamental mechanisms in cell biology and to develop selective cytotoxic therapies' in clinical medicine. the mechanism of internalisation of the active a under the acidic conditions of the endosome, some dt-a is released by reduction, dt-b undergoes conformational change and interacts with the endosomal membrane resulting in the opening of cation channels. the latter are not absolutely necessary, but their formation is associated with times greater uptake of dt-a. in contrast, most other toxins which enter the cytosol appear to do so by more complicated routes which initially involving endocytosis. shiga toxin (and ricin, a toxic plant lectin believed to have been located in the tip of an umbrella and used to poison a bulgarian spy!) are not translocated across the endosomal membrane but are transported to the trans-golgi network and all the way back to the endoplasmic reticulum. somewhere in the exocytotic pathway conditions exist for the translocation of shiga toxin (sht) into the cytoplasm. sht acts by modifying s ribosomal rrna. for cholera toxin, it used to be thought that a l ( fig. . ) moved down through the central hole of the ring-shaped complex of ct-b which was anchored to the membrane by five ganglioside gm binding sites. structural work on this family of proteins shows that this is not physically possible. the finding that the c-terminal sequence lysine-aspartateglutamate-leucine (the kdel* motif) in the a subunit of cholera toxin and related sequences in e. coli heat labile toxin and pseudomonas exotoxin a raises the possibility that transport to the endoplasmic reticulum may also be necessary for these a subunits to reach the cytoplasm. a new and exciting development is beginning. for decades, attempts have been made to make immunotoxins by coupling native toxins to antibodies specific to some surface antigen on tumour cells, with little practical success, as yet. however, scientists have now genetically engineered dt by substituting that portion of the dt structural gene encoding the native toxin receptor-binding domain with modified cdna encoding one of a variety of cytokines and growth factors including il- , il- , il- , egf and α-msh. the resultant fusion toxin bears a new 'cellular address', but retains all of the other biological properties of the native dt molecule as well as a three-dimensional structure nearly identical with native dt. the il- receptor-binding domain fusion toxin has undergone phase i/ii clinical trials for the treatment of il- receptor positive haematological malignancies (and other il- related conditions) and has been shown to be safe and well tolerated. such constructs are potent against a murine model of il- receptor positive lymphoma, and the hope is that a durable remission of disease will be achieved in humans. (an era of new 'magic bullets'?) p. aeruginosa exotoxin a. p. aeruginosa, already alluded to above, is common in soil and water and can occasionally be isolated from the faeces of normal, healthy individuals. it is virtually harmless for healthy adults, but its ability to multiply in almost any moist environment and its resistance to many antibiotics have made the bacterium a major cause of * the kdel motif is normally found in proteins which having been processed in the golgi are trapped by the endoplasmic reticulum which recognises the kdel motif. this prevents the protein being lost to the cell via exocytotic trafficking and results in its translocation into the cytoplasm. the schema shows a round of peptide elongation and illustrates the key role played by two enzymes, ef and ef . dta and pea each adp-ribosylates diphthamide (a modified histidine) in ef -gtp which can no longer translocate the newly elongated peptide from the a site to the p site. sht a fragment is a specific n-glycosidase which cleaves an adenine residue from near the '-prime end of the s ribosomal rna. this depurination results in failure of ef -dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis. poliovirus achieves selective inhibition of host protein synthesis at an earlier stage than is depicted here. host mrna is first modified (capped) then bound to the small ribosomal subunit; poliovirus mrna is not capped. the function of a cap-binding protein, which recognises and binds host mrna to the ribosome, is inhibited by a poliovirus virion protein thereby allowing differential translation of virus messenger rna. ef-Ια: nucleotide-binding protein. hospital-acquired infection, particularly among patients with impaired host defence mechanisms such as those with chronic illness, genetic immunodeficiencies, those under treatment with immunosuppressive drugs, or patients suffering from extensive burns. p. aeruginosa causes localised infection in the urinary tract, respiratory tract, burns and wound infections. in severely debilitated patients these localised infections may develop into general septicaemia, with mortality in such cases approaching %. the mechanisms of pathogenicity of p. aeruginosa are complicated and unclear because (unlike c. diphtheriae) the organism elaborates several potentially toxic extracellular products, including a phospholipase, several proteases, lipase, haemolysin, enterotoxin, lipopolysaccharide endotoxin, elastase, exoenzyme s (pes), and exotoxin a (pea); evidence definitely implicates the last three. elastase has already been mentioned above (p. ). pes is without question a virulence determinant, but at present its biological mode of action is not wholly understood. pea is in many ways like dt. it inhibits protein synthesis by cleavage of nad + and subsequent adp-ribosylation of ef at the same diphthamide residue, is synthesised predominantly during the decline phase of cell growth, and its production appears to be dependent upon the concentration of iron in the medium. its m r is , and it can be activated in cell-free systems by proteolysis, or by reduction with thiols in the presence of urea and other chaotropic agents. all the enzymic activity of the toxin resides in a proteolytically derived fragment (m r approximately ). however, dissimilarities exist between the two toxins. firstly, the active a fragment resides in the cooh-terminal portion of the molecule, whereas the diphtheria a fragment is at the nh -terminal end. secondly, the b fragment of pea recognises a different receptor to that of dt, and there are no sequence homologies or serological crossreactivity between the two toxins. shiga toxin. this toxin is a subunit protein with an ab type structure ( a subunit; b, an oligomer of subunits). certain biotypes of e. coli make vero toxins (vts) or shiga-like toxins (slts) which are identical (slt ) or near identical (slt ) with shiga toxin. the role of shiga toxin in bacillary dysentery is discussed below. slt-producing strains of e. coli are responsible for haemorrhagic colitis and haemolytic urea syndrome. very recent crystallographic studies have shown a remarkable similarity in organisational structure of the b subunits of shiga toxin and those of the cholera toxin family discussed below, despite the near complete absence of sequence homology. the b subunits attach to a ganglioside structure gb or gb (cholera toxin attaches to gm ) on susceptible cell surfaces. sht-a fragment is a specific n-glycosidase which cleaves an adenine residue from near the '-prime end of s ribosomal rna. this depurination results in failure of efl-dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis ( fig. . ). this is based on studies on cultured cells, and how such cytotoxic activity gives rise to the enterotoxic activity-watery diarrhoea seen in cases of dysentery preceding the bloody mode of action of actin-adp-ribosylating toxins. c. botulinum c toxin component binds to the cell membrane followed by c i. the latter is internalised and upsets the equilibrium between polymerisation and depolymerisation of actin. adp-ribosylation of actin inhibits its polymerisation and turns g-actin into a capping protein which binds to the fast growing (concave) ends of actin filaments. capping of the concave ends increases the critical concentration for actin polymerisation. since the slow-growing (pointed) ends of actin filaments are free, depolymerisation of actin occurs at these ends. released actin is substrate for the toxin and will be withdrawn from the treadmilling pool of actin by adpribosylation, i.e. trapped. both reactions will finally induce the breakdown of the microfilament network. (taken with permission of authors (k. aktorieseia/.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from botulinum c toxin. clostridium botulinum c toxin is not a neurotoxin but belongs to a family of cytotoxins {clostridium perfringens iota toxin, clostridium spiroforme toxin, clostridium difficile adp ribosyltransferase) whose common features are that they are binary toxins and that they target cytoskeletal actin. of these c. botulinum c is the best studied at present. component c i is the equivalent of the a subunit and c ii, the b subunit. they are produced in parallel although different strains produce different ratios. more c is produced during sporulation than in the vegetative phase of growth. c ii is activated by proteolysis to produce a m r protein which binds to cell surfaces and thereby exposes a binding site for c i (m r ca. ) either on the cell surface or on attached c ii. once inside, c i adp-ribosylates monomeric actin and not polymerised f actin. the substrate specificity is high. the toxin will modify a specific arginine residue present in certain isoforms of actin. neither subunit is toxic on its own, but together the ld dose for a mouse is ca. - ng. (other examples of such 'binary' activity include anthrax toxin and staphylococcal leucocidin). the two components may be injected into different sites or by different routes but toxic effects always occur at the site of injection of c ii. this mode of action, summarised in fig. . , is believed to underlie the biological manifestations of toxicity which, in rats, include hypotension, haemorrhaging in and fluid accumulation around the lungs as a result of vascular permeability changes. the toxin is currently being used as a tool in cell biology to study cytoskeletal systems and dissect mechanisms of cell signal transduction. cholera and related toxins. there is a growing number of toxins in this category. easily the most studied is cholera toxin (ct), a major research area for three decades, which was originally stimulated by the desire to make a more effective vaccine against cholera. ironically, effective immunity is best mediated by antibacterial antibodies which prevent colonisation of the gut with v. cholerae rather than antitoxin-neutralising antibodies. cholera toxin is an ab subunit toxin ( fig. . ). enterotoxigenic e. coli (etec) strains produce two heat labile toxins: lti and ltii. ltii exists as two minor variants, ltiia and ltiib, the latter requiring trypsin treatment for full activation in the y- adrenal cell assay. the b subunits of ct, ltiia and ltiib recognise and are bound to cell surface gangliosides gm , gdlb and gdi a respectively and mediate the translocation of a l subunits into the cytoplasm; lti binds to either gm or a glycoprotein. the same reaction occurs as was described above for dt: nad + is cleaved into nicotinamide and adp-ribose (adpr) moieties, with two important differences: ( ) adpr transferase is greatly enhanced by a group of proteins (both membrane bound and soluble) of ca. m r designated adpribosylation factors (arfs; of as yet unknown physiological significance); ( ) adpr is transferred to the a s subunit of the g s regulator of adenylate cyclase as illustrated in fig. . . this results in the elevation of camp levels in the cell with the consequences described below in the section on diarrhoea. bordetella pertussis toxin (pertussigen) and adenylate cyclasehaemolysin (ac-hly). whooping cough (pertussis) is a severe respiratory tract infection characterised by prolonged paroxysmal coughing, attacks of which continue long after infection has cleared. the disease is capable of striking all ages but is particularly prevalent and severe in young children, where hospitalisation is required in about % of cases. the causative agent of pertussis, bordetella pertussis, is transmitted aerially from the respiratory tract of an infected individual to that of a susceptible host. the organism attaches (probably via its filamentous haemagglutinin, pili, and a m r outer membrane protein) to, and colonises the mucosal surface between the cilia, and multiplies there during the incubation period of the disease which is commonly around seven days. the infection then manifests as a slight fever and catarrh which is often indistinguishable from a common cold. however, one to two weeks later bouts of uncontrollable coughing begin. it is this paroxysmal coughing, along with the notorious 'whoop' as the child attempts to draw breath, which characterises the disease. the paroxysmal coughing stage often lasts for several weeks and no treatment fully effective in controlling the symptoms. the only proven means of controlling whooping cough is vaccination but, in the uk at least, sporadic reports of vaccine-induced brain damage in infants has diminished public acceptance of the vaccine. it should be noted that permanent encephalopathy (brain damage) is a recognised though rare consequence of whooping cough infection. much current work is being devoted to producing immunogenic, completely nontoxic preparations of pertussis toxin by genetic manipulation of t h e gene encoding t h e s i s u b u n i t ( fig. . ). in clinical trials in italy, such engineered vaccines h a v e been shown to be both safe a n d effective a s j u d g e d by antibody titres to pertussis toxin. several toxic substances h a v e been isolated from bordetella pertussis including a h e a t labile toxin, tracheal cytotoxin, endotoxin, adenylate cyclase-haemolysin (ac-hly), a n d pertussis toxin. t h e latter h a s m a n y biological activities-histamine sensitisation, leucocytosis promotion, islet (a) the production of cyclic amp by adenyl cyclase. cyclic amp is an important second messenger invoked in the intracellular amplification of many cellular responses to external signals including hormones. the nature of the physiological response reflects the biochemical differentiation of the cell responding to the stimulus. for example, in gut cells the response would be altered ion transport and hence fluid secretion; in muscle cells it would be glycogen breakdown in response to the call for more energy. the production of camp is controlled both positively and negatively at two different levels. the central cycle represents a normal membranebound hormone receptor and the heterotrimeric (aß ) g protein regulator complex which is activated upon binding of hormone to the receptor. there are two receptors, each with regulatory g proteins: one system responds to stimulatory and the other responds to inhibitory stimuli; only one system is shown. in gut cells these receptors would be on the basolateral (nonluminal) side of enterocytes enabling responses to stimuli from the circulation. (b) the second level of control involves endogenous gtpase properties of both stimulatory (a s ) and the inhibitory (a a ) subunits of the g protein regulator which may be outlined as follows. stimulation: agonist stimulation of its receptor results in the dissociation of the a s ß g protein from the receptor and of the subunits from each other, and the binding of gtp to a s . a s -gtp will productively interact with the catalytic unit of adenyl cyclase (not shown) and stimulate the production of camp from atp. endogenous gtpase activity in a s results in a delayed conversion of a s -gtp to inactive a s -gdp which can no longer stimulate the cyclase but allows the reassociation of the trimeric g protein and loss of gdp. inhibition: antagonist stimulation of its receptor results in an analogous situation for inhibiting cyclase activity via c^: inhibitory cti-gtp inactivates the cyclase; cti-gdp does not inactivate the cyclase. (c) the level of camp may be affected by physiological stimuli (which stimulate removal and modification of the g-protein complex) or by perturbation of the normal regulatory cycle as illustrated, by cholera toxin (ct) or pertussis toxin (ptx). (d) cholera toxin acts first by interacting with its receptor resulting in the internalisation of the active a l subunit. a l adp-ribosylates a s -gtp which promotes continued dissociation of the heterotrimer; also the endogenous gtpase is no longer functional hence the stimulation of the cyclase continues. lt toxins act in a similar manner. (e) pertussigen acts first by interacting with its receptor resulting in the internalisation of the active si subunit. si adp-ribosylates the ai-gdpß -heterotrimer which can no longer associate with the receptor (or lose gdp) to undergo another cycle of gtp activation; activated cyclase can no longer be turned off. (adapted, with kind permission of author (gierschik, p.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from figure in 'adp-ribosylation of signal-transducing guanine nucleotide-binding proteins by pertussis toxin', current topics in microbiology andlmmunology ( ) , , edited by klaus aktories. activation and adjuvanticity-and is believed to be the most important but by no means the only virulence determinant of b. pertussis: it is called pertussigen and is the basis of the vaccine against whooping cough. pertussigen is a complex subunit toxin whose biochemical mode of action is identical to that of ct except that the target is the aj subunit of the gj regulator of adenylate cyclase (see fig. . ). it is not yet clear how such biochemical activity relates to the clinical syndrome. ac-hly has two functional domains. one confers adenylate cyclase activity and another haemolytic activity. its haemolytic activity arises from its pore-forming properties in cell membranes. pore formation is probably responsible for the translocation of the adenylate cyclase portion of the complex. ac-hly is also known as cyclolysin and belongs to the group of toxins whose common feature is the presence of an array of a nine amino acid repeats; they have been designated rtx (repeats in toxin) toxins. the prototype is the haemolysin of e. coli which is important in extraintestinal infections caused by this organism. other toxins of this group include the haemolysin from proteus vulgaris, and the leukotoxin from pasteurella haemolytica. anthrax toxin. anthrax is a disease of animals, particularly sheep and cattle, and to a lesser extent man, caused by infection with bacillus anthracis. infection takes place following the ingestion of spores, the inhalation of spores, or by the entry of spores through abraded skin. the spores germinate and then the bacteria form a toxin that increases vascular permeability and gives rise to local oedema and haemorrhage. infection of the skin in man leads to the formation of a lesion (malignant pustule; a black eschar, hence b. anthracis; gr. anthrakos = coal) consisting of a necrotic centre surrounded by vesicles, blood-stained fluid and a zone of oedema and induration. in severe infections there is septicaemia with toxic signs, loss of fluid into tissues, with widespread oedema and eventually death. anthrax toxin, the discovery of which by smith and keppie in the early s was a major milestone in our subject, consists of three components, none of which are toxic by themselves: factor i (oedema factor or ef), factor ii (protective antigen or pa) and factor iii (lethal factor or lf). the toxin is largely responsible for local lesions, kills phagocytes, enters the circulation and accounts for the toxicity, oedema and death caused by b. anthracis. virulent strains also have a capsular polypeptide composed of d-glutamic acid, which inhibits opsonisation and phagocytosis (see ch. ). anthrax in man occurs mainly in those whose work brings them into contact with infected animals. it is not a common disease in the uk, and the usual source of infection is imported bones, hides, skins, bristles, wool and hair, or imported fertilisers made from the blood and bones of infected animals. most of what has just been described was discovered more than three decades ago but more recently new insights have been obtained as to the biochemical basis of the mode of action of anthrax toxin. ef was shown to be an inactive form of adenylate cyclase which is activated by calmodulin; pa and lf were not active. however combinations of pa and ef (but not pa and lf) caused an elevation of camp. thus anthrax toxin (at least the pa/ef combination) is a binary toxin. the response observed is more rapid and greater than that induced by cholera toxin. in contrast to cholera toxin, the anthrax complex is the cyclase, acts without a lag phase and its effects are instantly reversible upon washing toxin-treated cells. this suggests that the enzyme is either rapidly degraded after internalisation, or that it is not completely internalised but wsfhiw ii camp atp fig. . mode of action of anthrax toxin. factor ii (pa) interacts with the cell membrane. after proteolytic cleavage sites are exposed which bind factor i (ef) and facilitate internalisation of factor i (ef). this internalisation takes place via an endocytic step, with factor i being released into the cytosol. factor i must be rapidly inactivated since washing toxin-treated cells results in a rapid loss of adenylate cyclase activity. factor i interacts with calmodulin (cal) to become an active adenylate cyclase enzyme. interaction of factor i with factor ii and subsequent internalisation is blocked by prior binding of factor iii (lf) to factor ii. associated with the cell membrane in a manner allowing it to be readily removed. these facts together with the observations that lf would block the activity of ef are summarised in fig. . . this explains how lf blocks the effect of ef. they either compete for the same sites on pa, in which case the explanation is self-evident, or they bind to separate sites on pa which are situated such that occupancy of one occludes access to the other. one can extend these observations made in vitro to the in vivo situation. the model provides a basis for understanding the protective function of pa. antibodies to pa could either block the initial attachment to the cell membrane of target organs in vivo, or prevent the binding of ef to pa, or both. the fact that camp is known to be a potent secretagogue could explain how the oedematous reaction occurs when pa and ef are injected into test animals, i.e. ef is, after activation, an oedema factor. moreover, in biological tests, addition of lf (factor iii) depresses oedema production as would be predicted by this model. the observation that pa was probably fixed first may now be placed on a firmer basis since binding of pa is a prerequisite for the expression of ef (and by implication) lf activities. the case for the view that, in experimental anthrax in guinea-pigs, animals died (as do humans) of secondary shock, is also placed on a firmer basis with perhaps one important reservation. smith and coworkers claimed that factor iii (lf) decreased oedema but increased lethality; the former but not the latter observation is explicable by the model which is therefore too simplistic to explain the effects of the holotoxin. it is likely that lf exerts its activity together with pa on other cells in vivo. it has in fact been shown that lf in combination with pa is cytolytic for macrophages and macrophage-like cell lines-the probable basis of the earlier observation that anthrax toxin kills phagocytes and perhaps justification for talking about a plurality of anthrax binary toxins. clostridium tetani spores germinate in an infected wound and produce their toxin. spores are ubiquitous in faeces and soil, require the reduced oxygen tension for germination provided locally in the wound by foreign bodies (splinters, fragments of earth or clothing) or by tissue necrosis as seen in most wounds, the uterus after septic abortion, or the umbilical stump of the newborn.* the site of infection may be a contaminated splinter just as well as an automobile or battle injury. all strains of clostridium tetani produce the same toxin. it also reaches the central nervous system by travelling up other peripheral nerves following bloodborne dissemination of the toxin through the body. the motor nerves in the brain stem are short and therefore the cranial nerves are among the first to be affected, causing spasms of eye muscles and jaw (lockjaw). there is also an increase in tonus of muscles round the site of infection, followed by tonic spasms. in generalised tetanus there is interference with respiratory movements, and without skilled treatment the mortality rate is about %. botulism* caused by clostridium botulinum, a widespread saprophyte present in soil and vegetable materials. c. botulinum contaminates food, particularly inadequately preserved meat or vegetables, and produces a powerful neurotoxin. the toxin is destroyed at °c after min-of great importance to the canning industry-and there are at least seven antigenically distinct serotypes (a-g) produced by different strains of bacteria but which have a pharmacologically similar mode of action. it is absorbed from the intestine and acts on the peripheral nervous system, interfering with the release of acetylcholine at cholinergic synapses of neuromuscular junctions. somewhere between and hours after ingestion there are clinical signs suggesting an acute neurological disorder, with vertigo, cranial nerve palsies and finally death a few days later with respiratory failure. * botulus (latin) = sausage. in a large sausage was eaten by people in wildbad in germany; all became ill, and six died. the disease was subsequently referred to as botulism. t in recent years a less typical form of botulism has been described in small infants. the spores, present in honey applied to rubber teats, appear to colonise the gut, so that the toxin is produced in vivo after ingestion. tetanus and botulinum toxins are two of the most toxic substances known to man: g of botulinum toxin will kill mice. a great deal is now known about the genetics of both these toxins. tetanus toxin and botulinum toxin type g are encoded by plasmids, botulinum toxins types c and d are encoded by phages that infect clostridia, and botulinum type a is encoded in the bacterial genome; the genes have been sequenced. both toxins are synthesised as single peptides and are released from bacteria upon lysis. they are proteolytically activated by endogenous proteases to yield dichain derivatives consisting of -s -s -linked l (m r ) and h (m r ) chains. the recently adopted nomenclature reflects our current understanding of their peptide structures ( fig. . ). very recently, huge strides have been made in our knowledge of the mode of action of these toxins ( fig. . ). there is very little overall sequence homology between bonts and tetx; this could account for their immunological characteristics and the animal types normally affected. however, there is one structural feature common to all these neurotoxins located in the middle of the light chain and that is a zincbinding motif. in fact these neurotoxins are zinc endopeptidases. their targets are protein constituents of the synaptic fusion machinery responsible for the exocytotic release of neurotransmitters. the intracellular target cleaved by bonts b, d, and f and tetx is synaptobrevin (vamp, vesicle-associated membrane protein) present in small synaptic vesicles. bonts a and e cleave snap- (synaptosomal associated protein of m r ) a highly conserved protein known to be involved in exocytosis. bont c acts on syntaxin, a synaptic membrane protein also involved in exocytosis. however, some puzzles remain to be resolved. tetanus and botulinum toxins enter neuronal tissue preferentially at motoneuronal endplates, but the nature of the 'physiologically relevant ectoacceptors' is still not known. it remains unclear why botulinum toxin acts directly at the site of uptake and not, as observed with tetanus toxin, in the central nervous system, although a considerable amount of botulinum toxin (like tetanus toxin) is retrogradely transported. botulinum toxin blocks the release of acetyl choline at neuromuscular junctions to cause flaccid paralysis. likewise, one might ask why tetanus toxin fails to act at the motoneuronal junction at concentrations which would completely block release of neurotransmitter from gabaergic synapses. to reach inhibitory interneurons-its principal site of action-tetanus toxin must leave α-motoneurons after the primary uptake step, traverse the synaptic cleft to interneurons, leave those again in order to become finally internalised again from presynaptic membranes-a route identical to that of several neurotropic viruses. it acts by blocking the release of inhibitory transmitters (glycine or gab a) resulting in a failure to relax the affected muscle-pathophysiological 'tetanus'. only in rare cases does it act peripherally like botulinum toxin. the most toxic substances known to man are now being used as therapeutic agents to treat focal dystonias such as neck twists and eye squints, or eyelid closure and, more recently, some childhood palsies. the preparations are made from bont a and consist of toxin-haemagglutinin complex-the form in which the toxin is usually produced by the organism. this complex is less toxic than purified neurotoxin when administered parenterally but relatively more toxic when given orally; the haemagglutinin apparently protects the neurotoxin from proteolytic degradation in the gut. the effects of bont in relieving muscle spasms are not permanent but last for several months. treatment has to be repeated. to date the successes significantly outweigh the failures and no long-term adverse effects have been reported. antibody to the toxin has not so far been detected in the sera of the majority of patients. some toxins destroy membranes by virtue of their proteolytic activities, and some by their ability to degrade lipid components, while others are pore-forming or detergent-like in their mode of action. in addition to their action on protein components of lung connective tissue referred to above, pseudomonas aeragmosa-elastase and the zinc metalloprotease of legionella pneumophila are believed to destroy cell membranes by their proteolytic activity. this is the probable reason for the haemorrhage associated with lung infections caused by these pathogens, i.e. effects on type i alveolar epithelial and endothelial cells. clostridium perfringens α-toxin. a large number of bacterial enzymes are phospholipases some of which, but by no means all, are important toxins. the best example is the α-toxin of clostridium perfringens, the organism most commonly associated with gas gangrene. it is strictly anaerobic and occurs as a normal inhabitant in the large intestines of man and animals; its spores are ubiquitous in soil, dust and air. c. perfringens does not multiply in healthy tissues, but grows rapidly when it reaches devitalised and therefore anaerobic tissues. this could be after contamination of a natural wound with soil or dust, particularly on battlefields or in automobile accidents, or after contamination of a surgical operation site with clostridia from the patient's own bowels or skin. after abortions, particularly in the old days before antibiotics, intestinal clostridia often gained access to necrotic or devitalised tissues in the uterus and set up life-threatening infections. invasion of the blood was common and soon resulted in death, the clostridia localising and growing in internal organs such as the liver after death. c. perfringens has various enzymes that enable it to break down connective tissue materials, including collagen and hyaluronidase, thereby facilitating spread of the infection along tissue planes. most of the enzymes are toxic (a) reflex arc (top). mechanism for inhibiting the antagonists to a muscle contracting in response to stretch. muscles are reciprocally innervated with sensory and motor neurons, although for clarity this is shown only for the protagonist muscle. on stretch, the stretch receptors generate an impulse which is transmitted along the afferent sensory (s) neuron of the protagonist (p) muscle. this sp neuron enters the spinal cord by the dorsal root and synapses with the motor neuron supplying the protagonist muscle (mp) and with an interneuron (i) which in turn synapses with the motor neuron supplying the antagonist muscle (ma); the efferent motor neurons leave the spinal cord by the ventral root. at the sp/mp synapse an excitatory transmitter is released which induces an impulse in mp which leads to contraction of protagonist muscle. however, excitation of i causes release of an inhibitory transmitter at the i/ma synapse which leads to relaxation of the antagonist muscle. note that the basic reflex arc has been shown for simplicity but tetx acts mainly on voluntary muscles. (b) a simplified version of the biochemical events occurring in synapses (lower left). excitatory and inhibitory synapses, neurotransmitter release and action. gly, glycine; r, receptors of neuro transmitters; x, hitherto uncharacterised (candidates include glutamate, dopamine, atp, substance p, and somatostatin). to host cells and tissues, but α-toxin is easily the most important one. it is dermonecrotic, haemolytic (a feature seen mainly in tissues close to the focus of infection but sometimes responsible for large-scale intravascular haemolysis in infected patients), causes turbidity in lipoprotein-rich solutions and is lethal. while it is still true that these activities are all due to one molecular species, they are not different expressions of the one enzymic activity. historically, c. perfringens α-toxin was the first bacterial toxin to be characterised as an enzyme: it is a phospholipase c (plc) which removes the head group, phosphoryl choline, from phosphatidyl choline and from sphingomyelin. it is of undoubted importance in gas gangrene. toxoid prepared by formalin-treated toxin will protect sheep against infection caused by c. perfringens. however, one might ask why all enzymes with such biochemical specificity are not equally toxic or important in determining virulence; there are several reasons which can be put forward. by the combined use of monoclonal antibodies and molecular genetic analyses we now know that there are at least two functional domains in this c. perfringens α-toxin. if one compares c. perfringens α-toxin with the phosphatidyl choline-preferring, nontoxic phospholipase of bacillus cereus, one finds that two-thirds of the n-terminal sequence shows homology with the entire sequence of b. cereus plc. this portion of c. perfringens α-toxin retains its plc activity but not its haemolytic and lethal activities. the c-terminal part is not haemolytic, not enzymatically active and not cytotoxic for mouse lymphocytes, but is necessary for conferring toxicity on the n-terminal part of the protein. in fact, the c-terminus is a potent immunogen that will solidly protect mice -and hopefully man-against experimental infection with c. perfringens. surprisingly, the c-terminal domain of the nontoxic c. bifermentans enzyme shows sequence similarity with that of its c. perfringens α-toxin counterpart. the nontoxicity of this enzyme is ascribed to its comparatively much lower turnover rate, i.e. it is a much less efficient enzyme. recently, attention has been drawn to the distinct possibility that while haemolysis may be initially induced by the plc activity of c. perfringens α-toxin, it is possible that actual disruption of the membrane is due to the activation by the toxin of other phospholipases (phosphatidylinosotol- , -biphosphate (pip ) specific phospholipase and phospholipase d) present in the erythrocyte membrane. some now believe (c) sites of neurotoxin action (lower right). the predominant site of action of tetx is the intermotor neuron synapse; the exocytotic machine is interfered with by the endopeptidase action of tetx on vamp. bont acts at the neuromuscular junction inhibiting the release of acetyl choline (ach) by its proteolytic action on vamp (types b, d and f), or snap (types a and e), or syntaxin (type c). (amplified from figs and , 'bacterial toxins', nd edition, by j. stephen and r. a. pietrowski ( ), pp. and , van nostrand reinhold (uk).) that the basis of toxicity is not simply a cytolytic one, but rather a consequence of its ability, in sublytic doses, to release inosotol triphosphate (ip ) and activate the arachidonic acid cascade. there are other pathogenic clostridia that cause gas gangrene and produce similar toxins. infected tissues show inflammation, oedema and necrosis, not necessarily with the formation of gas, and the illness can be mild or very severe according to the extent of bacterial spread and the nature and quantity of toxins that are formed and absorbed. since the bacteria grow and produce their toxins only in devitalised tissues, the most important form of treatment is to remove such tissues. clostridia are strictly anaerobic, and exposure of the patient to hyperbaric oxygen (pure oxygen at - atmospheres in a pressure chamber) has been found useful in addition to chemotherapy. staphylococcal ß-toxin. staphylococcal ß-haemolysin is known to be produced in vivo. in ch. studies with isogenic mutants were described which indicate that it is important in killing neutrophils. it probably has the narrowest substrate specificity among the phospholipases, and is a hotr-cold haemolysin: lysis of erythrocytes occurs only on cooling after incubation at °c. the phenomenon, though of doubtful significance in vivo, has attracted attention and generated speculation about its mechanism. perhaps the most likely explanation is that, when cooled below their phase-transition temperature, the remaining phospholipids undergo quasi-crystalline formation, thereby generating intramembranous stresses incompatible with structural integrity. these proteins (made by some species, not all of which are pathogens) have been called oxygen-labile haemolysins because they are reversibly inactivated on standing in air, and their most studied property is their ability to lyse red cells. sh-activation of crude preparations is necessary for the expression of haemolytic activity but they are also active towards a variety of cell types. thiol-activated cytolysins share the following properties: they are cross-neutralised by hyperimmune sera, lyse a wide range of species of erythrocyte, exhibit similar ph and temperature optima for cytolytic activity, are lethal and cardiotoxic, and lose activity on incubation with erythrocyte ghosts. they are inactivated irreversibly by small amounts of cholesterol. interaction with cholesterol is the key primary event in their interaction with susceptible membranes which leads to the impairment of the latter; cholesterol plays no further part in the subsequent damage process. a schematic outline of how they damage cell membranes is shown in fig. . . several facts have recently reopened considerable interest in this group. first, the recognition of what these substances do to host defence cells when presented in sublethal doses. for example, pneumolysin from s. pneumoniae inhibits the respiratory burst in neutrophils, inhibits antibody synthesis in b cells and activates the classical complement pathway in the absence of antibodies. since complement is an important defence mechanism against the pneumococcus in vivo this could lead to depletion of complement levels and abrogate protection. in fact, immunisation of mice against pneumolysin affords some protection against challenge infection. second, in addition to its role in promoting invasive bacteraemia, pneumolysin has also been implicated in causing sensorineural deafness associated with meningitis caused by the pneumococcus; this perception is based on very recent work with a guinea-pig other pore-forming toxins t h e r t x toxins h a v e a l r e a d y b e e n referred to above in connection w i t h b. pertussis a c -h l y . b u t t h e r e a r e o t h e r pore-forming toxins. staphylococcal δ-toxin. this toxin acts in a manner similar to that of the sh-activated cytolysins, with an important difference: there is no initial specific binding. δ-toxin initially forms small pores and then islands of membrane or large micelles; this gives rise to its perceived detergent-like properties. there is a family of closely related δ-toxins which inhibit the growth of gonococci. it is not often that one can ascribe a positive function to a toxin which is beneficial for the organism producing it. in this case -toxin(s) could have important ecological significance in the mixed culture situation that is characteristic of the real microbial world. of great interest is the synergy that δ-toxin displays. sublytic amounts of δ-toxin causes release of cell constituents without lysis. but, only . haemolytic units of staphylococcal ß-toxin will cause lysis of cells in the presence of . lytic units of δ-toxin. this synergistic interaction could be the way in which staphylococcal toxins, which rarely exert their lethal effects in the majority of infections, exercise important cytolytic effects. of less obvious significance is the fact that δ-toxin is a poor antigen; for a long time its antigenicity was controversial. if δ-toxin were to prove of crucial importance as a cytolytic potentiator, then this could also partly explain why natural acquired immunity to staphylococcal infection is either non-existent or sufficiently low as to be easily overcome. staphylococcal a-and -toxins. staphylococci produce a range of toxins several of which we have already met. the α-toxin is considered as the main cytolysin produced by s. aureus. like streptolysin- and staphylococcal δ-toxin, it is secreted as a water-soluble protein and undergoes self-induced oligomerisation on cell membranes to form pores. it has recently been suggested that transmembrane channels formed by the hexameric form allow the penetration of the monomeric form which interferes with certain steps in translation. in systemic staphylococcal infections death is most probably due to the potent α-toxin but in localised pyogenic infections-such as mastitis in cattle, goats, rabbits and mice-its role is most likely one of killing phagocytes or conferring survivability on intracellular bacteria. -toxin is a binary toxin with a haemolytic action on rabbit erythrocytes. there is evidence that α, β and haemolysins and leucocidin are produced in vivo during natural and experimental infections. this is based on the presence of antibodies to these haemolysins in subjects undergoing staphylococcal infection and the extraction of toxin from peritoneal abscesses in mice. however, the precise roles that these or any other staphylococcal toxins play in localised infections is not clear. atrophie rhinitis is a disease of pigs in which the principal clinical sign is atrophy of the nasal turbinate bones and shortening of the snout. scientists have isolated and purified a toxin believed to be important in the causation of this disease. this is a remarkable toxin on at least two counts. it shows an incredible predilection for the target tissue in that it causes turbinate atrophy in gnotobiotic pigs, even when injected intraperitoneally; it also causes necrosis of spleen and thymus. perhaps even more surprising is the fact that it is a potent mitogen, hardly the kind of activity that one would associate with atrophy! one can only assume that it upsets the dynamic balance between the laying down and resorption of mesenchymal bone tissue by osteoblasts and osteoclasts respectively. the remainder of this section deals with other important (or potentially important) toxins which have not already been referred to in earlier chapters or in the section above. several staphylococcal toxins have already been referred to above. staphylococci are ubiquitous and responsible for a large group of diseases which range from the relatively harmless skin pimple through abscesses, impetigo, food poisoning, osteomyelitis, mastitis, primary pneumonia, staphylococcal scalded skin syndrome (ssss) and toxic shock syndrome (tss) to fatal septicaemias. in the case of ssss a toxin is known and is described on p. . toxic shock syndrome toxin (tsst- ). tss is seen characteristically in menstruating women whose tampons harbour multiplying staphylococci. it is due to a toxin called toxic shock syndrome toxin (tsst- ; one of the so-called staphylococcal enterotoxins, see next paragraph). toxic shock syndrome is characterised by sudden onset of fever, vomiting, diarrhoea, an erythematous rash followed by peeling of the skin, hypotensive shock, impairment of renal and hepatic functions and occasionally death. the main symptoms of the disease have been reproduced in rabbits by implanting chamber-enclosed tss-strains in the rabbit uterus or peritoneum or by injection of tsst- into rabbits. complex changes are observed including: haemorrhage in kidney and liver; congestion and haematomas in the lungs; leakage of blood into the thymus; and fluid in the pericardial sac and in the gut lumen. these effects in rabbits are very similar to those seen in humans and would certainly explain the shock and diarrhoeal syndrome so characteristic of the disease. all of this indicates that the primary effect of tsst- is on the integrity of capillary walls. the lethal effect of tsst- is enhanced considerably by endotoxin (see below). such synergy is also known for streptococcal erythrogenic toxin and lps, but the mechanism(s) involved in this complex synergy is not clear. although relatively uncommon in the uk, staphylococcal food poisoning accounts for over % of all cases of food poisoning in the united states. the disease is characterised by vomiting and diarrhoea commencing - h after consumption of contaminated food, especially dairy produce. symptoms usually last no longer than h. like botulism, staphylococcal food poisoning is commonly caused by the ingestion of food containing preformed toxins, known collectively as the staphylococcal enterotoxins. they are serotypically heterogeneous single-chain globular proteins, of molecular weight between and kda, secreted by certain strains of s. aureus. one of these, serotype f is identical to tsst- and staphylococcal pyrogenic exotoxin c. these toxins are not classical enterotoxins acting on intestinal cells but 'neurotoxins' activating receptors on the abdominal viscera, the stimulus reaching the vomiting centre via the vagus nerve. several of the staphylococcal toxins referred to above (tss- and enterotoxins), and also the erythrogenic toxins a and c of streptococcus pyogenes (see below) have an additional important action. they are among the most powerful t cell mitogens known, acting at picomolar concentrations. they bind to mhc class ii molecules on antigenpresenting cells and interact with the vß chain of the t cell receptor (see chs and ). this causes proliferation and cytokine release by the entire subset of t cells bearing that particular vß chain- - % of all t cells are affected, whereas only . - . % of t cells respond to a given regular antigen. this represents an important interference with a coordinated immune response, and the widespread polyclonal activation and cytokine release can be regarded as a microbial strategy, a 'diversion' of host immune defences. in addition, if the superantigen reacts with developing t cells (with the correct vß chain) in the thymus, these cells are deleted. it seems probable, therefore, that this is a more important biological function of these toxins than the one responsible for the characteristic disease, which may be no more than an 'accidental' phenomenon. it turns out that similar molecules are formed by mycoplasma and by certain retroviruses (e.g. the mis antigen of mouse mammary tumour virus). scarlet fever is one of the many conditions caused by streptococcal infection and may accompany a streptococcal sore throat or occasionally a streptococcal wound infection. there is a generalised erythematous rash together with fever and a sore throat. the rash is due to an erythrogenic toxin produced by certain strains of streptococcus pyogenes. erythrogenic toxin is a low molecular weight protein produced in a complex form with hyaluronic acid, which acts as a carrier. the protein consists of two parts. one is heat labile, carries the determinants of immunological specificity which give rise to three serotypes, a, b and c, and is responsible for primary toxicity manifestations, including pyrogenicity, low lethality, cytotoxic effects on cultured spleen macrophages, and suppression of the reticuloendothelial and immune systems. the second part is heat stable, antigenically common to types a, b and c, and is responsible for secondary toxicity; that is, hypersensitivity effects, including skin hyperreactivity (the basis of the rash), myocardial necrosis, enhancement of pyrogenicity and lethality, and enhanced host response to other injurious agents. thus, it is not possible to define the biological activity of 'erythrogenic' toxins without considering the immunological state of the host. an individual may suffer no reddening of the skin upon intradermal injection of erythrogenic toxin (negative dick test) because a high neutralising titre of antibody blocks the primary toxiphore or because of a lack of hypersensitivity to the common antigen. streptolysins o and s are dealt with in ch. . there is a plethora of toxins produced by numerous clostridial pathogens important in both human and veterinary medicine. the clostridial genus comprises a large number of toxigenic species, some of which are known to produce several toxins, extracellular enzymes, and other factors which are as yet recognised only by a letter of the greek alphabet. all the α-toxins are dermonecrotic and the others have haemolytic, enzymatic properties. toxin production in vitro is used as the basis of typing clostridia, and the picture is complex. no attempt will be made to describe every disease in man or animals associated with clostridia; only those are selected which best serve to illustrate the involvement of some recognisable toxins. in the case of sheep diseases-lamb dysentery, struck, enterotoxaemia, black disease, braxy, black quarter-some of the best evidence for implicating relevant toxins comes from field studies using multivalent vaccines (based on toxoids of these toxins). clostridium perfringens type c causes pig bei in man, essentially due to the production of ß-toxin. this is a rare disease in developed societies but a public health hazard in papua new guinea. four factors are responsible: the ubiquity of c. perfringens type c in the soil and faeces of man and pigs; the relatively low immunogenicity of ß-toxoids in young children, the group most at risk; the high carbohydrate, low protein nature of the staple diet; and the sporadic consumption of large quantities of pork on occasions of celebration. the latter dietary change promotes a proliferation of clostridia in the intestine which may lead to intestinal gangrene and death. ß-toxin damages the mucosa, reduces mobility of villi and causes more bacteria to become attached to the villi. more toxin is absorbed and the mucosa and underlying the intestinal wall become necrotic, leading to death in many cases. the influence of diet is additionally important in t h a t low protein diets cause decreased secretion of pancreatic proteolytic enzymes and sweet potato contains a trypsin inhibitor. these conditions promote the survival of ß-toxin which is highly sensitive to proteolytic inactivation. immunisation with ß-toxoid preparations has dramatically lowered the incidence of this fatal disease in children. gas gangrene in man may be caused by several bacterial species separately or in concert. these include clostridium perfringens type a, c. novyi types a and b, and c. septicum; c. perfringens and its a-toxin have already been discussed. far less is known about c. novyi and c. septicum and their toxins in gas gangrene in man. much more is known about the role these organisms play in diseases of animals, and multivalent vaccines confer a very high degree of immunity (particularly to sheep) against several clinically identifiable but separate diseases. a few of these diseases are described below. clostridium perfringens type b causes lamb dysentery. this is an acute, fatal disease of young lambs occurring during the first week of life and caused by absorption of toxin(s) generated by c. perfringens type b in the small intestine. clostridium perfringens type c causes struck in sheep, a disease occurring in the romney marshes of kent, but rare in other areas in the world. the pathological changes observed differ markedly from other enterotoxaemias and include enteritis. clostridium perfringens type d enterotoxaemia in sheep is another acute fatal disease. the most constant lesion is subendocardial haemorrhage around the mitral valve. c. perfringens type d e-toxin or its protoxin are recoverable from intestinal contents. clostridium novyi type b causes black disease of sheep or infectious necrotic hepatitis. this is an acute infectious disease of sheep (occasionally cattle) caused by the absorption of the α-toxin elaborated by the organism in necrotic foci in the liver, and is nearly always associated with invasion of the liver by immature liver flukes. how c novyi gets to the liver in the first place is not known but it is readily demonstrable in livers of normal sheep in areas where the disease is prevalent. experimental reproduction in guinea-pigs of a similar disease is possible by the combined action of c. novyi spores and liver fluke infestation. clostridium novyi type d causes a rapidly fatal disease in cattle (redwater disease) similar to, and regarded by some as an atypical manifestation of, black disease. the characteristic lesions include jaundice, various haemorrhagic manifestations, and anaemic infarcts in the liver; active liver fluke infestation may or may not be present. in culture this organism produces ß-toxin, which explains the haemoglobinuria, but no a-toxin. clostridium septicum causes braxy in sheep. the role of c. septicum in this acute, fatal disease is assumed because of its association with the characteristic haemorrhagic inflammatory lesion in the abomasum. the disease has not been reproduced experimentally with c. septicum but can be prevented by immunisation with sterile toxoids derived from this organism. clostridium chauvoei causes black quarter in sheep and cattle. this is a gas gangrene type infection of muscles and associated connective tissues in cattle and sheep; c. chauvoei is also the causative agent of parturient gas gangrene in sheep. the initial stimulus which activates the infection in cattle is not known, since the disease is hardly ever associated with any overt wounding. washed spores alone do not cause disease when injected, but do in conjunction with a tissue-necrotising agent. in sheep, wounding caused by parturition, castration, tailing, shearing, vaccination, as well as accidental damage will create a focus within which c. chauvoei can multiply. there are numerous examples now known of synergistic reactions between toxins of the same or different species. bacillus cereus makes a phosphatidyl choline-preferring phospholipase and a sphingomyelinase which are separately nontoxic; in concert they are haemolytic and termed cereolysin a-b. the examples of staphylococcal a-and δ-toxins and of staphylococcal tsst- and endotoxin, have already been alluded to above. streptococcal erythrogenic toxins increase sensitivity to other streptococcal factors (for example, streptolysin-o) and also to gramnegative endotoxin. the susceptibility of rabbits to endotoxin is increased times, and adult cynomolgus monkeys die within h, when injection of low levels of steptococcal toxin is followed h later by an otherwise sublethal dose of endotoxin. this may be because streptococcal toxin creates a state of hypersusceptibility to a wide variety of stressful agents. perhaps, in view of the high incidence of exposure of man and his domestic animals to streptococci, we should actively investigate possible toxin-mediated synergy in mixed infections involving streptococci. another entirely different type of example is that of the increase in toxicities of staphylococcal a-and -toxins, diphtheria toxin and endotoxin for neonatal ferrets preinfected with influenza virus. increases were -, -, -and -fold respectively. no increase in viral replication was observed. neonates died suddenly without clinical symptoms as in human babies dying from sudden infant death syndrome (sids). pathological examination showed inflammation of the upper respiratory tract, lung oedema and collapse, and early bronchopneumonia in animals receiving the dual challenge but not those receiving either toxin or virus on their own. thus, some bacterial toxins in conjunction with influenza virus could be one of the several causes of sids. many fungi contain substances that are harmful when taken by mouth, and there are two diseases that result from the ingestion of food containing preformed fungal toxins. as with c. botulinum, the disease is caused without the need for infection. aspergillus flavus infects ground nuts (monkey nuts) and produces a very powerful toxin (aflatoxin). contaminated (badly stored) ground nuts used to prepare animal feeds caused the death of thousands of turkeys and pigs in the uk in and the survivors of intoxication nearly all developed liver cancer. human disease has not yet been associated with this toxin. clavicepspurpurae is a rust fungus affecting rye, and it produces toxins (ergotamine especially) that give rise to ergot poisoning when contaminated grain is eaten. mushrooms and toadstools have long been recognised as sources of poisons and hallucinogens. unlike the toxins already discussed in this chapter, there is a group of toxins which are distinct structural components and are not released into the surrounding medium in any quantity except upon death and lysis of the bacteria. several protein toxins (e.g. clostridium difficile toxin a, tetanus toxin) are released during the decline phase of batch culture-probably on autolysis-and these are classified as exotoxins. here we deal with toxins which are known to comprise well-recognised structural entities which on a priori grounds must have key functions in the organism: they are found in the outer membranes of gram-negative organisms. there are two chemically distinct types of toxin considered: lipopolysaccharide (endotoxin; lps) and protein. the bulk of this section is taken up with endotoxin. many pathogenic organisms, however, are pathogenic by virtue of possessing various types of surface structure important in conferring virulence. these include, for example, adhesins which are important in colonising body surfaces or a variety of surface molecules (which may or may not be inside capsules) which render them resistant to phagocytosis. but the majority of adhesins and anti-phagocytic determinants are themselves nontoxic. the gram-negative bacterial cell wall is subject to considerable variations in both the composition of lps and in the number and nature of the proteins found in the outer cell membrane. apart from the examples given in ch. in relation to the gonococcus, such phenotypic variation in lps has rarely been examined in the context of pathogenicity. however, the examination of cell-bound proteins of yersinia pestis from organisms grown in vivo led to the discovery of a toxin lethal for mice and guinea-pigs. plague is one of the most deadly diseases of man and has, over several thousands of years, claimed millions of lives. in the fourteenth century, 'the black death' wiped out a quarter of the population of europe before spreading through the middle east and asia. fortunately, however, the last years or so have seen a drastic decrease in outbreaks of plague, though the threat of another epidemic is still with us. the causative organism of plague, yersinia pestis, is primarily a parasite of rodents in which it is endemic in many areas of the world. only when man comes into close proximity with infected rodents do outbreaks of human plague occur. the disease is spread from rat to rat and from rat to man by fleas. rodents in the terminal stages of infection with y. pestis suffer massive bacteraemia and so when a flea sucks the blood of an infected animal it swallows large numbers of bacteria. when the rodent eventually dies, the flea leaves the corpse and awaits a new host. in the meantime, however, y. pestis multiplies rapidly in the alimentary tract of the insect, often completely blocking the proventriculus. the flea becomes voraciously hungry and feeds on any suitable host, which sooner or later is man. however, because the flea's alimentary canal is blocked by bacteria it cannot feed efficiently and usually succeeds in imbibing blood only to regurgitate it, now contaminated with y. pestis, back into the wound. during the next - days, organisms become localised and multiply in the regional lymph nodes, causing considerable swelling. these swellings, which are often in the axillary and inguinal regions because the most common sites for flea-bites are the arms or legs, are referred to as primary buboes (greek boubon = groin); hence the name bubonic plague. secondary buboes may develop, followed by necrosis of the lymphoid tissue and vascular damage giving rise to haemorrhaging in many organs and tissues. these pathological changes are accompanied by prostration, high fever and delirium, followed in the terminal stages of the disease by shock and death. it is also possible for the disease to be transmitted from human to human by the aerosol route: exhaled organisms from heavily infected patients are capable of infecting others giving rise to pneumonic plague. the principal features of human plague can be reproduced in guineapigs and mice. monkeys show shock-like signs only during the terminal period of - h, when they become quiet, progressively weak, prostrate and hypothermic; for the previous - days infected animals are lively and vigorous. in the terminal stages blood pressure drops rapidly but there is no evidence of oligaemia (low blood volume) caused by haemorrhage, or of oedema, suggesting that vascular collapse must be associated with a vasodilatory factor(s), resulting in pooling of blood. in this respect monkeys differ from humans and guinea-pigs. the symptoms of plague-high fever and vascular damage -are characteristic of intoxication with endotoxin. however, it is extremely unlikely that endotoxin alone is the main toxin involved in plague. it is much more likely to act in conjunction with one or more other potentially toxic fractions from y. pestis. plague murine toxin is a protein which, although highly lethal for mice and rats, is relatively nontoxic for guinea-pigs, rabbits, dogs and monkeys. a completely separate guinea-pig toxin complex exists comprising at least two cell wall/membrane protein components, one of which will kill mice, although both are needed to kill guinea-pigs. however, the nature of the toxin or toxins of y. pestis and their role in the human disease syndrome are still far from clear. the classical approach to such a problem is to prepare a specific toxoid and to determine whether injection of this confers immunity to the disease. needless to say, the severity of human plague renders such experiments impossible. until such questions can be answered, we are left to argue whether, in the context of plague, man resembles a mouse, a monkey or a guinea-pig. endoxotins are part of the outer membrane of gram-negative bacteria. it has been known for many years that the cells (alive or dead) or cell extracts of a wide variety of gram-negative bacteria are toxic to man and animals. the literature on this subject is vast, sometimes confusing and often controversial; here we can give no more than a brief outline. some of the diseases in which endotoxin may play an important role include typhoid fever, tularaemia, plague and brucellosis, and a variety of hospital-acquired infections caused by opportunistic gram-negative pathogens which include escherichia coli, proteus, pseudomonas aeruginosa, enterobacter, serratia and klebsiella. in addition, endotoxin has been intensively studied as a possible causative agent of shock arising from post-operative sepsis or other forms of traumatic injury in which the normal flora of the gut is often the source of endotoxin. the toxins we have considered so far have been protein (or at least part protein) in nature but, in contrast, endotoxin is a complex lipopolysaccharide. it is also much more heat stable than protein toxins and much less easily toxoided. in addition to lethality, endotoxin displays a bewildering array of biological effects. the complex nature of the multi-layered gram-negative bacterial envelope is shown in fig. . (see also fig. . ) . the outer membrane is composed of a bimolecular leaflet arrangement as are other membranes but has a different composition from the cytoplasmic membrane. the lipopolysaccharide (lps) is unique in nature, only found in gramnegative bacteria, and is, or contains within it, what we designate endotoxin. immunoelectron microscopy indicates that lps exists in the outer leaflet of the membrane and extends outward up to nm; it is on, rather than in, the cell. thus it is evident that the term endotoxin is a misnomer which derives from the era when toxins were considered to be either exotoxins, which were synthesised and secreted by the viable organism, or endotoxins, which were intracellular and released only upon lysis. moreover, extraction with edta shows that approximately % of lps is held noncovalently linked in the membrane. extraction with a variety of different solvents yields material which is highly heterogenous and of apparent molecular weight - x . however, treatment with pyridine or addition of detergents reduces the polydispersity. the endotoxic glycolipid from the rough mutant of salmonella minnesota, r , has an m r of for the basic unit, from which complex aggregate structures are derived. lipopolysaccharide consists of three regions: polysaccharide side chains, core polysaccharide, and lipid a which consists of a di-glucosamine backbone to which long chain fatty acids are linked (fig. . ). the relationship of this type of molecule to the outer membrane is also shown in fig. . . the long chain fatty acids interdigitate between the phospholipids in the outer leaflet and may also be linked (or interact) with lipoproteins, which in turn may or may not be covalently anchored to the rigid peptidoglycan (pg). the polysaccharide side chains project outwards. this structure is not invariant. for example, many organisms when first isolated give rise to colonies with a smooth appearance on agar but on subculture produce colonies with a rough appearance. in general, 'smooth' strains of pathogenic species are more virulent than rough strains. this s->r conversion is accompanied by a loss of region i side chains, which contain the deoxy and dideoxy sugars found in these lps complexes. in addition to these somewhat drastic changes involving loss of side chains, it is possible to induce major compositional changes by manipulating the growth rate of these organisms in a chemostat. thus the lps of salmonella enteriditis, when grown with a mean generation time of min is nearly totally deficient in tyvelose (a dideoxy sugar), possesses % of the galactose and % of the glucose contents of lps obtained when the generation time is min. these genotypic s organisms exhibit an r-phenotype in terms of their vastly reduced o-agglutinability (see below); such observations are potentially very important in the context of the in vivo phenotype and pathogenicity, since it is well known that the growth rate of salmonella typhimurium in mice is - times lower than in vitro. examples have already been given in ch. of changes in lps structures in vivo in relation to neisseria gonorrheae and neisseria meningitidis. the extent to which lipid a is common between different genera is uncertain, but it is not likely to vary tremendously. the core polysaccharide structure is the same or very similar within groups of the enterobacteriaciae: thus polysaccharides from salmonellae are similar to each other, but differ from those of e. coli strains. however, within a group such as the salmonellae, there is a wide variation in the composition and detailed structures of the side chains, a fact which is exploited in the kauffman-white scheme for classifying salmonellae, giving rise to several thousand serotypes. the side chains carry the o-somatic antigen specificities of which there are far more than can readily be accounted for on the basis of the known number of sugars involved in the basic repeating units. in the side chains are found a range of depxy and dideoxy sugars. the general principles governing the relationship between the various chemotypes and serotypes are now well understood; the multiplicity of antibody specificities evoked may be explained in terms of antibodies which can recognise different aspects of one three-dimensional structure. lipid a is the primary toxiphore, but the polysaccharide plays an important part in conferring solubility upon, and optimising the size of micellar aggregates of lps, hence affecting biological activity. however, the immune status of the test animal may affect toxicity: as normal animals produce antibodies to the antigenic determinants on the surface of normal gut organisms (including o-somatic antigens), some of the biological effects of endotoxin may be mediated by hypersensitivity mechanisms. the most powerful evidence that lipid a is the primary toxiphore comes from studies on smooth (s) and rough (r) mutants whose biosynthetic capabilities are blocked at various points. this established that neither the o-side chains, nor the core polysaccharide are necessary for endotoxicity. pure lipid a can be made toxic by complexing it to a hydrophilic carrier like bovine serum albumin. one can make synthetic lipid a preparations which are toxic and assume space-filling configurations, which make it easy to see how they could fit into and be part of a bimolecular leaflet arrangement in the outer membrane. the range of biological properties of endotoxin is quite bewildering and the mode(s) of action very complicated. included among those effects which might play a role in gram-negative bacterial infections, are abortion, pyrogenicity, tolerance (not immune tolerance), the schwartzmann phenomenon, hypotension and shock, and lethality, but the precise part played by lps in these phenomena in gram-negative infections is far from clear. lps causes the release of vasoactive substances, activates the alternative pathway of the complement cascade, and also activates factor xii (hageman factor), the first step of the coagulation cascade, which sometimes results in disseminated intravascular coagulation (p. ). many, perhaps nearly all, the actions of lps are due to the stimulation of cytokine release from macrophages and other cells. there is an effect on the circulation, leading ultimately to vascular collapse. the vascular regions most affected differ from species to species; in man and sheep the main changes are found in the lungs. lps has powerful immunological actions, which is surely no accident; as well as activating the complement system, it induces il- production and is a potent b cell mitogen. man is one of the most sensitive of all species to the pyrogenic action of endotoxin. a dose of n g per kg of body weight injected intravenously into man causes the release of an endogenous pyrogen (interleukin- , see glossary) and tumour necrosis factor (tnf) from macrophages, which act on the hypothalamus to give an elevation of body temperature within an hour. it is possible that the pyrogenic action of lps helps to generate fever in gram-negative bacterial infections, but lps is not the only bacterial factor capable of inducing a febrile response. in spite of all these toxic actions, there have been suggestions that some of the responses to lps (by macrophages, polymorphs) could be advantageous to the host, possibly assisting in the recognition and destruction of bacteria. could it be that host responses to lps are, like the complement or the clotting systems, useful in moderation but harmful in excess? there are reports that when animals with less vigorous responses to lps are infected they suffer fewer symptoms, but permit greater growth of bacteria. very large numbers of gram-negative bacteria are normally present in the intestines (see ch. ), their continued death and exit in the faeces being balanced by multiplication in the lumen. there is a continuous, inevitable low-grade absorption of endotoxin from the intestine.* absorbed (endogenous) endotoxin enters the portal circulation and is taken * in addition, various antigens are absorbed in small quantities from the intestine, and in normal individuals antibodies are formed against various food proteins and to some extent against resident intestinal bacteria (see ch. ). kupffer cells remove any antigen-antibody complexes formed locally in the intestine and prevent them from entering the systemic circulation. up and degraded by reticuloendothelial cells, mainly kupffer cells in the liver. continuous exposure to endotoxin probably has profound effects on the immune system and on the histology of the intestinal mucosa, stimulating development of the immune system in the immature individual, but there are no obvious pathogenic consequences. normal people have low levels of antibody to endotoxin as a result of this continuous exposure. the sick individual may be much more susceptible to endogenous endotoxin, perhaps because of defects in removal by kupffer cells. after trauma or after genito-urinary instrumentation endotoxin is detectable in peripheral blood by the limulus test,* but this leads to no particular signs or symptoms. when large amounts of endotoxin enter the blood there are profound effects on blood vessels with peripheral vascular pooling, a drastic fall in blood pressure, collapse and sometimes death. thus, if enough endotoxin enters the blood during massive gramnegative bacterial sepsis, the vasomotor action of endotoxin becomes important and shock intervenes.! in experimental animals endotoxin also causes vasodilation and haemorrhage into the intestinal mucosa, and sometimes haemorrhage into the placenta with abortion, but these actions do not appear to be important in all gram-negative bacterial infections. to summarise, endotoxin, although studied so carefully and for so long, has not yet been shown to play a definitive role as a toxin in the pathogenesis of any infectious disease. however, there is a growing body of opinion that seeks to implicate endotoxin in the inflammatory response induced in meningitis caused by gram-negative bacteria. but, in spite of its effects on various host defence systems including polymorphs, lymphocytes, macrophages, complement, and on endothelial cells and platelets, its overall role in infection is still not clear. it can, however, cause shock when gram-negative bacteria invade the blood. it is for this reason that considerable effort in recent years has gone into the development of antilipid a antibodies for use as therapeutic agents to combat shock in such situations; the success rate is only partial and the expense enormous. for that reason several groups are seeking to exploit the wealth of chemical and biophysical information available on lps in attempts to develop synthetic derivatives which would neutralise the biological activity of lipid a. we await the outcome of such research. however, the characteristics of the o-antigen polysaccharide are sometimes important in determining virulence (pp. - ). * a sensitive test for endotoxin based on the ability of endotoxin to induce gelation of a lysate obtained from the blood cells of the horseshoe crab, limulus polyphemus. considerable space has been given to toxins because they are being intensively investigated as possible virulence determinants. the account illustrates the complexity of host-microbe interactions when analysed at the molecular level. most toxins are liberated from the microbial cell and can be studied with greater facility than many of the more elusive determinants of pathogenicity. but remember that microbes that replicate inside host cells are less likely to form powerful toxins because they cannot afford to damage at too early a stage the cell in which they are multiplying. thus, toxins are not prominent products in intracellular infections due to mycobacteria, brucella, rickettsiae, or chlamydia, and viruses do not form toxins. although a single molecule of a toxin like diphtheria toxin is enough to kill a cell, other toxins may do no more than impair cell function when present in sublethal concentrations. this can lead, for instance, to defective function in immune or phagocytic cells. low concentrations of the streptococcal streptolysins* will inhibit leucocyte chemotaxis. the ability to form toxins, whether encoded by plasmids or the microbial genome, is subject to selective forces. if toxin production puts a microorganism at a serious disadvantage it will tend to disappear. if it is advantageous it will be maintained, and will spread through the microbial population, just as the genetic changes that confer resistance to antimicrobial drugs are selected for when these drugs are widely used. it is therefore not unreasonable to ask how many of the well-known toxins are actually useful to the microbe as well as being important in causing disease in the host (table . ). however, microbes that multiply extracellularly must produce a variety of enzymes and other molecules involved in nutrition, adherence to substrate, and so on. in the case of free-living microbes these substances, as well as substances that damage or interfere with competing organisms, are of major importance. probably they cannot all be discarded when the parasitic mode of life is adopted. many will have a toxic action. yet, for the infecting microbe, these substances remain as unfortunate necessities, of no particular advantage and perhaps a disadvantage, in the parasitic way of life. in infectious diseases there is nearly always a certain amount of direct microbial damage to host tissues, as discussed above. host cells are destroyed or blood vessels injured as a direct result of the action of * the streptolysins are responsible for ß-haemolysis. most haemolysins will kill phagocytes. ) . inflammatory materials are liberated from necrotic cells, whatever the cause of the necrosis. also many bacteria themselves liberate inflammatory products and certain viruses cause living infected cells to release inflammatory mediators. therefore it is not always clear how much of the inflammation is directly microbial rather than host in origin.* but inevitably the host (see ch. ) generates inflammatory and other tissue responses, and these responses sometimes account for the greater part of the tissue changes. pathological changes can then be regarded as occurring indirectly as a result of these responses to the infection. inflammation causes redness, swelling, pain and sometimes loss of function of the affected part (see ch. ) and is generally a major cause of the signs and symptoms of disease. indirect damage attributable to the host immune response is discussed separately below. in most diseases direct and indirect types of damage both make a contribution to pathological changes, but in a given disease one or the other may be the most important. in a staphylococcal abscess the bacteria produce inflammatory materials, but they also kill infiltrating polymorphs whose lysosomal enzymes are thereby liberated and induce further inflammation. this type of indirect nonimmunological damage is sometimes important in streptococcal infections. virulent streptococci produce various toxins that damage phagocytes, and also bear on their surfaces substances that impede phagocytosis (see ch. ). nevertheless, with the help of antibody, all streptococci are eventually phagocytosed and killed and the infection terminated. unlike the staphylococci, however, killed group a streptococci pose a digestive problem for phagocytic cells. the peptidoglycan component of the streptococcal cell wall is very resistant to digestion by lysosomal enzymes. when streptococci are injected into the skin of a rabbit, for instance, streptococcal peptidoglycans persist in macrophages for as long as days. hence macrophages laden with indigestible streptococcal cell walls tend to accumulate in sites of infection. lysosomal enzymes, including collagenase, leak from these macrophages, causing local destruction of collagen fibres and the connective tissue matrix. macrophages secrete many other substances some of which may contribute to cell and tissue damage (see also p. ). many macrophages eventually die or form giant cells, sometimes giving rise to granulomatous lesions (see p. ). in this way persistent streptococcal materials sometimes cause chronic inflammatory lesions in the infected host. an additional immunopathological contribution to the lesions is to be expected if the host is sensitised to peptidoglycan components. other pathogenic microorganisms that are digested with difficulty by phagocytes include listeria, shigella, candida albicans and, of course, mycobacteria, but the importance of this in the pathogenesis of disease is not generally clear. the expression of the immune response necessarily involves a certain amount of inflammation, cell infiltration, lymph node swelling, even tissue destruction, as described in ch. . such changes caused by the immune response are classed as immunopathological. sometimes they are very severe, leading to serious disease or death, but at other times they play a minimal part in the pathogenesis of disease. with the possible exception of certain vertically transmitted virus infections and the transmissible 'prion' dementias (see ch. ), there are signs of an immune response in all infections. therefore it is to be expected that there will nearly always be some contribution of the immune response to pathological changes.* often the immunological contribution is small, but sometimes it forms a major part of the disease. for instance, in tuberculosis the pathological picture is dominated by the operation of a strong and persistent cmi response to the invading bacillus. in the classical tubercle a central zone of bacilli with large mononuclear and giant cells, often with some necrosis, is surrounded by fibroblasts and lymphocytes. mononuclear infiltrations, giant cells and granulomatous lesions (see p. ) are characteristic pathological features of tuberculosis. there are no recognised toxins formed by tubercle bacilli, and there seems to be no single antigen or other component that accounts for virulence. bacterial glycolipids (e.g. 'cord factor'), resistance to h (see pp. - ) and ability to utilise host fe (see p. ) have been correlated with pathogenicity, and inhibition of phagosome-lysosome fusion in macrophages (see pp. - ) by release of unidentified bacterial components would also contribute to pathogenicity. however, none of these factors is by itself absolutely necessary for virulence, which in such a complex, ancient parasite is likely to be multifactorial. as a gene library for m. tuberculosis is slowly built up there will be opportunities for clearer definition of virulence determinants. when macrophages are killed by intracellular mycobacteria the lysosomal enzymes and other materials released from the degenerating cell contribute to chronic inflammation as in the case of the streptococcal lesions referred to above. * a number of different microbial antigens are produced during most infections (see ch. ) and the possible immunological reactions are therefore numerous. for instance, at least types of circulating malarial antigen are found in heavily infected individuals. the mere enlargement of lymphoid organs during infectious diseases is a morphological change that can often be regarded as pathological. the lymph node swelling seen in glandular fever, for instance, is an immunopathological feature of the disease, and the same can be said of the striking enlargement of the spleen caused by chronic malaria and other infections in the condition known as tropical splenomegaly. as often as not the relative importance of direct microbial damage as opposed to immune and nonimmune inflammatory reactions have not yet been determined, but the picture is clearer in most of the examples given below. in one important human disease, pathological changes are certainly immunopathological in nature, but not enough is known about it to classify the type of reaction (see table . ). this disease is rheumatic fever, which follows group a streptococcal infections of the throat. it is the commonest form of heart disease in many developing countries. antibodies formed against a streptococcal cell wall or membrane component also react with the patient's heart muscle or valves, and myocarditis develops a few weeks later. many strains of streptococci have antigens that cross-react with the heart, and repeated infections with different streptococci cause recurrent attacks of rheumatic fever. there is genetic predisposition to the disease, based either on a particular antigen present in the heart of the patient or on a particular type of antibody response. chorea, a disease of the central nervous system, is a rare complication of streptococcal infection and antistreptococcal antibodies have been shown to react with neurons in the caudate and subthalamic nuclei of the brain. a number of microorganisms have antigens similar to host tissue components (p. ) so that in the course of responding immunologically to such infections the host is vulnerable to autoimmune damage (see ankylosing spondylitis, p. ). the antibodies to host components such as dna, igg, myofibrils, erythrocytes etc. that are seen in trypanosomiasis, mycoplasma pneumoniae, and eb virus infections appear to result from polyclonal activation of b cells (see p. ). it is not clear how important these autoimmune responses are in pathogenesis, but they reflect fundamental disturbances in immunoregulation. four types of immunopathology can be distinguished according to the classification of allergic reactions by coombs and gell, and microbial immunopathology will be described under these headings (see table . ). these depend on the reactions of antigens with reaginic (ige) antibodies attached to mast cells, resulting in the release of histamine, leukotrienes (see p. ) and heparin from mast cells, and the activation of serotonin and plasma kinins. if the antigen-antibody interaction takes place on a large enough scale in the tissue, the histamine that is released can give rise to anaphylactic shock, the exact features depending on the sensitivity and particular reaction of the species of animal to histamine. guinea-pigs suffer from bronchospasm and asphyxia, and in man there are similar symptoms, sometimes with a fall in blood pressure and shock. this type of immunopathology, although accounting for anaphylactic reactions to horse serum or to penicillin, is not important in infectious diseases. when the antigen-ige antibody interaction takes place at the body surface there are local inflammatory events, giving rise to urticaria in the skin, and hayfever or asthma in the respiratory tract. this local type of anaphylaxis may play a part in the pathogenesis of virus infections of the upper respiratory tract (e.g. common cold, respiratory syncytial virus infections of infants), or in skin rashes in infectious diseases. type reactions are common in helminth infections perhaps because ige antibodies have an important role in protection against these parasites. a dramatic type reaction can follow rupture of a hydatid cyst of echinococcus granulosus (the dog tapeworm). slow leakage of worm antigens means that mast cells are sensitised with specific ige antibody, and the sudden release of antigen can cause life-threatening anaphylaxis. when the larvae of ascaris lumbricoides pass through the lung on their journey from blood to intestine, they can give rise to igemediated respiratory symptoms, with infiltration of eosinophils. reactions of this type occur when antibody combines with antigen on the surface of a tissue cell, activates the complement sequence or triggers cytotoxicity by k cells (nk cells or phagocytes with fc receptors). k (killer) cell cytolysis is referred to as antibody-dependent cellular cytotoxicity (adcc). the antibody-coated cell is destroyed. as discussed in ch. , the same reaction on the surface of a microorganism (e.g. enveloped virus) constitutes an important part of antimicrobial defences, often leading to the destruction of the microorganism. cells infected with viruses and bearing viral antigens on their surface are destroyed in a similar way. clearly the antibody-mediated destruction of infected cells means tissue damage, and it perhaps accounts for some of the liver necrosis in hepatitis b, for instance, and probably in yellow fever. infected cells can also be destroyed by sensitised lymphocytes or nk cells independently of antibody (see below). in certain infections antibodies are formed against host erythrocytes and these cells are particularly sensitive to lysis. the haemolysis in malaria is caused by antibodies to parasite-derived antigens that have attached to red cells, rather than by autoantibodies to red cells themselves. in pneumonia due to mycoplasma pneumoniae (atypical pneumonia), antibodies (cold agglutinins) are formed against normal human group o erythrocytes. haemolytic anaemia is occasionally seen, and there is reticulocytosis (see glossary) in % of patients. the lesions in the lungs are perhaps based on cell-mediated immunopathological reactions. the combination of antibody with antigen is an important event, initiating inflammatory phenomena that are inevitably involved in the expression of the immune response. in the infected host, these inflammatory phenomena are most of the time of great antimicrobial value (see ch. ). but there are nevertheless immunopathological features of the infection, and immune complex reactions sometimes do a great deal of damage in the infected individual. the mechanisms by which antigenantibody reactions cause inflammation and tissue damage are outlined in fig. . . iga immune complexes are less harmful. antigens absorbed from the intestine can combine locally with iga antibody and the complex then enters the blood, to be filtered out in the liver and excreted harmlessly in bile (see p. ). when the antigen-antibody reaction takes place in extravascular tissues, there is inflammation and oedema with infiltration of polymorphs. if soluble antigen is injected intradermally into an individual with large amounts of circulating igg antibody, the antigen-antibody reaction takes place in the walls of skin blood vessels, and causes an inflammatory response. the extravasating poly morphs degenerate and their lysosomal enzymes cause extensive vascular damage. this is the classical arthus response. antigen-antibody reactions in tissues are not usually as serious as this, and milder inflammatory sequelae are more common as in the case of allergic alveolitis (see below), or the red zone seen round the borders of a smallpox vaccination site after seven or eight days. in the latter example circulating antibodies pass through vessel walls, meet vaccinia virus antigen in the dermal tissues and an inflammatory response is generated. a similar mild response can be induced experimentally to cause a reaction known as cutaneous anaphylaxis (see glossary), the test antigen being injected into the skin and reacting with blood-borne antibody. the resulting inflammation is detected by the visible local leakage of plasma proteins from blood vessels, circulating plasma proteins having been coloured by the intravenous injection of evans' blue. when the antigen-antibody reaction takes place in the blood to give circulating immune complexes, the sequelae depend to a large extent on size and on the relative proportions of antigen and antibody. if there is a large excess of antibody, each antigen molecule is covered with antibody and is removed rapidly by reticuloendothelial cells, which have receptors for the fc portion of the antibody molecule (see ch. ). when equal amounts of antigen and antibody combine, lattice structures are produced, and these form large aggregates whose size ensures that they are also rapidly removed by reticuloendothelial cells. if, however, complexes are formed in antigen excess, the poorly coated antigen molecules are not removed by reticuloendothelial cells. they continue to circulate in the blood and have the opportunity to localise in small blood vessels elsewhere in the body. the mechanism is not clear, but complexes are deposited in the glomeruli of the kidneys, the choroid plexuses, joints and ciliary body of the eye. factors may include local high blood pressure and turbulent flow (glomeruli), or filtering function of vessels involved (choroid plexus, ciliary body). in the glomeruli the complexes pass through the endothelial windows (fig. . ) and come to lie beneath the basement membrane. the smallest-sized complexes pass through the basement membrane and seem to enter the urine. this is probably the normal mechanism of disposal of such complexes from the body. immune complexes are formed in many, perhaps most, acute infectious diseases. microbial antigens commonly circulate in the blood in viral, bacterial, fungal, protozoal, rickettsial etc. infections. when the immune response has been generated and the first trickle of specific antibody enters the blood, immune complexes are formed in antigen excess. this is generally a transitional stage soon giving rise to antibody excess, as more and more antibody enters the blood and the infection is terminated. sometimes the localisation of immune complexes and complement in kidney glomeruli is associated with a local inflammatory response.* there is an infiltration of polymorphs, swelling of the glomerular basement membrane, loss of albumin, even red blood cells, in the urine and the patient has acute glomerulonephritis. this is seen following streptococcal infections, mainly in children (see below). as complexes cease to be formed the changes are reversed, and complete recovery is the rule. repeated attacks or persistent deposition of complexes leads to irreversible damage, often with proliferation of epithelial cells following the seepage of fibrin into the urinary space. under certain circumstances complexes continue to be formed in the blood and deposited subendothelially for long periods. this happens in certain persistent microbial infections in which microbial antigens are continuously released into the blood but antibody responses are only minimal or of poor quality (see below). complexes are deposited in glomeruli over the course of weeks, months or even years. the normal mechanisms for removal are inadequate. the deposits, particularly larger complexes containing high molecular weight antigens or antibodies (igm) are held up at the basement membrane and accumulate in the subendothelial space together with the complement components. as deposition continues, they gradually move through to the mesangial space ( fig. . ) where they form larger aggregates. mesangial cells, * see also footnote p. ; cells in kidney glomeruli, in joint synovium and in choroid plexuses bear fc or c b receptors. this would favour localisation in these tissues. of cell and tissue damage one of whose functions is to deal with such materials, enlarge, multiply and extend into the subepithelial space. if these changes are gradual there are no inflammatory changes, but the structure of the basement membrane alters, allowing proteins to leak through into the urine. later the filtering function of the glomerulus becomes progressively impaired. in the first place the glomerular capillary is narrowed by the mesangial cell intrusion. also, the filtering area is itself blocked by the mesangial cell intrusion, by the accumulation of complexes (fig. . ) , and by alterations in the structure of the basement membrane. the foot processes of epithelial cells tend to fuse and further interfere with filtration. the pathological processes continue, some glomeruli ceasing to produce urine, and the individual has chronic glomerulonephritis. circulating immune complex deposition in joints leads to joint swelling and inflammation but in choroid plexuses there are no apparent pathological sequelae. circulating immune complexes are also deposited in the walls of small blood vessels in the skin and elsewhere, where they may induce inflammatory changes.* the prodromal rashes seen in exanthematous virus infections and in hepatitis b are probably caused in this way. if the vascular changes are more marked they give rise to the condition called erythema nodosum, in which there are tender red nodules in the skin, with deposits of antigen, antibody and complement in vessel walls. erythema nodosum is seen following streptococcal infections and during the treatment of patients with leprosy. when small arteries are severely affected, for instance in some patients with hepatitis b, this gives rise to periarteritis nodosa. immune complex glomerulonephritis occurs as an indirect immunopathological sequel to a variety of infections. first there are certain virus infections of animals. the antibodies formed in virus infections generally neutralise any free virus particles, thus terminating the infection (see ch. ), but the infection must persist if antigen is to continue to be released into the blood and immune complexes formed over long periods. non-neutralising antibodies help promote virus persistence because they combine specifically with virus particles, fail to render them noninfectious, and at the same time block the action of any good neutralising antibodies that may be present. immune complexes in antigen excess are formed in the blood when the persistent virus or its antigens circulates in the plasma and reacts with antibody which is present in relatively small amounts. virus infections with these characteristics are * it is not clear how inflammation is caused. complement activation would presumably take place while complexes were circulating in the blood. perhaps the complexes bind more antibody after they have localised, or alternatively it is possible that free antigen circulates in the first place, localises, and later binds antibody to generate mediators of inflammation. included in table . . in each instance complexes are deposited in kidney glomeruli and sometimes in other blood vessels as described above. in some there are few if any pathological changes (ldv and leukaemia viruses in mice) probably because there is a slow rate of immune complex deposition, whereas in others glomerulonephritis (lcm virus in mice, adv in mink) or vasculitis (adv in mink) is severe. a persistent virus infection that induces a feeble immune response forms an ideal background for the development of immune complex glomerulonephritis, but there are no known viral examples in man. there are one or two other microorganisms that occasionally cause this type of glomerulonephritis and it is seen, for instance, in chronic quartan malaria and sometimes in infective endocarditis. in both these examples microbial antigens circulate in the blood for long periods. but immune complex deposition does not necessarily lead to the development of glomerulonephritis, and immune complexes are detectable in the glomeruli of most normal mice and monkeys. even in persistent virus infections the rate of deposition may be too slow to cause pathological changes as with ldv and leukaemia virus infections of mice (see table . ). during the acute stage of hepatitis b in man, when antibodies are first formed against excess circulating viral antigen (hepatitis b surface antigen), immune complexes are formed and deposited in glomeruli. but the deposition is short-lived and there is no glomerulonephritis. persistent carriers of the antigen do not generally develop glomerulonephritis, because their antibody is usually directed against the 'core' antigen (nephrotic syndrome in secondary syphilis) unknown causative agents man + + + + of chronic glomerulonephritis of the virus particle, rather than against the large amounts of circulating hepatitis b surface antigen. kidney failure in man is commonly due to chronic glomerulonephritis, and this is known to be mostly of the immune complex type, but the antigens, if they are microbial, have not yet been identified. immune complex glomerulonephritis occurs in man as an important complication of streptococcal infection, but this is usually acute in nature with inflammation of glomeruli, as referred to above. antibodies formed against an unknown component of the streptococcus react with circulating streptococcal antigen, perhaps also with a circulating host antigen, and immune complexes are deposited in glomeruli. streptococcal antibodies cross-reacting with the glomerular basement membrane may contribute to the picture. deposition of complexes continues after the infection is terminated, and glomerulonephritis develops a week or two later. the streptococcal infection may be of the throat or skin, and streptococcus pyogenes types and are frequently involved. when certain antigens are inhaled by sensitised individuals and the antigen reaches the terminal divisions of the lung, there is a local antigen-antibody reaction with formation of immune complexes. the resulting inflammation and cell infiltration causes wheezing and respiratory distress, and the condition is called allergic alveolitis. persistent inhalation of the specific antigen leads to chronic pathological changes with fibrosis and respiratory disease. exposure to the antigen must be by inhalation; when the same antigen is injected intradermally, there is an arthus type reaction (see p. ). there are a number of microorganisms that cause allergic alveolitis. most of these are fungi. a disease called farmer's lung occurs in farm workers repeatedly exposed to mouldy hay containing the actinomycete micromonospora faeni. cows suffer from the same condition. a fungus contaminating the bark of the maple tree causes a similar disease (maple bark stripper's disease) in workers in the usa employed in the extraction of maple syrup. the mild respiratory symptoms occasionally reported after respiratory exposure of sensitised individuals to tuberculosis doubtless have the same immunopathological basis. in addition to their local effects, antigen-antibody complexes generate systemic reactions. for instance, the fever that occurs at the end of the incubation period of many virus infections is probably attributable to a large-scale interaction of antibodies with viral antigen, although extensive cmi reactions can also cause fever. the febrile response is mediated by endogenous pyrogen (interleukin- ) and tumour necrosis factor (tnf) liberated from polymorphs and macrophages, as described on p. . probably the characteristic subjective sensations of illness and some of the 'toxic' features of virus diseases are also caused by immune reactions and liberation of cytokines. systemic immune complex reactions taking place during infectious diseases very occasionally give rise to a serious condition known as disseminated intravascular coagulation. this is seen sometimes in severe generalised infections such as gram-negative septicaemia, meningococcal septicaemia, plague, yellow fever and other haemorrhagic arthropod-borne virus diseases. immune complex reactions activate the enzymes of the coagulation cascade ( fig. . ), leading to histamine release and increased vascular permeability. fibrin is formed and is deposited in blood vessels in the kidneys, lungs, adrenals and pituitary. this causes multiple thromboses with infarcts, and there are also scattered haemorrhages because of the depletion of platelets, prothrombin, fibrinogen etc. systemic immune complex reactions were once thought to form the basis for dengue haemorrhagic fever. this disease is seen in parts of the world where dengue is endemic, individuals immune to one type of dengue becoming infected with a related strain of virus. they are not protected against the second virus, although it shows immunological cross-reactions with the first one. indeed the dengue-specific antibodies enhance infection of susceptible mononuclear cells, so that larger amounts of viral antigen are produced (see p. ). it was thought that after virus replication, viral antigens in the blood reacted massively with antibody to cause an often lethal disease with haemorrhages, shock and vascular collapse. however, it has proved difficult to demonstrate this pathophysiological sequence, and the role of circulating immune complexes and platelet depletion remains unclear. perhaps in this and in some of the other viral haemorrhagic fevers the virus multiplies in capillary endothelial cells. disease seems due to cytokines liberated from infected mononuclear cells. immune complex immunopathology is probable in various other infectious diseases. for instance, the occurrence of fever, polyarthritis, skin rashes and kidney damage (proteinuria) in meningococcal meningitis and gonococcal septicaemia indicates immune complex deposition. circulating immune complexes are present in these conditions. certain african arthropod-borne viruses with exotic names (chikungunya, o'nyong-nyong) cause illnesses characterised by fever, arthralgia and itchy rashes, and this too sounds as if it is immune complex in origin. immune complexes perhaps play a part in the oedema and vasculitis of trypanosomiasis and in the rashes of secondary syphilis. sensitive immunological techniques are available for the detection of circulating complexes and for the identification of the antigens and antibodies in deposited complexes. the full application of these techniques will perhaps solve the problem of the aetiology of chronic glomerulonephritis in man. the mere expression of a cmi response involves inflammation, lymphocyte infiltration, macrophage accumulation and macrophage activation as described in ch. , and can therefore by itself cause pathological changes. the cmi response to infection dominates the pathological picture in tuberculosis, with mononuclear infiltration, degeneration of parasitised macrophages, and the formation of giant cells as central features. these features of the tissue response result in the formation of granulomas (see glossary) which reflect chronic infection and accompanying inflammation. there is a ding-dong battle as the host attempts to contain and control infection with a microorganism that is hard to eliminate. the granulomas represent chronic cmi responses to antigens released locally. various other chronic microbial and parasitic diseases have granulomas as characteristic pathological features. these include chlamydial (lymphogranuloma inguinale), bacterial (syphilis, leprosy, actinomycosis), and fungal infections (coccidiomycosis). antigens that are disposed of with difficulty in the body are more likely to be important inducers of granulomas. thus, although mannan is the dominant antigen of candida albicans, glucan is more resistant to breakdown in macrophages and is responsible for chronic inflammatory responses. the lymphocytes and macrophages that accumulate in cmi responses also cause pathological changes by destroying host cells. cells infected with viruses and bearing viral antigens on their surface are targets for cmi responses as described in chs and . infected cells, even if they are perfectly healthy, are destroyed by the direct action of sensitised t lymphocytes, which are demonstrable in many viral infections. in spite of the fact that the in vitro test system so clearly displays the immunopathological potential of cytotoxic t cells, this is not easy to evaluate in the infected host. it may contribute to the tissue damage seen, for instance, in hepatitis b infection and in many herpes and pox virus infections. antigens from trypanosoma cruzi are known to be adsorbed to uninfected host cells, raising the possibility of autoimmune damage in chagas' disease, caused by this parasite.* it is also becoming * chagas' disease, common in brazil, affects million people, and is transmitted by blood-sucking bugs. after spreading through the body during the acute infection, the parasitaemia falls to a low level and there is no clinical disease. years later a poorly understood chronic disease appears, involving heart and intestinal tract, which contain only small numbers of the parasite but show a loss of autonomic ganglion cells. an autoimmune mechanism is possible (see p. ), because a monoclonal antibody to t. cruzi has been obtained that cross-reacts with mammalian neurons. clear that cells infected with certain protozoa (e.g. theileria parva in bovine lymphocytes) have parasite antigens on their surface and are susceptible to this type of destruction. little is known about intracellular bacteria. the most clearly worked out example of type (cmi) immunopathology is seen in lcm virus infection of adult mice. when virus is injected intracerebrally into adult mice it grows in the meninges, ependyma and choroid plexus epithelium, but the infected cells do not show the slightest sign of damage or dysfunction. after - days, however, the mouse develops severe meningitis with submeningeal and subependymal oedema, and dies. the illness can be completely prevented by adequate immunosuppression, and the lesions are attributable to the mouse's own vigorous cd + t cell response to infected cells. these cells present processed lcm viral peptides on their surface in conjunction with mhc i proteins, and sensitised cd + t cells, after entering the cerebrospinal fluid and encountering the infected cells, generate the inflammatory response and interference with normal neural function that cause the disease. the same cells destroy infected tissue cells in vitro, but tissue destruction is not a feature of the neurological disease. in this disease the cd + t cells probably act by liberating inflammatory cytokines. it may be noted that the brain is uniquely vulnerable to inflammation and oedema, as pointed out earlier in this chapter. the infected mouse shows the same type of lesions in scattered foci of infection in the liver and elsewhere, but they are not a cause of sickness or death. lcm infection of mice is a classical example of immunopathology in which death itself is entirely due to the cell-mediated immune response of the infected individual. this response, although apparently irrelevant and harmful, is nevertheless an 'attempt' to do the right thing. it has been shown that immune t cells effectively inhibit lcm viral growth in infected organs. however, a response that in most extraneural sites would be useful and appropriate turns out to be self-destructive when it takes place in the central nervous system. another type of t cell-mediated immune pathology is illustrated by influenza virus infection of the mouse. when inoculated intranasally, the virus infects the lungs and causes a fatal pneumonia in which the airspaces fill up with fluid and cells. the reaction is massive and the lungs almost double in weight. effectively the animal drowns. the cause is an influx of virus-specific cd + t cells. normally when an appropriate number of t cells had entered the lungs, the t cells would issue a feedback response to prevent such over-accumulation, but it is thought that influenza virus infects the t cells and inhibits this control process, so that the lungs are eventually overwhelmed. the action of the virus is subtle as it does not multiply in or kill the infected t cells, and it is presumed that it undergoes limited gene expression. one human virus infection in which a strong cmi contribution to pathology seems probable is measles. children with thymic aplasia show a general failure to develop t lymphocytes and cell-mediated immunity, but have normal antibody responses to most antigens. they suffer a fatal disease if they are infected with measles virus. instead of the limited extent of virus growth and disease seen in the respiratory tract in normal children, there is inexorable multiplication of virus in the lung, in spite of antibody formation, giving rise to giant cell pneumonia. this indicates that the cmi response is essential for the control of virus growth. in addition there is a total absence of the typical measles rash, and this further indicates that the cmi response is also essential for the production of the skin lesions. there is evidence that cell-mediated immune responses also make a contribution to the rashes in poxvirus infections. sometimes in infectious diseases there are prominent pathological changes which are not attributable to the direct action of microbes or their toxins, nor to inflammation or immunopathology. the stress changes mediated by adrenal cortical hormones come into this category. stress is a general term used to describe various noxious influences, and includes cold, heat, starvation, injury, psychological stress and infection. an infectious disease is an important stress, and corticosteroids are secreted in large amounts in severe infections (see also ch. ). they generally tend to inhibit the development of pathological changes, but also have pronounced effects on lymphoid tissues, causing thymic involution and lymphocyte destruction. these can be regarded as pathological changes caused by stress. it was the very small size of the thymus gland as seen in children dying with various diseases, especially infectious diseases, that for many years contributed to the neglect of this important organ, and delayed appreciation of its vital role in the development of the immune system. appreciation of the effects of stress on infectious diseases and the immune response in particular has led to the establishment of the science of neuroimmunology. properly controlled experiments are difficult to mount but there is a persuasive and growing body of evidence which shows that the nervous system affects the functioning of the immune system. the pathways of this communication are still poorly understood. work on mycobacterium bovis grew out of observations from the turn of the century that stress appears to increase the death rate in children with tb. in one type of experiment mice were stressed by being kept in a restraining device where movement was virtually impossible. this resulted in the reduction of expression of mhc class ii antigens on macrophages, which correlated with increased susceptibility to infection. similarly stressing mice infected with influenza virus caused several immunosuppressive events including reduction of inflammatory cells in the lung, and decreased production of interleukin- . suppression of antibody responses is found in people suffering a type of stress familiar to students-examinations! the best responses to hepatitis b vaccine in students immunised on the third day of their examinations were found in those who reported the least stress. finally, in a double blind trial at the common cold research unit in england with five different respiratory viruses, it was ascertained in human volunteers that stress gave a small but statistically significant increased likelihood of an individual developing clinical disease. pathological changes are sometimes caused in an even more indirect way as in the following example. yellow fever is a virus infection transmitted by mosquitoes and in its severest form is characterised by devastating liver lesions. there is massive mid-zonal liver necrosis following the extensive growth of virus in liver cells, resulting in the jaundice that gives the disease its name. destruction of the liver also leads to a decrease in the rate of formation of the blood coagulation factor, prothrombin, and infected human beings or monkeys show prolonged coagulation and bleeding times. haemorrhagic phenomena are therefore characteristic of severe yellow fever, including haemorrhage into the stomach and intestine. in the stomach the appearance of blood is altered by acid, and the vomiting of altered blood gave yellow fever another of its names, 'black vomit disease'. haemorrhagic phenomena in infectious diseases can be due to direct microbial damage to blood vessels, as in certain rickettsial infections (see p. ) or in the virus infection responsible for haemorrhagic disease of deer. they may also be due to immunological damage to vessels as in the arthus response or immune complex vasculitis, to any type of severe inflammation, and to the indirect mechanism illustrated above. finally there are a few infectious diseases in which platelets are depleted, sometimes as a result of their combination with immune complexes plus complement, giving thrombocytopenia and a haemorrhagic tendency (see also disseminated intravascular coagulation, p. ). thrombocytopenic purpura is occasionally seen in congenital rubella and in certain other severe generalised infections. infection during pregnancy can lead to foetal damage or death not just because the foetus is infected (pp. - ), but also because of infection and damage to the placenta. this is another type of indirect pathological action. placental damage may contribute to foetal death during rubella and cytomegalovirus infections in pregnant women. certain viruses undoubtedly cause tumours (leukaemia viruses, human papillomaviruses, several herpes viruses in animals) and this is to be regarded as a late pathological consequence of infection. as was discussed in ch. the tumour virus genome can be integrated into the host cell genome whether a tumour is produced or not, so that the virus becomes a part of the genetic constitution of the host. sometimes the host cell is transformed by the virus and converted into a tumour cell, the virus either introducing a transforming gene into the cell, activating expression of a pre-existing cellular gene, or inactivating the cell's own fail-safe tumour suppressor gene. the transforming genes of dna tumour viruses generally code for t antigens which are necessary for transformation, and the transforming genes of rna tumour viruses are known as one genes.* transformation has been extensively studied in vitro, and the features of the transformed cell described (changed surface and social activity, freedom from the usual growth restraints). simultaneous infection with two different microorganisms would be expected to occur at times, merely by chance, especially in children. on the other hand, a given infection generates antimicrobial responses such as as interferon production and macrophage activation which would make a second infection less likely. dual infections are commonest when local defences have been damaged by the first invader. the pathological results are made much more severe because there is a second infectious agent present. this can be considered as another mechanism of pathogenicity. classical instances involve the respiratory tract. the destruction of ciliated epithelium in the lung by viruses such as influenza or measles allows normally nonpathogenic resident bacteria of the nose and throat, such as the pneumococcus or haemophilus influenzae, to invade the lung and cause secondary pneumonia. if these bacteria enter the lung under normal circumstances, they are destroyed by alveolar macrophages or removed by the mucociliary escalator. in at least one instance the initial virus infection appears to act by interfering with the function of alveolar macrophages. mice infected with parainfluenza (sendai) virus show greatly increased susceptibility to infection with haemophilus influenzae, and this is largely due to the fact that alveolar macrophages infected with virus show a poor ability to phagocytose and kill the bacteria. specialised respiratory pathogens such as influenza, measles, parainfluenza or rhinoviruses damage the naso-* one genes (oncogenes) are also present in host cells, where they play a role in normal growth and differentiation, often coding for recognised growth factors (e.g. human platelet-derived growth factor). they can be activated and the cell transformed when tumour viruses with the necessary 'promoters' are brought into the cell. the one genes of the rna tumour viruses themselves originate from cellular oncogenes which were taken up into the genome of infecting viruses during their evolutionary history. pharyngeal mucosa and can lead in the same way to secondary bacterial infection, with nasal catarrh, sinusitis, otitis media or mastoiditis. the normal microbial flora of the mouth, nasopharynx or intestine are always ready to cause trouble if host resistance is lowered, but under normal circumstances they hinder rather than help other infecting microorganisms (see ch. ). one interesting example of exacerbation of infection occurs in mice dually infected with influenza virus and microorganisms such as streptococcus aureus or serratia marcescens. under these conditions animals suffer a more severe viral infection. this results from the need to proteolytically cleave the viral haemagglutinin protein which is done by a cellular enzyme. if the appropriate protease is in short supply or lacking completely, virions are formed but they are not infectious. under these circumstances the haemagglutinin can be cleaved extracellularly by microbial proteases with resulting increased amounts of infectious virus and disease. as a final example of dual infections, microorganisms that cause immunosuppression can activate certain pre-existing chronic infections. in measles, for instance, there is a temporary general depression of cmi; tuberculin-positive individuals become tuberculin negative, and in patients with tuberculosis the disease is exacerbated. in aids (see p. ) immunosuppression by hiv activates a variety of pre-existing persistent infections. diarrhoea deserves a separate section, since it is one of the commonest types of illness in developing countries and a major cause of death in childhood. particularly in infants, who have a very high turnover of water relative to their size, the loss of fluid and salt soon leads to life-threatening illness. it is estimated that on a global scale diarrhoea is responsible for - million deaths per year in children under five years old. in villages in west africa and guatemala the average - -year-old child has diarrhoea for about two months in each year.* diarrhoea also interacts with malnutrition and can cause stunted growth, defective immune responses and susceptibility to other infections (pp. - ). diarrhoea is also a common affliction of travellers from developed countries, * diarrhoea on a massive scale is not always confined to developing countries. there was a major outbreak of cryptosporidium infection in milwaukee, usa, in with more than cases; of these were diagnosed in the laboratory and they suffered watery diarrhoea (mean stools a day) for a mean of nine days. the small ( - μιη) oocysts, probably from cattle, had entered lake michigan, and then reached the community water supply because of inadequate filtration and coagulation treatment. and business deals, athletic successes and holiday pleasures can be forfeited on the toilet seats of foreign lands. the most reliable prophylaxis is to 'cook it, peel it, or forget it'. most attacks of diarrhoea are self-limiting. fluid and electrolyte replacement is a simple, highly effective, life-saving treatment that can be used without determining the cause of the diarrhoea. oral rehydration therapy (orf) means giving a suitable amount of salt and sugar in clean water and this is something that can be done by the mother. diarrhoea means the passage of liquid faeces,* or faeces that take the shape of the receptacle rather than have their own shape. this could arise because of increased rate of propulsion by intestinal muscles, giving less time for reabsorption of water in the large bowel, or because there was an increase in the amount of fluid held or produced in the intestine. in many types of infectious diarrhoea the exact mechanism is not known. diarrhoea, on the one hand, can be regarded as a microbial device for promoting the shedding and spreading of the infection in the community, or on the other hand as a host device to hasten expulsion of the infectious agent. diarrhoea is a superb mechanism for the dissemination of infected faeces (see p. ) and there is no doubt that strains of microbes are selected for their diarrhoea-producing powers. the advantages to the host of prompt expulsion of the infectious agent was illustrated when volunteers infected with shigella flexneri were given lomotil, a drug that inhibits peristalsis. they were more likely to develop fever and had more difficulty in eliminating the pathogen. in recent years significant strides have been made in our understanding of the pathophysiology of diarrhoeal disease. first, a quick resume of the normal structure and function of gut before attempting to understand the processes whereby it may be perturbed. the main function of the gut is the active inward transport of ions and nutrient solutes which is followed by the passive movement of water ( fig. . ). the driving force is the na + /k + atpase situated in the basolateral membrane of enterocytes on the villus (fig. . ) which maintains a low intracellular [na" "] thus creating the electrochemical gradient favourable for na + entry and, a high regional [na + ] in the intercellular spaces; cl~ follows na + . a similar situation exists in crypt cells: na + /k + atpase drives secretion. the key difference is the location of the carrier systems responsible for the facilitated entry of the actively transported species. in villus cells the carriers are present in the brush border, whereas in crypt cells they are located in the basal membrane: this is responsible for the vectorial aspects of ion/fluid traffic in villus/crypt assemblies. however, it is clear that several factors in addition to enterocytes are involved in * liquid faeces are not abnormal in all species. the domestic cow experiences life-long diarrhoea, but presumably does not suffer from it. (a) two methods of n a + cotransport are shown involving a glucose-linked symport and two coupled antiports; the latter results in the cotransport of cl~. the coupled antiports are functionally linked via h + and hc ~, the relative concentrations of which are a reflection of metabolic activity. these processes occur within the same cells but are shown separately for clarity. the driving force for n a + uptake is the low na + concentration maintained by the na + /k + pump (atpase) which creates the electrochemical gradient which promotes the inward movement of na + ; cl~ follows n a + by diffusion. water is drawn osmotically across the epithelium paracellularly (i.e. across tight junctions) and/or transcellularly, the former pathway accounting for approximately % of fluid movement. (b) secretion is the result of the coupled entry of n a + and cl~ across the basolateral membrane. n a + is recycled by the na + /k + pump and cl~ exits by diffusing down an electrochemical gradient and across the undifferentiated crypt cell apical membrane; n a + follows cl~ and water follows passively. if by whatever means, the level of nacl were to increase in such cells (as for example in rotavirus infection of neonatal mice, see below) then one could perceive how this could give rise to a cl~ driven hypersecretion of water. note, (i) the driving force results from the same mechanism that powers absorption i.e. the n a + / k + pump located in the basolateral membrane; it is the location of the 'port' 'diffusion' systems that determines the vectorial aspects of ion movement, (ii) the tight junctions are less tight in the crypts than villi. (iii) the apical membrane of the crypt cell is undifferentiated and only acquires micro villi during ascent into villous regions, φ : na + /k + pump; o: sym-, anti-port or diffusion channel. r e g u l a t i n g fluid t r a n s p o r t in t h e gut; t h e s e include t h e enteric n e r v o u s s y s t e m a n d t h e a n a t o m y of t h e microcirculation. t h e l a t t e r plays a profoundly i m p o r t a n t role in t h e u p t a k e of fluid. this is i l l u s t r a t e d in fig. . , which shows t h e existence of zones of g r a d e d osmotic fig. . schematic representation of a villus. note the central arterial vessel (av) which arborizes at the tip into a capillary bed drained by a subepithelial venous return (vr). movement of sodium into vr creates a concentration gradient between vr and av causing absorption of water from av and surrounding tissue. this results in a progressive increase in the osmolarity of incoming blood moving into the tip region through to vr. tip osmolarity is about three times higher than normal. this counter current system has been demonstrated in man and can be inferred in mice from the morphology of red blood cells which changes during ascent of the same vessel from base to tip regions of villi. the shaded areas indicate a vertical increase in osmolarity. left crypt: represents normal physiological secretion. right crypt: represents hyper secretion. ens, enteric nervous system, depicted schematically and not anatomically. potential. at the tips of villi in adult human gut, osmolalities range from to mosmkg _ h , which would generate huge osmotic forces. thus, current perceptions are that enterocytes are responsible for generating this gradient and the blood supply acts as a countercurrent multiplier which amplifies the gradient in a manner analogous to the loops of henle in the kidney. the hypertonic zone has been demonstrated directly in whole villi of infant mice in terms of the changing morphology of erythrocytes: in the lower regions of villi they show characteristic discoid morphology, whereas in the upper region they are crenated, indicating a hyperosmotic environment. the hypertonicity is dissipated if the blood flow is too slow and washed out if too fast. it is the villus unit rather than enterocytes by themselves which is responsible for fluid uptake. another consequence of the microcirculatory anatomy is that villus tip regions are relatively hypoxic. in addition, neonatal brush borders contain disaccharidases (principally lactase) which break down nonabsorbable disaccharides (e.g. lactose) into constituent absorbable monosaccharides. villus tips and crypts are regarded as the anatomical sites of physiological absorption and secretion respectively. fluid transport is a bidirectional process in the healthy animal with net absorption in health and net secretion in disease. the balance between absorption and secretion is poised at different points throughout the intestinal tract reflecting differences in both structure and function. proximal small intestine is relatively leaky; in contrast the colon is a powerfully absorptive organ. finally, crypts are the principal sites of cell regeneration, replacing cells which migrate up the epithelial escalator. the epithelium is renewed in approximately - days. at villus tips senescent cells are shed. diarrhoeal disease can result from interference with almost any one, or combination of these systems. diarrhoea-producing microbes are listed in table . . some examples are considered in detail below. rotaviruses are known to invade intestinal epithelial cells and cause diarrhoea in man, foals, dogs, pigs, mice etc. extensive multiplication takes place and very large amounts of virus ( particles g _ ) are shed in faeces. the conventional wisdom is that tips of villi especially are affected, leading to reduced absorption of fluid from the lumen. in addition destruction of enterocytes leads to a loss in lactase resulting in an accumulation of lactose in the gut causing an osmotic flux of fluid into the intestine. a major study of rota virus-induced diarrhoea in neonatal mice provides a different model of this important disease of children. oral infection of baby mice with a murine strain of rotavirus resulted in virus replication in enterocytes of the small intestine. before there had been detectable virus replication, villi became ischaemic, enterocytes severely damaged and villi shortened. in gut enterocytes this virus was demonstrably not cytopathic; the cell damage and villus responses were almost identical to that seen in experimentally induced ischaemia in physiological experiments. at the peak of virus replication, which was coincident with maximum shortening of villi, the gut was still absorptive, glucose transport intact and sufficient lactase activity remained to deal with the normal lactose load delivered to the stomach. after this the gut became secretory and diarrhoea clinically evident. villi were rapidly regenerated, but hyperaemia and diarrhoea persisted until the regeneration of the hypertonic zones as judged by the morphology of the red blood cells. peak diarrhoea coincided with the resynthesis of new villi (as judged by thymidine kinase levels, increased mitotic activity and morphometric analyses), and increased levels of intracellular nacl in the zone of cells where cell division was most active. transiently high levels of nacl have also been observed in dividing cells in culture. since ischaemic and hyperaemic villi were occasionally seen in control villi it may well be the case that the pathological changes reflect an exaggerated synchronised response of basic circulatory control mechanisms, which are part of the normal homeostatic mechanisms of villus physiology. thus, the pathophysiology of rotavirus-induced diarrhoea in neonatal mice may be summarised as follows: the reduction of red blood cells flowing through villi in the early stages of infection instigates hypoxia and hence atrophy of villi. the degree of atrophy in this infection is not associated with lack of absorption. the ensuing villus repair processes induce hypersecretion. the increase in blood flow throughout the remaining course of the infection reduces the hypertonicity of villi which impairs water absorption and thus prolongs diarrhoea. the preceding description of the self-limiting diarrhoea induced by rotavirus in neonatal mice is probably applicable to many diarrhoeas. however, the observed pathology may be different according to age, species, or the inducing pathogen. for example, in rotavirus-infected lambs, villus atrophy and crypt hypertrophy occur (the latter indicative of crypt cell division) but as in mice, infected lambs are not lactose intolerant. in rotavirus-infected piglets, crypt hypertrophy occurs but villus atrophy is severe, the animals are lactose intolerant and mortality high; a similar situation exists for the coronavirus, transmissible gastroenteritis (tge) virus. the latter has often been used as the model for infantile diarrhoea but the question is whether human infants are more like piglets or lambs. clinical studies have shown that recovery from mild, acute gastroenteritis of rota virus origin occurs within two weeks irrespective of the carbohydrate ingested. clearly, the severity of disease and the clinical outcome will depend on the extent of vertical' villus/ crypt involvement and the regions of intestine infected. when villus erosion is severe, then lactose may cause an 'osmotic' purge or be fermented by intestinal bacteria to short chain fatty acids which stimulate secretion in the colon. astroviruses, norwalk virus, certain coronaviruses, certain adenoviruses and probably toroviruses all cause gastroenteritic disease by infecting enterocytes. however, parvoviruses cause severe intestinal disease in dogs by virtue of their predilection for the mitotically active crypt cells; this causes the near complete erosion of villi similar to that seen after exposure to sublethal doses of irradiation. the classic paradigm for bacterial watery diarrhoea is cholera caused by vibrio cholerae in the small intestine. v. cholerae attaches to enterocytes of the proximal small bowel and is capable of producing at least three toxins: classical cholera toxin (ct); a toxin which disrupts the zonula occludens tight junction (designated zot); and another less well defined, auxilliary cholera enterotoxin (designated ace). as a result of toxic action, water and electrolytes are lost through the intact epithelial cells into the small intestine. as the multiplying bacteria increase in numbers and more and more epithelial cells are affected, the absorptive capacity of the colon is overwhelmed and there is profuse watery diarrhoea, as much as l l h - in severe cases.* the massive loss of isotonic fluid with excess of sodium bicarbonate and potassium leads to hypovolaemic shock, acidosis and haemoconcentration. anuria develops, and the collapsed, lethargic patient may die in - h. lives are saved by replacing the lost water and salts. the effect of toxin on an intestinal epithelial cell is long lasting, but the patient recovers as affected cells are shed and replaced in the normal fashion. the infection is particularly severe in children who easily develop low levels of plasma potassium. however, on a global scale this greatly feared disease, cholera, is only responsible for less than % of the total deaths due to diarrhoea. administration of as little as μg of purified ct alone to a healthy volunteer will reproduce the hugely dramatic, potentially lethal, purge of near isotonic fluid emphasising the central importance of ct in the causation of disease. the effect of zot has been demonstrated by electron microscopy in human biopsies, but the relative importance of zot and ace in human disease has not been quantified. how does ct work? as already outlined (pp. - ) the a subunit of this toxin is an adpribosyl transferase whose target is the a s subunit of the regulatory g protein which governs the expression of adenylate cyclase and hence the production of camp. this in turn results in the perturbation of the transport systems leading to a net efflux of ions and hence water. things are slightly more complicated, because elevation of camp occurs in villus tip cells (hence affecting absorption) but not in crypt cells. probably a signal is generated in intoxicated tip enterocytes which is transmitted to the crypt region by the ens causing release of neurotransmitters (e.g. serotonin, acetyl choline, and vasoactive peptide) which act on enterochromaffin (secretory) cells and on intestinal smooth muscle thereby augmenting crypt secretion and increasing peristalsis. studies have been made of sequential histological changes in human biopsies, taken from a series of patients from the onset of symptoms to recovery. it was clear that fluid secretion was not the result of massive desquamation of the epithelium. histological changes included 'engorgement of the capillaries' (i.e. interference with the blood supply), 'dilation of lacteals, vacuolation of enterocytes, exhaustion atrophy of enterocytes' (remember this is a noninvasive pathogen), 'accelerated shedding of cells, and increased mitotic activity'. most of these changes are similar to those described above for experimental murine rotavirus at post-peak diarrhoea and it seems that the range of histological reactions that can occur in the small intestine are limited so that qualitatively the picture is similar in different diarrhoeas. despite the undoubted importance of ct in the causation of the disease, and the potent antigenicity of ct, it is now recognised that protective immunity is very largely antibacterial. it is stopping effective colonisation which is important rather than neutralisation of the toxin. this has been partially achieved by using killed whole cell vaccines. several attempts have been made in the laboratory to genetically manipulate virulent strains (in practice this means deleting or inactivating the known toxin genes) such that the attenuated strain will colonise the gut and stimulate local immune responses and thereby prevent colonisation of the gut by virulent strains. to date, attenuated strains have been developed which fulfil these criteria but induce a mild transient diarrhoea which has prevented their adoption into vaccination programmes. however the recent emergence of v. cholerae strain (a new, third type) in india and bangladesh is a reminder that today's solution may not be adequate for tomorrow's problems. current vaccines are ineffective against this new strain. the picture withi£. coli is complicated since there are several biotypes of this pathogen which include: etec (enterotoxigenic e. coli, the principal cause of travellers' diarrhoea), epec (enteropathogenic e. coli), eiec (enteroinvasive e. coli), and ehec (enterohaemorrhagic e. coli) variants (see ch. ). the situation with etec is very similar to v. cholerae as far as heat labile toxin (lt, which is very closely related to ct) is concerned. it is additionally complicated by the fact that some strains of etec also make heat stable toxins (sts), which are nonantigenic low molecular weight peptides which activate membrane-bound guanylate cyclase which causes the production of cgmp which is the functional equivalent of camp. the 'classical' epec lesion is the formation of a characteristic pedestal-like lesion in the brush border of enterocytes with only limited invasion of the mucosa. just how it causes diarrhoea is not wholly clear. eiec is almost indistinguishable from shigella, and causes similar disease. ehec strains, which belong mainly (but not exclusively) to serogroup :h , cause a spectrum of intestinal disease ranging from asymptomatic carriage through mild diarrhoea to severe haemorrhagic colitis. they also cause extraintestinal infections such as haemolytic uraemic syndrome (hus). the initial lesion seen in the gut is similar to that caused by epec but ehec do not remain localised there: they multiply in the lamina propria and glandular crypts. while there is no conclusive proof of the involvement of slt in the pathogenesis of ehec diarrhoea there is a correlation between slt production and the severity of diarrhoeal disease or hus caused by this organism. salmonella spp. cause acute gastroenteritis and systemic typhoid disease in man, as well as other important infections in domestic animals. virulence in salmonella, especially in their natural hosts (see table . , footnote) isxomplex and mediated by numerous genes, some of which are present in otherwise nonpathogenic organisms such as e. coli. presumably all these bacteria share nutritional and environmental problems, whether inside or outside a host, that call for such genes. those restricted to salmonella are more likely to be critical in vivo. the clinical features of salmonella-induced diarrhoea (gastroenteritis) in humans and systemic typhoid infections are quite different. for example, gastroenteritis may follow - h after ingestion of contaminated food, whereas typhoid follows an incubation period of - days. diarrhoea, (which is usually watery, but may be severe, and sometimes bloody) is the predominating feature of gastroenteritis, whereas in adults, constipation is seen in the early clinical stages of typhoid; diarrhoea may occur much later. although fever may occur in gastroenteritis, in typhoid this may be so severe as to cause delirium. current perceptions are that gastroenteritis results from the initial interactions of salmonella (any one of many serotypes but frequently typhimurium or enteriditis, and/or their products) with the gut mucosa, whereas typhoid fever is produced by s. typhi organisms which translocate the mucosa, survive within macrophages, multiply and release endotoxin which triggers the highly complex endotoxin cascade. gastroenteritis is usually self-limiting, whereas in untreated typhoid mortality can be as high as %. the pathophysiology of fluid secretion caused by salmonellae is highly complex and as yet incompletely understood. the only two biological parameters which have been shown to correlate with induction of fluid secretion in a rabbit model (the best available small laboratory animal for human gastroenteritis) is the ability to invade the gut mucosa, and the induction of a leucocyte (mainly polymorphonuclear cell) influx into the mucosa and lumen of the gut. fluid secretion does not correlate per se with the ability to make an enterotoxin: avirulent as well as virulent strains make a cholera-like toxin though neither appear to release the toxin readily. the latter is therefore either not primarily involved, or is involved only after release from bacteria by means other than normal protein secretion mechanisms. these questions are as yet unresolved and the reader is referred to the reference list for a fuller discussion of these complex matters. currently, the major diarrhoeagenic pathogen of the small intestine in the developed world is campylobacter jejuni. the latter are bacteria present in wild birds, chickens and in the faeces of up to % of healthy cows in the uk. milk becomes contaminated and those who drink raw (unpasteurised) infected milk develop diarrhoea, sometimes with dysentery (blood and pus in the stools) and fever. the incubation period is - days and diarrhoea occurs after bacterial replication in the upper small intestine (jejunum). however, we are only at the beginning of understanding the detailed mechanisms and determinants responsible for disease causation by this pathogen. giardia lamblia is a noninvasive protozoan pathogen of the human small bowel which causes a spectrum of infection ranging from asymptomatic carriage through acute watery diarrhoea to chronic diarrhoea and malabsorption. giardia has a simple life cycle, existing in two forms: the multiplying trophozoite which infects mammalian hosts to cause disease and the environmentally resistant cyst. after ingestion, excystation is triggered by the ph in the stomach, and the trophozoite colonises the small bowel; the putative adherence factors have been described in ch. . the precise mechanisms involved in subsequent stages responsible for diarrhoea, malabsorption and cystation are less well understood. entamoeba histolytica causes lysis of target cells apparently by direct contact with the cell membrane. this pathogen produces under in vitro conditions a spectacular array of potential (but as yet unproven) virulence determinants including: proteases that round up cells, poreforming proteins, collagenases and oligosaccharidases and neurotransmitter-like compounds; the latter can induce intestinal fluid secretion. some of these factors have been implicated as the determinants responsible for liver abscess formation. not all diarrhoeal disease is the result of small bowel dysfunction. two important pathogens of the colon will be considered-clostridium difficile and shigella dysenteriae. c. difficile causes a spectrum of disease varied hepatitis a there are more than serotypes of salmonella, distinct from salmonella typhi and salmonella paratyphi. they are primarily parasites of animals, ranging from pythons to elephants, and their importance for man is their great tendency to colonise domestic animals. pigs and poultry are commonly affected, and human disease follows the consumption of contaminated meat or eggs. b other campylobacters cause sepsis, abortion and enteritis in animals. ranging from asymptomatic carriage through mild antibiotic-associated diarrhoea (aad) to fatal pseudomembranous colitis (pmc). normal flora play a major part-by means which are still not completely understood-in suppressing outgrowth of resident c. difficile spores. disruption of the normal flora, by antibiotic treatment for example, results in vegetative growth and production of several toxins of which c. difficile toxin a seems to be the most important in disease. this toxin is responsible for the secretory and inflammatory response in the intestine, and epithelial disruption. it probably acts by disrupting tight junctions between cells, and by causing chemotactic and other responses in polymorphs. diarrhoea could be due to the destruction of the 'absorptive epithelium' and failure to cope with the normal fluid load delivered by the small intestine, or an active type of secretion caused by replacement of the damaged epithelium, or both. the second example is that of dysentery caused by shigellae spp., in particular s. dysenteriae and s. flexneri. we have already dealt with the capacity of shigellae to invade (ch. ). invasiveness is a vital part of the virulence armoury of shigellae spp.; non-invasive mutants are a virulent. the pathogen invades the gut via m cells in peyer's patches and thence adjacent microvillus-bearing colonocytes. what is the role played by shiga toxin? mutants of s. dysenteriae devoid of the ability to make shiga toxin still retained the ability to cause lethal fulminant dysentery (low volume fluid production, pus cells, mucus) in macaque monkeys, and they invaded, multiplied within and rapidly killed hela cells. the major difference between the mutants and the toxin-positive wild type was that the latter caused a more severe disease with more haemorrhage, giving rise to blood in the stools, and, in some instances greater destruction of mucosal tissues. evidently the production of shiga toxin is not a sine qua non for causing bacillary dysentery: it exacerbates the disease. although much research has been focused on toxins, their mode of action, and their role in disease, it is useful to compare different types of intestinal infection and to refer to the concept of food poisoning. types of intestinal infection are set out in table . . food poisoning is a loosely used term, and usually refers to illnesses caused by preformed toxins in food, or sometimes to illnesses that come on within a day or so after eating contaminated food. food may be contaminated with plant poisons, fungal poisons (e.g. poisoning due to amanita phalloides), fish poisons,* heavy metals, as well as with bacterial toxins or bacteria. sourcebook of bacterial toxins the virology and immunology of lymphocytic choriomeningitis virus infection * ingestion of scombroid fish (mackerel etc.) containing large amounts of histamine or similar substances leads to headache, flushing, nausea and vomiting within an hour response of man to infection with vibrio cholerae. . clinical, serologic and bacteriologic responses to a known inoculum epitopes of streptococcal m proteins shared with cardiac myosin bacterial infections of respiratory and gastrointestinal mucosae pathogenesis and immunology of treponema pallidum t-lymphocyte stimulation by microbial antigens lessons from diarrhoeal diseases, demography to molecular pharmacology clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use disseminated intravascular coagulation: a review campylobacterpyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium typhoid fever: pathogenesis and immunologie control comparison of the alpha-toxin genes of clostridium perfringens type a and c strains: evidence for extragenic regulation of transcription pathogenic mechanism ofneisseriagonorrhoeae: observations on damage to human fallopian tubes in organ cultures by gonococci of colony type i or type rift valley fever virus in mice vi: histological changes in the liver in relation to virus multiplication viral aetiology of diseases of obscure origin campylobacter jejuni. current status and future trends cytolytic pore-forming proteins and peptides: is there a common structural motif? treatment of spasmodic torticollis with local injections of botulinum toxin pathogenesis of diseases caused by entamoeba histolytica: studies of adherence, secreted toxins and contactdependent cytolysis pituitary dwarfism in mice persistently infected with lymphocytic choriomeningitis virus tetanus and botulinum-b neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc experimental salmonella typhimurium induced gastroenteritis bacterial phospholipases c the development of respiratory syncytial virus-specific ige and the release of histamine in naso-pharyngeal secretions after infection immune complexes in human diseases key: cord- -o mh wz authors: olano, juan p.; peters, c.j.; walker, david h. title: distinguishing tropical infectious diseases from bioterrorism date: - - journal: tropical infectious diseases doi: . /b - - - - . - sha: doc_id: cord_uid: o mh wz nan bioterrorism can be defined as the intentional use of infectious agents or microbial toxins with the purpose of causing illness and death leading to fear in human populations. the dissemination of infectious agents with the purpose of attacking livestock and agricultural resources has similar motives. many of the agents that could potentially be used in bioterror (bt) attacks are also responsible for naturally occurring infectious diseases in the tropics. as such, naturally occurring outbreaks must be differentiated from bt attacks for public health, forensic, and security reasons. if a bt attack occurs in tropical underdeveloped countries, owing to their weak public health infrastructure, the public health implications would be even more dramatic than in developed countries. an outbreak of smallpox due to a bt attack would probably require vaccination and mandatory quarantine of millions of people in order to control the outbreak and quell global public unrest. this chapter will concentrate on selected infectious agents that have the potential to be used as bioterror agents in human populations. the first step in managing the damage from a covert biological dissemination is recognition of the attack and the organism(s). as in most emerging infections, we predict that in bioterrorist attacks the etiological diagnosis will be made by a clinician or pathologist and the recognition of a bioterrorist event will be through geographical and epidemiological anomalies. we have very limited environmental detection capability at this time, and there are no comprehensive pointof-care diagnostics for most of the high-impact bt agents. some diseases such as inhalational anthrax or smallpox may be relatively readily recognized by an alert clinician because of their very distinctive presentation in many cases. however, the leading edge of a bt epidemic may arrive on a pathologist' s doorstep without prior suspicion. for example, individual cases of pneumonic plague as the earliest harbingers of an attack will presumably present as community-acquired pneumonia and probably die without clinical diagnosis. given the short window available for successful treatment, the recognition of these earliest cases is paramount. sartwell has demonstrated empirically that incubation periods follow a log-normal distribution, which results in "front-loading" of cases ( fig. - ). delay in recognizing the epidemic through reliance on syndromic surveillance or other surrogates will likely result in most of the cases of diseases such as plague and tularemia being well into their disease course and perhaps unsalvageable. bioterrorist events will enlarge our knowledge of tropical diseases. for example, inhalational anthrax and several viral hemorrhagic fevers (vhf) thought to be transmitted mainly by aerosol are under-represented in naturally occurring case series, and a bt attack would provide an opportunity to answer questions about the underlying host factors and pathogenesis. indeed, the extension of the risk population to include children, the elderly, and the immunosuppressed is likely to provide considerable insight into these oftenunderstudied groups. it is also likely that our lack of information about them will challenge our current diagnostic algorithms. in october , anthrax spores were distributed covertly in the u.s. postal service, leading to cases of human anthrax and billions of dollars spent on controlling the potentially devastating effects of a small inhalational anthrax epidemic. , this attack was by no means the first intentional attempt to use infectious agents as weapons of terror. ever since the times of the ancient greeks and romans, humans have tried to inflict damage by the use of contagion on other populations. , less than % of the people or groups responsible for terrorist attacks on human populations take responsibility for their actions. therefore, the use of biological weapons is ideal to conduct covert attacks. in addition, it has been estimated that to kill the same number of human beings with biological weapons as compared to chemical or nuclear weapons, the cost is far less with biological weapons ($ /human casualty) compared with chemical ($ / human casualty) and nuclear ($ , , /human casualty) weapons. hypothetical bt attacks would range from an overt attack of a large city with a bomb containing several kilograms of an agent (weaponized bacteria, viruses, or toxins) to discrete or covert intentional release of the infectious agent through a delivery system, such as spray devices, postal service, ventilation ducts, water supplies, and food supply. based on transmissibility, severity of morbidity and mortality, and likelihood of use (availability, stability, weaponization), potential bt agents are divided into three categories (table - ) . this chapter will concentrate on selected agents from categories a and b and on the diagnostic challenges posed by illnesses caused by such agents. table - are capable of producing illness under natural circumstances. therefore, the first challenge is to identify the infectious agent responsible for a certain disease correctly, followed by a thorough epidemiologic and microbiologic analysis of the epidemic or outbreak. in some circumstances, the identification of a bt attack would be obvious. a case of smallpox in any human population is an international emergency that would trigger a massive response of the public health systems around the world. sophisticated epidemiological investigations would follow in order to characterize the outbreak, identify the source, and possibly label it "intentional." in other cases, the identification of the outbreak as secondary to intentional dissemination of an infectious agent will require the use of sophisticated epidemiological and molecular tools, especially for diseases endemic to the area where the outbreak occurs. the need to use genetic sequences as markers has spawned a new discipline referred to as microbial forensics, sister to phylogenetics and "molecular epidemiology." differentiation between natural infections and a biological warfare attack rests firstly on disease patterns given by several epidemiological clues. they include presence of disease outbreaks of the same illness in noncontiguous areas, disease outbreaks with zoonotic impact, different attack rates in different environments (indoor versus outdoor), presence of large epidemics in small populations, increased number of unexplained deaths, unusually high severity of a disease for a particular pathogen, unusual clinical manifestations owing to route of transmission for a given pathogen, presence of a disease (vector-borne or not) in an area not endemic for that particular disease, multiple epidemics with different diseases in the same population, a case of a disease by an uncommon agent (smallpox, viral hemorrhagic fevers, inhalational anthrax), unusual strains of microorganisms when compared to conventional strains circulating in the same affected areas, and genetically homogenous organisms isolated from different locations. , these are a few guidelines that could prove helpful when investigating an outbreak, but it has to be kept in mind that the deduction will not be based on any single finding but rather the pattern seen in its totality. first and foremost, the possibility of an attack must be ever in mind, or differentiation of a covert bt attack and a natural outbreak of an infectious disease may not be made. in fact, the outbreak of salmonellosis in oregon in was due to a covert attack planned by the rajneeshee leadership and accompanied by distinctive epidemiological clues. it was not labeled as intentional until somebody came forward with the information leading to the responsible group; as in most of medicine, the unsuspected diagnosis is the easiest to miss. an increasing number of public health departments are now acquiring the technology necessary to perform syndromic surveillance. this new method of surveillance is based on syndromic disease rates such as respiratory, gastrointestinal, and neurological syndromes or analysis of other health-related activities such as laboratory test requests and results, purchasing rates for certain pharmaceutical agents, unexplained death rates, and veterinary surveillance. , , the purpose of syndromic surveillance is to detect a bt attack as early as possible by analyzing the previously mentioned variables by extracting and analyzing data through computer networks. the rationale behind syndromic surveillance is the nonspecific nature of early signs and symptoms of many of the illnesses caused by bt agents. examples of proposed syndromes are as follows: gastroenteritis of any apparent infectious etiology, pneumonia with the sudden death of a previously healthy adult, widened mediastinum in a febrile patient, acute neurologic illness with fever, and advancing cranial nerve impairment with weakness. a key component factors affecting syndromic surveillance include selection of data sources, definition of syndrome categories, selection of statistical detection thresholds, availability of resources for follow-up, recent experiences with false alarms, and criteria for initiating investigations. it must be emphasized that these systems are experimental and not yet of proven value in managing bt attacks. they are expensive, require follow-up confirmation, have unproven sensitivity and specificity, and ultimately depend on the clinician. they may prove to be more useful in managing an event than in expeditiously detecting one. conventional epidemiological investigations are by no means obsolete with the availability of more sophisticated methods to study possible bt attacks. they include the confirmation of an outbreak once it is suspected. confirmation is based in many cases on laboratory analysis of patients' samples or autopsy material. a case definition is constructed to increase objectivity of the data analyzed and to enable determination of the attack rate. other variables are included in the analysis, such as time and place, and an epidemiological curve can be constructed. epidemiological curves are an important tool to analyze epidemics and suggest the mode of transmission and propagation. a point source epidemic curve is classically log-normal in distribution and would suggest a common exposure of a population to an infectious agent. of course, there can be variations depending on the presence of susceptible subpopulations (e.g., children, immunosuppressed, aged) and on varying doses of the agent. propagative curves are more characteristic of highly communicable agents such as smallpox. a short description of selected category a and b agents follows. all these pathogens are addressed as naturally occurring disease agents in other chapters of this book. bacillus anthracis (anthrax) b. anthracis (see chapter ) is without a doubt the microorganism that has received the most attention as a bt agent due to its high lethality (inhalational form), ease of propagation, and high environmental stability. fortunately, the disease is not transmitted from person to person. however, the first three characteristics make it one of the ideal bioweapons. anthrax presents in humans as four different clinical syndromes, depending on the portal of entry: cutaneous (the most common form of the disease resulting from contact with infectious animal products), gastrointestinal and oral/oropharyngeal (both secondary to ingestion of contaminated meat), and inhalational (woolsorter' s disease), secondary to inhalation of spores from the environment. in the event of a bioterror attack, either overt or covert, the clinical presentation of the patients affected by the attack would be that of inhalational anthrax. this form of anthrax is so rare that a single case of inhalational anthrax should raise immediate suspicion, as dramatically demonstrated during the bt attacks in the fall of . [ ] [ ] [ ] during those attacks, % of cases were cutaneous anthrax thought to be secondary to handling of anthraxlaced mail envelopes or environmental surface contamination in the presence of minor cutaneous lesions, providing a portal of entry for the spores. an outbreak of inhalational anthrax also took place in sverdlovsk (former soviet union) as a result of an accidental release into the air of b. anthracis spores from a facility producing anthrax for the bioweapons program in the ussr. , [ ] [ ] [ ] inhalational anthrax should be suspected clinically in any individual presenting with fever and a widened mediastinum on chest radiograph (due to hemorrhagic mediastinitis). , the incubation period is normally to days, but in some cases it can be as short as days and as long as days depending on inoculum and the time of germination of the spore. based on research performed on rhesus monkeys, the ld is estimated to be to , spores. [ ] [ ] [ ] however, as few as to spores may be capable of producing a fatal outcome in approximately % of those exposed to these quantities. the initial symptoms are nonspecific and consist of fever, malaise, anorexia, fatigue, and dry cough. these symptoms are followed in to days by an abrupt onset of respiratory insufficiency, stridor, diaphoresis, and cyanosis. the subsequent clinical course is rapid, and patients usually die within to hours after clinical deterioration. mortality is % without antibiotic therapy. , [ ] [ ] [ ] early diagnosis, aggressive treatment with antimicrobial agents to which the bacteria are susceptible, and aggressive supportive therapy decreased the mortality to % in the attacks. pathologic studies performed on the sverdlovsk victims confirmed some of the findings in animal models of inhalational anthrax, such as hemorrhagic lymphadenitis and mediastinitis. however, many patients also developed hematogenous hemorrhagic pneumonia. pleural effusions were usually large and frequently led to severe lung atelectasis. in about half of cases, hemorrhagic meningitis developed, leading rapidly to central nervous system (cns) manifestations terminating in coma and death. , , yersinia pestis (plague) y. pestis (see chapter ) is a gram-negative, aerobic, nonsporulating coccobacillus, member of the enterobacteriaceae with a wide host range, including rodents, felines, and humans. the most important reservoirs are urban rats, and its main vector is the rat flea. in rural epizootics, reservoirs include prairie dogs and squirrels in the united states. y. pestis has been responsible for some of the most devastating pandemics in human history in the preantibiotic era ( th, th, and th centuries). public health measures have made this disease a rarity in the united states (around cases/year) and around the world, although approximately cases are reported to the world health organization (who) every year (countries reporting plague include madagascar, tanzania, and peru, among others). clinical presentation in naturally acquired infections takes five forms, namely bubonic, septicemic, pneumonic, cutaneous, and meningeal. the pneumonic form is the most likely presentation in a case of plague due to a bt attack. it is worth mentioning that plague has already been used as a bt agent when japan dropped thousands of y. pestis-infected fleas over china leading to small outbreaks of bubonic plague in continental china during world war ii. , the incubation period for pneumonic plague is short, ranging from to days. it is the rarest form in natural infections ( % or less) but has the highest mortality, reaching % in untreated patients. the initial presentation is nonspecific and consists of cough, fever, and dyspnea. cough may be productive (bloody, purulent, or watery in the initial phases). this is followed by a rapid clinical course leading to respiratory failure and the patient' s demise if not treated with antibiotics early in the course of the disease. , , the factors that led to the severe manchurian pneumonic plague outbreaks in the early th century are unknown, but weather, hygiene, and crowding were important factors. more recent outbreaks worldwide and particularly in the united states have been much smaller and readily controlled. pneumonic cases are common in the united states, but secondary transmission has been rare in the last years. modeling of pneumonic transmission using eight small outbreaks to derive the parameters find average of secondary cases per primary case (ro) to be approximately . prior to any control measures. this is one of the most scientifically neglected microorganisms with bt potential. tularemia is a zoonotic infection caused by a strictly aerobic, gram-negative, nonsporulating small coccobacillus. two subspecies are recognized, namely f. tularensis subspecies holarctica (jellison type b) and f. tularensis subspecies tularensis (jellison type a). type a is by far the more virulent and is present only in north america. of the bacteria with potential as bt agents, f. tularensis has by far the widest host range, including wild and domestic animals, humans, fish, reptiles, and birds. vectors are also numerous and include ticks, fleas, mosquitoes, and biting flies. , this is an impressive range for any human pathogen. in contrast to other diseases described in this chapter, tularemia does not have the remarkable history that some of the other pathogens have. in europe, tularemia was first described in ; in the united states, it was first described in in california in the aftermath of the san francisco earthquake. in natural infections, the most common source of infection is a tick bite and manipulation of infected animals such as wild rabbits. six different clinical syndromes have been described as follows: ulceroglandular, glandular, oculoglandular, pharyngeal, pneumonic, and typhoidal. marked overlap exists among all these forms, and for practical purposes two syndromes (ulceroglandular and typhoidal) have been proposed. [ ] [ ] [ ] as a bt agent, f. tularensis will most likely cause a disease with a primary pulmonary component with secondary dissemination (typhoidal/systemic). in natural infections, both ulceroglandular and typhoidal forms can have a hematogenous pulmonary component, although it is more common in typhoidal forms. pulmonary features include cough, pleural effusions, and multifocal bronchopneumonic infiltrates. if not treated promptly, patients usually develop adult respiratory distress syndrome leading to respiratory insufficiency and the patient' s demise. case-fatality rate approaches % if not treated with appropriate antibiotics. smallpox eradication remains the single most important victory in the war against infectious diseases. smallpox (see chapter ) is the only disease so far eradicated from the face of the earth due to human intervention. the who declared smallpox eradicated in after the last case of natural disease was diagnosed in somalia in , and vaccination ceased around the world, rendering humankind vulnerable to reintroduction of the virus. [ ] [ ] [ ] a laboratory accident was responsible for two more cases in in england. this accident prompted the who to restrict the frozen virus to two places in the world: the cdc in atlanta, georgia, and the institute for polyomyelitis and viral encephalitides in moscow, later moved to npo vector, novosibirisk, russia. however, it is suspected that secret military repositories exist after the fragmentation of the soviet union and the subsequent exodus of scientists involved in its bioweapons program (biopreparat). , the agent responsible for this disease is an orthopox virus with no known animal reservoir, but high aerosol infectivity, stability, and mortality. although not a category a agent, monkeypox is responsible for outbreaks in africa and is the only other member of the orthopox genus capable of producing systemic disease in humans. the clinical disease is potentially indistinguishable from smallpox, where mortality rates in tropical africa are around % to %. in may and june , an outbreak of monkeypox occurred in the united states. thirty-seven infections were laboratory-documented and involved humans exposed to infected prairie dogs that had become infected because of contact with infected gambian rats and dormice, two animal species shipped from africa earlier that year. infected humans included veterinarians, exotic pet dealers, and pet owners. the clinical spectrum in this outbreak ranged from asymptomatic seroconversions to febrile illness with papulovesicular rash. no deaths were associated with this outbreak. however, phylogenetic analysis of the virus placed it in the west africa clade as opposed to the central africa clade which carries the previously mentioned case-fatality rate of % to %. a single case of smallpox would trigger a massive public health response in order to contain the outbreak. an outbreak in germany in resulted in cases with , people vaccinated to contain the infection. in , yugoslavia underwent an epidemic with a total of cases ( deaths) and a vaccination program that included million people in order to contain the outbreak and obtain international confidence. vaccination with the vaccinia virus (a related orthopox virus) is the most effective way to prevent the disease and can be administered up to days after contact with ill patients. strict quarantine with respiratory isolation for days is also mandatory. the newer generation of antivirals that have been developed after the disease was eradicated has never been tested in human populations, but in vitro data and experiments in animal models of poxvirus disease suggest some antiviral activity for the acyclic nucleoside phosphonates such as cidofovir. the only vaccine available in the united states is dryvax, and sufficient doses have been manufactured to cover the entire u.s. population. however, newer vaccines that may have fewer side effects are being developed. the clinical presentation is characteristic. the incubation period ranges from to days. the initial phase is nonspecific, common to other viral syndromes, and is characterized by abrupt onset of fever, fatigue, malaise, and headaches. during this prodromal phase in % of patients with fair complexion, a discrete erythematous rash appears on the face, forearms, and hands. the typical smallpox rash has a centrifugal distribution (that is, more abundant on the face and extremities than on the trunk and abdomen). an enanthem also develops with presence of oral ulcerations by the time the exanthem appears. systemic manisfestations begin to subside once the rash appears and can reappear with superinfection of skin lesions or superimposed bacterial bronchopneumonia. progression of the lesions is synchronous (maculopapules, vesicles, pustules). after pustules rupture, scabs form and detach in to weeks, leaving depigmented, scarred areas. this form of the disease, called variola major, is fatal in up to % of unvaccinated patients and % of vaccinated individuals. various hemorrhagic forms exist. in some cases, the rash progresses very slowly and hemorrhage develops into the base of the lesions, which remain flat and soft instead of tense, carrying a bad prognosis. in some other cases, the disease is hemorrhagic from the beginning, leading to death to days after the initial symptoms appear (case-fatality rate: %). finally, in some cases, a severe and overwhelming illness is followed by dusky skin lesions; these patients have a large quantity of virus and are extremely dangerous epidemiologically. previously vaccinated individuals usually develop a milder disease that consists of a mild pre-eruptive phase followed by few skin lesions that appear more superficial, evolve more rapidly, and are not as synchronous as the classical type. viral hemorrhagic fever (vhf; see chapter ) is caused by a heterogenous group of rna viruses that belong to several different families. the cdc identified filoviruses (ebola and marburg viruses), arenaviruses (lassa, junin, machupo, guanarito, and sabia), and bunyaviruses (crimean-congo hemorrhagic fever [cchf] and rift valley fever [rvf]). [ ] [ ] [ ] the common denominator in these infections is the increased vascular permeability in the microcirculation leading to hemorrhagic diathesis and systemic manifestations such as pulmonary edema and cerebral edema related to leaky capillaries. these viruses usually have a very narrow geographic range determined by their natural reservoirs and vectors. humans are accidental hosts. these diseases have caught great public attention due to their high mortality. this, combined with their aerosol infectivity, has led to the use of biosafety level laboratories in their study. clinical presentation is usually nonspecific and consists of fever and malaise, followed by signs of increased vascular permeability and circulatory compromise. vhf usually terminates in shock, generalized mucocutaneous hemorrhages, and multiorgan failure. differences exist among the clinical details and pathogenesis of the different viruses (see chapter for an overview and the individual chapters for details). for example, vhf due to filoviruses usually have prominent hemorrhagic manifestations and disseminated intravascular coagulation (dic) as a terminal event. rvf virus leads to liver damage, dic, and hemorrhagic manifestations in approximately % of patients with severe disease. cchf also behaves like the filoviral infections with prominent hemorrhagic manifestations. lassa fever has few neurologic or hemorrhagic manifestations. the south american arenaviral hemorrhagic fevers usually have hemorrhagic and neurologic components. toxins in the context of bt agents are substances of biologic origin that are capable of producing human illness. toxins are usually proteins synthesized by living bacteria, fungi, or plants. toxins are generally less dangerous than infectious agents. the most potent biological toxin is that from clostridium botulinum and it is -fold or more less lethal than anthrax on a weight basis. other toxins such as ricin are more than a -fold less toxic than botulinum toxin and sarin is -fold less toxic than ricin. there are seven similar toxins produced by seven different serotypes of c. botulinum (a to g), all leading to the same clinical manifestations and with the same lethality. the toxins have a molecular weight of approximately kda and block neurotransmission at the presynaptic level in cholinergic neurons including the neuromuscular junction, leading to progressive palsies of cranial nerves and skeletal muscle. botulinal toxins are among the most lethal substances known to mankind with ld of . μg/g of body weight when administered parenterally. , , the aerosol route decreases its lethality to times. both aerosol attacks and contamination of food supplies are potential bt scenarios. clinical manifestations consist of progressive bulbar and skeletal paralysis in the absence of fever, including diplopia, dysphagia, blurred vision, ptosis, dysarthria, dysphonia, mydriasis, dry mucosae, and descending paralysis. , the cause of death in lethal cases is respiratory insufficiency due to paralysis of respiratory muscles. onset of symptoms is variable and depends on the inoculum, ranging from hours to several days after exposure. most cases of naturally occurring intoxication are related to consumption of improperly sterilized canned food or ingestion of preserved fish. rare cases of inhalational botulism were documented in germany in the early s due to accidental laboratory exposure. the rapid absorption through the respiratory tract may offer a different pathogenesis and it is not known if antitoxin is useful in therapy, although animal models show efficacy in prophylaxis. all the agents in category a are generally recognized as serious threats for causing extensive casualties. categories b and c are much more heterogeneous. they were considered to provide significant threat potential but there are continuing reassessments. these conditions are caused by the genus alphavirus, family togaviridae (eastern, western, and venezuelan equine encephalitis [vee] viruses; see chapter ). natural infections are usually transmitted by mosquitoes, but aerosol transmission is the notorious cause of numerous laboratory infections and is the basis of its historic weaponization. , most of these viruses cause systemic illness characterized by fever, myalgias, and prostration. clinically apparent involvement of the central nervous system is present in some cases and varies among the different viruses. eastern equine encephalitis (eee) is by far the most virulent, leading to case-fatality rates of % to %, and survivors usually have severe neurologic sequelae. , vee, in contrast, leads to cns manifestations in no more than % of cases and almost all vee infections are symptomatic even in the absence of cns involvement. [ ] [ ] [ ] rickettsia prowazekii (epidemic typhus) and r. rickettsii (rocky mountain spotted fever) typhus (see chapter ) is another disease that has played a historic role in human populations. [ ] [ ] [ ] [ ] millions of people perished in world war i and world war ii due to epidemic, louse-borne typhus. large outbreaks of the disease still occur in tropical regions around the world in areas stricken by war, famine, and poverty. rocky mountain spotted fever (rmsf), on the other hand, is transmitted by tick bites and occurs endemically in south and central america as well as north america. rickettsiae target the microvascular endothelium leading to leaky capillaries systemically. the main causes of morbidity and mortality are noncardiogenic pulmonary edema and cerebral edema leading to diffuse alveolar damage and meningoencephalitis. clinical manifestations are nonspecific and include fever, malaise, headache, myalgias/arthralgias, cough, nausea, vomiting, confusion, stupor, and coma in severe cases. skin rash ranges from maculopapular to petechial, depending on the severity, and is observed in around % of patients with rmsf and % to % of cases of epidemic typhus, depending on the darkness of cutaneous pigmentation. rickettsiae are remarkably underestimated biothreats as they are highly infectious by low-dose aerosol exposure, possess a stable extracellular form, and are resistant to most empirically administered antibiotics, including β-lactams, aminoglycosides, and macrolides, and are exacerbated by sulfonamides. case-fatality rates can be as high as % to % without antibiotic therapy and % to % with adequate antibiotic coverage. lethal cases are usually due to delayed diagnosis. , , these rickettsiae are highly infectious by aerosol and are potent bt agents. they are often discounted because of their susceptibility to tetracycline and chloramphenicol. however, the severity of the illness, the exhaustion of antibiotics in the face of a mass attack, and the existence of antibiotic-resistant organisms suggest they are still formidable players. this gram-negative, obligately intracellular bacterium has a high degree of infectivity (one organism is capable of causing infection by inhalation) and low lethality. [ ] [ ] [ ] [ ] the distribution of q fever is worldwide and results from exposure to animals such as sheep, cattle, goats, cats, rabbits, and others. c. burnetii has spore-like characteristics that can withstand harsh environmental conditions and be transported by wind to other places. in natural infections, % of cases are asymptomatic and are diagnosed by seroconversion. in symptomatic cases, the presentation is nonspecific and includes malaise, fever, myalgias, cough, chills, headaches, anorexia, weight loss, and in some cases pleuritic chest pain. hepatomegaly and splenomegaly are sometimes observed, although not frequently. transmission occurs by exposure to infected animal products (meat, milk). less common routes of infection are inhalational and cutaneous. the clinical presentation of brucellosis is highly variable, even after inhalational exposure. the clinical spectrum ranges from asymptomatic seroconversion to severe acute systemic disease. intermediate forms include undulant fever or chronic disease, characterized by presence of brucella in virtually any organ. acute systemic disease is highly incapacitating with high fever, headache, nausea, vomiting, chills, severe sweating, and, in very severe cases, delirium, coma, and death. undulant fever is characterized by relapses of fever, weakness, generalized aching, and headache. chronic infections may have manifestations related to several organ systems such as the gastrointestinal and genitourinary tracts, cns, joints, and bones. [ ] [ ] [ ] developing countries with insufficient water treatment and food security are more vulnerable to enteric bt attack. these agents include shigella dysenteriae, salmonella spp., enterohemorrhagic e. coli, vibrio cholerae, and cryptosporidium parvum. shigella and salmonella have in fact already been used as agents of biorevenge or biopolitics in small-scale attacks: one (shigella) in an office setting by a disgruntled employee and one in oregon by a religious sect that led to nearly cases of salmonella-related gastroenteritis. , these agents are indeed ideal for small-scale attacks since large-scale attacks would require contamination of large water supplies which, because of enormous dilution factors and susceptibility of all these agents (except for c. parvum) to standard chlorinating procedures, would decrease the number of bacteria to below that required to infect large numbers of people. occasional outbreaks of nontyphoidal salmonella and shigella infections occur in the united states. shigella is a highly infectious organism that requires very low numbers ( - organisms) to provoke clinical disease. the illness caused by shigella and enterohemorrhagic e. coli is explosive and starts with fever, vomiting, severe abdominal cramping, bloody diarrhea, and systemic manifestations such as hypotension, and circulatory collapse if not treated rapidly. both microorganisms produce an exotoxin responsible for most of the systemic manifestations associated. a distinct complication, hemolytic uremic syndrome, occurs in a small percentage of cases, being more common in children younger than years of age, leading to renal failure and hemolysis. salmonella is less infectious and less explosive than shigella, and leads to fever, vomiting, diarrhea, abdominal cramping, and in some cases to typhoidal manifestations. imported cases of v. cholerae have been diagnosed in the united states in the past. however, the disease occurs in southern asia and latin america as large outbreaks. the clinical illness is characterized by explosive watery diarrhea that leads to rapid dehydration and circulatory collapse. c. parvum infections are characterized by watery diarrhea and abdominal cramping for to weeks. the disease is self-limited except in patients with acquired immunodeficiency syndrome (aids) or other conditions of compromise, in whom illness can last for months or years if immune function is not restored. c. parvum is resistant to standard chlorine concentrations in water supplies. the largest outbreak in this country occurred in milwaukee in the early s and was responsible for thousands of cases and increased mortality among those with aids. , , this section addresses other toxins considered of potential bt use, such as staphylococcal enterotoxin b (seb) and ricin toxin (derived from castor beans, which in turn are the fruit of the ricinus communis plant). the ricin toxin is composed of two glycoproteins of approximately , kda. the toxin inhibits protein synthesis by blocking elongation factor (ef ) at the ribosomal level. ricin toxin is not a weapon of mass destruction since its lethal dose in humans is much higher than previously believed. however, the use of the toxin in small bt attacks is possible in the tropics because of its ready availability and relatively easy extraction from the beans. clinical presentation depends on the route of administration as does the ld . in cases where large amounts of the toxin are ingested, the manifestations include nausea, vomiting, severe abdominal cramping, rectal hemorrhage, and diarrhea. as the clinical course progresses, anuria, mydriasis, severe headaches, and shock supervene leading to the patient' s demise in to days. clinical manifestations usually appear within hours after ingestion of the toxin. inhalational exposure leads to prominent pulmonary manifestations to hours after exposure and fever, dyspnea, progressive cough, cyanosis, and death. histologically, there is widespread necrosis of pulmonary parenchyma and pulmonary edema. a single case of parenteral intoxication was documented. a defector from bulgaria was injected with a pellet containing ricin from a weapon disguised in an umbrella, resulting in local necrosis, regional lymphadenopathy, gastrointestinal hemorrhage, liver necrosis, nephritis, and dic. staphylococcus aureus enterotoxin b (seb) is a -kda, heatstable exotoxin produced by certain strains of s. aureus and is responsible for food poisoning after ingestion of the preformed exotoxin in improperly handled food. in bt scenarios, exposure can occur either by inhalation or ingestion leading to seb food poisoning or seb respiratory syndrome. the toxin is highly incapacitating and not very lethal. the dose that causes symptoms in half of exposed persons and ld differ by a magnitude of log scales for inhalational exposure. thus, it is thought of as an incapacitating agent. incubation time after ingestion is short ( - hours) followed by explosive vomiting that persists for several hours. weaponization of the toxin as an aerosol is possible due to its high stability. manifestations after inhalation of the seb are related to the respiratory system and consist of fever, cough, chills, myalgias, chest pain, and pulmonary insufficiency due to alveolar edema. general symptoms and signs are universal and consist of multiorgan failure secondary to a cytokine storm. these toxins are superantigens due to their ability to bind to major histocompatibility complex (mhc) class ii molecules on large numbers of lymphocytes and macrophages, leading to a hyperactivation of the immune system and massive cytokine release including interferon-gamma (ifn-γ), tumor necrosis factor-alpha (tnf-α), interleukin (il- ), and other mediators such as leukotrienes and histamine. the role of the clinical laboratory in the diagnosis of possible cases related to a bt attack is of utmost importance. , on the one hand, standard clinical microbiology laboratories will be receiving specimens for diagnostic purposes, and communication with clinicians regarding their suspicions is critical. certain isolates in the laboratory are not pursued further (bacillus spp. is a classic example) unless specifically requested due to the frequent isolation of contaminants with similar characteristics. in addition, handling of certain specimens will require added biosafety level requirements due to their infectivity (table - ) . certain samples will have to be shipped to highly specialized laboratories for initial or further work-up. environmental testing is challenging due to the complexity of the samples to be analyzed. , this type of testing takes place in highly specialized laboratories and is not undertaken by the standard clinical microbiology laboratory. the bacterial diseases caused by the bt agents outlined in this chapter, with the exception of c. burnetii and rickettsia spp., can be diagnosed by standard isolation techniques in clinical microbiology laboratories. isolation of rickettsiae and the bt viruses requires specialized laboratories with bsl- or bsl- biocontainment. serological assays are available for detection of antibodies against all bt agents. however, for many organisms serological assays require the presence of rising antibody titers, and therefore the serologic diagnosis is usually retrospective in nature. for some viral diseases, a reliable diagnosis can be established based on elevation of immunoglobulin m (igm) titers in the acute phase of the disease. with the advent of molecular techniques, rapid and sensitive diagnostic tests are becoming available for bt agents during the acute phase of the disease. [ ] [ ] [ ] this is of utmost importance in a bt event since identification of the first cases would be critical for a rapid and effective public health response. in addition, treatment and prophylactic measures can also be initiated as quickly as possible. molecular diagnostic techniques can be applied to potential bt agents in an additional setting: as part of the epidemiological and forensic investigations that a bt attack would immediately trigger. postmortem diagnosis is also possible by analysis of frozen or paraffin-embedded tissues by immunohistology or nucleic acid-based amplification techniques. rapid diagnosis of the initial case (cases) in a bt event requires a high degree of clinical suspicion from the physicians having contact with such patients in the emergency room or outpatient setting. the clinical laboratories would then play a critical role in detecting the suspected agent and/or referring the appropriate specimens to higher level laboratories for specialized testing (table - ) . , , several of the agents discussed in this chapter are zoonotic diseases. therefore, diagnosis of certain zoonotic diseases in animals may be important in identifying some bt attacks. in such situations, animals could be seen as either direct victims of the attack or as sentinel events in a human outbreak. there are currently efforts to establish a network of laboratories dedicated to diagnosis of veterinary agents. bsl- suitable for work involving well-characterized agents not known to cause disease in healthy bacillus subtilis adult humans and of minimal potential hazard to laboratory personnel and the environment. naegleria gruberi canine hepatitis virus bsl- suitable for work involving agents of moderate potential hazard to personnel and the measles virus environment. laboratory personnel have specific training in handling pathogenic agents salmonella spp. and are directed by competent scientists; access to the laboratory is limited when work toxoplasma spp. is being conducted; extreme precautions are taken with contaminated sharp items; and hepatitis b virus certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. bsl- suitable for work with infectious agents which may cause serious or potentially lethal coxiella burnetii disease as a result of exposure by the inhalation route. in addition to the requirements rickettsia spp. described for work in bsl- environment, all procedures are conducted within biological m. tuberculosis safety cabinets, or other physical containment devices, and by personnel wearing alphaviruses appropriate personal protective clothing and equipment. laboratory should be located in a separate building or an isolated zone within a building. laboratories are equipped with double door entry, directional inward flow, and single-pass air. bsl- required for work with dangerous and exotic agents that pose a high individual risk of filoviruses aerosol-transmitted laboratory infections and life-threatening disease. members of the arenaviruses laboratory staff have specific and thorough training in handling extremely hazardous infectious agents. they are supervised by competent scientists who are trained and experienced in working with these agents. access to the laboratory is strictly controlled by the laboratory director. the facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. all activities are confined to class iii biological safety cabinets, or class ii biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. the biosafety level laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. the diagnosis of inhalational anthrax is based on isolation and identification of b. anthracis from a clinical specimen collected from an ill patient. in cases of inhalational anthrax, samples of sputum, blood, or cerebrospinal fluid (csf) may yield growth of the agent. demonstration of b. anthracis from nasal swabs has more epidemiological and prophylactic implications than clinical importance. standard diagnostic techniques are based on visualization and isolation in the clinical microbiology laboratory and serological demonstration of antibodies against b. anthracis. [ ] [ ] [ ] [ ] [ ] visualization of b. anthracis from clinical specimens (blood cultures, csf, and cutaneous lesions) by gram stains is not difficult. b. anthracis appears as large gram-positive, spore-forming rods with a bamboo appearance. isolation is achieved by inoculating standard sheep blood agar plates, and colonies appear as small, gray-white, nonhemolytic colonies. a selective medium (polymyxin-lysozyme-edta-thallous acetate agar) is available mostly for environmental samples and inhibits the growth of other bacillus spp., such as b. cereus. growth is rapid ( - hours) . confirmatory tests include γ-phage lysis, detection of specific cell wall and capsular antigens, and polymerase chain reaction (pcr) amplification of dna followed by sequencing. serological tests available for clinical diagnosis are based on detection of antibodies directed against protective antigen (pa). cross-reactive antibodies decrease the specificity of this test. assays based on toxin detection are available in specialized centers and are based on capture of anthrax toxins by using antibodies. antibody-coated immunomagnetic beads are then analyzed by electrochemiluminescence technology. the analytical sensitivity of this technique for detection of anthrax toxin is at the picogram to femtogram level ( − to − ). , immunoliposomal technology combined with real-time pcr (for a dna reporter sequence) is also in the early stages of development for several toxins (ricin, cholera, and botulinum) and appears promising with analytical sensitivity in the attomolar to zeptomolar ( − to − ) range for cholera toxin. the specificity of this assay is given by the toxin-capturing antibody. nucleic acid amplification techniques (pcr) are also available both in standard format and real-time format. extraction of dna from spores is challenging and requires modification of dna extraction protocols in order to facilitate release of dna from spores or induction of germination prior to dna extraction. real-time pcr tests have been developed by applied biosystems (taqman ' nuclease assay) and roche applied science (lightcycler). [ ] [ ] [ ] the analytical sensitivity of both techniques is extremely high, and testing times have been decreased to to hours. portable pcr instruments are being developed for rapid deployment to the field. examples include the rugged advanced pathogen identification device (rapid), the smartcycler (cepheid, ca), and the miniature analytical thermal cycler instrument (matci) developed by the department of energy' s lawrence livermore national laboratory. this instrument later evolved into the advanced nucleic acid analyzer (anaa) and handheld advanced nucleic acid analyzer (hanaa). molecular subtyping of b. anthracis is also possible by using the s ribosomal rna (rrna) subunit gene, multiplelocus vari-able number tandem repeat analysis of eight genetic loci, and amplified fragment length polymorphism (aflp) techniques. , environmental testing also plays a role in the investigation of a bt event. in this setting, detection of b. anthracis relies heavily on molecular techniques for confirmation of potentially contaminated samples (e.g., surfaces, air). , postmortem diagnosis is also possible by using gram stains on paraffin-based tissues or immunohistochemical procedures using polyclonal or monoclonal antibodies against various anthrax antigens. diagnosis of y. pestis is based on demonstration of the bacillus in blood or sputa from patients. standard diagnostic techniques in the laboratory include visualization of gramnegative coccobacilli, which by giemsa, wright, or wayson stains reveal a "safety pin" appearance. isolation is performed in blood and mcconkey agar plates on which colonies appear as nonlactose fermentors. the organisms are identified preliminarily by direct immunofluorescent assay with y. pestisspecific antibodies, with final identification based on biochemical profiles in clinical microbiology laboratories. molecular diagnostic techniques based on real-time pcr have become available in recent years and involve detection of y. pestis genes such as plasminogen activator (pla), genes coding for the yop proteins and the capsular f antigen, and the s rrna gene, which allows distinction from other yersinia spp. [ ] [ ] [ ] assays have been developed to detect resistance to particular antibiotics. the importance of these diagnostic techniques in a disease such as plague is evident. the log-normal epidemic curve with a narrow dispersion of the incubation periods (see fig. - ) and the short interval for successful antibiotic therapy mandate recognition of the earliest cases if the bulk of the exposed are to be saved. molecular subtyping of y. pestis is also possible by analyzing polymorphic sites in order to identify the origin of strains in the event of a bt attack. diagnosis is made in the clinical laboratory by demonstration of the microorganisms in secretions (sputa, exudates) by direct immunofluorescence or immunohistochemically in biopsy specimens. isolation in the clinical laboratory may be achieved by using regular blood agar plates, posing a risk to laboratory personnel not employing bsl- facilities and procedures. the procedure for isolation of f. tularensis in the laboratory is very similar to that described for y. pestis. final identification in the clinical laboratory is based on the biochemical profile. molecular diagnostic techniques are based on pcr detection of f. tularensis by using primers for different genes such as outer membrane protein (fop) or tul and real-time detection systems. , , smallpox virus diagnosis of variola major is suggested by its clinical presentation and the visualization of guarnieri bodies in skin biopsy samples. preliminary confirmation requires visualization of the typical brick-shaped orthopox virus by electron microscopy, followed by isolation from clinical specimens and accurate molecular identification to differentiate it from the morphologically (and sometimes clinically) similar monkeypox virus. confirmation of this diagnosis is performed only under bsl- containment facilities at the cdc. molecular techniques are based on pcr amplification using real-time or standard technology followed by sequencing or use of restriction fragment length polymorphism (rflp) for accurate identification. technologies so far developed for smallpox molecular testing include taqmanand lightcycler-based assays with primers designed for the hemagglutinin gene and a-type inclusion body proteins. [ ] [ ] [ ] [ ] sequencing of the smallpox genome has been completed for some asian strains of variola major and one of variola minor. other strains are being sequenced and will provide more information for probe design and treatment targets. diagnosis of these diseases is performed in highly specialized centers in the united states because special isolation procedures and highly contained laboratories are required. initial diagnosis of these diseases is suspected on clinical and epidemiologic grounds. laboratory diagnosis involves isolation, electron microscopy, and serological assays. immunohistochemical detection of hemorrhagic fever viral antigens in paraffin-embedded tissues is also performed in highly specialized centers such as the cdc. [ ] [ ] [ ] [ ] [ ] molecular diagnostic techniques have also improved dramatically during the last few years. serum or blood is the most common specimen used for reverse transcriptase-pcr amplification of viral nucleic acids. both standard and realtime techniques are available. design of primers for this heterogenous group of rna viruses that are highly variable is one of the limitations. therefore, multiplex pcr techniques are required to detect as many targets as possible in a single assay. , real-time pcr based on detection of the target sequence using fluorescent probes therefore limits the number of targets that can be identified because of the limited wavelength range for fluorescent applications (usually only four different wavelengths can be detected at the same time). [ ] [ ] [ ] the use of microchips containing several thousands of oligonucleotides from all viruses known to be pathogenic to humans is an encouraging development. in fact, the rapid identification and characterization of the novel human coronavirus responsible for the sars outbreak in is an excellent example of the power of hybridization-based microchips. the creation of an automated and easily deployable instrument capable of detecting all possible potential bt agents based on highly sensitive techniques such as electrochemoluminescence (ecl) or pcr would be ideal. the nonspecific nature of presenting symptoms is a major problem with several of the agents. the rapid recruitment of cases into the infected cohort requires that an early diagnosis of the epidemic be established, particularly for organisms such as y. pestis in which there is only a short window for successful treatment. in fact, such projects are already in the making. an example of this system is the automated biological agent testing system (abats) that combines the techniques mentioned previously. the system is the result of integrating several commercially available technologies into a single automated and robotized instrument for detection of viruses, bacteria, and parasites considered potential bt agents. the technologies incorporated into this "super system" include automated specimen preparation (both nucleic acid-based and protein-based such as immunodiagnostics), thermocyclers for pcr detection, chemiluminescent detectors for immunobased assays, sequencers, and software programs for sequence analysis. rickettsia prowazekii (epidemic typhus) and r. rickettsii (rocky mountain spotted fever) diagnosis of these infections in the clinical microbiology laboratory currently rests on the identification of antibodies in serum during the acute and convalescent period in order to demonstrate seroconversion or rising titers. the diagnosis is therefore retrospective. , detection of rickettsial dna from blood or skin samples during the acute phase of the disease is possible via pcr assays. however, these assays are not standardized and are not commercially available. primers have been designed for amplification of several rickettsial genes including citrate synthase, -kda protein gene, ompa, and ompb. [ ] [ ] [ ] [ ] [ ] the clinical sensitivity and specificity of standard or real-time pcr techniques have not been determined. most likely real-time pcr is superior due to the higher analytical sensitivity of this technique and low risk of sample contamination with dna amplicons when compared to standard pcr amplification methods. isolation of rickettsiae from clinical specimens is performed in very few specialized laboratories in the nation and requires the use of cell monolayers, embryonated eggs, or animals. detection of rickettsial antigens or whole bacteria in blood specimens is theoretically possible by using ultrasensitive methods, but such assays are currently only in the early phases of development. immunohistochemical detection of rickettsiae in paraffin-embedded tissue has also been applied to tissue samples obtained pre-or postmortem. [ ] [ ] [ ] salmonella spp., shigella dysenteriae, vibrio cholerae, and cryptosporidium parvum (acute enteric syndromes) diagnosis of salmonella, shigella, and vibrio infections is based on isolation of the offending agent on standard microbiological media in the clinical laboratory, followed by specialized confirmatory tests to identify the specific serotype involved. diagnosis of c. parvum is based on visual identification of the protozoan in fecal specimens by using modified trichrome stain. the diagnosis rests on serological demonstration of antibodies by immunofluorescent assay (ifa) or enzyme-linked immunosorbent assay (elisa). antibodies remain elevated for years after the acute infection, and therefore a fourfold rise in titers is the gold standard for diagnosis. pcr detection of c. burnetii dna from blood or tissues also yields a diagnosis of q fever. brucella spp. diagnosis of brucellosis requires a high degree of clinical suspicion due to the protean manifestations related to this disease. laboratory diagnosis is based on isolation of the microorganism from blood, bone marrow, or other tissue samples. isolation is not easy due to the slow-growth of brucella spp. colonies usually appear after to weeks, and therefore communication with the clinical laboratory is important so that appropriate media will be used and the cultures will be held long enough for colonies to be detected. serologic assays for demonstration of rising antibody titers are available, although the diagnosis is retrospective. pcr detection is promising, but it is not standardized. [ ] [ ] [ ] alphaviruses (encephalitic syndromes: venezuelan, eastern, and western equine encephalomyelitis) diagnosis is based on isolation of the virus from serum or brain (postmortem specimens) in a bsl- environment. pcr detection of viral sequences is also possible. serologic diagnosis is based on demonstration of antibodies in acute and convalescent sera. [ ] [ ] [ ] botulinum toxins the diagnosis of botulism relies heavily on clinical parameters. an afebrile patient with signs and symptoms of progressive bulbar palsies and descending neuromuscular paralysis is highly suspected of having botulism. demonstration of the toxin in cases of botulism due to ingestion of contaminated food is made from gastric samples, feces, blood, and urine. however, detection of minute amounts of toxin (and contacts with samples from cases may prove fatal due to the toxin' s potency) would be difficult by current immunoassay systems such as elisa platforms. detection techniques based on electrochemiluminescence and immunoliposomes are currently under development. , pcr assays can be performed in cases of ingestion of contaminated food in order to detect the genetic material present in c. botulinum. if weaponized toxin is used in the absence of c. botulinum organisms, detection of the genetic material would be difficult and would rely on the presence of residual dna after toxin purification procedures. if inhalational botulism is suspected, respiratory secretions and nasal swabs should be obtained as early as possible. postmortem samples of liver and spleen can be used for detection of botulinum toxins. diagnosis is also based on clinical presentation and requires a high index of suspicion due to the nonspecific nature of the signs and symptoms. laboratory diagnosis rests on detection of the toxin in body fluids by immunoassays (capture elisa and igg elisa). a new generation of tests using more sensitive detection methods is under development (see preceding discussion). diagnosis is also suspected on clinical grounds and confirmed by demonstration of the toxin in nasal swabs early in the disease process, feces, and, in fatal cases, from kidney and lung tissue. serum can be analyzed by elisa, and pcr can be performed for detection of toxin genes of s. aureus if present. the distribution and incubation periods of infectious diseases 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group on civilian biodefense monkeypox transmission and pathogenesis in prairie dogs potential antiviral therapeutics for smallpox, monkeypox, and other orthopox virus infections viral hemorrhagic fevers including hantavirus pulmonary syndrome in the americas viral agents as biological weapons and agents of bioterrorism hemorrhagic fever viruses as biological weapons: medical and public health management [see comment role of the endothelium in viral hemorrhagic fevers the action of botulinum toxin at the neuromuscular junction botulinum toxins venezuelan equine encephalitis an outbreak of eastern equine encephalomyelitis in jamaica, west indies. i: description of human cases eastern and western equine encephalitis an outbreak of venezuelan equine encephalomeylitis in central america. evidence for exogenous source of a virulent virus subtype an epidemiologic study of venezuelan equine encephalomyelitis in costa rica venezuelan equine encephalitis zinsser h: rats, lice and history rickettsial infections. in lack ee rocky mountain spotted fever and other rickettsioses typhus and its control in russia rickettsia conorii infection of c h/hen mice. a model of endothelial-target rickettsiosis rickettsioses as paradigms of new or emerging infectious diseases threats in bioterrorism ii: cdc category b and c agents q fever - . clinical and epidemiologic features of , infections epidemiologic features and clinical presentation of acute q fever in hospitalized patients: french cases human brucellosis an overview of human brucellosis medical aspects of chemical and biological warfare an outbreak of shigella dysenteriae type among laboratory workers due to intentional food contamination viability of cryptosporidium parvum oocysts in natural waters surveillance for waterborne-disease outbreaks-united states cryptosporidiosis in children during a massive waterborne outbreak in milwaukee, wisconsin: clinical, laboratory and epidemiologic findings medical aspects of chemical and biological warfare georgi markor--death in a pellet staphylococcal enterotoxin b and related pyrogenic toxins bioterrorism: implications for the clinical microbiologist the role of the clinical laboratory in managing chemical or biological terrorism diagnostic analyses of biological agent-caused syndromes: laboratory and technical assistance automated biological agent testing systems department of health and human services applying molecular biological techniques to detecting biological agents current laboratory methods for biological threat agent identification molecular diagnostic techniques for use in response to bioterrorism a national laboratory network for bioterrorism: evolution from a prototype network of laboratories performing routine surveillance definitive identification of bacillus anthracis-a review pc bacillus and other aerobic endospore-forming bacteria mabs to bacillus anthracis capsular antigen for immunoprotection in anthrax and detection of antigenemia specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin g antibodies to anthrax toxin protective antigen comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin g antibodies to anthrax toxins an enzymatic electrochemiluminescence assay for the lethal factor of anthrax comparative studies of magnetic particle-based solid phase fluorogenic and electrochemiluminescent immunoassay high-sensitivity detection of biological toxins a field investigation of bacillus anthracis contamination of u.s. department of agriculture and other washington, dc, buildings during the anthrax attack of sensitive and rapid identification of biological threat agents detection of bacillus anthracis dna by lightcycler pcr a handheld real time thermal cycler for bacterial pathogen detection real-time microchip pcr for detecting single-base differences in viral and human dna rapid pathogen detection using a microchip pcr array instrument genetic sleuths rush to identify anthrax strains in mail attacks sequencing of s rrna gene: a rapid tool for identification of bacillus anthracis environmental sampling for spores of bacillus anthracis bacillus anthracis contamination and inhalational anthrax in a mail processing and distribution center manual of clinical microbiology development of rrnatargeted pcr and in situ hybridization with fluorescently labelled oligonucleotides for detection of yersinia species ' nuclease pcr assay to detect yersinia pestis yersinia pestis-etiologic agent of plague francisella and brucella detection of francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction detection of francisella tularensis within infected mouse tissues by using a hand-held pcr thermocycler pcr strategy for identification and differentiation of small pox and other orthopoxviruses real-time pcr system for detection of orthopoxviruses and simultaneous identification of smallpox virus gene for a-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses the potential of ′ nuclease pcr for detecting a single-base polymorphism in orthopoxvirus detection of smallpox virus dna by lightcycler pcr comparative pathology of the diseases caused by hendra and nipah viruses a novel immunohistochemical assay for the detection of ebola virus in skin: implications for diagnosis, spread, and surveillance of ebola hemorrhagic fever. commission de lutte contre les epidemies a kikwit immunohistochemical and in situ localization of crimean-congo hemorrhagic fever (cchf) virus in human tissues and implications for cchf pathogenesis retrospective diagnosis of hantavirus pulmonary syndrome, - : implications for emerging infectious diseases hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease molecular diagnostics of viral hemorrhagic fevers rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr quantitative real-time pcr detection of rift valley fever virus and its application to evaluation of antiviral compounds development and evaluation of a fluorogenic ' nuclease assay to detect and differentiate between ebola virus subtypes zaire and sudan rickettsioses as paradigms of new or emerging infectious diseases laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae diagnosis of mediterranean spotted fever by cultivation of rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of r. conorii in circulating endothelial cells: a -year follow-up differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of pcr-amplified dna of the gene encoding the protein rompa differentiation among spotted fever group rickettsiae species by analysis of restriction fragment length polymorphism of pcr-amplified dna diagnostic tests for rocky mountain spotted fever and other rickettsial diseases immunohistochemical diagnosis of typhus rickettsioses using an anti-lipopolysaccharide monoclonal antibody monoclonal antibody-based immunohistochemical diagnosis of rickettsialpox: the macrophage is the principal target investigation of foodborne and waterborne disease outbreaks rapid laboratory confirmation of human brucellosis by pcr analysis of a target sequence on the -kilodalton brucella antigen dna the -kda cytoplasmic protein of brucella species-an antigen useful for diagnosis-is a lumazine synthase characterization of an -kilodalton brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections toxins as weapons of mass destruction: a comparison and contrast with biological warfare and chemical warfare agents sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor key: cord- -s d ew authors: e. newcomer, christian; g. fox, james title: zoonoses and other human health hazards date: - - journal: the mouse in biomedical research doi: . /b - - / - sha: doc_id: cord_uid: s d ew zoonoses refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated hazards. in addition to the zoonoses, mice are capable of inflicting bites on inadequately trained personnel and are a rich source of allergens for a substantial number of persons predisposed to develop mouse-associated allergic sensitivities. this chapter discusses the mouse-associated zoonotic diseases and other health hazards and explains the strategies that are helpful for reducing or eliminating the risk of personnel exposure to these conditions. zoonoses is derived from the greek words zoon (meaning animals), and noses (meaning disease), and refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. however, when these essential animal program quality measures fail or are not incorporated into the animal facility operations, zoonotic pathogens may be unwittingly introduced and perpetuated, placing personnel at increased risk of exposure. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated implications in the new world serocomplex group are present among the wild rodents endemic to the united states such as neotoma spp. and peromyscus spp. buchmeier et al. ; fulhorst et al. ) . research animal programs that import wild rodents for laboratory studies should stay abreast of the developments on the identification and host-range characteristics of the new world arenaviruses of emerging importance. this section will focus only on lymphocytic choriomeningitis (lcm), a naturally occurring viral zoonosis of the laboratory mouse. many published reports of human lcm infection are associated with laboratory animal and pet contact, particularly mice and hamsters, and these studies now span many decades (armstrong and lillie ; bowen et al. ; dykewicz et al. ; jahrling and peters ; lehmann-grube et al. ; rousseau et al. ) . there seems to be a resurgent awareness among physicians that lcm should be sought as an etiology in human neurological disease and in pediatric congenital brain disorders (barton and hyndman ; barton et al. barton et al. , romero and newland ) . lcmv is widely distributed among the wild mouse population throughout most of the world presenting a zoonotic hazard (childs et al. ; childs and peters ; morita et al. ; smith et al. ). surveys conducted within the urban environment of baltimore, maryland, reported that % of house mice and . % of persons tested had measurable lcmv antibody titers (childs et al. (childs et al. , . a similar serological survey conducted in spain across urban and rural ecological settings also found a % prevalence in mice and a . % prevalence among persons by immunofluorescence assay (lledo et al. ) . the recent serological detection of lcmv in five mice on the treeless, sub-antarctic, macquarie island of australia, indicates the extent of distribution of this agent to the remotest areas of our planet (moro et al. ) . the apparent ease with which lcmv is transmitted to humans also occurs in a variety of other laboratory animal species; hamsters, guinea pigs, swine, dogs, and nonhuman primates, especially callitrichids, which readily sustain natural infections. in the case of the callitrichids, there have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) due to lcmv, with a high mortality rate in zoological parks in both the united states and england over the past two decades (lucke and bennett ; montali et al. ; stephensen et al. stephensen et al. , stephensen et al. , . rodent (mouse) infestations of these zoos and/or the supplementation of the diets of tamarins and marmosets with suckling mice, a common practice (richter et al. ) , are potentially rich sources for lcmv. in the research animal facility environment, the laboratory mouse continues to merit attention as the species of primary concern as a reservoir for cases of human lcm (dykewicz et al. ). in the laboratory mouse, and to a lesser degree the hamster, breeding colonies can become endemically infected when the virus is transmitted to pups in utero or early in the neonatal period, producing a tolerant subclinical infection characterized by chronic viremia and viruria. when infected, athymic and other immunodeficient mouse strains may be predisposed to harboring silent, persistent infections and present a higher risk to personnel (dykewicz et al. ) . under some circumstances lcmv also produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. bedding material and other fomites contaminated by lcmv-infected animals can also be important sources of infection for humans, as demonstrated in a recent outbreak among laboratory animal technicians and on many previous occasions (biggar et al. ; dykewicz et al. ) . the experimental passage of tumors and cell lines contaminated with lcmv has long been recognized (haas and stewart ) and represents one of the biggest threats for the introduction of lcmv into animal facilities at the present time (bhatt et al. ; dykewicz et al. ; nicklas et al. ) . reported that of rodent transplantable tumors screened were positive for lcmv and identified contamination in of hamster tumors and of mouse tumors that had been propagated in animals (nicklas et al. ) . the growth of lcmv in insect cell lines has also been demonstrated (rahacek ) , and the article by hotchin summarized the work of others indicating that numerous experimentally infected, bloodsucking ectoparasites are capable of transmitting the disease to laboratory rodents (hotchin ) . lcm virus also has been recovered from cockroaches (armstrong and lillie ) . the diagnosis and control of lcmv infection in mouse colonies has been reviewed in chapter of this volume. tumor and cell-line screening before animal passage, the control of wild rodent infestations in areas where animals are housed or used, and the early detection of colony infections through sound colony health surveillance practices are of critical importance to the prevention of infection in mouse colonies. once established in mouse breeding colonies, the high viral load characteristically shed by mice infected congenitally or neonatally represents a very serious hazard to personnel. most of the cases of human infection, whether involving exposure in the home, agricultural, or laboratory setting, have involved contact with live mice and their excreta or mouse carcasses (dykewicz et al. ; havens ; morbidity and mortality weekly report ) . several authors have emphasized the association between the actual handling of infected mice and the contraction of the disease by humans (dykewicz et al. ; havens ; smithard and macrae ) . several cases of human infection have suggested the possibility that infected rodent tissues can serve as a source of infection for laboratory personnel (baum et al. ; dykewicz et al. ; tobin ). humans may be infected by inhalation or by the contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. the transmission of lcmv by the bite of an infected mouse can also occur (scheid et al. ) . also, hotchin reported the findings of other researchers that lcmv was transmissible experimentally through the intact skin of the guinea pig, but this finding has not been reported in humans (hotchin ) . airborne transmission is well documented and plays a very important role in human infections, especially through the ready dispersion and inhalation of viral-contaminated dust from the animal cage or room (biggar et al. ; hinman et al. ) . following an incubation period of to weeks, humans may experience asymptomatic or a mild febrile disease ranging to a serious flu-like illness characterized by anorexia, malaise, diffuse myalgia and arthralgia, fever, chills, vomiting, headache, stiff neck, and photophobia. some patients enter a second stage of the disease several days after the resolution of early mild symptoms, developing meningoencephalitis and exhibiting additional signs such as drowsiness, confusion, sensory disturbances, and motor abnormalities. patients can also develop more serious nonneurological manifestations of the disease such as maculopapular rash, lymphadenopathy, parotitis, orchitis, arthritis, and epicarditis (peters ). central nervous system involvement has resulted in death in several cases. infections during pregnancy pose a risk of infection for the human fetus (wright et al. ). wright et al. reported cases in human infants, with lcmv confirmed serologically over a two-year period in a major u.s. medical center (wright et al. ) . these infants presented with ocular abnormalities, macrocephaly, and hydrocephalus with microcephaly. fifty percent of the mothers reported having had illnesses compatible with lcmv infection, and over half reported exposures to rodents during their pregnancies. the diagnosis of lcm infection in humans is currently made by serological testing using either the immunofluorescent antibody (ifa) test or the enzyme-linked immunosorbent assay (elisa) (barton et al. ) . both of these tests are available through the centers for disease control and prevention and are superior to the complement fixation test that is widely available commercially. although there are no proven effective antiviral therapies for lcm infection, intravenous ribavirin therapy reduces mortality in patients infected with lassa fever virus (a member of the old world arenavirus serocomplex) and may be of some benefit in patients with severe lcmv infections (andrei and de clercq ; mccormick et al. ). this disease can be prevented in the laboratory through periodic serological surveillance using elisa and ifa tests of newly introduced animals with inadequate disease profiles and of resident animal colonies at risk. thorough screening of all tumors and cell lines intended for animal passage using the highly sensitive mouse antibody production assay or newer pcr-based laboratory tests for the presence of lcmv is another crucial element in the program to prevent the introduction of lcmv into established animal colonies (besselsen et al. and chapter of this volume). sound animal facility sanitation practices and the use of microbarrier caging systems with proper infection-control techniques should prevent or suppress the spread of lcmv if present in the environment. the elimination of wild rodent infestations in animal facilities is very important to prevent the introduction of lcmv into the animal facility environment. also, facilities with wild rodent infestations may encounter the relatively common, free-living, bloodsucking mite of the rodent, ornithonyssus bacoti, in abundance (personal communication). although the natural lcmv transmission to humans from bloodsucking ectoparasites is unproven, the control of potential ectoparasitic vectors of this type would be a prudent measure. rabies is an acute, almost invariably fatal disease that occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. neither the laboratory mouse nor other small wild rodent hosts appear to be important as reservoirs of natural rabies infection. hence, the principal reason for our discussion of rabies as a zoonotic disease of the laboratory mouse is to provide information that should quickly allay the fears of uninformed research and animal facility staff who suffer the bite of a mouse. on the other hand, the experimental use of mice in the study and characterization of rabies virus and in rabies vaccine development is an important component of some animal care and use programs that deserves special attention by the institution during all phases of research planning and implementation. there are no known cases of human rabies from rodents in the united states ( ). the incidence in larger wild rodent species within the united states has increased in recent years, however. during the interval - , a total of cases of rabies in rodents were recorded, but from to approximately cases were reported in large rodents annually ( ) . the rodents involved were woodchucks and beavers, both species presumably large enough to survive the chance encounter and attack by a wild rabid carnivore such as the raccoon, skunk, fox, or feral cat. earlier literature on the federal republic of germany summarized findings from to , which indicated that three mice, one rat (species not given), nine norway rats, and three muskrats were infected with rabies and had bitten humans (scholz and weinhold ) . despite rare reports of this nature, rodents are not a proven source of rabies transmission to humans (johnson ). all mammals are generally regarded as susceptible to rabies. in humans, the course of the disease proceeds through several phases: incubation, prodromal, acute neurological, coma, and rarely, recovery (johnson ) . the incubation period varies from days to over months. during the prodromal stage lasting to days, patients experience a period of apprehension and develop headache, malaise, and fever. an abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. patients also may develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. further progression of the disease involves paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. adult mice used experimentally in rabies studies usually exhibit clinical signs between and days following inoculation and die within days of the onset of clinical signs coinciding with the period of viral shedding. clinical signs in the mouse consist of muscular tremors, incoordination, excitation, or paralysis. certain rabies virus isolates from skunks produce a spastic paralysis in adult mice followed by recovery in a high percentage of infected mice. also, infant mice inoculated with certain strains of street rabies virus are capable of full recovery (johnson ). personnel working with mice experimentally infected with rabies virus should adhere to the well-established and detailed procedures that have been described in other sources for animal inoculation, husbandry, and tissue harvest procedures (johnson ) . vaccination of personnel involved in rabies studies with laboratory animals also is clearly indicated, regardless of the animal species involved. four other viruses that produce natural infections in the mouse have been implicated previously or are known to be infectious for humans. these include hantavirus, sendai virus, reovirus , and mouse hepatitis virus. for none of these viral agents is there documented evidence of zoonotic transmission of the agent from naturally infected laboratory mice to personnel in the laboratory. hantaviruses are zoonotic viruses comprising at least species that are maintained among natural rodent reservoirs, despite the presence of neutralizing antibody in the rodent host (elliott et al. ; meyer and schmaljohn ) . approximately half of the hantaviruses are known human pathogens producing virus-specific patterns of disease that include hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and its benign form, nephropathia endemica (mills and childs ) . although mus musculus can exhibit the pattern of persistent hantavirus infection in the presence of neutralizing antibody when induced experimentally (araki et al. ) , this does not occur in natural infections of the mouse (meyer and schmaljohn ) . this is the likely reason that the wild mouse apparently is not important as a natural reservoir for the hantaviruses. this interpretation is supported by serological studies of wild mus musculus that detected either no or a very low incidence of serological evidence to hantavirus exposure even when other wild rodent species in the vicinity had a high level of endemic infection (kantakamalakul et al. ; meyer and schmaljohn ; pacsa et al. ; zuo et al. ) . mus musculus is used in the laboratory as an animal model to study various aspects of hantavirus infection ranging from vaccine development (choi et al. ) , viral pathogenicity (ebihara et al. ; kim and mckee ) , and immunological aspects of persistence (araki et al. ) , and it is primarily in this context that hantavirus deserves mention as a zoonotic infection in the mouse. the control of wild rodent infestations in animal facilities, quarantine and testing of wild caught rodent species during importation, and proper observance of animal biosafety guidelines for hantavirus-infected animals would be expected to virtually eliminate the risk of this zoonosis in laboratory maintained mice. mouse hepatitis virus (mhv) remains a prevalent infection in many mouse colonies where it potentially impacts colony health and disrupts experimental studies (see chapter ). earlier studies have demonstrated that human sera contained complement-fixing and neutralizing antibodies to mhv (hartley et al. ) . later studies suggested that this was most likely due to cross-reactive antibodies from human cold virus infections (bradburne ; mcintosh et al. ). mouse hepatitis virus and the two prototype human cold viruses (oc and e) are members of the antigenic group coronaviruses, and it is now known that members of this group share four, and in some cases five, structural genes that could account for this crossreactivity (navas-martin and weiss ). the coronaviruses have a very narrow host range and generally replicate only in the cells of the host species (navas-martin and weiss ). however, under unique laboratory conditions involving persistent cell culture infection, the use of mixed cell cultures of murine and of a nonpermissive species, or the use of cells possessing modified receptors, mhv has been adapted to grow in human, nonhuman primate, and hamster cells. also, the many strains of mhv in combination with use of targeted rna recombinant system have also been very useful for experimental study of the molecular basis of coronavirus pathogenicity (masters ) . application of the targeted rna recombinant system to mhv for exploring of emerging coronavirus infections such as sars may delineate the molecular basis for expanding of host range. these studies may warrant the reader's future attention, but at the present, mhv can be reasonably dismissed as a zoonotic infection. sendai virus was once a prevalent agent in mouse colonies but has become a rarity in most institutions. this is due to its ease of eradication through the use of temporary cessation of breeding to eliminate a naive population that is susceptible to infection and through the use of caging systems that prevent transmission (see chapter ). sendai virus was originally isolated and described in the s during the investigation of cases of human respiratory illness (gerngoss ; kuroya et al. a kuroya et al. , b sano et al. ; zhandoff et al. ). in the original report involving the isolation of sendai virus from japanese newborn human infants suffering from fatal pneumonitis, lung suspensions from the newborns were inoculated intranasally into mice, producing lung consolidation and death in several cases (kuroya et al. b) . in later studies, sendai virus isolates were reported to produce disease in human volunteers (kuroya et al. a; yamada ) , and reports from many countries indicated that serological evidence of sendai virus infection was associated with outbreaks of human respiratory illness (demeio and walker ; gardner ; jensen et al. ). tennant et al. demonstrated that personnel working with laboratory animals had antibody titers to sendai virus, but personnel with no known exposure to laboratory animals also had significant titers to the agent (tennant et al. ) . recombinant sendai virus is widely used for gene transfer experiments, and these vectors can readily infect human airway epithelium and a variety of other human tissues under experimental conditions (nagai ; pinkenburg et al. ) . although these recent studies have clearly demonstrated that sendai virus is capable of infecting human tissues, the initial evidence for its role as a human pathogen remains doubtful. the mice used for the early isolations of the agent may have already been endemically infected with sendai and served as the source, or it may be that other serologically cross-reactive parainfluenza viruses, which were not characterized during this era, were responsible for producing false positive reactions to sendai virus (ishida and homma ) . reoviruses are generally regarded as the cause of childhood infections producing asymptomatic or very mild disease, and there are few reports linking these infections with a particular disease (tsai ) . reovirus was originally isolated from the feces of a clinically ill child (stanley et al. ) , and it continues to receive attention as a possible etiology for neonatal hepatitis and extrahepatic biliary atresia in infants (richardson et al. ; steele et al. ) . reovirus is highly infectious for infant laboratory mice and still receives some attention in the health-screening programs for the mouse and laboratory rodent species. jacoby and lindsey ( ) reported that mouse colonies in the united states continue to have a to % prevalence of reovirus infection. although there are no confirmed reports of reovirus transmission from mice (or other laboratory animal species) to humans, the laboratory mouse should be considered a possible source for this infectious agent for humans and other susceptible species. the rickettsiae are fastidious, small pleomorphic coccobacilliary organisms maintained in nature in a cycle of infection involving mammalian hosts and arthropod vectors as reservoirs (saah ) . in most rickettsial infections, humans serve only as an incidental host and do not contribute to the propagation of the organism in nature. such is the case for rickettsialpox, a nonfatal, self-limiting zoonotic disease caused by rickettsia akari, which is perpetuated in a cycle of infection involving mus musculus as the primary host and a mite vector (liponyssoides sanguineus). isolation of the organisms from rats (rattus) and voles (microtus) has also been reported. the first cases of human rickettsialpox were described in patients in new york city (huebner et al. a (huebner et al. , b , and outbreaks of the disease have generally remained clustered within large urban areas of the united states and in rural north carolina, utah, south africa, korea, and the former soviet union . according to koss et al. approximately cases of rickettsialpox have been reported in the literature, with nearly of these within the three years following the original description of the disease . prior to the report by koss et al. the largest case study included patients accumulated over a -year period. the recent report by koss et al., however, included patients in new york city reporting over only a -month period in the wake of the september , attacks, suggesting that perhaps the heightened sensitivities to possible bioterrorism events stimulated an upsurge in the reporting of cases as a byproduct of increased patient concerns . authors have commented on a variety of other social and demographic factors that may also be contributing to the noticeable increase in the incidence of rickettsialpox and murine typhus in the urban environment (comer et al. ; paddock et al. ) . there are no reported cases of rickettsialpox in personnel related to exposure to naturally infected laboratory mice. the mite vector l. sanguineus has not been reported in laboratory mouse colonies either historically or contemporarily. however, the tropical rat mite (ornithonyssus bacoti) can be experimentally infected with r. akari but has not been shown to play a role in the natural cycle of infection. in the authors' experience of ornithonyssus bacoti infestations of laboratory mouse or rat colonies are still seen with some frequency in facilities that have resident wild or feral mouse populations and should be addressed in the institution's pest control and infection control programs. rickettsialpox has an incubation period of to days following the bite of the infected mite (saah mild to severe with an abrupt onset, and it typically presents with a classic triad of an eschar, fever, and a papulovesicular rash. the papule develops at the site of the bite and later ulcerates and progresses to an eschar, . to cm in diameter, as r. akari proliferates locally in the skin and vasculitis develops saah ) . the rash begins with firm, generally nonpruritic, erythematous papules, to mm in diameter, that develop into vesicles and heal by crusting. in addition to fever, patients may experience chills and headache, and less commonly, backache, myalgia, and photophobia. the disease is mild and self-limiting within to days, and serious complications or death have rarely been reported (saah ) . patients typically respond quickly after the initiation of antirickettsial therapy with tetracycline, doxycycline, or other appropriate agent saah ) . however, following resolution of the infection, headache and lassitude can persist for to weeks. the reader should refer to koss et al. for information on other rash-producing or eschar-related diseases that should be considered in the differential diagnosis to rickettsialpox . rickettsia akari are diagnosed by complement fixation tests or the more sensitive indirect immunofluorescent antibody test. serum antibody to r. akari generally takes weeks to develop, and paired sera are needed to confirm a four-fold rise in antibody titer (saah ) . during the acute phase of the infection, immunohistochemistry or pcr analysis can be used for a rapid diagnosis on biopsy material obtained from the papulovesicular rash or eschar paddock et al. ) . laboratory mice infected with r. akari develop fatal pneumonia with intranasal inoculation and severe illness and death with intraperitoneal inoculation. mice develop anorexia, depression, and dyspnea. peritonitis, splenomegaly, and lymphadenitis are found upon necropsy examination. subcutaneous inoculation produces an active infection for month, with organisms being recovered from the spleen but not the feces or urine (bell ) . the control and eradicaton of r. akari infections depend on the prevention of wild mice and the mite vector from entering laboratory animal facilities and human dwellings. murine typhus is a rickettsial disease caused by rickettsia typhi (previously r. mooseri) that occurs worldwide with epidemics or with high prevalence in particular geographic areas (dumler and walker ) . in the united states, most human cases of the disease are concentrated in texas and southern california. the disease is now predominantly associated with the rat as the primary host species for the oriental rat flea (xenopsylla cheopis), which serves as the principal ectoparasitic vector transmitting the disease to humans. however, the mouse can also serve as a host for this flea, as well as for the northern rat flea (nasopsyllus fasciatus) and the mouse flea (leptosylla segnis), which also bite man and can be involved in the transmission of r. typhi (flynn ; pratt and wiseman ; yunker ). an early report in the literature indicated that x. cheopis was easily established in an animal facility inhabiting rooms used for housing laboratory mice (yunker ) . it is now also known that the cat and opossum and the cat flea (ctenocephalides felis) can be involved in sustaining the cycle in some geographic localities (azad et al. ) . clark and will ( ) reported on use of the laboratory mouse as an experimental host for rearing x. cheopis, but there have been no reports of natural infestations of mouse colonies with any of the flea vectors of r. typhi for several decades. also, r. typhi has not been isolated from natural infections in laboratory mice. murine typhus is a more serious disease than rickettsialpox and presents with fever, headache, chills, nausea, and vomiting. splenomegaly, hepatomegaly, central nervous system involvement, and multiorgan failure can occur as severe and potentially fatal complications. a skin rash, which is typically maculopapular in the case of murine typhus, occurs much less commonly than in rickettsialpox, and its absence should not dissuade the clinician from making a diagnosis of murine typhus and from promptly instituting therapy due to the potential severity of the disease (dumler and walker ) . the methods used for the laboratory diagnosis and treatment of the disease in humans and the principles of preventing the introduction of r. typhi into laboratory animal colonies are similar to those for r. akari. leptospirosis microorganisms were discovered in when they were isolated from jaundiced patients (inada et al. ) ; after further study they were named in (noguchi ) . leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. rodent reservoir hosts of leptospirosis include, in addition to rats, mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (fox and lipman ; torten ) . human to human transmission is extremely rare. leptospira interrogans (comprising more than serovars) have been isolated worldwide (tappero et al. ) . l. interrogans contains serogroups with strains pathogenic for amphibians, reptiles, and mammals including humans. serovars australis, ballum, bataviae, hardjo, grippotyphosa, icterohemorrgagiae, javanica, and pomona are associated with rodent infections. leptospira serovars, including l. australis, bataviae, grippotyphosa, hebdomidis, icterohaemorrhagiae, pomona, and pyrogenes, are found in the house mouse (torten ) . leptospira ballum has also been reported from mice and is e. newcomer and james g. fox most commonly associated with zoonotic outbreaks (borst et al. ; friedmann et al. ; stoenner and maclean ) . although particular serovars usually have distinct host species, most serovars can be carried by several hosts. leptospira are well adapted to a variety of mammals, particularly wild animals and rodents. in the chronic form, the organism chronically infects the host and is shed in the urine inconspicuously for long periods of time. rodents are the only major animal species that can shed leptospires throughout their lifespan without clinical manifestations (fox and lipman ; torten ) . active shedding of leptospires by rodents can go unrecognized until personnel handling the animals become clinically infected or are infected by exposure to water or food contaminated by urine. rats and mice are common animal hosts for serotype, l. ballum, although it has been found in other wildlife as well. water can often be contaminated with infected rodent urine. the infection can persist unnoticed in laboratory rodents, though their carrier rates for laboratory-maintained rodents in the united states are unknown, but it is probably low. the organism is not routinely screened on health surveillance protocols for mouse colonies; however, there was a report of leptospirosis in in a research colony of mice in the united states being housed in a large research institution (alexander ) . l. icterohaemorrhagiaes antibody when compared to children living in the detroit suburbs. therefore, children living in rodent-infested tenements may be at increased risk of infection (demers et al. ) . in europe and more recently in north america, rodents including house mice have provided a source of leptospira infection for swine and by extension could also infect personnel working in swine production units (galton ; smith et al. ) . leptospira interrogans serovar bratislava is commonly reported in these mice. the disease may vary from unapparent infection to severe infection and death. a self-limited systemic illness is seen in approximately % of infected humans. the incubation period is usually to days. individuals infected with leptospira experience a biphasic disease (faine ; sanger and thiermann ; stoenner and maclean ) . they become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. during the second phase of the disease, conjunctival suffusion and a rash may occur. upon examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. intravenous penicillin is the drug of choice in treating early-onset and late-stage leptospirosis infection (faine ; taber and feigin ; watt et al. ). ampicillin and doxycycline also have been effective in treating people with mild to moderate forms of leptospirosis. because leptospirosis in humans is often difficult to diagnose, the low incidence of reported infection in humans may be misleading. from to , only cases were reported, for an incidence of . per , people per year (sanger and thiermann ) . leptospirosis was removed from the reportable disease category in the united states in because of the small number of cases reported. outbreaks have been documented in the united states from personnel working with laboratory mice (barkin et al. ; stoenner and maclean ) . in one study, of employees handling the infected laboratory mice ( % of breeding females were excreting l. ballum in their urine) contracted leptospirosis (stoenner and maclean ) . infection with leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning (barkin et al. ; friedmann et al. ; stoenner and maclean ) . skin abrasions or exposure to mucous membranes may serve as the portal of entry. all secretions and excretions from infected animals should be considered infective. in one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage (boak et al. ) . rodent bites can also transmit the disease (looke ). in detroit, children from the inner city had a significantly higher because of the variability in clinical symptoms and the lack of pathognomonic pathologic findings in humans and animals, serologic diagnosis or actual isolation of leptospires is imperative (faine ) . as an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions (sulzer et al. ). the definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in -to -week-old hamsters) and subsequent culture and isolation. culture media with long-chain fatty acids with % bovine serum albumin are routinely used as a detoxicant (faine ) . serologic assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (faine ) . the serologic test most frequently used is the modified microtiter agglutination test. titers of : or greater are considered significant. molecular techniques including pcr and randomly amplified polymorphic dna fingerprinting are used for identification of serovars (tappero et al. ) . in mouse colonies infected with l. ballum, antibodies against l. ballum were detected in sera of mice of all ages, but . zoonoses and other human health hazards leptospires could be recovered only from mature mice. progeny of seropositive females had detectable serum antibodies at days of age but not at days. it was also reported that progeny of seropositive female mice, which possessed antibody at birth and acquired additional antibody from colostrums, remained free of leptospires if isolated from their mothers at days of age, despite exposure during the nursing period (stoenner ) . studies in mice experimentally infected with l. grippotyphosa demonstrated that maternal antibodies, whether passed through milk or placental transfer, conferred protection of long duration against the carrier state and shedding of leptospires. thus, serologically positive immune mothers do not transmit the disease to their offspring. however, mice born to nonimmune mothers, if infected at day postpartum, become carriers with no trace of antibodies. thus a population of carrier pregnant mice without antibody could serve as a precipitator in outbreaks among susceptible mouse populations (birnbaum et al. ) . field surveys have supported this data in that a significant percentage of carrier mice do not have antibodies. this led to the diagnostic approach, which specifies that both serologic and isolation methods must be utilized to determine the rate of leptospiral infection in rodents (galton et al. ) . leptospira ballum is frequently found in the common house mouse (m. musculus) (brown and gorman ; yager et al. ) . therefore, eradication of infected colonies, use of surgically derived and barrier-maintained mice or of conventional laboratory mice free of leptospira infection, coupled with the prevention of ingress of wild rodents, should effectively preclude introducing of the organism into research and commercial laboratories (loosli ) . leptospira ballum has been eliminated from a mouse colony by administration of feed containing gm chlorotetracycline hydrochloride per ton for days. after days of antibiotic therapy, mice were transferred to clean containers and administered clean water, both having been sterilized by steam. mouse traps and rodenticides were used to destroy escaped mice and to prevent reintroduction of l. ballum by the common house mouse . commercial animal colonies maintained in research vivarium today are not routinely screened for leptospirosis, assuming that the organism has been effectively eliminated from commercial and research-maintained mice. rat bite fever can be caused by either of two microorganisms: streptobacillus moniliformis or spirillum minus. streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat bite fever, or streptobacillosis. haverhill fever and epidemic arthritic erythemia are diseases associated with ingestion of water, food, or raw milk contaminated with str. moniliformis. sodoku, derived from the japanese words for rat (so) and poison (doku), spirilosis, and spirillary rat bite fever are caused by another bacterium, spirillum minus. the bite of an infected rat is the usual source of infection. in some cases, other animal bites, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact, cause the infection. in a retrospective analysis coveting three decades of s. moniliformis isolates ( % from humans) from the department of public health in berkeley, california, % of the isolates were from children < years of age (graves and janda ) . in % of the human infections where a diagnosis was made, rat bite fever (rbf) was suspected; % of those suspected cases involved either known rat bite or exposure to rodents. two cases of rbf were attributed to exposurem in one case a squirrel, and in the second a mouse (graves and janda ). interestingly, > % of the cases could not be attributed to a rat bite or scratch, indicating that close contact with infected rodents can be sufficient to become infected (graves and janda ) . other reports have indicated that the disease can occur in individuals who have no history of rat bites, but reside or work in rat-infested areas or have pet rats with whom they have close contact (fordham et al. ; holroyd et al. ; rumley et al. ) . rat scratches can also be the source of the organism (edwards and finch ; shanson et al. ) . exposure to cats and dogs that prey on wild rodents may also be the source of the organisms. these organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (wilkins et al. ). mice can be infected with resulting morbidity and mortality due to arthritis and pneumonia. in one study, streptobacillus moniliformis was isolated as the predominant microorganism from the upper trachea of laboratory rats (paegle et al. ) . presumably the incidence of str. moniliformis is now lower in high-quality, commercially reared specific pathogen-free rats. surveys in wild mice indicate to % infection with spirillum minus (hull ) . ( ). streptobacillus moniliformis incubation varies from a few hours to to days, whereas spirillum minus incubation ranges from to weeks (table - ) . fever is present in either form. inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills (arkless ; cole et al. ; gilbert et al. ; mcgill et al. ) . the discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. arthritis occurs in % of all cases of s. moniliformis but is less common in spirillum minus. streptobacillus moniliformis may be cultured from serous to purulent effusion, which is recovered from affected larger joints. a total of cases of endocarditis due to s. moniliformis were reported from to (shvartsblat et al. ). death has occurred in cases of s. moniliformis involving preexistent valvular disease or as a result of endocarditits in a previously healthy individual. infants can also die of the infection (sens et al. ) . if antibiotic treatmentmusually penicillin at doses of , to , daily for daysmis not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (richter ). if endocarditis is present, the penicillin should be given parenterally at doses of to million units daily for for weeks. streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-associated allergies. addition of streptomycin to standard therapy is also advised in cases where isolates of sir.. moniliformis are cell wall deficient (rupp ) . spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. streptobacillus moniliformis grows slowly on artificial media but only in the presence of % blood and sera, usually % to % rabbit or horse serum incubated at reduced partial pressures of oxygen. sodium polyanethol sulfonate sometimes found in blood-based media because of its properties as a bacterial growth promoter should not be used due to its inhibitory effects on sir. moniliformis (lambe et al. ; shanson et al. ) . growth on agar consists of to mm gray, glistening colonies. the api zym diagnostic system can be used for rapid biochemical analysis and diagnosis. a pcr-based assay has also been described to diagnose sir. moniliformis (berger et al. ) . the genus salmonella are gram-negative bacteria with approximately serotypes. nontyphoidal salmonellosis is caused by any of these serotypes. other than salmonella typhi, the causative agent of typhoid fever, salmonellosis occurs worldwide and is important in humans and animals. s. typhi and salmonella choleraesuis have only one serotype, whereas the remaining serotypes are within the species salmonella enteritidis. references to the salmonella enteritidis serotypes are abbreviated such that "enteritidis" is dropped; for example, s. enteritidis serotype typhimurium is called salmonella typhimurium. salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. other serotypes most commonly reported from humans and animals are salmonella heidelberg, salmonella agona, salmonella montevideo, and salmonella newport. salmonellae are pathogenic to a variety of animals. salmonella are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. fecal-oral transmission is the primary mode for spreading infection from animal to animal or to humans. transmission is enhanced by crowding and poor sanitation. during the early s, rodenticides containing live cultures of s. enteritidis were distributed on a large-scale basis by commercial and public health organizations in an attempt to eliminate feral rats. these cultures were known as "rat viruses" and were widely used in europe, england, and the united states as "rat poisons" (weisbroth ). however, enthusiasm for their use waned when it was discovered that the spread of the organisms couldn't be limited; predictably, the baiting program was implicated in several epidemics among exposed human populations (weisbroth ) . surprisingly, as late as the s in england, s. enteritidis (serovar danzy) was isolated from adults living four miles apart. the source of infection was traced to contaminated cakes from a local bakery. mice that had acquired the (brown and parker ) . as with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with salmonella or animal reservoirs excreting the organism. salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. fat and moisture in food promote survival of salmonella. pasteurization of milk and proper cooking of food ( ~ for to minutes) effectively destroys salmonella. municipal water supplies should be routinely monitored for coliform contamination (pavia and tauxe ) . clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. diarrhea and anorexia may persist for several days. organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. as with other microbial infections, the severity of the disease relates to the organism's serotype, the number of bacteria ingested, and the host's susceptibility. in experimental studies with volunteers, several serovars induced a spectrum of clinical disease ranging from brief enteritis to serious debilitation. incubation varied from to hours. cases of asymptomatic carriers, persisting for several weeks, were common (hull ) . salmonella are flagellated, nonsporulating, aerobic gramnegative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. salmonella serotyping requires antigenic analysis (fox ) . salmonella gastroenteritis is usually mild and self-limiting. with careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. in humans, antimicrobial therapy may prolong rather than shorten the period that salmonella are shed in the feces (nelson et al. ; pavia and tauxe ) . in one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of salmonella carriage. bacteriologic relapse after antibiotic treatment occurred in % of the patients, and % of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (nelson et al. ). tick-borne relapsing fever occurs primarily in foci in the western part of the united states, as well as other parts of the world. the disease is caused by at least borrelia species and is transmitted to humans from a variety of rodents (chipmunks, squirrels, rats, mice, prairie dogs, hedgehogs) via soft ticks of the genus ornithodorus. of recent interest are the increasingly recognized enterohepatic helicobacter spp., which cause both hepatic and intestinal disease in mice (whary and fox ) . one of these, h. bilis, isolated routinely from mice, has been found using pcr-based assays in bile and gallbladder of chilean patients with chronic cholecystitis and in biliary cancers in japanese patients (fox et al. ; matsukura et al. ) . whether these new helicobacters will be linked to zoonotic transmission from wild or laboratory rodents will require further studies. pathogenic staphylococcus aureus of human phage type can cause clinical disease in mice and rats. this organism has been (davies and shewell ) lab worker % not determined, alopecia, increased scaling on head (booth ) and back, mice bacteriologist of , crusted or crustless plaques, circular with (cetin et al. ) prominent periphery; general alopecia; mortality in some mice technician % colony with alopecia and scaly skin (dolan et al. ) technician alopecia with crusting an erythema (povar ) introduced into spf barrier-maintained mouse colonies and spf rats and guinea pigs; the same phage type was isolated from their animal caretakers (davey ; shults et al. ) . colonization by normal s. aureus strains in the nasopharyngeal area of humans presumably minimizes the zoonotic potential of animal-originated s. aureus. as reviewed comprehensively in blank, reports of ringworm (favus) in the mouse began to appear in the european literature in the mid-nineteenth century and in the north american literature during the early twentieth century (blank ) . several of the early authors noted the similarities between the lesions of favus in the mouse and in humans. quincke, who is generally credited with isolating the causative agent which he named ~-pilz (now trichophyton mentagrophytes), suggested that the infection in the mouse was also a source of infection of the cat, and thereby, of humans. earlier reports of murine ringworm referred to the causative agent as t. quinckeanum, but the successful crossing of t. quinckeanum with the perfect state of t. mentagrophytes, arthroderma behamiae, indicates that t. quinckeanum is not a distinct species (ajello et al. ) . a later study of the two varieties, t. mentagrophytes var. mentagrophytes and t. m. var. quinckeanum, noted that the conidia from both produced two morphological variants on cultivation (granular and fluffy), and these variants were a. behamiae type + and pathogenic (hejtmanek and hejtmankova ) . in addition to t. mentagrophytes, epidermophyton floccosum, mircrosporum gallinae, m. gypseum, m. canis, t. erinacai, t. schoenleini, and t. (keratinomyces) ajello have been reported as zoophilic dermatophytes that can infect mice and cause ringworm in humans (dvorak ; krempl-lamprecht and bosse ; marples ; papini et al. ; refai and ali ) . the dermatophytes are distributed worldwide and can involve a variety of small animal host species in addition to the mouse. chmel et al. ( ) conducted field studies in a wooded farm setting in czechoslovakia and detected an overall prevalence rate of . % ( positive of ) for t. mentagrophytes infection in of species sampled; the prevalence in mus musculus was . %, with mice comprising . % of the infections detected. of the species that harbored the infection, all frequented the barn or granary area; the seasonal incidence was highest during the winter months when the rodent carriers were more likely to seek harborage indoors. chmel et al. ( ) also analyzed patient data and demonstrated that t. mentagrophytes was the predominant isolate from those who did agricultural work, while t. verrucosum was the main isolate from individuals who worked with farm animals. also, human t. mentagrophytes infections were most common on the hands, wrist, forearm, face, and neck, unprotected skin sites readily contaminated by fodder, litter, or other materials while working in the barns. ringworm infections associated with the handling of bags of grain in which mice had been living have also been reported (alteras ; blank ) . ringworm infection in laboratory mice is often asymptomatic, remaining unrecognized until laboratory personnel become infected. early reports in the literature indicated that the prevalence of t. mentagrophytes was to % among some laboratory mouse stocks (davies and shewell ; dolan et al. ). however, these reports predated the era of modem laboratory animal colony management marked by the commercial availability of cesarean-derived, barrier-maintained rodents. moreover, the modem production practices that have been universally adopted by the industry for decades and the use of microbarrier cages with appropriate technique have further reduced the opportunity for ringworm to become a significant problem in contemporary colonies. in recognition of this fact, none of the major commercial vendors in the united states survey their colonies for dermatophyte infections as part of routine health monitoring. sporadic cases of ringworm infections in rodents have been reported in the past three decades (hironaga et al. ; mizoguchi et al. ; papini et al. ) . programs involved in importing mice from sources that fail to meet contemporary rodent production and husbandry practices should consider screening mice for dermatophytes during the quarantine period. the ease of transmission of dermatophytes from animals to humans is well known and is a significant health hazard. laboratory mice, as well as other laboratory animal species, can harbor dermatophyte infection, with few or no visible skin lesions transmitting the infection to unsuspecting personnel (dolan et al. ) . transmission can occur through direct contact with the infected animal or through indirect contact with animal bedding or other materials in the environment of the contaminated animal room. rigorous facility and equipment sanitation has long been recognized as an essential element of an effective control program and should be undertaken in conjunction with efforts to treat valuable animals or to repopulate previously contaminated areas of a facility (davies and shewell ; dolan et al. ; mizoguchi et al. ). the importance of barrier protections by donning appropriate clothing, using gloves and other personal protective equipment, and modifying work practices to minimize skin exposure to dermatophytes has also been acknowledged for the prevention of transmission (dolan et al. ) . when prevention methods fail, allowing the introduction of dermatophyte infection into a mouse colony, and when transmission to humans occurs, clinical cases of dermatophytosis routinely respond well either to topical or systemic antifungal therapy. *found in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature (see yunker ) . **wee, western equine encephalitis. +sle, st. louis equine encephalitis. ++rmse rocky mountain spotted fever. dermatophytosis or ringworm in humans can be asymptomatic and minor, often self-limiting and drawing little attention from the affected individual. the infection usually causes an expanding, scaly and erythematous inflammatory plaque on the skin that occasionally contains fissures or vesicles when severely eczematous. on the trunk and extremities, the lesion may consist of one or more circular lesions with a central clearing and sharply defined margins, forming a ring, and hence the name "ringworm" (fig. - ) (merlin et al. ) . other dermatophytes are named according to the sites of involvement on the body (e.g., tinea pedis for foot infections, tinea capitis for scalp infections). the dermatophyte infections of humans associated with direct or indirect contact from mice usually involve the body or extremities, especially the arms and hands. zoophilic t. mentagrophytes infection usually produces a highly inflammatory lesion and often undergoes rapid resolution. however, it can also produce furunculosis--deep infection of the hair follicles or widespread tinea corporism which is also seen in infections of e. f l o c c o s u m . in a laboratory-acquired infection with t. (keratinomyces) ajelloi, mice were the source of infection for a laboratory technican who developed small, grayish-white, scaly lesions on both hands. hand lesions yielded the organism, as did of apparently health mice (refai and ali ) . a. tapeworms a. reservoir and incidence although this parasite occurs in the mouse intestine, it is more commonly associated with rats and is especially common in wild norway (rattus norvegicus) and black (rattus rattus) rats throughout the world (faust and russell ; stone and manwell ; wardle and mcleod (voge and heyneman ) . larval development in tribolium sp. at ~ requires days. therefore, humans become infected only through ingestion of infected insects, such as flour beetles, which may contaminate rodent food or cereal marketed for human consumption. c. clinical signs the infection in humans is usually asymptomatic, but in moderate to heavy infections it may cause headaches, dizziness, abdominal discomfort, and diarrhea. the greatest length of an adult parasite removed from a patient was meter. usually, adult parasites are to cm long and as much as mm wide (markell et al. ). a. reservoir and incidence the dwarf tapeworm is a common parasite of both the wild house mouse and the laboratory mouse. as indicated earlier in the text, in most well-managed mouse colonies, r. nana incidence is low compared to earlier reports of its high incidence in rodent colonies (wescott ) . the estimate that million humans in the world are infected was made many years ago but probably is an underestimate (markell et al. ) . surveys conducted in central europe report that this tapeworm in humans is more prevalent in warm than in temperate regions. an incidence of % has been noted in some south american countries (jelliffe and stanfield ) . it is most commonly diagnosed in children. diagnosis is made by observing characteristic eggs in the feces. b. mode of transmission r. nana is unique among tapeworms in that the adult worm develops after the egg is ingested. the hooked oncosphere then invades the intestinal mucosa and develops into a cysticeroid larva. rodentolepsis nana eggs can contaminate hands, be trapped on particulate matter, or be aerosolized, and then accidentally ingested. since no intermediate host is required, the eggs are readily infective for the reciprocal hosts (faust and russell ) . precautions against infection include strict personal hygiene, appropriate laboratory uniforms, and use of disposable gloves and face masks when handling contaminated bedding and feces. c. clinical signs the clinical picture of r. nana infection is quite cosmopolitan. in well-nourished persons, essentially no symptoms occur; the infection is noted when the proglottids or ova are seen in the stool. in other persons, the symptoms include headaches, dizziness, anorexia, inanition, pruritis of the nose and anus, periodic diarrhea, and abdominal distress. a tapeworm identified as r. nana was found in a tumor removed from the chest wall (jelliffe and stanfield ) . the diagnosis is based on identification of the characteristic eggs or proglottids in the stool. d. treatment praziquantfel, given orally in a single dose of mg/kg body weight is the drug of choice. alternatively, niclosamide is given daily for days because of the tissue phase of the parasite. the dose is gm for adults and . gm for children > kg, and . gm for children between and kg (markell et al. ) . recently, a parasite known to naturally colonize mice, r. microstoma, has been identified in the feces of humans living in the northwest of western australia (macnish et al. ) . although r. nana was the most common enteric parasite based on microscopic examination of feces, r. microstoma was identified as a mixed infection in of individuals by using a molecular-based assay consisting of restriction fragment length polymorphism of tapeworm dna as well as a sequencing of the pcr product of the internal transcribed spacer region of ribosomal dna (macnish et al. ) . given that r. microstoma requires an intermediate host, tribolium confusum for its life cycle, it is understandable why it was not as common as r. nana in this study. however, given the morphological similarities of the eggs of r. nana and r. microstoma, the true prevalence of r. microstoma in humans won't be known until molecular techniques to differentiate the two species are utilized in future studies. syphacia obvelata is an ubiquitous parasite in both wild and laboratory mice. although parasitology texts report that syphacia is infectious to humans, this citation originates from a publication in , in which two s. obvelata adult worms and eggs reportedly were found in the formalin preserved feces of a filipino child whose entire family of five was infected with h. nana (riley ) . no mention is made of the method of collecting the feces, nor is it known whether the feces could have been contaminated with murine feces or with the parasite and/or eggs. the only other report is an unpublished finding of s. muris eggs in the feces of two children and two rhesus monkeys, cited in a personal letter from dr. e. e. faust of tulane university, dated january , (stone and manwell ) . both of these cases may therefore be examples of spurious parasitism, but definitive information for that conclusion is lacking. regardless, no published information indicates that laboratory personnel have been infected by working with syphacia-infected mice. contamination of food or utensils or accidental ingestion of syphacia ova (e.g., via contaminated hands) could result in infection of humans. people working with infected mice probably ingest ova occasionally, but there is no evidence that this exposure results in active infection. because syphacia infection in humans has not been described, clinical signs have not been noted. there are striking differences in size between specimens of female s. obvelata and those of enterobius vermicularis, the pinworm, in humans (markell and voge ) . syphacia is . to . mm long, whereas the enterobius sp. female reaches a length of to mm. the male syphacia sp. measures . to . mm compared to . mm for enterobius. the size difference between the eggs of the two species is also marked: syphacia eggs are more than twice as long ( gm versus /am as those of enterobius). it is unlikely therefore that syphacia sp. would be misdiagnosed as enterobius sp., assuming, of course, that the observer was aware of the size difference and measured the eggs. although many species of mites are found on laboratory mice, only ornithonyssus bacoti, the tropical rat mite, and liponysoides sanguineus, the house mouse mite, are vectors of human disease. ornithonyssus bacoti is seen in laboratory mice (fox ) ; l. sanguineus has been identified only on wild mice. bites from these mites, as well as from another mouse mite, haemalaelaps casalis, are responsible for allergic dermatitis, or local inflammation, in humans. ornithonyssus bacoti can be found on many rodents; the brown norway rat and the black roof rat are probably the primary host species (beaver and jung ) . since the time of the first report of human ornithonyssus bacoti-associated dermatitis in australia in , and a report in humans in the united states, many other cases have continued to be described throughout the world (see table - ) (charlesworth and clegern ; chung et al. ; dove and shelmire ; dowlati and maguire ; engel et al. ; fox ; haggard ; hetherington et al. ; riley ; theis et al. ; wainschel ; weber ) . ornithonyssus bacoti is an obligate bloodsucking parasite, usually tan but red when engorged with blood. both the male and female feed on a rodent as their preferred host. the female is ~tm to mm in length; the male is smaller (fig. - ) . eggs are laid in bedding or wall crevices by the female, which survives for about days and feeds about every two days during this period. the mite has five developmental stages: adult, egg, nonfeeding larva, bloodsucking protonymph, and nonfeeding deutonymph. after feeding, the adults and protonymphs leave their host and seek refuge in cracks and crevices. the life cycle from adult to egg requires to days at room temperature. unfed protonymphs have survived for days (brettman et al. ) . the mite often gains access to the premises on wild rodents and lives in crevices. if wild rodents are not readily available or are captured, the mite will seek blood elsewhere, either from the wild or laboratory rodent (if in an animal research facility) and/or humans. in some infestations, the rodent shows no clinical signs. however, in more chronic cases, dermatitis and anemia may develop. historically, this mite has been a troublesome parasite in certain laboratory animals, especially rats, mice, and hamsters (fox ; keefe et al. ). a. clinical signs tropical rat mites produce painful, pruritic lesions in humans. examination of patients often discloses papular lesions on the wrists, arms, abdomen, and chest. raised erythematous papules and nodules several millimeters to greater than cm in size occur singly or in linear configuration ( fig. - ) . epidemiologically, cases usually occur in clusters that involve a common source of exposure to the mite. experimentally, cases have been shown to be a vector of pathogens. in the laboratory, mite transmission of various rickettsial species, pasteurella tularensis, and coxsackie virus between different laboratory animals has been shown (hopla ; petrov ; philip and hughes ; schwab et al. ) . affected individuals may be treated with topical lindane or treated symptomatically, given that the mite does not reside on humans for any extended periods. papular dermatitis will regress after a period of to days post-therapy. recurrence of ornithonyssus bacoti infestations is common unless the premises have been treated with an appropriate insecticide, and any feral rodents eradicated (engel et al. ). fleas are seldom found in laboratory mice but are common parasites of feral rodents. the oriental rat flea, xenopsylla cheopis, and another flea, nasopsyllus fasciatus, both naturally infest mice and rats; they are vectors for murine typhus. apparently, x. cheopis is easily established in animal facilities. at a midwestern u.s. university, it inhabited a room housing laboratory mice where, on two separate occasions, the flea caused distress by biting students (yunker ) . leptopsylla segnis, the mouse flea, bites humans and is a vector for plague and typhus, serious diseases in humans. also, l. segnis can serve as an intermediate host for the rodent tapeworms r. nana and r. diminuta, which can infect people. the flea's bite can also be irritating and cause allergic dermatitis. epidemiological perspective on the transmission of infectious diseases, principally rabies. the bite of the rat is far more powerful and more likely to be disfiguring than that of the mouse, and rat bites are known to be associated with the transmission of bacterial zoonoses such as rat bite fever and leptospirosis (see elsewhere in this chapter). one should assume that the mouse is also capable of transmitting these agents via bite. rabies transmission from small rodents in the wild occurs but is exceedingly rare (gdalevich et al. ) ; therefore, rabies is of concern only if experimental studies with the virus are being conducted in mice. mice can also transmit hantavirus infection and lymphocytic choriomeningitis virus infection via bites. anecdotally, most animal care and use programs report that rodent bites among personnel are a reasonably common occurrence that often are unreported to an institution's occupational medical service, despite the fact that bites inflict pain, produce anxiety, and may have significant health consequences. in addition to the hazard of zoonotic disease or local wound infection with pyogenic or toxin producing bacteria such as clostridium tetani, staphylococcus spp., streptococcus spp., escherichia coli, and bacillus subtilis, rodent bites, including those of the mouse, can induce a severe local allergic reaction or anaphylaxis in individuals previously sensitized to allergen (hesford et al. ; teasdale et al. ; thewes et al. ) . thus, bite wounds from mice should be immediately cleaned thoroughly and reported to the institutional occupational medical service to permit evaluation of the person's tetanus immunization status and need for additional local wound or other medical care. the need for additional training of bitten persons in animal handling may also be indicated. authoritative information on the incidence and impact of animal bites in the general population over the past several decades is scant, and reliable data on the incidence of mouse bites among personnel who work in laboratory animal facilities or among the general populace is lacking. there have been occasional studies on the occurrence and clinical characteristics of rat bites within urban populations, including a recent investigation of bites over the period - that associated this phenomenon with urban blight, poverty, and unemployed populations (hirschhorn and hodge ) . traditionally, animal bites have received attention from the clinical perspective of wound management and complications and from the allergic skin and respiratory reactions to laboratory mice are very common in laboratory animal caretakers and technicians who work with these animals. a survey by lutsky ( ) demonstrated that three-fourths of all institutions with laboratory animals had animal care personnel with allergic symptoms. the prevalence of symptoms of laboratory animal-associated allergy (laa) among personnel working with laboratory animals has been estimated as between and % in numerous recent studies, and among these individuals, approximately % are estimated to eventually proceed to the development of asthma (chan-yeung and malo ; eggleston and wood ; hollander et al. ; hunskaar and fosse ; knysak ; renstrom et al. ) . furthermore, other sources have suggested that among atopic individuals with preexisting allergic disease, up to % of persons exposed to lab animal allergens may eventually develop laa (committee on occupational health and safety in research animal facilities/national research council allergens ). the population at risk for work-related exposure to rodents was estimated at , (newill et al. ); this population has likely grown in the intervening years to the expanding populations of genetically modified mice that are used in contemporary biomedical research programs and require care. moreover, a recent study would seem to suggest that the risk of exposure to mouse allergens is not confined to those working in the laboratory animal facility environment. data analyzed from the first national survey of lead and allergens in housing in the united states demonstrated that % of homes of diverse types and income levels across geographic locations had evidence of mouse allergen; % had detectable levels on the kitchen floors specifically; and % had allergen concentrations greater that . ~tg/g of dust collected, a level previously correlated with the significantly increased rate of sensitization to mouse allergen (cohn et al. ) . the large number of staff at risk of exposure in the workplace or already presensitized, in combination with the substantial added costs to employers for the medical management, operational disruptions, and retraining efforts related to employees who develop laa and later proceed to asthma, should provide the impetus for many institutions to pay grater attention to this element of the occupational health and safety program (schweitzer et al. ) . the major allergen of the laboratory mouse is the mus m protein, a member of the mouse major urinary proteins encoded by a multigene family consisting of approximately genes (clark et al. a (clark et al. , b . the earlier literature on the subject of mouse allergy referred to the mouse urinary proteins (mups), whereas recent literature cites the specific protein (mus m ) that is now known to be the primary offending allergen in the mup multigene family. the mus m protein is in the lipocalin family of proteins that are produced in the saliva and liver and are excreted in the urine at levels times higher than are present in mouse serum. lipocalins serve to bind small hydrophobic molecules and function biologically to transport vitamins, small volatile odorants, and pheromones conferring the characteristic odor to mouse urine (cavaggioni et al. ; flower et al. ; konieczny et al. ; santa et al. ; virtanen et al. ) . several studies have indicated that production of mus m is under hormonal control and that the urine of male mice contains four-fold higher levels than the urine of female mice (hastie et al. ; lorusso et al. ; price and longbottom ) . in addition to being present in the saliva and urine, mus m in the serum becomes incorporated into the pelt, conferring the allergenic property to mouse dander. the main allergens of many furred animals are structurally similar proteins within the lipocalin family, including those of the cow (bos d ), horse (equ c ), dog (can f ), and rat (rat n ) (virtanen et al. ) . the mus m and rat n lipocalin allergens, to which % of mouse and rat allergic individuals react, respectively, are closely related, sharing a % homology (clark et al. a) . some have proposed that personnel exposed to laboratory animal allergens can be categorized into four basic risk groups based on their history of allergic disease and sensitization to animal proteins (committee on occupational health and safety in research animal facilities/national research council allergens ). these risk groups are ( ) normal individuals, ( ) atopic individuals with preexisting allergic disease, ( ) asymptomatic individuals with ige antibodies to allergic animal proteins, and ( ) symptomatic individuals with clinical symptoms upon exposure to animal allergens. individuals in the normal risk group do not have a history of allergic disease, and % will never develop symptoms of laa. if laa develops in individuals in the normal risk group, it usually appears during the first three years of exposure. however, infrequently individuals in this risk group who have remained free of laa for or more years of exposure have developed a delayed onset of the condition (department of health and human services, national institute of occupational safety and health ). atopic individuals have a genetic predisposition for an exaggerated tendency to mount ige responses to common environmental allergens. atopic individuals have higher total levels of ige in the circulation and higher blood eosinophil counts compared to normal individuals, possibly as a result of the activation of cytokines involved in ige isotype switching, eosinophil survival, and mast cell proliferation (janeway et al. ). among atopic individuals, up to % of those exposed to allergenic animal proteins eventually develop symptoms (agrup et al. ) . in asymptomatic individuals with elevated circulating ige antibodies to animal allergens, up to % are at risk of developing allergic symptoms. of the individuals in risk groups that are already symptomatic for laa, approximately % will develop chest symptoms and % are likely to develop occupational asthma and face the prospect that continued exposure will result in permanent impairment. allergic rhinitis, allergic conjunctivitis, and contact urticaria are the most common disorders seen in laa (committee on occupational health and safety in research animal facilities/ national research council allergens ). clinically, allergic rhinitis and conjunctivitis present with the symptoms of sneezing, clear nasal discharge, nasal congestion, itchiness, and watery eyes. contact urticaria presenting as raised, circumscribed, erythematous lesions may also be present in laa patients who report an intense itchiness to the skin in the area of contact with the allergen. figs. - and - (fox and brayton ) illustrate the typical wheal and flare reaction on the skin provoked in an individual who had developed hypersensitivity to mouse urine over a period of several years and who was exposed by having a mouse with urine-contaminated feet walk over his arm (ohman ) . one large survey of laboratory animal workers summarized in the niosh alert (department of health and human services, national institute of occupational safety and health ) reported that of animal workers from animal facilities, % developed allergic symptoms related to laboratory animals. of the workers reporting symptoms, % had nasal or eye symptoms, % had skin complaints, and % had asthma. patients who develop asthma as a more serious complication of laa manifest symptoms of wheezing, intermittent dyspnea or shortness of breath, cough, often nocturnal or in the early morning, and tightness of the chest. the key clinical sign in these patients is wheezing on auscultation, and physiological abnormalities include airflow obstruction, which may vary over time, bronchodilator responsiveness, and increased airway responsiveness (airway hyperreactivity) (tang et al. ) . though quite rare, generalized anaphylactic reactions that are potentially life threatening can occur in individuals highly sensitized to animal allergens. anaphylaxis may manifest as diffuse itching, hives, and swelling of the face, lips, and tongue. in some individuals, breathing becomes difficult owing to laryngeal edema, and others develop asthma and wheezing. laboratory animal-associated allergy is an example of the type i, ige antibody-mediated, immune reaction, and the reader should refer to other sources for a detailed discussion of the molecular mechanisms involved in developing this reaction (janeway et al. ). in the case of animal allergens, the usual route of initial exposure is airborne, although bite exposures (saliva) and direct contact with the skin can also become important in later clinical symptoms. in the type i reaction, upon exposure to antigen, which is often a protein or glycoprotein, the allergen is taken up and processed by cells of the innate and adaptive immune systems and by dendritic cells located in the mucosal-associated lymphoid cells, gut-associated lymphoid cells, and/or the dermis. the cytokine profiles of these cells favor the development of na'fve cd t cells into th cells that induce b cells to produce ige specific for the allergen. once the ige response is initiated, it can be further enhanced by basophils, mast cells, and eosinophils that also drive allergen-specific ige production (janeway et al. ) . ige is normally found only in low levels in the circulation because it binds to tissue mast cells and circulating basophils. in the sensitized individual, restimulation with the sensitizing allergen results in allergen binding to ige and the release of histamine and other chemical mediators from the mast cells and basophils. these mediators produce the array of clinical signs and symptoms that are characteristic of the allergy: itchiness, nasal congestion, sneezing, nasal and ocular drainage, coughing, wheezing, and shortness of breath. to establish the diagnosis of laa related to mouse exposure, the physician should begin by considering the strength of the history, physical examination findings, the temporal relationship between the patient symptoms and the environmental exposure to mice, and possibilities of alternative explanations for the patient's problems such as exposure to other potential allergens in the workplace or allergens of a nonoccupational nature. the development of clinical symptoms concomitant with or following exposure to an environment containing mice or mus m laden mouse products should help in narrowing the number of allergens tested. the patient's family history of allergy is also very important to consider because atopy is a proven risk factor in developing laa (botham et al. ; meijer et al. ; venables et al. ). physical examination of the patient and clinical monitoring for the progression of allergic disease incorporate a number of approaches. pulmonary function tests such as bronchial hyperresponsiveness (in response to pharmacologic challenge with methacholine and not the specific allergen) and the forced expiratory volume in one second (fev ) are commonly used to evaluate the degree of airway impairment and the response to bronchodilators, glucocorticoids, and other therapeutic agents. radiographs may also be useful in patients with pulmonary involvement. routine laboratory tests may also aid in the characterization and management of the patient's condition, such as complete blood count and nasal smears for eosinophilia which is common in allergic individuals but also can be seen in those with perennial nonallergic rhinitis (dykewicz et al. ) . measurement of total serum ige has little value to the physician as an aid in distinguishing whether a particular patient has allergic disease, but it may offer some potential for the identifying of populations at risk for developing of laa as indicated in both prospective and cross-sectional studies of laboratory animal workers (hollander et al. ; renstrom et al. ) . use of the radioallergosorbent test (rast) for the detection of human ige antibodies of defined allergen specificity is also available for patient evaluation. however, the quality of the laboratory performing the in vitro rast assays, the specificity of the allergens used, and the potential for cross-reactivity are important considerations in adopting the rast as a diagnostic tool (hamilton ) . even when properly conducted, in vitro tests usually fail to detect a modest number of skin testpositive individuals, and on a per-test basis, skin tests have lower time and reagent costs (hamilton ) . clinicians agree that when properly performed, prick-puncture skin tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in most patients (demoly et al. ) . the valid interpretation of these tests relies on standardized allergens and methods, and negative prick-puncture tests may be confirmed by more sensitive intradermal techniques. even after falsepositive and false-negative tests have been eliminated, the proper interpretation of results requires thorough knowledge of the patient's history and physical findings. a positive skin test alone does not confirm a definite clinical sensitivity to an allergen in the asymptomatic patient but possibly predicts the onset of allergic symptoms. a positive skin test in conjunction with a history suggestive of clinical sensitivity strongly indicates the allergen as the cause of the disease (horak ) . strong positive skin tests along with a suggestive clinical history also correlate well with results of bronchial or nasal challenges with the antigen. the animal facility conditions and practices that contribute to mouse-associated laa as a serious and prevalent workplace hazard have received considerable study over the past several decades, enabling effective strategies for achieving control of exposures in most research animal care and use settings. in summary, these strategies involve exposure reduction through source reduction, containment of hazard through the use of modern equipment and engineering controls, and barrier protection with personal protective equipment. the mus m allergen load in the environment is markedly increased when male mice are used in studies due to the fact that they excrete -fold higher levels of allergen in the urine than do female mice (lorusso et al. ). therefore, sources have recommended, that whenever scientifically possible, use of only female mice would be a means of reducing allergen load in the environment and minimizing the exposure of personnel (department of health and human services, national institute of occupational safety and health ; renstrom et al. ). furthermore renstrom et al. ( ) reported a three-fold higher rate of allergic sensitization in technicians who worked with male rodents. although this approach may be useful in a few studies, this method of source reduction would appear to have only very limited applicability across the broad scope of contemporary studies using mouse models. source reduction of mouse allergen is also achieved through reduction of animal density within an animal room (the number of animals per room volume) and through use of frequent, effective facility sanitation practices (department of health and human services, national institute of occupational safety and health ). the risk of exposure to mouse allergen varies by the type of animal-related activities conducted by personnel and by the type of animal housing systems and equipment containment devices employed in the use and maintenance of laboratory mice (gordon et al. (gordon et al. , schweitzer et al. ; thulin et al. ) . many studies have examined the different caging systems used for mouse housing, and the ability of each cage system design to reduce environmental allergen is well known (gordon et al. ; schweitzer et al. ) . the application of just a simple filter sheet top or fitted filter bonnet to an open cage is effective in reducing ambient allergen levels (reeb-whitaker et al. ) . however, studies indicate the clear superiority of individually ventilated caging (ivc) systems run under negative pressure for the purpose of controlling room allergen levels (gordon et al. ; reeb-whitaker et al. ; schweitzer et al. ) . gordon et al. ( ) suggested that the use of efficient negative ivc in combination with other engineering controls for allergen containment during procedures and waste processing would potentially produce a virtually allergen-free work environment. when negative pressure ivc housing is not available, the placement of cages in a hepafiltered, ventilated cabinet is effective at reducing room allergen loads (thulin et al. ) . gordon et al. ( ) reported that individuals who have direct contact with mice (animal technicians) have the highest exposure, followed by those who have intermittent contact with anesthetized animals or mouse tissues (scientists and necropsy technicians), followed by those with indirect contact (office workers or histology technicians). the specific animal husbandry activities that are known to result in high exposure of personnel to mouse allergen are cage-changing activities, including animal transfer, stacking dirty cages, and manual emptying of cages; handling animals directly (particularly males); and room sweeping (gordon et al. ) . for each of these activities, use of improved containment equipment and changes in equipment handling procedures are effective in the controlling the allergen hazard and should be encouraged. for example, use of ventilated cabinets or biological safety cabinets during cage changing and animal handling is effective in conjunction with the use of microisolation cages (gordon et al. ; schweitzer et al. ; thulin et al. ) . containment equipment has also been designed for the capture of airborne allergens generated when the bottom of one dirty cage is placed into the opening of another to stack the cages for transport to the cage wash area or when dirty bedding is removed prior to cage washing (gordon et al. ; kacergis et al. ) . room cleaning with a vacuum equipped with hepa filtration followed by mopping with a damp mop also aids in reducing the environmental allergen load and personnel exposure (kacergis et al. ) . use of personal protective equipment and dedicated work clothing for personnel involved in high-exposure activities is an important asset in reducing allergen exposure. it is important for the work clothing to remain at work, as evidenced by the finding that children of laboratory animal workers had a higher incidence of clinical signs during provocative testing, positive skin tests, and ige specific to laboratory rodents than did the children of parents who worked in other occupations (krakowiak et al. ) . full sleeve protection and gloves should be worn to prevent the urticarial reactions in persons who are highly sensitive to the mus m allergen. personnel should also be provided with respiratory protection and eye or face protection when warranted. either filtering facepiece particulate respirators (n equivalent) or powered air purified respirators are effective in reducing exposure and alleviating clinical symptoms (schweitzer et al. ; thulin et al. ) . special attention must be paid to the selection and fitting of n filtering facepiece particulate respirators to ensure proper function (morbidity and mortality weekly report ). when the elimination of mouse allergen exposure in the workplace is not achieved through the use of engineering controls, work practices, and personal protective equipment, allergic reactions in persons sensitive to mus m can be managed with pharmacological agents that have a long history of use for this condition. these include antihistamines, topical ~-adrenergic agents (bronchodilators), cromolyn sodium as a nasal spray, and intranasal potent glucocorticoids (austen ) . prophylaxis in patients with mild symptoms is often provided by topical cromolyn sodium on a continuous basis, supplemented with the intermittent use of antihistamines often at bedtime. the selection of the antihistamine has been an area of considerable discussion, and the reader should refer to casale et al. ( ) for further insights into this matter. in more serious cases, potent topical glucocorticoids may be necessary for alleviating clinical signs. immunotherapy, or hyposensitization, is typically reserved for patients who are unable or unwilling to escape the allergen provoking the response. although allergy to the dog or the cat can be ameliorated by immunotherapy (norman (norman , , the infrequent reports in the literature of immunotherapy for allergy to mice and other small laboratory animals have failed to establish the usefulness of this approach for the control of allergy to these species (sorrell and gottesman ; wahn and siraganian ) . the progress made in the past two decades in the evolution of health care systems responsible for the monitoring, control, and elimination of infectious diseases in laboratory mice as well as the advancements in the facilities, equipment, and techniques used to maintain mice in contemporary animal care and use programs, has vastly reduced the likelihood that personnel will encounter zoonotic diseases or other health hazards in the laboratory under most circumstances. continued program success in the control of mouse-associated hazards relies on the use of well-designed and maintained animal facilities, exclusion of wild rodents and other vermin, and quality control in animal care and veterinary care practices. in unique experimental situations that place personnel at a high risk of exposure to mouse-associated hazards, institutional review should ensure that procedures are carefully planned and conducted using personal protective equipment for worker safety. allergy to laboratory animals in laboratory technicians and animal keepers the relationship of trichophyton quinckeanum to trichophyton mentagrophytes alteras, i. 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transmissible from laboratory animals to man leptospirosis. in crc handbook series in zoonoses orthoreoviruses. in principles and practices of infectious diseases laboratory animal allergy in a pharmaceutical company important animal allergens are lipocalin proteins: why are they allergenic? development of hymenolepis nana and hymenolepis diminuta (cestoda: hymenolepididae) in the intermediate host tribolium confusum efficacy and specificity of immunotherapy with laboratory animal allergen extracts rat mite bite the zoology of tapeworms placebo-controlled trial of intravenous penicillin for severe and late leptospirosis bacterial and mycotic diseases helminths natural and experimental helicobacter infections rat-bite fever in a gerbil breeder congenital lymphocytic choriomeningitis virus syndrome: a disease that mimics congenital toxoplasmosis or cytomegalovirus infection natural occurrence of leptospira ballum in rural house mice and in an opossum on the biological character of hvj akitsugu strain and its pathogenicity in human beings as revealed after experimental inoculation of it in volunteers infections of laboratory animals potentially dangerous to man: ectoparasites and other arthropods, with emphasis on mites influenza d in early infancy key: cord- - xw qeku authors: julian, richard j. title: the peritoneum, retroperitoneum, and mesentery date: - - journal: pathology of domestic animals doi: . /b - - - - . - sha: doc_id: cord_uid: xw qeku nan the peritoneal cavity is a single unit incompletely divided into compartments by the mesentery, omentum, and ligaments of double peritoneal membrane. this, with the assistance of fibrin in inflammatory reactions, can result in localization of lesions within the abdomen, although peritonitis can also be more or less generalized. omental bursitis is an example of peritonitis localized within a compartment, and the peritonitis of traumatic reticulitis is frequently localized by fibrin and adhesions. blood or fluid in the abdomen can occasionally be localized within a cystic structure but is usually generalized. the peritoneal membrane forms "ligaments" between organs surrounded by it, and in some cases these ligaments are involved in bowel entrapments, particularly the nephrosplenic ligament in the horse. normal holes, such as the epiploic foramen, and congenital or acquired defects in the double layer of peritoneum making up the mesentery can also be involved in entrapments. the retroperitoneum is the areolar and adipose tissue immediately outside the peritoneal lining of the abdominal cavity. its volume is small except when adipose tissue accumulates, as it normally does in the dorsal retroperitoneum, pelvic cavity, and mesenteries. the normal peritoneum is a smooth, shiny membrane with just enough fluid present to keep it moist. it becomes dry in animals that die of, or with, severe dehydration and is then clammy, sticky, and slightly opaque. it seems that a horse's peritoneum dries out shortly after death because if examination is postponed for a few hours, apposed serous surfaces stick to each other so that when separated the serosa is drawn into tags; this is especially evident on the diaphragm and viscera touching it. these changes should not be taken as residue of peritoneal inflammation or confused with the fibrous tags of scar tissue that occur on the diaphragmatic surface of livers that have been traversed by many larval nematodes. distinction should also be made between antemortem and postmortem effusions and discolorations. some lymph accumulates in the peritoneal cavity after death, and this becomes stained with hemoglobin as soon as erythrocytes in the serosal vessels begin to undergo lysis. such fluid does not clot and is also present in other serous cavities. its nature is usually evident because gaseous changes of putrefaction accompany the lysis of erythrocytes. diffusion of bile pigments through the wall of the gallbladder, the biliary ducts, or the duodenum will stain adjacent viscera. where hydrogen sulfide from the intestine meets hemoglobin from lysed erythrocytes, greenish black sulfmethemoglobin is produced to discolor the tissues. congenital anomalies of the peritoneal membranes are rare and when present are most frequently associated with embryonic remnants of vitelline structures. a persistent vitelline duct may form a fibrous ligament between the intestine and the umbilicus, or a meckel's diverticulum and the umbilicus. the remnant may be partial, not reaching the umbilicus, and attached to the mesentery or a loop of intestine to form a band that could act as an internal hernial ring. either form of band may become involved in strangulation of the intestine. a persistent vitelline artery results in an anomaly, classified as a mesodiverticular band. this is a fold of mesentery, occasionally carrying a patent vitelline artery in its free edge, that extends from the cranial mesenteric artery to the antimesenteric side of the intestine (to the site of meckel's diverticulum). the pocket formed between this fold and the normal mesentery may entrap intestine, or defects may develop in it that allow passage of intestinal loops and strangulation. occasionally, double (both left and right) mesodiverticular bands are present. occasionally, fibrous cords of mesenteric tissue may be found that do not appear to be part of embryonic remnants of vitelline structures. foreign bodies occur commonly in the peritoneal cavity, and obviously, there is potentially a wide variety in both objects and circumstances. foreign materials of endogenous origin are important because their presence requires an abnormality of another organ. ingesta is frequently found in the peritoneal cavity of horses, cattle, and swine, seldom in sheep and goats, and rarely in dogs and cats. the origin of the ingesta is the stomach or intestine, and the site of leakage is usually easy to find, especially when the animal dies before there is time for peritonitis to develop; such is the case, for instance, in gastric rupture in the horse. once peritonitis has developed and matted the intestines and mesenteries, the site of perforation may be very difficult to find, especially as severe peritonitis devitalizes segments of the intestinal wall and, occasionally, causes perforation of the bowel from the serosal surface. digestion of the abomasal wall postmortem will release gastric contents in calves and lambs fed on milk-replacement diets, especially if the amounts ingested are excessive. the margins of the abomasal defect and the peritoneum do not show evidence of reactionary changes. in horses, cecal rupture following impaction usually occurs on the medial side of the dorsal cecum following ischemic pressure necrosis. perforation of the colon or rectum following impaction probably has a similar pathogenesis. rectal perforation may be secondary to accidental injury, or occasionally in foals, to focal ischemic necrosis from adherent meconium. hemoperitoneum is the presence of blood in the peritoneal cavity. the amount present at death is not an indication of the volume of bleeding during life because the blood is removed quite rapidly by diaphragmatic lymphatics. the blood in the cavity may be fluid or partially clotted. animals occasionally die from bleeding into the peritoneal cavity, but the outcome will depend on the rate and volume of bleeding, the site of hemorrhage, the cause, and the initial health of the animal. hemoperitoneum is seen most commonly in the dog and cat as a result of traumatic injury to the liver, spleen, and kidney. rupture of the liver and hemorrhage occur in infectious canine hepatitis. manual efforts at artificial resuscitation, especially if vigorous, will rupture the liver, particularly in anesthetized dogs, if the anesthetic is one causing hepatic congestion and turgidity. repeated splenic ruptures at the site of hemangiomas are well known in dogs past middle age. spleen and liver enlarged and tensed by infiltrating leukemic cells, fat, or amyloid are predisposed to rupture; the volume of hemorrhage in these cases may be very small. warfarin poisoning in several species may result in nonclotting blood in the abdomen. manual squashing of a corpus luteum is a source of hemorrhage in cattle. in cattle and horses, laceration of the uterus or rupture of a uterine artery can result in a sometimes fatal hemorrhage. calves born of cows fed moldy sweet-clover hay bleed from the umbilical vessels into the peritoneal cavity (as well as elsewhere). hemorrhage on or beneath the peritoneum without free blood in the cavity is commonly observed in many acute infectious toxemias and noninfectious conditions that interfere with vascular integrity or the hemostatic mechanisms. peritoneal hemorrhage must be differentiated from hemorrhagic peritonitis, which is an important lesion in many diseases. hemorrhage into an omental or mesenteric cyst can cause sudden abdominal enlargement without free blood in the abdomen. hydroperitoneum (ascites), is the accumulation of noninflammatory transudate in the peritoneal cavity. it is arguable that the term ascites should be reserved for peritoneal lymphedema of hepatic origin, whether the issue is primarily hepatic, as in hepatic fibrosis, or secondary to a sustained increase in central venous pressure with chronic hepatic congestion. the fluid of ascites is watery, clear, or straw colored and will contain few leukocytes but large numbers of desquamated mesothelial cells. the serosal lining is normal unless fluid has been present for some weeks, when the serosa acquires a flat, whitish semiopacity. the principles governing the transfer of fluid (lymph) between blood vessels and lymphatics across the peritoneal membrane are, so far as known, basically the same as those governing fluid exchange in tissues, and there is normally a very rapid turnover of peritoneal fluid. in tissues generally, there is a small excess of filtration at the arteriolar end of the capillary bed over absorption at the venous end of the capillary bed; the excess of fluid is drained from the tissue spaces by lymphatics. these mechanisms are operative in abdominal viscera, but it is to be noted that free fluid in the peritoneal cavity drains through the ventral diaphragm and the sternal lymphatics. the accumulation of excess peritoneal fluid can be viewed most simply as the result of diminished removal or overproduction of fluid. the relative importance of the various pathways for the removal of fluid from the peritoneal cavity has not been studied in detail for the various species, but the following schema is generally applicable. in spite of the large area of peritoneum, fluid absorption from the cavity is virtually limited to those diaphragmatic lymphatics that form an abundantly anastomotic plexus in the muscular portion of the right side of the diaphragm. vessels penetrate the diaphragm to form a corresponding pleural plexus from which the lymph is conveyed in the sternal ducts to the anterior sternal node, and then via the right lymphatic duct to the vena cava. there are subsidiary pathways that will deliver some peritoneal lymph to the cisterna chyli or thoracic duct, but % or more of peritoneal lymph will follow sternal lymphatics. the smallness of the area of lymphatic absorption provides an explanation for the ease and rapidity with which peritoneal drainage can be obstructed. obstruction to diaphragmatic lymphatics leading to ascites is best exemplified in peritoneal carcinomatosis. implanta-tion metastasis of carcinomas usually and perhaps always, if given time, implant most extensively on the diaphragm in the region of the lymphatic exits, and there, permeation of the lymphatic vessels is easily demonstrated microscopically. it is probable that the neoplastic cells are carried to the anterior abdomen by the same forces that propel the lymph in that direction, namely, normal intestinal movements and normal respiratory excursions. the restriction of the area through which lymphatic absorption can take place is compensated by the high efficiency of the mechanism, a level of efficiency not satisfactorily explained. carcinomatous implants on the peritoneum, in addition to obstructing diaphragmatic lymphatics, may provoke fluid production. this has been argued for papillary adenocarcinoma of the ovary, which in the bitch provides the best example of implantation and ascites. it will be apparent that ascites may develop if there is obstruction to sternal pathways anterior to the diaphragm, and this condition seems often to be provided in lymphomatosis of adult cattle when there is massive neoplastic involvement of the anterior mediastinal lymph nodes. the overproduction of peritoneal lymph is related to the mesenteric circulation and the hepatic circulation. this excludes the rare chylous ascites, which results when the cisterna chyli is ruptured, the chylous origin of the fluid indicated by the high content of chylomicron fat and the milkiness of the fluid. the filtration pressures in mesenteries and omentum appear to differ quantitatively from those in other tissues, with the effective filtration occurring along the entire capillary bed. that all of the filtered fluid must return to the circulation via the lymphatics attests to the capacity for lymphatic drainage. increased venous pressure in the portal venous system is not in itself expected to lead to ascites. acute portal vein obstruction leads rapidly to death. chronic obstruction to prehepatic portal flow leads quickly to the development of collateral venous circulation, with connections to the abdominal vena cava. the interstitial tissues of the mesentery and omentum are, as are interstitial tissues generally, rather noncompliant, and small increases in interstitial fluid volume produce marked increases of tissue hydrostatic pressure, which limit transudation. the balance of oncotic and hydraulic pressure that regulates fluid exchange between blood vessels and interstitial tissue depends on the integrity of vascular structures and on the maintenance of their permeability characteristics. vascular injury that allows increased permeability to plasma protein substantially alters the balance of forces and favors transudation. small amounts of ascitic fluid may be generated under these circumstances in a variety of systemic illnesses, such as the clostridial intoxications, acute uremic syndromes in ruminants and pigs, and exudative diathesis of pigs deficient in vitamin e. ascites resulting from overproduction of fluid is usually an expression of hepatic lymphedema. it appears that the sine qua non of hepatic ascites is that there should be obstruction to the intrahepatic veins or the suprahepatic veins with congestion and edema of the liver; obstruction to the portal veins with increased portal venous pressure will not do, except perhaps if there is concurrently a critical degree of hypoproteinemia. the usual conditions providing obstruction to the efferent hepatic vessels are fibrosis of the liver and congestive heart failure. there is, of course, a variety of additional causes, including primary neo- . plasms of the liver, principally those of cholangiocellular type, which tend to be diffuse; secondary tumors, especially those of lymphocytic type; extensive infestation with hydatid cysts; chronic fascioliasis; and so on. when the liver is congested, there is, as in any acutely congested tissue, an increased turnover of protein and fluid and an increased flow of hepatic lymph with a high concentration of protein. the bulk of hepatic lymph comes from the space of disse, which is separated from the capillary lumen by a fenestrated endothelium that is freely permeable to plasma constituents, including protein of high molecular weight. oncotic pressures, normally dependent on proteins of low molecular weight, are not as important in the liver as they are elsewhere in the regulation of fluid exchange. instead, the formation of hepatic lymph is sensitive to small changes in hydraulic pressure in the sinusoids, which accounts for the frequency with which ascites is associated with those diseases leading to increased central and hepatic venous pressure. if the ordinary capacity of the hepatic lymphatics is not equal to the challenge provided in hepatic venous stasis, then lymph high in protein oozes from the hepatic capsule, presumably from the rich lymphatic plexus there, and spills from the efferent lymphatics that pass from the porta hepatis to the cisterna chyli; in hepatic ascites, these efferent lymphatics become very large, numerous, and thick walled, and presumably, the same changes take place in all efferent hepatic lymphatics, some of which follow different routes, for example, through the suspensory ligaments. the fluid that accumulates in the peritoneal cavity is not static but dynamic, continually produced and removed. the efficiency of normal removal has already been indicated, and it will be evident therefrom that it is only in instances of severe increased pressure in the hepatic vessels that gross ascites will develop. in all considerations of hepatic ascites, the thoracic duct, which is the main channel for removal of hepatic lymph, is assumed to be completely permissive. the conducting capacity of the duct is, however, probably quite limited, even though the duct is quite distensible. concurrent increase of venous pressure at the thoracic inlet may impair lymph drainage; of greater importance may be altered configuration, when the duct is distended, of the opening between duct and vein. in experimental ascites, the flow of lymph in the hepatic lymphatics can be increased in rate to five or more times the normal, and since only a part of this flow oozes into the peritoneal cavity, some additional factors would seem to be necessary before the drainage capacity is overwhelmed, if indeed it is overwhelmed, an event that seems unlikely in view of calculations purporting to show a complete turnover of the albumin in the ascitic fluid in ~ days and a turnover of ~ % of the volume of ascitic fluid each hour. thus it seems clear that although severe obstruction to the intrahepatic circulation is necessary to initiate hepatic ascites, the gross and persistent accumulation of fluid depends on additional factors. chief among these is the retention, by diminished urinary excretion, of salt and therefore also of water. there is much evidence connecting the retention of sodium and water, and the resultant expansion of plasma volume, with the development of edema and ascites in congestive heart failure and hepatic venous obstruction. the mechanisms remain ob-scure but may involve receptor sites for sensing volume-pressure changes in the systemic circulation. the effector mechanisms are renal and probably regulated by a variety of neural and humoral pathways that influence renal hemodynamics, the composition of tubular fluid, and ionic gradients. the principal diseases of animals in which ascites occurs have been indicated in the foregoing discussion, but there are some others that ought to be mentioned. the ascites of congestive heart failure is presumably of the same pathogenesis as in humans, whatever that might be, and as indicated above, it may be related in part to the hepatic congestion that is part of the syndrome of cardiac failure. there is the additional possibility of retarded lymphatic flow, and although there appear to be no measurements of this, the tortuous dilatation of lymphatics in some cases of congestive heart failure are quite suggestive. ascites occurs in congestive heart failure without edematous transudations elsewhere, especially in dogs and cats, but it should be pointed out that only a minority of cases of congestive failure in dogs and cats is associated with ascites. excess peritoneal fluid is also part of a generalized dropsical condition in cachectic diseases, anemia, and starvation. although the mechanisms have not been intensively studied, hypoproteinemia has been given consideration as one of the principal factors. very probably it is also in those diseases that are known, or presumed, to result in chronic protein leakage and loss, such as gastrointestinal trichostrongylosis and johne's disease. hypoproteinemia appears not, however, to be of initiating significance in the edema and ascites of chronic starvation without other accompanying disease; it is suggested in such cases that the fluidretention serves largely to replace wasted tissues, especially adipose tissue. effusion of fluid, sometimes massive, occurs in the peritoneal cavity, thorax, and ventral body wall in some instances of uremia in sheep and cattle. it is part of the postmortem picture in sheep and cattle dying of urethral obstruction by calculus, and the fluid may have a distinct uriniferous odor. the pathogenesis of this fluid accumulation has not been examined, and although in a few cases there is a rupture of the lower urinary tract (which might provide the fluid by leakage), in many cases a rupture cannot be demonstrated. renal uremia also is associated with similar fluid accumulations in cattle; this is evident in some dying with renal amyloidosis, and the contributing factor here is probably hypoproteinemia consequent on prolonged massive proteinuria. acute toxic nephrosis, of which certain plant poisonings and mycotoxicoses provide the best examples in ruminants and pigs, may also be accompanied by massive effusions, and as is usually the case with ascites of urinary tract disease, the mesenteries and retroperitoneum are also saturated. necrosis of the omental or other abdominal or retroperitoneal fat is a frequent finding at autopsy. the pathogenesis is poorly understood, but there appear to be a number of causes. in pancreatic necrosis, enzymatic necrosis of fat occurs constantly, and indeed, peripancreatic necrosis of fat may be the initial morphologic change. enzymatic necrosis of fat may extend throughout the abdomen. the acute lesions are discrete foci or confluent masses of yellow, necrotic adipose tissue surrounded by a zone of intense hyperemia with fibrin deposited on the surface. free droplets of fat can be found in the peritoneal fluid. the lesion has been attributed to lipolytic enzymes from the acinar pancreas, but there is no agreement on which enzymes initiate the reaction. pancreatic amylase, elastase, lipase, trypsin, and phospholipase a are examples of enzymes that may be involved, but some of these would have to overcome local enzyme inhibitors before becoming activated. kinins, hageman factor, and complement have been suggested as possible causes of fat necrosis once the process has been initiated. neutrophils respond in large numbers, and their degeneration may aid the ongoing process. lipase released from degenerating fat cells also contributes to fat necrosis. microscopically, the acute lesion is made up of necrotic fat cells containing acidophilic, opaque, amorphous or lacy substance or basophilic fibrillar or granular mineralized material. masses of degenerating neutrophils and necrotic debris are present. fibroplasia and vacuolated macrophages are features of the chronic lesion, along with necrotic fat and, occasionally, dystrophic mineralization. widespread or isolated focal necrosis of abdominal and retroperitoneal fat is frequently found in sheep and sometimes in horses, pigs, and other animals. this necrotic fat is usually seen only at the chronic stage in the form of small, dry, firm or gritty plaques. a flat white color gives the plaques clear distinction from surrounding normal fat. there is no grossly apparent inflammatory reaction, and histologically the lesions resemble the chronic lesion described above, with occasional large lipid vacuoles containing necrotic debris encapsulated by connective tissue. the pathogenesis of this form of fat necrosis has not been explained. the focal lesion may be due to avascular necrosis of fat from pressure ischemia, perhaps due to differences in the texture or composition of the fat, or some other circulatory deficit in the small capillaries that nourish the large masses of fat, since the lesion does appear more frequently in excessively fat animals. it is also possible that the lesions are initiated by intracellular lipolytic disturbances in circumstances in which there is accelerated mobilization of fat to meet acute metabolic demands. the third form, not uncommon and perhaps the most curious, is massive fat necrosis in cattle. although this condition has been reported to occur more frequently in channel island breeds, there is more evidence for herd than breed predisposition. it does appear to be associated with excessively fat and fattening cattle. both sexes are affected, but probably not clinically before the second year of life. the disease has two significant features. first, although it is seen more often in the slaughterhouse than in the postmortem room, it is frequently fatal, perhaps always progressive and potentially fatal, and second, the hard masses of necrotic fat have been, and will be, mistaken for fetal prominences in the diagnosis of pregnancy by perrectal palpation. the fatal outcome in this disease is usually by intestinal obstruction, the intestine compressed by the expanding lumps of necrotic fat (fig. . ib), but other complications also occur, such as compression and stenosis of the ureters. affected animals may exhibit a variety of clinical signs such as anorexia, diarrhea, constipation, colic, or bloat. they may become emaciated prior to death. the pathologic process occurs in any portion or all of the omental, mesenteric, and retroperitoneal fat. in the early stage of development there is evidence of acute inflammation, and the hard necrotic masses are surrounded by a zone of hyperemia, the overlying peritoneum may be necrotic, and the necrotic margins umbilicate. the masses may vary from small nodules to large solid masses encapsulated by fibrous tissue and that on cut section are firm, dry and caseous, or moist, and deep yellow. the inner surfaces of such masses are firmly molded to the contours of enclosed organs. because of the unusual bulk of the necrotic tissue, and because fat is sometimes found in unusual locations, such as under the serosa of the intestine, the condition has been called lipomatosis, but there is no indication that the lesions are neoplastic. on microscopic examination the tissue resembles a mixture of acute and chronic enzymatic fat necrosis with fewer neutrophils, some lymphocytes, plasma cells, and eosinophils, and more macrophages and giant cells. crystal-shaped clefts are present in fat cells and in macrophages and giant cells. the pathogenisis of this diffuse lipogranulomatosis in cattle is not clear, but there is accumulating evidence of a dietary cause somewhat similar to that of steatitis as it occurs in horses, pigs, and other species. the different lesions in cattle may be due to the different chemical composition of fat or to a different host response to material recognized as ' 'foreign'' (which might also allow for breed predisposition, as might breed differences in texture and amount of abdominal fat). the lesion is possibly related to ingestion, or production in the rumen, of high levels of saturated fatty acids, which form long-chain compounds that are solid at normal body temperature. chemical change or avascular necrosis may initiate a reaction that results in the formation of insoluble salts, cholesterol, or other material recognized by the body as foreign. this material provokes the inflammatory response, with fibrosis, collagen formation, and the accumulation of materials in fat cells, giant cells, and macrophages. steatitis (yellow-fat disease) occurs in many species of animals except ruminants, affecting the abdominal and retroperitoneal fat along with other body fat. the condition is described (as panniculitis) with the skin and appendages (volume ) and is caused by a diet high in polyunsaturated fat and low in tocopherols, allowing oxidation of body fatty acids. peroxidation creates free radicals, which damage tissue and provoke the characteristic inflammatory response in the adipose tissue. several types of steatitis, all vitamin e responsive, occur in newborn or young, as well as in mature animals. lipofuscin, which is not present in all forms of steatitis, is responsible for the yellow color and often fills the macrophages, which are a feature of this form of steatitis. peritonitis is very common in the large domestic animals but uncommon in dogs and cats. it may be serofibrinous, fibrinopurulent, purulent, or hemorrhagic, and whatever the type, it may be localized or more or less generalized. differences in type and distribution can be more or less anticipated from the origin and the causes; the latter are numerous and varied, so only some of the more common and important of them will be considered. most cases of peritonitis are caused by bacteria and their toxins, some by helminth infestations, a few are viral, and a few chemical. the intraperitoneal injection of a number of therapeutic agents causes a mild, and usually inconsequential, peritonitis. the most devastating forms of chemical peritonitis are endogenous and are caused by bile and pancreatic enzymes. the peritonitis caused by bile is intense, shock inducing, and may be rapidly fatal. there is very little exudate unless the leakage is minor and infected, and then it may become purulent. biliary peritonitis is readily recognized by the typical staining. the peritonitis of pancreatic necrosis is also acute and is rather common in dogs, rare in horses, and virtually never occurs in other species. the reaction about the pancreas, particularly its head, is liquefactive and purulent, and the exudate mats the lesser omentum to the pancreas and the adjacent liver and other organs. this local peritoneal reaction resolves completely if the animal survives, and only very minor adhesions or slight puckering of the mesentery persist. the peritoneal exudate is usually scant but is distinctive because, as well as pus, it contains white droplets of fats and soaps released from the surrounding adipose tissues by the pancreatic enzymes. bacterial peritonitis may be regarded as primary in the sense that the bacteria are implanted directly on the peritoneum by perforating lesions from a contaminated surface, either a hollow viscus or the skin. it is secondary when it reaches the peritoneum from an adjacent focus by direct extension, or when the organisms are carried there in the blood or lymph streams. primary peritonitis need not be discussed further; it is most commonly associated with the lesions described above as being the source of peritoneal contamination by ingesta but may also rise from parasites penetrating the intestinal wall in some species. in females, the abdominal cavity is open to the exterior through the reproductive tract, and infection may enter the abdomen through the oviduct as well as by rupture of the uterus or laceration of the vagina. secondary bacterial peritonitis is also common either as an extension from localized inflammation in an abdominal viscus or as a typical part of the morbid picture in a number of specific diseases. acute serofibrinous peritonitis occurs by extension through the wall of a gangrenous intestine or uterus prior to rupture, or death from toxemia may intervene before rupture occurs. secondary peritonitis occasionally results by extension from retroperitoneal infection or, in ruminants, from omental bursitis. in those cases in which the peritonitis develops by direct extension, there is little difficulty in ascertaining its origin, even when the process becomes diffuse. the sources of secondary peritonitis are so varied that detailed consideration cannot be given to them here. instead, some of the features of peritonitis in the different species are given with an indication of the specific diseases in which peritonitis is a feature, although not necessarily a consistent one. the incidence and nature of peritonitis in specific infections are discussed more fully with the specific diseases. diffuse peritonitis is usually fatal in horses, chronic diffuse peritonitis virtually never occurring. in almost all cases it is caused by rupture or perforation of the stomach or intestine. acute or chronic local peritonitis does occur occasionally from castration wounds or other penetration from the skin, or streptococcal abscess in the mesentery. it may be due to local verminous lesions, suppurative gastritis in habronemiasis, or from perforating gasterophilus. abscesses or ulcerations attributable to strongylus are more common in the colon than elsewhere. migrating s. equinus or s. edentatus larvae cause retroperitoneal lesions in the flank, perirenal fat, and diaphragm, perihepatitis with fibrin tags on the liver capsule, and a chronic diffuse thickening and inflammation in the mesentery, omentum, and hepatorenal ligament, with occasional caseous nodules. copious purulent peritonitis is often seen in infections of foals by corynebacterium equi. intestinal infection of foals by actinobacillus equuli causes fibrinous mesenteric lymphadenitis and peritonitis. acute diffuse purulent peritonitis in cattle is common and usually the result of perforation of a viscus, especially the reticulum or uterus. both may also result in local acute, and then chronic, peritonitis. perforation of the abomasum or intestine is more likely to give fibrinohemorrhagic peritonitis. a serofibrinous peritonitis, sometimes with very copious exudate (and with similar lesions on other serous membranes), is typical of sporadic bovine encephalomyelitis. a diffuse fibrinohemorrhagic peritonitis occurs in most cases of clostridial hemoglobinuria and in some cases of blackleg and septicemic pasteurellosis; a more localized peritonitis of this type occurs in some cases of clostridial enterotoxemia caused by clostridium perfringens types b and c. tuberculosis causes white, nodular granulomas (pearls), and actinobacillosis, although rather rare, produces the usual heavily scarified granulomas, especially about the peritoneum of the forestomachs. extension of infection from the umbilicus of the newborn produces a fibrinopurulent peritonitis not localized but most severe along the ventral abdominal wall, or up the urachus to the bladder. peritonitis of specific cause is uncommon in sheep except for the very local variety that accompanies penetration of the intestine by the larvae of oesophagostomum columbianum. the uterus is probably the usual site in adults from which local spread occurs to the peritoneum, the antecedent lesion in most cases either a postpartum septic metritis or so-called blackleg of the fetus; in either event the peritonitis is fibrinosuppurative and hemorrhagic. a serofibrinous peritonitis is a feature of contagious agalactia, or any variant of it, caused by mycoplasma. mycoplasma mycoides may cause an acute fibrinous peritonitis in goats, although acute death from septicemia or joint and mammary gland disease is more common. paratuberculosis (johne's disease) frequently produces a nodular granulomatous lymphangitis in the mesentery, and sometimes caseous or calcified lymphadenitis. . a few filmy strands of fibrin frequently overlie the intestine and the borders of the mesentery in many acute infectious diseases of swine, and in conditions that result in vascular damage such as edema disease and vitamin e/selenium-responsive conditions; this does not qualify as peritonitis. a diffuse suppurative and adhesive peritonitis is common in pigs; in these cases the intestines are so matted that they cannot be dissected. corynebacterium pyogenes, escherichia coli, or a miscellany of organisms are frequently present in these, and in some the extension of the inflammatory process can be traced up the inguinal canals from castration wounds; in other cases of similar type, the peritonitis is localized to the inguinal and pelvic regions, is adhesive, and causes death from intestinal obstruction. occasionally, c. pyogenes produces discrete encapsulated abscesses as profuse implants on both visceral and parietal peritoneum. a serofibrinous peritonitis, more fibrinous than serous, and with similar lesions on other serous membranes, is almost pathognomonic for glasser's disease caused by haemophilus suis. mycoplasma hyorhinis and possibly other mycoplasmas may produce a serofibrinous peritonitis that becomes fibrous, with adhesions to a thickened serous membrane; this disease is to be distinguished from glasser's disease. small, firm, lemon yellow nodules and flattened disks of inspissated fibrin often are found free in the peritoneal cavity in chronic mycoplasma infections. rectal strictures in swine cause a very dilated colon and cecum full of ingesta. the serosa is frequently thickened, white, and covered with fibrin tags, resembling infectious serositis. the thickening is probably caused by subserosal edema and fibrosis but may be due to passage of bacteria or by-products through the intestinal wall. stephanurus dentatus larvae cause subserosal focal hepatitis and a mild reaction with edema in the perirenal fat and retroperitoneal tissue, and sometimes in the mesentery and local lymph nodes as they move to the kidney. in intestinal anthrax in swine there is an acute gelatinous hemorrhagic peritonitis very typically localized to the mesentery between the intestine and the mesenteric node. the distribution of the lesion is due to the lymphatic spread of the infection from the intestine. tuberculous peritonitis in swine is localized and characterized by adhesions to the spleen. a fibrinohemorrhagic peritonitis, slight in degree and easily overlooked, is common in infectious canine hepatitis. the peritoneum, especially that covering the intestine, is gray and granular like ground glass, and there are a few reddish strands of fibrin, most of them about the liver. there is edema of the intestinal subserosa and, frequently, petechiae or larger hemorrhages. in parvovirus infection, similar lesions may be present on the serosa of the affected portion of intestine. putrid peritonitis occurs when the uterus ruptures either as a result of pyometra or septic metritis with fetal putrefaction. nocardiosis, which may be infection by bacteria of the genera nocardia or actinomyces, produces very characteristic lesions on the peritoneum (fig. . b ). it is, however, more common on the pleura. sometimes there are purulent granulomas, but most frequently there is a profuse, pink mush. the color is from admixture of copiously exuded cells and blood; the blood is derived from a tremendous proliferation of thin-walled capillaries from serous surfaces. purulent peritonitis in the dog is rare; it has been observed as an extension from umbilical and hepatic abscesses in puppies and is caused by streptococcus canis. putrid peritonitis occurs when the uterus ruptures in consequence of pyometra or fetal putrefaction. peritonitis also occurs from penetrating wounds or by extension from retroperitoneal tissues. nocardiosis similar in appearance to the disease in dogs frequently complicates myelodysplastic disease in cats. itonitis is a relatively recently recognized coronavirus infection of felidae, particularly domestic cats. it has a worldwide distribution, but the incidence of disease is low and sporadic even in the countries where it is most common. the disease is in part immune mediated, and the lesions are for the most part the result of deposition of immune complexes leading to arthus-type reactions. the naturally occurring disease tends to be chronic and results in death after one to several months, although there may be nonclinical carriers. most cases have a relatively easily recognized clinical course with typical gross and histologic findings. close contact appears to be necessary for the spread of the disease and is likely to occur by way of ingestion or inhalation of infective material from body secretions and excretions. cats in catteries, pet shops, and households with multiple cats are at greatest risk. cats of all ages can be affected, but there may be a higher incidence in two groups; young, entire adults and aged cats. feline infectious peritonitis has an insidious onset, and since cats have a propensity to mask serious illness, the disease may be well advanced before clinical signs are recognized. the early signs are nonspecific, consisting of decreased activity, depression, anorexia, or diarrhea. the increasing abdominal distension present in the effusive form of the disease may hide the progressive weight loss. a variety of mild to severe neurologic signs develop in up to % of affected cats, and it is these signs, and panophthalmitis (which is also relatively common), that are likely to attract attention. affected cats are usually pyrexic and often hypergammaglobulinemic, with total serum protein increased and the albumin/globulin ratio lowered. feline infectious peritonitis is unresponsive to antibiotics but may show a temporary response to corticosteroids. occasionally, respiratory signs with cough or dyspnea are present, and in atypical or noneffusive forms, neurologic or ophthalmic signs may be the first noted. a variety of serologic tests are available to detect feline infectious peritonitis antibody. antibody levels are usually high (titer > : ) in cats with clinical infectious peritonitis, but interpretation is complicated because current tests do not distinguish between antibodies formed in response to the virus and those stimulated by the feline enteric coronavirus. for descriptive purposes, the disease has been divided into effusive " w e t " and noneffusive " d r y " forms, but since the only distinction between these forms is the extent of fibrinous peritonitis and exudate, the division serves little useful purpose. all serous surfaces throughout the body may be involved in the inflammatory response, and pleurisy with pleural effusion is present in - % of cases. peritonitis is present in most natural cases, although this may be obvious grossly in only to %. the tunica vaginalis is affected, resulting in periorchitis in entire males. up to liter of abdominal exudate may be present. the fluid is usually clear and pale to deep yellow, although it may be flocculent and contain strands of fibrin. the serosal surfaces may be covered with fibrin exudate, giving them a granular appearance. fibrin is frequently prominent over the visceral peritoneum (fig. . c) , and fragile adhesions may be present. there are white foci of necrosis or raised granulomatous cellular infiltrations on the serosa and extending into the organs or wall of the intestine from the serosa. these vary in size from a few millimeters to a centimeter in diameter. the kidneys may be enlarged and nodular with single or multiple, small to large, white, granulomatous nodules protruding from the cortex (fig. . ie). severe or mild granulomatous hepatitis and pancreatitis may also be present. small white foci of inflammation may be found in the organs. fibrin is usually less prominent in the thorax, but white foci may be visible under the pleura, and the lungs may be dark, firm, and rubbery. hydropericardium and fibrinous epicarditis occur less frequently but are similar in type to other serosal reactions. abdominal and thoracic lymph nodes are enlarged and have a lobulated pattern. in the effusive form of the disease, visceral lesions are sometimes minimal, and in the noneffusive form, peritonitis may be mild or not obvious on gross examination, and in these cats the lesions can be in the abdominal or thoracic organs, as described above, or in the eyes or nervous system. the lesions in the eyes begin as a diffuse uveitis progressing to a panophthalmitis, with fibrin usually seen in the anterior chamber. lesions in the central nervous system can involve the leptomeninges, spinal cord, or brain but usually are visible grossly only on the leptomeninges as thickenings or white streaks. a subacute form of feline infectious peritonitis with a shorter clinical course is seen occasionally, primarily in weaned kittens. it occurs as a generalized systemic infection, and the lesions are more fibrinonecrotic than granulomatous. an acute hepatic form of the disease resembling suckling mouse hepatitis (coronavirus infection of mice) has been described in experimentally infected kittens. the basic histopathological lesion of the disease is a generalized vasculitis and perivasculitis (fig. . a) and focal pyogranulomatous reaction that occurs in the serous membranes, the meninges, and in the connective tissue of the parenchymatous organs. this results in the serofibrinous and cellular exudate on the visceral and parietal serosal surfaces of the body cavities, and the fibrinonecrotic and pyogranulomatous reaction around affected vessels. the small, random, necrotic foci in organ parenchyma may be part of the same process and due to thrombophlebitis, or they may develop as a result of a separate mechanism, such as disseminated intravascular coagulation, or as the direct effect of the virus. the vascular lesion begins as a proliferation and desquamation of vessel endothelium followed by medial necrosis, narrowed vascular lumina, and thrombophlebitis. there are accumulations of neutrophils, lymphocytes, plasma cells, and . macrophages in and around the affected vessels. occasionally, adventitial fibrosis occurs with little cellular infiltrate. the changes in the omentum, mesentery, and serosal tissues may be mild or severe. the mild changes are proliferation of mesothelial cells, slight fibrin exudate with fibroblast proliferation, and scattered neutrophils and mononuclear cells. the severe changes result in a thick layer of fibrin adherent to the serosa, with necrosis and cuboidal metaplasia with syncytial cell formation of the serosa. large numbers of neutrophils and mononuclear cells and necrotic debris are embedded in the fibrin. the focal necrosis and pyogranulomatous reaction may be present in the parenchyma of the organs and extend into the intestine, affecting the muscularis, peripheral nerve ganglia, submucosa, and mucosa. lesions in the various organs are caused by the vascular damage that occurs in the capsule and connective-tissue stroma. they may be found throughout the body. subcapsular infiltrations occur particularly in the liver, lung, and pancreas, and the pyogranulomatous reaction can develop deep in the parenchyma, particularly of the kidney. in the spleen and lymph nodes there are histiocytic and fibroblastic proliferation and either depletion or hyperplasia of lymphoid follicles. in addition to focal lung lesions, there may be a diffuse interstitial pneumonia, sometimes most severe close to the visceral pleura. similarly, a severe focal or generalized lymphocytic and plasmacytic interstitial nephritis may develop. cellular infiltrations in the spinal or cerebral meninges and perivascular spaces tend to be more mononuclear and diffuse, with only occasional focal pyogranulomatous lesions. degenerative and necrotic lesions in the parenchyma of the central nervous system appear to be related to the prominent vasculitis. experimental studies have clarified the pathogenesis of the disease and the role that the immunopathologic mechanisms play in the production of the lesion. the sequence of events proposed is as follows. the virus is phagocytized after introduction and transported to the regional node, where the primary viral replication takes place, with the generation of a primary viremia. this results in generalized infection of the monocytemacrophage system and a secondary cell-associated viremia. the development of nonneutralizing antibody results in deposition of antigen-antibody complexes that fix complement and trigger the vascular and pyogranulomatous changes. acute generalized peritonitis is a catastrophic development in many local diseases of the abdominal cavity, but it may be an inconsequential feature of the generalized infections because, obviously, peritonitis does not significantly affect the outcome of a strangulated loop of intestine or clostridial infections such as blackleg, hemoglobinuria, or braxy. toxins from peritonitis may be absorbed directly through the peritoneum into retroperitoneal venules and lymphatics or may be carried from the peritoneal cavity via the diaphragmatic lymphatics. death from toxemia may result before obvious peritonitis develops. the peculiarly efficient diaphragmatic lymphatics remove not only fluid but also particulate matter, including bacteria, and in this way the inflammation may spread via the lymphatics to the pleura and the mediastinal nodes. of all the abdominal viscera, the intestine is most affected by inflammation of its serosa. the typical clinical syndrome includes, within the first few hours of generalized peritonitis, intestinal hypermotility with diarrhea. by hr there is absence of bowel movement and, on auscultation, complete abdominal silence; by this time the serosal irritation has caused reflex paralysis of the gut, so-called ileus. the development of ileus has the advantage that exudates are no longer distributed by intestinal movements, but there is also the disadvantage that fibrinous adhesions develop between loops of intestine, and if the animal recovers, they scarify and produce fibrous adhesions that may cause intestinal stenosis. assessing the age of peritonitis may have significance, such as in iatrogenic rectal perforation, and requires careful gross and microscopic examination of the serosal surface and the adherent fibrin. the proliferation and age of fibroblastic components in the fibrin, on the serosa, and in the edge of the wound will give some indication of the age of the lesion. not all cases of generalized peritonitis are immediately fatal; depending on the nature of the exudate, the lesions may resolve completely, be converted to diffuse adhesions, or persist in some localized areas as active or adhesive peritonitis. it is remarkable how adhesions may become attentuated and even removed under the stress of continual tension. the main defenses in the usual sorts of peritonitis are cellular, and this seems to be equally true whether or not the animal has an acquired immunity to the bacteria present. the removal and destruction of any particulate matter, including bacteria, from the abdominal cavity is one function of the lowly omentum; it is so generously endowed with phagocytes that their clusters are often visible to the naked eye as small milky spots. these fixed histiocytes are very efficient at ingesting bacteria and, if exposed to opsonins, of destroying them. in the case of johne's disease, the organisms may be ingested by the macrophages but protected from destruction. when the bacteria are sufficiently virulent, they may proliferate in and destroy the phagocytes. this, no doubt, is why in many instances of acute peritonitis the omentum is first and most severely involved in the inflammatory reaction. it is also a ' 'purpose'' of the omentum to seal off foci of inflammation, and this it does with exceptional efficiency. the omentum is continually moved about the abdominal cavity by bodily and intestinal movements, and it sticks to areas of inflamed peritoneum. such local omental adhesions may persist indefinitely. most of the parasites found in the peritoneal cavity have their final habitat elsewhere, and entry on to the peritoneum occurs in the normal course of migrations or as an accident. thus various cysticerci may be found on the peritoneum during their normal development, dioctophyma renale, the young fasciola hepatica, and a variety of nematode larvae pass this way, and an example of accidental entry is that of ascaris equorum through an intestinal perforation. their significance and the lesions they produce are described in other chapters in these volumes with the parasite in question, and for the most part they are readily recognized at autopsy. filariae, which are occasionally found as young or adult worms in the abdomen of dogs, may be assumed to be dirofilaria immitis. two parasites are unusual and deserve special attention. young liver flukes, fasciola hepatica, can cause acute and chronic peritonitis in cattle and sheep, and the inflammation involves the parietal peritoneum and sometimes the visceral peritoneum, especially that of liver, spleen, and omentum. the changes may consist of many tags of fibrin or a more diffuse, crusty thickening, and the young flukes may be found in the inflammatory lesions both on and beneath the peritoneum. stephanurus dentatus larvae may produce severe peritonitis in the course of their migration across the peritoneal cavity of swine (see the urinary system, this volume). plerocercoids, larval forms of mesocestoides (dithyridiumtetrathyridium) or spirometra (sparganum), occur as bladder worms or solid, straplike bodies in the peritoneal cavity of some carnivores, rodents, and reptiles in some countries. a local reaction may occur around these intermediate forms in tissues and in the peritoneal cavity, and ascites may be present. some of these larvae can multiply by transverse division or budding, so very large numbers may be present. some parasites use the peritoneal cavity as their final habitat. parasites of the subfamily setariinae, family onchocercidae, superfamily filarioidea inhabit the peritoneal cavity of many domestic and wild ungulates, such as horses, cattle, buffalo, camels, sheep, goats, swine, deer, and antelope. they are commonly found at autopsy, particularly in cattle. there are only two genera, papillo setaria, a monotypic genus found in thagulas, which will not be referred to again, and setaria (= hyraconema = artionema), in which there are many species. some species of setaria have a cosmopolitan distribution and may be found in several species of wild and domestic ungulates within the same family ( . equina in equidae, and s. labiatopapillosa in cattle, buffalo, and perhaps deer and antelope), while others are restricted geographically (s. digitata, asia) perhaps by the distribution of intermediate hosts. all members of the genus live as well-adjusted symbionts as adults in their normal host and do not cause peritoneal lesions. the larval form of s. digitata can produce a mild fibrous peritonitis and granulomas in the retroperitoneum and bladder of cattle, and the larvae of s. equina and perhaps others that may normally spend part of their time in the central nervous system may occasionally penetrate the parenchyma and cause lesions. adults in the peritoneal cavity are oviparous, and the microfilariae can be found in the blood. the intermediate host may be one of a variety of genera of mosquitoes, or for some setaria species, biting flies (hemotobia spp.). the microfilariae develop into infective larvae in to weeks, depending on ambient temperatures, and may survive another to weeks in the intermediate host. they are released from the mosquito and penetrate the final host. setaria digitata is normally found as an adult in the peritoneal cavity of cattle and buffalo in asia. the migration of immature forms in aberrant hosts, such as horses, camels, sheep, and goats, is an important cause of neurologic disease called kumri in asia. the migratory pattern of the larvae is not known, but sensitive organs receive little or no damage in the natural host. in some aberrant hosts, however, infective larvae may invade the brain and spinal cord, usually about - weeks after infection. the sites of penetration and subsequent cerebral migrations are variable, as are the clinical signs produced. characteristically, neurologic signs are of ataxia, weakness, or paralysis. the severity of the clinical signs varies from slight weakness to quadriplegia, depending on the number and location of the wandering parasites; however, affected animals may remain bright and alert. the lesions produced are fundamentally traumatic; the inflammatory component is less conspicuous. a careful gross examination of brain and spinal cord may show the areas of damage as brown foci or streaks. these are more prominent as black spots in formalin-fixed tissue. the lesions are apt to be confined to relatively small foci, so it is important that the examination be made carefully and the material for microscopic examination taken from suspicious areas. the clinical signs may suggest which parts of the brain and cord should receive the most attention. apart from the foci of malacia, the remainder of the nervous system may be normal. at low power, the most obvious feature of the lesions is the microcavitation that is caused mechanically by the migration of the larvae; hemorrhage is variable. surrounding the areas of cavitation, there is loss of myelin, gitter-cell formation, and fragmentation and beading of axons. in cross section this beading may appear as swollen basophilic masses, or alternatively as loss of axis cylinders. gemastocytic astrocytes are present in older lesions. occasionally the parasites can be found in section and made available for proper identification, but the cerebral lesions produced by aberrant parasitic migration, irrespective of the parasite, are distinctive and suggest the diagnosis. they differ from more conventional lesions in that all neural structures (myelin, axis cylinders, nerve cells, and glia) are involved, the patterns of damage are completely random, and frequently, eosinophils are the most common inflammatory cell. neutrophils and macrophages are also frequently present, along with a mild meningitis and vascular cuffing. the term cerebrospinal nematodiasis has been applied to the group of nervous diseases resulting from aberrant larval migrations (see the nervous system, volume ); many parasites such as strongylus vulgaris may be occasional culprits, but only setaria digitata and a few other nematodes, such as pneumostrongylus tenuis in moose, elaphostrongylus cervi in deer, and baylissascaris procyonis in rodents, have tropism for the nervous system and produce the syndrome in epizootic proportions. setaria cervi, which may occur normally in deer and a variety of other cloven-hoofed animals, has been reported as causing cerebrospinal nematodiasis in deer, although elaphostrongylus larvae produce similar signs and lesions, and differentiation may be difficult. setaria digitata larvae frequently invade the eyes of horses, as do the microfilariae of s. equina. primary tumors of the peritoneum may arise from the serosa itself, from the subserous connective tissues, and from the various differentiated special tissues, such as nerve sheaths. tumors arising from the serosa are called mesotheliomas, sometimes . qualified as malignant. the qualification is unnecessary as virtually all are malignant, although the malignant capability is nearly always exhibited as implantation rather than metastasis. mesotheliomas are not common. they occur with greatest frequency in cattle and dogs but occasionally in other species. interest in mesotheliomas has increased since the association between asbestos fiber and mesothelioma was discovered in humans. this association has not been made in animals, in which the tumor has the distinction of occurring most frequently as a congenital neoplasm in fetal or young cattle (fig. . a,d) . mesotheliomas arise from the cells of the serous linings of pericardial, pleural, and peritoneal cavities, frequently involving all three locations. they appear as multiple small firm nodules or villous projections on a thickened mesentery or serosal surface, although fibrous or sclerosing forms have occasionally been reported. the tumor frequently is associated with a milky effusion, and in sclerosing tumors in which adhesions occasionally occur, the lesion might resemble chronic granulomatous peritonitis. ascites as the result of effusion and blocked lymphatics is nearly always present with peritoneal tumors. mesotheliomas of the pleura, pericardium, or peritoneum may assume a variety of histologic patterns. this is not surprising given the diverse potentialities of the tissue that lines the embryonic celomic cavity. the tumors usually take either of two histologic forms, the one predominantly fibrous and resembling fibrosarcoma, and the other papillary and resembling adenocarcinoma. the most common tumor is a solid mass made up of single layers of dark, plump, cuboidal or columnar, neoplastic mesothelial cells with a distinct border and abundant pink cytoplasm, over a proliferating fibrocellular stroma. the mesothelial cells form loops and festoons in a papillary pattern, or line cystic spaces and tubular structures. there may be a mucinous matrix in this acinar pattern. such malignant mesotheliomas, that is, those resembling adenocarcinoma, can mimic implantation of a true carcinoma so completely that an adequate differentiation may rest on very careful examination and exclusion of some primary focus of carcinoma. special stains or electron microscopic examination may be required to identify the cell type. in mesotheliomas that are predominantly fibrous, the cells may be spindle-shaped and resemble a fibrosarcoma. in the sclerosing forms there may be a thick, fibrous serosa with adhesions, and large anaplastic cuboidal cells may be found in clusters or lining cystic spaces within the fibrous tissue. of the retroperitoneal tumors, the lipoma is most frequent. these benign tumors are well known in horses, in which they originate usually in the mesenteries. they may reach enormous size, but their special significance is that they tend to become pedunculated and occasionally cause acute intestinal obstruction when the pedicle winds about a-loop of intestine. in the dog, these tumors arise in the omentum rather than the mesenteries and settle on the abdominal floor. they may attain a very large size but tend not to be pedunculated. they do not, therefore, cause acute distress, and although they may be histologically malignant, metastases are unusual. they develop a pseudocapsule and central areas of necrosis. tumors of the subserosal connective tissues, myxomas, fibromas, and their malignant counterparts, are rare, although fibrosarcomas are observed in dogs. tumors of differentiated retroperitoneal tissues are also uncommon. involvement of the abdominal nerves and plexuses occurs in neurofibromatosis of cattle, and ganglioneuromas have also been observed in this species. extramedullary pheochromocytomas have been observed in cattle and dogs, and nonchromaffin paragangliomas occur in dogs, the latter usually in association with similar tumors elsewhere. occasional adenocarcinomas of high malignancy, but of obscure histogenesis and origin, have been discovered in the dorsal retroperitoneum in dogs. secondary tumors of the peritoneum are not common but are to be expected in any abdominal neoplasia. these arise as direct implantations or as lymphogenous or hematogenous metastases. they are more usually carcinomas than sarcomas. secondary carcinomas may be very scirrhous and, when accompanied by ascites, may closely resemble chronic peritonitis, although a relative or complete absence of adhesions is often a helpful distinguishing feature at autopsy. there are obviously many possibilities for the origin of secondary tumors; several common types are listed below. the cholangiocellular and ovarian carcinomas have already been mentioned. those of bile duct tend to be scirrhous, as do intestinal adenocarcinomas in cattle and sheep. squamous-cell carcinomas of the equine stomach form rather discrete implants, which may be partially caseated, but they are likely to be differentiated enough to be recognizably keratinized on gross inspection. implants of ovarian carcinoma tend to be papillary. vesical tumors developing in cattle in enzootic hematuria implant locally on pelvic epithelium, and rectal adenocarcinoma in dogs tends to confine its implants to the pelvic peritoneum. malignant melanomas of perineal origin in horses produce flattish, black smudges on the peritoneum. cysts of the peritoneum are rather common but insignificant. those associated with genital adnexa are described with those systems, and those associated with intermediate-stage tapeworms have been mentioned, except for echinococcus, which, following the rupture of a mature hydatid into the abdomen, may develop cysts on the peritoneum. small cysts, sometimes multiple, which are observed in the omentum, may be either inclusion cysts or local lymphatic ectasias. they are inert. the normal flat, pavement-type cells of the serosa may undergo metaplasia to a cuboidal or columnar type resembling epithelium. such metaplasia is probably the mildest response of the peritoneum to irritation but may also be a response to estrogen. inflammatory metaplasia leading to ossification can occur in peritoneal scars, especially in swine, but may also be found in the mesenteries and the dorsal retroperitoneum without obvious antecedent change, although ossification may occur following fat necrosis as well. the newly formed bones are flat and of variable size and shape and are usually found in adipose tissue. the assistance of jean middlemiss, sandra brown, and e. w. eaton in the preparation of the manuscript and illustrations is gratefully acknowledged. we thank dr. louise laliberte for information obtained epidemiological study of coronavirus infection in cats with special reference to feline infectious peritonitis" and her supervisor, dr. r. c. povey, for advice on the section on that topic pathogenesis of salt retention in dogs with chronic bile-duct ligation diagnostic abdominal paracentesis and lavage in the evaluation of abdominal injuries in dogs and cats: clinical and experimental investigations renal sodium handling in cirrhosis: a reappraisal renal sodium retention and ascites formation in dogs with experimental cirrhosis but without portal hypertension or increased splanchnic vascular capacity mesothelial cells in peritoneal fluid macrophage functions in antimicrobial defense enzyme histochemical studies of adipose tissue in porcine yellow fat disease fish oil induced yellow fat disease in rats. . histologic changes biology of disease. free radicals and tissue injury a pathological study on fat necrosis in swine pathological studies on fat necrosis (lipomatosis) in cattle muscular dystrophy and yellow fat disease in shetland pony foals studies on fat necrosis of beef cattle in japan. i. occurring aspects. ii. an area study of the outbreak of fat necrosis disseminated necrotizing panniculitis and pancreatic nodular hyperplasia in a dog clinical and pathological observations on generalized steatitis in foals chemical composition of necrotic fat lesions in beef cows grazing fertilized 'kentucky- ' tall fescue studies on bovine fat necrosis. i. epidemiological observations steatitis in two donkey foals lipomatosis (fat necrosis) in cattle and pigs non-extractable lipids in the adipose tissue of horses and ponies affected with generalized steatitis feline infectious peritonitis-proteins of plasma and ascitic fluid systemic vascular lesions in feline infectious peritonitis enteritis due to feline infectious peritonitis virus the virology and pathogenesis of feline infectious peritonitis: brief review immune complexes and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis feline infectious peritonitis: experimental evidence for its multiphasic nature pathogenesis of feline infectious peritonitis: nature and development of viremia pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence feline infectious peritonitis feline infectious peritonitis: review of gross and histopathologic lesions the pathogenesis and transmission of neurotropic and accidental nematode parasites of the central nervous system of mammals and birds. review article. helminthol abstr r part . filarioidea, aproctoidea and diplotriaenoidea. cih keys to the nematode parasites of vertebrates the first report of lumbar paralysis in sheep due to nematode larvae infestation in iran kumri-so-called lumbar paralysisof horses in ceylon (india and burma), and its identification with cerebrospinal nematodiasis strongylus edentatus: development and lesions from ten weeks postinfection to patency t h e zoology of tapeworms sclerosing mesothelioma in five dogs congenital tumors of calves (mesothelioma) smooth muscle differentiation in genital tract disorders mesothelioma in the dog: six case reports an ultrastructural study of canine mesothelioma key: cord- - l fyy authors: nan title: cornea date: - - journal: jubb, kennedy & palmer's pathology of domestic animals doi: . /b - - . - sha: doc_id: cord_uid: l fyy nan presumed allergic conjunctivitis occurs in all species but is most likely to be investigated in dogs (most examples of conjunctivitis in cats are assumed to have an infectious pathogenesis). rarely is a specific allergen identified and, like its counterparts in allergic skin diseases, the diagnosis is based upon the failure to demonstrate infectious or mechanical causes, response to corticosteroid therapy, and sometimes a convincing association with environmental changes. biopsy is rarely warranted, but when taken during the acute disease may show epithelial changes ranging from erosion to hyperplasia to squamous metaplasia, with eosinophils around dilated subepithelial blood vessels and percolating throughout the epithelium. eosinophils are much more likely to be identified in cats than in dogs, a species difference that is also true of allergic skin disease in general. more chronic lesions, which are the more usual to be biopsied, have squamous metaplasia and lymphocytic-plasmacytic subepithelial infiltrates and the formation of lymphoid nodules. there are a few histologically distinctive examples of conjunctivitis that are assumed to represent immune-mediated disease, mostly because they respond only to aggressive immunosuppressive therapy. in some dogs with chronic conjunctivitis (perhaps particularly german shepherd dogs), the infiltrate sometimes becomes particularly plasmacytic, diffuse, and thick in a fashion resembling an interface dermatitis. the bulbar surface of the third eyelid is the favorite location, and many believe this lesion (sometimes referred to as "plasmoma") to be the conjunctival variant of pannus keratitis. cats and horses may develop a severe eosinophilic conjunctivitis that is thought, by some, to be a conjunctival counterpart of the eosinophilic keratitis syndrome. lesions may be unilateral or bilateral, and at least in cats there is almost always a concurrent ulcerative marginal blepharitis. histologic changes include ulceration, epithelial hyperplasia, squamous metaplasia, and a hea w lymphocytic infiltration with a large proportion of eosinophils. lesions in cats contain no detectable infectious agent, and no herpesviral dna can be detected (the role ofherpesviral infection in the pathogenesis of histologically similar eosinophilic keratitis in cats remains controversial). the clinical syndrome is rapidly responsive to topical corticosteroid administration. ligneous conjunctivitis is a distinctive clinical and histologic entity, thus far described only in doberman pinscher dogs.the clinical disease is bilateral and characterized by marked thickening and opacity of the palpebral conjunctiva and conjunctiva of the third eyelid. histologically, the conjunctival lamina propria is thickened by massive deposition of hyaline material and a diffuse scattering of mononuclear leukocytes. most of the leukocytes are t-lymphocytes, and the hyaline material stains weakly for igg and iga. feline lipogranulomatous conjunctivitis is probably the feline counterpart of canine chalazion. the lesion occurs almost exclusively in the lamina propria of the palpebral conjunctiva adjacent to the margin of either the upper or lower eyelid. the histology is very repeatable, consisting of a nodular accumulation of clear lipid lakes intermingled with large foamy macrophages and a few small or mononuclear leukocytes. although the original report contained no mention of adjacent meibomian lobules, the similarity between this entity and some cases of canine chalazion (or granulomatous dermatitis adjacent to injured cutaneous sebaceous glands) is striking and impossible to ignore (see fig. . b). the cornea of domestic mammals is a horizontal ellipse varying from . - . mm in thickness among the various species. in general, the larger and older the animal, the thicker is the cornea. it appears as a structural and physiologic modification of sclera, and when chronically injured may lose the specialized features of cornea and resemble sclera both ophthalmoscopically and histologically. embryologically, the epithelium is derived from surface ectoderm; the stroma and corneal endothelium are from neural crest mesenchyme. the major attribute of cornea is its clarity, and it is the loss of clarity that is the most obvious indicator of corneal disease.the clarity results from several highly specialized anatomic and physiologic features: an unusually regular, nonkeratinized and nonpigmented surface epithelium; an avascular, cell-poor stroma composed of very thin collagen (mostly type i) fibrils arranged in orderly lamellae separated by a critical distance to allow the uninterrupted passage of light ( - angstroms); and a high degree ofstromal dehydration maintained by the presence of epithelial tight junctions, endothelial tight junctions, and a na-k-dependent atp-ase pump in the cell membrane of the corneal endothelium ( fig. . a) . the cornea exists in a privileged environmental niche, bathed in the nurturing and antimicrobial saline of the tear film and protected from other irritation by the movable eyelids. within this protected niche, the cornea does therefore not require the protective attributes of skin (keratinization, leukocytes, blood vessels) to protect itself from the harsh external environment. if there is rapid deterioration in any component of this protective environment, the cornea is most likely to respond with ulceration and subsequent wound healing. if, on the other hand, the shift is of gradual onset, then the more likely response is adaptive metaplasia in which the cornea reaches into its embryologic cutaneous heritage and becomes skin-like. corneal injury may result from physical or chemical trauma, microbial agents, increased intraocular pressure and, rarely, from inborn errors of metabolism. specific features of some of these injuries will be discussed later, but the general reactions to most corneal injuries are presented here. corneal edema occurs rapidly following injury and results from imbibition of lacrimal water through damaged corneal epithelium, absorption of anterior chamber water at a site of corneal endothelial damage, or failure of electrolyte (and thus water) extrusion by the corneal endothelium. if the epithelial or endothelial defect is focal, the resultant edema is limited to the stroma adjacent to the defect. the edematous cornea is clinically opaque, and may be up to five times its normal thickness ( fig. . b ).at least experimentally, edema subsequent to endothelial damage tends to be more severe than edema secondary to epithelial injury. edematous stroma stains less intensely than normal, and collagen lamellae are separated into a fine feltwork of pale-staining fibrils by excessive hydration of the glycosaminoglycan ground substance (principally keratan sulfate and chondroitin sulfate). percolation of stromal fluid into the epithelium results in the intercellular and intracellular edema known as bullous keratopathy (fig. . c) . edema may also be part of more chronic corneal disease. corneal vascularization in response to severe injury is accompanied by edema, as the porous new capillaries leak fluid into the interstitial spaces.a small amount of perivascular corneal edema frequently accompanies the deep stromal vascularization seen in chronic anterior uveitis of any cause. sometimes the edema is unexpectedly diffuse, severe, and may persist even after the uveitis itself has subsided. such eyes have a neutrophilic or lymphocytic destructive endothelialitis, with leukocytes interspersed among the vacuolated, pyknotic endothelial cells (see later section uveitis). other examples of corneal edema are seen in glaucoma and anterior segment anomalies. in the former, it is assumed that the high aqueous pressure drives fluid into the hydrophilic corneal stroma to a degree that overcomes the endothelial ion pump that dehydrates the stroma under normal conditions. in anterior segment anomalies, persistent pupillary membranes or congenital anterior synechiae cause focal defects in endothelial continuity. persistent corneal edema seems to predispose to stromal vascularization and fibrosis, but numerous experimental models show that edema itself is not the stimulus. instead, it is probably the infiltrating neutrophils that provide most of the angiogenic and fibroblastic cytokines. damaged epithelium and stromal fibroblasts are alternative sources. a natural example of virtually permanent corneal edema, without accompanying vascularization, occurs in boston terrier and chihuahua dogs with endothelial dystrophy. injury to the corneal surface that exceeds the homeostatic ability of that epithelium resulting in corneal ulceration is described below. less drastic change to the local environment that results in sublethal injury to the surface epithelium (qualitative or quantitative inadequacy of tears, irritation from misdirected eyelashes or from entropion) will result in adaptive cutaneous metaplasia. the chronically irritated epithelium undergoes reactive hyperplasia with the appearance of rete ridges, melanin pigmentation, and suocace keratinization. the adjacent stroma undergoes dermis-like irregular fibroplasia and acquires vascularization via capillary migration from the limbus.these changes, while they enable the cornea to survive in a hostile environment and to combat the inflammatory stimulus, also deprive it of its transparency ( fig. . ). corneal ulceration represents a loss of the surface epithelial barrier. it causes rapid osmotic imbibition of the tear film water, resulting in corneal edema. neutrophils may be absorbed as well, and if present in large numbers they may initiate stromal lysis as they release their cytoplasmic enzymes. persistent recruitment of neutrophils is usually a manifestation of sepsis, and is discussed further in the sections dealing with keratitis. in any corneal wound, however, there will be at least a few neutrophils. the mechanisms of healing of corneal wounds vary with the severity of the injury, and involve an extremely complex interaction of epithelium, stroma, and innumerable cytokine growth factors derived from tear film, infiltrating leukocytes, and resident epithelium and stroma. only the major elements will be described here, with an emphasis on histologically detectable events rather than on chemical mediators. it is probably true that virtually any corneal injury results in at least some degree of both epithelial and stromal injury, but from a purely histologic perspective one can sort corneal injuries into those involving primarily the epithelium and those involving a combination of the epithelium and underlying stroma. those nonseptic defects involving epithelium alone, or epithelium and supeoqcial stroma, heal by epithelial sliding followed after about hours by mitosis. one cannot claim that depth alone is the deciding factor, since even very deep wounds will sometimes heal just with epithelial sliding and eventual mitotic rebuilding, as long as the epithelium is satisfied with the quality of the underlying stroma. the sliding begins within a few hours, initially from wing cells from the immediately adjacent, viable cornea. migration by basal cells rapidly follows. the sliding is preceded by lysis of the hemidesmosomes. adhesion of the sliding epithelium is initially to adhesion molecules like fibronectin and laminin deposited along the exposed stromal surface. reformation of the hemidesmosomes and their anchoring filaments may take weeks or even months, during which time epithelial adhesion remains precarious. healing of shallow, uninfected corneal ulcers is rapid; experimentally induced -ram ulcers in horses heal within a mean of days. persisting or reoccurring ulcers that cannot be healed just by sliding and replication of adjacent corneal epithelium may require the recruitment of cells from the epithelium at the corneoscleral junction, which is the site of the permanent replicative population. such cells, when recruited for corneal wound healing, seem prone to retaining a conjunctival phenotype that includes pigmentation and a tendency to form rete ridges. one of the characteristics by which we recognize chronic, stubborn corneal ulceration is to observe this conjunctival "metaplasia" of the suocace epithelium.another is to recognize a thickened basement membrane, as the regenerating epithelium always seems to produce its own new basement membrane even if preexisting basement membrane still seems available. if the injurious stimulus disappears, the conjunctival character of the epithelium slowly fades, being replaced within a few weeks by a normal corneal epithelial configuration. epithelial adhesion to the underlying stroma remains fragile for - weeks until the hemidesmosomal attachments of epithelium to basal lamina reform, and until the new epithelium secretes type vii collagen fibrils that anchor the basal lamina to the stroma. in the interim, the cells adhere to a mixture of fibrin and fibronectin derived from the inflamed conjunctival vessels via the tear film or from the injured cornea itself. in many cases, the only evidence of previous shallow ulceration is a thickened basal lamina resulting from secretion by the regenerating epithelium, and gentle undulation of the normally flat epithelial-stromal interface. stromal damage may be a direct result of the severity of the initial injury, but more often it is the result of neutrophil-mediated stromal lysis in those corneal injuries that were initially, or later became, septic. shallow nonprogressive defects in the superficial stroma may be ignored, or may become filled by a thickened plaque of epithelial cells that create an epithelial facet. deeper defects that include more than the outer third of stroma will usually require rebuilding of the damaged stroma before epithelial sliding and regeneration can occur. within a few hours of the insult, neutrophils enter the wound via the tear film.they migrate into the stroma and control bacterial contamination, degrade damaged collagen, and stimulate both fibroplasia and vascularization via production of various cytokines, especially basic fibroblast growth factor derived from injured epithelium, stromal neutrophils, and injured stromal fibroblasts themselves. viable stromal cells (keratocytes) adjacent to the wound undergo fibroblastic metaplasia and secrete large amounts of sulfated ground substance, particularly chondroitin sulfate. most of the new stroma is usually produced by fibroblasts recruited from the limbus.their ingrowth is always accompanied by a similar ingrowth of new blood vessels.this ingrowth begins about days after substantial corneal injury, and migrates from the limbus centrally at a maximal rate of about mm per day. this -day lag time is, presumably, a period of grace in which small or shallow defects can heal with epithelial regeneration alone, without the visual impairment that inevitably follows stromal fibroplasia. once the epithelium seals the defect, there is immediate cessation of neutrophilic influx and, presumably, a similarly abrupt drop in the production of fibroblastic/angioplastic stimulatory cytokines. the scarring and vascularization that are the manifestations of stromal rebuilding are permanent, even though the fibrous tissue gradually becomes less cellular, the collagen fibrils reorient to resemble more closely the parallel arrays of normal stroma, and the ground substance gradually reverts from an embryonic configuration dominated by chondroitin sulfate to the normal predominance of keratan sulfate. complete restitution of normal stroma, however, never occurs, although the residual scar may be subtle and better detected by clinical examination than by histology. undesirable though such scarring may be, it is certainly better than the alternative of ineffective corneal healing and inevitable corneal rupture. healing of a corneal perforation involves the same events as does healing of a deep corneal ulcer, but there are some added challenges and complications.the cut edges of descemet's elastic membrane retract from the wound and the transcorneal gap is initially plugged with fibrin and, sometimes, by prolapsed iris. if the gap is not closed by suturing or by a provisional matrix supplied by fibrin and/or iris stroma, there is the risk that the surface epithelium will grow downward along the cut surface of the stroma and into the anterior chamber ( fig. . a). its migration will be inhibited only by contact with viable corneal endothelium. if it does not encounter that endothelium, there is nothing to stop the epithelium from growing as a layer of stratified squamous epithelium all over the inside of the globe ( fig. . b, c). obstruction of the filtration angle inevitably causes glaucoma. as with the surface epithelium, the corneal endothelium at the deep edge of the perforation attempts to bridge the defect by sliding over the fibrin scaffold to restore endothelial continuity. replacement by mitosis begins within about hours in some experimental models, but the regenerative capability of the corneal endotkelium in adult animals of most domestic spedes is very limited, and repair occurs by endothelial sliding and hypertrophy. so potent is this capability that normal stromal dehydration can be maintained even in the face of a % reduction in endothelial cell density. the cut ends of descemet's membrane make no apparent effort at regrowth, but rather the endothelium gradually secretes a new membrane that may eventually fuse with the old or remain separated from it by a layer of fibrous tissue. the sequence of epithelial sliding and regeneration, remodeling stromal fibrosis and endothelial repair is not uniformly successful. large gaping wounds fill with proliferating epithelium and stromal fibrous tissue that may protrude into the anterior chamber.the fibroblasts, most of which are probably derived from keratocytes but which may also evolve via endothelial metaplasia, tend to grow along the posterior surface of descemet's membrane. regenerating or sliding endothelium is then separated from the coiled remnants of the original descemet's membrane by a dense fibrous layer, called a retrocorneal fibrous membrane. eventually, the corneal endothelium may regain continuity on the posterior surface of this membrane, secrete a new descemet's membrane, and result in a cornea with two separate descemet's membranes. those perforations leading to iris prolapse will usually heal with permanent incorporation of the iris into the huge corneal scar, creating a permanent anterior synechia ( fig. . ). corneal dystrophies are bilateral, inherited (but not necessarily congenital) defects in structure or function of one or more corneal components. they are subclassfed as epithelial, stromal, or endothelial.they are all uncommon, and almost all examples have been described in dogs. the list grows daily, with more than different breeds affected. the least infrequent are the stromal dystrophies characterized by the deposition of lipids and/or minerals within an otherwise normal-appearing stroma. the deposition of mineral or lipid secondary to inflammatory disease or systemic metabolic abnormality should not be interpreted as corneal dystrophy. chihuahuas, dachshunds, and several other dog breeds, and causes slowly progressive bilateral corneal edema in mature dogs.the edema usually begins adjacent to the lateral limbus and may initially be unilateral and unaccompanied by other clinical signs. later, epithelial fluid bullae may rupture to cause painful corneal ulcers and associated inflammation. despite the persistent stromal edema, fibrosis and vascularization do not occur unless rupture of epithelial bullae initiates keratitis. the primary lesion is spontaneous necrosis of corneal endothelium followed by hypertrophy, fibroblastic metaplasia, and sliding of viable endothelium.a marked progressive decrease in overall endothelial cell density results, eventually, in what usually is severe bilateral corneal edema. the reason for the endothelial cell death is unknown. focal irregularities in descemet's membrane occur in areas of endothelial loss, presumably a result of new basement membrane production by adjacent reactive endothelium. posterior polymorphous dystrophy is characterized by multifocal random degeneration of corneal endothelial cells, accompanied by compensatory endothelial hypertrophy. cellular loss causes exposure of descemet's membrane, and adjacent deep stromal patchy edema. a rare,juvenile-onset, genetically transmitted endothelial dystrophy in manx and domestic shorthair cats is manifest as bilateral, progressive central epithelial and stromal edema. fluid accumulates within superficial stroma and within the epithelium. primary morphologic abnormalities are not described in the manx, but in shorthairs there is irregularity and vacuolation of corneal endothelium. corneal stromal dystrophies include a wide range of breed-specific lipid and/or mineral deposits within the corneal strorna, with the exact age of onset, location, and clinical appearance being relatively specific in each breed. specimens are rarely available for histologic assessment since the disease does not cause blindness and is not associated with any systemic abnormality. in most, the chemical nature of the deposit and character of the metabolic abnormality have not been determined. deposition of mineral, lipid, or pigment within the cornea may occur secondary to chronic corneal injury or to systemic metabolic disease in any species. corneal hypermelanosis often accompanies chronic corneal irritation in dogs and less frequently in other species, particularly horses. the pigment is found in the basal layer of the corneal epithelium and in the superficial stroma. it is the result of progressive ingrowth of new germinai cells that have retained pigment from the bulbar conjunctiva. the clinical name, "pigmentary keratitis;' is purely descriptive. the corneal epithelium is invariably hyperplastic and often has the other features of corneal cutaneous metaplasia, such as rete ridge formation, kerafinization, and abnormally thick basement membrane.there is usually evidence of chronic stromal inflammation, including vascuiarization. corneal stromal pigmentation without evidence of epithelial cutaneous metaplasia occurs infrequently, associated with previous iris prolapse that has contributed uveal melanin to the corneal stromal scar. other types of corneal pigmentation are rare. hemosiderin will be found within corneal endothelial cells subsequent to anterior chamber hemorrhage or within stromal macrophages if there has been hemorrhage into the corneal stroma itself. similar pigment may occur following implantation of corneal foreign bodies containing iron or other metals. diets high in cholesterol produce diffuse corneal stromat iipidosis, as well as focal lipid deposits in uveal epithelium and stroma. while hyperlipemia is not a documented prerequisite for most cases of corneal lipidosis in dogs (most of which are spontaneous dystrophies), circumferential peripheral stromal lipidosis is reported in dogs with hyperlipoproteinemia resulting from hypothyroidism and other causes. regardless of pathogenesis, the histologic lesion is similar. cholesterol crystals and lipid vacuoles are found principally in anterior stroma, and are sometimes surrounded by lipid-laden macrophages and variable numbers of other leukocytes (fig. . ) . vascularization is often present, but its pathogenesis is unknown. it appears that corneal vascularization can predispose to stromal lipidosis in animals with hyperlipemia, but it is also true that some animals with primary corneal lipidosis will develop secondary inflammation and vascularization. mineral deposition occurs primarily in the anterior stroma and the epithelial basement membrane. predisposing corneal changes include desiccation, anesthesia, edema, or inflammation. there are many methods for inducing deposition of calcium salts, but stromal edema seems to be the common denominator in almost all cases. the edema may result from corneal epithelial desiccation (exposure keratitis), uveitis, corneal trauma, or chemical injury. hypercalcemia from vitamin d toxicity or hyperparathyroidism exacerbates the mineralization and is essential to lesion development in some experimental models. an unidentified corneal deposition is often seen in canine eyes suffering from multiple anomalies, particularly those involving uvea. similar deposits are seen, with less regularity, in the horizontal midportion of the cornea of many normal puppies. fine basophilic periodic acid-schiff positive linear deposits are associated with the epithelial basement membrane or superficial stroma. there is some disarray of superficial stromal fibers but no inflammation.the nature and pathogenesis of the deposit are unknown, but most disappear after a few months. "corneal degeneration" is a vague term sometimes used to describe those corneal lesions characterized by noninflammatory loss of epithelial or stro-mai viability. diseases such as keratoconjunctivitis sicca and pannus keratopathy are sometimes considered primary degenerative lesions but their principal manifestation is inflammatory and they are discussed under keratitis. the only degenerative, noninflammatory, acquired corneal lesions presented here are corneal sequestrum in cats and horses, and canine persistent erosion syndrome. it is quite possible that all three diseases have a similar pathogenesis, but for the moment they will be listed as three different diseases because of differences in clinical presentation. feline corneal sequestrum is recognized clinically as a discrete orange-brown discoloration of the central cornea, affecting one or both eyes (fig. . ) . persian and himalayan cats are more frequently affected than other breeds. histologically, the lesion is noninflammatory necrosis of stroreal keratocytes, accompanied by pallor, hyalinization, and slight orange discoloration of the affected stroma. the discoloration may be absent in very early cases. the overlying epithelium may be ulcerated or intact, but in those cases with an intact epithelium there is virtually always histologic evidence of previous ulceration. in older lesions, the periphery of the sequestrum may be marked by a zone of reactive mononuclear leukocytes and, perhaps, a few giant cells.the pigment is derived from porphyrins within the tear film, absorbed into the cornea as part of corneal edema that follows ulceration. the sequestrum will eventually slough, and the defect heals by granulation (although most lesions are treated by excision before that stage is reached). the pathogenesis remains controversial. in flat-faced persian and himalayan cats, the pathogenesis probably involves corneal ulceration secondary to desiccation because of abnormal facial configuration. in non-persian cats, there is a loose statistical association with herpesviral infection, and it is reasonable to propose that corneal sequestrum is an uncommon sequel to any corneal ulceration in cats.as will become clearer below, the brown discoloration is unique to cats and, for a long time, caused us to overlook the histologic similarity between the feline disease and similar histologic entities in horses and dogs. not all feline cases acquire the characteristic brown pigmentation, and indeed some examples are virtually indistinguishable from canine and equine persistent ulcers described below. canine persistent (recurrent) ulcer syndrome was first described in boxer dogs (hence the name "boxer ulcer"). although boxers and related breeds may be predisposed, similar recurrent erosions are encountered in a wide variety of breeds. the clinical syndrome is distinctive, characterized by a shallow central corneal erosion with scant edema and (at least initially) no vascularization. the lesion refuses to heal, or repeatedly re-ulcerates, because of poor adhesion of the epithelium to the underlying stroma. the defect appears not to be in epithelial healing per se, since sliding and mitotic activity are normal in affected dogs. keratectomy specimens reveal poorly adherent hyperplastic epithelium at the ulcer margins, usually with multiple clefts separating epithelium from stroma even in areas distant from the obvious ulcer (fig. . ) . the basal lamina is usually not visible with light microscopy, and the epithelium appears to be attempting to adhere to a thin zone of hypocellular, pale-staining stroma that could correctly be interpreted as a very shallow sequestrum qualitatively similar to what was described above in cats. the observation of pyknotic and lyric keratocyte nuclei within this supegtcial zone suggests that the basic defect is degeneration of the supe~cial stroma, so that epithelial hemidesmosomes and anchoring collagen fibrils attempting to reform after ulceration have no substrate in which to anchor.very chronic cases usually acquire superficial stromal granulation tissue appropriate to any chronic ulceration, but its onset is much delayed in comparison to infectious or traumatic ulcers. the lesion in horses is less frequent and less well characterized than in dogs or cats. it is histologically identical to what occurs in dogs, although it seems to be more often complicated and thus disguised by superimposed fungal infection (see fig. . b). morgan rv. feline cornea[ sequestration: a retrospective study of cases ( ) ( ) ( ) ( ) ( ) corneal inflammation is called keratitis and has traditionally been divided into epithelial, stromal (interstitial), and ulcerative keratitis. it is probably time to abandon this arbitrary classification, at least when dealing with ocular histopathology. in realistic terms, almost all corneal inflammatory lesions reaching a pathologist are chronic and severe, and it is difficult to determine how they started. by the time we see them, almost all are ulcerated or show extensive stromal scarring below a healed ulcer. regardless of cause, corneal inflammation initially follows the stereotyped sequence of edema and leukocyte immigration from tears and distant limbic venules.with severe lesions, corneal stromal vascularization, fibrosis and epithelial metaplasia with pigmentation may occur. keratitis usually results from physical, chemical, or microbial injury to the cornea, but the cornea may also be affected by extension of disease from elsewhere in the eye, adnexa, or conjunctiva. the stroma and endothelium may become involved in diseases of the uvea by extension via the aqueous or by direct extension from iris root or ciliary apparatus across the limbus. purely epithelial keratitis is rarely encountered in histologic preparations, either because the clinical lesion is transient and so mild that eyes are unavailable for histologic examination, or because the lesion progresses to ulceration (as in acute keratoconjunctivitis sicca). ulcerative keratitis includes a large group of lesions caused by physical and chemical trauma, desiccation, bacterial, fungal or viral infection, and rarely from primary degeneration of the corneal epithelium itself. regardless of cause, the loss of epithelium initiates a predictable series of corneal reactions caused by tear imbibition, local production of cytokines, and opportunistic microbial contamination of the wound. imbibition causes superficial stromal edema below the ulcer and is followed by immigration of neutrophils from the tear film and, later, from the limbus. the leukocytes, while somewhat protective against opportunistic pathogens, also add their collagenases, proteases, and stimulatory cytokines to the wound and thereby may contribute to its progression. epithelial and stromal repair proceeds as already described for corneal wound healing, but the repair fails in those cases in which microbial contamination is well established or in which the cause of the initial ulceration has not been corrected. common examples of the latter are found in dogs in which corneal trauma by misdirected cilia or facial hair, or desiccation due to lacrimal gland dysfunction, persists. the usual role of bacteria and fungi in the pathogenesis of corneal ulceration is opportunistic. however, these opportunists contribute significantly to the perpetuation and worsening of the lesion. proteases and collagenases of microbial, leukocytic, or corneal origin progressively liquefy corneal stroma, a process termed keratomalacia (fig. . ) . ulcers contaminated by pseudomonas and streptococcus spp. are particularly prone to rapid liquefaction because of the potent collagenases and proteases produced by these organisms. pseudomonas ulcers have been extensively investigated because of the devastating liquefaction of cornea that commonly accompanies this infection. the bacteria themselves produce numerous proteases and other toxins that may be important in the establishment of the early infection, but most of the characteristic stromal malacia results from the action of proteases originating from leukocytes, reactive corneal epithelium, or injured stroma. the stroma contains a variety of proenzymes (for collagenases, elastases, gelatinases, and other stromal lysins) that are cleaved by the pseudomonas toxins to produce the active enzymes. which toxins are produced, and in what quantities, is very strain dependent. the stepwise degradation of stroma is seen histologically as a featureless eosinophilic coagulum, which occurs with progressive septic ulcers regardless of the species of bacterium. neutrophils may encircle the liquefying focus as a thick wall of live and fragmented cells. the resulting localized suppurative keratomalacia is called a ring abscess (fig. . ) , although that terminology is rarely used today. it is seen more commonly in cattle than any other species, perhaps because of the prevalence of untreated, contaminated corneal ulcers in that species and the prevalence of septic corneal perforation. the sequelae of ulcerative keratitis involve cornea, conjunctiva, and uvea. the ulcer itself may heal with vascularization and scarring proportional to the severity of the initial lesion. it may persist as a stubborn but nonprogressive lesion, or it may progress to involve more of the stroma and epithelium. stromal liquefaction that reaches descemet's membrane results in its forward bulging as a descemetocele. this membrane, while resistant to penetration of the microbial agents themselves, is apparently permeable to inflammatory mediators and microbial toxins that diffuse into the anterior chamber. these chemicals, combined with a vasoactive sensory neural reflex from irritated cornea, are responsible for the vasodilation and exudation in anterior uvea that are seen histologically in virtually all globes with deep ulcerative keratitis. even in nonpefforating keratitis, the anterior uveal inflammation may result in sufficient fibrin exudation so as to predispose to focal adhesions of iris to lens or (rarely) to cornea. in the case of corneal perforation, the iris flows forward to plug the defect (iris prolapse) and may subsequently become incorporated into the corneal scarring. this outcome is usually called anterior staphyloma, meaning a focal defect in the ocular fibrous tunic (i.e., cornea) that becomes lined by uvea (fig. . a, b) . the distinction from anterior synechia is of little significance. the conjunctiva is involved in almost all instances of keratitis, either as a victim of the same injury or as the nearest vascularized tissue to the diseased cornea. hyperemia, cellular exudation, and lymphofollicular hyperplasia are common as the conjunctiva responds to the diffusion of inflammatory mediators of microbial, leukocytic, and tissue origin from the injured cornea. there are some instances in which the lesions are found primarily within the stroma. examples of bacterial or fungal keratitis in which the organisms were implanted into the stroma may cause chronic suppurative keratomalacia with negligible involvement of superficial stroma or epithelium. alternatively, deep ulcerative septic keratitis may heal superficially, yet persist deep within the stroma. in either of these two situations, the deep lesion is referred to as stromal abscess. midstromal corneal vascular ingrowth from the limbus is a very common lesion in response to vascular endothelial growth factors elaborated in the course of chronic uveitis of virtually any cause. it appears to be a purely accidental lesion with no obvious purpose, but it does serve as a valuable and permanent histologic marker for previous or ongoing intraocular inflammatory disease. its liability as a marker for subacute or chronic intraocular inflammation is probably not absolute, because similar midstromal vascularization can probably occur in response to growth factors liberated from detached retina or intraocular neoplasms (see significance of uveitis). the only credible candidate for a genuine stromal keratitis, in which the primary target for the inflammatory disease is the stroma itself, is pannus keratitis. this is an idiopathic disease seen most frequently in german shepherds and phenotypically similar breeds. its prevalence and severity are directly correlated with altitude, suggesting that sunlight exposure is part of the pathogenesis. the clinical disease is distinctive. the early lesion is seen in dogs of either sex, usually in early middle age, as a vascularized opacity growing into the corneal stroma from the limbus. the ingrowth is bilateral although not always of simultaneous onset, and most frequently originates from the ventrolateral limbus. there is no ulceration, but pigmentation is often marked. the untreated lesion eventually infiltrates the entire cornea, converting the superficial stroma to an opaque membrane resembling granulation tissue.at one time, superficial keratectomy was the recommended therapy and so histologic specimens were quite often available. today, most cases are treated with potent immunosuppressive therapy, and the need to perform a keratectomy to restore vision is rare indeed. the histologic appearance varies with the duration of the lesion. the initial lesion is supe~cial stromal infiltration of mononuclear cells, especially plasma cells. subsequently, there is progressive vascularization and fibroplasia in the superficial third of the stroma, accompanied by epithelial hyperplasia and pigmentation that may include the stroma. the deep stroma is never affected. the pathogenesis of the condition is unknown, but an immune reaction to altered corneal epithelial antigens is hypothesized. its response to continuous corticosteroid administration supports this hypothesis, as does it striking histologic similarity to discoid lupus and other lupoid dermatoses. despite the similarity, immunofluorescence tests for intraepithelial or basement membrane immunoglobulin are negative. infectious agents are not consistently isolated.a histologically similar lesion of the bulbar conjuctiva of third eyelid occurs in the same breed (so-called "plasmoma") and may reflect the same mysterious pathogenesis. the response of the cornea to desiccation depends on the rapidity of onset and the severity of the desiccation. it is seen as a consequence of inadequacy in the quantity or quality of the tear film (usually called keratoconjunctivitis sicca). it also occurs as a consequence of exophthalmos, improper eyelid closure because of eyelid developmental anomaly, acquired eyelid disease, nerve injury to prevent blinking, profound cns depression in which the blink reflex is lost, or conditions such as glaucoma or orbital mass that prevent proper eyelid closure because of abnormal ocular size or position. under such circumstances, the corneal lesion is usually referred to as desiccation keratitis, although the effect upon the cornea is exactly the same as in acute keratoconjunctivitis sicca. in those cases in which the desiccation occurs only in a horizontal band not adequately covered by the eyelids for whatever reason, the lesion is sometimes referred to as band keratopathy. if the desiccation is profound and occurs rapidly, the cornea has no time to adapt and the outcome is acute ulceration. it can be distinguished from other types of corneal ulceration histologically because it is the only example of corneal ulceration that occurs in the absence of edema or neutrophilic infiltration (because there is no tear film to provide either the water or the leukocytes). if the desiccation is only mild, or occurs over a long interval that allows corneal adaptation, the resulting lesion is corneal cutaneous metaplasia. desiccation keratitis (either acute or chronic) may follow destruction or denervation of lacrimal or accessory lacrimal gland by orbital inflammation, drugs, neoplasia, or trauma. squamous metaplasia with resultant inadequacy of secretion may be seen with chronic deficiency of vitamin a. specific lacrimal adenitis with subsequent atrophy is well recognized with coronavirus infection in rats and may be seen in the acute or chronic phases of canine distemper. similar adenitis probably occurs with other viruses and in other species but such lesions are poorly documented. transient keratoconjunctivitis sicca may accompany acute herpetic keratoconjunctivitis in cats. keratoconjunctivitis sicca, as a specific disease entity, is encountered more commonly in dogs than in any other species, with an overall prevalence in north america of about %. most cases are chronic, progressive, and idiopathic. the reason for greater than expected prevalence in certain breeds (english bulldog, lhasa apso, shih tzu, west highland white terrier, and others) is unknown. because the disease is amenable to medical or surgical management, few specimens are available for histologic examination until the very chronic stages. at this time, the lacrimal gland is atrophic with interstitial lymphoid infiltration and fibrosis, but provides no clue as to the initial lesion. the ability of certain immune modulators, notably cyclosporine, to reverse the disease, points to some kind of immune-mediated phenomenon, perhaps autoimmunity. the corneal changes vary with the severity and rapidity of onset of lacrimal deficiency. in acute disease with marked lacrimal deficiency, clinical signs of ulcerative keratitis may occur. the corneal epithelium is thinned, has numerous hydropically degenerate cells, and may suffer full-thickness ulceration. the accompanying stromal changes, including eventual vascularization and fibrosis, are those of ulcerative keratitis. more commonly in dogs, however, the desiccation is not absolute (at least initially) and the epithelial response is protective epidermalization without prior ulceration. keratinization, marked hyperplasia with rete ridge formation, and pigmentation are commonly seen. stromal inflammation and vascularization are usually superficial, resulting in a lesion very similar to pannus keratitis. squamous metaplasia may also occur in the bulbar conjunctiva.the conjunctivitis that clinically is the earliest lesion of keratoconjunctivitis sicca is rarely available for histologic examination. feline herpetic keratitis, caused by felid herpesvirus , is seen either as the sole ocular lesion or in concert with conjunctivitis. clinical signs associated with herpesviral infections in cats include conjunctivitis, keratitis, rhinotracheitis and, in neonates, systemic disease with encephalitis and necrosis in visceral organs. acquired immunity alters the manifestations of the disease and results in different lesions predominating in different age groups. keratitis is commonest in adult cats and seems to result from activation of latent infection during concurrent immunosuppressive disease or corticosteroid therapy. concurrent mild respiratory disease may be present, but in adults the disease is often purely corneal and may even be unilateral. in contrast, the infection in adolescent cats causes nonspecific bilateral erosive conjunctivitis without keratitis. intranuclear inclusions are numerous within cells prior to sloughing, and leukocytes are sparse until ulceration permits opportunistic contamination. upper respiratory disease is almost always present. the corneal lesions fall into two very different categories: shallow transient erosions and ulcers that represent the direct cytopathic effect of acute viral infection, and more severe stromal keratitis that is probably an immune response to viral antigen in persistent or recurring infections. the typical acute superficial corneal lesions are multifocal minute corneal erosions and ulcers that have a tendency to coalesce into branching dendritic ulcers. early in the disease, typical herpesviral inclusions may be seen with histology, and herpesviral antigen can be demonstrated with immunofluorescence or other techniques. severe or recurrent lesions in immunosuppressed cats may result in underlying stromal keratitis with lymphocytic infiltration, persistent edema, and vascularizati°n" there is much more written about the clinical features and clinical diagnosis of herpesviral keratitis than there it is about its pathology, simply because most cases are never subjected to histologic evaluation. by the time a sample of conjunctiva or cornea is taken for histologic assessment in cases that have been therapeutically resistant, histologic detection of inclusion bodies is futile and immunofluorescence is usually negative.virus can usually be detected with pcr, but interpretation of that result is almost impossible because of the high prevalence of carriage in asymptomatic, healthy cats. for the same reason, attempts to link persistent herpesviral infection with feline corneal sequestrum or feline eosinophilic keratitis have been less than convincing. another uniquely feline ocular lesion is seen clinically as unilateral or bilateral proliferative, 'fluffy" white stromal keratitis. there is no breed, age, or sex predilection, and no proven association with other ocular or systemic disease. since the diagnosis is made by cytologic evaluation of superficial scrapings or (occasionally) by histologic examination of surgical keratectomy specimens, this disease is more likely to be seen by pathologists than most other corneal disorders. scrapings of the surface of the lesion reveal numerous eosinophils and fewer mast cells and other mononuclear leukocytes. eosinophils may be less conspicuous on histologic examination of keratectomy specimens, perhaps because most seem determined to emigrate through the epithelium and into the tear film rather than remain within the tissue. instead, the stromal lesion is a mixture of macrophages, plasma cells, fibroblasts and, unpredictably, mast cells and at least a few eosinophils.the latter are least frequent in older lesions, either because of time alone or because older lesions are more likely to have received a lot of corticosteroid therapy. a characteristic lesion, not present in every case, is a dense granular eosinophilic coagulum along the surface of the keratectomy specimen. it seems to consist of free eosinophil granules. no bacterial or fungal agents have been seen.while there are histologic similarities to cutaneous eosinophilic ulcer and linear granuloma, no statistical association has been proven and the lack of understanding even of the cutaneous eosinophilic lesions makes such attempted comparisons of very limited value. while much speculation exists about the relationship between persistent herpesviral infection and eosinophilic keratitis, there is no proven etiologic link. mycotic keratitis is a destructive, suppurative, ulcerative and deep stroreal keratitis most commonly seen in horses, but occasionally encountered in all domestic species. the offending fungus is usually a member of the normal conjunctival flora, and its role in the disease is always that of opportunistic contaminant. aspergillus is the most frequent isolate, but cases caused by other common conjunctival fungi like alternaria, penicillium, and cladosporium are not rare. most cases are probably iatrogenic, occurring in animals in which a corneal ulcer, laceration, or penetrating wound had been treated with long-term antibiotics and/or corticosteroids.the latter is a particularly common villain in this context. horses seem particularly prone to mycotic keratitis, perhaps related to the mold-laden, dusty environment in which many horses are housed; only rarely does the lesion occur in dogs or cats. since virtually all stabled horses have fungi as part of their conjunctival flora, seeing hyphae within the corneal stroma is required for the diagnosis. isolation from a corneal swab or shallow scraping is not adequate. because the disease is much more prevalent in horses than any other species, most of the description below is derived from equine cases. the histologic changes in other species, however, are very similar to what occurs in horses.there appears to be a difference in the typical lesion seen in temperate climates and what occurs in horses in very warm and humid environments. in the latter, there are cases in which the fungi are found throughout the cornea and are easily identified by even shallow scraping.that is not the case in those examples of the disease diagnosed in cooler climates, which i will consider the "typical" disease. the typical early lesion is deep ulcerative keratitis with suppurative keratomalacia. some chronic lesions are exclusively stromal because of successful epithelial and superficial stromal healing of the initial penetration (or perhaps because therapy eliminated the infection in the superficial stroma). for whatever reason, the typical equine eye enucleated for mycotic keratitis has an intense neutrophil-rich deep stromal keratitis with several characteristic features: neutrophils are karyorrhectic, inflammation is most intense immediately adjacent to descemet's membrane, and frequently there is lysis of the normally resistant descemet's membrane with spillage of the corneal inflammation into the anterior chamber. fungi are numerous within the malacia of the deep stroma and within descemet's membrane itself, but rarely if ever are seen within the anterior chamber (fig. . a ). when they occur within the anterior chamber, they are always anchored to the nearby descemet's membrane. despite ample opportunity, there has never been a reported case of disseminated intraocular mycosis as a sequel to mycotic keratitis. fungi are sparse or absent within the superficial half of the stroma, which explains why corneal scrapings or even keratectomy specimens may fail to reveal the agent. the reason for the apparent targeting of descemet's membrane is not known, but the presence of the tropism even in untreated eyes suggests that it is a genuine tropism and not just persistence of a previously generalized stromal infection in the site least likely to be reached by topical fungicides. in horses in tropical and near-tropical climates (many reports, for example, come from florida), the fungi are more diffusely distributed within the cornea and are thus more easily captured by routine cytology or culture swabs. while the distribution of the lesion within the cornea is also more diffuse, its fundamental lytic character remains the same. in horses, cats, and dogs, we will occasionally see corneal sequestra contaminated with fungal hyphae; the fungi are easily found on scraping, leading to the mistaken impression that this is true mycotic keratitis ( fig. . b ). one could debate the issue, but it seems better to keep this as a separate syndrome quite different from the lesion described above. infectious bovine keratoconjunctivitis ("pinkeye") vies with squamous cell carcinoma as the most important disease of the bovine eye. it occurs worldwide, is most prevalent in summer due to the increase in fly vectors, and has a clinical expression that ranges from initial conjunctivitis and ulcerative keratitis to iris prolapse, glaucoma, and phthisis bulbi. the prevalence of severe sequelae reflects inadequate management of the disease rather than any special virulence of this agent as compared to other infectious causes of keratitis in other species. the disease behaves as an infectious epizootic within a susceptible population, frequently affecting over % of the cattle at risk within weeks of the initial clinical case. shedding of virulent organisms by a carrier animal is thought to be the usual route of introduction into a previously unexposed group, although a role for various mechanical or biological vectors is also assumed. moraxella bovis has been confirmed as the most important causative agent, and the only one for which koch's postulates have been fulfilled. concurrent infection with other agents such as mycoplasma bovoculi, mycoplasma conjunctivae, acholeplasma laidlawii, and bovine herpesvirus may contribute to lesion severity. earlier skepticism about the virulence of m. boris, based upon the unreliability of reproduction of the disease, isolation of the organism from apparently healthy cattle, and failure of isolation from some overtly affected cattle, has been overcome by detailed information on the pathogenesis of the disease. it is now clear that virulence of m. boris is associated with hemolytic, leukocytolytic, piliated strains that predominate only in the eyes of affected cattle. pathogenic isolates of m. boris express a calcium-dependent transmembrane pore-forming cytotoxin. nonpiliated, nonhemolytic strains predominate in healthy cattle and are probably part of the normal conjunctival flora. the use of immunofluorescence has demonstrated m. boris in many of the naturally occurring cases for which the results of culture were negative. in naturally occurring outbreaks, the number of isolations of hemolytic m. boris falls to almost zero as the outbreak wanes, but a few chronically affected carriers remain as the most important source of virulent bacteria for outbreaks of disease in the next summer. in addition to variation in the virulence of different strains of m. boris, sunlight, dust and, perhaps, concurrent infection with infectious bovine rhinotracheitis virus (bovine herpesvirus ) increase the severity of the disease. calves are usually affected more severely than cattle over years of age, although absolute resistance to infection seems fragile. the protective effect of serum antibody against the disease is controversial. specific iga is found in tears of infected calves, and there is substantial evidence that locally produced iga is strongly protective. following experimental inoculation of virulent m. bovis onto the cornea, pilus-mediated adhesion and production of bacterial cytotoxin result in microscopic ulceration in as little as hours. initial adhesion is to older surface epithelium ("dark cells") and results in the development of microscopic pits in the cell surface. moraxella is found within degenerate epithelial cells, but it is not known whether invasion is necessary for subsequent cellular destruction. infield epizootics, the earliest lesion is bulbar conjunctival edema and hyperemia, followed in - hours by the appearance ofa shallow central corneal ulcer. the ulcer is a small (< . cm) focus of epithelial necrosis that may appear as erosion, vesicle, or full-thickness epithelial loss. in untreated animals destined to develop the full clinical expression, the ulcer enlarges, deepens, and frequently attracts enough neutrophils to qualifj as a corneal abscess. stromal liquefaction ensues, probably as a result of neutrophil lysis, which is itself initiated by moraxella-derived leukotoxins. by the end of the first week, there is extensive stromal edema and vascularization extending from the imbus.a~ with any severe ulcerative keratitis, the subsequent progression or regression of the lesion varies with each case as modifications by therapy, opportunistic bacterial and fungal contamination, trauma, inflammation, and immunity interact. keratomalacia frequently leads to forward coning of the weakened cornea (keratoconus). in most instances, whether treated or not, the cornea heals by sloughing of necrotic tissue and filling of the defect by granulation tissue. re-epithelialization may take up to a month, leaving a cornea that is slightly coned and variably scarred. the scarring often is scant and interferes little with vision in spite of the severity of the primary lesion. less satisfactory sequelae, while not common in relation to the overall disease prevalence, are still relatively common. sterile anterior uveal inflammation may result in focal or generalized adherence of iris to cornea (anterior synechia) or lens (posterior synechia). descemetocele may progress to corneal rupture, which in turn may lead to phthisis bulbi or resolve by sealing with prolapse of the iris. synechia and staphyloma may lead to impairment of aqueous drainage and thus to glaucoma. epizootics of conjunctivitis and keratitis in sheep and goats share many of the features of the bovine disease: summer prevalence, rapid spread, and exacerbation by dust, sunlight, and flies. feedlot lambs seem particularly susceptible. unlike bovine keratoconjunctivitis, the range of clinical signs and proposed causes suggests that there may in fact be several different diseases. many agents including bacteria, mycoplasmas, chlamydiae and rickettsiae have been suggested as causes, but various mycoplasmas and chlamydophila psittaci may be the most important agents. the lesions caused by mycoplasma mycoides var. capri in goats and mycoplasma conjunctivae var. ovis in sheep are similar but usually milder than those caused by moraxella bovis in cattle. this is particularly true of goats in which deep corneal ulceration is uncommon. keratoconjunctivitis associated with chlamydophila psittaci is usually predominantly conjunctivitis. initial chemosis and reddening are followed by massive lymphofollicular hyperplasia in bulbar conjunctiva and nictitating membrane. keratitis may occur but ulceration is seldom prominent. animals with conjunctivitis may have concurrent polyarthritis from which chlamydiae can be isolated. the lens is a flattened sphere of epithelial cells suspended in the pupillary aperture by a large number of transparent elastin-like fibers known as lens zonules. these originate from the lens capsule near the equator, and fuse with the nonpigmented ciliary epithelium along the lateral surfaces of the ciliary processes or in the valleys between adjacent ciliary processes. the range ofhistologic reaction of lens to injury is very limited due to the simplicity of its structure and physiology, and its lack ofvascularity. the lens is entirely epithelial. outermost is a thick, elastic capsule, which is the basement membrane produced by the underlying germinal epithelial cells.the capsule is thickest at the anterior pole and becomes progressively thinner over the posterior half of the lens. the capsule in the neonate is thin, but it thickens progressively throughout life. below the capsule is a layer of simple cuboidal lens epithelium that, in all but fetal globes, is found below the capsule of only the anterior half of the ens.the apex of these cells faces inward toward the lens nucleus. at the equator, these germinal cells extend into the lens cortex as the nuclear bow, an arc of cells being progressively transformed from cuboidal germinal epithelium to the elongated spindle shape of the mature lensfibers (fig. . ) .the bulk of the lens is composed of onion-like layers of elongated epithelial cells anchored to each other by interlocking surface ridges, grooves, and ball-and-socket protuberances. these elongated fibers contain no nucleus and few cytoplasmic organelles, relying almost entirely on anaerobic glycolysis for energy. since the lens cannot shed aging fibers as does skin or intestine, these cells are compacted into the fetine eosinophiuc conjunctivitis diseases of the eyelids (part i) eyetid pyogranutomas in four dogs studies on specific ocub-vascutar myiasis (uitpeubog)in sheep onchocerciasis in two dogs occurrence of thelazia lacrymalis (nematoda, spirurida, thetaziidae) in native horses in abruzzo region (centrat eastern itaty) ophthatmomyiasis interna anterior associated with cuterebra spp in a cat keratoconjunctivitis associated with infectious bovine rhinotracheitis keratoconjunctivitis sicca: histopathotogic study of nictitating membrane and [acrimat grands from dogs lipogranubmatous conjunctivitis in cats diseases of the eyetids (part ii) motecutar characterization of the first interna[ transcribed spacer of ribosoma[ dna of the most common species of eyeworms cutaneous and ocutar habronemiasis in horses: cases ligneous conjunctivitis in four doberman pinschers diseases of the equine eyeud lipogranubmatous conjunctivitis: clinical findings from eyes of cats the identification of mycoplasmo conjunctivae as an aetio-[ogica[ agent of infectious keratoconjunctivitis of sheep in south africa canine and feline primary ocutar bacteriat infections keratoconjunctivitis sicca in dogs pl, dice pf cornea[ dystrophy in the dog and cat. vet c indolent-like cornea[ ulcers in three horses crystalline cornea[ dystrophy in the dog. histochemica[ and ultrastructural study cornea ouantitation of excess stromal cho[estero[ and phospho[ipid cornea[ necrosis in the cat recurrent cornea[ erosions and epithelial dystrophy in the boxer dog mineralized cornea[ sequestrum in a cat bilateral ocu{ar [ipidosis in a cottontail rabbit fed an all-milk diet primary canine cornea[ endothelial ceil dystrophy: specular microscopic evatuation, diagnosis and therapy chronic superficial erosions with anterior stroma[ sequestration in three horses. vet comp ophtha[mo idiopathic persistent cornea[ erosions: clinical and patho[ogicat findings in dogs nonhea[ing cornea[ ulcers in cats: cases ( - ) cornea[ stroma[ sequestration and keratoconjunctivitis sicca in a horse. vet phtha[mo crystalline cornea[ opacities in the siberian husky cornea[ endothe[iat dystrophy in the dog ocular manifestations of feline herpesvirus corneal pannus (chronic superficial keratitis) in the german shepherd dog superficial pigmentary keratitis in the dog ulcerative keratitis caused by hemolytic streptococcus equi in eleven horses pathogenesis of cornea[ damage from pseudomonas exotoxin a. invest ophtha[mo the cornea[ response to pseudomonas aeruginosa: histo-pathological and enzymatic characterization. invest ophtha[mo detection of feline herpesvirus dna in corneas of cats with eosinophi[ic keratitis or cornea[ sequestration bacteria[ cornea[ diseases in dogs and cats. c[in tech small anim pract pathogenesis of cornea[ infection: binding of pseudomonas aeruginosa to specific phospho[ipids feline eosinophilic keratitis: a review of clinical cases u[trastructura[ study of unidentified inclusions in the cornea and iridocorneal angle of dogs with pannus relevance of host-derived and bacteria[ factors in pseudornonas aeruginosa cornea[ infections. invest ophtha[mo histological and immunohistochemica[ evaluation of canine chronic superficial keratitis histopathology of mycotic diseases and horses and a review of the literature clinical appearances, healing patterns, risk factors, and outcomes of horses with funga[ keratitis: cases ( - ) equine keratomycosis: clinical and laboratory findings in cases canine mycotic keratoconjuntivitis caused by acremonium kiliense prevalence of ocular microorganisms in hospitalized and stabled horses key: cord- -t f vr w authors: dowers, kristy l; lappin, michael r title: the pyrexic cat date: - - journal: problem-based feline medicine doi: . /b - - - - . - sha: doc_id: cord_uid: t f vr w nan • temperature > . ˚c ( . ˚f). • true fever results from a cascade of events, which starts with activation of leukocytes. pyrogenic factors released from the leukocytes increase the thermoregulatory set point in the hypothalamus. signs that may be associated with fever include: • elevated body temperature. • reluctance to move. • anorexia. • depression. • hyperpnea. • muscle or joint stiffness/discomfort. • shivering. • inflammation anywhere in the body can result in elevation of core body temperature above . ˚c ( . ˚f). • the most common etiology for fever in the cat is percutaneous cellulitis or abscess. viral diseases such as fiv, felv and fip are important diseases to consider. conjunctivitis is the predominant sign and is often initially unilateral and becomes bilateral. ocular discharge is serous initially then mucopurulent, but is usually mild. fever, anorexia and lethargy may occur. true fever must be differentiated from hyperthermia, which can be caused by increased muscle activity, increased environmental temperature and stress. true fever results from activation of leukocytes that release factors (pyrogens) such as interleukin- and tumor necrosis factor. • these factors cross the blood-brain barrier and increase the thermoregulatory set point in the hypothalamus. • leukocytes are activated by a multitude of infectious agents, neoplasia, tissue necrosis and immune-mediated diseases. fever is defined as systemic elevation of core body temperature above . ˚c ( . ˚f). the most accurate measurement of core body temperature is obtained rectally. aural temperature is approximately − . ˚c ( . ˚f) lower than the rectal temperature. fever is a general clinical sign that can be associated with many different diseases. the most common disease causing fever in the cat is percutaneous cellulites or abscess. many viral and bacterial diseases cause fever because leukocytes are recruited and activated as part of the general immune response. organ inflammation, such as pancreatitis, cholangiohepatitis and myocarditis, can be associated with an elevated temperature even when an infectious agent is not present. classical signs • fever. • anorexia (partial or complete). • reluctance to move, lethargy and depression. • pain, heat or swelling at site of abscess or cellulitis. cellulitis usually precedes an abscess, and if treated appropriately, the abscess may not even form. cellulitis may be the only evidence of a previous abscess. an abscess may rupture spontaneously, and the owner may notice foul-smelling, purulent discharge on the fur. • some abscesses resolve on their own with or without rupture, if they have been present long enough. regional lymphadenopathy may occur near the affected site. cellulitis spreads rapidly with the development of multiple fistulae and a febrile response. • lameness from septic arthritis is a common sequelae to infection with l forms. joints are affected by the hematogenous route and may be distant to the initial site. lower limbs (tarsus and carpus) are most commonly affected. the joints often ulcerate with a grayish mucinous exudate. infection remains confined to subcutaneous tissues and joints without systemic spread to internal organs. history supports access to outdoors or conflict with other cats indoors. palpation reveals a tender area or fluctuant swelling, with or without evidence of puncture wounds. microscopic examination of a fine-needle aspirate of the abscess reveals a heterogeneous population of bacteria, numerous degenerate neutrophils and intracellular bacteria. a complete blood count will generally show neutrophilia. l forms are not visible in tissue samples even with special stains, nor do they grow on culture. on electromicroscopy, organisms are visible intracellularly within phagocytes. diagnosis is often made by response to tetracyclines in a therapeutic trial (doxycycline mg/kg po, q h). response is rapid and evident within h. non-healing abscesses should have histopathology and culture of tissue. causes include nocardia, fungi, mycobacteria, and tumors. see page , the cat with non-healing wounds. in plague-endemic regions, yersinia pestis (plague) must be considered, if the swelling is predominately in the neck region and the cat's fever is in the region of . ˚c ( ˚f). cautionary measures such as gloves, masks and isolation of the suspect cat should be taken until diagnosis established. (see below for discussion of y. pestis infections). fracture. ligament/tendon injury. neoplasia. clip area looking for evidence of puncture wounds. drainage of the purulent material is the key to treatment. surgical drainage can be done under sedation or general anesthesia with a # blade. make a / - / " incision over the dependent area, or the area most likely to allow for continued drainage. flush the wound thoroughly with sterile saline or a saline/betadine mixture. explore the wound with a sterile cotton swab or hemostats to assess the extent of dead-space and to look for a possible foreign body. leave the wound open to allow drainage of further purulent material. do not suture incision closed, as this will only allow the abscess to reform. a penrose drain may be placed for - days to allow maximum drainage for abscesses that close too early. antibiotic therapy for - days directed against anaerobes: penicillins, cephalosporins, clindamycin and metronidazole are reasonable choices. most abscesses respond extremely well to drainage and amoxicillin at - mg/kg po q hours for days or amoxicillin/clavulonic acid ( . mg/kg po q hours). l-forms and mycoplasma spp. respond to doxycycline or tetracycline within hours, but not other antibiotics. if the wound is not healing well, or the cat has had recurrent abscesses, felv/fiv testing is recommended to rule out an underlying immunodeficiency. further considerations are inappropriate antibiotics (consider culture and sensitivity testing) or the presence of an undetected foreign body (consider surgical exploration of the area) or involvement of underlying bone (osteomyelitis). prognosis is good unless there is an underlying immunodeficiency. restrict the cat to an indoor environment only; although less effective, confine cat indoors at least from dusk to dawn. neuter male intact animals to decrease territorial behavior. felv and fiv serology should be repeated - months following bite wounds. classical signs acute onset of sneezing followed by oculonasal discharge. discharge progresses from serous to mucoid to mucopurulent. severe conjunctivitis with tearing, photophobia and chemosis. hypersalivation may occur as an initial sign before the classic signs of upper respiratory tract appear. punctate corneal ulcers that may coalesce to larger ulcers or perforation. fever of - days duration, anorexia and depression. retching or coughing may occur. cats with anterior uveitis have occasionally have herpesvirus in the aqueous humor. presumptive diagnosis can be made on the basis of history and clinical signs because treatment for feline herpes virus- and calicivirus are similar. ocular ulcerations and chemosis are more suggestive of fhv- . definitive diagnosis is by direct ifa of cells obtained from conjunctival or nasal scrapings, or by viral isolation or polymerase chain reaction assays from oropharyngeal or nasal swabs. sudden onset of serous ocular discharge and mild conjunctivitis; these signs may begin unilaterally, but often progress bilaterally. initial signs are rapidly followed by sneezing, which are not paroxysmal and are less prominent than in herpesvirus. nasal discharge is primarily serous to mucoid and rarely progresses to purulent. oral ulcerations are common, especially on the tongue, and may be associated with drooling or hypersalivation. ulcers may also occur at the mucocutaneous junction, hard palate and nose. fever generally spikes initially after infection prior to onset of signs, and returns with onset of clinical signs. viral pneumonia occurs occasionally with certain strains, and may produce significant mortality. death is often sudden and preceded by laboured respiration. a rare variant strain (fcv-ari) reported from the united states, produces a high fever, facial and paw edema ( % of cats), ocular and nasal discharge, conjunctivitis and ulcerative stomatitis ( % of cats), hemorrhage from the nose, git, etc. ( - % of cats), icterus ( % of cats) and rapid death. mortality is high ( - %). presumptive diagnosis can be made on basis of history and clinical signs because treatment for feline herpes virus- and calicivirus are similar. oral ulcerations or clinical signs of pneumonia are more suggestive of calicivirus. definitive diagnosis is by viral isolation or reverse transcriptase polymerase chain reaction assays from swabs taken from the oropharynx, ideally in the first week of illness. demonstration of increasing serum antibody titers to feline calicivirus in paired samples is also useful, whereas measurement of a single titer is not useful because many cats have titers from vaccination. identification of fcv-ari is based on the clinical syndrome, pathology and culture of virus from blood, nasal or ocular discharge, spleen or lungs. clinical signs are often non-specific and include fever, anorexia and weight loss. dyspnea and harsh lung sounds without coughing is common. peripheral and visceral lymphadenopathies are frequently present. pale mucous membranes, icterus, hepatomegaly or splenomegaly may be evident. ocular signs are uncommon, but can occur. gastrointestinal signs are uncommon in cats compared to dogs, and include chronic diarrhea, mesenteric lymphadenopathy and anorexia. osseous lesions produce soft tissue swelling and lameness. diagnosis is by demonstration of the organism in lymph nodes, draining tracts, bone lesions or vitreous humor. the organism has a thin capsule and is intracellular within macrophages. no reliable serologic test available. genetic predisposition appears to play a role. fip is most common in catteries and multi-cat households. there are two clinical forms of fip, effusive or wet form and non-effusive or dry form. both are characterized by a fluctuating fever unresponsive to antibiotics, anorexia, lethargy and weight loss. typical age of onset is months to years, but any age can be affected. the effusive form may have any of the following signs: • abdominal effusion that is non-painful but progressive. the amount of effusion varies from volumes causing abdominal enlargement, to amounts only detectable by abdominocentesis. fluid is straw-colored and highly viscous, like egg white. • pleural effusion resulting in dyspnea occurs in % of cats with the effusive form. pericardial fluid may be evident on ultrasound. usually it not associated with clinical signs, but occasionally can produce cardiac tamponade. • male cats may present with scrotal swelling. the non-effusive form may have any of the following signs: • ocular signs result from pyogranulomatous inflammation of the iris and ciliary body. they include bilateral uveitis, perivascular exudates (cuffing), retinal hemorrhage, retinal detachment. • neurologic signs include cerebral and cerebellarvestibular signs such as seizures, personality changes, nystagmus, head tilt, circling, head tremor and hyperesthesia. • dysfunction of any organ system may result from granuloma formation within the tissue of that organ, e.g., liver, kidney, spleen, intestines, lungs, etc., however, organ failure producing clinical signs only rarely occurs, and most dysfunction is only detected on biochemical tests. • granulomatous masses may be palpable in abdominal viscera especially mesentery, mesenteric lymph nodes and omentum as tender, irregular masses. occasionally vomiting or diarrhea results from extensive lesions on the bowel wall. jaundice may occur with either form of the disease. histopathology of affected tissues provides the only definitive antemortem diagnosis. the classic fip lesion is pyogranulomatous infiltration around venules. the following are typical abnormalities associated with fip. all asterisked items must be present for a high likelihood of fip; if any one parameter is not present, fip is unlikely. a negative coronavirus ("fip") titer suggests fip is not the cause of the fever, although a few cats with the effusive form of the disease are titer negative. lymphopenia (< . × cells/μl).* occurs in many cats with fip, and many cats without fip. except where the classical effusive fluid is present, definitive diagnosis of fip requires organ biopsy and demonstration of classical histopathological lesions. various non-specific abnormalities may be evident on laboratory tests, including increased total white cell count, mild to moderate anemia, and increased concentrations of bilirubin, liver enzymes, bun, creatinine, fibrinogen, globulin and mild proteinuria. csf typically has increased protein (> g/l) and cell counts (> cells/ml) which are predominantly nonlytic neutrophils. ocular signs: toxoplasmosis, fungal agents. neurologic signs: toxoplasmosis, neoplasia (e.g., lymphoma), trauma, congenital abnormalities in young cats. other clinical signs: rule out other diseases associated with the apparent organ dysfunction. lymphocytic, plasmocytic cholangiohepatitis occasionally produces a high protein abdominal fluid similar to that of effusive fip. fip is a fatal disease with no known treatments. the therapies listed below have been used in an attempt to slow progression and/or to improve quality of life. glucocorticoids at immunosuppressive doses (prednisolone mg/kg/day). cyclophosphamide ( - mg/m q - weeks or . mg/kg daily for days each week) or chlorambucil ( mg/m q - weeks). +/− broad-spectrum antibiotics to control secondary bacterial infections while the cat is immunosuppressed. prognosis is poor. the mortality is > %. fecal-oral transmission is most likely; transplacental transmission is rare. fomites, e.g., food bowls and litter trays, may be an important mode of transmission, as some strains of fcov survive in dried secretions for several weeks. a seronegative cat introduced into a household where coronavirus is endemic has a in chance of developing fip; a seropositive cat under the same conditions has a in chance. both young and old animals seem to be most susceptible due to vulnerable immune systems. maternal antibodies that protect kittens wane at approximately - weeks of age. reduce fecal-oral contamination by providing one litterbox for every - cats, cleaning litterboxes daily, and placing litterboxes away from feeding areas. minimize stress, especially crowding in catteries. do not introduce fcov-positive cats into a multi-cat household. wean kittens at weeks and remove from the queen's environment if she is seropositive. an intranasal vaccine is available for use in seronegative cats. however, efficacy has not yet been demonstrated against wild strains. classical signs see main reference on page for details (the anemic cat). onset of illnesses occurs over an extended period of time (months to years), although young kittens can become acutely ill. chronic, opportunistic infections occur that do not respond to appropriate antibiotic therapy and are primarily due to immunosuppression. fever may occur in any age cat but is primarily seen initially in the viremic stage or later in response to neoplastic, inflammatory or immunosuppressive effects. chronic fever occurs in later stages of disease. weight-loss/cachexia. non-regenerative anemia. thrombocytopenia. lymphoma is associated with felv-positive cats, especially thymic and multicentric forms. history and clinical signs may be suggestive. complete blood count showing anemia, thrombocytopenia, leukemias, increased mcv and leukopenia are supportive. bone marrow aspirate may show myeloproliferation and arrested erythroid differentiation. a positive felv antigen test (viral core antigen p ) on whole blood using an ifa (can also be done on bone marrow sample) or an elisa test (also on serum, plasma, saliva, tears). see page for interpretation. polymerase chain reaction is available from some laboratories. • pale mucous membranes. see main reference on page for details (the anemic cat). classical signs are pale mucous membranes and/or icterus primarily from extravascular hemolysis due to complement binding of infected erythrocytes. severe, regenerative hemolytic anemia may ensue. anorexia and depression are typical. fever occurs in % of cats in the acute phase, and may occur intermittently in chronic infections. history and clinical signs are suggestive, especially if an immunosuppressive disorder is present concurrently. diagnosis is via demonstration of the organism on the surface of erythrocytes. use a marginated blood sample for diagnosis, e.g., ear vein. multiple blood smears over a number of days may be required as most of the organisms are removed from circulation by the time clinical signs are apparent. infected cats may be coomb's positive. a polymerase chain reaction test is available in some laboratories for diagnosis. classical signs gastrointestinal signs, primarily abdominal discomfort and small bowel diarrhea, are due most likely to replication of the organism (tachyzoites) in enteroepithelial cells resulting in necrosis. clinical signs in the acute, fatal form of extraintestinal disease are caused primarily by tissue damage from the rapidly dividing tachyzoites. tachyzoites begin to disappear from tissues approximately weeks after infection. the organism may persist in tissues as tissue cysts containing bradyzoites. chronic disease may be a result of delayed hypersensitivity reactions and tissue reaction to antibody-antigen complex deposition. concomitant illness, such as felv, fiv and immunosuppression with glucocorticoids, has been reported in some cases. gastrointestinal disease. • mild, self-limiting small bowel diarrhea may occur in the definitive host (cats), but only after ingestion of tissue cysts, oocysts or sporulated oocysts. • young kittens are more likely to have gastrointestinal signs, although mild clinical disease has been reported in adult cats as well. all newborn kittens experimentally infected developed severe diarrhea - days later. • fatal extraintestinal disease is most likely to occur in transplacentally infected kittens. • kittens may be stillborn or exhibit signs that are severe and rapidly progressive and reflect involvement of the lungs, liver and cns tissues. these signs may also be observed in postnatally infected kittens and include: -a distended abdomen from an enlarged liver and/or ascites. -icterus from hepatitis or cholangiohepatitis. -dyspnea is present in most kittens and cats with signs of acute infection. -neurologic deficits; continuous vocalization; excessive sleeping. -fever, anorexia, depression often accompanies the tissue-specific signs. • cats may have a moderate fever, lethargy and depression that waxes and wanes. • hyperesthesia and stiff painful joints or shifting lameness may be evident, presumably due to an immune-mediated process. • unilateral or bilateral anterior or posterior uveitis may occur with possible sequelae of lens luxation, glaucoma or retinal detachment. • seizures and ataxia may be present if cns tissues are involved. • rarely, a toxoplasma granuloma (tissue cyst) forms in the gastrointestinal tract or pancreas causing chronic vomiting. clinical signs consistent with toxoplasmosis are suggestive, especially when other causes of the signs have been ruled out. igm titers > : and a four-fold increase in igg:igm titers within weeks correlate best with clinical toxoplasmosis. however, some cats do not develop detectable igm titers, and in other cats, positive igm titers can persist for months to years after infection. elevated ocular and csf titers relative to serum titers in cats with ocular or neurologic signs, respectively, are very suggestive. coefficient values > . are highly suspect and > . strongly suggest local production of t. gondii antibodies. response to therapy for toxoplasmosis is a useful indicator of infection. definitive diagnosis requires demonstration of the organism in inflamed tissues by histology, immunohistochemistry or polymerase chain reaction assay. rule out diseases associated with affected organs, e.g., fip for neurologic and ocular signs. clindamycin at - mg/kg orally q hours for weeks is usually effective. • cats should respond within several days of treatment. • if no response is evident after weeks of antibiotic therapy, reconsider the diagnosis. • the chronic form may recur even after successful treatment, as drugs tend to suppress replication rather than kill the parasite. other systemic drugs with potential efficacy include the trimethoprim sulfas combination, doxycycline, minocycline, azithromycin and clarythromycin. cats with ocular lesions should also be treated with corticosteroids, either topically (e.g. topical . % prednisolone acetate drops applied q - h) or systemically to control inflammation and its sequelae (glaucoma, lens luxation). gastrointestinal disease has a good prognosis, although it may lead to inflammatory bowel disease in rare cases. acute extraintestinal disease has a guarded to poor prognosis. chronic extraintestinal disease has a fair to good. • the placenta or milk with tachyzoites. • ingestion of meat infected with tissue bradyzoites, e.g., rodents. • ingestion of sporulated oocysts in food or water. t. gondii has a zoonotic potential. infection of humans can occur via: • ingestion of undercooked meat containing tissue bradyzoites (most common mode of transmission). • ingestion of sporulated oocysts from the environment. • transplacentally, if first-time exposure to the organism occurs during pregnancy. only cats host the sexual replication that results in oocysts in the feces. • oocysts are shed for - weeks. • most seropositive cats do not shed oocysts on repeat exposure. oocysts must sporulate to be infectious: • sporulation occurs - days after environmental exposure, thus handling individual cats rarely results in infection of humans. transplacental transmission occurs in cats and people after primary exposure. discourage cats from going outdoors and hunting behavior. do not feed cats undercooked meat. • cook meat at ˚c ( ˚f) for minutes. • use gloves when gardening or changing the litterbox, and wash hands well. • change litterboxes daily. use litterbox liners or clean with scalding water. • lethargy and anorexia. see main reference on page for details (the cat with depression, anorexia or dehydration). the classical signs are not as well-defined for cats as for dogs for the following reasons: • cats tend to have intermittent bouts of chronic pancreatitis. • diagnostic tests for pancreatitis are not as reliable in cats. • there is poor correlation of biochemical parameters with pancreatitis in the cat. lethargy and anorexia is variable depending on chronicity. vomiting only occurred in % of cases in one study. dehydration occurred in % of cases in the same study. abdominal pain is quite variable. fever is variably present, and generally mild. in severe acute pancreatitis it may progress to hypothermia, which is a poor prognostic sign. diagnosis is unreliable based on a biochemistry panel. lipase may be increased or normal in pancreatitis. • hyperbilirubinemia and elevated liver enzymes may be present. • hypocalcemia occurs in % (total serum calcium) or % (plasma ionized calcium concentration) of cats due to soponification of fat. cats with a plasma ionized calcium concentration < . mmol/l (< . mg/dl) have a grave prognosis ( % mortality) and aggressive medical treatment is indicated. pancreatic lipase immunoreactivity is probably a more sensitive diagnostic tool for confirming pancreatitis in cats than measurement of plasma lipase concentration or trypsin like immunoreactivity. a feline-specific assay must be used. abdominal ultrasound to visualize an enlarged pancreas or heterogeneous echogenicity in the area of the pancreas is considered by many to be most sensitive. demonstration of higher lipase levels in abdominal fluid compared to those of the serum is suggestive. diagnostic peritoneal lavage may be necessary to obtain a fluid sample. clinical signs may be acute, chronic or intermittent. typically, there is anorexia and depression together with icterus or increased bilirubin and liver enzymes on a biochemistry panel. vomiting and dehydration may be present. fever, especially in the suppurative form occurs in approximately % of the cases. chronic cholangiohepatitis may lead to end-stage liver disease and the cat may present with ascites and hepatic encephalopathy. multiple causes include bacterial, protozoal (t. gondii) and immune-mediated disease. complete blood count may show neutrophilia with a left shift, and mild non-regenerative anemia. biochemistry panel shows hyperbilirubinemia, elevated liver enzyme activities (alp, alt, ggt), +/− elevated serum bile acids. • signs of late-stage liver disease are occasionally present, such as decreased bun, glucose and albumin concentrations. abdominal ultrasound should be performed to evaluate the gall bladder and bile duct for cholelithiasis, bile sludging and cholecystitis. liver aspirates/biopsy allows for differentiation of suppurative from non-suppurative forms of cholangiohepatitis. clinical signs are primarily due to immunosuppression, i.e., chronic recurring infections that do not respond to appropriate therapy. gingivitis, stomatitis and peridontitis are more common findings in fiv infections than in felv, although one study suggests that these signs may be to an effect of age, rather than a consequence of fiv infection. fever is chronic and is related to production of tumor necrosis factor and/or il- in infected cats. weight loss/cachexia are common in the late stages of fiv, as in human hiv infections. diarrhea resembles a panleukopenia-type syndrome that may be due to actual enterocyte infection by the virus or secondary to inflammation. cats are often thin and scruffy with an unkempt haircoat, and may have miliary dermatitis. diagnosis may be suspected based on history and clinical signs, but requires antibody or antigen tests for confirmation. virus isolation and polymerase chain reaction for virus detection is available at some research facilities. cats infected with fiv can be co-infected with felv. chronic nasal discharge can be unilateral or bilateral and is generally serosanguineous. sneezing and stertorous breathing is often present. facial deformity may occur due to invasion of the surrounding bone. chronic low-grade fever may be present. depression, anorexia and weight-loss are signs of disseminated disease. neurologic signs occur via hematogenous spread or invasion into the cns through the cribiform plate but are uncommon. signs include seizures, blindness, depression and ataxia. the skin form typically produces nodules which often ulcerate. diagnosis is by demonstration of narrow-based budding yeast with a very thick capsule from affected tissue or by culture of affected tissue or csf. demonstration of cryptococcus antigen in serum, urine or csf is also diagnostic. occurs in cats of all ages, with and without outdoor access. progressive clinical signs occur over a period of - weeks. according to one study, non-supportive meningoencephalitis may be the most common cause of seizures in cats. systemic signs, which are not present in all cats, include fever, anorexia, lethargy, vomiting, diarrhea and lymphadenopathy. the condition, however, does not appear to be contagious to other cats. csf tap can be very useful to rule out other causes of cns signs, specifically toxoplasmosis and fip; csf analysis reveals a normal or mild protein elevation (typically < g/l) and/or an increased white cell count (< cells/μl). complete blood count findings are non-specific and may include leukopenia or leuko-cytosis, eosinophilia and anemia. • uniphasic or biphasic fever. • depression. • lethargy. • mild generalized lymphadenopathy. • +/-signs of cardiac failure. • viral, e.g., fip has been shown to cause cardiac infection. • trypanosoma cruzi, which causes chagas' disease in humans. • streptococcus and borrelia (lyme's disease) in certain geographic areas. no single agent has been identified, and the disease may be multifactorial. fever is biphasic in % of the cats; if biphasic: • first fever occurs approximately days after exposure, lasts - days and peaks at . - . ˚c ( . - . ˚f). • second fever occurs - weeks after the first fever (at - weeks post-exposure), lasts days and peaks at . - . ˚c ( . - . ˚f). appetite is mildly decreased in some cats, but most continue to eat and drink. some animals exhibit mild generalized lymphadenopathy. irritable disposition and hyperesthesia may occur, and are most likely due to fever and malaise. in a few case reports, cats have died from peracute cardiac failure, but this outcome is not common. myocarditis/diaphragmitis is a diagnosis of exclusion. biochemistry and complete blood counts are unremarkable, except for a mild to moderate increase in ck in less than % of experimentally infected cats. definitive diagnosis can only be made at necropsy. histopathology shows a neutrophilic infiltrate with a foci of myonecrosis in myocardium and diaphragm. any other causes of fever should be ruled out including infectious, inflammatory, immune-mediated, drugs, neoplasia and metabolic. other causes of cardiac failure that should be ruled out include congenital deformities, hypertrophic cardiomyopathy, restrictive cardiomyopathy and dilatative cardiomyopathy. supportive therapy is indicated if dehydration or cardiac disease are present. broad-spectrum antibiotics are indicated if complete blood count supports an infectious cause. fever and depression resolve spontaneously in the majority of cats. prognosis is poor if peracute cardiac failure is present with systemic signs of fever and depression. although an infectious agent is suspected, no single etiologic agent has been identified, making recommendations for prevention difficult. • cardiovascular or respiratory compromise. • external signs of injury. cardiovascular compromise may result in tachycardia, hypovolemia or hypotension. respiratory compromise may produce dyspnea/ tachypnea due to pneumothorax, hemothorax or pyothorax. internal injuries may result in abdominal pain from organ rupture, bone/joint pain or focal swelling. diagnosis is based on clinical signs and history. radiographs of the chest, abdomen and/or limbs may be required to characterize the injury. complete blood count and biochemistry panel is indicated to rule out specific organ injury and primary infection. • alert, febrile cat being treated with antibiotics or antifungal agents. history of treatment with antibiotics or antifungal agents. fever does not correspond to clinical appearance of animal. cats are bright, alert and responsive, despite a temperature in the range of . - ˚c ( - ˚f). onset of fever is idiosyncratic and variable, but the fever is generally present for the duration of the drug treatment. tetracycline is the most common antibiotic cause of drug-induced fever in cats. amphotericin b can cause fever by disrupting cell membranes and releasing pyrogens into circulation. be aware that other drugs (griseofulvin, chloramphenicol and chemotherapeutic drugs) can cause bone marrow suppression leading to a cat with fever, neutropenia and secondary bacterial infection. these cats are obviously sick, whereas the drug-induced fever animals are bright and alert in comparison. • history is of treatment with fever-inducing drugs, especially tetracycline and amphotericin b. • clinical signs are inappropriate, that is, the cat appears bright and alert although febrile. temperature normalizes after drug is discontinued. classical signs • sneezing. • conjunctivitis and ocular discharge. see main reference on page for details (the cat with acute sneezing or nasal discharge). marked conjunctivitis is the predominant sign, which often starts unilaterally, but usually progresses to both eyes. classic triad of upper respiratory infection signs including oculonasal discharge and sneezing. serous ocular discharge accompanied by blepharospasm, chemosis and conjunctival hyperemia are initial signs. discharge becomes mucopurulent over the course of the disease. mild to moderate fever can be seen in the acute phase. pneumonia is rarely associated with this infection. history and clinical signs are highly suggestive. cytology of conjunctival scrapings reveal dark blue inclusion bodies (giemsa stain). immunofluorescent antibody staining or polymerase chain reaction assay to demonstrate the organism in conjunctival scrapings is available from some laboratories. • acute onset of depression. • acute onset of vomiting. rapid onset of depression, anorexia, and vomiting especially in peracute and acute disease. fetid diarrhea (may be hemorrhagic) typically follows - days after initial onset of signs. severe dehydration and electrolyte abnormalities. initial fever followed by hypothermia as the disease progresses. high mortality rate when signs are severe. the disease should be suspected in cats less than one year of age with no history of vaccination and a rapid clinical course. panleukopenia evident on hematology. parvoviral antigen can be detected in feces using the canine parvoviral antigen tests or electron microscopy. histopathologic changes include denuded intestinal crypts and blunted villi (often a post-mortem diagnosis). classical signs francisella tularensis is a gram-negative coccobacillus. clinical signs are associated with gram-negative endotoxins and bacteremia. there are two main strains of the organism, both of which have been isolated from cats. • associated with tick-rabbit cycle. • found only in north america. • highly virulent for laboratory rabbits. • associated with more severe disease in humans. • associated with a more complex cycle involving rodents, ticks, mosquitoes, mud and water. • found throughout the northern hemisphere. • avirulent for laboratory rabbits. history of contact with rabbits, especially if the cat is a hunter. any age of cat can be infected, but younger cats are more susceptible to developing septicemia. the spectrum of illness varies from severely affected to asymptomatic. fever is generally > ˚c ( ˚f). marked depression, anorexia and lethargy, with or without vomiting are typical. on physical examination, peripheral lymphadenopathy, icterus and palpable splenomegaly and hepatomegaly are reported. oral, lingual or pharyngeal ulcers may be present. clinical signs together with a history of exposure to wild rabbits is highly suggestive. hematologic and serum biochemical abnormalities may include panleukopenia, with severe toxic changes in neutrophils, high band neutrophil count, thrombocyto-penia and hyperbilirubinemia. definitive diagnosis is via identification of the bacterial agent by ifa or bacterial culture, but should only be performed in a qualified laboratory. • samples can be obtained from affected lymph nodes, bone marrow, urine or blood. serum antibody titers > : or a four-fold increase in serum antibodies in samples collected during acute and convalescent phases ( - days) are considered diagnostic. fip, fiv, panleukopenia. toxoplasmosis. multicentric lymphoma. antimicrobial efficacy studies have not been done in the cat, therefore therapy is derived from case reports and/or human therapy regimens. enrofloxacin ( mg/kg q hours iv or po). tetracycline and chloramphenicol may be effective, but because they are bacteriostatic for f. tularensis, relapses can occur. in humans, the drugs of choice are streptomycin and gentamycin. prognosis is poor to fair as mortality rate varies across case reports. f. tularensis has a serious zoonotic potential if there is contact with infected animal tissue. bites from infected ticks, deer flies or mosquitoes are the most common method of transmission. infection can also occur via ingestion of infected meat. • this is the most common method of transmission to humans in cat-associated cases. • the infected cat may have no obvious signs of illness, but have a history of hunting wild animals, especially rabbits. inhalation of aerosolized organisms may also transmit the disease. care should be taken by veterinary and laboratory personnel handling suspected animals or samples being prepared for ifa or culture. discourage hunting behavior in cats. ectoparasite control, especially tick control. onset of illness occurs - hours after exposure to the organism. transmission to cats is either via ingestion of infected rodents or a fleabite from infected fleas. rapid multiplication of organism causes tissue damage and necrosis. the host immune response contributes to pathology. three forms of the plague exist: bubonic (local infection), bacteremic/septicemic and pneumonic. bacteremia occurs in many cases, resulting in the septicemic or pneumonic form of plague. endemic regions of the world include the western usa, south america, africa, asia, eastern europe. history of hunting rodents, especially in known endemic areas. current flea infestation is evident. acute onset of fever, anorexia, depression over a period of - days. the clinical course may last - days. submandibular or cervical swelling associated with lymph nodes (can be unilateral or bilateral). the inflamed, swollen lymph node is referred to as a bubo. subcutaneous abscessation may occur and appear similar to a cat bite abscess. in the pneumonic form (~ % of cases), upper and lower respiratory signs may be present, including sneezing, nasal discharge, coughing, dyspnea/tachypnea. initially, microscopic examination of a lymph node aspirate, especially a markedly swollen lymph node (bubo) should reveal a homogeneous population of bipolar-staining coccobacilli. • blood should be examined in cats with the bacteremic/septicemic form. fluorescent antibody testing of sample provides a definitive diagnosis. culture of organism should be performed by a qualified laboratory only. a four-fold rise in antibody titers (taken - days apart) is suggestive of plague. these results must be interpreted carefully, as high titers can persist for up to one year after infection. chest radiographs may reveal patchy, nodular lesions if the pneumonic form is present. be aware that the risk of exposing other staff members to the disease should be weighed against the benefit of the diagnostic test. reactive lymph nodes from a percutaneous abscess or tooth-root abscess. • aspirates of cat bite abscesses contain a mixed bacterial population compared to y. pestis, which is homogeneous. neoplasia, although it is less common in the us for cats with lymphoma to have peripheral lymphadenopathy. respiratory signs may be due to other upper respiratory infections (calicivirus, herpesvirus, chlamydophila) or lower respiratory disease (parenchymal lung disease, pleural disease). other diseases which cause high fever (tularemia, toxoplasmosis, fip, etc.). absolute caution must be practiced in all suspect plague cases. cautionary measures include gloves, mask, isolation of animal and limited exposure to other staff members. doxycycline/tetracycline: ( ) doxycycline at mg/kg q hours po for - days or ( ) tetracycline mg/kg q hours po. • begin treatment immediately after samples for diagnosis have been collected. • doxycycline is preferred as tetracycline has been associated with relapse. consider aminoglycosides or enrofloxacin ( mg/kg im q hours) for the first days to avoid placing hands into the cat's mouth (see transmission section below). prognosis for bubonic plague is fair to good. prognosis for the pneumonic form is guarded to fair. prognosis for septicemic form is guarded. persistent fever > ˚c ( ˚f) despite treatment is associated with a poor prognosis. y. pestis has a serious zoonotic potential, and great care should be taken in suspect cats to prevent transmission to humans and other cats. • infected cats are no longer a zoonotic risk after days of antibiotic therapy. infection can also occur via inhalation of aerosolized organism, either from aspirates of infected tissue or nasal discharge/sneezing of cats with pneumonic form. discourage hunting behavior especially during the peak flea season (april to october). provide effective flea control to prevent flea bites. • anorexia and weight loss. see main reference on page for details (the anemic cat). this disease is uncommonly reported in cats and is difficult to diagnose because of its vague and variable clinical signs. age range of cats with documented disease was - years of age, with no breed or sex predilection reported. infection has a variable effect on appetite, from mild inappetence to anorexia and mild to moderate weight loss. chronic intermittent fever in the moderate range is common. lymphadenopathy was reported in three of cats. hyperesthesia, joint pain or irritable disposition is common. complete blood counts may show a non-regenerative anemia with a leukopenia or a leukocytosis; thrombocytopenia is present in about % of the cats. biochemistry abnormalities are uncommon, except for hyperglobulinemia in about % of documented cases. a complete blood count and biochemistry panel consistent with chronic ehrlichia spp. infection is suggestive. diagnosis is by demonstrating e. canis and/or anaplasma phagocytophilum serum antibody titers or a positive ifa test. demonstration of morulae in mononuclear cells, neutrophils or eosinophils (rare) is diagnostic. pcr assays can be positive. • anorexia, lethargy, weight loss. • ± fever. • signs depend on tumor type and organ system involved. anorexia, lethargy and weight loss. poorly groomed coat. some cats have a fever associated with neoplasia, which is generally a secondary neoplastic syndrome. tumors which destroy the bone marrow and result in neutro-penia are classically associated with fever. fever may occur with other tumors via other mechanisms, including antibody stimulation from tumor antigens, and tissue necrosis which activates leukocytes to release pyrogenic factors. signs are specific to the organ system involved. lymphoma (mediastinal, gi, renal) , mammary adenocarcinoma, squamous cell carcinoma (nasal, oral) and mast cell tumor are the most common tumors in cats. hematology, biochemistry panel, radiology, ultrasound and/or bone marrow aspirates may be necessary to provide evidence that a tumor is present, especially if it involves the hematopoietic system (leukemia) or is located internally (splenic mast cell tumor). identification of the tumor type is via fine-needle aspirates and/or biopsies. organ dysfunction due to infectious or degenerative disease process. felv/fiv or other immunosuppressive illness. benign masses (granulomas, abscesses, reactive lymph nodes, benign tumors). treatment involves surgical excision of identifiable masses +/− regional lymph nodes, especially for mammary adenocarcinoma, nasal squamous cell carcinoma, splenic mast cell tumor, etc. chemotherapy may be effective and needs to be based on tumor type, e.g., cop (cyclophosphamide, vincristine, prednisolone) protocol in lymphoma cases. radiation therapy is used for local disease only, and response to radiation therapy is tumor dependent (e.g., squamous cell carcinoma). • radiation therapy is most effective after surgical debulking of the primary mass. the effectiveness of radiation therapy may be enhanced with concurrent chemotherapy. classical signs acute onset of fever and malaise are initial clinical signs. vomiting, diarrhea and abdominal pain may occur, however, approximately % do not have gastrointestinal signs. dehydration. shock may occur if septicemia/bacteremia develops. mortality rate approaches % and may be higher if the cat is concurrently immunosuppressed. typically, the cat is an outdoor cat with a history of hunting behavior, especially of birds. complete blood count and biochemistry panel supports infectious diarrhea or septicemia, e.g., neutropenia with a left shift, bacterial rods in blood leukocytes if overwhelming sepsis present, hypoglycemia, hypoproteinemia, pre-renal azotemia. blood cultures provide the best definitive diagnosis if positive. three separate samples over a - hour period should be taken during febrile episodes using aseptic techniques. fecal cultures may isolate salmonella organisms, but because many animals are subclinical carriers, positive culture does not prove that the organism is the cause of the clinical signs. • skin lesions. • respiratory signs. • ocular lesions. • fever, anorexia, depression. the geographical distribution includes south-west usa, central america and south america in areas that have sandy soil with low rainfall and high temperatures. soil is the reservoir for infection, and the highest frequency of cases occur when the soil is dry and dusty, and organisms are disseminated in the wind. most humans and animals in endemic areas become infected, but the majority of infections are subclinical or cause only mild, transient clinical signs. cats are more resistant to infection and signs are less common than in dogs. infection is contracted via inhalation, and only a few organisms are required to produce signs, which occur after - weeks. initial infection is confined to the respiratory tract, but dissemination may occur resulting in chronic disease over months or years with signs referable to bones, eyes, central nervous system and abdominal organs. localized infection following a penetrating skin wound appears to be rare. cats appear to be resistant to clinical disease. skin lesions are the most frequent types of infection in cats and were reported in % of cats in one study. • lesions begin as small bumps and progress to abscesses, ulcers or draining tracts. • in cats, underlying bone involvement is uncommon. systemic signs such as fever, anorexia and depression are commonly reported ( % of cats) and can be seen with skin lesions. respiratory signs such as coughing and wheezing are less common in cats and occur in approximately % of cases. musculoskeletal signs such as lameness, with or without painful bone swelling, were reported in % of cats. ocular lesions are seen infrequently and include chorioretinitis and anterior uveitis. ocular or cns signs were reported in % of cats. most cats have clinical signs for less than weeks prior to diagnosis. hyperproteinemia is present in approximately % of cats. definitive diagnosis is by identification of the organism via biopsy of lesions. antibody detection is available using latex agglutination (igm), agid (igm) or elisa (igm or igg). tube precipitin (tp) for igm and complement fixation (cp) for igg were previously thought to be less reliable in cats, but have been subsequently demonstrated to detect feline infections. itraconazole ( mg/kg po if possible, q h or mg/kg q h) is the treatment of choice. treatment is required for - months and must be continued for at least months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at / the dose for - days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective ( . mg/kg in ml dextrose % iv over minutes q h or given subcutaneously) -see page , for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of mg/kg is given or until bun > . mmol/l ( mg/dl). amphotericin has the disadvantage of requiring frequent parenteral or subcutaneous administration and causes significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at - mg/kg iv days per week for a total of - treatments (cumulative dose of - mg). dilute to a concentration of mg/ml in dextrose % and infuse over - hours. if the titer has decreased four-fold and there is a similar improvement in physical and radiographic signs, treatment can be stopped after - months. antibodies may persist for long periods and obtaining a zero titer is not a useful treatment goal. classical signs • fever. • respiratory signs. • ocular signs. • lymphadenopathy. the geographical distribution includes north america, central america and africa. soil is believed to be the reservoir for infection, and living near a lake or river increases the risk of infection in dogs. signs are more common in dogs than in the cats. disseminated disease is primarily contracted via inhalation. respiratory signs include coughing, dyspnea and harsh lung sounds. ocular disease, such as uveitis, glaucoma and retinal detachment, is a frequent finding. fever, anorexia, depression, weight loss and lymphadenopathy are systemic signs associated with disseminated disease. draining skin lesions may occur and are usually a manifestation of systemic disease rather than local disease. neurological signs are associated with cns involvement of the brain or spine and include circling, disorientation, anisocoria, paresis, decreased conscious proprioception, or upper motor neuron signs, hyperesthesia and extensor rigidity. definitive diagnosis is by demonstration of an extracellular, broad-based budding yeast in aspirates or biopsies from lymph nodes, draining tracts, bone lesions or vitreous humor. an antibody detection test is available, but may be negative. itraconazole ( mg/kg po if possible, q h or mg/kg q h) is the treatment of choice. treatment is required for - months and must be continued for at least months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at / the dose for - days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective ( . mg/kg in ml dextrose % iv over minutes q h or given subcutaneously) -see page , for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of mg/kg is given or until bun > . mmol/l ( mg/dl). amphotericin has the disadvantages of requiring frequent parenteral or subcutaneous administration and causing significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at - mg/kg iv days per week for a total of - treatments (cumulative dose of - mg). dilute to a concentration of mg/ml in dextrose % and infuse over - hours. classical signs see main reference on page for details (the anemic cat). primarily found in the south-central and southeast united states. the north american bobcat is the natural host. there is usually a history of exposure to ticks in the previous - days (incubation period is - days). the clinical course of disease is approximately week and often ends in death. clinical signs are the result of an overwhelming hemolytic crisis. rapid onset of fever, dyspnea, anorexia, pale mucous membranes, icterus and dark-colored urine are typical. collapse and death occur - days after the fever peak. hypothermia occurs in the terminal stages. there appear to be non-pathogenic strains as well. a complete blood count reveals regenerative anemia, hemoglobinemia and neutrophilia or neutropenia. the biochemistry panel commonly has hyperbilirubinemia. urinalysis may show evidence of hemoglobin and bilirubin. demonstration of the organism in erythrocytes (merozoite stage) is possible only relatively late in the disease, approximately - days before death. parasitemic cats usually have only - % of rbcs affected, and up to % of cats have parasitemias that are very low or undetectable. demonstration of the organism in macrophages from bone marrow, spleen, liver or lymph node aspirates may be possible even when organisms are not evident in blood. serum antibody levels and direct fa test for detection of tissue phase are available through some labs. weight loss in spite of normal to increased appetite. polyuria/polydipsia. behavioral changes which often include hyperactivity and aggression. unkempt, rough hair coat and sometimes overgrown nails. tachycardia accompanied by a "gallop" rhythm and/or a systolic murmur. mild fever which may be intermittent in nature and reflect the increased metabolic rate in this disease. these cats are easily stressed and may present dyspneic and tachycardic with a mildly elevated temperature, usually not greater than . ˚c ( . ˚f). enlarged thyroid glands are often evident on palpation of the neck. diagnosis is based on clinical signs and history and confirmed by demonstrating increased thyroid hormone concentrations (total t , free t ). thyroid glands can be palpated in approximately % of cats with hyperthyroidism, and are unilaterally or bilaterally enlarged. • enlarged thyroid glands may not be palpable if the abnormal thyroid tissue is within the thoracic inlet. complete blood count and a biochemistry panel are required to rule out diseases such as diabetes mellitus, renal disease, etc. a trh stimulation test may be necessary when clinical signs are highly suggestive and total and free t are in the upper region of the reference range for normal cats. thyroid radionuclide uptake and imaging with pertechnetate ( m tc) is also available at some institutions. response to therapy with anti-rickettsial drugs (tetracycline or doxycycline) is highly suggestive. • subclinical or mild fever and occasional ocular signs. bartonella henselae is an intracellular bacterium within erythrocytes. bacteremia is present in many healthy cats in the population, and cats are reservoirs for infection. b. henselae is an important pathogen because of its zoonotic potential in immunocompromised humans. • humans may develop fever, malaise, lymphadenopathy and skin eruptions following cat scratches or bites. • b. henselae causes bacillary angiomatosis, bacillary peliosis and encephalitis in human aids patients. naturally infected cats usually only develop subclinical infection. mild, self-limiting fever lasting - hours has been documented in some experimentally infected cats. anterior uveitis and fever were documented in naturally exposed cats. lymphadenopathy. atypical seizures occur in some cats. antibody titers are prevalent in healthy cats, but there is a poor correlation with blood culture and pcr assay results. intermittent bacteremia may occur for longer than one year following infection, with - % of healthy cats bacteremic for up to months. the organism is present within erythrocytes, therefore, hemolyzing red blood cells increases the sensitivity of the culture. other causes of mild transient fever need to be considered, such as mild cellulitis following a cat fight. other infectious causes of anterior uveitis need to be ruled out, such as toxoplasmosis, fungal diseases, felv, fiv, cuterebra or dirofilaria. antimicrobial efficacy has not been clearly demonstrated. clinical signs of disease have resolved when the cats are administered doxycycline at - mg po q h for days. azithromycin is used in humans and is a safe alternative in cats when administered at mg/kg po q h for days fluoroquinolones also may be effective. while clinical signs resolve, bacteremia is usually only temporarily suppressed. b. henselae has very low pathogenicity in cats. once cleared of infection, cats are resistant to re-infection by innoculation, but are still susceptible if transmitted via blood transfusion. transmission is via arthropod vectors. in endemic areas, cats infested with fleas and/or ear mites are more likely to be seropositive. the organism survives in flea feces for at least days. because of the frequency of bacteremia in healthy cats, blood transfusions are a likely route of infection. primary immune-mediated disease is extremely rare in cats. stimulation from primary infectious disease antigens is the most common cause of immune-mediated disease in cats, and is most often associated with hemobartonella (mycoplasma) and calicivirus. systemic lupus erythematosus is rare in cats. a multitude of signs may occur including fever, weight loss and cutaneous lesions. immune-mediated hemolytic anemia is most commonly associated with hemobartonellosis. signs include anemia, icterus, fever and anorexia. cats with immunosuppressive disorders such as felv may be more susceptible. immune-mediated thrombocytopenia is rarely reported in cats. felv-positive cats, however, may have thrombocytopenia that is thought to be the result of an immune-mediated response. immune-mediated polyarthritis is uncommon in cats, but has been documented in kittens and adult cats with post-calicivirus vaccination. -the pyrexic cat a bacteriologic investigation of subcutaneous abscesses in cats clinical, clinicopathologic, and pathologic features of plague in cats: cases ( - ) bacterial diseases; fungal diseases; and protozoal diseases feline toxoplasmosis: interpretation of diagnostic test results bartonella spp. antibodies and dna in aqueous humor of cats feline toxoplasmosis and the importance of the toxoplasma gondii oocyst feline infectious peritonitis. part i. etiology and diagnosis feline infectious peritonitis. part ii. treatment and prevention consensus statement of ehrlichial disease of small animals from the infectious disease study group of the acvim feline infectious myocarditis/diaphragmitis diagnostic approach and medical treatment of seizure disorders an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis tularemia in two cats key: cord- -ebj v o authors: marini, robert p.; otto, glen; erdman, susan; palley, lori; fox, james g. title: biology and diseases of ferrets date: - - journal: laboratory animal medicine doi: . /b - - / - sha: doc_id: cord_uid: ebj v o nan ferrets (mustela putorius furo) belong to the ancient family mustelidae, which is believed to date back to the eocene period, some million years ago. the taxonomic groups in the family mustelidae, as recognized by corbet and hill ( ) , include species from north, central, and south america, eurasia, and africa. no other carnivore shows such diversity of adaptation, being found in a wide variety of ecosystems ranging from earlier references to ferrets are probably the basis of the belief that ferrets originated in north africa (thomson, ) . evidently they were bred specifically for rabbiting (rabbit hunting) and were muzzled before being sent into rabbit burrows. this practice was later introduced into europe, asia, and the british isles, where the sport is still practiced today. although the ferret has been historically used for hunting, more recently it has been increasingly used in biomedical research and is popular in north america as a pet. it is most likely a domesticated version of the wild european ferret or polecat (m. putorius or m. furo) (thomson, ) . alternatively, it may be related to the steppe polecat (m. eversmanni), which it closely resembles in skull morphology (walton, ) . the domesticated ferret, although introduced to north america by the early english settlers some years ago, has not established feral colonies on this continent. the ferret was not recognized as having potential as an animal model for biomedical research until the s. early studies utilized the ferret in classic experiments with influenza virus pathogenesis (pyle, ) . its use was cited infrequently; an article published in , detailing the use of ferrets in research, cited only publications (pyle, ) . literature reviews undertaken in , , , and , however, revealed an increasing appreciation for the ferret's usefulness and versatility in the study of human physiologic, anatomic, and disease mechanisms (hahn and wester, ; marshall and marshall, ; shump et al., ; frederick and babish, ) . in , a bibliography containing "selected" literature citations on the ferret and its use in biomedical research was published (clingerman et al., ) . the document was designed to serve as a reference tool for individuals involved in the care or use of ferrets in the laboratory setting. although not comprehensive, the document provides extensive coverage of ferret biology, diseases, and use as an animal model. the domesticated ferret has been and continues to be used extensively in studies involving virology, reproductive physiology, anatomy, and endocrinology, as well as other areas of biomedical research (morgan and travers, ) . the ferret is also being used to replace the cat in some types of neuroendocrinology, neuroanatomy, and cardiology experiments. the ferret's increasing popularity in research and as a pet is mainly a result of large-scale commercial production. for example, commercial farms have been raising ferrets for almost years. biomedical researchers in the united states can request animals of a specific sex, weight, and age for individual experiments. investigators in other countries may acquire fer-rets from fur operations or may make arrangements with commercial vendors in the united states. even though the ferret is nonstandardized with regard to exact genotype and pedigree, its routine availability in a clinically healthy state has aided immeasurably its acceptance as a research animal. readily available commercial stocks, based on coat color, are albino, sable (or fitch), siamese, silver mitt, and siamese-silver mitt (siamese with white chest and feet) (mclain et al., ) . the fitch or so-called wild coat color is the most common, recognized by yellow-buff fur with patches of black or dark brown, particularly on the tail and limbs (andrews and illman, ) . the production of ferrets by large commercial operations has raised concern by some that inbreeding of these animals has made the ferret more susceptible to diseases, e.g., endocrinerelated disorders. this topic is covered in more detail in chapter . housing of ferrets in a research facility is similar to that of other small carnivores such as cats (fox, c) . ferrets tolerate low temperatures well and high temperatures poorly; the recommended temperature range for juvenile and adult animals is - ~ (hammond and chesterman, ) . ferrets less than weeks of age should be housed at > ~ c. kits under this age require a heat source if separated from the dam; older kits that are group-housed do not. elevated temperatures (> ~ cannot be tolerated by ferrets, because they have poorly developed sweat glands and are susceptible to heat prostration. signs of hyperthermia include panting, flaccidity, and vomiting. the preferred humidity is - %. for nonbreeding animals that will remain in the facility for a short time, a conventional dark-light cycle at : hr is adequate. lighting may be altered to control breeding cycles. breeding and lactating jills should be exposed to hr of light daily. ferrets that are maintained for breeding or for use beyond months should be exposed to "winter" lightw weeks per year of hr of dark dailywto maintain physiologic normalcy. it is also essential that researchers receiving time-pregnant jills preserve the photoperiod to which jills were exposed prior to shipment. failure to do so may cause inappetence, with subsequent negative energy balance and pregnancy toxemia. similar to other laboratory animal species, ferrets should be housed with - air changes per hour (usdhhs, ) . it is important to use nonrecirculated air because of the strong odor of ferrets and the susceptibility to respiratory tract infections. the ferret odor should not overlap into any rodent housing areas, because rodents have an instinctive fear of ferrets, and the ferret scent can disrupt rodent breeding and physiology (fox, c) . female ferrets can be housed singly or in groups, but estrous females that are cohoused may become pseudopregnant (beck et al., ) . males should be housed individually after weeks of age. molded plastic caging used to house rabbits works very well for ferrets. the solid bottom is perforated with holes and is readily sanitizable. an absorbable paper liner may be used in the pan beneath the cage to facilitate daily disposal of urine and feces. in a research setting, the plastic caging should be washed weekly to avoid excessive soiling. the spacing of grid walls should be . x . inches apart, or . inch if using wire mesh. ferrets like to lick and bite at their enclosures, so sharp edges and galvanized metal should be avoided. zinc toxicosis has been reported from licking galvanized bars from which metals had leached during steam sterilization (straube and walden, ) (table i) . ferrets can be trained to use a litter box because they repeatedly urinate or defecate in one corner of the cage. clay litters have been reported to cause chronic upper respiratory irritation parameter (jenkins and brown, ) . ferrets prefer sleeping in a soft isolated area, and in a research facility this can be accomplished by providing a washable "snooze tube" (fox, c) . the thorax of the ferret is narrow and elongated, and as a result the trachea is proportionally long. this makes the ferret an ideal species for studies of tracheal physiology. the tracheal size and laryngeal anatomy make endotracheal intubation somewhat challenging, and as a result the ferret has been advocated as a species suitable for use in pediatric intubation training (powell et al., ) . the lungs are relatively large, and the total lung capacity is nearly times that which would be predicted based on body size, as compared with other mammals. this characteristic, together with a higher degree of bronchiolar branching and more extensive bronchial submucosal glands (as compared with the dog), makes the ferret an attractive model for pulmonary research studies (vinegar et al., ) . although a previous report (willis and barrow, ) commented that the carotid arterial branching pattern in the ferret is unusual, it is actually typical for a carnivore. as is the case in the dog and the cat, the paired common carotid arteries arise from the brachiocephalic trunk (sometimes called the innominate artery) at the level of the thoracic inlet (andrews et al., b) . the ferret's gastrointestinal tract is specialized to fit its carnivorous nature. the simple monogastric stomach is similar to that of the dog. there is no cecum present, and the indistinct ileocecal transition makes it difficult to identify the junction of the small and large intestines during a gross examination. the overall length of the alimentary tract is very short relative to the body size, resulting in a gastrointestinal transit time as short as hr (bleavins and aulerich, ) . as in other mustelids, the paired anal scent glands of the ferret are well developed. although not as potent as those of the skunk, the secretions of the ferret are sufficiently odoriferous that many pet or research ferrets are descented. surgical techniques for this procedure have been described (creed and kainer, ; mullen, ) . ferrets, especially intact males and estrous jills, may possess a distinctive musky odor even after a successful descenting, because of normal sebaceous secretions. ferrets lack well-developed sweat glands for use in thermal regulation, and as a result they are predisposed to heat prostration when ambient temperatures reach ~ ( ~ f) (ryland et al., ) . extramedullary hematopoiesis is commonly found during histological examination of the spleen, and in some cases it may result in a grossly evident splenomegaly (erdman et al., ) . this must be differentiated from splenomegaly that can arise from a variety of pathologic conditions or from isoflurane administration (see section iii,e). experimental evidence suggests that ferrets have no naturally occurring antibodies against unmatched erythrocyte antigens, and that none develop even in the face of repeated transfusions . ferrets are seasonal breeders, and the resulting pronounced physiological variations in body weight, behavior, and gametogenesis are utilized in scientific studies of photoperiod responses and neuroendocrine control. prolonged estrus in unbred females can cause an aplastic anemia, an effect that can be reproduced with exogenous estrogen administration . the male has a radiographically evident os penis, and, contrary to some earlier reports, a prostate gland is present in males (evans and an, ) . newborn ferret kits weigh - gm at birth and will grow to gm by the time they are weaned at - weeks (shump and shump, ) . in sexually intact populations, males ( . - . kg) can be twice the size of females ( . - . kg). the adult weight of nonobese male and female ferrets that have been gonadectomized prior to weaning and raised in captivity will generally fall between . and . kg (brown, a) . adult animals (especially those that are sexually intact) may be subject to seasonal fluctuations in body fat percentage, which can cause body weight to fluctuate by - % (fox and bell, ) . the approximate life span for the ferret is - years, but on rare occasions they may live as long as years (table ii) . normal hematology and serum chemistry values have been reported for the ferret (thornton et al., ; lee et al., ; fox, e) . these values are not greatly dissimilar from those of other domestic carnivores. one distinctive hematological characteristic of the ferret is the presence of a relatively robust erythron, characterized by hematocrit, hemoglobin, and total erythrocyte and reticulocyte counts that are generally higher than those of the dog or cat. reported neutrophil-lymphocyte ratios range from . : to . : . representative hematology and chemistry ranges from one of our studies (fox et al., b) are shown in tables iii and iv, but for diagnostic purposes any laboratory that evaluates ferret samples should develop its own set of specific normal ranges. a low-grade proteinuria may be identified by urinalysis in normal, healthy ferrets (thornton et al., ) (table v) . ferret diets have been formulated both empirically and based upon the nutrient requirements of other mustelids (fox and mclain, ) . specific requirements for various life-cycle stages have not been determined experimentally. available commercial diets are certainly capable of supporting growth, reproduction, and maintenance in conventional settings. in the (fox et al., b) . bfour-to -month-old ferrets (loeb and quimby, ) . cnd, not done. absence of careful analysis, however, it is uncertain whether the proportion and quantity of ingredients in these diets is optimal. ferrets are strict carnivores with a high requirement for dietary fat and protein. their short digestive tract and rapid gastrointestinal transit time ( - hr) require protein to be readily digestible. there is general agreement that ferrets should not be given diets high in complex carbohydrates or fiber. diets that are high in fish products are also not recommended for ferrets (fox and mclain, ) . the use of any raw chicken, beef, or other meats is strongly discouraged because of the potential contamination by campylobacter, salmonella, listeria, mycobacterium, and streptococcus (fox, a) . daily maintenance energy consumption for ferrets is - kcal/kg body weight. calorie-percent protein ratios have been determined for mink (mustela vison) kits up to and after weeks of age (sinclair et al., ; allen et al., ) . a ratio of and a caloric density of kcal/ gm of feed, corresponding to % protein, provided optimum growth for male kits up to weeks. after weeks, ratios of and , corresponding to % and % protein, respectively, were recommended. diets containing - % fat and - % carbohydrate have been used successfully to maintain ferrets. one author recommends - % protein and - % fat for adult, nonbreeding animals and a minimum of % protein and % fat for reproductively active animals and those that have notreached sexualmaturity (brown, a) . the long-term impact of diets containing high levels of fat and protein are unknown. ferrets have been used to investigate the absorption, metabolism, and interaction of the dietary micronutrients [ -carotene and vitamin e. ferrets, like humans, convert [ -carotene to vitamin a in the gut and absorb ~-carotene intact (fox and mclain, ) . in intestinal perfusion experiments in ferrets, it was demonstrated that [ -carotene, retinol, and retinyl esters are absorbed intact into lymph and that cleavage products, including [ -apo- '-carotenal, [ -apo- '-carotenal, and retinoids, accumulate in the intestinal mucosa (wang et al., ) . the intestinal mucosa is capable of converting [ -carotene into retinoic acid and other polar metabolites, which are then transported via the portal vein to the liver (wang et al., ) . [ -carotene absorption is enhanced by co-perfusion with a-tocopherol, and the perfusion of the latter is unaltered by the presence of [ -carotene. the conversion of [ -carotene into retinol is also enhanced by the presence of a-tocopherol (wang et al., ) . these and other findings have established the ferret as an important model for the study of these antioxidants. adult ferrets drink - ml of water daily, depending on the dry-matter content of the feed (andrews and illman, ) . fresh water can be provided ad libitum in stainless steel bowls or water bottles with sipper tubes. ferrets are playful and will overturn bowls or water bottles that are not well secured. features of ferret reproduction may be found in table vi for males, corresponding temporally to increasing day length. ferrets born in the late spring or early summer and maintained under natural lighting will not assume an adult pattern of gonadal activity (i.e., puberty) until the following season (baum, ) . under artificial illumination, jills that are maintained at hr light- hr dark reach puberty at - months. stimulatory photoperiods may be used, however, in the laboratory or intensive production setting, as a method of breeding ferrets out of the natural season. however, the transfer from short to long photoperiods should not occur prior to days of age, because jills that are prematurely transferred will remain anestrous (hammond and chesterman, ) . management practices in one breeding facility are such that jills commence breeding at - months, average . litters a year, and are cycled out of reproduction after litters. in another strategy, ferrets are exposed to a : hr photoperiod at weeks of age, are bred at weeks during their first estrus, and whelp at v months. vulvar swelling is the hallmark of estrus in jills. the ease with which estrus is detected in the ferret, as well as the size of the ferret and ease of its maintenance in captivity have made the ferret a model for study of neuroendocrine events and their gonadal correlates. along with the hamster, the ferret has contributed extensively to an understanding of the photoperiodic influences on the hypothalamic-pituitary-gonadal axis (baum, ) . as in females of other species, estradiol concentrations are responsible for controlling the development of the female reproductive tract and secondary sexual characteristics, and the tonic inhibition of luteinizing hormone (lh) secretion by the anterior pituitary during both prepubertal life and anestrus. the sensitivity of the hypothalamic gonadostat to negative feedback inhibition by estradiol changes at the time of puberty, and under the influence of increasing light exposure, lh levels rise despite estradiol (ryan, ) . similarly, age differences in the sensitivity of negative feedback inhibition of the hypothalamic secretion of gonadotropin-releasing hormone (gnrh) by testosterone, or to estrogenic compounds derived from the aromatization of testosterone, appear to be essential in determining puberty and seasonality of reproduction in the male (baum, ) . estrus in jills is characterized by dramatic vulvar swelling from an anestrous diameter of - mm to an estrous diameter of - mm. changes in vaginal cytology have also been described for the ferret and other mustelid species, but these changes are seldom used to determine onset of estrus or to schedule breeding (williams et al., ) . after a -to -week proestrus, estrus occurs. estrus onset is not associated with elevated serum fsh in the ferret, as it is in the rodent. once estrus has occurred, it may terminate in coitus-induced ovulation and pregnancy, pseudopregnancy after infertile mating, pharmacologic termination (by injection of human chorionic gonadotropin (hcg) or gnrh), death due to estrogen-induced aplastic anemia, or spontaneous remission and anestrus due to reduced photoperiod. waves of follicular development occur in estrus, and - ova are ovulated approximately - hr after coitus. female ferrets are brought to the male approximately days after vulvar enlargement. females and males copulate many times and for prolonged periods of time; they are typically left together for days. both intromission and neck restraint by the male are apparently required for induction of ovulation (baum, ). an lh surge accompanies coitus in females, but the same is not true of males (carroll et al., ) . implantation occurs days after mating; both a functional corpus luteum and the anterior pituitary are required for implantation and maintenance of pregnancy. placentation is typical of carnivores and is zonary and endotheliochorial (morrow, ) . pregnancy may be detected by ultrasonographic demonstration of - discrete nonechogenic structures as early as day (peter et al., ) , by palpation as early as day , or by radiographic demonstra-tion of calcified fetal skeletons at approximately days of gestation. jills within weeks of parturition should be singly housed and provided with a secluded place in which to deliver their kits. when rabbit cages are used for housing, nest boxes may take the form of polypropylene rat cages or other plastic boxes (cat litter box or dishpan). nest boxes should have bedding provided for warmth and comfort. materials suitable for bedding include pieces of fabric (towels), ripped cageboard, shredded paper, or cotton batting. the nest box should be at least inches deep and should prevent the kits from wandering from the jill. entrance to the nest box should be smooth, to avoid injury to the teats and mammary gland. at our institution, jills are provided a stainless steel rectangular box with a smooth-surfaced plastic entrance ( fig. ) . a retractable steel roof panel and a guillotine side panel exposing a plexiglas sidewall allow access to the jill and permit observation with minimal disturbance. one major supplier of ferrets uses sunken tubs filled with bedding to promote a sense of security and isolation of the jill. most jills will leave the nest box to eat and drink. if the jill will not leave, however, low-sided food bowls should be placed within the nest box. parturition occurs rapidly in ferrets and may last as little as - hr. primiparous jills typically deliver on day of gestation whereas multiparous jills deliver on day . there are few signs of impending parturition, although abdominal enlargement and mammary development do occur in the last week or two. small litters (fewer than three) may result in inadequate stimulus for parturition. jills that pass their due date without delivery should be palpated for fetuses. kits remaining in utero beyond the rd day typically die; kits with congenital malformations such as cyclopia and exencephaly may also delay the initiation of labor. dystocia is common in ferrets because of positional abnormalities and fetal oversize and should be treated by cesarean section. jills tolerate cesareans well and will nurse kits delivered in this way. if small littel: size is responsible for delayed parturition, prostaglandins ( . - . mg lutalyse) may be used, followed by . ml oxytocin ( u) after hr (fox and bell, ) . failure to deliver within hr of administration of prostaglandin is an indication for cesarean section. jills should be provided heat, energy, hydration, and analgesia following cesarean. kits will attempt to nurse soon after parturition, but jills experiencing difficult labor may not allow them to nurse until all kits are delivered. jills that are not attentive to their kits should be palpated for the presence of additional, undelivered kits. oxytocin may be used to facilitate delivery of remaining kits. offering the jill regular chow mixed with warm water may promote maternal acceptance. kits should be kept warm pending acceptance by the jill. jills should be left undisturbed for the first several days postpartum to avoid their cannibalizing the litter. cross-fostering to other jills may be successfully accomplished, provided that the kits are warm and that the foster jill has kits of similar age. kits to be fostered should be allowed to mingle with the foster jill's own kits while their dam is absent so that rejection due to olfaction will not occur. kits are born in an altricial state, covered by lanugo hair and with their eyes closed. by days of age, albino ferrets retain their white hair whereas pigmented ferrets acquire a gray coat. they are completely dependent on the jill for the first weeks of life. defecation and urination are stimulated by jills through anogenital licking of the kits. kits are born weighing - gm, double their weight in days, and triple it in days to a weight of gm. the -week-old male kit should weigh at least gm. sexual dimorphism in size is apparent by week and persists into adulthood. developmental landmarks include ability to hear at days, opening of the eyes at days, eruption of deciduous teeth at days, eruption of permanent canines at - days, and displacement of deciduous canines by - days (fox and bell, ) . gender may be distinguished in neonatal ferrets, as in other species, by anogenital distance, with the distance being much shorter in females than in males. in males, the urogenital opening is seen just caudal to the umbilicus. the prominent midline raphe penis overlying the palpable os penis is also a distinctive feature in the male. ferrets are typically weaned at weeks of age. early weaning may be encouraged by making a slurry of the jill's chow available at - weeks; fat may be added to achieve a fat content of %. the fatty acid supplement linatone (lambert kay, cranberry, new jersey) is recommended by one author (brown, a) . the diet should contain approximately % fat and % protein. the slurry should be fed twice daily for a restricted time and then removed to avoid having kits walking through and defecating in the diet. unthrifty kits over days of age may be supplemented with canine or feline milk replacers administered per os by tygon-tipped pasteur pipette . weaned ferrets are best housed in groups until sexually mature. males over weeks old may begin to fight if exposed to greater than hr light per day. jills may return to estrus during the second or third week of lactation if they have fewer than kits or weeks after weaning if the litter is of normal size. jills should be rebred or administered hcg to terminate estrus, even if still lactating. a highquality, calorie-dense diet is required for lactation and to maintain pregnancy. if maintained on a stimulatory photoperiod and adequate nutrition, jills may have - liters of or more kits yearly until they are years old (fox and bell, ) . a nonstimulatory photoperiod should be used weeks per year to rest the ferret and preserve maximum fertility; a maintenance diet can be given at this time. jills return to estrus approximately weeks after reinstitution of the longer photoperiod. artificial insemination is not commonly performed in ferrets but has been studied in the context of providing strategies for species perpetuation of the endangered black-footed ferret (wildt et al., ) . synchronization of estrus as practiced in rodent production is not used as a tool of reproductive management in the ferret. synchronization ofjills may be approximated, however, by manipulation of photoperiod. with natural illumination in outdoor housing, jills all come into estrus within a -to -week period (baum, ) . in the laboratory setting, when jills are maintained in a nonstimulatory photoperiod ( hr light- hr dark) for - weeks, followed by reversal of the cycle ( hr light- hr dark), estrus will follow in weeks (immature jills) or weeks (mature jills) after the change (carroll et al., ) . this correlates with follicular development and increased plasma estradiol. the occurrence of infectious disease affects animal health and well-being and may complicate research efforts. a program combining good animal husbandry, optimal nutrition, health monitoring practices, and clinical care is essential to maintaining a healthy ferret colony. etiology. the etiologic agent is clostridium perfringens type a (clostridium welchii). epizootiology and transmission. clostridium perfringens is ubiquitous and is present in the intestinal contents of humans and animals. clostridium perfringens type a has been associated with the occurrence of acute abdominal distension, dyspnea, and cyanosis in weanling ferrets (field and laboratory service veterinary staff, ) and an outbreak of gastroenteritis in weanling black-footed ferrets (schulman et al., ) . the exact cause of these conditions is uncertain, but predisposing factors such as overeating, sudden changes in diet, the prolifer-ation of c. perfringens type a, and the production of overwhelming amounts of toxins are suspected (field and laboratory service veterinary staff, ; schulman et al., ) . the alpha toxin is the principal lethal toxin. it is hemolytic and necrotizing and possesses the ability to split lecithin or lecithinprotein complexes, leading to destruction of cell membranes and subsequent necrosis. reported cases have involved weanling animals exclusively. clinical signs. ferrets may present with acute abdominal distension, dyspnea, and cyanosis or may be found dead and bloated (field and laboratory service veterinary staff, ; schulman et al., ) . diagnosis. isolation of c. perfringens type a from gastric and small-intestinal contents is required. toxin identification may be performed by the use of a mouse protection assay (smith, ) . necropsy findings. gross findings include markedly distended stomachs and intestines containing a large amount of gas and a moderate amount of brown, semiliquid ingesta, and subcutaneous emphysema with minimal or no putrefaction (field and laboratory service veterinary staff, ; schulman et al, ) . histologic findings observed in weanling black-footed ferret cases included the observation of abundant gram-positive bacilli in smears of gastric and intestinal contents. other findings included varying degrees of gastrointestinal mucosal necrosis, numerous gram-positive bacilli lining the denuded mucosal surface and extending into the gastric glands and intestinal crypts; lymphoid necrosis of lymph nodes, spleen, and thymus; mild to moderate dilatation of central hepatic sinusoids with mild, acute, centrilobular hepatocellular dissociation and multifocal aggregates of small numbers of necrotic neutrophils within portal areas (schulman et al., ) . and feeding practices is the primary means of control. in the reported cases of c. perfringens type a-associated gastroenteritis in black-footed ferret weanlings, supportive care and gastric trocharization were unrewarding. the occurrence of the condition was eliminated by restricting feeding of weanlings to twice a day instead of times daily. etiology. campylobacteriosis is caused by infection with campylobacter jejuni. epizootiology and transmission. campylobacter jejuni is a gram-negative, spirally curved microaerophilic bacterium that is recognized as a significant cause of human enteritis and is as-sociated with diarrheic illness in several animal species, including dogs, cats, cows, goats, pigs, mink, ferrets, and sheep (carter et al., ) . it also known to cause mastitis in cows, infectious hepatitis of chickens, and abortion in cattle, sheep, goats, dogs, and mink (carter et al., ) . the organism may also be cultured from the feces of normal asymptomatic dogs, cats, and ferrets carter et al., ) . transmission occurs by ingestion of organisms through direct contact with feces or contaminated food and water (carter et al., ) . there have been reports linking the disease in humans to pets. many of these outbreaks were associated with dogs, puppies, and kittens recently obtained from animal shelters or pounds and displaying diarrhea before the human illness occurred . isolation of campylobacter jejuni from asymptomatic ferrets also implies a potential for zoonotic transmission (fox et al., . clinical signs. experimental oral inoculation of ferret kits with various strains of c. jejuni produced a self-limiting diarrhea that ranged in character from very mild to watery manning, a, ) . the presence of mucus and/or blood was also noted in the feces of affected animals. anorexia, dehydration, and tenesmus with watery diarrhea were also observed. intravenous inoculation of pregnant mink and pregnant ferrets resulted in reproductive failure, ranging from fetal resorption to expulsion of dead or premature living kits . oral inoculation resulted in abortion in a majority of the infected animals . diagnosis. diagnosis is based on history, clinical signs, and culture of affected animals. reports of spontaneous cases in ferrets require diagnostic confirmation and differentiation from cases of proliferative bowel disease and other infectious and noninfectious causes of diarrhea. campylobacter jejuni grows slowly and has specific culture requirements that involve the use of selective media or filtration techniques, and a requirement for thermophilic ( ~ and microaerophilic conditions (fox, a) . cultures should be examined every hours for round, raised, translucent, and sometimes mucoid colonies (fox, a) . campylobacter jejuni revealed small focal neutrophilic infiltrates in the lamina propria of the colon of relatively few infected animals . bell and manning ( ) noted mild to moderate enterocolitis with neutrophilic infiltration of the lamina propria, which was most severe in kits with concurrent cryptosporidiosis. placentitis was the most notable histologic finding in pregnant ferrets and mink after experimental inoculation of a strain of an abortion storm-associated isolate of c. jejuni . erythromycin is the drug of choice for treatment of human campylobacteriosis (fox, a) . in a study to eliminate the carrier state in ferrets, erythromycin was ineffective even though in vitro isolates of c. jejuni were sensitive to the antibiotic . according to the author, reasons for therapeutic failure included dose selection, interspecies differences in pharmacokinetics and possible reinfection. supportive care should be instituted, and choice of antibiotic therapy in confirmed diarrheic cases should be based on culture and sensitivity. in addition, because of its zoonotic potential, isolation of affected animals and good hygienic practices are recommended. reculture of animals after treatment to ensure elimination of the organism is recommended. epizootiology and transmission. in , a gastric helicobacter-like organism was isolated from the margins of a duodenal ulcer of a ferret and named helicobacter mustelae (fox et al., a (fox et al., , a . subsequently, in the united states, gastritis and peptic ulcers have been routinely reported in ferrets colonized with h. mustelae (fox et al., b (fox et al., , a . every ferret with chronic gastritis is infected with h. mustelae, whereas specific pathogen-free (spf) ferrets not infected with h. mustelae do not have gastritis, gastric ulcers, or detectable igg antibody to the organism (fox et al., , a . helicobacter mustelae has also been isolated from the stomachs of ferrets living in england, canada, australia and, most recently, from ferrets in new zealand (forester et al., ; tompkins et al., ) . koch's postulates have been fulfilled: by oral inoculation of h. mustelae into naive ferrets uninfected with h. mustelae, the infection induced a chronic, persistent gastritis similar to that observed in ferrets naturally infected with h. mustelae (fox et al., b) . it is now known that h. mustelae colonizes nearly % of ferrets shortly after weaning. feces from weanling and adult ferrets have been screened for the presence of h. mustelae to determine whether fecal transmission could explain the % prevalence observed in weanling and older ferrets (fox et al., b (fox et al., , b . helicobacter mustelae was isolated from the feces of of nine-week-old and of eight-month-old ferrets. ferrets placed on proton pump inhibitors, which raise gastric ph, have a statistically higher recovery of h. mustelae from feces when compared with age-matched untreated control ferrets . clinial signs and pathology. helicobacter mustelae-infected ferrets examined in our laboratory are usually asymptomatic. ferrets with gastric or duodenal ulcers can be recognized clinically by vomiting, melena, chronic weight loss, and lowered hematocrit. clinical signs in ferrets with h. mustelae-associated gastric adenocarcinoma have consisted of vomiting, anorexia, and weight loss, signs that may be confused with gastric foreign body. diagnosis. gastric and duodenal ulcers are observable endoscopically. it is interesting that the ferret is the only domesticated animal to date that has naturally occurring helicobacterassociated ulcer disease. the h. mustelae isolated from ferrets has similar but not identical biochemical features to those of h. pylori, particularly in regard to the production of large amounts of urease. gastric samples collected by endoscopy or necropsy are minced with sterile scalpel blades and inoculated onto blood agar plates supplemented with trimethoprim, vancomycin, and polymixin b (remel, lenexa, kansas). the plates are incubated at ~ or ~ in a microaerobic atmosphere ( % n , % h , and % co ) in vented jars for - days. bacteria are identified as h. mustelae on the basis of gram-stain morphology; production of urease, catalase, and oxidase; resistance to cephalothin; and sensitivity to nalidixic acid. necropsy and findings. the histopathological changes occurring in the stomach closely coincided in topography with the presence of h. mustelae . a superficial gastritis present in the body of the stomach showed that h. mustelae was located on the surface of the mucosa but not in the crypts. inflammation occupied the full thickness of the distal antral mucosa, the so-called diffuse antral gastritis described in humans (fig. a,b) . in this location, h. mustelae was seen at the surface, in the pits, and on the superficial portion of the glands. in the proximal antrum and the transitional mucosa, focal glandular atrophy, a precancerous lesion, and regeneration were present, in addition to those lesions seen in the distal antrum. also, deep colonization of h. mustelae was observed focally in the affected antral glands. animals infected with helicobacter spp. may also be susceptible to gastric cancer yu et al., ) . there is recent documentation of the presence of argyrophilic bacteria, compatible in location and morphology to h. mustelae, within the pyloric mucosa of male ferrets with pyloric adenocarcinoma . in humans, epidemiologic data strongly support the association between h. pylori and development of gastric adenocarcinoma. similarly, we have recently documented a series of h. mustelae-infected ferrets with gastric mucosa-associated lymphoid tissue (malt) lymphoma that parallels the same syndi'ome found in humans. lymphoma was diagnosed in the wall of the lesser curvature of the pyloric antrum, corresponding to the predominant focus ofh. mustelaeinduced gastritis in ferrets. gastric lymphomas demonstrated characteristic lymphoepithelial lesions, and the lymphoid cells were igg positive in all ferrets (erdman et al., ) . these findings and their parallels in h. pylori-infected humans implicate the involvement of h. mustelae in the pathogenesis of gastric cancer in ferrets. treatment. studies in ferrets indicate that triple therapy consisting of oral amoxicillin ( mg/kg), metronidazole ( mg/ kg), and bismuth subsalicylate ( . mg/kg) (pepto-bismol original formula, procter and gamble) times a day for - weeks has successfully eradicated h. mustelae . clinical improvement, including increased appetite and resolution of melena, may occur within hr of initiation of triple therapy. a new treatment regimen being used to eradicate h. pylori in humans has also been used successfully for eradication of h. mustelae from ferrets . ferrets received mg/kg ranitidine bismuth and . mg/kg clarithromycin per os times daily for weeks. culture of tissue collected by gastric endoscopic biopsy at , , and weeks after termination of treatment indicated that long-term eradication was achieved in all ferrets. eradication was associated with decrease in anti-h, mustelae igg antibody titers, results that are consistent with findings in humans after h. pylori eradication. omeprazole in ferrets at an oral dose of . mg/kg once daily effectively induces hypochlorhydria and may be used in conjunction with antibiotics to treat h. mustelae-associated duodenal or gastric ulcers. cimetidine at mg/kg tid per os can also be used to suppress acid secretion. acute bleeding ulcers must be treated as emergencies, and fluid and blood transfusions are essential. etiology. proliferative bowel disease is caused by intracellular campylobacter-like organisms, closely related to desulfovibrio spp., that are now classified as lawsonia intracellularis in proliferative enteropathy of swine (fox, a) . the organisms are gram-negative, comma-to spiral-shaped bacteria. epizootiology and transmission. proliferative bowel disease is a common clinical disease observed in young ferrets. fecaloral spread is suspected. the disease typically involves the large bowel, although it has been observed to affect the small bowel (rosenthal, ) . campylobacter species, coccidia, and chlamydia have been isolated from some cases of proliferative bowel disease in ferrets (li et al., b) . the role, if any, of copathogens in this disease is unclear. clinical signs. clinical signs include chronic diarrhea, lethargy, anorexia, weight loss (which is often marked), and dehydration. diarrhea may be blood-tinged, may contain mucus, and is often green in color. rectal prolapse may be observed in affected animals. ataxia and muscle tremors have also been observed (fox et al., ) . diagnosis. diagnosis is based on clinical signs, a palpably thickened colon, and colonic biopsy. it is important to rule out other causes of diarrhea and weight loss through diagnostic tests that include but are not limited to a complete blood count, chemistry profile, radiographs, and fecal analysis and culture. necropsy findings. gross findings include a segmented, thickened lower bowel, usually the terminal colon but occasionally including the ileum and rectum (fox et al., ; fox, a) . histologic examination consistently reveals marked mucosal proliferation and intracytoplasmic l. intracellularis demonstrated with silver stain within the apical portion of epithelial cells in the hyperplastic epithelial cells (fox et al., ; fox, a) (fig. a,b) . other common histologic changes observed include the presence of a mixed inflammatory infiltrate that is variable in severity, reduced goblet cell production, hyperplasia of the glandular epithelium, glandular irregularity with penetration of the mucosal glands through the muscularis mucosa, and an increase in thickness of the tunica muscularis (fox et al., ; fox, a) . translocation of proliferating glandular tissue to extraintestinal sites, including regional lymph nodes and liver, has been described in two ferrets (fox et al., b) . differential diagnosis. proliferative bowel disease should be differentiated from other diseases that may cause diarrhea and wasting, including dietary changes, eosinophilic gastroenteritis, gastric foreign bodies, lymphoma, aleutian disease, and gastric ulcers (bell, b) . a complete physical exam that includes palpation of the abdomen should reveal a palpably thickened intestine in cases of proliferative bowel disease. treatment and control. supportive care, including fluid therapy and nutritional support, should be provided. treatment with chloramphenicol ( mg/kg bid po, sq, im) or metronidazole ( m/kg bid po) for weeks is reported to be effective (krueger et al., ; bell, b) . clinical improvement may be apparent within hr. etiology. tuberculosis can be caused by a variety of mycobacteria, including mycobacterium bovis, m. avium, and m. tuberculosis. epizootiology and transmission. mycobacteria are aerobic, gram-positive, nonbranching, non-spore-forming, acid-fast rods. natural infections with mycobacterium bovis and m. avium have been reported in the ferret. ferrets are also susceptible to experimental infection with human tubercle bacillus. most reports of tuberculosis in ferrets are in animals that were used for research in england and the rest of europe between the years of to and were likely related to the feeding of raw poultry, raw meat, and unpasteurized milk to ferrets during this time (fox, a) . the feeding of commercially prepared diets and widespread tuberculosis testing and elimination in livestock and poultry have resulted in the reduced incidence of the disease in ferrets. mycobacterium avium-infected wild clinical signs and necropsy findings. clinical signs and lesions are dependent on the infective strain. systemic infection with the bovine strain in ferrets results in disseminated disease with weight loss, anorexia, lethargy, death, and miliary lesions involving the lungs and other viscera (fox, a) . progressive paralysis has also been reported in a case of spontaneously occurring bovine tuberculosis in a ferret (symmers and thomson, ) . mycobacterium bovis lesions contain numerous acid-fast bacilli within macrophages with little cellular reaction (fox, a) . in contrast, infection of ferrets with the human tubercle bacilli results in localized infection, often confined to the site of injection and adjacent lymph nodes; microscopically few organisms are observed. an impaired cell-mediated response may account for the large number of organisms observed in m. bovis lesions. vomiting, diarrhea, anorexia, and weight loss were observed in a pet ferret with granulomatous enteritis caused by m. avium (schultheiss and dolginow, ) . granulomatous inflammation characterized by large numbers of epithelioid macrophages containing numerous acid-fast bacilli were present in the lamina propria and submucosa of the jejunum and pylorus. other sites of granulomatous inflammation included peripancreatic adipose tissue, mesenteric lymph nodes, spleen, and liver. a source of infection was not identified in this report. pulmonary infection with m. avium has also been reported in ferrets in a zoo in france (viallier et al., ) . diagnosis. definitive diagnosis of tuberculosis requires isolation and identification of the organism from suspect tissue specimens. great care should be exercised in handling suspect clinical specimens, and an appropriately equipped laboratory should be identified for culture and identification of the organism. although there has been some experimental work in the area of the intradermal tuberculin skin-test response in ferrets and its apparent use in controlling tuberculosis in a breeding colony of ferrets, a tuberculin skin-testing regimen, including dose and type, has not been definitively characterized for clinical use in ferrets (kauffman, ) . treatment and control. because of the zoonotic risk, ferrets infected with m. bovis and m. tuberculosis should be euthanized (fox, a) . recurrent m. bovis infection involving the palmar aspect of the wrist of a -year-old man, which developed after he was bitten by a ferret at the age of , was reported and demonstrates the zoonotic potential (jones et al., ) . mycobacterium avium infection is not reportable but may pose a risk to immunocompromised patients (fox, a) . personnel at risk should be followed up by a physician for appropriate diagnostic testing (fox, a) . etiology. salmonellosis is caused by infection with organisms of the genus salmonella. epizootiology and transmission. salmonella is a gram-negative, non-spore-forming, facultative anaerobic rod in the family enterobacteriaceae (carter et al., ) . infection is by the oral route. transmission may be direct from infected carrier animals or humans or through contaminated food products or water (carter et al., ) . several salmonella serovars have been isolated from mink with gastroenteritis and abortion (gorham et al., ) . contaminated raw meat products were suspected as the source in one outbreak. salmonella typhimurium was isolated in ferrets in an outbreak of clinical disease (coburn and morris, ) and several serotypes including s. hadar, s. enteritidis, s. kentucky, and s. typhimurium were isolated from the feces of ferrets surveyed in a research colony (fox et al., a) . clinical signs and necropsy findings. clinical signs of an outbreak of s. typhimurium in ferrets included conjunctivitis, rapid weight loss, tarry stools, and febrile temperature fluctuations (coburn and morris, ) . gross findings in ferrets days after inoculation with s. typhimurium of ferret origin included marked tissue pallor, petechiae in the gastric mucosa, and the presence of melena in one and a dark-colored fibrinous exudate in the large intestine of the other ferret (coburn and morris, ) . studies involving experimental inoculation with s. enteritidis, s. newport, and s. choleraesuis via the oral route to healthy, distemper-infected, and feed-depleted ferrets and mink showed a fairly high resistance to infection (gorham et al., ) . only animals of in the diet-restricted group-- ferret and minknshowed clinical signs of infection after feeding s. newport culture. signs included lethargy, anorexia, trembling, and fecal blood. the gastrointestinal tract showed a large amount of mucus containing red blood cells; bits of desquamated epithelium and few mononuclear cells overlying the gastric mucosa; an exudate in the small intestine consisting of mu-coid material, red blood cells, and desquamated small intestinal villi; edematous villi in the ileum; and a diffuse infiltrate of the small intestinal mucosa with lymphocytes and macrophages. necrotic foci in the liver, spleen, and, less commonly, the kidney, as well as splenomegaly and visceral lymphadenopathy, were observed in chronic fatal infections (coburn and morris, ) . abortion and gastroenteritis have been reported in mink (gorham et al., ) . diagnosis. diagnosis is based on history, clinical signs, and isolation of the organism. the organism can be cultured on enrichment and selective media and then characterized serologically. samples of blood, feces, exudates, tissues, and intestinal material may be cultured. treatment and control. coburn and morris ( ) treated of ferrets experimentally infected with s. typhimurium with sulfathalidine in the feed (coburn and morris, ) . salmonella typhimurium was isolated in of control animals and none of the treated animals days after the administration of the last dose. sulfathalidine was administered by the same authors to a colony of ferrets in which an outbreak of salmonella occurred. the group was surveyed days after sulfathalidine treatment and showed weight gain, improvement in condition, and a reduction in the number of salmonella-infected ferrets (coburn and morris, ) . salmonella spp. isolated from ferrets may show resistance to a number of antibiotics (fox, a) . treatment includes appropriate use of antimicrobials and supportive care, which may include fluid therapy, nutritional support, maintenance of electrolyte balance, treatment of concurrent diseases, recognition of and attention to shock, and reduction of stress (fox, a) . etiology. streptococcus zooepidemicus and other group c and g streptococci, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and bordetella bronchiseptica have been reported as primary and secondary bacterial pathogens in pneumonia in ferrets (fox, a) . epizootiology and transmission. bacterial pneumonia may occur secondary to megaesophagus in the ferret. an influenza virus-bacteria synergism has been the subject of several studies in ferrets (fox, a) . debilitated and immunosuppressed animals and animals with concurrent diseases such as influenza may be more susceptible to bacterial pneumonias (fox, a) . clinical signs. clinical signs may include nasal discharge, dyspnea, lethargy, anorexia, increased lung sounds, cyanosis, and fever (rosenthal, ) . fulminant pneumonia may progress to sepsis and death (fox, a) . diagnosis. diagnosis is based on history, clinical findings, a complete blood count, culture and cytology of a tracheal wash or lung wash, and radiographs (rosenthal, ) . differential diagnosis. diagnostic rule-outs include dilatative cardimyopathy, heartworm disease, mycotic pneumonia, pneumocystis pneumonia in immunosuppressed animals, neoplasia, and influenza. treatment and control. treatment should consist of appropriate antimicrobial therapy and supportive care, which may include the administration of oxygen, fluid therapy, and force feeding (rosenthal, ) . etiology. a variety of bacteria have been associated with abscesses and localized infection of the lung, liver, uterus, vulva, skin, mammary glands, and oral cavity. these include staphylococcus spp., streptococcus spp., corynebacterium spp., pasteurella, actinomyces, hemolytic escherichia coli, and aeromonas spp. (fox, a) . epizootiology and transmission. abscesses in ferrets may result from wounds that are inflicted secondary to biting during fighting, playing, mating, or chewing sharp objects. clinical signs. localized or subcutaneous abscesses present as swellings with or without draining tracts. the swelling may be fluctuant. in most cases, the abscess is walled off and does not result in systemic signs (fox, a) . abscesses or infection involving visceral organs may give rise to organ-specific and/or systemic signs. diagnosis. cytologic and gram staining of an aspirate of a suspect subcutaneous swelling will aid in the definitive diagnosis. culture and sensitivity of the aspirate should also be performed to identify the causative organism and guide appropriate antibiotic therapy. differential diagnosis. differential diagnosis of a subcutaneous swelling in a ferret should include myiasis, granuloma, hematoma, and neoplasia. treatment and control. prevention of ferrets from exposure to sharp objects in the cage and feed, and limiting the exposure of male and female during breeding, can minimize the occurrence of abscesses. treatment of localized abscesses should include appropriate antibiotic therapy and establishment of drainage and debridement if necessary. bacterial culture and sensitivity of the exudate should be performed. a broad-spectrum antimi-crobial may be used pending results of culture and sensitivity (orcutt, ) . etiology. gram-positive cocci such as streptococcus spp., staphylococcus aureus, and coliforms such as hemolytic e. coli are the most frequently associated organisms (bernard et al., ; bell, a) . although the exact pathogenesis of mastitis in ferrets is not clear, a number of factors may play a role and include the stress of lactation, injury to mammary glands by the kits' teeth, environmental contamination, and the virulence of the organism. in one report, the causative organism, hemolytic e. coli, was cultured from the feces of mastitic and healthy ferrets and the oral cavity of suckling kits (liberson et al., ) . the high level of perineal contamination and the presence of the organism in the oral cavity of suckling kits may enhance transmission and introduction of this organism into mammary tissue. in another outbreak, the causative organisms were cultured from bovine meat fed prior to the outbreak, and the meat was suspected as a possible source. clinical signs. mastitis occurs in nursing jills and has been characterized as acute or chronic (bell, a) . the acute form is reported to occur soon after parturition or after the third week of lactation. examination of affected jills reveals swollen, firm, red or purple, and painful glands. affected glands may quickly become gangrenous. the chronic form, which may occur when kits are weeks old or as a sequela to the acute form, is characterized by glands that are firm but not painful or discolored. diagnosis. diagnosis is based on history, clinical signs, physical examination findings, and isolation of the causative organism. necropsy findings. in acute mastitis, grossly affected glands are swollen, and the skin overlying the gland may be discolored. surgical biopsies and necropsies of ferrets with mastitis caused by hemolytic e. coli (liberson et al., ) revealed extensive edema, hemorrhage, and coagulative and liquefactive necrosis involving the glandular tissue as well as surrounding subcutaneous tissue. other findings included the presence of a mixed leukocytic infiltrate composed primarily of polymorphonuclear leukocytes; large numbers of bacteria; and thrombosis and necrosis of vessels within and immediately adjacent to areas of inflammation (liberson et al., ) . in an outbreak of mastitis in mink due to staphylococcus aureus and escherichia coli, histologic examination of affected glands revealed an acute suppurative mastitis with desquamation of alveolar epithelium, edema of the connective tissue stroma, alveoli filled with neutrophils and cellular debris, and lactiferous ducts filled with purulent exudate and mats of bacteria within lobules (trautwein and helmboldt, ) . treatment. broad-spectrum antibiotic therapy may be instituted pending culture and sensitivity results of the milk. enrofloxacin ( . mg/kg bid po after a loading dose of . mg/kg im) is often effective. jills may require aggressive care, because acute mastitis may progress rapidly and animals may become septicemic and moribund (liberson et al., ) . oral antibiotic administration to kits nursing affected jills is recommended (bell, a) . surgical resection and debridement of affected glands and supportive care may be necessary for jills with acute mastitis. supplementation of kits with milk replacer may also be necessary, because jills with acute mastitis are reluctant to nurse, and jills with the chronic form have diminished lactation as milk-producing tissue is replaced by scar tissue (bell, a) . maintaining thorough personal hygiene practices when handling affected jills is important in minimizing spread to other lactating jills. cross-fostering kits may be required; however, kits may spread infection to healthy jills. it is reported that jills with the chronic form of mastitis should be culled (bell, a ). etiology. canine distemper (cd) is caused by a paramyxovirus of genus morbillivirus that is related to measles and rinderpest (budd, ) . there are several strains, including a ferret-adapted strain of canine distemper virus (cdv), that vary in incubation, clinical signs, and duration . the virus can be inactivated by heat, light, and various chemicals, including phenol, roccal, sodium hydroxide, and formalin (shen and gorham, ; budd, ) . infectious virions have been recovered from fomites after min at room temperature. canine distemper is the most serious viral infection of ferrets. mortality approaches %, making appropriate husbandry and vaccination imperative. the disease has a catarrhal phase and a neurological, or central nervous system (cns), phase. the catarrhal phase is - days postinfection and involves anorexia, pyrexia, photosensitivity, and serous nasal discharge. an erythematous pruritic rash spreads from the chin to the inguinal region. it is suspected that the rash results from cell-mediated immunity to infected endothelial cells, similar to the response seen in humans with measles (norrby and oxman, ) . hyperkeratosis of footpads, called hard pad, is an inconsistent feature. secondary bacterial infections result in mucopurulent ocular and nasal discharge and possibly bacterial pneumonia. the cns phase, with ataxia, tremors, and paralysis, may or may not be preceded by the catarrhal phase. death occurs in - days from ferret strains of cdv and up to days with canine strains. infection is uniformly fatal. epizootiology and transmission. virus is shed from infected hosts from conjunctival, nasal, and oral exudates, urine, feces, and sloughed skin (gorham and brandly, ) . transplacental infection is not reported in ferrets. attenuated cdv vaccine strains have not been recovered from the body secretions of ferrets following vaccination (shen et al., ) . unvaccinated dogs and other canids, mustelids, and procyonids may serve as reservoirs of infection. viremia is detectable days postinfection and persists until the ferret dies or mounts a neutralizing antibody response (liu and coffin, ) . the primary site of replication is the respiratory and lymphatic systems, and cdv has been recovered from the nasal secretions of ferrets - days postinfection. a decrease in lymphocyte subsets is detectable - days postinfection. clinical signs and necropsy findings. histologically, intracytoplasmic and intranuclear inclusion bodies may be observed in tracheal, bronchial, epithelia, and bile duct as well as transitional epithelium in the bladder (liu and coffin, ) (fig. ) . the eosinophilic (hematoxylin-eosin) inclusions appear orange using pollack's trichrome stain. diagnosis and differential diagnoses. presumptive diagnosis is based on clinical observation, questionable vaccination history, and exposure. a fluorescent antibody test can be used on peripheral blood and conjunctival mononuclear cells to detect infection. reverse transcriptase-polymerase chain reaction (rt-pcr) has also been used to detect experimental infection (stephensen et al., ) . differential diagnoses should include infection with influenza virus or bordetella bronchiseptica. influenza does not rapidly progress to mucopurulent ocular and nasal discharge as cd does. during an outbreak, clinically affected ferrets should be isolated and the remainder of the colony vaccinated. distemper infection can be prevented by vaccination with modified live vaccine of chicken embryo tissue culture origin (cetco) administered subcutaneously or intramuscularly. kits should be vaccinated every - weeks, starting at age weeks, until weeks and annually thereafter . it is important to adhere to the prescribed vaccination protocol, because ferret deaths have been reported following double-dose vaccination (carpenter et al., ) . inactivated distemper vaccines do not elicit consistent, effective immunity and are not recommended. it is important to know the vaccination schedule of your ferret supplier and to vaccinate supplementally as appropriate. new ferrets should be held in quarantine for weeks prior to introduction into the resident colony. ferrets have been experimentally infected with feline panleukopenia, canine parvovirus, canine parainfluenza virus, mink enteritis virus, respiratory syncytial virus, transmissible mink encephalopathy, and pseudorabies, but natural infection with these viruses has not been reported . etiology. aleutian disease virus (adv) is a parvovirus with strains of varying virulence and immunogenicity. mink-derived strains are more virulent to mink than are ferret-derived strains . epizootiology and transmission. aleutian disease (ad) is a chronic progressive illness that was first described in mink (oxenham, ) . it was originally named hypergammaglobulinemia (hgg) because of this remarkable finding. infection may be subclinical for years. because the immunomodulation associated with adv infection is disruptive to biomedical research, it is important to seek sources of adv-free ferrets . transmission between ferrets may be direct or via aerosol of urine, saliva, blood, feces, and fomites (kenyon et al., ; gorham et al., ) . vertical transmission is established in mink but is unproven in ferrets. clinical signs. ferrets infected with adv as adults develop persistent infection but rarely disease, although chronic progressive weight loss, cachexia, malaise, and melena have been described (porter et al., ) . ad may also cause ataxia, paral-ysis, tremors, and convulsions (oxenham, ; welchman et al., ) . the lesions are typically immune-mediated, and there is elevation of the gammaglobulins to generally greater than % of the total proteins (porter et al., ; fig. ). the precise mechanism of immunomodulation is unknown, but in mink there is depression of b-and t-cell responses. necropsy. ferrets may have no lesions upon necropsy, or infrequently they may have hepatosplenomegaly and lymphadenopathy. the most consistent histological finding is periportal lymphocytic infiltrates (fig. ). bile duct hyperplasia and periportal fibrosis have also been reported. membranous glomerulonephritis has been described (ohshima et al., ) . although lesions are subtle, use of adv-infected ferrets in biomedical research is contraindicated because histological lesions interfere with the interpretation of study results . diagnosis and differential diagnoses. presumptive diagnosis is based on hgg and chronic weight loss. diagnosis is confirmed by immunofluorescent antibody (ifa) or counterimmunoelectrophoresis (ciep) for antibody to adv antigen (palley et al., ) . pcr-based assays have also been used (erdman et al., b; saifuddin and fox, ; erdman et al., ) . differential diagnoses include the neurotropic form of cd, as well as chronic wasting diseases such as neoplasia, malabsorption, maldigestion, and bacterial enteritis . vaccination against adv would be contraindicated because of the immune-mediated reaction, and a vaccine is not available. chemical disinfection may be achieved tp - . g/dl /globulin = . g% , .. with formalin, sodium hydroxide, and phenolics (shen et al, ) . there is no treatment for ad, and infected ferrets should be culled from the colony. etiology. influenza is caused by an orthomyxovirus that is transmissible from humans to ferrets and ferrets to humans (smith and stuart-harris, ) . human influenza viruses a and b are pathogenic to ferrets . ferrets are also susceptible to avian, phocine, equine, and swine influenza, although only porcine influenza causes clinical signs. because the viruses can be readily transmitted from humans to ferrets, handling precautions such as wearing masks and gloves should be in place to minimize transmission. epizootiology, transmission, and clinical signs. influenza virus generally remains localized in nasal epithelium in ferrets but may cause pneumonia. clinical signs appear hr postinfection and include anorexia, fever, sneezing, and serous nasal discharge. conjunctivitis, photosensitivity, and otitis are also sometimes seen . secondary bacterial infection by streptococcus sp. and occasionally bordetella bronchiseptica may prolong recovery. transmission occurs via aerosol and direct contact. diagnosis. diagnosis is based on typical clinical presentation and recovery within days, unlike with cd, which progresses to more severe disease and death. hemagglutination inhibition antibody titers on acute and convalescent sera are rarely needed. treatment and control. antibiotic therapy may be instituted to preclude secondary bacterial infection. animal technicians and investigators suffering from influenza should avoid contact with ferrets. ferrets have been used extensively as a model for influenza research because the biological response to infection is similar to that in humans . ferrets have been used in influenza a research to study pathogenesis, to investigate reye's syndrome, and to evaluate vaccine trials (deshmukh, ; sweet et al., ) . etiology. rabies is caused by a rhabdovirus. rabies infection is infrequently reported in ferrets, and until recently, research on rabies in ferrets was lacking . ferrets in a well-managed facility would have low risk of exposure to rabies virus. a usda-approved, killed rabies vaccine given subcutaneously at ages months and year and annually thereafter is recommended to protect ferrets against rabies (rupprecht et al., ) . modified live vaccine (mlv) is not recommended, because there is at least one case of rabies in a ferret that was vaccinated with mlv rabies vaccine . there is no treatment for rabies. clinical signs and pathogenesis. clinical signs of rabies infection in ferrets may include anxiety, lethargy, and posterior paresis. in one experimental infection, of ferrets died, and negri bodies were seen in the brain of only of the (blancou et al., ) . there is conflicting data on the isolation of rabies virus from the salivary glands following experimental infection. in one study using raccoon variant of rabies for infection, more than half of the ferrets had rabies isolated from the salivary glands . ferrets at risk for exposure to rabies virus that bite or scratch a human should be placed under quarantine for not less than days of observation. veterinarians and facility managers should seek assistance from state public health officials. diagnosis and differential diagnoses. differential diagnosis includes the neurotropic form of cd. diagnosis is based on direct ifa of brain tissue. because rabies in ferrets is poorly understood, the head from ferrets that exhibit signs compatible with rabies and that have exposure histories that raise concerns about rabies should be shipped to the state public health authority for confirmation. etiology. rotaviruses cause diarrhea in young of many species, including humans, calves, pigs, sheep, and rats. diarrhea in ferret kits is thought to be caused by a poorly characterized, atypical rotavirus that has not been cultivated in vitro (torres-medina, ) . atypical rotaviruses lack the rotavirus common antigen. epizootiology, transmission and clinical signs. clinical disease may occur in kits as young as - days old or in older animals up to weeks of age. diarrhea soils the perineum and possibly the fur and nest material. mortality rates are agedependent, with high mortality occurring in young kits and lower mortality occurring in kits over days of age (bell, a; fox et al., b) . secondary bacterial infection may influence the severity of diarrhea. necropsy andpathogenesis. lesions are restricted to the gastrointestinal tract. yellow-green liquid or mucous feces may be seen in the terminal colon on necropsy. subtle small-intestinal villous atrophy and epithelial cell vacuolation are detectable histologically. diagnosis and differential diagnoses. clinical diagnosis can be confirmed by using clarified and ultracentrifuged fecal pel-lets for electron microscopy. the ferret rotavirus does not crossreact with commercially available enzyme immunoassays (torres-medina, ) . it is desirable to avoid sources that are known to be infected with ferret rotavirus. affected kits may be supplemented with kitten milk replacer, using a medicine dropper. mortality is reduced if the kits continue nursing. treatment of secondary bacterial infections may reduce severity of the diarrhea, and supportive care, including subcutaneous fluid administration for young kits, may be required . jills develop immunity to rotavirus infection, and subsequent litters are protected. infectious bovine rhinotracheitis (ibr) was isolated from the liver, spleen, and lung of clinically normal ferrets (porter et al., ) . raw beef was suspected as the source of infection, reinforcing the need to exclude raw meat products from the diet of ferrets used for research. ibr does cause significant respiratory pathology in experimentally infected ferrets (porter et al., ) . a transmissible diarrhea, referred to as epizootic catarrhal enteritis, has been observed in adult ferrets several days after direct contact and fomite exposure to affected ferrets . clinically the diarrhea is green and bile-tinged, and the ferrets become rapidly dehydrated. mortality is low. some ferrets develop elevated liver enzymes. treatment involves aggressive oral and systemic fluid therapy. a recent study implicates a coronavirus as the cause of this disease (williams et al., ) . a. protozoa i. enteric coccidiosis etiology. three species of the genera isospora and eimeria have been reported to infect the ferret: isospora laidlawi, eimeria furonis, and e. ictidea (blankenship-paris et al., ) . tion of sporulated oocysts. clinical signs. coccidiosis in ferrets is usually subclinical but has been reported to be associated with diarrhea, lethargy, and dehydration in one ferret (blankenship-paris et al., ) . clinical signs are often seen in young, newly acquired ferrets and are more common after a stressful event (rosenthal, ) . rectal prolapse can also develop in association with coccidial infection (rosenthal, ) . diagnosis. diagnosis is generally made by any of the fecal flotation methods commonly used in veterinary practice or by direct wet mount of feces and microscopic examination for sporulated or unsporulated oocysts. because coccidial oocysts are small, slides should be examined under higher magnification. necropsy findings. diagnosis is usually performed antemortem. pathologic lesions associated with enteric coccidiosis in a laboratory-reared ferret that was euthanized were described in one published report (blankenship-paris et al., ) . microscopic lesions were confined to the jejunum and ileum and consisted of villous and epithelial thickening. parasitic cysts and microorganisms within epithelium, and a mild granulomatous inflammation in the villar lamina propria, were also observed. a recent report documents clinical and anatomic pathology associated with biliary coccidiosis in a weanling ferret (williams et al., ) . differential diagnosis. diarrhea may be observed in ferrets that present with gastroenteritis secondary to gastrointestinal foreign bodies and dietary indiscretion, as well as other nutritional, inflammatory, infectious, or other systemic diseases. infectious causes such as proliferative colitis, salmonellosis, giardiasis, rotavirus, and campylobacteriosis should be considered. diarrhea may also be seen in eosinophilic gastroenteritis, an uncommonly reported condition in ferrets. good husbandry practices that include sanitation and frequent disposal of feces reduce the number of oocysts in the environment. cleaning cages with a strong ammonium hydroxide solution is reported to be effective (kirkpatrick and dubey, ) . heat treatment of surfaces and utensils may also be effective (kirkpatrick and dubey, ) . treatment of ferrets with sulfadimethoxine at mg/kg orally once and then mg/kg orally every hr for days is recommended (rosenthal, ) . as in dogs and cats, the complete elimination of a coccidial infection requires an immunocompetent host. ii. cryptosporidiosis etiology. cryptosporidiosis is caused by infection with cryptosporidium spp. epizootiology and transmission. cryptosporidium is a protozoan in the class sporozoa, subclass coccidia, that inhabits the respiratory and intestinal epithelium of birds, reptiles, mammals, and fish (regh et al., ) . it is known to cause gastrointestinal tract disease in many species, including rodents, dogs, cats, calves, and people (hill and lappin, ) . it has a life cycle similar to other coccidian parasites and is transmitted by ingestion of sporulated oocysts. autoinfection is also a characteristic of the life cycle. transmission may occur through consumption of contaminated food or water. cattle, dogs, and cats, shedding oocysts, are reported to be potential sources of human infection (hill and lappin, ; fox, g) . immunosuppressed people are at greatest risk of developing severe fulminating gastrointestinal disease (hill and lappin, ) . the finding of cryptosporidiosis in two ferrets that died from unrelated causes in one animal facility resulted in a survey of the existing ferret population and new arrivals into the facility to determine the prevalence and incidence of infection (regh et al., ) . findings indicated that % of the resident population and - % of new arrivals had oocysts in their feces but showed no clinical signs. clinical signs. only subclinical infection has been reported in both immunocompetent and immunosuppressed ferrets (regh et al., ) . diagnosis. diagnosis is based on the identification of the organism in feces. the oocysts are small when compared with other coccidia and may be overlooked or mistaken for yeasts (kirkpatrick and dubey, ) . yeasts are oval, whereas cryptosporidium oocysts are spherical or ellipsoidal. additionally, yeasts will stain with iodine and are not acid-fast, whereas cryptosporidium has the opposite staining characteristics. the oocyst residuum is seen as a refractive dot under phase-contrast microscopy, a structure lacking in yeast (kirkpatrick and dubey, ) . sugar-solution centrifugation and fecal sedimentation using formalin-ether or formalin-ethyl acetate are effective di-agnostic concentration techniques (hill and lappin, ) . oocysts may then be viewed with phase-contrast or bright-field microscopy of specimens stained with an acid-fast method. a direct fecal smear may be methanol-or heat-fixed and stained with an acid-fast method (hill and lappin, ) . necropsy findings. histologic evaluation reveals the presence of organisl ns, spherical to ovoid in shape and from to ~tm in diameter, associated with the brush border of the villi. a mild eosinophilic infiltrate was observed in the lamina propria of the small intestine in most animals. the ileum was the most common and heavily infected section of small intestine (regh et al., ) . there is no known definitive treatment for cryptosporidiosis (fox, g) . supportive and symptomatic care should be provided in clinical cryptosporidiosis. infections are self-limiting in immunocompetent patients (fox, g) . control is aimed at eliminating or reducing infective oocysts in the environment and avoidance of contact with known sources. because of the potential for zoonotic transmission, restricting contact of children and immunosuppressed individuals with infected ferrets and practicing good hygiene may help reduce the potential for infection. drying, freeze-thawing, and steam cleaning inactivate the organism (hill and lappin, ) . there are few effective commercial disinfectants. i. sarcoptic mange etiology. sarcoptic mange is caused by infection with sarcoptes scabiei. epizootiology and transmission. transmission occurs through direct contact with infected hosts or contact with fomites. this parasitic infection is rare under research conditions. clinical signs. infection of ferrets with s. scabiei may occur in a generalized or a pedal form (bernard et al., ) . in the generalized form, lesions consist of focal or generalized alopecia with intense pruritus. in the pedal form, lesions are confined to the toes and feet, which become swollen and encrusted with scabs. nails may be deformed or lost if the condition is left untreated. diagnosis. diagnosis is made by finding the mites in skin scrapings or removing crusts, breaking them up, and clearing with % koh for microscopic examination (phillips et al., ) . false-negative results are possible; multiple scrapings may be necessary. differential diagnosis. differential diagnosis should include other pruritic external parasitic conditions, including flea infes-tation. demodicosis has been reported to cause mild pruritus and alopecia in ferrets (noli et al., ) . in the pedal form, treatment consists of trimming the claws and removing the scabs after softening them in warm water (bernard et al., ) . treatments that have been used include ivermectin, . - . mg/kg, administered subcutaneously and repeated every - days until mites are gone; shampoos or soaks to reduce the pruritus; and topical or systemic antibiotic administration for treatment of secondary bacterial dermatitis (hillyer and quesenberry, b) . alternatively, weekly dips in % lime sulfur until weeks after clinical cure have been shown to be effective (fox, a) . treatment of all affected animals as well as contact animals, and decontamination of enclosures and bedding, are recommended. ii. demodicosis etiology. demodicosis is caused by infection by demodex spp. epizootiology and transmission. the parasite is found in normal skin of almost all dogs and is not considered contagious. predisposing factors such as immunologic or genetic conditions have been suggested (kwochka, ) . one clinical report describes demodicosis in two adult ferrets that had been treated with an ear ointment containing triamcinolone acetonide for recurrent ear infections daily for periods of months each during the course of a year (noli et al., ) . clinical signs. in the report mentioned above, the ferrets presented with alopecia, pruritus, and orange discoloration of the skin behind the ears and on the ventral surface of the abdomen and an accompanying seborrhea (noli et al., ) . diagnosis. deep skin scrapings should be performed to demonstrate mites. finding a large number of live adult mites or immature forms and eggs is necessary to confirm the diagnosis. in very chronic cases, the skin may be so thickened that scrapings may be unrewarding. in these cases, a skin biopsy may be diagnostic (kwochka, ) . necropsy findings. histologic evaluation of skin biopsies obtained in the case report described above revealed mites with a short, blunted abdomen similar to that of demodex criceti and located in the infundibulum of hairs. the epidermis was slightly hypertrophic, and there was a mild superficial orthokefatotic hyperkeratosis. a very mild superficial and perivascular mixed cellular infiltrate was also observed in the dermis. differential diagnosis. generalized demodicosis should be differentiated from sarcoptic mange and flea infestation. primary or secondary bacterial dermatitis or pyoderma should also be considered. treatment and control. the ferrets in the above-mentioned clinical report were treated initially with a suspension of . % amitraz applied as a dip times at -day intervals for treatments. two drops of the same solution were applied in each ear every other day. after the initial treatment, the ferrets were reexamined, and treatment was continued with the same concentration of solution applied once every days, while the tail was washed with a higher concentration of amitraz ( . %) once every other day. thereafter, final treatments with . % amitraz every days for the body, and every other day for the ears and tail, were administered. the ferrets were evaluated and skin scrapings were performed regularly during treatment and posttreatment to monitor response to therapy. treatment of any associated pyodermas, systemic illnesses, or management problems should also be included as part of the therapeutic regimen. iii. ear mites etiology. the ear mite, otodectes cynotis, which commonly infects dogs and cats, is also a common clinical problem in ferrets (fox, g) . epizootiology and transmission. ear mites are transmitted through direct contact with infested ferrets, dogs, or cats (fox, g) . the entire life cycle is completed in weeks. clinical signs. ear mite infestation in the ferret is usually asymptomatic (orcutt, ) . however, clinical signs may include head shaking; mild to severe pruritus with inflammation and excoriation; secondary otitis interna with ataxia; circling; torticollis; and horner's syndrome (orcutt, ; fox, g) . a brownish black waxy discharge is often present. diagnosis. diagnosis is based on direct observation of mites via otoscopic examination or microscopic identification of the ear mite or any of the life-cycle stages of the mite in exudate from the ear canal. treatment and control. several treatment regimens, including topical and injectable mitocidal treatments, have been recommended (orcutt, ; fox, g) . a recent study using three treatment regimens--two topical and one injectable--revealed that topical treatments were more efficacious than the injectable in reducing or eradicating ear mites (patterson et al., ) . efficacy was evaluated by microscopic evidence of ear mites in debris from aural swabs taken weekly for an -week period. topical % ivermectin (ivomec, merck agvet division, rahway, new jersey), diluted " in propylene glycol at a dosage of ~tg/kg body weight divided equally between the two ear canals and administered on days and of the study, was the most effective treatment. all susceptible animals in a household should be treated. ears should be gently cleaned prior to initiating treatment (orcutt, ) . high doses of injectable iver-mectin ( . ml of % ivermectin) administered to jills at - weeks of gestation resulted in high rates of congenital defects (orcutt, ) . iv. fleas etiology. ctenocephalides species can infest ferrets. epizootiology and transmission. transmission requires direct contact with another infested animal or a flea-infested environment. clinical signs. flea infestation may be asymptomatic or may cause mild to intense pruritus and alopecia of the dorsal thorax and neck (timm, ) . differential diagnosis. sarcoptic and demodectic mange should be included in the differential diagnosis of pruritic skin disease in the ferret. close examination of the pelage for fleas or flea excrement should be performed. skin scrapings may be indicated. as with flea infestation in dogs and cats, concurrent treatment of the environment, as well as all animals in the household, is essential for effective flea control. compounds approved for flea control in cats such as rotenone or pyrethrin powders or sprays may be used in ferrets (hillyer and quesenberry, a) . ferrets may develop systemic disease from blastomyces, coccidioides, cryptococcus, and histoplasma. the reservoir of most of these fungi is the soil, however, making infection unlikely in a research facility. in production facilities, exposure can be minimized through careful selection of source animals, appropriate sanitation, and control of pests, particularly birds. pneumocystis carinii has been recently reclassified as a fungus. although p. carinii inhabits the lungs of many different species, recent transmission studies suggest that these fungi are highly species-specific (gigliotti et al., ; fox et al., b) . clinical disease is evident only in immunocompromised ferrets and can be induced using high doses of exogenous steroids (stokes et al., ) . lesions include interstitial pneumonitis with mononuclear cell infiltrates; cysts and trophozooites are evident with gomori methanamine-silver nitrate and giemsa on bronchoalveolar lavage. treatment with trimethoprim sul-famethoxazole probably controls but does not eliminate infection . ferrets are susceptible to secondary fungal infection of the outer ear canal with absida corymbifera or malassezia spp. (dinsdale and rest, ; fox, d) . the fungi are widespread in the environment and will cause a secondary fungal infection in the ears of ferrets infested with otodectes cynotis. the yeasts can be visualized by impressions of ear exudates. treatment involves eradication of the underlying mite infestation followed by oral and topical ketoconazole, miconazole, and polymyxin b. dermatomycoses in ferrets are caused by microsporum canis and trichophyton mentagrophytes. dermatophytes are transmissible to humans and are a zoonosis; thus affected animals should be quarantined and removed from the facility to minimize risk (dinsdale and rest, ; scott et al., ; fox et al., b) . control of infection includes general disinfection and destruction of contaminated bedding. lesions are circumscribed areas of alopecia and inflammation, which begin as small papules that spread peripherally in a scaly inflamed ring. the yellow-green fluorescence of m. canis under ultraviolet light helps distinguish it from t. mentagrophytes. skin scrapings digested with % potassium hydroxide reveal characteristic arthrospores. treatment with griseofulvin causes clinical remission but may not clear infection. other ectoparasitic infections observed to occur in ferrets include cutaneous myiasis and tick infestation. granulomatous masses in the cervical region caused by the larval stage of hypoderma bovis have been reported in ferrets (fox, g) . cuterebra larvae, although uncommonly observed in ferrets, may cause subdermal cysts found in the subcutis of the neck (orcutt, ) . infestation with the flesh fly has been reported as a problem in commercially reared mink and ferrets housed outdoors (fox, g) . ticks may be found on ferrets housed outdoors or on those used for hunting rabbits (fox, g) . ticks should be removed carefully with hemostats or tweezers, ensuring that the entire head and mouthparts are removed from the skin. appropriate caution should be exercised in tick removal, because ticks are responsible for transmission of various zoonotic pathogens; gloves should be worn. etiology. the ferret is susceptible to natural and experimental infection with dirofilaria immitis. epizootiology and transmission. dirofilaria immitis is a filarial parasite that is transmitted by mosquitoes, which serve as the intermediate host and vector. microfilaria are ingested by mosquitoes and, after two molts, become infective third-stage larvae. infective larvae are deposited onto the skin when mosquitoes feed, and larvae find their way into the body of the final host through the bite wound and migrate subcutaneously to the thorax and eventually to the heart (knight, ) . the primary reservoir of infection is dogs, but heartworm may be found in a variety of mammals, including humans. all other species except wild and domestic canids, domestic felines, ferrets, and the california sea lion are considered aberrant hosts (knight, ) . clinical signs. the following clinical signs have been reported in clinical reports describing cases of d. immitis in the ferret: weakness, lethargy, depression, dyspnea, cyanosis, anorexia, dehydration, cough, and pale mucous membranes (miller and merton, ; parrott et al., ; moreland et al., ) . moist lung sounds and/or muffled heart sounds were revealed by thoracic auscultation in many of these cases. pleura/or abdominal effusion may be observed radiologically. the ferrets described in these cases were housed outdoors and either died or were euthanized. diagnosis. diagnosis of heartworm is based on clinical signs, radiographic findings, and testing for circulating microfilariae and heartworm antigen. microfilaremia is not consistently observed in naturally occurring and experimental cases of heartworm infection in ferrets (fox, g) . testing for heartworm antigen appears to be more diagnostically useful (stamoulis et al., ) . in a study to determine the minimum oral dose of ivermectin needed for monthly heartworm prophylaxis in ferrets, the use of an antigen test (uni-tec canine heartworm test, pitman-moore co., mundelein, illinois) detected infection in more untreated control animals than did the modified knott test for detection of circulating microfilaria in the same ferrets (supakorndej et al., ) . necropsy findings. cardiomegaly, pleural and/or abdominal fluid, and pulmonary congestion are common findings at necropsy. grossly, adult worms have been observed in the right atrium, right ventricle, pulmonary artery, and cranial and caudal vena cava. microscopically, microfilaria may be seen in small and large vessels of the lung. differential diagnosis. differential diagnosis should include primary cardiac diseases, such as dilatative cardiomyopathy, and other systemic or pulmonary diseases. control is best directed at prevention through the administration of heartworm preventative and it is recommended that ferrets in heartworm-endemic areas receive monthly oral ivermectin throughout the year (stamoulis et al., ; fox, g) . the dosage recommended for ferrets by the american heartworm society is . mg/kg body weight monthly (fox, g) . housing ferrets indoors, particularly during the mosquito season, would help minimize exposure. successful adulticide treatment in ferrets has been described and includes the administration of thiacetarsemide, with the same precautions used in dogs: antithrombotic therapy, treatment for heart failure, and strict cage confinement (stamoulis et al., ) . one should follow up with heartworm antigen tests until negative and resume heartworm prevention month after adulticide treatment (stamoulis et al., ) . ferrets are also susceptible to infection with the following nematodes: toxascaris leonina; toxocara cati; ancylostoma spp.; dipylidium caninum; mesocestoides spp.; atriotaenia procyonis; trichinella spiralis; filaroides martis; and spiroptera nasicola (rosenthal, ; fox, g) . a day of their due date should include cesarean section and intensive postoperative support, including force-feeding a gruel of high-quality cat food and ferret chow, nutritive pastes, intravenous fluids containing glucose, and supplemental heat. cesarean section should be performed under isoflurane anesthesia because hepatic dysfunction prolongs the metabolism of injectable agents. agalactia is common after cesarean section, and kits may require hand feeding with kitten or puppy milk replacers, administered per os by fine-tipped syringe times daily for the first hr. cross fostering is an effective method of enhancing kit survival; hand rearing of kits if the jill fails to nurse within a day postoperatively is energy-consuming and generally unrewarding. for jills that develop pregnancy toxemia before day of gestation, fluids and nutritional support must be provided until viable kits can be delivered by cesarean. pregnancy toxemia may be avoided by close monitoring of appetite of jills in late gestation, provision of a highly palatable diet with > % fat and > % crude protein, and avoidance of stress and dietary change. water should be made available in both bowls and water bottles, and food should be provided ad libitum in several bowls. pregnancy toxemia in the ferret occurs predominantly in primiparous jills carrying large litters. an inadvertent fast in late gestation is sometimes implicated. at least % of jills carrying more than kits will develop pregnancy toxemia if subjected to hr of food withdrawal in late gestation (bell, a; batchelder et al., ) . any jill with or more kits may develop pregnancy toxemia because abdominal space is not adequate for both the gravid uterus and the volume of food required to support it. pregnancy toxemia of the ferret is of the metabolic type and shares features with similar conditions in pregnant sheep, obese cattle, pregnant camelids, obese guinea pigs, and starved pregnant rats, as well as with the condition feline idiopathic hepatic lipidosis. it is characterized by abnormal energy metabolism with consequent hyperlipidemia, hypoglycemia, ketosis, and hepatic lipidosis. in this condition, energy demand exceeds intake, leading to excessive mobilization of free fatty acids and a chain of metabolic events that culminates in a shift from fatty acid metabolism and export to ketosis and hepatic lipidosis. clinical signs include anorexia, lethargy, melena, dehydration, and easily epilated hair. differentials include dystocia, metritis, pyometra, septicemia, renal failure, and helicobacter mustelae-induced gastric ulcer. in a recent study of ferrets with pregnancy toxemia, consistent clinical chemistry abnormalities included azotemia ( %), hypocalcemia ( %), hypoproteinemia ( %), and elevated liver enzymes ( %) (batchelder et al., ) . anemia was found in % of ferrets tested. necropsy findings include tan or yellow discolored liver, gastric hemorrhage, and gravid uterus. treatment forjills within ferrets are induced ovulators and may remain in persistent estrus if they are not bred or if estrus is not terminated chemically or via ovariohysterectomy (bell, a) . jills that remain in estrus for more than month are at risk for developing estrogeninduced anemia. hyperestrogenism from persistent estrus causes bone marrow hypoplasia of all cell lines in approximately half of ferrets in prolonged estrus (ryland et al., ) . clinical signs include vulvar enlargement, bilaterally symmetric alopecia of the tail and abdomen, weakness, anorexia, depression, lethargy, weight loss, bacterial infection, and mucopurulent vaginal discharge. hematology findings may vary from an initial neutrophilia and thrombocytosis early in the disease course to lymphopenia, thrombocytopenia, neutropenia, and anemia. the anemia begins as normocytic normochromic but progresses to macrocytic hypochromic (sherrill and gorham, ) . coagulopathy associated with hepatic dysfunction and thrombocytopenia combine to produce extensive manifestations of bleeding, pallor, melena, petechiation or ecchymosis, subdural hematoma, and hematomyelia (hart, ; fox and bell, ) . at necropsy, tissue pallor, light tan to pale pink bone marrow, hemorrhage, bronchopneumonia, hydrometra, pyometra, and mucopurulent vaginitis may be seen. histopathology may reveal cystic endometrial hypoplasia, hemosiderosis, diminished splenic extramedullary hematopoiesis, and mild to moderate hepatic lipidosis (sherrill and gorham, ; bell, a) . treatment consists of terminating estrus while supporting the animal with antibiotics, blood transfusion, b vitamins, and nutritional supplementation. estrus may be terminated by injection with - iu of human chorionic gonadotropin (hcg) or ~tg of gonadotropin-releasing hormone (gnrh), repeated week after initial injection if required. ovariohysterectomy may be considered for ferrets that are stable and have adequate numbers of platelets and red cells. ferrets with a packed cell volume (pcv) of % or greater have a good prognosis and require only termination of estrus for resolution of aplastic anemia. jills with a pcv of - % may require blood transfusions and have a guarded prognosis. ferrets with a pcv of less than % have a poor prognosis and require aggressive therapy with multiple transfusions. the lack of identifiable blood groups in ferrets makes multiple transfusions uncomplicated by potential transfusion reactions . estrogen-induced anemia may be avoided by ovariohysterectomy. of nonbreeding females, use of vasectomized hobs, or pharmacologic termination of estrus initiated days after estrus onset. a -to -day pseudopregnancy then follows, except in the case of ovariohysterectomy. repeated administration of hcg may result in sensitization and anaphylaxis. after several administrations, hcg is unlikely to be effective in termination of estrus. anaphylaxis is manifest as incoordination, tremor, vomiting, and diarrhea and may be reversed by prompt administration of diphenhydramine. arginine-free diets are unlikely to be fed in the laboratory setting, but administration of such a diet to young ferrets fasted for hr leads to hyperammonemia and encephalopathy within - hr (thomas and desmukh, ) . exacerbation of signs may be achieved by challenging young ferrets with influenza virus and aspirin (desmukh et al., ) and constitutes a model of reye's syndrome in children. lethargy and aggressiveness yield to prostration, coma, and death in affected ferrets. hyperammonemia presumably occurs because of the inability of ferrets to produce adequate amounts of ornithine from non-arginine precursors. detoxification of ammonia is thereby compromised. ferrets more than months old are unaffected by arginine-free diets. ferrets of all ages are susceptible to zinc toxicosis, and the condition has been documented in two ferret farms in new zealand (straube and walden, ) . leaching of zinc from steam-sterilized galvanized food and water bowls was implicated. clinical signs included pallor, posterior weakness, and lethargy. definitive diagnosis requires demonstration of elevated concentrations of zinc in kidney and liver. at necropsy, kidneys are enlarged, pale, and soft; livers are orange, and gastric hemorrhage may be seen. histopathology reveals glomerular collapse, tubular dilation, tubular proteinaceous debris, focal cortical fibrosis, hepatic periacinar infiltration, and depression of the erythroid series. avoidance of galvanized materials precludes the development of zinc toxicosis. umbilical entanglement may occur in ferrets on the day of parturition and has been associated with fine-particle bedding, large litters, and short kit-birth intervals (bell, a; fox et al., a) . jills may neglect to clean placentas from their kits, or kits may be born so rapidly that there is not adequate time for the jill to clean the kits of placental membranes, thereby predisposing to entanglement. entangled kits may succumb to dehydration, hypothermia, and hypoglycemia because they are unable to nurse and the jill cannot curl around them. detailed dissection with fine scissors and forceps under a heat lamp or on a heated surface can free the kits. occasionally, kits may need to be rotated on their umbilical pedicle to achieve adequate clearance to cut the cord; cords should be cut as far from the umbilicus as possible. the use of warm saline or water may help soften the mass. some kits in the tangle may present with dark, swollen extremities or prolapsed umbilical cords and may require euthanasia. parturition should be supervised, if possible, to avoid umbilical entanglement. hydronephrosis may occasionally occur in the ferret and is most commonly associated with inadvertent ligation of the ureter during ovariohysterectomy. ovarian remnants are another potential sequela to ovariohysterectomy. ovarian remnants in ferrets may be associated with estrus, vulvar enlargement, and alopecia. appropriate diagnostic procedures include ultrasonography and plain and contrast radiography for hydronephrosis and ultrasonography and serum hormone concentrations for ovarian remnants. exploratory celiotomy confirms the diagnosis, and unilateral nephrectomy or ovariectomy is indicated if the remaining kidney is normal and the ferret is otherwise healthy. over the last few decades, increasing numbers of ferrets have been used in research or kept as pets, and as these animals have received veterinary care, it has become evident that ferrets are subject to a wide variety of neoplastic conditions . however, four categories of cancer account for the majority of ferret neoplasms: pancreatic islet cell tumors, adrenocortical cell tumors, lymphoma, and skin cancers. functional pancreatic islet cell tumors (insulinomas) are the most common neoplasm diagnosed in ferrets . disease may be evident in ferrets as young as years old, but later onset (at - years of age) is typical (caplan et al., ; ehrhart et al., ) . nonspecific presenting signs include weight loss, vomiting, and ataxia. weakness is often evident, ranging from lethargy to posterior paresis or outright collapse (caplan et al., ) . hypoglycemia caused by excess production of insulin by neoplastic cells may cause tremors, disorientation, or seizures (fox and marini, ) . excessive salivation (ptyalism) or pawing at the mouth is a frequent finding. clinical signs are often intermittent or episodic. other common findings include splenomegaly and lymphocytosis. presumptive diagnosis is made based on clinical signs in conjunction with the demonstration of hypoglycemia. blood glucose determinations for the diagnosis of insulinoma are most useful when taken after a hr fasting period. fasting glucose concentrations below may be diagnostic for the condition (quesenberry and rosenthal, ) , whereas values between and are suspect and the test should be repeated (fox and marini, ) . other potential causes for hypoglycemia should be ruled out, including anorexia, starvation, hepatic disease, sepsis, and nonpancreatic neoplasia (antinoff, ) . demonstration of concurrent hyperinsulinemia aids the diagnosis (caplan et al., ) . medical management using prednisone and/or diazoxide along with dietary modification such as frequent feeding of high-protein meals can minimize or control clinical signs but will not affect the underlying tumor (quesenberry and rosenthai, ) . surgical exploration of the pancreas and tumor excision are recommended for animals that are healthy enough to be subjected to anesthesia and surgery. histological examination of the tissue removed can provide a definitive diagnosis, and although the effect may be transient, clinical signs are often reduced or eliminated after surgical debulking (figs. and ) (ehrhart et al., ) . histologically, these tumors reveal ma-lignant proliferation of pancreatic cells, and local recurrence or metastasis to lymph nodes, mesentery, spleen, or liver may occur (caplan et al., ) . adrenocortical cell tumor is the second most common type of neoplasia in ferrets and is generally diagnosed between and years of age. if clinical signs are present, they often include weight loss and a bilateral, symmetric alopecia. pruritus is a variable finding (quesenberry and rosenthal, ) . although ferrets with this syndrome have been called "cushingoid," it is rare to diagnose elevated resting levels of glucocorticoids or an abnormal response to adrenocorticotropic hormone (acth) stimulation or dexamethasone suppression testing. elevation of adrenal sex hormones (e.g., androstenedione, -hydroxyprogesterone, and/or estradiol) is more likely, and these may lead to characteristic changes such as estruslike vulvar swelling in spayed females and prostatic changes in males coleman et al., ) . rule-outs for enlarged vulva include estrus in an intact female or functional ovarian remnants in a spayed female. abdominal palpation may reveal cranial abdominal masses, and ultrasound may be useful (barthez et al., ) . serum assay for abnormal levels of the sex hormones listed above should be considered (lipman et al., ; wagner and dorn, ; rosenthal and peterson, ) . in many cases the alopecia begins as a seasonally intermittent partial hair loss that becomes more severe as time goes on (fig. ) . even severe manifestations of this endocrine alopecia can spontaneously reverse in the absence of specific therapy, as demonstrated in a group of ferrets referred to our facility for diagnostic workup. in each of these ferrets, near total alopecia resolved within a few months of being housed in a research environment. despite being asymptomatic at the end of the study, all were shown to have histologic evidence of adrenocortical neoplasia. although this phenomenon is mediated by hormonal effects, anecdotal reports such as this suggest that the alopecia may be significantly modulated by environmental factors (e.g., photoperiod or diet). surgical exploration and removal of enlarged adrenals are commonly performed to establish the diagnosis and to remove hyperfunctional tissue. unilateral adrenalectomy early in the disease may be curative, but because bilateral neoplastic involvement is not uncommon, full or partial removal of both glands may be required. adrenolytic agents such as mitotane have been used with limited success (quesenberry and rosenthai, ) . histologically, adrenocortical adenomas are generally cm or less in diameter and are composed of well-differentiated cells with a granular or vacuolated cytoplasm. adrenal cell carcinomas are less commonly found and are larger, with a more pleomorphic and invasive character . metastasis to nearby tissues can occur. in our experience, adrenal cortical hyperplasia with or without neoplasia is an extremely common finding in aging ferrets, even in those not showing clinical signs. in one retrospective survey of our necropsy records it was found that more than % of ferrets greater than years of age had hyperplastic or neoplastic adrenal changes when examined (data not shown). for this reason, careful considerations of other possible disease processes should be made before attributing clinical signs solely to adrenal enlargement. lymphoma can affect ferrets of almost any age. ferrets younger than years of age often present with mediastinal lymphoma and/or leukemia, whereas those older than years of age often develop multicentric solid tumors . the early age of onset in some ferrets and reports of case clustering have led to investigation into potential infectious etiologies for lymphoma in the ferret (erdman et al., b) . earlier reports of feline leukemia virus (felv) seroconversion in affected animals have not been substantiated. however, experimental and epidemiological evidence suggests that a retrovirus that is distinct from felv may be involved . in one study, whole or filtered lymphoma cells from a -year-old ferret with spontaneous lymphoma were injected ip into recipient ferrets . two of the ferrets were euthanized after months, but the remaining developed splenomegaly, lymphocytosis, and lymphoma. one ferret that received cell-free materials developed multicentric lymphoma with prominent cutaneous lymphoma nodules. elevated reverse transcriptase activity and retrovirus-like particles evident by electron microscopy were seen in the donor and all of the affected recipient ferrets. other potential etiologies that have been considered include two infectious agents that are known to cause chronic immune stimulation in affected ferrets, the aleutian disease virus (adv) and helicobacter mustelae. a link with adv has not been proven, but h. mustelae seems to be responsible for the development of a very specific type of gastric b-cell lymphoma (erdman et al., ) . affected ferrets may exhibit localizing signs (e.g., dyspnea in a ferret with mediastinal involvement or peripheral lymphadenopathy in an animal with a multicentric distribution) but as is the case in many species, lymphoma is a "masquerader," and affected ferrets often present with chronic, nonspecific signs. weight loss, anorexia, and lethargy are often reported. splenic and/or hepatic enlargement may be evident. cutaneous involvement has been documented (li et al., ; rosenbaum et al., ) . although hematological examination typically reveals anemia and lymphopenia, lymphocytosis may be found, especially in younger ferrets. atypical lymphocytes are identified in the circulation in some cases. antemortem definitive diagnosis of lymphoma can be made by cytological examination of specimens obtained via fine-needle aspiration or excisional biopsy. tan-colored masses involving lymph nodes, spleen, liver, or other organs are commonly found at necropsy (fig. ) . diffuse involvement may lead to uniform enlargement of these organs or to a thickening of the wall of the stomach or intestines. as in other species, histological evaluation reveals neoplastic lymphocytes in affected tissues, generally evident as a monomorphic population (fig. ) . although surgery and radiation therapy may be useful in certain cases, most attempts to treat ferret lymphoma have utilized chemotherapeutic regimens with dosages extrapolated from other domestic animals or humans. treatment generally results in a remission that may last from months to years (brown, b; erdman et al., ) . mast cell tumors are among the most commonly reported integumentary tumors in ferrets (parker and picut, ; . cutaneous mastocytomas may occur anywhere on the body and present as firm, nodular skin lesions - mm in size that are often associated with alopecia or crusty ulceration of the overlying skin. pruritis is common (stauber et al., ) . histologically, they are composed of well-differentiated mast cells with metachromatic cytoplasmic granules that may be difficult to detect in sections stained with hematoxylin-eosin, but are more evident in toluidine bluestained sections. a variety of tumors of epithelial origin occur in ferrets, and they can appear at any site on the body. the most common are the basal cell tumors, which present as firm plaques or pedunculated nodules that are white or pink (parker and picut, ) . they may grow rapidly and become ulcerated. the percentage of basiloid cells present in these tumors, and the degree of associated squamous or sebaceous differentiation can vary, resulting in a spectrum of tumor subtypes and associated histological diagnoses (orcutt, ) . however, as is the case with mastocytomas, most are benign and will not recur after excision. resected tumors should be examined histologically to rule out less common tumors that might have a more guarded prognosis, such as squamous cell carcinoma or apocrine gland adenocarcinoma. chordomas are not epithelial tumors, but they often present as readily evident firm masses on the tail that may cause ulceration of the overlying skin. these neoplasms arise along the axial skeleton from notochord remnants and are typically slow-growing (dunn et al., ) . tumors involving the tail generally do not recur after amputation of the affected region, but a wide surgical margin should be maintained by removing several vertebrae proximal to the tumor. the prognosis is guarded for those rare chordomas that arise in the cervical region, and metastasis has been documented (williams et al., ) . congenital defects identified in ferrets include a variety of neural tube defects, gastroschisis, cleft palate, amelia, corneal dermoids, cataracts, and supernumerary incisors (willis and barrow, ; ryland and gorham, ; mclain et al., ; besch-williford, ) . cystic or polycystic kidneys have been observed (andrews et al., a; dillberger, ) . cystic genitourinary anomalies associated with the prostate, bladder, and/or proximal urethra most likely develop secondary to aberrant hormone secretion by adrenocortical tumors coleman et al., ) . newborn ferrets are normally born with a closed orbital fissure and are prone to developing subpalpebral conjunctival abscesses. treatment involves surgically opening the lids (a minor procedure) to establish drainage and to allow topical antibiotics to be administered (bell, a) . cardiomyopathy is a common cause of disease in aging ferrets. the dilatative form of disease is most commonly diagnosed. affected animals commonly present with lethargy, weight loss, and anorexia. physical examination may reveal signs of congestive heart failure such as hypothermia, tachycardia, cyanosis, jugular distension, and respiratory distress (lipman et al., ) . auscultation may reveal a heart murmur and/or muffled cardiac sounds. hepatomegaly and splenomegaly are often identified. radiographs may reveal an enlarged cardiac silhouette and evidence of pulmonary edema or pleural effusion (greenlee and stephens, ) . electrocardiography and echocardiography can help make the definitive diagnosis. medical therapy (supportive care, diuretics, and inotropic drugs) may relieve clinical signs and improve the qual-ity of life for a period of months (stamoulis et al., ) . the long-term prognosis for survival is guarded to poor. splenomegaly is a common finding in ferrets. in many cases the enlarged spleen appears to be a secondary manifestation of another disease (e.g., insulinoma, cardiomyopathy, or adrenal tumor) and is of unknown significance (stamoulis et al., ) . histologic examination of affected organs has revealed that the most common cause for splenic enlargement (in the absence of a neoplastic infiltrate) is extramedullary hematopoiesis (emh) (erdman et al., ) . this may be an incidental finding, but it has been suggested that in some cases a pathologically enlarged spleen may play a role in chronic anemia that may respond to splenectomy, a syndrome known as hypersplenism (ferguson, ) . splenomegaly can also be commonly found in conjunction with lymphoma, with or without intrasplenic neoplastic lymphoid accumulations. in anesthetized ferrets, splenomegaly may be caused by splenic sequestration of erythrocytes (marini et al., (marini et al., , . because this is a transient effect, the normalization of splenic size upon recovery from anesthesia can help in the differentiation of anesthetic-induced splenomegaly from that due to other causes. eosinophilic gastroenteritis is an idiopathic disorder characterized by peripheral eosinophilia ( - % of circulating leukocytes), hypoalbuminemia, and diffuse infiltration of the gastrointestinal tract with eosinophils (fox et al., a) . presenting signs for this syndrome generally include chronic weight loss, anorexia, diarrhea, and occasionally vomiting. eosinophilic granulomas have been found in the mesenteric lymph nodes of most affected ferrets, and in some cases other organs (e.g., lung or liver) may be involved. an interesting finding in many ferrets is the presence of splendore-hoeppli material in the inflamed lymph nodes, a histological phenomenon that has been associated in other species with helminths, bacteria, fungi, and foreign bodies (fig. ). an etiological agent has not been identified; consequently, therapy consists largely of supportive care to treat the chronic enteritis (fox, b) . based on the biology of eosinophils, however, the use of corticosteroids or ivermectin has been attempted and may be beneficial (bell, b) . megaesophagus has been diagnosed in ferrets presenting with a variety of signs, including weight loss, anorexia, difficulty in eating, or repeated regurgitation. the cause is generally unknown, and the prognosis is poor, despite efforts at supportive care 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alopecic ferrets with adrenal gland tumors the ferret intestinal uptake and lymphatic absorption of [ -carotene in ferrets: a model for human [ -carotene metabolism intestinal perfusion of [ -carotene in the ferret raised retinoic acid level in portal blood vitamin e enhances the lymphatic transport of [ -carotene and its conversion to vitamin a in the ferret aleutian disease in domestic ferrets: diagnostic findings and survey results semen characteristics and testosterone profiles in ferrets kept in a long-day photoperiod, and the influence of hcg timing and sperm dilution medium on pregnancy rate after laporoscopic insemination biliary coccidiosis in a ferret (mustela putoriusfuro) cervical chordoma in two ferrets (mustela putorius furo) coronavirus-associated epizootic catarrhal enteritis in ferrets comparative vaginal cytology of the estrous cycle of black-footed ferrets (mustela nigripes) the ferret (mustela putorius furo) as a laboratory animal effect of helicobacter mustelae infection on epithelial cell proliferation in ferret gastric tissues key: cord- -hdmwv authors: nan title: gastrointestinal disease date: - - journal: equine neonatal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: hdmwv nan straining to defecate. ' rockn'roll at the time of presentation to the veterinary teaching hospital at approximately hours of age. the foal is exhibiting signs of straining to defecate: raised tail head, slightly arched back, hindlimbs slightly under the trunk. note the meconium staining on the tail and perineum. the white tape on the foal's back was used as a landmark to serially measure the circumference of the foal's abdomen. compare this foal's stance to the stance in united states in and , the incidence of colic in foals less than six months of age was approximately times less than the incidence in mature horses. however, when considering aliments affecting foals, gastrointestinal tract problems and infection were most commonly reported. , because some etiologies of colic are unique in the neonatal period, special consideration must be given to the evaluation of abdominal pain in this age group. in some respects, the diagnostic approach to colic in neonatal foals is similar to that used in mature horses: rarely will any single fact be useful in determining the exact etiology. however, careful and simultaneous inspection of multiple historical, physical, and diagnostic findings may be formative in determining the anatomical location of the lesion (stomach, small intestine, large intestine, peritoneal cavity), the etiologic category (congenital, nonstrangulating obstruction, strangulating obstruction, inflammatory, or other), or even possibly the specific diagnosis (table - ). in obtaining a history, particular attention should be given to the farm history, use of medications (especially analgesics), risk factors for septicemia or failure of passive transfer, and problems with other foals on the farm. as some differentials are strictly age-dependent (see table - ), knowing the age of onset of clinical signs is important. in the neonatal period, commonly reported causes of abdominal pain are meconium impaction, small-intestinal volvulus, enteritis or colitis, uroperitoneum, intussusception, gastric ulcers, and ileus secondary to prematurity, septicemia, or neonatal encephalopathy. [ ] [ ] [ ] [ ] [ ] clinical signs of lethal white syndrome, meconium impaction, and uroperitoneum most commonly manifest in the first to hours, to hours, and to hours of life, respectively. however, if uroperitoneum is the result of urachal or urinary bladder infection, clinical signs may be delayed until to days of age. although the potential spaces created by congenital or traumatic umbilical, inguinal, and diaphragmatic hernias may be present since birth, incarceration of bowel into these spaces may occur at any age, if at all. enteritis, colitis, intussusception of small intestine, small-intestinal volvulus, and clinically significant gastric ulcers may develop at any age. the breed and sex of the foal may provide supportive evidence for certain differential diagnoses. lethal white syndrome is most commonly reported in all-white to almost-all-white offspring of overo cross overo paint horses. in one study of horses with congenital umbilical hernias, the incidence was two times greater in fillies compared to colts, and thoroughbreds were twice as likely to have an umbilical hernia compared to standardbreds. in this later study, incarceration of bowel into the umbilical hernia was not reported. scrotal hernias most commonly occur in standardbred and tennessee walking horse colts, and fecaliths are frequently reported in american miniature foals. , in one study, meconium impactions occurred twice as often in colts compared to fillies. although the incidence of uroperitoneum is often quoted to occur more commonly in colts, in one recent study, the incidence was approximately equal among the sexes. additional historic information that should be carefully scrutinized is the general health of the dam, the foal's gestational age, the foaling history and general perinatal health, such as ingestion of colostrum, age at passage of meconium, nursing frequency (see chapter for review of normal perinatal health), and farm history of potentially infectious causes of enteritis or colitis (salmonella, rotavirus, clostridium). most foals will have passed meconium within to hours of life; however, the gastrocolonic reflex stimulated by ingestion of colostrum frequently initiates earlier passage of meconium. evacuation of meconium may be delayed (meconium retention) as the result of ileus secondary to another primary nongastrointestinal disease, such as septicemia or neonatal encephalopathy. in these cases, although passage of meconium may be slower than expected, the delay in passage may not be accompanied by clinical signs of abdominal pain. passage of "milk feces" or yellow pasty feces does not necessarily indicate that all meconium has been removed from the colon. clinical signs of abdominal pain in foals can be highly variable, and the intensity of the signs is not necessarily indicative of the etiology. it is important to note that foals with inflammatory lesions of the intestinal tract or those suffering from general functional ileus secondary to systemic disease can act as violently painful as foals with obstructive or strangulating lesions of bowel. however, in general, foals suffering from uroperitoneum and gastric ulcers have less intense abdominal pain than do foals with inflammatory or obstructive lesions. repeatedly thrashing or rolling from side to side is generally accepted as a highly representative clinical sign of abdominal pain. however, early signs of abdominal pain in neonatal foals may only manifest as reduced frequency of nursing and prolonged recumbency. other subtle but significant signs of abdominal pain in foals include general restlessness, especially in recumbency, and/or frequent adjustment of recumbent positions (figure - ). often recumbent foals with abdominal pain will stretch their limbs, twist their head or neck, roll into dorsal recumbency ( figure - ) , and make frequent attempts or strain to either defecate and/or urinate. in the standing foal, signs that are classically associated with straining to defecate include frequent tail swishing, a "water spout" tail, and a "camped under" leg stance with a dorsiflexed back (figure - ) . in contrast, a flat or ventroflexed back with the hindlimbs stretched backward and the tail held up is associated with urination ( figure - ) . other signs of abdominal pain include lip curling, flank biting or watching, pawing at the ground, and kicking at the abdomen. it is important to recognize that often critically ill foals that are stuporus will not overtly demonstrate classic signs of the colicky neonatal foal should receive a thorough general physical examination with careful attention given to identification of clinical signs or physical evidence of sepsis (see chapter ) . sepsis may be directly associated with the primary etiology of the abdominal pain or it may develop secondary to bacterial translocation if the integrity of the gastrointestinal mucosa is compromised. the presence of fever, depression, petechiae, injection, synovitis, uveitis, or diarrhea may be cardinal clues that the abdominal pain has an inflammatory etiology. although the presence of diarrhea may be an important sign of enterocolitis, intense abdominal pain frequently precedes the onset of diarrhea. tachycardia and/or tachypnea are expected findings despite etiology. persistent tachycardia (heart rate > beats per minute) has been suggested to be more commonly associated with surgical disorders of the abdomen, compared to medical etiologies. gross abdominal distension will develop with gas or fluid accumulation in the bowel or abdomen. severe abdominal distension with intense pain that elicits high-pitched "pings" with percussion is consistent with gas-distended large intestine or cecum. repeated measurement of the abdominal circumference may be helpful in more objectively determining the course of progression of abdominal distension. typically, the absence of intestinal sounds is not helpful in determining etiology; however, increased borborygmi may be associated with enteritis or colitis. if diarrhea is present with signs of abdominal pain in a foal, additional fecal diagnostics may be indicated (see . unlike mature horses, transabdominal wall palpation can be performed in the neonate and may identify gastric distension, hepatomegaly, thickened or distended bowel, meconium, and the urinary bladder. the ventral abdomen and inguinal rings should be carefully examined for evidence of herniation. ballottement or succussion of the abdomen may verify a fluid wave compatible with excess fluid in the peritoneal cavity. with adequate restraint or sedation, a careful digital rectal examination may identify meconium or ingesta in the pelvic inlet. a nasogastric tube should be passed in all foals with clinical signs of abdominal pain. compared to mature horses, successful retrieval of gastrointestinal reflux can be frustrating in the neonate, even in the presence of proximal intestinal disease. frequently, nasogastric tubes that are designed for neonatal enteral feeding are of insufficient size for gastric decompression. with this in mind, the largest-bore tube that can be comfortably passed should be used, and persistent attempts should be made to obtain gastric reflux, either by suction or priming the tube with water. a complete blood count and serum biochemical profile were obtained from ' rockn'roll. the pcv was normal at %. the total white blood cell count was , /ml, characterized by a normal neutrophil count ( , (figure - changes in the leukogram or serum biochemical analysis may provide indirect etiologic evidence. neutropenia, neutrophilia, a significant left shift, toxic changes within the neutrophils, and hyperfibrinogenemia are indicative of an inflammatory or infectious etiology and if present in the equine neonate, aseptic collection of additional blood for culture is recommended. hypoglobulinemia is indicative of failure of passive transfer and should be verified by specific igg testing (see case - ). panhypoproteinemia is indicative of loss and may accompany severe inflammatory lesions of bowel. azotemia, hyponatremia, hypochloremia, and hyperkalemia may occur with enteritis, colitis, or uroperitoneum. marked acidosis most commonly occurs in foals with inflammatory lesions of the bowel. many clinicians are hesitant to perform an abdominocentesis on a neonatal foal as the risk for complications such as inadvertent enterocentesis or omental prolapse occur more commonly in foals undergoing an abdominocentesis, as compared to adult horses. many clinicians perform a transabdominal ultrasound examination first to assess potential risks of performing an abdominocentesis versus the likelihood of successfully obtaining peritoneal fluid. if an ultrasound examination reveals widespread and/or grossly distended loops of intestine, and/or free peritoneal fluid is difficult to identify, the chances of an uncomplicated and successful abdominocentesis are reduced. an abdominocentesis may be completed on the standing foal or with the foal in lateral recumbency. in either position, proper restraint is imperative and is best facilitated by light sedation (table - ). the procedure for performing an abdominocentesis in a foal is similar to the adult horse, using either a -to -gauge . -inch needle or a teat cannula to collect the peritoneal fluid. first, the hair from the ventral midline of the rostral abdomen should be clipped and the skin sterilely prepared. the subcutaneous tissue of the abdominocentesis site in the rostral midline of the abdomen should be infiltrated with . ml of % lidocaine. when a teat cannula is used, entry through the abdominal wall must be preceded by a stab incision made with a number blade through the skin and external portion of the linea alba. although use of an -gauge needle is expeditious, there may be a greater risk of inadvertent enterocentesis or bowel laceration; however, enterocentesis may also occur with a teat cannula if the intestine is grossly distended. localized or generalized peritonitis may develop subsequent to an enterocentesis, and prophylactic use of parenteral antimicrobials is recommended. fig. - ultrasonographic image obtained on ' rockn'roll at hours of age with acute abdominal pain. this image was obtained using a - mhz curvilinear probe set to a depth of cm. note the four "balls" of meconium of mixed echogenicity in small colon ( , , , ) . this image was obtained from the left caudoventral abdomen. more commonly, use of a teat cannula results in inadvertent prolapse of omentum through the abdominocentesis site upon collection of peritoneal fluid or removal of the cannula. if this happens, the omentum should be sharply transected with a sterile blade at the abdominal wall and tucked back into the abdominal cavity with another sterile teat cannula. normal peritoneal fluid should be light yellow in color and clear. the normal mean nucleated cell count in the peritoneal fluid of healthy foals was reported to be cells/μl (range to , cells/μl) for foals that were to days of age and , cell/μl (± ) for thoroughbred foals that were to days of age. both of these reference ranges for nucleated cell counts are lower than means reported for healthy adult horses. the mean protein concentration by biochemical biuret determination and refractive index in foals was reported to be . g/dl and . g/dl, respectively. the interpretation of peritoneal fluid nucleated cell count and protein concentration is similar to adult horses with abdominal pain and does not necessarily definitively distinguish etiology or medical from surgical causes of colic. sanguinous fluid can be present with either severe enterocolitis or strangulating lesions of bowel. special attention should be given to the cytologic examination. the presence of degenerative neutrophils or bacteria is worrisome (figure - ) and is indicative of loss of the mucosal integrity, bowel rupture, or primary sepsis in the peritoneal cavity. in one study, % of colicky foals with peritoneal fluid protein concentrations > . g/dl that subsequently underwent an exploratory laparotomy were euthanized. when uroperitoneum is suspected, the creatinine concentration of both peritoneal fluid and serum should be simultaneously assessed. a peritoneal fluid creatinine to serum creatinine ratio > is supportive evidence of uroperitoneum. when determining peritoneal fluid creatinine concentration on an automated chemistry analyzer, it is important to use plasma or serum methodology on the abdominal fluid. if urine methodology is used to determine the creatinine concentration on a peritoneal fluid sample, erroneously increased creatinine concentration may lead to an incorrect diagnosis of uroperitoneum. unlike mature horses, abdominal radiography can provide useful information in a colicky neonatal foal. grid, rare-earth screens and sufficient mas ( to ) and kvp ( to ) should be used. radiographs may be taken with the foal either standing or in lateral recumbency; however, keep in mind that fluid-gas interfaces are easier to detect in the standing foal, when the radiographic beam is horizontal or perpendicular to the dorsoventral plane of the gas/fluid interface in the abdomen. both right and left lateral views should be obtained as some structures will be more obvious, depending upon which side of the abdomen is closer to the film. ventrodorsal views may only be possible in small foals or those that are stuporus, sedated, or anesthetized. this view will optimize identification of the pylorus, the descending duodenum, the base of the cecum, the left colon, and the transverse colon. some degree of gas is normally visible in the stomach, small intestine, cecum, and small colon. in general, plain film abdominal radiology is more likely to provide information on the anatomic location of the problem than information directly leading to the exact etiology. , , for example, gas distension of the small intestine is a nonspecific finding that may occur with enteritis, functional obstruction (ileus), or mechanical obstruction (figure - ) . however, if small-intestinal distension is accompanied by "hairpin" or "u-shaped" turns of the bowel ( figure - ) or multiple, uneven intraluminal gas-fluid interfaces ( figure - ), it is more likely associated with mechanical obstruction, though these findings can also occur with enteritis or functional ileus. thickened walls of small intestine may appear uneven or "corrugated" when caused by enteritis (figure - ). severe generalized gas distension of large intestine is more commonly associated with mechanical obstruction than with inflammatory lesions of large colon ( figure - ) . meconium frequently appears as granular contents in the ascending or descending colon ( figure - ) . pneumoperitoneum suggestive of bowel this neonatal foal had radiographic signs of smallintestinal obstruction, as evidenced by isolated loops of gasdistended small intestine that form a u-turn or "hairpin" turn (arrows). an exploratory celiotomy confirmed a jejunal intussusception. fig. - radiographic appearance of small-intestinal disease. the presence of multiple gas-fluid interfaces at different levels is often considered radiographic evidence of smallintestinal obstruction; however, it can also be seen in foals with intense ileus from enteritis, as was the situation for the foal depicted in this figure. rupture should be suspected if there is a gas cap in the dorsal aspect of the abdominal cavity, if the serosal surfaces of bowel are enhanced, or if visualization of the renal silhouettes is improved. iatrogenic pneumoperitoneum should be considered if an abdominocentesis was performed prior to abdominal radiography. finally, abdominal radiography obtained on foals with necrotizing enterocolitis may demonstrate the pathognomonic sign of pneumatosis intestinalis, or intramural air. radiographic signs of pneumatosis intestinalis are spectacular and include localized cystic collections that appear as radiolucent "bubbles" in the bowel wall, or diffuse linear strips or flat oval-shaped areas of radiolucency in the bowel wall flanked by the radiopaque serosa and mucosa, when the bowel wall is viewed end-on. contrast radiography also has some specific indications in neonatal foals. an upper gastrointestinal contrast study can be used to document delayed gastric emptying of older foals with suspected pyloric outflow obstruction. foals less than two weeks of age ideally should be fasted for four hours, and foals consuming solid feeds should be fasted for hours prior to contrast radiography. barium is administered by gravity flow via a nasogastric tube ( ml/kg as a % weight/volume solution), and abdominal radiographs are obtained every minutes. if barium remains in the stomach for longer than two hours, delayed gastric emptying should be suspected but does not necessarily distinguish between mechanical or functional obstruction. barium should normally reach the cecum and transverse colon by two hours and three hours, respectively, in -to -day-old foals. , barium transit time to the transverse colon is five to eight hours in foals one to two months of age. in addition to documenting transit time, stenosis or abnormal bowel wall may be highlighted by the contrast (figure - ). lower-intestinal contrast studies (i.e., barium enema) have been reported to have % sensitivity and % specificity for identifying mechanical obstruction (meconium impaction, atresia coli) of the transverse colon or small colon in foals less than days of age ( figure - ) . the foal should be restrained or lightly foals with enteritis may have numerous nonspecific radiographic findings, such as gas-distended small intestine and multiple gas-fluid interfaces. in the radiograph depicted here, the finding of individual loops of mildly to moderately gas-distended small intestine with irregular or "corrugated" walls is highly indicative of enteritis. fig. - radiographic appearance of a large-or smallcolon obstruction. unlike small-intestinal gas distension, generalized gas distension of large colon is considered to be fairly specific for large-or small-colon obstruction. this foal had a distal meconium impaction. sedated and placed in lateral recumbency. a -french foley catheter is placed into the rectum and the bulb gradually inflated. by gravity flow, administer up to ml/kg of % weight/volume barium. two-dimensional transabdominal ultrasonography is a valuable diagnostic tool in the colicky neonatal foal. scanning techniques for the foal abdomen are extensively reviewed elsewhere, though a brief review follows here. standard linear array to mhz transducers are sufficient for visualization of most intra-abdominal structures in the neonatal foal; however a curvilinear transducer will optimize image quality. the foal may be scanned in either lateral recumbency or standing; however keep in mind that fluid-filled, thickened, or enlarged structures may descend to the dependent portion of the abdomen and could be overlooked if only the nondependent side of a recumbent foal is scanned. furthermore, gas in nondependent bowel, especially gas in large colon, may preclude examination of deeper structures in laterally recumbent foals. visualization of intra-abdominal structures is facilitated by clipping the abdominal hair; however thorough wetting of the hair with water or alcohol, in addition to acoustic coupling gel, may be sufficient. in the colicky neonate, transabdominal ultrasonography can be used to identify fluiddistended structures (i.e. stomach, small intestine, large intestine, urinary bladder), gastric or intestinal wall thickness, abnormal intestinal contents (i.e. meconium, fecaliths, phytobezoars, or trichophytobezoars), peritoneal fluid, and to determine intestinal motility. normal gastric wall thickness in foals should be less than mm, whereas intestinal wall thickness is to mm. foals with gastritis or gastric ulcers may have a thickened or irregular gastric wall. likewise, foals with enteritis or colitis will frequently have diffusely increased small and/or large intestine wall thickness ( figure - ). this feature alone does not definitively identify enteritis, as edematous bowel that develops as the result of strangulation (volvulus or intussusception) will also appear regionally thickened ( figure - ), with distended fluid-filled intestine proximally. the presence of gas echoes in the intestinal wall, a thickened hypoechoic wall that appears "wavy," or the presence of sloughed mucosa in the lumen are more consistent with a diagnosis of enteritis and should be administration of barium as an enema (see text) reportedly has % sensitivity and % specificity for identification of small or large-colon obstructions. the abrupt end of contrast in the small colon in this foal was highly indicative of atresa coli (thin arrow), which was later confirmed at necropsy. also note the stenosis of the rectum above the pelvis (thick arrow). considered as distinguishing features from strangulating lesions (figure - ). foals with enteritis or colitis may demonstrate either hypermotile or hypomotile bowel, whereas functional ileus and mechanically obstructed intestine more often will appear hypomotile. neonatal foals with mechanical obstruction of small intestine may have hairpin or u-shaped turns of the small intestine when viewed in long axis ( figure - ). the unique regional presence of a double intestinal wall or what appears to look like a "bull's-eye" of multiple concentric rings in a short axis view of small intestine is consistent with an intussusception (figure - ) , which most commonly is seen in the dependent portion of the abdomen. meconium may appear hyperechoic, hypoechoic, or as a mixture of echogenicities in hypomotile intestine. fluid-or gas-distended intestine may be present proximal to the obstruction ( . when present in the small colon, retained or impacted meconium often appears as a row of "balls" and is most easily identified in the dependent portion of the left caudal abdomen in the standing foal. it may be traceable dorsal to the urinary bladder and often is surrounded by a thin layer of hyperechoic gas in sacculated intestine (small colon). meconium in large colon typically is more amorphous. extensive gas in the large colon frequently hinders visualization of other intra-abdominal structures and can be associated with mechanical obstruction, though it may also occur with acute colitis. the sonographic appearance of abdominal abscesses is variable, but in the neonatal foal, intra-abdominal abscesses are often associated with umbilical remnants, previously devitalized bowel, or mesenteric lymph nodes. finally, the presence of excessive anechoic fluid may be indicative of uroperitoneum (see chapter ), though anechoic transudate may also develop as a result of enteritis, colitis, or strangulation. hyperechoic densities within peritoneal fluid may represent fibrin strands, leukocytes, or adhesions ( figure - ) . the presence of gas echoes free within the peritoneal fluid is indicative of a ruptured viscus. gastroscopy may be accomplished with a -meter endoscope in a neonatal foal and is covered elsewhere in more detail later in this chapter (see . for most neonatal foals, suckling can be allowed prior to gastroscopy, but solid-feed consumption should be withheld for to hours. caution should be exercised in the interpretation of the clinical significance of gastric ulcers in foals, as up to % of young asymptomatic foals have gastric ulcers along the margo plicatus of the greater curvature of the stomach, and gastric ulcers may develop secondary to another primary gastrointestinal disorder. , however, multiple deeper ulcers, bleeding ulcers, ulcers along the lesser curvature or in the glandular mucosa, or those accompanied by clinical signs that are consistent with gastric ulceration should be considered clinically significant. most information on colic in the neonate is obtained from referral institutions or, more often, from foals undergoing exploratory celiotomy for acute abdominal pain; therefore the data may not precisely reflect the true incidence of each reported etiology. the only published study on foals less than two weeks of age with acute abdominal pain reported that an exploratory celiotomy revealed functional ileus ( %), meconium impaction ( %), large-colon displacement ( %), small intestine displaced around the base of the cecum ( %), ruptured gastric ulcer, and small colon obstructed by the ovarian ligament. other retrospective studies of abdominal surgery in the foal include foals three to six months of age and thus it was difficult to ascertain the etiology of abdominal pain exclusive to the neonatal period. in foals that underwent an exploratory celiotomy from birth to three months of age, meconium impaction, uroperitoneum, enteritis, small-intestinal strangulation (herniation with incarceration, small-intestinal volvulus, and intussusception), and enteritis accounted for % of the total cases. in a study that reviewed foals up to six months of age, the most commonly reported diseases identified at surgery were small-intestinal volvulus, meconium impaction, and intussusception. these reports underscore the difficulty in definitively identifying the cause of abdominal pain prior to exploratory celiotomy in neonatal foals, as clearly some of these cases, such as enteritis and functional ileus, would not be considered to be predominantly surgical diseases. furthermore, it is noteworthy that if the etiology of the abdominal pain cannot be ascertained in the violently painful patient, an exploratory celiotomy may be indicated solely as a diagnostic tool. the purpose of this section is to provide a brief review of the conditions most commonly reported in the neonatal foal. meconium is the sticky caramelized feces of the newborn foal that comprises intestinal secretions, swallowed amniotic fluid, and cellular debris. in one study of newborn foals, it was reported that the total weight of meconium is equal to % of the foal's body weight. most foals will start to evacuate meconium shortly after the first ingestion of colostrum, which acts both as a laxative and stimulator of the gastrocolonic reflex. the majority of the meconium is evacuated within the first hours of birth and is replaced by "milk" feces, which are pasty and yellow in appearance. however, concurrent disease such as neonatal asphyxia, prematurity, septicemia, and encephalopathy may delay passage. meconium impaction implies failure to evacuate sufficient quantities of meconium with subsequent development of signs of colonic obstruction: pain, straining to defecate, and abdominal distension secondary to accumulation of gas in bowel proximal to the impacted meconium. it has been suggested that meconium impaction is more likely to occur in colts and in foals greater than days of gestational age. to the author's knowledge, prophylactic use of an enema at birth has not been shown to prevent meconium retention or impaction. foals with meconium impaction are disinterested in nursing, strain to defecate, and typically stand with a slightly arched back and frequently "swish" their tail ( figure - ). digital examination may detect meconium within the rectum. colic, abdominal distension, tachypnea, general restlessness, and tachycardia are frequently reported clinical signs. intestinal borborygmi are usually present and are not a reliable sign of obstruction. meconium can usually be identified by either plain or contrast abdominal radiography or ultrasonography (see "diagnostic approach to colic in neonatal foals" section above). in more severe obstructions, excessive amounts of gas may accumulate in the large colon, proximal to the obstruction. rupture of the urinary bladder may occur in foals that strain excessively from meconium impaction. furthermore, extensive mural damage lends to bacterial translocation and secondary septicemia. other causes of acute abdominal pain that closely mimic meconium impaction and primarily manifest during the first to hours of life are the rarely reported cases of ileocolonic aganglionosis, atresia coli, and atresia recti. ileocolonic aganglionosis, or "lethal white" syndrome, is a fatal autosomal recessive disorder principally of overo cross overo paint horses that is caused by a point mutation in amino acid in endothelin receptor b. the endothelin receptor is critical for the proper development and migration of cells from the neural crest that ultimately form melanocytes in the skin as well as neurons in the intestinal tract. thus, foals that are homozygous for the mutation are essentially all white (though some small areas of pigmentation can occur) and develop signs of functional ileus in the first few hours of life ( figure - ). color patterns in the dam and sire that have the highest incidence of heterozygote carriers of the mutation are frame overo, highly white calico overo, and frame blend overo horses. however, the heterozygote mutation has been occasionally rarely detected in other white-patterned paints and "nonpaint" whitepatterned bloodlines. it has not been detected in solidcolored breeding-stock paint horses without white, but heterozygote adult solid-colored miniature horses of paint lineage and white-patterned horses of breeds other than the paint horse have been rarely identified. lethal white foals typically are born "normal" in appearance and behavior, with the exception of the mostly white coat. clinical signs of abdominal pain usually develop in the first few hours of life, after ingestion of colostrum, and progressively develop gross abdominal distension. evidence of passage of meconium is often missing, though some meconium may be passed. the only way to definitively identify a lethal white foal is by dna testing for the mutation or histopathologic demonstration of insufficient intestinal ganglia. unfortunately, it can take weeks to obtain the results of dna testing and hours to days to obtain the results of intestinal biopsies. diagnosis is often initially presumptive, based on signalment, heterozygote parents, lack of response to symptomatic treatment, and rule out of other causes by additional diagnostics or surgical exploratory. affected foals and heterozygote adults can be identified by submitting plucked hair with intact root bulbs from the mane (preferred sample) to the veterinary genetics laboratory at the university of california, davis, available at www.vgl.ucdavis.edu/service/horse/coatcolor.html. it should be noted that not all white foals of paint lineage are lethal whites and when tested, these unaffected, rare, all-white paint horses are not homozygous for the mutation. atresia coli, recti, or ani are rarely reported causes of colic in the newborn foal that may mimic meconium impaction, based on age of onset and signs of obstructive disease. it may be possible to identify atresia recti on visual or digital examination of the rectum or by protoscopy. retrograde contrast barium enema usually identifies an abrupt obstruction (see , but definitive identification may require an exploratory celiotomy. successful surgical repair has been described; however, other congenital abnormalities have been simultaneously reported and should be ruled out prior to consideration of surgery. the heritability of these defects is not fully known. fecaliths are hard concretions of ingesta ( figure - ) that may also contain undigested material, such as hair. they most commonly occur in the american miniature breed and have been reported in foals as young as days of age. , affected foals typically present with progressive unresponsive abdominal pain that is accompanied by gross abdominal distension. as fecaliths obstruct the small colon or rectal lumen, abdominal radiographs typically demonstrate gas distension of the large colon. successful treatment almost inevitably involves a celiotomy with a small colon enterotomy. strangulating lesions of the small intestine that require surgery occur more frequently in neonatal foals, as compared to strangulating lesions of the large intestine. volvulus of the small intestine is the most commonly reported strangulating lesion in foals less than three months of age. factors leading to development of these lesions are undetermined, but alterations in motility may contribute. intussusceptions are most commonly reported in three-to five-week-old foals and may be acute or chronic and intermittent. smallintestinal intussusceptions are most frequent, but ileocecal and cecocolic intussusceptions have been reported in young foals. most inguinal and umbilical hernias in foals resolve spontaneously without incarceration of intestine. , [ ] [ ] [ ] [ ] in a large retrospective study of foals with umbilical hernias, only required surgical repair. only of the foals that needed surgery had incarcerated bowel in the umbilical hernia. although the hernias were present since birth, and strangulation may rarely occur shortly after birth, the majority of foals with strangulating umbilical hernias were not neonates, but most often, affected foals were presented in the first six months of life. the most common presenting complaint in foals with an umbilical hernia with incarcerated bowel was an acute firm enlargement of the hernia that was sensitive upon palpation. only % of foals with strangulated umbilical hernias presented with signs of abdominal pain. in separate retrospective reports, inguinal hernias in neonatal foals that resulted in incarceration of small intestine that required surgical correction were exclusively found in newborn foals as a result of rents in the vaginal tunic (direct inguinal hernia). , onset of clinical signs was typically within hours of birth. diagnosis should be suspected by the presence of an irreducible mass in the scrotal sac, edema in the scrotum and prepuce, and colic. diagnosis of either an umbilical or scrotal hernia with incarceration can usually be confirmed by palpation and ultrasonography. it has been suggested that elective herniorrhagy should be considered if an umbilical hernia has been present in foals greater than six months of age or if the defect is larger than cm. diaphragmatic hernias are rare in foals and can result from failure of fusion of its embryonic components or from traumatic rupture of the diaphragm in utero, at birth, or after birth. affected foals can remain asymptomatic for prolonged periods of time, but the presenting complaint typically is acute abdominal pain. the intensity and onset of clinical signs is related to the amount of intestine that has herniated into the thoracic cavity and whether the herniated bowel is simply displaced or strangulated. in addition to abdominal pain, tachypnea and/or dyspnea may be present. the diagnosis can be confirmed by either radiographic or ultrasonographic evidence of an incongruous diaphragmatic line, the presence of gas-fluid interfaces indicative of intestine in the thoracic cavity, and/or pleural effusion. enterocolitis, gastric ulceration, and uroperitoneum are commonly reported causes of abdominal pain in the neonate and are described separately later in this chapter and in chapter . functional ileus secondary to prematurity, sepsis, neonatal asphyxia, neonatal encephalopathy, electrolyte abnormalities, concurrent gastrointestinal disease, hypoxic or ischemia bowel, overfeeding, use of milk replacer, botulism, and many other concurrent diseases may induce significant abdominal pain in the neonatal foal. if these underlying conditions are present, careful consideration to additional diagnostics should be used to rule out more serious gastrointestinal disorders (see "diagnostic approach to colic in neonatal foals" section above). simple solutions, such as reducing or eliminating enteral feeding, changing the source of milk, or correction of electrolyte derangements and other underlying disorders should be tried first. however, it may be necessary to consider an exploratory celiotomy for definitive diagnosis and for therapeutic decompression of bowel before initiation of prokinetic therapy. cecal impaction, large-colon volvulus, large-colon displacement, intestinal infarction, and ileal impaction are considered to be rare disorders of the neonatal foal. medical therapy for meconium impaction includes judicious use of analgesics, intravenous polyionic isotonic fluids, oral laxative therapy, and enemas. foals with meconium impactions are expected to exhibit some degree of pain. judicious use of analgesics (table - ) is required to balance the necessity to provide relief from pain and the ability to appropriately assess the patient's progress. mineral oil ( to ounces administered via a nasogastric tube) is used for its lubricating effect. milk of magnesia ( to ounces) provides an osmotic laxative effect, but should be used sparingly as it may be dehydrating. the detergent dioctyl sodium sulfosuccinate can be quite irritating, and it should be avoided in both oral and rectal therapy. castor oil therapy has been described, but can provoke violent abdominal pain in the foal. enemas are a mainstay of treatment for small-colon meconium impactions. warm-water liquid detergent (i.e., palmolive ® ) enemas ( / teaspoon liquid detergent to ml water) are purportedly gentle to the rectal mucosa and effective. commercial phosphate enemas (i.e., fleet ® ) can also be used, but repeated administration may increase the risk of phosphate toxicity. recently, acetylcysteine retention enemas have been reported to be a highly successful treatment for meconium impactions in foals. it is hypothesized that the acetylcysteine cleaves disulphide bonds in the mucoprotein molecules in meconium, decreasing its overall tenacity. a % acetylcysteine solution, ph . , is made by adding g of baking soda and g of acetylcysteine to ml of water. a -french foley catheter with a ml bulb is inserted approximately . to cm into the rectum and, the bulb is slowly inflated to occlude the rectum. one hundred to ml of the % acetylcysteine solution is administered by gravity flow and retained for to minutes. the acetylcysteine retention enema was effective in eliminating % of meconium impactions within hours. if needed, the acetylcysteine therapy can be repeated in hours, and was repeated up to three times in some cases before resolution of the impaction. occasionally, repeated use of an enema generates significant rectal and small colon mucosal irritation that sustains signs of straining despite effective removal of the impaction. this continued straining may confound determination of successful treatment. surgical intervention should be considered if medical therapy is unsuccessful. , although there often is no single criterion that distinguishes the course of treatment in any particular case, an exploratory celiotomy should be considered in a foal with abdominal pain (of any etiology) if there is: the prognosis for foals with a meconium impaction is generally considered good to excellent with short-term survival reported to be % and long-term survival, following either medical or surgical treatment, reported to be % to %. , most meconium impactions will resolve with medical intervention. at the university of california, davis, from to , out of foals ( %) with meconium impactions were successfully treated medically with acetylcysteine enemas. about % of these former cases required more than one acetylcysteine enema, and in about % of cases it took more than hours for the impaction to resolve. once hospitalized, ' rockn'roll received one acetylcysteine enema and oral laxative and intravenous fluid therapy over hours. prior to surgery, the foal exhibited only mild to moderate signs of intermittent pain (straining to defecate with recumbency and rare dorsal recumbency) and his abdominal size did not significantly increase. the discovery of pneumoperitoneum before celiotomy was worrisome. however, because an abdominocentesis had been performed several hours prior to taking the abdominal radiograph, it was speculated that the pneumoperitoneum was iatrogenic from introduction of room air through the abdominocentesis needle into the peritoneal cavity. the subsequent detection of a ruptured transverse colon during surgery was an unexpected complication. spontaneous rupture of the colon as a sequela to meconium impaction must indeed be rare, as there are no reports of its occurrence in the american literature. it remains unclear as to when the colon rupture occurred. at presentation, the foal did not exhibit clinical signs of sepsis, though a small but significant left shift was present on the leukogram. this may have been a result of early sepsis from an unrelated cause or may have been the result of bacterial translocation across compromised mucosa along the colonic impaction. the foal did not exhibit intense signs of abdominal pain, in fact, he only showed signs of straining to defecate during the first hours of hospitalization. although he received an analgesic dose of flunixin meglumine prior to referral, he only received one other shortacting analgesic prior to surgery: mg of butorphanol. the abdominocentesis was performed at approximately hours of hospitalization, after the foal was observed to roll into dorsal recumbency. although the nucleated cell count and protein concentration were both increased, there were no signs of sepsis cytologically. either rupture had not yet occurred or it had already occurred and the peritoneal fluid sample obtained by abdominocentesis was not representative. in retrospect, the pneumoperitoneum seen radiographically at hours of hospitalization most likely was indicative of a localized rupture. the decision to perform an exploratory celiotomy was based upon the lack of passage of a significant amount of meconium, the presence of extensive amounts of meconium in the colon (as seen ultrasonographically and radiographically), the presence of free air in the peritoneal cavity on abdominal radiographs, and the development of persistent tachycardia, with a sustained heart rate greater than beats/minute. although the majority of foals with meconium impactions are treated medically, surgery is indicated in some cases. postoperative intra-abdominal adhesion formation is the most common anticipated complication, thus surgery is often delayed for fear of poor long-term survival. two short-term survival (i.e., survival until discharge) studies on foals undergoing exploratory celiotomy for meconium impactions had a combined survival rate of % ( out of foals). , long-term survival to maturity was % ( out of ) for foals undergoing surgery with either enterotomy or manual reduction. in a retrospective study in germany on foals undergoing an enterotomy for a meconium impaction, long-term survival to six months was %. in general, for foals undergoing an exploratory celiotomy, the survival rate is better for foals with lesions of the large colon, compared to the small intestine. although studies before indicate that survival rate for foals undergoing surgery for colic is lowest for neonates, data from the early s showed significantly higher survival rates, as compared to the s. [ ] [ ] [ ] with advancements in diagnostics and critical care, assuredly the long-term survival has continued to improve, though specific data is unavailable for recent years. (figure - ) . over the next several hours, the foal could rise with assistance and would attempt to nurse, but would stop nursing shortly after beginning. dorsal recumbency became increasingly frequent. gastric ulceration has been diagnosed in foals as young as hours of age and can been seen throughout the neonatal period and beyond. the incidence of gastric ulceration in foals has been cited as high as % in normal foals less than days of age. , because of this, it is difficult to know what role it plays in the health of the asymptomatic foal. stress has been implicated as a cause for gastric ulceration in the sick foal. in the neonate, ulcers are often present with concurrent diseases, such as enteritis or sepsis. the history should be utilized to determine if the foal had any prior illnesses, including diarrhea, colic, lethargy, or anorexia. furr found that foals stressed by disease were more likely to have abnormal cortisol, t , t , and reverse t levels than age-matched normal controls foals. these stressed foals had a higher incidence of gastric ulceration than the normal foals. the clinical findings associated with ulcers can range from asymptomatic to death secondary to severe peritonitis from gastric rupture. a study in japan of foals with gastric ulceration found that the most prevalent clinical signs were depression and intermittent nursing ( . % of affected foals) followed by diarrhea ( %), colic ( . %), bruxism ( %), and ptyalism ( . %). affected foals will often lay in dorsal recumbency trying to be more comfortable. spontaneous gastric reflux from the nostrils can be seen on rare occasion. foals with suspected or confirmed ulcers should be evaluated closely for sepsis or signs of peripartum asphyxia, as ulcers may develop secondary to these conditions. some clinicians associate the stress of orthopedic disease in foals to be a risk factor for the development of gastric ulcerations. because of the role that other diseases may play in the development of gastric ulcers, the diagnostics surrounding the neonate with gastric and/or duodenal ulceration should include a thorough investigation of the systemic status of the foal, including complete blood count, serum biochemistries, and urinalysis. blood cultures should be taken to evaluate for concurrent sepsis. because of the potential role for hypoperfusion in the development of ulcer disease, perfusion should be monitored through serial blood pressure measurements (direct or indirect) and blood or plasma lactate concentrations. specific diagnostics for ulcer disease include gastroscopy or gastroduodenoscopy (including esophagoscopy for reflux esophagitis) as the most sensitive and specific means of diagnosing ulcer disease in the newborn. usually this can be accomplished with a -meter endoscopy. the foal should be fasted for approximately two hours before scoping so that the gastric mucosa can be viewed better. the examination should be systematic being sure to view the distal esophagus and the squamous and glandular portions of the stomach. lesions may vary from a mild hyperemia or gastritis to deep bleeding ulcerations ( figure - , a and b) . another finding of gastroscopy in young foals is a desquamation of the squamous epithelia. this occurs in % of foals less than days of age. sheets of squamous epithelia shed during this time period. this can occur with or without ulceration (figure - ). radiographs of the abdomen may be useful in ruling out other causes of colic in the foal. the presence of free gas in the abdomen may be suggestive of a gastric perforation. contrast radiography should be performed in foals with suspected gastric outflow obstruction (emptying disorders) such as those with a b fig. - a, endoscopic view of ulceration of the squamous mucosa of a foal's stomach. b, ulcerative esophagitis from gastric reflux. note the "cobblestone" appearance of the mucosa. pyloric or duodenal ulceration (see . delayed gastric emptying should be suspected if the barium is still in the stomach after two hours. foals with ulcer disease should also have abdominal ultrasonography performed to evaluate for bowel wall thickness, ileus, and the possibility of peritoneal effusion. ascites due to peritonitis in response to ulcers is a negative prognostic sign, and can mean perforation is imminent or has occurred. fecal or gastric occult blood may be suggestive of bleeding ulcers, but these tests are neither sensitive nor specific. neonatal ulcer disease occurs in a number of different syndromes, including silent ulcers, clinical or active ulcers, perforating ulcers, and duodenal ulcers. silent ulcers are subclinical, but on occasion they have gone on to perforate. they may also be found incidentally on gastroscopy or postmortem examination. clinical ulcers are consistent with the foal of this case; clinical signs included depression, partial anorexia, bruxism, ptyalism, dorsal recumbency, and colic. ulcers can develop both in the glandular and nonglandular regions of the stomach. duodenal ulcers, most common in the older (two-to five-month-old) foal, can result in pyloric strictures and gastric outflow obstruction and appear to represent a different syndrome than neonatal ulcers. these foals may develop reflux esophagitis as a result of the obstructive nature of this problem. healthy neonatal foals have an acidic baseline gastric ph, with an average ph of approximately . to . . they intermittently develop a gastric ph level below . prolonged recumbency, greater than minutes, results in a more acid gastric ph, often below . for prolonged periods. suckling milk raises the gastric ph to above . , and sustains this high ph for several minutes. this emphasizes the importance of frequent feedings in minimizing the potential for development of ulcers in the critically ill foal that is unable or not allowed to nurse. the acid secretory profile of the critically ill foal is highly variable and differs widely from foal to foal. in one study, % of hospitalized foals demonstrated alkaline profiles continuously, while another % had profiles typical of healthy foals. the etiology of alkaline ph is unknown, but may represent parietal cell dysfunction as a consequence of hypoperfusion, or may alternatively reflect ileus with enterogastric reflux from the duodenum. approximately % of foals have atypical profiles with periods of marked acidity. the pathophysiology of gastric ulcer disease is multifactorial. in human infants, concurrent illness is strongly associated with gastric ulcers, with risk factors in some studies including mechanical ventilation (one of the most consistent risk factors), abnormal mode of delivery, delayed delivery, and hypotension. luminal factors that may predispose to ulcer formation include hydrochloric acid (hcl), pepsin, bile acids, and volatile fatty acids. additional pathophysiologic mechanisms for ulcer development include impairment of mucosal perfusion, reduction in mucus or bicarbonate secretion, neutrophil-and inflammation-mediated injury, and inhibition of nitric oxide. normal defenses of the mucosa vary anatomically. the squamous regions, including the esophagus, cardia, and fundus, are protected from acids by intercellular tight junctions. the glandular epithelium is protected by a mucous and bicarbonate barrier, prostaglandins, a rich mucosal blood supply, and rapid cellular restitution upon injury. mucosal blood flow is highly dependent upon prostaglandins e and e as well as nitric oxide. normal gastric and duodenal motility and duodenal sphincter tone are other protective mechanisms that minimize duodenal reflux of bile acids and other cytotoxins. ulceration in the squamous mucosa, particularly the region adjacent to the margo plicatus, must be interpreted with caution in terms of clinical significance because of the finding that up to % of clinically normal foals may have ulcers in this area. , only % of these healthy foals had ulcers in the glandular mucosa. however, up to % of critically ill foals had glandular ulcers. it appears that concurrent illness (sepsis, diarrhea, pneumonia) increases the incidence of glandular ulcers in the neonatal foal. while luminal factors such as hcl may be the primary mechanisms of nonglandular mucosal injury, damage to the glandular mucosa may be multifactorial. hypoperfusion and reduced oxygen delivery, as may occur with septic shock or hypovolemia, are believed to be important in the pathogenesis. hyposecretion of sodium bicarbonate or mucus is also important, and these often occur secondary to hypoperfusion. inflammatory mediators also play a role in mucosal injury. the use of nonsteroidal anti-inflammatory drugs, such as phenylbutazone, may lead to gastric ulceration as a consequence of prostaglandin e inhibition, leading to a reduction in mucosal blood flow, and bicarbonate or mucus secretion. , candidiasis has been associated with gastric ulcers in neonatal foals, however in the author's opinion it is not clear whether they were primary or secondary invaders. ' belly dancer was treated with a combination of supportive intensive care and gastric protectants. supportive care consisted of crystalloid administration and parenteral dextrose administration, with a conservative approach to enteral feeding. blood pressure was monitored and maintained with crystalloid therapy. urine output was also monitored closely as a marker of renal perfusion. treatment treatment of the neonatal foal with gastric ulcer syndrome consists of supportive care and the use of antiulcer medications. supportive care consists of maintenance of fluid volume, pressure support, and supplementation of oxygenation through insufflation as necessary for hypoxemia. acid-base and electrolyte disorders should be corrected. antimicrobials should be administered to treat and/or prevent sepsis in the neonate. nutritional support is also critical to the management of the neonate with gastric ulcers. small, frequent meals of milk should be provided in order to buffer gastric acids and to maintain higher intraluminal ph. continuous rate infusions of milk can also be provided, but caution must be taken that the foal's stomach does not become overdistended. the nasogastric tube should be intermittently checked for gastric residual accumulation. specific therapy of gastric ulcers includes mucosal adherents, histamine type receptor antagonists, and proton pump inhibitors. sucralfate, a hydroxy aluminum salt of sucrose called sucrose octasulphate, is a mucosal adherent. at acid ph (< ), sucralfate forms a viscous gel that binds ulcers. sucralfate also inhibits pepsin, buffers acid, and stimulates the production of prostaglandin e. this latter action stimulates bicarbonate and mucus secretion. sucralfate also binds epidermal growth factor and thus may play a role in epithelial restitution. suggested doses for sucralfate include to mg/kg po q to h (table - ) . histamine- antagonists include cimetidine, ranitidine, and famotidine. these drugs decrease acid secretion by competitively binding to the histamine receptor, thereby reducing stimulation of gastric acid secretion by histamine (see table - ). suggested dosages include the following: cimetidine mg/kg po q h or . mg/kg iv q h; ranitidine . mg/kg po q h or . mg/kg iv q h; famotidine . mg/kg po q h or . mg/kg iv q h. because cimetidine is less potent, has variable absorption in horses, and is associated with inhibition of hepatic microsomal enzymes, it may be the least preferred drug compared to the other h- antagonists. intravenous or oral administration of ranitidine significantly increased intragastric ph for four and eight hours, respectively, in healthy experimental neonatal foals. however, in critically ill foals, ranitidine may have a blunted duration in terms of alkalinizing response. in addition, these drugs will only be beneficial when gastric ph is acidic, which may not be the case in all foals. ranitidine and sucralfate have been shown to provide partial protection against clinical, clinicopathologic, and pathologic manifestations of phenylbutazone in foals. proton pump inhibitors have been more recently added to the treatment options for gastric ulcers in horses. the most commonly used agent is omeprazole, which decreases acid production through irreversible binding to the hydrogen-potassium atp pump of the parietal cell. omeprazole has been studied in healthy neonatal foals. , an oral dose of mg/kg resulted in an increase in gastric ph within two hours of administration (see table - ). for foals with delayed gastric emptying, prokinetic therapy may be in order. metaclopramide is a dopaminergic receptor antagonist that can be given as a constant rate infusion ( . - . mg/kg/hour). its actions are to tighten the esophageal sphincter, increase gastric motility, and relax the pyloric sphincter. caution should be used, as overdosage can result in neurologic excitement (see table - ). while the therapy of ulcers certainly includes pharmaceuticals to raise gastric ph, prevention of ulcers through their use is controversial. , some clinicians feel that alkalinizing the foal's stomach without evidence of ulceration may lead to increased survival of ingested bacteria and a risk of translocation of bacteria through the gastrointestinal mucosa. also as stated above, the acid-secreting profile in the stomach of the critically ill foal may already be alkaline. excellent supportive care along with intensive management of hemodynamics for the at-risk foal is at least as important to prevention of ulcers. until research studies are available for defining the risks and benefits of the prophylactic use of antiulcer medications, definitive recommendations cannot be given. the outcome of foals with gastric ulcers is highly variable. in a retrospective study by taharaguchi, mortality from gastric ulceration ranged from . % to . % of deaths in foals between and . foals with silent ulcers due to hemodynamic perturbations that go on to perforate have a grave prognosis. foals with ulcers that do not progress to the point of perforation have a better prognosis, particularly those whose primary problem responds to therapy. foals with duodenal ulcers have a guarded prognosis in general, because of the potential for duodenal strictures, fibrosis, and gastric outflow obstruction. (figure - ) . the peripartum history and history of the days prior to the development of diarrhea are important in differentiating the causes of diarrhea in foals. the potential for peripartum asphyxia, immature gestational age, and sepsis can be assessed through history. foals with perinatal hypoxic-ischemic injury are at significant risk for gastrointestinal dysfunction, including diarrhea. premature foals are predisposed to necro-tizing enterocolitis. additional information that is pertinent to the workup and support of the diarrheic foal include nursing ability and appetite, mentation and activity level, housing environment, and character and frequency of the diarrhea. assessment of passive transfer of colostral antibodies is also important, as foals with failure of passive immunity are at increased risk of developing sepsis and possibly infection with pathogenic enteric organisms. on the other hand, foals with high intakes of colostrum and milk may be at increased risk for clostridial enteritis, as it has been hypothesized that colostral trypsin inhibitors may protect clostridial toxins from acid degradation in the stomach. ingestion of large quantities of milk from heavy lactating mares may provide for additional enteral substrate for proliferation of clostridial organisms. additional historic information centers on diet. foals provided milk replacer or a nonequine source milk rather than mare's milk are at increased risk for development of osmotic diarrhea. foals from poor milkers may be ingesting water or foreign material such as dirt or bedding and are at risk for diarrhea associated with pica. some level of epidemiologic data is also very useful in determining the etiology of diarrhea in foals. information as to whether the foal is part of an outbreak situation (suggesting an infectious, nutritional, or toxic cause) or the sole foal with diarrhea on a farm is useful in directing the diagnostic approach. hygiene and husbandry practices should be assessed for aiding in education toward minimizing the incidence of diarrhea on farms. stall versus pasture housing and number of foals per paddock or pasture should also be considered. neonatal foals can present with diarrhea at any age between hours and several days of age. early diarrhea is consistent with hypoxic-ischemic damage or sepsis. diarrhea in the slightly older foal is indicative of infectious or dietary causes. the neonate with diarrhea should be evaluated for the following: ) circulatory volume and hydration status, ) clinical signs consistent with systemic inflammation (e.g. sirs, sepsis) such as mucosal hemorrhages, congested or hyperemic mucous membranes, injection of vessels, tachycardia, tachypnea, or fever, ) nutritional status through assessment of body condition or weight, and ) indicators of concurrent disease such as bacteremia, signs of asphyxial injury, or congenital defects. circulatory status should be assessed in the physical examination by close attention to mentation status, capillary refill time, jugular vein fill time, extremity temperature, mucous membrane color, heart rate, and pulse quality. heart rate can be misleading in the neonatal foal, however, as those with hypoxemia, hypothermia, or hypoglycemia can be bradycardic despite significant hypovolemia. hydration status, reflecting interstitial and intracellular volumes, is indicated by skin turgor, mucous membrane texture, sunken eyes, and corneal quality. the abdomen should be evaluated for distention. ballottement can indicate ascites or gas distention. the perineum should be assessed for scalding and dermatitis associated with fecal adherence to hair and skin. a cautious digital examination can reveal sand or dirt, rectal impactions, and rectal edema. body condition scoring can be performed in the neonatal foal, and a subjective evaluation should be performed as a minimum because many diarrheic foals lose substantial amounts of weight (see chapter ). because foals with enterocolitis are at risk for bacterial translocation and subsequent sepsis, they should be evaluated for signs of localized sepsis such as effusive joints or enlarged external umbilical stumps. the pathophysiology of diarrhea is multifactorial. factors resulting in diarrhea from enterocolitis include hypersecretion, osmotic draw, altered motility, altered starling's forces (as with increased vascular permeability or hydrostatic pressure, or decreased colloid osmotic pressure), maldigestion/malabsorption, and inflammation. though one mechanism may predominate in some forms of diarrhea, most of them contribute. foals with enterocolitis often exhibit signs consistent with abdominal pain. differential diagnoses that should be considered in foals with acute abdominal disease include strangulating and nonstrangulating obstructions, uroabdomen, and other nongastrointestinal causes of colic, such as liver disease. foals with impending enterocolitis can be very difficult to differentiate from those with obstructive diseases, because they often exhibit signs of abdominal pain and distention prior to the onset of diarrhea. the presence of fever and leukopenia are suggestive of enterocolitis, but not specific. abdominal ultrasonography and radiography can help in differentiation of these conditions. causes of diarrhea in neonatal foals include both infectious and noninfectious causes. noninfectious causes include "foal heat diarrhea," pica, dietary intolerance, gastric ulcer disease, hypoxic-ischemic injury, and necrotizing enterocolitis. infectious causes include bacterial, viral, parasitic, and protozoal causes (table - ) . foals with foal heat diarrhea are otherwise bright and nursing well. they do not experience a systemic inflammatory response, and therefore have normal hematologic and vital sign findings. , the name foal heat diarrhea is a misnomer because the diarrhea does not appear to be a result of hormonal changes in the mare. rather, it is a result of intestinal maturational changes. it usually occurs between seven and days of life and is self-limiting. pica results from ingestion of sand, dirt, bedding, or hair. inflammatory or motility alterations secondary to the presence of foreign material can result in obstructions or impactions as well as diarrhea. another dietary cause of diarrhea in the foal is dietary intolerance. it occurs when either a foal is lactoseintolerant (secondary to infectious diarrheas such as clostridiosis or rotaviral infection) or is an orphan foal with diarrhea resulting from feeding excessive amounts of milk replacer or milk replacers mixed at higher than % total solids. milk replacers with sucrose or maltose are particularly offensive. gastric ulcer disease is associated with diarrhea in some neonatal foals. clinical signs can be inapparent or include bruxism, ptyalism, dorsal recumbency, diarrhea, or colic (see . hypoxic-ischemic insult as occurs with peripartum asphyxia can result in gastrointestinal injury, leading to diarrhea. hypoxic and subsequent reperfusion injury may both contribute to mucosal compromise. smooth muscle and serosal dysfunction may also result. energy depletion as occurs secondary to hypoxic-ischemic injury leads to cell membrane pump failure. subsequent reperfusion injury leads to oxidant injury as well as inflammatory cell activation and "no-reflow" phenomenon. necrotizing enterocolitis (nec) is a state of gastrointestinal necrosis that occurs from mucosal injury, as might occur with asphyxial injury, the presence of enteral nutrition, and bacterial invasion of the gi wall. ileus, gastric reflux, abdominal distention, intestinal perforations, or diarrhea can result from nec. prematurity is a risk factor for development of necrotizing enterocolitis. infectious causes of diarrhea often occur in large groups of foals that are housed together; sporadic cases can also occur, particularly with clostridial agents. viral etiologies include rotavirus and coronavirus. rotavirus infects and denudes small intestinal microvilli, resulting in maldigestion and malabsorption. it thus interferes with lactase function and sodium-glucose cotransport. because of relative sparing of crypt epithelium, the virus also results in increased net secretion. clinical signs vary from mild to severe diarrhea, requiring minimal to intensive therapeutic intervention, respectively. experimentally, the incubation period is as short as two days. most affected foals are five to days of age, although younger and older foals can be affected. older foals (up to days of age) usually have mild diarrhea, and asymptomatic animals can shed the virus. , vaccination of gravid mares may reduce the incidence of disease on farms, as well as the severity of clinical signs. , phenolic compounds are the optimal disinfectant type used, as rotavirus is resistant to many others. coronavirus has been associated with diarrhea in foals. , it infects the small intestine during the first few days of life, with persistence of viral antigen in the crypt cells for three to four days after onset of diarrhea. complications of coronaviral infections can include coronitis and limb edema associated with inadequate perfusion of the extremities. other potential viral etiologies of diarrhea include adenovirus and parvovirus, although their exact role in foal diarrhea has not been clearly defined. the most common bacterial causes of diarrhea include clostridial agents, especially clostridium difficile and c. perfringens, and salmonella. the clostridial infections can result in outbreaks or sporadic cases of diarrhea. c. difficile is a primary pathogen in neonatal foals and does not require prior antimicrobial administration as a risk factor for development of diarrhea as is usually the case with adult horses. , the primary virulence factors for pathogenicity include two large exotoxins, toxins a (enterotoxin) and b (cytotoxin). c. difficile-associated disease ranges from mild diarrhea to highly fatal, hemorrhagic, or necrotizing diarrhea. it has also been associated with lactose intolerance in foals. it should be noted that toxigenic clostridium difficile can be cultured from the feces of clinically normal foals, just as it can from healthy infants, and it is unknown what circum-stances or agent-host interactions lead to clinical disease. , clostridium perfringens types a and c have been reported to cause enterocolitis and diarrhea in neonatal foals. as with c. difficile, the pathogenicity of c. perfringens a and c is dependent upon virulence factors, especially the production of enterotoxins. in a recent epidemiological study, c. perfringens was found in % of normal three-day-old foals and % of normal foals at eight to hours of age. the most common genotype was type a ( %), and c. perfringens type c was found in less than % of the foals. these results suggest that type a is likely part of the normal microflora of the neonatal foal (and thus the clinical relevance of positive cultures without presence of enterotoxin are questionable), whereas type c is rarely found in the normal neonatal and has been associated with the clinical signs of watery to hemorrhagic diarrhea, but also includes abdominal distention, colic, and ileus. therefore, typing of isolates is critical to pursuing positive cultures, because the type of c. perfringens varies between clinically normal and affected foals. foals with c. perfringens, type c infection can develop rapidly progressive obtundation and colic and may die before the onset of diarrhea. c. perfringens-associated diarrhea is associated with a high mortality rate, % in one study, and is highly associated with fatal diarrhea in another study. , adult horses may serve as reservoirs for spores in the environment. salmonellosis is another bacterial form of enterocolitis that can affect the neonatal foal as it can adult horses. concurrent sepsis is a concern with salmonellae, due to invasiveness and the potential for translocation across the compromised gut barrier. studies have found an association between isolation of salmonellae from foals with diarrhea and fatality. the severity of the disease, in part, relates to the serotype involved, with group b salmonellae, such as s. typhimurium, being among the most pathogenic. salmonella of other groups can also cause disease, for example, a group c salmonella ohio has been reported to cause an outbreak of neonatal salmonellosis. mares of infected foals may be asymptomatic carriers of salmonella and the source of the foal's infection. other bacteria that can cause diarrhea sporadically in the neonate include actinobacillus sp. (in association with bacteremia), enterotoxigenic e. coli, bacteroides fragilis, clostridium sordelli, aeromonas hydrophila, and streptococcus durans. [ ] [ ] [ ] [ ] [ ] [ ] the significance and pathogenesis of these agents in neonatal foal diarrhea requires further study. the primary parasitic agent that has been associated with diarrhea in the young foal is strongyloides westeri. it appears that heavy infestations with more than eggs per gram of feces are required for development of diarrhea, and it is generally not a cause of severe diarrhea. strongyloides westeri is transmitted to foals through suckling milk. the primary protozoal enteric pathogen of foals is cryptosporidium parvum. it was initially believed that cryptosporidium infections were limited to immunocompromised foals such as arabians with severe combined immunodeficiency syndrome. more recently, it has been identified as a pathogen of immunocompetent foals as well. the organism invades the microvillus, occupying an intracellular but extracytosolic space. it thus causes villus blunting and maldigestion with a secondary osmotic diarrhea. most affected foals are five days of age and older, but younger foals can develop disease with high-level exposure. diarrhea usually lasts between five and days, with recovering animals and asymptomatic older foals and adult horses shedding oocysts. one study identified cryptosporidium parvum causing an outbreak of neonatal diarrhea to be genotypically identical to that associated with bovine diarrhea. however, exposure to cattle was not found to be an important source of infection for foals in another epidemiologic study. sources of cryptosporidial infection in foals have not been definitively elucidated. mares, cattle, and municipal water sources have all been suspected as the source of infection in foals, but these remain speculative. because of the zoonotic risk associated with cryptosporidium, affected foals should be handled carefully with particular attention to good hygiene. the role of other protozoal agents, including eimeria leukarti, trichomonas equi, and giardia sp., is poorly understood and requires further study. daily fecal cultures for salmonella sp. were negative. immunoassays for clostridium difficile toxin a were negative, as was fecal culture for c. difficile. the clinical pathology of foals with diarrhea can vary depending upon whether there is a concurrent systemic inflammatory response syndrome. the hematologic (cbc) findings can vary from unremarkable to those of hemoconcentration, leukopenia, and hypoproteinemia. a left shift with immature (band, metamyelocytes) neutrophils, with variable toxic cytologic changes, may also be present, reflecting a profound inflammatory response such as sepsis. hypoproteinemia often reflects protein-losing enteropathy, and hyperfibrinogenemia may be present in the face of inflammatory mediators induced by endotoxemia. thrombocytopenia occurs when coagulopathies such as disseminated intravascular coagulation are triggered. as for hematology, serum biochemistry analyses are normal when diarrhea occurs without gastrointestinal inflammation. enteritis or enterocolitis with subsequent activation of sirs can lead to organ dysfunction as might occur with severe sepsis. increases in creatinine and/or bun and liver enzymes or bilirubin concentrations reflect renal or hepatic dysfunction, respectively. hypoalbuminemia usually reflects gastrointestinal protein loss. similarly, hypoglobulinemia may be present, although it may be due to failure of passive transfer of colostral antibodies as well as gi loss. a variety of electrolyte derangements may reflect enteric losses, renal dysfunction, or acid-base derangements and often include hyponatremia, hypochloremia or hyperchloremia, hypokalemia or hyperkalemia, and an increased anion gap. decreases in total co concentrations reflect metabolic acidemia. anion gap may also be increased with hyperlactatemia. metabolic acidemia results from both organic and inorganic acidosis. lactate is the primary organic anion contributing to acidemia. hyperlactatemia results from a combination of hypoperfusion associated with hypovolemia and hypotension, hypermetabolism, increased glycolysis from inhibition of pyruvate dehydrogenase by inflammatory mediators, enhanced activity of na-k atpase due to circulating catecholamines, and reduced hepatic or renal clearance of lactate. inorganic causes of acidemia in diarrhea cases include hyponatremia or hyperchloremia resulting in a decrease in strong ion difference. the acid-base status is reflected on blood gas analyses as reduced ph and increased base deficit. as part of intensive management of foals with severe enterocolitis, additional diagnostic monitoring should include indirect blood pressure, urine output measurements, and urinalysis with fractional excretion of electrolytes. because foals with diarrhea and sirs are at risk for coagulopathies, monitoring should also include clotting times (pt, aptt) and measurement of fibrin degradation product or d-dimer concentrations. antithrombin concentrations may be low because of induction of coagulopathies or loss of this small protein through the compromised gi mucosa. imaging should be a part of the diagnostic workup of these foals with diarrhea. abdominal ultrasonography and radiography can aid in differentiating causes of acute abdominal disease. ultrasound evaluation should include assessment of bowel wall thickness, luminal diameter, and small-intestinal motility (figures - and - ) . ascites or effusion can also be detected. radiography can be utilized to assess the degree of gastrointestinal distention (figures - , - , and - ) . it can also be utilized to detect pneumatosis intestinalis, or linear gas shadows within the wall of the gi tract, a hallmark of necrotizing enterocolitis. sand and radiopaque foreign material will also be apparent on abdominal radiographs. specific diagnostic evaluation of the neonatal foal with diarrhea includes a number of tests to rule out pathogenic agents (table - ) . fecal samples can be screened for viral particles with electron microscopy. fecal elisa and immunoassays are available for rotavirus and coronavirus. fecal cultures can be performed for salmonella sp. and clostridial organisms. to increase the sensitivity of identifying salmonella shedders, repeated daily cultures should be performed (up to five consecutive samples). alternatively, pcr techniques are available for salmonellae, although the findings must be interpreted in light of the high-level sensitivity of this test. positive cultures for c. difficile and c. perfringens must be interpreted with caution as nontoxigenic and incidental colonization is possible. the diagnosis of c. difficile-associated disease should be coupled to toxin assays. commercial immunoassays test for toxin a or both toxin a and b. these kits are easy and rapid, however they have variable sensitivities. stool cytotoxin assays for toxin b are also available and are considered the gold standard for toxin testing, but are time-consuming. pcr techniques for c. difficile toxin gene sequences are currently moving from the experimental arena to the clinical setting. diagnosis of c. perfringens enterocolitis is difficult to make. unfortunately, immunoassays are not available for most toxins, except for enterotoxin. therefore, cultured isolates must be typed using pcr techniques, such as the multiplex pcr. the diagnosis is made from positive cultures of toxigenic and pathogenic isolates of c. perfringens (e.g., type a or c), as well as exclusion of other pathogenic agents. gram staining of feces can serve as a rapid screen for clostridial overgrowth, however this technique is neither sensitive nor specific (figure - ) . there are a number of diagnostic tools available for diagnosis of cryptosporidium parvum infection. immunofluorescence assay (ifa), acid-fast (af) staining, and flow cytometry have been developed for identification of oocysts. in one study, af and flow cytometry were determined to be more sensitive than ifa. af staining was less specific, however. because of their small size, oocysts are very difficult to visualize microscopically after fecal flotation. fecal flotation is useful for identifying strongyloides westeri ova, with the mcmaster method used for quantification. if one is concerned that lactose intolerance is playing a role in the continuation of a foal's diarrhea, then the use of a lactose tolerance test may be indicated. after a four-hour fast, the foal is administered lactose ( g/kg) through a nasogastric tube. blood samples collected in fluoride oxalate tubes are taken for glucose levels at preadministration, immediately postadministration, and every minutes for three hours. a normal absorption curve would show a doubling of glucose in to minutes. an absence of increase in glucose or a delay in the increase would indicate a problem with lactose digestion and absorption. tiofur ( mg/kg, iv, q h) and amikacin ( mg/kg, iv, q h) were administered to provide broad-spectrum antimicrobial coverage with potent gram-negative efficacy. kaolin pectate and bismuth subsalicylate were utilized intermittently as gastrointestinal protectants. the treatment of foals with diarrhea is highly variable, depending upon the severity of clinical signs and physiologic derangements. in general, the management of enterocolitis includes a number of goals. these include hemodynamic, metabolic, and nutritional support; gastrointestinal protectants; antibiotic coverage; and infectious disease control. if a specific agent is identified, then specific therapy for that agent can be instituted. hemodynamic disturbances are common among foals with enterocolitis, particularly those with sirs or sepsis syndrome. hypoperfusion results from hypovolemia, altered myocardial contractility, inappropriate vasomotor responses, and hypooncotic states associated with hypoalbuminemia. hemodynamic support occurs primarily in the form of intravenous fluid therapy, consisting of both crystalloids and colloids. a number of crystalloids are appropriate for foals with diarrhea, but because of the tendency for metabolic acidemia, fluids with higher strong ion differences such as normosol r, plasma-lyte , or lactated ringers solution are more ideal than . % saline. volume replacement in the neonatal foal should be done with great attention; overzealous administration of crystalloids may potentiate gastrointestinal edema. monitoring of central venous pressure, urine output, and serial lactate concentration measurements can aid in guiding volume replacement. colloid therapy is indicated in the hypooncotic and hypovolemic foal. plasma or synthetic colloid solutions, such as hetastarch, can be used. plasma has the advantages of providing not only albumin, but also immunoglobulin, antithrombin, clotting factors, and other proteins. if hetastarch is used, platelet counts and clotting times should be monitored, as coagulopathies associated with reduced concentrations of von willebrand's factor and factor viii may develop. once volume replacement has occurred and the central venous pressure (cvp) is at maximum ( cm h o), hypotension should be addressed with inotropes, such as dobutamine ( to μg/kg/minute, iv cri). if hypotension and clinical parameters of hypoperfusion persist after initiation of inotropy, then vasopressors should be considered (norepinephrine or vasopressin cri-see chapter ). metabolic support occurs in the form of managing acid-base and electrolyte balance. metabolic acidemia is treated depending upon the cause of acidosis; organic acidoses (lactic acidosis) should be treated with optimizing peripheral oxygen delivery. inorganic acidoses, as occur with hyponatremia or hyperchloremia associated with electrolyte loss into the gi tract, can be treated by correcting the underlying electrolyte disorder. sodium bicarbonate is indicated in these situations. potassium, calcium, and magnesium may need to be supplemented in foals with severe diarrhea. glucose concentrations should be monitored frequently in the neonatal foal, particularly if the foal is anorexic or receiving parenteral dextrose. both hypoglycemia and hyperglycemia should be avoided. recently, tight glucose regulation with insulin has received a great deal of study in human critical care and is associated with improved survival in septic patients (see chapter ) . in cases with severe diarrhea, ileus and abdominal distention, abdominal discomfort, or suspected osmotic diarrhea, partial or complete withholding of milk should be considered. parenteral nutritional support is required in these foals (see chapter ) . antimicrobial therapy should be provided in order to minimize the risk for bacterial translocation across the compromised gut barrier. broad-spectrum coverage, as with a beta lactam and aminoglycoside combination or second-or third-generation cephalosporins, is indicated (see chapter ). gastrointestinal protectants include di-tri-octahedral smectite and activated charcoal, which act as adsorbents. smectite has been shown to bind clostridial toxins in vitro. however, to the author's knowledge, this product has not been evaluated in neonatal foals and should be used with caution. other protectants include kaolin/pectin combinations and bismuth subsalicylate. nonspecific therapy for diarrhea may include antiulcer medication and probiotics. sucralfate is also protective, stimulating mucosal blood flow and mucus production because of increased local prostaglandin synthesis. treatment of gastric ulcers with proton pump inhibitors and/or histamine type receptor antagonists is indicated when ulcers are suspected from clinical signs (bruxism, ptyalism, dorsal recumbency) or documented via gastroscopy (see . probiotics are often utilized to modulate colonic flora. however, evidence documenting their efficacy in horses is lacking, and their use in neonatal foals prone to bacteremia warrants further study. if a specific offending agent is identified, then treatment may be more directed. metronidazole, administered orally or intravenously ( to mg/kg, q to h), is indicated in clostridiosis cases. the early use of metronidazole in even suspect cases of c. perfringens is important because foals can die before the onset of diarrhea. metronidazole is also the first-line therapeutic for treating c. difficile infection in foals, however, cases of metronidazole resistance have been documented. , in these cases, vancomycin can be used, although it should be restricted to foals in isolation and foals that have severe clinical scores. unfortunately, bacitracin is uniformly ineffective against equine isolates of c. difficile. , ivermectin is effective in treating strongyloides westeri infestations. paromomycin has been suggested as a treatment for cryptosporidium infections, although its efficacy and safety are unproven in foals. exogenous lactase enzyme ( to u/ kg foal, po, q to h) should be provided to foals with rotaviral, cryptosporidial, and streptococcal infections and those with osmotic diarrheas to aid in digestion and prevention of chronic osmotic diarrhea. nursing care is very important for the diarrheic neonate. cleanliness and hygiene are important not only for infectious disease control, but also for preventing secondary complications such as thrombophlebitis in the patient. the foal with diarrhea should be isolated so as to prevent spread of infectious agents. consideration must be given to efficacy of particular disinfectants for specific agents. though bleach is effective against most pathogens, rotavirus is best killed with phenolic compounds. cryptosporidium is resistant to most disinfectants. high heat and ultraviolet light may be helpful in eliminating this organism. owners should be warned about the potential zoonotic nature of salmonella and cryptosporidium. frequent handwashing and use of separate clothing when working with sick foals are prerequisites of curtailing the spread of the infection. cryptosporidium can also cause disease through any mucous membrane contact. protective eyewear and the use of surgical masks may be an important part of disease prevention in caretakers. physical examination revealed the foal to be severely depressed and responsive only to noxious stimuli. the foal exhibited a systemic inflammatory response syndrome, with tachycardia, tachypnea, and pyrexia. clinical signs compatible with sepsis were found on ophthalmologic exam, including yellow-discolored irides, hyphema, hypopyon, and aqueous flare. the foal was markedly icteric (mucous membranes and sclera) and was in hypovolemic shock as exhibited by prolonged capillary refill time, tachycardia, poor pulse quality, and cold extremities. important historical information for the foal with icterus and altered mentation includes farm epidemiologic data such as the number and percentage of affected foals. the spectrum of clinical signs and course of the disease encountered in outbreak situations are important, as is the age range. administered supplements or medications should be evaluated for potential hepatotoxicity. for isolated cases, the history leading up to the disease should be investigated. age of onset is important; for example tyzzer's disease is limited to foals a few days of age to six weeks of age. , management practices, including housing, diet, peripartum history, and additional concurrent disease in the herd, should be considered. treatment prior to veterinary examination or therapies provided by the referring veterinarian, in the case of specialty practices, should be evaluated. a detailed physical examination of the foal with liver dysfunction is important not only for elucidating the etiology, but also for management and therapy. with acute hepatic disease, foals may exhibit a systemic inflammatory response syndrome with tachycardia, tachypnea, and/or pyrexia or hypothermia. hypovolemia and dehydration are common due to lack of nursing, third space losses (diarrhea or ascites), or vascular permeability alterations. icterus or jaundice is a key finding with liver disease, and mucous membranes, including gingiva, vulvar, and ocular mucous membranes, as well as sclera should be evaluated closely for yellow discoloration (figure - ) . fecal character should be evaluated, as some forms of liver disease can cause diarrhea or changes in fecal color. for example, foals with biliary atresia may have gray feces while those with tyzzer's disease can exhibit diarrhea. [ ] [ ] [ ] abdominal distention may be present, and abdominal ballottement can be used to reveal ascites or hepatomegaly. foals with subacute or chronic liver disease may exhibit weight loss or poor growth, which may be apparent as poor body condition. those with acute hepatopathy, such as tyzzer's disease, have a normal body weight and appear normal until acute onset of signs. , the perinatal foal with hepatopathy should be closely evaluated for concurrent signs of hypoxicischemic injury, including encephalopathy, enteropathy, and nephropathy. hepatic dysfunction may occur in these foals as well. similarly, foals with sepsis can develop hepatitis. hepatoencephalopathy is present in many foals with acute liver disease or end-stage liver failure. signs range from subtle to severe and fulminate. subtle signs include mild depression and yawning. more obvious signs include central blindness, circling, head pressing, seizures, behavioral changes, and obtundation to coma. fundic examinations should be performed in icteric foals because herpes-positive cases often demonstrate dark and red-discolored optic discs and irregularly dilated vessels. the pathophysiology of liver disease in the neonatal foal is multifactorial and complex. because of a large number of etiologies, insult or abnormalities may occur in the form of congenital, genetic, toxic, infectious, and hemodynamic causes. the differential diagnoses for foals with icterus include fasting hyperbilirubinemia, increased erythrocyte destruction, hepatocellular disease, and cholestatic disorders. causes of hemolysis include neonatal isoerythrolysis, drug-induced lysis, and increased red blood cell turnover from sepsis or peripartum asphyxial injury. sepsis may impair bilirubin metabolism due to the presence of endotoxemia contributing to hyperbilirubinemia. liver disease occurs from a number of causes. infectious causes include bacterial hepatitis secondary to sepsis, and infection with actinobacillus equuli or clostridium piliformis. bacteremia and sepsis in general can cause hepatic dysfunction by means of inducing hypoperfusion and tissue hypoxia. inflammatory mediators stimulated by the systemic inflammatory response syndrome (sirs) will contribute to hepatic damage. cholangiohepatitis from enteric microbes can result from ileus, enteritis, or ulcerative duodenitis/ stricture syndrome. bacterial hepatopathy may also occur as extension of umbilical vein phlebitis or abscessation. hepatoencephalopathy was associated with rhodococcus equi infection in one foal. although not cultured, a liver biopsy suggested a bacterial etiology and the authors speculated that it may have been due to r. equi bacteremia or ascending biliary infection. tyzzer's disease, caused by clostridium piliformis, formerly known as bacillus piliformis, is an acute and multifocal hepatic necrosis associated with a high mortality rate. most commonly associated with sporadic cases on farms, outbreaks of disease can also occur. foals present with a rapidly progressive clinical course including recumbency, hyperthermia or hypothermia, and stupor or coma, icterus, and seizures with marked hypoglycemia and metabolic acidosis. some foals may be found dead without preceding clinical signs. viral hepatitis from equine herpes virus (ehv- ) neonatal infection can occur in association with respiratory distress, neurologic signs, and bone marrow necrosis resulting in leukopenia. foals are infected in-utero from repeated cell-associated viremia in the dam, so signs are seen immediately at birth. icterus is a common finding in neonatal herpes infections as a result of multifocal and acute hepatic necrosis. despite profound necrosis, liver enzymes were not increased in one retrospective report. in the report, herpes-positive foals were more likely to have total white blood cell counts less than , /μl and to be icteric (and hyperbilirubinemic) compared to septic or premature foals. foals with neonatal herpes infections have a poor prognosis; however, those with milder infections may survive. treatment with acyclovir can be attempted. foals with hypoxic-ischemic injury from peripartum asphyxia most commonly exhibit clinical signs consistent with encephalopathy, nephropathy, or gastroenteropathy, however hepatopathies may also result. such foals exhibit icterus from biliary stasis and hepatic dysfunction, and have increased concentrations of liver enzymes. foals with neonatal isoerythrolysis sporadically develop hepatopathies with increases in direct bilirubin concentration in addition to indirect hyperbilirubinemia and increased liver enzymes. liver disease is suspected to occur as a result of tissue hypoxia, iron overload from hemolysis, or bile stasis caused by the increased amounts of conjugated bilirubin. another form of liver disease occurs secondary to gastrointestinal disease, primarily strangulating or obstructive lesions and ileus. endotoxin or bacteria can enter the portal circulation from translocation. congenital causes of hepatopathies include portacaval shunts and biliary atresia. , portosystemic vascular anomalies include both intrahepatic and extrahepatic shunts. [ ] [ ] [ ] persistent vitelline arteries have been reported in conjunction with intrahepatic portosystemic shunts. foals with portosystemic shunts have recurrent episodes of blindness, seizures, or altered mentation associated with hepatoencephalopathy. the shunts may not be clinically apparent until affected foals are eating grain or hay. congenital hepatic fibrosis and cystic bile duct formation is a syndrome suspected to be an autosomal recessive genetic trait of swiss freiberger horses. this report included foals that demonstrated jaundice, neurologic signs, colic, and unthriftiness. hyperammonemia of morgan foals and equine glycogen storage disease iv are syndromes suspected to have a genetic basis with some degree of hepatopathy. [ ] [ ] [ ] persistent hyperammonemia in morgan horses is suspected to be similar to a syndrome of hyperornithinemia, hyperammonemia, and homocitrullinuria (hhh) in humans. these foals exhibit neurologic signs including seizure activity, aimless wandering, yawning, and circling. supportive care, lactulose, and a low-protein diet resulted in temporary clinical improvement in one of two foals that was treated, however the foal relapsed and was euthanized. glycogen-branching enzyme deficiency has been recently attributed to a mutation in the glycogen branching enzyme (gbe ) gene in quarter horses. the syndrome has recently been termed glycogen storage disease iv and is believed to be inherited as an autosomal recessive trait, with clinically affected foals being homozygous for the mutation. , affected foals are sometimes stillborn, and otherwise exhibit flexural deformities, seizure activity, respiratory or cardiac failure, or recumbency. leukopenia, hypoglycemia, and high serum ck, ast, and ggt concentrations were common, and all foals died by seven weeks of age. the livers of affected foals contained periodic acid schiff's (pas)-positive intracellular inclusions and no gbe activity. an additional consideration for congenital hepatic disease is hepatoblastoma. polycythemia, hyperbilirubinemia, and increased liver enzymes occur with this liver neoplasm. these foals may survive to weaning or adulthood, but hepatoblastoma has been reported in an aborted fetus and may thus be congenital. toxic hepatopathies have been reported in neonatal foals following administration of oral pastes contain-ing ferrous sulfate in the first one to two days of life. experimental administration of iron to foals confirmed that the hepatotoxicity from the oral paste was indeed due to the contained iron. , pathologic changes included liver atrophy, bile duct hyperplasia, lobular necrosis, cholestasis, and periportal fibrosis. druginduced hepatopathies can also occur during this time period because of relatively immature hepatic function and increased absorption of macromolecules during colostral igg absorption. oral medications should be used with caution in foals less than hours of age because of the potential for increased bioavailability with subsequent hepatotoxicity. other potential hepatotoxins reported in horses include aflatoxins, pyrrolizidine alkaloids, leukoencephalomalacia, and chlorinated hydrocarbons, although these are considered unlikely in the young foal because of lack of ingestion of feedstuffs by neonates. steroid hepatopathy has been reported in three-and -year-old horses, secondary to intramuscular administration of high doses of triamcinolone acetonide in both cases. , (figure - ) . clinical pathologic findings in foals with liver disease include increases in concentrations of both hepatocel- lular and biliary enzymes. aspartate aminotransferase (ast) and sorbitol dehydrogenase (sdh) are examples of hepatocellular enzymes. ast is not liver-specific, however, having significant components originating from both skeletal and cardiac muscle. sdh is considered to be liver-specific and has a short (two-to three-hour) half-life. biliary-derived enzymes include γ-glutamyl transferase (ggt) and serum alkaline phosphatase (sap). ggt is nearly biliary-specific, however pancreatic origins must be considered when concentrations are increased. sap has isoenzymes from multiple sources, including fast-growing bone in the neonatal foal. it should be noted that serum biochemical indicators of liver status in neonatal foals have some distinct features as compared to adult horses. serum alkaline phosphatase ( to iu/liter), ggt ( to iu/liter), and sdh ( . to . iu/liter) activities are increased during the first two weeks of life. serum cholesterol, triglycerides, and total and unconjugated bilirubin concentrations peak during this same neonatal period. foals with cholangiohepatitis and infectious hepatopathies usually have increases of both hepatocellular and biliary enzymes. similarly, those with toxic insults and hypoxic-ischemic disease also have increased liver enzymes. glycogen-branching enzyme deficiency results in increased ggt concentrations. those with portacaval shunts often have normal liver enzymes, but liver function tests are abnormal. foals with herpes hepatopathies also usually have normal enzymes, but increased bilirubin concentrations. both indirect-reacting (unconjugated) and directreacting (conjugated) bilirubin concentrations increase with liver disease. cholestatic disease results in increases in both components of bilirubin as well, while the percentage attributed to direct bilirubin is usually higher than with primary hepatocellular disease, often exceeding % to % of the total bilirubin concentration. on the other hand, fasting hyperbilirubinemia and that resulting from hemolysis are due exclusively to indirect bilirubin. serum bile acid concentrations increase with liver dysfunction, as occurs with portacaval shunts. they represent a good screening test of liver function. bile acids, such as choleic and chenodeoxycholic acids, are synthesized by the liver from cholesterol, and are excreted into bile after conjugation with amino acids. these conjugated forms form micelles with fat in the intestine and are reabsorbed and recirculated via enterohepatic circulation. serum concentrations of bile acids increase in the face of hepatocyte damage, biliary stasis, and shunting of portal blood to the systemic circulation as occurs with portosystemic shunts. hyperammonemia is often present, also reflecting liver dysfunction. the liver is the primary site of ammonia removal by converting it to urea for renal excretion. foals with portacaval shunts commonly have high resting ammonia concentrations. morgan foals with hhh syndrome have very high concentrations of blood ammonia (> μmol/liter), while liver enzymes are only mildly to moderately increased and bilirubin is normal to mildly elevated. proper handling procedures are critical to accurate measurement and interpretation of blood ammonia. heparinized blood should be used, and the sample should be collected gently (to avoid hemolysis) and kept on ice. the sample should be run within one hour of collection or otherwise spun in order to freeze the plasma at − °c for subsequent measurement within hours. blood from aged-matched control foals should be run concurrently. additional biochemical indicators of liver function include serum glucose, bun, albumin, and cholesterol concentrations. glucose concentrations are variable, and reflect the degree of hepatic necrosis and milk intake. foals with severe hepatitis or necrosis, such as those with tyzzer's disease, have profound hypoglycemia. those with portacaval shunts also commonly exhibit hypoglycemia. as bun, albumin, and cholesterol are synthesized by the liver, dysfunction is reflected by reduced concentrations of these analytes. however, hypoalbuminemia is uncommon with liver disease, as only % and % of horses with chronic and acute liver disease had albumin concentrations below the reference value in one report. it has been estimated that greater than % of liver mass must be lost for longer than three weeks before hypoalbuminemia develops. fractionation of blood amino acids and determination of the branch chain amino acid (bcaa) to aromatic amino acid (aaa) ratio can aid in determining hepatic function. decreases in the ratio indicate insufficiency, with normal ratios falling between . and . . because of liver dysfunction and reduced conversion of blood ammonia to bun, a decrease in bun concentration is often observed. it should be noted, however, that bun concentrations in the normal neonatal foal are lower than those in the adult horse and low concentrations are therefore difficult to interpret in the neonatal liver patient. bromsulphalein (bsp) and indocyanine green are dyes that are excreted primarily by hepatic clearance. clearance and half-life reflect hepatic excretory function and are prolonged in animals with biliary obstruction or hepatocellular failure. it should be noted that alterations in hepatic blood flow alter interpretation of bsp clearance; significant decreases in blood flow as occurs with portosystemic shunts will reduce the delivery of bsp and hence cause an increase in halflife. high bilirubin and low albumin concentrations will result in increased and decreased bsp half-life, respectively. quantification of serum bile acids has largely replaced the use of bsp clearance as an indicator of hepatic function in horses. the concentration of triglycerides may increase in foals with liver disease because of reduced hepatic clearance and increased mobilization from adipose tissue associated with a catabolic state. cholesterol and vldl concentrations may decrease, however, because of reduced hepatic synthesis. it should again be noted that triglyceride and cholesterol concentrations are higher in healthy neonatal foals compared to adults, and vary with age. in one report, foals had concentrations of to and to mg/dl for triglycerides in < -hour and in one-day-old foals, respectively. cholesterol concentrations were to and to in those same age groups. cbc findings are dependent upon the cause of liver disease. leukocytosis may be present with cholangiohepatitis. when liver disease is secondary to sepsis or hypoxic-ischemic disease, however, leukopenia with band neutrophils is more common. hyperfibrinogenemia is usually present with infectious or inflammatory hepatopathies, however fibrinogen may be decreased when coagulopathies such as disseminated intravascular coagulation exist. blood cultures should be performed in the neonatal foal with liver disease because of the possibility of sepsis causing hepatitis. clotting times may be prolonged with liver failure. prolongation of prothrombin time (pt) and partial thromboplastin (ptt) occur due to a lack or reduction of synthesis of several clotting factors, including factors ii, v, vii, ix, and x. this is an important clinical finding because liver biopsies may have to be delayed in hypocoagulable animals. for similar reasons, plasma antithrombin concentrations may be reduced, leading to coagulopathies such as disseminated intravascular coagulation. in one review, almost half of horses with liver disease had a prolonged pt or ptt. coagulopathies are common in horses with liver dysfunction due to concurrent systemic inflammatory response syndrome, and fibrin degradation products may be increased and thrombocytopenia may be present. the acid-base status of foals with liver disease should be evaluated closely through blood gas analysis. with liver dysfunction, acidemia may occur as a result of increased formation and reduced clearance of organic acids such as lactate, pyruvate, and amino acids. impaired urea synthesis may reduce renal buffering through urine acidification. serial monitoring of lactate should be included as part of the diagnostic monitoring of the foal with liver disease because lactate clearance may be reduced. ultrasonography is a critical component of the diagnostic workup of foals with liver disease. it can be utilized to determine the size of the liver, changes in parenchyma, and dilated bile ducts. with infectious etiologies, such as cholangiohepatitis, hepatitis secondary to sepsis, and tyzzer's disease, the liver appears subjectively enlarged. biliary distention is evident in some cases. evidence of fibrosis (hyperechogenicity) may be present in subacute to chronic cases. hepatic scintigraphy will confirm the diagnosis of portacaval shunts. injection of technetium m-labeled sulfur colloid demonstrates alterations in blood flow or the presence of masses. biliary scans can also be performed looking for biliary obstruction or atresia. portograms are necessary in cases of portosystemic shunts where surgical correction is contemplated. these are done intraoperatively using radiopaque agents that are injected into a mesenteric vein with subsequent radiography. simultaneous filling of the portal and azygous veins and caudal vena cava, and lack of intrahepatic filling indicate portacaval shunting. histopathology provides the most specific information as to the etiology of liver disease and can aid in diagnostic, prognostic, and therapeutic directions. the safest means of performing liver biopsies is under ultrasonographic guidance and using an automated needle biopsy instrument. it should be pointed out that focal lesions such as neoplasia are easily missed by liver biopsy. special stains (silver or warthin-starry stains) may be helpful in specific diagnoses such as tyzzer disease. fine-needle aspirates may also be helpful in the diagnosis of a specific etiology ( figure - ) . culture of liver biopsies is indicated when infectious etiologies are suspected. cholangiohepatitis is usually associated with gram-negative enteric aerobes and gram-negative or gram-positive anaerobes. foals with tyzzer's disease develop a peracute to acute multifocal necrotizing hepatitis. grossly, hepatomegaly is evident. these foals generally have marked increases in ast and sdh concentrations. other liver enzymes, both hepatocellular and biliary (ldh, sap, ggt), are also increased, as are both components of bilirubin. striking hypoglycemia should be a clue to the presence of infection with clostridium piliformis. these foals often have a metabolic acidosis, with hyperlactatemia due to increased production (hypoperfusion, inflammation, catecholamine surges) and reduced hepatic clearance. complete blood counts often reveal leukopenia with a left shift and cytologic evidence of toxicity, although leukocytosis is present in some cases. hemoconcentration reflects hypovolemia and dehydration. hyperfibrinogenemia is common. physical examination is consistent with septic shock, with petechial hemorrhages and injection being common. hypovolemic shock is also evident. mentation is usually depressed, ranging from lethargy to comatose, and icterus is a prominent finding. clinical signs of hepatoencephalopathy may include depression, weakness, and seizures. diarrhea is an inconsistent finding. fever is usually present, although hypothermia from shock may be predominating when initially examined. affected foals are often found dead. serologic testing for recovered or suspected cases can be helpful. pathology reveals hepatomegaly with rounded margins (figure - ). histopathologic findings with tyzzer's disease include multifocal and random necrosis with neutrophilic inflammation (figure - ). long bacilli are often but not always seen, particularly in hepatocytes at the periphery of necrotic areas. they are best delineated with warthin-starry stains (figure - ). an intranuclear location of the microbe has been found using electron microscopy. myocardial necrosis with warthin-starry stained bacilli has also been reported. , intestinal necrosis may also be present. and organisms consistent with c. piliformis may occasionally be observed in sections of intestine, particularly the large intestine. clostridium piliformis is an obligate intracellular spore-forming anaerobe. it is very difficult or nearly impossible to culture in vitro using routine microbiologic methods. inoculation of embryonated eggs has been one means of trying to assess antimicrobial susceptibility. penicillin, tetracycline, erythromycin, and streptomycin appear to be effective in vitro, while sulfonamides and chloramphenicol do not. treatment with dextrose, volume replacement, and sodium bicarbonate can result in temporary improvement of mentation, however most foals are reported to die. the exact pathophysiology of infection is unknown but is believed to be oral with subsequent distribution to the liver. ingestion of spores from feces shed by subclinical carriers may play a role in development of disease, and the disease has been experimentally reproduced. it has also been suspected that rabbit and rodent populations could serve as reservoirs of infection. most reported cases have been sporadic, however a number of cases have been noted on particular premises. preventive measures are unknown, however efforts should be directed at environmental hygiene on affected farms, including the use of . % sodium hypochlorite to eliminate spores from barns housing affected foals. risk factors for clostridium piliforme infection in foals include foaling season, farm residency and age of mare. in one study, foals born between march and april were . times as likely to develop infection as those born at any other time of the foaling season. foals of nonresident visiting mares were . times as likely to develop the disease as were foals born to mares that were permanent residents of the farm. foals born to young mares were . times as likely to develop tyzzer's disease as those born to mares ≥ years of age. ( mg /kg iv, q h) and gentamicin ( . mg/kg iv, q h) . seizure activity was controlled with phenobarbital. ( . ml/kg po, q treatment treatment of liver disease is largely supportive, with specific therapies available for certain diseases. foals with acute hepatopathies often present in hypovolemic crises. a combination of crystalloids and colloids is indicated in volume replacement. ideal crystalloids for liver disease include those containing acetate and/or gluconate instead of lactate, such as plasma-lyte or normosol-r (abbott laboratories, abbott park, il). the liver is the primary site of lactate clearance, whereas muscle and renal metabolize acetate and gluconate is utilized by most tissues. though accumulation of lactate from lrs does not lead to lactic acidosis (the cation associated with lactate in lrs is sodium as opposed to hydrogen ion), lactate is depressive to the myocardium; therefore iatrogenic increases in lactate concentration should be avoided. foals with septic shock, such as those with tyzzer's disease, should be treated with aggressive fluid therapy. boluses of to ml/kg of isotonic replacement crystalloids or to ml/kg of colloids should be administered over to minutes until the hypovolemic crisis is improved or fails to improve with further volume. if hypotension persists despite volume resuscitation, inotropes and pressors are indicated. if systemic blood pressure does not normalize in the face of a maximum or high cvp (> cm h o), dobutamine should be administered as a β-agonist inotrope ( to μg/kg/ minute). lastly, vasopressors should be administered. norepinephrine ( . to μg/kg/minute) or vasopressin ( . to mu/kg/minute) can be used in an attempt to normalize blood pressure and perfusion (see chapter ). oxygen insufflation ( to liters/ minute) should be provided to foals with central depression, hypoventilation, or concurrent respiratory tract disease. because hepatic biotransformation and elimination of drugs is reduced, medications metabolized by the liver should be used judiciously and dosage alterations should be considered. antimicrobial therapy is indicated in cases of bacterial cholangiohepatitis or hepatitis. broad-spectrum antimicrobials should be administered to foals with cholangiohepatitis. a combination of beta lactam, such as penicillin or ampicillin, and aminoglycoside, such as amikacin or gentamicin, is a reasonable initial choice. ceftiofur is also a good choice, and potentiated sulfonamides can be used for long-term therapy. foals with tyzzer's disease may be treated with any of a number of antimicrobials as the agent appears very susceptible to penicillin, gentamicin, tetracycline, and metronidazole. plasma transfusions ( to ml/kg) can be beneficial to foals with liver disease. plasma provides additional colloid support, albumin and immunoglobulin replacement, and provision of antithrombin or clotting factors for foals with coagulopathies. dextrose supplementation in polyionic nonlactated fluids should be provided. potassium chloride should be supplemented even if potassium concentrations are normal because it aids in renal excretion of ammonia. neomycin ( to mg/kg po, once) or metronidazole (low doses because of reduced hepatic metabolism ( mg/kg po q h) may be administered to reduce enteric bacterial production of ammonia. alternatively, lactulose can be used and is preferred by the author ( . to . ml/kg po q to h). lactulose is a synthetic disaccharide that bypasses the small intestine. fermentation in the colon results in increased nitrogen incorporation into the enteric flora, with less available for absorption. lactulose also reduces the ph of ingesta, resulting in inhibition of ammonia generation and increased ionization of ammonia with ion trapping within the gi lumen. for severe neurologic signs, mannitol ( . to . g/kg) may be administered if increased intracranial pressure is suspected. sodium bicarbonate should not be administered unless a significant inorganic acidosis is present; rapid correction of acidemia can increase the concentration of ionized ammonia and exacerbate clinical signs. benzodiazepines, such as diazepam, should be avoided in foals with hepatic insufficiency as they can potentiate clinical signs of hepatic encephalopathy by enhancing the effects of gaba. seizures should be controlled with phenobarbital or pentobarbital. flumazenil ( . to . mg/kg slowly iv), a benzodiazepine antagonist, can be tried in foals with clinical signs of hepatic encephalopathy that are unresponsive to other therapeutics. nutritional support of foals with hepatic disease is vital to a successful outcome. parenteral nutrition should be administered to foals with fulminate hepatitis or liver dysfunction, including those with tyzzer's disease. formulations for patients with hepatic failure should be utilized. protein requirements should be met but not exceeded in order to avoid contributing to hyperammonemia. formulations with increased branch chain amino acids (valine, isoleucine, leucine) and decreased aromatic amino acids (phenylalanine, tryptophan, tyrosine, methionine) are being evaluated for use in human liver patients. vitamin k should be administered in foals with long-term liver disease. since the liver is the primary source of vitamin c synthesis and storage of vitamins a, d, and riboflavin, a multivitamin should be provided to foals with chronic liver disease. b vitamins should be administered diluted in fluids. vitamin e can be supplemented as an antioxidant. foals with portacaval shunts should be stabilized for hepatic encephalopathy, and then surgical correction can be attempted after diagnostic venography to localize the shunt. gastric ulcer prophylaxis is indicated in select cases, particularly those with hypovolemic or hypotensive shock. sucralfate is advantageous in that it does not undergo hepatic biotransformation. for treating ulcers, famotidine or ranitidine should be utilized, because unlike cimetidine, they are excreted primarily in the kidneys and do not inhibit hepatic metabolism of other drugs. tyzzer's disease, which is uniformly fatal. the outcome in tyzzer's disease is generally grave. there are currently no reports of survival in documented (definitively diagnosed) cases. some cases will improve temporarily with volume replacement and administration of dextrose and sodium bicarbonate. however, there are three reported cases and a few anecdotal reports of survival in presumptive cases of clostridium piliforme infection. , early therapy with antimicrobials and aggressive intensive care including parenteral nutrition appear to be key to a successful outcome. gastric ulceration in an equine neonate endoscopic appearance of gastric lesions in foals: cases pathophysiology of peptic disorders in foals and horses: a review the effects of stress on gastric ulceration, t , t , reverse t and cortisol in neonatal foals gastric ulceration in foals in the hidaka district gastroduodenal ulceration effect of ranitidine on intragastric ph in clinically normal neonatal foals intragastric ph in critically ill neonatal foals and the effect of ranitidine stress-induced gastric findings in critically ill newborn infants: frequency and risk factors gastric and duodenal ulceration in the critical neonate. proc international veterinary emergency and critical care society clinical and pathological effects of flunixin meglumine administration to neonatal foals phenylbutazone toxicosis in the foal gastroesophageal ulceration and candidiasis in foals nutritional support: enteral and parenteral treatment and prevention of equine gastric ulcer syndrome the protective effects of sucralfate and ranitidine in foals experimentally intoxicated with phenylbutazone effect of omeprazole paste on intragastric ph in clinically normal neonatal foals safety, acceptability, and endoscopic findings in foals and yearling horses treated with a paste formulation of omeprazole for twenty-eight days is prophylaxis for gastric ulcers necessary in critically ill equine neonates: a retrospective study of necropsy cases - . proc th american college veterinary internal medicine scientific forum ulcerative duodenitis in foals nutritional support: enteral and parenteral faecal composition in foal heat diarrhea mares' milk composition as related to "foal heat" scours sand-induced diarrhea in a foal effect of protein source in liquid formula diets on food intake, physiologic values, and growth of equine neonates necrotizing enterocolitis in two equine neonates rotavirus infection in foals foal diarrhea between and in the united kingdom associated with clostridium perfringens, rotavirus, strongyloides westeri, and cryptosporidium spp field study of the safety, immunogenicity, and efficacy of an inactivated equine rotavirus vaccine prevention of rotavirus diarrhoea in foals by parenteral vaccination of the mares: field trial neonatal enterocolitis associated with coronavirus infection in a foal: a case report characterization of a coronavirus isolated from a diarrheic foal adenovirus in the intestinal epithelium of a foal with prolonged diarrhea association of clostridium difficile with enterocolitis and lactose intolerance in a foal isolation of clostridium difficile and detection of cytotoxin in the feces of diarrheic foals in the absence of antimicrobial treatment hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals asymptomatic neonatal colonization by clostridium difficile clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility enterocolitis associated with clostridium perfringens infection in neonatal foals: cases populationbased study of fecal shedding of clostridium perfringens in broodmares and foals an outbreak of equine neonatal salmonellosis actinobacillus sp. bacteremia in foals: clinical signs and prognosis experimental inoculation of foals and pigs with an enterotoxigenic e. coli isolated from a foal isolation of enterotoxigenic escherichia coli from a foal with diarrhea streptococcus durans: an unexpected enteropathogen of foals clostridium sordelli isolated from foals diarrhea associated with enterotoxigenic bacteroides fragilis in foals cryptosporidium in immunodeficient arabian foals prevalence of and risk factors for fecal shedding of cryptosporidium parvum oocysts in horses identification of cryptosporidium parvum "cattle" genotype from a severe outbreak of neonatal foal diarrhea foal diarrhea; pathogenesis, etiology and therapy volume replacement in the neonatal icu: crystalloids and colloids intensive insulin therapy in the critically ill patient evaluation of the ability of di-tri-octahedral smectite to adhere to clostridium difficile toxins and clostridium perfringens enterotoxin in vitro. proc th annual convention of the american association of equine practitioners characterization of clostridium difficile isolates from foals with diarrhea: cases antimicrobial susceptibilities of equine isolates of clostridium difficile and molecular references . brown cm, ainsworth dm, personett la et al: serum biochemical and haematological findings in two foals with focal bacterial hepatitis (tyzzer's disease) bacillus piliformis infection (tyzzer's disease) in foals in northwestern united states: a retrospective study of cases the pathology of a case of biliary atresia in a foal tyzzer's disease in an -day-old foal tyzzer's disease in a foal nutritional support: enteral and parenteral a comparison of the clinical, clinicopathological, and bone marrow characteristics of foals with equine herpes and neonatal septicemia pulmonary abscessation, hepatoencephalopathy and igm deficiency associated with rhodococcus equi in a foal four possible causes of hepatic failure and/or icterus in the newborn foal clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus- infection compared with septic and premature foals neonatal equine herpesvirus type infection on a thoroughbred breeding farm urinary and hepatic disorders in neonatal foals the diagnosis and surgical correction of congenital portosystemic vascular anomalies in two calves and two foals clinical signs and radiographic diagnosis of a portosystemic shunt in a foal clinical and diagnostic features of portosystemic shunt in a foal persistent vitelline arteries in a foal congenital hepatic fibrosis and cystic bile duct formation in swiss freiberger horses persistent hyperammonemia in two related morgan weanlings glycogen branching enzyme deficiency in quarter horse foals glycogen branching enzyme (gbe ) mutation causing equine glycogen storage disease iv hepatoblastoma in a foal sm: hepatoblastoma in an equine fetus iron toxicity in neonatal foals hepatotoxicosis in neonatal foals (letter) steroid hepatopathy in a horse with glucocorticoid-induced hyperadrenocorticism glucocorticoid-induced laminitis with hepatopathy in a thoroughbred filly serum biochemical indicators of liver function in neonatal foals liver failure and hemolytic anemia serum protein concentrations in horses with severe liver disease: a retrospective study and review of the literature disorders of the liver clinicopathological features of equine primary hepatic disease: a review of cases clinical and clinicopathologic findings in two foals infected with bacillus piliformis tyzzer's disease in a foal tyzzer's disease in foals multifocal hepatic necrosis and hepatitis in foals caused by bacillus piliformis naturally-occurring tyzzer's disease (bacillus piliformis infection) in horse foals tyzzer's disease risk factors for clostridium piliforme infection in foals tyzzer's disease in an -day-old foal neonatal hepatic failure in a thoroughbred foal: successful treatment of a case of presumptive tyzzer's disease key: cord- -rrverrsj authors: delano, margaret l.; mischler, scott a.; underwood, wendy j. title: biology and diseases of ruminants: sheep, goats, and cattle date: - - journal: laboratory animal medicine doi: . /b - - / -x sha: doc_id: cord_uid: rrverrsj nan since the first edition of this book, the use of ruminants as research subjects has changed dramatically. formerly, large animals were primarily used for agricultural research or as models of human diseases. over the past decade, ruminants have continued in their traditional agricultural research role but are now extensively used for studies in molecular biology, genetic engi-british stock with egyptian and indian goats. this breed is relatively heat tolerant and produces milk with the highest butterfat (about - %). fiber breeds include the angora and the cashmere. the angora, the source of mohair, originated in turkey. the cashmere breed is found primarily in mountainous areas of central asia. the la mancha, a newer breed of dairy goat first registered in the united states in , has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. the meat breeds include the boer, sapel, ma tou, kambling, and pygmy. the pygmy goat is small and is sometimes used for both meat and milk. the mubend of uganda and the red sokoto of west africa produce quality skins for fine leather (smith and sherman, ) . most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." common dairy breeds in the united states include holstein-friesian, brown swiss, jersey, ayrshire, guernsey, and milking shorthorn. holsteins have the largest body size, whereas jerseys have the smallest. of breeds in temperate regions, jerseys have been considered to be the most heat tolerant, but holsteins have been found to adapt to warmer climates. there are many beef breeds. the more common in the united states include angus (also called aberdeen-angus), hereford (both polled and horned), and simmental (briggs and briggs, ; schmidt et al., ) . breeds indigenous to other continents, such as the cape buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis (muranjan et al., ) . more detailed information regarding these and other ruminant breeds is available in briggs and briggs ( ) . "rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. discussions of these and efforts at conservation are described in detail elsewhere (national research council, ) . several terms are unique to ruminants. in relation to sheep, a ewe is the female, and a ram is the adult intact male. a lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. a wether is a castrated male. the birthing process is referred to as lambing. with respect to goats, a doe or nanny is the female. a buck or billy is the adult intact male. a kid or goatling is a young goat. a young male may be referred to as a buckling, and a young female may be referred to as a doeling. a castrated male in this species is also called a wether. the birthing process is called kidding. with respect to cattle, an adult female is a cow, and an adult male is a bull. a calf is a young animal. a heifer is a female who has not had her first calf. a steer is a castrated male. calving refers to the act of giving birth. ruminants have been used as research models since the inception of the land grant college system, first in production agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves (salerno et al., ) . for many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle (wall et al., ) . several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species (ebert et al., ; schnieke, ; cibelli et al., a,b) (see fig. ). one of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated (ebert et al, ; memon and ebert, ) . healthy sheep and goats are also often used for antibody production (hanly et al., ) . genome mapping developed rapidly during the s; extensive information is available and is increasing for sheep and cattle (broad et al., ; womack, ) . sheep are often selected for studying areas such as ruminant physiology and nutrition. these animals provide obvious bene-fits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. sheep are also widely used models for basic and applied fetal and reproductive research (buttar, ; rees et al., ; ross and nijland, ) . the species is used for investigating circadian rhythms related to day length (lehman et al., ) , and the interaction between olfactory cues and behavior (kendrick et al., ) . the number and diversity of natural-and induceddisease research models in sheep are great and increasing. natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (dubin-johnson syndrome) in the corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (gilbert syndrome) in the southdown breed, glucose- -phosphate dehydrogenase deficiency in the dorset breed, gm~ gangliosidosis in the suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (hegreberg, l a) . induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (wilson's disease), and metabolic toxocosis (hegreberg, lb) . goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. vascular researchers select the goat because of the large, readily accessible jugular veins. goats with inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (thomsen's disease) (kuhn, ) . a line of inbred nubians serves as models for the genetic disease [ -mannosidosis and prenatal therapeutic cell transplantation strategies (lovell et al., ) . (these disorders are discussed in more detail in section iii,b, .) goats are used as a model for osteoporosis research (welch et al., ) . cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid (dougherty, ) . cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. this species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. bovine trichomoniasis, caused by tritrichomonas (trichomonas)fetus, has been identified as a useful model for the human infection by trichomonas vaginalis (corbeil, ) . inherited cardiomyopathies have been found in the holstein-friesian, simmental-red holstein, black spotted friesian, and polled hereford with woolly coat (weil et al., ) . lipofuscinosis has been identified in ayrshires and friesians, and glycogenesis in shorthorns and brahmans. metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in holstein-friesian or friesian cattle. holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome (afip, ) . common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and -h networks, regional ruminant breeders' associations, and farm bureaus. commercial sources of purposebred animals are found in technical publications and annual listings of research animal vendors. breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the armed forces institute of pathology. organizations such as the institute for laboratory animal research (ilar), national center for research resources (ncrr), or the animal welfare information center (awic) may also serve as information sources about the animals needed. purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. these commercial animals are frequently described as specific pathogen-free (spf) and housed as biosecure or closed flocks. animal health programs are in place, and health reports or other quality assurance reports are usually available on request. agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. management practices exist, however, to extend the breeding and milking seasons. most cattle used as animal models in research in the united states are from one of the dairy breeds, usually holstein, because this breed is now the most common. purpose-bred, specific pathogen-free research cattle are not typically available. because of selection and the management of dairy production units, calves and young stock are available year-round. availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. auction barns or sales are not appropriate sources for research ruminants. many of these animals are culls and will be poor-quality research subjects. they may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. selection of the suppliers should be made only after research needs have been carefully considered. consistently working with and buying directly from as few sources as possible are best. certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. the selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. preliminary and periodic visits to the source farms should be conducted. it is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. they may need to be oriented on the specific requirements of animal research. creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. supplying the vaccines or dewormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. a set of testing standards can be developed based on one high-quality supplier, and then flocks or herds can be "qualified" based on those standards. qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. the testing regimen itself should be carefully developed and evaluated. once qualified, each source farm should be reevaluated periodically to maintain its status. slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. vaccination and deworming regimens can be instituted during these quarantine periods. a second quarantine should occur when animals arrive at the research facility. the animal screening process also depends on the origin of the animal (state, country) and the scientific program. federal and state regulations must be followed. socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. for example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. these references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ("current veterinary therapy," , "council report," ; "large animal internal medicine," ; smith and sherman, ) when designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. it is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. typical health screening programs for sheep include q fever (coxiella burnetii); contagious ecthyma; caseous lymphadenitis (corynebacterium pseudotuberculosis); johne's disease (mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intes-tinal worms; and external parasitism such as sheep keds. each supplier should be queried about vaccination programs for bluetongue, brucella ovis, campylobacter spp., chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza , pasteurella haemolytica, and p. multocida), ovine ecthyma, rabies, dichelobacter (bacteroides) nodosus, arcanobacterium pseudotuberculosis, bacillus anthracis, and fusobacterium necrophorum. because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ("council report," javma, ) . in some cases, approved feed additives, such as coccidiostats, are fed to sheep. the basic screening profile for goats should include q fever (coxiella burnettii), caprine arthritis encephalitis (cae), brucellosis, tuberculosis, and johne's disease (mycobacterium paratuberculosis) . goats may also be tested for caseous lymphadenitis, contagious ecthyma, or mycoplasma as needed. herd vaccination programs may include immunizations against tetanus and other clostridial diseases, chlamydia, campylobacter, contagious ecthyma, caseous lymphadenitis, corynebacterium pseudotuberculosis, and escherichia coli. cattle herds should be screened for johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. determination of the status of the herd with respect to bovine leukemia virus (blv) may be worthwhile. herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), bovine respiratory syncytial virus (brsv), parainfluenza (pi- ), leptospira pomona, tritrichomonas fetus, rotavirus, coronavirus, campylobacter (vibrio) , pasteurella haemolytica and p. multocida, and brucella abortus. other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the clostridial diseases, moraxella bovis (pinkeye), fusobacterium necrophorum (foot rot), staphylococcus aureus (mastitis), haemophilus somnus, rabies, tetanus, bacillus anthracis, enterotoxigenic e. coll anaplasma, and other leptospira species. some products considered to have limited efficacy include vaccines against salmonella dublin and s. typhimurium. some autogenous vaccines may be more effective than the commercially available products--for example, the bovine papillomavirus (warts) vaccines. rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by hours after birth. in the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminal organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. use varies by the segment of the industry, and regulations vary by country. subcutaneous hormonal implants (such as estradiol benzoate and progesterone combined, zeranol, or [~-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. it is best to have the animals transported in vehicles regularly utilized by the source farm. if commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. the loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("livestock handling and transport," ). recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the u.s. department of agriculture (usda) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. the usda animal care policy notes that the "guide for the care and use of agricultural animals in agricultural research and teaching" and the "guide for the care and use of laboratory animals" provide additional information to supplement the existing animal welfare act regulations (cfr, ; fass, ; hays et al., ; nrc, a; usda, ) . in all cases, stress should be considered and minimized in the husbandry and handling of ruminants. animals need to be provided adequate time to adapt to new surroundings. stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. reproductive soundness and rumen function are affected by transport and similar stresses. standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors (houpt, ) . animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. ap-preciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published (houpt, ; grandin, ) . when ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. ammonia buildup and other waste gases may induce respiratory problems. in cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. waterers should not create a muddy environment in paddocks or pens. there should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. forelimbs may be caught easily in the mesh. floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. all ruminants are social and herding animals. therefore, they should be housed in groups or at least within eyesight and hearing of other animals. singly housed animals should have regular human contact. environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. calves, in particular, that must be singly housed or that have been recently weaned, need play objects (morrow-tesch, ) . because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. normally, sheep and goats should be maintained on a cycle comparable to natural conditions. light intensity should be maintained at about lux (ilar, ; fass, ) . light cycles can be manipulated for experimental reasons. the development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. there is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). the mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. digestion is also aided by other microorganisms, such as protozoa ( - /ml) and bacteria ( - ~ that contribute to rumen fermentation. the result is the production of volatile fatty acids (acetic, propionic, and butyric) . unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. the main sources of energy for ruminants are volatile fatty acids (vfas) rather than glucose. glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (cns), uterus, and mammary glands. plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. the rumen microorganisms also synthesize vitamins, such as b and k, and provide protein that is used by the animals' systems. large amounts of fermentation gases such as co and methane, and small amounts of nitrogen, are naturally eructed (hecker, ; schimdt et al., ) . intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. this transfer mechanism is functional for approximately the first hr after birth. neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (ibr) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mamrnary gland (hecker, ) . in addition to normal lymph node chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. inadvertently named hemal "lymph nodes," they contain numerous red blood cells. sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. three major ovine histocompatability classes have been identified and designated as ovar (ovis aries) classes i, ii, and iii (franz-werner et al., ) . bovines are recognized as having several unique aspects involving their immune systems. the bovine lymphocyte antigen (bola) system ranks after the hu-man (hla) and murine (h- ) systems in terms of depth of knowledge (lewin, ) . cattle are considered free of autoimmune diseases (schook and lamont, ) . the complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described (sordillo et al., ) . hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables (duncan and prasse, ; jain, ; kaneko et al., ) . these references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. in addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see table i ) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. normal hematologic and clinical biochemistry data are presented in tables ii and iii. some general statements apply to most ruminants. most ruminants have fewer neutrophils than lymphocytes. the blood urea nitrogen (bun) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. severe dehydration can occur quickly, however, in animals that are ill. urine ph is generally alkaline in adult ruminants. ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. increased red-cell fragility is also associated with the smaller erythrocyte. rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. in addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins (hecker, ) . blood coagulation in sheep is similar to that in humans. (di / , dc / , dp / ) = (di / , dc / , dp / ) = (di / , dc / , dp / ) = permanent dental formula ( / , c / , m / ) = ( / , c / , m / ) = ( / , c / , m / ) = avital sign data for goats are from "large animal internal medicine" ( ) . sheep weight data represent weights of feeder lamb and adult dry ewe (federation of animal science societies [fass], ) . goat weight data are for a large-breed male goat. cattle weight data represent weights of female holstein or guernsey dairy cattle (fass, ) . life span data for sheep and cattle are from brooks et al. ( ) . erythrocytes in pygmy and toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. normal caprine erythrocytes lack central pallor because they are fiat and lack biconcavity. normal caprine erythrocytes may exhibit poikilocytosis. at least five blood groups have been reported in goats: b, c, m, r-o, and x. because transfusion reaction rates may be as high as - %, cross-matching is advisable although not always practical (smith and sherman, ) . blood loss of up to % of the red cell mass at a single time point can be tolerated by healthy goats. blood may safely be obtained in volumes of ml/kg body weight and given in volumes of - ml/kg. in general, aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) are not liverspecific in goats, and alanine aminotransferase (alt; formally serum glutamic-pyruvic transaminase, or sgpt) cannot be used to evaluate hepatic disease in goats. ~,-glutamyltransferase (ggt) and alkaline phosphatase (ap) are associated with biliary stasis, and elevations in ggt are generally associated with hepatic damage. the nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. for example, mineral requirements and other nutritional requirements vary even among breeds of cattle. several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available (jurgens, ; "large animal clinical nutrition," ; nrc, nrc, , nrc, , nrc, , b "large animal internal medicine," ) . preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. concentrate mixtures contain salt, minerals, and other elements. concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. computer programs are also readily available for those who may need to formulate and balance rations. the palatability of feeds should be taken into account. mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility (nrc, ) . good-quality pasture can support ruminants under certain circumstances. lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. when ruminants have access to pasture, it is important to be aware of different eating habits. sheep and cattle are grazers. goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. therefore, goats have a tendency to "waste" hay. other eating habits should also be considered. finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large particles in mixes. generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. when given access to a salt block, goats generally are self-regulating. grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not (bretzlaff et al., ) . horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses (bretzlaff et al., ) . the copper content of horse feed is not excessive for goats, as it is for sheep. pelleted horse feeds with - % fiber and - % protein are good goat rations. goats will consume - % of body weight in dry-matter intake (whereas cattle will usually consume only % of body weight). goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. these may also occur when forage grasses are high in silicates and oxalates. to increase ovulation rate in does, some producers "flush" females by feeding . - lb concentrate per head per day for several weeks before and after the initiation of the breeding season. thin pregnant dairy goats should be fed lb concentrate per ) . - . ( . ) . - . potassium (k; mmol/l) hp . - . ( . ) . - . ( . ___ . ) . - . ( . adata presented as ranges with mean and standard deviation in parentheses, s, serum; p, plasma; hp, heparinized plasma. clinical biochemistry data from kaneko et al. ( ) . day, with the amount increasing to . lb per head per day during the last weeks of gestation. forage should be fed ad libitum during this time. all newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as igg ), functional leukocytes, cytokines, vitamins, minerals, and protein. colostrum also has laxative properties. trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. the quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. most neonatal ruminants can suckle well within hr of birth. those that do so have been shown to have significantly less diarrhea (naylor, ) . neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. if necessary, the dam should be hand-milked and the newborn fed colostrum (for example, - ml for kids) every - hr for the first - days. in typical management situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about hr and suckle fresh colostrum during this time. dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted : with warm water times a day to the calves during the next - days. extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. holstein calves, for example, should receive a minimum of - liters within hr of birth and then be fed about - % of body weight in colostrum by hr of age. after days, calves are then placed on milk replacers. although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. containers used to prepare and feed these replacers should be sanitized daily. the fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed. young ruminants can be offered good-quality hay (such as second cutting) to nibble on by week of age. calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at - weeks of age. they can be weaned off milk replacer by - weeks of age. young ruminants ( - months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. in farm management situations, forage can be silage, pasture, and hay. in a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. animals should not be overfed and should be offered a mineral mix free-choice. in contrast to dairy calves, beef calves remain with their mother cows until weaning at months of age. calves tend to suckle many times per day. as they mature, calves are creepfed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. several useful references addressing ruminant reproduction in detail are available ("current veterinary therapy: food animal practice," practice," , practice," , "large animal internal medicine," ; "current therapy in large animal theriogenology," ; hafez, ) . sheep are seasonally polyestrous; most breeds will express estrus in the fall (northern hemisphere) and subsequently lamb in the spring. some breeds of sheep may cycle in both the fall and the spring. between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. in a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in hr of light and hr of dark for - weeks. puberty is reached at about - months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. ewes will display signs of estrus for about - hr and will ovulate spontaneously at the end of estrus. the estrous cycle length is - days, with an average of about days. following breeding, the average length of gestation is - days. slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from merinos. prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). the finn and dorset breeds are especially prolific. lambs vary in size at birth from about - lb up to lb. factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. they are shortday breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. in temperate climates of the northern hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. the actual length of the sexual cycle depends on day length, breed, and nutrition. most dairy goats cycle between august and february or march. nubians often have extended breeding cycles, and the sexual season of some breeds, including the alpine, can be extended by artificial means. the caprine gestation length averages days with a variation of - days. does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. cows are polyestrous. domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. in spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. reproductive physiology in cattle is influenced by many factors. the reproductive programs in source herds and at well-managed facilities will be production-related. extensive coverage of both physiologic basics and specific industry-related criteriamfor retention of a cow as a breeder, for examplenare addressed in detail in texts and references oriented toward herd and production management ("current veterinary therapy," ). gestation in cattle is approximately days, with a range of - days. the length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. as noted, these factors are also important in sheep and goats. cows usually bear single calves, although twin births do occur. when twins are combinations of male and female calves, the female should be evaluated for freemartinism. ovine estrus detection is usually accomplished by the ram. nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. ewes may isolate from the flock and appear anxious. it is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. two mating systems commonly employed include hand mating and group mating. with hand mating, ewes are placed either singly or in small groups with the ram of choice. ewes are removed as serviced. group mating involves placement of a mature ram with approximately - ewes for the entire -week breeding season. in either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. this is important so that parturition dates can be calculated. an easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. any animal marked by the ram probably has not conceived. ultrasound scanners are also used for pregnancy detection. the ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. claims of % accuracy at weeks postbreeding have been made, although accuracy is generally best beyond days of gestation. interrectal doppler ultrasound probes detect fetal pulses. fetal heart rate is in the range of - beats per minute, whereas maternal heart rates tend to be - beats per minute. accuracy is best beyond days of pregnancy. rectal-abdominal palpation is an inexpensive alternative. a plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. the plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. the age of the doe when she first expresses heat varies with breed. some does will express signs of heat between and months old. however, does should be - months old or at least - lb in weight before being bred. the caprine estrous cycle lasts - days. the duration of estrus is - hr but averages about hr. the estrous cycle can be more erratic in the beginning than in the end of the breeding season (smith, season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). in some cases, the presence of mature cycling cows influences heifer puberty. with adequate nutrition, dairy breeds will reach puberty at - months and beef breeds at - months, and estrous cycles will occur regularly after the pubertal (first) estrus, maturing heifers will often have one or more ovulations before showing overt signs of estrus. only one follicle usually ovulates per estrous cycle (hafez, ) estrus, or standing heat, in cattle averages - hr in length, with a range of - hr ("large animal internal medicine," ) . detection of standing heat is important because it is closely related to the time of ovulation. ovulation occurs approximately - hr after estrus. detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. teaser animals outfitted with marking devices are also used. other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal ph; and body temperature changes (hafez, ) . estrous cycles are usually days in length, with a range of - days. it is recommended that a heifer deliver her first calf by years of age. after successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the ). "standing heat" is usually - hr but can be as short corpus luteum of pregnancy, and they decline only during the as a few hours. signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. a doe that is not in heat will not stand to back pressure or for attempts to hold her tail. does can be induced to show signs of heat by buck exposure and will ovulate within - days after introduction of the buck. goats ovulate during the later part of the estrous cycle, most between - hr after the onset of estrus. nevertheless, goats should be mated once signs of estrus are recognized and every hr until the end of estrus. most goats kid only once a year, although some goats near the equator may kid twice. once bred successfully, a goat will only rarely show signs of heat again. in fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. pregnancy can be affirmed by a variety of means. goats will generally decrease milk production with pregnancy and should have at least a -to -week dry period for the udder to fully involute and prepare for the next milking period. in cattle, age of first estrus is dependent on the breed, the final month. conceptus implantation occurs beginning at about day . if the pregnancy fails before this time, the cow will begin to cycle again between days - , but if the pregnancy ends after day , there may be a delayed return to estrus. realtime ultrasonography can be used to determine pregnancy as early as days after insemination, with embyros seen by days - . fetal gender can also be determined by experienced personnel by this method by about day . detection of pregnancy can be successful by - days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days - and/or amniotic vesicle by days - ). palpation of the fetus is possible by day and placentomes by approximately days - . palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days - after insemination. levels of bovine pregnancy-specific protein b may also be measured; this is produced by trophoblastic cells and is detectable by days - and elevated throughout pregnancy. placentation in sheep, goats, and cattle is epitheliochorial and ft. evaluation of a cow's udder prior to breeding and especotyledonary, in contrast to the diffuse or microcotyledonary cially as parturition approaches is important in order to assure placentas of horses and pigs. the placentomes, the infolded adequate nutrition and success of passive transfer by the functional units of the placenta, are formed as the result of fu-neonate. if the udder is edematous or if mastitis is present, for sion of the villi of the fetal cotyledons projecting into the crypts example, an alternate source of colostrum (such as frozen reof the maternal caruncles (specialized projections of uterine " serves) must made be available. poor udder conformation may mucosa). caruncles of sheep and goats are concave in shape, whereas those of cows are convex. the placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are - . in cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. the placentomes in the nongravid horn will be smaller than in the gravid horn. the total number will be - . the best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. if possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. the birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. outdoor parturition in a small birthing pasture has advantages. there is less stress and less intensity of pathogens. indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. moving the female immediately before or during parturition may delay the birthing process. in goats, furthermore, in utero death may occur if parturition is unduly delayed. dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. prior to parturition, ewes should be sheared or crutched. crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. foot trimming can be done at this time as well. the tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. in general, the pregnant doe needs a ft ( . m x . m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. each cow should have a minimum pen area of ft x also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. the pelvic region will appear relaxed, and milk will be present in the udder. once hard labor contractions begin, lambs will usually be born quickly. animals that do not appear to be progressing correctly should be examined for dystocia. most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. occasionally cesarean sections will be necessary. sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. for about a week before parturition, rectal temperature of the doe will be above normal, or about ~ depending on environmental temperatures. approximately hr prior to birth, rectal temperature will fall to slightly below normal. many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. the drug of choice to induce parturition in the goat is prostaglandin f ~ (pgf ~) (ott, ) . on day of gestation, goats given pgf ~ ( . - mg) will deliver kids within - hr. most goats prefer to kid alone and do so unaided. human interaction can actually interfere with normal birthing, especially in young or nervous does. some does may reject kids if extensive human interference occurs. does nearing parturition have an obviously swollen udder and a red, swollen vulva. pelvic ligaments at the base of tail relax. the doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. eight to hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. kids should present within - hr in either anterior or posterior position. a posterior presentation can be recognized by the presence of upward-pointing feet. most does will rest between fetuses and are best left alone. however, if labor is prolonged more than hr, a vaginal exam is indicated. if the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. goats are not prone to retained placenta. normal kids will be quite active and will quickly attempt to stand and nurse. weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. the goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. this is a fairly common condition. does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. subsequent pregnancies can be normal. goats should be tested for pregnancy by days of age. veterinary use of prostaglandins has been successful in treating this condition. as in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably acth (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within days of birth. administration of acth to a fetus, or administration to the dam, results in premature birth. pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. the fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. coincident with this, maternal progesterone levels fall. the rising levels of estrogen cause release of maternal pgf ~ and induction of oxytocin receptors. most cows will separate themselves from the rest of the herd. a cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. typically, the whole birthing process takes about min. the length of labor of cows carrying larger calves also will be longer. nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. all postparturient animals should be monitored for successful passage of these fetal membranes within hr of birth. veterinary intervention is required if not. cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. for cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. in a well-managed flock, many lambs and ewes will not need much assistance. when assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. the lamb should be dried off and stimulated through rubbing to aid its breathing. the lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. and the lamb should be identified by the application of an ear tag or ear notch. it is extremely important that the lamb be supplied with highquality colostrum within the first hr of birth. lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. passive transfer can be assessed by measuring serum y-glutamyltransferase (ggt) levels (tessman et al., ) . after the first few days, colostrum changes over to milk. nursing lambs will ingest increasing amounts of milk as they grow. if the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. one report notes that - % of lamb deaths occur during the first week of life and up to % occur within the first month. good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses (ross, ) . immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. kids do not usually need assistance. if kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. to control caprine arthritis encephalitis (cae), kids should be immediately removed from the dam and hand-fed heattreated colostrum. colostrum should be heat-treated for hr at ~ e the first feeding can be up to ml of colostrum. kids should receive a total of ml colostrum within the first - hr of birth. after day , kids can be placed on milk replacer. milk replacers should contain - % fat and - % milkbased protein. by days of age, kids should be consuming approximately . - . liters of milk per day. kids should be introduced to forages as soon as possible and may be weaned by - weeks or - lb body weight. milk that is fed can be reduced by weeks of age by decreasing either the volume fed or the number of feedings. as with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. calves typically are standing by hr after birth and are suckling within hr. as noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. a stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) (house, ; kersting, ; kasari and roussel, ) . because newborn calves can be deficient in vitamin a and iron, these may be injected to improve disease resistance (wikse and baker, ) . in cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at day old and followed with boosters at regular intervals. dehorning is performed when horn buds appear. castration is performed between and weeks of age or later. sexing the young in any of the ruminant species is straightforward. the vulva of the female young is located just ventral to the anus. the genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. the phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of bloodforming cells and germ cells results in the xx/xy chimeras. this occurs in - % of phenotypic bovine females born as co-twins with males. the female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. sometimes singleton freemartins are born if the male fetus is lost after days' gestation. multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. twinning is common in goats, and freemartinism occurs in about % of male-female pairs of twins. intersexes are seen in some goat breeds and when polled goats are mated. proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. to prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. therefore, the lambs will be accustomed to the new ration through this creep-feeding process. if lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. lambs that are consuming . - lb of creep feed per day may be weaned. depending on the individual program, lambs may be weaned as early as weeks of age, although - weeks of age is more common. if ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. about - days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. at weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. the ewes should be monitored for postweaning mastitis and treated as necessary. ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. the lambs should be monitored to assure that they continue to gain weight and are eating the new ration. kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. kids can be weaned by - weeks or - lb. hand-fed milk should be reduced by weeks of age by reducing the volume fed or by decreasing the number of feedings. dairy calves are now usually removed from their dams immediately after birth. it is less common now to allow the calves to remain with their dams for about hr and suckle fresh colostrum during this time, because their intake will be inadequate. dairy producers refrigerate and/or freeze the colostrum produced during the first hr and feed this, diluted : with warm water, twice a day to the calves during the next - days. holstein calves, for example, should receive a minimum of - liters within hr of birth and then be fed about - % of body weight in colostrum by hr of age. after days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. milk replacers are commercially available and should be fed according to manufacturer's recommendations vaccination programs for calves vary with the preventive medicine program for the overall herd. passive immunity provided by colostrum from cows on sound management programs will last until a calf is about - months old; normally vaccinations are not necessary and are contraindicated during those first months. the duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at - months of age and continue with monthly booster immunizations until the animals are months old, when passive immunity is no longer a possibility. artificial insemination (ai) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. breeding animals artificially with fresh semen produces pregnancy rates averaging % (not unlike that of cattle); artificial insemination with frozen semen is less successful. several artificial insemination techniques have been used. laparoscopic ai involves the surgical instillation of semen into the uterus through a small abdominal opening. the procedure is successful but is technically involved and costly. cervical ai involves the transvaginal introduction of semen into the cervix. a modification of this technique (transcervical ai) allows for penetration through the cervix into the uterus. this method (called the guelph system for transcervical ai) leads to successful penetration into the uterus in up to % of ewes when performed by an experienced inseminator. artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. technicians performing the ai technique are available through commercial enterprises. dairy production employees are also trained. information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere (nebel, ) . because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. progesterone suppresses follicle-stimulating hormone (fsh) secretion, preventing animals from developing follicles and exhibiting estrus. artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. the progesterone is withdrawn in about - days, after which the fsh secretion will initiate the process of follicle development (trower, ) . estrus usually will occur in - hr (average is hr). a natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. it should be noted that introduction of a male during late anestrus will often stimulate ovulation in about days; however, this cycle will generally be without clinical signs of estrus (silent heat). vasectomy of rams is one method of producing sterile "teaser rams." introduction of the buck to a group of does will induce ovulation and may even synchronize does. does that are kept separate from the buck will show signs of estrus, will ovulate within - days, and will have normal pregnancies when introduced to a buck. bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. goats in the luteal phase of the estrous cycle, days - , are sensitive to pgf ~ ( . - mg im) and will show estrus in - hr postinjection (bretzlaff, ) . dosing cycling animals twice days apart will synchronize goats, and artificial insemination using this method has resulted in - % conception rates (bretzlaff, ; greyling and van niekerk, ) . programs for timed breeding have been described and involve administering progestogens (bretzlaff, ) . vaginal pessaries of fluorogestone acetate left in place for days in the doe followed by an injection of pregnant mare serum gonadotropin (pmsg) at the time of pessary removal have proven successful. also, when primed by pgf ~, an day regimen of fluorogestone acetate with pmsg given on day has been successful. synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. the methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. the options and dosing regimens are described in detail in veterinary clinical texts (wenzel, ; vanderboom et al., ) . in synchronization, the principle is lysis of the existing corpus luteum. the more common practices involve the use of products approved for use in cattle such as pgf ~, one of its analogs, or products containing estradiol valerate. progestogens are also used in conjunction with estradiol valerate. other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. superovulation regimens involve injections of fsh either alone or with pgf ~ at timed internals. estrus is expected hr after the final injection, and two inseminations are performed at hr intervals after estrus detection. preparation of recipients involves injection of pgf ~ or progestogens with gonadotropins such as pmsg. for greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. this method maximizes the genetic potential of the donor animal. the donor animal is hormonally superovulated and inseminated. in sheep, about week after breeding, the embryos are surgically removed from the donor's uterus. in cattle, the procedure is nonsurgical. about % of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. furthermore, not all collected embryos are of transferable quality. recipients are hormonally synchronized with the donor animals. on the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. pregnancy rates average about %. if recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. in sheep flocks and goat herds, as noted, male young are usually castrated by month of age. the elastrator method is the more popular for animals less than week of age. other methods include the emasculatome (crushing) and surgical removal ("knife method"). the distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently (kent et al., ) . as noted, male calves are usually castrated as early as possible and no later than month of age. in some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. smaller farms often borrow breeding males. breeding males are typically selected by production record, pedigree, and/or breed. vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. the vasectomy technique for both species is comparable (smith and sherman, ) . rams may be housed together for most of the year, whereas bucks are penned separately. because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. furthermore, it is impor-tant to know the semen quality of the ram as one indicator of fertility. semen can be collected via electroejaculation or by use of a teaser mount. once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between . and . ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately %); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain . x sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed % in high-quality semen). the extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. breeding bulls are maintained in beef production establishments. breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. tails are typically docked to about inches in length. the practice is more popular in certain regions in the united states. to date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. even adult animals, when provided sufficient space, will play. sheep and goats have tidy "pelleted" dark green feces. cattle have pasty, moist, dark green-brown feces. ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. excessive, strained vocalizations are often a sign of stress in cattle. "bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. unhealthy ruminants may be thin, may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. all ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. animals should be provided sufficient time to acclimate to handlers and research staff. cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. generally, fine-wooled breeds, such as rambouillet, are the most gregarious and are best handled in groups. the meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary (ross, ; asia, ) . nonetheless, movement of animals is simplified by proper facility design. sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. sheep should be moved slowly and gently. to capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. this minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. goats exhibit behavioral characteristics that make them quite distinct from other ruminants. their browsing activity makes them quite orally investigative. goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. a herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. if maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. goats with horns will use them to advantage, and horns may also become entangled in fencing. although less strongly affected by flock behavior, goats are social animals. most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. an understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result (houpt, ) . cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (grandin, ) . groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. in contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. beef breeds, such as angus, are known for their independent natures and protective maternal instincts. all cattle respond well to feed as a reward for desired behavior. healthy cattle typically are very curious and watchful and are alert to sounds and smells. when not grazing or eating, they hold their heads up. when sleeping, the head and neck may be tucked back. because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. occasionally, adult cows sit upright like dogs. cattle maintained inside tend to be more docile. in addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. these stressors may exacerbate signs of systemic illnesses. calves are known for non-nutritive suckling, bar licking, and tongue rolling. non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. it is best to provide nipples and other clean noninjurious materials for the animals to suck. non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. environmental enrichment devices have been developed to cope with this behavior. the behavior diminishes as the animals are weaned onto solid food (morrow-tesch, ) . play activity and vocalizations of calves mimic adult dominance behaviors. play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. horns, age, and weight have been reported to be the most important determi-nants. aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. aggression is more common among intact adult males. this section focuses primarily on the more common diseases affecting sheep, goats, and cattle in the united states and elsewhere in north america and those that are reportable. for detailed information not included in this limited overview and for diseases of importance internationally, the authors recommend several excellent comprehensive and focused veterinary clinical texts and periodicals that address ruminant diseases, preventive medicine, and individual and flock or herd management. these are listed under "major references" in the reference list at the end of this chapter. recommendations for current drug therapies, both approved and off-label use in ruminants, including withholding prior to slaughter, formularies, and related information can be found in the references noted above and in formularies (hawk and leary, ; plumb, ) . in addition, the food animal residue avoidance databank (farad), accessible on the internet <http://www.farad.org>, should be used as a resource. farad is a food safety project of the u.s. department of agriculture and is an information resource to prevent drug and pesticide residues in food animals and animal products. food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. diagnosis is made based on clinical signs and is confirmed by culture. epizootiology and transmission. the organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. rough feed material and foreign bodies may play a role in causing abrasions. actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. necropsy findings. purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. exudates will also contain characteristic small white-gray (sulfurlike) granules. the pus is usually nonodorous. differential diagnosis. contagious ecthyma and caseous lymphadenitis are the primary differentials. diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. treatment. animals should be fed softer feeds. antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. penicillin is not effective. weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. surgical excision and drainage are not recommended. etiology. actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. in sheep and cattle, a. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. this disease, called wooden tongue, has not been documented in goats. clinical signs. skin lesions are common. tongue lesions are more common in cattle than in sheep. lip lesions are more common in sheep. soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. animals may have difficulty prehending prevention and control. because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. arcanobacterium infection (formerly actinomycosis, or "lumpy jaw") etiology. arcanobacterium (formerly known as actinomyces or corynebacterium) pyogenes and a. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. this organism has also been associated with pharyngitis and mastitis in cattle. clinical signs and diagnosis. arcanobacterium bovis causes mandibular lesions primarily. the mass will be firm, nonpainful, and immovable. draining tracts may develop over time. if teeth roots become involved, painful eating and weight loss are evident. radiographic studies are helpful for determining fistulas. diagnosis is based on clinical signs, and culture is required to confirm arcanobacterium. the prognosis is poor for lumpy jaw. epizootiology and transmission. these organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. necropsy. draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. ious degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. research complications. young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. pathogenesis. arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. differential diagnosis. actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. a major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. there are many differentials for septic joints and polyarthritis: chlamydia spp., mycoplasma spp., streptococci, coliforms, erysipelothrix rhusiopathiae, fusobacterium necrophorum, and salmonella spp. tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. prevention and control. arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. treatment. penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. extended antibiotic therapy may be necessary. surgical excision is an option. in addition to medications noted above, isoniazid is somewhat effective for a. bovis infections in nonpregnant cattle. research complications. the possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. most cases occur within the first months of age, and animals are presented with a painful enlargement of the umbilicus. animals may exhibit var- etiology. bacillus anthracis is a nonmotile, capsulated, sporeforming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. clinical signs and diagnosis. anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. the incubation period is day to weeks. bloody secretions such as hematuria and bloody diarrhea often occur. abortion and blood-tinged milk may also be noted. the disease is usually fatal, especially in sheep and goats, after - days. death is the result of shock, renal failure, and anoxia. diagnosis is based on the clinical signs of peracute deaths and hemorrhage. stained blood smears may show short, single to chained bacilli. blood may be collected from a superficial vein and submitted for culture. epizootiology and transmission. cattle and sheep tend to be affected more commonly than goats, because of grazing habits. older animals are more vulnerable than younger, and bulls are more vulnerable than cows. although the disease occurs worldwide, and even in cold climates, most cases in the united states occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. spores survive very well in the environment. the anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. abrasive forages may play a role in infection. transmission via insect bites or through skin abrasions rarely occurs. necropsy. necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. pathogenesis. the rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. encapsulation protects the organisms from phagocytosis. liberated toxins cause local edema. differential diagnosis. although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. prevention and control. outbreaks must bereported to state officials. anthrax is of particular concern as a bioterrorism agent. any vaccination programs should also be reviewed with regulatory personnel. herds in endemic areas and along waterways are usually vaccinated routinely with the sterne-strain spore vaccine (virulent, nonencapsulated, live). careful hygiene and quarantine practices are crucial during outbreaks. dead animals and contaminated materials should be incinerated or buried deeply. biting insects should be controlled. the disease is zoonotic and a serious public health risk. treatment. treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. during epidemics, animals should be vaccinated with the sterne vaccine. research complications. natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. the organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. etiology. brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. brucella abortus is one of several brucella species that infects domestic animals but cross-species infections occur rarely. brucella abortus or b. melitensis may cause brucellosis in sheep, cattle, and goats. brucella melitensis (biovar , , or ) is the primary cause of sheep disease (garin-bastuji et al., ) . brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. in the united states, clusters of brucellosis are still found in western areas contiguous to yellowstone national park. bang's disease is the common name given to the disease in ruminants. clinical signs and diagnosis. brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about months. however, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. abortion usually occurs in the third trimester, after which the ewe will appear to recover. it has been reported that up to % of infected ewes may abort more than once. rams will also be infected and may develop orchitis or pneumonia. the disease caused by b. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. brucella ovis causes decreased fertility. brucella melitensis is the more common cause of brucellosis in goats. brucella abortus has been shown to infect goats in natural and experimental infections, and b. ovis has also been shown to infect goats experimentally. does infected with b. melitensis will also abort during the third trimester. infections with b. abortus in cattle produce few clinical signs. there may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. in cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. infection may cause abortions after the fifth month, with resulting retained placentas. permanent infection of the udder is common and results in shedding of organisms in milk. in bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. organisms may be in the semen. in infected herds, lameness may also be a clinical sign. diagnosis of brucellosis can be made by bacterial isolation of the brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (elisa), polymerase chain reaction (pcr), and others. test selection is often dependent on state requirements in the united states. epizootiology and transmission. the primary route of transmission of b. abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. necropsy findings. a sheep fetus aborted due to brucella will exhibit generalized edema. the liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. peritoneal and pleural cavities often contain serofibrinous exudates. the placenta will be leathery. pathogenesis. ruminants are considered especially susceptible to brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. brucella utilizes erythritol preferentially over glucose as an energy source. placentas and male genitalia also contain high levels of erythritol. brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. abortion is the result of placentitis, typically during the third trimester of gestation. brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. the organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. orchitis may be a sequelae of the disease. epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to b. ovis, and by semen cultures. differential diagnosis. differential diagnoses include all other abortion-causing diseases. many other agents, such as actinobacillus spp., arcanobacterium (actinomyces) pyogenes, eschericia coli, pseudomonas spp., proteus mirabilis, chlamydia, mycoplasma, and others may be associated with ovine epididymitis and orchitis. a clinically and pathologically similar agent, actinobacillus seminis, has been isolated from virgin rams. this organism has morphological and staining characteristics similar to those of b. ovis and complicates the diagnosis (genetzky, ) . prevention and control. the rev vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the united states. separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with b. ovis bacterin can prevent the disease. over the past years, aggressive federal and state regulatory and cattle herd health programs in the united states have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. strain and the recently validated attentuated strain rb are live vaccines and can be used in healthy heifer calves - months old. vaccination for older animals may be done under certain circumstances. vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. the strain vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. the vaccine will not, however, protect the animals from becoming infected with b. abortus. strain vaccine induces an antibody response in cattle. the rb vaccine does not result in antibody titers and therefore is advantageous because infection with brucella can be determined serologically. the rb vaccine has been designated as the official calfhood bovine brucellosis vaccine in the united states by the u.s. department of agriculture's animal and plant health inspection service (aphis) (stevens et al., ) . brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. the rb is regarded as less pathogenic and abortigenic in cattle. clinical signs and diagnosis. ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. the organism inhabits the intestines and gallbladder in subclinical carriers. abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. it is reported that - % of the flock may become infected and up to % of the ewes will die (jensen and swift, ) . some lambs may be born alive but will be weak, and dams will not be able to produce milk. diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. tentative identification of the organism can be made by observing curved ("gull-wing") rods in giemsa-stained or ziehl-neelsen-stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. epizootiology and transmission. campylobacteriosis occurs worldwide. campylobacter spp., such as c. jejuni, normally inhabit ovine gastrointestinal tracts. transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. in abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. ewes may have active campylobacter organisms in uterine discharges for several months after abortion. the bacteria will also be shed in feces, and feed and water contamination serve as another source. there is no venereal transmission in the ovine. necropsy. aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. the liver may contain - cm pale foci. placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. the placental cotyledons may appear gray. pathogenesis. the organism enters the bloodstream and causes a short-term bacteremia ( - weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. should be considered in late gestation ovine abortions. a bacterin is available to prevent the disease. carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral chlortetra-cycline. aborting ewes should be isolated immediately from the rest of the flock. after an outbreak, ewes will develop immunity lasting - years. treatment. infected animals should be isolated and provided with supportive therapy. prompt decontamination of the area and disposal of the aborted tissues and discharges are important. research complications. losses from abortion may be considerable. campylobacter ssp. are zoonotic agents, and c. fetus subsp, intestinalis may be the cause of "shepherd's scours." ii. clinical signs and diagnosis. preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. this will be particularly apparent in virgin heifers that may return to estrus by days after breeding. long interestrous intervals also serve an indication of a problem. spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. severe endometritis may lead to salpingitis and permanent infertility. demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. the vaginal mucous agglutination test is used to survey herds for campylobacteriosis. serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. culture from breeding animals may be difficult because campylobacter will be overgrown by faster-growing species also present in the specimens. epizootiology and transmission. the bacteria is an obligate, ubiquitous organism of the genital tract. transmission is from infected bulls to heifers. older cows develop effective immunity. necropsy findings. necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. in addition, bronchopneumonia and hepatitis will be seen histologically. pathogenesis. campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. the resulting endometritis prevents conception or causes embyronic death. differential diagnosis. the primary differential diagnosis for campylobacteriosis is trichomoniasis. other venereal diseases should be considered when infertility problems are noted in a herd. these include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv), and bovine virus diarrhea (bvd). leptospirosis should also be considered. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. prevention and control. killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. the former is preferred because of duration of immunity but causes granulomas. that vaccine also has specific recommendations regarding administration several months before the breeding season. the latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. in both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. only one bacterin product is approved for use in bulls. many combination vaccine products contain only the aluminum hydroxide adsorbed product. artificial insemination (ai) is particularly useful at controlling the disease, but bulls used for ai must be part of a screening program for this and other venereal diseases such as trichomoniasis. treatment. cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. etiology. the most common caprine bacterial skin infection is caused by staphylococcus intermedius or s. aureus and is known as staphylococcal dermatitis (smith and sherman, ) . the staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. staphylococcus aureus and s. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. clinical signs and diagnosis. small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. staphylococcal dermatitis may occur secondary to other skin lesions. diagnosis is based on lesions. culture will distinguish s. aureus. pathogenesis. simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. differential diagnosis. the presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. treatment. severe infections should be treated with antibiotics based on culture and sensitivity. severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent (smith and sherman, ) . h. clostridial diseases i. clostridium perfringens type c infection (enterotoxemia and struck) etiology. clostridium perfringens is an anaerobic, grampositive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. the bacteria is categorized by toxin production. toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. types of c. perfingens are a, b, c, d, and e. this is a common and economically significant disease of sheep, goats, and cattle. clinical signs and diagnosis. the beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first hr of a young ruminant's life. many animals may be found dead, with no clinical presentation. affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. feces may range from loose gray-brown to dark red and malodorous. morbidity and mortality may be nearly %. a similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. struck is rare in the united states. the disease is also caused by the beta toxin of c. perfringens type c and is often associated with rapid dietary changes or shearing stresses in sheep. although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. mortality is usually less than %. diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. necropsy findings. necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. pulmonary and brain edema may also be seen. histologically, the gram-positive c. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. in cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. in late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. pathogenesis. hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of clostridium perfringens type c. neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. the trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. the toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. the intestinal injury may result in diarrhea, with small amounts of hemorrhage. associated electrolyte and water loss result in dehydration, acidosis, and shock. differential diagnosis. differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. mature affected sheep may be blind and anorectic and may head-press. necropsyfindings. necropsy findings are similar to those seen with c. perfringens type c. additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (this phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. shock, probably through vascular damage. the noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. the largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. the hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. should be administered to the pregnant animals prior to parturition. an alternative includes administration of an antitoxin to the newborn lambs. the disease may become endemic once it is on the premises. treatment. treatment is difficult and usually unsuccessful. antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. differential diagnosis. tetanus, enterotoxigenic e. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. prevention and control. vaccination prevents the disease. maternal antibodies last approximately weeks postpartum; thus young animals should be vaccinated at about this time. feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. research complications. this disease can be costly in losses of neonates and younger animals. treatment. treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. toxin that is proteolytically activated by trypsin. this disease caused by c. perfringens tends to be associated with sheep and is of less importance in goats and cattle. clinical signs. the peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. because the disease progresses so rapidly to death (within - hr), clinical signs are rarely observed. hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. in acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. etiology. clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. at least serotypes of c. tetani exist. clinical signs. infection by c. tetani is characterized by a sporadic, acute, and fatal neuropathy. after an incubation period of days to weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. the animal may convulse. death occurs within - days, and mortality is nearly %, primarily from respiratory failure. diagnosis is based on clinical signs. musclerelated serum enzymes such as aspartate aminotransferase (ast), creatinine kinase (ck), and lactate dehydrogenase (ldh) might be elevated. (jensen and swift, ) . serum cortisol may also be elevated, and stress hyperglycemia may be evident. permanent lameness may result in survivors. contaminant and is often found as part of the gut microflora of herbivores. the organisms sporulate and persist in the environment. all species of livestock are susceptible, but sheep and goats are more susceptible than cattle. individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. mouth wounds may also be sites of entry. pathogenesis. tetanus, or lockjaw, is caused by the toxins of c. tetani. all serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. a nonspasmogenic toxin is also produced. contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. the toxin inhibits the release of glycine and y-aminobutyric acid from renshaw cells; this resuits in hypertonia and muscular spasms. proliferation of c. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. the uterus is the most common site of infection in postparturient dairy cattle with retained placentas. differential diagnoses. early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. polyarthritis of cattle is a differential for the gait changes in that species. necropsy findings. findings are nonspecific except for the inflammatory reaction associated with the wound. because of the low number of organisms necessary to cause neurotoxicosis, isolation of c. tetani from the wound may be difficult. administering tetanus antitoxin (e.g., at least iu in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. supportive fluids and glucose must be administered until the animal is capable of feeding. if the animal survives, revaccination should be done days after the previous dose. prevention and control like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. the disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. tetanus antitoxin can be administered ( iu in lambs) as a preventive or in the face of disease as an adjunct to therapy. both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered weeks later. animals should be vaccinated or times during the first year of life. does and ewes should receive booster vaccinations within months of parturition to ensure colostral antibodies. research complications. unprotected, younger ruminants may be affected following routine flock or herd management procedures. contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. etiology. clostridium novyi, an anaerobic, motile, sporeforming, gram-positive bacteria, is the agent of bighead and black disease. clostridium novyi type d (c. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." clostridium chauvoei is the causative agent of blackleg. clinical signs. bighead is a disease of rams characterized by edema of the head and neck. the edema may migrate to ventral regions such as the throat. additional clinical signs include swelling of the eyelids and nostrils. most animals will die within - hours. black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with c. novyi. it is more common in cattle and sheep but may be seen in goats. the clinical course is - days in cattle and slightly shorter in sheep. otherwise healthy-appearing adult animals are often affected. clinical signs are rarely seen, because of the peracute nature of the disease. occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. animals that survive a few days will develop icterus. mortality may be high. blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema (jackson et al., ) . blackleg is more common in cattle than in sheep. the incubation period is - days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. the clinical course is short, - hr, and untreated animals invariably die. blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. most lesions are associated with muscles of the face, neck, perineum, thigh, and back. epizootiology and transmission. bighead is caused by the toxins of c. novyi, which enters through wounds often associated with horn injuries during fighting. the c. novyi type b organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. clostridium novyi type d is endemic in the western united states. it is hypothesized that the c. chauvoei organisms enter through the gastrointestinal tract. black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. the diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. birds and wild animals may be vectors of the pathogen. ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. necropsy. diagnosis of black disease is usually based on postmortem lesions. subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. carcasses putrefy quickly. in addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. blood coagulates slowly in affected animals. pathogenesis. the propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. diagnosis is based on clinical signs. in black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. the toxins produced by c. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. differential diagnosis. differential diagnoses include other clostridial diseases as well as photosensitization. hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. treatment. for c. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. treatment for black disease is not rewarding even if the animal is found before death. carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. prevention and control. vaccinating animals with multivalent clostridial vaccines can prevent these diseases. subcutaneous administration of vaccine material is recommended over intramuscular. vaccinations may be useful in an outbreak. careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. for bighead, mature rams penned together should be monitored for lesions, especially during breeding season. control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. etiology. clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as c. chauvoei, c. novyi, c. sordellii, and c. perfringens may occur. clostridium spp. are motile (c. chauvoei, c. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. clinicial signs. malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by clostridium spp. the incubation period is approximately - days. the affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. regional lymphadenopathy and fever may occur. the animal becomes anorexic, severely depressed, and possibly hyperthermic. edema and crepitation may be noted around the wound; death occurs within hr to days. epizootiology and transmission. the organisms are ubiquitous in the environment and may survive in the soil for years. the disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. necropsy findings. the tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. pathogenesis. in most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. necropsy. spreading, crepitant lesions around wounds are suggestive of malignant edema. affected tissues are inflamed and necrotic. gas and serosanguineous fluids with foul odors infiltrate the tissue planes. large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. organisms can be cultured from guinea pig tissues. treatment. infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. prevention and control. proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. multivalent clostridial vaccines are available. research complications. morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. etiology. escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. escherichia coli organisms have three areas of surface antigenic complexes (o, somatic; k, envelope or pili; and h, flagellar), which are used to "group" or classify the serotypes. colibacillosis is the common term for infections in younger animals caused by this bacteria. clinical signs. presentation of e. coli infections vary with the animal's age and the type of e. coli involved. enterotoxigenic e. coli infection causes gastroenteritis and/or septicemia in lambs and calves. colibacillosis generally develops within the first hr of life when newborn animals are exposed to the organism. the enteric infection causes a semifluid, yellow to gray diarrhea. occasionally blood streaking of the feces may be observed. the animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." hyperthermia is rare. severe acidosis, depression, and recumbancy ensue, and mortality may be as high as %. the septicemic form generally occurs between and weeks of age. animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. opisthotonos, depression, and death follow. occasionally, swollen, painful joints may be observed with septicemic colibacillosis. blood cultures may be helpful in identifying the septicemic form. in ruminants, e. coli is is a less common cause of cystitis and pyelonephritis. the cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. the infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. in chronic cases, animals will be polyuric and undergo weight loss. escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. epizootiology and transmission. escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. zeman et al. ( ) classify e. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. enterotoxigenic e. coli (etec) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. fimbrial (pili) antigens associated with ovine disease include k and f . enterohemorrhagic e. coli (ehec) attach and efface the microviuus, produce verotoxins, and occasionally cause disease in humans and animals. enteropathogenic e. coli (epec) colonize and efface the microvillus but do not produce verotoxins. epec are associated with disease in humans and rabbits and cause a secretory diarrhea. enteroinvasive e. coli (eiec) invade the enterocytes of humans and cause a shigella-like disease. overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. the organism will be endemic in a contaminated environment and present on dams' udders. the bacteria rapidly proliferate in the neonates' small intestines. the bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. if the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. necropsy findings. swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. isolation and serotyping of e. coli confirm the diagnosis. elisa and latex agglutination tests are available diagnostic tools. differential diagnosis. differential diagnoses include the enterotoxemias caused by c. perfringens type a, b, or c; campylobacter jejuni; coccidia, rotavirus, coronavirus, salmonella, and cryptosporidia. other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. prevention and control. the best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. attention to colostrum feeding techniques and colostral quality are important means of preventing disease. treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. treatment. antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. vaccines are available for prevention of colibacillosis in cattle. etiology. corynebacterium pseudotuberculosis (previously c. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. caseous lymphadenitis (cla) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. the disease has been reported occasionally in cattle. clinical signs and diagnosis. abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. radiographs may be helpful in identifying affected central nodes. peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. the incubation period may be weeks to months. over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. fever, increased respiratory rates, and pneumonia may also be common signs. exotoxin-induced hemolytic crises may occur occasionally. morbidity up to % is common, and morbid animals will often eventually succumb to the disease. diagnosis is based on clinical lesions; elisa serological testing is also available. smears of the exudate or lymph nodes aspirates can be gram-stained. lymph node aspirates may also be sent for culturing. epizootiology and transmission. the organism can survive for months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. necropsy findings. disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. cut surfaces of the affected lymph nodes may appear lamellated. lungs, liver, spleen, and kidneys may also be affected. cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. pathogenesis. corynebacterium pseudotuberculosis produces an exotoxin (phospholipase d) that damages endothelial and blood cell membranes. this process enhances the organisms' ability to withstand phagocytosis. the infection spreads through the lymphatics to local lymph nodes. the necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. differential diagnosis. differentials include pathogens causing lymphadenopathy and abscessation. treatment. antibiotic therapy is not usually helpful. abscesses can be surgically lanced and flushed with iodinecontaining and/or hydrogen peroxide solutions. abscessing lymph nodes can be removed entirely from valuable animals. during warmer months, an insect repellent should be applied to and around healing lesions. all materials used to treat animals should be disposed of properly. because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from cla-negative animals at least until healed. commercial vaccines are available (piontkowski and shivvers, ) . minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. research complications. this pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. etiology. corynebacterium renale, c. cystitidis, and c. pilosum are sometimes referred to as the c. renale group. these are piliated and nonmotile gram-positive rods and are distinguished biochemically. corynebacterium renale causes pyelonephritis in cattle, and c. pilosum and c. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. in many references, all these clinical presentations are attributed to c. renale. clinical signs and diagnosis. acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. relapses are common, and some infections are severe and fatal. diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). urine culturing may not be productive. in chronic cases, e. coli and other gram-negatives may be present. posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. the area may have a distinct malodor. necrosis and scarring may be sequelae of more severe infections. fly-strike may also be a complication. diagnosis is based on clinical signs and on investigation of feeding regimens. epizootiology and transmission. ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. the vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. posthitis occurs in intact and castrated sheep and goats. necropsy findings. pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. of bovine genitourinary tracts. the pilus mediates colonization. conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. in addition, more basic ph urine levels may block some immune defenses. infections ascend through the urinary tract. the bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. corynebacterium cystitidis and c. pilosum are normally found around the prepuce of sheep and goats. high-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. the ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. differential diagnosis. urolithiasis is a primary consideration for these diseases. contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. prevention and treatment. because high-protein feed is often associated with posthitis and vulvovaginitis, feeding prac-tices must be reconsidered. clipping long wool and hair also is helpful. treatment. long-term ( weeks) penicillin treatment is effective for pyelonephritis. reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. surgical debridement or correction of scarring may also be indicated in severe cases. etiology. erysipelothrix rhusiopathiae is a nonmotile, nonspore-forming, gram-positive rod that resides in alkaline soils. clinical signs. erysipelothrix causes sporadic but chronic polyarthritis in lambs less than months of age. in older goats, erysipelas has been associated with joint infections. epizootiology and transmission. the disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. following wound contamination and a -to -day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. necropsy findings. thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. the joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. differential diagnosis. differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. other bacteria causing septic joints include areanobacterium pyogenes and fusobacterium necrophorum. caprine arthritis encephalitis (cae) should also be considered. prevention and control. proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. screening of goat herds for cae is recommended. therapy. erysipelas is sensitive to penicillin antibiotic m. etiology. dermatophilus congolensis is an aerobic, grampositive, filamentous bacterium with branching hyphae. dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers (scanlan et al., ) . clinical signs. animals will be painful but will not be pruritic. two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. strawberry foot rot is rare in the united states but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. animals will be lame. in goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. lesions in younger goats are seen along the tips of the ears and under the tail. diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. epizootiology and transmission. the disease occurs worldwide, and the dermatophilus organism is believed to be a saprophyte. transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. biting insects may aid in transmission. necropsy findings. lymphadenopathy as well as liver and splenic changes may be observed. histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. pathogenesis. lesions typically begin around the muzzle and hooves and the dorsal midline. prevention and control. potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. minimizing moist conditions is helpful in controlling and preventing the disease. in addition, controlling external parasites or other factors that cause skin lesions is important. lesions will resolve during dry periods. treatment. animals can be treated with antibiotics such as penicillin and oxytetracycline. treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. etiology. two bacteria, dichelobacter (bacteroides) nodosus and fusobacterium necrophorum, work synergistically in caus-ing contagious foot rot in sheep and goats. other organisms may be involved as secondary invaders. both dichelobacter and fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues (bulgin, ) . it is the leading cause of lameness in sheep. at least serotypes of dichelobacter are known. arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. foot scald, an interdigital dermatitis, is caused primarily by d. nodosus alone. clinical signs. varying degrees of lameness are observed in all ages of animals within - weeks of exposure to the organisms. severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. the interdigital skin and hooves will be moist, with a distinct necrotic odor. morbidity may reach % in susceptible animals. diagnosis is based on clinical signs. smears and cultures confirm the definitive agents. clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. epizootiology and transmission. fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. in contrast, dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about weeks. some animals may be chronic carriers. overcrowded, warm, and moist environments are key elements in transmission. outbreaks are likely in the spring season. shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. pathogenesis. both organisms are transmitted to the susceptible animal by direct or indirect contact. the organisms enter the hoof through injuries or through sites where strongyloides papillosus larvae have penetrated. fusobacterium necrophorum initiates the colonization and is followed by d. nodosus. the latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. the pathogenicity of the serotypes of d. nodosus is correlated with the production of these proteases and numbers of pili. additionally, f. necrophorum causes a severe, damaging inflammatory reaction. differential diagnosis. foot abscesses, tetanus, selenium/ vitamin e deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. treatment. affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. goats have improved dramatically when given a single dose of penicillin ( , u/kg) (smith and sherman, ) . footbaths containing % zinc sulfate, % copper sulfate, or % formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. affected animals should be separated from the flock. vaccination has been shown to be effective as part of the treatment regimen. some breeds of sheep and some breeds and lines of goats are resistant to infection. individual sheep may recover without treatment or are resistant to infection. epizootiology and transmission. cases may be sporadic, or epizootics may occur. bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. the factors here are comparable to those present in foot rot of smaller ruminants. necropsy findings. findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. prevention and control. prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. biannual vaccinination in wet areas may be essential. some breeds may develop vaccination site lumps. footbaths of % zinc sulfate, % formalin (where allowed by state regulations), or % copper sulfate are also considered very effective preventive measures. goats are less sensitive than sheep to the copper in the footbaths. treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. etiology. interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by fusobacterium necrophorum and bacteroides melaninogenicus. like f. necrophorum, b. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. this is a common cause of lameness in cattle. clinical signs. clinical signs include mild to moderate lameness of sudden onset. hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. the interdigital space will be swollen, as will be the coronet and bulb areas. characteristic malodors will be noted, but there will be little purulent discharge. in more severe cases, animals will have elevated body temperature and loss of appetite. the les~ons progress to fissures with necrosis until healing occurs. the diagnosis is by the odor and appearance. anaerobic culturing confirms the organisms involved. pathogenesis. the bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. colonization leads to cellulitis. in addition, f. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by b. melaninogenicus. zinc deficiency may play a role in the pathogenesis in some situations. differential diagnoses. the most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. bluetongue virus should also be considered. grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. the exotic footand-mouth disease virus would be considered in areas where that pathogen is found. prevention and control. as with foot rot in smaller ruminants, management of the area and herd are important. paddocks and pens should be kept dry, well drained, and free of material that will damage feet. footbaths and chlortetracycline in the feed have been shown to control incidence. affected animals should be segregated during treatment. chronically affected or severely lame animals should be culled. new cattle should be quarantined and evaluated. ing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as % zinc sulfate, . % formalin, or % copper sulfate) twice a day. in severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. animals can recover without treatment but will be lame for several weeks. acquired immunity is reported to be poor. research complications are comparable to those noted for foot rot in smaller ruminants. fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. only one claw of the affected hoof may be involved. the animals will be three-legged lame, and the affected hoof will be hot. pockets of purulent material may be in the heel or toe. etiology. bacteria such as fusobacterium spp., bacteroides spp., and dichelobacter nodosus have been isolated from bovine heel lesions. spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (pdd), in the united states and europe; these have culturing requirements similar to those of treponema species. treatment. antibiotic and antiseptic regimens have been used successfully for this problem. antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. the footbaths must be well maintained, minimizing contamination by feces and other materials. tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. research complications. infectious, contagious ppd is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. the outbreaks are generally worse in younger animals in chronically infected herds. the immune response is not well understood, and it may be temporary in older animals. clinical signs. all lesions occur on the haired, digital skin. one or all feet may be affected. most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. progression of lesions, typically over - weeks, includes erect hairs, loss of hair, and thickening skin. moist plaques begin as red and remain red or turn gray or black. exudate or blood may be present on the plaque. plaques enlarge and "hairs" protrude from the roughened surface. lesioned areas are painful when touched. the lesions may or may not be malodorous. epizootiology and transmission. facility conditions and herd management are considered contributing factors. the following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. these interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. the disease is seen only in cattle. pathogenesis. the organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. differential diagnosis. differentials for lameness will include sole abscesses, laminitis, and trauma. prevention and control. each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. all equipment used for hoof trimming must be cleaned and disinfected after every use. trucks and trailers should also be sanitized between groups of animals. etiology. haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. diseases caused by this organism include thromboembolic meningoencephalitis (teme), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. haemophilus somnus is a also major contributor to the bovine respiratory disease complex. haemophilus spp. have been associated with respiratory disease in sheep and goats. clinical signs. the neurologic presentation may be preceded by - weeks of dry, harsh coughing. neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. high fever, extreme morbidity, and death within hr may occur. respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza , mycoplasma, and pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (brdc). in acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. in all cases, asymptomatic infections may also occur. diagnosis based on culture findings is difficult because h. somnus is part of the normal nasopharyngeal flora. paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. in cases of abortion, other causes should be eliminated from consideration. because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. outbreaks are associated with younger cattle in feedlots in western united states, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. adult cattle have also been affected. vaccination for viral respiratory pathogens may increase susceptibility. transmission is by respiratory and genital tract secretions. the organism does not persist in the environment. times of stress to the cattle is worthwhile. killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. control of other clinical aspects of the h. somnus disease by these bacterins has not been well described. treatment. rapid treatment at the first signs of neurologic disease is important in an outbreak. haemophilus somnus is susceptible to several antibiotics, such as oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. necropsy findings. pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. many thrombi with bacterial colonies will be seen in these affected areas. ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. usually animals with neurological disease will not have respiratory tract lesions. the respiratory tract lesions include bronchopneumonia and suppurative pleuritis. when combined with pasteurella infection, the pathology becomes more severe. aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. pathogenesis. inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. the anatomical location of bacterial residence within the carriers has not been identified. after gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. the bacteria are phagocytosed by neutrophils but are not killed. the thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. other pathogens associated with neurological disease and respiratory disease such as pasteurella hemolytica, p. multocida, and p. aeruginosa. in smaller ruminants, corynebacterium pseudotuberculosis should be considered. prevention and control. stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). the late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. planning vaccination programs carefully will decrease chances of outbreaks. for example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during clinical signs. leptospirosis is a contagious but uncommon disease in sheep and goats. the disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. after a -to -day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. leptospiremia may last up to days. immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; crossserovar protection does not occur. during active bacteremia, hemolysis may result in hemoglobin levels of % below normal. hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. abortion usually occurs only once. mortality rates of above % have been reported in infected ewes and lambs (jensen and swift, ) . subclinical infection is more common in nonpregnant and nonlactating animals. sheep infected with leptospirosis may display a hemolytic crisis associated with igm acting as a cold-reacting hemagglutinin. acute and chronic infections in cattle are more common than infections in sheep and goats. acute forms in cattle display signs similar to those in sheep. acute infection in calves may progress to meningitis and death. lactating cows will have severe drops in production. chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. infertility may also be a sequela. epizootiology and transmission. leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. the organism survives well in the environment, especially in moist, warm, stagnant water. cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. organisms are shed in urine, in uterine discharges, and through milk. animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-hostadapted serovar (heath and johnson, ) . infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. placental or venereal transmission may occur. as the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). chronically infected animals may shed the organism in the urine for days or longer. necropsy. diagnosis is confirmed by identification of leptospires in fetal tissues. the leptospires are visible in silver-or fluorescent antibody-stained sections of liver or kidney. leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. differential diagnosis. more than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. prevention and control. polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. immunity is serovar specific. because serological titers tend to diminish rapidly ( - days in sheep [jensen and swift, ] ), frequent vaccination may be necessary. other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. treatment. antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. treatment methods for acute leptospirosis include oxytetracycline for - days. addition of oxytetracycline or chlortetracycline to the feed for week may be helpful. these antibiotics are considered best for removal of the carrier state of some serovars. vaccination and antibiotic therapy can be combined in an outbreak. research complications. leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. etiology. listeria monocytogenes is a pleomorphic, motile, non-spore-forming, [ -hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. of the known serovars, several produce clinical signs in ruminants. listeria ivanovii (associated with abortions in sheep) is serovar . clinical signs. listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. the overall case rate is low. the disease may present as an isolated case or with multiple animals affected. three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. the encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs (scarratt, ) . clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of - weeks. as the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves v-xii. the neck will by flexed away from the affected side. facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. the hypersalivation may lead to metabolic acidosis in advanced cases in cattle. anorexia, prostration, coma, and death follow. the placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. the affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. abortion usually occurs within weeks of listeria infection. in cattle, abortion occurs during the last months of gestation and has been induced experimentally - days after exposure. cows present with the range of clinical signs seen in smaller-ruminant dams. there is no long-term effect on the fertility of affected dams. epizootiology and transmission. the organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. by the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. when severe outbreaks occur, feedstuffs should be assessed for spoilage. listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. diagnosis and necropsy findings. diagnosis is usually made from clinical signs. culture confirms the diagnosis (cold enrichment at ~ is preferable but not essential for isolation). impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. tissue fluorescent antibody techniques may also be utilized. gross lesions are not observed with the encephalitic form. microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. the pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. microabscesses of the midbrain are characteristic of listeria encephalitis in sheep. aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. necrotic lesions of the fetal spleen and lungs may also be seen. in goats, listeria-induced neurological lesions occur only in the brain stem. placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. pathogenesis. with the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. the listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). in the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. after infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). a toxin produced by listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. differential diagnoses. rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. in sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to clostridium perfringens type d. in goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. in both species, scrapie is a differential. in cattle, aberrant parasite migration or hemophilus somnus infection must also be considered. prevention and control. affected dams should be segregated and treated. other animals in the group may be treated with oxytetracycline as needed. aborted tissues should be removed immediately. proper storage of fermented feeds minimizes this source of contamination. when silage spoils, the ph increases, producing a suitable growth environment for the organism. commercial vaccines are not available in the united states. treatment. affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. exceptionally high levels of penicillin are required for treating affected cattle. severely affected animals should receive appropriate fluid support and other nursing care. treatment is less successful, and mortality is especially high in sheep. recovered animals tend to resist reinfection. research complications. in addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. listeriosis in humans is a reportable disease. etiology. lyme disease is caused by the spirochete borrelia burgdorferi. clinical signs and diagnosis. reports in ruminants indicate seroconversion to b. burgdorferi, but there are few definitive correlations to the arthritis that is present. diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. epizootiology and transmission. the organism is present throughout much of the northern hemisphere and has been reported in many mammals and also in birds. ticks of the ixodes ricinus complex are the major vectors of the spirochete and must be attached for hr for successful transmission. pathogenesis. the ixodes ticks have three life stages: larval, nymphal, and adult. feeding occurs once during each stage, and wild animals are the source of blood meals. the larval stages feed from rodents, such as the white-footed deer mouse, peromyscus leucopus, from which they acquire the spirochete. the nymphal stage is that which usually infects other animals. the adult ticks are usually found on deer. differential diagnosis. seroconversion to b. burgdorferi does not necessarily confirm the cause of arthritis. other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, mycoplasma spp., chlamydia psittaci, erysipelothrix spp., arcanobacterium pyogenes, brucella spp., and rickets. prevention and control. control of the tick vector is the most important factor in preventing the possibility of exposure or disease. treatment. antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected lyme arthritis. research complications. lyme disease is zoonotic, and the lxodes ticks transmit the disease to humans. v. mastitis i. ovine mastitis mastitis in ewes may be acute, subclinical, or chronic. acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. pasteurella haemolytica is the most common cause of acute mastitis. additional isolates may include, in order of prevalence, staphylococcus aureus, actinomyces (corynebacterium) spp., and histophilus ovis. escherichia coli and pseudomonas aeruginosa have also been found to cause acute mastitis. as many as six serotypes of pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes (watkins.and jones, ) . subclinical mastitis is detected only indirectly, by counting somatic cells. the most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. other isolates include actinomyces bovis, streptococcus uberis, s. dysgalactiae, micrococcus spp., bacillus spp., and fecal streptococci. most of these organisms are commonly found in the environment. diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. both glands are usually affected, but no inflammation is present. serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (opp or maedi/visna virus). other bacterial agents or mycoplasma have not usually been isolated from udders with this type of mastitis. acute mastitis occurs in approximately % of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are - months old (lasgard and vaabenoe, ) . subclinical mastitis occurs in - % of lactating ewes (kirk and glenn, ) . subclinical mastitis is more common in ewes from high-milk-producing breeds. skin or teat lesions and dermatitis increase the prevalence of disease. acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. subclinical mastitis is often suggested by somatic cell counts elevated above x cells/ml. when high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. the california mastitis test may also be helpful as an indicator of mastitis. manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. it is may be helpful to milk out the infected ud-der frequently; oxytocin injections preceding milking will improve gland evacuation. because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. there is currently no treatment available for diffuse chronic mastitis. ii. caprine mastitis lactating goats are subject to inflammation of mammary gland, or mastitis. the primary causative organisms are staphylococcus epidermidis and other coagulasenegative staphylococcus spp. clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. large abscesses may be present in the affected gland. staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. this organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. diagnosis is based on careful culture of mastitic milk. treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. oxytocin ( - u) may help milk letdown for frequent strippings. bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. in severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. other less common causes of mastitis in goats include streptococcus spp. (s. agalactiae, s. dysgalactiae, s. uberis, and zooepidemicus). gram-negative causes of caprine mastitis include escherichia coli, klebsiella pneumoniae, pasteurella spp., pseudomonas, and proteus mirabilis. corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas mycoplasma mycoides may cause agalactia and systemic disease. "hard udder" can be caused by caprine arthritis encephalitis virus (caev). brucellosis and listeriosis can cause a subclinical interstitial mastitis (smith and sherman, ) . iii. bovine mastitis mastitis is the disease of greatest economic importance for the dairy cattle industry. the majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. the most common bovine mastitis pathogens include staphylococcus aureus and streptococcus agalactiae, s. dysgalactiae, and s. uberis; coliform agents such as escherichia coli, enterobacter aerogenes, serratia marcescens, and klebsiella pneumoniae; mycoplasmal species such as mycoplasma bovis, m. bovigenitalium, m. californicum, m. canadensis, and m. alkalescens; and salmonella spp. such as s. typhimurium, s. newport, s. enteritidis, s. dublin, and s. muenster. many of these agents such as staphylococcus spp., salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. these must be regarded as herd and environmental pathogens in terms of treatment and prevention. the pathogenesis of staphylococcal infections is comparable to that in goats. staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. in contrast, s. uberis and s. dysgalactiae are environmental organisms and can be highly resistant to pencillin. mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. treatments for mycoplasmal mastitis are not successful; culling is recommended. there are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. herd management will diminish teat lesions. bacterin vaccines are available for preventing and controlling coliform mastitis and s. aureus mastitis. at the time of dry-off, all cows must be treated by intramammary route. some infections can be successfully cleared during this time. younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. in that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. etiology. moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (ibk) in cattle. this organism is not a cause of keratoconjunctivitis in sheep and goats. the disease includes conjunctivitis and ulcerative keratitis. the pathogenic m. bovis strain is piliated, and at least seven serotypes exist. clinical signs. lacrimation, photophobia, and blepharospasm are seen initially. conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. corneal vascularization begins by days after onset. reepithelialization of the corneal ulcers occurs by - weeks after onset. diagnosis is usually based on clinical signs, but culturing is helpful and fluoroscein staining is useful for demonstrating corneal ulceration. epizootiology and transmission. the disease is more severe in younger cattle. the clinical signs of ibk tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (ibr) virus or those that have been vaccinated recently with modified live ibr vaccine. the bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. transmission is by fomites, flies, aerosols, and direct contact. incidence in winter months is very low. nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. necropsy findings. necropsy is not typically performed on these cases. corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. pathogenesis. the pili ofm. bovis bind to receptors of corneal epithelium. the virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. differential diagnoses. infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of ibk is not seen with m. bovis infections. mycoplasma, listeria, branhamella (neisseria) , and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. prevention and control. cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during ibk outbreaks; this will likely exacerbate the infection. new animals should be quarantined and treated prophylactically before introduction to herds. the available vaccines, containing. m. bovis pili or killed m. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the m. bovis strain may not be homologous to that used for the vaccine preparation. other preventive measures include % permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as % coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. treatment. cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. these should be administered twice per day. subconjunctival injections of antibiotics, such as penicillin g, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and doses hr apart eliminate carriers. third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. epizootiology and transmission. although m. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. clinically normal animals may serve as carriers. the bacilli stimulate an initial neutrophilic tissue response. neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called langhans' giant cells. an outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. necropsy findings. yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. research complications. this pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. the overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. mycobacterium bovis infection (tuberculosis) etiology. mycobacteria are aerobic, nonmotile, non-sporeforming, acid-fast pleomorphic bacteria. most cases of tuberculosis in sheep are related to mycobacterium bovis or m. avium. cases in goats have been attributed to m. bovis, m. avium, or m. tuberculosis. mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. other agents of mammalian tuberculosis include m. microti and m. africanum. clinical signs. tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. the infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. the respiratory system (m. bovis) or the digestive system (m. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. locations of the characteristic tubercles will determine whether clinical signs are seen. respiratory signs may include dyspnea, coughing, and pneumonia. digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. lymphadenopathy occurs in advanced cases. fever and generalized disease may be seen after calving. infected goats lose weight and develop a persistent cough. prevention and control. significant progress has been made in eradication programs in the united states during the past several decades, but during the s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. the intradermal tuberculin test, using purified protein derivative (ppd), is usually used as a diagnostic indicator in live animals. this test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. notification to state officials is required following identification of intradermal-positive animals. great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. no treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. slaughter is preferred, to prevent potential transmission to humans. paratuberculosis, or johne 's disease (mycobacterium paratube rculo sis) etiology. mycobacterium paratuberculosis, the causative agent of johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. the organism is actually a subspecies of m. avium, but m. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of m. avium. clinical signs and diagnosis. johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. in sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. in cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. although clinical signs are nonspecific, johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. the disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than % sensitive. in addition, the sensitivity of the serological tests differs between species. the standard is the fecal culture that takes - weeks. theenzyme-linked immunosorbent assay (elisa) is now considered the most reliable serological test, but false negatives do occur. other serological tests such as agar gel immunodiffusion (agid) and complement fixation are useful. herd screening may be done using the agid or elisa serological tests. identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. some animals serologically negative for johne's disease, however, have been found to be positive on fecal culture. commercial agid tests approved for use in cattle may be useful in diagnosing johne's disease in sheep (dubash et al., ) . serological tests cross-react with other species of mycobacterium, especially m. avium. epizootiology and transmission. the organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. chronic carriers may intermittently shed the organisms. parasite that grows only in macrophages of infected animals. nursing infected dams are a primary source of infection of neonates. if the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. the organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. after an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. necropsy and diagnosis. the ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. differential diagnosis. diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. these include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. deer can be infected, and the lesions can be confused with those of tuberculosis. prevention and control. prevention is the most effective method to manage this pathogen. efforts should be focused on eliminating the disease through test and slaughter. neonates should not be reared by infected dams. some states have johne's disease eradication programs. facilities and pastures where animals testing positive for johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. other considerations. mycobacterium paratuberculosis is being investigated as a factor in the development of crohn's disease in humans. etiology. the most common organism causing infection of the umbilicus is arcanobacterium (formerly actinomyces, corynebacterium) pyogenes; other bacteria may be present. arcanobacterium spp. are anaerobic, nonmotile, non-sporeforming, gram-positive, pleomorphic rods to coccobacilli. other environmental contaminants are also associated with this disease, such as escherichia coli, enterococcus spp., proteus, streptococcus spp., and staplylococcus spp. clinical signs and diagnosis. navel ill is an acute localized inflammation and infection of the external umbilicus. animals present with fever and painful enlargement of the umbilicus. animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. neonates, and most cases occur within the first months of age. cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. navel ill is diagnosed by typical clinical signs. the presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. differential diagnosis. the major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. mycoplasmal arthritis is a differential in kids. in the past, erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. treatment. omphalitis can be treated with a to day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. if an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. the prognosis for recovery is good if systemic involvement has not occurred. prevention and control. the disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. may invade the bloodstream, causing disseminated septicemia. clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. with the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. the disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. in cultures, p. hemolytica is distinguished from p. multocida by hemolysis on blood agar; only p. multocida produces indole. epizootiology and transmission. the organism is ubiquitous in the environment and in the respiratory tracts of these animals. younger ruminants, between and months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. the pneumonic form appears as a complex associated with concurrent infections such as parainfluenza , adenovirus type , respiratory syncytial virus, mycoplasmas, chlamydia, pasteurella multocida and bordetella parapertussis (martin, ; brogden et al., ) . the organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. necropsy findings. necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal mm lesions distributed on the surfaces of the lungs and liver. with the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident.. the disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. etiology. pasteurella hemolytica and p. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. biotype a serotypes are associated with pneumonia and septicemia in all ruminants (ellis, ) . serotype of p. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. clinical signs. pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. the organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. the lungs and liver are frequent areas of formation of microabscesses. acute rhinitis or pharyngitis often precedes the respiratory form. the organism also pathogenesis. a leukotoxin is considered to be a key factor in the pathogenesis of the p. hemolytica infection. macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. treatment. treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. in outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. prevention and control. the incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. early pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. in young animals, passive immunity is protective. preventive measures also include maintaining good ventilation in enclosures and barns. new animals to the flock or herds should be quarantined for at least weeks before introduction. etiology. salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. it is a common inhabitant of the gastrointestinal tract of ruminants. current nomenclature categorizes s. typhimurium as a serovar within the species s. enteritidis (the other two species are s. typhi and s. choleraesuis). salmonella typhimurium, s. dublin, and s. newport are the common species seen in bovine cases. salmonella typhimurium, s. dublin, s. anatum, and s. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. ovine abortions due to various salmonella species are not reported in the united states but are enzootic in other countries. salmonella serotypes have been associated with aborted fetuses in all ruminant species. clinical signs and diagnosis. salmonellosis causes acute gastroenteritis, dysentery, and septicemia (anderson and blanchard, ) . clinically, the animals become anorexic and hyperthermic. diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. animals become severely depressed and weak, losing a high percentage of their body weight. animals may die in - days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. morbidity may be %, and mortality may be high. septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. chronically infected animals may have intermittent diarrhea. in goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. morbidity and mortality will be highest in neonates, and some may simply be found dead. the older animals generally tend to fare better during the disease. abdominal distension with profuse yellow feces is common. kids become severely depressed, anorexic, febrile (with temperatures as high as ~ dehydrated, acidotic, recumbent, and comatose. salmonella abortions may occur throughout gestation. there may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. hemorrhage, placental necrosis, and edema will be present. metritis and placental retention may occur. some mortality of dams may occur. diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. leukopenia and a degenerative shift to the left are not uncommon hematological findings. epizootiology and transmission. stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. birds and rodents may be natural reservoirs of salmonella in external housing environments. transmission is fecal-oral. after ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally (arora, ) . fecal-oral transmission is also associated with salmonella abortion; veneral transmission has not been reported. necropsy findings and diagnosis. animals will have noticeable perineal staining. intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. the wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. this membrane is not normally seen in the goat (smith and sherman, ) . splenomegaly may be present. aborted fetuses will often be autolysed. placentitis, placental necrosis, and hemorrhage are commonly seen. serologic evidence of recent infection can be demonstrated in the dam. salmonella can be isolated from the aborted tissues. pathogenesis. after ingestion, the organism proliferates in the intestine. damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. although the salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. septicemia is a common sequela, with the bacteria localizing throughout the body. in latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal ph. carriers may develop clinical disease when stressed. differential diagnoses. in young animals, differentials include other enteropathogens: escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. these pathogens may also be present in the affected animals. differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. prevention and control. affected animals should be isolated during herd outbreaks. samples for culture should include herdmates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. the bacteria survive for about a week in moist cow manure. vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the j- bacterin is now considered better. treatment. nursing care includes rehydration and correction of acid-base abnormalities. antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. negative, rod-shaped bacterium. type a is more virulent than type b. clinical signs. although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. the disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. infected animals move stiffly, are depressed, and are hyperthermic. anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. mortality may reach %. animals that recover will have immunity of long duration. epizootiology and transmission. the disease is most commonly transmitted by ticks or biting flies. the wood tick, dermacentor andersoni, is an important vector in transmitting the disease in the western united states, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. research complications. salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. attempts should be made to identify and cull carrier animals. pathogenesis. the organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. the organisms can also be transmitted orally through contaminated water. etiology. spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. the disease has been reported only in the foothills bordering the central valley of california. necropsy findings. ticks may also be present on the carcasses. suppurative, necrotic lymph nodes are typical. lungs will be congested and edematous. diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; p. tularensis does not survive for long periods in carcasses. serological findings may also be helpful. clinical signs. cows that become infected with the causative agent before months of gestation abort or give birth to weak calves without any clinical sign of infection. cows infected after months of gestation give birth to normal calves. affected cows rarely abort in subsequent pregnancies. the tick vector is ornitho- necropsy. fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. the calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. treatment. chlortetracycline treatment has been effective in controlling this disease. etiology. tularemia is caused by pasteurella (francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-control and prevention. eliminating the tick vectors can prevent tularemia. animals should be provided with fresh water frequently. the organism can survive in freezing conditions and in water and mud for long periods of time. caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. research complications. the disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the united states should be aware of it. the organism is antigenically related to brucella spp. etiology. yersiniosis is caused by infections with yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family enterobacteriaceae. there are serotypes reported for y. enterocolitica. yersinia pseudotuberculosis infections have also been seen in ruminants. enteric infections predominate in the diseases caused by these bacteria. clinical signs and diagnosis. clinical disease may be seen rarely in many groups of ruminants. goats of - months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting or more days. spontaneous abortions and weak neonates are also clinical manifestations of infection. lactating does may have mastitis that becomes chronically hemorrhagic. bacteremia results in internal abscesses, abortion, and acute deaths. yersinia pseudotuberculosis has been associated with laboratory goat epizootics (obwolo, ) . diarrhea in pastured sheep, stressed by other factors, has also been reported. diagnosis is based on culture and serology. epizootiology and transmission. the bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. research complications. yersinia is zoonotic. prevention and control. control measure are not well defined, because the epidemiology of the disease is poorly understood (smith and sherman, ) . tissues from affected goats must be handled and disposed of properly. areas housing affected goats must be thoroughly sanitized. treatment. in case of an abortion storm, treatment of goats with tetracycline has been useful. other broad-spectrum antibiotics may also be useful. clinical signs. contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever (mcmartin et al., ) . infections with other mycoplasma species also have similar clinical signs. septicemia without respiratory involvement may also be a presentation. epizootiology and transmission. this disease is highly contagious, with high morbidity and mortality. transmission is by aerosols. mycoplasma mycoides subsp, mycoides has become a serious cause of morbidity and mortality of goat kids in the united states. necropsy. large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. some lung consolidation may be present. meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. differential dagnosis. in the united states, the principal differential for m. mycoides subsp, mycoides is caprine arthritis encephalitis. treatment. tylosin and oxytetracycline are effective. some infections are slow to resolve. prevention and control. vaccines are available in some areas. infected herds are quarantined. new goats should be quarantined before introduction to the herd. research complications. the worldwide distribution of the f biotype, as well as the aerosol transmission and high mor-bidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) etiology. mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. mycoplasma mycoides subsp, mycoides, m. agalactiae, m. arginini, and acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. respiratory disease and other infections, such as mastitis, may also be observed. clinical signs and diagnosis. all ages of animals may be affected. initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. sometimes uveitis is evident. although the presentation is usually unilateral, bilateral involvement is possible. recurring infections are common. culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. ily between animals by direct contact. animals can become reinfected, and carrier animals may be a factor in outbreaks. necropsy. it is unlikely that animals would die or be euthanized and undergo necropsy for this problem. conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. epithelial cell cytoplasm should be examined for organisms. differential diagnosis. the primary differential in sheep and goats is chlamydia, as well as branhamella, rickettsia (colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. it is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. treatment. animals do recover spontaneously within about weeks. tetracycline ointments and powders are also used. third-eyelid flaps may be necessary if corneal ulceration develops. prevention and control. new animals should be quarantined and, if necessary treated, before introduction to the flock or herd. etiology. eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent eperythrozoon. host-specific species of importance are e. ovis, the causative species in sheep and goats, and e. wenyoni, e. tegnodes, and e. tuomii, the causative agents in cattle. although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. clinical signs and diagnosis. the disease is more severe in sheep. following an incubation period of - weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. losses may be greater in younger lambs. cattle are usually latently infected but may have swollen and tender teats and legs. fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. epizootiology and transmission. the rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. necropsyfindings. necropsy findings include splenic enlargement and tissue icterus. has resulted in transient hyperthermia, mild respiratory disease, and mastitis. abortions, stillbirths, and births of weak lambs are also seen. epizootiology and transmission. coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. the organism is associated with either a free-living or an arthropod-borne cycle. coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. once introduced into a mammal, coxiella may be maintained without a tick intermediate. the organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. additionally, the organism is shed in milk, urine, feces, and oronasal secretions. necropsy findings. no specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. the placenta will contain white chalky plaques and a red-brown exudate. the disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. the organism has been found in the placentas of clinically normal animals. the organism stains red with modified ziehl-neelsen and macchiavello stains and purple with giemsa stain. pathogenesis. the organism invades and destroys red blood cells. it is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. as with other red blood cell parasites, splenectomy aggravates the disease. differential diagnosis. because of the organisms' similarity to chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, elisa, and complement fixation tests. differential diagnosis. clontridium novyi type d, babesiosis, and leptospirosis are the primary differentials. prevention and control. following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. treatment. treatment is not usually recommended, but oxytetracycline has been used. sheep will develop immunity if supported nutritionally during the disease. research complications. splenectomized animals are the experimental models used to study these diseases. ii. q fever, or query fever (coxiella burnetii) etiology. coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. clinical signs. infection of ruminants with c. burnetii is usually asymptomatic. experimental inoculation in other mammals treatment. coxiella can be treated with oxytetracyclines. a vaccine is not commercially available. prevention and control. any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. serologic screening of ruminant sources should be performed routinely. barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. all placentas and all aborted tissues should be handled and disposed of carefully. q fever has been reported in many mammalian species, including cats. research complications. coxiella burnetii-free animals are particularly important in studies involving fetuses and placentation. because of its zoonotic potential, c. burnetii presents a unique problem in the animal research facility environment. a single organism has been shown to cause disease. some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. etiology. the ruminant adenoviruses are dna viruses that cause respiratory and reproductive tract diseases. nine antigenic types of the bovine adenovirus have been identified, with type associated with respiratory disease. two of the ovine and two of the caprine antigenic types have been identified. clinical signs. signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. respiratory tract and intestinal tract diseases may be concurrent. infections caused by this virus are often found associated with other viral and bacterial infections. epizootiology and transmission. the virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. necropsy findings. lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. etiology. the bluetongue virus is an rna virus in the orbivirus genus and reoviridae family. five serotypes ( , , , , and ) have been identified in the united states, where it is seen mostly in western states. bluetongue is an acute arthropodborne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities (bulgin, ) . clinical signs and diagnosis. sheep are the most likely to show clinical signs. clinical disease is less common in goats and cattle. early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. the virus name, bluetongue, is associated with the typical cyanotic membranes. the fever may subside, but tissue lesions erode, causing ulcers. increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. chorioretinitis and conjunctivitis are also common signs in cattle and sheep. lameness may be observed associated with coronitis and is evident in the rear legs. skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. secondary bacterial pneumonia may also occur. animals may also develop severe diarrhea and become recumbent. sudden deaths due to cardiomyopathy may occur at any time during the disease. hematologically, animals will be leukopenic. the course of the disease is about weeks, and mortality may reach %. if animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. abortions may occur at any stage of gestation in cattle. prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. fluorescent antibody tests, elisa, virus neutralization tests, pcr, and agar gel immunodiffusion (agid) tests are also used to confirm the diagnosis. necropsy findings. at necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. ulceration or hyperemia of the coronary bands may also be seen. many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. pathogenesis. the virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. after entering the host, the virus causes prolonged viremia. the incubation period is - days. the virus migrates to and attacks the vascular endothelium. the resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. the effects on fetuses appear to be due to generalized infections of developing organs. differential diagnosis. differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. rinderpest is a differential in countries where it is endemic. photosensitization should be considered. foot rot is a differential for the lameness and coronitis. differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as charolais cattle and merino sheep. prevention and control. cellular and humoral immunity are necessary for protection from infection. the bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. in addition, there is little to no cross protection between strains. modified live vaccines are available in some parts of the united states but should not be used in pregnant animals. vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating lategestation ewes may cause birth defects or abortions. congenital defects are more common from vaccine use than from naturally occurring infection. minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. treatment. supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. nonsteroidal anti-inflammatory drugs are often administered. for the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. research complications. this is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. etiology. bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (blv) infection, and those in older cattle that are associated with b lv. b lv is a b lymphocyte-associated retrovirus (johnson and kaneene, a,b,c) . clinical signs. forms of bovine lymphosarcoma that are not associated with blv infection are calf, or juvenile; thymic, or adolescent (animals months to years old); and cutaneous (any age). the calf form is rare and characterized by generalized lymphadenopathy. onset may be sudden, and the disease is usually fatal within a few weeks. signs include lymphadenopathy, anemia, weight loss, and weakness. some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. the adolescent form is also rare, the course rapid, and the prognosis poor. the disease is seen most often in beef breeds such as hereford cattle and is characterized by space-occupying masses in the neck or thorax. these masses are also often present in the brisket. secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. the cutaneous presentation has a longer course and may wax and wane. the masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. the animals are anemic, and neoplastic involvement may affect cardiac function. generalized or limited lymphadenopathy may be apparent. only the adult, or enzootic, form of bovine lymphosarcoma is associated with blv infection. many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. some cows manifest disease only during the periparturient period. malignant lymphoma is the more common, whereas leukosis, due to b-lymphocyte proliferation, is rare. clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. tumors are associated with lymphoid tissues. common sites also include the abomasum, spinal canal, and uterus. cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. the common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. many internal organs may be involved, and tumors may be palpable per rectum. secondary infections will be due to immunosuppression and the weakened state of the animal. sheep have acquired blv infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. goats seroconvert but do not develop the clinical syndromes. diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. kits are available for running agid, for which the blv antigens gp- and gp- are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. elisa and pcr diagnostic aids will also be helpful. worldwide. it is estimated that at least % of the cattle in the united states are infected with blv. as few as % of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the united states. larger herds tend to have higher rates. genetic predisposition may be involved; in addition to the presence of blv, the type of bovine lymphocyte antigen (bola) may be correlated to resistance or susceptibility and to the course of the disease. transmission is believed to be by inhalation of blv in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). natural-service bulls may transmit the infection to cows. cows infected with blv may transmit the infection to their calves in utero. tabanid and other flies also serve as vectors, but these represent a minor means of transmission. necropsy findings. neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. tumors may be local or widely distributed in the enzootic form. definitive diagnosis of neoplastic tissue specimens is by histology. pathogenesis. as with other retroviruses, the blv integrates viral dna into host target cell dna by means of the reverse transcriptase enzyme, creating a provirus. epizootiology and transmission. the virus is reported to be widespread. occurrence is often seasonal, and biting insects may be vectors. transmission with successful infection requires deep penetration of the skin. transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. differential diagnosis. differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. there is no vaccine for this disease. development and maintenance of a blv-free herd, or controlling infection within a herd, requires financial and programmatic commitments: blv-positive and blv-negative animals maintained separately; serologic testing (such as at least every months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile singleuse products), and equipment for ear tagging and dehorning and other such equipment between animals. a fresh rectal exam sleeve and lubricant should be used for each animal examined. otherwise serologically positive cows may have undetectable antibodies during the periparturient period. embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. calves should be fed colostrum from serologically negative cows. treatment. treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. in cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. research complications. the united states and several countries, some in europe, have official programs for eradication of enzootic bovine leukosis. prevention and control. established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. treatment. there is no treatment, and affected animals should be separated from the herd and milked last. lesions can be cleaned and treated with topical antibacterials. etiology. the bovine viral diarrhea virus (bvdv) is a pestivirus of the flaviviridae family. the flaviviridae include hog cholera virus and border disease virus of sheep. the virus contains a single strand of positive-sense rna. a broad range of disease and immune effects is produced by b vdv only in cattle. in addition, this virus is important in the etiology of bovine pneumonias. bovine viral diarrhea/mucosal disease (bvd/md) is one of the most important viral diseases and one of the most complex diseases of cattle. strains of bvdv are characterized as cytopathic (cp) and noncytopathic (ncp), based on cell-culture growth characteristics. the virus has also been categorized as type and type isolates. heterologous strains exist that may confound even sound vaccination programs. etiology. bovine herpesvirus causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. clinical signs and diagnosis. lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. the udder lesions may extend to the perineum. the lesions progress to vesicles, then to ulcers; these may take weeks to heal. lesions rarely may also develop focally around the mouth and generally on the skin of the udder. secondary mastitis may occur, because of bacteria associated with the scabs. diagnosis is by clinical signs and serologically. clinical signs and diagnosis. signs of bvdv infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (pi) with gradual debilitation, and acute and fatal disease. the presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. an acute form of the disease, caused by type bvdv, occurs in cattle without sufficient immunity. after an incubation period of - days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. the disease course may be shorter with hemorrhagic syndrome and death within days. clinical signs of b vdv in calves also include severe enteritis and pneumonia. when susceptible cows are infected in utero from gestational be found extending throughout the gastrointestinal tract to the days - , or gestational cows are vaccinated with a modi-cecum. the respiratory tract lesions will often be complicated fled live vaccine, abortion or stillbirth result. congenital defects caused by bvdv during gestational days - include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. some severely affected calves will not be able to stand. ophthalmic effects include retinal degeneration and microphthalmia. fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (pi). the term mucosal disease is commonly associated with this form of the infection. many pi animals do not survive to maturity, however, and many have weakened immune systems. the pi animals are important because they shed virus and will probably show the clinical signs of mucosal disease (md) caused by a cp b vdv strain derived from an ncp b vdv strain. these md clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to b vdv in precolostral serum. viral culturing from blood may be useful. in older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. leukopenia, and especially lymphopenia, are seen. serology must be interpreted with the awareness of the possibility of pi immunotolerant animals. vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against cp or ncp strains. the condition of the animals is also a variable. epizootiology and transmission. bvdv is present throughout the world. transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. pi females transmit the virus to their fetuses. semen also is a source of virus. necropsy findings. in affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. older calves may have areas of intestinal necrosis. in cases where oral erosions occur, erosions will by secondary bacterial pneumonia. when the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. pathogenesis. the cp and ncp strains are thought to be related mutations of the bvdv; the cp short-lived isolates are believed to arise from the ncp strains. the ncp strains are those present in the pi animals, and the strains are maintained in cattle populations. cp and ncp isolates vary in virulence, and classification of these types is based on viral surface proteins. considerable antigenic variation also exists between strains and types. other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. the pathology caused by b vdv is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. in type bvdv hemorrhagic syndrome, death results from viral-induced thrombocytopenia. in fetuses, the virus infects developing germinal cells of the cerebellum. the purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. the immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. all of these predispose the affected animals to bacterial infections with pasteurella haemolytica. b vdv damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. differential diagnosis. many differentials must be considered for the clinical manifestations of b vdv infections. differentials for enteritis of calves include viral infections, cryptosporidia, escherichia coli, salmonella, and coccidia. salmonella, winter dysentery, johne's disease, intestinal parasites, malignant catarrhal fever (mcf), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. respiratory tract pathogens such as bovine respiratory syncytial virus, pasteurella, haemophilus, and mycoplasma must be considered for the respiratory tract manifestations. oral lesions are also produced by mcf, vesicular stomatitis, bluetongue, and papular stomatitis. infectious bovine herpesvirus , leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. prevention and control. combined with sound management in a typical cattle herd, vaccination is the best way to prevent b vdv and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. vaccine preparations for b vdv are modified live virus (mlv) or killed virus. each has advantages and disadvantages. the former induces rapid immunity (within week) after a single dose, provides longer duration of immunity against sev-eral strains, and induces serum neutralizing antibodies. mlv vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. the immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. the mlv strains may cross the placenta, resulting in fetal infections. the killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given - times per year, and do not induce cell-mediated immunity. passive immunity may protect most calves for up to - months of age. subsequent vaccination with mlv may provide lifelong immunity, but this is not guaranteed. annual boosters are recommended to protect against vaccine breaks. the virus persists in the environment for weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. other management strategies may require a program for testing and culling pi cattle. this can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. no specific treatment is available. supportive care and treatment with antibiotics to prevent secondary infection are recommended. animals that survive the infection should be evaluated a month after recovery to determine their status as pi or virus-free. etiology. cache valley virus (cvv), of the arbovirus genus of the bunyaviridae family, is a cause of congenital defects in lambs. cvv infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. stillbirths and mummified fetuses are seen. lambs will be born weak and will act abnormally. diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. western united states, although it has been isolated in a few midwestern states. although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. transmission is by arthropods during the first trimester of pregnancy. etiology. caprine arthritis encephalitis virus (caev) occurs worldwide, with a high prevalence in the united states. caprine arthritis encephalitis (cae) is considered the most important viral disease of goats. the caev is in the lentivirus genus of the retroviridae family. it causes chronic arthritis in adults and encephalitis in young. caev is in the same viral genus as the ovine progressive pneumonia virus (oppv). clinical signs and diagnosis. the most common presentation in goats is an insidious, progressive arthritis in animals months of age and older. animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." in dairy goats, milk production decreases, and udders may become firmer. this retrovirus also causes neurological clinical signs in young kids - months old. kids may be bright and alert, afebrile, and able to eat normally even when recumbent. some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. older animals in the group may experience interstitial pneumonia or chronic arthritis. the pneumonia is similar to the pneumonia in sheep caused by oppv; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. some animals in a herd may not develop any clinical signs. diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to caev. an agar gel immunodiffusion (agid) test identifies antibodies to the virus and is used for diagnosis. kids acquire an anti-caev antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. the antibody does not prevent viral transmission. ep&ootiology and transmission. the virus is prevalent in most industrialized countries. the common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-caev antibody in the colostrum. transmission may occur among adult goats by contact. intrauterine transmission is believed to be rare. transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. necropsy findings. necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. in severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. tem, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. the infection is lifelong. differential diagnosis. the differential diagnosis for the neurologic form of caev should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. the differential diagnosis for caev arthritis should include chlamydia and mycoplasma. prevention and control. herds can be screened for cae by testing serologically, using an agid or an enzyme-linked immunosorbent assay (elisa) test. the elisa is purported to be more sensitive, whereas the agid is more specific. individual animals show great variation in development of antibody. because cae is highly prevalent in the united states, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. recently, an immunoprecipitation test for cae has been developed that has high sensitivity and specificity. control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum ( ~ for hr). caev-negative goats should be separated from caevpositive goats. treatment. there is no treatment for caev. is also referred to as bovine herpesvirus (bhv- ) and is an alphaherpesvirus. ibrv causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. it is one of the primary pathogens in the bovine respiratory disease complex. strains include bhv-i. (associated with respiratory disease), bhv . (associated with respiratory and genital diseases), and bhv . (associated with neurological diseases), which has been reclassified as bovine herpesvirus . clude conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. the respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. the mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. neonatal calves may develop respiratory as well as general systemic disease. in these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. this form is usually fatal within days. abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as months after the respiratory tract disease signs. infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. the cow will often carry her tail elevated away from these lesions. these soon coalesce, and a fibrous membrane covers the ulcerated area. if uncomplicated, the infection lasts about - days, and lesions heal in weeks. younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. epizootiology and transmission. ibrv is widely distributed throughout the world, and adult animals are the reservoirs of infection. the disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. modified live vaccines are capable of causing latent infections. necropsy findings. fibrinonecrotic rhinotracheitis is considered pathognomic for ibrv respiratory tract infections. there will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. when there are secondary bacterial infections, such as pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). intranuclear inclusion bodies are not a common finding with this herpesvirus. pathogenesis. in the encephalitic form, the virus first grows in nasal mucosa and produces plaques. these resolve within days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. latent infections are also established in neural tissue. differential diagnosis. the severe oral erosions seen with bvdv infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv). the conjunctivitis of ibr may initially be mistaken for that of a moraxella bovis (pinkeye) infection; the ibr will be peripheral, and there will not be corneal ulceration. bovine viral diarrhea virus and ibrv are the most common viral causes of bovine abortion. differentials for balanoposthitis include trauma from service. vated, attenuated, modified live, and genetically altered preparations. some are in combination with parainfluenza (pi- ) virus. the mlv preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. treatment. uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. the encephalitic animals may need to be treated with anticonvulsants. etiology. parainfluenza , an rna virus of the family paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. the viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. viral strains are reported to vary in virulence. serotypes seen in the smaller ruminants are distinct from those isolated from cattle. clinical signs and diagnosis. infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. in pregnant animals, exposure to pi- can result in abortions. clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as pasteurella haemolytica infection, is involved. greater morbidity and mortality will be sequelae of the bacterial infections. viral isolation or direct immunofluorescence antibody (ifa) from nasal swabs can be used for definitive diagnosis. presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. for an infection of pi- only, findings will be negligible. some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. immunohistochemistry may also be used. pathogenesis. pi- infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. differential diagnosis. differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: ibrv, bvdv, bovine respiratory syncytial virus, and type bovine adenovirus. prevention and control. immunization, management, and nutrition are important for this respiratory pathogen, as for others. in cattle, modified live vaccines for intramuscular (im), subcutaneous (sc), or intranasal (in) administration are available. the im and sc routes provide immune protection within week after administration but will not provide protection in the presence of passively acquired antibodies. it is contraindicated for pregnant animals because it will cause abortion. the in route immunizes in the presence of passively acquired antibodies, provides immunity within days of administration, and stimulates the production of interferon. other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered im, and followup immunizations are needed within weeks. booster vaccinations are recommended for all preparations within - months after the initial immunization. all presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. the humoral immunity protects against pi- abortions. there is no approved pi- vaccine for sheep and goats. the use of the cattle formulation in these smaller ruminants is not recommended. sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. treatment. uncomplicated disease is not treated. etiology. the respiratory syncytial viruses are pneumoviruses of the paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. two serotypes of the bovine respiratory syncytial virus (brsv) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. clinicalfindings and diagnosis. infections may be subclinical or develop into severe illness. clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. secondary bacterial pneumonia, especially with pasteurella haemolytica, with morbidity and mortality, is also a common sequela. abortions have been assciated with brsv outbreaks. diagnosis is based on virus isolation and serology (acute and convalescent). nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. prevention and control. vaccination should be part of the standard health program, and all animals should be vaccinated regularly. vaccinations should be administered within - months of stressful events, such as weaning, shipping, and introduction to new surroundings. currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. but this immunity suppresses the mucosal iga response and serum antibody responses. the basis for successful immune protection is the mucosal memory iga, but this is difficult to achieve with present vaccine formulations. the virus is easily inactivated in the environment. preventive measures in preweaning animals should include preconditioning to minimize weaning stress. treatment. recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. in severe cases, antihistamines and corticosteriods may also be necessary. use of vaccine during natural infection is not productive and may result in severe disease. etiology. ulcerative dermatosis is a contagious disease of sheep only. it is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ("current veterinary therapy," ). epizootiology and transmission. these viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. teroventral lung lobes. edema and emphysema are present. as the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. crusts associated with the skin and mucous membranes of the genitalia, face, and feet (bulgin, ) . genital lesions are much more common than the facial or coronal lesions. discomfort may be associated with the lesions. paraphimosis occasionally occurs. these lesions are painful; during breeding season, animals will avoid coitus. morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. diagnosis is based on clinical signs. pathogenesis. the severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. virulence may vary greatly among viral strains. united states, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. necropsy findings. necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. findings will be similar to those described for contagious ecthyma. when no contact with cattle has occurred. persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. pathogenesis. following an incubation period of - days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. pustules rapidly break, forming weeping ulcers. the ulcers scab over and eventually form a fibrotic scar. the disease usually resolves in - weeks. rarely, the disease will persist for many months to more than a year. differential diagnosis. the main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. this is also a feature of ulcerative dermatosis. imals, especially males, should not be used for breeding. treatment. affected animals should be separated from the rest of the flock. treatment is supportive, including antiseptic ointments and astringents. research complications. breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. etiology. border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern united states), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (bvdv), a pestivirus of the togaviridae family. goats are also affected. the virus causes few pathogenic effects in cattle. clinical signs and diagnosis. border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. the virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. diagnosis includes the typical signs described above, as well as serological evidence of viral infection. virus isolation confirms the diagnosis. wide, and reports of disease are sporadic. disease has occurred necropsy findings. lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. pathogenesis. the virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. infection during the first days of gestation causes embryonic death. in lambs infected between and days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. infection after days of gestation results in lambs that are born persistently infected. infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. prevention and control. border disease can be prevented by vaccinating breeding ewes with killed-bvdv vaccine. congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. new animals to the flock should be screened serologically. if cattle are housed nearby, vaccination programs for bvdv should be maintained. treatment. there is no treatment other than supportive care for affected animals. etiology. contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or off, is an acute dermatitis of sheep and goats caused by a parapoxvirus. this disease occurs worldwide and is zoonotic. naturally occurring disease has also been reported in other species such as musk ox and reindeer. other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. clinical signs and diagnosis. the disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. off is usually found in young animals less than year of age. younger lambs and kids will have difficulty nursing and become weak. lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. the scabs may appear nodular and raised above the surface of the surrounding skin. morbidity in a susceptible group of animals may exceed %. mortality is low, but the course of the disease may last up to weeks. diagnosis is based on characteristic lesions. biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. electron microscopy will reveal the virus itself. disease is confirmed by virus isolation. epizootiology and transmission. all ages of sheep and goats are susceptible. seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. older animals develop immunity that usually prevents reinfection for at least or more years. resistant animals may be present in some flocks or herds. the virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. necropsy findings. necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. secondary bacterial infection may also be evident. pathogenesis. the virus is typical of the poxviridae, resembling sheep poxvirus (not found in the united states) and vaccinia virus and replicating in the cytoplasm of epithelial cells. following an incubation period of - days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. the vesicles form pustules that rupture and finally scab over. virus should be considered in both sheep and goats. an important differential in goats is staphylococcal dermatitis. prevention and control. individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. colostral antibodies may not be protective. vaccinating lambs and kids with commercial vaccine best prevents the disease. dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. animals newly introduced to infected premises should be vaccinated upon arrival. precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. affected dairy goats should be milked last, using disposable towels for cleaning teat ends. treatment. affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. treatment for myiasis may also be necessary. the viral infection is self-limiting, with recovery in about weeks. research complications. carrier animals may be a factor in flock or herd outbreaks. contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. the virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as weeks. lesions heal without scarring. etiology. foot-and-mouth disease (fmd) is caused by the foot-and-mouth disease virus, a picornavirus in the aphthovirus genus. the disease is also referred to as aftosa or aphthous fever. seven immunologically distinct types of the virus have been identified, with subtypes within those . epidemics of the disease have occurred worldwide. north and central america have been free of the virus since the mid- s. this is a reportable disease in the united states; clinical signs are very similar to other vesicular diseases. cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. clinical signs and diagnosis. in addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. vesicles may be as large as cm, rupture after days, and subsequently erode. secondary bacterial infections often occur at the erosions. anorexia is likely due to the pain associated with the oral lesions. high morbidity and low mortality, except for the high mortality in young cattle, are typical. diagnosis must be based on elisa, virus neutralization, fluorescent antibody tests, and complement fixation. epizootiology and transmission. domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. greater mortality occurs in younger animals. the united states, great britain, canada, japan, new zealand, and australia are fmd-free, whereas the disease is endemic in most of south america, parts of europe, and throughout asia and africa. the virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. transmission may also occur by fomites, such as shoes, clothing, and equipment. human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. necropsy findings. vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. myocardial and skeletal muscle degeneration (zenker's) is most common (and accounts for the greater mortality) in younger animals. histological findings include lack of inclusion bodies. vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. virus shedding begins about hr before clinical signs are apparent. vesicles result from the separation of the superficial epithelium from the basal epithelium. fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. differential diagnosis. vesicular stomatitis is the principal differential. other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. products from endemic areas is regulated. quarantine and slaughter are practiced in outbreaks in endemic areas. quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated or times per year to be effective and will provide only partial protection. autogenous vaccines are best in an outbreak. passive immunity protects calves for up to months after birth. the virus is inactivated by extremes of ph, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. treatment. nursing care and antibiotic therapy to minimize secondary reactions help with recovery. humoral immunity is considered the more important immune mechanism, with cellmediated immunity of less importance. research complications. rare cases in humans have been reported. importation into the united states of animal products from endemic areas is prohibited. etiology. malignant catarrhal fever (mcf) is a severe disease primarily of cattle. the agents of mcf are viruses of the gammaherpesvirinae subfamily. alcelaphine herpesvirus and and ovine herpesvirus are known strains. the alcelaphine strains are seen in africa. the ovine strain is seen in north america. the alcelaphine and ovine strains differ in incubation times and duration of illness. disease may occur sporadically or as outbreaks. clinical signs and diagnosis. signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. sudden death may also occur in cattle. presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. as the disease progresses, cattle may shed horns and hooves. in north america, cattle will also have severe diarrhea. the course of the disease may extend to week. recovery is usually prolonged, and some permanent debilitation may occur. the disease is fatal in severely affected individuals. history of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. serology, pcr-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. because of the susceptibility of rabbits, inoculation of this species may be used. in less severe outbreaks or individual animal disease, definitive diagnosis may never be made. necropsy. gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. lymph nodes should be submitted for histological examination. histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. pathogenesis. the incubation period may be up to months. vascular endothelium and all epithelial surfaces will be affected. the virus is believed to cause proliferation of cytotoxic t lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. differential diagnoses. the differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. in africa, rinderpest is also a differential. other differentials are arsenic toxicity and chlorinated naphthalene toxicity. in north america, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. if there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. lambs can be free of the infection if removed from the flock at weaning. the virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. lobes; and hematological findings indicate anemia and leukocytosis. the rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. death occurs in weeks to months. the disease can be serologically diagnosed with agar gel immunodiffusion (agid) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (elisa) tests. sixty-eight percent of sheep in some states have been infected with the virus (radostits et al., ) . it is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. in addition, transmission through the milk or colostrum is considered common (knowles, ) . necropsy findings. lesions are observed in lungs, mammary glands, joints, and the brain. pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (cns). histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the cns. affected and any exposed animals should be isolated from healthy animals. there is no specific treatment for mcf; supportive treatment may improve recovery rates. corticosteroids may be useful. etiology. an rna virus in the lentivirus group of the retroviridae family causes ovine progressive pneumonia (opp), or maedi/visna. maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in iceland. visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. clinical signs and diagnosis. opp is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia (pepin et al., ; de la concha bermejillo, ) . clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to years. respiratory rate and dyspnea gradually increase as the disease progresses. the animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. additionally, mastitis is a common clinical feature. thoracic auscultation reveals consolidation of ventral lung pathogenesis. the virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. after initial infection, the virus integrates into the dna of mature monocytes and persists as a provirus. later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. the virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. secondary bacterial pneumonia may contribute to the animal's death. pulmonary adenomatosis is the differ-prevention and control. isolating or removing infected animals can prevent the disease. facilities and equipment should also be disinfected. ii. proliferative stomatitis (bovine papular stomatitis) etiology. a parapoxvirus is the causative agent of bovine papular stomatitis. this virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. it is also a zoonotic disease. the disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. in addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. the disease occurs worldwide. clinical signs and diagnosis. raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. in some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. lesions persist or may come and go over a span of several months. morbidity among herds may be %. mortalities are rare. bovine papular stomatitis is associated with "rat tail" in feedlot cattle. animals continue to eat and usually do not show a fever. no lesion is seen on the feet. the infection may also be asymptomatic. diagnosis is based on clinical signs, histological findings, and viral isolation. epizootiology and transmission. cattle less than year of age are most commonly affected, and disease is rare in older cattle. transmission is by animal-to-animal contact. necropsy findings. raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. pathogenesis. following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. these become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. differential diagnosis. pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. the differential for the "rat tail" clinical sign is sarcocystis infection. there is no vaccine available for bovine papular stomatitis. because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. vaccination using the local strain, and the skin scarification technique for off, have been protective. handlers should wear gloves and protective clothing. treatment. cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. their hands at sites of contact with lesions of cattle. iii. pseudocowpox etiology. pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see sections iii,a, ,m and iii,a, ,q,ii). lesions are confined to the teats. this is also a zoonotic disease. clinical signs and diagnosis. minor lesions are usually confined to the teats. these are distinctive because of the ring-or horseshoe-shaped scab that develops after days. additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. the teat lesions may predispose to mastitis. etiology. pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. pulmonary adenomatosis is caused by a type d retrovirus antigenically related to the mason-pfizer monkey virus. jaagsiekte was the designation when the disease was described originally in south africa. progressive respiratory signs such as dyspnea, rapid respiration, and wasting. the disease is diagnosed by these chronic clinical signs and histology. epizootiology and transmission. the disease is transmitted by aerosols. body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. necropsy. the adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. the adenocarcinomas metastasize to regional lymph nodes. pathogenesis. as with ovine progressive pneumonia (opp), the incubation period is up to years long. adenocarcinomatous lesions arising from type ii alveolar epithelial cells may be discrete or confluent and involve all lung lobes. with or is a differential diagnosis for opp. etiology. cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. the disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. these tumors are caused by a papillomavirus (dna virus) of the papovaviridae family, and the viruses are host-specific and often body site-specific. most are benign, although some forms in cattle and one form in goats can become malignant. in cattle, the site specificity of the papillomavirus strains are particularly well recognized. designations of the currently recognized bovine papillomavirus (bpv) types are bpv- through bpv- . clinical signs and diagnosis. the papillomas may last up to months and are seen more frequently in younger animals. lesions have typical wart appearances and may be single or multiple, small ( mm) or very large ( mm). the infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. in addition, when infections are severe, weight loss may occur. when warts occur on teats, secondary mastitis may develop. in cattle, bpv- and bpv- cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. bpv- causes flat warts that occur in all body locations, b pv- causes warts in the gastrointestinal tract, and b pv- causes small white warts (called rice-grain warts) on teats. warts caused by bpv- and bpv- do not regress spontaneously. prognosis in cattle is poor only when papillomatosis involves more than % of the body surface. in sheep, warts are the verrucous type. the disease is of little consequence unless the warts develop in an area that causes dis-comfort or incapacitation such as between the digits, on the lips, or over the joints. in adult sheep, warts may transform to squamous cell carcinoma. in goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. warts on goat udders tend to be persistent. diagnosis is made by observing the typical proliferative lesions. epizootiology and transmission. older animals are less sensitive to papillomatosis than young animals, although immunosupressed animals of any age may develop warts as the result of harbored latent infections. the virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. pathogenesis. the incubation period ranges from to months. the virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. the disease is generally self-limiting. differential diagnosis. in sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. autogenous vaccines are generally considered more effective. some vaccine preparations are effective at prevention but not treatment of outbreaks. viricidal products are recommended for disinfection of contaminated environments. minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. halters, brushes, and other items may also be sources of virus. treatment. warts will often spontaneously resolve as immunity develops. in severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. etiology. pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus . one serotype is recognized, but strain differences exist. the disease has worldwide distribution. it is a primarily a clinical dis-ease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. during the rapid course of this usually fatal disease. at the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. pruritus will not be asymmetric. animals will also become hyperthermic and will vocalize frantically. other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. nystagmus and strabismus are also seen. animals will be fearful or depressed, and aggression is sometimes seen. recumbency and coma precede death. diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. serology of affected animals is not productive, because of the rapid course. if swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. epizootiology and transmission. swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. the infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. other animals are dead-end hosts. the unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. when the virus is inhaled, the clinical signs of pruritus are less likely to be seen. transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. transport vehicles used for swine may also be sources of the virus. raccoons are believed to be vectors of the virus. horses are resistant to infection. there is no pathognomonic gross lesion. definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. eosinophilic intranuclear inclusion bodies (cowdry type a) may be present in some affected neurons. methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus . pathogenesis. the incubation period is - hr and duration of the illness is - hr. the longest duration is seen in animals with pruritus around the head. differential diagnoses. differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. prevention and control. pseudorabies is a reportable disease in the united states, where a nationwide eradication program exists; states are rated regarding status. effective disinfectants include sodium hypochlorite ( % solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. five minutes of contact time is required, and then surfaces must be rinsed. other disinfectant methods for viral killing include hr of formaldehyde fumigation, or min of ultraviolet light. transport vehicles should be cleaned and disinfected between species. serological screening for pseudorabies of swine housed near ruminants is essential. there is no treatment, and most affected ani-research complications. swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. humans have been reported to seroconvert. the porcine herpesvirus shares antigens with the infectious bovine rhinotracheitis virus. etiology. rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. the rabies virus is a neurotropic rna virus of the lyssavirus genus and the rhabdoviridae family. sheep, goats, and cattle are susceptible. the zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. rabies is endemic in many areas of the world and within areas of the unites states. this is a reportable disease in north america. clinical findings and diagnosis. animals generally progress through three phases: prodromal, excitatory, and paralytic. many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. during the short prodromal phase, animals are hyperthermic and apprehensive. animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. the final paralytic stage, with recumbency and death, occurs over several hours to days. this paralytic stage is common in cattle, and animals may simply be found dead. the clinical course is usually - days. diagnosis is based on clinical signs, with a progressive and fatal course. confirmation presently is made with the fluorescent antibody technique on brain tissue. epizootiology and transmission. the rabies virus is transmitted via a bite wound inflicted by a rabid animal. cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in north america. virus is also transmitted in milk and aerosols. necropsy findings. few lesions are seen at necropsy. many secondary lesions from manic behaviors during the course of disease may be evident. histological findings will include nonsuppurative encephalitis. negri bodies in the cytoplasm of neurons of the hippocampus and in purkinje's cells are pathognomonic histologic findings. pathogenesis. after exposure, the incubation period is variable, from weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. the rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). the fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. differential diagnosis. rabies should be included on the differential list when clinical signs of neurologic disease are evident. other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. prevention and control. vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the united states for goats. ruminants in endemic areas, such as the east coast of the united states, should be routinely vaccinated. any animals housed outside that may be exposed to rabid animals should be vaccinated. vaccination programs generally begin at months of age, with a booster at year of age and then annual or triennial boosters. awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. the virus is fragile and unstable outside of a host animal. research complications. aerosolized virus is infective. personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (tse), is not known to occur in the united states, where since it has been listed as a reportable disease. the profound impact of this disease on the cattle industry in great britain during the past two decades is well known. the disease may be caused by a scrapielike (prion) agent. it is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. the incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other tse diseases such as scrapie, which affects sheep and goats. in addition, the infectious agent is extremely resistant to dessication and disinfectants. confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. molecular biology techniques, such as western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. there is no vaccine or treatment. prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent usda regulations prohibit feeding any mammalian proteins to ruminants. etiology. scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. scrapie is a reportable disease. it is much more common in sheep than in goats. the disease is similar to transmissible mink encephalopathy, kuru, creutzfeldt-jakob disease, and bovine spongiform encephalopathy (mad cow disease). prions are nonantigenic, replicating protein agents. clinical signs and diagnosis. during early clinical stages, animals are excitable and hard to control. tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. in advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. blindness and abortion may also be seen. morbidity may reach % within a flock. most animals invariably die within - weeks; some animals may survive months. in goats, the disease is also fatal. pruritus is generally less severe but may be localized. a wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. as with sheep, the disease gradually progresses to anorexia and debilitation. diagnosis can be made by clinical signs and histopathological lesions. a newer diagnostic test in live animals is based on sampling from the third eyelid. tests for genetic resistance or susceptibility require a tube of edta blood and are reasonably priced. epizootiology and transmission. the suffolk breed of sheep tends to be especially susceptible. scrapie has also been reported in several other breeds, including cheviot, dorset, hampshire, corriedale, shropshire, merino, and rambouillet. it is believed that there is hereditary susceptibility in these breeds. targhees tend to be resistant. genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons (q, r, or h genes can be present) and (a or v genes can be present). of the five genes, r genes appear to confer immunity to clinical scrapie in suffolks in the united states. affected suffolks in the united states that have been tested have been aa qq. the disease is also enzootic is many other countries. the disease tends to affect newborns and young animals; however, because the incubation period tends to range from to years, adult animals display signs of the disease. scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. vertical transmission is questioned, and transplacental transmission is considered unlikely. necropsy findings. at necropsy, no gross lesion is observed. histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. inflammatory lesions are not seen. pathogenesis. replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. differential diagnosis. in sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. prevention and control. if the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. the u.s. department of agriculture has approved the use of % sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. prions are highly resistant to physicochemical means of disinfection. artificial insemination or embryo transfer has been shown to decrease the spread of scrapie (linnabary et al., ) . research complications. as noted, this is a reportable disease. stringent regulations exist in the united states regarding importation of small ruminants from scrapie-infected countries. etiology. vesicular stomatitis (vs) is caused by the vesicular stomatitis virus (vsv), a member of the rhabdoviridae. three serotypes are recognized: new jersey, indiana, and isfahan. the new jersey and indiana strains cause sporadic disease in cattle in the united states. the disease is rare in sheep. clinical signs and diagnosis. adult cattle are most likely to develop vs. fever and development of vesicles on the oral mucous membranes are the initial clinical signs. lesions on the teats and interdigital spaces also develop. the vesicles progress quickly to ulcers and erosions. the animal's tongue may be severely involved. anorexia and salivation are common. weight loss and decreased milk production are noticeable. morbidity will be high in an outbreak, but mortality will be low to nonexistent. diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. virus isolation, enzyme-linked immunosorbent assay (elisa), competitive elisa (celisa), complement fixation, and serum neutralization are used for diagnosis. epizootiology and transmission. this disease occurs in several other mammalian species, including swine, horses, and wild ruminants. vsv is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of ph, and low temperatures. outbreaks of vs occur sporadically in the united states, but it is not understood how or in what species the virus survives between these outbreaks. incidence of disease decreases during colder seasons. equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. transmission may also be from contaminated water and feed. transmission is also believed to occur by insects (blackflies, sand flies, and culicoides) that may simply be mechanical vectors. it is believed that carrier animals do not occur in this disease. necropsy. it is rare for animals to be necropsied as the result of this disease. typical vesicular lesion histology is seen, with ballooning degeneration and edema. there is no inclusion body formation. pathogenesis. lesions often begin within hr after exposure. the virus invades oral epithelium. injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. differential diagnosis. foot-and-mouth disease lesions are identical to vs lesions. other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. prevention and control. quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. vaccines are available for use in outbreaks and have decreased the severity of lesions. phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. treatment. affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. these animals should be cared for after unaffected animals. any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. topical or systemic antibiotics control secondary bacterial infections. cases of mastitis secondary to teat lesions must be treated as necessary. any abrasive materials that could cause further trauma to the animals should be removed. research complications. animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. vsv causes a flulike illness in humans. x. viral diarrhea diseases i. ovine. rotavirus, of the family reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. four antigenic groups (a-d) have been identified by differences in capsid antigens vp and vp . primarily group a, but also groups b and c, have been isolated from sheep. the disease is characterized by yellow, semifluid to watery diarrhea occurring - days after infection. the disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. the virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. the villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. virus may remain in the environment for several months. the disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal elisa tests (marketed tests generally detect group a rotavirus), and fecal latex agglutination tests. rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. a rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ("current veterinary therapy," ). coronavirus, of the family coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. clinical signs are similar to above, although the incubation period tends to be shorter ( - hr), and animals exhibit less anorexia than those with rotavirus. additionally, mild respiratory disease may be noted (janke, ) . like rotavirus, coronavirus also destroys enterocytes of the villus tips. the virus can be visualized with electron microscopy. treatment is supportive; close consideration of hydration and acid-base status is essential. bovine vaccines are available. ii. caprine. rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. it appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. bovine. rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (bvdv) are associated with diarrheal disease in calves. each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. these viral infections may be complicated by parasitic infections (e.g., cryptosporidium, eimeria) or bacterial infections (e.g., escherichia coli, salmonella, campylobacter). treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in section ii,b, . combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic e. coli. additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. rotaviruses of serogroup a are the most common type in neonatal calves; -to -day old calves are typically affected, but younger and older animals may also be affected. the small intestine is the site of infection. antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. transmission is likely from other affected calves and asymptomatic adult carriers. the diarrhea is typically a distinctive yellow. colitis with tenesmus, mucus, and blood may be seen. this virus may be zoonotic. coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small-and large-intestinal epithelial cells. the virus infection may extend to mild pneumonia. transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. calves that appear to have recovered continue to shed virus for several weeks. parvovirus infections are usually associated with neonatal calves. b vdv infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in section iii,a, ,e. iv. winter dysentery. winter dysentery is an acute, winterseasonal, epizootic diarrheal disease of adult cattle, although it has been reported in -month-old calves. the etiology has not yet been defined, but a viral pathogen is suspected. coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. individual cows are ill for only a few days. the incubation period is estimated at - days. the outbreaks of disease are often seen in herds throughout the local area. clinical signs include explosive diarrhea, anorexia, depression, and decreased production. the diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. animals will become dehydrated quickly but are thirsty. respiratory symptoms such as nasolacrimal discharges and coughing may develop. recovery is generally spontaneous. mortalities are rare. diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as salmonella or mycobacterium paratuberculosis, and viruses such as b vdv. pathology is present in the colonic mucosa, and necrosis is present in the crypts. etiology. chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. clinical signs. enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids (rodolakis et al., ) . the only presenting clinical sign may be serosanguineous vulvar discharges. other animals may present with arthritis or pneumonia. infection of animals prior to about days of gestation results in abortion, stillbirths, or birth of weak lambs. infection after days results in potentially normal births, but the dams or offspring may be latently infected. latently infected animals that were infected during their dry period may abort during the next pregnancy. ewes or does generally only abort once, and thus recovered animals will be immune to future infections. and specific antigens associated with the cell surface. the group antigen is common among all chlamydia; the specific antigen is common to related subgroups. two subgroups are recognized, one that causes eae and one that causes polyarthritis and conjunctivitis. the disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. necropsy. placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. impression smears in placental tissues stained with giemsa, gimenez, or modified ziehl-neelsen can provide preliminary indications of the causative agent. immunofluorescence, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr) methods also aid in diagnosis. differential diagnosis. q fever will be the major differential for late-term abortion and necrotizing placentitis. campylobacter and toxoplasma should also be considered for late-term abortion. treatment. animals may respond to treatment with oxytetracycline. abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. this is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. research complications. in addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. etiology. chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness (bulgin, ) , and conjunctivitis (see section iii,a, ,c) in growing and nursing lambs. clinical signs. clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. lambs may be anorexic and febrile. animals frequently also exhibit concurrent conjunctivitis. the disease usually resolves in approximately weeks. joint inflammation usually resolves without causing chronic articular changes. epizootiology and transmission. the disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. the organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. the organism causes acute inflammation and associated fibrinopurulent exudates. necropsy findings. lesions are found in joints, tendon sheaths, conjunctiva, and lungs. pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. lesions will be infiltrated with mononuclear cells. lung lesions include atelectasis and alveolar inspissation. diagnosis is based on clinical signs. synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. treatment. animals respond to treatment with parenteral oxytetracycline. etiology. chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. chlamydia and mycoplasma are considered to be the most common causes of this disease in goats. chlamydial conjunctivitis is not a disease of cattle. clinical signs. infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. perforation may result from the ulceration. animals will be photophobic. in less severe cases, corneal healing associated with fibrosis and neovascularization occurs in - days. lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. morbidity may reach - %. bilateral and symmetrical infections characterize most outbreaks. relapses may occur. other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. epizootiology and transmission. direct contact, and mechanical vectors such as flies easily spread the organism. necropsy. if the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. conjunctival smears are also useful. pathogenesis. the pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. the infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. the chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. diagnosis is suggested by the clinical signs. cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. differential diagnosis. nonchlamydial keratoconjunctivitis also occurs in sheep and goats. the primary agents involved include mycoplasma conjunctiva, m. agalactiae in goats, and branhamella (neisseria) ovis. a less common differential for sheep and cattle is listeria monocytogenes. other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. should be minimized whenever possible. quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. shade should be provided for all animals. treatment. the infections can be self-limiting in - weeks without treatment. treatment consists of topical application of tetracycline ophthalmic ointments. systemic or oral oxytetracycline treatments have been used with the topical treatment. atropine may be added to the treatment regimen when uveitis is present. shade should be provided. a. protozoa i. anaplasmosis etiology. anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan anaplasma marginale. anaplasma is a member of the anaplasmatacae family within the order rickettsiales. in sheep and goats, the disease is caused by a. ovis and is an uncommon cause of hemolytic disease. anaplasmosis has not been reported in goats in the united states. some controversy exists regarding the classification. most recently it is classified as a protozoal disease because of similarities to babesiosis. it has also been classified as a rickettsial pathogen. this summary addresses the disease in cattle with limited reference to a. ovis infections, but there are many similarities to the disease in cattle. clinical signs and diagnosis. acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. four disease stagesnincubation, developmental, convalescent, and carriermare recognized. the incubation stage may be long, - weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. most clinical signs occur during the -to -day developmental stage, with hemolytic anemia being common. death is most likely to occur at this stage or at the beginning of the convalescent stage. death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. reticulocytosis characterizes the convalescent stage, which may continue for many weeks. morbidity is high, and mortality is low. the carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and anaplasma organisms and any parasitemia are not discernible. common serologic tests are the complement fixation test and the rapid card test. these become positive after the incubation phase and do not distinguish between the later three stages of disease. definitive diagnosis is made by clinical and necropsy findings. staining of thin blood smears with wright's or giemsa stain allows detection of basophilic, spherical a. marginale bodies near the red blood cell peripheries. evidence will most likely be found before a hemolytic episode. a negative finding should not eliminate the pathogen from consideration. epizootiology and transmission. the disease is common in cattle in the southern and western united states. anaplasma organisms are spread biologically or mechanically. mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. biological transmission occurs when the tick stage of the organism is passed by dermacentor andersoni and d. occidentalis ticks. the carrier stage covers the time when discernible anaplasma organisms can be found on host blood smears. recovered animals serve as immune carriers and disease reservoirs. necropsy. pale tissues and watery, thin blood are typical findings. splenomegaly, hepatomegaly, and gallbladder distension are common findings. pathogenesis. the parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. the four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. differential diagnosis. the clinical disease closely resembles the protozoal disease babesiosis. whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. vaccinated animals can still become infected and become carriers. the cattle vaccine has shown no efficacy in smaller ruminants, and there is no a. ovis vaccine. identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. removing carriers to a separate herd is also an approach. interstate movement of infected animals is regulated. treatment. oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. other tetracycline treatment programs have been described to help control carriers. ii. babesiosis (red water, texas cattle fever, cattle tick fever) etiology. babesia bovis and ba. bigemina are protozoa that cause subclinical infections or disease in cattle. these are intraerythrocytic parasites. babesia bovis is regarded as the more virulent of the two organisms. this disease is not seen in the smaller ruminants in the united states. clinical signs and diagnosis. the more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. hemoglobinuria indicates a poor prognosis. acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. the encephalitic form generally also has a poor prognosis. sudden death may occur. thin blood smears stained with giemsa will show babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. the trophozoites occur in a variety of shapes, such as piriform, round, or rod. complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. babesiosis is present on several continents, including the americas. in addition to domestic cattle, some wild ruminants, such as white-tailed deer and american buffalo, are also susceptible. bos indicus breeds have resistance to the disease and the tick vectors. innate resistance factors have been found in all calves. if infected, these animals will not show many signs of disease during the first year of life and will become carriers. stress can cause disease development. prevention and control. offspring of immune carriers resist infection up to months of age because of passive immunity. vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. there is no entirely effective means, however, to prevent and control the disease. vaccination (killed necropsy findings. signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. pathogenesis. the protozoon is transmitted by the cattle fever ticks boophilus annulatus, b. microplus, and b. decoloratus; these one-host ticks acquire the protozoon from infected animals. it is passed transovarially, and both nymph and adult ticks may transmit to other cattle. only b. ovis is transmitted by the larval stage. clinical signs develop about weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. hemolysis is due to intracellular reproduction of the parasites and occurs intra-and extravascularly. in addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. the development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. differential diagnosis. in addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. several differentials in the united states for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. prevention and control. control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in section iii,a, ,a,i, are important preventive measures. some vaccination approaches have been effective, but a commercial product is not available. treatment. supportive care is indicated, including blood transfusions, fluids, and antibiotics. medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. research complications. this is a reportable disease in the united states. iii. coccidiosis etiology. coccidiosis is an important acute and chronic protozoal disease of ruminants. in young ruminants, it is characterized primarily by hemorrhagic diarrhea. adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. coccidia are protozoal organisms of the phylum apicomplexa, members of which are obligatory intracellular parasites. there are at least reported species of coccidia in sheep, of which several are considered pathogenic: eimeria ashata, e. crandallis, and e. ovinoidalis (schillhorn van veen, ). at least species of eimeria have been recognized in the goat (foreyt, ) . eimeria ninakohlyakimovae, e. arloingi, and e. christenseni are regarded as the most pathogenic. eimeria bovis and e. zuernii (highly pathogenic), and e. auburnensis and e. alabamensis (moderately pathogenic), are among the species known to infect cattle. eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." clinical signs and diagnosis. hemorrhagic diarrhea develops days to weeks after infection. fecal staining of the tail and perineum will be present. animals will frequently display tenesmus; rectal prolapses may also develop. anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. the diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. the intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. concurrent disease with other enteropathogens may also be part of the clinical picture. in sheep, secondary bacterial infection with organisms such as fusobacterium necrophorum may ensue. young goats may die peracutely or suffer severe anemia from blood loss into the bowel. older goats may lose the pelleted form of feces. cattle may have explosive diarrhea and develop anal paralysis. the disease is usually diagnosed by history and clinical signs. numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. laboratory results are usually reported as number of oocysts per gram of feces. coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. epkzootiology and transmission. as noted, coccidiosis is a common disease in young ruminants. in goats, young animals aged weeks to months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. coccidia are host-specific and also host cell-specific. the disease is transmitted via ingestion of sporulated oocysts. coccidial oocysts remain viable for long periods of time when in moist, shady conditions. necropsy. necropsies provide information on specific locations and severity of lesions that correlate with the species involved. ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. mucosal scrapings will frequently yield oocysts. various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. fibrin and cellular infiltrates will be found in the lamina propria. pathogenesis. this parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes (speer, ) . the severity of the disease is correlated primarily with the number of ingested oocysts. specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. in most cases, the disease is well established by the time clinical signs are seen. oocysts must undergo sporulation over a -to -day period in the environment. after ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. the organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. differential diagnosis. differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, yersinia, viruses, and other intestinal parasites such as helminths. in cattle, for example, bovine viral diarrhea virus and helminthiasis caused by ostergia must be considered. management factors, such as dietary-induced diarrheas, are also differentials. in older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. prevention and control. good management practices will help prevent the disease. oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. proper sanitation of animal housing and minimizing overcrowding are essential. coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. treatment. affected animals should be isolated. on an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). immunity may result when low numbers are ingested and there is only mild disease. immunity also may develop after more severe infections. iv. cryptosporidiosis etiology. cryptosporidium organisms are a very common cause of diarrhea in young ruminants. four cryptosporidium species have been described in vertebrates: c. baileyi and c. meleagridis in birds and c. parvum and c. muris in mammals. cryptosporidium parvum is the species affecting sheep (rings and rings, ) . debate continues regarding whether there are definite host-specific variants. clinical signs and diagnosis. cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. the diarrhea may last only - days or may be persistent and fatal. the diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. in relapsing cases, animals become cachectic. overall, morbidity will be high, and mortality variable. mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. the disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-schiff stain or methenamine silver. cryptosporidium also stains red on acid-fast stains such as kinyoun or ziehl-neelsen. fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of . (foryet, ) . fecal immunofluorescent antibody (ifa) techniques have also been described. epizootiology and transmission. younger ruminants are commonly affected: lambs, kids (especially kids between the ages of and days old), and calves less than days old. like other coccidians, cryptosporidium is transmitted via the fecal-oral route. in addition to local contamination, water supplies have also been sources of the infecting oocysts. the oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. necropsy findings. the lesions caused by cryptosporidium are nonspecific. animals will be emaciated. moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. cecal and colonic mucosae may sometimes be involved. gastrointestinal smears may be made at necropsy and stained as described above. pathogenesis. although cryptosporidium infections are clinically similar to eimeria infections (moore, ) , cryptosporidium, in contrast to eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. there is no intermediate host. the oocysts are half the size of eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. within - days of exposure, diarrhea and oocyst shedding occur. the diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. autoinfection within the lumen of the intestines may also occur and result in persistent infections. in addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. environmental stressors such as cold weather increase mortality. intensive housing arrangements increase morbidity and mortality. differential diagnosis. other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic escherichia coli; clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). in addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. eimeria is more likely to cause diarrhea in calves and lambs at - weeks of age. giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. blood. animals exhibit fever, dehydration, and depression. chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. oval cysts can be floated with zinc sulfate solution ( %). standard solutions tend to be too hyperosmotic and to distort the cysts. newer enzyme-linked immunosorbent assay (elisa) and ifa tests are sensitive and specific. epizootiology and transmission. giardia infection may occur at any age, but young animals are predisposed. chronic oocyst shedding is common. transmission of the cyst stage is fecaloral. wild animals may serve as reservoirs. necropsy findings. gross lesions may not be evident. villous atrophy and cuboidal enterocytes may be evident histologically. prevention and control. precautions should be taken when handling infected animals. affected animals must be removed and isolated as soon as possible. animal housing areas should be disinfected with undiluted commercial bleach or % ammonia. formalin ( %) fumigation has proven successful (foryet, ) . after being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. no known drug treatment is available. the disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. supplementation with vitamin a may be helpful. age resistance begins to develop when the animals are about month old. research complications. cryptosporidiosis is a zoonotic disease. it is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. the disease can be life-threatening in immunocompromised individuals. v. giardiasis etiology. giardia lamblia (also called g. intestinalis and g. duodenalis) is a flagellate protozoon. giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds (kirkpatrick, ), but it not considered to be a significant pathogen in ruminants. clinical signs and diagnosis. diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain pathogenesis. following ingestion, each giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. the organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. disturbances in intestinal motility may also occur (rings and rings, ) . prevention and control. intensive housing and warm environments should be minimized. cysts can survive in the environment for long periods of time but are susceptible to desiccation. effective disinfectants include quaternary ammonium compounds, bleach-water solution ( : or : ), steam, or boiling water. after cleaning, areas should be left empty and allowed to dry completely. treatment. giardia has been successfully treated with oral metronidazole. benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. should be taken when handling infected animals. etiology. neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species neospora caninum. abortions have also been reported in sheep and goats. neonatal disease is seen in lambs, kids, and calves. until , these infections were misdiagnosed as caused by toxoplasma gondii. some similarities exist between the life cycles and pathogeneses of both organisms. clinical signs and diagnosis. abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. although cows that abort tend to be culled after the first or second abortion, repeated n. caninum-caused abortions will occur progressively later in gestation (up to about months) and within a shorter time frame in the same cow (thurmond and hietala, ) . although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. many neospora-infected calves will be born asymptomatic. weakness will be evident in some infected calves, but this resolves. rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. serology (ifa and elisa) is useful, including precolostral levels in weak neonates, but this indicates only exposure. titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. earlier and rapid infections are less likely to yield antibodies against neospora. none of the currently available tests is predictive of disease. epizootiology and transmission. the parasite is now acknowledged to be widespread in dairy and cattle herds. the life cycle of n. caninum is complex, and many aspects remain to be clarified. the definitive host is the dog (mcallister et al., ) . placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. the many intermediate hosts include ruminants, deer, and horses. transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. a less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. seropositive immunity does not protect a cow from future abortions. many seropositive cows and calves will never abort or show clinical signs, respectively. some immunological cross-reactivity may exist among neospora, cryptosporidia, and coccidium. necropsy findings. aborted fetuses will usually be autolysed. in those from which tissue can be recovered, tissue cysts are most commonly found in the brain. spinal cord is also useful. histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. pathogenesis. as with toxoplasma, cell death is the result of intracellular multiplication of neospora tachyzoites. neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. the intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. infections of this type are latent and lifelong. except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. it usually takes - weeks for a fetus to die and to be expelled. many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of neospora abortions remain to be elucidated. differential diagnosis. even when there is a herd history of confirmed neospora abortions, leptospirosis, bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), salmonellosis, and campylobacteriosis should be considered. bvdv in particular should be considered for abortion storms. differentials for weak calves are b vdv, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, toxoplasma, exposure to teratogens, or congenital defects. prevention and control. the primary preventive measure is preventing contact with contaminated feces. oocysts will not survive dry environments or extremes of temperature. dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. dogs should also be restricted from feed bunks and other feed storage areas. preventive culling is not economically practical for most producers. a vaccine recently became available. if embryo transfer is practiced, recipients should be screened serologically before use. laxis. there is no known treatment or immunoprophy- clinical signs and diagnosis. clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. often the infections are asymptomatic. fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. abortions in cattle occur during the second trimester and in smaller ruminants days after ingestion of the sporulated oocysts. definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. sarcocystis-specific igg will increase dramatically by - weeks after infection. there is no cross-reaction between sarcocystis and toxoplasma. epizootiology and transmission. infection rates among cattle in the united states are estimated to be very high. transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. dogs are the definitive hosts for the species that infect the smaller ruminants. cats, dogs, and primates (including humans when s. hominis is involved) are the definitive hosts for the species that infect cattle. necropsy. aborted fetuses may be autolysed. lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. cysts in cardiac and skeletal muscles are common incidental findings during necropsies. pathogenesis. ingestion of muscle flesh from an infected ruminant results in sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. the sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. prevention and control. feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. these animals should be controlled and must also not have access to carcasses. in larger production situations, monensin may be fed as a prophylactic measure. treatment. monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. etiology. toxoplasmosis is caused by the obligate intracellular protozoon toxoplasma gondii, a coccidial parasite of the family eimeridae. cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. the disease is a major cause of abortion in sheep and goats and less common in cattle. clinical signs and diagnosis. clinical signs depend on the organ or tissue parasitized. toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young (underwood and rook, ; buxton, ) . it has also been shown to cause pneumonia and nonsuppurative encephalitis. the enteritis at the early stage of infection may be fatal in some hosts. hydrocephalus does not occur in animals as it does in human fetal toxoplasma infections. rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. in sheep, weak young will develop normally if they survive the first week after birth. infected adult sheep show no systemic illness. infected adult goats, however, may die. diagnosis may be difficult, and biological, serological, and histological methods are helpful. serological tests are the most readily available. complement fixation and the sabin-feldman antibody test may assist in diagnosis. antibodies found in fetuses are indicative of congenital infection and are typically detectable days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. epizootiology and transmission. this protozoon is considered ubiquitous. fifty percent ( %) of adult western sheep and % of feedlot lambs have positive hemagglutination titers ( : or higher) (jensen and swift, ) . transmission among the definitive host is by ingestion of tissue cysts. necropsy findings. at necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. histologically, granulomas with toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. impression smears of these tissues, stained appropriately (e.g., with giemsa), provide a rapid means of diagnosis. identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. pathogenesis. the protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. the definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. cats rarely show clinical signs of infection. one cat can shed millions of oocysts in gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. oocysts sporulate in cat feces after day. ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. as in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. the organism migrates to tissues such as the brain, liver, muscles, and placenta. placental infection develops about days after ingestion of the oocysts. the damage caused by an infection is due to multiplication within cells. toxoplasma does not produce any toxin. campylobacter, chlamydia, and q fever. prevention and control. feline populations on source farms should be controlled. eliminating contamination of feed and water with cat feces is the best preventive measure. sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. vaccines for abortion prevention in sheep are available in new zealand and europe. treatment. toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful (underwood and rook, ) . (monensin is not approved for this use in the unites states.) weak lambs that survive the first week after birth will mature normally and will not deliver toxoplasmainfected young. research complications. because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. infections in immunocompromised humans have been fatal. etiology. trichomoniasis is an insidious venereal disease of cattle caused by tritrichomonas (also referred to as trichomonas) fetus, a large, pear-shaped, flagellated protozoon. the organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. in the united states, it is now primarily a disease seen in western beef herds. there are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. clinical signs and diagnosis. clinical signs include infertility manifested by high nonpregnancy rates as well as periodic py-ometras and abortions during the first half of gestation. often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. the abortion rate varies from % to %, and placentas will be expelled or retained. tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. other than these manifestations, infection with t. fetus causes no systemic signs. diagnosis is based on patterns of infertility and pyometras. for example, pyometras in postcoital heifers or cows are suggestive of this pathogen. diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. other nonpathogenic protozoa from fecal contamination may be present in the sample. the trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. culturing must be done on specific media, such as diamond's or modified pastridge. real exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. necropsy findings. nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. pathogenesis. tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. tritrichomonas fetus does not interfere with conception. embryonic death occurs within the first months of infection. affected cows will clear the infection over a span of months and maintain immunity for about months. infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. older bulls become chronic carriers, probably because of the ability of t. fetus to colonize deeper epithelial crypts of the prepuce and penis. differential diagnosis. campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. heifers, cows, and breeding bulls are vaccinated subcutaneously twice at to week intervals, with the booster dose administered weeks before breeding season starts. similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. bulls used for artificial insemination (ai) are screened routinely for t. fetus (and campylobacter) . ai reduces but does not eliminate the disease. the use of younger, vaccinated bulls is recommcmded in all circumstances. new animals should be tested before introduction to the herd. control measures also include culling affected cows or else removing them from the breeding herd for months to rest and clear the infection. culling chronically infected bulls is strongly recommended. treatment. imidazole compounds have been effective, but the use of these is not permitted in food animals in the united states. therapeutic immunizations are worthwhile when a positive diagnosis has been made. these will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. research complications. trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. the major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. mixed infections of these parasites are common. generally, older animals develop resistance to some of the species; thus, animals between about months and years of age are most susceptible to infection. because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (baermann technique). a number of anthelmintics can be used to interrupt nematode life cycles. see zajac and moore ( ) and pugh et al. ( ) for comprehensive reviews of treatment and control of nematodiasis. i. haemonchus contortus, h. placei (barber's pole worm, large stomach worm) . haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. haemonchus contortus and h. placei infections do occur in younger cattle and are similar to the disease in sheep. haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. severe anemia may lead to death. weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. diarrhea is not seen in all cases but may sometimes be severe or chronic. the life cycle is direct. under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in weeks. embryonated eggs may develop into infective larvae within a week. hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of dosings given - weeks apart. levamisole is also used. in severe cases, animals may benefit from blood transfusions and iron supplementation. because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. ostertagia (teladorsagia) circumcincta (medium stomach worm). ostertagia circumcincta is also highly pathogenic for sheep and goats and, like haemonchus, attaches to the abomasal mucosa and ingests blood. the life cycle is comparable to that of haemonchus, including the phenomenon of hypobiosis. larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric ph from about . to near . , leading to decreased digestive enzyme activity and malnutrition. clinical syndromes are categorized as type or type . the former type is associated with infections acquired in fall or spring and is seen in younger animals. the latter type is associated with emergence of the arrested larvae during spring or fall. clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. diarrhea is usually seen in type only; the symptoms of type are comparable to those of haemonchus infections. anthelmintic drug therapy is comparable to that for haemonchus, and drug resistance is also a problem with ostertagia. iii. ostertagia ostertagi (cattle stomach worm). ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. ostertagia leptospicularis and o. bisonis also cause disease. the life cycle is direct, and egg shedding by the cattle may occur within - weeks of ingestion of infective larvae. hypobiosis is also a characteristic of o. ostertagi. in the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. cycles of infection and morbidity depend on geographic location, climate, and production cycles. type cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than years old, and causes diarrhea and anorexia. type ostertagiasis occurs in cattle - years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type , hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. ostertagia is susceptible to desiccation but is resistant to freezing. iv. trichostrongylus vitrinus, t. axei, t. colubriformis (hair worms) . trichostrongylus species favor cooler conditions, and some larvae may overwinter. although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. trichostrongylus vitrinus and t. colubriformis infect the small intestine of sheep and goats. trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal ph similar to those seen with ostertagia. mucosal hyperplasia is not seen. the prepatent period is about weeks. affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. these worms show intermediate resistance to freezing temperatures and dry conditions. v. nematodirus spathiger, n. battus (thread-necked worms vii. strongyloides papillosus. strongyloides papillosus is a small-intestinal parasite of sheep and cattle. strongyloides has a different life cycle from that of many nematodes. the eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. the parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. adult females may reproduce in the small intestines by parthenogenesis. clinical signs associated with strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. the current broad-spectrum anthelmintics are effective against strongyloides. strongyloides, bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). the larvae mature in the small intestines and suck blood. larvae are susceptible to desiccation and freezing. heavy infection with bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. oesophagostomum columbianum, o. venulosum (nodule worms) . oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. oesophagostomum columbianum and o. venulosum infect sheep and cattle. these nematodes may affect sheep from months to years of age, and the prepatent period is about weeks. larvae are highly sensitive to freezing and desiccation and rarely overwinter. larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. the resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. nodular lesions are typical at necropsy. x. chabertia ovis (large-mouth bowel worm). chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. signs of infection are not usually seen in cattle. prepatent periods are up to days. heavy infection, which may result from as few as worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. trichuris (whipworms). trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. trichuris has a rather long prepatent period, extending from to months. the oval eggs are double-operculated and survive well in pasture environmental extremes. the adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. the nematodes cause a minor cecitis and will feed on blood. clinical infection is rare and results in diarrhea with mucus and blood. treatment and prevention methods are similar to those for other nematodes. xii. dictyocaulus (lungworms). dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. in sheep, dictyocaulus filaria, protostrongylus rufescens, and muellerius capillaris cause disease; dictyocaulus is the most pathogenic. goats are infected by the same species as sheep, but infections are uncommon. dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." infections with these parasites in the united states tend to be associated with cooler, moister climates. lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately and months of age. sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. coughing and dyspnea are symptoms in goats. diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. the baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. dictyocaulus has a direct life cycle. the adult worms reside in the large bronchi. dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. the expelled larvae are infectious in about - days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about weeks. dictyocaulus filaria causes an especially severe bronchitis in sheep. protostrongylus inhabits smaller bronchioles. muellerius is of minor pathogenicity. protostrongylus and muellerius require the snail or slug as an intermediate host. infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of dictyocaulus larvae. immunity wanes over a year. viral and bacterial respiratory tract infections may be associated with the parasitic infection. more severe illness is seen after infections with cooperia and ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. hypobiosis (arrested development of immature worms in lung tissue) is associated with dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. pastures can be heavily contaminated during the next grazing season. necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. prevention and control of the disease involve appropriate pasture management. elimination of intermediate hosts is important in sheep and goat pastures. in a laboratory setting, animals may be procured that are already harboring the disease. infected animals can be treated with anthelmintics such as ivermectin or levamisole. muellerius tends to be resistant to levamisole. there is no anthelmintic currently approved for goats, but fenbendazole, administered weeks apart, has been effective for all three tapeworms are rarely of clinical or economic importance. in younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. moniezia expansa, and less commonly moniezia benedini, inhabit the small intestines of grazing ruminants. moniezia expansa has the widest distribution of the tapeworm species in north america. soil mites (galumna spp. and oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to weeks. cysticercoids released from the mites are grazed, pass into the small intestines, and mature. no clinical or pathological sign is usually observed with moniezia infection; diagnosis is made by observing the characteristic triangularshaped eggs in fecal flotation examinations. infection is treated with cestocides. thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the mississippi river in the united states. thysanosoma is of the family anoplocephalidae. the life cycle is indirect, and the intermediate host is the psocid louse. larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. typically, no clinical signs are observed with thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. necropsy lesions include bile and/or ductal hyperplasia and fibrosis. thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. abdominal or visceral cysticercosis. abdominal or visceral cysticercosis is an occasional finding at slaughter. the socalled bladder worms typically affect the liver or peritoneal cavity and are the larval form of taenia hydatigena, the common tapeworm of the dog family. taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. after ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. the larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next - months into small fluid-filled bladders. the life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. at necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. migration tracts may result in fibrosis and inflammation. diagnosis is usually made at necropsy. because of the migration through the liver, fasciola hepatica is a differential diagnosis. minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. research animals may have been exposed prior to purchase. echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. the hydatid cyst is the larval intermediate of the adult tapeworm echinococcus granulosus, which resides in the small intestines of dogs and wild canids. embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. the eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. the cystic structure develops and potentially ruptures, forming new cystic structures. clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. cysts are typically observed at necropsy. prevention should be aimed at decreasing fecal contamination of feed and water by canids. additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. treatment of infected ruminants is uncommon. iv. gid. coenuris cerebralis, the larval form of the canid tapeworm taenia (multiceps) multiceps, is the causative agent of the rare condition called gid. the disease occurs in ruminants as well as many other mammalian species. the larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. the resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. diagnosis is usually made at necropsy. eliminating transfer from the canid hosts prevents the disease. the cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. the metacercariae may remain in a dormant stage on the grass for months or longer until ingested by a ruminant. the ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. there they locate in a bile duct, mature, and remain for up to years. acute liver fluke disease is related to the damage caused by the migration of immature flukes. migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. in cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. liver fluke damage may predispose to invasion by anaerobic clostridium species such as c. novyi that could lead to fatal black disease or bacillary hemoglobinuria. chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. blood loss into the bile may lead to anemia and hypoproteinemia. liver damage also is evidenced by increases in liver enzymes such as y-glutamyl transpeptidase (ggt). persistent eosinophilia is also seen with liver fluke disease. other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. at necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. bile ducts will be enlarged, and areas of fibrosis will be evident. diagnosis can be made from clinical signs and postmortem mites cause a chronic dermatitis. the principal symptom of these infections is intense pruritus. in addition, papules, crusts, alopecia, and secondary dermatitis are seen. anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. mites are rare in ruminants in the united states, but infections of sarcoptes and psorergates mange must be reported to animal health officials. ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. diagnosis is based on signs, examination of skin scrapings, and response to therapy. no effective treatment for demodectic mange in large animals has been found. the differential for mite infestations is pediculosis. several genera of mites may affect sheep. these have been eradicated from flocks in the united states or are very rare and include psoroptes ovis (common scabies), sarcoptes scabiei (head scabies, barn itch), psorergates ovis (sheep itch mite), chorioptes ovis (foot scabies, tail mange), and demodex ovis (follicular mange). goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. the scabies mite sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. the chorioptic mite, either chorioptes bovis or c. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. the chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruri-tus. the psoroptic mite psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. repeated treatments of lime sulfur, amitraz, or ivermectin may be effective (smith and sherman, ) . goats are also susceptible to demodectic mange caused by demodex caprae. adult mites invade hair follicles and sebaceous glands. pustules may develop with secondary bacterial infection. psoroptes bovis continues to be present in cattle in the united states, although it has been eradicated from sheep. chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. the sarcoptic mange mite s. scabei can survive off the host, so fomite transmission is a factor. the mange usually begins around the head but then spreads. this parasite can be transmitted to humans. demodex bovis infects cattle; nodules on the face and neck are typical. demodex bovis infections may resolve without treatment. lindane, coumaphos, malathion, and lime sulfur are used to treat psoroptes and psorergates. ivermectin is effective against sarcoptes and is approved for use in cattle. lice that infect ruminants are of the orders mallophaga, biting or chewing lice, and anoplura, sucking lice. these are wingless insects. members of the mallophaga are colored yellow to red; members of the anoplura are blue gray. lice produce a seasonal (winter-to-spring), chronic dermatitis. in sheep, biting lice include damalinia (bovicola) ovis (sheep body louse). sucking lice that infect sheep include linognathus ovillus (blue body louse) and l. pedalis (sheep foot louse). in goats, biting lice infection are caused by d. caprae (goat biting louse), d. limbatus (angora goat biting louse), and d. crassipes. suckir/g louse infections in goats are caused by l. stenopis and l. africanus. damalinia bovis is the cattle biting louse. sucking lice include l. vituli, solenopotes capillatus, haematopinus eurysternus, and h. quadripertusus. pruritus is the most common sign and often results in alopecia and excoriation. the host's rubbing and grooming may not correlate with the extent of infestation. hairballs can result from overgrooming in cattle. in severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. young animals with severe infestations of sucking lice may become anemic or even die. pregnant animals with heavy infestations may abort. in sheep infected with the foot louse, lameness may result. lice are generally species-specific. those infecting ruminants are usually smaller than mm. goats may serve as a source of infection for sheep by harboring damalinia ovis. transmission is primarily by direct contact between animals. transmission can also occur by attachment to flies or by fomites. some animals are identified as carriers and seem to be particularly susceptible to infestations. biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. sucking lice inhabit the host's neck, back, and body region and feed on blood. lice eggs or nits are attached to hairs near the skin. three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about month for all species. lice cannot survive for more than a few days off the host. all ruminant mite infestations are differentials for the clinical signs seen with pediculosis. animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. label directions should be read and adhered to, including withdrawal times. products should not be used on female dairy animals. treatments must be repeated at least twice at intervals appropriate for nit hatches (about every days) because nits will not be killed. fall treatments are useful in managing the infections. systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (hypoderma lineatum and h. bovis). back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. sustained-release insecticide-containing ear tags are approved for use in cattle. etiology. ruminants are susceptible to many species of ixodidae (hard-shell ticks) and argasidae (softshell ticks). many diseases, including anaplasmosis, babesiosis, and q fever are transmitted by ticks. clinical signs and diagnosis. tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. feeding sites on the host vary with the tick species. ticks are associated with an acute paralytic syndrome called tick paralysis. this disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. diagnosis is based on identification of the species. epizootiology and transmission. ticks are not as host-specific as lice. ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. pathogenesis of tick infestations. patterns of feeding on the host differ between argasidae and ixodidae. the former feed repeatedly, whereas the latter feed once during each life stage. pathogenesis of tick paralysis. following a tick-feeding period of - days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. in these cases, death due to respiratory failure occurs. insecticides. ticks can be treated using systemic or topical h. other parasites i. nasal bots (nasal myiasis, head grubs). nasal myiasis causes a chronic rhinitis and sinusitis. the disease is caused by the larval forms of the botfly oestrus ovis. the botfly deposits eggs around the nostrils of sheep. the ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. in - months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. the mature larvae penetrate the soil and pupate for - . months and emerge as botflies. clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. hypersensitivity to the larvae occurs (dorchies et al., ) . later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. at necropsy, larvae will be observed in the nasal cavity or sinuses. mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. the disease is diagnosed by observing the behaviors or identifying organisms at necropsy. up to % of a flock will potentially be infected; treatment should be employed on the rest of the flock. ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. fly repellents may be helpful at preventing additional infections. ii. screwworm flies. cochliomyia hominivorax (callitroga americana) is the the screwworm that causes occasional disease in the southwestern united states along the mexico border. eradication programs have been pursued, and the disease is reportable. large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. eggs hatch within hr. larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. larvae eventually drop off, pupate best in hot climates, and hatch in weeks. large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. single animals or entire herds may be affected. treatment is intensive, with dressings and larvicidal applications. if there is no intervention, the host succumbs to secondary infections and fluid loss. effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. sheep keds ("sheep ticks"). in sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. the disease is caused by melophagus ovinus, which is a fiat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. the adult fly lives entirely on the skin of sheep. females mate and produce - larvae following a gestation of about - days. the larvae attach to the wool or hair and then pupate for about weeks. the adult female feeds on blood and lives for - months; the life cycle is completed in about - weeks. infection is highest in fall and winter. pruritus develops around the neck, sides, abdomen, and rump. in severe cases, anemia may occur. keds can transmit bluetongue virus. keds are diagnosed by gross or microscopic identification. ivermectin or other insecticides are useful treatment agents. portant, other immune mechanisms are not well understood. immunity may not be of long duration. recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. a number of topical treatments, such as - % lime-sulfur solution, % captan, iodophors, thiabendazole, and . % sodium hypochlorite, can be used. in severe cases, systemic therapy with griseofulvin may be successful. prevention and control. the animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. pens should be thoroughly cleaned and disinfected. research complications. ringworm is a zoonotic disease. etiology. dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera trichophyton and microsporum. the primary causes in sheep are t. mentagrophytes and t. verrucosum. in goats, the agents are t. mentagrophytes, m. canis, m. gypseum, t. verrucosum, t. schoenleinii, and epidermophyton floccosum. in cattle, t. verrucosum is the primary causative agent. dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. clinical signs and diagnosis. multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. lesions will vary in size. in all ruminants, lesions will be around the head, neck, and ears. in goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. cattle lesions are unique in the marked crustiness, which progressively appears wartlike. hair shafts become brittle and break off. intense pruritus is often associated with the alopecic lesions. the disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and % potassium hydroxide digestion. dermatophyte test media (dtm) cultures are the most reliable means to diagnose the fungus. broken hairs from the periphery of the lesion are the best sources of the fungus. epizootiology and transmission. younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. pathogenesis. incubation can be as long as weeks. the organisms invade and multiply in hair shafts. treatment. spontaneous recovery occurs in all species in - months. although cell-mediated immunity is considered im- inverted eyelids are a common inherited disorder of lambs and kids of most breeds. generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. young animals will display tearing, blepharospasm, and photophobia initially. if the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. the procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a -day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. boric acid or % argyrol solutions have been used as treatments. because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. [ -mannosidosis is an autosomal recessive lysosomal storage disease of goats. the disease affects kids of the nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge (smith and sherman, ) . carrier adults can be identified by plasma measurements of [ -mannosidase activity. caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. goats with this disease are commonly known as fainting goats. "fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli (smith and sherman, ) . muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing (smith and sherman, ) . contractions of skeletal muscle are sustained for up to min. kids exhibit the condition by weeks of age, and males appear to exhibit more severe clinical signs than females (smith and sherman, ) . electromyographic studies produce an audible "dive-bomber" sound characteristic of hyperexcitable cell membranes (smith and sherman, ) . i. congenital erythropoietic porphyria. congenital erythropoietic porphyria (cep) is an autosomal recessive disease of cattle seen primarily in holsteins, herefords, and shorthorns. the disease also occurs in limousin cattle, humans, and some other species. in the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. a reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. bones are more fragile compared with bones of normal animals. a regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. the genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen iii synthase, in the porphyrin-heme synthesis pathway in erythrocytic tissue. the ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a wood's lamp. diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. heterozygotes may have milder symptoms. many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ("large animal internal medicine," ) . in many cases, the genetic basis has been clarified, and associated defects also noted. many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. the bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming (womack, ) . some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of holstein cattle. some of the more commonly reported defects include syndactyly in holsteins and other breeds and polydactyly in simmentals; lysosomal storage diseases such as a-mannosidosis in some beef breeds; enzyme deficiencies such as citrullinemia in holsteins; and progressive degenerative myeloencephalopathy ("weaver") in brown swiss. ii. goiter of sheep. a defect in the synthesis of thyroid hormone has been identified in merino sheep (radostits et al., ) . lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. immaturity of the lungs at birth causes neonatal respiratory distress and resuits in dyspnea and respiratory failure. spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in suffolk and hampshire breeds. severely affected lambs die shortly after birth. animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. with time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. radiologically, secondary ossification centers--especially the physis, subchondral areas, and cuboidal bonesmare affected. abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. muscle atrophy is common. diagnosis can be based on typical clinical signs, which are similar to those seen with marfan syndrome in humans (rook et al., ) . long-term survival is rare; treatment is unsuccessful. i. abomasal and duodenal ulcers. abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. copper deficiency, dietary changes, mycotic infections, clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. in older adult cattle, abomasal lymphosarcoma may be the underlying condition. gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. chronic hemorrhage may lead to anemia. although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. dark feces or melena and abdominal pain may be observed. arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. fecal occult blood is as an easy diagnostic test. treatment includes gastrointestinal protectants and histamine antagonists. anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. abomasal emptying defect. abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. suffolks tend to be especially predisposed, although the disease has been diagnosed in hampshires, columbias, and corriedales. the mechanism of the disease is unknown. affected animals will exhibit a gradual weight loss with a history of normal appetites. feces will continue to be normal. ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. diagnosis is primarily based on history and clinical signs. elevations in rumen chloride concentrations (> meq/liter) are commonly found. radiography or ultrasonography may be helpful at identifying the distended abomasum. abomasal emptying defect is usually eventually fatal. medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. abomasal displacement. displaced abomasum (da) is a sporadic disorder usually associated with multiparous -to year-old dairy cows in early lactation, but the condition can occur even in young calves. displacement to the right (rda) may be further complicated by torsion (rta), a surgical emergency. left displacement (lda) is more common than rda. clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. da occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. no exact cause of da has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. factors such as body size and conformation indicate the possibility of genetic predisposition. treatments include surgical and nonsurgical techniques for lda; the former has a better chance of per-manent correction. emergency surgery is necessary for rta; the disorder is fatal within hr. recurrence is rare after surgical correction. electrolyte and acid-base imbalances are likely in severe cases and especially with rta. prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above (rohrbach et al., ) . fat cow syndrome is seen in peri-or postparturient overconditioned or obese multiparous dairy cows. factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. the possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. signs are nonspecific and include depression, anorexia, and weakness. prognosis is usually guarded. diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. in prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (nefa) greater than ~teq/liter and - ~teq/liter, respectively, are abnormal (gerloff and herdt, ) . triglyceride analysis of liver biposy specimens are useful. in affected cows, body fat is mobilized, in the form of nefa in response to the energy demands. hepatic lipidosis occurs rapidly as the nefa are converted into hepatic triglycerides. the ability of the liver to extract the albumin-bound nefa from the blood is better than that of other tissues that need and can also use nefa as an energy source. treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease nefa production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. therapeutic measures include intravenous glucose drips, insulin (nph or lente) injections every hr, and transfaunation of ruminal fluid from a normal cow. prevention includes minimizing stress to lategestation cows. dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. i. bloat. bloat or tympanites refers to an excessive accumulation of gas in the rumen. the condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. bloat is classified into two broad categories: frothy bloat and free-gas bloat. frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. fermentation gases such as co , ch , and minor gases such as n , , h , and h s incorporate into the froth, overdistend the rumen, and eventu-ally compromise respiration by limiting diaphragm movement. the froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. additional signs may include colic-like pain of the abdomen and dyspnea. passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. frothy bloat is more difficult to treat, because the foam blocks the stomach tube. addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. in acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. lactic acidosis. lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. the disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive streptococcus and lactobacillus species. the gram-positive organisms efficiently convert the starches to lactic acid. the lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. clinically, animals will become anorexic, depressed, and weak within - days after the initial insult. incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. diagnosis is based on history and clinical signs. blood, urine, or milk ketones can be detected (moore and ishler, ) . additionally, rumen ph, which is normally above . , will drop to less than . and in severe cases may achieve levels as low as . . similarly, urine ph will become acidic, blood ph will drop below . , and hematocrit will appear to increase due to the relative hemoconcentration. necropsy findings will be determined by secondary conditions. the primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. treatment must be applied early in the syndrome. in early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. the t patient should be given mineral oil and antlfermentatlves to prevent the continued conversion of starches to acids and the absorption of metabolic products. bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen ph. furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. rumen parakeratosis. parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium excessive and continuous feeding of diets low in roughage causes the mucosal changes generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. at necropsy, papillae will be thickened and rough. they will frequently be dark in color, and multiple papillae will clump together. abscessation may be observed. histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. chronic laminitis may be observed. however, diagnosis of parakeratosis is generally made at necropsy. feeding adequate roughage, such as stemmy hay, will prevent the disease. antibiotics may be administered to prevent secondary liver abscess formation. iv. rumenitis. rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis in addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. the inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. endogenous rumen bacteria such as fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. clinically, the animals will appear depressed and anorexic. rumen motility will be decreased, and animals will lose weight. the disease may resolve in a week to days; mortality may reach %. necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. granulation tissue and scarring may be observed following healing. rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. the disease can be prevented by minimizing the incidence of lactic acidosis. etiology. traumatic reticulitis-reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. the disease is rarely seen in smaller ruminants. clinical signs. clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. the prognosis is poor when peritonitis becomes diffuse. sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. epizootiology and transmission. this is a noncontagious disease. the occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. necropsy findings. in severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. cardiac puncture will be present in those animals succumbing to sudden death. pathogenesis. consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. this sets off a localized inflammation or a localized or more generalized peritonitis. the inflammation may also temporarily or permanently affect innervation of local tissues and organs. further damage may result from migration and penetration of the diaphragm, pericardium, and heart. diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. radiographs and abdominocentesis may be useful. differential diagnosis. differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. infectious diseases that are differentials include systemic leptospirosis and internal parasitism. diseases causing sudden death may need to be considered. prevention and control. this problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a bailing gun in young stock at - months of age) are also significant prevention measures. treatment. provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. in severe cases, rumenotomy may be considered. etiology. pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. beef heifers are susceptible to protein energy malnutrition (pem) syndrome, which is also referred to as pregnancy toxemia. clinical signs. in sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. they become anorexic and act uncoordinated, frequently leaning against objects. advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. body temperature, heart rate, respiratory rate, and rumen motility continue normally. up to % of infected ewes may die from the disease. the course of the disease may last up to a week. in goats, the disease usually occurs in the last weeks of gestation, especially in does carrying triplets. pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. the doe may separate herself from the herd, stagger, or circle and may appear blind. appetite is poor, and tremors may be evident. a rapid metabolic acidosis results in subsequent recumbency. urinalysis will readily reveal ketonuria. if fetal death occurs, acute toxemia and death of the doe may result. in beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. some cows develop diarrhea. because the catabolic state is often so advanced, most affected heifers die even if treated. pregnancy toxemia is diagnosed by evidence of typical clinical signs. sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. blood glucose levels found to be below mg/dl and ketonuria are good diagnostic indicators. in cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. that are obese or bearing twins or triplets. the disease develops during the last weeks of pregnancy. pem most frequently occurs in heifers during the final trimester of pregnancy. necropsy findings. at necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. the liver will be enlarged, yellow, and friable, with fatty degeneration. the adrenal gland may also be enlarged. in cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. pathogenesis. rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. the ewe develops a severe hypoglycemia in early stages of the disease. the ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. when the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. glycerol is converted to glucose while the fatty acids are metabolized for energy. the oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and [ -hydroxybutyric acid), thus causing the condition ketoacidosis. heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. marginal diets and poor-quality forage will place the cows in a negative energy balance. differential diagnosis. hypocalcemia is a common differential diagnosis. in cattle, differentials include chronic or untreated diseases such as johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. prevention and control. pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. in late pregnancy, the dietary energy and protein should be increased . - times the maintenance level. pem can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. treatment. in sheep, because the morbidity may be as high as %, treatment should be directed at the flock rather than the in-dividual. treating the individual is usually unsuccessful. oral administration of ml of propylene glycol or % glucose twice a day, anabolic steroids, and high doses of adrenocorticosteroids may be helpful. if ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. in goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. parturition can be induced in does by either dexamethasone ( mg) or pgf a ( ~tg). in addition, goats may be treated with % dextrose ( to ml iv) or propylene glycol ( ml per os or times a day). adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin b and transfaunation. heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on iv % glucose drips, and treated for concurrent disease. research complications. in research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f hypocalcemia (parturient paresis, milk fever) etiology. hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. clinical signs and diagnosis. in sheep, the disease is seen in ewes during the last weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. as calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. later, muscular tremors, muscular weakness, and recumbency will ensue. animals will frequently be found breathing rapidly despite a normal body temperature. morbidity may approach %, and mortality may reach as high as % in untreated animals. affected does become bloated, weak, unsteady, and eventually recumbent. cows are affected within - hr before or after parturition. cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. the terminal stage of disease is a rapid progression from coma to death. heart rates will be high, but pulse may not be detectable. hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. it is later confirmed by laboratory findings of low serum calcium. with hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of - mg/dl to values of - mg/dl. in cattle, plasma levels below . mg/dl are hypocalcemic; at the terminal stages levels may be mg/dl. ewes during the last weeks of pregnancy or during the first few weeks of lactation. the disease is not as common in the dairy goat as in the dairy cow. high-producing, older, multiparous dairy cows are the most susceptible, and the jersey breed is considered susceptible. cows that have survived one episode are prone to recurrence. in addition, dry cows must be managed carefully regarding limiting dietary calcium. the disease is not common in beef cattle unless there is an overall poor nutrition program. ing at necropsy. there is no pathognomonic or typical find-pathogenesis. during the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. after parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation (rings et al., ) . thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. prevention and control. maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. in cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. treatment. hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. response will often be rapid, with the resolution of the animal's dull mentation. less severely affected animals will often try to stand in a short time. relapses are common, however, in sheep and cattle. hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. these imbalances should be considered when animals appear to be unresponsive to treatment. hypocalcemia in the goat can be treated with - ml of calcium borogluconate. heart rate should be monitored closely throughout calcium administration. if an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. calcium gels and boluses are also available for treatment (rings et al, ) . prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. etiology. urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (murray, ) . the disease occurs rarely in female ruminants. clinical signs and diagnosis. affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. these postures may be mistaken for tenesmus. male cattle may develop swelling along the ventral perineal area. affected animals will not stay with the herd or flock. small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. the pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. as the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. the abdomen will swell as the bladder enlarges, and rupture can occur within hr after development of clinical signs. bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. the disease may progress over a period of - weeks, and the mortality is high unless the blockages are reversed. diagnosis is made by the typical clinical signs. abdominal taps may yield urine. calculi are usually composed of calcium phosphate or ammonium phosphate matrices. clinical disease is usually seen in growing intact or castrated males. the disease may be sporadic or there may be clusters of cases in the flock or herd. necropsy findings. necropsy findings include urine in the abdomen with or without bladder or urethral rupture. renal hydronephrosis may be evident. calculi or struvite crystal sediment will be observed in the bladder and urethra. histologically, trauma to the urethra and ureters will be present. etary, anatomical, hormonal, and environmental factors. male sheep and goats have a urethral process that predisposes them to entrapment of calculi. in cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. this physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. grains fed to growing animals tend to be high in phosphorus and magnesium content. these calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). differential diagnosis. grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. trauma to the urethral process should be considered. urinary tract infections are uncommon in ruminants. prevention and control. one case often is indicative of a potential problem in the group. urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. the normal ratio should be : . additionally, increasing the amount of dietary roughage will help balance the mineral intake. increasing the amount of salt (sodium chloride, - %) in the diet to increase water consumption, or adding ammonium chloride to the diet, at gm/head/day or % of the ration, to acidify the urine, will aid in the prevention of this disease. palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. treatment. treatment is primarily surgical (van metre et al. ) . initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. as the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. in more advanced stages, perineal urethrostomy may yield good results. the prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. research complications. young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. rickets is a disease of young, growing animals but rarely occurs in goats. it is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. the condition can occur as an absolute deficiency in vitamin d , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. the syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. spontaneous fractures of long bones may occur. animals will recover when dietary phosphorus is provided and if joint damage is not severe. a. copper deficiency (enzootic ataxia, swayback) etiology. chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. in goats, this deficiency also causes swayback in the fetuses. clinical signs and diagnosis. this disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about months of age. additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. when the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. diagnosis is based on clinical signs, feed analysis, and pathological findings. epizootiology and transmission. enzootic ataxia is rarely seen in western states; most north american diets have sufficient copper levels to prevent this disease. copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. pathogenesis. the maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth (smith and sherman, ) . differential diagnosis. the differential diagnosis for newborns includes [ -mannosidosis, hypoglycemia, and hypothermia. for older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. prevention and control. copper deficiency can be prevented by providing balanced nutrition for pregnant animals. necropsy findings. gross encephalomalacia has been noted. histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. extensive nerve demyelination and necrosis are evident. postmortem lesions include extensive demyelination and neuronal degeneration. treatment. because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. necropsy findings. common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. hemoglobin-stained urine will be visible in the bladder. copper accumulations in the liver reaching - ppm are toxic. pathogenesis. hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. etiology: acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. growing lambs may be the most susceptible. copper toxicosis is rare in goats. differential diagnosis. other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. clinical signs and diagnosis. the clinical course in sheep can be as short as - days, and mortality may reach %. hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (ast) and y-glutamyltransferase (ggt) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. and goats is the range of - mg/kg, and for cattle it is - mg/kg. chronic poisoning in sheep may occur when . mg/kg is ingested. copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. phytogenous sources include certain pastures such as subterranean clover. feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. a common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. pregnant dairy cattle may be more susceptible to copper toxicity. rare sources of copper ingestion may include copper sulfate footbaths. control and prevention. the disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. molybdenum may be administered to animals considered at high risk. molybdenum-deficient pastures may be treated with molybdenum superphosphate. herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. treatment. oral treatment for sheep consists of ammonium or sodium molybdenate ( - mg/day), and sodium thiosulfate ( . - . mg/day) for weeks aids in excretion of copper. oral d-penicillamine daily for days ( mg/kg) has also been shown to increase copper excretion in sheep. ammonium molybdenate has been administered intravenously to goats at . mg/kg for treatments on alternate days. cattle have been treated orally with sodium molybdenate ( gm/day) or sodium thiosulfate ( gm/day). treatment for anemia and nephrosis may be necessary in severe cases. merino crosses and the british breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. (nutritional muscular dystrophy, nutritional myodegeneration, white muscle disease, stiff lamb disease) etiology. white muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin e. clinical signs and diagnosis. clinically two forms of the disease have been identified: cardiac and skeletal. the cardiac form occurs most commonly in neonates. in these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. young will be able to nurse when assisted. in slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. mortality may reach % (jensen and swift, ) . paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. with the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. young will be reluctant to get up but will readily nurse when assisted. peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. serum selenium levels are usually below ppb (normal is - ppb) (nelson, ) . diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin e. glutathione peroxidase levels in red blood cells can be measured as an indirect test. clinical biochemistry findings of significant elevations of aspartate aminotransferase (ast) in creatinine kinase (ck) are also supportive of the diagnosis. epizootiology and transmission. selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. necropsy findings. necropsy lesions include petechial hemorrhages and muscle edema. hallmarks are pale white streaking of affected skeletal and cardiac muscle. these are due to coagulation necrosis. pale striated muscles of the limb, diaphragm, and tongue are also seen. antioxidants that protect lipid membranes from oxidative destruction. selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. lack of one or both results in loss of membrane integrity. differential diagnosis. in neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. prevention and control. awareness of regional selenium deficiencies is important. control involves providing good-quality roughage, vitamin e and selenium supplementation, and parenteral injections prior to parturition and weaning. treatment. affected animals may be treated by administering vitamin e or selenium injections. administering vitamin e or selenium to ewes in late pregnancy can prevent white muscle disease (kott et al., ) . the label dose for selenium is . - mg/ kg of body weight. combination products are available and can be used in goats at the sheep dose (smith and sherman, ) . proper mineral balance in the diet is critical. selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. the main preventive measure for the former is the use of the appropriate product for the species. secondarily, the concentration of the available product should be double-checked. in the united states, ruminants in the midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. signs of overdosing include weakness, dyspnea, bloating, and diarrhea. shock, paresis, and death may occur. initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. etiology. polioencephalomalacia (pem) is a noninfectious, noncontagious disease characterized by neurological signs. growing and adult ruminants on high-concentrate diets are typically affected. animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. clinical signs and diagnosis. an early sign may be mild diarrhea. acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. in subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. hypersalivation may be present, but body temperatures and ocular reflexes are normal. morbidity and mortality may be high, especially in younger animals. diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. epizootiology and transmission. pem is caused by a thiamin deficiency. the disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. pastured animals are also vulnerable if grain is feed. thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. sherman, ) . vitamin a deficiencies associated with hyperkeratosis have been reported, as well as vitamin e-responsive and selenium-responsive dermatitis. necropsy signs. cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. gliosis and cerebral capillary proliferation may be observed. a lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. differential diagnosis. several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin a deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type d clostridial enterotoxemia. prevention and control. the disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of b vitamins. if excess sulfur is the primary factor, immediate removal of the source is critical. neonatal ruminants are born without immunoglobulins and must receive colostrum by hr after birth. the morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. measures to assure passive immunity for neonatal ruminants are covered in section ii,b, , and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., escherichia coli infections) and, of viral diseases (e.g., diarrheas) in section iii,a, and iii,a, . generally, transfer of less than mg/dl of immunoglobulins in the serum is classified as failure of transfer, - mg/dl is partial, and above mg/dl is complete transfer. methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, y-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). treatment. early aggressive treatment is essential to save animals. the disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. dexamethasone, b vitamins, and diazepam may also be required. treatment is less successful when sulfur plays a prominent role in the etiology. research complications. this disease is preventable. although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. vitamin d toxicity can result either from iatrogenic overadministration or ingestion of the plant trisetum flavescens. serum calcium levels may be high enough that blood in edta tubes will clot. laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). laminitis is also associated with mastitis and metritis. facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. the complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. chronically, the hoof wall takes on a "slipper" appearance. treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. in goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. zincresponsive dermatitis has been reported in goats (smith and otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. only milk replacer formulated for the particular species should be used. once nutritional imbalances are corrected, the feces readily return to normal. sudden changes in diet can also result in loose feces. photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. the photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. animals with a lack of pigment are more susceptible to the disease. three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. the only example of aberrant photosensitization is congenital porphyria of cattle (see section iii,b, ). pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. the activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. the vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. the facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. with secondary photosensitization, icterus is also common. necrosis and gangrene may occur. diagnosis is based on clinical lesions and exposure to the photosensitive chemi-cals and sunlight. treatment is symptomatic. the prognosis for hepatogenous type may be guarded if hepatic disease is severe. from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. if the prolapse obstructs subsequent urination, rupture of the bladder may occur. the vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. the animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (buhner suture). alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. these animals should be culled. vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body (ross, ) . uterine prolapses occur sporadically in postpartum ewes and cattle. the gravid horn invaginates after delivery and protrudes from the vulva. the cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. in cattle, the uterine prolapses usually develop within week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. cows may also have concurrent parturient paresis. initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. if identified early, the uterus can be replaced as for vaginal prolapses. electrolyte imbalances should be corrected if present. additional supportive therapy, including the use of antibiotics should always be considered. tetanus prophylaxis should be included. oxytocin should be administered to induce uterine reduction. vaginal closures are less successful at retaining uterine prolapses. preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. vaginal and uterine prolapses occur in ewes, does, and cows. the conditions are not common in does. vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. in sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. overconsumption of roughages, which distends the rumen, and lack of exercise leading to intraabdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. the condition may result f rectal prolapse rectal prolapse is common in growing, weaned lambs and in cattle from months to years old. the physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. urolithiasis may result in prolapses as the animal strains to urinate. any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. diagnosis is based on clinical signs. early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. the mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. in all cases of treatment, determination and elimination of the underlying cause are essential. gastrointestinal accumulations or obstructions of hair (and/ or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. in addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal tric. hobezoars as well. clinical signs may be mild or severe according to size, number, and location. ruminal trichobezoars rarely result in clinical signs. obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. prevention includes providing good-quality roughage and treating lice and mange infestations. wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. predators will usu-ally be sources of bite wounds. standard veterinary wound assessment and care are essential for wounds or bites. tetanus antitoxin may be indicated. use of approved antibiotics may be appropriate. the lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. abscesses may also occur in the soft tissues of the hooves (sole abscesses; see section iii,c, ) because of entrapped foreign bodies or hoof cracks that fill with dirt. preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations. acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. the animals will also be anxious and shivering and will become hyperthermic. salivation, diarrhea, and bloat also occur. immediate therapy must include epinephrine by intravenous infusion at ( ml of : per kg of body weight for goats and : , ( . mg/ml) or . mg/kg (about ml) for adult cows.) furosemide ( mg/kg) may be beneficial to reduce edema. prognosis is usually guarded. recovery can occur within hr. in a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. traumatic injuries to peripheral nerves can cause acute lameness. improper administration of therapeutics can easily cause this type of lameness. injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. contraction of the quadriceps results in the limb being pulled forward. injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. traumatic injury to the radial nerve can result in a "dropped elbow" (nelson, ) . husbandry procedures such as tail docking, castration, dehorning, dosing with a bailing gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. bailing-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. standard veterinary assessment and care are essential for these cases. local and systemic antibiotics with supportive care may be indicated. swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammato-ries. mild cases of peripheral nerve damage may recover in - days. personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. albumin values and foaming urine. the proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. prognosis is poor, and no treatment is reported. neoplasia and tumors are relatively rare in ruminants. lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. virus-induced papillomatosis (warts), discussed in section iii,a, ,s, and squamous cell carcinomas have also been reported in sheep. in goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. persistent udder papillomas may progress to squamous cell carcinoma. lymphosarcoma is reported rarely in goats. although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or oscc) are the most commonly reported cancers in cattle. lymphosarcoma is described in section iii,a, ,c. lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in oscc. genetic factors may also play a role. many cases occur in herefords. this is a disease of older cattle; no case has been reported in animals less than years of age. the cancer metastasizes through the lymph system to major organs. treatment in either lymphosarcoma or oscc is recommended only as a palliative measure. the extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. papillomatosis (warts) are common in cattle (see section iii,a, ,s). dental wear is seen most commonly in sheep. as sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. several factors predisposing to dental wear should be considered. the diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. sheep older than about years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. managing the content and consistency of the diets can best prevent the disease. of the ruminants, cows are the most frequently affected by subsolar absesses. dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. fusobacterium necrophorum is often the pathogen involved. separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated. amyloidosis amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. the disease has been classified as aa type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. the loss of protein in the urine contributes to abnormal plasma advances in sheep and goat medicine animals and animal products, subchapter a, animal welfare formulary for laboratory animals domestic animal behavior for veterinarians and animal scientists schlam's veterinary hematology diseases of sheep animal feeding and nutrition guide for the care and use of laboratory animals veterinary drug handbook veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goats, and horses sheep production and management animal and plant health inspection service (aphis), policy # , farm animals used for nonagricultural purposes goats the clinical syndromes caused by salmonella infection armed forces institute of pathology (afip) ( ) the effect of stress on the carrier state of salmonella typhimurium in goats bibliography of naturally occurring models of human disease clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: cases control of the estrous cycle the goat industry: feeding for optimal production neurologic disease in sheep and goats modern breeds of livestock the sheep gene map pasteurella haemolytica complicated respiratory infections in sheep and goats ungulates as laboratory animals diagnosis of lameness in sheep an overview of the influence of ace inhibitors on fetalplacental circulation and perinatal development protozoan infections (toxoplasma gondii, neospora caninum, and sarcocystis spp.) in sheep and goats: recent advances cloned transgenic calves produced from nonquiescent fibroblasts transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells use of an animal model of trichomoniasis as a basis for understanding this disease in women council report: vaccination guidelines for small ruminants (sheep, goats, llamas, domestic deer, and wapiti) ( ) maedi-visna and ovine progressive pneumonia pathophysiology of oestrus ovis infection in sheep and goats: a review experimental surgery in farm animals evaluation of an agar gel immunodiffusion test kit for detection of antibodies to mycobacterium paratuberculosis in sheep veterinary laboratory medicine induction of human tissue plasminogen activator in the mammary gland of transgenic goats pasteurella haemolytica infections in sheep coccidiosis and cryptosporidiosis in sheep and goats the major histocompatibility complex region of domestic animal species brucella melitensis infection in sheep: present and future hemoglobin switching epididymitis in rams current veterinary therapy: food animal practice livestock handling guide: management practices that reduce livestock bruises and injuries, and improve handling efficiency. livestock conservation institute synchronization of oestrus in the boer goat doe: dose effect of prostaglandin in the double injection scheme. south afr guide to the dissection of domestic ruminants reproduction in farm animals review of polyclonal antibody production procedures in mammal and poultry considerations in the design and construction of facilities for farm species clinical update: leptospirosis the sheep as an experimental animal bibliography oflnduced animal models of human disease bibliography of naturally occurring models of human disease postpartum assessment and care of the newborn ruminant animal genetics guide for the care and use of laboratory animals blackleg: a new perspective on an old disease protecting calves from viral diarrhea bovine leukemia virus. part : descriptive epidemiology, clinical manifestations, and diagnostic tests bovine leukemia virus. part : risk factors of transmission bovine leukemia virus. part : zoonotic potential, molecular epidemiology, and an animal model. in "infectious disease in food animal practice bovine leukemia virus. part : economic impact and control measures brucella abortus strain rb vaccine: its advantages and risks current veterinary therapy: food animal practice neural control of maternal behavior and olfactory recognition of offspring comparison of the burdizzo and rubber ring methods for castrating and tail docking lambs postpartum care of the cow and calf advances in the control of foot rot in sheep mastitis in ewes giardiasis in large animals laboratory diagnostic tests for retrovirus infections of small ruminants effects of dietary vitamin e supplementation during late pregnancy on lamb mortality and ewe productivity myotonia congenita (thomsen) and recessive myotonia genetic and environmental causes of variation in mastitis in sheep the gnrh system of seasonal breeders: anatomy and plasticity genetic organization, polymorphism, and function of the bovine major histocampaticulity complex scrapie in sheep biochemical and morphological expression of early prenatal caprine beta-mannosidosis respiratory infections of sheep dogs are the definitive hosts of neospora caninum a century of classical contagious caprine pleuropneumonia from original description to aetiology sheep and goat practice gene manipulation in goats through biotechnology minimizing morbidity and mortality from cryptosporidiosis managing dairy cows during the transition period: focus on ketosis environmental enrichment for dairy calves and pigs the trypanocidal cape buffalo serum protein is xanthine oxidase oral rehydration therapy for diarrheic calves neonatal ruminant diarrhea techniques for artificial insemination of cattle with frozenthawed semen noninfectious causes of lameness neosporosis: its prevalence and economic impact a review of yersinosis (yersinia pseudotuberculosis infection) dairy goat reproduction maedivisna virus in sheep: a review evaluation of a commercially available vaccine against corynebacterium pseudotuberculosis for use in sheep ruminant production management: control programs for gastrointestinal nematodes in sheep and goats fetal brain injury following prolonged hypoxemia and placental insufficiency: a review managing cryptosporidium and giardia infections in domestic ruminants milk fever: seeking new solutions to an old problem recent advances on ovine chlamydial abortion risk factors for abomasal displacement in dairy cows the spider syndrome: a report on one purebred flock development of ingestive behavior current state of in vivo preclinical heart valve evaluation dermatophilus congolensis infections in cattle and sheep ovine listeric encephalitis coccidiosis in ruminants principles of dairy science human factor ix transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts the major histocompatibility complex region of domestic animal species clinical reproductive anatomy and physiology of the doe immunobiology of the mammary gland coccidiosis brucella abortus strain rb : a new brucellosis vaccine for cattle use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs effect of congenitally acquired neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle artificial control of breeding in ewes toxoplasmosis infection in sheep bovine reproductive biotechnology transgenic dairy cattle. genetic engineering on a large scale the effect of intra-mammary inoculation of lactating ewes with pasteurella haemolytica isolates from different sources bovine surgery and lameness reduction of myocardial myoglobin in bovine dilated cardiomyopathy intraosseous infusion of prostaglandin e prevents disuse-induced bone loss in the tibia estrous cycle synchronization the bronchopneumonias (respiratory disease complex of cattle, sheep, and goats) the cattle gene map treatment and control of gastrointestinal nematodes in sheep diagnosis, treatment, and management of enteric colibacillosis key: cord- -ybj lwdb authors: platt, simon r. title: vestibular disorders date: - - journal: consultations in feline internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: ybj lwdb nan cats have the ability to control posture and movements of the body and eyes relative to their external environment. the vestibular system mediates these activities through a network of receptors and neural elements. this system integrates peripheral sensory information from vestibular, somatosensory, and visual receptors, in addition to motor information from the cerebellum and cerebral cortex. central processing of these inputs occurs rapidly and provides coordinated relevant muscle movements. although the vestibular system is considered as a special sense, most vestibular activity is conducted at a subconscious level. disease leading to dysfunction of the vestibular system can lead to dramatic signs of dysequilibrium. treatment and prognosis for causes of dysequilibrium differ, depending on whether the peripheral or central components of the system are affected. this chapter outlines relevant anatomy of the vestibular system with emphasis on the clinical signs of vestibular dysfunction. additionally, an overview of the diseases responsible for vestibular dysfunction in cats is provided. the vestibular system can be divided into peripheral components located in the inner ear, and central nervous system (cns) components. three major cns areas receive projections from the peripheral sensory receptors of the vestibular system: the cerebral cortex, the spinal cord, and the cerebellum. projection pathways to the cerebral cortex incorporate extensions to the extraocular muscles. three neurons make up the pathway responsible for the sensory input of head and body position and movement to the cerebral cortex ( figure - ). the location for the first-order neuron is within the vestibular ganglion of cranial nerve viii or the vestibulocochlear nerve. the axon projects to the ipsilateral vestibular nuclei. these neurons receive input from the vestibular receptors contained within the membranous labyrinth that is surrounded by a bony labyrinth located in the petrous temporal bone. the membranous labyrinth consists of four fluid-filled, communicating compartments; these include the saccule and utriculus, three semicircular ducts, and a cochlear duct (figure - ). [ ] [ ] [ ] [ ] specifically, the vestibular portion of the eighth cranial nerve innervates five sites: the crista of the ampulla of each of the three semicircular ducts and the maculae of the utricle and saccule. each semicircular duct is orientated at right angles to the others and connects at both ends with the utriculus, which in turn communicates with the saccule. movement of endolymph contained within the membranous labyrinth is responsible for stimulation of the receptors; the endolymph is thought to be derived from blood. the crista detects head movement. neuroepithelial cells are stimulated by the movement of the crista's gelatinous cupula secondary to the flow of endolymph as the head turns; any movement deflects the cupula and cilia, which leads to their depolarization and propagation of nerve impulses to the vestibular neurons. primary function of the crista involves dynamic equilibrium with regard to acceleration and deceleration. [ ] [ ] [ ] [ ] , the maculae detect head orientation; the macula of the utricle is parallel to the ground with its "hairs" pointing dorsally, and the macula of the saccule is vertical with its "hairs" pointing laterally. constant input from gravity acts upon the neuroepithelial cells of each macula, subsequently causing them to fire. these slow-adapting receptors are responsible for sensing static position of the head and linear acceleration and deceleration. [ ] [ ] [ ] , the sensory neurons of the vestibulocochlear nerve consist of cell bodies in the spiral ganglion located within the modiolus of the cochlea. the vestibulocochlear nerve and the facial nerve exit the petrous temporal bone through the internal acoustic meatus. the nerve enters the medulla of the brainstem at the ventrolateral margin of the trapezoid body. a branch of the vestibulocochlear nerve enters the medulla directly, and a branch travels within the acoustic stria on the dorsal surface of the medulla and caudal cerebellar peduncle before entering the brainstem. the course of the vestibulocochlear nerve stays within the cranium. , , , the location of the cell bodies for the second-order neuron is the vestibular nuclei in the medulla oblongata. four paired vestibular nuclei exist: the caudal vestibular nucleus located medial to the caudal cerebellar peduncle, the medial vestibular nucleus that lies medial to the caudal nucleus, the lateral vestibular nucleus positioned dorsal to the caudal nucleus, and the rostral vestibular nucleus (figure - ) . the lateral and the rostral nuclei are juxtapositioned dorsally to the cerebellar peduncles. , , , similar to the auditory pathway, axons from the vestibular nuclei have ipsilateral and contralateral projections. some axons course within the medial longitudinal fasciculus and project to the contralateral medial geniculate nucleus of the thalamus. ascending fibers within the fasciculus give off numerous branches to the motor nuclei of cranial nerves iii, iv, and vi before synapsing in the medial geniculate nucleus. this pathway coordinates conjugate eye movements associated with changes in position of the head. the medial longitudinal fasciculus also contains fibers that descend to the spinal cord. some axons have afferent projections from the vestibular nuclei to the vomiting center located within the reticular formation. , , , neuron cell bodies for the third-order neuron are located in the medial geniculate nucleus, within the medial geniculate body. these axons project to the cerebral cortex via the internal capsule and via a poorly defined pathway to the temporal lobe. , , two vestibulospinal pathways, termed lateral and medial vestibulospinal tracts, correspond with their origin from the vestibular nuclei. fibers from the lateral vestibular nucleus descend ipsilaterally the entire length of the spinal cord in the ventral funiculus to synapse with alpha and gamma motor neurons of the extensor muscles. this pathway is facilitatory to ipsilateral extensor muscles and inhibitory to ipsilateral flexor muscles and contralateral extensor muscles. fibers from the medial vestibular nucleus descend the spinal cord in the medial longitudinal fasciculus located in a dorsal area of the ventral funiculus. these fibers synapse in the cranial area of the thoracic spinal cord with cervical motoneurons that control head position and maintain equilibrium. projection pathways between the vestibular nuclei and the cerebellum course through the caudal cerebellar peduncle. fibers from the vestibular nuclei synapse in the ipsilateral flocculonodular lobe (the flocculus of the hemisphere and the nodulus of the caudal vermis) and the fastigial nucleus of the cerebellum. fibers from the fastigial nucleus of the cerebellum synapse in the vestibular nuclei. projections from the cerebellum have a strong influence over the activity of the vestibular nuclei. the vestibular system maintains equilibrium through ipsilateral tonic input to the muscles of the head, neck, and torso. an asymmetrical lesion causes loss of the ipsilateral extensor system and causes the extensor system on the contralateral side to become functionally "dominant." clinical signs are recognized as ipsilateral hypotonicity and contralateral hypertonicity. unilateral vestibular disease produces ipsilateral dysfunction. common clinical signs of vestibular disease are head tilt, nystagmus, and ataxia; these may be present as single entities or as a combination of signs ( figure - ) . the primary aim of the neurological examination is to determine if these vestibular signs are due to a peripheral vestibular system (inner ear) disease or a central vestibular system (brainstem and/or cere-bellum) disease. neuroanatomical localization determines the most appropriate diagnostic tests, the differential diagnoses, and the prognosis. essential determination of whether these signs are due to a peripheral or central disease may be possible by the identification of associated neurological signs that are associated only with central disease. , signs of central vestibular syndrome suggest brainstem involvement and are not present in patients with inner ear disease except in cases of direct extension of the disease process, such as can be seen with otitis media/interna and neoplasia. loss of equilibrium most commonly is represented clinically as a head tilt (figure - and table - ) . a head tilt may be present with either central or peripheral vestibular disease. the head tilt is always toward the side of the lesion with peripheral disease but may be to either side with central disease. a head tilt that is opposite to the side of the lesion is paradoxical. this can be seen with lesions of the flocculonodular lobe of the cerebellum or the supramedullary part of the caudal cerebellar peduncle, with sparing of the vestibular nuclei in the rostral medulla. the head tilt often is accompanied by ipsilateral cerebellar signs, paresis, and postural reaction deficits. , , , the mechanism by which the paradox of vestibular signs is contralateral to the lesion is not well understood. a loss of cerebellar inhibition over intact vestibular nuclei could result in hyperactivity of the latter, which simulates a relative loss of function on the other side. cats with bilateral peripheral vestibular disease do not have asymmetrical lesions such as a head tilt, but have a characteristic "side-to-side" head movement (figure - ) . pathological or spontaneous nystagmus is an involuntary rhythmic oscillation of both eyes, which can occur when the head is still or can be induced with a change in head position. this is a sign of altered vestibular input to neurons of cranial nerves that innervate the extraocular eye muscles. this is in contrast to physiological nystagmus, which can be induced in normal cats by moving the head from side to side, best achieved by holding the cat in the air and moving the whole cat's body the fast phase is toward the direction of the head movement and represents the corrective repositioning of the eye as the extraocular muscles reach their stretch threshold after the slow phase. delayed physiological nystagmus can be seen with peripheral or central vestibular disease. pathological nystagmus may be horizontal, rotatory, or vertical in direction (figure - ) . vertical nystagmus implies a central vestibular lesion. if nystagmus of any direction is induced only when the head is placed in an unusual position, it is known as positional nystagmus ( figure - ) , which may be more common with, but not specific for, central disease; this term also may refer to nystagmus that changes its predominant direction with altered head positions. a reliable way to induce positional nystagmus is to decompensate the cat by quickly positioning the cat on its back. eye movements typically are described to have a slow and fast phase. damage to the vestibular system on one side impedes the resting baseline activity on this side, with the normal side continuing to emit baseline activity, now interpreted as head rotation to the normal side. , , , therefore, the nystagmus occurs with the fast phase away from the damaged side and with the slow phase directed commonly toward the affected side; the exception is in the case of paradoxical disease (see section on head tilt above). the direction also can depend on whether the lesion is irritative or destructive to the vestibular system. with acute onset nystagmus, the eyelids may be seen to contract at a rate corresponding to that of the nystagmus. nystagmus may disappear in chronic lesions as a result of adaptation, particularly with peripheral disease; however, its presence usually indicates an active disease process within the vestibular apparatus. cats with bilateral vestibular disease do not have pathological or physiological nystagmus. caloric nystagmus is a type of physiological nystagmus that can be induced by irrigating the ear canal with ice-cold water ( ∞ c) or warm water ( ∞ c) for to minutes. the water causes the flow of endolymph within the ducts. absence of response or asymmetry between sides may indicate vestibular dysfunction, but this often is too unreliable to use in the clinical case. , , ataxia is a loss of muscular coordination or an irregularity of muscle action. it generally is associated with an abnormality of the cerebellar, vestibular, or proprioceptive pathways. cats with vestibular dysfunction assume a wide-based stance and may lean or drift toward the side of a lesion.* with disease of the flocculonodular lobe of the cerebellum or the supramedullary part of the caudal cerebellar peduncle, the ataxia may be directed to the side opposite the lesion as part of the paradoxical central vestibular syndrome. cats with bilateral vestibular disease usually have a symmetrical ataxia and may fall to either side. strabismus is an abnormal position of the eye and often is present in cats with vestibular disease. strabismus can be induced when the head is moved dorsally and is thus termed positional; normally, when the head and neck are extended, the eye should remain centered within the palpebral fissure. the deviation often is ventral and lateral on the ipsilateral side but is not due to paralysis of any of the cranial nerves innervating the extraocular muscles of the eye.* the eyeball occasionally can be noted to deviate without extension of the head and neck, which appears as a lower motor neuron strabismus, corrected by inducing the patient to move its eyeballs to gaze in different directions. the presence of positional strabismus does not help with the determination of a peripheral or central vestibular disease. dysconjugate strabismus implies deviation of both eyes in different directions and is an uncommon finding, which may be more common with central disease. rarely, the opposite eyeball exhibits a dorsal strabismus. cranial nerve vii, the facial nerve, enters the internal acoustic meatus of the petrosal bone, and courses through the facial canal to exit the stylomastoid foramen located dorsal to the tympanic bulla. its course is near the components of the peripheral vestibular system and is affected commonly with destructive lesions to the peripheral vestibular system. the resulting signs are those of facial paresis, paralysis, or more rarely spasm. the owners may report that the patient drools excessively or drops food from the mouth on the affected side. the menace response and palpebral and corneal reflexes often are reduced or absent because of an inability to close the eyelid. , , , because the facial nerve also supplies preganglionic parasympathetic fibers to the lacrimal gland and salivary glands, neurogenic keratoconjunctivitis sicca may accompany facial nerve paralysis associated with middle ear disease, in addition to the presence of xeromycteria. , hemifacial spasm may be seen early in the course of middle ear diseases. inflammation of the facial nerve may cause the facial muscles on the affected side to become hypertonic, causing the face and nose to be pulled caudally. a narrowed palpebral fissure may exist, which is caused by partial closure of the eyelids, elevation of the ear, and wrinkling of the face. these signs may precede those of facial paresis and paralysis. horner's syndrome (miosis, ptosis, enophthalmos, and protrusion of the third eyelid) of the ipsilateral eye may be present with middle or inner ear disease, causing peripheral vestibular dysfunction ( figure - ). , , , this association is seen because the vagosympathetic trunk synapses in the cranial cervical ganglion deep to the tympanic bulla. the postganglionic fibers pass with the internal carotid artery into the middle ear cavity through the tympano-occipital fissure, which is in close proximity to the vestibulocochlear nerve ( figure - ) . , horner's syndrome is associated rarely with central vestibular syndrome. , , sympathetic hyperirritability has been reported in early otitis media, because of disease of the post-ganglionic sympathetic fibers resulting in dilation of the pupil , , and exophthalmos. this has been likened in human beings to pourfour du petit syndrome. paresis suggests abnormal neurological function (weakness) without complete paralysis, which implies that some voluntary motion remains. locomotion is thought to be initiated in the brainstem of animals, and so paresis usually is seen with any lesion within the neuraxis caudal to the level of the red nucleus in the midbrain. with unilateral focal central vestibular diseases, paresis of the ipsilateral limbs (hemiparesis) may be seen if the motor pathways in the medulla oblongata also are affected. large or multifocal lesions can cause an asymmetric tetraparesis. strength always is maintained with peripheral vestibular dysfunction, which is a key finding on neurological examination. a tremor is an involuntary, rhythmic, oscillatory movement of all or part of the body. it results from alternating contraction of antagonistic muscles of variable frequencies. localized tremor usually involves the head and in most cases this is an intention tremor. intention tremors occur commonly with goaloriented tasks such as when an animal "intends" to perform a task such as eating or drinking. these tremors indicate underlying cerebellar dysfunction. cerebellar dysfunction in conjunction with vestibular dysfunction implies central vestibular disease. disorders causing central vestibular dysfunction may be accompanied by altered mentation. the reticular activating system of the brainstem facilitates the alert-awake state in animals. damage to this area may cause disorientation, stupor, or coma. , , peripheral vestibular disease often causes disorientation, which makes the assessment of mentation more difficult. central vestibular syndrome may be accompanied by other cranial nerve dysfunction. cranial nerves v, vi, vii, ix, x, and xii may be affected. clinical signs suggesting involvement of these cranial nerves include ipsilateral facial hypalgesia, atrophy of the masticatory muscles, reduced jaw tone, facial paralysis, tongue weakness, and loss of the swallow or gag reflex. an ipsilateral loss of menace response accompanying vestibular dysfunction usually implies cranial nerve vii dysfunction, or multifocal disease affecting the forebrain or optic nerve. a loss of menace response also can be associated with cerebellar dysfunction. possible causes include an alteration of the menace response pathway from the visual cortex to the facial nucleus through the cerebellum or a loss of cerebellar influence on the cerebrocortical neurons. falling or leaning toward the side of the lesion indicates asymmetrical vestibular disease. cats with unilateral vestibular dysfunction show reduced extensor tone ipsilaterally and increased extensor tone contralaterally. this is manifested clinically as leaning, falling, and a tendency for tight circling toward the side of the lesion.* shaking the head induces falling or leaning. decerebellate posturing can be observed with severe and acute central vestibular dysfunction. this posture is characterized by opisthotonus with thoracic limb extension, normal mentation, and flexion of the pelvic limbs. decerebellate posturing can be intermittent and misinterpreted as seizure activity. dorsiflexion of the neck sometimes elicits this posture in cats with cerebellar dysfunction. the vomiting center is located within the reticular substance of the medulla, with direct connections to and from the vestibular nuclei. [ ] [ ] [ ] [ ] [ ] , , vomiting can occur in cats with acute vestibular dysfunction. middle and/or inner ear disease also may cause hearing loss through conductive or sensorineural impairment, respectively. conductive deafness occurs with impedance of sound wave transmission through the middle ear caused by structural defects such as ceruminoliths, a ruptured tympanum, bony ossicle damage, fluid accumulation, or aural neoplasms. , , external ear canal lavage can affect hearing thresholds in dogs and the same is assumed for cats. sensorineural deafness results from abnormalities of the inner ear structures, cochlear nerve, or central auditory pathway. , , deafness associated with central disease is considered rare. the diagnostic approach for a cat with vestibular dysfunction depends upon whether the neuroanatomical localization is peripheral or central (figure - ) . signalment, assessment of the clinical history, and thorough physical and neurological examinations are essential. peripheral vestibular dysfunction results from disease of the middle and inner ear affecting the receptors in the labyrinth and the vestibular portion of cranial nerve viii. central vestibular dysfunction results from disease affecting the brainstem and or the cerebellum. testing procedures are performed in a logical sequence, which depends on the cost expenditure and amount of invasiveness. diagnosis of a central vestibular disorder may require performance of most of the testing procedures (see figure - ). hematology, serum biochemistry, thyroid hormone testing, and urinalysis are useful to screen for other underlying metabolic disorders. thoracic radiography and abdominal ultrasound are recommended in older cats or in cats with central vestibular dysfunction to evaluate for multisystemic disease or metastatic neoplasia. an ophthalmological examination may reveal evidence of inflammatory cns disease. serology can assist with the diagnosis of some infectious diseases. cats with peripheral vestibular disease require examination of the ears and pharynx under general anesthesia. both ears should be examined with an otoscope. the tympanum is examined for color, texture, and integrity. otitis media is suspected when the tympanum is dark gray or brown. an intact tympanum does not rule out otitis media; visualization of a ruptured tympanum without other associated abnormalities also is unreliable for diagnosis of otitis media. bulging (convex appearance) of the tympanum can indicate fluid accumulation within the middle ear (see figure - ), whereas retraction (and a concave appearance) suggests a partially filled middle ear with obstruction of the auditory tube. examination of the pharynx may reveal evidence of inflammation, polyp formation from the eustachian tube, or other masses associated with the choanae. radiography is useful for evaluation of the osseous tympanic bulla. skull radiographs are performed under general anesthesia to achieve adequate positioning. lateral, dorsoventral, ventrodorsal, and oblique views are advised for tympanic bullae *references , , , , , . assessments. positioning for radiography of the bullae has been described. the normal tympanic bulla is a thin-walled gas-filled structure with well-defined, smooth borders. bilateral sclerosis of the bullae can be normal in older animals or a residual finding of previous ear disease. the external acoustic meatus is rounded with distinct smooth margins. myringotomy is the deliberate puncture or incision through the tympanic membrane. a -gauge spinal needle is used to puncture the ventrocaudal part of the tympanic membrane. the needle is connected to a -ml or -ml syringe, and fluid is aspirated for cytological analysis and culture. , , purulent or particulate matter within the middle ear may prevent needle aspiration and a larger hole may be needed for adequate drainage. a myringotomy knife can be used to make a curvi- linear or radial incision. care must be taken not to incise the tympanum too deeply and damage contents within the middle ear. similarly, forceful flushing of the middle ear should be avoided. a normal tympanum heals within to days. brainstem auditory evoked potential (baep) testing is used to assess the integrity and function of the peripheral and central auditory pathways, and to evaluate the closely associated vestibular pathways indirectly. baep are recordings of sound-evoked electrical activity in the auditory pathway between the cochlea and the auditory cortex. because of the level of patient "cooperation" with cats, sedation or a light plane of general anesthesia often is needed for this test to be performed and interpreted properly. small ( -gauge) needle electrodes are placed subcutaneously in the scalp and connected to sensitive amplifiers that can record signals in the microvolt range. the electrodes are arranged with the positive electrode over the bregma on the dorsum of the skull, the negative electrode just rostral to the base of the pinna of the ear to be tested, and the reference electrode in the same position relative to the untested ear (figure - ) . the brain activity, resulting from broad-spectrum sounds, such as clicks delivered at to hz through earphones inserted into the external ear canal, usually is averaged for milliseconds (ms) for the early latency or brainstem potentials averaging for ms includes a record of middle latency responses, but these are not as well documented in cats. the baep recording consists of six to seven positive time-locked peaks (i through vii) beginning at approximately ms after the stimulation (figure - ). wave i represents acoustic nerve activity, and subsequent waves mark peak activities as sound is being processed through ascending portions of the auditory pathway ( figure generally, cerebrospinal fluid (csf) analysis is a useful adjunctive test for determination of the cause of central vestibular disease but rarely is specific. risk of iatrogenic cns trauma or cerebellar herniation after cisterna magna puncture in cats with space-occupying lesions should not be underestimated. obtaining advanced imaging studies of the brain (see below) before csf tapping is recommended, especially if a caudal fossa lesion is suspected. i frequently use a hypodermic needle for csf acquisition in cats rather than a spinal needle and stilet to lessen risks of iatrogenic cns damage. serology is useful for determining titers for presence of antigens but nonspecific for evaluation of antibody. polymerase chain reaction analysis of csf is now performed in specialized laboratories to evaluate for the presence of some infectious agents. computed tomography (ct) and magnetic resonance imaging (mri) have revolutionized the diagnosis of vestibular diseases. the physics and interpretation details of both of these modalities have been described in detail. ct evaluation of the peripheral vestibular system is particularly useful if radiographs have not determined an underlying cause, if nasopharyngeal polyps or neoplasia are suspected, or if the patient is a potential surgical candidate. the same interpretive principles used for the radiographic diagnosis of peripheral vestibular diseases apply to ct. findings are more apparent on transverse ct images, however, because of reduced superimposition of structures in comparison to radiographs. ct can allow for an earlier diagnosis of subtle lesions. on a well-positioned study, both bullae should appear symmetrical, although subtle variations occur. lumina of the tympanic bulla and the external ear canals are gas filled (see figure - ). the tympanic bulla has a thin well-defined wall. optimal resolution of the inner ear is achieved with high-resolution ct, but it still may be inferior to high-field mri. ct evaluation for central vestibular diseases is less helpful because of beam hardening artifacts. the density of the petrous temporal bones obliterates the visualization of the medulla. mri is used less than radiography and ct for the diagnosis of peripheral vestibular disease because of its comparative limited availability and high cost. mri allows for multiplanar views when compared to ct. improved soft tissue resolution allows for better assessment of neoplastic and inflammatory processes that affect the vestibular system. a typical mri study consists of t -weighted (t w), t -weighted (t w), and proton density-weighted sequences. transverse, sagittal, and dorsal planes are used to evaluate the brain and cranium. a t w sequence is obtained after intravenous administration of a gadolinium-based contrast agent. transverse and dorsal planes with t w and t w sequences are suggested for mri of the middle ear in cats. post-contrast sequences are recommended if a mass is present in the tympanic bulla or external ear canal. congenital vestibular disorders have been reported in siamese and burmese kittens (tables - and - ). signs of peripheral vestibular dysfunction and concurrent deafness may be detected by to weeks of age and show clinical improvement within to months. a hereditary abnormality has not been proven. diagnosis is based on history, excluding other causes, and baep results. neoplasms that involve the peripheral vestibular system include squamous cell carcinoma, fibrosarcoma, osteosarcoma, chondrosarcoma, and ceruminous gland and sebaceous gland adenocarcinoma. squamous cell carcinoma is the most common malignant tumor of the middle and inner ear in cats. nonkeratinizing squamous epithelial cells are found normally in the eustachian tube and the middle and inner ear. clinical signs of peripheral vestibular dysfunction have been documented but vary depending upon lesion extension. , neoplasms of the middle/inner ear also can cause oropharyngeal signs that present with pain on palpation of the bulla or when manipulating the jaw. in addition to an examination of the external ear cavity and the tympanum for masses, the oropharynx should be examined for swelling or deviations of the soft palate. suspicious lesions should be aspirated for cytological analysis. radiography of the skull can reveal soft tissue opacity in the tympanic bulla, osteolysis, and periosteal reaction (figure - ) . , ct is a more accurate method for determining lesion extent. opacity within the tympanic bullae can indicate fluid or a soft tissue mass effect. lesion extent within the horizonal and vertical ear canals is identified. bony lysis involving osseous bulla, petrous temporal bone, and adjacent calvarium may be visualized with aggressive neoplasms. some neoplasms contrast enhance. mri characteristics described for neoplasms of the middle ear include lysis of the osseous tympanic bulla and petrous temporal bone that can extend to adjacent structures. however, a malignant melanoma involving the external ear canal and dorsalateral compartment of the tympanic bulla has been described in the cat, in which destruction of the bulla was not present and contrast enhancement of the mass did not occur. radical surgical resection and adjunctive radiotherapy often is recommended as a treatment for neoplasms involving the middle ear. median disease-free interval of months has been reported for cats with ceruminous gland adenocarcinoma after surgery alone. bacterial otitis interna or labyrinthitis can cause clinical signs of peripheral vestibular dysfunction. often otitis interna and media occur concurrently. organisms isolated commonly from the bullae include staphylococcus spp., streptococcus spp., pasteurella spp., proteus spp., escherichia coli, enterococcus spp., pseudomonas spp., and obligate anaerobes. yeast infections are an uncommon cause of otitis media. diagnosis is based on otoscopic examination, myringotomy, and imaging. otitis externa may be evident but is not necessarily the origin of the bacterial infection. bulging and discoloration of the tympanum may be identified if the bulla contains fluid or an exudate. fluid within the middle ear can be collected by myringotomy for cytological examination and anaerobic and aerobic culture/sensitivity. the external ear canal also is cultured. skull radiography is performed with the cat under general anesthesia. the latero- -degree ventrolaterodorsal oblique and rostral- -degree ventral-caudodorsal open-mouth oblique views are best for evaluation of the tympanic bullae. common radiographic findings associated with otitis media/interna include soft tissue opacity in the bulla and/or petrous temporal bone and bony proliferation of the petrous temporal bone (figure - ) . if the infection is severe enough, lysis of the tympanic bullae also can be visible. ct findings with otitis media/interna include thickening and irregularity of the tympanic bulla wall, lysis of the bulla, and radiopacity within the bulla, which suggests fluid or a soft tissue mass (see figure - , b) . a study that compared ct with radiography for diagnosis of otitis media/interna found ct to have per cent false-positives and per cent false-negatives for diagnosis confirmed by surgical findings. ct was a more sensitive but less specific technique than skull radiography. , neither radiography nor ct was able to detect early lesions associated with otitis media/interna when no osseous involvement occurred. otitis interna is difficult to assess with ct except in cases of severe destruction of the inner ear. mri findings that are compatible with otitis media include mediumsignal intensity material in the tympanic bulla on a t w sequence and hyperintense on a t w sequence. the inner margin of the tympanic bulla also may enhance after gadolinium administration. osseous lesions of the tympanic bulla are more difficult to assess with mri. an mri finding of otitis interna is a lack of signal intensity of the labyrinthine fluid on t w sequences. this may represent replacement of the fluid with fibrous tissue; however, similar findings are seen in normal ears. meningeal enhancement on post-contrast t w sequences also has been described secondary to otitis interna. treatment consists of long-term ( to weeks) antibiotic therapy and prognosis usually is good. improvement often occurs within to weeks of therapy. refractory cases may require surgical drainage of the tympanic bulla. , cryptococcosis more often causes central vestibular dysfunction. however, three cats have been reported with peripheral vestibular disease referable to otitis media/interna because of cryptococcosis. the infection was isolated from the tympanic bulla in two cats and the eustachian tube in one cat. all cats responded well to surgical drainage and medical therapy. nasopharyngeal polyps are pedunculated masses that can arise from the epithelial lining of the tympanic cavity, eustachian tube, or nasopharynx. , , nonseptic otitis media/ interna may occur secondary to occlusion of the eustachian tube because of a nasopharyngeal polyp, and polyps may occur as a result of chronic middle ear infection or from ascending infection from the nasopharynx. , polyps are especially common in young adult to middle-age cats, with no apparent gender or breed predisposition. clinical signs include peripheral vestibular dysfunction, head-shaking, aural discharge, facial nerve paralysis, and horner's syndrome. clinical signs of nasopharyngeal involvement include dysphagia, stertorous respiration, respiratory distress, and change in phonation. a secondary suppurative meningoencephalitis has been documented in a young cat with lesion extension of an inflammatory polyp within the tympanic bulla. inflammatory polyps of the middle ear can be visualized using otoscopy or by inspection of the oropharynx with the cat under general anesthesia. a lateral skull radiograph can reveal a soft tissue mass in the nasopharyngeal area and assist with the identification of nasopharyngeal polyps (figure - ) . other radiographic findings associated with polyps include unilateral or bilateral soft tissue opacity within the tympanic bulla and sclerosis of the osseous bulla. , transverse, sagittal, and parasagittal ct images of nasopharyngeal polyps in cats have been described. , ct can lateralize the lesion and assess the lesion extent. mri of polyps is recommended because of the superior soft tissue resolution of this modality. two cases of inflammatory polyps have been described in which signal intensity on post-contrast t w sequences was increased. in one cat, a nonuniform increase occurred in signal intensity on t w sequences. treatment involves traction and avulsion of the mass through the external acoustic meatus or from the nasopharyngeal cavity. bulla osteotomy can facilitate polyp removal from the tympanic bulla. prognosis usually is good, although a residual head tilt is not uncommon. the recurrence rate after polyp removal is approximately per cent. recurrence is more likely in cats with aural polyps and more severe signs of otitis externa and less likely if treated with steroids after surgery. idiopathic feline vestibular syndrome (ifvs) is a disease of peracute peripheral vestibular dysfunction (less than hours). the incidence is highest during the months of july and august in the united states. no sex predilection exists, and the median age of affected cats in one study was years. no confirmed cause exists; however, as in meniere's disease in human beings, abnormal endolymphatic flow or electrolyte aberrations in the perilymph have been hypothesized. with lack of a structural lesion, other associated neurological deficits such as horner's syndrome or facial nerve paralysis would not be expected. bilateral disease can occur but this is uncommon (less than per cent). clinical signs of ifvs often are preceded by upper respiratory tract disease ; additionally, excessive vocalization can be seen, which probably is due to the generalized feeling of disorientation. diagnosis of ifvs is made through exclusion of other causes of peripheral vestibular disease (see table - ). no specific treatment exists for ifvs besides managing the clinical signs such as anorexia, which may accompany this condition. prognosis for spontaneous recovery is good although this may take to weeks, and per cent of affected cats may have residual deficits such as a head tilt. i have seen recurrence of signs with ifvs to be more common in cats than dogs with idiopathic vestibular disease. cats with cns cuterebriasis have been documented to present most commonly during the months that coincide with the occurrence of ifvs. , this similarity has led to the hypothesis that cuterebra larval migration may account for some idiopathic vestibular cases in cats in the united states. however, clinical signs of cns cuterebriasis and idiopathic vestibular disease are dissimilar, and most cats with idiopathic vestibular disease recover in a few weeks, which makes this hypothesis less plausible. migration of a cuterebra larva through the ear canal to the peripheral vestibular apparatus still remains as a potential cause of peripheral vestibular disease. peripheral vestibular disease can be caused by ototoxic agents. an ototoxic agent is a substance that can produce cochlear or vestibular damage by causing unilateral or bilateral damage to structures of the inner ear. parenteral or oral administration of ototoxic drugs reaches the structures of the inner ear by the hematogenous route. topical drugs applied into the external ear canal reach the middle ear through a ruptured tympanic membrane and subsequent penetration into the inner ear via the round or oval window. the membrane of the round window is more permeable to macromolecules when otitis media is present. the ototoxic substance passes into the perilymph, which is contiguous within the osseous labyrinths of the cochlea and vestibule. many agents are listed in the literature as "potentially" ototoxic, but much of the information is based on anecdotal reports. studies also are extrapolated from species other than cats, and use dose formulations that far exceed the concentrations in proprietary medication. as an example, chlorhexidine and gentamycin often are quoted as ototoxic drugs when administered topically; however, no vestibular abnormalities were seen when these drugs solutions were administered at . per cent and . per cent concentrations, respectively. a list of potential ototoxic agents for cats is shown in table - . aminoglycosides can damage the neuroepithelium of the macule and crista of the vestibular apparatus, in addition to the hearing apparatus. the severity of vestibular toxicity may be directly proportional to the duration and concentration of aminoglycoside given. , other antibiotics, such as erythromycin, minocycline, chloramphenicol, vancomycin, and topical polymyxin b, have been reported to cause vestibular damage in human beings, but this has not been observed in cats. loop diuretics (e.g., furosemide) cause ototoxicity in human beings, but this has not been reported in cats when standard clinical doses have been prescribed. regarding antiseptics, many studies have been performed to document the ototoxic effect of intratympanic application of chlorhexidine. at per cent concentration, chlorhexidine is obviously ototoxic to the cochlea and vestibular system, but the damage is much more subtle at . per cent, and no clinical effects are seen. peripheral vestibular disease has been reported after the offlabel use of intraaural per cent fipronil solution for otoacariosis in two cats. the cats developed vestibular dysfunction and signs of horner's syndrome within hours after two drops of the solution were administered in each ear. both cats showed signs of improvement within days, but one of the cats had a residual head tilt. diagnosis of toxicity in peripheral vestibular disease is based on history and results of otoscopic examination and baep testing. treatment consists of cessation of the ototoxic agent and initiation of supportive care. prognosis for recovery from the vestibular signs is good in most instances. cranial trauma. peripheral vestibular signs may follow any trauma to the head, secondary to a fracture of the petrosal part of the temporal bone or tympanic bulla. this often is accompanied by facial paresis/paralysis. skull radiography or advanced imaging will be necessary for an accurate diagnosis (figure - ) . treatment is supportive and should be focused on any concurrent injuries sustained during the trauma. iatrogenic trauma. peripheral vestibular disease can be seen immediately after a bulla osteotomy, especially in cases of vigorous curettage of the petrous temporal bone. supportive care and appropriate antibiosis are necessary, but resolution usually occurs because of compensation by the animal. three cats with signs of unilateral ocular sympathetic hyperactivity (mydriasis and exophthalmos) have been reported after middle ear flushing procedures; however, the cats had signs of peripheral vestibular dysfunction because of otitis media/interna before the procedure. cerebellar cortical abiotrophy. in contrast to dogs, this condition in cats is exceedingly rare. sporadic anecdotal cases have been mentioned in the literature. late-onset cerebellar abiotrophy has been documented in adult cats, but it would be expected primarily in kittens. , lysosomal storage diseases. specific lysosomal storage disorders have been reviewed (see consultations in feline internal medicine, volume , chapter ). lysosomal storage diseases documented to cause central vestibular disease include gm -gangliosidosis, niemann-pick disease type c (sphingomyelinosis), and alpha-mannosidosis. hydrocephalus. this disease is not common in cats but may be the result of obstructive processes such as neoplasia or inflammation elsewhere in the neuraxis. enlargement of the fourth ventricle may cause central vestibular dysfunction because of the anatomical location of the vestibular nuclei. diagnosis requires advanced imaging ( figure - ) , but a csf tap also would be warranted to rule out an underlying inflammatory disease. treatment is possible with either the use of prednisone ( . mg/kg po q h) or surgical placement of a ventriculoperitoneal shunt. thiamine deficiency. this is the most common nutritional deficiency affecting the cns, usually resulting in lesions of the oculomotor and vestibular nuclei, the caudal colliculus, and lateral geniculate nucleus. the earliest neurological sign is vestibular ataxia, progressing to seizures, dilated nonresponsive pupils, and ultimately coma. treatment is administration of thiamine, parenterally ( to mg q h) or intravenously. , neoplasms neoplasms can affect the medulla of the brainstem or vestibular pathways associated with the cerebellum directly (parenchymal compression or invasion) or indirectly to cause central vestibular dysfunction. neoplasms can affect these regions indirectly by ( ) causing an obstructive hydrocephalus affecting the fourth ventricle and/or ( ) increasing intracranial pressure, causing a rostrocaudal shift of the forebrain and/or hindbrain with subsequent cerebellar herniation through the foramen magnum. space-occupying lesions in the region of the cerebellomedullary pontine angle often can be responsible for paradoxical vestibular syndrome. , rarely, middle ear tumors in cats may extend medially to involve the brainstem. the most common neoplasms in cats that affect this region are meningioma and lymphoma, but a cerebellar oligodendroglioma causing paradoxical signs also has been described in the cat. , [ ] [ ] [ ] in a study of intracranial tumors in cats, five meningiomas, lymphomas, and three glial cell tumors were documented to occur in the region of the cerebellomedullary angle and the region of the fourth ventricle. although meningiomas have been observed in cats from to years of age, the majority of cats are older than years. , the imaging characteristics of feline meningiomas have been well described ( figure - ) . , surgical resection of tumors in this area is challenging but can be achieved with improvement of the clinical signs, although recurrence is common (see chapter ) . a -year-old cat has been diagnosed with a medulloblastoma, a type of primitive neuroectodermal tumor. the cat presented with a -month history of an ipsilateral ataxia, which progressed to develop nystagmus, ipsilateral paresis, and dysmetria. magnetic resonance imaging using a t w sequence demonstrated an irregularly shaped hypointense mass within the cerebellar parenchyma that contrast-enhanced and was irregularly hyperintense on t w images. surgical resection was possible but no follow-up was documented. the same cat seems to have been described in another report, which documented a -day postsurgery survival. any inflammatory disease that affects the cns has the potential to cause central vestibular signs, usually as part of a multifocal syndrome. these diseases have been discussed in detail and are documented in table - . the more common infectious agents are discussed briefly. bacterial meningoencephalitis/abscessation from otitis media and otitis interna can extend into the intracranial cavity and result in bacterial meningoencephalitis (see chapter ) . seven such cats with otitis media/interna have been documented, in one study, with cns dysfunction that included central vestibular signs. mri was extremely effective in demonstrating the location, extent, and relationship to normal structures of inflammation of the middle ear and brainstem in all cases. a mild to severe neutrophilic pleocytosis was present in the csf of four of five cats tested. marked neurological improvement was seen in all the cats, which underwent surgical drainage in addition to prolonged antibiotic therapy. extension of bacterial infection into the cns also has been documented in a -month-old male maine coon cat with an inflammatory polyp of the middle ear. the cat required a ventral bulla osteotomy to remove the polyp in addition to broad-spectrum antibiotic therapy for the secondary suppurative meningoencephalitis but made a good recovery with a residual head tilt. mri is useful in detecting brain abscessation secondary to otitis media/interna. abscessation with extension of an inner ear infection can affect the brainstem and has a heterogenous signal intensity on t w and t w images (figure - ) . a ring-enhancing lesion with extension into the tympanic bulla can be seen after intravenous contrast administration. feline infectious peritonitis. feline infectious peritonitis (fip) results from infection with a mutated form of feline enteric coronavirus and represents the most common cause of inflammatory brain disease in cats. neurological disease usually is seen with the noneffusive form of fip, and up to a third of cats with this form of disease develop neurological disease. some affected cats have evidence of disease only localized to the cns. insidious multifocal or diffuse cns clinical signs are seen, which commonly include vestibular dysfunction. analysis of csf is the most useful antemortem diagnostic test, which often reveals a neutrophilic pleocytosis with a marked protein elevation (more than mg/dl). however, this test cannot be relied upon to be either sensitive or specific for fip. positive coronavirus antibody titers in the csf are the most reliable indicator of the disease, but only if an albumin quotient and igg index rule out serum protein translocation across a disrupted blood-brain barrier. polymerase chain reaction (pcr) testing of the csf recently has become available; unfortunately, only a third of cats with neurological fip have positive csf pcr results, and only two thirds of brain tissue specimens actually are pcr-positive. advanced imaging reveals the presence of hydrocephalus in the majority of affected cats. no documented effective treatment exists, and the prognosis is poor. toxoplasmosis. cats are the definitive hosts of toxoplasma gondii. occasionally, cats develop central neurological disease because of this organism. after the initial enteroepithelial life cycle, tachyzoites are disseminated through the blood and lymph. the immune system generally can suppress proliferation of tachyzoites with subsequent development of cysts. these cysts remain dormant for long periods and have a predilection for sites such as the brain. diseases associated with toxoplasmosis can be due to recrudescence of local infection. multifocal neurological signs are a common clinical manifestation. a definitive diagnosis is difficult. csf analysis reveals a mixed pleocytosis and elevated protein levels. comparison of csf and serum antibody titers may aid in the diagnosis of the disease. pcr analysis for protozoal disease on the csf is now available but may not be highly sensitive. advanced imaging can reveal multifocal areas of irregular contrast-enhancing lesions within the brain parenchyma. clindamycin ( . mg/kg po q h for to weeks) is advocated for treatment of this disease; however, the prognosis is guarded and residual signs and recrudescence may be common. cryptococcosis. cryptococcosis is the most common systemic mycosis of cats. feline cryptococcosis has been reviewed extensively. more than per cent of cats present with signs of nasal cavity disease, including sneezing, nasal discharge, respiratory stridor, and subcutaneous masses at the nostrils (see figure - ). the cns occasionally is involved, manifesting with multifocal neurological signs, including central vestibular dysfunction. csf analysis is the most helpful diagnostic test in cats with cns cryptococcosis. the organism may be identified cytologically or cultured from the csf. a positive capsular antigen test can provide a definitive diagnosis. treatment consists of triazole drugs (fluconazole, itraconazole) for at least months beyond resolution of the clinical signs. fluconazole crosses the blood-brain-barrier readily and is the preferred antifungal agent. the decision to discontinue therapy is based upon repeat csf analysis results, serology, and resolution of clinical signs. often patients require long-term antifungal therapy. prognosis is considered guarded. although not common, central vestibular signs have been reported in cats after chronic high-dose therapy with metronidazole. clinical signs reversed in two of the cats reported within a few days of drug withdrawal and with appropriate supportive care. diazepam administration has improved the recovery time in dogs with metronidazole toxicity; this remains to be determined for cats. unlike metronidazole toxicosis in dogs, nystagmus is an uncommon clinical finding. lead. the most common clinical signs of lead toxicosis in cats are anorexia, vomiting, and seizures. central vestibular abnormalities, including vertical nystagmus and ataxia, have been reported. old paint is the most common source of exposure for cats. recovery can be complete after standard treatment. central vestibular signs subsequent to head trauma often imply brainstem involvement; occasionally, the signs may be due to elevated intracranial pressure, causing a rostrocaudal transtentorial herniation or a cerebellar herniation through the foramen magnum. diagnosis is supported by history and skull fractures on radiographs; however, it is not necessary for the skull to be fractured for central vestibular damage to occur. advanced imaging studies can be used to assess for intracranial hemorrhage and edema. principles for management of head trauma address the pathophysiologic sequelae to traumatic brain injury such as edema. feline ischemic encephalopathy (fie). fie is a poorly understood syndrome of brain infarction in cats. onset of clinical signs is peracute. fie affects cats of all ages and most commonly in the months of july and august. the main clinical signs are acute in onset, focal, and lateralizing; these include depression, blindness, circling, and central vestibular dysfunction. this has been associated with cuterebra spp. migration. although central vestibular signs have been reported with this abnormality, other neurological signs supportive of forebrain disease are more common. diagnosis is based on focal lesions identified by advanced imaging and csf analysis. treatment is supportive care. gradual improvement of clinical signs can occur over several months, but residual signs are likely. severe cases can be fatal. the damaged vestibular system can compensate over time with central reprogramming of eye movements and postural responses in addition to reliance on visual and other sensory input that replaces lost vestibular input. , , histamine is thought to be involved in the recovery of vestibular function, although the mechanism is unclear. if the underlying disease process can be targeted, the prognosis for a functional recovery can be good. residual signs, such as a head tilt, are not uncommon. recurrence of vestibular dysfunction can occur at times of stress, recurrent disease, or after an anesthetic episode. supportive care often is essential in cats with vestibular dysfunction, because anorexia is a frequent complication. vomiting, salivation, and possible nausea associated with vestibular disease can be treated medically. drugs used commonly include the phenothiazine derivative chlorpromazine ( . to . mg/kg sq q h); and the antihistamines diphenhydramine ( to mg/ kg po or im q h), dimenhydrinate ( to mg/kg po q h), and meclizine ( . mg q h). betahistine dihydrochloride is a histamine-like substance that is used in human beings with meniere's syndrome and also has been shown to accelerate the recovery process from a central vestibular syndrome in experimental cats when used at daily doses of mg/kg. clinical use has not been documented. vestibular system-special proprioception cortical and brainstem control of motor function vestibular dysfunction disorders of the vestibular system king as: special senses otitis media and otitis interna recognition and localization of intracranial disease neurologic evaluation of the ear meningoencephalitis secondary to bacterial otitis media/interna in a dog feline vestibular diseases-new developments cerebellar meningioma with paradoxical vestibular signs vestibular diseases strain gm: aetiology, prevalence and diagnosis of deafness in dogs and cats facial neuropathy in dogs and cats: cases ( - ) cranial nerves and cutaneous innervation of the head essentials of veterinary ophthalmology horner's syndrome in dogs and cats: cases ( - ) iatrogenic pupillary dilation resembling pourfour du petit syndrome in three cats paradoxical vestibular syndrome in a cat with a cerebellar meningioma effects of otitis on hearing in dogs characterized by brainstem auditory evoked response testing healing of experimentally perforated tympanic membranes demonstrated by electrodiagnostic testing and histopathology congenital deafness and its recognition deafness in the dog and cat the normal ear diagnostic imaging of the canine and feline ear imaging techniques in the diagnosis of middle ear disease microbial flora and antimicrobial susceptibility patterns of isolated pathogens from the horizontal ear canal and middle ear with otitis media otitis externa and otitis media: diagnostic and medical aspects insertion of a transtympanic ventilation tube for the treatment of otitis media with effusion electrodiagnostic evaluation of hearing and vision middle and long latency auditory evoked potentials in cats. ii. component distributions and dependence on stimulus factors use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs advanced imaging concepts: a pictorial glossary of ct and mri technology intracranial neoplasia magnetic resonance imaging of the normal and diseased feline middle ear feline vestibular disorders. part ii: diagnostic approach and differential diagnosis adenocarcinoma of the middle ear with osteolysis of the tympanic bulla in a cat vestibular dysfunction caused by squamous cell carcinoma involving the middle and inner ear in two cats peripheral vestibular disease in a cat with middle and inner ear squamous cell carcinoma oral and peripheral vestibular signs in a cat with squamous cell carcinoma the cranial and nasal cavities-canine and feline results of magnetic resonance imaging in dogs with vestibular disorders: cases ( - ) results of surgery in cats with ceruminous gland adenocarcinoma clinical neurology in small animals: localization, diagnosis and treatment. international veterinary information service a comparison of radiographic versus surgical diagnosis of otitis media radiographic and computed tomographic evaluation of otitis media meningeal enhancement on magnetic resonance imaging in dogs and cats feline vestibular disorders. part i: anatomy and clinical signs peripheral vestibular disease associated with cryptococcosis in three cats tumors of the ear canal inflammatory polypoid growths in the ear canal of cats nasopharyngeal diseases in cats: a retrospective study of cases ( - ) inflammatory polyp in the middle ear with secondary suppurative meningoencephalitis in a cat computed tomographic appearance of inflammatory polyps in three cats feline nasopharyngeal polyps results of surgery and long-term follow-up in cats with nasopharyngeal polyps feline inflammatory polyps: historical, clinical, and pcr findings for feline calicivirus and feline herpesvirus- in cases review of idiopathic feline vestibular syndrome in cats clinical and clinicopathological features in cats with cuterebra larvae myiasis of the central nervous system gentamicin tympanoclysis: effects on the labyrinthine cells off-label treatment for otoacariosis current trends in the treatment of sarcoptes, cheyletiella and otodectes mite infestations in dogs and cats tympanic bulla osteotomy for treatment of middle-ear disease in cats: cases ( - ) adult-onset cerebellar cortical abiotrophy and retinal degeneration in a domestic shorthair cat late onset cerebellar abiotrophy in a siamese cat cerebellar cortical atrophy in a kitten study of hereditary cerebellar degeneration in cats degenerative brain disease meningiomas in cats: a retrospective clinical study of cases clinical and pathological features of a cerebellar oligodendroglioma in a cat cerebral meningiomas: diagnostic and therapeutic considerations magnetic resonance imaging features of feline intracranial neoplasia: retrospective analysis of cats feline intracranial neoplasia: retrospective review of cases early diagnosis of feline medulloblastoma in the vermis medulloblastoma in a cat: clinical and mri findings inflammatory disorders of the central nervous system otitis media/interna with central extension in dogs and cats: clinical signs, magnetic resonance imaging features, and outcome after surgical intervention magnetic resonance imaging features of brainstem abscessation in two cats diagnostic features of clinical neurologic feline infectious peritonitis cryptococcosis: new perspectives on etiology, pathogenesis, diagnosis, and clinical management metronidazole neurotoxicosis in two cats succimer for treatment of lead toxicosis in two cats feline ischemic encephalopathy in a cat cerebrospinal cuterebriasis in cats and its association with feline ischemic encephalopathy vestibular compensation in the cat: the role of the histaminergic system betahistine dihydrochloride treatment facilitates vestibular compensation in the cat key: cord- -fkddo n authors: griffin, brenda title: population wellness: keeping cats physically and behaviorally healthy date: - - journal: the cat doi: . /b - - - - . - sha: doc_id: cord_uid: fkddo n nan o u t l i n e whereas feline practitioners are usually well versed in the creation of wellness programs tailored to individual cats, optimizing the health of a population of cats requires additional knowledge and poses unique challenges. these challenges will vary depending on many factors, including the nature and purpose of the population itself. indeed, veterinarians may be tasked with developing health care programs for cat populations in a wide spectrum of settings-from facilities housing laboratory animals, to animal shelters, home-based rescue and foster providers, care-for-life cat sanctuaries, breeding catteries, or large multicat households. regardless of the setting, a systematic approach to the health of the clowder is crucial for success. merriam-webster's dictionary defines wellness as "the quality or state of being in good health especially as an actively sought goal." ensuring population health requires careful planning and active implementation of comprehensive wellness protocols that address both animal health and environmental conditions ( figure - ). addressing physical health alone is not sufficient to ensure wellness. for example, a cat may be in proper physical condition and free from infectious or other physical disease, yet suffering from severe stress and anxiety. in this case, the patient cannot be assessed as healthy, because its behavioral (emotional) state is compromising its health and well-being. thus physical health and behavioral health are both essential components of wellness, and preventive health care must actively address each of these. addressing the environment of the population is also critically important when considering wellness. even the best-designed facilities cannot favor good health in a multicat environment without thoughtful implementation of environmental wellness protocols. in small animal practice, environmental wellness is frequently not emphasized simply because many owners are accustomed to providing a reasonably healthy environment for their pets. in contrast, a structured program to address environmental wellness is essential in the more specific goals will vary depending upon the given population and its purpose. for example, in an animal shelter, specific goals of the wellness program might include decreasing the incidence and prevalence of infectious diseases in the shelter and following adoption, decreasing the incidence of problem behaviors in the shelter, decreasing the rate of return of cats to the shelter for problem behaviors, increasing the adoption rate, and so forth. in the context of a breeding colony, the goals might include increasing kitten birth weights, decreasing neonatal mortality, or improving socialization of kittens. by identifying and tracking measurable factors (often called performance targets in large animal medicine), it is possible to measure progress toward these goals. once baseline data (such as disease rates) are established, it is possible to measure the impact of protocol changes on population health by evaluating these performance targets. both medical records and a system for regular surveillance and reporting are required to accurately track and access trends in animal health. early recognition is crucial for effective control of infectious disease and problem behavior in a group. therefore a regular system of health surveillance must be in place to monitor every individual. in a population setting, daily "walk-through rounds" represent the foundation of an effective animal health care program. rounds should be conducted at least once daily (preferably twice a day or more often, depending upon the needs of individual cats) for the purpose of monitoring and evaluating both physical and behavioral health. medically trained caregivers should visually observe every animal and its environment, taking note of food and water consumption, urination, defecation, attitude, behavior, ambulation, and signs of illness, pain or other problems. monitoring should take place before cleaning so that food intake and the condition of the enclosure, including the presence of feces, urine, or vomit can be noted. alternatively, observation logs can be completed by caregivers at the time of cleaning and reviewed during walk-though rounds. any cat that is observed to be experiencing a problem, whether it be signs of respiratory infection, diarrhea, anxiety, or obvious pain, suffering, or distress must be assessed and treated in a timely manner. regardless of length of stay, regular daily assessment is imperative to identify new problems (medical or behavioral) that may develop so that they can be identified and addressed in a timely fashion to ensure the welfare of the individual animal as well as that of the population. context of a population, regardless of the actual physical facility. proactive measures to maintain clean, sanitary environments that are not overcrowded-where cats are segregated by age and health status and provided with regular daily schedules of care by well-trained dedicated caregivers-are essential. simply stated, the overarching goals of a population wellness program are to optimize both the physical and behavioral health of the cats as well as preventing transmission of zoonotic diseases. in other words, a population wellness program should be designed to keep animals "healthy and happy" while keeping human caregivers safe. it is not difficult to identify a healthy population of cats: when wellness protocols are successful, cats "look healthy" and "act like normal cats." in other words, they appear in good physical condition and display a wide variety of normal feline behaviors, including eating, stretching, grooming, scratching, playing, rubbing, resting, and if allowed, courtship and breeding. just as changes in a cat's physical appearance should alert the clinician to potential problems, so should the absence of such normal feline activities and behaviors by members of the group. wellness goals must include maintaining the health of individual animals as well as that of the population as a whole. in the context of the population, the individuals that are physically or behaviorally ill serve as indicators or "barometers" of the health care and conditions of the population. when individuals are ill, their health and well-being is always a priority; however, it should also immediately trigger the clinician to ask, "why is this individual sick? what is the cause of its illness, and how can i prevent this from affecting others?" to optimize feline health, wellness programs must be carefully structured to address both the physical and behavioral health of the animals, which are intimately linked to their environment, making it crucial to systematically address environmental conditions as well. behavioral health . freedom from thirst, hunger, and malnutrition by providing ready access to fresh water and a diet that maintains full health and vigor . freedom from discomfort by providing a suitable environment, including shelter and a comfortable resting area . freedom from pain, injury, and disease by prevention or rapid diagnosis and treatment . freedom to express normal behavior by providing sufficient space, proper facilities, and company of the animals' own kind . freedom from fear and distress by ensuring conditions that avoid mental suffering in addition to early recognition of health problems, timely action is crucial to effectively limit their morbidity. ideally, all facilities that house multiple cats should have written policies and protocols in place that detail how medical and behavioral problems will be handled. , , a committee or team of individuals composed of medical staff, managers, and caregivers can establish and oversee these policies and protocols. such protocols serve as guidelines for systematic triage and care of animals and help to prevent delays in care that may otherwise arise if such plans were not in place. policies and protocols should be based on medical facts, taking into account the entity's purpose or mission and the availability of resources for care. they should include a definition or description of the disease or condition in question, a description of the methods that will be used for diagnosis, and a general policy regarding the handling and disposition of affected cats. in addition, protocols should include details on notification, housing, decontamination, treatment, and documentation (box - ). just as quality-of-life assessment is the responsibility of every veterinarian as they guide the medical care of individual animals, quality-of-life assessment is also a critical part of population health care and monitoring. the factors that affect physical and mental well-being are broad, complex, and often vary substantially among individuals. exacting criteria are lacking for the objective measurement of quality of life of cats. however, subjective assessments can and should be made by medical and behavioral personnel at regular intervals (weekly or even daily, as indicated) considering the most information possible. , the "five freedoms," which were originally described by the farm animal welfare council in the s, represent a benchmark for ensuring quality of life or animal welfare (box - ). these principles provide a useful framework that is applicable across varying situations and species and have been widely accepted and endorsed by animal care experts. many agencies have used the five freedoms as the basis of recommendations for minimum standards of care for many species, including cats housed in catteries, shelters, and research facilities. , , , the tenets of the five freedoms define essential outcomes and imply criteria for assessment but do not prescribe the methods by which to achieve those outcomes. regardless of the setting, population wellness programs should ensure the five freedoms for all cats. wellness always starts with prevention: it is far more time and cost efficient than treatment, and it is kinder to the animals and their caregivers. with this in mind, population wellness programs should provide broad-based, holistic approaches to preventive care, rather than being based on the control of a single disease or problem, regardless of the setting. maintenance of good health or wellness is especially challenging in populations with high turnover and interchange of cats of varying ages and susceptibilities, such as animal shelters. infectious diseases can become endemic in facilities where populations of animals are housed. even in closed populations, certain pathogens can be difficult to exclude or to eliminate once introduced. notably, upper respiratory viruses, dermatophytes, and coccidia are among the most difficult pathogenic agents to control because of their persistence in the environment through carrier states and/or resistance to environmental disinfection. in particular, upper respiratory disease is the most common endemic disease in cat populations and is impossible to completely prevent in an open population. feline herpes virus type (fhv- ) and feline calicivirus (fcv) have been implicated as the causes of most infections: both viruses induce persistent carrier states and are widespread in the cat population. cats that recover from fhv- remain latently infected and shed virus intermittently, especially following periods of stress. fcv carriers shed continuously for months to years following infection. a variety of other viral and bacterial pathogens may also contribute to feline upper respiratory disease, and bordetella, chlamydophila, and mycoplasma are problematic in some populations. feline infectious peritonitis (fip) is another disease that is nearly impossible to eradicate from a multicat environment, and sporadic cases can be expected to occur, especially in young cats. fortunately, proper wellness programs can greatly limit the incidence and severity of diseases, even for pathogens that are difficult to control. the multicat environment also presents enormous opportunities for inducing stress. because of their unique biology, cats are particularly prone to experiencing acute stress and fear in novel environments. anything unfamiliar to a cat can trigger apprehension, activating the stress response. confinement in a novel environment can result in a wide variety of behavioral indicators of stress including hypervigilance, feigned sleep, constant hiding, activity depression, and loss of appetite, among others. in the long term, if cats are unable to acclimate or cope in their environments, chronic stress, fear, frustration, or learned helplessness may result. in group settings, signs of social stress may also manifest with medical decisions must be weighed in the context of the health of the population as well that of the individual, while considering animal welfare and the availability of resources for care. when large numbers of animals are involved, situations may arise in which animal health and welfare cannot be managed in the case of every individual animal. this may be due to physical or behavioral illness, or environmental conditions that negatively impact animal health, such as crowding. regardless of the cause, it may be necessary to euthanize affected individuals if no other remedies exist to relieve animal suffering or to protect population health. these decisions can be difficult and emotionally challenging, especially in instances where the individual could easily be treated or otherwise accommodated if adequate resources were available. however, such decisions may be crucial for disease control, animal welfare, and population health. that being said, euthanasia should never be used as a substitute for providing proper husbandry and care. indeed, a critical need for a comprehensive wellness program exists in every multicat setting. it is unacceptable to house animals under conditions likely to induce illness and poor welfare, and such conditions can be expected when wellness programs are not in place and carefully monitored. when facilities elect to house cats with medical or behavioral problems, appropriate veterinary care must be provided. it is imperative that a humane plan for diagnosis, treatment/management, monitoring, and housing be implemented in a timely fashion. when determining if cats with special needs can be humanely cared for in a population setting, the following goals and considerations should be addressed: what measures must be implemented to prevent transmission of disease to other cats or people? can appropriate care realistically be delivered? will the care provided result in a cure or adequate management of the disease or problem behavior? can the facility afford the cost and time for care? how will it impact resources available for other cats? in the case of animal shelters, additional considerations should include will the cat be adoptable? what steps can be taken to minimize the holding time required for treatment? if the cat is not adopted, do humane long-term care options exist in the shelter? what welfare assessment will be used to measure quality of life in the shelter? disease control efforts when disease is present. however, the best method of disease control is always prevention. when creating preventive medicine programs for a population, consideration must be given to all components of wellness: physical, behavioral, and environmental health. with regard to promoting physical health, wellness programs should address the following essential elements: implementing population wellness protocols and ensuring quality and timely care require reliable systems for medical record keeping and animal identification. regardless of the system used, medical record keeping procedures should comply with state and local practice acts, guidelines provided by state and national veterinary medical associations, and, in the case of laboratory animals, regulations as prescribed by federal law, the increases in problem behaviors, including urine marking, spraying, or other inappropriate elimination; constant hiding; and/or aggression. stress not only has the potential to negatively impact behavioral health but also physical health as well. the intimate link between stress and immunity has been well described. in fact, stress is a leading factor in the development of infectious disease and is particularly important in the pathogenesis of feline upper respiratory infections. , wellness programs that reduce stress will also serve to minimize the morbidity of infectious disease. despite the fact that infectious agents can never be completely eliminated from the environment, it is still possible to maintain good health. this is because the development of disease is determined by a complex interaction of many factors surrounding the host, the infectious agent, and the environment. keeping these factors in mind provides a rational context for many of the recommendations in this chapter. some of the host factors that influence health and the development of disease include age, sex and reproductive status, immune status, body condition, stress, and genetics. the amount and duration of exposure to an infectious agent (i.e., the "dose effect"), as well as its virulence and route of inoculation, also influence the likelihood and severity of disease. in addition, environmental conditions contribute to the development of infectious disease, including such factors as housing density, sanitation, and fluctuations in temperature or air quality. the fact that disease results from such a large combination of factors underscores the importance of a holistic and broad-based approach to population wellness. when infectious disease does occur in a population, general principles of infectious disease control should guide the response. these include . some facilities prefer to use safety collars that are designed to break away should the collar become caught on something. even for kittens, collars can be used and may be especially beneficial, because they will learn to wear them from an early age. microchips may also be used for identification and are safe and simple to implant (figure - ) . the procedure is well tolerated by the vast majority of cats without the need for sedation. unlike visual means of identification, a scanner is necessary for positive identification of a microchipped animal. for this reason, microchips are often used in conjunction with a visual means of identification and serve as important permanent means of backup identification. box - describes the proper technique for scanning for a microchip. during the last decades, microchips of varying radiofrequencies ( , , and khz) have been introduced in the united states. the -khz chips have historically been the most common, whereas the accepted standard in the rest of the world is the -khz chip. because some scanners read only certain radiofrequencies, it is possible to miss detecting a microchip that is present, depending on the scanner being used. currently, there are efforts to standardize microchipping in the united states, including widespread distribution of universal (global) scanners to ensure that all implanted microchips can be reliably identified. once global scanners are widely available, the american veterinary medical association (avma) recommends adoption of the -khz (iso) microchip as the american standard, because this frequency is recognized as the international standard for microchips institute for laboratory animal research and institutional animal care and use committees. computerized records are preferred; however, written records may also be used. computerized records offer the advantage of mechanized reporting, which facilitates detection and monitoring of health trends in the population. a medical record should be prepared for each cat and should include the cat's entry date, identification (id) number, date of birth, gender, breed, and physical description, as well as historical and physical/behavioral examination findings. in addition, it should contain the dosages of all drugs administered and their routes of administration, including vaccines, parasite control products, other treatments, and anesthetic agents; the results of any diagnostic tests performed; any surgical procedure(s) performed; and other pertinent information regarding the animal's condition. standardized examination and operative reports may be used, but should allow for additions when necessary. identification of cats in the form of a neckband, collar and tag, tattoo, earband, and/or a microchip is also essential for preventive health care and ongoing surveillance of individuals. whenever possible, some form of identification should be physically affixed to every individual cat. in addition, enclosures should be labeled with the cats' unique identification number and/or name. contrary to popular belief, most cats can reliably wear collars safely and comfortably. many facilities use disposable collars, including commercially available plastic or paper neckbands made for animals or hospital-type wristbands made for human patients (figure - ). commercially available cat collars with an id tag affixed in the rest of the world. efforts have also focused on improving, updating, and centralizing microchip registries. this is extremely important in the context of animal shelters. box - contains information on the use of collars and microchips as tools for improving cat-owner reunification. in laboratory settings, tattoos may be used as a means of permanent identification of cats ( figure - ). tattoos are most commonly applied to the inner pinna of the ear using a tattoo machine with multiple needles. care must be taken to properly disinfect the needles between patients. a significant disadvantage of tattooing is that tattoos can sometimes be difficult to read because of the presence of hair, fading, or distortion that may occur as the cat grows. in addition, their application requires anesthesia or heavy sedation. small stainless steel ear tags manufactured for wing banding of birds are especially useful for identifying newborn kittens in some settings and are highly economical (figure - ). they can be placed without the need for anesthesia or sedation when kittens are less than to days old. placing earbands requires skill and experience. they must be positioned in such a way as to provide adequate space for growth of the ear, while seating them deeply enough in the ear margin to ensure a secure piercing far enough away from the edge. if placed too close to the ear margin, the ear flap may tear, resulting in loss of the band. other complications include local inflammation or infection at the site of the piercing. ear tags are a practical method for identifying individual kittens in institutional or commercial breeding colonies, because when applied skillfully, they are seldom lost and provide reliable, long-lasting visual identification. in contrast, private breeding catteries and animal shelters generally prefer to use methods that will not alter the cat's cosmetic appearance long term. colored ribbon, nail polish, or clipping of hair in various areas of the body can all be useful means of temporary kitten identification in the neonatal stage, especially when coat color or patterns do not easily allow individuals to be distinguished. every cat, including those surrendered by their owners, should be systematically scanned for the presence of a microchip at the time of intake, as well as prior to being made available for adoption or being euthanized. proper technique and scanning more than once are crucial to avoid missing microchips. , a universal (global) scanner (e.g., one that will read all microchip frequencies that are currently in use) should be used to ensure that all microchip frequencies are detected. at this time, the only universal scanners available in the united states are the new home again global world scanner (schering plough, whitehouse station, ny) and the imax black label resq scanner (bayer animal health, shawnee mission, kans.). one of the most common causes of scanner failure is weak batteries; therefore it is imperative that batteries be checked and replaced regularly. to ensure a thorough scan and avoid missing chips, cats must be removed from carriers or cages prior to scanning. metal and fluorescent lighting may interfere with chip detection. metal exam surfaces should be covered with a towel or other material prior to scanning to minimize interference. the entire animal should be scanned using a consistent speed, scanner orientation, scanning pattern, and distance. • scanner orientation: the scanner should be held parallel to the animal. rocking the scanner slightly from side to side will maximize the potential for optimal chip orientation and successful detection. the button on the scanner should be depressed continuously during the entire scanning procedure. • scanning distance: the scanner should be held in contact with the animal during scanning such that it is lightly touching the hair coat. • scanner speed: the scanner should not be advanced any faster than . m/second ( . ft/second). scanning slowly is crucial, because universal scanners must cycle through various modes to read all possible chip frequencies. • areas of animal to scan: the standard implant site is midway between the shoulder blades, and scanning should begin over this area. if a microchip is not detected here, scanning should proceed systematically down the back, on the sides, neck, and shoulders-all the way to the elbows in the front and the hindquarters in the rear. • scanning pattern: the scanner should be moved over the scanning areas in an "s"-shaped pattern in a transverse direction (from side to side). if no microchip is detected, the scanner should be rotated degrees, and then the "s"-shaped pattern should be repeated in a longitudinal direction (e.g., the long way) on both sides of the animal. this pattern of scanning will maximize the ability of the scanner to detect the microchip, regardless of its orientation. • less than % of cats are reunited with their owners, compared to as many as % to % of lost dogs. • the use of collars and tags as visually obvious forms of identification is extremely valuable, although overlooked by many cat owners. • cats wearing collars are more likely to be identified as owned and not mistaken for strays. • even indoor cats require identification in case they escape, and studies clearly demonstrate that visual identification improves the odds of pet-owner reunification. • the provision of permanent identification in the form of a microchip represents an important backup, further improving the odds of pet-owner reunification because collars and tags can be lost. • because owners and shelter staff often describe cat coat color and patterns differently, photographs that can be posted online are a useful method of improving lost-pet matching and enabling owners to look for their pet, even if they are physically unable to come to the shelter. • adopted animals should be sent home with id collars and microchips. • shelter staff should always register microchips before the cat leaves the shelter, because many owners will neglect to do so following adoption, making the microchip an ineffective means of identification. • web-based search engines for pet microchip identification numbers (http://www.checkthechip.com and http://www.petmicrochiplookup.org) have been established in an effort to functionally centralize microchip registries by linking existing national databases. facility that houses cats establish a formal relationship with one or more veterinarians who have direct knowledge of their animal population. this is essential to ensure that medical protocols are established with the proper professional oversight, and helps to ensure compliance with local veterinary practice acts that restrict the practice of veterinary medicine to licensed veterinarians. in facilities such as animal shelters, trained shelter staff can carry out preventive health care under the instructions of a veterinarian. the success or failure of a population wellness program hinges in large part on its implementation and oversight. a knowledgeable, cohesive, and dedicated team, where accountability, responsibility, and lines of authority are well defined, is crucial for management success. as a part of the management structure and plan, veterinarians must be involved in the oversight of all aspects of animal care and must be given direct authority for the oversight of medical decisions. this requires that every physical examination is the clinician's single most important tool for evaluating health. following a standardized physical examination form will ensure a complete and systematic review of all body systems. a veterinarian should carefully examine any new cat entering a closed population prior to admittance. in the context of animal shelters, every cat that is safe to handle should receive a physical examination at or as close to the time of admission to the shelter as possible. in many shelters, a veterinarian may not be available to examine incoming animals. however, staff can and should be trained to perform basic evaluations including sexing, aging, body condition scoring, and looking for evidence of fleas, ear mites, dental disease, overgrown claws, advanced pregnancy, or other obvious physical conditions. of particular importance in the shelter physical examination are an accurate physical description of the animal and careful inspection for the presence of identification, both of which may aid in pet-owner reunification. the gold standard for maintaining the health of a population is through exclusion of pathogens in combination with implementation of comprehensive wellness protocols. this requires that members of a population be free from specific pathogens when the group is established and that the colony be closed to any new individuals that do not meet the health standards of the group. this is the foundation of disease control procedures in a laboratory animal setting, and these concepts should be applied to other population settings whenever possible. consideration should be given to testing for the following: feline leukemia virus (felv), feline immunodeficiency virus (fiv), dermatophytosis, intestinal parasites and infections (e.g., campylobacter, giardia, coccidia), as well as other endoparasites and ectoparasites. the setting and resources available, as well as the individual's history and physical examination findings, should guide the clinician's decisions regarding selection of testing for cats entering a specific population. when new stock is added to a closed colony, disease testing is imperative. the american association of feline practitioners (aafp) maintains detailed professional guidelines for the management of felv and fiv infections. identification and exclusion of infected cats is the most effective method of preventing new infections. cats and kittens should always be tested prior to entry to a closed population. those that test negative should be retested, because it the clinician should develop a program for physical health for the population that addresses all of the essential elements as noted. none of these should be considered as optional, but their implementation will depend on the setting, purposes, and resources of the group. the value of obtaining an accurate medical history on any cat entering a population is immeasurable, because it will often alert the clinician to the presence of potential problems. in a laboratory setting, obtaining cats from commercial purpose-bred colonies or institutional breeding colonies ensures that an accurate history will be available, maximizing the odds that only healthy cats will be added to the population. likewise, private breeding catteries should always strive to obtain an accurate medical history on any cat that may be accepted into the cattery. the introduction of cats from random sources to closed populations of cats risks the health of the population and should be avoided whenever possible. in contrast, by their very nature, animal shelters must frequently receive cats from multiple random sources, and it will not always be possible to obtain accurate histories. in some cases, cats are brought in by animal control officers or good samaritans who have little if any information about them. furthermore, some shelters provide a location (e.g., drop-off cages) where cats can be relinquished after business hours. this practice should be discouraged; however, if facilities elect to do this, every effort must be made to obtain a history through questionnaires that can be completed when the cat is left. the presence of staff to directly accept cats and obtain a history at the time of relinquishment is greatly preferred. even so, surrendering owners may or may not provide complete or accurate information, fearing that if they are honest about a pet's problems, the pet may be euthanized. nonetheless, when available, a history can be extremely valuable, saving time and money as well as preventing unnecessary stress for cats and staff alike. intake procedures should be in place to capture basic patient information, including both physical and behavioral data as well as the reason(s) for relinquishment. the importance of obtaining historical information cannot be overemphasized. in many cases, historical information may be used to expedite the disposition of the cat in the shelter. can be problematic. in relation to population health, testing is of little value, because infected cats pose no risk to other cats. nonetheless, a clinician may elect testing as part of an initial database for individual cats, especially if they will be used for breeding. with heartworm tests readily available in combination with pointof-care felv/fiv tests, many animal shelters have been faced with determining whether or not to perform routine screening of cats in their care. to answer this question, it is helpful to consider the following: in consideration of these facts, the author does not recommend routine screening of cats for heartworm disease in shelters. monthly chemoprophylaxis, however, is a safe and effective option for cats sheltered in areas where heartworm infection is considered endemic. dermatophytosis or ringworm, the most common skin infection of cats, is a known zoonosis. it is caused by infection of the skin, hair, and nails with microscopic fungal organisms that cause varying degrees of hair loss and dermatitis. the dermatophyte that causes the majority of cases in felines is microsporum canis, which is responsible for greater than % of all cases. if left untreated, most infections will spontaneously resolve within to weeks postinfection. however, during this time, the infected cat will infect the surrounding environment and other animals or humans in the area. not all cats infected with dermatophytosis develop lesions, and some may become chronic carriers. control of dermatophytosis is difficult, because the spores formed by m. canis can survive in the environment for up to months or longer and are extremely resistant to disinfectants and detergents. in addition, the presence of asymptomatic carriers makes it difficult to readily recognize all infected cats. for this reason, consideration should be given to culturing all cats prior to entry to a closed colony. in particular, persian cats may be predisposed to dermatophyte infection and can be particularly difficult to clear once infected. in closed colony settings, dermatophyte testing by culture is highly recommended unless the source of the cat excludes the possibility of infection (e.g., specific pathogen-free [spf] cats, purposebred laboratory cats). to screen cats using cultures, may take as long as days following exposure for a cat to test positive. , in the context of animal shelters, testing decisions are often influenced by the availability of resources. the aafp's guidelines include recommendations specifically for shelters. they state that all cats should ideally be tested at the time of entry and again in days in case of recent exposure. when cats test positive on screening tests (e.g., point-of-care enzyme-linked immunosorbent assay [elisa] tests), the aafp recommends that the results be confirmed by additional testing, including testing over an interval of time, because false positives can occur. however, such confirmatory testing requires substantial time and monetary investment and may not be feasible in many shelters. in recognition of this, the association of shelter veterinarians established a policy statement on "management of cats who test positive for felv and fiv in an animal shelter," which states that the logistics and cost of holding and retesting unowned cats may be an ineffective use of resources. in addition, it can be difficult to find homes for retroviruspositive cats, which in many instances translates into stressful, prolonged shelter stays. such long-term confinement may compromise quality of life and may compound the emotional stress of caregivers who may later be faced with euthanizing cats that have been held for long periods awaiting confirmatory testing or adoption opportunities. for all of these reasons, many shelters elect to euthanize cats that test positive on retrovirus screening tests. although it may be ideal for shelters to test cats on entry, it is not always feasible because of financial constraints. the next best practice might be to test cats prior to adoption as well as those that are housed in the shelter long term. in addition, cats should be tested prior to placement in group housing with unfamiliar cats and prior to investment, such as foster care, treatment, or spay/neuter surgery. however, given the limited resources of many shelters, the relatively low prevalence in healthy cats and the fact that transmission can be prevented by housing cats separately, it may not be cost effective for all shelters to screen every cat before selection for adoption. each shelter should evaluate its own resources and determine their best use. when testing is performed, samples must never be pooled, and the negative results of one cat (such as a mother cat) should not be extrapolated to other cats (such as her kittens). these practices are invalid and can falsely lead to misidentification of a cat's true infection status. , if testing is not performed prior to adoption, adopters should be advised to have their new pet tested and to keep them separate from any other cats they may own prior to doing so. point-of-care heartworm tests for cats have recently become more widely available, but interpreting results vaccination protocols are typically applied uniformly to all of the individuals comprising the population. this simplifies their application and helps to afford the best possible protection for the group. detailed vaccination records should be maintained for each cat, including vaccine name, manufacturer and serial number, date, the initials of the person who administered it, and any adverse reactions. proper vaccination can substantially reduce disease in cat populations, and serious adverse reactions are relatively rare. for this reason, vaccination against certain core diseases is recommended in all population settings. although exclusion of infectious disease is always a goal of health management, certain pathogens are so widespread that even with careful biosecurity in a closed population, an infection may be introduced to susceptible cats. only in the case of specific pathogen-free colonies, where there may be a compelling reason not to vaccinate as dictated by the purposes of the research, should vaccination be foregone. the aafp maintains published guidelines for vaccination of cats in a variety of settings and includes detailed recommendations for cats in animal shelters. although many vaccines are commercially available for cats, only a few are recommended for routine use in populations. unnecessary use of vaccines should be avoided to minimize the incidence of adverse reactions and reduce cost. core vaccines involve diseases that represent significant morbidity and mortality and for which vaccination has been demonstrated to provide relatively good protection against disease. core vaccines for cats in a population setting include feline parvovirus (fpv or panleukopenia), fhv- (feline herpes virus type or feline rhinotracheitis virus), and feline calicivirus (fcv). these vaccines are usually given in a combination product commonly referred to as an fvrcp vaccine (feline viral rhinotracheitis, calicivirus, panleukopenia). in most cases, timely vaccination against panleukopenia will prevent the development of clinical disease. in contrast, vaccination against the respiratory viruses (fhv- and fcv) does not always prevent disease. in many instances, it affords only partial protection, lessening the severity of clinical signs but not preventing infection. to optimize response, modified live vaccines (mlv) should be used in most cases, because they evoke a more rapid and robust immune response and are better at overcoming maternal antibody interference than killed products. this is especially important in multicat environments in which the risk of infection is high, such as animal shelters, foster homes, as well as any population setting where upper respiratory disease is endemic. a samples should be collected using the mckenzie toothbrush method, where a new toothbrush is used to brush the cat's entire body, giving special attention to the face, ears, and limbs. in addition, if skin lesions are present, hair should be plucked around these areas for culture as well. campylobacter, salmonella, giardia, coccidia, tritrichomonas, and other gastrointestinal parasites and pathogens are common in some cattery situations and can be very difficult to eliminate once they are introduced. in fact, in some settings, these pathogens may become endemic and nearly impossible to eliminate. treatment of coccidia in shelter kittens is described in although clinical signs, such as diarrhea, may be associated with infection, some cats remain asymptomatic. these pathogens have the potential for high morbidity in a population (especially in young kittens), and some possess zoonotic potential. therefore routine fecal examinations, cultures, and/or empirical treatments should be considered prior to the introduction of new cats. it is well recognized that vaccination plays a vital role in the prevention and control of infectious diseases. protocols should be established in the context of the population's exposure risk, which will vary depending upon the setting. in the context of population medicine, ponazuril is a metabolite of toltrazuril that has proven activity against coccidia.* because there is no approved product for use in cats, the equine product marquis oral paste ( % w/w ponazuril; bayer healthcare) may be dosed at mg/kg, po, once daily for to days. prophylactic treatment may be instituted in high-risk situations, such as young kittens in environments with documented infection. proper hygiene, including the use of disposable litter boxes and frequent removal of feces, is also necessary. oocysts survive in the environment and are not treated by routine disinfectants, such as bleach and quaternary ammonium compounds. with a history of upper respiratory infection) may benefit from vaccination prior to breeding to maximize passage of maternal antibody to their kittens. for pregnant cats in such environments, administration of mlv should be avoided, because the potential risk of injury to the developing kittens may outweigh the risk of infection in this case. vaccination of lactating queens should also be avoided in a low-risk environment. a series of vaccinations should be administered to kittens less than months of age to minimize the window of susceptibility to infection and ensure that a vaccine is received as soon as possible after maternal antibodies have decreased sufficiently to allow vaccine response. for kittens, vaccines should be administered every to weeks until they are weeks (e.g., months) of age or their permanent incisors have erupted. the minimum interval of weeks is recommended in high-risk settings to narrow the window of susceptibility as maternal antibody wanes. a vaccination interval of less than weeks is not recommended, because it may actually blunt the immune response from previous vaccination. in the case of an outbreak of panleukopenia, extending vaccination to months of age may be warranted to ensure than no animal remains susceptible. although the vast majority will respond by months of age, a few may fail to respond, while others are provided with a boost to enhance the immune response. just as in owned pets, booster vaccines are generally not required until year later for modified live vaccines but should ideally be administered once in to weeks whenever resources permit. this may be especially important for cats that were ill at the time of initial vaccination, as may be the case in an animal shelter. revaccination in long-term shelter facilities should follow the guidelines set forth for pets: boost at one year, then every years for fvrcp. vaccination against rabies virus is regarded as a core requirement for pet cats and is required by law in some jurisdictions. thus vaccination against rabies is recommended in the context of private catteries. in contrast, rabies vaccination may be considered optional in most closed laboratory settings, because the risk of exposure should be absent and legal requirements may not apply. in animal shelters, vaccination against rabies is not generally recommended at the time of admission, simply because there is no benefit in terms of disease prevention or public health. vaccination on admission will not provide protection against an infection acquired prior to entry, nor will it limit concern if a cat with an unknown health history bites someone soon after admission. rabies vaccination is recommended for cats prior to adoption when a veterinarian is available to administer it (or as otherwise legally prescribed by state laws). alternatively, rabies vaccination may be administered as single modified live fvrcp vaccine will usually afford protection to cats that are at least months of age. in contrast, killed products require a booster in to weeks to confer immunity, making their use largely ineffective in such environments. to ensure rapid protection against panleukopenia, injectable fvrcp vaccines are preferred, but intranasal vaccines may offer advantages for feline respiratory disease, because they have been shown to rapidly induce local immunity at the site of exposure. furthermore, intranasal vaccines may be better at overriding maternal antibody in young kittens. for this reason, they are often used to reduce the morbidity and severity of upper respiratory infection (uri) in preweaningage kittens. when intranasal vaccines are used in animal shelters, they should be used in combination with injectable fvrcp vaccines to ensure and optimize response against panleukopenia as well as the respiratory infections. ideally, all cats should receive a mlv fvrcp vaccine at least week prior to entering a population. in the context of an animal shelter setting, this is seldom feasible. vaccination immediately upon entry is the next best practice and can provide clinically significant protection for the majority of cats. if neither maternal antibody nor another cause of vaccine failure interferes, modified live vaccinations against panleukopenia will often confer protection against disease in only days. intranasal vaccines against respiratory infections, including fhv and fcv, typically provide partial protection within to days. , in animal shelters, all incoming cats and kittens weeks of age and older that can be safely handled should receive an injectable mlv fvrcp vaccine immediately upon entry. a delay of even a day or two significantly compromises the vaccine's ability to provide timely protection. even injured cats, those with medical conditions, and those that are pregnant or lactating should be vaccinated on entry, because vaccination will likely be effective and the small risk of adverse effects is outweighed by the high risk of disease exposure and infection in the shelter. when vaccination of all cats on entry is not financially feasible, the next best practice is to vaccinate all those that are deemed adoptable at the time of entry or that are likely to be in the shelter long term. whenever possible, vaccinated cats should be separated from those that will remain unvaccinated (e.g., those that will be euthanized following a brief holding period) as soon as that determination can be made. in contrast, in lower-risk settings, ensuring that cats are in good health prior to vaccination should be a priority. vaccination of kittens with injectable fvrcp vaccinations may be delayed to to weeks of age. however, when respiratory disease is endemic, administration of intranasal vaccines beginning at weeks of age may be beneficial. in breeding catteries, queens (especially those control and prevention of internal and external parasites represent another important component of a population wellness program. common products used for their management are described elsewhere in this book. of particular importance are roundworms and hookworms, common intestinal parasites with zoonotic potential (see chapter ) . although uncommon, the risk of human infection from contaminated environments is real and can result in organ damage, blindness, and skin infections. for this reason, the centers for disease control and prevention and the companion animal parasite council strongly advise routine administration of broad-spectrum anthelminthics for their control. , pyrantel pamoate is one of the most costeffective and efficacious drugs for treatment and control of roundworms and hookworms. in both shelter and cattery settings, the author recommends administration of pyrantel pamoate at a dosage of mg/kg to all cats with re-treatment in weeks and then at monthly intervals. in shelters, if it is not possible to treat all cats at the time of entry, at a minimum, all cats that are deemed adoptable should be treated as soon as possible. in addition, kittens should be treated at -week intervals until months of age. for cats with diarrhea, fecal examination (e.g., flotation or centrifugation, direct fecal smear and cytology) should be performed with treatment according to results. even if results are negative, the administration of broad-spectrum anthelminthics should be strongly considered. in animal shelters, ectoparasites, particularly ear mites and fleas, are also very common in cats and kittens. shelter staff should be trained to recognize infestation and protocols should be established for treatment. in terms of shelter treatment protocols, the author recommends treating ear mites with ivermectin, because it is highly efficacious and costs only pennies per dose. the recommended dosage is . mg/kg subcutaneously. for fleas, the author recommends topical treatment with fipronil (frontline, merial, duluth, ga.) as a spray or top spot. in particular, the spray is very cost effective. it is safe for use in cats of all ages, including pregnant and nursing mothers and neonatal kittens. in addition, fipronil also has activity against ear mites, cheyetiella, chewing lice, and ticks. , spaying and neutering is another important consideration in the context of population wellness. reproductive stress from estrous cycling in queens and sex drive in tomcats can decrease appetite, increase urine spraying/ marking and intermale fighting, and profoundly increase social and emotional stress in the group. for these reasons, spaying and neutering cats that will not be used soon as possible following adoption. the latter may encourage new owners to establish a relationship with a private veterinarian. rabies vaccination is warranted when cats are housed long term in shelter facilities. in addition, if individual cats must be held for bite quarantines, they should be vaccinated against rabies in accordance with the current compendium of animal rabies prevention and control. noncore vaccines include those that may offer protection against disease, but because the disease in question is not widespread or only poses a risk of exposure in certain circumstances, vaccination is only recommended based on the individual risk assessment of a population of animals. noncore vaccines include felv, fiv, chlamydophila, and bordetella. vaccination against felv is not warranted in a closed population of cats in which there is no risk of exposure (e.g., most laboratory animal settings). in private catteries, a risk assessment should be done to determine if vaccination is warranted (e.g., cats permitted in outdoor enclosures, frequent introduction of cats from external sources, other opportunities for exposure). special consideration should be given to vaccinating kittens because of their high susceptibility to felv infection and the high likelihood that they will become persistently infected if exposed. in general, felv vaccination is not recommended in animal shelters when cats are housed short term. however, its use is warranted when cats are group housed when resources permit. fiv vaccination is not generally recommended in population environments. a confounding feature of fiv vaccination is that vaccinated cats develop false-positive test results on most commercially available tests (see chapter ) . if fiv vaccination is elected, vaccinated cats should be permanently identified (e.g., by use of a microchip) to help clarify their status. chlamydophila felis (c. psittaci) and bordetella bronchiseptica vaccines may be of benefit when clinical signs of these diseases are present in the population and diagnosis is confirmed by laboratory evaluation. their efficacy is moderate, and reactions are more common than with most other feline vaccines; therefore ongoing use should be periodically reassessed. some vaccines are not generally recommended for use because of undemonstrated efficacy, such as the feline infectious peritonitis (fip) vaccine. wants whenever he or she chooses. dry food is used for this method of feeding, because canned products left at room temperature are prone to spoiling. the major advantage of free choice feeding is that it is quick and easy: caregivers simply need to ensure that fresh dry food is always available. major disadvantages include the fact that cats that are not eating may remain unrecognized for several days, especially when more than one animal is fed together, and some cats may choose to continually overeat and become obese. free choice feeding is an excellent method for cats that require frequent food consumption. these include kittens up to to months of age, queens in late gestation, and those that are nursing. unlike dogs, who are competitive eaters by nature, free choice feeding may benefit cats that are group housed, because it ensures that there will be ample time for all members to eat, provided that dominant members of the colony do not block the access of subordinate cats. meal feeding using controlled portions of dry and/or canned food may be done as an alternative to or in conjunction with free choice feeding. when used alone, a minimum of two meals should be fed per day. meal feeding is ideal for any cat that requires controlled food intake and facilitates monitoring of appetite. meal feeding also has the benefit of enhancing caregiver-cat bonding and provides a pleasant and predictable experience for cats when done on a regular daily schedule. using a combination of free choice plus once daily meal feeding takes advantage of the positive aspects of both methods and works well for most cats in a population setting. typically, dry food is available free choice, and a small meal of canned food is offered once daily. this combination approach accommodates the normal feeding behavior of cats by allowing them to eat several smaller meals throughout the day while allowing caregivers to monitor the cat's appetite at least for the canned food meal. as necessary for the individual cat, some may be fed additional meals of canned food to ensure adequate nutritional support. good body weight and condition and a healthy hair coat are evidence of an adequate nutritional plane and proper nutritional management. both appetite and stool quality should be monitored daily. normal stools should be well-formed and medium to dark brown. adult cats typically defecate once daily, although healthy adults may defecate anywhere between twice a day and twice a week. kittens tend to produce a larger volume of stool more frequently, which is often lighter in color and softer in form than that of adults. simple scales can be used for monitoring appetite (e.g., good, some, none), and fecal scoring charts are available. the author recommends the purina fecal scoring system chart available from nestlé purina petcare company (figure - ) . for breeding is recommended. in animal shelters, spaying and neutering cats prior to adoption will ensure that they do not reproduce and contribute to the surplus of community cats. this will also serve to enhance husbandry, because the procedures rapidly decrease spraying, marking, and fighting; eliminate heat behavior and pregnancy; and greatly mitigate stress. in addition to reducing stress and odor, spaying and neutering sexually mature cats will facilitate group housing, which is often beneficial for cats, especially when housed longer term (see below). the medical benefits of spay/neuter have also been well described, including dramatic reductions in the risk of mammary carcinoma, elimination of cystic endometrial hyperplasia, pyometra and ovarian cancer in queens, and decreased risk of prostate disease in toms. thus spaying and neutering favors both individual as well as population health. proper nutrition has a profound impact on wellness. not only is it essential for management of healthy body weight and condition, good nutrition is also known to support immune function. a regular diet of palatable commercial food consistent with life stage should be offered, and fresh water must always be available. although some cats tolerate changes in food without apparent problems, it is important to recognize that for others, changing from one diet to another can cause loss of appetite and/or gastrointestinal upset. for this reason, it is generally best to provide the most consistent diet possible. whereas this may be relatively easy to do in a laboratory or cattery setting, it can be more challenging in a shelter environment. some pet food companies offer feeding programs for animal shelters, providing a consistent food for purchase at a special rate for shelters. however, some shelters rely heavily on donations of food. in this case, by requesting donation of certain brands of food, shelters are able to provide a consistent diet whenever possible. it is also feasible to mix donated foods with the shelter's usual diet to minimize problems caused by abrupt diet changes while taking advantage of other donated products. the wild ancestors of domestic cats hunted to eat, feeding up to times in a -hour period. this style of feeding behavior is preferred by many domestic cats that would nibble throughout the day and night, consuming many small meals if left to their own devices. although this is true, most cats are capable of adapting to either free choice or meal feeding as their daily feeding pattern. , there are advantages and disadvantages to each in a population setting. with free choice or ad libitum feeding, food is always available such that a cat can eat as much as he or she trends in body weight, because both weight loss and gain can compromise health and well-being. appropriate grooming is also essential to ensure wellness and must never be considered as optional or purely cosmetic. most cats require minimal grooming because of their fastidious nature. however, long-haired cats are notable exceptions, often experiencing matting of the hair coat without regular grooming sessions. matted hair coats are not only uncomfortable for the animal, but may lead to skin infection. overgrown nails can also be a problem for some cats, particularly those that are geriatric or polydactyl. the provision of appropriate surfaces for scratching will encourage cats to condition their own claws; and a system for regular inspection of the hair coat and nails should be established. in addition to ensuring proper coat and nail maintenance, regular grooming sessions provide an excellent opportunity to monitor body condition; and some cats enjoy the physical contact and attention. in high-risk settings, the use of stainless steel combs or undercoat rakes that can be readily disinfected are generally preferable to the use of in addition to appetite and stool quality, it is essential to monitor body weight and condition. body condition can be subjectively assessed by a process called body condition scoring, which involves assessing fat stores and, to a lesser extent, muscle mass. fat cover is evaluated over the ribs, down the top line, tail base, and along the ventral abdomen and inguinal (groin) areas. body condition score charts have been established on scales of to and to . the author recommends use of the purina body condition score chart which is based on a scale of to with being emaciated and being severely obese (see figure - ). cats should be weighed and their body condition scored at routine intervals. ideally, body weight should be recorded at entry to the population and then weekly during the initial month of care, after which it could be recorded once a month or more often as indicated based on the individual's condition. this is especially important for cats, because significant or even dramatic weight loss may be associated with stress or illness during the first few weeks of confinement in a new setting. on the other hand, in long-term-housed cats, excessive weight gain may occur in some individuals. therefore protocols must be in place to identify and manage unhealthy score -very moist (soggy); distinct log shape visible; leaves residue and loses form when picked up. score -very moist but has distinct shape; present in piles rather than as distinct logs; leaves residue and loses form when picked up. score -has texture, but no defined shape; occurs as piles or as spots; leaves residue when picked up. score -watery, no texture, flat; occurs as puddles. score -firm, but not hard; should be pliable; segmented appearance; little or no residue left on ground when picked up. score -log-like; little or no segmentation visible; moist surface; leaves residue, but holds form when picked up. fecal scoring system bristle brushes because the latter are impossible to disinfect and have the potential to spread common skin infections such as ringworm. dental health is another component of wellness. in the context of population wellness, it may not be the highest priority; however, it should always be a consideration in terms of individual health care and well-being. this is important because periodontal disease will occur unless it is actively prevented, and plaque and tartar buildup may contribute to serious health concerns, ranging from oral pain to chronic intermittent bacteremia and organ failure. feline tooth resorption and gingivostomatitis are also common conditions of the feline oral cavity that can lead to chronic pain, affecting the cat's appetite and ability to self-groom, and negatively impacting quality of life. when painful dental disease is present, a plan for timely treatment should be identified and implemented. preventive dental care may include tooth brushing, dental-friendly diets, and treats and chew toys in combination with periodic professional dental care. these should be tailored to meet the needs of individuals in the population to optimize dental health. cats with stomatitis should be removed from breeding programs. wellness protocols may also be dictated by the specific needs of certain breeds of cats. for example, persian, himalayan, and other brachycephalic cats are predisposed to respiratory disease and tend to be more severely affected than other cats because of their poor airway conformation. because of the high likelihood of exposure in a shelter setting, these cats should be housed in highly biosecure areas that are well ventilated and should be prioritized for immediate adoption or transfer to foster care or rescue. in the author's experience, even intranasal vaccination of these breeds can result in severe clinical signs of respiratory disease and is best avoided. just as a physical wellness program must be tailored to the population in question, a behavioral wellness program, composed of all of the essential elements, should be created to meet its specific needs as well. even when animals will only be housed for short periods, considerations for behavioral care are essential to ensure humane care. short-term confinement can induce severe stress and anxiety, and when confined long term, cats may suffer from social isolation, inadequate mental stimulation, and lack of exercise. a behavioral wellness program should strive to decrease stress from the moment cats arrive at a facility until the moment that their stay ends. as previously described, a thorough behavioral history will provide an important baseline for action and follow-up. understanding the importance of minimizing stress in cats and possessing the ability to recognize and respond to it are essential to facilitate a cat's transition into a population. , staff should be trained to evaluate cats beginning at intake and to recognize and respond to indicators of stress. active daily monitoring of cats for signs of stress or adjustment should be performed, and staff should record their findings daily, noting trends and making adjustments in the care of individual cats and the population as indicated. in animal shelter environments, proper behavioral care of cats also requires an understanding of the wide spectrum of feline lifestyles and an approach tailored to the individual needs of each group. domestic cat lifestyles and levels of tractability range from the most docile, sociable housecat, to free-roaming strays and truly unsocialized feral cats that will not allow handling. stray cats include those that may have been previously owned or are "loosely owned" neighborhood or barn cats. because of their lack of socialization, capture, handling, and confinement are especially stressful for feral cats. however, fearful cats may resort to overt aggressive or may "teeter on the edge" of defensive aggression regardless of their socialization status. in fact, even the tamest house cats may exhibit the same behaviors as feral cats when they are highly stressed (figure - ) . , these responses can compromise cat welfare and staff safety and hinder adaptation to a new environment. regardless of their demeanor, all cats and kittens should be provided with a hiding box in their enclosure at the time of entry, because the ability to hide has been shown to substantially reduce feline stress. for those cats that are severely stressed or reactive, covering the cage front, in addition to providing a hiding box, and posting signage to allow the cat "chill out" time for several hours or even a few days can facilitate adaptation. this is important because, once highly stressed or provoked, cats often remain reactive for a prolonged time and may become more reactive if they are stimulated again before they have been allowed a period of time to calm down. soft bedding should be available for comfort and so that cats may establish a familiar scent, which aides in acclimation to a new environment. care should be taken during cleaning procedures to minimize stress and noise, behavioral evaluation may be useful, especially for cats that will be re-homed. several evaluations have been recommended, but none are scientifically validated for predicting future behavior with certainty. , , nonetheless, this form of evaluation may be useful for determining behavioral needs while cats remain in a facility, as well as guiding appropriate placement. box - describes common components of a feline behavioral evaluation (figure - ) . housing design and operation can literally make or break the health of a population. regardless of the species in question, housing should always include a comfortable resting area and allow animals to engage in species-typical behaviors while ensuring freedom from fear and distress. it is not sufficient for the design to address only an animal's physical needs (e.g., shelter, warmth). it must meet their behavioral needs as well, and both the structural and social environment are essential considerations for housing arrangements. furthermore, the environment must provide opportunities for both physical and mental stimulation, which become increasingly important as length of stay increases. a sense of control over conditions is well recognized as one of the most critical needs for behavioral health. thus housing design must provide cats with a variety of satisfying behavioral options. specifically, housing arrangements must take into account the following feline behavioral needs : • opportunities for social interactions with humans and/or other compatible cats and cats should be allowed to hide while their cage is quietly tidied and replenished around them as needed. commercially available "cat dens" are ideal for this purpose, because they can be secured from a safe distance such that the cat is closed inside a secure, familiar hiding place during cleaning procedures (figure - ) . cats should be returned to the same cage and only spot cleaning should be performed to preserve their scent, which is necessary for stress reduction. if it becomes necessary to house the cat in another location, the den and towel should accompany the cat to ease the transition. finally, the use of commercially available synthetic analogues of naturally occurring feline facial pheromones (feliway, veterinary product laboratories, phoenix, ariz.) have been shown to be useful for stress reduction in cats during acclimation to new environments and can be sprayed onto bedding and allowed to dry prior to use or dispersed in the room using plug-in diffusers. the way in which cats are handled at intake has a profound impact on their behavior, health, and wellbeing and will impact the cat's ability to adapt to its new environment. when stress is successfully mitigated, cats are more likely to adapt and to "show their true colors" rather than reacting defensively. during a period of a few days, many cats that did not appear to be "friendly" at intake will become tractable and responsive to their human caregivers, facilitating care. aside from informally "getting to know" cats during their initial acclimation period in a facility, a systematic • the ability to create different functional areas in the living environments for elimination, resting, and eating • the ability to hide in a secure place • the ability to rest/sleep without being disturbed • the ability to change locations within the environment, including using vertical space for perching • the ability to regulate body temperature by moving to warmer or cooler surfaces in the environment • the ability to scratch (which is necessary for claw health and stretching, as well as visual and scent marking) • the ability to play and exercise at will • the ability to acquire mental stimulation because these needs will vary depending upon such factors as life stage, personality, and prior socialization and experience, facilities should maintain a variety of housing styles in order to meet the individual needs of different cats in the population (figure - ) . managing housing arrangements for a population of cats of varying ages, genders, personality types, social experiences, and stress levels requires knowledge of normal feline social behavior and communication. during the past decades, knowledge of feline social structure has evolved from the widespread belief that cats are generally an asocial and solitary species to the realization that they are social creatures. , with the exception of solitary hunting, free-roaming cats perform responses are observed and recorded for each of the following: • the tester approaches cage, stands quietly for seconds, then offers verbal encouragement. • if deemed safe to proceed, tester opens the cage door and calmly extends an open hand towards the cat, then attempts to gently touch the cat's head. • if the caregiver is unsure if this is safe to do, a plastic hand may be used to gauge the cat's receptiveness to touch (see figure - ). • if the cat allows handling, the cat is gently lifted and carried to a secure, quiet room for further observation. • the tester sits quietly on a chair and/or the floor; the tester calls and solicits the cat's attention. • the tester pets the cat on the head. • the tester strokes the cat down the back several times. • the tester picks up the cat and hugs it for seconds. • with the cat standing on the floor, the tester strokes the cat down the back and firmly but gently grasps the base of the tail and lifts the cat off of its hind feet for second. the tester repeats this a second time. • the tester engages the cat in play with an interactive toy. in some instances, it is difficult to determine if a cat will accept handling. to prevent injury to staff, a plastic hand (assess-a-hands; great dog productions, accord, ny) is used to approach this cat. as the hand approaches, the cat appears tense (a) but begins to relax and accepts petting (b and c) . the absence of normal behaviors (e.g., grooming, eating, sleeping, eliminating, stretching, greeting people). defensive behavior may involve characteristic postural and/or vocal responses, and is usually motivated by fear. disruptive behavior involves destruction of cage contents and creation of a hiding place. stereotypic behaviors (e.g., repetitive pacing, pawing, and circling) may also develop as a result of stress but generally occur less commonly. as an illustration of these feline behaviors, consider the responses of a typical social domestic cat when caged in a novel environment (box - and figures - to - ) . behavioral signs of stress may be further classified as active communication signals or passive behaviors. , signals of anxiety, fear, aggression, and submission may be subtle or obvious and include vocalization (growling, hissing), visual cues (facial expression, posturing of the body, ears, and tail), and scent marking (urine, feces, various glands of the skin). passive signs of stress include the inability to rest/ sleep, feigned sleep, poor appetite, constant hiding, absence of grooming, activity depression (decreased play and exploratory behavior), and social withdrawal. high-density housing exacerbates these signs. lowsocial-order cats in such an environment may exhibit decreased grooming, poor appetite, and silent estrus. cats that are consistently fearful or anxious may hide, most of their activities within stable social groups where cooperative defense, cooperative care of young, and a variety of affiliative behaviors are practiced. affiliative behaviors are those that facilitate close proximity or contact. cats within groups commonly practice mutual grooming and allorubbing (e.g., rubbing heads and faces together). this may serve as a greeting or as an exchange of odor for recognition, familiarization, marking, or development of a communal scent. cats of both genders and all ages may exhibit affiliative behaviors, and bonded housemates often spend a large proportion of their time in close proximity to one another. maternal behavior is the primary social pattern of the female cat, and cooperative nursing and kitten care are common. if allowed, queens form social groups along with their kittens and juvenile offspring. , tomcats typically reside within one group or roam between a few established groups. within groups of cats, a social hierarchy or "pecking order" forms. , once established, this hierarchy helps to support peaceful co-existence of cats within a stable group, minimizing agonistic behaviors between members. social hierarchy formation occurs within groups of cats that are sexually intact, as well as in those that are neutered. knowledge of behavioral signaling is critical for successful management of housing arrangements. manifestations of both normal and abnormal behavior indicate how successfully an animal is coping with its environment. common behavioral expressions of feline anxiety may manifest with inhibited or withdrawal behavior, defensive behavior or disruptive behavior. , inhibited or withdrawal behavior refers to activity depression or • fear is typically the initial response, and if threatened by the proximity of unfamiliar caregivers, defensive aggression may be displayed. alternatively, the cat may freeze or appear catatonic. • if provided with a box for concealment, the cat will hide or otherwise slink against the back of the enclosure, behind the litter box, or disrupt the cage and hide under the paper. • given time, most cats become more active and engage in greeting behaviors, coming to the front of the cage and pawing or mewing as caregivers approach. • if the cat remains confined with time without adequate periods of exercise, mental stimulation, and social companionship, stress and frustration will manifest with activity depression and withdrawal (lying in the litter box, failure to groom, failure to greet caregivers, and, in some cases, displaying aggression towards caregivers). • displays of stereotypic behavior (such as pacing) may occur; however, inhibited or withdrawal behaviors are much more common (see unfamiliar or new cats entering the group. within an established group, however, most social conflicts are not characterized by overt aggression. instead, the main mode of conflict resolution is avoidance or deference (figure - ) . , , deference behaviors include looking away, lowering the ears slightly, turning the head away, and leaning backward. large numbers of cats peacefully co-exist together, using such strategies for avoidance provided ample space and resources are available for all members of the group. signs of social stress within groups of cats may manifest with overt aggression, increased spraying and turn their back, huddle, and avert their eyes from the gaze of other cats. hiding is a normal and important coping behavior; however, when hiding is occurring with increased frequency or in response to stimuli that did not previously cause hiding, it should be recognized as a sign of stress. , in group settings, the complexity of the social structure cannot be overestimated. the internal structure of social groups rarely represents a straightforward linear hierarchy, except in very small groups of less than four to five animals. in larger groups of cats, there are usually one or two top-ranking individuals and one or two obvious subordinates, while the remaining cats share the middle space. , most cats within the group form affiliative or friendly relationships; however, some may fail to form such relationships and remain solitary. colony members commonly display aggression toward c marking, or constant hiding. , lower-ranking cats may spend little time on the floor, remaining isolated on single perches or other locations where they may even eliminate, while higher-ranking cats remain more mobile, controlling access to food, water, and litter resources. high-density housing conditions frequently result in such abnormal behaviors and are associated with increases in transmission of infectious diseases and reproductive failure as well. cats are commonly housed in three basic arrangements: cage or condo units, multiple runs within a room, or free ranging in a room. cage housing of cats should be avoided unless necessary for short periods for intake observation, legal holding periods in shelters as required by local ordinances, medical treatment or recovery, or to permit sample collection. although space recommendations vary substantially in the literature, common sense dictates that a determination of necessary housing space should take into account the cat's length of stay. in the author's opinion, it is neither appropriate nor humane to house cats in traditional cage housing long term (e.g., more than to weeks). the design of short-term housing should include provisions for housing individual animals, litters, families, or bonded housemates for intake evaluation and triage. housing must be easy to clean and sanitize, well ventilated, and safe for animals and caregivers. short-term housing should provide sufficient space to comfortably stand, stretch, and walk several steps; sit or lay at full body length; and separate elimination, feeding, and resting areas. litter boxes should be of appropriate size to comfortably accommodate the cats for which they are intended ( figure - ) . resting areas should include comfortable surfaces, soft bedding, and a secure hiding place to provide a safe refuge. a hiding place is essential, because it reduces stress by allowing cats to "escape," facilitating adaptation to a new environment. the addition of a sturdy box to a cage will provide a hiding place as well as a perch (figures - and - ) . in addition, cages should be elevated off of the floor by at least . m ( . feet), because this serves to reduce stress as well. in most instances, cage or condo style housing is used in most facilities for short-term holding at intake for observation, acclimation, and/or triage. runs or small rooms are also appropriate for intake housing, and offer cats the obvious benefit of additional space to meet their behavioral needs (figure - ) . regardless of their configuration, enclosures for short-term housing of cats should be large enough to allow them to stretch, groom, and move about while maintaining separate functional areas, at least . m ( feet) apart, for sleeping, eating, and elimination. , , laboratory guidelines in the united states call for a minimum floor area of . m ( ft ) for cats weighing less than kg and . m ( ft ) for cats weighing kg or more, with a minimum height . m ( ft). a resting upper respiratory infections. double-sided enclosures (e.g., cat condos) are ideal for meeting these specifications and have the benefit of easily allowing cats to remain securely in one side of the enclosure while the opposite side is cleaned (figure - ) . this helps to minimize stress, prevent exposure to infectious disease, perch is also required. current guidelines (european convention for the protection of vertebrate animals used for experimental and other scientific purposes, ets ) promulgated by the council of europe (http:// www.coe.int) for laboratory cats are similar, but proposed revisions call for substantially more floor space for cats, at . m ( . ft ) per adult cat with a height of at least m ( . ft). , the revisions, which have not been approved to date, also call for the provision of shelves, a box-style bed, and a vertical scratching surface. animal shelter facilities in the united states have traditionally been equipped with small perchless cages (e.g., . to . m or . to . ft long) that are poorly designed for housing cats. the association of shelter veterinarians (http://www.sheltervet.org) recommends a minimum enclosure size of m ( ft ) for adult cats. commercially available cages are typically approximately . m ( . ft) deep (e.g., an arm-length deep so that they can be readily accessed); therefore a cage with a length of . m ( ft) is required to provide this approximate square footage, and it will also allow for adequate separation of food, water, and litter ( figure - ) . similarly, the cat fanciers' association (http:// www.cfa.org) recommends a minimum of . m ( ft ) of space per cat for those weighing kg or more. cubic measurements take into account the use of vertical space in addition to floor space, which is crucial for improving the quality of the environment. for example, a . -m ( - larger enclosures also allow for better air circulation, which is an important consideration for control of feline housing for a single cat. note the large -ft long cage, provision of a secure hiding place and perch with bed, separation of litter from resting and feeding areas, and appropriately sized litter box for this large cat. housing units are available for cats and serve to separate functional living areas and provide improved opportunities for exercise and exploration. this unit (tristar metals, boyd, tex.) is constructed of powder-coated stainless steel, which is highly durable and easy to disinfect but less noisy than uncoated stainless steel. note the elevation from the floor and the grills on both the front and back, which allows flow-through ventilation. is both mentally and physically stimulating for cats and preferably that which is esthetically pleasing to humans. the latter is an important consideration to facilitate adoption in animal shelters. and, even in other types of facilities, it is important to create a pleasant environment not only for the animals, but also for their caregivers. studies indicate that employee satisfaction improves animal care and staff retention, both of which may positively impact population health and well-being. for long-term housing of cats alternatives to traditional cage housing should be afforded. , , at an absolute minimum, cats that are cage housed must be released each day and allowed an opportunity to exercise and explore in a secure enriched setting. for long-term housing, most cats will benefit from colony-style housing, provided there is sufficient space, easy access to feeding and elimination areas, an adequate number of comfortable hiding, and resting places and careful grouping and monitoring to ensure social compatibility among cats. not every cat, however, will thrive in a group setting, and certain individuals will require enriched single housing, depending on their unique physical or behavioral needs. these may include cats that bully other cats or are otherwise incompatible and those with special medical needs. it is important to recognize that such singly housed cats will require more regular contact with their human caregivers and higher levels of mental and physical stimulation in order to maintain behavioral health during long-term confinement. whenever possible, long-term housing of such individuals should be avoided. when cats are housed in amicable groups, it is easier to maintain proper behavioral welfare in the long term, because many of their social and emotional needs can be met by conspecifics. group housing affords cats with opportunities for healthy social contact with others, which, in turn, provides additional mental and physical stimulation. when properly managed, this housing arrangement enhances welfare.* insufficient space and crowding or poor compatibility matching of cats serves to increase stress and negates the benefits of the colony environment. group housing should never be used as a means of simply expanding the holding capacity of a facility. in animal shelters, the high turnover rate of cats contributes substantially to feline stress levels, especially in the context of groups of unfamiliar animals. because it may take days to weeks to acclimate to a group environment, enriched individual housing may be preferable when a brief stay is anticipated. however, the benefits of enriched social group housing become evident when stays extend beyond a few weeks. and preserve staff safety, which are especially crucial for newly arrived cats. traditional cages can be modified into condo-style enclosures by creating portals to adjoin two or three smaller cages (figure - ) . regardless of the precise specifications of the enclosures, the importance of the overall quality of the living environment cannot be overemphasized. this includes a holistic approach to husbandry, with careful attention to the way in which cats are handled, noise levels, the provision of creature comforts, positive contact with caregivers, and strict avoidance of overcrowding, as well as good sanitation, medical protocols, and careful monitoring to ensure health and welfare. for long-term housing (e.g., greater than weeks), consideration should also be given to providing space that a b *references , , , , , , , , , , . breeding age should be avoided whenever possible. at a minimum, mature tomcats should be neutered to prevent intermale aggression, urine spraying, and breeding. reproductively intact females may be co-housed with other intact females or with neutered males. in contrast, in breeding colonies, harem-style housing may be used to facilitate breeding (e.g., a few queens with a tomcat). it is also advantageous to house compatible pregnant queens together before delivery, because they will usually share nursing and neonatal care ( figure - ) . after delivery, pairing of queens becomes more difficult. when tomcats are not breeding, they can usually be co-housed with a spayed female, a neutered male, or a compatible juvenile for companionship. other recommended groupings in the context of a breeding colony include postweaning family groups, prepubertal juveniles, or compatible single-sex adults. personality type there are two basic feline personality types: cats that are outgoing, confident, and sociable and those that are relatively timid and shy. cats with bold, friendly temperaments tend to cope and adapt more readily than shy, timid cats. a subset of the bold, friendly personality type is the "assertive" or "bully" cat. bully cats constantly threaten other cats in a group setting in order to control access to food, litter, perches, or the attention of human caregivers. to maintain harmony, removing cats of this personality type from a colony is usually necessary. reassignment is possible, but may prove difficult, necessitating single housing. shy, timid cats sometimes have difficulty interacting successfully with more dominant members of a group or may fall victim to a bully, resulting in chronic stress and increased hiding. placement of shy cats in smaller groups or with calm juvenile cats, where they will not be intimidated or harassed, is generally rewarding and often helps them to "come out of their shells." , similarly, dominant cats will often accept calm, younger cats, as opposed to other adults by whom they may feel threatened. and finally, in the case of some dominant males, the introduction of a female cat will be more likely to be successful. , , the precise space requirements for long-term housing of cats will vary, because it is dependent on many factors (box - ). , of paramount importance is that group size must be small enough to prevent negative interactions among cats and to permit daily monitoring of individuals. cats typically prevent social conflict through avoidance, and adequate space must be available so that cats can maintain social distance as needed. crowding can make it impossible for animals to maintain healthy behavioral distance, creating situations where individuals may not be able to freely access feeding, resting, or elimination space because of social conflicts over colony careful attention to groupings of cats is essential for success. family groups and previously bonded housemates are natural choices for co-housing, , but unfamiliar cats may also be grouped using careful selection criteria. many cats do have preferences for housemates, necessitating conscientious compatibility matching combined with the provision of a high-quality environment. groupings of unfamiliar cats should always be given priority for the largest available enclosures. in addition, cats should always receive appropriate health clearances prior to admission to a group. these should be determined by the specific protocols of the facility; but in most cases, minimum requirements would include that cats be free of signs of contagious disease, tested for felv and fiv, vaccinated against fvrcp, and treated for parasites. in addition to prior relationships, selection criteria for groupings should include age, reproductive status, and personality. age age is an important consideration regarding housing arrangements. to ensure proper social and emotional development, kittens should be housed with their mother at least until they are weaned. because it can be behaviorally beneficial, it is desirable for them to remain with her for a longer period of time when this is feasible. in fact, queens frequently do not fully wean their kittens until to weeks of age if left to their own devices. if older kittens are housed with their mother, it is important to provide a perch that allows her the option of periodically resting away from them if desired. most queens will accept the kittens of another cat; therefore young orphan or singleton kittens should be housed with other lactating queens and/or kittens of similar age/size. in a shelter setting where there is a high turnover of cats, it may be beneficial to house young kittens up to to months of age in large cages or condos for biosecurity purposes. juveniles and adults can be housed in colony rooms or runs but should be segregated by age (e.g., juveniles to months old, young adults, mature adults, geriatrics). well-socialized juveniles tend to adapt quickly in a group setting with other cats of similar ages and exhibit healthy activity and play behavior. in contrast, mature adults and geriatric cats often have little tolerance for the high energy and playful antics of many younger cats, which can cause them substantial stress. for this reason, adult cats should be kept separate from juvenile cats, and aging or geriatric cats separate from other age groups. in animal shelters, compatible cats that enter the shelter together should be housed together regardless of age, whenever possible. unless cats will be used for breeding, group housing of sexually intact cats of all of these reasons, housing cats in small groups is preferred. , , in most instances, the author recommends housing cats in compatible pairs or small groups of not more than three to four individuals. housing cats in runs is ideal for this purpose (figure - ) . a well-equipped, . -× . -m ( -× -ft) run can comfortably house two to three adult cats depending on their familiarity and compatibility, or up to four juveniles (e.g., to months old). juveniles tend to accept a slightly higher housing density than adults. likewise, previously bonded housemates and families will generally peacefully co-exist at a higher density than will unfamiliar cats. when runs are used, they must have a top panel and should be at least . m ( ft) high to allow caregivers easy access for cleaning and care. if chain-link is used, . -cm ( -inch) mesh is ideal, but larger mesh can be used. existing dog kennel runs can be converted into areas for cat housing. this is an important and practical consideration in animal shelters, because many shelters have experienced a decrease in dog intake, while the need for improved cat housing is great. cats and dogs should never be co-housed in the same area; thus conversion should result in an exclusive cat housing area. for colony rooms, the author recommends a minimum enclosure size of approximately to . m × to . m ( to ft × to ft) for colonies of up to a maximum of eight adult cats, or in the case of juveniles, a few more. doubling the size of an enclosure does not necessarily allow a twofold increase in the number of cats that can be properly housed. another author recommends . m ( ft ) per cat as a general guideline for group housing, resources. both crowding and constant introduction of new cats induce stress and must be avoided to ensure proper welfare. the addition of new cats always results in a period of stress for the group, and if there is constant turnover within the group, cats may remain stressed indefinitely. high turnover also increases the risk of infectious disease. if cat group numbers are small, disease exposure will be limited, facilitating control. for • length of stay • overall quality of the environment, including use of vertical space • overall quality of behavioral care • physical and behavioral characteristics of the cat (e.g., age, personality type, prior experience, and socialization) • individual relationships between cats (e.g., family groupings, previously bonded housemates, versus unfamiliar groupings and degree of social compatibility among cats) • turnover of cats (e.g., frequency of introduction of new members) • total room size • absolute number of cats • individual needs and levels of enrichment being used to meet these needs enabling caregivers to better monitor individual appetites and litter box results while allowing cats a period of rest away from one another. alternatively, individual enclosures may only be used for brief periods for meal feedings of canned food, with dry food available free choice in the colony. this sort of arrangement can also be used to facilitate introduction of new cats to the group and represents a desirable option. if design and biosecurity procedures permit, portable intake enclosures could even be transferred to group rooms to smooth the transition of new cats from intake to long-term housing areas. tremendous individual variation exists among cats in the context of social relations with other cats. although introduction of some previously unfamiliar cats will seem effortless and uneventful, introduction of others will result in considerable stress, not only for the new cat but for the entire group as well. for this reason, introductions should always be done under supervision, and whenever possible, they should be gradual. to accomplish this, a new cat can be kept in a separate cage within or adjacent to the group enclosure equipped with food, water, litter, and a hiding box. usually, within a few days, it will be evident by the behaviors of the cats whether or not the new cat can be transferred into the group enclosure without risk of fighting. wellsocialized kittens and juvenile cats frequently adapt readily to group accommodations, and prolonged introductions may not be necessary unless they are shy or undersocialized. in established groups of cats, the introduction or removal of individuals will require a period of adjustment and may result in signs of social stress for members of the colony. these signs usually subside once a new social hierarchy and territorial limits (usually favored resting places) are established. in some cases, arrangement of incompatible cats, even within visible distance of one another, may create substantial anxiety, necessitating rearrangement (figure - ) . in the case of animal shelters, where population interchange is high, it is generally not feasible to maintain consistent groupings of cats. this underscores the absolute necessity of careful selection and compatibility matching, as well as maintaining a variety of housing styles. even in modestly populated, carefully introduced, environmentally enriched colonies, behavior problems may occur. for this reason, some facilities elect to use an "all in-all out" approach to avoid repeated introductions of new cats into stable groups. in animal shelters, bonded pairs and family groupings of cats frequently enter the shelter together and are usually perfect choices for co-housing. because cats do have strong preferences for new roommates, caregivers must expect to find many that are incompatible as roommates. if only one or two cats are responsible for social destabilization of a group, they can acknowledging that many factors influence the spatial needs of cats, including the overall quality of the environment as well as the relationships of the individual animals. in sanctuary and laboratory situations where cats are housed for months to years in stable colonies, larger groupings of cats may be feasible, provided ample space is available. housing arrangements can also be created in which individual enclosures are maintained within a colony room. in this case, cats could be allowed to wander and interact freely in the colony room by day but be confined to their respective enclosures at night, for pair-housing of two adult cats. note the multiple separate areas for resting, perching, hiding, feeding, eliminating, scratching, and playing. b, cats enjoy the increased behavioral options provided by run-style housing. should exceed the number of cats and should be arranged in as many locations within the enclosure as possible. open single perches should be separated by at least . m ( ft) or staggered at different heights to ensure adequate separation, while larger perches should be available for cats who choose to rest together in close proximity. many cats enjoy hammock-style perches or semienclosed box-style perches where they can hide. if there are not enough comfortable, desirable resting and hiding places, cats may choose to lie in litter boxes. comfortable bedding (that is either disposable or can be easily laundered) should be provided. not only do cats demonstrate preferences for resting on soft surfaces, they experience longer periods of normal deep sleep with soft bedding. the environmental temperature should be kept comfortable and constant, and living quarters should be well ventilated, without drafts. by changing location within the colony (e.g., from the cooler surface of the floor to a sunny window), cats should be able to choose the environmental condition they prefer (figure - ) . in colony rooms, installation of stairs, shelves, and walkways are ideal for increasing the use of vertical space (figure - ) . in larger rooms, installation of freestanding towers provides additional living and activity space and contributes to functionally reducing overcrowding (figure - ) . depending on the setting, it may not be desirable for cats to access areas above the level of an arm's reach so that cleaning is easy and cats can be easily retrieved from the highest perches if needed. colony room design should also ensure that cats cannot easily escape. in some cases, constructing a foyer at the entrance to the room will be necessary to minimize the risk of escape when the room is entered (figure - ) . in addition, ceilings should be constructed of solid surfaces, because cats can easily dislodge the usually be reassigned to another colony, because it is often the social grouping, not the individual, that is the problem. if a cat shows persistent incompatibility with other cats, he or she should be housed singly. studies indicate that cats that fight at the time of initial introduction are nearly times more likely to continue fighting in the following weeks and months. if overt fighting occurs, cats should be permanently separated. cohousing of incompatible cats or cats that fight is unacceptable. the success of group housing depends not only on selection of compatible cats and the size of the enclosure but also on the quality of the environment.* a variety of elevated resting perches and hiding boxes should be provided to increase the size and complexity of the enclosure and to separate it into different functional areas, allowing a variety of behavioral choices. the physical environment should include opportunities for hiding, playing, scratching, climbing, resting, feeding, and eliminating. whenever possible, a minimum of litter box and food and water bowl should be provided per to cats and arranged in different locations of the colony space, taking care to separate food and water from litter by at least . m ( ft). in addition, placement should allow cats to access each resource from more than one side, whenever possible, without blocking access to doorways. litter boxes should not be covered, to allow easy access and to prevent entrapment or ambush by other cats. the number of resting boards and perches the importance of a cat-savvy staff that enjoys working with cats cannot be overemphasized. animal care staff must be willing to spend quality time interacting with cats to assure socialization and tractability. , whenever possible, caregivers should be assigned to care for the same cats on a regular basis so that they become aware of the personality of each individual cat, which is necessary for detection of health problems, incompatibilities between cats, and, in the case of breeding colonies, estrous cycling. this is also important, because not all cats uniformly enjoy human companionship and will be more likely to be stressed by the presence of different caregivers, rather than becoming familiar and more at ease with one. in general, regular daily contact and socialization is essential to ensure that cats are docile, easy to work with, and have no fear of humans. caregivers should schedule time each day to interact with "their" cats aside from the activities of feeding and cleaning. some cats may prefer to be petted and handled, while others prefer to interact with caregivers by playing with toys (figure - ) . in particular, human contact is essential for proper socialization of young kittens. a sensitive period of socialization occurs during the development of all infant animals, during which social attachments to members of the same species and other species form easily and rapidly. in kittens, the sensitive period of socialization occurs between and weeks of age, and cats not properly socialized to humans during this time may never permit handling. , beginning shortly after birth, kittens should be handled daily, talked to in a soothing panels typically used for dropped ceilings, and escape into the rafters (figure - ) . in addition to contact with conspecifics, cats must be afforded time for pleasant daily contact with human caregivers. as previously discussed, daily social contact and exercise sessions with humans are especially important for individually caged cats. although social contact is usually highly desirable, it is not invariably pleasant for all cats. personality, socialization, previous experience, and familiarity contribute to whether or not social interactions are perceived as pleasurable, stressful, or somewhere in between. , a b voice, gently petted, and held. interactions should include play (stimulated with toys) as the kittens become ambulatory. for kittens housed in a shelter, socialization must always be balanced with infectious disease control, and caregivers should take precautions accordingly. other forms of stimulation, including those that engage the various senses, are important methods of enriching the living environment by promoting healthy mental and physical activity. for singly housed cats and longterm residents, appropriate levels of additional enrichment should be provided on a daily basis. the provision of birdfeeders, gardens, or other interesting stimuli in the external environment can enhance the internal environment of the colony. resting perches in view of windows or other pleasant areas of the facility are especially desirable. other novel and enriching visual stimuli include cat-proof aquariums with fish, water fountains, bubbles, perpetual motion devices, and videos especially designed for cats (figures - to . a radio playing soft, low music in the room provides a welcome distraction and important source of play items that stimulate prey drive and physical activity, such as plastic balls, rings, hanging ropes, springmounted toys, plastic wands, and catnip toys, should also be provided but must be either sanitizable or disposable. empty cardboard boxes and paper bags are inexpensive, disposable, and stimulate exploration and play behavior as well as scratching. cats tend to be most stimulated by active toys, including wiggling ropes, wands with feathers, kitty fishing poles, and toys that can be slid or rolled to chase. many cats enjoy chasing stimulation. in addition, it may help to habituate cats to human voices and prevent them from being startled by loud noises. most caregivers also enjoy listening to the radio, and happy caregivers create a relaxed environment. the provision of scratching boards is especially important, and a variety of sturdy surfaces, both horizontal and vertical, should be provided for scratching. sisal rope, the backs of carpet squares, and corrugated cardboard are all useful (figure - ) . many cats like to smell and chew grass, and containers of cat grass or catnip can be introduced for brief periods to stimulate activity (figure - ) . providing novel sources of food is another important source of stimulation and can be easily accomplished by hiding food in commercially available food-puzzle toys or in cardboard boxes or similar items with holes such that the cat has to work to extract pieces of food ( figure - ) . , positive reinforcement-based training obedience training using clickers with food rewards is an excellent form of enrichment, combining social contact with caregivers together with both mental and physical stimulation. positive reinforcement training using a target stick is a powerful tool for teaching shy cats to approach the front of an enclosure. teaching cats awaiting adoption to perform tricks is not only stimulating for them, but it often makes them more attractive to potential adopters (figure - ) . a cardboard tube, and a plastic container with holes). treats are hidden inside, and they will have to work to extract pieces of food. novel feeding is an excellent source of enrichment for cats that are housed long term. (e.g., feeding, cleaning, enrichment activities), and unpredictable caregiving has been shown to dramatically increase stress. if events that are perceived as stressful (such as cleaning time) occur on a predictable schedule, cats learn that a predictable period of calm and comfort will always occur in between. cats also respond to positive experiences in their daily routines. for example, feeding and playtime may be greatly anticipated; thus scheduling positive daily events (e.g., a treat at : pm every day) should also be a priority. erratic periods of light and darkness are also known to be significant sources of stress for cats. animals possess natural circadian rhythms and irregular or continuous patterns of light or darkness are inherently stressful. lighting should be maintained on a regular the beams of laser pointers, small flashlights, or suspended rotating disco balls. commercially available electronic toys that stimulate play are especially useful in long-term settings (figure - ) . varied toys should be substituted regularly to ensure continued interest. in some climates, cats may be housed comfortably in outdoor enclosures where fresh air, sunshine, and other stimuli can help to create a healthy environment ( figures - and - ) . , , when indoor group enclosures connect to outdoor enclosures, it is important to have ample space for passage between them (e.g., more than one doorway) so that cats can pass freely. cats will also benefit greatly from consistent daily routines of care. they become entrained to schedules of care adoption, or euthanasia (when no other options exist) may be necessary to ensure cat welfare. achieving population wellness requires a healthy environment. thus the clinician's final task in creating a population wellness program is to develop tailored schedule, with lights on by day and off by night. whenever possible, full-spectrum and/or natural lighting is ideal. housed cats require active daily monitoring by staff trained to recognize signs of stress and social conflict. to the inexperienced observer, such signs may appear subtle (figure - ) . it is often the absence of normal behaviors (such as engaging in grooming or exercise) or subtle social signals (such as covert guarding of resources or dominant staring) that signify problems. careful observers will note these behaviors and respond accordingly to ensure that stress or conflicts do not persist. when cats are well adjusted and housing arrangements meet their behavioral needs, they display a wide variety of normal behaviors, including a good appetite and activity level, sociability, grooming, appropriate play behavior, and restful sleeping (figure - ) . , ultimately, the success of adaptation of cats to a new environment will depend on both the quality of the environment and the adaptive capacity of the individual. although most adapt to new environments with time, some never adjust and remain stressed indefinitely, ultimately resulting in decline of physical as well as emotional health. when cats fail to adjust to their environment and remain markedly stressed and fearful despite appropriate behavioral care, every effort must be made to prevent long-term stays. depending on the circumstances, transfer to another colony room, foster care, a shelter will result in saving more lives. to the contrary, euthanasia rates are highly correlated to intake rates, regardless of the number of animals that a facility houses. in many instances, keeping more animals in the shelter may actually reduce the organization's ability to help animals, because time and resources are tied up caring for a crowded, stressed population, rather than focusing on adoption or other positive outcomes. in shelter medicine, the term population management is used to refer to an active process of planning, ongoing daily evaluation, and responding to changing conditions as an organization cares for multiple animals. , the major goal of population management is to minimize the amount of time any individual animal spends confined in the shelter, while maximizing the organization's lifesaving capacity. moving animals through the system efficiently is the foundation of effective population management. to move animals through the shelter more quickly, delays in decision making and the completion of procedures (e.g., intake processing, transfer from holding to adoption areas, spay/neuter surgery) must be eliminated or minimized whenever possible. in openadmission shelters, even delays of to days can have a dramatic effect on the shelter's daily census, particularly for shelters handling thousands of animals per year. this, in turn, affects the ability to provide adequate care. it is important to recognize that effective population management does not change the final disposition of an animal. it does mean that determinations are made as soon as possible, which serves both the individual animal as well as the population as a whole. for wellness programs to be effective, a clean and sanitary environment must be maintained. not only does this promote cat and human health, but it also promotes staff pride as well as public support. in addition to protocols for routine daily cleaning and disinfection procedures, protocols should be in place for periodic deep cleaning and disinfection as well as procedures to be used in the event of disease outbreaks. when crafting protocols, it is important to recognize that cleaning and disinfection are two separate processes. the cleaning process involves the removal of gross wastes and organic debris (including nonvisible films) through the use of detergents, degreasers, and physical action. although this process should result in a visibly clean surface, it does not necessarily remove all of the potentially harmful infectious agents that may be present. disinfection is the process that will destroy most of these agents, but it cannot be accomplished until surfaces have been adequately cleaned. disinfection is usually accomplished through the application of chemical compounds or disinfectants. the most commonly used of these are reviewed in protocols focused on optimizing environmental conditions that favor cat health. once again, all essential elements as noted should be addressed. perhaps the most critical aspect of environmental management is to ensure a modest population density. high population density increases opportunities for introduction of infectious disease while increasing the contact rate among members of a group. both the number of asymptomatic carriers of disease, such as those with upper respiratory infection, as well as susceptible cats in a given group are likely to increase, enhancing the odds of disease transmission among group members through both direct contact as well as fomites. in addition, crowding also increases the magnitude of many environmental stressors (e.g., noise levels, air contaminants) and compromises animal husbandry, all of which induce unnecessary stress and further inflate the risk of disease in the population. indeed, crowding is one of the most potent stressors recognized in housed animals. although adequate space for animals is essential, it is crucial to recognize that crowding is not solely dependent on the amount of available space. it is also a function of the organization's ability to provide proper care that maintains animal health and well-being. every organization has a limit to the number of animals for which it can provide proper care. when more animals are housed than can be properly cared for within the organization's capacity, caregivers become overwhelmed, and animal care is further compromised. , , in animal shelters, crowding may also negatively impact adoption rates, because potential adopters often find crowded environments to be overwhelming and uninviting. if disease spread results as a consequence of the environmental conditions, animal adoptions may be further disrupted. although unexpected shelter intake may occasionally result in temporary crowding, a good wellness program dictates that protocols must be in place to alleviate crowding and maintain a modestly populated environment for the health and protection of the animals and staff. regardless of the setting, facilities must limit the number of animals housed to the number for which they can provide proper space and care. there are three basic methods of reducing crowding: ( ) limiting the admission (or births) of new animals into a population, ( ) increasing release of animals from a population, and ( ) euthanasia. in animal shelters, management practices that minimize each animal's length of stay and programs that speed or increase adoption, owner reunification, or transfer (e.g., to rescue or foster care) help to minimize crowding and maximize the number of animals that an organization can serve. it is a common misperception that housing more animals in • although commonly used, they must be applied to clean hands and allowed seconds of contact time to be effective. • they are highly effective against bacteria, but have only moderate activity against viral agents, including feline calicivirus (fcv). • they should not be used as a substitute for hand washing or the use of gloves. • chlorhexidine is the most commonly used biguanide and is relatively expensive. its major use is as a surgical preparation agent. • although biguanide compounds have broad antibacterial activity, they have limited efficacy against viruses and are ineffective against nonenveloped viruses, such as panleukopenia and fcv. therefore they are not recommended as general-purpose environmental disinfectants. • household bleach ( . % sodium hypochlorite) is the most commonly used chlorine compound and is an excellent, safe, and highly cost-effective disinfectant when used correctly. • at a dilution of : , bleach is highly effective against bacteria and viruses, including nonenveloped viruses, such as panleukopenia and fcv. • solutions must be made fresh daily and stored in opaque containers, because bleach is highly unstable once mixed with water and degrades in the presence of ultraviolet light. • surfaces must be thoroughly cleaned with a detergent, rinsed, and dried prior to the application of bleach, because it is ineffective in the presence of detergents and organic material. • proper disinfection requires minutes of contact time with a bleach solution. • although bleach is not effective when mixed with detergents, it can be safely and effectively mixed with quaternary ammonium compounds, which do provide some cleaning activity. therefore this combination can be used for cleaning and disinfection, provided gross organic material is first removed and adequate contact time is allowed. the addition of bleach improves the disinfection properties of the solution, making it effective against nonenveloped viruses, including panleukopenia and fcv. • concentrations stronger than a : dilution can result in respiratory irritation for both animals and people, as well as increased facility corrosion, and are therefore not recommended for routine use. • at a dilution of : , bleach will destroy dermatophyte spores. however, cats must be removed from the environment prior to application of this concentration. • the use of calcium hypochlorite (wysiwash, st. cloud, fla.) is becoming more common and offers the potential advantages of reduced contact time and a neutral ph, which prevents corrosion. oxidizing agents quarantine involves the holding of healthy-appearing animals. it is most useful when animals enter a closed population to ensure that they are not incubating disease when they are introduced into the general population. quarantine areas, with rigid biosecurity procedures in place, should be used to segregate healthy animals for observation. the use of such areas not only allows apparently healthy animals to be observed for developing signs of infectious disease, but it also allows time for response to vaccination in a highly biosecure environment where exposure risks are minimized. the use of quarantine is a mainstay of effective infectious disease control programs and is intended to prevent the introduction of disease into a population. it should be used whenever it is feasible to implement effectively, such as in a laboratory setting, a private cattery, or a low-volume, limited-admission sanctuary setting. however, quarantine practices are not effective in most animal shelters, because the high volume and turnover of animals precludes proper implementation of a true quarantine where an "all in-all out" system is used. instead, incoming animals are usually added to the "quarantine group" on a daily basis, effectively defeating the purpose of true quarantine and simply prolonging the animal's stay. this is especially concerning given the fact that a cat's length of stay in a shelter is a major risk factor for development of upper respiratory infection. for this reason, the use of quarantine is not recommended in most shelter settings. instead, high biosecurity areas are recommended for housing the most susceptible animals (e.g., kittens less than to months of age). on the other hand, quarantine is warranted when a serious disease is discovered in a shelter population. if healthyappearing animals are exposed during a serious outbreak, quarantine procedures should be used to stop the movement of animals and prevent further spread of disease. if possible, temporary closure to admittance is also recommended. quarantine may also be required in bite cases to ensure compliance with state rabies laws. the particular population setting will guide the clinician's determination regarding implementation and length of quarantine, if any. a -day quarantine is sufficient to determine that cats are not incubating many common infectious diseases, including feline panleukopenia. however, other diseases, including feline leukemia virus and dermatophytosis, can have longer incubation periods and will therefore require a longer quarantine period. , in breeding colonies, early weaning and quarantine have been advocated to prevent infection of kittens with feline coronavirus. pregnant queens are isolated, and product selection should take into consideration the conditions present in a given environment (e.g., the type of surface and the presence of organic matter) and the compound's activity against the pathogens for which the animals are at greatest risk. the nonenveloped viruses, panleukopenia and feline calicivirus, are of particular concern. it is important to note that despite product label claims to the contrary, multiple independent studies have consistently shown that quaternary ammonium disinfectants do not reliably inactivate these important feline pathogens. , , in addition to selecting effective agents, ensuring adequate contact time followed by thorough drying of surfaces is essential for achieving proper disinfection. protocols should include detailed methods for achieving both cleaning and disinfection. when performed properly and regularly, these practices decrease both the dose and duration of exposure to infectious agents. box - outlines essential considerations for the development of cleaning and disinfection protocols. segregation refers to the separation of animals from the main group or into subpopulations as necessary to promote health. segregation of cats by physical and behavioral health status is essential for infectious disease control, stress reduction, and safety. in animal shelters, segregation may also be required to ensure compliance with animal control procedures as prescribed by state or local ordinances. depending on the setting, consideration should be given to separating cats by gender and reproductive status (e.g., intact, neutered, in heat, pregnant, nursing), physical and behavioral health status (e.g., apparently healthy, signs of contagious disease, reactive, feral), and life stage. a variety of separate areas will be necessary, depending on the needs of the given population and the context of the setting. the wellness program should define these areas in order to optimize cat health, while providing for the necessary functions of the facility. depending on the setting, consideration should be given to establishing areas for quarantine, isolation, general holding, adoption, and long-term housing, as well as to tailoring these by life stage. for example, the very young and very old typically require more specialized care than healthy juveniles and adults. kittens less than to months of age are particularly susceptible to infectious disease, and extra care must be taken to heighten biosecurity and limit their exposure. in particular, geriatric cats require comfortable, quiet quarters with careful attention to stress reduction (e.g., the provision of a secure hiding place and a dedicated caregiver to enhance bonding and comfort). if cats are used for breeding, functional areas will be required to facilitate mating, queening, and kitten care. and, as previously • staff must wear personal protective equipment, as necessary, to prevent exposure to chemicals and/or pathogens. • thorough cleaning and disinfection of enclosures should occur between occupants and as part of periodic deep cleaning procedures. • cats must be removed from enclosures during these procedures. • "spot cleaning" is generally sufficient for apparently healthy cats that will continue to occupy the same enclosure. • the cat remains in the enclosure while it is cleaned and soiled material is removed. • this method is often less stressful for cats (see figure - ). • separate staff should clean and care for animals in areas with highly susceptible or sick animals, whenever possible. • at minimum, attention should be given to the order of cleaning. • the least contaminated areas should be cleaned before those that are the most contaminated. kittens are weaned as early as possible (e.g., to weeks of age) and placed in strict quarantine. in this manner, as maternal antibody wanes and kittens become susceptible to the virus, exposure and infection are prevented. however, it is important to note that the level of biosecurity required for success is difficult to achieve. furthermore, eventual exposure and infection are highly likely because of the ubiquitous nature of coronaviruses. in addition, because of the importance of the mother-kitten relationship to normal social and emotional development, this management practice may not always be desirable. healthy environmental conditions in isolation areas promote recovery, and their importance cannot be overemphasized. crowding, noise, and stress must be avoided, and facilities must be easy to clean and disinfect. room temperature should be warm and comfortable with good air quality. windows are ideal, because natural sunlight is always beneficial to animal health and healing. strict biosecurity in quarantine and isolation areas, with attention to traffic patterns and the use of protective clothing, such as gowns and shoe covers, is essential. footbaths are insufficient to prevent transfer of infectious agents on shoes. this is because disinfectants typically require minutes of contact time and may be poorly effective in the presence of organic debris. in fact, footbaths may even contribute to the spread of disease. dedicated boots or shoe covers should be used when entering contaminated areas. , in addition, separate, designated staff should care for animals in high biosecurity areas whenever possible. by design, traffic patterns should move from the healthiest and most susceptible groupings to the least susceptible, and finally to isolation areas housing sick animals. observation windows and signage are useful to reduce traffic flow into high-risk areas. staff hygiene is extremely important, and the importance of diligent hand washing cannot be overemphasized. where space or facilities are not available, foster care may represent a viable and medically sound option for quarantine or isolation in some settings. for instance, in animal shelters, foster care is particularly useful for the care of preweaning-age kittens. foster homes must be monitored to ensure that cats receive proper care and that resident animals are protected from disease exposure. in addition to ensuring proper population density, segregation, and sanitation procedures, there are several other essential aspects of facility operations that must be incorporated into a population wellness program. these include heating, ventilation, and air conditioning (hvac) considerations, noise and pest control, general facility maintenance, and staff training. extremes or fluctuations in temperature and humidity, as well as poor ventilation and air quality, can compromise animal health. poor ventilation and high humidity favor the accumulation of infectious agents, while dust and fumes may be irritating to the respiratory tract. many cats are particularly sensitive to drafts and chilling, both of which can predispose to upper respiratory general holding, housing, adoption, and other areas in many settings, general holding areas are used for housing healthy juvenile and adult cats at intake. in animal shelters, it is important to consider that length of stay is associated with an increased risk of feline upper respiratory disease and that vaccination against core diseases often rapidly confers immunity. for these reasons, holding periods should be minimized whenever possible. in some cases, holding times will be influenced by legally required holding periods prescribed by state laws that allow owners a chance to reclaim lost pets. legal holding periods are usually not required for owner-relinquished pets and preweaning-age animals, but a brief medical hold (e.g., to days) for evaluation and triage is usually warranted. regardless, management practices that reduce length of stay are generally best for population health in a shelter setting. immunity is often strengthened with time through a combination of both active and passive immunity resulting from vaccination and exposure. upper respiratory disease is often endemic in cat populations, and in open populations, constant introduction of large numbers of carriers and susceptible cats make exposure likely. as length of stay increases, many cats develop and recover from respiratory disease. as animals acclimate to their environment and gain immunity, less stringent biosecurity requirements may be required for long-term housing areas, depending on the particular setting. in animal shelters, the public is usually allowed to interact with cats in adoption areas, which is another management consideration. isolation areas are used to segregate sick animals from the general population. immediate isolation of animals with signs of infectious disease is critical to effective control. isolation should be targeted by age and disease. for example, separate isolation areas should be available for cats and kittens with respiratory disease and those with gastrointestinal disease, whenever possible. in populations where upper respiratory infection is problematic, having two isolation areas for cats with respiratory infections is ideal: one area for those cats with moderate to severe signs that will require more intensive monitoring and treatment, and a separate area for those cats with only mild clinical signs and those that have been treated and are nearly recovered. when mildly symptomatic cats can be housed separately from those that are very ill, staff compliance with isolation procedures are often improved. cats with non-infectious conditions should also be housed in separate areas for treatment, and, in some cases, housing in the general population is appropriate. prevents exchange of air among them and is recommended, because air pressure gradients that recirculate or cause exchange of air between rooms have been associated with the spread of disease by aerosols. when applying these standard recommendations to a particular setting, there are some practical considerations that should be taken into account. first, even when ventilation systems provide to room air changes per hour, airflow may be restricted inside of cages or other enclosures within the room. in other words, the body of the room may be well ventilated, yet inside the cages, the air may remain relatively stagnant. in this case, ventilation may be improved by altering the housing design or arrangement; for example, the use of flow-through cages, runs, free-range rooms, or outdoor access may result in improved air quality. when considering the recommendations for % fresh, nonrecycled air with separate ventilation systems in various areas of the facility, consideration should be given to the fact that respiratory pathogens in cats are not aerosolized because of the cats' small tidal volume. although droplet transmission is possible, droplet spray does not extend more than feet, and most transmission of respiratory disease in cats is through direct contact with infected cats, carriers, or fomites. although this recommendation seems prudent to consider, especially for isolation areas, it is very expensive to install and operate this type of ventilation system throughout a facility, especially in very cold or very hot climates. if air quality remains good and the facility maintains effective comprehensive wellness protocols, it may not be necessary for animal health. more research is needed on the impact of such air exchange, but in the meantime, the author recommends consulting with an hvac specialist to establish effective and efficient settings to suit the specific needs of the given population. in addition to ensuring good ventilation, it is imperative to use other strategies for maintaining good air quality, including regular maintenance of filters, control of dust and dander through routine vacuuming and periodic deep cleaning, and the use of dust-free litter. noise control is another important consideration. it is crucial to keep cats out of auditory range of dogs, because many are profoundly stressed by the sounds of barking. also, staff should be trained to reduce or avoid other sources of noise whenever possible. the installation of sound-proofing systems may be necessary for noise abatement and stress reduction. routine pest control may also be required, depending on the setting. it may be necessary to treat the environment for fleas, ticks, or other insects or ectoparasites. products used to treat the environment must be selected carefully, because cats are extremely sensitive to the toxic effects of many insecticides. in many instances, it will infection. heating, ventilation, and air conditioning (hvac) specialists are uniquely qualified to help establish and maintain the environmental conditions required for animal health. when facilities are designed specifically for housing animals, these specialists should be consulted beforehand to ensure installation of the most effective and efficient systems possible. in reality, many facilities that house cats, including private catteries and shelters, among others, were not originally built for this purpose. retrofitting existing facilities with the ideal hvac system is often neither logistically nor financially feasible. regardless, consultation with hvac specialists is recommended in order to maximize the potential of the facility's existing system. the recommended temperature range for cats is between ° c and ° c ( ° f and ° f) with a temperature setting in the low-to mid- s celsius ( s fahrenheit) being typical. the temperature setting should be determined according to the specific animals' needs. for example, neonatal kittens are more susceptible to hypothermia and generally require warmer temperatures than healthy adult cats. the location of the cats may also be a consideration. for example, enclosures located closer to floor level are often a few degrees cooler than those at higher levels. the exact temperature setting may also vary somewhat based on the season of the year. for instance, power companies typically recommend keeping the temperature between ° c and ° c ( ° f and ° f) during hot weather to conserve electricity and reduce power bills. laboratory guidelines recommend % to % humidity for cats. higher humidity (e.g., %) may be advantageous in areas housing cats with respiratory disease because moist air may be beneficial to the respiratory passages, whereas lower humidity (e.g., % to %) may be beneficial in other areas in order to reduce survival of infectious agents in the environment. although the range considered acceptable is large, a given room should have a relatively constant humidity (i.e., it should not have large fluctuations). hosing or even mopping a room usually results in temporary spikes in humidity, but these will be short lived in a well-ventilated room. adequate ventilation is crucial for good air quality. this is especially important for cats, because good air quality is essential for control of upper respiratory disease. ten to fifteen air changes per hour is the standard recommendation for an animal room, but more or less airflow may be acceptable or necessary depending on the housing density. theoretically, the best case scenario, and what is typical in laboratory animal settings, is for the hvac system to allow for % fresh (e.g., nonrecycled) air in each room so that the air entering a given room is exhausted out of the building and not recirculated to another room. maintaining separate ventilation systems for various rooms or areas of a facility ensure both the physical and behavioral health of cats, as well as a healthy environment. a proactive, holistic approach coupled with compassion is required. when these are combined with careful attention to the unique needs and stress responses of cats, the result will be "healthy, happy cats." be necessary to remove cats during their application and only return them to the environment once it is thoroughly dried and ventilated. if rodent control is necessary, the use of rodenticide baits should be avoided, because cats can be exposed even if the bait is not within their reach. rodents that have ingested the poisonous bait may enter an animal enclosure and, if the animal ingests the rodent, the poison will affect that animal. humane live traps can be used to capture rodents for removal from a facility. food containers should be kept tightly sealed, and clutter should be minimized to discourage pests in the environment. general building maintenance procedures (e.g., regular inspection and servicing with repairs as needed) are also important considerations for the maintenance of a healthy environment. for example, periodic resealing of floors may be required as well as maintenance of plumbing fixtures to repair leaks or other problems. developing and following written standard operating procedures and daily, weekly, monthly, and quarterly checklists will ensure that systematic schedules of maintenance are carried out in a timely fashion. regular staff training is essential for implementing effective population wellness programs. simply stated, staff caring for animals must be qualified to do so. to a large extent, their knowledge and skill will determine the success or failure of the wellness program. embracing a culture of training promotes high-quality animal care as well as human safety. both formal and on-thejob training should be provided to ensure that a staff has the knowledge and skills required to perform their assigned tasks. protocols should be established for all levels of training, and a system should be in place to ensure proficiency. staff training should be documented, and continuing education should be provided to maintain and improve skills. finally, training must include the provision of information about zoonoses and 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welfare five key population management factors affecting shelter animal health cat housing in rescue shelters: a welfare comparison between communal and discrete-unit housing recognizing and managing problem behavior in breeding catteries quality of life in long-term confinement maddie's infection control manual for animal shelters a review of feline infectious peritonitis virus observations on the epidemiology and control of viral respiratory disease in cats employee reactions and adjustment to euthanasia related work: identifying turning points through retrospective narratives the aafp feline vaccine advisory panel report welfare of cats in a quarantine cattery recommendations for the housing of cats in the home, in catteries and animal shelters, in laboratories and in veterinary surgeries comfortable environmentally enriched housing for domestic cats efficacy of fipronil in the treatment of feline cheyletiellosis virucidal disinfectants and feline viruses the influence of food presentation on the behavior of small cats in confined environments validation of a temperament test for domestic cats evaluation of the effects of footwear hygiene protocols on nonspecific bacterial contamination of floor surfaces in an equine hospital the human-cat relationship editor: cattery design: the essential guide to creating your perfect cattery intercat aggression in households following the introduction of a new cat feline leukemia virus and feline immunodeficiency virus american association of feline practitioners feline retrovirus management guidelines intercat aggression: a retrospective study examining types of aggression, sexes of fighting pairs and effectiveness of treatment evaluation of collars and microchips for visual and permanent identification of pet cats characterization of animal with microchips entering shelters in vitro sensitivity of commercial scanners to microchips of various frequencies sensitivity of commercial scanners to microchips of various frequencies implanted in dogs and cats search and identification methods that owners use to find a lost cat provision of environmentally enriched housing for cats environmentally enriched housing for cats when singly housed managing oral health in breeding catteries assessment of stress levels among cats in four shelters enriching the environment of the laboratory cat the impact of paternity and early socialisation on the development of cats' behaviour to people and novel objects quality of life in animals development of a mental wellness program for animals toxicology brief: the most common toxicoses in cats the domestic cat. perspective on the nature and diversity of cats definition of wellness behavioral effects of cage enrichment in single-caged adult cats treatment of dermatophytosis in dogs and cats: review of published studies development of an in vitro, isolated, infected spore testing model for disinfectant testing of microsporum canis isolates quality-of-life assessment in pet dogs aafp-aaha. feline life stage guidelines meet your match and feline-ality adoption program key: cord- -f o owg authors: navarre, christine b.; pugh, d.g. title: diseases of the gastrointestinal system date: - - journal: sheep & goat medicine doi: . /b - - - / - sha: doc_id: cord_uid: f o owg nan the gastrointestinal system is, arguably, more prone to disease than any other part of the sheep or goat. gastrointestinal parasitism alone is the most significant cause of production and animal losses in much of north america. , there is no substitute for a thorough physical examination when trying to determine the affected body systems of a sick animal; this is especially true in diseases of the gastrointestinal system. a complete physical examination should include palpation for body condition, assessment of abdominal shape and rumen motility, observation of the consistency of the stool, and evaluation for the presence of bloat. however, because rectal palpation cannot be performed in sheep and goats, diagnosis of disease in a particular segment of the gastrointestinal system can be difficult. therefore, the clinician may have to perform ancillary diagnostic procedures to characterize gastrointestinal diseases properly. clinicopathologic data consisting of a complete blood count (cbc), serum biochemical evaluation (sbe), and urinalysis can be helpful in differentiating gastrointestinal diseases, developing a prognosis and plan for treatment, and monitoring treatment. a cbc rarely identifies a specific disease, but it can be helpful in evaluating the severity of dehydration, anemia, and hypoproteinemia. the clinician must take care to interpret the packed cell volume (pcv) and total protein in light of the hydration status of the animal as noted on physical examination. an anemic or dehydrated hypoproteinemic animal may have normal pcv and total protein values. both the cbc and sbe can be helpful in characterizing the presence and severity of an inflammatory disease process. changes in the total and differential white blood cell count indicate acute or chronic inflammation; increases in globulins or fibrinogen suggest a chronic inflammatory disease. low protein levels, especially albumin, can point to chronic blood loss from intestinal parasitism or infiltrative bowel disease. liver disease should be suspected if liver enzymes or bilirubin are elevated. however, liver enzymes can be normal in chronic liver disease. also, albumin levels rarely drop in ruminants with liver disease, as they do in other species. changes in electrolytes can occur with gastrointestinal disease, especially if the animals are anorexic. electrolyte measurements also are helpful in formulating a treatment plan. hypochloremia and metabolic alkalosis occasionally occur in abomasal disease. a mild hypocalcemia may be encountered in some small ruminants with gastrointestinal atony. because many animals with gastrointestinal disease are dehydrated and therefore azotemic and possibly hypoproteinemic, urinalysis is helpful to eliminate urinary disease as a cause of these pathologies. normal ranges for clinicopathologic values are included in this textbook (see appendix iii) and also have been published in several other textbooks. [ ] [ ] [ ] [ ] however, clinicians would do well to learn the normal values, especially serum biochemistry values, established by the laboratory most commonly used for analysis. analysis of rumen fluid can help differentiate diseases of the forestomachs. an appropriately sized orogastric tube can be passed through the oral cavity for fluid collection (figure - ) . the clinician must properly restrain the animal, using a mouth speculum (figure - ) to prevent tube chewing. if the tube is chewed, its roughened surface may damage the esophagus; parts of a broken tube can be swallowed. rumen fluid also can be collected using percutaneous rumenocentesis , [ ] [ ] [ ] [ ] [ ] (figure the clinician then aspirates fluid with a syringe. local anesthesia and sedation of the animal may be necessary. this technique avoids the saliva contamination that can occur during collection with an orogastric tube, and it appears to be less stressful. rumenocentesis presents a slight risk of peritonitis, but this risk can be minimized with proper restraint. percutaneous rumenocentesis should not be performed on pregnant females. after the fluid is collected, it can be analyzed for color, odor, ph, protozoal species and motility, methylene blue reduction time (mbr), gram's staining characteristics, and chloride levels. normal values are listed in table - . anorexia may cause the fluid to appear darker, the ph to increase, and the number and motility of protozoa to decrease. a gray color, low ph, and dead or no protozoa are seen in rumen acidosis from grain overload. the mbr is prolonged with any type of indigestion. large numbers of gram-positive rods (lactobacillus species) also may be seen in rumen acidosis. elevated rumen chloride indicates an abomasal or proximal small intestinal obstruction (either functional or mechanical). the most important reason for examining feces in sheep and goats is to determine the presence and relative number of nematode parasites infesting an animal or flock. the quantitative technique for determining eggs per gram of feces (epg) is shown in box - . fecal epg values of more than to indicate serious infestation and the need for intervention. fecal occult blood testing and acid-fast staining of fecal smears also can be performed. fecal occult blood tests can detect microscopic amounts of blood in the feces. however, they cannot indicate which part of the gastrointestinal tract is bleeding. acid-fast stains of fecal smears that reveal clumps of acid-fast rods usually indicate infection with mycobacterium paratuberculosis ( johne's disease). generally, individual acid-fast rods found on fecal examination are nonpathogenic. abdominocentesis is useful in discerning the causes of fluid distention in the abdomen. two methods can be used. the first technique involves tapping the lowest point of the abdomen slightly to the right of midline; it is useful in ruling out a ruptured bladder as the cause of general ascites (figure - ) . , the clinician should take care to avoid the prepuce in males. the second technique is useful if peritonitis is suspected. because localized peritonitis is more common than generalized peritonitis, four sites are tapped. the two cranial sites are slightly caudal to the xyphoid and medial to the milk veins on the left and right sides. the two caudal sites are slightly cranial to the mammary gland and to the left and right of midline. for either technique, manual restraint with sedation is recommended; the use of real-time ultrasonography may help locate fluid pockets. a -gauge needle or teat cannula can be used for fluid collection. the clinician should prepare the site using sterile technique and provide local anesthesia when employing a teat cannula. fluid should be collected in a small ethylenediamine tetra-acetic acid (edta) tube for analysis and a sterile tube for culture. abdominal fluid can be difficult to obtain because of the small amounts normally present in both sheep and goats. the clinician should minimize the ratio of edta to fluid because edta can falsely elevate protein levels. using edta tubes made for small animals or shaking excess edta out of large tubes resolves this problem. normal culture values are similar to those for cattle (clear, colorless to slightly yellow, to g/dl protein, less than , cells). cytologic examination . weigh g of feces and thoroughly mix with ml of water. this is the preferred method. however, if a gram scale is not available, feces can be added to the ml of water until the water level indicates ml. this approximates g of feces. . remove ml of well-mixed fecal-water suspension, add to ml of sheather's solution,* and mix well. is needed to characterize the cell population and assess for the presence of phagocytized bacteria. radiography of the abdomen can be performed in small ruminants using small animal techniques. in adults, the rumen normally fills the entire abdomen. radiography can detect gas distention of the small intestine, abdominal fluid, and foreign bodies. , contrast techniques are useful for diagnosing atresia of the rectum or colon. unlike in other small animals, contrast techniques are not practical for characterizing small intestinal problems in sheep and goats because the rumen dilutes and slows passage of the contrast media. ultrasonography ultrasonography can be used to provide better characterization of abdominal distention, internal and external abdominal masses, and gross lesions of the liver. ascites may be differentiated from fluid in the intestinal tract, and gas distention of the intestines can be differentiated from fluid distention. normal ultrasonographic examination of the liver in sheep has been described. the liver can be viewed on the right side from the seventh or eighth rib caudally to the thirteenth rib ( . ultrasonography can be used to perform biopsies of organs or masses and to locate pockets of fluid. laparoscopy is more commonly used as a reproductive tool, but it also can be used diagnostically as an alternative to exploratory laparotomy in small ruminants. , general anesthesia is recommended. the technique for laparoscopic exploration of the abdomen used for cattle can be modified for use in sheep and goats. the clinician inserts a cannula in the caudal abdomen and carefully inflates the abdomen with carbon dioxide (co ). with the animal restrained in dorsal recumbency and either sedated or anesthetized, the clinician places the cannula in the inguinal area as described for laparoscopic insemination in chapter . entrance on the right side allows visualization of most of the abdominal organs. the clinician should avoid the rumen when introducing the laparoscope into the abdomen. this procedure may be enhanced by lowering the head or rear of the animal, allowing better visualization of the entire abdomen. animals should be properly ventilated during this procedure because inflation of the abdomen and lowering of the head can put pressure on the diaphragm. exploratory laparotomy can be a valuable diagnostic tool in evaluating gastrointestinal diseases when other tests indicate abdominal disease. in some cases, therapeutic surgical techniques can be performed at the same time. the technique of exploratory laparotomy used in cattle can be adopted for sheep and goats as long as the clinician keeps in mind that these animals are more likely to lie down during surgery and standing surgery should only rarely be attempted. small ruminants should be heavily sedated or placed under general anesthesia during this procedure. they may show signs of postoperative pain, anorexia, and depression and should be treated accordingly with a nonsteroidal antiinflammatory drug (nsaid) (flunixin meglumine . to . mg/kg intravenously [iv] ). the decision to use perioperative and postoperative antimicrobial agents should be based on the conditions under which the surgery is performed and the diagnosis made at surgery. sheep the area should be clipped, but in goats alcohol can be applied to the overlying hair and skin. if the area is clipped, the clinician should apply a bland coupling material (e.g., methyl cellulose, vegetable oil) between the skin and the transducer. liver biopsy in sheep and goats is performed using the same technique and instruments as in cattle. however, sedation and ultrasound guidance are recommended. the biopsy can be performed in the ninth to tenth intercostal space slightly above an imaginary line from the tuber coxae to the point of the elbow (figure - ) . the site should be surgically prepared, and a local anesthetic ( % lidocaine hydrochloride) infused subcutaneously. a small scalpel blade is used to make a stab incision through the skin. a -gauge, . -cm liver biopsy instrument is inserted through the incision and the intercostal muscles and into the liver. the biopsy instrument should be directed toward the opposite elbow in most cases, but the use of real-time ultrasonography can help determine the direction and depth needed ( to cm). the clinician should avoid the vessels along the caudal border of the ribs. on reaching the liver, the clinician will note a slight increase in resistance. samples can be submitted for culture (in a sterile plastic or glass vial or tube), histopathology (in formalin at a Ϻ ratio of formalin to tissue); and/or mineral analysis (in a plastic tube). when performing a liver biopsy for mineral analysis, the clinician should rinse the biopsy site with distilled and deionized water after sterile preparation to minimize sample contamination. samples for mineral analysis should not be placed in formalin. the skin incision can be sutured, and aseptically prepared, the surgeon makes a stab incision in the skin and introduces a -gauge biopsy needle. bloat is less common in small ruminants than in cattle, with goats having the condition less commonly than sheep. bloat is the accumulation of either free gas or froth in the rumen, which causes rumen distention. the causes of bloat can be divided into three categories , : . frothy bloat-caused by diets that promote the formation of stable froth . free gas bloat-caused by diets that promote excessive free gas production . free gas bloat-caused by failure to eructate pathogenesis. frothy bloat is usually associated with the ingestion of legume forages or hay (particularly alfalfa) and with grazing on lush cereal grain pastures, but it also may occur with high-grain diets. in the case of frothy bloat from a finely ground diet (usually corn), mucoprotein released from rumen protozoa stabilizes the foam at a low ph. in legume-associated frothy bloat, plant chloroplasts released into the rumen trap gas bubbles. regardless of the form of frothy bloat, the small bubbles fill much of the rumen, preventing clearance of the rumen's cardia and resulting in a cessation of eructation. free gas bloat also occurs with grain diets, especially if the animals are not adapted to the diet. failure to eructate has a variety of causes. physical obstructions of the esophagus such as choke or swollen mediastinal lymph nodes can cause free gas bloat. any disease of the rumen wall can interfere with rumen contractions and eructation. hypocalcemia, endotoxemia, pain, peritonitis, and some pharmaceutical agents (especially xylazine) can all interfere with rumen function and eructation. , , , clinical signs. clinical signs of frothy bloat and free gas bloat from either food intake or physical obstruction of the esophagus are usually more severe and immediately life-threatening than bloat seen from rumen wall diseases and systemic influences. abdominal enlargement occurs, particularly in the dorsal left paralumbar fossa. this may be subtle in sheep or angora goats with full fleece. signs of colic and anxiety are common. the rumen may be either hypomotile or hypermotile. respiratory distress is evident, with some animals breathing through their mouths; death can ensue if the bloat is not treated. this condition is a medical emergency, and therefore diagnosis and treatment should occur almost simultaneously. if the animal is not in immediate danger of dying, an orogastric tube can be passed. most cases of free gas bloat are relieved with passage of the tube. a clinician should then take a thorough history and perform a complete physical examination to find the cause of the bloat. if the bloat is not relieved with an orogastric tube, the tube should be removed and examined for evidence of froth. frothy bloat can be treated with poloxalene ( mg/kg) or dioctyl sodium sulfosuccinate (dss) ( cc [ oz]) delivered by orogastric tube. the froth encountered in frothy bloat caused by the ingestion of finely ground grain has a ph of less than . . if frothy bloat occurs while animals are being fed concentrates, mineral oil ( ml) may work better. peanut oil ( to mg/kg), vegetable oil ( to ml), and hand soap ( ml) also have been recommended in emergency situations. if the animal is in severe respiratory distress, the clinician should insert a trocar or large needle into the rumen at the paralumbar fossa. if gas does not escape, or froth is seen coming out of the trocar, an emergency rumenotomy should be performed (see the rumenotomy section of this chapter). if several cases of bloat are encountered in a group of pastured animals, the entire group should be removed from the pasture and reintroduced slowly after gradual acclimation. if only one or two cases of bloat are encountered, the healthy animals can remain on the offending pasture, but grazing should be limited to ensure gradual acclimation. prevention. prevention of frothy bloat involves limiting access to offending pastures or feedstuffs; providing supplemental feed and providing poloxalene in mineral supplements; and adding ionophores to the ration or supplement. when grazing or consuming legumes as "greenchop," animals should be introduced to the feed or pasture slowly, preferably over to weeks. animals should be closely monitored after a frost and during the rapid growth phase of plants because legumes, particularly alfalfa, may be more likely to cause bloat at this time. certain varieties of legumes that are designed for intensive grazing systems (e.g., alfagraze) should be planted and managed in a manner that decreases the incidence of bloat (limited or creep grazing). feeding dry, stemmy hay for to hours before allowing access to the legume pasture also may help minimize bloat. grass-legume pastures in which legumes are limited to less than % of the forage are safer but can still pose a problem for animals that are selective grazers. grazing legumes with high leaf tannin concentrations (e.g., arrowleaf clover, kudzu) is usually safer because tannins help break down rumen foam. the inclusion of poloxalene ( to mg/kg daily) in the feed or mineral supplement is useful in preventing frothy bloat. if poloxalene supplements are used, keepers should feed them for to weeks before moving animals onto a problem pasture. free gas bloat from concentrate feeds can be controlled by slow introduction to these feeds to allow for rumen adaptation and by the inclusion of ionophores in the diet. monensin ( mg/head/day in ewes, mg/kg/day in goats) and lasalocid ( . to mg/kg/day in sheep and goats) both decrease the formation of free ruminal gas. by enhancing propionic acid formation, these drugs not only reduce the amount of methane produced in the rumen, they also improve the efficiency of nutrient assimilation from feedstuffs. bloat in lambs and kids can have the same causes as in adults but also can be caused by improper milk feeding. overfeeding, feeding of large infrequent meals, and feeding spoiled or cold milk have all been associated with bloat in lambs and kids. rapid overdistention of the abomasum and improper chemical or physical composition of milk replacers inhibit rumen motility, leading to bloat. even though the feeding of cold milk has been associated with bloat, the practice can be used effectively in orphan feeding programs. lambs and kids tend to limit their intake of cold milk after they have become accustomed to cold milk in a free-choice feeding system. milk is usually placed in the rumen when animals are tube-fed; this may result in milk spoilage. , simple indigestion is a mild form of upset of the reticulorumen caused by a change in feeding routine. it can be caused by an alteration in the type of feed or in the amount of feed offered. the most common causes of simple indigestion are the addition of grain to the diet, an increase in the amount of grain fed, and an increase in the energy density of the diet. examples of such dietary changes are replacing oats with corn or changing from whole to ground corn. if the changes are drastic, rumen acidosis can occur (see the following section). other common causes are changes in hay or pasture, consumption of moldy hay, and ingestion of weeds and toxic plants after overgrazing or droughts. clinical signs include mild anorexia that lasts for to days. mild diarrhea and bloat also may occur. rumen fluid ph can be unchanged, increased, or decreased depending on the inciting cause. most animals improve with no treatment. pathogenesis. rumen acidosis is caused by the rapid rumen fermentation of highly digestible carbohydrates that are ingested in excessive amounts. although corn is commonly implicated, other cereal grains (oats, wheat, barley) may be involved, particularly if they are finely ground. the smaller the particle size, the more quickly rumen bacteria are able to ferment the carbohydrates contained in the feed. the common name of this condition is "grain overload," but breads, candy, apples and other fruits, beets, and potatoes also can cause this condi-tion. rumen acidosis usually occurs in animals that have been fed predominantly forage-based rations and are suddenly given access to large amounts of highly fermentable concentrates or concentrated forms of energy. it also can occur in animals that have been receiving concentrates previously, if the amount is suddenly and drastically increased; if access is denied for a time, then suddenly returned (e.g., during weather changes and alterations in water availability); or if ration mixing errors occur (e.g., leaving out monensin and rumen buffers) as highly digestible carbohydrates are fermented, rumen ph drops. lactobacillus species, which are lactic acid producers, proliferate in the acidic rumen environment and further lower rumen ph. as the rumen ph drops, rumen protozoa and many of the lactate users begin to die. lactic acid production causes the osmotic pressure in the rumen to increase. fluid is drawn from the systemic circulation into the rumen, resulting in dehydration and possibly hypovolemic shock. lactate concentrations increase in the blood and may cause systemic lactic acidosis. the lactic acid in the rumen also is toxic to the rumen epithelium. damage to the epithelium can result in leakage of bacteria and toxins into the portal and systemic circulation. chronic sequelae to rumen acidosis include fungal rumenitis and occasionally liver abscesses. , liver abscesses are less commonly encountered in sheep and goats than in cattle. laminitis also can occur, but may be more of a problem in sheep than in goats. the severity of the disease depends on the composition of the feed, particle size, amount of feed consumed, and the period of adaptation to the diet. clinical signs. clinical signs vary with the amount and type of feed ingested and the time since ingestion. signs first appear to hours after ingestion of the offending feed; they vary from anorexia, depression, and weakness to a down animal suffering from severe circulatory shock. dehydration is usually severe and evidence of toxemia is present (e.g., injected mucous membranes, increased scleral injection). colic, bilateral ventral abdominal distention, rumen stasis, and a "splashy" feel to the rumen also may be present. diarrhea can develop, adding to dehydration. , , the diarrhea can range from a pastelike feces to very watery droppings with foam and occasionally pieces of grain easily recognized. dehydration, lactic acidosis, and toxemia result in neurologic signs, including ataxia, head pressing, opisthotonos, and seizures. the body temperature is initially elevated but may drop as the condition worsens or the animal becomes toxic. some animals develop polioencephalomalacia and appear blind. diagnosis. the rumen fluid ph may fall below . . the fluid itself is milky gray and particles of the inciting feed may be noticed. protozoa are usually reduced in number or absent, and large gram-positive rods (lactobacillus species) may be seen on gram's stain. clinicopathology is consistent with dehydration (increased pcv and total protein, prerenal azotemia) and metabolic acidosis. liver enzymes (gamma-glutamyl transpeptidase [ggt], aspartate aminotransferase [ast], lactate dehydrogenase [ldh]) may be elevated on serum biochemical analysis. , the leukogram can vary from normal to a degenerative left shift, depending on the severity of the case. urinalysis reveals an increased specific gravity. treatment. treatment is aimed at correcting cardiovascular shock, dehydration, acidosis, and toxemia and removing or neutralizing the offending feedstuffs. iv fluids containing % sodium bicarbonate should be administered. , oral fluids are contraindicated because they cannot be absorbed and may increase the rumen distention and discomfort of the animal. nsaids are indicated for toxemia (flunixin meglumine, . to . mg/kg iv). , oral administration of magnesium hydroxide and magnesium oxide ( g/kg) may neutralize the acidic ph and is sufficient in mild cases. however, if much of the feed is still in the rumen, these two alkalinizing agents will only work temporarily. oral antibiotics have been recommended to kill rumen microflora and stop fermentation. however, the authors of this chapter feel they are contraindicated because the gram-negative anaerobes that need to flourish to reestablish normal rumen microflora are susceptible to most antimicrobials effective against lactobacillus species. removing the substrate for the lactobacillus species is more effective. because orogastric tubes with large enough bores to reflux feedstuffs are too large for sheep and goats, rumenotomy is indicated in severe cases to remove the feed (see the section on rumenotomy in this chapter). after the rumen ph is cor-rected, transfaunation of the rumen microflora with about qt of rumen fluid from a small ruminant is beneficial (box - ). thiamine supplementation (vitamin b , mg/lb subcutaneously [sc] three times a day [tid] to four times a day [qid]) is indicated until rumen function returns. in certain instances, calcium may be indicated and can be included in the iv fluids (calcium gluconate). the clinician should avoid mixing calcium salts and sodium bicarbonate. bacterial leakage into the rumen wall, liver, and systemic circulation makes antimicrobial therapy necessary. the systemic antimicrobial agent of choice is penicillin (procaine penicillin g, , iu/kg im bid) because anaerobes are the most likely offending organisms. if treated aggressively, the prognosis for immediate survival is good. feed (grass hay only) and water should be limited until rumen contractions return to prevent overdistention of the rumen. the chronic sequelae discussed previously influence long-term survival. prevention. prevention involves introducing concentrate feeds slowly to allow rumen microflora adaptation. dietary change from a lower to a higher fermentable energy concentration should occur slowly and preferably over a -to -week period. in the case of animals being fed high-grain rations (e.g., club lambs, feedlot lambs, dairy goats), buffering agents can be added to the diet. rumen buffers may improve milk production, increase feed intake, and increase rate of gain. the crude fiber content should comprise a minimum of % of the diet's total digestible nutrients (tdn). for example, the tdn is %, the minimum acceptable crude fiber is %. crude fiber levels lower than this can be fed for short periods if the rumen is properly adapted, but problems may nevertheless occur. sodium bicarbonate is probably the most commonly used buffer; it can be offered free choice or included in the diet as % of dry matter intake. calcium carbonate or limestone (which both have low can be collected through a weighted orogastric tube. alternatively, fluid can be collected from any normal ruminant at slaughter. handling rumen contents collected from a fistulated cow or at slaughter can be strained through gauze or cheesecloth to separate the fluid from the fibrous contents. fluid collected through a weighted tube should be ready for storage. rumen fluid should ideally be administered immediately. however, it can be stored for to hours. the surface of the fluid should be covered with a layer of mineral oil to maintain an anaerobic environment and stored at refrigerator temperature. caution: do not store rumen fluid in a closed container because it may explode. rumen solubility) and magnesium oxide (which has poor palatability) also can be included in the feed. magnesium oxide should be limited to . % to . % of the dry matter intake. pathogenesis. reticulitis and rumenitis can result from chemical or mechanical damage to the mucosal lining of the reticulorumen. the most common cause of chemical damage is rumen acidosis. however, ingestion of caustic toxins also can damage the mucosa. mechanical damage can occur from ingested foreign bodies or the formation of rumen bezoars. in cattle, some viruses such as the ones that cause bovine virus diarrhea and infectious bovine rhinotracheitis can infect the rumen wall. similar viruses have yet to be identified in sheep and goats. after the mucosa has been damaged, secondary infection by bacteria or fungi can occur. previous treatment with oral antibiotics may predispose to fungal infections of the rumen wall, especially if the mucosa is already damaged. actinobacillosis, actinomycosis, and tuberculosis rarely affect the rumen wall. tumors of the rumen wall also have been reported. , not all of these causes of reticulitis and rumenitis have been reported in sheep and goats, but all are potential problems. clinical signs. the clinical signs of these diseases are vague. anorexia and forestomach hypomotility may be the only clinical signs. confirming a diagnosis also may prove difficult. samples of rumen fluid may only show changes associated with anorexia (alkaline ph, decreased numbers and motility of protozoa, prolonged mbr time; see table - for normal values). occasionally fungal organisms may be seen on diff quik stained slides of rumen fluid. in these cases a diagnosis of fungal rumenitis should be made. an exploratory laparotomy and rumenotomy may be required to diagnose foreign bodies or masses. rumen parakeratosis is characterized by dark, thickened, and clumped rumen papillae. it is seen mainly in feedlot lambs that consume finely ground or pelleted rations. the parakeratotic rumen papillae are fragile and predisposed to damage, which can increase the chances of rumenitis. treatment and prevention. treatment depends on the inciting cause. dietary changes should be made to decrease energy density and increase fiber intake. mild rumenitis may improve with time and supportive care (transfaunation, fluid support, high-quality feed). fungal rumenitis can be treated with thiabendazole ( mg/kg orally). severe changes may lead to scarring and permanent impairment of rumen function. traumatic reticuloperitonitis is not as common in small ruminants as in cattle, but it has been reported. goats are affected more commonly than sheep. this is probably because of the dietary habits of small ruminants; they tend to be selective grazers and do not "vacuum" the ground as cattle do. offending foreign bodies that cause traumatic reticuloperitonitis include pieces of wire and needles. , the clinical signs are identical to those in cattle and may include anorexia, depression, colic, signs of heart failure, and evidence of draining tracts from the chest cavity. treatment is usually difficult. rumen impaction can occur after dehydration, blockage of the omasal orifice by a foreign body, sand ingestion, or consumption of diets high in fiber and low in digestibility. clinical signs are nonspecific, but the firm rumen can usually be palpated in the left flank. the feces may be scant and dry. oral fluids containing magnesium sulfate ( g) may loosen impactions, but a rumenotomy is required in severe cases. to reduce rumen fill, sheep or goats should ideally have feed withheld for hours before rumenotomy. however, this is usually impossible because in most cases rumenotomy is an emergency procedure. the perioperative administration of antimicrobial agents is essential because even with meticulous technique some contamination of the incision site and possibly the peritoneal cavity is inevitable. because the rumen microflora is predominantly composed of anaerobic bacteria, penicillin ( , iu/kg) is the antimicrobial agent of choice and should be administered to hours before surgery. if the rumenotomy is being performed in an emergency situation, penicillin salts (potassium or sodium) that can be given iv provide therapeutic concentrations more rapidly than procaine penicillin. nsaids (flunixin meglumine, . to . mg/kg iv) also are recommended before surgery. if necessary, treatment of cardiovascular shock and dehydration with iv fluids also should begin before surgery and continue until the animal is rehydrated and in stable condition (see appendix ii). general anesthesia is recommended, but heavy sedation and local anesthetic infiltration of the incision site can be efficiently used (see chapter ) . the clinician should clip and surgically prepare a square area from cm in front of the last rib to the tuber coxae, and from the dorsal midline to the lower abdomen, encompassing the entire left paralumbar fossa. the surgeon makes a skin incision approximately cm longer than the width of the hand cm caudal and parallel to the last rib. the incision is continued through the muscle layers into the abdomen. because the abdominal wall is relatively thin, the surgeon should take care not to enter the rumen or bowel. the surgeon grasps the rumen wall and pulls it through the incision; suturing it to the skin with a simple continuous circular pattern around the entire incision. this forms a seal that minimizes rumen content contamination of the deep layers of the incision and peritoneal cavity. the rumen wall is then incised inside the circle of sutures. the incision in the rumen wall should be large enough for the surgeon to put his or her hand inside the rumen without traumatizing the rumen wall. after the rumen has been explored and emptied and the primary reason for doing the procedure has been completed, the surgeon closes the rumen wall in a continuous inverting pattern (cushing, lembert, or guard's rumen stitch) with absorbable suture ( catgut). the area should be rinsed with copious amounts of sterile isotonic fluids, and a new set of sterile instruments, sterile gloves, and surgical attire should be used for the remainder of the surgery. the surgeon then removes the suture securing the rumen to the skin and rinses the area again before performing routine closure of the abdominal muscles and subcutaneous layers with absorbable suture ( catgut) in simple continuous patterns, taking care to close dead space between layers. the skin is closed with a continuous pattern (ford interlocking) using a nonabsorbable suture material. the sheep or goat should be observed closely by the clinician for signs of complications, including peritonitis, incisional dehiscence, incisional hematoma, abscess, and hernia formation. penicillin therapy (procaine penicillin g, , iu/kg bid) should continue for at least days. the skin sutures can be removed to days after surgery. abomasitis and abomasal ulcers in adult sheep and goats are associated with rumen acidosis or chronic rumenitis but also can be caused by infections. [ ] [ ] [ ] [ ] finely ground feeds, pelleted rations, systemic stress, and feeding lush forages have all been implicated. anecdotal associations with mineral deficiency (copper) have gone unproved. clinical signs and diagnosis. this disease often goes unnoticed in mild cases, and the most common signs are anorexia and colic. no definitive antemortem diagnostic tests are available. fecal occult blood is often absent. occasionally dark stool, altered appetite (wood chewing), and bruxism are seen. therefore other causes of colic should be eliminated. diagnosis is based on clinical signs. effective therapy can be difficult. oral medications such as coating agents must first pass through the rumen, and therefore arrive at the abomasum diluted. iv (not oral) ranitidine ( mg/kg once a day [sid]) may be beneficial. herd problems of rumen acidosis may be addressed with buffers in the feed. a syndrome of abomasal hemorrhage, bloat, and ulceration is seen in lambs and kids to weeks of age. sarcina-like bacteria, clostridium falax, clostridium sordelli, and clostridium septicum have been isolated from many of these cases. [ ] [ ] [ ] [ ] c. septicum infections of the abomasum are commonly called braxy. the feeding of milk replacer free choice, iron deficiency, and bezoars have been implicated as predisposing factors. , clinical signs. the signs of this syndrome are severe, acute abdominal distention; colic; and death. [ ] [ ] [ ] [ ] diagnosis and treatment. the diagnosis of this condition is by postmortem examination. treatment in suspected antemortem cases is unsuccessful. prevention. adding formalin to milk replacers and vaccinating for clostridial diseases may decrease the occurrence of this disease. , lambs or kids on problem farms can be vaccinated for clostridium species during the first week of life with multivalent bacterins. similar to rumen impaction, abomasal impaction usually occurs when poor-quality roughage is fed, but it also can be seen with foreign body obstruction of the pylorus. , , goats appear to be more commonly affected than sheep, and boer goats are more commonly affected than angora goats. pregnant animals may be more prone to this condition. clinical signs and diagnosis. affected animals are usually anorexic. they have mild distention of the ventral abdomen, and in some cases the firm abomasum can be palpated through the abdominal wall on the right side. weight loss may be apparent. clinicopathologic evaluation may be normal, or mild hypochloremic metabolic alkalosis may be present, with elevated rumen chloride concentrations (more than meq/l). treatment. diet changes and mineral oil by mouth (po) are the most commonly employed treatments. abomasotomy can be attempted, but it has rarely been reported in small ruminants and does not usually improve the animals' long-term prognosis. when attempting abomasotomy, the clinician should perform the procedure with the animal in dorsal recumbency and under general anesthesia. the abomasum can best be visualized through an incision parallel and to the right of midline, caudal to the xyphoid process. the prognosis is poor. prevention. dietary manipulation to improve feed or forage quality is the best mode of prevention. abomasal emptying defect is a disease that presents similarly to abomasal impaction but is recognized only in suffolk sheep. the underlying cause is unknown. unlike abomasal impaction, this disease is associated with concentrate feeding and often occurs around lambing time. the clinical signs are chronic weight loss, abdominal distention, and anorexia. clinical pathology and rumen chloride levels are the same as described for abomasal impaction. on necropsy the abomasum is greatly distended, and the contents may be liquid or dry. treatment with laxatives, cathartics, motility modifiers, and abomasotomy has been mostly unsuccessful. [ ] [ ] [ ] azalea, laurel, and rhododendron toxicity members of the azalea, laurel, and rhododendron plant group produce andromedotoxins that alter sodium metabolism, resulting in prolonged nerve depolarization. these plants are cardiotoxic, but affected animals generally exhibit acute gastrointestinal upset. these evergreen shrubs produce thick, dark green leaves. they also have five-lobed, white to pink, saucer-shaped flowers that bloom around july. some of these plants are grown as ornamental shrubbery around homes, whereas others grow wild along streams, cliffs, and rocky slopes. they can be short or tall (as large as m) and can form thickets. all parts of these plants are toxic. clinical signs. animals browsing a new area, those fed clippings from trimmed azalea hedges, and underfed, hungry animals given access to these plants are likely candidates for intoxication. animals that ingest as few as two or three leaves may show signs of salivation, grinding teeth, nasal discharge, colic, epiphora, and acute digestive upset within hours of ingestion. as the intoxication progresses, animals become depressed and exhibit projectile vomiting, frequent defecation, and a slowed pulse. terminally intoxicated animals become paralyzed and comatose. some sheep and goats develop aspiration pneumonia secondary to intoxication. diagnosis. the diagnosis of this condition is usually based on clinical signs coupled with a history of ingestion of one of these plants and/or the discovery of these plants in the gastrointestinal tract. treatment. intoxicated animals may recover in to days without any therapy if the offending plants are removed from the diet. however, the administration of charcoal ( to g/kg po), atropine ( . to . mg/kg iv), other antiarrhythmic drugs, and iv fluids all may be indicated. to manage the aspiration pneumonia, the administration of antibiotics (penicillin , units/kg bid im) and oral magnesium hydroxide also may be beneficial. obviously, any existing dehydration should be corrected (see appendix ii). mountainous or hilly areas should be fenced. feeding shrubbery clippings is discouraged. diarrhea in lambs and kids is a complex, multifactorial disease involving the animal, the environment, nutrition, and infectious agents. decades of research have been devoted to the study of the pathophysiology of infectious diarrhea of calves; the pathology in lambs and kids is quite similar. despite improvements in management practices and prevention and treatment strategies, diarrhea is still the most common and costly disease affecting neonatal ruminants. [ ] [ ] [ ] [ ] some general preventive measures (e.g., improved sanitation) decrease disease no matter the cause. however, specific control measures such as vaccination require the definition of a specific cause of diarrhea. table - lists the agents most likely to cause diarrhea in lambs and kids, tissues or other samples required for diagnosis, and commonly employed test methods. the color and consistency of the feces and any gross lesions can appear similar no matter the cause. therefore laboratory identification of infectious agents and tissue histopathology are key to establishing a diagnosis. because autolysis and secondary bacterial invasion of the gut begins within minutes of death, necropsy samples taken immediately from euthanized lambs and kids yield the most reliable diagnostic material. mixed infections with two or more pathogens are common, and pathogens that are a problem on a farm change from year to year. , , in some cases an underlying nutritional deficiency or excess may occur concurrently with an infectious agent. therefore the clinician should be careful to take a variety of samples to ensure that all pathogens and predisposing factors involved are recognized; continued reevaluation of the causes of diarrhea is crucial. examination of several cases, with a focus on those in the acute phases, is important. although examination of antemortem fecal samples can be diagnostic, laboratory testing of tissue samples may yield better results. treatment and preventive measures specific to a particular disease are discussed with that disease in the following paragraphs. general supportive treatment and control measures are covered at the end of this section. four major pathogens cause diarrhea in lambs and kids during the first month of life: enterotoxigenic escherichia coli (etec), rotavirus, cryptosporidium species, and salmonella species. the relative prevalence of these infectious agents varies greatly among studies. this variance most likely results from differences in location, season, diagnostic techniques, and the occurrence of mixed infections. other, less common causes of diarrhea in neonates are giardia infections and nutritional diarrhea. pathogenesis. etec employs two virulence factors to cause disease. the first is the ability to attach and colonize the intestinal villi, which is accomplished via fimbria or pili. the most important fimbria in lambs are k and f . , the fimbrial antigens can be recognized from samples sent to most diagnostic laboratories and are im- portant in diagnosing this agent as a cause of diarrhea. after the organism attaches to the villi, it produces the second virulence factor, enterotoxin. enterotoxin interferes with the normal physiology of the gut, with resultant diarrhea. calves have an age-associated resistance, most likely related to the blocking of fimbrial attachment to the gut, so etec occurs mainly in calves less than a week old. , the mode of infection is fecal-oral. clinical signs. etec is seen in lambs and kids less than days of age but is most common at to days of age, so age-related resistance also may occur in these animals. , it usually presents as an outbreak in lambs and kids between and hours of age. because etec causes a "secretory" diarrhea, bicarbonate loss in the diarrhea leads to severe acidosis, with lambs and kids quickly becoming dehydrated and recumbent. however, many infected animals die before developing diarrhea. affected neonates are depressed, stop nursing, and may show excessive salivation. fluid sequestration in the abomasum causes a "splashing" sound on movement.this condition results in high mortality if animals are not treated promptly. diagnosis. fecal culture and serotyping for the k and f fimbrial antigens are the basis for diagnosis. because many nonpathogenic e. coli are normal gut inhabitants, simply culturing this organism is usually insignificant. occasionally the bacteria do not express the fimbrial antigens in culture, so etec cannot be ruled out if the culture is negative for k and f . histologic evidence of colonization of the small intestine can support a diagnosis. treatment. supportive care consisting of fluid therapy with either oral, iv, or sc administration of a polyionic solution is the mainstay of therapy. the use of oral antimicrobial agents is controversial. although antibiotics may kill the etec, they also may interfere with normal gut flora. if fluid support is provided, the diarrhea usually subsides without antibiotic treatment. still, oral neomycin ( to mg/kg bid) or trimethoprim sulfa ( mg/kg po) and systemic ampicillin ( to mg/kg im bid) or amoxicillin ( to mg/kg im tid) may be beneficial. nsaids are indicated to decrease inflammation of the gut and provide some analgesia. the use of flunixin meglumine ( to mg/kg im) has been shown to decrease fecal output in etec infections in calves and appears to be beneficial in lambs. prevention. it is recommended that clinicians vaccinate ewes and does with bovine etec vaccine before they give birth to increase passive immunity. , , monoclonal and polyclonal antibody products for calves may be beneficial during an outbreak if it can be given to lambs or kids within the first hours of life. the use of neomycin ( to mg/kg po bid) in lambs that appear normal may help stop the progression of an outbreak. shearing ewes prepartum to minimize fecal ingestion by neonates and ensuring that newborns ingest adequate colostrum both help decrease the incidence of this disease. making sure that ewes and does give birth at a . to . body condition score increases the chance of adequate colostrum manufacture by the dam. pathogenesis. lambs and kids are infected with group b rotaviruses, whereas most other animals and human beings are infected with group a rotaviruses. rotaviruses infect villus tip cells of the small intestine, which results in villus atrophy and malabsorptive diarrhea. clinical signs. rotavirus generally causes diarrhea in lambs and kids to days old, but older animals also can be affected. young animals can become very depressed and dehydrated. , , , diagnosis. detection of the organism by electron microscopy of fecal or colonic samples or by immunologic techniques on feces or tissue sections is the basis of diagnosis. , because these organisms are sloughed with the villus tip cells they infect, and viral antigens are complexed with the lambs' and kids' antibodies, tissue samples from acutely infected animals are best. rotavirus has been detected in animals without diarrhea, so other causes of diarrhea should be investigated as well. , treatment and prevention. rotavirus is treated with supportive care. prevention by vaccination of ewes and does with bovine rotavirus vaccines before they give birth is recommended to increase passive immunity. clinical signs. cryptosporidia can cause diarrhea in lambs and kids to days of age. , , affected animals are often active, alert, and nursing. the diarrhea is usually very liquid and yellow. diarrhea can vary from mild and self-limiting to severe, especially with mixed infections. , , , relapses are quite common, and this organism usually occurs as a component of mixed infections. diagnosis. acid-fast staining of air-dried fecal smears is a quick and easy method of diagnosis. examination under ϫ to ϫ magnification reveals round protozoa that have taken up the red color of the carbol fuchsin portions of the stain on a green background (figure - ). although they can be diagnosed by fecal flotation, their very small size ( - mm) makes this method difficult and subject to false negative results. , both immunologic and polymerase chain reaction (pcr) techniques have been developed to improve detection limits. , cryptosporidia also can be identified with histology. cryptosporidiosis is a zoonotic disease, and people can easily become infected from handling infected animals or feces. prevention. no consistently effective treatment for cryptosporidiosis in ruminants has been identified. anecdotal reports suggest that decoquinate and monensin sodium may be useful in control of cryptosporosis. decoquinate ( . mg/kg po) may be very useful in prevention of cryptospirosis in goats and possibly kids. during an outbreak affected animals should be isolated from the rest of the flock. no new animals should be added to a pen in which the disease has been diagnosed. keepers should depopulate pens in which the disease has been diagnosed and attempt to clean the environment. cryptosporidiosis can be particularly difficult to control because of the organisms' persistence in the environment and resistance to most chemical disinfectants. however, ammonia ( % to %) and formalin ( %) seem to be most effective. , feeders should be constructed to minimize fecal contamination. studies are currently underway to develop a vaccine for cryptosporidiosis in cattle. early results are favorable, and this may prove the best way to control the disease in the future. this is potentially a zoonotic disease, and therefore clinicians and keepers should exercise great caution when handling affected animals. pathogenesis. the bacterial genus salmonella has thousands of serotypes, and all can potentially cause diarrhea in animals. salmonella can cause diarrhea in lambs and kids of any age. , the microbes produce enterotoxins, are invasive, and cause severe inflammatory disease and necrosis of the lining of the small and large intestines. clinical signs. animals less than week old are more likely to die acutely without clinical signs, whereas animals older than week are more likely to have diarrhea. , , an acute onset of fever, depression, tenesmus, and shock is occasionally observed. salmonella-induced diarrhea is more likely to contain blood. this also is a zoonotic disease that warrants protective measures. a diagnosis of this condition is based on culture of the organism in feces or tissues and histologic examination of the small and large intestine. more sensitive pcr techniques for identifying salmonella species in feces are being developed. the diarrhea may occa-sionally contain fibrin, but many animals die before this is observed. clinicians may note leukopenia or leukocytosis in the cbc results. treatment. therapy for salmonella-induced diarrhea involves supportive care and possibly parenteral antimicrobial therapy. the use of antimicrobial agents is controversial and probably does not influence the gastrointestinal infection. however, because this is an invasive organism, parenteral use of antimicrobial agents may be beneficial in preventing septicemia. antimicrobial susceptibility patterns are difficult to predict for salmonella species, so antimicrobial therapy should be based on culture and sensitivity results. ceftiofur sodium ( . to . mg/kg im bid) or trimethoprim sulfadiazine ( mg/kg sc sid) can be administered until antimicrobial sensitivity results are known. prevention. latent carriers of salmonella can potentially shed organisms to other animals, particularly when they are stressed. newly introduced animals should be isolated for month, and fecal culture should be considered. bleach is an effective disinfectant to use during an outbreak. identification of carrier animals by fecal culture is recommended for herd problems. vaccine efficacy is questionable, and to date its effects have not been thoroughly evaluated in sheep and goats. giardia: giardia-induced diarrhea is more commonly seen in but not limited to -to -week-old lambs and kids. , the diarrhea is usually transient, but infected animals can continue to shed cysts for many weeks, even when they are clinically normal. , , therefore simply finding the agent in feces does not mean it is the cause of diarrhea, especially in older animals. however, these animals may be a source of infection for other animals and possibly humans. , iodine-stained wet mounts of feces or tissue is the classic method of diagnosing giardiasis, but more sensitive immunologic techniques are now available. , infected animals can be treated effectively with fenbendazole ( to mg/kg bid for days or sid for days). giardia has historically been treated with metronidazole ( mg/kg po sid for days). however, use of this drug class in food animals is currently illegal in the united states.this is potentially a zoonotic condition. infectious agents are not the only cause of diarrhea in neonates. nutritional problems can result in diarrhea, but these causes are overshadowed in the literature because the resulting diarrhea is usually mild and subsides without treatment. nutritional diarrhea is most common in orphaned animals as a result of keepers offering poorquality milk replacers, making mixing errors, or feeding large amounts infrequently (see chapter ) . diarrhea re- sulting from consumption of lush pasture or high-energy rations is a common occurrence. in most cases such diarrhea is self-limiting. the incidence of this form of gastric upset can be minimized by a slow introduction (over to weeks) to energy-dense diets. calves with infectious diarrhea that develop maldigestion or malabsorption can have secondary nutritional diarrhea from an inability to digest carbohydrates (lactose, xylose). , this has been reported in goats, and also is probably a cause of diarrhea in lambs. diarrhea resulting from primary lactose deficiency also has been reported in calves. calves on poor-quality milk replacers can develop an overgrowth of normal enteric e. coli, resulting in diarrhea. if lactose intolerance is suspected, decreasing the amount of lactose fed and using commercially available lactose enzymes may alleviate signs. the most common cause of diarrhea in older lambs and kids is nematode infestation. this condition is discussed later in this chapter in the section on causes of adult diarrhea. other major causes of diarrhea in older lambs and kids are c. perfringens and coccidiosis. c. perfringens types a, b, c, and d can all cause diarrhea in lambs and kids, but type d is the most common agent. , , pathogenesis. the disease occurs in peracute, acute, and chronic forms and is commonly called enterotoxemia or overeating disease. in the case of type c infection, a beta-toxin can cause acute hemorrhagic enteritis. type c infection is seen mostly in lambs or kids younger than weeks of age. an epsilon-toxin is responsible for pathology in type d infections. enterotoxemia is usually seen in rapidly growing feedlot lambs on high concentrate rations. it also is associated with other feeding changes, including changes in type of pasture. however, it occasionally occurs with no reported dietary changes, particularly in goats. , , this disease usually occurs in the fastest-growing and most well-conditioned animals. it can occur in vaccinated herds (again, more commonly in goats) so it should not be ruled out if a history of previous vaccination is present. clinical signs. the peracute form of clostridial infection is characterized by the rapid onset of severe depression; abdominal pain; profuse, bloody diarrhea; and neurologic signs. death occurs within hours of the onset of signs. sudden death may occur without signs of diarrhea. the onset of neurologic signs followed by sudden death is more common in sheep, whereas goats are more likely to show signs of diarrhea before death. similar but less severe signs are seen in the acute form of the disease. the chronic form occurs more commonly in goats. , diagnosis. antemortem diagnosis is based on clinical signs. at necropsy, c. perfringens can be cultured from intestinal tissue samples. however, the significance of a positive culture can be difficult to interpret because these organisms can be present in the gut normally and then proliferate after death. histologic examination of sections of the gut can be helpful. identification of the toxins (namely the epsilon-toxin) in intestinal contents is required for a definitive diagnosis. , because the toxin degrades within several hours of death, not finding the toxin does not preclude enterotoxemia as a diagnosis. treatment. treatment is rarely effective but consists mainly of aggressive supportive care. c. perfringens type d antitoxins ( to ml sc) can be administered to animals during an outbreak of enterotoxemia if clinical signs are noted before death. the antitoxin may be more effectively used as a preventive in the face of an outbreak. during an outbreak any animals that have not been vaccinated should be given the antitoxin and vaccinated with the toxoid simultaneously; those previously vaccinated should receive a booster vaccination. prevention. routine vaccination should start at to weeks of age and be followed by a booster to weeks later. however, on farms where the disease has become endemic, lambs or kids can be vaccinated and given antitoxin during the first week of life. yearly vaccination, preferably a few weeks before the ewes and dams give birth increases colostral immunity in neonates and improves prevention programs. goats may not respond as well to vaccination as sheep, so biannual or triannual vaccination is recommended, especially in problem herds. , vaccination with only c. perfringens types c and d and tetanus is superior to the use of more polyvalent clostridial vaccines. reducing the energy density of the diet and avoiding sudden dietary changes or alterations of the feeding routine are crucial to prevention. reducing internal parasites, particularly tapeworms, may further reduce the incidence of these disorders. pathogenesis. coccidiosis is a protozoan parasitic disease that is a common cause of diarrhea in lambs and kids. it also may cause subclinical production losses. clinical disease is often seen when some form of stress (e.g., dietary change, weather changes, parturition, weaning) is occurring on the farm or in the flock. eimeria species cause the disease in sheep and goats; each is infested with its own host-specific species. unlike cryptosporidium, which can be shed in feces in the infective stage, coccidia must sporulate outside the host to become infective. sporulation occurs under moderate temperatures and high moisture conditions. the nonsporulated and sporulated oocysts can survive a wide range of temperatures and may survive for years under certain conditions. clinical signs. lambs and kids are most susceptible to the problem at approximately to months of age, although younger animals may become infected. clinical disease is common after the stress of weaning, feed changes, or shipping. crowded conditions result in excessive manure and urine contamination, which is ideal for the buildup and sporulation of the oocysts. under these conditions, animals may be exposed to high numbers of infective organisms and develop diarrhea.the diarrhea in lambs and kids is usually not bloody, but it can contain blood or mucus and be very watery. anorexia, dehydration, weakness, rough hair coat, and death all may occur. weight loss is common, and constant straining can result in rectal prolapse. in severe cases the disease becomes protracted because of necrosis of the mucosal lining. even if these animals are treated appropriately, the diarrhea continues until the intestinal mucosa heals, which can take several days to weeks. permanent scarring can result in chronic poor development, even if the diarrhea subsides. , [ ] [ ] [ ] diagnosis. acute coccidiosis can be easily diagnosed from a direct smear or flotation of feces (figure - ) . in the chronic stages, most of the organisms have been shed and very low numbers are seen on fecal examination. because normal animals can shed small numbers of pathogenic species or large numbers of nonpathogenic species, interpretation of fecal examinations in the chronic stages of coccidiosis or in animals with diarrhea from other causes can be difficult. [ ] [ ] [ ] in these cases the clinician should rule out other diseases before making a diagnosis of coccidiosis. blood analysis may show both anemia and hypoproteinemia. treatment. treatment of affected animals with clinical signs includes supportive care and administration of coccidiostats. all animals in the group should be treated during an outbreak. the use of coccidiostats has little effect on the existing infection, but it does prevent the spread of the disease from continued exposure to infective organisms. many coccidiostats inhibit coccidia development and prevent disease if given prophylactically. they are of little value if they are given after the onset of clinical disease. sulfa drugs appear to be clinically beneficial, but they may simply decrease secondary or concurrent bacteria-induced diarrhea. because coccidia develop some resistance to coccidiostats, these drugs should be administered only before stressful events (e.g., shipping, weaning, parturition). the drugs listed in table - and trimethoprim sulfa ( mg/kg orally sid for days) are approved for use in the united states. , prevention. control involves improved sanitation and possibly the use of coccidiostats. preventing overcrowding decreases the buildup of manure and infective oocysts. exposure to sunlight and desiccation are two of the most effective means of killing the organisms. minimizing stress and optimizing nutritional intake also are important. coccidiostats available in the united states are shown in table - and appendix i. to avoid toxicity in growing animals, the clinician or keeper must carefully adjust dosages to the changing levels of feed intake as animals grow. all agents except amprolium should be fed for at least weeks. , this allows exposure and subsequent development of immunity to occur while preventing the detrimental effects of clinical disease. however, coccidia can become resistant to coccidiostats; fecal samples should be periodically evaluated after prolonged use of a particular product. anecdotal reports suggest amprolium resistance may occur on some farms. moreover, if amprolium is offered with a creep feed rich in thiamine, its ability to act as a thiamine antagonist may be compromised. year-round use of coccidiostats increases the potential for resistance. therefore they should be fed only during times of expected risk. the inclusion of lasalocid ( kg of % premix) or decoquinate ( kg of % premix) in kg of trace mineralized salt fed as the only source of salt for days prepartum can reduce the number of oocysts shed in ewe or dam feces. this practice can reduce the coccidia contamination of pasture and thereby remove a source of infection for kids and lambs. the benefits of administering lasalocid and monensin beyond coccidia control include increased feed efficiency, enhanced growth rate, and decreased incidence of free gas bloat. however, if coccidiostats are included in either mineral or feed supplements, inconsistent or depressed intake may result in subtherapeutic drug dosing. lambs are resistant to infection in the first few weeks of life. exposure to the protozoa during this time confers immunity and resistance to later infections. , adenovirus, caprine herpesvirus, coronavirus, campylobacter jejuni, yersinia species, and strongyloides papillosus can cause diarrhea in lambs and kids of various ages. , , enterohemorrhagic e. coli (ehec) and enteropathogenic e. coli (epec) also have been isolated in the feces of kids with diarrhea. , these e. coli types are k and f -negative. culture and serotyping of these organisms from feces and tissue samples with typical histopathologic lesions is diagnostic. although etec is not zoonotic, ehec and epec can potentially affect humans. although some causes of diarrhea have specific treatments, many animals need to be treated for dehydration and metabolic acidosis regardless of the inciting cause. animals with only mild diarrhea, especially mild nutritional diarrhea, may not require therapy unless they become dehydrated. if kids or lambs become less than % dehydrated and are only mildly depressed but still willing to nurse, they can be treated with oral electrolytes designed for calves. fluids can be administered by bottle or by tube if the animal will not nurse. the keeper or clinician should carefully adjust the amount of fluids for lambs and kids ( to ml, or to oz, as opposed to l in a calf ). because most electrolyte solutions designed for calves contain glucose, after they have been mixed they should be refrigerated and any leftovers discarded within hours. iv fluids may be needed to treat more severe dehydration. if the lamb or kid is too weak to stand, iv fluids are indicated. isotonic fluids containing electrolytes should be given to replenish losses. glucose can be added to fluids to make a % to . % solution. sodium bicarbonate also may be administered, especially if the dehydration is severe. a rule of thumb is to give one fourth of the calculated fluid need (see appendix ii) as isotonic bicarbonate ( . %). extra potassium ( to meq/l) can be added to fluids because most animals are severely dehydrated from diarrhea and depleted in potassium, even though their blood potassium levels may be elevated. if extra potassium is added, acidosis must be corrected concurrently. after correcting the dehydration, the keeper or clinician can offer oral electrolyte-enriched fluids to replace ongoing losses caused by continued diarrhea. removing milk or milk replacer from the diet is not recommended. young animals need nutrients, and even high-energy, glucose-containing electrolyte solutions are no substitute for milk. animals should continue to receive milk replacer in normal amounts or be allowed to nurse; they can be supplemented with oral electrolytes if necessary. animals being hand fed should be offered small amounts frequently to help minimize problems. electrolytes should never be mixed with milk, but should instead be given in separate feedings. if lactose deficiency is suspected, lactase drops or capsules (available in health food stores) can be added to milk or milk replacer. nsaids (flunixin meglumine, . to . mg/kg iv; ketoprofen, . to . mg/kg iv) are beneficial, especially if toxemia is involved, as in etec, enterotoxemia, and salmonellosis. it is the authors' opinion that antimicrobial agents should be reserved for proven outbreaks of salmonellosis and for animals with other causes of diarrhea that do not respond to fluid therapy and nsaids; these drugs should only be administered parenterally. the use of oral coating agents and antacids is popular, but it has not been shown to be beneficial and is not therapeutically logical in light of the pathogenesis of these diseases. probiotics may be beneficial in reestablishing the normal flora of the small intestine. the authors' rule of thumb is that nothing should be given orally except milk, oral electrolytes, and probiotics. ensuring adequate intake of high-quality colostrum and minimizing stress are important for prevention of all neonatal diseases. a normal lamb or kid will stand and nurse within minutes to hour of birth. the ingestion of colostrum within to hours is essential in preventing hypothermia and hypoglycemia and decreasing the incidence of various diseases. lambs or kids born as twins or triplets, weak or injured neonates, those born during severe weather, those born from a dam with dystocia, and those delivered by cesarean section are all candidates for colostrum supplementation. if supplemental colostrum is provided, it should be good-quality colostrum from females that have tested negative for johne's disease, ovine progressive pneumonia (opp), and caprine arthritis-encephalitis (cae). mixing colostrum from several cows decreases the incidence of the "cow colostrum-associated" hemolytic disease sometimes seen in lambs. if the lamb or kid is unable to nurse, it should be tube fed ml/kg of colostrum. the veterinarian or animal handler can sit comfortably holding the lamb or kid in sternal recumbency in the lap. a to french soft feeding tube is then lubricated, inserted into the side of the mouth, and passed slowly. if the tube is placed in the trachea, the lamb or kid will become uncomfortable and may shake and cough. the tube may be palpated on the left side of the throat. after the tube has been slowly passed to the thoracic inlet, colostrum can be administered by gravity flow (see chapter ) . prepartum shearing of the dam may decrease the ingestion of feces by lambs. good sanitation of lambing and kidding areas is paramount in management programs that stress prevention. the presence of organic matter interferes with the effectiveness of many disinfectants, so removal and proper disposal of feces, carcasses, and placentas are essential. when disposing of waste material containing either cryptosporidium or giardia, the keeper should be careful to avoid contaminating water sources. infected animals should be isolated to prevent spread of the infection throughout the flock. in general, infected animals should remain in the environment where the infection was first diagnosed, because it is already contaminated. removing pregnant ewes or dams to a clean area before lambing or kidding helps minimize the continued spread of disease. if possible, lambs and kids already born but not showing clinical signs should be removed to a third area. if "safe" pastures are maintained for internal nematode control, they are ideal for use in an emergency situation to control these diseases. although some animals may appear normal, they may be incubating and possibly shedding the infective agents of a disease. if such animals are moved with pregnant females, they can be a source of contamination in a clean area. if healthy lambs and kids cannot be moved to a third, relatively safe area, they should be left with the clinically infected animals because they have already been exposed. . schultheiss p: diarrheal disease in calves, large anim vet ( ) the differential diagnosis list for acute and chronic diarrhea in small ruminants is very long. the most common cause of diarrhea in adult sheep and goats is parasitism; another major cause is johne's disease. both of these diseases are discussed in the following sections. other causes of acute diarrhea include rumen acidosis, peritonitis, endotoxemia, and ingestion of toxins. the list of toxins that cause diarrhea also is very long, and often the diarrhea is not the primary clinical sign. some of the more common toxins that produce diarrhea are arsenic, toxic amounts of salt, levamisole, copper, oak, selenium, and pyrrolizidine alkaloids. salmonella species and chronic enterotoxemia can cause diarrhea in adult animals. coccidiosis can occur in adults under severe stress or in animals that possess limited immunity because of lack of exposure. hepatic and renal disease and copper deficiency are sometimes accompanied by chronic diarrhea, but weight loss is a more common sign in adults. etiology and pathogenesis. sheep and goats are infested with many of the same gastrointestinal nematode parasites as cattle, but these parasites tend to either be species-specific or have some amount of host specificity. sheep and goats are susceptible to the same nematodes and tend to share resistance to those that infect cattle and horses. the major gastrointestinal nematodes that parasitize pastured sheep and goats are haemonchus, ostertagia, trichostrongylus, cooperia, nematodirus, oesophagostomum, and bunostomum species. the acronym hotc comes from the first letter of each of the first four genera of parasites listed. the specific parasites that produce disease vary from flock to flock. climate usually determines which parasites are of clinical significance on a farm, and the weather determines when the parasites will be transmitted and infective. in much of the united states, haemonchus is the most significant parasite with respect to both clinical disease and anthelmintic resistance. most of these parasites affect the abomasum or small intestine of young, recently weaned animals and occasionally adult animals. sheep (and, to a lesser extent, goats) that are older than months may be less susceptible. overcrowding and overgrazing with concurrent pasture mismanagement and malnutrition usually increase susceptibility to these parasites. inadequate nutrient or protein intake may result in greater susceptibility. the life cycle appears to be similar in most of these parasite species. adults lay eggs that are passed in the feces; except for nematodirus species, the eggs hatch under favorable environmental conditions. the larvae go through several free-living developmental stages becoming infective. when the infective larvae are ingested by the host, the parasite completes its life cycle as an adult. trichuris eggs are the infective stage and can survive for extended periods in dry lots or barns. however, trichuris is associated with minimal pathology. during dry environmental conditions, fecal pellets tend to trap the nematode larvae, whereas in wet conditions, larvae are released onto the pasture. therefore drought conditions followed by rain can result in devastatingly high rates of pasture contamination as larvae that have remained in fecal pellets are released. very high environmental temperatures result in shorter survivability of some stages of infective larvae. most of the larvae have adapted the ability to over-winter, but can survive only for short periods outside the host during spring. nematodirus is an exception in that the developmental stages leading to infective larvae occur while the microbe is still encapsulated in the egg. however, compared with other species of parasite, nematodirus is of minor importance. nematodirus battus may pose a threat to young, newly weaned and therefore immunologically naive grazing lambs. the hookworm bunostomum also is different, as it may infect the host by either oral ingestion or percutaneous penetration. with the exception of the small intestinal parasite strongyloides, lambs or kids fed indoors or in dry pens tend to be free of parasites. clinical signs. all intestinal nematode infections produce similar signs, although infection with the more rarely encountered bunostomum may perhaps result in more profound anemia. if they infest the animal in sufficient numbers, all nematodes may cause poor growth, decreased feed conversion, decreased milk production, weight loss, diarrhea, anemia, ventral edema (bottle jaw), midline edema, and death. again, all these parasites can potentially result in disease, but haemonchus is the most devastating, particularly in more temperate regions. diagnosis. antemortem diagnosis of nematode infestation is made by examining the feces for nematode eggs. although a direct fecal smear can be examined, the mere presence of parasite eggs is not helpful in determining the parasite load of an animal or animals. quantifying of the epg of feces is the best way of estimating parasite loads. the quantitative mcmaster's technique for determining the epg of feces is shown in box - . common nematode eggs are shown in figure - . treatment and control program. after taking a thorough history of the previous parasite control program used on a farm and determining its effectiveness, the clinician can design and implement a new control program. , however, before deciding to implement a deworming program, the clinician should decide which parasites are in need of control and whether control of these parasites is cost effective in a particular flock. whenever possible, a dewormer that can reduce epg counts on the farm by % should be identified and used for at least a year. of all the parasite prevention programs, strategic deworming or a combination of strategic and tactical programs appear to produce the best results. , , strategic deworming is used when most of the parasites are inside the animals and not on the pasture. in northern climates, strategic deworming can best be carried out during the winter, when the nematode parasites are in a hypobiotic state. when environmental conditions are inhospitable for the survival of the infective larvae, some of the most pathogenic nematodes (e.g., haemonchus) may become hypobiotic; that is, they assume a state of arrested development. they may then mature to the adult stage when environmental conditions become conducive for the survival of their eggs or larvae. preventing or decreasing the numbers of maturing adults by killing the larvae before the periparturient rise in parasite egg production and pasture contamination is an excellent management tool. , , unfortunately, in warmer, more temperate to subtropical environments, this method is less effective because larvae can survive the environment for longer periods. the addition of a protein supplement overlapping the expected periparturient rise has been shown to decrease the number of parasite eggs shed around the time of parturition. however, the cost of the protein supplement may outweigh its benefits. a strategic program entails the use of an anthelmintic agent that is capable of killing encysted larvae. animals are then moved to parasite-free or safe pastures-areas in which the level of parasite contamination is too low to result in infection of grazing animals. examples of safe pastures include pastures where sheep or goats have not grazed for to months in the spring or fall, respectively (and depending on the climate); pastures used for hay production; new pastures (i.e., those used for corn, cotton, or other crops); and pastures grazed by horses or cattle. the use of safe pastures is paramount in any de-worming program. rotating pastures after less than months during the warm part of the year or less than months during cooler months is ineffective. however, if pastures are tilled and replanted, by the time new grazeable forage is available, infective parasite larvae will be dead or significantly decreased. an alternative to pasture rotation is to perform an initial strategic deworming before lambing or kidding and follow it with two to four more dewormings at week intervals throughout the lambing and kidding period. [ ] [ ] [ ] treating lambs or kids at weaning and moving them to a safe pasture is a form of strategic deworming. in lambs or kids to be sold at an early age, the administration of a single anthelmintic treatment followed by a move to a safe pasture may be all that is required. a "double treat and move" system is required for lambs kept for to weeks after weaning, particularly during the summer. , this form of strategic deworming requires two treatments to weeks apart as well as two safe pastures. in northern climates where animals are moved to a dry lot or barn for the winter, a strategic anthelmintic administration as animals are moved off pasture can help reduce the parasite burden through the winter. if this deworming is followed by minimal or no exposure to grazing areas and another dewormer is administered before the spring rise in fecal egg counts, the total parasite burden on spring pasture can be drastically reduced, effectively controlling parasites until summer or fall. tactical deworming programs are used to remove parasites from their hosts before they enter their reproductive phase and can contaminate the pasture. an example of tactical deworming is treating animals to days after a rain, particularly if the rain has followed a drought. parasite transmission is worse in most flocks during this time as pastures become heavily contaminated. mcmaster's counts of more than epg in the spring or more than epg in the fall warrant tactical deworming. , , opportunistic deworming and salvage deworming are usually less effective in long-term flock management. many times salvage deworming programs are used to save the lives of heavily parasitized animals. if animals are dewormed only after showing signs of parasitism (e.g., bottle jaw, anemia), animal and flock productivity have already been depressed. deworming during handling for other procedures (e.g., castration, vaccination, shearing) is an example of an opportunistic program. it is convenient but is not conducive to long-term flock health. flock work should be scheduled around parasite management programs, not vice versa. , suppressive deworming programs entail the use of anthelmintics at regular intervals, usually every to weeks. suppressive programs are labor-intensive, tend to be very expensive, fail to identify animals with superior immunity to parasites, and ultimately result in anthelmintic resistance despite initial effectiveness. , as a general rule, the more frequently deworming occurs, the more quickly resistance is attained to anthelmintics. after deworming, only resistant parasites remain to infect the animal and they are able to reproduce freely, resulting in proliferation of resistant strains. , using drugs that remain in tissues at inappropriately low concentrations and treating and retaining immunocompromised animals encourage the development of anthelmintic resistance. practices that ensure adequate dosages, proper treatment techniques, and appropriate types of anthelmintics should be emphasized. , the clinician should do everything in his or her power to minimize the incidence of anthelmintic resistance, both through their own actions and by counseling owners in proper use of deworming drugs. the product development market for anthelmintics is the cattle industry. the small sheep and goat markets simply use drugs made available for cattle. because most available anthelmintics are highly effective in controlling parasites, anthelmintic resistance in sheep and goats must be avoided. the anthelmintics that have been used previously on a flock, the route of administration (e.g., po, sc, im, pour-on), and the length of use should be determined. few dewormers are approved for use in sheep and goats, but many approved for use in cattle and horses may be effective. , if sheep graze with goats that harbor anthelmintic-resistant parasites, the sheep also may become infected. however, if sheep are allowed to graze while the goats browse and the two groups rarely mingle, less parasite movement will occur between these species. resistance to macrolides (e.g., ivermectin, doramectin, moxidectin) does occur. resistant worms are generally not very tolerant of cold temperatures and therefore resistance to this drug class in northern environments is not as large a problem as it is in more temperate or subtropical zones. although moxidectin is not approved for use in sheep and goats in the united states, it has been shown to be effective in cases where ivermectin resistance is encountered. still, this drug should be avoided until all other anthelmintics have failed. craig , has suggested that clinicians refrain from injecting or using pour-on macrolide preparations designed for cattle in small ruminants. this practice may enhance the development of resistant strains of some internal parasites because of inappropriately low drug absorption (with pour-on use) or long-term subtherapeutic levels (with injection). if resistance to tetrahydropyrimidines (e.g., morantel, pyrantel) occurs in a flock, levamisole also may be ineffective. morantel and levamisole resistance in parasites appears to be sex-linked. therefore if animals are not exposed to these drugs for several years, reversion to susceptibility can occur. , if resistance to one of the benzimidazole dewormers has been documented in a flock, some resistance to all members of that class is likely. benzimidazole-resistant haemonchus species appear to be more virulent, produce more eggs, cause greater environmental contamination, and survive in the environment as free-living larvae for longer periods. benzimidazole-resistant parasites apparently do not revert to susceptible forms, even over long periods. therefore the clinician or keeper should exercise caution to minimize resistance. benzimidazole efficacy can be improved by increasing dosages, dividing dosages into two treatments administered at -hour intervals, and instituting pretreatment fasting. if resistance to numerous classes of anthelmintics occurs on a farm, combining two of the resistant classes of dewormers (fenbendazole and levamisole) has proven effective. when using combined dewormers, the clinician should administer the full therapeutic dosage of each. anthelmintics are metabolized at different rates by sheep and goats. goats may require larger dosages of some dewormers than sheep. craig has suggested that if no dose rate is known for a particular anthelmintic for sheep or goats, the animals should be treated at twice the suggested cow dosage. pour-on anthelmintics designed for cattle tend to be of limited value when used topically on either goats or sheep. table - . to maximize a parasite control program, anthelmintics that appear effective should be used for only year before a new class of deworming drug is used. more frequent rotation (after less than year) of anthelmintic agents hastens resistance and should be avoided whenever possible. whenever a flock is dewormed, animals should be treated based on the heaviest animal in the group and not on the group's average weight. underdosing can hasten the formation of parasitic resistance and therefore should be avoided. holding the sheep or goats in a dry lot overnight or feeding only dry hay for to hours before and hours after deworming appears to improve the efficacy of some orally administered anthelmintic agents (benzimidazole). limiting feed intake before deworming slows the rate of passage of ingesta through the bowel, enhancing drug effectiveness. , , feed should never be withheld from sick or debilitated animals or late-term females. , , most dewormers may effectively control adult or larval parasites but are ineffective against eggs. therefore animals should be kept on a dry lot for as long as days after deworming, then moved to a safe pasture. use of this procedure minimizes parasite egg contamination of the new pasture because most of the egg-contaminated feces is voided within hours of deworming. if more than one dosage appears on the drug label, the larvacidal dose should be used (fenbendazole at mg/kg rather than mg/kg). anthelmintic effectiveness can be determined by comparing a mcmaster's fecal epg on the day of deworming with one taken to days later. if less than a % drop in epg is found, anthelmintic resistance exists and the animals should be switched to another class of dewormer. although it is a controversial method, the authors have used this technique to identify anthelmintic effectiveness for many years and on many farms and ranches. , the authors randomly collect feces from % to % (or a minimum of animals) of the sheep or goats on the farm. a composite sample is prepared by combining equal amounts of stool from all animals. craig has suggested that combining stool samples from many animals alters the accuracy of the tests because great individual variation in fecal egg counts occurs among animals. composite egg counts more accurately reflect parasite burdens in groups of young animals, and individual fecal examinations are more accurate in adults. , still, the authors prefer to use composite samples unless obvious differences in stool character or body condition score exist among the sampled animals. anthelmintic resistance can be minimized by using drugs that reduce fecal egg counts by %. pre-and postdeworming changes in epg should be evaluated yearly or whenever resistance is suspected. in vitro methods of assessing flock parasite resistance also are available at some diagnostic laboratories. in most in vitro tests, larvae are hatched from collected feces and the sensitivity of different anthelmintics is determined by larval exposure. these tests are very accurate but tend to be quite expensive. the most effective method to prevent anthelmintic resistance is to not use deworming drugs at all. one of the most overlooked management procedures is the identification and selection of parasite-resistant sheep and goats. some breeds or familial lines within breeds have excellent parasite resistance (e.g., gulf coast native and barbados sheep, some strains of spanish, pygmy, and tennessee myotonic-fainting goats). one study comparing boercrossed goats with non-boer crosses found that the boer crosses had significantly more parasite infestations. only a small number of flock members contribute the greatest amount of environmental parasite contamination because susceptible animals shed the most eggs in their feces. animals with the lowest epg in a flock may be those that possess the most parasite resistance. salvage deworming programs should generally be avoided, but they may be used as aggressive selection criteria. that is, animals that do well with little or no deworming, particularly those grazing heavily contaminated pastures, should be identified and retained in the breeding flock. those that become infected should be dewormed to salvage them or save their lives and then sold when possible. proper record keeping and identification of all animals is paramount in selecting for parasite resistance. , this aggressive approach can yield excellent results if it is carefully implemented, but devastating losses can occur if it is poorly managed. when introducing new animals to a flock, keepers should have biosecurity programs in place to limit the introduction of new or potentially anthelmintic-resistant parasites. new flock additions should be kept in a dry lot for weeks and dewormed at least twice with two different classes of dewormers during this period. the effectiveness of the anthelmintic agent used should be deter-mined by fecal examination before the animal is allowed contact with the rest of the flock. other nontraditional chemical methods of parasite control are used by some owners. some appear to be worthless (e.g., diatomaceous earth), but others (e.g., nematophagous fungi, herbal dewormers) may prove effective in some situations. pathogenesis. the most common gastrointestinal tapeworm of sheep and goats seen in north america belongs to the genus moniezia. cestodes (tapeworms) are usually of more concern to owners than clinicians, who generally consider them only incidental low-grade pathogens, particularly in adult animals. still, several to -foot-long tapeworms can compete with the host for nutrients, hinder normal gut motility, and excrete some toxic wastes into the host's gastrointestinal tract. mature tapeworm eggs are passed in the feces individually or protected in proglottides, which are usually visible to the owner. the eggs embryonate and infect a mite, a small pasture-living arthropod that serves as the intermediate host. a sheep or goat ingests the mite while grazing, allowing the tapeworm to complete its life cycle. clinical signs. tapeworms may rarely cause disease in lambs and kids less than months of age. anecdotal reports suggest a cause-effect relationship between heavy tapeworm infestation and an increased incidence of c. perfringens enteritis, digestive disturbances (e.g., diarrhea, constipation), poor condition, and anemia. ulceration at the site of attachment may be seen on necropsy. rarely species of trypanosoma, the fringed tapeworm, may cause liver condemnation. a presumptive diagnosis can be made by finding proglottides in the stool, eggs on direct smears, or eggs on fecal flotations (see figure - ).treatment with albendazole ( mg/kg), fenbendazole ( to mg/kg), or praziquantel ( to mg/kg) may be effective either with a single treatment or with daily therapy (e.g., fenbendazole daily for to days). because of the free-living nature of the arthropod intermediate host, animals are readily reinfected after treatment, which may give rise to the false assumption that the therapy was ineffective. again, tapeworm infestation may result in disease, but often it is easier to blame the tapeworm segment seen in the stool as a cause of disease than to implicate the unseen thousands of hotc complex parasites in the abomasum and small intestine of the animal. , johne's disease johne's disease (also called paratuberculosis) is a chronic wasting and diarrheal disease caused by the bacteria my-cobacterium avium subspecies paratuberculosis. transmission of the organism is primarily by the fecal-oral route. young animals are more susceptible to infection than adults. it can be transmitted through milk and placenta. pathogenesis. bacterial shedding in feces and milk and transplacental transmission is more common in animals showing clinical signs. [ ] [ ] [ ] therefore the offspring of infected animals and especially the offspring of animals showing clinical signs are most likely to acquire the infection. after an animal is exposed, it will either clear the organism or develop a chronic, persistent infection. the infection is most commonly isolated to the ileal regions of the small intestine, where it causes granulomatous thickening of the intestine and subsequent malabsorptive diarrhea. infected animals may be asymptomatic for years. clinical signs. morbidity rates are low (approximately %), but for every animal with clinical signs, several exist in the subclinical state, and may be a source of both horizontal and vertical transmission. both sheep and goats appear to remain asymptomatic until to years of age. the most consistent clinical sign in sheep and goats is chronic weight loss. chronic diarrhea occurs in approximately % of cases. signs may appear with or be exacerbated by stress, especially after parturition. , hypoproteinemia and chronic mild anemia are the only consistent clinicopathologic findings. because of their low protein levels, infected animals can develop submandibular edema. diagnosis is by culture of the organism from feces. unfortunately, this testing takes between and weeks, but it can detect % to % of clinically infected goats. sheep strains of johne's disease and some goat variant strains seem to be more difficult to culture in media used to identify cattle strains of the disease.therefore fecal culture in sheep and goats appears to be of limited benefit. , a relatively inexpensive and easily performed method of identifying approximately % of all clinically infected animals is acid-fast staining of fecal smears. , a pcr test of feces also is available, but its sensitivity is lower than that of fecal culture. good diagnostic results can be obtained with serologic testing for antibodies (e.g., agar gel immunodiffusion [agid], enzyme-linked immunospecific assay [elisa], complement fixation) in animals showing clinical signs. the specificity of all the serologic tests is greater than % in sheep and goats with signs of clinical disease, although the sensitivity is not as high. [ ] [ ] [ ] therefore a positive serologic test in an animal showing clinical signs indicates that the animal has johne's disease. however, the disease cannot be ruled out with a negative test. sheep and goats appear to respond differently in regard to the formation of antibodies. sheep tend to develop antibodies in the later stages of the disease, whereas antibodies may be detected much earlier in the goat. the agid test appears to be the best serologic test currently available. , the elisa and complement fixation tests can cross-react with corynebacterium pseudotuberculosis, making them of limited value in flocks with caseous lymphadenitis infections. , necropsy diagnosis is based on the finding of thickened, corrugated intestines, especially in the area of the ileum. acid-fast staining of impression smears (taken from the ileum and ileocecal lymph nodes) can help yield a quick diagnosis. the staining of numerous clumps of acid-fast rods is highly suggestive of johne's disease. prevention. johne's disease has no effective treatment, so prevention and control are imperative. however, preventing the introduction of johne's disease into a herd can be difficult. because animals with subclinical infection may not shed the organism or may shed only small quantities of it, fecal culture is helpful only if a positive culture is obtained. the sensitivity of serologic tests of animals with subclinical disease is low and variable among flocks. , negative test results in subclinically infected animals are common. however, the specificity of serologic tests remains high, and therefore a positive test is a valid reason to not purchase an animal. because johne's disease also occurs in cattle, supplemental colostrum supplies should come only from dairy herds with no history of johne's disease. after johne's disease is diagnosed in a herd, several control measures can be taken. sanitation is important because the organism is highly resistant in the environment (able to survive more than year under most conditions). reduced stocking rates, frequent cleaning of pens, and use of automatic waterers decrease fecal transmission. keepers should cull the offspring of infected animals. culling animals based on the results of agid tests or fecal culture of the flock is recommended. animals should be tested at least once a year. more frequent testing as resources allow speeds the identification of infected animals. a vaccine for cattle is only available in some locales and clinicians or keepers may require official permission to use it. vaccine use does not eliminate infection, but it can decrease herd prevalence, delay the onset of clinical signs, and decrease cross-transmission by infective bacterial shedding in the feces. any cause of intestinal obstruction that occurs in other ruminants may occur in sheep and goats. most of these diseases produce abdominal discomfort and occasionally abdominal distention. diagnosis can be difficult because rectal palpation cannot be performed on small ruminants. abdominal radiographs and ultrasonography may help differentiate these diseases, but exploratory surgery may be required to obtain a definitive diagnosis and select appropriate treatment. intussusception is more common in young animals, but it can occur in adults. it occurs when one segment of the intestine telescopes into an adjacent segment. any portion of the intestine can intussuscept, but the ileum and ileocecal junction are the most common areas involved. when intussusception occurs, the lumen of the intestine narrows to the point of obstruction. the initiating cause is not always known. , it is associated with an intestinal mass in adults and enteritis in young animals. oesophagostomum infestations have been implicated as a cause in sheep. clinical signs. the initial complaint is colic (manifested as kicking at the abdomen, repeated rising and lying down, and vocalization) followed by low-grade pain. true colic signs are variable in lambs and kids. in some cases, after the initial colic episode subsides, animals show no evidence of pain until the abdomen becomes distended. the time between the initial intussusception and abdominal distention depends on where the blockage occurs. intussusception of the ileal area may take several days to cause bilaterally symmetric abdominal distention. fecal output is scant, and what little there is may be dark or tarry, or may contain mucus. dehydration becomes evident, hypochloremic metabolic alkalosis may develop and rumen chloride levels may increase with obstructions of the duodenum. diagnosis. abdominocentesis may yield fluid compatible with a transudate (increased protein concentration and leukocyte numbers). radiography and ultrasonography reveal fluid-distended intestinal loops. occasionally the intussusception itself can be visualized with ultrasonography or palpable through the abdominal wall. if the disease is not treated, intestinal rupture and peritonitis can occur. treatment. surgical correction is required. if the intussusception is corrected early, the prognosis is good in the absence of peritonitis. fluid support is needed to correct dehydration and metabolic abnormalities. fluids should be administered iv until rumen function returns. ringer's solution with added calcium (approximately ml calcium borogluconate per liter) and potassium ( to meq/l) is a good choice for fluid therapy. ingested foreign bodies or bezoars can obstruct portions of the intestines. , the signs are similar to those of obstruction caused by intussusception and depend on the part of the intestine that is blocked. in some cases the obstructing body can be seen with radiography or ultrasonography. surgical removal is required for treatment. cecal volvulus and torsion of the root of the mesentery occur sporadically in sheep and goats. , extreme abdominal pain, rapid abdominal distention, and circulatory collapse are typical signs. immediate surgical correction and circulatory support are needed. atresia of the colon, rectum, and anus can all occur as congenital problems. the clinical sign of progressive abdominal distention usually is noted in the first week of life. atresia of the anus can be diagnosed on physical examination, but atresia of the colon and rectum may require contrast radiography for a definitive diagnosis. surgical establishment of anal patency can be performed for atresia ani. a permanent colostomy may be required for atresia of the colon and rectum. atresia of the anus and rectum are considered heritable in cattle. in the authors' experience, atresia ani is more common in sheep than in goats. if surgical correction of atresia ani is attempted, the animal should be neutered or kept out of the breeding program because of the potential genetic basis for this condition. occasionally a slight bulge in the skin may occur where the anus should be located, especially in male lambs. ultrasonography can be used to locate a fecesfilled rectum. for surgical correction, the clinician should locate the area where the anus should be, prepare it with sterile technique, and infiltrate it with a local anesthetic. the surgeon then makes a circumferential incision to remove the overlying skin covering the rectum. an alternative is to make an x-shaped incision into the rectum. treated animals should be given mineral oil, dss, or stool softeners as needed. ileus of the small intestine is a pseudo-obstruction that occurs when there is an absence of intestinal motility. the animal's failure to pass ingesta leads to signs similar to intussusception. the cause of ileus is usually unclear, but it often occurs secondary to systemic diseases. the same elements that cause rumen stasis may potentially result in intestinal stasis and ileus. symptomatic treatment with nsaids for pain and inflammation and fluids for dehydration is usually curative. however, if signs persist, surgical exploration is indicated to rule out true obstructive diseases. pathogenesis. infection of the peritoneal lining of the abdominal cavity may lead to septic peritonitis. common causes include uterine tears; rupture of the rumen or abomasum secondary to rumenitis, abomasitis, or abomasal ulcers; trocarization of the rumen for bloat; and rupture of the intestine secondary to obstruction. clinical signs. signs depend on the severity of the condition. abdominal discomfort and distention, dehydration, injected mucous membranes, depression, and death can all occur in cases of peritonitis. the presence of a fever is variable, both heart rate and respiration rate are usually elevated, and respiratory effort may be guarded. animals may be febrile early, but have a normal to low body temperature as the condition progresses. abdominal ultrasound can be useful in locating pockets of fluid for abdominocentesis, which usually yields fluid with increased protein concentration and leukocyte numbers. on occasion, intracellular bacteria are observed on cytologic examination. the presence of extracellular bacteria is not diagnostic because accidental enterocentesis can occur. culture of abdominal fluid and subsequent antimicrobial sensitivity tests are indicated for the implementation of proper treatment. the causative organisms vary depending on the source of the bacteria. rumen bacteria are typically gram-negative anaerobes, and e. coli and other enteric bacteria are common if the intestine is the source of infection. exploratory surgery may be required to diagnose a gastrointestinal rupture. the cbc can be normal but often shows an inflammatory leukogram and, in severe cases, a degenerative left shift. treatment. treatment includes the prescription of appropriate antimicrobial agents, the administration of nsaids for pain and endotoxemia, and fluid support for dehydration. the prognosis is guarded, especially if an intestinal rupture has occurred. pathogenesis, clinical signs, and diagnosis. rectal prolapse is more common in sheep than in goats. this evagination of the rectal mucosa and rectal structures (and possibly the descending colon) is usually associated with excessive straining. straining is seen in lambs with diarrhea caused by coccidiosis, salmonella, or dietary imbalances, in ewes or ewe lambs with vaginal prolapse, in males with urolithiasis, and in animals grazing lush forage (particularly legumes such as alfalfa and clover). it also can occur secondary to chronic coughing, short tail docking, and the use of growth implants. [ ] [ ] [ ] rabies also can cause chronic straining and rectal prolapse. [ ] [ ] [ ] [ ] [ ] regardless of the cause, after the rectal mucosa becomes everted and exposed, irritation of the mucosa causes further straining, which exacerbates the problem. venous drainage of the prolapse may be compromised, but the arterial supply usually remains intact and contributes to the swelling. rectal prolapses are graded as type i to iv, based on the portion of rectum and distal colon that is everted. a description of these grades in shown in table - . treatment. correction may be cost prohibitive for feedlot lambs, and immediate slaughter is recommended. in more valuable animals, very mild, early cases can be treated with frequent application of hemorrhoidal ointment designed for humans and manual replacement of the prolapsed mucosa into the anus. the authors try to avoid applying purse-string sutures in the anus because they tend to serve as a nidus and result in further straining. however, if less aggressive therapies do not relieve the problem in hours, a purse-string suture may become necessary, particularly in type i and ii prolapse. in all cases and modes of treatment, restricting feed for to hours and administering mineral oil is recommended. dusty feedstuffs (concentrates, pellets, hay) should be avoided because they may contribute to coughing, which exacerbates this condition. adding molasses to feeds and lightly wetting hay may help reduce problems with dust. purse-string suture is easily performed. the prolapsed tissue and perineal area are washed with mild soap and lubricated with petroleum jelly or hemorrhoidal ointment before the prolapse is replaced. , after replacing the prolapsed mucosa, the clinician inserts a tubular object (syringe case, wooden dowel, gloved finger) into the rectum. he or she then places a purse-string suture of nonabsorbable suture material ( - nylon) in the skin around the anus, tightens it around the tubular object, and ties it off. the suture should be placed around the anus using a cutting needle, and entering and exiting at the o'clock position. tying the knot above the anus ensures that less fecal soiling of the suture will occur. the clinician should tie the suture in a bow knot to allow easy identification over the next few days and then remove the tubular object. the suture should be tight enough to prevent prolapse but loose enough to allow feces to pass. the clinician should regularly reevaluate the animal and if possible loosen the purse-string suture at -hour intervals until no tension exists. after a full day of no tension, the suture can be removed. if animals continue to strain, an epidural anesthetic can be administered. petroleum jelly and hemorrhoid gels should be placed on the anus daily. , the injection of counterirritants around the rectum ( ml or less of lugol's iodine) either alone or in conjunction with anal purse-string suturing is a quick and inexpensive treatment. , , the clinician inserts an -gauge needle ( cm) deeply into the skin around the anus at , , and o'clock. an injection at the o'clock position should be avoided because swelling around the urethra can result in obstruction. for more severe cases, submucosal resection or rectal amputation of tissue may be necessary. , rectal amputation can be performed with a prolapse ring or suture technique. prolapse ring usage is a salvage technique. the clinician inserts the prolapse ring into the rectum and places an elastrator band or suture around the area to be amputated to induce vascular compromise and necrosis of tissue. if a ligature is used, it should be tightened to allow purchase on the tube or ring. a fibrosis is induced just proximal to the band or suture, and mucosa subsequently grow across the areas. strictures, peritonitis, and abscesses are possible complications, but this technique may be useful as a field procedure. submucosal resection can be performed under epidural analgesia after the prolapse and the perineal area have been surgically prepared. the clinician places two spinal needles ( to cm) at -degree angles to each other to mm distal to the anal sphincter and through the entire prolapse. a circular incision is made to mm distal to the spinal needles through the mucosa and around the outside of the anus. another circular incision is made just distal to the caudal extent of the prolapse into the point where the mucosa reflects on itself on the innerside of the prolapse. the clinician connects these two incisions with a longitudinal incision parallel to the prolapse and dissects the mucosa between the circumferential incisions. the mucosal edges are then sutured with a simple interrupted pattern using a suitable absorbable suture material. after completely suturing the mucosal surfaces, the clinician removes the two spinal needles and places a purse-string suture in the anal sphincter. placement of the suture and follow-up care are the same as described for the purse-string suture technique. submucosal resection decreases the incidence of both peritonitis and stricture formation compared with other surgical techniques, but it is expensive. in all of these techniques, a caudal epidural anesthetic ( % lidocaine, . ml per kg) is recommended to decrease straining and ease pain from the procedures. , a xylazine epidural ( . to . mg/kg as sufficient [qs] to ml with % lidocaine) may give longer relief (approximately to hours) from straining than lidocaine. an alcohol epidural also may prevent straining for extended periods. either isopropyl alcohol or ethanol can be used to demyelinate the motor and sensory nerves. this type type i small, circular amount of submucosal swelling good prognosis if there is no damage to mucosa; protrudes through anus; probing reveals a pocket purse-string suture, iodine injection, submucosal or fornix just inside anus resection type ii slightly more circular submucosal and mucosal good prognosis if treated quickly and no mucosal swelling, possibly containing retroperitoneal rectal damage; purse-string suture, iodine injection, tissue from anus; probing reveals a pocket just submucosal resection, rectal amputation inside anus type iii complete prolapse containing part of the if there is vascular injury to the descending colon, retroperitoneal structures of the rectum and the prognosis is guarded to poor; submucosal descending colon; probing reveals a fornix just resection or rectal amputation are the methods of inside anus; the affected portion of the descending choice colon does not prolapse through the anus type iv the descending colon appears as a tube, and has if there is vascular injury to the descending colon, intussuscepted through the rectum and anus; prognosis is poor; abdominal exploration may unlike the previous types, in this case a probe be required to determine the extent of damage to or finger can be inserted into the prolapse through the descending colon the anal sphincter for a distance of to cm of anesthesia can be permanent and therefore should be used only for animals intended for slaughter. because of the potential for some loss of sciatic nerve function, the clinician should perform a test injection of a local anesthetic ( % lidocaine) before using alcohol. if the epidural appears effective and no ataxia or muscle weakness of the rear limbs occurs, the clinician can inject a mixture of equal parts of lidocaine and alcohol into the sites where the test epidural was performed. possible problems with alcohol epidural anesthesia include injection site necrosis, sciatic nerve dysfunction, and the inability to void feces. regardless of the type of epidural used, the clinician clips, washes, and dries the area before placing a small needle ( -to -gauge [ . cm]) in the most cranial yet moveable intracaudal vertebral space-usually c to c or c to c . the needle is placed on the dorsal midline, with the needle degrees to the skin and the hub moved slightly caudal, and then slowly advanced. liver abscesses usually occur as a result of chronic rumenitis in cattle, but they are rare in sheep and goats. they can occur in feedlot lambs and kids and other animals fed rations high in grain. in lambs and kids, septicemia or extension of an umbilical vein infection can cause liver abscesses. in most cases, however, liver abscesses are an incidental finding. weight loss, anorexia, depression, and decreased production (growth, milk) may occur. in adults, corynebacterium pseudotuberculosis is the most common cause. actinomyces pyogenes and fusobacterium necrophorum also are cultured from abscesses. [ ] [ ] [ ] liver enzymes may or may not be elevated. diagnostic ultrasonography of the liver may help detect abscesses, especially if they are numerous and widespread. however, no specific treatment or control measure is available. many of the preventive protocols used for feeder cattle apply to the control of abscesses in sheep and goats. these include slowly introducing concentrates into the diet, offering long-stemmed hay free choice, and including rumen buffers (alkalinizing agents) and antimicrobial agents in the feed. pathogenesis. fatty liver occurs in conjunction with pregnancy toxemia in ewes and does during the last month of gestation. it is most common in thin or obese ewes or does with a single large fetus, twins, or triplets. during late gestation, particularly in obese females, the abdominal space is filled with accumulated fat and an ever-expanding uterus. because of the lack of rumen space, these females have difficulty consuming enough feedstuffs to satisfy energy requirements. in most management systems, late gestation occurs during the winter months, when less pasture is available and poorer-quality feedstuffs are offered. energy requirements for ewes and does carrying twins or triplets is greatly increased during the final months of gestation because % to % of fetal growth occurs during this time. ewes with twins require % more energy, and those with triplets need % to % more dietary energy. pregnancy toxemia also occurs in association with anorexia caused by other diseases (foot rot, opp, cae) or sudden stresses (feed or weather changes, predator attacks, hauling). whatever the initiating cause, a period of anorexia and lack of sufficient energy intake result in a negative energy balance. these animals begin to mobilize body stores of fat and transport them to the liver. in the liver, fat is catabolized to glycerol and free fatty acids (ffas). ffas can be used in the citric acid cycle (krebs cycle) as an energy source, but not in the direct formation of glucose. anorexic animals have less ruminal substrate available for production of the glucose precursor propionic acid. however, oxaloacetate, which is an integral part of the citric acid cycle, is removed from the cycle and converted into glucose. depletion of oxaloacetate inhibits the normal citric acid cycle's function, inhibiting the use of ffas. as the pool of ffas increases, they are converted to ketone bodies or repackaged into lipoproteins. because ruminants are not efficient at transporting lipoproteins out of the liver and back to the adipose stores, the lipoproteins overwhelm the liver's ability to handle this massive buildup, resulting in a fatty liver. because less substrate is available for glucose formation, more oxaloacetate is "cannibalized" from the citric acid cycle, further inhibiting the body's ability to use ffas. this in turn causes the continued accumulation of ketone bodies. hypoglycemia, hy-perketonemia, and potentially uremia and death can occur. clinical signs. animals suffering from fatty liver or pregnancy toxemia become anorexic and depressed, display altered behavior, and become recumbent. some are constipated, grind their teeth, have a ketone smell to their breath, and suffer from dystocia. neurologic signs include blindness, circling, incoordination, star-gazing, tremors, and convulsions. , death can occur if the condition is left untreated. in the case of in-utero fetal death, maternal septicemia-endotoxemia and death are common sequelae. diagnosis. diagnosis is based on clinical signs, the presence of multiple fetuses, and typical clinicopathologic findings. cbc results may be normal or show an eosinophilia, neutropenia, and lymphocytosis. these animals may or may not be hypoglycemic, but ketoacidosis, hypocalcemia, and hypokalemia are common. , liver enzymes are usually within normal limits but occasionally may be increased. azotemia, both from dehydration and secondary renal disease, is a common finding, and a fatal uremia may occur. blood concentrations of ß-hydroxybutyric acid greater than mmol/l are consistent with pregnancy toxemia. urinalysis will be positive for both ketones and protein. urine is collected from sheep by holding the nares and from does by frightening them and then allowing them a perceived escape when they stop, squat, and void. although not commonly performed, liver biopsy can help determine the extent of fatty infiltration. this syndrome must be differentiated from hypocalcemia, hypomagnesemia, polioencephalomalacia, encephalitis, lead toxicity, and cerebral abscesses. treatment. very early cases (before the animal exhibits recumbency) may be treated with oral or iv glucose. a balanced electrolyte solution with extra calcium ( ml of a % calcium borogluconate per liter), potassium ( to meq/l), and % dextrose is needed. in some cases, sodium bicarbonate is valuable in treating acidosis (see appendix ii). energy intake must be increased, and propylene glycol can be administered ( to ml every hours) as a glucose precursor. rumen transfaunation and supplementation with vitamin b complex (including vitamin b , biotin, niacin, and thiamine) also are recommended. after females become recumbent, treatment must be very aggressive. removal of the fetuses is crucial in these cases. chemically inducing parturition (by administering . to mg of prostaglandin f a or . mg/ kg of cloprostenol in does and to mg of dexamethasone in ewes) and giving the ewe or doe medical support (fluids, b vitamins, glucose) while waiting is a useful protocol in some cases. unfortunately, during the time before parturition, endotoxemia from dead fetuses further compromises the female. for this reason, the authors recommend immediate cesarean section on depressed moribund animals (see chapters and ) . the owner should be forewarned of the poor prognosis for animals already in a moribund state. fluid support during and after surgery is crucial. regardless of the therapeutic plan, the animal should be offered a palatable, energy-rich, highly digestible feedstuff. the keeper and clinician should take care to minimize the risk of a confounding disease during convalescence (e.g., lactic acidosis, polioencephalomalacia). prevention. fatty liver and pregnancy toxemia can be prevented through proper nutrition. maintaining animals in proper body condition throughout the year and making sure energy and protein levels are adequate in late gestation (see chapter ) are two key preventive measures. , the owner/manager should be taught to assess body condition in individual animals and should maintain emergency stores of feed in case of severe weather or natural disasters. the requirement for energy may be one and a half to two times maintenance for single fetuses and two to three times maintenance for multiple fetuses. prevention of concurrent disease that may further increase energy demands or cause anorexia (e.g., intestinal parasitism, foot rot) is crucial. the keeper should take care to increase the grain portion of the diet slowly because anorexia from rumen upset can lead to this disease. ewes should be offered . to kg of a cereal grain (corn, oats, barley, or a combination) every day during the final months of gestation; does can be offered ⁄ to kg of grain. keepers should maintain ewes and does at a body condition score of . to (see chapter ) throughout gestation and evaluate the animals' energy every to weeks. ultrasonography can help identify females with multiple fetuses. these animals should be separated into groups and fed accordingly. ultrasonographic determination of fetal numbers is best accomplished between days and after breeding with a . mhz transducer; a mhz transducer produces better results between days to . either type of transducer may be of value and these windows of time may be expanded by the ability of the operator (see chapter ) . determination of fetal numbers may be enhanced by shearing the hair or fiber in front of the udder, applying a coupling substance to the skin, and viewing as much of the abdomen as possible, building a mental image of its structures and the number of fetuses while systematically moving from one side of the posterior abdomen to the other. keepers and clinicians should ensure that ewes are healthy and free of chronic diseases (e.g., opp, cae, foot rot, chronic parasitism) and that a good-quality trace mineral salt mixture is available free choice. the addition of lasalocid ( . to mg/kg/day) or monensin ( mg/kg/day) to the feed or mineral mixture enhances the formation of the glucose precursor propionic acid and improves the efficiency of feed use. however, monensin should be used with caution because toxicity may occur; the agent should comprise no more than ppm of the complete diet. the inclusion of niacin ( g/head/day) in a feed supplement or mineral mixture helps prevent pregnancy toxemia. supplementation with lasalocid, monensin, or niacin should begin to weeks before the females give birth. shearing in the last trimester also is recommended in ewes. many sheep producers routinely clip the wool around the vulva. if complete body shearing is performed, the incidence of fatty liver or pregnancy toxemia may be decreased. sheared sheep require less energy to walk and graze. sheared ewes also tend to shiver on cold days, exercising the enzyme systems that promote the more efficient use of ffas as energy substrate. these ewes tend to seek shelter during cold weather, which may decrease lamb losses resulting from hypothermia. obviously, if ewes are to be shorn, keepers should make adequate shelter available. keepers should avoid hauling or moving females during late gestation. proper predator control measures should be maintained. good hoof care programs should be in place on farms or ranches where grazing is the predominant form of nutrient intake. sheep and goats should have their teeth checked to ensure good dentition before the breeding season. animals with poor teeth should be culled. measuring serum b-hydroxybutyric acid concentrations is useful in assessing energy status in ewes. values of . to . mmol/l suggest a negative energy balance. keepers should take steps to correct the problem by feeding better-quality, more digestible feedstuffs. white liver disease is a form of fatty liver disease reported only in angora and angora-cross goats and sheep. it is associated with cobalt deficiency. pathogenesis. cobalt is needed by rumen microflora to produce cyanocobalamin, or vitamin b , which is a coenzyme for methylmalonyl-coa mutase. in turn, this enzyme is needed to convert propionate to glucose through the krebs cycle. cobalt deficiency leads to the accumulation of methylmalonyl-coa, or methylmalonic acid, which is converted to branched chain fatty acids that accumulate in the liver. , high-grain diets that are fermented to propionate coupled with deficient or marginal cobalt intake may predispose to this condition. , white liver disease has not been reported in the united states, but ill thrift from cobalt deficiency has been observed. it is therefore possible that the disease goes unrecognized. clinical signs. signs are most commonly seen in young animals, and include ill thrift, anorexia, and diar-rhea; sheep may exhibit photosensitization. clinicopathologic findings include a macrocytic, normochromic anemia and hypoproteinemia. , diagnosis. abnormal serum or liver concentrations of vitamin b or liver cobalt are the basis of diagnosis. liver cobalt concentrations on a dry matter basis of . Ϯ . ppm were reported in goats with white liver disease, compared with . Ϯ . ppm in controls. treatment and prevention. sheep can be treated with oral cobalt ( mg/head/day) or vitamin b injections. the condition can be prevented by including cobalt in the ration by feeding a good-quality trace mineral salt. both fasciola hepatica and fascioloides magna can infest sheep and goats. the disease occurs along the gulf coast and in the pacific northwest and great lakes areas. clinical signs. f. hepatica infestation usually causes acute disease in sheep and goats but can present as a chronic condition. chronic disease is the result of the mature flukes in the bile ducts and is manifested in depressed growth and milk production. acute disease occurs when large numbers of immature flukes migrate at once, particularly in animals with limited immunity to flukes. signs include anorexia, depression, weakness, dyspnea, anemia, ascites, colic-like signs, dry feces, and sudden death. the clinical signs are identical to those of nematode infestations (i.e., chronic weight loss, ill thrift, diarrhea, anemia, hypoproteinemia). similar but more severe signs occur with f. magna infection, which is usually fatal. , , diagnosis. antemortem diagnosis of fluke infestation can be difficult. finding eggs in feces is diagnostic for f. hepatica. eggs are only produced by adults and not in great numbers, so a negative fecal test cannot preclude acute or chronic fascioliasis. fluke eggs do not float in routine fecal flotation methods used for nematode diagnosis; a sedimentation technique should be used for suspected fluke infestations. to perform a sedimentation test, the clinician mixes to g of feces with ml of tap water and strains the mixture through a tea strainer into a beaker. the sediment can be examined minutes later under a dissecting microscope. eggs are light yellow to golden and have an operculum at one end (see figure - ). f. magna does not mature, so eggs are not produced and fecal examination is of no value. most fluke infestations are discovered by finding the flukes at necropsy or slaughter. an elisa test may be available in the future. , , cbcs of affected animals may indicate eosinophilia and anemia. increased liver enzymes and hypoalbuminemia also are occasional findings. because antemortem diagnosis is difficult, the clinician should institute fluke treatment after ruling out other differential diagnoses if the possibility of fascioliasis exists. if fascioliasis is diagnosed at necropsy, the remaining animals in the herd should be treated. because flukicides available in the united states are highly effective only against mature flukes, the timing of treatment is important. in the southern portions of north america the snails are ingested in the spring and the flukes migrate in the summer and mature in the fall. in cooler, northern climates, snails may remain active during summer, so flukes can mature in the fall and into the winter. clinicians should begin treatment in the southern united states in the late summer or early fall. a single treatment in late winter or early spring is commonly used in the northern climates of north america. albendazole ( - mg/kg orally) and clorsulon ( mg/kg orally, mg/kg sc) are very effective against adult f. hepatica. , , clorsulon has no efficacy against nematode parasites but is highly efficacious against both adult and late-stage immature flukes. albendazole ( mg/kg orally) is somewhat useful in controlling f. magna at weeks after infestation, and clorsulon is effective only at very high dosages. [ ] [ ] [ ] unfortunately, neither agent can kill % of f. magna, and only a few remaining flukes can be fatal. control of fluke infestations is difficult, although timely treatment of animals can decrease infec-tions in successive years. decreasing exposure is the key to control. eliminating the snail is impractical, but fencing off low-lying areas may prevent ingestion. depending on local fluke life cycles, keepers should avoid grazing animals on areas with high fluke populations during peak infection times. areas where water stands or flows over grazing pastures, streams, and irrigation ditches (particularly those with clay soil) are high-risk zones. cysticercus tenuicollis is the larval stage of the dog tapeworm taenia hydatigena, of which sheep and goats are intermediate hosts. the larval stage migrates through the liver, then attaches to the liver or other abdominal organs and causes black, winding tracts and cysts in the liver. acute disease occurs only with large numbers of cysticerci and is characterized by depression and weakness resulting from liver damage. the chronic cystic stage is usually asymptomatic. no treatment is available and control is problematic because it requires treating infestation in dogs and preventing contact with dogs. , , pathogenesis. copper (cu) toxicosis is more common in sheep than in goats. goats appear closer to cattle than sheep in their ability to store and handle cu and resist toxicosis. toxicity results from chronic accumulation in the liver from the ingestion of excess cu in relation to molybdenum (mo) or sulfate in the diet. in sheep, a cuto-mo ratio greater than Ϻ leads to the accumulation of excess cu. the most common sources of excess cu in sheep and goats are trace mineral mixtures and feeds formulated for cattle or horses. clinical signs are often absent during the chronic accumulation phase. acute disease is seen when cu is suddenly released from the liver in large amounts. stress usually precipitates this acute phase. acute release and subsequent high blood cu concentrations cause an acute hemolytic crisis, resulting in anemia, hemoglobinuria, and acute renal failure. existing hepatic disease (such as that caused by liver flukes) may predispose animals to this condition. some breeds seem to be prone to cu absorption and storage problems (merino sheep), whereas others tend to be more resistant and prone to deficiency (pygmy goats) (see chapters and ). clinical signs. anorexia, depression, diarrhea, and weakness are all signs of cu toxicity. many affected animals are found dead with hemolysis and icterus. signs of abdominal pain and diarrhea are sometimes present. port wine-colored urine is evidence of hemoglobinuria. hemoglobinemia produces icterus of the mucosal membranes and fever. diagnosis. on clinicopathologic examination, anemia, hemoglobinemia, hyperbilirubinemia, increased liver enzymes, and azotemia are present. urinalysis reveals hemoglobinuria and isosthenuria. the combination of azotemia and isosthenuria indicates acute renal failure. definitive diagnosis of acute disease requires measurement of cu concentrations in serum. normal blood cu concentrations are approximately to mg/dl in sheep and goats. , , these concentrations increase to -fold with an acute hemolytic crisis. on necropsy, kidney cu concentrations are the most diagnostic because liver concentrations may be normal from release into the bloodstream. generally kidney concentrations greater than ppm and liver concentrations greater than ppm on a dry matter basis are diagnostic. , if tissue copper is reported in wet weight, the conversion to dry tissue weight can be estimated by multiplying the tissue concentration by a factor of . treatment. treatment of acutely affected animals is often futile. it consists of supportive therapy for the acute renal failure and anemia and attempts to lower liver cu stores. fluid therapy for the acute renal failure (see appendix ii) is of therapeutic value, and a blood transfusion may be needed if the pcv drops precipitously. ammonium tetrathiomolybdate ( . mg/kg iv or . mg/kg sc on alternate days for three treatments) is the most economical agent for treatment for acute cases. in valuable animals, d-penicillamine ( to mg/kg bid or mg/kg sid po for days) increases urinary cu excretion. trientine is used in human beings, but has shown variable results in sheep. treatment of the remainder of the flock should include the administration of ammonium molybdate ( to mg/head/day po) and sodium thiosulfate ( to mg/head/day po) for weeks. stress should be minimized, so keepers and clinicians should delay routine maintenance procedures such as deworming and hoof trimming until after treatment. the offending source of cu should be eliminated. prevention. avoiding high dietary cu (more than ppm), a high cu-to-mo ratio (greater than Ϻ ) in the feed, cu-containing foot baths, and other sources of cu is crucial. including supplemental mo in the diet to lower the cu-to-mo ratio to Ϻ to Ϻ is beneficial. this requires to ppm of mo in many instances. often too much emphasis is placed on the trace mineral component of the diet. the clinician should be aware that even if no cu is added to the trace mineral mixture and the element does not appear on the product label, the mineral mixture may still contain cu. many components of mineral mixes are contaminated with cu (zinc sulfate may contain ppm of cu, dicalcium phosphate may contain more than ppm of cu). therefore the clinician needs to perform a dietary analysis to find and correct the problem. pathogenesis. the liver is vulnerable to toxic insult because one of its major functions is detoxification. the most common plants that are gastrointestinal and liver toxins are shown in table - . clinical signs depend on the cause. acute, severe toxicity is more common with chemical toxicosis, whereas plant toxins usually cause chronic disease. a thorough history is important and in many cases inspection of the animals' environment is required. clinical signs. the clinical signs of toxic hepatitis can be vague. animals may only show anorexia and depression. icterus is more common with hemolytic diseases and is not always seen with liver disease. photosensitivity is a common clinical feature in ruminants and hepatoencephalopathy also can occur. clinicopathologic data are more helpful in diagnosing acute toxicity. serum ast and ldh levels can increase with hepatocellular necrosis but are not liverspecific, so muscle injury and disease must be ruled out. these enzymes also increase if serum is not separated from a blood clot in a timely fashion. increased levels of alkaline phosphatase (ap) and ggt indicate biliary stasis. ap also is not liver-specific, but increased serum levels of ggt are very specific for liver disease. ggt also increases in some hepatocellular diseases, so testing for its normal concentrations is important. unfortunately, all of these enzymes can be normal with liver disease, especially if it is chronic. hyperbilirubinemia, hypoglycemia, low blood urea nitrogen (bun), and hypoalbuminemia are not always evident as classically taught. if hepatoencephalopathy is suspected, blood ammonia concentrations may be elevated. blood ammonia analysis may be impractical in the field because the blood should be kept on ice, and the test should be performed within minutes of collection. to enhance the accuracy of blood ammonia analysis, the clinician should collect blood from a normal control animal for comparison. ammonia concentrations three times those of the control animal are diagnostic. liver biopsy remains the most valuable tool in diagnosing liver disease. although clotting dysfunction may occur in liver disease, it is an uncommon complication in ruminants and should not discourage the clinician from performing a liver biopsy. treatment. if the intoxication is caught in the acute stage, activated charcoal ( g per adult animal) can be given. supportive care, especially fluid support with dextrose solutions, is the mainstay of therapy. low-protein diets may suppress ammonia production temporarily, but they can be detrimental over time depending on the production status of the animal. if photosensitization occurs, animals should be housed indoors if possible, and broad-spectrum (systemic or topical) antibiotics may be necessary to control secondary bacterial dermatitis. corticosteroids (dexamethasone . to mg/kg iv or im) may be indicated in early cases of photosensitization to decrease inflammation. neurologic signs can be controlled with phenobarbital (initial dose: to mg/kg iv diluted in saline and administered over minutes; subsequent doses: to mg/kg iv diluted in saline, as needed up to tid). diazepam (valium) is contraindicated in hepatoencephalopathy because it may worsen signs. congenital hyperbilirubinemia, or black liver disease, occurs in mutant corriedale sheep (dublin-johnson syn-drome). this is a genetically recessive condition. it is characterized by an abnormality in the excretion of conjugated bilirubin and phylloerythrin and is often seen in animals consuming green forage. clinical signs include anorexia, photodermatitis, and icterus. liver biopsy of affected animals reveals dark to black granules in otherwise normal hepatocytes. the syndrome first manifests itself in lambs around months of age. a similar condition occurs in southdown lambs around months of age (gilbert's syndrome). this too is a recessive condition that causes decreased hepatic uptake of phylloerythrin and bilirubin, with concurrent renal failure. signs include icterus, photodermatitis, and ulceration around the ears and mouth. a liver biopsy reveals normal hepatic tissue. in both of these conditions, animals should be kept out of sunlight and fed minimal various tumors of the liver, including fibrosarcoma, lymphosarcoma, and cholangiocellular carcinoma, have been reported. , the use of ultrasonography and ultrasound-guided liver biopsy may aid in diagnosis. the umbilicus is an opening in the ventral abdominal wall that allows passage of the umbilical vessels and allantoic stalks. this opening should close within a few day of birth. the failure of this opening to close properly is termed umbilical hernia. the hernial sac has an inner peritoneal layer and an outer layer of skin. these hernias are probably of genetic origin but may occur as sequelae to umbilical remnant infection. the opening in the abdominal wall is perceived as a ring on palpation. if the clinician can insert more than one finger into the hernial ring or if the hernia persists for more than to weeks, surgical intervention is indicated. penning. clamps or rubber bands may be of value for closing small hernias (those less than cm in diameter). the clinician should either lightly sedate the animal or infiltrate the skin around the hernia with a local anesthetic ( % lidocaine). the animal should be placed on its back and held by a technician-helper. any viscera prolapsing into the hernial sac should be replaced into the abdomen. the clinician then inserts two metal pins (baby diaper pins can be used) through the skin and on opposite sides of the hernial ring, just on the edge of the linea alba. the pins should be placed deep enough to sit next to the abdominal wall. slight tension is placed on the skin in the center of the umbilical sac, pulling it away from the abdomen. when the clinician is confident that all viscera have been cleared from the hernial sac, he or she places an elastrator band between the pins and the abdominal wall. this results in ischemic necrosis of the skin. the skin will slough and the abdominal defect will heal in to days. lambs should be given tetanus prophylaxis. this procedure and other clamping techniques are useful in females and some males. however, urine scalding of the skin may occur in some males. clinicians should closely monitor animals that have undergone clamping. surgical resection. in cases in which the hernial ring is larger than cm, surgical intervention should be carried out. animals can be sedated and then infiltrated with a local anesthetic or placed under general anesthesia. the area around the hernia is clipped and surgically prepared. the clinician opens the hernial sac and introduces a finger into the abdomen to ensure that no viscera have adhered to the inner lining of the ring and that no enlarged or infected umbilical remnants are present. he or she then carefully excises the ring and closes the defect in the abdominal wall. this closure can be made by simply opposing the abdominal wall with a horizontal mattress pattern stitch (absorbable suture). an alternate closure of the abdominal wall is to suture the peritoneal lining in a separate pattern and close the abdominal wall defect so one side of the defect is pulled to overlap the other side. the upper free edge is sutured to the opposite wall with a near-far-far-near pattern. the authors choose not to employ surgical techniques that slow this procedure. the subcutaneous tissue can be closed with simple interrupted pattern using absorbable suture and the skin should be closed with whatever pattern the clinician prefers. animals should be given tetanus prophylaxis and antibiotics. they should be closely monitored for signs of sepsis and surgical failure. exercise should be limited for to days after surgery. infections of the umbilical arteries (omphaloarteritis) and veins (omphalophlebitis) and urachal disease can occur because of failure or partial failure of passive transfer of colostral antibodies and subsequent sepsis. contamination of the umbilicus, retracting of these structures after stretching and breaking, and chemical damage (from strong tincture of iodine) to the amniotic remnants are other possible causes. , , dipping the umbilicus with iodine or iodine-chloriodine substances is a common practice. aggressive use of these chemicals may precipitate serious inflammation of the cord. excessive torsion of the umbilical cord, distention of the proximal urachus, and some genetic factors may all be associated with patent urachus, which also may occur as a sequela to omphaloarteritis or omphalophlebitis. clinical signs and diagnosis. the clinical signs include umbilical swelling, pain, and occasionally drainage or discharge of the umbilical stump. palpation and transabdominal ultrasonographic evaluation reveal an enlarged cord-like structure ascending from the umbilicus cranially (umbilical vein) or caudally (urachus or um-bilical artery). ultrasonographic evaluation may indicate an abscess or thickened tissue. patent urachus is associated with dermatitis, urine scalding of the ventral abdomen, and urine dribbling. if the urachus becomes infected it may leak urine intraperitoneally or subcutaneously. both of these developments may be identified with abdominal palpation, ballottement, ultrasonographic evaluation, and, when indicated, paracentesis. the cbc may indicate neutrophilia. blood culture is indicated if sepsis occurs simultaneously. occasionally infection of the internal structures may occur with no outward umbilical swelling. deep abdominal palpation and/or the use of real-time ultrasound are necessary to attain a diagnosis. animals with umbilical infections also may have signs of septicemia, anorexia, depression, joint distention, and fever. treatment. if a patent urachus occurs without inflammation of the associated tissues, it can be cauterized daily with iodine or silver nitrate. however, if it remains patent for more than days, it should be surgically closed. the animal should be placed under general anesthesia (see chapter ) . the area around the umbilicus should be clipped and surgically prepared, and the animal should be placed on a broad-spectrum antimicrobial agent to hours before surgery. the clinician opens the abdomen lateral to the umbilicus and digitally explores the adjacent area for adhesion formation. the urachus should be identified and followed to the urinary bladder. after this, the clinician should amputate the urachal attachment to the bladder and close the bladder with a double-layered inverting pattern (cushing). the abdominal wall, subcutaneous tissue, and skin are closed as described for umbilical hernia repair. on occasion some cases of omphalophlebitis-omphaloarteritis can be treated medically. prolonged antibiotic therapy with a broad-spectrum antimicrobial agent (ceftiofur . mg/kg sid or oxytetracycline mg/kg sc every hours) may be attempted. however, if medical therapy is ineffective, the infected umbilical remnants should be marsupialized or excised. the authors prefer more aggressive, surgical removal of the umbilical rem-nants. as with urachal surgery, the abdomen should be opened lateral to the umbilicus. depending on the severity of infection and the amount of tissue involved, the clinician may need to perform extensive dissection of necrotic tissue and possibly intestinal resection. if the infection of the umbilical vein extends to and involves the liver, marsupialization of the umbilical vein is an effective method of therapy. , the clinician can pull the vein to the most cranial portion of the abdominal incision and suture it to the muscle layers and skin before closing the abdomen as described for umbilical hernia repair. however, a preferable method is to close the abdominal wall, pull the transected umbilical vein through, and suture it to a separate stab incision. this may help minimize the incidence of abdominal wall herniation. only monofilament, absorbable, non-gut suture material should be used. the venous stump should be flushed daily with antiseptic solution ( % chlorhexidine, . % povidone iodine), and the animal should be maintained on antibiotics for more than days. the venous stump usually closes within a month. prevention. umbilical infections can be prevented or drastically reduced by ensuring adequate intake of goodquality colostrum. lambs and kids also should be only minimally stressed (particularly during the first to days of life) to enhance colostral absorption. in some management scenarios, proper dipping of the navel with non-caustic materials also helps reduce the incidence of this disease. indigestion in ruminants bloat or ruminal tympany diseases of the goat on the effect of xylazine on forestomach motility in sheep anorexia during febrile conditions in dwarf goats: the effect of diazepam, flurbiprofen and naloxone bloat in kids experimentally induced lactic acidosis in nubian goats: clinical, biochemical, and pathological investigations diagnosis of enteric disease in small ruminants ruminal lactic acidosis in sheep and goats biochemical alterations in serum and cerebrospinal fluid in experimental acidosis in goats commonly encountered diseases of goats generalized aspergillosis in dairy sheep rumen papillomas in sheep effect of monensin on development of ruminal parakeratosis in fattening lambs, zentralblatt fur veterinarmedizin lactic acidosis foreign body syndrome in goats-a report of five cases traumatic gastritis in sheep and goats goat medicine references . sherman dm: causes of kid morbidity and mortality: an overview, proceedings of the fourth international conference on goats enteric infections in young goats and their control enteritis and diarrhea goat medicine infectious gastrointestinal diseases of young goats occurrence of cryptosporidia, rotaviruses, coronavirus-like particles and k ϩ escherichia coli in goat kids and lambs jensen and swift's diseases of sheep escherichia coli in domestic animals and humans development of resistance with host age to adhesion of k ϩ escherichia coli to isolated intestinal epithelial cells references . smith bp: alterations in alimentary and hepatic function control programs for gastrointestinal nematodes in sheep and goats epidemiology of internal parasites: effects of climate and host reproductive cycles on parasite survival anthelmintic resistance: the selection and successful breeding of superior parasites control and prevention of specific diseases of sheep and goats herd rp: control of periparturient rise in worm egg counts of lambing ewes production medicine and health programs for goats the effects of dietary protein on establishment and maturation of nematode populations in adult sheep nematode infections-cattle, sheep, goats, swine parasites affecting goats in the southeast, proceedings of goat production and marketing opportunities in the south grazing management strategies for the control of parasitic diseases in intensive sheep production systems goat medicine helminth parasites of the gastrointestinal tract. nematode infections in cattle, sheep, goats, and swine parasite control programs in sheep and goats epidemiology and control of trematodes in small ruminants use of anthelmintic combinations against multiple resistant haemonchus contortus in angora goats preliminary investigation of anthelmintic efficacy against gi nematodes of goats and susceptibility of goat kids to gastrointestinal nematode infection johne's disease in sheep and goats paratuberculosis in small ruminants, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference paratuberculosis in small ruminants, deer, and south american camelids comparison of the absorbed elisa and agar gel immunodiffusion test with clinicopathologic findings in ovine clinical paratuberculosis paratuberculosis in a large goat herd serodiagnosis of paratuberculosis in sheep by use of agar gel immunodiffusion corynebacterium pseudotuberculosis infection in sheep and the complement fixation test for paratuberculosis obstructive intestinal diseases intussusception in goats goat medicine duodenal obstruction by a phytobezoar in a goat general surgical techniques for small ruminants: part ii, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference rectal prolapse in ruminants and horses jensen and swift's diseases of sheep rectal prolapse in food animals. part : cause and conservative management rectal prolapse in food animals. part ii: surgical options, comp cont ed pract vet : , . references . fetcher a: liver diseases of sheep and goats goat medicine a retrospective study of hepatic abscesses in goats: pathological and microbiological findings hepatic lipidosis jensen and swift's diseases of sheep hepatic lipidosis associated with cobalt deficiency in omani goats epidemiology of internal parasites: effects of climate and host reproductive cycle on parasite survival epidemiology and control of trematodes in small ruminants efficacy and safety of albendazole against experimentally induced fasciola hepatica infections in goats efficacy of an injectable ivermectin/clorsulon combination against fasciola hepatica in sheep albendazole treatment of experimentally induced fascioloides magna infection in goats efficacy of clorsulon against fascioloides magna infection in sheep evaluation of clorsulon against fascioloides magna in cattle and sheep clinical biochemistry of domestic animals metals and other inorganic compounds interpreting a bovine serum chemistry profile: part i large animal internal medicine large animal internal medicine neonatal conditions, with emphasis on equine neonate umbilical hernia, umbilical abscess, and auricle fistula general surgical techniques for small ruminants: part ii, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference key: cord- -y agnh authors: nan title: oral research communications of the (nd) ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: y agnh nan sidestream dark field imaging (sdf) is a technically relatively simple method to visualize the microcirculation. however, current gold-standard, or 'consensus' analysis (ca) of sdf films takes approximately hour per film limiting the application of sdf in a clinical setting, and leading to increased expense due to analysis of low quality films. we developed a subjective point of care scoring system (bedside evaluation of the microcirculation, bem) that could provide real-time intervention points and optimize care for the critical patient. the objective of this study is to evaluate whether the bem-score correctly identifies films of sufficient and insufficient diagnostic quality as defined by ca.twenty variable-length microcirculation films taken at the level of the canine tooth were selected from a database of films with available ca. before the study, three observers were trained using an instruction video to evaluate five quality parameters: stability, content, illumination, focus and pressure. the bem was performed by viewing and scoring each film four times in immediate succession. all five quality parameters were scored ( perfect, sufficient and insufficient) according to ca analysis. only the fourth viewing was considered for analysis. bem quality analysis was only considered sufficient if no parameter was scored insufficient. repeatability and reproducibility were assessed by assessing all films in a random order three times daily for three days.bem pass-fail assessment matched ca . % of the time with individual observer agreement of . - . %. agreement of bem with ca did not change over the study period ( . %, . % and . % on days , and respectively) indicating accurate quality analysis after a single bem-score of the training video. the mean cumulative bem quality score ( . , sd . ) was very similar to ca (mean . , sd . ). however mean individual bemparameter scores differed from ca reflecting differences in observer interpretation.high levels of inter-observer agreement and the strong correlation with ca for pass-fail assessment demonstrate that bem quality evaluation can produce repeatable and reliable results. variation in individual parameter scores may reflect systematic erroneous assignment of certain parameters or individual bias towards assessment of certain features. nevertheless, this did not impact on the overall evaluation of film quality. this study provides a platform to investigate whether rapid semi-quantitative analysis of the microcirculation itself is similarly feasible. sidestream dark field imaging (sdf) is a straightforward technique to evaluate microcirculation. however, current 'consensus' analysis (ca) of sdf films is time-consuming, thereby restricting the clinical application of sdf. a subjective bedside scoring system (bem) is proposed that could rapidly provide a semi-quantitative assessment. we have previously shown that observers could accurately evaluate film quality parameters. the objective of the present study is to assess the correlation of bem with ca for quantitative microcirculation parameters. three observers were trained using an instruction video to evaluate four quantity parameters: total vessel density (tvd), capillary vessel density (cvd), perfused vessel density (pvd) and microvascular flow index (mfi). fifteen variable-length microcirculation films of sufficient quality, taken at the level of the canine tooth, were selected from a database of films with available consensus analysis. each parameter was scored ( lowest - highest) . the bem was performed by viewing and scoring each film four times in immediate succession with the final score being considered for analysis. bem was performed on each film in random order three times daily for three days. ca scores were divided into quintiles for each parameter and mean bem score were calculated for each. conversely, for each bem score, mean ca scores were calculated, per observer or per day.the mean tvd and pvd bem scores for the % quintile were lower than mean bem scores for other quintiles. mean bem score for the - % quintile was the highest mean bem score for pvd only. mean cvd, pvd and mfi values for bem score were lower than mean values for other bem scores. in parallel, although tvd never received a bem score of , the mean ca value for films with bem score were lower than for higher bem scores. the mean tvd and mfi values for bem score of were higher than mean values for bem scores [ ] [ ] [ ] [ ] . similar values were obtained on each individual study day.this study demonstrates that rapid semi-quantitative assessment of the microcirculation using the bem-score can produce repeatable and reliable results, although significant overlap exists. further studies are required to evaluate the value of this technique in a clinical setting. objective: compare two non-invasive blood pressure (nibp) measurement devices (petmap and doppler) with invasive blood pressure (ibp) measurement in normotensive, anesthetised and awaken dogs. design: prospective clinical study animals: ten female dogs aged between to months (average . months), and weighting . to . kg (average . kg) undergoing a routine spay. interventions: blood pressure measurement procedures: after the induction of general anesthesia, a catheter ( g) was placed in the dorsal pedal artery and invasive systolic (sap i ), diastolic (dap i ) and mean (map i ) arterial blood pressures were obtained. the nibp cuffs were placed on the ipsilateral front limb. five consecutive measurements were obtained with each indirect device and considered as a mean measure. the ibp was obtained simultaneously to the five nibp measurements and also considered as a mean measurement. measurements on awaken dogs were obtained four hours after surgery. doppler's systolic (sap d ) and petmap's systolic (sap o ), diastolic (dap o ) and mean (map o ) arterial blood pressure were evaluated and compared to the corresponding ibp using the bland-altman analysis. the percentage of paired measurements with a mean difference of and mmhg was also evaluated for the sap o , dap o and sap d . results: agreement between ibp and nibp measurements obtained with the petmap and the doppler was assessed with the bland-altman analysis. on anesthetised dogs, both indirect devices underestimated all direct blood pressures. the petmap bias (standard deviation) were - . mmhg ( . mmhg), - . mmhg ( . mmhg), and - . mmhg ( . mmhg) for sap o , dap o and map o respectively. the doppler bias was - . mmhg ( . mmhg) for sap d . on awaken dogs, the petmap underestimated sap i and overestimated dap i and map i . the bias were - . mmhg ( . mmhg), . mmhg ( . mmhg) and . mmhg ( . mmhg) for sap o , dap o and map o respectively. the doppler underestimated the ibp and the bias was - . mmhg ( . mmhg).on anesthetised and awaken patients, the percentage of values lying within and mmhg of the ibp was higher (or equal) for the petmap compared to the doppler. conclusion: results suggest a better performance of petmap device to predict the ibp in normotensive-anesthetised and normotensive-awaken dogs. intracranial hypertension (ich) is associated with high morbidity and mortality in canine veterinary medicine, yet remains difficult to clinically diagnose. the lack of an easily feasible and available diagnostic method has prevented clinical studies on the prevalence, cause and treatment of ich. recently, transcranial doppler ultrasonography (tcd) has been reported as a noninvasive diagnostic method in humans. however, only a few preliminary reports on the use of this technique in dogs have been published. this study evaluated the repeatability and reproducibility of tcd of the basilar and left and right cranial, cerebral arteries in healthy beagle dogs.tcd was performed using a standard ultrasound machine at the level of the basilar artery, and left and right cranial cerebral artery, in six adult beagle dogs. systolic, diastolic and average velocity, resistance (ri) and pulsatility indexes (pi) were assessed for each vessel. repeatability was evaluated by calculating the intra-class correlation coefficient (icc) between three separate, consecutive measurements in every dog. an icc was also determined for the reproducibility of these measurements on three consecutive days. during the procedures, ecg, blood pressure and clinical parameters were monitored.statistical analysis showed a highly significant repeatability of all measured parameters (systolic, diastolic and average velocity, ri and pi) for all blood vessels (n = , icc = . - . , p < . ). systolic velocity, ri and pi were also significantly reproducible for the basilar artery (n = , icc = . - . , p < . ), and the left and right cranial cerebral arteries (n = , icc = . - . , p < . ). however, no significant correlation was found between basilar and cerebral blood flow velocities.measurement of pi and ri of the basal and cranial cerebral arteries using tcd appeared to have high intra-operator repeatability and reproducibility. however, measurements performed on the basilar artery had higher between day reproducibility. since the technical skill required to assess these parameters subjectively appeared to be less complicated at the level of the basilar artery, tcd in a clinical setting is probably advocated at the basal artery.in conclusion, tcd is characterized by high intra-observer repeatability and reproducibility, making this technique a promising tool for the measurement of pi and ri, which have both been reported to be correlated with intracranial pressure. intracranial hypertension (ich) is associated with high morbidity and mortality in canine veterinary medicine, yet remains difficult to clinically diagnose. furthermore, many patients suspected to suffer from ich are treated with sedatives or anticonvulsants. besides the impact of these molecules on the patients' neurological examination, they variably influence the cardiovascular system. this study evaluated the effect of various sedatives and anticonvulsants on the pulsatility and resistance index (pi) and (ri), as measured by transcranial doppler ultrasonography (tcd) in healthy beagle dogs. additionally, we evaluated the effect of ivabradine, a specific negative chronotrope, on tcd findings. tcd was performed at the level of the basilar artery in six adult, beagle dogs. this technique was performed prior to the injection of molecules (v ), and after administration of acepromazine (ace, lg/kg iv), diazepam (dia, . mg/kg iv), medetomidine (med, lg/kg im) or ivabradine (iva, mg/kg iv). a wash-out period of at least hours was respected between the administration of each drug. during the procedure, ecg, heart rate (hr) and blood pressure (bp) were monitored. results were analyzed using repeated measures anova. correlations between the clinical parameters, and pi and ri were investigated using pearson's correlation. results were considered significant when p < . . significantly lower pi and ri values [(mean pi; % ci), (mean ri; % ci)] were obtained for med [( . ; . - . ( . ; . - . ) ]. pi for dogs after iva was significantly increased compared to dia, but did not differ significantly from other groups.hr was significantly lower after med compared to other groups, and after iva, compared to v . bp was significantly lower after ace than after iva, med or on v . these clinical parameters were not significantly correlated with pi or ri. dia and ace did not significantly influence ri or pi, however med significantly decreased ri and pi. these findings agree with previous reports describing a decrease in icp provoked by med, as pi is positively correlated with icp. this effect appears to be independent of the effect of med on hr, since iva also significantly decreased hr, yet did not significantly affect pi or ri. in conclusion, blood pressure and heart rate did not significantly affect pi and ri. hypertrophic cardiomyopathy (hcm) is the most commonly diagnosed heart disease in cats with little documentation of the effects of treatment on outcome in cats with preclinical disease. therefore, this prospective cohort study was undertaken to evaluate the effects of treatment with atenolol on outcome in cats with asymptomatic hcm. we hypothesized that ) -year mortality would be increased in cats with hcm compared to a matched control group of healthy cats, and ) administration of atenolol would reduce -year cardiac mortality in occult feline hcm.cats were enrolled over a -year time period ( to ) in a prospective, open-label, observational study. diagnosis of hcm was based on transthoracic echocardiography. cats were either treated with atenolol ( . to . mg/cat, q h, po) or did not receive treatment. decision to treat was based on owner preference, suggestion by the clinician, and animal compliance with regard to pill administration. baseline echocardiograms were analyzed, and morbidity and mortality were monitored at and months and thereafter by annual rechecks, and by phone interviews of referring veterinarians and owners over a -year time period. groups were compared by a non-paired t-test, introduction: systolic anterior motion of the mitral valve (sam) is a dynamic left ventricular outflow obstruction frequently observed in feline hypertrophic cardiomyopathy (hcm). however, several cases of sam have also been observed in cats without left ventricular hypertrophy (lvh) and this finding could be interpreted as an early stage of the disease not yet accompanied by lvh. alternatively, it could be speculated that the dynamic outflow obstruction could cause intraventricular pressure overload sufficient enough to induce lvh at a later stage. this study was conducted to test the hypothesis that sam can potentially cause myocardial stress and myocardial damage by measuring plasma ntprobnp and serum troponin-i levels in cats with sam not associated with lvh. methods: the study was based on a retrospective analysis of cats who underwent cardiac investigation of a heart murmur, performed by a boarded cardiologist. these cats were diagnosed with sam not associated with lvh (ivsd and lvfwd < . mm, both on bmode and mmode measurements) via colour doppler echocardiography. plasma ntprobnp (cardiopet test, idexx laboratories, inc.) and serum high sensitivity troponin-i (hsctni hs, idexx laboratories, inc) were measured in these patients, as well as serum urea, creatinine and thyroxin concentrations. the association between biomarkers (ntprobnp and hsctni) concentration and echocardiographic values (myocardial thickness, left atrial dimension and aortic peak velocity) was determined by using the spearman correlation coefficient (rho). results: all cats were euthyroid and did not show evidence of renal disease. mean hsctni measurement was not performed in one cat due to an unsuitable sample. eleven out of cats ( %) showed ntprobnp values above the normal reference limit ( pmol/l) with a median concentration of pmol/l (± , range - ) . five out of cats ( %) showed hsctni values above the normal reference limit ( . ng/ml) with a median concentration of . ng/ml (± . , range . - . ). there was a significant positive correlation between aortic peak velocity and ntprobnp (rho= . , p= . ). conclusion: ntprobnp is increased in cats with sam without lvh, suggesting the presence of myocardial stress, which appears to be proportional to the degree of outflow obstruction derived by the aortic peak velocity. some of these cats also present a degree of myocardial insult suggested by the increased serum hsctni. whether the increased biomarker concentrations are related to sam-induced pressure overload or to an early stage of primary myocardial disease needs to be evaluated with appropriate longitudinal studies. heart murmurs are caused by turbulent blood flow or by vibration of cardiac structures. turbulent blood flow in young animals may originate from congenital structural heart disease or from physiological phenomena. the prevalence of heart murmurs and congenital heart disease in the general (i.e. non referral) feline population are unknown. the aims of this prospective study were to determine the prevalence of heart murmurs in young cats and to determine the prevalence of congenital heart disease in young cats with heart murmurs.in total domestic shorthair cats aged to months underwent a routine physical examination prior to vaccination between may st until march st . cats were from adoption programs run by either dierenasiel breda e.o. ( / ) or dierenopvangcentrum tilburg ( / ). in cats with murmurs, the murmur was timed, graded and the point of maximum intensity was determined. subsequently, d, m-mode and doppler transthoracic echocardiography with continuous ecg monitoring was performed.heart murmurs were detected in animals ( . %). congenital heart disease was detected in animals ( . %), acquired heart disease in animals ( . %) and no identifiable heart disease in animals ( . %) with murmurs.in the animals without heart disease dynamic right ventricular outflow tract obstruction was the cause of the murmur in cases, turbulence within the left ventricle was the cause in cases.in animals with acquired heart disease, pulmonary hypertension associated with a. abstrusus was diagnosed.in the cats with congenital heart disease, isolated defects were found in cats, being tricuspid valve dysplasia in cats, dynamic left ventricular outflow tract obstruction associated with mitral valve dysplasia in cats, ventricular septal defects in cats, double chambered right ventricle in cats and pulmonic stenosis in cat. combination defects were found in cats, being ventricular septal defect and tricuspid valve dysplasia in , ventricular septal defects and double chambered right ventricle in , ventricular septal defect and atrial septal defect in cat, a ventricular septal defect and pulmonic stenosis in cat and mitral and tricuspid valve dysplasia in cats.in conclusion in this prospective study we found the prevalence of heart murmurs to be . % and the prevalence of congenital heart disease to be . % with atrioventricular valve abnormalities and ventricular septal defects being the most common. dynamic left ventricular outflow tract obstruction (dlvoto) is a common cause of heart murmurs in adult cats. the obstruction is caused by systolic anterior motion (sam) of the mitral valve. sam is typically observed in adult cats and humans with hypertrophic cardiomyopathy (hcm). many studies support the concept that structural deformities of the mitral valves and the papillary muscles could be primary causes of sam. congenital malformations of the mitral valve causing sam and dlvoto have been observed in dogs and people and are believed to exist in cats, but have not yet been reported. the aim of this study was to report the clinical and echocardiographic findings in young cats with dlv-oto.thirteen domestic shorthair kittens, between - weeks of age, presented with a systolic heart murmur between may st until march st . all cats underwent a physical examination preceding d-, m-mode and doppler echocardiography. kittens were from adoption programs run by either dierenasiel breda e.o. (n= / ) or by dierenopvangcentrum tilburg e.o. (n= / ) nine kittens were male, four were female. all kittens were asymptomatic. dlvoto caused by sam was present in all animals, uninterruptedly in eleven, only at high heart rates in two. seven cats had normal left ventricular dimensions (nlvd), six cats had concentric left ventricular hypertrophy (clvh). in five cats papillary muscle abnormalities were noted being an accessory papillary muscle in two and enlarged papillary muscles in three, two of which had clvh.twelve animals were reexamined - months after the initial examination. all animals were still asymptomatic. a heart murmur and dlvoto could no longer be detected in nine animals, five with initial clvh and four with initial nlvd. in three animals a heart murmur and dlvoto were still present. two of the cats with nlvd developed clvh. one cat with initial clvh continued to have clvh.treatment with atenolol was instigated in three cats which continued to have dlvoto and clvh. two cats were reexamined. treatment with atenolol led to complete reversal of clvh in both cases. interventional radiology techniques have been used to palliate both malignant and non-malignant causes of vascular obstruction for both intrinsic and extrinsic lesions.three dogs presented for with large, non-resectable cardiac masses obstructing venous return to the right atrium. venous return to the heart was severely obstructed leading to congestion with subsequent ascites ( ) or head swelling and pleural effusion ( ) . due to the extensive nature of the disease, an interventional palliative approach was pursued. transatrial self-expanding metallic nitinol stents were placed from the cdvc to the crvc in order to restore venous return to the heart via blood flow through the stent interstices.in all cases, stent placement was successful and resolution of clinical signs achieved. two dogs required additional stent placement in months and months, respectively, for stent occlusion. in both cases, restenting resulted in ascites resolution or substantial reduction. one dog was euthanized months following initial stent placement for general systemic decline and return of moderate ascites. the second dog remains alive months following initial stent placement on diuretic therapy with moderate ascites present. the third died of undetermined causes / months later.to the authors' knowledge, this is the first report of longterm palliative transatrial stenting for cardiac tumors affecting venous return to the heart. the stents were well tolerated in these canine patients for which surgical options were not possible. the irish wolfhound (iw) dog has a high prevalence of heart diseases, particularly dilated cardiomyopathy (dcm) and atrial fibrillation (af).during a prospective longitudinal study, irish wolfhounds were investigated by one veterinary cardiologist ( ) between and . dcm was diagnosed in about % of dogs. in % of cases, dcm was accompanied by af. af without evidence of dcm was diagnosed in dogs ( %).for pathological investigations, hearts of iws were collected and fixed in % buffered formalin. based on the most recent results from cardiovascular examination, five groups were established: normal hearts (group , n= ), dcm with sinusrhythm (group ; n= ), dcm with af (group ; n= ), dcm with af and congestive heart failure (chf) (group ; n= ), af and left ventricular reverse remodeling (lvrr) due to medical therapy after diagnosis of dcm (group ; n= ).all hearts were evaluated by one pathologist ( ) who was blinded to the clinical diagnosis. gross inspection included measurments of weight, size, and architecture of left (lv) and right (rv) ventricles. three sites of each lv, rv, and interventricular septum (ivs) were histologically evaluated, and the extend of myocardial fibrosis, adipocyte infiltration, and angiosclerosis were graded semiquantitatively.iws with dcm and chf (group ) had died significantly younger ( . ± . years) than dogs with normal hearts ( . ± . years) (p= . ). concerning gross pathology findings in hearts with clinical dcm diagnosis, lv chambers were dilated in / cases of group , while in groups and , the papillary muscles appeared grossly prominent and not flattened as in group .histopathologically, in control dogs lv and rv myocardium showed no ( / ) or mild ( / ) interstitial collagen deposits, no or single adipocytes, and normal vessels. in contrast, heterogeneous findings were seen in groups - : most hearts ( / ) showed mild to moderate multifocal myocardial fibrosis and up to moderate diffuse infiltration of adipocytes within the lv myocardium, and mild angiosclerosis, but five hearts were histologically normal. only in one dog attenuated wavy fibers were seen in the apical region of lv. ivs was normal in out of cases. rv showed mild interstitial fibrosis and mild to moderate adipocytes in most cases ( / ) of all groups.in conclusion, pathological findings in hearts of iws affected with dcm are different from other breeds. furthermore, the gross and histological findings are variable and do not correspond to the clinical diagnosis in all cases. the irish wolfhound (iw) dog has a high prevalence of heart diseases, particularly dilated cardiomyopathy (dcm) and atrial fibrillation (af). during a prospective longitudinal study, irish wolfhounds were investigated by one veterinary cardiologist ( ) between and . dcm was diagnosed in about % of dogs. in % of cases, dcm was accompanied by af. in addition, af without evidence of dcm was diagnosed in dogs ( %).for pathological investigations, hearts of iws were collected and fixed in % buffered formalin. based on the most recent results from cardiovascular examination, three groups were established: dcm with af (group ; n= ), af without evidence of dcm (group ; n= ), normal hearts (group , n= ).aim of this study was to investigate the histopathological findings in left and right atria of iws with atrial fibrillation compared to normal hearts of iws. all hearts were evaluated by one pathologist ( ) who was blinded to the clinical diagnosis. hearts were inspected grossly and two cross sections of each left (laa) and right (raa) atrial appendage were embedded in paraffin wax and stained with h&e and picrosirius red. myocardial fibrosis and adipocyte infiltration in both atria were graded semiquantitavely.mean age ± sd of all dogs at time of death was , ± . yrs. the gross and histopathological findings of the left atrial appendage (laa) were not significantly different among groups, but dogs in both af groups had raa dilation ( . to . ml, median ml volume) compared to controls ( . to . ml volume, median . ml). histopathologically, raa in control dogs showed small amounts of interstitialcollagen and single adipocytes. in contrast, raa in dogs affected with af with andwithout evidence of dcm, had mild to moderate multifocal or diffuse myocardial fibrosis, and diffuse infiltration of adipocytes which was statistically significant different from normal hearts (p= . ). in general, fibrosis and number of adipocytes were significantly increased in raa compared to laa in both groups with af (p= . ).fibrosis and the accumulation of adipocytes within the myocardium are described toresult in electrical inhomogeneity predisposing to arrhythmia.on a cellular level, right atrial fibrosis and adipocyte accumulation might be the changes responsible for the development of atrial fibrillation and atrial dilatation in this breed of dogs with an exceptional high prevalence of af and dcm. asynchronous ventricular contraction causes deterioration in cardiac function and reduces the response to medical therapy in people with heart disease. various echocardiographic techniques have been used in humans for evaluation of left ventricular (lv) synchronization in order to assess whether cardiac resynchronization therapy (crt) would be beneficial. prior studies using tissue doppler imaging (tdi) derived strain imaging have not detected lv dyssynchronization in doberman pinschers with dilated cardiomyopathy (dcm). a newer d imaging modality, speckle tracking strain analysis, is more effective in detecting lv dyssynchrony in humans. this technique has not been previously evaluated in doberman pinschers with dcm. this study therefore aims to evaluate lv synchrony in doberman pinschers with dcm using d speckle tracking strain. client-owned doberman pinschers were included. standard echocardiography was used to evaluate systolic function and left ventricular dimension in order to categorize the dogs as normal or abnormal, depending on systolic and diastolic ventricular dimensions, ejection fraction and/or shortening fraction. the operator was blinded to the echocardiographic diagnosis of each dog. each parameter was measured in triplicate. radial (rs) and circumferential strain (cs), using right parasternal short axis at the level of the papillary muscles were calculated. synchrony was assessed by measuring the difference in the qrs onset time-to-peak strain between the septal anterior and posterior left ventricular segments. to evaluate differences between groups a nested effect anova (beat within group) was performed. a p< . was considered significant. based on the echocardiographic parameters mentioned above, / dogs were considered normal and / had dcm. significant differences were found in the time to peak rs (p= . ) between normal dogs and dogs affected with dcm. mean in time to peak rs was . ms (+/- . ) in normal dogs and . ms (+/- . ) in dogs with dcm. time to peak cs did not exhibit significant differences between groups (p= . ) and was . ms (+/- . ) in normal dogs and . ms (+/- . ) in dogs with dcm. the results of this study show that the delay between the anteroseptal-to-posterior wall peak radial strain is greater in doberman pinschers with dcm than in normal dogs. further studies would be required to evaluate whether there is a difference in synchrony between dogs in occult stage and overt dcm. ventricular dyssynchrony could negatively affect systolic dysfunction as it does in people, and crt may therefore be helpful for the treatment of dobermans with dcm. patent ductus arteriosus is often treated with intra-arterial coilembolization or implantation of an 'amplatz canine duct-occluder'. for both procedures an arterial access is necessary. because of the high risk of developing a fatal hemorrhage from the arterial puncture site after removal of the large-bore introducer sheath upon completing the intervention, cardiologists generally use surgical cut-down with subsequent ligation of the femoral artery instead of percutaneous arterial puncture using seldinger's technique. the effect of commercially available chitosan patch has been tested in experimental dogs with the approval of the institute's ethical committee. on the first beagles the committee required a terminal experiment. for the selection of an appropriate introducer-sheath (i.e. smaller than the arterial diameter), the femoral artery was first imaged with ultrasound. under general anesthesia introducer-sheaths were placed in both femoral arteries using seldinger's technique. after their removal a chitosan-patch was applied on the wound according to the manufacturer's instructions: -minute manual pressure. the dogs were monitored with direct arterial blood pressure measurement, ecg, pulse oxymetry and capnography. all dogs were kept under general anesthesia for several hours and the legs were moved vigorously every minutes to mimic movements of awake animals. no macroscopic hemorrhage was noticed on the puncture sites and no signs of severe subcutaneous bleeding was suspected as the blood pressure and heart rate remained stable. after several hours the dogs were euthanized. after having shown the effective working of chitosan-patch, a permission was granted for survival experiments in beagles. in of these dogs one femoral artery was punctured with an introducer whose thickness exceeded the diameter of the femoral artery. in both dogs an uncontrollable subcutaneous hemorrhage occurred immediately after the -minute manual compression time with severe drop of blood pressure and development of tachycardia. both dogs were euthanized during anesthesia. in another beagles the size of the introducer was smaller than the diameter of the artery ( - %). no relevant hemorrhage took place in these dogs and they recovered from the procedure without any complications. similarly good outcome was found in two -month-old boerboels. from this pilot study we concluded that chitosan-patch can effectively control hemorrhage from a femoral arterial puncture site if the introducer-sheath is thinner than the artery's lumen. using seldinger's technique allows a less invasive and quicker cardiac catheterization and preservation of the femoral artery for the leg's blood supply and for repeated intra-arterial interventions. objectives were to examine longitudinal change in echocardiographic left heart chamber dimensions and cardiomyopathy classification in cats with primary myocardial disease.clinical records from - were reviewed for cats with or more echocardiographic examinations at least months apart and a diagnosis of primary myocardial disease. for each study ( from each cat), left heart chamber dimensions were measured by a single blinded trained observer in random order, and the cardiomyopathy type was classified according to predefined criteria. paired comparisons were made using the wilcoxon signed rank test and fisher's exact test. cats met the inclusion criteria. cardiomyopathy type at entry consisted of hypertrophic cardiomyopathy (hcm, n= ), dilated cardiomyopathy (dcm, n= ), arrhythmogenic right ventricular cardiomyopathy (arvc, n= ) and normal cat. overall, median left ventricular (lv) diastolic diameter and median long axis left atrial (la) diameter both increased (p= . , . respectively) whereas lv diastolic wall thickness and lv fractional shortening did not change, but changes in dimensions of > % were seen in both directions. change in cardiomyopathy type was documented in cats: hcm to normal; normal to hcm; hcm to the endomyocardial form of restrictive cardiomyopathy.the inclusion criteria led to a bias towards clinically stable cats with hcm. despite this, changes in cardiomyopathy phenotype were observed. either our criteria for the different feline cardiomyopathies should be defined more precisely, or phenotypic expression in cats with cardiomyopathy changes over time. transesophageal echocardiography (tee) has proven useful in evaluating patent ductus arteriosus (pda) morphology thereby guiding appropriate device selection. additionally, tee, in combination with fluoroscopy, has been used to guide the transcatheter coil embolization and for deployment of amplatz canine ductal occluder (acdo) in dogs. recently, we described the use of transthoracic echocardiography (tte) guidance during transcatether pda occlusion with acdo without the use of fluoroscopy, but observed problems of deployment in patients with sub-optimal acoustic windows. however, tee, can overcome issues of suboptimal tte acoustic windows and provides higher image resolution of cardiac and vascular regions. therefore, we hypothesized that tee could be used to successfully visualize the vascular structures and interventional devices to safely perform pda occlusion with acdo without requiring fluoroscopy.we recruited dogs with patent ductus arteriosus (pda) for tee-guided percutaneous ductal occlusion with an acdo. dogs were anesthetized, positioned in right lateral recumbency and the right femoral artery was accessed percutaneously (modified seldinger technique). the tee probe was advanced to a midesophageal position with minimal force to obtain a long axis -chamber view (transverse plane). the probe was then retroflexed and withdrawn to a cranial esophageal position until a cross section of the descending aorta was seen. to visualize pda to the probe was slightly straightened and turned counterclockwise, and the ultrasonic beam was oriented between and degrees.in all dogs, the guide wire and a long introducer-sheath were guided from the aorta through the pda into the main pulmonary artery by tee monitoring. the acdo was advanced through the introducer-sheath until the flat distal disk was visualized within the main pulmonary artery by tee monitoring. the distal disk was positioned against the pulmonic ostium and the coupled proximal disk was deployed within the ductal ampulla while being monitored by tee visualization.the guide wires, long introducer-sheath and acdo appeared hyperechoic on tee images and tee guidance provided images of sufficient quality to clearly monitor the procedures in real-time. real-time monitoring also allowed for immediate corrections to guide wire, catheter or device positioning. the procedures were successful and without complications in all patients.we have demonstrated that tee monitoring, like tte monitoring, can guide every step of transcatheter acdo embolization procedures without requiring fluoroscopy, thereby avoiding radiation exposure, and provides an alternative to tte-based guidance, especially when tte visualization of the pda is insufficient for safe and timely acdo deployment. right ventricular (rv) dysfunction occurs in human patients with left-sided cardiac disorders because of the mechanism of ventricular interdependence. doppler echocardiographic indices of diastolic function of the right ventricle are good prognostic markers during left ventricular (lv) failure secondary to ischemic and dilated cardiomyopathy.the aims of the present study were: to assess lv and rv diastolic function by conventional doppler and pulsed-wave tissue doppler imaging (pw-tdi) in dogs with mitral valve disease (mvd), with or without pulmonary hypertension (ph); to test if echocardiographic parameters of lv and rv diastolic dysfunction correlate to the doppler-estimated pulmonary artery systolic pressure (pasp). dogs were prospectively evaluated, including dogs with mvd. for each dog, a complete echocardiographic evaluation was carried out. dogs with mvd were divided in groups according to the acvim classification of heart failure. using the cut-off value of tricuspid regurgitation (tr) peak velocity of . m/s, presence or absence of ph was considered, standard echocardiographic and mitral and tricuspid doppler parameters (e wave and a wave), and pw-tdi parameters (systolic wave, sa; early diastolic wave, e'; late diastolic wave, a'; e'/a' ratio; e/e' ratio) for lateral and septal mitral annulus, and lateral tricuspid annulus were measured. the echocardiographic data were compared by use of anova and multiple contrast t-test with bonferroni correction. the relationship between left-sided echocar-diographic parameters and rv diastolic parameters was examined by correlation analysis. the correlation of pasp with lv and rv diastolic parameters was examined by multiple linear regression. a value of p< . was considered significant.dogs were classified as follows: healthy dogs; dogs in class b ; dogs in class b ; dogs in class c and d. no differences were found among groups regarding rv conventional doppler and pw-tdi parameters. however, a significant, weak correlation was found between some left-sided echocardiographic parameters (left atrial dimension; end diastolic and end systolic volume indexes; peak e wave and a wave velocities and e:a ratio) and some rv pw-tdi parameters. ph was diagnosed in dogs, while dogs were deemed without ph. dogs with ph had significantly different trans-mitral e and a wave peak velocity and e/e' ratio of lateral and septal mitral annulus. these two latter parameters were also correlated with pasp (r = . and . , respectively).our findings highlight the importance of considering ventricular interdependence in dogs with mvd, particularly those with ph. during the cardiac cycle, the left ventricle (lv) undergo a complex deformation, consisting of overall shortening accompanied by increase in wall thickness and twisting due to the helical orientation of the myocardial fibers. using speckle tracking echocardiography (ste), the circumferential strain (cs) as well as the twisting motion of the lv, calculated as the net difference between lv apical and basal rotation angles during the cardiac cycle, can be quantified by post-processing -dimensional short axis images of the lv. thus, the aim of this study was to evaluate the global cs and the twisting motion in small-medium sized dogs with varying severity of mr attributable to myxomatous mitral valve disease (mmvd). using a vivid-i ultrasound system, all dogs underwent echocardiography including parasternal short axis views at the basal and apical level for offline analysis of rotation, and at the midpapillary muscle level for analysis of global cs, using commercially available software (echopac). twisting motion during systole (twist sys ), early (untwist early ) and late (untwist late ) diastole were calculated as well as their rates computed as the time derivatives. furthermore, time to onset of untwist, calculated from start of electromechanical activation until peak twist sys was measured. associations between twist and untwist variables, global cs and conventional echocardiographic indices of mr severity and lv remodeling were examined by multiple linear regression analyses including dog characteristics such as heart rate (hr), sex, breed, body weight and age. global cs increased with increasing mr (p < . ). untwist early (p = . ) and its rate (p = . ) also increased with increasing mr, albeit in co-variation with certain baseline dog characteristics. time to onset of untwist increased with mr (p = . ), left atrium to aortic root ratio (p = . ) and lv internal diameter in diastole (p < . ). in conclusion, untwist early and global cs gradually increased and the onset of untwist appeared to be delayed with increasing mr severity in small-medium sized dogs with spontaneous mmvd. this hyperdynamic stage with a delay in untwist may represent lv adaptation to loading conditions in mmvd, but might also indicate mid-and subepicardial compensation for an early lv dysfunction, as reflected by delayed onset of relaxation, as timing of contraction-relaxation cross over is the most vulnerable period of myocardial fiber mechanics. systolic anterior motion (sam) of the mitral valve is the mechanical correlate of left ventricular outflow tract dynamic obstruction (lvotdo) and has been associated with hypertrophic cardiomyopathy (hcm) in most instances. however, sam without left ventricular hypertrophy or with regressing hypertrophy is increasingly recognised.the echocardiographic studies of cats, diagnosed between / and / , with sam without hcm ( cats) or sam with regressing hypertrophy ( cats) were reviewed for evaluation of the anatomic and mechanic alteration contributing to lvotdo .ventriculo-aortic and aorto-septal malalignment were suggested by the significantly narrower aorto-mitral (am) and aorto-septal (as) angle compared to a group of control cats (as = versus -p = . ; am = versus -p = . ).other anatomic alterations of the lvot constituents included false tendon inserted on the septal crest ( / ), basal septal angulation ( / ), apical displacement of the postero-medial or antero-lateral papillary muscle ( / ), bifid/accessory papillary muscle ( / ), aberrant chordal insertion on the septal crest ( / ), severe papillary muscle hypertrophy ( / ), and thickened aortic cusps with sub-aortic ancillary echoes ( / ).mechanisms of lvotdo included obstructive mitral septal leaflet ( / ), protrusion of a displaced papillary muscle ( / ), impingement of an angulated basal septum into the lvot ( / ), and mechanical lvot narrowing from hypertrophic papillary muscle ( / ). basal septal impingement into the lvot was associated with apical ballooning ( / ) without obvious apical akinesis.out of cats with lvotdo without hypertrophy, had follow-up studies; only cat responded positively to atenolol treatment. all the cats with initial hypertrophy had reverse remodelling and normalised aortic ejection velocities within month to month after atenolol treatment initiation; cat developed aortic regurgitation.these data indicate that sam is not exclusively related to hcm and may be a cause, not a complication, of left ventricular hypertrophy. therapies that delay the onset of congestive heart failure (chf) in dogs with dmvd at risk of disease progression would be clinically beneficial. increases in la/ao, lveddn and serum nt-probnp and ctni concentrations are associated with decreased survival times. activation of the renin-angiotensinaldosterone system is implicated in cardiac remodeling in canine dmvd. we hypothesised that administration of spironolactone to dogs with compensated dmvd demonstrating the above risk factors would reduce the rate of cardiac remodeling associated with progressive dmvd. dogs with acvim class b dmvd were recruited to a randomized, blinded, placebo-controlled pilot study. no dogs were receiving medications for cardiac disease. all dogs demonstrated at least one of the following risk factors: echocardiographic evidence of cardiomegaly, nt-prob-np> pmol/l, ctni> . ng/ml. no dogs had evidence of other cardiac disease or renal disease, hypoadrenocorticism, hyperkalaemia, or hyponatraemia. dogs were randomized to receive spironolactone ( mg/kg orally) or placebo sid for months. comparisons between groups were made using mann-whitney tests. repeated measures linear models were constructed to compare the rate of change of variables over time. significance was set at p< . . data were analysed based on the intention to treat. twenty dogs of varying breeds were enrolled. ten dogs demonstrated risk factors, dogs risk factors and dogs risk factor. ten dogs received placebo; age range . - . years (mean ±sd, . ± . years), body weight range . - . kg ( one dog in this group died suddenly, progressed to chf and received suboptimal spironolactone dosage. nt-probnp was significantly higher in the spironolactone group at baseline. (p= . ). nt-probnp (p= . ), la/ao (p= . ) and lveddn (p= . ) increased over time in the placebo, but not in the spironolactone, group. the rates of change of nt-probnp (p= . ), la/ao (p= . ) and lveddn (p= . ) approached, but did not reach, significant differences between groups. in conclusion, treatment with spironolactone might slow the rate of increase in cardiac size in dogs with acvim class b dmvd showing risk factors for poor outcome. decreasing the rate of increase in cardiac size might delay the onset of chf. further studies are warranted to investigate these hypotheses. cardiac dysfunction is a concern in human systemic inflammatory response syndrome (sirs) patients, where increased cardiac biomarkers and decreased cardiac function have already been described. in a previous canine sirs study, an increase of cardiac biomarkers (nt-probnp, ctnt and lactate) and their prognostic value has been established. the present study evaluated the kinetics of basic echocardiographic parameters (fractional shortening (fs) and left ventricular ejection fraction (lvef), which both reflect systolic function; and the ratio of the left atrium to the aorta (la/ao), which reflects preload) in canine sirs. our hypotheses were that ( ) fs, lvef and la/ao are altered in canine sirs and ( ) that these parameters carry prognostic information.dogs with sirs, without primary cardiac disease, presenting to the emergency service were prospectively included from january until august . cardiac ultrasonography was performed by two veterinarians in a standardized fashion at initial presentation, after (t ), (t ), (t ), (t ) hours of hospitalization until discharge or death and at a control visit (t m) over one month after discharge. dogs were classified according to their underlying disease process: infection, neoplasia, trauma, gastric-dilation and volvulus (gdv), other gi diseases, and miscellaneous diseases. statistical analysis was performed with sas. univariate analysis was used to assess normal distribution. a mixed procedure and a logistic procedure was performed accordingly (p < . ).thirty seven dogs (infection, n= ; neoplasia, n= ; trauma, n= ; gdv, n= ; other gi, n= and miscellaneous diseases, n= ) were included. twenty-eight patients survived, while did not (died, n= ; euthanasia for financial reasons, n= ; euthanasia for prognostic reasons, n= ). eleven dogs had control visits, owners declined a control echocardiography, patients were lost to follow-up and died before control visit. fs and la/ao were significantly correlated with survival to discharge, however lvef was not. additionally, lvef and fs did not change significantly during hospitalization; neither compared to t m. la/ao did however increase significantly during hospitalization. la/ao at t ( . ; . - . ) differed significantly from values at t ( . ; . - . ), t ( . ; . - . ) and t m ( . ; . - . ).unexpectedly, surviving dogs had lower fs ( . %; - ) than non-survivors ( . %; - ). la/ao was associated with survival and increased rapidly after hospitalization to values similar to t m, which probably reflects the efficacy of fluid therapy in emergency cases.in this population of canine sirs patients, no echocardiographic evidence of cardiac dysfunction was demonstrated. reports from first-opinion practice of feline arterial thromboembolism (ate) are scarce. our aim was to describe and evaluate the outcome in cats with ate presenting to three first-opinion clinics.clinical records of cats presenting with ate between - were reviewed for history, clinical findings, presence of congestive heart failure (chf) and outcome. kaplan-meier and log rank analysis was performed to evaluate associations with survival.during the study period, cats presented with ate; an overall feline incidence of . %. most cats were male ( . %) and non-pedigree ( . %). signs of cardiovascular disease prior to ate included cardiomyopathy ( %), a heart murmur ( . %), a gallop sound/arrhythmia ( . %) and hyperthyroidism ( . %). most cats presented within hours of clinical signs ( . %). median age at presentation was atrioventricular block (avb) is an arrhythmia resulting from conduction abnormalities through the atrioventricular node that leads to severe signs and sudden death.the aim of this study was to evaluate long-term intrinsic rhythm variations in dogs undergone pacemaker (pm) implantation. ninety-two dogs of different breeds with rd degree avb ( avb) ( . %), advanced nd degree avb ( avb)( . %), paroxysmal avb ( %), : avb ( . %) and avb with atrial fibrillation ( . %) were retrospectively analyzed. forty-nine ( . %) were males and ( . %) females with a mean age of . + . (sd) years and a mean body weight of . kg + . . the intrinsic rhythm was evaluated the day of pm implantation (t ), after day (confidence interval % [ci %] - ) (t ), days (ci % - ) (t ), days (ci % - ) (t ), days (ci % - ) (t ). according to the avb grade at different controls, the rhythm disturbance was considered advanced, regressed or unchanged. shapiro-wilk and kolmogorov-smirnov tests were used to test normalcy, f-test to compare means in a generalized linear model and chi-squared to examine the association between categorical variables and status at each control. sixty ( . %) dogs had no intrinsic rhythm changes, ( . %) had avb progression and ( %) had avb regression. forty-eight cases of avb remained unchanged, while regressed to sinus rhythm, to : avb and to advanced avb. eight advanced second degree avbs progressed to complete avb, regressed to sinus rhythm, to : avb and remained unchanged. five paroxysmal avbs progressed to complete avb, to : avb and remained unchanged. four : avbs progressed to complete avb, regressed to sinus rhythm and remained unchanged. all avbs with atrial fibrillation remained unchanged. chi-square test showed that changes of intrinsic rhythm were associated with the type of avb (v . , p<. ) and the time of controls (v . , p<. ), while other factors were not statistically significant. regression occurred within days while progression occurred at any times. the results showed that the degree of avb at the moment of pm implantion should not be considered a definitive diagnosis since more than / of the cases could present progression or regression. because of their potential progression, pm implantation should be considered as first choice treatment also in cases of low avbs, and further studies are needed to evaluate the cause of transitory high grade avbs. tissue doppler imaging (tdi) is a complement to conventional echocardiography for assessment of myocardial function. the aims of the study were to investigate breed differences and intraobserver-variability of colour tdi variables in healthy dogs.fifty-three privately-owned male dogs were prospectively recruited. dogs were declared healthy by physical examination, blood pressure measurement, ecg, analyses of urine and blood (haematology and biochemistry), and conventional echocardiographic d and doppler examination, as part of the eu-funded lupa-project. directly following these extensive examinations, the tdi acquisition was performed by the same experienced echocardiographer using a philips hd xe with a s - (small dogs) and s - (large dogs) mhz probe. cineloops were acquired from the right short-axis view and radial colour tdi variables at the endocardium and epicardium of the left ventricular free wall were later analysed. six other healthy dogs were included in a substudy aimed at evaluating the effect of sources of variation using a hierarchial random-effects model.ten of the examined dogs were excluded due to breathing artifacts and poor quality of the curves, leaving dogs in the study; labrador retrievers ( ), cavalier king charles spaniels ( ), and dachshunds ( ), with a mean age of . ± . years (sd). a p-value < . was considered significant in the statistical analyses. kruskal-wallis one-way analysis of variance showed that labrador retrievers had significantly higher values for endocardial and epicardial systolic (s) waves, and longer time to peak for both endocardial and epicardial e-and a-waves, compared to the other two breeds. labrador retrievers also had significantly lower heart rate (hr). further analysis of the breed differences using multiple regression analysis showed major effects of body weight and hr on endocardial and epicardial s waves, while time to peak, both endocardial and epicardial, for the e-and a-waves, were primarily affected by hr. dog was the variance component having the major effect on variability of tdi variables.in conclusion, time to peak of both diastolic waves was longer in labrador retrievers compared to the small-breed dogs. furthermore, higher s-wave values in labrador retrievers might indicate a different contractility pattern in largebreed dogs, and warrants further investigation. mitral regurgitation (mr) is the most common heart disease in dogs.dogs with a more advanced stage of this disease are likely to develop pulmonary edema of heart failure. the aim of this study was to evaluate the plasma c-reactive protein (crp) concentra-tion in dogs that underwent mitral valve repair for mr.all dogs were operated between october and october . the dogs were categorized according to the international small animal cardiac health council (isachc) classification, and physical examination, thoracic radiography, and d color flow doppler echocardiography were performed before and after surgery. the plasma crp concentration and white blood cell counts were also determined before and after surgery. cardiogenic pulmonary edema was diagnosed on the basis of clinical examination and thoracic radiography.overall, dogs (mean body weight, . ± . kg and mean age, . ± . years) were enrolled; of these dogs had cardiogenic pulmonary edema. the dogs breeds were chihuahua (n = ), cavalier king charles spaniel (n = ), maltese (n = ), yorkshire terrier (n = ), shih tzu (n = ), miniature dachshund (n = ), and others (n = ). no significant difference was found for age and body weight. the vertebral heart size and la/ao ratio significantly decreased after surgery compared with the preoperative values. before the operation, crp concentration and white blood cell counts in isachc class iiib dogs ( . ± . mg/dl and ± /ll, respectively) were higher than those in class ib ( . ± . mg/dl and ± /ll, respectively), class ii ( . ± . mg/dl and ± /ll, respectively), and class iiia ( . ± . mg/dl and ± /ll, respectively) dogs. additionally, crp concentration and white blood cell counts in class iiib dogs significantly decreased after surgery compared with preoperative values. crp concentration and white blood cell counts in the dogs with cardiogenic pulmonary edema significantly increased compared with those with non-pulmonary edema. furthermore, cardiogenic pulmonary edema disappeared within months after surgery, and the crp concentrations and white blood cell counts became normal.in conclusion, crp concentration increases in dogs with mr and cardiogenic pulmonary edema. it is widely recognized that inflammatory reaction plays a key role in the development of heart failure. consequently, these data indicate the importance of strict management for pulmonary edema and inflammation. the neurotransmitter serotonin ( -hydroxytryptamine, ht) has recently been suggested to have a role in development of myxomatous mitral valve disease (mmvd) in dogs.the aim of this study was to investigate whether serum ht concentration was associated with mmvd severity in dogs, and to assess potential associations between serum ht concentrations and dog characteristics, echocardiographic variables, heart rate, systolic blood pressure, and platelet size (mean platelet volume) in the study population. client-owned dogs with naturally acquired mmvd of varying severity were prospectively recruited for the study. dogs were classified according to mmvd severity (breeds predisposed to early onset of mmvd, but without echocardiographic evidence of the disease, or mild, moderate or severe disease). serum ht concentrations were analyzed using an elisa assay.lower serum ht concentrations were shown in dogs with severe mmvd, compared with dogs predisposed to mmvd (p = . ) and dogs with mild mmvd (p = . ). unilinear and multiple regression analyses showed that serum ht concentrations decreased with increasing left atrial to aortic root ratio (la/ao), were higher in cavalier king charles spaniel (ckcs) dogs compared to dogs of other breeds, and were higher in female dogs than in male dogs. the la/ao was the variable most strongly associated with serum ht concentration.in conclusion, the finding of higher serum ht concentrations in dogs predisposed to mmvd (ckcs) and dogs with mild mmvd suggests that alterations in ht signaling might play a role in progression of early stages of mmvd. hypersomatotropism (hs) can be a common reason for development of diabetes mellitus in the cat. remission of diabetes can be achieved with an accurate diagnosis of the hs, although diagnosis is hampered by the relative complexity of confirming hs, requiring a combination of insulin growth factor- (igf- ) or feline growth hormone measurement and intracranial imaging. unfortunately, all three have limitations as diagnostic aid and when evaluating the success of therapy, particularly radiotherapy (rt). consequently, more precise markers of hs are required. the current study aimed to evaluate physiological behaviour and diagnostic potential of serum ghrelin in feline hs. as ghrelin is an endogenous ligand of the gh secretagogue receptor, it was hypothesised production of ghrelin might be suppressed in hs and subnormal serum levels could be a marker for increased gh activity.fasted (pre-insulin) serum samples were collected from normal (age matched control), uncomplicated diabetic (dm) and hypersomatotrophic diabetic (hsdm) cats. cats were categorized into the hsdm-group on the basis of elevated igf- (> ng/ml) and demonstration of a pituitary lesion on imaging. in cats additional serum samples were obtained following rt. cats were categorized into the dm-group on the basis of low igf- (< ng/ml) and normal insulin requirements (< . iu/kg). serum ghrelin was determined using a total ghrelin elisa system validated for the cat. data were tested for normality and concentrations compared between groups using unpaired t-tests and a paired t-test for the before and after rt hsdmgroups.serum total ghrelin was not different between the hsdm ( . ng/ml+/- . ) and dm-group ( . ng/ml+/- . , p= . ). a significant difference was present between the control group ( . ng/ml+/- . ) and both the hsdm (p= . ) and the dmgroup (p= . ). serum ghrelin concentrations in the hsdm cats undergoing rt (n = ) were significantly higher following completion of treatment ( . ng/ml+/- . versus . ng/ml+/- . , p= . ).the results suggest feline serum total ghrelin is suppressed to similar levels in both the diabetic and diabetic hypersomatotrophic state compared to healthy subjects. consequently, it appears serum ghrelin levels are not helpful in determining the presence of hs in the diabetic cat. however, the results do indicate treatment of hs with rt results in an increase in serum ghrelin, suggesting the presence of an independent inhibitory effect of excess gh on serum ghrelin production. this potential inhibitory effect of gh might render serum ghrelin measurement useful as an additional tool to assess hypersomatotrophic remission after rt. further studies are however indicated. to date only few studies characterized histopathological features of the endocrine and exocrine pancreas in cats with diabetes mellitus. loss of b-cells is a consistent finding but no detailed data about the presence and types of inflammatory cells are available. we recently observed that hyperglycemia increases neutrophils in the exocrine pancreas. the aims of the present study were to assess whether diabetic cats have pathological evidence of islet inflammation or pancreatitis and to define islet lesions in comparison to a well-matched control population.formalin-fixed, paraffin-embedded pancreatic samples were collected from post-mortem examination performed on diabetic and control cats died due to any disease at the clinic for small animal internal medicine, university of zurich (switzerland) between and . control cats were selected to be matched for age, sex, breed and body weight. sections were routinely stained with hematoxilin-eosin, and doublelabeled immunohistochemistry was performed for the following markers: insulin and myeloperoxidase (neutrophils), insulin and cd (t-lymphocytes), insulin and cd (b-lymphocytes), insulin and pcna (proliferation marker), and glucagon and ki- (proliferation marker). light-microscopic cell counting and morphometric analyses were performed manually and with software (image-j), respectively. data were analyzed with contingency tables and ttests.thirty-seven diabetic cats and controls were included. the mean insulin-positive cross sectional area was approximately % lower in diabetic than control cats (p< . ), that of glucagon was similar. proliferation of insulin-positive and glucagon-positive cells and the average counts of neutrophils, t-and b-lymphocytes in the islets did not differ between groups. interestingly, the presence of (t and b) lymphocytes in general tended to be more frequent in diabetic ( / = . %) than control ( / = . %) cats. in the exocrine pancreas, a trend towards increased presence of necrosis and fibrosis was observed in diabetic cats ( / = . % vs. / = %; p= . ) but inflammatory infiltration did not differ. proliferation of acinar cells was -fold increased in diabetic cats (p< . ), notably nearby islets ( -fold, p< . ).the results confirm previous observations that loss of b-cells occurs in diabetic cats. in addition, a subset of diabetic cats shows lymphocytic infiltration of the islets that might have contributed to b-cell loss. increased necrosis and fibrosis of the exocrine tissue may suggests that the diabetes leads to pancreatitis in some cats. the increased proliferation rate of acinar cells deserves further investigation. in humans this finding has been associated with chronic pancreatitis as well as transdifferentiation into islet cells. on the basis of a relatively high prevalence of hypersomatotropism (hs) amongst cats with a diagnosis of diabetes mellitus, as well as the possible subtle phenotype of these patients and the significant implications on prognosis and treatment, screening diabetic cats for hs could be advocated. for most veterinarians serum total insulin-like growth factor- (igf- ) assessment represents the most feasible and accessible means of performing screening. however, hepatic igf- production is dependent on presence of sufficient portal insulin, which can be deficient in newly diagnosed diabetic cats, resulting in false negative results. additionally, elevation of igf- has been reported in non-acromegalic diabetic cats. alternative or additional diagnostic tests for hs, as well as to evaluate the success of treatment of hs, are therefore desirable. since feline hs is associated with tissue growth, serum type iii procollagen propeptide (piiip), a peripheral indicator of collagen turnover, was hypothesised to be a useful indicator of active disease or growth hormone bioactivity. fasted, pre-insulin, serum samples were prospectively collected from uncomplicated diabetic (dm) and hypersomatotrophic diabetic (hsdm) cats. cats were categorised into the hsdm-group on the basis of elevated igf- (radioimmunoassay, > ng/ml), followed by demonstration of a pituitary lesion on intracranial imaging and into the dm-group on the basis of low igf- (< ng/ml) and modest insulin requirements (< . iu/kg). an elisa system for piiip was developed for use in the cat. data were tested for normality and concentrations compared using the mann whitney test. correlation with serum igf- was assessed by calculating a spearman's correlation coefficient (rho).dilutional parallelism using cat serum with high and low piiip-activity indicated validity of the elisa system. intra-assay coefficient of variation calculation proved adequate precision at high and low concentrations ( . % and . %) and the assay detection limit was found to be . ng/ml. median serum piiip was . ng/ml (range: < . - . ) in the hsdm group, versus . (range: < . - . ) in the dm group (p< . ). there was a significant correlation between serum igf- and piiip (rho= . , p< . ).in conclusion, serum piiip can be measured in the cat. additionally, serum piiip seems an alternative measure of growth hormone bioactivity in cats, given the significant elevation in concentration in feline hs. further evaluation of piiip in cases with hs will help determine the exact added value of evaluating this parameter in the diagnosis of hs, as well as in the assessment of treatment efficacy. glucose and galactose are transported across the brush border membrane (bbm) of enterocytes by sodium/glucose cotransporter- (sglt ), coded for by slc a . sglt is the sole route for intestinal glucose absorption and its level of expression dictates bbm transport capacity for glucose. the relevance of sglt expression in predisposition to diabetes mellitus and obesity was investigated in dogs. the aims were to assess the effect on promoter function of known snps in the ' flanking region of canine slc a , and to search for novel snps in well defined samples of dogs with varying risk for diabetes or obesity. caco- /tc cells were shown to express sglt in vitro. the kbp fragment of canine slc a ' flanking region from - to + relative to the transcription start site was cloned from canine genomic dna, ligated into pgl basic plasmids bearing firefly luciferase as a reporter gene, used for transient transfection of caco- /tc cells, and shown to drive luciferase production significantly above control (p< . ). to determine the effect of the three known snps in this region on promoter function, new promoter/reporter constructs (all possible permutations of these three snps) were created using site-directed mutagenesis. these constructs were used for transient transfection of caco- /tc cells using renilla luciferase as an internal control. no significant differences in promoter function were seen, suggesting that these three snps do not have a significant effect on the constitutive transcription of sglt mrna in dogs.a search for novel snps in this region in dogs was made in two breeds predisposed to diabetes mellitus (samoyed, cairn terrier), two breeds that rarely develop diabetes (boxer, german shepherd dog), and two breeds predisposed to obesity (labrador retriever, cocker spaniel). genomic dna from healthy individuals of each of these breeds was obtained from the uk companion animals dna archive, with kind permission. the slc a ' flanking region was amplified from each individual by high-fidelity pcr using breed-labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing ( sequencing, gs flx, roche). the sequence of the slc a ' flanking region in all individuals of all breeds tested was identical. on this evidence, variations in slc a promoter sequence between dogs do not influence the pathogenesis of diabetes or obesity in these breeds. remission of diabetes mellitus may be achieved in up to % of cats. for remission to occur, recovery of b-cell function and possi-bly of b-cell mass is required. a novel class of antidiabetic drugs that act via the incretin system increase b-cell proliferation and glucose-stimulated insulin secretion in rodents. two strategies of incretin-based therapies, glp- analogues (e.g. exenatide shortacting (ex-sa), exenatide long-acting (ex-la)), and dpp- inhibitors (e.g. sitagliptin), are successfully used in human diabetics. knowledge about the use of incretins in cats is scarce. it was demonstrated that ex-sa and a dpp- inhibitor (nvp-dpp ) increase insulin secretion after intravenous glucose stimulation in healthy cats. the effects of these drugs after meal stimulation and the use of ex-la have not been explored in cats so far.the aims of the study were to test whether ex-sa (byetta ® , q h, sc), ex-la (bydureon ® , q d, sc) and sitagliptin (januvia ® , q h, po) can be safely used in cats, and to identify the most effective drug and dose in a dose-escalation study.nine healthy cats were used. ex-sa was given to cats at . , . , and lg/kg for consecutive days each. ex-la was given to other cats at , , and lg/ kg with single injections each. sitagliptin was given to cats at , , and mg/kg for consecutive days each. a washout period of weeks was allowed between doses. on day of each treatment block, a meal response test (mrt) was performed in all cats after a h fastby feeding % of daily energy intake with subsequent blood sampling at timepoints , , , , and minutes. insulin and glucose area under the curves (auc) were calculated for each drug dose.gastrointestinal side effects of to days duration were observed in cats of each group, irrespective of the dose. well-being and appetite were otherwise conserved. ex-sa increased insulin auc by %, %, % and %, respectively, compared to insulin auc during mrt without drug administration. ex-la and sitagliptin increased insulin auc by %, %, %, % and %, %, %, %, respectively. auc for glucose was similar in all cats, irrespective of the drug and dose. we conclude that ex-sa, ex-la and sitagliptin can be safely used in healthy cats and that ex-sa increases insulin secretion more effectively than ex-la and sitagliptin. insulin detemir is a synthetic long acting insulin analogue designed to maintain basal levels of insulin in humans with diabetes mellitus (dm).pharmacokinetic studies in dogs indicate that insulin detemir has a greater effect than other types of insulin, requiring a lower dose. the objective of our study was to evaluate its efficacy and the frequency of hypoglycemia in dogs with dm treated with insulin detemir. eight dogs were included into the study. median (range) age was years ( - ), were female ( intact, spayed), and was intact male; median (range) body weight was . kg ( . - . ). dogs with relevant concurrent diseases (e.g. hypothyroidism, hypercortisolism, neoplasia, renal insufficiency) and dogs with prior administration of diabetogenic drugs were excluded. all dogs received insulin detemir bid for at least months, re-evaluations were performed after , , , and weeks and included clinical signs, blood glucose curves (bgc) and fructosamine concentrations. median (range) insulin dose was . u/kg ( . - . ) bid at admission which was not significantly different after weeks of therapy ( . u/kg, range . - . ). initially, all dogs had markedly elevated blood glucose ( . mmol/l, . - . mmol/l), and elevated fructosamine concentrations ( lmol/l, - lmol/l). mean(±sd) glucose concentrations (mmol/l) of the bgc at each re-evaluation were . ± . , . ± . , . ± . , . ± . , . ± . ; median (range) of the glucose nadir values (mmol/l) were . ( . - . ), . ( . - . ), . ( . - . ), . ( . - . ), . ( . - . ) and fructosamine concentrations (lmol/l) were ( - ), ( - ), ( - ), ( - ), ( - ), respectively. glucose concentrations (mean) were significantly (p< . ) lower after weeks therapy than before treatment. hypoglycemia (glucose nadir < mmol/l) was a consistent problem, identified in dogs ( % of the bgc of the dogs). there were episodes (in dogs) of owners reporting clinical signs (lethargy, weakness, unsteady gait) that could have been caused by hypoglycemia. based on owner opinion, clinical assessment of the veterinarian and bgc after months of therapy, good control of the disease was obtained in ( %) and moderate control in dogs ( %). according to our preliminary results insulin detemir is effective in controlling hyperglycemia in dogs with dm. it is more potent than other types of insulin which are also used bid and therefore lower doses were used. nevertheless hypoglycemia was a common finding especially in small dogs. therefore insulin detemir should be used with great caution and dilution could be considered. spontaneous hypercortisolism is due to acth-independent hypersecretion of cortisol by an adrenocortical tumor (at) in about % of the cases. although the understanding of growth and hormonal activity of ats has expanded in recent years, the pivotal factors/acts in the pathogenesis of the at remain undisclosed.the canonical wnt-pathway plays a role in cell survival and cell cycle progression and has been shown to be involved in many different tumor types, including human and mouse ats. central in this pathway is ß-catenin. cytoplasmic and/or nuclear accumulation of ß-catenin has been demonstrated in human cortisol-secreting ats. this has been partly explained by mutations in exon of ß-catenin.in our study, the activation of the canonical wnt-pathway was investigated in adrenal adenomas and carcinomas of dogs with acth-independent hypercortisolism. fifteen normal canine adrenals served as control tissue. the mrna expression was measured for wnt-ligands (wnt , wnt , wnt , wnt a, wnt b variants and , wnt , wnt a, wnt b), wnt-ligand inhibitors (dkk , wif , sfrp ) and for wnt target genes (cmyc, axin and cyclind ). in addition, the coding region of the mrna of ß-catenin was sequenced and the localization of ß-catenin was evaluated by immunohistochemistry (ihc).the results of expression analysis of wnt-ligands and wnt-ligand inhibitors demonstrated a significant downregulation of wnt and wnt b variants and . wnt target gene cyclin d was significantly downregulated in adenomas, while cmyc and axin mrna expression did not differ between ats and normal adrenals.sequence analysis of ß-catenin revealed a mutation in / of the ats: silent mutations, most probably single nucleotide polymorphisms (snps), neutral mutation (arginine to histidine), nonsense mutation resulting in a premature stop codon and deletion of basepairs combined with a neutral mutation. the amino acid change appeared not to be tumorassociated whereas the stop codon was. there were no activating ß-catenin mutations. the ihc demonstrated accumulation of ß-catenin in the cytoplasm of part of the ats, while nuclear staining for ß-catenin was rarely present.we conclude that the canonical wnt-pathway is most likely not involved in the pathogenesis of canine cortisol-secreting ats. trilostane is the treatment of choice for canine pituitary-dependent hyperadrenocorticism (pdh) however there is controversy about the ideal treatment regime. the objective of this study was to evaluate efficacy and safety of sid vs bid trilostane treatment for canine pdh.this prospective randomised study included dogs with pdh, dogs treated with trilostane sid (initial dose - mg/kg/ hrs) and with trilostane bid ( . - mg/kg/ hrs). a history, physical exam, haemogram, biochemical profile, acth stimulation test and urinary cortisol to creatinine ratios (uccr) were performed before treatment, and at one week (sid - hrs post trilostane; bid - hrs) and , , and months after treatment ( - hrs post medication)the median (±sd) daily dose throughout the study was similar in dogs receiving sid ( . ± . mg/kg/day) or bid ( . ± . mg/kg/day) trilostane. baseline cortisol concentration (mean±sd) before trilostane and at the five reevaluations in the sid group were . ± . , . ± . , . ± . , . ± . , . ± . and . ± . and in the bid group were . ± . lg/dl, . ± . , . ± . , . ± . , . ± . and . ± . . baseline cortisol was significantly higher (p= . ) in the sid group only at six-month evaluation. post acth cortisol concentrations in the sid group were . ± . , . ± . , . ± . , . ± . , . ± . and . ± . and in the bid group were . ± . , . ± . , . ± . , . ± . , . ± . and . ± . . no statistically significant differences were found at any evaluation. uccr in dogs in the sid group were ± , ± , ± , ± , ± and ± . and in the bid group ± , ± , ± , ± , ± and ± . similarly no statistically significant differences were found throughout the study. in each of the five re-evaluation times, clinical signs (polyuria, polydipsia and/or polyphagia) persisted in % ( / ), % ( / ), % ( / ), % ( / ) and in % ( / ) of dogs in the sid group. persistence of clinical signs occurred in % ( / ), % ( / ), % ( / ), % ( / ) and % ( / ) of dogs in the bid group. mild adverse effects occurred in / and in / of the dogs treated with sid and bid trilostane, respectively.based on this study, we conclude that the results of acth stimulation test and uccr in dogs with pdh treated with both protocols are similar at most re-evaluation times. however, clinical signs resolved in a greater proportion of dogs receiving trilostane bid. using trilostane twice daily will help to reduce the number of dogs that do not have a good clinical response. the effectiveness of trilostane therapy is monitored by regular acth stimulation, which is time-consuming and expensive. therefore, a monitoring system without a stimulation protocol and with less client expense would be preferable.the aim of our study was twofold: firstly, to evaluate, if baseline cortisol, endogenous acth (cacth) or the baseline cortisol to acth ratio (cortisol/acth ratio) could replace the acth stimulation test; secondly, to evaluate, if baseline cortisol provides additional information than post-acth cortisol alone or if its measurement could be abandoned.forty-one trilostane-treated dogs with pdh diagnosed between april and december were included in the study. a total of acth stimulation tests with baseline, post-acth and delta cortisol (post-acth cortisol minus baseline cortisol) and cacth results and cortisol/acth ratios were analyzed.control of adrenal gland function was classified according to the target range of post-acth cortisol concentration as: excessive (< nmol/l; group ), optimal ( - nmol/l; group ), or inadequate (> nmol/l; group ). in a second step, control of adrenal gland function was reclassified according to baseline cortisol values only and the new classification was compared with the original one.there was a significant correlation between baseline cortisol and post-acth cortisol and a significant difference of baseline cortisol between the groups, however, with a large overlap. reclassification of the adrenal gland function on the basis of baseline cortisol revealed a misclassification in / ( %) tests.endogenous acth did not correlate with baseline or post-acth cortisol and did not differ between the groups. the baseline cortisol:acth ratio differed significantly between group and and between group and , but again with a large overlap.to determine if measurement of baseline cortisol gives additional information than post-acth cortisol alone, the delta cortisol values were analysed. delta cortisol correlated significantly with post-acth cortisol but not with baseline cortisol and differed significantly between the groups, the overlap however was large.the large overlap using only single values without taking post-acth cortisol into consideration leads to an unacceptably low correct differentiation of control of adrenal gland function. therefore, the acth stimulation test and determination of post-acth cortisol cannot be replaced by baseline cortisol, endogenous acth or the cortisol/acth ratio. however, as baseline cortisol concentration does not give additional information than post-acth cortisol alone, its determination can be abandoned. in humans, recombinant human thyrotropin (rhtsh) enhances radioactive iodine uptake (raiu) in patients with differentiated thyroid cancer. this property is particularly interesting in dogs because high doses of radioiodine- ( i) are used for the treatment of this disease. no studies have been performed in veterinary medicine to optimize i treatment of thyroid cancer.the aim of this study was to evaluate the effect of rhtsh on the uptake of i in dogs with thyroid tumors.nine dogs with thyroid neoplasia were included in this prospective cross-over study. six dogs had unilateral tumors, dog had bilateral tumors and dogs had ectopic tumors. diagnosis was based on physical examination, cytology, cervical scintigraphy and, when available, histopathology. in dogs i was administered for a baseline raiu determination in week . in week (after a wash out period of weeks), these dogs received rhtsh ( lg iv) h before i injection. in patients the order of the protocol was reversed. for each scan, the dogs received mbq ( mci) of i iv and planar scintigraphy was performed h and h thereafter for tumor raiu calculation. blood samples were taken at baseline and at , , and h after rhtsh administration for measurement of serum total thyroxine (tt ) and serum thyrotropin (tsh) concentrations.rhtsh caused no statistical significant change on thyroid tumor raiu at h (p= . ) or at h (p= . ). despite the lack of overall statistical significance, after rhtsh administration the h raiu increased in tumors and the h raiu increased in tumors. when an increased raiu was observed, i uptake with rhtsh ranged . to . times baseline uptake. in patients, the post-rhtsh raiu more than doubled compared to baseline raiu. the raiu of thoracic metastases from patients could be calculated. in thoracic metastasis the raiu doubled after rhtsh; in the other the raiu decreased after rhtsh.in euthyroid patients, rhtsh induced a significant increase in tt concentrations (p= . ), confirming the biological activity of rhtsh.this study suggests that iv administration of lg rhtsh h before i has an inconsistent effect on thyroid tumor raiu, with a marked increase in uptake in some tumors and a decrease in others. further studies are necessary to determine the best dosage, route and timing of rhtsh to optimize thyroid tumor raiu.this study was partly funded by the dutch animal cancer foundation transdermal methimazole has been suggested as an alternative treatment option of hyperthyroid cats. in a previous study we could show a good clinical effectiveness of a pleuronic lecithin organogel (plo-) based product on a twice-daily basis. a reduced dose frequency is known to improve owner compliance, however no study has yet evaluated long-term treatment responses after once daily administration.objectives of the present study were to assess whether once daily administration of transdermal methimazole in its original formulation of (plo) was an effective alternative to the twice-daily treatment during a follow-up period of up to months and to evaluate t courses during a -hour period after methimazole application in selected cats.twenty client-owned cats with newly diagnosed hyperthyroidism and with available follow-up information were included in the study. methimazole was formulated in plobased vehicle and was applied to the pinna of the inner ear at a starting dose of mg/cat q hours. cats were rechecked - weeks, - weeks, - months, - months, - months, - and - months after starting therapy. additionally, in cats t concentrations were measured every hours after gel application over a hour period week after starting therapy. after - weeks, clinical improvement was observed in all animals. a change of treatment to oral medication due to erythema of the internal pinna of the ears was necessary in one cat, while none of the other animals showed any side effects during the follow-up period. significant decreases in t concentrations were determined at all rechecks compared to pre-treatment concentrations. methimazole dosage was increased in , decreased in and remained unchanged in cats. two cats with a decrease in the dose later had to be re-increased, while one cat was changed to q hours. there was no significant change in t during the -hour period and fluctuations corresponded to variations of precision in series.these results are in accordance with those of our previous study using the twicedaily regimen. we could show that once daily administration of transdermal methimazole in its original formulation of plo is an effective treatment option for long-term management of feline hyperthyroidism. further, timing of blood sampling after gel application is not important when assessing response to treatment. increase in the prevalence of large thyroid tumors, intra-thoracic thyroid thyroid scintigraphy provides valuable information regarding both thyroid anatomy and physiology and plays an integral role in the diagnosis, staging, and management of feline thyroid disease. in this study, we performed thyroid imaging on , consecutive hyperthyroid cats that were referred for radioiodine therapy between january and december . scintigraphy was performed as part of our staging protocol in which thyroid volume is estimated for i dose estimation (vet radiol ultrasound ; : ) . in each cat, the location of each area of increased radionuclide uptake (iru) was also recorded (cervical, thoracic inlet, chest). finally, each scan was evaluated for features suggesting malignancy (multiple, extensive areas of iru, heterogeneous pattern of iru with irregular, spiculated margins, extension of tumor through thoracic inlet into the thorax, and metastasis to regional lymph nodes or lung).of the , cats, most had been recently diagnosed. in cats, however, the interval between diagnosis and i treatment ranged from > to . years; almost all of these cats had received long-term antithyroid drug treatment. the , cats were divided into groups based on interval from diagnosis to i treatment: group ( - year), , cats; group (> - years), cats; group (> - years), cats; group (> - years), cats; and group (> - . years), cats. when the estimated thyroid volumes in the groups of cats were compared, a progressive, significant (p< . ) increase in median tumor volume occurred: . cm (group ); . cm (group ); . cm (group ); . cm (group ); and . cm (group ). the prevalence of cats with areas of iru within the thoracic cavity also increased progressively: . % (group ); . % (group ); . % (group ); . % (group ); and . % (group ). finally, the prevalence of suspected thyroid carcinoma ( of the , cats) also increased progressively: . % (group ); . % (group ); . % (group ); . % (group ); and . % (group ). in contrast, no increase in prevalence of ectopic thyroid tissue was found: . % (group ); . % (group ); . % (group ); . % (group ); and . % (group ). in conclusion, our results indicate that hyperfunctional thyroid tissue continues to grow and enlarge over time. thyroid carcinoma is extremely rare in cats with recently diagnosed hyperthyroidism, but the prevalence increases dramatically over time, suggesting that transformation from benign disease is common in cats controlled medically. measurement of plasma renin activity (pra) is considered the gold standard for monitoring mineralocorticoid substitution in humans with primary hypoadrenocorticism (ph). it is the most sensitive parameter to reflect insufficient as well as inappropriate high replacement. in dogs with ph mineralocorticoid substitution is currently monitored mainly by serum potassium and sodium concentrations. the role of pra for monitoring mineralocorticoid replacement has not been investigated. the aims of the study were to measure and compare pra in dogs with newly diagnosed ph and dogs with diseases mimicking ph, and to evaluate pra in dogs with ph treated with different mineralocorticoid substitution regimes.the following groups of dogs were included in the study: dogs with newly diagnosed ph (group ), dogs that were already treated for ph (group ), and dogs with diseases mimicking ph (group ). in group pra was measured before treatment and - weeks, - weeks, and - weeks after start of therapy. in group pra was measured at least twice every to months. in group pra was measured once at initial presentation. three dogs of group and dogs of group were treated with fludrocortisone (florinef ® , bristol-myers squibb). two dogs of group and dogs of group were treated with docp (percorten ® -v, novartis). pra was measured with an enzymatic assay via trapping of angiotensin i in the service d'angiologie, chuv-nes, lausanne. results were analysed by means of non-parametric methods (p < . ).pra before treatment was significantly higher in group ( . - . ng/ l/h, median . ) than in group ( . - . ng/l/h, median . ). average pra during therapy ranged from . to ng/ml/h (median ). pra did not decrease significantly in dogs treated with fludrocortisone. pra of dogs treated with docp ( . - . ng/ml/h, median ) was significantly lower compared to dogs treated with fludrocortisone ( . - . ng/ml/h, median . ). all dogs treated with docp had normal serum sodium and potassium at all re-checks, whereas dogs treated with fludrocortisone had mild to severe electrolyte abnormalities at several occasions. there was a weak correlation between pra and serum potassium.measurement of pra is a promising tool for monitoring mineralocorticoid substitution in dogs with ph. according to our preliminary results docp is superior to fludrocortisone for mineralocorticoid replacement. phenobarbital is widely used to control epilepsy in dogs. use of phenobarbital induces hepatic enzyme activity, and may decrease serum total and free thyroxine (tt and ft ), with the exact mechanisms and prevalence of this phenomenon being unknown. the aim of the present retrospective study therefore was to investigate how many dogs treated with phenobarbital show a decrease in thyroid hormones and to give insight into potential mechanisms. for this, tt , ft , and tsh were measured in canine serum samples submitted for assessment of therapeutic concentrations of phenobarbital. in a smaller subset of dogs, albumin, total protein, and transthyretin (ttr) were also measured at the diagnostic center for population and animal health, michigan, usa.according to thyroid results, dogs were classified as 'non thyroidal illness' (nti) (tt < nmol/l, ft ! pmol/l, tsh . ng/ml); 'equivocal' (e) (tt < nmol/l, ft < pmol/l, tsh . ng/ml); 'hypothyroid' (ht) (pattern of low tt and ft , tsh > . ng/ ml); and 'normal' (n) (tt ! nmol/l; ft ! pmol/l and tsh . ng/ml).forty-five dogs were classified as nti ( . %), dogs were classified as e ( . %), were classified as ht ( . %), and as n ( %); dogs did not fit in any defined category. there was no statistically significant difference in mean phenobarbital concentrations between nti, e, ht, and n ( . ± . , . ± . , . ± . , and . ± . lmol/l, respectively). twenty dogs of each group were analyzed for albumin and total protein and compared to healthy dogs that were not receiving phenobarbital. no statistically significant difference was noticed (p> . ). the attempt to measure ttr concentrations via commercially available elisa (tsz elisa, framingham, ma, usa) gave us inconsistence performance. in conclusion, % of dogs on phenobarbital treatment were classified as nti. if group e would be included, this value could be as high as %. neither phenobarbital, nor total protein or albumin concentrations had predictive value for thyroid hormone concentrations. this study was able to define the prevalence of the phenomenon that some dogs on phenobarbital therapy have low thyroid hormone concentrations, but did not give insight into the pathogenesis. ideally, demonstration of hypothyroidism in dogs receiving phenobarbital should include assays of thyroid hormones and tsh. pheochromocytoma (pheo) is a rare malignant catecholamine-secreting tumor of the adrenal medulla. catecholamines and metanephrines in plasma and in -h urine are approved biomarkers for the detection of the disease in humans, however, the question which of the tests is best is controversial. we previously demonstrated that measurement of urinary catecholamine and metanephrine to creatinine ratios is helpful for the diagnosis of pheo in dogs and that urinary normetanephrine to creatinine ratio may be the best test to discriminate between pheo and hypercortisolism (hc).knowledge on plasma catecholamines and metanephrines in dogs is scarce and no comparison between urinary and plasma parameters has been performed. the objective of the study was to measure urinary and plasma catecholamines and metanephrines in dogs with pheo, hc and in healthy dogs and to determine the test with the least overlap between the groups. six dogs with pheo, dogs with hc ( with ath, with pdh) and healthy dogs were included. urine samples were collected into hcl containing tubes to ensure a ph < , blood samples were collected on ice, centrifuged at °c and immediately snap frozen in liquid nitrogen. all samples were stored at - °c. urinary epinephrine (u-epi), norepinephrine (u-norepi), metanephrine (u-meta) and normetanephrine (u-normeta), and plasma epinephrine (p-epi), norepinephrine (p-norepi), free and total metanephrine (pf-meta and pt-meta) and free and total normetanephrine (pf-normeta and pt-meta) were analysed by hplc. urinary catecholamines and metanephrines were expressed as ratios to urine creatinine concentrations. data were analysed by non-parametric tests (p< , ).similar to our previous findings u-epi, u-norepi, u-meta and u-normeta were significantly higher in dogs with pheo and u-norepi and u-normeta were significantly higher in dogs with hc compared to healthy dogs. comparison between dogs with hc and dogs with pheo revealed significantly higher u-meta and u-normeta in the latter group. u-normeta was the only parameter with no overlap.in dogs with pheo p-norepi, pf-meta, pt-meta, pf-normeta, pt-normeta were significantly higher and in dogs with hc p-norepi, pf-normeta and pt-normeta were significantly higher than in healthy dogs. comparison between dogs with hc and dogs with pheo showed significant higher pf-meta, pt-meta, pf-normeta, pt-normeta in the pheo group. overlap was present with all parameters, but was least with pf-normeta and pt-normeta.according to our preliminary results u-normeta, pf-normeta and pt-normeta are valuable parameters for the diagnosis of pheo, so far u-normeta has performed better than the plasma parameters. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to ckd progression. vascular endothelial growth factor (vegf) is a marker of hypoxia. the aim of this study was to evaluate urinary vegf as a prognostic marker in cats with ckd compared with the established progression factors, proteinuria and hyperphosphataemia. cats were recruited through geriatric clinics held at two first opinion london practices between and . diagnosis of ckd was based on concurrent findings of plasma creatinine > mg/dl and usg < . , with persistence of azotaemia for at least weeks. vegf was measured in urine samples taken from cats at diagnosis of ckd and indexed to creatinine giving vegf: creatinine ratios (uvc). survival was compared among low (< . lg/g), medium ( . - . lg/g) and high (> . lg/g) categories of uvc using the log-rank test. multivariable binary logistic regression was used to assess whether uvc was associated with an increase in plasma creatinine concentration of at least % within year of diagnosis. cats which did not demonstrate progression but were followed for < year were excluded from the study. cases which developed hyperthyroidism, received ace inhibitors, had gross haematuria, urinary tract infections, nephrotic syndrome or evidence of bladder neoplasia were also excluded. survival data are presented as median [ % confidence interval] and other descriptive data are presented as median ( th , th percentile). significance was set at p< . . cats with low uvc (n= ) had survival of [ , ] days, while those with medium (n= ) and high uvc (n= ) had survival of [ , ] days and [ , ] days respectively. there was no difference in survival time between cats with low and medium values of uvc (p= . ), but cats with both low (p< . ) and medium (p= . ) uvc had significantly longer survival than cats with high uvc. plasma creatinine concentration increased by ( , )% in cats which progressed (n= ) and ( , )% in cats with stable renal function in the year following diagnosis (n= ). uvc was also associated with progression of azotaemia (p= . ) independently of upc and plasma phosphate concentration. the progressive group had uvc of . ( . , . ) lg/g, while the stable group had uvc of . ( . , . ) lg/g. if high uvc indicates renal hypoxia, the results of this study support the hypothesis that hypoxia is associated with progression in cats with ckd. however, further studies evaluating renal hypoxia directly would be required to verify this. the term 'triaditis' is used to describe concurrent inflammation of the liver, pancreas, and small intestine, although occasionally only two of these organs may participate. the aim of this study was to investigate the frequency of coexistence of different combinations of cholangitis, pancreatitis and/or inflammatory bowel disease in cats, and describe the clinical, clinicopathological, and histopathological findings.initially cats were included in the study. thirty-nine cats had a suspicion of 'triaditis' based on clinical signs (depression, anorexia or polyphagia, vomiting, diarrhea, and/or weight loss), while cats clinically healthy, were considered as controls. each cat on presentation underwent cbc, biochemistry profile, serum total t , spec fpl ® and ftli blood examinations. cats diagnosed with intestinal parasitism, infectious disease, neoplasia, and/or hyperthyroidism were excluded from the study. biopsies from the liver, pancreas and small intestine (duodenum, jejunum, and ileum) were collected from each sick cat during laparotomy as part of the diagnostic investigation. biopsies from healthy cats were collected during laparotomy for ovariohysterectomy. all owners had signed a consent form and the study protocol was approved by the university's and state's ethics committee.of the sick cats with a suspicion of triaditis, were excluded because of neoplasia or other conditions. of the clinically healthy cats, had histopathological evidence of inflammation in their liver, pancreas, and/or intestine and were eventually grouped together with the sick cats. collectively, histopathological evaluation of the biopsies revealed cats ( with clinical signs and without) with inflammatory lesions in at least one organ. of those cats, ( . %) had histopathological evidence of ibd, ( . %) of cholangitis, and ( . %) of pancreatitis. thirteen cats ( . %) had only ibd, ( . %) of which were symptomatic. six ( . %) cats had only cholangitis, ( . %) of which were symptomatic. one ( . %) had only pancreatitis and was symptomatic. sixteen cats ( %) had concurrent ibd and cholangitis, ( . %) of which were symptomatic; ( . %) had ibd in combination with pancreatitis, ( . %) of which were symptomatic, while ( %) had ibd, cholangitis and pancreatitis and were all symptomatic. common biochemical findings were increased activities of alt ( / ), alp ( / ), and increased total bilirubin ( / ), ftli ( / ), spec fpl ( / ) concentrations.the results of our study indicate that different combinations of concurrent inflammation of the liver, pancreas, and intestine do exist in cats. it also appears that these conditions may be subclinical in many cases. several potential pathogens are found in feline faeces, ranging in significance from incidental to pathogenic. treatment recommendations are based on accompanying clinical signs. real time pcr has enabled rapid screening of small quantities of faeces for potential pathogens, with high sensitivity and specificity. co-carriage of feline faecal pathogens may reflect a symbiotic relationship or related pathogenesis, in which case identification of common cocarriage patterns may influence treatment decisions and prognosis. the primary objective of this study was to identify co-carriage of selected feline faecal pathogens. secondary objectives were to evaluate the prevalence of individual pathogens in feline faeces, and their association with pedigree status, and a history of diarrhoea. results of a commercial -way feline diarrhoea realpcr tm panel (idexx reference laboratories, uk) from june to january were evaluated. real time pcr was performed for tritrichomonas foetus, giardiaspp., cryptosporidiumspp., toxoplasmagondii, salmonellaspp., clostridium perfringens enterotoxin a gene, feline coronavirus, and feline panleukopenia virus. additional data was recorded when available, including age, gender, breed, and history of diarrhoea. weak or borderline positive results and those from pooled faecal samples were excluded. associations of the carriage of pairs of pathogens were evaluated with a chi-squared test, statistical significance set at p < . .results of pcr panels were evaluated. the prevalence of faecal pathogens was . % (tritrichomonas foetus), . % (giardia), . % (cryptosporidium), . % (toxoplasma gondii), . % (salmonella), . % (clostridium perfringens), . % (feline coronavirus), and . % (feline panleukopenia virus).salmonella was the only faecal pathogen significantly associated with a history of diarrhoea. faecal samples from pedigree cats were significantly more likely than dsh to be positive for tritrichomonas foetus, giardia, clostridium perfringens and feline coronavirus.significant co-carriage was identified for feline coronavirus with all other pathogens except salmonella. there was significant co-carriage of tritrichomonas foetus with clostridium perfringens and giardia, and also for giardia with panleukopenia virus, cryptosporidium, and clostridium perfringens. finally, there was significant co-carriage of cryptosporidium with clostridium perfringens, panleukopenia virus with cryptosporidium, and toxoplasma gondii with salmonella.in conclusion there was a moderate to high prevalence of all feline faecal pathogens tested except toxoplasma gondii and salmonella. positive pcr results were more likely in pedigree cats, and salmonella was associated with a history of diarrhoea. significant co-carriage of faecal pathogens was common, possibly reflecting common environmental risk factors, a shared pathogenesis, or a symbiotic relationship. mycoplasma spp have been identified as causative pathogen in feline lower airway disease and are not thought to colonize the lower airways of clinically healthy cats.to challenge this hypothesis, domestic shorthair cats aged - years (median, . years), without signs of respiratory disease, housed in a shelter, underwent transoral lower airway washing with sterile saline under general anaesthesia. retrieved bronchalveolar lavage fluid (balf) was subjected to microbiological and cytological analysis.during preanaesthetic clinical examination, only minor alterations were discovered: tartar in , conjunctivitis in , flea infestation in and dirty auditory canals in cats. one animal each showed dandruff, corneal ulceration and a heart murmur / .balf-cultures of animals ( . %) were positive for mycoplasma spp. . % of examined cats had mycoplasma felis in their balf (light growth in , moderate growth in and heavy growth in animals), % ureaplasma felinum/cati (light growth in , moderate growth in cases), . % mycoplasma gateae (light growth in , moderate growth in and heavy growth in cat) and . % mycoplasma feliminutum (light growth in animal). using aerobic bacterial culture, pasteurella multocida ( . %), a-haemolytic streptococci ( . %), haemolytic e. coli ( . %) and acinetobacter iwoffii ( . %) were detected, with only samples ( . %) yielding negative results.median balf-total nucleated cell count was /ll (range, - ) with a median mononucleated cell percentage of %, a median neutrophil fraction of . % and a median eosinophil proportion of . %. only samples were neutrophil-dominated ( . and . %), indicating purulent inflammation. both specimens were positive for mycoplasma felis ( light growth, heavy growth), and one showed moderate growth of pasteurella multocida. like all of the animals included in this study, the corresponding cats remained clinically healthy during a -week followup period.balf-samples showed neither cytological nor microbiological signs indicative of upper airway contamination.in contrast to earlier studies, we conclude that -at least in cats housed in shel-ters and subjected to high infection pressure -in addition to bacteria like pasteurella spp, streptococcus spp and e. coli, mycoplasma spp can occur in the feline lower airways without causing respiratory signs. inflammasomes are intracellular multi-protein complexes that coordinate the maturation of interleukin (il)- b and il- in response to pathogens and metabolic danger signals. both cytokines are vital for the maintenance of the intestinal homeostasis and have been linked to chronic intestinal inflammation in humans. both il- b and il- are produced as inactive proforms and undergo subsequent maturation through cleavage into their active forms by caspase (casp)- , which is in turn activated by the inflammasome complex. the best characterized inflammasome subtype in human inflammatory bowel disease (ibd) is nlrp , which seems to be crucial for the regulation of intestinal homeostasis. defective nlrp signaling has been suggested to contribute to ibd. additionally, il- b, il- and casp- are upregulated on the mrna and protein level in human ibd. so far, no study has investigated the role of the inflammasome and respective down-stream cytokines in canine ibd. thus, the goal of the current study was to investigate the expression of inflammasome components in duodenal tissues from dogs with ibd compared to healthy controls. rna extraction from endoscopic biopsies (ibd group n = , control group n = ) and reversetranscriptase quantitative pcr was performed in a sybrgreenbased assay using specific primers for the following canine genes: nlrp , casp- , il- b and il- . a -fold dilution of plasmid controls for each gene was used to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). comparison between groups was performed using mann whitney u tests (graph pad prism ). significance was set at p < . . eight samples ( ibd, controls) had to be excluded due to poor cdna quality (inadequate expression of reference genes). when comparing the remaining samples in both groups, casp- (p = . ) and nlrp (p < . ) expression was significantly lower in ibd dogs than controls. in contrast, il- b (p = . ) and il- (p = . ) expression was not different between groups.down-regulation of nlrp and casp- could be part of a negative feedback loop in the pro-inflammatory environment in ibd. alternatively, this could represent general disturbances in intestinal homeostasis or failure to up-regulate "danger-signaling pathways"in inflammation. final conclusions might be difficult to draw without matching protein data, as assessing il- b and il- mrna levels cannot give insight into the ratio of "pro-cytokines"to active cytokines. thus, investigating il- b and il- protein content of canine ibd tissues is warranted in the future. the trefoil factor family (tff) comprises a group of small peptides produced in goblet cells, which are crucial for epithelial restitution and maintenance of tight junction function in the gut. in humans with inflammatory bowel disease (ibd), tff expression is up-regulated, which is thought to represent an unspecific repair mechanism. however, tffs have also been shown to be involved in the local control of disease in rodent models and in humans. so far, there has been no study investigating tff expression in the canine intestine. thus, the goal of this study was to assess tff expression in gastrointestinal tissues from dogs with ibd and healthy dogs. rna was extracted from endoscopic duodenal (ibd n = , healthy controls n = ) and colonic biopsies (ibd n = , controls n = ) and cdna generated. quantitative reverse-transcription pcr was performed for canine tff and tff in a sybr-green-based assay. a -fold dilution of plasmid controls for each gene was used to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). for statistical analysis, significance was set at p < . . overall, tff expression was significantly different across groups (kruskal wallis p < . ): in control dogs, tff expression was higher in the colon than in the duodenum (mann whitney p < . ). when comparing ibd cases to controls, duodenal tff was significantly up-regulated (mann whitney p = . ). tff expression was not different across groups (kruskal wallis p = . ). however, separate analysis of the intestinal location showed significant down-regulation of tff in the colon of ibd dogs compared to controls (t-test p = . ).this study demonstrates evidence for dysregulation of tff gene expression in canine ibd. a lack of tff could contribute to defective epithelial barrier function, causing "leakiness"of tight junctions. this increased intestinal permeability could lead to an increased antigenic load from microbes or food antigens, which could perpetuate faulty immune recognition of microbe-associated molecular patterns, thus leading to increased intestinal inflammation. up-regulation of tff in the colon could in turn signify a compensatory repair-mechanism in inflammation. further investigation of tff gene or protein expression is warranted in canine ibd. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb e (ef) on gene expression and protein production in canine duodenal biopsies. samples from healthy beagles and ibd dogs were cultured ex-vivo with ef ( x cfu/ml) or sterile nutrient broth/ pbs (negative control) for hours. rna extraction from biopsies and reverse-transcriptase quantitative pcr was performed in a sybrgreen-based assay using specific primers for the following canine genes: tlr , tlr , tlr , tlr , il- a, il- , il- , tgfb, il- , ifnc and tnfa; using a -fold dilution of plasmid controls for each gene to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). protein content of ifnc, il- a and tnfa was measured in culture supernatants using commercially available canine-specific elisas. a linear mixed model for each genewith disease group and treatment set as fixed parameters -was chosen for statistical analysis using spss software. significance was set at p < . .no significant interaction between disease group and treatment was observed for any gene. only expression of il- was significantly increased in ibd dogs compared to healthy dogs (p = . ) in unstimulated samples. all other significant differences were independent of disease group, but depen-dent on treatment with ef. expression of the following genes was reduced by ef treatment: tlr (p = . ), tlr (p = . ), tlr (p = . ), tgfb (p = . ), ifnc (p = . ) and tnfa (p = < . ). no significant amounts of ifnc, il- a or tnfa protein were detected in culture supernatants.this is the first study demonstrating a profound effect of ef treatment on gene expression in ex vivo cultured canine duodenal biopsies. down-regulation of several genes of innate immune receptors and pro-inflammatory cytokines was observed, with the most significant effect on suppression of tnfa expression. effects were seen both in healthy and in ibd dogs. whether this translates to a beneficial effect in a clinical situation needs further investigation. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of cats. there is mounting evidence that microbes play an important role in the pathogenesis of chronic enteropathies, as compositional changes of the intestinal bacterial ecosystem have been associated with chronic intestinal inflammation in humans and dogs. the aim of this study was to characterize the bacterial microbiota in cats with chronic enteropathies.fecal samples were obtained from healthy cats (n= ) and cats with histologically confirmed chronic enteropathies (n= ). the bacterial composition was analyzed by massive parallel s rrna gene -pyrosequencing (yielding , sequencing tags), and selected quantitative pcr assays. differences in microbial community structure between healthy and diseased cats were assessed by the phylogenetic unifrac distance method, followed by analysis of similarity (anosim) of the distance matrix. differences in bacterial groups between the disease groups were analyzed using mann-whitney u tests. the resulting p-values were corrected for multiple comparisons using the benjamini & hochberg's false discovery rate. an adjusted p< . was considered for statistical significance.the unifrac distance metric indicated no significant clustering according to disease status. however, the relative proportions of sequences belonging to bacteroides spp. were significantly decreased in the cats with chronic enteropathies compared to the healthy cats (p= . ). the employed qpcr assays confirmed the sequencing results and also showed a significant decrease in proportions of bacteroides spp. in the diseased cats. in addition, qpcr revealed also a significant decrease in turicibacter spp. in cats with chronic enteropathies compared to the healthy cats (p= . ). no significant differences in diversity indices were observed between healthy and diseased cats.in conclusion, the here used molecular approach revealed significant reductions in bacteroides and turicibacter spp. in cats with chronic enteropathies. future studies are necessary to evaluate if these microbial changes correlate with functional changes in the intestinal microbiota. campylobacter spp. represent a common cause of gastroenteritis in humans with c. jejuni and c. coli considered responsible for the majority of clinical cases. many human diagnostic laboratories use the prospect ® campylobacter microplate assay (eia) as a sole or screening test for detection of c. jejuni and c. coli as this technique is easier, faster and cheaper than campylobacter spp. culture. c. upsaliensis can cause a diarrhoeal illness similar to that caused by c. jejuni in humans but methods routinely used for c. jejuni and c. coli detection, including the eia, are not optimised or have not been evaluated for the detection of other campylobacter species. c. upsaliensis and c. helveticus have been more commonly isolated from dogs and cats than c. jejuni and c. coli in most studies. campylobacter spp. from pets are potentially zoonotic while the pathogenicity in pets remains uncertain. the significance of c. upsaliensis and c. helveticus infections may be underestimated in human medicine due to detection methods applied.the aim of this pilot study was to assess if the eia detects c. upsaliensis and c. helveticus isolated from different pets in spiked human clinical samples. in addition, the ability of eia to detect campylobacter spp. in dog and cat faeces was assessed. ten clinical human faecal samples and two healthy dog and cat faeces confirmed negative by eia were tested by a range of culture methods to exclude presence of campylobacter spp. dilutions of eight c. upsaliensis, five c. helveticus cultures, and one c. jejuni and c. coli culture, all pcr confirmed, were added to aliquots of faecal samples to obtain a range of viable bacteria from to cfu/ml. eia was performed following the manufacturer's instructions and in duplicates. fifty out of samples tested positive by eia at various dilutions: / c. upsaliensis, / c. helveticus, / of each c. jejuni and c. coli. detection limit varied between the isolates and was lower in watery than semi-solid faeces.in conclusion, eia detected c. upsaliensis and c. helveticus in spiked faeces from human clinical cases and all campylobacter spp. tested in healthy pets. eia should not be used as a sole detection method and culture methods selected following a positive eia result should enable detection of a wide range of campylobacter spp. in addition, eia may be a useful test in pets to rule out campylobacter spp. infection and in epidemiological investigations. high-resolution manometry (hrm) is the gold standard for the evaluation of functional esophageal motility disorders in humans. no information on this non-invasive technique is available in dogs. the aims of this study were to evaluate the feasibility of hrm in dogs, generate first normal values, and to examine the influence of a standard sedation on the esophageal pressure profile.the study population consisted of healthy adult beagle dogs (mean bw . kg), the study protocol was approved by the local ethics committee. hrm was performed in sitting position after a -h fast, lidocaine jelly at % was used as a lubricant. a solid-state catheter (sierra scientific, . mm diameter) with circumferential pressure sensors spaced cm apart was inserted intranasally, measurements were started after minutes adaptation time. real-time pressure imaging during catheter intubation enabled accurate placement. each manometric protocol included water and canned food bolus swallows. the procedure was repeated minutes after standard im sedation with acepromazine and buprenorphine. data were analysed using manoview software and results (awake vs. sedated) were compared using the wilcoxon test. statistical significance was set at p< . .hrm could successfully be performed in / dogs. reasons for unsuccessful examinations were: defence reactions ( ), inability to pass through the ventral meatus ( ), reverse sneezing ( ). upper esophageal sphincter (ues) characterisation comprised: baseline pressure, residual pressure (nadir of the ues relaxation during swallowing), and relaxation duration. tubular esophageal function characterisation comprised: peristaltic con-tractile integral (pci; amplitude x duration x length of the contraction wave) and the bolus transit time (btt). lower esophageal sphincter (les) characterisation comprised: baseline pressure and residual pressure (lowest continuous second mean les pressure relative to intragastric pressure during swallow induced relaxation).the median values for water/food bolus swallows in awake (sedated) dogs were calculated i) ues: baseline pressure . mmhg ( . ), residual pressure - . / . mmhg (- . / . ) and relaxation duration . / ms ( . / ) ii) tubular esophagus function: pci . / mmhg-cm-s ( . / . ) and btt . / . ms ( . / . ) iii) les: baseline pressure . mmhg ( . ), residual pressure . / . mmhg ( . / ). significant differences were found for the ues relaxation duration (water) (p= . ) and btt (food bolus) (p= . ).in conclusion, hrm is a feasible technique for the evaluation of esophageal function in dogs. patients that require sedation can still be examined, however at this point it is not clear if sedation would affect the assessment of motility disorders in dysphagic dogs. in humans ambulatory intraesophageal ph-monitoring utilizing the bravo ® capsule is the standard test for establishing pathological gastroesophageal reflux (ger). this technique not only provides information on esophageal acid exposure, but is also able to assess symptoms associated with ger. in dogs ger is poorly understood and it is not clear if ger actually represents a clinically relevant problem.the goals of this study were to examine the canine esophageal ph milieu in health and to examine esophageal ph in dogs presenting with signs commonly attributed to ger in the veterinary literature.thirteen client-owned dogs (cod) of various breeds (median bw . kg, . - ; median age y, - ) were included. clinical signs ultimately leading to ph-monitoring comprised: lipsmacking ( ), repeated swallowing motions ( ), chronic vomiting ( ), cough ( ), retching ( ), regurgitation ( ), sudden discomfort ( ), excessive surface-licking ( ), ptyalism ( ), presumed postprandial pain ( ), refusal to eat despite interest ( ), history of esophageal foreign bodies ( ), halitosis ( ) . each dog showed a median of ( - ) signs, dogs had additional diarrhea. six healthy beagles (median bw . kg, median age . y) with unremarkable gastroduodenal evaluation served as controls (c). no prior antacid or prokinetic treatment was allowed. the capsule was endoscopically placed cm above the lower esophageal sphincter, ph data were transmitted every s to a receiver attached to the dog's collar. owners were instructed to press the individually predefined symptom-buttons on the receiver whenever indicated, and not to change the daily routine. data were analysed using the rapidph ® software, reflux was defined as a single ph-measurement < . results between groups were compared using non-parametric tests.the median ph-monitoring period (cod/c) was . / . h. the following parameters (median, range for cod/c) were evaluated: number of refluxes: ( - )/ . ( - ), number of longest (> min) reflux: ( - )/ ( - ), duration of longest reflux (min): ( - )/ . , and fraction time ph < (%): . ( . - . )/ . ( - . ). there were no differences between groups. the median number of button pushes was ( - ), dogs had reflux-positive pushes ( . , , and . % of pushes). mild distal esophagitis was noted in dog. final diagnoses were: food-responsive ibd ( ), steroid-responsive ibd ( ), allergic skin disease ( ), chronic laryngotracheobronchitis ( ), muscular dystrophy ( ) .dogs presenting with historical and clinical signs interpreted as ger may not have relevant reflux episodes. considering normal values established in humans, none of the dogs would have been classified as abnormal. the aim of the present study was to evaluate the changes of some biochemical and ultrasonographic (us) parameters in a group of dogs with naturally occurring acute pancreatitis (ap) during the therapeutic follow-up.dogs with clinical signs and abdominal us findings suggestive of ap associated with increased serum canine pancreatic lipase (cpl) activity were included into the study. in these dogs, the serum concentration of c-reactive protein (crp), amylase and lipase were also measured. severity indexes were established to semi-quantitatively evaluate the severity of clinical and us findings. in particular, a clinical score ( - ) for each of the following clinical parameters was given: presence and frequency of vomiting, appetite and general condition; an us score ( =normal, =abnormal) was assigned per each of the following parameters: pancreas (echogenicity, volume, echotexture and echogenicity of the mesentery), gastrointestinal tract, biliary ducts, lymph nodes and abdominal effusion (total score - ). all dogs were treated with fluid therapy, ampicillin-sulbactam, mg/kg iv q h, buprenorphine, . mg/kg q h, and, if needed, maropitant mg/kg sc q h. the two severity scores, serum crp, amylase and lipase concentrations were measured at diagnosis (t ) and after (t ), (t ), and (t ) days, and at discharge (td) and week after discharge (td ).nine client-owned dogs were included with a median (range) age of years ( - years). median (range) clinical and us scores were ( - ) and . ( - ), respectively, at t , and ( - ) and ( - ), respectively, at td . a significant, positive correlation was found between the clinical and us score (p< . , r= . ). the median (range) serum concentration of crp (mg/dl), amylase (u/l) and lipase (u/l) was . ( . - . ), , ( - , ) and ) , respectively, at t , and . ( . - . ), ( - , ) and ( - ), respectively, at td . on admission, serum crp, amylase and lipase levels were increased in %, %, and % of dogs, respectively while they were increased in %, . % and % of dogs, respectively, at td . serum crp and amylase, but not lipase, concentrations decreased during the follow up and were significantly (p< . ) lower at td compared to t .results suggest that us findings, and crp and amylase concentrations are correlated with the recovery from the ap. further studies are warranted to evaluate the usefulness of these parameters in the follow-up of ap in a wider population of dogs. enzyme replacement therapy is the mainstay therapy for exocrine pancreatic insufficiency (epi) in dogs. 'enteric-coated' preparations have been developed to protect the enzyme from degradation in the stomach, but their efficacy has not been critically evaluated. the hypothesis of the current study was that enteric coating would have no effect on the efficacy of pancreatic enzyme supplements for dogs with epi.thirty-eight client-owned dogs with naturally occurring epi were included in this multicentre, blinded, randomised controlled trial. dogs received either an enteric-coated enzyme preparation (test group) or an identical preparation without the enteric coating (control group) over a period of days.there were no significant differences in baseline characteristics between test and control treatment groups. body weight and body condition score increased in both groups during the trial (p< . ) but the magnitude of increase was greater for the test treatment compared with the control treatment (p< . ). by day , mean body weight increase was % ( % confidence interval - %) in the treatment group and % ( % confidence interval - %) in the control group. the dose of enzyme used increased over time (p< . ) but there was no significant treatment group difference at any time point (p= . ). clinical disease severity score decreased significantly over time for both groups (p= . ) and no significant difference was noted between groups (p= . ). no significant adverse effects were reported, for either treatment, for the duration of the trial.adding an enteric coating to a pancreatic enzyme supplement conveys a therapeutic advantage, when treating dogs with epi. when liver injury is caused by toxins that inhibit hepatocyte replication, or when the proliferative potential of hepatocytes is exhausted due to the chronic condition of the disease, hepatocyte-dependent liver regeneration is strongly impaired. in this situation another cell compartment is activated to regenerate the liver: the hepatic progenitor cell compartment. bipotent hepatic progenitor cells (hpcs) can differentiate into hepatocytes or cholangiocytes. not only are hpcs activated during severe liver disease, they may well be the cells of origin for subtypes of hepatocellular carcinoma (hcc).the polycomb group protein bmi is involved in murine hpc activation and has prognostic relevance in hcc. therefore we investigated the expression of bmi in canine liver diseases including hcc. functional consequences of deregulated bmi expression are included to reveal its feasibility in liver regenerative medicine.immunohistochemistry (ihc) and laser microdissection followed by gene expression analysis were used to investigate the expression of bmi in activated hpcs and hcc. to elucidate the role of bmi in hpcs, in vitro gene silencing experiments followed by gene expression analysis, western blot analysis, and a proliferation assay (edu-incorporation) were performed in the human hpc-like cell-line heparg.ihc and gene expression showed a strong nuclear expression of bmi in activated hpcs in canine liver diseases. keratin is a marker for hpcs and cholangiocytes, and high expression is associated with poor prognosis in hccs. bmi staining was more intense in highly malignant hepatocellular carcinomas positive for keratin compared to keratin negative hccs. bmi -silencing in vitro studies revealed a significantly reduced hpc proliferation and suggested a role of bmi in differentiation (gene expression) of hpcs.these results indicate that bmi is expressed in activated hpcs in all liver diseases tested. bmi is needed for proliferation of hpcs in vitro and is potentially involved in hpc differentiation. expression of bmi in hccs suggests a role in tumour development, potentially due to a persistent activation of hpcs. therefore, enhanced activation of bmi will lead to more progenitor cells (beneficial) but most likely also to more aggressive hcc with a hpc-signature. hepatic progenitor cells (hpc) are bipotential, forming cholangiocytes or hepatocytes. as a result, they express markers of both cell types. various markers have been used to select for hpc's yet there is no specific cell marker. cd , a transmembrane glycoprotein, has been identified as a organ stem cell marker and cancer stem cell marker, and used to select hpc's in rodents and humans. canine hpc have been shown to have gene expression of cd . this study aimed to assess if cd selection could enrich for canine hpcs.a wedge of liver from a healthy dog euthanased for behavioural reasons was digested by collagenase perfusion. hepatocytes were removed by pelleting at g for mins. the remaining non-parenchymal cells (npc) were then pelleted at g for mins. magnetic assisted cell sorting (macs) for cells positive and negative for cd was performed according to the manufacturer's instructions. cd positive and negative npc were cultured on bovine type i collagen-coated plates with hepatocyte culture media (lonza). cell morphology was monitored. rna was extracted and cdna produced from cell pellets immediately after separation and also after weeks of culture. relative gene expression of cd , cholangiocyte marker keratin (k ) and hepatocytes markers k , albumin and cyp a was performed using b mg and rpl as reference genes.the cd positive fraction was % of the npcs. morphologically, cd positive cells formed more colonies of cells with a small cytoplasm to nucleus ratio, which then transformed to hepatoblastic appearance while the negative cells produced more fibroblastic cells. compared to cd negative cells, initially cd positive cells showed a -fold increase in cd , and . -fold increase in cholangiocyte marker k as well as modest increases in hepatocyte markers albumin and cyp a and a modest decrease in k . after weeks culture, positive cells expressed more albumin, cyp a , k & k compared to cd negative cultures. cd expression had reduced to a fold increase after culture.the cd positive fraction expressed cholangiocyte and hepatocytes markers compared to negative cells initially after sorting. after weeks culture, cd positive cells formed hepatocyte-like colonies, as well as continuing to have greater hepatocyte and cholangiocyte gene expression. cd expression decreased, consistent with differentiation.a large percentage of the npc were positive for cd . cd sorting also labels haematopoietic and mesenchymal stem cells therefore subsequently using facs for other markers may further enrich for specifically the hpc compartment. until now the hepatic neoplasms in dogs are classified as hepatocellular adenomas and carcinomas, cholangiocellular adenomas and carcinomas, mixed hepato-and cholangiocellular carcinomas and hepatic carcinoids. over the past decade, many advances have been made in the characterization of primary liver tumours in man. this knowledge has resulted in a proposal for a new morphological and immunohistochemical classification of primary liver tumours which facilitates the diagnosis and categorization of these tumours including their aggressiveness and prognosis. the purpose of this study was to investigate the presence and relative incidence of the various morphological types of primary hepatic neoplasms in the dog and to determine whether the new human classification also can be applied to canine hepatic neoplasms. for this study canine primary liver tumours were examined histologically and classified using several immunohistochemical markers including keratin(k) (hepatic progenitor (stem) cell/bile duct epithelium marker), heppar- (hepatocyte marker), ema (muc ; mucine producing biliary epithelium marker), pcea (canalicular, ductular, and bile duct epithelium marker), nse and chromogranin-a (neuro-endocrine markers). in addition, the tumours were graded according to cellular and nuclear pleiomorphism and mitotic index (grade: - ) and staged with respect to absence or presence of invasive growth, intrahepatic and/or distant metastases (stage: - ). of the primary liver tumours, had a hepatocellular origin ( %). these hepatocellular tumours could be subdivided in hepatocellular tumours with < % positivity for k ( %) and tumours with > % positivity for k ( %). the hepatocellular tumours with > % positivity for k were histologically poorly differentiated and often revealed lymphatic and vascular invasion in portal tracts and all showed intrahepatic and/or distant metastasis. in contrast the hepatocellular tumours with < % positivity for k were almost always well differentiated and well demarcated and did not have evidence for vascular invasion, intrahepatic and/or distant metastasis. eight of the tumours were from cholangiocellular origin ( %). all of the cholangiocellular tumours were poorly differentiated (grade and ) and showed intrahepatic and/or distant metastases. the third group of primary liver tumours had neuroendocrine characteristics, consisted of five tumours ( %) and were classified as carcinoids. they all were histologically poorly differentiated and had intrahepatic and/or distant metastases. in conclusion, the morphological types of the primary hepatic neoplasm in the dog, including their aggressiveness and prognosis, are highly comparable to the situation in man. these finding indicate that the new human classification of primary hepatic neoplasms is applicable in the dog. doberman hepatitis (dh) is a rare inflammatory liver disease characterized by female preponderance, elevated serum transaminase activity and increased hepatic copper content. immune system involvement is suggested by the presence of lymphocyte infiltration, female predisposition, abnormal expression of major histocompatibility complex (mhc) class ii antigens by hepatocytes and association of homozygosity for the mhc ii risk allele drb * of the dog leukocyte antigen system genotype.we investigated the possibility that autoantibodies are involved in dh. serum samples from subclinical and clinical dh patients and clinically healthy control dobermans were included in an elisa assay for detection of igg autoantibodies against histones (aha). the cut-off value for positivity in the anti-histone elisa was . , determined using the mean absorbance + sd of samples from healthy controls. the values for subclinical dh cases (mean ± sd: . ± . ; % confidence interval (ci) . to . ) and clinical dh cases (mean ± sd: . ± . ; %ci . to . ) were both significantly higher than values for controls (mean ± sd: . ± . ; %ci . to . ; p< . ). no seropositivity was noted in the control group.autoantibodies are the serological hallmark of autoimmune disease. normally, the immune system has an extraordinary capacity for preventing self-antigens to stimulate an inflammatory reaction. the presence of autoantibodies is therefore the consequence of a breakdown or failure of b-cell tolerance toward the corresponding autoantigens. since the appearance of aha indicates possible presence of autoimmunity, our results support the assumption that dh is an autoimmune disease. man's best friend (canis lupus familiaris) is an ideal model organism for a broad variety of naturally occurring diseases. dog breeds have a population structure suited for genetic studies of complex disorders. haploblocks within dog breeds extend up to times longer distances than in human populations. related dog breeds often share phenotypes and causative mutations, allowing for finemapping and validation. the inbreeding and bot-tlenecks of dog populations has led to an increased incidence of genetic diseases which remained incidental in the panmictic human populations.extrahepatic portosystemic shunts (ehpss) are large abnormal venous blood vessels connecting the portal vein with a major systemic vein. it results in almost complete diversion of the portal blood past the liver, leading to lack of liver function and liver growth, and hepatic encephalopathy due to brain neurotransmitter dysfunctions. there are only reported human cases, whereas the disease is widespread in dogs. shunts are more frequently diagnosed in purebred dogs than in crossbred dogs. the inheritance is complex without sex effect.a genome wide association studied with k snps was peformed on cairn terrier cases and controls of the same breed. after quality control cases and controls were analyzed in snps. allelic association was calculated with plink and grammas of the genabel package was used to correct for genetic kinship. several chromosome regions were detected with close to significant association. overlap between the two analysis methods was found on three genomic regions covering about . mb. finemapping was conducted by analyzing more snps in a larger group of cairn terriers and in cases and controls of other breeds. the finemapping resulted in two genomic regions covering and kb that were associated with significant pvalues. comparison of haplotypes of cases from different breeds confirmed the association signals. the regions of interest are analyzed by ngs of cases and controls. historically, doxycycline has been the first line drug for the treatment of canine monocytic ehrlichiosis (cme). some studies have shown that dogs may remain carriers despite doxycyxline treatment, therefore investigation for other anti-ehrlichial agents may be warranted. rifampin was suggested as a promising alternative for the treatment of cme, though its efficacy in clearing the infection has not been thoroughly evaluated. the purpose of this study was to assess the efficacy of rifampin in achieving clinicopathological recovery and clearing the ehrlichial infection from the blood and other tissues in dogs with experimentally-induced acute cme. of the purpose-bred beagle dogs that were included in the study, dogs with acute experimental cme were treated with rifampin ( mg/kg, sid, po, for weeks), infected dogs received no rifampin (infected controls) and two dogs served as uninfected controls. fourteen days after the completion of rifampin treatment, dexamethazone ( . mg/kg, iv, once) was given to the rifampin-treated dogs. clinical score, platelet counts, anti-e. canis immunofluorsecent antibody titers (ifa) and polymerase chain reaction (pcr) detection of e. canis-specific deoxyribonucleic acid in the blood, bone marrow and spleen aspirates were evaluated between the treated and untreated infected dogs on day post-inoculation (pi) (start of rifampin), on day pi (end of rifampin) and day pi (end of post-treatment monitoring). by day pi, all infected dogs became clinically ill and thrombocytopenic, seroconverted and became pcr-positive in at least one tissue. the median clinical score and ifa titers did not differ between the treated and untreated dogs at any of the three time points. Μedian platelet counts were significantly higher in the treated compared to the untreated infected dogs on day pi ( , /ll versus , , p= . ) and day pi ( , /ll versus , /ll, p= . ) (two-sample wilcoxon rank-sum [mann-whitney] test). on day pi, treated and untreated infected dogs were pcr-positive, while on day pi, treated and untreated infected dogs remained pcr positive in at least one of the tissues tested. as administered in this study, rifampin hastened hematological recovery of the infected dogs, but was inconsistent in clearing the experimentally-induced acute e. canis infection. acute phase proteins (app) are considered one of the hallmarks of the inflammatory response. among their major functions, apps seem to modulate innate immune system efficiency. in cats, serum amyloid a (saa) and a glycoprotein (agp) are two major positive apps that are increased during inflammation. this rise is presumed to be secondary to various cytokines that are involved in the innate inflammatory response.recombinant feline interferon-x (rfeifn-x) is an immune-modulator drug that is commonly used in cats naturally infected with retroviruses, namely feline immunodeficiency virus (fiv) and feline leukemia virus (felv). several studies have been performed to clarify the clinical benefits of rfeifn-x therapy in naturally infected fiv and/or felv cats. our group has previously described that c-reactive protein (crp) increased in naturally retroviralinfected cats under rfeifn-x therapy. however, the role of apps such as saa, agp and crp in the innate immuneresponse, remains unknown.the aim of this study was to evaluate saa, agp and crp serum levels in naturally retroviralinfected cats under rfeifn-x therapy.sixteen naturally retroviral infected cats ( fiv, felv and co-infected fiv/felv stray cats) housed in a lisbonanimal rescue shelter were submitted to rfeifn-x therapy. the licensed protocol was used: courses of mu/kg sc administered once daily for days, beginning on days , and . blood samples were collected for saa, agp and crp quantification before, during and after treatment (at d , , , ) . saa was quantified by elisa (phase saa, tridelta) and agp was determined by single radial immunodifusion (agp,tridelta). feline crp was quantified by elisa (kamiya biomedical company).app serum levels were compared before and after rfeifn-x therapy. a statistically significant increase of saa and agp (p= . and p= . respectively -friedman test) was observed at d in comparison to d . these findings corroborate the significant increase of crp serum levels previously described (p < . -friedman test).all the apps tested behaved similarly, showing an evident increase in their serum values after rfeifn-x therapy. these results suggest a possible immune modulation effect induced by rfeifn-x which seems to maximize the efficiency of innate immune response. further studies correlating these findings with the cytokine profile will extend our knowledge about the efficiency of rfeifn-x therapy in naturally retroviral infected cats. canine theileriosis is a tick-born disease caused by protozoan parasites of the genera theileria that has been associated with anaemia and/or thrombocytopaenia. the clinical manifestation of this disease in the dog is poorly described. this disease of emerging importance has been diagnosed in several dogs, emphasizing the need to elucidate the specific pathogenic species and characterize the clinical manifestations of the disease.this retrospective study describes the clinical characteristics, diagnostic tests, treatment and outcome of six client owned clinically ill dogs diagnosed with canine theileriosis at the onderstepoort veterinary academic hospital in south africa during - . canine theileriosis was diagnosed by polymerase chain reaction (pcr) on whole-blood followed by a reverse line blot (rlb) hybridization assay. other tickborn diseases and neoplastic conditions were excluded.the most common clinical findings were pale mucous membranes ( / dogs), lethargy ( / dogs) and oral bleeding ( / dogs). all dogs had thrombocytopaenia with a median of . x /l (range: - ) and / dogs had anaemia with a median haematocrit of % (range: - ). the anaemia was regenerative in / dogs and non-regenerative in / dogs. one dog was positive on an in saline auto-agglutination test. bone marrow cytology and core biopsies were performed in two dogs with severe non-regenerative anaemia and showed myelofibrosis. theileriasp. were detected in four dogs and theileria equi in two dogs. imidocarb dipropionate was administered in all dogs as treatment of choice for the theileriosis. five dogs that received the complete treatment achieved clinical cure. pcr post-treatment was performed in three dogs and was negative. prednisolone and azathioprine was administered in all dogs for suspected immune-mediated haematological disorders secondary to the theileriosis. one dog was euthanased one week after diagnosis.canine theileriosis should be considered a differential diagnosis for dogs with thrombocytopaenia and/or anaemia in endemic tick-born disease areas. pcr is a versatile tool for diagnosis and treatment monitoring in theileriosis. in our study, imidocarb dipropionate was effective in sterilizing the parasitic infection. the bleeding tendency seen in the theileriosis cases is most likely secondary to the thrombocytopaenia and/or concurrent thrombocytopathy as described in canine ehrlichiosis. further studies are required to determine the possible links between thrombocytopaenia, anaemia and myeloproliferative disorder observed in canine theileriosis. a.l. proksch , s. unterer , u. truyen , r.s. mueller , k. hartmann . clinic of small animal medicine, lmu university of munich, munich, germany, institute for animal hygiene and veterinary public health, university of leipzig, leipzig, germany canine parvovirosis still is a common and severe disease, especially in puppies, and there is a need for drugs that can decrease severity of symptoms and accelerate recovery. the paramunity inducer pind-orf stimulates the immune system. therefore, the aim of the study was to investigate whether pind-orf, used as an additional drug, leads to faster recovery in dogs with parvovirosis.in total, dogs with parvovirosis were randomly assigned to two groups ( dogs each). in all dogs, infection with canine parvovirus was diagnosed by fecal elisa or polymerase chain reaction (pcr). also, all dogs had clinical signs. the study was performed as prospective placebo-controlled, double-blinded trial. all dogs received either pind-orf or placebo, and additional standardized treatment for canine parvovirosis. clinical scores, complete blood count, and serum protein and albumin were evaluated daily (day - ) and at day . viral shedding was measured on day , , , and by fecal pcr and virus isolation.no significant difference could be found in clinical scores, most blood parameters, and duration of virus shedding when comparing dogs receiving pind-orf and dogs receiving placebo. the only significant difference was an increase in lymphocyte counts observed in the pind-orf group. three dogs receiving placebo did not survive, but no significant difference between groups was determined concerning survival rate.in this study, no significant influence of the paramunity inducer pind-orf on the course of parvovirosis was determined. therefore, there is no indication to recommend pind-orf therapy in canine parvovirosis. the microscopic agglutination test (mat) is currently considered as the gold standard for the diagnosis of leptospirosis in dogs. however, it is not a perfect tool to predict the infecting serogroup as cross-reactions and paradoxical reactions do exist. the objective of this study was to determine the infecting serovar in dogs with a positive polymerase chain reaction (pcr) on one or more biological sample(s) by molecular typing and to compare the results with those provided by the highest mat titer for each dog.forty-one positive pcr biological samples ( urine samples, blood samples, one kidney sample and one cerebrospinal fluid -csf-sample) from dogs with a clinical suspicion of leptospirosis between and and at least one positive pcr on blood, urine, kidney or csf (kit taqvet pathogenic leptospira, lsi, france) were submitted to molecular typing. the genomospecies were first determined by partial rpob or partial s rrna gene sequencing. the serovar was further identified by multiple loci variable number tandem repeat analysis (mlva). three variable number tandem repeat (vntr) loci were used as markers for serovar identification.the genomospecies were leptospira interrogans sensu stricto in samples belonging to dogs, leptospira borgpetersenii in three samples (three dogs) and leptospira kirschneri in two samples (two dogs). interestingly, for one dog, we found a different species in the blood and in the urine. mlva could unequivocally determine infecting serovar in samples belonging to dogs: australis in one dog, muenchen in three dogs, fugis in one dog, canicola in two dogs, autumnalis in six dogs. for only two dogs among those , the infecting serovar identified by mlva belonged to the infecting serogroup identified by mat. for samples belonging to dogs, mlva failed to identify the infecting serovar because one or more marker(s) could not be amplified. for samples belonging to dogs, amplification profile suggested coinfection or infection with a serovar that had not been previously characterized by mlva. it must be noted that for some dogs, we were able to characterize the serovar in a single kind of sample.despite lack of sensitivity of mlva applied to clinical strains in our study, this rapid method could contribute to a better knowledge of the epidemiology of canine leptospirosis with some adaptations in the choice of markers. recently, two trichomonads have been identified in diarrheal stool of puppies: pentatrichomonashominis (ph) and tritrichomonas foetus (tf) [ ] . tf infection in cats results in a chronic colitis. however in dogs, pathogenicity of tf and ph has never been studied. so the objective of this study was to evaluate the association between trichomonads' infection in puppies and the presence of diarrhea.faecal samples were collected prospectively from puppies ( to weeks of age) in french breeding kennels. for each puppy, a rectal swab was performed and the fecal consistency was evaluated using a -point numerical scale ( = liquid feces, = hard feces) [ ]. detection of trichomonads was performed by using a commercially available system "in pouchtm tf test"(biomed diagnostics, oregon usa). evaluation of this media was done as already described [ ] . observation of motile trichomonads organisms in the system culture was considered as a positive result. nine positive culture systems, from one kennel, were frozen and single-tube nested pcr assays were performed on them in order to sequence and identify the trichomonads. . % ( / ) of the cultures were positive. . % of the puppies ( / ) had gastrointestinal troubles. puppies infected by trichomonads had significantly more digestive problems than puppies not infected ( . % vs . %; p < . ). ph was systematically isolated in the positives cultures.ph was the only trichomonads isolated in the nine culture system tested by single-tube nested pcr assay. these results show the poor specificity of the medium to distinguish tf from ph. this observation underlines the necessity to use pcr for precise identification of type of trichomonads. trichomonads are significantly associated to the presence of digestive disorders in puppies. some etiological studies on the subject (by considering the co-infection with fly snapping, fly-biting or jaw snapping are names given to a syndrome in which dogs appear to be watching something then suddenly leaping and snapping at it. fly-biting dogs are generally referred to neurologists or behaviourists because the abnormalities are often interpreted as focal seizures or as obsessive compulsive disorder (ocd). there is one published case report of fly biting presumably caused by dietary intolerance in a cavalier king charles spaniel.the aims of this case series were ) to characterize fly biting, ) perform a complete medical evaluation of dogs presented with fly biting, and ) evaluate the outcome of this behaviour following appropriate treatment of the underlying medical condition.seven dogs presented for fly-biting behaviour (fb) were assessed. all dogs underwent a complete medical and behavioural history as well as physical and neurological examinations. further investigation was performed if an abnormality was found on examination or if the history was suggestive of an underlying problem. based on clinical presentation, physical examination, neurologic examination, and laboratory test results, a diagnosis was made and a specific treatment recommended. response to treatment was monitored and evaluated following phone conversations with owners at day , and from onset of treatment. many gastrointestinal disorders were found in fb dogs which included eosinophilic and lymphoplasmacytic infiltration of the stomach and small bowel, delayed gastric emptying and gastroeosophageal reflux. complete resolution of the fb was observed in / dogs diagnosed and specifically treated for the underlying gastrointestinal (gi) disease. one dog was diagnosed with chiari malformation and responded temporarily to pain management.in conclusion, this prospective case series indicates that fly biting behaviour may be caused by an underlying medical disorder, gi disease being the most common. resolution of this behaviour is possible following specific treatment of the underlying medical condition. although several diagnostic tests have been developed to diagnose fip, there are still difficulties to differentiate between fip and diseases with similar clinical appearence in vivo. fip is an inflammatory disease, therefore, as in other inflammatory conditions, the concentrations of apps are expected to be increased. the aim of this study was to evaluate the ability of the apps to distinguish fip from other diseases.serum samples from cats presented with effusion were obtained for measurement of three apps. the diagnostic work-up was performed with respect to fip (jvim ; : ) and further diagnostic procedures were performed depending on the medical condition. cats were diagnosed as having fip and cats had another disease (cardiac , tumor , other ). serum amyloid a (saa) and haptoglobin (hp) were measured by automated analyser using feline validated assays (eiken and tridelta, respectively), alpha- -acid glycoprotein (agp) was measured using a manual method based on single radial immunodiffusion (tridelta).the median concentrations of saa, hp and agp were significantly (p< . ) higher in cats with fip compared to cats without fip (saa: . ; range . - . lg/ml; . ; range . - . lg/ml,), (hp: . ; range . - . mg/ml; . ; range . - . mg/ml,), (agp: ; range - lg/ml; , range - lg/ml).all major feline apps seem to be useful diagnostic tools to help in differentiating fip from other diseases. however, the concentration of apps in some diseases (septic processes, disseminated neoplasias) was as high as in fip. protothecosis is an uncommon disease of people and animals caused by prototheca spp., an unicellular aerobic algae. to date, about cases have been described in dogs in north america and australia. in europe, only cases have been documented in the last decades (poland, italy, greece and spain). affected dogs show signs referable to the gastro-intestinal tract, particularly the colon, but ocular and neurologic signs are also reported. the disease has an insidious onset, a slow progression and fatal course. the aim of the present study is to describe clinical and laboratory findings in cases of canine protothecosis from the north of italy, diagnosed between - . medical records were retrieved and information pertaining history, clinical and instrumental data, as well as follow-up, were collected.the median age of the dogs was years (range: - ), of them were female and were boxers. major complaints were chronic large bowel diarrhea with hematochezia and weight loss observed since a median time of months (range: to ). previous treatment with gastrointestinal diets, antiparasitic drugs and antibiotics yielded no improvement. additionally, dogs developed uveitis during the disease course. in all dogs a complete blood count, a serum biochemical profile, including protein electrophoresis, and abdominal ultrasound were performed; serum ctli, folate and cobalamin were available in dogs and urinalysis in one. the results of the above laboratory tests were normal. ultrasonography was unremarkable in dogs and showed increased colon wall thickness in the other . definitive diagnosis was obtained from endoscopic biopsies of the colon and/or rectal scrapings in dogs and from biopsies of the colon at necropsy in one. in each case spheroid, ovoid or irregularly-shaped organisms suggestive of prototheca spp. were observed. different treatments were attempted without benefit in dogs. in one dog transient improvement was obtained with itraconazole. the median survival time was months (range: - ).the present work indicates that protothecosis should be included in the list of differential diagnosis in dogs with large bowel diarrhea, especially in those with chronic refractory colitis or developing ocular signs. dogs infected with prototheca spp. have a guarded prognosis. diagnosing protothecosis in dogs over a -year period may suggest that the disease is emerging in some southern european countries. idiopathic pulmonary fibrosis (ipf) is an interstitial fibrotic pulmonary disease, mainly described in the west highland white terrier (whwt). the diagnosis is challenging and ultimately relies on lung histopathology. identification of biomarkers specific for the disease would be very helpful. ccl (mcp- ) is a chemotactic cytokine for monocytes. it is a known biomarker in human ipf. in dogs with ipf, increased ccl gene expression has been described in lung tissue. the aim of the present study was to compare serum ccl concentration in dogs with ipf versus healthy dogs and dogs with other chronic pulmonary diseases.thirteen dogs with ipf (ten whwts, two scottish terriers, one yorkshire) mean age years, range - ), nine dogs with eosinophilic bronchopneumopathy (ebp) (various breeds, years, - ), ten dogs with chronic bronchitis (cb) (various breeds, years, - ) and ten healthy whwts ( years, - ) entered the study. diagnosis was established after clinical, radiographical, bronchoscopic (and balf analysis) examinations, as well as, in dogs with ipf, either lung high resolution computed tomography (six dogs) or histopathology (three dogs) or both (four dogs). ccl concentration in serum was determined by elisa (canine cll /mcp- quantikine ® , r&d sytems). results in the different groups were then compared using non-parametric test (mann-whitney rank sum test).serum ccl concentration was elevated in dogs with ipf (median; interquartile range = . pg/ml; . - . ) compared to healthy whwts ( . ; . - . ), (p< . ). serum ccl value in ipf dogs was higher than in ebp dogs ( . ; . - . ) (p= . ) and than in cb dogs ( . ; . - . ) (p= . ).the present study shows that ( ) idiopathic pulmonary fibrosis (ipf) is a progressive interstitial fibrotic disease, described in humans and in dogs. etiology and pathogenesis of ipf are poorly known in both species, even if a genetic basis is suspected in dogs because of the predisposition of the west highland white terrier (whwt). serum transforming growth factor beta (tgfb ) concentration is elevated in both healthy whwts and whwts with ipf, as compared to healthy dogs of various breeds. in human ipf, pathways involving tgfb , a cytokine with profibrotic properties, seem to be central in the pathogenesis and are considered as potential therapeutic targets. tgfb is produced as a pro-protein and usually stored as a latent complex. activation of the latent complex is an important step that regulates tgfb function. multiple activation mechanisms have been identified including binding to integrins and thrombospondin (thbs ).the aim of the present study was to quantify tgfb expression, as well as expression of proteins involved in tgfb activation, by quantitative rt-pcr, in lung tissue from dogs with ipf versus control dogs.total rna was extracted from lung tissues from dogs with ipf ( whwts, scottish terrier, lhassa apso) and control dogs (various breeds). ipf was confirmed by histopathology on all samples. expression of tgfb , integrins (itgb and itgb ) and thbs was measured by qrt-pcr. for each gene, a relative copy number was calculated for each sample and results were normalised using two stably expressed housekeeper genes (rps and tbp). statistically significant differences between the groups were assessed using a student t-test or a mann-whitney rank sum test with significance defined as a p < . .expression of tgfb and itgb was not statistically different between the two groups. expression of itgb was significantly lower (p< . ) while thbs expression was significantly higher (p= . ) in the ipf group relative to controls.results of the present study could not confirm that increased gene expression of tgfb by lung tissue is the source of the high circulating tgfb level in ipf. this study highlights different activating pathways of tgfb in ipf lungs compared to control lungs with a shift toward an increased activation via thbs in canine ipf. obesity is the most common nutritional problem in dogs, and its detrimental effect on basal lung function parameters has been recently shown using whole-body barometric plethysmography. the -minute walk test ( mwt) has been recently demonstrated to be a non-invasive easy-to-perform test in clinical settings, able to discriminate between healthy dogs and dogs with pulmonary disease.the aim of this study was to investigate the effect of body weight loss (bwl) on pulmonary function assessed by mwt and arterial blood gas values.six experimental beagles and privately-owned dogs, all obese but otherwise healthy, were enrolled in a diet-induced bwl program. physical examination, bw and body condition score (bcs) assessment, arterial blood gas analysis and mwt were performed when dogs were obese (bcs - / ), and repeated with animals in the middle of their bwl program (overweight, bcs - / ) and at the end of it (lean, bcs / ). for the mwt, dogs were walked for minutes, along an inside m-long hallway. heart rate (hr) and oxygen saturation (spo ) were measured by pulse oximetry before the test (pre-test value), after three minutes of walk (mid-test value) and at , , , and minutes post-test.all dogs concluded the bwl program (initial bw: , ± , kg; final bw: , ± , , means±se, p , ). bwl caused a significant increase in the walked distance (lean: , ± , m; overweight: , ± , m; obese: , ± , m; means±se, p , ) and a decrease in pretest respiratory rate (rr) (lean: ± , /min; overweight: ± , / min; obese: ± , /min; means±se, p , ). resting arterial blood gas results were not influenced by bwl and neither did the pre-test hr and spo values measured by pulse oximetry. obese dogs showed significant higher hr mid-test values compared to overweight and lean dogs (lean: , ± , /min; overweight: , ± , /min; obese: , ± , /min; means±se, p , ). moreover, spo values recorded at and minute post-test were significantly higher in overweight and lean dogs, compared to obese dogs. also, hr values registered at , , and minutes post-test were all lower in overweight and lean dogs.in conclusion, obesity negatively affects the blood oxygenation level during and shortly after physical exercise in dogs, with subsequent hr increase. bwl induces a significant decrease in resting rr and it improves pulmonary function during exercise, even before achieving the targeted ideal bw. the mwt, but not pre-test arterial blood gas values, is an efficient tool to demonstrate the efficacy of bwl. there are few diagnostic laboratory methods available for evaluation of platelet function and contribution to thrombotic events in the clinical setting. the impedance whole blood platelet aggregometer multiplate ® has recently become available. although it is being marketed for monitoring effect of antiplatelet therapy, it can also be used for assessment of platelet aggregation in response to various agonists, reflecting platelet function, activity and reactivity in response to disease. the purpose of this study was therefore to investigate multiplate ® as a diagnostic tool for detection of variations in platelet aggregation in dogs with diseases known to predispose to hypercoagulability and thrombosis and to evaluate whether there is a correlation between multiplate aggregation response and the maximal amplitude (ma) measured by thromboelastography (teg).twenty clinically healthy dogs and eighteen diseased dogs with neoplasia, generalized inflammation or protein losing enteropathy ornephropathy admitted to the university hospital for companion animals, university of copenhagen, were included in the study. citrated and heparinised blood samples were collected. multiplate ® aggregations were performed on diluted heparinised whole blood for minutes using adp, collagen (col) and arachidonic acid (aa) as agonists and nacl as buffer control. results were recorded as area under the curve (auc). dilute ( : ) tissue factor teg analyses were performed on citrated whole blood.diseased dogs had significantly increased auc compared to healthy dogs for nacl buffer control (p= . ), adp (p< . ) and col (p= . ) whereas no significant difference was obtained for aa as agonist (p= . ). teg-ma was significantly higher (p= . ) in diseased dogs compared to healthy dogs. a significant correlation was not found between teg-ma and multiplate auc using adp (p= . , r=- . ), col (p= . , r=- . ) or aa (p= . , r=- . ) .these results demonstrate that multiplate ® aggregation responses are significantly increased in a population of diseased dogs with diseases known to predispose to hypercoagulability and thrombosis, but results are not significantly correlated to teg-ma. this suggests that the multiplate method can be used to detect increased platelet reactivity in dogs with diseases known to predispose for hypercoagulability and thrombosis and that multiplate provides additional information on platelet function than teg alone in this patient group. further studies are needed to determine how multiplate and teg-ma results correlate to thrombosis and whether there may be an added benefit of using them in combination. hypoalbuminaemia is a commonly identified biochemical dyscrasia. the clinical impact of this can be far reaching, particularly in severely affected animals. problems recognised to ensue include altered colloid osmotic pressure and cavity effusions, clotting abnormalities, altered carriage of drugs, hormones and electrolytes, along with acid-base disturbances.the aim of the study was to determine the incidence of side effects encountered when administering % intravenous human albumin to dogs suffering with hypoalbuminaemia. animals were presented to a specialist referral centre in hampshire, uk for various conditions resulting in hypoalbuminaemia. hypoalbuminaeamia was considered to be present when the serum albumin concentration was < g/l. a total of albumin infusions were given to dogs presenting with a serum albumin concentration of g/l or lower. albumin was given as an intravenous infusion of mg ( ml)/kg following premedication with chlorphenamine. the duration of administration was between minutes and hours. the dogs were monitored for potential adverse reactions during the administration of albumin and in the post-infusion period. potential adverse reactions included hypotension, hyperthermia, tachycardia, tachypnea, peripheral oedema, agitation/restlessness and collapse. none of the animals showed adverse reactions during the administration of albumin or in the post-infusion period. one animal demonstrated mild hyperaesthesia that resolved prior to completion of the infusion.of the animals presented, survived to discharge, animals were euthanased and died. each dog received an average of . (sd . ) albumin infusions during the period of hospitalisation. there was no significant difference in the starting albumin concentrations between the survivors and nonsurvivors. there was no significant difference in the increase in albumin concentration post-infusion between survivors and non-survivors. there was no significant difference in the number of albumin infusions between the survivors and non-survivors. the concentration of albumin pre-infusion did not negatively impact on survival and discharge from the hospital. the results of this study demonstrate no significant complications during or following administration of % human albumin solution in dogs. there was no association between the administration of albumin, number of albumin infusions or amount of albumin administered in any patient and outcome. albumin infusion may improve the chances of severely hypoalbuminaemic dogs surviving to be discharged from the hospital. the use of human albumin for this purpose enables the limited supplies of canine plasma to be reserved for dogs needing plasma transfusion for other reasons. recently, metabolic syndrome has gained attention in human medicine given its associations with development of diabetes mellitus and cardiovascular diseases. canine obesity is associated with the development of insulin resistance, altered lipid profiles, and mild hypertension, but the authors are not aware of any existing studies examining the existence of ms in obese dogs. thirty-five obese dogs were assessed before and after weight loss. the guidelines of the international diabetes federation were modified in order to produce a definition for canine metabolic syndrome (cms), which included a measure of adiposity (using a -point body condition score [bcs]), systolic blood pressure, plasma cholesterol, plasma triglyceride, plasma glucose, and urine protein:creatinine ratio (upcr). by way of comparison, total and regional body fat mass were assessed by dual-energy x-ray absorptiometry, whilst adiponectin, insulin, and high-sensitivity c-reactive protein (hscrp) were also assessed with validated assays.systolic blood pressure (p= . ), cholesterol (p= . ), triglyceride (p= . ), insulin (p< . ), and upcr (p= . ) all decreased after weight loss, whilst plasma adiponectin increased (p= . ). however, hscrp did not change with weight loss. prior to weight loss, dogs were defined as having cms. there was no difference in total or regional body fat mass between these dogs and those who did not fit the definition of cms. however, plasma adiponectin concentration was less (p= . ), and plasma insulin concentration was greater (p= . ) in cms dogs.in this study, up to a third of obese dogs suffer from cms, and this is characterized by hypoadiponectinaemia and hyperinsulinaemia. these studies can form the basis of further investigations to determine pathogenetic mechanisms and the health significance for dogs, in terms of disease associations and outcomes of weight loss. cystourethroscopy has greatly enhanced the diagnosis and treatment of numerous conditions in veterinary medicine. rigid telescopes used in female cytoscopy provide well-illuminated highly detailed images using rod lens system technology. the flexible ureteroscope has been used for male urethrocytoscopy but is limited in image quality and procedural abilities due to the fiberoptic system, diminished illumination, and smaller working channel. the purpose of this study was to describe the technique, efficacy, and complications using a novel percutaneous perineal approach to the male urethra in order to gain access for rigid cystoscopy.the perineal approach was performed ten times in nine dogs for ectopic ureter laser ablation of idiopathic renal hematuria sclerotherapy. the dogs were placed in dorsal recumbency. using fluoroscopic guidance, an gauge renal access needle was advanced trans-perineally into the pelvic urethra. guidewire access was obtained and the access site dilated to accept a fr peel-away sheath. a rigid cystoscope was then placed through the sheath to perform the procedures. urinary catheters were placed following three of ten procedures for three to eighteen hours. the only identified peri-operative minor complication included urination form the perineal site approximately six hours post-operatively once in a single dog. no signs of stranguria or pollakiuria or incisional complications were identified in any of the dogs post-operatively at follow-up examination or contact (range to days).the percutaneous perineal approach in male dogs for rigid cystoscopy appears to be a safe and effective means of facilitating endoscopic procedures. irh results in chronic upper urinary tract bleeding. in humans, ruptured renal pelvic hemangiomas/angiomas are typically the cause. although benign, anemia, ureteral and urethral obstruction(s) can ensue. with the advent of endourology renalsparing therapies like ureteropyeloscopic-guided electrocautery or sclerotherapy has replaced ureteronephrectomy. the objective is to describe the use of endoscopic-fluoroscopic-guided sclerotherapy for the treatment of irh in dogs and report the first clinical outcomes.each uvj was identified cystoscopically. once the bleeding was confirmed a retrograde ureteropyelogram was performed. a ureteropelvic junction balloon was used for ureteral occlusion and pelvis filling volumes were recorded. four dwells were performed ( % povidone iodine mixture; and sterile liquid . - % silver nitrate). a double-pigtail ureteral stent was placed.seven dogs had sclerotherapy. five unilateral, bilateral, and developed contralateral bleeding (n= units). five were right and left-sided. there were males and female. the median age and weight was years and . kg, respectively. median procedure time was minutes. there was complication of severe renal discomfort and pyelectasia in an unstented dog.cessation of hematuria occurred in / renal units (median hours). two had recurrence within weeks; both resorted to intermittent mild hematuria. two failed treatment. median follow-up time was months (range, . - ) .overall, topical sclerotherapy for irh can be safe and effective. this is the first report of local sclerotherapy for irh in dogs and could be considered a valuable endoscopic-guided therapy prior to ureteronephrectomy. further investigation is required. percutaneous nephrolithotomy (pcnl) is considered the standard -of-care for removal of nephroliths > . cm in people, minimizing morbidity and preserving renal function. success rates are reported to be - %. most veterinary nephroliths remain clinically silent and removal is only recommended for complicated stones. morbidity of nephrotomy can be severe. the objective is to describe endoscopic-guided nephrolithotomy (enl) in canine and feline patients and report clinical outcomes, hypothesizing it is safe and effective. patients that had either pcnl or surgicallyassisted endoscopic nephrolithotomy (senl) were retrospectively evaluated. a renal puncture needle and balloon-dilation-sheath combination was used for tract formation. a nephroscope provided visualization for intracorporeal lithotripsy. stone fragments were removed and a ureteral stent was placed. nine dogs and cat ( renal units) were included. four had pcnl and senl. indications included recurrent utis ( ), worsening azotemia ( ), and ureteral-outflow obstructions ( ). median weight was . kg ( . - . ). stone composition was calcium oxalate ( ), mixed struvite ( ), urate ( ), and cystine ( ). median stone size was cm ( . - ). median pre-and month post-operative creatinine was . ( . - . ) and . mg/dl ( . - . ), respectively. the median procedure time was minutes. successful removal of all stones were documented in / ( . %). procedure-related complications occurred in units, all were easily managed. median follow-up time was days ( - ). four patients are still alive. no patient died from the procedure. overall, enl can be safely performed in dogs and cats, yielding similar success rates to people. advanced endourologic experience is recommended. fgf- is a key regulator of plasma phosphate concentration. it is elevated in cats with naturally occurring ckd and increases as renal function declines. elevated fgf- concentrations are an independent predictor of survival time in human haemodialysis patients, but this association has not previously been examined in cats with ckd. this study investigated if fgf- was independently associated with survival time (all cause mortality) in cats with newly diagnosed ckd.cats diagnosed with ckd at two london-based first opinion practices between and were identified. ckd was defined as plasma creatinine concentration > lmol/l with concurrent urine specific gravity (usg) < . or plasma creatinine concentration > lmol/l on two consecutive visits. cats were excluded if no residual edta plasma was available to measure plasma fgf- concentration within days of the date of diagnosis of ckd. fgf- concentrations were measured using a previously validated human intact fgf- elisa. plasma fgf- , creatinine, phosphate and total calcium concentrations, packed cell volume (pcv), systolic blood pressure (sbp), usg and age were entered into univariable cox regression models of survival time. fgf- concentrations were logarithmically transformed due to a highly skewed distribution. statistical significance was defined as p< . . variables with p< . were carried forwards into a backwards, stepwise multivariable cox regression analysis of survival time. ninety-one cats were included in the study. at the end of the follow-up period (february ), cats had been euthanased or died (median (range) survival time ( - ) days) and cats were alive or lost to follow-up (median (range) survival time ( - ) days). median (range) age at diagnosis (n= ) was . ( . - . ) years. univariable cox regression analysis indicated that plasma creatinine (p< . ), phosphate (p< . ) and log-fgf (p< . ) concentrations were negatively associated with survival time, and that pcv (p< . ) and usg (p< . ) were positively associated with survival time. in the multivariable model (n= ), plasma logfgf (hazard ratio (hr)= . , % confidence interval (ci) for hr= . - . ; p= . ) and creatinine concentration (hr= . , % ci for hr= . - . ; p< . ) were negatively associated with survival time, and pcv (hr= . , % ci for hr= . - . ; p= . ) was positively associated with survival time.plasma fgf- concentration is a novel prognostic indicator in feline ckd, independent of other factors including plasma creatinine concentration and pcv. future studies should investigate whether belgium ragdoll breeder organizations often forewarn ragdoll cat owners that renal problems may develop due to polycystic kidney disease (pkd), chronic interstitial nephritis, familial renal dysplasia or nephrocalcinosis. in several european countries, screening of ragdoll cats for kidney disease is already performed for years, without scientific evidence. therefore, we aimed to investigate if ragdoll cats are predisposed for kidney disease based on the laboratory parameters, one ragdoll cat was diagnosed with iris stage chronic kidney disease (ckd) one of these six cats was the ragdoll cat with iris stage ckd. in one ragdoll cat, pkd could not be excluded on ultrasonography because one cyst was detected in one kidney. however, none of the ragdoll cats was genetically positive for pkd.based on this study, pkd and ckd appear to be uncommon in ragdoll cats residing in belgium and the netherlands. however, renal infarcts were seen more commonly in ragdoll cats compared to an age-matched control group spain urine markers are advocated to early detect kidney damage in the clinical practice, nevertheless histology remains the gold standard. the aim of this study was to evaluate quali-quantitative proteinuria and possible renal damage using different non-invasive tests in dogs affected by leishmaniasis.based on clinical signs and serology/cytology, affected dogs (leish) were included. fifteen healthy, non-proteinuric dogs were selected as control. upon admission, all dogs underwent to physical examination, systolic blood pressure (sbp) measurement, clinicopathological evaluation urine high resolution agarose and silver staining sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (hre; sds-page) were performed. a cut-off of kda was selected to classify bands in high or low molecular weight (hmw; lmw). data were analyzed with non-parametric statistics and roc curve analysis (roc). a difference was considered significant for p< . leish dogs presented significantly higher upc (mean , median . ) and uac (mean . , median . ). rri values were significantly higher in leish (mean . ) than control (mean . ). rri was significantly correlated to wbc (r= . ), hemoglobin (r= . ) and albumin concentrations (r= . ), usg (r= . ) and upc (r= . ). hre and sds-page protein patterns allow to distinguish p from np and control dogs. sds-page revealed a significantly higher number of bands in leish dogs ( - ) than in control ( - ). np and blp dogs presented a significantly lower number of lmw bands than p. number of bands was significantly correlated to upc (r= . ) and uac (r= . ) in humans, diabetes mellitus (dm) is an important cause of renal damage. main lesions include thickening of the glomerular basement membrane and mesangial expansion, whereas tubular atrophy and vascular hypertrophy are less frequent. in cats, although diabetes is a common endocrinopathy, it is yet unknown whether dm causes renal damage. the aim of the present study was to compare renal histopathological features and clinical parameters of kidney function in diabetic cats against a well-matched control population.formalin-fixed, paraffin-embedded kidney samples were retrieved from diabetic and control cats that died between and due to any disease at the clinic for small animal internal medicine, university of zurich (switzerland), and in which a post-mortem examination was performed. control cats were selected to be matched for age, sex, breed and body weight. serum creatinine and urea levels were analyzed if they had been measured within days before death. kidney sections were stained with haematoxilin-eosin, periodic acid-schiff (pas), masson's trichrome, acid fuchsine orange-g (afog), and periodic acid methenamine silver (pams). with optical microscopy glomerular, tubulointerstitial and vascular parameters were identified and scored using a grading scale. data were analyzed with contingency tables and t-tests.thirty-two diabetic cats and matched controls were included. with optical microscopy, scores of glomerular lesions (i.e., sclerotic glomeruli, mesangial or endocapillary hypercellularity, increased mesangial matrix, immunodeposits, glomerular basement membrane thickening, mesangial interposition), tubulointerstitial lesions (i.e., inflammation, fibrosis, tubular atrophy, necrosis and lipidosis, intratubular mineralizations) and vascular lesions (i.e., small or large artery hypertrophy) did not differ between the groups. overall, glomerular, tubulointestitial and vascular lesions were observed in . %, . % and . % of diabetic cats and in . %, . % and . % of the controls. similarly, serum creatinine and urea levels were not different between groups (creatinine: ± vs. ± lmol/l, reference: - lmol/l; urea: . ± . vs. . ± . mmol/l, reference: . - . mmol/ l).the results suggest that dm in cats does not lead to microscopically detectable renal lesions or clinically relevant renal dysfunction when compared to a well-matched control group. we hypothesize that the short life expectancy of diabetic cats and the low prevalence of hypertension are main reasons for the difference to human diabetics.early diagnosis of aki and differentiation from non-renal disease or ckd remains challenging in veterinary medicine. in human medicine ngal is used as a real time indicator of aki but few data exist in veterinary medicine. in this study plasma and urine ngal was measured in healthy dogs with normal gfr (plasma inulin clearance) and dogs with renal azotemia (creatinine > . mg/dl and/or urea > mg/dl persisting at least hours after correction of prerenal factors). based on history, clinical course, laboratory and ultrasonographic findings, azotemic dogs were diagnosed with aki (n= ) or ckd (n= ). urine and plasma ngal was measured with a dog ngal elisa kit (bioporto ® diagnostics a/s, gentofte, denmark). intra-assay variability for plasma and urine ngal was . % and . %, respectively. azotemic dogs had significantly higher plasma ngal concentrations and urine ngal-creatinine ratios compared to healthy dogs (p< . , mann-whitney u-test). median (min-max) plasma ngal concentration in healthy dogs, dogs with aki and ckd was . ( . - . ) ng/ml, . ( . - . ) ng/ml and . ( . - . ) ng/ml, respectively. using a multiple linear regression model in the azotemic dogs with ngal as dependent and age, weight, sex, aki vs. ckd, dialysis and survival as independent variables revealed a significant differ-ence only for aki vs. ckd (p = . ). in conclusion, ngal can be measured successfully in plasma and urine of healthy dogs and dogs with kidney disease. dogs with aki had significantly higher plasma ngal concentration compared to dogs with ckd. xanthine urolithiasis is a rare condition accounting for . % of all canine urolithiasis in one study. this pathology has been reported as a primary disorder in dogs, most notably in cavalier king charles spaniels (ckcs). xanthine is an intermediate product of purine metabolism, which is converted from hypoxanthine by xanthine oxidase. xanthine is only slightly soluble in urine and therefore hyperxanthinuria may lead to urolith formation. it has been speculated that some ckcs have an inherited mutation in the xanthine oxidase gene. in humans, isolated deficiency of xanthine oxidase occurs rarely and approximately % of individuals are asymptomatic, despite having significant xanthinuria. therefore we hypothesised that asymptomatic xanthinuria may be commonplace in the uk population of ckcs. in support of this, a previous case report of a symptomatic ckcs reported significant xanthinuria occurring in an asymptomatic sibling. in order to examine the prevalence of xanthinuria in ckcs, urine concentrations of hypoxanthine and xanthine metabolites as well as creatinine were measured in clientowned cavalier king charles spaniel dogs and dogs of other breeds from three first-opinion veterinary practices in the uk. urine samples were collected by free catch and purine metabolites were measured by high-performance liquid chromatography. ratios of xanthine/creatinine and hypoxanthine/creatinine from the two populations were compared by mann whitney u test and were found not to be significantly different (p= . and p= . respectively). in the control population, the xanthine/creatinine ratio ranged from . to . (median . ), while in the ckcs population it ranged from . to . (median . ). these results are markedly lower than the previously reported case of xanthine urolithiasis in a uk ckcs dog, which utilised the same reference laboratory (xanthine/creatinine ratio . ). these data suggest that asymptomatic xanthinuria is not prevalent in the uk ckcs population. key: cord- -awivedxp authors: diaz, james h. title: ticks, including tick paralysis date: - - journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: . /b - - - - . - sha: doc_id: cord_uid: awivedxp nan ticks are the most competent and versatile of all arthropod vectors of zoonotic infectious diseases for several reasons. first, ticks are not afflicted by most of the microorganisms that they may transmit or the paralytic salivary toxins that they may transfer during bloodfeeding. second, and unlike mosquitoes, ticks can transmit the broadest range of infectious microbes among all arthropods, including bacteria, viruses, and parasites. in addition, tick-transmitted coinfections appear to be increasing and complicate differential diagnosis and antimicrobial treatment. third, ticks can vertically transmit infectious microorganisms congenitally to their offspring of both genders (transovarian transmission) and then disseminate carrier state infections among all generational growth stages (trans-stadial transmission). tick-borne infectious diseases can also be transmitted to humans by blood transfusions and organ transplants, and babesiosis, a tick-borne infection caused by malaria-related parasites, can be transmitted congenitally. fourth, ticks have capitalized on many competitive advantages afforded them by evolving changes in climate and human lifestyle, including the following: wider geographic distributions and longer active breeding and blood-feeding seasons as a result of increases in global mean temperatures and humidity; greater abundance of wild animal reservoir hosts no longer effectively controlled, especially deer, rabbits, and rodents; greater residential construction in recently cleared woodlands adjacent to pastures and yards frequented by wildlife, domestic animals, and humans; and more vacation and leisure-time activities enjoyed by humans and their pets during prolonged tick host-questing and blood-feeding seasons from earlier springs through later falls and milder winters. in short, ticks of all ages and both genders may remain infectious for generations without having to reacquire infections from host reservoirs and environmental and behavioral changes now place humans and ticks together outdoors for longer periods for tick breeding, blood-feeding, and infectious disease transmission. with the exception of toothed hypostomes for blood-feeding and clawless palps, adult ticks resemble large mites with eight legs and diskshaped bodies. there are four stages in the tick life cycle-egg, six-legged larva, nymph, and adult. ticks are classified into three families: the ixodidae, or hard ticks; the argasidae, or soft ticks; and the nuttalliellidae, a much lesser known family, with characteristics of both hard and soft ticks. ixodid ticks have a hard dorsal plate or scutum, which is absent in the soft-bodied, argasid ticks. ixodid ticks also exhibit more sexual dimorphism than argasid ticks, with both genders looking alike. however, all blood-fed ticks, especially females, are capable of enormous expansion and engorged ixodid females are often confused with engorged argasid females. although ticks from all families may serve as disease vectors, the ixodid or hard ticks are responsible for most tick-borne diseases in the united states. ixodid ticks have mouth parts that are attached anteriorly and visible dorsally. they live in open exposed environments, such as woodlands, grasslands, meadows, and scrub brush areas. argasid ticks are leathery and have subterminally attached mouth parts that are not visible dorsally. argasid ticks prefer to live in more sheltered environments, including animal nests, caves, crevices, woodpiles, and uninhabited rural cabins. all ticks feed by cutting a small hole in the host's epidermis with their chelicerae and then inserting their hypostomes into the cut, with blood flow maintained by salivary anticoagulants. ticks are attracted to warm-blooded hosts by vibration and exhaled carbon dioxide. ixodid ticks actually "quest" for hosts by climbing onto vegetation with their forelegs outstretched; waiting to embrace passing hosts ( fig. - ). ticks spend relatively short periods of their lives mating and blood-feeding on hosts: soft ticks feed rapidly for hours and then drop off, whereas hard ticks blood-feed for days ( to ) before dropping off for egg laying. • ticks can transmit the broadest range of infectious microbes among all arthropods, including bacteria, viruses, and parasites. • gravid ticks may also transmit paralytic salivary toxins during blood-feeding. • ticks are among the most competent and versatile of all arthropod vectors of infectious diseases. • tick-transmitted lyme borreliosis or lyme disease is now the most common arthropodborne infectious disease in the united states and europe. • most tick-borne infectious diseases can also be transmitted to humans by blood transfusions and organ transplants, and babesiosis can be transmitted congenitally. • ticks of all ages and both genders may remain infectious for generations without having to reacquire infections from host reservoirs. • new tick-transmitted pathogenic species are constantly being described in the united states. • ticks can transmit several pathogens during one blood-feeding, resulting in coinfections that can complicate differential diagnosis and treatment. • the diagnosis of tick-transmitted infectious diseases is based on combinations of tick-bite history and characteristic lesions, such as erythema migrans and eschars, microscopic identification of pathogens in blood and tissue biopsy specimens, serologic and immunocytologic tests, and nucleic acid serotyping. • most tick-transmitted bacterial diseases remain sensitive to doxycycline, amoxicillin, and chloramphenicol. • the tick-transmitted viral diseases can be managed only supportively. • babesiosis is caused by a malaria-like parasite and must be treated with combinations of antimalarial agents and azithromycin or clindamycin. • combinations of immunization, prophylactic antibiotics, personal protective measures, landscape management, and wildlife management are all effective strategies for the prevention and control of tick-borne infectious diseases. • a single -mg dose of doxycycline administered within hours of a tick bite is more than % effective in preventing lyme disease. short view summary keywords anaplasmosis; argasid ticks; babesia; babesiosis; borrelia; borreliosis; ehrlichiosis; francisella; ixodid ticks; lyme disease; rickettsialpox; rocky mountain spotted fever; tick paralysis; tick-borne coltiviruses; tick-borne encephalitis viruses; tick-borne hemorrhagic fever viruses; tick-borne relapsing fever viruses; tick-borne rickettsioses; ticks; tularemia by the s and s, the causative agents of the ehrlichioses were stratified as newly emerging, rickettsia-like species, and later ( ) were completely reorganized into separate genera, ehrlichia and anaplasma. , in , kirkland and colleagues described a new erythema migrans-like rash illness in north carolina, a nonendemic region for lyme disease, transmitted by the lone star tick, amblyomma americanum (see fig. - ) . this new borreliosis would soon be named the southern tick-associated rash illness (stari) or masters' disease, but its causative agent, b. lonestari, a new borrelia species, would not be identified until (see fig. - ). , by , ticks were recognized as the most common vectors of all arthropod-borne infectious diseases in europe, five new spotted fevercausing rickettsiae were described, four new subspecies of the lyme disease-causing b. burgdorferi complex were identified, a new relapsing fever borrelia species was isolated, and anaplasmosis was exported to europe from the united states. in a seemingly unending era of new discoveries in tick-transmitted diseases, another new and unanticipated vector for rmsf, rhipicephalus sanguineus, the brown dog tick, was identified in the united states in ( fig. - ) . in , the first human cases of relapsing fever caused by ticktransmitted borrelia miyamotoi were reported from russia, and by , % to % of surveyed residents of new england states where lyme disease is endemic were seropositive for prior b. miyamotoi infection. in , a new pathogenic ehrlichia species in addition to endemic ehrlichia chaffeensis and ehrlichia ewingii was identified in four febrile patients in minnesota or wisconsin and presumed to be related to ehrlichia muris. because most tick-borne diseases are caused by obligate intracellular organisms, many of which infect erythrocytes, granulocytes, or vascular endothelial lining cells, many tick-borne infections may also be transmitted congenitally (e.g., babesiosis) and by blood product transfusions and organ transplants. blood product-transmitted infections have now been described for the tick-borne rickettsial diseases (including q fever), babesiosis, and ehrlichiosis. in , the centers for disease control and prevention (cdc) reported the first case in which transfusion transmission of anaplasma phagocytophilum, the tick-borne causative agent of anaplasmosis (formerly, human granulocytic ehrlichiosis [hge]) was confirmed microscopically and serologically by testing of both the recipient and donor. today, the seroprevalence of tick-borne diseases is increasing significantly among blood and organ donors in the united states, tick-borne infectious diseases have challenged researchers and physicians since dr. howard t. ricketts identified the wood tick, dermacentor andersoni, as the vector of rocky mountain spotted fever (rmsf) in and firmly established the insect vector theory of infectious disease transmission. the emergence and recognition of lyme disease in the early s in the united states, whose causative agent, the spirochete borrelia burgdorferi, was not identified until , sparked renewed interest in tick-borne diseases in the united states and europe ( fig. - ) . by the early s, lyme borreliosis had become the most common arthropod-borne infectious disease in the united states and europe. since the s, every decade now describes emerging or rediscovered tick-borne infectious disease and new vectors for previously described tick-borne diseases, such as rmsf. these latest discoveries have been spawned by new immunodiagnostic technologies, especially by nucleic acid identification technologies, particularly the polymerase chain reaction (pcr) assay. states and has been exported to europe, most cases of lb in europe and northern asia are caused by b. afzelii and b. garinii (see table - ) . collectively, the three borrelia species are often referred to as b. burgdorferi (sensu lato). ticks usually acquire borrelia infections as larvae or nymphs by blood-feeding on small reservoir hosts, most commonly birds and rodents, and may transmit lb to humans during blood-feeding, which may go unnoticed (see fig. - ) . borrelia organisms are further maintained in nature as infected adult ixodes ticks blood-feed on larger mammals, especially deer. unlike argasid or soft ticks, ixodes ticks prefer temperate ecotonal zones of canopied forests abutting cleared scrub or grasslands and transmit b. burgdorferi to humans during outdoor exposures in such habitats. because borrelia spirochetes must migrate from the tick's midgut to the salivary gland during blood-feeding, tick attachments for less than hours rarely result in lb in humans. after an incubation period of to weeks, the hallmark of spirochete transmission manifests as solitary erythema migrans, a maculopapular erythematous rash with a bull's eye pattern, at the site of tick attachment . erythema migrans also occurs in stari at the site of amblyomma americanum or lone star tick attachment and results from the subcutaneous centrifugal movement of the spirochetes from the bite sites to the central circulation and target organs (see fig. - ) . in a meta-analysis of longitudinal studies of lb in the united states and europe, tibbles and edlow have reported that many patients do not recall a tick bite ( % in the united states, % in europe), constitutional symptoms of low-grade fever (< ° c [ . ° f]) and headache are common but nausea and vomiting are rare, and a solitary erythema migrans lesion is the most common initial presentation of lb ( % in the united states, % in europe). although deaths from lb are rare, the greatest morbidity from target organ damage in lb occurs in patients with prolonged or untreated infections, with % to % developing cardiac manifestations, % to % developing neurologic manifestations, and % to % developing chronic arthritis. [ ] [ ] [ ] however, if lb is recognized and treated early in the erythema migrans stage, cure rates will exceed %, late manifestations of chronic arthritis will be avoided, and outcomes will be excellent (see table - ). combined tick-transmitted coinfections have been described in regional u.s. populations, and an unexplained increase in the virulence of tick-borne infectious diseases has been described in the united states (rmsf), europe, and north africa (mediterranean spotted fever) and australia (queensland tick typhus). several tick-borne infectious diseases have now been reclassified by the cdc as potential biologic terrorism agents, including the following: francisella tularensis (tularemia), a category a agent (highly likely microorganism to be weaponized); coxiella burnetii (q fever), a category b agent (less likely to be weaponized); and the tick-borne encephalitis and hemorrhagic fever viruses, category c agents (least likely to be weaponized). in the future, the tick-transmitted infectious diseases will increase in prevalence over wider distributions at higher altitudes in a warmer world. unexpected tick vectors of emerging infections caused by obligate intracellular microorganisms will continue to be discovered as people spend more leisure times outdoors in temperate climates in tickpreferred ecosystems. the borrelioses are a large group of tick-borne spirochetal diseases caused by several species of borrelia, with unique geographic distributions, tick vectors, and host animal reservoirs ( lyme borreliosis (lb) or lyme disease is now the most common tick-borne infectious disease in the northern hemisphere and the most common arthropod-borne infectious disease in the united states. , in the united states, lb is caused by borrelia burgdorferi (sensu stricto), first identified as a novel bacterial spirochete in , and transmitted to humans by ixodes spp. hard ticks in u.s. regional pockets, specifically the northeast and co-workers have now dispelled the former concept of chronic persisting b. burgdorferi infections and have demonstrated that repeat episodes of pathognomonic erythema migrans in appropriately treated lb patients were due to reinfections and not to recurrences. , the jarisch-herxheimer reaction (jhr), an inflammatory cytokinemediated reaction to dying spirochetes with a worsening of presenting symptoms, vasodilatation, and myocardial dysfunction, may occur during antibiotic treatment for lb but is more common after antibiotic therapy for tick-borne relapsing fevers. , there have been no reported deaths from jhr during antibiotic therapy for lb, and the very rare case fatalities from lb have been attributed to cardiac conduction abnormalities from myocarditis in untreated cases. first recognized in , stari manifests initially as erythema migrans, as in lb, but occurs in regions in which b. burgdorferi is not endemic and follows the prolonged attachment of blood-feeding lone star ticks, amblyomma americanum, more abundant in the southeastern and south central united states (see figs. - and - ). , patients who are bitten by lone star ticks may develop lb-like erythema migrans rashes and occasionally develop milder constitutional symptoms than in lb, including fever, headache, fatigue, and generalized myalgias. however, unlike lb, stari is not a reportable infectious disease and has no diagnostic serologic tests, such as enzyme-linked immunoassays (elisas), immunofluorescent assays (ifas), and western immunoblot assays. in addition, a microbiologic analysis of skin biopsy specimens obtained from the rashes of patients in missouri with clinical diagnoses of stari failed to detect b. lonestari, suggesting that stari could be caused by other pathogens. because some patients have recovered from stari without antibiotic treatment in lb-untreated patients, recurrent attacks of chronic arthritis were formerly referred to as chronic lb. , later, as all patients in whom lb was diagnosed were treated with antibiotics, synovitis persisting for months to years after initial treatment was renamed "antibioticrefractory arthritis. " antibiotic-refractory arthritis was attributed to a combination of retained spirochetal antigens and postinfectious autoimmune reactions. however, recent investigations by nadelman tbrf is defined clinically by the sudden onset of two or more episodes of high fever (> ° c [ . ° f]) spaced by afebrile periods of to days, with the first febrile episode lasting to days and the relapsing episodes lasting to days each. , , the first episode ends with a -to -minute "crisis" with tachycardia, hypertension, hyperpyrexia (as high as ° c [ . ° f]), and rigors, followed by diaphoresis and defervescence. , , all febrile episodes are accompanied by nausea, headache, neck stiffness, myalgia, and arthralgia. the relapsing febrile episodes result from the growth of new spirochete populations in the blood to replace those killed by macrophages and cytokines. most patients have splenomegaly, % will have hepatomegaly, and most will have elevated aminotransferase levels, unconjugated bilirubin, and prolonged prothrombin and partial thromboplastin times. , direct neurologic involvement is more common than in lb and may include cranial nerve neuritis (especially cranial nerves vii and viii), radiculopathy, and myelopathy. myocarditis is also more common than in lb; may be complicated by adult respiratory distress syndrome (ards), pulmonary edema, and cardiomegaly; and is often fatal. diagnostic and treatment strategies for tbrf are outlined in table the jhr is much more common, although rarely fatal, during treatment of tbrf than during treatment of lb and occurs in % to % of patients with tbrf. at present, no prophylactic strategies to reduce the severity of the jhr have proved beneficial or have been adequately tested in multiple clinical trials, including therapy with antipyretics, corticosteroids, or naloxone. treatment with penicillin instead of tetracycline has a slightly lower risk for causing jhr during antibiotic therapy for tbrf. the family rickettsiaceae contains two genera, the spotted fevercausing genus rickettsia and the typhus-causing genus orientia (see chapters and ). the rickettsiae may be further stratified clinically into the tick-borne spotted fever group and mouse mitetransmitted rickettsialpox caused by rickettsia akari (see chapter ). the rickettsiae are obligate intracellular, gram-negative bacteria that thrive in ixodid tick salivary glands and are transmitted during bloodfeeding. once injected into the host, rickettsiae are initially distributed regionally via lymphatics, with some species causing marked regional lymphadenopathy (e.g., rickettsia slovaca). within to days (mean, days), rickettsiae are disseminated hematogenously to vascular endothelial lining cells of target organs, including the central nervous system (cns), lungs, and myocardium. rickettsiae gain entry into host endothelial cells in a trojan horse-like manner by using their outer membrane proteins (ompa and ompb) to stimulate endocytosis. once within phagosomes, rickettsiae escape to enter the cytosol or nucleus for rapid replication by binary fission, safe from host immune attack. the tick-borne rickettsial diseases that cause spotted fevers (sfs) are compared in a descending order of clinical severity of infection by preferred tick vectors and wild animal reservoirs in table - the global epidemiology of the tick-borne sf-causing rickettsiae has dramatically evolved since the transmission cycle of rmsf was first described by ricketts in with the following: emerging new strains and diseases (r. slovaca-associated lymphadenopathy); greater understanding of the highly conserved genome of several related species (r. africae-r. parkeri and the r. conorii subspecies); wider geographic distribution and greater virulence of existing strains (r. rickettsii, r. conorii subspecies, r. australis); unanticipated new tick vectors for some sfs (rhipicephalus sanguineus for rmsf in the united states); cluster outbreaks of tick-borne rickettsioses in returning travelers (r. africae causing african tick-bite fever); and regional clusters and epidemic cycles of more severe sfs worldwide (rmsf in the united states, mediterranean sf [msf] in europe, and queensland tick typhus [qtt] in australia).* the reasons for such changes in rickettsial sf epidemiology are unclear and may include warming temperatures and increasing humidity, more frequent drought-rain cycles, residential development in preferred tick ecosystems, more competent tick vectors given competitive advantages by environmental and and there have been no long-term sequelae reported in stari cases, some have questioned whether antibiotic therapy is indicated in stari. because distinguishing stari from lb may be difficult, wormser and co-workers have recommended that the differential diagnosis rely on a combination of regional exposures, clinical presentations, serologic results, and potential for long-term sequelae based on their comparison of lb cases from new york and stari cases from missouri. the investigators noted that the timing of rash onset was shorter ( days) in stari compared with lb ( days) and that stari patients were less likely to be symptomatic than lb patients. in addition, the stari rash was more often circular with central clearing than the lb rash. most authorities recommend antibiotic therapy for stari with oral doxycycline or amoxicillin following the same regimen as for lb to cover any missed diagnoses of lb with potential for chronic arthritic and cardiac sequelae (see table - ). the tick-borne relapsing fevers (tbrfs) comprise a worldwide group of serious bacterial infections by borrelia spirochetes after brief, painless, and usually unnoticed bites by ornithodoros spp. argasid or soft ticks. these ticks prefer indoor living-in cabins, caves, and crevices-and quickly abandon warm-blooded rodent hosts for egg laying (see table - ). , unlike the ixodid ticks, ornithodoros ticks feed very briefly, usually for less than minutes, and at night. , adults can live for as long as to years and survive without blood meals for several years. transovarian transmission of the tbrf spirochetes occurs commonly among all species and, unlike lb-causing borrelia species, tbrf spirochetes are already present in the salivary glands at the onset of blood-feeding and do not need time to migrate from the gut to the mouth parts. the wild animal host reservoirs of tbrf are maintained in birds and several mammals, most commonly rodents. the bite of a tbrf-infected tick is painless, and the bite site is marked after a few days by a small red to violaceous papule with a central eschar. , one spirochete is sufficient to initiate tbrf, and the infection rate after a single bite by an infected tick is more than %. the incubation period to onset of the first febrile episode is to days. evolving to petechial rash in % to % of cases in to days. the pathognomonic rash starts distally on the wrists and ankles and then spreads centripetally up the limbs (see fig. - ). the pathophysiologic mechanisms of petechial rashes and target organ system damage (cns, lungs, heart) in the sf rickettsioses include vascular endothelial cell damage by microbial replication, vascular inflammation (vasculitis), and increased widespread vascular permeability, which may result in hypovolemic shock, oliguric prerenal failure from acute tubular necrosis, cerebral edema, and noncardiogenic pulmonary edema. distal, digital skin necrosis may occur in severe cases of rmsf and qtt from hypoperfusion. cardiac vasculitis may manifest as myocarditis with intraventricular conduction blocks. aside from petechial rash and thrombocytopenia, other hemorrhagic manifestations in rmsf and other sfs are rare. cns complications in rmsf and other severe sf infections may include ataxia, photophobia, transient deafness, focal neurologic deficits, meningismus, meningoencephalitis, seizures, and coma. pulmonary complications may include cough, alveolar infiltrates, interstitial pneumonitis, pleural effusions, pulmonary edema, and ards. , [ ] [ ] [ ] initially, msf caused by r. conorii was thought to be a more benign disease than rmsf. in , severe cases of msf with multiple eschars and multisystem disease similar to rmsf with cns, renal, and pulmonary complications were first reported and now appear to be increasing across europe. in a outbreak of msf in portugal, case-fatality rates (cfrs) of % were recorded and exceeded those of untreated rmsf of %. qtt, african tick bite fever (atbf), and r. slovacaassociated lymphadenopathy are generally milder diseases than rmsf and msf. however, severe cases of qtt with rmsf-like complications, including renal insufficiency and pulmonary infiltrates, were recently reported from australia. although atbf caused by r. africae, a similar tick-bite fever in north america caused by r. parkeri, and r. slovaca infections may all cause multiple necrotic eschars and painful regional lymphadenopathy, these sf infections are often spotless (≥ %) and follow typical rickettsial sf prodromes. , a history of tick bites, eschars, and painful regional lymphadenopathy helps to establish the correct diagnosis, especially in the absence of adequate diagnostic laboratory services. the precise laboratory diagnosis of tick-borne rickettsial sfs may be established by microbiologic isolation of the causative organisms from skin biopsy specimens or blood cultures, nonspecific immunofluorescent antibody tests that cross react with many sf antigens, other immunocytologic techniques to demonstrate intracellular rickettsiae, and pcr assay to identify and speciate rickettsial dna or rna. genetic changes, more frequent contact between ticks and humans outdoors, and international trade and travel distributing tick vectors and their preferred animal hosts quickly and widely. the tick-borne sf rickettsioses share many common features in clinical presentations, including incubation periods of approximately week, flulike prodromes of fever, headache, myalgia, nausea, vomiting, and abdominal pain (that may mimic acute appendicitis in rmsf), spotty rashes within to days of fever onset, and necrotic eschars at tick-bite sites (fig. - ). some sf rickettsial diseases may be "spotless, " including rmsf in % to % of cases, complicating early differential diagnosis. the tick-borne rickettsial infections that can cause spotty rashes include r. rickettsii (rmsf), r. conorii (msf), r. australis (qtt), and r. africae-r. parkeri (african-north american tick bite fever) in about % of cases (see fig. - ) . , the tick-borne rickettsial infections that are associated with one or more necrotic eschars at tick-bite sites include r. conorii, r. australis, r. africae-r. parkeri, r. japonica, r. slovaca, r. aeschlimannii, and r. honei. the sf rickettsioses may vary in severity from causing multisystem organ failure (rmsf, msf) to painful lymphadenopathy (r. africae-r. parkeri, r. slovaca) to mild to subclinical disease (r. aeschlimannii). , , after an average incubation period of week, rmsf starts with a flulike, febrile prodrome followed by a characteristic maculopapular tularemia, also known as rabbit fever or deer fly fever, was first described as a zoonosis in squirrels in tulare county, california, in . its causative agent, francisella tularensis, was later identified as a gram-negative coccobacillus by dr. edward francis during an investigation of deer fly fever in utah in . tularemia occurs in regional pockets worldwide, has a very large wild and domestic animal reservoir, and is seasonally transmitted to humans by ixodid tick and deer fly bites and by contact with infected animals, especially rabbits and muskrats. the primary tick vector of tularemia in the united states is the american dog tick, dermacentor variabilis ( fig. - ) . ticktransmitted tularemia is most commonly reported during the spring and summer (may to august) worldwide. tularemia transmitted through contact with an infected animal occurs more often during the fall through hunting and trapping seasons, especially among male hunters who field-clean infected animal carcasses. f. tularensis is an extremely stable microorganism in nature, surviving in soil, water, and animal carcasses for months to years. in addition to fecal or vomit contamination of tick bites and direct inoculation of intact skin or mucosal surfaces when crushing ticks or skinning animals, tularemia may be transmitted by ingesting raw or undercooked infected game or bush meats, drinking contaminated water, or inhaling aerosolized microorganisms. [ ] [ ] [ ] in , a cluster outbreak of primary pneumonic tularemia in patients (with one fatality) was reported from martha's vineyard, massachusetts. a case-control investigation of the outbreak implicated aerosolized exposure to f. tularensis during summertime brush cutting and lawn mowing as significant (odds ratio [or], . ; % confidence interval [ci], . to . ) risk factors for pneumonic tularemia. concerns about inhalation transmission and potential biologic weaponization of f. tularensis led to the reinstatement of tularemia as a nationally notifiable infectious disease in . , the cdc reported a total of cases of tularemia from states from to (period prevalence, cases/yr; range, to cases/yr), with most cases occurring in males during may to august in regional pockets, antibiotic treatment mainstays for the tick-borne rickettsial sfs remain the tetracyclines for most cases and chloramphenicol for severe multisystem disease and during pregnancy. although the quinolones, azithromycin, and clarithromycin may be as effective as tetracyclines and chloramphenicol for rapidly managing some sfs, they are not recommended for initial therapy at this time. although short, -to -day courses of doxycycline have been reported to be as successful as -day courses in some sf infections (e.g., msf), such treatment strategies have not been tested in randomized controlled trials in other sf infections and are also not recommended at this time. most authorities now recommend that tetracycline, chloramphenicol, or ciprofloxacin for tetracycline-allergic patients be continued for a minimum or days or until the patient has been afebrile for at least hours and is improving clinically. q fever q (query) fever was first described in australia in , and its causative organism, coxiella burnetii, was isolated shortly thereafter. c. burnetii is a gram-negative, intracellular, spore-forming bacterium that is the sole species of its genus. c. burnetii is genetically related to legionella pneumophila and, like l. pneumophila, c. burnetii is usually transmitted to humans by inhalation of contaminated aerosols. q fever is a zoonosis with worldwide distribution and extensive domestic animal (cattle, sheep, goats, cats, dogs), wild animal (birds, rabbits, reptiles), and arthropod (ticks) reservoirs. in most cases, humans are not infected by tick bites but by inhaling spores or bacteria in aerosols contaminated with infectious particles in dried animal feces, milk, or products of conception. , q fever may also be transmitted by ingestion of contaminated milk, by vertical transmission from mother to fetus, by contaminated blood product transfusion, and even percutaneously by crushing infected ticks near breaks in the skin barrier. c. burnetii is reactivated during pregnancy and multiplies extensively in the placenta, exposing abattoir workers, veterinarians, researchers (especially those working with parturient sheep), and domestic pet owners (especially of cats) to highly infectious aerosols during delivery. , recently, several cases of q fever were reported among u.s. military personnel deployed to iraq and afghanistan and in travelers returning from asia, latin america, and sub-saharan africa. , c. burnetii has long been considered a potential bioterrorism weapon for several reasons, including its environmental stability, spore-forming capability, ease of aerosolized dispersal, and high pathogenicity, with an ability to initiate infection with a single microorganism. after an average -week incubation period (range, to days), q fever may manifest as a wide variety of illnesses in humans, including the following: acute q fever, a self-limited febrile illness with severe headache, retro-orbital pain, and nonproductive cough; q fever pneumonia with consolidated opacities, pleural effusions, and hilar lymphadenopathy on chest radiographs; q fever granulomatous hepatitis, usually after ingestion of contaminated milk; cns q fever with protean manifestations ranging from aseptic meningoencephalitis and transient behavioral and sensory disturbances to cranial nerve palsies and hemifacial pain mimicking trigeminal neuralgia; and chronic q fever endocarditis, especially in predisposed patients with congenital valvulopathies, prosthetic heart valves, aortic aneurysms, or vascular grafts. , , patients who are immunocompromised by pregnancy, congenital immunodeficiency disorders, cancer, hiv infection/aids, organ transplant antirejection therapy, renal dialysis, or prolonged corticosteroid therapy are at greater risk for acquiring more severe and chronic q fever infections. because the isolation of c. burnetii requires biosafety level , most diagnostic laboratory strategies for q fever rely on microscopic detection on giemsa-stained smears of blood or sputum or tissue biopsies (liver, excised heart valves), on antibody detection by immunofluorescent assays, or on dna detection by pcr assay. , the prognosis is usually excellent in the acute q fever illnesses, and mortality is rare after appropriate antibiotic therapy with tetracyclines (doxycycline is preferred- mg po twice daily for days) or fluoroquinolones. chronic q fever endocarditis will require prolonged treatment with two antibiotics, either rifampin ( mg po twice daily) and genera that are tick-borne bacterial infections of many mammals, including humans, ehrlichia and anaplasma (fig. - ) . , like rickettsiae, the anaplasmataceae attach to molecular ligands on phagocytic cells to gain trojan horse-like entry into leukocytes and then trick intracellular phagosomes into releasing them into the cytosol for replication (see fig. - ) . , the tick-borne anaplasmataceae are now endemic in the united states and have preferred geographic distributions, tick vectors, and wild and domestic animal reservoirs (table - ). they spread from the infected tick's gut to its salivary gland, are inoculated over to hours into the host's dermis, and cause subclinical (especially in children) to severe and potentially fatal infections (especially in immunocompromised adults) within to weeks. because transovarian transmission in ticks has not been observed, the major reservoirs of the anaplasmataceae in nature are wild and domestic animals. , although the presenting clinical manifestations are similar among anaplasmataceae infections, the potential multisystem complications and resulting cfrs from these diseases are ultimately determined by the immunocompetence of human hosts (see table - ). the human anaplasmataceae are resistant to fluoroquinolones but remain susceptible to tetracyclines, which are now recommended for children and adults. because there are no vaccines for the tick-borne ehrlichioses and anaplasmosis, the best preventive measures are tick avoidance and control and rapid removal of blood-feeding ticks by hours or less. , including arkansas and missouri, eastern oklahoma and kansas, southern montana and south dakota, and martha's vineyard. there are two biovars of f. tularensis, with biovar a (f. tularensis biogroup tularensis) causing % to % of tularemia cases in north america and biovar b (f. tularensis biogroup palearctica) causing a milder disease throughout europe and asia. , , the presenting clinical manifestations of infection depend on the virulence of the biovars (a > b), route of entry of microorganisms, multisystem infections, and immunocompetence of infected hosts. the portal of entry of f. tularensis has historically been used to classify the clinical manifestations of tularemia, with untreated pneumonic tularemia having the highest cfrs of % to % (table - ) . [ ] [ ] [ ] , the differential diagnosis of ulceroglandular tularemia, the most common presentation, is extensive and includes other arthropod bites, bacterial and viral infections, and fungal diseases capable of causing skin ulcers with painful regional lymphadenopathy. diagnostic strategies for tularemia include the following: microscopic identification or culture in biosafety level facilities of microorganisms from blood, sputum, gastric lavage fluid, lung biopsy, or lymph node aspirates (sensitivity, % to %); acute and convalescent serology comparing antibody titers (sensitivity, > %); direct immunofluorescent antibody testing; and antigen detection by pcr assay (sensitivity, % to %). frequently accompanying laboratory abnormalities in tularemia include significant elevations in the erythrocyte sedimentation rate (esr), significant leukocytosis (> , /µl), often with normal differential counts, and thrombocytosis. the recommended treatment strategies for tularemia have evolved considerably from historical treatments with painful intramuscular injections of streptomycin to oral therapy with the aminoglycosides and fluoroquinolones, which are effective in % of cases and may result in resolution of ulcers within hours. most cases in adults, including pneumonic tularemia, may be managed with fluoroquinolones alone (ciprofloxacin, mg iv or mg po twice daily for to days, or levofloxacin, mg iv or po twice daily for to days), with aminoglycosides (gentamicin or amikacin, to mg/kg/day for to days) reserved for pediatric infections and widely disseminated systemic infections. relapse rates are highest with oral tetracyclines, including doxycycline, and chloramphenicol, which may still be indicated for cases with cns dissemination despite its potential for bone marrow toxicity. the human ehrlichioses and anaplasmosis (formerly known as human monocytic and human granulocytic ehrlichiosis, respectively) are classic examples of emerging tick-borne infectious diseases. since , four new tick-borne bacterial species have been identified and classified into a new family, anaplasmataceae. the four genera of anaplasmataceae comprise obligate, intracellular, gram-negative bacteria, closely related genetically to the family rickettsiaceae. the anaplasmataceae include two genera that are synergistic parasites of flatworms (neorickettsia sennetsu) and filarial worms (wolbachia spp.) and two cases, usually in elderly, immunocompromised, or splenectomized human hosts, massive hemoglobinuria may be associated with severe anemia, jaundice, acute renal failure, and increased cfrs. babesiosis is now reemerging as an arthropod-borne parasitic disease, as confirmed by increasing numbers of reported cases in the northeast united states and increasing seroprevalence rates there and in california. [ ] [ ] [ ] human babesiosis may be divided into two epidemiologic and clinical patterns based on the causative babesia species, their regional endemicity, and the immunocompetence of their human dead-end hosts (see table - ). the first pattern is caused by babesia divergens and related species or subspecies and occurs in immunocompromised, and often splenectomized, human hosts. it includes b. divergens babesiosis, first in eastern and now in western europe, a b. divergens-like babesiosis in the midwest caused by a babesia species designated mo- , and a babesiosis along the pacific coast caused by b. divergenslike species designated as wa- and as ca types (e.g., ca- , ca- ). [ ] [ ] [ ] [ ] [ ] babesiosis is a tick-borne, malaria-like zoonosis that usually causes subclinical infections with prolonged parasitemias in humans and can be transmitted vertically in utero and horizontally by blood product transfusion. [ ] [ ] [ ] [ ] [ ] babesiosis was initially described in cattle with red water (hemoglobinuric) fever in , when victor babes observed inclusions within bovine erythrocytes. theobald smith later identified the causative agent of bovine red water fever in as babesia bigemina, accurately described the parasite's life cycle, and demonstrated for the first time the arthropod-borne transmission of an infectious disease to a mammal. although more than species of babesia have now been identified as zoonoses in domestic and wild mammals, only a few species can cause babesiosis in humans, a disease characterized by fever, intravascular hemolysis, and hemoglobinuria (table - ). in severe for specific igm antibodies in acute infections and pcr-based assays to detect babesia dna and species-specific dna sequences. quinine ( mg orally three times daily) and clindamycin ( . g iv twice daily or mg orally three times daily), continued for week or until parasitemias are in remission, can be used to treat babesiosis caused by all species. quinine and clindamycin are preferred therapies for wa- babesiosis and for severe b. microti infections, especially the b. divergens-related species are maintained in tick vectors by transovarial and trans-stadial transmission of the parasites, and most infections are transmitted by diminutive and usually unidentified and unnoticed nymphal ticks. the human b. divergens-like cases occur primarily in cattle-ranching regions during the summer months, when tick vectors are most active and the incidence of bovine red water fever is greatest. these are the more severe cases of babesiosis, with hemolytic anemia, hemoglobinuria, and renal failure, usually in splenectomized persons. the second and more common pattern of babesiosis in the united states occurs in regional pockets on the northeast coast (new york, massachusetts, rhode island, connecticut, new jersey, and offshore islands [block island, long island, nantucket]) and upper midwest (minnesota, wisconsin) and is caused by babesia microti, a rodent babesia species transmitted to humans by the same ixodid ticks (blacklegged deer ticks) that transmit lyme disease (see fig. - ) . thus, b. microti babesiosis in the united states parallels the distribution of lyme disease and its tick vectors, occurs in clusters in the same regional pockets as lyme disease, and may coexist with lyme disease in an increasing number of cases. , , , b. microti-induced babesiosis occurs during the warmest months, with % of cases reported between may and august, when deer ticks are most active. humans are usually infected by unnoticed bites by nymphal deer ticks from rodent reservoirs in mice, especially the white-footed mouse (peromyscus leucopus), rather than deer. diagnostic strategies for babesiosis include the demonstration of characteristic intraerythrocytic and extraerythrocytic organisms on giemsa-stained thin smears and subinoculation of human blood samples into hamsters for suspected b. microti infections or into gerbils for suspected b. divergens-related infections (fig. - ) . , , the serologic methods, especially useful when microscopic methods fail in low parasitemias, include indirect immunofluorescent antibody testing in older adults and splenectomized or immunosuppressed individuals. , for non-life-threatening b. microti infections, a -week course of oral atovaquone ( mg twice daily) and azithromycin ( mg on day , followed by to mg/day for week) cleared parasitemias as effectively as quinine and clindamycin, with fewer side effects. for coinfections with b. burgdorferi, patients should be treated specifically for lyme disease with doxycycline ( mg orally twice daily for weeks) and with antimalarial agents for babesiosis. , , the tick-borne viral infections are caused primarily by flaviviruses and may be divided into two separate clinical presentations, each with preferred tick vectors and wild animal reservoirs-the viral encephalitides and viral hemorrhagic fevers (table - ) . the tick-borne viral infections share several common clinical and epidemiologic characteristics, including the following: incubation periods of approximately week; biphasic illnesses separated by symptom-free periods beginning with flulike viremic stages and ending with cns or hemorrhagic manifestations with increased cfrs; nonspecific serodiagnosis by comparing acute and convalescent sera for increased antibody titers or by hemagglutination inhibition; specific serodiagnosis by enzymelinked immunosorbent assay (elisa) and antigen detection from blood or cerebrospinal fluid (csf) by reverse-transcriptase (rt)-pcr; no specific treatments other than supportive therapy; and significantly increased postinfection morbidity. from a global distribution perspective, the tick-borne encephalitis viruses (tbevs) are separated into the old world (eastern hemisphere) and new world (western hemisphere) strains, with the old world strains having significantly higher cfrs ( % to %) and permanent neurologic morbidity rates ( % to %) than the new world strains (cfr, % to %; morbidity rate < %). although additional old world flaviviral strains have now been discovered in sheep reservoirs, the most common old world tbevs have been further stratified regionally into three major subtypes-european or central european (tbev-eu), siberian or russian spring-summer (tbev-sib), and far eastern (tbev-fe; see table - ) . except for the old world tbevs with sheep reservoirs, all the tbevs are transmitted by the injection of infected saliva from viremic ixodid ticks. during blood-feeding, viruses in tick saliva increase up to -fold and render early removal of the feeding tick ineffective in preventing disease. the preferred wild animal reservoirs for tbevs include rodents, insectivores, medium-sized mammals, deer and other ungulates, birds, and, less often, domestic animals (see table - ) . powassan encephalitis, first isolated in , typifies a new world tbev with a confined regional distribution in the new england states and eastern canada, several ixodid tick vectors, primarily ixodes spp., an extensive wild animal reservoir in rodents and medium-sized mammals, especially woodchucks and skunks, and a seasonal occurrence. cases occur from may to december and peak during june to september, when ticks are most active. patients with powassan encephalitis present with somnolence, headache, confusion, high fever, weakness, ataxia, and csf lymphocytosis. transient improvement may be followed by neurologic deterioration, evidence of ischemia or demyelination on magnetic resonance imaging, and slow recovery, often with permanent deficits including memory loss, weakness, ophthalmoplegia, and lower extremity paraparesis. unlike the old world tbevs, powassan encephalitis is uncommon, with only confirmed cases reported by the cdc from to . because there is no vaccine or specific therapy for powassan encephalitis, the best means of prevention is protection from tick bites. since , powassan encephalitis cases historically confined to the northeastern united states and canada have been increasingly confirmed farther westward in minnesota and wisconsin, with fatal cases reported in the elderly. the old world tbevs remain common causes of permanent neurologic morbidity from scandinavia to eastern japan, with more than , cases reported per year, a third of which result in permanent neurologic deficits. in addition to tick bites, the old world tbevs may occasionally be transmitted by ingestion of unpasteurized milk products from viremic livestock (especially goats), breast-feeding, and slaughter of viremic animals. old world tbev is typically biphasic in over % of cases, with an initial febrile flulike presentation followed by a -week (range, to days) symptom-free interval. this honeymoon or recovery period is followed by meningoencephalitis with csf pleocytosis, with or without myelitis, and a poliomyelitis-like flaccid paralysis that targets the arms, neck, and shoulders. magnetic resonance imaging and electroencephalographic abnormalities are common but nonspecific. other acute neurologic complications may diagnoses for the tick-borne coltiviruses are other tick-borne febrile diseases, most commonly rmsf in north america, which may be distinguished from ctfv and srv infections by its characteristic rash and leukocytosis. serologic diagnostic methods to detect anticoltivirus antibodies include complement fixation, seroneutralization assay, immunofluorescence assay, elisa, and western immunoblot. the most specific and confirmatory laboratory diagnostic methods include rt-pcr assays to identify ctfv-rna (or the rna of its crossreacting serotypes, ctfv-ca and srv) or the isolation of coltiviruses after intracerebral inoculation of infected human blood into suckling mice. treatment of all tick-borne coltivirus infections is entirely supportive, and long-term complications are rare in uncomplicated cases. first described in in australia, canada, and the united states, tick paralysis is a rare, regional, and seasonal cause of acute ataxia and ascending paralysis with an incubation period of to days after female tick attachment, mating, and blood-feeding. [ ] [ ] [ ] [ ] although species of ticks have been implicated in tick paralysis cases worldwide, most cases occur in the united states and canadian pacific northwest (washington state and british columbia) and in australia. in the u.s. pacific northwest, tick paralysis is caused by the american dog tick, d. variabilis, or the rocky mountain wood tick, dermacentor andersoni, during april through june, when dermacentor ticks emerge from hibernation to mate and to seek blood meals (see fig. - ) . [ ] [ ] [ ] [ ] [ ] [ ] the mechanism of neurotoxic paralysis in dermacentor tick paralysis is unknown, but neuroelectrophysiologic studies have suggested that sodium flux across axonal membranes is blocked at the nodes of ranvier, leaving neuromuscular transmission unimpeded. in australia, the marsupial ixodid tick, ixodes holocyclus, can cause a more severe form of ascending neuromuscular paralysis by producing a botulinum-like neurotoxin that blocks neuromuscular transmission by inhibiting the presynaptic release of acetylcholine. most cases of tick paralysis in north america have occurred sporadically in young girls with long hair concealing ticks feeding on the scalp or neck. however, a four-patient cluster of dermacentor tick paralysis, including a -year-old girl with a tick on her hairline, and three adults with ticks on the neck (n = ) and back (n = ), was reported from colorado in . although botulism causes a descending neuromuscular paralysis with a preserved sensorium, tick paralysis, guillain-barré syndrome, acute poliomyelitis, and spinal cord tumors may all cause acute ascending paralysis with preserved mental status and must be differentiated from each other (table - ) . [ ] [ ] [ ] because poliomyelitis has been nearly eradicated by vaccination worldwide, tick paralysis is frequently misdiagnosed as guillain-barré syndrome, and the correct diagnosis is made accidentally by finding an engorged, usually female, tick on the scalp, head, or neck during hair combing or when applying electroencephalographic electrodes (see table - ) . before , postmortem examinations of persons who died suddenly of unexplained paralytic illnesses demonstrated attached ticks on their heads and necks. in a review of canadian tick paralysis cases in the s before the widespread availability of mechanical ventilation in intensive care units, rose reported a cfr of % to % without tick removal. in a review of tick paralysis cases in washington state over the period to , dworkin and co-workers reported a cfr of up to %, with most deaths occurring in the s. in a -year meta-analysis of confirmed tick paralysis cases in the united states, diaz reported a cfr of % in the first years, a seasonal pattern of case clusters in children and adults in urban and rural locations, and a significant increase in initial misdiagnoses of tick paralysis as guillain-barré syndrome in more recently reported cases. in addition, the misdiagnoses of tick paralysis cases as guillain-barré syndrome often directed unnecessary therapies, such as central venous plasmapheresis with immunoglobulin g, and delayed correct diagnosis and treatment by tick removal. in all cases, the diagnosis of tick paralysis was later established when attached ticks were either discovered by caregivers or by cranial neuroimaging studies. the cfr from tick paralysis has steadily declined over the past years, with almost all deaths in canada and the united states reported in the s and s. [ ] [ ] [ ] include altered consciousness, seizure activity, cranial nerve palsies, and an often fatal bulbar syndrome with cardiorespiratory failure. because no specific treatments other than supportive therapy exist, tick avoidance and immunization remain the best preventive measures. effective vaccines have now been developed for the three subtypes of old world tbevs, and some have been shown to even provide crossprotection among the subtypes in experimentally infected animals. the tick-borne hemorrhagic fever (tbhf) viruses are maintained in nature in extensive wild and domestic animal reservoirs and are transmitted by infected ixodid tick bites, squashing infected ticks, creating infective aerosols, direct contact with blood or tissues from infected animals or humans, or nosocomial spread among medical personnel. tbhfs may be caused by flaviviruses and bunyaviruses, which are distributed throughout eastern europe, africa, and asia. they are characterized clinically by biphasic illnesses that present as febrile flulike symptoms and end as hepatomegaly and hemorrhagic manifestations (petechiae, purpura, subconjunctival and pharyngeal hemorrhage, thrombocytopenia, cerebral hemorrhage, disseminated intravascular coagulation) separated by a few afebrile days. cfrs range from % to over %, with most deaths occurring within to days of symptom onset during hemorrhagic stages. diagnoses may be confirmed by immunologic techniques, such as antibody increases in paired sera and elisa, and by molecular techniques, such as rt-pcr. although ribavirin can inhibit crimean-congo hemorrhagic fever (cchf) virus replication in animal models, it has not been tested in clinical trials in humans with cchf. nevertheless, if tbhf is suspected in the tropics and laboratory confirmation is unavailable, intravenously administered ribavirin ( mg/kg initially, followed by mg/kg four times daily for days, and then mg/kg three times daily for days) is recommended for severe cases, and oral ribavirin is recommended for high-risk contacts. all patients with tbhfs should be placed in isolation, and strict universal precautions should be practiced by all medical personnel. a mouse brain-derived cchf vaccine has been developed in bulgaria but is not available elsewhere. in the absence of a universal vaccine, the best preventive measures for the tbhfs are tick avoidance and control, rapid burial of dead animals, and personal protective equipment for abattoir workers and medical personnel. the tick-borne coltiviruses of the family reoviridae are all doublestranded rna viruses of the genus coltivirus and include colorado tick fever virus (ctfv), which is endemic in the united states and canadian rocky mountain regions; the california tick fever virus (tfv) of rabbits (ctfv-ca); the salmon river virus (srv) of idaho, a serotype of the ctfvs; and the european eyach virus (eyav). the ixodid or hard ticks are the only vectors of the coltiviruses, with dermacentor ticks (mainly d. andersoni) being the principal vectors of ctfv and srv in the rocky mountains and ixodes ticks (i. ricinus, i. ventalloi) being the only vectors of eyav throughout europe. among the coltiviruses, ctfv has the widest host range, which includes squirrels, other rodents, rabbits, porcupines, marmots, deer, elk, sheep, and coyotes. the remaining coltiviruses have fewer, more specific wild animal hosts, including the black-tailed jackrabbit (lepus californicus) for ctfv-ca and primarily the european rabbit (oryctolagus cunniculus) but also rodents, deer, domestic goats, and sheep for eyav. the coltiviruses are maintained in nature by ixodid ticks that blood-feed on wild animal hosts with prolonged viremias and then transmit coltiviruses trans-stadially but not transovarially. infected nymphs hibernate over winter, and previously infected nymphs and newly infected adults then transmit coltiviruses to human dead-end hosts during spring-summer blood-feeding. ctfv has also been transmitted by blood transfusion and congenitally. both ctfv and srv can cause biphasic to triphasic febrile illnesses that mimic mild cases of rmsf without rash. leukopenia and thrombocytopenia are common laboratory manifestations of coltivirus infections. complications are rare but may include meningoencephalitis, orchitis, hemorrhagic fever, pericarditis, and myocarditis. eyav infections are more often complicated by cns manifestations than american strain coltivirus infections. the most common differential performing regular tick checks. wearing long pants tucked into socks, long-sleeved shirts, and light-colored clothing can help keep ticks off the skin and make them easier to spot on clothing. impregnating clothing with permethrin, routinely performed by the military on maneuvers, is a highly effective repellent against ticks and other insects. the topical application of insect repellents containing % to % formulations of n,n-diethyl-meta-toluamide (deet) directly on the skin is another effective and recommended measure. most patients with lyme disease, tbrf, babesiosis, ehrlichioses, and anaplasmosis will not recall tick bites because these diseases are often transmitted by diminutive nymphal ticks. nevertheless, tick localization and removal as soon as possible, preferably within hours, remain recommended strategies to prevent the rickettsial and viral ixodid tick-borne diseases and to reverse tick paralysis. ticks should always be removed with forceps (or tweezers), not fingers (because squashing ticks can transmit several tick-borne diseases across dermal barriers or create infectious aerosols), and in contiguity with their feeding mouth parts, rather than burning ticks with spent matches or painting embedded ticks with adhesives or nail polishes. landscape management strategies to prevent tick-borne diseases include widespread application of acaricides over tick-preferred ecosystems, removal of vegetation and leaf litter near homes and recreation sites, and creation of dry barriers of gravel, stone, or wood chips between forested areas and yards or playgrounds. wildlife management strategies to prevent tick-borne diseases include encouraging the development of better veterinary vaccines for tick-borne diseases with large domestic animal reservoirs, applying acaricides actively to domestic animals and passively to deer and cattle at baited feeding and watering stations or salt licks, and setting out acaricide-baited rodent houses for rodents to occupy or acaricide-baited cotton balls for rodents to adopt as nesting materials, especially in crawl spaces under homes and near playgrounds. most emerging infectious diseases today, such as west nile virus and severe acute respiratory syndrome, arise from zoonotic reservoirs, and many are transmitted by arthropod vectors. because ticks are the most common insect vectors of zoonotic diseases, ticks have become common arthropod vectors of emerging zoonotic diseases, including lyme disease, ehrlichiosis, and anaplasmosis. ticks are highly competent and versatile vectors of infectious diseases because ticks of all ages and both genders may remain infectious for generations, without having to reacquire infections from host reservoirs. recent environmental changes and human behaviors now place the treatment of dermacentor tick paralysis simply requires removing the tick with forceps (or tweezers) to restore neuromuscular function within hours. although i. holocyclus tick paralysis is also treated by tick removal, transient neuromuscular deterioration may occur for - hours after tick removal. the administration of i. holocyclus antitoxin before tick removal and prolonged observation for hypoventilation have been recommended. there are a number of strategies that can be used in the prevention and control of tick-borne infectious diseases, including immunization, personal protective measures, landscape management, and wildlife management. in the s, a lyme disease vaccine was developed for the united states, but it was withdrawn from the market in because of poor sales. immunization strategies to prevent tick-borne infectious diseases have proved far more effective in europe and asia than in the united states, where neurologic complications from tbevs are second only to japanese encephalitis as causes of permanent paraparesis. current immunization programs for tick-borne viral diseases now provide primary prevention of tbev-eu in europe, tbev-sib in russia and the middle east, tbev-fe in china and the far east, and cchf in bulgaria. a canine antitoxin for i. holocyclus-induced tick paralysis has been used to reverse tick paralysis in animals and humans in australia. in addition to immunization, antibiotic therapy after presumed ixodid tick bites with erythema migrans has been recommended as a prophylactic therapeutic strategy for the primary prevention of some tick-borne infections. a randomized clinical trial found that a single -mg dose of doxycycline administered within hours of a tick bite was % effective in preventing lyme disease. finally, because most tick-borne infectious diseases may also be transmitted by blood product transfusions, screening blood product donors in high seroprevalence areas for lyme disease and other borrelioses, babesiosis, ehrlichioses, and anaplasmosis would eliminate transfusion-transmitted cases. physicians are encouraged to order leukocyte-reduced blood components for blood product transfusions to potentially reduce the risks for ehrlichiosis and anaplasmosis, especially in regions that are highly endemic for leukocytotropic tick-borne infectious diseases. personal protective measures to prevent tick-transmitted diseases include wearing appropriate clothing, using insect repellents, and -transmitted microorganisms reassort their nucleic acids with their hosts and develop antimicrobial resistance (especially to tetracyclines) or superpathogen capabilities, either by nature's own design or human terrorist intent humans and ticks together outdoors for longer periods in welcoming ecosystems for breeding, blood-feeding, and infectious disease transmission. better prevention and treatment strategies for tick-borne diseases are indicated, before the highly conserved genomes of key references the complete reference list is available online at expert consult. . dumler js, walker dh. rocky mountain spotted feverchanging ecology and persisting vigilance rocky mountain spotted fever from an unexpected tick vector in arizona human borrelia miyamotoi infection in the united states emergence of a new pathogenic ehrlichia species, wisconsin and minnesota southern tick-associated rash illness: erythema migrans is not always lyme disease does this patient have erythema migrans? a critical appraisal of "chronic lyme disease reinfection versus relapse in lyme disease failure to isolate borrelia burgdorferi after antibiotic therapy in culture-documented lyme borreliosis associated with erythema migrans: report of a prospective study differentiation of reinfection from relapse in recurrent lyme disease the epidemiology of tick-borne relapsing fever in the united states microbiological evaluation of patients from missouri with erythema migrans questions on mediterranean spotted fever a century after its discovery q fever: epidemiology, diagnosis, and treatment an outbreak of primary pneumonic tularemia on martha's vineyard ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment epidemiology and impact of coinfections from ixodes ticks. vector borne zoonotic dis coltiviruses and seadornaviruses in north america a six-year-old girl with tick paralysis a -year meta-analysis of tick paralysis in the united states: a predictable, preventable, and often misdiagnosed poisoning clinical and neurophysiological features of tick paralysis prophylaxis with single dose doxycycline for the prevention of lyme disease after an ixodes scapularis tick bite what makes ticks tick? climate change, ticks, and tick-borne diseases goddard j. physician's guide to arthropods of medical importance rocky mountain spotted feverchanging ecology and persisting vigilance tick-borne bacterial diseases emerging in europe lyme disease-united states emerging tick-borne infections: rediscovered and better characterized, or truly "new"? ehrlichiosis and anaplasmosis reorganization of genera in the families rickettsiaceae and anaplasmataceae in the order rickettsiales: unification of some species of ehrlichia with anaplasma, cowdria with ehrlichia, and ehrlichia with neorickettsia, descriptions of six new species combinations and designation of ehrlichia equi and hge agent as subjective synonyms of ehrlichia phagocytophila erythema migrans-like rash at a camp in north carolina: a new tickborne disease? stari, or masters disease-tick-vectored lyme-like illness first culture isolation of borrelia lonestari, putative agent of southern tick-associated rash illness tick-borne rickettsial diseases: emerging risks in europe rocky mountain spotted fever from an unexpected tick vector in arizona human borrelia miyamotoi infection in the united states emergence of a new pathogenic ehrlichia species, wisconsin and minnesota anaplasma phagocytophilum transmitted through blood transfusion-minnesota southern tick-associated rash illness: erythema migrans is not always lyme disease relapsing fever and other borrelia diseases does this patient have erythema migrans? a critical appraisal of "chronic lyme disease reinfection versus relapse in lyme disease failure to isolate borrelia burgdorferi after antibiotic therapy in culture-documented lyme borreliosis associated with erythema migrans: report of a prospective study differentiation of reinfection from relapse in recurrent lyme disease the epidemiology of tick-borne relapsing fever in the united states microbiological evaluation of patients from missouri with erythema migrans tick-borne relapsing fever in the northwestern united states and southwestern canada acute respiratory distress syndrome in persons with tickborne relapsing fever-three states spotted fever group rickettsioses targeting rickettsia questions on mediterranean spotted fever a century after its discovery african tick bite fever severe spotted group rickettsiosis in australia tropical infectious diseases: principles, pathogens, and practice q fever: epidemiology, diagnosis, and treatment q fever in returned febrile travelers the occurrence of tularemia in nature as a disease of man ulceroglandular tularemia in a nonendemic area tick-borne bacterial, rickettsial, spirochetal, and protozoal infectious diseases in the united states: a comprehensive review an outbreak of primary pneumonic tularemia on martha's vineyard ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment ehrlichioses and anaplasmosis tropical infectious diseases: principles, pathogens, and practice seroepidemiology of emerging tickborne infectious diseases in a northern california community vertically transmitted babesiosis transfusiontransmitted babesiosis in washington state: first reported case caused by a wa -type parasite investigation of transfusion transmission of a wa -type babesial parasite to a premature infant in california enzootic transmission of babesia divergens among cottontail rabbits on persistent parasitemia after acute babesiosis epidemiology and impact of coinfections from ixodes ticks. vector borne zoonotic dis tick-borne encephalitis outbreak of powassan encephalitis-maine and vermont powassan virus encephalitis bunyaviral fevers: rift valley fever and crimean-congo hemorrhagic fever coltiviruses and seadornaviruses in north america acute ascending paralysis, or tick paralysis tick bite in british columbia injuries and diseases of man in australia attributable to animals (except insects) cluster of tick paralysis cases-colorado tick paralysis presenting in an urban environment a six-year-old girl with tick paralysis tick paralysis: electrophysiologic studies a review of tick paralysis tick paralysis: human cases in washington state a -year meta-analysis of tick paralysis in the united states: a predictable, preventable, and often misdiagnosed poisoning clinical and neurophysiological features of tick paralysis prophylaxis with single dose doxycycline for the prevention of lyme disease after an ixodes scapularis tick bite key: cord- -vrs je x authors: powers, karen s. title: acute pulmonary infections date: - - journal: pediatric critical care study guide doi: . / - - - - _ sha: doc_id: cord_uid: vrs je x acute lower respiratory infection is a common cause of morbidity in infants and children, and at times, requires intensive care and mechanical ventilation. viral bronchiolitis and bacterial pneumonia account for the majority of lower respiratory tract infections that lead to respiratory insufficiency and pediatric intensive care admission. twenty-seven percent of children who require mechanical ventilation for at least h in pediatric intensive care units are diagnosed with bronchiolitis and % have the diagnosis of pneumonia. the median length of time intubated for an acute pulmonary infection leading to respiratory failure is approximately days. acute lower respiratory infection is a common cause of morbidity in infants and children, and at times, requires intensive care and mechanical ventilation. viral bronchiolitis and bacterial pneumonia account for the majority of lower respiratory tract infections that lead to respiratory insuffi ciency and pediatric intensive care admission. twenty-seven percent of children who require mechanical ventilation for at least h in pediatric intensive care units are diagnosed with bronchiolitis and % have the diagnosis of pneumonia. the median length of time intubated for an acute pulmonary infection leading to respiratory failure is approximately days. viral bronchiolitis remains the leading cause for hospital admission in infancy and the most frequent cause of acute respiratory failure in children admitted to pediatric intensive care units in north america. pneumonia in children younger than years of age has an annual incidence karen s. powers of - cases per , . community acquired pneumonia can also lead to severe respiratory compromise especially in children with pre-existing disease. a detailed understanding of the diverse etiologies and distinct clinical courses of acute pulmonary infections is essential for the pediatric critical care practioner. this chapter will focus on bronchiolitis and pneumonia as the two leading causes of pulmonary infections leading to picu admission. approximately one third of children develop bronchiolitis during the fi rst years of life. of these, only in ( % of all infants in the united states) will require hospitalization. although hospitalization rates have increased over the last three decades, mortality remains low. overall mortality rate is - %, but as high as % in high risk infants. most deaths occur in infants younger than months of age with co-morbidities such as prematurity, congenital heart disease, congenital or acquired lung disease or immunodefi ciency. respiratory syncytial virus (rsv) was fi rst isolated in and still represents the major cause of bronchiolitis. other causative viruses include parainfl uenza, adenovirus, enterovirus, infl uenza and most recently human metapneumovirus and human bocavirus (hbov). in the northern hemisphere, rsv outbreaks occur from october to june. human metapneumovirus (hmpv) recently has been identifi ed as the causative agent in - % of bronchiolitis cases, possibly surpassing parainfl uenza as the second most common etiology. its prevalence is slightly higher in the late winter and spring. parainfl uenza infections peak at months of age, representing approximately - % of cases of bronchiolitis. parainfl uenza (piv- ) is endemic throughout the year, but especially common in the late spring. males are . - times more likely to require hospitalization for bronchiolitis and are likely to have more severe disease. an x-linked genetic trait that results in a reduced tolerance to hypoxia has been postulated and would be consistent with the observation of increased mortality in newborn males with infant respiratory distress syndrome. virtually all children by the age of two will have been infected with rsv, all children by the age of fi ve will have been infected with hmpv, and all children by the age of nine will have been infected with hbov. the remainder of the discussion on bronchiolitis will be divided into rsv and non-rsv bronchiolitis. although etiologic agents may differ, clinical courses are often similar. respiratory syncytial virus (rsv) accounts for - % of bronchiolitis, infecting one-half of all infants within the fi rst year of life and hospitalizing approximately , infants yearly (about % of affected infants). approximately % of these infants require mechanical ventilation. co-infection with either hmpv or rhinovirus occurs in - % of young children. two types of rsv exist -types a and b. type a is more common and is believed to cause more severe disease, although data is not conclusive. both types may exist simultaneously in the community. infants less than year will typically shed the virus for about days. children with immunodefi ciencies may shed the virus for months. the immune response varies with age and contributes to both termination of the disease and its pathologic features. the virus is transmitted from respiratory secretions by close contact with infected persons or by contact with contaminated objects or surfaces. there is a % rsv transmission rate within families and about one-half of hospital workers will acquire rsv. therefore, hand washing and the wearing of gowns and gloves is of primary importance to attenuate transmission. mortality from rsv bronchiolitis continues to decline with better intensive care and the use of preventive therapies. male infants are more likely to require hospitalization and usually manifest more severe disease. about ½ of all infants will be infected with rsv bronchiolitis in their st year of life; % will be hospitalized; % of hospitalized infants will require mechanical ventilation. antibody-mediated immunity rsv introduced onto the nasal or conjunctival mucosal surface causes profuse rhinorrhea within a few days. during the fi rst months of life, passively acquired maternal antibodies are protective. however, as maternal antibody titers gradually decrease, infants become susceptible to severe disease. cell-bound iga may develop to help clear the virus. circulating igg directed against the glycoprotein (g) and fusion (f) proteins (operative in syncytia formation) on the viral surface will develop several days later. infants less than months of age appear to induce a weaker antibody response likely due to the presence of maternal antibodies. virus-specifi c ige in the respiratory tract is associated with disease severity. often, complete and effective immune responses are not induced, thus re-infections are possible even during the same season. epithelial cells and alveolar macrophages are key activators of cellular immunity. although these cells enhance viral clearing, they also contribute to airway infl ammation through the release of cytokines and chemokines. these include interleukin (il)- , tumor necrosis factor-alpha, il- , il- , macrophage-infl ammatory protein (mip)- -alpha and rantes (regulated upon activation, normal t cell expressed and secreted). release of these cytokines and chemokines are believed to be partially responsible for airway infl ammation and hyperreactivity. the effects of these mediators persist beyond the acute infection and contribute to prolonged pulmonary dysfunction. children who require mechanical ventilation have lower peripheral t cell counts compared to hospitalized infants not requiring mechanical ventilation. these infants demonstrate low t cell proliferative responses and interferon (ifn-g ) production. il- is required for the initiation of cellular immunity. the length of time requiring mechanical ventilation has been found to be inversely related to il- production. the role of th /th -like cytokine profi les, expressed as ifn-g /il- ratios, is controversial. in some studies, these ratios decreased after polyclonal stimulation in hospitalized infants with rsv. however, more recent studies have shown normal ratios following polyclonal stimulation. neutrophils are the predominant cell found in the airways of infants with rsv bronchiolitis. elevated levels of il- are found in high concentrations in the nasal secretions of infected children and act as a neutrophil chemoattractant. further evidence of cellular induced injury is seen in post-mortem examination where peribronchial lymphocyte infi ltration with bronchial epithelial necrosis is typically present. infants typically present with tachypnea, rhinorrhea, cough, low-grade fever, irritability, poor feeding and vomiting. respiratory rates greater than breaths per minute are often associated with room air saturations of less than %. infants may also have tachycardia, mild conjunctivitis, otitis media, or pharyngitis. low-grade fever usually persists for - days. in addition, infants may develop a metabolic acidosis from poor caloric and fl uid intake. apnea often is the fi rst presenting symptom of rsv bronchiolitis in small infants. the etiology of apnea remains unknown; however, is likely related to the immaturity of the respiratory control center in the brainstem. the incidence of apnea in infants with bronchiolitis is approximately - %. the heterogeneous nature of rsv induced lung disease can cause atelectasis in some areas and overdistension in others. chest roentgenograms often show hyperinfl ation with fl attening of the diaphragms and patchy or peribronchial infi ltrates. atelectasis, especially of the right upper lobe, is often seen. infants may have high lung volumes with the functional residual capacity often being twice normal. the decrease in dynamic compliance and increase up regulation of the infl ammatory cascade with release of chemokines and cytokines are contributory to the airway infl ammation and hyperreactivity. c hapter • ac ute pu lmonary i n fections in airway resistance leads to marked increase in work of breathing, often worse during expiration from lower airway obstruction. alterations in gas exchange and hypoxemia are secondary to a ventilation-perfusion mismatch. the anatomical differences between young infants and older children contribute to the severity of the disease in the young. due to the highly compliant cartilaginous chest wall and poor thoracic musculature, the infant's chest wall has diffi culty countering the lung's inherent tendency towards collapse. this leads to a greater propensity of small infants towards atelectasis compared with older children. the absence of effective collateral ventilation in infants also contributes to the development of atelectasis and impaired gas exchange. cellular debris in small airways and peribronchial edema increase airways resistance leading to wheezing as the predominant symptom in some infants. despite the potential for severe impairment in lung function, most hospitalized infants improve within - days. typically, by weeks, they have normal respiratory rates, oxygenation, and ventilation. chest radiographs usually normalize by day . however, about % of infants will have a protracted course, with some mild respiratory symptoms persisting for months. viral respiratory infections have been linked to the development of asthma later in childhood. the tucson children's respiratory study group prospectively followed for years, infants who had bronchiolitis and found an increased risk for subsequent wheezing episodes. some infants are at an increased risk for severe rsv disease such as those with chronic lung disease due to prematurity (bronchopulmonary dysplasia), cystic fi brosis, congenital heart disease, and immunodefi ciencies. in children with cystic fi brosis, rsv accounted for % of symptomatic infections, % of hospitalizations for infants less than year, and % of infants requiring mechanical ventilation. in a study of hospitalized infants with congenital heart disease infected with rsv, % required intensive care, % received mechanical ventilation, and . % died. children having undergone hematopoietic stem cell transplants who develop rsv infections have an extremely high mortality of - % despite mechanical ventilation and antiviral therapy. environmental factors such as crowding, passive exposure to tobacco smoke, and lack of breast-feeding are associated with the development of severe disease. compared to national averages, native american and alaskan children younger than year of age have higher rates of infections. there are three subtypes of human parainfl uenza viruses. hpiv- is most frequently isolated from children with bronchiolitis, while piv- and piv- most commonly cause croup. similar to rsv, both cell-mediated hyper-responsiveness to viral antigen and virus-specifi c ige responses are observed in children with parainfl uenza bronchiolitis. upper airway edema with concomitant obstructive symptoms may be present. children that are infected with parainfl uenza have a signifi cant likelihood of developing asthma later in life. the human metapneumoviruses (hmpv) are a group of rna viruses of the paramyxoviridae family identifi ed in humans in . hmpv appears to be the second most common cause of bronchiolitis in children throughout the world. the majority of children are born with maternal hmpv specifi c igg which wanes to around % by - months of age. by age fi ve, essentially % of children have been exposed to hmpv and will have neutralizing antibody to hmpv. there are two subgroups, a and b, with group a having more severe clinical symptoms. clinical presentation of children with this virus is similar to rsv. the pulmonary infl ammation generally peaks on day which includes interstitial edema and infl ammatory cell infi ltrates of the bronchioles and alveoli. these infl ammatory changes can persist for up to days. about half of infected children are - months of age, and infection is primarily in the winter months. human bocavirus (hbov) was recently discovered in . with amino acid sequencing, this new member of the parvoviridae family was found to be closely related to the bovine parvovirus and the canine minute virus, hence the name bocavirus (bo for bovine and ca for canine). detection of the hbov from the respiratory tract in symptomatic children and its absence of detection in non-symptomatic controls strongly suggest the virus to have a role in respiratory infections in children. co-infection is commonly described in up to % of samples. it remains unclear if hbov is a primary pathogen or acts to exacerbate other viral illnesses. the pathogenesis of hbov has not been well described, but with the high occurrence of wheezing and lower respiratory tract symptoms in children infected with the virus, it is speculated that this virus may be a signifi cant contributor to asthma exacerbations. the majority of infected children have rhinorrhea, cough, and wheezing, however, diarrhea has been reported in up to % of these children. in children with high viral loads, hbov has been detected in the serum suggesting the potential for disease beyond the respiratory tract. both infl uenza a, including novel infl uenza strains such as h n , and infl uenza b can cause a clinical picture consistent with bronchiolitis in the small infant. these viruses may cause severe multisystem disease and are discussed in greater detail in the viral pneumonia section. rapid diagnostic assays are available for early detection of many viruses. the older assays are antigen-based and include indirect immunofl uorescence/direct immunofl uorescence (ifa/ dfa), enzyme immunoassay (eia), optical immunoassay (oia), and neuraminidase activity assays. although still widely used because they are inexpensive and technically simple, they have a low specifi city and sensitivity. molecular assays are becoming the new "gold standard" for respiratory virus detection -replacing tissue culture that may take days. the published sensitivities and specifi cities approach % when compared to tissue culture or antigen assay. these assays generally use polymerase chain reaction (pcr) amplifi cation. signifi cant advancements in these assays are being made to simplify the performance of the assay and decrease the required time. the most important cause of false negative test results remains poor specimen handling or inadequate sample collection. other than aiding with cohorting of hospitalized patients, serologic detection of respiratory viruses is rarely clinically useful. regardless of the viral etiology of bronchiolitis, supportive care remains the mainstay of treatment. supplemental humidifi ed oxygen is frequently needed. due to many infants being obligate nasal breathers, frequent nasal suctioning may be benefi cial to maintain an unobstructed upper airway. the affected infant or child is often unable to take adequate fl uids complicated by increased insensible losses from the respiratory tract; hence, intravenous fl uids may be required. infants and children with severe respiratory distress should be kept npo in the event respiratory failure ensues and endotracheal intubation is required. antibiotics are not routinely indicated in previously healthy children infected with rsv. progressive disease, leukocytosis, persistent fever, consolidation on radiograph or systemic toxicity should prompt an evaluation of bacterial co-infection and the use of empiric antibiotics. high risk patients often require close monitoring and care in an intensive care unit. these include infants less than weeks of age or infants with a history of prematurity, congenital heart disease, bronchopulmonary dysplasia, immunodefi ciency or neurologic disease. infants with rsv bronchiolitis typically have a combination of hyperinfl ation, pulmonary infi ltrates, supportive therapy is the mainstay of treatment for bronchiolitis. ribavirin, bronchodilators, and corticosteroids have not shown to be of benefi t. secondary bacterial infections are rare. and atelectasis. therefore, no one mode of ventilation can be recommended for all infants. non-invasive positive pressure (niipp) modes (cpap or bipap) may be attempted in infants where their primary respiratory embarrassment is secondary to atelectasis. however, this may not be suitable if the disease process appears severe or protracted as prolonged use of nipp may make feeding diffi cult, cause breakdown of facial tissue, or be diffi cult to maintain without signifi cant sedation that further compromises ventilation. if an infant requires endotracheal intubation, the mode of mechanical ventilation should be tailored to the predominant lung pathology present (i.e. atelectasis versus hyperinfl ation). children with signifi cant air trapping may need mechanical ventilation similar to a child with asthma, providing low respiratory rates and longer inspiration and exhalation times. the more typical infant will lose functional residual capacity (frc) because of atelectasis and alveolar infi ltrates. therefore, despite having some air trapping, these infants often need peep to be adjusted to recruit alveoli and return frc to normal. in the setting of elevated pulmonary vascular resistance (pvr) which may occur in infants with congenital heart disease or bronchopulmonary dysplasia, lowering pvr by traditional methods such as maintaining oxygenation, deep sedation, muscle relaxation and even nitric oxide may be indicated. ribavirin is the only fda-approved antiviral drug for rsv. ribavirin inhibits viral replication and is active against rsv, infl uenza a and b, adenoviruses, and hepatitis viruses. for lower respiratory tract diseases, ribavirin is typically administered via aerosolization. in , a meta-analysis of studies involving ribavirin was discouraging and was consistent with the common clinical experience that ribavirin did not improve clinical outcomes. therapy targeted at attenuating the virus-induced infl ammatory cascade has also been disappointing. corticosteroid administration was not associated with reduction in clinical scores, the need for hospitalization, or the length of hospitalization. routine use of any corticosteroid given via any route (intravenous, enteral or aerosolized) is not indicated, except in patients with pre-existing chronic lung disease. bronchodilators have not shown a clear benefi t in patients with acute rsv bronchiolitis. in randomized control trials, involving infants, evaluating the effect of salbutamol or albuterol on bronchiolitis, ( %) showed no effect. the remaining three studies demonstrated only a small transient improvement in the acute clinical score. although the routine use of bronchodilator therapy cannot be recommended, it has become acceptable practice to attempt to see if individual infants are beta agonist responsive or not. if no clinical response is seen after a trial of a beta agonist, its use should be discontinued. in the s, fi ve randomized trials involving infants, evaluating the effect of nebulized adrenaline (epinephrine) on bronchiolitis showed clinical improvement, with reductions in oxygen requirement, respiratory rate, wheezing, and decrease in pulmonary vascular resistance. two of these studies showed lower hospital admission rates and earlier discharge. a cochrane systematic review suggested a potential benefi t with epinephrine administration. however, subsequent studies have not supported its routine use. as with albuterol, a clinical trial in selected infants seems reasonable. nebulized hypertonic saline has been used for treating hospitalized, as well as ambulatory, children with viral bronchiolitis with variable success. a recent cochrane meta-analysis of nebulized hypertonic saline has shown an improvement in clinical scores and decrease in hospital duration. several studies have evaluated the benefi t of surfactant and nitric oxide for severe respiratory distress. the results have been inconclusive and do not currently support their routine use. heliox, a mixture of oxygen ( - %) and helium ( - %) with lower viscosity than air has been used successfully in cases of airway obstruction, croup, airway surgery, and asthma to reduce respiratory effort during the period of airway compromise. several studies have shown improved respiratory distress scores in patients on heliox with continuous positive airway pressure obviating the need for intubation and mechanical ventilation. palivizumab is a neutralizing humanized mouse monoclonal antibody directed against the rsv-f glycoprotein. it was licensed by the food and drug administration (fda) in for premature infants and infants with bronchopulmonary dysplasia. the randomized, double blind, placebo controlled impact-rsv trial involving , high risk infants found a signifi cant reduction of % in hospitalizations. with the exception of very rare anaphylaxis, no signifi cant adverse effects have been observed. palivizumab has been approved for use in infants with congenital heart disease. the cardiac synagis study group included , children with congenital heart disease in a randomized, double blind, placebo controlled trial; it found a % relative reduction in rsv associated hospitalizations with no deaths attributable to the palivizumab. since cardiopulmonary bypass can decrease serum drug concentrations by about %, it is recommended that an additional dose be given following surgery, if continued protection is desired. palivizumab should be administered intramuscularly as mg/kg every days for a total of fi ve doses during rsv season, which is generally from november through march, to high risk infants. infants or children that develop an rsv infection should continue to receive prophylaxis following recovery because the naturally acquired antibodies are not fully protective. motavizumab, a new, enhanced potency, humanized rsv monoclonal antibody has demonstrated - times greater neutralizing activity against rsv. in completion of a phase iii trial, motavizumab was found equal to palivizumab for the prevention of rsv hospitalization and superior to palivizumab for reduction of rsv-specifi c outpatient medically attended lower respiratory tract infections (malris). pneumonia describes any infl ammatory condition of the lung in which the alveoli are compromised by aspirated foreign matter, infl ammatory fl uid, or cellular debris. infection is the primary cause of parenchymal injury to the lung. pathogens include viruses, bacteria and fungi. signs and symptoms of pneumonia are non-specifi c and may be occult in the young infant. children often have fever, chills, headache, malaise, restlessness, and irritability. gastrointestinal complaints such as abdominal pain, distention, or emesis may also be present in young children. the symptoms are often preceded by minor upper respiratory tract infections characterized by low-grade fever and rhinorrhea. with more signifi cant involvement of the lower respiratory tract, tachypnea, dyspnea, cough, nasal fl aring, grunting, or retractions may be seen. the older child may demonstrate productive sputum and complain of pleuritic chest pain. on auscultation of the chest, rales and/or decreased breath sounds might be heard over areas of consolidation or pleural effusions. however, due the short path for transmission of breath sounds and the small chest size in infants, breath sounds may not be decreased, even in the presence of effusions. children with pleural irritation might prefer to lie on the affected side with legs fl exed and may complain of radiating pain to the neck and shoulder or into the abdomen. community acquired pneumonia (cap) is a common, and at times, a serious infection in children. the incidence of cap is - cases per , children less than years of age and - cases per , children - years of age. the exact prevalence of the etiologic agents causing pediatric pneumonia is diffi cult to ascertain. it is often diffi cult to differentiate viral from bacterial pneumonia based solely on clinical examination. specifi c pathogens causing cap can be determined in only approximately one-third of children using commonly available cultures, antigen detection, or serologic techniques. blood cultures yield pathogens in only about - % of infants and children with bacterial cap and many children do not undergo viral testing as it is often unnecessary. with these inherent limitations, it is generally thought that viruses account for approximately % of cap in children under the age of years and approximately % of cap in preschool children ages - years. palivizumab should be used as preventive therapy in infants with chronic lung disease and congenital heart disease. cardiopulmonary bypass signifi cantly lowers the serum level of palivizumab, so it should be redosed following surgery if continued protection desired. viral causes decline in the school age and adolescent child and bacterial causes such as streptococcus pneumoniae and mycoplasma become important pathogens ( fig. - ) . overall, bacteria account for - % of community-acquired pneumonias. the likelihood of infection with different bacteria varies by age. in the newborn period, organisms from the maternal genital tract are likely causes and include group b streptococcus , escherichia coli , enteric gram-negative bacilli, listeria , and chlamydia . in older infants, streptococcus pneumoniae becomes a signifi cant cause and remains so until years of age. group a streptococcus and staphylococcus aureus are uncommon causes. moraxella catarralis is a common cause of upper respiratory tract disease, but rarely causes pneumonia. about % of infants with pertussis will have bacterial co-infection. in children older than years of age, streptococcus pneumoniae remains the most common cause. hemophilus infl uenzae type b (hib), and most recently streptococcus pneumoniae , have decreased signifi cantly as causes of cap due to the widespread use of effective vaccines. in the older child and young adolescent, the atypical pneumonias, mycoplasma and chlamydia , become more prevalent and viral causes less common. rare bacterial pneumonias can occur with animal contact and include: francisella tularensis (rabbits); chlamydia psittaci (parrots and birds); coxiella burnetii (sheep); and salmonella choleraesuis (pigs). children with congenital anatomical defects, immunodefi ciencies, and genetic disorders are at increased risk for bacterial, viral and fungal pneumonia. the airways are normally sterile below the sublaryngeal area to the lung parenchyma. there are several protective mechanisms that include anatomic and mechanical factors, local immune defenses, and the systemic immune response. microbes are fi ltered by nasal hairs or are expelled from the airways by the epiglottic refl ex, cough refl ex, and mucociliary apparatus. immunoglobulin a (iga) is the predominant immunoglobulin present in the upper respiratory tract. iga is able to bind two antigens simultaneously, forming large antigen-antibody complexes. in this manner, the microbes are neutralized and removed by ciliary clearance, thus preventing microbial binding to the epithelium. in the lower tract, immunoglobulin g (igg) provides humoral protection by opsonizing microbes for phagocytosis by neutrophils and macrophages, activating the complement cascade, and by neutralizing bacterial it is diffi cult to determine the etiologic agent causing pneumonia, but when microbial agents are identifi ed, bacteria are isolated in - %. etiology of community acquired pneumonia based on age endotoxin. alveolar macrophages produce superoxide anions, hydrogen peroxide, and hydroxyl radicals that serve an important role in the host defense; however, uncontrolled production can lead to lung injury. in addition to oxygen radicals, a number of cytokines are produced by the alveolar macrophages. these include il- , il- , tnf, transforming growth factor-β (tgf-b ), chemotactic factors, platelet derived growth factor, and m-csf. these cytokines play a central role in phagocytic recruitment and activation. infection occurs when one or more of the defense mechanisms is altered or if the inoculum is too large. pathogens typically gain entry through inhalation of aerosolized material or through aspiration of resistant organisms inhabiting the upper airways. less frequently, pneumonia can occur via hematogenous spread. in children with bacterial pneumonia, a signifi cant portion will have a concurrent or preceding viral infection. viral infection may predispose to bacterial superinfection by reducing clearance mechanisms and by weakening the host immune response. pathogens entering the lower airways evoke an exudative consolidation of pulmonary tissues. initially, there is hyperemia of lung parenchyma due to vascular engorgement and capillary leak causing exudation and intra-alveolar fl uid accumulation. fibrin is then deposited and the airways are infi ltrated with neutrophils. consolidation causes a decrease in lung compliance and vital capacity and a total reduction in the surface area available for gas exchange. a physiologic shunt (v/q mismatch) occurs as there is increased blood fl ow through poorly ventilated segments of lung, resulting in hypoxia. compensatory hypoxic vasoconstriction may occur in an attempt to reduce v/q mismatch and hypoxia, especially in localized areas of consolidation. with treatment, resolution of consolidation will occur in - days. the exudate undergoes enzymatic digestion and is either reabsorbed or removed by coughing. if the bacterial infection extends into the pleural cavity, an empyema may result. streptococcus pneumoniae is a gram-positive diplococcus that is frequently found in the upper respiratory tract. there are over capsular serotypes with % of infections caused by serotypes. it is the most common bacterial cause for pneumonia occurring at a peak age of - months. typically, it causes a lobar or segmental consolidation, but it may manifest as patchy infi ltrates in infants. pleural effusions occur in up to % of children that require hospitalization (fig. - ) . pneumatocoele formation is rare. hemolytic uremic syndrome is associated with neuraminidase-producing strains. treatment is typically with a penicillin or cephalosporin. emerging resistance may require initial therapy with vancomycin. in hospitalized patients, parenteral therapy is generally needed for - h after fever resolves, followed by completion of - days of enteral therapy. pneumococcal conjugate vaccines (pcv) have been developed that confer immunity against and serotypes. the -valent pcv (prevnar) was licensed for use in the united states in . a -valent pcv has been recently introduced and will replace the -valent pcv. the pcvs have been highly effective at reducing hospitalizations among children younger than years for pneumococcal pneumonia. pcv is now recommended universally for children younger than months of age and older children at high risk due to underlying diseases. high risk children include those with sickle cell disease and other types of functional asplenia, human immunodefi ciency syndrome, primary immunodefi ciency, children receiving immunosuppressive therapy, and children with chronic pulmonary or cardiac disease. a -valent pcv is available for pneumonia occurs when one or more of the host defense mechanisms are altered. viruses enhance the host susceptibility to bacterial pathogens by affecting clearing mechanisms and by weakening the host immune response. streptococcus pneumoniae is the most common bacterial cause for pneumonia. c hapter • ac ute pu lmonary i n fections high risk children who need expanded serotype coverage. children with sickle cell disease or functional asplenia should continue to receive antibiotic prophylaxis regardless of whether or not they have received pneumococcal vaccines. approximately - % of infants born to chlamydia trachomatis -infected mothers will become infected at one or more anatomical site, including conjunctiva, nasopharynx, rectum, and vagina. about % of infants with nasopharyngeal infections will develop pneumonia. the infants usually present at about - weeks of age with cough and congestion, but an absence of fever. the cough often interferes with the ability to feed. infants generally have tachypnea and rales on examination and chest x-ray frequently shows hyperinfl ation. a peripheral eosinophilia may be present. c . trachomatis is susceptible to macrolides, tetracyclines, quinolones, and sulfonamides. erythromycin for - weeks is the treatment of choice for neonatal pneumonia. mycoplasma pneumoniae and chlamydia pneumoniae play a greater role in causing respiratory tract disease in children then previously thought. an indolent course that develops over - days manifested by low-grade fever, scratchy sore throat, aches, and headaches characterizes both pathogens. after a few days, rales may be heard, particularly in the bases where the infi ltrates tend to occur. these organisms have been associated with the initiation, promotion, and exacerbation of asthma in children. in addition, a pertussis-like illness with acute bronchitis has been described. a recent study has shown that nearly half of the cases of community-acquired pneumonia in children aged - years were associated with m . pneumoniae or c . pneumoniae . classic atypical pneumonias caused by these organisms are usually mild and self-limited. however, a number of studies have suggested that severe pulmonary infection may occur in otherwise healthy children. pleural effusions, pneumatocoeles, lung abscesses, pneumothoraces, bronchiectasis, chronic interstitial fi brosis, and acute respiratory distress syndrome although rare complications, have all been reported. serological testing is the most common means of diagnosis, but this is often retrospective. cultures obtained from swabbing the nasopharynx may take several days to grow. pcr techniques are currently being refi ned and standardized. treatment with antibiotics reduces the rate of recurrent wheezing episodes, decreases morbidity, and shortens the duration of symptoms. the organisms are susceptible to tetracyclines, macrolides, and quinolones. the optimal doses and duration of treatment is unclear; however, some data suggest that prolonged treatment for greater than weeks may be more desirable to decrease symptoms and eradicate the organism from the nasopharynx. chlamydia pneumoniae have an increased prevalence in older children. chest radiograph of year old female with streptococcus pneumoniae pneumonia. note the combination of consolidation and effusion affecting the right lung. (image provided courtesy of fa maffei) staphylococcus aureus is a gram-positive organism that can be found on the skin, nasal mucosa, and other mucus membranes. about - % of children are carriers. it is generally spread by direct contact or by respiratory particles. s . aureus is an unusual cause of lower airway disease in otherwise healthy children. it is more typically isolated from infants and young children with debilitating conditions. primary s . aureus pneumonia presents in the winter or early spring with a short febrile prodrome and a rapid onset of pulmonary symptoms. blood cultures are positive in - % of patients. secondary staphylococcal pneumonia will have a more prolonged prodrome with no seasonal predilection, but is often seen after infl uenza infections. as this secondary pneumonia is usually a result of hematogenous spread, blood cultures are positive in about % of patients. unilateral lobar disease is more typical with primary disease, while diffuse bilateral infi ltrates are more frequent with secondary pneumonia. effusions can be diagnosed in about % of children at presentation, but ultimately will develop in about % of cases. pneumatocoeles occur in up to - % of children. treatment is with nafcillin or oxacillin, but more organisms are becoming resistant and require therapy for serious or invasive disease with vancomycin, linezolid, daptomycin, or quinupristin-dalfopristin. methicillin resistant staphylococcus aureus (mrsa) was once considered to be restricted to hospitals and long-term care facilities. however, community acquired mrsa (ca-mrsa) is now a signifi cant cause of a variety of infections (including pneumonia) in children without prior health care facility exposure. the majority of community acquired mrsa infections involve minor skin and soft tissue infections, but invasive and sometimes fatal infections can occur in otherwise healthy individuals. ca-mrsa and healthcare-associated mrsa (ha-mrsa) can be distinguished by several important features. patients with ca-mrsa by defi nition have not had recent hospitalization (acute or chronic care), prolonged antibiotic use or chronic underlying disease. toxin production also distinguishes ca-mrsa from ha-mrsa. panton valentine leukocidin (pvl) is a toxin which is present in most ca-mrsa isolates, but rarely in ha-mrsa isolates. pvl toxin lyses white blood cells leading to leukopenia and a decreased ability to kill s . aureus . its production has been implicated as a contributor to the development of ca-mrsa necrotizing pneumonia. ca-mrsa isolates, unlike ha-mrsa, lack multi-drug resistance. ca-mrsa is generally more susceptible to clindamycin, trimethoprim-sulfamethoxazole and doxycycline than ha-mrsa, probably because ha-mrsa has developed resistance to survive in the healthcare setting. group a betahemolytic streptococcus (gabhs) is a gram-positive organism responsible for about % of pharyngitis and tonsillitis in children. it is rare as a primary cause of pneumonia. when it does occur, the children generally have high fever and appear toxic. the pneumonia is typically lobar. associated empyemas are common and pneumatocoeles may develop. there are several virulent toxin-producing gabhs m-serotypes that are associated with toxic shock syndrome. pre-existing varicella disease with disruption of skin and soft tissue as the port of entry is reported approximately - % of the time. an associated pneumonia occurs in - % of children with toxic shock syndrome. gabhs are highly susceptible to penicillins and cephalosporins. in cases of toxic shock, clindamycin is often added to inhibit the production of streptococcal pyrogenic exotoxins a (spe-a) and b (spe-b). about - % of infants with perinatally acquired group b streptococcus (gbs) infections will have pneumonia. the infant usually has systemic disease and blood cultures are frequently positive. late-onset gbs is predominantly caused by the type iii serotype. in these infants, the infection is usually manifest as bacteremia without a focus or with meningitis. pneumonia is rare in late-onset disease. gbs is uniformly sensitive to penicillin. while staphylococcus aureus pneumonia is uncommon, effusions ultimately develop in about % of cases and pneumatocoeles occur in - %. pertussis, or "whooping cough" is a highly contagious respiratory tract infection caused by the gram-negative pleomorphic bacillus bordetella pertussis and less commonly bordetella parapertussis . with the development and widespread use of a vaccine in the s, a significant and sustained decrease in incidence has occurred. however, despite immunization rates greater than %, cyclical recurrences of the disease have occurred every - years since the s. this is likely secondary to the waning of immunity in adolescents and young adults. under-immunized or unimmunized infants are the most vulnerable. nearly all deaths reported from pertussis occur in infants younger than months of age. pertussis is often divided into catarrahal (fever, rhinnorhea and initiation of cough), paroxysmal (severe coughing episodes, lymphocytosis, potential for complications) and convalescent stages (slow waning of cough over weeks to months). complications include secondary bacterial or viral pneumonia, apnea, malnutrition, pulmonary hypertension and neurologic involvement including seizures and encephalopathy. infants less than months of age are at highest risk for complications and mortality. characteristic paroxysms of cough with an end inspiratory whoop occur in children. infants may present with a nonspecifi c cough with associated apnea and cyanosis, without a whoop. adolescents may be asymptomatic or have only a mild prolonged cough. an increased white blood count up to , with a lymphocytosis is characteristic early in the course of the disease. the preferred test for laboratory confi rmation is the detection of b. pertussis dna by pcr assay. bacteriologic culture provides a defi nitive diagnosis. if administered during the early stages of the disease (fi rst - days of illness), erythromycin for days may decrease symptoms and reduce the risk of spread. a day course of azithromycin or a - day course of clarithromycin has been found to be as effective with less gastrointestinal symptoms. corticosteroids, bronchodilators, or intravenous immunoglobulins have not demonstrated effi cacy. supportive care with supplemental oxygen, mechanical ventilation, intravenous fl uids, maintenance of adequate caloric intake, and treatment of secondary bacterial infections are the mainstay of therapy. the use of extracorporeal membrane oxygenation in infants with hypoxemia, pulmonary hypertension and right heart failure refractory to conventional mechanical ventilation has resulted in poorer outcomes than expected. vaccination in infancy with booster doses in adolescence is preventative. about - % of pneumonias in children are caused by viruses. there is considerable evidence that viral infections often precede bacterial pneumonias and cause weakening of the host defenses. viral pneumonias with rsv and parainfl uenza are discussed in more detail in the bronchiolitis section. infl uenza is the main viral cause of pneumonia in school-aged children requiring hospitalization. there are three serotypes, a, b, and c which are further divided into subtypes based on the hemagglutinin and neuraminidase genes. hemagglutinin , , and and neuraminidase and typically infect humans. the gene segments for the surface glycoproteins are unstable, so mutations, called antigenic shift, occur regularly. epidemics occur annually during the winter months with a short, - day incubation period. the virus causes destruction of the ciliated respiratory epithelium within day of symptoms. airway edema and infi ltration with infl ammatory cells into the airway mucosa and epithelium follows. slow repair occurs over - weeks. a severe fulminating pneumonia may result in hemorrhagic exudates that contain many polymorphonuclear and mononuclear cells. destruction of the respiratory epithelium often leads to secondary bacterial infections. during the - infl uenza season, infl uenza-related deaths occurred in children; of these, % were less than years of age. forty-fi ve percent of the older children ( - years of age) did not have an underlying medical condition. rare complications of although death from infl uenza pneumonia is uncommon, a signifi cant number of the children that died were previously healthy. infl uenza include acute myositis, rhabdomyolysis, myocarditis, pericarditis, reye syndrome, encephalitis, transverse myelitis, and guillain-barré syndrome. children may present with an abrupt clinical course manifested by high fever, myalgias, headaches, scratchy sore throats, and dry cough. peripheral white blood counts are usually less than , . pulmonary infi ltrates often involve multiple lobes. bacterial co-infection, especially with mrsa, increases morbidity and mortality signifi cantly. rimantidine and amantadine can shorten the course for infl uenza type a disease by limiting viral replication, but only if given within the fi rst h of the disease. prophylactic dosing is - % effective and does not interfere with antibody production from the vaccine. both drugs have central nervous system and gastrointestinal side effects, including an increase in the incidence of seizures. oseltamivir and zanamivir have recently been approved for the treatment of infl uenza infections in children. they inhibit neuraminidase, an enzyme produced by infl uenza a and b. the course of disease in healthy adults can be reduced by - days, if started within h of the onset of symptoms. zanamivir is a dry powder aerosol that must be delivered by a special breath-activated device. bronchospasm in patients with asthma has been reported. aspirin or aspirin-containing products should be avoided due to the risk of reye syndrome. immunoprophylaxis is the most effective strategy for the prevention of infl uenza infection. inactivated vaccines have effi cacy rates from % to %. currently, the inactivated vaccine is recommended for all children older than months of age with high risk conditions including chronic pulmonary or cardiac disease, immunosuppressive disorders, sickle cell disease and other hemoglobinopathies, diseases requiring long-term aspirin therapy, chronic metabolic and renal diseases; healthy children aged - months; and household contacts over the age of months of high risk persons. a live, attenuated infl uenza vaccine was licensed in . it is administered by the intranasal route and is approved for healthy children aged - years. avian infl uenza viruses do not normally infect species other than birds and pigs. however, in , the fi rst human death from avian infl uenza occurred in hong kong in a year old with reye syndrome. subsequently, an epidemic occurred among humans in hong kong with close contact to live, infected poultry. the subtype h n appears to be the most ominous due to its ability to rapidly mutate and infect new species. the overall mortality rate is greater than %. the avian viruses are not believed to be transmissible from person-to-person, but some recent cases are being investigated for this possibility. children uniformly present with fever and cough. symptoms range from typical infl uenza-like symptoms to conjunctivitis to respiratory disease and failure. signifi cant laboratory data include leukopenia and thrombocytopenia. all children who developed pneumonia and progressed to ards died. diagnosis remains diffi cult, as no tests are widely available. of the antiviral drugs available for infl uenza a, the most recent h n strains in southeast asia are resistant to rimantadine and amantadine. therefore, treatment is mainly supportive. a prototype h n vaccine was made available to manufacturers in april , but production is diffi cult because the standard means of producing infl uenza vaccines from specially grown chicken eggs is not feasible. h n kills the embryo before enough viruses can be harvested for vaccine production. in april, , the centers for disease control confi rmed the emergence of a novel infl uenza a (h n ) virus with genes from swine viruses of the eurasian lineage and genes from avian infl uenza viruses. by june, , the fi rst infl uenza pandemic since was declared, affecting over countries and territories. in comparison to illnesses with seasonal infl uenza, the majority of cases occurred in individuals younger than years of age, with nearly half of the cases occurring in children under years of age . the clinical symptoms can be typical for infl uenza; fever, sore throat, cough, and muscle aches with the addition of vomiting and diarrhea in children. a wide range of complications although antiviral medications may attenuate the course of infl uenza when given early, immunoprophylaxis with vaccines is the most effective strategy for the control of infl uenza infections. avian infl uenza has occurred in epidemics among persons with close contact to live, infected poultry. all children with pneumonia that progressed to ards succumbed to the disease. have been reported that include mild-to-moderate (otitis media, sinusitis, myositis, and febrile seizures) to more severe complications such as myocarditis, rhabdomyolysis or encephalitis. severe complications may frequently involve invasive bacterial co-infection (i.e. mrsa) and/or exacerbation of underlying medical conditions in particular asthma. children who present initially with uncomplicated infl uenza may have rapidly progressive hypoxemic respiratory failure and multiorgan system dysfunction that is refractory to all therapies ( fig. - ) . of reported h n deaths, approximately % were in children. the majority of these children had comorbid asthma, neuro-developmental conditions, or obesity. an american academy of pediatrics work group identifi ed children at greatest risk for life-threatening h n infl uenza disease (table - ) . the centers for disease control has recommended prompt empiric antiviral therapy for infants, children, and adolescents of any age presenting with suspected or confi rmed h n infl uenza and any of the following conditions: illness requiring hospitalization ■ progressive, severe, or complicated illness, regardless of previous health ■ presence of signifi cant risk factors (see table ■ - ) the h n strain has been found to be resistant to amantadine and rimantadine, but is usually sensitive to neuraminidase inhibitors, specifi cally oseltamivir or zanamir. in , oseltamivir was emergently approved for treatment in children less than months of age. resistance to oseltamivir has been reported and is thought due to the h y mutation. interestingly, the mutation confers resistance to oseltamivir, but not to zanamivir. peramivir, a neuraminidase inhibitor, an unapproved (investigational) antiviral available in an intravenous formulation received an emergency use authorization permit from the fda for use in children with confi rmed severe refractory h n infl uenza. its use should be restricted to children that are not responding to either oral or inhaled antiviral drugs or if the parenteral route is the only dependable method of drug delivery. a vaccine was manufactured and licensed using the same standards as seasonal infl uenza by late . a single dose was found to provide adequate protection in children older than years of age, younger children requiring two doses separated by at least days. adenoviruses have been implicated in - % of pneumonias in children. adenoviruses are classifi ed into serotypes with types , , a, , and being the most common etiologic agents of lower respiratory disease and causing a severe necrotizing pneumonitis. these serotypes are associated with serious pulmonary sequelae, such as bronchiectasis, bronchiolitis obliterans, unilateral hyperlucent lung, and persistently abnormal pulmonary function tests. adenovirus infections peak between months and years of age. mortality from severe respiratory infections can be high, because the disease often involves multiple organ systems. survivors may have permanent lung injury often in the form of bronchiolitis obliterans. in the immunocompromised host, mortality rates are as high as - %. cidofovir has in vitro activity against adenovirus, but proof of effi cacy is limited. therapy is supportive. severe acute respiratory syndrome is a newly described pulmonary infection caused by a novel sars-associated coronavirus. sars-cov is highly contagious and was coined "the fi rst plague of the twenty-fi rst century". the disease rapidly spreads among household contacts and healthcare personnel. children less than years of age account for only approximately % of those affected, with a mean age of years. no deaths were reported among children in the outbreak. children and adults present with fever, malaise, cough, coryza, chills or rigor, sputum production, headache, myalgia, leukopenia, lymphopenia, thrombocytopenia, mildly elevated activated partial thromboplastin times, and elevated levels of lactate dehydrogenase. radiographs of the chest show non-specifi c infi ltrates. apart from diarrhea, patients have minimal extrapulmonary symptoms. early diagnosis by reverse transcription-polymerase chain reaction (rt-pcr) can be made with % sensitivity on nasopharyngeal aspirates within the fi rst days of the illness. the clinical course follows a triphasic pattern. there is an incubation period of - days with a prodrome of high fever, chills, malaise, headache, and myalgias. diarrhea occurs in up to % of adults. after - days, the disease progresses to involve the lower airways with a dry non-productive cough and dyspnea. in - % of cases, acute respiratory distress syndrome (ards) follows and often patients require mechanical ventilation. deaths occur from respiratory failure. young children run a milder and shorter biphasic clinical course. cough is found in approximately half the children, and crackles are rarely heard despite radiographic evidence of infi ltrates. a regimen of antibiotics, ribavirin, and corticosteroids was proposed based on initial anecdotal success. however, ribavirin has demonstrated minimal activity against sars-cov isolates in vitro . non-randomized studies of corticosteroids have reported favorable outcomes. a pediatric series of children with confi rmed sars treated with ribavirin and corticosteroids showed no adverse effects and all survived. mortality from adenovirus infections remains high because of multiple organ system involvement. sars rarely affects children, and when it does, morbidity is less, with no reported mortalities. . neurological disorders, such as epilepsy, cerebral palsy, developmental delay and neuromuscular disorders . chronic respiratory diseases associated with impaired pulmonary function and/or diffi culty handling lung secretions, moderate and especially severe persistent asthma, technology-dependent children (e.g., those requiring oxygen, tracheostomy, or a ventilator) . primary immunodefi ciencies or conditions that require medications or treatments that result in secondary immunodefi ciencies . congenital heart disease . metabolic (e.g., mitochondrial) or endocrine disorders, especially if cardiopulmonary function is impaired adapted from http://www.aap.org/new/swinefl u.htm hantavirus cardiopulmonary syndrome is a viral zoonotic disease that affects healthy children and adolescents who are exposed to aerosols of rodent excreta. the deer mouse is the main rodent reservoir. most cases occur in the southwestern united states, but cases have been confi rmed in states. hcps presents with a prodrome of fever, chills, myalgia, headache, and gastrointestinal symptoms. respiratory compromise requiring supplemental oxygen generally occurs within h. the disease can progress to respiratory distress and ards. the majority of deaths result from hypoxemia and cardiac dysfunction with marked hypotension and ventricular arrhythmias. in adults, the case fatality rate is approximately %. a recent case series of children aged - years, revealed that % of infected children developed hcps, % died, and % were critically ill and required mechanical ventilation. treatment is supportive as ribavirin has not been proven to reduce mortality. extracorporeal membrane oxygenation was used on two patients, one of which survived. laboratory evaluation reveals thrombocytopenia, leukocytosis, and circulating immunoblasts. an elevated prothrombin time of ³ s is predictive of severe disease. no deaths were reported in children younger than years of age. diagnosis can be made by detection of hantavirus-specifi c immunoglobulin m, hantavirus-specifi c rna by polymerase chain reaction, or hantavirus antigen by immunohistochemistry. respiratory infections in children with primary or acquired immunodefi ciencies requiring intensive care are not uncommon. these infants and children are susceptible to many organisms that are rarely pathogenic in a normal host. primary immunodefciencies include abnormalities or defi ciencies in immunoglobulins and antibodies, t and b cells, phagocytes, natural killer cells, and complement. acquired immunodefi ciencies include asplenia, human immunodefi ciency virus (hiv), corticosteroid therapy, and immunosuppresion used for marrow or solid organ transplants. immunocompromised children can present with attenuated signs and symptoms of respiratory infections. in addition to physical examination and chest roentgenograms, these children often require chest computed tomography to better delineate the extent of disease. bronchoalveolar lavage, needle aspiration, or lung biopsies might be required to make a defi nitive diagnosis. pulmonary specimens should be tested for common bacteria as well as for pneumocystis carinii, acid-fast bacilli, nocardia, legionella, crytococcus, aspergillus, candida, histoplasma, coccidioides, and blastomyces. viruses such as cytomegalovirus, varicella, herpes virus, and measles should be considered. pneumocystis carinii (now known pneumocystis jiroveci ) is an opportunistic pulmonary pathogen in infants and children with human immunodefi ciency virus (hiv) and other primary immunodefi ciencies, malnutrition, hematological malignancies, solid organ and bone marrow transplant recipients, and patients on high dose corticosteroid therapy for infl ammatory and collagen-vascular diseases. it is a unicellular organism that exists as a cyst (the diagnostic form). the organism attaches to the type i alveolar cells resulting in an alveolitis characterized by ventilation-perfusion mismatch and decreased pulmonary compliance. if untreated, pcp carries a mortality rate of - %, and nearly % in the hiv-seropositive child. fortunately, the incidence has markedly decreased with the administration of chemoprophylactic agents to high risk patients. children typically present with fever, tachypnea, non-productive cough, and hypoxia with an absence of rales on auscultation of the chest. initially, they may have an elevated ph and low carbon dioxide levels. lactate dehydrogenase levels are generally elevated. bilateral diffuse alveolar infi ltrates are seen with initial hilar involvement subsequently spreading to the periphery (fig. - ) . diagnosis is made by demonstrating the organism with the methenamine silver nitrate stain on pulmonary tissue, respiratory secretions, or lung fl uid. bronchoalveolar lavage is the most widely used technique to obtain lung fl uid for diagnosis. treatment consists of supportive therapy hantavirus is rare in infants and school-aged children. no deaths have been reported in children less than years of age. with supplemental oxygen; ultimately continuous positive airway pressure or mechanical ventilation may be necessary if respiratory failure occurs. trimethoprim-sulfamethoxazole (tmp-smx) is the recommended initial treatment. in patients that cannot tolerate tmp-smx, then pentamidine isoethionate should be used. corticosteroids in anti-infl ammatory doses as an adjunct to antimicrobial therapy have improved clinical outcomes. concurrent pulmonary infections were found in % of patients, most frequently bacterial or cytomegalovirus pneumonia. determination of the etiologic agent in pneumonia is diffi cult. fortunately, in most community-acquired pneumonias, identifi cation of the specifi c causative organism is not critical. however, in children with a complicated course that fails to respond to standard therapies, defi nitive diagnosis of the etiologic agent is essential. complete blood counts, infl ammatory markers, and chest radiographs do not differentiate the causative agents for pneumonia. blood cultures are rarely positive outside of the neonatal period. rapid antigen tests are available for rsv, parainfl uenza, infl uenza, and adenovirus. nasopharyngeal swabs for viral cultures generally take - days to become positive, and in one study, % of the patients had been discharged prior to the positive results. older children and adolescents might be able to produce sputum for gram stain and culture. an adequate specimen should contain more the leukocytes and fewer than squamous epithelial cells per low-power fi eld. in the intubated patient, sputum can be more easily acquired. however, interpretation of the results of gram stains and cultures is at times diffi cult in differentiating colonizing from pathologic organisms. colonization of the endotracheal tube may occur as early as h, but most frequently between and h. the oropharynx becomes colonized within h, the stomach at - h, and the lower respiratory tract between and h. in addition, a comparison of infectious agents isolated by both tracheal aspirates and bronchoalveolar lavage found only % concordance. bronchoalveolar lavage (bal) can be safely used to obtain secretions from the lower airways for gram stain and culture. it is especially useful in the diagnosis of pneumonia in the immunocompromised child. however, bal performed directly through the bronchoscope carries a risk of contamination. the smallest bronchoscope that can accommodate a protected specimen brush is . mm and requires a . mm endotracheal tube for passage. the smallest fl exible fi beroptic bronchoscope with a suction channel has an external chest radiograph of severe pneumocystis carinii pneumonia in a month old male with combined immunodefi ciency. note the diffuse alveolar involvement and air bronchograms. (image provided courtesy of fa maffei) diameter of . mm and is too small to admit a double-sheathed brush. non-bronchoscopic double-lumen plugged catheters can be inserted blindly through the endotracheal tube to obtain a non-contaminated specimen. the sensitivity and specifi city of these samples are similar to those obtained by a bronchoscopic guided protected specimen. transthoracic needle aspirations are performed in some centers with good results. one study reported a diagnostic success rate in % of patients. the incidence of pneumothorax was approximately %, but none required subsequent placement of a pleural drainage catheter. a lung biopsy is rarely needed to make a defi nitive diagnosis. supportive treatment with oxygen and intravenous fl uids are often standard therapies. as both pneumonia and mechanical ventilation can cause an elevation in anti-diuretic hormone levels, careful fl uid monitoring is essential to avoid overhydration, excessive lung water and hyponatremia. initial antibiotic choices should be empiric and based upon the likely organisms for each age group, because of the diffi culty in identifying the causative agent. the child's respiratory status including respiratory rate, work of breathing, pulse oximetry, and central nervous system response should be closely monitored. non-invasive bi-level positive airway pressure (bipap) has been effective for use in children with mild to moderate respiratory insuffi ciency, defi ned as an a-a gradient > and < or pao /fio ratio < but > mm hg. serial evaluation of mask-face contact areas is essential to avoid skin breakdown. children with moderate or severe respiratory insuffi ciency often require intubation and mechanical ventilation. children with respiratory failure secondary to pneumonia often require increased positive end expiratory pressure (peep), increased inspiratory time, and aggressive pulmonary toilet to recruit alveoli. for patients requiring high levels of peep, adequate sedation is often required to prevent patient/ventilator asynchrony and barotrauma. spontaneous respirations should be encouraged while on mechanical ventilation. rarely, the use of neuromuscular blockade is required to allow mechanical ventilation. prone positioning may improve ventilation/perfusion (v/q) mismatching in dependent lung regions. lung protective strategies allowing permissive hypercapnea with small lung volumes to ventilate and appropriate peep to maintain alveolar recruitment is recommended for children with pneumonia. high frequency oscillatory ventilation can also be utilized to maintain mean airway pressure and alveolar recruitment. airway pressure release ventilation (aprv) provides recruitment of alveoli while allowing spontaneous respirations. in children with severe respiratory distress syndrome, treatment with bovine surfactant may improve oxygenation. extracorporeal life support continues to have a role in children with reversible severe acute hypoxemic respiratory failure refractory to mechanical ventilation. pneumonias can often be complicated by the development of pleural effusions and empyemas. these occur when the fl uid production by the interstitial lung tissue exceeds the maximum pleural lymphatic fl ow. parapneumonic effusions often occur from pneumonia as white blood cells and other debris of infection block the lymphatics resulting in elevation of protein in the pleural space, increase in colloid osmotic pressure, and consequent failure of fl uid reabsorption. on physical exam, the child will have decreased breath sounds over the effusion. in older children, auscultatory percussion changes might be appreciated. plain chest radiographs can reveal most clinically signifi cant effusions. ultrasound and chest computed tomograms are useful in determining the volume and quality of the fl uid and the presence of loculations. simple parapneumonic effusions or transudates can also be differentiated from exudates by using the criteria of light et al. (table - ). a pleural fl uid ph less than . indicates a complicated effusion that is likely exudative and requires drainage whereas a pleural fl uid ph more than . suggests that the effusion may be managed with systemic antibiotics alone. complicated parapneumonic effusions or empyemas occur when the fl uid becomes purulent. during this stage, the effusions undergo a fi brinopurulent stage with many polymorphonuclear leukocytes, bacteria, and cellular debris entering the fl uid. fibrin is deposited over the pleural surfaces and loculations begin to form. the ph and glucose levels fall as the ldh levels rise. if untreated, they often progress to a third organizing stage in which the exudate non-invasive bipap ventilation can be effective for children with moderate respiratory insuffi ciency. develops into an inelastic, fi brotic peel that restricts the lung. simple parapneumonic effusions usually resolve with thoracentesis or tube thoracostomy and antibiotic treatment of the pneumonia. more complicated parapneumonic effusions have been successfully treated with thoracotomy tubes and fi brinolytics. however, although risks for bleeding are reportedly low, this therapy requires close monitoring of chest tube drainage and instillation of expensive medications with intermittent clamping of the chest tube. no single recommendation for the choice of fi brinolytic agent or dosage has been established. also, if tried late in the organizing phase, this is often unsuccessful due to loculations and the high viscosity of the purulent fl uid. surgical debridement either by open procedure or by video-assisted thorascopic surgery (vats) is often needed for organizing, complicated parapneumonic effusions. multiple studies have reported that early vats or thoracotomy for empyema leads to a shorter hospital stay. the treatment modality is best determined by the temporal stage and nature of the effusion. acute pulmonary infections are common diagnoses that require admission to the pediatric intensive care units. understanding the pathophysiology of lower respiratory infections enables the intensivist to tailor therapy to the individual child and pathogen. early establishment of a specifi c etiology and the selection of the correct treatment plan directly impacts clinical outcome. video-assisted thorascopic surgery (vats) for the treatment of empyemas has been associated with shorter hospital stay. which of the following therapies have been proven to be a consistent benefi t for rsv bronchiolitis? a. aminophylline b. bronchodilators c. corticosteroids d. ribavirin e. supportive care . palivizumab is indicated for which of the following children? a. a month old, former week premature infant who just underwent surgical repair of a large ventricular septal defect who received palivizumab weeks ago b. a month old, former week premature infant with mild bronchopulmonary dysplasia who received palivizumab weeks ago c. a month old, former week premature infant with peripheral pulmonic stenosis who has never received palivizumab d. a month old full term infant with a urea cycle defect who has never received palivizumab e. an month old, former week premature infant with bronchopulmonary dysplasia who received his fi fth dose of palivizumab a month ago pleural fl uid may be classifi ed as exudative, if one or more of the following criteria are met: ■ pleural fl uid protein divided by serum protein > . (sensitivity %, specifi city %) ■ pleural fl uid lactate dehydrogenase (ldh) divided by serum ldh > . (sensitivity %, specifi city %) ■ pleural fl uid ldh is more than two-thirds of the upper limit of normal for serum ldh (sensitivity %, specifi city %) adapted from light ( ) human bocavirus and acute wheezing in children american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis. diagnosis and management of bronchiolitis the yield of fl exible fi beroptic bronchoscopy in pediatric intensive care patients immunological mechanisms of severe respiratory syncytial virus bronchiolitis human metapneumovirus infection in young children hospitalized with acute respiratory tract disease: virologic and clinical features a multicenter, randomized, controlled trial of dexamethasone for bronchiolitis natural infection of infants with respiratory syncytial virus subgroups a and b: a study of frequency, disease severity, and viral load the use of albuterol in hospitalized infants with bronchiolitis advances in the treatment and prevention of severe viral bronchiolitis the presence and sequence of endotracheal tube colonization in patients undergoing mechanical ventilation palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically signifi cant congenital heart disease systemic corticosteroids in infant bronchiolitis: a meta-analysis drainage, fi brinolytics, or surgery: a comparison of treatment options in pediatric empyema does vats provide optimal treatment of empyema in children? a systematic review intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children clinical picture, diagnosis, treatment, and outcome of severe acute respiratory syndrome (sars) in children noninvasive therapy with helium-oxygen for severe bronchiolitis pleural effusion pleural effusions: the diagnostic separation of transudates and exudates nebulized % hypertonic saline solution treatment in hospitalized infants with viral bronchiolitis development of wheezing disorders and asthma in preschool children heliox therapy in infants with acute bronchiolitis nasal continuous positive airway pressure with heliox versus air oxygen in infants with acute bronchiolitis: a crossover study diagnosis and management of pneumonia in children community-acquired pneumonia in children selected populations at increased risk from respiratory syncytial virus infection human metapneumovirus and human bocavirus in children mixed respiratory virus infections mycoplasma pneumoniae and chlamydia pneumoniae cause lower respiratory tract disease in paediatric patients children's hospital respiratory syncytial virus database: risk factors, treatment and hospital course in infants and young children infection with sin nombre hantavirus: clinical presentation and outcome in children and adolescents ribavirin for respiratory syncytial virus lower respiratory tract infection: a systematic overview risk of bacterial infection in previously healthy respiratory syncytial virus-infected young children admitted to the intensive care unit infl uenza in pediatric intensive cure unit do bronchodilators have an effect on bronchiolitis? comparison of conventional viral cultures with direct fl uorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease respiratory syncytial virus in early life and risk of wheeze and allergy by age years h n infl uenza the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis bench-to-bedside review: ventilator strategies to reduce lung injury -lessons from pediatric and neonatal intensive care etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods a multicenter, randomized, double-blind, controlled trial of nebulized epinephrine in infants with acute bronchiolitis respiratory syncytial virus and other respiratory viruses effect of exogenous surfactant (calfactant) in pediatric acute lung injury. a randomized controlled trial current concepts on pulmonary host defense mechanisms in children nebulized hypertonic saline solution for acute bronchiolitis in infants bronchiolitis: recent evidence on diagnosis and management key: cord- -afgvztwo authors: nan title: engineering a global response to infectious diseases: this paper presents a more robust, adaptable, and scalable engineering infrastructure to improve the capability to respond to infectious diseases.contributed paper date: - - journal: proc ieee inst electr electron eng doi: . /jproc. . sha: doc_id: cord_uid: afgvztwo infectious diseases are a major cause of death and economic impact worldwide. a more robust, adaptable, and scalable infrastructure would improve the capability to respond to epidemics. because engineers contribute to the design and implementation of infrastructure, there are opportunities for innovative solutions to infectious disease response within existing systems that have utility, and therefore resources, before a public health emergency. examples of innovative leveraging of infrastructure, technologies to enhance existing disease management strategies, engineering approaches to accelerate the rate of discovery and application of scientific, clinical, and public health information, and ethical issues that need to be addressed for implementation are presented. powerful antibiotics and vaccines helped mitigate the threat from infectious diseases for several generations. in , most human deaths were associated with infectious diseases like tuberculosis and influenza. as recently as , the worldwide mortality associated with infectious diseases was . percent of deaths from all causes. in , this had continued to decline to . percent. unfortunately, this means there were still over million deaths associated with infectious diseases [ ] . a recent review examined antimicrobial resistance and predicted that by , the impact would include a reduction of the world's potential gross domestic product by % to . % and cause an additional million premature deaths a year [ ] . although beyond the scope of this paper, it is worth noting that microorganisms have also been implicated as contributing to or causing many chronic diseases, including some forms of cancer, arthritis, and neurological disease [ ] . it is tempting to approach the infectious disease challenge as doing battle with a pathogen enemy where brigades of combatant bacteria or viruses are held back or even defeated by increasingly sophisticated pharmaceutical weapons. there is certainly a place for improved pharmaceuticals; however, a sustainable approach will need to be much more sophisticated. microbes and their hosts form a complex and dynamic ecosystem, and a long-term strategy for infectious disease control must take into account the fact that diseases can result from changes in the microbe, the host, or the environment. it is time to move beyond the simple war metaphor [ ] . to compound the challenge, microbes are notoriously fast in adapting to new environments. this can include bacteria developing antibiotic resistance or acquiring metabolic traits that allow them to thrive in a new environmental niche, and viruses evolving to reduce the effectiveness of antivirals and vaccines. in addition to the evolution of existing pathogens, like the seasonal influenza virus, there are emerging pathogens that are often the result of changing or encroachment upon new ecosystems and the ''leap'' from a conventional host to a new host species. an example from recent headlines is the middle east respiratory syndrome coronavirus. mers-cov appears to have reached humans by direct contact and potentially airborne transmission through animal hosts including camels [ ] . the mers-cov is related to the coronavirus that caused severe acute respiratory syndrome (sars). the sars outbreak began in and likely spread to humans via bats [ ] . although the viruses are similar, this does not guarantee that utilization of the same medical and public health intervention techniques will be effective. there are many examples of emerging infectious disease outbreaks, including the on-going hiv/aids pandemic that has already caused million deaths [ ] . each pathogen involved in an infectious disease outbreak provides an opportunity to identify what scientific data are needed to support effective interventions. quoted in the global health security agenda [ ] , u.s. president obama said in ''. . . we must come together to prevent, and detect, and fight every kind of biological dangervwhether it's a pandemic like h n , or a terrorist threat, or a treatable disease.'' the agenda complements and supports existing international health regulations of the world health organization [ ], u.s. public health [ ] , [ ] , and biodefense objectives [ ] - [ ] . the framework for data requirements and response priorities used in this paper integrates across these initiatives and regulations. specifically, in order to manage infectious diseases, capabilities are required for: preparedness, detection, characterization, response, and support for the return to normal, see fig. . this framework is analogous to homeostasis in living organisms. these capabilities are relevant for addressing health interests from the global to the individual organism, e.g., human, animal, plant, or bacterium. the global perspective is studied and implemented by public health, ecological, industrial, and other communities with the principal foci of public benefit, humanitarian needs, scaling, and statistical measures. for an individual human, the perspectives are from medical, economic, relationship, and other personal priorities with the foci of individual health, quality of life, and gaining access to effective care. integrating frameworks are needed to support optimization of technical, economic, medical, and ethical components of this complex system. infectious diseases are a major cause of death and economic impact worldwide. a more robust, adaptable and scalable infrastructure would improve the capability to respond to epidemics. because engineers contribute to the design and implementation of infrastructure, there are opportunities for innovative solutions to infectious disease response within existing systems that have utility, and therefore resources, before a public health emergency. examples of innovative leveraging of engineered infrastructure are provided throughout the paper. the next section of this paper discusses opportunities for technology to improve on current approaches to infectious disease management and the following section discusses engineering challenges to accelerate the application of science to infectious disease planning and response at the global scale. i conclude with a brief discussion of the importance and opportunity for engineers to address the ethical issues needed to leverage traditional infrastructure for infectious disease response and help nurture a global culture of responsibility in both healthcare and technical applications. leveraging their significant investment in planning and response experience, i adopted from the u.s. pandemic influenza plan [ , p. g- ], infectious disease management goals to provide: public health policy-makers with data to guide response, and clinicians with scientific data to justify recommended treatments, vaccines, or other interventions. i have integrated priorities from the influenza plan with a more pathogen-centric approach from the food industry [ ] in table to provide descriptions of priority data to support infectious disease outbreak response. health have parallels shown in the inner and outer rings, respectively. because traditional diagnostics and treatments have long lead development, regulatory approval, and manufacturing lead times, it is challenging to provide timely and effective interventions at a public health scale for an outbreak caused by an emerging or novel pathogen. approaches to achieving robust, economically viable scaling include improved leveraging of existing infrastructure, establishment of an integrating framework like the digital immune system for optimization, and spiral development processes similar to homeostasis. these data can be provided with currently available technologies. however, there are several recurring issues that inhibit global utilization. the issues that can be addressed, even if only partially, by technology are discussed. the key recurring issue is availability. limited availability is driven by many factors including cost, appropriate sharing of data and materials, and timely manufacture and distribution. on a more basic level, one of the key factors in sustainable preparedness is infrastructure, and availability is a challenge here as well. malnutrition due to starvation, unsafe water, and insufficient sanitation all impact infectious disease mortality. in , over half of the deaths of children under five years old were associated with infectious diseases and the significant contributing factor for many of these deaths was under nutrition [ ] . building the infrastructure to eliminate these hazards has historically been the domain of civil and agricultural engineers. electrical and computer engineers are now providing valuable low-cost information linkages across systems so that weather satellite data can be utilized to help increase local crop yields and prepare water treatment and sanitation plants for adverse weather. the computational algorithms and information networks can be applied worldwide for irrigation and weather prediction for storm and drought management [ ] . remote sensing has been utilized to indirectly detect vibrio cholera [ ] and predict a rift valley fever (rvf) outbreak [ ] . in both of these examples, the disease and environmental biology were shown to correlate with changes that could be measured by air and space borne sensors. for v. cholerae, sea-surface temperature and sea height were linked to the inland incursion of water with commensal plankton. satellite measurements of seasurface temperature, rainfall, and vegetation changes were used to predict the areas where outbreaks of rvf in humans and animals occurred in africa. the techniques and data used for rvf may be more broadly applicable to other vector-borne diseases. in regions with limited infrastructure, it is often difficult or impossible to provide the refrigeration required to maintain the ''cold chain'' for life-saving vaccines and other medicines. an inspirational consortium of industry, churches, and nonprofits in zimbabwe, africa, leveraged the reliable power requirements of cellphone towers to help address the refrigeration storage needs for many vaccines [ ] . the initiative has included innovative contributions by wireless providers, refrigerator manufacturers, and others in order to help provide immunizations against polio, measles, and diphtheria. another area in which technology is poised to impact infectious disease management is in gaining timely situational awareness of outbreaks. global and regional travel often make this a difficult task, and data collection and sharing for epidemiologists, care providers, patients, and the public is also limited by other factors such as privacy concerns for individual patients' medical data, governmental goals to protect tourism and other local-to-national interests, the lack of recognized standards for sharing protected data, and an accepted international norm for transfer of public and commercial material during and in response to an infectious disease. improved network, encryption and access information technologies are also necessary to support managed care organizations and telemedicine applications. a global system addressing these issues and capable of operating on time scales relevant to controlling an epidemic is needed. a comparison of five outbreak detection algorithms was conducted using a surveillance case study of the seasonal ross river virus disease [ ] . challenges were identified for making quantitative comparisons of the algorithms as well as in evaluating the performance of each algorithm. a network model has been proposed that has the potential to address algorithm shortcomings for outbreak localization and performance under changing baselines [ ] . this is accomplished through modeling the relationships among different data streams rather than only the time series of one data stream compared to its historical baseline. using measured and simulated data, this approach showed promise for addressing shifts in health data that occur due to special events, worried well, and other population shifts that happen during significant events like pandemics. another study compared animal and public health surveillance systems, finding challenges due to a limited number of common attributes, unclear surveillance objectives of the design, no common [ ] . privacy issues pose challenges that are difficult to address with technology, but they have been addressed in some applications through voluntary enrollment. for example, the geographic location capability in many cell phones allows applications to push public health and animal disease outbreak information to users based on location. the u.s. centers for disease control and prevention (cdc) has the fluview application that provides geographic information on influenza-like illness activity [ ] . obviously, the pervasiveness of cell phones improves timely reporting from the field for both the public and the public health profession. moving forward, addressing privacy issues will be critical so that geographic tracking of a phone's location could be used to help inform an individual of potential contact with infected persons or animals and support automated, anonymous, electronic integration of those data to accelerate the epidemiological detective work of identifying and surveying those same individuals for public health benefit. electronic health information systems have made significant progress. however, even as recently as , it was noted: ''despite progress in establishing standards and services to support health information exchange and interoperability, practice patterns have not changed to the point that health care providers share patient health information electronically across organizational, vendor, and geographic boundaries. electronic health information is not yet sufficiently standardized to allow seamless interoperability'' [ ] . the u.s. has taken a risk-based approach to health information technology (it) regulation [ ] . safety in health it has been recognized as part of a larger system that needs to consider not just specific software, but how it interacts with the it system and how it will be used by clinicians [ ] . continued development of quality management, human factors, and other standards to support usability and regulatory review are needed. as the large-scale systems that require computer algorithms to scan and integrate data into summary reports continue to progress, significant benefit is being derived from systems with fairly simple technology. in , promed was started as the first e-mail reporting system with curation. it has grown to over , subscribers in over countries and has roles in outbreak detection including sars in [ ] . there are many health alert networks (han) that utilize websites and electronic communications [ ] , [ ] . a study in new york city showed that most physicians ( %) received health department communications, but less than half of those ( %) received the information through the han [ ] . an important trend is that % prefer e-mail distribution of communications and this preference trends with younger respondents. looking to the future, achieving the benefits of distributed diagnostics and electronic reporting will depend on both technical and clinical integration. this would improve individual care as well as reduce or eliminate separate data entry and reporting for public health surveillance. in order to realize the benefits of personalized medicine with treatment customized to the individual, the costs, scalability, and compatibility across all data sources must be addressed. personalized infectious disease medicine might include customization of antimicrobials to an individual patient to improve care and help reduce overuse and misuse of antibiotics. similarly at the global health scale, timely identification of appropriate virus strains coupled with rapid manufacturing for seasonal influenza vaccination would reduce the disease burden of thousands. in personalized and global applications, the approaches will need to move from diagnostics, characterizations, and interventions aimed at a single specific disease causing pathogen to robust methods that can be adapted for safe and effective use in a timely manner for broad classes of disease. fully realizing these goals will require improved scientific understanding and new engineering and computational approaches. there are significant challenges in utilizing traditional engineering approaches in the life sciences. living organisms are complex by most machine standards. individual organisms are also typically influenced by peer organisms forming a community, by other living organisms that may be beneficial, neutral or detrimental, and by the environment. there are opportunities for engineers to develop improved measurement, analysis, and model systems to better characterize, predict and manage infectious diseases. given the complexity of these interacting systems, there are significant challenges to the reductionist approaches familiar to design-based engineering. fortunately, as the history of vaccination demonstrates, a comprehensive knowledge of the biology is not always required in order to provide healthcare benefits. with the advent of deoxyribonucleic acid (dna) sequencing and the efficient detection and laboratory replication of dna through polymerase chain reaction (pcr) amplification, there are opportunities to organize scientific and medical data using dna-based indices. the field that has grown up around these technologies, genomics, is an excellent example of how engineering can enable profound advances in biological research. a significant contributor to the organizing principles and demonstrator of this dnabased approach, carl woese, summarized in the impact as providing ''a new and powerful perspective, an image that unifies all life through its shared histories and common origin, at the same time emphasizing life's incredible diversity and the overwhelming importance of the microbial world,'' [ ] . here, i build on this insight as well as our previous assessment in of the engineering contributions and opportunities related to dna sequence fitch: engineering a global response to infectious diseases data that describe an organism's genome, transcriptome and proteome [ ] . as depicted in fig. , nucleic acid sequence provides a common framework to organize data related to infectious diseases. today's dna sequencing instruments can produce terabases of data in a few days with per-base costs a million-fold lower than a decade ago. recent studies have demonstrated the power of personalized approaches to major human diseases like cancer. it appears likely that the next few years will bring the personalized human genome and the miniaturized sequencing instrument, each for less than one thousand dollars. now is the time to innovate and apply the engineering approaches needed to utilize these and other data. dna sequencing and the associated bioinformatics tools for analysis provide a powerful methods-based approach to monitoring living systems. dna and ribonucleic acid (rna) provide data that help characterize an individual's health status as well as the status of the surrounding environment. because sequencing converts biological information to digital data, computer networks, data management, integrity, scaling, analysis, privacy, and affordability are keys to expanding access. the ''digital immune system'' is a powerful concept that generalizes the method to population dynamics and public health [ ] . basing the system on dna allows correlation techniques to identify patterns in the sequence datavrecurring and deviate patterns. these patterns can be indicative of healthy or pathologic host status as well as the absence or presence of pathogens in clinical and environmental samples. the growth of sequence databases with appropriate clinical and environmental metadata will improve the potential quality of the analyses and the impact on individual and public health. growing databases will also need to address the scaling and privacy issues. one of the most powerful features of the digital immune system approach is the potential to detect a novel pathogen, i.e., one that is not already in the database. the flexibility inherent in this methodbased approach is a huge strength and distinguishes it from many of the traditional methods which are not able to detect or characterize a new or emerging pathogen. seasonal influenza provides an example of a system of method-based opportunities. the influenza virus changes genetically as it uses an error-prone enzyme to replicate its genome, and mutates further as it migrates from host-tohost, across speciesve.g., waterfowl to humans, and across ecosystems of the environment. this mutation process gives rise to a different population of viruses in circulation each year, and poses a challenge to vaccine manufacturers, as one year's vaccine will typically have little value when confronted with the next year's viral strains. each year's vaccine is constructed specifically to protect against the strains that are projected to be dominant in the upcoming flu season. the current process uses egg-based techniques for manufacturing influenza vaccine and has been successfully utilized for decades as have the techniques for isolating and identifying emerging strains of the virus. unfortunately, the combined pipeline is relatively slow and does not typically allow for vaccine manufacturing to be based on strains that have been detected at the start of the flu seasonvvaccine production must be started earlier, and so relies heavily on imperfect predictions as to which strains of influenza will dominate. rna sequence data provide a method-based framework for managing influenza response at the global scale. in addition to the detection and identification of the virus, sequence data can be utilized to compare and predict the performance of egg and other manufacturing approaches. as the sequence, clinical, animal, and environmental data are accumulated, there is also the potential to support computational safety and efficacy screening. shortening the current timeline from detection through vaccination would have significant positive health benefits. there are method-based approaches to vaccine manufacturing with significant potential to improve upon the egg-based approach. even though the influenza virus has only eight genes and an ominous history of multimillion death pandemics, the scientific understanding is not yet sufficient to avoid the thousands of deaths annually from seasonal influenza nor to mitigate the potential from a pandemic strain. complex samples to be converted to nucleic acid sequence data that are easily represented in a digital computer. these data can be used as a framework or index for health and disease related metadata supporting correlative studies across species and providing insight into infectious diseases. because genetic material is traded among organisms and is often part of complex nonlinear networks within an organism and beyond, increased collection and interpretation of the associations across dna sequence and metadata are needed. a digital immune system for individuals and populations is envisioned that identifies causation and intervention options to support patient-specific and public health interventions. biocontainment laboratories are needed to safely conduct the research needed to understand pathogens as well as analyze clinical samples. characterization of existing pathogens increases understanding of current diseases and also helps to prepare for emerging diseases. laboratories that work with infectious agents are categorized by biosafety level (bsl) ranging from a basic biomedical laboratory (bsl- ) to bsl- laboratories (fig. ) that can safely handle untreatable disease agents [ ] . the engineering systems for automatically controlling airflow and other facility safety components are critical for supporting clinical and research laboratories. infectious disease research is benefiting from ''omic'' methods for characterizing proteins (proteomics), metabolites (metabolomics), messenger rna (transcriptomics), etc. in order to safely produce genomic data more quickly, dna sequencing instruments have been moved into our biocontainment laboratories. these methods produce large volumes of data, requiring research and clinical labs to have access to traditional engineering disciplines in data management and analysis. data management is not the only challenge. so far, our descriptions have implied a single host interacting with a single invading pathogenvthe war metaphor. it is not that the metaphor does not work in many cases. for instance, the eradication of smallpox was accomplished through an aggressive worldwide campaign as was the animal and livestock disease rinderpest [ ] . it is that the war metaphor is an oversimplification with an often unrealizable aspiration for victory. consider that most hosts, including bacteria, have associated pathogens and symbiotic microbes. for example, a tropical grass, fungus, and virus have been found to have a three-way symbiosis that confers heat tolerance [ ] . when the virus is removed from the fungus, thermal tolerance is lost by the grass. if the virus is reintroduced to the fungus, thermal tolerance is conferred to the grass. viruses can also integrate their genes into animal genomes as part of the animal's nuclear dna affecting inherited traits [ ] . virus infection is one of several naturally occurring changes in the nucleic acid of a genome. even simple nucleic acid transfers among different biological entities often have difficult to predict results. the complexity compounds as the number of entities increases and community behaviors emerge. for each of the approximately one trillion human cells in our bodies, there are about ten microbes. the microbes are distributed in different, highly specialized, communities in the gut, mouth, skin, etc. collectively referred to as the microbiota [ ] , [ ] . given a global population over billion, there are approximately cells/microbes associated with people on the planet. for comparison, it has fig. . engineering, process, and other controls allow important infectious disease experiments to be conducted safely. representative biosafety level (bsl) labs are shown. bsl- is for agents not known to cause disease in normal, healthy humans. bsl- is for moderate-risk agents that may cause disease of varying severity through ingestion or percutaneous or mucous membrane exposure. bsl- is for agents with a known potential for aerosol transmission and that may cause serious and potentially lethal infections. bsl- is for agents that pose a high individual risk of life-threatening disease resulting from exposure to infectious aerosols, or for agents where the risk of aerosol transmission is unknown. agents appropriate for this level have no vaccine available, and infection resulting from exposure has no treatment other than supportive care. fig. . the global ecosystem is a highly interconnected network of hosts and environments each with its own associated microbiome. nucleic acids, chemicals, and energy are shared within each environment as well as across the global network. new approaches are needed to visualize and understand the relationships among these environments to improve infectious disease management. fitch: engineering a global response to infectious diseases been estimated that there are cells of prochlorococcus bacteria in the ocean [ ] . equally impressive, there are viruses in the ocean causing roughly viral infections every second [ ] . as represented in fig. , whether, grass, animals, or humans, the microbes, the communities, and their host community are sharing chemicals, energy, and nucleic acids. it is no surprise that clinical, animal, environmental, and other samples often have significant genetic complexity. information visualization tools like krona (fig. ) , support analysis of complex metagenomic data [ ] . these types of data can be used to support many applications including investigating food borne disease outbreaks [ ] . genomics helped explain the initiation of the ebola outbreak in sierra leone in [ ] . genomes from virus samples from patients provided evidence that the index case and associated thirteen initial cases in sierra leone had attended the funeral of a traditional healer that had been seeing ebola patients from nearby guinea. the sequence data also showed mutations in parts of the genome that might impact diagnostics, vaccines and therapies. even though the west africa ebola virus disease (evd) epidemic is by far the largest to date, fortunately ''the clinical course of infection and the transmissibility of the virus are similar to those in previous evd outbreaks'' [ ] . the goal is to be able to make these clinically relevant assessments from the virus genome prospectivelyvi.e., before the epidemic provides the observations. computational algorithms and tools are available to find signatures of existing pathogens as well as recognize patterns that help identify previously unidentified pathogens. moving forward, computational models for the interactions among the different genes are needed that provide a context for the correlations in data as well as improve predictive capabilities. for instance, going beyond detecting disease outbreaks to being able to assess and predict disease risks is desired. these much more complicated models offer the potential to accelerate disease understanding and the development and safe utilization of medical interventions at both the individual and the global scales. there are multiple initiatives underway that will lay the foundation and strengthen the underlying science. the foundation will enable new engineering approaches that will achieve the greater goals for infectious disease management. using an approach that parallels the evolution of electrical circuit design and fabrication, the biobricks approach is having increasing impact [ ] . perhaps most famous for its student genetic engineering competition , the biobricks approach of utilizing a library of well-characterized, interconnectable parts is powerful. just as early circuit designers benefited from standards for circuit fabrication, design, interfaces and modules, aspects of biology are amenable to these approaches. not only does the approach provide validation of reductionist concepts of each ''brick,'' but the innovative integration of parts also contributes to the characterization and future sharing of more complicated parts. just as circuit designers can incorporate existing designs or entire functional units like memory or analog to digital conversion, biologists are increasingly able to import others' designs and achieve significantly more functionality. for instance, a rewritable digital memory system has been demonstrated that writes and rewrites nucleic acid bases in a chromosome [ ] . just as dna sequencing brought a methods-based structure to pathogen detection, approaches like biobricks bring structure to biological circuits. the electronic circuit analogy continues with the opportunity for software to facilitate design and accelerate testing and evaluation. just as in our early example for influenza vaccine development, the components of the biobricks approach are amenable to spiral engineering for both performance and cost. building computer architectures and software that can implement ab initio test and evaluation calculations for infectious disease trials would require significant advances in algorithms, capabilities, and fundamental biochemical data. while exciting progress is being made in these areas, an alternative approach is to consider the network of interactions among fundamental building blocks and identify correlations with disease and health. this is the domain of systems biology and the building blocks can include dna, rna, and peptides. progress in systems biology is often paced by access to appropriately curated and calibrated datavnot just the underlying nucleic acid content but the associated metadata that describe the host and the relevant associated microbial communities. one of the multiple approaches to address data needed is the hundred person wellness project of the institute for systems biology. this project is measuring multiple indicators of health over a nine month period and will include lifestyle coaching as part of the study [ ] . if successful, the project already has plans to scale to a thousand and then thousand participants. there are similar concepts underway at organizations as diverse as google x and human longevity. even though these studies do not focus on infectious diseases, they are of significant value in helping to define the baselines for ''normal'' at different ages, genders, and many other variations that may affect health. infectious disease is a global issue that remains a significant cause of death and economic impact. engineers and engineering have many opportunities to help mitigate these diseases ranging from using cell tower power to help deliver vaccines to providing new network analysis software to identify novel viruses in an outbreak. the joint evolution of engineering and life sciences brings expanded availability and opportunity to understand and design living systems. these are attributes that will be needed to address the medical and infrastructure needs for effective global infectious disease management. however, the zeal to innovate needs to be mediated by a culture of responsibility [ ] where the benefits and the risks are considered. the ieee code of conduct [ ] is consistent with this ethos and addresses quality of life and privacy topics that have been discussed in this paper. engineers have an opportunity to provide innovative application of existing infrastructure to infectious disease management and to help nurture a global culture of responsibility in both healthcare and technical applications. h global health estimates summary tables: deaths by cause, age, and sex antimicrobial resistance: tackling a crisis for the health and wealth of nations enhancing the research, mitigating the effects ending the war metaphor: the changing agenda for unraveling the host-microbe relationship detection of the middle east respiratory syndrome coronavirus genome in air sample originating from a camel barn owned by an infected patient. mbio learning from sars: preparing for the next disease outbreak emerging infectious diseases: threats to human health and global stability available: http:// www.globalhealth.gov/global-health-topics/ global-health-security/ghsagenda.html fitch: engineering a global response to infectious diseases department of health and human services implementation plan for the national strategy for pandemic influenza hspd- : biodefense for the st century defense of united states agriculture and food food safety management tools global health risks: mortality and burden of disease attributable to selected major risks global agricultural report: sustainable pathways to sufficient nutritious and affordable food climate and infectious disease: use of remote sensing for detection of vibrio cholerae by indirect measurement prediction of a rift valley fever outbreak power from cellphone towers keeps vaccines cool outbreak detection algorithms for seasonal disease data: a case study using ross river virus disease. bmc medical informatics and decision making an epidemiological network model for disease outbreak detection evaluation of animal and public health surveillance systems: a systematic review. epidemiology infection report to congress: update on the adoption of health information technology and related efforts to facilitate the electronic use and exchange of health information the office of the national coordinator for health information technology, food and drug administration and federal communications commission building safer systems for better care global infectious disease surveillance and health intelligence: the development of effective, interconnected systems of infectious disease surveillance is essential to our survival nyc health alert network putting public health into practice: a model for assessing the relationship between local health departments and practicing physicians ( s ), pp. s -s interpreting the universal phylogenetic tree genomic engineering: moving beyond dna sequence to function the rise of a digital immune system. gigascience department of health and human services oie world organization for animal health a virus in a fungus in a plant: three-way symbiosis required for thermal tolerance endogenous viral elements in animal genomes human microbiota human microbiome project present and future global distributions of the marine cyanobacteria prochlorococcus and synechococcus marine virusesvmajor players in the global ecosystem interactive metagenomic visualization in a web browser whole-genome sequencing expected to revolutionize outbreak investigations. food safety news genomic surveillance elucidates ebola virus origin and transmission during the outbreak the who ebola response teamonline. ebola virus disease in west africavthe first months of the epidemic and forward projections rewrittable digital data storage in live cells via engineered control of recombination directionality medicine gets up close and personal guidance for enhancing personnel reliability and strengthening the culture of responsibility the author thanks dr. k. bernard for his suggestion to consider the broader utility of pandemic influenza plans to other diseases, dr. n. bergman for his valuable suggestions and comments, and ms. c. conrad for her expert assistance preparing the manuscript. key: cord- - a o authors: nan title: ii. topic sessions date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: a o nan although the majority of children with asthma achieve symptom control on low or moderate doses of maintenance inhaled steroids, there is a small proportion that remain uncontrolled despite high doses of prescribed maintenance therapy. these children are prescribed treatments equivalent to stage / of the british thoracic society (bts) guidelines for asthma management, and either need at least this amount of therapy to achieve control, or have persistent symptoms and frequent exacerbations despite maximal treatment. children with poor control despite maximal prescribed therapy have problematic severe asthma . however, the reasons for poor control may be very varied and can broadly be divided into two sub-categories. the first, "difficult asthma" is the term used to describe patients whose asthma is difficult to control because of a failure to address the basics of asthma management, an incorrect diagnosis has been made, or there has been a failure to address associated comorbidities. underlying reversible and modifiable factors that can result in poor control include poor adherence, unfavourable environmental exposures such as tobacco smoke and aero-allergens to which the patient is sensitised, poor inhaler technique and psychosocial issues . if modifiable factors are successfully identified and addressed, then control can be achieved in children with difficult asthma without the need for escalating therapy or additional invasive investigations. a multidisciplinary team (mdt) is critical to enable modifiable factors to be identified and addressed in children with difficult asthma. the team must include specialist respiratory nurses, a psychologist, pharmacist, physiotherapist and medical staff. significant resources are therefore required to manage paediatric difficult asthma optimally and only specialist centres should be tasked with the assessment of these patients. although this may have an impact on healthcare resources, long term benefits for lung health are significant. the second sub-category of children that have poor asthma control despite maximal therapy are those with true severe asthma. these patients remain with persistent symptoms, or can only be controlled on maximal doses of maintenance therapy, often including oral steroids, after underlying reversible or modifiable factors have been identified and addressed . importantly, more than half of all children with problematic severe asthma have difficult asthma because of underlying modifiable or reversible factors preventing asthma control . therefore, the overall approach to managing a child with problematic severe asthma includes an initial step to identify and treat difficult asthma, and if symptoms persist after this, true severe asthma can be confirmed, which requires additional investigation and management . very clear criteria and definitions that allow distinctions between difficult and severe asthma have been specified for both adults and children aged six years and above by the european respiratory society and american thoracic society . an important point to consider when faced with a child that has poor asthma control despite maximal doses of prescribed maintenance therapy is that once above a threshold of treatment (> mcg/day or equivalent of budesonide), the child should be referred to a specialist for further management. the national review of asthma deaths in the uk identified % of asthma deaths occurred in patients who should have been referred to a specialist for management of problematic asthma . the modifiable factors that result in a child having difficult asthma may be identified extremely efficiently if the mdt approach described is adopted. however, what remains equally important is the continuing assessment and follow-up of patients with difficult asthma in order to ensure: . maintenance therapy is reduced to the minimal amount needed to achieve control . symptoms do improve after all modifiable factors have been addressed, and there is no progression to true severe asthma À either after short term follow-up or in the longer term . the basics of inhaler technique / device / adherence / allergen exposure are all being maintained a retrospective analysis of follow-up of children with difficult asthma for up to six years revealed that those in whom underlying modifiable factors were identified and addressed had an improvement in lung function and reduction in exacerbations over time, while being able to reduce maintenance dose of inhaled steroids such that the majority fell below the threshold for problematic severe asthma . however, there was a large drop out in the number of patients that could be traced for the full six years, highlighting the need for better prospective longitudinal data of outcomes for children with difficult asthma. these missing data are essential in light of recent cohort studies that have followed children with severe asthma to adulthood and shown the irreversible reduction in lung function and prevalence of copd . asthma is one of the most common chronic diseases in children, with a high prevalence in many developed and developing countries. worldwide prevalence of asthma in children varies from . - . % according to the international study of asthma and allergies in childhood (issac) study. ( ) despite its high prevalence, information about the prevalence of severe asthma in children is unknown, particularly in countries in transition. some estimates come from different studies that have shown that the prevalence of severe asthma in a general population is approximately . - % among children with asthma, however its true prevalence in a low-income country is unknown. ( ) ( ) ( ) according to issac phase iii, the centers with the highest prevalence of severe asthma symptoms were mostly from english language countries, latin america, africa, the indian subcontinent and the eastern mediterranean. ( ) lack of control of the disease has been attributed to various factors such as low accessibility to basic medications, weak healthcare services, poor compliance with prescribed therapy, lack of asthma education, and social and cultural factors. in general, asthma in both children and adults represents a significant problem in public health given the reduced quality of life, school or work absenteeism and increased healthcare costs, especially in countries in transition. in addition, asthma severity and control in childhood are of particular importance as they have been shown to translate into asthma morbidity in adulthood. ( ) practical guidelines addressing the management of severe asthma in children have pointed out various aspects important in the development of this condition: medication issues, the environment, asthma education, comorbidity, and psychological problems. worldwide, but particularly in countries in transition, both intrinsic (race, ethnicity, weight) and extrinsic (exposure to allergens, indoor or outdoor pollutants) factors may overlap in a single child to enhance or diminish asthma control and severity. different to many developing countries from other continents, asthma is highly prevalent in latin america. moreover, issac phase iii showed that asthma prevalence in this region is still on the rise. furthermore, evidence suggests that poorly controlled asthma in some areas of latin america leads to significant economic costs attributed to emergency and unscheduled visits, and high mortality rates from asthma. ( ) similar to other regions, asthma control is not obtained in most patients, despite available management guidelines and evidence of ics as controllers. several surveys have shown that close to . % of all patients met all the gina criteria for total asthma control, proposing under-recognition of uncontrolled asthma, underuse of appropriate controller treatment, inadequate patient education, and patient denial as possible explanations. ( ) also, several risk factors such as poverty, environmental factors, diet, genetics, vitamin d deficiency and tobacco smoking have detrimental effects on asthma control. cross-sectional data from children with asthma in costa rica suggested that low serum vitamin d detected in children with mild to moderate asthma is associated with asthma severity. ( ) since the development of worldwide guidelines on the diagnosis and management of asthma, special attention on achieving and maintaining asthma control as the key goal in asthma treatment has been a priority. in clinical studies of children with asthma, satisfactory asthma control can be achieved and maintained in most patients by regular treatment with ics. nevertheless, large population-based surveys consistently show that poor asthma control is common in many children with asthma, despite ics treatment. ( ) other several studies have shown a reduction in the number of hospitalizations caused by asthma in various countries in transition when effective preventive and controller measures are implemented, ( ) mainly avoidance of risk factors, importance on the use of basic medications and patient education. patients should be educated about the cause of asthma, what triggers the condition, how it should be monitored and managed and, importantly, the outcomes that can be expected and when to recognize lack of asthma control. moreover, health professionals should also be educated regarding under-recognition and under-treatment of asthma, as patients or parents tend to deny the severity of symptoms. in a recent study performed in costa rica ( ) , we aimed to examine trends in hospitalization and mortality due to asthma over a -year period ( - ) , in particular following a national asthma plan (nap). this nap consisted of education meetings at all major public health care centers, emphasizing early diagnosis, early treatment using ics as first-line therapy for asthma control, early use of reliever medication to treat exacerbations, appropriate referral to specialists for asthma care, and avoidance of common allergen sources (e.g. dust-mite and cockroaches) or tobacco smoke. concurrent with this program, general practitioners, pediatricians and internists were first allowed to prescribe ics for asthma (only pulmonologists or allergists could prescribe ics before ). as a result of the implementation of the nap, the total number of asthma hospitalizations in costa rica in both children and adults decreased by approximately % over this period. in children younger than years, hospitalizations for asthma were reduced by % in boys and % in girls between and . in addition, the number of deaths due to asthma decreased by % over the -year period, with a more marked reduction occurring after implementation of the nap. in parallel with the decrement in asthma hospitalization and mortality, the number of prescriptions for ics (beclomethasone) increased by %. in summary, asthma prevalence in deprived regions is high and shows increased severity. reasons for inadequate asthma control in poor populations include low accessibility to effective controller medications, weak infrastructure of health services for the management of chronic disease, poor adherence to therapy, lack of educational approaches, and social, cultural and language barriers. however, recent studies have shown several alternatives to control its burden and improve outcomes. there is urgent need for more research into severe asthma, in particular in children in countries in transition. it has now become a much used adage that asthma is not a single disease but rather multiple diseases which present with common symptoms [ , ] . this paradigm has been fundamental in shaping the way we think about asthma and possible approaches to treatment and management strategies. if one is not treating a single disease when we talk about what is commonly known as the syndrome of asthma, then we need a more personalised medication strategy to treat these different syndromes. alongside this acknowledgment of moving medicine towards more personalised treatment and management strategies, statistics and machine learning have been instrumental in helping us to shape the face of medicine by fostering engagement between clinicians, basic scientists, statisticians and mathematical modellers in order to attain a more unbiased approach to classifying different subgroups of patients using probabilistic models. the proliferation of genetic, molecular, clinical and biological data has made it necessary to use a cross-disciplinary approach to understanding the underlying mechanisms which precipitate distinct profiles of asthma and allergic disease during childhood. statistical analysis to understand subtypes of childhood wheezing the seminal paper by martinez et al. [ ] was the first to propose the existence of different subgroups of childhood wheezing. based on visual assessment of patterns of wheeze during childhood using data from the tuscan children's respiratory study, they identified four groups of wheezers: "no wheeze", "transient early wheeze", "late-onset wheeze" and "persistent wheeze". this classification has been used as a classical basis for subsequent definitions of distinct subgroups of wheeze and has provided the building block for statistical pattern recognition-based methods to identify heterogeneous groups of children based on probabilistic modelling of the longitudinal profiles of asthma and wheeze over time. one such statistical technique is latent class analysis. latent class analysis assumes that the longitudinal fluctuation observed in data is measured with uncertainty. some of this uncertainty is due to random error, but another element of this uncertainty may be due to the existence of a subgroup or latent class which explains some of the heterogeneity in clinical measures which is not directly observed. henderson et al. were the first to apply such models using a data-driven approach based on wheeze observations from the avon longitudinal study of parents and children [ ] . using latent class analysis based on parental reporting of wheeze, this group identified two additional phenotypes to those identified by martinez et al.: "prolonged early" and "intermediateonset" wheeze. this classification has been replicated in other studies. [ ] one of the caveats of basing these modelling strategies on parental reporting of wheeze is that parents may not be able to correctly ascertain a clinical diagnosis of wheeze [ ] . in light of this, belgrave et al. extended these methods by jointly modelling data from both parental questionnaires and general practitioner records which provided complementary data to give a more accurate measure of wheeze [ ] . this model identified two classes of persistent wheeze: a "persistent controlled wheeze" group and a "persistent troublesome wheeze" group who had poorer lung function and more reactive airways compared to the other wheeze groups, including the "persistent controlled wheeze" group. where machine learning begins and statistical modelling ends identifying consistently defined and optimal numbers of subgroups of wheeze across different cohorts is challenging. within the era of "big data" rather than focusing on traditional statistical methodology, the medical field is looking towards data science as a means to extract knowledge and meaning from the vast quantity of information provided by clinical data. to achieve this, both traditional statistical inference methods based on robust assumptions and machine learning models which are more amenable to data complexity, breadth and depth. although there is overlap between the functionality of machine learning and statistics, the flexibility of machine learning is driven towards learning from data and integrating new information in order to update models and create more accurate models with better model performance. the programmatic focus of machine learning which incorporates vast amounts of computational power provides an excellent framework where tools traditionally used for statistical modelling would be unable to accommodate large, multi-scale datasets. in the near future, the capability of machine learning to be able to learn from data interactively may facilitate computer-assisted reasoning in identifying subgroups of patients. identifying such subgroups may be crucial in proposing effective personalised treatment strategies. such an approach will also allow us to capitalise on the existent data. as data-transparency and data-sharing become more widespread in the global community, we will have a better understanding of the evolution of asthma and allergic diseases. research into identifying heterogeneous subgroups of asthma and allergic disease has reached crucial milestones. we have moved from a subjective approach to classifying subgroups of wheezers, whereby the clinician gives a clinical assessment or diagnosis of the most likely subgroup based on observed clinical history, and we are moving towards computer-assisted reasoning, whereby we can use new information to predict the most likely class assignment based on models derived from prospective data. such reasoning would also allow us to model the evolution of asthma and allergic diseases in the future. populations of microbes (such as bacteria and yeasts) inhabit the skin and all mucosal surfaces. healthy individuals host thousands of different types of bacteria and different body sites have their own distinctive communities, with estimates suggesting that %- % of all the cells in the human body are microbes. the highest density and greatest diversity of bacteria is found within the gastrointestinal tract. research suggests that the relationship between the microbiome and humans is not only commensal (a non-harmful coexistence), but is a mutualistic, symbiotic relationship with benefits for both ( ) . even though we live in such a "dirty" bacterial world, infections due to bacteria are relatively very rare in individuals with a competent immune system. the microorganisms that make up a microbiome perform a wide range of useful functions, such as fermenting unused energy substrates, educating the immune system, preventing growth of pathogens, regulating the development of organs such as the gut, producing vitamins for the host and producing hormones to influence host metabolism such as directing the host to store fats. in particular, specific microbe-host interactions are thought to be critical for inducing mucosal tolerance and immune regulatory cells such as tregs. why do we develop "tolerance" to the microbes living in us and on us? perhaps we should consider tolerance as an alternative defense strategy. the continuous effort involved in destroying the microbes that surround us would impair organ function and require vast amounts of energy, which is not compatible with life. for this reason, it makes much more sense to have robust tolerance mechanisms that work in tune with potent effector responses, to ensure optimal host fitness. an intriguing question is that posed by the concept of the hygiene hypothesis in that altered exposure to microbes may influence the induction of tolerogenic immune responses, thereby making individuals more susceptible to react aggressively to nondangerous encounters with antigens such as allergens. the balance between immune tolerance and inflammation is regulated through the crosstalk between epithelial and immune cells with the microbiome involving many signaling pathways and molecules. direct contact with bacterial-associated structures can activate receptors (e.g. tlrs) on host cells, which induce signaling cascades resulting in both innate and adaptive polarized immune responses. the microbiome is also metabolically active and microbial metabolites have been shown to exert significant effects on host immune signaling networks (e.g. scfas and biogenic amines). the biogenic amine histamine can promote either pro-or anti-inflammatory effects depending on which of its four receptors are activated ( ) . some, but not all, commensal bacteria express histidine decarboxylase (the enzyme needed to convert histidine to histamine). lactobacillus saerimneri a produces high levels of biologically active histamine and feeding this strain to mice resulted in a deterioration in health, particularly in histamine receptor knock-out mice ( ). significant efforts are underway to determine the positive and negative health effects associated with production of histamine by the microbiota ( ). abnormalities in microbiome composition and/or metabolic activity have been shown in a wide range of disease states including type- diabetes, obesity, inflammatory bowel disease, colorectal cancer and allergies. efforts to use microbiome-associated therapeutics (e.g. probiotics) have clearly shown beneficial effects in animal models, with inconsistent findings in humans probably due to differences in the bacterial strains used. one probiotic bacterium that has shown consistent immunoregulatory effects in murine models and humans is b. longum subsp. longum . murine models have demonstrated that oral consumption of this strain results in the induction of treg cells and these treg cells dampen nfkb activation, preventing excessive inflammation induced by salmonella infection ( , ) . similarly, in humans, oral consumption induces treg cells, which is associated with increased secretion of il- by peripheral blood cells ( ) . interestingly, this strain reduces systemic pro-inflammatory biomarkers in patients with psoriasis, ibs patients with chronic fatigue syndrome and patients with ulcerative colitis ( ) . the mechanism involved includes the recognition of this bacterium via tlr- / and dc-sign by myeloid dendritic cells and tlr- by plasmacytoid dendritic cells, resulting in changes in dendritic cell cytokine secretion and the production of metabolites such as retinoic acid ( ) . however, these effects and mechanisms are not seen even with closely related bacterial strains, suggesting that the careful selection of microbes is essential for the future clinical development of immunotherapeutic microbes for allergy and asthma. overall, it can be concluded that the vast majority of microbes, which interact continuously with the host, are not bad. certain specific microbes can positively influence the host, while there is a minority that can have negative effects on the host. gwas findings are based upon association p-values below à À , socalled "genome-wide significance", as well as replication in independent populations. this generally requires very large sample sizes and in order to obtain these, a 'team science' approach has been used where several studies have combined their data in meta-gwas. the largest gwas on asthma to date combined data from different studies involving more than , individuals from the gabriel consortium and identified genome-wide significant asthma loci.( ) similar meta-gwas have been conducted, for example by the eagle consortium, revealing a number of susceptibility loci for asthma-related traits, including feno,( ) eczema,( ) and allergic sensitization.( ) it could be expected that the large heterogeneity in disease phenotypes introduced by combining many different studies in meta-gwas would preclude valid discoveries. nevertheless, gwas on asthma and the related traits have resulted in identification of relatively few, but robust, loci with more consistency between studies compared to previous candidate gene studies. one example of this is the first large-scale gwas on allergic sensitization.( ) by meta-analysis of data from different studies, it included a discovery phase of approximately , cases and , controls and a similar-sized replication phase. allergic sensitization was assessed objectively and defined by elevated levels of allergen-specific ige and/or a positive skin prick test. this study identified loci associated with allergic sensitization at the genome-wide significant level and with robust replication. simultaneously, another large gwas was performed on allergic symptoms including approximately , individuals. ( ) in spite of the large phenotype differences between the two studies, there was a high agreement in results with all of the genome-wide significant loci from the sensitization study also showing strong association in the study on allergic symptoms, and previous gwas findings were confirmed. there has been some disappointment with the results from gwas. the identified loci only explain a minor part of the heritability, and the susceptibility variants identified in gwas are mainly common variants with relatively small effect sizes (often with odds ratios around . per risk allele) with no clinical relevance on the individual level. ( , ) on the other hand, gwas have identified novel and robust susceptibility loci, with the potential to provide important understanding of disease mechanisms. also, comparison of results from gwas on different diseases and traits have increased the understanding of the mechanistic relationship between these, for example the relationship between allergic sensitization and asthma,( ) between allergen-specific ige and total ige levels, ( ) and between atopy and autoimmunity. ( ) ( ) ( ) larger, consortium-based studies on asthma and the related phenotypes are ongoing and are expected to identify many novel susceptibility loci. novel loci discovered from these larger studies are likely to have even smaller effect sizes than the ones previously found but, from the perspective of understanding disease pathology, each novel locus may potentially pinpoint a novel mechanism and a potential treatment target. furthermore, the era of genome-wide nucleotide sequencing applied on gene expression-and epigenome-profiling has brought new possibilities of combining gwas data with data from large public 'omics repositories. these data will increase the usefulness of gwas data by providing understanding of functional effects related to susceptibility loci, and future gwas on asthma and related diseases will be a part of integrated approaches to discover how different molecular layers modulate the genetic effect on disease, and will thereby be a central component in the attempt to tailor and improve medical treatment. asthma is a highly heterogeneous disease probably consisting of several subtypes of disease associated with different functional mechanisms. genetic loci may be involved in specific disease mechanisms and thereby help understanding this heterogeneity. for example, the strongest asthma locus identified in gwas, the q - locus, seems strongly associated with an asthma phenotype characterized by onset in early childhood ( ) and recurrent, severe exacerbations ( ) and was stronger associated with asthma than allergic rhinitis. ( ) in contrast, another locus at chromosome q has been associated with multiple allergy-related phenotypes, including allergic sensitization,( ) allergic symptoms,( ) eczema,( ) and asthma, suggesting a different, allergy-related, disease mechanism. the heterogeneous nature of asthma suggests that an alternative to increasing sample size in genetic studies is to focus on more specific phenotypes. such phenotypes are likely closer associated to specific mechanisms and the genetic substrate and might therefore increase study power. this was demonstrated by a gwas focusing on a specific asthma phenotype characterized by onset in early childhood and recurrent, severe exacerbations. ( ) in spite of the relatively small sample size, this study resulted in association results of the same magnitude as previous much larger gwas ( ) and with much larger effect sizes, particularly for the children with the highest number of exacerbations. one novel asthma gene, cdhr , was identified, and it was confirmed, in a collaborative effort involving several birth cohort studies, that the cdhr locus was strongly associated with asthma exacerbations in the first years of life, both in individuals of european and non-european ancestry. these results highlight the potential of future genetic studies focusing on more homogenous phenotypes. one important future step is the translation of genetic associations to disease mechanisms. a major limitation of gwas is that they often merely identify a susceptibility locus without any clear relationship to a specific gene or biological function. two examples of this are the q - and q loci mentioned above, where the underlying mechanisms are still poorly understood several years after their discovery, even though these loci are strong and probably central to the pathogenesis of asthma and allergy. one example of a gwas discovery where the functional mechanism might have been identified is cdhr . in the discovery study ( ) , it was suggested that the association to asthma was caused by a specific functional variant affecting surface expression of cdhr . a later study reported that cdhr functions as a rhinovirus c receptor and showed that the functional variant associated with asthma exacerbations increases rhinovirus c binding and replication. ( ) this potentially explains the underlying mechanism of this locus and identifies a target for future asthma and virology research. another major future challenge is to understand how genetic susceptibility interacts with environmental factors. gene-environment interactions are not accounted for in normal gwas and that might be one reason for the large heritability not explained by gwas findings. one important environmental risk factor for childhood asthma and other wheezing disorders is viral infections, and focusing on this environmental factor might be a tool to understanding mechanisms of asthma genes. ( ) as an example, children with q - risk variants seem more susceptible to rhinovirus infections, ( ) and the finding that cdhr seems to be a rhinovirus c receptor ( ) indicates that children carrying cdhr risk variants will have a specific susceptibility to rhinovirus c infections, a hypothesis that is currently being tested. only a few genome-wide gene-environment interaction studies have been performed, and the results of these have generally been disappointing without convincing findings. there are many inherent challenges in such studies. first, they might require even larger sample sizes than normal gwas, and exact information on environmental exposures is difficult to obtain in such large-scale studies. furthermore, the effect of a specific environmental exposure can be difficult to disentangle from that of other related environmental factors. an alternative approach is to perform cell or animal models where specific exposures can be controlled.( ) a recent study investigated the potentially protective effect of endotoxin and farm dust exposure in a mouse model of house dust mite-sensitized asthma.( ) it was found that a was an important mediator of the protective effects of endotoxin exposure, and this was validated in human bronchial epithelial cells. furthermore, a potential modifying effect of a was supported by 'look up' of snps located near the human tnfaip gene using data from an earlier genome-wide interaction study. this potential gene-environment interaction needs to be replicated, but this study exemplifies how mechanistic studies targeting specific environmental exposures and the use of experimental models can facilitate identification of genes involved in gene-environment interactions. in conclusion, improved understanding of the genetic architecture of asthma and other childhood wheezing disorders will require a combination of gwas focusing on more homogeneous subtypes of disease, geneenvironment interaction studies in birth cohorts and in cell models, and integration with other types of omics data. this challenge can only be overcome by a 'team science' approach bringing together many studies to provide sufficient statistical power and bringing together researchers from many disciplines to translate clinical associations to mechanistic understanding. such studies present great challenges but also the opportunity to understand asthma pathogenesis and heterogeneity, and ultimately to improve prevention and treatment of disease. recently, who definitions have changed to classify children with lower chest indrawing as having pneumonia rather than severe pneumonia and recommending treatment with oral antibiotics as ambulatory cases. [ ] however, a recent meta-analysis reported that no single clinical feature is sufficient to accurately diagnose radiological pneumonia and that the who recommended diagnostic signs alone lack sufficient sensitivity or specificity, particularly for identifying children who need antibiotics. [ ] radiological diagnosis of pneumonia has relied largely on changes on chest x-ray, principally consolidation or interstitial infiltrates. [ ] however, chest x-rays are subject to variable interpretation, expose a child to ionizing radiation and require infrastructure and skill to do. recently, chest ultrasound has been suggested as a feasible imaging modality for diagnosis of childhood pneumonia. ultrasound has several advantages including that it can be used as a point-of-care test, can be taught to non-radiologists, is quick to perform and does not involve exposure to radiation. initial studies suggest that it has high sensitivity and specificity for pneumonia compared to chest x-rays. [ , ] diagnosis of the etiology of pneumonia remains challenging as bacteremia is rare, distinguishing colonizing from pathogenic organisms may not be possible on respiratory specimens and co-infections are common. improvements in specimen collection and improved molecular techniques for detection of organisms have enabled more accurate detection of organisms, however ascribing etiology may be difficult unless the organism is invariably pathogenic. advances in specimen collection include the use of induced sputum in infants and young children, which provides a better specimen for detection of specific pathogens such as b. pertussis or m. tuberculosis. [ ] urine antigen detection has not proven to be useful for pneumococcal pneumonia or for pulmonary tuberculosis in children. [ , ] for induced sputum, testing of sequential, repeat specimens provides a higher yield for pathogens such as m. tuberculosis. [ ] careful attention to specimen collection methods and use of different specimens may maximize the yield especially in the context of new sensitive molecular detection techniques. [ ] with the availability of improved tools for etiological diagnosis, and with better vaccine coverage for conjugate vaccines, including pneumococcal conjugate vaccine, viral pathogens especially rsv and other bacteria, such as s. aureus or pertussis, are emerging as prominent causes of childhood pneumonia. [ ] [ ] [ ] in areas of high tb prevalence, m. tuberculosis has been reported to be associated with acute pneumonia in children, with culture confirmed disease occurring in approximately % of cases. [ ] however, better tools for detection of potential pathogens have also provided data on the complexity of etiology, with several potentially pathogenic organisms frequently identified in a single pneumonia episode. further delineation of the interactions between different organisms and pneumonia pathogenesis is needed. asthma affects as many as million people of all ages in all parts of the world and is the commonest long-term respiratory condition affecting children in developed countries, the prevalence and morbidity varying by ethnic group . accurate diagnosis and effective management of respiratory diseases such as asthma requires objective measures of lung function, but reliable use of such measures is only possible if appropriate normative ranges are available to distinguish the effects of disease and treatment from those of growth and development. evidence for ethnic differences in lung function ethnic differences in lung function have been well documented . in the past, attempts to interpret observed ethnic differences in lung function were often confounded by selection bias related to use of small population samples that were not necessarily representative or generalizable, use of different methods, equipment and quality control (qc) criteria, failure to adjust for other important determinants of lung function, including socio-economic circumstances and/or inappropriate statistical analyses. in recent years, many of these problems have been addressed by applying standard methodology, inclusion criteria and qc to large, ethnically homogenous groups. current research shows that after adjusting for age, sex and standing height, forced expired volume in sec (fev : a measure of airway calibre) and forced vital capacity (fvc: a measure of lung size) are both reduced by approximately % in individuals of african ancestry (black) across the entire life span when compared with those of european ancestry (white) [ ] [ ] [ ] [ ] . similar though smaller reductions have been observed among south asian (from indian subcontinent) [ ] [ ] [ ] and south-east asian (e.g. china, thailand, malaysia, etc) subjects. since these "ethnic" reductions in fev and fvc are generally proportional, the fev /fvc ratio, which is the most commonly used outcome to assess airways obstruction, is usually independent of ethnic background [ ] [ ] [ ] , suggesting that there are no structural or functional ethnic differences in lung design. thus the observed ethnic differences in lung function appear to be primarily limited to lung size rather than airway or dynamic respiratory characteristics. however, the same adjustment factor cannot be used for all lung volume outcomes. for example, there is evidence that the lower fvc found among black children can be attributed at least in part to a relatively high residual volume, suggesting that factors such as anatomic differences in diaphragmatic position or respiratory muscle strength might contribute to some of the observed differences . furthermore, lung function indices that are internally adjusted for the size of the individual's resting lung volume, such as the lung clearance index (lci: a measure of gas mixing efficiency) , or specific airways resistance (sraw: a measure of airway calibre adjusted for lung volume) , do not appear to be influenced by ethnic background. nevertheless, since larger sample sizes will be required to confirm these findings, data interpretation of lci and sraw from non-white subjects should currently be undertaken with caution. recently, the global lung function initiative (gli) collated results from > , healthy non-smokers aged - years to create the first allage, multi-ethnic reference equations for spirometry with appropriate age dependent lower limits of normal . prediction equations were derived using the lms method, which allows simultaneous modelling of the mean (mu), the coefficient of variation (sigma) and skewness (lambda) of the distribution, and reference equations were derived for caucasians (white); african-americans (black), north-and south-east asians. these equations enable assessments to be evaluated over the entire age range using a single reference data set, thereby avoiding the errors that can occur when switching between equations, particularly during the transition between paediatric and adult care . defining ethnicity ethnicity is extremely difficult to define. self-assigned ethnicity may differ from observer-assigned ethnicity and in certain countries it is against the law to record ethnic origin for any purpose. furthermore, in recent censuses in both the uk ( ) and us ( ), mixed-race populations have been shown to be the fastest-growing ethnic group. thus, classifying ethnicity may become an increasingly complex task! could differences in body proportions explain the ethnic differences in lung function? standing height is a major determinant of lung volumes, reflecting the fact that lung size is adapted to our metabolic needs. however this is not ideal since the size of the lungs is more closely related to thoracic size than leg length and differences in body proportions may underpin much of the observed ethnic variation in lung function. the size and lung function in children (slic) study was designed to improve normative reference ranges for lung function by taking differences in body physique into account to facilitate early diagnosis and treatment of lung disease in all children, irrespective of ethnic background . however, of the numerous additional anthropometric measurements undertaken to quantify body physique, only sitting height and chest width significantly contributed to the prediction of spirometric lung function. chest dimensions and lean mass also significantly predicted fev and fvc within each ethnic group, but did not affect differences between groups. the persistence of ethnic differences after adjustment for sitting height, chest dimensions, body composition and socio-economic factors may reflect the fact that some factors affecting chest size such as diaphragmatic position or muscle strength cannot be assessed by anthropometry, and emphasises the importance of taking ethnicity into account when interpreting spirometry data . while some studies have shown an association between socio-economic conditions (sec) , and lung function and suggested that this is a key factor in explaining ethnic differences in lung function , there is increasing evidence that the contribution of sec to variability of lung function is very small except under the most adverse of conditions , , . a recent study in india , using identical equipment and techniques as those used in the slic study found that while average fev and fvc in urban indian children were similar to those in indian children residing in the uk, they were significantly higher than in semiurban and rural indian children (by $ % and % respectively). these results probably reflect the marked differences in the degree of social deprivation between the uk and india , and suggest that there may be a threshold effect of poverty on lung function. adjusting for sitting height has been shown to reduce the contribution of sec to ethnic variability , . the use of inappropriate reference equations and misinterpretation can lead to serious errors with respect to both under-and over-diagnosis. in the past, attempts to correct for ethnic differences, if made at all, tended to apply the same fixed adjustment factor across all ages , all ethnic groups, both sexes and all spirometric outcome measures, an approach now shown to be oversimplistic , . in addition to errors relating to ethnic differences in lung function, misdiagnosis may also occur when fixed cut-offs, such as % predicted fev or . fev /fvc are used; particularly in young children and elderly adults. while %predicted has historically been used to interpret lung function results, z-scores are more appropriate as they take into account the between-subject variability of measurements for any given outcome at any given age, as well as the predicted value . similarly, use of < . as a fixed threshold for abnormal fev /fvc can lead to gross under-diagnosis of airway obstruction in the young and over-estimation in the elderly . with exception of extreme deprivation, ethnic differences in lung function cannot be explained by socio-economic factors. after adjusting for confounders, genetic factors do contribute to ethnic differences in body physique and lung function. given the marked ethnic differences in lung function, the magnitude of which are similar across the entire life span, it is essential that lung function results in children are interpreted using ethnic specific equations whether in clinical practice or epidemiological research. although gli- do not and never will cover all ethnic groups, appropriate use of age, height and sex adjusted values of fev /fvc ratio derived from these equations (which is consistent across all ethnic groups) will facilitate better identification of airway obstruction in children irrespective of ethnic background. failure to adjust lung function for ethnic differences will result in overestimation of both the severity of airway obstruction and the severity and prevalence of restrictive lung disease. it is now recognised that asthma is a complex, heterogeneous disease. therefore, we need to move away from offering a single approach to management for all children and consider the identification of individual phenotypes for each child to enable optimal treatment and control. the specific facets of the disease that need to be considered and defined in each child include: i. an accurate description of symptom pattern (exacerbations alone, or persistent symptoms with and without exacerbations), ii. the nature of airway inflammation (eosinophilic, neutrophilic or non-inflamed), iii. the type and degree of structural airway changes (remodelling). although asthma control can be achieved in most children with low-moderate doses of inhaled steroids, we remain unclear about the choice of optimal maintenance therapy for each child. how should a decision between regular inhaled steroids, or leukotriene receptor antagonists be made? when additional therapies are required to achieve control, a scientific rationale for add-on therapies also is unavailable. the majority of decisions about therapies are made using a "trial of treatment" approach . if one approach is not successful, then another is adopted without a clear thought process dictating choice of treatments. it is apparent that we need to change our current one size fits all approach to the management of asthma in children. although perhaps less important for children with mild or moderate disease, this becomes extremely important when we consider those with more severe disease. personalised medicine for severe asthma although atopy, airway hyperresponsiveness, eosinophilic inflammation and remodelling are the cardinal pathophysiological features of paediatric asthma, we now know that each of these features can be present to very different degrees in the individual child - . pathology has been most studied in children with severe disease, and although features such as eosinophilic inflammation and increased airway smooth muscle represent the patients as a group, there is huge overlap between children with and without asthma, and a huge spread of severity of these features within the group of children with severe asthma . this within-group variability means assessments need to be made in the individual before deciding on the most appropriate add-on therapy. a proposed approach to identifying the "individual phenotype" in children with severe asthma is to split response to steroids into different domains (bossley c et al. j allergy clin immunol , in press). not all children with asthma have abnormal lung function, not all have inflammation or remodelling, the response to a trial of systemic steroids can therefore be split into the following: i. lung function response, ii. inflammation response (exhaled nitric oxide and sputum eosinophils) and iii. symptom response. we have analysed this approach in patients with severe therapy resistant asthma and shown a similar proportion of children (approx. %) responded to systemic corticosteroids in each domain, but there were no reliable predictors of a response pattern. furthermore, only % were complete responders (response in all domains), % were nonresponders (no response in any domain) and the majority ( %) were partial responders (response in > domain). these data highlight that childhood severe asthma is heterogeneous and a complete response in symptoms, inflammatory and physiological parameters is rare (bossley c et al. j allergy clin immunol , in press). individual response patterns to systemic steroids need to be applied in the future to guide the choice of addon therapies in each child as a step towards achieving personalised medicine. subsequently, this multi-domain approach was applied clinically to identify characteristics of responders to the add-on therapy omalizumab. it became apparent that only those with a positive response in the inflammation domain (a significant reduction in exhaled nitric oxide after a trail of systemic steroids) had a beneficial response from omalizumab . as increasing numbers of add-on therapies become available for use, specifically in the context of severe asthma, we need to better define pathophysiological phenotypes in individual patients and we need to understand the mechanisms mediating disease in children. in addition, we now need to incorporate individual genotypes into our definition of phentoypes to more accurately define treatment responses , as has been successfully done for response to montelukast in preschool wheeze . not only will this individualised approach allow us to discover novel molecular targets that will be effective specifically in the paediatric population, but it will also enable us to objectively choose the best therapy tailored to the individual child. europe consistently report that - % of children with a recognized asthma diagnosis require acute medical care yearly. this is a reflection of the inadequacy of the available treatment options for prevention and treatment of exacerbations, suggesting that asthma with severe exacerbations may represent a distinct subtype of disease and demonstrating a need for improved understanding of its pathogenesis. asthma heritability is estimated at - %. a number of genes have been verified in genome-wide association studies (gwas), but still the genetic background of asthma remains poorly understood. larger gwas may reveal new susceptibility loci with smaller effects, but due to the large heterogeneity of asthma ( ), an alternative strategy may be to increase phenotype specificity. a specific phenotype is likely to be closer related to a specific pathogenetic mechanism and may therefore markedly increase the power of genetic studies. this was the background for a gwas focusing on a particular asthma phenotype defined by repeated, severe exacerbations in early childhood. ( ) a sufficient number of cases were obtained by identification of children with recurrent acute hospitalizations for asthma between and years of age in the danish national patient register, and extraction of dna from dried blood spots from the danish newborn screening biobank. the case phenotype was rare with only / of children born in denmark between and fulfilling the inclusion criteria. the final study comprised , children with repeated hospitalization and , healthy controls. five loci were identified with genome-wide significant association (pvalue < à - ): gsdmb, il , rad , il rl and cdhr . even though the sample size of this gwas was less than one fifth of the largest published gwas on asthma from the gabriel consortium,( ), it identified a similar number of genome-wide significant loci with similar statistical significance. the effect estimates were remarkably high with odds ratios between . and . per risk allele, compared to the odds ratios around . - . usually found in gwas on complex traits. further increasing phenotype specificity by stratified analysis in the children with the highest number of exacerbations resulted in a further increase in effect estimates, with odds ratios between . and . per risk allele, and strong statistical significance. these strong results demonstrate the value of focusing on a more specific phenotype in asthma genetics. furthermore, it indicates that studies on this severe and early-onset phenotype is a "cost effective" approach whereby methodologies requiring large resources and/or strong statistical power can be applied in a limited number of individuals and still provide powerful results. the top-locus in this study, at chromosome q - near gsdmb/ ormdl , has consistently been associated with childhood onset asthma. ( ) ( ) ( ) the effect size in the present study was remarkable with an or of . (p-value ¼ . à À ) and increasing to . for the children with highest number of exacerbations. this suggests an important role for this locus in severe exacerbations in early childhood in line with a previous report from the copsac birth cohort study.( ) cdhr had not previously been associated with asthma or any other disease. the association with asthma was replicated in the publically available gabriel results ( ) protein structure modeling showed that the risk-associated variant is located at the interface between two domains where it could be involved in disulfide rearrangement and interfere with inter-domain stabilization, overall protein stability or conformation, in agreement with the observation in experimental studies of altered cell surface expression. ( ) the biological function of cdhr is unknown but it seems to be a highly plausible asthma gene. it belongs to the cadherin gene family of transmembrane proteins involved in several cellular processes including epithelial polarity, cell-cell interaction, and differentiation ( ) and is highly expressed in the lungs. also, other members of the cadherin family have been associated with asthma and related traits, including e-cadherin. ( ) recently, it was reported that cdhr functions as a receptor for rhinovirus c. ( ) cdhr was differentially expressed in epithelial cells susceptible to rhinovirus c infection compared to unsusceptible cells, and its expression on epithelial cells enabled rhinovirus c binding and replication. importantly, introduction of the risk variant at rs by transfection resulted in -fold increased rv-c binding and progeny yield compared to the non-risk variant. these data provide strong evidence that cdhr is a rhinovirus c receptor and that the association signal in the cdhr gene might result from increased susceptibility to rv-c infections. this finding is in line with the exacerbationrelated phenotype from the discovery gwas, since rhinovirus c has been reported to be the most common viral trigger of severe asthma exacerbations in children and associated with more severe disease and higher rates of hospital readmissions compared to other respiratory viral infections.( , ) if correct, this would indicate that children with the cdhr risk variants are specifically susceptible to rhinovirus c infections compared to illnesses triggered by other viruses, a hypothesis that is currently being tested. in conclusion, the strong results found in this gwas on childhood asthma with severe exacerbations demonstrate the value of specific phenotyping in the search for asthma genes. focusing on this extreme subtype of disease might reveal mechanisms that would not be revealed in studies of milder disease, but might also increase the understanding of general asthma mechanisms. identification of cdhr as a risk gene might be one of the first examples where the underlying mechanism of an asthma gwas finding is understood. future studies of this gene may improve understanding and treatment of asthma exacerbations in childhood. the timing of bacterial colonization early in life is thought to be important for appropriate immune education and the transmission from mother to the fetus during pregnancy and birth is being better described. cultures of meconium have shown diverse groups of gram-positive and gram-negative bacteria, possibly not all derived post-delivery. the development of the gut microbiome is a dynamic process and early colonization with bacteroides and bifidobacterium species might play a crucial role in the development of immune regulation ( ) . factors that can influence early life colonization include antibiotic treatment, method of delivery, maternal and infant diet and biodiversity in the home, surrounding environment and in family members. the gut microbiome increases in diversity during the first years of life. germ-free mice, which are not exposed to live bacteria, display exaggerated th and ige responses, associated with diminished polarization of treg cells. monocolonization of the mice with specific microbes, but not all microbes, suppresses the ige response and promotes treg differentiation ( ) . however, certain immunological changes, such as increased inkt numbers in the mucosa, cannot be reversed following colonization of mice later in life ( ). interestingly, more severe allergic responses and anaphylaxis were observed in mice who received a microbiome transplant from allergic animals, suggesting that certain microbial species can actually promote allergic responses ( ). the immune system at birth is dominated by th cells. however, the human fetus has a functional immune system at a relative early status of development comprising cd þ and cd þ t cells but also foxp þ treg cells. one concept gaining support is that the developing fetus may become educated by whole bacteria or their genetic material that is provided via maternal serum. dna from bifidobacteria and lactobacilli, two genera typically used as probiotics, are found in human placenta. in contrast, in utero exposure to potentially pathogenic bacteria such as ureaplasma species leads to immune dysregulation commonly ending in fatal complications. maternal consumption of probiotic-containing food components may reduce the risk for childhood allergic diseases and mouse models demonstrate a reduced risk of inflammatory bowel diseases. epigenetic mechanisms may be critical since application of acinetobacter lwoffii to pregnant mice reduced the airway hypersensitivity response of the offspring. the promoter region of ifn-g in cd þ t cells of the offspring had high levels of histone- acetylation, associated with enhanced transcription, while the il- promoter region had lower levels of histone- acetylation ( ). moreover, exposure of pregnant mothers to the farm environment, which have high levels of acinetobacter lwoffii, was associated with dna demethylation of the foxp locus and methylation of the th -associated genes rad and il- . since gut microbiota composition during the first months of life seems to be important for development of appropriate immune regulatory networks and thereby influence later life disease risk, intervention with probiotics, prebiotics or synbiotics might be most effective at this age or even during pregnancy. probiotics can be defined as live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host. notably, the definition of a probiotic does not differentiate between the wide range of potential health benefits and it is clear that not all probiotics will influence the immune system in the same way. findings observed with one probiotic strain cannot be extrapolated to other probiotic strains. current evidence does not indicate that the probiotics clinically tested to date reduce the risk of children developing allergy but there are significant differences between studies such as the use of different probiotic strains, different age groups and different endpoints ( ) . despite very poor quality of evidence, it has been suggested that there may be benefits in specific high risk groups, such as pregnant women at high risk of having an allergic child, in women who breastfeed infants at high risk of allergy and in infants at high risk of developing allergy. in general, probiotic-supplemented formula was found to be well tolerated and safe for infants. in conclusion, a better description of the bacterial strains and metabolites, which influence immune function, is required in order to allow for the improved design and selection of future probiotic strains for prevention and treatment of allergic disorders ( ). people with cystic fibrosis (cf) are living longer lives than ever in the past. the median predicted survival in developed countries is now above years of age and adults with cf are outnumbering pediatric patients in several regions. various reasons may explain such improvement in life expectancy, including the establishment of cf-dedicated and multidisciplinary centers; greater attention to nutritional issues and use of pancreatic enzymes replacement therapy; airway clearance techniques tailored to individual needs and attitudes; infection control measures; use of antibiotics both chronically by inhalation and aggressively to treat pulmonary exacerbations; mucolytic and airway hydration therapies; and liver and lung transplantation ( , ). on account of the overwhelming evidence that organ impairment begins very early, even in asymptomatic cf infants, there is now general consensus that at least some of these strategies of care should be implemented as soon as possible in order to prevent or delay irreversible structural lung damage. indeed, this has possibly been the main argument in favor of cf nbs ( ). the strength of such argument has been tested by several studies and considering different approaches. randomized studies À only two randomized trials on newborn screening for cf have been completed ( , , ). these evaluations need many years of follow-up and, given the high degree of evidence in favor of cf newborn screening presently available, further implementation of similar studies seems improbable and possibly non ethical. observational studies À although most of these studies confirm clinical benefits from early diagnosis of cf, their results are hampered by several biases inherent to the methodological approach. the constant improvement in treatment and the consequent longer survival has an influence on the comparison of screened individuals and unscreened historical controls. on the other hand, examining the clinical evolution of screened infants and unscreened controls from different geographical areas but born in the same years may be affected by different care practices. finally, ascertainment biases may also have an impact on the assessment of outcomes, as patients presenting clinically are likely to have more severe cf than those identified through screening or unscreened patients with very critical disease may have died before being diagnosed. health economics studies À these studies use surrogate end-points, such as the quantity of treatment needed to remain healthy, and are based on the assumption that the optimal management offered to cf patients makes it harder to detect evidence of better clinical outcome in those diagnosed by screening. late-diagnosed patients may show clinical pictures similar to those diagnosed early, but at the expense of a considerably heavier burden of care ( ) . most of these studies have focused on respiratory and nutritional outcomes and on hta assessments. their overall results clearly point in the direction of a positive effect on height and weight, of longer survival and of health service savings in populations screened at birth for cf. positive effects may also be obtained in several other domains, namely: -the prevention of salt loss syndrome thanks to early beginning of salt supplementation - the opportunity of surveying from birth the natural history of cf -a better understanding of the early stages of cf. - the possibility of testing presymptomatic therapeutic strategies, both conventional and patient targeted. cystic fibrosis (cf) is associated with the presence of two cf-causing mutations, one in each parental cftr gene, resulting in the absence or abnormality of the cftr protein and defect in electrolyte transport across epithelial membranes, the most well known being sweat chloride > mmol/l. even in , cf remains by essence a clinical diagnosis. the wide range and severity of symptoms/ organs involved between and within individuals makes it a clinical decision as to whether or not a person should be managed as a cf patient. this is especially the case in a small number of ambiguous or atypical cases. in , a first diagnosis consensus listed criteria for cf diagnosis: (i) one or more of the phenotypic features of the disease or (ii) cf in a sibling or (iii) a positive immunoreactive trypsin (irt), in association with at least one other feature, including a positive sweat test result on two occasions, a cf-causing mutation in each cftr gene or an abnormal nasal potential difference (npd) ( ). this consensus statement of the us cystic fibrosis foundation was later modified in europe based on the concept of cftr dysfunction included in the diagnosis algorithm ( ) . most atypical cf patients are diagnosed based on sweat tests and/or genetic analysis. these "mild cf" individuals usually present later in their lives with pancreatic sufficiency and milder respiratory disease. they frequently carry wide clinical spectrum mutations. the difficulty occurs when patients present with clinical symptoms suggestive of cf and a sweat chloride value in the intermediate range ( - mmol/l). among these subjects, those with abnormalities in npd measurement or identified cftr mutations have, on average, more severe lung disease than the remaining subjects, although their disease symptoms are milder than those in subjects with a sweat chloride concentration above mmol/l. therefore, from a physician's and also from a patient's perspective, these individuals must be differentiated from subjects with the classical life-shortening form of cf. the remaining cases, termed "possible" or "borderline", are difficult to classify because there is poor agreement between sweat test results and prognosis on the one hand and the frequent presence of at least one cftr mutation of uncertain clinical relevance on the other. the term "cftr-related disorders" (cftr-rds) designates these varied conditions, which include multi-system disease and monosymptomatic disorders associated with cftr dysfunction but which do not fulfill the diagnostic criteria for cf ( ). this encompasses main clinical entities with cftr dysfunction: cbavd (congenital bilateral absence of the vas deferens), acute recurrent or chronic pancreatitis and disseminated bronchiectasis. diagnosis of cbavd is based on impalpable vas deferens on scrotal examination. even if in a proportion of men scrotal palpable vas deferens are present, surgical exploration reveals a fibrous cord or a non-permeable duct. cbavd males have either a severe and a mild/variable ( %) or two mild/ variable ( %) cftr mutations ( ) . approximately % of men with cbavd have a cftr mutation in one gene and the splicing variant ivs - t on the other allele, often in association with a longer polymorphic dinucleotide repeat, a combination that does not result in cf, but reduces levels of functional cftr protein in wolffian tissues, which constitutively produce less full-length cftr mrnas than other tissues ( ) . about % of patients with idiopathic chronic pancreatitis or recurrent acute pancreatitis are found to carry cftr mutations. no specific cftr mutations have been reported, but rare class or class mutations are often found ( ) . an increased incidence of cftr gene mutations has been found in bronchiectasis. according to the studies, at least cftr mutation is found in - %, and mutations in - % of cases. mutations found are mostly uncommon and likely to result in residual cftr function ( ) . no specific cftr mutation is associated directly with bronchiectasis. ( ) . these patients must be monitored carefully for development of any complications and appropriate therapy implementation. it should be pointed out, however, that labeling patients with mild or unclear manifestations with a cf diagnosis may have negative implications such as psychological, reproductive, social, employment, and insurance issues. therefore the explanation of the diagnostic challenge, including also prognosis, must be fully and honestly explained to the patient and or his family. department of pediatrics, cf and pcd center, hadassah hebrew-university medical center, mount scopus, jerusalem, <eitank@hadassah.org.il> effective mucociliary clearance (mcc) in the respiratory system requires proper mucus production and functioning airway surface fluid layer as well as competent and coordinated ciliary beating. the vital role of these systems is best demonstrated in patients with genetic defects such as primary ciliary dyskinesia (pcd) and cystic fibrosis (cf), both of which are characterized by impaired mcc leading to acute and chronic sino-pulmonary infections. pcd is caused by defects in genes that encode the structure or regulate the movement or function of the respiratory cilia. cf is caused by mutations in the cftr gene causing abnormality in the airway surface fluid layer, with production of thickened and viscous mucus leading to impaired mcc. in both diseases, recurrent and chronic respiratory infections and persistent inflammation cause progressive lung damage. most patients with cf suffer from pancreatic insufficiency (cf-pi); however, approximately % have sufficient pancreatic enzyme production to maintain normal fat absorption (cf-ps). patients with pcd are similar to patients with cf-ps in that they have normal pancreatic function, and are usually without the nutritional deficiencies that are typically associated with more severe pulmonary disease in cf. in addition, pcd and cf-ps are often diagnosed at a later age and have better survival compared to cf-pi ( , ). therefore when comparing cf and pcd, one should differentiate between patients with cf-pi and cf-ps. santamaria et al. compared chest hrct scan scores for patients with pcd and a group of age-and gender-matched cf patients and showed that patients with pcd had significantly less structural damage than cf patients ( ) . a recent study comparing between pcd and cf-ps and cf-pi revealed that patients with pcd had disease severity in terms of pulmonary function and structural abnormality similar to patients with cf-ps, which was significantly less severe when compared to patients with cf-pi ( ). furthermore, when comparing structural abnormalities by hrct, there was a significant disparity in the distribution of the structural changes in the lungs between the three groups of patients: in pcd, the upper lung zones were relatively preserved and most changes were localized to the middle and lower lobes, whereas in cf-pi, the upper lobes were remarkably involved. in cf-ps, there was no characteristic distribution of the structural damage ( ) . other studies showed that in pcd, contrary to cf groups, there was no correlation between fev and ct score and between fev and age ( ) ( ) ( ) ( ) ( ) ( ) , which provides further support to the understanding that, in pcd, lung function is not a strong indicator of severity of lung disease and therefore, follow-up by low radiation chest hrct scans should be considered. it is important to note that, in general, patients with pcd receive less intensive therapy ( ) . they are not always followed regularly in specialized centers, and many are not adherent to routine treatments. the most common bacterial infection in pcd patients is h. influenzae, which is significantly less common in older cf patients ( , ) . in cf, chronic infection with p. aeruginosa is associated with a more severe lung disease ( ) . however, among patients with pcd, there was no correlation between p. aeruginosa infection and pulmonary function or hrct severity score, suggesting a different role for this microorganism in the pathogenesis of pulmonary disease in pcd ( ). bush et al. compared the mucous properties in both diseases and demonstrated that inflammation, measured by il- concentration, was greater in pcd sputa, and that there were no significant differences in biophysical or transport properties of sputum between the two groups; however, survival in patients with pcd was generally better ( ). ratjen et al. ( ) assessed the inflammatory response in the airways of cf and pcd patients during pulmonary exacerbation. in stable pcd patients, no significant differences were found in sputum inflammatory markers between individuals colonized with different bacterial pathogens. however, higher bacterial density for s. aureus and h. influenzae was found in patients with cf versus pcd, and the absolute neutrophil counts were higher in pcd patients. while sputum elastase activity was similar in pcd and cf at the time of exacerbation, it decreased with antibiotic therapy in pcd but not cf patients. thus, pcd patients differ from those with cf in their responses to treatment of pulmonary exacerbations, with higher neutrophil elastase activity persisting in the cf airways at the end of treatment. joenesen et al. ( ) measured the difference in breath profiles of patients with pcd and cf, with and without distinct chronic lung infections, using an electronic nose. no significant difference was found between the breath profiles of pcd patients with a chronic pa infection and pcd patients without a chronic infection. however, there was a significant difference between the breath profiles of cf patients with a chronic pa infection and cf patients without a chronic pa infection, suggesting a different response to infection between pcd and cf. in conclusion, although pcd and cf are both characterized by impaired mcc and respiratory infections, patients with pcd have a different lung disease expression compared to patients with cf-ps and with cf-pi, as assessed by fev , hrct, nutritional status and bacterial infection on sputum cultures. in pcd, normal fev can be maintained over time in spite of severe structural damage. this suggests a greater involvement of the large airways in pcd and the small airways in cf. furthermore, p. aeruginosa infection is less common in pcd than in cf. bronchopulmonary dysplasia (bpd) is the most important complication following mechanical ventilation in preterm infants and no definite therapy can eliminate this complication. although the mechanism is not completely clear, pulmonary inflammation is believed to play a central role in the pathogenesis. glucocorticoid is one of the most effective therapies to treat or prevent bpd. however, systemic glucocorticoid therapy is not generally recommended because of long-term adverse events ( , ). our previous pilot study in neonates and studies in animals indicated that surfactant can be used as a vehicle to deliver a topical glucocorticoid, budesonide, to the lung periphery and effectively suppress lung inflammation and lung injury ( . . ). the mechanism for the effective delivery of budesonide using surfactant as vehicle is based on a physical phenomenon, the "marangoni effect": in the interface between high and low surface tension, a convection force is generated and this force can be used as a vehicle to facilitate the delivery of medication ( ) . this is an important delivery method because inhaled glucocorticoid is technically difficult and the effect has been shown to be limited ( , there was no significant difference between the groups during the study in serum electrolytes, glucose, bun and in blood pressure, and in physical growth. there was no significant difference between the groups in neuromotor function, and in mdi, pdi and in neurodevelopmental impairment (ndi) score when examined at - years of corrected age. we concluded that in very low birth weight infants with severe respiratory distress syndrome, intra-tracheal administration of surfactant/budesonide compared with surfactant alone significantly decreased the incidence of bpd or death without apparent short term or long term adverse effect. further large-sample, double-blind trials are warranted. measuring lung function in "non-collaborating" children has always been one of most difficult tasks for pediatric pulmonologists. this is because young children are not able to perform the voluntary forced expiratory maneuvers generally used in adults and schoolchildren. in infants and children up to years, this problem has been generally overcome by the use of sedation, although this contributes to make lung function measurements less suitable for routine clinical use in this age group. preschool children ( - years) are too old to be sedated and yet too young to properly perform the forced expiratory maneuvers required for spirometry. for this age group, several techniques that just require tidal breathing have been implemented during the past decades. the american thoracic society/european respiratory society (ats/ers) working group on lung function in young children has published technical recommendations for most infant ( , ) and preschool techniques ( ) and their clinical applications have also been recently summarized ( ) . this lecture will focus on the most used pulmonary function tests (pfts) in infants and preschool children. chloral hydrate ( - mg/kg, maximum g) is commonly used to sedate infants and young children up to years for performing lung function testing. however, chloral hydrate is no longer available in the u.s.a. and the use of other sedatives might lead to different results ( ). the most commonly used pfts in infants are the raised volume rapid thoracoabdominal compression and infant plethysmography. other pfts that are performed during tidal breathing (e.g.: tidal breathing measurements, multiple breath washout, forced oscillation technique) are more suitable to be used without sedation, especially in younger infants. the raised volume rapid thoracoabdominal compression (rvrtc) allows for the measurement of forced expiratory flow and volume in sedated infants ( ) . repeated inflations using a pressure of cmh o are applied through a facemask and an inflatable jacket is then activated to rapidly compress the infant's chest and abdomen to obtain forced vital capacity (fvc), forced expiratory volume in . seconds (fev . ) and forced expiratory flow (fef) at defined proportions of fvc. to ensure that flow limitation has been reached, the inflation pressure of the jacket is increased at each maneuver until no further increase in flow is noticed. recently published reference equations using a current commercially available device ( ) will improve the interpretation of the results. rvrtc has been successfully used in children with all kinds of respiratory diseases, including children with cystic fibrosis (cf), children born prematurely, and those with recurrent wheezing ( ), showing its capability to distinguish disease populations from healthy control subjects and to detect lung function changes in clinical intervention trials. however, its long-term clinical utility still remains to be established. moreover, the need for sedation along with the time and resource intensity required are other important limitations for its use in routine clinical practice ( ) . infant plethysmography is used to measure functional residual capacity (frcpleth) in sedated infants ( ) . specific airway resistance (sraw) can also be measured, provided that a proper electronic thermal compensation is applied to the system to account for thermal artifacts. this technique is based on the same principle (boyle's law) as plethysmography for older subjects and uses an infant whole body plethysmograph where the infant lies supine breathing through a facemask sealed with silicon putty ( ). infant plethysmography has been successfully applied to children with lung disease, especially cf and bronchopulmonary dysplasia (bpd) ( ). however, as for rvrtc, its long-term clinical utility remains to be ascertained and its role in routine clinical practice is hence very limited. preschool children ( - years) are too old to be sedated, but also too young to properly perform the forced expiratory maneuvers required for spirometry. for this age group, several techniques that just require tidal breathing have been implemented during the past decades, allowing for lung function to be measured in awake children ( ) . also, modified acceptability criteria for spirometry have been proposed for the use in preschool children ( ) . it is important to highlight that the feasibility of any lung function technique in preschool children strongly depends on the capability of the operator of keeping the child quiet and focused ( ). spirometry has been proposed for preschool children using modified acceptability criteria ( ). since the forced expiratory volume in second (fev ) often cannot be obtained in preschoolers due to their different lung physiology, the use of fev in . (fev . ) or . seconds (fev . ) is recommended in this age group. also, fvc should not be reported if flow stops at more than % of peak flow (early termination), but fev may still be reported. less stringent repeatability criteria have also been proposed in preschool children: at least two acceptable maneuvers should be obtained with the two fvc and fev within ml or %, but in case of a single acceptable maneuver, this should be recorded nevertheless ( ) . spirometry is reported to be feasible in - % of - year old children, but its feasibility tends to be much lower in younger children ( ) . global multiethnic reference equations including preschool children have recently be published ( ) . spirometry has been reported to discriminate healthy controls from preschool children with cf and with recurrent wheezing, although substantial overlap between groups may occur and bronchodilator response appears to be more sensitive than baseline values ( ). however, a careful and rigorous approach to the use of spirometry must be taken in preschool children and several gaps in our knowledge still limit the application of this technique to clinical practice in this age group ( ). the interrupter technique is based on the principle that a sudden flow interruption at the mouth during tidal breathing would make alveolar pressure rapidly equilibrate with mouth pressure, thus allowing an estimation of alveolar pressure by measuring mouth pressure. the interrupter resistance (r int ) is then calculated dividing the change in mouth pressure by the flow measured immediately before the interruption ("classical" technique) or immediately after the interruption ("opening" technique). measuring r int has been proved to be particularly suitable for preschool children, its feasibility being generally higher than % in this age group ( ). proper reference values have been published ( ) and cut-off values for the bronchodilator response have also been reported. r int is able to detect changes in the airway caliber and has been successfully used in preschool children with recurrent wheezing ( ). however, its utility in clinical care remains to be established, especially by longitudinal studies ( ) . the forced oscillation technique (fot) is used to measure the impedance of the respiratory system (z rs ) during tidal breathing by applying, through a mouthpiece and a filter, low-frequency pressure oscillations generated by a loudspeaker (usually - hz) ( ) . changes in flow and pressure measured at the mouth are used to calculate z rs and its two components, resistance (r rs , reflecting frictional losses) and reactance (x rs , reflecting elastic properties at low frequencies and inertial forces at higher frequencies). forcing signals based on sinusoidal waves or impulses have been used, both as single-frequency or composite signals. frequencies between and hz are considered to reflect the mechanical properties of the total airways. fot has a good feasibility in preschool children (> %) and several reference equations have been published ( ) . fot has been used in many studies on children with recurrent wheezing, showing a good capability in discriminating health from disease, especially when bronchodilator response is used ( ). however, for this technique as well, longitudinal studies on its clinical utility in young children are still needed ( ). the multiple breath washout (mbw) is based on the washout of an inert gas (typically n washout using % o ) to measure ventilation inhomogeneity and frc during tidal breathing ( ). non-resident inert gases have also been used. the lung clearance index (lci, the number of lung volumes expressed as frcs required to washout the inert gas) is the most commonly used mbw index. the general standard operating procedure for this technique has been recently reported ( ) . lci has a good feasibility in preschool children (nearly %). lci has been successfully used in preschool children with cf ( ), proving to be more sensitive than spirometry and plethysmography in detecting abnormal lung function. however, longitudinal studies on the clinical utility of mbw in preschool children are lacking ( ) and more data are needed before lci or other mbw indices can be recommended in the routine clinical management of patients with cf ( ). specific airway resistance (sraw) can be measured at tidal breathing in preschool children using a whole body plethysmograph. since sraw is the product of airway resistance by the thoracic gas volume, it can be calculated without the need to breathe against a closed valve ( ), provided that a proper electronic thermal compensation is applied to obviate the need for the panting maneuver. the measurement of sraw has a good feasibility in young children and reference values are also available ( ) . however, the lack of consensus on measurement methods and outcome measures makes it difficult to compare results among centers and methodological techniques are urgently needed for this technique. an accurate assessment of pulmonary function is now possible in infants and preschool children using a number of techniques. although these techniques have proven to be powerful research tools, further studies are needed to ascertain their utility in the clinical care of infants and young children with lung disease. . past studies have shown that persistent echocardiographic evidence of ph beyond the first few months of life is associated with up to % mortality in infants with bpd. the association of ph with poor survival in bpd has continued into the recent era of the "new bpd," especially in infants with severe disease who require prolonged support with mechanical ventilation. thus, developing insights into the pathogenesis and pathobiology of ph and related pulmonary vascular disease (pvd) in bpd continue as an important challenge and may help to improve early and late cardiopulmonary outcomes after preterm birth. mechanisms that coordinate normal vascular growth and alveolarization during development or cause abnormal lung growth in bpd are poorly understood. disruption of key signals between airway epithelium and endothelial cells can alter vascular and alveolar growth, resulting in decreased arterial and airspace structure. for example, hyperoxic lung injury in newborn animals decreases expression of the critical proangiogenic and endothelial cell survival factor, vascular endothelial growth factor (vegf). early impairment of vegf production inhibits vascular growth and impairs endothelial function, which leads to ph. in addition, disruption of angiogenesis due to adverse antenatal factors, such as chorioamnionitis, preeclampsia or maternal smoking, and postnatal events after premature birth, can cause vascular injury that not only lead to ph but can also impair distal lung growth. ongoing laboratory studies suggest that the developing endothelial cell plays a key role in the regulation and coordination of epithelial growth and distal airspace structure through the production of critical "angiocrines," such as nitric oxide (no), hepatocyte growth factor, vitamin a, insulin growth factor- and others. thus, since angiogenesis is necessary for normal alveolarization, it has been suggested that protecting the developing pulmonary vasculature from early injury may not only lower pvr and improve gas exchange, but may enhance distal lung growth and improve long term outcomes. abnormalities of the pulmonary circulation in severe bpd include altered tone and reactivity, structure and growth, which can cause right heart failure, impaired gas exchange, pulmonary edema, decreased exercise capacity and other clinical problems. physiologic abnormalities of the pulmonary circulation in bpd include elevated pulmonary vascular resistance (pvr) and abnormal vasoreactivity, as evidenced by the marked vasoconstrictor response to acute hypoxia and by impaired gas exchange due to abnormal distribution of lung blood flow. abnormal pulmonary vascular structure also contributes to high pvr due to increased smooth muscle cell hyperplasia and altered vascular compliance caused by increased production of an abnormal extracellular matrix. growth of the distal lung circulation is abnormal in infants with severe bpd, and decreased arterial growth (angiogenesis) reduces vascular surface area that further impairs gas exchange and increases the risk for the development of ph and impaired exercise capacity in older children. prominent bronchial or other systemic-to-pulmonary collateral vessels were noted in early morphometric studies of infants with bpd, and can be readily identified in many infants during cardiac catheterization. although these collateral vessels are generally small, large collaterals may contribute to significant shunting of blood flow to the lung, causing edema and need for higher fio . in addition, recent autopsy studies suggest the presence of striking intrapulmonary anastomotic, or "shunt," vessels that link the distal pulmonary and bronchial vessels, and may contribute to poor oxygenation. past clinical studies have further shown that metabolic function of the pulmonary vasculature is impaired in bpd, as reflected by the lack of pulmonary clearance of circulating norepinephrine during passage through the lung, which may contribute to left ventricular dysfunction and systemic hypertension. clinical studies have recently shown that early echocardiographic findings of pvd after preterm birth are strongly associated with the development and severity of bpd and ph at weeks corrected age. interestingly, these findings were not only associated with a worse respiratory course during the initial hospitalization, but also late respiratory outcomes, including respiratory exacerbations, hospitalizations and the need for asthma medications. ongoing studies are exploring the impact of ph-specific drug therapies, such as sildenafil and other agents, on ph and related complications. thus, pvd in preterm infants with bpd is characterized by altered lung vascular development, growth, structure, and function, which precede the onset of measureable ph. pvd due to disruption of normal pulmonary vascular development in association with preterm birth is an important determinant of the pathobiology of bpd and contributes significantly to morbidity and mortality. exposure to adverse stimuli during the antenatal and/ or early postnatal periods impairs normal pulmonary vascular development and creates an imbalance between risk and resiliency factors. recent studies have revealed the magnitude of ph in preterm infants, but many aspects of pvd remain understudied, and ongoing investigations continue to explore risk factors, mechanisms of disease, and long-term outcomes. prospective studies are needed to definitively establish standardized clinical criteria for pvd and ph in bpd, and to determine the best methods for early diagnosis, risk stratification and disease monitoring. larger collaborative studies and improved clinical infrastructure to conduct these important investigations will provide answers to these critical questions. recent evidence suggests that cftr does not act as a pure ion channel but as a platform for multiple cellular signaling pathways. importantly, the protein interactomes of wt-and f del-cftr are rather different, and there is growing consensus that indirect measures that avoid the enhanced degradation of f del-cftr may restore its function. recently, we discovered that cftr orchestrates a proteostatic network that influences multiple cellular functions by acting as a hub protein. this hub-dysfunction model proposes that the proteostasis network is widely deranged, both in transgenic cf mice and in primary nasal epithelial cells freshly collected from cf patients bearing f del-cftr either in homozygous or compound heterozygous form, at two levels. firstly, autophagy, the major mechanism determining cytoplasmic protein turnover, is blocked due to tissue transglutaminase (tg )-mediated depletion of the essential autophagy-related protein beclin (becn ), leading to secondary accumulation of the autophagic substrate sqstm /p . secondly, peptide fragments released from proteolytically-cleaved f del-cftr provoke an over-activation of a pleiotropic protein kinase (protein kinase ck ), which in turn contributes to f del-cftr degradation. combined inhibition of tg by cysteamine, which is fda-approved for the treatment of cystinosis, and over-active ck by the over-the-counter greentea flavonoid epigallocatechin-gallate (egcg) respectively rescue and stabilize a functional f del-cftr protein at the pm, both in mice and in primary nasal cells from cf patients bearing f del-cftr or other class ii-cftr mutations. pre-clinical evidence on transgenic mice has provided the mechanistic proof-of-concept for using this combination of proteostasis regulators as an alternative cftr-repairing therapy. moreover the combination treatment reduces lung inflammation and this beneficial effect persists up to weeks following cysteamine withdrawal provided that egcg was administered during washout. this prompted an open-label phase- trial to assess the individual response to the synergistic combination of cysteamine and egcg in cf patients bearing different cftr mutations. the combination treatment was well tolerated and decreased sweat chloride from baseline while increasing the abundance and function of cftr protein and restored autophagy in nasal cells. notably, the treatment decreased cxcl and tnf-a in the sputum and improved respiratory function. these positive effects were particularly strong in patients carrying f del-cftr (or other class ii) mutations in homozygosity or heterozygosity, whereas patients with class i cftr mutation failed to respond to therapy. altogether, these results suggest that the combination treatment acts "on target", according to the hypothesis underpinning our drug design. discordance in therapeutic response rate complicates mutation-specific approaches, thus entailing the need of patient-centered (personalized) approaches to assess drug efficacy. testing the putative individual responsiveness to treatment by appropriate biomarkers before in vivo therapy should support the decision to treat. we show that restoring cftr function in vitro in nasal cells in response to cysteamine plus egcg, is highly predictive of whether the combination treatment will restore cftr function in vivo. hence, this in-vitro assay may constitute a tool to guide the clinical development of cf treatments, allowing to select patients for new therapeutic options. general frame for care infants with cf must receive care in an accredited cf care center. they must be reviewed in clinic frequently after diagnosis, for example once a month during the first months of age, every months until year of age, and every months thereafter ( ). after initial diagnosis, the cf center should contact the primary care professionals for regular ambulatory follow-up to implement therapeutic strategy. parents of infants with cf should be offered access to genetic advice and counseling. the standard childhood immunization schedule must be applied in accordance with national guidelines. anti-influenza vaccination is recommended for the infant from the th month of life and for all household members and healthcare providers. according to french guidelines, vaccination against chicken pox could be recommended. growth targets should reflect genetic potential, sibling height and local population demographics ( ) . french guidelines recommend to catch-up birth weight percentile at months ( ). at years, weight-for-height should be at the th percentile and height at the target height percentile (target height: average of the height of the parents plus . cm for boys and minus . cm for girls) ( ). energy intake evaluation should be performed by a dietician on a regular basis and adapted to achieve the objectives of weight-for-height growth. energy intake could be as much as % of the daily recommended calorie intake for the same age in the general population ( ). breast feeding is encouraged, all the more that recent data acknowledge its protective effect against pseudomonas aeruginosa infection ( , ). formula with hydrolyzed cow's milk protein is recommended in infants with risks of malabsorption, or severe undernourishment. sodium chloride supplementation is systematic, particularly in the case of breast feeding and should be adapted to natriuresis ( ) . it should be increased during periods of hot weather and all other causes of high salt loss (diarrhea, fever, ileostomy, etc.). at initial diagnosis, infants must have pancreatic function assessed by stool fecal elastase. if elastase is normal, repeat assessment is recommended. pancreatic enzyme replacement therapy should be started at diagnosis in case of clinical symptoms of exocrine pancreatic insufficiency even before obtaining the results of the elastase assay. the starting dose could be . iu lipase per ml of milk. in case of persistence of symptoms of pancreatic insufficiency despite a maximum dose of . ui/kg/day of lipase, it may be necessary to evaluate the patient's compliance and the methods of conservation and administration of the pancreatic extracts. in case of poor weight-for-height growth despite an adapted substitutive pancreatic opotherapy, an evaluation is necessary including a dietetic review, a search for sodium insufficiency and other etiologies of malabsorption. in case of persistence of symptoms of exocrine pancreatic insufficiency despite a maximum dose of . ui/kg/day of lipase and in the absence of other etiologies, the administration of gastric secretion inhibitors may be envisaged. bacterial cultures of bronchial flora should be performed at each session of physiotherapy or, in case of abnormal clinical status, ideally on bronchial secretions expectorated or obtained by sputum induction ( ). a chest x-ray should be performed at baseline and annual assessment, and, in case of clinical abnormality. high resolution computed tomography should complete the assessment in case of clinical or radiological abnormality and/or at initial assessment according to local practice to detect early bronchiectasis ( ) . systematic respiratory physiotherapy is recommended from the time of diagnosis. the frequency of sessions of physiotherapy depends on the clinical status of the infant. regular therapy might be recommended even in the asymptomatic infant ( ). any evidence of respiratory infection justifies performing a respiratory culture and adapted antibiotic treatment of the isolated pathogens. infection by staphylococcus aureus sensitive to meticillin should be treated by adapted antibiotherapy. in case of isolation of s. aureus resistant to meticillin, a treatment aiming eradication is recommended. evidence of p. aeruginosa justifies systematic antibiotic treatment, even in the asymptomatic infant. although there is still no consensus, treatment might begin with an inhaled antibiotic, eventually associated with oral ciprofloxacin. in case of persistence of p. aeruginosa after initial therapy, or if the infant presents with severe clinical signs, intravenous antibiotics should be considered ( , , ) . for other pathogens, there is less clear agreement and treatment should be guided by local policies. in the absence of clinical improvement despite an adapted antibiotherapy, bronchial sampling by bronchoalveolar lavage should be considered and non-infectious causes should be searched for, including gastroesophageal reflux, asthma and an ent cause. respiratory syncytial virus (rsv) may have adverse effects on respiratory status in patients with cf ( ) . there is insufficient evidence to support systematic recommendation of palivizumab in the cf infant even if some small studies suggest that there could be benefit from the use of rsv prophylaxis in infants with cf ( ). us and french guidelines state that palivizumab could be discussed, namely for the infant of less than months of age during an epidemic period ( , ). finally, dornase alfa, % hypertonic saline might be used in symptomatic infants ( ) . with increasing numbers of infants with cf being diagnosed by newborn screening across most of europe and in north america, we will have the opportunity for large cohort follow-up and randomized controlled trials. this will help to establish still lacking best available evidence to harmonize therapeutic strategy in infants newly diagnosed with the final aim of improving clinical status at later ages. department of pediatrics, cf and pcd center, hadassah hebrew-university medical center, mount scopus, jerusalem, israel <eitank@hadassah.org.il> bronchiectasis is the distraction of the normal anatomy of conducting airways that results in impaired mucociliary clearance leading to chronic cough, sputum production, and recurrent infections and inflammation that cause further damage to the bronchial and bronchiolar walls leading to a vicious cycle of airway injury. the prevalence of non-cf bronchiectasis (ncfb) in children differs between developed and poor countries. in the developed world, the most common cause of bronchiectasis in children is cystic fibrosis (cf), followed by primary ciliary dyskinesia and immune deficiencies. however, up to half of cases remain without a known etiology. in developing countries, a systematic review of children ( ) demonstrated that an etiology was identified in % of children, with a previous severe pneumonia of bacterial or viral etiology and b-cell defects as the most common identified disorders. bronchiectasis should be suspected in patients who present with chronic productive cough of mucopurulent sputum. physical findings in bronchiectasis patients are nonspecific but may include crackles and wheezes on lung examination and clubbing of the digits. pulmonary function testing results generally show airflow obstruction. the diagnosis of bronchiectasis is confirmed by hrct scan which is now the gold standard for diagnosis. these include bronchial dilatation (an internal bronchial diameter greater than the diameter of the accompanying bronchial artery [i.e., the "signet ring" formation]) and a lack of bronchial tapering on sequential slices ( ) . patients in whom bronchiectasis has been diagnosed should be evaluated for potential underlying causes. they need to undergo chest ct scan to define the extent of their disease. patients with focal disease require bronchoscopy to evaluate for a localized airway obstruction as the cause of the bronchiectasis. patients with diffuse bronchiectasis should be assessed for underlying systemic abnormalities including congenital disorders, chronic aspiration, impaired mucociliary clearance and systemic or local innate immune dysfunction. all patients with bronchiectasis should have a regular routine microbiological examination of their sputum for routine bacterial and ntm organisms. pulmonary exacerbations of ncfb are known to be associated with poor outcomes, and infections are common causes. gram-negative bacteria are isolated more frequently in patients with ncfb, with h. influenzae and p. aeruginosa representing the majority of identified species. however, up to % of sputum samples fail to grow any pathogenic bacteria ( ). patients with sputum samples dominated by p. aeruginosa (pa) had a higher frequency of exacerbation and poorer lung function compared to patients whose samples were dominated by other organisms ( ). nontuberculous mycobacteria (ntm) are opportunistic pathogens that afflict patients with preexisting lung disease; in particular those with ncfb, shown in a meta-analysis by chu et al. to be prevalent in nearly % of the patients ( ). respiratory viruses were found in nearly % of exacerbations. the goals of bronchiectasis treatment are to reduce the number of exacerbations and to improve quality of life. if an underlying systemic etiology such as immune deficiency is identified, it should be addressed. pharmacologic agents and the mechanical mobilization of secretions have been evaluated to a limited degree in patients with non-cf bronchiectasis. short-acting or long-acting bronchodilator adrenergic and anticholinergic agents are commonly prescribed, but there have been no randomized controlled trials to support their use. pulmozyme had adverse effects when studied in patients with non-cf bronchiectasis. inhaled mannitol showed improved time to first exacerbation and quality of life. nebulized hypertonic saline solution ( %) have shown promise in the treatment of patients with both cf and non-cf bronchiectasis, but long-term prospective trials are needed. the role of the use of maintenance antibiotic therapy is uncertain in patients with non-cf bronchiectasis. rotating oral antibiotic strategies have been commonly used. for exacerbations, antibiotic therapy should be tailored to their sputum microbiology results. severe exacerbations, particularly in patients who are infected with organisms that are resistant to therapy with oral quinolones, require iv antibiotic therapy. azithromycin has been shown to attenuate muc ac and muc gene expression, thereby suppressing the synthesis of mucin on human airway epithelial cells. clinically, this was demonstrated in a study that found that mean -hour sputum volume and qol were significantly lower in patients with bronchiectasis after weeks of azithromycin compared with control subjects ( ) . a recent randomized, double-blind, placebo-controlled trial in adults assigning patients to receive mg azithromycin or placebo three times a week for months, showed that azithromycin significantly reduced the exacerbation rate with no significant effect on fev ( ) . based on the above and other studies, it is recommended that all patients with ncfb be treated with azithromycin. long-term inhaled antibiotics are used for patients with uncontrolled ncfb, but until more recently, data on their efficacy have been lacking. the use of mechanical aids, including chest physical therapy with postural drainage, active cycle of breathing, oscillatory positive expiratory pressure devices, and high frequency assisted airway clearance, also constitute potential adjunct therapies for patients with bronchiectasis. though these modalities are considered to be standard therapy for patients with cf bronchiectasis, their utility is less well proven in patients with non-cf bronchiectasis. it was shown that comprehensive medical care in children with ncfb was associated with a decrease in exacerbation rates ( ) . these findings further exemplify the importance not only of identifying ncfb in pediatric patients, but also of ensuring that they receive close surveillance. treatment burden with lack of immediate apparent outcomes cause patients to avoid daily therapy and seek therapy only for exacerbations. resectional surgery and lung transplantation are rarely required. surgical treatment has classically been an option for patients who have localized bronchiectasis with persistent symptoms despite maximal therapy, or recurrent infections with resistant pathogens ( ). the prognosis for patients with bronchiectasis is variable given the heterogeneous nature of the disease. because there are so few randomized controlled trials of therapies for non-cf bronchiectasis, patients must be evaluated and treated on an individual basis in a tailored, patient-focused approach in a specialized center to optimally evaluate and treat individuals with bronchiectasis. in humans, the dominant innervation to the airways is provided by the parasympathetic vagus nerve, whose activation induces the release of acetylcholine [ ] . acetylcholine, the primary parasympathetic neurotransmitter in the airways, interacts predominantly with nicotinic receptors and the five muscarinic receptor subtypes. in addition to its well-known functions, i.e. bronchoconstriction and mucus secretion regulation, there is evidence that acetylcholine might also modulate inflammatory cell chemotaxis and activation and participate in signaling events that lead to airway wall remodeling [ ] . these findings can have significant implications for anticholinergic therapy of diseases characterized by airway inflammation, bronchial obstruction and mucus hypersecretion, since a variety of data indicate that the function of muscarinic receptors is altered in these patients. nicotinic acetylcholine receptors (nachrs) are ligand-gated ion channels, formed by five homologous or identical subunits, arranged to form a central ion channel [ , ] . depending on the subunit composition, nachrs show different kinetics and pharmacological properties. in lung tissues, the "muscle" nachrs are localized at the neuromuscular junctions of the smooth muscle cells, whilst the "neuronal" nachrs are expressed by autonomic ganglia, but also by almost every cell type, including bronchial and alveolar epithelial cells, endothelial cells, pulmonary neuroendocrine cells, submucosal glands, airway and vascular smooth muscles, fibroblasts and alveolar macrophages [ ] . although nachrs are classically linked to the depolarization of the plasma membrane required for neurotransmission, non-neuronal nachrs in the lung act most frequently as calcium channels and have been linked to regulatory proteins controlling cell proliferation [ ] [ ] [ ] . the functional role of nachrs is particularly complex and depends on subunit composition, dose response, and duration of ligand stimulation. although nachr activation often leads to a positive feedback loop that induces receptor expression, chronic stimulation of nachrs can produce channel desensitization and decreased activity. the majority of studies of nachr function in the lung are related to the effects of nicotine, i.e. to tobacco-induced mutagenesis and lung carcinogenesis, whilst little is known on the physiological functions in regulating lung growth and repair, airway epithelial cell proliferation and differentiation and electrolyte transport [ ] . muscarinic receptors belong to the large family of g protein-coupled receptors, characterized by seven transmembrane domains. out of the five subtypes identified, only m , m and m receptors have been detected in the airway and lung tissues of most mammals, including humans. almost all airway and lung cell types express muscarinic receptors. m receptors are present mainly in the peripheral lung tissue and in the alveolar walls: they are expressed by airway epithelial cells, where they modulate electrolyte and water secretion, by goblet cells, where they regulate mucus production, and by the ganglia, where they facilitate parasympathetic neurotransmission. m and m receptors represent the major populations in the large airways. m receptors are expressed by neurons, where they function as autoreceptors inhibiting the release of acetylcholine from both preganglionic nerves and from parasympathetic nerve terminals. in airway smooth muscles, they modulate different ion channels involved in cell contraction, effects that require concomitant m receptor-mediated release of calcium from intracellular stores. in fibroblasts and smooth muscles, m receptors stimulate cell proliferation and modulate cellular responses associated with airway remodeling [ , ] . m receptors are the dominant receptor subtype in the regulation of airway smooth muscle contraction and of mucus secretion from submucosal glands and goblet cells [ ] . m receptors can also favor airway smooth muscle proliferation, increasing the responses to epidermal growth factor and platelet-derived growth factor [ ] . acetylcholine, in addition to the parasympathetic nerve, is also synthesized and released by a large number of non-neuronal cells, including neuroendocrine, ciliated, basal and secretory epithelial cells where it can act as an autocrine or paracrine signaling molecule. secretory and ciliated cells release acetylcholine into the luminal periciliary fluid, whereas endocrine and basal cells secrete acetylcholine basally [ ] . current knowledge suggests that the local auto/paracrine production of acetylcholine by epithelial cells may play a role in regulating various aspects on the innate mucosal defense mechanisms, including mucociliary clearance. acetylcholine is known to increase ciliary beat frequency in the airways and to modulate the release of inflammatory mediators by these cells through m receptors and to affect inflammatory cells involved in the pathogenesis of obstructive airway diseases [ ] . expression of muscarinic receptors has also been shown by most inflammatory cells, including macrophages (m -m ) , t-and b-lymphocytes (m -m ), mast cells (m ), neutrophils (m -m ) and eosinophils (m ). in these cells, muscarinic receptors appear to be involved in cell proliferation and release of pro-inflammatory mediators [ , ] . arteries, veins and bronchopulmonary anastomoses also express muscarinic receptors (m ) and dilate in response to acetylcholine released by vagal nerve stimulation. the postganglionic nerve fibers do not form defined synapses to their target cells but a terminal meshwork called 'autonomic plexus' with numerous varicosities, called sites of transmitter release, in variable and only rarely close contact to cells, such as airway smooth muscle [ ] . release of acetylcholine from the parasympathetic nerve terminals in the airways appears to be under complex prejunctional regulatory mechanisms. the available data indicate that acetylcholine release can be enhanced by a variety of pro-inflammatory mediators (histamine, bradykinin, neuropeptides) and by b -adrenergic agents, whist it is under the inhibitory control of muscarinic autoreceptors and downregulated by eicosanoids, such as pge , opioids, nitric oxide and a -adrenergic agents [ ] . the activity of m receptors in smooth muscle appears to be spared or even increased in asthmatics, possibly because of a greater affinity of the acetylcholine binding site. there is also no evidence that muscarinic receptors are overexpressed or upregulated in airway smooth muscle in disorders characterized by bronchial obstruction or hyperresponsiveness although an acquired loss or impairment of neuronal m receptor function may be involved in their pathogenesis [ ] . these functional changes occur after exposure to allergens, infectious agents (viruses) or pollutants (ozone) and result in increased acetylcholine release from parasympathetic nerves [ ] . m autoreceptors dysfunction in allergic asthma is caused by the eosinophil basic protein released by activated eosinophils that, upon binding to m autoreceptor sialic acids, acts as an allosteric antagonist [ ] . with the same mechanism, an early recruitment and activation of eosinophils is thought to cause the airway hyperreactivity that follows environmental ozone exposure [ ] . in contrast, viral respiratory infections are purported to induce bronchial hyperresponsiveness through different mechanisms, including: a) the inhibition of m receptor synthesis, mediated by the release of interferon-g by activated cd þ t-lymphocytes; b) the production of neuraminidase, that determines functional impairment of m receptor activity by cleaving their sialic acid; c) m receptor dysfunction, caused by the activation of the substance p (nk ) receptor overexpressed by influenza, parainfluenza and respiratory syncytial virus [ ] . interestingly, increased substance p production has been reported in patients with asthma and gastroesophageal reflux, a disorder that recognizes vagus-mediated oesophageal-tracheobronchial reflexes in its pathogenesis. experiments performed in humans have corroborated the relevance of pathogenesis of m autoreceptors in generating airflow limitation showing that m receptor selective agonists inhibit cholinergic-induced bronchoconstriction in normal individuals but not in asthmatic patients [ ] . defects in m autoreceptor activity may also explain bronchoconstriction induced by b-blockers in asthma. these drugs can increase cholinergic tone downregulating the action of endogenous catecholamines on b -adrenoceptors present on cholinergic nerves [ ] . thus, in extreme synthesis, the three muscarinic receptor subtypes expressed in the airways have different, somehow conflicting functions: m and m receptors facilitate cholinergic-induced events, including bronchoconstriction and mucus glands secretory activities, whilst m receptors have a feedback inhibitory function, regulating the release of acetylcholine from cholinergic nerve endings. this information is of great importance to understand the activity of the three anti-cholinergic agents that can be used to treat patients with reversible airway obstruction. two of these, ipratropium and oxitropium bromide, are short-acting and non-selective muscarinic antagonists. because of the lack in selectivity, they also block m receptors, increasing acetylcholine release, and therefore reducing the degree of their "useful" action on m and m receptors [ ] . in contrast, the more recent longacting anticholinergic drug tiotropium bromide is characterized by a kinetic selectivity for m and m receptors over m receptors: it dissociates rapidly from m receptors and very slowly from m and m receptors [ , ] . to date, the anti-cholinergic agents most commonly used to treat respiratory disorder in childhood is the "non-selective" ipratropium bromide which, alone or associated with inhaled b -adrenoceptors agonists, has been demonstrated to significantly improve pulmonary function and clinical outcomes in acute asthma, in preschool wheezing, although no long-term assessments have been included [ , ] . interestingly, preliminary data show that inhaled tiotropium bromide, once daily, is well tolerated and also improves lung function in pediatric patients with cystic fibrosis [ ] and in asthmatic adolescents, symptomatic despite inhaled corticosteroids [ ] . evidence from experimental models also suggest that tiotropium bromide may also modulate the acetylcholine-induced inflammatory and remodeling changes induced in the airways by a variety of stimuli, leading to hopes of having favorable clinical responses in other respiratory disorders. a relevant role in the pathogenesis of obstructive airway disorders is thought to be played by an increased acetylcholine release, at least in part due to m receptor dysfunction. the most commonly prescribed short-acting anticholinergic drug, ipratropium bromide, is not selective for muscarinic receptor subtypes. despite some efficacy in the most common pediatric airway diseases such as asthma and pre-school wheeze and cystic fibrosis, ipratropium bromide is not commonly prescribed as a standalone medication. the more recently introduced anticholinergic drug, tiotropium bromide, has advanced pharmacologic properties such as long duration of action and a functional selectivity for m and m receptors over m receptors, and has shown a good efficacy and safety profile in adult respiratory disorders, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. ongoing studies are now under way to define its therapeutic role for pediatric airway diseases. inhalation of smoke from datura strammonium, a member of the deadly nightshade family, was recommended for the treatment of asthma in th century ayuverdic literature. general gent, himself an asthmatic, on return from india in the early th century, was reported to have brought this therapy to england. strammonium and belladonna cigarettes were widely used to treat respiratory disease until the middle of the th century. however there were frequent side effects, including tachycardia, hallucinations, and even addiction. with the introduction of synthetic atropine derivatives with fewer side effects, there has been a renewed interest in anticholinergic therapy for asthma. bronchial smooth muscle tone is predominantly set by cholinergic activation. patients with asthma have increased bronchial smooth muscle tone and mucus hypersecretion, likely as a result of cholinergic activity. anticholinergic medications can relax smooth muscle in children with acute asthma, these drugs also appear to have anti-inflammatory properties, and may reduce goblet cell hyperplasia driven by neutrophil elastase À a feature of severe asthma known to be resistant to steroid therapy. the short-acting anticholinergic agents, ipratropium bromide and oxitropium bromide, have been used in asthma for many years, primarily for acute asthma in the emergency department. paradoxically, although the addition of an anticholinergic medication to a beta agonist can decrease acute asthma severity and hospital admission, studies suggest that continuing the anticholinergic while the patient is in hospital does not hasten recovery or decrease length of hospital stay. however these studies have been small and potentially underpowered. until the past decade, these results have dampened enthusiasm for studying anticholinergic medications as maintenance asthma therapy. this has changed with long-acting anticholinergic (lama) bronchodilators under investigation or are available for treating lung disease: these include tiotropium, aclidinium, glycopyrronium, glycopyrrolate and umeclidinium. the once-daily lama, tiotropium bromide, is demonstrated to improve lung function and decrease the risk of exacerbation in adolescents and adults with moderate to severe asthma, despite the use of inhaled corticosteroids (ics) and long-acting b -agonists (labas). in september , the fda in the united states approved tiotropium for the long-term, maintenance treatment of asthma in patients years of age and older. tiotropium by respimat soft-mist inhaler is now included in the global initiative for asthma report (gina) global strategy for asthma management and prevention. in phase studies, tiotropium improved asthma symptoms in % of enrolled subjects and decreased exacerbations by % whilst having a safety profile similar to that of placebo. studies also show that tiotropium was effective in improving pulmonary function (fev ) and decreasing asthma attacks in children age - with poor asthma control despite use of a medium dose of ics with or without a leukotriene modifier. there was no difference in effectiveness when comparing the fda-approved dose or . mcg (  . mcg) once daily tiotropium to a higher dose of mcg. initial studies in children younger than years do not appear to show benefit. with increasing knowledge about the diverse actions of the cholinergic system in asthma and the role of muscarinic receptors in the airway, we are gaining an increased appreciation of how anticholinergic medication can play an important role in treating children and adults with chronic and poorly-controlled asthma. the ers task force opted to not use the term asthma to describe preschool wheezing illness since there was insufficient evidence showing that the pathophysiology of preschool wheezing illness is similar to that of asthma in older ages. the task force referred to pre school wheezing and described episodic (viral wheeze) for children who wheeze intermittently and are well between episodes versus multiple-trigger wheeze for children who wheeze both during and outside discrete episodes.( ) we will therefore in our current discussion refer to this young age morbidity as an entity that should be discussed separately from asthma, acknowledging that much has yet to be learned on the nature of this entity. amongst the many mechanism of virus-induced airway hyperreactivity; a common phenomenon in pediatric practice related to this young age group, studies have shown that cholinergic overactivity such as through the modulation of substance p may mediate virus-induced airway hyperreactivity. virus-specific cd þ t lymphocytes may induce cholinergic activation through m receptor dysfunction.( ) hence anticholinergic medications may have a role in viral-induced wheeze with compounds that display selectivity for m and m muscarinic receptors over m receptors having advantages over nonselective compounds. a number of small studies addressed the role of anticholinergics in acute bronchiolitis but failed to show a role for this acute intervention. a study on infants who were randomly assigned to receive nebulized salbutamol, ipratropium bromide or placebo resulted in faster improved clinical scores and oxygen saturation levels in the bronchodilator groups than in the placebo, but no effect to change the natural course of the disease. ( ) in studies on this topic from , inhaled ipratropium bromide administered to wheezy children ( - months of age) improved lung function when measured by total body plethysmography and forced oscillation technique. ( ) the authors were unable to differentiate between responders and nonresponders by clinical or by physiological parameters, but submitted that the differential distribution of obstruction between small and large airways may underlie response or lack thereof; and that subjects with a predominance of large airways obstruction were the responders to inhaled ipratropium. a logical if unproven additional speculation was that anticholinergics decrease airway secretions and with it reduce large airway resistance. a cochrane review examining the effect of adding ipratropium bromide to b -agonists in wheezy infants ( ) suggested that the combined therapy improved symptom scores after hours compared to the use of jb -agonist alone. the ers task force cited above( ) offered evidence-based recommendations on the definition, assessment and treatment of wheezing disorders in preschool children. addition of ipratropium bromide to short acting b -agonists was suggested for patients with severe wheeze. in the review of the task force recommendations no reference was made to the use of anticholinergic medications. ( ) tracheobronchomalacia it is widely believed amongst pediatric pulmonologists that administration of b -agonists in infants with airway structural instability, predominantly tracheobronchomalacia is detrimental, while the use of anticholinergics for bronchodilatation is safe. this notion derives from a study of only infants with intrathoracic tracheomalacia, using infant pulmonary function testing and demonstrating that flows improved significantly after administration of metacholine but worsened after administration of albuterol.( ) these results suggest that in patients with abnormally collapsible tracheas or large bronchi, stimulation of the smooth muscle can improve airway stability, thereby increasing forced expiratory flows, while relaxation of airway smooth muscle by bronchodilators can exacerbate obstruction. the sole support for this observation comes from a review of a series of patients with tracheobronchomalacia from chile, in whom beta-agonist medications were discontinued while the anticholinergics were not. ( ) the effect of anticholinergic medication has not been assessed directly in any study, and thus whether this class of medications may have a different effect compared to beta -agonists in such pathology has not been established. further studies on the effect of the various bronchodilators for such pathologies using newer technologies to assess airway resistance (e.g., forced oscillation) should be undertaken. while more invasive and challenging, a technique of direct quantitative assessment of tracheal collapsibility in infants with tracheomalacia has been described, and may be the most adequate technique to answer this important clinical question. ( ) tiotropium bromide in pediatric use -asthma and the asthma-copd overlap syndrome ipratropium bromide has a limited role in childhood asthma, largely due to lack of selectivity. the more recently introduced long-acting muscarinic antagonists/anticholinergic (lama), tiotropium bromide, presents advanced pharmacologic properties such as selectivity for m muscarinic receptors over m receptors and long duration of action. a high safety profile and increasing evidence of efficacy have rendered it a mainstay medication for copd with an emerging role in adult asthma. few studies have emerged on its role in the treatment of childhood asthma and defining its therapeutic niche for pediatric airway diseases. in a recent -year randomized controlled trial, tiotropium add-on therapy in adolescents with moderate asthma, ( ) significantly improved lung function and was safe and well tolerated when added to at least ics maintenance therapy. a study of pediatric patients with asthma and chronic cough from an asthma center ( ) concluded that tiotropium can be beneficial in distinct patient populations: add-on therapy to asthmatics on maximal maintenance medication, an alternative to highdose inhaled steroids in patients who are experiencing significant side effects, and patients with bronchorrhea as their predominant symptom manifested by a chronic productive cough, the latter population is most likely explained by its drying effect on airway secretions. a recent editorial ( ) states "approximately in people worldwide are affected by asthma or chronic obstructive pulmonary disease (copd); once regarded as two distinct disease entities, these two conditions are now recognized as heterogeneous and often overlapping conditions. the term "asthma-copd overlap syndrome" (acos) has been applied to the condition in which a person has clinical features of both asthma and copd". in recent years multiple reports describing this interface between asthma and copd have been published recognizing that the demarcation line between these two entities is difficult to define. while the precise definition in various populations is still being worked out, and it is obvious that the majority of such patients are adults, there is early recognition that some pediatric populations, who are viewed as asthmatic, yet have no airway reversibility, may constitute an early presentation of the overlap syndrome. the mainstay therapies for copd are long-acting inhaled bronchodilators, including longacting b -agonists (labas) and lamas, with its characteristic member being tiotropium bromide. in patients with copd they are recognized as being equally effective because they reduce air trapping by relaxing airway smooth muscle as a result of reducing the effects of intrinsic cholinergic tone. it is therefore intriguing to speculate that once a better definition of the overlap syndrome emerges in pediatrics, an important role for tiotropium is likely to emerge particularly as a potential steroid sparing medication. peter d sly, ao mbbs, md, dsc, fracp, fers, f thor soc, fapsr, fahms p.sly@uq.edu.au the measurement of lung function is of major importance in clinical practice or respiratory medicine and in respiratory research. much has been learned about the risk factors underlying respiratory disease by measuring lung function in patients and comparing it with that in healthy controls. however, for managing an individual patient or assessing risk of disease onset or progression, it is necessary to know whether an individual's lung function is "normal" or "abnormal". over the years, a number of sets of normative equations have been produced by individual research groups in different parts of the world. these have been incorporated into commercially-available spirometers and used in populations other than those in which the data were collected. this situation was far from ideal, especially as some of the normative equations were many decades old. what is the gli? data were obtained from centers in countries (n ¼ , ) ; however not all could be used due to lack of data on ethnicity (which is illegal in france!), small numbers, missing data, lack of quality control and other factors. data were also pooled by region with data from europe, israel, while fev and fvc varied between ethnic groups, they did so proportionally, meaning that fev /fvc was independent of ethnicity. the lower limit of normality for fev and fvc showed age dependence that differed between males and females, reaching % by mid-childhood and falling progressively below % from approximately years of age. the rate of fall in the lower limit of normal for fev and fvc was identical for women but fvc declined more slowly in males. a ratio of fev / fvc > . is taken to indicate pathological airflow limitation; however, the proportion of the healthy non-smoking population with fev /fvc > . rises steadily to - % at years of age. how well do the gli reference equations predict lung function in people in individual countries? given that the gli reference equations were compiled by pooling data from a variety of sources, one might expect that the equations would provide good estimates of lung function for populations that were well represented in the pooled data whereas they may not for populations either not included or underrepresented in the pooled data. indeed this appears to be the case, with the gli equations adequately representing lung function in australasian caucasians , but not performing as well for adults in brazil , north africa , madagasca , and children in poland and peri-urban and rural india . further study is required to ascertain how widely the gli reference equations can and should be applied. what constitutes "normal" data? an important consideration when creating reference equations is what characterizes a "normal" population and who should be excluded? the dataset used to construct the gli reference equations excluded ever smokers, but is this reasonable? if - % of an adult population smoke, should they be excluded from equations designed to the lung function of that population? maternal smoking during pregnancy results in long-term reduction in lung function but is not generally taken into consideration when defining a healthy population. "healthy" children are often defined as those with no prior asthma or hospitalization for respiratory problems, born full term with birth weight ! . kg and asymptomatic at the time of testing . however, lum et al. recently demonstrated that with the exception of clear-cut factors such as current and chronic respiratory disease, including children born prematurely or with low birth weight, prior asthma and mildly symptomatic made little difference to the reference equations but increased the generalizability to the target population. this debate continues! the implications of switching to the gli equations will depend on how well the gli equations represent lung function of the local population. in poland, a switch from the polish reference values to the gli would see an increase in diagnosis of obstructive lung disease from . % to . % and an increase in diagnosis of restrictive lung disease from . % to . %. whether this represents an over-diagnosis with gli or an under-diagnosis with the old equations is a matter of clinical judgment. the impact on parents and children with cystic fibrosis is likely to be substantial as families tend to focus on lung function, especially fev expressed as a percent of predicted as evidence of the state of the child's lung disease. a change in number for a technical reason must be balanced against the likelihood of creating anxiety in the clinic population. respiratory symptoms are very frequent in infants and young children. special emphasis has been put on symptoms signaling bronchial obstruction and bronchial hyperresponsiveness as these may be associated with early onset of asthma. since the early s, several research groups have been focusing on early events in the development of asthma, especially seeking potential risk factors for predicting persistent symptoms. structural changes in the bronchial mucosa and lung function impairment in children with early obstructive symptoms have also been studied. it was documented that eosinophilic inflammation and remodeling (particularly epithelial basement membrane thickening and increased airway smooth muscle mass) are consistently present in patients with persistent asthma. interestingly, some markers of inflammation and even those of initial remodeling have already been described in children before the clinical diagnosis of asthma could be confirmed . this finding supports the hypothesis of remodeling not being a late consequence of a long lasting eosinophilic inflammation but that it may run in parallel with the development of asthma, if not even precede or initiate inflammation in the bronchial mucosa. this hypothesis was later supported by further research based on bronchial biopsies in infants. eosinophilic inflammation and some markers of remodeling have been documented in the bronchial mucosa of symptomatic children as early as in the second year of life . in a recent study, we were able to show that basement membrane thickening could be found even in young children at risk of developing asthma even without a history of recurrent wheeze . however, the significance of these findings in terms of long term prognosis still remains less documented. it is known that airway hyperresponsiveness in infancy is associated with persistent symptoms later in childhood . also, reduced airway patency at birth was shown to be linked to an increased risk of developing asthma and severe bronchial hyperresponsiveness by the age of years . long-term follow up of children investigated in infancy and reassessed in later childhood have so far showed that reduced baseline lung function in symptomatic infants was significantly associated with subsequent respiratory morbidity as well as with the need of anti-asthma medication at the age of years. in addition, the usage of inhaled corticosteroids at the age of years also seems to be in positive correlation with basement membrane thickening and increased number of mast cells in bronchial mucosa in bioptic samples taken earlier in infancy . this study has thus suggested that early morphological changes in the airway wall might indeed play a role in determining subsequent respiratory morbidity. on the other hand, at the next follow-up of these children at the age of eight years, the positive correlation between current respiratory symptoms and markers of inflammation and remodeling described in infancy was no longer found . this finding is consistent with the results of the follow-up of our group of children where we did not find a significant correlation between lung function (both fev and fvc) measured in preschool age and basement membrane thickness measured earlier in infancy and toddler's age both in the risk group and control group of children (unpublished data). more recently, airway smooth muscle mass has come into the center of interest of many researchers in respiratory medicine. smooth muscle hyperplasia and hypertrophy in the bronchial wall of patients with asthma are considered to be a consistent feature of bronchial remodeling. it is notably a possible dysfunction of newly formed smooth muscle bundles that deserves attention and more studies in this area are urgently required. the first works in children have shown that the increase in the airway smooth muscle mass in the bronchial wall might be associated with school age asthma . lately, another study has described a negative correlation between the airway responsiveness at the age of years and airway smooth muscle mass in infancy . however, this area of airway remodeling still remains poorly understood, especially with regard to its role in childhood asthma. based on currently available data, reduced lung function at birth or in early childhood is apparently associated with the persistence of symptoms and the decrease in lung function in later life. however, it still has not been reliably confirmed whether this low lung function has any correlation with early signs of airway remodeling. more long-term follow-up studies are needed in pediatric patients comprising both tissue biopsies taken in early age followed by longitudinal long-term lung function monitoring. nasal and sinus disease is universal in cystic fibrosis (cf). because nasal and sinus disease usually coexist, we will refer to this as "sinonasal disease". since the mucosa of the sinuses and upper respiratory tract and the mucosa of the lower respiratory tract are similar, disease may be similar in both locations and sinonasal disease could influence the severity of pulmonary disease. this view of the "universal airway" has been demonstrated in patients with pulmonary conditions, such as asthma and copd. in these diseases, an improvement in sinus health is reflected by an improvement in the lower airway disease. this has not been well studied in cf but the implications of this relationship combined with increasing life span makes an understanding of sinonasal disease important to the care of these patients. mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene in cf carriers appear to be independently associated with a higher prevalence of sinonasal disease; % of carriers reported chronic rhinosinusitis compared to the - % in the general population. the bacterial flora of the sinuses changes with patient age, can include anaerobes and fungi, and often mirrors the organisms present in the lower respiratory tract. a link between sinus infection and lower respiratory tract infection may contribute to morbidity following lung transplantation and immunosuppression. somewhat surprisingly, the prevalence of otitis media in cf appears to be no greater than in an age-matched general population. endoscopy and computerized tomography have broadened our understanding of how cf affects the sinuses. endoscopic sinus exams are almost always abnormal and give a better indication of the presence of nasal polyposis than physical examination of the nose alone. nasal polyps become more common with age and may represent a proliferative airway repair mechanism. sinus ct has demonstrated several anomalies characteristic of sinonasal disease in cf such as bulging or displacement of the lateral nasal wall, demineralization of the uncinate process, and hypoplasia or aplasia of the paranasal sinuses. serious complications of sinonasal disease in cf are rare and include mucoceles and periorbital abscesses. these usually require surgery. there are few randomized, controlled trials evaluating medical or surgical treatments of cf sinus disease. sinus surgery may provide some benefit, though there are no established selection criteria for appropriate candidates. the trend today in neonatal intensive care units (nicus) is to be as gentle and less invasive as possible in the care of neonates. this attitude takes place in every field of neonatology, and will discuss its implementation specifically in the respiratory care administered to premature infants with respiratory distress syndrome (rds). , prenatal corticosteroid therapy is recommended in all pregnancies with threatened preterm labor below weeks' gestation. recently, it was shown that such therapy could also be beneficial in late preterm infants as it significantly reduced the rate of a neonatal composite of respiratory treatments in the first hours or stillbirth or neonatal death within hours after delivery. at delivery, the term stabilization and not resuscitation is preferred for the vast majority of very preterm infants. only a minority of babies should require delivery room intubation. neopuff can be helpful in the delivery room and the transport to the nicu, and enables the administration of continuous positive airway pressure (cpap) and intermittent positive pressure ventilation under controlled conditions. recent large trials that reflect current practice (including greater utilization of maternal steroids and routine post delivery stabilization on ncpap) demonstrated less risk of bronchopulmonary dysplasia (bpd) or death when using early stabilization on ncpap with selective surfactant administration to infants requiring intubation. the comprehensive strategy to prevent bpd in the nicu is based on ventilatory and non-ventilatory measures. the ventilatory route allows an individualized endotracheal intubation approach. recent studies concluded that early nasal cpap (ncpap) is a safe alternative to immediate intubation even in extremely low birth weight (elbw) infants. , , endotracheal intubation and ventilation can result in significant damage to premature lungs and are independently associated with cerebral palsy. furthermore, despite new modes of ventilation and surfactant, bpd remains a significant morbidity and its incidence was correlated with the use and length of endotracheal mechanical ventilation. bpd in itself is associated with adverse neurodevelopmental outcome. thus, we need to avoid endotracheal ventilation, if possible. when the infant requires nasal respiratory support (nrs), we should aim for adequate oxygenation (spo of - %), permissive hypercapnia (paco of - mm hg, ph > . ) and gentle ventilation, similarly as in endotracheal ventilation. , ncpap is recommended as the early primary treatment of active respiratory distress syndrome (rds) (to avoid intubation or as part of the insure [intubation surfactant extubation] approach), or later, post extubation at rds resolution, in order to allow shortening of the duration of endotracheal ventilation and to treat apnea of prematurity. , recent studies , report comparable rates of bpd in elbw infants treated initially with ncpap as compared to endotracheal ventilation with surfactant administration. can we enhance ncpap and get better outcome for nrs by using nasal intermittent positive pressure ventilation (nippv)? nippv was defined as a method of augmenting ncpap by delivering ventilator breaths via nasal prongs. the rationale behind the use of nippv is the administration of "sigh" to the infant, thus opening microatelectasis and recruiting more ventilation units. it was shown that synchronized nippv (snippv) compared with ncpap may improve the patency of the upper airway, could activate the respiratory drive, improves thoraco-abdominal synchrony, stabilizes the chest wall, improves lung mechanics and decreases the work of breathing in premature infants. when nippv was compared to ncpap for the different indications of nrs, it was shown to enhance the potential of nrs. a recent meta-analysis demonstrated a relative risk reduction for intubation in the first hours in the nippv group compared with ncpap (rr . , % ci . , . ). the nippv trial was a large international multicenter randomized trial powered to study the important outcome of bpd, recruiting , extremely low birth weight babies, and it showed no difference between babies randomized to nippv compared with cpap. snippv vs. ncpap for later use, post extubation at rds resolution, as a "bridge" to spontaneous unsupported breathing, was shown to be more effective than ncpap. a pooled meta-analysis showed that snippv was more effective than ncpap in preventing failure of extubation [rr . ( . , . )] and the number needed to treat was only infants to prevent one extubation failure. snippv vs. ncpap, post extubation, also tended to decrease the rate of bpd. snippv may also be more effective than ncpap for apnea of prematurity. a meta-analysis regarding apnea of prematurity suggests that snippv is more efficacious with apnea that is frequent or severe. however, the studies performed addressed short-term outcomes and as such could not properly address the incidence of requirement for reintubation. thus, more studies are needed before recommending snippv as standard of care for apnea of prematurity. while non-invasive ventilation is probably safe, its success depends on gestational age. the data indicate that surfactant may still have a significant role in the treatment of rds, especially in elbw infants. recent studies reported on an intubation rate of $ % in their ncpap group in elbw infants. , , this leads us to the insure approach. this approach may allow the infant to benefit from both surfactant and nrs. a cochrane review concluded that the insure approach with ncpap compared with later selective surfactant administration, continued mechanical ventilation, and extubation from low respiratory support was associated with less need for mechanical ventilation, lower incidence of bpd and fewer air leak syndromes. another option for surfactant application to the trachea without endotracheal intubation was described by using a thin catheter in spontaneously breathing preterm infants receiving ncpap. this technique was reported to reduce the need for mechanical ventilation. there are ongoing trials with inhaled surfactant. to summarize, ncpap is still the most common mode of non invasive respiratory support worldwide. , the available evidence supports the preference of early or later use of nippv/snippv compared to ncpap because of minimizing the use and the length of endotracheal ventilation. there are data to suggest that this approach may also reduce the rate of bpd, however this has yet to be shown. the results of a large international rct comparing both primary and post-extubation use of nippv with ncpap, with a composite primary outcome of death or bpd at weeks' corrected age, indicate no additional benefit, or risk, conferred by nippv in comparison to ncpap. whether nippv/snippv is more beneficial than ncpap within the insure approach needs to be shown. recently, heated, humidified high-flow nasal cannula (hhhfnc) is frequently used as a mode of nrs. high flows result in washout of anatomical and physiological dead space and contribute to improved fractions of alveolar gases with respect to carbon dioxide as well as oxygen and decrease the work of breathing and the energy cost of gas conditioning. hhhfnc probably creates positive end expiratory pressure (peep) that may contribute to its beneficial effect. however, the peep that is not monitored had raised concerns regarding the safety of hhhfnc in terms of air leak. recent prospective studies support the notion that hhhfnc is as effective as ncpap for early stages of rds, post extubation and for apnea of prematurity. yet, more studies, especially in the initial treatment of rds and in elbw infants, are needed before adopting hhhfnc as an alternative mode of nrs in these conditions. new modes of nrs such as neurally adjusted ventilator assist (nava), and nasal high frequency ventilation, need to be further studied before concluding on benefits for the short and long term outcomes in premature infants. non-ventilatory measures in the treatment of rds, such as caffeine, nutrition, fluid and pda management and postnatal steroids in certain conditions should be included in the care of premature infants with rds in order to minimize the rate of bpd. , the noninvasive ventilator strategy needs to be confirmed by large prospective randomized controlled trials (with long-term follow up) in order to assure it is applicable to most elbw infants. furthermore, the strategy needs to be tailored to individualized infants according to the infant's maturation; antenatal steroid treatment and severity of rds; general condition; and to certain practical nicu conditions such as experience, personnel and timing during the day. for many years, it has been generally accepted that the pathophysiology of rsv bronchiolitis is driven by the inflammatory response evoked by horizontal (i.e., interpersonal) transmission of the virus in the first few months after birth ( ). however, a recently published study has brought to the forefront a striking new idea: rsv may be transmitted vertically from the respiratory tract of the mother to the lungs of the fetus ( ) . until now, we believed that when a pregnant woman got a cold, the developing fetus was protected by the placenta from rsv and other respiratory viruses. in this study, pregnant rats were inoculated with a recombinant rsv strain that could be tracked through expression of a red fluorescent protein (rrrsv). the same virus was subsequently found in percent of fetuses exposed in utero, as well as in the lungs of percent of newborn rats and percent of rats born to inoculated mothers when tested in adulthood. these data provide proof of concept for the transplacental transmission of rsv from mother to offspring and the persistence of vertically transmitted virus in lungs after birth. notably, the intrauterine rsv infection changed expression and function of critical neurotrophic pathways that control the development of cholinergic nerves in the budding airways and lung tissues ( ). these changes in cholinergic innervation of the fetal respiratory tract resulted in the development of postnatal airway hyperreactivity upon reinfection with the same virus ( ) . the airway smooth muscle tone was normal in the absence of stimulation and its contraction was normal in the absence of either maternal or neonatal infection. but in pups reinfected with rsv after prenatal exposure to the virus, markedly potentiated contractile responses were measured after either electrical nerve stimulation or methacholine inhalation, suggesting the involvement of both pre-and postjunctional mechanisms. these findings are consistent and provide a plausible mechanism to the epidemiologic evidence that early-life rsv infection -or possibly reinfection -predisposes a subpopulation of children to recurrent wheezing and asthma that typically spans through the first decade of life even in the absence of atopic phenotype ( ). to our knowledge this is the first report of vertical transmission of rsv, or for that matter any common respiratory virus. a number of infectious agents, including herpesviruses and retroviruses, have been shown to cross the placenta and establish persistent infection in offspring. the new evidence extends this possibility to other infections, such as rsv, once regarded as temporary and localized and that instead may be longer lasting and more pervasive than we thought. also, as shown for other viral pathogens, if rsv seeds the fetus before full t-cell maturation, this could lead to induction of prenatal tolerance and justify the limited synthesis of interferon and other inflammatory cytokines that have been noted when newborns develop severe infections ( ) . vertical rsv and asthma -the general concept that we have been working under for decades is that nothing bad happens in the lungs until the baby is born -even with serious conditions such as cystic fibrosis -and that the lungs are "clean" of pathogens at birth. but if human studies replicate the findings from animal models outlined above, our understanding of the pathogenesis of rsv infections would be completely changed. it would turn back the clock of respiratory developmental diseases by months and mean that we would need to start thinking about lung development and pathology during pregnancy rather than at birth. this could create a paradigm shift by extending our focus on prevention from the first few years after birth to also include the last few months before birth. this new paradigm is in line with the emerging evidence that many (or most) chronic inflammatory, degenerative, and even neoplastic diseases plaguing adults have their origins from often-subtle events occurring during fetal life. the "foetal programing hypothesis" was originally formulated by dr. david barker more than two decades ago to explain the extensively reproduced and confirmed epidemiologic evidence that low birth weight predisposes to cardiovascular disease in late adulthood ( ) . dr. barker died aged in september , leaving the legacy of this initially controversial, but now widely accepted, idea that common chronic illnesses such as cancer, cardiovascular disease and diabetes result not always from bad genes and an unhealthy adult lifestyle, but from poor intrauterine and early postnatal health. in one of his last public speeches, he argued: "the next generation does not have to suffer from heart disease or osteoporosis. these diseases are not mandated by the human genome. they barely existed years ago. they are unnecessary diseases. we could prevent them had we the will to do so." we believe the same concepts can be extended to chronic obstructive airway diseases like asthma and copd. asthma is the final product of complex interactions between genetic and environmental variables. prenatal events like the intrauterine exposure to viruses with specific tropism for the developing respiratory epithelium ( ) or imbalanced maternal diet ( ) will cause a shift in the trajectory of structural and functional airway development towards a hyperreactive phenotype. the same intrauterine exposures can affect gene expression via epigenetic modifications like dna methylation, histone acetylation, and by altering the relative expression of regulatory micro-rnas ( ) . the resulting neonatal phenotype will predispose the child to aberrant responses to common respiratory infections and airborne irritants, thereby increasing the risk of obstructive lung disease later in life. postnatal events, such as exposure to indoor and outdoor pollutants and allergens, can further shift the equilibrium of the adult phenotype by exacerbating airway inflammation and hyperreactivity ( ) . the continuous range of possible developmental trajectories and multiple sequential events acting during development will define the severity and duration of disease. the incidence, severity and mortality from childhood pneumonia has declined substantially in the last decade due to improved socioeconomic conditions, better access to care, wider implementation of effective management and preventative strategies and development and availability of improved vaccines, particularly the pneumococcal (pcv) and h influenzae type b (hib) conjugate vaccines. [ ] however, pneumonia remains the leading cause of childhood mortality globally outside the neonatal period and a major cause of morbidity and hospitalization despite good immunization coverage. [ , ] further, early childhood pneumonia has increasingly been associated with the development of chronic noncommunicable respiratory diseases into childhood and adulthood, such as asthma or chronic obstructive airways disease (copd). [ , ] with improved global coverage of the newer conjugate vaccines, it is likely that viral causes of pneumonia may be responsible for an increasing proportion of pneumonia cases. [ ] however, defining the etiology of pneumonia may be challenging as it can be difficult to distinguish colonizing from pathogenic organisms in respiratory specimens, blood culture rarely is positive and pneumonia, especially severe disease, may frequently be due to multiple co-pathogens. the development of better methods for specimen collection and of molecular diagnostics have provided more sensitive techniques to define potential etiologic agents but further compound the difficulty of ascribing pathogenicity. [ , ] despite these limitations, studies in the post-pcv era have reported an increasing predominance of viruses in childhood pneumonia cases, with a virus identified in - % of cases. [ , ] in children vaccinated with valent pcv (pcv ), rsv has been reported to be the predominant pathogen in case control analyses from both high income countries and lowmiddle country settings. however, there is frequent co-occurrence of other potential pathogens with rsv, including bacteria and other viruses. [ ] children under months of age are at highest risk of rsv disease. [ ] to adequately interpret data on viruses in the context of childhood pneumonia, the prevalence of these in healthy control children must be considered. using case control designs, viruses identified in association with pneumonia have been rsv, influenza virus and human metapneumovirus (hmpv); adenovirus, parainfluenza virus and coronavirus have been variably associated with pneumonia while the prevalence of rhinovirus has consistently been similar in cases and controls. [ , , , ] the use of quantitative measurements of viral load has not shown to be useful in distinguishing cases from controls except for rsv and for hmpv, but the presence of these alone is sufficient to ascribe etiology. these studies indicate that rsv is a major cause of pneumonia in the era of conjugate vaccines for bacterial pathogens, particularly in young infants. however they also highlight the limitations of current diagnostic strategies, particularly the poor sensitivity of current tests for bacterial etiology and the potential for incorrectly assigning etiology based on molecular diagnostics. they also provide further data on the complexity of ascribing pneumonia etiology, showing interactions between multiple potential pathogens. despite these limitations, the emerging data indicate that a key strategy for reducing the burden of childhood pneumonia lies in prevention of rsv disease in young children. identifying the etiology of pneumonia is key for initiating appropriate management strategies particularly use of antibiotics and to guide development of new vaccines. the reduction in bacterial pneumonia through conjugate vaccines underscores the need to reconsider the empiric treatment of pneumonia in settings where there are strong immunization programs. case management with antibiotics for pneumonia or severe pneumonia in the world health organization integrated management of childhood illness (imci) program has been a highly effective strategy for reducing mortality prior to widespread conjugate vaccine availability [ ] , but defining the residual burden and identifying clinical or laboratory features that distinguish bacterial from viral pathogens will be important before any change in pneumonia strategy can be recommended globally. late preterm (lp) newborns (born at - / to - / weeks gestational age) comprise the fastest growing subset of neonates, accounting for approximately % of all preterm births and about - % of total births in the us [ ] . "late preterm" infants are born near term, but are "immature". the late premature birth interrupts normal in utero fetal development during the last weeks of gestation that are probably a "critical period" of growth and development of the fetal lungs [ ] . three factors play a role in the respiratory vulnerability of lp infants [ ] : . prematurity with its developmental and consequently physiologic components; . heightened rate of respiratory morbidity in the neonatal period; . short-term pulmonary outcome respiratory complications are the prime morbidities of lp infants [ ] . a large retrospective study [ ] found that the odds of respiratory morbidity (respiratory distress syndrome [rds] , transient tachypnea of the newborn [ttn], pneumonia, respiratory failure, surfactant administration, and mechanical ventilation) decreased significantly with each advancing week of gestation up to weeks compared with to weeks. despite a relatively low absolute risk for rds or ttn at weeks compared with more premature infants, this rate poses an increased risk for lp infants when compared with term infants [ ] . acknowledgement of these morbidities led to studies aiming to decrease this burden. a recent large randomized controlled study [ ] showed that administration of betamethasone to women at risk for late preterm delivery significantly reduced the rate of a neonatal composite of respiratory treatments in the first hours (the use of continuous positive airway pressure or high-flow nasal cannula for at least hoursr, supplemental oxygen with a fraction of inspired oxygen of at least . for at least hours, extracorporeal membrane oxygenation, or mechanical ventilation) or stillbirth or neonatal death within hours after delivery. of note, neonatal hypoglycemia was more common in the betamethasone group than in the placebo group. late prematurity may affect the respiratory system in the long term [ ] . several studies reported an association of preterm birth ( - weeks' ga) without clinical lung disease with altered lung development and function [ ] . friedrich et al. [ ] in a longitudinal study found that despite normal lung volume, healthy preterm infants had persistently reduced airflow through the age of months and concluded that preterm birth in itself was associated with altered lung development. a single study [ ] showed a potential improvement, especially for large airway function, with advancing age. a recent large prospective cohort study showed that the number of hospitalizations caused by respiratory problems during the first year of life was doubled in moderately/late preterm ( - weeks' ga) compared with term infants [ ] . at preschool age, moderately preterm infants revealed more nocturnal cough or wheeze during or without a cold and increased use of inhaled steroids. at the age of years, rates of respiratory symptoms between moderate and early preterm born (< weeks' ga) children were similar; both were higher than in term born children. whether lp birth is associated with airway disease such as asthma in early childhood remains controversial [ ] . different findings in published studies could result from the different methods of asthma diagnosis, age groups at diagnosis, and from the difficulties in diagnosing asthma in early childhood. a recent study [ ] found that late preterm birth history is not independently associated with childhood asthma until years of age. lp infants are more vulnerable to viral respiratory infections, particularly rsv, which are more severe in these infants vs. term infants. the pernicious combination of rsv bronchiolitis affecting an a priori compromised lung/ airways of lp infants may have a lasting effect on respiratory function and consequent long-term morbidity [ ] . long-term persistence of an early decrease in pulmonary function tests (pft) was demonstrated by a longitudinal follow-up into early adulthood for an unselected random population in the tucson children's respiratory study [ ] . these observations suggest that the notion of a "critical developmental period" for the respiratory system does exist. deficits in lung function during early life, especially if associated with lower respiratory illnesses (especially rsv), increase the risk for chronic obstructive pulmonary disease later in adult life [ ] . summary lp infants are born during a "critical developmental time period" for the lungs. this may result in short and long-term pulmonary consequences. in addition, to screen the population at high-risk for disease. therefore, the effectiveness of early case finding should be a priority, but it depends on several factors such as health care system, contact tracing, and laboratory diagnosis. the diagnosis of tb in children is a common clinical challenge, and relies on a careful assessment of history of exposure, clinical examination, and relevant investigations. the most recommended approach to the diagnosis of tb in both children and adults is based on the who guidelines recommendations from and (table ) . ( , ) important factors to consider in all children with suspected tb is the endemic setting as well as the age and immune status of the child. in countries with a low incidence of tb, a positive contact with a case in combination with suggestive symptoms makes diagnosis more straightforward. in high tb endemic areas, a history of tb contact remains important, but is much less sensitive, given that transmission often occurs through unknown source cases. ( ) laboratory tests for the diagnosis of infections can be grouped into two groups: detection of microbes (or components) and detection of components of the immune response to the microbe. the sensitivity of the first group will depend on the quality of the specimen and the concentration of microorganisms. this group includes microscopy, culture, elisa, and nucleic acid detection (pcr). the second group measures the activity of the immune system against microbe-specific antigens in the possibly infected host. this category includes antibody detection and activated t cells. the gold standard for the diagnosis of tb is bacillary detection by smear or culture. in adults, microscopy can detect up to % À % of culture-positive samples. in children, this does not work as well due to limited access to appropriate body specimens, and also because children usually have paucibacillary disease, since cavitating disease is rare in children. studies have shown that under best circumstances, acid-fast bacilli sputum smear is positive in only about - % of children with tb while culture gives a better yield of - %. until recently, the diagnosis of ltbi has been based exclusively on the tst, which has relatively poor sensitivity and specificity. despite these limitations, it remains the standard of care for diagnosis of ltbi worldwide, particularly in low-income countries. interferon gamma release assays (igras) measure the in vitro response to specific m. tuberculosis antigens. although they offer several advantages over tst such as better specificity, single visit, little inter-observer variability, and no booting effect; they have not been found better than tst, and are not able to predict the risk of infected individuals developing active tb disease. given their increased cost, replacing tst by igras as a public health intervention in resourceconstrained setting is not recommended. novel approaches to confirmation of tb have been developed. these include methods based on rapid culture techniques and genotypic techniques that improve detection of m. tuberculosis. an example is the xpert mtb/rif assay, which is a fully automated realtime dna based test that can detect both tb and rifampicin resistance in less than two hours. ( , , ) as expected, it should be used rather than conventional microscopy and culture in children suspected of having mdr-tb. the clinical diagnosis of primary tb in children remains challenging because of non-specific signs and symptoms and difficulty with acquiring diagnostic specimens. because of this, the diagnosis of primary tb in practice, relies on a combination of clinical features and chest x-ray (cxr) findings. the detection of lymphadenopathy in the hilar and para-tracheal regions on the frontal cxr, supported by identification of subcarinal lymphadenopathy on the lateral cxr, represent a useful surrogate marker of tb at relatively low cost. however, sensitivity and specificity for identifying lymphadenopathy on cxr in children is relatively poor with significant inter-observer variation in the interpretation of radiographs, complicated further by poor quality of radiographs. affecting both accuracy and observer agreement is the lack of standardized imaging criteria and lymph nodes sizecriteria for a positive diagnosis of primary tb. attempts are therefore being made to establish 'objective' chest radiograph signs backed up by a standard set of images as a guide. ultrasound is an especially attractive imaging alternative to cxr as it does not involve radiation or require sedation and because it is relatively cheap and mobile. ultrasound of the mediastinum has been used to detect mediastinal lymphadenopathy and can also be used to detect extrapulmonary tb through abdominal imaging, at the same sitting. it is particularly useful in rural settings where no other imaging is available. the ability to store digital ultrasound images and cine-loops also enables teleradiology support by expert interpretation and opinion, from a distance. computed tomography (ct) and magnetic resonance imaging (mri) are obvious diagnostic imaging considerations that will improve diagnostic accuracy of primary tb, but the radiation dose in ct, the need for anesthesia in mri, the limited availability and high cost are real barriers to their clinical utility. mri is preferred to ct because it does not involve ionizing radiation. however, the disadvantages of mri for lung imaging (poor signal generated from the air in the lungs and movement artefacts from breathing), the cost and the requirement for the child to keep still for a prolonged period (requiring anesthesia) have slowed its use in thoracic infections. yet, whole body mri, including thoracic imaging is mainstream for detecting lymphadenopathy in childhood lymphoma. the preferred imaging technique varies with the suspected pathology and available equipment. dynamic imaging techniques such as inspiratory/ expiratory cxr, fluoroscopy, and inspiratory/expiratory or cine ct permit the lungs and airways to be imaged at different phases of the respiratory cycle. inspiratory/expiratory cxr and inspiratory/expiratory chest ct have long been the preferred initial imaging methods for detecting foreign body aspiration or bronchiolitis obliterans, respectively, on the basis of air trapping rather than direct visualization of the airway obstruction. fluoroscopy has historically been the preferred noninvasive method for diagnosing tracheobronchomalacia due to its ease of performance, even in uncooperative patients, and its high specificity, but it is limited by its subjective interpretation, low sensitivity, poor depiction of the paratracheal structures, and inability to simultaneously display the anteroposterior and lateral walls of the airway and quantify luminal cross-sectional area . in infants and children too young to comply with breath-hold instructions, inspiratory/expiratory phases can be simulated by imaging during right/left lateral decubitus or prone/supine positioning. controlled-ventilation ct under sedation or anesthesia also permits inspiratory/expiratory imaging of the lungs and airways in uncooperative patients. dynamic cine ct technique allows the airways to be imaged sequentially during successive phases of the respiratory cycle, but coverage was initially limited to short ( cm or less) segments of the airway, resulting in sampling misregistration and preventing synchronous evaluation of the true extent and severity of airway collapse during the same phase of the respiratory cycle . made possible by recent technologic advances including more rapid gantry rotation and wider detector arrays (up to cm craniocaudal coverage), dynamic volumetric cine ct now allows all or nearly all of the lungs and central airways to be imaged rapidly and sequentially throughout the respiratory cycle without the need for sedation or intubation. this technique is capable of providing multiplanar, d and d information about the airways during normal tidal breathing or forced expiratory maneuvers, as well as depicting the relationship of the airways to the adjacent vasculature if intravenous contrast is administered . with dynamic volumetric cine ct, intrinsic and extrinsic causes of airway narrowing can be distinguished and fixed airway stenosis can be differentiated from expiratory central airway collapse due to tracheobronchomalacia (softening of tracheobronchial cartilage) or excessive dynamic airway collapse (inward bulging of the posterior membrane) . tracheobronchomalacia is primary (congenital) in approximately / children and often resolves in isolated mild to moderate cases by years of age as the cartilage geometry and composition matures and posterior membrane tone develops. tracheobronchomalacia is often accompanied by gastroesophageal reflux disease and is associated with other foregut anomalies, especially esophageal atresia and tracheoesophageal fistula. tracheobronchomalacia can be secondary to extrinsic compression, chronic airway inflammation, intubation, or positive pressure ventilation and is identified in about onefourth of children with chronic respiratory symptoms or signs such as wheezing, barking cough, recurrent respiratory tract infection, apnea, cyanotic spells, or difficulty weaning from respiratory support . tracheobronchomalacia was originally defined as > % reduction in airway cross-sectional diameter during coughing, but false positives are very common with this definition, especially for the bronchi in which physiologic expiratory airway narrowing is more pronounced than for the trachea. the shape and cross-sectional area of the airway lumen can be precisely determined by ct, but there is no current consensus on the optimal threshold degree of expiratory airway collapse for a diagnosis of tracheobronchomalacia among children of varying ages with or without coexisting lung disease during either tidal breathing or forced expiration. expiratory collapse of normal airways can occur in the setting of obstructive lung disease such as asthma or bronchopulmonary dysplasia due to increased pleural pressure and increased peripheral airways resistance that reduces airway transmural pressure . dynamic volumetric cine ct provides objective information to classify expiratory central airway collapse according to the femos (functional status, extent, morphology, origin, severity) system , but it should be noted that the degree of luminal narrowing is only one factor in airflow limitation. evidence of airway compression or expiratory collapse on imaging does not necessarily indicate a condition requiring therapeutic intervention, and correlation with the clinical symptoms, signs, risk factors, and pulmonary function tests is necessary to determine the functional significance , . in addition to the noninvasive nature, the advantages of dynamic volumetric cine ct over bronchoscopy include the ability to directly evaluate for vascular structures or soft tissue masses that impinge on the airway, depict the airways distal to a narrowing impassable by bronchoscope, and assess the lung parenchyma for conditions such as air trapping that may be associated with dynamic central airway collapse . a disadvantage of ct is the exposure to ionizing radiation. for perspective, dynamic airway ct incurs a radiation dose similar or less to than that from a year of natural background radiation exposure . dynamic cine magnetic resonance imaging (mri) avoids exposure to ionizing radiation and is capable of imaging the central airways and vasculature , but is limited by a longer scan time, more frequent need for sedation/anesthesia and less detailed depiction of the lung parenchyma compared to ct. additional studies in children are needed to determine how the anatomic and functional information provided by dynamic ct is best applied to the diagnosis, treatment planning, and post-therapeutic monitoring of pediatric airway disorders. the main driving force to develop sophisticated mri sequences for pediatric chest imaging is that mri is a radiation-free technique. this is especially important for children who are more sensitive to ionizing radiation than adults [ ] . this justifies the use of chest mri for short-and long-term follow-up of chronic lung diseases such as cystic fibrosis (cf), so as to reduce the lifelong cumulative radiation dose [ ] . chest mri is challenging because of the magnetic heterogeneous environment in the chest region [ ] . lung parenchyma is a low proton density structure and hence has a reduced signal-to-noise ratio [ ] . in addition, the numerous airtissue interfaces within a voxel induce strong localized microscopic magnetic field gradients, which produce extensive mri signal dephasing leading to extremely short t star (t à ) and geometric distortions. these effects become stronger at higher magnetic field strengths (i.e. t), which are increasingly used in clinical settings for enhanced signal-to-noise ratio [ ] . however, signalto-noise ratio in cases of lung pathology, such as pneumonia, edema, tumors and atelectasis, is increased by higher fluid content and amount of tissue. these conditions result in higher proton density and improved visualization [ ] . moreover, mri has the advantage of integrating anatomical and functional information in a single examination, a possibility not as readily available with other imaging modalities. mri can provide functional information regarding lung perfusion using gadolinium contrast [ ] , lung mechanics using dynamic acquisitions [ ] , and ventilation using inhaled hyperpolarized gases [ ] , oxygen enhancement or dynamic motion-based methods [ ] . moreover, dwi is able to give new insight in the management of pneumonia, especially in cf patients [ ] . chest mri has reached the point where it can be used in routine clinical practice. although mri cannot yet be compared to ct for anatomical detail, new sequences allow acquisition of lung images with high diagnostic quality in less than s, which makes mri feasible in a clinical setting. mri can be considered an alternative to ct for the diagnosis of lung diseases and for monitoring response to treatment in pediatric lung disease. moreover, in some diseases that require long-term follow-up, such as cystic fibrosis, mri can play an important role in reducing lifelong radiation exposure related to repeated ct scans. furthermore, mri has the ability to offer functional information: information regarding lung mechanics, perfusion and ventilation can provide new insight in different pediatric lung diseases. this functional information can not only improve our understanding with regard to the pathophysiology of pediatric lung diseases, it can also open new diagnostic and therapeutic options. obstructive sleep apnea syndrome (osas) is characterized by prolonged partial airway obstruction and/or intermittent complete obstruction (obstructive apnea) during sleep, affecting about % to % of children [ ] . osas is a complex syndrome with multiple etiologic factors: the main causative factor is adenotonsillar hypertrophy while other conditions, such as craniofacial dysmorphism, obesity, hypotonic neuromuscular diseases, despite inducing reduction of the caliber of the upper airways, are commonly mistreated [ ] . adenotonsillectomy has been considered for many years the only treatment in children with osas although its efficacy remains uncertain, depending on the severity and on the presence of other co-morbidities, [ ] . since a residual osa is reported in a large proportion of children after adenotonsillectomy [ ] , and children with osa display a complex phenotype (mild or major craniofacial anomalies, and/or comorbid obesity, and/or adenotonsillar enlargement), a multi-therapeutic approach to pediatric osas and a defined timing of therapy are required [ , ] . a narrow upper airway accompanied by maxillary constriction and mandibular retrusion is commonly reported in children with osas [ ] . the skeletal conformation showing hyperdivergent skeletal growth pattern associated with posterior displacement of the tongue base, increases the upper airway narrowing and craniomandibular, intermaxillary, goniac and mandibular angles leading to a high-arched (ogival) palate [ ] . rapid maxillary expansion (rme) is the most common dento-facial orthopedic procedure used in young patients to treat maxillary transverse deficiencies, starting up to years of age. recently, it has been demonstrated to be efficacious to treat osas in children with a narrow palate and malocclusion: a significant reduction in the apnea-hypopnea index and in diurnal symptoms after six months of therapy with rme [ ] , and positive long-term effects in children with osa and malocclusions treated with rme have been reported [ ] . similar results were obtained after one year of treatment with rme in preschool and school-aged non obese children with osas and dental malocclusions with a significant drop in clinical symptoms as well as apnea-hypopnea index [ ] . this study also demonstrated that starting treatment early when the bone is still extremely plastic and its growth rate is maximum increases the percentage of success of rme treatment. a two-year follow up after the end of the rme application was performed in the same population of children confirming a stable decrease in apnea-hypopnea index, an increase of mean overnight oxygen saturation and a persistent improvement in clinical symptoms [ ] . finally, a recent randomized study showed preliminary results regarding the effect of rme applied before adenotonsillectomy compared to the effect of rme applied after surgery, in children with osa. no significant differences between the two different approaches were described [ ] . in conclusion, orthodontic treatment is a valid treatment for osa, improving clinical symptoms, respiratory parameters measured during psg with long lasting effect. the widening of the maxilla, the corrections of dental malocclusions and the correct relationships between maxillary and mandibular arches with respect to the anterior cranial base, are the main craniofacial changes induced by rme that may explain the efficacy of orthodontic therapy. orthodontic therapy should be encouraged in pediatric osas, and an early approach may permanently modify nasal breathing and respiration, thereby preventing obstruction of the upper airway. towards the turn of the century, david gozal's group published a series of papers that raised important questions. in a sample of st grade pupils whose school performance was in the lowest decile of their class ranking, they found that % had sleep-associated intermittent hypoxia and/or hypercapnia; school performance improved in those whose parents had opted for adenotonsillectomy ( ) . they then showed that % of - year olds with poor school performance had parent-reported snoring at age - , compared to only % among those with good school performance ( ). finally, a group of first graders with snoring, but no obstructive sleep apnea, i.e. an obstructive apnea index < , performed worse on measures related to attention, social problems and visuospatial function than non-snorers, suggesting that simple snoring may not be as benign as hitherto widely believed ( ) . against this background, we set out to perform the hannover study on sleep apnea in childhood (hassac), a community-based cross-sectional study on several aspects of sleep-disordered breathing (sdb) in primary school children incorporating a two-phase sequential screening procedure: participants were screened for symptoms and signs of sdb using an sdbquestionnaire and home pulse oximetry (hpo), those with outlying results on either screening method subsequently underwent an abbreviated home polysomnography (hpsg) for a final diagnosis of obstructive sleep apnea syndrome (osas). overall, participants were representative of the underlying population of third-graders in the study region. we found that . % of this cohort were habitual snorers, while the population prevalence for osas was . % ( , ) . we then wanted to know how these symptoms affected behavior and academic achievements. for this, we used parental questionnaires and collected teachers' ratings, and defined poor school performance as grade or worse in the last school report form, or requirement for special assistance, with this classification roughly corresponding to the lowest quintile of a class. we found that children with habitual snoring, compared to those who never snored, had - times the odds for daytime symptoms such as hyperactivity, difficulty concentrating, falling asleep while watching tv or at school or having peer problems, and - times the odds for poor school performance in mathematics, science and spelling ( , ) . there was a clear dose-effect gradient, i.e. the proportion of children with poor school performance increased with increasing frequency of parent-reported snoring. considering its high prevalence, and assuming a causal link to disturbed behavior, habitual snoring appeared to be a substantial public health problem in primary school children. given this association, we wanted to know how this is mediated, i.e. whether this is mainly through detrimental effects of intermittent hypoxemia or more likely due to recurrent arousal. contrary to our hypothesis, the increased odds for poor school performance or daytime symptoms associated with habitual snoring stayed the same once children exhibiting intermittent desaturation in their overnight pulse oximetry recording had been excluded, suggesting that even so-called benign snoring, i.e. snoring without hypoxemia, may in fact not be benign. if not via intermittent desaturation, could the relationship with poor school performance be mediated via frequent arousals elicited by recurrent obstructive apnea? to address this question, we took advantage of the fact that children with an abnormal questionnaire score in our hassac study also underwent hpsg. thus, we re-analyzed our data on the relationship of snoring with daytime symptoms and poor school performance after excluding all children with a mixed-obstructive apnea/hypopnea index (maohi) ! . , but again, the risk for poor school performance was not reduced among snorers after excluding those with recurrent apneas. given that simple snoring has such a strong association with daytime symptoms À are these reversible? in our hassac study, we could collect year follow-up data in snorers and controls. among these, snorers ( %) had stopped snoring. while their scores for emotional problems, hyperactivity and problems with peers improved, their school performance did not ( ) . this is in line with other data suggesting that reduced scores in executive functioning and iq seen in children prior to adenotonsillectomy may not improve following this operation ( ) . similarly, in the avon longitudinal study on parents and children (alspac), even those whose sdb symptoms peaked at age months and abated thereafter still had almost twice the odds for hyperactivity and % higher odds for behavioral problems at age years ( ). taken together, there is now a growing body of evidence that frequent snoring in children may not be as benign as previously thought, but may instead be associated with impaired behavior and poor academic achievements. these problems may even persist after snoring ceases, which À if these statistical associations were confirmed as causal À would argue for their early recognition and treatment. here, it is encouraging to see that in another longitudinal study on snoring and daytime symptoms, the proportion of children who did not snore at age and years was % in those who were breastfed for less than month, but % in those who were breastfed for months or longer ( ) , suggesting that breastfeeding may reduce the risk of snoring during early childhood. in addition, given the limited availability of sleep labs, we urgently need better and easier-toperform screening methods to identify those who may need treatment for their snoring, e.g. in whom poor school performance can be predicted from a screening test ( ) . also, interventions such as nasal steroids, montelukast or orthodontic treatment may deserve further study. diagnosis of osas using home respiratory polygraphy (hrp). alonso-alvarez et al. prospectively assessed the diagnostic reliability of hrp in children aged to years with a clinical suspicion of osas. they found a sensitivity of % and a specificity of % and concluded that hrp emerges as a potentially useful and reliable approach for the diagnosis of moderate/ severe osas in children. drug-induced sedation endoscopy (dise) aims to reproduce upper airway obstruction during sleep and is gaining increasing popularity, with the hope of guiding efficient surgery and cure osdb children. in a meta-analysis, galluzi et al. concluded that dise may benefit a minority of children with osas, and should only be used in children with unremarkable clinical evaluation or upon persistent osas after at. obstructive sleep-disordered breathing and obesity pathogenesis of osas in obese adolescents. literature on the pathogenesis of osas in adolescents is very limited. schwab et al. prospectively compared upper airway magnetic resonance imaging in adolescents aged to years. results indicated that lymphoid tissue, rather than other soft tissue components (tongue, lateral pharyngeal walls, parapharyngeal fat pads), are the primary upper airway anatomical risk factors for osas. while the pathogenesis of osas is clearly multifactorial (e.g., decreased upper airway reflexes in osas obese adolescents) and often require additional treatment, the results are clinically important since they suggest that at should still be considered as the first-line treatment in adolescents with osas. osdb and metabolic syndrome. in a systematic assessment of the literature on the interactions between sleep, osdb, obesity and disruptions of metabolic homeostasis in children and adolescents, hakim et al. concluded that obesity and osdb appear to contribute to the initiation and progression of each other, and that both are linked to the metabolic phenotype . one intriguing mechanism postulates that osdb/ disrupted sleep as well as other factors favoring obesity, such as high-fat/ fructose diet, disrupt the gut microbiome and lead to increased systemic levels of lipopolysaccharides, in turn promoting inflammation and metabolic dysfunction. treatment of sleep-disordered breathing in children watchful waiting. chervin et al. followed children aged to years with mild/moderate osas after seven months of watchful waiting only. they found resolution of osas in % of the children. independent predictors of resolution were lower ahi and normal waist circumference. the authors concluded that, in practice, a baseline low ahi and normal waist circumference, or low pediatric sleep questionnaire and snoring score, may help identify an opportunity to avoid at. myofunctional therapy (mt). camacho et al. performed a meta-analysis of the use of mt as a treatment for osas in adults and children. although the total number of patients (especially children, n ¼ ) was low, the effects were highly significant. overall, mt decreased ahi by - % in pediatric and adult patients. in children, a positive effect was reported when used as the only treatment in mild osas as well as to consolidate osas cure after at þ rapid maxillary expansion. the authors concluded that mt could be an adjunct to other osas treatments in patients of all ages. evolution of obstructive sleep-disordered breathing in children evolution in preschool children with osdb. walter et al. investigated the long-term evolution of osdb in preschool-aged children with normal weight. half of the preschoolers with osdb were treated, most often by adenoidectomy and/or tonsillectomy. overall, osdb resolved in half of the children, either spontaneously ( %) or with treatment ( %). however, % still had osas, similarly to observations in school-aged children. intriguingly, complete resolution of osdb at three years post-treatment was more likely in preschoolers with moderate/severe osas compared to those with mild osas or primary snoring. long-term evolution of osas. spilsbury et al. reported results on both remission and incidence of osas in participants who underwent psg at - and - years of age. the authors first observed that osas in middle childhood usually remitted by adolescence. secondly, while habitual snoring and obesity predicted osas at each time point, distinct additional risk factors for osas were found in middle childhood vs. adolescence. hence, prematurity, a disadvantaged neighborhood or african-american origin also predicted osas in middle childhood, while risk factors in adolescents included male sex and previous at. finally, obesity, but not habitual snoring, in middle childhood predicted adolescent osas. these results confirm that prevention and treatment of obesity appears of utmost importance in the fight against pediatric osas. evolution of osas after treatment. lee et al. performed a meta-analysis of psg 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perfusion in children with cystic fibrosis-initial results spirometer-controlled cine magnetic resonance imaging used to diagnose tracheobronchomalacia in paediatric patients imaging of lung function using hyperpolarized helium- magnetic resonance imag-ing: review of current and emerging translational methods and ap-plications epidemiology of pediatric obstructive sleep apnea upper airway collapsibility in children with obstructive sleep apnea syndrome adenotonsillectomy outcomes in treatment of obstructive sleep apnea in children: a multicenter retrospective study pediatric obstructive sleep apnea: complications, management, and long-term outcomes craniofacial morphology in preschool children with sleep-related breathing disorder and hypertrophy of tonsils craniofacial modifications in children with habitual snoring and obstructive sleep apnoea: a case-control study randomized controlled study of an oral jaw-positioning appliance for the treatment of obstructive sleep apnea in 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children with sleep disordered breathing sleep-disordered breathing in a population-based cohort: behavioral outcomes at and years persistent snoring in preschool children: predictors and behavioral and developmental correlates predicting poor school performance in children suspected for sleep-disordered breathing utility of symptoms to predict treatment outcomes in obstructive sleep apnea syndrome pediatric osas: oximetry can provide answers when polysomnography is not available reliability of home respiratory polygraphy for the diagnosis of sleep apnea in children drug induced sleep endoscopy in the decision-making process of children with obstructive sleep apnea understanding the anatomic basis for obstructive sleep apnea syndrome in adolescents obesity and altered sleep: a pathway to metabolic derangements in children? childhood adenotonsillectomy trial. prognosis for spontaneous resolution of osa in children myofunctional therapy to treat obstructive sleep apnea: a systematic review and meta-analysis long-term improvements in sleep and respiratory parameters in preschool children following treatment of sleep disordered breathing remission and incidence of obstructive sleep apnea from middle childhood to late adolescence polysomnographic findings after adenotonsillectomy for obstructive sleep apnea in obese and non-obese children: a systemic review and meta-analysis this last year has seen a number of significant advances in the field of pediatric sleep-disordered breathing. the following is a personal selection of a few publications. overnight polysomnography (psg) is considered necessary to diagnose children suspected of sleep-disordered breathing (sdb). in practice, however, most children do not have access to overnight psg, due to the lack of sleep laboratories worldwide. the quest for a simpler means to diagnose sdb, or at least to prioritize children for referral to a sleep laboratory, remains a high priority. questionnaire. in a prospective study in children aged to years with obstructive sleep apnea syndrome (osas), rosen et al. found that, conversely to psg, the pediatric sleep questionnaire results reflect osasrelated impairment in behavior, quality of life and sleepiness as well as predict their improvement post-adenotonsillectomy (at). the authors concluded that while psg is needed to diagnose osas, results from a careful clinical assessment provide important adjunctive information on comorbidities and their improvement after surgery. overnight oximetry in osdb children. kaditis et al. performed a systematic analysis of the literature on the use of nocturnal oximetry in children with obstructive sdb (osdb ¼ from primary snoring to osas). their conclusion confirmed that overnight oximetry (spo ) is useful for diagnosing osdb and for predicting post-at complications in a child with a history suggestive of osdb. overall, a desaturation index (! %) higher than episodes/hour can predict both mild and moderate-to-severe osdb, while criteria based on clusters of desaturation such as the mcgill oximetry score can predict moderate-to-severe osdb. key: cord- -cd adns authors: thachil, jecko; owusu-ofori, shirley; bates, imelda title: haematological diseases in the tropics date: - - journal: manson's tropical infectious diseases doi: . /b - - - - . - sha: doc_id: cord_uid: cd adns nan haematological disorders are common in low-income countries. they make a substantial contribution to morbidity and mortality of individuals in these regions and have a negative impact on the growth and development of under-resourced nations. genetic red cells abnormalities are common in lowincome countries because they provide protection against malaria and they often co-exist with other causes of anaemia such as malnutrition and chronic illnesses. there is a close association between haematological abnormalities and infections which are a major cause of illness and death in these populations. morphological abnormalities of blood can often provide clues about the underlying diagnosis and blood film examination is particularly important where diagnostic facilities are limited. abnormal blood counts can manifest as various combinations of alterations of numbers of red cells, white cells or platelets. this section will outline some of the most common causes of abnormal blood counts likely to be encountered in clinical practice in low-income countries. anaemia is one of the most common causes of morbidity in the world and its impact is reflected in several of the health-related millennium development goals. although anaemia by itself is not a diagnosis, it suggests that there is an underlying disease state which needs to be recognized and treated. it is also a useful indicator of the general health of the population. the causes of anaemia may be identified systematically by considering the life cycle of the red cells (figure . ). nutrients necessary for red cell production are absorbed from the gastrointestinal tract and carried through the portal vein to the liver and ultimately reach the bone marrow where erythropoiesis occurs. this process is regulated by erythropoietin, a hormone released from the kidneys mainly in response to hypoxia. mature • africa and asia have more than % of the world's anaemic populations and anaemia burden is highest among children and women of reproductive age. • the accurate diagnosis of anaemia has been neglected; clinical assessment of anaemia is unreliable unless the anaemia is severe. • in low-income countries, anaemia in an individual is often due to multiple interdependent factors. removing or treating a single factor may not resolve the anaemia. • early diagnosis of sickle cell disease and rapid access to a specialist centre for emergencies such as severe pain crises, strokes and acute chest syndrome, can help to prevent permanent long-term complications. • beta-thalassaemia major is fatal in the first few years of life unless regular blood transfusions are given; unless they are accompanied by iron chelation, these transfusions will eventually cause death due to irreversible organ damage from iron overload. • malarial anaemia is a particular problem for children and pregnant women and severe anaemia can be caused by p. falciparum and p. vivax. malarial anaemia can be reduced with chemoprophylaxis and intermittent treatment, and by anti-mosquito measures such as insecticidetreated bed nets and vector control. • anaemia occurs in % of hiv-infected patients and is an independent risk factor for death. prompt treatment of factors associated with anaemia, such as infections and poor nutrition, and commencement of antiretroviral treatment will reduce deaths. • blood shortages are common in tropical countries. to increase the availability of blood, transfusions should be prescribed in accordance with guidelines and efforts made to encourage blood donors to donate regularly as repeat donors are the safest type of donor. reaction' , characterized by circulating myelocytes and metamyelocytes, can be mistaken for leukaemia but, unlike leukaemia, there is an orderly maturation and proliferation of neutrophils. leukaemoid reactions have also been described in patients with tuberculosis, juvenile rheumatoid arthritis and dermatitis herpetiformis. , decreased margination of neutrophils with egress of cells into the circulation can occur with exercise, adrenaline (epinephrine) injection, emotional stress and postoperatively or in response to drugs (e.g. steroids, β-agonists). other drugs, such as lithium and tetracycline, produce neutrophilia through increased production. neutrophilia is also a feature of bone marrow proliferation which occurs in myeloproliferative neoplasms, particularly chronic myeloid leukemia and myelofibrosis. teardrop cells and nucleated red blood cells are features of myelofibrosis on the blood film; basophilia and eosinophilia are common with chronic myeloid leukaemia. molecular testing for the jak- mutation or bcr-abl fusion gene can also help to differentiate between myeloproliferative neoplasms. rebound neutrophilia can occur following treatment of megaloblastic anaemia or after recovery from neutropenia induced by drugs. acute haemorrhage can cause neutrophilia, especially if bleeding occurs into the peritoneal cavity, pleural space, joints or adjacent to the dura. this is possibly due to the release of adrenaline and chemokines in response to local inflammation. the presence of neutrophilia can be useful in raising suspicions about the onset of complications in infections that are not primarily associated with neutrophilia. examples include meningitis in tuberculosis, orchitis in mumps, bowel perforation in typhoid fever and superadded bacterial infection in measles. the absence of neutrophilia can be helpful in differentiating typhoid and paratyphoid fever from pyogenic infections. neutropenia is defined as an absolute neutrophil count < . × /l. it is usually classified into severe (< . × /l), moderate red cells are released into the circulation from the bone marrow and percolate through the tissues and organs. anaemia can result from defects in any of these stages. inadequate production of red cells in the bone marrow can be due to lack of nutrients (e.g. iron, b , folate, vitamin a, copper or zinc), abnormal haemoglobin synthesis (i.e. haemoglobinopathies) or ineffective erythropoeisis from myelodysplasia or infections. red cells can be lost from the body (e.g. gastrointestinal bleeding) or removed prematurely if they are abnormal or the spleen is enlarged (i.e. haemolysis). kidney disease can result in decreased erythropoietin. anaemia of chronic disease (or 'anaemia of inflammation') is due to an inadequate response to erythropoieitin or to increased cytokine-induced hepcidin release in inflammatory states which interferes with iron absorption or iron utilization. diagnostic algorithms to determine the cause of anaemia are usually based on a combination of the mean cell volume of the red cells, the reticulocyte count and blood film appearance (figures . , . ). this approach is based on the availability of a haematology analyser and an experienced microscopist. several conditions which cause anaemia may co-exist in the same individual (e.g. intestinal parasites, malaria and sickle cell disease) and hence a thorough investigation is crucial to identify all potential causes of anaemia. neutrophils released from the marrow after maturation can either enter the 'circulating pool' or they can remain in the 'marginal pool' where they are loosely attached to the blood vessel wall. cells in the marginal pool are not sampled when blood is taken for a full blood count. neutrophilia can therefore result from increased bone marrow synthesis and also from decreased margination which increases the circulating pool. there are many causes of neutrophilia (box . ) but the commonest is bacterial infection in which there is increased bone marrow production of neutrophils and release of neutrophil precursors into the peripheral blood. this 'leukaemoid ( . - . × /l) or mild ( . - . × /l). the propensity to develop infections is related to the degree and duration of neutropenia, with higher risk associated with counts below . × /l. africans, african americans, yemenite jews, palestinians and saudi arabians generally have slightly lower neutrophil counts compared with other races. this is thought to be due to an increase in the bone marrow storage pool as ethnic neutropenia is associated with good neutrophil responses to infections. neutropenia can be due to impaired or ineffective (intramedullary death of neutrophil precursors despite normal bone marrow production) synthesis by the bone marrow (e.g. myelodysplasia, megaloblastic anaemia, treatment with phenytoin or methotrexate); a shift from the circulating pool to marginated pool (pseudoneutropenia) and increased peripheral destruction (e.g. secondary to antibodies against the neutrophils or increased reticulo-endothelial activity in sepsis or haemophagocytic syndrome) (box . ). increased consumption of neutrophils can result from increased attachment of cells to endothelium or other leukocytes in inflammatory states. neutropenia is often the result of a combination of several of these mechanisms. infants of hypertensive mothers may have moderate to severe neutropenia, which can last for several days. this is probably related to bone marrow suppression. moderate to severe neutropenia can also occur in newborn infants as a result of the transfer of maternal igg anti-neutrophil antibodies in a manner similar to rhesus haemolytic disease of the newborn. although neutropenia has been described with typhoid fever, minimum neutrophil count seldom falls below . × /l and the box neutropenia may not develop until after the first week of illness. infectious hepatitis and yellow fever can both cause neutropenia. overwhelming infections can lead to a failure of bone marrow production of neutrophils, especially in undernourished individuals and alcoholics. individuals with severe neutropenia can develop lifethreatening septicaemia, often from endogenous flora (e.g. oral cavity), and stringent measures should be taken to avoid situations which may predispose these individuals to infections. they may need prophylactic antimicrobials and should have rapid access to medical care. fungal infections are less common than bacterial infections in neutropenic individuals, and viral or parasitic infections rarely occur with isolated neutropenia. granulocyte colony stimulating factor (gcsf) injections can be helpful in raising the neutrophil count in patients with complicating infections since it stimulates the release of neutrophils from the marrow, but gcsf is only useful if there is some bone marrow reserve. patients with some congenital or immune forms of neutropenia can tolerate persistently low counts without any increase in the incidence of infections. monocytosis occurs in chronic infections and inflammatory conditions. protozoan infections such as typhus, trypanosomiasis and kala-azar may be associated with monocytosis. chronic and juvenile myelomonocytic leukaemias are malignant disorders in which monocytosis may be severe; acute monocytic leukaemias may present with mild to moderate monocytosis. monocytosis, and particularly a monocyte : lymphocyte ratio greater than . - . , may indicate active progression of tuberculosis and an unfavourable prognosis. the normal ratio of . or less is restored when the healing process is complete. a decreased absolute monocyte count occurs in bone marrow failure states such as aplastic anaemia or after chemotherapy. low monocyte counts can occur with overwhelming sepsis and with splenomegaly. monocytopenia is a characteristic feature of hairy cell leukaemia and is considered to be a diagnostic hallmark of this disease. peripheral blood contains only around % of the total body lymphocyte population since these represent the cells present in the blood during their transit into secondary lymphoid organs. wide variations exist in lymphocyte counts between individuals especially in childhood. lymphocyte counts exhibit a diurnal pattern; peaking at night with a nadir in the morning. lymphocytosis is characteristic of infectious mononucleosis and many atypical and large lymphocytes can be seen in the peripheral blood film. these atypical cells can also occur in cytomegalovirus infection and infectious hepatitis. absolute lymphocytosis can occur with chronic infections such as brucellosis and in the recovery stages of tuberculosis. lymphocytosis is unusual in bacterial infections except in the case of pertussis. heavy smoking is also an often overlooked cause of lymphocytosis and is probably one of the commonest reasons for a mild to moderate increase in the lymphocyte count. malignant bone marrow disorders, predominantly acute lymphoblastic and chronic lymphocytic leukaemia and non-hodgkin's lymphomas, can cause lymphocytosis. these lymphocytes may have characteristic morphological changes identifiable in the blood film (e.g. smear cells with chronic lymphocytic leukaemia) and the correct diagnosis can be confirmed by immunophenotyping for specific combinations of cell markers. lymphopenia is due to decreased production, redistribution or increased rate of death of lymphocytes. decreased production usually results from cytotoxic drugs and radiotherapy, while increased lymphocyte death can occur in infections such as influenza and hiv. occasionally, an isolated low lymphocyte count in the context of an otherwise normal full blood count can be a clue to the diagnosis of hiv. this reflects the destruction of cd + t cells by the virus although an expansion of cd + t cells may raise the total lymphocyte count to normal levels. redistribution rather than depletion of total body lymphocyte numbers occurs with steroid treatment or with endogenous secretion of corticosteroids during acute illnesses due to the retention of lymphocytes in secondary lymphoid organs. eosinophilia eosinophils are involved in innate immunity and hypersensitivity. their number in the circulation is relatively small compared to other leukocytes because they predominantly reside in tissues such as the gut, skin and lungs which are entry points for allergens and infections. the commonest causes of eosinophilia are helminthic infections, atopy and allergic diseases, and adverse drug reactions. less common causes are classified under the umbrella term of hypereosinophilic syndromes (table . ). since parasitic infections are likely to be the commonest cause of eosinophilia in the tropics and in returning travellers, an extensive search for such infections should be undertaken in patients with persistent eosinophilia; initial investigations should be determined by the patient's history of geographical exposure (figure . ) . [ ] [ ] [ ] the absolute number of eosinophils in the peripheral blood may not correlate with their tissue distribution or with their potential to cause tissue damage from their granule release. this is because the degree of eosinophilia depends on the extent of tissue invasion and is therefore modest with tapeworms and roundworms resident in the bowel but much higher where invasion occurs, for example with, toxocara canis or filaria. schistosomiasis almost always causes eosinophilia. strongyloides stercoralis has the capacity to remain in the host for decades after initial infection and causes varying degrees of eosinophilia, with or without other symptoms. steroid treatment, which may be necessary in cases of eosinophilic tissue damage, can exacerbate clinical problems in patients with strongyloides infection so this parasitic infestation should be excluded before starting steroids for hypereosinophilia. mild to moderate eosinophilia is common in asthma although a very high count should prompt a search for churg-strauss syndrome or allergic bronchopulmonary aspergillosis. most drugs including penicillins can cause eosinophilia but the diagnosis can only be made by noting recovery when the drug is discontinued. eosinophilia can be a feature of hodgkin's lymphoma. it signifies a more favourable prognosis and may precede the original diagnosis of lymphoma or relapses. in immunocompromised patients, such as those with hiv infection, the finding of eosinophilia may be crucial since the success of antiretroviral treatment may depend on concomitant eradication of parasites. thrombocytopenia is often discovered incidentally in patients during full blood count estimation. a platelet count above - × /l is usually not associated with any symptoms such as bleeding. if clinically evident haemorrhage does occur at counts above this level, other conditions such as coagulation defects, vascular problems or rarely platelet dysfunction should be suspected. although the prime role of platelets is in haemostasis, several other important roles have been recognized in recent years including wound repair, tissue healing, antimicrobicidal properties, lymphangiogenesis, tumour metastasization and maintenance of blood vessel integrity. congenital platelet disorders are often part of a syndrome. patients with wiskott-aldrich syndrome have small platelets in association with eczema and recurrent infections. other congenital platelet disorders, such as myh -related disorders, can present with deafness or cataracts while skeletal deformities and oculocutaneous albinism are common in other syndromic presentations. blood film morphology can provide important clues about the causes of thrombocytopenia (figure . ). fragmented red cells (schistocytes) increase the possibility of microangiopathic haemolytic anaemia, where an altered vessel wall and fibrin formation in the blood vessels shred the erythrocytes and consume platelets. thrombotic thrombocytopenia purpura, haemolytic uremic syndrome and disseminated intravascular coagulation can all present with thrombocytopenia. dysplastic red or white cells should raise the suspicion of myelodysplasia which can be confirmed by bone marrow examination and cytogenetic analysis. it is important to exclude in vitro platelet agglutination as a cause for apparent thrombocytopenia. this can be an anticoagulant (edta)-dependent phenomenon so a repeat sample should be examined using citrate anticoagulant. rarely, platelet satellitism where the platelets clump round the neutrophils, can cause artefactual thrombocytopenia. anaemia affects nearly two billion people globally with a much higher prevalence in developing countries compared with more wealthy nations ( % vs %). the continents of africa (highest prevalence) and asia (greatest absolute burden) account for more than % of the anaemic population. anaemia burden is highest among children and women of reproductive age. anaemia contributes to more than maternal deaths and perinatal deaths globally per year. who have defined anaemia according to various haemoglobin concentrations (table . ) but the appropriateness of these thresholds has been questioned because there are wide variations in haemoglobin concentration among people of different races. the prevalence of anaemia can be a useful indicator of public health status of a nation because: • the prevalence of anaemia is objective and quantifiable • anaemia is a major complication of several infections, including malaria, hiv, tuberculosis, and the neglected tropical diseases, which are among the commonest problems in most tropical countries • the incidence of anaemia changes in a predictable fashion with alterations in disease burden • the prevalence of anaemia can be used to assess whether an intervention has reached the poorest communities. haemoglobin concentration of < g/l has been recommended for disease surveillance in high-prevalence countries where changes in haemoglobin are used for monitoring the impact of interventions. anaemia in tropical countries (box . ) is often due to infections but chronic health problems, such as diabetes and chronic respiratory disease, and cancer and related complications are increasing as causes partly due to lifestyle changes. the body to compensate for the drop in haemoglobin content. for this reason the haemoglobin level can drop to extremely low levels before symptoms develop. anaemia presents with symptoms such as exertional breathlessness, palpitations and in some cases, syncopal attacks. patients with chronic anaemia may also have a multitude of nonspecific symptoms including poor concentration, decreased work performance and easy exhaustion (table . ). a thorough history and clinical examination may provide clues about the cause of anaemia but further investigations are often necessary to confirm the diagnosis and guide treatment. however, in many resource-poor settings, access to routine biochemical and haematological testing is scarce, so much reliance is placed on clinical examination. the international guidelines for the integrated management of childhood illness recommend that a diagnosis of anaemia in sick children is based on the assessment of palmar pallor. for pregnant women, symptoms of fatigue and dyspnoea, combined with signs of conjunctival and palmar pallor, and increased respiratory rate suggest anaemia. however, making a diagnosis of anaemia based on clinical assessment alone is unreliable unless the anaemia is severe. no specific anatomical site is particularly accurate for the prediction of anaemia though sensitivity may be increased by using multiple sites. most central laboratories in low-income countries have automated haematology analysers and several manual methods exist for assessment of haemoglobin concentration, which are suitable for rural areas where there is no mains electricity (e.g. haemoglobin colour scale; hemocue technique). [ ] [ ] [ ] haemoglobin colour scale principle. the colour of a finger-prick blood sample, soaked into special chromatography paper, is compared with the clinical symptoms and signs of anaemia vary and depend on the cause and the speed of onset. a rapid drop in haemoglobin is much more likely to cause symptoms of anaemia than chronic anaemia. slowly developing anaemia allows time for in many cases, there will be more than one of these conditions coexisting in the same individual. an adequate response to the treatment of anaemia requires management of all the contributory factors. intrauterine growth. thus, low birth weight and prematurity are both associated with iron depletion in the postnatal period. several interventions have been suggested to improve infantile iron deficiency, including: [ ] [ ] [ ] • delayed cord clamping at delivery; the short delay of - minutes allows a small but important amount of blood to continue to flow to the foetus from the placenta • improvement of infant feeding practices • prevention and treatment of infectious diseases • interventions to prevent low birth weight, such as maternal nutritional supplementation, the control of infections and chronic health problems in pregnancy. anaemia in young children can be due to increased nutrition requirements during periods of rapid growth; these requirements may be up to times higher per kilogram of body weight than that of an adult male. in addition, infant and toddler diets often lack bio-available iron. a case-control study of preschool children in malawi with severe anaemia (haemoglobin concentration, < g/l) identified bacteraemia, malaria, hookworm, hiv infections and deficiencies of vitamins a and b as the commonest causes of anaemia. lack of folate and iron were uncommon. in low-income countries multiple interdependent causes of anaemia often operate in one individual so rectifying a single factor is unlikely to make a big impact on resolving anaemia. interventions which are useful in preventing anaemia in younger children include micronutrient supplementation (food fortification), de-worming, prevention and treatment of infectious diseases, school nutrition programmes and community-based nutrition promotion. who defines anaemia of pregnancy as a haemoglobin level less than g/l, or haematocrit less than %, at any time during pregnancy. about one-fifth of maternal mortality is attributable to anaemia in pregnancy and anaemia affects nearly half of all pregnant women worldwide. maternal anaemia is associated with many factors that might also be causally associated with mortality including poverty, infections and inadequate health-seeking behaviour. globally, the most important cause of anaemia in pregnancy is iron deficiency although hookworm, malaria, hiv infection, and deficiencies in folate and other micronutrients may contribute. pregnancy-associated complications, including septicaemia, pre-eclampsia and other obstetric problems can precipitate anaemia. it is important to note that a diagnosis of iron deficiency in pregnancy which relies on ferritin measurements may be misleading because of high-quality digital examples of known haemoglobin concentration. the colours are represented in g/l increments from g/l to g/l. this method is inexpensive, does not depend on skilled scientists, is durable in dusty, hot, dry and humid conditions and is probably better than clinical diagnosis for detecting mild and moderate degrees of anaemia. the disadvantages are that it requires specific chromatography paper and good natural light and it cannot detect changes in haemoglobin less than g/l. this is a small battery-or mains-operated machine, which uses a drop of blood in a plastic cuvette to produce a direct read-out of haemoglobin in a few seconds. it is simple to use, produces accurate and consistent results to one decimal place and it has an in-built quality-checking mechanism. the hemocue hb- has been specifically designed for tropical conditions and operates in temperatures up to °c, in dusty and humid conditions. however the recurrent costs associated with disposable plastic cuvettes mean there is little opportunity for cost-saving with high-volume workloads. the iron status of an infant is directly proportional to its body mass and blood volume, both of which are reflections of the major cause of anaemia in most of these cases is iron deficiency. some of the effects have been described in individuals with iron deficiency without obvious features of anaemia. there are three intervention strategies recommended by who to prevent anaemia in pregnancy: . weekly iron and folic acid supplementation in women of reproductive age . daily iron and folic acid supplementation during pregnancy . presumptive treatment of hookworm infection during pregnancy in areas where hookworm infection is known to be endemic. several factors may interfere with the efficacy of these interventions. under-participation in antenatal care may be common due to factors such as geographic distance, low motivation and poor interpersonal skills of health staff, poor quality of supplies and facilities, insufficient supply of iron and folic acid pills and womens' poor understanding about the daily use of supplements, especially in the face of common side effects. in sub-saharan africa, the acute shortage and high turnover of health workers, and lack of time have also been shown to contribute to ineffective antenatal measures for reducing anaemia. interestingly, a study from bangladesh showed that the first pills (whether taken on a daily basis or less frequently) yielded most of the benefit for raising haemoglobin levels, which suggests that currently recommended doses may be higher than necessary to achieve optimal outcomes, except when anaemia is very severe. the global burden of iron deficiency has been estimated from anaemia prevalence surveys, which include many different causes of anaemia so data may be unreliable as they are often not based on proven cases of iron deficiency. who estimates that globally % of women and % of pre-school children are affected by iron-deficiency anaemia, making it number of selected risk factors for preventable death and disability worldwide. iron deficiency begins in childhood, worsens during adolescence in girls and is aggravated during pregnancy. poor iron stores at birth, low iron content of breast milk and low dietary iron intake throughout infancy and childhood result in high prevalence of anaemia in childhood. anaemia is exacerbated by increased requirements during adolescence and iron loss from menstruation and is often compounded by the lack of adequate nutrition. the situation is worsened by pregnancy when iron requirement is approximately two times higher than in a nonpregnant state. iron deficiency should not be considered a diagnosis but a secondary outcome due to an underlying medical condition. although it may be a physiological response to rapid growth or increased requirements during childhood and pregnancy, it still requires treatment due to potential deleterious consequences. many of the chronic effects of iron deficiency may develop before the clinical and laboratory evidence of anaemia becomes apparent. the biochemical evidence for iron deficiency occurs in several steps. initially, iron stores in the bone marrow are depleted as reflected by a decreased serum ferritin. the total iron-binding capacity then starts to rise, while the serum iron saturation begins to fall before microcytosis and a drop in haemoglobin ensue. there have been attempts to identify this early iron deficiency before anaemia develops in order to improve neurological and psychomotor functions in children and work performance in adults through widespread iron supplementation. however, there are concerns that iron excess may promote infections, especially in malarious areas. a range of laboratory investigations are usually necessary if iron deficiency is suspected (table . ) - because once the diagnosis is confirmed, a search for the precise cause is necessary. a systematic approach to the investigation of iron deficiency (see below) is required based on an understanding of alterations in the iron absorption and transport cycle. • deficient intake (cow's milk has poor iron content and can cause gut blood loss in some infants) • rare defects of haem biosynthesis and iron transport. iron-deficient individuals may have no symptoms. excessive fatigue and other nonspecific signs of anaemia become more pronounced as anaemia develops. consumption of unusual 'foods' such as ice and paint or 'pica' only occurs in a minority of individuals. physical examination may reveal stomatitis, glossitis, koilonychia (spoon-shaped nails) and hair loss. oesophageal webs have been described in the plummer-vinson syndrome but are rare and may respond to iron replacement. since iron is important in neuromuscular development, several features of anaemia described in table . may be related to iron deficiency. treatment of iron deficiency is with dietary modifications and oral or parenteral iron. blood transfusions should be reserved for those with severe symptoms especially if the anaemia developed rapidly. haemoglobin levels alone should not be considered as a criterion for transfusion since very low levels (e.g. - g/l) may be appropriately treated with oral iron if anaemia has developed slowly. intravenous iron should only be considered in cases of poor response or intolerance to oral iron. cereals, poultry and green leafy vegetables, contain non-haem iron, which is often poorly absorbed. if dietary history suggests a deficiency, diet with foods rich in haem iron, such as red meat or liver should be recommended if social and religious customs and financial status allow, ideally with a drink containing vitamin c to facilitate iron absorption. absorption is also facilitated by taking supplements on an empty stomach although side effects of dyspepsia may not always allow this strategy. heavy tea intake can interfere with iron absorption and should be avoided. multivitamin or dietary supplements containing calcium, zinc or copper can also interfere with iron absorption. absorption may be delayed by tetracyclines, milk and soft drinks. since acid is necessary for iron absorption, antacids may account for a poor response to oral iron. iron is usually prescribed as a daily dose of - mg of elemental iron, commonly ferrous sulphate, tablet three times daily. the dose in children is - mg/kg per day split into divided doses. assuming good compliance and absorption, this should result in an increase in haemoglobin within weeks. once the haemoglobin is normalized, iron should be continued for months to replenish the iron stores. the major problem with oral iron is upper gastrointestinal side-effects, which can be dose-dependent. a reduction in the dose or change in the formulation to gluconate or fumarate or even liquid forms, may be successful. liquid iron preparations may stain the teeth and should therefore be taken through a straw. oral iron can also cause constipation or diarrhoea which is not dose-dependent. parenteral iron is best given intravenously because intramuscular iron is painful and has been associated with development of soft tissue sarcomas. high-molecular-weight iron dextran carries a low but significant risk of anaphylaxis, but the newer formulations including low-molecular-weight iron dextran, iron sucrose, ferumoxytol and iron gluconate have minimal risks. vitamin b or cobalamin deficiency is a well-recognized cause of macrocytic anaemia (box . ). although some microorganisms can synthesize cobalamin, humans need to obtain this essential vitamin from foods, mainly meat, poultry and dairy products. vitamin b is an essential co-factor in dna synthesis, serving as a co-factor in two key biochemical processes involving methylmalonic acid and homocysteine as precursors. consequently vitamin b deficiency can interfere with dna synthesis. clinical manifestations include haematological (megaloblastic anaemia and pancytopenia), and neuropsychiatric disorders (paraesthesia, peripheral neuropathy, psychosis and dementia) and an increased risk of cardiovascular disease because of hyperhomocystinaemia. [ ] [ ] [ ] a systematic approach to the investigation of vitamin b deficiency requires an understanding of the absorption cycle. ingested vitamin b is broken down in the acidic environment of the stomach. it binds to r-binders in gastric secretions and saliva which stabilize the vitamin b . in the alkaline environment of the small intestine, vitamin b is released from r-binders to bind to intrinsic factor, synthesized in the gastric parietal cells. this vitamin b -intrinsic factor complex is absorbed from the terminal ileum. recently, an alternative absorption system independent of intrinsic factor and the terminal ileum has been postulated which provides a rationale for mean cell volume useful as a diagnostic clue but not confirmatory can also be low in thalassaemia, sideroblastic anaemia and rarely lead poisoning can be falsely normal in the presence of iron deficiency in older people or with coexistent megaloblastic anaemia anaemia of chronic disease can occasionally cause microcytosis serum ferritin the most useful laboratory measure of iron status low value is diagnostic in the presence of anaemia very high values (> µg/l) usually exclude iron deficiency' being an acute-phase protein, it increases in inflammatory conditions, and certain malignancies, making it unreliable also increased in tissue damage especially of the liver levels are falsely decreased in vitamin c deficiency and hypothyroidism erythrocyte zinc protoporphyrin an intermediate in haem biosynthesis and elevated concentrations indicate interrupted haem synthesis due to iron deficiency when zinc is incorporated in place of iron can be measured on a drop of blood with a portable haematofluorometer small sample size makes it very useful as a screening test in field surveys, particularly in children, and pregnant women where inflammatory states may not co-exist red cells should be washed before measurement (serum bilirubin and fluorescent compounds like some drugs can give falsely high values) although not often done lead poisoning can give falsely high values rarely acute myeloid leukaemia and sideroblastic anaemia give slightly high values useful in that it is not increased in thalassaemias who recommends normal level > µmol/mol haem iron studies serum iron concentration represents the iron entering and leaving the circulation. its range varies widely with age, circadian rhythm, infections and iron ingestion total iron binding capacity measures iron bound to transferrin. raised levels are suggestive of iron deficiency transferrin saturation is the ratio of serum iron and the tibc expressed as a percentage -it is probably more useful in detecting iron overload rather than low levels. sensitive indicator that falls within days of onset of iron-deficiency reduced levels shown to be predictor of iron deficiency especially in the setting of renal insufficiency false normal values can occur when mcv is increased or in thalassaemia serum transferrin receptor it is not increased in inflammatory conditions may be upregulated by increased erythropoiesis (haemolytic diseases) giving falsely high values -serum transferrin receptor to ferritin ratio has been suggested in these cases bone marrow examination with special iron staining (perl's) absence of stainable iron in a sample that contains particles can establish the diagnosis without other laboratory tests a simultaneous control specimen containing stainable iron should also be assessed useful in differentiating from anaemia of chronic disorders or α-thalassaemia or milder forms of thalassaemia can help in identifying the sideroblastic anaemias (ring sideroblasts with perls stain), and some forms of congenital dyserythropoietic anaemia which can also cause microcytosis. an improvement in haemoglobin and clinical symptoms with iron replacement is probably the simplest way to diagnose iron deficiency. peripheral smear may help by demonstrating pencil cells, anisopoikilocytosis and high platelet number in cases of blood loss. the treatment of vitamin b deficiency can be by the oral or parenteral route. increasing evidence suggests that oral supplementation may be adequate even in the presence of malabsorption or pernicious anaemia. , the recommended initial oral replacement dosage is - mg but higher doses may be needed for malabsorption or pernicious anaemia. for patients with severe anaemia and/or neurological disease, daily or alternate day intramuscular injections should be initiated for the first - weeks before reverting to the maintenance threemonthly dose. reticulocytosis is an early marker of response to treatment and is noticeable within - weeks. folic acid deficiency causes similar haematological manifestations to vitamin b deficiency though neuropsychiatric manifestations are less common. the ability of nerve tissue to concentrate folate to levels five times greater than those in the plasma has been suggested as a reason for the absence of neuropathy in folate deficiency. folic acid deficiency is associated with fetal neural tube defects, and possibly with an increase in atherosclerosis and arteriovenous thrombosis, dementia and colonic cancer. dietary folic acid is present in the form of polyglutamates, which are converted to folate monoglutamates by the enzyme folate conjugase in the intestinal brush border, prior to absorption. the monoglutamates function as a carbon transporter and are essential for dna biosynthesis. folate is found in green vegetables and fruits and deficiency can result from decreased intake, impaired absorption and increased utilization, although the commonest cause is dietary insufficiency. in some wealthy countries, cereals have been fortified with folic acid to successfully prevent vitamin deficiency. however folate deficiency continues to be a problem in less wealthy countries and particularly among children and pregnant women. , exclusive feeding of goat's milk to infants can lead to folate deficiency. other causes include alcoholism, excessive cooking of vegetables, and malabsorption (e.g. abnormalities of the small bowel). increased demand for folic acid occurs in pregnancy because the growing foetus has a high avidity for folate. for this reason, folate supplementation has been widely recognized as an essential part of routine antenatal care to reduce the risks of neural tube defects. high folate utilization also occurs in haemolytic anaemias such as sickle cell disease due to high red cell turnover and exfoliative dermatitis. several drugs, including sulfasalazine, trimethoprim, methotrexate, pyrimethamine and phenytoin, can also interfere with folate metabolism. folate-deficient individuals develop a macrocytic anaemia with peripheral blood and bone marrow findings similar to that found in vitamin b deficiency. diagnosis of folate deficiency is confirmed by the presence of low serum folate. red cell folate levels decrease more slowly than serum levels during the -day turnover of the red cells. red cell folate levels may be a better indicator of tissue folate levels than serum folate, although red cell folate can be more expensive and falsely low in vitamin b deficiency. , treatment of folate deficiency is with oral folate ( mg daily) which is sufficient even in malabsorptive states. it is crucial that any co-existing vitamin b deficiency is ruled out before initiating folic acid therapy, otherwise the neurological manifestations of b deficiency may deteriorate rapidly. it is also important increasingly popular oral replacement therapies. once absorbed, vitamin b binds to transcobalamin ii to be transported around the body. the diagnosis of vitamin b deficiency is based on the measurement of serum vitamin levels in a patient with clinical evidence of deficiency. a note of caution is that folic acid deficiency can cause falsely low serum vitamin b levels. diagnostic clues for vitamin b deficiency include marked macrocytosis (often > fl), neutrophil nuclear hypersegmentation and oval macrocytes in the peripheral blood film. blood tests may demonstrate increased lactate dehydrogenase and low haptoglobin levels due to haemolysis within the bone marrow. the cause of the macrocytosis can be confirmed by bone marrow examination which reveals a megaloblastic picture. although macrocytic anaemia is a typical feature of vitamin b deficiency, it can be absent in older individuals who may only have neuropsychiatric features. measurements of methylmalonic acid and homocysteine levels, two markers which are very sensitive for detecting b deficiency, have shown that vitamin b deficiency can occur with normal haemoglobin levels and without macrocytosis. pernicious anaemia is probably the commonest cause of vitamin b deficiency. the presence of parietal cell or intrinsic factor antibodies supports a diagnosis of pernicious anaemia. [ ] [ ] [ ] [ ] schilling tests are rarely performed because of the unavailability of the radio-labelled vitamin b and the difficulty in interpreting the results in the presence of renal insufficiency. • pernicious anaemia (begins after ), increased risk of gastric carcinoma and carcinoid tumours • rare congenital disorders, e.g. imerslund-grasbeck syndrome. the neglected tropical diseases are a group of infections which are endemic in developing countries. several of these neglected tropical diseases cause anaemia and many can be managed using inexpensive interventions to treat the underlying parasitic infections. the mechanisms of anaemia in these conditions are predominantly blood loss from the gastrointestinal or genitourinary tracts but also poor nutrition, bone marrow suppression, inflammation, hypersplenism and haemolysis. anaemia is a common consequence of infections with soiltransmitted helminths or schistosoma with a strong correlation between haemoglobin level and worm load or faecal egg count. even mild infections can lead to anaemia. polyparasitism (i.e. infection with several parasites simultaneously) can be responsible for unresponsiveness of the anaemia to eradication of one organism. treatment of communities at high risk of soiltransmitted helminths improves growth and iron stores in children and reduces anaemia in pregnant women. the treatment of anaemia due to neglected tropical diseases depends on eradication of the parasite with drugs such as albendazole and praziquantel though anaemia resolution may be less successful if it is due to trichuriasis. - the addition of iron to anthelmintic treatment has met with variable success rates probably because there is associated anaemia related to inflammation. however it is still generally recommended that iron supplementation should be included with anthelmintic therapy in treatment programmes for neglected tropical diseases. [ ] [ ] [ ] introduction haemoglobin s (hbs) has a prevalence of - % in many parts of africa and also some areas in the middle east ( figure . ). hbs tends to be common among ethnic groups that have traditionally had high exposure to plasmodium falciparum malaria. in sub-saharan africa approximately infants are born with sickle cell disease each year, mostly with hbss. sickle cell disease (scd) is an autosomal recessive disorder characterized by production of an abnormal haemoglobin, sickle haemoglobin. sickle haemoglobin (hbs) arises from a mutation in codon of the β-globin gene resulting in replacement of the normal glutamic acid residue by a valine. scd is most commonly caused by the co-inheritance of two sickle cell genes (homozygous hb ss disease) but patients who are heterozygous for hbs and for another haemoglobin mutation such as hbc (haemoglobin sc disease) or β-thalassaemia (sβ and sβ + ) can also present with features of scd. ss disease and sβ disease are more severe than sc disease and sβ + disease (box . ). scd can affect multiple organs and its clinical course is punctuated by episodes of acute illness on a background of progressive organ damage, especially of the central nervous system and the lungs. the first description of scd was in in an anaemic grenadian dental student and over the next years it was that the underlying cause of folate deficiency is identified and treated. vitamin a is important in erythropoiesis, iron metabolism (enhances iron absorption and its release from stores to the bone marrow) and for decreasing the risk of infections. vitamin a deficiency is a major public health problem in lowincome countries, with an estimated million preschool children affected. pregnant women and women of childbearing age also constitute high-risk groups for vitamin a deficiency. vitamin a given to thai school children with conjunctival xerosis led to a significant increase in haemoglobin level and in anaemic school children in tanzania, vitamin a supplementation produced a marked increase in haemoglobin which was enhanced by co-administration of iron. vitamin a can also improve anaemia in pregnant women, depending on the local prevalence of deficiency [ ] [ ] [ ] [ ] though the response may be suboptimal in pregnant women infected with hiv. copper is a trace element necessary for normal haematopoiesis and myelopoiesis. anaemia in copper deficiency is due to decreased activity of the copper-dependent enzymes, hephaestin, ceruloplasmin and cytochrome c oxidase. these are important in ferrous-ferric iron conversions and their decrease leads to abnormalities in iron absorption and its incorporation into the haemoglobin molecule. acquired copper deficiency occurs with malnutrition and gastrointestinal malabsorption syndromes. coeliac disease, cystic fibrosis and individuals who have had gastrectomy or surgery resulting in 'short bowel' are also at risk. copper deficiency has also been described in persons ingesting excessive amounts of zinc-containing supplements and those who have swallowed zinc-containing coins. , anaemia related to copper deficiency is normocytic or macrocytic and can be associated with neutropenia; thrombocytopenia is rare. bone marrow findings are characteristic with cytoplasmic vacuolization of both erythroid and myeloid precursor cells with ringed sideroblasts and an unusual finding of iron granules in plasma cells. these findings may be misdiagnosed as myelodysplastic neoplasm. measurement of serum copper levels is helpful in confirming the diagnosis although the test is fairly insensitive. since almost complete haematological recovery can occur with copper replacement, this may be a useful diagnostic test. oral copper supplements can be started with mg of elemental copper a day slowly decreasing over the next few weeks to mg until a good response is noted. although low zinc levels do not cause anaemia they have been linked to growth retardation, heightened susceptibility to infection and male hypogonadism in relation to sickle cell disease. zinc deficiency has been described in nearly half of children and % of adults with sickle cell disease possibly due to increased loss of zinc in the urine and high cell turnover with decreased dietary intake. in contrast zinc excess can cause anaemia through interference with copper absorption by sequestering it in the gut lumen. for this reason, zinc compounds have been used to treat wilson's disease which is characterized by copper excess. however repeated sickling and unsickling eventually causes irreversible changes, so early management to avoid repeated crises is important to prevent disease progression. polymerization, and therefore the clinical features of scd, are influenced by three main factors ; hypoxia, the intracellular hbs concentration and the co-existence of other genetic haemoglobin abnormalities (e.g. α-thalassaemia or hereditary persistence of fetal haemoglobin-haemoglobin f). sickled red cells lead to vaso-occlusion and haemolysis due to the entrapment of sickled erythrocytes in the microvasculature and upregulation of adhesion receptors. , , white blood cells contribute to this process by providing an inflammatory discovered hypoxia led to red cell sickling scd arises from the tendency of hbs to polymerize in hypoxic states. this phenomenon occurs where there is deoxygenation and is due to the binding between β and β chains of two haemoglobin molecules, a property unique to haemoglobin variants that have the glu- -val substitution. the polymerized haemoglobin fills the erythrocyte and deforms its architecture and flexibility to form a sickle shape. this alteration in the structure promotes cellular dehydration, , , upon reoxygenation, the polymers dissolve thus reversing the sickling process. exposure to cold, fever, menstruation, alcohol intake and dehydration can precipitate pain crises. unlike acute pain crises, chronic pain in scd usually has an identifiable basis such as femoral head necrosis, osteoarthritis or chronic skin ulcers. sickle erythrocytes have an average life span of days and anaemia can be due to several causes (box . ). red cell haemolysis causes anaemia and gall stones and can cause fatigue out of proportion to the anaemia. , there are suggestions that patients with low haemoglobin concentrations and high haemolytic rates are more likely to develop vascular problems compared with those with higher haemoglobin concentrations. splenic sequestration with a sudden rapid drop in haemoglobin occurs in those who have not yet developed autosplenectomy so it can occur in young children with hbss and adults with hbsc disease or sickle cell-β + -thalassaemia. treatment may require blood transfusion and in rare cases, sequestration can be fatal. splenectomy may be needed for recurrent severe sequestration. parents can be taught to feel their infant's abdomen for an enlarging spleen and report to hospital if there is a sudden increase in spleen size. red cell aplasia can develop due to secondary parvovirus infection which has a predilection for erythroid progenitors. alloimmunization is common in scd patients who have had frequent transfusions so, if possible, extended red cell phenotyping should be undertaken. hyperhaemolytic crisis is suspected when there is sudden exacerbation of anaemia with increased reticulocytosis and bilirubin level. infectious complications of scd are a major cause of morbidity and mortality, even with adequate vaccination and prophylactic antibiotic regimens. this propensity to infection is related to impaired splenic function although tissue ischaemia, especially in the lungs and renal system, can contribute. hyposplenism is demonstrable in the peripheral blood film by the presence of howell-jolly bodies. most children with scd have undergone autosplenectomy by the age of years and therefore have increased risk of infection from encapsulated microorganisms. typical infectious complications include pneumococcal sepsis, neisseria meningitis, osteomyelitis caused by salmonella species, urinary tract infections and pyelonephritis due to escherichia coli. anatomical abnormalities such as renal papillary necrosis can predispose to urinary complications which may require long-term antibiotics. acute chest syndrome (acs) is defined as a new pulmonary infiltrate on the chest radiograph combined with one or more environment. activation of platelets and the coagulation system also contribute to the vaso-occlusion in scd. [ ] [ ] [ ] [ ] [ ] infants with scd are protected during the first few months of life by the high levels of haemoglobin f in the red cells. anaemia usually develops by months. at all ages, chronic haemolysis of abnormal red cells means that scd is associated with steady state haemoglobin levels of - g/l. although any organ can be affected by scd and complications can occur at any age, certain features tend to predominate in different age groups (box . ). pain is the hallmark of scd and four different patterns of pain have been described with scd each with different underlying mechanisms: • vaso-occlusive (acute and intermittent) • pain from bone and tissue necrosis (chronic) • neuroplasticity (chronic, neuropathic) -functional brain changes • opioid-induced hyperalgesia (acute or chronic). painful crises often start in young children as dactylitis or handfoot syndrome, in which painful swelling of the hands and feet results from the inflammation of metacarpal and metatarsal periosteum. these crises are the result of vaso-occlusion of the bone marrow causing bone infarction and release of mediators that activate pain receptors. the number, severity and frequency of painful episodes vary widely in individuals. half may never have any episodes whereas about % may need hospital admission up to times a year. more than three pain episodes requiring hospitalization per year is associated with increased mortality among patients over years old. in under-resourced settings, hospital visits underestimate the frequency of pain box manifestations such as fever, cough, sputum production, tachypnoea, dyspnoea or new-onset hypoxia. acs is the most common cause of death in scd patients and a frequent cause of hospitalization, second only to painful crisis. mortality in patients with acs in a wealthy country setting is % in children and . % in adults. the peak incidence for acs is - years of age and gradually declines to . per patient-years in subjects older than years. , fever and cough are more common in children with acs and chest pain and dyspnoea are more common in adults. acs is often preceded by febrile pulmonary infection in children and by vaso-occlusive pain crisis and lung infarction in adults. it is important to note that although tachypnoea, wheezing and features of chest infection may be identified, a third of the patients may have a normal physical examination. more than one-third of patients with acs are hypoxaemic (oxygen saturation < %). chest radiography is essential although infiltrates may lag behind clinical symptoms by up to days. repeat chest x-rays are recommended if there is a strong clinical suspicion of acs. bilateral infiltrates or involvement of multiple lobes may predict a poorer prognosis. risk factors for acs (box . ) include fat embolus which can be confirmed by finding stainable fat in pulmonary macrophages. chronic complications such as pulmonary hypertension occur in as many as % of patients and do not appear to be associated with prior episodes of acs. high serum phospholipase a , and the surrogate marker c-reactive protein, have been noted in patients admitted with vaso-occlusive crisis - hours before the development of acs. , stroke neurological complications occur in at least % of patients with scd and scd is one of the most common causes of stroke in children. , in scd, the risk of having a first stroke is % by the age of , % by age years and % by age years. both thrombotic and haemorrhagic strokes occur, although the former is more common in children and those over years of age, whereas the latter is more common between the ages of and years. this age-specific pattern may be related to the higher cerebral flow rates in early childhood. although the prevalence of clinically overt stroke is of the order of %, clinically silent infarction, detectable by magnetic resonance scans, affect nearly double this number by the age of . silent infarcts are associated with cognitive impairment and the majority of these children require lifelong specialist care. cerebral thrombosis, which accounts for - % of all strokes in scd, results from large-vessel occlusion whereas silent infarcts are the result of microvascular occlusion or thrombosis or hypoxia secondary to large-vessel disease. in a third of scd patients, major-vessel stenosis is accompanied by collateral vessels that appear as 'puffs of smoke' (moyamoya) on angiography. risk factors for ischaemic strokes in scd include increased cerebral blood flow velocity, previous silent infarcts, nocturnal hypoxaemia, severe anaemia, acute chest syndrome and elevated systolic blood pressure. an elevated leukocyte count is a risk factor for haemorrhagic stroke. [ ] [ ] [ ] [ ] diagnosis often the family history and clinical findings clearly point towards a diagnosis of scd and during an acute crisis, abundant sickled red cells can be seen on a blood film. white cell counts are higher than normal in scd disease, particularly in patients under age years. the presence of sickle haemoglobin in different sickle syndromes (e.g. hbas, hbss, hbsc) ( table . ) can be confirmed by a simple sickle slide or solubility test. haemoglobin electrophoresis will distinguish between many of these variants but high-performance liquid chromatography and iso-electric focusing are preferred for a definitive diagnosis. haemoglobin mass spectrometry and dna analysis are being increasingly used. antenatal screening is available to women in some countries to help to identify couples who are at risk of having a baby with scd. community acceptance of reproductive genetic services however depends on the effectiveness of education and counselling. the use of prophylactic penicillin and the provision of comprehensive medical care during the first years of life have reduced mortality related to scd from % to less than %. management (box . ) individuals with scd are best managed by a multidisciplinary team as they may require a variety of specialist inputs including haematology, ophthalmology, nephrology, obstetrics, orthopaedics and physiotherapy. the cornerstones of scd therapy are disease modification and prompt and effective management of crises. severe pain crises generally require intravenous fluids and adequate, often opiate, analgesia (box . ), while disease modification is based on interventions to increase hbf levels. in steady state it is usual practice to give sickle cell patients folate supplements ( - mg/day) because their high rates of haemopoiesis put them at risk of deficiency. scd is associated with functional asplenia so patients should also receive prophylactic oral penicillin ( mg twice a day) and vaccinations against encapsulated organisms. hydroxycarbamide is the main agent used to increase hbf (box . ) and is associated with significant reductions in acute pain crises, hospitalization rate, time to first and second pain crises, episodes of acute chest syndrome, and the need for transfusions and the number of units transfused. other beneficial effects of hydroxycarbamide, which are independent of the increase in hbf, include reduced neutrophil count, increased cellular water content, decreased hbs concentration, changing expression of adhesion molecules and nitric oxide generation. hydroxycarbamide may also be an alternative to frequent blood transfusions for the prevention of recurrent stroke in children as it can lower transcranial doppler velocities. , under-use of this cheap, effective drug is related to concerns about leukaemogenicity but this has not been shown to be a problem when used for a non-malignant condition like scd. the two main approaches to transfusion in scd are simple top-up transfusion and exchange transfusion. target haemoglobin level in scd therapy is g/l or a haematocrit of %; higher target levels are associated with hyperviscosity and box . management of complications of sickle cell disease • inability to maximally concentrate urine (hyposthenuria) in response to water deprivation is an early finding • renal tubular acidosis • increased urinary tract infections • glomerular hyperfiltration, increased creatinine secretion, and a very low serum creatinine are characteristic of young patients with sickle cell anaemia, so renal dysfunction can be present even with normal serum creatinine values • microalbuminuria is common in childhood and up to % of adults develop nephrotic-range protein loss • gross haematuria can develop due to microthrombin in renal vessels, renal medullary carcinoma, and nocturnal enuresis • treatment is based on the early use of hydroxycarbamide and angiotensin-converting enzyme inhibitors in children with clinically significant albuminuria. • noted in up to % of scd cases • no relationship to acute chest syndrome (different pathophysiology) • mortality risk with even mild pulmonary hypertension is high • regular blood transfusions and long-term anticoagulation have been tried • hydroxycarbamide may decrease the risk • prostacycline analogues (epoprostenol, and iloprost), endothelin- receptor antagonists (bosentan), phosphodiesterase inhibitors (including sildenafil), and calcium channel blockers are being evaluated. • brief but recurrent (stuttering); may occasionally last for many hours and can lead to impotence • usually ischaemic, or low-flow, priapism • patients should be educated to seek medical attention if more than hours duration • detumescence within hours is necessary to retain potency • intravenous hydration and analgesia initially with consideration for α-adrenergic agonists (etilefrine or phenylephrine) • penile aspiration and irrigation with saline and α-adrenergic agents or shunting may be required in severe cases in combination with an exchange transfusion. • assess pain intensity • choose the analgesic, dosage, and route of administration • paracetamol and hydration should be considered in all patients • oral, sustained-release morphine is as good as intravenous morphine infusion in children and young adults • manage mild pain with rest, hydration, and weak opioids (such as codeine). admit patients in whom pain that does not subside promptly or require opioid treatment; fever, pallor, or signs of respiratory compromise; a low likelihood of receiving appropriate care at home • pain management should be individualized and dosing should take into account prior pain management and use of opioids • the pain pathway should be targeted at different points with different agents, avoiding toxicity with any one class • always look for a cause, e.g. infection, dehydration, etc. • education about avoiding exposure to precipitants • be empathetic, reassuring, and supportive • benzodiazepines may be helpful to reduce anxiety • re-examine the patient often to ensure adequate pain relief, to assess sedation and respiratory rate (to avoid opioid overdose). in assessing patient responses to conventional doses of analgesia, it must be remembered that those with sickle cell disease metabolize narcotics rapidly • re-search for evidence of any complications such as acute chest syndrome or anaemia • always look for a cause, e.g. infection. of multi-organ failure. both simple transfusion and exchange transfusions have been used and neither appears to be superior. a short course of steroids may attenuate acs but it may also increase the risk of re-hospitalization. bronchodilators may help patients with wheezing but inhaled nitric oxide has not shown any clear benefits. since coagulation activation is important in the pathophysiology of acute chest syndrome, treatment with low-molecular-weight heparin may reduce clinical complications. transcranial doppler measurement of cerebral blood flow has been a major step forwards in identifying individuals with an increased risk of ischaemic stroke. a value more than cm/ second imparts a % risk of stroke within the next years. regular blood transfusions can reduce the incidence of stroke in children. due to a high recurrence of stroke ( %) on stopping transfusions, continuation of transfusions should be guided by transcranial doppler measurements. , once a stroke has developed, the best therapeutic strategy is exchange transfusion which probably needs to be done monthly. , neurosurgical re-vascularization should be considered for moyamoya-like syndromes when new strokes occur despite transfusion. haemoglobin sc results from the co-inheritance of hbs and hbc and has its highest prevalence in west africa. clinical features and disease management are similar to those of hbss disease but splenomegaly, splenic infarcts and splenic sequestration may occur into adulthood. proliferative retinopathy necessitates regular ophthalmic review in those aged over years. compared with hbss, anaemia is less marked in hb sc ( - g/l) and there are fewer sickle cells and more target cells on the blood film. the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focussing. worsening of complications. in exchange transfusion, the aim is to achieve an hbs% of < %. complications of transfusion in scd include alloimmunization, delayed haemolytic transfusion reactions and iron overload. the high rates of red cell antibody formation ( %) noted in wealthy countries are due to minor blood group incompatibilities between the recipient and the blood donor who is often of a different ethnicity. leukocyte reduction of transfused blood, routine abo, rh and kell matching for all patients and extended phenotype matching for those with alloantibodies may be useful for reducing transfusion reactions. treatment for acs is predominantly supportive and includes adequate pain relief, antibiotics (e.g. a macrolide with a cephalosporin), continuous pulse oximetry and delivery of supplemental oxygen to patients with hypoxaemia. incentive spirometry can prevent atelectasis and infiltrates and blood transfusion is indicated when a patient develops respiratory distress, a clinically significant fall in the haematocrit or signs the following predict a more severe clinical course and are additional reasons to consider offering hydroxyurea: hb < g/l, wbc > × /l, hbf < % and renal insufficiency due to scd. • start at - mg/kg per day (to the nearest mg/day) • if no or poor response, increase dose by increments of mg/ kg per day every weeks (max: mg/kg per day). most good responses require about - g/day in adults • monitor fbc, hbf%, and reticulocytes every or weeks initially, then every weeks when on a stable dose • monitor biochemistry profile (hydroxyurea has renal excretion and hepatic toxicity). • less pain • persistent increase in hbf (usually measured every - weeks) or mean cell volume • persistent increase in haematocrit if severely anaemic • decrease in ldh • acceptable toxicity. improvement in symptoms and blood parameters may take - months of therapy, but can be seen after approximately weeks. if the reticulocyte count is less than expected for the degree of anaemia, erythropoietin deficiency should be considered. • aim in all cases to reduce hbs level to < % • exchange transfusions may be considered in cases of stroke, acute chest syndrome not responding to top-up transfusion and major surgeries • target haemoglobin concentration of g/l may be considered in cases of organ failure and surgery. individuals with sickle cell trait (hb as) have -fold protection against severe malaria compared to individuals with normal haemoglobin (hbaa) probably due to both innate and immune-mediated mechanisms. individuals with sickle cell trait (hbas) are generally asymptomatic and they have a normal haemoglobin and normal life expectancy. uncommonly, complications such as poor perfusion of the renal papillae and increased bacteruria may occur. the blood film is generally normal and the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focusing. the original descriptions of thalassaemia originated from areas round the mediterranean and the term derives from the greek thalassos (sea) and haima (blood). [ ] [ ] [ ] epidemiology thalassaemia is one of the most common single gene disorders and approximately - % of the global population are carriers. α + -thalassaemia occurs throughout the tropics, whereas α thalassaemia, which is responsible for haemoglobin bart's hydrops fetalis, is concentrated predominantly in south-east asia and to a lesser extent around the mediterranean. , β-thalassaemia is common in the mediterranean countries, parts of africa, throughout the middle east, the indian subcontinent and south-east asia. haemoglobin e prevalence is highest in cambodia, laos and thailand and can reach - % with lower prevalence rates in indonesia, malaysia, singapore and vietnam. β-thalassaemia β-thalassaemia is an inherited quantitative deficiency of β-globin chains which are required to make normal adult haemoglobin. more than mutations have been associated with the development of β-thalassaemia (a complete list is available at the globin gene server website, at: http://globin.cse. psu.edu) and they affect protein synthesis , leading to reduced (designated β + ) or absent (designated β ) production of the β-globin chains. the clinical severity of thalassaemia can be lessened by co-existing haemoglobin abnormalities such as the co-inheritance of α-thalassaemia and increased production of haemoglobin f. , α-thalassaemia normal α-globin synthesis is regulated by duplicate α-globin genes on chromosome . the genotype is usually represented as αα/αα and α-thalassaemia usually results from deletion of one or both α-genes. occasionally point mutations in critical regions of the α-genes may cause non-deletional α-thalassaemia (α t ). mutations can completely abolish expression of the αgenes (i.e. α -thalassaemia) or partially down-regulate expression (α + -thalassaemia). both α and α + thalassaemias can occur in the heterozygous or homozygous state or as a compound α /α + heterozygote form (table . ). underproduction of α-globin chains due to three or four gene deletions gives rise to excess γ (fetal) or β (adult) globin chains which form tetramers, called hb bart's (fetal) or hbh (adult). rare forms of α-thalassaemia occur in association with other conditions such as mental retardation and myelodysplastic/ leukaemia syndrome. , pathophysiology β-thalassaemia (figure . ) thalassaemias , , cause an imbalance of αand β-globin chain synthesis. in homozygous β-thalassaemia, excess α-chains precipitate in the red cell precursors and up to % of cells are destroyed in the bone marrow resulting in ineffective erythropoiesis and a shortened red cell survival. the red cells released from the bone marrow contain abnormal α-chains and these inclusions promote destruction of the cells by the spleen leading to clinical symptoms and signs of haemolysis. in heterozygotes, the α-chain excess and the degree of inadequate erythropoiesis is much less than in homozygous β-thalassaemia. hbf production normally tails off within a few months of birth but in β-thalassaemia hbf production can continue into adulthood. the effect of increased hbf production is to prevent precipitation of the excess globin chains and consequent ineffective erythropoiesis. however hbf has a high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion. the pathophysiology of α-thalassaemia, and hence the clinical manifestations, is quite different from β-thalassaemia. the excess non-α-globin chains form soluble tetramers rather than precipitates so there is only minimal ineffective erythropoiesis. the only clinical abnormality in those with hbh may be splenomegaly secondary to increased work load from destruction of red cells containing inclusions. rarely anaemia may be severe enough to require blood transfusions. classification of α-thalassaemia divide β-thalassaemia into thalassaemia major (transfusiondependent), thalassaemia intermedia (able to maintain adequate haemoglobin without transfusions or requiring less than units/year) and thalassaemia minor (asymptomatic). infants with β-thalassaemia are protected from severe anaemia by the presence of haemoglobin f and are usually asymptomatic. clinical manifestations of thalassaemia major depend on whether adequate blood transfusions are available and the stringency with which iron chelation is undertaken. untreated patients with thalassaemia major will die in late infancy or early childhood from the effects of severe anaemia. those who receive sporadic transfusions may survive longer but suffer from the secondary effects of anaemia, bony deformities and growth retardation. the clinical features of β-thalassaemia major are divided into those resulting from anaemia, bony changes and iron overload. anaemia from defective erythropoeisis, decreased red cell survival and increased haemolysis in thalassaemia major leads to cardiac decompensation, failure to thrive and growth retardation in children. splenomegaly, from the increased work load of culling red cells with inclusion bodies, can cause dilutional anaemia and a further drop in haemoglobin. compensatory extra-medullary haematopoiesis can lead to hepatomegaly and occasionally vertebral compression and neurological defects. haemolysis from increased red cell destruction is associated with gall stones in up to % of individuals with β-thalassaemia. another consequence of accelerated haemolysis is the increased incidence of thromboembolism ( % in thalassaemia major and % with intermedia) from the exposure of negatively charged phospholipids on the red cell membrane and the generation of red cell and platelet microparticles. splenectomy with postoperative thrombocytosis is a risk factor for thrombosis especially if combined with endothelial oxidative stress from iron overload, or procoagulant co-morbid conditions such as diabetes mellitus, hormone therapy, thrombophilic mutations and atrial fibrillation. folate deficiency, hyperuricaemia and occasionally gout have been observed in thalassaemia major due to the high turnover of red cells. the enhanced erythropoietic drive from anaemia in thalassaemia can lead to increased marrow expansion with in homozygous β-thalassemia, β-globin synthesis is markedly reduced or absent. the excess α-chains cannot form a tetramer but form a precipitate in the red cell precursors leading to intra-medullary destruction of these cells. this destructive process of the red cell membrane occurs from the formation of α-chain hemichromes (shown as red cell inclusions) and degradation products of the excess α-chains. the red cells which may be released from the bone marrow are destroyed by the spleen leading to clinical symptoms and signs of haemolysis. since only the β-chain is affected in these individuals, the synthesis of hbf and hba continues unabated. these haemoglobins have very high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion splenomegaly, which may be massive, and growth retardation in children. bony changes are unusual. other complications include infections, leg ulcers, gall stones and acute haemolysis in response to drugs and infections. the severity of the clinical features is related to the molecular basis with non-deletional types of hbh disease more severely affected. haemoglobin bart's (−/−) occurs almost exclusively in asians, especially chinese, cambodian and thai populations. an infant with hb bart's hydrops fetalis syndrome has pallor and gross oedema with signs of cardiac failure, marked hepatosplenomegaly and skeletal and cardiovascular deformities. there is often gross hypertrophy of the placenta. many of the clinical manifestations of this condition can be explained by the characteristic bossing of the skull and overgrowth of maxillary region, radiologically noted as 'hair on end' or 'sun-ray' appearance. metatarsal and metacarpal bones are the first to expand so measurement of the metacarpal bones has been considered a good indicator for initiation of transfusion therapy. other skeletal deformities include shortening of long bones due to early epiphyseal fusion and overgrowth of the maxilla causing dental malocclusion. the marrow expansion can also lead to pathological fractures, early bone thinning and osteoporosis , while ineffective drainage of the sinuses and middle ear from skull bone overgrowth can cause chronic sinus and ear infections. growth retardation is primarily the result of anaemia with contributions from iron overload, hypersplenism, deficiencies of thyroid and growth hormone, hypogonadism, zinc deficiency, chronic liver disease, malnutrition and psychosocial stress. patients with β-thalassaemia have increased iron absorption mediated by reduced hepcidin and those who receive regular transfusions may also develop transfusion siderosis if they are inadequately chelated. the iron is deposited in the parenchymal tissues with a variety of clinical consequences (box . ), [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] a process which may be modulated by variants in the haemochromatosis (hfe) gene. thalassaemia intermedia is characterized by haemoglobin concentrations of - g/l and children usually present at around - years of age with symptoms of anaemia, jaundice and hepatosplenomegaly. there may also be skeletal changes such as expansion of the facial bones and obliteration of the maxillary sinuses. several molecular factors including: (a) coinheritance of α-thalassaemia; (b) hereditary persistence of haemoglobin f; (c) δβ-thalassaemia and (d) the specific gγxmn polymorphism contribute to the 'conversion' of thalassaemia from major to intermedia type. in contrast to patients with thalassaemia major, iron loading in thalassaemia intermedia occurs mainly as a result of increased intestinal iron absorption rather than transfusion therapy. ineffective erythropoiesis with resultant chronic anaemia and hypoxia can suppress hepcidin, the regulator of iron metabolism, leading to increased iron absorption. the excess iron tends to accumulate in the liver rather than the heart. other clinical complications in thalassaemia intermedia include gallstones, extramedullary haemopoiesis leg ulcers, thromboembolic events and pulmonary hypertension, which is the major cause of heart failure in these individuals. although individuals with thalassaemia intermedia do not usually need regular blood transfusions, there is some evidence that complications, particularly later in life, may be less common in regularly transfused patients. α-thalassaemias , carriers of α-thalassaemia (traits, with loss of or α genes) are usually asymptomatic and may only be detected through a routine blood count which shows mild to moderate microcytic, hypochromic anaemia. antenatal counselling may be indicated if the mother has αα/− as there is a possibility that the fetus may be at risk of having haemoglobin bart's. haemoglobin h disease occurs mainly in asians and occasionally in the mediterranean population. it is the result of deletion of three α genes (α−/−) and can produce anaemia varying from - g/l. there is usually associated • hypogonadism is the most frequent complication in patients with prevalence over % in both males and females. it is usually hypogonadotrophic suggesting iron damage to the anterior pituitary or hypothalamus. the features range from total absence of sexual development to delayed puberty. in females with normal menstrual function, fertility is normal with the ovarian function preserved in most although secondary amenorrhoea can develop. damage of the ovaries is rare and is more likely to appear in older women (around ) because of high vascular activity on the ovaries at this age. secondary hypogonadism is common ( %) in older men. serum ferritin > ng/ml is a risk factor. • hypothyroidism is the second most common endocrine disorder (about %) although many of them may have the subclinical variety. most commonly hypothyroidism is of the primary type with secondary, central hypothyroidism increasingly being diagnosed in recent years. • the prevalence of diabetes mellitus is around % with the mean age of diagnosis being years. impaired glucose tolerance occurs first with microvascular damage like retinal changes being less common than the conventional form. erythroid precursor destruction. osmotic fragility is reduced, sometimes strikingly so since in some cases the red blood cells do not haemolyse even in distilled water. for this reason, if sophisticated tests are not available, osmotic fragility can be used as a screening test for thalassaemia trait. serum zinc levels may be low and this may be related to abnormal growth. vitamin c levels may also be low due to its increased conversion to oxalic acid in the presence of iron overload. care may be needed if folic acid is commenced on a background of bone marrow failure due to folate deficiency as it may precipitate painful erythropoietic crises. management a comprehensive management plan for patients with thalassaemia may involve transfusion therapy, iron chelation, splenectomy, prevention or early treatment of complications and stem cell transplant. the mainstay of treatment for the severe forms of thalassaemia is blood transfusion with the aim of reducing anaemia and erythropoietic drive. however, in many low-income settings blood supplies are inadequate and many thalassaemic patients are chronically under-transfused (table . ). transfusion frequency should be guided by clinical symptoms and signs such as poor growth and facial or other bone abnormalities, and should take into account any potential disease-modifying comorbidities. although the decision to transfuse should not be based purely on haemoglobin levels, a value of < g/l is often used as a trigger for regular transfusions. to prevent alloimmunization, extended red cell antigen typing for c, e and kell in addition to abo and rh(d) typing should be carried out prior to the first transfusion, and before each transfusion, full cross-match and screening for new antibodies should be undertaken. the risk of alloimmunization appears to be greater in patients who begin transfusion therapy after the first few years of life. development of alloantibodies and autoantibodies may result in increased transfusion requirements or haemolysis. use of leukodepletion techniques can result in less alloimmunization and fewer febrile transfusion reactions. since storage of red cells in anticoagulant solutions may decrease their efficacy, the use of blood that has been stored for less than - days may be beneficial for patients who require frequent transfusions. the use of st-degree relatives as blood donors should be discouraged, especially if the patient is a candidate for stem cell transplant. patients with thalassaemia major need lifelong regular blood transfusions, ml/kg per month or - units of blood every - weeks, to maintain the pre-transfusion haemoglobin level above - g/l. the clinical benefits of this regular transfusion programme include normal growth, suppression of erythropoiesis and bone marrow expansion, reduced hepatosplenomegaly and an overall sense of wellbeing, which allows normal age-appropriate activities. a higher target pretransfusion haemoglobin level of - g/l may be necessary for patients with heart disease or other medical conditions and for those patients who do not achieve adequate suppression of bone marrow activity at the lower haemoglobin level. shorter intervals between transfusions may reduce overall blood requirements but need to be balanced against the patient's work or school schedule and other lifestyle issues. iron chelation therapy , has improved survival rates for thalassaemic patients, and prevented hepatic fibrosis and ironinduced cardiac disease; most patients who are compliant with chelation therapy have normal growth and sexual development. iron chelators (box . ) are usually initiated in children over years who have received units of blood and/or have a steady-state serum ferritin level above ng/ml on at least two occasions. this level of iron overload typically occurs after - years of transfusions. desferrioxamine is started at - mg/kg per day in these children initially, to avoid toxicity due to over chelation. marked splenomegaly, often treated with splenectomy, was common in thalassaemia patients before the advent of regular transfusion programmes. severe haemolysis in thalassaemia is related to a hyperactive spleen, which aggravates anaemia and can increase transfusion requirements. although early the initiation of regular transfusion therapy for severe thalassaemia usually occurs in the first years of life. some patients with thalassaemia intermedia who only need sporadic transfusions in the first two decades of life may later need regular transfusions because of a falling haemoglobin level or the development of serious complications. haemoglobin should be monitored to assess the rate of fall in the haemoglobin level between transfusions and this can be used to indicate the frequency of transfusions. exchange transfusions have been tried as a way of reducing iron loading and are associated with a reduction in blood requirements by about one-third. (table . since each unit of red cells can contain up to mg of iron, cumulative iron burden is an inevitable consequence of a longterm transfusion programme. in addition there is increased iron absorption from the gut ( . - . mg/kg per day) as a response to severe anaemia and down-regulation of hepcidin. transfusion therapy can avert splenomegaly, hypersplenism still can develop, usually in children between and years of age. in these individuals, splenectomy can limit the complications from extramedullary hematopoiesis. splenectomy should be considered when the annual transfusion requirement reaches - ml red blood cells/kg per year and usually results in a halving of the transfusion requirements. splenectomy complications include opportunistic infections with encapsulated organisms. patients should therefore receive appropriate vaccinations preoperatively and should be advised to seek medical advice at the first sign of infection. it is advisable to delay splenectomy until patients are at least years old because of the increased risk of overwhelming sepsis below this age. thalassaemia patients can develop thromboembolic complications and pulmonary hypertension after splenectomy so partial splenectomy and splenic embolization have been attempted to minimize these complications but have not been studied in large trials. iron overload can occur in any organ in thalassaemia patients but particularly affects the heart, liver, the endocrine system, the bone and occasionally the pancreas and lungs. iron overload needs to be detected early and treated to prevent long-term damage. annual assessment of the iron loading of the liver and heart can be achieved using non-invasive methods such as magnetic resonance scanning to detect early changes. children should have regular growth and endocrine assessments and appropriate investigations should be carried out if there are any signs of developmental delay or hormonal deficiencies. osteoporosis is increasingly being recognized and should be prevented by ensuring adequate dietary calcium intake and sun exposure. vitamin supplementation with folic acid, zinc, vitamin e and vitamin c may be useful although the combination of vitamin c and desferrioxamine carries a risk of cardiac toxicity. allogeneic stem cell transplant , is currently the only means of curing thalassaemia. the outcome in carefully selected patients, measured by overall event-free survival, is around % with a transplant-related mortality of %. hepatomegaly, liver fibrosis, and inadequate iron chelation therapy predict a poor outcome. the best results from transplant have been obtained with hla-matched siblings. umbilical cord blood is a useful source of stem cells for young children. other potential treatment options for thalassaemia are outlined in box . . [ ] [ ] [ ] [ ] prevention of severe thalassaemia births by prenatal diagnosis and termination of pregnancies has been successful in countries with a high prevalence of thalassaemia. early identification of couples at risk and culturally sensitive genetic counselling facilitate decision-making for termination or continuation of pregnancy. the mean corpuscular haemoglobin (mch) is used to screen for the presence of thalassaemia using a cut-off of less than pg. rarely, silent β-thalassaemia mutation may present with an mcv over pg and should be considered in those with a positive family history. at-risk couples should be referred for detailed counselling on the options for prenatal diagnosis. these include chorionic villous sampling or amniocentesis, which are used to obtain fetal dna samples for genetic analysis. polymerase chain reactions and precise hybridization assays to detect single point mutations using very small dna samples have also been developed. a less invasive and less risky option is to isolate fetal dna circulating in the maternal blood for genetic analysis. pre-implantation genetic diagnosis is a newer technique where dna from the blastomere is used for genetic diagnosis. ultrasound can be used from the nd trimester for fetuses suspected of having α-thalassaemia to detect signs of hydrops fetalis and enlarged placenta (figure . ) . • hydroxyurea -helpful in some patients with β-thalassaemia intermedia, but not as effective in thalassaemia major • histone deacetylase inhibitors -derivatives of butyric acid; intermittent pulses with hydroxycarbamide has been tried • kit ligand • decitabine • knockdown of bcl a (regulator of γ-globin expression) • erythropoietin. • vitamins c and e • fermented papaya preparations. • successful in β-thalassaemia animal models using a retroviral vector transferring the human β-globin gene sequence and its promoter region into mice stem cells • β-globin gene transfer into progenitor hematopoietic cells of humans is also being studied • other molecular approaches being tried include using different mutations of stop codons and aberrant splicing. partner testing in all cases β-thalassaemia trait protecting red cells. red cells lack any other source of nadph and are solely dependent on the pentose phosphate pathway so g pd deficiency leaves these cells with no defence against oxidative damage. oxidative damage results in denatured haemoglobin aggregates which form heinz bodies (denatured haemoglobin precipitates). these damaged cells bind to the membrane cytoskeleton resulting in decreased cellular deformability, and are also destroyed in the spleen, resulting in haemolysis. the level of enzyme activity is higher in young erythrocytes than in more mature cells so older cells are more susceptible to haemolysis. the global distribution of g pd deficiency mirrors that of malaria, and where malaria has historically been prevalent, and it provides a degree of protection against malaria. • class iv -normal ( - % enzyme activity) • class v -increased activity (> % enzyme activity). g pd enzyme variants can be distinguished by their electrophoretic mobility. g pd b, the wild-type enzyme, and g pd a + , a common variant in populations of african descent, demonstrate normal enzyme activity and are not associated with haemolysis. g pd a − is the most common variant associated with mild to moderate haemolysis with approximately - % hb e is caused by a substitution of glutamic acid by lysine at codon of the β-globin gene. this causes reduced synthesis of the β-e chain and leads to a thalassaemia phenotype. hb e β-thalassaemia affects at least a million people worldwide and is an important health problem particularly in the indian subcontinent and south-east asia. in some areas, it has replaced β-thalassaemia as the most common thalassaemia disorder. the frequency of hbe reaches % in many regions of thailand, laos and cambodia with estimates of at least new cases of hbe β-thalassaemia expected in the next few decades in thailand alone. the natural history of hbe thalassaemia is highly variable; some patients are asymptomatic (e.g. heterozygotes, hbe - % or homozygotes hbe, - %) while others (e.g. hbe with β-thalassaemia) may be transfusiondependent. pathophysiology glucose- -phosphate dehydrogenase (g pd) deficiency was originally recognized through its association with haemolysis related to eating fava beans ('favism') and primaquine ingestion. g pd deficiency is the most common enzyme defect in humans and is present in about million people worldwide (figure . ). , it is an x-linked, hereditary defect caused by mutations in the g pd gene. g pd is an enzyme that catalyses the first reaction in the pentose phosphate pathway, to produce nadph, which is an important antioxidant used to preserve the reduced form of glutathione. , reduced glutathione acts as a scavenger for oxidative metabolites thereby < . % . - . % - . % - . % - . % - % of africans carrying this variant. g pd mediterranean, present in all countries surrounding the mediterranean sea, middle east, india and indonesia, has the same electrophoretic mobility as g pd b but the enzyme synthesis and catalytic activity are reduced. in several populations, g pd a − and g pd mediterranean co-exist. the clinical manifestations of g pd deficiency can be classified into: (i) asymptomatic; (ii) acute haemolytic anaemia; (iii) favism; (iv) neonatal jaundice; and (v) chronic non-spherocytic haemolytic anaemia. acute haemolytic anaemia in g pd deficiency can be secondary to infection (e.g. pneumonia, hepatitis a and b, and typhoid fever) or oxidant drugs, or may be precipitated by diabetic ketoacidosis, myocardial infarction and strenuous physical exercise. , a list of the drugs which may cause haemolysis in g pd-deficient individuals (table . ) can be obtained from: http://www.g pd.org/favism/english/index.mv. a drug which is deemed to be safe for some g pd-deficient individuals may cause haemolysis in others due to the heterogeneity of the underlying genetic variants. haemolysis typically occurs within - days after commencing the drug and can produce intense haemoglobinuria. fortunately, the disorder is self-limiting and most patients do not develop renal impairment or anaemia requiring transfusion. the spontaneous recovery reflects replacement of the older, enzyme-deficient red cells by younger reticulocytes which can withstand oxidative injury. if the precipitating cause has been removed the haemoglobin begins to recover after - days. acute renal failure due to acute tubular necrosis and tubular obstruction by haemoglobin casts can develop as a complication of haemolysis in g pd deficiency. this occurs more often in adults than children and may require haemodialysis. this occurs predominantly in boys aged - years in mediterranean countries, but it has also been observed in the middle east, asia and north africa. both intravascular and extravascular haemolysis, occasionally severe enough to cause renal impairment, can occur after eating fresh or cooked fava beans, and favism has been reported in breastfed babies of mothers who have eaten fava beans. divicine and isouramil have been implicated as the toxic components of fava beans. neonatal jaundice this occurs in one-third of male babies in areas where g pd deficiency is common and is likely due to g pd deficiency. it presents - days after birth and can lead to kernicterus. , maternal exposure to oxidant drugs, and even naphthalenecamphor mothballs, can precipitate haemolysis in affected babies. breast-feeding mothers should therefore be warned to avoid offending drugs, umbilical potions containing fava, triple dye or menthol, and should not apply henna to the skin or use clothes that have been stored in naphthalene. premature infants and babies who have co-inherited the mutation for gilbert's syndrome are at particular risk. phototherapy and exchange transfusion therapy may be required to reduce the level of unconjugated bilirubin. the diagnosis may be easily missed so assessment of g pd status should be undertaken for any jaundiced infant whose family history or ethnic or geographic origin suggest the likelihood of g pd deficiency, and in infants who respond poorly to phototherapy. this is an unusual manifestation of g pd deficiency and usually presents in childhood. , there may be a history of severe neonatal jaundice, episodic or worsening anaemia which requires blood transfusions, and complications from gallstones. although these individuals usually have a well-compensated anaemia, and require transfusions only for exacerbations, rarely some may become transfusion-dependent. antioxidants such as vitamin e and selenium may be of benefit in some cases. the haemolysis does not resolve following splenectomy. folic acid supplementation is necessary to support the increased compensatory erythropoiesis. the diagnosis of g pd deficiency is usually suspected when neonatal jaundice occurs in an area where g pd deficiency is drugs which may cause haemolysis in g pd-deficient individuals common or when an episode of non-immune haemolytic anaemia occurs in association with an infection or drug. the appearance of the red cells on the blood film is characteristic because denatured haemoglobin concentrates in one area within the cell creating 'helmet' or 'bite' cells. denatured haemoglobin precipitates in peripheral red blood cells as heinz bodies which can be detected by staining with methyl violet. definitive diagnosis of g pd deficiency is by quantitative spectrophotometric analysis of the rate of nadph production. point of care tests for g pd deficiency are being developed but have not yet been validated for routine use. measuring enzyme activity during an episode of acute haemolysis is not helpful since reticulocytosis, which is a feature of acute haemolysis, produces a false-negative result because of the high enzyme levels in younger erythrocytes. , management the most effective management strategy for g pd deficiency is to prevent haemolysis by avoiding triggering agents like infections, drugs and fava beans. for the milder variants (e.g. class iii and iv), drugs known to trigger haemolysis may be given to individuals with g pd deficiency if the benefits outweigh the risks and the blood count is closely monitored (e.g. use of low-dose primaquine for individuals with g pd avariant). screening programmes have been established in some mediterranean and other populations where g pd deficiency is prevalent. haematological complications of malaria (see chapter ) the pathophysiology of anaemia in malaria is multi-factorial and influenced by the age of the individual and their antimalarial immune status. anaemia mechanisms in malaria involve: • haemolysis with increased red cell destruction of both infected and bystander erythrocytes • dyserythropoiesis • hypersplenism • haemolysis • co-existent conditions which can cause anaemia. haemolysis is more common in non-immune individuals with acute malaria, whereas dyserythropoiesis is the predominant mechanism for anaemia in recurrent falciparum malaria. , haemolysis is the result of red cell phagocytosis by the reticuloendothelial system and is triggered by damage to the red cell membranes and exposure of abnormal surface antigens on their surface. [ ] [ ] [ ] [ ] ten uninfected red cells are removed from the circulation for each infected red cell destroyed, possibly related to loss of red cell complement regulatory proteins and increased levels of circulating immune complexes. this may partly explain the persistent or worsening anaemia following parasite clearance and the poor correlation between parasitaemia and the severity of anaemia noted in some studies. an increased incidence of anaemia has been noted in malaria vaccine trials possibly due to enhanced clearance of uninfected red blood cells. decreased erythropoeisis with abnormalities in red cell precursors and reticulocytopenia is found consistently on examination of bone marrow from malaria-infected patients. the decreased erythropoiesis is due to many factors including low levels of tnf-α, high levels of interleukin- , abnormalities of erythropoietin, a decrease in burst colony forming units, cytokine-induced suppression of red cell production and the inhibitory effect of the malarial pigment haemozoin. [ ] [ ] [ ] [ ] epidemiology malaria-related anaemia is most commonly seen in children and pregnant women. the prevalence of malarial anaemia in sub-saharan africa in children is - % and in pregnant women it is - %. the highest prevalence is in infants and children less than years of age. infants may acquire malaria through the placenta. , individuals living in malarious areas may have multiple reasons for anaemia such as bacteraemia, hookworm infections and vitamin a deficiency making it difficult to assign anaemia solely to malaria. however, animal studies and the fact that anaemia improves with anti-malarial treatment suggest a direct relationship between malaria infection and anaemia. , for example, in tanzanian children about % of anaemic episodes were thought to be caused by malaria. who defines severe anaemia attributable to malaria as: (i) haemoglobin concentration < g/l or haematocrit < %; (ii) parasitaemia with > parasites/µl of blood and (iii) normocytic blood film (to exclude other common causes of anaemia). however, aspects of this definition have been criticized because blood films are not examined routinely and parasite density varies with endemicity and age. although traditionally it is p. falciparum that has been associated with the most severe malaria-related anaemia, p. vivax is also a major risk factor for severe anaemia especially in young children or those with chronic and recurrent infections. p. vivax anaemia is associated with recurrent bouts of haemolysis of predominantly uninfected erythrocytes with increased fragility. symptoms of malarial anaemia can vary from negligible to profound depending on the degree of anaemia and the rapidity of onset. splenomegaly is a common feature of malarial anaemia because of the role of the spleen in the removal of both infected and uninfected red cells. blackwater fever, characterized by intense intravascular haemolysis with haemoglobinuria and occasionally renal failure in a patient with malaria, may be related to underlying glucose- -phosphate deficiency. , factors such as poor nutrition, deficiencies of vitamins and micronutrients, bacteraemia, and hookworm or hiv infection may co-exist with malaria and contribute to anaemia so nonmalarial causes of anaemia should be considered in patients whose anaemia does not respond to malaria treatment. the management of severe malarial anaemia involves supportive care and treatment of the malaria and any other underlying conditions. recovery from malaria-associated anaemia can be slow, taking weeks or even longer if there are episodes of re-infection. in children, blood transfusion is usually reserved for those with haemoglobin levels of less than g/l (< g/l if there are complications such as respiratory distress ). there of parasitized red cells and the release of von willebrand factor multimers which cause widespread platelet aggregation leading to thrombocytopenia , , platelet synthesis by the bone marrow is relatively well maintained during infection , but antiplatelet antibodies, immune complexes and splenomegaly all contribute to thrombocytopenia. thrombocytopenia occurs in - % of individuals infected with malaria irrespective of the species of plasmodium. , thrombocytopenia in febrile patients in an endemic area increases the probability of malaria by a factor of and in individuals returning from tropical countries with a fever, thrombocytopenia is highly specific for malaria infection. profound thrombocytopenia is unusual and malaria-associated thrombocytopenia is rarely associated with haemorrhagic manifestations. the clinical consequences of platelet aggregation and endothelial binding are primarily microvascular ischaemia. this may manifest as renal impairment, cerebral ischaemia, and occlusion of retinal vasculature or even in some cases, skin necrosis. bleeding is unlikely, although in severe thrombocytopenia, petechiae or purpura may develop which denotes extravasation of red cells into the subcutaneous tissue. continued platelet activation and consumption can exacerbate bleeding and decreased circulating platelets are associated with increased vascular leakage and the development of oedema. platelet transfusions are rarely required because the platelet count generally rises rapidly on treating the underlying malaria. coagulopathy is a disturbance of the whole coagulation system involving not just coagulation factors but platelets, anticoagulant factors, fibrinolytic system and, in the case of malaria, the parasitized red cells and the vascular endothelium. parasitized red cells induce expression of tissue factor on endothelial cells and monocytes, release of microparticles, cytokine release and platelet clumping, all of which initiate blood coagulation and tilt the balance towards the pro-coagulant state ( figure . ) . , [ ] [ ] [ ] [ ] [ ] [ ] anticoagulant factors are severely depleted in malaria. protein c and antithrombin levels are inversely correlated with severity of falciparum malaria and return to normal with treatment of the malaria. have been some concerns about a possible increased risk of infection associated with iron supplementation for children in malarious areas , but current recommendations advocate that where iron deficiency and malaria are common, iron supplements should not be withheld and appropriate anti-malarial treatment or prevention should also be offered. the best way to prevent malarial anaemia is to prevent malaria infection by avoiding mosquito bites (e.g. through the use of bed nets) or through chemoprophylaxis. malaria chemoprophylaxis during infancy can reduce both malaria and anaemia. children who have been hospitalized with severe malarial anaemia may benefit from intermittent preventive malarial therapy after discharge to prevent recurrence of anaemia. daily co-trimoxazole prophylaxis which is used for hiv-infected individuals has been shown to reduce malaria parasitaemia and anaemia. the normal platelet life span of - days is reduced to less than days in malaria infection. several factors are responsible for thrombocytopenia in malaria infection, the most common being increased platelet activation and aggregation ( figure . ). platelet activation is by parasitized red cells which express surface tissue factor and initiate coagulation and platelet aggregation. the resultant activated endothelium binds platelets and sequesters them in vascular beds including in the cerebral vasculature. , these platelets facilitate the adhesion and to % after a year. , although transfusions may be required in severe life-threatening cases of anaemia, aggressive transfusion therapy has been associated with fatal pulmonary emboli due to accelerated haemolysis and disseminated intravascular coagulation. in those who do not respond to art, erythropoietin may be considered since reduced responsiveness to this hormone and antierythropoietin antibodies have been noted in hiv patients. erythropoietin is particularly useful in individuals whose erythropoietin levels are less than iu/l because in addition to increasing the haemoglobin it can also improve the quality of life. erythropoietin may take several weeks to achieve full effect and patients should be replete in haematinics. erythropoietin can very rarely be associated with thrombosis or pure red cell aplasia. thrombocytopenia is a common finding in hiv-infected patients and it may be the initial manifestation of hiv infection in as many as % of patients. data from wealthy countries demonstrate platelet counts less than × /l in % of patients, and less than × /l in . %. overall the -year coagulopathy in malaria infection is unusual, occurring in less than % of cases. it appears to be most common in adults with cerebral malaria who may present with gastrointestinal bleeding or with microvascular ischaemia in the brain, kidneys, retina and occasionally, the dermal vasculature. prolongation of prothrombin time and activated partial thromboplastin time only occur in - % of patients with p. falciparum infection and coagulopathy does not appear to be a feature of p. vivax infection. since coagulation factors need to be depleted to less than % of normal to prolong the clotting times, these tests can be normal despite active coagulopathy. management of coagulopathy aims to restore the balance between pro-and anticoagulant processes. this is complex and requires input from a coagulation specialist and ideally, access to plasma, heparin and factor concentrates and a well-equipped coagulation laboratory. anaemia anaemia is very common in hiv-infected individuals occurring in up to % at initial presentation and about % at some stage during their disease. thirty-seven percent of patients with clinical aids have a -year incidence of anaemia (haemoglobin < g/l) and high rates of anaemia persist despite combination anti retroviral treatment (art). anaemia is directly related to mortality in hiv infection and is independent of other risk factors including cd count. there are multiple reasons for anaemia in hiv-infected patients (box . ), which often co-exist in individual patients. bone marrow infection by mycobacteria species, histoplasma, cryptococcus and penicillium marneffei can all decrease red cell production and can be detected by bone marrow examination and cultures. parvovirus has a predilection for the erythroid progenitor cells and can cause severe anaemia in hiv-infected patients. serological tests for parvovirus are unhelpful in hivinfected patients and viral polymerase chain reaction is needed to confirm the diagnosis. the likelihood of parvovirusinduced anaemia increases with the severity of anaemia and has been found in % of individuals with hiv and haemoglobin less than g/l. haemophagocytosis occurs in hiv infections and may be secondary to co-infection with mycobacteria, cytomegalovirus, epstein-barr or other herpesviruses. poor nutrition due to socioeconomic reasons, hiv-related anorexia, malabsorption from conditions affecting the gastrointestinal tract, and achlorhydria may contribute to anaemia. haemolytic anaemia occurs secondary to drugs or concomitant glucose- phosphate dehydrogenase deficiency and because reticulocytopenia is common in those with hiv infection, reticulocyte counts cannot be used to exclude haemolysis. although the direct coombs test may be positive in patients with hiv infection, autoimmune haemolysis is not a common cause of anaemia. a reduction in red cell precursors has also been noted in children in africa with severe anaemia. treatment of hiv-related anaemia should focus on starting art and eliminating any other factors, such as infections or vitamin deficiencies, which may contribute to the anaemia. in wealthy countries art has been shown to reduce anaemia prevalence from % to % within months of starting treatment, non-hodgkin's lymphoma (nhl) was noted to be associated with hiv infection early in the epidemic and is an aidsdefining illness. the incidence of nhl is up to times greater in hiv-infected adults than in those who are not infected, and it is responsible for nearly one-sixth of the deaths attributable to aids. since the introduction of haart, the incidence of all types of nhl has decreased by approximately - % , and the outcome of hiv-infected patients with lymphoma has improved. in the setting of clinical trials, the % -year survival rate is comparable to those without hiv infection. the incidence of hodgkin's lymphoma has increased in the post-haart era, possibly due to immune reconstitution and increased cd cells. , evidence of epstein-barr virus (ebv) infection can be found in virtually all cases of hodgkin's disease. hiv-related lymphomas (box . ) (see also lymphomas, below), are broadly divided into systemic lymphomas ( %) and primary central nervous system lymphomas. the incidence of highly aggressive lymphomas, either burkitt's lymphoma (approx. %) or diffuse large b-cell lymphoma (approx. %), is much higher in hiv-infected patients than in those without infection. although t-cell lymphomas are uncommon in hiv disease ( %), there has been an increase in recent years. the incidence of primary central nervous system lymphoma in hiv-affected individuals is - % and it is times more common than in the general population. the pathogenesis of nhl in hiv infection is related to the inadequate host immune responses to viruses with oncogenic potential, predominantly ebv and human herpesvirus (hhv )/kaposi's sarcoma-associated herpesvirus. this allows unregulated lymphoid growth and an accumulation of genetic abnormalities in b cells. markers of b-cell activation such as serum immunoglobulins and free light chains, and cd cell count have been suggested as predictive markers for the development of nhl in hiv infection. , extranodal and leptomeningeal involvement, and b-symptoms occur in the majority of hiv-infected patients with nhl and the bone marrow is commonly involved. the most common extranodal site to be involved is the incidence of moderate thrombocytopenia (< × /l) is . %, though this is higher in those with clinical aids ( . %). , thrombocytopenia is more common in those who abuse drugs, have opportunistic infections and malignant disorders of the bone marrow (e.g. lymphoma), and it may also be a side-effect of therapeutic drugs. the most common cause of thrombocytopenia in hiv infection is immune thrombocytopenia which may be associated with hepatitis c co-infection, and produces decreased platelet survival, particularly at cd counts below /µl. the anti-platelet antibodies, immune complexes and cross-reacting antibodies to hiv envelope proteins and platelets, which occur in hiv-associated thrombocytopenia , may also contribute to generation of reactive oxygen species. platelet production can also be affected in hiv infection and may explain the high levels of thrombopoietin that have been documented in hivrelated thrombocytopenia. some cases of hiv-related thrombocytopenia may undergo spontaneous remission so treatment of thrombocytopenia is usually only initiated if it is associated with bleeding, which is unusual. the first line of treatment involves antiretroviral therapy with the aim of achieving undetectable plasma hiv viraemia. , any drugs that may be associated with causing thrombocytopenia should be withdrawn and opportunistic infections or secondary malignancies treated. the treatment of immune thrombocytopenia is the same as in non-hiv cases and options include a short course of steroids, intravenous immunoglobulin (short-lived response), anti-d, interferon-α or splenectomy. although there are multiple causes of thrombocytopenia in hiv-positive individuals, one of the most devastating is the thrombotic microangiopathy of thrombotic thrombocytopenic purpura (ttp). this is because the combination of haemolytic anaemia and microthrombi has a very poor prognosis. symptoms are nonspecific and may include fever, headache, bleeding and changes in consciousness. if ttp is suspected, an urgent blood film should be requested and the combination of thrombocytopenia with red cell fragmentation is highly suggestive of ttp. ttp associated with hiv infection was more frequent before the introduction of art and is more common if adherence to treatment is poor or resistance to therapy has developed. ttp is thought to be due to endothelial damage, but unlike the situation in non-hiv-infected individuals, low levels of adamts- are not a useful predictor of outcome. treatment of ttp involves plasma exchange, and although refractoriness may occur, this can be corrected by art in some cases. if art is administered in these cases it is important to maintain adherence throughout the period of plasma exchange. if apheresis facilities are limited, plasma infusions alone ( ml/ kg per day) may also produce a response. art should also be administered immediately after plasma exchange to minimize drug removal. patients with a viral load of less than copies/ml generally require fewer plasma exchanges for remission than those with a higher load. survival of patients with hiv-associated ttp in the pre-art era was rarely longer than years, even with plasma exchange and steroid treatment, but for patients who are compliant with art the mortality is around %. which immediately limits the amount of blood loss. exposure of the subendothelial space releases factors such as von willebrand factor multimers which bind to platelets and initiate platelet adhesion to the endothelium. the adherent platelets release their granules and attract more platelets, which in combination with fibrinogen, form an aggregate. the activated platelets also attract coagulation factors thereby promoting the clotting process. the critical parts of clot formation are the conversion of prothrombin to thrombin and the thrombinfacilitated conversion of fibrinogen to fibrin (figure . ) . haemostatic control mechanisms operate throughout the clotting process to prevent excessive clot formation and involve proteins c and s, and anti-thrombin and antifibrinolytic systems. any alteration in these regulatory pathways can lead to either bleeding or thrombotic complications. bleeding can result from: • inadequate vasoconstriction, due to vascular problems which can be acquired (e.g. viral haemorrhagic fevers or immune vasculitis) or congenital (e.g. collagen vascular disorders) • qualitative or quantitative abnormality of von willebrand factor causing von willebrand's disease • decreased number or function of platelets which can be either acquired (e.g. aspirin, nsaids) or congenital (e.g. platelet function defects) • qualitative (e.g. caused by inhibitors to coagulation factors, commonly factor viii) or quantitative (e.g. haemophilia) abnormality of coagulation factors • increased fibrinolysis (e.g. viral haemorrhagic fevers, snake bites). acquired bleeding disorders are commonly caused by vitamin k deficiency, disseminated intravascular coagulation (dic) or platelet disorders (box . ) but may sometimes be due to acquired inhibitors of coagulation factors. the initial laboratory tests in a patient with excessive bleeding should therefore include a platelet count, clotting screen (prothrombin time (pt) and activated partial thromboplastin time (aptt)), and gastrointestinal tract, often the stomach or the perianal region. hepatic involvement, seen in a quarter of cases, is associated with a particularly poor prognosis. cns disease may be asymptomatic so diagnostic lumbar puncture may be required. hiv-related lymphomas frequently present with poor prognostic features such as elevated serum lactate dehydrogenase levels. , older age, lowest nadir cd cell counts prior to nhl diagnosis, developing nhl while on art, and cumulative hiv viraemia are also poor prognostic features. a formal prognostic scoring system has been developed which takes into account the cd count (< cells/µl). some types of hiv-related lymphoma are associated with characteristic clinical and laboratory features. primary effusion lymphoma is an aggressive lymphoma characterized by effusions in serosal cavities in the absence of any other tumour masses. , it is strongly associated with hhv infection and the virus can be identified in the nuclei of the malignant cells. plasmablastic lymphoma mainly affects the oral cavity and the mucosa of the jaw and is typically associated with epstein-barr virus. histological examination of biopsied tissue is necessary to confirm the diagnosis and type of lymphoma. diagnostic difficulties may arise because hiv-related hyperplasia in lymph node biopsies may be confused with lymphoma, the histological appearance of hiv-related lymphomas may be different from those of non-infected individuals and many opportunistic pathogens may mimic the appearances of nhl, or co-exist with it, and will need to be identified or excluded before making a diagnosis of lymphoma. prior to the widespread use of art, conventional lymphoma chemotherapy resulted in considerable toxicity, increased opportunistic infections and high mortality. art has facilitated the use of conventional doses of chemotherapy in conjunction with haematopoietic growth factor support. this has markedly improved the outcome of patients with hiv-related lymphomas who now have overall response rates of %. the concomitant use of art and chemotherapy is therefore recommended, especially in those with cd counts of less than /µl. anti-cd antibody is now included in treatment regimens for nhl, and studies that include patients with hivrelated lymphomas all report favourable outcomes. , some antiretroviral agents such as zidovudine are best avoided in combination with chemotherapy, because it adds to the myelosuppression of chemotherapy. didanosine may worsen the peripheral neuropathy caused by taxanes and vinca alkaloids. hiv-infected patients undergoing chemotherapy should receive adequate anti-infective prophylaxis due to the high risk of opportunistic infections such as pneumocystis, herpes simplex and zoster and candida. consolidation chemotherapy and stem cell transplant have been used successfully in relapsed hivrelated lymphomas. haemostasis is maintained by interactions between vessel walls, platelets and a balance between pro-and anticoagulant factors. although the process of haemostasis is usually considered to occur in a stepwise fashion, in vivo the steps happen virtually simultaneously. activation of the lining of the endothelium by trauma, cancer cells or cytokines triggers vasoconstriction, the tests for each pathway is given with arrows corresponding to each box complications, malignancies and infections and is a serious condition with a high mortality. patients present with spontaneous bruising or excessive bleeding from minor wounds such as venepuncture sites, and they may also have signs of complications such as renal failure, acute respiratory distress syndrome and microangiopathic haemolytic anaemia. dic is associated with a combination of depleted clotting factors (i.e. prolonged pt and aptt), a falling platelet count, red cell fragments on the blood film, raised d-dimers or fibrin degradation products, and reduced fibrinogen levels. management of disseminated intravascular coagulation includes treating or removing the underlying cause, and correcting the haemostatic abnormalities with combinations of platelets, cryoprecipitate and fresh frozen plasma. although bleeding due to thrombocytopenia is unusual unless the platelet counts falls below - × /l, bleeding may occur with a normal platelet count and normal clotting screening tests (i.e. pt and aptt) if platelet functions are impaired (e.g. myelodysplastic syndromes). platelet transfusions are generally not required unless there is active bleeding or prior to surgery. idiopathic thrombocytopenic purpura. idiopathic thrombocytopenic purpura is due to immune destruction of platelets. it is usually primary but can be associated with conditions such as lymphomas and infections including hiv. it may present incidentally or with petechiae, bruising or bleeding from the nose or gums, especially if the platelet count is less than × fibrinogen levels, which may be helpful in cases of excessive fibrinolysis (table . ). a difficult venepuncture can cause in vitro activation of the clotting system resulting in a shortened pt or aptt. similar findings may occur in chronic dic due to in vivo activation. the pt and aptt are not necessarily good predictors of the bleeding risk because some clotting disorders associated with thrombosis (e.g. anti-phospholipid antibodies) can prolong the aptt. a shortened aptt can be associated with marked elevation of factor viii levels (e.g. pregnancy) and may be a predictor of deep vein thrombosis. a prolonged thrombin time is caused by quantitative or qualitative fibrinogen deficiency, heparin and fibrin degradation products. reptilase time is helpful to distinguish between fibrinogen abnormalities (prolonged reptilase time) and heparin therapy (normal reptilase time). deficiency of vitamin k can be due to poor diet, small bowel disease or bile flow obstruction. clotting factors (ii, vii, ix and x) are dependent on vitamin k which is a fat-soluble vitamin. vitamin k deficiency therefore causes prolongation of the pt and aptt. in newborn infants, vitamin-k-dependent clotting factors can drop precipitously within a couple of days of birth. this causes haemorrhagic disease of the newborn which particularly affects infants that are premature, exclusively breast fed or have been exposed to drugs for tuberculosis, convulsions or anticoagulation in utero. these babies develop bleeding into the skin and gut, or bleeding from the umbilical stump or circumcision. vitamin k deficiency will respond to intravenous vitamin k ( mg/day for days orally or by intravenous injection) and in severe bleeding the clotting abnormality can be treated with fresh frozen plasma. haemorrhagic disease of the newborn can be prevented with mg of intramuscular vitamin k given at delivery. disseminated intravascular coagulation (dic) is characterized by activation of haemostasis with widespread fibrin formation, activation of fibrinolysis and consumption of platelets and clotting factors. it may be precipitated by tissue injury, obstetric desmopressin (ddavp) is a relatively inexpensive drug that increases fviii levels and vwf activity within minutes of administration. it is useful in mild haemophilia and mild von willebrand's disease. the major side effects are headaches and hyponatraemia so fluid intake should be restricted to . l/ day. tranexamic acid mouthwashes may be helpful for oral mucosal bleeding. danazol can increase both factor viii and ix levels within - days and has therefore been recommended for patients with recurrent haemarthrosis or with central nervous system bleeding which both carry a high risk of recurrence. most thromboembolic episodes are single events and may be associated with precipitating events or underlying risk factors. thrombophilia is the clinical state of hypercoagulability and should be suspected in patients who have a strong family history of thrombosis, or who have recurrent or unusual thromboses. increasing affluence and consequent lifestyle changes mean that the prevalence of thromboembolism is rising in some low-and middle-income countries. risk factors such as sedentary work, obesity, excessive alcohol intake, smoking and additional cardiovascular risk factors are compounded by other /l. spontaneous recovery occurs less commonly in adults than in children. it is important to exclude other causes of thrombocytopenia such as drugs, dic or sepsis. the diagnosis can be suspected from a bone marrow examination which shows increased numbers of platelet precursors. treatment with prednisolone ( . - . mg/kg) is usually only necessary if there is bleeding or excessive bruising and the dose should be reduced slowly once the platelet count improves. second-line treatments include immunosuppressive agents and danazol. splenectomy may also be beneficial but carries an increased risk of infection. platelet transfusions or intravenous gammaglobulin can temporarily increase the platelet count in an emergency or prior to surgical procedures. inherited bleeding disorders can be classified broadly into coagulation factor deficiencies (e.g. factor viii and factor ix deficiencies), von willebrand's disease and platelet disorders. the frequency of genes for inherited bleeding disorders is the same throughout the world. haemophilia a has a prevalence of about / , von willebrand's disease of > / and haemophilia b of < . / . these conditions occur more frequently among populations where consanguineous marriage is common and where prenatal diagnostic facilities are unavailable. in general, individuals with inherited coagulation factor deficiencies present with soft tissue bleeds such as haemarthroses or intramuscular bleeds. those with platelet disorders or von willebrand's disease tend to present with mucosal bleeds, however severe (type iii) von willebrand's disease can present with severe soft tissue bleeds. many of these conditions are diagnosed following excessive and uncontrolled bleeding after trauma or surgical procedures. menorrhagia and delayed severe postpartum haemorrhage may be presenting features of bleeding disorders, particularly von willebrand's disease or hypothyroidism, which can cause decreased synthesis of von willebrand factor. some inherited platelet function disorders are associated with characteristic syndromes (e.g. oculocutaneous albinism or skeletal defects) which may provide a clue to the diagnosis. early recognition of symptoms by clinicians, teachers and the public is important so that early treatment can be established. patients with inherited bleeding disorders are usually managed with blood products (box . ) or chemotherapy designed to reduce bleeding and associated complications. , , , clotting factor concentrates may be imported or produced locally by fractionation of plasma and are included in the who list of essential medicines. , one international unit (iu) of fviii clotting factor concentrate per capita is recommended as the minimum requirement for countries wishing to achieve optimal survival for their haemophilia population but only about % of the estimated people in the world with haemophilia receive adequate treatment. management of patients with bleeding disorders relies on a wellequipped and quality assessed laboratory for accurate diagnosis and monitoring of treatment and access to plasma and components for replacement therapy. appropriate support services such as physiotherapy, orthopaedics and counselling should also be available. in many countries inherited bleeding disorders are associated with stigma, which is particularly directed against the mothers of affected children, acute and chronic leukaemias are usually associated with a high white cell count but acute leukaemias can present with normal or even sub-normal white cell counts. morphology of peripheral blood and bone marrow specimens is crucial to confirm the diagnosis. this is particularly important in the case of acute leukaemia in children which may be mistaken for an acute viral infection. staining methods including sudan black b, myeloperoxidase and nonspecific esterase are important to distinguish between the different subtypes of acute myeloid and lymphoid leukaemias and therefore to guide treatment. acute myeloid leukaemia (aml). prevalence of this increases with age and the success rate with chemotherapy protocols is not high even in the most sophisticated centres. neutropenia and myelosuppression requiring intensive blood component support occur during chemotherapy and bone marrow transplantation offers the best option for cure for patients who relapse. management of aml is therefore complex and expensive. hydroxycarbamide or subcutaneous cytarabine may be used as a palliative treatment. acute promyelocytic leukaemia (aml subtype m ). this must be distinguished from other types of acute myeloid leukaemia because it has a high cure rate with early treatment. it predominantly affects young adults and it has a high incidence in certain ethnic groups especially those of latin american descent. a treatment protocol which includes all-transretinoic acid with combination chemotherapy has been developed which is feasible in low-income countries. , another regimen based on intravenous arsenic trioxide has been developed in india, , which has an % response rate with good diseasefree and overall survival. conditions that are associated with thrombosis such as hiv infection, and chronic infections including tuberculosis , and helminth-induced eosinophilic myocarditis. african americans are more likely to be diagnosed with pulmonary embolism rather than deep-vein thrombosis compared to other racial groups and african patients with thrombosis tend to be younger than those reported in literature with higher mortality rates (around %) possibly due to late presentation and poor access to health facilities. asian populations - seem to have a lower prevalence of symptomatic venous thrombosis compared to african americans. very little is known about the prevalence of prothrombotic factors such as mutations of the prothrombin gene or deficiencies of antithrombin, protein c and protein s in tropical countries, although high rates of factor v leiden, a risk factor for venous thrombosis, have been described in tunisia. , lupus anticoagulant and anti-phospholipid syndrome, which are associated with increased thrombosis risk, are increased in afro-caribbean populations, especially in the presence of hiv, and have also been described in nigerian women with pre-eclampsia. , the management of venous thrombosis is initially with heparin and then with warfarin for - months. compliance may be difficult in low-resource settings because of the requirement for regular monitoring of warfarin. it is therefore important to try to prevent thromboses by removing any underlying risk factors and by treating individuals at risk of thrombosis with a short course of prophylactic heparin to cover procedures known to be associated with thrombosis risk. this can present as venous or arterial thromboembolism and it may be inherited (e.g. deficiencies of thrombin, protein s or protein c) or acquired (e.g. antiphospholipids). the patient's personal and family history, and the results of clinical and imaging examinations to confirm thrombosis, may suggest the diagnosis. the laboratory tests needed to determine the cause and classify the type of thrombophilia, and their interpretation, are complex, so patients with recurrent or unusual thromboses should be referred to a specialist centre. haematological malignancies are predominantly leukaemias, lymphomas and myelomas. some of the general approaches for managing these conditions in low-income countries are outlined in box . but definitive treatment should be undertaken by a specialist haematology unit. leukaemias can be broadly classified as acute or chronic, and lymphoid or myeloid. the presenting symptoms and signs are related to the disturbed blood cell production from the bone marrow due to the effects of the malignant cell clone (box . ) . acute leukaemias are characterized by rapid progression and poor prognosis if left untreated whereas chronic leukaemias generally follow a much slower course. • mobilization of the community (especially parents and families) to raise awareness among local councils and government bodies about the treatability of the cancers and benefits from curing them • find an external partner unit locally, nationally or internationally which is already well-established and willing to help but will not dictate terms • improvement of supportive care facilities, especially protection from those with infectious diseases • development of a safe and reliable blood transfusion service • provision of subsidized travel, and satellite clinics to lessen the burden • development of appropriate protocols for each disease entity which is locally practicable with minimum cost and maximum efficacy • development of medical, nursing and paramedical expertise in the diseases to be treated -initially by offering visiting fellowships and in the long term for the trained individuals to arrange regional and local teaching programmes • formation of a cooperative group bringing together all the professionals involved in the speciality within a country or region to share expertise and develop training programmes. acute lymphoblastic leukaemia (all). this is the most common type of leukaemia in children. it has a good prognosis when treated with modern chemotherapy protocols with cure rates in the best centres exceeding %. in low-income countries, cure rates are much lower at around % primarily because of failure to complete therapy and deaths caused by treatment. considerable improvements in all outcomes have been achieved by twinning institutions in developing countries with specialist centres elsewhere in the country or internationally. measures that may improve outcomes focus on preventing abandonment of therapy (e.g. providing funding for transport, satellite clinics and support groups) and prompt treatment of infection. treatment in a dedicated paediatric oncology unit using a comprehensive multidisciplinary team approach and protocol-based therapy, is also associated with improved outcomes in resource-poor settings. chronic myeloid leukaemia (cml). management has been revolutionized by tyrosine kinase inhibitors (e.g. imatinib) which can produce complete remission in over % of cases. once the diagnosis of cml is established, hydroxycarbamide can be used to reduce the white cell count, followed by treatment with a tyrosine kinase inhibitor. manufacturers will provide the drug free of charge to patients in low-income countries with confirmed cml and generic forms of tyrosine kinase inhibitors are now becoming available. chronic lymphocytic leukaemia (cll). this occurs predominantly in older people and usually presents with lymphadenopathy and recurrent infections. treatment is with chlorambucil and prednisolone although aggressive forms require combination therapy with rituximab, fludarabine and cyclophosphamide. treatment is generally not curative but the disease may be indolent and drugs may only be required if the patient has symptoms or if there is a risk of hyperviscosity from a very high lymphocyte count. approximately cases of non-hodgkin lymphoma (nhl) occur in the equatorial belt of africa each year (table . ). there are marked geographical variations in prevalence but up to % are thought to be related to hiv infection. burkitt's lymphoma, a b-cell nhl, was originally described in children from africa and has an estimated incidence of - per million. lymphomas are broadly classified into hodgkin's lymphoma and nhl; nhl are divided into b-cell, t-cell and nk-cell, and immunodeficiency-associated types. the clinical presentation of lymphomas is characterized by enlargement of the lymphoid organs and subsequent compression of the adjacent structures, infiltration of organs by the malignant lymphoid cells and a dysfunctional immunological system which can manifest as immunosuppression or excessive but dysregulated immune activation associated with, for example, autoimmune conditions. the diagnosis and management of the various types of lymphomas are complicated and should be undertaken in a specialist box . clinical features of leukaemias • fatigue and cardiac symptoms from anaemia • bleeding from thrombocytopenia • increased risk of infections despite a higher number but dysfunctional white cells • lymphadenopathy and hepatosplenomegaly occur with all although lymphadenopathy may be observed in the monocytic variety of aml • blindness due to hyperviscosity from hyperleukocytosis • tumour lysis syndrome due to spontaneous cell lysis presents as renal failure • pustules or pyogenic infections of the skin from minor wounds • bleeding gums are a characteristic feature of acute monocytic leukaemia • disseminated intravascular coagulation can occur with acute promyelocytic leukaemia • gout can arise from breakdown of the excess white cells and release of uric acid • oral aphthous ulceration is seen with severe neutropenia in both aml and all • granulocytic sarcoma or chloroma represent extramedullary deposits of leukaemic cells in any organ but mainly the skin. this may occur in the absence of peripheral blood involvement and is more common with chromosomal translocation ( ; ) of aml • central nervous system manifestations due to sludging of the cerebral circulation by the malignant cells or increased intracranial pressure due to ventricular blockade can occur. monocytic myeloid leukaemia can also involve the meninges • intracranial haemorrhage can occur in all with very high white cell counts (> × /l) • bone pain and arthralgia can be a presenting feature of all in children in more than a quarter. these children may present with a limp or unwillingness to walk due to marrow infiltration by leukaemic cells. rarely, they may have normal blood counts delaying the diagnosis of all • anterior mediastinal mass (thymus enlargement) can also occur in children and young adults with all which may present as superior venocaval obstruction • painless enlargement of scrotum is a sign of testicular leukaemia or hydrocele from lymphatic obstruction. priapism can result from hyperleukocytosis rarely. • most often asymptomatic and usually suspected on blood counts • the chronicity of cml or cll tends to cause gradual-onset symptoms since the patients get adjusted to the slowly developing anaemia • abdominal discomfort and early satiety are a feature of cml due to excessive splenomegaly compressing the stomach and reducing the luminal volume • sternal tenderness may be noted in cml • hyperleukocytosis in cml can occur more often than with aml or all due to the gradual increase in white cells. this can cause symptoms like hyperuricaemia and gout, tinnitus, priapism or central nervous system disturbances • left shoulder tip pain can arise from splenic infarction from the massive splenomegaly in cml • cml can rarely present with features of thyrotoxicosis (heat intolerance, weight loss and excessive sweating) due to hyper-metabolism • cll is often associated with lymphadenopathy and rarely with mild to moderate splenomegaly. all, acute lymphoid leukaemia; aml, acute myeloid leukaemia; cll, chronic lymphocytic leukaemia; cml, chronic myeloid leukaemia. centre. diagnosis depends on clinical history and examination, radiological investigations to document the extent of disease, and morphology, immunohistochemistry and molecular studies on tissue samples to confirm the lymphoma subtype. guidance on the diagnosis and treatment of lymphoma in settings where resources are limited includes recommendations about panels of immunostains and chemotherapy regimens that minimize the need for supportive care. tele-pathology, which involves transmitting histological images via the internet to experts overseas, may be helpful in certain circumstances though it is dependent on the quality of the histology preparations and the images of appropriate diagnostic regions in the sample. treatment regimens for lymphomas differ according to the subtype but may involve chemotherapy and radiotherapy. high remission rates can be achieved in burkitt's lymphoma with a combination of cyclophosphamide, vincristine and methotrexate and progressive disease can be managed with ifosfamide, mesna and cytosine arabinoside. , adult t-cell leukaemia-lymphoma (atll) adult t-cell leukaemia-lymphoma (atll) is an uncommon lymphoid malignancy which occurs in patients infected with human t-lymphotropic virus type i (htlv-i). htlv- is endemic in the caribbean, western africa, peru and southern japan. less than % of those infected with htlv-i develop atll and up to years can elapse between the primary infection and the development of atll suggesting additional factors are needed for malignant transformation. atll presents acutely in approximately % of cases, although chronic forms have also been described. the clinical presentation is with generalized lymphadenopathy in most cases and hepatosplenomegaly in over half. atll is associated with a high risk of hypercalcaemia which occurs in more than two-thirds of patients during the course of their disease and may be associated with central nervous system disturbances and renal impairment. lytic bone lesions occur as a para-neoplastic types of lymphomas identified from selected countries in sub-saharan africa phenomenon due to production of parathormone-like peptides. as with other t-cell disorders, atll can involve the skin, producing, e.g. erythrodermic plaques. the diagnosis of atll can be suspected from a high peripheral blood white blood cell count in combination with hypercalcaemia and characteristic lymphocytes with convoluted and hyperlobulated nuclei. the diagnosis is confirmed by histological examination of a tissue (lymph node or bone marrow), immunophenotyping for specific cell markers and proof of htlv infection, usually by serological methods. management of atll is primarily with combination chemotherapy with intrathecal prophylaxis. , a combination of zidovudine and interferon, as agents against htlv, has also been tried with some success. hypercalcaemia and opportunistic infections should be sought and treated early in these patients. the high white cell count is associated with a significant risk of tumour lysis syndrome and should be prevented by adequate hydration and the judicious use of allopurinol and other urate-reducing agents. myeloma is a monoclonal proliferation of plasma cells and it particularly affects older people. myeloma appears to be less common in asian countries than elsewhere, although during the last years, an almost four-fold increase in incidence of myeloma has occurred in taiwan. in the united states, the incidence of multiple myeloma in the black population is twice that of the white population. the abundant plasma cells infiltrate the bone marrow and interfere with normal haematopoiesis. this leads to anaemia, which is a presenting feature in % of individuals. bony infiltration by the malignant plasma cells can produce osteoporosis, lytic lesions and pathological fractures in % of patients with myeloma. involvement of the bones can lead to hypercalcaemia, which may be a presenting feature, and vertebral fracture leading to spinal cord compression. the malignant plasma cells produce a paraprotein which can cause renal impairment in - % and hyperviscosity may ensue in % of patients if the paraprotein production is not controlled. patients with myeloma may need a variety of supportive interventions including management of anaemia, renal failure, hypercalcaemia, hyperviscosity, infections and bone pains. specific anti-myeloma treatment should be managed within a specialist unit and has undergone a radical change in the last decade with the use of thalidomide and its newer formulations, and the more expensive, proteasome inhibitors (e.g. bortzomib). thalidomide is relatively safe and effective although somnolence and constipation can sometimes be troublesome. there is a risk of thrombosis with thalidomide especially at the initiation of therapy, and prophylaxis with heparin, warfarin or antiplatelet agents, depending on an assessment of the risk, may be warranted. melphalan may also be useful, particularly if resources are limited and there is no specialist centre. however it is myelosuppressive, so regular monitoring of the blood count is essential. maintaining an adequate blood supply is a major challenge for low-income countries. only % of the global blood supply is donated in the poorest countries where % of the world's population lives. blood transfusion is a vital component of every country's health service. it can be a life-saving intervention for illnesses such as severe acute anaemia, but mistakes in the transfusion process can be life-threatening, either immediately or years later through transmission of infectious agents. clinicians need to understand how blood is acquired and its risks and benefits, and to use it appropriately. governments and transfusion services need to put measures in place to ensure that blood is safe for transfusion and that it reaches those who need it in a timely manner. only % of member states meet all the world health organization's (who) recommendations for a national quality blood transfusion system. at the national level the transfusion service should have a director, an advisory committee and clear transfusion policies and strategies (table . ). who recommend standardization of blood collection, testing and distribution. although centralization of these services may offer the best guarantee of quality, it is often not practical in countries with poorly developed communications and transport infrastructure. two systems, centralized and hospital-based, exist in lowincome countries for managing blood supply. in the centralized system, voluntary blood donors are recruited, screened and bled by regional centres and the blood collected is distributed to peripheral hospitals. hospital-based systems are the predominant source of blood across sub-saharan africa. hospital-based systems obtain blood predominantly from relatives of patients, and blood is screened and used within the local vicinity. blood from the centralized system costs at least three times as much per unit as that from a hospital-based system. although centralized systems can save costs through batching and bulk purchasing, the quality assurance processes and donor recruitment components are expensive and difficult to maintain without dependence on external funds. in hospital-based transfusion services, testing quality is variable and the families of patients bear the cost of finding blood donors. the vast majority of blood in low-income countries is transfused as whole blood. in high-income countries it is standard practice to optimize the use of each donation of blood by separating it into individual components but whether this approach is cost-effective in low-income countries, where indications for transfusion are different, is not known. these components, which may include plasma, platelets and cryoprecipitate, are prepared by centrifugation using a closed, sterile system and each component has different storage requirements. plasma and cryoprecipitate are kept frozen, red cells are stored at - °c, and platelets at - °c with constant agitation. recent evidence suggests that warm, fresh, whole blood may be better than component therapy for resuscitation of acidotic, hypothermic and coagulopathic trauma patients and for patients needing massive transfusions. many infections can be transmitted through blood transfusions and transfusion of infected blood causes morbidity and mortality in the recipients, and has an economic and emotional impact on their families and communities. those who become infected through blood transfusion are infectious to others and contribute to the spread of disease thereby increasing the burden on health services and reducing productive labour. strategies for recruiting blood donors have to provide blood for all who need it in a timely manner while ensuring that the blood is as safe as possible. the safest type of blood donor is one who donates regularly (i.e. repeat donors). who states that the safest source of blood is altruistic, voluntary, unpaid donors. only % of who member states report having at least % of their blood supply from voluntary donors, and low-income countries have not been able to increase the recruitment of voluntary donors for several years. recent evidence from sub-saharan africa indicates that the focus on voluntary donors may be misplaced since first-time voluntary donors have a similar prevalence of transfusion-transmitted infections as family replacement donors. in order to limit blood shortage and maintain constant blood supply in poorer countries, both voluntary and replacement donors should be accepted and encouraged to donate regularly. mechanisms to convert family replacement donors into repeating voluntary donors have the potential to significantly increase blood donations in africa. political will and open-mindedness about ways to improve the supply and safety of blood are essential to promote more evidence-based approaches to blood transfusion practice in poorer countries. supporting strategy in wealthy countries, the majority of transfusions are carried out electively. by contrast, in poorer countries, and particularly those where the malaria transmission rate is high, most transfusions are given for life-threatening emergencies. in low-income countries, - % of transfusions are administered to children, predominantly for malaria-related anaemia, and pregnant women. transfusion can significantly reduce the mortality of children with severe anaemia within the first days of hospital admission and successful malaria control can reduce paediatric transfusion requirements. in sub-saharan africa, % of in-hospital maternal deaths from severe bleeding were due to lack of blood for transfusion. other specialities which are significant users of blood are surgery, trauma, emergency medicine and general medicine. in low-income countries the most effective way to avoid transfusions is to reduce the prevalence of anaemia. more studies on the efficacy and cost of combinations of interventions including insecticide-treated bed nets, nutritional supplements and anthelmintic drugs to prevent anaemia are needed. when resources are very limited, governments may need to make some difficult decisions in order to achieve an equitable balance between investing in a transfusion service and public health measures to reduce anaemia. whether a patient needs a blood transfusion or not is ultimately a clinical decision. emergency transfusions can be lifesaving for patients in whom anaemia has developed too quickly to allow physiological compensation, as in severe malariarelated anaemia in children, and sudden, severe obstetric bleeding. in contrast, if the anaemia has developed slowly, for example due to hookworm infestation or nutritional deficiency, patients can generally be managed conservatively by treating the cause of the anaemia and prescribing haematinic replacements. iron supplements should be continued for at least months after the haemoglobin has returned to normal, so that body stores can be replenished. clinical guidelines. it is possible to avoid unnecessary transfusions by adhering to clinical transfusion guidelines. most institutions have developed guidelines to help clinicians make rational decisions about the use of blood transfusions (box . ) , and strict enforcement of transfusion protocols can significantly reduce avoidable transfusions. the principles underlying most transfusion guidelines are similar and combine a clinical assessment of oxygenation, with haemoglobin measurement being used as a surrogate measure for intracellular oxygen concentration. increasingly, transfusion guidelines are making use of evidence which shows that adequate oxygen delivery to the tissues can be achieved at haemoglobin levels that are significantly lower than the normal range. implementation of transfusion guidelines is particularly difficult if clinicians do not have access to reliable haemoglobin high-risk donors, such as commercial sex workers and their contacts, intravenous drug abusers, or those with an itinerant lifestyle such as traders, drivers and military personnel, should be deterred from donating. even in areas where hiv infection rates in the general population are high, donor deferral can be effective in excluding hiv-infected donors. the whole donation process, including tests for hiv and other infections, should be explained to the donor before blood is collected and donors should have the option of knowing the results and receiving counselling. it is imperative that complete confidentiality is maintained throughout all procedures. infections with organisms such as hiv, hepatitis viruses, cytomegalovirus, syphilis, lyme borreliosis, malaria, babesiosis, american trypanosomiasis (chagas disease) and toxoplasmosis can all be acquired through blood transfusions. some - % of hiv infections worldwide are thought to have been transmitted through the transfusion of infected blood and blood products. there have also been reports of transmission of variant creutzfeldt-jakob disease through blood transfusion and there is a theoretical risk of transmission of severe acute respiratory syndrome (sars). , who recommends that all donated blood should be screened for hiv, hepatitis b and syphilis and, where feasible and appropriate, for hepatitis c, malaria and chagas disease. malaria can be transmitted by blood transfusion and, depending on the local infection prevalence, - % of blood donors in africa screen positive for malaria. however, there is very little evidence to suggest that these donors transmit malaria to transfusion recipients. although who recommends screening donors in endemic areas for malaria, none of the screening methods that would be practical for transfusion services are sufficiently sensitive. furthermore, in some countries with high malaria transmission, exclusion of parasitaemic donors could result in deferral rates exceeding % which would have a major impact on blood supply. there is no evidence to support the widespread practice of routine treatment of transfusion recipients for malaria. fresh blood is potentially infectious for syphilis, but storage at °c for more than days can inactivate treponema pallidum. the high demand for blood in low-income countries means that blood is generally not stored for long enough to inactivate t. pallidum and syphilis seroconversion associated with transfusion has been reported from africa. globally, the prevalence of hepatitis c, htlv- and - and chagas disease is variable and the decision to introduce donor screening for these infections should be based on local assessments of the risks, benefits, feasibility and costs. blood should not be separated into components if the residual risk of infection is high, as this will increase the number of potentially infected recipients. a unit of blood is usually stored until screening tests for infections have been completed. this means that potentially infected blood may be mixed up with units that have already been screened, and costly blood collection bags are wasted. screening potential donors before venesecting a unit of blood may therefore be a more cost-effective way of ensuring safe blood. tests for screening blood donors need to be highly sensitive, and infected blood should be rejected. before informing the donor of the outcome, all positive results should be confirmed using a test with a high degree of specificity. where blood or that the blood may become infected with bacteria during the process. intraoperative blood salvage. this involves collecting blood lost during the operation and reinfusing it into the patient either during or after surgery. although this technique is practical and safe, and reduces the need for donor blood by - %, it requires specialized equipment and training, and may be more expensive than routinely donated blood. other measures. normal saline or intravenous replacement fluids can be used judiciously in acute blood loss, and in certain circumstances may be as effective as whole blood, red cells or plasma. erythropoietin, which stimulates endogenous red cell production is well-established for use in chronic anaemias such as those due to renal failure, cancer and hiv infection but its delayed action makes it unsuitable for use in acute anaemias. synthetic oxygen carriers, such as perfluorocarbons, are not yet routinely available. in low-income countries, the recommended haemoglobin threshold for transfusions is often well below that which would be accepted in more wealthy countries. randomized controlled studies in wealthy countries indicate that for most adults and children undergoing critical care, a haemoglobin threshold of g/l for transfusion is safe whereas paediatric blood transfusion protocols in sub-saharan africa often recommend transfusions for stable children only when the haemoglobin level is less than g/l. complications such as cardiac failure or infection may necessitate transfusion at a higher haemoglobin level. transfusion should be combined with adequate haematinic replacements and underlying conditions should be treated. early evidence suggests that intermittent preventive treatment with anti-malarials may reduce the high hospital readmission rates experienced by children post-transfusion. complications can occur immediately during transfusion, within a few hours of its completion, or be delayed for many years, as in the case of viral infections (box . ). measurements. when they doubt the haemoglobin result, clinicians rely entirely on clinical judgement to guide transfusion practice which can lead to significant numbers of inappropriate transfusions. a lack of investment in the quality of a critical test, such as haemoglobin measurement, can waste significant resources downstream in the transfusion process, and unnecessarily expose recipients to the risk of transfusion-related infections. minimizing surgical blood loss. where blood is in short supply, it is particularly important to ensure that the best anaesthetic and surgical techniques are used, to minimize blood loss during surgery. drugs which improve haemostasis or reduce fibrinolysis, such as aprotinin and cyklokapron, and fibrin sealants, can be effective in reducing perioperative blood loss. these drugs can therefore reduce the need for blood transfusion but they may be too expensive for use in low-income countries. a cost-effectiveness study of surgical bleeding in four sub-saharan countries indicates that the antifibrinolytic, tranexamic acid, could save lives in countries with blood shortages, reduce healthcare costs and prevent transmission of infections. preoperative autologous blood deposit. patients undergoing planned surgery who are likely to require a blood transfusion can have units of their own blood removed and stored in case they have significant intraoperative blood loss and need a transfusion. this process, known as preoperative autologous donation, can reduce the need for allogeneic transfusions by - % but it requires careful organization: the surgeon needs to predict how much blood will be required, the patient has to be fit enough to withstand removal of one or more units of blood over the weeks preceding the surgery and the surgery must take place within the shelf-life of the blood. as the blood has to be stored in the blood bank there is still a risk that the patient may receive blood which is not their own box . prescribing blood: a checklist for clinicians always ask yourself the following questions before prescribing blood or blood products for a patient: . what improvement in the patient's clinical condition am i aiming to achieve? . can i minimize blood loss to reduce this patient's need for transfusion? . are there any other treatments i should give before making the decision to transfuse, such as intravenous replacement fluids or oxygen? . what are the specific clinical or laboratory indications for transfusion in this patient? . what are the risks of transmitting hiv, hepatitis, syphilis or other infectious agents through the blood products that are available for this patient? bacterial contamination and should be investigated and managed accordingly. allergic reactions are due to infusion of plasma proteins and manifestations include erythema, rash, pruritus, bronchospasm and anaphylaxis. the transfusion should be stopped and the patient treated with antihistamines. if the reaction is mild and the symptoms and signs completely disappear, the transfusion can be restarted. if this type of mild reaction occurs repeatedly with more than one unit of blood, the red cells can be washed before transfusion. this should only be done if absolutely necessary, as it carries the risk of introducing potentially fatal bacterial infection. severe allergic reactions with evidence of systemic toxicity should be managed as acute anaphylaxis. blood should always be transfused slowly to avoid overloading the circulation, unless the patient has active and severe bleeding. fluid overload may be a particular problem when paediatric blood bags are not available, as children may be over-transfused due to miscalculation of the required volume, lack of accurate infusion devices or inadvertent administration of an adult-sized unit of blood. four units of blood contain the equivalent amount of iron stored in bone marrow (approx. g). repeated transfusions for chronic haemolytic anaemia, as in thalassaemia major and sickle cell disease, lead to iron deposition in parenchymal cells. eventually failure of the heart, liver and other organs supersedes. adequate doses of iron chelators, such as injectable desferrioxamine or oral deferiprone, are able to maintain acceptable iron balance in patients with chronic anaemia who need regular transfusions. it is not usually necessary to warm blood unless large quantities are transfused rapidly. this may lower the temperature of the sino-atrial node to below °c at which point ventricular fibrillation can occur. if blood needs to be warmed, an electric blood warmer specifically designed for the purpose should be used. this keeps the temperature below °c and avoids the haemolysis associated with overheating blood. graft-versus-host disease occurs when donor lymphocytes engraft in an immune-suppressed recipient. the lymphocytes recognize the recipient's bone marrow as foreign and induce aplasia. graft-versus-host disease is almost universally fatal and can be prevented by irradiating the donor blood, which inactivates the donor lymphocytes. transfusion of blood into a recipient who possesses antibodies to the donor's red cells can cause an acute, and occasionally fatal, intravascular haemolysis. this could occur, e.g. if group a cells are transfused into a group o recipient who has naturally occurring antibodies to group a cells. the profound haemolysis induces renal vasoconstriction and acute tubular necrosis. treatment involves stopping the transfusion, cardiorespiratory support and inducing a brisk diuresis. in addition to abnormalities indicating renal failure, laboratory findings include haemoglobinuria and haemoglobinaemia. proof of the diagnosis involves rechecking the whole transfusion process including all documentation stages, regrouping the donor and the recipient, and screening for antibodies on red cells with a direct antiglobulin test. these tests are usually available in any hospital laboratory capable of providing a transfusion service. delayed haemolysis has a similar physiological basis to acute intravascular haemolysis but it tends to be less severe, it occurs - days after the transfusion and it is less likely to present as a clinical emergency. limited data from sub-saharan africa show rates of bacterial contamination in donated blood of around % , but the clinical consequences for transfusion recipients are unknown. bacteria can enter the blood bag during venesection or if the bag is breached, e.g. when reducing the volume for a paediatric recipient or during component preparation. gram-negative bacteria, including pseudomonas and yersinia, grow optimally at °c and infected blood may not necessarily appear abnormal to the naked eye. reactions following infusion of infected blood are often due to endotoxins and may occur several hours after the transfusion has finished. although these reactions are rare, they can be severe and fatal. if bacterial contamination is suspected, the transfusion should be stopped and samples from the patient and the blood bag sent to the laboratory for culture. cardiorespiratory support may be needed and broad-spectrum antibiotics should be started immediately and continued until culture results are available. non-haemolytic febrile reactions are episodes of fever and chills associated with transfusion and for which no other cause can be found. they are due to the recipient's antibodies reacting against antigens present on the donor's white cells or platelets. these reactions are most common in patients who have had transfusions in the past and have therefore been exposed to allo-antigens. mild febrile reactions usually respond to simple antipyretics such as paracetamol. more 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contribution of ineffective blood transfusion services press release who/ . . whd/ information sheet for clinicians development and evaluation of a new paediatric blood transfusion protocol for africa electrocardiographic st-segment changes during acute, severe isovolemic hemodilution in humans use of clinical judgement to guide administration of blood transfusions in malawi giving tranexamic acid to reduce surgical bleeding in sub-saharan africa: an economic evaluation autologous transfusion techniques: a systematic review of their efficacy intraoperative autologous blood management artificial o carriers: status in red blood cell transfusions in acute paediatrics survival and haematological recovery of children with severe malaria transfused in accordance to who guidelines in kilifi intermittent preventive therapy for malaria with monthly artemether-lumefantrine for the post-discharge management of severe anaemia in children aged - months in southern malawi: a multicentre, randomised, placebocontrolled trial bacterial contamination of pediatric whole blood transfusions in a kenyan hospital bacterial contamination of blood and blood components in three major blood transfusion centres in accra, ghana access the complete references online at www.expertconsult.com key: cord- - khcmta authors: delaney, martha a.; treuting, piper m.; rothenburger, jamie l. title: rodentia date: - - journal: pathology of wildlife and zoo animals doi: . /b - - - - . - sha: doc_id: cord_uid: khcmta this chapter includes diseases of animals in the order rodentia, in which there are over species representing % of all mammals. this incredibly diverse order includes members inhabiting every continent, either naturally or in human-made environments. while rodents have been the cause or implicated in disease transmission that has lead to human pandemics, such as the black death, and the decimation of certain animal species, like island-dwelling birds; genetically modified rodents have contributed significantly to the advancement of biomedical research and human health. there are more than species of endangered rats, mice, voles, squirrels, and marmots. the recent extinction of the bramble cay melomys represents the first human-induced rodent extinction linked to climate change. rodents are the reservoir host of several human and domestic pathogens of concern listed by oie. herein, we highlight those diseases of rodents that lead to clinically important gross and microscopic lesions. the order rodentia includes families, genera, and over species. two separate systems exist to classify rodents (carleton and musser, ) . the first classification scheme by j.f. brandt ( ) is based on morphology of the jaw and skull together with masticatory muscle position and has three suborders: sciuromorpha (squirrellike); myomorpha (mouse-like); and hystricomorpha (porcupine-like). the second separates rodents into two suborders, sciurognathi and hysticognathi, based on the position of incisors and the angle of the jaw (carleton and musser, ) . molecular studies yield seven clades containing three phylogenetic lineages: squirrel-related (sciuriodea and gliridae); mouse-related (anomaluromorpha, castoridae, geomyoidae, and myodonta); and ctenohystrica. with diverse ecologic strategies, rodents are found naturally in a variety of habitats globally except antarctica. the natural history and biology of members of this extensive order exhibit substantial variation. nevertheless, similarities exist among rodents including dentition, anatomy, and disease susceptibility. the unifying characteristic of all rodents is their dentition, which includes two pairs of continuously growing (elondont) incisors. brachydonts have elondont incisors with brachydont molars; in hystricomorphs, all of the teeth are elondont. dentition is important when considering the diet and husbandry needs of these groups. brachydonts, which include mice, rats, hamsters, and gerbils, need high calorie diets with low amounts of fiber, whereas the hystricomorphs (porcupine-like; guinea pigs, chinchillas, agoutis) are strictly herbivores and require ample abrasive roughage. dental formulae vary but in general, rodents have four incisors, no canines, - premolars, and numerous (up to ) molars. as most rodents are omnivorous, their digestive system typically consists of a simple stomach (monogastric) and a variably adapted intestinal tract depending on the degree of hind gut fermentation (stevens and hume, ) . most practice coprophagy or cecotrophy to maintain normal microflora and absorb microbially derived amino acids and vitamins (e.g., b and k). as such, herbivorous, omnivorous, insectivorous, and carnivorous rodents have different gastric and large intestinal anatomy. some rodents lack gall bladders (e.g., rats and pocket gophers). rats and hamsters are unable to vomit due to a muscular sphincter at the esophageal-gastric junction. members of caviidae lack the enzyme l-gulonolactone oxidase, making vitamin c (ascorbic acid) a necessary dietary requirement for this group (see scurvy in section, nutritional diseases). rodents are typically small (as small as g) and rotund with short legs, though the largest rodents are almost kg (e.g., capybaras). rodents are nocturnal and though some species hibernate (e.g., woodchucks) but most of the species do not. there are numerous specializations among rodents including cheek pouches (food transport), extremity and claw development (e.g., burrowing, jumping, climbing, flying), sensory organ variations (e.g., vibrissae and large ears or eyes), special integument and pelage (e.g., porcupine quills and chinchilla fur), and tail adaptations (e.g., prehensile, fat storage). rodents have a strong sense of smell and a well-developed vomeronasal organ, which is important for reproduction and social behavior through the sensing of pheromones. since rodents lack sweat glands, they are susceptible to overheating. reproductive anatomy varies though most female rodents ovulate spontaneously and are polyestrous. like rabbits, chinchillas have two cervices that communicate with individual uterine horns. vaginas are imperforate prior to sexual maturing and during nonbreeding and/or postpartum intervals. placentation is discoid and hemochorial. mammary gland anatomy varies among species. the suborders pathology of wildlife and zoo animals scuirognatha and stricognatha have altricial and precocial young, respectively. some male rodents have testes within a scrotum, though the testes of porcupines, agoutis, chinchillas, cavies, and capybaras are located within the inguinal canal, which remains open in most male rodents. male rodents also have well developed accessory sex glands and an os penis. prairie dogs have perianal sacs similar to canids and felids, which are important for sexual communication. male beavers have persistent paramesonephric ducts that resemble a uterus and are referred to as masculine uterus (doboszynska and zurowski, ; meier et al., ) . south american rodents including guinea pigs and related species have unique pulmonary anatomy as do burrow-dwelling, subterranean mole-rats that live in low oxygen tension environments, for example, large and dilated, primitive bronchi and bronchioles (ilgun et al., ; widmer et al., ) . lung morphology of different rodent species representing families is well described (wallau et al., ) . common microscopic findings in rodents that may be misinterpreted as lesions include: multinucleated, karyomegalic, and cytomegalic hepatocytes are common in several rodent species and can increase with age ( fig. . ); hepatocellular intranuclear cytoplasmic invaginations (pseudoinclusions) (fig. . ); eosinophilic cytoplasmic spherical inclusions in renal tubular epithelial cells and hepatocytes seen predominantly male mice, rats, and hamsters; splenic extramedullary hematopoiesis, which is very common in healthy rodents of all ages (fig. . ); hemosiderin, lipofuscin, ceroid, and melanin (in dark or black coated animals) are commonly detected in various tissues, such as spleen, liver, kidney, and adrenal glands; cardiac muscle in the tunica of pulmonary veins in the lung is a normal finding in mice; male rodents may have refluxed seminal coagula in the urinary bladder and urethra that is thought to occur peri mortem; and adrenal x-zone vacuolation in female mice. furthermore, there are evident sexual dimorphisms of some organs and glands, such as the cuboidal parietal epithelium of the glomeruli in male rodents. hematological reference ranges have been established for some common domestic, zoo-housed, and ubiquitous free-ranging species. in general, the lifespan of rodent erythrocytes is shorter than other mammals, thus, higher levels of circulating reticulocytes ( %- %), seen as polychromasia and anisocytosis, is considered normal. howell-jolly bodies may also be present. in all age groups, lymphocytes are more numerous than neutrophils. in fact, lymphocytes are the predominant white blood cells in guinea pigs ( %- %). in addition, guinea pigs and capybaras have kurloff cells (fig. . ), which are lymphocytes containing a single, large intracytoplasmic mucopolysaccharide inclusion body, known as a kurloff body (jara et al., ) . though the actual function of these cells is unknown, they are presumed to be natural killer cells. the number of kurloff cells in females fluctuate with the reproductive cycle stage, thus they are considered to be estrogen-dependent. pregnant females may have %- % kurloff cells in circulation while males typically have fewer. guinea pigs have heterophils similar to rabbits. rats, mice, and hamsters commonly have neutrophils with ring-form nuclei. chinchillas and porcupines have welldefined and segmented neutrophil nuclei. band neutrophils may be a normal finding in some species including new world porcupines, coypu, and agoutis. serum biochemical parameters have been compiled for some rodent species (kurtz et al., ; . of note is that serum levels of alanine aminotransferase (alt) are not sensitive biomarkers of liver health in guinea pigs and figure . normal liver in an aged mouse. common findings inlcude anisocytosis, anisokaryosis, and eosinophilic intranuclear pseudoinclusions of invaginated cytoplasm. within the red pulp are hematopoietic elements including erythroid and myeloid lineages. note the large multinucleated megakaryocytes, which are readily visible at this magnification. some other rodents as they normally have low hepatic levels of this enzyme. in contrast, gamma glutamyl transferase is present in the liver of all rodents and can be evaluated to determine the hepatic injury or disease. hamsters and other desert species may have high levels of serum calcium. rodents like mice and rats, and specifically those evolved in arid regions, have a profound ability to concentrate their urine, thus it is often turbid and contain calcium crystals. the following list of diseases of rodents is not exhaustive but instead highlights the more common findings in species of which there is published literature or knowledge. a plethora of information exists regarding the anatomy, physiology, and diseases of rodents used as animal models in biomedical research (i.e., laboratory animals) and is a valuable resource when examining lesser-studied rodent species using a comparative approach greaves, ; suckow et al., ; treuting and dintzis, ) . as many rodents are important prey species, diseased or dead rodents are exceedingly difficult to detect in nature using the passive surveillance techniques of traditional wildlife disease monitoring. thus, less is known about the diseases and pathology of free-ranging rodents as compared to larger and charismatic mammals. one of the most well described nutritional diseases in rodents is hypovitaminosis c or scurvy. guinea pigs and a number of other species including capybaras and humans, lack the enzyme l-gulonolactone oxidase which is required for l-ascorbic acid (vitamin c) production therefore, these species require dietary vitamin c (cueto et al., ) . vitamin c is necessary for the formation of collagen via hydroxylation of proline/lysine for cross linking of fibrillar collagen. hypovitaminosis c results in decreased collagen fibril cross linking, leading to the increased fragility of blood vessels, cartilage, and osteoid. in affected individuals, gross and histologic lesions are characteristic and include capillary hemorrhage and lack of bone deposition and remodeling as a result of poorly formed and weak collagen. other gross lesions include evidence of anemia (e.g., pallor, reactive bone marrow), periarticular hemorrhage, and swollen joints, specifically at the costochondral junctions (scorbutic lattice). characteristic oral lesions include gingival swelling, erythema, hemorrhage, erosion, ulceration, and tooth loss. histological examination is necessary to confirm and define bone lesions, which include: dilation of metaphyseal blood vessels, irregular columnization and hypertrophy of physeal cartilage, calcification of metaphyseal cartilage with reduced or absent osteoid, microfractures with surrounding and subperiosteal hemorrhage, medullary fibrosis, and decreased osteoclastic activity and remodeling. tooth loss may result from a combination of abnormal odontoblasts, dentin resorption, pulp fibrosis, and a lack of/ abnormal alveolar bone remodeling. like lagomorphs, some hindgut-fermenting rodents are susceptible to gut stasis and related diseases if they are not able to obtain enough fiber from the diet. obesity can be common in rodent species if fed a high caloric diet and can predispose to diabetes mellitus, cardiac disease, pregnancy toxemia, and hepatic lipidosis in females. dystrophic mineralization occurs in guinea pigs, hamsters, and free-ranging rats (rothenburger et al., a) . mineralization most often affects the myocardium but can also occur in other tissues, such as the diaphragm, tongue, liver, kidney, lungs, cornea, and aorta (see section congenital/ genetic). though the pathogenesis is not fully elucidated, mitochondria appear to be preferentially mineralized based on ultrastructural studies. gross lesions may include cardiomegaly with chalky to gritty white material and streaking within the myocardium of the atria and ventricles. histologically, affected cardiac myocytes are degenerative and/ or necrotic with rupture of the sarcolemma. damaged myocytes will have variable and progressive mineralization until the entire myofiber is densely mineralized. variably mature fibrosis surrounds individual and clusters of dead and mineralized myocytes. satellite cells are frequently hypertrophic. pregnancy toxemia occurs in overweight, pregnant, and lactating female guinea pigs and hamsters. there are two forms in guinea pigs, a fasting type that results from decreased carbohydrate intake and subsequent mobilization of fat stores and a circulatory type that occurs when the gravid uterus compresses the aorta and causes reduced blood flow to abdominal organs. characteristic gross and histologic lesions include hepatic lipidosis and fat deposition in renal tubular epithelial cells. similarly, hepatic lipidosis and associated metabolic effects occasionally affect overweight nongravid sows and boars. urolithiasis is common in guinea pigs, rats, and mice due to high urine ph and calcium concentrations. obstructive urolithiasis has also been reported in captive male agoutis resulting in bilateral hydronephrosis, urinary bladder rupture, and peritonitis (batista et al., ) . a number of other endocrine diseases have rarely been documented in various rodent species. they have typically been associated with hormone-producing neoplasms (adrenal, islet, thyroid, parathyroid, and pituitary tumors). fluorosis is well studied in rats as they are used as models in toxicology studies. guinea pigs and rabbits are also susceptible and fluorosis has occurred in free-ranging cotton rats in sites contaminated by petrochemical waste (rafferty et al., ) . pathognomonic lesions occur in teeth and bones. periosteal hyperostosis occurs first along the medial surface of the metatarsals, then progresses to affect the mandible, metacarpals, and ribs though all bones can be affected in severe, chronic disease. bones are brittle and chalkywhite. pathological fractures are common but joints are typically spared. although characteristic, enamel hypoplasia only occurs when fluorosis is present during tooth development. tooth lesions include dull, dry, and chalky hypomineralized or hypoplastic enamel with pitting, grooves, and discoloration (due to oxidation); excessive attrition affects the incisors first. irregular wear, malocclusion, and fractures may be present. histologically, ameloblasts are small while osteoblasts are vacuolated and disorganized. there is abundant globular dentin and hypomineralization of the outer enamel layer. a differential for enamel hypoplasia in young syrian hamsters is hamster parvovirus (hapv); concurrent testicular hypoplasia, and cerebral malacia is present with this viral infection. cholecalciferol (vitamin d) is used as a rodenticide. the deliberate poisoning of free-ranging rats and mice results in inadvertent toxicity of nontarget animals including other rodents, such as squirrels or carnivorous predators that consume affected carcasses. multisystemic mineralization occurs prior to death in affected individuals. unlike other rodents, naked mole-rats, beavers, and woodchucks do not require dietary vitamin d and toxicity can develop if they are fed rodent chow or diets supplemented with vitamin d. lesions consistent with calcinosis cutis and circumscripta can develop in naked mole-rats fed such diets. soft tis-sue calcification is not limited to pressure points and can be found throughout the body in severe cases. resolution occurs with correction of the diet (delaney et al., ) . anticoagulant rodenticides including warfarin and its derivatives are frequently used to control free-ranging mice and rat populations. these compounds antagonize vitamin k and thus inhibit production of vitamin k-dependent clotting factors (i, ii, vii, ix, x). gross lesions include multisystemic hemorrhage, particularly subcutaneous and periarticular hematomas. "bait," some of which are brightly colored (e.g., teal-green), may be present within the intestines. inadvertent consumption by nontarget wildlife may have fatal consequences. hindgut fermenting rodents are susceptible to similar antibiotic-related toxicities as rabbits (see chapter ). nephrotoxicosis can be the result of exposure to numerous agents and has been extensively described in laboratory animals. for example, swainsonine toxicity occurs following the ingestion of plants from the genus swainsona and results in acquired alpha-mannosidosis in rats. histologically, renal tubular epithelial cells are severely and diffusely vacuolated. dystrophic mineralization, also known as dystrophic cardiac calcinosis is considered as a genetic disease in inbred mouse strains, particularly in aged individuals (eaton et al., ) . cardiac mineralization is detected grossly as pinpoint foci to larger irregular crusts composed of white, gritty material within and overlying the epicardium. histologic lesions vary depending on severity and chronicity with degeneration and necrosis of individual myocytes to extensive regions of myocyte loss and replacement by deeply basophilic granular material. in severe cases, mineralization extends into the myocardium resulting in scar formation and can also be seen in the kidneys and lungs. the prevalence of this disease in wild mice from which these strains originated is unknown. diabetes mellitus (dm) in chinese hamsters is a heritable, autosomal recessive disease (green et al., ) . symptomology and lesions are similar to those found in humans and include hydropic degeneration and degranulation of pancreatic islet β-cells with secondary vascular lesions, such as arteriosclerosis and glomerulosclerosis. in addition, affected hamsters are predisposed to pancreatic adenocarcinomas. serum levels of α- globulins are a useful indicator of risk for diabetes mellitus development in these populations. polycystic kidney disease has been diagnosed in captive populations of brazilian agoutis (müller et al., ) . most affected individuals have no clinical signs and are potentially related genetically. grossly, affected agoutis have a range of bilateral renal changes including atrophy, roughened granular cortical surfaces, and multiple cysts. histologically, there is cystic dilatation of renal tubules and bowman's capsules with mesangial and capsular thickening accompanied by mononuclear interstitial nephritis and fibrosis. many degenerative and age-related diseases are described in rodents used in toxicology studies pettan-brewer and treuting, ) . a significant disease confounding these studies is chronic progressive nephropathy, which is best described in rats though mice, naked mole-rats, and australian rodents, particularly the sticknest rat, are also affected (delaney et al., a) . the exact pathogenesis is unknown; however genetic factors may play a role. grossly, kidneys are bilaterally pale and either enlarged or shrunken with numerous pinpoint to large, mm cortical microcysts creating a nodular surface. histologically, renal tubules are markedly ectatic and tortuous and many contain brightly eosinophilic fluid (proteinosis) ( fig. . ). tubules have a range of degeneration, necrosis, and regeneration with occasional tubular hyperplasia and adenoma formation (predominantly in rats). glomeruli are variably affected by membranous and/or proliferative changes with thickened basement membranes, sclerosis, and obsolescence. synechiae, dilated bowman's capsules, and hypertrophied parietal epithelium are common. the interstitium is expanded by lymphoplasmacytic infiltrates with pigment-laden macrophages, mild hemorrhage, and edema. there is often substantial interstitial fibrosis and in severe cases, infarction. diagnosis is based on histologic evaluation though in some species, clinical pathology may be preferentially used to determine renal function. cardiomyopathy is common in aged mice, rats and hamsters; it is particularly common in syrian hamsters and is reported in woodchucks (chanut et al., ; rothenburger et al., a; roth and king, ) . lesion severity varies though it appears to increase with age and is more common in males. though considered strain and thus possibly genetically related in laboratory rats, free-ranging rats develop similar disease (rothenburger et al., a) . grossly, there may be cardiomegaly but macroscopic changes are not always apparent. histologically, myocardial lesions consist of multifocal and perivascular lymphoplasmacytic infiltrates accompanied by fibrosis, myocyte degeneration, and drop out ( fig. . a,b). trichrome, movat pentachrome, or other stains that highlight the fibrosis are useful to evaluate severity and chronicity. syrian hamsters develop cardiomyopathy associated spontaneous atrial thrombosis and subsequent coagulopathy that is often fatal (fig. . ). females appear to be affected earlier in the life than males. like other species, rodents are susceptible to skeletal degeneration and related diseases. guinea pigs, specifically the duncan-hartley breed and syrian hamsters, frequently develop degenerative joint disease. gross lesions include thickened joint capsules, variable osteophyte formation, roughened cartilage surfaces, eburnation, joint effusion, and synovial proliferation. depending on which joints are affected and chronicity, crepitus may be present when joints are manipulated. microscopic examination of joints reveals degeneration, necrosis, and ulceration of the articular cartilage with proliferation and fibrosis of the synovium and joint capsule, periarticular and periosteal bone formation, and remodeling. there may also be variable inflammation and hemorrhage within joint spaces. amyloidosis is typically seen in aged animals. amyloid is an extracellular accumulation of insoluble protein. excessive amyloid deposits can lead to tissue atrophy and dysfunction. amyloidosis is most common in myomorphs (mice, rats, hamsters, gerbils) and appears in the kidneys, spleen, adrenals, and liver as well as the gastrointestinal lamina propria (mouse) (fig. . ). there is a higher prevalence in females. of note is the eosinophilic substance within the nasal planum of aged mice that was once thought to be amyloid, through histochemical and ultrastructural studies, has instead been shown to consist of collagen and complex carbohydrates produced by the nasal gland epithelial cells (doi et al., ) . hemosiderosis is the accumulation of iron within various tissues, most commonly the liver. it is described in numerous species including primates, birds, rhinoceros, hyraces, and rodents. in one zoological institution, hemosiderosis was present in approximately % of adult naked mole rats (delaney et al., ) . grossly, livers appear bronze. iron accumulation appears histologically as zonal to diffuse hepatocellular accumulation of brown granular to figure . chronic progressive nephropathy in a naked molerat. renal tubules are variably ectatic, atrophied, degenerate, necrotic, and replaced by collagenous fibrous tissue. within the interstitium are accumulations of mononuclear cells (primarily lymphocytes and fewer macrophages). there is also sclerosis of glomeruli and periglomerular fibrosis. globular pigment. pigment is most intense in the periportal hepatocytes. the pigment stains positively with prussian blue and is not refractile with polarized light. aged mice can develop intrapulpal denticles, which are dysplastic tooth-like growths arising in the pulp of incisors. these may result in abnormal wear and or breakage of the incisors contributing to periodontitis and decreased ability to effectively gnaw (pettan-brewer and treuting, ). despite their protective quills, porcupines are particularly susceptible to cutaneous trauma and tears due to an exquisitely fragile integument. secondary infections resulting in pyoderma and persistent ulceration may occur. stress dermatosis (or dermatitis) is a condition of several south american rodents including, the agouti, acouchis, and capybaras. this syndrome is seen in captive and free-ranging individuals in densely populated groups. it presents as alopecic skin lesions and lacerations along the lumbosacral spine. stress is thought to increase skin fragility and overcrowding with intraspecific aggression contributes to lesion development. sun exposure, pruritus, and self-mutilation result in large patches of alopecia with erythema and excoriated skin with loss of the long hairs along the dorsum and tail. microscopic lesions correlate to severity and chronicity and include variable epidermal degeneration, necrosis, erosion/ulceration, and associated dermatitis with hemorrhage and edema. diagnosis is based on clinical history, demonstrated absence of a parasitic etiology in skin scrapings and histopathology. malocclusion is common especially in hysticomorphs, though it is also seen in brachydonts. it is a significant disease in captivity and may be related to genetics, nutrition and diet, and husbandry practices (losco, ) . ovarian cysts are common in rodents. cystic rete ovarii are prevalent in female guinea pigs (veiga-parga et al., ) and can be distinguished from other ovarian cysts as they arise from the rete and compress adjacent ovarian tissue ( fig. . ). other ovarian cystic structures reported in female rodents include: bursal cysts, epithelial cysts, follicular cysts, luteal cysts, and paraovarian cysts . the hemochorial placentation in rodents predisposes them to trophoblast emboli. this incidental lesion is best described in chinchillas and is not associated with clinical ramifications or gross lesions. histologically, intravascular trophoblasts are present in the lung, uterus, spleen, liver, and other sites, such as the adrenal glands with no associated inflammation. trophoblasts are large (> µm) with abundant amphophilic granular cytoplasm and large nuclei with prominent nucleoli. many neoplasms of inbred mice and rats appear to be strain related. nevertheless, certain neoplasms occur more frequently among free-ranging and domesticated outbred rodents than in other mammalian orders. several of them are described below. a list of other common neoplasms in rodent species is listed in the supplemental materials (table e ) . naked mole-rats are a popular zoo species and are heralded as animal models for cancer and aging due to their extreme longevity and purported cancer resistance. rare cases of neoplasia have been reported in naked mole-rats and in the related damaraland mole-rat, which is also longlived and appears to be relatively resistant to cancer and agerelated diseases (delaney et al., b; taylor et al., ,) . other subterranean rodents used as animal models for carcinogenesis research include some members of spalacidae (mole-rats, blind mole-rats, zokors, and bamboo rats). there are currently no reported spontaneous tumors in this group; however, subcutaneous tumors (fibromas and fibrosarcomas) may be induced with application of carcinogenic compounds. odontoma, or more aptly named odontogenic dysplasia, is described in several rodent species (rats, mice, guinea pig, prairie dogs, degus, chinchilla, chipmunk, squirrels; . most odontomas are not true neoplasms and instead represent dysplastic changes resulting from chronic inflammation or trauma and associated regional tissue response. as there is no demonstrated metastatic potential or criteria for malignancy, these lesions are increasingly identified as hamartomas versus neoplasms (and also referred to as pseudo-odontomas). nevertheless, these toothy masses are regionally destructive and almost exclusively affect the incisors. grossly, odontogenic dysplasia presents as nodular swellings around the maxillary or mandibular incisors. microscopically, these lesions are composed of disorganized and dysplastic proliferations of odontogenic epithelium, enamel matrix, and mineralized enamel, dentin, pulp, and cementum. in rodents, odontomas have been described as complex because they contain fully differentiated tooth elements but do not form the tooth-like structures grossly present in compound odontomas seen in other species (e.g., horse). histologically, rodent odontomas are composed of welldifferentiated odontogenic epithelium among an enamel organ with dental pulp mesenchyme and a variably mineralized enamel matrix. several important infectious and zoonotic diseases of rodents described are later with a focus on those that cause significant lesions in rodent species. additional pathogens, including zoonotic pathogens that occur in rodent species are listed in the supplemental materials (table e ). several references exist pertaining to rodent reservoirs of emerging and reemerging zoonotic pathogens ). poxviral diseases in rodents are caused by members of orthopoxviridae, which includes mousepox and cowpox (oie listed reportable zoonotic diseases). several species of rodents serve as the reservoir for cowpox virus, which results in cutaneous lesions in humans. in laboratory mice, mousepox, more commonly known as ectromelia, is an important disease. this infection can be acutely fatal with minimal lesions. in the chronic form, gross lesions include crusting lesions of face, legs, and tail, conjunctivitis, an enlarged, swollen and friable liver and spleen, and in cases of recovery, splenic fibrosis. necrosis of the kidney, thymus, and lymph nodes may also be grossly evident. the small intestine, urinary bladder, and vagina can have mucosal erosions and hemorrhage. histologically, skin and mucosal lesions include epithelial ballooning degeneration, hyperplasia, and erosions with characteristic intraepithelial, intracytoplasmic inclusion bodies. within the parenchymal organs, splenic and hepatocellular necrosis is typically coagulative and multifocal to coalescing. diagnosis is based on characteristic lesions with viral inclusions. electron microscopy, pcr, and virus isolation are useful for confirmation of the viral strain. in , an outbreak of monkeypox (orthopoxviridae), a zoonotic virus, occurred in prairie dogs sold as companion animals in the united states (gaurner et al., ) . fatal infections in infected prairie dogs were characterized by necrotizing bronchopneumonia, conjunctivitis, and glossal ulcers. viruses and viral antigens were detected in multiple tissues and cell types via immunohistochemical staining, pcr, and electron microscopy. infected humans developed fever and vascular rashes. forty seven confirmed and probable cases of monkeypox were reported from six states. the prairie dogs contracted the virus following exposure to infected african rodents. other rodents that also developed disease and died included: rope squirrels, tree squirrels, gambian giant rats, brush-tailed porcupine, dormice, and striped mice. fibromas and fibromatosis of gray squirrels are potentially fatal conditions caused by squirrel fibroma virus (leporipoxviridae). disease is mainly reported in the eastern north america (terrell et al., ) but also occurs in red squirrels and has been reported in a fox squirrel (wilcoxen et al., ) . transmission is likely through biting arthropods (fleas, ticks, and mosquitoes). gross and histologic lesions are similar to shope fibromas in rabbits (both are leporipoxviruses). multifocal to coalescing tan, firm nodules may cover all parts of the body and begin as alopecic nodules with progression to plaque-like to pedunculated masses ( fig. . a ). systemic disease manifests as renomegaly and multiple pulmonary nodules . histologically, the skin nodules exhibit marked epidermal hyperplasia with ballooning degeneration of keratinocytes, spongiosis, and intracytoplasmic eosinophilic viral inclusions (fig. . b,c) . pulmonary nodules correlate histologically to adenomatous hyperplasia with similar viral inclusions. other microscopic lesions may include atypical mesencyhmal proliferation in the liver and seminal vesicles, and renal tubular epithelial hyperplasia with viral inclusions. diagnosis is based on characteristic lesions and detection of virus by pcr or isolation. parapoxvirus is an important cause of disease in red squirrels in the united kingdom and ireland where it is a suspected factor in severe population declines tompkins et al., ) . the causative agent is referred to as red squirrel parapoxvirus. grey squirrels are the likely maintenance hosts since a large percentage of healthy gray squirrels are positive for the virus and their range overlaps with that of red squirrels. lesions in red squirrels include exudative dermatitis with crusting that is similar to other poxviral diseases. viral inclusions can be detected histologically within the cytoplasm of infected cells. electron microscopy and pcr are used to detect the virus. woodchuck hepatitis virus (whv; hepadnaviridae) is a disease in captive and free-ranging animals . although not zoonotic, this disease is a valuable animal model for viral hepatitis and hepatocarcinogenicity associated with hepatitis b in humans. it affects both sexes and is typically found in animals greater than years of age. activated t cells (cd +) cause cellular and dna damage leading to persistent hepatitis. simultaneously, viral dna integrates into the host genome at loci containing oncogenes, creating the potential for carcinogenic transformation. hepatocellular carcinomas are reported in % of experimentally infected woodchucks. grossly, most carcinomas are solitary and involve one hepatic lobe. histologically, neoplastic masses have differing growth patterns: trabecular, adenoid, or solid with and without cystic spaces, hemorrhage, and necrosis. an interesting feature of these tumors is the presence of atypical, bizarre, and giant neoplastic cells. adenomas may be present and are considered a precursor to carcinomas. mitotic index and cellular morphology are used to classify tumors as adenomas versus carcinomas. the remaining hepatic parenchyma often contains acute and/or chronic hepatitis with neutrophilic to mononuclear inflammation, variable hepatocellular degeneration, necrosis, fibrosis, and biliary changes. electron microscopic identification of the virus is definitive. beechey ground squirrels have a similar virus with variable hepatic lesions (cullen and marion, ) . hamster polyomavirus (hapyv), a papovavirus, causes cutaneous trichoepitheliomas in syrian hamsters . the virus is transmitted via urine and is highly contagious, with reported % morbidity in affected breeding colonies. early lesions consist of wartlike growths and alopecic nodules of the face and perineum with progression to severe, multifocal to coalescing nodules throughout the body. histologically, these characteristic skin tumors contain multiple islands of basilar to variably differentiated follicular epithelium and abrupt central keratinization with faded anuclear keratinocytes (ghost cells). rudimentary hairs may be present and follicular rupture may be associated with inflammation. other polyomaviruses-associated neoplasms include other cutaneous tumors, lymphomas and salivary gland tumors. in virally induced lymphomas, affected tissues are effaced by diffuse sheets of neoplastic lymphocytes among scant stroma. viral inclusions are not present. in a case of polyomavirus-induced, multicentric lymphoma in an -week old syrian hamster, the virus was detected in tumor cells, renal tubular epithelial cells, and enterocytes via in situ pcr indicating some degree of tissue tropism . diagnosis is based on clinical history, gross and histologic lesions, and detection of the virus via pcr and/or electron microscopy. mouse hepatitis caused by mhv (coronaviridae) is enzootic in some colonies of inbred laboratory mice . mouse hepatitis may also occur in nonlaboratory strains of mice in zoo settings. mhv causes polytropic and enterotropic disease syndromes, both of which occur following highly contagious viral infection by the oronasal route. grossly, there is hepatomegaly, splenomegaly, lymphadenomegaly, and lymphadenopathy with jaundice and ascites. there may be gross evidence of intestinal (ileal and cecal) disease with thickened and necrotic mucosa. within the liver, spleen, and lymph nodes, there are multiple coalescing and random white necrotic foci. histologically, polytropic disease includes necrotizing hepatitis with syncytial cells within necrotic foci, and demyelination in cns in immunodeficient mice. the enterotropic disease lesions are age-dependent and may include villous attenuation and syncytial cell formation with eosinophilic intracytoplasmic inclusion bodies in enterocytes, mesenteric lymph nodes, and endothelial cells; granulomatous serositis may also be present. diagnosis is based on histologic features and immunohistochemical demonstration of viral antigens within lesions. virus isolation, pcr, and serology are used to confirm infection and exposure, respectively. sendai virus infection (parainfluenza- ; paramyxoviridae) is common in wild and pet rodents and is actively excluded from laboratory populations of mice, rats, and gerbils (easterbrook et al., ) . disease manifestations occur quickly following the aerosol inhalation, direct contact, or in utero exposure. the virus targets respiratory epithelium and type ii pneumocytes, which may impair ciliary activity and predispose affected individuals to secondary mycoplasma pulmonis and other bacterial infections. gross lesions include dark purple discoloration, atelectasis, and consolidation of the cranioventral lung lobes and diffuse pulmonary edema. splenomegaly and lymphadenomegaly are common and if infection occurs in a gravid female, fetal death may occur. histologically, lesions include necrotizing rhinitis, laryngotracheitis, bronchitis, and bronchointerstitial pneumonia. in immunocompromised animals (e.g., athymic and scid mice), intracytoplasmic and intranuclear inclusion bodies may be present. diagnosis is based on supportive clinical history, histologic findings, and pcr detection of the virus. sialodacryoadenitis is a disease specific to rats and is caused by sialodacryoadenitis virus (sdav; coronaviridae). the virus may be enzootic in some research colonies and in pet and free-ranging rats (easterbrook et al., ) . virus is shed in nasal secretions and/or saliva. sdav infects and replicates in the respiratory tract and spreads to salivary and lacrimal glands resulting in secondary lesions of the nasopharynx, respiratory tract, and eyes. grossly, the parotid and submandibular salivary glands, harderian glands, exorbital glands, and cervical lymph nodes are enlarged, pale, and edematous. the harderian glands may exhibit brown pigmentation, which correlates to accumulations of porphyrin within acini. histologically, there is acute necrotizing adenitis. in chronic cases, there is fibrosis and extensive squamous metaplasia of glandular tissue. there may be rhinitis, tracheitis, bronchitis, and bronchiolitis in severe and chronic cases. histologic lesions are characteristic and supportive of the diagnosis. within this section are several zoonotic and notable nonzoonotic bacteria that infect rodents. several others that are notable but not associated with disease in their rodent host are listed in the supplemental materials (table e ). plague is an oie-listed and who-reportable zoonotic disease that is important to consider when working with rodents. fleas spread the causative agent, yersinia pestis, between hosts. cats and other rodent-eating predators can also contract infection through ingestion and rarely via inhalation. historically, plague has been implicated in the deaths of millions of humans, most notably in th century europe during the black death. there are two main host types: enzootic reservoirs (voles and deer mice) and epizootic amplifiers (prairie dogs, rats, and squirrels). with close to % mortality, plague outbreaks in prairie dog colonies are a devastating and significant threat not only this species but to endangered black-footed ferrets, who are susceptible to disease because prairie dogs are their primary food source (cully et al., ) . there are three disease forms: bubonic (lymphadenomegaly with abscessation); pneumonic ( necrotizing pneumonia with fibrinous pleuritis); and septicemic (multifocal necrosis of liver, lung, spleen, kidney, eye, brain). gross lesions include multifocal pulmonary hemorrhage (fig. . ) , hemorrhagic lymph nodes, erythema of axillary and inguinal skin, splenomegaly and localized hemorrhage and necrosis in the dermis and subutis (suspected site of flea bite). histologic lesions of bubonic disease include necrosuppurative lymphadenitis; in the pneumonic form, necrotizing pneumonia; and in the septicemic form, multifocal abscessation. all lesions contain gram and giemsa positive coccobacilli with characteristic bipolar morphology resembling a safety pin. bacterial culture must be completed at a certified, biosecure laboratory; identification using immunohistochemistry or immunofluorescence is a beneficial diagnostic aid. yersiniosis (pseudotuberculosis) is caused by y. pseudotuberculosis and y. entercolitica. rodents may serve as carriers of this zoonotic bacterial agent, though some species, including mice, voles, beavers, and muskrats may succumb to acutely fatal disease or develop more characteristic lesions and symptoms. gross and histologic lesions include multifocal necrotizing hepatitis and splenitis and/or fibrinonecrotizing and ulcerative enterocolitis (fig. . ). agoutis appear to be uniquely susceptible in zoo-settings (k. terio, personal communication). tularemia caused by francisella tularensis is an important zoonotic and oie listed reportable disease that is maintained by wild rodents and lagomorphs. disease occurs in many rodents including mice, beavers, and muskrats, while voles appear to be subclinical carriers and serve as a reservoir (nelson et al., ; rossow et al., ) . lesions of affected species are similar to those described in rabbits (see chapter ). miliary to multifocal and coalescing hepatic necrosis with variable inflammation (depending on chronicity) characterize the disease (fig. . ) . unlike yersiniosis and similar to listeriosis, bacteria are not readily detected but can be better visualized with special stains. listeriosis is a common zoonotic bacterium in rodent species. it can cause variable, yet similar lesions as for rabbits (see chapter ) . the systemic form is most common. during an outbreak in bushy-tailed jirds, death occurred without clinical signs (tappe et al., ) . salmonellosis is reported in guinea pigs, rats, and mice, though any rodent species can be infected and develop disease. salmonella enterica enteritidis or typhimurium are the most common zoonotic serovars. guinea pigs become septicemic and often die acutely. other rodents may develop gastrointestinal disease that includes diarrhea and abdominal pain prior to systemic spread. in hamsters, salmonellosis must be considered and ruled out in cases of "wet tail," which is typically associated with infection by lawsonia intracellularis. gross lesions of salmonellosis include cyanosis of mucous membranes and polyserositis with splenomegaly. hepatic, splenic, and lymph node necrosis are typical, as are multifocal necrosis and button ulcers in the colon with segmental intestinal infarction. histologic lesions correlate to gross findings and include small foci of hepatocellular necrosis containing necrotic cell debris and variable numbers of macrophages, lymphocytes, and fewer neutrophils (paratyphoid nodules) and kupffer cell hyperplasia; granulomatous hepatitis, splenitis, and lymphadenitis; and fibrinonecrotic ileotyphlocolitis. intestinal infarction can be attributed to vasculitis and thrombosis of the mesenteric or mesocolic vessels. culture of the causative agent and histologic lesions confirm the diagnosis. mycobacteriosis is a zoonotic disease that is rarely reported in rodents, though some species may serve as reservoirs. there are reports of mycobacteriosis (mycobacterium microti) in british voles and wood mice (cavanagh et al., ) , hamsters, and korean and richardson's ground squirrels in spain. chlamydophila caviae and less frequently c. psittaci cause disease in guinea pigs, primarily juveniles. conjunctivitis, bronchitis, and pneumonia may occur and result in debilitation and death. histologically, there is heterophilic keratoconjunctivitis and uveitis. pneumonia has a cranioventral pattern and is heterophilic. some animals may be asymptomatic. diagnosis is achieved through cytology of ocular discharge and/or pcr of affected tissues. immunohistochemistry of tissue sections is also available. bordetella bronchiseptica causes bordetellosis (epizootic pneumonia), a common respiratory disease in guinea pigs though is also reported in free-ranging squirrel populations . similar disease can occur in other rodents, particularly in the context of immunosuppression or coinfection. grossly, there is cranioventral pneumonia with multifocal to coalescing, discrete, reddish-gray consolidated regions affecting multiple lobes, and pleuritis. mucopurulent exudate may be present in the nares, nasal passages, trachea, and tympanic bullae. there is often crusting conjunctivitis. females may have metritis and pyosalpinx. histologically, lung lesions consist of necrotizing and heterophilic bronchopneumonia with obliteration of the airways. diagnosis is confirmed with bacterial culture or pcr. other predisposing pathogens (e.g., adenovirus, mycoplasma spp.) must be ruled out. ciliated associated respiratory (car) bacillus is an unclassified bacterium that colonizes the ciliated epithelium of the respiratory tract of rats, mice, rabbits, and a variety of other species. this agent acts synergistically with other respiratory pathogens including mycoplasma pulmonis, sendai virus, coronavirus, streptococcus pneumoniae, corynebacterium kutscheri, bordetella bronchiseptica, klebsiella pneumoniae, or pasteurella spp. lesions include suppurative to mucopurulent bronchopneumonia, perivascular and bronchiolar lymphoid hyperplasia, rhinitis and lymphoplasmacytic tracheitis in free-ranging, pet and laboratory rats rothenburger et al., b) . coinfection of car bacillus and mycoplasmas has been reported in rats and spinifex hopping mice with pneumonia (mackie et al., ) . murine respiratory mycoplasmosis is caused by mycoplasma pulmonis . gross lesions include catarrhal exudate in the upper respiratory tract with cranioventral bronchopneumonia and mucopurulent exudate in airways with atelectasis. histologic lesions include peribronchial lymphoid cuffing with balt hyperplasia; neutrophilic bronchopneumonia; type ii pneumocyte hyperplasia of alveolar epithelium; and emphysema; bronchiectasis is a characteristic finding. histology alone cannot differentiate m. pulmonis and car bacillus respiratory infections (rothenburger et al., b) . other lesions associated with m. pulmonis include endometritis, salpingitis, and perioophoritis. diagnosis is based on histologic features, culture or pcr detection, and elisa. pasteurellosis caused by p. multocida affects young and immunocompromised rodents, most frequently following direct transmission from the dam. strain virulence and species determine lesions, which in general, are similar to those reported in other species, including rabbits (see chapter ) . prairie dogs develop upper respiratory disease and pneumonia, while conjunctivitis is more common in rats and mice. hamsters have variable susceptibility. cervical lymphadenitis, a disease classically described in guinea pigs, is caused by streptococcus equi subsp. zooepidemicus. infection is associated with coarse foods that cause traumatic lesions of the oral mucosa; bite wounds may serve as another route of entry. grossly, the cervical and submandibular lymph nodes are markedly enlarged and contain thick purulent yellow-white to red-gray exudate. lymph nodes in severely affected and chronic cases may rupture. histologically, affected lymph nodes are effaced by heterophilic lymphadenitis with central necrosis and peripheral fibrosis. thoracic lesions include fibrinous pleural adhesions, fibrinosuppurative bronchopneumonia, pleuritis, and pericarditis. chains of gram-positive cocci are present within lesions. diagnosis is based on gross and histologic lesions and culture of causative agent. tyzzer's disease, caused by clostridium piliforme, is a potentially fatal disease of many rodent species. it has been reported in captive gerbils, hamsters, and spinifex hopping mice (stannard et al., ) . infection results in a triad of gross lesions including icterus and hepatomegaly with military gray foci, congestion, and edema of the intestine, and lymphadenomegaly with edema and hemorrhage. sudden death without clinical disease or lesions is possible in peracute cases. histologically, multifocal random to coalescing hepatic necrosis is surrounded by hemorrhage, heterophils, and macrophages (fig. . a ). at the necrotic margins, hepatocytes may accumulate characteristic criss-crossed bundles of faintly staining bacilli that are best visualized with silver stains (fig. . b ). proliferative ileitis (wet tail) due to lawsonia intracellularis is most commonly reported in hamsters. similar to this infection in other species, there is segmental thickening of ileum with serosal nodules. histologically, there is marked crypt hyperplasia and herniation and villous elongation, hyperplasia, and fusion with variable necrosis and hemorrhage. silver stains and pcr are useful to confirm infection. a diverse array of pneumocystis spp. are carried without clinical signs by a variety of wild rodents; these organisms are thought to be highly host-specific. pneumocystis spp. are found within alveoli and alveolar macrophages and appear as small, eosinophilic round organisms with a small central body. in severe cases, organisms within alveolar spaces and alveolar macrophages can obliterate alveoli; in some cases they can be associated with interstitial pneumonia (fig. . a,b) . this fungus can cause significant opportunistic infections in immunocomromised individuals. cryptococcosis (cryptococcus neoformans) can be a significant pathogen in some rodent species. for example, approximately % of slender-tailed cloud rats at one zoological institution died with or as a result of cryptococcal pneumonia over a -year period (berliner et al., ) . these large, arboreal rodents were likely infected by contaminated substrates and may be a highly susceptible species, similar to some species of nonhuman primates and felids. lesions included none or mild to severe, chronic granulomatous pneumonia with or without necrosis and regional lymphadenitis. disseminated disease can produce subcutaneous nodules ( cryptococcoma) and meningoencephalitis. microscopically, characteristic fungal yeasts with narrow-based budding are present in cytologic preparations and histologic sections. gms staining can enhance detection of fungal yeasts, which are - µm diameter with a distinct, thick ( - µm), nonstaining mucopolysaccharide capsule, the latter of which is best visualized using the pas reaction and mucin stains. culture can confirm infection ( fig. . ) adiaspiromycosis is a systemic fungal disease reported in several captive and free-ranging rodent species. it is caused by the saphrophytic fungi chrysosporium parum and c. crescens. once inhaled, these fungi cause pulmonary granulomas with fungal spherules and conidia. additional diagnostic tests may be useful, including immunodiffusion and complement fixation. these fungi are zoonotic. other systemic mycoses of rodents include infections caused by the endemic (and zoonotic) fungi blastomcyes dermatitides and histoplasma capsulatum. reported cases include juvenile mice and chinchillas. immunity to fungal pathogens appears limited in mice. dermatophytosis is a cutaneous fungal disease that can be found in any rodent species and is similar to that of other mammalian species. causative agents include trichophyton mentagrophytes, microsporum canis, m. gypseum, and epidermophyton. of note, these fungi are zoonotic and can cause skin lesions in other animals. additionally, sporothrix schneckii associated cutaneous disease is reported in several rodent species and can be zoonotic. dermatitis lesions vary from suppurative and necrotizing to granulomatous and fibrosing depending on chronicity. paspositive yeast are present within macrophages. character-istic organisms on cytologic preparations of skin scrapes prepared with potassium hydroxide support the diagnosis. capillaria hepatica (synonymn calodium hepaticum) affects a broad host range worldwide, including rodents and humans. it is a ubiquitous parasite of globally invasive norway and black rat populations . gross lesions consist of multifocal to coalescing, tortuous, white tracks in the liver parenchyma ( fig. . a ). histologically, varying combinations of lymphoplasmocytic and granulomatous inflammatory infiltrates, fibrosis, and bioperculate eggs and occasionally adult parasites replace hepatic parenchyma (fig. . b ). cross sections of viable or mineralized capillarid nematodes may be present. eggs are only released from the liver following carcass decomposition or ingestion by a predator/cannibalistic conspecific. therefore, diagnosis is based on the presence of the nematode and/or eggs in the liver and not fecal parasitology. many species of free-ranging rodents are susceptible to fatal visceral larval migrans associated with the raccoon nematode baylisascaris procyonis (fig. . ). significant clinical disease is often related to parasite migration in the brain, where they may be accompanied by low numbers of eosinophils, gliosis, spheroids, and rarefaction or necrosis of the neuropil. rodents, particularly squirrels are prone to notoedric mange cornish et al., ; . mites burrow into the stratum corneum inducing marked hypekeratosis, which results in patchy alopecia, and crusting and thickening of the skin (lichenification), which can appear yellow to gray. gross lesions are predominantly found on the ears, nose, tail, external genitalia, inguinal and perianal regions, and feet though they can cover the entire body in severe cases. peripheral lymphadenopathy may also be seen. skin scrapings and fecal floats are beneficial to best examine mite features and presence of eggs, respectively. histologically, there is marked epidermial hyperplasia with parakeratotic hyperkeratosis forming caps over tunnels containing mites with superficial, perivascular eosinophilic dermatitis ( fig. . a,b) . secondary bacterial infections are common. additional notable parasites of rodents are listed in the supplemental materials (table e ) . chronic progressive nephropathy (cpn), naked mole-rat, kidney. in the kidney, there is marked ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. . ). hepatic hemosiderosis is seen in sections of the liver. eslide: vm .e chronic progressive nephropathy (cpn), naked mole-rat, kidney. in the kidney, there is marked ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. . ). hepatic hemosiderosis is seen in sections of the liver. eslide: vm .e chronic progressive nephropathy (cpn), naked mole-rat, kidney. there is mild to moderate ectasia of tubules and bowman's spaces forming numerous microcysts seen at low magnification. some ectatic tubules contain luminal proteinaceous material and there is multifocal tubular degeneration, necrosis, and regeneration. interstitium has areas of mixed inflammation, fibrosis and edema. glomeruli have segmental to global membranous change and multifocal sclerosis. (see fig. . ). eslide: vm .e hepatic hemosiderosis, naked mole-rat, liver. prussian blue staining highlights bright blue granular intracytoplasmic (iron) pigments throughout the liver. the kidney has changes consistent with chronic progressive nephropathy. eslide: vm .e yersiniosis, beaver, liver and diaphragm. yersiniosis in the liver and diaphragm of a free-ranging north american beaver. bacterial infection causes multifocal random,necrosis throughout the hepatic parenchyma. large, central colonies of the causative agent, yersinia pseudotuberculosis are surrounded by mild neutrophilic inflammation. (see fig. . ). eslide: vm .e tularemia, beaver, liver and spleen. tularemia in the liver and spleen of a free-ranging north american beaver. large, multifocal random areas of coagulative necrosis are present throughout the hepatic and splenic parenchyma. note the relative lack of inflammation. unlike yersiniosis, bacterial colonies are not readily detected with routine hematoxylin and eosin staining. (see fig. . ). eslide: vm .e pneumocystis murina pneumonia, mouse, lung. multifocally, alveoli are partially to completely filled with large macrophages containing small round yeasts.(see fig. . ). eslide: vm .e pneumocystis murina pneumonia, mouse, lung, gms. gomori methenamine silver (gms) staining highlights the intrahistiocytic yeasts within the alveoli. (see fig. . ). eslide: vm .e pulmonary cryptococcosis, slender-tailed cloud rat, lung. the interstitium and air spaces are moderately to markedly expanded and distorted by myriad fungal yeasts that are associated with histiocytic to granulomatous inflammation. aggregates of lymphoplasmacytic inflammation are present but infrequent. (see fig. . ). eslide: vm .e pulmonary cryptococcosis, slender-tailed cloud rat, lung. gms. intralesional yeasts are hhighlighed gray/ blue with silver staining. gms (see fig. . ). eslide: vm .e pulmonary cryptococcosis, slender-tailed cloud rat, lung. mucicarmine. mucicarmine staining highlights the thick mucopolysaccharide capsule of the crytpococcal yeast and stains it bright pink. mucicarmine (see fig. . ). eslide: vm .e capillaria hepatica (calodium hepaticum), norway rat, liver. capillaria hepatica in the liver of a freeranging norway rat. adult nematodes and eggs invade and efface the liver parenchyma. there are examples of viable and degenerative adults. the eggs are characteristically bioperculate with a thick shell. inflammation is lymphoplasmacytic to granulomatous and is associated with fibrosis indicating chronic infection. in some areas, there is multifocal mineralization adjacent to eggs. (see fig. . ). eslide: vm .e baylisacaris visceral larval migrans, groundhog, brain. multifocally, multiple sections of larval nematodes with prominent lateral alae and lateral cords, a thick cuticle, and coelomyarian musculature, features consistent with larval ascarids, are present in the brain. inflammation, including eosinophils, gliosis, and spheroids and rarefaction of the neuropil due to parasite migration, are multifocal and occasionally associated with intralesional nematodes. (see fig. . ). eslide: vm ( ); krinke ( ) , krinke ( ) mice, rats mammary fibroadenoma rudman ( ); krinke ( ) , krinke ( ) gray squirrel mammary (mixed malignant) williams ( ) mice, rats zymbal gland adenoma/carcinoma rudman ( ); krinke ( ) , krinke ( ) mice, rats preputial/clitoral adenoma/carcinoma rudman ( ), , krinke ( ) , krinke ( ) female reproductive ovarian adenocarcinoma , krinke ( ) , krinke ( ) mice teratoma dixon ( ), mice, rats uterine decidual reaction/deciduoma dixon ( ) woodchuck, mice, rats uterine leiomyoma foley ( ) , mice, rats uterine leiomyosarcoma dixon ( ) woodchuck adenoma of the rete testis foley ( ) rat, woodchuck interstitial cells tumors foley ( ) , , creasy ( ) woodchuck sertoli cell tumors foley ( ) woodchuck seminoma foley ( ) woodchuck, mice teratoma foley ( ) , , creasy ( ) hepatobilliary foci of cellular alteration thoolen ( ) woodchuck, rat, mice hepatocellular adenoma roth ( ) , thoolen ( ) , krinke ( ) , krinke ( ) woodchuck, rat, mice hepatocellular carcinoma , thoolen ( ) , krinke ( ) , krinke ( ) hemolymphatic mice, rats histiocytic sarcoma krinke ( ) , krinke ( ) , kogan ( ) , guinea pig, rat lymphocytic leukemia yarto-jaramillo ( ), guinea pig, hamster, mice, woodchuck, rat lymphoma nagy ( ) , , yarto-jaramillo ( ), kogan ( ) , woodchuck, mice, rats myeloproliferative disease roth ( ) , kogan ( ) , ( ), , krinke ( ) , krinke ( ) ground squirrel integumentary gland adenocarcinoma carminato ( ) damaraland mole rat melanomas sura ( ) yellow cheeked vole sebacious gland adenoma williams ( ) capybara, tundra vole, arctic ground squirrel,woodchuck squamous cell carcinoma hamanno ( ) , takahisa ( ), anderson ( ) beaver, insular vole, porcupine squamous papilloma williams ( ) hamsters, red squirrels trichoepithelioma , guinea pig trichofolliculoma ( ) beaver rhabdomyoma williams ( ) red backed vole rhabdomyosarcoma williams ( ) tundra vole osteoma williams ( ) guinea pig alveologenic carcinoma yarto-jaramillo ( ) guinea pig bronchogenic carcinoma yarto-jaramillo ( ) guinea pig papillary adenoma yarto-jaramillo ( ) gray squirrels pulmonary adenomatosis red squirrels, mice pulmonary carcinoma , , renne mice, rats pulomnary adenoma , renne ( ) red and gray squirrels, mice, rats renal adenoma , williams ( ) , frazier spontaneous neoplastic and hyperplastic skin lesions of the woodchuck cutaneous and systemic poxviral disease in red (tamiasciurus hudsonicus) and gray (sciurus carolinensis) squirrels pathology of laboratory rodents and rabbits non-proliverative and proliferative lesions of the cardiovascular system of the rat and mouse endocrine system odontoma-like tumours of squirrel elodont incisors -elodontomas spontaneous hibernomas in sprague-dawley rats adenocarcinoma of the dorsal glands in european ground squirrels (spermophilus citellus) synhimantus (nematoda) associated with gastric squamous tumors in muskrats proliferative and nonproliferative lesions of the rat and mouse male reproductive system nonproliferative and proliferative lesions of the rat and mouse female reproductive system neoplastic and nonneoplastic lesions of the reproductive tract of the woodchuck (marmota monax) polyomavirus infection in hamsters and trichoepitheliomas/ cutaneous adnexal tumors proliferative and nonproliferative lesions of the rat and mouse urinary system proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium squamous cell carcinoma in a capybara (hydrochoerus hydrochaeris) malignant pleural mesothelioma in a woodchuck (marmota monax) oral leiomyosarcoma in a woodchuck (marmota monax) changes in the major ocular glands nonneoplastic and neoplastic changes in the harderian and lacrimal glands pulmonary lesions produced by fibromas viruses in squirrels and rabbits bethesda proposal for classification of nonlymphoind hematopoietic neoplasms in mice lymphosarcoma in the laboratory woodchuck obstructive respiratory disease in prairie dogs with odontomas proliferative and nonproliferative lesions of the rat and mouse respiratory tract harderian gland neoplasms in captive, wild-caught beechey ground squirrels (spermophilus beecheyi) chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection hepatic lesions in woodchucks (marmota monax) serongative for woodchuck hepatitis virus lesions associated with eucoleus sp. in the non-glandular stomach of wild urban rats (rattus norvegicus) proliferative and nonproliferative lesions of the rat and mouse mammary, zymbal's preputial and clitoral glands hamster polyomavirus infection in a pet syrian hamster (mesocricetus auratus) causes of mortality and pathological lesions observed post-mortem in red squirrels (sciurus vulgaris) in great britain endocrine system. endocrine system neoplasia and granulomas surrounding microchip transponders in damaraland mole rats (cryptomys damarensis) squamous cell carcinoma in a capybara (hydrochoerus hydrochaeris) comparative histomorphological review of rat and human hepatocellular proliferative lesions spontaneous tumors of free-ranging terrestrial mammals of north america fowler's zoo and wild animal medicine leptospira and leptospirosis natural history of plague: perspectives from more than a century of research plague and yersiniosis bartonella infection in rodents and their flea ectoparasites: an overview. vector borne zoonotic dis rodent reservoirs of future zoonotic diseases a review of listeria monocytogenes and listeriosis yersiniosis in wildlife and its public health implications rodent-borne diseases and their risks for public health the ecology of tularaemia tularemia in deer mice (peromyscus maniculatus) during a population irruption in saskatchewan *this list includes the zoonotic pathogens carried by rodents that cause disease in wild animals and those deemed most relevent to wildlife and zoo animal specialists ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver nathural pathogens of laboratory mice, rats, and rabbits and their effects on research toxoplasmosis in a woodchuck (marmota monax) and two american red squirrels (tamiasciurus hudsonicus) baylisascariosis-infections of animals and humans with 'unusual'roundworms echinococcus multilocularis detection in live eurasian beavers (castor fiber) using a combination of laparoscopy and abdominal ultrasound under field conditions synhimantus (nematoda) associated with gastric squamous tumors in muskrats besnoitia jellisoni (sporozoa: toxoplasmea) in rodents from utah and california a survey of hemoparasite infections in free-ranging mammals and reptiles in french guiana systemic toxoplasmosis in a five month old beaver, (castor canadensis) echinococcus vogeli rausch and bernstein, , from the paca, cuniculus paca l.(rodentia: dasyproctidae), in the departamento de santa cruz pathomorphologic findings in short-tailed voles (microtus agrestis) experimentallyinfected with frenkelia microti pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus more than a rabbit's tale-encephalitozoon spp. in wild mammals and birds studies on the nematode parasite, gongylonema neoplasticum (spiroptera neoplasticum) and avitaminosis a in the forestomach of rats: comparison with fibiger's results echinococcus multilocularis in a european beaver from switzerland frenkelia sp. from the brain of a porcupine (erethizon dorsatum) from alberta the role of wildlife rehabilitation as sentinels for one health issues at the wildlife and public health interface: reports of taenia crassiceps cysticercosis in woodchucks (marmota monax) and squirrels (sciurus carolinensis) in maryland and virginia cysticerci of taenia mustelae in the fox squirrel characteristics of natural infections of the stomach worm, obeliscoides cuniculi (graybill), in lagomorphs and woodchucks in canada first identification of echinococcus multilocularis in rodent intermediate hosts in sweden parasites of ferrets, rabbits, and rodents klossiella infection of the guinea pig the taxonomic status of echinococcus cruzi brumpt and joyeux, (cestoda: taeniidae) from an agouti (rodentia: dasyproctidae) in brazil capillaria hepatica in wild norway rats (rattus norvegicus) from vancouver lesions associated with eucoleus sp. in the non-glandular stomach of wild urban rats (rattus norvegicus) cutaneous leishmaniasis in the amazon: isolation of leishmania (v.) lainsoni rodent agouti paca (rodentia: dasyproctidae) in the state of para causes of mortality and pathological lesions observed post-mortem in red squirrels (sciurus vulgaris) in great britain pathologic findings in western gray squirrels (sciurus griseus) from a notoedric mange epidemic in san bernardino moutnains, california natural infection of the ground squirrel (spermophilus spp.) with echinococcus granulosus in china ear mange mites (notoedres muris) in black and norway rats (rattus rattus and rattus norvegicus) from inner-city vancouver. can nathural pathogens of laboratory mice, rats, and rabbits and their effects on research cutaneous and systemic poxviral disease in red (tamiasciurus hudsonicus) and gray (sciurus carolinensis) squirrels pathology of laboratory rodents and rabbits diseases of agouti (dasyprocta agouti) rained in captivity diagnosed by pathological examination cryptococcus neoformans pneumonia in slender tailed cloud rats (phloeomys pallidus): a review of seven cases odontoma-like tumors of squirrel elodont incisors -elondontomas order rodentia mycobacterium microti infection (vole tuberculosis) in wild rodent populations spontaneous cardiomyopathy in young sprague-dawley rats: evaluation of biological and environmental variability notoedric mange in western gray squirrels from washington scurvy in capybaras bred in captivity argentine non-neoplastic liver disease associated with chronic ground squirrel hepatitis virus infection disease limits populations: plague and black-tailed prairie dogs. vector-borne zoonotic dis spontaneous histologic lesions of the adult naked mole-rat (heterocephalus glaber): a retrospective survey of lesions in a zoo population renal pathology in a non-traditional aging model: the naked mole-rat (heterocephalus glaber) initial case reports of cancer in naked mole-rats (heterocephalus glaber) nonproliferative and proliferative lesions of the rat and mouse female reproductive system anatomical studies of the male genital organs of the european beaver eosinophilic substanceis "not amyloid" in the mouse nasal septum a survey of rodent-borne pathogens carried by wild-caught norway rats: a potential threat to laboratory rodent colonies dystrophic cardiac calcinosis in mice polyomavirus infection in hamsters and trichoepitheliomas/cutaneous adnexal tumors monkeypox transmission and pathogenesis in prairie dogs histopathology of preclinical toxicity studies prediction of spontaneous hereditary diabetes mellitus in chinese hamsters by means of elevated alpha- serum levels rodent reservoirs of future zoonotic diseases macroscopic anatomy of the lower respiratory system in mole rats (spalax leucodon) kurloff cells in peripheral blood and organs of wild capybaras pulmonary lesions produced by fibromas viruses in squirrels and rabbits the clinical chemistry of laboratory animals dental dysplasia in rats and mice concurrent infection with cilia-associated respiratory bacillus and mycoplasmas in spinifex hopping-mice (notomys alexis) with pneumonia rodent-borne diseases and their risks for public health persistent paramesonpehric ducts (masculine uterus) in the male north american beaver (castor candadensis) polycystic kidney disease in adult brazilian agoutis (dasyprocta leporina) francisella tularensis infection rodentia chapter | without lesions in gray tree squirrels (sciurus griseus): a diagnostic challenge practical pathology of aging mice fluorosis risks to resident hispid cotton rats on land-treatment facilities for petrochemical wastes detection of francisella tularensis in voles in finland. vector borne zoonotic dis congestive cardiomyopathy in the woodchuck, marmota monax chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection capillaria hepatica in wild norway rats (rattus norvegicus) from vancouver survey of cardiovascular pathology in wild urban rattus norvegicus and rattus rattus respiratory pathology and pathogens in wild urban rats (rattus norvegicus and rattus rattus) hamster polyomavirus infection in a pet syrian hamster (mesocricetus auratus) causes of mortality and pathological lesions observed postmortem in red squirrels (sciurus vulgaris) in great britain pathologic findings in western gray squirrels (sciurus griseus) from a notoedric mange epidemic in san bernardino moutnains, california contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients the laboratory rabbit, guinea pig, hamster, and other rodents neoplasia and granulomas surrounding microchip transponders in damaraland mole rats (cryptomys damarensis) four cases of spontaneous neoplasia in the naked mole-rat (heterocephalus glaber), a putative cancer-resistant species an epizootic of fibromatosis in gray squirrels (sciurus carolinensis) in florida parapoxvirus causes a deleterious disease in red squirrels associated with uk population declines listeriosis in seven bushy-tailed jirds spontaneous reproductive pathology in female guinea pigs lung morphology in rodents (mammalia rodentia) and its implication for systematics working underground: respiratory adaptations in the blind mole rat fibroma virus infection in an eastern fox squirrel (sciurus niger) from sangamon county illinois fowler's zoo and wild animal medicine chirodiscoides caviae skin none guinea pigs morrisey ( ) demodex skin alopecia, dermatitis, pruritis gerbil, hamster morrisey ( ) gliricola porcelli skin alopecia, crusting dermatitis, pruritis guinea pigs morrisey ( ) gyropus ovalis skin alopecia, crusting dermatitis, pruritis guinea pigs morrisey ( ) myobia musculi skin alopecia, pruritis, selfmutilation mice myocoptes musculinis skin alopecia, pruritis, selfmutilation mice morrisey ( ) notoedres sp. skin alopecia, crusting dermatitishamster, grey squirrel, ratsanholt ( ), , morrisey ( ) , ornithonyssus bacoti skin anemia rats morrisey ( ) polyplax serrata skin anemia, pruritis, dermatitis mice morrisey ( ) polyplax spinulosa skin anemia, pruritis rats morrisey ( ) psoregates simplex skin follicular nodules mice morrisey ( ) radfordia affinis skin alopecia, pruritis, selfmutilation mice morrisey ( ) trixacarus caviae skin alopecia, crusting dermatitis guinea pigs morrisey ( ) key: cord- -rv j authors: boes, katie m.; durham, amy c. title: bone marrow, blood cells, and the lymphoid/lymphatic system date: - - journal: pathologic basis of veterinary disease doi: . /b - - - - . - sha: doc_id: cord_uid: rv j nan within the marrow spaces, a network of stromal cells and extracellular matrix provides metabolic and structural support to hematopoietic cells. these stromal cells consist of adipocytes and specialized fibroblasts, called reticular cells. the latter provides structural support by producing a fine network of a type of collagen, called reticulin, and by extending long cytoplasmic processes around other cells and structures. both reticulin and cytoplasmic processes are not normally visible with light microscopy but are visible with silver reticulin stains (e.g., gordon and sweet's and sometimes with periodic acid-schiff). bone marrow is highly vascularized but does not have lymphatic drainage. marrow of long bones receives part of its blood supply from the nutrient artery, which enters the bone via the nutrient canal at midshaft. the remaining arterial supply enters the marrow through an anastomosing array of vessels that arise from the periosteal arteries and penetrate the cortical bone. vessels from the nutrient and periosteal arteries converge and form an interweaving network of venous sinusoids that permeates the marrow. these sinusoids not only deliver nutrients and remove cellular waste but also act as the entry point for hematopoietic cells into blood circulation. sinusoidal endothelial cells function as a barrier and regulate traffic of chemicals and particles between the intravascular and extravascular spaces. venous drainage parallels that of the nutrient artery and its extensions. • bleeding time (template bleeding time, buccal mucosal bleeding time). this assay assesses primary hemostasis (platelet plug formation) by measuring the time interval between inflicting of standardized wound and cessation of bleeding. sedation may be required. in small animals the test is usually performed on the buccal mucosa; in large animals it may be performed on the distal limb. prolonged bleeding time may be because of a platelet function defect, von willebrand disease, or a vascular defect. the sensitivity of this test is low; reference intervals are species and site dependent (can perform test on a normal animal as a control). this test is contraindicated in cases of thrombocytopenia because significant thrombocytopenia can cause a prolonged bleeding time (invalidates interpretation of test results). • clot retraction test. this assay assesses retraction of a clot, in which platelets play an essential role. this is a crude test that is rarely performed. different protocols are described. significant thrombocytopenia invalidates interpretation of test results. • tests to characterize platelet function abnormalities more specifically are available through specialized laboratories. • aggregometry-to assess platelet aggregation in response to different physiologic agonists. • adhesion assays-to assess the ability of platelets to adhere to a substrate (e.g., collagen). • flow cytometry-to assay for expression of surface molecules. • pfa- -an instrument that simulates a damaged blood vessel, by measuring time for a platelet plug to occlude an aperture; to date, this instrument has mainly been used in research applications. • thromboelastography (teg)-global assessment of hemostasis (platelets, coagulation, and fibrinolysis) based on viscoelastic analysis of whole blood. • tests for immune-mediated thrombocytopenia (imt). • flow cytometry-to detect immunoglobulin bound to the platelet surface, using a fluorescent-labeled antibody. • bone marrow immunofluorescent antibody (ifa) test-to detect bound immunoglobulin. sometimes referred to as the "antimegakaryocyte antibody test," this assay actually detects the presence of immunoglobulin nonspecifically: a smear of a bone marrow aspirate is incubated with a fluorescent-labeled antibody to species-specific immunoglobulin. other components of the marrow include myelinated and nonmyelinated nerves, as well as low numbers of resident macrophages, lymphocytes, and plasma cells. of note, the macrophages play an important role in iron storage and erythrocyte maturation. the following basic concepts provide a framework for understanding the mechanisms of injury and diseases presented later in the chapter. • hematopoietic tissue is highly proliferative. billions of cells per kilogram of body weight are produced each day. • pluripotent hematopoietic stem cells are a self-renewing population, giving rise to cells with committed differentiation programs, and are common ancestors of all blood cells. the process of hematopoietic differentiation is shown in fig. - . • hematopoietic cells undergo sequential divisions as they develop, so there are progressively higher numbers of cells as they mature. cells also continue to mature after they have stopped dividing. conceptually, it is helpful to consider cells in the bone marrow as belonging to mitotic and postmitotic compartments. examples of developing hematopoietic cells are shown in fig. - . • mature cells released into the blood circulation have different normal life spans, varying from hours (neutrophils), to days (platelets), to months (erythrocytes), and to years (some lymphocytes). • the hematopoietic system is under exquisite local and systemic control and responds rapidly and predictably to various stimuli. • production and turnover of blood cells are balanced so that numbers are maintained within normal ranges (steady-state kinetics) in healthy individuals. • normally the bone marrow releases mostly mature cell types (and very low numbers of cells that are almost fully mature) into the circulation. in response to certain physiologic or pathologic stimuli, however, the bone marrow releases immature cells that are further back in the supply "pipeline." the composition of the marrow changes with age. the general pattern is that hematopoietic tissue (red marrow) regresses and is replaced with nonhematopoietic tissue, mainly fat (yellow marrow). thus in newborns and very young animals the bone marrow consists largely of hematopoietically active tissue, with relatively little fat, whereas in geriatric individuals the marrow consists largely of fat. in adults, hematopoiesis occurs primarily in the pelvis, sternum, ribs, vertebrae, and the proximal ends of humeri and femora. even within these areas of active hematopoiesis, fat may constitute a significant proportion of the marrow volume. immature hematopoietic cells can be divided into three stages: stem cells, progenitor cells, and precursor cells. hematopoietic stem cells (hscs) have the capacity to self-renew, differentiate into mature cells, and repopulate the bone marrow after it is obliterated. progenitor cells and precursor cells cannot self-renew; with each cell division, they evolve into more differentiated cells. later-stage precursors cannot divide. stem cells and progenitor cells require immunochemical stains for identification, but precursor cells can be identified by their characteristic morphologic features (see fig. - ). control of hematopoiesis is complex, with many redundancies, feedback mechanisms, and pathways that overlap with other physiologic and pathologic processes. many cytokines influence cells of different lineages and stages of differentiation. primary growth factors for primitive cells are interleukin (il) , produced by t lymphocytes, and stem cell factor, produced by monocytes, macrophages, fibroblasts, endothelial cells, and lymphocytes. interleukin is an early lymphoid growth factor. lineage-specific growth factors are discussed in their corresponding sections. hematopoiesis occurs in the interstitium between the venous sinusoids in the so-called hematopoietic spaces. there is a complex functional interplay among hematopoietic cells with the supporting connective tissue cells, extracellular matrix, and soluble factors, which form the hematopoietic microenvironment. behavior of hematopoietic cells is influenced by direct cell-to-cell and cellmatrix interactions and by soluble mediators, such as cytokines and hormones that interact with cells and with matrix proteins. cells localize to specific niches within the hematopoietic microenvironment via adhesion molecules, such as integrins, immunoglobulins, lectins, and other receptors, which recognize ligands on other cells or matrix components. cells also express receptors for soluble molecules such as chemokines (chemoattractant cytokines) and hormones that influence cell trafficking and metabolism. iron is essential to hemoglobin synthesis and function. it is acquired through the diet and is transported to the bone marrow via the iron transport protein, transferrin. central macrophages either store iron as ferritin or hemosiderin, or transfer the iron to erythroid precursors for hemoglobin synthesis. hemosiderin is identifiable in routinely stained marrow preparations as an intracellular brown pigment. however, perls's prussian blue stain is more sensitive and specific for iron detection. the earliest erythroid precursor identifiable by routine light microscopy is the rubriblast, which undergoes maturational division to produce to progeny cells. late-stage erythroid precursors, known as metarubricytes, extrude their nuclei and become inhibiting apoptosis of developing erythroid cells. the stimulus for increased epo production is hypoxia. within the bone marrow, erythroid precursors surround a central macrophage in specialized niches, termed erythroblastic islands . the central macrophage, also known as a nurse cell, anchors the precursors within the island niche, regulates erythroid proliferation and differentiation, transfers iron to the erythroid progenitors for hemoglobin synthesis, and phagocytizes extruded metarubricyte nuclei. although erythroblastic islands occur throughout the marrow, those with more differentiated erythroid cells neighbor sinusoids, whereas nonadjacent islands contain mostly undifferentiated precursors. as erythroid cells mature from a rubriblast to a mature erythrocyte, their nuclei become smaller and more condensed. the nucleus is eventually extruded to form a polychromatophil. erythroid cells also become less basophilic and more eosinophilic as more hemoglobin is produced and as rna-rich organelles are lost during maturation. (hemoglobin stains eosinophilic, and rna stains basophilic with routine romanowsky's stains.) as granulocytes (e.g., neutrophils, eosinophils, and basophils) mature from a myeloblast to their mature forms, their nuclei become dense and segmented. granulocytes acquire their secondary or specific granules during the myelocyte stage and can be morphologically differentiated starting at this stage. neutrophils have neutral-staining secondary granules, eosinophil secondary granules have an affinity for acidic or eosin dyes, and basophil secondary granules have an affinity for basic dyes. monoblasts differentiate into promonocytes with ruffled nuclear boarders and then into monocytes. in most mammals, mature erythrocytes have a biconcave disk shape, called a discocyte. microscopically, these cells are round and eosinophilic with a central area of pallor. however, the central concavity may not be microscopically apparent in species other than the dog. camelids normally have oval erythrocytes, termed ovalocytes or elliptocytes, which facilitate better gas exchange at high altitudes. the erythrocytes of some animals are prone to in vitro shape change, including those of cervids, pigs, and some goat breeds (e.g., angora). erythrocyte size during health depends on the species, breed, and age of the animal. in dogs, some breeds have relatively smaller (e.g., akitas and shibas) or larger (e.g., some poodles) erythrocytes. akitas and shibas also have a high concentration of potassium, unlike erythrocytes in other dogs. juvenile animals may have larger erythrocytes because of the persistence of fetal erythrocytes, which is followed by a period of relatively smaller cells before reaching adult reference intervals. mature mammalian erythrocytes lack nuclei and organelles and are thus incapable of transcription, translation, and oxidative metabolism. however, they do require energy for various functions, including maintenance of shape and deformability, active transport, and prevention of oxidative damage. red blood cells generate this energy entirely through glycolysis (also known as the embden-meyerhof pathway). except in pigs, glucose enters erythrocytes from the plasma through an insulin-independent, integral membrane glucose transporter. within circulation the erythrocyte mean life span varies between species and is related to body weight and metabolic rate: approximately days in horses and cattle, days in dogs, and days in cats. when erythrocytes reach the end of their life span, they are destroyed in a process termed hemolysis. hemolysis may occur within blood vessels (intravascular hemolysis) or by sinusoidal macrophages (extravascular hemolysis). during intravascular hemolysis, erythrocytes release their contents, mostly hemoglobin, directly into blood. however, during extravascular hemolysis, macrophages phagocytize entire erythrocytes, leaving little or no hemoglobin in the blood. normal turnover of erythrocytes occurs mainly by extravascular hemolysis within the spleen, and to a lesser extent in other organs such as the liver and bone marrow. the exact controls are not clear, but factors that likely play a role in physiologic hemolysis include the following: • exposure of membrane components normally sequestered on the inner leaflet of the erythrocyte membrane, particularly phosphatidylserine. reticulocytes, and subsequently mature erythrocytes. the normal transit time from rubriblast to mature erythrocyte is approximately week. reticulocytes start maturing in the bone marrow but finish their maturation in the blood circulation and spleen. horses are an exception in that they do not release reticulocytes into circulation, even in situations of increased demand. unlike mature erythrocytes, which lack organelles, reticulocytes still contain ribosomes and mitochondria, mainly to support completion of hemoglobin synthesis. these remaining organelles impart a bluish-purple cast (polychromasia) to reticulocytes on routine blood smear examination. the resultant cells are termed polychromatophils. because older reticulocytes do not exhibit polychromasia, more sensitive laboratory techniques must be used for accurate reticulocyte quantification. when a blood sample is incubated with new methylene blue stain, the reticulocytes' ribosomal rna precipitates to form irregular, dark aggregates . cats also have a more mature form of reticulocyte, termed punctate reticulocyte, which is stippled when stained with new methylene blue. punctate reticulocytes indicate prior, not active, regeneration and do not appear polychromatophilic on routine blood smear evaluation. storage pool, which consists of a reserve of fully mature neutrophils. the size of the storage pool varies by species; it is large in the dog, but small in ruminants. in homeostasis mostly mature segmented granulocytes are released from the marrow into the blood. the first monocytic precursor identifiable by morphologic features is the monoblast, which develops into promonocytes and subsequently monocytes (see fig. - ). unlike granulocytes, monocytes do not have a marrow storage pool; they immediately enter venous sinusoids upon maturation. after migrating into the tissues, monocytes undergo morphologic and immunophenotypic maturation into macrophages. within blood vessels there are two pools of leukocytes: the circulating pool and the marginating pool. circulating cells are free flowing in blood, whereas marginating cells are temporarily adhered to endothelial cells by selectins. in most healthy mammals there are typically equal numbers of neutrophils in the circulating and marginal pools. however, there are threefold more marginal neutrophils relative to circulating neutrophils in cats. only the circulating leukocyte pool is sampled during phlebotomy. the concentration of myeloid cells in blood depends on the rate of production and release from the bone marrow, the proportions of cells in the circulating and marginating pools, and the rate of migration from the vasculature into tissues. the fate of neutrophils after they leave the bloodstream in normal conditions (i.e., not in the context of inflammation) is poorly understood. they migrate into the gastrointestinal and respiratory tracts, liver, and spleen and may be lost through mucosal surfaces or undergo apoptosis and be phagocytized by macrophages. lymphopoiesis. lymphopoiesis-from lympha (latin, water)refers to the production of new lymphocytes, including b lymphocytes, t lymphocytes, and natural killer (nk) cells. b lymphocytes primarily produce immunoglobulins, also known as antibodies, and are key effectors of humoral immunity. they are distinguished by the presence of an immunoglobulin receptor complex, termed the b lymphocyte receptor. plasma cells are terminally differentiated b lymphocytes that produce abundant immunoglobulin. t lymphocytes, effectors of cell-mediated immunity, possess t lymphocyte receptors that bind antigens prepared by antigen-presenting cells. a component of innate immunity, nk cells kill a variety of infected and tumor cells in the absence of prior exposure or priming. main growth factors for b lymphocytes, t lymphocytes, and nk cells are il- , il- , and il- , respectively. lymphocytes are derived from hscs within the bone marrow. b lymphocyte development occurs in two phases, first in an antigenindependent phase in the bone marrow and ileal peyer's patches (the site of b lymphocyte development in ruminants), then in an antigendependent phase in peripheral lymphoid tissues (such as spleen, lymph nodes, and mucosa-associated lymphoid tissue [malt] ). t lymphocyte progenitors migrate from the bone marrow to the thymus, where they undergo differentiation, selection, and maturation processes before migrating to the peripheral lymphoid tissue as effector cells. unlike granulocytes, which circulate only in blood vessels and migrate unidirectionally into target tissues, lymphocytes travel in both blood and lymphatic vessels and continually circulate between blood, tissues, and lymphatic vessels. also in contrast to nonlymphoid hematopoietic cells, blood lymphocyte concentrations in adult animals are primarily dependent upon extramedullary lymphocyte production and kinetics, and not lymphopoiesis by the marrow. in healthy nonruminant mammals, lymphocytes are the second most numerous blood leukocyte. according to conventional wisdom, • decreased erythrocyte deformability. • binding of immunoglobulin g (igg) and/or complement to erythrocyte membranes. complement binding may be secondary clustering of the membrane anion exchange protein, band . • oxidative damage to erythrocytes. macrophages degrade erythrocytes into reusable components, such as iron and amino acids, and the waste product bilirubin. bilirubin is then exported into circulation, where it is transported to the liver by albumin. the liver conjugates and subsequently excretes bilirubin into bile for elimination from the body. intravascular hemolysis normally occurs at only extremely low levels. hemoglobin is a tetramer that, when released from the erythrocyte into the blood, splits into dimers that bind to a plasma protein called haptoglobin. the hemoglobin-haptoglobin complex is taken up by hepatocytes and macrophages. this is the major pathway for handling free hemoglobin. however, free hemoglobin may also oxidize to form methemoglobin, which dissociates to form metheme and globin. metheme binds to a plasma protein called hemopexin, which is taken up by hepatocytes and macrophages in a similar manner to hemoglobin-haptoglobin complexes. free heme in the reduced form binds to albumin, from which it is taken up in the liver and converted into bilirubin. the concentration of circulating erythrocytes typically decreases postnatally and remains below normal adult levels during the period of rapid body growth. the age at which erythrocyte numbers begin to increase and the age at which adult levels are reached vary among species. in dogs, adult values are usually reached between and months of age; in horses, this occurs at approximately year of age. granulopoiesis is the production of neutrophils, eosinophils, and basophils, whereas monocyte production is termed monocytopoiesis. granulocytic and monocytic cells are sometimes collectively referenced as myeloid cells. however, the term myeloid and the prefix myelo-can be confusing because they have other meanings; they may reference the bone marrow, all nonlymphoid hemic cells (erythrocytes, leukocytes, and megakaryocytes), only granulocytes, or the spinal cord. the main purpose of granulocytes and monocytes is to migrate to sites of tissue inflammation and function in host defense (see chapters and ). briefly, these cells have key immunologic functions, including phagocytosis and microbicidal activity (neutrophils and monocyte-derived macrophages), parasiticidal activity and participation in allergic reactions (eosinophils and basophils), antigen processing and presentation, and cytokine production (macrophages). neutrophils are the predominant leukocyte type in blood of most domestic species. primary stimulators of granulopoiesis and monocytopoiesis are granulocyte-macrophage colony-stimulating factor and il- , il- , and il- (granulocytes and monocytes), granulocyte colonystimulating factor (granulocytes), and macrophage colonystimulating factor (monocytes). in general, these cytokines are produced by various inflammatory cells, with or without contribution from stromal cells. the earliest granulocytic precursor identifiable by routine light microscopy is the myeloblast, which undergoes maturational division over days to produce to progeny cells (see fig. - ). these granulocytic precursors are conceptually divided into those stages that can divide, including myeloblasts, promyelocytes, and myelocytes (proliferation pool), and those that cannot, including metamyelocytes, and band and segmented forms (maturation pool). within the neutrophil maturation pool is a subpool, termed the platelet aggregation and adherence to subendothelial collagen. expansion of surface area and release of granule contents is aided by a network of membrane invaginations known as the open canalicular system. this system is not present in horses, cattle, and camelids. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. mechanisms of bone marrow disease are summarized in box - . hematopoietic cells' response to injury is dependent upon whether the insult is on the marrow or within extramarrow tissues. in general, marrow-directed injury or disturbances result in production of abnormal hematopoietic cells (dysplasia), fewer hematopoietic cells (hypoplasia), or a failure of hematopoietic cell development (aplasia). dysplasia, hypoplasia, and aplasia may be specific for one cell line, such as pure red cell aplasia, or affect multiple lineages, as seen with aplastic anemia. accordingly, decreased blood concentrations of the involved cell types are expected with hypoplasia or aplasia. erythroid, myeloid, and megakaryocytic hypoplasia or aplasia causes nonregenerative anemia, neutropenia, and thrombocytopenia, respectively. bicytopenia is used to describe decreased blood concentrations of two cell lines, whereas pancytopenia indicates decreased blood concentrations of all three cell types. bicytopenia or pancytopenia may indicate generalized marrow disease, such as occurs with aplastic anemia or marrow malignancies (leukemia), necrosis, fibrosis (myelofibrosis), or inflammation (myelitis). replacement of hematopoietic tissue within the bone marrow by abnormal tissue, including neoplastic cells, fibrosis, or inflammatory cells, is termed myelophthisis. cattle normally have higher numbers of lymphocytes than neutrophils in circulation. however, recent studies suggest that is no longer the case, most likely due to changes in genetics and husbandry. in most species the majority of lymphocytes in blood circulation are t lymphocytes. the concentration of blood lymphocytes decreases with age. thrombopoiesis. thrombopoiesis-from thrombos (gr., clot)refers to the production of platelets, which are small ( to µm), round to ovoid, anucleate cells within blood vessels. platelets have a central role in primary hemostasis but also participate in secondary hemostasis (coagulation) and inflammatory pathways (see chapters and ). thrombopoietin (tpo) is the primary regulator of thrombopoiesis. the liver and renal tubular epithelial cells constantly produce tpo, which is then cleared and destroyed by platelets and their precursors. therefore plasma tpo concentration is inversely proportional to platelet and platelet precursor mass. if the platelet mass is decreased, less tpo is cleared, and there is subsequently more free plasma tpo to stimulate thrombopoiesis. the earliest morphologically identifiable platelet precursor is the megakaryoblast, which undergoes nuclear reduplications without cell division, termed endomitosis, to form a megakaryocyte with to nuclei. as the name suggests, megakaryocytes are very large cells, much larger than any other hematopoietic cell ; also see fig. - ). megakaryocytes neighbor venous sinusoids, extend their cytoplasmic processes into vascular lumens, and shed membranebound cytoplasmic fragments (platelets) into blood circulation. orderly platelet shedding is partially facilitated by β -tubulin microtubules within megakaryocytes. platelets circulate in a quiescent form and become activated by binding platelet agonists, including thrombin, adenosine diphosphate (adp), and thromboxane. platelet activation causes shape change, granule release, and relocation of procoagulant phospholipids and glycoproteins (gps) to the outer cell membrane. specific procoagulant actions include release of calcium, von willebrand factor (vwf), factor v, and fibrinogen, as well as providing phosphatidylserine-rich binding sites for the extrinsic tenase (factors iii, vii, and x), intrinsic tenase (factors ix, viii, and x), and prothrombinase (factors x, v, and ii) coagulation complexes. platelet gp surface receptors include those for binding vwf (gpib-ix-v), collagen (gpvi), and fibrinogen (gpiib-iiia), which facilitate increased destruction hemorrhage (especially erythrocytes) consumption (platelets) neoplasia altered distribution abnormal function bone marrow is not routinely sampled during postmortem examinations. however, indications for bone marrow evaluation include suspected leukemia, metastatic neoplasia within bone marrow, or infectious myelitis, as well as cytopenia(s) or hematopoietic dysplasia of unknown cause. multimodal evaluation is ideal, including a recent (< hours) complete blood count with bone marrow cytologic and histopathologic examination. however, antemortem blood analyses are not always available, and interpretation of hematopoietic cytomorphologic examination results becomes difficult to impossible shortly after death. postmortem bone marrow should be collected as soon as possible after death or euthanasia, preferably within minutes. samples may be collected from the proximal femur, rib, sternum or vertebrae. when collecting from the femur, the femoral neck is removed with a bone saw, or a fragment of the shaft is removed with bone-cutting shears. cytologic samples are first collected using the paintbrush technique: gently sample the red marrow with a clean, dry, naturalbristle brush, and then carefully brush the material onto a clean glass microscope slide in two to four parallel wavy lines. the brush should be cleaned and dried before its use on a different animal. the slide is then air dried, stored away from formalin fumes, and then stained with a routine (romanowsky) stain. for histologic evaluation the entire femoral head or femoral shaft or rib fragment with exposed red marrow is submersed in % neutral buffered formalin. for cosmetic necropsies, samples may be obtained by antemortem techniques, such as needle biopsies for cytologic examination and core biopsies for histopathologic examination. the complete blood count (cbc) is the cornerstone for diagnosis of hematologic disturbances and is often part of a minimum database in sick patients. the cbc includes numeric data indicating the concentration of different cell types, as well as other estimations of red blood cell mass (hemoglobin concentration, packed-cell volume, and hematocrit), red blood cell volume (mean cell volume), and red blood cell hemoglobin content (mean cell hemoglobin and mean cell hemoglobin concentration). cell morphologic features and the presence or absence of hemic parasites are assessed upon microscopic review of a blood smear and are also included in a cbc report. (note: some parasites may infect blood cells, such as hepatozoon organisms within circulating neutrophils or monocytes or bartonella organisms within erythrocytes, but mainly cause disease in other body systems and are therefore not discussed in this chapter.) learning to evaluate blood smears is a valuable skill for any practicing veterinarian. the cbc also may include the plasma protein concentration, as measured with a refractometer. it is important to remember that changes in hydration status and in the distribution of body fluids between the vascular and extravascular compartments affect the concentration of both cells and proteins in the blood. other tests that may help with evaluation of the hematopoietic system include cell or tissue biopsies, the direct antiglobulin test, flow cytometry, immunophenotyping, and polymerase chain reaction (pcr). aspiration cytology and/or histopathology of organs other than the bone marrow can be pursued to assess for the presence of emh, increased destruction of erythrocytes, neoplasia, or infection. the coombs test, or direct antiglobulin test, detects excessive antibody or complement bound to red blood cells' surfaces and is the standard assay for immune-mediated hemolytic anemia. flow cytometry and immunofluorescent antibody tests may also be used to detect autoantibody bound to erythrocytes or other hematopoietic cells. immunophenotyping and pcr are further discussed in the section on hematopoietic neoplasia. structural or functional abnormalities of blood vessels, platelets, or coagulation factors may result in a tendency toward hypocoagulability (bleeding), hypercoagulability (inappropriate thrombosis), or both. in veterinary medicine there has been a great deal of work on specific mechanisms of hypocoagulability, whereas mechanisms of hypercoagulability are less fully characterized. disorders of primary hemostasis typically result in "small bleeds" (e.g., petechiation, mild ecchymosis, bleeding from mucous membranes, bleeding immediately after venipuncture), whereas disorders of secondary hemostasis typically result in "big bleeds" (e.g., hemorrhage into body cavities/ joints, marked ecchymosis, large hematomas, delayed bleeding after venipuncture). this chapter concentrates on primary disorders of hemostasis and also covers disseminated intravascular coagulation, which is the secondary condition. however, it is important to note that coagulation disorders can also result from other underlying disease processes. for example, advanced liver disease can lead to abnormal hemostasis through decreased or defective synthesis of coagulation factors or impaired clearance of fibrinolytic products that inhibit coagulation reactions and platelet function. vascular disorders may also result in a bleeding tendency because of abnormalities of endothelial function or collagen-platelet interactions. specific diseases involving abnormal structure or function of hematopoietic or hemostatic elements are discussed later in this chapter. the cbc provides basic information about platelets, including numeric values for platelet concentration and mean platelet volume (mpv), subjective assessment of platelet morphologic features (size, shape, and granularity), and a rough estimation of platelet numbers based on examination of a blood smear. some laboratories measure reticulated platelets (platelets recently released from the bone marrow), although this test is mostly used in the research setting at present. increased mpv and increased numbers of reticulated platelets tend to indicate increased thrombopoiesis. bone marrow examination is indicated with any unexplained cytopenia, including thrombocytopenia, to evaluate production. tests to evaluate the components of the hemostatic process are described and listed in e-appendix - . secondary myelofibrosis is the enhanced deposition of collagen within the marrow by nonneoplastic fibroblasts and reticular cells. disease pathogenesis is unclear, but there are two leading theories. first, it may represent scar formation after marrow necrosis, as previously presented. and second, high concentrations of growth factors present during times of marrow injury or activation may stimulate fibroblast proliferation. in particular, stimulated megakaryocytes and macrophages produce fibrogenic cytokines, including plateletderived growth factor, transforming growth factor-β, and epidermal growth factor. early in disease there is reticulin deposition without reduction of hematopoietic elements. however, fibrous collagen replaces hematopoietic cells with disease progression. histologic identification of reticulin and collagen fibers can be aided with reticulin silver and masson's trichrome stains, respectively. in animals, secondary myelofibrosis occurs most commonly with leukemias, extramarrow malignancies, and chronic hemolytic anemias, but many cases are idiopathic. experimental whole-body gamma irradiation, dietary strontium- exposure, and certain drugs and toxins can also induce myelofibrosis. the responses of marrow adipocytes to systemic and localized disease are under current investigation, especially in relation to energy metabolism, inflammation, and bone trauma. during times of severe energy imbalance, such as cachexia, the marrow may undergo serous atrophy of fat, also known as gelatinous marrow transformation (e- fig. - ). the pathogenesis of this phenomenon is unknown, but it is characterized by adipocyte atrophy, hematopoietic cell hypoplasia with subsequent cytopenias, and replacement of the marrow with extracellular hyaluronic acid-rich mucopolysaccharides. positive alcian blue staining identifies the extracellular material as mucin. marrow adipocytes secrete adipose-derived hormones, termed adipokines, including leptin and adiponectin. in general, leptin is proinflammatory, prothrombotic, and mitogenic for various cell types, including lymphocytes, hematopoietic progenitors, and leukemic cells. conversely, adiponectin has antiinflammatory and growth inhibitory properties. during times of inflammation and infection, leptin production is increased. in response to marrow trauma, such as orthopedic surgery, fat may enter the vasculature, embolize to various tissues, and cause tissue ischemia. the severity of tissue injury caused by fat embolism is dependent upon the quantity of fat entering circulation and the tissue's susceptibility to ischemia (see chapter ). responses of circulating blood cells to injury include decreased survival (destruction, consumption, or loss), altered distribution, and altered structure or function (see box - ). these responses are not mutually exclusive-for example, altered erythrocyte structure may lead to decreased survival. often, but not always, these responses result in decreased concentrations of blood cells in circulation. abnormal concentrations of blood cells. the concentration of blood cells may be decreased, termed cytopenia (from kytos [gr., hollow vessel] and penia [gr., poverty]) or increased, designated cytosis (from osis [gr., condition]). a specific blood cell type is denoted as being decreased by using the suffix -penia (table - ) . a decreased concentration of erythrocytes is the exception and is termed anemia (from a [gr., without] and haima [gr., blood]). decreased concentrations of blood basophils are not recognized in domestic animals because the lower reference interval is typically zero. an increased blood cell type is denoted with the suffix -osis or -philia (see table - ). postmortem quantification of blood cell insults to extramarrow tissues and cells tend to cause increased production of the involved cell types (hyperplasia) with or without dysplasia. loss of erythrocytes from blood vessels (hemorrhage), or premature destruction of erythrocytes (hemolysis) causes erythroid hyperplasia. tissue inflammation may cause neutrophilic, eosinophilic, basophilic, and/or monocytic hyperplasia, depending on the type of inflammation. megakaryocytic hyperplasia may occur with increased platelet use during hemorrhage or disseminated intravascular coagulation (dic) or with immune-mediated platelet destruction. exceptions to these generalizations, such as anemia of chronic disease, iron deficiency anemia, and anemia of renal failure, are discussed in more detail later. endothelial cell response to injury specifically within the marrow is poorly characterized, but it is likely similar to that of endothelial cells elsewhere, playing active roles in coagulation and inflammation (see chapters and ). however, one potential sign of marrow sinusoidal injury is the presence of circulating nucleated erythrocytes in the absence of erythrocyte regeneration, termed inappropriate metarubricytosis. it is proposed that injured marrow endothelial cells allow premature passage of metarubricytes into blood circulation during times of stress. however, a conflicting theory proposes that marrow stress causes decreased metarubricyte attachment to central macrophages, and subsequent release into circulation. specific causes of marrow injury-induced metarubricytosis include sepsis, hyperthermia, malignancies, hypoxia, and certain drugs and toxins. inappropriate metarubricytosis may also occur with erythroid dysplasia and splenic disorders. in addition to a suspected role in inappropriate metarubricytosis, marrow macrophages are integral to altered iron metabolism, including anemia of chronic disease and hemosiderosis. anemia of chronic disease is a mild to moderate nonregenerative anemia observed in animals with a variety of inflammatory and metabolic disorders. this anemia is discussed in more detail later, but briefly, it is primarily a result of iron sequestration within macrophages. hemosiderosis is the excessive accumulation of iron in tissues, typically macrophages. accumulation of iron in parenchymal organs, leading to organ toxicity, is termed hemochromatosis. in animals, iron overload due to blood transfusions or chronic hemolytic anemias may cause marrow hemosiderosis and hemochromatosis. myelitis can take different forms. granulomatous myelitis occurs with systemic fungal infections (e.g., histoplasmosis) or mycobacteriosis. acute or neutrophilic myelitis may occur with lower-order bacterial infections or those with an immune-mediated component. dogs and cats with nonregenerative immune-mediated hemolytic anemia (imha) often have myelitis, in addition to myelofibrosis and necrosis. the inflammation is evident as fibrin deposition, edema, and multifocal neutrophilic infiltrates; immune-mediated cytopenias may also concurrently occur with bone marrow lymphocytic and/or plasma cell hyperplasia. bone marrow necrosis is the necrosis of medullary hematopoietic cells, stromal cells, and stroma in large areas of bone marrow. potential causes include leukemias, extramarrow malignancies, infection (bovine viral diarrhea virus [bvdv] , ehrlichia canis, and feline leukemia virus [felv]), sepsis, drugs or toxins (carprofen, chemotherapeutic agents, estrogen, metronidazole, mitotane, and phenobarbital), and irradiation. direct hematopoietic or stromal cytotoxicity and altered marrow microvasculature (disseminated intravascular coagulation) are proposed pathogeneses. extensive marrow necrosis results in decreased hematopoiesis and subsequent blood cytopenias, including anemia, neutropenia, and thrombocytopenia. if the animal survives the initial insult, the marrow may recover and resume normal hematopoiesis, or it may undergo scar formation, termed myelofibrosis. concentrations is not possible due to perimortem coagulation. however, a complete blood count (cbc) with microscopic blood smear evaluation is the foundation for antemortem assessment of blood cells. anemia. anemia causes clinical signs referable to decreased red hemoglobin pigment (e.g., pale mucous membranes), decreased oxygen-carrying capacity (e.g., depression, lethargy, weakness, and exercise tolerance), and decreased blood viscosity (e.g., heart murmur). recumbency, seizures, syncope, or coma may occur with severe anemia. anemia is confirmed by identifying a decreased hemoglobin concentration or reduced erythrocyte mass, as measured by the packed-cell volume, hematocrit, or red blood cell concentration. the three general causes of anemia are blood loss (hemorrhage), red blood cell destruction or lysis (hemolysis), and decreased red blood cell production (erythroid hypoplasia). classifying anemia as regenerative or nonregenerative is clinically useful because it provides information about the mechanism of disease; regenerative anemia indicates hemorrhage or hemolysis, whereas erythroid hypoplasia or aplasia causes nonregenerative anemia (table - ). the hallmark of regenerative anemias, except in horses, is reticulocytosis (i.e., increased numbers of circulating reticulocytes [immature erythrocytes]), which is evident as polychromasia on a routinely stained blood smear (see fig. - ). reticulocytosis indicates increased bone marrow erythropoiesis ( fig. - ) and release of erythrocytes before they are fully mature. reticulocytosis is an appropriate marrow response to anemia and is often seen with hemorrhage or hemolysis. on a cbc a strong regenerative response may produce an increased mean cell volume (mcv) and decreased mean cell hemoglobin concentration (mchc) because reticulocytes are larger and have a lower hemoglobin concentration than mature erythrocytes. horses are an exception to this classification scheme because they do not release reticulocytes into circulation, even with erythroid hyperplasia. horses with a regenerative response may have an increased mcv and red cell distribution width (an index of variation in cell size). but definitive determination of regeneration in a horse requires demonstration of erythroid hyperplasia via bone marrow examination or an increasing red cell mass over sequential cbcs. in addition to reticulocytosis there may be increased numbers of nucleated red blood cells (nrbcs) in circulation with erythrocyte regeneration, termed appropriate metarubricytosis. when nrbcs are present as part of a regenerative response, they should be in low numbers relative to the numbers of reticulocytes. however, the presence of circulating nrbcs is not in itself definitive evidence of regeneration and may signify dyserythropoiesis (e.g., lead poisoning or bone marrow disease) or splenic dysfunction. these processes should be suspected when nrbcs are increased without reticulocy- such as into the peritoneal cavity, because iron is not lost from the body and can be reused for erythropoiesis. in hemolytic anemia, erythrocytes are destroyed at an increased rate. whether the mechanism is intravascular or extravascular, or a combination, depends on the specific disease process (specific diseases are discussed later in this chapter). some clinical indicators of hemolytic anemia and their pathogeneses are summarized in fig. - and are further described in the following discussion. a classic sequela of hemolytic anemias in general is hyperbilirubinemia, which is an increase in the plasma bilirubin concentration. bilirubin is a yellow pigment, which explains why hyperbilirubinemia, if severe enough, causes icterus-the grossly visible yellowing of fluid or tissues ( fig. - ) . icterus, also known as jaundice, is usually detectable when the plasma bilirubin concentration exceeds mg/dl. however, it is important to note that hyperbilirubinemia and icterus are not pathognomonic for hemolysis and may also occur with conditions of impaired bile flow (cholestasis), such as hepatopathy or cholangiopathy. in addition to icterus, hemolytic anemia often results in splenomegaly , which is secondary to extravascular hemolysis and macrophagic hyperplasia within the spleen, as well as splenic emh. splenomegaly may also occur in other conditions, as discussed elsewhere in this chapter. intravascular hemolysis is grossly evident as pink-tinged plasma or serum, termed hemolysis or hemoglobinemia. hemolysis is not apparent until the concentration of extracellular hemoglobin is greater than approximately mg/dl. cell-free hemoglobin is scavenged by haptoglobin until haptoglobin becomes saturated with hemoglobin at a concentration of approximately mg/dl. when haptoglobin is saturated, any remaining free hemoglobin has a low enough molecular weight to pass through the renal glomerular filter into the urine. this imparts a pink or red discoloration to the urine, called hemoglobinuria. thus extracellular hemoglobin can cause gross discoloration of the plasma, where it is bound to haptoglobin, before becoming grossly visible in urine. the half-life of haptoglobin is markedly decreased when bound to hemoglobin, so when large amounts of haptoglobin-hemoglobin complex are formed, the concentration of haptoglobin in the blood decreases and hemoglobin can pass through the glomerulus at even lower concentrations. hemoglobinuria is a contributing factor in the renal tubular necrosis (hemoglobinuric nephrosis) that often occurs in cases of acute intravascular hemolysis (see chapter ). a similar lesion occurs in the kidneys of individuals with marked muscle damage and resulting myoglobinuria (see chapters and ). hemoglobinuria cannot be distinguished grossly from hematuria (erythrocytes in the urine) or myoglobinuria (myoglobin in the urine), and all three processes cause a positive reaction for "blood protein" on urine test strips. comparing the colors of the plasma and the urine may be informative. in contrast to hemoglobin, myoglobin causes gross discoloration of the urine before the plasma is discolored. this is because myoglobin is a low-molecular-weight monomer, freely filtered by the glomerulus, and does not bind plasma proteins to a significant degree. hematuria can be distinguished from hemoglobinuria on the basis of microscopic examination of urine sediment (i.e., erythrocytes are present in cases of hematuria). in addition to red plasma and urine, hemoglobinemia may also be identified by increased mch or mchc values on a cbc. this is because the hemoglobin concentration is measured by lysing all erythrocytes in the sample and then measuring the total hemoglobin via spectrophotometry. by this method, hemoglobin that originated within or outside of erythrocytes is measured together. however, calculations for mch and mchc, which include results for the hemoglobin and red blood cell concentrations, assume that all of tosis, or their numbers are high relative to the degree of reticulocytosis, termed inappropriate metarubricytosis. in ruminants, reticulocytosis is often accompanied by basophilic stippling (fig. - ) . however, like metarubricytosis, basophilic stippling without reticulocytosis is concerning for lead poisoning or other causes of dyserythropoiesis. recall that the stimulus for increased erythropoiesis is increased secretion of epo in response to tissue hypoxia. although the action of epo on erythropoiesis is rapid, evidence of a regenerative response is not immediately apparent in a blood sample. one of the main effects of epo is to expand the pool of early-stage erythroid precursors, and it takes time for these cells to differentiate to the point where they are released into circulation. in a case of acute hemorrhage or hemolysis, for example, it typically takes to days until reticulocytosis is evident on the cbc and several more days until the regenerative response peaks. the term preregenerative anemia is sometimes used to describe anemia with a regenerative response that is impending but not yet apparent on the cbc. confirming a regenerative response in such cases requires either evidence of erythroid hyperplasia in the bone marrow or emergence of a reticulocytosis on subsequent days. hemorrhage results in escape of erythrocytes and other blood components, such as protein, from the vasculature. as a result, a decreased plasma or serum protein concentration, termed hypoproteinemia, may be evident on a cbc or chemistry panel. if the hemorrhage is into the gastrointestinal lumen, some of the protein may be resorbed and converted to urea, resulting in an increased urea nitrogen concentration relative to creatinine in plasma. hemorrhage within the urinary tract may cause red urine with erythrocytes observed in the urine sediment. causes of hemorrhage include trauma, abnormal hemostasis, certain parasitisms, ulceration, and neoplasia. hemorrhage may be acute or chronic, or internal or external. during acute hemorrhage, there are ample iron stores within the body for hemoglobin synthesis and erythrocyte regeneration. however, with chronic external hemorrhage, continued loss of iron may deplete the body's iron stores. as iron stores diminish, so does erythrocyte regeneration, eventually leading to iron deficiency anemia. iron deficiency anemia is either poorly regenerative or nonregenerative and is discussed in more detail later in the chapter. iron deficiency anemia does not occur with chronic internal hemorrhage, spherocytes form when macrophages (mainly in the spleen) phagocytize part of an erythrocyte plasma membrane bound with autoantibody ( fig. - ) . the remaining portion of the erythrocyte assumes a spherical shape, thus preserving maximal volume. this change in shape results in decreased deformability of the cells. erythrocytes must be extremely pliable to traverse the splenic red pulp and sinusoidal walls; spherocytes therefore tend to be retained in the spleen in close association with macrophages with risk for further injury and eventual destruction. in the dog, spherocytes appear smaller than normal and have uniform staining ( fig. - , a), in contrast to normal erythrocytes, which have a region of central pallor imparted by their biconcave shape. this difference in staining between spherocytes and normal erythrocytes is not consistently discernible in many other domestic animals (including horses, the hemoglobin originated within erythrocytes. in the case of hemoglobinemia, the excess extracellular hemoglobin may cause an artifactual increase in the calculated mch and mchc. it is important to remember that similar artifactual increases may also occur with lipemia. once hemolytic anemia has been identified, the specific cause for hemolysis should be investigated based on signalment, clinical history, and microscopic blood smear evaluation. the most common causes of hemolytic anemia in domestic animals are immunemediated, infectious, oxidative, and mechanical fragmentation (i.e., microangiopathic) disorders (table - ) . spherocytosis and autoagglutination are hallmarks of immunemediated hemolytic anemia, either primary (also known as idiopathic) or secondary to infectious disease, drugs/toxins, or neoplasms. several initiating processes can cause intravascular hemolysis; formation of the complement membrane attack complex is pictured. with intravascular hemolysis, free hemoglobin is release directly into the plasma, where it is scavenged by haptoglobin and hemopexin. when haptoglobin and hemopexin are saturated, the cell-free hemoglobin causes red discoloration of the plasma (hemolysis) and is excreted in the urine (hemoglobinuria; dark red urine). the liver clears haptoglobinhemoglobin and hemopexin-methemoglobin complexes from plasma and converts hemoglobin to unconjugated bilirubin and then conjugated bilirubin. conjugated bilirubin is normally excreted in the bile and then converted to urobilinogen (yellow) and subsequently stercobilinogen (dark brown). however, excessive bilirubin will spill over into the plasma, resulting in hyperbilirubinemia, icteric plasma (if severe enough), and urinary excretion of bilirubin (bilirubinuria; icteric urine). extravascular hemolysis: during extravascular hemolysis, erythrocytes are phagocytized by macrophages, which digest erythrocytes, and convert hemoglobin to unconjugated bilirubin. excessive bilirubin in plasma causes hyperbilirubinemia with or without icteric plasma. unconjugated bilirubin is processed and excreted by the liver (as previously described) and in dogs, the kidney. kidney u-bilirubin cattle, and cats), whose erythrocytes differ from those of the dog in that they are smaller and have less pronounced biconcavity and therefore less pronounced central pallor. autoagglutination occurs because of cross-linking of antibodies bound to erythrocytes (see . autoagglutination is evident macroscopically as blood with a grainy consistency (see fig. - , b) , and microscopically as clusters of erythrocytes (see fig. - , c). autoagglutination may also result in a falsely increased mcv and decreased red blood cell concentration when clustered cells are mistakenly counted as single cells by automated hematology analyzers. when autoagglutination is present, the packed-cell volume is the most reliable measurement of red blood cell mass. ghost cells are ruptured red blood cell membranes devoid of cytoplasmic contents (see a) . they indicate intravascular hemolysis and may be seen with a variety of hemolytic disorders, including those with immune-mediated, infectious, oxidative, or fragmentation causes. in the case of immune-mediated hemolytic anemia, antibody or complement binds to red blood cell membranes and activates the complement membrane attack complex (see fig. - ). this causes pore formation in the red blood cell membrane and release of cytoplasmic contents into the plasma. ghost cells are eventually cleared from circulation by phagocytic macrophages, mainly within the spleen. oxidative damage to erythrocytes occurs when normal antioxidative pathways that generate reducing agents (such as reduced nicotinamide adenine dinucleotide [nadh] , reduced nicotinamide adenine dinucleotide phosphate [nadph] , and reduced glutathione [gsh]) are compromised or overwhelmed, resulting in hemolytic anemia, abnormal hemoglobin function, or both. hemolysis caused by oxidative damage may be extravascular or intravascular, or a combination. evidence of oxidative damage to erythrocytes may be apparent on blood smear examination as heinz bodies or eccentrocytes or on gross examination as methemoglobinemia. heinz bodies are foci of denatured globin that interact with the erythrocyte membrane. they are usually subtly evident on routine wright-stained blood smears as pale circular inclusions or blunt, rounded protrusions of the cell margin but are readily discernible on smears stained with new methylene blue . cats are particularly susceptible to heinz body formation and may have low numbers of heinz bodies normally. there is no unanimity of opinion, but some clinical pathologists believe that the presence of heinz bodies in up to % of all erythrocytes in cats is within normal limits. this predisposition is believed to reflect unique features of the feline erythrocyte, whose hemoglobin has more sulfhydryl groups (preferential sites for oxidative damage) than do erythrocytes of other species and may also have lower intrinsic reducing capacity. it is also possible that the feline spleen does not have as efficient a "pitting" function (splenic structure and function are discussed in more detail later in this chapter). eccentrocytes, evident as erythrocytes in which one side of the cell has increased pallor ( fig. - , a) , are another manifestation of oxidative damage. they form because of cross-linking of total hemoglobin), methemoglobin imparts a grossly discernible chocolate color to the blood. by itself, mechanical fragmentation hemolysis tends to cause mild or no anemia. mechanical fragmentation results from trauma or shearing of erythrocytes within blood vessels. normal erythrocytes may be flowing through abnormal vasculature, such as with heart valve defects, intravascular fibrin deposition (e.g., disseminated intravascular coagulation), vasculitis, or hemangiosarcoma. alternatively, the red blood cells may be particularly fragile within normal blood vasculature, as occurs with iron deficiency. in either instance, microscopic evidence of mechanical fragmentation includes the presence of erythrocyte fragments (schistocytes [see fig. - , c]), erythrocytes with irregular cytoplasmic projections (acanthocytes), erythrocytes with blister-like projections (keratocytes), or ghost cells (see figs. - , a, - , d, and - , e). membrane proteins, with adhesion of opposing areas of the cell's inner membrane leaflet, and displacement of most of the hemoglobin toward the other side. the fused membranes may fragment off of the eccentrocyte, leaving a slightly ruffled border; this cellular morphologic abnormality is called a pyknocyte (see fig. - , b) . oxidative insult may also result in conversion of hemoglobin (iron in the fe + state) to methemoglobin (iron in the fe + state), which is incapable of binding oxygen. methemoglobin is produced normally in small amounts but reduced back to oxyhemoglobin by the enzyme cytochrome-b reductase (also known as methemoglobin reductase). methemoglobinemia results when methemoglobin is produced in excessive amounts (because of oxidative insult) or when the normal pathways for maintaining hemoglobin in the fe + state are impaired (as in cytochrome-b reductase deficiency). when present in sufficiently high concentration (approximately % of degradation. antierythrocyte antibodies bind rbc surface antigens, resulting in rbc opsonization by immunoglobulins (mainly immunoglobulin g [igg] ) and complement (primarily c b). immunoglobulin-or c b-bound rbcs are phagocytized and digested by sinusoidal macrophages. , spherocytes. spherocytes form when the membrane of immunoglobulin-or c b-bound rbcs are phagocytized by macrophages, without removing the entire rbc from circulation. compared to normal erythrocytes, spherocytes appear smaller, more eosinophilic, and lack central pallor. , rbc aggregation (agglutination). rbc aggregation occurs when antierythrocyte immunoglobulins (immunoglobulin m [igm] or high concentrations of igg) bind multiple erythrocytes simultaneously. , ghost cells. antierythrocyte antibodies bind rbc surface antigens, resulting in complement activation and formation of the membrane attack complex (mac). macs form membrane pores, resulting in rupture of rbcs, and the release of hemoglobin into the circulation. ghost cells are rbc membrane remnants that lack cytoplasm (hemoglobin normocytic, normochromic anemia). it has long been known that patients with inflammatory or other chronic disease often become anemic, and that this condition results in increased iron stores in the bone marrow. sequestration of iron may be a bacteriostatic evolutionary adaptation because many bacteria require iron as a cofactor for growth. in recent years, investigators have begun to elucidate the molecular mechanisms underlying anemia of inflammation. hepcidin, an acute phase protein and antimicrobial peptide synthesized in the liver, is a key mediator that limits iron availability. hepcidin expression increases with inflammation, infection, or iron overload and decreases with anemia or hypoxia. hepcidin exerts its effects by causing functional iron deficiency. it binds to and causes the degradation of the cell surface iron efflux molecule, ferroportin, thus inhibiting both absorption of dietary iron from the intestinal epithelium and export of iron from macrophages and hepatocytes into the plasma ( fig. - ). anemia of inflammation involves factors besides decreased iron availability. inflammatory cytokines are likely to inhibit erythropoiesis by oxidative damage to and triggering apoptosis of developing erythroid cells, by decreasing expression of epo and stem cell factor, and by decreasing expression of epo receptors. in addition, experimentally induced sterile inflammation in cats resulted in shortened erythrocyte survival, indicating that anemia of inflammation is likely also a function of increased erythrocyte destruction. other causes of decreased erythropoiesis are listed in table - . specific examples of diseases causing nonregenerative anemia by these mechanisms are discussed later in this chapter. neutropenia. neutropenia refers to a decrease in the concentration of neutrophils in circulating blood. neutropenia may be caused by decreased production, increased destruction, altered distribution, or a demand for neutrophils in tissues that exceeds the rate of granulopoiesis. decreased production is evident on bone marrow examination as granulocytic hypoplasia. this usually results from an insult that affects multiple hematopoietic lineages, such as chemical insult, radiation, neoplasia, infection, or fibrosis, but may also be caused by a process that preferentially targets granulopoiesis. in marked contrast to erythrocytes, neutrophils have a very short life span in circulation. once released from the bone marrow, a neutrophil is in the bloodstream only for hours before migrating into the tissues. when neutrophil production ceases, a reserve of mature neutrophils in the bone marrow storage pool may be adequate to maintain normal numbers of circulating neutrophils for a few days; however, after the bone marrow storage pool is depleted, neutropenia rapidly ensues. immune-mediated neutropenia is a rare but recognized condition in domestic animals. bone marrow findings range from granulocytic hypoplasia to hyperplasia, depending on where the cells under immune attack are in their differentiation programs. neutropenia with no evidence of decreased production and in which other causes of neutropenia have been excluded may be a result of destruction of neutrophils before they leave the bone marrow, a condition known as ineffective granulopoiesis. like other forms of ineffective hematopoiesis, this condition is often presumed to be immune mediated; in cats this condition may occur as a result of infection of hematopoietic cells with felv. as presented in the earlier section on granulopoiesis and monocytopoiesis (myelopoiesis), neutrophils within the blood vasculature are in two compartments: a circulating pool, consisting of those cells flowing freely in the blood, and a marginating pool, consisting of those cells transiently interacting with the endothelial surface. (in reality, neutrophils are constantly shifting between these two pools, but the proportion of cells in either pool normally remains fairly schistocytes are the only red blood cell morphologic abnormality specific for mechanical fragmentation because all other morphologic abnormalities can be seen with other disease processes. for example, ghost cells may be observed with other types of hemolysis. nonregenerative anemia is characterized by a lack of reticulocytosis on the cbc; however, reticulocytosis does not occur in horses even in the context of regeneration. most often this is a result of decreased production in the marrow (i.e., erythroid hypoplasia). erythrocytes circulate for a long time, so anemias caused by decreased production tend to develop slowly. the most common form of nonregenerative anemia is known as anemia of inflammation or anemia of chronic disease. in this form of anemia, erythrocytes are decreased in number but are typically normal in size and hemoglobin concentration (so-called c or because of one specifically depressing thrombopoiesis. in either case, decreased thrombopoiesis is evident as megakaryocytic hypoplasia upon bone marrow examination. general causes of decreased hematopoiesis outlined earlier in the sections on anemia and neutropenia also apply to thrombocytopenia. increased platelet destruction due to immune-mediated thrombocytopenia (imtp) is a fairly common disease in dogs and may also occur in other species. thrombocytopenia with immune-mediated thrombocytopenia is often severe (e.g., < , platelets/µl), resulting in spontaneous multisystemic hemorrhage. increased use of platelets occurs with hemorrhage and disseminated intravascular coagulation. thrombocytopenia secondary to hemorrhage is often mild to moderate, whereas disseminated intravascular coagulation may cause mild to severe thrombocytopenia, often with evidence of mechanical fragmentation hemolysis (e.g., schistocytes). disseminated intravascular coagulation is a syndrome in which hypercoagulability leads to increased consumption of both platelets and coagulation factors in the plasma, with subsequent hypocoagulability and susceptibility to bleeding. risk factors for developing disseminated intravascular coagulation include severe inflammation, such as sepsis or pancreatitis, neoplasia, and organ failure. the spleen normally contains a significant proportion of total platelet mass (up to one-third in some species), and abnormalities involving the spleen may result in changes in the number of circulating platelets. for example, splenic congestion may result in platelet sequestration and thrombocytopenia, and splenic contraction may cause thrombocytosis. lymphopenia. lymphopenia refers to a decreased concentration of lymphocytes in blood. it is a common hematologic finding in sick animals. usually the precise mechanism of lymphopenia is not clear but is often presumed secondary to endogenous glucocorticoid excess that occurs with stress. excess glucocorticoids, either endogenous or exogenous, cause an altered distribution of lymphocytes; there is increased trafficking of lymphocytes from blood to lymphoid tissue, and decreased egress of lymphocytes from lymphoid tissue to blood. at higher concentrations of glucocorticoids, lymphocytes are destroyed. other causes of lymphotoxicity include chemotherapeutic agents, radiation therapy, and some infectious agents. lymphopenia may occur with various mechanisms, including loss of lymphocyte-rich lymphatic fluid (e.g., gastrointestinal disease, repeated drainage of chylous effusions), and disruption of the normal lymphoid tissue architecture because constant in any given species.) circulating neutrophils are part of the blood sample collected during routine venipuncture and are thus counted in the cbc, whereas marginating neutrophils are not. pseudoneutropenia refers to the situation in which there is an increased proportion of neutrophils in the marginating pool. this may occur because of decreased blood flow or in response to stimuli, such as endotoxemia, that increase expression of molecules promoting interaction between neutrophils and endothelial cells. this mechanism of neutropenia is rarely observed in clinical practice. neutropenia may also result from increased demand for neutrophils in the tissue. how rapidly such a situation develops depends not only on the magnitude of the inflammatory stimulus but also on the reserve of postmitotic neutrophils in the bone marrow. the size of this reserve, or storage pool, is species dependent. in dogs this pool contains the equivalent of days' normal production of neutrophils. cattle represent the other extreme in that they have a small storage pool and thus are predisposed to becoming neutropenic during times of acute inflammation. horses and cats are somewhere between the two extremes, closer to cattle and dogs, respectively. it stands to reason that the clinical significance of neutropenia because of a supply and demand imbalance is also species dependent. in dogs, neutropenia as a result of inflammation is an alarming finding because it is evidence of a massive tissue demand for neutrophils that has exhausted the patient's storage pool and is exceeding the rate of granulopoiesis in the bone marrow. however in cows, neutropenia is commonly noted in a wide range of conditions involving acute inflammation and does not necessarily indicate an overwhelming demand. eosinopenia/basopenia. eosinopenia and basopenia are decreased concentrations of blood eosinophils and basophils, respectively. in many laboratories, cbc reference values for eosinophils and basophils are as low as zero cells per microliter, precluding detection of eosinopenia or basopenia. when detectable, eosinopenia is often a result of stress (i.e., glucocorticoid mediated). monocytopenia. monocytopenia denotes a decreased concentration of monocytes in blood; it is of little to no pathologic significance by itself. thrombocytopenia. thrombocytopenia refers to a decrease in the concentration of circulating platelets. mechanisms of thrombocytopenia include decreased production, increased destruction, increased consumption, and altered distribution. decreased production may occur because of a condition affecting cells of multiple hematopoietic lineages, including megakaryocytes, is often used interchangeably with erythrocytosis, but technically and for the purposes of this chapter, polycythemia refers to a specific type of leukemia called primary erythrocytosis or polycythemia vera. causes of erythrocytosis are either relative or absolute. relative erythrocytosis results from a fluid deficit or an altered distribution of erythrocytes within the body (i.e., the body's total erythrocyte mass of generalized lymphadenopathy (e.g., lymphoma, blastomycosis). some hereditary immunodeficiencies, such as severe combined immunodeficiency or thymic aplasia, can cause lymphopenia due to lymphoid aplasia. erythrocytosis. an increase in the measured red cell mass above the normal range is known as erythrocytosis. the term polycythemia rarely, an epo-secreting tumor may cause inappropriately elevated levels of epo in the absence of hypoxia. absolute erythrocytosis, whether primary or secondary, causes increased viscosity of the blood, resulting in impaired blood flow and microvasculature distention. affected individuals are at increased risk for tissue hypoxia, thrombosis, and hemorrhage. clinical signs of hyperviscosity syndrome may include erythematous mucous membranes ( fig. - ) , prolonged capillary refill time, prominent scleral vessels, evidence of thrombosis or hemorrhage, and secondary signs related to specific organ systems affected (e.g., neurologic and cardiovascular signs). neutrophilia. neutrophilia, an increased blood concentration of neutrophils, occurs in response to a number of different stimuli, which are not mutually exclusive. major mechanisms of neutrophilia are shown in fig. - . understanding the cbc findings characteristic of these responses is an important part of clinical veterinary medicine. inflammation can result in neutropenia, as discussed earlier, or neutrophilia, as discussed next. however, before moving on to a discussion of inflammatory neutrophilia and the so-called left shift, it is important to mention two other common is not increased). it occurs most frequently with dehydration, when the decreased proportion of water in the blood results in hemoconcentration. it is observed less frequently with epinephrine-mediated splenic contraction, wherein erythrocytes move from the spleen into peripheral circulation. erythrocytosis from splenic contraction occurs to the most pronounced degree in horses and cats, especially in young, healthy animals. absolute erythrocytosis is a true increase in red blood cell mass due to erythroid neoplasia or hyperplasia and includes causes of primary and secondary erythrocytosis. primary erythrocytosis, or polycythemia vera, is a neoplastic proliferation of erythroid cells with a predominance of mature erythrocytes. diagnosis is based on a marked increase in red cell mass (hematocrit in normally hydrated dogs ranges from % to > %), an absence of hypoxemia, an absence of other tumors, and a normal or decreased plasma epo concentration. secondary erythrocytosis refers to epo-mediated erythroid hyperplasia causing an increased red blood cell mass. the erythroid hyperplasia may be an appropriate response to chronic hypoxia, such as occurs with right-to-left cardiac shunts or chronic pulmonary inflammatory mediators, including interleukin- (il- ), interleukin- (il- ), interferon (inf), and tumor necrosis factor (tnf), cause anemia of inflammatory disease due to oxidative hemolysis, iron sequestration within enterocytes and macrophages, and impaired erythroid responsiveness to erythropoietin (epo). during homeostasis the membrane transport molecule, ferroportin, transports iron from the cytosol to the extracellular space. the iron is then used for various physiologic processes, including hemoglobin production within bone marrow erythroid precursors. during times of inflammation the liver increases production of hepcidin, which binds ferroportin and causes its internalization and lysosomal degradation. with fewer membrane ferroportin molecules, less iron is absorbed from the diet and mobilized from macrophages. rbc, red blood cell. ( of course, neutrophilia may also indicate inflammation, and inflammatory stimuli of varying magnitude and duration produce different patterns of neutrophilia. a classic hematologic finding in patients with increased demand for neutrophils is the presence of immature forms in the blood, known as a left shift. not all inflammatory responses have a left shift, but the presence of a left shift almost always signifies active demand for neutrophils in the tissue. the magnitude of a left shift is assessed by the number of immature cells and their degree of immaturity. the mildest form is characterized by increased numbers of band neutrophils, the immediate predecessor to the segmented neutrophil normally found in circulation. progressively immature predecessors are seen with increasingly severe inflammation. a left shift is considered orderly if the number of immature neutrophils in circulation decreases as they become progressively immature. the term degenerative left shift is sometimes used to describe cases in which the number of immature forms exceeds the number of segmented neutrophils. as with glucocorticoidmediated neutrophilia, the typical magnitude of neutrophilia caused by inflammation varies by species, with dogs having the most pronounced response. it might be useful to think of neutrophil kinetics in terms of a producer-consumer model in which the bone marrow is the factory, and the tissues (where the neutrophils eventually go) are the customers. the bone marrow storage pool is the factory inventory, and the neutrophils in the bloodstream are in delivery to the customer. within the blood vessels, circulating neutrophils are on the highway, and marginating neutrophils are temporarily pulled off to the side of the road. during health, there is an even flow of neutrophils from the factory to the customer. thus the system is in steady state, and neutrophil numbers remain relatively constant and within the normal range. however, disease states may perturb this system at multiple levels. decreased granulopoiesis is analogous to a factory working below normal production level. ineffective granulopoiesis is analogous to goods that are produced at a normal to increased rate but are damaged during manufacturing and never leave the factory. a left shift is analogous to the factory meeting increased customer demand by shipping out unfinished goods. cases of persistent, established inflammation are characterized by bone marrow granulocytic hyperplasia and mature neutrophilia, analogous to a factory that has had time to adjust to increased demand and is meeting it more efficiently by increasing its output. eosinophilia/basophilia. eosinophilia and basophilia are increased concentrations of blood eosinophils and basophils, respectively. they may occur with parasitism, hypersensitivity reactions, paraneoplastic responses (e.g., lymphoma, mast cell neoplasia, or leukemia), and nonparasitic infectious disease. eosinophilia has also been documented with hypoadrenocorticism and rare idiopathic conditions (e.g., hypereosinophilic syndrome). most cases of eosinophilia and basophilia are due to eosinophilic and basophilic hyperplasia within the bone marrow in response to inflammatory growth factors. however, cortisol deficiency is thought to cause eosinophilia in dogs with hypoadrenocorticism. monocytosis. monocytosis is an increased concentration of monocytes in blood. it most commonly occurs with excessive glucocorticoids or inflammation and uncommonly to rarely with monocytic leukemia, immune-mediated neutropenia, and cyclic hematopoiesis. with excessive endogenous or exogenous glucocorticoids, monocytes shift from the marginating pool to the circulating pool. this stress monocytosis is most common in dogs, less frequent in cats, and rare in horses and cattle. inflammatory diseases cause monocytosis by cytokine-mediated monocytic hyperplasia in the bone marrow. causes of neutrophilia: glucocorticoid excess and epinephrine excess. less common causes of neutrophilia, such as leukocyte adhesion deficiency and neoplasia, are discussed later in the chapter. glucocorticoid excess, either because of endogenous production or exogenous administration, results in a cbc pattern known as the stress leukogram, characterized by mature neutrophilia (i.e., increased concentration of segmented neutrophils without immature neutrophils) and lymphopenia, with or without monocytosis and eosinopenia. mechanisms contributing to glucocorticoid-mediated neutrophilia include the following: • increased release of mature neutrophils from the bone marrow storage pool • decreased margination of neutrophils within the vasculature, with a resulting increase in the circulating pool • decreased migration of neutrophils from the bloodstream into tissues the magnitude of neutrophilia tends to be species dependent, with dogs having the most pronounced response (up to , cells/µl) and in decreasing order of responsiveness, cats ( , cells/µl), horses ( , cells/µl), and cattle ( , cells/µl) having less marked responses. with long-term glucocorticoid excess, neutrophil numbers tend to normalize, whereas lymphopenia persists. epinephrine release results in a different pattern, known as physiologic leukocytosis or excitement leukocytosis, characterized by mature neutrophilia (like the glucocorticoid response) and lymphocytosis (unlike the glucocorticoid response). this phenomenon is short lived (i.e., < hour). neutrophilia occurs primarily because of a shift of cells from the marginating to the circulating pool. physiologic leukocytosis is common in cats (especially when they are highly stressed during blood collection) and horses, less common in cattle, and uncommon in dogs. of a regenerative response in patients recovering from thrombocytopenia, as a result of redistribution after splenic contraction, or within the several weeks after splenectomy. in these cases, thrombocytosis is transient. in the case of splenectomy, thrombocytosis may be marked but normalizes after several weeks. because the body's total platelet mass regulates thrombopoiesis, and a significant portion of the platelet mass is normally in the spleen, it makes sense that splenectomized animals develop thrombocytosis. however, the reason that the number of circulating platelets normalizes in these individuals in the weeks after splenectomy is not thrombocytosis. thrombocytosis, or an increased concentration of platelets in the blood, is a relatively common, nonspecific finding in veterinary patients. in the vast majority of cases, thrombocytosis is reactive-a response to another, often apparently unrelated, disease process. examples of conditions having reactive thrombocytosis include inflammatory and infectious diseases, iron deficiency, hemorrhage, endocrinopathies, and neoplasia. factors that may contribute to reactive thrombocytosis include increased plasma concentration of thrombopoietin, inflammatory cytokines (e.g., il- ), or catecholamines. thrombocytosis may also occur as part , neutrophils and their precursors are distributed in five pools: a bone marrow precursor pool, which includes mitotically active and inactive immature cells; a bone marrow storage pool, consisting of mitotically inactive mature neutrophils; a peripheral blood marginating pool; a peripheral blood circulating pool; and a tissue pool. the relative size of each pool is represented by the size of its corresponding wedge. the peripheral blood neutrophil count measures only neutrophils within the circulating peripheral blood pool, which can be enlarged by ( ) increased demargination, ( ) diminished extravasation into tissue, ( ) increased release of cells from the marrow storage pool, and ( ) cells. the preceding section focused on abnormalities in the number of blood cells. there are also various acquired and congenital conditions involving abnormal structure or function of blood cells. this section briefly discusses abnormal blood cell structure or function occurring secondary to other underlying disease. primary disorders of blood cells are discussed later in the chapter in the section on specific diseases. morphologic abnormalities detected on routine microscopic examination of blood smears may provide important clues about underlying disease processes. poikilocytosis is a broad term referring to the presence of abnormally shaped erythrocytes in circulation. e- table - lists conditions with and mechanisms involved in the formation of a number of specific types of erythrocyte morphologic abnormalities, and fig. - shows some examples. the acquired neutrophil morphologic abnormality known as toxic change (fig. - ) reflects accelerated production of neutrophils as part of the inflammatory response. features of toxic change include increased cytoplasmic basophilia, the presence of small bluegray cytoplasmic inclusions known as döhle bodies (often noted incidentally in cats), and in more severe cases, cytoplasmic vacuolation. although not causing impaired neutrophil function, toxic change occurs during granulopoiesis and thus is technically a form of dysplasia (e.g., döhle bodies are foci of aggregated endoplasmic reticulum). toxic change may accompany any inflammatory response, but in general the more marked the toxic change, the higher the index of suspicion for infection or endotoxemia. other secondary changes to neutrophils may not be evident morphologically. for example, studies in human beings and dogs have shown that individuals with cancer have abnormal neutrophil function (including phagocytic activity, killing capacity, and oxidative burst activity) before initiation of therapy. the clinical significance of this finding is not clear. platelet function disorders, also known as thrombopathies or thrombopathias, may be primary or secondary. many conditions are known or suspected to cause secondary platelet dysfunction (hypofunction or hyperfunction) by altering platelet adhesion or aggregation or by mechanisms that are not fully understood. box - shows underlying conditions having secondary platelet dysfunction. clear. there is also a rare form of megakaryocytic leukemia known as essential thrombocythemia, which is characterized by marked thrombocytosis. lymphocytosis. lymphocytosis refers to an increase in the concentration of lymphocytes in blood circulation. there are several causes of lymphocytosis, including age, excessive epinephrine, chronic inflammation, hypoadrenocorticism, and lymphoid neoplasia; lymphoid neoplasms are presented later in the chapter. young animals normally have higher concentrations of lymphocytes than older animals, and normal healthy young animals may have counts that exceed adult reference values. because this is not pathologic lymphocytosis, but normal physiologic variation, it is often termed pseudolymphocytosis of young animals. as discussed earlier in the section on neutrophilia, lymphocytosis is also a feature of epinephrine-mediated physiologic leukocytosis, resulting from redistribution of lymphocytes from the blood marginating pool into the blood circulating pool. epinephrine-mediated lymphocytosis may be more marked than neutrophilia, particularly in cats (lymphocyte counts of > , /µl are not uncommon). antigenic stimulation may result in lymphocytosis, which may be marked in rare cases (up to approximately , /µl in dogs and , /µl in cats); however, this is not usually the case, even when there is clear evidence of increased immunologic activity in lymphoid tissues. in cases of antigenic stimulation, it is common for a minority of lymphocytes to have "reactive" morphologic features-larger lymphocytes with more abundant, deeply basophilic cytoplasm and more open chromatin ( fig. - ). just as glucocorticoid excess can cause lymphopenia, glucocorticoid deficiency (hypoadrenocorticism) can cause lymphocytosis, or lack of lymphopenia during conditions of stress that typically result in glucocorticoid-mediated lymphopenia. a condition known as persistent lymphocytosis (pl) occurs in approximately % of cattle infected with the bovine leukemia virus (blv). the condition is defined as an increase in the blood concentration of lymphocytes above the reference interval for at least months. this form of lymphocytosis is a nonneoplastic proliferation (i.e., hyperplasia) of b lymphocytes. in the absence of other disease, cattle with persistent lymphocytosis are asymptomatic. however, cattle infected with blv, especially those animals with persistent lymphocytosis, are at increased risk for developing b lymphocyte lymphoma. aplastic anemia (aplastic pancytopenia) aplastic anemia, or more accurately aplastic pancytopenia, is a rare condition characterized by aplasia or severe hypoplasia of all hematopoietic lineages in the bone marrow with resulting cytopenias. the term aplastic anemia is a misnomer because affected cells are not limited to the erythroid lineage. many of the conditions reported to cause aplastic anemia do so only rarely or idiosyncratically; more frequently, they cause other hematologic or nonhematologic abnormalities. a partial list of reported causes of aplastic anemia in domestic animals includes the following: most of these causes, especially the chemical agents, are directly cytotoxic to hscs or progenitor cells, resulting in their destruction. however, another proposed mechanism is disruption of normal stem cell function because of mutation or perturbation of hematopoietic cells and/or their microenvironment. this pathogenesis is mostly recognized in retroviral infections. aplastic anemia occurs in both acute and chronic forms. most of the chemical causes result in acute disease. grossly, affected animals may show signs of multisystemic infection and hemorrhage due to severe neutropenia and thrombocytopenia, respectively. severe neutropenia typically develops within week of an acute insult to the bone marrow, and severe thrombocytopenia occurs in the second week. this sequence is a result of the circulating life spans of each cell type; in health, neutrophils have a blood half-life of to hours, whereas platelets circulate for to days. the development of signs of anemia, such as pale mucous membranes, is more variable. the presence and severity of anemia depends on how rapidly the marrow recovers from the insult and the erythrocyte life span of the particular species. microscopically, bone marrow is hypocellular with markedly reduced hematopoietic cells. hematopoietic cells are replaced with adipose tissue, and there is a variable inflammatory infiltrate of lymphocytes, plasma cells, and macrophages. in addition, there may be necrosis, hematopoietic cell apoptosis, and an increase in phagocytic macrophages. fig. - shows bone marrow aspirates from a dog with pancytopenia from acute -fluorouracil toxicosis, before and during recovery. many inherited or presumably inherited disorders of blood cells have been recognized in domestic animals, including rare or sporadic cases and conditions that are of questionable clinical relevance. this section and the later sections covering species-specific disorders are invading cells or microorganism gain access to the bone marrow or blood circulation either hematogenously or by trauma. trauma may be as obvious as a gaping wound or as subtle as the bite of an insect. portals of entry for the bone marrow are summarized in box - . diseases that arise from the bone marrow, such as leukemia, typically spread to other tissues hematogenously. the bone marrow is encased by a protective shell of cortical bone, and blood supply to the marrow provides access to systemic humoral and cellular defenses. of course, leukocytes themselves function as an essential part of inflammation and immune function, as discussed briefly in the section on granulopoiesis and monocytopoiesis (myelopoiesis) and in greater detail in chapters and . biochemical steps in the glycolytic pathway or linked to it generate antioxidant molecules that enable erythrocytes to withstand oxidative insults throughout their many days in circulation. in addition to producing energy in the form of adenosine triphosphate (atp), glycolysis generates nadh, which helps convert the oxidized, nonfunctional form of hemoglobin, known as methemoglobin, back to its active, reduced state. another antioxidant erythrocyte metabolic pathway, the pentose shunt or hexose uremia antiplatelet antibodies (also cause immune-mediated thrombocytopenia) infection (bvdv, felv) hyperglobulinemia increased fibrinolytic products hypoammonemia snake envenomation platelet inhibitors nsaids-irreversible (aspirin) or reversible inhibition of cyclooxygenase colloidal plasma expanders (e.g., hydroxyethyl starch) other drugs and exogenous agents (many) hematogenously direct penetration (trauma) including hypercellular glomeruli, thickened glomerular and tubular basement membranes, and tubular epithelial lipidosis, degeneration, and necrosis. other porphyrias have been diagnosed in cattle, pigs, and cats but are not known to cause hemolytic anemia. pyruvate kinase deficiency. pyruvate kinase (pk) deficiency is an inherited autosomal recessive condition due to a defective r-type pk isoenzyme that is normally present in high concentrations in mature erythrocytes. to compensate for this deficiency, there is persistence of the m -type pk isoenzyme, which is less stable than the r-type isoenzyme. the disease is reported in many dog breeds and fewer cat breeds (e.g., abyssinian, somali, and domestic shorthair). erythrocyte pk deficiency results in decreased atp production and shortened erythrocyte life spans. in dogs the hemolytic anemia is typically chronic, moderate to severe, extravascular, and strongly regenerative. with chronicity, hemolytic anemia causes enhanced intestinal absorption of iron and subsequent hemosiderosis, especially of the liver and bone marrow. dogs typically die at to years of age of hemochromatosis-induced liver and bone marrow failure. however, cats with pk deficiency typically show no clinical signs, have milder anemia, and do not develop organ failure. grossly, affected animals have lesions attributed to hemolytic anemia, including splenomegaly, pale mucous membranes, and rarely icterus. dogs with end-stage disease have cirrhosis, myelofibrosis, and osteosclerosis. dogs with pk deficiency do not necessarily have the same genetic defect, so mutation-specific dna-based assays are required. in contrast, a single dna-based test is available to detect the common mutation affecting abyssinian, somali, and domestic shorthair cats. cytochrome-b reductase deficiency. deficiency of cytochrome-b reductase (cb r, also known as methemoglobin reductase), the enzyme that catalyzes the reduction of methemoglobin (fe + ) to hemoglobin (fe + ), has been recognized in many dog breeds and in domestic shorthair cats. it is probably an autosomal recessive trait. affected animals may have cyanotic mucous membranes or exercise intolerance but usually lack anemia and clinical signs of disease. life expectancies are normal. glucose- -phosphate dehydrogenase deficiency. deficiency of glucose- -phosphate dehydrogenase (g pd), the ratecontrolling enzyme of the pentose phosphate pathway (ppp), has been reported in an american saddlebred colt, its dam, and one male dog. the ppp is an antioxidative pathway that generates nadph, which maintains glutathione in its reduced form (gsh). therefore in animals with g pd deficiency, oxidants are not scavenged, and erythrocyte oxidative injury occurs. the colt with g pd deficiency had severe oxidative hemolytic anemia with eccentrocytes on blood smear evaluation. however, the colt's dam only had eccentrocytes, and showed no hematologic signs of disease. leukocyte adhesion deficiency. leukocyte adhesion deficiency (lad) is a fatal autosomal recessive defect of leukocyte integrins, in particular the β chain (also known as cluster of differentiation [cd] [cd ]). disease has been recognized in holstein cattle (known as bovine leukocyte adhesion deficiency [blad] ) and irish setter dogs (known as canine leukocyte adhesion deficiency [clad]) (see chapter ). without normal expression of this adhesion molecule, leukocytes have severely impaired abilities to migrate from the blood into tissues. as a result, animals with leukocyte adhesion deficiency have marked neutrophilia with nonsuppurative multisystemic infections. blood neutrophils often have nuclei with greater than five nuclear segments, termed not comprehensive but instead focus on the more common, wellcharacterized, or recently reported conditions. erythropoietic porphyrias. porphyrias are a group of hereditary disorders in which porphyrins accumulate in the body because of defective heme synthesis. inherited enzyme defects in hemoglobin synthesis have been identified in holstein cattle, siamese cats, and other cattle and cat breeds, resulting in bovine congenital erythropoietic porphyria and feline erythropoietic porphyria, respectively. accumulation of toxic porphyrins in erythrocytes causes hemolytic anemia, whereas accumulation of porphyrins in tissues and fluids produces discoloration, including red-brown teeth, bones, and urine (see fig. - ). because of the circulation of the photodynamic porphyrins in blood, these animals have lesions of photosensitization of the nonpigmented skin. all affected tissues, including erythrocytes, exhibit fluorescence with ultraviolet light. histologically, animals may exhibit perivascular dermatitis, as well as multisystemic porphyrin deposition, hemosiderosis, emh, and marrow erythroid hyperplasia. cats may show evidence of renal disease, a b syndrome exhibit oculocutaneous albinism (due to altered distribution of melanin granules) and are prone to infection and bleeding. blood smear evaluation reveals granulocytes with large cytoplasmic granules. glanzmann thrombasthenia. glanzmann thrombasthenia (gt) is an inherited platelet function defect caused by a mutated α iib subunit of the integrin α iib β (also known as glycoprotein iib-iiia [gpiib-iiia]). the disorder has been recognized in great pyrenees and otterhound dogs and several horse breeds, including a quarter horse, a standardbred, a thoroughbred-cross, a peruvian paso mare, and an oldenburg filly. the α iib β molecule has multiple functions but is best known as a fibrinogen receptor that is essential for normal platelet aggregation. bleeding tendencies vary widely between affected individuals but mainly occur on mucosal surfaces. the condition is characterized by an in vitro lack of response to all platelet agonists and severely impaired clot retraction (i.e., whole blood samples without anticoagulant often fail to clot). molecular testing is available to detect diseased or carrier states in dogs and horses. thrombopathia. calcium diacylglycerol guanine nucleotide exchange factor i (caldag-gefi) is a molecule within the signaling pathway that results in platelet activation in response to platelet agonists. mutated caldag-gefi has been documented in basset hound, eskimo spitz, and landseer dogs, and simmental cattle. all reported mutations have a bleeding tendency. in vitro platelet aggregation responses to platelet agonists, such as adp, collagen, and thrombin, are absent or impaired. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. oxidative agents. a variety of oxidative toxins cause hemolytic anemia and/or methemoglobinemia in domestic species. more common or well-characterized oxidants are listed here: • horses-acer rubrum (red maple) • ruminants-brassica spp. (cabbage, kale, and rape), copper hypersegmented neutrophils, due to neutrophil aging within blood vessels ( fig. - ). these animals are highly susceptible to infections and usually die at a young age. pelger-huët anomaly. pelger-huët anomaly (pha) is a condition of hyposegmented granulocytes due to a lamin b receptor mutation. it has been described in dogs, cats, horses, and rabbits, especially in certain breeds. in australian shepherd dogs the mode of inheritance is autosomal dominant with incomplete penetrance. most cases of pelger-huët anomaly are the heterozygous form and of no clinical significance. however, skeletal abnormalities, stillbirths, and/or early mortality may accompany pelger-huët anomaly in rabbits and cats, especially homozygotes. in pelger-huët anomaly the nuclei of neutrophils, eosinophils, and basophils fail to segment, resulting in band-shaped, bean-shaped, or round nuclei. although the nuclear shape is similar to that of an inflammatory left shift, healthy animals with pelger-huët anomaly do not have clinical signs or other laboratory findings indicating inflammation. for example, neutrophils in healthy animals with pelger-huët anomaly have mature (clumped) chromatin and do not show signs of toxicity ( fig. - ). an acquired, reversible condition mimicking pelger-huët anomaly, known as pseudo-pelger-huët anomaly, is occasionally noted in animals with infectious disease, neoplasia, or drug administration. chédiak-higashi syndrome. chédiak-higashi syndrome (chs) is a rare autosomal recessive defect in the lysosomal trafficking regulator (lyst) protein. the syndrome has been identified in hereford, brangus, and japanese black cattle, persian cats, and several nondomestic species. the defective lyst protein results in granule fusion in multiple cell types, including granulocytes, platelets, and melanocytes, as well as abnormal cell function. individuals with chédiak-higashi syndrome have severely impaired cellular innate immunity because of neutropenia, impaired leukocyte chemotaxis, and impaired killing by granulocytes and cytotoxic lymphocytes. platelets lack the dense granules that normally contain key bioactive molecules involved in hemostasis, including platelet agonists, such as adp and serotonin. in vitro platelet aggregation is severely impaired. as a result, animals with chédiak-higashi .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system von willebrand disease (vwd) is the most common canine hereditary bleeding disorder and has also been described in many other domestic species. the disease actually refers to a group of inherited conditions characterized by a quantitative or qualitative deficiency of vwf. this factor is a multimeric glycoprotein that is stored in platelet α-granules and endothelial cells and circulates as a complex with coagulation factor viii. its primary functions are to stabilize factor viii and mediate platelet binding to other platelets and subendothelial collagen. although not technically a platelet disorder, von willebrand disease is often classified as such because it results in a loss of normal platelet function. different types of von willebrand disease vary in terms of mode of inheritance and severity of clinical disease. type i von willebrand disease is characterized by low plasma vwf concentration but normal multimeric proportions and a mild to moderate clinical bleeding tendency; it has been reported in many dog breeds. type ii von willebrand disease is characterized by low vwf concentration, absence of large multimers, and a moderate to severe bleeding tendency; it has been reported in german short-haired pointer and german wirehaired pointer dogs. type iii von willebrand disease is characterized by absence of vwf and a severe bleeding tendency; familial and sporadic cases have been reported in numerous dog breeds. the buccal mucosal bleeding time is prolonged with von willebrand disease, often with adequate concentrations of platelets and normal prothrombin time and partial thromboplastin time (ptt). however, ptt may be mildly prolonged because vwf stabilizes factor viii, and deficiency of vwf results in enhanced factor viii degradation. grossly, affected animals exhibit bleeding tendencies, especially in the form of inherited coagulation factor deficiencies have been documented in most domestic species, including deficiencies of prekallikrein and factors i, ii, vii, viii, ix, x, xi, and xii. of these disorders, hereditary coagulation factor viii (hemophilia a) and factor ix (hemophilia b) deficiencies are most common. hemophilia a has been recognized in horses, cattle, dogs, and cats, and hemophilia b occurs in dogs and cats. both disorders have an x-linked recessive mode of inheritance, meaning that clinical disease is more common in males. affected males have variable tendencies to bleed, depending on the severity of the deficiency, exposure to trauma, and size and activity level of the affected individual. carrier females are usually asymptomatic. laboratory tests often reveal adequate platelets, normal prothrombin times, and prolonged partial thromboplastin times. hereditary defects in γ-glutamyl carboxylase, the enzyme required for normal carboxylation of vitamin k-dependent coagulation factors, have been recognized in a flock of rambouillet sheep and two devon rex cats from the same litter. the genetic defect is not known in cats, but in sheep it is an autosomal recessive trait that results in a premature stop codon and truncated γ-glutamyl carboxylase. in sheep there is increased lamb mortality with excessive bleeding during parturition, especially through the umbilicus or into subcutaneous tissues. gingival bleeding, epistaxis, and hematuria or at sites of injections, venipuncture, or surgery. of loss is through the gastrointestinal tract (e.g., neoplasia in older animals or hookworm infection in puppies). chronic blood loss may also be caused by marked ectoparasitism (e.g., pediculosis in cattle or massive flea burden in kittens and puppies), neoplasia in locations other than the gastrointestinal tract (e.g., cutaneous hemangiosarcoma), coagulation disorders, and repeated phlebotomy of blood donor animals. rapidly growing nursing animals may be iron deficient when compared with adults because milk is an iron-poor diet. in most cases this has little clinical significance (and in fact is normal). an important exception is piglets with no access to iron, which may cause anemia, failure to thrive, and increased mortality. neonatal piglets are routinely given parenteral iron for this reason. copper deficiency can cause iron deficiency in ruminants and may occur because of copper-deficient forage or impaired usage of copper by high dietary molybdenum or sulfate. it is believed that copper deficiency impairs production of ceruloplasmin, a copper-containing enzyme involved in gastrointestinal iron absorption. iron deficiency causes anemia by impaired hemoglobin synthesis. iron is an essential component of hemoglobin, and when it is absent, hemoglobin synthesis is depressed. because erythrocyte maturation is dependent upon obtaining a critical hemoglobin concentration, maturing erythroid precursors undergo additional cell divisions during iron-deficient states. these additional cell divisions result in small erythrocytes, termed microcytes (see fig. - , g). however, erythrocytes with low hemoglobin concentrations are produced when microcyte formation can no longer compensate for iron deficiency. the classic hematologic picture with iron deficiency anemia is microcytic (i.e., decreased mcv), hypochromic (i.e., decreased mchc) anemia. microcytes and hypochromasia (see fig. - , g) may also be discernible on blood smear examination as erythrocytes that are abnormally small and paler-staining, respectively. early iron deficiency anemia is poorly regenerative, whereas continued hemorrhage and iron loss cause nonregenerative anemia. additional hematologic changes may include evidence of erythrocyte mechanical fragmentation (e.g., schistocytes) and reactive thrombocytosis. hypophosphatemic hemolytic anemia. marked hypophosphatemia is recognized as a cause of intravascular hemolytic anemia in postparturient dairy cows and diabetic animals receiving insulin therapy. in postparturient cows, hypophosphatemia results from increased loss of phosphorus in their milk. insulin therapy may cause hypophosphatemia by shifting phosphorus from the extracellular space to the intracellular space. in either case, marked hypophosphatemia (e.g., mg/dl in cows, or ≤ . mg/dl in cats) is thought to decrease erythrocyte production of atp, leading to inadequate energy required for maintenance of membrane and cytoskeletal integrity. an accompanying decrease in reducing capacity and increase in methemoglobin concentration have also been noted in experimental studies of hypophosphatemic hemolytic anemia in dairy cattle, suggesting that oxidative mechanisms may also contribute to anemia. affected animals are anemic and hemoglobinuric. gross postmortem findings include pallor, decreased viscosity of the blood, and lesions arising from the underlying metabolic derangement (e.g., discolored pale yellow and swollen liver due to hepatic lipidosis). renal tubular necrosis and hemoglobin pigment within the tubules is evident microscopically. this section covers infectious agents within the same genus that are recognized to cause disease in multiple species. other infectious agents with more limited host specificity (e.g., cytauxzoonosis in cats, feline and equine retroviruses) are covered in later sections on species-specific diseases. throughout both sections, diseases are • dogs-acetaminophen, propofol, zinc • cats-acetaminophen, propofol, propylene glycol • all species-allium spp. (chives, garlic, and onions) in horses, red maple leaves and bark are toxic, especially wilted or dried leaves. the toxic principle is believed to be gallic acid. plants that contain high concentrations of nitrates, such as cabbage, kale, and rape, may cause oxidative injury to erythrocytes; cattle are more susceptible than sheep and goats. however, sheep are more prone to copper toxicosis relative to other ruminants. the condition occurs in animals that have chronically accumulated large amounts of copper in the liver through the diet. the copper is then acutely released during conditions of stress, such as shipping or starvation. continuous rate infusions of the anesthetic propofol may cause oxidative hemolytic anemia in dogs and cats, but single or multiple single doses are not expected to cause clinical hemolysis. zinc toxicosis has been identified in a wide range of animals; however, it is most common in dogs due to their indiscriminate eating habits. common sources include pennies, batteries, paints, creams, automotive parts, screws, nuts, and coating on galvanized metals. propylene glycol is an odorless, slightly sweet solvent and moistening agent in many foods, drugs, and tobacco products. although it is "generally recognized as safe" for animal foods other than for cats by the food and drug administration, it has been banned from cat food since . grossly and microscopically, animals show varying signs of oxidative hemolysis and/or methemoglobinemia, as previously presented in the section discussing anemias (see bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in sheep with copper toxicosis, hemoglobinuric nephrosis, frequently described as gunmetal-colored kidneys with port wine-colored urine, is a classic postmortem lesion. snake envenomation. hemolytic anemia from snake envenomation has been reported in horses, dogs, and cats. it is most commonly reported with viper and pit viper envenomations, including those from rattlesnakes. hemolysins within viper venom directly injure erythrocytes, causing intravascular hemolysis. other mechanisms of hemolysis include the action of phospholipase a on erythrocyte membranes and erythrocyte mechanical fragmentation due to intravascular coagulation and vasculitis. nonhemolytic lesions depend on the venom's additional components and may include hemorrhage, paralysis, and/or tissue edema, inflammation, and necrosis. on blood smear evaluation, animals with snake envenomation may have ghost cells, spherocytes, and/or echinocytes (see figs. - and - ). information on this topic is available at www.expertconsult.com. severe malnutrition is probably a cause of nonregenerative anemia in all species attributable to combined deficiencies of molecular building blocks, energy, and essential cofactors. by far the most commonly recognized specific deficiency that results in anemia is iron deficiency. other specific nutritional deficiencies causing anemia in animals are uncommon or rare. acquired cobalamin (vitamin b ) and folate deficiencies are recognized as causes of anemia in human beings but are rare in animals. iron deficiency anemia. iron deficiency is usually not a primary nutritional deficiency but rather occurs secondary to depletion of iron stores via chronic blood loss. the most common route .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system antagonism of vitamin k leads to production of a nonfunctional form of some coagulation factors and resulting coagulopathy; a similar condition results from vitamin k deficiency. conditions with avitaminosis k include poisoning with coumarin-related molecules, fat malabsorption (vitamin k is a fat-soluble vitamin) caused by primary intestinal disease or impaired biliary outflow (uncommon), dietary deficiency (rare), and antibiotics that interfere with vitamin k absorption or usage. a number of coagulation factors-factors ii, vii, ix, and x (collectively known as the vitamin k-dependent factors), as well as the regulatory molecules protein c and protein s-must undergo carboxylation to be functional. this posttranslational modification is catalyzed by the enzyme γ-glutamyl carboxylase, and requires vitamin k as a cofactor. vitamin k is oxidized during the carboxylation reaction and is converted back into its active reduced form by the enzyme vitamin k epoxide reductase. coumarin-related rodenticides, such as warfarin, act by inhibiting vitamin k epoxide reductase, resulting in an absence of vitamin k in its active reduced form (e- fig. - ). this inhibition lasts until the rodenticide is metabolized and cleared. how long this takes depends on the half-life of the rodenticide and dose, but it may take many weeks. secondgeneration rodenticides, such as bromadoline and brodifacoum, are more potent than warfarin, with longer half-lives. spoiled sweet clover contains dicumarol, which causes coagulopathy by the same mechanism. e- figure - mechanism of anticoagulant rodenticide toxicity. anticoagulant rodenticides inhibit the enzyme that converts vitamin k back to its active reduced form. active factors (carboxylated) inactive factors (ii, vii, ix, x) = inactivation of vitamin k epoxide reductase, the enzyme that maintains vitamin k in the active form vitamin k epoxide (inactive) x x laboratory findings include prolonged coagulation times (prothrombin time [pt] , ptt, and activated clotting time [act] ). early in the course of rodenticide and related toxicoses, pt may be the only one of these tests that is prolonged because factor vii has the shortest half-life of the vitamin k-dependent factors. however, the other tests become prolonged as nonfunctional forms of the other factors accumulate. in uncomplicated cases, patients are not thrombocytopenic. a wide range of hemorrhagic lesions may occur in affected individuals, including ecchymoses, epistaxis, gingival bleeding, hematomas, hemoptysis, melena or hematochezia, hematuria, and other forms of hemorrhage. there are also lesions with regenerative anemia, such as pale mucous membranes and splenomegaly. histologically, there is hemorrhage, emh, and marrow erythroid hyperplasia. the treatment of cases of rodenticide and related toxicoses is regular administration of exogenous vitamin k until the toxin is cleared (determined by repeat coagulation testing after withholding treatment). babesiosis may cause intravascular and extravascular hemolytic anemia via direct red blood cell injury, the innocent bystander effect, and secondary immune-mediated hemolytic anemia. infection with highly virulent strains may cause severe multisystemic disease. in these cases, massive immunostimulation and cytokine release cause circulatory disturbances, which may result in shock, induction of the systemic inflammatory response, and multiple organ dysfunction syndromes. babesia organisms can usually be detected on a routine blood smear in animals with acute disease. infected erythrocytes may be more prevalent in capillary blood, so blood smears made from samples taken from the pinna of the ear or the nail bed may increase the likelihood of detecting organisms microscopically. buffy coat smears also have an enriched population of infected erythrocytes. pcr-based tests are the most sensitive assay for detecting infection in animals with very low levels of parasitemia. at necropsy, gross lesions are mainly related to hemolysis and include pale mucous membranes, icterus, splenomegaly, dark red or black kidneys, and reddish-brown urine. the cut surface of the congested spleen oozes blood. the gallbladder is usually distended with thick bile. less common lesions include pulmonary edema, ascites, and congestion, petechiae, and ecchymoses of organs, including the heart and brain. parasitized erythrocytes are best visualized on impression smears of the kidney, brain, and skeletal muscle. microscopic findings in the liver and kidney are typical of a hemolytic crisis and include anemia-induced degeneration, necrosis of periacinar hepatocytes and cholestasis, and hemoglobinuric nephrosis with degeneration of tubular epithelium. erythroid hyperplasia is present in the bone marrow. in animals that survive the acute disease, there is hemosiderin accumulation in the liver, kidney, spleen, and bone marrow. in chronic cases there is hyperplasia of macrophages in the red pulp of the spleen. theileriosis (piroplasmosis). theileria spp. are tick-borne protozoal organisms that infect many domestic and wild animals worldwide. numerous theileria spp. have been documented, but only the more economically or regionally important species are mentioned here. diseases with the greatest economic impact in ruminants are east coast fever (theileria parva infection) and tropical theileriosis (theileria annulata infection). • horses-theileria equi (formerly babesia equi) • cattle-theileria annulata, theileria buffeli, t. parva • sheep and goats-theileria lestoquardi (formerly theileria hirci) like babesiosis, theileriosis is generally restricted to tropical and subtropical regions, including parts of africa, asia, the middle east, and europe. except for t. buffeli, all previously listed species are exotic to the united states. infection is characterized by schizonts within lymphocytes or monocytes, and pleomorphic intraerythrocytic piroplasms (merozoites and trophozoites). within host leukocytes the parasite induces leukocyte cellular division, which expands the parasitized cell population. infected cells disseminate throughout the lymphoid system via the lymphatic and blood vessels. the infected leukocyte may block capillaries, causing tissue ischemia. later in infection some schizonts cause leukocyte lysis and release of merozoites. merozoites then invade and parasitize erythrocytes, causing hemolytic anemia. possible mechanisms of anemia in theileriosis include invasion of erythroid precursors by merozoite stages and associated erythroid hypoplasia (as occurs with t. parva infection), immune-mediated hemolysis, mechanical fragmentation because of vasculitis or microthrombi, enzymatic destruction by proteases, and oxidative damage. gross and microscopic lesions are similar to those of babesiosis, except that cattle with east coast fever tend not to develop organized by taxonomy (protozoal, bacterial and rickettsial, and viral). babesiosis (piroplasmosis). babesia spp. and theileria spp., presented in the next section, are members of the order piroplasmida, and are generally referenced as piroplasms. these organisms are morphologically similar but have different life cycles; babesia spp. are primarily erythrocytic parasites, whereas theileria spp. sequentially parasitize leukocytes and then erythrocytes. both are protozoan parasites spread by ticks, but other modes of transmission are possible (e.g., biting flies, transplacental, and blood transfusions). evidence is accumulating that dog fighting also transmits babesia gibsoni infection. babesia organisms are typically classified as large ( to µm) or small (< µm) with routine light microscopy ( fig. - ). over babesia species have been identified, some of which are listed here, along with their relative microscopic size in parentheses: geographic distributions vary with the species, but most have higher prevalences in tropical and subtropical regions. for example, equine and bovine babesiosis are endemic in parts of africa, the middle east, asia, central and south america, the caribbean, and europe. both were eradicated from the united states and are now considered exotic diseases in that country. of the previously mentioned species, only agents of canine babesiosis are thought to be endemic in the united states. fever. anaplasmosis, ehrlichiosis, heartwater, and tick-borne fever are tick-borne diseases caused by small, pleomorphic, gramnegative, obligate intracellular bacteria within the order rickettsiaceae, also colloquially known as rickettsias. as a group, rickettsias primarily infect hematopoietic cells and endothelial cells. rickettsias that predominantly infect endothelial cells (e.g., rickettsia rickettsii [rocky mountain spotted fever]), or cause gastrointestinal disease (e.g., neorickettsia helminthoeca [salmon poisoning disease] and neorickettsia risticii [potomac horse fever]) are discussed elsewhere (see chapters and ). less commonly, transmission may occur via blood transfusions or blood-contaminated medical supplies. rickettsias that infect erythrocytes include the following species (the disease name follows in parentheses): • cattle-anaplasma marginale, anaplasma centrale (bovine anaplasmosis) • sheep and goats-anaplasma ovis (ovine and caprine anaplasmosis, respectively) anaplasma marginale and a. ovis have worldwide distributions, but a. centrale is mostly restricted to south america, africa, and the middle east. hemolytic anemia. in acute east coast fever, lymph nodes are enlarged, edematous, and hemorrhagic. but with chronic cases they may be shrunken. there is often splenomegaly, hepatomegaly, and hemorrhagic enteritis with white foci of lymphoid infiltrates (pseudoinfarcts) in the liver and kidney. microscopically, infected leukocytes may block capillaries. african trypanosomiasis. trypanosomes are flagellated protozoa that can infect all domesticated animals. the most important species that cause disease are trypanosoma congolense, trypanosoma vivax, and trypanosoma brucei ssp. brucei. disease is most common in parts of africa where the biologic vector, the tsetse fly, exists. however, t. vivax has spread to central and south america and the caribbean, where other biting flies transmit the parasite mechanically. in africa, cattle are mainly affected due to the feeding preferences of the tsetse fly. african trypanosomiasis must be distinguished from nonpathogenic trypanosomiasis, such as trypanosoma theileri infection in cattle. animals become infected when feeding tsetse flies inoculate metacyclic trypanosomes into the skin of animals. the trypanosomes grow for a few days, causing a localized chancre sore, and then sequentially enter the lymph nodes and bloodstream. trypanosomal organisms do not infect erythrocytes but rather exist as free trypomastigotes (i.e., flagellated protozoa with a characteristic undulating membrane) in the blood ( fig. - , a) or as amastigotes in tissue. the mechanism of anemia is believed to be immune mediated. cattle with acute trypanosomiasis have significant anemia, which initially is regenerative, but less so with time. the extent of parasitemia is readily apparent with t. vivax and t. theileri infections because the organisms are present in large numbers in the blood. this is in contrast to t. congolense, which localizes within the vasculature of the brain and skeletal muscle. chronically infected animals often die secondary to poor body condition, immunosuppression, and concurrent infections. gross examination of animals with acute disease often reveals generalized lymphadenomegaly, splenomegaly, and petechiae on serosal membranes. an acute hemorrhagic syndrome may occur in cattle, resulting in lesions of severe anemia (e.g., pale mucous membranes) and widespread mucosal and visceral hemorrhages. main lesions of chronic infections include signs of anemia, lymphadenopathy (e.g., enlarged or atrophied lymph nodes), emaciation, subcutaneous edema, pulmonary edema, increased fluid in body cavities, and serous atrophy of fat. trypanosoma cruzi is the flagellated protozoal agent of american trypanosomiasis. infections have been reported in more than mammal species in south america, central america, and the southern united states, but dogs and cats are among the more common domestic hosts. infected triatomine insects, or "kissing bugs," defecate as they feed on their mammalian host, releasing infective t. cruzi organisms. the parasite then enters the body through mucous membranes or breaks in the skin. like the other trypanosomes described previously, t. cruzi lives in the blood as extracellular trypomastigotes (see fig. - , b) and in the tissues as intracellular amastigotes. trypanosoma cruzi primarily causes heart disease. lesions of acute disease include a pale myocardium, subendocardial and subepicardial hemorrhages, and yellowish-white spots and streaks. there may also be secondary lesions, such as pulmonary edema, ascites, and congestion of the liver, spleen, and kidneys. in chronic disease the heart may be enlarged and flaccid with thin walls. microscopically, there is often myocarditis and amastigotes within cardiomyocytes. most of these rickettsias have worldwide distributions. however, e. ewingii has been reported only in the united states, and e. ruminantium is endemic only in parts of africa and the caribbean. although a. phagocytophilum has a wide geographic distribution, strain variants are regionally restricted. for example, a. phagocytophilum causes disease in ruminants in europe, but it has not been documented in ruminants in the united states. reservoirs of disease vary, depending upon the rickettsial species. cattle are the reservoir host for e. ruminantium, canids are the reservoir host for a. platys and e. canis, and the other rickettsias have wildlife reservoirs. pathogenesis of disease involves endothelial cell, platelet, and leukocyte dysfunction. those agents that infect endothelial cells cause vasculitis and increased vascular permeability of small blood vessels. if only plasma is lost, then there is hypotension and tissue edema. however, more severe vasculitis causes microvascular hemorrhage with the potential for platelet consumption thrombocytopenia, disseminated intravascular coagulation, and hypotension. infection of platelets may cause thrombocytopenia by direct platelet lysis, immune-mediated mechanisms, or platelet sequestration within the spleen. pathogenesis of leukocyte dysfunction is unclear, but may involve sepsis, inhibited leukocyte function, endothelial cell activation, and platelet consumption. chronic e. canis infection may cause aplastic anemia with pancytopenia by an unknown mechanism. some studies indicate that german shepherd dogs with ehrlichiosis are predisposed to have particularly severe clinical disease. some breeds of cattle (bos taurus), sheep (merino), and goats (angora and saanen) are more susceptible to heartwater. upon blood smear evaluation, thrombocytopenia is the most common hematologic abnormality; anemia and neutropenia occur less frequently. in early stages of infection, blood cells may contain morulae, which are clusters of rickettsial organisms within cytoplasmic, membrane-bound vacuoles ( fig. - ). examination of buffy coat smears increases the probability of detecting the organism. chronic infection may cause lymphocytosis, particularly of granular lymphocytes. anaplasma platys causes recurrent marked thrombocytopenia. in general, more common gross lesions are splenomegaly, lymphadenomegaly, and pulmonary edema and hemorrhage. more severe cases may also exhibit multisystemic petechiae, ecchymoses, and bovine anaplasmosis causes anemia mainly by immune-mediated extravascular hemolysis. the severity of disease in infected animals varies with age. infected calves under year of age rarely develop clinical disease, whereas cattle years of age or older are more likely to develop severe, potentially fatal, illness. the reason for this discrepancy is not clear. indian cattle (bos indicus) are more resistant to disease than european cattle (bos taurus). surviving cattle become chronic carriers (and thus reservoirs for infection of other animals) and develop cyclic bacteremia, which is typically not detectable on blood smears. splenectomy of carrier animals results in marked bacteremia and acute hemolysis. pcr testing is the most sensitive means of identifying animals with low levels of bacteremia. grossly, acute disease causes lesions of acute hemolytic anemia, including pale mucous membranes, low blood viscosity, icterus, splenomegaly, hepatomegaly, and a distended gallbladder. in animals with acute disease it is usually easy to detect a. marginale organisms on routine blood smear evaluation ( fig. - ) or impression smears from cut sections of the spleen. however, in recovering animals, the organisms may be difficult to find. rickettsias that infect leukocytes are broadly divided into those that preferentially infect granulocytes ( in addition to anemia, common findings in animals with leptospirosis-induced hemolysis include hemoglobinuria and icterus. on necropsy, renal tubular necrosis, which occurs in part because of hemoglobinuria (hemoglobinuric nephrosis), may also be present. hemotropic mycoplasmosis (hemoplasmosis). the term hemotropic mycoplasmas, or hemoplasmas, encompasses a group of bacteria, formerly known as haemobartonella or eperythrozoon spp., that infect erythrocytes of many domestic, laboratory, and wild animals. hemotropic mycoplasmas affecting common domestic species are as follows: • cattle-mycoplasma wenyonii • camelids-"candidatus mycoplasma haemolamae" • sheep and goats-mycoplasma ovis • pigs-mycoplasma suis (e- fig. - ) • dogs-mycoplasma haemocanis, "candidatus mycoplasma haematoparvum" • cats-mycoplasma haemofelis, "candidatus mycoplasma haemominutum," "candidatus mycoplasma turicensus" like other mycoplasmas, hemoplasmas are small ( . to µm in diameter) and lack a cell wall. they are epicellular parasites, residing in indentations and invaginations of red blood cell surfaces. the mode of transmission is poorly understood, but blood-sucking arthropods are believed to play a role; transmission in utero, through biting or fighting, and transfusion of infected blood products are also suspected. effects of infection vary from subclinical to fatal anemia, depending on the specific organism, dose, and host susceptibility. most hemoplasmas are more likely to cause acute illness in individuals that are immunocompromised or have concurrent disease. however, m. haemofelis is an exception and tends to cause acute hemolytic anemia in immunocompetent cats. anemia occurs mainly because of extravascular hemolysis, but intravascular hemolysis also occurs. although the pathogenic mechanisms are not completely understood, an immune-mediated component is highly probable, as well as direct red blood cell injury by the bacteria and the innocent bystander effect. hemotropic mycoplasmas induce cold agglutinins in infected individuals, although it is not clear whether these particular antibodies are important in the development of hemolytic anemia. when detected on routine blood smear evaluation, the organisms are variably shaped (cocci, small rods, or ring forms) and sometimes arranged in short, branching chains ( fig. - ) . the organisms may also be noted extracellularly, in the background of the blood smear, especially if the smear is made after prolonged storage of the blood in an anticoagulant tube. in animals dying of acute hemoplasma infection, the gross findings are typical of extravascular hemolysis, with pallor, icterus, splenomegaly, and distended gallbladder ( fig. - ). additional lesions documented in cattle include scrotal and hind limb edema and swelling of the teats. microscopic lesions in the red pulp of the spleen include congestion, erythrophagocytosis, macrophage hyperplasia, emh, and increased numbers of plasma cells. bone marrow has varying degrees of erythroid hyperplasia, depending on the duration of hemolysis. immune-mediated hemolytic anemia. immune-mediated hemolytic anemia is a condition characterized by increased destruction of erythrocytes because of binding of immunoglobulin to red blood cell surface antigens. it is a common, life-threatening condition in dogs but also has been described in horses, cattle, and cats. immune-mediated hemolytic anemia may be idiopathic (also called edema, cavitary effusions, and effusive polyarthropathy. hydropericardium gives heartwater its name but is more consistently found in small ruminants than in cattle. chronically infected dogs are emaciated. the bone marrow is hyperplastic and red in the acute disease but becomes hypoplastic and pale in dogs with chronic e. canis infection. equine anaplasmosis is often mild but may cause edema and hemorrhages. disease in cats is rare and poorly documented. histologic findings include generalized perivascular plasma cell infiltration, which is most pronounced in animals with chronic disease. multifocal, nonsuppurative meningoencephalitis, interstitial pneumonia, and glomerulonephritis are present in most dogs with the disease. rickettsial organisms are difficult to detect histologically; examination of wright-giemsa-stained impression smears of lung, liver, lymph nodes, and spleen is a more effective method for detecting the morulae within leukocytes. heartwater is often diagnosed by observing morulae in endothelial cells of giemsastained squash preparations of brain. rickettsial diseases are often diagnosed on the basis of serologic testing, but pcr testing is more sensitive. clostridial diseases. certain clostridium spp. may cause potentially fatal hemolytic anemias in animals; nonhemolytic lesions are presented elsewhere (see chapters , , , and ) . clostridium haemolyticum and clostridium novyi type d cause the disease in cattle known as bacillary hemoglobinuria. (the phrase "red water" has also been used for this disease and for hemolytic anemias in cattle caused by babesia spp.) similar naturally occurring disease has been reported in sheep. in cattle the disease is caused by liver fluke (fasciola hepatica) migration in susceptible animals. ingested clostridial spores may live in kupffer cells for a long time without causing disease. however, when migrating flukes cause hepatic necrosis, the resulting anaerobic environment stimulates the clostridial organisms to proliferate and elaborate their hemolytic toxins, causing additional hepatic necrosis. the mechanism of hemolysis involves a bacterial β-toxin (phospholipase c or lecithinase), which enzymatically degrades cell membranes, causing acute intravascular hemolysis. bacillary hemoglobinuria also occurs with liver biopsies in calves. clostridium perfringens type a causes intravascular hemolytic anemia in lambs and calves-a condition known as yellow lamb disease, yellows, or enterotoxemic jaundice because of the characteristic icterus. the organism is a normal inhabitant of the gastrointestinal tract in these animals but may proliferate abnormally in response to some diets. c. perfringens causes intravascular hemolytic anemia in horses with clostridial abscesses, and clostridial mastitis in ewes. c. perfringens type a produces hemolytic α-toxin, which also has phospholipase c activity. leptospirosis. leptospirosis is recognized as a cause of hemolytic anemia in calves, lambs, and pigs. specific leptospiral organisms that cause hemolytic disease include leptospira interrogans serovars pomona and ictohaemorrhagiae. leptospira organisms are ubiquitous in the environment. infection occurs percutaneously and via mucosal surfaces and is followed by leptospiremia; organisms then localize preferentially in certain tissues (e.g., kidney, liver, and pregnant uterus). proposed mechanisms of hemolytic disease include immune-mediated (immunoglobulin m [igm] cold agglutinin) extravascular hemolysis and enzymatic (phospholipase produced by the organism) intravascular hemolysis. leptospirosis can also cause many disease manifestations besides hemolysis (e.g., renal failure, liver failure, abortion, and other conditions) that are not discussed here. primary immune-mediated hemolytic anemia or autoimmune hemolytic anemia) or secondary to a known initiator, termed secondary immune-mediated hemolytic anemia. although the cause of idiopathic immune-mediated hemolytic anemia is unknown, certain dog breeds (e.g., cocker spaniels) are predisposed to developing disease, suggesting the possibility of a genetic component. causes of secondary immune-mediated hemolytic anemia include certain infections (e.g., hemoplasmosis, babesiosis, and theileriosis), drugs (e.g., cephalosporins, penicillin, and sulfonamides), vaccines, and envenomations (e.g., bee stings). immune-mediated hemolysis directed at nonself antigens, such as in neonatal isoerythrolysis, is presented later. in most cases of idiopathic immune-mediated hemolytic anemia, the reactive antibody is igg, and the hemolysis is extravascular (i.e., erythrocytes with surface-bound antibody are phagocytized by macrophages, mainly in the spleen). igm and/or complement proteins may also contribute to idiopathic immune-mediated hemolytic anemia. complement factor c b usually acts as an opsonin that promotes phagocytosis and extravascular hemolysis. however, formation of the complement membrane attack complex on red blood cell surfaces causes intravascular hemolysis; this mechanism more commonly occurs with igm autoantibodies. most immunoglobulins implicated in immune-mediated hemolytic anemia are reactive at body temperature (warm hemagglutinins). a smaller portion, usually igm, are more reactive at lower temperatures, young (hours to days old) with typical gross and microscopic changes of immune-mediated hemolytic anemia. pure red cell aplasia. pure red cell aplasia (prca) is a rare bone marrow disorder characterized by absence of erythropoiesis and severe nonregenerative anemia. primary and secondary forms of pure red cell aplasia have been described in dogs and cats. primary pure red cell aplasia is apparently caused by immune-mediated destruction of early erythroid progenitor cells, a presumption supported by the response of some patients to immunosuppressive therapy and by the detection of antibodies inhibiting erythroid colony formation in vitro in some dogs. administration of recombinant human erythropoietin (rhepo) has been identified as a cause of secondary pure red cell aplasia in dogs, cats, and horses, presumably caused by induction of antibodies against rhepo that cross-react with endogenous epo. experimentation with the use of speciesspecific recombinant epo has produced mixed results. dogs treated with recombinant canine epo have not developed pure red cell aplasia. however, in experiments reported thus far involving cats treated with recombinant feline epo, at least some animals have developed pure red cell aplasia. parvoviral infection has been suggested as a possible cause of secondary pure red cell aplasia in dogs. infection with felv subgroup c causes secondary erythroid aplasia in cats, probably because of infection of early-stage erythroid precursors. grossly, animals with pure red cell aplasia have pale mucous membranes without indicators of hemolysis (e.g., icterus). microscopic examination of the bone marrow shows an absence or near absence of erythroid precursors with or without lymphocytosis, plasmacytosis, and myelofibrosis; production of other cell lines (e.g., neutrophils and platelets) is normal or hyperplastic. immune-mediated neutropenia. immune-mediated neutropenia is a rare condition that has been reported in horses, dogs, and cats. this disease is characterized by severe neutropenia from immune-mediated destruction of neutrophils or their precursors. the range of causes is presumably similar to that of other immunemediated cytopenias (e.g., immune-mediated hemolytic anemia, pure red cell aplasia, and immune-mediated thrombocytopenia). affected animals may have infections, such as dermatitis, conjunctivitis, or vaginitis, which are secondary to marked neutropenia and a compromised innate immune system. microscopically, there may be neutrophil hyperplasia, maturation arrest, or aplasia in the bone marrow, depending on which neutrophil maturation stage is targeted causing a condition known as cold hemagglutinin disease. this results in ischemic necrosis at anatomic extremities (e.g., tips of the ears), where cooling of the circulation causes autoagglutination of erythrocytes and occlusion of the microvasculature. typically immunemediated hemolytic anemia targets mature erythrocytes, causing a marked regenerative response. however, as discussed earlier in the chapter, immune-mediated destruction of immature erythroid cells in the bone marrow may also occur, resulting in nonregenerative anemia. pathogenesis of secondary immune-mediated hemolytic anemia is dependent upon the cause. erythrocytic parasites may cause immune-mediated hemolysis by altering the red blood cell surface and exposing "hidden antigens" that are not recognized as selfantigens by the host's immune system. alternatively, the immune attack may be directed at the infectious agent, but erythrocytes are nonspecifically destroyed because of their close proximity-this is called the innocent bystander mechanism. certain drugs, such as penicillin, may cause immune-mediated hemolytic anemia by binding to erythrocyte membranes and forming drug-autoantigen complexes that induce antibody formation, termed hapten-dependent antibodies. other proposed mechanisms include binding of drugantibody immune complexes to the erythrocyte membrane, or induction of a true autoantibody directed against an erythrocyte antigen. hematologic, gross, and histopathologic abnormalities are typical of those of hemolytic anemia, as presented in the earlier section on bone marrow and blood cells, dysfunction/responses to injury, blood cells, abnormal concentrations of blood cells, anemia). in brief, there may be spherocytes and autoagglutination on blood smear evaluation, icterus and splenomegaly on gross examination, and emh, erythrophagocytic macrophages, and hypoxia-induced or thromboemboli-induced tissue necrosis on histopathologic examination. dogs with immune-mediated hemolytic anemia also frequently develop an inflammatory leukocytosis and coagulation abnormalities (prolonged coagulation times, decreased plasma antithrombin concentration, increased plasma concentration of fibrin degradation products, thrombocytopenia, and disseminated intravascular coagulation). intravascular hemolysis plays a relatively insignificant role in most cases of immune-mediated hemolytic anemia, but evidence of intravascular hemolysis (e.g., ghost cells, red plasma and urine, dark red kidneys) is noted occasionally, presumably in those cases in which igm and complement are major mediators of hemolysis. neonatal isoerythrolysis. neonatal isoerythrolysis (ni) is a form of immune-mediated hemolytic anemia in which colostrumderived maternal antibodies react against the newborn's erythrocytes. it is common in horses ( fig. - ) and has been reported in cattle, cats, and some other domestic and wildlife species. in horses, neonatal isoerythrolysis occurs as a result of immunosensitization of the dam from exposure to an incompatible blood type inherited from the stallion (e.g., transplacental exposure to fetal blood during pregnancy or mixing of maternal and fetal blood during parturition). a previously mismatched blood transfusion produces the same results. some equine blood groups are more antigenic than others; in particular, types aa and qa are very immunogenic in mares. in cattle, neonatal isoerythrolysis has been caused by vaccination with whole blood products or products containing erythrocyte membrane fragments. neonatal isoerythrolysis has been produced experimentally in dogs, but there are no reports of naturally occurring disease. in cats the recognized form of neonatal isoerythrolysis does not depend on prior maternal immunosensitization but on naturally occurring anti-a antibodies in queens with type b blood. affected animals are s l rather a secondary complication of many types of underlying disease, including severe inflammation, organ failure, and neoplasia. it is included in the section on inflammatory disorders because the coagulation cascade is closely linked to inflammatory pathways. information on this topic, including e- fig. - , is available at www.expertconsult.com. as well as in chapter . the term hematopoietic neoplasia encompasses a large and diverse group of clonal proliferative disorders of hematopoietic cells. historically, numerous systems have been used to classify hematopoietic neoplasms in human medicine, some of which have been applied inconsistently to veterinary species (examples include the kiel classification and national cancer institute working formulation). the world health organization (who) classification of hematopoietic neoplasia was first published in (updated in ) and is based on the principles defined in the revised european-american classification of lymphoid neoplasms (real) from the international lymphoma study group. the who classification system is considered the first true worldwide consensus on the classification of hematopoietic malignancies and integrates information on tumor topography, cell morphology, immunophenotype, genetic features, and clinical presentation and course. a veterinary reference of the who classification system, published in , was later validated in using the canine model of lymphoma. this project, modeled after the study to validate the system in human beings, yielded an overall accuracy (i.e., agreement on a diagnosis) among pathologists of %. currently this classification system is accepted as the method of choice in both human and veterinary medicine. the who classification broadly categorizes neoplasms primarily according to cell lineage: myeloid, lymphoid, and histiocytic. this distinction is based on the fact that the earliest commitment of a pluripotent hsc is to either a lymphoid or nonlymphoid lineage. many pathologists and clinicians distinguish leukemias from other hematopoietic neoplasms. leukemia refers to a group of hematopoietic neoplasms that arise from the bone marrow and are present within the blood. leukemia may be difficult to differentiate from other forms of hematopoietic neoplasms that originate outside of the bone marrow but infiltrate the bone marrow and blood. for simplicity, cases of secondary bone marrow or blood involvement may not be considered leukemia but rather the "leukemic phase" of another primary neoplasm. it is now recognized that certain lymphomas and leukemias are different manifestations of the same disease (e.g., chronic lymphocytic leukemia and small lymphocytic lymphoma), and the designation of lymphoma or leukemia is placed on the tissue with the largest tumor burden. based on their degree of differentiation, leukemias are classified as acute or chronic. acute leukemias are poorly differentiated or undifferentiated, meaning that there are high percentages of early progenitor and precursor cells, including lymphoblasts, myeloblasts, monoblasts, erythroblasts, and/or megakaryoblasts. in contrast, well-differentiated cells predominate in chronic leukemias. because well-differentiated cells also predominate with nonneoplastic proliferations, chronic leukemias must be differentiated from reactive processes, such as those cells that occur in chronic and/or granulomatous inflammation. diagnosis of chronic leukemia is often made by excluding all other causes for the proliferating cell type. for example, causes of relative and secondary erythrocytosis are excluded to be able to diagnose polycythemia vera. furthermore, the designation of acute or chronic also refers to the disease's clinical course. acute leukemias tend to have an acute onset of severe and rapidly progressive clinical signs, whereas animals with chronic leukemia for destruction. marrow lymphocytosis and plasmacytosis may be marked (e.g., > % of nucleated cells). the diagnosis may be supported by flow cytometric detection of immunoglobulin bound to neutrophils but is most often made on the basis of exclusion of other causes of neutropenia and response to immunosuppressive therapy. immune-mediated thrombocytopenia. immune-mediated thrombocytopenia (imtp) is a condition characterized by immunemediated destruction of platelets. it is a fairly common condition in dogs and is less frequent in horses and cats. the disease is usually idiopathic but may be secondary to infection (e.g., equine infectious anemia and ehrlichiosis), drug administration (e.g., cephalosporins and sulfonamides), neoplasia, and other immunemediated diseases. when immune-mediated thrombocytopenia occurs together with immune-mediated hemolytic anemia, the condition is called evans's syndrome. the thrombocytopenia is often severe (e.g., < , platelets/µl), resulting in varying degrees of bleeding tendencies, mainly in skin and mucous membranes. microscopically, there are multifocal perivascular hemorrhages in multiple tissues, and the bone marrow exhibits megakaryocytic and erythroid hyperplasia. rarely, immune-mediated destruction of megakaryocytes may cause megakaryocytic hypoplasia, termed amegakaryocytic thrombocytopenia. neonatal alloimmune thrombocytopenia. a form of immune-mediated thrombocytopenia, known as neonatal alloimmune thrombocytopenia, is recognized in neonatal pigs and foals. the pathogenesis of this disease is virtually identical to that of neonatal isoerythrolysis as a cause of anemia: a neonate inheriting paternal platelet antigens absorbs maternal antibodies against these antigens through the colostrum. in principle, a similar situation may occur after platelet-incompatible transfusion of blood or blood products containing platelets. gross and microscopic changes are similar to those of immune-mediated thrombocytopenia except that the animal is young (e.g., to days). hemophagocytic syndrome. hemophagocytic syndrome is a term used to describe the proliferation of nonneoplastic (i.e., polyclonal), well-differentiated but highly erythrophagic macrophages. the condition is rare but has been recognized in dogs and cats. unlike hemophagocytic histiocytic sarcoma, which is a neoplastic proliferation of phagocytic macrophages, hemophagocytic syndrome is secondary to an underlying disease, such as neoplasia, infection, or an immune-mediated disorder. the primary disease process causes increased production of stimulatory cytokines, which results in macrophage proliferation and hyperactivation. these activated macrophages phagocytize mature hematopoietic cells and hematopoietic precursors at an enhanced rate, resulting in one or more cytopenias. affected animals usually have lesions of the primary disease, as well as signs of the anemia (e.g., pale mucous membranes), neutropenia (e.g., bacterial infections), and thrombocytopenia (e.g., petechiae and ecchymoses). microscopically, phagocytic macrophages are found in high numbers in the bone marrow and commonly in other tissues, including lymph nodes, spleen, and liver. additional bone marrow findings reported in animals with hemophagocytic syndrome vary widely, ranging from hypoplasia to hyperplasia of cell lines with peripheral cytopenias. disseminated intravascular coagulation. disseminated intravascular coagulation is a syndrome characterized by continuous activation of both coagulation and fibrinolytic pathways and is also known as consumptive coagulopathy. it is not a primary disease, but .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system disseminated intravascular coagulation is a consumptive coagulopathy resulting from activation of both coagulation and fibrinolytic pathways. it is a secondary complication of many types of underlying disease, including many infectious diseases, trauma, burns, heat stroke, immune-mediated disease, hemolysis, shock, neoplasia, organ failure, obstetric complications, and noninfectious inflammatory disease, such as pancreatitis. it is common in critically ill domestic animals. disseminated intravascular coagulation involves an initial hypercoagulable phase, resulting in thrombosis and ischemic tissue damage, and a subsequent hypocoagulable phase as a result of consumption of coagulation factors and platelets, resulting in hemorrhage (e- fig. - ) . the pathogenesis of disseminated intravascular coagulation typically involves the release of tissue factor (thromboplastin) and subsequent activation of coagulation pathways and platelets but may also involve defective normal inhibition of coagulation or defective fibrinolysis. classically diagnosis of disseminated intravascular coagulation is based on clinical evidence of hemorrhage and/or thromboembolic disease and a triad of laboratory findings: thrombocytopenia, usually moderate (below the lower reference value but above , /µl); prolonged coagulation times (prothrombin time and/or partial thromboplastin time); and decreased fibrinogen or increased concentration of plasma fibrin degradation products or d-dimer. milder forms of disseminated intravascular coagulation that do not meet all of the diagnostic criteria also occur. decreased plasma antithrombin (antithrombin iii) concentration and schistocytosis are other laboratory abnormalities often found in patients with disseminated intravascular coagulation. dysplasia of myeloid cells, and fewer than % myeloblasts and "blast equivalents." acute myeloid leukemia. acute myeloid leukemia (aml) is uncommon in domestic animals but most frequently occurs in dogs and cats. in veterinary species, acute myeloid leukemia is most commonly of neutrophil, monocyte, and/or erythroid origin, with rare reports of eosinophil, basophil, or megakaryocytic lineages. it is caused by felv infection in cats. evaluations of blood smears show many early myeloid precursors, including myeloblasts and blast equivalents ( fig. - , a) . in dogs the total leukocyte concentration averages approximately , /µl; anemia, neutropenia, and thrombocytopenia commonly occur. grossly, animals show lesions attributed to anemia, neutropenia, and thrombocytopenia, such as pale mucous membranes, secondary infections, and multisystemic bleeding, respectively. neoplastic cells often infiltrate tissues, resulting in splenomegaly, hepatomegaly, and lymphadenomegaly. microscopically, myeloid cells efface (replace) the bone marrow and infiltrate extramedullary tissues, especially lymphoid tissue. chronic myeloid leukemia. chronic myeloid leukemia (cml), also called chronic myelogenous leukemia or myeloproliferative neoplasia, is rare in animals. most reported cases occur in dogs and cats. there are various subclassifications of chronic myeloid leukemia, including excessive production of erythrocytes (polycythemia vera), platelets (essential thrombocythemia), neutrophils (chronic neutrophilic leukemia), monocytes (chronic monocytic leukemia), neutrophils and monocytes (chronic myelomonocytic leukemia), eosinophils (chronic eosinophilic leukemia), or basophils (chronic basophilic leukemia). complete peripheral blood count analysis often reveals very high concentrations of the neoplastic cells, such as greater than , to , leukocytes/µl (see fig. - , b) or , , platelets/µl. cellular morphologic features are often normal, but slight dysplasia may be observed. later in the disease there may be cytopenias of nonneoplastic cell types. animals with polycythemia vera often have red mucous membranes and lesions of hyperviscosity syndrome, such as bleeding and dilated, tortuous retinal vessels. essential thrombocythemia results in multisystemic bleeding due to dysfunctional platelets, or multisystemic infarcts from hyperaggregability and excessive platelets. chronic myeloid leukemias of leukocytes often result in splenomegaly, hepatomegaly, and lymphadenomegaly because of infiltration by the neoplastic cells. histologically, the bone marrow shows proliferation of the neoplastic cell type characterized by dysplasia and low numbers (e.g., < %) of myeloblasts and blast equivalents. mast cell neoplasia. mast cell tumors (mcts) of the skin and other sites are common in animals (see chapters , , and ), but mast cell leukemia is rare. in cats, mcts are the most common neoplasm in the spleen (e- fig. - ) . mast cells normally are not present in the blood vascular system, but the finding of mast cells in the blood (mastocytemia) is highly suggestive of disseminated mast cell neoplasia (systemic mastocytosis) in cats. however, mastocytemia does not necessarily indicate myeloid neoplasia in dogs. in fact, one study found that the severity of mastocytemia in dogs was frequently higher in animals without mcts than those with mcts and that random detection of mast cells in blood smears usually is not the result of underlying mct. granulocytic sarcoma. granulocytic sarcoma is a poorly characterized extramedullary proliferation of myeloid precursors, most typically have indolent, slowly progressive disease. this classification scheme is summarized in table - . subcategories exist within each of these groups, as discussed further later. information on this topic is available at www.expertconsult.com. this section discusses examples of myeloid neoplasms, including myelodysplastic syndrome, myeloid leukemias, and mast cell neoplasms (technically a form of myeloid neoplasia), and lymphoid neoplasms, including lymphoid leukemias and multiple myeloma. other lymphoid neoplasms, such as the numerous subtypes of lymphoma and extramedullary plasmacytomas (emps), as well as histiocytic disorders are described in the section on lymphoid/lymphatic system, disorders of domestic animals, neoplasia. additional discussion of hematopoietic neoplasia occurs in the species-specific sections at the end of this chapter. myelodysplastic syndrome. myelodysplastic syndrome (mds) most commonly occurs in dogs and cats and may be caused by felv infection in cats. the disease refers to a group of clonal myeloid proliferative disorders with ineffective hematopoiesis in the bone marrow, resulting in cytopenias of more than one cell line. hematopoietic proliferation in bone marrow with concurrent peripheral blood cytopenias is likely a result of increased apoptosis of neoplastic cells within the bone marrow, before their release into circulation. clinical illness and death often result from secondary manifestations, such as secondary infections or cachexia, attributable to the effects of cytopenias and/or transformation of the neoplasm into acute myeloid leukemia. gross lesions are dependent upon the type and severity of the cytopenias. however, essential microscopic findings within the bone marrow are normal or increased cellularity, "blast equivalents" include other stages of immature myeloid cells, such as abnormal promyelocytes, monoblasts, promonocytes, erythroblasts, and megakaryoblasts. before the discussion of specific diseases, it is worthwhile to describe the diagnostic techniques required to classify hematopoietic neoplasms that are becoming increasingly available for routine use in veterinary medicine. immunophenotyping refers to the use of antibodies recognizing specific molecules expressed on different cell types to determine the identity of a cell population of interest. immunophenotyping on the basis of these lineage-specific or lineage-associated markers can be performed on histologic sections (immunohistochemistry [see fig. - ]), air-dried cytologic examination smears (immunocytochemistry), or by laser analysis of cells in suspension in blood or buffer solutions (flow cytometry). in cases of lymphoid neoplasia, immunophenotyping most routinely refers to determination of b or t lymphocyte origin. clonality assays, pcr for antigen receptor rearrangement (parr), can help identify neoplastic lymphoid proliferations on the basis of clonal rearrangements of genes encoding lymphocyte antigen receptors. in terms of practical application the parr assay is most useful in helping to distinguish lymphoid neoplasms from those nonneoplastic lymphoid proliferations mimicking neoplasia. cytogenetic testing has not been routinely used in veterinary medicine, though several genetic mutations have been identified in dogs. for example, breakpoint cluster region-abelson (bcr-abl) translocations have been identified in some canine leukemias, including acute myeloblastic leukemia and chronic monocytic leukemia. dogs with burkitt-like lymphoma have a translocation leading to constitutive c-myc expression. a b thrombocytopenia commonly occur. gross and microscopic lesions also are similar to those that occur in cases of acute myeloid leukemia, except that neoplastic cells may differentiate into morphologically identifiable lymphoid cells. chronic lymphocytic leukemia. chronic lymphocytic leukemia (cll) is uncommon in veterinary medicine. it is predominantly a disease of middle-aged to older dogs but is also documented in horses, cattle, and cats. most canine chronic lymphocytic leukemia cases are of t lymphocyte origin, typically cytotoxic t lymphocytes expressing cd . in cats the majority of chronic lymphocytic leukemia cases have a t helper lymphocyte immunophenotype. a cbc often shows very high numbers of small lymphocytes with clumped chromatin and scant cytoplasm. proliferating cytotoxic t lymphocytes frequently contain a few pink cytoplasmic granules when stained with most methanol-based romanowsky stains (e.g., wright-giemsa). however, these granules may not be appreciated with some aqueous-based romanowsky stains (e.g., diff-quik). although the number of total blood lymphocytes is often greater than , /µl, relatively mild lymphocytosis (e.g., , /µl) has been reported. seventy-five percent of affected dogs also have often of eosinophilic or neutrophilic cell lines. although rare, there are reports of granulocytic sarcoma in dogs, cats, cattle, and pigs, and it may arise in a number of sites, such as lung, intestine, lymph nodes, liver, kidney, skin, and muscle. acute lymphoblastic leukemia. acute lymphoblastic leukemia (all) is uncommon in dogs and cats, and rare in horses and cattle. in a recent immunophenotype study of cases of acute lymphoblastic leukemia in dogs, arose from b lymphocytes and arose from double-negative t lymphocytes that were immunonegative for cd and cd markers. in the blood of animals with acute lymphoblastic leukemia, there are typically many medium to large lymphoid cells with deeply basophilic cytoplasm, reticular to coarse chromatin, and prominent, multiple nucleoli (see fig. - , c) . in affected dogs the mean blood lymphoid concentration is approximately , /µl, but cats with acute lymphoblastic leukemia often have low numbers of neoplastic cells in the circulation. as with animals with acute myeloid leukemia, anemia, neutropenia, and c d m the light chains deposit as nonamyloid granules, it is termed light chain deposition disease. light chains are low-molecular-weight proteins that pass through the glomerular filter into the urine, wherein they are also known as bence jones proteins. they tend to not react with urine dipstick protein indicators and are most specifically detected by electrophoresis and immunoprecipitation. in addition to aiding in the diagnosis of multiple myeloma, paraproteins have an important role in pathogenesis of disease. these proteins may inhibit platelet function, increase blood viscosity, deposit in glomerular basement membranes (see chapter ; see figs. - and - , or precipitate at cool temperatures, which results in bleeding tendencies, hyperviscosity syndrome, glomerulopathies, and cryoglobulinemia, respectively. hyperviscosity syndrome refers to the clinical sequelae of pathologically increased blood viscosity, which are slowed blood flow and loss of laminar flow. clinical signs include mucosal hemorrhages, visual impairment due to retinopathy, and neurologic signs, such as tremors and abnormal aggressive behavior. cryoglobulinemia is the condition in which proteins, typically igm, precipitate at temperatures below normal body temperature (cold agglutinins). precipitation often occurs in blood vessels of the skin and extremities, such as the ears and digits, and results in ischemic necrosis. in multiple myeloma the neoplastic proliferation of plasma cells results in osteolysis. work with human cell cultures has shown that anemia, and % have thrombocytopenia. autopsy findings depend on the stage of disease. in advanced cases with marked infiltration of organs with neoplastic cells, there is often uniform splenomegaly, hepatomegaly, and lymphadenomegaly, and the bone marrow is highly cellular (e- fig. - ; see fig. - , d) . other lesions depend on whether there are concurrent cytopenias, such as anemia, neutropenia, and thrombocytopenia, and if the neoplastic cells produce excessive immunoglobulin. lesions caused by excessive immunoglobulin are further discussed in the section on multiple myeloma. histologically, the bone marrow is densely cellular with welldifferentiated lymphocytes. small lymphocytes infiltrate and often efface in the architecture of the lymph nodes and spleen. the liver may have dense accumulations of neoplastic cells in the connective tissue around the portal triad. plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone; the latter is discussed in the section on lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia. multiple myeloma. multiple myeloma (mm) is a rare, malignant tumor of plasma cells that arises in the bone marrow and usually secretes large amounts of immunoglobulin. the finding of neoplastic plasma cells in blood samples or smears is rare. dogs are affected more frequently than other species, but multiple myeloma has also been reported in horses, cattle, cats, and pigs. diagnosis of multiple myeloma is based on finding a minimum of two or three (opinions vary) of the following abnormalities: • markedly increased numbers of plasma cells in the bone marrow ( fig. - , a) • monoclonal gammopathy • radiographic evidence of osteolysis • light chain proteinuria the classic laboratory finding in patients with multiple myeloma is hyperglobulinemia, which results from the excessive production of immunoglobulin or an immunoglobulin subunit by the neoplastic cells. this homogeneous protein fraction is often called paraprotein or m protein. paraproteins produced from the same clone of plasma cells have the same molecular weight and electric charge. therefore they have the same migration pattern using serum protein electrophoresis, which results in a tall, narrow spike in the globulins region, termed monoclonal gammopathy (see fig. - , b) . the term gammopathy is used because most immunoglobulins migrate in the γ-region of an electrophoresis gel. however, some immunoglobulins, especially immunoglobulin a (iga) and igm, migrate to the β-region. occasionally, biclonal or other atypical electrophoretic patterns may be seen with multiple myeloma as a result of protein degradation, protein complex formation, binding to other proteins, or when the tumor includes more than one clonal population. it is important to note that monoclonal gammopathy is not specific to multiple myeloma but has also been reported with lymphoma, chronic lymphocytic leukemia, canine ehrlichiosis, and canine leishmaniasis. definitively distinguishing monoclonal from polyclonal gammopathy requires immunoelectrophoresis or immunofixation using species-specific antibodies recognizing different immunoglobulin subclasses and subunits. occasionally, multiple myeloma cells produce only the immunoglobulin light chain. an immunoglobulin monomer consists of two heavy chains and two light chains connected by disulfide bonds. these light chains may deposit in tissues and cause organ dysfunction, especially renal failure. when the light chains form amyloid deposits, the disease is called amyloid light chain amyloidosis. but if marrow is dark red as a result of replacement of fat by hematopoietic tissue; the extent of replacement is an indication of the duration of the anemia. the severity of microscopic lesions is dependent on the chronicity of the disease, and they are most significant in the spleen, liver, and bone marrow. as would be anticipated, microscopic findings of the spleen are predominantly influenced by the number and activity of macrophages, which is a reflection of the duration of the disease and the frequency of hemolytic episodes. hemosiderin-laden macrophages persist for months to years; therefore large numbers are consistent with chronicity. kupffer cell hyperplasia with hemosiderin stores and periportal infiltrates of lymphocytes are the most significant changes in the liver. bone marrow histologic findings vary depending on the duration of the disease. in most animals the marrow is cellular because of the replacement of fat by intense, orderly erythropoiesis. granulocytes are relatively less numerous, and plasma cells are increased. as in the spleen, hemosiderin-laden macrophages are present in large numbers in chronic cases. emaciated animals with chronic disease have serous atrophy of fat (see e- fig. - ) . clinical findings with viremic episodes include fever, depression, icterus, petechial hemorrhages, lymph node enlargement, and dependent edema. equine infectious anemia infection is diagnosed on the basis of the coggins test, an agarose gel immunodiffusion test for the presence of the antibody against the virus. congenital dyserythropoiesis in polled herefords. a syndrome of congenital dyserythropoiesis and alopecia occurs in polled hereford calves. the cause and pathogenesis of this often fatal disease are unknown. early in disease there is hyperkeratosis and alopecia of the muzzle and ears, which progresses to generalized alopecia and hyperkeratotic dermatitis. histologically, there is orthokeratotic hyperkeratosis with dyskeratosis, as well as erythroid hyperplasia, dysplasia, and maturation arrest in the bone marrow. ineffective erythropoiesis results in nonregenerative to poorly regenerative anemia. erythrocyte band is integral membrane protein that connects to the cytoskeleton and aids in erythrocyte stability. a hereditary deficiency of this protein has been identified in japanese black cattle, resulting in increased erythrocyte fragility, spherocytosis, intravascular hemolytic anemia, and retarded growth. affected calves show lesions consistent with hemolytic anemia, including pale mucous membranes, icterus, and splenomegaly. histologically, there are bilirubin accumulations in the liver, and hemosiderin in renal tubules. bovine leukemia virus. bovine leukemia virus is discussed in the later section on lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). bovine viral diarrhea virus. bvdv infection may cause thrombocytopenia in cattle, and a thrombocytopenic hemorrhagic syndrome has been specifically caused by type ii bvdv infection. investigations of the mechanism of bvdv-induced thrombocytopenia have resulted in varying, sometimes conflicting, conclusions. more than one study has shown viral antigen within bone marrow megakaryocytes and circulating platelets. evidence of impaired thrombopoiesis (megakaryocyte necrosis, megakaryocyte pyknosis, osteoclasts support the growth of myeloma cells, and that direct contact between the two cell types increases the myeloma cell proliferation and promotes osteoclast survival. increased osteoclast activity causes osteolysis, but the exact mechanism is not known. osteolysis often results in bone pain, lytic bone lesions on radiographs, hypercalcemia, and increased serum alkaline phosphatase activity. later in disease, osteolysis may cause pathologic fractures. morphologically, myeloma cells tend to grow in sheets that displace normal hematopoietic cells in the bone marrow. a proposed diagnostic criterion of multiple myeloma is that plasma cells constitute % or more of the nucleated cells in the marrow. welldifferentiated plasma cells are round with abundant basophilic cytoplasm (due to increased rough endoplasmic reticulum) and a perinuclear pale zone (enlarged golgi apparatus for the production of immunoglobulin); anisocytosis and anisokaryosis are often mild but may be marked. some plasma cell neoplasms have a bright eosinophilic fringe due to accumulated iga (see fig. - , a) . nuclei are round with clumped chromatin and often peripherally placed with the cytoplasm; binucleation and multinucleation are common. poorly differentiated myeloma cells may lack and/or display less characteristic features. osteolysis of bone may be present microscopically. common sites of metastasis include the spleen, liver, lymph nodes, and kidneys. flavin adenine dinucleotide deficiency. flavin adenine dinucleotide (fad) is a cofactor for cytochrome-b reductase, the enzyme that maintains hemoglobin in its functional reduced state, and for glutathione reductase, an enzyme that also protects erythrocytes from oxidative damage. reported in a spanish mustang mare and a kentucky mountain saddle horse gelding, erythrocyte fad deficiency is a result of an abnormal riboflavin kinase reaction, which is the first reaction in converting riboflavin to fad. clinicopathologic changes include persistent methemoglobinemia of % to %, eccentrocytosis, a slightly decreased or normal hematocrit, and erythroid hyperplasia in the bone marrow. equine infectious anemia virus. equine infectious anemia virus (eiav), the agent of equine infectious anemia, is a lentivirus that infects cells of the monocyte-macrophage system in horses (also ponies, donkeys, and mules). the virus is mechanically transmitted by biting flies, such as horseflies and deer flies. less common routes of transmission include blood transfusions, contaminated medical equipment, and transplacentally. disease may present in acute, subacute, and chronic forms and is potentially fatal. after an acute period of fever, depression, and thrombocytopenia that lasts to days, there is a prolonged period of recurrent fever, thrombocytopenia, and anemia. in most cases, clinical disease subsides within a year, and horses become lifelong carriers and reservoirs of eiav. eiav causes anemia by both immune-mediated hemolysis and decreased erythropoiesis. hemolysis is typically extravascular but may have an intravascular component during the acute phase. decreased erythropoiesis may result from direct suppression of earlystage erythroid cells by the virus, as well as anemia of inflammation. thrombocytopenia likely results from immune-mediated platelet destruction and suppressed platelet production. animals dying during hemolytic crises are pale with mucosal hemorrhages and dependent edema. the spleen and liver are enlarged, dark, and turgid, and they and other organs have superficial subcapsular hemorrhages. petechiae are evident beneath the renal capsule and throughout the cortex and medulla. the bone affected animals are not necessarily anemic. however, acute intravascular hemolytic episodes may occur with hyperventilationinduced alkalemia. lesions are typical of hemolytic anemia and include pale mucous membranes, icterus, hepatosplenomegaly, and dark red urine with microscopic emh and marrow erythroid hyperplasia. a single dna-based test is available to detect the common mutation. erythrocyte structural abnormalities. congenital erythrocyte structural abnormalities may occur with abnormal membrane composition or defective proteins within the membrane or cytoskeleton. some of these morphologic changes occur concurrently with clinical disease, but others do not. hereditary stomatocytosis is recognized in alaskan malamutes, drentse patrijshonds, and schnauzers. the specific defects are not known, but they are likely different in the various dog breeds. however, all affected dogs have stomatocytes on blood smear evaluation, as identified by their slit-shaped area of central pallor. erythrocytes also have increased osmotic fragility and decreased survival. schnauzers are clinically healthy and not anemic but do have reticulocytosis, suggesting that the hemolytic anemia is compensated by erythroid hyperplasia. mild to marked hemolytic anemia is documented in alaskan malamutes and drentse patrijshonds. alaskan malamutes have concurrent short-limb dwarfism, and drentse patrijshonds have hypertrophic gastritis and polycystic kidney disease. other (presumably heritable) erythrocyte abnormalities in dogs that do not have clinical signs include elliptocytosis caused by band . deficiency or β-spectrin mutation, and familial macrocytosis and dyshematopoiesis in poodles. scott's syndrome. an inherited thrombopathy resembling scott's syndrome in human beings, in which platelets lack normal procoagulant activity, has been recognized in a family of german shepherd dogs. the specific defect in these dogs has not been identified on the molecular level but involves impaired expression of phosphatidylserine on the platelet surface. affected dogs have a mild to moderate clinical bleeding tendency characterized by epistaxis, hyphema, intramuscular hematoma formation, and increased hemorrhage with surgery. macrothrombocytopenia. macrothrombocytopenia is an inherited condition in cavalier king charles spaniels in which there are lower than normal concentrations of platelets with enlarged and giant platelets. the condition is caused by defective β -tubulin, which results in impaired microtubule assembly. affected dogs are asymptomatic but may have abnormal platelet aggregation in vitro. canine distemper. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells and is presented in greater detail in the lymphoid section. canine distemper virus may also infect other hematopoietic cells, including erythrocytes, nonlymphoid leukocytes, and platelets ( fig. - ) , and can cause decreased peripheral blood concentrations of neutrophils, lymphocytes, monocytes, and platelets during viremia. the thrombocytopenia is a result of virus-antibody immune complexes on platelet membranes and direct viral infection of megakaryocytes. increased erythrocyte osmotic fragility. a condition characterized by increased erythrocyte osmotic fragility has been described in abyssinian and somali cats. the specific defect has and degeneration) and increased thrombopoiesis (megakaryocytic hyperplasia, increased numbers of immature megakaryocytes) in the bone marrow has been reported in type ii bvdv-infected animals, including concurrent megakaryocyte necrosis and hyperplasia in some experimental subjects. calves infected with type ii bvdv also have impaired platelet function. cattle with the hemorrhagic syndrome are severely thrombocytopenic and neutropenic with multisystemic hemorrhages, particularly of the digestive tract, spleen, gallbladder, urinary bladder, and lymph nodes. histologic lesions include hemorrhage, epithelial necrosis of enterocytes, intestinal erosions, crypt proliferation with microabscesses, and lymphoid depletion of the gut-associated lymphoid tissue, peyer's patches, and spleen. lesions of the bone marrow are variable, as previously described. bovine neonatal pancytopenia. bovine neonatal pancytopenia (bnp) is caused by alloantibodies absorbed from colostrum, resulting in a hemorrhagic syndrome in calves. the syndrome was first recognized in europe in the early s and has since been experimentally correlated with prior vaccination of affected calves' dams with a commercial bvdv vaccine (pregsure bvd; pfizer animal health). the vaccine has since been voluntarily recalled from the market. it is thought that vaccination induces alloantibody formation by the dam. the alloantibodies are ingested by the calf and bind to the calf's hematopoietic progenitor cells, resulting in functional compromise of those cells. acutely affected calves are less than a year of age and have peripheral thrombocytopenia and neutropenia. death results from thrombocytopenia-induced hemorrhages or neutropenia-induced secondary infections, including pneumonia, enteritis, and septicemia. within the bone marrow there is erythroid, myeloid, and megakaryocytic hypoplasia. cyclic hematopoiesis. cyclic hematopoiesis (also known as lethal gray collie disease) is an autosomal recessive disorder of pluripotent hscs in gray collie dogs. a defect in the adaptor protein complex (ap ) results in defective intracellular signaling and predictable fluctuations in concentrations of blood cells that occur in -day cycles. the pattern is cyclic marked neutropenia, and in a different phase, cyclic reticulocytosis, monocytosis, and thrombocytosis. production of key cytokines involved in regulation of hematopoiesis is also cyclic. neutropenia predisposes affected animals to infection, and many die of infectious causes. affected animals have dilute hair coats and lesions with acute or chronic infectious disease, especially of the lungs, gastrointestinal tract, and kidneys. dogs older than weeks of age have systemic amyloidosis, which occurs because of cyclic increases in concentration of acute phase proteins during phases of monocytosis. phosphofructokinase deficiency. inherited autosomal recessive deficiency of the erythrocyte glycolytic enzyme, phosphofructokinase (pfk), is described in english springer spaniel, american cocker spaniel, and mixed-breed dogs. there are three genes encoding pfk enzymes, designated m-pfk in muscle and erythrocytes, l-pfk in liver, and p-pfk in platelets. a point mutation in the gene coding for m-pfk results in an unstable, truncated molecule. erythrocytes in pfk-deficient dogs have decreased atp and , -diphosphoglycerate ( , -dpg) production and increased fragility under alkaline conditions. the disease is characterized by chronic hemolysis with marked reticulocytosis. the marked regenerative response may compensate for the ongoing hemolysis; therefore erythrophagocytosis, thrombosis, and histologic changes of ischemia are common, especially within the spleen, liver, and lungs. affected cats typically become acutely ill with fever, pallor, and icterus and usually die within to days. for many years, cytauxzoonosis was considered to be almost always fatal. however, a recent not been identified, but pk deficiency (which has been reported in these breeds) was excluded as the cause. affected cats have chronic intermittent severe hemolytic anemia and often have other lesions secondary to hemolytic anemia (e.g., splenomegaly and hyperbilirubinemia). cytauxzoonosis. cytauxzoonosis is a severe, often fatal disease of domestic cats caused by the protozoal organism, cytauxzoon felis. disease is relatively common in the south central united states, particularly during summer months. bobcats (lynx rufus) and other wild felids are thought to be wildlife reservoirs of disease. c. felis is transmitted by a tick vector, dermacentor variabilis, which is probably essential for infectivity of the organism. cytauxzoonosis has a schizogenous phase within macrophages throughout the body (especially liver, spleen, lung, lymph nodes, and bone marrow) that causes systemic illness. these schizontcontaining macrophages enlarge and accumulate within the walls of veins, eventually causing vessel occlusion, circulatory impairment, and tissue hypoxia. later in disease, merozoites released from schizonts enter erythrocytes, resulting in an erythrocytic phase of infection. infected domestic cats often have nonregenerative anemia, but the pathogenesis for the anemia is unclear. however, it likely represents preregenerative hemolytic anemia because erythrocyte phagocytosis is a prominent finding in many organs. infected cats often also develop neutropenia and thrombocytopenia, which likely result from inflammation and disseminated intravascular coagulation, respectively. on blood smear evaluation, signet ring-shaped erythrocytic inclusions (piroplasms) may be observed during the erythrocytic phase of disease ( fig. - ) . these inclusions closely resemble small-form babesia (see fig. - , a) and some theileria organisms. postmortem examination typically shows pallor, icterus, splenomegaly, enlarged and red lymph nodes, diffuse pulmonary congestion and edema, and multisystemic petechiae and ecchymoses. vascular obstruction may cause marked distention of abdominal veins. cavitary effusions are present in some cats. microscopically, large, schizont-laden macrophages accumulate within venous and sinusoidal lumens and often completely occlude the lumens (fig. - ) . which the b and t lymphocytes proliferate, differentiate, and mature. in mammals, lymphocytes arise from hscs in the bone marrow, and b lymphocytes continue to develop at this site. ruminants also have b lymphocyte proliferation and maturation within their peyer's patches. progenitor t lymphocytes migrate from bone marrow to mature and undergo selection in the thymus. the spleen, lymph nodes, and lymph nodules are secondary lymphoid organs and are responsible for the immune responses to antigens, such as the production of antibody and cell-mediated immune reactions. at these sites, lymphocytes are activated by antigens and undergo clonal selection, proliferation, and differentiation (see also chapter ). in addition, the spleen and lymph nodes contain cells of the monocyte-macrophage system and thus also participate in the phagocytosis of cells and materials. the bone marrow is described in the first section of this chapter. the remaining primary lymphoid organ, the thymus, is described first in this section, followed by the secondary lymphoid organs: spleen, lymph nodes, and diffuse and nodular lymphatic tissues. errors from selection of inappropriate sampling sites and artifacts from compression and incorrect fixation for histopathologic and immunohistochemical examinations are common in routine veterinary pathologic analysis. the identification and remedies for these problems are discussed in e-appendix - . the thymus is essential for the development and function of the immune system, specifically for the differentiation, selection, and maturation of t lymphocytes generated in the bone marrow (see also chapter ). the basic arrangement of the thymus in domestic animals consists of paired cervical lobes (left and right), an intermediate lobe at the thoracic inlet, and a thoracic lobe, which may be bilobed. the cervical lobes are positioned ventrolateral to the trachea, adjacent to the carotid arteries, and extend from the intermediate lobe at the thoracic inlet as far cranially as the larynx. the intermediate lobe bridges between the cervical and the thoracic lobe. the right thoracic lobe is usually small or completely absent. the left lobe lies in the ventral aspect of the cranial mediastinum (except in the ruminant, where it is dorsal) and extends caudally as far as the pericardium. horse-the cervical lobes in foals are small, and the thoracic lobe constitutes the bulk of the thymus. ruminant-the cervical lobes are large. the left and right thoracic lobes are fused and unlike other domestic animals, lie in the dorsal aspect of the cranial mediastinum. pig-the cervical lobes are large. dog-the cervical lobes regress very early and thus appear absent. the thoracic lobe extends caudally to the pericardium. cat-the cervical lobes are small, and the thoracic lobe, which forms the majority of the thymus, extends caudally to the pericardium and molds to its surface. the thymus is referred to as a lymphoepithelial organ and hence is composed of epithelial and lymphoid tissue. formed from the endoderm of the third pharyngeal pouch in the fetus, the thymic epithelium is infiltrated by blood vessels from the surrounding mesoderm, resulting in the development of the thymic epithelial reticulum. the lymphocyte population consists of bone marrow-derived progenitor cells, which fill spaces within the epithelial network. a connective tissue capsule surrounds the thymus, and attached thin septa subdivide the tissue into partially separated lobules. each report, in which numerous cats from a subregion of the endemic area in the united states survived infection with an organism with greater than % homology to cytauxzoon felis, suggests the emergence of a less virulent strain. felv is an oncogenic, immunosuppressive lentivirus that causes hematologic abnormalities of widely varying types and severity. manifestations of disease caused by felv infection vary depending on dose, viral genetics, and host factors, but normal hematopoiesis is probably suppressed to some degree in all cases. felv infects hematopoietic precursor cells soon after the animal is exposed and continues to replicate in hematopoietic and lymphatic tissue of animals that remain persistently viremic. the virus disrupts normal hematopoiesis by inducing genetic mutations, by other direct effects of the virus on infected hematopoietic cells, or by an altered host immune system. hematologic changes include dysmyelopoiesis with resultant cytopenias or abnormal cell morphologic features, and neoplastic transformation of hematopoietic cells (leukemia). a notable form of dysplasia is the presence of macrocytic erythrocytes (macrocytes) and metarubricytosis in the absence of erythrocyte regeneration (inappropriate metarubricytosis). the relatively uncommon subgroup c viruses cause erythroid hypoplasia, probably because of infection of early-stage erythroid precursors. felv may be detected in megakaryocytes and platelets in infected cats and may result in platelet abnormalities, including thrombocytopenia, thrombocytosis, increased platelet size, and decreased function. proposed mechanisms of felv-induced thrombocytopenia include direct cytopathic effects, myelophthisis, and immunemediated destruction. platelet life span and function have been shown to be decreased in felv-positive cats. persistently viremic cats are immunosuppressed and are prone to developing other diseases, including infectious diseases, bone marrow disorders, and lymphoma. cbc abnormalities attributed to felv infection include various cytopenias, especially nonregenerative anemia, which may be persistent or cyclical. regenerative anemia may also occur with felv infection, often because of coinfection with m. haemofelis. hematopoietic cell dysplasia or neoplasia may also be evident. grossly, infected cats are often pale, but other lesions are dependent upon the presence of other cytopenias or concurrent disease. microscopically, the bone marrow is hypocellular, normocellular, or hypercellular. there may be erythroid hypoplasia, erythroid hyperplasia with maturation arrest, or acute leukemia. feline immunodeficiency virus. feline immunodeficiency virus (fiv), another feline lentivirus, causes anemia in a minority of infected cats. immunosuppressive effects of fiv from thymic depletion are discussed elsewhere. it is generally accepted that anemia does not result directly from fiv infection but instead develops because of concurrent disease such as coinfection with felv or hemotropic mycoplasma, other infection, or malignancy. the severity and type of anemia in fiv-infected cats depends on the other specific disease processes involved. the thymus, spleen, lymph nodes, and lymph nodules, including malt, are classified as part of both the lymphoid and immune systems. the lymphoid system (also known as lymphatic system in some texts) is broadly categorized into primary and secondary lymphoid organs. the main primary lymphoid organs include thymus, bone marrow, and bursa of fabricius in birds and are the sites at .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system because the thymus involutes after sexual maturity, evaluation of whether it is smaller than normal is difficult to discern unless the change is extreme or age-matched control animals are available. before sexual maturity the thymus is easily identified as a lobular white to gray organ with a thin capsule. after sexual maturity the gland is often grossly indistinguishable from adipose connective tissue within the cranial mediastinum, although microscopic remnants may remain. an extremely small thymus in a neonatal animal should be considered abnormal and may indicate a primary immunodeficiency or secondary lymphoid depletion caused by extreme stress, often due to infectious diseases. enlargement of the thymus is most often due to neoplasia. serial sectioning of the thymus allows for gross identification of neoplasms, cysts, or hematomas. spleens vary in size within the same species and among the different species of domestic animals. the spleen can be enlarged (splenomegaly), normal in size, or small (atrophy), and the surface can be smooth, wrinkled, or nodular. the appearance of the cut surface of the spleen in normal animals depends on the amount of stroma (e.g., trabeculae are prominent in ruminants); the size and visibility of the white pulp, which reflects the amount of lymphoid tissue; and whether the red pulp is congested with blood. during an autopsy (syn: necropsy), the spleen is dissected free and checked for torsion of the gastrosplenic ligament (in nonruminants). the spleen is then sliced transversely at approximately -mm intervals (serial sectioning), and the cut surfaces are checked for lesions. specimens are taken for tests that require fresh tissue (e.g., bacteriologic and virologic examinations), and the remaining cross-sections are placed in fixative ( % buffered neutral formalin). a diffusely enlarged spleen should be serially sectioned to determine if the splenomegaly is due to congestion. the cut surface of severely congested spleens is red to bluish-black and exudes blood (bloody spleens), whereas cut surfaces of noncongested enlarged spleens ooze little blood (meaty spleens) and the color depends on how much of the normal parenchyma is replaced by inflammatory cells, stored materials, or neoplastic cells (see splenomegaly and table - ). the spleen may be measured and weighed, but because of the wide variation in the dimensions and weight of normal spleens and the amount of blood stored, this information is difficult to interpret. it is essential that spleens with one or more nodules also be serially sectioned and the nodules evaluated for size, shape, and consistency. nodules may be dark red and ooze blood on cut surface, white-tan with a more firm texture, or a mixture of both. multiple wedge sections that include the interface between a nodule and the adjacent nonmass spleen should be collected, because the center of the nodules often consists only of hemorrhage and necrosis, and neoplasms may be missed. the color of the capsular surface of the spleen also varies among species of domestic animals and depends on the opacity or translucence of the splenic capsule. the degree of opacity of the capsule is a function of its thickness and the amount of collagen. the splenic capsules of horses and ruminants are thick and usually appear gray because the color of the red pulp is not visible through the capsule. in the pig, dog, and cat, the splenic capsule is thin, and thus the surface of the spleen is red. the tenseness of the capsule depends on how much the splenic parenchyma is distended; storage spleens devoid of blood usually have a wrinkled surface. irregular contraction of storage spleens is common, especially in dogs, and consists of nonuniform areas of congestion with intermingled contracted and wrinkled regions. lymph nodes should be dissected free of fat and connective tissue, and any firm attachment to adjacent tissues should be noted because these attachments may indicate neoplastic infiltration through the capsule. gross examination includes evaluating size (measurement or weight), shape, and whether the capsule is intact. the cut surface is examined for the presence of bulging tissue, edema, congestion, exudate, discoloration (see pigmentation), obscuration of the normal architecture, and masses such as abscesses, granulomas, and discrete neoplasms. cytologic evaluation of superficial lymph nodes through fine-needle aspirates provides excellent cellular detail and often yields a diagnosis. however, diagnosis of certain diseases (including lymphomas for complete world health organization [who] classification) requires architectural assessments, and therefore cytologic or small histologic samples are not sufficient. tru-cut biopsies are not ideal, but a -mm tru-cut needle may provide adequate tissue. the surgeon or pathologist must handle lymph nodes carefully to minimize artifacts. compression (e.g., squeezing with forceps) may cause crush artifacts, usually resulting in nuclear "streaming." immediately after removal, imprints/impression smears should be prepared and then kept away from formalin fumes. formalin fixation (in this case by formaldehyde fumes) destroys the differential staining seen with romanowsky stains such as wright's and giemsa and results in diffuse blue staining. prompt transfer of biopsy or postmortem specimens into fixative is crucial because delayed fixation can lead to numerous artifacts, including an artificial decrease in mitotic index (up to % reduction with a more than -hour delay in fixation); this reduction can alter tumor classification and grade. the current recommendation for the duration of formalin fixation is to hours; complete fixation of -to -mm thick tissues is likely to be achieved after to hours. both underfixation and overfixation may lead to difficulties with antigen retrieval for immunohistochemistry, though underfixation is considered the more common and serious problem. thinly slicing some nodes may be difficult, and allowing the node to fix for hour before slicing may help. some pathologists prefer not to incise very small lymph nodes to avoid compression artifacts, but instead nick the capsule to allow formalin penetration. however, fixation of unincised lymph nodes can also cause compression artifacts because the fibrous capsule contracts in the fixative. once fixed, nodes should be cut in uniformly thick cross section to include both the cortex and medulla. transverse sections are usually sufficiently small to allow the entire cross section of most lymph nodes to fit on one microscopic slide, which facilitates histologic interpretation. the longitudinal plane is preferred for porcine lymph nodes because the location and amount of cortex and medulla vary at different sites in transverse sections. molecules) but not self-antigens are permitted to mature by a process called positive selection. cells that do not recognize mhc molecules are removed by apoptosis. those t lymphocytes that recognize both mhc molecules and self-antigens are removed by macrophages at the corticomedullary junction, a process called negative selection. because of the rigid differentiation requirements attributable to mhc restriction and tolerance (positive and negative selection, respectively), only a small fraction (< %) of the developing t lymphocytes that arrive at the thymus from the bone marrow survive. mature naïve t lymphocytes exit the thymus through postcapillary venules in the corticomedullary region, enter the circulation, and recirculate through secondary lymphoid tissues, primarily located in the paracortex of lymph nodes and the periarteriolar sheaths of the spleen. in these specialized sites, the mature naïve t lymphocytes are activated upon exposure to their specific antigens and undergo additional phases of development to differentiate into effector and memory cells. the thymus attains its maximal mass relative to body weight at birth and involutes after sexual maturity; the rate of involution may vary among domestic species. the lymphoid and epithelial components are gradually replaced by loose connective tissue and fat, although remnants remain histologically, even in aged animals. lobule is composed of a central medulla and surrounding cortex (fig. - ) . the thymic cortex consists mainly of an epithelial reticulum and lymphocytes ( fig. - ) . the stellate cells of the epithelial reticulum have elongate branching cytoplasmic processes that connect to adjacent epithelial cells through desmosomes, thus forming a supportive network (cytoreticulum). the lymphoid component is composed of differentiating lymphocytes derived from progenitor (also known as precursor) t lymphocytes in the bone marrow. the medulla is composed of similar epithelial reticular cells, many of which are much larger than those in the cortex and have a more obvious epithelial structure. some of the epithelial reticular cells form thymic corpuscles, also called hassall's corpuscles, which are distinctive keratinized epithelial structures (see fig. - ). interdigitating dendritic cells (dcs) are also present within the medulla, but there are far fewer lymphocytes than in the cortex. the progenitor t lymphocytes released from the bone marrow into the blood enter the thymus in the subcapsular zone of the cortex and begin the differentiation and selection processes, developing into mature naïve t lymphocytes as they traverse the thymic cortex to the medulla. in the cortex, t lymphocytes that recognize self-molecules (major histocompatibility complex [mhc] the responses of the thymus to injury and causes are listed in boxes - and - . the most common change is lymphoid atrophy caused by physical and physiologic stresses, toxins, drugs, and viral infections. atrophy. because the thymus does not contain any lymphopoietic tissue, it depends on the bone marrow for the supply of progenitor t lymphocytes. thus thymic lymphoid atrophy can be the result of either an inadequate supply of lymphocytes from the bone marrow or lysis of lymphocytes (lymphocytolysis) in the thymus. thymic aims to define the terms used in this chapter. the term splenic sinusoid is used to describe a vascular structure present in the sinusal spleen (also known as sinusoidal spleen); dogs are the only domestic animal with true splenic sinusoids. the term red pulp vascular spaces is used (as opposed to "sinus") to describe the vascular spaces in the red pulp of both the nonsinusal and nonsinusoidal spleens of all domestic animals. the other terms used here include marginal sinus, marginal zone, periarteriolar lymphoid sheath (pals), periarteriolar macrophage sheath (pams), and splenic lymphoid follicles. the spleen is located in the left cranial hypogastric region of the abdomen, where it is typically suspended in the gastrosplenic ligament between the diaphragm, stomach, and the body wall. the exception is in domestic ruminants, where it is closely adhered to the left dorsolateral aspect of the rumen. the gross shape and size of the spleen vary markedly among domestic animals, but generally it is a flattened, elongated organ. some species, notably birds, demonstrate seasonal variation in splenic shape and size. the spleen is covered by a thick capsule composed of smooth muscle and elastic fibers, from which numerous intertwining fibromuscular trabeculae extend into the parenchyma. these trabeculae and reticular cells form a spongelike supportive matrix for the parenchyma of the mammalian spleen in all domestic species. in cattle and horses the three muscular layers of the capsule lie perpendicular to each other, forming a capsule thicker than that of carnivores. carnivores, small ruminants, and pigs have interwoven smooth muscle within the splenic capsule, and pigs also have abundant elastic fibers within the capsule. the spleen differs from many other organs in the organization of its parenchyma. instead of a cortex and medulla, the spleen is divided into two distinct structural and functional components: the red pulp and white pulp (fig. - ) . with hematoxylin and eosin (h&e) staining, red pulp appears red-pink because of the abundance of red blood cells, whereas white pulp appears blue-purple because of the heavy concentration of lymphocytes. the white pulp consists of splenic follicles, populated by b lymphocytes; the pals, inhabited by t lymphocytes; and the marginal zone at the periphery of follicles. macrophages, antigen-presenting cells, and trafficking b and t lymphocytes populate the marginal zone. the radial arteries, branches of the central artery (also known as central arteriole), and capillaries from both red and white pulp drain into the marginal sinus of the marginal zone, although the latter has not been shown to be the case in all species to the same degree (e.g., the cat has a small marginal sinus but a well-developed pams) (figs. - and - ). the red pulp consists of cells of the monocyte-macrophage system, pams, sinusoids (dogs, rats, and human beings only), red pulp vascular spaces, and associated stromal elements such as reticular cells, fibroblasts, and trabecular myocytes. the labyrinth of the splenic red pulp vascular spaces serves as both a functional and physical filter for circulating blood cells. the blood circulation of the spleen is particularly suited to enable its functions, namely, ( ) filtering and clearing the blood of atrophy must be differentiated from involution, which normally begins at sexual maturity. this distinction is difficult to make, unless the change is extreme or age-matched control animals are available for comparison. inflammation. inflammation of the thymus is rare. neutrophils and macrophages are often present within keratinized hassall's corpuscles during involution and should not be mistaken for a true thymitis. thymitis has been reported in salmon poisoning disease of dogs (see chapter ), epizootic bovine abortion (see chapter ), and in pigs infected with porcine circovirus type (pcv ). necrosis and secondary infiltrates of neutrophils and macrophages may be seen in other infectious diseases (e.g., equine herpesvirus [ehv- ]). the main portal of entry to the thymus is hematogenous. portals of entry used by microorganisms and other agents and substances to access the lymphatic system are summarized in box - . these portals include the blood vessels (hematogenous spread by microorganisms free in the plasma or within circulating leukocytes or erythrocytes), afferent lymphatic vessels (lymphatic spread), direct penetration, or through m (for "microfold") cells and dcs in malt. defense mechanisms used by the thymus to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . viruses, bacteria, and particles arriving in the lymph and blood interact with cells of the monocyte-macrophage system through phagocytosis and antigen processing and presentation. hyperplasia of the macrophages often occurs concurrently. antigen processing and presentation are followed by an immune response resulting in proliferation of b lymphocytes, plasma cells, and the subsequent production of antibody; proliferation of t lymphocytes may also occur. the relationships between anatomic structures and the different functions of the spleen are complicated. there are also anatomic differences among domestic animal species and confusion about the correct and up-to-date terminology. the following brief discussion malt, mucosa-associated lymphoid tissue. there are numerous synonyms and misuse of terms within the literature, which have contributed to the confusion over terminology for red pulp vascular spaces. these terms include reticular space, red pulp, splenic cords, sinuses, red pulp sinuses, sinus spaces, pulp spaces, mesh space of the spleen, reticular cell-lined meshwork, interstices of the reticulum network, bloodfilled reticular meshwork of the red pulp, chordal spaces, splenic cords, and cords of billroth. the latter two terms are defined as the red pulp between the sinusoids, which most domestic animals do not have (except the dog). therefore the term red pulp vascular spaces is more appropriate. as a result of this pattern of blood flow, macrophages in the marginal sinus have the first opportunity to phagocytize antigens, bacteria, particles, and other material before macrophages in the sinusoids (in the dog) or in the pams and red pulp vascular spaces (all other domestic animals). in the dog the marginal sinus drains into the sinusoids, but in other domestic animals it drains into the red pulp vascular spaces. the central arteries leave the white pulp, enter the red pulp, and branch into smaller penicillar arterioles. each arteriole is surrounded by a sheath of macrophages known as periarteriolar macrophage sheaths (pams, previously known as ellipsoids), which are notably prominent in pigs, dogs, and cats. in horses, cattle, pigs, and cats the terminal branches of the penicillar arterioles empty into the red pulp vascular spaces lined by reticular cells. because the red pulp vascular spaces are not lined by endothelium, this type of circulation is known as an open system. this system is in contrast to the sinusoidal spleen of the dog (also of the rat and human beings), where the branches of the central artery of the white pulp and vessels from the marginal sinus enter into the sinusoids, which are lined by a discontinuous endothelium, and these empty into splenic venules. this type of circulation is known as a closed system because the blood flow is through blood vessels (arterioles, capillaries, sinusoids, and venules), all of which are lined by endothelium. although circulation in the red pulp is anatomically open in nonsinusoidal spleens, under certain conditions (e.g., during splenic contraction) the circulation is functionally closed, and the blood in the red pulp is particulate matter and senescent cells; ( ) transporting recirculating lymphocytes and naïve b and t lymphocytes to the follicle and pals, respectively, to fulfill their specific immune functions; and ( ) storage of blood in some domestic animal species (dog, cat, and horse) (fig. - ) . phagocytosis is particularly effective in the spleen because blood flows through areas within the red pulp that are populated with increased concentrations of macrophages, namely, within the marginal sinuses, in cuffs around the penicillar arteries (pams), diffusely on the reticular walls of the red pulp vascular spaces, and along the sinusoids in dogs. trafficking of naïve and recirculating lymphocytes is facilitated by the proximity of the marginal sinus to the follicular germinal centers and pals. maps of the vascular blood flow in sinusoidal and nonsinusoidal spleens are illustrated in figures - to - . the celiac artery is the major branch of the abdominal aorta from which the splenic artery arises. the splenic artery enters the splenic capsule at the hilus, where it branches and enters the fibromuscular trabeculae as trabecular arteries to supply the splenic parenchyma. trabecular arteries become the central arteries of the white pulp and are surrounded by cuffs of t lymphocytes forming the pals. the splenic follicles, populated by b lymphocytes, are eccentrically embedded within or just adjacent to the pals. the central arteries send branches-the radial arteries-to supply the marginal sinus surrounding the splenic follicles. thus the cells at the circumferences of the follicles are brought into intimate contact with blood-borne antigens and trafficking b and t lymphocytes in the marginal sinus. diverted into "channels" lined by reticular cells. because the dog has both sinusoids and red pulp vascular spaces, it has both open and closed splenic circulations, which may allow for both fast and slow flows of blood depending on the physiologic need of the animal. blood flowing through the sinusoids or red pulp vascular spaces is under the surveillance of macrophages. in dogs the pseudopodia of these perisinusoidal macrophages project into the sinusoidal lumen through the spaces in the discontinuous endothelium. in all domestic animals, blood in the red pulp vascular spaces is under surveillance of macrophages attached to the reticular walls. blood from the red pulp vascular spaces and sinusoids then drains into the splenic venules, splenic veins, and ultimately into the portal vein, which empties into the liver. the spleen filters blood and removes foreign particles, bacteria, and erythrocytes that are senescent, have structural membrane abnormalities, or are infected with hemotropic parasites. as a secondary lymphoid organ, its immunologic functions include the activation of macrophages to process and present antigen, the proliferation of b lymphocytes and production of antibody and biologic molecules, and the interaction of t lymphocytes and antigens. in some species the spleen stores significant quantities of blood (box - ). the functions of the spleen are best considered on the basis of the two main components of the spleen: the red and white pulp and the anatomic systems contained within them (monocyte-macrophage system, red pulp vascular spaces, and hematopoiesis in the red pulp, and the b and t lymphocyte systems within the white pulp). monocyte-macrophage system. within the red pulp, macrophages are located in the marginal sinus, pams, and attached to the reticular walls of the red pulp vascular spaces. in the dog, macrophages are also located perisinusoidally. the supportive reticular network of the red pulp vascular spaces is composed of a fine meshwork of reticular fibers made of type iii collagen, on which macrophages are dispersed. exactly in which of these concentrations of macrophages phagocytosis of blood-borne particles takes place depends upon ( ) the sequence in which they are exposed to the incoming blood, ( ) the concentration of macrophages in these areas (e.g., the cat marginal sinus is small and thus not a major site of clearance; there is a compensatory increase in pams for phagocytosis), and ( ) the functions of the macrophages. some of the macrophages in the marginal sinus and marginal zone are responsible for phagocytosis of particulate matter and others for the trapping and ingestion of antigens and antigen-antibody complexes. macrophages responsible for phagocytosis of blood-borne foreign material (fig. - ) , bacteria, and senescent and/or damaged erythrocytes (e.g., as seen in immune-mediated anemias and infections with hemotropic parasites) are also found in the red pulp. in the dog, sinusoidal macrophages remove entire erythrocytes (erythrophagocytosis), as well as portions of an erythrocyte's membrane and cytoplasmic inclusions, such as nuclear remnants like heinz bodies, by a process called pitting. as such, the presence of large numbers of nuclear remnants in erythrocytes in canine blood smears may indicate malfunction of the sinusoidal system. the normal rate of removal of senescent erythrocytes from the circulating blood does not cause an increase in size of the spleen; however, splenomegaly can be observed when large numbers of defective erythrocytes must be removed, as in cases of severe acute hemolytic anemia. nonsinusoidal spleens lack the fenestrated endothelium and perisinusoidal macrophages of canine sinusoids that allow for slow processing of red blood cells to determine which are to be returned to the equine, canine, and feline spleens all have considerable storage and contractile capacity because of their muscular capsule, increased numbers of trabeculae, and the relatively small amount of splenic parenchyma devoted to white pulp. the storage capacity in dogs and horses is remarkable: it has been claimed that the canine spleen can store one-third of the dog's erythrocytes while the animal sleeps and the equine spleen holds one-half of the animal's circulating red cell mass (which is considered advantageous because it reduces the viscosity of the circulating blood). storage spleens expand and contract quickly under the influence of the autonomic nervous system, via sympathetic and vagal fibers in the trabeculae and reticular walls of the red pulp vascular spaces and other circulatory disruptions, such as hypovolemic and/or cardiogenic shock. thus storage spleens may be either grossly enlarged and congested or small with a wrinkled surface and a dry parenchyma depending on whether the spleen is congested from stored blood or shrunken from contraction (see uniform splenomegaly and small spleens). hematopoietic tissue. in the developing fetus the liver is the primary site of hematopoiesis, with the spleen making a minor contribution. shortly before or after birth, hematopoiesis ceases in the liver and spleen, and the bone marrow becomes the primary hematopoietic organ. under certain conditions, such as severe demand due to prolonged anemia, splenic hematopoiesis can be reactivated; this outcome is called extramedullary hematopoiesis (emh). studies have indicated that splenic emh in dogs and cats most commonly occurs with degenerative or inflammatory circulation, pitted, or phagocytized. instead, the macrophages of the red pulp perform these functions, and phagocytized cells remain in the red pulp vascular spaces. the location of the primary sites of pitting in nonsinusoidal spleens is unclear, but it is likely that most erythrophagocytosis takes place in the red pulp vascular spaces. the cat's spleen is deficient in pitting, and removal of heinz bodies is slow; however, some erythrophagocytosis does occur in the marginal sinus. the macrophages of the sinusoids, marginal sinus, and red pulp vascular spaces are of bone marrow origin. from the bone marrow these cells circulate in the blood as monocytes and migrate into the spleen. some macrophages are replenished by local proliferation. for example, after phagocytizing large amounts of material from the blood, the macrophages of the pams migrate through the wall of the cuff into the adjacent red pulp, denuding the pams of macrophages. after hours, local residual macrophages have proliferated to repopulate the pams. the fixed macrophages elsewhere in the body, namely, those in connective tissue, lymph nodes (sinus histiocytes), liver (kupffer cells), lung (pulmonary intravascular macrophages and pulmonary alveolar macrophages), and brain (resident and perivascular microglial cells), are also derived from bone marrow (see chapters , , , and ) . storage or defense spleens. spleens are also classified as either storage or defense spleens, based on whether or not they can store significant volumes of blood. the ability to store blood in the spleen depends on the fibromuscular composition of the splenic capsule and trabeculae. splenic capsules and trabeculae with a low percentage of smooth muscle and elastic fibers cannot expand and contract and are designated as defense spleens. these are found in rabbits and human beings. the spleens of other domestic animal species have distention of the red pulp from stored blood separates the foci of white pulp (pals and lymphoid follicles), making white pulp appear sparser. splenic white pulp is organized around central arteries in the form of pals, which are populated primarily by t lymphocytes (see . primary splenic follicles are located eccentrically in pals and are primarily composed of b lymphocytes. when exposed to antigen, the splenic lymphoid follicles develop germinal centers (see lymphoid/ lymphatic system, lymph nodes, function) . macrophages in the white pulp follicles remove apoptotic b lymphocytes not selected for expansion because of low binding affinity for antigen. failure of these macrophages to phagocytize has been experimentally correlated with decreased production of growth factors like tgf-β and increased production of inflammatory cytokines that predispose the animal to autoimmune conditions. the marginal zone surrounds the marginal sinus at the interface of the white and red pulp and consists of macrophages, dcs, and t and b lymphocytes. the blood supply of the marginal sinus is from conditions (e.g., hematomas, thrombosis) and may occur without concomitant hematologic disease (see uniform splenomegaly with a firm consistency). it is also found in splenic nodular hyperplasia (see splenic nodules with a firm consistency). in some species, such as the mouse, emh is a normal function of the adult spleen and not necessarily a response to disease or hypoxic challenge. the splenic red pulp also contains large numbers of monocytes, which function as a reserve for generating tissue macrophages in response to ongoing tissue inflammation in the body. white pulp. white pulp consists of pals, each with a splenic lymphoid follicle surrounded by a marginal zone. normally these foci of white pulp are so small that they may not be visible on gross examination of a cross section of the spleen. however, if nodules are enlarged either by lymphoid hyperplasia, amyloid deposits, or a neoplastic process (e.g., lymphoma), they can become grossly visible on the cut surface, initially as . -to . -mm white circular foci scattered through the red pulp. in animals with storage spleens, the the splenic artery enters at the hilus and divides into arteries, which enter the trabeculae. when a trabecular artery emerges from a trabecula it becomes the central artery and is encased in a periarteriolar lymphoid sheath (pals), which is composed of t lymphocytes. it then enters the splenic follicle and gives off branches-the radial arteries, which supply the marginal sinus and marginal zone. the central artery emerges from the splenic follicle to enter the red pulp and branches into the penicillary arterioles, which are enclosed in a cuff of macrophages-the periarteriolar macrophage sheath (pams). the emerging penicillar arteries branch into arterioles and capillaries that supply the red pulp vascular spaces (see fig. - ) . the red pulp vascular spaces also receive blood from capillaries draining from the marginal sinus and drain into the splenic venules and then into the trabecular veins and splenic vein. b, sinusoidal spleen, dog. the blood flow is essentially the same but with the additional feature that arterioles from the marginal sinus drain into the sinusoids and some blood from the red pulp vascular space passes through slits in the sinusoidal wall to enter the sinusoid (see fig. - ) . this is the site of pitting and erythrophagocytosis. note that the major flow in a is sequentially past concentrations of macrophages in the marginal sinus, pams, and red pulp vascular spaces. in b there is the additional route from the marginal zone into the sinusoids. dog. red pulp vascular spaces macrophages in the marginal zone are phenotypically distinct from those in the red pulp. the red pulp macrophages function primarily to filter the blood by phagocytizing particles and by removing senescent or infected erythrocytes and pathogenic bacteria and fungi. marginal zone macrophages are divided into two types based on their location and the type of cell surface receptors they possess. the first group is positioned toward the periphery of the marginal zone, whereas the second group, the marginal metallophilic macrophages (so called for their silver staining positivity), is at the inner margin of the marginal zone closer to the splenic follicle and pals. it has been difficult to generate mammalian models that eliminate one of the two classes of marginal zone macrophages, so the degree to which one group specializes in a particular function is not clear. some marginal zone macrophages actively phagocytize particulate matter or bacteria (e.g., septicemias caused by streptococcus pneumoniae, listeria monocytogenes, campylobacter jejuni, or bacillus anthracis) in the blood (see fig. - ) . they also play a similar role in limiting the spread of viral infections. other marginal zone macrophages phagocytize and process antigens. thus macrophages of the marginal zone serve to bridge the innate and adaptive immune responses by secreting inflammatory cytokines to activate other immune cells and providing receptor-based activation of marginal zone lymphocytes. studies have shown that a loss of marginal zone macrophages coincides with decreased antigen trapping by resident b lymphocytes of the marginal zone and consequently a decrease in the early igm response to antigens. the responses of the spleen to injury (box - ) include acute inflammation, hyperplasia of the monocyte-macrophage system, hyperplasia of lymphoid tissues, atrophy of lymphoid tissues, storage of blood or contraction to expel reserve blood, and neoplasia. these responses are also best considered on the basis of the two main components of the spleen, the red and white pulp, and the anatomic systems associated with each. monocyte-macrophage system. the distribution and function of macrophages in the spleen is described earlier in the section on structure and function. these interactions are complex, and their relationships to both innate and adaptive immunity are areas of intense study (see also chapter ). to facilitate filtering, all of the blood in the body passes through the spleen at least once a day, and % of the cardiac output goes to the spleen. in dogs, blood flow and transit time depend on whether the spleen is contracted or distended; blood flow is slower in the distended spleen. the extent to which macrophages of the monocyte-macrophage system phagocytize particles depends to a large degree on the sequence in which they receive blood. in most species, macrophages of the marginal sinus are the first to receive blood, and consequently phagocytized particles and bacteria tend to be more concentrated here initially. however, there are differences among domestic animal species; the cat, for instance, has a comparatively small marginal sinus, and thus the pams play a larger role in phagocytosis. the spleen is able to mount a strong response to blood-borne pathogens, which has been demonstrated in several studies. the blood of immunized rabbits injected intravenously with pneumococci cleared % of those bacteria within minutes and % within an hour. the blood of dogs injected with billion pneumococci per pound of body weight into the splenic artery was cleared of all bacteria in minutes. after splenectomy, blood-borne the radial branches of the central artery, and it serves as the portal of entry into the spleen for recirculating b and t lymphocytes. from here, t lymphocytes migrate to the pals and b lymphocytes to the germinal centers. macrophages in the marginal zone capture bloodborne antigens, process them, and present them to the lymphocytes. senescent erythrocytes damaged erythrocytes (e.g., immune-mediated anemias) parasitized erythrocytes (e.g., hemotropic parasites) storage of blood (in storage spleens) extramedullary hematopoiesis severe demand (e.g., anemias) degenerative/inflammatory conditions without concomitant hematologic disease incidental (e.g., within nodules of hyperplasia) monocytes within splenic cords reserve for generating tissue macrophages in response to inflammation homing of circulating lymphocytes in the blood phagocytosis and processing of antigen macrophage activation inflammation, which may be diffuse or multifocal/focal (e.g., blastomycosis and tuberculosis, respectively). red pulp vascular spaces. the main response to injury of the red pulp vascular spaces is congestion (see uniform splenomegaly with a bloody consistency), as well as the storage of blood or contraction to expel reserve blood. white pulp. the responses to injury within the white pulp are most pronounced in the splenic lymphoid follicles. lymphoid follicular hyperplasia is a response to antigenic stimuli and results in the formation of secondary follicles; marked hyperplasia may be grossly evident. hyperplasia of splenic lymphoid follicles follows a similar sequence of events and morphologic changes as seen in other secondary lymphoid organs and is discussed in more detail in lymphoid/lymphatic system, lymph nodes, dysfunction/responses to injury. similarly, atrophy of splenic lymphoid follicles has similar causes as lymphoid atrophy in other lymphoid organs (see box - ). briefly, atrophy occurs in response to lack of antigenic stimulation (e.g., from regression after antigenic stimulation has ceased), from the effects of toxins, antineoplastic chemotherapeutic agents, microorganisms, radiation, malnutrition, wasting/cachectic diseases, or aging, or when the bone marrow and thymus fail to supply adequate numbers of b and t lymphocytes, respectively. the follicles are depleted of lymphocytes, and with time, germinal centers and follicles disappear. the amount of the total lymphoid tissue is reduced, and the spleen may be smaller. the response to injury of the monocyte-macrophage system in the marginal sinus and marginal zone is also phagocytosis and proliferation. capsule and trabeculae. lesions in the capsule and trabeculae are uncommon and include splenic capsulitis secondary to peritonitis, and complete or partial rupture of the splenic capsule, usually due to trauma. the two main portals of entry to the spleen for infectious agents are hematogenous spread and direct penetration. the splenic capsule is thick, and thus direct penetration is less common. inflammation from an adjacent peritonitis is unlikely to penetrate the capsule into the splenic parenchyma. cattle with traumatic reticulitis may have foreign objects migrate into the ventral extremity of the spleen, causing a splenic abscess. splenic abscesses also develop secondary to perforation of the gastric wall in horses, due to foreign body penetration, gastric ulcers, or gastric inflammation. portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box - . defense mechanisms used by the spleen to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsule of the spleen. lymph nodes are soft, pale tan, round, oval or reniform organs with a complex three-dimensional structure. on gross examination of a cross section of lymph nodes, two main areas are visible: an outer rim of cortex and an inner medulla (fig. - ) . to understand the pathologic response of the lymph node, it is important to consider its anatomic components and their relationship with antigen processing (fig. - ) : organisms multiply rapidly and may disseminate widely in the body to cause an overwhelming postsplenectomy infection. studies have also shown that the phagocytic function of the spleen is critical in the control of plasmodium (causative agent of malaria) in human beings and babesiosis in cattle. if the number of pathogenic bacteria in the circulation exceeds the capacity of the splenic macrophages, as in cases of severe septicemia, it may result in acute splenic congestion (see uniform splenomegaly with a bloody consistency). this may be followed by inflammation with areas of necrosis, fibrin deposition, and infiltration by neutrophils in bacteremias of pyogenic bacteria. the marginal zone can be the initial site of response to blood-borne antigens and bacteria delivered by the radial branches of the central arteries to the marginal sinus. similar to the response of the red pulp vascular spaces, the marginal zone can become congested and with time (only hours with highly pathogenic organisms) may contain aggregates of neutrophils and macrophages. histologically, the congestion and inflammation form a complete or partial concentric ring around the circumference of the splenic nodule (see anthrax). hyperplasia of the red pulp macrophages is also seen in chronic hemolytic diseases, because there is a prolonged need for phagocytosis of erythrocytes. similarly, chronic splenic congestion, usually the result of portal or splenic vein hypertension, can lead to proliferation of the macrophages present on the walls of the red pulp vascular spaces and results in thickening of the reticular walls between the red pulp vascular spaces. macrophages in the red pulp also proliferate in response to fungi and facultative intracellular pathogens (e.g., mycobacterium bovis) arriving hematogenously to the spleen. the number of red pulp macrophages may be augmented by monocytes recruited from the blood to form granulomatous acute inflammation with fibrin and necrosis abscesses, microabscesses granulomatous inflammation (diffuse, multifocal, focal) fibroblastic reticular cells and fibers. besides providing structural support, this reticulum helps form a substratum for the migration of lymphocytes and antigen-presenting cells to the follicles and facilitates the interaction with b and t lymphocytes. cortex. the outer/superficial cortex contains the lymphoid follicles (also referred to as lymphoid nodules) (see . the follicles are designated as primary if they consist mainly of small lymphocytes: mature naïve b lymphocytes expressing receptors for specific antigens exit the bone marrow and circulate through the bloodstream, lymphatic vessels, and secondary lymphoid tissues. on their arrival at lymph nodes, b lymphocytes exit through hevs in the paracortex and home to a primary follicle (which also contains follicular dcs in addition to the resting b lymphocytes). lymphoid follicles with germinal centers are designated as secondary follicles: b lymphocytes that recognize the antigen for which they are expressing receptors are activated and proliferate to form the secondary lymphoid follicles characterized by prominent germinal centers. germinal centers are areas with a specialized • stroma-capsule, trabeculae, and reticulum • cortex-"superficial" or "outer" cortex (lymphoid follicles, b lymphocytes) • paracortex-"deep" or "inner" cortex (t lymphocytes) • medulla-medullary sinuses and medullary cords • blood vessels-arteries, arterioles, high endothelial venules (hevs), efferent veins • lymphatic vessels-lymphatic afferent and efferent vessels; lymphatic sinuses (subcapsular, trabecular, and medullary) • monocyte-macrophage system-sinus histiocytes stroma. the lymph node is enclosed by a fibrous capsule penetrated by multiple afferent lymphatic vessels, which empty into the subcapsular sinus (see also . at the hilus, efferent lymphatic vessels and veins exit, and arteries enter the node. fibrous trabeculae extend from the capsule into the parenchyma to provide support to the node and to house vessels and nerves. the lymph node is also supported by a meshwork of medulla. the medulla is composed of medullary cords and medullary sinuses (see . the medullary cords contain macrophages, lymphocytes, and plasma cells. in a stimulated node the cords become filled with antibody-secreting plasma cells. the medullary sinuses are lined by fibroblastic reticular cells and contain macrophages ("sinus histiocytes"), which cling to reticular fibers crossing the lumen of the sinus. these macrophages phagocytize foreign material, cellular debris, and bacteria from the incoming lymph. vasculature: blood vessels, lymphatic vessels, and lymphatic sinuses. the blood vessels of the lymph node include arteries, arterioles, veins, and postcapillary venules (hevs) lined by specialized cuboidal endothelium (see figs. - and - ) . microenvironment that support the proliferation and further development of b lymphocytes to increase their antigen and functional capacity (see lymphoid/lymphatic system, lymph nodes, function). the mantle cell zone surrounds the germinal center and consists of small inactive mature naïve b lymphocytes and a smaller population of t lymphocytes (approximately %). paracortex. the diffuse lymphoid tissue of the paracortex (also referred to as the deep or inner cortex) consists mainly of t lymphocytes, as well as macrophages and dcs (see . this region contains the hevs through which b and t lymphocytes migrate from the blood into the lymphoid follicles and paracortex, respectively. t and b lymphocytes may also enter the lymph node via the lymphatic vessels. and larger molecules, small molecules and free antigens, and antigen within dcs. it is helpful to consider the paths taken by particles, molecules, antigens, and cells arriving at a lymph node. the following account describes the journey of an antigen as it enters a lymph node to trigger an immune response. antigen in the lymph arriving in the afferent lymphatic vessels empties into the subcapsular sinus. hydrostatic pressure here is low, and reticular fibers crossing the sinus impede flow, and thus particles tend to settle, which facilitates phagocytosis by the sinus macrophages. lymph then flows down the trabecular sinuses that line the outer surface of fibrous trabeculae, to the medullary sinus, and eventually exits via efferent vessels. as antigens within the lymph travel through the sinuses, they are captured and processed by macrophages and dcs. alternatively, dcs charged with antigen can migrate within blood vessels to the node and enter the paracortex via the hevs. circulating b lymphocytes also enter across the hevs, and if they encounter antigen-bearing dcs, there is a local reaction involving the appropriate t helper lymphocytes, b lymphocytes, and dcs. this results in the migration of the activated b lymphocytes to a primary follicle, where they initiate formation of a germinal center. germinal centers, upon migration of antigen-activated b lymphocytes, develop a characteristic architecture. distinct polarity composed of a superficial or light zone and a deep dark zone is present in cases of antigenic stimulation. the light zone, orientated at the source of antigen, consists mainly of small lymphocytes, called centrocytes, which have moderate amounts of pale eosinophilic cytoplasm. the cells of the dark zone, called centroblasts, are large, densely packed lymphocytes with scant cytoplasm, giving this area a darker appearance on h&e staining. the centroblasts undergo somatic mutations of the variable regions of the immunoglobulin gene, followed by isotype class switching (from igm to igg or iga). during this process most centroblasts undergo apoptosis, and cell fragments are phagocytized by macrophages, which are then termed tingible (stainable) body macrophages. the cells that have survived the affinity maturation process are now called centrocytes and along with t lymphocytes and follicular dcs, populate the germinal center light zone. these post-germinal center b lymphocytes leave the follicle as plasma cell precursors (immunoblasts or plasmablasts) and migrate from the cortex to the medullary cords, where they mature and excrete antibody into the efferent lymph. some of these cells may colonize the region surrounding the mantle cell zone to form a marginal zone. marginal zones are apparent only in situations of prolonged and intense immune stimulation and serve as a reservoir of memory cells. the elliptical mantle cell cuff is wider over the light pole of the follicle, though in instances of strong antigenic stimulation, the cuffs can completely encircle the germinal center. responses to injury are listed in box - , and the responses are discussed on the basis of the following systems: sinus histiocytes of the monocyte-macrophage system, cortex, paracortex, and medulla (medullary sinuses and medullary cords). generally, enlarged lymph nodes can be distributed in several different patterns in the body. first, all lymph nodes throughout the body (systemic or generalized) may be enlarged (lymphadenopathy or lymphadenomegaly). this pattern is usually attributed to systemic infectious, inflammatory, or neoplastic processes. if a single lymph node or regional chain of nodes is enlarged, then the area drained by that node should be checked for lesions (e.g., evaluate the oral cavity if the mandibular lymph nodes are enlarged). thus it is important to know the area drained by specific lymph nodes. mesenteric lymph nodes are normally larger because of follicular approximately % to % of lymphocytes enter lymph nodes through the hevs, which also play an important role in lymph fluid balance. the lymphatic vasculature consists of afferent lymphatic vessels, which pierce the capsule and drain into the subcapsular sinus. lymph continues to drain through the trabecular sinuses to the medullary sinuses and finally exits at the hilus via efferent lymphatic vessels. all lymph nodes receive afferent lymphatic vessels from specific areas of the body. the term lymphocenter is often used in veterinary anatomy to describe a lymph node or a group of lymph nodes that is consistently present at the same location and drains from the same region in all species. for example, the popliteal lymph node, caudal to the stifle, drains the distal hind limb. the tracheobronchial nodes (bronchial lymphocenter), located at the tracheal bifurcation, collect lymph from the lungs and send it to the mediastinal nodes or directly to the thoracic duct. because lymph from a single afferent lymphatic vessel drains into a discrete region of a lymph node, only these regions of the node may be affected by the contents of a single draining lymph vessel (e.g., antigen, infectious organisms, or metastatic neoplasms [ fig. - ] ). the lymph node of the pig has a different structure. the afferent lymphatic vessels enter at the hilus instead of around the periphery of the node and empty lymph into the center of the node. the lymph drains to the "subcapsular" sinus (the equivalent of the medullary sinuses of other domestic animals) and then into several efferent lymphatic vessels, which pierce the outer capsule. this reversal of flow is the result of an inverted nodal architecture, with the cortex in the middle of the node surrounded by the medulla at the periphery. thus a pig lymph node that is draining an area of hemorrhage will have blood accumulate in the periphery (subcapsular) instead of in the center of the node (which may be grossly visible). the functions of the lymph node are ( ) to filter lymph of particulate matter and microorganisms, ( ) to facilitate the surveillance and processing of incoming antigens via interactions with b and t lymphocytes, and ( ) to produce b lymphocytes and plasma cells. material arriving in the lymph can be subdivided into free particles that is undergoing follicular hyperplasia is enlarged and has a taut capsule, and the cut surface may bulge. histologically, the follicles contain active germinal centers with antigenic polarity (light and dark zones) (figs. - and - ; also see fig. - ) . depending on the duration and continued exposure to the antigen, there may also be concomitant paracortical hyperplasia and medullary cord hyperplasia and sinus histiocytosis, because these nodes continuously receive and respond to barrages of antigens and bacteria from the intestinal tract. sinus histiocytes (macrophages) are part of the monocyte-macrophage system and the first line of defense against infectious and noninfectious agents in the incoming lymph. in response to these draining agents, there is hyperplasia of the macrophages ("sinus histiocytosis"), most notable in the medullary sinuses ( fig. - ) . leukocytes, often monocytes, may harbor intracellular pathogens (e.g., mycobacterium spp., cell-associated viruses such as parvovirus), arrive in the blood or lymph, infect the lymph node, and then are disseminated throughout the lymphoid tissues of the body via the efferent lymph and circulating blood. cortex (lymphoid follicles). follicular hyperplasia of the cortex is discussed in the section lymphoid/lymphatic system, lymph node, function. an antigenically stimulated lymph node the two main portals of entry to the lymph node for infectious agents and antigens are afferent lymphatic vessels (lymphatic spread) and blood vessels (hematogenous spread). portals of entry used by microorganisms and other agents and substances to access the lymphoid/lymphatic system are summarized in box - . infectious microorganisms, either free within the lymph or within lymphocytes or monocytes, are transported to regional lymph nodes through lymphatic vessels. agents may escape removal by phagocytosis in one lymph node and be transported via efferent lymphatic vessels to the next lymph node in the chain and cause an inflammatory or immunologic response there. this process can continue serially down a lymph node chain, and if the agent is not removed, it may eventually be transported via the lymphatic vessels to either the cervical or thoracic ducts and then disseminated throughout the body. although most pathogens are transported to lymph nodes via afferent lymphatic vessels, bacteria can be transported to lymph nodes hematogenously (free or within leukocytes such as monocytes) in septicemias and bacteremias. direct penetration of a lymph node is uncommon, because it is protected by a thick fibrous capsule. occasionally, inflammatory cells or neoplasms can extend directly into nodal parenchyma from adjacent tissues. defense mechanisms used by the lymphatic system to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . other defense mechanisms are structural in nature to protect against external trauma and include the thick fibrous capsules of lymph nodes. hemal nodes are small, dark red to brown nodules found most commonly in ruminants, mainly sheep, and have also been reported in horses, primates, and some canids. their architecture resembles that of a lymph node with lymph follicles and sinuses, except that in the hemal node, sinuses are filled with blood (e- fig. - ) . because erythrophagocytosis can be present, it is presumed that hemal nodes can filter blood and remove senescent erythrocytes, but as their blood supply is small, their functional importance is not clear. malt is the initial site for mucosal immunity and is crucial in the protection of mucosal barriers. malt is composed of both diffuse lymphoid tissues and aggregated lymphoid (also known as lymphatic) nodules, which can be subcategorized based on their anatomic location: ( ) bronchus-associated lymphoid tissue (balt), which is often at the bifurcation of the bronchi and bronchioles; ( ) tonsils (pharyngeal and palatine) form a ring of lymphoid tissue at the oropharynx; ( ) nasal-, larynx-, and auditory tube-associated lymphoid tissues (nalt, lalt, and atalt, respectively) within the nasopharyngeal area; ( ) gut-associated lymphoid tissue (galt), which includes peyer's patches and diffuse lymphoid tissue in the gut wall; ( ) conjunctiva-associated lymphoid tissue (calt); ( ) other lymphoid nodules (e.g., genitourinary tract) (fig. - ) . diffuse lymphoid tissue consists of lymphocytes and dcs within the lamina propria of the mucosa of the alimentary, respiratory, and genitourinary tracts. these cells intercept and process antigens, which then travel to regional lymph nodes to initiate the immune response, leading ultimately to the secretion of iga, igg, and igm. plasmacytosis. less florid follicular reactions will have smaller separated germinal centers, whereas nodes receiving persistent high levels of antigen stimulation may have coalescing germinal centers (termed "atypical benign follicular hyperplasia"). in such cases of chronic strong antigenemia, the highly reactive nodes may also exhibit colonization of lymphocytes into perinodal fat, and germinal centers may contain irregular lakes of eosinophilic material, known as follicular hyalinosis. as the immune response declines, there is follicular lymphoid depletion and the concentration of lymphocytes in the germinal centers is reduced, allowing the underlying follicular stroma (including dcs and macrophages) to become visible. with ongoing lymphocyte depletion, the mantle cell zones are thinned, less populated, and discontinuous. eventually, residual mantle cells collapse into the follicular stroma, forming clusters of small dark cells within the bed of dcs and macrophages, referred to as fading follicles. paracortex. paracortical atrophy may result from a variety of causes, including deficiency in lymphocyte production in the bone marrow, reduced differential selection of lymphocytes in the thymus, or destruction of lymphocytes in the lymph node by viruses, radiation, and toxins directly on the lymphocytes in the lymph node (see box - ). examination of h&e-stained sections allows evaluation of follicular activity in the cortex and the concentration of plasma cells in the medullary cords, which serve as a reasonable estimate of b lymphocyte activity for comparison. paracortical hyperplasia may have a nodular or diffuse appearance depending on which and how many afferent lymphatic vessels are draining antigen. this reaction may precede or be concurrent with the germinal center reaction of follicular hyperplasia. proliferation of t lymphocytes has been reported in the paracortex (and pals of the spleen) in malignant catarrhal fever (mcf) in cattle and in pigs with porcine reproductive and respiratory syndrome. pcv can cause a diffuse proliferation of macrophages within the paracortex. responses to injury by the medullary sinuses are dilation of the sinuses and proliferation of histiocytes ("sinus histiocytosis"). sinus macrophages proliferate in response to a wide variety of particulate matter in the lymph, including bacteria and erythrocytes (erythrophagocytosis) draining from a hemorrhagic area (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, pigmentation of lymph nodes). dilation of the sinuses due to edema occurs with many underlying conditions, including chronic cardiac failure or drainage from an acutely inflamed area. as the inflammation progresses, the sinuses become filled with neutrophils, macrophages, and occasionally fibrin, in addition to the hyperplastic resident sinus histiocytes (see fig. - ) . depending on the intensity of the inflammation, the adjacent parenchyma may become affected (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly], acute lymphadenitis). as pointed out in the section on lymph nodes, function, after activation and proliferation of b lymphocytes in the follicle, the immunoblasts formed there move to and mature in the medullary cords, which as a result are distended with plasma cells that secrete antibody into the efferent lymphatic vessels ("medullary plasmacytosis"). the concentration of medullary plasma cells correlates with the activity of the germinal centers. as the immune response subsides, the number of plasma cells decreases and the medullary cords return to their resting state populated by few lymphocytes and scattered plasma cells. chapter bone marrow, blood cells, and the lymphoid/lymphatic system composition. for instance, m cells increase in animals transferred from pathogen-free housing to the normal environment. m cells may also be exploited as a portal for entry by some microbes (see lymphoid/lymphatic system, portals of entry/pathways of spread). table - lists the interactions of the malt with different microorganisms. the responses of malt to injury are similar to those of other lymphoid tissues: hyperplasia, atrophy, and inflammation (box - ). hyperplasia. hyperplasia of lymphoid nodules is a response to antigenic stimulation and consists of activation of germinal centers with subsequent production of plasma cells (see fig. - , b) . lymphoid nodule hyperplasia is often present in chronic disease conditions, such as balt hyperplasia in chronic dictyocaulus spp. (horses, cattle, sheep, and goats) or metastrongylus spp. (pigs) associated bronchitis or bronchiolitis. mycoplasma spp. pneumonias of sheep and pigs display marked balt hyperplasia that can encircle bronchioles and bronchi ("cuffing pneumonia"). hyperplastic lymphoid nodules can be so enlarged that they become grossly visible as discrete white plaques or nodules (see fig. - , a). they can be seen in the conjunctiva of the eyelids and the third eyelid in chronic conjunctivitis, the pharyngeal mucosa in chronic pharyngitis, the gastric mucosa in chronic gastritis, and the urinary bladder in chronic cystitis (follicular cystitis). the normal fetus has no detectable balt, though it may be present in fetuses aborted due to infectious disease. atrophy. atrophy of the diffuse lymphoid tissue and lymphoid nodules has the same causes as atrophy affecting other lymphoid tissues (see box - ) and includes lack of antigenic stimulation, cachexia, malnutrition, aging, viral infections, or failure to be repopulated by b lymphocytes from the bone marrow or t lymphocytes from the thymus. lymphocytolysis of germinal center lymphocytes of peyer's patches is a characteristic lesion in bvdv infection in ruminants and canine and feline parvovirus infections ("punchedout peyer's patches) (see chapters and ). the main portals of entry to malt for infectious agents are hematogenous spread and through migrating macrophages, dcs , and m cells. pathogenic bacteria such as escherichia coli, yersinia pestis, mycobacterium avium ssp. paratuberculosis (map), l. monocytogenes, salmonella spp., and shigella flexneri can invade the host from the lumen of the intestine through dendritic or m cells. some viruses (e.g., reovirus) may be transported by m cells. the scrapie prion protein (prp sc ) may also accumulate in peyer's patches. many viruses, such as bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus, cause lymphocyte depletion within the malt. portals of entry used by microorganisms and other agents and substances to access the lymphoid system are summarized in box - . defense mechanisms used by malt to protect itself against microorganisms and other agents are the innate and adaptive immune responses, discussed in chapters , , and . congenital disorders of the thymus are discussed in detail in chapter . summaries of the gross and microscopic morphologic changes are solitary lymphoid nodules are localized concentrations of lymphocytes (mainly b lymphocytes) in the mucosa and consist of defined but unencapsulated clusters of small lymphocytes (primary lymphoid nodule). they are usually not grossly visible in the resting or antigenically unstimulated state, but upon antigenic stimulation, they proliferate and form germinal centers and surrounding mantle cell zones (secondary lymphoid nodules). aggregated lymphoid nodules consist of groups of lymph nodules, the most notable of which are the tonsils and peyer's patches. the aggregated lymphoid follicles of the peyer's patches are most obvious in the ileum. the latter are covered by a specialized epithelium, the follicle-associated epithelium (fae). the fae is the interface between the peyer's patches and the luminal microenvironment and consists of enterocytes and interdigitated m cells. m cells transport (via endocytosis, phagocytosis, pinocytosis, and micropinocytosis) antigens, particles, bacteria, and viruses from the intestinal lumen to the underlying area rich in dcs , which deliver the material to the lymphoid tissue of the peyer's patches. m cells also express iga receptors, which allows for the capture and transport of bacteria entrapped by iga. the proportion of enterocytes and m cells within the fae is modulated by the luminal bacterial a b mercury have a suppressive effect on the immune system. halogenated aromatic hydrocarbons cause dysfunction of dcs through several mechanisms that lead to atrophy of the primary and secondary lymphoid organs. heavy metals, such as lead, mercury and nickel, are immunosuppressive and generally affect the levels of b and t lymphocytes, nk cells, and inflammatory cytokines. other metals, such as selenium, zinc, and vanadium, may be immunostimulatory at low doses. the immunotoxic mechanisms may differ and include chelation of molecules and effects on protein synthesis, cell membrane integrity, and nucleic acid replication. the toxic effects of mycotoxins such as fumonisins b and b (secondary fungal metabolites produced by members of the genus fusarium) and aflatoxin (produced by aspergillus flavus) include lymphocytolysis in the thymic cortex. box - responses of mucosa-associated lymphoid tissue to injury described in the sections on disorders of horses and disorders of dogs. thymic cysts can be found within the developing and mature thymus and in thymic remnants in the cranial mediastinum. thymic cysts are often lined by ciliated epithelium and represent developmental remnants of branchial arch epithelium and are usually of no significance. thymitis is an uncommon lesion and may be seen in pcv infection (see disorders of pigs and also chapter ), enzootic bovine abortion (see chapter ), and salmon poisoning disease of dogs (see chapter ). infectious agents more commonly cause thymic atrophy. variable degrees of acquired immunodeficiency can be also be caused by toxins, chemotherapeutic agents and radiation, malnutrition, aging, and neoplasia. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv- in aborted foals (fig. - ) , classic swine fever virus, bvdv, canine distemper virus, canine and feline parvovirus, and fiv; severe thymic lymphoid depletion is an early lesion in fiv-infected kittens. environmental toxins, such as halogenated aromatic hydrocarbons (e.g., polychlorinated biphenyls and dibenzodioxins), lead, and thymic hyperplasia. asymptomatic hyperplasia may occur in juvenile animals in association with immunizations and results in symmetrical increase in the size of the thymus. autoimmune lymphoid hyperplasia of the thymus has germinal center formation and occurs with myasthenia gravis. asplenia or the failure of a spleen to develop in utero occurs rarely in animals, and the effect on the animal's immune status is uncertain. (splenic aplasia is present in certain strains of mice, but because these are usually maintained under either germ-free or specific pathogen-free [spf] conditions, the effect of asplenia cannot be evaluated.) congenital immunodeficiency diseases are described in detail in chapter , and in the sections on disorders of horses and disorders of dogs. gross examination of the spleen involves deciding whether the spleen is enlarged (splenomegaly), normal, or small (see e-appendix - ). diffuse enlargement of the spleen may be due to congestion (termed bloody spleen) or other infiltrative disease (termed meaty spleen). the cut surface of congested spleens will exude blood, whereas meaty spleens are more firm and do not readily ooze blood. the diseases and disorders having splenomegaly are discussed using the following categories, which list the common causes of uniform splenomegaly (table - ): • uniform splenomegaly with a bloody consistency (bloody spleen) ( fig. - , a) • uniform splenomegaly with a firm consistency (meaty spleen) (see fig. - , b) • splenic nodules with a bloody consistency • splenic nodules with a firm consistency uniform splenomegaly with a bloody consistency-bloody spleen. the common causes of a bloody spleen are ( ) congestion (due to gastric volvulus with splenic entrapment, splenic volvulus chemotherapeutic drugs inhibit the cell cycle through various mechanisms, and thus all dividing cells, including lymphocytes, bone marrow cells, and enterocytes, are sensitive to their effects. as such, bone marrow suppression, immunosuppression, and gastrointestinal disturbances are common side effects of anticancer drugs. purine analogues (e.g., azathioprine) compete with purines in the synthesis of nucleic acids, whereas alkylating agents like cyclophosphamide cross-link dna and inhibit the replication and activation of lymphocytes. cyclosporin a specifically inhibits the t lymphocyte signaling pathway by interfering with the transcription of the il- gene. methotrexate, a folic acid antagonist, blocks the synthesis of thymidine and purine nucleotides. the immunosuppressive effects of some of these agents is desirable for the treatment of immune-mediated disease (e.g., immune-mediated hemolytic anemia) or to prevent allograft rejection after transplantation. corticosteroids may be given at an immunosuppressive dose, though the degree of suppression is highly variable among species. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissue is within the therapeutically irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. malnutrition and cachexia, which may occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. thymic function may be impaired in young malnourished animals, resulting in a decrease in circulating t lymphocytes and subsequent depletion of t lymphocyte regions of secondary lymphoid organs. lymphoid atrophy may result from physiologic and emotional stress, which can cause the release of catecholamines and glucocorticoids. as part of the general effects of aging in cells (see chapter ), all lymphoid organs decrease in size (atrophy) with advancing age. in the case of the thymus this reduction in size occurs normally after sexual maturity and is more appropriately termed thymic involution. the term involution should be reserved for normal physiologic processes in which an organ either returns to normal size after a period of enlargement (e.g., postpartum uterus) or regresses to a more primitive state (e.g., thymic involution). because the thymus has both lymphoid and epithelial components, neoplasms may arise from either component. thymic lymphoma arises from the t lymphocytes in the thymus (and very rarely b lymphocytes). it is most often seen in young cats and cattle and less frequently in dogs (fig. - ) (see hematopoietic neoplasia). thymomas arise from the epithelial component and are usually benign neoplasms that occupy the cranial mediastinum of older animals. histologically, these neoplasms consist of clustered or individualized neoplastic epithelial cells, often outnumbered by nonneoplastic small lymphocytes ("lymphocyte-rich thymoma"). thymomas are common in goats and often contain large cystic structures. immunemediated diseases, including myasthenia gravis and immunemediated polymyositis, occur with thymomas in dogs, and also rarely in cats. myasthenia gravis is caused by autoantibodies directed toward the acetylcholine receptors, which lead to destruction of postsynaptic membranes and reduction of acetylcholine receptors at neuromuscular junction. megaesophagus and aspiration pneumonia are common sequelae to this condition. (syn: necropsy) in horses and dogs that have been euthanized or anesthetized with barbiturates. grossly, the spleen is extremely enlarged (fig. - ) , and the cut surface bulges and oozes copious blood. because of the splenic distention, the splenic capsule can be fragile and easily ruptured. histologically, the red pulp is distended by erythrocytes, and the lymphoid tissues of the white pulp are small and widely separated (fig. - ). electric stunning of pigs at slaughter may result in a large congested spleen; the mechanism is unknown, but it should not be confused with a pathologically congested spleen. splenic congestion in acute cardiac failure is rarely seen in animals. acute congestion/hyperemia. acute septicemias may cause acute hyperemia and concurrent acute congestion of marginal zones and splenic red pulp. microbes are transported hematogenously to these sites, where they are rapidly phagocytized by macrophages. enormous numbers of intravenous bacteria can be cleared by the spleen from the blood in to minutes, but when this defensive mechanism is overwhelmed, the outcome is usually fatal. the response of the spleen depends on the duration of the disease. in acutely fatal cases, such as anthrax and fulminating salmonellosis, distention by blood may be the only gross finding. if the animal survives longer, as in swine erysipelas and the less virulent forms of [all of which compress the splenic vein], and barbiturate euthanasia, anesthesia, or sedation), ( ) acute hyperemia (due to septicemia), and ( ) acute hemolytic anemia (due to an autoimmune disorder or an infection with a hemotropic parasite). splenic torsion. torsion of the spleen occurs most commonly in pigs and dogs; in dogs this usually involves both spleen and stomach and is seen more often in deep-chested breeds (see chapter ). in contrast to ruminants, in which the spleen is firmly attached to the rumen, the spleens of dogs and pigs are attached loosely to the stomach by the gastrosplenic ligament. it is the twisting of the spleen around this ligament that results initially in occlusion of the veins, causing splenic congestion, and later in occlusion of the artery, causing splenic infarction. in dogs the spleen is uniformly and markedly enlarged and may be blue-black from cyanosis. it is often folded back on itself (visceral surface to visceral surface) in the shape of the letter "c." treatment for this condition is most often splenectomy. barbiturate euthanasia, anesthesia, or sedation. intravenous injection of barbiturates induces acute passive congestion in the spleen due to relaxation of smooth muscle in the capsule and trabeculae. this phenomenon is seen most dramatically at autopsy only histologic lesion may be marked congestion of the marginal sinuses and the splenic red pulp vascular spaces. at low magnification, congestion of the marginal sinus may appear as a circumferential red ring around the splenic follicle, and there is marked lymphocytolysis of follicles and pals. intravascular free bacilli are noted and may be seen in impression smears of peripheral blood, presumably because death is so rapid from the anthrax toxin that there is insufficient time for phagocytosis to take place. if the animal lives longer, scattered neutrophils are present in the marginal sinuses and red pulp vascular spaces (fig. - ). anthrax cases are not normally autopsied because exposure to air causes the bacteria to sporulate-anthrax spores are extremely resistant and readily contaminate the environment. acute hemolytic anemias. hemolytic diseases, including acute babesiosis, hemolytic crises in equine infectious anemia, and immune-mediated hemolytic anemia, can cause marked splenic congestion. the splenic congestion is due to the process of removal (phagocytosis) and storage of large numbers of sequestered parasitized and/or altered erythrocytes from the circulation. histologically, there is dilation of the red pulp vascular spaces with erythrocytes and erythrophagocytes. with chronicity there is hyperplasia of the red pulp macrophages, hemosiderosis, and reduced congestion because the number of sequestered diseased erythrocytes is diminished. spleen. the three general categories of conditions leading to uniform splenomegaly with a firm meaty consistency are ( ) marked phagocytosis of cells, debris, or foreign agents/material; ( ) proliferation or infiltration of cells as occurs in diffuse lymphoid and histiocytic hyperplasia, diffuse granulomatous disease (e -table - ), emh, and neoplasia; ( ) storage of materials in storage diseases or amyloidosis. it is important to recognize that more than one of these processes can occur in the same patient (e.g., dogs with immunemediated hemolytic anemia may have both marked erythrophagocytosis and emh). the appearance of the cut surface of a meaty spleen depends on the underlying cause. in diffuse marked lymphoid hyperplasia, large, disseminated, discrete, white, bulging nodules are visible. spleens with diffuse infiltrative neoplasms, such as lymphoma, are pink-light purple on cut surface. diffuse lymphoid hyperplasia. lymphoid hyperplasia has been described in detail in the section on dysfunction/responses to injury. in cases of prolonged antigenic stimulation the lymphoid salmonellosis, there may be sufficient time for neutrophils and macrophages to accumulate in the marginal sinuses, marginal zones, and splenic red pulp vascular spaces. anthrax. b. anthracis, the causative agent of anthrax, is a grampositive, large, endospore-forming bacillus, which grows in aerobic to facultative anaerobic environments. anthrax is primarily a disease of ruminants, especially cattle and sheep (see chapters , , , and ) . once the spores are ingested, they replicate locally in the intestinal tract, spread to regional lymph nodes, and then disseminate systemically through the bloodstream, resulting in septicemia. b. anthracis produces exotoxins, which degrade endothelial cell membranes and enzyme systems. grossly, the spleen is uniformly enlarged and dark red to bluishblack and contains abundant unclotted blood. in peracute cases the .e chapter bone marrow, blood cells, and the lymphoid/lymphatic system diffuse granulomatous disease. chronic infectious diseases may cause a uniformly firm and enlarged spleen, mostly due to macrophage hyperplasia and phagocytosis, diffuse lymphoid hyperplasia, or diffuse granulomatous disease. diffuse granulomatous diseases (see e- table - ) occur in ( ) intracellular facultative bacteria that infect macrophages (e.g., mycobacterium spp., brucella spp., and francisella tularensis); ( ) systemic mycoses (e.g., blastomyces dermatitidis, histoplasma capsulatum) (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, enlarged lymph nodes [lymphadenomegaly]) ( fig. - , a and b) , and ( ) protozoal infections that infect macrophages (e.g., leishmania spp.). some of these organisms may also produce nodular spleens with the formation of discrete to coalescing granulomas (e.g., m. bovis) (see splenic nodules with a firm consistency). follicles throughout the splenic parenchyma can become enlarged and visible on gross examination (fig. - ), leading to diffuse splenomegaly. in contrast to b lymphocyte hyperplasia of the lymphoid follicles, certain diseases (e.g., malignant catarrhal fever in cattle) may lead to t lymphocyte hyperplasia of the pals. diffuse histiocytic hyperplasia and phagocytosis. splenomegaly from hyperplasia and increased phagocytosis of splenic macrophages is a response to the need to engulf organisms in prolonged bacteremia or parasitemia from hemotropic organisms. whereas acute hemolytic anemias cause splenomegaly with congestion (bloody spleen), with chronicity there is decreased sequestration of diseased erythrocytes and hence less congestion. therefore in cases of chronic hemolytic disease, splenomegaly is attributed to diffuse proliferation of macrophages, phagocytosis, and concurrent hyperplasia of the white pulp due to ongoing antigenic stimulation. for example, equine infectious anemia has cyclical periods of viremia, with immune-mediated damage to erythrocytes and platelets, and phagocytosis to remove altered erythrocytes and platelets. these cycles result in proliferation of red pulp macrophages, hyperplasia of hematopoietic cells (emh) to replace those lost, and hyperplasia of lymphocytes in the white pulp. in animals less than year of age. in general, these substrates are lipids and/or carbohydrates that accumulate in the cells, the result of the lack of normal processing within lysosomes. major categories of stored materials include mucopolysaccharides, sphingolipids, glycolipids, glycoproteins, glycogen, and oligosaccharides. macrophages are commonly affected by storage diseases, and thus extramedullary hematopoiesis. emh is the development of blood cells in tissues outside the medullary cavity of the bone (e- fig. - ). the formation of single or multiple lineages of hematopoietic cells is often observed in many tissues and commonly in the spleen. the ability of blood cell precursors to home, proliferate, and mature in extramedullary sites relies on the presence of hscs and pathophysiologic changes in the microenvironment (i.e., extracellular matrix, stroma, and chemokines). in the spleen, hscs have been found within vessels and adjacent to endothelial cells to form a vascular niche; thus splenic emh occurs in the red pulp, both within the red pulp vascular spaces and sinusoids (of the dog). the predilection for emh to occur varies among species (for instance, splenic emh persists throughout adulthood in mice), and the underlying mechanisms are not completely understood, but four major theories to explain the causes of emh are ( ) severe bone marrow failure; ( ) myelostimulation; ( ) tissue inflammation, injury, and repair; and ( ) abnormal chemokine production. because splenic emh is often observed in animals without obvious hematologic abnormalities, tissue inflammation, injury, and repair is the most likely mechanism of emh in this organ. in dogs and cats emh occurs most frequently with degenerative and inflammatory disorders, such as lymphoid nodular hyperplasia, hematomas, thrombi, histiocytic hyperplasia, inflammation (e.g., fungal splenitis), and neoplasia. emh in multiple tissues may be observed in chronic cardiovascular or respiratory conditions, chronic anemia, or chronic suppurative diseases in which there is an excessive tissue demand for neutrophils that exceeds the supply available from the marrow (e.g., canine pyometra). primary neoplasms. primary neoplastic diseases of the spleen arise from cell populations that normally exist in the spleen and include hematopoietic components, such as lymphocytes, mast cells, and macrophages, and stromal cells, such as fibroblasts, smooth muscle, and endothelium. the primary neoplasms that result in diffuse splenomegaly are the round cell tumors, including lymphoma ( fig. - ) , leukemia, visceral mast cell tumor, and histiocytic sarcoma. it is important to note that all of these types of neoplasms can produce nodular lesions instead of-or along with-a diffusely enlarged spleen. the different types of lymphoma in domestic animals are discussed in the section on hematopoietic neoplasia. secondary neoplasms of the spleen are due to metastatic spread and most often form nodules in the spleen, not a uniform splenomegaly. amyloid. the accumulation of amyloid in the spleen may occur with primary (al) or secondary (aa) amyloidosis (see chapters and ). rarely, severe amyloid accumulation may cause uniform splenomegaly ( fig. - ) , in which the spleen is firm, rubbery to waxy, and light brown to orange. microscopically, amyloid is usually in the splenic follicles, which if large enough, are grossly visible as approximately -mm-diameter gray nodules. amyloid deposition can also be seen within the walls of splenic veins and arterioles. plasma cells tumors within the spleen may also be associated with amyloid (al) deposits. lysosomal storage diseases. storage diseases are a heterogeneous group of inherited defects in metabolism characterized by accumulation of storage material within the cell (lysosomes). genetic defects, which result in the absence of an enzyme, the synthesis of a catalytically inactive enzyme, the lack of activator proteins, or a defect in posttranslational processing, can lead to a storage disease. acquired storage diseases are caused by exogenous toxins, most often plants that inhibit a particular lysosomal enzyme (e.g., swainsonine toxicity due to indolizidine alkaloid found in astragalus and oxytropis plant spp.). storage diseases typically occur diagnosis, it may be difficult (and futile) to determine the primary site. histologically, hemangiosarcomas are composed of plump neoplastic endothelial cells, which wrap around stroma to form haphazardly arranged and poorly defined blood-filled vascular spaces ( fig. - ). splenic nodules with a firm consistency. the most common disorders of the spleen with firm nodules are ( ) lymphoid nodular hyperplasia, ( ) complex nodular hyperplasia, ( ) primary neoplasms, ( ) secondary metastatic neoplasms, ( ) granulomas, and ( ) abscesses. lymphoid and complex nodular hyperplasia. see lymphoid/ lymphatic system, disorders of dogs. primary neoplasms. the primary neoplastic diseases of the spleen that result in firm nodules include lymphoma (multiple subtypes), histiocytic sarcoma, leiomyoma, leiomyosarcoma, fibrosarcoma, myelolipomas, liposarcomas, myxosarcomas, undifferentiated pleomorphic sarcomas, solid hemangiosarcomas, and rare reports of primary chondrosarcomas. these locally extensive neoplasms may be solitary or multiple, raised above the capsular surface, but usually confined by the capsular surface. the consistency and cut surface appearance varies depending on the type of neoplasm; spindle cell tumors like leiomyosarcomas and fibrosarcomas will be white and firm, liposarcomas and myelolipomas are soft and bulging, and myxomatous neoplasms are gelatinous. it is important to remember that many round cell neoplasms, such as lymphoma, mast cell tumors, plasma cell tumors, myeloid neoplasms, and histiocytic sarcomas, can form nodules or diffuse splenic enlargement (or both). metastatic neoplasms. neoplasms that metastasize to the spleen usually result in enlarged nodular spleens (fig. - ) and include any number of sarcomas, carcinomas, or malignant round cell tumors. metastatic sarcomas can include fibrosarcomas, leiomyosarcomas, chondrosarcomas, and osteosarcomas. mammary, prostatic, pulmonary, anal sac gland and neuroendocrine carcinomas may metastasize widely to abdominal viscera, including the spleen. granulomas and abscesses. microorganisms that cause diffuse granulomatous splenitis and uniform splenomegaly may also cause focal to multifocal nodular lesions (e.g., mycobacterium spp., fungal organisms) (see diffuse granulomatous diseases of the spleen and also enlarged lymph nodes). although there are a large number of abscesses bulge from the capsule and cut surfaces, and the exudate can vary in amount, texture, and color depending on the inciting organism and the age of the lesion. the most common diseases or conditions that have small spleens are ( ) developmental anomalies, ( ) aging changes, ( ) wasting and/or cachectic diseases, and ( ) splenic contraction. splenic hypoplasia. primary immunodeficiency diseases can result in splenic hypoplasia, as well as small thymuses and lymph nodes (which may be so small as to be grossly undetectable in some diseases). these diseases affect young animals and involve defects in t and/or b lymphocytes (fig. - ) . spleens are exceptionally small, firm, and pale red and lack lymphoid follicles and pals. these diseases and their pathologic findings are discussed in chapter and in the sections on disorders of horses and disorders of dogs. congenital accessory spleens. accessory spleens can be either congenital or acquired (see splenic rupture). congenital accessory spleens are termed splenic choristomas, which are nodules of normal splenic parenchyma in abnormal locations. these are usually small diseases and conditions commonly caused by bacteremia (e.g., navel ill, joint ill, chronic respiratory infections, bacterial endocarditis, chronic skin diseases, castration, tail docking, and ear trimming and/ or notching), these rarely result in visible splenic abscesses. pyogranulomas and abscesses in the spleen (multifocal chronic suppurative splenitis) that do develop after septicemia and/or bacteremia are usually caused by pyogenic bacteria such as streptococcus spp., rhodococcus equi (fig. - ) , trueperella pyogenes ( fig. - ) , and corynebacterium pseudotuberculosis. cats with the wet or dry form of feline infectious peritonitis virus may have nodular pyogranulomatous and lymphoplasmacytic inflammatory foci throughout the spleen. splenic abscesses due to direct penetration by a migrating foreign body are reported in cattle (from the reticulum) and less commonly in the horse (from the stomach). perforating gastric ulcers in horses due to gasterophilus and habronema spp. have also reportedly led to adjacent splenic abscesses. granulomas and a b and may be located in the gastrosplenic ligament, liver, or pancreas (see fig. - , b) . splenic fissures. fissures in the splenic capsule are elongated grooves whose axes run parallel to the borders of the spleen. this developmental defect is seen most commonly in horses but also occurs in other domestic animals and has no pathologic significance. the surface of the fissure is smooth and covered by the normal splenic capsule. aging changes. as part of the general aging change of cells as the body ages, there is reduction in the number of b lymphocytes produced by the bone marrow and decline of naïve t lymphocytes due to age-related thymic involution. consequently, there is lymphoid atrophy in secondary lymphoid organs. the spleen is small, and its capsule may be wrinkled. microscopically, the white pulp is atrophied, and splenic follicles, if present, lack germinal centers. sinuses may also collapse from a reduced amount of blood, possibly because of anemia, which makes the red pulp appear fibrous. wasting/cachectic diseases. any chronic disease, such as starvation, systemic neoplasia, and malabsorption syndrome, may produce cachexia. starvation has a marked effect on the thymus, which results in atrophy of the t lymphocyte areas in the spleen and lymph nodes, which is in part mediated by leptin. b lymphocyte development is also diminished, because b lymphocytes require accessory signals from helper t lymphocytes to undergo somatic hypermutation and immunoglobulin isotype switching. splenic contraction. contraction of the spleen is a result of contraction of the smooth muscle in the capsule and trabeculae of storage spleens. it can be induced by the activation of the sympathetic "fight-or-flight" response and is seen in patients with heart failure or shock (cardiogenic, hypovolemic, and septic shock) and also occurs in acute splenic rupture that has resulted in massive hemorrhage (hemoabdomen/hemoperitoneum). the contracted spleen is small, its surface is wrinkled, and the cut surface is dry. hemosiderosis. hemosiderin is a form of storage iron derived chiefly from the breakdown of erythrocytes, which normally takes place in the splenic red pulp. thus some splenic hemosiderosis is to be expected, and the amount varies with the species (it is most extensive in the horse). excessive amounts of splenic hemosiderin are seen when erythropoiesis is reduced (less demand for iron) or from the rapid destruction of erythrocytes in hemolytic anemias (increased stores of iron), such as those caused by immune-mediated hemolytic anemias or hemotropic parasites. excess splenic hemosiderin may also occur in conditions such as chronic heart failure or injections of iron dextran or as focal accumulations at the sites of old hematomas, infarcts, or trauma-induced hemorrhages. hemosiderin is also present in siderofibrotic plaques. siderofibrotic plaques. siderofibrotic plaques are also known as siderocalcific plaques and gamna-gandy bodies. grossly, they are gray-white to yellowish, firm, dry encrustations on the splenic capsule. usually they are most extensive along the margins of the spleen but can be elsewhere on the capsule (fig. - ) and sometimes in the parenchyma. with h&e staining these plaques are a multicolored mixture of yellow (hematoidin), golden brown (hemosiderin), purple-blue (hematoxylinophilic calcium mineral), and pink (eosinophilic fibrous tissue) (fig. - the diseases or conditions with small lymph nodes are ( ) congenital disorders, ( ) lack of antigenic stimulation, ( ) viral infections, ( ) cachexia and malnutrition, ( ) aging, and ( ) radiation. congenital disorders. primary immunodeficiency diseases are described in detail in chapter and in the sections on disorders of horses and disorders of dogs. neonatal animals with primary immunodeficiency diseases often have extremely small to undetectable lymph nodes. in dogs and horses with severe combined immunodeficiency disease (scid), lymphoid tissues, including lymph nodes from affected animals, are often grossly difficult to identify and characterized by an absence of lymphoid follicles. congenital may represent sequelae to previous hemorrhages from trauma to the spleen. splenic rupture. splenic rupture is most commonly caused by trauma, such as from an automobile accident or being kicked by other animals. thinning of the capsule from splenomegaly can render the spleen more susceptible to rupture, and this may occur at sites of infarcts, hematomas, hemangiosarcomas, and lymphoma. in acute cases of splenic capsular rupture, the spleen is contracted and dry and the surface wrinkled from the marked blood loss (fig. - ) . in more severe cases the spleen may be broken into two or more pieces, and small pieces of splenic parenchyma may be scattered throughout the omentum and peritoneum (sometimes called splenosis) (fig. - , a) . clotted blood, fibrin, and omentum may adhere to the surface at the rupture site. if the rupture is not fatal, the spleen heals by fibrosis, and there may be a capsular scar. occasionally there are two or more separate pieces of spleen adjacent to each other and sometimes joined by scar tissue in the gastrosplenic ligament. the functional capabilities of the small accessory spleens are questionable, although erythrophagocytosis, hemosiderosis, hyperplastic nodules, emh, and neoplasia can be present in these nodules. accessory spleens due to traumatic rupture should be distinguished from peritoneal seeding of hemangiosarcoma and the developmental anomaly splenic choristomas (see fig. - , b) , which are nodules of normal splenic parenchyma in abnormal locations (such as liver and pancreas). chronic splenic infarcts. in the early stage, splenic infarcts are hemorrhagic and may elevate the capsule (see splenic nodules with a bloody consistency). however, as the lesions age and fibrous connective tissue is laid down, they shrink and become contracted and often depressed below the surface of the adjacent capsule. conditions causing lymphadenomegaly include ( ) lymphoid hyperplasia (follicular or paracortical), ( ) hyperplasia of the sinus histiocytes (monocyte-macrophage system), ( ) acute or chronic lymphadenitis, ( ) lymphoma, and ( ) metastatic neoplasia. macrophage system. detailed descriptions of lymphoid follicular hyperplasia, paracortical hyperplasia, and hyperplasia of sinus histiocytes are in the sections on lymph nodes, function, and lymph node, dysfunction/responses to injury. follicular lymphoid hyperplasia can involve large numbers of lymph nodes, as in a systemic disease, or can be localized to a regional lymph node draining an inflamed or antigenically stimulated (e.g., vaccine injection) area. acute lymphadenitis. lymph nodes draining sites of infection and inflammation may develop acute lymphadenitis (e.g., retropharyngeal lymph nodes draining the nasal cavity with acute rhinitis, tracheobronchial lymph nodes in animals with pneumonia ( fig. - ), and mammary [supramammary] lymph nodes in animals with mastitis). grossly, affected lymph nodes in acute lymphadenitis are red and edematous, have taut capsules, and may have necrotic areas ( fig. - ). in some instances the afferent lymphatic vessels may also be inflamed (lymphangitis). the material draining to the regional lymph node may be microorganisms (bacteria, parasites, protozoa, and fungi), inflammatory mediators, or a sterile irritant. in septicemic diseases, such as bovine anthrax, the lymph nodes are markedly congested and the sinuses filled with blood. examination of these lymph nodes should include culturing for bacteria and the examination of smears and histologic sections for bacteria and fungi. pyogenic bacteria, such as streptococcus equi ssp. equi in horses , streptococcus porcinus in pig, and trueperella pyogenes in cattle and sheep, cause acute suppurative lymphadenitis (see disorders of horses and disorders of pigs). hereditary lymphedema has been reported in certain breeds of cattle and dogs. grossly, the most severely affected animals have generalized subcutaneous edema (see fig. - ) and effusions. in severe cases the peripheral and mesenteric lymph nodes are hypoplastic and characterized by an absence of follicles. nodes draining an edematous area may be grossly enlarged from marked sinus edema. lack of antigenic stimulation. the size of the lymph node depends on the level of phagocytosis and antigenic stimulation; lymph nodes that are not receiving antigenic stimuli (e.g., spf animals) will be small with low numbers of primary lymphoid follicles and few, if any, secondary follicles or plasma cells in the medullary cords. conversely, nodes receiving constant antigenic material (such as those draining the oral cavity or intestines) are large with active secondary lymphoid follicles. the number of follicles increases or decreases with changes in the intensity of the antigenic stimuli, and the germinal centers go through a cycle of activation, depletion, and rest, as described previously (see lymph nodes, function). as the antigenic response wanes, germinal centers become depleted of lymphocytes, and lymphoid follicles become smaller. viral infections. many viral infections of animals target lymphocytes and cause the destruction of lymphoid tissue. of infectious agents, viruses most commonly infect and injure lymphoid tissues and include the following: ehv- in aborted foals, classic swine fever virus, bvdv, canine distemper virus, and canine and feline parvovirus. although some viruses destroy lymphoid tissue, others can lead to lymph node hyperplasia (e.g., follicular b lymphocyte hyperplasia in fiv and paracortical t lymphocyte hyperplasia in malignant catarrhal fever virus) or cause neoplasia (e.g., felv, blv, and marek's disease). cachexia and malnutrition. malnutrition and cachexia, which occur with cancer, lead to secondary immunosuppression through several complex metabolic and neurohormonal aberrations. starvation has marked effect on the thymus with resultant atrophy of the t lymphocyte areas in the spleen and lymph nodes and may also affect b lymphocyte development. lymphoid atrophy may result from physiologic and emotional stress and the concurrent release of catecholamines and glucocorticoids. glucocorticoids reduce b and t lymphocytes via redistribution of these cells and glucocorticoidinduced apoptosis. t lymphocytes are more sensitive to glucocorticoid-induced apoptosis than are b lymphocytes. aging. as part of the general aging change of cells as the body ages, there is reduction in the number of lymphocytes produced by the bone marrow and regressed thymus, and consequently a reduction in the b and t lymphocytes in secondary lymphoid organs, resulting in lymphoid atrophy. consequently, lymph nodes are small, with loss of b and t lymphocytes and plasma cells in the cortical follicles, paracortex, and medullary cords, respectively. radiation. local or palliative treatment of cancer may include radiotherapy (ionizing radiation) to target and damage the dna of the neoplastic cells. although some immunosuppression may be noted, particularly if bone marrow or lymphoid tissues are within the irradiated field, mounting evidence suggests that radiotherapy can induce a cascade of proimmunogenic effects that engage the innate and adaptive immune systems to contribute to the destruction of tumor cells. fibrosis of tissues within the irradiated field also occurs as mainly a late effect of chronic radiation. bouts of chronic lymphadenitis (e.g., regional lymph node draining chronic mastitis in cows) lead to fibrosis and lymphoid hyperplasia, in addition to chronic abscesses. the classic example of chronic suppurative lymphadenitis with encapsulated abscesses is caseous lymphadenitis, a disease of sheep and goats caused by c. (also see disorders of ruminants). it is also the cause of ulcerative lymphangitis in cattle and horses and pectoral abscesses in horses. classic examples of focal to multifocal granulomatous lymphadenitis are mycobacterium tuberculosis complex, which includes m. bovis among others. members of m. avium complex cause similar lesions and have been described in a number of species, including dogs, cats, primates, pigs, cattle, sheep, horses, and human beings. infection may begin by inhalation of aerosol droplets containing the bacilli, which may spread via the lymphatic vessels to regional lymph nodes, resulting in granulomatous lymphangitis and lymphadenitis (fig. - ) . initially lesions in the lymphatic system are confined to the lymphatic vessels (granulomatous lymphangitis) and regional lymph nodes (e.g., the tracheobronchial lymph nodes in the case of pulmonary histologically, the subcapsular, trabecular, and medullary sinuses and the parenchyma of the cortex and medulla have focal to coalescing foci of neutrophilic inflammation, necrosis, and fibrin deposition ( fig. - ). if inflammation in the lymph node continues for several days or longer, the lymph node is further enlarged by follicular hyperplasia and plasmacytosis of the medullary cords from the expected immune response. chronic lymphadenitis. the types of chronic lymphadenitis include chronic suppurative lymphadenitis, diffuse granulomatous inflammation, and discrete granulomas. in chronic suppurative inflammation, abscesses range in size from small microabscesses to large abscesses that occupy and obliterate the whole node. recurrent figure - acute lymphadenitis, lymph node, dog. acute lymphadenitis usually occurs when a regional lymph node drains a site of inflammation caused by microorganisms and subsequently becomes infected. the lymph node is firm and enlarged with a tense capsule. the cut surface bulges and is wet with blood, edema, and an inflammatory cell infiltrate. histoplasmosis (see disorders in dogs). in feline cryptococcosis (most often cryptococcus neoformans), the inflammatory response may be mild due to the thick polysaccharide capsule, which has strong immunomodulatory properties and promotes immune evasion and survival within the host. therefore the nodal enlargement is due mainly to a large mass of organisms (see e- fig. - ) . pigs with pcv infection may have a multifocal to diffuse infiltrate of macrophages and multinucleated giant cells of varying severity (see disorders of pigs). secondary (metastatic) neoplasms. carcinomas typically metastasize via lymphatic vessels to the regional lymph node. other common metastatic neoplasms include mast cell tumor and malignant melanoma. although sarcomas most often metastasize hematogenously, some more aggressive sarcomas (e.g., osteosarcoma) may spread to regional lymph nodes. histologically, single cells or clusters of neoplastic cells travel via the afferent lymphatic vessels and are deposited in a sinus, usually the subcapsular sinus (see fig. - ). here the cells proliferate and can ultimately occupy the whole lymph node, as well as drain to the next lymph node in the chain. tuberculosis), but once disseminated in the lymph or blood, lymph nodes throughout the body will have lesions. well-organized granulomas consist of a central mass of macrophages with phagocytized mycobacteria, surrounded by epithelioid and foamy macrophages and occasional multinucleated giant cells (langhans type). these inflammatory nodules are surrounded by a layer of lymphocytes enclosed in a fibrous capsule. over time the center of the granuloma may undergo caseous necrosis due to the high lipid and protein content of the dead macrophages (see chapter ). in bovine johne's disease the mesenteric lymph nodes draining the infected intestine can have noncaseous granulomas (fig. - ). diffuse granulomatous lymphadenitis. coalescing to diffuse granulomatous lymphadenitis is seen in disseminated fungal infections such as blastomycosis, cryptococcosis (e- fig. - ) , and extent of the emphysema. in severe cases the lymph node is light, puffy, and filled with discrete gas bubbles, and the cut surface may be spongy. histologically, the sinuses are distended with gas and lined by macrophages and giant cells. this change has been considered a foreign body reaction to the gas bubbles. macrophages and giant cells are also seen in afferent lymphatic vessels (granulomatous lymphangitis). vascular transformation of lymph node sinus (nodal angiomatosis). vascular transformation of the sinuses is a nonneoplastic reaction to blocked efferent lymphatic vessels or veins. this pressure-induced lesion results in the formation of anastomosing vascular channels and may be confused with a nodal vascular neoplasm. these proliferative but noninvasive masses usually begin in the subcapsular sinuses and may be followed by lymphoid atrophy, erythrophagocytosis/hemosiderosis, and fibrosis. the blockage may be caused by malignant neoplasms of the tissues that the lymph node drains (e.g., thyroid carcinoma with nodal angiomatosis of the mandibular lymph node). see section on bone marrow, disorders of domestic animals, hematopoietic neoplasia for a discussion of the who classification of hematopoietic neoplasia that predominantly arise and proliferate within bone marrow. this section will cover neoplasms of lymphoid tissue(s) arising outside of bone marrow. lymphoma. the term lymphoma (also known as lymphosarcoma) encompasses a diverse group of malignancies arising in lymphoid tissue(s) outside of bone marrow. grossly, there may be diffuse to nodular enlargement of one or more lymph nodes (fig. - ) , and the cut surface is soft, white, and bulging with loss of normal corticomedullary architecture. there is great variation in the clinical manifestations and cytopathologic features of lymphoma, which underlie the importance of classification to better predict the clinical behavior and outcome. an understanding of lymphocyte maturation is crucial, because the who classification of lymphoma postulates a normal cell counterpart for each type of lymphoma (when possible). in other words, lymphoma can arise at any stage in the development/maturation of a lymphocyte-from precursor lymphocytes (b or t lymphoblasts) to mature lymphoid b and t, lymphocytes and nk cells (table - and box - ). pathologists use gross features, histomorphologic features, immunophenotype (b or t lymphocyte), and clinical characteristics to red discoloration is caused by ( ) draining erythrocytes from hemorrhagic or acutely inflamed areas, ( ) acute lymphadenitis with hyperemia and/or hemorrhage, ( ) acute septicemias with endotoxininduced vasculitis or disseminated intravascular coagulation, and ( ) dependent areas in postmortem hypostatic congestion. blood in pig lymph nodes is especially obvious due to the inverse anatomy (the equivalent of the medullary sinuses are subcapsular and thus readily visible in the unsectioned node). initially erythrocytes fill trabecular and medullary sinuses and then rapidly undergo erythrophagocytosis by proliferating sinus macrophages. hemosiderin deposition occurs within to days in these macrophages, imparting a brown discoloration of the node. black discoloration is often present in the tracheobronchial lymph nodes due to draining of carbon pigment (pulmonary anthracosis, see chapter ). black ink from skin tattoos will drain to the regional lymph node. these pigments are usually noted within the medullary sinus macrophages. brown discoloration may be due to melanin, parasitic hematin, or hemosiderin. melanin pigment is seen in animals with chronic dermatitis when melanocytes are damaged and their pigment is released into the dermis and phagocytized by melanomacrophages (pigmentary incontinence) and drained to the regional lymph node. the mandibular lymph nodes often contain numerous melanomacrophages in animals with heavily pigmented oral mucosa, presumably due to chronic low levels of inflammation. this must be distinguished from metastatic malignant melanomas. lymph nodes draining areas of congenital melanosis may have melanin deposits. parasitic hematin pigment is produced by fascioloides magna (cattle) and fasciola hepatica (sheep) in the liver and then transported via the lymphatic vessels to the hepatic lymph nodes. hemosiderin, an erythrocyte breakdown product, may form in a hemorrhagic node or arrive in hemosiderophages draining from congested, hemorrhagic, or inflamed areas. drainage of iron dextran from an intramuscular injection may also cause hemosiderin pigment accumulation within the draining lymph node. green discoloration is rare and may be caused by green tattoo ink (often used in black animals); ingestion of blue-green algae, which drain to mesenteric lymph nodes; massive eosinophilic inflammation; and in mutant corriedale sheep, which have a genetic defect that results in a deficiency in the excretion of bilirubin and phylloerythrin by the liver. the phylloerythrin or a metabolite stains all the tissues of the body a dark green, except for the brain and spinal cord, which are protected by the blood-brain barrier. miscellaneous discolorations of lymph nodes may be seen with intravenously injected dyes (e.g., methylene blue or trypan blue) or subcutaneous drug injections. lymph nodes may be yellow in severely icteric patients. the pigmented strain of map (johne's disease) may impart an orange discoloration in the mesenteric lymph nodes of sheep. inclusion bodies. many viruses produce inclusion bodies, and some of these occur in lymph nodes. these viruses include ehv- in horses, bovine adenovirus, cytomegalic virus in inclusion body rhinitis and pcv , herpesvirus of pseudorabies in pigs, and rarely parvovirus in dogs and cats. emphysema. emphysema in lymph nodes is a consequence of emphysema in their drainage fields and is seen most frequently in tracheobronchial lymph nodes in bovine interstitial emphysema and in porcine mesenteric lymph nodes in intestinal emphysema (see chapter ). the appearance of the lymph node varies with the immunohistochemistry (mum /irf is particularly sensitive and specific for plasma cell neoplasms). histiocytic disorders. histiocytic disorders are frequently diagnosed in dogs and occur less often in cats. briefly, histiocytes are categorized as macrophages and dcs, the latter of which are subdivided into langerhans cells (lcs), found in skin, gastrointestinal, respiratory, and reproductive epithelia (mucosae), and interstitial dcs (idc), located in perivascular spaces of most organs. the term interdigitating dcs describes dcs (either resident or migrating) found in t lymphocyte regions of lymph nodes (paracortex) and spleen (pals); interdigitating dcs consist of both lcs and idcs. these lineages can be differentiated using immunohistochemical stains. histiocytic disorders that are diagnosed in veterinary medicine at this time include the following: canine cutaneous histiocytoma, canine lc histiocytosis, canine cutaneous and systemic histiocytosis, feline pulmonary lc histiocytosis, feline progressive histiocytosis, dendritic cell leukemia in the dog, and histiocytic sarcoma and hemophagocytic histiocytic sarcoma in both dogs and cats. lymph node involvement is seen in many of these conditions. rare reports of regional lymph node metastasis in cases of solitary canine cutaneous histiocytoma have been published. lymphatic invasion with subsequent regional nodal involvement may be seen in dogs with lc histiocytosis, which is a poor prognostic indicator and likely reflects systemic infiltration. the normal architecture of tracheobronchial lymph nodes is often effaced in cats with pulmonary lc histiocytosis. canine reactive histiocytoses are not clonal neoplastic proliferations but likely reflect an immune dysregulation consisting of activated dermal idcs (and t lymphocytes). they are categorized as cutaneous histiocytosis (ch), involving skin and draining lymph nodes, and a more generalized systemic histiocytosis (sh), affecting skin and other sites (e.g., lung, liver, bone marrow, spleen, lymph nodes, kidneys, and orbital and nasal tissues). histiocytic sarcoma complex. histiocytic sarcomas (hss) are neoplasms of idcs and therefore can arise in almost any tissue, frequently the spleen, lung, skin, meninges, lymph nodes, bone marrow, and synovium. secondary involvement of the liver is common as the disease progresses. this neoplasm is most commonly diagnosed in dogs, and a lower incidence is seen in cats. localized histiocytic sarcoma may be a focal solitary lesion or multiple nodules within a single organ. disseminated histiocytic sarcoma describes classify lymphomas. the morphologic features used in histopathologic classification are the following: • histologic pattern-nodular or diffuse. • cell size-the nuclei of the neoplastic lymphocytes are compared to the diameter of a red blood cell (rbc ≅ µm). small is less than . times the diameter of an rbc; intermediate is . to . times the diameter of an rbc; large is more than . times the diameter of an rbc. • grade-mitotic figures are counted in a single high-power ( ×) field. indolent is to ; low is to ; mid is to ; high is more than . although there are numerous subtypes of lymphoma recognized under the who system, a detailed discussion of each subtype is outside the scope of this textbook. however, a select number of subtypes are more commonly seen in domestic animals (see table - ) and currently best described in the dog (see disorders of dogs, neoplasms, lymphomas). the most common types in dogs are large cell lymphomas and include diffuse large b cell lymphoma and peripheral t cell lymphoma. t cell-rich large b cell lymphoma is thought to be a variant of diffuse large b cell lymphoma with a distinctive reactive t lymphocyte infiltrate. intermediate cell lymphomas include b or t lymphocyte lymphoblastic lymphomas and burkitt-like lymphoma (both high grade), marginal zone lymphoma, and the intermediate cell variant of t zone lymphomas (both indolent and nodular). small cell lymphomas most commonly diagnosed in domestic animal species include enteropathy-associated t cell lymphoma, commonly seen in the cat, t zone lymphoma (small cell variant), and small cell lymphoma. cutaneous lymphomas are most often of t lymphocyte origin and may be epitheliotropic or nonepitheliotropic, and a distinct entity of inflamed t cell lymphoma has been recently described in dogs (see chapter ). plasma cell neoplasia. plasma cell neoplasms are most easily categorized as myeloma or multiple myeloma, which arises in the bone marrow, and extramedullary plasmacytoma, which as the name implies involves sites other than bone. multiple myeloma. see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, plasma cell neoplasia. extramedullary plasmacytomas. extramedullary plasmacytomas are most commonly diagnosed in the skin of dogs (also cats and horses), where they constitute . % of all canine cutaneous tumors (see chapter ). the pinnae, lips, digits, and chin are the most commonly affected locations, and most lesions are solitary, though multiple plasmacytomas are infrequently diagnosed. other tissues affected include the oral cavity, intestine (colorectal in particular), liver, spleen, kidney, lung, and brain; of these, the oral cavity and intestine (colorectal) are involved most often. in one study, extramedullary plasmacytomas represented % of all canine oral tumors and % of all extramedullary plasmacytomas diagnosed. most cutaneous extramedullary plasmacytomas are benign, and complete excision is usually curative; oral cavity and colorectal extramedullary plasmacytomas are likely to behave in a similar manner. more aggressive forms may occur at any site. as with multiple myeloma, the neoplastic cells composing the tumor may vary from well differentiated to pleomorphic, often within the same tumor. the cells often have a characteristic perinuclear golgi clearing or "halo," and the more pleomorphic cells exhibit karyomegaly and binucleation (fig. - ) . extramedullary plasmacytomas may produce monoclonal immunoglobulins with resulting monoclonal gammopathy. amyloid deposition (which may mineralize) is also observed in a proportion of cases. differentiation from other round cell tumors may be aided by severe combined immunodeficiency disease of arabian foals is an autosomal recessive primary immunodeficiency disorder characterized by the lack of functional t and b lymphocytes caused by a genetic mutation in the gene encoding for dna-dependent protein kinase catalytic subunit (dna-pkcs). this enzyme is required for receptor gene rearrangements involved in the maturation of lymphocytes, and the resulting loss of functional t and b lymphocytes leads to a profound susceptibility to infectious diseases. though normal at birth, these foals develop diarrhea and pneumonia by approximately days of age, often due to adenovirus, cryptosporidium parvum, and pneumocystis carinii infections. affected foals often die before months of age. lymph nodes and thymus are small and often grossly undetectable, and the spleen is small and firm due to the absence of white pulp (see fig. - ) . the development of genetic tests to identify carriers of the disorder has led to a decrease in the prevalence of severe combined immunodeficiency disease. recently, severe combined immunodeficiency disease was diagnosed in a single caspian filly, though the exact genetic defect was not determined. congenital immunodeficiency diseases are also discussed in detail in chapter . streptococcus equi ssp. equi, the etiologic agent of equine strangles, is inhaled or ingested after direct contact with the discharge from infected horses or from a contaminated environment. the bacteria attach to the tonsils, penetrate into deeper tissues, enter the lymphatic vessels, drain to regional lymph nodes (mandibular, retropharyngeal, and occasionally parotid and cervical lymph nodes), and cause large abscesses (see fig. - ). retropharyngeal enlargement from abscesses may lead to compression of the pharynx and subsequent respiratory stridor and dysphagia. abscesses may rupture and discharge pus through a sinus to the skin surface or spread medially into guttural pouches, where residual pus dries and hardens to form chondroids (which serve as a nidus for live bacteria to persist in carrier animals). in up to % of these cases, ruptured abscess material may spread via blood or lymph to other organs (metastatic abscess formation, bastard strangles), including lung, liver, kidney, synovia, mesenteric and mediastinal lymph nodes, spleen, and occasionally brain. purpura hemorrhagica, a type iii hypersensitivity reaction, may result in necrotizing vasculitis in some horses with repeated natural exposure to s. equi ssp. equi or after vaccination in horses that have had strangles. the typical manifestation of r. equi infection is chronic suppurative bronchopneumonia with abscesses (see chapter ). approximately % of foals also develop intestinal lesions characterized by pyogranulomatous ulcerative enterotyphlocolitis, often over peyer's patches, and pyogranulomatous lymphadenitis of mesenteric and colonic lymph nodes (see chapter ; see fig. - ) . large abdominal abscesses may be the only lesion in the abdomen and presumably originate from an infected mesenteric lymph node. the diffuse lymphatic tissue in the lamina propria may contain granulomatous inflammation with the phagocytized bacteria. mediastinal pyogranulomatous lymphadenitis may compress the trachea, causing respiratory distress. r. equi lesions also can develop in the liver, kidney, spleen, or nervous tissue. lymphoma is the most common malignant neoplasm in horses and mostly affects adult animals (mean age to years) with no lesions that involve distant sites and has replaced the term malignant histiocytosis. breed predispositions to histiocytic sarcoma complex are seen in bernese mountain dogs, rottweilers, golden retrievers, and flat-coated retrievers, though the disease can occur in any breed. histiocytic sarcoma complex is considered to have a rapid and highly aggressive course, and the clinical signs depend on the particular organ(s) involved. grossly, affected organs may be uniformly enlarged and/or contain multiple coalescing white-tan nodules. tissue architecture is effaced by sheets of pleomorphic round to spindle-shaped cells. there is marked cellular atypia with numerous karyomegalic and multinucleated neoplastic cells (fig. - ) . hemophagocytic histiocytic sarcoma. hemophagocytic histiocytic sarcoma is seen in dogs and cats and is a neoplasm of macrophages of the spleen and bone marrow. clinically, dogs present with hemolytic regenerative anemia and thrombocytopenia, thus mimicking evans's syndrome, though they are coombs negative. this form of histiocytic sarcoma carries the worst prognosis of the histiocytic sarcomas, which is likely in part related to the severe anemia and coagulopathy. it is characterized by a non-mass forming infiltrate of histiocytes within the bone marrow and splenic red pulp, causing diffuse splenomegaly. the neoplastic cells exhibit marked erythrophagocytosis, but the severe cellular pleomorphism seen in the histiocytic sarcoma complex may be lacking. the neoplastic cells are often intermixed with emh and plasma cells. metastasis is frequently to the liver, where the cells concentrate within the sinuses. tumor emboli within the lung are often present. malt is involved in a variety of ways with bacteria and viruses, and these are summarized for large animals in table - . these interactions include being a portal of entry for pathogens (e.g., salmonella spp., yersinia pestis, map, and l. monocytogenes); a site of replication for viruses (e.g., bvdv); a site for hematogenous infection (e.g., panleukopenia virus and parvovirus); and a site of gross or microscopic lesions in some viral diseases. bovine coronavirus, bvdv, rinderpest virus, malignant catarrhal fever virus, feline panleukopenia virus, and canine parvovirus cause lymphocyte depletion within the malt. anthrax is caused by b. anthracis, a gram-positive bacillus found in spore form in soil. cattle, sheep, and goats become infected when grazing on infected soil, and infection causes fulminant septicemia. the spleen in infected animals is markedly enlarged and congested (see uniform splenomegaly with a bloody consistency; see also chapter ). bovine viral diarrhea is caused by bvdv, a pestivirus. cattle are the natural host, but other animals such as alpacas, deer, sheep, and goats are also affected. bvdv preferentially infects cells of the immune system, including macrophages, dcs, and lymphocytes. the associated lesions in lymphoid tissues are severe lymphoid depletion in mesenteric lymph nodes and peyer's patches, whose intestinal surface may be covered by a fibrinonecrotic membrane. histologically, there is marked lymphocytolysis and necrosis of germinal centers in peyer's patches and cortices of lymph nodes. there is thymic atrophy because the thymus is markedly depleted of lymphocytes and may consist of only collapsed stroma and few scattered lymphocytes. bvd is discussed in detail in chapters and . apparent breed or sex predisposition. the most frequent anatomic locations of equine lymphoma are multicentric, cutaneous, and gastrointestinal tract. multicentric lymphoma, defined as involving at least two organs (excluding the regional lymph nodes), is the most common manifestation, followed by skin and gastrointestinal tract types. solitary locations have been reported in the mediastinum, lymph nodes, ocular/orbital region, brain, spinal cord, oral cavity, and spleen. of the multicentric lymphomas, the most frequently observed type is t cell-rich large b cell lymphoma (tcrlbcl), reportedly in one study affecting % of the cases. peripheral t cell lymphoma (ptcl) was the second most common, followed by diffuse large b cell lymphoma (dlbcl). the most common lymphoma type in the gastrointestinal tract is also t cell-rich large b cell lymphoma, followed by enteropathyassociated t cell lymphoma. cutaneous lymphomas in horses account for up to % of all equine skin tumors. t cell-rich large b cell lymphoma is again the most common lymphoma subtype in the skin, representing up to % of all cutaneous lymphomas, and most frequently presents clinically as multiple skin masses. cutaneous t cell lymphoma (ctcl) is the second most common form and arises as smaller solitary nodules. thoroughbreds may have a higher incidence of cutaneous t cell lymphoma compared to other breeds. overall, horses with cutaneous t cell-rich large b cell lymphoma appear to have a longer survival time than horses with other types of lymphoma of the skin. progesterone receptor-positive lymphomas have also been identified in horses, and there is one report of subcutaneous tumor regression following removal of an ovarian granulosa-theca cell tumor. there may be an increased frequency of lymphoma in horses diagnosed with equine herpesvirus (ehv- , gammaherpesvirus), when compared to healthy horses, although the exact cause-effect role of this observation in lymphomagenesis is not yet known. histologically, the hallmark features of t cell-rich large b cell lymphoma include a majority of small (nuclei approximately the size of an rbc), reactive, mature t lymphocytes admixed with a neoplastic population of large b lymphocytes whose nuclei are two to three times the diameter of an equine rbc. these large atypical cells are often binucleated and have prominent eosinophilic nucleoli ( fig. - ). the large cells may be observed in mitosis or in necrosis as single cells with retracted cytoplasm and pyknotic nuclei. t cell-rich large b cell lymphoma is often accompanied by the presence of a dense fibrovascular network. johne's disease primarily affects domestic and wild ruminants (and rarely pigs and horses) and is due to infection by map. the characteristic lesions include granulomatous enteritis usually confined to the ileum, cecum, and proximal colon; lymphangitis; and lymphadenitis of regional lymph nodes (see fig. - ) . the bacteria are ingested, engulfed by the m cells overlying peyer's patches, and then transported to macrophages in the lamina propria and submucosa. among cattle, sheep, goats, and wild ruminants, there is wide variation in the severity, distribution of lesions, primary inflammatory cell type (lymphocytes, epithelioid macrophages, multinucleated giant cells), and numbers of bacteria within lesions (multibacillary or paucibacillary). histologically, the architecture of the ileocecal lymph nodes may be partially replaced by aggregates of epithelioid postweaning multisystem wasting syndrome pcv , a small single-stranded dna virus, is highly prevalent in the domestic pig population. several clinical syndromes are attributed to pcv infection and collectively termed pcv-associated diseases (pcvads). these include postweaning multisystemic wasting syndrome (pmws), porcine respiratory disease complex (prdc), porcine dermatitis and nephropathy syndrome, and enteric disease (see chapters and ). the major postmortem findings of postweaning multisystemic wasting syndrome are poor body condition, enlarged lymph nodes, and interstitial pneumonia. the lesions of the lymphoid system are commonly observed in the tonsil, spleen, peyer's patches, and lymph nodes. some pigs have all lymphoid tissues affected, whereas others may have only one or two affected lymph nodes. the characteristic microscopic lesions are lymphoid depletion of both follicles and paracortex with replacement by histiocytes, mild to severe granulomatous inflammation with multinucleated giant cells, and intrahistiocytic sharply demarcated, spherical, basophilic cytoplasmic inclusion bodies. necrosis of prominent lymphoid follicles (necrotizing lymphadenitis) is occasionally observed, and pcv can be detected within the necrotic regions. the loss of lymphocytes may be due to reduced production in the bone marrow, decreased proliferation in the secondary lymphoid organs, or necrosis of lymphocytes. splenic abscesses can be the result of bacteremia (see fig. - ) or direct penetration by a foreign body from the reticulum (see spleen and also portals of entry/pathways of spread). c. pseudotuberculosis is a gram-positive intracellular bacterium that causes caseous lymphadenitis, a chronic suppurative disease of sheep and goats. the bacterium may enter through skin wounds (e.g., shearing cuts in sheep, tagging, tail docking, or castration), drain to the regional lymph node, and then be disseminated in lymph and circulating blood to external and internal lymph nodes, as well as other internal organs, including lung. external abscesses are most often detected in the "jaw and neck" region, specifically in the mandibular and parotid lymph nodes. on gross examination the abscesses are encapsulated and filled with greenish semifluid pus due to an infiltrate of eosinophils (see fig. - ) . over time the abscesses lose the greenish hue, and contents become inspissated to form the characteristic concentric laminations (see fig. - ); old abscesses may reach a diameter of to cm. bovine lymphoma is broadly classified into enzootic and sporadic forms. the enzootic form, called enzootic bovine leukosis (ebl), is caused by blv, a retrovirus common in cattle. there is a higher prevalence in dairy cattle compared to beef breeds. blv is transmitted horizontally (e.g., blood, milk/colostrum, saliva) or iatrogenically (e.g., rectal sleeves, instruments/equipment). following infection, blv invades and integrates into the genome of infected b lymphocytes, resulting in a polyclonal b lymphocyte lymphocytosis in approximately % of cattle. in approximately % to % of blv-infected cattle, a single clone will emerge, leading to the development of b lymphocyte leukemia/lymphoma. the average incubation period between infection and development of lymphoma is to years, and this low conversion rate suggests that the latency period may be longer than the life span of most animals (dairy cattle seldom live to the -to -year peak incidence of lymphoma occurrence). other contributing variables, such as genetic background, coinfections, and environmental factors, may also play a role in lymphomagenesis. the exact mechanism of blv-induced tumorigenesis is poorly understood. recently blv micrornas (mirnas) were identified in preleukemic and malignant b lymphocytes, which showed repression of structural and regulatory gene expression. these findings suggested that mirnas may play a key role in tumor onset and progression. grossly, multiple tissues may be affected in cattle that develop lymphoma, including peripheral lymph nodes (cephalic, cervical, sublumbar) ( fig. - ) , abdominal lymph nodes, retrobulbar region, abomasum, liver, spleen, heart, urogenital tract, bone marrow, vertebral canal ( fig. - ) , and spinal cord. one study indicates most of these high-grade lymphomas are diffuse large cell lymphomas ( %), and approximately % are intermediate cell lymphomas (burkitt-like and lymphoblastic lymphomas). the sporadic form of bovine lymphoma is most often of t lymphocyte immunophenotype and has three subcategories: cutaneous, calf, and thymic. there is no known viral cause for the sporadic form, and each subcategory has a much smaller prevalence compared to the enzootic form. of the three sporadic forms, the cutaneous form seems to be the most common and manifests itself as multiple skin nodules in -to -year-old cattle. the calf form presents as generalized lymphadenopathy with weight loss, lethargy, and weakness in calves less than months old. the thymic form is reportedly more common in beef cattle, to months of age. lymphoma is the most frequently reported cancer of pigs based on abattoir surveys. affected pigs are typically less than year of age, and there is no reported breed predisposition, although a hereditary basis is suspected in cases arising in inbred herds. the two main forms of porcine lymphoma are thymic/mediastinal and multicentric; the latter is more common. spleen, liver, kidney, bone marrow, and lymph nodes are affected in the multicentric form, with visceral lymph nodes reportedly more commonly involved than peripheral nodes. a recent study of lymphoma in pigs found the majority to be multicentric, and subtypes included the following: b lymphoblastic leukemia/lymphoma, follicular lymphoma, diffuse and intestinal large b cell lymphoma, and peripheral t cell lymphoma. one case each of thymic b cell and t cell lymphomas were also described. several types of severe combined immunodeficiency diseases have been described in dogs. a mutation in dna-pkcs (similar to arabian horses) with an autosomal recessive mode of inheritance is seen in jack russell terriers. an x-linked form of severe combined immunodeficiency disease is well described in basset hounds and is caused by mutations in the common γ-chain (γc) subunit of the receptors for il- , il- , il- , il- , il- , and il- . a similar disease is seen in cardigan welsh corgi puppies, though it is an autosomal mode of inheritance in this breed. the mutation inhibits the signal transduction pathways initiated by any of these cytokines, which are critical for the proliferation, differentiation, survival, and function of b and t lymphocytes. affected dogs have normal numbers of circulating b lymphocytes that are unable to class switch to igg or iga and reduced numbers of t lymphocytes, which are nonfunctional due to the inability to express il receptors. affected puppies are remarkably susceptible to bacterial and viral infections and rarely survive past to months of age. the thymus of these dogs is small and consists of only small dysplastic lobules with a few of hassall's corpuscles. tonsils, lymph nodes, and peyer's patches are often grossly unidentifiable due to the severe lymphocyte hypoplasia. congenital immunodeficiency diseases are also discussed in detail in chapter . thymic hemorrhage and hematomas have been reported in dogs and are most often seen in young animals. a variety of causes are described, including ingestion of anticoagulant rodenticides (warfarin, dicumarol, diphacinone, and brodifacoum), dissecting aortic aneurysms, trauma (e.g., automobile accident), and idiopathic/ spontaneous. histologically, hemorrhage variably expands the thymic lobules and septa, and in severe cases the lobular architecture is obscured by hemorrhage. in cases of anticoagulant rodenticide toxicosis, the medulla appears to be the main site of hemorrhage. see uniform splenomegaly with a bloody consistency (also see figs. - and - ). see the section on splenic nodules with a bloody consistency for discussion on splenic hematomas (including those induced by nodular hyperplasia or occurring with hemangiosarcoma), incomplete splenic contraction, acute splenic infarcts, and hemangiosarcomas. porcine reproductive and respiratory syndrome (prrs) is caused by an arterivirus and causes two overlapping clinical syndromes: reproductive failure and respiratory disease. the virus is transmitted by contact with body fluids (saliva, mucus, serum, urine, and mammary secretions and from contact with semen during coitus), but often it first colonizes tonsils or upper respiratory tract. the virus has a predilection for lymphoid tissues (spleen, thymus, tonsils, lymph nodes, peyer's patches). viral replication takes place in macrophages of the lymphoid tissues and lungs, though porcine reproductive and respiratory syndrome virus antigen is found in resident macrophages in many tissues and may persist in tonsil and lung macrophages. the result of this infection is a reduction in the phagocytic and functional capacity of macrophages of the monocytemacrophage system. as a consequence, there is reduction in resistance to common bacterial and viral pathogens. most porcine reproductive and respiratory syndrome-infected pigs are coinfected with one or more pathogens, including streptococcus suis and salmonella choleraesuis. infection with bordetella bronchiseptica and mycoplasma hyopneumoniae appear to increase the duration and severity of the interstitial pneumonia. the major lesions are interstitial pneumonia and generalized lymphadenopathy, and tracheobronchial and mediastinal lymph nodes are most commonly affected. coinfections often complicate the gross and histopathologic changes. lymph nodes are enlarged, pale tan, occasionally cystic, and firm; some strains of virus also cause nodal hemorrhage. microscopically, the lesions in the lymph nodes, tonsils, and spleens consist of varying degrees of follicular and paracortical hyperplasia and lymphocyte depletion in follicular germinal centers. streptococcus porcinus causes jowl abscesses in pigs. the bacteria colonize the oral cavity and spread to infect tonsils and regional lymph nodes. the mandibular lymph nodes are the most often affected and have multiple, -to -cm abscesses; the retropharyngeal and parotid lymph nodes may also be involved (fig. - ). this once-prevalent disease is now rare, presumably due to improvements in husbandry, feeder design, and hygiene. it is occasionally isolated in pigs with bacteremia. disseminated histoplasmosis include wasting, emaciation, fever, respiratory distress, diarrhea with hematochezia or melena, and lameness. the clinicopathologic changes of disseminated histoplasmosis may include neutrophilia, monocytosis, nonregenerative anemia in chronic infections, changes in total serum protein level, and liver enzyme level elevations with hepatic involvement. the anemia is likely a result of chronic inflammation, histoplasma infection of the bone marrow, and/or intestinal blood loss in dogs with gi disease. cytologic examination is useful for the diagnosis of histoplasmosis (tracheal wash preparations, aspirates of bone marrow and lymph nodes), where organisms are often visible in macrophages (e- fig. - ) . grossly, there is hepatosplenomegaly, the intestines are thickened and corrugated, and the lymph nodes are uniformly enlarged (fig. - ) with loss of normal architecture (somewhat similar to siderofibrotic plaques, splenic rupture, and accessory spleens see the section on miscellaneous disorders of the spleen for discussions on siderofibrotic plaques, splenic rupture, and accessory spleens. splenic nodular hyperplasia is common in dogs and categorized based on their cellular components as lymphoid nodular hyperplasia or complex nodular hyperplasia. hematomas may arise within nodules of hyperplasia (see splenic nodules with a bloody consistency). lymphoid (or simple) nodular hyperplasia consists of a focal well-demarcated mass composed of discrete to coalescing aggregates of lymphocytes. the lymphocytes may form follicular structures with germinal centers and/or consist of a mixture of lymphocytes with mantle and marginal zone cell morphologic features. the intervening tissue is often congested and may contain plasma cells, but stroma is not observed (fig. - ; e-fig. - ) . complex nodular hyperplasia is a focal mass that contains two proliferative components: lymphoid and stroma (e- fig. - ) . the lymphoid component resembles lymphoid nodular hyperplasia described above. there is proliferation of the intervening stromal tissues with fibroplasia, smooth muscle hyperplasia, and histiocytic hyperplasia; emh and plasma cells may also be present. it has recently come to light that the entity splenic fibrohistiocytic nodule (sfhn), first described in , is not a single condition, but in fact a complex group of diseases. our better understanding of the spectrum of diseases once described under the term splenic fibrohistiocytic nodule is due to increasing knowledge of histiocytic disorders and immunochemistry. the original definition of splenic fibrohistiocytic nodule is a nodule characterized by a stromal population of histiocytoid and spindle cells intermixed with lymphocytes. grading was based on the lymphocyte percentage of the population (e.g., > % lymphocytes = grade ; < % lymphocytes = grade ); dogs with grade splenic fibrohistiocytic nodule had a much better -year survival rate, and dogs with grade nodules may develop sarcomas (often malignant fibrous histiocytoma, a now outdated term). with our increasing knowledge of histiocytic disorders and additional immunohistochemical stains, diseases that likely were encompassed by the term splenic fibrohistiocytic nodule include the following: complex and lymphoid nodular hyperplasia (see earlier), stromal sarcoma, histiocytic sarcoma, marginal zone hyperplasia, marginal zone lymphoma, and diffuse large b cell lymphoma (see lymphoid/lymphatic system, disorders of domestic animals: lymph nodes, neoplasia, lymphoma). histoplasma capsulatum can cause a disseminated fungal disease that is widely endemic, particularly in areas with major river valleys and temperate or tropical climates (e.g., midwestern and southern united states). free-living organisms in the mycelial phase produce macroconidia and microconidia that are inhaled and converted to the yeast phase in the lung. yeasts are phagocytized and harbored by macrophages of the monocyte-macrophage system. in some dogs the disease is limited to the respiratory tract and causes dyspnea and coughing. however, in most dogs, the disease is disseminated throughout the body, predominantly affecting the liver, spleen, gastrointestinal tract, bone marrow, skin, and eyes; primary gastrointestinal disease is also reported. the clinical signs in cases of and hepatosplenomegaly (e- fig. - ) . histologically, the lymph node sinuses and splenic red pulp are filled with macrophages that contain intracytoplasmic, round, -µm-diameter organisms with a small kinetoplast. though there is an initial stage of lymphoid hyperplasia in the spleen and lymph node, subsequent lymphoid atrophy occurs with chronicity. the atrophy is due to impairment of follicular dcs, b lymphocyte migration, and germinal center formation. there may be lymphoid atrophy of the spleen and lymph nodes in severe chronic infections. canine distemper virus preferentially infects lymphoid, epithelial, and nervous cells (see chapter ). dogs are exposed through contact with oronasal secretions, and the virus infects macrophages within the lymphoid tissue of the tonsil and respiratory tract (including tracheobronchial lymph nodes) and later disseminates to the spleen, lymph nodes, bone marrow, malt, and hepatic kupffer cells. the virus causes necrosis of lymphocytes (especially cd t lymphocytes) and depression of lymphopoiesis in the bone marrow, leading to severe immunosuppression. dogs are therefore susceptible to secondary infections, including bordetella bronchiseptica, toxoplasma gondii, nocardia, salmonella spp., and generalized demodicosis. canine parvovirus type (cpv- ) is a highly contagious disease of dogs spread through the fecal-oral route or oronasal exposure to contaminated fomites. the virus has tropism for rapidly dividing cells, and replication begins in the lymphoid tissues of the oropharynx, thymus, and mesenteric lymph nodes and then is disseminated to the small intestinal crypt epithelium. by infecting lymphoid tissues, canine parvovirus type causes immunosuppression directly through lymphocytolysis and indirectly though bone marrow depletion of lymphocyte precursors. there is marked lymphoid atrophy of thymus and follicles of the spleen, lymph nodes, and maltparticularly of peyer's patches to produce the classic gross lesion of depressed oval regions of the mucosa (so-called punched-out peyer's patches). thymomas. see lymphoid/lymphatic system, disorders of domestic animals: thymus, neoplasia. lymphomas. lymphoma is the most common hematologic malignancy in the dog. using the who classification scheme, several lymphoma subtypes are identified in dogs and clinically range from slow-growing indolent tumors to highly aggressive tumors. of all domestic animal species, lymphoma is the most extensively studied in dogs. the most common clinical presentation in dogs is generalized lymphadenopathy, with or without clinical signs such as lethargy and inappetence. the majority of lymphomas in dogs are large cell mid-to highgrade lymphomas, and up to half of all lymphoma cases are subtyped as diffuse large b cell lymphoma. diffuse large b cell lymphomas are further subdivided into centroblastic or immunoblastic based on nucleolar morphologic features (see table - and box - ), although it is unclear if this difference has any prognostic significance. histologically, lymph node architecture is most often completely effaced by sheets of large neoplastic cells, which may invade through the capsule and colonize the perinodal tissue. these dogs are often treated with chemotherapy and achieve remission. the overall median survival time for dogs with diffuse large b lymphocyte lymphoma is approximately months, although this number lymphoma, though the nodes tend to be more firm in histoplasmosis). histologically within the node, there is a multifocal to coalescing infiltrate of epithelioid macrophages with intracytoplasmic, small ( to µm in diameter) yeast organisms with spherical basophilic central bodies surrounded by a clear halo (fig. - ) . leishmaniasis is a disease of the monocyte-macrophage system caused by protozoa of the genus leishmania. it occurs in dogs and other animals and is endemic in parts of the united states, europe, mediterranean, middle east, africa, and central and south america. the protozoa proliferate by binary fission in the gut of the sand fly and become flagellated organisms, which are introduced into mammals by insect bites, where they are phagocytized by macrophages and assume a nonflagellated form. cutaneous and/or visceral forms of the disease are observed. in the visceral form, dogs are emaciated and have general enlargement of abdominal lymph nodes numerous other subtypes of lymphoma have been reported in dogs, including several forms of cutaneous lymphomas, most often of t lymphocyte origin and epitheliotropic (see chapter ). hepatosplenic t cell lymphoma, thought to be of γ/δ t lymphocyte origin, affects the liver and spleen without significant nodal involvement. hepatocytotropic t cell lymphoma is a distinct form of lymphoma with tropism for the hepatic cords; clusters or individual neoplastic lymphocytes invade the hepatic cords, without hepatocyte degeneration. intravascular lymphoma is a proliferation of large neoplastic lymphocytes within blood vessels of many tissues, leading to progressive occlusion and subsequent thromboses and infarcts. this neoplasm does not form an extravascular mass, and neoplastic cells are not found in peripheral blood smears or bone marrow. indolent lymphomas constitute up to % of all canine lymphomas. indolent lymphomas in dogs, in descending order of frequency, include t zone lymphoma (tzl), marginal zone lymphoma (mzl), mantle cell lymphoma (mcl), and follicular lymphoma (fl). mantle cell lymphoma and follicular lymphoma are less commonly diagnosed than t zone lymphoma and marginal zone lymphoma; therefore the reader is referred to the suggested readings to learn more on mantle cell lymphoma and follicular lymphoma. t zone lymphoma. t zone lymphoma is the most common indolent lymphoma in dogs ( fig. - ) . it presents as a solitary or varies based on the study and the grade of the tumor (as determined by mitotic figures). peripheral t cell lymphomas-not otherwise specified are the second most common subtype in dogs. this category includes all t cell lymphomas that do not fit into the other categories (e.g., t zone lymphoma, enteropathy-associated t cell lymphoma, and hepatosplenic t cell lymphoma). peripheral t cell lymphoma also effaces nodal architecture, and when compared to diffuse large b cell lymphoma, there is more variation in nuclear size and morphologic features. dogs with this subtype tend to have shorter survival times. intermediate cell size, high-grade lymphomas are less common in dogs, and the two most frequently encountered subtypes are lymphoblastic lymphoma (lbl) and burkitt-like lymphoma (bll). lymphoblastic lymphoma may be of b or t lymphocyte origin, though t cell lymphoblastic lymphoma is more common of the two. it is important to recognize a common misuse of the term "lymphoblast" in lymphoblastic lymphoma-by definition in lymphoblastic lymphoma, lymphoblasts are intermediate-sized cells with a distinct dispersed chromatin pattern, and not the large lymphocytes seen in cases of diffuse large b cell lymphoma or peripheral t cell lymphoma. t lymphocyte lymphoblastic lymphoma is an aggressive disease that is often resistant to treatment. burkitt-like lymphoma is a high-grade lymphoma of b lymphocytes. consequently there is marked lymphoid atrophy of thymus, spleen, lymph node, and malt (particularly peyer's patches). see bone marrow and blood cells, disorders of domestic animals, types of hematopoietic neoplasia, myeloid neoplasia, mast cell neoplasia and see e- fig. - . lymphoma is the most commonly diagnosed neoplasm in cats, and the incidence is reportedly the highest for any species. mediastinal or multicentric lymphomas are seen in young, felv-infected cats (see fig. - ). with the advent of felv vaccine and routine testing, the prevalence of felv-associated lymphoma is decreased. currently the alimentary tract is the most commonly affected site, and typically occurs in cats greater than years of age . other miscellaneous sites commonly affected are brain, spinal cord, eye, kidney, and nasopharynx. the retrovirus felv has long been recognized as a cause of lymphoma in cats-the risk for lymphoma is increased sixtyfold in infected cats. before the advent of a vaccine in , approximately % of cats (mainly young animals) with lymphoma were felv positive. felv infects t lymphocytes and can cause myelodysplastic syndrome, acute myeloid leukemias (see myeloid neoplasia), and t lymphocyte leukemia/lymphoma. in the latter the mediastinum (thymus, mediastinal, and sternal lymph nodes) is the site most commonly involved, although a multicentric distribution also occurs. routine felv vaccination has led to a significant decrease in the prevalence of felv infection, which has resulted in a decrease in the proportion of mediastinal lymphomas. the risk for developing lymphoma in fiv-infected cats is fivefold to sixfold higher than in uninfected cats. cats that underwent kidney transplantation and thus received immunosuppressive drug therapy had a similar risk for developing lymphoma. both fivinfected and posttransplantation cats predominantly developed extranodal, high-grade, diffuse large b lymphocyte lymphomas. this form is also the most common subtype in human immunodeficiency virus and posttransplantation patients caused by the epstein-barr virus (ebv). therefore it is reasonable to question whether these two groups of immunosuppressed cats may be more prone to infection by a gammaherpesvirus similar to ebv, leading to lymphoma. recently a novel feline gammaherpesvirus (fcaghv ) was multiple peripheral lymphadenomegaly (often mandibular lymph nodes) in otherwise healthy-appearing dogs. the characteristic histopathologic architecture is a nodular expansion of the paracortex by neoplastic cells, which push atrophied "fading" cortical follicles against the thinned capsule and trabeculae. this unique architectural feature is best highlighted with immunohistochemical stains (often cd for t lymphocytes and cd a, pax , or cd for b lymphocytes). the neoplastic cells are small to intermediate in size with pale eosinophilic cytoplasm and oval nuclei with sharp shallow indentations. mitotic figures are rare. dogs with this lymphoma subtype tend to be diagnosed with an advanced stage of the disease, likely because they present clinically healthy, without loss of appetite or activity level. even so, dogs with t zone lymphoma have a relatively long survival time compared to other lymphomas: reports on median survival time range from to months, and data suggest that dogs who do not receive chemotherapy actually have longer median survival times. marginal zone lymphoma. marginal zone lymphoma is an indolent b lymphocyte neoplasm derived from the cells of the marginal zone of lymphoid follicles. most marginal zone lymphomas (and mantle cell lymphomas) are assumed to originate in the spleen with slow spread to lymph nodes and often present as a mottled white-red smooth spherical splenic mass. histopathologic assessment of tissue architecture is needed for a diagnosis of marginal zone lymphoma and is characterized by a distinct nodular pattern in which the lighter-staining neoplastic marginal zone cells form a dense cuff around small foci of darkly stained mantle cells (fading follicles). the neoplastic marginal zone lymphocytes are intermediate in size and have a single prominent central nucleolus. mitotic figures are often rare or absent early on and increase with disease progression. differentiating between marginal zone lymphoma and marginal zone hyperplasia (which refers to a proliferation of marginal zone cells and contains a mixture of small and intermediate lymphocytes) is challenging because marginal zone lymphoma arises on the background of marginal zone hyperplasia. additionally, lymphoid and complex nodular hyperplasia are common in the dog spleen (see disorders of dogs), and it is possible that many cases of nodular hyperplasia contain areas of marginal zone lymphoma. therefore immunophenotyping and molecular clonality are ultimately required for a definitive diagnosis of marginal zone lymphoma. the overall median survival time in dogs with splenic marginal zone lymphoma after splenectomy is approximately months (even longer if it is diagnosed as an incidental finding). plasmacytomas. see disorders of domestic animals: lymph nodes, neoplasia, plasma cell neoplasia, extramedullary plasmacytomas (see fig. - ). feline panleukopenia, caused by the single-stranded dna virus feline parvovirus (fpv), is a highly contagious and often lethal disease of cats and other felidae, as well as other species (including raccoons, ring-tailed cats, foxes, and minks). fpv is transmitted by the fecal-oral route through contact with infected body fluids, feces, or fomites. following intranasal or oral infection, the virus initially replicates in the macrophages in the lamina propria of the oropharynx and regional lymph nodes, followed by viremia, which distributes the virus throughout the body. because fpv requires rapidly multiplying cells in the s phase of division for its replication, replication occurs in mitotically active tissues (lymphoid tissue, bone marrow, and intestinal mucosa). by infecting lymphoid tissues, fpv causes immunosuppression directly through lymphocytolysis and of the small intestine. the neoplastic cells are small (nuclei are equal to the diameter of a feline rbc), mitotic figures are infrequent (low grade), and mucosal and crypt epitheliotropism is common (see fig. - ) . a diagnosis of this subtype of lymphoma may be difficult (particularly in endoscopic biopsy samples), because this disease often is multifocal and concurrent with or arises within lymphoplasmacytic inflammatory bowel disease (ibd). the neoplastic lymphocytes are morphologically similar to the inflammatory lymphocytes. early small cell mucosal t cell lymphomas often require additional diagnostic testing, namely, immunohistochemistry and molecular clonality testing (pcr for antigen receptor rearrangement [parr]) to confirm a clonal neoplasm. transmural t cell lymphomas also occur focally or multifocally in the small intestine of cats (best classified as enteropathy-associated t cell lymphoma type i) and by definition must extend into the submucosa and muscularis. some tumors invade the serosa and adjacent mesentery. t cell large granular lymphocyte (lgl) lymphoma is often diagnosed, and the intestinal segments orad and aborad to the transmural mass may also have mucosal lymphoma. gastrointestinal b cell lymphomas are less prevalent in cats but occur in the stomach, jejunum, and ileocecocolic region as transmural lesions. most are diagnosed as diffuse large b cell lymphomas. lymphomas in other sites also occur less frequently in cats. the upper respiratory tract (nasal and/or nasopharyngeal region) is a relatively rare site for lymphoma. however, lymphoma is the most common primary nasal tumor, and diffuse large b cell lymphomas (of immunoblastic type) are the predominant subtype. both cutaneous (cutaneous t cell lymphoma) and subcutaneous lymphomas (usually large cell lymphomas) are rare. presumed solitary ocular lymphomas have also been reported. t cell-rich large b cell lymphoma, also referred to as feline hodgkin-like lymphoma in some studies, is composed of a mixture of reactive small lymphocytes and large neoplastic b lymphocytes, many of which may be binucleated and/or have prominent nucleoli (thus resembling the reed-sternberg cells of human hodgkin's lymphoma). this disease is typically characterized by a distinctive clinical presentation of an indolent unilateral neoplasm of the cervical lymph nodes, which spreads slowly to adjacent nodes within the chain. however, a proportion of cases may go on to develop into a more aggressive multicentric large to anaplastic b lymphocyte lymphoma that can affect peripheral and central nodes and multiple organs. suggested readings are available at www.expertconsult.com. a b discovered in domestic cats with a % prevalence in north america, and further studies to investigate its role in lymphomagenesis are needed. the overall incidence of feline lymphomas has increased, mainly due to an increase in gastrointestinal lymphomas. mucosal t cell lymphoma, also known as enteropathy-associated t cell lymphoma (eatcl type ii), is the most common and arises from diffuse malt partial thromboplastin time (aptt or ptt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of a contact activator, calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in intrinsic and/or common coagulation pathway (all factors except for vii and xiii) causes prolongation of ptt • insensitive test-prolongation requires % deficiency. • other causes of prolongation include polycythemia (less plasma per unit volume, so excess amount of citrate is available to chelate calcium) and heparin therapy activated clotting time (act) • required sample: nonanticoagulated whole blood in special act tube (diatomaceous earth as contact activator) used in practice setting-performed by warming sample to body temperature, monitoring for clot formation; normal clotting times are within to seconds in dogs, seconds in cats • less sensitive version of ptt-prolongation requires % deficiency severe thrombocytopenia may cause prolongation. • one-stage prothrombin time (ospt or pt) • required sample: citrated plasma • measures time for fibrin clot formation after addition of tissue factor (tf; thromboplastin), calcium, and a substitute for platelet phospholipid • deficiencies/dysfunction in extrinsic (factor vii) and/or common coagulation pathway cause prolongation of pt • insensitive test-prolongation requires % deficiency proteins induced by vitamin k antagonism or absence (pivka) test • required sample: citrated plasma • essentially a version of the pt using an especially sensitive thromboplastin reagent • pivka are inactive (uncarboxylated) vitamin k-dependent factors; an increase in pivka is not specific for vitamin-k antagonism but may be an earlier and more sensitive detector than pt or ptt • thrombin time (tt) • required sample: citrated plasma • measures time for fibrin clot formation after thrombin (factor iia) is added • defects directly involving formation and/or polymerization of fibrin prolong this test (i.e., if the lesion is upstream of the conversion of fibrinogen to fibrin, the tt will be normal). hypofibrinogenemia or dysfibrinogenemia causes prolongation of the tt required sample: citrated plasma. • fibrinogen concentration measured based on time to clot formation after addition of thrombin; this is essentially the same as the tt mentioned earlier and is a more accurate method than the heat precipitation method • decreased fibrinogen may be because of increased consumption (disseminated intravascular coagulation) or decreased production (liver disease) increased fibrinogen is associated with inflammation, renal disease, and dehydration required sample: special fdp tube. • used in the practice setting. • performed by adding blood to a special tube containing thrombin and a trypsin inhibitor (sample clots almost instantly in normal dogs and cats) and incubating two dilutions of serum ( : and : ) with polystyrene latex particles coated with sheep anti-fdp antibodies (should be negative in normal dogs and cats required sample: citrated plasma. • latex agglutination test. • to date, only validated in dogs and horses assay detects a specific type of fdp resulting from breakdown of cross-linked fibrin; concentration of plasma d-dimer indicates the degree of fibrinolysis; often used as part of a disseminated intravascular coagulation panel • required sample: citrated plasma. • decreased because of decreased production (liver disease), loss (protein-losing nephropathy or enteropathy) • specific factor assays • required sample: citrated plasma. • performed at specialized laboratories intermediary spleen microvasculature in canis familiaris-morphological evidence of a closed and open type molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states characteristics, diagnosis, and treatment of inherited platelet disorders in mammals making sense of lymphoma diagnostics in small animal patients canine granulocytic anaplasmosis: a review lymphoid tissues and organs lymph nodes and thymus diagnostic cytology and hematology of the dog and cat two hundred three cases of equine lymphoma classified according to the world health organization (who) classification criteria hepcidin: a key regulator of iron metabolism and mediator of anemia of inflammation changes to bovine hematology reference intervals from to atlas of veterinary hematology: blood and bone marrow of domestic animals pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses feline leukemia virus infection and diseases tumors of the hemolymphatic system proposed criteria for classification of acute myeloid leukemia in dogs and cats immunobiology: the immune system in health and disease extramedullary hematopoiesis: a new look at the underlying stem cell niche, theories of development, and occurrence in animals lineage-specific hematopoietic growth factors hepatosplenic and hepatocytotropic t-cell lymphoma: two distinct types of t-cell lymphoma in dogs histology and cell biology: an introduction to pathology lymphoid neoplasms in swine jc: robbins & cotran pathologic basis of disease the spleen ehrlichiosis and related infections hemotropic mycoplasmas (hemoplasmas): a review and new insights into pathogenic potential clinical, laboratory, and histopathologic features of equine lymphoma classification and clinical features in cases of equine cutaneous lymphoma histologic and immunohistochemical review of splenic fibrohistiocytic nodules in dogs feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality current state of knowledge on porcine circovirus type -associated lesions molecular pathology of severe combined immunodeficiency in mice, horses, and dogs hematologic abnormalities associated with retroviral infections in the cat pathology of the pig: a diagnostic guide pathologic and prognostic characteristics of splenomegaly in dogs due to fibrohistiocytic nodules: cases splenic myeloid metaplasia, histiocytosis, and hypersplenism in the dog ( cases) fundamentals of veterinary clinical pathology feline parvovirus infection and associated diseases novel gammaherpesviruses in north american domestic cats, bobcats, and pumas: identification, prevalence, and risk factors palmer's pathology of domestic animals veterinary comparative hematopathology histologic classification of hematopoietic tumors of domestic animals. in world health organization international histological classification of tumors in domestic animals, second series canine lymphomas: association of classification type, disease stage, tumor subtype, mitotic rate, and treatment with survival classification of canine malignant lymphomas according to the world health organization criteria canine indolent nodular lymphoma the revision of the world health organization (who) classification of myeloid neoplasms and acute leukemia: rationale and important changes the coombs' test in veterinary medicine: past, present, future a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital ( - ) schalm's veterinary hematology chronic lymphocytic leukemia in dogs and cats: the veterinary perspective grossly, incompletely contracted areas are characterized by multiple, variably sized and irregularly shaped, dark red to black, raised, soft, blood-filled "nodules." these areas are usually at the margins of the spleen, and the intervening tissues are depressed and pink-red, corresponding to the contracted portions of red pulp devoid of blood. incompletely contracted areas may be confused with acute splenic infarcts or hematomas on gross examination.acute splenic infarcts. splenic infarcts are wedge-shaped or triangular hemorrhagic lesions that occur primarily at the margins of the spleen. in dogs, splenic infarcts most often occur with hypercoagulable states (e.g., liver disease, renal disease, cushing's disease), neoplasia, and cardiovascular disease. splenic vein thrombi may occur in association with traumatic reticulitis, splenic abscesses, portal vein thrombosis, and arterial thrombosis in bovine theileriosis in cattle. valvular endocarditis may also lead to multiorgan infarcts, including the spleen. splenic infarcts are common in pigs with classical swine fever.acute splenic infarcts may not always be grossly visible in the early stages but develop into discrete, dark red and blood-filled, bulging, wedge-shaped foci with the base toward the splenic capsule ( fig. - , a) . with chronicity the lesion becomes gray-white and contracted due to fibrosis (see fig. - , b).hemangiosarcoma. hemangiosarcoma is a malignant neoplasm of endothelial cells and is a common primary tumor of the spleen, especially in dogs. benign splenic hemangiomas are extraordinarily rare. grossly, hemangiosarcomas may appear as single, multifocal, or coalescing dark red-purple masses and cannot be easily differentiated from a hematoma ( fig. - ). on cut surface they are bloody with varying amounts of soft red neoplastic tissue; in more solid areas the neoplasm can be slightly more firm and whitetan. metastatic spread occurs early in the disease process. seeding of the peritoneum results in numerous discrete red-black masses throughout the omentum and serosa of abdominal organs, and hematogenous spread to liver and lung are common. hemangiosarcomas in dogs also occur in the right atrium of the heart, retroperitoneal fat, and skin (dermal and/or subcutaneous) and multiorgan hemangiosarcomas are described in horses, cats, and cattle. because hemangiosarcomas have often metastasized at the time of initial accumulations of macrophages within several organs, including splenic macrophages, kupffer cells of the liver, and macrophages in the brain are often observed.splenic nodules with a bloody consistency. the most common disorders of the spleen with bloody nodules are ( ) hematomas, including those induced by nodular hyperplasia or occurring with hemangiosarcoma, ( ) incompletely contracted areas of the spleen, ( ) acute splenic infarcts, and ( ) hemangiosarcomas. the term nodule has been applied rather loosely here. in some of these conditions, such as incompletely or irregularly contracted areas of the spleen, the elevated area of the spleen is not as well defined as the term nodule would imply.hematomas. bleeding into the red pulp to form a hematoma is confined by the splenic capsule, and produces a red to dark red, soft, bulging, usually solitary mass of varying size ( to cm in diameter) (fig. - ). resolution of a splenic hematoma progresses over days to weeks, through the stages of coagulation and breakdown of the blood into a dark red-brown soft mass (fig. - , a) , infiltration by macrophages that phagocytize erythrocytes and break down hemoglobin to form hematoidin and hemosiderin (see fig. - , b), and repair leading to fibrosis. on occasion the capsule (splenic capsule and visceral peritoneum) over the hematoma can rupture, resulting in hemoperitoneum, hypovolemic shock, and death.the origin or cause of many hematomas is unknown. some are due to trauma, and others may also be induced by splenic nodular hyperplasia. it is postulated that as the splenic follicles become hyperplastic they distort the adjacent marginal zone and marginal sinus, which compromises their drainage into sinusoids and red pulp vascular spaces. the result is an accumulation of pooled blood surrounding the hyperplastic nodule, which leads to hematoma formation. splenic hematomas can also occur secondary to the rupture of hemangiosarcomas within the spleen.incompletely contracted areas of the spleen. incompletely or irregularly contracted areas of the spleen are caused by failure of the smooth muscle to contract in response to circulatory shock (hypovolemic, cardiogenic, or septic) or sympathetic "fight-or-flight" response, resulting in a lack of splenic evacuation of stored blood. key: cord- -ujflw b authors: newcomer, benjamin w.; cebra, chris; chamorro, manuel f.; reppert, emily; cebra, margaret; edmondson, misty a. title: diseases of the hematologic, immunologic, and lymphatic systems (multisystem diseases) [image: see text] date: - - journal: sheep, goat, and cervid medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ujflw b nan in this chapter, multisystemic diseases are discussed in small ruminants (sheep, goats, and cervids). these include diseases of the hematologic, immunologic, and lymphatic systems. in general, species will be discussed together, but when pertinent data are available, each species will be considered separately. the terms "cervid" and "deer" have been used interchangeably in parts of this chapter by the authors. an adequate volume of blood for hematologic and biochemical analysis is best obtained from the jugular vein. a docile animal may be restrained in a standing position or tipped up (sheep only) with the head turned away from the jugular vein to be used. wilder ones, such as some cervids, may require restraint devices or chemical sedation. ideally, the animal should be restrained by someone other than the blood collector, although the same person may be able to both restrain a sheep and collect blood if the animal is tipped up or a halter is used (see chapter ) . the animal should be at rest, with minimal excitement. the collector parts or clips the wool or hair to visualize the jugular vein and then uses the hand not holding the needle to apply digital pressure proximally just above the thoracic inlet to block blood movement through the vein. the vessel may take a second or more to distend after pressure is applied. the collector may then use the needlebearing hand to "strum" the vessel and cause the blood to oscillate. if in doubt about whether the distended vessel is the jugular vein, the collector can release the hand placing pressure on the vessel and observe whether the distended vessel disappears; if it does, the distended vessel was probably the jugular vein. the collector should avoid vessels that pulsate because these are likely to be the carotid arteries. the area should be cleaned with alcohol or other disinfectant, water, or a clean, dry gauze sponge. an -or -gauge, -to . -inch needle is usually adequate to collect blood from an adult, whereas a -gauge needle may be used in a neonate. the skin of adults or males may be thicker and more difficult to penetrate with the needle. a syringe or evacuated tube attached to a vacutainer (becton dickinson inc., rutherford, nj) can be used to collect blood. the needle should be plunged through the skin into the vein at an approximate -degree angle. the blood should not come out of the vessel in pulsatile waves; this is suggestive of an arterial stick. after aseptically obtaining an adequate volume of blood, the collector removes the needle and releases the pressure on the vessel near the thoracic inlet. pressure should be applied to the site of puncture for a minute or more to prevent extravascular leakage of blood and hematoma formation. the blood should be carefully transferred to a vial containing the appropriate anticoagulant to prevent red blood cell (rbc) rupture. goat erythrocytes are small and particularly prone to hemolysis. to minimize this problem, goat blood should be collected with a needle and syringe, not a vacutainer. white blood cell (wbc) differential distribution, individual blood cell staining characteristics, and morphology may be assessed by microscopic examination of a stained blood film. the differential distribution provides more information than total wbc count because inflammatory conditions in artiodactyls often result in a shift in neutrophil populations toward more degenerate, toxic, or immature forms without changing the overall wbc count. the preferred anticoagulant for a complete blood count (cbc) is ethylenediaminetetraacetate (edta), and tubes should be filled to ensure the proper blood-to-anticoagulant ratio. blood samples should be processed as soon as possible after collection. if a delay is anticipated, the blood sample should be refrigerated ( ° c) and an air-dried blood smear should be made because prolonged contact of blood with edta causes changes in wbc morphology and the separation of some rbc parasites. blood can be refrigerated for hours and still yield an accurate cbc. a reference range for hematologic data for sheep and goats is provided in table . (see appendix , tables and ) . goats tend to have a low mean corpuscular volume (mcv) because of their small erythrocytes. sheep and goats younger than months old tend to have lower hematocrit, rbc count, hemoglobin, and plasma protein concentrations, as well as a higher total wbc count. neonates often have a high hematocrit at birth that decreases with colostral ingestion. lactating animals may have decreased hematocrits, rbc counts, and hemoglobin concentrations. animals grazing at high altitude (mountain goats and bighorn sheep) tend to have increased rbc counts, hematocrits, and hemoglobin concentrations. interpreting hematologic changes in cervids is more complex. restraint method affects a variety of parameters in non-acclimated individuals. physical restraint yields red cell counts and hematocrit and hemoglobin concentrations that are to % higher than animals immobilized chemically. , neutrophil, lymphocyte, monocyte, and total white cell counts are also to % higher in physically restrained cervids (see appendix , tables and ) . adult deer also have seasonal variations in their hemogram. red cell numbers and related values are highest during midsummer and late winter. white cells, especially neutrophils, are also highest in midsummer, and platelet counts are highest in spring and fall. these changes may relate to diet or to seasonal activities, such as antler growth and rutting conflicts, which increase the chance of trauma. red cell stickling has also been reported in a variety of deer species. this appears to relate to a mutation in hemoglobin's b-globin component, similar to the disorder in people, but no pathologic role has been described. bone marrow aspirates and core biopsy samples taken from sites of active erythropoiesis can be useful to evaluate erythrocyte production and determine the cause of anemia and other hemogram abnormalities. the sites of biopsy include the sternebrae, femur, and ileum. the procedure should be done under chemical sedation or anesthesia (see chapter ) . the area over the biopsy site is clipped and surgically prepared; the sampler should wear sterile gloves to maintain asepsis. aspirates can be obtained by inserting a sterile needle attached to a -or -cc syringe containing one or two drops of edta through the bone and into the bone marrow. drawing back on the syringe plunger several times may aid in the procurement of an acceptable sample; such a sample may consist of as little as . ml of bone marrow. if the sample is going to be processed immediately, no anticoagulant is required. core biopsies are obtained using a jamshidi or westerman-jensen biopsy needle. the skin is incised with a scalpel and the biopsy needle is inserted into the bone and turned several times to obtain a core sample. more than one site may be used. the sampler then closes the skin with sutures or staples. biopsy samples are preserved by placing them in % neutral buffered formalin solution. impression smears can be made from these samples by gently rolling them on a clean glass slide before placing them in the formalin solution. information obtained from bone marrow samples includes subjective data regarding cell density, megakaryocyte numbers, abnormal cells, maturation patterns of rbcs and wbcs, and the ratio of erythroid to myeloid cells. prussian blue stain can be used on bone marrow to demonstrate iron stores. bone marrow aspirates and biopsies are painful and invasive procedures. therefore, animals should be placed on antibiotics and antiinflammatory drugs prophylactically. blood cultures can be useful in diagnosing bacteremia in an intermittently or persistently febrile animal or one with numerous sites of organ infection. ideally, the clinician should obtain the sample before instituting antimicrobial therapy. however, if this is impossible, antimicrobial therapy should be discontinued to hours before sampling. samples should be taken before and during febrile episodes. the jugular vein is most commonly used to attain a blood culture. as described previously, the skin over the jugular vein should be clipped and surgically prepared. the person collecting the blood sample should wear sterile gloves and use a sterile needle and syringe. blood samples should be placed immediately in a blood culture flask. the chances of attaining a positive culture from bacteremic animals increase with the size of the sample up to about ml, but adding more than the recommended amount to any single culture vial may overwhelm the capacity of the specialized antibiotic-absorbing resins within the flasks. the clinician should change the needle on the sample syringe after collecting the blood and before putting the sample in the culture medium. samples should be refrigerated until they can be sent to a diagnostic laboratory, where aerobic and sometimes anaerobic cultures are made. as an alternative to hematologic testing, comparing conjunctival color to swatches on a standardized famacha chart has been used as a rapid and inexpensive assessment of anemia in whole flocks, primarily to assess the impact of haemonchus contortus and other blood-sucking parasites. , results from a number of trials have yielded fair to good sensitivity to packed cell volume and h. contortus load in both sheep and goats. similar to body condition scoring systems, it is essential to calibrate assessors to ensure consistency when using this system. also, some breeds hematocrit (%) - - hemoglobin (g/dl) - . [ ] [ ] [ ] [ ] [ ] red blood cell count ( /ml) - . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mean corpuscular volume (fl) - - mean corpuscular hemoglobin concentration (g/dl) - [ ] [ ] [ ] [ ] [ ] [ ] [ ] platelet count ( /ml) . - . . - . total white blood cell count (/ml) - , - , segmented neutrophils (/ml) - - band neutrophils (/ml) lymphocytes (/ml) - - monocytes (/ml) - - eosinophils (/ml) - - basophils (/ml) - - total plasma protein (g/dl) . - . . - . fibrinogen (mg/dl) - read differently on the cards, and use of an electronic color analyzer, while more expensive and less field-friendly, may detect anemia earlier (see chapter , figure . a, b, and chapter ) . easy use of this technique in deer is limited by their intractability and has not been reported. the most common and significant abnormality of the hemogram is anemia. anemia occurs most commonly after blood loss, hemolysis, or chronic disease. blood loss is usually covert and commonly caused by gastrointestinal or external parasites. overt blood loss is usually caused by major trauma such as that caused by dog bites, severe lacerations, male rivalry fighting, or complications of castration or dehorning. cbc values appear normal immediately after acute blood loss. however, after a few hours of fluid redistribution, anemia and hypoproteinemia are evident. evidence of red cell regeneration (macrocytosis, reticulocytosis, and nucleated red cells) should appear within a day or two of the blood loss. hemolysis occurs most commonly after ingestion of toxic plants, rbc parasitism, intravenous (iv) injection of hypotonic or hypertonic agents, contact with bacterial toxins, water intoxication, or immune-mediated destruction of opsonized erythrocytes. ingested toxins include sulfur compounds from onions and brassica plants (kale and canola), [ ] [ ] [ ] [ ] nitrates, nitrites, and copper. [ ] [ ] [ ] [ ] except for that caused by copper, hemolysis usually occurs within a day or two after ingestion. copper toxicosis can occur after acute overingestion but more commonly is seen in animals that are chronically overfed copper and suffer some stressful event. goats are more tolerant of excess copper than sheep are, and certain breeds of sheep, particularly the suffolk, are highly sensitive to copper toxicosis (see chapters and ) . hemolytic bacterial toxins include those from clostridium perfringens type a, clostridium haemolyticum, and leptospira interrogans. , intraerythrocytic parasites include anaplasma species, mycoplasma (eperythrozoon) species, and babesia species. [ ] [ ] [ ] [ ] [ ] immune-mediated rbc destruction is very uncommon except with parasitemia, the administration of certain drugs (penicillin), or bovine colostrum to small ruminant neonates. rapid reduction of plasma osmolality can lead to osmotic lysis of erythrocytes. this can occur locally as a sequela to rapid iv injection of hypotonic substances or after ingestion of a large quantity of water following a period of water deprivation and dehydration (water intoxication). selenium and copper deficiency have also been associated with heinz body anemia. parasite infestation, opsonization, and ingestion of toxic plants typically cause extravascular hemolysis. in these cases, damaged erythrocytes are removed by cells of the reticuloendothelial system, resulting in anemia, pallor, weakness, depression, icterus, and dark urine. bacterial toxins, changes in plasma osmolality, and copper toxicosis cause intravascular hemolysis, resulting in the additional signs of hemoglobinemia and hemoglobinuria. other signs such as fever, neurologic symptoms, and sudden death may be seen with specific diseases. signs of regeneration should be seen on the hemogram to days after the onset of hemolysis. anemia that is not related to the loss or destruction of erythrocytes usually results from a lack of production and thus are nonregenerative. although mild forms may exist in pregnant sheep and goats and those deficient in vital minerals (e.g., iron, selenium, copper, and zinc), the most common cause of nonregenerative anemia is chronic disease. under these conditions, iron is sequestered in an unusable form in the bone marrow; staining a marrow sample with prussian blue stain reveals large iron stores, differentiating this disease from iron-deficiency anemia. the causes of anemia of chronic disease are numerous and include infectious conditions (e.g., pneumonia, foot rot, and caseous lymphadenitis), malnutrition, and environmental stressors. most anemia does not require treatment. unless loss of rbc mass is rapid and severe, the animal is usually able to compensate to the decreased oxygen-carrying capacity by decreasing activity. it is important to remember in this regard that anemia often first becomes apparent to the manager of a flock or herd when animals appear overly stressed or die during movement or handling. if possible, the cause of the anemia should be addressed. this can involve trying to control internal and external parasites, changing the diet, and treating infectious diseases. maintaining adequate hydration is essential in animals with intravascular hemolysis to avoid hemoglobin-induced renal tubular damage. specialty compounds such as molybdenum salts, such as ammonium molybdate, and sulfur or penicillamine for copper toxicosis and methylene blue ( mg/kg in a % solution in % dextrose or normal saline intravenously) for nitrate toxicity are usually too expensive or difficult to be used on a flock-wide basis but may be useful in valuable individual animals. veterinarians should be aware that methylene blue is no longer approved for use in food-producing animals. animals with severe acute blood loss or hemolysis may benefit from a whole blood transfusion. because transfusion reactions are rare and strong erythrocyte antigens have not been identified in small ruminants (including cervids), almost any donor of the same species is acceptable for a first transfusion. cross-matching can be done to ensure compatibility, which becomes more important if the animal receives more than one transfusion. blood should be withdrawn aseptically from the donor and collected by a bleeding trocar into an open flask or by a catheter into a special collection bag. blood should be mixed at a . : ratio with acid-citrate dextrose, or : with % sodium citrate, or another suitable anticoagulant and administered through a filtered blood administration set. if the jugular vein is not accessible, blood may be infused into the peritoneal cavity, but the slower absorption from that site makes it less effective for treating acute blood loss. the first to minutes of administration should be slow. if no reaction is seen (fever, tenesmus, tachypnea, tachycardia, and shaking), the rate may be increased. transfused erythrocytes may only survive a few days, and therefore, the original cause of the anemia must be addressed. peripheral wbcs include granulocytes (neutrophils, eosinophils, and basophils) and mononuclear cells (lymphocytes and monocytes). immature forms of neutrophils and lymphocytes may be seen during severe inflammatory diseases. abnormalities of the neutrophil line are usually the best cellular evidence of inflammation in small ruminants, and inflammation is almost always a sequela of infection. an increase in neutrophil numbers and their proportional contribution to the total wbc count is usually seen in mild gram positive, subacute, or chronic bacterial infections. animals with more severe disease may exhibit high or normal counts, but a greater proportion of the neutrophils will have toxic changes or be immature forms (band cells, metamyelocytes, or myelocytes). in severe, acute inflammation and many diseases caused by gram negative bacteria, a temporary reduction in neutrophil numbers is observed, often with a concurrent shift toward more toxic or immature forms. if the animal survives the peracute disease, neutropenia should resolve over to days, first through an increase in immature cells, and later through a mature neutrophilic response. another important cause of increased total and relative neutrophil counts is stress (or glucocorticoid administration), which inhibits neutrophil margination and extravasation and thereby increases the number of these cells in the midstream blood. increases in eosinophil counts are usually related to exposure to eukaryotic parasites. decreases are rarely of clinical significance and may be part of the stress response. idiopathic allergic-type reactions also are indicators of pathology but are very rare. increases in basophils are rarely clinically significant. increases in lymphocyte counts often reflect chronic inflammatory disease such as that seen with internal abscesses. in rare cases, lymphocytosis may consist of abnormal, blast-type cells and indicate a lymphoproliferative neoplasm. lymphopenia is an important part of the stress response; nevertheless, the clinician must keep in mind that many diseases stimulate a stress response. therefore, lymphopenia and neutrophilia may represent either stress or inflammation, and an examination of neutrophil morphology and plasma fibrinogen concentrations may be useful in distinguishing the two situations. a high fibrinogen concentration, toxic changes, and high counts of immature neutrophils indicate inflammation under those circumstances. blood monocyte counts also may indicate stress or chronic inflammation. the difficulties in interpreting individual cell count abnormalities highlight the importance of obtaining a differential wbc count and description of cellular morphology in assessing sick sheep and goats. leukogram abnormalities are rarely given specific treatment. it is far more common and useful to use the information from the leukogram to develop a plan to treat the disease responsible for the abnormality. palpation of external lymph nodes is part of the thorough physical examination. lymph nodes that can be found in normal sheep and goats include the submandibular, prescapular, and prefemoral nodes. none of these should be prominent or painful on palpation. additional nodes that may be palpated occasionally in normal animals include the parotid, retropharyngeal, supramammary, perirectal, and popliteal nodes. internal lymph nodes that may be identified during specialized diagnostic procedures include the mediastinal, mesenteric, and other abdominal nodes. enlargement of lymph nodes may be focal, multifocal, or generalized. identification of a single enlarged superficial node does not always rule out a multifocal or generalized disorder because the status of the internal nodes often cannot be determined. enlargement generally indicates either inflammation or neoplasia. inflammatory enlargement is generally related to an associated disease with an infectious component. small ruminants are particularly sensitive to lymph node-based infections (e.g., caseous lymphadenitis), so the search often does not extend beyond aspirating or draining the lymph node itself. neoplastic enlargement almost always results from lymphosarcoma. lymphosarcoma pathogenesis. neoplastic transformation of a member of the lymphocyte cell line leads to unregulated clonal expansion of that cell. the cause of transformation is usually unknown; in rare cases, especially in flock outbreaks in sheep, it can be linked to exposure to the bovine leukemia virus, which has occurred experimentally and as a result of the administration of whole blood anaplasma vaccines. whether the bovine leukemia virus can induce lymphosarcoma in goats and cervids is still unclear. multicentric lymphosarcoma has been reported sporadically in white-tailed deer (odocoileus virginiatus) and other deer, but bovine leukemia virus infection has not been diagnosed in cervids. in one study of neoplastic diseases affecting goats from to , lymphoma was identified as the most common neoplasm, accounting for . % of the assessed tumors. in contrast to other species such as cattle, sheep, and horses, lymphomas in goats are predominantly t-cell lymphomas affecting the mediastinum. a recent study attempted to classify the type of lymphoma affecting goats. using immunohistochemistry (ihc), it was determined that % (n ) of affected goats had t-cell lymphoma and only % (n ) had b-cell lymphoma. proliferation of t or b lymphocytes leads to mass lesions and infiltration of viscera. these changes cause physical obstruction (to breathing, blood flow, urination, defecation, etc.), ulceration of mucosal surfaces (blood loss, bacterial invasion), immune system dysfunction, organ failure, and generalized malaise and cachexia. tissue masses may be internal or visible on external examination. clinical signs. clinical signs in affected animals vary according to the type of lymphoma (t-or b-cell) and the location of the masses. t-cell lymphomas in goats are usually localized in the thoracic cavity and/or neck, suggesting thymic origin or homing. in contrast, b-cell lymphomas tend to have a multicentric distribution. lymphoma in small ruminants usually presents with non-specific signs that can mimic other respiratory or gastrointestinal conditions. slowly progressive weight loss is the most common finding. in some cases, generalized peripheral lymphadenopathy and expansile masses are noted ; at first, they usually are presumed to be caseous lymphadenitis abscesses. progressive chemosis and exophthalmos have been reported in a sheep and a goat with multicentric b-cell lymphoma. , most masses form at the sites of internal or external lymph nodes. in sheep, masses in the brain, skin, joint, and lymphoid tissue have been reported. leukemia is rare. the most common abnormalities are those of chronic disease and cachexia and include nonregenerative anemia and hypoalbuminemia. bone marrow examination may reveal clonal expansion of lymphoid precursor cells. in cervids, lymphadenopathy and multifocal masses affecting the heart, blood vessels, kidney, urinary bladder, and peritoneum have been reported. a more recent report described a subcutaneous maxillary mass in a -year-old captive-born, female whitetailed deer. the mass was diagnosed as focal lymphosarcoma with local metastasis. diagnosis. history and clinical signs are important in the diagnosis of lymphoma in small ruminants. age of affected animals ranges from to years and no gender or breed predisposition has been reported. final diagnosis of affected animals is achieved through necropsy, histopathology, and ihc. lesions seen at necropsy include homogeneous white to tan masses that bulge on the cut surface. they may be small or large. less commonly, diffuse paleness of the reticuloendothelial organs is noted. microscopic examination of these tissues reveals infiltrates of abnormal cells of the lymphocyte line. prevention. avoiding exposure to the bovine leukemia virus and restricting the use of instruments to one animal between cleaning procedures may help prevent the spread of lymphosarcoma. in most animals, however, this neoplasm appears to develop spontaneously. pathogenesis. lambs, kids, and fawns are born with functional lymphocytes that can produce endogenous immunoglobulin. these cells develop the ability to respond to foreign antigens in the fetus during mid to late gestation. because of a lack of in utero exposure, however, basal concentrations of immunoglobulin are very low at birth. these cells therefore are naïve to foreign antigens and unable to develop protective immunity through specific cellmediated and immunoglobulin production. additionally, as with other ruminants, no transplacental passage of maternal immunoglobulin to fetal sheep, goats, and fawns occurs. lambs, kids, and fawns depend exclusively on intestinal absorption of maternally derived colostral antibodies, immune cells (t-lymphocytes), and other immune factors to provide a ready supply of specific immunity and allow opsonization of pathogens for the first months of life. adequate passive transfer requires delivery of a sufficient quantity of good-quality colostrum (immunoglobulin g [igg] concentration in mg/ml) into the gastrointestinal tract, as well as adequate absorption of antibodies (timely) from the colostrum into the blood. however, the amount of maternal colostrum produced by the dam, and its composition, as well as the ability of the newborn to stand and nurse in a timely manner, can be affected by several factors. colostrum igg concentration and volume of production can be influenced by breed, age, nutrition, body condition score (bcs) at parturition, and vaccination status of the dam. the igg concentration in colostrum samples from ewes of different breeds can vary between and mg/ml. one study demonstrated that primiparous ewes with low bcs (, . ) at lambing produced less colostrum compared with multiparous ewes with similar bcs. additionally, ewes with higher bcs (. . ) tended to produce higher volumes of colostrum compared with ewes with lower bcs. another study suggested that undernutrition of ewes during late gestation can affect colostrum quality and immune development and function in newborn lambs. it has been suggested that at least g of total igg should be fed to newborn lambs and kids during the first hours of life to reach adequate transfer of passive immunity. adequate transfer of passive immunity in small ruminant neonates has been suggested as serum igg levels at hours of life of  mg/ml. one study indicated that lambs that nurse low-quality colostrum (igg , mg/ml) had lower serum igg concentrations compared with lambs that that nurse colostrum of higher quality (igg . mg/ml), indicating that the concentration of igg in colostrum is a determining factor for the presentation of failure in the transfer of passive immunity. other factors such as pregnancy toxemia, gastrointestinal parasitism, excess of iodine intake during pregnancy, and inadequate vaccination of the dam can result in poor colostrum synthesis and quality. timely consumption of maternal colostrum during the first hours of life is essential to achieve adequate transfer of passive immunity. in small ruminants, cells of the small intestine are able to internalize and transfer igg into the blood during the first hours of life; however, the absorption efficiency of igg is higher during the first to hours of life. , factors associated with the neonate, such as weakness, inability to stand, and congenital abnormalities, will prevent timely nursing of maternal colostrum and lead to failure of passive transfer (fpt). litter size and body weight (bw) of the kid(s) have also been correlated with inadequate absorption of igg from colostrum. one study demonstrated that litter sizes of three light goat kids (, . kg bw) or more had significantly lower mean serum igg levels at hours of life when compared with litter sizes of one or two heavier kids ( . versus . mg/ml, respectively). this suggests that special attention and monitoring should be paid to multiple fetus gestation as the risk of fpt under these circumstances at kidding is higher; however, the quality of colostrum, amount ingested, and adequacy of absorption are rarely monitored by small ruminant producers in natural or artificial rearing systems. the use of monitoring tools to evaluate colostrum quality and igg absorption is common in modern dairy cattle operations, and these tools are readily available for small ruminant production systems. recent reports have presented the use of %brix in maternal colostrum and neonate serum and its positive correlation with serum total proteins (stps) at hours as effective monitoring tools of fpt in lambs and goat kids. [ ] [ ] [ ] the use of stp has also been used to monitor colostrum deficiency intake in mule deer fawns ; however, adequate values of serum igg for cervid neonates have not been established yet. inadequate colostrum intake and low serum igg at to hours of life have been consistently associated with higher morbidity and mortality rates in lambs, goat kids, and fawns. one study reported that % of lamb mortality between hours and weeks of age can be attributed to fpt. another study suggested that colostrum deficiency and low serum igg in goat kids resulted in higher mortality rates at weeks and of life due to chronic infections with pasteurella multocida and escherichia coli. other reports demonstrated that % of lambs with a serum igg of , mg/ml at hours died before weeks of age compared with only % of the lambs with a serum igg of . mg/ml at hours. in a previous report, mule deer (odocoileus hemionus) fawns with a stp of # g/dl between days and of age developed diarrhea and died before days of age compared with fawns with stp . g/dl. in a more recent report, a -day-old formosan sambar deer (rusa unicolor swinhoei) with a history of colostrum deprivation died due to severe suppurative meningitis caused by e. coli infection. in addition to immunoglobulins, colostrum also contains large quantities of fat-soluble vitamins that do not cross the placenta. the most important of these are vitamins a, d, and e, which are important in bone development and the immune or inflammatory response. neonates that have not ingested enough colostrum are likely to be deficient in these vitamins. diagnosis. history of dam dystocia, inadequate colostrum nursing, complete colostrum deprivation, and signs of undernourishment or sepsis in the first few days after birth are usually a presumptive indication of failure in the transfer of passive immunity. a high prevalence of diarrhea and respiratory disease in neonates should prompt investigation and evaluation of passive transfer of immunity in affected herds or flocks. owners occasionally evaluate lambs or kids for adequate intake by picking up the animal and holding it at ear level, while carefully cradling the head and neck, and then shaking the abdomen to hear milk in the abomasum; however, this is not a reliable indication of adequate transfer of passive immunity. a definitive diagnosis of fpt can be made by direct laboratory measurement (single radial immunodiffusion [srid]) of igg in serum at hours of life. although some practitioners use the value of igg used in dairy calves ( mg/ml), others have suggested an igg value , mg/ml to establish the presence of fpt in small ruminants. numerous semiquantitative methods of estimating igg are available and are easy to use in sheep, goats, and cervids. the most common is the measurement of serum total solids or stp values at hours of life through an optical refractometer. the stp at hours of life in a well-hydrated animal has demonstrated correlation with serum igg in calves, lambs, and goat kids. studies in goat kids indicated that an stp between . and . g/dl was associated with adequate transfer of passive immunity. , another study demonstrated fpt in lambs with stp values , . g/dl at hours of life. a study in mule deer suggested that fawns with an stp # g/dl had inadequate colostrum intake and fpt. recently, the measurement of %brix in maternal colostrum and serum with a digital brix refractometer has become an alternative method to evaluate colostrum quality and fpt in dairy operations. colostrum %brix . % and serum %brix . . % have been associated with adequate transfer of igg in calves and goat kids. other qualitative methods to assess the transfer of passive immunity in large animals include various agglutination (glutaraldehyde), precipitate assays (sodium sulfate), and measurement of g-glutamyl transferase (ggt) in serum. these methods may be relied on to give an overall flock assessment of adequacy of passive transfer, but they are rarely accurate enough to provide definitive information on individual animals. treatment. fpt is not in itself pathologic, but it greatly increases the neonate's susceptibility to infectious diseases. the amount of colostrum absorbed across the gut decreases with time, especially in animals that have been ingesting other proteins (e.g., the casein in milk); it also decreases with illnesses that decrease gastrointestinal function. neonatal small ruminants should receive at least g of igg/kg of bw or ideally g of total mass of igg from a good-quality colostrum source (. mg/ml of igg) during the first hours of life. other authors recommend an intake of to ml of colostrum/kg during the first hours of life. in artificial rearing systems or lamb feedlots, feeding of colostrum every hours until hours of life is recommended. when same species' maternal colostrum is unavailable, goat colostrum or bovine colostrum/colostrum replacers or are a good alternative; however, hemolysis has been reported in lambs receiving cattle colostrum. one study demonstrated that there was no difference in serum igg levels of lambs that received the same volume of sheep or goat colostrum at birth. another study demonstrated that lambs that received ml of a bovine colostrum replacer at birth in addition to ml of stored sheep colostrum at hours of life had higher serum %brix values at hours and had less incidence of disease during the preweaning period compared with lambs that received the same volume of stored sheep colostrum at birth and at hours of life. since igg absorption cannot be extended more than hours after birth, administration of an oral colostrum source is the best treatment in the immediate postpartum period in still-healthy neonates. after the window for immunoglobulin absorption has closed, plasma, serum, or whole blood administered by the iv or intra-peritoneal route is the best way to raise the neonate's blood immunoglobulin concentrations. adult donor plasma contains approximately . to . g of immunoglobulin/dl, so administration of a volume equivalent to % of bw or a dose of to ml/kg has been recommended for the treatment of large animal neonates. if plasma is used instead of colostrum, administration of vitamins a, d, and e also may be beneficial. if colostrum and plasma are unavailable or cost-prohibitive, "closing" the gut as quickly as possible with milk, maintaining high standards of hygiene, and possibly administering prophylactic antibiotics offer the greatest prospects for preventing infectious disease. vaccination of the neonate or the administration of antitoxin hyperimmune serum should not be considered protective but may be of value. prevention. prevention of fpt should be based on the establishment of an adequate colostrum program managing the previously mentioned factors that affect production, quality, and absorption of maternal colostrum components in lambs, goat kids, and fawns. ensuring colostral quality is best done through good nutrition, health care, and vaccination of dam (see chapters and ) . administration of vaccines weeks before parturition, followed in weeks with a booster, provides the highest quantity of protective immunoglobulin in the colostrum. antepartum leakage is rarely the problem in small ruminants that it is in horses and cattle. however, in a flock or herd environment, still-pregnant dams may steal babies from other sheep or goats. to prevent such theft and the resultant loss of colostrum by the "adopted" neonate, owners may choose to keep pregnant animals separate from those that have already delivered. if complete separation is not possible, the dam and her offspring should be allowed to bond with each other in a private pen ("jug" or "crate") for at least hours before being placed back with the flock. clipping excessive wool or mohair from around the perineal area and udder before lambing or kidding, expressing the teats to ensure they are not plugged, and having extra colostrum available when pregnant females are placed in jugs or crates are other good preventive measures. etiology and pathogenesis. uncomplicated diarrhea in lambs, goat kids, and fawns may be caused by infectious agents such as viruses, bacteria, and protozoa. in goat kids and elk calves, metabolic causes of diarrhea have been described. , group b and a rotavirus, enterotoxigenic e. coli k , cryptosporidium parvum, and other cryptosporidium spp. have been commonly identified as causal agents of diarrhea in small ruminant neonates. [ ] [ ] [ ] [ ] with recent advances in diagnostics and metagenomics of the enteric environment of large animals, novel viruses have been identified as potential causal agents of diarrhea in lambs and goat kids. adenovirus, astrovirus, calicivirus, coronavirus, and picornavirus have been identified in feces of diarrheic lambs and goat kids ; however, their role in the pathogenesis of neonatal diarrhea is still uncertain. these organisms differ from the agents of complicated diarrhea in that they do not invade beyond the gut wall or result in systemic toxemia (see chapter ) . additional causes of diarrhea reported in goat kids and elk include lactose intolerance and hypernatremia, respectively. , less frequently, bacteria such as c. perfringens, clostridium difficile, and attaching and effacing e. coli have been associated with complicated diarrhea in small ruminant neonates. , the net result of such an infection is that a large volume of water and electrolytes are lost into the bowel due to malabsorptive, hypersecretory, or hyperosmolar processes. if enough fluid and electrolytes are lost, dehydration and metabolic acidosis arise, inducing systemic clinical signs of depression and weakness in association with diarrhea. in goats, this clinical entity is one component of the floppy kid syndrome. clinical signs. profuse, watery, yellowish-green to brown diarrhea without fever is the hallmark clinical sign. with severe dehydration and acidosis, affected lambs, kids, and fawns become weak and dull and lack appetite. [ ] [ ] [ ] excessive salivation and loss of suckle reflex have also been reported in affected lambs and kids. , mucous membranes become tacky, and skin tenting times are prolonged. shock signs may develop. physical assessment often must take the place of clinicopathologic analysis in affected neonates. mild, nonclinically complicated diarrhea is characterized by profuse diarrhea with minimal systemic signs. the affected animal is bright and alert, with minimal skin tenting, and can stand and eat readily, with a strong suckle reflex. it is less than % dehydrated, with a blood ph of . to . , and bicarbonate deficit is minimal. moderate uncomplicated diarrhea is characterized by profuse diarrhea in a dull but responsive animal. skin tenting is prolonged, but eye luster is normal. the affected animal is able to stand and eat but eats slowly and has a weak suckle reflex. the head typically is held down. it is to % dehydrated, with a blood ph of . to . and a bicarbonate deficit of to meq/l. severe uncomplicated diarrhea is characterized by profuse diarrhea. the affected animal is dull and minimally responsive, with a very long skin tent time and dull, sunken eyes. it can stand only with assistance and prefers to stay in sternal recumbency with its head up. the animal eats very slowly, if at all, and has a minimal suckle reflex. it is to % dehydrated, with a blood ph of . to . and a bicarbonate deficit of meq/l. very severe uncomplicated diarrhea is characterized by profuse diarrhea and profound weakness. the animal's skin remains tented for more than minute, and its eyes are very sunken and dull. it is nonresponsive with no suckle response. it is unable to maintain sternal recumbency, lying on its side instead. the animal is to % dehydrated, with a blood ph of . to . and a bicarbonate deficiency of to meq/l. epidemiology. morbidity and mortality of uncomplicated diarrhea in small ruminants and fawns vary depending on the cause. reports of rotaviral diarrhea in newborn lambs indicate morbidity rates between % and % and mortality rates between and % , ; however, one study reported a % case fatality rate in lambs affected with types b and a rotavirus diarrhea. another study reported mortality rates between % and % in lambs and kids affected with c. parvum diarrhea. most of infectious agents associated with uncomplicated neonatal diarrhea in small ruminants are shed by adult animals and older lambs/kids around stressful events such as lambing/kidding and extreme weather conditions. one study reported that pregnant does shed to times more oocysts during the weeks around kidding compared with other time periods. additionally, poor husbandry/hygiene of lambing/kidding sheds, fecal soiling, flock size (. animals), lambing/kidding season (winter/spring), and the presence of c. perfringens type a in feces have been suggested as potential risk factors for uncomplicated diarrhea in small ruminant neonates. [ ] [ ] [ ] clinical pathology. the leukogram should be normal or show abnormalities compatible with stress. serum biochemical or blood gas analysis may reveal evidence of intestinal malabsorption, electrolyte loss, metabolic acidosis (hypoglycemia, hyponatremia, hypochloremia, hyperkalemia, low bicarbonate, and increased anion gap), and dehydration (hyperalbuminemia and increased blood urea nitrogen [bun] and creatinine). in contrast with the common leukogram and biochemical abnormalities found in calves, lambs, and goat kids with uncomplicated diarrhea, elk calves with diarrhea develop leukocytosis, hyperchloremia, and hypernatremia (serum na . meq/l). additionally, increased anion gap, bun, creatinine, and albumin concentrations have been reported in affected elk calves. a presumptive diagnosis may be based on the characteristic history and clinical signs. response to conservative treatment also is supportive of this diagnosis. identification of the specific causative agent is less important than proper treatment of affected animals; however, feces or intestinal contents from affected animals can be submitted for electron microscopy, reverse-transcription polymerase chain reaction (pcr), and cell culture immunofluorescent assays to identify viruses. [ ] [ ] [ ] additionally, intestinal tissue can be submitted for ihc for rotavirus and c. parvum. , feces of affected animals can also be submitted for enzyme-linked immunosorbent assay (elisa), ziehl-neelsen staining technique, light or fluorescence microscopy, sugar flotation, and auramine or fluorescent antibody staining for the diagnosis of c. parvum infection. fecal culture to determine a bacterial cause is recommended. treatment. the immediate goals of treatment are rehydration, replacement of lost electrolytes, and restoration of acid-base balance as these are usually the leading causes of death in affected neonates. less immediate goals are provision of nutrition and replacement of ongoing losses. the aggressiveness of treatment is dictated by the severity of the condition, as well as economic considerations. . rehydration: calculate the percent dehydration and use to calculate fluid requirements for a -hour period. example: % dehydration in a -kg lamb: dehydration: . kg kg/l . l or ml. maintenance: ml/kg/day . l or ml. total fluids to replace in hours . l or ml fluid loss due to dehydration ( ml in this case) should be replaced during the first hours and the rest can be replaced in the next hours. . replace lost electrolytes: sodium, chloride, and bicarbonate are lost roughly in proportion to extracellular fluid (ecf) in the acute phase of diarrhea ( - days) in untreated animals. potassium tends to be increased in this phase due to the presence of metabolic acidosis and care should be taken when selecting fluids containing potassium to treat affected animals at this time. in chronic cases of diarrhea, and especially in cases where the owner/producer has given oral milk replacer or electrolyte supplements/replacements to affected animals before veterinary evaluation, the serum concentration of sodium, potassium, and bicarbonate might be variable or increased. special care should be taken in these cases when selecting fluids to treat affected animals as the risk of causing hypernatremia is higher. in cases of diarrhea in elk calves, hypernatremia is common, and fluids should be selected accordingly. in the majority of cases, initial replacement of sodium, chloride, and bicarbonate with fluids containing proper composition is recommended. example: assessment suggests a bicarbonate deficit of meq bicarbonate in a -kg, comatose lamb with prolonged skin tenting ( . is the multiplier for ecf in a neonate): . ( meq) kg meq bicarbonate. commercial iv . % sodium bicarbonate solutions contain meq of bicarbonate per milliliter and could be added directly to iv fluids in severely dehydrated and acidotic animals. therefore, the immediate goal is to provide ml of fluid and meq of bicarbonate to this lamb in a formulation that resembles normal ecf. fluids can be given by various routes. selection of route of administration of fluids depends on degree of dehydration, presence or not of a strong suckle reflex, and degree of depression. neonates with advanced degrees of dehydration, depression, and absence of suckle reflex will benefit from iv fluid therapy. in contrast, neonates with mild dehydration and active suckle reflex can be effectively treated with oral electrolytes ; however, if oral fluids have not produced an improvement within to hours, iv treatment should be strongly considered. other routes such as subcutaneous, intra-peritoneal, and intra-osseous can also be used for fluid administration to neonates. • advantages: oral fluids are inexpensive (nonsterile) and easy to give. they are less likely to cause fatal arrhythmias or neurologic disease than iv fluids. • disadvantages: an animal receives a maximum of its gastric volume ( % of bw), and good gastric motility is required. oral fluids may not be well absorbed by a damaged gut. absorption also is slow. intravenous • advantage: this method allows rapid correction of all deficits, even in moribund animals. • disadvantages: it is expensive (sterile), requires venous access, and can rapidly lead to overcorrection. subcutaneous • advantages: this method does not require venous access or good gut motility. • disadvantages: it is expensive (sterile), and the fluids may not be well absorbed in very dehydrated animals. absorption is not as quick as by iv administration. animals should be given only hypotonic or isotonic fluids. intra-peritoneal • advantages: this method does not require venous access or gut motility. fluids are absorbed quickly by this route. • disadvantages: it is expensive (sterile) and can cause peritonitis. isotonic fluids are best used in this route. only a limited volume can be given. many commercial oral electrolyte solutions for neonatal ruminants are available; however, not all of them fulfill the requirements to adequately replace fluids and electrolytes in neonatal ruminants with diarrhea. oral electrolyte solutions must contain enough sodium , provide agents that increase absorption of water (glycine, glucose, and acetate), provide an alkalinizing agent (bicarbonate, propionate, acetate, and citrate; acetate has demonstrated best results), and an energy source (glucose). the amount of carbohydrates might vary and is usually higher in "high-energy" solutions specifically used for severely affected neonates that are not eating and develop negative energy balance. less carbohydrate is needed in less severely affected animals because they are usually eating some and are less likely to have severe negative energy balance. fluids to be avoided include medicated milk replacers and unbuffered saline solutions. iv treatment should be provided with a sterile commercial product. such preparations typically contain to meq/l of base. additional sodium bicarbonate solution or sterile powder can be added to fluid therapy based on the bicarbonate deficit ( meq/ml of . % solution and meq of bicarbonate/g of powder, respectively). the bicarbonate deficit should be over the first hours. after deficits are replaced, the following continued treatments and adjuncts may be considered: . continued administration of fluids (oral rather than iv, if possible) to replace ongoing losses: • oral electrolytes at a volume equal to % of the bw per feeding can be given; the number of feedings can be increased from two (normal) to three to six per day. • iv fluids can be continued at twice the maintenance fluid rate until appetite is restored. • more bicarbonate may be necessary. . consideration of addition of milk to the treatment regimen: • milk or milk replacers should be added to the therapy of neonates with diarrhea. they provide nutrition to the affected neonate, preventing negative energy balance and promoting intestinal healing. • care should be taken to not mix oral electrolyte solutions with milk or milk replacers in the same container as the concentration of sodium and overall osmolarity of the solution can dramatically increase, leading to hypernatremia or other metabolic abnormalities. • milk or milk replacers should be given in small volumes (, % of total requirements) but at a higher frequency (every - hours) to avoid overloading the abomasum and intestine of affected animals. lambs fed milk lose less weight with scours. • free water helps prevent hypernatremia. • milk is a good potassium source (see chapter ). elk deer calves. elk deer calves commonly develop diarrhea with hypernatremia (serum na . meq/l) compared with other large animal neonates, where hyponatremia is more common. therefore, administration of oral electrolyte solutions designed for other ruminants (calves, lambs, and kids) should be avoided in these animals. a dilution ( : or : ) of commercially available bovine calf electrolyte solutions to reduce sodium content is recommended for the treatment of elk calf diarrhea. the use of lactated ringer's solution, which has a low sodium concentration in addition to a very low reduction rate of serum sodium (, . meq/l/hour) has been advocated in the fluid therapy of hypernatremic elk calves with diarrhea. additional therapy. dextrose ( . - %) solutions can be added to the fluid therapy of hypoglycemic animals. the use of nonsteroidal antiinflammatory drugs (nsaids) in neonatal ruminants with diarrhea is controversial due to the risk of renal damage and abomasal ulceration; however, in cases of diarrhea complicated by septicemia or endotoxemia, nsaids should be used to reduce the effects of systemic inflammation. flunixin meglumine at a dose of . to . mg/kg is the only nsaid approved for food animal use. similarly, the use of oral or systemic antibiotics in cases of uncomplicated diarrhea is controversial due to its potential effect on the intestinal microbiota and development of bacterial resistance; however, their use is warranted in the presence of septicemia or endotoxemia in addition to diarrhea. in these cases, b-lactams such as oral amoxicillin or systemic ceftiofur are usually good choices. the effect of mucosal protectants and probiotics in cases of diarrhea is unknown in small ruminant neonates, and their use is left to practitioners based on their own experiences (see appendix ) . prevention. prevention of uncomplicated diarrhea in small ruminant neonates is based primarily on the timely feeding of adequate amounts of good quality maternal colostrum or colostrum replacer (see "failure of passive transfer" section). vaccination of dams with antigens of common infectious agents associated with uncomplicated neonatal diarrhea before parturition has demonstrated to be effective increasing colostrum immunity and prevention of diarrhea in lambs. maintenance of adequate husbandry and hygiene conditions in lambing/kidding sheds or barns is necessary to reduce neonatal exposure to infectious agents normally shed in feces of dams during parturition such as rotavirus and c. parvum. ruling out infectious causes of depression and weakness is difficult, and clinicians often do well to assume that an infectious disease is contributing to clinical signs when making treatment decisions. however, several noninfectious systemic disturbances also can depress neurologic and muscular function. successful treatment often requires identification and correction of each of these disturbances. among the more common abnormalities leading to depression in neonates are hypoxemia, metabolic or respiratory acidosis, hypothermia, hyperthermia, hypoglycemia, dehydration, azotemia, and some electrolyte imbalances. hypothermia and hyperthermia can easily be diagnosed by measuring body temperature with a rectal thermometer. hypothermia is far more common and can result from weakness, shock, and environmental stress. cold, windy weather or tube feeding with cold milk replacer or fluids can lead to a rapid drop in core body temperature, especially in neonates that are small or weak or have been inadequately licked off or were rejected by their dams. strong, vigorous neonates usually are protected by heat produced during muscular activity and are able to seek food and shelter. clinical signs appear when the rectal temperature drops to ° f ( . ° c) or below. protection from wind and cold such as with an individual ewe jug or pen, heat lamps (positioned far enough away so as not to burn the neonate), hot water bottles, blankets, and administration of warm fluids is helpful in treating and preventing hypothermia. shearing the ewe before lambing is of value because it forces the ewe to seek shelter. if this management technique is used, care should be taken to avoid inducing severe hypothermia in the dam. environmental hyperthermia is much less common than fever in neonates. therefore, treatment for infectious diseases in young animals with high temperatures usually is warranted. providing cool shelter with good ventilation, minimizing stressful events, ensuring adequate fluid intake, and shearing the adults are the best defenses against environmental heat stress. hypoglycemia also is easy to diagnose with the aid of an inexpensive, portable glucose meter. lambs and kids typically develop hypoglycemia under the same circumstances as those leading to hypothermia. administering ml/kg of dextrose (approximately . fl oz/lb, or % of bw) in warm milk replacer or ml/kg of % dextrose, by either the iv or oral route (diluted to % dextrose), should provide ample energy to correct hypoglycemia. iv administration may be necessary if gut motility is absent. follow-up treatment may be necessary if the neonate does not regain its appetite. except during severe conditions, normal lambs and kids should be able to maintain normal body core temperature. they should therefore be examined for an underlying disorder if they exhibit signs of hypothermia or hyperthermia. clinicians and owners should not assume that warming and feeding a cold, weak neonate will always correct the problem. hypoxemia is much more difficult to diagnose. portable blood gas meters for arterial analysis and radiography units for thoracic imaging are available but are still not in common use in small ruminant practice. for those reasons, hypoxemia usually is underdiagnosed. hypoxemia can result from prematurity or dysmaturity, infection, depression or weakness (decreased ventilation), meconium aspiration, bullous emphysema, hernias, and other thoracic fluid or tissue masses. it is likely to be a contributing factor in illness and death in most weak neonates younger than days of age. such animals benefit from the provision of supplemental oxygen, either through a nasal insufflation tube or by oxygen tent. in addition to its direct effect on general wellbeing and behavior/ attitude, hypoxemia at birth leads to poor gut function and subsequent poor colostral absorption. many animals that exhibit fpt and subsequent sepsis had a previous bout of hypoxemia. azotemia, metabolic acidosis, and electrolyte imbalances are difficult to diagnose without clinicopathologic analysis. therefore, these problems are best treated in animals showing signs of dehydration with the administration of a balanced, physiologic electrolyte solution. metabolic acidosis usually is accompanied by either obvious evidence of bicarbonate loss (diarrhea) or severe dehydration. however, neither of these conditions is present with floppy kid syndrome. this descriptive title is applied to muscle weakness, anorexia, and depression in kids observed in the first weeks of life. by its strictest definition, floppy kid syndrome refers to metabolic acidosis with a high anion gap without dehydration or any known cause in young kids that were normal at birth. a variety of disorders and conditions have been proposed as the cause of metabolic acidosis without dehydration, including intestinal fermentation of milk in well-fed kids with subsequent absorption of volatile fatty acids, transient neonatal renal tubular acidosis, and lactic acidosis secondary to toxic impairment of cardiovascular function. overgrowth of c. perfringens type a often is suggested as a source of the toxin. with a high anion gap, a pathologic condition that leads to overproduction of an organic acid is more likely than one that leads to bicarbonate loss. the disease can occur in individual animals or in outbreaks; although parity of the dam and number of offspring have not been associated with this metabolic disturbance, aggressively feeding kids are more likely to suffer from milk fermentation or clostridial overgrowth. an infectious etiology appears to be more likely in herds displaying an increased incidence of this metabolic disturbances as the kidding season progresses. the disease also is reported to be more common in meat goats than in dairy goats. the prevalence can vary tremendously from year to year in a single flock or region. a similar disease has been reported in calves and llama crias, and lambs are likely to be susceptible under the right conditions. because blood gas analysis and exclusion of other diseases often are impractical, the term floppy kid syndrome frequently is used by owners to refer to any kid that is weak and does not have an overt, organ-specific sign (e.g., diarrhea). different pathologic processes are grouped together by their common clinical endpoint (as with "thin ewe syndrome"), and the veterinarian is charged with determining the etiology in a specific flock. most possible causes are found in the previous list of conditions that cause weakness and depression in neonates. among these entities, sepsis and hypoxemia are the most important items and therefore must also be considered possible causes of floppy kid syndrome. treatment and prevention of floppy kid syndrome currently follow the same lines as for treatment and prevention of neonatal sepsis or enteritis. spontaneous recovery of animals with floppy kid syndrome may occur. however, in valuable kids, quick assessment of blood chemistry and base deficits will allow requisite correction of electrolyte and blood ph abnormalities with . % sodium bicarbonate. tissue-invading clostridia are large, straight, gram positive rods that are to mm in length. c. perfringens and c. haemolyticum are smaller bacteria, and clostridium novyi, clostridium chauvoei, and clostridium septicum are larger. the bacteria grow best under anaerobic conditions and produce waste gases. clostridia bear spores, which may be the only viable form in the environment (soil and decomposed organic matter). identification of these spores within bacteria on microscopic examination is useful to identify clostridia, but it is not diagnostic of disease. spores in c. perfringens are central and do not affect the shape, whereas most other species have the spore toward one end and appear slightly club shaped. clostridia cause infectious, noncontagious disease. the bacteria inhabit the intestinal tract and are present in the feces of ruminants. small numbers of organisms in their dormant spore form also may reside in tissues such as liver and skeletal muscle. they can be isolated from soil, where most are thought to have short life spans. soil concentrations are highest in locations recently contaminated with ruminant feces, especially crowded, overused facilities such as feedlots and lambing sheds. environmental contaminations are associated with cool, damp times of the year such as late winter and spring. the concentration of organisms and their toxins found in the feces, gut contents, and internal organs of most adult ruminants usually is small. competition and peristalsis prevent overgrowth in the gut, and aerobic conditions prevent overgrowth in other tissues in live animals. however, rapid overgrowth and tissue invasion ensue after death, making rapid postmortem examination essential to ascertain whether clostridial organisms are responsible for the death. pathogenic clostridial organisms all produce heat-labile protein exotoxins. most make a variety of toxins, and the relative contribution of each toxin to the disease state is not known. c. perfringens is a normal commensal of the intestinal tract of clinically healthy large animals, including cervids; however, the number of bacteria and their toxin production within the intestine usually remain low due to peristalsis and normal homeostasis. c. perfringens is classified into five biotypes (a, b, c, d, and e) based on the production of four major exotoxins, namely alpha (cpa), beta (cpb), epsilon (etx), and iota (itx); however, the production of more than different exotoxins in various combinations has been associated with these bacteria, including perfringolysin o (pfo), enterotoxin (cpe), and beta toxin (cpb ). the different biotypes of c. perfringens cause different diseases in relation with the exotoxins they produce. the major effect of the phospholipase/ sphingomyelinase cpa, produced by all c. perfringens biotypes, is cell lysis and hemolysis, and its role on intestinal disease of large animals is not well understood. however, this toxin has been associated with hemolytic disease and hemorrhagic enteritis in large animals; cpb, produced by c. perfringens types b and c, is a trypsinlabile toxin associated with necrotizing enteritis and enterotoxemia in large animal neonates; etx, produced by c. perfringens types d and b, is a trypsin-activated necrotizing toxin associated to vasculitis, edema, and necrosis of the cns and enterotoxemia; and itx, another trypsin-activated necrotizing toxin produced by c. perfringens type e, has also been associated to intestinal disease in small ruminants. , c. perfringens types c and d are considered the most important types in veterinary medicine as they can cause disease in most farm animals. severe clinical disease due to bacteria sporulation and massive toxin production only occurs when the normal intestinal environment and microbial balance are disrupted in affected individuals. decreased peristalsis and poor ruminal and abomasal function have also been proposed as factors that contribute to disease presentation. weather and handling stresses, feed changes, and an overabundance of high-energy feeds such as milk, bakery products, and cereal grains might promote bacteria overgrowth and exotoxin synthesis and release. additionally, other enteric infections that disrupt the mucosal border may increase systemic absorption of toxins and promote severe disease. c. perfringens type a is a normal inhabitant of the intestinal tract of large animals and is ubiquitous in the environment (soil). one study reported c. perfringens type a as the most common isolate among other clostridia from healthy young lambs. c. perfringens type a has been associated with a fatal hemolytic syndrome in younger lambs and cattle but not goats ("yellow lamb disease"), , acute hemorrhagic enteritis and hemolytic enterotoxemia in cattle (hemorrhagic bowel/jejunal syndrome) and goats, , , and intestinal hemorrhage and splenomegaly in farmed deer. , risk factors for infection have not been established; however, high soluble carbohydrate diets and high bcss have been associated with clinical disease. , , this disease occurs most commonly in lambs to months old. under favorable conditions, the organisms proliferate and cause a corresponding increase in alpha toxin production. the alpha toxin (cpa), in synergy with the beta toxin (cpb ), is responsible for hemolytic crisis, vasculitis, and gastrointestinal lesions. the clinical course usually is less than hours. clinical signs. in most cases, sudden death or history of found dead is common. clinical signs observed usually include weakness, depression, fever or hypothermia, icterus, anemia, hemoglobinuria, tachypnea, colic, hemorrhagic diarrhea or absence of feces, and terminal recumbency. , , [ ] [ ] [ ] adult animals also are susceptible to hemolytic disease and vasculitis caused by c. perfringens type a infection. fatal abomasitis and rumenitis in neonates and juveniles also have been blamed on c. perfringens type a, but the rapid postmortem proliferation of the organism makes substantiation of this claim difficult. morbidity in a flock is lower than for many of the other enteric clostridial diseases, but the mortality rate is very high. diagnosis. the most characteristic clinicopathologic change is neutrophilic leukocytosis with a left shift. other evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. laboratory evaluation reveals evidence of intravascular hemolysis. necropsy in sheep, goats, and cervids usually reveals evidence of hemolysis, pallor, jaundice, hemoglobinuria, hyperemic and edematous intestines, splenomegaly, gastrointestinal serosal and mucosal hemorrhage, and multifocal internal petechial hemorrhages. , , [ ] [ ] [ ] the isolation of c. perfringens type a from necropsied animals is not itself diagnostic. definitive diagnosis can be made based on identification of the alpha toxin and the absence of other toxins by elisas or older, live animal assays. more recently, multiplex pcr techniques are replacing immunodiffusion assays for the identification of a specific toxin-producing gene isolate, typing of bacteria, and demonstration of toxins or toxin genes. gut content and intestinal samples collected from freshly dead animals make the most meaningful samples for diagnosis. treatment. administration of high doses (. , iu/kg bid) of penicillin and clostridium antitoxin ( - ml subcutaneously [sc] or orally [po] ) is the mainstay of treatment, although animals may die acutely before therapies can be instituted. prevention. a conditionally licensed toxoid against the clostridial alpha toxin is available for cattle in the united states. a recent report demonstrated that a new vaccine including recombinant cpa, cpb, and etx was effective at inducing protective antibodies to c. perfringens biotypes in cattle, sheep, and goats. this could be an alternative for the prevention of morbidity and mortality caused by c. perfringens type a. prevention efforts should focus on environmental hygiene and avoiding gut conditions favorable for proliferation of the organism (high content of soluble carbohydrates in the diet). because this type appears to survive better in soil than other types, preventing ingestion of soil may be important in preventing disease. c. perfringens types b and c occur in the soil and the animals' housing environment and can be shed by asymptomatic individuals. the reported geographic range of both diseases is limited (type b to the united kingdom and south africa and type c to the united kingdom, australia, and north america), even though infection with c. perfringens type c appears to occur worldwide. these organisms cause very similar diseases called lamb dysentery and hemorrhagic enterotoxemia, respectively. very young lambs and kids ( - days to - weeks of age) are usually affected due to the presence of trypsin inhibitors in colostrum. older animals may become susceptible as a result of overwhelming infection or trypsin inhibition by some soy and sweet potato products or temporary suppression of pancreatic trypsin production (struck in adult sheep). with both diseases, the beta toxin (cpb) is a required pathophysiologic factor, and inactivation of this toxin after maturation of pancreatic trypsinogen secretion is what commonly limits the susceptible population to neonatal animals. the cytolytic and necrotizing effects of the beta toxin (cpb), in synergy with the beta toxin (cpb ), cause necrosis and ulceration of the intestinal mucosa and are translocated into circulation, causing severe toxemia and death. the diseases initially affect lambs and kids younger than days of age, with illness occasionally occurring in older lambs. the incidence of disease in lambs and kids can be around to %, with a case fatality rate of %. high stocking density in lambing areas, cold weather, single-born lambs, and high milk production of dams have been suggested as potential risk factors for type b and c enterotoxemia. because of management practices in young animals and age-related vulnerability, fecal contamination of teats, hands, and equipment that enter the mouths of the neonates (orogastric tubes and nipples) is a major cause of infection. clinical signs. severely affected animals or those at the beginning of an outbreak usually are found dead. less acutely affected animals expel initially yellow, fluid feces that progressively become brownish and/or hemorrhagic. feces may also contain flecks of blood and show splinting of the abdomen, especially when handled, along with signs of colic and feed refusal. the clinical course usually is short, and the disease is almost always fatal. one study reported acute abdominal pain, hemorrhagic diarrhea, and death within hours of experimental oral inoculation of three goat kids with a field strain of c. perfringens type c. dehydration, anemia, and severe weakness are also common clinical signs in affected animals. terminal convulsions and coma occasionally are noted, especially in outbreaks in the united states. c. perfringens type c in older sheep causes the disease known as "struck." affected animals usually are found dead or with signs of toxemia. specific antemortem signs of gastrointestinal disease are rare. specific antemortem signs of gastrointestinal disease are rare. clinical pathology changes observed in these animals include neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. necropsy findings. postmortem examination reveals focal hemorrhagic ulcers (up to . cm in diameter) in the small intestine (mostly in the ileum) with type b infection and diffuse reddening with hemorrhage and necrosis of the abomasum and the entire segments of the intestine with type c infection. type c infections in ruminants can also present with generalized peritonitis, subendocardial and subepicardial hemorrhages, and hemorrhagic lymph nodes. animals that die very rapidly may exhibit minimal or no gross abnormalities of the intestine. a similar syndrome of type c enterotoxemia has been previously reported in a sika deer (cervus nippon). sudden death, severe hemorrhagic gastritis including forestomach and abomasum, and catarrhal enteritis was observed in the affected animal. diagnosis. diagnosis of these diseases is made by identification of characteristic history, clinical signs, postmortem lesions, and positive toxin assays. because the beta toxin is very labile, negative toxin assays are less significant than negative assays for presence of other tissue-invading clostridia. the isolation of c. perfringens type b or c from necropsied animals is not itself diagnostic. immunodiffusion assays or multiplex pcr of intestinal contents for specific isolate and beta toxin (cpb) identification are recommended to obtain final diagnosis (see "diagnosis" in "c. perfringens type a" section). treatment. if the infection is identified early in the disease course, high doses of oral and parenteral penicillin and c. perfringens c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. usually, the condition is not recognized early enough, and animals are found dead or dying. prevention. a beta toxoid is available in the united states and other countries. it usually is packaged with an epsilon toxoid. the best protection is achieved by vaccinating pregnant dams twice, with the second dose administered approximately to weeks before lambing or kidding and annual booster. deer does should receive double the dose of sheep as low antibody responses to clostridia have been reported in these animals. , vaccination of pregnant dams is directed to increase specific colostrum antibodies to protect neonates. juveniles also should be vaccinated twice or three times at months, months, and months. adults, including males, should receive an annual booster. in the face of an outbreak, the lambing area should be moved to a different place. additionally, vaccination of dams and newborns with a beta toxoid and administration of c. perfringens c and d antitoxin can be carried out in the face of an outbreak to reduce morbidity and mortality. c. perfringens type d produces epsilon toxin (etx), which is responsible for causing type d enterotoxemia in sheep, goats, calves, and deer. , other common names for the disease include "overeating disease" or "pulpy kidney disease." the disease has a worldwide distribution and occurs primarily in suckling lambs of to weeks of age, although it has also been reported in weaned lambs up to months of age and adult sheep. the disease is also common in grazing goats and deer. the prevalence of disease has been reported from . to . %, with a % case fatality rate in feedlot lambs. one study on proportional distribution of goatherd mortality in the province of quebec, canada, reported a . % mortality of goats to c. perfringens type d enterotoxemia. the disease is more common in feedlot lambs after they enter the lot. tail docking, castration, and other management interventions are thought to decrease the incidence of this disease by temporarily decreasing appetite. the disease also affects unvaccinated adult sheep, even without any history of stressors or feed changes. sudden changes in the diet are the main predisposing factor in goats. the disease can occur in vaccinated goats, as vaccination has not demonstrated to be completely protective in this species. , c. perfringens type d is normally found in the gastrointestinal tract of healthy ruminants, but the acid environment of the abomasum and continuous peristalsis help to keep numbers of bacteria and levels of toxin production low. however, under specific conditions such as overingestion of high-energy feeds (milk, grain, and lush pasture), excess of fermentable starches in the intestine, and intestinal stasis, the organism proliferates rapidly, producing lethal quantities of epsilon toxin. these conditions are usually triggered in well-conditioned, fast-growing animals that are on a highly nutritious diet. the epsilon toxin, once produced, acts locally, causing increasing gut permeability and widespread tissue damage. epsilon toxin and other exotoxins are then absorbed through the intestinal tract into systemic circulation and transported to the brain, lungs, and kidneys, causing increased endothelial permeability, perivascular edema, and generalized necrosis. , the characteristic increased vascular permeability and perivascular edema in the kidney and brain are responsible for the name of "pulpy kidney disease" and "focal symmetric encephalomalacia." clinical signs. the course of the disease is usually very short ( . - hours), so sudden or spontaneous death is a common clinical sign across affected small ruminant species. , - natural disease caused by c. perfringens type d differs between sheep and goats, possibly because of a difference in relative local and systemic actions of the epsilon toxin, although experimental models have demonstrated that both species develop similar lesions. , , in sheep, systemic actions of the toxin leads to mostly neurological signs such as dullness, depression, ataxia, trembling, stiff limbs, opisthotonus, convulsions, frothy mouth, and rapid death. in goats, actions of the toxin appear to be more localized to the intestinal tract, causing enterocolitis, colic, diarrhea, dehydration, and occasional neurological signs. , necropsy findings. postmortem findings in sheep are characterized by edema of the brain, lungs, and heart in addition to hydropericardium. edema of the kidneys (pulpy kidney lesion) is inconsistent. sheep usually demonstrated minor and inconsistent intestinal changes. other lesions reported in cattle and deer include hemorrhages on the epicardium, thymus, and diaphragm and petechial hemorrhages in the jejunal mucosa. , necropsy lesions reported in goats include pseudomembranous enterocolitis with mucosal ulceration, as well as fibrin, blood clots, and watery contents in the bowel lumen. evidence of systemic toxemia, including multifocal petechial and ecchymotic hemorrhage, proteinaceous exudates in body cavities, pulmonary edema, hydropericardium, and cerebral malacia with perivascular cuffing, have also been reported in goats and affected deer. , , , , clinical pathology. characteristic clinicopathological changes include pronounced hyperglycemia and glucosuria, which are considered a hallmark of c. perfringens d enterotoxemia. additionally, neutrophilic leukocytosis with a left shift and evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. treatment. in general, the course of disease is too acute for the establishment of any treatment. however, as with infections with types b and c, if the disease is identified early in the disease course, high doses of oral and parenteral penicillin in addition to clostridium c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. prevention. vaccination of pregnant ewes with two doses of toxoid, with the second dose given to weeks before lambing, and adequate ingestion of colostrum are the best methods of protecting newborn lambs. vaccination of older lambs should occur before exposure to diets rich in carbohydrates (grain-feedlot settings) or lush pastures. in these cases, lambs should be vaccinated twice or three times around , , and months of age. males and adult females that are not part of the breeding program may be vaccinated annually. vaccination has been shown to protect goats from experimental disease, but clinical evidence suggests that well-vaccinated goats are still susceptible to developing clostridial enteritis. the toxoids may not protect against local action of the toxins in the goat, which appears to play a greater role in their disease than it does in the sheep. , , more frequent vaccination (every months) in goats is suggested to increase protection. the adjuvant present in some multivalent clostridial vaccines may cause subcutaneous reactions that may lead to abscess formation. in the face of an outbreak, immediate mass administration of c and d antitoxin ( iu/kg) in addition to vaccination is recommended. nonenteric clostridial infections c. novyi, c. septicum, c. chauvoei, and c. sordelli have been identified as causal agents of severe muscle, liver, and abomasal necrosis in small ruminants and cervid species. , [ ] [ ] [ ] these organisms are usually present in the soil and environment and in the gastrointestinal tract and liver of healthy ruminants. pathogenesis is usually facilitated by trauma of affected tissues, local multiplication of the organism, local and systemic damage by exotoxin production, and ultimately death. , four types of c. novyi have been described, a, b, c, and d. c. novyi type c is considered nontoxigenic and therefore is not associated with disease. c. novyi type a produces alpha toxin and is associated with wound infections and myonecrosis in cases of "bighead" and "malignant edema." c. novyi type b produces alpha and beta toxins and is associated with infectious necrotic hepatitis or "black disease." , the temporal and geographic distributions of black disease resemble those of fascioliasis, with the highest incidence of disease in milder, moister months in many countries. black disease is less common in sheep than in cattle and is rare in goats. , c. novyi type d (c. haemolyticum) produces beta toxin and is associated with bacillary hemoglobinuria (red water disease). c. septicum produces alpha toxin and is associated with malignant edema and necrotic abomasitis (braxy). c. chauvoei produces alpha and beta toxins and is associated with severe myonecrosis observed in blackleg and c. sordelli produces a hemolytic toxin associated with myonecrosis in cases of malignant edema and blackleg. , pathogenesis. spores of the organism shed in feces of carrier animals contaminate the environment and are ingested with feed/ grass and stored within kupffer cells. , liver damage caused by migrating liver fluke larvae (fasciola hepatica, fasciola gigantica, and cysticercus tenuicollis) create perfect ischemic conditions that induce germination of c. novyi type b spores and toxin synthesis and production. , , the alpha toxin is necrotoxic and causes liver necrosis and diffuse damage of the vascular system. the beta toxin is produced in smaller amounts and contributes to vascular damage and systemic toxemia. infective organisms also may be brought into the liver by the flukes. clinical signs. the course of disease from first illness to death is short and never lasts more than a few hours in sheep. therefore, peracute or sudden death is not uncommon in this species. wellnourished adult sheep between and years are more commonly affected. the disease course is a little longer ( - days) in cattle and deer. , affected sheep are debilitated, fail to keep up with the flock, and exhibit generalized weakness, sternal recumbency, separation, and anorexia. tachypnea and tachycardia may be seen; high fever ( - ° f) occurs early in the disease. clinical signs observed in cattle, goats, and deer are similar and may include severe depression, anorexia, abdominal distention, colic, ruminal stasis, and lateral recumbency. , , , , , a report of black disease in a forest reindeer (rangifer tarandus fennicus) described serosanguinous discharge from mucocutaneous orifices (nostrils and anus), periorbital edema, and nystagmus in addition to other clinical signs. necropsy findings. necropsy might be difficult due to rapid autolysis of tissues in affected animals. severe venous congestion usually darkens the underside of the skin of affected animals, giving this disease its common name of "black disease." fluid in the pericardial sac, pleural space, and peritoneal cavity is usually present. endocardial and epicardial hemorrhages are a common finding. the liver is swollen and congested and on its diaphragmatic surface presents pale foci of coagulation necrosis; however, solid organs such as liver and kidneys could be in an advanced state of autolysis. characteristic lesions of black disease in the liver are single or multiple yellow to white areas ( - cm in diameter) of necrosis surrounded by a bright hyperemic zone. a recent report of black disease in a reindeer described moderate amounts of dark red thoracic and pericardial fluid, edema of the lungs and upper respiratory tract, swollen spleen, and several well-circumscribed areas of black discoloration in the liver. diagnosis. the most characteristic clinicopathological change is neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen; however, diagnosis of black disease is based on characteristic history (endemic liver fluke areas), clinical signs, and postmortem findings and testing. an impression smear of the margins of the liver might reveal large numbers of gram positive rods, but this is not definitively diagnostic. anaerobic culture of c. novyi from typical liver lesions, in addition to demonstration of the alpha/beta toxins from peritoneal fluid or liver (fresh-refrigerated), through elisa or pcr is required to establish final diagnosis. , the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smear samples or other liver (formalin-fixed) samples have also been described. , treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium ( , - , iu/kg) iv every hours or oxytetracycline mg/kg iv every hours should be initiated. supportive care, including iv fluids, nutritional support, and stress reduction, may be beneficial. in the face of an outbreak, vaccination of the whole herd/flock should be initiated immediately. efforts to control fluke infestation constitute the most effective approach to prevention of this disease. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every months starting around to months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given month before expected fluke exposure may provide additional protection. , deer should be vaccinated in the same fashion as sheep but double the vaccine dose for sheep should be used for these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. , efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis. c. novyi type d (c. haemolyticum) is the etiologic agent associated with red water disease. c. haemolyticum is similar to other clostridial species in its life cycle and appears to thrive on alkaline soils and pastures with standing water. the disease tends to be seasonal occurring at times of high larval fluke migration. similar to c. novyi b, c. haemolyticum colonizes the livers of healthy animals and proliferates after liver damage, including damage caused by migrating flukes (f. hepatica, fascioloides magna, dicrocoelium dendriticum, and c. tenuicollis), liver abscessation (fusobacterium necrophorum or trueperella pyogenes), or damage incurred during liver biopsy. , under ischemic conditions of the liver, spores of c. haemolyticum germinate and produce high amounts of beta toxin. the beta toxin causes localized hepatic necrosis and after reaching circulation induces severe intravascular hemolysis and damage of the capillary endothelium. intravascular hemolysis leads to rapid anemia and death due to anoxia. the disease is seen worldwide and is more commonly reported in sheep than in goats. bacillary hemoglobinuria has been reported in a free-ranging elk calf (cervus elaphus roosevelti) found dead in the southwest of the state of washington, united states. clinical signs. bacillary hemoglobinuria usually affects wellnourished animals older than year of age. , in most cases, the disease is per-acute and sudden dead or found dead is the only sign. in cases where signs are recognized antemortem, affected animals appear weak, depressed, and febrile ( - ° f); blood or blood-tinged froth may be present in the nostrils; rectal bleeding and bloody feces may be present; and severe hemoglobinuria (dark red, port wine-colored urine) is usually observed. , blood appears thin and watery and mucous membranes are pale and icteric. heart and respiratory rates are high and become much higher with any sort of effort or stress. other terminal signs include bloat and the presence of blood in the nostrils, mouth, vagina, and rectum. death occurs within hours to a few days after onset of clinical signs. necropsy findings. gross lesions include jaundice of mucous membranes and tissues and subcutaneous petechial/ecchymotic hemorrhages, edema, and emphysema. marked autolysis of internal organs might prevent identification of typical lesions. dark red urine is present in the bladder. lymph nodes and spleen are congested and hemorrhagic. hemorrhagic abomasitis and enteritis might occur, as well as the presence of hemoglobin-stained transudate in pleural and peritoneal cavities and pericardial sac. pulmonary edema is common. the pathognomonic lesion is the ischemic hepatic infarcts ranging from to cm in diameter with a hyperemic interface with healthy liver tissue. , diagnosis. clinicopathological abnormalities usually include anemia, leukocytosis with mature neutrophilia, and degenerative left shift (immature forms of neutrophils and toxic changes) often is present. , serum biochemical evaluation may reveal increased levels of liver enzymes such as sorbitol dehydrogenase, ggt, aspartate aminotransferase, and increased indirect total serum bilirubin. [ ] [ ] [ ] presumptive diagnosis can be made on history, clinical sigs, clinicopathological abnormalities, and postmortem findings; however, similar to black disease, final diagnosis should be based on anaerobic culture of c. novyi from typical liver lesions in addition to demonstration of the beta toxins from peritoneal fluid or liver (fresh-refrigerated) through elisa or pcr techniques. , , , the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smears or other liver (formalin-fixed) samples has also been described. , more recently, a pcr assay for the detection of c. novyi type d in cattle has been reported. treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium ( , - , iu/kg) iv every hours or oxytetracycline mg/kg iv every hours should be initiated. supportive therapy should include the administration of iv fluids, blood transfusions, and antiinflammatory agents. efforts to control liver flukes and prevent other causes of liver damage are most important. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every months starting around to months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given month before expected fluke exposure may provide additional protection. deer should be vaccinated in the same fashion as sheep, but double the vaccine dose for sheep should be used as these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. , efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis and clinical signs. fecal and soil contamination of wounds received during fighting (head-butting) or dehorning (disbudding) leads to proliferation of c. novyi type a in damaged head and neck tissues. accumulation of secreted toxins leads to swelling, edema, serohemorrhagic exudates, and local tissue necrosis. wounds appear and smell gangrenous. systemic toxemia may affect internal organs, leading to the death of the animal. c. sordelli causes identical disease. diagnosis. laboratory analysis may reveal an increase in enzymes of muscle or liver origin as well as neutrophilic leukocytosis with many immature and toxic neutrophils. postmortem findings include local necrosis around the injury site. diagnosis usually is made by characteristic clinical signs and lesions. treatment. wound management (disinfection, debridement) and administration of high doses of penicillin g sodium ( , - , iu/kg) iv every hours are important treatment considerations. prevention. ram management may aid in the prevention of head-butting wounds. vaccination with multivalent clostridial toxoids starting around weaning time ( - months of age) and with annual boosters also may be helpful. in flocks with a high prevalence of this disorder, a booster vaccine given to rams month before the breeding season and to ewes/does before parturition may provide additional protection. pathogenesis. c. septicum is the most important agent in the pathogenesis of malignant edema and braxy. in the case of malignant edema, other tissue-invasive clostridia (c. chauvoei, c. sordelli, and c. perfringens a) have also been associated with this disease, and mixed infections are common. the pathogenesis of infection is often similar to that seen with bighead and blackleg: soil or fecal clostridial invasion of a contaminated wound. in sheep and goats, this disease has been reported following lambing/kidding, after shearing of tail docking. c. septicum can also invade the abomasal lining of lambs, causing severe hemorrhagic, necrotic abomasitis known as braxy. activation of dormant bacteria in previously damaged tissue (myositis/abomasitis) similar to that seen in clostridial necrotic hepatitis also occurs. in both cases (malignant edema and braxy), bacterial toxins precipitate local tissue necrosis and systemic toxemia. the alpha, beta, gamma, and delta toxins produced by c. septicum are lecithinase, deoxyribonuclease, hyaluronidase, and hemolysin, respectively. commonly affected sites of malignant edema include castration, dehorning, and injection sites; the umbilicus; and the postpartum uterus. factors that promote braxy have not been identified, although it usually affects weaned and yearling lambs in the winter after ingestion of frozen feedstuffs implicated as initial causes of abomasitis. , both forms of the disease have worldwide distribution and are described more in sheep than in goats. , clinical signs. malignant edema is characterized by local lesion (wound) or regional pain characterized by swelling and edema that progressively becomes tense and dark (skin). high fever, signs of shock/toxemia, and frothy exudation of the wound are usually present. evidence of subcutaneous gas production is less common in this infection than in blackleg. uterine infection may cause a fetid vaginal discharge. death occurs within hours to a few days after onset of clinical signs. braxy usually causes death before any abnormalities are noted. on rare occasions, signs of sudden onset of illness with high fever, abdominal distention, depression, colic, and recumbency may be seen before death. diagnosis. characteristic clinicopathologic changes include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. examination of a gram-stained smear from the edematous swelling(s) or wound swabs could give an early diagnosis. one study reported the successful use of a pcr assay for the identification of bacteria associated with malignant edema in cattle, sheep, and other ruminants. postmortem changes with malignant edema include dark red, swollen muscle filled with hemorrhagic, proteinaceous exudate and little or no gas. with braxy, the abomasal wall is hemorrhagic and necrotic. both diseases are associated with rapid postmortem decomposition of the carcass. treatment and prevention. wound management and the rapid administration of high doses of penicillin (penicillin g sodium at , - , iu/kg iv every hours) are important in treating malignant edema. local treatment consists of surgical incision of the affected area to provide drainage and irrigation with peroxide. injection of penicillin directly into or in the periphery of the lesions may help. ancillary treatments such as iv fluids, antiinflammatory agents (e.g., flunixin meglumine, mg/kg iv), and nutritional support may be necessary. maintenance of good hygiene during procedures such as lambing, tail docking, shearing, castration, obstetric manipulation, and administering injections is helpful in preventing malignant edema. multivalent clostridial toxoids may provide some protection and should be given annually to animals at risk for the disorder. pathogenesis. several species of clostridial organisms can cause myonecrosis in small ruminants. , , the disease is acute to per-acute, has a short course of duration, and is usually fatal. c. chauvoei, c. septicum, and c. sordelli are commonly involved with clostridial myonecrosis in ruminants. [ ] [ ] [ ] blackleg can be enzootic in some areas or farms because of increased bacterial contamination and occurs more commonly in the warm months of the year. [ ] [ ] [ ] animals between months and years of age can be affected. , c. chauvoei is the most important cause of blackleg. c. sordelli tends to be involved in the myonecrosis of older feedlot animals. these organisms are found in the soil and can gain access to muscles after translocation from the gastrointestinal tract and liver into systemic circulation. additionally, direct inoculation of the organisms by penetrating wounds or intramuscular injections has been suggested. local tissue trauma, wounds, unsanitary procedures (i.e., shearing, tail docking, and castration), umbilical infection (neonates), or vaginal trauma from lambing can create perfect conditions for the germination of clostridial spores inducing rapid toxin synthesis and production. in some cases, bacterial proliferation appears to occur in a site distant from the original wound (i.e., fetal infections after shearing of a ewe and myocardial necrosis in cattle and sheep). bacterial toxins cause severe local tissue necrosis, systemic toxemia, and ultimately death. as with braxy, several other strains of tissue-invasive clostridia can cause this disease and mixed infections are common. clinical signs. clostridial myonecrosis usually progresses rapidly and sudden death or history of found dead is not uncommon. [ ] [ ] [ ] clinical signs in animals who are still alive include local to regional painful, edematous swelling most commonly in the limbs or trunk muscles. skin of the affected area can become discolored and crepitus; however, in affected sheep, subcutaneous edema and gaseous crepitation are uncommon and cannot be felt before death. other signs might include stiff gait, lameness, fever, and signs of shock. in cases where the infection occurred through a wound, there is extensive local damage and malodorous serosanguinous fluid discharge. c. chauvoei also causes uterine infection and severe gangrenous mastitis in postparturient ewes. , in these cases, uterine and mammary infections may cause fetid vaginal and mammary discharge, respectively. death often occurs within to hours after onset of clinical signs. necropsy findings. rapid tissue autolysis is not uncommon in animals that succumb to clostridial myonecrosis. bloodstained fluid and froth can be observed discharging from nostrils and anus. in small ruminants and especially sheep, affected muscle areas are more localized and deeper, brown to black discoloration is present, the subcutaneous edema is not as severe, and, although there is gas present, is not in such large amounts as in cattle. in cases of infection from skin wounds, the area demonstrates subcutaneous edema, swelling, and underlying muscle discoloration. in cases of infection through the urogenital tract, typical lesions are found in the perineal area, vagina, uterus, and fetus. lung congestion, fibrinohemorrhagic pleuritis, pericarditis, myocardial damage, and bloat are also common findings. , diagnosis. it is rarely possible to obtain samples for clinicopathological analysis due to the per-acute course of the disease. if samples can be obtained, common findings include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia-metabolic acidosis, azotemia, and increases in liver and muscle enzymesalso may be seen. presumptive diagnosis can be made from history, characteristic clinical signs, and gross pathology findings; however, aspirates or tissue specimens from affected muscles for direct smear examination, fluorescent antibody testing, or anaerobic culture are required for definitive diagnosis. , a multiplex pcr is available for identification of pathogenic clostridia on fluid and tissue samples. treatment and prevention. aggressive antibiotic therapy (e.g., penicillin g sodium or potassium penicillin at , - , iu/kg iv every hours), in combination with surgical debridement of affected tissues (fasciotomy), and supportive care (nutritional support, iv fluids, and antiinflammatory agents) are important within the treatment plan for clostridial myositis. prognosis for treatment of all types of clostridial myositis cases is usually guarded to poor and depends on the duration and extension of the lesions. maintaining excellent hygiene during invasive procedures such as castration, obstetric manipulation, shearing, tail docking, and administering injections is helpful in preventing blackleg. multivalent clostridial toxoids may provide some protection and should be given to all animals starting at weaning time, before parturition, and annually. , both tetanus and botulism are important diseases in small ruminant medicine. these two diseases are covered elsewhere in this book (see chapters , , , , and ) . older animals are generally more resistant to sepsis than neonates because they have larger amounts of circulating antibodies. however, this resistance can be overwhelmed by aggressive bacteria, or loss of immune function can allow invasion by opportunistic bacteria. malnutrition, parasitism, transport, overcrowding, other diseases, extreme weather conditions, and other stressors are the major causes of immune suppression. sepsis may produce peracute, acute, or chronic disease signs. peracute signs include fever, injected mucous membranes (including the sclera), tachycardia, tachypnea, dyspnea, swollen joints, lameness, splinting of the abdomen, weakness, depression, anorexia, recumbency, seizures, coma, and sudden death. acute signs are similar, except that they persist for a longer period and therefore are more likely to be noticed. chronic signs usually result from the partial clearance of infection after an acute episode, which may be clinical or inapparent. pathogenesis. gram negative bacteria and their toxins gain access to the blood from a site of proliferation or destruction. the most important toxin is endotoxin, a group of lipopolysaccharide molecules that reside within the wall of the bacteria. bacteria or endotoxins incite a systemic inflammatory response, chiefly through activation of host macrophages and stimulation of host cytokine release. these cytokines cause inflammation, produce leukocyte recruitment, increase capillary permeability, induce fever through stimulation of the hypothalamus, and have regional or diffuse vasomotor effects. because the ruminant gut has a plentiful population of gram negative bacteria, it is implicated as the source of most cases of gram negative sepsis. grain overload causes a die-off of the normal gram negative ruminal flora, ulcerative enteric disease allows invasion of bacteria or absorption of their toxins, and ingestion of pathogens provides a suitable place for proliferation and route for invasion of the body. gram negative sepsis caused by opportunistic organisms is best recognized in immunocompromised neonates but also can be seen in stressed or immunocompromised animals of all ages. e. coli is commonly found in fecal material, klebsiella pneumoniae is found in feces and wood products, f. necrophorum lives in the gastrointestinal tract and in soil and invades through compromised gastric mucosa or foot-rot lesions, and pseudomonas aeruginosa is commonly found in water and wash solutions. primary pathogens are most common in adults. although some coliform bacteria may fit into this category, by far, the most important genus is salmonella. sources of salmonella infection are numerous and include carrier animals of the same species, cattle, rodents, birds, other animals, environmental contamination, and possibly feedstuffs. only one serotype of salmonella is specifically adapted to sheep (salmonella abortus ovis), and it is not found in north america. no strain is known to be host-adapted to goats or cervids. therefore, all infections in sheep, goats, and cervids have the potential to spread to and from other species, including humans. serotypes of salmonella that have caused important infections in sheep or goats include salmonella typhimurium, salmonella dublin, and salmonella montevideo. most of these infections lead to bacteremia with mild systemic signs, followed by abortion. s. dublin and s. typhimurium tend to cause more illness in adults because of fibrinonecrotic enteritis. clinical signs. affected animals can exhibit anything, from mild depression with a low-grade fever to shock. common signs include fever, tachycardia, tachypnea, depression with slow or absent eating and drinking, weakness or recumbency, and injection or cyanosis of mucous membranes. organ-specific signs may betray the source or at least the primary location of the infection. fetid discharge may be seen with metritis or abortion; dyspnea and abnormal lung sounds may be seen with pulmonary infection; and bloat, ruminal atony, abdominal distention, and diarrhea may be seen with gastrointestinal infections. diagnosis. the most common abnormality identified on a cbc with peracute gram negative sepsis is panleukopenia. over the course of several days, this condition may resolve, first through an increase in immature neutrophils and later through an increase in mature neutrophils and restoration of lymphocyte counts. very immature cells, severe toxic changes, and persistence of neutropenia suggest a poor prognosis. serum biochemical changes often reflect the severity of the condition. the greater the evidence of shock or tissue damage, the worse the prognosis. metabolic acidosis with a large anion gap and azotemia suggest advanced disease. necropsy findings include diffuse evidence of inflammation, including pulmonary congestion, and polyserositis with body cavity exudates. hemorrhagic pneumonia or fibrinonecrotic enteritis may be seen and reflect the source of bacterial invasion. in all cases, diagnosis is best confirmed by bacteriologic culture of body tissues or fluids. in the live animal, culture of blood, feces, or tracheal fluid yields the best results. when several animals are infected, environmental samples (including feed, water, and bedding) should be tested for the presence of the bacteria. bacteriologic culture of aborted fetuses or placentas frequently yields heavy growth of the organism. prevention. maintaining overall good health and hygiene is the best means of preventing gram negative sepsis. antiendotoxin bacterins are available for cattle in the united states, but their use in small ruminants has been too limited to assess their efficacy. during a flock outbreak, the use of autogenous bacterin may help prevent the spread of disease on a farm. actinobacillus seminis is a gram negative bacillus or coccobacillus that affects primarily the male and female reproductive tracts. infection causes posthitis, epididymitis, and orchitis in rams and metritis and abortion in ewes. other sites of infection, including rare occurrences of chronic sepsis, also are possible. serologic tests are much more useful for identifying infected flocks than infected individuals within flocks. definitive diagnosis depends on bacteriologic culture of the organism and differentiation of it from brucella ovis. the bacillus is common in sheep in some parts of the world but is uncommon in north american sheep and goats. t. pyogenes is best known as an abscess-forming bacterium because of the thick pus formed in response to infection by it and the fibrinous response it elicits. it occasionally also causes sepsis. its association with chronic sepsis lends credence to the belief that trueperella is often a secondary invader that colonizes tissues damaged by another bacterium (see chapter ) . bacillus anthracis is a large, gram positive, anaerobic bacillus that causes anthrax. it forms spores under aerobic conditions (such as on culture plates) but rarely does so when oxygen tensions are low, as in carcasses. the organism affects most mammals, with herbivores being most susceptible. it is usually carried from one area to another by shedding or dying animals and also can multiply in alkaline, nitrogenous soils. periods of heat and intermittent flooding promote overgrowth of the organism. b. anthracis spores may be inhaled or ingested; in rare cases, the bacillus itself may be spread by biting flies. after local replication, the organism gains access to the blood, where it multiples readily. large numbers of the organisms colonize the spleen. b. anthracis secretes a holotoxin made of edema factor), protective antigen, and lethal factor. this toxin impairs phagocytosis, increases capillary permeability, and inhibits clotting. splenic engorgement, generalized edema, circulatory shock, and bleeding diathesis are the most common lesions and signs of anthrax. generalized infection should be considered uniformly fatal. death may occur before or within hours of initial recognition that the animal is sick. prophylactic antibiotic treatment of healthy animals (oxytetracycline mg/kg iv sid) may decrease spread and mortality during outbreaks. the disease is reportable in many areas. local forms of anthrax also occur, most commonly after transmission through a skin wound or fly bite. local heat, pain, swelling, and necrosis are seen first, and the generalized syndrome often follows. bacterial organisms are rarely identified before important treatment decisions must be made. therefore, treatment should follow general principles and have a wide spectrum of efficacy. antimicrobial drugs are the cornerstone of treatment. in meat-or milkproducing animals, the veterinarian must be careful to use drugs within label directions or have a rational plan for extra-label drug use. the issue of extra-label drug use is especially important in small ruminants and cervids because very few pharmaceutical products have been licensed for them in north america. unless a specific organism is suspected (clostridiosis or anaplasmosis), a single antibiotic or combination of antimicrobial drugs to provide a broad spectrum of coverage should be selected. penicillins, macrolides, tetracyclines, and cephalosporins all provide effective coverage against gram positive pathogens. the newer third-generation cephalosporins are effective against many systemic and enteric gram negative pathogens. the gram negative pathogens of the respiratory tract are often sensitive to other classes of antibiotics. macrolides and tetracyclines also are effective against mycoplasma species and rickettsial organisms. nsaids are almost always beneficial in severe infectious conditions because of their antiinflammatory, antipyretic, and antiendotoxic effects. they are likely to be more effective than corticosteroids because they provide benefits without suppressing the immune response. all such drug use should be considered extralabel and administered accordingly with appropriate withdrawal times established. specific antisera are available for some of the clostridial diseases and may be beneficial if given before widespread tissue necrosis has occurred. severely compromised animals should be treated with fluids for shock (see chapter ). the most common zoonotic disease risk posed by exposure to small ruminants is orf, also known as contagious ecthyma in animals (see chapter ) . the disease is caused by an epitheliotropic poxvirus and is transmitted to humans by direct contact with infected animals. skin trauma is a significant risk factor for transmission in both humans and animals. in humans, erythematous macules or papules appear at the site of infection to days following exposure. the infection is generally self-limiting in immunocompetent individuals with complete healing occurring within weeks. brucella melitensis. apart from contagious ecthyma, the greatest risk of zoonotic disease from small ruminants is due to pathogens typically found in the reproductive tract that are transmitted to humans through contact with aborted fetuses, the placenta, or birthing fluids or through the consumption of raw or improperly pasteurized dairy products. b. melitensis is more common in goats than sheep (see chapter ) . swine, cattle, and other ruminants are common hosts. infection in animals usually causes inapparent mammary infection and abortions; infection in humans is characterized by undulant fever, myalgia, and fatigue. coxiella burnetii. c. burnetii is a rickettsial organism that is an important cause of abortion in sheep and goats (see chapter ) . wildlife and farm-raised deer may serve as reservoir hosts for infection in other ruminants and humans. infection is a documented cause of reproductive failure in farmed deer and prolonged shedding of the organism is an important source of environmental contamination. in addition to abortion, newly infected sheep and goats occasionally have mild, transient fevers. c. burnetii is far more important as the cause of q fever in humans, who become infected after inhaling particles, handling contaminated animals, or coming into contact with contaminated body fluids (uterine fluid, milk) from infected animals. infection in humans may be asymptomatic, present with flu-like symptoms, or, in the chronic form, present as granulomatous hepatitis, osteomyelitis, or bacterial endocarditis. chlamydophila spp. chlamydophila abortus (previously chlamydia psittaci) is an obligate intracellular parasite and the cause of enzootic abortion of small ruminants (see chapter ) . chlamydophila pecorum may cause polyarthritis and keratoconjunctivitis (see chapter ) in sheep and goats. transmission between animals and to humans most commonly occurs through direct contact with infected tissues or materials. infection in humans results in an acute febrile syndrome or respiratory symptoms. chlamydial diseases are more commonly reported in sheep than in goats. chlamydial diseases are suspected to cause disease in other species, including deer. recent serologic evaluation of wild ungulates identified multiple species of deer with antibodies against several chlamydial species. the clinical significance of serological infection in these species remains undetermined. francisella tularensis. f. tularensis is more common in sheep than goats. the organism has many hosts, of which the most important are wild rabbits and rodents. it can contaminate water sources. transmission to sheep is usually through biting arthropods that have previously fed on an infected wild mammal. acute or chronic sepsis may be seen, with more widespread and severe disease occurring in sheep with poor immune function. at necropsy, the disease is characterized by military foci of necrosis in the liver, and less commonly in the lymph nodes, spleen, and lungs. most cases in humans result in acute onset of flu-like symptoms a few days after exposure. l. interrogans. pathogenesis. leptospira spp. are spirochete bacteria that live in moist environments. their survival time outside of hosts is usually short, so their most important reservoirs are the kidneys of infected animals, especially rodents. infected animals shed the organisms through urine and most other body fluids. organisms enter new hosts through mucous membranes and skin breaks and cause bacteremia. signs of sepsis range from inapparent to severe, with more severe signs predominating in neonates. intravascular hemolysis may result. in animals that survive the acute stage, infection may localize in sites such as the kidneys, eyes, and fetoplacental unit. abortion may occur a month or more after acute signs first become evident while renal shedding may occur for several months. leptospirosis is zoonotic. in most cases, infections in humans are asymptomatic and selflimiting. however, in approximately % of cases, severe, and potentially fatal, systemic disease may develop, including jaundice, renal failure, and pulmonary hemorrhage. clinical signs acute leptospirosis causes signs of sepsis, including fever, depression, dyspnea, exercise intolerance, weakness, and death (see chapter ) . additionally, many affected animals show signs of intravascular hemolysis such as anemia, icterus, and hemoglobinuria. diagnosis evidence of intravascular hemolysis such as anemia, hyperbilirubinemia, hemoglobinuria, and hemoglobinemia is suggestive of this disease. in chronic infection, non-specific inflammatory changes and azotemia may be seen. animals dying in the acute hemolytic stage are likely to have dark, discolored urine, bladder, and kidneys. spirochetes can be identified on dark-field microscopy of fresh urine or plasma from infected animals and may be cultured with special techniques. in animals with less severe infection, a rise in antibody titers can be used to support a diagnosis of leptospirosis. prevention numerous vaccines are available for sheep. because protection is serotype specific, it is important to vaccinate against common serotypes in the area. leptospira pomona is the most consistent isolate from sheep and goats; leptospira hardjobovis is the predominate serovar in deer. vaccination immunity is thought to be short lived; boosters should be given at least twice a year in endemic areas. vaccination of deer against serovars hardjobovis and pomona has been associated with decreased urine shedding and increased growth rate in young animals. pathogenesis. l. monocytogenes causes disease with similar frequency in sheep and goats (see chapter ) . the organism is a common soil and fecal contaminant. it also proliferates in silage that is not properly acidified and in rotting, woody debris. risk of exposure depends on the feed and environment of the animals. environmental and fecal contamination is a more common source than silage in small ruminants overall because most sheep and goats throughout the world are not fed silage. infection in humans almost always results from ingestion of contaminated food products or unpasteurized milk. clinical signs. nervous system dysfunction and abortion are the most common manifestations of the disease. animals with the brainstem form of the disease display signs reflective of cranial nerve dysfunction, including drooped ears or eyelids, decreased facial sensation, and deviated nasal septum. a head tilt and circling may be present; in advanced cases of the disease, the animal is recumbent. clinical signs are mainly unilateral, occasionally bilateral, according to the nerve nuclei affected. diagnosis. antemortem diagnosis of listeriosis is difficult. a presumptive diagnosis is made based on history, clinical signs, and potential response to treatment. histopathologic identification of microabscesses in the brainstem and culture of the organism from affected tissues can be used to confirm the diagnosis. pathogenesis. p. multocida is a small, gram negative, bipolar, ovoid rod that inhabits the pharynx of healthy ruminants. it can survive in soil and water for varying amounts of time after contamination with ruminant nasal secretions. healthy ruminants shed p. multocida much more frequently than mannheimia haemolytica. disease occurs when bacteria colonize the lower respiratory tract or enter the blood. risk factors for pulmonary and systemic infection include viral or mycoplasmal respiratory diseases, temperature extremes, respiratory tract irritants, transport, overcrowding, changes to higher-energy feeds, and handling stress. these factors are thought to both increase bacterial replication in the airway and suppress mechanisms to clear the infection. pasteurellosis is a major problem in feedlot sheep but less common in small breeding or hobby flocks. pasteurellosis also is a significant disease in certain wild small ruminants such as bighorn sheep. direct spread of the organism between animals occurs with nasal contact, and indirect spread occurs after contact with infected nasal secretions. the organism persists in the environment for longer periods during warm, moist weather. p. multocida produces a polysaccharide capsule that inhibits phagocytosis and an endotoxin that contributes to clinical signs. the major disease caused by p. multocida is pneumonia (see chapter ). however, pasteurella spp. also are capable of entering the blood to cause septicemia in young lambs and hemorrhagic septicemia in adults. occasionally, focal infections such as septic arthritis and mastitis are found. clinical signs. clinical signs of pneumonic and septicemic pasteurellosis include severe depression, bilateral purulent nasal discharge, coughing, diarrhea, anorexia, high fever, and edema of the head, neck, and brisket. the disease course can be short with septicemic pasteurellosis and is usually more insidious with p. multocida pneumonia. pasteurella mastitis is characterized by the bluebag condition or gangrene of the udder. diagnosis. inflammatory changes in the leukogram and hyperfibrinogenemia are the most frequent abnormalities. with severe disease and in the septicemic form, immature neutrophils may predominate over mature cells. inflammation of the intestine and abomasum also may be seen. hemorrhage and fibrin are usually absent or less prominent than in pneumonia caused by m. haemolytica. samples for bacteriologic culture are usually obtained postmortem. blood or tracheal fluid may be obtained before death if the value of the animal warrants it. m. haemolytica is a gram negative rod that is a common commensal inhabitant of the tonsils of young animals. disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lower respiratory tract. clinical signs and diagnosis. the most common syndrome is enzootic pneumonia, which is seen in young lambs and their dams (see chapter ) . hemorrhagic bronchopneumonia is the major lesion and respiratory signs predominate. gangrenous mastitis (bluebag) is seen in some of the dams, presumably after they have been nursed by infected offspring. factors that promote respiratory disease, including viral infections, airborne irritants, high stocking density, and stress, are thought to promote invasion of the lower airway by these bacteria. b. trehalosi is a gram negative rod that is a commensal inhabitant of the upper respiratory tract (see chapter ) . disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lung or blood. replication occurs in the lung and systemic toxemia or bacteremia resulting in septicemic pasteurellosis. septicemic pasteurellosis is a significant cause of mortality in young lambs and in some farms is the leading cause of death in the age group. clinical signs. septicemic pasteurellosis occurs most commonly in weaned lambs, often following some form of stress such as transport, marketing, or weaning itself. the course of the disease is relatively rapid, and animals may be found dead within hours without showing premonitory clinical signs. when observed, clinical signs include depression, recumbency, and signs of toxemia. diagnosis. septicemic pasteurellosis should be suspected when presented with a dead, recently weaned, sheep with a recent history of stress. diagnosis is best confirmed by typical lesions at necropsy and culture of the organism from bodily tissues. demonstration of b. trehalosi in nasal swabs is of limited value due to the high prevalence of upper respiratory tract colonization in healthy lambs. at necropsy, there may be no evidence of pneumonia, but blood-stained foam can be found in the upper respiratory tract. ulceration of the pharynx and esophagus is commonly present as is subcutaneous hemorrhage of the neck and thorax. prevention. treatment is difficult due to the rapid course of disease. efforts should be made to minimize stressors, particularly during and following weaning, and to manage management factors that may contribute to the disease. vaccination with pasteurella bacterins is rarely effective at controlling natural outbreaks of disease. pathophysiology. abscess-forming bacteria are usually able to survive phagocytosis and thereby avoid destruction by cells of the immune system. alternatively, they invoke such an inflammatory response that the host body "walls off" the entire region with fibrous tissue. abscesses may occur locally, frequently after a wound infection, or at numerous or distant sites from the point of infection. for abscesses to occur at the latter sites, the organism must travel either by way of the blood or within leukocytes. disease characterized by multifocal or internal abscesses usually results from a low-grade, transient event of bacteremia. the best known and most important abscess-forming bacterium in small ruminants is corynebacterium pseudotuberculosis, the gram positive, facultative anaerobic coccobacillus that causes caseous lymphadenitis. infection is usually maintained in a flock by infected animals that spread the organism to others through purulent material draining from open abscesses. the organism is very hardy, so infection can occur through direct contact or indirect contact with contaminated common instruments and facilities. infection is usually introduced into a flock through acquisition of an infected animal, although it also can occur when a naive flock is moved into a contaminated area. horses, cattle, and humans also are minor hosts. infection is thought to occur after ingestion, inhalation, or wound contamination. except for lower respiratory tract invasion, a surface break is thought to be necessary. contaminated shears, tail-docking knives, and emasculators readily spread the organisms through a flock. abscesses can form at the site of invasion or more commonly at the site of the local lymph node. clinical signs. clinical signs of external abscesses include surface swellings and draining lesions. drainage may be intermittent and usually consists of thick, yellow-white purulent material. internal abscesses are more difficult to diagnose. thoracic masses may cause inspiratory dyspnea or occlude venous return to the heart. abdominal lesions may cause tenesmus, stranguria, and occasionally colic. the most common sign of internal abscesses is weight loss with or without intermittent fever. common external sites include the submandibular or retromandibular space and the preinguinal, prefemoral, and supramammary lymph nodes. head and neck lesions are more common in goats, whereas sheep have a more even distribution of cranial and caudal lesions, presumably as a result of shearing wounds. external infections rarely cause clinical illness beyond the draining abscess, although some degree of cachexia may be present. diagnosis. diagnosis is often made by the characteristic lesions with their thick, nonmalodorous pus. bacteriologic culture provides a definitive diagnosis, which may be important for flock management. serologic tests have been developed to identify carrier animals and may be useful if the manager wishes to eliminate infection from the flock. treatment. treatment is often unrewarding: antibiotic sensitivity profiles do not reflect the degree of protection afforded the organisms within the abscesses. long-term treatment with antibiotics and drainage of any compromising masses may lead to some degree of resolution, but internal abscesses are likely to persist. prevention. prevention through the use of vaccines has been attempted. vaccines appear to reduce the severity of the disease but do not completely prevent infection. moreover, live attenuated bacterins lead to de facto infection of all vaccinated animals and therefore should not be used in naïve flocks. other abscess-forming bacteria are most important as differential diagnoses for caseous lymphadenitis. t. pyogenes is another wound contaminant that affects focal areas or regional external lymph nodes. it also commonly colonizes damaged internal tissues such as postpneumonic lungs, postacidotic livers, and damaged feet and heart valves. it is thought to be ubiquitous and poorly invasive in ruminants and therefore does not have the same flock significance as c. pseudotuberculosis. flocks with outbreaks of this infection often have suboptimal management. f. necrophorum causes similar disease and often coinfects with t. pyogenes. it is generally more necrotizing and leads to greater systemic signs of acute illness, including death. f. necrophorum also produces fetid pus, whereas t. pyogenes usually does not. rhodococcus equi is a rare cause of pulmonary abscesses in sheep. numerous small, coalescent, nodular skin abscesses may result from pseudomonas pseudomallei infection (melioidosis). infection usually occurs after the sheep or goat is bitten by an insect that previously fed on an infected rodent. this organism is found in many subtropical regions, including the caribbean, but is not reported in north america. f. necrophorum causes or is associated with a variety of diseases in sheep and is likely to cause many similar diseases in goats. it is best known as a cause of foot rot and hepatic abscesses and appears to be important in lip-leg ulceration. it is an enteric gram negative anaerobe and as such can cause gram negative sepsis after entrance of the bacteria or its toxins into the circulation. f. necrophorum has a poor ability to invade healthy tissue. however, it readily colonizes regions damaged by trauma, persistent moisture, and infection. in addition to endotoxin, the bacterium produces leukocidal and cytolytic toxins that form zones of necrosis around bacterial colonies. this tissue necrosis and the foul-smelling waste gases produced by the bacteria are characteristic of necrobacillosis, or f. necrophorum infection. clinical signs include necrotic, fetid lesions, usually of the mouth or feet, that can cause ingestion or lameness problems. efforts to maintain good hygiene are helpful in preventing fecal contamination. additionally, preventing trauma to foot and mouth tissues through good surface choices and proper pasture drainage is important. pathogenesis. yersinia spp. are gram negative bacteria. yersinia enterocolitica and yersinia pseudotuberculosis both have many mammalian and avian hosts, including humans, and cause clostridial enteritis-like disease in goats. rodent and bird hosts may be important reservoir populations for infections in domestic animals. kids younger than months develop enteritis, bacteremia, and diarrhea that is watery but not bloody. severe toxemia and sudden death can occur. older kids and flocks with chronic exposure tend to have less severe acute disease. instead, chronic diarrhea and weight loss are seen, usually in association with gut wall and abdominal abscesses. sheep, deer, and wild ungulates are rarely affected. clinical signs. signs of enteritis or sepsis predominate in acute disease, whereas signs of wasting are more common in chronic disease. diagnosis. evidence of acute or chronic inflammation is provided by blood work. characteristic necropsy lesions include numerous microabscesses in the gut wall and mesenteric lymph nodes, as well as other evidence of enteritis or sepsis. culture of lesions and demonstration of a rising antibody titer are diagnostic. prevention. avoiding exposure to sources and maintaining overall flock health are helpful in preventing losses due to yersiniosis. pathogenesis. mycobacteria are small, aerobic, straight or curved pleomorphic rods with thick lipid cell walls. they can be stained with acid-fast stains and are usually gram positive. the bacteria live within infected animals of many mammalian species and survive for several years in warm, moist environments. infection occurs after ingestion or inhalation. an identifying characteristic of the mechanism of infection by mycobacteria is the bacteria's ability to survive within macrophages by preventing fusion of phagosomes and lysosomes. the organisms are carried to local lymphatic vessels or lymph nodes, where they form granulomas. as they enlarge, granulomas may develop necrotic or mineralized centers surrounded by macrophages and giant cells. disease can be local, regional, or generalized, depending on the distance the organism is carried from the original site of infection. granulomatous pneumonia, enterocolitis, and lymphadenitis are the most common local and regional forms of the disease. organisms from ruptured granulomas may be spread in contaminated respiratory secretions and feces. mycobacterial infections of all types are uncommon in north american sheep, goats, and cervids, and these species are considered to be relatively resistant to infection. mycobacterium bovis is the most common organism associated with ovine tuberculosis in other countries (see chapter ), but mycobacterium avium is more common in the united states. the most common mycobacterial infection is johne's disease (paratuberculosis) caused by the etiologic agent m. avium subsp. paratuberculosis (see chapter ) . mycobacterium tuberculosis is rare in the united states. mycobacterial infections are reportable in most parts of the united states. some debate is ongoing about human susceptibility to m. avium subsp. paratuberculosis; the other organisms are known to be pathogenic in people. clinical signs. the most common clinical sign is emaciation. diarrhea may be seen terminally in both tuberculosis and paratuberculosis. the disease is insidious, with signs becoming more apparent over several weeks to months. respiratory signs may be seen, especially with infection by m. bovis or m. avium subsp. diagnosis. reports of clinicopathologic abnormalities are rare. hypoalbuminemia and hypoproteinemia are likely to be common with chronic enterocolitis caused by either tuberculosis or paratuberculosis. the most common necropsy lesions seen in tuberculosis are nodular lesions of the lung, liver, lymph nodes, spleen, and intestines. histologic evaluation reveals the nodules to be granulomas with giant cells and acid-fast organisms. frequently, the center of the lesion is necrotic and mineralized. intestinal lesions appear to be more common than pulmonary lesions in goats. the lesions of paratuberculosis are centered around the ileocecocolic junction and the adjacent mesentery. the regions may appear normal or be notably thickened. thickening of bowel or nodular infiltrates of lung or liver may be detected antemortem using imaging modalities, such as ultrasonography or computed tomography. postmortem diagnosis is made by identifying characteristic lesions and culturing the organisms. antemortem diagnosis of tuberculosis is best achieved by observing the reaction to intradermal injection of tuberculin with or without comparative injection of purified protein derivatives of m. bovis and m. avium subsp. paratuberculosis. all tuberculosis testing should be done in accordance with local regulations. antemortem diagnosis of johne's disease can be achieved by fecal culture of the organism, but this test takes several weeks to months to complete and is far less reliable in sheep or goats than cattle, with a sensitivity as low as . . serologic tests (e.g., elisa) appear to be sensitive and specific for johne's disease in animals demonstrating clinical disease rather than preclinical infection. serologic detection of clinical johne's disease in cervids has been shown to be highly sensitive and specific while the sensitivity of fecal culture is low in both sheep and goats. the recommended organism detection method in both species is fecal pcr. fecal or milk pcr can be used on pooled samples for flock identification and to type the organism. prevention. tuberculosis should not be endemic in flocks in the united states because positive animals are quarantined or destroyed. preventing exposure to wild ruminants and other possible sources is crucial. except in goat flocks raised for the production of milk that is to be sold unpasteurized, testing is uncommon, so animals are usually not identified until they develop overt disease. paratuberculosis is much more common and may be maintained in flocks by carrier animals. no effective treatment is available for either disease, nor should any be encouraged because efforts should be concentrated on eliminating infection from the flock or herd. vaccination of sheep is used extensively in australia to control paratuberculosis. prolonged vaccination has been shown to decrease fecal shedding in infected animals over time. pathogenesis. mycoplasma spp. are very small, simple bacteria that parasitize cells of higher species. they are common inhabitants of mucous membranes and can have either a commensal or pathogenic relationship with the host. transmission between animals is most likely through direct or indirect contact with body fluids from infected animals, inhalation of respiratory droplets, and arthropod vectors. common sites for superficial infection include the ocular membranes, lung, mammary gland, and female reproductive tract. the organisms can also enter the blood and cause septicemia, abortion, pleuritis, and polyarthritis. flare-ups often occur during times of crowding and during parturition, when neonates can spread the organisms from the mother's mouth to her udder and in turn become infected by ingesting contaminated milk. the most important mycoplasma species in the united states are mycoplasma conjunctivae, mycoplasma capricolum, and the less pathogenic mycoplasma ovipneumoniae. they are most commonly associated with keratoconjunctivitis, acute or chronic sepsis, and pneumonia, respectively. m. conjunctivae and c. abortus are the most common causes of pinkeye in north american small ruminants. mycoplasma spp. are thought to inhibit tracheal ciliary function and thus may have a role similar to viruses in "shipping fever pneumonia" in facilitating lower respiratory tract invasion by primary bacterial pathogens. many of the major pathogenic serotypes found in other countries (some of which cause severe pleuropneumonia without the participation of another bacteria), including mycoplasma mycoides subsp. mycoides, mycoplasma mycoides subsp. capri, mycoplasma agalactiae, and strain f , are not found in or have been eradicated from north america clinical signs. keratoconjunctivitis, mastitis, exudative vulvovaginitis, fever, cough, dyspnea, exercise intolerance, abortion, lameness, swollen joints, neonatal death, and depression may all be seen with mycoplasma infections. diagnosis. no specific clinical pathologic findings occur with these diseases. mycoplasma infection should be suspected in sheep and goats with severe exudative pleuropneumonia in some parts of the world. mycoplasma can be identified by bacteriologic culture or staining of exudates. examiners must take care in interpreting positive cultures from body surfaces because nonpathogenic mycoplasma are common. prevention. vaccines against mycoplasmal infections are available in some parts of the world, but not in the united states. providing fly control, preventing stress and overcrowding, and isolating sick animals from healthy ones may help prevent the spread of disease. anaplasma ovis, mycoplasma ovis, and babesia spp. a. ovis and m. ovis are small bacteria that lack cells walls and parasitize erythrocytes. these and similar organisms have undergone recent reclassification following molecular analysis. other species of hemotropic mycoplasmas may affect sheep and cervids. the organisms are spread from animal to animal by insect or mechanical vectors. known arthropod vectors for a. ovis include ticks and horseflies; other biting flies may be more important with m. ovis infection. hypodermic needles and equipment used for tail-docking, castrating, or disbudding animals may be important in iatrogenic transmission. after being introduced into a naive host, the organisms proliferate, and the number of red cells infected increases rapidly until an effective immune response begins to weeks later. a similar proliferation of organisms may occur in chronically infected animals after temporary immune suppression. the humoral and cellular immune responses against a. ovis lead to opsonization of parasitized erythrocytes and their removal by cells of the reticuloendothelial system; m. ovis infection is thought to cause more intravascular hemolysis. the result in both cases is hemolytic anemia. the protozoon parasites babesia ovis and babesia motasi have similar life cycles and cause similar diseases, but they have been eradicated and are reportable in the united states. babesia spp. affecting small ruminants are generally less pathogenic than are their bovine counterparts. animals surviving acute hemolytic crisis reduce the parasites to low numbers but rarely clear the infection completely; they serve as sources of infection for other animals. sheep and goats are susceptible to infection by either organism; goats generally appear to be more resistant to the development of severe parasitemia and clinical signs. clinical signs. signs present during hemolytic crises include fever, weakness, pale mucous membranes, and pigmenturia. urine discoloration results from increased amounts of bilirubin in most cases, although hemoglobinuria may be seen in some sheep with m. ovis infection. icterus is usually present only after the acute hemolytic crisis. clinical signs are exacerbated during times of stress, and infection is often first noted when the animals are moved or handled. chronically infected animals may appear clinically normal, may have recrudescence of infection after stress, or may display signs of ill-thrift such as poor body condition and fleece. babesiosis occasionally causes concurrent central neurologic signs. diagnosis. the major clinical laboratory finding is regenerative anemia with detection of the intraerythrocytic bodies. chronically infected sheep often have high counts of nucleated erythrocytes. because m. ovis consumes glucose, hypoglycemia and metabolic acidosis may be detected, especially in blood samples that are not processed immediately. diagnosis is by identification of the organisms on blood smears. special stains are available to make the organisms more visible. postmortem lesions include pallor or icterus of membranes and splenomegaly. some evidence of vasculitis, including edema or exudates in body tissues or cavities, may be seen with m. ovis infection. treatment. mycoplasma spp. and anaplasma spp. are sensitive to tetracycline antibiotics. babesiosis is more difficult to treat. effective drugs include diminazene, pentamidine, and imidocarb dipropionate. supportive care for all blood parasite infections includes whole blood transfusions, nutritional support, and administration of fluids. prevention. prevention in most cases involves maintaining low levels of parasites rather than eliminating them entirely. this method ensures continual stimulation of the immune response, whereas eradication often leaves the animal susceptible to another bout of acute infection. vector control can also be important in management of the disease. pathogenesis. two organisms belonging to the anaplasmataceae family, ehrlichia ovis and anaplasma phagocytophilum, infect ovine wbcs, causing fever, immune suppression, and some organ damage. a. phagocytophilum is the causative agent of tick borne fever in sheep and granulocytic anaplasmosis in horses, dogs, and humans. the organism is transmitted by ticks (ixodes spp.) and maintained in the environment by asymptomatic carrier animals. the distribution and incidence of disease is seasonal with the life cycle of the tick. the organism infects cells of the granulocytic lineage, leading to severe persistent neutropenia and acute lymphopenia. fever occurs to weeks after infection, lasts as long as weeks, and occasionally relapses. chronic infection is common. spleen, lung, liver, and kidney tissue may show some damage because of immune destruction of infected cells, but organ-specific signs are usually the result of secondary infection. secondary bacterial joint infections in lambs infected with a. phagocytophilum develop debilitating lameness known as tick pyemia. e. ovis causes fever (benign ehrlichiosis) to weeks after infection. because of this organism's predilection for mononuclear cells, the degree of immunosuppression and subsequent importance of this disease are much less than for a. phagocytophilum infection. diagnosis. specific diagnosis is best made by identifying darkly stained bodies at the periphery of granulocytic cells, as well as occasional large bodies deep within the cytoplasm of some cells. stained bodies also can be seen on the periphery of mononuclear cells from a blood smear during the acute febrile stage or in tissues during chronic infection. serologic tests are available for detection of anaplasmosis. the available celisa is incapable of distinguishing species of anaplasma and serologic results must be interpreted appropriately, and the species confirmed by pcr. both infections affect sheep and goats (a. phagocytophilum also affects many other ruminants, including white-tailed deer), but neither has been reported in north america. a recent study demonstrated that sheep are capable of being experimentally infected with a human isolate a. phagocytophilum. interestingly, the sheep did not develop clinical disease. such findings suggest that sheep could serve as asymptomatic carriers and potential reservoirs for humans. a. phagocytophilum is widespread in northwestern europe, including the united kingdom, scandinavia, and india, and e. ovis is found mainly in countries bordering the indian ocean. in spite of documented seropositive status of animals, there have been no reports of sheep or goats naturally infected with a. phagocytophilum in the united states developing clinical disease. treatment and prevention. treatment and prevention efforts should focus on reducing vectors and bacterial counts during vector season. both organisms are susceptible to treatment with tetracycline. people and animals can become infected with trypanosome protozoa. the trypanosomes can complete their developmental cycle only in tsetse flies (glossina species). trypanosomes multiply in blood, tissues, and body fluids of their vertebrate hosts and are transmitted between vertebrate hosts in the saliva of blood-sucking flies as they feed. the trypanosome species that are known to infect goats and sheep include trypanosoma congolense, trypanosoma vivax, trypanosoma brucei subsp. brucei, trypanosoma evansi, and trypanosoma simiae. pathogenesis. after entering through the skin, trypanosomes reach the bloodstream by way of the lymphatic system. the parasites multiply, and the prepatent period lasts for to days after infection. the infection is characterized by periods of parasitemia, followed by the absence of parasites. this pattern of infection occurs because of antigenic variation: trypanosomes vary the antigenic nature of their glycoprotein surface coat to evade the host's immune system. this immune system-evasive maneuver prolongs infection and is responsible for chronic disease. some trypanosomes tend to invade extravascular spaces, such as the ocular aqueous humor and cerebrospinal fluid. the pathogenicity of trypanosomes varies with the different host species. trypanosomes may produce a hemolysin early in the course of the disease that causes anemia in the host. later, increased phagocytic activity results in massive erythrocyte destruction. clinical signs. the clinical signs are variable and non-specific and depend on the speed of onset of anemia and the degree of organ impairment. entire herds may be affected. all aspects of production are impaired-fertility, birth weight, lactation, weaning weight, growth, and survival. trypanosomiasis may predispose the animal to the development of other diseases that mask the underlying trypanosome infection. trypanosomiasis may be acute, subacute, or chronic, with chronic infection occurring most commonly. acute disease often causes abortion. dairy goats may show a sudden drop in milk production. depression, anorexia, and a stiff gait may be present. physical examination reveals tachycardia, tachypnea, and a slight fever. hyperemic mucous membranes and excessive lacrimation may be noted. affected animals often become recumbent and anorexic and die within to weeks of onset of clinical signs. if the animal survives, progression to the subacute phase, characterized by listlessness, weight loss, enlargement of superficial lymph nodes, and a dull, dry hair coat, may occur. in such cases, auscultation findings are similar to those in other forms of acute cardiac disease, as well as pale mucous membranes and a pronounced jugular pulse. the animal may linger for several weeks or months, or the chronic form of the disease may develop. affected animals show ill-thrift: dull and dry hair coat, inelastic skin, lethargy, emaciation, peripheral lymphadenopathy, pale mucous membranes, and exercise and stress intolerance. death may occur many months or even years after infection and usually results from congestive heart failure. subclinical trypanosomiasis causes acute episodes when animals are stressed by inadequate nutrition, increased production demands, or concurrent disease. diagnosis. diagnosis is difficult because the parasitemia is intermittent, clinical signs are non-specific, and infection is not always synonymous with disease. a pcr assay is gaining acceptance as the most sensitive diagnostic modality, but not all infected animals exhibit clinical disease. although a tentative diagnosis of pathologic trypanosomiasis can be made on the basis of history, clinical signs, and the presence of appropriate vectors, a definitive diagnosis requires identification of trypanosomes on a fresh blood smear, a giemsa-stained blood smear, or less commonly, a lymph smear. examination of the buffy coat of centrifuged blood with darkfield phase-contrast spore illumination is the most sensitive direct microscopic method and is useful when parasite numbers are low. pathogenic trypanosomes must be distinguished from more ubiquitous, nonpathogenic species particularly common in cattle, such as trypanosoma theileri. repeated blood sampling in individual animals often is necessary, because as noted, parasitemia is intermittent. the diagnosis is supported by evidence of anemia on a cbc. indirect diagnostic methods include an indirect fluorescent antibody test and the elisa. these tests are less helpful for diagnosis of a single clinical case but are useful in assessment for herd infection. both t. congolense and t. brucei readily infect rats and mice, and detection of these pathogens can be used to diagnose the infection indirectly. treatment. treatment consists of the use of trypanocidal agents and supportive care. animals with acute, subacute, and subclinical disease respond better to treatment than those with chronic disease because of the irreversible damage to hematopoiesis associated with chronic infection. with most trypanocides, the therapeutic index is low and varies with the host species. trypanocide efficacy also varies with the species of trypanosome present; resistance to agents is common. some trypanocides are irritating to the skin and may cause severe inflammation at the injection site. in sheep and goats with t. brucei infection, the trypanocide of choice is diminazene aceturate, which should be used at a higher dosage rate ( mg/kg given intramuscularly [im] or sc) than that recommended for cattle. protection after trypanocide use usually lasts to months, depending on the season. animals must be rested before and after treatment. supportive care consists of providing fluids, an environment conducive to rest, good nutrition, and possibly blood transfusions. prevention. vector control, stress and nutrition management, and selection of trypanosome-tolerant breeds of sheep and goats all help control or prevent trypanosomiasis. no vaccine is available. animals can be treated with insecticides (pyrethroids) to prevent bites by tsetse flies and other flies. control is accomplished by strategic use of trypanocides during the peak season. continued parasitologic and clinical surveillance is essential to determine the efficacy of control measures. pathogenesis. sarcocystis spp. are protozoon parasites that have a two-host life cycle. sexual reproduction occurs in the bowel of a carnivore (mainly dogs and wild canids) after the carnivore ingests cysts in the muscles of sheep, goats, and cervids. sporocysts are passed in the carnivore's feces and later ingested by a sheep, goat or cervid. the sporocysts hatch in the ruminant gut and invade the vascular endothelium during three phases of asexual reproduction. after the third phase (approximately to weeks after ingestion), merozoites enter the ruminant's muscle tissue and encyst. clinical signs are uncommon but can occur during the stages of reproduction and muscle invasion of the host. n. caninum has a similar life cycle and causes similar disease, except that it appears more likely to cause abortion and affect the central nervous system. clinical signs. most infections are asymptomatic. however, if a large number of sporocysts are ingested, tissue damage may occur during the intestinal, vascular, and muscle stages of the sarcocystis life cycle. fever, lameness or a stiff gait, reluctance to move, and diarrhea may be seen. central neurologic signs (blindness, changes in mentation, and seizures) may occur if the organisms invade the brain or interrupt blood flow to it. abortion can occur as early as weeks after ingestion. with severe chronic infections, emaciation and anorexia are seen. diagnosis. the most characteristic abnormality is an increase in muscle enzyme activity in the blood. anemia is common and may result from extravascular hemolysis. cerebrospinal fluid may show mild mononuclear pleocytosis or may appear normal. on necropsy, muscles may display pale streaks or macroscopic cysts throughout. other evidence of vasculitis includes hemorrhagic serosal surfaces, body cavity fluids, and lymphadenopathy. microscopic or ultrastructural examination of affected tissues should reveal the presence of organisms. specific antibody tests are available and do not cross-react with t. gondii antibodies. blood antibody titers often peak around the onset of clinical signs and should be markedly higher than baseline values. antibody preparations also are available for identification of organisms in tissue preparations. treatment. sheep infected with sarcocystis species can be treated with salinomycin ( ppm in complete feed), monensin ( . - mg/kg po), or amprolium ( - mg/kg po). drugs such as sulfadiazine or trimethoprim ( - mg/kg im sid), pyrimethamine ( . - mg/kg po sid), and clindamycin have shown some success in treating neospora infections. these treatments are off-label and thus are governed by regulations regarding extra-label drug use. prevention. preventing contamination of feedstuffs with the feces of infected carnivores and preventing ingestion of raw meat by carnivores are most important, but these measures may not be possible in flocks handled with dogs or those living on range land. anticoccidial drugs appear to decrease the chance of clinical disease. pathogenesis. t. gondii is a protozoon parasite with a life cycle very similar to sarcocystis, except that the definitive host is the cat and that a wider range of mammalian and avian species, including humans, appear to be capable of acting as intermediate hosts. sporocysts are infective a few days after passage in cat feces, and most ruminants are infected by eating feed contaminated with cat feces. people can become infected by ingesting raw meat or milk from infected animals. abortion, stillbirth, and neonatal death are the most common forms of clinical disease in sheep and goats, and toxoplasma should be considered one of the most common causes of perinatal losses in small ruminants (see chapter ) . abortion usually occurs during the final month of pregnancy. fever, vasculitis-induced disease, and neurologic disease are less common manifestations. clinical signs. beyond abortion, clinical disease is rare in adults and resembles systemic sarcocystosis. clinical signs include fever, dyspnea, depression, and anorexia. neurologic signs are more common than with sarcocystis infection, especially in lambs and kids infected in utero. diagnosis. no specific laboratory abnormalities are associated with toxoplasmosis. nodular lesions similar to sarcocysts may be seen in various tissues, including the brain. aborted or stillborn fetuses may appear normal except for histologic lesions in the brain, liver, or lung, but more commonly fetuses are macerated. the placenta is usually abnormal, with gross and microscopic evidence of necrosis of the cotyledons. microscopic identification of the organism in body tissues is the most common means of diagnosis. serologic tests also are available. treatment and prevention. drugs similar to those used to treat neospora may be effective against toxoplasma. preventing contamination of feeds with cat feces and preventing ingestion of dead animals by cats are the most important ways of stemming the spread of this organism. both methods are likely to be difficult in most flocks. direct spread from one animal to another is rare. clinical signs. bluetongue disease has two different manifestations-reproductive problems (see chapter ) and acute vasculitis of several organ systems. with vasculitis, a spiked fever often precedes depression, anorexia, and rapid weight loss. leukopenia is present. affected animals may develop edema of the lips, tongue, throat, ears, and brisket. other signs include excessive salivation and hyperemia or cyanosis of the oral mucosa, including the tongue (hence the name bluetongue). affected sheep often produce profuse serous nasal discharge that soon becomes mucopurulent and produces crusts and excoriations around the nose and muzzle. oral lesions progress to petechial hemorrhages, erosions, and ulcers. pulmonary edema is often severe, and pneumonia may develop. skin lesions can progress to localized dermatitis. affected sheep may exhibit stiffness or lameness because of muscular changes and laminitis. cyanosis or hemorrhagic changes of the skin of the coronet can extend into the horny tissue. after recovery, a definite ridge in the horn of the hoof may be present for many months. in severe cases, the hoof sloughs. mortality varies widely. in africa, the virus is much more virulent than in the united states, and mortality ranges from to %. the reproductive or teratogenic form of the disease varies greatly with strain, host, and environmental factors. teratogenic effects include abortions, stillbirths, and weak, live "dummy lambs." congenital defects may include hydranencephaly. diagnosis. in parts of the world where the disease is common, the diagnosis is usually based on clinical signs alone. the virus can be isolated from blood, semen, or tissues (spleen and brain from aborted fetuses). viral isolation from blood obtained during the viremic state is the most definitive means of diagnosis. serologic evaluation involves two types of viral antigen groups called p and p . the former is found in all bluetongue viruses, and the latter determines the serotype. sera are commonly tested with complement fixation, agar gel immunodiffusion (agid), or one of several elisa tests. a competitive elisa is considered the best serologic test for detecting group antibodies to bluetongue virus. a direct fluorescent antibody test is available. molecular tests (e.g., pcr) for bluetongue have recently become available and are extremely sensitive and specific. they can be useful for distinguishing serotypes. other clinicopathologic signs that aid in diagnosis include leukopenia during the early febrile stage of the disease and an increase in serum ck corresponding to the latter phase of muscle stiffness and lameness. treatment. treatment is non-specific and consists of nursing care. because of the reluctance of animals to eat, they should be fed a gruel of alfalfa pellets by stomach tube or encouraged to eat soft feeds and green grass. broad-spectrum antimicrobials are often used to treat secondary pneumonia and dermatitis. animals should be kept on soft bedding with good footing. water and shade should be readily available. nsaids are commonly used. prevention. the culicoides vector is difficult to eliminate, so animals should be kept indoors during periods of peak gnat activity (dusk and early evening). owners should attempt to eliminate gnat breeding grounds such as overflowing watering troughs and shallow septic systems and should limit exposure of sheep to gnats with the use of repellent sprays. modified live vaccines based on local strains and serotypes are available in some parts of the world. some cross-protection among serotypes does occur. the vaccine should be administered at least weeks before breeding season to prevent teratogenic effects. vaccinated breeding rams may have a slight risk of decreased fertility. lambs can be vaccinated in the face of an outbreak. pregnant animals cannot be vaccinated with modified live vaccines. sheep that have recovered from an attack of bluetongue are solidly resistant for months to infection by the same viral strain and to some other viral types. active immunity in sheep requires both humoral and cellular immunity. etiology. epizootic hemorrhagic disease virus (ehdv) is an orbivirus belonging to the family reoviridae. the virus is structurally related to bluetongue virus, and the pathogenesis and clinical signs of disease resulting from these two viral infections are very similar. at least seven distinct serotypes of ehdv are recognized, although formal classification of serotypes has yet to be finalized. only two serotypes (ehdv and ehdv ) have historically circulated throughout north america, and those serotypes are largely considered to be endemic in almost all areas of the united states, with the exception of the northeast and arid areas of the southwest. however, in , ehdv was isolated from surveillance efforts in dead white-tailed deer. since then, ehdv has been increasingly identified from both surveillance samples and clinical cases and is also believed to be endemic in several regions. pathogenesis. epizootic hemorrhagic disease (ehd) is a noncontagious disease that is transmitted by the culicoides biting midges. culicoides sonorensis is the primary vector of ehdv in the united states, although other species are also suspected to transmit the disease based on the geographic distribution of clinical cases, although this has yet to be formally shown. due to the vector-borne route of transmission, peak incidence of the disease is closely associated with peak vector population, namely, in the late summer and fall of the year. although capable of infecting a wide range of wild and domestic ruminants, ehdv is largely a pathogen of wild cervids, particularly white-tailed deer. episodes of clinical disease are less common in mule deer, pronghorn antelope, and bighorn sheep and have lower morbidity and mortality. sheep are only rarely infected with the virus and goats appear to be resistant to the virus. cattle are commonly infected based on seroprevalence surveys, but overt clinical disease is uncommon. as a rule, infection in livestock is usually asymptomatic except for periodic epidemics. the last major ehd epidemic in the united states occurred in and affected a variety of captive and wild ruminant species. in endemic areas, seroprevalence in cervids and other ruminants is high, but clinical disease is not commonly seen. conversely, where seroprevalence is low, introduction of the virus results in widespread infection, where morbidity and mortality can reach % and %, respectively. following transmission of the virus by biting midges, ehdv replicates in the endothelial cells of the lymphatics surrounding the site of the bite. a primary viremia allows for systemic spread of the virus and secondary replication in lymph nodes throughout the body and the spleen. viremia is important for disease propagation and generally lasts no more than weeks following infection, although the virus can occasionally be isolated from deer infected days previously. antibodies to ehdv are first detected to days following infection but are not always capable of completely neutralizing the infection. thus, it is possible to find both neutralizing antibodies and live virus in the same animal. passive antibodies in fawns can be found up to approximately months of age. as in adults, antibodies in fawns may not protect from infection but generally protect from severe clinical signs. clinical signs. clinical disease in white-tailed deer can be peracute, acute, or chronic. the course of the peracute syndrome of diseaseis relatively short, with death often occurring within hours of infection, with or without the presence of clinical signs. when present, clinical signs include severe edema of the head and neck, swelling of the tongue and conjunctiva, anorexia, fever, weakness, and respiratory distress. hemorrhagic diatheses are not present antemortem but may occur after death. in contrast, in the acute form of the disease, the clinical signs of the peracute form are accompanied with bleeding throughout body tissues (figure . a, b) . ulcers may be evident in the oral cavity and throughout the upper gastrointestinal tract, forestomachs, and abomasum. case fatality rates are high for both the peracute and acute forms. deer that recover after several weeks of illness are said to suffer from the chronic form of the disease. signs of previous illness may include breaks or rings in the hoof horn due to interrupted growth and synthesis leading to lameness, sometimes severe. ulceration and scarring of the rumen and gastrointestinal tract may result in loss of body condition despite a seemingly normal appetite and ample nutrition. widespread evidence of vasculitis may be observed histopathologically. diagnosis. the gold standard for ehdv diagnosis is virus isolation. demonstration of neutralizing antibodies to ehdv reference strains is evidence of previous infection but may be of limited value in endemic areas where seroprevalence levels are expected to be high. also, all potentially suspected serotypes must be used when testing the sample, thereby increasing the time and cost involved with the test. continued research and refinement of molecular techniques, including pcr, are ongoing and are attractive due to the short turnaround times and the potential for high throughput of samples. however, it is important to remember that a positive result using molecular techniques does not equate to the presence of infectious virus, and thus, interpretation of results must be done with caution. control. control of ehd is difficult and relies on a combination of disease surveillance, vector control, and potentially, vaccination. eradication of vector-borne diseases from endemic areas is difficult and time-consuming, and thus, disease control is likely more attainable than strict eradication. vector control is more important in the late fall and summer, when populations are at peak levels and viral transmission is more likely. midge-proofed housing and the treatment of animals with pyrethroid insecticides have been attempted but may be logistically challenging and have yet to have been demonstrated efficacious. vaccine availability in north america is limited, but inactivated autogenous vaccines have been developed from isolates obtained from ill or recently diseased animals. autogenous vaccines are tested for purity but not necessarily for efficacy. vaccine usage must be approved by the u.s. department of agriculture prior to administration. etiology. peste des petits ruminants (ppr) is an acute or peracute, febrile, often fatal disease of ruminants caused by a virus in the family paramyxoviridae and genus morbillivirus. sheep are less susceptible than goats and white-tailed deer. cattle are only subclinically infected, and some wild ungulates, as well as camels, appear to suffer the occasional epizootic. the virus (pprv) is serologically related to the virus that causes rinderpest. geographically, the virus is found throughout northern africa, the middle east, and adjacent regions of asia, with possible movement into southern africa and europe noted. pathogenesis. the main route of infection is respiratory, and ppr is spread by airborne droplets. all secretions and excretions of infected animals are contagious throughout the course of the disease, but no carrier state exists. the virus targets lymphoid tissue. lymphocytes are destroyed in germinal centers in lymph nodes, peyer's patches, tonsils, splenic corpuscles, and cecal lymphoid tissue. immunosuppression results from lymphoid destruction. lymphocytes are partially replaced by plasma cells, macrophages, an eosinophilic acellular matrix, and occasionally neutrophils. the epithelial lining of the mouth and digestive tract is highly vulnerable to the pprv. with the loss of the alimentary tract mucosa, weight loss and diarrhea become severe. the incubation period is usually to days, with up to days possible. clinical signs. the clinical disease produced by pprv in sheep and goats closely resembles that of rinderpest, but the course is much more rapid. with the acute form, sheep and goats typically display an abrupt rise in temperature to ° to ° f ( °- ° c). within a few days, infected animals develop nasal and lacrimal discharge, depression, thirst, anorexia, and leukopenia. congestion of the conjunctival and other mucous membranes occurs, followed by serous and mucopurulent exudates. sheep and goats develop oral erosions with necrotic foci, which results in excessive salivation. diarrhea that may be profuse but rarely hemorrhagic develops within to days and is accompanied by abdominal pain, tachypnea, emaciation, and severe dehydration. bronchopneumonia, particularly that caused by pasteurella spp., may be a terminal • fig. . a. the lungs of the adult pen-raised, white-tailed deer, have been retracted to reveal to ecchymoses on the ventral surface of the "ribcage." petechiae and ecchymoses can occur anywhere within the carcass in cases of epizootic hemorrhagic disease (ehd), but common locations are on the epicardium, on the pleural surface the ribs, subcutaneously, and on the surface of the spleen. b. ecchymoses over the surface of the reticulum (bottom right of photo) and the surface of the rumen (left side of photo). in addition to ehd, this deer also had bronchopneumonia (fibrin overlying consolidated lung can be seen in the far right of photo). (courtesy dr. kelley steury, auburn, al.) a b sequela. death usually occurs to days after the onset of fever. pregnant sheep or goats with ppr may abort. diagnosis. a presumptive diagnosis of ppr can be made on the basis of clinical, pathologic, and epizootiologic findings. the diagnosis can be confirmed by isolating the virus from blood or tissues, including lymph nodes, tonsils, spleen, and lung. immunocapture elisa or pcr may be used to detect infection several days before the development of clinical disease. most serologic tests (complement fixation or agid) cannot differentiate between ppr and rinderpest. characteristic postmortem findings include necrotic stomatitis that is generally confined to the inside of the lower lip and adjacent gum, the cheeks near the commissures, and the ventral surface of the free portion of the tongue. abomasal erosions are often present. in the small intestine, peyer's patches are markedly affected, particularly in the first portion of the duodenum and terminal ileum. the large intestine may be severely affected. lesions occurring near the ileocecal valve, at the cecocolic junction, and in the rectum are often described as zebra stripes that indicate areas of congestion along the folds of the mucosa. treatment and prevention. infection with pprv has no specific treatment. mortality can be reduced by supportive care, including the administration of antimicrobial and antiinflammatory agents, as well as nutritional support. in the united states, state and federal veterinarians should be notified if pprv is suspected. methods used to eradicate rinderpest are useful in the eradication and control of ppr. all sick sheep and goats and those exposed should be slaughtered and disposed of by burning, burying, or rendering. the premises should be decontaminated, and the area quarantined. sheep and goats can be protected against ppr by immunization with rinderpest vaccines or by the simultaneous administration of ppr hyperimmune bovine serum and virulent pprv. pathogenesis. louping ill is a tickborne disease caused by a flavivirus. it affects mainly lambs but occasionally also affects other livestock species and infrequently affects deer, camelids, and humans. transmission is most common during tick season, and ixodes ricinus is thought to be the most important infective host. many sheep clear the infection after a few days of fever and viremia, but others develop severe, fatal viral encephalitis. the virus is shed in many secretions, including milk, which is an important source of infection for other animals (and humans). the severity of the disease depends on herd immunity because previous exposure gives long-lasting immunity. colostrum from immune females is protective for the neonate. high antibody titers also appear to shorten the duration and level of viremia and thereby prevent invasion of the central nervous system. naïve flocks may have fatality rates as high as %. clinical signs. high biphasic fever, anorexia, and depression are seen in most infected sheep. lambs may die quickly before illness is noted. some sheep also develop central neurologic signs, including hyperexcitability, muscle tremors, and rigidity. abnormal coordination and muscle activity may cause sheep to move with a bounding gait (hence the name louping ill). diagnosis. the condition has no characteristic gross lesions. microscopic examination of animals with neurologic signs reveals evidence of viral meningoencephalitis. diagnosis is made by history (based on location, signs, and time of year), the identification of characteristic lesions, virus isolation, or fluorescent antibody staining of fresh brain tissue. a demonstrated increase in specific antibody titers in survivors strongly suggests the presence of this infection. prevention. vaccines are available in endemic areas to control infection. vector control during tick season also is important. lambing season should also be timed so that lambs have high colostral antibody protection at the time of exposure to ticks. pathogenesis. foot-and-mouth disease is caused by a highly contagious picornavirus and has been eradicated from the united states. vesicular stomatitis is caused by a rhabdovirus and is intermittently eradicated from the united states. both diseases are highly contagious, nearly indistinguishable from each other clinically, and reportable. foot-and-mouth disease has a broad host range that includes most hoof stock (including pigs but not horses) and several other mammalian species. vesicular stomatitis also affects many species of hoof stock, including both pigs and horses. sheep and goats are relatively less susceptible than cattle, particularly to vesicular stomatitis. the viruses are spread by aerosol and mechanical vectors and primarily colonize skin or mucous membranes. milking machines, flies, birds, and humans all may be important mechanical vectors. vesicular stomatitis tends to remain at the site of infection, and colonization is facilitated by damage to the skin. oral mucous membranes, coronary bands and interdigital skin, and teat-end skin are common sites of lesions. vesicular stomatitis outbreaks in the united states tend to occur in the summer or fall and end with the first killing frost. viremia plays more of a role with foot-and-mouth disease. the virus is present in most body tissues and fluids in infected animals and can be transmitted through milk, meat, bone, and hide products; semen; equipment that pierces the skin; and biting arthropods. it also tends to spread through the circulation from the site of infection to other susceptible tissues, including the sites of vesicular stomatitis, as well as to the nasal cavity, mammary glandular epithelium, and ruminal pillars. the basic lesion for both diseases are the vesicles that form in the oral cavity and on the teats and coronary band. the vesicles quickly rupture and may not be visualized before forming erosions. ruptured vesicles leave deep erosions on the skin or mucous membranes and appear to cause pain. tissue damage and inflammation are often compounded by secondary bacterial infection, which can cause greater morbidity and mortality than the original viral infection. morbidity is related to feed refusal, increased recumbency, and secondary infections of the mouth, udder, and feet. clinical signs. sheep and goats usually develop minor lesions, if any, and are more important in many outbreaks as transport or multiplying hosts than as primary clinical cases. however, identification of lesions should raise suspicion of this disorder. in the worst cases, vesicles, erosions, and ulcers are seen at target sites. they may appear mildly inflamed and erythematous; if they are infected, they may appear severely inflamed with hemorrhage and necrosis. other signs vary according to the location and severity of the lesions. lingual and buccal lesions cause salivation, dysphagia, and feed refusal. foot lesions, which are the most common clinical manifestation in small ruminants, cause lameness and recumbency. teat lesions cause reluctance to be milked or nursed and a decrease in production. fever also may be seen early in the disease, when vesicles are most apparent. the fever then usually abates, and vesicles are replaced by erosions or ulcers. abortion may occur, especially with foot-and-mouth disease, and is probably related to the fever rather than to fetal infection. the disease is usually self-limiting; most animals recover within to weeks. shedding of the virus causing vesicular stomatitis is thought to subside soon after healing of lesions. foot-and-mouth disease virus may be shed for as long as months, and all body secretions and tissues should be considered contagious, including milk, semen, meat, and offal. both viruses have zoonotic potential and cause a disease in humans that resembles mild influenza. the diseases are self-limiting, but people can shed the viruses in sufficient quantities to infect other animals. diagnosis. no characteristic clinicopathologic changes are reported for either virus. gross lesions resemble those seen before death and include vesicular, erosive, and ulcerative lesions of the mouth, feet, and teat ends; foot-and-mouth disease also causes lesions of the mammary gland and ruminal epithelium. microscopic findings include hydropic degeneration of cells of the stratum spinosum of the epidermis without inclusion bodies. secondary bacterial infection may lead to deeper ulcers and complicate identification of the viral etiology of these lesions. myocarditis lesions may be seen with some forms of foot-and-mouth disease. a presumptive diagnosis may be made by identifying characteristic lesions during a season and in an area at risk for one of these infections. in north america, bluetongue should be considered as an important differential diagnosis for ulcerative oral lesions in sheep. a confirmed diagnosis of foot-and-mouth disease is achieved by a combination of virus isolation (from vesicles), ihc, and serology by regulatory officials. identifying the source of infection also is very important. diagnosis of vesicular stomatitis is achieved by complement fixation or fluorescent antibody staining of virus in vesicular fluid or detection of a rise in antibody titers. flocks with either of these diseases in the united states are subject to quarantine and possible destruction (especially for foot-and-mouth disease). prevention. meticulous personal hygiene and avoidance of contact with new animals are important during outbreaks to prevent spread between flocks. vaccines against foot-and-mouth disease are available in many parts of the world, but not in the united states. most nations slaughter or quarantine affected animals. vaccines against vesicular stomatitis are available and are most commonly used if the risk of outbreak is high, but vaccination does not prevent infection or shedding. good hoof and teat care and soft feeds may help prevent spread of the virus by providing a healthy, intact barrier against invasion. pathogenesis. sheep and goat pox are caused by two closely related poxviruses. some strains are infective to both sheep and goats; most are species specific. they are maintained in populations by infected animals, and transmission occurs by aerosol or direct or indirect contact. flies may play an important role as mechanical vectors in some flocks. viruses remain infective in the environment for as long as months. after infection, viremia and inflammation of the oral, nasal, and ocular mucous membranes occur. erythematous papular pox lesions appear a few days later. severity varies according to strain pathogenicity, breed susceptibility, and immune status. mild infections are characterized by lesions concentrated in the non-wooled or hairless regions of the skin. severe infections produce lesions throughout the oral cavity, respiratory tract, and peritoneal cavity. secondary infection is common with the severe form and mortality is high. if the animal survives, lesions heal in to weeks. both diseases have been eradicated from the united states and are reportable. people can develop mild disease on exposure to these viruses. clinical signs. fever, inappetence, conjunctivitis, and upper respiratory signs are seen in the initial stages. pox lesions are visible shortly thereafter. secondary infection can lead to a variety of more serious signs indicative of respiratory disease, sepsis, and shock. diagnosis. characteristic pox lesions are highly suggestive of this disease. microscopic analysis reveals eosinophilic intracytoplasmic inclusion bodies, acantholysis, and pustule formation within the epidermis and occasionally the dermis. viral particles may be seen on ultrastructural examination. gross and microscopic lesions are characteristic with the severe form, but mild disease may produce mild lesions that are difficult to differentiate from other viral diseases that cause oral proliferative or ulcerative lesions. virus can be isolated from blood or tissues (mainly skin) during the acute viremic stage and identified by antibody staining of more chronic lesions. serologic tests are available to detect rising titers in convalescent animals. treatment and prevention. no specific treatment is available for sheep or goat pox. antibacterial drugs may be useful to treat secondary infection. judicious use of insecticides and confinement of affected animals may prevent spread. vaccines are available in some countries, but not in the united states. infected flocks are placed under quarantine or destroyed in regions where the diseases are not endemic. these viruses are difficult to eradicate from flocks because of their environmental persistence and the constant supply of susceptible hosts. caprine arthritis-encephalitis virus (caev) is an enveloped, singlestranded retrovirus in the lentivirus genus. like other retroviruses, caev integrates into the host chromosomal dna before replicating. the virus is able to remain latent or undergo sporadic bouts of productive viral replication. caev is closely related to ovine lentiviruses. clinical signs. clinical disease may be evident in only % of goats from a caev-infected herd at any given time. as many as % of seropositive goats may be clinically normal. caev produces four clinical syndromes: encephalomyelitis, arthritis, interstitial pneumonia, and indurative mastitis. the pattern of disease usually varies with age. arthritis is generally seen in sexually mature goats, whereas encephalomyelitis is generally seen in kids to months old. interstitial pneumonia and indurative mastitis are more common in adult goats. some goats suffer from a wasting disorder characterized by poor body condition and rough hair coat. diagnosis. a presumptive diagnosis of caev can be made on the basis of history and clinical signs suggestive of one or more of the syndromes. in general, elisa tests are better for detecting disease in an individual animal because the sensitivity of the test is higher than that of the agid, whereas the agid is better for herd screening that requires high specificity. with the agid test, false negatives may occur in goats that have not yet seroconverted to recent infection. individual goats may take months or years to seroconvert or may never do so. parturition or advanced stages of disease also may contribute to a false-negative result. false positives may occur in goats younger than days old that have colostral antibodies. for this reason, it is often suggested that kids be at least months old before they are tested. pcr testing has high specificity and sensitivity and can detect infection within a day of exposure. other less commonly used tests include a western blot to detect antibodies and a northern blot to look for mitochondrial rna. because of the limitations in interpreting serologic results, caev-induced disease can only be definitively diagnosed by identification of characteristic lesions from examination of biopsy specimens or postmortem viral isolation. treatment. no specific treatments are available for any of the syndromes associated with caev. young goats suffering from encephalomyelitis may benefit from physical therapy if they are recumbent, and bottle feeding may help maintain hydration and caloric intake. antibiotics may be beneficial to goats affected with interstitial pneumonia or mastitis if secondary bacterial infection is present. generally, the prognosis is poor for the encephalitic form and guarded for the other forms. prevention. prevention of caev is crucial because infection is lifelong. infected colostrum and milk are the most important sources of infection. newborn kids should be prevented from ingesting colostrum from infected does and should instead be fed pasteurized goat's milk or milk from caev-negative goats. all goats in a herd should undergo serologic testing twice yearly; seropositive goats should be segregated or culled to prevent direct contact between infected and uninfected animals. ovine progressive pneumonia (opp) is an ultimately fatal retroviral disease that causes chronic, progressive, debilitating inflammatory conditions of the lungs (united states) and central nervous system (other parts of the world). it also is called maedi-(maeði is icelandic for "shortness of breath") visna (meaning "wasting"). the virus is a member of the lentivirus genus of retroviruses and is closely related to caev. recombination between opp and cae viruses has been observed. the virus primarily affects sheep and rarely goats and has been identified worldwide, except in australia and new zealand. the disease has a long incubation period and protracted clinical course. pathogenesis. only sheep older than years of age are affected by opp virus (oppv). the virus is spread by direct contact, probably in respiratory and salivary secretions, and by excretion in the milk and colostrum. transplacental transfer is of minor importance. virus is excreted by animals that exhibit clinical signs and asymptomatic animals. infection is established in the monocyte and macrophage cell line and spread by these cells to the lungs, lymph nodes, choroid plexus, spleen, bone marrow, mammary gland, and kidneys. like caev, oppv evades the cellular and humoral immune system of the host by incorporation of its provirus in host dna, low-grade replication of virus only when monocytes differentiate into macrophages (restricted replication), and production of antigenic variants that are not neutralized by existing antibodies. continual antigenic stimulation of the host by low-grade replication of oppv results in chronic inflammation and resultant lymphoid proliferation in various target tissues. the virus may prevent b lymphocytes from differentiating into plasma cells in lymph nodes and may thereby impair immunoregulation. seroconversion occurs within to weeks after infection. clinical signs. in the united states, serologic surveys reveal infection rates of between and % but rarely is more than % of a flock lost to oppv. icelandic, texel, border leicester, and finnish landrace appear to be susceptible sheep breeds. more resistant sheep breeds include rambouillet, suffolk, and columbia. various clinical syndromes are associated with oppv and include wasting (thin ewe syndrome), dyspnea occasionally with a dry cough, pneumonia, mastitis ("hard bag"), posterior paresis, arthritis, and vasculitis. in north america, pneumonia and indurative aseptic mastitis are common sequelae of infection. coinfection with the jaagsiekte virus (the cause of pulmonary adenomatosis) worsens respiratory signs. visna, the neurologic form, is more common in goats. over the course of up to a year, subtle signs such as a head tilt or hindlimb weakness progress to gross incoordination, whole body tremors, and rarely more profound cranial nerve signs. diagnosis. a presumptive diagnosis can be made on the basis of clinical signs, poor response to treatment, characteristic postmortem findings, and serologic testing. definitive diagnosis requires pcr or isolation of the virus from wbcs (buffy coat of whole blood sample) or tissues. less expensive and faster serologic tests include agid, elisa, and an indirect immunofluorescence test. the agid test is frequently used as a flock screening test, but the elisa is more sensitive on an individual basis and can detect antibodies earlier in the course of the disease. as with caev, false negatives and false positives are possible. characteristic postmortem lesions include generalized wasting and firm, noncollapsing lung or firm, mottled mammary glands, both with regional lymphadenopathy. microscopic evaluation of those tissues reveals interstitial non-septic, mononuclear cell infiltrates, although these may be complicated by secondary infections. histopathology of nervous tissue reveals meningoleukoencephalitis. treatment. no effective treatment is available for oppv. supportive therapy that includes appropriate husbandry and control of secondary infection with antibiotics may prolong life for a few weeks or months but, ultimately, the disease is fatal. because of the poor prognosis and risk of exposure of naive animals to clinical disease, long-term treatment is not recommended. prevention. the only known method of preventing oppv infection in a flock is to prevent exposure to the virus. management practices that help decrease the incidence of horizontal transmission include disinfection of milking equipment, dehorning instruments, and tail docking and castration tools before use and between animals. contaminated feed and water also are potential routes of infection and should not be shared among infected and uninfected animals. serologic testing and separation or culling of seropositive animals may help reduce infection. although oppv can readily be isolated from ewe colostrum, colostral transmission of oppv has not been definitively established. however, many prevention guidelines recommend that offspring from infected dams be separated from the dam before they nurse and then be fed cow colostrum and artificially reared. quarantine and serologic testing of flock additions before placing them with the current flock and purchase of sheep only from oppv-free flocks are important to prevent the introduction of new infections. because of the potential cross-species spread, all precautions taken for sheep also apply to contact goats. serologic testing should be performed at least annually in a flock until two consecutive negative test results are obtained. border disease virus (bdv) is in the genus pestivirus and family flaviviridae, which also includes the two genotypes of bovine viral diarrhea virus (bvdv) and classical swine fever virus. it rarely causes disease in adults and is most important as a cause of in utero infection of lambs and kids. the condition gets its name from the fact that it was first reported in sheep along the welsh border of the united kingdom. other names such as "hairy shakers" and "fuzzy lamb disease" refer to some of the clinical signs seen in affected newborns. it is important to recognize that although bdv is genetically distinct from the two types of bvdv, sheep and goats also are susceptible to some strains of bvd. pathogenesis. horizontal transmission of bdv occurs through contact with secretions and excretions of body fluids and tissues from infected animals. the virus crosses intact mucous membranes and can spread rapidly through a flock. the major reservoir is the persistently infected sheep or goat. these reservoirs are usually asymptomatic, congenitally infected, and often seronegative animals that shed large quantities of virus. these may be residents of a flock with an ongoing problem or bought in as replacement animals to a naïve flock. some cross-infection from other species is possible, particularly from cattle. adult, immunocompetent sheep rarely show any signs of acute infection. however, if a pregnant ewe or doe is infected, the virus may be transmitted vertically to the embryo or fetus. depending on the stage of gestation, embryonic or fetal infection may have different outcomes ranging from embryonic reabsorption to normal birth. these infections are the most important aspect of border disease. the major organ system targeted by bdv is the fetal central nervous system. the hallmark lesion is hypomyelination, or degeneration of oligodendroglial cells. three factors contribute to this lesion. the first is direct viral damage. the second is viral-induced inhibition of the thyroid gland that causes decreased secretion of thyroid hormones. in the absence of these hormones, a resultant lowered concentration of a specific nucleotide in the central nervous system also contributes to the hypomyelination. the third factor is altered immune function. the virus causes the host to produce a virus-specific delayed hypersensitivity reaction that causes inflammation in the central nervous system. it also causes immunosuppression. death often results from opportunistic conditions such as parasitism, diarrhea, and bronchopneumonia. clinical signs. clinical signs depend on the time during gestation when the fetus or embryo is exposed to the virus. clinical signs also may vary in severity from animal to animal because different fetuses develop competent immune systems at different times. if the fetus or embryo is exposed to the virus within days of conception, it dies and is resorbed or aborted. these losses are not usually noticed by the flock manager. the principal manifestation in the flock is a large number of open ewes and a small lamb crop. infection of the fetus between days and of gestation causes damage to rapidly growing systems such as the skin and nervous, lymphoid, thyroid, and skeletal systems. congenital malformations are seen at birth. lambs have abnormal fleece characteristics (hairy rather than woolly in consistency), small stature, domed heads, shortened legs, and dark pigmentation of the skin, particularly on the dorsal aspect of the neck. the lamb may exhibit tonic-clonic tremors ("hairy shakers") when awake, which may prevent standing or suckling. most of these lambs die within a few days of birth. if they survive, the hair changes disappear in to weeks and the central nervous system signs resolve by weeks. goats infected at this time have similar symptoms except that they rarely exhibit hair coat changes. if kids are infected before day of gestation and are still viable, they may become persistently infected and immunologically compromised. they are small at birth and generally weak. typical outbreaks of border disease cause abortions and birth of weak lambs in the first year as the virus rapidly spreads throughout a susceptible flock and then insignificant losses in the succeeding years as adult sheep develop immunity. however, if new naïve ewes are introduced in the flock, substantial losses may occur in perpetuity. diagnosis. border disease viral antigens can be demonstrated in abomasum, pancreas, kidney, thyroid, skin, and testicle tissues from aborted fetuses and persistently infected animals using fluorescent antibody tests. however, ihc on ear notch samples is not considered as reliable for detecting persistently infected small ruminants as it is for cattle. the virus can be isolated, or viral antigen detected by elisa, from serum, heparinized whole blood, and tissue taken from brain, spinal cord, spleen, and bone marrow from affected lambs. whole blood is better than serum if colostral antibodies are likely to be high; serum is an adequate sample in neonates and juveniles that have not suckled. antibodies to the virus may be quantified by serum neutralization, agid, and complement fixation with hyperimmune bvd antiserum. serologic tests are useful to detect exposure in lategestation (after day ) neonates and unvaccinated animals but may be confounded by colostral antibodies in suckling neonates, previous exposure, and vaccination in older animals. any titer in a presuckling neonate indicates in utero exposure, whereas a serum neutralization titer of : to : suggests infection in adults. the presence of specific antibodies in the cerebral spinal fluid suggests bdv infection. negative presuckling serologic tests do not rule out exposure because persistently infected lambs tend to be immunotolerant to the bdv and therefore are born without an antibody titer. these animals may subsequently develop a titer that is indistinguishable from that of a normal animal. although persistently infected animals do not respond immunologically to the strain of the virus they carry, they may respond to other strains of the virus, including vaccine strains. as with bvd, pcr assays are gaining popularity for the detection of bdv in fluids and tissue samples. these assays appear to be superior to other techniques, except in autolyzed tissues. realtime pcr may also be used to differentiate bdv from bvd and to type isolates. gross postmortem findings include hydranencephaly, porencephaly, microcephaly, cerebellar hypoplasia, abnormal rib curvature, brachygnathia, doming of the frontal bones of the skull, narrowing of the distance between the orbits, shortening the crown-to-rump length, shortening of the diaphyseal length, retention of secondary hair fibers, and abnormal skin pigmentation. the major histopathologic changes include hypomyelination and hypercellularity of the white matter. glial cells appear normal. treatment. no treatment is available for border disease infection. supportive care may include assistance in nursing and standing for affected lambs, provision of good bedding and solid footing, and treatment of secondary opportunistic infection. prevention. control is primarily achieved by eliminating persistently infected carrier animals from the flock and preventing the addition of new carrier animals. this is easiest in a closed flock but especially difficult in small ruminant flocks because of the frequent desire to import new genetics. to identify carriers, virus isolation must be performed on every animal in the flock; carrier animals must be culled. additionally, all unborn animals must be considered potential carriers and should be tested at birth. an alternative solution in hobby flocks is to arrest breeding activity until all animals have been shown to be free of infection. new animals should be quarantined and tested before admission to the flock. herd screening with the ear skin biopsy test using fluorescent antibody staining to detect virus is less expensive and more convenient than the whole blood virus isolation test. the role of vaccination in preventing infection is still unclear. no vaccine against bdv is available, but some reports suggest that bvdv vaccines for cattle may be helpful for sheep at risk. however, these vaccines have proven to be more effective at preventing clinical disease in vaccinated animals than in preventing in utero infection because they do not prevent transient viremia. vaccination decreases viremia and fetal infection but does not eliminate them. therefore, vaccines play a role in decreasing economic loss but do not replace culling of carrier animals as the major method of control. another member of the slow infection group of diseases of small ruminants is scrapie. it is an afebrile, chronic, progressive degenerative disorder of the central nervous system of sheep and occasionally of goats (see chapter ) . scrapie is caused by a prion and, as such, is one of the transmissible spongiform encephalopathies. sheep (and goats and mouflon to a lesser degree) are the natural hosts for scrapie. clinical signs often do not usually appear until animals are years old, and animals as old as years may exhibit clinical disease. both vertical and horizontal transmission have been demonstrated experimentally in sheep and goats. abnormal scrapie protein has been identified in milk, urine, and seminal plasma of sheep up to months prior to the development of clinical signs. also, new evidence from deer with chronic wasting disease, a similar disorder, suggests that infective prions are excreted in the saliva and feces well before the development of clinical signs. these new revelations may help explain horizontal transmission of infection. clinical signs. the onset of scrapie is insidious. initially, sheep show subtle changes in behavior such as mild apprehension, staring or fixed gaze, failure to respond to herding dogs, and boldness around humans. several months later, the animals become intolerant of exercise and develop a clumsy, unsteady gait and floppy ears. later, the sheep develop itchy skin that causes them to rub themselves excessively against firm, immobile objects (origin of the name scrapie). this leads to excoriations and wool damage. there is a general decline in body condition and coordination. diagnosis. histologically, the only consistent lesions are degenerative changes in the central nervous system consisting of bilaterally symmetric vacuolation of the neurons in the brainstem and spinal cord with accompanying spongy degeneration. as a preclinical test, ihc may be performed in lymphoid tissue from the tonsils, third eyelid, or rectoanal mucosa, but none of these methods is foolproof. cwd is discussed in chapters , , and . testing for clinical anaemia caused by haemonchus spp. in goats farmed under resource-poor conditions in south africa using an eye colour chart developed for sheep validation of the fama-cha eye color chart for detecting clinical anemia in sheep and goats on farms in the southern united states validation of the famacha © eye colour chart using sensitivity/ specificity analysis on two south african sheep farms is the famacha chart suitable for every breed? correlations between famacha scores and different traits of mucosa colour in naturally parasite infected sheep breeds rumen bacteria are involved in the onset of onion-induced hemolytic anemia in sheep the role of free radicals in brassicainduced anaemia of sheep: an esr spin trapping study kale poisoning: the brassica anaemia factor hemolytic anemia in sheep fed wild onion (allium validum) copper toxicosis in sheep: a case report chronic copper poisoning in sheep. i. the relationship of methaemoglobinemia to heinz body formation and haemolysis during the terminal crisis chronic copper toxicity of ruminants copper poisoning in a flock of sheep. copper excretion patterns after treatment with molybdenum and sulfur or penicillamine evaluation of mechanisms of leptospiral hemolytic anemia fatal hemolytic anemia attributed to leptospirosis in lambs studies on eperythrozoon ovis-infection in sheep eperythrozoon ovis-a blood parasite of sheep experimental eperythrozoon ovis infection of sheep mycoplasma ovis comb. nov. (formerly eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia and concomitant anaplasma ovis infection bovine colostrum as a cause of hemolytic anemia in a lamb heinz body anaemia in lambs with deficiencies of copper or selenium maxillary lymphosarcoma in a white-tailed deer (odocoileus virginianus) large animal internal medicine effect of physical restraint and xylazine sedation on haematological values in red deer (cervus elaphus) seasonal variations in red deer (cervus elaphus) hematology related to antler growth and biometrics measurements the genetic basis and evolution of red blood cell sickling in deer one hundred two tumors in goats lymphoma classification in goats exophthalmos due to multicentric b-cell lymphoma in a goat ocular involvement of multicentric malignant b-cell lymphoma in a ewe. a case report diseases and parasites of white-tailed deer, miscellaneous publication no. . tall timbers research station colostrum composition of santa inês sheep and passive transfer of immunity to lambs effects of maternal undernutrition during late gestation and/or lactation on colostrum synthesis and immunological parameters in the offspring failure in passive transfer of immunoglobulin g to lambs: measurement of immunoglobulin g in ewe colostrums iodine supplementation of the pregnant dam alters intestinal gene expression and immunoglobulin uptake in the newborn lamb short communication: apoptosis regulates passive immune transfer in newborn kids effects of newborn characteristics and length of colostrum feeding period on passive immune transfer in goat kids a field trial evaluating the health and performance of lambs fed a bovine colostrum replacement use of a digital brix refractometer to estimate serum immunoglobulin in goat kids field methods for estimating serum immunoglobulin concentrations in newborn kids colostrum deficiency in mule deer fawns: identification, treatment and influence on neonatal mortality passive transfer of colostral immunoglobulins from ewe to lamb and its influence on neonatal lamb mortality transfer of maternal passive immunity to kids in goat herd suppurative meningitis in a -day-old formosan sambar deer (cervus unicolor swinhoei) caused by escherichia coli factors affecting igg concentration in day-old lambs effects of maternal nutrition on udder development during late pregnancy and on colostrum production in scottish blackface ewes with twin lambs the effect of colostrum source (goat vs. sheep) and timing of the first colostrum feeding ( h vs. h after birth) on body weight and immune status of artificially reared newborn lambs bovine neonatal pancytopenia and anaemia in lambs caused by feeding cow colostrum secondary lactose intolerance in a neonatal goat hypernatremia in neonatal elk calves: cases ( - ) group b rotavirus associated with an outbreak of neonatal lamb diarrhea rotaviruses associated with neonatal lamb diarrhea in two wyoming shed-lambing operations novel group a rotavirus g p[ ] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain enteric viral infections in lambs or kids suspected clostridium difficile-associated hemorrhagic diarrhea in a -week-old elk calf observations and immunohistochemical detection of coronavirus, cryptosporidium parvum and giardia intestinalis in neonatal diarrhoea in lambs and kids giardia duodenalis and cryptosporidium parvum infections in adult goats and their implications for neonatal kids case control study of diarrhoea and faecal soiling in two-to six-month-old lambs comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in cyprus fluid therapy in calves passive immunisation of neonatal lambs against infection with enteropathogenic escherichia coli via colostrum of ewes immunised with crude and purified k pili floppy kid syndrome (metabolic acidosis without dehydration in kids clostridium perfringens toxins involved in mammalian veterinary diseases first isolation of clostridium perfringens type e from a goat with diarrhea clostridial enteric diseases of domestic animals isolation and molecular characterization of clostridium perfringens from healthy merino lambs in patagonia region lamb losses associated with clostridium perfringens type a hemorrhagic bowel syndrome in dairy cattle: cases clostridium perfringens type a and beta toxin associated with enterotoxemia in a -week-old goat investigation of a syndrome of sudden death, splenomegaly, and small intestinal hemorrhage in farmed deer gastric mucormycosis in a sika deer (cervus nippon) associated with proliferation of clostridium perfringens the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves multiplex pcr method for genotyping clostridium perfringens the effect of clostridium perfringens type c strain cn and its isogenic beta toxin null mutant in goats beta toxin is essential for the intestinal virulence of clostridium perfringens type c disease isolate cn in a rabbit ileal loop model clostridial diseases vaccines for control, prevention and eradication of disease in farmed deer development and application of an oral challenge mouse model for studying clostridium perfringens type d infection enterotoxaemia caused by clostridium perfringens type d in farmed fallow deer rates of diseases and their associated costs in two colorado sheep feedlots ( - ) proportional mortality: a study of goats submitted for necropsy from goat herds in quebec, with a special focus on caseous lymphadenitis the pathology of experimental clostridium perfringens type d enterotoxemia in sheep enterotoxaemia in goats: a review diagnosis of clostridium perfringens intestinal infections in sheep and goats clinico-pathological findings of clostridium perfringens type d enterotoxaemia in goats and its hemolytic activity in different erythrocytes experimental clostridium perfringens type d enterotoxemia in goats clinical signs, treatments, and postmortem lesions in dairy goats with enterotoxemia: cases epsilon toxin is essential for the virulence of clostridium perfringens type d infection in sheep, goats, and mice clinicopathologic features of experimental clostridium perfringens type d enterotoxemia in cattle ulcerative enterocolitis in two goats associated with enterotoxin-and beta toxin-positive clostridium perfringens type d the passive protection of lambs against clostridium perfringens type d with semi-purified hyperimmune serum blackleg in deer bacterial diseases of farmed deer and bison black disease in a forest reindeer bovine vaccines and herd vaccination programs toxigenic clostridia characterization of the catalytic domain of clostridium novyi alpha-toxin first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan clostridium novyi (myonecrosis, black disease, and bacillary hemoglobinuria) and clostridium septicum (braxy) infections first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan liver and biliary system bacillary hemoglobinuria: induction by liver biopsy in naturally and experimentally infected animals bacillary hemoglobinuria in a free-ranging elk calf bacillary hemoglobinuria in dairy cows an outbreak of bacillary haemoglobinuria in sheep in india successful treatment of bacillary hemoglobinuria in japanese black cows acute abomasitis due to clostridium septicum infection in experimental sheep rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the s- s rdna spacer region suppurative abomasitis associated with clostridium septicum infection clostridial myocarditis in lambs outbreak of clostridial myocarditis in calves clostridial myositis in cattle: bacteriology and gross pathology clostridial vaccination efficacy on stimulating and maintaining an immune response in beef cows and calves failure of clostridium chauvoei vaccines to protect against blackleg prevalence of coxiella burnetti infection in wild and farmed ungulates coxiella burnetii shedding by farmed red deer (cervus elaphus) high prevalence of antibodies against chlamydiaceae and chlamydophila abortus in wild ungulates using two regional seroprevalence of leptospirosis on deer farms in new zealand growth response and shedding of leptospira spp. in urine following vaccination for leptospirosis in young farmed deer corynebacterium pseudotuberculosis paratuberculosis (johne's disease) in cattle and other susceptible species efficacy of a killed vaccine for the control of paratuberculosis in australian sheep flocks detection of a novel reassortant epizootic hemorrhagic disease virus (ehdv) in the usa containing rna segments derived from both exotic (ehdv- ) and endemic (ehdv- ) serotypes the first years ( - ) of epizootic hemorrhagic disease virus serotype in the usa review of the epizootic hemorrhagic disease outbreak in domestic ruminants in the united states peste des petits ruminants demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats key: cord- -a ie fs authors: nan title: digestive system, liver, and abdominal cavity date: - - journal: the cat doi: . /b - - - - . - sha: doc_id: cord_uid: a ie fs nan these complex pathways highlight the need to consider the whole cat and not just the cat's gastrointestinal vomiting can be defined as the ejection of part or all of the contents of the stomach and/or upper intestine through the mouth, usually in a series of involuntary spasmodic movements. the disturbances in gastrointestinal (gi) motility are coordinated with respiratory and abdominal muscle contractions and mediated by the central nervous system (cns). vomiting begins with retching, a series of brief negative intrathoracic pressure pulses that coincide with positive abdominal contractions. these pressure changes occur as a result of repeated herniations of the abdominal esophagus and cardiac portion of the stomach into the esophagus. during retching, food freely moves back and forth in the esophagus, which is now dilated because of the ingesta. ultimately, the diaphragm rapidly moves cranially, resulting in positive intrathoracic pressure that leads to expulsion of these contents. vomiting is such an active process that it seems to involve the whole cat, and so it is little wonder that it concerns owners so much. since vomiting is mediated by the cns with input and influence from just about anywhere in the body, it is important to summarize this physiology so it can be appreciated when managing clinical cases. vomiting results from stimulation of the "vomiting center," which is located in the brainstem; there are four main pathways that stimulate the vomiting center, and these are summarized below and in figure (though not all older cats have grown out of this habit). some extragastrointestinal problems, such as hyperthyroidism and renal disease are more likely to occur in older cats. most texts and references instruct clinicians to distinguish between vomiting and regurgitation, with the latter noted as being quite passive. , , in practice, it can be hard to make this distinction, because it is the author's experience that cats with esophageal disease can have quite forceful, spasmodic movements when ejecting ingesta by regurgitation-although it is also possible for regurgitation to be a passive process. given that the physiology of vomiting, as described above, results in ingesta being forced to and then evacuated from the esophagus, it is hardly surprising that it can resemble regurgitation. fortunately, regurgitation and esophageal disease do vary from vomiting in other ways! vomiting . blood and urine testing . imaging (radiography, ultrasonography) . biopsy samples . treat and manage underlying problem the decision to proceed to steps and is based on the assumption that the prior steps have narrowed down the underlying cause as gastrointestinal, pancreatic, or hepatic in origin. the important aspects of the clinical history are given in chronic kidney disease. the author has found that some cats with dental disease can gorge their food, resulting in vomiting; so, paying attention to the state of the teeth and gums is important. of course, some cats have multiple problems, and correction of dental disease may not resolve vomiting if there is another process. in the examination, it is also important to note consequences of both the underlying process and the vomiting itself; these include the demeanor of the cat, hydration status, and abdominal pain. the physical examination findings, together with the clinical history, help determine the next appropriate steps. well cats that are not continually vomiting and are appropriately hydrated, with no other specific signs, may be treated as outpatients by fasting them for hours, then returning to food with a bland diet, such as plain cooked chicken or commercial, low-residue prescription diets designed for this purpose. follow-up is important to ensure signs do not progress. cats with nonspecific signs may require supportive care with subcutaneous or intravenous fluids and perhaps analgesia (with opioids). if clinical signs do not resolve, the pursuit of a specific diagnosis should be attempted. the practitioner must ask the following important questions: • are ancillary tests appropriate? • is supportive care necessary? • are any medications required? routine serum/plasma biochemistries, hematology, urinalysis, and total thyroxine (t ) (for older cats) testing is not only important to distinguish primary from secondary gastrointestinal disease but to look for consequences of vomiting that may need to be addressed, such as hydration status and electrolyte abnormalities. careful interpretations should be made. severe azotemia, even with hyperphosphatemia, can occur as a result of primary gastrointestinal disease, and the distinction from renal disease usually requires an assessment of urine specific gravity. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity (fpli) tests are useful markers of intestinal and pancreatic disease, , , , but it is important to note that they mostly do not give a precise diagnosis. more detail about the utility of these tests is noted below in the section approach to the cat with diarrhea. is usually preceded by the cat licking its lips, salivating, or making attempts to swallow. regurgitated ingesta is often in a tubelike structure and if undigested can be covered with frothy saliva. partially digested food suggests vomitus, and the presence of bile or digested blood confirms this. it is important to determine if the cat vomits regularly. many owners have seen their cats vomit on a regular basis with no evidence of the cat being unwell, and this is noted frequently in the veterinary literature. , hairballs can cause gastric irritation, and it may be that eating quickly also stimulates the peripheral sensory receptors that contribute to vomiting. if a cat does vomit regularly, it is important to assess if the cat is presenting for a change in the vomiting pattern (e.g., frequency or timing in relation to eating) and if the cat is unwell in any way, such as anorexia or weight loss. the pattern of vomiting is important in all cases, because cats presenting with acute gastritis usually have a sudden onset of frequent vomiting compared with those with chronic disease processes that may vomit every few days. the timing in relation to eating can be helpful, because the stomach should empty by to hours after a meal; so, vomiting longer than hours after a meal can suggest motility or retention disorders. the description of the vomitus can be helpful. if bile is present, the pylorus is not obstructed; the presence of blood (digested or fresh) indicates ulceration. hair in the vomitus can indicate hairball gastritis, and the possibility of trichobezoar obstruction should be considered. access to foreign bodies or toxins is an important aspect of the clinical history. has the cat been seen playing with an insect, mouse, or other prey? are there any medications unaccounted for (e.g., a dropped aspirin tablet)? are lilies present in the house? vomiting is the major sign of gastric disease, but given the number of potential organ systems that can be involved, a thorough physical examination should be undertaken. because linear foreign bodies are a common cause of vomiting, all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of string caught there. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see . a thorough examination may reveal specific signs, such as a palpable thyroid nodule and tachycardia in the case of hyperthyroidism or palpably small kidneys with and analgesia, many cats recover uneventfully. one survey assessed that % of cats undergoing exploratory laparotomy survived the hospitalization, and although complications occurred in % of cats, these were more likely to be associated with the underlying disease process and not surgery or anesthesia. laparoscopy is not readily available in all veterinary clinics. this alternative is less invasive and allows exploration of the abdomen but not as thoroughly as with laparotomy. organs are usually exteriorized for biopsy. there is the possibility of anesthetic complications associated with insufflating the abdomen. endoscopy is the least invasive procedure and is the only alternative that allows examination of the intestinal lumen. this option limits the parts of the gastrointestinal tract that can be biopsied; it does not allow examination or sampling of any other part of the gastrointestinal tract and does not enable full-thickness biopsy samples. one study found that, of cats investigated for gastrointestinal disease, of cats ( %) had no pathology recognized proximal to the jejunum (i.e., the effective length of diagnostic endoscopes would have precluded diagnosis), and other organs were affected in of cats with inflammatory bowel diseases and of cats with intestinal small cell lymphoma. careful case selection for endoscopy from survey ultrasonography can reduce the number of missed diagnoses from endoscopy, but the possibility still remains. the quality of endoscopically obtained biopsy samples varies greatly with the skill of the endoscopist. it has been stated that "it is exceedingly easy to take inadequate tissue samples with a flexible endoscope." in an assessment of endoscopically obtained biopsy samples, two laboratories were compared, one that received samples from any practitioner and the other that received samples only from practitioners trained to take, mount, and submit endoscopy samples. all slides were reviewed by three pathologists who found that, of samples from the first laboratory, % of the slides were considered inadequate for diagnosis, % were considered questionable, and only % were adequate. by comparison, in the second laboratory (with samples from experienced practitioners) % of slides were inadequate, % were questionable, and % were considered adequate for diagnosis. in the case of distinguishing between lymphocytic intestinal infiltrates (commonly known as inflammatory bowel disease) and lymphocytic neoplasia (small cell lymphoma), endoscopically obtained samples can give an incorrect diagnosis. many of these problems can be minimized with experienced operators and careful case selection from prior ultrasonography. radiography is most useful for identifying foreign bodies or signs of intestinal obstruction from other causes. the major findings are noted below in the section intestinal obstruction. contrast radiography can aid the diagnosis for both discrete and linear foreign bodies but should be used with caution, because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium irritates the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. ultrasonography is a useful diagnostic adjunct and helps to detect and characterize localized thickening of the stomach or intestinal wall, lymphadenopathy, radiolucent foreign bodies, and changes in the size and echogenicity of the pancreas, liver, kidneys, or spleen. abdominal effusions can be assessed and sampled. ultrasound-guided fine-needle aspiration can be used to sample masses, bile, or peritoneal fluid. it should be recognized that in most cases of gastrointestinal disease, imaging will not give a definitive diagnosis and biopsy will be required, usually using either endoscopy or laparotomy. ultrasonography can be a considered as a means to "survey the field," assessing • the nature of the underlying disease, such as • thickened intestines with or without discrete layers • lymph node involvement • other organ involvement • the location of disease, for example, • diffuse or focal • proximal duodenum (reachable by endoscope) versus distal ileum these factors may be used to assess the appropriateness of endoscopy versus laparotomy to obtain diagnostic samples. most commonly used antiemetics all control vomiting by different mechanisms and include mirtazapine, metoclopramide, dolasetron/ondansetron, maropitant, and the phenothiazines (tables - and - ) . metoclopramide functions both as an antiemetic and prokinetic in cats, while cisapride functions solely as a prokinetic. mirtazapine, a piperazinoazepine, antagonizes the presynaptic alpha -adrenergic receptor, increasing noradrenergic and serotonergic neurotransmission; the primary mechanism targeted for its use is as an antidepressant in humans. mirtazapine is also a potent antagonist of the postsynaptic serotonergic receptors ( -ht and -ht ) and histamine h receptors. because of its antiserotonergic and antihistaminic effects, mirtazapine is used as an entiemetic and appetite stimulant in cats. anorexia is a common clinical problem in ill cats, and in some anorexic or partially anorexic cats the use of an appetite stimulant as adjunctive therapy to nutritional support (i.e. feeding tubes) may be of clinical benefit. prior to the development of mirtazapine, cyproheptadine was used as an appetite stimulant in cats, with variable clinical results. recently, the pharmacokinetics and pharmacodynamics of mirtazapine have been reported in cats. in a group of healthy cats, mirtazapine was found to be an effective appetite stimulant, with a shorter half-life than that reported in humans. the recommended oral dose is . mg/cat every hours. a in humans, age and kidney and liver dysfunction affect mirtazapine metabolism (hepatic cyp enzymes) and clearance (excreted in urine and feces), suggesting that dose adjustment may be necessary. a side effects reported in cats treated with mirtazapine include behavior changes (vocalization and interaction), tremors, muscle twitching, and hyperactivity. a, a metoclopramide is both an antiemetic and prokinetic drug that acts peripherally on the gastrointestinal tract and centrally within the central nervous system (cns). at low doses metoclopramide inhibits dopaminergic (d ) transmission, and at higher doses it inhibits serotonergic -ht receptors in the chemoreceptor trigger zone (crtz). , metoclopramide also acts peripherally as a prokinetic at the level of the gastrointestinal smooth muscle of the stomach and duodenum, triggering gastric emptying and duodenal contractions. multiple mechanisms mediate metoclopramide's prokinetic activity, including augmentation of acetylcholine release and increased smooth muscle sensitivity to cholinergic neurotransmission, which may in part be because of antagonism of dopamine, but more recently, serotonergic ht receptor activation has been suggested. , metoclopramide has been reported to increase the lower esophageal sphincter tone in humans, although in cats metoclopramide's affect on the lower esophageal sphincter is reported to be weak. adverse central nervous system, extrapyramidal signs occur secondary to dopamine (d ) antagonism, including excitement and behavior changes. extrapyramidal signs are most often seen at the higher doses needed to block -ht receptors. because of metoclopramide's prokinetic properties, an intestinal obstruction should be ruled out prior to its use. dopamine is a less important neurotransmitter in the chemoreceptor trigger zone of cats than alpha adre nergic and -ht -serotonergic receptors, suggesting that d -dopaminergic antagonist may be a less effective antiemetic in cats. clinically metoclopramide commonly controls vomiting in cats, although this clinical response may be secondary to -ht antagonism and/ or its prokinetic effects. , extrapolated from the short elimination half-life of metoclopramide in dogs ( minutes), frequent be clinically significant side effects. phenothiazines have the potential to lower the seizure threshold; their use is not recommended in patients with a known seizure history. other cns-associated side effects linked to d antagonism occur at higher doses and produce extrapyramidal signs, including rigidity, tremors, weakness, and restlessness. antagonism of the histaminergic receptors carries the risk of sedation. because of the need for frequent dosing ( . to . mg/ kg subcutaneously every hours) and the risk of hypotension and sedation, the clinical use of phenothiazine antiemetics is limited to hospitalized patients with refractory vomiting and should be avoided in patients who are dehydrated or hypotensive. cisapride is a serotonergic -ht agonist that increases propulsive gastrointestinal motility from the lower esophageal sphincter to the colon. cisapride binds serotonergic -ht receptors in the myenteric plexus, increasing the release of acetylcholine in gastrointestinal smooth muscle. in dogs cisapride has greater prokinetic activity in the stomach relative to metoclopramide. cisapride has no direct antiemetic effect, although it is indicated in a vomiting cat with colonic dysmotility secondary to megacolon. colonic distention can trigger the vomiting reflex in cats. cisapride induces colonic smooth muscle contractions in cats with megacolon that is dependent on the influx of extracellular calcium and is only partially cholinergic dependent. other potential indications include refractory generalized ileus or gastroesophageal reflux. dosage recommendations based on the pharmacokinetics in healthy cats is . mg/kg orally every hours. prior to the use of cisapride, an intestinal obstruction should be ruled out because of its strong prokinetic effects. side effects reported in humans are cramping and diarrhea. potentially life-threatening side effects include qt prolongation and ventricular arrhythmias, the primary concern in humans that led to cisapride's removal from the market in the united states. in cats qt prolongation associated with cisapride administration requires times the therapeutic dose. because of the risk of prolongation of the qt interval and ventricular arrhythmias, the concurrent use of cisapride and dolasetron is not recommended. other potential drug interactions associated with cisapride include concurrent therapy with azole antifungals (ketoconazole and itraconazole), because of their inhibition of hepatic cyp a isoenzyme system and the inhibition of cisapride metabolism. diet trials are commonly used in cats with idiopathic gastrointestinal signs or in cats with suspected or known intermittent dosing or delivery by a constant rate infusion (cri) is necessary. empirical dosing in cats is . to . mg/kg subcutaneously or orally every hours or to mg/kg/day as a cri. approximately % of metoclopramide is excreted in the urine, thus dose reduction is recommended in cats with underlying renal azotemia. dolasetron and ondansetron are selective serotonin antagonists that inhibit central and peripheral -ht receptors. their main antiemetic effect is through antagonism of the peripheral -ht receptors in the gastrointestinal tract. in cats -ht antagonism of the crzt is also likely important in the antiemetic effect of dolasetron and ondansetron. dolasetron and ondansetron were originally used for vomiting secondary to chemotherapy because of their superior clinical efficacy. the clinical use of dolasetron and ondansetron in cats has not been associated with reported side effects, and experimental studies report minimal toxicity in animals at doses times the antiemetic dose. side effects reported in humans include headaches, elevated liver enzymes, rare hypersensitivity reactions, prolongation of the qt interval, and arrhythmias. , dolasetron is commonly used for parenteral administration and ondansetron for oral administration, dictated primarily based on the tablet sizes available and cost. recommended dosing of dolasetron is . to mg/kg intravenously every hours and ondansetron . mg/ kg orally every hours. maropitant is a neurokinin- (nk- ) receptor antagonist, blocking the binding of substance p to the nk- receptors located in the emetic center, crtz, and the enteric plexus. in cats maropitant has been reported to be efficacious in treating xylazine-induced vomiting and motion sickness. recommended dosing in cats is mg/ kg intravenously, subcutaneously or orally every hours for up to days. maropitant is reported to be well tolerated in cats. prochlorperazine and chlorpromazine are considered broad-spectrum antiemetics by antagonism of d dopaminergic, histaminergic (h and h ), and cholinergic (muscarinic) receptors within the crtz and, at high doses, the alpha-adrenergic receptors (alpha and alpha ) within the vomiting center. in cats alpha -receptors play a key role in emesis (recall xylazine is the emetic of choice in cats), suggesting cats may be more sensitive to the antiemetic effects of the phenothiazines. prochlorperazine and chlorpromazine produce an antiemetic effect at relatively low doses, thus avoiding profound sedation; although, because of antagonism of the alpha-receptors, vasodilation and hypotension can hyperacidity alone is not considered a common cause for vomiting in cats, but famotidine is effective in treating vomiting in cats associated with gastric ulcers or gastritis. recommended dosage in cats is . mg/kg every to hours. ranitidine is also a competitive inhibitor of the h receptor associated with gastric parietal cells. in addition, ranitidine increases lower esophageal sphincter tone and functions as a prokinetic agent (increasing gastric emptying and stimulating intestinal motility, including colonic motility), because of its anticholinesterase food hypersensitivities. dietary strategies used to control vomiting in cats focus on either a highly digestible diet or an elimination (novel protein/carbohydrate or hydrolyzed protein) diet. the empirical use of elimination diets in cats is reported to be relatively successful, with approximately % of cats with idiopathic gastrointestinal signs responsive to a novel protein/carbohydrate diets within to days. interestingly, traditional diet trials are recommended for a minimum of to weeks, but in this group of diet-responsive cats with chronic gastrointestinal disease, clinical improvement was reported within days. thus if a cat is going to be diet responsive, clinical improvement to a diet trial should be noted relatively early. highly digestible diets enable more effective absorption and assimilation of nutrients in the face of a compromised digestive tract. these diets contain highly digestible proteins and carbohydrates, moderate to low fat, soluble fiber but low concentrations of insoluble fiber, and are supplemented with omega- fatty acids. these diets are recommended when food allergy or intolerance is suspected. these diets contain a single highly digestible novel carbohydrate source and novel protein source. alternatively, diets formulated with hydrolyzed proteins can be used as an alternative to novel protein/carbohydrate diets. see tables - and - for information on gastrointestinal ulcers. famotidine has no direct antiemetic effect but is a competitive inhibitor of the histamine (h ) receptors associated with the gastric parietal cells. the h -receptor is the dominant receptor involved in gastric acid secretion. h receptor antagonism is reported to result in a % to % reduction in acid production. famotidine is more effective at suppressing gastric acid secretion relative to ranitidine. famotidine is well tolerated, although, with chronic therapy, there is the potential for hypoacidity and gastric bacterial overgrowth. in humans dose reduction is recommended in association with renal dysfunction. famotidine is not an inhibitor of the hepatic microsomal cytochrome p- enzyme system, therefore significant drug interactions are not anticipated. activity. , significant drug interactions associated with hepatic microsomal cytochrome p- enzyme system inhibition are not a clinical concern with ranitidine. an adverse effect to be aware of in cats treated with ranitidine is transient hypotension associated with ranitidine administered as an iv bolus. in humans dose reduction is recommended in patients with renal azotemia. ranitidine is effective in decreasing gastric acid in cats. ranitidine would be a logical choice in a cat with gastrointestinal ulceration and/or atony. the reported dosage recommendation for ranitidine in cats is . mg/ kg orally every hours or . mg/kg intravenous every hours. omeprazole omeprazole is a proton pump inhibitor that targets the h + /k + atpase pump on the luminal surface of partial cells. omeprazole is effective at suppressing parietal cell acid secretion, and its effects persist for ≈ hours after drug withdrawal because of drug accumulation in the parietal cell (by ion trapping). indications for omeprazole therapy are for the treatment and prevention of nonsteroidal antiinflammatory drug (nsaid)-induced ulcers. omeprazole is enteric coated to prevent its degradation by gastric acid; therefore oral formulations should not be crushed. based on human studies, omeprazole is a hepatic microsomal cytochrome p- enzyme inhibitor with known drug interactions with diazepam. the extent of clinically significant drug interactions in cats has yet to be studied. omeprazole is reported to be effective in reducing gastric acid secretion in cats. the recommended empirical dosage in cats is . to mg/kg orally once daily. long-term use in humans and dogs is associated with gastric polyps and parietal cell hyperplasia, respectively, but the effect of long-term use in cats is currently unknown. sucralfate is a disaccharide complexed with aluminum that dissociates to sucrose octasulfate and aluminum hydroxide upon exposure to gastric acid. the sucrose octasulfate spontaneously polymerizes, producing a viscous material capable of binding ulcerative lesions in the gastric mucosa. once bound to the exposed mucosa, it prevents back diffusion of h + , inactivates pepsin, absorbs bile acids, and increases mucosal prostaglandin synthesis, collectively supporting ulcer healing. sucralfate is not systemically absorbed but does prevent the absorption of drugs capable of chelating with aluminum, including fluoroquinolones, tetracyclines, and digoxin. if sucralfate is indicated in a cat being treated concurrently with fluoroquinolones, tetracyclines, or digoxin, the recommendation is to administer the other drug hours prior to the administration of sucralfate to optimize drug absorption. clinical indications for the use of sucralfate in cats are for the treatment of gastric ulcers and esophagitis. dosage recommendation in cats is mg orally every hours. sucralfate can be crushed, suspended in water, and administered as slurry. diet trials are used in some cats with diarrhea if the underlying cause is from known or suspected food hypersensitivities. dietary management includes either a highly digestible diet, an elimination (novel protein/ carbohydrate or hydrolyzed protein) diet (see above for both), or a diet high in fiber. high-fiber diets contain a mixture of both soluble and insoluble fiber that can be beneficial in patients with signs of large bowel diarrhea. insoluble fiber, such as cellulose, functions to increase the bulk of the stool, bind fluid, and regulate intestinal motility. soluble fiber, including fruit and vegetable pectins and beet pulp, functions as a source of butyric acid that can be used by the colonic mucosa and decreases proinflammatory cytokines. , cobalamin cobalamin (vitamin b ) is an essential vitamin needed by a number of different enzymes, including key enzymes involved in methionine metabolism and the conversion of methylfolate to tetrahydrofolate needed for dna synthesis. cobalamin and folate are intimately linked, and hypocobalaminemia can lead to a functional deficiency of folate. ingested cobalamin requires intrinsic factor binding for enterocyte absorption at the level of the ileum. hypocobalaminemia is commonly associated with distal small intestine diseases in cats, including inflammatory bowel disease. in addition, low cobalamin has a negative impact on enterocyte function; therefore in many cats with intestinal disease and hypocobalaminemia, cobalamin supplementation is necessary for resolution of clinical signs. , quantification of serum cobalamin levels is recommended in cats with clinical signs of small bowel diarrhea, ones suspected to have an infiltrative disease of the small intestine (inflammatory bowel disease or gastrointestinal lymphoma), or ones with pancreatic dysfunction. when hypocobalaminemia is identified, supplementation is recommended mannanoligosaccharides, inulin, chicory, and lactosucrose. reports on the use of prebiotics in cats are limited to their use in healthy cats; healthy cats fed fructooligosaccharides were reported to have a trend toward an increase in fecal concentrations of lactobacilli and a decrease in concentration of c. perfringens and e. coli relative to the controls. to date no reports are available on the use of prebiotics in cats with gastrointestinal disease. probiotics and prebiotics potentially have a supportive role in the treatment of gastrointestinal disease in cats. the important clinical consideration in the use of probiotics as an adjunctive therapy is to ensure the use of live nonpathogenic microorganisms that have been documented to colonize the intestinal tract of cats. gastrointestinal flora co-evolve with their host. gastrointestinal microorganism colonization varies among species and within each individual animal. the distribution of fecal microflora for a given individual is considered unique but stable over time. antimicrobial and antiparasitic therapies for the treatment of feline diarrhea are indicated based on the specific diagnosis of infectious diarrhea, bacterial enteritis, or as adjunctive therapy for inflammatory bowel disease. infectious pathogens more commonly associated with feline diarrhea include bacterial enteropathies (clostridium, campylobacter), protozoal enteropathies (tritrichomonas foetus, giardia spp.), and helminthic enteropathies associated with ascarids, hookworms, whipworms, and tapeworms. only the more common anthelminthic, antimicrobial, and antiprotozoal therapies are discussed below (tables - and - ) . more information about antimicrobials and antiparasitics is found under specific infections in the discussions of infectious enteritis and gastrointestinal parasites. fenbendazole is an anthelmintic used to treat common helminth infections, including ascarids, hookworms, whipworms, and a single species of tapeworm, taenia pisiformis. giardia spp. are also considered susceptible to fenbendazole. fenbendazole binds beta-tubulin subunits of microtubules, interfering with their polymerization. side effects include vomiting and diarrhea, although both are considered rare. fenbendazole is not approved for use in cats in north america but is commonly used clinically, and an empirical dosage of mg/kg ( µg/cat every days) while the underlying cause of cat's malabsorption is being investigated and at initiation of targeted therapy. probiotics probiotics are ingested live microorganisms intended to benefit the host, specifically to support the microflora environment of the gastrointestinal tract as well as to provide an overall benefit to the body's immune function by immunomodulation. , , probiotics chemically modify ingesta and intestinal mucus, as well as affect immune cells, enterocytes, and goblet cells within the intestinal mucosa through direct receptor interactions and indirectly through the action of cytokines. the microorganisms commonly used are nonpathogenic bacteria and yeast that have a vital role in gastrointestinal health, including lactobacillus spp., enterococcus faecium, bifidobacterium spp., and saccharomyces spp. for example, lactobacilli synthesize b vitamins, digestive enzymes, and folate coenzymes. clinical indications for the use of probiotics are diverse, including primary gastrointestinal disease, chronic renal disease, and pancreatitis. the rational use of probiotics in the treatment of gastrointestinal diseases include their ability to modulate gastrointestinal flora, minimize colonization by pathogenic bacteria, and decrease the likelihood of bacterial translocation. in healthy cats, lactobacillus acidophilus is reported to reduce fecal clostridium counts. when lactobacillus acidophilus was used adjunctively with antimicrobial therapy, fecal shedding of campylobacter was reduced in cats with campylobacter-induced diarrhea relative to cats treated with antimicrobials alone. specifically, in cats with gastrointestinal disease, available research supports the probiotic enterococcus faecium as clinically beneficial in resolving diarrhea in kittens. relative to the control group, the kittens treated with probiotics had increased fecal bifidobacteria and blood iga concentrations and decreased fecal counts of clostridium perfringens. prebiotics are dietary supplements used to select for the more beneficial enteric flora, support gastrointestinal function, and prevent the overgrowth of pathogenic bacteria, including salmonella, escherichia coli, clostridium, or campylobacter. for a food additive to be considered a prebiotic, it must be nondigestible by the gastrointestinal tract (resistant to gastric acidity, gastrointestinal hydrolysis and absorption), yet fermentable by gastrointestinal microflora to short-chain fatty acids to stimulate the growth of "good" intestinal bacterial. prebiotics include nondigestible oligosaccharidescommonly, oligofructose, fructo-oligosaccharides, pyrantel pamoate is a nicotinic anthelmintic used primarily for the treatment of ascarids, but its spectrum of activity also includes hookworms and the stomach worm, physaloptera spp. pyrantel is toxic to susceptible parasites through its selective action on their nicotinic acetylcholine receptors, resulting in depolarization and spastic paralysis. pyrantel is not approved for use in cats but is considered safe in cats and is commonly used clinically. the dosage recommendation in cats is mg/ kg orally once, repeat in weeks, and finally repeated in months. metronidazole is a nitroimidazole antibiotic with an anaerobic antibacterial spectrum with antiprotozoal activity against giardia spp. in an anaerobic environment, metronidazole is converted to unstable intermediates (nitroso free radicals) that disrupt bacterial dna synthesis. immunomodulatory properties capable of inhibiting cell-mediated immunity have been described for metronidazole, although its immunomodulatory properties are reported at dosages well beyond what is recommended for clinical use, raising questions about the clinical use of metronidazole as an adjunctive therapy for treating inflammatory bowel disease. , resistance to metronidazole is considered rare. the most common adverse reaction is gastrointestinal upset, including inappetence, anorexia, nausea, and vomiting. profuse salivation can occur in cats after oral administration of metronidazole base (formulation used in standard tablets), which has lead to the use of metronidazole benzoate (a compounded formulation not approved by the food and drug administration) in some cats because of its better oral palatability. at high doses (> mg/ kg/day) benzoic acid is reported to be neurotoxic in cats, but with appropriate clinical dosing of metronidazole benzoate benzoic acid toxicity is unlikely. dose-related metronidazole toxicity in cats results in cerebellovestibular ataxia secondary to gamma-aminobutyric acid (gaba) inhibition at dosages greater than or equal to mg/kg/day , ; clinical signs include nystagmus, head tilt, ataxia, seizures, and obtundation. in cats with inflammatory bowel disease, the dosage recommendation for the metronidazole base is to mg/kg/day. metronidazole benzoate contains approximately % metronidazole base by weight, translating to an empirical dosage of mg/kg/day of metronidazole benzoate (equivalent to . mg/kg/day of metronidazole base). little is known about the safety of chronic metronidazole use in cats, but oral metronidazole has been reported to disrupt dna within feline peripheral mononuclear cells following days of therapy. this metronidazole-induced genotoxicity is reversible and is no longer detected days after antibiotic therapy is discontinued. ronidazole is a nitroimidazole antibiotic (similar to metronidazole) and available as a powder-on-feed antibiotic. ronidazole is not approved for use in cats but has immunosuppressive therapies used in cats with inflammatory bowel disease include glucocorticoids, cyclosporine, and chlorambucil (tables - and - ). more information on the treatment of inflammatory bowel disease is found elsewhere in this chapter. glucocorticoids are considered first-line therapy in the treatment of cats with inflammatory bowel disease. glucocorticoids bind their intracellular glucocorticoid receptors, modifying the expression of genes with glucocorticoid response elements. immunomodulation is achieved through inhibition of cytokine release and response, including decreasing leukocyte phagocytosis, chemotaxis, and antigen expression. the more common side effects in cats include gastrointestinal ulceration, opportunistic infections (e.g., urinary tract infections), pancreatitis, and diabetes mellitus. cats are less susceptible to iatrogenic hyperadrenocorticism than dogs. initial therapy is usually with oral prednisone or prednisolone. prednisone is a prodrug that is metabolized to its active form prednisolone. cats are reported to be less efficient in the conversion of prednisone to prednisolone ; therefore prednisolone may be preferred in cats, especially in cats refractory to prednisone therapy. been used off-label to effectively treat tritrichomoniasis in naturally and experimentally infected cats ( mg/kg orally every hours for days). t. foetus reduces nitroimidazoles to their nitroso free radicals. ronidazole has been reported to have better in vitro and -fold higher in vivo activity against t. foetus relative to metronidazole. , , ronidazole resistance is beginning to be reported in t. foetus isolates from cats with diarrhea. side effects include hepatoxicity and neurotoxicity. neurotoxicity is associated with high doses and has been reported in cats. the use of ronidazole is recommended only for confirmed cases of t. foetus, and dosing should not exceed mg/kg once daily in cats, especially in cats at risk for neurotoxicity. ronidazole is not registered for human or veterinary use in the united states; therefore its use in cats requires owner informed consent and client education of the potential human hazards. immunosuppressive therapies are considered the standard of care for cats with gastrointestinal biopsies consistent with inflammatory bowel disease (lymphoplasmacytic or eosinophilic inflammation). the common alternative forms of glucocorticoids can be considered in specific patient populations. in patients with severe malabsorption, injectable dexamethasone may provide improved bioavailability and clinical response. also dexamethasone maybe preferred in patients with a history of heart failure, fluid retention, or hypertension because of its lack of mineralocorticoid activity relative to prednisone/prednisolone. dexamethasone's potency is to times that of prednisolone; therefore a dose reduction is necessary when prescribing dexamethasone (the dexamethasone dose is one seventh that of prednisolone). , budesonide is an oral, locally active, highpotency glucocorticoid that is formulated to be released in the distal gastrointestinal tract (based on the ph differential between the proximal and distal small intestine), where it is absorbed and is locally immunomodulating at the level of the enterocyte. the amount of systemically absorbed budesonide is minimized, because % to % of the budesonide absorbed from the gastrointestinal tract undergoes first-pass metabolism in the liver. some systemic absorption does occur, as evidenced by a blunted adrenocorticotropic hormone (acth) stimulation test in dogs treated with budesonide at mg/m for days. , the use of budesonide in cats remains anecdotal, with a suggestive empirical dose of . to mg/cat/day. initial glucocorticoid therapy for cats with inflammatory bowel disease consists of antiinflammatory ( . to mg/kg/day) to immunosuppressive ( to mg/kg/ day) dosages, with dosages based on the potency of prednisone/prednisolone. the goal of therapy is to achieve clinical remission and slowly taper the dose of glucocorticoids to the lowest dose that will control the cat's clinical signs. some cats may be completely weaned off therapy, while others require long-term lowdose therapy. the tapering of therapy should be slow, with a % to % dose reduction every to weeks. cyclosporine is considered a second-tier immunosuppressive drug used to treat inflammatory bowel disease in cats. use of cyclosporine in the treatment of diarrhea associated with inflammatory bowel disease in cats is extrapolated from its use in dogs to treat glucocorticoid refractory inflammatory bowel diarrhea. cyclosporine suppresses t-lymphocyte-mediated inflammation in the gastrointestinal tract secondary to suppression of inflammatory cytokines. specifically, cyclosporine attenuates t-lymphocyte activation and proliferation through the inhibition of interleukin- (il- ) production. side effects of cyclosporine in cats include dose-dependent inappetence and vomiting, which may occur at the onset of therapy and are generally responsive to dose reduction. other less common side effects reported in cats are opportunistic infections, including toxoplasmosis and hepatoxicity. the microemulsion formulation of cyclosporine has higher oral bioavailability and less variable pharmacokinetics. a suggested initial dosage of cyclosporine is mg/kg every or hours. serum cyclosporine levels can be used to monitor for excessive trough plasma concentration (> ng/ml) as determined using a highperformance liquid chromotography (hplc) analytical method. chlorambucil is a slow-acting nitrogen mustard that alkylates and effectively cross links dna, leading to altered protein production. the immunosuppressive effects of chlorambucil are the result of its cytotoxic effect on lymphocytes, similar to other nitrogen mustards. bone marrow suppression is considered mild to moderate and is rapidly reversible. neurotoxicity and myoclonus has been reported in a cat accidently overdosed with chlorambucil. chlorambucil is used as a second-tier drug in cats to treat immune-mediated disorders, in part because of ease of administration and its low risk of myelosuppression. for the treatment of inflammatory bowel disease, the recommended dosing in cats is mg/cat every hours in cats greater than kg and mg/cat every hours in cats less than kg. chlorambucil is commonly used in combination with glucocorticoids in the treatment of immune-mediated diseases, including inflammatory bowel disease, overlooked. awareness about feline esophageal diseases is low, the clinical signs are often not specific, and imaging beyond survey radiographs may be required for diagnosis. the esophagus is composed of four layers (from inner to outer): mucosa, submucosa, muscularis, and adventitia (there is no serosal layer). in the dog, the muscle layer is entirely composed of skeletal muscle, but in cats, the distal third of the esophagus is composed of smooth muscle. the upper esophageal sphincter prevents reflux of esophageal contents into the pharynx and minimizes aerophagia. the lower esophageal sphincter prevents gastroesophageal reflux and relaxes during swallowing to allow food and fluid to enter the stomach. clinical signs of esophageal disease include drooling, dysphagia, pain on swallowing (odynophagia), and, most classically, regurgitation. weight loss may occur secondary to inadequate food intake when disease is severe or chronic. other clinical signs, such as anorexia, cough, dyspnea, and fever, may occur if complications such as aspiration pneumonia or esophageal perforation occur. regurgitation is passive expulsion of food or fluid from the esophagus. the food is undigested and often accompanied by mucus and saliva. mucosal erosions may produce frank blood in the regurgitated material. regurgitation must be differentiated from vomiting (table - esophageal disease is uncommon in the cat when compared with dogs, but it is also likely that problems such as esophagitis and esophageal strictures are often salivation, retching, and abdominal contractions. the vomitus consists of partially digested food from the stomach and/or intestines and may be mixed with bilestained fluid. some cats will have both vomiting and regurgitation. expectoration may also be confused with vomiting or regurgitation. expectoration is associated with coughing, but cats that cough excessively may also stimulate vomition so that a careful history is needed to characterize the clinical signs correctly. coughing may also occur in cats that have aspirated as a result of regurgitation. drooling, dysphagia, and odynophagia are most commonly seen with conditions of the oropharynx and/or proximal esophagus. odynophagia is most commonly associated with esophagitis and foreign bodies. dysphagia and regurgitation together most commonly indicate oral or pharyngeal dysfunction; if regurgitation is not accompanied by dysphagia, esophageal dysfunction is likely. regurgitation in cats with esophageal disease is caused by obstruction or muscular dysfunction. causes of obstruction include vascular ring anomaly, foreign object, stricture, and neoplasia. causes of muscular dysfunction include congenital disease, esophagitis, myopathies, neuropathies, and dysautonomia. regurgitation may occur immediately after eating if the lesion is in the proximal esophagus. however, a dilated esophagus provides a reservoir for food and fluid so that regurgitation may not be associated in time with eating. young cats with signs of esophageal disease should be suspected of congenital defects, such as vascular ring anomaly, or a foreign body. adult cats with esophageal disease may have a recent history of general anesthesia, administration of certain oral medications, or ingestion of irritant chemicals. acute onset of clinical signs may suggest a foreign body, while chronic, slowly worsening signs may indicate a stricture or tumor. all cats suspected of esophageal disease should have a minimum database as part of the diagnostic plan (complete blood cell count, serum chemistries, urinalysis, and other tests as indicated by age or concurrent diseases, such as serum total t and blood pressure measurement). an important part of diagnosis is observation of the cat while eating food, to localize the location of the dysfunction. if the cat is unwilling to eat while in the veterinary clinic, the owner can make a video of the cat eating at home for the clinician to view. the general diagnostic approach to regurgitation in cats is found in figure - . plain and contrast radiography and endoscopy are important diagnostic tools for esophageal disease. fluoroscopy is valuable for the diagnosis of motility disorders, but availability is limited to universities and referral centers because of the cost of equipment. ultrasonography is limited to evaluation of * references , , , , , . the cervical esophagus and a small segment of abdominal esophagus between the cardia of the stomach and the diaphragm. the entire esophagus should be evaluated with cervical and thoracic radiographs. thoracic radiographs may also show evidence of complications such as aspiration pneumonia or esophageal perforation. the normal esophagus is not visualized on plain radiographs, but may be seen if food or fluid are retained or a foreign body or mass is present. radiographic contrast agents useful for esophagrams in cats include liquid or paste barium. a water-soluble iodinated contrast agent (e.g., iohexol, gastrografin) is preferred if there is any risk the esophagus is perforated, because these agents are less irritating and more rapidly reabsorbed. esophagrams are most useful for diagnosis of luminal obstructions, extraluminal compression, mucosal irregularities, and possibly alterations in motility. dilute liquid barium can be administered with a syringe or it may be mixed with canned food, especially if a motility disorder or stricture is suspected. multiple lateral radiographs are taken rapidly, starting within seconds of swallowing the contrast agent. contrast is rapidly cleared from the normal esophagus by peristalsis. if the contrast in the esophagus terminates abruptly, an obstruction is likely. if the contrast is retained throughout the esophagus, muscular dysfunction is suspected. some conditions, such as esophagitis, are difficult to diagnose radiographically, because contrast agents may or may not adhere to ulcerated mucosa. flexible endoscopy is a noninvasive diagnostic tool for esophageal disorders and is often used if plain and contrast radiographs have failed to establish a diagnosis. it is most sensitive for diagnosis of masses, ulcers, perforations, and obstructions. in addition, it is often possible to retrieve foreign bodies using endoscopy as well as to assist with dilatation of strictures or placement of gastrostomy feeding tubes if required. biopsy of the esophageal mucosa is more difficult than biopsy of gastric or intestinal mucosa and is not commonly performed with the exception of mass lesions. esophagitis may result from various causes of inflammation, such as contact irritation from foreign bodies (including trichobezoars lodged in the esophagus), chemical irritants or caustic medications, gastroesophageal reflux, persistent vomiting, hiatal hernia, or general anesthesia. inflammation disrupts the esophageal mucosa and exposes the submucosa. an important part of the treatment plan is identification and treatment of the underlying cause. clinical signs include dysphagia, regurgitation, salivation, and repeated swallowing, although signs may be absent in cats with mild esophagitis. cats with odynophagia may repeatedly extend the head and neck while swallowing. if the esophagitis or underlying disease is severe, weight loss and dehydration may occur secondary to anorexia. if the submucosa and muscularis are damaged, strictures may form as a result of the production of fibrous connective tissue and compromise the esophageal lumen. neoplasia is an important cause of esophageal stricture in humans, but not in cats. most cases have single strictures, but multiple strictures are possible. in two studies, the mean stricture diameter was reported as mm. , most strictures are less than cm in length. clinical signs associated with strictures appear to days after the esophageal injury and may be present for weeks before definitive treatment is pursued. regurgitation typically occurs immediately after eating, although if the stricture is long standing, a pouch may form cranial to the lesion where food accumulates. survey radiographs may be normal in cats with esophagitis and strictures, but are useful to rule out other causes for the clinical signs, such as a foreign body, or to detect related problems, such as aspiration pneumonia. in some patients, dilation of the esophagus with fluid or air may be seen. a contrast esophagram may disclose irregularities of the mucosa in cats with severe esophagitis. segmental dilation may occur with severe inflammation. strictures may be diagnosed with an esophagram (figure - ) ; however, in some cases, it may be difficult to differentiate a stricture from intramural thickening (e.g., because of neoplasia). endoscopy is useful for diagnosis of esophagitis; findings include mucosal erythema, hemorrhage, and erosions or ulcerations. if gastroesophageal reflux is present, the lesions will be most severe in the distal esophagus, and the lower esophageal sphincter may be dilated. endoscopy is often used for definitive diagnosis of esophageal stricture as well as to visualize the lesion during treatment by bougienage or balloon catheter dilation. strictures appear as a ring of white fibrous tissue that narrows the esophageal lumen. if endoscopy is performed after a barium esophagram, hours should be allowed to elapse between the procedures or the barium will obscure visualization with the endoscope. general anesthesia is an important cause of esophagitis (sometimes leading to stricture formation) in cats, probably because gastroesophageal reflux appears to occur commonly in anesthetized cats.* for example, in a series of seven cats with benign esophageal stricture, recent anesthesia for ovariohysterectomy was the suspected cause in five cases. clinical signs appeared up to days after anesthesia. abnormal esophageal tissue was performed in two cases. the authors noted that the esophageal mucosa may appear grossly normal, but submucosal inflammation may be found on histopathologic examination of biopsies. a consequence of chronic severe gerd in humans is the development of metaplastic columnar epithelium (barrett esophagus) that replaces the normal squamous epithelium. one case series reported on barrett-like esophagus in three cats. two cases were associated with hiatal hernia and one with cardial incompetence. drug-induced esophageal damage and stricture formation is well known in humans and cats (see . in humans over drugs have been implicated, and most are antibacterials or nsaids. implicated drugs in the cat include tetracycline, doxycycline, and clindamycin in tablet or capsule form administered without a food or water bolus. , , , , clinical signs (dysphagia, regurgitation, salivation, anorexia) appear to days after drug treatment is started. strictures commonly form in the midcervical esophagus or over the heart base in the thoracic esophagus. doxycycline hyclate is most commonly associated with esophageal strictures in cats, and the principle reason for its irritating properties is an acidic ph. the monohydrate salt of doxycycline is less irritating and is marketed as tablets and a palatable paste licensed for use in dogs and cats in some countries. in humans esophageal ulceration after doxycycline therapy is more common than stricture formation. although the development of strictures in cats would appear to be uncommon, it seems possible the incidence of esophagitis is underestimated, because the clinical signs (e.g., odynophagia, chest pain) may go unrecognized. esophageal transit studies of normal cats have shown that the passage time of dry-swallowed tablets and capsules is often prolonged (longer than seconds). , complete entrapment (retention for more than minutes) in the midcervical region occurs commonly. however, a small bolus of food or water is sufficient to ensure immediate passage of the medication into the stomach. , the risk of esophageal retention can also be lessened by coating a tablet or capsule with butter or a gel dietary supplement (nutri-cal; vétoquinol, fort worth, tex.). one study determined that tablets or capsules administered using a one-step pill gun with flavored liquid (flavorx pill glide; flavorx, columbia, md.) or a pill delivery treat (greenies pill pockets; nutro products, franklin, tenn.) ensured an average transit time of seconds or less. delayed esophageal transit of medications allows tablets and capsules to disintegrate within the esophagus, exposing the mucosa to irritating chemicals. cats may be at risk of delayed esophageal transit, because they do not typically drink water with medication, and they do not have an upright posture. in addition, medications are often given to sick or dehydrated patients many preanesthetic drugs and induction agents reduce lower esophageal sphincter pressure. , other predisposing factors may be intraabdominal surgery and a head-down position on the surgery table. reflux fluid with a ph less than is likely to cause esophageal mucosal damage, as is prolonged contact time. esophageal defense mechanisms include clearance of the reflux fluid by peristalsis and neutralization of the acidic ph by the bicarbonate present in saliva. in a study of kittens less than weeks of age, risk of gastroesophageal reflux during anesthesia was evaluated with use of a laryngeal airway mask versus endotracheal intubation. gastroesophageal reflux was observed in % of kittens with use of the laryngeal airway mask but more importantly in % of kittens with endotracheal intubation. the reflux episodes occurred shortly after anesthesia induction. in a study of cats anesthetized with thiopentone or propofol, gastroesophageal reflux occurred in %. reflux also occurred shortly after anesthesia was induced and lasted for a mean of minutes. it is unknown why esophageal strictures form only in a small number of cats that experience gastroesophageal reflux during anesthesia. gastroesophageal reflux disease (gerd) is a commonly reported cause of esophagitis in humans, but it is rarely reported in cats when not associated with general anesthesia. , the true incidence is unknown, and diagnosis may be hampered by scant knowledge about the clinical presentation and diagnosis. clinical signs and diagnostic procedures are as for other causes of esophagitis. in one case series of three cats, diagnosis of gerd was based on clinical signs, contrast radiography, and endoscopic findings. biopsy and histopathology of obtained from a compounding pharmacy in most countries and can only be given orally. h -receptor antagonists are competitive inhibitors that block parietal h receptors and decrease the amount of gastric acid produced. proton pump inhibitors are noncompetitive inhibitors that act on the h + /k + atpase enzyme system at the secretory surface of gastric parietal cells. they are considered superior for decreasing gastric acid secretion and are therefore the first choice, despite their greater cost. a drawback of proton pump inhibitors is that they must be administered orally. sucralfate may be beneficial for reflux esophagitis, because it binds to mucosal erosions in an acid environment and provides a protective barrier. it is given as oral slurry, ideally separate from meals or other medications. antibiotics are not commonly recommended unless aspiration pneumonia is present or the eroded mucosa is at risk of bacterial infection in a patient with severe disease or a compromised immune system. corticosteroids are often recommended for cats with esophagitis to reduce esophageal inflammation and impair the formation of fibrous connective tissue. however, the benefit of corticosteroids in cats with esophagitis has not been investigated and administration must be weighed against potential adverse effects, especially in patients with aspiration pneumonia. treatment of esophageal stricture typically requires dilation with either bougienage or a balloon catheter; both are used with endoscopic visualization under general anesthesia. appropriate analgesia should be provided, because dilating the stricture is painful. it does not appear that placement of a gastrostomy feeding tube is specifically required to recover from dilation procedures, although a tube may be placed in some anorexic cats to ensure nutritional intake and administer oral medications. a bougie is a long, narrow, oblong, mechanical dilator available in various sizes (typically -to -mm sizes are used in cats) that is gently passed through the stricture, usually over a guide wire. established criteria for selection of bougie diameter and dilation end points are not available. in one study, the initial bougie chosen was approximately the same size as the estimated diameter of the stricture, or no more than mm larger. once the first bougie is passed, subsequent bougies of increasing diameter are employed. two to four bougies of increasing size may be passed in a single session, with the goal of dilating the stricture without causing esophageal tear or perforation. determining when dilation should be stopped is a matter of clinical judgment. the procedure may be repeated as needed to maintain improvement; the total number of procedures required is variable. in one retrospective case series of eight cats treated with bougienage, the median number of procedures was . , and a good outcome was achieved in % of the cases. in some cases, the endoscope tip itself has been used for that may be at greater risk of esophageal retention of medication. all oral medication given to cats in tablet or capsule form should be followed with food or a liquid. mild esophagitis will resolve on its own, especially if an underlying cause can be removed or treated. frequent meals of canned food should be provided. cats with moderate to severe esophagitis will require medical therapy, and those with difficulty eating or weight loss may also require gastrostomy tube feeding. esophagostomy or pharyngostomy feeding tubes should be avoided in these patients. treatment is provided to control inflammation and promote healing while reducing gastric acid secretion and increasing lower esophageal sphincter tone. the length of medical treatment will vary from about one week to several weeks, depending on the underlying cause and severity of disease. medications indicated for esophagitis include prokinetics, h -receptor antagonists, proton pump inhibitors, and sucralfate (table - ) . prokinetic drugs enhance gastric emptying and increase lower esophageal sphincter tone. metoclopramide also has antiemetic effects, which may be beneficial in patients with chronic vomiting. it can be administered by the subcutaneous (sc) route, an advantage in a vomiting or regurgitating patient. cisapride may be more effective at enhancing both gastric emptying and lower esophageal sphincter tone, but it must be stent placement has recently been described in cats with esophageal strictures with variable results. a -year-old cat presented with a -week history of dysphagia and regurgitation caused by a single cervical esophageal stricture after treatment with oral clindamycin. guided balloon dilation was performed times over a period of weeks, but stricture formation always recurred. a self-expanding metal stent was placed using endoscopy and fluoroscopy after another dilation procedure. the cat did well eating a canned diet from an elevated position for months, but by months, the cat was no longer able to eat even liquid food and was euthanized. on necropsy, the stent had migrated and was obstructed by swallowed hair. in another case, a biodegradable self-expanding stent was used to successfully treat an -year-old cat that presented with a stricture in the cervical esophagus after anesthesia for dentistry. balloon dilation was performed twice, but regurgitation recurred days after the last procedure. the stricture was dilated a third time with a balloon catheter, and a tubular selfexpanding polydioxanone stent was placed with fluoroscopic guidance. the life span of the stent was estimated to be to weeks, sufficient time to allow healing of the esophagus. foreign bodies are less commonly found in the esophagus of the cat than in other gastrointestinal locations. reported foreign bodies include string, needles, fish hooks, and bones. trichobezoars may cause obstruction when they become lodged in the esophagus during vomiting ( figure - ). recurrent esophageal trichobezoars have been infrequently reported in the literature. , it is not known if an esophageal motility disorder is the underlying cause for recurrent obstructions. in one case, an esophageal diverticulum developed in association with recurrent trichobezoars. treatment for recurrent trichobezoars includes prokinetic drug therapy (e.g., cisapride), moderate to high-fiber diets, and shaving of long-haired cats. common areas for foreign bodies to lodge include the thoracic inlet, the heart base, and the esophageal hiatus in the diaphragm. obstruction of the esophageal lumen may be complete or partial. clinical signs include acute onset of gagging, salivation, repeated swallowing, dysphagia, and regurgitation. however, chronic esophageal foreign bodies have been reported in cats with dysphagia, intermittent regurgitation, and weight loss over a period of weeks or months. bougienage when bougies or balloon catheters were not available. balloon catheter dilation has become a popular method in recent years. , , although some clinicians feel this is a safer procedure than bougienage, there is no data in the literature to support this assumption. the catheter can be placed through the endoscope biopsy channel, alongside the endoscope, or with the aid of a preplaced guide wire. as for bougienage, established criteria for selection of balloon diameter and dilation end points are not available, and the clinician's best judgment must be used. various balloon sizes are available; in one study, the size was selected so that the inflated diameter was mm larger than the stricture diameter. the balloon is passed into the stricture with endoscopic guidance. it is then inflated to a predetermined pressure for to minutes to stretch the stricture, usually with saline, but contrast agents may also be used if fluoroscopy is used. as for bougienage, some cases may require more than one dilation procedure (typically two to four). cuffed endotracheal tubes are not appropriate substitutes for balloon catheters. regardless of the method used, after the dilation procedure, the endoscope should be used to look for other strictures and should be passed into the stomach to look for potential causes, such as causes of chronic vomiting. after treatment, medical management to decrease ongoing gastroesophageal reflux, resolve inflammation, and prevent further stricture formation should be instituted (as described previously). most cats are able to eat the day following the dilation procedure. corticosteroid treatment after dilation is controversial, and no controlled studies in animals are available. antibiotics are not routinely recommended. the prognosis for cats undergoing esophageal dilation is generally good based on the ability to eat canned food with minimal episodes of regurgitation. however, published studies show % to % of cats died or were euthanized despite multiple episodes of dilation, and up to % could only be fed liquid diets. , , , even among cats with good outcomes, a return to a dry kibble diet may not be possible. the dilation technique employed may be dictated by the clinician's experience, the equipment available, and the cost. potential complications of both methods include esophageal tear or perforation, hemorrhage, infection, and aspiration. esophageal tears or perforations may lead to pneumothorax or pneumomediastinum. repeated stricture formation is also possible, leaving only less desirable treatment options, such as long-term percutaneous gastrostomy tube feeding or surgery. esophageal surgery is generally avoided whenever possible, because it is difficult and invasive (requiring a thoracotomy), with risk of serious complications, such as failure of anastomosis, necrosis, and stricture formation. closure of incisions in the esophagus is following uncomplicated foreign body removal, the esophagus should be carefully inspected for lesions and bleeding before the endoscope is withdrawn. food and water should be withheld for to hours. supportive care includes fluid therapy and analgesia; a gastrostomy feeding tube may be required in selected cases for nutritional support. broad-spectrum antibiotics are administered to control bacterial infection and therapy for esophagitis should be instituted as described previously. careful follow-up should include evaluation for stricture formation. if an esophageal perforation has occurred, conservative management may be sufficient if the defect is small. a broad-spectrum antibiotic should be administered along with other supportive care, such as fluid therapy and analgesia. feeding through a gastrostomy tube for several days is recommended as well as close monitoring for complications such as pleuritis. large perforations require thoracotomy for surgical repair. megaesophagus is a diffuse hypomotility disorder that may be classified as congenital versus acquired or idiopathic versus secondary to other diseases. it is uncommon in cats compared with dogs. at least two dog breeds have been identified with heritable congenital megaesophagus. a heritable form of megaesophagus has been suggested for cats, particularly for siamese cats, although no detailed studies have been performed. , it is often frustrating to determine the underlying cause of acquired megaesophagus. megaesophagus may be a manifestation of neuromuscular diseases, such as dysautonomia or myasthenia gravis (see chapter ) . megaesophagus may also develop secondary to esophagitis from chronic vomiting or gerd. , other uncommon causes of megaesophagus are found in the literature. one case report describes a young cat with megaesophagus secondary to a large nasopharyngeal polyp that extended into the cervical esophagus. megaesophagus resolved once the polyp was removed. in another report, a young cat with diaphragmatic hernia was diagnosed with megaesophagus and gastric dilation. megaesophagus resolved with medical treatment and surgical correction of the diaphragmatic defect. clinical signs are typically those of esophageal dysfunction; regurgitation is the most consistently found sign. regurgitation may not be closely related in time to eating if the esophagus is markedly distended and holds food. cats with long-standing disease may suffer from weight loss or secondary rhinitis. the appetite is typically normal or increased. additional signs may occur if systemic neuromuscular disease is present. aspiration pneumonia may cause fever, dyspnea, and cough. two case reports describe cats with idiopathic cough, mucopurulent nasal discharge, and fever may be found if aspiration has occurred. trauma to the esophagus may cause esophagitis and even esophageal stricture. perforation of the esophagus by the foreign body may lead to pneumothorax, pneumomediastinum, or pyothorax with signs of depression, anorexia, fever, and dyspnea. if the perforation occurs in the cervical esophagus, swelling, cellulitis, and drainage of serous or purulent material may be noted. many foreign bodies are readily diagnosed with survey radiographs, especially if they are radiopaque. other radiographic findings include an esophagus dilated with fluid or air. radiolucent objects may be detected with an esophagram. care must be taken when performing esophagrams on cats that may have an obstruction, because aspiration is a concern. if abnormalities that could be consistent with an esophageal perforation (e.g., periesophageal gas or fluid, pleural effusion) are detected on survey radiographs, an aqueous iodine contrast solution should be used. removal of esophageal foreign bodies should be performed as soon as possible to minimize esophageal trauma and pressure necrosis. endoscopy can be used to confirm the diagnosis and often to remove the object. both rigid and flexible endoscopes may be used along with accessories such as various forceps and foley catheters. care should be taken to remove the object as atraumatically as possible, especially if the object is sharp or pointed. if the object is in the caudal esophagus and it cannot be grasped and removed, an attempt should be made to gently push it into the stomach, where it can be retrieved using laparotomy and gastrotomy. if esophageal perforation has occurred, esophagotomy is recommended and is described elsewhere. , removal of fish hooks may require a combination of surgery and endoscopy. , a surgical approach to the esophagus is made, but the esophagus is not incised; rather, the portion of the hook protruding through the esophagus is cut and removed, and the endoscope is used to retrieve the remainder. clinical sign is regurgitation, and most patients are underweight. a distended cervical esophagus may be palpated, and secondary aspiration pneumonia may occur. a history of regurgitation since weaning is very suggestive of a vascular ring anomaly, but other causes of regurgitation must be ruled out. survey radiographs show a dilated esophagus cranial to the heart, while the caudal esophagus is usually normal. the bulge of the aortic arch normally seen on a ventrodorsal radiographic view is absent. an esophagram is used to confirm the location of the obstruction and the severity of disease. definitive treatment is surgical repair of the vascular defect (i.e., ligation and transection of the ligamentosum arteriosum). some patients will require nutritional support through gastrostomy tube feeding and treatment for aspiration pneumonia before surgery. early diagnosis and surgical intervention brings the best prognosis for return of normal esophageal function. some affected cats are left with residual esophageal hypomotility, which is managed as for idiopathic megaesophagus. esophageal neoplasia is rare in the cat as in the dog. although parasitic granulomas caused by spirocerca lupi are associated with esophageal neoplasia in dogs, this parasite does not infect cats. both primary and metastatic esophageal tumors can occur in the cat. squamous cell carcinoma is the most common primary esophageal tumor in cats and is often found in the caudal two thirds of the esophagus. , , , affected cats are middle aged or older. clinical signs are typically those associated with esophageal obstruction, such as regurgitation, dysphagia, odynophagia, and salivation. patients with advanced disease may suffer anorexia, depression, and weight loss. on physical examination, an esophageal mass may or may not be palpable. survey and contrast radiographs reveal esophageal dilation, a soft tissue mass, or periesophageal lesions that displace the esophagus. computed tomography is useful to identify periesophageal or intraluminal masses. definitive diagnosis is made with endoscopy and biopsy. mucosal biopsies are difficult to obtain, because the esophageal mucosa is tough; exfoliative cytology may also be helpful. treatment is rarely undertaken, because disease is often advanced at the time of diagnosis, and many patients have complications such as aspiration pneumonia. palliation may be attempted with chemotherapy or radiation, although data on efficacy is unavailable. in general, squamous cell carcinomas in other anatomic locations respond poorly to treatment. surgical resection may be attempted if anastomosis can be accomplished without excessive tension. megaesophagus and chronic vomiting associated with intermittent gastroesophageal intussusception. , survey and contrast radiographs may identify a dilated esophagus (figure - ), but contrast fluoroscopy is the diagnostic tool of choice when available, because it allows for assessment of peristalsis. care must be taken with contrast studies because of the risk of aspiration. treatment of megaesophagus is largely symptomatic and supportive unless an underlying disorder can be identified and treated. frequent small meals are offered with the cat feeding in an upright position. the upright position should be maintained for at least minutes after eating to allow for gravity-assisted passage of food into the stomach. this is best accomplished by having the owner hold the cat over their shoulder so that the esophagus is in a vertical position. different types of diets should be offered to determine which is best for the individual patient; calorically dense diets may be beneficial for patients with weight loss. prokinetic drugs, such as cisapride, stimulate smooth muscle, but since most of the esophagus is skeletal muscle, the efficacy of such drugs is questionable for treatment of megaesophagus. prokinetic drugs also increase lower esophageal sphincter tone and may increase esophageal transit time, neither of which is desirable in patients with megaesophagus. vascular ring anomalies are congenital malformations of the great vessels that entrap the thoracic esophagus and cause obstruction. the most commonly reported anomaly is persistent right aortic arch. the esophagus is entrapped by the aorta on the right, the ligamentum arteriosum and the pulmonary trunk on the left, and the heart base ventrally. other vascular anomalies are rarely described in cats, such as a double aortic arch described in a siamese cat. onset of clinical signs occurs around the time of weaning to solid food so that most affected cats are presented at less than months of age. the most common surgery. surgery is the treatment of choice for large defects, especially in young cats with congenital disease or cats that have failed medical management. various reconstructive surgical techniques have been described. disorders of the hiatus are rare in cats. hiatal hernia is protrusion of the distal esophagus and stomach through the esophageal hiatus of the diaphragm into the thoracic cavity; the protrusion may be intermittent ("sliding") or persistent. other organs are occasionally involved, such as the omentum. this is distinct from a gastroesophageal intussusception where the stomach is prolapsed into the lumen of the distal esophagus. , both congenital and traumatic hiatal hernias have been described in cats. , , , , congenital hernias appear to be more common than acquired hernias, and affected cats typically present with clinical signs before year of age. it is suspected that increased inspiratory effort associated with upper airway obstruction, such as a nasopharyngeal polyp, may also lead to development of hiatal hernia. hiatal herniation reduces lower esophageal sphincter pressure. clinical signs associated with hiatal hernia, such as intermittent vomiting and regurgitation, may be because of reflux esophagitis, hypomotility, or obstruction. large hernias and secondary aspiration pneumonia may be associated with respiratory distress. survey radiographs may reveal a gas-filled soft tissue density in the caudal dorsal mediastinum. an esophagram will show the gastroesophageal junction and gastric rugae cranial to the diaphragm (figure - ) . both fluoroscopy and endoscopy may be useful for diagnosis but are not typically necessary. the prognosis for cats with hiatal hernia is considered to be good. a trial of medical management (as for reflux esophagitis) for month has been recommended before the stomach is a frequent site for gastrointestinal problems in cats, and the most common gastric problems are described in this chapter. some conditions such as gastric dilatation-volvulus are often reported in dogs but rarely reported in cats. in one report of three feline cases, all were associated with diaphragmatic hernia. gastric parasites, the diagnostic approach to the vomiting cat, the gastric emptying time of normal cats is shorter than that of other mammals. in one study, the gastric emptying half-time for solid food in normal cats was . to . hours. this implies prolonged fasting (longer than hours) in preparation for anesthesia and surgery is unnecessary. the main clinical sign of gastric disease is vomiting, but it is important to note that vomiting is also associated with many nongastric problems, including concurrent intestinal disease, such as enteritis or colitis. vomiting patients therefore require a thorough physical examination and diagnostic plan to determine the cause. vomiting must be distinguished from regurgitation, which is primarily associated with esophageal disease (see table - ). vomitus often contains food, hair, refluxed bile, and therapeutics for vomiting are covered elsewhere in this chapter. the anatomy of the feline stomach is similar to that of other mammals having a simple glandular stomach. most of the stomach is situated on the left side of the abdominal cavity. it has five regions, starting from the lower esophageal sphincter: cardia, fundus, body, antrum, and pylorus ( figure - ) . the pylorus of the cat is unique compared with other species in that it is narrow and has high resistance in order to maintain a tight seal ( figure involve a wide variety of objects, including linear objects (e.g., dental floss, thread with or without a needle, tinsel, string). the owner may or may not be aware of the ingestion. ingestion of multiple foreign bodies may be seen in cats with pica ( figure - ). in one case report, a young domestic shorthair cat required gastrotomy for removal of copper pennies. some patients require multiple surgeries, because of repeated foreign body ingestion. in such cases, a behavioral diagnosis should be sought and treatment instituted (see chapter ) . trichobezoars (large masses of hair) also represent a type of foreign object. both long-and shorthaired cats may be affected. hair is normally ingested during grooming and is eliminated in vomitus and feces. cats lack the strong peristaltic contractions ("housekeeper" contractions) that clear the stomach of undigested contents normally found in other species. this may explain why cats seem to be susceptible to gastric trichobezoars. gastric motility dysfunction is suspected to cause repeated gastric trichobezoars in some cats. intestinal , and esophageal , obstruction with trichobezoars has also been documented. traditional treatments for cats with recurrent trichobezoars include regular grooming, shaving the hair coat of long-haired cats, flea control, or blood. fresh blood may appear as large or small clots. older blood clots have a brown "coffee ground" appearance. gastric bleeding may also cause melena. other clinical signs may be associated with gastric disease, such as anorexia, weight loss, pain, lethargy, bloating, and nausea. gastritis may be acute or chronic in nature, and this distinction may be useful in assessing the potential cause. for example, cats with acute gastritis may be suspected of foreign body or plant ingestion, drug or toxin exposure (see chapter ), or dietary indiscretion. cats with chronic gastritis may be suspected of parasitism, helicobacter spp. infection, or dietary intolerance or hypersensitivity (see chapter ) . chronic lymphocytic plasmacytic gastritis of unknown etiology is also a common cause of chronic vomiting. whenever possible, a specific underlying cause should be sought and treated. patients with sudden onset of vomiting may have an obvious cause in the history (e.g., dietary indiscretion), but in many cases, the cause is not apparent. abdominal radiographs should be taken if foreign body ingestion is possible, especially in a young cat. if the patient is systemically well, further diagnostic testing may be postponed pending response to therapy. treatment for uncomplicated acute gastritis is symptomatic and supportive. clinical signs are expected to resolve in to hours; if signs persist, re-evaluation and further investigation is warranted. subcutaneous fluid therapy using an isotonic balanced electrolyte solution may be used to correct mild fluid deficits (< %). oral intake of fluids and food should be discontinued for up to hours. a highly digestible diet, either commercial or homemade, is introduced with a gradual transition back to the normal diet over the next several days. antiemetic therapy may be indicated for acute uncomplicated gastritis if the vomiting is frequent or the cat has signs of nausea (see table - ). protectants, such as kaolin and pectin, are difficult to administer to cats and are without proven efficacy. bismuth subsalicylate is controversial; it is considered contraindicated by some experts, because of the cat's sensitivity to salicylates, yet is commonly used in clinical practice. cats ingest foreign bodies less commonly than dogs. in one study of cases of gastrointestinal foreign body ingestion, only % were in cats. foreign body ingestion is most likely to be seen in young cats and may a b taken just before surgery to ensure the object has not moved further down the gastrointestinal tract. postoperative management after gastrotomy includes maintenance of hydration and electrolyte balance. hypokalemia is common with anorexia and vomiting and should be treated by supplementation of iv fluids with to meq/l potassium chloride (not to exceed . meq/kg/hour). refractory vomiting should be treated with an antiemetic. a highly digestible diet can be introduced the day after surgery. in general, the prognosis for recovery is good. in one study, % of cats with gastrointestinal foreign bodies survived to discharge. those cats that did not survive had linear foreign bodies of long-standing duration with subsequent peritonitis. helicobacter are spiral or curved gram-negative bacteria that inhabit the glands, parietal cells, and mucus of the gastric antrum and fundus. helicobacter contain large amounts of urease, which alters the ph in the vicinity of the bacteria and allows for colonization of the acidic environment of the stomach. in the early s, the discovery of the association of helicobacter pylori with gastric disease (gastritis, peptic ulcers, and neoplasia) in humans revolutionized treatment of those diseases. since then, helicobacter spp. have been associated with gastric disease in various veterinary species, including cats and dogs. several helicobacter spp. (e.g., h. heilmannii, h. bizzozeronii, h. felis) have been identified in cats, some of which have the potential to infect humans, although transmission is thought to be rare. , the prevalence of helicobacter infection in cats varies geographically and may be very high (> %) in some locations. , , , , the importance of helicobacter as a cause of gastric disease is cats is unclear; the bacteria may be found in the stomach of both clinically normal cats and cats with gastritis. the prevalence of helicobacter infection is not higher in cats with gastritis compared with normal cats. determination of the role of helicobacter is also hampered by the paucity of controlled clinical trials that evaluate eradication of gastritis and clinical signs in infected cats. an immune response to infection characterized by gastric lymphoid hyperplasia is common, although the local immune response in cats is generally less severe than the response in humans infected with h. pylori. to date gastrointestinal ulcers have not been associated with helicobacter infection in cats. recent studies have suggested a possible association between helicobacter infection and gastric lymphoma in cats, although more research is needed to confirm the association and understand the pathogenesis. , helicobacter spp. may be commensal in most cats, and perhaps loss of tolerance explains the development of gastritis in some individuals. another possibility is that the inflammatory response is normally well managed and disease may treatment of underlying dermatologic disorders, and administration of semisolid petroleum laxatives. more recently, commercial diets have been formulated for control of trichobezoars. cats with recurrent trichobezoars causing illness and suspected motility disorders may benefit from treatment with prokinetic drugs such as cisapride. clinical signs of gastric foreign bodies are variable but typically involve intermittent or persistent vomiting because of gastric outflow obstruction, distention, and mucosal irritation. gastric obstruction may be partial or total. patients with complete obstruction will present with more dramatic signs, including anorexia and depression. the base of the tongue should always be examined, because linear foreign bodies are sometimes anchored either in this location, or they may be lodged in the pylorus, causing intestinal plication. gastric foreign bodies may also be asymptomatic and found incidentally. physical examination may be unremarkable or may reveal dehydration or abdominal pain. if the stomach is markedly distended, the foreign body may be palpable in some patients. survey radiographs are always indicated when foreign body ingestion is suspected. radiopaque foreign bodies may be readily diagnosed, although some, along with radiolucent objects, will require a contrast study for diagnosis ( figure - ) . barium is commonly used as a contrast agent, although if gastric perforation is suspected, an aqueous iodinated agent is preferred. ultrasonography is also useful for detection of gastrointestinal foreign bodies. removal of some foreign bodies can be attempted endoscopically, particularly if the object does not have sharp edges and is not too large. successful removal of fish hooks, particularly single-barb hooks, using endoscopy has been described. otherwise, foreign objects are best removed using gastrotomy through a ventral midline laparotomy. a radiograph should always be to know when treatment should be attempted. one expert recommends treating only patients with clinical signs of gastritis that have biopsy-confirmed helicobacter infection with a treatment regimen of amoxicillin ( mg/kg, every hours, po), clarithromycin ( . mg/ kg, every hours, po) and metronidazole ( mg/kg, every hours, po) for days. a common dilemma would be determining the treatment of choice for patients with lymphoplasmacytic inflammation of the stomach and small intestine and confirmed helicobacter infection. are such patients best treated for inflammatory bowel disease, helicobacter infection, or both? currently, guidelines for determining the best treatment approach are lacking. also, few studies on the efficacy of combination therapy have been conducted in cats. long-term eradication of infection may be difficult, and histopathologic resolution of gastritis may not be possible, which raises the question of whether helicobacter is the true underlying cause. , in one study, two cats with clinical gastritis and helicobacter infection were treated with oral metronidazole, amoxicillin, and bismuth subsalicylate for weeks and were also fed a commercial elimination diet. posttreatment gastric biopsies were obtained a mean of weeks after the cessation of treatment. resolution of clinical signs occurred rapidly, and clearance of helicobacter spp. was achieved at that time point, but gastric inflammation persisted in post-treatment biopsies. in another study, cats with asymptomatic helicobacter infection were treated with oral omeprazole, amoxicillin, metronidazole, and clarithromycin for days. treatment failed to eradicate infection in of the cats based on molecular analysis of post-treatment gastric biopsies. it is unclear if treatment failure is because of recrudescence or reinfection. the reader is referred to excellent reviews of helicobacter in cats for more information. , , chronic gastritis chronic gastritis is common in cats with chronic intermittent vomiting. ollulanus tricuspis is a worm that infects the stomach of cats, causing chronic gastritis, and it is difficult to diagnose (see below, gastrointestinal parasites). the worm is occasionally found on histologic examination of gastric biopsy samples. it is reasonable to treat empirically (fenbendazole mg/kg, once daily, po × days) for this parasite when the cause of gastritis is not apparent. the frequency of vomiting in cats with chronic gastritis is highly variable, ranging from once or twice per week (and not necessarily every week) to more than once daily. most patients are otherwise well, although other clinical signs (inappetence, anorexia, depression, or weight loss) are possible depending on disease severity. results of routine laboratory testing are typically normal but may show neutrophilic leukocytosis, result when there is an abnormality of the immunoregulatory system. the most commonly used methods for diagnosis of helicobacter infection in cats are based on gastric specimens obtained during endoscopy (or laparotomy): exfoliative cytology, histopathologic examination of biopsy specimens, and rapid urease testing of biopsy specimens. however, it is important to note that even when helicobacter organisms are identified, the infection may not be the cause of the patient's clinical signs, and other causes of vomiting should always be evaluated. exfoliative cytology is the least expensive and most easily performed diagnostic test. in one study, it was also the most sensitive diagnostic method when compared with urease testing and histologic examination. brush cytology samples gathered during endoscopy are airdried on microscope slides and stained with wright's stain. the slide is examined at × magnification under oil immersion. spiral bacteria are readily seen if present. at least oil-immersion fields on two slides should be examined before determining a specimen is negative for helicobacter-like organisms. since helicobacter produce abundant urease, a rapid urease test (e.g., clotest, ballard medical products, draper, utah) may be used for diagnosis. the kit consists of an agar gel impregnated with urea and a ph indicator. a gastric biopsy sample is applied to the gel, and if urease is present, ammonia will form and change the ph (and thus the color) of the gel. the gel may change color rapidly (within minutes), but hours must elapse before the test can be considered negative. the more rapidly the color changes, the higher the bacterial load. both false-positive and false-negative results are possible with rapid urease testing for various reasons, giving the test a sensitivity of % to %. , histopathologic examination of gastric biopsy samples using hemotoxylin and eosin (h&e) or silver stains is highly sensitive and specific in human studies for detection of helicobacter-like organisms. the organisms are not equally distributed; so, examination of biopsy specimens from multiple sites will increase sensitivity. the bacteria may be seen in mucus on the surface epithelium as well as in the gastric pits, glandular lumen, and parietal cells. organisms may also be seen submucosally within gastric lymphoid follicles. histopathologic examination of biopsy samples also allows for assessment of other abnormalities. mild to severe lymphocytic-plasmacytic or lymphocytic gastritis may be present. in humans combination therapy with antibiotics and antisecretory drugs is recommended to reduce the risk of gastric ulcers and cancer from h. pylori infection. treatment is highly successful at eradicating both clinical signs and histologic changes in the gastric mucosa. since helicobacter infection is common in cats, yet no clear pathogenic role has been established, it is difficult cases. depending on the underlying cause and severity of disease, abdominal pain, anorexia, lethargy, pale mucous membranes, and drooling may also be seen. cats with neoplastic disease may have prolonged clinical signs and are more likely to present with anorexia and weight loss. cats with perforated ulcers may or may not present with signs of shock. diagnosis may be problematic because the clinical signs and physical examination findings are often not specific, even in cats with perforated ulcerations. the causes of gastric ulceration in cats are not well characterized. in dogs the most common cause is the administration of ulcerogenic drugs, particularly nsaids, either alone or in combination with corticosteroids. several cases of nsaid-induced gastroduodenal ulceration or perforation have been reported in cats. , , additional cases may be reported in the future, because long-term administration of these drugs is gaining in popularity for treatment of chronic diseases such as osteoarthritis. nsaids cause direct mucosal damage and interfere with prostaglandin synthesis. although inhibition of the cox- enzyme is thought to be the cause of adverse effects, such as gastric ulceration, even cox- -selective drugs have been associated with adverse effects, and safety in sick cats is not well evaluated. recently, guidelines for the long-term use of nsaids in cats were published by the international society of feline medicine and the american association of feline practitioners. the recommendations include administering nsaids either with or shortly after food, withholding therapy if inappetence or anorexia develops, determining dose based on lean body weight, and titrating to the lowest effective dose. neoplastic causes of gastric ulceration include systemic mastocytosis, mast cell tumor, lymphosarcoma, adenocarcinoma, and gastrinoma (zollinger-ellison syndrome). cats with chronic renal disease may suffer mucosal damage from uremic toxins and increased gastric acid production secondary to hypergastrinemia (because of decreased renal metabolism of gastrin). hepatic disease is a cause of gastric ulceration in dogs but is uncommonly reported in cats. recent anesthesia and surgery have been implicated as a cause of gastric ulceration and perforation, perhaps through hypovolemia, hypoperfusion, or stress. , other non-neoplastic causes reported for gastric or gastroduodenal ulceration in cats include parasites (e.g., ollulanus tricuspis, toxocara cati, aonchotheca putorii, gnathostoma spp.), bacterial infections, toxins, inflammatory bowel disease, and foreign bodies. one case report describes a cat with severe gastric ulceration caused by intoxication with dieffenbachia leaves. in some case reports, the cause for the gastric ulcerations could not be determined. a minimum database should be collected for cats suspected of gastric ulceration, to identify underlying diseases. anemia, usually regenerative, may be present. eosinophilia, or hypoproteinemia. survey and contrast radiographs are often normal. the most common finding on histopathologic examination of biopsy samples is lymphocytic plasmacytic (lp) gastritis ( figure - ) . some patients will also have concurrent evidence of lp inflammation in the small intestine, pancreas, and/or liver. such patients will be treated for their concurrent problem; treatment of inflammatory bowel disease, pancreatitis, and cholangiohepatitis is covered elsewhere in this chapter. some cats with chronic lp gastritis respond to treatment for dietary intolerance or hypersensitivity with a limited antigen diet (see chapter ) . patients with moderate to severe lp gastritis may be best treated with a limited antigen diet and immunosuppressive therapy (prednisolone to mg/kg/day, po tapering to every other day at the lowest dose that controls clinical signs). patients that fail this initial treatment approach may require additional immunosuppressive therapy, such as chlorambucil (see table - ) . occasionally, cats with chronic gastritis are diagnosed with eosinophilic inflammation on histopathologic examination of biopsy specimens. treatment is similar to that for lp gastritis, although such patients should be evaluated for evidence of hypereosinophilic syndrome and eosinophilic enteritis. eosinophilic fibrosing gastritis was suspected to be caused by toxoplasmosis in one case report. gastric or gastroduodenal ulcerations are uncommon in the cat compared with the dog and may be caused by a variety of disorders, both gastric and nongastric. classical clinical signs include vomiting, hematemesis, and melena. however, in one review of eight cats, hematemesis and melena were present in less than one third of suturing of the ulcer site as well as collection of biopsy samples for histopathologic examination. the prognosis for recovery was excellent in two studies, particularly for cats with non-neoplastic causes of gastric or gastroduodenal ulceration. , in one study of seven cats with perforated gastric or duodenal ulcers, the survival rate was low ( %). disorders of gastric motility are better characterized in dogs than in cats. the most common clinical sign is vomiting of undigested food hours or more after a meal. if outflow obstruction is present, vomiting may be projectile. there may also be a history of recurrent trichobezoars. various disorders are associated with impaired gastric motility, such as chronic gastritis, drug therapy (e.g., anticholinergic and narcotic drugs), dysautonomia, gastric neoplasia, metabolic disorders (e.g., hypokalemia), and temporary postsurgical gastroparesis. in some cases of chronic motility dysfunction, no cause can be identified. outflow obstruction may be caused by neoplasia, foreign bodies, and extragastric masses. pyloric stenosis is infrequently documented in young cats, often siamese cats. , , since the range of underlying disorders is diverse, the diagnostic approach should allow for detection of both gastric and nongastric disorders. a minimum database (cbc, serum chemistries, urinalysis, feline leukemia virus [felv] and feline immunodeficiency virus [fiv] serology) is used to establish overall health status. radiographs are used to confirm presence of food in the stomach for longer than hours. ultrasonography may detect gastric lesions, such as masses. endoscopy is used to identify outflow obstruction as well as other lesions, such as ulcers, and evidence of gastritis. assessment of gastric emptying using nuclear scintigraphy is the most accurate method but is limited to referral centers. gastric emptying times for liquids, canned food, and dry diets have been established using nuclear scintigraphy. , , however, emptying times are variable, depending on the amount and type of diet fed as well as the amount of water ingested. even the shape of kibble affects emptying time. radiographic contrast series are widely used, but gastric emptying times are variable for barium in either liquid form or mixed with canned food. contrast radiography using liquid barium ( to ml/kg) is performed in a fasted patient. radiographs are taken immediately after administration of the barium and again at and minutes, in some cases, also at and hours. liquid barium is expected to enter the duodenum no more than minutes after administration, and the stomach should be completely empty of barium within hours. the clinician should be aware that some cats with gastric motility disorders will have other findings will be dependent on the presence of underlying diseases; for example, azotemia and isosthenuria may indicate renal disease. electrolyte and acidbase abnormalities may be because of chronic vomiting and anorexia. survey and contrast radiographs and ultrasonography are primarily useful to rule out other causes for the clinical signs, such as foreign bodies. cats with perforated ulcers may have evidence of pneumoperitoneum (sometimes severe) on plain radiographs or ultrasonographs, and this is an indication for surgical exploration. , , , , evidence of peritonitis on imaging studies should be followed with peritoneal fluid analysis. a definitive diagnosis may be made using endoscopy, which allows direct visualization of lesions and collection of biopsy samples. however, some cats with gastric ulceration present in poor condition, which may preclude the use of endoscopy because of anesthetic risk and risk of ulcer perforation. the location of ulcers is typically pyloroantral or fundic in cats with nonneoplastic disease. , areas of erosion may appear pale or hemorrhagic; the mucosa is often friable and bleeds easily. fresh or clotted blood may be seen in the stomach lumen. in some cases, mucosal ulceration must be distinguished from ulcerated tumors. nsaid-induced ulcers are typically found in the antrum and do not have marked mucosal thickening; ulcerated tumors frequently have thickened edges and surrounding mucosa. biopsy samples should be taken at the periphery of the ulcer to avoid perforation. treatment should be directed at any underlying disorder. treatment for nsaid toxicity is described in chapter . general supportive measures include fluid therapy and electrolyte replacement; blood transfusion may also be required (see chapter ) . gastric acid production can be decreased with the use of h -receptor blockers or proton pump inhibitors, and sucralfate is used as a mucosal protectant (see table - ). sucralfate may inhibit absorption of other oral medications and should be given hours apart from other drugs. if vomiting is severe or persistent, antiemetic therapy is warranted (see table - ). analgesia should be provided for painful patients; a good choice is the opioid buprenorphine (see table - ). broad-spectrum antibiotic therapy is indicated for patients with significant mucosal barrier dysfunction, perforation, leukopenia and/or neutrophilia, fever, and melena. surgical intervention is warranted for patients with life-threatening hemorrhage, failure to respond to medical management, or evidence of perforation. the entire abdominal cavity and gastrointestinal tract should be thoroughly explored to locate extragastrointestinal lesions, non-perforated ulcers, and multiple ulcers. in one case series, nonperforated ulcers were detected at laparotomy by association with adhesions or a gastric mass. surgical management includes débridement and months. physical examination findings are nonspecific, although occasionally a gastric mass or gastric thickening may be palpated if the stomach is markedly enlarged. results of routine diagnostic testing are generally nonspecific; anemia may be associated with ulceration. survey or contrast radiography may reveal a mass ( figure - , a); other findings include delayed gastric emptying, impaired motility, and mucosal ulceration. ultrasonography is also useful for diagnosis and can be used to guide needle aspirates of masses ( figure - , b). endoscopy allows for visualization of lesions as well as the ability to obtain partial thickness biopsy samples. problems with interpretation of endoscopic biopsy samples include detection of necrosis, inflammation, and ulceration rather than the primary lesion. in dogs some neoplastic lesions are submucosal, making it very difficult to obtain diagnostic samples by endoscopy. therefore several biopsies should be taken and masses should be biopsied multiple times in the same place to sample deeper tissues. the center of ulcerated lesions should not be biopsied. surgical biopsies are more reliable for diagnosis. a normal gastric emptying time with liquid barium. barium can also be mixed with canned food and fed as a meal; retention of barium-containing food in the stomach for more than to hours is abnormal. gastric emptying time may also be established with the use of barium impregnated polyspheres (bips; med i.d. systems, grand rapids, mich.) and radiography. gastric emptying times for bips have been established in healthy fasted and fed cats as well as in sedated cats, , but the values do not correlate well with scintigraphic studies. a mixture of small ( . mm) and large ( mm) spheres are administered with food, and two to four radiographs are taken over the next hours. the small spheres are intended to mimic liquid transit time and the large spheres solid transit time. however, studies assessing the clinical relevance of this method are lacking. one review concluded that bips are probably sufficiently sensitive to detect grossly delayed gastric emptying. treatment of gastric emptying disorders is directed at identifiable causes. treatment for gastric ulcers, chronic gastritis, and foreign bodies is described elsewhere in this chapter. pyloric stenosis is managed surgically. if no outflow obstruction exists, treatment with prokinetic agents, such as metoclopramide or cisapride, may be beneficial (see table - ). gastric tumors account for less than % of malignancies in dogs and cats. benign gastric tumors are even less common than gastric malignancies. gastric smooth muscle hamartoma has been reported in one -year-old cat. although adenocarcinoma is the most common gastric cancer of the dog, lymphoma is the most common gastric cancer in the cat. feline gastrointestinal lymphoma occurs as two major types: small cell (lymphocytic) and the more aggressive large cell (lymphoblastic) form. small cell lymphomas are more frequently enteric. in one study of cats with gastric lymphoma, diffuse large b-lymphocyte tumors of immunoblastic nuclear type predominated. gastric lymphoma is not associated with felv, and the role of helicobacter in the development of gastric lymphoma in cats requires investigation. adenocarcinoma, , , plasmacytoma, and gastric carcinoid have also been described. the siamese cat may be predisposed to adenocarcinoma. , as would be expected, most cats with gastric neoplasia are older cats. as for most gastric diseases, vomiting is the most common clinical sign of neoplasia. the vomitus may contain blood and melena may be present. other clinical signs include anorexia, weight loss, bloating, and depression. perforation of the tumor may occur, leading to pneumoperitoneum or septic peritonitis. clinical signs present gradually and are often present for weeks to surgical resection is the most common treatment for gastric neoplasia other than lymphoma ( figure - ). the prognosis for most patients is poor, typically because of debilitation, concurrent diseases, and recurrent or metastatic disease. the success of chemotherapy for lymphoma depends on cell type, with small cell tumors carrying a better prognosis than large cell tumors. in diarrhea can be defined as increased volume and/or increased frequency of defecation of stools with increased water content. approaches to diarrhea, as for any clinical sign, need to take into account the individual animal. for example, neoplasia is much less likely to occur in a kitten than in a geriatric cat. in many cases, the precise diagnosis of gastrointestinal disease cannot be reached without biopsy samples. the decision to obtain biopsy samples should follow a logical pathway that is appropriate to the cat's condition. these are summarized in figure - . for example, many cases of acute diarrhea in a well cat can resolve with limited or no intervention, and so do not require a precise diagnosis. the diagnostic steps are . signalment and clinical history . physical examination . fecal assessment . blood and urine testing . imaging (radiography, ultrasonography) . biopsy samples these steps do not include treatment/diet trials or other empiric therapies that are appropriate in many cases. steps and are often undertaken at the same time, and there is no definite order for these steps. they are divided here for reasons of clarity. in a younger cat, where infectious causes are more likely, thorough fecal testing is more important; in an older cat, extragastrointestinal diseases, such as hyperthyroidism, are more likely; so, blood and urine testing is more important, but fecal assessment should not be neglected. the decision to proceed to step (and each subsequent step) should take into account several considerations. the main considerations in assessing and managing a cat with diarrhea are • is there an acute onset or a chronic time course? • are there any dietary changes or indiscretion? consider treatment trials: antibiotics (e.g., amoxicillin/ clavulanate ؉ metronidazole). food trials with novel proteins (e.g., rabbit, kangaroo, venison). • is the cat well or unwell? • is there primary or secondary gastrointestinal disease? • is there small or large bowel diarrhea? the components of the clinical history for cats with diarrhea are detailed in table - . after establishing the cat's age, breed, vaccination, and deworming history, it is important to establish the duration and nature of the diarrhea. chronic diarrhea is usually defined as greater than weeks in duration and mostly warrants at least some degree of a diagnostic workup, whereas acute diarrhea is often self-limiting in a well cat. a description of the feces helps determine whether the diarrhea is small or large bowel in origin (table - ); this will affect how any investigations might proceed. important questions to ask concern frequency of defecation (and how this compares with the normal state), tenesmus (straining usually indicates large bowel diarrhea, since an irritated colon leads to urgency), volume of feces (smaller volumes are typical of large bowel diarrhea; larger volumes are more typical of small onset and duration of diarrhea acute versus chronic? acute diarrheas are abrupt in onset and of short duration, and generally they are self-limiting. chronic diarrheas persist usually longer than weeks and fail to respond to symptomatic therapy. appearance of diarrhea quantity and quality of the stool (color, consistency, character, presence of blood or mucus)? loose to watery feces that contain fat droplets, undigested food, melena, and variable colors suggests small intestinal disease. the volume is always increased with small intestinal disease. loose to semisolid feces containing excess mucus and fresh blood (hematochezia) indicates large intestinal disease. the volume may be normal to slightly decreased with large intestinal disease. description of defecation process tenesmus (straining) and dyschezia (painful defecation)? these are hallmarks of large intestinal disease (e.g., inflammatory or obstructive lesions of the colon, rectum, or anus). frequency is normal to slightly increased with small bowel disease, but greatly increased with large bowel disease. associated physical signs vomiting, anorexia, weight loss, and dyschezia may help localize the disorder to a specific part of the gastrointestinal tract. clinical signs relating to problems in other organs or body systems should be noted and may suggest a more generalized disease. vomiting may occur as a consequence of small intestinal inflammation in some cats with diarrhea. weight loss may result from decreased caloric intake (anorexia), decreased nutrient assimilation (maldigestion/malabsorption), or excessive caloric loss (protein-losing enteropathy or nephropathy). weight loss is observed uncommonly with large bowel disease. in many cases, the answers to these questions are obvious. for example, a cat may seem well but has had access to lilies (the author has seen diarrhea as a primary presenting sign for this!) or has a palpable abdominal mass. substantial weight loss is an indicator that further investigations are warranted sooner rather than later. if the decision is made for empiric management and outpatient care, it is vital to follow up either by scheduling a recheck visit or calling the client, because simple acute problems can turn into complicated chronic problems. if the diarrhea has been present for less than a week and the cat has no weight loss, dehydration, fever, or palpable abdominal abnormalities, it is appropriate to manage the cat as an outpatient. even in the absence of fecal testing, it is appropriate to deworm the cat (see the section gastrointestinal parasites). the cat should be fasted for hours ( hours, if less than months old) and then fed a bland diet (such as plain, cooked, skinless chicken, or low-residue prescription diets designed for cats with gastrointestinal problems). it is appropriate to maintain the cat on the low-residue diet for at least to days and then slowly reintroduce the regular diet. fecal assessment is mostly used to assess infectious agents, such as parasite-associated diarrhea, but the importance of assessing feces, even when parasitic or bacterial infections are not suspected, should not be underestimated. gross examination of feces can determine if melena or fresh blood or mucus are present to help distinguish large from small bowel disease when the owner's observations may be misleading. occult fecal blood can be an indicator of gastrointestinal inflammation in cases of subtle disease, and undigested starches and fats can indicate maldigestion or malabsorption. for assessment of feces for parasites, the fecal sample should ideally be fresh (< hour old). refrigeration (for no longer than one week) can preserve ova, oocysts, and cysts but not protozoal trophozoites. feces should be assessed by a. to assess for trophozoites bowel), how formed the stool is (from soft stool to cow-pat consistency to liquid tea; usually more watery stool relates to small intestinal disease), color (darker indicates digested blood), and presence of any mucus or blood (presence relates to large bowel). most household toxins, such as plants, cause signs additional to diarrhea such vomiting or neurological signs, but it is important to ascertain if the cat has had access to anything unusual. likewise, it is important to find out if the cat has had any possible exposure to dietary indiscretions; this can include if the cat has been seen with or is known to hunt prey including insects. cockroaches carry pathogenic bacteria , and other prey such as birds and rats can carry salmonella; salmonellosis in cats has been dubbed songbird fever. simple causes of self-limiting diarrhea include dietary change (either a new flavor or a new style of food, such as dry food for the first time); so, the owner must also be quizzed if anything new has been offered, either new cat food or treats (such as greasy fish or chicken). although the physical examination will usually determine how unwell a cat is, the owner's impressions are also important, because cats can hide signs from strangers, particularly in a practice setting. lethargy and inappetence are important signs, as ill cats typically do not eat well. the cat's general demeanor can be an indicator of how unwell a cat is and therefore dictate the extent of diagnostic testing required. this can be noted by assessing how interested the cat is in its surroundings or any behavior changes from previous visits, such as if a normally difficult-to-handle cat is placid. body weight should be assessed and, if possible, compared with that of previous visits (even those noted on a clinical record from another veterinarian). the body condition score (bcs) should also be assessed and can be very important when there is no prior weight information. dehydration is usually a sign that a cat needs more involved management. abdominal palpation should be performed to assess pain (where?), any masses (foreign bodies, lymph nodes, or even focally thickened intestines, such as with neoplasia), or turgid intestines. fever often indicates infection but can also reflect neoplasia or other inflammatory changes. a thorough examination of all body systems should always be performed, no matter what a cat presents for. in the case of diarrhea, extragastrointestinal signs can be of vital importance, such as a palpable thyroid and tachycardia suggesting hyperthyroidism. after the clinical history has been taken and the physical examination performed, the veterinarian must make the important decisions of whether any interventions are required and whether the patient should be factors affecting interpretation include whether the growth is a heavy and pure growth of a known pathogen, such as salmonella, campylobacter, yersinia, or clostridium difficile. further information about the relevance of culture and pcr results is contained below in the section infectious enteritis. investigations begin by assessing if the diarrhea is the result of primary gastrointestinal disease or secondary to another process, by performing routine serum/plasma biochemistries, hematology, urinalysis, and total t (for older cats). in most cases of secondary gastrointestinal disease, diarrhea is not usually the primary presenting complaint, but since the approach to investigations and management diverge so much, this is an important step to take. biochemistry and urine tests may also show the consequences of diarrhea, such as dehydration and electrolyte abnormalities. a. can aid in the visualization of internal structures of some protozoa . fecal flotation (preferably with centrifugation) a. to find cysts, oocysts, and ova fecal culture should be undertaken with the understanding that bacteria will be cultured; so, interpretation is based on the relevance of the positive culture result. used to evaluate the smear for the presence of trophozoites, such as giardia spp. and tritrichomonas foetus. . place peppercorn size amount of feces on a warm slide and mix with a drop of . % saline (smear must not be too thick, because trophozoites will be easily missed). . apply coverslip. . evaluate systematically for motile organisms using the × magnification. . confirmation at × magnification. adding iodine to a wet mount through the edge of the coverslip can aid in the visualization of internal structures of some protozoa. the direct wet preparation must be examined without any stain for motility first, because staining the preparation kills the organism. methylene blue is useful for identifying trophozoites, particularly those of entamoeba histolytica. this method has little to no diagnostic value for the diagnosis of bacterial-associated diarrhea. used to find cysts, oocysts, and ova in feces. standing (gravitational) flotation methods are easier and quicker but have much poorer sensitivity than centrifugation methods. solutions used in centrifugation flotation methods include zinc sulfate and sheather sugar. . weigh out to g of feces. . mix feces with approximately ml of flotation solution. . pour mixture through a tea strainer into a beaker or fecal cup. . pour strained solution into a -ml centrifuge tube. . fill tube with flotation solution so that a slight positive meniscus forms, being sure not to overfill the tube. . place a coverslip on the tube, and put the tube in the centrifuge. . make sure the centrifuge is balanced. . centrifuge at rpm ( × g) for minutes. . remove the tube and let stand minutes. . remove the coverslip, and place it on a glass slide. systematically examine the entire area under the coverslip at × magnification (i.e., × objective). you may wish to use the × objective lens to confirm your diagnosis and make measurements; however, with practice, most parasites can be identified using the × objective ( × magnification). (fpli) are useful markers of intestinal and pancreatic disease, [ ] [ ] [ ] [ ] but it is important to note that they typically do not give a precise diagnosis. cobalamin and folate are water-soluble vitamins and are readily found in commercial cat foods so that dietary insufficiency is rare, and decreased levels are almost always because of gi disease. these vitamins are taken up by specific receptors in different areas of the small intestine. chronic inflammatory gastrointestinal disease may damage the receptors and lead to decreased serum concentrations of one or both vitamins, provided the disease process is severe and long standing enough to deplete body stores. serum cobalamin and folate concentrations may also be decreased in cats with exocrine pancreatic insufficiency (epi). trypsin-like immunoreactivity is a pancreasspecific marker, and assessment of serum tli is used for diagnosis of epi and pancreatitis in the cat, although the sensitivity of the assay for pancreatitis is low. pli is a marker for pancreatic inflammation and is more sensitive than tli for the diagnosis of pancreatitis. since inflammation of the small intestine may be seen concurrently with pancreatitis, serum tli and pli are useful adjunctive tests in the diagnosis of diarrhea. tli, pli, and cobalamin are stable in serum at room temperature for several days, but folate is unstable so that samples for cobalamin/folate analysis should be frozen (table - ) . samples submitted for folate concentration should not be hemolyzed, because red blood cells contain high levels of folate. in addition, folate is light-sensitive, and samples should be wrapped to exclude light. severe lipemia may interfere with common assays for tli and pli. the main utility of these tests are to indicate that further investigation of gastrointestinal disease is warranted. when a cat presents for weight loss with no overt signs of gi disease, decreased cobalamin or folate can indicate that further investigations with imaging and, ultimately, biopsy sampling are warranted. many clients are more willing to proceed with hematology can be normal in some cats, with changes expected, and so should not be used to rule out any condition. it can be useful, for example, if there is a left shift neutrophilia, indicating acute infection, or eosinophilia, reflecting parasitism. monocytosis can suggest chronic disease that was not suggested by the clinical history. in the case of acute onset diarrhea, the cat may be unwell as a consequence of the diarrhea (e.g., from dehydration) and not because of the cause of the diarrhea. if rehydration is required (with intravenous or subcutaneous fluids, depending on severity of illness), then it is important that biochemistry tests are performed before fluid administration so that any diagnostic clues are not lost by alteration of the profile from the fluid therapy. fever and neutrophilia may indicate the need for antibiotic therapy. if infection is suspected, fecal sampling (see step ) should occur before starting antibiotics. if a cat is unwell from dehydration, then further testing may not be warranted. the clinician should be alert that linear foreign bodies can result in diarrhea (see the section intestinal obstruction). diarrhea of chronic duration (greater than weeks) does require a more thorough investigation at the outset. however, if clinically well, the cat can be managed as an outpatient in the first instance, at least while waiting for results of diagnostic testing. a diet trial with a novel protein is appropriate for a well cat with stable weight. as with any patient managed as an outpatient, follow-up is vital and, in this scenario, includes scheduling revisits. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity invasive diagnostics when a specific marker of the disease in the organ involved has been recognized. caution should be exercised, because either cobalamin or folate may not be reduced with gi disease. in one study of small cell lymphoma, only % of cats were hypocobalaminemic, meaning that if this was the only instigating factor to investigate, nearly one fourth of cats would not have been investigated further. also, cobalamin may be reduced in nonalimentary illness. . to detect hypocobalaminemia that may indicate the need for supplementation for clinical improvement. to recognize pancreatic pathology when fpli is increased. it is important to note that an elevated value gives no indication of the nature of the pancreatic pathology. . to make a diagnosis of epi when the ftli is low. it should be noted that epi can result from other pathology that may require further investigations. veterinarians if the patient is new to the practice) are usually helpful. body condition scoring (using a -point or -point scale) for every cat seen is helpful in recognizing those that are underweight. weight loss often occurs with loss of muscle mass in cats, and muscle mass can be assessed over the ribs and pelvis as well as scapulae and nuchal crest. thickened intestines are also a subjective finding; it is the author's opinion that thickened intestines are actually intestines with increased turgidity, since differences between normal intestines and those with inflammatory infiltrates can be as little as . mm. perhaps more important during the history taking and physical examination are those signs that can point to extragastrointestinal disease. when confronted with a cat showing weight loss or vomiting or diarrhea (or a combination of signs), the clinician should start with trying to distinguish the signs as being either primary gastrointestinal or secondary signs. examples of clues pointing to extragastrointestinal diseases include tachycardia and palpable thyroid nodule, indicating hyperthyroidism, or polydipsia/polyuria, which has a variety of causes but is not typical of primary intestinal disease. inflammatory bowel disease has traditionally been considered an immune-mediated disease. the local immune system of the intestinal mucosa no doubt plays an important role, but recent work has also shown the importance of the normal bacterial population in perpetuating and, perhaps, even initiating pathology. it is known for certain that ibds are an expression of an overanxious immune response, with a recent study indicating increases in inflammatory (il- ), type- immunity (il- p ), and immunomodulatory (transforming growth factor [tgf]-beta, il- ) cytokines. other researchers have found an association with bacterial counts (enterobacteriaceae, e. coli, and clostridium spp.) and abnormalities in mucosal architecture, indicating that mucosal bacteria are involved in the etiopathogenesis. we can summarize these theories by saying that ibds are likely to be a consequence of hypersensitivity reactions to antigens from the intestinal lumen (e.g., bacterial, parasitic, or dietary antigens). this hypersensitivity may occur because of failed immunoregulation (suppressive function) of the gut-associated lymphoid tissue (galt). it is known that granulomatous colitis in boxer dogs is associated with infection, and pathogens may well be found in at least some cases of ibd in cats that cause the immune response and subsequent inflammatory infiltrate of the lamina propria typically seen. although not described specifically in cats, chronic intestinal inflammatory change can impair motility. inflammatory bowel disease (ibd) refers to intestinal inflammatory infiltrates of the small or large intestine (or both) of unknown etiology. the term ibd should strictly be applied to mean idiopathic ibd, thus excluding inflammatory enteritis because of food sensitivities, although common usage has led to ibd referring to intestinal inflammatory infiltrates of both known and unknown causes. ibd is not a diagnostic end point but a description of a series of intestinal diseases that have similar histopathology. recent efforts by the world small animal veterinary association (wsava) gastrointestinal standardization group have led to both diagnostic and classification guidelines , , that encompass chronicity, nonresponse to symptomatic treatment, no specific cause found, as well as histologic confirmation of non-neoplastic intestinal inflammatory changes. there are no obvious breed or gender predispositions, and although cats of any age can be affected, inflammatory intestinal diseases are more likely to occur in middle-aged to older cats ( to years of age or older) than in younger cats. presenting clinical signs include vomiting, diarrhea, and weight loss with increased or decreased appetite. these signs can occur in isolation or together. weight loss without vomiting or diarrhea deserves special mention because not only have several studies , shown this to be the most common presenting sign for ibd, but many veterinarians do not consider primary intestinal disease without the presence of vomiting or diarrhea. weight loss despite normal to increased caloric intake can represent poor absorption of food because of small intestinal disease, although it can also represent maldigestion associated with exocrine pancreatic insufficiency or increased metabolism associated with hyperthyroidism, or even lack of energy utilization associated with diabetes mellitus. conversely, appetite may be reduced, most likely because of nausea. if the large bowel is affected, signs are typically discomfort when defecating, resulting in frequent small volumes of diarrhea, often with mucus and blood; if the large bowel alone is affected, there may be no weight loss. physical examination findings are often nonspecific, but the most consistent findings for small intestinal disease are weight loss (or being underweight in a cat not seen previously) and palpably "thickened" intestines. noting a cat as underweight can be subjective, and prior recorded weights (even from previous paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma than those with ibd, but this change was also seen in % of cats with normal small intestine. inflammatory bowel diseases require histologic findings obtained from biopsy samples for diagnosis, but diagnosis should not be made solely on these findings. the wsava international gastrointestinal standardization group has proposed "an all encompassing definition of inflammatory bowel disease" that comprises clinical criteria, imaging criteria, as well as pathophysiologic criteria. the clinical criteria for the diagnosis of ibd include there are no typical laboratory findings in ibd, and many cats may have entirely normal results from routine biochemical and hematologic investigations. moderate liver enzyme elevations may be seen , , , even in the absence of recognizable hepatic pathology, and this may reflect subclinical secondary hepatic disease, secondary cholestasis, or showering of the liver with inflammatory cells from the small intestine through the portal circulation. other changes can reflect consequences of the intestinal disease, such as azotemia or hemoconcentration reflecting dehydration, or hypokalemia reflecting inappetance. the chronic inflammation may be reflected by neutrophilia, monocytosis, , , , or hyperglobulinaemia. hypocobalaminemia can reflect ileal inflammation, and low serum folate can reflect proximal small intestinal inflammation. typical ultrasonographic findings consistent with ibd are focal or diffuse intestinal wall thickening ( figure - ) ; normal wall thickness is less than or equal to . mm for the duodenum and less than or equal to . mm for the ileum, and large mesenteric lymph nodes with hypoechoic changes may be seen. one study found that ultrasonographic findings correlated with histologic grade of ibd. there is no clear distinction between ultrasonographic changes from ibd and those from small cell lymphoma. one recent therapeutic trial, follow-up visits are vitally important. many cats with small intestinal disease may show initial improvement simply because of the diet having lower residue, since there is decreased substrate for intestinal bacteria to digest and lower osmotic potential. the corollary of this is that failure of one novel protein diet does not mean that all novel protein diets will fail. when food sensitivities are responsible for gastrointestinal clinical signs in cats, the responsible food ingredient is usually a dietary staple. commonly incriminated ingredients are beef, fish, wheat, and corn gluten. a careful dietary history is therefore important. large bowel inflammation typically improves with higherfiber diets, , and attempting a trial with such a diet is certainly appropriate. immune suppressive therapy is the mainstay of ibd treatment, and glucocorticoids, such as prednisolone, are most commonly used. sulfasalazine use for large bowel signs has not been critically evaluated but seems safe and effective. in cats with substantial weight loss or severe clinical signs, such as chronic diarrhea, the author prefers to start with corticosteroid therapy, even if dietary causes have not yet been ruled out. the diet should also be changed to one containing a novel protein, and, if and when clinical signs resolve, an attempt is made to wean the cat from corticosteroid therapy, hopefully to the point of being discontinued. a diet challenge can then be used to confirm the diagnosis of food sensitivity. there are no universal guidelines for doses of corticosteroids. the author prefers the use of orally administered prednisolone to reduce the chance of side effects and will choose the starting dose based on the severity of disease. the starting dose is usually mg/kg, once daily, po ( mg/cat/day for most cats) starting days after biopsies have been obtained to allow time for the mucosa to heal. if there is an improvement noted after a recheck at weeks, the higher dose is maintained for a further to weeks, at which point, many cats are back to their normal weight and are not exhibiting clinical signs. if this is the case, the corticosteroid dose can be weaned down to mg/kg, po (often mg/cat/day) for several months, with continued rechecks scheduled to assess weight, clinical signs, and diet. the goal is to wean down to the lowest effective dose. if hypocobalaminemia is present, cobalamin supplementation may be required. cobalamin is administered parenterally at µg/cat subcutaneously weekly for weeks, then every second week for weeks, then monthly. owners can be shown how to inject their cats (as practitioners routinely do with diabetics). clinicians often consider the assessment of histologic samples to be out of their hands; however, it is important to work with the pathologist by providing good quality samples and a good clinical history, as well as having an open dialogue if the findings are not within expectations. for example, with lymphocytic/plasmacytic infiltrations, the pathologist has the difficult task of distinguishing diseased from normal tissue in a site that is laden with lymphocytes in the healthy state. once deciding the tissue has pathology, the pathologist's next task is distinguishing inflammatory infiltrate from neoplastic infiltrate with normal, mature lymphocytes (as seen in small cell lymphoma). inflammatory change also results in changes to normal tissue architecture, with thickened villi, edema, or erosion of the epithelium being typical changes. clinicians should expect morphologic descriptions as well as assessments of degree and type of inflammation. these difficulties are further compounded with the recognition that histologic grading of mild, moderate, or severe does not necessarily correlate with severity of clinical signs. this means that a cat with severe clinical signs of weight loss and vomiting or diarrhea may have only mild histologic changes (and vice versa). concurrent inflammation of the pancreas and liver with intestinal inflammation was first described in the mid- s, and despite constant reference to this phenomenon at conferences and veterinary websites, there has been little description since then, though one study found % of ibd cases had liver inflammation and % had pancreatic inflammation. the term "triaditis" has frequently been used, but the author prefers to spell this "tri-iditis" to distinguish it from inflammation of the hepatic portal triads. there has been no assessment of prognosis when the pancreas and/or liver are involved, but the author has found no difference in prognosis. many cases diagnosed with intestinal inflammatory infiltrates have these changes because of dietary sensitivity. in one study, % of cats with histologic gastrointestinal changes improved with dietary elimination therapy alone. interestingly, improvement was noted within days compared with the longer duration of weeks often recommended for improvement of dermatologic manifestations of food sensitivities. this careful study made note of the cat's prior diets and likely dietary causes of sensitivities. another study found dietary therapy to be unsuccessful in of cats but no specifics of diets tried are noted. as with any national cancer institute working formulation (nci wf) system. for most veterinarians in practice, the most important distinction is the histologic grade, because low-grade (lymphocytic or small cell) lymphoma has a much better prognosis (and requires different treatment) compared with high-grade (often lymphoblastic) or intermediategrade lymphoma. for the purposes of simplicity and practicality, only small cell lymphoma and high-grade lymphoma will be addressed here. the prognosis and treatment for intermediate-grade intestinal lymphoma should be considered as for high-grade lymphoma. small cell lymphoma was first described in human pathology in . earlier, small lymphocytes were considered end-stage cells without the ability to divide. in cats small cell lymphoma is most commonly associated with the gastrointestinal tract or skin. small cell neoplasia can be a confusing concept, since our traditional ideas of malignant neoplasia focus on rapidly dividing cells. the confusion is compounded by various terms used in the literature, such as lymphocytic lymphoma, low-grade lymphoma, well-differentiated lymphoma, or diffuse lymphoma; another term, epitheliotropic malignant lymphoma predominantly applies to small cell lymphoma, and other papers fail to distinguish these lymphomas from lymphoblastic lymphosarcoma (the traditional, aggressive form). "small cell lymphoma" seems to be most widely used term, though the author prefers "lymphocytic lymphosarcoma," since it is more descriptive. intestinal small cell lymphoma can be considered as a severe lymphocytic intestinal infiltrate, the most common form of which is commonly called ibd. not only is lymphocytic ibd hard to distinguish histologically from lymphocytic lymphosarcoma, but the approaches and treatments are similar. several reports have suggested a relationship between the two conditions in that inflammatory infiltrates may become neoplastic over time. , , prevalence the true prevalence of intestinal small cell lymphoma is unknown, but several recent studies have indicated similar rates to inflammatory bowel diseases, with kleinschmidt et al noting small cell lymphoma cats compared with with intestinal lymphocytic infiltrates, evans et al reporting cases compared with with ibds , and baral et al diagnosing cases compared with with ibds. traditionally, % of feline lymphosarcoma is regarded as intermediate or high grade, but this may not be the case within the gastrointestinal tract. fondacaro et al found % of gastrointestinal lymphoma to be lymphocytic ; a more recent paper found some cats seem resistant to conventional therapy. if this is the case, the diagnostic findings should be re-assessed to ensure no steps were missed or findings disregarded; the cat should be reexamined to look for emergence of other signs; and the pathologist who reads the histology should be contacted to recheck the findings. some cases of apparently resistant ibd are actually food sensitive, but it can be difficult to find the incriminating diet source, and commercial diets are not always effective. if underlying infectious causes have been entirely ruled out and the practitioner is certain of the diagnosis of idiopathic disease, immune suppressive therapy can be increased by either increasing the dose of prednisolone or using other agents, such as chlorambucil, typically at mg/cat, po, every second day. it has been suggested that cats with eosinophilic inflammation may be more likely to be refractory to standard therapy. side effects of immunosuppressive therapy are rare but include inducing diabetes mellitus, immune suppression, delayed healing, and gastrointestinal ulceration. reported doses of sulfasalazine to manage large bowel ibd are to mg/kg, po, once daily for to days. because this drug is usually only available as mg tablets, one eighth of a tablet, providing a dose of . mg, is usually appropriate for most cats. in some countries, it is possible to have a compounding pharmacist formulate the drug into more convenient tablet sizes or as an oral suspension. cats are generally regarded as susceptible to salicylates, and possible side effects include vomiting or diarrhea, or anemia. the exact pharmacodynamics of this drug are not known; so, caution for extended use should be exercised and the drug withdrawn if any possible adverse signs are noted, but there are anecdotal reports of extended use of this drug without adverse consequences. a survey of the online veterinary cancer registry (http://www.vetcancerregistry.com) identified % of all submitted feline tumors to be intestinal tumors. approximately % of reported feline small intestinal tumors were lymphomas. adenocarcinomas accounted for %, and other tumor types reported included mast cell tumors and leiomyosarcomas. "lymphoma in veterinary medicine: no longer a oneword diagnosis" was the title of an editorial in a recent issue of the veterinary clinical pathology journal, and this is nowhere truer than in the feline gastrointestinal tract! a recent study classified cases of feline gastrointestinal lymphoma both histologically and immunophenotypically, and it found eight different categories according to the revised european and american lymphoma/world health organization (real/who) classification system and six categories according to the approximately equal numbers of high-grade and lowgrade gastrointestinal lymphoma. older cats are more at risk of small cell lymphoma, with mean or median ages reported from to years. younger cats with the disease have, however, been recognized. , , , no breed or gender predispositions have been definitively recognized. two larger studies have suggested a skew to males with males compared with females in one report, and males compared with females in the other ; most other studies looking at gender and breed did not clearly distinguish between lymphoblastic and lymphocytic neoplasia. clinically, it is impossible to distinguish cats with ibds from cats with small cell lymphoma. this is hardly surprising when even histologic distinction can be difficult! therefore cats will present with weight loss or vomiting or diarrhea at a similar frequency to those with ibd. weight loss has been recognized as a presenting sign in % to % of cases, diarrhea in % to % of cases, and vomiting in % to % of cases, with various combinations of these signs also possible. other variable signs are lethargy and inappetence or, conversely, polyphagia. , , , these findings can be summarized by stating that cats with gastrointestinal small cell lymphoma can present with any combination of signs relating to the gastrointestinal tract. intestinal small cell lymphoma is typically a diffuse disease, and therefore multiple areas of the alimentary tract are usually affected. in studies where different locations of the small intestine were assessed, the jejunum was most commonly affected ( %), with the ileum frequently affected ( % to %), and duodenal pathology slightly less prevalent ( % to %). , although the numbers of cats assessed in these studies are small, the important fact that the duodenum is not always affected needs to be recognized, which has important implications for how biopsy samples are obtained, because lesions beyond the duodenum are likely to be beyond the reach of an endoscope. further difficulties in precise diagnosis may arise, since non-neoplastic lymphocytic infiltrates (e.g., ibd) are often found in other locations along the intestinal tract. , , the stomach is also affected in % to % of small cell lymphoma cases. , , although not fully assessed, involvement of the colon appears rare. local lymph node involvement is common, being noted in up to % of cases. this percentage may be even higher, because many studies assessed lymph node cytology from ultrasound-guided fine-needle aspirates, which may miss spread to the lymph node, because the population of neoplastic lymphocytic cells is indistinguishable from the normal population of lymph node cells. histology is required to assess changes in lymph node architecture. liver involvement is not uncommon but not thoroughly assessed. one study noted liver lymphocytic neoplasia in of cats with small intestinal lymphocytic neoplasia, another found of affected cats in which the liver was biopsied, another noted of cats had liver involvement, and a further study detected neoplasia "in the lymph nodes, liver, or both" in all cats with intestinal small cell lymphoma. the pancreas may also be involved. , this may be akin to the noted association of lymphocytic inflammation of intestine, pancreas, and liver that has been dubbed tri-iditis. ultrasound findings may not suggest extragastrointestinal involvement. in the case of liver pathology, ultrasonography may show no changes in as many as % to % of cases. , focal nodular changes and he patomegaly have been recognized as ultrasonographic signs of hepatic small cell lymphoma. both lymphocytic ibd and lymphocytic neoplasia are often recognized simultaneously in the same cat, , and numerous authors have suggested that lymphocytic ibd may be a precursor to intestinal lymphoid neoplasia. , if this is the case, then antigenic factors, such as bacterial population changes or food sensitivities, could be considered primary initiating factors for small cell lymphoma since they are potential underlying etiologies of ibds. however, neoplasia also requires genetic mutations to occur (often affecting regulation of cell death and cell survival), and these may be initiated by the inciting antigenic factors or the ongoing inflammatory changes. as opposed to other feline lymphoid neoplasia, no association has been made with felv infection. , , , intestinal lymphocytic lymphosarcoma begins in the superficial mucosa and progresses to involve the entire mucosa and submucosa; then advancing in a perivascular pattern into the tunica muscularis, eventually infiltrating all four intestinal tunics. lymph node and other organ (such as liver or pancreas) involvement likely represent metastasis through lymphatics and perhaps hematogenously. more distant metastasis is not reported. serum or plasma biochemistry and hematologic findings are typically nonspecific. however, this testing is important as part of the diagnostic workup to rule out extra-gi disease, such as hyperthyroidism or diabetes mellitus. common biochemistry findings are mild to moderate increase of liver enzymes, such as alanine aminotransferase (alt), aspartate aminotransferase (ast), and/or alkaline phosphatase (alp). , , , as with ibds, these liver enzyme changes may or may not represent overt hepatic disease. albumin may be reduced but is normal in most cases , , ; azotemia may be present and may be of prerenal origin or represent concurrent renal disease. in one study, of cats were hypocobalaminemic; of cats had low folate, but of had elevated folate; and of cats had increased ftli. hematologically, a mature neutrophilia with or without monocytosis is sometimes present, representing the inflammatory response; lymphopenia may be present as a stress response. anemia may be present and may occur as a result of chronic slow gi blood loss, and in some cases, ulceration, or it may be because of chronic disease; hemoconcentration is also possible, reflecting dehydration. , , , palpable or ultrasonographically visible thickened intestines ( % to % of cases) , , , or mesenteric lymph nodes ( % to % of cases) , , , are no more or less likely to be present in comparison with ibds. there are no defined ultrasound guidelines for cats with intestinal small cell lymphoma, because most prior papers do not distinguish between small cell and lymphoblastic neoplasia. , a more recent paper found of cats undergoing ultrasound examination had diffuse small intestinal wall thickening, with a mean of . mm (range, . to . mm; median, . mm), and focal mural thickening of mm was noted in one cat. in many cases, against expectations, intestinal wall layering was preserved. these findings also mean that of cats had ultrasonographically normal intestinal wall thickness (≤ . mm for the duodenum and ≤ . mm for the ileum). if affected, jejunal lymph nodes may appear as hypoechoic and enlarged; in the same study, of cats had lymph node changes with a mean diameter of . mm (range, . to mm; median, mm) compared with the normal diameter of less than or equal to . mm. none of these findings can definitively distinguish small cell lymphoma from ibds; although one recent paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma (figure - ) than those with ibd, this change was also seen in % of cats with a normal small intestine. however, thickening of the muscularis layer together with lymphadenopathy was recognized in % of those cats with small cell lymphoma compared with % of those with ibd and % of cats with no small intestinal pathology. biopsy samples and histopathology are required for definitive diagnosis. an example of jejunal and mesenteric lymph node appearance at laparotomy is shown in figure it is difficult to distinguish between lymphocytic inflammation and small cell lymphocytic neoplasia in any location; some histopathologic features that might help in differentiating the ends of the spectrum may include therefore become known as the fondacaro protocol. this consists of a combination of prednisolone and chlorambucil given orally by the client at home (table - ). the rationale is that a slow alkylating agent, such as chlorambucil, is more appropriate to use for the slowly dividing, well-differentiated lymphocytes that cause disease. this can be contrasted to the aggressive chemotherapeutic agents required for the rapidly proliferating cells in lymphoblastic neoplasia that is typically associated with lymphosarcoma. reported response rates to this protocol are excellent, with % to % of cats achieving complete clinical remission, reported median survival times ranging from to months for those cats responding to therapy, and reports of individual cats surviving as long as months. , , the original reported protocol comprised prednisolone ( mg/cat, po or mg/kg, po) given daily with chlorambucil pulsed by administration of mg/m for days every weeks. a more recent study dosed prednisolone similarly, but chlorambucil was given as continuous therapy of mg/cat, po every second or third day. no mucosal congestion, edema, or fibrosis in lymphocytic neoplasia, compared with ibd . epitheliotropism, or homing of neoplastic t lymphocytes to the mucosal epithelium in lymphocytic neoplasia these features can be seen in figure - . each of these criteria may be useful but are unlikely to be definitive. further studies that may not be routinely available but which may be helpful are immunophenotyping; most reports have found purely t lymphocytes in most cases of intestinal small cell lymphoma , , , (figure - ). . clonality; the detection of a clonal population of cells, as recently described for intestinal lymphocytic lymphosarcoma, would be closest to providing the basis for definitive diagnosis. effective treatment of feline intestinal small cell lymphoma was brought to light by fondacaro et al and has cat to be weaned off corticosteroids, with chlorambucil continued as monotherapy (as is often the case with humans). iatrogenic diabetes mellitus usually needs to be managed with insulin therapy, at least initially (see chapter ) . high-grade lymphoma or lymphosarcoma is the traditional style of aggressive, rapidly dividing lymphoid neoplasia that carries a much poorer prognosis than small cell lymphoma. most early studies do not distinguish grade of neoplasia; so, the prevalence of low-grade and high-grade alimentary lymphoma are difficult to assess. several recent studies found a similar prevalence of each, , but the seminal paper describing small cell lymphoma found only cases of lymphoblastic lymphoma compared with cases of small cell lymphoma. this ratio of approximately one high-grade gi lymphoma case for every three low-grade cases more closely approximates the rate found in the author's practice. the reported median ages of affected cats range from to years, but cats as young as year old have been diagnosed. most papers note that males are overrepresented, and siamese cats may also be overrepresented although most affected cats are domestic shorthairs. , , , , precise signalment is difficult to determine from the literature, because many papers assess all anatomic locations of lymphoma without necessarily breaking down epidemiologic data for each anatomic site. also, there are few comparisons to a reference population. the association of lymphoma with felv infection is well established and documented and is covered in chapter ; fiv has also been shown to be lymphomagenic. , since the control of felv through vaccination began in the s, nonretroviral-associated lymphoid neoplasia has become more common, and the rates of intestinal lymphoma have, in fact, increased since felv infection rates have decreased. the underlying causes for this increase are not known. the association with inflammation from ibds was noted for small cell lymphomas, and perhaps there is a spectrum from lymphocytic ibd to small cell lymphoma to high-grade lymphoma. that some cats are more likely to have inflammatory changes become neoplastic is suggested by a paper noting higher lymphoma rates in cats with vaccine-associated sarcomas (a neoplastic condition where the role of chronic inflammation is well noted). similar protocols are used in humans with both lowgrade (i.e., lymphocytic) lymphosarcoma and chronic lymphocytic leukemia. , some studies with humans have indicated that continuous therapy with chlorambucil results in prolonged survival, although metaanalyses have not been able to determine optimum dosing and scheduling of administration of chlorambucil or other alkylating agents in these conditions in humans. , although we do not have enough data to critically compare pulsed therapy to continuous dosing, the study assessing continuous dosing appeared to have a lower number of cats completely responding, although those cats that did respond had a longer median survival than those in the studies assessing pulsed chlorambucil dosing. , the differences may also relate to the definitions used for complete response. the chlorambucil dose of mg/cat, po every second day (or third day) is often chosen because of the ready availability of mg coated tablets, the breaking of which can expose the owner to these cytotoxic medications. chlorambucil can be compounded into smaller doses, thus allowing daily dosing of mg capsules. the author has used this dose to apparent good effect, but there has been no critical assessment. it is unknown whether involvement of lymph nodes or other organs, such as the liver, affects prognosis. the only study of substantial size to include extra-gi locations found anatomic location was not prognostic for response or survival time. in another study, of the five cats with liver involvement, two cats did not survive more than months, yet the other three lived longer than years, with two surviving longer than years. a study of hepatic small cell lymphoma suggests the density of neoplastic lymphocytes may influence survival, and density may relate to the stage of the disease when diagnosis occurs. adverse effects of chlorambucil are rare, but gastrointestinal signs, myelosuppression, and myoclonus have all been reported. gastrointestinal signs, such as vomiting, diarrhea, or inappetence, can be difficult to distinguish from continuation of the gastrointestinal disease diagnosed. these signs are usually self limiting. myelosuppression is also possible with thrombocytopenia reported. , monoclonus has been reported on one occasion. it is ideal to check hematologic parameters every months for cats receiving chlorambucil. continuous therapy using lower doses of chlorambucil may be less likely to lead to these adverse effects. high doses of corticosteroids can induce diabetes mellitus, and thus blood glucose should be checked regularly. if diabetes occurs, the author has found that budesonide ( mg budesonide is generally considered to be equivalent of mg prednisolone) can be substituted for prednisolone, since it has reputed lower systemic effects (though no assessments of this drug's effectiveness in cats have been made). an alternative is for the of distinction of intestinal layering as shown in figure - . the area of lymphomatous infiltration is hypoechoic, because it contains a uniform cell population without much reactionary fibrous tissue. mesenteric lymphadenomegaly is common (figure - ) , as are changes in other organs, such as kidney, liver, or pancreas. ascites may also be seen. , although ultrasonographic distinctions predominate, there is considerable overlap between ultrasonographic findings with small cell lymphoma and high-grade lymphoma. the clinician must not lose sight of the fact that microscopic distinctions are required to diagnose either condition. cytologic diagnosis of high-grade lymphoma from fine-needle aspirates (fna) is much more likely than with small cell lymphoma. this is because there is usually a focal lesion, and the neoplastic cells are a monomorphic population of large, immature cells (i.e., that are not normally seen in tissue). sometimes, mixed lymphoid whether the underlying cause is retroviral or chronic inflammation or anything else, the pathogenesis of highgrade intestinal lymphoma, as with small cell lymphoma and other neoplasia, depends on chromosomal changes that affect regulation of cell growth and death, resulting in malignant transformation and clonal expansion of immature lymphocytes. metastasis can occur in one third to two thirds of cases, , with involvement of mesenteric lymph nodes most commonly noted, but spread to liver, spleen, kidneys, and thorax is also possible. a recent survey of gastrointestinal lymphoma found that most cases ( of ) involved the small intestine (including that also involved the stomach and that also involved the large intestine), and of cases involved the large intestine only. cats with high-grade alimentary lymphoma often present similarly to those with other gastrointestinal diseases. typical clinical signs are weight loss, anorexia, lethargy, vomiting, diarrhea, or a combination of these signs. repeated studies have found cats with no vomiting or diarrhea; in one study, of cats had only anorexia or weight loss on presentation. cats with large bowel pathology usually present, as with other causes of colitis, with increased urgency, and small, frequent amounts of diarrhea, often with blood or mucus. cats with large bowel neoplasia of any form can present for constipation caused by intestinal obstruction. palpation of an abdominal mass has been recognized in % to % of cases, , but the corollary of this is that % to % of cases did not have a palpable mass. it is also important to note that up to % of cats with intestinal small cell lymphoma, and a number with ibds, have palpable mesenteric lymph nodes; so, a palpable abdominal mass is not a specific indication of high-grade neoplasia. many cats have palpably thickened bowel loops. hematology and plasma or serum biochemistry findings are also nonspecific. increased liver enzymes may or may not indicate liver involvement. anemia may be recognized and can be non-regenerative, reflecting chronic disease or slow blood loss, or regenerative if there is more substantial blood loss associated with mucosal ulceration. hypoalbuminemia can be because of blood loss or intestinal protein loss. hypercalcemia of malignancy is a possibility but not commonly reported. despite nonspecific signs, laboratory testing is important to rule out extra-gi diseases and help manage consequences of enteric disease, as with small cell lymphoma and ibds. ultrasonography commonly shows a focal intestinal thickening (of to mm) with partial or complete loss although noted as the next most common intestinal neoplasia, after the various forms of lymphoma, adenocarcinoma is seen relatively infrequently in practice. most cats are more than years old, , , males may be overrepresented, and several studies have recognized a distinct overrepresentation of siamese cats. , three distinct forms have been described : cats typically present with nonspecific signs of gastrointestinal disease but can present with obstructive signs. cats with large bowel neoplasia can present for tenesmus or hematochezia and even constipation, if the lesion is obstructive (or partially obstructive). on physical examination, an abdominal mass is palpable in approximately % of cases, but other findings are usually nonspecific. anemia can be found if mucosal ulceration has occurred, but there are no distinctive laboratory findings. lesions can occur anywhere along the intestinal tract. one study of cases found % of feline intestinal adenocarcinoma lesions were present at the ileum or ileocolic junction. twenty-five to percent of cases have metastasis at the time of the diagnosis, and this is a poor prognostic indicator. , , radiology may show a mass lesion or intestinal obstruction, and ultrasonography can localize lesions to an intestinal origin. the ultrasonographic appearance of the proliferative, circumferential, outwardly expansile populations (of immature lymphoblasts and mature lymphocytes) are seen if a germinal lymphoid follicle is aspirated, and precise diagnosis may be difficult if there are a large number of lymphoblasts. fna samples are best obtained with ultrasound guidance. the cytologic sample quality is greatly improved by not aspirating when the needle is visualized in the mass but merely "pecked" into the mass so that the needle is merely acting to finely "core" the mass. on removing the syringe and needle, the hub of the needle is removed before drawing air into the syringe, the hub is replaced, and the sample within the needle is expressed onto a slide. usually, the decision to diagnose by cytology from fna is based on the ultrasonographic appearance of a mass. since there is substantial crossover of ultrasonographic appearance of intestinal masses, laparotomy for excision is often performed with the affected bowel submitted for histology. except when intestinal obstruction has resulted, there is no therapeutic benefit of excising a gastrointestinal lymphoma (which requires excision and anastomosis), but there is minimal room for doubt when a histologic diagnosis is achieved. the response to therapy for high-grade intestinal lymphoma is significantly worse than that for small cell lymphoma. , further, response to therapy for highgrade intestinal lymphoma appears to be worse than for lymphoma in other anatomic locations. precise remission rates and survival times are difficult to quantify, because many studies assess lymphoma from multiple locations and do not necessarily differentiate response of gastrointestinal lymphoma or report the grade of lymphoma. with remission rates reported from % to % , , and a median survival time of up to weeks (range, to weeks), it can be said with some certainty that some cats respond to therapy for reasonable durations. multiple authors have noted that the best prognostic indicator is response to an initial treatment cycle, , , which should prompt clinicians to encourage owners to start therapy and decide whether to continue based on the cat's response. there are several published chemotherapeutic protocols, , , , , but all follow the same principles of using medications to target specific phases of the cell division cycle (such as l-asparaginase and vincristine) with other medications that interrupt multiple phases of the cell cycle (cyclophosphamide and doxorubicin). targeting the cancer cell in different ways enables more cells to be killed, reduces the toxicity of the individual drug used, and reduces the likelihood of resistance to a specific drug. several authors have noted increased success with the addition of l-asparaginase and doxorubicin to protocols. , , chemotherapy for lymphoma is covered in more detail in chapter , oncology. look promising, but only two cats assessed had gastrointestinal mast cell neoplasia. lomustine was used unsuccessfully in one cat with sclerosing mct; another cat with sclerosing mct received eight treatments of vinblastine and had a survival time of greater than years. adenomatous polyps have been reported in the duodenum and ileum and can result in intussusception. cats of asian ancestry, predominantly siamese, are greatly overrepresented, and most reported cases have been males. cats usually present for vomiting or hematemesis that, surprisingly, can be very acute in onset; complete intestinal obstruction may result. , , resection is curative, with survival times of more than years reported. eosinophilic sclerosing fibroplasia has recently been described in a series of cases and is not strictly neoplasia. the ulcerating mass lesions that can occur anywhere from the stomach to the colon are often grossly and histologically mistaken for neoplasia. there appears to be no breed predisposition or age predisposition (with ages ranging from weeks to years), but of cases ( %) were castrated males cats compared with of ( %) female spayed cats. eighty-four percent of cats presented for vomiting, % presented for weight loss, and of ( %) cats had peripheral eosinophilia. all cases had a palpable abdominal mass. the pyloric sphincter was the most common site, and lesions in this location were mostly considered unresectable. fourteen of cats ( %) had bacterial colonies within microabscesses and necrotic foci within the lesion. the bacteria recognized were predominantly gram-negative rods, but antibiotics did not seem to be clinically effective. the bacteria are suspected to initiate the lesions, having been embedded after foreign body penetration. there are no specific treatment recommendations, but excision, where possible, would be prudent; corticosteroids appear to be helpful adjunctive therapy. survival times are difficult to estimate since many cats were euthanized because neoplasia was suspected and follow-up times were short (up to months) for the remaining cats. there are very few reports of intestinal leiomyosarcomas in cats, , which have been reclassified as gastrointestinal stromal tumors. these tumors may be more likely to arise from the ileocecocolic junction. resection, if possible, is usually recommended, with survival times of to months reported before recurrence. the author owns a cat with this tumor where resection was not form is better described than the annular, constrictingband form with minimal outward enlargement. in these cases, sonographically, a solitary segmental intestinal mural mass is present and characterized by circumferential bowel wall thickening with transmural loss of normal sonographic wall layers. the thickening can vary in echogenicity but may be hypoechoic and may be symmetric or asymmetric. there is no definitive distinction, however, from lymphosarcoma, mast cell tumor, smooth muscle origin tumors, or even segmental benign inflammatory bowel disease. surgical resection is the treatment of choice. there seem to be two distinct groups in terms of survival time postresection: . short-duration survival (euthanasia or death within weeks of surgery) . long-duration survival (mean survival time of months, with a number of cats surviving greater than years) , because clean margins improve prognosis, for large bowel adenocarcinoma, subtotal colectomy may be required for complete excision. because of the potential for success after resection, it is recommended to excise unidentified masses at the time of surgery. other forms of intestinal neoplasia are recognized infrequently, and include intestinal mast cell tumors, adenomatous polyps, eosinophilic sclerosing fibroplasia, gastrointestinal stromal tumors (leiomyosarcoma), and hemangiosarcoma. mast cell tumors (mct) are often cited as the third most common form of feline gastrointestinal tumor, but the intestines are a far less common site than cutaneous, splenic, or hepatic mast cell neoplasia. , masses are usually segmental nodular thickenings that occur in older cats. the masses are indistinguishable ultrasonographically from other tumors, such as lymphoblastic lymphosarcoma. a recent series of cases described a variant of feline intestinal mast cell tumor, dubbed sclerosing mast cell tumor, for which neoplastic cells form a trabecular pattern with dense stromal collagen. additionally, eosinophilic infiltrates were moderate to marked in most cases. these cases can be confused histologically with eosinophilic enteritis, gastrointestinal stromal tumor, or fibrosarcoma. surgical resection is recommended, but lesions are commonly infiltrative or metastasize widely, and there are few reports of successful treatment. lomustine (dosed to mg/m , po every to weeks) has recently been assessed as adjunctive chemotherapy for mast cell neoplasia in various locations, and results recognized in that many intestinal bacteria can be found in healthy animals. further antibiotic administration can result in increase of other bacteria. fungal causes of diarrhea are usually recognized from histology of biopsy samples. it remains to be seen whether the recent ready availability of pcr panels looking at a number of infectious causes of diarrhea will be beneficial for recognizing pathogens that had previously been misdiagnosed or a hindrance for readily recognizing commensal organisms not necessarily causative of the clinical signs being investigated. the most common viral, bacterial, and mycotic causes of diarrhea in cats are described below. parasitic gastrointestinal diseases are covered later in this chapter. viral causes of diarrhea are not usually specifically diagnosed, since, with the exception of the canine fecal elisa possible, and the cat appears healthy months past diagnosis (figure - ). cats with intestinal hemangiosarcoma often present with anemia, and the disease appears to be highly metastatic. the intestines appear grossly thickened by dark red tissue. the small and large intestines seem to be affected with similar frequency. removal of macroscopic disease is recommended, but often the full extent of the severity is only recognized at surgery. the prognosis is poor. suspicions of infectious causes of diarrhea should be aroused in younger cats, cats from shelters, or cats with immune suppression. when considering infectious causes of diarrhea, clinicians should assess whether the diarrhea is large bowel or small bowel in origin and correlate this with specific pathogens that are likely to cause clinical signs as shown in table - . to increase the diagnostic yield of fecal examination for parasitic causes of diarrhea, wet smears and appropriate fecal flotations should be performed on fresh fecal samples (< hour old). it is appropriate to administer broad-spectrum anthelminthics, even if fecal tests are negative. bacterial and viral causes of diarrhea should be considered when the cat is systemically unwell with fever. fecal culture should be performed in these circumstances, but the limitations of this testing need to be yersinia enterocolitica current theory of fip pathogenesis involves initial infection with fecv and then mutation to fipcv in small numbers of susceptible individuals. , routine serologic testing for fecv in cats with diarrhea would neither prove correlation with the clinical signs nor affect how the disease is managed and so is not recommended. cats with fecv diarrhea should be managed with symptomatic therapy of fasting, then reintroducing a bland diet and supportive care with fluid therapy if necessary. other viruses, such as astrovirus, reovirus, rotavirus, and torovirus-like agent, have been recognized to cause diarrhea in cats, but their roles as pathogens are unclear. they are not routinely recognized in practice, since electron microscopy of fecal samples is necessary for diagnosis and is not routinely performed. management is supportive care with appropriate fasting, then reintroduction of bland diets and fluid and electrolyte replacement if necessary. successful identification of a known bacterial pathogen from a fecal sample does not necessarily mean that the agent found is the cause of disease in the cat. although a number of bacterial pathogens have been demonstrated to cause diseases when specific pathogen-free (spf) cats are experimentally infected, these same organisms can be found in healthy cats. the differences between healthy and diarrheic cats that have bacteria found in their feces may relate to virulence factors of the organism, or host factors (local or systemic immunity) of the cat. there is no definitive answer for this quandary. the author's opinion is that • if a diarrheic cat is systemically unwell and has a fever, then feces should be cultured. • if an organism is isolated that is known to cause signs consistent with those the cat is showing, the cat should be treated appropriately. campylobacter diarrhea is usually caused by c. jejuni. clinical signs of infection are poorly documented, but most cats are asymptomatic. younger cats are more likely to have clinical signs and hemorrhagic, mucoid diarrhea has been reported. diagnosis can be from culture of feces or swabs, and the organism is quite hardy; so, it usually survives transport to the laboratory. in individual cases, the organism has not been cultured after antibiotic treatment, , but it is not definitively proven that antibiotic therapy affects the natural course of the disease. antibiotics that can be used are amoxicillin-clavulanate ( mg/kg, every hours, po) for parvovirus, routine definitive tests are not available. clinical signs of panleukopenia (feline parvovirus infection) are more likely to occur in kittens, with the highest morbidity and mortality occurring between and months of age. subclinical cases in older (susceptible) cats probably go unrecognized. the organism is very stable in most environments, and infections mostly occur from environmental contact. peracute cases can result in death within hours, with little or no warning signs. acute cases often have fever, depression, and anorexia, with signs beginning approximately to days before presentation. vomiting is usually bile tinged and unrelated to eating. diarrhea does not always occur, and when it does, it is usually later in the course of the illness. leukopenia is not pathognomonic, because this can also occur with acute bacterial infection (e.g., salmonellosis can present identically). commercially available elisa tests for canine parvovirus antigen in feces can detect feline parvovirus; however, shedding may have ceased by the time clinical signs occur, and vaccination can result in positive test results for up to weeks. aggressive fluid therapy, usually at twice maintenance rates, is usually required. broad-spectrum antibiotic coverage is used to prevent or treat secondary bacterial infection from viral injury of intestinal mucosa. parenteral antibiotics are preferred to prevent the possibility of further gastrointestinal irritation. the author recommends calculating iv doses and introducing appropriate amounts of antibiotics to the fluids bag to create a constant rate infusion (cri); cefazolin can be used in this way at mg/kg/ hours, and betalactam cris are commonly used in human medicine. aminoglycosides or fluoroquinolones can be used concurrently at routine doses if fever persists after hours or the cat is moribund on presentation, but care must be used with these agents. aminoglycosides are potentially nephrotoxic, and fluoroquinolones have been reported to result in cartilage damage in growing animals, although this has not been demonstrated clinically in cats. fluoroquinolone retinal toxicity has been seen in all animals. cats that survive the first week usually recover, and prior infection imparts lifelong immunity. vaccinations are highly effective for disease prevention. feline enteric coronavirus (fecv) mostly causes mild, self-limiting diarrhea and must be distinguished from feline infectious peritonitis coronavirus (fipcv), which is essentially always fatal and for which diarrhea is not a typical sign (but is possible). the most widely accepted or fluoroquinolones, such as enrofloxacin ( mg/kg, once daily, po) for durations of to days. macrolides, such as erythromycin ( to mg/kg, every hours, po), are regarded as the drug of choice for humans but can cause gastrointestinal side effects. clostridium difficile has been recognized in up to % of diarrheic cats. clinical signs are typically acute onset watery diarrhea and anorexia. diagnosis has been made with detection of toxin a or toxin b in fecal samples using elisa. although these tests have not yet been validated for cats, they may prove to be a useful aid to diagnosis and are available for testing of equine feces at some commercial laboratories. nontoxigenic strains exist; so, positive culture alone does not ensure diagnosis. metronidazole ( mg/kg, every hours, po) for approximately days is the treatment of choice. clostridium perfringens typically results in large bowel diarrhea with tenesmus, mucus, and hematochezia, but small bowel signs can also be seen. pcr testing for enterotoxin a is commercially available and may prove to be a useful adjunct in diagnosis. antibiotics that can be used include metronidazole ( mg/kg, every hours, po), tylosin ( to mg/kg, twice daily, po), or amoxicillin-clavulanate ( mg/kg, every hours, po) for days. escherichia coli is a ubiquitous organism within the feline intestinal tract, and it would be unusual not to successfully culture e. coli from the feces of both healthy and unwell cats. when e. coli is associated with clinical signs of gastrointestinal disease, it is mostly as an opportunistic pathogen, with overgrowth resulting from changed environmental conditions, such as inflammation from other pathology or another pathogen. there are also specific strains of e. coli that are true pathogens because of virulence factors not present in commensal e. coli; these include enteropathogenic e. coli and enterotoxigenic e. coli, which both induce a watery diarrhea, and enterohemorrhagic e. coli, which produces a diarrheal syndrome with copious bloody discharge and no fever. pcr testing is commercially available to identify pathogenic strains of e. coli , ; although not offered at routine veterinary laboratories, this testing is available to veterinarians, and laboratories offering these services can readily be found online. diagnosis should also document histologic lesions corresponding to the strain of e. coli identified. there is emerging resistance to e. coli worldwide in all species of animals, including humans. this includes the typical therapies for gram-negative bacteria of beta-lactamenhanced penicillins and fluoroquinolones. a major risk factor is prior antibiotic usage, because commensal organisms are exposed to antibiotics. pcr testing does not enable antibiotic sensitivity testing, and fecal culture may not be able to distinguish pathogenic from nonpathogenic strains; so, sensitivities may not be an accurate reflection of the pathogenic organism. pcr testing for genes that impart resistance to e. coli have recently been described but are not yet commercially available. in some circumstances, supportive care with fluid and electrolyte replacement may be all that is required while the cat's immune system combats the infection. empiric therapy could include beta-lactam-enhanced penicillins (such as amoxicillin-clavulanate at mg/kg, every hours, po), fluoroquinolones (such as enrofloxacin at mg/kg, once daily, po), or cefovecin ( mg/kg, every weeks, sc), but the clinician must be aware of possible drug resistance. salmonella typhimurium infection is possible from ingestion of infected prey, infected food sources, or from a contaminated environment, including the veterinary hospital. the resulting clinical signs depend on the number of infecting organisms, the immune status of the cat, and the presence of concurrent diseases. infection rates in cats (and humans) have been correlated with seasonal bird migrations, and the illness has been dubbed songbird fever, but there is no distinction between this and other salmonella infections. clinical signs usually begin to days after exposure, starting with fever (often > ° c [ ° f]), malaise and anorexia, and progressing to diarrhea, vomiting, and abdominal pain. hematology can show leukopenia with a left shift and nonregenerative anemia, and biochemistry results are usually nonspecific. diagnosis is based on isolation of the organism by culture or identification with pcr, but care should be taken to correlate pathogen identification with clinical signs since, as with most gi pathogens, the organism can be isolated from healthy animals. as with e. coli, antibiotic resistance is widespread, with one united kingdom survey finding the multiple drugresistant strain dt to be the most frequent bacteriophage type identified. treatment should be reserved only for those cats showing systemic signs, because routine antibiotic use in treating salmonellosis induces drug-resistant strains and prolongs the convalescent excretion period. antibiotic choice should be based solely on sensitivity findings, since resistances are so widespread and unpredictable. this means that if the organism has been identified by pcr, then culture of feces must also be undertaken. the duration of treatment must be long enough to eliminate fecal excretion of the organism, prevent the chance of relapse, and reduce the chance of resistance developing; up to days has been advocated. , these cautions are particularly important because of the zoonotic potential of salmonellosis. linear foreign bodies have traditionally been considered more common than discrete foreign bodies in cats, , , but a study from a primary care facility indicated only % of foreign body cases were because of linear foreign bodies. the larger case load of linear foreign bodies at referral institutions noted in earlier studies may indicate the abilities of primary care practitioners to recognize and effectively deal with discrete foreign body obstructions. most studies have found that cats with intestinal foreign bodies are generally younger (mean, . to . years), with a notable exception being obstruction from trichobezoars where three of five cats in one study were years or older; the greatest risk factor appears to be length of hair coat. no specific breed predispositions have been described but siamese and siamese-related cats have been noted to have oral fixations and so may be expected to be overrepresented with intestinal foreign bodies. clinical signs will vary depending on the type of foreign body (linear or discrete), the position of obstruction, and the time since obstruction. most cats present for anorexia or vomiting. partial obstruction can result in diarrhea (which can be bloody). foreign body obstruction is typically considered an acute condition, with duration of obstruction because of a linear foreign body, measured from the onset of clinical signs to diagnosis, reported to range from to days. , , however, one paper demonstrated chronic, intermittent, gastrointestinal disease from a linear foreign body of a -month duration demonstrating that partial obstruction can result in a chronic course. physical examination may or may not reveal abdominal pain, palpable abdominal mass (or plication), dehydration, or fever. all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of a linear foreign body. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see figure - ) . life-threatening consequences can result from the interactions of local and systemic factors that arise from intestinal obstruction. locally, damage to the mucosa from traction and pressure of the foreign object can cause hemorrhage, ischemia, and necrosis. systemically, hypovolemia, toxemia, and acid-base and electrolyte imbalances can ensue. complete intestinal obstruction by discrete masses results in gas and fluid distention of the lumen proximal other bacterial causes of diarrhea have been reported in cats, such as yersinia enterocolitica, yersinia pseudotuberculosis, clostridium piliforme (tyzzer's disease), and anaerobiospirillum sp. specific diagnosis of these (and other bacterial infections) may be found in the course of investigation. management follows the principles of supportive care and appropriate antibiosis based on sensitivity testing. small intestinal bacterial overgrowth (sibo) has not been specifically described in cats. the criteria defined for dogs is a fasting bacterial count in duodenal juice of greater than organisms/ml and is often recognized with other chronic gastrointestinal diseases. healthy cats appear to have at least this number of upper intestinal bacterial with a range of to /ml recognized. bacterial overgrowth could potentially occur with ileus or intestinal inflammation of any underlying cause. foul-smelling small bowel diarrhea with no specific pathogen recognized may be an indicator of this condition, as could an increase in bacterial metabolites, such as folate. if suspected, it is appropriate to manage with broad-spectrum antibiotics, such as metronidazole ( to mg/kg, every hours, po) or amoxicillin ( mg/ kg, every hours, po) for an extended duration such as to days. alterations in intestinal flora have been recognized after such treatment ; however, any advice for this "condition" is entirely empirical. all efforts should be directed at identifying a precise underlying cause. mycotic and other infectious agents are only rarely recognized as intestinal pathogens in cats. diagnosis is made by histologic and microbial analysis of samples obtained at biopsy. possible agents include histoplasma capsulatum, aspergillus spp., candida albicans, and pythium insidiosum. intestinal obstructions arise most commonly as a result of neoplasia in older cats and foreign body ingestion predominantly in younger cats. , , less common causes include intussusception and granulomatous inflammation (e.g., from fip) ; tapeworm infection, with greater than worms acting as a linear foreign body, has also been reported. other listed causes are volvulus, intestinal torsion, incarceration of bowel in a hernia, adhesions or stricture, intramural abscess or hematoma, and congenital malformations. may not occur if the obstruction is partial or intermittent, or if vomiting results in less fluid present. since most foreign body obstructions in cats are proximal, identifiable dilatation may not be recognized for this reason. linear foreign bodies present further challenges for radiographic recognition; the following typical radiographic signs , may or may not be present: to the obstruction. most gas accumulation is a result of swallowed air, which is predominantly nitrogen that cannot be absorbed by the intestinal mucosa. gas also arises from bacterial fermentation. fluid accumulates as a result of increased secretions (saliva, bile, and secretions of gastric, pancreatic, and small intestinal origin) and retention of fluid already ingested, and it can be augmented by local hemorrhage. since most intestinal obstructions in cats do not reach the midjejunum, reabsorption of fluids that normally occurs at the jejunum and ileum is impaired. linear foreign bodies, such as string, dental floss, or elastic toys, require proximal anchoring, usually under the tongue or in the pylorus (for example, by part of a toy attached to elastic). peristalsis moves the free end of the "string" through the intestinal tract, resulting in pleats of intestines around the foreign body. as the foreign body is forced against the intestinal mucosa, the mucosa becomes edematous, and even partial penetration affects mucosal integrity, allowing systemic entry of bacteria. intraluminal bacterial populations increase for both discrete and linear foreign bodies as a result of stasis. mucosal permeability can be affected by prolonged luminal distention, allowing entry of bacteria and toxins systemically or into the peritoneal cavity. direct entry of bacteria to the peritoneal cavity, causing septic peritonitis, can result from perforation of the intestinal wall from linear foreign bodies or sharp discrete foreign bodies, such as toothpicks or plastic toys. definitive diagnosis requires identification of the foreign body retrieved at surgery or in some cases, by endoscopy. this may be aided greatly prior to surgery by diagnostic imaging. however, imaging findings, particularly in the case of partial obstructions, may be subtle enough that obstruction of no identifiable cause is recognized or no overt signs are apparent. laboratory findings are not helpful in the precise diagnosis but are important to assess fluid and electrolyte balances that must be corrected. cats rarely help practitioners by ingesting radiopaque objects, but on the rare occasions that they do, these can be observed easily on plain radiographs. nonopaque foreign bodies depend on dilatation of the intestine from gas and fluid accumulation proximal to the obstruction for radiographic recognition (figure - ) . one study has suggested that if the jejunal diameter is greater than . times the length of the cranial end plate of the second lumbar vertebra, then intestinal obstruction is the most likely abnormality. care must be taken that the jejunal and not duodenal diameter is measured and that the radiographs must be positioned strictly lateral, because an oblique view can alter the measurement of the lumbar vertebra. however, dilatation of an obstructed intestine successful treatment of foreign body obstruction requires evacuation or removal of the foreign body as well as correction of any bacteremia or endotoxemia, acid-base or fluid imbalances. discrete foreign body obstruction requires surgery or endoscopy to remove the object. in some specific circumstances, linear foreign body obstruction may be managed conservatively by cutting the anchor point below the tongue and allowing the cat to pass the foreign body by peristalsis. however, the decision to manage a cat conservatively must be done with the cat hospitalized, with fluid therapy and antibiotic coverage and a clear recognition on behalf of the practitioner and the owner that surgery may subsequently be required. cutting a sublingual linear foreign body may be achieved in a conscious cat by applying pressure with the thumb of one hand in the intermandibular space to elevate the tongue and gently grasping it using gauze with the other hand while a second person cuts the line with a suture cutter. there is a chance of a small nick on the sublingual surface. if the cat will not tolerate the procedure, sedation is appropriate. when cutting the line, the nature of the linear foreign body should be assessed (i.e., is it more or less likely to cut mucosa). in one study, cats with linear foreign bodies were managed conservatively with cats subsequently requiring surgery. the authors of that paper created guidelines that will be adapted here. conservative management should be attempted if the cat • is presented acutely (within days) after known ingestion of a linear foreign body • has a sublingually fixed linear foreign body that can be cut • has no overt signs of peritonitis • altered gas pattern with luminal gas collecting in small bubbles instead of normal curved tubular columns. this can be subtle when there is only minimal involvement of the intestine but overt when involving the entire small intestine. commashaped gas patterns are more likely to occur with linear foreign bodies. contrast radiography can aid diagnosis for both discrete and linear foreign bodies but should be used with caution because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium is irritating to the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. ultrasonography is a very useful diagnostic tool, particularly for discrete foreign bodies, where, in most cases, there is overt distention of the small intestines with intraluminal fluid apparent (figure - ) . this modality has not been extensively assessed as an adjunct to diagnosis of foreign body intestinal obstruction in cats specifically, although there are several papers assessing dogs and small numbers of cats that agree with its utility. , , linear foreign bodies are more difficult to assess ultrasonographically, but plicated bowel can be recognized, sometimes with the foreign body seen as a hyperechoic line centrally. si a technique has been described for removal of linear foreign bodies by making a single enterotomy incision proximally and passing a red rubber catheter over the linear foreign body aborally, milking the foreign body within the catheter through the colon for retrieval from the cat's anus by an assistant. this technique is not always effective, because it can be hampered if the foreign body is knotted or does not run smoothly through the red rubber catheter. if the affected bowel segment demonstrates evidence of necrosis or perforation on the mesenteric border of the intestine, resection and anastomosis should be performed. necrosis is indicated by dark discoloration, thin intestinal wall, poor arterial pulsation, poor capillary bleeding, or lack of peristalsis. end-to-end anastomosis can be accomplished using a simple interrupted appositional pattern or a modified simple continuous appositional pattern with the same type of suture material used for enterotomy closure. , intraabdominal masses causing intestinal obstruction are often presumed to be neoplastic but can also be of infectious origin. resection, where possible, is always recommended, because resection of neoplasia (if no metastasis) can offer a good prognosis, , , , and infectious causes may be managed with adjunctive therapy after definitive diagnosis. intestinal obstruction in older cats is more likely to be secondary to neoplasia. any neoplasia can cause obstruction, but adenocarcinoma , and adenomatous polyps , are reported to cause obstruction more often surgical intervention is mandatory if • clinical signs (e.g., vomiting or anorexia) persist or deterioration occurs with conservative management • the cat has overt signs of peritonitis • the linear foreign body is fixed at the pylorus some authors disagree with attempting conservative management, since a perforated intestine from a linear foreign body reportedly carries a % mortality rate, , and early surgical intervention is never an incorrect decision. this should be balanced with the observation that cats can carry a linear intestinal foreign body, such as an elastic cord for a -month duration without intestinal perforation. however, fishing line, for example, would not be so forgiving! surgery to remove an intestinal foreign body (figures - and - ) should be considered an exploratory laparotomy. that is, the aim of the surgery is not only to remove the foreign body but to assess the entire intestinal tract and abdomen for other foreign bodies or pathology. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction, thus delaying healing and creating the potential for surgical dehiscence. linear foreign bodies require multiple enterotomy incisions, since pulling the object out through a single incision could create iatrogenic intestinal perforation. the anchor point (either sublingual or pylorus by gastrotomy) must be released in the first instance. enterotomy incisions are closed with / synthetic, monofilament, absorbable suture material, such as polydioxyanone (pds) or equivalent, in either a simple interrupted or simple continuous pattern. , removal of a discrete foreign body (a piece of leather) at laparotomy. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction. this is the same cat as in the radiology image in figure - . affected bowel is required, with anastomosis of the healthy tissue. there appears to be no benefit to enteroplication, which can result in significant ileus. there is no benefit to performing resection-anastomosis if the intussusception does reduce manually. , the prognosis depends on the underlying disease process and the chronicity of the intussusception, and therefore how debilitated the cat is at presentation, however, prognosis is mostly good, with survival reported in up to % of cases, though recurrence can occur in some cats with idiopathic disease, often at different locations of the intestinal tract. constipation is defined as infrequent or difficult defecation associated with retention of feces within the colon and rectum. prolonged constipation results in harder than other types of neoplasia. please refer to the sections on intestinal neoplasia earlier in this chapter for more details. granulomatous inflammation causing a single focal intestinal lesion can lead to obstruction in the same way that neoplastic change can. feline infectious peritonitis (fip) can present as focal lesions, often in the colon or ileocecocolic junction. in the case of fip, the focal lesion is usually an indicator of multisystemic disease; so, resection does not help prognosis. the fungus-like organism, pythium insidiosum has also been reported to cause granulomatous lesions, resulting in intestinal obstruction from large extraluminal masses that are approximately fist sized. resection with adjunctive itraconazole ( mg/kg) for months after surgery was a successful treatment. intussusception refers to invagination or prolapse of one portion of the intestine into the part of the tract that either precedes or follows it. there is a bimodal age distribution with intussusceptions in older cats, most likely associated with neoplasia (or ibd in some cases) ; underlying causes for younger cats are ill defined and may be idiopathic in many cases, , but associations with parasitism and, in one case, a linear foreign body, have been made. siamese and burmese cats seem to be overrepresented. the most common locations are the ileocolic region and the jejunum. , , , affected cats present with nonspecific signs of gastrointestinal disease, such as anorexia and lethargy. vomiting is not necessarily a presenting sign; diarrhea may occur. abdominal palpation reveals a mass in most cases. plain and contrast radiography only show evidence of obstruction and usually do not help define that the bowel has intussuscepted. , , , ultrasonography is very useful for diagnosis, because a distinctive pattern of alternating hypoechoic and hyperechoic concentric rings (figure - ) is present in transverse sections. , sometimes, the target lesion seen can be hard to distinguish from the pathology of other intraabdominal masses, such as lymph nodes, and in these cases, the size of the lesions can help, because the width will always be greater than mm with an intussusception (because the sum of at least four intestinal wall widths cannot be less) and is often greater than mm. surgical correction is always required, and manual reduction is typically not possible because there is usually significant venous infarction, edema, and congestion (figure - ) as well as adhesions from fibrin and effusions from the affected bowel. , if the intussusception does not reduce manually, resection of the and drier feces that become impacted, and this is known as obstipation. chronic, recurrent constipation and obstipation can result in megacolon, which refers to persistent increased bowel diameter that is not responsive to therapy. megacolon is not a specific disease entity; it may be considered the most advanced stage in the spectrum of chronic constipation. in most cases, constipation can be managed quite simply if the underlying cause is determined and dealt with. a comprehensive list of causes of constipation is noted in table of course, multiple factors can interact. for example, an older cat may have renal disease and so will be dehydrated to some degree and have arthritic hips and so be reticent to squat. the presenting signs of constipation are usually evident to owners and include straining in the litter box and producing hard dry feces, if at all. sometimes, however, owners can misinterpret signs. cats can strain because of lower urinary tract problems, and, if no urine is produced, some owners assume the problem is because of constipation. some constipated cats can intermittently have diarrhea because of direct colonic irritation from hard dry feces and so may present for diarrhea and not constipation. cats can also present for less specific signs, such as anorexia, lethargy, weight loss, and even vomiting. , vomiting can occur because of colonic receptors stimulating vagal afferent endings, which, in turn, can stimulate the chemoreceptor trigger zone. sometimes owners are concerned that their cat is defecating less, but the cat has just changed its diet to a much lower-residue diet and so is producing less feces. a full dietary history is an important aspect of the initial assessment. physical examination should confirm presence of feces in the colon and assess the degree of impaction. the presence of feces can usually be confirmed by abdominal palpation. in constipated cats, the colon is often palpated as a long firm tube extending cranially; sometimes, feces can be palpated to and around the colic flexure. alternatively, the feces may be palpated as large, discrete fecal concretions (that can sometimes be hard to distinguish from intraabdominal masses such as lymph nodes). if there is any doubt of the presence or degree of fecal impaction, survey abdominal radiographs should be taken. a lateral view taken in a conscious cat should be adequate to confirm the diagnosis. the physical examination should also assess for contributing causes, including musculoskeletal conditions. any recent trauma should be taken into account. the hips and lumbosacral region should be assessed for pain. the degree of flexion and extension of the hips should be gently assessed. the lumbosacral spine can be assessed by running two fingers on either side of the spinous processes. the cat will flinch in painful areas. any arthritic change is magnified in an underweight cat, since there may be less muscle mass and the joints may bear a heavier load. any suspicions of underlying musculoskeletal abnormalities can be confirmed with radiographs. neurologic assessment should also be performed. subtle changes just affecting colonic innervation will not be apparent on physical examination alone. however, an assessment of proprioception, placing reflexes, and gait should at least be performed to assess for lumbosacral spinal cord disease. anorectal abnormalities or lesions should be evaluated. impacted or infected anal sacs can lead to reticence to defecate; and therefore anal sacs should be assessed and expressed. because this is painful for most cats, the cat should be held by an experienced assistant. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb positioned externally. a rectal exam can be performed with a well-lubricated (gloved!) middle finger, feeling over the pelvic rim for masses as well as assessing if the colon closes over (squeezes) the finger. if the colon feels open around the finger, this can be an indicator of impaired colonic innervation but does not imply that this is a permanent change. if there are impacted feces continuing to the anus, rectal examination is not possible until this has been cleared. if a cat finds anal gland expression or rectal examination too painful to tolerate (based on the clinician's judgment), these procedures should be done under sedation. hydration and electrolyte status are also important factors in the constipated cat. chronic renal disease is defined by azotemia (in conjunction with inadequately concentrated urine), which means the cat must be dehydrated to some degree. plasma or serum biochemistry and urinalysis can be used to diagnose renal disease, assess degree of renal disease, or recognize prerenal dehydration. electrolyte changes including hypokalemia and hypocalcemia may also contribute to reduced colonic smooth muscle function. in young to middleaged cats of apparent good health and hydration, blood difficulty defecating and pass hard, dry stools but do not have fecal impaction at the time of examination. after the obstructing feces have been removed, steps must be taken to ensure colonic motility and smooth passing of feces. medical management of constipation traditionally involves laxatives and prokinetic agents. these may not be required in straightforward cases. as long as there is no obstructive lesion, cisapride at . mg/ cat, every hours, po is very safe and can be instituted with a view to reducing the dose to once daily after to days and discontinuing if signs remain abated. doses of up to . mg/cat, every hours, po have been reported. cisapride is only available from compounding agencies in most countries. an osmotic or lubricant laxative (table - ) may be used concurrently at reduced doses as necessary. reducing the fecal bulk produced is an important part of long-term management. traditional dietary recommendations are to increase the amount of fiber. , , , increased dietary fiber results in production of shortchain fatty acids, which have been demonstrated to stimulate feline colonic smooth muscle contractions. however, dietary fiber is also classified as a bulk laxative and so, by definition, will increase fecal bulk. in humans dietary fiber has been considered a mainstay of therapy for constipation, but a recent review concluded that many patients with more severe constipation have worsening symptoms when increasing dietary fiber intake. because megacolon is believed to be the end result of chronic dilatation, , it is the author's firm belief that initial dietary efforts should be directed to reducing fecal bulk and thus introducing a low-residue diet. reduced dry matter intake reduces stool volume, and the author has found that recurrence rates of constipation reduce greatly when cats are transitioned to entirely wet food diets. wet food diets also help ensure adequate water and urine assessments are usually not required at an initial presentation for constipation. in all cases, the same principles of management apply: . ensure removal of obstructing feces . ensure colonic motility and smooth passage of feces . reduce fecal bulk . ensure adequate hydration . manage underlying problems the first step is to ensure obstructing feces are removed. in simple cases, the cat will evacuate feces after use of a glycerin or sorbitol pediatric rectal suppository. another option is administration of a microenema, such as microlax (mcneil consumer healthcare, fort washington, pa.), which contains ml of sodium lauryl sulfoacetate. these products act to lubricate the colon wall and therefore facilitate the passage of feces. the author prefers to use one or two of these within the consult room to observe the cat defecating (the cat must be provided with a litter tray!). the outside tube should be lubricated with the suppository contents before carefully inserting and then expressing the rest of the contents. there are also stimulant laxatives (containing bisacodyl) and emollient laxatives (containing sodium docusate) that have reportedly been used. if a rectal suppository vial cannot easily be inserted because hardened fecal content obstructs its entry, a more substantive enema will be required (sometimes requiring sedation or anesthesia), and this is covered in the next section on management. some cats present for the abdominal wall with the other hand, but great care must be taken with this maneuver, because the devitalized colon can be perforated more easily. , enemas as described are painful for the cat, and opioid analgesia is recommended at the time of anesthesia. opioids can reduce peristalsis in humans, but having evacuated the bowel, the pain relief is more important than this transient effect. an alternative to enemas is administration of an oral polyethylene glycol (peg ) solution (e.g., colyte, golytely). a nasoesophageal tube is placed and the solution is given as a slow trickle ( to ml/kg/hour) over to hours. defecation usually results in to hours. in a retrospective study of cats, median time to defecation was hours and the median total dose of peg was ml/kg. a no adverse effects were noted. a cat that has been obstipated needs supportive therapy when discharged. there are no controlled comparisons of the various therapies noted in table - ; the author prefers cisapride . mg, every hours to every hours, po (first thing in the morning, when the owner returns from work, when the owner goes to bed), and lactulose syrup ml/cat, every hours, po. a cat that has been so severely obstipated that an enema under anesthesia is required can be expected to continue these medications lifelong. to reduce fecal bulk and decrease the opportunity for recurrence, low-residue canned foods (or sachets) are preferred for cats that have become obstipated. some cats may benefit from high-fiber diets. as with simple initial episodes, canned food helps maintain adequate hydration, and at home subcutaneous fluids may be used additionally in cats with chronic kidney disease. with repeat episodes or severe obstipation, investigations for an underlying cause should be thorough and include evaluation for colonic mass obstructions. a review of published cases indicated that % of cases of megacolon are accounted for by idiopathic megacolon ( %), pelvic canal stenosis ( %), nerve injury ( %), or manx sacral spinal cord deformity ( %). although most cases are idiopathic, an attempt should be made to identify and treat any specific underlying causes. megacolon is not specifically defined in cats. it has been described as "generalized colonic dysfunction manifesting as severe colonic dilation and fecal impaction," or a "severely and irreversibly dilated and hypomotile" colon and "a subjective evaluation of the diameter of the colon, usually based on radiographic assessment." there are specific radiographic guidelines for humans with megacolon, in that a colonic diameter of more than . cm at the level of the pelvic brim is considered diagnostic. intake and therefore help maintain hydration. however, increased dietary fiber is beneficial for some cats, and trial and error may be required to determine whether a high-fiber or low-residue diet will be of benefit to each individual cat. in one report, cats with recurrent constipation refractory to traditional medical and dietary management were successfully treated with a psylliumenriched dry extruded diet. a after month on the diet, cats had no clinical signs of constipation. the remaining cat was clinically normal after months on the diet. improvement was noted in of cats after only days of dietary therapy. measures should be taken to ensure adequate hydration. maintaining adequate hydration is particularly relevant for cats with chronic kidney disease that have impaired ability to conserve water. changing to wet food diets helps increase water intake. some cats with chronic kidney disease may need additional fluid support, such as subcutaneous fluids administered by the owner at home on a regular basis. underlying problems may be minor and simple to manage, such as an anal gland abscess, or more involved, such as reduced pelvic outflow, as a result of prior trauma. arthritis is a common underlying factor in many older cats and may be managed with prudent use of nonsteroidal agents (see chapter ) . in cases of obstipation, the cat is more likely to be debilitated to some degree; so, laboratory investigations to assess plasma or serum biochemistry parameters as well as hematology and urinalysis are ideal. any hydration deficit or electrolyte abnormalities should be corrected before the anesthesia that is often required to remove the obstructing feces. rectal suppositories and microenemas are usually ineffective in obstipated cats. enemas are often required to remove impacted feces in such circumstances. the enema solution must be warmed and introduced slowly to avoid vomiting. the typical volume required is to ml/kg (so, up to approximately ml/cat). the enema solution can be an isotonic electrolyte solution or tap water, and mild soap can be added (but any soap used must not contain hexachlorophene, which is neurotoxic if absorbed); mineral oil can be used ( to ml/cat) as a lubricant or docusate as an emollient ( to ml/cat), but the two agents must not be used together since docusate promotes mucosal absorption. sodium phosphate-containing enemas must not be used, because they can induce severe hypernatremia, hyperphosphatemia, and hypocalcemia in cats. often, the enema solution alone is insufficient to reduce the fecal mass, and manual manipulation of the feces by abdominal palpation is required. sometimes the feces must be broken down by a gloved finger perrectum while the colon is massaged manually through radiographically, in the lateral view, the normal colon should be approximately the same diameter as the length of the body of the second lumbar vertebra. in cats, however, "there are no published guidelines for determining megacolon, so, diagnosis of abnormal colonic dilatation is subjective." however, one author has suggested that "as a rule of thumb, the diameter of the colon should be less than the length of the body of the seventh lumbar vertebra (l )." this author continues, "enlargement of the diameter of the colon beyond times the length of the body of l is indicative of chronic large bowel dysfunction and an explanation must be sought." a recent paper found that of cats with no gastrointestinal disease had a colon diameter greater than the length of l ; however, no assessment of constipated cats was made. in practice, many cats with megacolon have a colonic diameter far exceeding this guideline ( figure - ). one study of cats with megacolon found the mean diameter of the colon was . times greater than the length of the seventh lumbar vertebra (median, . ; range, . to . ), but in general, objective descriptions of this condition are lacking in the veterinary literature. the definition of megacolon in cats should include functional as well as radiographic guidelines. in the absence of broadly recognized radiographic recommendations, the author proposes that the o'brien rule-ofthumb guidelines (as noted above) be introduced until a more comprehensive study can establish other radiographic diagnostic criteria (or confirm these). the author therefore proposes to define megacolon as dilatation of the colon, to more than . times the length of the seventh lumbar vertebra, which is refractory to medical and dietary management. practitioners can expect the radiographic assessment of colonic dilatation to exceed this guideline in cats with megacolon and, conversely, there are likely to be cats having colonic distention greater than this amount that will respond to medical and dietary management and can therefore not be defined as having megacolon. by the definition used above, megacolon is refractory to medical and dietary therapy; so, to be defined as having megacolon, a cat may have had several episodes of obstipation managed by enema as well as dietary trials (with both low-residue and high-fiber diets) and medical therapy with cisapride and an osmotic or emollient laxative; yet the cat will still obstruct with feces. in these circumstances, the only possible therapy is subtotal colectomy. subtotal colectomy refers to surgical excision of % to % of the colon, whether it is grossly diseased or not with preservation of the ileocolic junction (icj). this approach has resulted in a more favorable clinical response than when the icj is also excised. , when preserving the icj, it has been noted that, in some rare cases, it can be difficult to join the proximal segment of colon to the distal piece of descending colon because of the tethering effect of the ileocecocolic blood vessels. in these cases, sacrificing these vessels and removing the icj (i.e., total colectomy) is recommended to facilitate approximation of the ileum to the distal colonic segment. a recently described technique using a biofragmentable anastomosis ring, compared with sutured anastomoses, showed no discernible effect on prognosis. prognosis following subtotal colectomy is generally good. a review of multiple papers, totaling over cats that had undergone subtotal colectomy, found the most commonly reported perioperative complication was diarrhea or loose stools immediately after surgery. in the majority of individuals, stool consistency improves without further treatment so that within to weeks of the surgery soft, formed stools are developed. diarrhea can persist in a small number of cases. in the longer term, in some cats, constipation can eventually return, but this can usually be managed by dietary and medical therapies. pathology of the rectum or anus is relatively rare in cats and therefore poorly described in the veterinary literature. consequently, published information is often not referenced, suggesting it expresses the authors' opinions. readers are directed to surgical texts for details and approaches about surgical corrections. the anal sacs are paired cutaneous evaginations situated between the internal and external sphincter muscles. these sacs store secretions from alveolar and sebaceous glands that reside within the sacs. each anal gland has an associated duct that opens to the skin surface just lateral to the anus. , normal anal gland secretions have only very recently been described and vary markedly; the color can be white, brown, orange, yellow, tan or gray, and consistency can range from watery to thick and creamy, with two thirds of cats having solid portions within the secretion. on microscopic examination, epithelial cells are commonly seen, with most cats having some neutrophils present. bacteria are commonly recognized as are, on some occasions, yeasts. bacteria seen in this study were mainly gram-positive cocci ( %) or gram-negative cocci ( %). gram-negative or grampositive rods were also seen but were rarely the dominant bacterial population. with such a wide range of normal secretions, it is difficult to diagnose any pathology from the nature of the secretion alone. however, blood is infrequently recognized, and neutrophils are typically present in only small numbers in normal secretions. anal sac diseases described in cats include impaction, inflammation (sacculitis), infection, abscessation, and neoplasia (essentially the same as in dogs). , it has been contended in dogs that sacculitis and abscessation are an extension of impaction. it is not known in dogs or cats what the predisposing causes are, but suggested underlying reasons are loose stools (that are less effective at expressing the sac during defecation), local swelling or edema occluding the duct, and obesity. the author's observations have also indicated that constipation can result in anal sac impaction because of less frequent expulsion of the sac contents; the resultant pain of the anal sac impaction can lead to further constipation, thus establishing a cycle. the retention of secretions may predispose to sacculitis, but impacted anal sacs do not always result in inflammation. abscessation is a likely sequel to sacculitis. cats usually present for licking, scratching, or biting at the perineal area and can present for scooting (or dragging their anus) as dogs do. other presenting signs can be inability to sit or settle, a lump seen by the owner, or a generally unwell state. expression is the only management required for impacted (and not infected) anal sacs. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb externally. this is painful for most cats; so, the cat should be held by an experienced assistant, and it is sometimes not possible without some degree of sedation. with frequent episodes, underlying causes should be investigated. sometimes, trial-and-error diet change to manipulate the nature of the feces to either more (highfiber diets) or less (low-residue diets) bulk help reduce the frequency of episodes. obesity should be managed by reduced caloric intake, but dietary management for this should also take into account the nature of the feces. overt infection may be recognized by pus secretion from the anal sacs, which will have a high numbers of neutrophils. this can be managed by broad-spectrum antibiotics, such as amoxicillin/clavulanate or cephalosporins. a single treatment with a nonsteroidal antiinflammatory drug, such as meloxicam, can be given in animals with appropriate hydration and without other illness. anal sac abscesses often present already open and draining. many heal well by secondary intention with antibiotic treatment until they are closed over; so rechecks are required before the completion of an antibiotic course. large abscesses may require surgical drainage with the insertion of a penrose drain and management as for a cat fight abscess. it must be remembered that wounds in this area are easily re-infected by fecal contamination. recurrent impaction, sacculitis, or infection may require anal sacculectomy (as in dogs). this procedure should be delayed until infection is cleared. the procedure is similar to that performed in dogs. reports of anal sac/gland neoplasia were confined to sporadic case reports, , until a large case series was recently published. in this study, cases of anal gland carcinoma were recognized at a private diagnostic laboratory during a -year period, with submissions from practices. this indicates that, for most practices, this condition will be seen, at most, once every years. affected cats ranged in age from to years (median and mean, years); female (mostly spayed) cats were overrepresented ( % of cases), and siamese cats may have been over-represented ( . % of cases). the number of siamese cats with anal sac neoplasia was times greater than the number of siamese cats in the laboratory reference population. affected cats presented for dyschezia, recurrent constipation, change in the nature or volume of feces, b). a low-residue wet diet is recommended to reduce fecal bulk during the healing period. rectal prolapse occurs as a result of a disease process that causes chronic straining, such as intestinal conditions that result in diarrhea and tenesmus . conditions that result in constipation or other intestinal obstruction . lower urinary tract diseases . dystocia and/or perineal swelling or ulceration, sometimes with purulent or hemorrhagic discharge. most tumors were originally interpreted as and initially managed as anal sac abscess. presumptive metastasis in liver, lung, or abdominal lymph nodes was recognized by physical examination or radiography in six cats; one cat was hypercalcemic. excision appeared to be curative (with a -to -year follow-up period) in of cats undergoing surgery for resection or debulking (others had only incisional biopsy performed). for the remaining cats with known postsurgical outcome, median survival was only months, with a % -year survival rate (with none of these cats surviving to years). atresia ani is a developmental defect of the anal opening or terminal rectum (see figure - ). kittens usually present within days or weeks of birth with abdominal distention, discomfort, tenesmus, restlessness, vomiting, and/or loss of appetite. there are several anatomic variations : • type i: a membrane over the anal opening remains, with the rectum ending as a blind pouch just cranial to the closed anus. • type ii: the anus is closed as in type i, but the rectal pouch is located somewhat cranial to the membrane overlying the anus. • type iii: the rectum ends as a blind pouch cranially within the pelvic canal (rectal atresia), whereas the terminal rectum and anus are normal. • type iv: occurs in females and atresia ani exists with a persistent communication between the rectum and the vagina (rectovaginal fistula). this fistula can occur with a normal anal opening as well. most reported cases have been type iv, , , , , and this has also been recognized with concurrent sacrococcygeal agenesis. surgical correction has been described for type ii and type iv , atresia ani in cats. the reader should consult these references for surgical advice; possible complications include megacolon after prolonged obstruction, postsurgical anal stricture, and fecal incontinence because of sphincter dysfunction. foreign bodies in cats rarely obstruct the gastrointestinal tract distal to the jejunum ; however, large fecal balls resulting from constipation can, additional to constipation or obstipation, cause distention of the anus. this distention can result in inflammation of the anal sphincter with loss of tone ( figure - , a) , which, in the author's experience, is temporary with correction of the underlying cause of constipation. it can take some weeks for the dilated anus to return to normal (figure - , with antibiotics, such as cephalosporins, and regular cleansing. prolapses are usually classified in three ways. first degree: prolapse of only mucous membrane . second degree: prolapse of full rectal wall thickness . third degree: prolapse is sufficient to bring mesorectum outside the anus the prolapsed rectum is obvious but must be differentiated from ileocolic intussusceptions, which have been described with neoplasia. this distinction can be made by inserting a thermometer through the anus alongside the prolapsed mass. insertion will not be possible for an intussusception but will be for an anorectal prolapse. the prolapsed tissue must be assessed for viability, and management must include determining and managing the underlying cause as well as management of the prolapse. in simple cases where the mucosa is viable, the prolapse can be reduced with lubrication and gentle pressure. a temporary purse-string suture may be required to prevent recurrence. perineal dermatitis is often confused with gastrointestinal or urogenital disease, because there are often copious sebaceous secretions that can mimic fecal or urinary secretions. perineal dermatitis can result from flea or other allergies but also fecal or urine scalding associated with diarrhea or urinary incontinence, respectively. skin fold dermatitis can also occur in obese cats ( figure - ). episioplasty has been described to correct this, but the author has found that stringent dieting can result in improvement while managing the skin fold dermatitis these populations. the reported prevalence for each parasite varies greatly with the population studied, the geographic location of the population, and the sensitivity of the diagnostic test used to study that population. the presence or absence of diarrhea is not a reliable predictor of whether a particular cat is infected with or shedding a parasite. in fact, most cats with diarrhea do not harbor enteric protozoa. on the other hand, most cats with diarrhea because of enteric pathogens will shed those organisms, often intermittently. it is important to remember that infection with most gastrointestinal parasites may not cause clinical signs. therefore detection of a pathogenic parasite in a cat with diarrhea does not necessarily prove causation. a search should always be undertaken to identify other causes of diarrhea prior to convicting a cat of having diarrhea because of a particular parasite. in addition, co-infections or the presence of other noninfectious causes of diarrhea can result in more severe diarrhea that is often refractory to treatment for the parasite. treatment will be more rewarding if all potential causes of diarrhea are identified in the patient. enteric parasites with zoonotic potential occur commonly enough that cats, particularly those with diarrhea and who are owned by immunocompromised persons, should be evaluated for those pathogens. , the following is a discussion of the most common enteric parasites found in cats. for more on parasite prevention and control, see chapter , and for more on zoonotic enteric parasites, see chapter . ollulanus tricuspis is an almost microscopic nematode worm infecting the stomach of domestic and wild cats. the worm measures less than mm long. the larvae of o. tricuspis develop and hatch within the uterus of the female worm. they develop to maturity in the stomach of the cat where it is capable of re-infecting the host. the worm is transmitted to other cats that ingest the vomitus of an infected cat. clinical signs shown by infected cats include vomiting, anorexia, and weight loss. , histologic findings in infected cats include lymphocytic-plasmacytic gastritis, lymphoid hyperplasia, and mucosal fibrosis. gross lesions may be absent, or the cat may develop nodular gastritis. one report suggested the parasite may have been a contributing factor in the carcinogenesis of a gastric adenocarcinoma in an infected cat. . a common theme when discussing the prevalence of most gastrointestinal parasites in cats is that they occur more commonly in younger cats and in cats housed in crowded conditions, such as catteries and shelters. it is likely an increased chance for transmission exists in lungs. after further development in the lungs, the parasite migrates up the trachea and is swallowed. adult s. felis and s. planiceps burrow into the wall of the small intestine, while adult s. tumefaciens lives in the colonic mucosa. ova may be shed in the feces or hatch in the intestinal tract. autoinfection occurs if larvae become infective and penetrate the intestinal wall before being shed. ova and larvae that are shed develop into freeliving adult worms. the prepatent period is between and days. , clinical signs and diagnosis signs of a strongyloides spp. infection are usually absent. , lung migration may cause cough or respiratory distress. the presence of the parasite in the intestinal tract may result in diarrhea and weight loss. strongyloides tumefaciens is associated with the formation of small, worm-filled nodules in the colon. identification of strongyloides spp. larvae using the baermann fecal concentration technique is required to diagnose most infections. unless the infection is heavy, examination of a fresh fecal smear is insensitive for identification of these larvae. the nodules formed by s. tumefaciens infection can be visualized during colonoscopy. histopathology of the biopsied nodules should reveal many adult worms. infection with strongyloides spp. can be treated with fenbendazole, pyrantel pamoate, thiabendazole, , or ivermectin. to evaluate efficacy, repeat a fecal examination to days after the treatment ends. because of the presence of free-living adult worms in the environment and the ability of larvae to cause infection by penetrating intact skin, prevention is difficult. keeping cats indoors in warm, humid climates may be an owner's only means of preventing infection with strongyloides spp. parasites. infections with trichuris vulpis rarely occur in cats and are considered to be clinically unimportant. , the two species of roundworms commonly infecting cats are toxocara cati (figure - ) and toxascaris leonina (figure - ) . the latter also has the ability to infect dogs. cats are infected with t. cati in several ways. most commonly, infection is by ingestion of contaminated food, water, or infected paratenic hosts such as rodents. transuterine transmission has not been reported. the diagnosis of infection with o. tricuspis is difficult, because ova are not shed in the feces; rather, the vomitus must be examined for worms or larvae. the worms may also appear in gastric mucosal biopsy samples. a report of cats undergoing endoscopic examinations found the parasite in gastric biopsy samples from cats. fenbendazole may be effective in treating infections with o. tricuspis. preparations with febantel may also be expected to successfully treat these infections. transmission can be prevented by appropriately treating infected cats. other cats should not be allowed to ingest infected vomit. this parasite is of no zoonotic concern. physaloptera another parasite rarely inhabiting the stomach in cats is in the genus physaloptera. larger than ollulanus tricuspis, this blood-sucking worm infects cats that have ingested intermediate hosts, such as cockroaches, crickets, or flour beetles. preying on transport hosts, such as mice that have eaten an intermediate host, is another way cats become infected with this parasite. clinical signs of infection with physaloptera spp. include vomiting, anorexia, and melena. a diagnosis of physaloptera infection can be made after identifying the ova in the patient's feces or adult worms in the vomitus. occasionally, the worms may be seen during gastroscopy. the adult worms must be differentiated from ascarids. infection can be treated with ivermectin, pyrantel pamoate, or fenbendazole. because there is no migratory phase of the life cycle, the treatment does not need to be repeated. three species of strongyloides infect cats. strongyloides felis infects cats in india and tropical australia, , s. tumefaciens is a rare parasite of cats in the southeastern united states, and s. planiceps is found in cats in malaya and japan. strongyloides stercoralis, found in dogs and humans, produces experimental infections in cats, but natural infection with this species has not been observed. feline infection with strongyloides spp. is considered by most to be rare. however, one report from australia identified s. felis in of necropsied cats. infection with strongyloides spp. occurs after ingestion of infective larvae. infection can also take place after the larvae penetrate the skin of the cat. ingested larvae penetrate the intestinal wall and migrate through the diaphragm into the lungs. after cutaneous penetration, the larvae enter the venous circulation and enter the clinical illness because of roundworm infection is uncommon. illness, when it does happen, most often occurs in kittens signs may be mild and can include vomiting, diarrhea, weight loss, poor growth, and a "pot belly." a heavy infection with t. cati can result in catarrhal enteritis. severe infections can lead to intestinal obstruction and, possibly, perforation. much less dramatic changes arise after infection with t. leonina, although enteritis may occur. roundworms are frequently diagnosed with a fecal floatation. the centrifugal floatation technique is more sensitive than the simple fecal floatation technique many hospitals use. occasionally, adult worms will be passed with the feces. the goals of treating roundworms include disease prevention in an individual cat or kitten, prevention of environmental contamination by cats defecating outside, and the prevention of zoonotic infections. many effective and safe anthelmintics are available (table - ) . benzimidazoles, such as fenbendazole, act on the parasite's microtubular structure, leading to disintegration of the worm's intestines, muscular layer, and hypodermis. pyrantel in the pamoate formulation is poorly absorbed and causes paralytic parasite death. macrocyclic lactones, such as milbemycin, also lead to paralytic parasite death. these compounds act on the parasite's gamma-aminobutyric acid (gaba)-and glutamate-controlled ion channels. these channels are lacking in tapeworms, accounting for the lack of efficacy against these parasites. lastly, emodepside (a cyclic octadepsipeptide) has been combined with praziquantel in the product profender (bayer animal health). this topical parasiticide has been shown to be both safe and effective. these drugs appear to be so safe that overdosing is almost impossible. kittens can be dewormed starting at two weeks of age and again at , , , , and weeks. older kittens and adults can be dewormed every month to months. because of the safety of these drugs, the possibility of false-negative tests and, more importantly, the zoonotic potential of these infections, perhaps all kittens should be dewormed, not just those testing positive. roundworm ova are very hardy and can remain infective for years. they survive sewage treatment and composting, and there is no practical means of decreasing the ova population once the environment is contaminated. thus it is best to attempt to prevent contamination in the first place. when practical, keeping cats indoors allows appropriate control of potentially transmammary infection occurs, but only if the queen is acutely infected late in pregnancy. chronically infected queens do not pass t. cati ova in their milk. after ingestion, t. cati larvae migrate through the small intestinal wall, into the liver, and then to the lungs where they are coughed up and swallowed. these larvae then infect the small intestine. some of the migrating larvae become encysted in the cat's muscle tissue. larvae from ova ingested through the milk tend not to undergo migration and mature directly in the small intestine. the prepatent period is approximately weeks. infection with t. leonina occurs after ingestion of infective ova or an infected paratenic host. unlike t. cati, very few t. leonina larvae migrate through the cat's tissues. most develop in the wall of the small intestine. the prepatent period is to weeks. toxascaris leonina ova can become infective within days of being passed in the feces when the ambient temperature is ° c but normally require to weeks. lungs, and kidneys. ocular larval migrans results in granulomatous retinitis that is often misdiagnosed as retinoblastoma in older children. this can lead to unnecessary enucleation. toxocara cati appears, however, to be less important than t. canis as an infection in humans. the species of hookworms that infect cats are ancylostoma tubaeforme and ancylostoma braziliense (see figure - ). they are reported to be an uncommon infection in cats. , ancylostoma braziliense can also infect dogs. hookworm infections occur after ingesting food or water contaminated with hookworm larvae or eating contaminated fecal material. if the pet cat is allowed outdoors, attempts at preventing hunting may reduce the possibility of infection. keep children's play areas, such as sand boxes, inaccessible to cats when children are not at play. feeding only well-cooked food can prevent infection by contaminated food. finally, empirical, preventative deworming for cats that go outdoors should be performed to times yearly. any less frequently does not lead to an appreciable decrease in the prevalence of the parasite. roundworms easily infect humans who ingest the ova, particularly children. visceral larval migrans occurs after infection with toxocara canis in humans. infection can lead to the formation of nodules in the brain, liver, tapeworm infections are well tolerated by the cat. usually there are no signs of infection other than finding segments on the feces or attached to perianal hair. because both d. latum and spirometra tapeworms absorb vitamin b across the cuticle, megaloblastic anemia is possible, but unlikely. tapeworm infections are diagnosed by identifying the typical appearance of the segments or the egg packets within the segments. the segments of t. taeniaeformis are flat, while those of d. caninum have been described as appearing like a grain of rice. the segments should be handled carefully, because they are friable and rupture may result in exposure of the handler. the operculated ova of d. latum and spirometra spp. must be differentiated from trematode ova. even though tapeworm infections are well tolerated, cats should be treated for reasons of owner discomfort and public health concerns (see table - ) . these infections are easily treated, because drug treatment is highly effective. re-infection must be controlled using preventative measures, especially flea control to prevent re-infection with d. caninum. praziquantel and infected paratenic hosts. the larvae can survive for months in the tissues of paratenic hosts. infection also occurs after larval migration through the skin. in either case, the worm matures in the small intestine. unlike dogs, transmammary infection has not been reported in cats. , the prepatent period is between and days, depending on the route of infection. the time to patency after transcutaneous infection is longer than for direct colonization. infective l larva develop to days after the ova are passed. developing larvae attach to the mucosa of the small intestine where they ingest copious amounts of blood. because the worms can remove a significant volume of blood from kittens, weakness from iron-deficiency anemia or blood-loss anemia may be noted. melena and diarrhea may also be recognized. signs are uncommon in adult cats. identification and treatment of hookworm infections are similar to that for roundworm infections (see table - ) . hookworm larvae are not as hardy as roundworm eggs. soil contamination may be a temporary problem in areas that experience a hard frost. hookworm larvae will not develop in temperatures less then ° c or greater than ° c. frequent, appropriate disposal of feces, cleaning surfaces with a % bleach solution, and deterring hunting may prevent infections. migration through the skin of persons coming into contact with the larvae of a. braziliense is the most common cause of cutaneous larval migrans, particularly in the southeastern united states. this is an erythematous, pruritic skin eruption often found on the soles of the feet of infected children. the tapeworms most commonly found in cats are dipylidium caninum and taenia taeniaeformis. diphyllobothrium latum, spirometra spp., and echinococcus multilocularis occasionally infect cats. the latter is important, because it can lead to alveolar echinococcosis in humans. spirometra tapeworms are found in north america (s. mansonoides) and far-east asia (s. mansoni and erinacei), while d. latum prefers temperate climates. unnoticed, but the cat may cough or experience hemoptysis. diagnosis involves demonstration of fluke ova in the feces. although therapy may be unnecessary, praziquantel or epsiprantel are effective in eliminating the intestinal population of the fluke. platynosomum spp. are flukes living in the gall bladder, bile ducts, and pancreatic ducts. these flukes are most prevalent in the southeast united states and caribbean islands and require two intermediate hosts. the first host is a snail, while the second intermediate host is a lizard, toad, gecko, or skink. cats become infected with this fluke after ingesting an infected second intermediate host. the prepatent period for the fluke is weeks. most infections are subclinical. if clinical signs do occur, they may include weight loss, vomiting, diarrhea, icterus, hepatomegaly, or abdominal distention. diagnosis involves identification of ova shed in the feces using a fecal sedimentation method or by finding adult flukes in the gall bladder or bile ducts during abdominal surgery. treatment involves administering praziquantel ( mg/kg, q h, po for to days) and/or surgical removal of the flukes. two species of coccidians are the most common to infect cats, isospora felis and isospora rivolta (figure - ) . the genus isospora may be renamed cystoisospora. these are epsiprantel are safe and effective. fenbendazole is effective against t. taeniaeformis, but not d. caninum. without controlling exposure to intermediate hosts, tapeworm infections are difficult to eliminate. flea control is imperative in eradicating infections with d. caninum. controlling predation helps prevent ingestion of t. taeniaeformis-infected rodents. infection with d. caninum occurs in young children who are most likely to eat fleas. infection results in only minimal signs of illness. the larval stage of t. taeniaeformis is of little zoonotic importance. although cats are uncommonly infected with echinococcus multilocularis, potentially life-threatening alveolar damage occurs in north american humans infected with this tapeworm. plerocercoids of spirometra spp. can penetrate the mucous membranes or open skin wounds of humans and migrate around the subcutaneous connective tissue, forming nodules, a condition called sparganosis. megaloblastic anemia, as a result of vitamin b deficiency, may occur in humans infected with d. latum or spirometra spp. tapeworms. alaria marcianae flukes reside in the intestinal tract of cats and the mammary glands of lactating queens. miracidia hatch underwater from ova shed in the feces and penetrate the skin of a snail. after further development, cercariae penetrate the skin of leopard frog tadpoles and are able to survive the metamorphosis to the adult frog. if the tadpole is eaten by a snake, bird, or mammal, the parasite enters the host's tissues but does not undergo further development. after a male or nonlactating female cat ingests the infected intermediate host, the parasite penetrates the wall of the small intestine, passes through the diaphragm, and enters the lungs for further development. finally, the parasite is coughed up and swallowed to complete maturation and reproduce in the small intestine. if, however, an infected host is ingested by a lactating queen, the parasite migrates through the tissues to the mammary glands, rather than the lungs. once shed in the milk, the parasites develop into mature adults in the kittens. some of the mesocercariae remain in the mammary glands to infect future litters. clinical signs associated with worms in the small intestine are uncommon. migration through the lungs often goes species-specific obligate intracellular parasites. , they are able to survive in the environment for months. a detailed description of the coccidial life cycle can be found elsewhere. , simply put, direct transmission is by ingesting oocyst-contaminated food or water or by grooming contaminated body parts. indirect transmission occurs after ingesting a mechanical vector or the infected tissues of paratenic hosts. after ingestion by a cat, the oocyst excysts in the small intestine and enters the enterocyte where further development occurs. the parasite may also migrate through the intestinal wall to form cysts in mesenteric lymph nodes. these cysts may serve as a source for reinfection. , the prepatent period is to days and the shed oocyst becomes infective after several days of exposure to warmth and moisture. infection with isospora spp. is usually subclinical. signs, if they occur, range from mild, transient watery diarrhea to severe mucohemorrhagic diarrhea with vomiting and resultant dehydration and weight loss. , signs are most commonly recognized in severely infected neonatal kittens, particularly those with concurrent illness, and arise because of small intestinal congestion, mucosal erosion, or villus atrophy. signs may also be noted in immunosuppressed adult cats. isospora species are readily found in fecal floatation or wet-mount examinations. shedding can be intermittent, but most cats with diarrhea caused by coccidial infection shed large numbers of oocsyts. fortunately, in most cats, the diarrhea from isospora spp. infection is self-limiting. in fact, if a kitten is persistently shedding oocysts despite appropriate treatment or the parasite is identified in an adult cat with chronic diarrhea, attempts should be made to identify co-infections or other diseases that may cause diarrhea. anticoccidial drugs are either coccidiostatic or coccidiocidal (table - ) . coccidiostatic drugs are the most commonly used drugs for individual pet cats. trimethoprim-augmented sulfadiazine (tribrissen; intervet/schering-plough animal health, summit, nj) or another sulfa-containing antibiotic, sulfadimethoxine (albon; pfizer animal health, madison, nj), can be used. supportive care for severely affected kittens, such as parenteral rehydration, should be used as needed. coccidiocidal drugs are often reserved for use in densely populated situations such as catteries or shelters. however, many veterinarians are now using them as a first-line defense against isospora spp. infection. ponazuril (marquis oral paste; bayer animal health, shawnee mission, kan.), formulated for horses, is effective and can be safely administered to cats. for more on the use of ponazuril in cats, see chapter . a related drug, diclazuril, is also available and may be administered once at mg/kg po. while not available in north america, toltrazuril (baycox, bayer animal health) may be administered once at mg/kg po or mg/kg po once daily for days. a a second course of therapy days later may be required to completely eliminate the oocysts. sanitation is very important, because the oocyst requires several days to become infective. frequent removal of feces, preferably daily, is recommended to prevent re-infection and transmission to other cats. controlling a cat's ability to hunt reduces the chance of ingesting an isospora-infected rodent. control of mechanical vectors, such as cockroaches and flies, is also useful. since a cat can become infected after grooming an infected cat's perineum, consideration should be given to treating all cats in contact with the patient. in addition, catteries and shelters should ensure all food is well cooked, litter boxes are cleaned daily, and surfaces are well cleaned with steam or % ammonia. where recurrent isospora spp. infections are a problem, prophylactic treatment of all -to -week-old kittens with ponazuril should be considered. despite all wellintentioned efforts at hygiene and treatment, isospora spp. infection can still be transmitted to other cats. because these are species-specific parasites, transmission of i. felis and i. rivolta from cats to humans does not occur. the flagellated protozoal parasite, giardia duodenalis, has seven microscopically indistinguishable genotypes or assemblages. assemblages a and b infect humans, while assemblage f is harbored by cats. cats will occasionally harbor assemblages a and b. infection with g. duodenalis occurs after ingesting cystcontaminated feces, by grooming an infected cat or from contaminated fomites. re-infection may occur by selfgrooming. only a small number of cysts need be ingested to establish an infection. in humans as few as cysts are required to cause infection. after ingestion of infective cysts, trophozoites begin to excyst in the stomach. this process is completed in the proximal duodenum. the trophozoites adhere to enterocytes along the length of the small intestine using the ventral suction disk. intermittent shedding of immediately infective cysts begins to days after infection. proteins released during encystment of the trophozoites are detected by the fecal antigen tests. cysts may adhere to the perianal region, facilitating re-infection by self-grooming. occasionally, trophozoites are found in examinations of fresh, watery feces. these do not survive for long and are not infective. the mechanisms of disease induced by g. duodenalis are still unclear. after the trophozoite attaches to the brush border of the enterocyte, the tight junction between cells is disrupted, increasing intestinal permeability. the brush border becomes attenuated, further exacerbating malabsorption of water, electrolytes, and other nutrients. the alteration in intercellular adhesion results in t-lymphocyte activation and mucosal cell injury. infection also promotes mucosal cell apoptosis (preprogrammed cell death). in addition, small intestinal bacterial overgrowth may accompany g. duodenalis infections, resulting in more severe clinical signs. fortunately, most cats infected with g. duodenalis show no clinical signs. , the most common sign is acute, transient, small bowel diarrhea without systemic illness, such as fever or vomiting. less commonly, a cat might have profuse, watery malodorous diarrhea with mucus. also possible, but uncommon, is weight loss , or abdominal pain. the severity of clinical signs exhibited in an individual cat depends on the age and general health of the cat. cats co-infected with cryptosporidium felis or tritrichomonas foetus may have more severe diarrhea that is more difficult to control, as will the presence of bacterial overgrowth. the diagnosis of g. duodenalis requires demonstration of trophozoites or cysts in a fecal examination, or detection of encystment proteins or giardial dna in a fecal sample. a reliable diagnosis may be difficult to obtain for several reasons. cysts are small, easily missed, and must be differentiated from plant debris or yeast. trophozoites are short lived outside the body and can only be found in very fresh, watery feces or, better yet, in diarrheic feces collected directly from the cat's rectum. shedding of cysts is usually intermittent, and the intensity of shedding varies greatly. , because of these difficulties, the absence of the organism in a fecal sample does not eliminate it as the cause of diarrhea. it is often necessary to test multiple fecal samples, using at least two different techniques in order to find the organism. , the easiest test to perform is a fecal smear or wet mount examination to identify trophozoites or cysts (figures - and - ). the sample examined should be very fresh, warm, diarrheic feces. one drop of feces is placed on a slide along with a drop of . % saline or lugol iodine. trophozoites are identified by their characteristic structure (table - ) . the motile trophozoites have a motion described as appearing like the back and forth rolling motion of a falling leaf. since lugol iodine stain kills the trophozoite, there will be no motion to detect. this test is not very sensitive; however, with trained examiners, the test has a high specificity. increased sensitivity can be gained by performing a centrifugal flotation using zinc sulfate. the sample should be warm, fresh feces or feces refrigerated for no more than days. the processed sample is examined for the same structures as the wet mount. the sensitivity of examining one sample is % and increases as more samples are examined. the sensitivity of looking at three samples is % , ; therefore the test is not considered negative until three specimens have been found free of the organism. a fecal antigen test that identifies the encystment protein is available. the snap giardia antigen test (idexx laboratories) uses fresh or frozen feces, or feces refrigerated for less than days. since the antigen is continuously shed, this test avoids the problem of intermittent shedding of the whole organism. the sensitivity of the test is %, with a specificity of %. by combining the antigen test with a zinc sulfate fecal centrifugal flotation, the sensitivity improves to . %. it is unknown how long the antigen remains in the feces after treatment. thus a zinc sulfate centrifugal flotation examination should be used to evaluate therapeutic efficacy. , the use of this test in cats without diarrhea is controversial, because these cats are unlikely to shed cysts. the zoonotic significance of a positive antigen test in a cat not shedding cysts is unknown and may cause confusion. polymerase chain reaction detection of giardia dna is available, but the test has not been standardized across all diagnostic laboratories. one needs to ensure the laboratory performing the test has validated it for assemblage f. the test may also be used to identify cats harboring the zoonotic assemblages a and b. the sensitivity of this test is unknown. two commonly available drugs are used most frequently to treat infections with g. duodenalis (see table - ) . fenbendazole may be effective and can be used in pregnant queens and in cats co-infected with roundworms, hookworms, and taenia spp. tapeworms. however, in one small study, only four of eight cats infected with both g. duodenalis and cryptosporidium felis stopped shedding giardia permanently after receiving fenbendazole. febantel, in the combination product drontal plus (bayer animal health), is converted to fenbendazole. when six experimentally infected cats received . mg/ kg of febantel q h po for days, four of them stopped shedding g. duodenalis cysts. metronidazole has been the traditional drug used to treat g. duodenalis in pets. the drug is also useful for treating concurrent small intestinal bacterial overgrowth and clostridial infections. the administration of metronidazole may eliminate shedding in % of cats. neurologic side effects may occur at the dose recommended for treatment of giardia (see above, therapeutics for vomiting and diarrhea). the use of a giardia vaccine was ineffective in clearing infection by itself. the combination of fenbendazole and metronidazole has been suggested as the initial treatment of choice for g. duodenalis infections. although controlled studies are lacking, they may work synergistically by acting on two different targets within the parasite. febantel would be expected to have the same synergism with metronidazole. drug therapy may not be necessary in cats without diarrhea that are infected with g. duodenalis, because it is uncommon for a cat to carry the assemblages required to infect humans. the veterinarian may be obligated to treat a healthy cat if the owner wants to treat, the owner is immunocompromised, or the goal is eradication of an infection from a multicat home or prevention of parasite transmission to giardia-naïve cats is attempted. what may appear to be treatment failure is more likely to be re-infection. in addition to drug therapy, steps should be taken to prevent re-infection. all cats with diarrhea positive for g. duodenalis should be treated along with their housemates. sanitation is imperative in the fight against re-infection and transmission of g. duodenalis. dispose of old litter pans and scoops and use disposable litter boxes during treatment. when the infection is eliminated, not just controlled, new litter boxes and scoops may be purchased. bathe all cats during treatment to remove cysts from the hair coat. since giardia spp. cysts are susceptible to desiccation, blowdry all cats using a warm air blower, paying particular attention to the perineal area. disinfect bowls, housing, and other utensils with bleach. in addition to antiprotozoal drugs and sanitation, supportive care may become necessary. probiotics and a highly digestible, bland diet may be offered to cats with small bowel diarrhea, while a high-fiber diet may be useful for those few cats with large bowel diarrhea. where required, hydration and electrolyte imbalances must be corrected and antiemetics used to control vomiting. therapy can be evaluated by retesting feces with a zinc sulfate centrifugal flotation examination to days after the end of treatment and again weeks later. a positive test immediately posttreatment is most likely because of therapeutic failure. if the cat is negative immediately after treatment ends, but is positive weeks later, re-infection is likely. since the fecal antigen test may remain positive long after the infection is eradicated, this test is inappropriate for evaluating therapy. , re-treatment of fecal flotation-positive, recovered cats may be handled in a manner similar to the positive healthy cat mentioned above. cats with diarrhea that continue to shed cysts may be re-treated for g. duodenalis infection along with dietary modification and empirical treatment for other common intestinal parasites. however, serious consideration should be given to investigation into other potential causes of diarrhea. the giardia vaccine has been found to be ineffective in preventing infection and production has been discontinued. this means prevention of giardia infection involves avoiding exposure, stress and re-infections. providing a clean environment, feeding only processed foods, and controlling potential transport hosts will help reduce the chances of exposure. isolation of cats with diarrhea may be important, too. municipal sanitation control is difficult as the cyst survives for weeks in cool, moist environments. cysts are also able to survive water treatment and can pass through attempts at water filtration. giardiasis is associated with debilitating diarrhea in some humans, particularly those who are immunocompromised. however, cats do not commonly carry the assemblages needed to infect humans. transmission of g. duodenalis from cats to humans is rare and unproven. still, it seems prudent to consider the owner's health when contemplating management of giardial infections in cats. to avoid human health risks, cats with diarrhea that test positive for g. duodenalis should be treated with the goal of controlling the diarrhea. since no treatment for g. duodenalis is completely effective or % safe, treatment of positive cats without diarrhea should only begin after a discussion of the benefits and risks of the treatment with the owner. tritrichomonas foetus is best known for causing bovine reproductive infections. it is an obligate anaerobic parasite that also colonizes the lower intestinal tract of cats. there are enough differences between the two isolates that the feline isolate does not cause disease in heifers and vice versa. the parasite depends on the host's normal intestinal flora and secretions for obtaining nutrition. a report from the united states of purebred cats tested at an international cat show found t. foetus in of the cats tested, a prevalence of %. this parasite seems to have a higher prevalence in purebred cats than nonpurebred cats. a study of pet cats visiting veterinary hospitals across the united states reported of purebred cats were positive for t. foetus, while only of nonpurebred cats were positive. in this same study, of the positive tests were from purebred cats. a study from the united kingdom of diarrheic fecal samples sent to a veterinary diagnostic laboratory reported similar results. purebred cats represented of the cats testing positive for t. foetus. the u.k. study also found the siamese and bengal breeds each represented of positive cats; only two other breeds tested positive. transmission like most other protozoal parasites, t. foetus is transmitted by ingestion of the parasite, in this case, the trophozoite. unlike most of the other parasites, t. foetus does not form cysts and only survives up to days outside the body in moist feces. a cat becomes infected through the use of a shared litter box with an infected cat. after walking into the box, the parasite is transferred from the infected feces of one cat to the paws of the other. infection then occurs through ingestion of the trophozoites during grooming. after infection, t. foetus colonizes the distal ileum and colon, followed by shedding of infective trophozoites to days later. there are several mechanisms by which t. foetus causes diarrhea. these include alteration of the cat's normal bacterial flora population, increases in local inflammatory cytokine concentrations, production of enzymes, and direct mucosal injury. the resulting injury leads to plasmacytic-lymphocytic and neutrophilic colitis. although most infections involve only the mucosa of the colon, one study reported two of seven cats with diarrhea and t. foetus infections as having trophozoites in deeper layers of the colonic wall. co-infection with cryptosporidium felis or giardia duodenalis can be associated with increased numbers of t. foetus trophozoites and increased severity of diarrhea. signs of infection are most frequent in kittens and young cats, although infections without clinical signs can occur. adult cats, however, may also show signs of t. foetus infection. the most common sign is a foulsmelling large bowel diarrhea with increased frequency of defecation, mucus, blood, and flatulence. the consistency of the diarrhea may wax and wane, but the presence of diarrhea does not. cats with diarrhea are otherwise in good health and maintain their body condition. , severe diarrhea can result in anal swelling and fecal incontinence. diarrhea may respond to the use of antibiotics because of changes in the cat's intestinal microbial flora. however, it always returns at the cessation of therapy. , many cats experience a spontaneous resolution of the diarrhea within years of diagnosis. , since t. foetus causes reproductive infections in heifers and bulls, there is speculation the parasite also infects the reproductive tract in cats. tritrichomonas foetus was found in the uterus of a queen with pyometra. however, in a study of breeding male and female cats from catteries, no cytologic or molecular evidence of t. foetus was found in the reproductive tract. the authors reported colonic infection with t. foetus in of the cats representing of the catteries. detection of the trophozoites in a sample of feces is the most expedient means of diagnosing an infection with t. foetus (figure - ). an index of suspicion is required, because the clinical presentation of t. foetus infection is often mistaken for infection with giardia duodenalis. if a cat is not responding to treatment for that parasite, consider t. foetus as a cause of the diarrhea. the sample required for the diagnosis of t. foetus is a fresh, nonrefrigerated sample of watery feces. refrigeration kills the trophozoites, and they are not found in normal feces. the sample may be freshly passed diarrhea, feces collected using a wire loop passed into the colon, or collected by a colonic flush using a red rubber catheter and ml of saline. a wet mount or smear examination of the feces should be performed on all cats with diarrhea. examination of multiple samples may be required to find the t. foetus trophozoites with this technique because it is insensitive. the trophozoites must be differentiated from giardia duodenalis based on structural differences and motility patterns (see table - ). the trophozoites of t. foetus can be cultured using the inpouch tf system (biomed diagnostics). this test is more sensitive than the fecal wet mount examination and detects trophozoites per sample. the number of parasites shed by a cat with diarrhea is high enough to be routinely detected with this method. the test should be performed in-house, because the parasite is unlikely to survive the trip to the laboratory. the test pouch is inoculated with µg of freshly collected feces, about the size of a peppercorn. any more than this increases the chances of bacterial overgrowth. the pouch is incubated at ° c and examined under the microscope for motile trophozoites every other day for days. the pouch should be tapped gently to dislodge the parasites, which tend to collect along the seams. the test is considered negative if parasites are not found after days. one benefit of this system is that it does not support growth of giardia duodenalis or pentatrichomonas hominis. if a fecal wet mount examination and culture are both negative and infection with t. foetus is still under consideration, a pcr test can be performed. this test detects dna from live or dead trophozoites, but is more expensive than other diagnostic methods. this test is more sensitive than the other two methods and can detect parasites per sample. the sample size is mg of feces not contaminated by litter preserved in to ml of rubbing alcohol shipped at room temperature. trophozoites of t. foetus are sometimes found in colonic biopsy samples adhered to the surface or in the lumen of crypts. the most effective drug for the treatment of t. foetus in cats is ronidazole. the drug has a bitter taste and should be compounded into capsules. veterinary staff and owners should use gloves when handling ronidazole. if a confirmed relapse occurs, another course of treatment may eliminate the parasite. diarrhea may take several weeks to resolve after elimination of the parasite, because significant colitis is often present. effectiveness of treatment can be evaluated by performing fecal pcr tests and weeks after the end of treatment. apparent treatment failures may occur because of re-infection, co-infection with giardia duodenalis or cryptosporidium felis, or the presence of another concurrent diarrhea-causing disorder. a more worrisome cause for treatment failure is a recent report of parasite resistance to ronidazole in two cats. fortunately, diarrhea ultimately resolved in both cats despite the continued presence of the parasite. if the cat retests negative and the diarrhea is not improving after weeks, consider the possibility that another disease may exist. nonspecific treatment for diarrhea is unhelpful and may prolong the duration of diarrhea. diarrhea may respond to antibiotics as they alter the intestinal flora population; however, once treatment is stopped, the diarrhea will return. an important and potentially serious adverse effect of ronidazole administration in cats is a reversible neurotoxicity. onset of signs often begins within week of the onset of therapy and may last between and weeks after cessation of therapy. these signs can include depression, ataxia, seizures, behavioral changes, weakness, hyperesthesia, and trembling. neurotoxicosis usually requires only supportive care along with discontinuation of the drug. the neurologically affected cat should be retested for the parasite, because it may have been eliminated. because of the potential for neurotoxicity, the use of ronidazole should be restricted to cats with confirmed infections with t. foetus. crowded conditions should be avoided, because transmission of t. foetus trophozoites is more efficient in these settings. cats testing positive should be isolated from other cats during treatment. providing a clean environment will help prevent transmission of trophozoites. although there is a report of an infection in one immunocompromised person, transmission of t. foetus trophozoites from cats to healthy humans has not been reported. still, prudence dictates handling feces infected with t. foetus trophozoites carefully. recent genetic evaluations have shown that most feline infections with cryptosporidium spp. are with c. felis; not, as previously thought, with c. parvum. cryptosporidium parvum seems to be limited to farm animals. cryptosporidium felis is an obligate intracellular parasite infecting the small intestine. infective oocysts are ingested from contaminated feces during self-grooming of contaminated body parts and from contaminated food and water. , after infection, the parasite attaches to the brush border of the enterocyte. the prepatent period is to days, and the oocysts are infective as soon as they are shed, making this a very contagious disease. like most intestinal parasites, shedding is often intermittent. the pathogenic effects of c. felis infections are not well understood. direct cytotoxicity and inflammation causes villus atrophy and decreased surface area for absorption of water, electrolytes, and other nutrients. , apoptosis (preprogrammed cell death) of the mucosal cells may be accelerated, adding to the malabsorption. most infections with c. felis are subclinical. signs, if present, range from a mild, self-limiting small bowel diarrhea to chronic intermittent small bowel diarrhea. severe diarrhea with weight loss and anorexia may also occur. , clinically apparent infections are most common in kittens, adult cats with concurrent gastrointestinal diseases, and cats co-infected with giardia duodenalis or tritrichomonas foetus. cats with co-infections may experience more severe clinical signs. a fecal flotation, which should be performed on all cats with diarrhea, may reveal c. felis if there are large numbers of oocysts (figure - ) . the fecal floatation test, however, is often negative because of intermittent shedding. the parasite is small and floats in a higher plane than helminth ova; the high-power lens and appropriate adjustment of the microscope stage is required to find the parasite. the small size of the oocyst makes identification difficult, particularly if the examiner is not specifically looking for them. a modified ziehl-neelsen stain of a thin fecal smear may help in the identification of the oocysts. this technique works well in humans with large numbers of oocysts. once signs resolve or the oocyst numbers decline, a single examination of a stained smear becomes insensitive. when only one sample is available, testing for c. felis antigen is a good choice. the prospect microplate assay (alexon biomedical, sunnyvale, calif.) is more sensitive and specific for the diagnosis of c. felis than is the examination of a stained smear. immunofluorescent antibody testing is available from some laboratories. fecal c. felis dna can be detected using pcr testing. this test is available at many veterinary diagnostic laboratories; however, at present, there is no test standardization among laboratories. the clinical and zoonotic significance of a positive pcr test combined with an oocyst negative test is unknown. therefore a positive pcr test in a cat without diarrhea presents a confusing situation for the attending veterinarian with regard to recommendations for the owner. unfortunately, there are no completely effective and safe treatment protocols available for c. felis. , a concerted attempt to find other causes of diarrhea should take place prior to convicting a cat of having diarrhea solely from c. felis infection. most reports on therapy for c. felis are uncontrolled and anecdotal. a number of drugs have been discussed. azithromycin for at least days appears safe but produces variable results. paromomycin, an oral aminoglycoside, may be effective. however, one study reported acute renal failure in of cats receiving the drug. deafness also occurred in three of those four cats. nitazoxanide is a drug approved for treating humans with diarrhea caused by cryptosporidium spp. infections. the administration of nitazoxanide to cats at mg/kg q h po for at least days up to days may be effective. however, nitazoxanide is a gastrointestinal irritant and commonly results in vomiting and foul-smelling diarrhea. co-infections with giardia duodenalis and/or tritrichomonas foetus are more difficult to control. if diarrhea from c. felis infection improves but does not resolve at the end of therapy, the duration of treatment may be prolonged. additional diagnostic testing should also be performed to ensure the only cause of the diarrhea is infection with c. felis. environmental control of c. felis is difficult, because it is extremely hardy. it is resistant to chlorination and most disinfectants. oocysts remain viable at temperatures above freezing up to ° c. the parasite is difficult to filter and survives treatment at municipal water treatment facilities. steam-cleaned housing and utensils may be beneficial in controlling parasite numbers, and they are susceptible to % ammonia solutions; however, the required contact time is hours. cryptosporidium spp. are relatively species specific, and there are no reports of waterborne outbreaks of human cryptosporidiosis associated with c. felis. cryptosporidiosis can cause life-threatening diarrhea in hivpositive persons. fortunately, humans are rarely infected with c. felis. in fact, the zoonotic species most commonly found in humans (often veterinary students), is c. parvum found in young heifers. regardless of a person's health, feces from a cat with diarrhea should be handled carefully. if a cat infected with cryptosporidium spp. is owned by an immunocompromised person, a pcr test may be useful in determining the species of the parasite and its zoonotic risk. like other coccidians, toxoplasma gondii is an obligate intracellular parasite. domestic cats and other felids are the only animals that shed oocysts. any warmblooded animal, including humans, can be infected with this parasite. toxoplasma gondii can be transmitted by ingestion of infective oocysts in fecally contaminated food or water after ingestion of tissue cysts through carnivorism, or by transplacental or trans-mammary transmission of the parasite. the parasite enters into one of two cycles, depending on the host species. the enteroepithelial cycle only occurs in cats and results in shedding of oocysts after sexual reproduction of the parasite. after a cat ingests an infective oocyst or a tissue cyst, the parasite enters the mucosal cells of the small intestine, where it may undergo development and sexual reproduction, after which oocysts are shed. the prepatent period after ingesting an infective oocyst is to days, while shedding after ingesting tissue cysts starts in to days. fecal shedding, which occurs only after initial infection, lasts for to weeks , and the oocysts become infective to days after they are shed. the extraintestinal cycle occurs in any animal, including cats. after ingestion, the parasite penetrates the cells of the small intestine and rapidly replicates in the enterocytes and associated lymph nodes into tachyzoites. after hematogenous and lymphatic spread, tachyzoites infect cells in all tissues of the body. tissues most commonly infected include the brain, liver, pancreas, and lungs. if a pregnant queen becomes infected, tachyzoites cause placentitis, after which they infect the fetus. in weeks, the host's immune response slows parasite replication, and the resultant bradyzoites form tissue cysts in the brain, striated muscle, and liver, and they remain viable for the life of the animal. immunosuppressive drugs or disease may dull the suppression of parasite division by the host immune system and allow the slowly dividing bradyzoites in tissue cysts to begin rapid division, thereby reactivating the infection with tachyzoites. none of the forms of t. gondii produces a toxin. rapid replication of tachyzoites within a cell leads to rupture of the cell and necrosis of the tissue in which they are located. the most commonly injured tissues are the brain, lungs, liver, and pancreas. prenatal infection leads to more severe illness, because the immature immune system is unable to slow down replication by tachyzoites, allowing continued damage to tissues. prenatal infection is more likely to result in ocular infections, and neonatal death is usually caused by pulmonary or hepatic infection. type ii and iv hypersensitivities may be involved in the pathogenesis of chronic disease from bradyzoites in tissue cysts. kittens infected perinatally can be stillborn or die shortly after birth. they may also suffer from hepatomegaly and ascites, central nervous system signs resulting from encephalitis, respiratory distress, or uveitis. , clinical signs of infection in healthy adult cats are uncommon (box - ). diarrhea from enteroepithelial development of the parasite is rare. cats that develop clinical disease often have an episodic course with vague signs that depend on the body system affected. onset of illness may be acute or chronic, and the most commonly affected organs include the brain, lungs, liver, heart, pancreas, and the eyes. signs are the result of spread of tachyzoites after initial infection or after reactivation of tissue cysts. cats suffering from uveitis may develop lens luxation and glaucoma. the best way to identify a cat shedding t. gondii oocysts is to demonstrate them with a centrifugal fecal flotation technique using sheather sugar solution. the oocysts are about a quarter of the size of isospora felis oocysts ( figure - ). oocysts of t. gondii are morphologically indistinguishable from hammondia or besnoitia spp. oocysts. detection of fecal t. gondii dna using a pcr test can be used to definitively differentiate t. gondii oocysts from similar coccidians. it is probably best, however, to assume suspicious oocysts are those of t. gondii until proven otherwise. proving infection with t. gondii is responsible for a cat's systemic illness is also difficult. finding tachyzoites in cytology samples is uncommon. they are most likely to be identified from body cavity effusions. the most common method of identifying an infected cat is by detecting t. gondii-associated immunoglobulins using immunofluorescent antibody or elisa techniques. since cats are infected for life, a seropositive cat has been infected at some point in its life. however, use of serology alone is insufficient to diagnose an active t. gondii infection. serum immunoglobulin m (igm) is produced within to weeks after infection, but increased igm titers may persist for months to years. serum immunoglobulin g (igg) begins to rise later; in some cats, igg may not be detectable for to weeks. by the time igg is detectable, shedding will have ceased. maternally acquired igg persists in kittens for to weeks. a rising igg titer is associated with an active infection, but the degree of increase is not associated with the severity of the clinical signs. if a cat becomes seronegative, it is more likely the titer has fallen below the sensitivity of the test rather than the parasite has been eliminated from the body. because of the vague nature of the clinical signs, many cats are presented later in the course of the disease. by this time, they may have switched from igm to igg production or passed the time of maximal igg production. thus a negative igm titer or a lack of rising igg titer does not rule out t. gondii infection. also, reactivation of tissue cysts is rarely associated with rising igg titers. ultimately, the diagnosis of an active systemic t. gondii infection requires demonstration of an igm titer greater than : or a fourfold increase in igg titers over a -to -week period along with signs consistent with toxoplasmosis, the exclusion of other disorders that may cause the clinical signs, and response to appropriate anti-t. gondii treatment. although serum igm titers may be increased in otherwise healthy cats, increased igm titers in cerebrospinal fluid or aqueous humor only occurs in cats with active cns or ocular infections. the goals of treating a cat infected with t. gondii are to reduce shedding of oocysts and to control the clinical signs in sick cats. shedding can be reduced by using ponazuril, toltrazuril, or high doses of clindamycin. the drug options for treating a sick cat include clindamycin, trimethoprim-augmented sulfadiazine, or azithromycin for at least weeks (see table - ) . recurrences are more common if the cat is treated for less than weeks. , the antifolate drug pyrimethamine may be more effective than trimethoprim, but megaloblastic anemia develops in many cats. supplementation with folinic acid ( mg/cat, once daily, po) or brewer's yeast ( mg/kg, once daily, po) may prevent or reverse the anemia. no drug clears all of the tissue cysts; so, cats remain infected for life. if uveitis is also present, use appropriate topical, oral, or parenteral corticosteroids. for a cat with proven t. gondii-associated uveitis alone, a topical ocular glucocorticosteroid is the only required treatment; no antibiotics are necessary unless the uveitis is persistent or recurrent. • wash hands after handling cats, especially if you are pregnant or immunocompromised. • remove fecal material from the home environment daily, since shed oocysts require a minimum of hours to become infective. • do not have immunocompromised persons clean the litter box. if they must clean the litter box, they should wear gloves and wash hands thoroughly when finished. • use litter box liners, and periodically wash the litter box with scalding water and detergent. • wear gloves when gardening, and wash hands thoroughly when finished. • cover children's sandboxes when not in use to avoid fecal contamination by outdoor cats. • only feed cats cooked or commercially processed food. • control potential transport hosts, such as flies and cockroaches, that may bring the organism into the home. • filter or boil water from sources in the environment. • cook meat for human consumption to ° c for minutes minimum (because of uneven heating, microwave cooking does not kill all t. gondii ). • freeze meat at − ° c for hours. • wear gloves when handling meat, and wash hands thoroughly with soap and water when finished. clinical signs such as malaise, fever, and muscle pain should begin to resolve in to days. if there is no response within days, switch to or add another drug. if there is still no response, search for another condition that may cause the observed clinical signs. however, ocular and cns signs resolve more slowly and thoracic radiographic changes may take weeks to resolve. some cns changes may never completely resolve. cats co-infected with feline immunodeficiency virus (fiv) do not respond to anti-t. gondii treatment as well as fivnegative cats respond. feeding cats commercially processed cat food and avoiding undercooked or raw meat can prevent exposure to t. gondii. controlling hunting reduces access to paratenic hosts with infective tissue cysts. access to mechanical carriers of t. gondii, such as earthworms or cockroaches, should be minimized. human infection with t. gondii is common, more so in warm, humid climates where the prevalence of t. gondii seropositive persons approaches %. the number of persons seropositive for t. gondii is estimated to be around , , worldwide. infective oocysts are hardy and may remain viable in the environment for up to months. human infection most often occurs after eating raw or undercooked meat infected with tissue cysts or by transplacental infection. seropositive cats are finished shedding and are unlikely to resume shedding even if the infection becomes reactivated. cats found to be shedding oocysts should be quarantined at a veterinary hospital until shedding ends. oocysts of t. gondii have not been found on the hair coat ; so, transmission of toxoplasmosis does not occur after touching a cat. pregnant women infected with t. gondii for the first time, or chronically infected women who are also hiv positive, can transmit the parasite to their unborn child. transplacental infection can result in stillbirths, cns, or ocular disease. more severe fetal disease may occur if the infection happens in the first half of the woman's pregnancy. toxoplasma gondii infection of immunocompetent humans usually results in a self-limiting fever and malaise. steps useful in preventing transmission of t. gondii to humans can be found in box - . pancreatitis refers to inflammation of the pancreas only, with no implication of the underlying cause or pathology. for example, acute necrotizing pancreatitis (anp) with pancreatic auto-digestion, requiring predominantly supportive care by maintaining fluid and electrolyte balances and pain relief, must not be confused with chronic pancreatitis (cp) caused by lymphocytic infiltration, and commonly associated with lymphocytic inflammatory bowel disease (ibd), and often requires corticosteroids to manage. these two conditions (and others) can only be definitively distinguished histologically. in many cases, the clinical signs of cats with acute pancreatitis will resolve with supportive care before a precise diagnosis is reached and will thus remain undiagnosed. there are no formal classifications for feline pancreatitis, but most authors , , use the terms • acute pancreatitis • acute necrotizing pancreatitis, characterized by severe peri-pancreatic fat necrosis • acute suppurative pancreatitis, characterized by neutrophilic infiltration • chronic pancreatitis, characterized by lymphocytic infiltration the exact prevalence of feline pancreatitis is unknown. necropsy studies from the s to s reported prevalence of feline pancreatitis ranging from . % to . %. , a more recent study found % of cats had evidence of pancreatitis. however, this included pancreatic pathology in % of apparently healthy cats, which suggests that mild pathology is unlikely to cause clinical signs. these studies all show lymphocytic pancreatitis to be significantly more prevalent than acute pancreatitis. this may underestimate the true prevalence of acute pancreatitis, since it is understood that no permanent histopathologic changes are present after resolution of acute pancreatitis. it is also possible that studies assessing pathology in necropsy cases do not reflect clinical practice. there are no specific age, breed, or sex predispositions. although one study reported siamese cats to be at increased risk of acute pancreatitis, subsequent studies have recognized the majority of cases are domestic shorthair cats, suggesting no specific breed predispositions. , , , most studies have indicated older cats ( to years of age) are more likely to be affected, , , , but these studies most likely underrepresent cats with less severe clinical disease for which definitive diagnosis may not be reached and which may be younger. no association has been made with a high-fat diet or obesity. in most cases of both acute and chronic pancreatitis, no specific cause is found, and the disease is primarily considered to be idiopathic. , there are, however, some specific underlying causes that are sporadically recognized. these include infections with herpesvirus, calicivirus, , feline infectious peritonitis (fip), liver fluke and pancreatic fluke, , and toxoplasmosis. however, a recent paper found no association between serum feline pancreatic lipase immunoreactivity (fpli) concentrations and toxoplasma gondii serology. pancreatitis has also been recognized subsequent to trauma and organophosphate poisoning. the association of pancreatitis with inflammatory bowel disease and cholangitis is frequently mentioned (triaditis) but poorly described in the literature. one study found % of ibd cases to have histologic evidence of pancreatic involvement, and another found fpli concentrations were elevated in % of cases with histologically confirmed ibd. it is the author's experience that many cases of pancreatitis recognized with ibd have no specific clinical signs attributable to pancreatitis and should therefore be diagnosed and treated as intestinal disease. diabetes mellitus is a recognized co-morbidity of pancreatitis in cats. a recent study found fpli concentrations were significantly higher in diabetic cats compared with non-diabetics. no association could be made between fpli concentrations and the degree of diabetic control. one study found of cats ( %) histologically diagnosed with hepatic lipidosis were also histologically diagnosed with acute pancreatitis. it is not known if pancreatitis is a cause, consequence, or coincident disease of hepatic lipidosis. for example, anorexia associated with acute pancreatitis could predispose to fatty infiltration of the liver. however, the high rate of concurrent disease has important implications for ensuring cats with pancreatitis receive adequate caloric intake. ongoing or recurrent pancreatitis may lead to pancreatic cysts or exocrine pancreatic insufficiency, which are both covered later in this chapter. although pancreatitis has been experimentally induced in cats, , , the pathophysiology of spontaneous pancreatitis remains unknown. acute pancreatitis is initiated by an increase in secretion of pancreatic enzymes that leads to inappropriate cellular activation of trypsin and subsequently other digestive zymogens. these activated digestive enzymes lead to local effects including inflammation, hemorrhage, acinar cell necrosis, and peripancreatic fat necrosis. , , chronic pancreatitis may result from any of several underlying processes: ongoing, low-grade acute pancreatitis episodes may instigate chronicity; chronic pancreatitis, with a predominance of lymphocytic inflammation has been induced experimentally within weeks by narrowing the main pancreatic duct to approximately % of its normal diameter ; and the association with ibd may suggest an immune-mediated cause. the clinical signs of pancreatitis in cats are nonspecific. a review of eight prior series totaling cases of acute pancreatitis in cats found anorexia ( % of cases) and lethargy ( %) to be the most common historical findings. vomiting was recognized in % of cases, diarrhea in %, and weight loss in %. physical examination findings were similarly nonspecific with dehydration ( %) being the major finding; fever was recognized in only % of cases and abdominal pain in %. it is important to note that vomiting and abdominal pain, key features of pancreatitis in dogs, are not consistently recognized in cats. similar, nonspecific findings indistinguishable from ibd are recognized in cats with chronic pancreatitis. , diagnosis because the presenting signs and physical examination findings are nonspecific, the diagnosis of pancreatitis can be challenging, requiring not only clinical suspicion but a combination of diagnostic modalities. for the most part, hematology and plasma biochemistry findings are unremarkable, although a combination of findings may increase clinical suspicion. for example, moderate elevations in liver enzymes, bilirubin, and glucose are present in approximately % of cases and hypocalcemia in approximately two of three of cases; hypocalcemia infers a poorer prognosis. hypoalbuminemia is seen in approximately one of three of cases and has important implications for fluid therapy. amylase and lipase elevations are not reflective of pancreatitis in cats. feline trypsinlike immunoreactivity (ftli) is the diagnostic test of choice of exocrine pancreatic insufficiency, but elevations in pancreatitis are not seen consistently enough to warrant use of this test for this purpose. , , the biggest recent advance in feline pancreatic diagnostics has been the characterization of feline pancreatic lipase, leading to the development of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fpli). it must be remembered, however, that an increase in fpli only tells the clinician that pancreatic pathology is present, but not the cause of pathology, which may be, for example, neutrophilic or lymphocytic pancreatitis or neoplasia, and it may or may not involve the intestines or liver. fpli should therefore be used as a screening test, with elevated results not suggesting a diagnostic end point. further, the high interassay variability of this test would suggest that mild cases may be missed as shown in one study and that the test may not be appropriate for serial monitoring. fpli is currently available as "spec fpl" from commercial laboratories and has a sensitivity of % and a specificity of % when . µg/l is used as the diagnostic cut off compared with . µg/l, which is the listed reference range high point. in an acutely unwell cat (less than days) with only mild to moderate signs of disease, further diagnostics may not be warranted, and many cats will improve with supportive therapy of balancing fluid and electrolytes, pain relief, and antinausea/vomiting therapy. cats with chronic duration of signs and acutely unwell cats that do not improve with supportive therapy warrant further diagnostics. the underlying disease process cannot be assumed from an elevated fpli; in one study of cases, acute necrotizing pancreatitis could not be distinguished from chronic nonsuppurative pancreatitis by signalment, duration of signs, or clinical findings. the major utility of diagnostic imaging is to rule out other differential diagnoses, such as an intestinal foreign body, and perhaps confirm that the pancreas is affected. radiography is non-specific for diagnosis of pancreatitis, but findings may include decreased abdominal detail (sometimes associated with ascites), soft tissue density in the right cranial quadrant of the abdomen, hepatomegaly, or gas-filled intestines , , (see . additionally, thoracic radiographs may show pleural effusion. one study found of cats with pancreatic necrosis had such a change ; the mechanisms resulting in pleural effusion are not precisely defined. ultrasonography has high specificity (> %) but low sensitivity (< %) for recognizing pancreatitis in cats, , , , with findings dependent on operator skills, quality of equipment, and severity of lesions. typical findings are hypoechogenicity of the pancreas, which may be enlarged or irregular; hyperechogenicity of the peripancreatic fat; the possible presence of abdominal effusion; and abnormal findings with other organs, such as liver or intestine, may add to the clinical picture , , , . one study indicated that contrast-enhanced doppler ultrasonography can provide further diagnostic insights. a recent study suggested that endosonography may be useful in cases where transabdominal ultrasonography is difficult, for example, because of obesity, hyperechoic mesentery, or excessive intestinal gas. for more than years, computed tomography (ct) has been a commonly used modality to confirm pancreatitis in humans, but this reliability has not been demonstrated in cats, where sensitivity may be as low as %. , definitive diagnosis of pancreatitis, including differentiation of the inflammatory process, can only be made by cytologic assessment of pancreatic tissue. in most cases, ultrasound-guided fine-needle aspiration (fna) of the pancreas is technically difficult because of the small dimension of the feline pancreas; there appears to be no assessment of feline pancreatic fna findings in the literature. gross inspection of the pancreas and samples for histologic assessment can be obtained during laparotomy , (see or laparoscopy. , because pancreatitis often occurs concurrently with pathology of other organs, thorough evaluation of the abdomen by ultrasonography or gross inspection is recommended, as are multiple biopsies of, for example, intestines, liver, and mesenteric lymph nodes, where appropriate. clinicians may be reluctant to biopsy the pancreas because of perceived risks of deleterious effects. studies of pancreatic biopsy in healthy cats dispel the concern that the pancreas is unforgiving to mild manipulation and biopsy , a and the author's clinical experience is consistent with these findings. supportive care comprising correction of fluid/ electrolyte imbalances, pain management, and nutritional support are the mainstay of therapy for cats with figure - gross appearance of pancreas at laparotomy; this was histologically diagnosed as chronic pancreatitis (i.e., lymphocytic infiltration was recognized). gross appearance of pancreas at laparotomy; this pancreas was found to be histologically normal. it does look smaller than is typically seen; pancreatic atrophy can look similar to this, grossly. pancreatitis. , , specific underlying causes, when diagnosed, should be managed, as should concurrent diseases. follow-up evaluation is determined on a case-by-case basis; reduction or resolution of clinical signs is the main criterion for success of therapy. serial fpli values may be monitored when initial results are extremely high but are of limited value for mild increases because of assay variability. dehydration, acid-base and electrolyte abnormalities should be corrected during the first to hours. hypocalcemia, if present, should be treated with a calcium gluconate infusion of to mg/kg during to hours, with continued assessment of plasma calcium concentrations. plasma transfusions can be considered in cats with hypoalbuminemia. , , although abdominal pain is not commonly described in cats with pancreatitis, it is likely to be present in most cases and may contribute to anorexia. historical concern about exacerbation of pancreatitis with opioids is no longer accepted, and this class of drugs is considered appropriate. meperidine ( to mg/kg sc or im) every to hours, butorphanol ( . to . mg/kg sc) every hours, or sustained-release buprenorphine ( µg/kg sc) every hours are alternatives. , , the author uses one dose of methadone ( . to . mg/kg sc, im, or iv) initially and places a fentanyl patch for longer-term pain management. the traditional recommendation for management of pancreatitis across all species has been nil per os for several days. this recommendation is appropriate for cats with severe vomiting, but there is no evidence to support this approach in cats that are not vomiting and that are eating normally. further, nutritional support is vital for those cats with concurrent hepatic lipidosis. if the cat is not eating voluntarily, nutritional support by tube feeding is often warranted. , , a recent paper found nasogastric tube feeding of cats with pancreatitis was tolerated well and resulted in few clinically significant complications. other reported nutritional strategies for cats with pancreatitis incorporate partial parenteral nutrition (ppn; . % amino acids, % lipids), or total parenteral nutrition (tpn; % amino acids, % lipids, % dextrose), or both instead of enteral feeding. , , cats do not seem to benefit from feeding of specially formulated low-fat diets; commercially available, veterinary liquefied diets appear to be well tolerated despite their high-fat contents. other therapy may be appropriate in individual cases. all cats with pancreatitis that are vomiting should be treated with antiemetics. examples of drugs that can be used are -ht antagonists, such as dolasetron ( . to . mg/kg iv or po, once to twice daily); ondansetron ( . to . mg/kg iv every to hours); and maropitant, an nk -inhibitor ( . to . mg/kg sc once daily). these drugs are covered in detail earlier in this chapter under therapeutics for vomiting and diarrhea. dopaminergic antagonists, such as metoclopramide, are less effective antiemetic agents in cats than the other choices mentioned. , in most cases, pancreatitis begins as a sterile process, and antibiotic therapy is controversial. pancreatic necrosis and inflammation may predispose to bacterial colonization of the pancreas as demonstrated in experimental models. , this has not been demonstrated in spontaneous disease, and no comparison of outcomes has been made of cats with pancreatitis treated with or without antibiotics. cefotaxime ( to mg/kg iv, im) has been used to prevent bacterial colonization in experimental models. other broad-spectrum cephalosporins or ampicillin may act similarly. antibiotic considerations are possibly more important for acute pancreatitis than for treatment of chronic disease. cats with demonstrated lymphocytic pancreatitis, with or without concurrent ibd or lymphocytic cholangitis, should be treated with corticosteroids (e.g., prednisolone, to mg/kg once to twice daily) with tapering to the lowest effective dose. there is no justification for use of corticosteroids in cats with acute necrotizing or acute suppurative pancreatitis, or cats for which the cause of pancreatitis has not been diagnosed histologically. use of corticosteroids in cats with pancreatic disease creates a risk of iatrogenic diabetes mellitus. surgical intervention is warranted to relieve any bile duct obstruction that may result or for the débridement of pancreatic abscesses or necrotic tissue; in many cases, cats will survive multiple years after such corrective surgery. pancreatic cysts, pseudocysts, and bladders have been described sporadically in cats.* pancreatic cysts are lined by a single layer of cuboidal epithelium and do not communicate with the pancreatic duct; pseudocysts are enclosed by a wall of fibrous tissue, lacking the epithelial lining characteristic of true cysts and can form secondary to pancreatic inflammation; cystic dilations of the pancreatic duct are referred to as pancreatic bladder. true pancreatic cysts have been described in three cats , , ; a congenital pancreatic cyst with associated inflammation was described as an incidental finding in an adult cat ; multiple pancreatic cysts were described in a cat with concurrent polycystic disease in the kidney and liver ; and a another cat had multiple recurrent pancreatic cysts with concurrent mild pancreatic inflammation and atrophy associated a with rapid clinical course resulting in diabetes mellitus. cysts, pseudocysts, and bladders may be identified ultrasonographically or by ct. they may be benign, but the associated pancreatic inflammation and other sequelae, such as diabetes mellitus, may need to be managed. pancreatic bladders may result in biliary obstruction, and surgical correction may be required. pancreatic nodular hyperplasia is recognized quite frequently as an incidental finding in older cats or at necropsy. neoplasia of the exocrine pancreas is rare in cats. its frequency was assessed in the s when one study estimated . cases per , patients per year at risk, and another found pancreatic tumors in of feline necropsies. a more recent study recognized, from , feline admissions over a -year study period, only two cats with pancreatic adenomas ( . % of admissions) and eight with pancreatic adenocarcinomas ( . % of admissions). adenomas appear as small, solitary or multifocal nodules and are not typically associated with adjacent pancreatic inflammation. they do not cause clinical signs, unless large, when any clinical signs result from the physical size and are usually an incidental finding. , few generalities can be made about the presentation for pancreatic adenocarcinoma. the age range is large ( to years), there is no sex predisposition, and no clear breed predispositions are present. , only cytology or histopathology can distinguish pancreatitis from pancreatic carcinoma in cats antemortem, yet it is important to differentiate the two conditions, because, in contrast to adenomas, pancreatic adenocarcinoma is associated with a grave prognosis. the presence of lesions consistent with metastases on radiography or ultrasonography may suggest malignancy, but one study could not distinguish neoplasia from pancreatic nodular hyperplasia ultrasonographically based on the appearance of the pancreas alone (figures - and - ) . pancreatic adenocarcinomas in cats can result in a paraneoplastic dermatologic condition consisting of nonpruritic, symmetric alopecia affecting the face, ventral body, and medial aspect of the limbs of cats. the skin is usually glistening but not fragile, and there can be crusty lesions on the footpads.* the pathogenesis of this dermatologic disease is unknown. in one case, surgical excision of the pancreatic carcinoma resulted in resolution of dermatologic disease, indicating that the process is reversible (although signs recurred as the tumor re-emerged). diabetes mellitus is a recognized complication of pancreatic adenocarcinoma. the mechanism is unknown and may simply be secondary to compression or invasion of islet cells by the tumor. in some cats, diabetes is recognized ahead of pancreatic neoplasia. , , obstructive jaundice has also been described with pancreatic adenocarcinoma. most cases of pancreatic adenocarcinoma in cats have metastasized by the time of diagnosis, and most reported cases die or are euthanized within days of diagnosis. surgical excision is a potential option if neoplasia is confined to one limb of the pancreas, but recurrence is possible even if there is no evidence of metastasis and excision seems complete at the time of surgery. exocrine pancreatic insufficiency (epi) is a condition caused by insufficient synthesis and secretion of pancreatic digestive enzymes from the exocrine portion of the pancreas. in humans it has been reported that % of pancreatic acinar cells must be lost before clinical signs of epi are seen. epi is considered rare in cats but is perhaps being recognized more frequently because of increased awareness. there are less than fifty cases described in the veterinary literature* with one of these papers describing only cases from five institutions, with prevalence described as . % to . % of cats seen over a -year period. in contrast to this, the gastrointestinal laboratory at texas a&m university recognized samples with serum ftli concentrations at or less than . µg/l, which is diagnostic for epi, out of , submissions, which equates to . % of cats with known or suspected gastrointestinal disease. all studies indicate a wide age range of cats can be affected, from kittens less than months of age to cats more than years old, with a median age of approximately years. there is no apparent breed predisposition. , , one paper recognized of ( . %) cats to be male, and another recognized of ( %) male cats, suggesting a possible sex predisposition. chronic pancreatitis is believed to be the most common cause of epi in cats, acinar atrophy (paa) is recognized as the most common cause of epi in dogs, and has been definitively described in two feline cases and mentioned as a cause for three other cases. other potential causes of epi include disruption of pancreatic enzyme flow at the duodenal papilla following duodenal resection and pancreatic fluke infection (eurytrema procyonis), , and amyloid deposition and neoplasia are other possible causes of pancreatic cell damage that have not definitively been described in cats. congenital pancreatic hypoplasia or aplasia has not definitively been reported in cats, but reports of epi in cats as young as months of age , suggest this possibility. since chronic pancreatitis is a common cause of epi and chronic pancreatitis has a strong association with ibd, many cats may have concurrent lymphocytic pancreatitis and enteritis. , , therefore cats failing to respond to therapy for epi may require further diagnostics and management of an underlying condition. further, destruction of functional exocrine pancreatic tissue can also affect pancreatic endocrine tissue, resulting in concurrent diabetes mellitus. several studies have indicated that all cats with epi will have weight loss when diagnosed, unless a kitten, in which case ill-thrift is recognized. , diarrhea is not necessarily present, being described in % to % of cats; the nature of feces can vary from voluminous, malodorous stools that can be discolored (yellow or pale), sometimes with steatorrhea, to normal feces in other cats. increased frequency of defecation and the presence of mucus in the feces of some cats can lead to the diarrhea being characterized as large bowel. only about % to % of cats are polyphagic, some described as having a ravenous appetite; conversely, some cats present with anorexia. vomiting has also been described. since cats with epi often have concurrent disorders, such as ibd, the clinical signs recognized may reflect the concurrent disease and not necessarily epi alone. physical examination findings are similarly nonspecific, with thin/ emaciated body condition being the most common finding. hematologic findings are non-specific, but a mild nonregenerative, normocytic, normochromic anemia may be recognized as well as lymphopenia or neutrophilia. plasma biochemistry results may show a mild to moderate increase in alanine aminotransferase (alt) and a mild increase in alkaline phosphatase in some cats. mild to moderate hyperglycemia may be seen, as may mild hypoglycemia or normoglycemia. , , hypocobalaminemia is recognized in nearly all cats with epi. , , , , this may be because of insufficient production of intrinsic factor, a cobalamin-binding protein only produced by the pancreas in cats and necessary for ileal absorption of cobalamin ; it may also be because of failure of pancreatic enzymes to liberate cobalamin from binding by r protein in the duodenum or small intestinal bacterial overgrowth (sibo), not yet specifically described in cats. folate concentrations may be reduced (because of concurrent intestinal malab sorption), normal, , or increased, which may relate to reduced pancreatic bicarbonate secretion, secondary to severe hypocobalaminemia, or associated with sibo. none of these presenting complaints, physical examination findings, or routine testing results are specific to epi. therefore epi requires a degree of clinical suspicion and/or thorough diagnostics to ensure the diagnosis is not missed. a low level of serum ftli is diagnostic for epi. , , samples can be sent to the gastrointestinal laboratory at texas a&m university from anywhere worldwide (with instructions about sample handling requirements on their website: http://vetmed.tamu.edu/gilab/). the reference range for serum ftli is to µg/l, with concentrations at or less than . µg/l diagnostic for epi. since the clinical signs and routine laboratory findings are nonspecific for epi, it is ideal to test serum for ftli in any cat with weight loss or ill-thrift. the texas a & m gastrointestinal panel also includes testing for levels of cobalamin, folate, and fpli, ensuring concurrent hypocobalaminemia will not be missed and potentially providing indications of other gastrointestinal disease. conversely, although a low level of serum ftli confirms a diagnosis of epi, it is not necessarily a diagnostic end point, since epi is so often recognized concurrently with other gastrointestinal disease. failure to respond to therapy should prompt the clinician to consider and investigate further for concurrent processes. most cats with epi can be successfully managed with dietary supplementation of pancreatic enzymes. commercial products (e.g., viokase [axcan pharma, birmingham, ala.], pancrezyme [virbac, fort worth, tex.], and creon [abbott laboratories, abbott park, ill.]) are available, and powder is considered more effective than tablets or capsules (some capsules can be opened and the contents sprinkled onto food, like powder). the required dose can vary quite substantially from cat to cat. it is appropriate to start with one teaspoon of powder with food twice daily, and adjustments can be made depending on the response; most cats accept the powder readily if it is mixed thoroughly through canned food, but other flavors (e.g., fish oil or brine from canned tuna) can be used to disguise the taste if necessary. raw pancreas (e.g., from beef or pork) may also be used, with to g twice daily an appropriate starting dose. since most cats with epi are hypocobalaminemic, supplementation by subcutaneous injection is required (oral supplementation is not effective since cobalamin deficiency leads to cobalamin malabsorption). an appropriate dose for most cats is µg, and it is usually given weekly for weeks, then every second week for a further six doses; it is appropriate to continue dosing every month beyond that. owners can be taught to inject their cats at home (as owners of diabetic animals are taught to do with insulin). because some cats may have sibo, antibiotics such as metronidazole ( to mg/kg po every hours for days) may be warranted. an elevation of folate may arouse suspicion of sibo, but it is appropriate to try antibiotics in a cat failing to respond to enzyme and cobalamin supplementation. concurrent diseases, such as lymphocytic, chronic pancreatitis, or ibd may need to be managed with corticosteroids, or diabetes mellitus with insulin. no studies have assessed specific dietary requirements in cats with epi. most cats respond to appropriate treatment, with a return to normal weight and normal feces. with ongoing therapy, cats can lead normal lives for a full life span. the feline liver is a large, complex organ involved in a variety of essential metabolic, functional, and detoxification processes that can be affected, individually or collectively, by disease or dysfunction. cats have a unique set of liver diseases that occur more commonly in this species compared with the typical diseases that occur in dogs, and these include hepatic lipidosis, feline cholangitis syndrome, and infectious hepatopathies (e.g., fip, flukes, histoplasmosis, toxoplasmosis). , , , , nevertheless, these conditions often present with characteristic clinical, laboratory, and histopathologic changes that are necessary for proper diagnosis and management. the goal of this section is to review the interpretation of clinical and laboratory changes that occur in these feline liver diseases, provide an approach for separating the more common diseases by their clinical footprint, and then discuss therapy of each liver disease based on our current level of understanding of hepatoprotectants, antioxidants, and drugs used for specific therapeutic purposes. the clinical signs of liver disease in cats are often vague and nonspecific; however, recognition of certain clinical and laboratory abnormalities and their association with liver disease can greatly aid the diagnostic process. the most common early clinical signs observed in cats with liver disease are anorexia, lethargy, and weight loss, which are signs present in many (if not most!) feline diseases. , because these early indicators of disease do not point specifically toward liver disease, a delay in diagnosis will occur unless the clinician carefully considers all possibilities and performs other tests to further evaluate the situation. for example, feline hepatic lipidosis is the most common form of liver disease in cats in the united states, united kingdom, japan, and western europe, occurring with a prevalence of nearly % in one study. however, the most common, and often only, clinical sign associated with onset of this condition is anorexia; the signs of serious hepatic disease (especially jaundice and vomiting) do not occur until later (days or weeks) in the course of the disease. , recognition that anorexia in a cat, even for a few days, is a risk factor for development of hepatic lipidosis is essential, and this risk is increased in obese cats. , further, the clinical signs of liver failure develop much more slowly; many cats with hepatic lipidosis present alert and responsive until much later in the course of the disease, thus delaying onset of appropriate therapy. a similar clinical situation exists for the second most common form of liver disease in cats, feline cholangitis syndrome. , , this complex of diseases in the cat can be associated with signs ranging from anorexia and lethargy to vomiting and jaundice, and these signs can vary in severity and prevalence. the key point is that except for development of jaundice, there is no constellation of clinical signs that are classic clinical indicators of liver disease in cats. , as with many feline diseases, the subtle clinical signs of anorexia, lethargy, or inactivity are often the only signs of illness and should be further investigated. there are few changes that occur in the complete blood count that are specific indicators of primary liver disease in cats. the most common finding is the presence of poikilocytes, which are red blood cells with an irregular shape, speculated to be caused by changes in membrane lipids as a result of liver dysfunction. other abnormalities may occur, such as anemia of chronic disease or neutrophilia, but these findings are nonspecific and occur with variable frequency. perhaps the most important reason for obtaining a hemogram is in icteric cats, because this test is essential to help rule out hemolysis as the cause of the hyperbilirubinemia. the serum chemistry profile can be very helpful, but there are several critical points in interpretation of these values that are important to review. the hepatic transaminases (alanine aminotransferase [alt] and aspartate aminotransferase [ast]) are leakage enzymes but do not discriminate among hepatobiliary disorders, nor do they provide an indicator of severity or disease origin. thus although increases in alt may be noted in cats with liver disease, they are also present in a variety of other systemic infectious, inflammatory, neoplastic, and and protein-losing nephropathies can also cause loss of albumin and affect cholesterol, it is essential to evaluate the cat for these problems when interpreting these results. finally, bilirubin metabolism is a critical function of the liver, but interpretation of hyperbilirubinemia requires a careful consideration of bilirubin disposition. hyperbilirubinemia develps because of one of three possible causes: ( ) excessive hemolysis of red blood cells (rbc) (also known as prehepatic icterus)-high bilirubin in the blood stream occurs because of an overload of the mononuclear/phagocyte system with heme pigments from rbc destruction, ( ) hepatic parenchymal disease or insufficiency (also known as hepatic icterus)-resulting in lack of normal bilirubin metabolism in hepatocytes and regurgitation of the pigments into the blood stream when they are not taken up into cells and excreted in bile, and ( ) disease of gall bladder, biliary tract, or pancreatic duct (also known as posthepatic icterus)resulting in obstruction of the bile ducts or loss of bile into the abdomen (duct or gall bladder rupture and bile peritonitis). the bottom line is that in any cat with hyperbilirubinemia, an assessment of the packed cell volume and rbc morphology should be completed to determine whether icterus is caused by hemolysis. once hemolysis is ruled out, then assessment of primary endocrine diseases, including hyperthyroidism, feline heartworm disease, fip, and neoplasia.* alternatively, the cholestatic membrane-associated enzymes alkaline phosphatase (alp) and gamma glutamyltransferase (ggt) are especially useful for recognizing disorders involving biliary or pancreatic ductal components. unlike the dog, these enzymes will only increase modestly in cats, even in severe disease, and there is no glucocorticosteroid or drug induction of the enzymes to influence interpretation. , thus increases in alp in the adult cat represent a release of enzyme from the hepatobiliary tree and should be considered clinically important. both alp and ggt are produced in other tissues than the liver, with the highest ggt activity present in the kidney and pancreas; however, sources other than the liver do not contribute to the activity of these enzymes in health. recent studies of the effects on these enzymes in cats with pancreatitis, cholangitis, extrahepatic bile duct obstruction (ehbdo), and hepatic lipidosis reveal some important characteristics in interpreting increases in these enzymes. first, both alp and ggt are increased in cats with pancreatitis, cholangitis, or ehbdo, because inflammation in the biliary tree also affects the pancreatic ducts (and vice versa, figure - ) , and if the fold increases in these enzymes are similar, the diagnosis is likely one of the three. conversely, in cats with hepatic lipidosis (without concurrent inflammatory disease of the biliary or pancreatic duct system), large increases in alp are observed, but ggt will remain normal or only slightly increased. thus if the increase in alp is to times, while ggt is not increased or is only increased to times, then the likely diagnosis is hepatic lipidosis. [ ] [ ] [ ] other than enzymes on the biochemistry panel, which are of limited value for assessing liver function, there are several key tests that can be used to help assess liver function cats with elevated liver enzymes. these five tests found on most routine biochemistry panels are helpful functional indicators: cholesterol, bilirubin, glucose, albumin, and urea nitrogen (bun). however, none are immune to outside influences on their interpretation, including bilirubin and cholesterol, which are the most liver specific. in cats with severe liver disease or failure, bilirubin levels tend to be quite elevated, while bun, albumin, cholesterol, and glucose concentrations tend to be significantly decreased, reflecting inability to metabolize urea (lack of arginine), inability to produce albumin or cholesterol, and abnormal metabolization of glucose. however, these changes represent severe loss of liver function and thus are not sensitive indicators of liver function because the changes occur quite late in the course of the disease. nevertheless, in cats with elevated liver enzymes and clinical signs of liver disease, these values should be carefully assessed. because gi disease of hepatic failure. in nonicteric cats with severe liver disease or in young cats suspected of having a portosystemic shunt, serum bile acids are the more reliable indicator of hepatic insufficiency. the measurement of serum bile acid concentrations, preprandially and postprandially, is the most reliable, readily available, and sensitive test of hepatic function in nonicteric cats. , that being said, although increases in bile acids are accurate indicators of hepatic insufficiency, the levels cannot be used to assess severity of disease or the type of dysfunction. further, bile acid assays are most effective when paired samples (preprandial-and postprandial) are compared, because single, fasting, or random bile acid samples can result in a falsenegative (normal) result. however, cats will often not eat in the hospital or when they are sick, and this prevents collection of a postprandial sample. however, this does not invalidate the results, because if the result of the single bile acid sample is abnormal, it does reliably indicate liver dysfunction. an alternative to using serum for testing bile acids in cats is urine bile acid analysis. healthy cats excrete a small percentage of conjugated bile acids in the urine ; however, in cats with liver disorders that cause increased serum bile acids (and especially cholestatic liver diseases) a significant increase in urine bile acid excretion occurs. when urine bile acids (uba) were collected to hours after a meal and measured (normalizing the value with urine creatinine: uba/ucr) and compared with serum bile acids in a study of cats with hepatic disease, cats with nonhepatic disease, and normal cats, the results were highly correlated. the utility of the urine bile acid test is that it does not require a paired sample (postprandial test), and it is not as affected as the serum test is by hemolysis or lipemia of the blood sample. normal cats will have an uba/ucr of less than . µmol/mg, while values greater than . are considered evidence of significant hepatic dysfunction. it is well known that the liver plays a central role in coagulation homeostasis and is the single site of synthesis of many coagulation proteins, anticoagulant proteins, and fibrinolytic factors. vitamin k is one of the most common factors found to be inactive or deficient in cats with liver dysfunction, and it is essential for normal functioning of factors ii, vii, ix, and x; protein c and s; and thrombin. insufficient or inactive vitamin k can occur for a variety of reasons, including dietary restriction (e.g., anorexia or diet deficiency), disruption of the enteric microflora that synthesize vitamin k (e.g., chronic antibiotic therapy), diseases causing fat malabsorption (e.g., ibd, exocrine pancreatic insufficiency), ingestion of vitamin k antagonists, or liver dysfunction. for example, in cats with hepatic lipidosis, approximately % will have an increased prothrombin time (pt), % will have an increased partial thromboplastin time (ptt), but % of cats will have increased pivka parenchymal disease versus disease of the biliary tree is completed by evaluating the clinical presentation, laboratory values, and imaging of the biliary tree and abdomen for possible evidence of biliary or pancreatic disease. a urinalysis is also an important part of the minimum database, and it is no different in a sick cat with suspected liver disease. in cats the presence of hyperbilirubinuria is abnormal at any urine concentration, because they do not conjugate bilirubin in their renal tubules. however, like bilirubinemia, presence of bilirubin in the urine can occur because of any of the three possible causes of hyperbilirubinemia: prehepatic, hepatic, and posthepatic; thus further evaluation is necessary once bilirubin is detected. ammonium biurate crystalluria suggests the presence of hyperammonemia, which in the cat is either because of a congenital portosystemic shunt (less common in cats than in dogs) or because of severe, end-stage liver disease resulting in portal hypertension, which is typically caused by cirrhosis or advanced polycystic liver disease. , the most common feline liver diseases are hepatic lipidosis and feline cholangitis syndrome, which are two diseases that often result in development of clinical or biochemical icterus. thus because hyperbilirubinemia is a more sensitive indicator of liver function than bile acids or other liver function tests, the need for further testing is moot. however, there will be circumstances when further assessment of liver function is indicated, and for this, serum bile acids, blood ammonia levels, and urine bile acids may be needed. there are several situations where liver function testing may be indicated, but the most common indications for additional testing would be a cat with persistently elevated liver enzymes of unknown origin, a cat that develops urethral obstruction because of urate stones (suggestive of portosystemic shunting) or a cat with possible polycystic liver disease. one of the oldest tests of liver function, because of its association with development of hepatoencephalopathy, is measurement of blood ammonia levels. however, although this test is the only practical way to diagnose hepatoencephalopathy in dogs, the test has a number of limitations, including differences in ammonia levels between arterial and venous (lower) samples and significant sample handling issues (ammonia is labile and results are affected by improper sample handling or lack of immediate measurement) that make its use difficult in practice. in cats hyperammonemia is even less common than in dogs likely because of their highfunctioning urea cycle pathways ; the assays have not been validated for feline blood in most laboratories, and as such, the test is not recommended as the sole indicator uncommon in cats, the most common causes are neoplasia (primarily of the pancreas, but cholangiocarcinomas can occur) or chronic pancreatitis, which can occur concurrently with cholangitis in cats, resulting in both intrahepatic and extrahepatic cholestasis in some cats. , the bile ducts are affected in cats with chronic pancreatitis, because the feline biliary system and pancreatic duct system merge at the level of the pancreas to form a single duct that empties into the duodenum. thus in cats with either pancreatitis or biliary disease, recent evidence has shown that the inflammation affects both organs. , further, in chronic pancreatitis, either persistent inflammation or development of fibrosis can result in dilation or obstruction of the common bile duct. in cats with chronic ehbdo, the common bile duct will become widely dilated and tortuous, a finding easily seen on abdominal ultrasonography but a problem not easily managed (figures - and - ) . interestingly, the gallbladder is often not enlarged, and may in fact be small in cats with this condition, because the remaining fluid in the gallbladder is white bile (highly concentrated mucinous bile from which the pigment has been resorbed). in addition, variable filling of the gall bladder is a normal phenomenon; thus gallbladder size is not an indicator of ehbdo. (proteins induced by vitamin k antagonists or absence). nevertheless, although pivka is a very sensitive test for abnormalities of vitamin k function, most cats with liver disease that have a normal pt/ptt, but abnormal pivka do not represent clinical evidence of bleeding. in any case, abnormalities in the clotting cascade related to vitamin k deficiency in cats with liver disease are common, whether or not they show evidence of active bleeding. and because the balance of the coagulation system in a cat with liver disease can be disrupted by a procedure that initiates small amounts of bleeding (e.g., a biopsy), all cats with liver disease should be given vitamin k as a precautionary measure before and after invasive procedures, even if the clotting times (pt and ptt) are normal. this may be especially important in cats with hepatic lipidosis, because their vitamin k clotting status is likely to be even more affected by the concurrent anorexia and disruption of enteric microflora. the dose of vitamin k (phytonadione, aquame-phyton [merck, west point, pa.]) used prophylactically is . mg sc, im, or po q h for to days, then weekly until recovered. see box - for a summary of the causes of icterus. cholestasis is the reduction of bile flow, which can occur at any point along the biliary tree; bile production occurs in hepatocytes, and flow is connected to the distal concentrating components (gallbladder and common bile duct) by the bile ductules. thus cholestasis can occur inside the liver's biliary tree (intrahepatic cholestasis) or outside the liver in the gallbladder and common bile duct (extrahepatic cholestasis). intrahepatic cholestasis most often occurs in diseases involving hepatocellular damage, leakage, or swelling, such as infections (e.g., bacterial cholangiohepatitis, toxoplasmosis, fip, or other diseases causing inflammation), infiltrative diseases (e.g., lymphoma), metabolic diseases (e.g., hepatic lipidosis), or diseases causing disruption of architecture (e.g., cirrhosis or severe polycystic disease). intrahepatic cholestasis occurs in zone of the liver lobules (periportal zone); at the level of hepatocytes, canaliculi or bile ductules; and is damaging to cells because of the emulsifying properties of lipid on membrane lipids. however, because the liver has a large reserve capacity, clinical icterus (e.g., jaundice) only occurs in the most severe cases when the liver is affected diffusely. thus severe or persistent intrahepatic cholestasis can serve to perpetuate the inflammation and cell damage if it is not corrected. extrahepatic cholestasis or extrahepatic bile duct obstruction (ehbdo) is less common than intrahepatic cholestasis and is most commonly associated with obstruction of the common bile duct. since gallstones are icterus is the result of cholestasis, and the underlying cause can be either hemolysis or hepatobiliary disease, for which further clinical examination will be needed to determine if rbc destruction or liver disease is occurring. in most hepatobiliary diseases of cats, cholestasis is occurring, but there may be no clinically apparent icterus because the degree of hyperbilirubinemia must be at least to times greater than the normal values to exceed the capacity of the liver to process the excess bilirubin. in cats with hyperbilirubinemia not caused by hemolysis, whether it is clinical or subclinical, there is no need for further evaluation of liver function (e.g., bile acid assays), because bilirubin is a more sensitive indicator of liver function than bile acids. the degree of hyperbilirubinemia does not suggest differentiation of intrahepatic versus extrahepatic cholestasis; however, the presence of acholic feces (white feces) is diagnostic for extrahepatic bile duct obstruction (ehbdo), because lack of stercobilinogen (the brown/black pigment in feces) is only found in cats with complete obstruction of the bile duct. finally, the presence of intrahepatic cholestasis and clinical icterus in a cat indicates a diffuse hepatobiliary disease, such as cholangitis or hepatic lipidosis, as focal liver disease, even if severe, will not cause clinical hyperbilirubinemia because of the tremendous reserve capacity of the liver for bilirubin uptake. portal hypertension is an abnormally high venous pressure in the portal system and is typically caused by increased resistance to portal blood flow. there are potentially three regional causes of portal hypertension: prehepatic (disease in the portal vein itself), hepatic (intrahepatic diseases causing compression or decreased flow), and posthepatic (diseases of the caudal vena cava, right heart or pulmonary vasculature). the most common cause of portal hypertension in the cat is cirrhosis or portal venous thrombosis, because portal vein hypoplasia (formerly known as microvascular dysplasia) is known to occur only in the dog, and the other causes of portal hypertension (budd-chiari syndrome, heartworm caval syndrome, pulmonary hypertension) are rare and more likely to occur in the dog. , in any case, the clinically recognizable effects of portal hypertension are development of ascites (unusual in the cat), acquired portosystemic shunting (reported in cats), and development of hepatic encephalopathy (less common in cats than in dogs, because of their profound ability to handle protein wastes). , , most cats and dogs that develop hepatic encephalopathy (he) secondarily to portal hypertension do so because of reduced liver function (because of portosystemic vascular shunting [pss] or cirrhosis and the acquired shunting that develops). cats can develop another form of chronic he because of hepatic lipidosis, but this is believed to be because of the combination of liver failure and prolonged fasting, resulting in arginine deficiency and impaired ammonia detoxification. portosystemic vascular anomalies, also called portosystemic shunts or portovenous shunts (pss), although less common than in dogs, also occur in cats. these vascular anomalies can be either congenital or acquired, single or multiple in number, and occur as extrahepatic vascular shunts or within the liver itself (intrahepatic shunts). the shunting of blood around the liver is the cause of hepatic atrophy and reduced hepatic function that results in an accumulation of toxins, particularly ammonia that leads to the development of hepatoencephalopathy. the two most common veins that serve as the connection point for the shunting portal venous blood are the caudal vena cava and the azygous. in cats a single, extrahepatic, portocaval shunt is the most commonly reported form, and occurs in % of cats with pss. as in dogs, specific breeds of cats may have pss more commonly, and these include domestic shorthair cats, burmese, siamese, persian, and himalayan breeds. in contrast to dogs, males may be more predisposed to pss than females, but the clinical signs relate to the three body systems most affected: the central nervous system, gi tract, and urinary tract. the most common presenting complaints in cats are weight loss or poor/stunted growth, and dull, bizarre or lethargic behavior, especially after eating. signs of gi disease common in dogs, such as vomiting, diarrhea, or inappetence, are less common in cats, but in one report, % of cats with pss drooled. finally, cats with pss often present with signs of lower urinary tract disease (e.g., hematuria, stranguria, or even obstruction) because of the development of urate uroliths (which are radiolucent, thus difficult to detect). because the most common signs of he are apathy, listlessness, and decreased mental alertness, they are often not recognized specifically as indicative of brain dysfunction but as part of the constellation of signs of the liver disease. however, with progression of the has not been reported. the clinical presentation is typically nonspecific (the most common signs are vomiting, lethargy, and anorexia), and there are no laboratory changes that are suggestive of hepatic neoplasia. thus the diagnosis must be made by identification of disease, other signs will develop, including ataxia, salivation, stupor, or coma. the best and only practical diagnostic test for he is plasma measurement of ammonia levels. however, as previously noted, the test has many technical issues that make its clinical utility in the practice setting difficult at best, and there are few laboratories that have validated ammonia measurement in the cat. cancer of the liver can occur as a primary disease (table - ) or as a result of metastasis of neoplastic disease occurring elsewhere and, most typically, the abdominal cavity. the most common neoplastic infiltration of the liver that is not a primary liver tumor is lymphoma (figures - and - ), followed by visceral mastocytosis. as with many other types of cancer, hepatobiliary neoplasia is most common in middle-aged to older cats, and it is relatively rare, with a reported incidence of . % to . %. benign tumors, such as biliary cystadenoma ( figure - ) , carry a good prognosis if they are amenable to surgical resection. the incidence of metastatic neoplasia (including lymphoma and mast cell tumors) the most common clinical signs are related to spontaneous rupture of the enlarged and friable liver. affected cats may present with lethargy, anorexia, pale mucous membranes, and a heart murmur secondary to anemia. clinical signs of liver disease are usually absent. hepatomegaly and hypotension may also be found. results of routine laboratory testing (mild to marked increases in alt and globulins while alp and ggt are typically normal) and ultrasonographic examination (hepatomegaly, generalized increase in hepatic parenchymal echogenicity) a of the liver may be supportive, but definitive diagnosis relies on histopathologic examination of a liver biopsy. fna of the liver is not helpful because amyloid is rarely detected with this method. hemostasis should be evaluated carefully before any biopsy procedure is planned. the most important differential diagnoses are fip, hepatic lipidosis, and hepatic lymphoma. scintigraphic imaging using i- serum amyloid p component has potential as a noninvasive test. a there is no specific treatment for amyloidosis in cats, so therapy is primarily supportive care (antioxidants, vitamin k, blood transfusion). attention should be paid to identification and control of any underlying chronic inflammatory disease. unfortunately, the long-term prognosis is poor as most affected cats die of intra-abdominal bleeding. survey abdominal radiography is the simplest and most readily available imaging modality to assess structures in the abdominal cavity. radiographs are most useful to assess liver size, will reveal large hepatic masses, and provide evidence of radiopaque masses or other abnormalities in the abdomen. however, the preferred imaging modality used to assess hepatic structures in cats with suspected liver disease is abdominal ultrasonography (aus). the reasons why ultrasonography is a more useful tool for assessment of the liver in cats are numerous, but because feline liver diseases are primarily diffuse, infiltrative, or metabolic diseases that also affect the biliary tree, ultrasonography is the only imaging tool that will give reliable diagnostic information. this widely available diagnostic tool can be helpful in determining liver size and parenchymal echogenicity, in identifying mass lesions, evaluating the biliary tree and gallbladder, quantifying flow (doppler techniques), and identifying vascular anomalies. as with all diagnostic modalities, the skill and experience of the operator is vital to accurate procurement and interpretation of the images. further, it is important to remember that although ultrasonographic images are extremely useful in the clinical evaluation of a cat with possible liver disease, the images themselves do not represent a histologic diagnosis. structural abnormalities by hepatobiliary imaging and subsequent examination of the tissue either by fna or biopsy techniques. historically, amyloidosis has been recognized as primarily a renal disease, especially in abyssinian cats. more recently, cases of hepatic amyloidosis without renal involvement have been diagnosed in siamese and related breeds, as well as in nonpedigreed cats. a, a, a the majority of cases have been described in australia, the united kingdom, and europe. amyloid a is deposited in the liver, probably in response to chronic inflammation in another organ. in the siamese breed, a genetic component may contribute. a the amyloid a protein occurring in the siamese breed differs from that known in the abyssinian breed. a contraindicated in cats, because they may cause a lethal shock reaction. a similar reaction may be seen with penetration of the larger bile ducts or gallbladder with a large-bore biopsy needle, because these tissues have a significant autonomic innervation in the cat that may result in bradycardia and shock following the procedure. , , it is particularly important to recognize this as a risk in cats with ehdbo or dilated bile ducts, and this risk factor reiterates the need for ultrasound examination of the liver prior to making biopsy decisions. nonetheless, owners should be informed of these potential risks, in addition to the risk of bleeding from biopsy sites in any cat undergoing liver sampling. , biopsy techniques liver biopsies, whether they are obtained by needle, laparoscopy, or surgical means, should be taken from a location that represents the primary liver pathology, handled appropriately to ensure accurate interpretation of the sample, and the histopathologic description should be interpreted according to the guidelines set by the wsava standards for clinical and histologic diagnosis of canine and feline liver disease. , guidelines for obtaining and handling surgical biopsies of the liver are reviewed elsewhere and will not be further discussed. because needle aspirates/biopsies, tru-cuttype biopsies, and laparoscopic biopsies are commonly used to obtain liver tissue in cats, the benefits and limitations of each of these techniques will be discussed. as a general rule, the more tissue that can be obtained, the better the pathologist's interpretation of the tissue abnormalities will be. for example, most pathologists believe that at least six portal areas are necessary to make a diagnosis of inflammation liver disease in cats. this will require either a -or -gauge needle size or larger piece of tissue than is obtained with smaller needles or an aspirate. the amount of tissues required to view at least six portal areas is approximately mg, and mg will be required for culture of the tissue. if other analyses of the tissues are considered (e.g., metal analysis), approximately to mg of liver is needed. a typical laparoscopic cup biopsy forceps will provide mg of liver tissue, a -g tru-cut-type biopsy needle provides to mg, and an -g needle biopsy provides only to mg of liver tissue. thus, depending on the clinical circumstances and considered differentials, the best approach for obtaining the needed tissue must be considered prior to planning the procedure. fine-needle aspiration to obtain liver tissue for cytologic examination is commonly performed in cats with liver disease for good reason. the procedure is inexpensive, easy to do, is relatively low risk, and often requires only sedation to complete. further, samples obtained by this method can be diagnostic for hepatic lipidosis, hepatic lymphoma or other round cell tumors, and in for the most common liver diseases of cats (hepatic lipidosis, feline cholangitis syndrome, and neoplasia/ lymphoma), aus examination provides a useful means of obtaining clinical clues and tissue to support or refute the differentials. for example, in cats with hepatic lipidosis, the liver is quite enlarged and typically diffusely hyperechoic, while in cholangitis or other inflammatory diseases, the liver is more often diffusely hypochoic. however, these sonographic findings are very nonspecific and can easily lead to errors in diagnosis if the tissue is not subsequently sampled for confirmation. , thus one of the most important utilities of the aus is the ability to obtain liver tissue (either by aspiration or guided-needle biopsy) and for aspiration of the gallbladder to obtain bile for culture. , these techniques alone have made the aus an extremely important diagnostic tool in the evaluation of liver disease in cats. the diagnosis of most liver diseases requires a histopathologic sample of liver tissue, and this is particularly true in the most common feline liver diseases, which tend to be diffuse diseases affecting the entire liver. cats with one of these diffuse diseases can be sampled randomly using any one of these commonly employed techniques: ultrasound-guided fine-needle aspirates (fna), ultrasound-guided needle biopsy, laparoscopic biopsies, or biopsies obtained surgically. some types of neoplasia (particularly round cell tumors) and vacuolar hepatopathies (hepatic lipidosis) can often be diagnosed by cytology using fna techniques. however, differentiation of liver cell tumors (adenomas and carcinomas) and inflammatory diseases of the liver cannot be diagnosed without a larger sample of tissue and histopathologic examination. , further, even in cats with classic hepatic lipidosis changes, concurrent diseases such as cholangitis or lymphoma can be missed if only fna techniques are employed. thus it is essential to consider that in many liver diseases the lesions, although typically diffuse, may also have focal components; for example, inflammation may be throughout the liver, but fibrosis will be present only in focal areas. thus the results of fna or tru-cut needle biopsies should always be considered in the light of the clinical, laboratory, and ultrasonographic evidence. prior to scheduling a cat for a biopsy, the risk-tobenefit ratio of performing a liver biopsy should always be considered. this is primarily because heavy sedation or anesthesia will be essential in most cats undergoing a liver fna, and for all cats undergoing a liver biopsy (needle or otherwise). in addition to anesthesia risks, the use of automatic spring-loaded biopsy guns to obtain ultrasound-guided biopsies of liver tissue is equipment, the interested reader is referred to several recent reviews on the subject. , to maximize the histopathologic accuracy, biopsies taken at laparoscopy or surgically should be taken from both normal-appearing and abnormal areas in the liver. further, if there is a need to obtain samples from the deeper tissues, the laparoscope can be used to direct a tru-cut needle biopsy to the best location for sampling. one of the major advantages of the laparoscopic technique is that it allows the operator to observe the biopsy sites for excessive bleeding, which is unusual, but if observed can be staunched by using pressure on the site, gelatin coagulation material placement, or electrocautery. with experienced operators, the complication rate for laparoscopy is very low (less than %), and most complications were because of anesthesia, bleeding, or air embolism. finally, although not necessary to have direct visualization to obtain an aspirate of gallbladder bile, laparoscopy allows easy sampling of bile for culture, which is important in all cats with suspected inflammatory liver disease or hepatobiliary disease. once a diagnosis of liver disease is made in the cat, specific therapy for the cause (if available) should be instituted; however, for many feline liver diseases, no specific therapy is available, and thus hepatoprotective therapy is used concurrently to aid in the recovery of the liver from the insult. in this section, therapy of two of the most common diseases of the feline liver will be considered, with a special emphasis on nutritional aspects of treatment, nutraceutical therapy, and the unique needs of cats. the most common liver disease of cats is idiopathic hepatic lipidosis (figures - and - ) , a disease that results in liver failure because of a combination of factors including hepatic lipid accumulation, insulin resistance, fasting, and protein (especially arginine) deficiency. , , , thus, unlike many diseases of the liver, the primary focus of therapy and the essential component for recovery is nutritional support. as in any patient with serious liver disease, initial therapy is always aimed at correction of any fluid or electrolyte abnormalities that may exist, because these may be profound if the cat has been vomiting. in addition, normalization of electrolytes is particularly important in cats that have been anorexic for an especially long time ( to weeks), because refeeding syndrome may be triggered with the initiation of feeding, resulting in sudden drops in potassium, phosphate, and magnesium. although this phenomenon is less common and usually less profound in cats fed enterally versus areas where appropriate, definitive diagnosis of certain infectious diseases (e.g., histoplasmosis). however, even with these relatively straightforward diseases, fna of liver tissue has significant limitations, the most important of which is the failure to accurately identify the primary disease. for example, although it is easy to make a diagnosis of hepatic lipidosis using this technique, a paper recently showed four cats that were incorrectly diagnosed with hepatic lipidosis instead of lymphoma because the fna samples were obtained from areas that did not have lymphoma infiltration. in another study, reviewing the agreement between fna cytologic samples of liver and the histopathologic diagnosis, only % of the cases had overall agreement. thus although cytology of fna samples of liver tissue in cats with diffuse hepatic disease remains a useful first step, it is important for the clinician to carefully interpret the results and discuss the potential limitations of this technique with owners. there are several needle biopsy techniques available for sampling liver tissue, but not all are suitable or safe for use in cats. the menghini technique is one such approach that is not suitable for use in cats, because it is a blind procedure using a large-bore needle that cannot be used with ultrasound guidance. the second option among the needle biopsy techniques that is not recommended for cats is the biopsy gun device. tru-cut biopsy guns are operated by a triggering device that can result in the induction of a lethal vagotonic shock reaction in the cat immediately following the procedure. for most ultrasound-guided liver biopsy procedures, either the manual or, preferably, the semiautomatic tru-cut device is recommended for use in obtaining needle biopsies from cats. as a general rule, the tru-cut device will advance into the liver to a depth of cm; so, it is essential to carefully note the amount of liver tissue available during the ultrasound assessment before advancing the needle for tissue collection. properly obtained tru-cut needle biopsies are a valuable technique for obtaining a representative sample of liver tissue ; however, because of the risk for bleeding or liver fracture with any movement, it is essential that cats be anesthetized for this procedure. laparoscopy is an intermediate step between needle biopsy and surgical laparotomy for obtaining liver tissue for histopathology in cats. , this technique is becoming more widely used as more specialists are trained for this procedure that allows visualization of tissues to be biopsied without opening the entire abdomen. although this technique does require general anesthesia, the limited degree of invasiveness, the large biopsy sample size, and rapid patient recovery make laparoscopy a valuable tool for obtaining liver tissue, and it can be used to obtain biopsies from the spleen, pancreas, kidneys, lymph nodes, or to aspirate the gallbladder. for a detailed discussion of laparoscopic techniques and lipidosis. the echogenicity of the parenchyma is uniformly increased, which is more apparent when compared with other ultrasonographic images presented in this chapter. additionally, the gall bladder is distended. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease. (courtesy dr. randolph baral.) liver gb figure - gross appearance of liver from a cat with hepatic lipidosis. note the pale tan and exaggerated reticular pattern. in most cases, the edges appear more rounded than is evident here. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease (same cat as in figure - ) . (courtesy dr. randolph baral.) those started on intravenous nutrition, it can be a significant source of morbidity if electrolyte replacement and monitoring are not carefully attended. once the cat is hemodynamically stable, the next step in treatment planning in cats with hepatic lipidosis is re-introduction of nutrition, which must include placement of a feeding tube (box - ). however, because many of these cats are extremely ill and are not good candidates for anesthesia, placement of a nasoesophageal (ne) tube to allow initiation of enteral feeding is often the most appropriate step for the first few days. when administering food through a feeding tube, there are several important points: . the food should be room temperature (not too hot or cold). . the tube should be flushed with water following feeding, to remove any particles of food or medication that may cause the tube to clog. . if the cat is volume sensitive, it is important to carefully calculate how much water is used for flushing the tube, because a significant volume of fluid can be infused, creating a potential fluid overload. if the cat is fluid sensitive, the total amount of fluids (amount in the food, amount added to food if blenderized, and amount of flush) must be determined, and the amount of fluid used in flushes or food preparation may have to be reduced. force feeding is to be strongly discouraged in these sick cats for several reasons: • it is highly stressful and will further increase the stress response and insulin resistance phenomena that are perpetuating the hepatic lipidosis. • it can be dangerous to the cat (aspiration) or operator (scratches/biting). • it is rarely able to meet the necessary nutritional goals set for the patient. • it may induce food aversion, a phenomenon unique to cats, but creating a profound aversion to the chosen food that can be lifelong. although ne tubes are excellent choices for short-term feeding of cats unwilling to eat, there are several disadvantages to their long-term use, including the nasal irritation that occurs, the relative ease with which cats can (and will) remove them, and the need to use liquid enteral diets. thus once the cat is deemed stable enough for general anesthesia, a long-term feeding tube solution is needed, and this typically is either an esophageal (e) tube ( figure - ) or percutaneous endoscopic gastrostomy (peg) tube. , both feeding options are generally well tolerated methods for providing long-term feeding, but e tubes have the advantage of being placed without the need for any specialized equipment, and if complications occur, they are generally easily addressed, because the most common complications are infection at the tube site or premature removal of the tube by the cat. placement of a peg tube, although relatively easy to learn to place, requires having the appropriate endoscopic equipment, and if complications occur as a result of infection or tube removal, more significant morbidity can result. because there is no advantage to placement of peg tubes easiest to use, and are an acceptable choice in most situations. finally, because many cat stomachs are volume sensitive with initiation of feeding, it is very important to start conservatively with small-volume feeding on a more frequent schedule. with prolonged fasting, the stomach volume of a cat with hepatic lipidosis may be reduced dramatically, preventing normal expansion and limiting intake to as little as % of normal. thus to avoid vomiting when feeding, the starting volume may have to be as small as to ml every to hours. a good rule of thumb is to start with estimation of resting energy requirement (rer) ( to kcal/kg is a good estimate of rer), and then attempt to meet % of rer the first day. if no problems are encountered, increase the amount to % of rer the second day, and so on, but during this period, keep the frequency as high as possible (feed four to six meals per day) so that the volume remains relatively small at each meal. once full rer has been achieved with multiple meals per day, the frequency of feeding can be gradually reduced to three to four meals per day. most cats will eventually tolerate three meals per day well, and some can tolerate two meals per day, but this is quite variable and should not be attempted during the first weeks of feeding. in general, most cats with hepatic lipidosis will require tube feeding for a minimum of to weeks before they will show interest in food and begin eating again on their own. the tube should be retained until the cat has been eating on his or her own for at least week or longer and can be maintained for a longer duration if it is being used to administer medications, because cats can eat normally with the e tube in place. the other therapeutic considerations for cats diagnosed with hepatic lipidosis are directed toward dealing with the complications of the disease and reducing the oxidative stress on the liver with hepatoprotective therapy (table - ) . in cats that are vomiting, in cats versus e tubes, placement of e tubes is advocated as the best approach for most practice situations. interested readers are referred to several recent reviews on tube placement for specific details on each method and to chapter . , diet selection is the next step in treatment planning for cats with hepatic lipidosis. in contrast to the belief that animals in liver failure need lower quantities of protein to reduce the workload on the liver, cats with hepatic lipidosis actually need protein to recover. in fact, the work of biourge and coworkers showed that protein was the essential nutrient in reducing hepatic lipid accumulation, was essential to eliminate the negative nitrogen balance, and also appeared to minimize muscle catabolism. further, diets high in protein can improve insulin sensitivity and assist weight loss in recovery from obesity. , conversely, although carbohydrates are a readily available energy source, they are often associated with gastrointestinal distress (diarrhea, abdominal cramping) and hyperglycemia (secondary to the insulin resistance in place as a result of obesity and hepatic lipidosis). thus diets selected for cats with hepatic lipidosis should ideally be high in protein (> % metabolizable energy [me]) and have lower amounts of carbohydrates (< % me), with the remaining calories coming from fat. the diets that best fit this profile are the diets formulated for diabetic cats; however, kitten food, many adult cat foods, and some of the enteral recovery diets have this high protein/low carbohydrate profile. many of the intestinal diets are not higher protein and are higher in carbohydrates, and so would not be the ideal choice. the key to using any of the foods that are not designed for use in a feeding tube is to blenderize them (and if necessary, strain the food) so that the food will easily go through the -or -g feeding tube without clogging it. enteral diets designed for use in feeding tubes are the because the primary starting point of the inflammatory disease in cats is the bile ducts (cholangitis), with inflammation extending to the hepatic parenchyma (cholangiohepatitis) only with time and severity, the term cholangitis syndrome has become the preferred terminology. the disease syndrome has been further classified by the wsava liver diseases group into one of three primary types: neutrophilic or suppurative, chronic lymphoplasmacytic (figures - and - ) , and lymphocytic (non-suppurative). each of the forms appears to behave quite differently clinically as well as in their progression and outcome. in general, cats with the suppurative form of cch typically have an acute onset of illness, which often includes fever, anorexia, and vomiting, and they may become icteric quite quickly (figure - ) . , the nonsuppurative form of cch (lymphocytic form) tends to be a more chronic condition, with affected cats showing nonspecific signs of illness that may include partial anorexia and lethargy, but the signs may wax and wane or are non-progressive. , because of the feline pancreatic and bile duct anatomy, it is common for cats with cch to have pancreatitis and vice versa, and in some cases, cats will also have concurrent ibd; the constellation of the three conditions occurring together is called triaditis. this combination is increasingly recognized in cats, and recent reports suggest from % to % of cats with one syndrome have all three diseases. , , , at this time, the etiology of each of these syndromes and the pathogenesis is not well understood; however, the enteric microflora are presumed to play an important role in the suppurative form, and immune mechanisms are presumed to be the cause of the chronic inflammation found in the chronic nonsuppurative antiemetic therapy is beneficial, because it is imperative that the cat continues to receive some food, and vomiting will complicate this. metoclopramide is often used in cats because of its ready availability and low cost, but it is a very weak antiemetic in cats and thus may not be the best choice. in most cats, the novel nk- receptor antagonist maropitant has been a safe and effective choice. the most commonly used antiemetics in the author's feline practice are maropitant ( mg/kg iv, sc or through the e tube q h), ondansetron ( . mg/kg iv q - h), or dolasetron ( . mg/kg iv, sc q h). in addition to control of vomiting, all cats with hepatic lipidosis should be given vitamin k ( . mg/cat po, sc) daily for a week, then weekly until the cat has recovered, and vitamin b (cobalamin) ( µg/cat sc) weekly for weeks, then monthly until blood values are normal. other vitamins may become deficient, such as some of the b vitamins and vitamin e; however, feeding is likely to rapidly replenish these deficiencies if they exist. this is also likely true of amino acid deficiencies, but supplementation of l-carnitine ( mg/day po) may be beneficial by improving fatty acid oxidation. finally, hepatoprotectant and antioxidant therapy with s-adenosylmethionine (same) ( mg/kg po q h) has been advocated to increase glutathione and may be beneficial in cats with hepatic lipidosis. , , it is important to note that if same is given through the tube (and thus the tablets must be crushed), the dose must be increased by approximately % to allow for the loss of absorption from loss of the enteric coating. because drug metabolism is often impaired in cats with hepatic lipidosis, appetite stimulants, such as mirtazapine, cyproheptadine, and clonazepam, should not be used in cats because dosing and side effects can be unpredictable. benzodiazepine agonist drugs (e.g., diazepam) should be completely avoided in cats with possible lipidosis-induced hepatoencephalopathy, because they will exacerbate the signs and may cause fulminant liver failure. , fortunately, most cats with idiopathic hepatic lipidosis that receive immediate and aggressive therapy and feeding for their disease will recover completely. cats that develop hepatic lipidosis secondary to other serious diseases (e.g., lymphoma) have a much lower chance of complete recovery and often die of their disease or its complications. the most common inflammatory liver disease in the cat is a complex syndrome with multiple subgroups of disease previously termed cholangitis/cholan giohepatitis complex (cch) but currently recognized under the terminology feline cholangitis syndrome. this disease is quite variable in both its presentation and severity, and it may occur as a primary process or secondary to/ concurrent with other diseases (e.g., pancreatitis, ibd). ultrasonographic appearance of liver with lymphocytic/plasmacytic inflammation. note the varying echogenicity throughout the hepatic parenchyma; areas of hypoechogenicity likely reflect inflammatory cell infiltration. the gall bladder is distended; its shape is distorted by pressure from the transducer. (courtesy dr. randolph baral.) liver gb [ mg/kg po q h]), and if pancreatitis is concurrent, pain control with opioid pain relievers (e.g., buprenorphine . to . mg/kg po, sq q - h). if culture is not possible, combination therapy with enrofloxacin ( mg/kg po q h) and metronidazole ( mg/kg po q h) is reasonable. in cats with chronic lymphoplasmacytic forms of cholangitis, management must be tailored to the individual situation and often requires therapy with either immunosuppressive doses of prednisolone ( to mg/kg po q h) or chlorambucil ( mg/m po q d), along with the hepatoprotectants and cholerectics, and concurrent treatment of other diseases (pancreatitis or ibd) that may be occurring. the lymphocytic or lymphoplasmacytic forms of cholangitis may wax and wane in intensity over time, and may require long-term continuous or intermittent therapy to control the disease. there is no specific diet that is recommended for cats with inflammatory liver disease, but protein restriction should not be initiated unless the cat has clear evidence of severe hepatoencephalopathy. the diet should be selected based on other conditions (such as ibd), for which the diet may be more critical in the management. monitoring of serum chemistry values (especially glucose), clotting times, cobalamin levels, and pli/tli concentrations are recommended every few months, as well as careful monitoring of the cbc for all cats on chlorambucil. in all cats with chronic inflammatory liver disease, prior to initiation of immunosuppressive therapy, a careful assessment of the cat for other possible causes of inflammation should be completed (box - ). as in dogs, if a cat with pss can have surgical closure of the shunting vessel (ligation, placement of an ameroid constrictor, intravenous coiling), the long-term forms. however, whether or not these syndromes are related, a continuum of disease or completely different diseases remains undetermined. once a definitive diagnosis is obtained by histopathology of the liver tissue and culture of bile, treatment can be tailored to needs of the cat. cats with the more aggressive suppurative form of cholangitis often require intravenous fluid therapy, antibiotic therapy (based on results of culture whenever possible), and supportive therapy (antiemetics, vitamin k , hepatoprotectants such as same [ important antioxidant and stabilizes membrane functions • n-acetylcysteine-a precursor to glutathione and antioxidant, also improves tissues oxygen delivery • ursodeoxycholic acid (tertiary bile acid)-used to replace hepatotoxic, hydrophobic bile acids and increase bile flow • silymarin (milk thistle)-a free radical scavenger and anti-inflammatory/antifibrotic agent • vitamin e-an antioxidant and antiinflammatory vitamin* although few clinical trials of these nutraceuticals have been performed in feline liver disease, a few studies have recently appeared showing that same, ursodeoxycholic acid, silymarin, and n-acetylcysteine all are hepatoprotective, have few adverse side effects, and may be beneficial in many types of liver disease in cats. , , , , feline liver disease is a common problem that requires careful consideration of the presenting complaint, clinicopathologic findings, imaging results, and, if available, histopathologic interpretation to be able to provide an accurate diagnostic and therapeutic plan. a variety of insults can be responsible for liver dysfunction or failure, but hepatic lipidosis and feline cholangitis syndrome remain the most common reasons for cats to present prognosis for function and quality of life is generally very good. however, even if surgical correction is anticipated, and especially if surgical correction is impossible or not completely successful, medical management of he is indicated. see table - for the basic therapeutic approach to medical management of cats with he resulting from pss. because hepatocytes, by their position in the body between the gi tract and rest of the body, as well as their critical role in metabolism and detoxification, are uniquely susceptible to oxidative injury and reactive intermediates of metabolism, they must be able to protect themselves. the natural defenses of the liver include superoxide dismutase and glutathione, free-radical scavengers such as vitamin e and ascorbate, and other prosurvival signaling pathways that are controlled by hormones and growth factors. however, in injury or overwhelming infection or inflammation, the natural defenses of the liver can be overwhelmed, and then it is essential for medicines and nutraceutical therapy to be included in the treatment plan to help reduce inflammation and fibrosis, protect against oxidant injury, and enhance bile flow. the cytoprotective agents most commonly used in liver diseases to assist in these processes (table - ) are: • s-adenosylmethionine (same)-a precursor in the synthesis of glutathione and an important methyl donor to dna and proteins, is an with icterus or liver failure. therapy must be tailored to the individual, but nutritional support is critical in the management of hepatic lipidosis, and appropriate supportive therapy with hepatoprotectants may be crucial to treatment success. resorption from that space. effusion accumulation is therefore correlated to increased capillary hydrostatic pressure, widening of the oncotic pressure gradient, increased endothelial permeability, increased interstitial hydrostatic pressure, or loss of effective lymphatic drainage or a combination of these factors. , , peritonitis of any cause results in vascular dilation, increased capillary permeability, and the migration of inflammatory cells into the peritoneum in response to immunomodulatory mediators. the inflamed peritoneum becomes a freely diffusible membrane, allowing a massive outpouring of fluid and plasma proteins from the circulation. , ascites is not commonly seen in practice; one study recognized ascites in only three cats out of admissions to an american veterinary teaching hospital, but the prevalence may be greater in primary care practice. in that study, dilated cardiomyopathy (dcm) was the most common disease associated with peritoneal effusion; however, dcm was diagnosed in most of these cats before , when taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. neoplasia was the most common cause after . feline infectious peritonitis (fip) was by far the most common cause of feline ascites diagnosed over a -year period at the feline centre at the university of bristol, comprising % of all cats with recognized ascites. cats with ascites usually present with nonspecific clinical signs, such as anorexia or lethargy. the owners may present the cat because they recognize abdominal enlargement ( figure - ), but in many cases, owners perceive this as weight gain. clinicians should be aware that sudden weight gain in a chronically underweight cat may be because of fluid accumulation (which can be intrathoracic fluid if ascites is not present), particularly if muscle mass seems reduced. ascitic cats presenting subsequent to trauma may have intraabdominal hemorrhage or urinary tract rupture. fever in a young ascitic cat will often suggest fip, and cats with fip may or may not be jaundiced. presence of jugular distention or even a jugular pulse can suggest right-sided heart failure. a palpable fluid thrill can help to distinguish ascites from other causes of abdominal enlargement, such as organomegaly, abdominal masses, bladder distention, abdominal wall weakness, obesity or, occasionally, accumulations of gas within the abdominal cavity (table - ) . recognizing a fluid thrill involves gently tapping one side of the abdominal wall with the fingers of one hand while feeling for a sensation of fluid movement the peritoneum is the serous membrane lining the abdominal cavity, as well as covering the organs of the abdomen. it comprises a single layer of squamous mesothelial cells resting on a deeper layer of loose connective tissue. the layer of peritoneum that lines the inner surface of the abdomen is called parietal peritoneum; the abdominal organs are lined by visceral peritoneum. the total surface area of the peritoneum is one to one-and-ahalf times that of the total cutaneous area of the body. , the peritoneal cavity contains a small amount of fluid (less than ml/kg body weight) that reduces friction between the abdominal organs as they slide over each other. the fluid is a pure transudate and contains solutes in the same concentration as serum (box - ). this fluid is absorbed from the abdominal cavity predominantly through lymphatic vessels lying beneath the mesothelial basement membrane on the surface of the diaphragm. lymphatic drainage occurs predominantly to the sternal lymph nodes. , ascites is the abnormal effusion and accumulation of fluid in the abdominal cavity. fluid exchange across the capillary bed is determined by starling forces, that is, the balance between hydrostatic pressure, which causes transudation of fluid out of blood vessels, and the colloid osmotic pressure, which acts to retain fluid within blood vessels. the amount of peritoneal fluid is therefore determined by the balance of these, as well as vascular permeability, with excess fluid drained by the lymphatic system. accumulation of fluid within a body cavity results when the rate of filtration of fluid into a space is greater than the rate of fluid routine laboratory findings are usually nonspecific but may provide clues to the underlying cause of ascites. for example, neutrophilia may point towards septic peritonitis but can also occur with fip; most cats with hemoperitoneum are anemic at presentation ; uroperitoneum often results in azotemia and electrolyte abnormalities; hypoglycemia may reflect sepsis with septic peritonitis, and a recent study recognized % of cases of septic peritonitis had ionized hypocalcemia ; elevated liver enzymes may be associated with inflammatory, infectious or neoplastic hepatopathies including fip; elevation of serum globulins occurs in many cats with fip but can also be associated with neoplasia or septic peritonitis; and a finding of hypoalbuminemia (which can cause a pure transudate) should prompt for an assessment of urine protein : creatinine ratio to assess if there is renal protein loss. imaging may be required to confirm the presence of fluid as well as to aid in diagnosis of the underlying cause. radiographic findings can vary greatly depending on the amount of abdominal fluid present and the underlying etiology. loss of normal detail or presence of a "ground glass" appearance to the abdominal cavity is suggestive of the presence of fluid (figures - and - ). very young, thin or dehydrated cats may also have a loss of detail that can mimic the presence of fluid. ultrasonography of the abdomen (figures - and if a large volume effusion causes discomfort because of abdominal distention, a three-way stopcock may be used so large volumes can be drained from one puncture (figure - ) . however, removal of large volumes of ascitic fluid can be detrimental, because it may prevent the subsequent reabsorption of valuable protein and/or red blood cells; the resulting reduction in intraabdominal pressure may encourage further accumulation of fluid; and rapid removal of large volumes can lead to fluid shifts causing cardiovascular collapse. fluid can be collected into ethylenediaminetetraacetic acid (edta) tubes (for total nucleated cell count, packed cell volume, total protein, and cytology), serum tubes (for biochemistry, such as albumin, bilirubin, creatinine, potassium, triglyceride, glucose, lactate, and lipase), sterile tubes for culture, and/or other tubes for effusion-specific tests such as pcr. samples should be prioritized according to the volume of fluid available and to the suspected underlying disease process. initial assessment of fluid retrieved is made on the basis of color and protein concentration, and much information can be gleaned from this simple assessment, even before cell numbers and types are assessed. although this brief, initial assessment is useful to refine the differential diagnoses, a thorough assessment based on underlying etiology and pathophysiology is required for definitive diagnosis and therefore appropriate management (table - ) . ascitic fluid, classified according to its pathophysiologic cause, can be divided into transudates, modified transudates, exudates (septic or nonseptic), or effusions (chylous or hemorrhagic). , can allow the detection of even very small volumes of fluid. it also enables evaluation of the size and structure of intraabdominal organs, such as the liver and spleen, which can help determine the underlying cause of ascites. abdominocentesis confirms the presence of abdominal fluid (in cases of low-volume effusion) and assessment of the fluid is required to diagnose the underlying cause of ascites. most cats tolerate abdominocentesis without sedation and the cat can be held in a standing position or in lateral recumbency (whichever is more comfortable for the cat and familiar to the clinician). the abdomen is clipped and aseptically prepared. a -to -gauge butterfly needle may be used with a -to -ml syringe. in cases with low-volume effusion, ultrasonography can help to guide fine needle aspiration from small pockets of abdominal fluid. diagnostic peritoneal lavage can be used if ultrasound-guided aspiration is unsuccessful. for this procedure, to ml/kg of warmed, sterile fluid is infused into the abdomen over to minutes after aseptic preparation of the site. the cat is gently rolled from side to side or allowed to stand; gentle massage of the abdomen also helps distribute the fluid. the fluid is allowed to dwell for a minimum of to minutes before aseptic preparation is repeated before paracentesis. no attempt is made to remove all the fluid. it must be remembered that, since the recovered fluid has been diluted by this procedure, cell counts and biochemical analyses will be affected. transudates are a consequence of altered fluid dynamics. protein-poor transudates (commonly referred to as pure transudates) form predominantly as a result of severe hypoalbuminemia, which causes a lowered colloid osmotic pressure. since there is no change in endothelial or mesothelial permeability, as fluid accumulates, there is no concurrent cell leakage; so, there is a decrease in the cell count through a dilutional effect. consequently, transudative effusions are typically clear and colorless. , , other pathologic causes of proteinpoor transudates include cirrhosis, lymphatic obstruction, and noncirrhotic portal hypertension (presinusoidal and sinusoidal). since hypoalbuminemia is the most common cause of transudates, serum albumin concentrations must be measured to guide further diagnostics. if the serum albumin concentration is normal (or only minimally decreased), then radiographs, abdominal ultrasonography, and/or echocardiography are indicated to assess cardiac function and for urinary bladder rupture. one review of feline ascitic cases found % of effusions were protein-poor transudates, of which % were the result of hepatic failure or primary renal disease. modified transudates can result from increased hydrostatic pressure within the postsinusoidal vessels of the liver secondary to right-sided congestive heart failure (e.g., tricuspid insufficiency) or potentially from mass lesions (such as neoplastic masses) obstructing blood flow from the hepatic vein or caudal vena cava into the right side of the heart. the increase in hydrostatic pressure within the vessels of the liver causes a protein-rich fluid to leach out of the liver into the abdominal cavity. since cell membrane permeability does not change, cells do not accumulate in the effusion. modified transudates can also result from increased vascular permeability in the early stages of an inflammatory process, in which case cellularity will be increased. modified transudates were described as the most common type of ascitic effusion identified in cats in one study, with most being resulting from neoplasia and congestive cardiac failure; however, this study partially included cases prior to , when right-sided heart failure associated with dilated cardiomyopathy (dcm) was prevalent. the recognition of the role of taurine deficiency in this condition and the subsequent addition of this amino acid to feline diets now means that right-sided heart failure is only rarely encountered as a cause of ascites in cats. exudates are a consequence of altered mesothelial and/ or endothelial permeability. this permeability results from a cytokine-mediated inflammatory response of any underlying cause (e.g., infectious, neoplastic, immune mediated). exudates have high protein and moderate to high cell concentrations and are classified as nonseptic or septic. exudates are often primarily composed of neutrophils. nondegenerate neutrophils (and the absence of organisms) points to a nonseptic exudate (mostly fip but also neoplasia). fip is the most common cause of exudative effusion in cats and was the most common cause of feline ascites diagnosed over a -year period at the feline centre at the university of bristol. the presence of neoplastic cells rules in neoplasia, but the absence of such cells does not rule out this diagnosis since many cases of neoplastic ascites are not associated with exfoliated neoplastic cells. other causes of nonseptic exudates include pancreatitis, lymphocytic cholangitis, and viscus rupture, such as the gall bladder or urinary bladder. degenerate neutrophils typify septic exudates (i.e., septic peritonitis), and their presence should instigate investigation for causes of infection (mostly leakage of gastrointestinal contents). chylous effusions appear as milky or pink opaque fluid, and small mature lymphocytes initially predominate in cell counts. after drainage, more macrophages and nondegenerate neutrophils may be found. chyle is typically classified as an exudate, but its physical characteristics can be consistent with a modified transudate (protein content between and g/l); biochemical analysis of triglyceride and cholesterol levels in the fluid are required to confirm the diagnosis. pseudochylous effusions resemble true chyle both in appearance and cytology but do not contain fat. similar conditions result in both chylous and pseudochylous effusions. chylous abdominal effusions are rarely reported in the cat and only accounted for % of cases of ascites in one study. the described causes of chylous ascites in cats are predominantly neoplastic. in a series of nine cats, chylous ascites was associated with nonresectable abdominal neoplasia in four cases (i.e., hemangiosarcoma and paraganglioma), with intestinal and mesenteric lymphoma in two cases and lymphangiosarcoma of the abdominal wall in another. one described case in a -year-old cat was thought to be because of fip. figure - shows an ultrasonographic image of a cat with chylous abdominal effusion associated with pancreatitis. other potential causes include right-sided congestive cardiac failure, steatitis (inflammation of fat), biliary cirrhosis, and lymphangiectasia. hemoperitoneum in companion animals is categorized as traumatic or spontaneous. traumatic hemoperitoneum is further divided into blunt causes of trauma (i.e., motor vehicle accidents and high-rise falls) and penetrating trauma (i.e., gunshot wounds and bite wounds). , inadvertent splenic aspiration, venipuncture, or acute severe hemorrhage should be suspected if the cytology is consistent with peripheral blood including platelets but without erythrophagocytosis or if the blood clots readily. when there is no history of trauma, coagulopathy or spontaneous rupture of a vascular neoplasm should be considered. in one study of feline cases of spontaneous hemoperitoneum, cases ( %) were associated with hepatic pathology such as neoplasia, necrosis, and amyloidosis. in another study of cases of spontaneous hemoperitoneum, % ( of ) of cats had abdominal neoplasia, and % ( of ) had non-neoplastic conditions. cats with neoplasia were significantly older and had significantly lower packed cell volumes (pcvs) than cats with non-neoplastic disease. hemangiosarcoma was the most often diagnosed neoplasm ( of , %), and the spleen was the most common location for neoplasia ( of , %). coagulopathies ( of , %) and hepatic necrosis ( of , %) were the most common causes of non-neoplastic hemoperitoneum. other nonneoplastic causes of hemoperitoneum include ruptured bladder, hepatic rupture secondary to hepatic amyloidosis, gastric/duodenal ulcer, hepatic hematoma, hepatitis, perinephric pseudocyst, feline infectious peritonitisinduced liver rupture, and feline infectious peritonitisinduced nephritis. , the prognosis of cats with spontaneous hemoperitoneum is poor. in two studies, only approximately % of cases survived to be discharged from hospital. , median survival time for cats that were discharged in one of those studies was days (range, to days). feline infectious peritonitis (fip) comprised % of cats with recognized ascites over a -year period at the feline centre at the university of bristol, and, as a rule of thumb, when ascites is recognized in a younger cat, fip should be considered the major rule-out. the abdominal effusion found with fip is typically straw to golden yellow (although the color can be very variable, for example, chyle may be present), may contain fibrin clots, and has a high protein concentration. the total protein content is greater than g/l and often greater than g/l, with globulins comprising % or more. one study described an effusion with total protein greater than g/l as % specific, % sensitive, and having a . positive predictive value to diagnose fip. the rivalta test evaluates the fluid's globulin content, and was found to be very sensitive but only % specific; this test is performed by adding one drop of acetic acid ( %) to ml of distilled water. this fluid is mixed thoroughly, and then one drop of effusion is gently placed on the surface of the mixture. if the drop stays at the top of the fluid or slowly floats to the bottom, the test is considered to be positive. this test can give inaccurate results if inappropriate technique is used or if there is a significant temperature difference between the fluid sample and the acetic acid solution. a positive rivalta test can result from lymphosarcoma, septic, or fip effusions; these can be distinguished by cytology and culture. immunofluorescence staining of coronavirus antigen in macrophages had a positive predictive value of . but a negative predictive value of . . the potential clinical presentations, diagnosis, and management of fip are covered in detail in chapter . one study found neoplasia to be the most common cause of ascites in cats, and neoplasia should be considered the major rule-out in older cats with ascites. the effusion from cats with ascites resulting from neoplasia may be a modified transudate, resulting from compression of hepatic veins or the caudal vena cava, or metastases to the peritoneum; hemorrhage from neoplasia can cause hemoperitoneum; chylous effusions may result from reduced lymphatic drainage or rupture of lymphatic vessels; and raised vascular permeability caused by neoplastic infiltration can result in an exudative effusion. carcinomas, mesotheliomas, and discrete (round) cell neoplasms (e.g., lymphoma, mast cell tumors, malignant histiocytosis) exfoliate cells into effusions more readily than sarcomas, and of these, lymphosarcoma is the most common malignancy of cats. cytology of ascitic fluid reveals neoplastic cells in less than a quarter of cases; so the absence of such cells does not rule out a diagnosis of neoplasia. in these circumstances, the diagnosis may be achieved by ultrasound-guided fine-needle aspiration of affected organs, or even biopsy samples obtained at laparotomy. the specific approaches will depend on the specific neoplasia diagnosed. exudates caused by septic inflammation usually result from bacterial contamination of the peritoneal cavity secondary to gastrointestinal tract leakage or penetrating wounds associated with trauma. gastrointestinal tract leakage may occur as a result of ulceration associated with neoplasia or inflammatory disease or as a result of penetration of a sharp object ingested (such as a toothpick), it can also occur subsequent to prior abdominal surgery. , , , primary septic peritonitis in which no apparent cause can be identified has also been described in cats. septic exudates are usually yellow to tan in color, with yellow particulate matter and are foul-smelling. microscopically, the fluid is characterized by the presence of degenerate neutrophils and bacteria. bacteria are often seen intracellularly within neutrophils. the condition is associated with high morbidity and mortality rates, with survival rates reported between % and %. , , , , the history and clinical signs are often vague and nonspecific but can include abdominal pain, vomiting, lethargy/depression, and anorexia. abdominal pain is an inconsistent finding, being recognized in only % of cats in one study and % in another. some cats may have an inappropriately low heart rate. , hematologic and serum biochemistry findings are also inconsistent; neutrophilia with a left shift may be present, as may neutropenia or a normal neutrophil count. similarly, cats may be hypoglycemic, hyperglycemic, or normoglycemic, and they may be hypoalbuminemic. , , one study recognized ionized hypocalcemia in % of cats with septic peritonitis at the time of diagnosis, and another suggested hyperlactatemia, when present, may be associated with a poorer prognosis. radiographic findings are usually typical of ascites of any cause, but presence of pneumoperitoneum in a cat that has not undergone recent surgery may suggest the presence of gas-forming bacteria or rupture of an abdominal viscus and warrants immediate surgical intervention. ultrasonography does not directly aid the diagnosis of septic peritonitis. exploratory laparotomy to determine and correct an underlying problem, such as full-thickness gastrointestinal perforation (often requiring partial resection) is required, as is copious abdominal lavage with sterile, warmed fluids ( figure - ). there are no statistically significant survival differences between postsurgical primary closure, open peritoneal drainage, or closed suction drainage postsurgical lavage; however, a trend toward a higher survival rate has been seen in cats treated with primary closure. , treatment also involves antibiotics, initially parenterally, based on culture and sensitivity findings. consistent with intestinal contents, most bacteria recognized are gram-negative aerobes, such as e. coli or enterobacter spp., but mixed infections are usually found. , anaerobes seem more common in cats with primary septic peritonitis, which perhaps suggests these cases may result from healed over-bite wounds into the abdomen. amoxicillin/ clavulanate would be an appropriate empirical choice of figure - fulminant peritonitis associated with gastrointestinal perforation. in this case, the effusion volume was low but the high degree of serosal inflammation is evident. antibiotics while awaiting sensitivity results. there are no definitive guidelines for duration of antibiotic treatment; the author uses extended treatment courses of to weeks. supportive care with intravenous fluids to maintain fluid and electrolyte balances is also required perioperatively. bile peritonitis is infrequently reported in cats but has been recognized in association with gunshot or motor vehicle trauma, with biliary obstruction from gall stones , and subsequent to percutaneous ultrasoundguided cholecystocentesis in a cat with infectious cholangitis. concurrent bacterial infection was recognized in each case; this increases severity of inflammation and worsens the prognosis, although full recovery was achieved in most reported cases. , , bile peritonitis has the potential to result in small-volume effusions; so, if abdominocentesis does not yield a sample of effusion but bile peritonitis is high on the differential list, then diagnostic peritoneal lavage is appropriate. since repair of or removal of the gall bladder and abdominal lavage are required, exploratory laparotomy is an appropriate means to diagnose this condition. management should be considered as for septic peritonitis of other causes. trauma, including blunt abdominal trauma, urethral catheterization, and bladder expression, is the most common cause of uroperitoneum in cats. it is also recognized as a complication of ureteral surgery. the bladder is the most frequent site of urine leakage after blunt abdominal trauma, whereas the urethra is most commonly injured following catheterization. cats with ruptured bladders may still have a palpable bladder and the ability to urinate. common historical complaints are anuria ( . %) and vomiting ( %). azotemia is a common finding, and hyperkalemia is seen in around % of cases. drainage of urine from the peritoneal cavity seems to improve patient stabilization. morbidity and mortality depended largely on the severity of associated injuries. regardless of the site of injury or the cause of uroabdomen, the first goal of treatment is patient stabilization. isotonic replacement fluids are used for initial resuscitation. treatment of hypovolemic shock, if present, is the first order of fluid therapy. after fluid resuscitation, drainage of urine from the abdomen should be established. continuous passive drainage of the urine is necessary for stabilization and allows effective diuresis to occur. indwelling catheterization of the urinary bladder is recommended to keep the bladder decompressed and reduce urine flow into the abdominal cavity in patients with bladder and proximal urethral injury. if the urethra is traumatized and a catheter cannot be placed, prepubic tube cystostomy may be necessary to achieve temporary urinary diversion. the decision to treat the uroabdomen patient surgically or conservatively should be based on the location and severity of the underlying injury, the condition of the patient at presentation, and the patient's response to initial stabilization. , congestive heart failure has become an uncommon cause of ascites in cats since the late s/early s, from which time dilated cardiomyopathy has been largely eradicated. , ascites does still result from rightsided congestive heart failure in conditions such as tricuspid insufficiency, arrhythmogenic right ventricular cardiomyopathy, myocardial fibrofatty infiltration, or restrictive cardiomyopathy. , concurrent pleural effusion or pulmonary edema is often, but not necessarily, present with cardiac induced ascites. a heart murmur is not necessarily noted. noting a jugular pulse or thrill is helpful diagnostically, if present. the ascitic fluid is typically a modified transudate, but a chylous effusion is also possible. cardiac diseases are covered in chapter . in some cases, hepatic lipidosis has been reported to cause ascites, particularly in association with pancreatitis. these cats are often hypoalbuminemic, with the possibility of intravenous fluid therapy contributing to the ascites by raising hydrostatic pressure. other liver diseases which can result in ascites include lymphocytic cholangitis, , neutrophilic cholangitis, cirrhosis, necrosis, neoplasia, and suppurative cholangiohepatitis. portosystemic shunts in cats rarely result in ascites, compared with dogs. hypoalbuminemia and hepatic failure result in transudates; portal hypertension and cirrhosis cause higher protein ascites because of raised capillary hydrostatic pressure causing leakage of high protein 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peritonitis: cases spontaneous hemoperitoneum in cats: cases primary bacterial peritonitis in dogs and cats: cases a review of the pathophysiology, classification, and analysis of canine and feline cavitary effusions feline hepatic lipidosis uroabdomen in dogs and cats chylous ascites in cats: nine cases congestive heart failure and atrial fibrillation in a cat with myocardial fibrofatty infiltration comparison of different tests to diagnose feline infectious peritonitis arrhythmogenic right ventricular cardiomyopathy in two cats retrospective study: ionized calcium concentrations in cats with septic peritonitis: cases management and outcome of cats with ureteral calculi: cases progressive lymphocytic cholangitis in the cat surgical treatment of bile peritonitis in dogs and cats: a retrospective study feline hemoperitoneum cases ( - ) duplex gall bladder associated with choledocholithiasis, cholecystitis, gall bladder rupture and septic peritonitis in a cat the cytologic examination of body cavity fluids a retrospective study of surgically treated cases of septic peritonitis in the cat chronic lymphocytic cholangitis in three cats primary bacterial septic peritonitis in cats: cases pneumoperitoneum in dogs and cats: cases chylous abdominal effusion in a cat with feline infectious peritonitis restrictive cardiomyopathy in a cat with hypereosinophilic syndrome kirk's current veterinary therapy xii: small animal practice feline infectious peritonitis: a review of clinicopathological changes in cases, and a critical assessment of their diagnostic value differential diagnosis of ascites in cats abdominal paracentesis and diagnostic peritoneal lavage peritoneal effusion in cats: cases ( - ) *when present for any length of time, a pure transudate will become modified. this is particularly true of transudates that develop slowly, such as those associated with congestive heart failure or portal hypertension. modified transudates are therefore more common than pure transudates. adapted from tasker s, gunn-moore d: differential diagnosis of ascites in cats, in practice : , . key: cord- -tvqpv fp authors: corrin, bryan; nicholson, andrew g. title: occupational, environmental and iatrogenic lung disease date: - - journal: pathology of the lungs doi: . /b - - - - . - sha: doc_id: cord_uid: tvqpv fp nan in practice the term is confined to the effects of mineral dust on the lungs. diseases caused by organic dusts are not included among the pneumoconioses and, in medicolegal practice at least, the presence of dust alone is insufficient to indicate pneumoconiosis: for compensation to be considered, the mineral dust must alter the structure of the lung and cause disability. the british industrial injuries advisory council defined pneumoconiosis as 'permanent alteration of lung structure due to the inhalation of mineral dust and the tissue reactions of the lung to its presence, excluding bronchitis and emphysema' . parkes recommends that cancer and asthma caused by mineral dust should also be excluded from the definition, an opinion with which we concur. to reach the lung, dust particles have to be very small. particle density and shape also affect the aerodynamic properties of dust. host factors such as airflow characteristics, airway branching patterns and airway disease also affect dust deposition. three deposition mechanisms are recognised ( fig. . . ): . inertial impaction: when air streams change direction or velocity, the inertia of the entrained particles causes them to maintain their original direction for a distance that depends upon their density and the square of their diameter. the same rules govern a car approaching a bend too fast: the car crashes into the outside of the bend. . sedimentation (gravitational settlement): under the influence of gravity, particles settle with a speed that is proportional to their density and the square of their diameter. . diffusion: very small airborne particles acquire a random motion as a result of bombardment by the surrounding gas molecules. inhaled dust particles are liable to sediment out in the alveoli if they have a diameter in the range of - µm, are roughly spherical in shape, and in density approximate to that of water. larger or denser particles impact or precipitate on the walls of the conductive airways and are rapidly removed by ciliary action. smaller particles may reach the alveoli but do not sediment so readily and many are therefore exhaled. very small particles are deposited on the walls of alveoli by diffusion but because they are so small the total amount of dust deposited in this way is insignificant compared with that deposited by sedimentation ( fig. . . ). direct measurement shows that most lung dust ( %) has a particle diameter less than . µm. fibrous dust particles behave differently. fibres over µm in length may reach the alveoli if they are very thin and remain aligned with the air stream. fibre penetration is inversely related to path length and the number of bifurcations. tall people have longer conductive airways and experience more deposition in these sites than short people who have greater alveolar deposition for the same level of exposure. slightly more dust is deposited in the right lung than the left, probably because the right main bronchus is more in line with the trachea, and is broader and shorter than the left, and carries % of the inhaled air. , dust clearance from the lung inhaled dust that settles in the conductive airways is removed within a day or two by ciliary action. only dust that reaches the alveoli is liable to cause pneumoconiosis and much of this is also removed, but the clearance rate here is much slower: many coalminers continue to expectorate mine dust years after retirement. alveolar clearance is gravity largely effected by macrophages, principally via the airways to the pharynx but also via lymphatics to the regional lymph nodes. the airway and interstitial routes interconnect at the bronchiolar level where some dust-laden macrophages leave the interstitium for the air space. this interconnection is probably the route utilised by circulating macrophages clearing other parts of the body of endogenous or exogenous particulate matter via the lung. long asbestos fibres present a particular problem to macrophage clearance. some minerals, notably chrysotile asbestos, undergo slow physicochemical dissolution in the lungs. only a small fraction of the inhaled dust gains access to the interstitium, a necessary step if it is to cause pneumoconiosis. some free dust enters through the bronchus-associated lymphoid tissue , and some is taken up by, or pierces, the alveolar epithelium ( fig. . , p. ). [ ] [ ] [ ] some of this is transported within hours to the hilar lymph nodes. so rapid is this translocation that it is thought not to involve most, the lesions are more numerous and better developed in the upper lobes than the bases but the reverse is true of asbestosis. the reasons for this are complex but undoubtedly involve the dust deposition:clearance ratio for the effect of the dust will depend upon both its amount and the duration of its stay in the lungs. there are well-recognised regional differences in the distribution and clearance of inhaled material, which in turn are dependent upon man's upright posture, the consequent gravitational forces being maximal at the apices. when standing at rest, the apices of the lungs are hardly perfused, so that lymph formation and clearance are much better at the bases. [ ] [ ] [ ] similarly, the apices are relatively less well aerated; alveoli in the lower lobes receive more air than those in the upper lobes. , the greater respiratory excursions at the bases are thought to promote macrophage mobility there. it is to be expected therefore that the bases would both receive and clear more dust than the apices, rendering it difficult to predict on theoretical grounds which parts of the lungs carry the heaviest dust burden. in fact, more dust of all types is found in the upper lobes, the part most severely affected by every type of pneumoconiosis except asbestosis. , the predilection of asbestos to affect the periphery of the lower lobes is attributed to the dangerous long asbestos fibres preponderating there. , pulmonary reactions to mineral dust the main tissue reaction to mineral dust is fibrosis. silica is highly fibrogenic and is therefore very likely to cause pneumoconiosis. carbon is non-fibrogenic and therefore, unless there are complications, coal pneumoconiosis causes little disability. tin too is harmless, and stannosis therefore unimportant, although the chest radiograph is highly abnormal because tin is very radiopaque. stannosis is one of several terms that specify pneumoconiosis due to a particular mineral, the best known being silicosis, asbestosis and anthracosis. the blackness of carbon and red-brown colour of iron give ample evidence, both naked-eye and microscopically, of the type and amount of these dusts when they are present in the lung ( fig. . . ), but other inorganic dusts may be more difficult to identify. however, a flick-out substage condenser and polaroid filters to test for refractility and birefringence respectively are useful adjuncts that are too often neglected by the histopathologist. crystalline silica is traditionally regarded as being only weakly birefringent, in contrast to silicates which generally show up brightly with simple crossed polaroid filters. however, with modern microscope lamps, if the light source is set at high intensity when using polaroid filters, both silica and silicates are birefringent. mineralogists use polarising microscopy for analysis, but only by studying large polished crystals with controlled orientation of the light. the small dust particles found in tissue sections are too small to permit analysis by this technique but it is nevertheless very useful for detecting their presence ( fig. . . ) . particle shape gives a useful indication of mineral type but appearances are sometimes deceptive: the plate-like crystals of talc are seldom observed as such, usually being viewed edge-on, when they appear to be needle-shaped. occasionally, stains can be used to identify minerals, e.g. a modified perls' reaction for inhaled iron, and irwin's aluminon stain for aluminium, but these too have largely been replaced by modern analytical techniques. ultrafine dust particles are particularly liable to be transported across the alveolar epithelium. the integrity of the alveolar epithelium is very important to dust translocation from the air spaces to the interstitium. much more dust reaches the interstitium if the epithelium is damaged. , it is widely thought that macrophages that have left the interstitium for the alveolar space never return, , but this is probably untrue. heavily laden macrophages accumulate in alveoli bordering the terminal and respiratory bronchioles, eventually filling them completely. erosion of the alveolar epithelium permits re-entry of these macrophages into the interstitium, very close to foci of bronchial mucosaassociated lymphoid tissue (malt), which are found near the terminal bronchioles. these aggregates guard the mouths of lymphatics, which commence at this point; alveoli are devoid of lymphatics. dustladen interstitial macrophages accumulate in and around the bronchial malt, which macklin therefore referred to as dust sumps. most pneumoconiotic lesions are found in the region of the dust sumps and are therefore focal. asbestosis is diffuse rather than focal because the long asbestos fibres are not readily mobilised and cannot be concentrated in the centriacinar dust sumps. this is also seen on occasion with platy non-fibrous dusts such as talc, mica, kaolinite and feldspar. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] within the dust sumps the dust particles are not static. they are constantly being freed and reingested by interstitial macrophages and, because these cells are mobile, successively inhaled dusts soon become intimately mixed. macrophages play an important role in pneumoconiosis and if the dust is fibrogenic the repeated phagocytosis of indestructible mineral particles results in constant fibroblast stimulation. the zonal distribution of pneumoconiosis pneumoconiosis affects both lungs but seldom evenly and some pneumoconioses show characteristic patterns of lung involvement. in microincineration combined with dark-field microscopy can also be used to demonstrate small particles. incombustible mineral particles that cannot be seen with bright-field or polarising microscopy are rendered visible by this technique and their position on the slide can be compared with tissue reactions evident in a serial section that has not been incinerated. microincineration has, however, also been largely replaced by modern analytical techniques that will now be considered. analytical electron microscopy is very helpful in identifying minerals, whether applied to lung digests or tissue sections. [ ] [ ] [ ] [ ] scanning electron microscopy permits the examination of thicker sections than transmission electron microscopy but does not detect very small particles. however, scanning electron microscopy allows more tissue to be examined and avoids the difficulty of cutting mineral particles with an ultramicrotome. mineral particles in a -µm thick deparaffinised section can be recognised in a scanning electron microscope set to collect the back-scattered electrons. the instrument can then be focused on points of potential interest and switched to x-ray diffraction, which provides information on crystal structure (fig. . . ) . alternatively, elemental analysis may be undertaken with either energy-dispersive or wavelength-dispersive x-ray spectroscopy. with energy-dispersive x-ray spectroscopy, all elements of atomic number above are identified, whilst with wavelength-dispersive x-ray spectroscopy the section can be scanned for one particular element. with the former technique different elements are shown graphically as individual peaks, the heights of which are proportional to the amounts of the different elements within the particle studied, thereby giving information on probable molecular formula ( fig. . . ) . thus, different silicates can be distinguished from each other and also from silica, which registers as pure silicon, oxygen (atomic number ) not being detected. the fact that the elements of low atomic number that constitute organic chemicals are not detected means that any minerals present (except beryllium, atomic number ) can be recognised easily in tissue sections. only particles can be analysed however: elements present in only molecular amounts cannot be detected by x-ray analysis. the detection of trace amounts of substances such as beryllium requires bulk chemical analysis or techniques that are not widely available such as atomic absorption spectrometry, neutron activation analysis and microprobe mass spectrometry. , the last of these techniques can also provide molecular (as opposed to elemental) analysis of organic as well as inorganic particles. another analytical technique of interest is microscopic infrared spectroscopy which provides data on the compound nature of microscopic particles in tissue sections ( fig. . . ) . micro-raman spectroscopy is also useful in this respect. some metals cause hypersensitivity, which can be identified by exposing the patient's lymphocytes to metals and measuring their reaction in vitro. radiological grading of pneumoconiosis a scheme for grading pneumoconiosis radiologically by comparison with standard radiographs has been adopted by the international labour organisation (ilo) and is widely used. small opacities (up to cm diameter) are graded by their profusion, , and indicating increasing numbers, and by their size, increasing through p, q and r if rounded and s, t and u if irregular. type p opacities are described as punctiform and measure up to . mm in diameter; larger lesions up to mm in diameter (type q) are described as micronodular or miliary; and those over mm and up to cm in diameter (type r) are described as nodular. irregular opacities cannot be sized so accurately, s, t and u indicating fine, medium and coarse respectively. large opacities (over cm diameter) are graded by their combined size, increasing through a, an opacity measuring between and cm in diameter; b, one or more opacities whose combined area does not exceed the equivalent of one-third of the area of the right lung field (when they are regrouped in the mind's eye or measured with a transparent ruler); and c, one or more opacities whose combined area exceeds one-third of the area of the right lung field (when similarly regrouped). in coalworkers, small opacities (up to cm diameter) correspond to simple coalworker's pneumoconiosis and large opacities (over cm diameter) to complicated coalworker's pneumoconiosis, which is also known as progressive massive fibrosis. silicotic lesions have been identified in the lungs of egyptian mummies, and the injurious effects on the lungs of inhaling mine dust have been recognised for more than years. as long ago as the sixteenth century in joachimsthal, bohemia (now jachymov, czech republic), diseases of miners' lungs were attributed to the dust the miners breathed. silicosis, tuberculosis and lung cancer are all now known to have been prevalent among the miners in this region, the cancer being largely attributable to the high level of radioactivity in the mines. silicosis was recognised in the uk soon after the discovery in that the addition of calcined flint to the clay from which china is made produced a finer, whiter and tougher ware. the preparation and use of this flint powder were highly dangerous, causing the condition known as potter's rot, one of the first of the many trade names by which silicosis has since been known. aluminium oxide (alumina) now provides a safe, effective substitute for flint in this industry. in it was noted that sheffield fork grinders who used a dry grindstone died early, and amongst other preventive measures it was recommended that the occupation should be confined to criminals: fortunately for them, the substitution of carborundum (silicon carbide) for sandstone was effective enough. however, silicosis still occurs in some miners, tunnellers, quarrymen, stone dressers and metal workers. silica in one form or another is used in many trades -in the manufacture of glass and pottery, in the moulds used in iron foundries, as an abrasive in grinding and sandblasting, and as a furnace lining that is refractory to high temperatures. rocks such as granite and sandstone are siliceous and their dusts are encountered in many mining and quarrying operations. in coal mining in the uk the highest incidence of the disease was in pits where the thinness of the coal seams required the removal of a large amount of siliceous rock, a process known as 'hard heading' . in south africa, silicosis causes a high mortality among the gold miners on the witwatersrand, where the metallic ore is embedded in quartz. slate is a metamorphic rock that contains both silica and silicates, and slateworkers develop both silicosis and mixed-dust pneumoconiosis. , nor are rural industries immune from the disease, particularly if ventilation is inadequate, as it is in certain african huts where stone implements are used to pound meal and the occupants develop mixed-dust pneumoconiosis. silicosis and mixed-dust pneumoconiosis have also been reported in dental technicians. desert sand is practically pure silica but the particles are generally too large to reach the lungs. however, silicosis has been reported in inhabitants of the sahara, libyan and negev deserts and those living in windy valleys high in the himalayan mountains, [ ] [ ] [ ] [ ] [ ] [ ] [ ] whilst in california the inhalation of dust raised from earth has led to silicate pneumoconiosis in farm workers, horses and a variety of zoo animals. the silica in rocks such as granite, slate and sandstone is largely in the form of quartz and this is therefore the type of silica encountered in most of the industries considered above. cristobalite and tridymite, which are possibly even more fibrogenic than quartz, are more likely to be encountered in the ceramic, refractory and diatomaceous earth industries where processing involves high temperatures. many workers with silicosis are asymptomatic. as a general rule, exposure to silica dust extends over many years, often or more, before the symptoms of silicosis first appear: by the time the disease becomes overt clinically, much irreparable damage has been inflicted on the lungs. the initial symptoms are cough and breathlessness. from then onwards, respiratory disability progresses, even if the patient is no longer exposed to silica dust. ultimately, there may be distressing dyspnoea with even the slightest exercise. silicosis sometimes develops more rapidly, perhaps within a year or so of first exposure. such 'acute silicosis' was observed in the scouring powder industry in the s when these cleansing agents consisted of ground sandstone mixed with a little soap and washing soda. , the additives were considered to have rendered the silica in the sandstone more dangerous but it is possible that the rapidity of onset of the disease merely reflected the intensity of the dust cloud to which the packers were exposed. confusingly, the term 'acute silicosis' has since been applied to a further effect of heavy dust exposure in tunnellers, sand blasters and silica flour workers, namely pulmonary alveolar lipoproteinosis (see below), , whilst the terms 'accelerated silicosis' or 'cellular phase silicosis' have been substituted for 'acute silicosis' in referring to the rapid development of early cellular lesions. , the time from first exposure to the development of symptoms (the latency period) is inversely proportional to the exposure level. however, it is evident that a certain amount of silica can be tolerated in the lungs without fibrosis developing, indicating either a time factor in the pathogenetic process or a threshold dust load that has to be reached before fibrosis develops. silica particles that are roughly spherical in shape and of a diameter in the range of - µm sediment out in the alveoli and are concentrated within macrophages at macklin's dust sumps, as explained previously (see p. ). early lesions, as seen in so-called accelerated or cellular phase silicosis, consist of collections of macrophages separated by only an occasional wisp of collagen. the early lesions have been likened to granulomas and on occasion have been mistaken for langerhans cell histiocytosis or a storage disorder, but langerhans cells are scanty and the histiocytes contain dust particles rather than accumulated lipid or polysaccharide. the macrophages of the early lesion are gradually replaced by fibroblasts and collagen is laid down in a characteristic pattern. the mature silicotic nodule is largely acellular and consists of hyaline collagen arranged in a whorled pattern, the whole lesion being well demarcated ( fig. . . ) and sometimes calcified. small numbers of birefringent crystals are generally evident within the nodules when polarising filters are used, but these mainly represent silicates such as mica and talc, inhaled with the silica. silica particles are generally considered to be only weakly birefringent, but fairly strong birefringence is evident in strong light (see above). silicotic nodules develop first in the hilar lymph nodes and are generally better developed there than in the lungs. [ ] [ ] [ ] indeed, silicotic nodules are occasionally found in the hilar lymph nodes of persons who have no occupational history of exposure to silica and whose lungs are free of such lesions, the silica in the nodes being presumed to represent inhaled particles derived from quartz-rich soil. severely affected lymph nodes often calcify peripherally, giving a characteristic eggshell-like radiographic pattern. this is sometimes the only radiological abnormality. such enlarged lymph nodes may occasionally press upon and obstruct adjacent large bronchi or result in a left recurrent laryngeal nerve palsy, so simulating malignancy. sometimes the nodules develop within the walls of major bronchi, occasionally causing a middle-lobe syndrome (see p. ). silicotic nodules are also found along the lines of the pleural lymphatics , where they have been likened to drops of candle wax on the visceral pleura. very rarely, silica-induced fibrosis is more pronounced in the pleura than in the lungs. lung tissue between the nodules is often quite normal and not until the process is very advanced is there any disability ( fig. . . b). in severe cases large masses of fibrous tissue are formed, which may undergo central necrosis and cavitation ( fig. . . ). on close inspection it is evident that these consist of conglomerations of many silicotic nodules closely packed together. in such severe cases cor pulmonale develops. occasionally, silicotic nodules develop in the abdominal as well as the thoracic lymph nodes, and in the liver, spleen, peritoneum and bone marrow. [ ] [ ] [ ] [ ] [ ] in about % of cases, the typical pulmonary nodules that predominantly affect the upper lobes are accompanied by diffuse fibrosis that is maximal in the lower lobes. , , [ ] [ ] [ ] the latter may show 'honeycombing' and closely resemble idiopathic pulmonary fibrosis. the association is too common to be explained by chance and the diffuse fibrosis is therefore regarded as a further manifestation of the pneumoconiosis, possibly due to an interaction between the dust and the immunological factors discussed below. the pathogenesis of silicosis has excited much interest and many different theories have been advanced over the years. an early theory held that the hardness of the silica was responsible, but this was discounted by the observation that silicon carbide (carborundum) is harder than silica but is non-fibrogenic. theories based on the piezoelectric property and on the solubility of silica were successively abandoned although the latter had a long period of popularity. it gained support from kettle's experiments which showed that fibrosis developed about chambers placed in an animal's peritoneal cavity if the chambers contained silica powder sealed in by a collodion membrane through which solutes such as silicic acid could pass. however, it was later shown that the pores in a collodion membrane are quite irregular in size and when the experiments were repeated using chambers guarded by millipore membranes, no fibrosis developed, despite solutes being able to diffuse out. the solubility theory also fails to take account of the differing fibrogenicity of the various forms of silica despite them being of similar solubility. furthermore, if the outer, more soluble layer of the particles is removed by etching, fibrogenicity is increased although solubility is decreased. in line with this, freshly fractured crystalline silica is more pathogenic in every respect than its aged equivalent, which may partly explain the severity of silicosis in trades such as sandblasting. these observations suggest that the fibrogenicity of silica is connected with its surface configuration. it is now known that uptake of the silica by macrophages is necessary for silicosis to develop. if silica and macrophages are enclosed together in peritoneal millipore chambers, a soluble product of the macrophages diffuses out and causes fibrosis. this observation led to the realisation that the fibrogenicity of the various crystalline forms of silica correlated well with their toxicity to macrophages and for a time macrophage death was thought to be necessary. it is now considered that before the macrophages are killed by the ingested silica, they are stimulated to secrete factors that both damage other con stituents of the lung and promote fibrosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] transforming growth factor-β is one fibrogenic factor that has been implicated in the pathogenesis of silicosis. [ ] [ ] [ ] toxic damage to macrophages is due to silica particles injuring the phagolysosomal membranes, so releasing acid hydrolases into the cytoplasm. it is important in the pathogenesis of the disease indirectly because when the macrophage crumbles, the silica particles are taken up by fresh macrophages and the fibrogenic process continues. it has been suggested that early involvement of the hilar lymph nodes in the fibrogenic process promotes the development of the disease in the lung by delaying dust clearance. immunological factors have been implicated in the pathogenesis of silicosis because many patients with silicosis have polyclonal hypergammaglobulinaemia, rheumatoid factor or antinuclear antibodies, and because there is a well-recognised association between autoimmune diseases such as systemic sclerosis and rheumatoid disease and exposure to silica. , [ ] [ ] [ ] [ ] the relation of immunity to dust exposure appears to be a reciprocal one: on the one hand, the presence of dust results in rheumatoid lesions in the lungs being more florid (see caplan's syndrome, p. ), whilst on the other, non-specific immunisation of rabbits with horse serum results in experimental silicotic lesions being larger and more collagenous. it is doubtful whether pneumoconiosis and autoimmune disease play a causative role in each other but one seems to aggravate the other and may lead to its earlier development. one of the commonest and most feared complications of silicosis is chronic respiratory tuberculosis. once this infection has been added to the silicosis, the prognosis rapidly worsens. it is thought that in the presence of silica, the tubercle bacilli proliferate more rapidly because the ingested silica particles damage phagolysosomal membranes and thereby interfere with the defensive activity of the macrophages. the synergistic action of silica dust has long been held responsible for the inordinately high incidence of respiratory tuberculosis in mining communities. many former south african gold miners now have acquired immunodeficiency syndrome (aids) as well as silicosis and tuberculosis has consequently reached almost epidemic proportions amongst these men. phagocyte damage by ingested dust particles may also cause some cases of chronic necrotising aspergillosis complicating pneumoconiosis. a series of studies suggesting that there might be a link between silica inhalation and lung cancer was reviewed by the international agency for research on cancer in , leading to the conclusion that the evidence for carcinogenicity of crystalline silica in experimental animals was sufficient, while in humans it was limited. subsequent epidemiological publications were reviewed in , when it was concluded that the epidemiological evidence linking exposure to silica to the risk of lung cancer had become somewhat stronger but that in the absence of lung fibrosis remained scanty. the pathological evidence in humans is also weak in that premalignant changes around silicotic nodules are seldom evident. nevertheless, on this rather insubstantial evidence, lung cancer in the presence of silicosis (but not coal or mixed-dust pneumoconiosis) has been accepted as a prescribed industrial disease in the uk since . some subsequent studies have provided support for this decision. in contrast to the sparse data on classic silicosis, the evidence linking carcinoma of the lung to the rare diffuse pattern of fibrosis attributed to silica and mixed dusts is much stronger and appears incontrovertible. , alveolar lipoproteinosis in response to heavy dust exposure a further complication of exposure to silica is the development of alveolar lipoproteinosis (see p. ). , , , very heavy experimental exposure to silica, and indeed other dusts, stimulates hypersecretion of alveolar surfactant to such an extent that the normal clearance mechanism is overwhelmed. [ ] [ ] [ ] [ ] [ ] [ ] [ ] alveolar macrophages are enlarged by numerous phagolysosomes distended by lamellar bodies that represent ingested surfactant. the alveoli are filled by such cells and, having a foamy cytoplasm, they produce the appearances of endogenous lipid pneumonia, similar to that more usually encountered as part of an obstructive pneumonitis distal to a bronchial tumour. the macrophages gradually disintegrate and the free denatured surfactant slowly becomes compacted, during which time its staining with both eosin and the periodic acid-schiff reagents intensifies until the appearances are finally those of alveolar lipoproteinosis. this process prevents the aggregation and concentration of the dust in the usual foci and thereby hinders the development of silicosis. lipoproteinosis and silicosis may be seen in conjunction but, more often, different areas of the lung show one or the other. the lipoproteinosis has its own severe impact on lung function, but, unlike silicosis, is potentially reversible (by massive alveolar lavage). occasional patients exposed to silica develop renal disease. [ ] [ ] [ ] [ ] two mechanisms appear to operate. first, translocation of silica particles from the lungs leads to their deposition in the renal interstitium with resultant nephrotoxity. second, silica stimulates an autoimmune response characterised by the formation of various antibodies, notably rheumatoid factor and antinuclear antibodies, which leads to the development of immune complex-mediated glomerulonephritis. , amorphous silica manmade submicron forms of silica, variously known as amorphous, vitreous, colloidal, synthetic or precipitated silica, are widely used in industry. they consist of pure non-crystalline silicon dioxide. particle size ranges from to nm but aggregates of the particles measure from to µm. industrial surveys suggest that inhalation of such dust is harmless, observations that are in accord with the results of animal experiments. an amorphous silica is the principal component of the fossilised remains of diatoms that constitute the sedimentary rock, diatomite ( fig. . . ). this is generally obtained by open-cast mining, following which the rock is crushed and calcined. the calcined product is used in filters, insulation material and as a filler. being amorphous, the silica in diatomite is harmless, but calcining (> °c) results in its conversion to crystalline forms of silica. diatomaceous earth pneumoconiosis is unusual and its risk appears to be related to the amount of cristobalite and tridymite (two forms of crystalline silica) produced in the calcining process. the silicates are complex compounds in which silicon and oxygen form an anion combined with cations such as aluminium and magnesium: talc, for example, is a hydrated magnesium silicate with the formula mg si o (oh) . silicates include fibrous forms (asbestos and the zeolites), plate-like forms (talc and mica) and clays (kaolinite and fuller's earth). in histological sections, the platy talc and mica particles are generally cut tangentially and therefore appear needleshaped (see fig. . . ). they are strongly birefringent whereas the clays are only weakly so. talc particles in the lung exceeding µm in length should arouse suspicion of intravenous drug abuse. of the fibrous silicates, zeolite is used as a building material in certain communities, notably in central turkey. pneumoconiosis is not a problem but zeolites are of medical interest because, like asbestos, they present a mesothelioma risk. asbestos is dealt with separately (see below). pneumoconiosis has been described with various non-fibrous silicates, notably in the rubber industry, which uses talc and, less commonly, mica as lubricants. other occupations posing a risk include the extraction of kaolinite from china clay (kaolin), , , and in the open-cast and underground mining of fuller's earth (montmorillonite, bentonite and attapulgite clays, which were originally used in 'fulling' (degreasing) wool). , however, all these substances are commonly contaminated with silica, asbestos or both, and it has been questioned whether in pure state they are at all fibrogenic. the modifying effect of inert substances such as iron on that of silica is well known (see mixed-dust pneumoconiosis, below) and it has been suggested that talc, mica and fuller's earth act in a similar way in regard to their more fibrogenic contaminants, the pneumoconioses attributed to them in reality representing mixed-dust pneumoconiosis or asbestosis. contrary evidence comes from reports of pulmonary fibrosis in persons heavily exposed to pure talc, mica or kaolin. all these silicates are evident in the tissues as plate-like birefringent crystals which often provoke a foreign-body giant cell reaction (see fig. . . ) and may result in fibrotic nodules. large focal lesions resembling the progressive massive fibrosis of coalworkers may be produced, and also a diffuse 'asbestosis-like' form of pneumoconiosis, the latter attributed to poor macrophage mobilisation of the plate-like particles. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it would appear therefore that silicates are indeed fibrogenic if enough is inhaled; they appear to vary in fibrogenicity but in all cases they are less fibrogenic than silica. inert dusts are non-fibrogenic and therefore of little clinical consequence, although elements of high atomic number can give rise to a striking chest radiograph. it should be noted however that inert or lowly fibrogenic materials may be associated with substances of medical importance, for example, kaolin, bentonite and barytes (barite) may all be contaminated with silica , , and talc may be contaminated with asbestos. the best known of the inhaled inert mineral dusts is carbon while, of the remainder, iron is the most widespread. others include tin and barium. with all these dusts, particles retained in the lung are gathered at macklin's dust sumps by heavily laden macrophages which are lightly bound together there by a few reticulin fibres. collagen is not formed and the worker suffers no ill-effects. the lungs take on the colour of the dust and in siderosis assume a deep brick-red hue. carbon deposition is commonly found in the lungs, particularly those of city dwellers and tobacco smokers. it is also the principal constituent of coal, which is dealt with separately below, and large amounts of pure carbon may be inhaled by workers involved in the manufacture of carbon black, carbon electrodes and charcoal. [ ] [ ] [ ] [ ] although carbon is regarded as being non-fibrogenic, the very heavy lung burdens encountered in industries such as these may lead to the complicated form of pneumonconiosis known as progressive massive fibrosis that is more commonly encountered in coal workers (see p. ). heavy pure carbon deposition may also be acquired domestically when wood is burnt in buildings devoid of a chimney, so-called 'hut lung', a term that is also applied to the domestic acquisition of carbon mixed with silica or silicates, resulting in forms of mixeddust pneumoconiosis. , , anthracofibrosis is a term introduced by chinese bronchoscopists for bronchial stenosis or obliteration associated with carbon pigmentation of the mucosa. although the original description incriminated tuberculosis, mixtures of various mineral dusts acquired at work or domestically are a more likely cause. [ ] [ ] [ ] [ ] iron dust in the lungs was first described by zenker in , when he also introduced the terms siderosis and pneumonokoniosis. zenker was describing a woman who coloured paper with iron oxide powder ('rouge'), a substance which is still encountered by some workers engaged in polishing silver, glass, stone and cutlery. siderosis is also found in welders, iron foundry fettlers, steel workers, boiler scalers and haematite miners and crushers. iron dust particles are reddish-brown but in the lung may be masked by carbon : when evident, or revealed by microincineration, they resemble haemosiderin and generally give a positive perls' reaction, but particularly with haematite, heat ( - o c) and concentrated ( n) hydrochloric acid may be necessary. haematite miners in both the uk (cumbria) and france (lorraine) have an increased risk of bronchial carcinoma, but radon gas rather than haematite is the suspected carcinogen. radon is a decay product of uranium. minute amounts are present in all rocks but local concentrations occur and these are liable to build up in mines if ventilation is limited. silver, as well as iron, is found in the lungs of silver polishers, where it stains elastin in alveolar walls and pulmonary vessels grey. such argyrosiderosis is as harmless as siderosis. tin miners are subject to silicosis but not stannosis because the ore, which is found in association with siliceous rocks, contains only low concentrations of the metal. tin smelters, on the other hand, and factory workers exposed to high concentrations of tin dust or fume, are liable to inhale large amounts of this inert metal and develop the striking chest radiograph of stannosis. they remain in good health however for tin is completely non-fibrogenic. tin particles in the lung resemble carbon but are strongly birefringent and remain after microincineration: microprobe analysis provides positive identification. other inert dusts include barium, which also has a high atomic number and is therefore radiopaque, and minerals of low radiodensity such as limestone, marble and cement (all chiefly composed of calcium carbonate) and gypsum (hydrated calcium sulphate). however, the extraction of barium ore (almost entirely in the form of barium sulphate, which is known as barytes in europe and barite in the usa) may entail exposure to silica and silicates. pure baritosis resembles stannosis and siderosis. the term 'mixed-dust pneumoconiosis' refers to the changes brought about by inhaling a mixture of silica and some other less fibrogenic substance such as iron, carbon, kaolin or mica. , , [ ] [ ] [ ] the proportion of silica is usually less than %. typical occupations include foundry work and welding and the mining of coal, haematite, slate, shale and china clay. the action of the silica is modified and, although fibrotic nodules are formed, they lack the well-demarcated outline and concentric pattern of classic silicosis. the lesions are found in a centriacinar position and are stellate in outline with adjacent scar emphysema. they are firm and generally measure no more than mm in diameter. they closely resemble the fibrotic nodules of simple coal pneumoconiosis (see below). confluent lesions also occur on occasions. these resemble the progressive massive fibrosis of coalworkers and appear to represent a single large lesion rather than a conglomeration of individual nodules, as in advanced silicosis. abundant dust is generally evident in lesions of all sizes; this consists of black carbon or brown iron mixed with crystals of varying degrees of birefringence, silicates generally being strongly birefringent and silica weakly so. calcification is unusual. mixed-dust pneumoconiosis carries an increased risk of pulmonary tuberculosis, but not to the same degree as silicosis. in some cases the stellate nodules are accompanied by diffuse fibrosis, as in silicosis and again possibly involving interactions between the dust and immunological factors. involvement of the bronchi with consequent stenosis (so-called anthracofibrosis) is described above. the term 'anthracosis' was initially applied to changes observed in a coalminer's lung but is now often extended to include the common carbon pigmentation of city dwellers' lungs, and the term 'coal pneumoconiosis' is more appropriate to a special form of pneumoconiosis to which coalworkers are subject, particularly those who work underground. the principal constituent of coal, carbon, is non-fibrogenic, so suspicion has naturally fallen on the ash content of mine dust, some of which derives from the coal, some from adjacent rock strata and some from stone dust laid in the roadways to minimise the risk of coal dust explosions. coal itself appears to be the responsible agent because coal-trimmers, working in the docks and not exposed to rock dust, also develop the disease. coalminers encountering siliceous rock are, of course, also liable to develop silicosis like other underground workers. coal consists largely of elemental carbon, oxygen and hydrogen with traces of iron ore and clays such kaolinite, muscovite and illite, but no silica. the mineral content varies with the type and rank (calorific value) of the coal. all coal derives from peat, the youngest type being lignite and the oldest anthracite, with bituminous (house) coal in between. as it ages, the oxygen and mineral constituents diminish and the coal hardens. lignite is soft and said to be of low rank, anthracite hard and of high rank, with bituminous coal intermediate. although high-rank coal is of low mineral content, its dust is more toxic to macrophages in vitro and is cleared more slowly in vivo. this observation may explain why, in the uk, high-rank coal is associated with a higher prevalence of coal pneumoconiosis. the low mineral content of high-rank coal is reflected in the mineral content of the lungs of those who hew such coal in the uk, but in the ruhr, in germany, and in pennsylvania, in the usa, anthracite miners' lungs contain more silica than those who hew bituminous coal, the silica presumably deriving from other sources. not surprisingly, the presence of silica is reflected in the tissue reaction to the inhaled dust, resulting in a more fibrotic reaction very analogous to mixed-dust pneumoconiosis. a spectrum of changes is therefore encountered in coalminers' lungs, ranging from coal pneumoconiosis through mixeddust pneumoconiosis to silicosis; the findings in any individual depend upon the nature of the coal being mined and the type of work undertaken. in high-rank british collieries the development of coal pneumoconiosis appears to depend on the total mass of dust inhaled, whereas in low-rank british collieries the mineral content of the lung dust appears to be more important. this may explain apparently contrary data drawn from different coalfields -data based on coals of different composition that are not strictly comparable. some workers have stressed the importance of silica in the dust whereas others, particularly in the high-rank coalfields of south wales, have been unable to detect any association between silica and the level of pneumoconiosis. both findings may be correct, but only for the particular group of miners examined in each case. the lesions of coal pneumoconiosis are generally focal and fall into one or other of two major types, simple and complicated, depending upon whether the lesions measure up to or over cm; simple corresponds to categories - of the ilo grading system (see p. ) and complicated, which is also known as progressive massive fibrosis, to ilo categories a-c. more diffuse interstitial fibrosis has been reported in about % of welsh and west virginian coalminers, usually involving those carrying a particularly heavy dust burden; it runs a more benign course than non-occupational interstitial fibrosis (idiopathic pulmonary fibrosis). similar findings have been reported from france. simple coal pneumoconiosis consists of focal dust pigmentation of the lungs, which may be associated with a little fibrosis and varying degrees of emphysema. its clinical effects are relatively minor. some degree of black pigmentation (anthracosis) of the lungs is common in the general urban population, especially in industrial areas, but much denser pigmentation is seen in coalminers, whose lungs at necropsy are black or slate-grey. black pigment is evident in the visceral pleura along the lines of the lymphatics and on the cut surface where it outlines the interlobular septa and is concentrated in macklin's centriacinar dust sumps ( fig. . . ). the dust is generally more plentiful in the upper parts of the lungs and in the hilar lymph nodes, possibly due to poorer perfusion and consequently poorer lymphatic drainage there (see p. ). two forms of coal dust foci are recognised, macules and nodules, the former being soft and impalpable and the latter hard due to substantial amounts of collagen. both lesions are typically stellate but the more fibrotic the nodules, the more rounded they become, until it is difficult to distinguish them macroscopically from those of silicosis. in these circumstances reliance has to be placed on the whorled pattern of the collagen that is evident microscopically in silicosis. the stellate nodules are analogous to those seen in mixed-dust pneumoconiosis caused by mixtures of silica and inert dusts other than carbon (see above). with polarising filters, small numbers of birefringent crystals may be seen in both macules and nodules, usually representing mica or kaolinite derived from rock that bordered the coal. macules consist of closely packed dust particles, free or within heavily laden macrophages, so that the lesion appears black throughout ( fig. . . ). appropriate stains show that the dust-laden macrophages and free dust are lightly bound by reticulin. very little collagen is evident. although striking in their appearance, dust macules are thought to have little effect on lung function. nodules contain substantial amounts of collagen and are thought to have an adverse, but limited, effect on respiration. they vary from a heavily pigmented, stellate lesion, which apart from its collagen content resembles the dust macule ( fig. . . ), to one that is less pigmented and more circumscribed. the stellate, heavily pigmented type of nodule is seen in lungs that have a relatively low ash content whilst the more rounded and less pigmented nodule is seen in lungs with relatively high ash loads. radiologically (see p. ), p-type opacities correspond to macules, q-type opacities to the stellate nodules that resemble those of mixeddust pneumoconiosis and r-type opacities to the rounded nodules that resemble those of silicosis. , thus, the radiological changes of simple coalworker's pneumoconiosis are due to the dust and the small amount of collagen present and do not reflect any emphysema that may also be present. however, pulmonary dust foci are often associated with emphysema ( fig. . . ) and the severity of the emphysema appears to correlate with the dust load. the prevalence of chronic bronchitis and emphysema is high in the coal industry and it has long been debated whether occupation or cigarette smoking is the major factor contributing to emphysema in coalminers. [ ] [ ] [ ] [ ] as well as mineral dust, nitrous fumes from shot-firing form another occupational hazard of coal mining. heppleston made a special study of the emphysema found in coalminers, claiming that it differs from centriacinar emphysema, as seen in smokers in the general population, and attributing it to the dust. he introduced the term 'focal emphysema of coalworkers' to describe this special process. others find it very difficult to identify any convincing difference between the emphysema of coalworkers and that encountered outside the industry but heppleston based his claims on the study of serial sections. by this means he showed that, although both forms affect respiratory bronchioles, the focal emphysema of coalworkers affects more proximal orders of these airways and is not associated with the bronchiolitis seen with centriacinar emphysema. furthermore, focal emphysema is a dilatation lesion whereas coniosis, also known as progressive massive fibrosis, can have very serious consequences. particularly when the lesions are large, it is associated with productive cough, breathlessness, significant impairment of lung function and premature death. the major factor accounting for the development of progressive massive fibrosis appears to be the sheer bulk of coal dust in the lung, rather than coal rank or the silica content of the mine dust. progressive massive fibrosis has occasionally been recorded in dockers loading silica-free coal into the holds of ships and in workers exposed to pure carbon in the manufacture of carbon black and carbon electrodes. [ ] [ ] [ ] progressive massive fibrosis is characterised by large (over cm) black masses, situated anywhere in the lungs but most common in the upper lobes. the lesions may be solitary or multiple and very large, occupying most of the lobe and even crossing an interlobar fissure to involve an adjacent lobe (figs . . b, . . ). they cut fairly easily, often with the release from a central cavity of black fluid flecked by cholesterol crystals. for many years it was believed that the condition was the result of synergism between mycobacterial infection and dust but the failure of the attack rate to decrease as tuberculosis declined negated this view. today, more emphasis is placed on total dust load for the lesions tend to affect lungs that carry an unduly heavy dust burden. if the remainder of the lung shows little evidence of dust accumulation, the possibility of the masses representing caplantype lesions (see below) should be considered. centriacinar emphysema involves destruction of adjacent alveolar walls. by definition, therefore, focal emphysema is not a true emphysema at all (see p. ). however, it has been shown that mineral dusts cause elastin and collagen breakdown in the rat lung. focal emphysema may progress to the destructive centriacinar form and this has strengthened claims that mine dust plays a causal role in centriacinar emphysema. , [ ] [ ] [ ] [ ] [ ] [ ] in the uk, these claims have been accepted and chronic bronchitis and emphysema in coalminers and metal production workers have been accepted as prescribed industrial diseases since . in germany too, chronic obstructive pulmonary disease is now compensatable as an occupational disease. the conditions for compensation in the uk were initially: • underground coal mining for a minimum of years in aggregate • forced expiratory volume in second at least litre below that expected or less than litre in total • radiological category of at least / . however the last of these criteria has now been dropped. the inclusion of a time element and the omission of some estimate of dust load (such as radiological category) have been criticised, with some justification. as with lung cancer caused by chromates benefit is paid irrespective of smoking habits. whereas simple coal pneumoconiosis, particularly the macular variety, has little effect on lung function, complicated coal pneumo- microscopically, the lesions consist of dust and connective tissue intermixed in a random fashion. central necrosis and cavitation commonly occur. the necrosis is thought to be ischaemic. it is amorphous or finely granular, and eosinophilic apart from abundant dust particles and cholesterol crystal clefts. the fibrotic component in a complicated pneumoconiotic lesion is rich in fibronectin, with collagen only more abundant at the periphery. two types of progressive massive fibrosis are recognisable, corresponding to the two types of nodule described in simple coal pneumoconiosis. the first appears to have arisen by enlargement of a single nodule, whereas the second is a conglomeration of individual lesions, each of which corresponds to the more circumscribed type of nodule seen in simple coal pneumoconiosis. the ash content of the lungs bearing these two types of progressive massive fibrosis varies in the same way as with the two types of simple pneumoconiotic nodules, the enlarged single lesion being found in lungs with a relatively low ash content, and the conglomerate lesion in lungs with a relatively high ash content. the second type resembles the conglomerate nodules of large silicotic lesions but lacks the characteristic whorled pattern of the latter. the diffuse interstitial fibrosis found in a minority of coalworkers is associated with heavy dust deposition. it may progress to honeycombing but, as with the focal forms and unlike idiopathic interstitial fibrosis, it is better developed in the upper zones, the reasons for which are discussed above (see the zonal distribution of pneumoconiosis, p. ). the pathogenesis of coal pneumoconiosis has much in common with that of silicosis, and indeed many other pneumoconioses. it involves the promotion of fibrogenic factor synthesis and release by cells phagocytosing the inhaled dust. several such factors have now been identified, the degree of fibrosis produced varying with the amount of dust inhaled and the ability of its constituents to promote the production of the responsible cytokines. these include plateletderived growth factor, insulin-like growth factors and , transforming growth factor-β and tumour necrosis factor-α. , , as with other minerals, the indestructability of the dust perpetuates the process. as in silicosis, immunological factors appear to be involved, for there is an increased prevalence of rheumatoid arthritis and of circulating autoantibodies [ ] [ ] [ ] in miners with coal pneumoconiosis. rheumatoid factor has also been demonstrated within the lung lesions. these abnormalities are generally more pronounced in miners with complicated pneumoconiosis but are also found in those with the simple variety. it is also possibly pertinent to the immunological basis of coal pneumoconiosis that some of the pulmonary manifestations of rheumatoid disease are more pronounced in coalminers. this was first pointed out by caplan and will be considered next. caplan described distinctive radiographic opacities in the lungs of coalminers with rheumatoid disease, and it is now recognised that similar lesions may develop in rheumatoid patients exposed to siliceous dusts. the development of such rheumatoid pneumoconiosis does not correlate with the extrapulmonary or serological activity of the rheumatoid process. nor is there a strong relation to dust burden: caplan lesions are characteristically seen in chest radiographs that show little evidence of simple coal pneumoconiosis. pathologists recognise the lesions as particularly large necrobiotic nodules similar to those seen in rheumatoid patients who are not exposed to dust (fig. . . ) . however, because of their large size (up to cm diameter) they may be confused with progressive massive fibrosis undergoing central ischaemic necrosis (see above) or silicosis complicated by caseating tuberculosis. such errors will be less likely if the radiological evolution of the lesions is considered for they tend to cavitate and undergo rapid remission, only to be succeeded by others. they are also well demarcated radiologically. pathologically, they resemble rheumatoid nodules in showing peripheral palisading but differ in their large size and the presence of dust. the dust accumulates in circumferential bands or arcs within the necrotic centres of the lesion (fig. . . ), an arrangement that suggests periodic episodes of inflammatory activity. caplan lesions differ from tuberculosis in lacking satellite lesions and tubercle bacilli, and from progressive massive fibrosis in showing characteristic bands of dust pigmentation (table . asbestosis is defined as diffuse interstitial fibrosis of the lung caused by exposure to asbestos dust. , it does not cover asbestos-induced carcinoma of the lung or asbestos-induced pleural disease. the development of asbestosis depends on the presence of fairly large dust burdens: this is in contrast to mesothelioma and other forms of asbestos-induced pleural disease, which, although also dose-related, occur following the inhalation of far smaller amounts of asbestos dust. asbestos is a generic term for more than naturally occurring fibrous silicates, fibre being defined as an elongated particle with a length-tobreadth (aspect) ratio of at least . asbestos fibres have a high aspect ratio, generally over . based on their physical configuration they can be divided into two major groups, serpentine and amphibole. the physical dimensions and configuration of asbestos fibres are strongly linked to their pathogenicity. chrysotile (white asbestos) is the only important serpentine form. it accounts for most of the world production of asbestos of all types ( being a serpentine mineral, chrysotile consists of long, curly fibres that can be carded, spun and woven like cotton ( fig. . . ). the curly chrysotile fibres are carried into the lungs less readily than the straight amphibole asbestos fibres, and once there undergo physicochemical dissolution and are cleared more readily. they readily fragment into short particles that are easily ingested by macrophages and in the acidic environment of the macrophage phagolysosome they are particularly unstable. the half-life of chrysotile in the lungs is estimated to be in the order of only a few months. , not surprisingly therefore chrysotile is the least harmful type of asbestos in respect of all forms of asbestos-induced pleuropulmonary disease. [ ] [ ] [ ] it may nevertheless cause pulmonary fibrosis if sufficient is inhaled. , in contrast to chrysotile, amphibole forms of asbestos consist of straight rigid fibres that are stable within the lung. they do not fragment, they are insensitive to chemical attack and their clearance halflives are in the order of decades rather than months. the main amphibole forms of asbestos of commercial importance are crocidolite (blue asbestos) and amosite (brown asbestos). crocidolite, reputedly the most dangerous in regard to all forms of asbestos-related disease, was formerly mined in western australia (wittenoom) and south africa (cape province and the transvaal); it was the principal amphibole used in the uk. amosite, the name of which derives from the acronym for the former asbestos mines of south africa company in the transvaal, was the principal amphibole used in north america. amphiboles are no longer imported by the developed countries but much remains in old lagging and presents a considerable dust hazard when this is removed. tremolite, a further amphibole asbestos, contaminates quebec chrysotile deposits, montana vermiculite and many forms of commercial (non-cosmetic) talc and is responsible for much of the asbestos-related disease in chrysotile miners and millers. another amphibole asbestos, anthophyllite, was formerly mined in finland. it causes pleural plaques (see p. ) but not lung disease, possibly because its fibres are relatively thick ( fig. . . ) . erionite is a zeolite rather than a type of asbestos but is comparable in form to amphibole asbestos and is also biopersistent. it is found . these coated structures are termed 'asbestos bodies' . because other fibres may gain a similar coat, the non-specific term 'ferruginous body' has been advocated. however, coated carbon fibres (so-called coal bodies) are easily recognised as such by their black core. in practice, ferruginous bodies with the appearance of asbestos bodies almost always prove to have an asbestos core. , long fibres are more likely to be coated than short ones, which are cleared more quickly: in one study few fibres less than µm in length were coated and few fibres over µm in length were uncoated. amphiboles form bodies more readily than chrysotile. a comparison of light and electron microscopic fibre counts found that . % of chrysotile, % of crocidolite and . % of amosite formed bodies. nevertheless, sufficient chrysotile fibres are coated to permit recognition of asbestosis by standard histological criteria (diffuse fibrosis and asbestos bodies), even if chrysotile is the only asbestos present. despite the biodegradability of chrysotile, asbestos body numbers do not materially diminish with time. very occasionally however a patient with diffuse pulmonary fibrosis and a history of asbestos exposure has no evident asbestos bodies but analysis shows a fibre burden within the range found in asbestosis, justifying fibre analysis in such cases. a there is evidence that alveolar macrophages are involved in the coating of asbestos fibres to form asbestos bodies and that the bodies are less harmful to the macrophages than uncoated fibres. asbestos bodies give a prussian blue reaction for iron when stained by perls' method and their yellow-brown colour makes them easily recognisable in unstained films of sputum or in unstained histological sections. sections may be cut µm thick to increase the yield and help identify bodies that lie at an angle to the microtome blade. there is a good correlation between the numbers of asbestos bodies seen in lung sections and those in tissue digests. , the bodies may be found singly or in irregular clumps or stellate clusters. they are unevenly distributed but in well-established asbestosis they are easily found. if they are not evident, asbestos burden may be assessed quantitatively in tissue digests (see below). their presence in lung tissue, sputum or bronchoalveolar lavage fluid merely confirms exposure, not the presence of disease. however, the number of asbestos bodies in lavage fluid correlates well with lung asbestos burden , and the number in sputum correlates with the duration and intensity of exposure. [ ] [ ] [ ] fibre counts , , [ ] [ ] [ ] [ ] [ ] quantitation is desirable in certain circumstances (box . . ), in which case it is best effected on -cm blocks of fixed or fresh lung tissue obtained from three different sites, avoiding tumour and thickened pleura. the tissue blocks are digested with caustic soda or bleach, following which the fibres may be collected on a millipore membrane or viewed in suspension in a red blood cell-counting chamber. if phase contrast optics are used both coated and uncoated fibres can be assessed. alternatively, dark ground illumination can be used to demonstrate uncoated fibres. however, electron microscopy is to be preferred as it detects far more fibres than are visible by light microscopy and can also provide information on fibre type. it is important that the laboratory is well practised in fibre analysis and has established its own control range for the general population as well as asbestosis as most lungs contain some asbestos. ambient fibres are generally shorter than µm and some workers therefore confine their counts to fibres that are at least as long as this. justification for this comes from animal experiments demonstrating that long fibres cause more inflammation, chromosomal damage, fibrosis, lung tumours and mesotheliomas than short fibres, [ ] [ ] [ ] [ ] and from studies in humans suggesting that long fibres in parts of central turkey where it causes both mesothelioma and a pattern of interstitial pulmonary fibrosis that is comparable to asbestosis. , asbestos use and exposure exposure to asbestos occurs in countries where it is extracted ( asbestos is used particularly for fireproofing, in heat and sound insulation and for strengthening plastics and cement. thus, unless adequate precautions are taken, exposure is experienced by dockers unloading asbestos in the close confines of a ship's hold, by thermal insulation workers (laggers and strippers) in shipyards, power stations, train maintenance depots, factories and other large buildings, by construction workers such as carpenters cutting asbestos building panels, and by workers making asbestos products such as fireproof textiles, brake and clutch linings, and specialised cement. as well as such direct exposure, exposure may also be: • indirect, as experienced by the families of asbestos workers • paraoccupational, as experienced by those working alongside an asbestos worker • neighbourhood, as experienced by those living downwind of an asbestos works or mine • ambient, as experienced by those living or working in a building containing asbestos. exposure to asbestos incorporated in the structure of a building carries a negligible health risk if the asbestos material is well maintained to prevent shedding of dust. stripping asbestos out is more dangerous than maintaining it in situ, but maintenance is sometimes neglected. the near indestructibility of asbestos accentuates the health problems that its ubiquity poses. because of their aerodynamic properties, fibres of µm or more in length may reach the finer bronchioles and alveoli. once impacted, the sharp asbestos fibres become coated with a film of protein that is rich in iron. the coating is thickest at the ends of the fibres, giving a other human studies have shown that, although asbestos load is maximal in the upper lobes, more long fibres are found at the bases, where fibrosis is most marked. , a further reason for limiting attention to the longer fibres is that the shorter ones are cleared more easily and their number therefore varies with the time lapsed since last exposure. for these reasons asbestos regulations in many countries now limit attention to fibres that are over µm in length and have a length-to-diameter (aspect) ratio greater than : such fibres have become known as regulatory or world health organization (who) fibres. values are best expressed as fibres/g dry lung. by light microscopy, normal values range up to : over is seen with mesotheliomas, and over in asbestosis (table . . ). , , , however, compared with electron microscopy, light microscopy is relatively insensitive, showing only . % of the amosite, % of the crocidolite and . % of the chrysotile. light microscopic counts correlate poorly with severity of asbestosis and electron microscopy non-asbestos fibres commonly found in the lung include mullite, which derives from fly ash. this may constitute up to % of the total fibre burden (see table . . ) and is thought to be harmless. there is no firm evidence that manmade fibres present a health hazard but in certain localities natural non-asbestos mineral fibres, zeolites for example, are important causes of mesothelioma (see p. ) and also cause interstitial pulmonary fibrosis. in contrast to the first half of the twentieth century, much of the asbestosis encountered today is asymptomatic, identified radiologically or histologically in lungs resected for carcinoma or removed at autopsy. symptomatic cases are characterised by an insidious onset of breathlessness, a dry cough and crackles over the lower lung fields. finger clubbing is a variable feature. lung function tests show a restrictive respiratory defect. radiology initially shows small irregular basal opacities that gradually coalesce to become linear, coarsen and eventually progress to a honeycomb pattern of small cysts. the principal differential diagnosis, both clinically and pathologically, is from idiopathic pulmonary fibrosis. this is aided by the slow progression of asbestosis, which often extends over years, as opposed to an average course of - years from presentation to death for the idiopathic condition. most cases of asbestosis are diagnosed solely on the occupational history and these clinicoradiological features. recourse to histology is unusual but biopsy (preferably as a wedge of lung) may be undertaken if the clinical features are atypical. histology also arises when the pathologist samples lung parenchyma remote from a resected carcinoma (the universal importance of which cannot be overemphasised). asbestosis (established) over over the light microscopic counts include total fibres (coated and uncoated). the electron microscopic counts include only amphibole asbestos. results from different laboratories vary and these figures, derived from several sources, , , provide only a general guide. reliable results depend upon counts being made regularly and the normal range from that laboratory being ascertained. ratios of counts obtained by electron and light microscopy vary greatly but approximate to . is better in this respect. [ ] [ ] [ ] by transmission electron microscopy, values may range up to in controls, with asbestosis generally above and mesotheliomas found at any level down to , all these figures representing amphibole fibres/g dried lung (see table . . ). , , it should be noted that counts from different parts of the same lung may vary widely; , - caution should therefore be exercised in interpreting a count obtained on a single sample. there is also wide discrepancy between laboratories, even when analysing the same sample. results obtained in an individual case therefore have to be evaluated against a standard set of values unique to that laboratory. electron microscopy also provides valuable information on the type of fibre. chrysotile differs physically from the amphiboles in two respects: its fibres are both curved and hollow (figs . . and . . ). with an electron microscope equipped for microprobe analysis, the various forms of asbestos may also be distinguished from other fibres and from each other (box . . ), , an important point as the amphibole forms of asbestos are far more dangerous than chrysotile (table . . ). [ ] [ ] [ ] coroners require autopsy verification of the diagnosis in all suspected cases and this also necessitates hystology. when the lungs from a patient with asbestosis are seen at autopsy, pleural fibrosis is often found, and although this may also be attributable to asbestos exposure it is to be regarded as an independent process and not part of the asbestosis: it is dealt with separately on page . slicing the lung affected by asbestosis shows a fine subpleural fibrosis, especially of the lower lobes ( fig. . . ). in severe cases the fibrosis often extends upwards to involve the middle lobe and lingula, and sometimes the upper lobes also. microcystic change associated with the fibrosis develops in advanced cases and in severe disease there may be cysts over cm in diameter. however, these classic changes are seldom seen in developed countries today. following decades of dust suppression in asbestos factories, current patients have mild to moderate asbestosis and are dying of related cancer or of non-pulmonary disease. in some of these cases the asbestosis is only detectable microscopically. fixation of the lungs through the bronchi and the use of heard's barium sulphate impregnation technique facilitate demonstration of the fibrosis (see p. and fig. . . ). the mild degree of asbestosis currently encountered is of little functional significance but is often critical in determining whether an associated carcinoma of the lung should be attributed to asbestos exposure (see below). the histological diagnosis of asbestosis requires an appropriate pattern of interstitial fibrosis associated with the presence of asbestos bodies. both components must be present. the fibrosis is paucicellular, lacking any significant degree of inflammation and being collagenous rather than fibroblastic. it is generally considered that asbestosis begins about the respiratory bronchioles and alveolar ducts where most of the asbestos fibres impact. alveolar walls attached to these bronchioles show fine interstitial fibrosis. however, this early lesion has to be interpreted with caution because it is not specific to asbestos, being found with other inhaled mineral dusts , and even in many cigarette smokers who have not been so exposed. it more likely represents a non-specific reaction to a variety of inhaled particles. it may cause mild airflow obstruction but is not associated with the radiographic, clinical or restrictive changes of classic asbestosis. as the disease progresses, the focal changes join up so that the basal subpleural regions show widespread interstitial fibrosis and eventually complete destruction of the alveolar architecture. in severe cases there may be honeycombing and metaplastic changes in the alveolar and bronchiolar epithelium. apart from the presence of asbestos bodies the changes resemble those of non-specific interstitial pneumonia, or more rarely usual interstitial pneumonia. fibroblastic foci may be found but they are uncommon. there is often an increase in alveolar macrophages but the desquamative interstitial pneumonia that has been reported in association with asbestos , is not to be regarded as a variant of asbestosis ; concomitant smoking is a more likely cause. a variety of other non-specific inflammatory processes such as organising pneumonia have been reported in asbestos workers and if localised some have been suspected of representing malignancy until biopsied. several schemes have been proposed for grading the extent and severity of asbestosis. these are of value in epidemiological studies but should only be applied to cases meeting the histopathological criteria for a diagnosis of asbestosis. one such scheme is shown in box . . . , , in well-established asbestosis asbestos bodies are numerous and easy to find, aggregates of them sometimes forming clumps ( fig. . . ) . in earlier lesions a detailed search may be necessary, in which fibrosis confined to the walls of respiratory bronchioles and the first tier of adjacent alveoli b extension of fibrosis to involve alveolar ducts and/or two or more tiers of alveoli adjacent to the respiratory bronchiole, with sparing of at least some alveoli between adjacent bronchioles fibrotic thickening of the walls of all alveoli between at least two adjacent respiratory bronchioles honeycomb change a an average score is obtained for an individual case by adding the scores for each slide ( - ), then dividing by the number of slides examined b grade and, to a lesser extent, grade need to be distinguished from smoking-induced peribronchiolar fibrosis and mixed-dust pneumoconiosis. case the examination of unstained or perls-stained sections facilitates their identification. minimum criteria for the diagnosis of asbestosis require the identification of diffuse interstitial fibrosis in well-inflated lung tissue remote from a lung cancer or other mass lesion and the presence of either two or more asbestos bodies in tissue with a section area of cm or a count of uncoated asbestos fibres that falls in the range recorded for asbestosis by the same laboratory. , there are marked variations in the concentration of asbestos fibres between samples from the same lung , and it is therefore recommended that at least three areas be sampled, the apices of the upper and lower lobes and the base of the lower lobe. the equivalent of mallory's alcoholic hyalin of the liver has been described in the lungs in asbestosis, , and subsequently in other [ ] [ ] [ ] [ ] it is seen as small eosinophilic cytoplasmic inclusions within hyperplastic type ii alveolar epithelial cells (fig. . . a ). electron microscopy shows that the inclusions consist of a tangle of tonofilaments ( fig. . . b ) and by immunocytochemistry a positive reaction is obtained with antibodies to cytokeratin, both these features being typical of mallory's hyalin in the liver. the inclusions also react for ubiquitin, the accumulation of which is indicative of cellular damage, in particular faulty proteinolysis. the differential diagnosis of asbestosis includes pulmonary fibrosis due to many other causes, any of which may of course affect an asbes-tos worker as much as members of the general population. the proportion of diffuse pulmonary fibrosis in asbestos workers that is not attributable to asbestos has been estimated to be as high as % and likely to rise as the risk of asbestosis diminishes with better industrial hygiene. the principal differential diagnosis of asbestosis is from idiopathic pulmonary fibrosis. both diseases affect the bases and periphery of the lungs predominantly. in the late stages, cystic change is more evident in idiopathic pulmonary fibrosis but this criterion is not totally reliable. nor is the presence of pleural fibrosis, although it is usually present in asbestosis and is seldom found in idiopathic pulmonary fibrosis. asbestosis seldom progresses or does so very slowly after exposure ceases , whereas idiopathic pulmonary fibrosis typically proves fatal within - years from onset. the fibrosis of asbestosis is generally paucicellular: inflammation is not a feature and the fibroblastic foci that characterise the usual interstitial pneumonia pattern of fibrosing alveolitis are seldom observed in asbestosis. very often the distinction from idiopathic pulmonary fibrosis has to be based on the amount of asbestos in the lung and, if asbestos bodies are not readily identifiable, this has to depend on fibre counts. errors are made both by overlooking substantial numbers of asbestos bodies completely and by ascribing undue importance to scanty bodies. if considering the possibility of minimal asbestosis (to justify attributing carcinoma of the lung to asbestos, for example) it should be remembered that a little peribronchiolar fibrosis is also characteristic of smokers' lungs, centriacinar emphysema and early mixeddust pneumoconiosis. [ ] [ ] [ ] as described above, at least two asbestos bodies/cm in the presence of interstitial fibrosis distant from any lung cancer or other mass lesion is required for a diagnosis of asbestosis. although the causes of asbestosis and idiopathic pulmonary fibrosis are very different, they resemble each other in several ways, suggesting that similar pathogenetic mechanisms may operate. , [ ] [ ] [ ] in both these diseases there is degeneration of the alveolar epithelium and capillary endothelium, with patchy loss of the former, and bronchoalveolar lavage shows an increase in macrophages that might perpetuate the damage by releasing lysosomal enzymes, nitric oxide and hydroxyl radicals. , [ ] [ ] [ ] both diseases are also characterised by an increased prevalence of circulating non-organ-specific autoantibodies. experimentally, asbestos exposure leads to the activation of a variety of fibrogenic cytokines at sites of lung injury. , [ ] [ ] [ ] [ ] [ ] [ ] inhaled asbestos activates a complement-dependent chemoattractant for macrophages and macrophage stimulation involves the secretion of fibroblast stimulating factors, [ ] [ ] [ ] asbestos being intermediate between haematite and silica in regard to macrophage-mediated fibrogenicity. the epithelial damage could be mediated directly by the needle-like asbestos fibres or indirectly through enhanced phagocyte generation of free radicals (which is much greater with amphibole asbestos than with either chrysotile or silica). , fibrogenic cytokines released by activated pulmonary phagocytes and regenerating alveolar epithelial cells in asbestosis include tumour necrosis factor-α and transforming growth factor-β, as in idiopathic pulmonary fibrosis. as a result of better industrial hygiene, asbestosis is less severe today than in earlier years when it followed much heavier exposure, with the consequence that death from respiratory failure and cor pulmonale is less common and sufferers are surviving longer. there is therefore now a greater risk of asbestos-related cancer eventually developing. asbestos exposure predisposes to two varieties of malignant neoplasm, carcinoma of the lung and mesothelioma of the pleura and peritoneum. the risk of malignancy increases with dose but the relative risk of carcinoma is much smaller than that of mesothelioma. for example, with heavy exposure, as in lagging, the risk of mesothelioma is increased -fold whereas it is increased only fivefold for lung cancer. hence, with light exposure there is a substantial risk of mesothelioma but only a small risk of lung cancer. asbestosis requires heavy exposure and in one group of patients with asbestosis, % died of pulmonary carcinoma, % of mesothelioma and % of other respiratory diseases. although there were many earlier reports, the link with carcinoma of the lung may be considered to have been firmly established by , that between crocidolite asbestos and mesothelioma by , and that between amosite asbestos and mesothelioma by . mesothelioma is considered on page . in regard to carcinoma of the lung, asbestos is not such a potent pulmonary carcinogen as cigarette smoke but together their effects are multiplicative rather than additive (table . . ). , however, the risk attributable to asbestos is the same regardless of smoking history, being increased fivefold in both smokers and non-smokers. there is usually a latent period in excess of years between first exposure to asbestos and the development of lung cancer and the risk increases the greater the cumulative exposure. the increased risk involves carcinomas of all the histological types encountered in the lung, although adenocarcinoma has been disproportionately overrepresented. , [ ] [ ] [ ] [ ] [ ] [ ] it is uncertain whether the increased risk of carcinoma is caused by the asbestos , [ ] [ ] [ ] [ ] [ ] [ ] or the asbestosis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the latter view envisages the carcinoma arising in the foci of alveolar epithelial hyperplasia and dysplasia that commonly accompany any interstitial fibrosis (see carcinoma complicating idiopathic pulmonary fibrosis, p. ). however, most carcinomas complicating asbestosis arise in the bronchi rather than the alveolar tissue. on the other hand, more arise in the sites worst affected by asbestosis, the lower lobes and the periphery of the lung, than in the general population ( fig. . . ) . , , [ ] [ ] [ ] [ ] [ ] the majority view has been that asbestosis is a necessary precursor of the carcinoma but evidence to the contrary is finding increasing support (table . . ). in the uk, industrial compensation was formerly only awarded to an asbestos worker for carcinoma of the lung if there was also asbestosis or diffuse pleural fibrosis but new rules were introduced in . asbestosis remains a sufficient criterion but diffuse pleural thickening is not and asbestosis is no longer a necessary criterion: asbestos is deemed to have been responsible if the patient worked in asbestos textile manufacture, spraying, lagging or gas mask manufacture for at least years before or years after . the basis for these changes is the premise that heavy asbestos exposure is sufficient in itself to account for carcinoma of the lung. together, these factors have a multiplicative rather than additive effect mortality ratio non-smoking controls non-smoking asbestos workers cigarette-smoking controls cigarette-smoking asbestos workers asbestosis associated with carcinoma of the lung. the asbestosis has been highlighted by barium sulphate impregnation and is seen as a grey subpleural band to the right of the picture. although the carcinoma has arisen in the same lobe as the asbestosis it has not obviously arisen in an area affected by asbestosis. . asbestosis diagnosed clinically, radiologically or histologically or a minimum count of asbestos bodies per gram dry lung tissue (/g dry), or an uncoated asbestos fibre burden of million amphibole fibres more than µm in length/g dry, or million amphibole fibres more than µm in length/g dry or estimated cumulative exposure to asbestos of at least fibres/ ml-years or an occupational history of year of heavy exposure to asbestos (e.g. manufacture of asbestos products, asbestos spraying) or - years of moderate exposure (e.g. construction or ship-building) and . a minimum lag time of years lung fibre counts in the asbestosis range (see table . . ) provide valuable evidence of such exposure. compensation standards for asbestos-associated lung cancer in different countries are shown in box . . . , asbestos-induced airway disease although asbestosis causes a restrictive respiratory defect, airflow limitation is also seen in this disease. much of the airflow limitation is attributable to cigarette smoking but it is also seen in non-smoking asbestos workers and is worse in those with asbestosis. the pathological basis of this appears to be small-airways disease (see p. ). it is possibly a non-specific reaction to inhaled dust or cigarette smoke. because it is not established that this lesion progresses to interstitial alveolar fibrosis (asbestosis) the term 'asbestos airways disease' is suggested. fibrosis limited to the bronchioles is specifically excluded from the definition of asbestosis in the latest guidelines (although these retain grade for fibrosis limited to the bronchiolar walls). it should also be noted that, although emphysema is considered to be a destructive rather than fibrotic condition, a little focal the presence of asbestos-related bilateral pleural plaques or asbestos-related bilateral pleural thickening and occupational exposure and a lag time of at least years the presence of asbestosis or pleural plaques or diffuse pleural thickening or fibre-years of exposure only fibre-years of exposure are taken into account exposure, at least years' latency and asbestos-related pleural or parenchymal changes asbestosis is not required but smoking is taken into consideration attempts are made to quantify separately the attributability to asbestos, smoking and other factors (e.g. radon) fibrosis is generally evident in this common condition and does not necessarily indicate early asbestosis. aluminium has been implicated in the development of respiratory disease during the refining of its principal ore, bauxite, to yield various aluminium oxides (aluminas), in the preparation of the metal by smelting alumina, in the production of corundum abrasive and in the production of special aluminium powders used in explosives. bauxite is a mixture of various aluminium oxides, hydroxides and silicates, iron oxide and titanium dioxide. the oxides of aluminium are obtained by differential heating of the ore and the respiratory effects of this work appear to be no more than mild airway irritation. it is generally accepted that aluminium oxide is inert. aluminium is prepared by the electrolytic reduction of its oxide dissolved in sodium aluminium fluoride (cryolite), a process releasing a considerable amount of fluoride-rich effluent. exposed workers have complained of what is termed pot-room asthma. the pathology of this condition is not well described but the pathogenesis is thought to involve irritation rather than allergy. the abrasive corundum is formed from bauxite mixed with coke and iron heated in an electric arc furnace, a process in which workers may be exposed to the fumes of alumina and free silica. in the past some of these workers developed diffuse pulmonary fibrosis (shaver's disease) and, although this was initially attributed to the aluminium, it is now agreed that the free silica was the responsible agent. the exposure to free silica has been reduced and the disease is now regarded as historic. aluminium powder holds a paradoxical position in regard to lung disease. in certain industries it has caused very severe pulmonary fibrosis, yet in others it has proved harmless. indeed, at one time canadian miners breathed aluminium dust before work, in the belief that this would reduce the danger of silica in the mine dust and more recently silicosis has been treated by such means in france. it is questionable whether this practice is effective but it at least appears to cause no harm. the explanation for these contradictory observations probably lies in differing methods of manufacture of aluminium powder. aluminium metal appears to be an inert substance but this is only because it has a high affinity for oxygen and the surface layer of aluminium oxide so formed is firmly bound to the underlying metal, unlike ferric oxide which permits further rusting of iron. granular aluminium powders, produced in a ball mill or from a jet of molten aluminium, therefore acquire a protective coat of surface oxide and are inert. with stamped aluminium powders, however, surface oxidation is prevented by lubricants added to aid the separation of these flake-like particles. the usual lubricant (stearin) contains stearic acid and this polar compound combines with the underlying metal, which is thereby protected from both atmospheric oxidation and the action of body fluids when such dust is inhaled. in certain circumstances, however, non-polar lubricants in the form of mineral oils have been substituted for stearin. this happened in germany during the second world war when munition production was stepped up but stearin was difficult to obtain, , and in the uk in the s to make the powder darker for purely commercial reasons. in vitro, oil-coated stamped aluminium powder reacts with water to produce aluminium hydroxide, which affords the underlying metal no protection against further attack, so that aluminium hydroxide continues to be formed. this substance is a protein denaturant, once used in the tanning industry, and it is believed that this property underlies the very ex ceptional cases of severe pulmonary fibrosis that have occurred in connection with stamped aluminium powder produced with mineral oil rather than stearin. , the fibrosis has a very characteristic pattern, affecting the upper lobes and progressing rapidly, the interval from onset of symptoms to death being as short as years. there is marked shrinkage of the lungs with gross elevation of the diaphragm and buckling of the trachea (fig. . . ). the lungs are grey ( fig. . . ) and microscopically, numerous small black jagged particles are seen. these can be shown to contain aluminium with irwin's aluminon stain or by microprobe analysis. what appears to be a different pathological effect of aluminium dust on the lungs is the rare development of granulomatous disease resembling sarcoidosis and berylliosis. , this represents hypersensitivity to the metal, amenable to confirmation with a lymphocyte transformation test similar to that used to diagnosis berylliosis (see below). rare cases of desquamative interstitial pneumonia and pulmonary fibrosis have been reported in aluminium welders. , elements with atomic numbers from (lanthanum) to (lutetium) are known as the lanthanides or rare earth metals. they are used in many manufacturing processes, including the production of hightemperature ceramics and the grinding of optical lenses. carbon arc lamps used in reproduction photography emit appreciable quantities of oxidised lanthanides, particularly cerium oxide, and there are reports of pneumoconiosis in exposed individuals. the pathological changes reported have varied from granulomatous nodules to diffuse interstitial fibrosis indistinguishable from the idiopathic variety except for the presence of rare earth elements (usually cerium) detected by polarising light microscopy and electron microprobe analysis. hard metal is a tungsten alloy containing small amounts of cobalt, titanium, molybdenum and nickel. it is exceptionally tough and once formed can only be worked with diamond. it is used in the tips of drill bits, on abrasive wheels and discs, and in armaments. interstitial lung disease is liable to arise in its manufacture or in those using hard metal as an abrasive. experimental work suggests that cobalt is the dangerous constituent but this element is soluble and, unless industrial contact has been recent, analysis of lung tissue usually shows tungsten and titanium but no cobalt. the role of cobalt is also indicated by the development of similar interstitial lung disease in diamond polishers using high-speed polishing discs made with a diamond-cobalt surface that lacked tungsten carbide and the other constituents of hard metal. , hard-metal lung disease and cobalt lung take two forms, an industrial asthma and interstitial fibrosis. the latter has a diffuse lower zonal distribution and the appearances mimic idiopathic pulmonary fibrosis. however, an unusual feature is the presence of moderate, or perhaps only small numbers, of giant cells (fig. . . a, b) . , not only are there multinucleate alveolar macrophages but syncytial cell forms develop in the alveolar epithelium. electron microscopy confirms that these are multinucleate type ii pneumocytes (fig. . . c ). such epithelial changes are well known in measles pneumonia but the viral inclusion bodies that characterise this infection are not found in hard-metal pneumoconiosis. the changes are those initially described as a particular pattern of idiopathic interstitial pneumonia termed giant cell interstitial pneumonia or gip (see p. ). elemental analysis shows that many, but not all, cases of gip represent hardmetal disease. the exceptions seldom give a history of cobalt exposure and must be presumed to represent true idiopathic cases. conversely, epithelial giant cells are not always found in hard-metal pneumoconiosis and so their presence, although highly characteristic, is neither totally specific nor totally sensitive. beryllium is the lightest of metals. it has an atomic weight of and special properties that make it especially useful in many applications. it is more rigid than steel, has a high melting point and is an excellent conductor of heat and elecricity. unfortunately, the inhalation of beryllium dust or fume is exceedingly dangerous. , those who worked with beryllium compounds before precautionary measures were taken suffered a high morbidity and mortality. sometimes, the escape of dangerous fumes from the factories was on such a scale that people living in nearby houses, downwind from the places in which these materials were being worked, contracted and occasionally died from berylliosis ('neighbourhood cases'). alternatively, contamination of a beryllium worker's clothes might lead to berylliosis in a temperatures. the alloys of beryllium are also now widely used, especially those with copper, on which it confers elasticity and resistance to fatigue. alloy manufacture and the machining of beryllium alloys are therefore further activities that entail a risk of berylliosis, as is the recovery of the metal in the recycling of scrapped electronic and computer parts. seemingly innocuous occupations such as dental laboratory technician are not without risk of chronic berylliosis. there are good grounds for regarding chronic berylliosis as being an allergic condition. many of those affected react strongly to skin tests with dilute solutions of beryllium salts, although these must be undertaken with care: occasionally in a highly sensitised person even so small an exposure may evoke a systemic reaction. the skin reaction is of the delayed type, occurs in only % of exposed individuals, is not associated with a clear-cut dose-response curve and represents a granulomatous response. further evidence for the disease having an allergic basis derives from bronchoalveolar lavage, which demonstrates an excess of t-helper lymphocytes that proliferate in vitro on exposure to beryllium salts. a positive transformation test given by these lymphocytes is a more reliable indicator of disease than in vitro blood lymphocyte transformation testing, which is safe but not wholly reliable and indicates only sensitization, rather than berylliosis. susceptibility to berylliosis varies widely from person to person and it is notable that chronic pulmonary disease is strongly associated with the hla antigen dpβ and the glu gene. , the importance of genetic factors is supported by a report of the disease in identical twins. chronic berylliosis is thought to be initiated by the metal binding to tissue proteins and acting as a hapten to initiate a delayed hypersensitivity response characterised by a proliferation of t-helper lymphocytes. these sensitised cells in turn secrete a variety of cytokines (e.g. interleukin- , tumour necrosis factor-α and interferon-γ) that recruit and activate macrophages, which mature into epithelioid cells. the resultant epithelioid cell granulomas destroy the lung tissue and lead to pulmonary fibrosis. if beryllium enters the subcutaneous tissues through a cut or abrasion, as often happened in the earlier days of fluorescent lamp manufacture, a sarcoid-like granuloma soon appears at the site; in time, the overlying epidermis may break down to form an ulcer. even more serious are the lesions produced by the inhalation of beryllium. chronic pulmonary berylliosis takes the form of a widespread granulomatous pneumonia with a histological picture identical to that of sarcoidosis (fig. . . a ). both berylliosis and sarcoidosis affect the upper lobes more than the lower (fig. . . b ) and in both diseases the granulomas are preferentially distributed along lymphatics and may involve adjacent blood vessels. in neither condition is there widespread necrosis but in both diseases the granulomas occasionally display a little central necrosis or hyalinisation. as in sarcoidosis, the hilar lymph nodes may be involved but, unlike sarcoidosis, not in isolation. over a period of many years, the sarcoid-like granulomas gradually undergo progressive fibrosis, with consequent impairment of pulmonary function. in the later stages, when the disease has become chronic, dispersal of beryllium from its site of initial absorption may lead to generalisation of the disease and to the appearance of similar granulomas elsewhere, particularly in the liver, kidneys, spleen and skin, but this is unusual. relative. beryllium compounds may also cause contact dermatitis and conjunctivitis. beryllium is also classified as a probable pulmonary carcinogen, but this is controversial. two forms of berylliosis are recognised, acute and chronic. acute berylliosis was first reported in germany in and is now largely of historical interest, being only encountered as a result of rare accidental or unexpected exposure. it follows the inhalation of a soluble beryllium salt and represents chemical injury, the pathology being that of diffuse alveolar damage (see p. ). further consideration will be confined to chronic berylliosis, which is allergic in nature. chronic berylliosis was first reported in in the fluorescent lamp industry. beryllium has now been replaced in this application but it has since proved to be of great value in the nuclear, electronic, computer and aerospace industries and the production of refractory materials and crucibles that are to be subjected to particularly high and there is a lifelong risk of disease. progression often entails alternating exacerbations and remissions, long after exposure has ceased. in keeping with the view that berylliosis is a hypersensitivity reaction, very little beryllium is necessary to cause the disease. particulate beryllium is so scanty in the affected tissues and the atomic number of beryllium so low that electron microprobe analysis is generally unsuitable for its detection. furthermore conventional detectors are protected by a beryllium window. however, the substitution of a polymeric window has enabled beryllium to be detected by electron microprobe analysis, presumably in a patient with fairly heavy exposure. ion or laser microprobe mass spectroscopy can also detect very small amounts of beryllium in tissue sections but these techniques are not widely available. the differential diagnosis of chronic berylliosis is from sarcoidosis, to which it is identical morphologically. [ ] [ ] [ ] however, as noted above, it is unusual for berylliosis to cause significant hilar lymphadenopathy in the absence of pulmonary disease, which is a common feature of sarcoidosis. extrathoracic granulomas, erythema nodosum and uveitis, which are all common in sarcoidosis, are unusual in berylliosis. however, one group found that % of patients initially diagnosed as having sarcoidosis actually had chronic berylliosis. similar findings have been reported by others. , any patient thought to have sarcoidosis who has worked with or near metals should be offered a beryllium lymphocyte transformation test. a list of laboratories performing this test can be found at www.dimensional. com/~mhj/medical_testing.html. although polyvinyl is not a mineral and the reaction of the lungs to its presence is therefore not a true pneumoconiosis, it is generally so termed and is dealt with here for convenience. workers are exposed to polyvinyl chloride dust in the milling and bagging of this plastic and micronodular opacities may be detected in their lungs radiologically. however, the material is non-fibrogenic and histology merely shows a foreign-body reaction to the dust particles. the radiological opacities may abate when exposure ceases. nevertheless, one polyvinyl chloride worker developed systemic sclerosis, which is a recognised complication of silicosis (see p. ). polyvinyl chloride is produced from vinyl chloride monomer, which has a causal association with angiosarcoma of the liver and probably other forms of cancer, including carcinoma of the lung (see p. ). in the late s a characteristic lung disease was identified in workers at several factories producing plush material by spraying nylon flock on to an adhesive backing material. [ ] [ ] [ ] [ ] the flock fibres are too large to be inspired but may be mixed with smaller nylon shards of respirable size. the workers complained of cough and breathlessness and were found to have a restrictive ventilatory defect with interstitial markings on radiography. their symptoms improved on removal from the workplace but relapsed on return to work. pathologically, there was lymphocytic bronchiolitis and peribronchiolitis with widespread lymphoid hyperplasia represented by lymphoid aggregates. chronic berylliosis is characterised by the gradual onset of cough, shortness of breath, chest pain, night sweats and fatigue. these symptoms may develop within a few weeks of exposure or many years later. once the worker is exposed, the beryllium is retained in the tissues granulomas were not identified. the histological appearances suggest a severe immunological reaction and raise possibilities such as rheumatoid disease and sjögren's syndrome but consideration of the clinical and serological setting and the occupation should permit recognition of the cause. the industrial production of popcorn and other foodstuffs appears to carry a risk of obstructive airway disease. [ ] [ ] [ ] [ ] biopsy of affected workers has shown peribronchiolar fibrosis and granulomas and air sampling has identified many volatile organic compounds, of which the flavouring agent diacetyl ( , -butanedione) is suspected of being responsible for the bronchiolitis. it is difficult to continue paint spraying (air brushing, aerographics) without adequate respiratory protection but in the early s several small aerographic factories operated in the neighbourhood of alicante, southeastern spain without any concern for the workers' health. the workers were required to paint patterns on textiles using a hand-held spray gun. the atmospheric pollution was intense but complaints of respiratory difficulties were met with reassurances and the workers urged to continue. this they did because of the otherwise poor economy, often returning to work when disabling breathlessness had settled down. a change of paint (to acramin f) may have contributed because the worst-affected workers were employed at two plants that had made this switch. their illness has been described as the 'ardystil syndrome' after the name of one of these factories. some workers were left with permanent respiratory disability. one required a lung transplant and others died. [ ] [ ] [ ] [ ] transbronchial biopsy showed organising pneumonia, which in the fatal cases had progressed to irreversible interstitial fibrosis. a similar outbreak of respiratory disease was subsequently reported in algerian textile factories where acromin f was applied by the same technique. , acromin f is marketed as a paste and used as such without ill-effect. its use in heavy spray form appears to be responsible for the 'ardystil syndrome' . workers in engineering workshops may be exposed to the prolonged inhalation of fine sprays or mists of the longer-chain hydrocarbons that constitute many mineral oils. this may result in exogenous lipid pneumonia, which is described on page , or extrinsic allergic alveolitis. [ ] [ ] [ ] the vapour of shorter-chain hydrocarbons such as paraffin oil (kerosene: c - ) and petrol (gasoline: c - ) and gaseous hydrocarbons such as propane may act as acute asphyxiants or central nervous system depressants but have negligible pulmonary toxicity. however, if they are ingested or aspirated in their liquid form they are acutely toxic to the lungs, producing a chemical pneumonitis with the features of diffuse alveolar damage. ingestion may be accidental or deliberate (see fig. . , p. ) whereas aspiration is generally inadvertent, occurring in siphoning accidents, such as those experienced by fairground operatives who 'breath or eat fire' ('fire-eater's lung'). , animal experiments involving the intratracheal injection of kerosene resulted in acute pulmonary exudates, which cleared except for residual bronchiolitis. welder's pneumoconiosis, first recognised in , essentially represents the fairly harmless deposition of iron in the lungs (siderosis -see p. ). however, welders may suffer various ill-effects from the inhalation of substances other than iron (table . . ). some of these are para-occupational risks, that is, encountered by welders because they work near another process and are inadvertently exposed: thus, shipyard welders may be exposed to asbestos, and those in foundries to silica. welders may therefore develop a mixed-dust pneumoconiosis (see p. ), rather than just siderosis. however, one analytical investigation identified excess amounts of iron alone in association with pulmonary fibrosis; the silicon content did not differ from that in controls. more directly, welders may be exposed to asbestos insulation that they themselves use, while welders of special steel alloys run the risk of metal-induced asthma, metal fume fever, polymer fume fever and the consequences of toxic metal fume inhalation, all of which are described separately in this chapter, as is lung disease in aluminium welders. chronic bronchitis has been attributed to the inhalation of low concentrations of irritants such as ozone and nitrogen dioxide by welders but this risk is unproven and the subject of much controversy. welders may also inhale carcinogenic hexavalent chromium compounds in the course of their work and therefore develop lung cancer. the term 'welder's lung' is often applied indiscriminately to any of these diseases and, as it has no specific meaning, is best avoided. dust, fume and gas are some of the terms used to describe different physical forms of respirable agents. they are defined in table . . on the finely divided fume of several metals is highly toxic to the lungs and capable of producing severe acute and chronic damage to both the conductive airways and the alveoli, resulting in acute tracheobronchitis and bronchiolitis, diffuse alveolar damage, obliterative bronchiolitis and pulmonary fibrosis. important metal fumes in this respect include aluminium, which is released together with silica fume in bauxite smelting (see shaver's disease, above), cadmium from welding or cutting special steels, chromium from cutting its alloys or in the manufacture of chromates, cobalt released in the production and use of its alloys (see hard-metal disease, above), mercury released in various industries and in the home, nickel carbonyl released during the purification of metallic nickel or the manufacture of nickel alloys and beryllium (see above). many irritant gases cause severe acute and chronic damage to both the conductive airways and alveoli. the changes are non-specific and similar to those wrought by toxic metal fumes (see above) and viruses amongst other agents. they consist of acute tracheobronchitis and bronchiolitis, obliterative bronchiolitis, diffuse alveolar damage and pulmonary fibrosis. the gases liable to produce such damage include oxides of nitrogen, sulphur dioxide, ozone, phosgene, chlorine, ammonia and various constituents of smoke, notably acrolein. some of these are also touched upon in chapter . because they are of general as well as occupational importance, although there is no rigid difference between general and occupational pollution. ozone, sulphur dioxide and nitrogen dioxide are oxidising gases that may be found together as industrial atmospheric pollutants. each is capable of producing diffuse alveolar damage by means of its oxidising properties and the release of free active radicals. in addition, they cause damage to distal airways, particularly terminal and respiratory bronchioles, with resulting bronchiolitis. oxides of nitrogen may be encountered with fatal consequences by farmhands seeking to free a blockage in a silo when they encounter pockets of this gas that have accumulated on top of the fermenting silage: the term 'silo-filler's disease' is generally applied to the initial haemorrhagic oedema or the obliterative bronchiolitis that develops in those who survive the initial chemical injury. [ ] [ ] [ ] [ ] asphyxia due to the farmhand encountering pockets of carbon dioxide is a further hazard within agricultural silos. other farmhands have suffered from the inhalation of toxic gases or bacteria when handling liquid manure. [ ] [ ] [ ] [ ] welding, which is considered below, may also involve exposure to toxic gases such as oxides of nitrogen. ozone, the principal oxidant gas of photochemical smog, produces pulmonary changes at relatively low levels and may be encountered at higher concentrations in various industries. potentially dangerous levels of ozone are produced from atmospheric oxygen by ultraviolet radiation given off in welding while ozone is used in industry to sterilise water, bleach paper, flour and oils, and mask the odour of organic effluents. the damage wrought by ozone is predominantly centriacinar in distribution, affecting terminal and respiratory bronchiolar epithelium and proximal alveolar epithelium. [ ] [ ] [ ] there is loss of cilia and necrosis of centriacinar alveolar type i epithelial cells. the changes are dose-dependent and, in one study, the youngest animals were most sensitive. in long-term experiments, hyperplastic bronchiolar clara and ciliated cells extended peripherally to line alveolar ducts. the role of granulocytes is stressed in some experimental studies and it is notable that neutrophil migration is prominent when the human lungs are damaged by ozone. aldehydes such as acetaldehyde, formaldehyde and acrylic aldehyde (acrolein) are widely used in the plastics and chemical industries. the first is a liquid and the others are water-soluble gases. pathologists are of course familiar with formaldehyde solution from its use as a disinfectant and histological fixative. all these aldehydes are intensely irritant and their acute effects generally prevent prolonged exposure to high concentrations. chronic effects include skin sensitivity and asthma, and in rats nasal carcinoma. however, the doses to which these experimental animals were exposed far exceed any that are likely to be encountered by humans, in whom there is no convincing evidence of aldehyde-induced cancer. ammonia gas is extensively used in industry as a raw material, notably in the manufacture of nitrogenous products such as fertilisers and plastics. it is highly soluble and its acute irritative effects are mainly felt in the eyes, nose and throat, but high levels affect the major airways, possibly leading to them being blocked by exudates. survival usually brings full recovery but bronchiectasis and obliterative bronchiolitis have been described. chlorine gas is widely used in the chemical industry. it is transported and stored under pressure in liquid form. heavy exposure through its accidental release or use as a war gas has proved fatal through its acute toxicity causing exudative airway occlusion and pulmonary oedema. survivors usually recover completely but, as with nitrogen dioxide and ammonia, there is a risk of obliterative bronchiolitis. phosgene (carbonyl chloride, cocl ) is a poisonous, colourless gas that was responsible for thousands of deaths during world war i, when it was used in chemical warfare. it is used industrially in the preparation of some organic chemical compounds and is formed, perhaps inadvertently, by the combustion of methylene chloride in products such as paint strippers. phosgene causes injury to terminal bronchioles and alveoli, with resulting oedema and hyaline membrane formation. the mechanism of cell damage is uncertain but it may depend on inactivation of intracellular enzymes by the gas. longterm problems are rare but chronic bronchitis and emphysema have been described in survivors. mustard gas (bichloroethyl sulphide, c h cl s) is a further agent that has been used in chemical warfare. it is primarily a skin vesicant but when inhaled it results in widespread epithelial destruction and pulmonary oedema. survivors may be left with irritant-induced asthma (reactive airways dysfunction), chronic bronchitis, tracheobronchomalacia, bronchiectasis and bronchiolitis obliterans. [ ] [ ] [ ] thionyl chloride is used in the manufacture of lithium batteries where it is liable to result in the release of sulphur dioxide and hydrochloric acid fumes. workers in such factories have developed lung injury varying from mild, reversible interstitial disease to severe obliterative bronchiolitis. hydrogen sulphide is the principal chemical hazard of natural gas production. high levels of the gas also buid up in sheds housing large numbers of pigs, the source here being the pig manure. once inhaled the gas is rapidly absorbed into the blood stream. the effects are therefore widespread but include the usual respiratory effects of irritant gases, varying from sneezing to pulmonary oedema and acute respiratory distress, depending upon the exposure. in alberta cases were identified over a -year period. the overall mortality was %; % of victims were dead on arrival at hospital. most required admission to hospital but the survivors experienced no long-term adverse effects. a the danger of asphyxia from the inhalation of gases devoid of oxygen is fairly widespread in industry. it generally arises from the use of inert gases, which, being non-toxic, give a false sense of security. pockets of these gases tend to form in confined spaces. anoxic death from the accumulation of methane is well known in mines and has also occurred in slurry pits and sewers. anoxic asphyxia in diving (and anaesthesia) has resulted from the incorrect connection of gas cylinders or failure to notice that a mixed gas contains insufficient oxygen. deaths have occurred in welding when argon or carbon dioxide has been used to shield the weld and prevent oxidation of the metals at the high temperatures employed. deaths have also resulted from inadvertent entry to discharged oil tanks filled with nitrogen to reduce the risk of explosions, or from the formation of pockets of nitrogen gas applied in liquid form to freeze the contents of damaged pipes so that they can be repaired without the necessity to drain down. the respiration of a gas devoid of oxygen causes loss of consciousness within seconds because it not only fails to provide oxygen but removes that present in the pulmonary arterial blood. the changes at autopsy are those common to cellular hypoxia. they include cerebral and serosal petechiae and pulmonary congestion and haemorrhage but these features are not specific and are not always present. the cause of death can generally only be surmised from the circumstances surrounding the death. occupational asthma is the commonest cause of work-related respiratory disease in many western countries (table . . ). [ ] [ ] [ ] the reported incidence ranges from per million workers in south africa to per million workers in finland. , it occurs in many industries (table . . ) and occupational factors can be identified as contributing to asthma in about % of adult cases. over aetio- in the uk a third are organic, a third chemical, % metallic and the rest miscellaneous. the commonest, in descending order, are isocyanates, flour and grain, laboratory animals, glutaraldehyde, solder or colophony and hardening agents. atopy appears to predispose to occupational asthma when the allergen is of high molecular weight but not when it is of low molecular weight. for example, atopic individuals are particularly prone to develop asthma if employed in the manufacture of biological detergents, whereas atopy does not increase the risk of asthma from sensitisation to toluene di-isocyanate, which is a serious health problem in the manufacture of polyurethane. similarly, platinum salts are such potent sensitising agents that nearly all those exposed to them develop asthma. asthma-provoking metals other than platinum include chromium, cobalt, nickel and vanadium, all of which are used in steel alloys, and possibly aluminium (see pot-room asthma, p. ). other asthma-inducing factors encountered in industry include grain and flour dust, certain wood dusts, soldering fluxes containing colophony (pine resin), epoxy resin hardeners such as phthallic anhydride, isocyanate-containing foams and paints, formaldehyde and the excreta of laboratory animals. contaminated humidifiers may cause occupational asthma as well as humidifier fever and extrinsic allergic alveolitis. pathologically, occupational asthma is identical to nonoccupational asthma (see p. ). byssinosis is a further form of occupational asthma, one encountered in the cotton industry. the sensitising agent is a component of the cotton bract, which is the part of the cotton harvest other than the cotton fibre. bract consists of dried leaf, other plant debris and soil particles and contains a variety of fungal and bacterial residues, including lipopolysaccharide endotoxin, but the exact nature of the sensitising agent remains unknown. the endotoxin is unlikely to be responsible for byssinosis but may be the cause of so-called mill fever, a self-limiting illness characterised by malaise, fever and leukocytosis that is experienced by many people on first visiting a cotton mill. dust levels and the risk of byssinosis are particularly high in the carding rooms where the raw cotton is teased out before it is spun. affected workers are worse when they return to work after the weekend break, a feature attributed to antibody levels having built up during this brief respite from the cotton dust. there is no link with atopy and the fluctuating antibodies are precipitins of the immunoglobulin g class. complement activation by both arms of the complement cascade has been reported. , when the lancashire economy was largely cotton-based, necropsies on workers suffering from byssinosis generally showed gross emphysema, and this came to be accepted as evidence of byssinosis. however, it is now realised that in this heavily industrialised part of the uk, emphysema is as common in the general population as in cotton workers and it can no longer be considered a component of byssinosis. other findings in byssinosis are more commensurate with asthma, namely an increase in bronchial muscle and mucous cells. no granulomas or other evidence of extrinsic allergic alveolitis are found. fever may be the predominant feature in a variety of occupational illnesses and the unifying term 'inhalation fever' has been proposed. however, the individual occupations are of interest and these conditions will therefore be considered separately. mill fever has been mentioned above under byssinosis. humidifier fever is an acute illness characterised by malaise, fever, myalgia, cough, tightness in the chest and breathlessness, all of which are worse on monday mornings if the humidifier responsible is at work rather than home. the chest complaints, and their aggravation on return to work after the weekend, are features shared with byssinosis (see above) but the general complaints fit better with extrinsic allergic alveolitis (see p. ). humidifier fever develops in circum-stances that also lead to the development of a form of extrinsic allergic alveolitis, and not surprisingly the same name has been extended to this latter condition, with inevitable confusion. both diseases are caused by microbiological contamination of humidifiers or air conditioners so that a fine spray of microorganisms is emitted into the office, factory or home. investigations have generally shown the baffle plates of the air conditioner to be covered with a slime of bacteria, fungi or protozoa (mainly amoeba and ciliates), and extracts of this have been used to identify precipitins in the patient's sera, as in extrinsic allergic alveolitis. however, unlike extrinsic allergic alveolitis, humidifier fever resolves within a day and leaves no permanent injury. for this reason there is seldom the opportunity to study the tissue changes, and partly for this reason it remains unclear whether the disease is mediated by immune complexes, as in extrinsic allergic alveolitis, or by endotoxins derived from the contaminants. a febrile illness occurring in precipitin-negative farm-workers after heavy exposure to fungi in their silos was attributed to inhaled fungal toxins and named pulmonary mycotoxicosis. it is also known as precipitin test-negative farmer's lung and organic dust toxic syndrome. the condition is generally self-limiting and is seldom biopsied but desquamative interstitial pneumonia and diffuse alveolar damage have been reported. , metal fume fever this is a self-limiting acute illness characterised by fever, sweating, myalgia, chest pain, headache and nausea, that comes on monday mornings when occupational exposure is experienced after a weekend's respite, as with bysinnosis and humidifier fever; during the week tolerance develops. , the disease involves the release of cytokines such as tumour necrosis factor and is presumed to have an allergic basis. the metals involved are chiefly zinc, copper and magnesium, and, to a lesser extent, aluminium, antimony, iron, manganese and nickel. occupations at risk include any that generate such metal fumes, but particularly welding. it is most commonly associated with welding zinc-coated surfaces. if the symptoms persist, alternative diagnoses, such as acute cadmium poisoning and other specific toxic metal fume diseases, should be suspected: these are not self-limiting and may cause severe bronchiolitis or diffuse alveolar damage (see above). this illness resembles metal fume fever except that it occurs without regard to previous exposure: no tolerance develops and there is therefore no particular susceptibility on mondays. the polymers concerned are quite inert, except when heated to produce fume: polytetrafluorethylene (ptfe, teflon, fluon, halon) is a notable example. as with other self-limiting diseases, little is known of the tissue changes. environmental irradiation chiefly affects the skin but in some parts of the world rocks near the surface release significant amounts of radon gas. this carcinogen is liable to accumulate in buildings and be inhaled, so subjecting the occupants to an increased risk of lung cancer. the installation of underfloor ventilation is therefore advocated in such areas. this subject is explored more fully on page . the body is vulnerable to both increases and decreases in pressure and it is the lungs that often bear the brunt of the damage. increased pressure may result in blast injury or crushing of the chest while decreased pressure may result in the lungs literally bursting or dissolved gases being released within the blood (caisson disease), or the vascular alterations that underlie mountain sickness developing. some of these pressure changes entail a risk of pneumothorax and it is essential that this is properly investigated postmortem by the chest being opened under a water seal. loud music has been incriminated as a specific form of air pressure change causing pneumothorax and metereologists have shown that 'spontaneous' pneumothoraces tend to occur in clusters associated with natural drops in atmospheric pressure. , explosions may cause injury by the body being violently thrown against a less moveable object, by objects being thrown against the body or by the blast wave hitting the body. these mechanisms often act together but sometimes there is only blast injury, to which the lungs are particularly vulnerable. for a time it was considered that the damage was direct, the blast wave travelling down the airways to injure the lungs. however, at the start of the second world war, experiments conducted in the uk showed that the lungs were injured indirectly, the blast wave being transmitted to them through the chest wall: pulmonary blast injury is worst on the side of the body towards the explosion, and can be reduced by protective clothing. underwater explosions are particularly dangerous because water is incompressible. there may be severe internal injury but no external evidence of damage other than a trickle of blood from the mouth or nose. this is because the injury is rate-dependent. quite small thoracic deform-ation may produce severe pulmonary damage if peak compression is attained very quickly, typically in less than ms. conversely, severe chest wall distortion may produce only minor pulmonary contusion if this time is extended beyond ms. at necropsy, the lungs are contused, with blood evident in the airways and parenchyma. depending on the force of the blast, the haemorrhage may be pinpoint, patchy or confluent. it tends to follow the lines of the ribs and may be accompanied by pleuropulmonary lacerations having the same distribution. in this case there will also be haemothorax, pneumothorax and possibly air embolism. patchy pulmonary haemorrhages cuff the blood vessels. , in patients who survive for a few days, the lungs resemble the liver macroscopically and histologically show chronic interstitial inflammation and fibrosis as well as haemorrhage. other injuries are often present and fat embolism, aspiration pneumonia, fluid overload and infection may all be added to the effects of the blast wave. 'chest squeeze' is another form of barotrauma caused by high pressure but here the body is compressed rather than subject to a sudden wave of pressure as in blast injury. it is experienced by divers who descend very deeply, thereby subjecting their bodies to such high pressure that their chest walls are literally crushed, so that their ribs break and their lungs are severely compressed. more common mishaps experienced by divers include drowning and decompression sickness, both of which are dealt with below, and neurological syndromes such as nitrogen narcosis, which will not be considered further. 'burst lung' is the most acute form of decompression sickness. it is experienced by divers and submariners making rapid ascents from depth and by aviators who ascend too rapidly in unpressurised aeroplanes, experience failure of a plane's pressure system or have to eject at high altitudes. injury to the lung is caused by trapped alveolar gas expanding so rapidly that it exceeds total lung capacity before it can escape through the trachea. the lungs literally burst: the alveolar walls rupture and blood mixes directly with alveolar air. the victim experiences chest pain and there may be blood-stained froth at the mouth or frank haemoptysis. air may enter the alveolar walls to cause interstitial emphysema or air embolism. asthmatics may be at particular risk because of regional air-trapping. . diving mammals such as porpoises and whales are protected from such dangers of peripheral air-trapping by cartilage extending far out into the finest conductive airways so that these passages never close, even at the end of full expiration (fig. . . ) . , patients requiring positive-pressure artificial respiration are also at risk of burst lung, but the complications of the resultant interstitial emphysema differ from those experienced by divers. in divers, the chest wall is buttressed by the surrounding water and air in the interstitium is liable to track towards the hilum of the lungs and enter pulmonary veins, with resultant cerebral and coronary air embolism, either of which may prove fatal. iatrogenic burst lung, on the other hand, takes place in patients whose chest wall is not so buttressed, and then outward rupture of the interstitial air is more likely, resulting in pneumothorax. extension of the interstitial emphysema to the mediastinum, neck and chest wall is also more likely in such patients, resulting in surgical emphysema at these sites. however, there are exceptional cases marked by both cerebral embolism and extensive air tracking. the same circumstances that lead to burst lung may also cause decompression sickness, which is also known as caisson disease. in this condition there is a sudden release of nitrogen gas that has gone into solution in the lipids of adipose tissue and of myelinated nervous tissue at the higher pressure: the released nitrogen gains access to the blood stream in which it forms bubbles. doppler ultrasound techniques show that this is quite customary when divers ascend from depth, but the lungs generally provide an effective filter so that there are no untoward systemic effects, although there may be sudden chest pain on deep inspiration ('the chokes'). gradual decompression permits the nitrogen to diffuse across the alveolar membranes and be exhaled. if, however, substantial amounts of nitrogen are released from solution, sufficient pulmonary arteries may be blocked to cause pulmonary hypertension, with resultant opening of arteriovenous communications or a patent foraman ovale, so permitting the gas to enter the systemic circulation. this is often followed by limb pains ('the bends') and perhaps cerebral symptoms ('the staggers'). fatal cases are characterised by gas bubbles within blood vessels throughout the body and froth in the heart chambers. delayed effects include ischaemic necrosis of bones and other tissues. deep-diving mammals are protected by the same mechanism that prevents them suffering from burst lung. they exhale before diving and during the dive the chest is compressed to the extent that virtually all the gas in the lungs passes into the cartilage-buttressed nonrespiratory airways (see fig. . . ) , resulting in very little to be absorbed by the blood. the pulmonary collapse also serves to reduce buoyancy. the distribution of the little gas that is absorbed is minimised by bradycardia. many viscera experience anaerobic respiration but hypoxia is minimised in the heart and musculature by high levels of haemoglobin and myoglobin. the brain is further protected by the supplying arteries drawing on oxygen stored in an unusual spongelike cervical organ known as the rete mirabilis. mountain sickness is due to reduced atmospheric pressure brought about more slowly than that responsible for decompression sickness . , it may be acute or chronic. acute mountain sickness is likely to be experienced by anyone who ascends above - m without a period of acclimatisation at intermediate levels. symptoms are as liable to occur in people born at high altitude who return after a few weeks spent at sea level as in those who go to the mountains for the first time: acclimatisation is obviously short-lived and is therefore necessary whenever an ascent is to be made. the ill-effects are commonly precipitated by exercise. in the susceptible, acute mountain sickness commonly appears within days of ascent. the basis of acute mountain sickness is tissue hypoxia. it results in deteriorating intellectual and psychological function, headache, nausea, vomiting, and more rarely pulmonary and cerebral oedema. high-altitude pulmonary oedema is characterised by increasing dyspnoea, cyanosis and a dry cough, and later the production of copious, frothy sputum, which sometimes becomes blood-stained. the pulmonary artery pressure is markedly raised but wedge pressures are normal, indicating that the left side of the heart is unaffected and that pulmonary venous constriction is unlikely to be an important contributory factor. the pulmonary oedema fluid has a high protein content and the condition has been characterised as a non-cardiogenic high-permeability oedema associated with excessive pulmonary hypertension. , hypoxia is a well-known cause of pulmonary arteriolar constriction but in acute mountain sickness the vascular response appears to be exaggerated for the pulmonary artery pressure is considerably higher than is usual for the altitude. an association with certain hla complexes (hla-dr and hla-dq ) suggests that this has a genetic basis. although arteriolar constriction only tends to protect the pulmonary capillaries, it could explain the oedema if the process was patchy -as is the resultant oedema -for patchy arteriolar constriction would subject the rest of the lung to abnormally high pressures and lead to capillary stress failure in these areas (see pp. and ). , measurements of capillary pressure suggest that this is indeed the case. furthermore, vasodilators such as calcium channel blocking agents and inhaled nitric oxide gas , , have been used with success to counter acute mountain sickness, supporting the idea that hypoxic vasoconstriction plays a central role. autopsy shows the lungs to be heavy and firm. the cut surface weeps oedema fluid, which is often blood-stained, but a striking feature is the patchy distribution of the changes. areas of haemorrhagic oedema alternate with others that contain clear oedema fluid and others that are normal apart from overinflation. pulmonary arterial thrombi are commonly found. microscopy confirms the presence of haemorrhagic oedema and may show neutrophils and hyaline membranes in the alveoli. the alveolar capillaries are congested and may contain thrombi. there may also be an increase in mast cells and rarely pulmonary infarction. the right ventricle is commonly dilated whereas the left ventricle is normal. highlanders generally show right ventricular hypertrophy and increased muscle in their pulmonary arteriesm, changes that are not apparent in lowlanders. , chronic mountain sickness prolonged residence at high altitude leads to hypoxic pulmonary hypertension (see p. ), an increase in red cell mass and cor pulmonale. livestock taken from lowland plains to high-altitude pastures suffer similarly but the natural stock of the himalayas and ethiopian highlands are apparently immune. so too are other species long established at high altitude such as the llama and yak. these species are said to have adapted to their climate, that is, the forces of natural selection have bred out the pulmonary vasoconstrictive response to hypoxia. cattle of european origin and humans acclimatise to high altitude by processes such as increasing their red cell mass but generally they are not adapted like native species and suffer hypoxic pulmonary hypertension at altitudes in excess of m. certain himalayan highlanders may be an exception to this in that their small pulmonary arteries are reported not to show the muscularisation that characterises hypoxic pulmonary hypertension. in cattle of european origin, the dependent oedema of right-sided cardiac failure caused by hypoxic pulmonary hypertension affects the breast (brisket) particularly and in the rocky mountains of north america such cattle are said to have 'brisket disease' . a human counterpart of this has been described in children of chinese ancestry who have been taken to reside in tibet and who have developed a fatal form of subacute infantile mountain sickness. a small minority of permanent residents in the andes develop the changes of chronic mountain sickness to a marked degree and are said to suffer from monge's disease. the basis of this is alveolar hypoventilation, which leads to a progressive fall in systemic arterial oxygen saturation and elevation of haemoglobin concentration to an unusually severe degree. the latter averages about g/dl, which exceeds even the g/dl found in healthy high-altitude residents. patients with monge's disease are so deeply cyanosed that their lips are virtually black. their pulmonary artery resistance is also markedly raised. the cause of the alveolar hypoventilation is uncertain but the only cases of monge's disease that have come to necropsy had conditions such as kyphoscoliosis that predispose to alveolar hypoxia. drowning is defined as suffocation by submersion, and usually occurs in water. it is the commonest cause of accidental death among divers but % of drowning accidents do not involve deep descents. falling into quite shallow water is a particularly common cause of drowning in young children. in adults, men outnumber women by to . more die in fresh water than the sea, not because it is more hazardous to the lungs than sea water, but because unguarded inland waters and swimming pools are visited more frequently. alcohol consumption contributes to many deaths by drowning. drowning is not simply a matter of being unable to keep one's head above water. this may be merely a secondary event. for example, the entry dive may result in underwater head injury, or the exertion of swimming may precipitate a heart attack. furthermore, the struggling swimmer going down for the third time ('drowning not waving') is the exception: most drowning is characterised by the swimmer failing to surface or quietly dropping beneath the surface without anyone noticing. swimming underwater can be extremely hazardous if it is preceded by hyperventilation, a danger that needs to be more widely appreciated. hyperventilation results in undue loss of carbon dioxide so that instead of hypercapnia forcing the swimmer to surface to breathe, progress under water may be continued until hypoxia causes sudden loss of consciousness. panic contributes to many swimming accidents and is often precipitated by the inadvertent aspiration of just a little water. most people are naturally buoyant, but only slightly so. with the lungs fully expanded the average adult has a positive buoyancy of about . kg, which is sufficient to keep the head out of the water if the rest of the body is submerged. if an arm (weight about kg) is raised to wave for help, the head will go down. if the swimmer shouts, exhalation reduces buoyancy to neutral at normal end-expiration and to negative at residual volume. buoyancy cannot be regained when the head is submerged and unless able to swim to the surface, the person will continue to sink. autopsy generally shows that the lungs are full of water, but some victims die of 'dry drowning' due to laryngospasm. events may also be modified by the temperature of the water. sudden immersion in cold water may result in tachycardia, hypertension and hyperventilation, making it difficult for the victim to keep the airways free of water. it may also result in sudden death due to ventricular fibrillation. even a good swimmer loses consciousness within an hour of immersion in very cold water. drowning is then inevitable unless a correctly fitted life jacket is worn, in which case there is a danger of death from hypothermia. however, as in open heart surgery, cold prolongs the interval before there is irreversible brain damage. if the person is rescued, water in the lungs is quickly absorbed, even if it is saline, and therefore hyperosmolar:aspirated sea water is quickly equilibrated by pure water joining it from the blood but the alveolar epithelial barrier remains impermeable to protein and once osmotic equilibrium is reached, all is quickly reabsorbed. [ ] [ ] [ ] fresh water is absorbed even more quickly. it is unnecessary to tip the patient to hasten this process. any water recovered in this way comes from the stomach and time that should be devoted to mouth-to-mouth breathing and cardiac massage is lost. these resuscitative efforts may need to be prolonged as fresh water in particular inactivates alveolar surfactant, leading to alveolar collapse which persists until the surfactant is replenished. very few victims who are resuscitated on site fail to survive, and very few who cannot be resuscitated on site recover later. interchange of fluid between the blood and air spaces may cause major fluctuations in plasma volume with consequent changes in ionic concentrations and haemolysis. hypervolaemia may cause circulatory problems but hyperkalaemia consequent upon the haemolysis is not thought to be as important as was formerly believed: ventricular fibrillation following submersion is more likely to be a complication of hypothermia than of electrolyte imbalance. circulatory collapse may ensue shortly after rescue. this is due to loss of the circulatory support provided by the pressure the water exerts on the body, which results in a considerable increase in cardiac output while the body is immersed. on leaving the water the loss of this support results in a tendency to venous pooling. although this is countered by baroreceptor responses, these are reduced by prolonged immersion in cold water. circulatory collapse is believed to be the cause of death in many persons who perish within minutes of rescue. to counter this effect, patients should be lifted out of the water in the prone position. it can be seen that, in fatal cases, the pathologist is faced with several possibilities. thus, death may have been due to: • natural causes before the body entered the water • unnatural causes before entry, the body merely being disposed of in the water • natural causes in the water • injuries received in the water from impact with rocks, a boat or a ship's propeller, or in tropical waters from predators such as a crocodile or a shark (any of which may also be incurred after death, as may disfigurement by fish and rats) • 'dry drowning' • true drowning • hypothermia • circulatory failure after rescue. true drowning is indicated by froth in the airways and heavy waterfilled lungs. both fresh and salt water contain numerous microscopic algae known as diatoms and those representative of the water in which the drowning occurred are found in the lungs. unless death occurred before submersion, diatoms are also found in other viscera because these tiny life forms easily enter the circulation. thus, the presence of diatoms in digests of organs such as the kidneys, liver, brain and bone marrow suggests that death was due to drowning. because they have a siliceous capsule, diatoms are resistant to putrefaction as well as digestion and can be identified in the body long after death. however, a positive test is not always accepted as proof of drowning and a negative test does not exclude drowning. the various physical forms in which respirable environmental agents may be encountered are defined in table . . . some effects of inhalant lung injury are recognised as distinct disease entities and are dealt with elsewhere: for example, the pneumoconioses on page , extrinsic allergic alveolitis on page , chronic bronchitis on page and lung cancer on page . other respirable agents, such as lead fume and carbon monoxide gas, exert their harmful effects elsewhere in the body and will not be considered further. this section is concerned with toxic substances that may be inhaled by the general public. those that are more likely to be encountered in the workplace or in war zones are considered on page . the lungs have a rather stereotyped pattern of response to inhaled toxins, displaying degenerative changes and inflammation of varying degree, the former sometimes amounting to necrosis. in general, the site of maximal absorption or injury is related to solubility (for gases and vapours) and particle size (for aerosols such as dusts, fog, fumes, mists, smog and smoke): the less water-soluble and the smaller the particle size, the further down the respiratory tract the agent will penetrate ( fig. [ ] [ ] [ ] thus, ammonia produces intense congestion of the upper respiratory passages and laryngeal oedema whereas phosgene has little effect on these sites but causes pulmonary oedema. air pollution [ ] [ ] [ ] [ ] [ ] the toxic (as opposed to allergenic) air pollutants thought to pose the greatest threat to the lungs comprise smoke particles, sulphur dioxide, oxides of nitrogen, various aldehydes and ozone. smoke and sulphur dioxide derive particularly from the combustion of fossil fuels in domestic fires and power stations, nitrogen dioxide is an important car exhaust and domestic gas appliance pollutant and ozone is the principal photochemical product of smog. aldehydes such as formaldehyde and acrylic aldehyde (acrolein) also contribute to general air pollution because they are released in the combustion of diesel oil and petrol. collectively, these pollutants have been incriminated in the exacerbation (rather than causation) of asthma. they also predispose to respiratory infection and result in airway inflammation and hypersecretion. , their effect on children is of particular concern because development of the lungs is known to continue well into childhood and damage to the lungs before their growth is complete is likely to be irreparable. at the other extreme of life episodes of severe air pollution are known to hasten the deaths of many patients with chronic airway disease. particularly high concentrations of the agents responsible for air pollution may be encountered in industry and their effects are therefore also considered in chapter . , on occupational diseases of the lung. many of the polycyclic hydrocarbons found in polluted air are carcinogenic (see p. ) and it is therefore not surprising that urban air pollution has been found to be associated with excess mortality from lung cancer. domestic air pollution is rife in many of the poorer parts of the world due to the burning of biomass (wood, dried cow dung, bagasse, straw) in unventilated living rooms for heating and cooking. the women are particularly at risk of developing chronic bronchitis while their children have an increased incidence of acute respiratory infections. , , a volcanic ash (tephra) irritates the eyes, skin and respiratory tract and in some eruptions may contain much free silica (e.g. montserrat in and mount st helens, washington state, usa in ) or be associated with the release of radon gas (e.g. the azores in ). the destruction of the world trade center in caused massive air pollution of new york city that had lasting respiratory effects on survivors, rescue workers and local residents. [ ] [ ] [ ] at the time of the disaster there was much smoke from combustion of aeroplane fuel and flammable materials in the building while the collapse of the twin towers released dust from cement and dry-wall partitions that was highly alkaline. [ ] [ ] [ ] this caused considerable irritation of the eyes and the conductive airways. a year later many victims were still suffering from bronchial hyperreactivity and poor ventilatory function, in a so-called reactive airways dysfunction syndrome , and there was continuing spirometric decline years later. the respirable portion of the dust formed only a small fraction of the whole but given the level of exposure its future effects cannot be discounted, particularly as it contained substances such as asbestos. unusual effects attributed to the disaster include acute eosinophilic pneumonia and granulomatous pneumonitis. , allergenic air pollutants are dealt with in detail in the sections on asthma (see p. ) and extrinsic allergic alveolitis (see p. ). allergenic air pollution is generally occupational or domestic but periodic widespread air pollution was responsible for the epidemics of asthma seen in barcelona in the s, which were eventually traced to ships discharging cargoes of soya flour (see p. ). smoking-related diseases figure large throughout this book and in this section they are merely summarised collectively. of the greatest importance, both in the number of patients they affect and in their clinical effects on the individual, are the various forms of chronic obstructive lung disease and lung cancer, but there are many other respiratory diseases associated with smoking, and a few that are less common in smokers (box . . ). not surprisingly, these diseases are often encountered in combination and sometimes one may obscure another. for example, a cigarette smoker may have emphysema in the upper lobes and idiopathic pulmonary fibrosis in the lower lobes. , alternatively, langerhans cell histiocytosis and desquamative interstitial pneumonia may affect the same parts of the lungs, in which case the focal lesions of the former may be masked by the latter condition. the term 'smoking-related interstitial lung disease' has been introduced to cover a spectrum of interstitial diseases related to smoking , , as well as being used in a more restricted sense to describe a combination of air space enlargement and interstitial fibrosis predominantly affecting the lower lobes. , , a quite advanced interstitial fibrosis has been reported in smokers with no clinical evidence of interstitial lung disease. b early changes detectable in smokers include chronic bronchiolitis, fibrosis of the bronchiolar wall and mild peribronchiolar interstitial fibrosis. , even earlier changes are detectable at the molecular level: as many as smoking-responsive genes that are significantly up-regulated or down-regulated have been identified in normal cigarette smokers. there is marked individual variation, which may explain why many lifelong heavy smokers experience no respiratory problems. histological evidence that a patient smokes is provided by an increase in the number of alveolar macrophages and a characteristic brown discoloration of cytoplasm due to the phagocytosis of tar and other particulate matter derived from tobacco smoke (fig. . . ) . cigarette smokers are at greater risk of lung disease than cigar and pipe smokers, probably because they inhale more deeply. they do this because cigarette smoke is more acid than cigar and pipe smoke and its nicotine content is therefore absorbed more easily through the lungs than the buccal mucosa. smokers obviously put their own health at greatest risk but the lesser hazards of passive smoking are now well recognized (see p. ). passive smoking involves both the smoke exhaled by others and that coming from smouldering tobacco between puffs, the latter being known as sidestream smoke. the harmful effects of maternal smoking on the unborn child also come in this category. they include increased airway responsiveness and reduced lung function during the neonatal period and an increased risk of sudden infant death syndrome. reduced numbers of alveolar attachments to the bronchioles have been demonstrated in such infants. smoking is also associated with disease of other organs (e.g. carcinoma of the oesophagus and bladder) but these are outwith the remit of this text. tobacco smoking by waterpipe (shisha, hubble-bubble) is enjoying a rise in popularity, both in its heartland, the middle east, and western countries, and wherever it is practised it is widely perceived as being less dangerous than smoking cigarettes. this is probably a mis conception. what evidence there is suggests that waterpipe tobacco smoking is just as harmful as cigarette smoking, if not more so. the lungs may be injured in burned patients in many ways (box . . ) , but an important consideration when a body is recovered from a fire is whether death was due to the fire or took place beforehand, the latter raising the possibility of foul play. a vital reaction to the skin burns and the presence of soot in the lower airways provide evidence that death occurred in the fire but an absence of soot from the airways may be due to death occurring rapidly, from asphyxia or poisoning by gases released in the conflagration. soot is cleared rapidly and if the patient survives a few days an absence of soot from the airways is to be expected. lung injury may result directly from heat and smoke inhalation or indirectly from the release of mediators associated with blast injury or shock. although air temperature in a fire may reach very high temperatures thermal injury seldom extends beyond the carina but more extensive injury from heat alone was seen in men exposed to steam escaping from a fractured boiler pipe. those dying immediately showed coagulative necrosis of the respiratory mucosa down to the level of the alveolar ducts and alveolar congestion and oedema, while those surviving a little longer exhibited diffuse alveolar damage. the diffuse alveolar damage probably represented a manifestation of shock from their extensive cutaneous scalding whereas the mucosal necrosis is directly attributable to heat. diffuse alveolar damage is extrinsic allergic alveolitis sarcoidosis blast injury asphyxia poisoning by combustion products (e.g. carbon monoxide, cyanide) direct thermal injury (largely limited to the trachea) irritant smoke, fume and gas (e.g. oxides of nitrogen, ammonia, acrolein, sulphur dioxide) hypovolaemic shock secondary to skin loss septicaemic shock from: infected skin burns infected central lines secondary viral and bacterial pneumonia fluid overload tracheostomy complications, including tracheobronchitis, pneumonia and barotrauma oxygen toxicity absorption of toxic topical disinfectants thromboembolism uraemia usually part of systemic multiorgan failure in these patients, and is the leading cause of death in burns. the ubiquity of plastics today means that smoke contains numerous irritants, including isocyanates, aldehydes and fluorinated organic chemicals. irritant smoke products have two principal effects. firstly, they cause an immediate painful stimulation of the eyes and respiratory tract which at low concentrations may prevent escape and at high concentrations may cause laryngeal spasm and death. secondly, they cause bronchopulmonary injury some hours after exposure. burned patients dying within - days often show tracheobronchial necrosis and diffuse alveolar damage with prominent hyaline membranes. , , secondary herpesvirus infection is often present. , the respiratory changes caused by heat and smoke are non-specific and careful consideration of the many causes of lung injury in burned patients listed in box . . and of the clinical circumstances and management is generally required. often it will be concluded that the cause of the lung injury is multifactorial. long-term consequences of smoke inhalation include bronchiectasis and obliterative bronchiolitis. methyl isocyanate, the chemical released at bhopal the bhopal catastrophe of was caused by the accidental release of tons of methyl isocyanate gas (ch -n=c=o) from a pesticide plant. over people were exposed, of whom died, mostly within hours of exposure, and were seriously injured. the victims complained of intense ocular and respiratory irritation. some survivors were left with persistent respiratory impairment, which was thought to be due to obliterative bronchiolitis. , methyl isocyanate is an extremely potent respiratory irritant, destroying the epithelium throughout the conducting airways, with comparatively less parenchymal injury. in survivors, epithelial regeneration, often involving squamous metaplasia, quickly commences, but not before endobronchial granulation tissue projections have developed, resulting in obliterative bronchiolitis. tear gases are chemical irritants delivered as an aerosol for the purpose of riot control. they react with mucocutaneous sensory nerve receptors causing intense irritation of the eyes, mucous membranes and skin. the respiratory effects are mainly concentrated on the upper tract so that there is violent sneezing, severe rhinorrhoea and cough but there may also be tracheobronchitis and rarely pulmonary oedema. patients with pre-existent asthma or chronic obstructive pulmonary disease are most severely affected while others may be left with reactive airways dysfunction. toxins reaching the lungs via the blood stream may be drugs, food contaminants, metabolites produced elsewhere in the body, or chemicals ingested intentionally or accidentally, either in the home or the workplace. the lungs are selectively damaged by certain blood-borne toxins for a variety of reasons. for example, the herbicide paraquat is preferentially taken up by the lungs because of its molecular homology with certain endogenous substances. as detailed below, the type i alveolar epithelial cells are the cells that bear the brunt of the damage in paraquat poisoning. on the other hand, the alveolar capillary endothelium has its own selective uptake mechanisms (see metabolic functions of the pulmonary endothelium, p. ) which may be responsible for it being selectively damaged by other chemicals. the bronchiolar clara cells are selectively injured by some ingested chemicals because they are equipped to deal with inhaled xenobiotics, but occasionally this activity results in metabolites that are extremely toxic. an example of this from veterinary medicine is provided by the furan-derivative -ipomeanol, which is found in mouldy sweet potatoes and results in acute pulmonary oedema in cattle fed such a diet. when this chemical is injected into mice, the bronchioles are denuded of clara cells whereas the intervening ciliated cells are completely unaffected. the selective damage to the bronchiolar clara cells appears to stem from the oxidative efficiency of their p- cytochromes, which is much higher than those of the liver. chemicals having a similarly selective effect on bronchiolar clara cells include -methylfuran, carbon tetrachloride, naphthalene and , -dichloroethylene, the last of which is a volatile compound that is widely used in the plastics industry. procarcinogens may be activated in the airways by similar mechanisms. paraquat is a dipyridylium compound that is widely used in agriculture as a herbicide. it kills all green plants but is inactivated on contact with the soil. it is applied as a spray and if the manufacturer's instructions are followed there is no danger to health. most fatal cases of paraquat poisoning, both accidental and suicidal, have been due to ingestion of the % aqueous solution gramoxone. the less concentrated granular form weedol is unlikely to be ingested accidentally but may be taken suicidally. paraquat is not absorbed by the intact skin but repeated or prolonged application damages the epidermis so that absorption into the blood stream with consequent systemic effects is possible, but rare. although paraquat has toxic effects on the liver, kidneys and myocardium, these are transient and attention has centred on the pulmonary changes, which are usually fatal. following suicidal ingestion of large amounts of paraquat, death from multiorgan failure and pulmonary haemorrhage occurs within a few days, whereas most victims of accidental paraquat poisoning die from progressive pulmonary fibrosis between and days after ingestion. in those who survive longer, a honeycomb pattern of pulmonary fibrosis may be apparent. paraquat is a powerful oxidant and owes its toxicity to the production of active oxygen radicals. the lungs are particularly susceptible because paraquat is concentrated there by an active uptake mechanism in the alveolar epithelium. the inadvertent uptake of paraquat probably stems from a similarity between the molecular arrangement of its quaternary nitrogen atoms and the amine groups of endogenous oligoamines such as putrescine, spermidine and spermine, which are concerned in alveolar epithelial cell division and differentiation (fig. . . ) . this results in paraquat levels being - times higher in the lung than in the plasma. once taken up by the lung, paraquat is not metabolised but participates in redox cycling so that superoxide radicals are constantly produced. epithelial injury is proportional to the concentration of paraquat, while it is lessened by hypoxia and antioxidants such as superoxide dismutase, and potentiated by increased concentrations of oxygen. [ ] [ ] [ ] [ ] the high concentration of oxygen in the alveoli is a further reason why the lungs are particularly vulnerable to paraquat. knowledge of the toxic effects of paraquat comes from observations on autopsy series , , and from experimental studies that have enabled the sequence of pulmonary changes to be observed. [ ] [ ] [ ] [ ] in accordance with paraquat being taken up by the alveolar epithelium, electron microscopy shows that these cells suffer more profound damage than the endothelium. type i epithelial cells swell and undergo necrosis (fig. . . ), whilst type ii cells, although remaining capable of proliferation, show ultrastructural evidence of damage with derangement of cell organelles. , histological changes in the lungs follow the pattern of diffuse alveolar damage, with a characteristic feature of the early exudative phase being intense vascular congestion and alveolar haemorrhage. , , hyaline membranes are most clearly seen by about days (fig. . . ) and epithelial proliferation and fibrosis are conspicuous by about days. the pattern of pulmonary fibrosis in paraquat poisoning has been disputed. some authors have stressed its interstitial position, whereas others have clearly demonstrated that it is intra-alveolar. , , [ ] [ ] [ ] [ ] however, as described on page , it generally assumes an obliterative pattern of intra-alveolar fibrosis in which the lumina of several adjacent alveoli are totally effaced, rendering them completely airless (see fig. . , p. ). a new multisystem disease appeared abruptly in the environs of madrid in . [ ] [ ] [ ] over people were affected and about in died. the disease was initially thought to be mycoplasma pneumonia but was soon found to be associated with the use of adulterated oil sold illicitly by door-to-door salesmen. although it was sold for culinary purposes the oil had been produced for industrial use in steel manufacture. it consisted of rapeseed and olive oil mixed with liquified animal fat, aniline and other organic chemicals. it has not been possible to identify the exact chemical responsible for the disease or to reproduce the changes in other species but the later induction of similar pathological changes by another substance contaminated with an aniline derivative is possibly relevant (see l-tryptophan-induced eosinophilia-myalgia syndrome, p. ). some clinical and pathological features of the disease suggest that immune mechanisms may also be involved. the initial clinical features included fever, respiratory distress, cough, haemoptysis, skin eruptions and marked eosinophilia. radiographs suggested pulmonary oedema and sometimes showed pleural effusion. about % of patients died at this stage but most recovered quickly. however, within a few weeks many were readmitted to hospital with nausea, vomiting, diarrhoea and abdominal pain. about a quarter then proceeded to develop weakness, myalgia, weight loss, scleroderma-like skin signs and pulmonary hypertension. , many of these patients died after a long, wasting illness or are permanently disabled with neurological and hepatic disorders. in the early phase the lungs showed the most severe changes, which consisted of a combination of diffuse alveolar damage, eosinophilic infiltrates and arterial luminal narrowing by endothelial swelling and vacuolation, intimal foam cell infiltration and a non-necrotising vasculitis. , , there was also capillary thrombosis, which later extended into arteries and veins, culminating in fibrosing obliteration of these blood vessels. in some patients dying of haemoptysis, dilated thin-walled blood vessels were identified in the mucosa of major blood-filled airways. late features in the lungs included plexogenic arteriopathy (see p. ), possibly secondary to changes in the liver. similar inflammatory and vascular changes were seen in many other tissues. notable extrapulmonary features included fasciitis, vasculitis, neuronal degeneration, perineuritis, hepatic injury and tissue eosinophilia. sauropus androgynus is a vegetable that is widely cultivated for the table in many south-eastern asian countries. it is apparently harmless when cooked but recently there has been a vogue in taiwan for consuming large amounts of its unprocessed juice, blended with that of guavas or pineapple, because of its supposed efficacy as a slimming aid and in blood pressure control. coincident with this fad there has been an upsurge in patients with symptoms of obstructive lung disease. within a -month period more than such patients were seen at one hospital. [ ] [ ] [ ] they had four features in common: recent consumption of uncooked s. androgynus juice, fixed ventilatory obstruction, radiological evidence of bilateral bronchiectasis and an absence of any previous chronic respiratory disease. four patients agreed to undergo open-lung biopsy. this showed chronic bronchiolitis or obliterative bronchiolitis of constrictive pattern. the lymphocytes were mainly t cells but immunofluorescent and electron microscopy showed no evidence of an immune process. four patients underwent single-lung transplantation. the excised lungs showed sclerotic obliteration of bronchial arteries in the walls of bronchi - mm in diameter with segmental necrosis of bronchi - mm in diameter. the changes were considered to fit best with segmental ischaemic necrosis of bronchi at the watershed zone of the bronchial and pulmonary vasculature. further patients have required lung transplantation but public education of the dangers of this herbal medicine now appears to have been successful. alcohol and nicotine outstrip all other recreational drugs in popularity and their effects are of course well known. those of tobacco smoking are summarised above and dealt with in detail in the chapters on obstructive lung disease (chapter ) and carcinoma of the lung (chapter . ). less well known is the lung disease that results from smoking blackfat tobacco, a practice popular with guyanese indians. blackfat is the trade name of a type of tobacco that is flavoured with mineral oil, some of which vaporises and is inhaled when the tobacco is smoked, to cause exogenous lipid pneumonia (see p. [ch ] ). in recent years the smoking of two other substances, marijuana and cocaine, has gained in popularity. it would not be surprising if the long-term effects of smoking these substances were similar to those of cigarette smoking but as yet it is too early to judge. however, the short-term effects are similar to those of tobacco smoking and this bodes badly for their ultimate effects. marijuana consists of the dried leaves of the cannabis plant, also known as hemp, as opposed to hashish, which is the plant's resin, and a further extract known as 'weed oil' . all these substances are smoked because they contain cannabis alkaloids which have psychoactive effects. however, this habit also exposes the lungs to many of the same respiratory irritants that are found in tobacco smoke. initial exposure to marijuana smoke often results in coughing while habitual smokers produce black sputum. bronchial biopsy shows inflammation and squamous metaplasia and bronchoalveolar lavage demonstrates increased numbers of cells, which are predominantly macrophages but also include neutrophils. [ ] [ ] [ ] [ ] [ ] these changes are virtually identical to the short-term effects of tobacco smoke and are therefore likely to be similarly followed by the development of chronic obstructive lung disease and lung cancer. indeed, the dangers of smoking marijuana are probably greater than those of smoking tobacco as compared with tobacco smoking it is associated with a fivefold greater increase in blood carboxyhaemoglobin and a threefold increase in the amount of tar inhaled. it is estimated that three cannabis cigarettes result in the same degree of bronchial damage as tobacco cigarettes. there is also evidence that the effects of smoking marijuana and tobacco are additive. not surprisingly therefore, epidemiological studies report a doserelated impairment of large-airway function in marijuana smokers. there are also several reports attributing pneumothorax to marijuana smoking ( fig. . . ) . , the pneumothorax may be spontaneous or develop during the deep, sustained inspiratory effort involved in smoking marijuana (or cocaine), which may be enhanced by a partner applying positive ventilatory pressure by mouth-to-mouth contact. thoracoscopy in such cases has shown predominantly apical, irregular bullous emphysema, while lung biopsy has demonstrated widespread alveolar filling by heavily pigmented macrophages. , evidence is also beginning to accumulate that long-term cannabis use increases the risk of lung cancer. smoking cannabis in the form of weed oil is also reported to result in exogenous lipid pneumonia. cocaine cocaine hydrochloride is a fine white powder derived from the leaves of the plant erythroxolon coca by a complex chemical process. it is heat-labile and therefore cannot be smoked. users inject it intravenously or inhale it unheated through the nose, the latter practice being known as 'snorting' . however, a heat-stable free-base form that can be smoked is easily prepared from the hydrochloride with baking powder and a solvent such as ether. this process results in a crystalline deposit that is known as 'rock' because of its appearance or 'crack' because of the crackling sound it emits when heated. when smoked, the cocaine is readily absorbed and an intense surge of euphoria is experienced within seconds. the intravenous route takes twice as long and 'snorting' several minutes. the hard addict therefore prefers to smoke 'crack' . a variety of pulmonary complications of smoking free-base cocaine has been reported. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] acute effects include cough, shortness of breath, chest pain and haemoptysis. asthma may be aggravated, black sputum is produced, and pneumothorax and interstitial emphysema have resulted from valsalva manoeuvres undertaken in the belief that they promote even more rapid absorption. biopsy has shown pulmonary congestion and oedema, organising pneumonia, haemorrhage, haemosiderosis, diffuse alveolar damage and interstitial pneumonia or fibrosis. less common effects include eosinophilic pneumonia, extrapulmonary eosinophilic angiitis, medial thickening of pulmonary arteries and the barotrauma described above (see fig. . . ) . severe burning of the airways has also been seen due to 'crack' being smoked before all the ether used in its preparation has evaporated. 'snorting' unheated cocaine has its own complications: substances such as cellulose or talc with which the drug is 'cut' (mixed as a diluent) are liable to provoke a foreign-body giant cell reaction in the lungs (fig. . . ). however, particles of foreign material larger than those in the usual respirable range (allowing for the fibrous shape of substances such as cellulose) should suggest intravenous use (see 'filler embolism', below). heroin is usually injected, but it may be smoked, when, as with marijuana, it is liable to lead to a very pronounced macrophage response. intravenous heroin abuse sometimes causes the sudden onset of a potentially fatal high-permeability pulmonary oedema (fig. . . ). intravenous abuse of heroin and other drugs is also liable to cause 'filler embolism', which will now be considered. 'filler embolism' is the result of illicit drug usage in which compounds designed for oral use are injected intravenously to heighten their effects. oral preparations consist largely of fillers such as talc or starch and this insoluble particulate matter accumulates in the pulmonary capillaries. it provokes a foreign-body giant cell reaction, thrombosis and fibrosis and may cause pulmonary hypertension ( fig. this 'designer' drug, taken for its central stimulant activity (street names 'ice' or 'u- -e-uh', pronounced euphoria), is related to the appetite suppressor aminorex, discussed on page , and has similarly been associated with pulmonary hypertension. assessing whether a particular clinical manifestation represents an adverse drug reaction considers previous experience with the drug, alternative aetiological agents, the timing of events, drug levels, and the effect of withdrawing the drug and rechallenge with the drug. it is worth bearing in mind that: • one drug may cause several patterns of disease. • one pattern of disease may be produced by a variety of drugs. • a drug reaction may develop long after the drug has been withdrawn. • a drug reaction may develop suddenly even though the dose of the drug has not been altered. • drug effects may be augmented by factors such as age, previous radiotherapy and elevated oxygen levels. • drug reactions may be localised. • many drugs cross the placenta to affect the fetus. an alternative classification of adverse drug reactions, which is more appropriate to pathology practice and which will be followed here, is one based on the pattern of disease. some pathological patterns of drug-induced lung disease are shown in table central depression of respiration occurs as a side-effect of barbiturates, morphine and its derivatives, and even mild sedatives, and may be particularly troublesome in patients suffering from chronic obstructive lung disease. ventilation in such patients may be largely dependent on hypoxic respiratory drive and treatment with oxygen may therefore also have an adverse effect on respiration by lowering the degree of hypoxia and so diminishing the stimulation of the respiratory centre. peripheral impairment of the respiratory drive may be brought about by aminosides and other antibiotics, while corticosteroids may result in a myopathy affecting the respiratory muscles. other iatrogenic hazards affecting the peripheral nerves controlling respiration include nerve root disease complicating immunisation and surgical damage to the spinal and phrenic nerves. asthmatic patients are particularly susceptible to exacerbations of their disease by drugs (box . . ). this effect may occur either as a predictable pharmacological side-effect of the drug or as an idiosyncratic response. examples of the former include β-adrenergicic antagonists and cholinergic agents while examples of the latter include sensitivity to the colouring agent tartrazine, for which reason many manufacturers have eliminated tartrazine from their red, orange and yellow tablets. allergic bronchoconstriction also forms part of generalised anaphylactic reactions induced by vaccines and antisera and occurs as a localised response to penicillin, iodine-containing contrast media, iron dextran and other medicaments. bronchospasm may also be initiated by the non-specific irritant effect of inhaling nebulised drugs if they are prepared as a hypotonic solution, a side-effect that is prevented by using isotonic solutions. aspirin and other non-steroidal anti-inflammatory agents aspirin-induced asthma has been recognised for many years and more recently several of the newer anti-inflammatory drugs have been found to exacerbate asthma in certain sensitive individuals. the basis for this is uncertain but the likelihood of an individual antiinflammatory drug provoking an asthmatic response is related to its potency as an inhibitor of prostaglandin cyclooxygenase pathway, resulting in the production of leukotrienes. [ ] [ ] [ ] as well as asthma being exacerbated by drugs, the disease has been caused by occupational exposure in the pharmaceutical industry to certain drugs which can be inhaled during manufacture, notably penicillin, cephalosporin, methyldopa, cimetidine and piperazine. obliterative bronchiolitis of the constrictive type has been reported with penicillamine , and gold , but in many cases it is possibly the underlying condition rather than the drug that is res ponsible (see p. ). this is often rheumatoid disease, which is sometimes complicated by bronchiolitis obliterans whether the patient is under treatment or not. organising pneumonia extending into peripheral bronchioles (see p. ) may be seen with a variety of drugs but results in a restrictive rather than obstructive lung defect and is to be regarded as a cytotoxic effect of the drug acting primarily at the alveolar level (see below). raw sancropus androgyns taken as a slimming aid causes severe obliterative bronchiolitis (see p. ). the cytotoxic effects of drugs may be acute or chronic, leading to changes as varied as pulmonary oedema, diffuse alveolar damage, pulmonary haemorrhage and haemosiderosis, organising pneumonia, interstitial pneumonitis and interstitial fibrosis. , some of the most severe acute effects are seen with the chemotherapeutic agents used in malignant disease but they are also recorded with drugs that are not traditionally thought to be cytotoxic, e.g. desferrioxamine administered as a prolonged intravenous infusion in acute iron poisoning. pulmonary toxicity due to busulphan was first described in , and has been the subject of several subsequent studies. [ ] [ ] [ ] [ ] it remains the mainstay of treatment for chronic myeloid leukaemia. like other alkylating agents, it acts by cross-linking dna strands. clinical estimates of the incidence of pulmonary toxicity vary around % but subclinical damage is thought to be much more common. although not strictly dose-dependent, toxicity is rarely seen with a total cumulative dose of less than mg. synergy with radiation and other cytotoxic drugs occurs. similar effects have been reported for most cytotoxic agents, particularly bleomycin. pulmonary toxicity is seen less commonly with other alkylating agents, such as cyclophosphamide and melphalan. [ ] [ ] [ ] [ ] bleomycin is a cytotoxic antibiotic derived from streptomyces species. it is widely used in the treatment of neoplasms such as lymphomas and germ cell tumours, and is thought to produce its therapeutic and toxic effects by altering the normal balance between oxidants (active oxygen radicals) and antioxidant systems. bleomycin produces superoxide radicals when incubated with oxygen and iron in vitro. oxygen enhances its effects, a fact well known to anaesthetists who accordingly take care to limit concentrations of inspired oxygen to % in patients on bleomycin who are undergoing surgery. [ ] [ ] [ ] radiotherapy and cytotoxic agents such as bleomycin are also synergistic. bleomycin is preferentially concentrated in the lungs and pulmonary fibrosis can be produced in animals when it is administered intravenously, intraperitoneally or by intratracheal instillation. electron microscopy shows that the early changes consist of swelling and vesiculation of endothelial cells, interstitial oedema and type i epithelial cell necrosis. , the reported incidence of bleomycin toxicity varies from to % depending on the type of patient being treated and on dosage. in general, toxic effects increase with age and cumulative dose: above a total dose of about units they rise significantly. the acute morphological changes attributable to drugs include pulmonary oedema and diffuse alveolar damage. acute pulmonary oedema is seen in heroin addicts who die while injecting themselves intravenously but it is also seen in patients administered a variety of drugs therapeutically, for example hydrochlorothiazide, salicylate, opiates, vinorelbine,and desferrioxamine. the oedema is of the high permeability type (see p. ), rich in protein, and is occasionally haemorrhagic or accompanied by the hyaline membranes of diffuse alveolar damage. diffuse alveolar damage has alveolar epithelial necrosis as its basis (figs . . and . . ). however, the continuing action of many cytotoxic drugs affects the regeneration process so that atypical type ii epithelial cells develop, a characteristic feature that was first described with busulphan and subsequently with bleomycin. , these two drugs differ chemically but both act (by different mechanisms) on dna. the atypical cells have abundant deeply eosinophilic or amphophilic cytoplasm and large nuclei, which may be multiple but are usually single. the nuclei measure up to µm and are densely stained throughout or contain either large homogeneous deeply eosinophilic inclusions or clear vacuoles (fig. . . ) . electron microscopy distinguishes the inclusions from nucleoli and shows them to consist of tubular aggregates derived from the internal nuclear membrane. airway epithelium shows similar nuclear changes and often undergoes squamous metaplasia. the presence of such cells in sputum specimens submitted for cytology can lead to a misdiagnosis of malignancy. fibrosis may follow diffuse alveolar damage or develop insidiously, perhaps many years after drug therapy ceased (fig. . . ) . it may be both interstitial and intra-alveolar. the interstitial component is often accompanied by a non-specific chronic inflammatory infiltrate. the proportions of inflammation, which is potentially reversible, and fibrosis, which when collagenous is irreversible, obviously bear on the prognosis. however, most case reports antedate the recent classification of interstitial pneumonia described in chapter and it is uncertain how their pathological appearances would now be classified. the majority lack the classic features of usual interstitial pneumonia and fibrotic non-specific interstitial pneumonia. many show overlapping patterns of intersitital pneumonia and this alone should arouse suspicion that a drug may have been responsible. however, some cytotoxic drugs result in pulmonary changes by more than one mechanism: for example, methotrexate may produce hypersensitivity reactions with granuloma formation [ ] [ ] [ ] [ ] or pulmonary eosinophilia as well as diffuse alveolar damage. pulmonary toxicity is also occasionally seen in patients undergoing treatment with gold salts for rheumatoid disease: in addition to diffuse alveolar damage, there may be eosinophilia and dermatitis in these cases, again indicating possible hypersensitivity. nitrofurantoin is another example of a drug resulting in a variety of patterns of alveolar injury: diffuse alveolar damage, desquamative interstitial pneumonia, giant cell interstitial pneumonia, organising pneumonia and eosinophilic pneumonia have all been recorded in association with this drug. [ ] [ ] [ ] it should also be noted that in patients with neoplastic disease, clinical features suggestive of a pulmonary drug reaction may be due to factors other than drugs. in leukaemic patients, for example, these include direct infiltration of the lungs by leukaemic cells, opportunist infection and, if bone marrow transplantation has been undertaken, the effects of irradiation and possibly graft-versus-host disease. phospholipidosis is encountered with drugs such as the antidysrhythmic agent amiodarone, which block lysosomal enzymes involved in the breakdown of complex lipids. this leads to their accumulation throughout the body but the effect is most marked in tissues that take up the drug and contain cells rich in lysosomes. the lung fulfils both these requirements through its rich complement of alveolar macrophages. these cells accumulate the enzyme substrate (phospholipid) in their cytoplasm with the result that large foam cells fill the alveoli (fig. . . ). the appearances are those of endogenous lipid pneumonia, similar to that seen in obstructive pneumonitis. however, with amiodarone cytoplasmic vacuolation is also seen in epithelial and interstitial cells. the phospholipid inclusions contained within the vacuoles are particularly well seen in unstained frozen sections viewed by polarised light. identical changes to those induced by amiodarone were seen in the lungs of rats exposed to very high levels of the antidepressant drug iprindole and the anorectic drug chlorphentermine. these three compounds, iprindole, chlorphentermine and amiodarone, all belong to the amphiphilic group of drugs which block lysosomal phospholipase and sphingomyelinase. although their pharmacological actions are very different, a molecular homology is apparent (fig. . . ) . it is likely that all patients receiving substantial amounts of amiodarone develop phospholipidosis throughout the body, but this is generally well tolerated. only a minority experience respiratory impairment and in these there is also evidence of pulmonary inflammation and fibrosis, which is possibly mediated immunologically. these patients generally have a restrictive lung deficit, the onset of which may be acute or chronic. bronchoalveolar lavage shows foamy macrophages but these cells indicate exposure to the drug rather than drug toxicity; nor are they specific to amiodarone, being observed on occasion with other drugs. lymphocytes of suppressor type may also be detected on lavage. histologically, amiodarone toxicity is diagnosed on a combination of phospholipidosis and interstitial pneumonia and fibrosis. occasionally the hyaline membranes of diffuse alveolar damage are superimposed on the interstitial changes (see fig. . . ) . [ ] [ ] [ ] in some patients the fibrosis is intraalveolar rather than interstitial and the appearances are those of organising pneumonia. the process may be localised and mimic a neoplasm radiologically. , amiodarone toxicity is probably dose-dependent but there is considerable individual variation in the amount required, , which appears to be under genetic control. amiodarone toxicity is uncommon in patients taking daily doses of mg or less whereas the there are drugs that undoubtedly cause a usual interstitial pneumonia pattern, for example the chemotherapeutic agents and nitrofurantoin (fig. . . ), while others, for example the statins, are recorded as having induced a non-specific interstitial pneumonia pattern. a drug history is therefore imperative when assessing any patient with diffuse parenchymal lung disease. organising pneumonia similar to the cryptogenic condition described on page , and probably similarly reversible with steroids, has been encountered with a variety of drugs, including amiodarone, sulphasalazine and pencillamine. penicillamine has also been incriminated in the development of both diffuse alveolitis and bronchiolitis obliterans, but both these changes could well be due to the underlying rheumatoid disease for which the pencillamine is administered. in busulphan lung there may be an organising intraalveolar fibrinous exudate, which at its most extreme results in irreversible effacement of the alveolar architecture by sheets of loose connective tissue (see p. ). with continued experimental administration of the drug iprindole mentioned above, the phospholipidosis it produced gradually evolved into alveolar proteinosis (more properly called lipoproteinosis; see p. ), but this has not been reported as a drug effect in humans. alveolar proteinosis has however been recognised in a number of patients receiving chemotherapy for conditions such as leukaemia. the mechanism here is probably based on the cytotoxic action of the drug and the material filling the alveoli may represent the detritus of degenerate alveolar cells rather than excess pulmonary surfactant, as in the primary auto-immune form of alveolar proteinosis. eosinophilic pneumonia, the pathology of which is described on page , may be caused by several drugs, including nitrofurantoin, para-aminosalicylic acid, sulphasalazine, phenylbutazone, gold compounds, aspirin and penicillin (see box . , p. ). , it may also follow radiation to the chest. the tissue eosinophilia is generally accompanied by a rise in the number of eosinophils in the blood. the clinical picture varies from transient asymptomatic opacities on a chest radiograph to a life-threatening illness with severe respiratory distress and hypoxaemia, so-called acute eosinophilic pneumonia (see p. ). the reaction is often associated with a florid rash. withdrawal of the drug may be all that is required to effect resolution but corticosteroids are usually given as they produce a marked improvement. this syndrome of necrotising granulomatosis, vasculitis and eosinophilia in asthmatic patients, which is described more fully on page , has been reported when leukotriene receptor antagonists have been used to treat asthma. however, it is likely that the syndrome has been merely unmasked by the antileukotriene permitting a reduction in corticosteroid dose rather than representing a direct effect of the antileukotriene. , mesalazine has also been implicated in inducing a vasculitis during treatment for inflammatory bowel disease. the eosinophilia-myalgia syndrome was identified in the usa in and quickly identified as being due to the ingestion of ltryptophan from one particular japanese supplier. withdrawal of this substance led to the virtual elimination of the disease, but not before patients had been affected, in fatally. [ ] [ ] [ ] [ ] [ ] cases were subsequently described in europe where there were further fatalities. l-tryptophan is an essential amino acid that is freely available to the public: its purchase does not require a medical prescription. it has been promoted as a dietary supplement and as an agent against insomnia and premenstrual tension. women in the reproductive years preponderated in the patients affected by the resultant eosinophiliamyalgia. the clinicopathological features of the syndrome are similar to those of the spanish toxic oil syndrome (see p. ) and differ more in degree than type. the discovery of an aniline-derived contaminant in the tryptophan-induced condition is a further link connecting these two syndromes. an immune basis is suggested by the identification of t lymphocytes activated against fibroblasts in the eosinophilia-myalgia syndrome. the illness is a multisystem disorder and besides blood eosinophilia and myalgia there may be arthralgia, fever, rash and involvement of the lungs, liver and central nervous system. as in the toxic oil syndrome, there is fasciitis, wasting and muscle pain associated with blood and tissue eosinophilia. the lungs are affected in % of cases. pulmonary symptoms have included cough, dyspnoea and chest pain. radiographs have shown diffuse bilateral infiltrates and pulmonary hypertension has been documented in a few cases. histology of the lungs shows an oedematous myxoid intimal thickening affecting small pulmonary blood vessels and a diffuse interstitial lymphocytic and eosinophilic infiltrate. , , , , these cells may also be seen within the walls of the thickened blood vessels (fig. . . ) . , massive ingestion of l-tryptophan has resulted in the appearances of an organising pneumonia. as an adverse drug reaction, granulomatous alveolitis is best exemplified by the extrinsic allergic alveolitis of pituitary snuff-takers, but it is also encountered on rare occasions with cytotoxic and other drugs, including methotrexate, bacille calmette-guérin (bcg) immunisation, interferons, ciprofloxacin, antiviral therapy and tumour necrosis factor antagonists. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the histological appearances may suggest extrinsic allergic alveolitis or sarcoidosis but the centri-acinar or lymphangitic concentration of these conditions is usually lacking. however, unless an infective agent can be demonstrated the diagnosis generally requires consideration of the clinical and environmental details, including any drug regimen. exogenous lipid pneumonia may result from the unintentional aspiration of various fat-based medicaments such as liquid paraffin, oily nose drops and petroleum jelly or of fat-rich dietary supplements in the form of ghee. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the consumption of liquid paraffin as an aperient is common in some countries and may be taking place without the knowledge of the patient's medical practitioner. regurgitation and aspiration of ingested oil are especially likely to happen during sleep in the presence of a hiatus hernia or when the oesophagus fails to empty completely into the stomach because of achalasia of the cardia. the aspiration of vegetable oil occurred in the past from the use of menthol in olive oil for the treatment of tuberculous laryngitis, and occasionally from the use of iodinated vegetable oils for bronchography. [ ] [ ] [ ] [ ] more recently exogenous lipid pneumonia has developed from the constant sucking of lollipops formulated for the administration of the analgesic fentanyl but also containing a stearate component. the treatment of epistaxis by nasal packing with paraffin gauze has also led to exogenous lipid pneumonia. the pathology of exogenous lipid pneumonia is described on page . other medicines may also be aspirated unwittingly, for example a ferrous sulphate tablet may cause brown iron staining and necrosis of the bronchus at the point of impact, progressing to bronchial stenosis. [ ] [ ] [ ] distal infection is then likely, as with any foreign body. barium sulphate aspiration may complicate gastrointestinal radiography. large amounts may impair ventilation but being inert there is no permanent injury to the lungs, although the striking changes are evident on the chest radiograph. an outbreak of pulmonary hypertension affecting many swiss, austrian and german patients in the period - was probably due to the anorectic drug aminorex, which was accordingly withdrawn with regression in the number of new cases. the pathology in these patients was identical to that of primary pulmonary hypertension (see p. ) and it proved impossible to reproduce the condition in laboratory animals but the epidemiological evidence that aminorex was to blame is very strong. fenfluramine and phentermine, further anorectic drugs that are chemically similar to aminorex, have also been associated with such plexogenic pulmonary hypertension, [ ] [ ] [ ] [ ] [ ] and with fibroproliferative plaque on the tricuspid valve and pulmonary arteries. pulmonary hypertension due to pulmonary veno-occlusive disease has sometimes complicated the use of cytotoxic chemotherapeutic agents or followed bone marrow transplantation. non-steroidal anti-inflammatory agents such as indomethacin and diclofenac cross the placenta and, if given in late pregnancy, may cause premature closure of the ductus arteriosus, resulting in severe neonatal pulmonary hypertension. , pulmonary hypertension is a well-recognised association of human immunodeficiency virus (hiv) infection but until recently has been unexplained. now, however, evidence is emerging that the highly active antiretroviral therapy administered to hiv-positive patients might be responsible for the pulmonary hypertension. the older high-oestrogen contraceptive drugs carried a slight risk of thromboembolism but this is not seen with the newer preparations. pulmonary thromboembolism has also occurred with a drug-induced lupus syndrome associated with anticardiolipin antibodies. chemotherapeutic drugs such as mitomycin may cause widespread smallvessel thrombosis resulting in the haemolytic-uraemic (thrombotic microangiopathic) syndrome. there is prominent involvement of pulmonary vessels and patients often suffer from respiratory as well as renal insufficiency, and pulmonary hypertension. the syndrome can develop during treatment or up to several months after the drug has been withdrawn. pulmonary thromboembolism is also recorded as a complication of immunoglobulin infusion. non-traumatic fat embolism has resulted from the agglutination or 'creaming' of fat emulsions administered intravenously as a source of calories to debilitated patients. [ ] [ ] [ ] [ ] [ ] [ ] the agglutinated liposomes occlude fine blood vessels throughout the body, causing effects such as priapism, osteonecrosis and pancreatitis. they may be demonstrated in the pulmonary capillaries but the lungs have considerable vascular reserve and it is uncertain what effect the vascular occlusion has on pulmonary function. agglutination of these fat emulsions is particularly common in severely ill patients and this has been attributed to the elevated blood levels of acute-phase proteins, especially c-reactive protein, that are found in the very ill. the agglutination is also induced by calcium and may be brought about by administering calcium and other mineral supplements through the same venous line as the fat. once agglutinated, the fat is less soluble and may be demonstrated in paraffin sections. sudan black is especially useful for this purpose (fig. . . ). microvascular crystal embolism is a further risk of parenteral nutrition, the crystals representing various calcium salts that may precipitate in the circulation. transient diffusion abnormalities attributed to oil embolism are very common in patients undergoing lymphangiography but serious respiratory impairment is limited to those patients with pre-existing lung disease or in whom substantial amounts of contrast medium are injected rapidly. [ ] [ ] [ ] [ ] other emboli of an iatrogenic nature described in pulmonary arteries include the broken-off ends of intravenous catheters and cannulas, particles from dialysis tubing, prosthetic implants of substances such as teflon and silicone , [ ] [ ] [ ] [ ] and various materials injected to occlude abnormal blood vessels. , diffuse pulmonary haemorrhage diffuse pulmonary haemorrhage may result from interference with the clotting mechanism by anticoagulants or from widespread pulmonary capillaritis, the latter reported in leukaemic patients treated with retinoic acid. pulmonary haemorrhage has also been reported as an idiosyncratic reaction to lymphangiography media and as a complication of immunoglobulin infusion, while the development of anti-basement membrane antibodies resulting in goodpasture's syndrome has been attributed to penicillamine. a infection is a common pulmonary hazard in any patient receiving corticosteroids, chemotherapy or any other immunosuppressant drug. viral, bacterial, fungal and protozoal infections, often in combination, may all develop in the lungs of such patients and tissue reactions may be atypical. pneumocystis jiroveci, for example, may elicit a granulomatous reaction or cause diffuse alveolar damage rather than the usual foamy alveolar exudate (see p. ). metastatic calcification, described on page , may result from any drug causing hypercalcaemia, e.g. high doses of vitamin d, calcium and inorganic phosphate or excessive alkali intake in the treatment of peptic ulceration. carcinoma of the lung may be promoted by drugs. arsenicals cause squamous metaplasia of the bronchi and occasionally squamous carcinoma, while peripheral scar cancers, usually adenocarcinomas, have developed in lungs showing fibrosis due to drugs such as busulphan. drugs may result in a variety of pleural diseases. common examples include effusions, chronic inflammation and fibrosis. these are usually encountered in isolation but may be associated with chronic interstitial pneumonia or fibrosis. sometimes there is also serological evidence of systemic lupus erythematosus: many drugs, including hydantoin, practolol, procainamide, hydralazine and sulphonamides, are associated with the development of a syndrome resembling systemic lupus erythematosus that includes pleural disease. whether the drugs are directly responsible for the syndrome or merely promote the development of latent natural disease is uncertain. ergotamine derivatives such as methysergide and bromocriptine are notable for the production of pleural fibrosis, which is sometimes associated with mediastinal and retroperitoneal fibrosis large amounts or prolonged treatment are generally required to produce this effect. [ ] [ ] [ ] in patients given practolol, pleural thickening has become evident several years after the drug was discontinued. this shows the need for a careful drug history in any patient with unexplained pleural fibrosis. reports of radiation-induced lung damage began to appear soon after ionising radiation became widely used in the treatment of malignant disease. [ ] [ ] [ ] despite refinements in radiotherapy techniques it is often impossible to avoid irradiating small areas of lung when treating cancer of the lung, breast, spine, thymus and oesophagus. parts of the lungs are also included in 'mantle' irradiation of mediastinal lymph nodes affected by lymphoma. occasionally, the whole of both lungs is irradiated, as in the treatment of widespread pulmonary metastases or as part of whole-body irradiation prior to marrow transplantation for the treatment of leukaemia. radiation pneumonitis, usually localised, is estimated to affect about % of patients. therapeutic irradiation is given as divided doses over several weeks in order to minimise damage to adjacent tissue. the effects of such fractionated treatment are cumulative. in the lungs an early exudative phase soon passes and progressive damage becomes apparent only after months or even years. , the changes are generally confined to the area of lung that is irradiated but are widespread when the whole body is irradiated prior to bone marrow transplantation or there is accidental whole-body irradiation. however, localised irradiation of the lung has been followed by abnormalities in non-irradiated areas. these include bilateral alveolar exudates, migratory organising pneumonia affecting both lungs , and fulminant bilateral interstitial pneumonia. the likelihood of lung injury is increased by the simultaneous use of cytotoxic drugs and oxygen therapy. furthermore, chemotherapy following irradiation may result in exacerbation of the injury in areas previously irradiated, a phenomenon termed 'recall pneumonitis' . , in the long term, irradiation also results in an increased incidence of lung carcinoma. this was seen in patients given therapeutic irradiation to the spine for ankylosing spondylitis and is still encountered on occasion following irra diation for breast cancer. the pathogenesis of radiation injury is described on page . radiation damage to the lung is traditionally separated into fulminant acute injury coming on within days, subacute pneumonitis developing within several weeks (typically - months) and interstitial fibrosis slowly evolving from the subacute stage or making itself apparent years later. the migratory organising pneumonia referred to above is an unusual further effect, as is chronic eosinophilic pneumonia. in the pleura, radiation causes fibrinous effusions and adhesions. pleural effusion and pulmonary oedema may be augmented by the long-term effects of radiation on the heart. fulminant acute injury is an unusual and unexpected effect of therapeutic radiation but one that is likely to come to the attention of the pathologist as an autopsy is often requested. the clinical features are those of acute lung injury and the pathological changes are those of diffuse alveolar damage. the cause is likely to be accidental overdosage, augmentation of the radiation damage by accompanying oxygen therapy or treatment with cytotoxic drugs. occasionally however these factors can be excluded, in which case the damage has to be ascribed to 'hypersensitivity' . subacute radiation pneumonitis is encountered more commonly. after an interval of about - months the patient complains of shortness of breath and a non-productive cough. the chest radiograph shows hazy opacification proceeding to more dense consolidation. lung biopsy shows alveolar and interstitial oedema, possibly with residual hyaline membranes, proliferation of atypical alveolar epithelial cells and interstitial fibroblasts and organising thrombosis. later, as the process advances, there is widespread fibrosis comparable to that illustrated in figure . on page and ultimately dense scarring (fig. . . ) . tracheal and aortic injury may complicate radiation treatment of tracheal lesions, sometimes resulting in an aortotracheal fistula. patients requiring mechanical ventilation are liable to suffer lung injury in a number of ways. in addition to effects of barotrauma such as pneumothorax and surgical emphysema, they often develop diffuse alveolar damage. the high oxygen tension that is often combined with mechanical ventilation is a major factor - but mechanical forces other than the high pressures responsible for barotrauma can also contribute to this form of lung injury, notably by resulting in excessive end-expiratory stretch and repeated collapse/recruitment of the alveolar walls. , low tidal volume ventilation is therefore a fundamental part of the management of diffuse alveolar damage. although oxygen is necessary to life, it is cytotoxic in high concentrations. severe hyperoxia damages dna, inhibits cellular proliferation and ultimately kills cells. its toxicity is thought to be due to the intracellular production of active oxygen radicals, some of which derive from activated neutrophils attracted to the site of injury. [ ] [ ] [ ] [ ] under normal conditions most of the oxygen is reduced to water by cytochrome oxidase, and any active radicals produced are eliminated by superoxide dismutase, catalase and other antioxidants. however, these defence mechanisms may prove inadequate when active radicals are produced in excess. problems are likely to arise in clinical practice when lung disease necessitates the concentration of oxygen in the inspired air being raised in order to maintain normal blood levels of oxygen and prevent cerebral hypoxia. [ ] [ ] [ ] a 'safe' level for oxygen administration is not firmly established and, because of species differences in susceptibility to oxygen, caution is needed in extrapolating from animal studies. however, animal experiments have shown that previous damage to the lungs renders them unduly sensitive to oxygen , and conversely that prior exposure to high levels of oxygen confers some resistance to subsequent oxygen exposure. clinical studies suggest that less than % oxygen (at atmospheric pressure) can be tolerated for long periods without ill effect. little, if any, serious lung damage results from administration of % oxygen for up to hours but concentrations between % and % carry a risk of damage if this period is exceeded. , extracorporeal oxygenation of the blood circumvents the problem but if it is to be prolonged it becomes a major undertaking that poses its own hazards; it is therefore generally reserved for patients who remain hypoxaemic despite other measures. intravenous blood oxygenators are employed to minimise the supplementation of inspired oxygen and partial liquid ventilation utilising perfluorocarbon has also been used. experimentally, disruption of cd binding to reduce the release of proinflammatory cytokines has shown promising results in blunting oxygen-induced lung injury. none of the morphological changes attributable to oxygen toxicity is specific. the earliest ultrastructural change in experimental oxygen poisoning is swelling of endothelial cells, the cytoplasm of which becomes grossly oedematous and vacuolated. swelling and fragmentation of type i epithelial cells follow and these cells become separated from their basement membrane, which is then coated by thin strands of protein. this coating is replaced by proliferating type ii cells by the th day. with recovery in room air the lungs practically return to normal. the full clinical picture of oxygen poisoning is the acute respiratory distress syndrome and the corresponding pathological changes are those of diffuse alveolar damage, as described on page . patients with hypovolaemic shock or undergoing major surgery often require massive blood transfusions and this provides another possible cause of pulmonary damage. although hypervolaemia is the commonest cause of pulmonary oedema after blood transfusion, transfusion-related acute lung injury is more often fatal. platelet and white cell aggregates are known to develop in stored blood, but a relationship between the number of microaggregates transfused and the degree of respiratory impairment has not been convincingly demonstrated. leukocyte antibodies are a more likely cause of lung injury in these patients. such antibodies are often found in multiparous female donors as a result of sensitisation by fetal white cells during pregnancy. alternatively, the recipient may have developed them during pregnancy or as a result of previous blood transfusions. the implicated antibodies are thought to initiate alveolar capillary damage within hours of transfusion by stimulating granulocyte aggregation. , electron microscopy has shown capillary endothelial damage with activated granulocytes in contact with alveolar basement membranes. cardiopulmonary bypass entails oxygenation and circulation of the blood by extracorporeal devices, so permitting major heart surgery. in the early days of such surgery it was not unusual for patients to develop fatal respiratory insufficiency in the postoperative period. this led to the term 'postperfusion lung' . electron microscopic studies showed alveolar damage with degranulation of neutrophils in pulmonary capillaries. , the syndrome is now less common but infants remain susceptible. the most likely explanation is that the synthetic materials with which blood comes into contact during the bypass procedure are able to activate complement. this is mediated by hageman factor (factor xii) and the alternative pathway. aggregation of neutrophils leads to their sequestration in the lungs and damage results from their release of lysosomal enzymes and active radicals. [ ] [ ] [ ] the process is delayed by hypothermia. a postcardiac injury syndrome develops after a variety of myocardial or pericardial injuries: it has been described after cardiac surgery (postpericardiotomy syndrome), myocardial infarction (dressler's syndrome), blunt trauma to the chest, percutaneous puncture of the heart and implantation of a pacemaker. there is a delay of anything between a few days and a few months between the cardiac injury and the onset of symptoms, which comprise chest pain, breathlessness, dyspnoea and fever. examination usually reveals haemorrhagic pleural or pericardial effusions and pulmonary infiltrates. the syndrome usually resolves spontaneously and few pathological studies have therefore been conducted. however, the changes of diffuse alveolar damage have been reported, principally hyaline membrane formation and type ii pneumocyte hyperplasia. the pathogenesis is obscure. antibodies reacting with myocardial antigens often develop after cardiac surgery but there is no relationship between these and the development of the syndrome. [ ] [ ] [ ] this minimally invasive technique is used to destroy lesions as varied as pulmonary metastases and the connection between the left atrium and ectopic foci in the muscular sleeves that surround the terminations of the pulmonary veins (see p. ). the former may be complicated by pneumothorax and the latter by pulmonary vein stenosis. , central venous cannulation (synonym: catheterisation) is widely used in treating seriously ill patients and may give rise to serious complications. the commonest early complications related to the respiratory tract are caused by local trauma: they include pneumothorax, subcutaneous emphysema, haemothorax and air embolism. infection occurs later, causing endocarditis, septic emboli and lung abscesses. thrombosis is another common late complication: one autopsy study of patients with central venous lines showed that % had major pulmonary emboli and % had microscopic emboli in their pulmonary arteries. pulmonary artery cannulation, for example with a swan-ganz catheter, may result in pulmonary infarction or any of the traumatic complications of central venous catheterisation mentioned above. , tracheotomy entails a small immediate risk of haemorrhage from damaged subthyroidal arteries, while an endotracheal tube predisposes to infection, as with all foreign bodies. infection is also promoted by the filtering action of the upper respiratory air passages being bypassed. the latter factor also necessitates humidification of the inspired air and on occasion the humidifier or ventilator has become contaminated so that an aerosol of bacteria is introduced directly into the lower respiratory tract. high-pressure ventilation may also lead to interstitial emphysema, pneumothorax and surgical emphysema. asphyxia may follow an endotracheal tube becoming blocked by secretions or through it being badly positioned. secretions need to be constantly removed yet repeated suctioning to achieve this has led to cardiac dysrrythmia and even cardiac arrest. if the balloon on the endotracheal tube is too near the tracheostomy it may act as a fulcrum, causing the tip of the tube to press into the tracheal wall. pressure necrosis and perforation may follow, leading to mediastinitis, tracheo-oesophageal fistula or erosion of a large blood vessel. these are also complications of tracheobronchial laser therapy. pressure from the balloon may lead to a tracheal diverticulum and after the tube is withdrawn the trachea may become narrowed at either the site of the incision or further down where the balloon on the tracheal tube causes pressure. small, shallow ulcers generally heal quickly but deeper ulcers cause necrosis of the tracheal cartilage, and healing is then often accompanied by fibrous stenosis (fig. . . ) or web formation. this results in wheezing and dyspnoea but not before the trachea has narrowed to % of its original size, which may take months. earlier narrowing may be caused by oedema or a fibrinous pseudomembrane. , sometimes the stenosis takes the form of a large mass of granulation tissue at the tracheostomy site, a so-called granuloma ball. in children especially, intubation may lead to tracheomalacia so that after the tube is removed the airway collapses. necrotising sialometaplasia is a further complication of prolonged intubation. the incidence of such posttracheostomy complications can be minimised by careful placement of the stoma and tube, avoidance of large apertures and high cuff pressures, elimination of heavy connecting equipment and meticulous care of the tracheostomy. nasogastric feeding tubes may of course lead to aspiration lesions in the lungs and even fatal asphyxia if they are inadvertently allowed to enter the trachea rather than the oesophagus. bronchoscopy is generally a safe, almost routine procedure. a review of patients who underwent bronchoscopy identified severe complications in ( . %), of whom three died. the fatal cases comprised a -year-old with coronary heart disease who developed cardiac arrest and two patients who had had tracheal transplantation for oesophageal cancer and required bronchoscopic laser treatment but died of airway obstruction. the pleural cavity is intubated in the treatment of pneumothorax and pleural effusions the tube being placed anteriorly to drain air and posteriorly to drain fluid. complications include laceration of an intercostal artery or vein, the lung, the diaphragm and the heart. pneumonectomy has been practised since the s, since when the mortality associated with this operation has dropped from over % to near zero in the best hospitals. risk factors include underlying lung disease, other medical conditions and more extensive procedures such as pleuropneumonectomy and pneumonectomy combined with chest wall resection. the anatomical changes that take place soon after pneumonectomy have been extensively studied by radiologists who describe the air-filled postpneumonectomy space gradually filling with fluid and contracting as the mediastinum shifts and the ipsilateral dome of the diaphragm rises. much of the space is filled by fluid within weeks but complete opacification may take up to months. rapid filling in the immediate postoperative period suggests haemorrhage or chylothorax. however, fluid accumulation is normally rapid after pleuro-pneumonectomy and may compromise the function of the other lung. pathologists conducting autopsies long after the operation may find complete fibrous obliteration of the postpneumonectomy space, coupled with mediastinal shift and elevation of the hemidiaphragm, but often there is persistent brown fluid, which may be clear, cloudy or occasionally purulent. the remaining lung is generally enlarged, with its volume greater than predicted. animal studies have shown that if one lung is excised early in life the enlargement is partly due to enhanced growth but later it represents only dilatation of existing air spaces. hepatocyte growth factor is thought to be involved in the proliferation of residual lung cells following pneumonectomy. pulmonary complications include those typically seen after other thoracic procedures, such as haemorrhage and infection, and those unique to the postpneumonectomy state, namely anastomotic dehiscence and postpneumonectomy pulmonary oedema. the latter presents as the acute respiratory distress syndrome and represents the early stages of diffuse alveolar damage. it follows severe shift of the heart and mediastinum, which is commoner in children and young adults, in whom the tissues are more compliant. [ ] [ ] [ ] [ ] the condition complicates up to % of lung resections , and is commoner following excision of the right lung when severe herniation of the left lung into the postpneumonectomy space stretches the trachea and left main bronchus and the latter is compressed between the left pulmonary artery in front and the arch of the aorta behind. in the long term the compression can result in bronchomalacia and postobstructive bronchiectasis. if postpneumonectomy oedema develops the immediate postoperative mortality is high - % following pneumonectomy, % following lobectomy and % following sublobar resections. , the pathogenesis is probably multifactorial but apart from factors such as fluid overload and high inspired oxygen concentrations there is probably an element of alveolar wall injury, induced by oxidant generation secondary to lung stretching and general surgical trauma. , occupational dust exposure and chronic obstructive pulmonary disease -a systematic overview of the evidence 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toxic oil syndrome pulmonary hypertension due to toxic oil syndrome. a clinicopathologic study pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome pulmonary vascular lesions in the toxic oil syndrome in spain outbreak of bronchiolitis obliterans associated with consumption of sauropus androgynus in taiwan histopathological study of sauropus androgynus-associated constrictive bronchiolitis obliterans: a new cause of constrictive bronchiolitis obliterans sauropus androgynus-constrictive obliterative bronchitis/bronchiolitishistopathological study of pneumonectomy and biopsy specimens with emphasis on the inflammatory process and disease progression segmental necrosis of small bronchi after prolonged intakes of sauropus androgynus in taiwan sauropus bronchiolitis -reply the lipoid pneumonia of blackfat tobacco smokers in guyana a morphometric analysis of the male and female tracheal epithelium after experimental exposure to marijuana smoke 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complications of cocaine abuse blackened bronchoalveolar lavage fluid in crack smokers -a preliminary study pulmonary histopathology in cocaine abusers pulmonary complications of crack cocaine: a comprehensive review cocaine-induced churg-strauss vasculitis pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization crack lung: an acute pulmonary syndrome with a spectrum of clinical and histopathologic findings effects of 'crack' cocaine on pulmonary alveolar permeability pulmonary hemorrhage and antiglomerular basement membrane antibody-mediated glomerulonephritis after exposure to smoked cocaine (crack): a case report and review of the literature cellulose granulomas in the lungs of a cocaine sniffer intravenous injection of talc-containing drugs intended for oral use. a cause of pulmonary granulomatosis and pulmonary hypertension autopsy findings in drug addicts pulmonary 'mainline' granulomatosis: talcosis of intravenous methadone abuse self induced pulmonary granulomatosis. a consequence of intravenous injection of drugs intended for oral use the pulmonary vascular lesions of intravenous drug abuse microcrystalline cellulose pulmonary embolism and granulomatosis talc granulomatosis: laboratory findings similar to sarcoidosis postmortem findings of pulmonary lesions of older datum in intravenous drug addicts. a forensic-pathologic study recreational use of aminorex and pulmonary hypertension fatal drug reactions among medical inpatients drug-related hospital admissions: a review of australian studies published - incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies (see comments) the quality in australian health care study adverse reactions to drugs drug-induced pulmonary disease -an update an algorithm for the operational assessment of adverse drug reactions. ii. demonstration of reproducibility and validity aspirin idiosyncrasy and tolerance aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks; diagnostic and physiopathological implications mechanism of aspirin sensitivity bronchiolitis and bronchitis in connective tissue disease. a possible relationship to the use of penicillamine bronchiolitis obliterans in a patient with localized scleroderma treated with d-penicillamine constrictive bronchiolitis obliterans following gold therapy for psoriatic arthritis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease progressive airway obliteration in adults and its association with rheumatoid disease drug-induced infiltrative lung disease drug-induced and iatrogenic infiltrative lung disease interstitial lung disease associated with drug therapy pulmonary toxic effects of continuous desferrioxamine administration in acute iron poisoning interstitial pulmonary fibrosis following busulfan therapy busulphan lung report of a case and review of the literature busulfan lung: report of two cases and review of the literature a -year-old man with pulmonary infiltrates after a bone marrow transplantation -busulfan pneumonitis drug-induced pulmonary disease lung parenchymal injury induced by bleomycin pathology of high dose intermittent cyclophosphamide therapy cyclophosphamide pneumonitis pulmonary histopathologic changes associated with melphalan therapy melphalan-induced pulmonary interstitial fibrosis potentiation of bleomycin-induced lung injury by exposure to % oxygen factors influencing postoperative morbidity and mortality in patients treated with bleomycin bleomycin therapy and anaesthesia risk factors of anesthesia and surgery in bleomycin-treated patients the pathogenesis of bleomycin-induced pulmonary damage in mice experimentally induced bleomycin sulfate toxicity origin and significance of intranuclear tubular inclusions in type ii pulmonary alveolar epithelial cells of patients with bleomycin and busulfan toxicity late bcnu lung -a light and ultrastructural study on the delayed effect of bcnu on the lung parenchyma statin-induced fibrotic nonspecific interstitial pneumonia bronchiolitis obliterans organizing pneumonia during treatment with acebutolol and amiodarone methotrexate-induced pneumonitis (baltimore) bronchoalveolar lavage findings suggest an immunologic disorder pneumonitis complicating low-dose methotrexate therapy for rheumatoid arthritis -discrepancies between lung biopsy and bronchoalveolar lavage findings methotrexate pneumonitis: review of the literature and histopathological findings in nine patients the pulmonary toxicity of antineoplastic agents relationship of gold and penicillamine therapy to diffuse interstitial lung disease desquamative interstitial pneumonia following long-term nitrofurantoin therapy two unusual pathological reactions to nitrofurantoin: case reports bronchiolitis obliterans organising pneumonia associated with the use of nitrofurantoin amiodarone lung toxicity: a human and experimental study effect of amiodarone on the lung shown by polarized light microscopy fine structural alterations in the lungs of iprindole-treated rats effects of chlorphentermine on the rat lung amiodarone-induced hypersensitivity pneumonitis. evidence of an immunological cell-mediated mechanism amiodarone pneumonitis: three further cases with a review of published reports amiodarone-associated pulmonary toxicity. a clinical and pathologic study of eleven cases amiodarone lung: pathologic findings in clinically toxic patients amiodarone-induced bronchiolitis obliterans organizing pneumonia (boop) amiodarone-related pulmonary mass and unique membranous glomerulonephritis in a patient with valvular heart disease: diagnostic pitfall and new findings nodular amiodarone lung disease amiodarone pneumonitis: no safe dose amiodarone pneumonitis: no safe dose susceptibility to amiodarone-induced pulmonary toxicity: relationship to the uptake of amiodarone by isolated lung cells acute amiodarone-induced pulmonary toxicity following lung resection pulmonary alveolar proteinosis developing from desquamative interstitial pneumonia in long term toxicity studies of iprindole in the rat eosinophilic lung diseases mesalazine-induced eosinophilic pneumonia chronic eosinophilic pneumonia after radiation therapy for breast cancer churg-strauss syndrome in patients receiving montelukast as treatment for asthma inhaled corticosteroids and churg-strauss syndrome: a report of five cases pulmonary migratory infiltrates and pachypleuritis in a patient with crohn's disease immunodeficiency virus-infected patients receiving antiretroviral therapy ciprofloxacin-induced acute interstitial pneumonitis pulmonary granulomas after tumour necrosis factor alpha antagonist therapy pulmonary sarcoidosis developing during infliximab therapy sarcoid-like granulomatous disease following etanercept treatment for ra pneumonia due to liquid paraffin: with chemical analysis a case of chronic paraffin pneumonitis liquid paraffin pneumonia -with chemical analysis and electron microscopy paraffinoma confirmed by infrared spectrophotometry foreign body granulomata of the lungs due to liquid paraffin exogenous lipoid pneumonia due to nasal application of petroleum jelly reaction of human lungs to aspirated animal fat (ghee): a clinicopathological study clinical reactions following bronchography the reaction of pulmonary tissue to lipiodol experimental study of bronchographic media on lung a method for the identification of lipiodol in tissue sections of lungs, lipids, and lollipops bronchial necrosis and granuloma induced by the aspiration of a tablet of ferrous sulphate syndrome of iron pill aspiration pulmonary disease associated with l-tryptophan-induced eosinophilic myalgia syndrome. clinical and pathologic features a case of the eosinophilia-myalgia syndrome associated with use of an l-tryptophan product an investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use l-tryptophan and the eosinophiliamyalgia syndrome: pathologic findings in eight patients histopathologic features of the l-tryptophan-related eosinophilia-myalgia (fasciitis) syndrome -(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome immunemediated mechanisms and immune activation of fibroblasts in the pathogenesis of eosinophilia-myalgia syndrome induced by l-tryptophan pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome tryptophan-induced lung disease -an immunophenotypic, immunofluorescent, and electron microscopic study bronchiolitis obliterans organizing pneumonia associated with massive l-tryptophan ingestion pulmonary disease complicating intermittent therapy with methotrexate case records of the massachusetts general hospital. a -year-old man with increasing dyspnea, dry cough, and fever after chemotherapy for lymphoma interferon-alpha therapy associated with the development of sarcoidosis proliferation and differentiation in mammalian airway epithelium sarcoid-like pulmonary disorder in human review of the literature severe barium sulfate aspiration into the lung: clinical presentation, prognosis and therapy aminorex and the pulmonary circulation pulmonary hypertension and fenfluramine irreversible pulmonary hypertension after treatment with fenfluramine dietary pulmonary hypertension appetite-suppressant drugs and the risk of primary pulmonary hypertension fatal pulmonary hypertension associated with short-term use of fenfluramine and phentermine autopsy findings of heart and lungs in a patient with primary pulmonary hypertension associated with use of fenfluramine and phentermine pulmonary veno-occlusive disease following therapy for malignant neoplasms pulmonary veno-occlusive disease in an adult following bone marrow transplantation: case report and review of the literature indomethacin in the treatment of premature labor. effects on the fetal ductus arteriosus prenatal diagnosis of intrauterine premature closure of the ductus arteriosus following maternal diclofenac application roles and mechanisms of human immunodeficiency virus protease inhibitor ritonavir and other anti-human immunodeficiency virus drugs in endothelial dysfunction of porcine pulmonary arteries and human pulmonary artery endothelial cells pulmonary embolism after intravenous immunoglobulin fat embolism in infancy after intravenous fat infusions pulmonary fat accumulation after intralipid infusion in the preterm infant intralipid microemboli the pathogenesis of fat embolism pulmonary lipid emboli in association with long-term hyperalimentation the impact of intravenous fat emulsion administration in acute lung injury microvascular pulmonary emboli secondary to precipitated crystals in a patient receiving total parenteral nutrition -a case report and description of the high-resolution ct findings pulmonary complications following lymphangiography with a note on technique changes in pulmonary function due to lymphangiography pulmonary complications of lymphangiography respiratory distress syndrome from lymphangiography contrast medium spallation and migration of silicone from blood-pump tubing in patients on hemodialysis acute pneumonitis after subcutaneous injections of silicone in transsexual men pulmonary granulomas secondary to embolic prosthetic valve material pulmonary teflon granulomas following periurethral teflon injection for urinary incontinence acute pneumonitis after subcutaneous injections of silicone for augmentation mammaplasty a pathological study following bronchial artery embolization for haemoptysis in cystic fibrosis isobutyl- -cyanoacrylate pulmonary emboli associated with occlusive embolotherapy of cerebral arteriovenous malformations hemorragie alveolaire diffuse secondaire a l'utilisation d'anticoagulants oraux diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome intrapulmonary hemorrhage with anemia after lymphangiography alveolar hemorrhage as a complication of treatment with abciximab d-penicillamine induced goodpasture's syndrome in wilson's disease drugs and the pleura pleuropulmonary changes induced by ergoline drugs pleuropulmonary disease as a side-effect of treatment with bromcriptine pleuropulmonary disease due to pergolide use for restless legs syndrome fibrosis of the lung following roentgen-ray treatments for tumor radiation reaction in the lung radiation pneumonitis: experimental and pathologic observations radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors the pathogenesis of radiationinduced lung damage radiation pneumonitis: a review adult respiratory distress syndrome after limited thoracic radiotherapy migratory bronchiolitis obliterans organizing pneumonia after unilateral radiation therapy for breast carcinoma migratory organizing pneumonitis 'primed' by radiation therapy hamman-rich syndrome 'primed' by radiation? recall' pneumonitis: adriamycin potentiation of radiation pneumonitis in two children recall lung pneumonitis due to carmustine after radiotherapy pulmonary radiation injury mortality from cancer and other causes after radiotherapy for ankylosing spondylitis increased risk of lung cancer after breast cancer radiation therapy in cigarette smokers aortotracheal fistula secondary to bacterial aortitis respirator lung -a misnomer pathology of adult respiratory distress syndrome pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project lung injury caused by mechanical ventilation ventilator-induced lung injury oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes in vitro damage of rat lungs by oxygen metabolites intercellular adhesion molecule- contributes to pulmonary oxygen toxicity in mice -role of leukocytes revised intercellular adhesion molecule- expression on the alveolar epithelium and its modification by hyperoxia normobaric oxygen toxicity of the lung oxygen pneumonitis in man pathology of pulmonary oxygen toxicity diffuse alveolar damage -the role of oxygen, shock and related factors diffuse interstitial pulmonary fibrosis. pulmonary fibrosis in mice induced by treatment with butylated hydroxytoluene and oxygen potentiation of diffuse lung damage by oxygen: determining values resistance and susceptibility to oxygen toxicity by cell types of the gas-blood barrier of the rat lung ultrastructural observations on the development of the alveolar lesions extracorporeal membrane oxygenation for adult respiratory failure pulmonary pathology of patients treated with partial liquid ventilation disruption of the cd -cd ligand system prevents an oxygen-induced respiratory distress syndrome pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. ii ultrastructural and morphometric studies diffuse alveolar damage, respiratory failure and blood transfusion pulmonary injury -secondary to extracorporeal circulation fine structural changes in the lungs following cardiopulmonary bypass complement and the damaging effects of cardiopulmonary bypass acute lung injury during cardiopulmonary bypass: are the neutrophils responsible? inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies the pleuropulmonary manifestations of the postcardiac injury syndrome the postmyocardial infarction syndrome. the nonspecificity of the pulmonary manifestations the postcardiac injury syndromes antiheart antibodies following open heart surgery: incidence and correlation with postpericardiotomy syndrome analysis of the factors associated with radiofrequency ablation-induced pneumothorax irreversible intrapulmonary vascular changes after pulmonary vein stenosis complicating catheter ablation for atrial fibrillation pulmonary venous stenosis after treatment for atrial fibrillation right sided infective endocarditis as a consequence of flow directed pulmonary artery catheterisation complications and consequences of endotracheal intubation and tracheotomy. a prospective study of critically ill adult patients pathologic changes of the trachea after percutaneous dilatational tracheotomy pseudomonas aeruginosa respiratory tract infections in patients receiving mechanical ventilation cardiac arrhythmias resulting from tracheal suctioning obstructive fibrinous tracheal pseudomembrane -a potentially fatal complication of tracheal intubation -h pretreatment with methylprednisolone versus placebo for prevention of postextubation laryngeal oedema: a randomised double-blind trial tracheobronchomalacia in children necrotizing sialometaplasia (adenometaplasia) of the trachea severe complications of bronchoscopy the postpneumonectomy state evaluation of post-pneumonectomy space by computed tomography the postpneumonectomy space: factors influencing its obliteration hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice severe airway obstruction caused by mediastinal displacement after right pneumonectomy in a child. a case report postpneumonectomy syndrome: diagnosis, management, and results treatment of left pneumonectomy syndrome with an expandable endobronchial prosthesis postpneumonectomy syndrome: another twist acute lung injury and acute respiratory distress syndrome after pulmonary resection the mortality from acute respiratory distress syndrome after pulmonary resection is reducing: a -year single institutional experience prevalence and mortality of acute lung injury and ards after lung resection lung injury following pulmonary resection in the isolated, blood-perfused rat lung the pathogenesis of lung injury following pulmonary resection it is estimated that % of all hospital admissions are due to effects of therapeutic drugs, that - % of inpatients experience a drug reaction and that % of deaths in hospital may be related to drug therapy. [ ] [ ] [ ] [ ] the lungs are often involved in these adverse reactions. the mechanism of an adverse drug reaction may be based on:• overdosage: toxicity linked to excess dose, or impaired excretion, one classification of adverse drug reactions is that based upon the type of drug (box . . ). this is not adopted here but in passing it is worth noting that pharmacists are generally very helpful in supplying details of adverse reactions to specific drugs. alternatively, information on the long list of potentially pneumotoxic drugs may be obtained at http://www.pneumotox.com. a useful scheme for chapter key: cord- -xz ftnat authors: moreno-lÓpez, j. title: acute respiratory disease in cattle date: - - journal: virus infections of ruminants doi: . /b - - - - . - sha: doc_id: cord_uid: xz ftnat nan acute respiratory disease in cattle is a serious problem in countries with intensive or semi-intensive systems of feeding and management. the etiology is complex and many reviews list the microorganisms incriminated. some of the viruses which can infect the respiratory tract of cattle are summarized in table . viruses and bacteria, together with physical stress, transport, overcrowding, irregular feeding and poor standard of hygiene as well as environmental factors (ventilation, temperature and humidity) play an important role in pathogenesis. the disease has world-wide distribution. it affects cattle of all races and ages, tends to recur and responds poorly to preventive and therapeutic treatment. even if the disease for the most part occurs during calfhood, adult cattle may also be involved. the symptomatology of acute respiratory disease is similar in different outbreaks and includes depression, inappétence, dullness, increased respiration and pulse rates in the early stages. the respiratory disorder may or may not be accompanied by diarrhea. acute respiratory disease in cattle is not attributable to a single etiological agent. in many instances, mixed infections with two or more agents occur, as shown in table . such mixed infections can contribute to the severity of the disease. moreover, the microorganisms involved appear to act synergetically. examples of synergism have been demonstrated in the usa (shipping fever, caused by parainfluenza- (pi ) virus and pasteurellae) and sweden (umeâ disease, caused by pi virus and bvdv). it is difficult to estimate the economic losses, but in terms of mortality, loss of milk production and delayed weight gains, the acute respiratory disease is considered as one of the most costly and troublesome problems for the cattle industry. it is generally accepted that viruses are the primary agents and that bacteria like pasteurellae may, as secondary invaders, accentuate the pathology of the respiratory tract, as in "shipping fever". some of the viruses are circulating in the cattle population, as for instance pi virus, which is common in the respiratory tract and is easily transmitted. high percentages of seropositive animals were found in various countries. serological surveys have shown that, on the contrary, bvdv was much less frequent on farms separated geographically than on farms with a close contact of animals on common pastures. collectivization of calves with a different immunological status like that occurring on breeder farms or in large feedlots, leads to accumulation of viruses introduced by animals in the incubation or convalescence stages or by latent carriers. under these circumstances, explosive outbreaks of respiratory and enteric disease may occur. it is unknown how stress factors during massive transport and crowding promote pathogenicity of various respiratory viruses, because it is not possible to mimic a natural outbreak by experimental infection. all too frequently only a mild disease is reproduced. some of the viruses per se have an adverse influence on defence mechanisms. it was shown that infection with pi virus may initially diminish the cell-mediated immune response; also, bvdv may act on lymphocytes and neutrophils, suppressing antibody production and phagocytosis, respectively. suppression may promote invasion of other agents, particularly through close contact and crowding in stables lacking proper ventilation. as regards diagnosis, a somewhat pathetic conclusion can be drawn: if foot-and-mouth disease virus is isolated from an animal, the disease has been identified. however, if pi virus or bvdv has been isolated, you have diagnosed the infection but not an "acute respiratory/enteric disease", because other viruses and also bacteria might be etiologically involved. as diagnostic techniques become more widely applied, an increasing number of viruses has been isolated from cattle with acute respiratory disease. at the end of the s, "shipping fever" was described as a disease following massive transports to or from the enormous feedlots of the usa, and pi virus was isolated as the first virus of a condition with a multiple viral etiology. in sweden, at about the same time, there were outbreaks of a severe acute respiratory-enteric syndrome on farms in the umeâ district in the northern part of the country. local veterinarians provisionally called the syndrome "umeâ disease". thus bvdv became the second member of a multiple etiology. at that time, pi virus was suspected to be the cause of the infection in the respiratory tract (manifested by tracheitis and pneumonia), and bvdv to be the cause of infection in the intestinal tract (manifested by erosions in the oral and intestinal mucosa). both viruses were indeed identified. however, it has to be kept in mind that the terms "parainfluenza" and "bovine viral diarrhea" are merely taxons by which these viruses are classified. during the following decade, the first two bovine adenoviruses (types and ) were described in the usa, and subsequently a third type was identified in the uk. these are the isolates that grow in kidney cells with a hexon antigen common to all mastadenoviruses. then hungarian workers reported the isolation of two new bovine adenoviruses, types and , which preferentially grow in cultured calf testicle cells. then additional adenoviruses, types through were reported as "testicle-cell types" from various countries and, finally, from bulgaria, a type being a "kidney-cell type". regarding the "testicle-cell type" adenoviruses, the proof for the presence of antigen(s) common to mastadenoviruses is equivocal at present. the types , and have been described each as a single causative agent of acute respiratory-enteric disease in hungary and japan, respectively. during the s the first records appeared on bovine respiratory syncytial virus (brsv), closely related to the human respiratory syncytial virus and causing pneumonia in cattle often less than years of age. brsv was found either as a single agent or as a member of a multiple etiology (as recently reported from uk and usa) condition. in some countries the bovid herpesvirus- , better known as infectious bovine rhinotracheitis (ibr) virus, is a "solo" pathogen. ibrv was the first viral agent definitely shown to cause respiratory infection in cattle. herpesvirus type (malignant catarrhal fever virus), with a low and sporadic incidence, is also incriminated in the bovine respiratory-enteric disease complex. the role of herpesvirus type (prototype strain movar / ) has not been elucidated. however, a respiratory illness caused by this virus in association with pasteurella multocida was quite severe. it is easy to extend this listing by adding, for example, bovine enteroviruses, rhino-and parvoviruses, coronaviruses and the reoviruses. the isolation of these viruses from apparently healthy cattle as well as from those with acute respiratory-enteric disease indicates that they are widespread. however, because they appear to be infrequently implicated in disease processes, it seems that they cause infection only under certain conditions. bacteria such as chlamydia, pasteurellae and mycoplasmas may influence the cycles of infection with viruses. young animals require more care than adult animals. calves fare best when confined individually or in very small groups. this facilitates individual observation, control of diseased animals and maintenance of a higher standard of hygiene. a large number of animals maintained in close contact provides ideal conditions for the spread of disease agents via the respiratory tract. the immunological status, especially passive immunity of the animals, is important in connection with respiratory disease. protective titers of serum antibodies have been calculated for some of the respiratory pathogens. pi virus infection seems to be prevented by a serum neutralizing antibody titer of and ibrv infection by a titer of . the significance of local nasal antibodies and cmi against respiratory disease is of special importance. with the complex etiology as that associated with respiratory infections it is logical that control measures are not easy. control of the disease may be attempted by diminishing the frequency of infections and by implementing a vaccination program against the most frequent agents. some authors have tried to suppress infections by interferon or interferon inducers, i.e. avirulent viruses known to stimulate nonspecific defence mechanisms, including interferon production; however, no efficient control procedures have become available. the development of effective and practical vaccines for the prevention of respiratory disease is notoriously difficult, due to the multiplicity of agents which are involved. as shown in table , at least distinct microorganisms have been identified. certain viruses, however, produce more severe disease than others; vaccines against them may be expected to break the chain of events leading to respiratory disease. the multiplicity of agents necessitates the use of polyvalent vaccines; polyvalence does not appear to affect the immunogenicity of the individual agents. although desirable, the incorporation of all potential pathogens in a single vaccine is difficult, if not impossible. the results of some limited field trials with a tetravalent vaccine have in no way been encouraging. finally, it must be reemphasized that a problem of this complexity is not solved by a single measure but demands that attention is given to both the infectious and environmental causes of the disease. a serosurvey of viruses during outbreaks of acute respiratory and/or enteric disease in swedish cattle key: cord- -ym ce ux authors: chawla, sonam; saxena, shailendra k. title: preparing for the perpetual challenges of pandemics of coronavirus infections with special focus on sars-cov- date: - - journal: coronavirus disease (covid- ) doi: . / - - - - _ sha: doc_id: cord_uid: ym ce ux covid- , arising from novel, zoonotic coronavirus- , has gripped the world in a pandemic. the present chapter discusses the current internationally implemented pandemic preparedness strategies succeeding/recommended to curb the covid- threat to humankind. the updated phase-wise categorization of a pandemic as recommended by the who is described, and associated innovations in surveillance, response, and medical measures/advisory in practice across the globe are elaborated. from a bird’s eye view, the covid- pandemic management relies on revolutionizing the disease surveillance by incorporating artificial intelligence and data analytics, boosting the response strategies—extensive testing, case isolation, contact tracing, and social distancing—and promoting awareness and access to pharmaceutical and non-pharmaceutical interventions, which are discussed in the present chapter. we also preview the economic bearing of the covid- pandemic. the present-day pandemic spotlight on covid- (coronavirus disease- ) was earlier placed on zika virus, h n , severe acute respiratory syndrome (sars), chikangunya, middle east respiratory syndrome (mers), and ebola. the "advancements" of the human race-increased urbanization, global travel, changes in land use, and fervent exploitation of the nature-are also the prime reasons for zoonosis and emergence of novel infectious diseases such as above (madhav et al. ; ahmed et al. ) . this rapid emergence of novel infectious diseases transmitting from surrounding animal life to humans and then from human to human, traveling quickly across the globe can trigger worldwide public health emergency situations, as prevalent today (https://www.who.int/dg/speeches/detail/who-director-general-sopening-remarks-at-the-media-briefing-on-covid- - march , http://www. emro.who.int/fr/about-who/rc /zoonotic-diseases.html). the world health organization (who) declared covid- a pandemic on march , . mesh database defines pandemics as-"epidemics of infectious disease that have spread to many countries, often more than one continent, and usually affecting a large number of people." such emergencies compromise human health, society, economics, and politics-a case in point: the covid- pandemic is forecasted to cost the global economy one trillion us dollars (https://www.ncbi.nlm.nih.gov/mesh/? term¼pandemics, https://news.un.org/en/story/ / / ). as against the earlier guidelines of who where it classified a pandemic into six stages, the revision in pandemic descriptors and stages stands today as follows: • predominantly animal infections, few human infections. this corresponds with the stages - of earlier classification, starting with phase where the virus is in its animal host and has caused no known infection in humans, phase where zoonosis has occurred and the virus has caused infection in humans, and phase where sporadic cases or clusters of infectious disease occur in humans. humanto-human transmission is limited in time and space and is insufficient to cause community-level outbreaks. • sustained human-human transmission. corresponds with the stage of the classical description wherein animal-human and human-human transmissions have sustained a community-level outbreak. the risk for pandemic is greatly increased. • widespread human infection or the stage - from the classical description where the same identified virus has caused a community-level outbreak in another country in another who region. • post-peak period where there exists a possibility of recurrence of infection. • post-pandemic phase when the disease activity is seasonal (https://web.archive. org/web/ /http://www.who.int/csr/disease/influenza/ gipa aidememoire.pdf, https://www.reuters.com/article/uk-china-health-who-idukkcn i pd). at the time of writing this chapter, coronavirus- (cov- )/covid- , though originated in wuhan, china, the first case being reported in november , had pervaded africa, americas, europe, south-east asia, eastern mediterranean, and the western pacific nations with , confirmed cases of covid- and claimed lives, globally (https://www.who.int/docs/default-source/coronaviruse/situa tion-reports/ -sitrep- -covid- .pdf?sfvrsn¼ _ ). europe was declared the new epicenter of the pandemic on march , . the number of new cases in china though declining and is believed to be in post-peak stage, the numbers are alarmingly increasing worldwide (https://www.nbcnews.com/health/ health-news/europe-now-epicenter-pandemic-who-says-n ). who and other leading epidemiology organizations unanimously agree on the indispensable role of pandemic preparation and planning at global and national levels to mitigate through the present public health emergency of covid- and any future outbreaks. pandemic preparation is not a job of single individual or organization. it requires inputs from each person susceptible to the infection agent as well as policy makers at national and international levels, frontline healthcare providers, infrastructure developers and maintenance personnel, pharmaceutical industry and researcher community, and so forth. moreover, the pandemic preparedness plan needs constant reviewing and improvisation (https://www.ecdc.europa.eu/ en/seasonal-influenza/preparedness/why-pandemic-preparedness). in line with the magnitude of the covid- pandemic, worldwide action plans have been activated on national and international levels. the united nations' strategic preparedness and response plan (sprs) against covid- , in layman terms, is designed to control human-human transmission, preventing outbreaks and delaying spread; provide optimal care for all patients; and minimize the impact on healthcare systems and socioeconomic activities. under sprs each nation is assessed for risk and vulnerability, and the resource requirements to support the country to prepare for and respond to covid- are estimated. several nations are well placed to implement this action plan with minimal support. however, otherwise partners are to be introduced to facilitate implementation of measures where there is a gap in capacity, on either a national or a subnational level, in additional support to national governments. thus, an extensive analysis and identification of an affected nation's gaps and needs shall be the basis to develop a covid- country preparedness and response plan (cprp). these cprps will need constant monitoring and reviewing using indicators charted in the sprp and updated as the situation evolves (https:// www.who.int/docs/default-source/coronaviruse/covid- -sprp-unct-guidelines.pdf). grossly, the extent of success of each pandemic action plan stands on the following pillars: • surveillance of coronavirus- and covid- infection: characterization of the virus, infection modes, diagnosing and detecting infection, contact tracing, annotation of data from confirmed cases, predicting mass infection outbreak, keeping a count, and estimation of mortality. • response management: bulk production and supply of protective/preventive pharmaceutical interventions or non-pharmaceutical interventions. • facilitating timely medical help: access to hospitals/healthcare providers, personal and public hygiene, disinfection, and quarantine services. • lesson learning from the present outbreak of covid- to facilitate future action plans and preparedness. hereafter we discuss the present salient strategies under the aegis of the covid- pandemic preparation plan, globally, which are helping the humankind mitigate through this emergency. we also discuss the impact of covid- on world economy and its bearing on future preparedness plans. as defined by the who, surveillance during pandemics is defined as "the ongoing collection, interpretation and dissemination of data to enable the development and implementation of evidence-based interventions during a pandemic event" (https:// www.who.int/influenza/preparedness/pandemic/who_guidance_for_surveil lance_during_an_influenza_pandemic_ .pdf). the present-day key worldwide surveillance activities against covid- include: (a) detection of coronavirus- and verification of covid- (b) risk and severity assessment (c) monitoring the pandemic the rapidly expanding array of pcr/reverse transcriptase pcr-based diagnostics which are quick and efficient in identifying the virus (pang et al. ; lake , https://www.finddx.org/covid- /) are basic requirements for surveillance at every phase of the pandemic for identifying and segregating the infected from non-infected and risk assessment, as well as monitoring recurrences and seasonal disease activity. covid- diagnostics have been discussed previously in this publication. an emerging exciting field of disease surveillance is infectious disease modeling and incorporation of artificial intelligence. notably, these are predictive techniques applicable to each of the three facets of disease surveillance (siettos and russo ) . a classical epidemiological surveillance parameter is quantitation of r (r nought, basic reproductive number) using mathematical models (https:// wwwnc.cdc.gov/eid/article/ / / - _article). r is a crucial metric indicating that on average the number of new infection cases are generated by a confirmed infection case, i.e., the potential transmissibility of an infectious disease. r of covid- infection is estimated as - . in the early phase, as even the asymptomatic patients or with mild pneumonia extruded large amounts of virus . important characteristics of r are: • it is a dynamic number and changes with -each stage of the disease -with interventions, e.g., vaccination, antivirals -precautionary measures such as personal/community disinfection, social distancing, and travel restrictions • with the knowledge of r , one can predict: -new cases expected on a daily basis, and hence facilitate arrangement of healthcare services and interventions locally -outbreak size and the dates of peak infection of the pandemic -probable decline timeline -the extent of vaccination coverage required to prevent future outbreaks • an r > indicates that each infected individual is transmitting the disease to more than new individual, and the infection is spreading increasingly. r ¼ indicates stable transmission and r < indicates the decline in disease transmission ( fig. . ). the aim of pandemic action plans is to monitor and depreciate the r . for instance, the median daily r in wuhan declined from . a week before the introduction of travel restrictions (january , ) to . one week later. the study used a stochastic transmission model of cov- transmission with four datasets from within and outside wuhan and estimated how transmission in wuhan varied between december and february (kucharski et al. ) . the most prominent mathematical model of covid- infection is the seir (susceptible-exposed-infected-recovered) model put forth by wu and coworkers and also endorsed by the who. this model estimates the size of epidemic in wuhan between december and january and forecasts the extent of domestic and global public health risks of taking into account for social and non-pharmaceutical prevention interventions. it is a compartmental model comprising four compartments and the individuals comprising the sample population move through each compartment-"susceptible" (not immune to infection) and get infected from other infection individuals and move to the "exposed" compartment for the incubation period. hereafter the infectious individuals move to the "infected" compartment and eventually to the "recovered" compartment after the disease has run its course, and they now have some immunity (fig. . ). the changes in the population in each compartment are estimated using ordinary differential equations to simulate the progression of an infectious disease. the critical parameters associated with this model are: • force of infection (λ) is the rate at which susceptible individuals are exposed. it depends on the transmission rate (β). • incubation rate (e) is the rate at which exposed people become infectious. • recovery rate (γ) is the rate at which infected individuals recover from the infection. through this model, it was predicted that the r was . , each confirmed case infected - other people, and the epidemic doubling time was . days. also, the size of the outbreak in wuhan was estimated to be up to , people (statistical uncertainty presented at % credible intervals). most striking feature of this model was that it took into account the travel data from and to wuhan over the period of study. thus, it was able to predict that multiple major chinese cities-guangzhou, beijing, shanghai, and shenzhen-had already imported the infection to trigger local epidemics. it also recommended that controlling the transmissibility by - % could eventually rein the local epidemics, and a control of % would phase out the epidemics . the application of artificial intelligence (ai) in close conjunction with technology in pandemic surveillance has demonstrated manifold advantages in surveillance activities as exemplified by china and other severely hit nations during the present covid- outbreak. ai, data analytics, and technological support amalgamated to facilitate: • track and forecast community outbreaks: bluedot is a canadian ai company using natural language processing and machine learning algorithms to monitor news outlets, worldwide official healthcare reports in several different languages, and air-travel data and flag the mention of contagious or novel diseases such as coronavirus. importantly this is followed by scrutiny by epidemiologists and thus also has a component of human analytics. bluedot alerted its clients to the potential outbreak in wuhan, china, on december , , days prior to the who recognized it as an epidemic (https://bluedot.global/products/). chest computed tomography (ct) scans have been endorsed as a primary diagnostic tool for covid- (ai et al. ). ali-baba group's research academy has developed a deep-learning ai-enabled system that can diagnose covid- in s with % accuracy and hence possibly automate the diagnosis activity in the face of overburdened healthcare systems. the ai system identifies an infectious individual based on the chest ct scans. the algorithm has been trained with data and ct scans from nearly confirmed coronavirus cases from across china. it can be used to track the efficacy of treatment during the course of infection as well as rapidly diagnose covid- (https://www.alizila. com/how-damo-academys-ai-system-detects-coronavirus-cases/). • implementing public hygiene guidelines: risk communication in places of potential communication is critical to alert the public and implement mass hygiene measures such as use of sanitizers and face masks when in public places. google trends was used in taiwan to monitor the public risk awareness following the first imported case of covid- which correlated with the increased search keywords "covid- " and "face masks." moreover, search for "handwashing" increased coinciding with the face mask shortage. high to moderate correlations between google relative search volume and covid- cases were evident in several major cities of taiwan (husnayain et al. ) . similarly, in china an ai-based company sensetime has developed a "smart ai epidemic prevention solutions"-a quick and effective system based on facial recognition and thermal imaging to screen for individuals with fever in a crowd without physical contact and hence preventing transmission. it can also monitor if any individual is wearing a face mask or violating the quarantine rules (https://www.sensetime.com/en/news/view/id/ .html). health code, a chinese government monitoring system, for which users can sign up through alipay or wechat, assigns individuals a color code (red- days self-quarantine/yellow- days self-quarantine/green-free movement) based on their travel history, time spent in outbreak hotspots and exposure to potential carriers of the virus. the software can be used to check the color of an individual on entering their identity numbers. • characterization of the cov- and vaccine/therapy development: the genome detective coronavirus typing tool is a web-based, user-friendly software application that can identify the novel severe acute respiratory syndrome (sars)-related coronavirus (sars-cov- ) sequences isolated in the original outbreak in china and later around the world. the tool accepts sequences per submission, analyzing them in approximately min. this tool facilitates tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs, and vaccines to stop the covid- disease (cleemput et al. ). google's ai platform deepmind-based protein structure prediction tool alphafold has predicted and released the d structures of several understudied proteins of the cov- as an open source. these can be useful in designing antivirals/vaccines against covid- in future outbreaks (jumper et al. ) . in summary, the new-age pandemic surveillance using ai, data analytics, mathematical modeling of infectious diseases, and risk prediction has significantly contributed to the management of the present covid- pandemic and is laying the foundation for future improvisation of the pandemic action plans. critical issues to be addressed while recruiting laboratory diagnostic services during the novel covid- pandemic are: • the authenticity of the diagnostic tool in light of the novelty of the coronavirus- • bulk of samples to be processed • the ease of obtaining the sample for use in the diagnostic tool the coronavirus causing covid- was first isolated from a clinical sample on january , , and within weeks, several reliable and sensitive diagnostic tools were developed and deployed. by mid-january, the first rt-pcr assays for covid- were accessible in hubei. the viral sequences and pcr primers and probe sequences were open-sourced and uploaded to public platforms by the centre for disease control, china. by february, there were ten kits for the detection of covid- approved in china by the national medical products association-six were rt-pcr kits, one isothermal amplification kit, one virus sequencing product, and two colloidal gold antibody detection kits (https://www.who.int/docs/defaultsource/coronaviruse/who-china-joint-mission-on-covid- -final-report.pdf). as of today, when the disease has assumed pandemic proportions, the volume of diagnostic tools needs to be multiplied, and hence, the united states food and drug administration (usfda) has provided regulatory relief to several testing companies like thermofisher, hologic, and labcorp under the emergency use authorizations, to facilitate ease of diagnosis. also it is keeping a tight watch on fraudulent companies claiming to sell interventions against covid- (https://www.fda.gov/ emergency-preparedness-and-response/mcm-issues/coronavirus-disease- covid- ). moreover, the world's first crispr (clustered regularly interspaced short palindromic repeats)-based diagnostic kit has been developed for covid- by mammoth biosciences, usa, and university of california. notably, the diagnostic kit is a simple strip-based assay and is easy to use and allows rapid detection without the need of transporting samples over long distances. the kit is still under approval evaluation by the fda (https://www.medrxiv.org/content/ . / . . . v ). nose and mouth swabs, the most widespread samples for cov- diagnostics, require trained personnel to procure samples. however, the study by to and coworkers recommends saliva as an easy-to-procure, noninvasive sample not requiring any trained personnel in covid- screening. they detected cov- in out of patient samples and also could trace the declining titers post-hospitalization . additionally, a long-term goal of evolving the diagnostics for this novel disease is to develop a prognostic marker of covid- . although it is too soon to say, qu and coworkers have proposed platelet counts and platelet to lymphocyte ratios as prognostic marker to distinguish between severe and non-severe patients. severe patients had higher platelet peaking and platelet to lymphocyte ratio correlating with deranged chest ct and longer hospital stays against the lower platelet peaks and platelet to lymphocyte ratios and lesser hospitalization stay (qu et al. ). vaccines and antiviral drugs are the prophylactic and therapeutic measures against a viral disease cov- . on march , , pfizer inc. and biontech announced to co-develop and distribute a potential mrna-based coronavirus vaccine which is likely to enter clinical testing by the end of april (https://www.pfizer.com/ news/press-release/press-release-detail/pfizer_and_biontech_to_co_develop_poten tial_covid_ _vaccine). however, in pandemic scenarios of new infectious diseases such as covid- , vaccine supplies will be limited or nonexistent at the early phase in lieu of the novelty of the disease and the unpredictability of the pandemic occurrence. thus, vaccines cannot be stockpiled, and production can only start once the novel virus has been recognized. with the current state-of the-art, the first doses of vaccine are not likely to become available in the early months of the pandemic. however, the pandemic action plans have accounted for forward planning to increase the likelihood that the vaccine will progressively become available as the pandemic unfolds. importantly, national or regional priorities need to be fixed in the action plan for the rational use of the building/limited supply of the novel vaccine. also, production and use of vaccines during the inter-pandemic period will influence their availability during a pandemic. thus, improving the infrastructure and logistics for vaccine production, administration, cold-chain, and professional training with the novel vaccines are important to avert/cruise through future outbreak events. the who advisory on prioritizing the population groups are as enlisted in descending order. however, the priorities need tailoring in each country/region according to local needs and epidemiological circumstances. recommended prioritizing of the groups is as follows: • healthcare workers and essential service providers • groups at high risk of death and severe complications requiring hospitalization • individuals (adults and children aged more than months) in the community who have chronic cardiovascular, pulmonary, metabolic or renal disease, or are immunocompromised • persons without risk factors for complications (https://www.who.int/csr/ resources/publications/influenza/ _ _ _a.pdf) antivirals are a crucial adjunct to vaccination as a potential strategy for managing covid- . several drugs such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical studies to establish their efficacy and safety against covid- . it is important to establish a regular supply chain of antivirals and a high surge capacity in the face of cov- pandemic and future outbreaks. antivirals have a significant impact in reducing morbidity and mortality in light of unlikelihood of availability of vaccine against in early phases of pandemic. it is important to evaluate the non-interference of the antiviral interventions with the eventual vaccination, as well as the epidemiology of the group of individuals most seriously affected. it is also advisable to make available the information about the performance characteristics, side effects, and costs of antiviral therapy to public. also the commonly used neuraminidase inhibitors in influenza pandemics are ineffective in the case of cov- mediated infection and outbreaks (pang et al. ; dong et al. a , https://www. who.int/csr/resources/publications/influenza/ _ _ _a.pdf). the incorporation of antiviral therapy can be categorized as a prophylactic and for treatment use. as with vaccines, prioritizing of groups for antiviral therapy is advised as follows: • essential service providers, including healthcare workers (prophylaxis or treatment). especially, healthcare providers are in a position to be in direct contact with infectious individuals and are thus entitled to priority antiviral therapy. other community services such as those responsible for vaccine manufacture and delivery and personnel responsible for enforcing law and order and public safety. • groups at high risk of death and severe complications requiring hospitalization. the goal of prophylaxis or treatment here is to rein the mortality and morbidity. thus, high-risk persons living in the community outbreaks, seriously ill hospitalized patients, patients for whom a potential cov- vaccination is contraindicated are prioritized. • persons without known risk factors for complications from covid- . here the approach is generally therapeutic and aims to rein the morbidity and rationalize the use of healthcare resources such as antibiotics. though, the logistics of this strategy are extensive and expensive (requires large quantities of antivirals and access to healthcare service providers), it is most likely to limit the economic and social destabilization associated with a pandemic (monto ; henry , https://www.who.int/csr/resources/publications/influenza/ _ _ _a.pdf). a major step in ensuring bulk supplies of antivirals and vaccines is the setting up of medical stockpiles as a part of the pandemic preparedness plans. the usa has constituted a strategic national stockpile which is the nation's largest supply of potentially life-saving pharmaceuticals and medical supplies-antibiotics, chemical antidotes, antitoxins, vaccines, life-support medication, iv administrations, airway maintenance supplies, and other emergency medical and surgical items, for use in a public health emergency severe enough to deplete the local supplies. the facility also houses a data bank of other stockpiles and supply agencies, so that any emergency requirements can be procured in the shortest possible time (https:// www.phe.gov/about/sns/pages/default.aspx). healthcare personnel, maintenance of hygiene and disinfection during pandemics • rapid facilitation of treatment/prophylaxis, hospital centers, quarantine centers • caring for the patients as well as the health service providers to ensure uninterrupted care • communicating awareness about public and personal hygiene and implementing measure to ensure personal and public hygiene in the face of a highly infectious disease causing novel virus, china has successfully executed an ambitious, swift, and aggressive disease containment effort in the history of mankind. the laudable of its responses to facilitate timely medical care for infected was construction of two dedicated hospitals- -bed huoshenshan facility and the -bed leishenshan hospital in weeks (https://www.wsj.com/arti cles/how-china-can-build-a-coronavirus-hospital-in- -days- ). moreover, it ensured coordinated medical supplies, reserve beds were used and relevant premises were repurposed medical care facilities, and prices of commodities were controlled to ensure the smooth operation of the society (https://www.who.int/docs/ default-source/coronaviruse/who-china-joint-mission-on-covid- -final-report.pdf). ensuring the care of the medical service providers is a key response strategy to warrant efficient care for the general public. under the china's response plan, the healthcare workers were facilitated with personal protective equipment. nosocomial infections accounted were reported to be nearly from hospitals across china. the majority of this nosocomial infection ( %) were reported from hubei. a deeper contact tracing indicates infection of the healthcare worker from households than the workplace and were pinpointed to the early stages of the epidemic when understanding of the covid- transmission and medical supplies were limited (https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mis sion-on-covid- -final-report.pdf). in fact a novel infection control system for averting nosocomial infections of covid- was proposed by chen and coworkers, titled "the observing system." designated personnel called "infection control observers" were appointed by the department of infection control and nursing in guangdong second provincial general hospital who underwent training to familiarize infection control requirements in the negative pressure isolation wards. the wards were under camera surveillance and the infection control observer monitors medical staff in real-time via computer monitors outside the ward. the observer ensures normal operation of the negative pressure isolation wards, supervise the implementation of disinfection, ensure a sufficient supply of protective materials, arrange specimens for inspection, and relieve anxiety of the medical personnel while treating patients . personal and public hygiene/disinfection implementation is a key step to control transmission and prevent community outbreaks. good hand hygiene and respiratory hygiene have been aggressively promoted by who worldwide in this covid- pandemic. the basic personal and public hygiene practices are depicted in fig. . . fig. . the basic personal and public hygiene practices a key parameter to consider while disinfecting in households and public places hence avoid contact with infected surfaces is the estimation of decay rates of cov- in aerosols and on various materials composing the surfaces. it has been estimated that cov- in an aerosol (< μm, similar to those observed in samples obtained from the upper and lower respiratory tract in humans) was viable for up to h, up to h on copper surfaces, up to h on cardboard, and - days on plastic and stainless steel (van doremalen et al. ). on the basis of these findings, disinfection protocols can be set up for public places/medical facilities depending on the surfaces involved, and even in households' frequently touched surfaces like door handles, slabs, and tables be disinfected. the exponential transmission of cov- -starting with few infectious individuals with covid- quickly increasing manifold in a geographical location within a short time-is a recurring observation (dong et al. b , https://www.who.int/ emergencies/diseases/novel-coronavirus- /situation-reports, https://www.ecdc. europa.eu/en/publications-data/download-todays-data-geographic-distributioncovid- -cases-worldwide). figure . depicts the exponential surge in new confirmed cases per day plotted against time lapse post outbreak. the highly implemented global covid- management strategies of social distancing, travel restrictions, implementation of personal and public hygiene (non-pharmaceutical interventions), and pursuit of pharmaceutical interventions aim to delay the peaking of the outbreak, avoid burden on the healthcare infrastructure and personnel to ensure quality care for all in need, and rein overall mortality and declined health effects. this phenomenon has been described as "flattening of the curve" and is much shared on social media platforms facilitating awareness in the public (https://www.cdc.gov/flu/pandemic-resources/ pdf/community_mitigation-sm.pdf). hereafter, we discuss the key, successful globally adopted strategies for covid- management. it is a non-pharmaceutical infection prevention and control intervention to avoid or control contact between infectious and uninfected individuals to rein the disease transmission in a community eventually culminating into decreased infection spread, morbidity and mortality. the individuals infected with cov- shed the virus from their respiratory tract during the early infection stage when there are minor clinical manifestations leading to the extensive community transmission. while practicing social distancing, each healthy individual behaves like an infected individual, self-restricting contact with others. the main advantage is gleaned when an individual in incubation period/early infectious stage of covid- restricts coming in contact with other healthy individuals. this strategy involves the policymakers, and strict advisories/orders are issued under the pandemic action plan to restrict public gatherings and events, shutting down of educational institutes/offices/restaurants, avoiding nonessential travel and use of public transport, maintaining a distance of m at least between two individuals, restricting visit to hospitals, and avoiding online shopping. additionally, the elderly and individuals with hypertension, cardiovascular disease, diabetes, chronic respiratory diseases, and cancer are at a higher mortality risk (https://www.mohfw.gov.in/socialdistancingadvisorybymohfw. pdf, https://onlinelibrary.wiley.com/doi/pdf/ . /ijcp. ; zou et al. ) . the who director-general at a media briefing on covid- on march , , recommends testing of any suspected case, isolation to break the chain of transmission, and contact tracing for the prior days the subject came in contact with and then testing them as well (https://www.who.int/dg/speeches/detail/who-director-gen eral-s-opening-remarks-at-the-media-briefing-on-covid- % d% d- -march- ). a case in point-south korea carried out , covid- tests until march , -second highest across the world trailing behind china (https:// ourworldindata.org/coronavirus-testing-source-data), combined with strict social distancing implementation and is now seeing a fall in the number of covid- cases (https://ourworldindata.org/grapher/daily-cases-covid- -who?time¼ .. & country¼kor, https://www.sciencemag.org/news/ / /coronavirus-caseshave-dropped-sharply-south-korea-whats-secret-its-success). social distancing combined with case isolation and contact tracing has demonstrated its effectiveness in controlling the transmission from imported infection cases to the community transmission scenario (wilder-smith and freedman ). the relevance of case isolation and contact tracing are highlighted in a study by hu and coworkers where they have performed a clinical characterization of asymptomatic patientsconfirming infection by a laboratory-confirmed positive for the covid- virus nucleic acid from pharyngeal swab samples. the asymptomatic cases did not present any obvious symptoms while nucleic acid screening. about . % developed classical symptoms of fever, cough, and fatigue, during hospitalization; . % presented ct images of ground-glass chest and . % presented stripe shadowing in the lungs; . % cases presented normal ct image and had no symptoms during hospitalization, comprising the younger subset (median age: . years). none developed severe covid- pneumonia, and no mortality was observed. however, epidemiological investigation indicated at typical asymptomatic transmission to the cohabiting family members, which even caused severe covid- pneumonia. this study puts a spotlight on close contact tracing and longitudinally surveillance via virus nucleic acid tests. case isolation and continuous nucleic acid tests are also recommended (hu et al. ) . de-isolating of suspect cases where the first confirmatory test has returned negative is also an emerging issue of concern and needs improvement in testing capacity as well as strict implementation of social distancing during pandemic scenarios (tay et al. ). approaching january , , the daily risk of exporting at least a one covid- infected individual from mainland china through international travel exceeded %. for containment of the global spread of cov- and a covid- epidemic, china implemented border control measures-airport screening and travel restrictions, and were later also adopted by several other countries. wells and coworkers in their study in proceedings of natural academy of sciences used daily incidence data of covid- outbreak from china from december , , to february , , as well as airline network data, to predict the number of exported cases with and without measures of travel restriction and screening. the group put forth that the lockdown of wuhan and more cities in hubei province on january - , , averted export of more covid- cases by mid-february. however, it is to be highlighted that these travel restrictions and lockdown measures only slowed the rate of infection exportation from china to other countries. the global spread of covid- was based on most cases arriving during the asymptomatic incubation period. the authors recommend rapid contact tracing at the epicenter and at importation sites to limit human-to-human transmission outside of the location of first outbreak (wells et al. ). the covid- pandemic in today's times of excessive electronic connectivity and several social media platforms has a potential to alleviate the global mental stress levels. especially, if local myths and rumors are circulated in the already homequarantined population, restriction in fear of the disease can lead to additional mass hysteria. thus it is important to create awareness and dispel any negative myth, as put forth by the who in context of cov- (https://www.who.int/emergencies/ diseases/novel-coronavirus- /advice-for-public/myth-busters). some myths dispelled are: • covid- virus can be transmitted in areas with hot and humid climates or cold and snowy climates. cov- can be transmitted in all areas. • hot bath cannot prevent cov- transmission. • cov- is not transmitted by mosquito bites. it is a respiratory illness and respiratory hygiene is a key protective strategy. • heat generated from hand dryers is not effective against killing cov- on your hands. the only way to disinfect hands is washing with soap-water or alcoholbased sanitizers. • thermal scanners are effective in detecting only the elevated body temperatures. a confirmatory nucleic acid test is the most guaranteed test. • all age groups can be infected by cov- . elderly people and people with pre-existing medical conditions-asthma, diabetes, cardiovascular disease-are more vulnerable to exhibit severe covid- . thus, who advises people from all age groups to protect themselves by implementing good hand hygiene and respiratory hygiene. • sars-cov- is not a bioengineered organism arising out of manipulations from earlier sars-cov. andersen and coworkers have analyzed the genome sequence of the novel coronavirus- and compared it with several other zoonotic viruses. they claim that the viral-human point of contact-the high affinity receptor binding domain of cov- binding with the angiotensin-converting enzyme (ace ) of the target host-is acquired through natural selection. also they have shed light of probable animal host being rhinolophus affinis, as another virus ratg , sampled from a bat, is~ % identical overall to cov- (https:// www.nature.com/articles/s - - - ). the covid- pandemic can cause short-term fiscal distress and longer-term damage to the global economic growth. if we trace the economic trajectory of a pandemic: • early phase measures to contain/limit outbreak-shutting down of workplaces and public businesses, mobilization of healthcare supplies and surveillance activities, contact tracing, social distancing by isolation of contacts/quarantines incur significant human resource and staffing costs (achonu et al. ). • as the scale of the epidemic expands, new medical infrastructure will need to be constructed to manage building number of confirmed infected individuals, the surge in demand for medical consumables can increase the health system expenditures (herstein et al. ). • the quarantine and lockdown impositions disrupt trans-national supply chains, transportation industry, agriculture, entertainment, and travel industry. hoarding and black marketing of essential and medical commodities are expected. • behavioral changes during pandemic-avoiding association with other people to avoid infection-are the major determinant for economic impact of pandemics ( fig. . ) and not mortality. the behavioral change fear driven which in turn is driven by awareness and ignorance (burns et al. risk_jonas.pdf). • the high-income countries in scenario of a moderate pandemic can offset fiscal distress by providing official development assistance to affected countries and budgetary support. however, during a severe pandemic, a high-income country will also confront the same fiscal stresses and may be unwilling to provide assistance. on march , , worldwide , confirmed cases were reported, mortality was against , confirmed cases, and mortality of on march , when the covid- crisis was declared a pandemic (https://www.who.int/emergen cies/diseases/novel-coronavirus- /situation-reports). it is increasingly becoming evident that no region of the world will remain untouched by cov- invasion. however, pandemic preparedness is the key to tackle the present-day covid- health emergency worldwide. the rapid and effective enforcement of existing international and national action plans, as well as parallel review and improvisation, is facilitating the affected countries to contain transmission and possibly delay the peak of outbreak and mortality and garner recovery. it is notable that in the present pandemic scenario, innovative ai-powered surveillance, quick and strategic response actions-the trinity of testing-isolation-contact tracing, committed social distancing measures-travel restrictions, self-isolation, implementation of personal and public hygiene, and extensive mobilization of medical care facilities are helping the world mitigate through. the most insightful trend emerging from the global clinical and epidemiological data is that the identification of asymptomatic infected individuals is a crucial step to contain community outbreaks. for preventing future outbreaks of cov- infection, high-volume cutting-edge investigations are warranted in understanding the covid- pathology, cov- origin, biology, structural data of potential surface antigens, and precise anti-cov- antiviral therapies. although the global economy is suffering at the hands of cov- , it is important to review the current action plans and suitably improvise the future action plans to avoid potential recurrence. pandemics are unforeseen. national and international preparedness are crucial to tackle a pandemic. world over, humanity is grappling with covid- . the pandemic preparedness charter prescribed by international and national agencies to tackle covid- is evolving on the go. the key facets of pandemic preparedness emerging from global success and failure scenarios are: • active surveillance employing state-of-the art technology: -development of mathematical models for simulating the infection of cov- in a given country. the models can help predict the basic reproductive number which in turn facilitates monitoring the pandemic on day-to-day basis. -incorporation of contactless artificial intelligence-based technologies for mass thermal screening, track and forecast community outbreaks, implement public hygiene and use of masks, and contact tracing. • expanding the diagnose capacity/testing for cov- (the infectious agent) to catch any asymptomatic carriers, but at the same time ensuring: -precision of the test. -test is adaptable to processing bulk samples. -easy procurement of sample from suspected infected individuals. • management of bulk antiviral interventions and vaccines: -national or regional priorities need to be fixed for rational use of antiviral/emerging vaccines -prioritizing the population has been recommended: the healthcare workers and essential service providers are top priority. -medical stockpiles should be established. • promoting facilities for healthcare/healthcare workers and facilitating public disinfection and hygiene via: -rapid expansion of hospitalization facilities for treatment and isolation/ quarantine centers. -ensuring minimal nosocomial infections to the healthcare workers. -issuing and implementation of guidelines for ensuring good hand and respiratory hygiene. (continued) -public disinfection (and personal household as well) based on the established life of cov- on different surfaces. • globally successful strategies are: -social distancing and lockdowns to ensure minimal human-human contact. -travel restrictions to facilitate containment. -trifecta of test-isolate-contact tracing. • the economic cost of the covid- pandemic management is expanding due to: -disruption of economic activities due to implementation of social distancing via lockdowns. -battling the diseased and the increasing mortality. -diversion of resources to expansion of healthcare systems. -international and national relief funds are being constituted. -fiscal reliefs and aid from developed nations to the developing or underdeveloped countries can release the surmounting economic pressure. the financial impact of controlling a respiratory virus outbreak in a teaching hospital: lessons learned from sars does urbanization make emergence of zoonosis more likely? evidence, 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structures associated with covid- . deepmind website - ) early dynamics of transmission and control of covid- : a mathematical modelling study what we know so far: covid- current clinical knowledge and research nugent r (eds) disease control priorities: improving health and reducing poverty, rd edn. the international bank for reconstruction and development/the world bank vaccines and antiviral drugs in pandemic preparedness potential rapid diagnostics, vaccine and therapeutics for novel coronavirus ( -ncov): a systematic review platelet-to-lymphocyte ratio is associated with prognosis in patients with corona virus disease- mathematical modeling of infectious disease dynamics pii: ciaa ) de-isolating covid- suspect cases: a continuing challenge pii: ciaa ) consistent detection of novel coronavirus in saliva unique epidemiological and clinical features of the emerging novel coronavirus pneumonia (covid- ) implicate special control measures pii: ) impact of international travel and border control measureson the global spread of the novel coronavirus outbreak isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study sars-cov- viral load in upper respiratory specimens of infected patients key: cord- - rgz t authors: radandt, siegfried; rantanen, jorma; renn, ortwin title: governance of occupational safety and health and environmental risks date: journal: risks in modern society doi: . / - - - - _ sha: doc_id: cord_uid: rgz t occupational safety and health (osh) activities were started in the industrialized countries already years ago. separated and specific actions were directed at accident prevention, and the diagnosis, treatment and prevention of occupational diseases. as industrialization has advanced, the complexity of safety and health problems and challenges has substantially grown, calling for more comprehensive approaches. such development has expanded the scope, as well as blurred the borders between specific activities. in the modern world of work, occupational safety and health are part of a complex system that involves innumerable interdependencies and interactions. these are, for instance, safety, health, well-being, aspects of the occupational and general environment, corporate policies and social responsibility, community policies and services, community social environment, workers’ families, their civil life, lifestyles and social networks, cultural and religious environments, and political and media environments. a well-functioning and economically stable company generates resources to the workers and to the community, which consequently is able to maintain a positive cycle in development. a high standard of safety and health brings benefits for everyone: the company, the workers and the whole community. these few above-mentioned interactions elucidate the need for an integrated approach, and the modelling of the complex entity. if we picture osh as a house, this integrated approach could be the roof, but in order to build a stable house, it is also necessary to construct a solid basement as a foundation to the house. these basement "stones" are connected to each other, and are described in more detail in sections . - . . section . focuses on the existing hazards, while section . mainly considers the exposure of workers to health hazards. health, due to its complexity, however, is not only influenced and impaired by work-related hazards, but also by hazards arising from the environment. these two sub-chapters are thus linked to section . . in addition, the safety levels of companies may affect the environment. the strategies and measures needed for effective risk management, as described in section . , therefore also contribute to reducing the risks to the environment. in the case of work that is done outdoors, the hazards arising from the environment understandably have to be given special attention. here, the methods applied to tackle the usual hazards at workplaces are less effective. it is necessary to develop protective measures to avoid or minimize hazards present in the environment. namely, agriculture, forestry and construction involve these types of hazards, and affect high numbers of workers on a global scale. finally, hazards in the environment or in leisure-time activities can lead to strain and injuries which -combined with hazards at work -may result in more severe health consequences. as an example one can mention the hazardous substances in the air causing allergies or other illnesses. another example is the strain on the musculoskeletal system from sports and leisuretime activities causing low back pain and other musculoskeletal disorders. depending on the type of hazard, the three topics, namely, safety, health and the environment, may share the common trait that the proper handling of risks, i.e., how to reduce probabilities and/or consequences of unwanted events is not always possible within a risk management system. this is true when one moves into the realm of uncertainty, i.e., when there is uncertain, insufficient or no knowledge of the consequences and/or probabilities (see chapter ). . integrated multi-sectorial bodies for policy design and planning (national safety and health committee). . comprehensive approach in osh activities. . multi-disciplinary expert resources in inspection and services. . multi-professional participation of employers' and workers' representatives. . joint training in integrated activities. . information support facilitating multi-professional collaboration. international labour office (ilo) ( ) international labour conference, st session, report vi, ilo standards-related activities in the area of occupational safety and health: an in-depth study for discussion with a view to the elaboration of a plan of action for such activities. sixth item on the agenda. international labour office, geneva. what are the main challenges arising from the major societal changes for business/companies and workers/employees? how can these challenges be met in order to succeed in the growing international competition? what is the role of occupational safety and health (osh) in this context? the above-mentioned changes create new possibilities, new tasks and new risks to businesses in particular, and to the workers as well. in order to optimize the relation between the possibilities and the risks (maximize possibilities -minimize risks) there is a growing need for risk management. risk management includes all measures required for the target-oriented structuring of the risk situation and safety situation of a company. it is the systematic application of management strategies for the detection, assessment, evaluation, mastering and monitoring of risks. risk management was first considered exclusively from the point of view of providing insurance coverage for entrepreneurial risks. gradually the demands of jurisdiction grew, and the expectations of users and consumers increased with regard to the quality and safety of products. furthermore, the ever more complex problems of modern technology and ultimately the socioeconomic conditions have led to the development of risk management into an independent interdisciplinary field of work. risks can be regarded as potential failures, which may decrease trust in realizing a company's goals. the aim of risk management is to identify these potential failures qualitatively and quantitatively, and to reduce them to the level of a non-hazardous and acceptable residual risk potential. the development and formulation of a company's risk policy is regarded as the basis of effective risk management. this includes, first and foremost, the management's target concept with respect to the organization of work, distribution of labour, and competence of the departments and persons in charge of risk management. risk issues are important as far as acceptance of technology is concerned. it is not enough to reduce the problem to the question of which risks are tolerable or acceptable. it appears more and more that, although the risks themselves are not accepted, the measures or technologies causing them are. value aspects have an important role in this consideration. a positive or negative view of measures and technologies is thus influenced strongly by value expectations that are new, contradictory and even disputed. comparing risks and benefits has become a current topic of discussion. the relation between risks and benefits remains an unanswered question. the general public has a far broader understanding of the risks and benefits of a given technology than the normal understanding professed by engineering sciences which is limited to probability x harm. the damage or catastrophe potential, qualitative attributes such as voluntary nature and controllability also play an important role in the risk assessment of a technology. a normative setting for a certain, universally accepted risk definition according to engineering science is therefore hardly capable of consensus at the moment. the balanced management of risks and possibilities (benefits) is capable of increasing the value of a company. it may by far surpass the extent of legal obligations: for example, in germany, there is a law on the control and transparency for companies (kontrag) . the respective parameters may be defined accurately as follows: • strategic decisions aim to offer opportunities for acquiring benefit, taking into consideration risks. • risks that can have negative consequences to the technological capacities, the profitability potential, the asset values and the reputation of a company, as well as the confidence of shareholders are identified and measured. • the management focuses on important possibilities and risks, and addresses them adequately or reduces them to a tolerable level. the aim is not to avoid risks altogether, but to create opportunities for promoting proactive treatment of all important risks. the traditional occupational health hazards, such as physical, chemical and biological risks, as well as accidents, will not totally disappear as a consequence of change, nor will heavy physical work. about - % of workers are still exposed to such hazards. there is thus need to still develop risk assessment, prevention and control methods and programmes for these often well-known hazards. in many industrialized countries, prevention and control programmes have had a positive impact by reducing the trends of occupational diseases and accidents, particularly in big industries. some developing countries, however, show an increase in traditional hazards. international comparisons, however, are difficult to make because of poor coverage, underreporting, and poor harmonization of concepts, definitions and registration criteria. statistics on occupational accidents are difficult to compare, and therefore data on their total numbers in europe should be viewed with caution. the majority of countries, however, have shown declining trends in accident rates irrespective of the absolute numbers of accidents. some exceptions to this general trend have nevertheless been seen. the accident risk also seems to shift somewhat as regards location, so that instead of risks related to machines and tools, particularly the risks in internal transportation and traffic within the workplace grow in relative importance. this trend may increase in future, particularly as the work place, as well as the speed and volume of material flows are increasing. a threat is caused by lengthened working hours, which tend to affect the vigilance of workers and increase the risk of errors. small-scale enterprises and micro-enterprises are known to have a lower capacity for occupational health and safety than larger ones. in fact, a higher accident risk has been noted in medium-sized companies, and a lower risk in very small and very large enterprises. we can conclude that this is due to the higher mechanization level and energy use in small and medium-sized enterprises (sme) compared with micro-enterprises, which usually produce services. on the other hand, the better capacity of very large enterprises in safety management is demonstrated by their low accident rates. the production of chemicals in the world is growing steadily. the average growth has been between - % a year during the past - decades. the total value of european chemical production in was about usd billion, i.e. % of the world's total chemical production, and it has increased % in the -year period of - . the european union (eu) is the largest chemical producer in the world, the usa the second, and japan the third. there are some , different chemical entities in industrial use, but only about , are so-called high-production volume (hpv) chemicals produced (and consumed) in amounts exceeding , tons a year. the number of chemicals produced in amounts of - , tons a year is about , . but volume is not necessarily the most important aspect in chemical safety at work. reactivity, toxicological properties, and how the chemicals are used, are more important. the european chemical companies number some , , and in addition there are , plants producing plastics and rubber. surprisingly, as many as % of these are smes employing fewer than workers, and % are micro-enterprises employing fewer than workers. thus, the common belief that the chemical industry constitutes only large firms is not true. small enterprises and self-employed people have much less competence to deal with chemical risk assessment and management than the large companies. guidance and support in chemical safety is therefore crucial for them. the number of workers dealing with chemicals in the european work life is difficult to estimate. the chemical industry alone employs some . million workers in europe, i.e. about % of the workforce of manufacturing industries. about %, i.e. over , work in chemical smes. but a much higher number of workers are exposed in other sectors of the economy. there is a distinct trend showing that the use of chemicals is spreading to all sectors, and thus exposures are found in all types of activities: agriculture, forestry, manufacturing, services and even in high-tech production. the national and european surveys on chemical exposures in the work environment give very similar results. while about % of the eu work-ers were exposed to hazardous chemicals, the corresponding figure in central and eastern european countries may be much higher. the workers are exposed simultaneously to traditional industrial chemicals, such as heavy metals, solvents and pyrolytic products, and to "new exposures", such as plastics monomers and oligomers, highly reactive additives, cross-linkers and hardeners, as well as to, for example, fungal spores or volatile compounds in contaminated buildings. this implies that some million people in the eu are exposed at work, and usually the level of exposure is one to three orders of magnitude higher than in any other environment. about the same proportion ( % of the workforce, i.e. , ) of the finnish workers in the national survey reported exposure. the chemicals to which the largest numbers of workers are exposed occur typically in smes; they are e.g. detergents and other cleaning chemicals, carbon monoxide, solvents, environmental tobacco smoke, and vegetable dusts. european directives on occupational health and safety require a high level of protection in terms of chemical safety in all workplaces and for all workers. risk assessment and risk management are key elements in achieving these requirements. the risk assessment of chemicals takes place at two levels: a) systems-level risk assessment, providing a dose-response relationship for a particular chemical, and serving as a basis for standard setting. risk assessment at the systems level is carried out in the pre-marketing stage through testing. this consequently leads to actions stipulated in the regulations concerning standards and exposure limits, labelling and marking of hazardous chemicals, limitations in marketing, trade and use. in this respect, the level of safety aimed at remains to be decided. is it the reasonably achievable level or, for example, the level achieved by the best available technology? the impact is expected to be system-wide, covering all enterprises and all workers in the country. this type of risk assessment is an interactive practice between the scientific community and the politically controlled decision making. a high level of competence in toxicology, occupational hygiene and epidemiology is needed in the scientific community. and the decision makers must have the ability to put the risk in concern into perspective. in most countries the social partners also take part in the political decision making regarding occupational risks. b) workplace risk assessment directed at identifying the hazards at an individual workplace and utilizing standards as a guide for making decisions on risk management. risk assessment at workplace level leads to practical actions in the company and usually ensures compliance with regulations and standards. risk assessment is done by looking at the local exposure levels and comparing these with standards produced in the type a) risk assessment. risk management is done through preventive and control actions by selecting the safest chemicals, by controlling emissions at their source, by general and local ventilation, and by introducing safe working practices. if none of the above is effective, personal protective devices must be taken into use. noise is a nearly universal problem. the products of technology, which have freed us from the day-to-day survival struggle, have been accompanied by noise as the price of progress. however, noise can no longer be regarded as an inevitable by-product of today's society. not only is noise an undesirable contaminant of our living environment, but high levels of noise are frequently present in a variety of work situations. many problems arise from noise: annoyance, interference with conversation, leisure or sleep, effects on work efficiency, and potentially harmful effects, particularly on hearing. in short, noise may affect health, productivity, and well-being. the selection of appropriate noise criteria for the industry depends on knowledge of the effects of noise on people, as well as on the activities in which they are engaged. many of the effects are dependent on the level, and the magnitude of the effects varies with this level. hearing damage is not the only criterion for assessing excessive noise. it is also important to consider the ability and ease of people to communicate with each other. criteria have therefore been developed to relate the existing noise environment to the ability of the typical individual to communicate in areas that are likely to be noisy. the effects of noise on job performance are difficult to evaluate. in general, one can say that sudden, intermittent, high-intensity noise impedes efficient work more than low-intensity and steady-state noise. the complexity of the task with which noise interferes plays a major role in determining how much noise actually degrades performance. two common ways in which noise can interfere with sleep are: delaying the onset of sleep, and shifting sleep stages. one effect of noise that does not seem to depend strongly on its level is annoyance. under some circumstances, a dripping water faucet can be as annoying as a jackhammer. there are no generally accepted criteria for noise levels associated with annoyance. if the noise consists of pure tones, or if it is impulsive in nature, serious complaints may arise. new information and communication technologies (ict) are being rapidly implemented in modern work life. about % of workers have computers at work, and about % are e-mail and internet users. there are three main problem areas in the use of new ict at work. these are: ) the visual sensory system, ) the cognitive processes, and ) the psychomotoric responses needed for employing hand-arm systems. all three have been found to present special occupational health and even safety problems, which are not yet fully solved. the design of new more user-friendly technology is highly desirable, and the criteria for such technology need to be generated by experts in neurophysiology, cognitive psychology and ergonomics. it is important to note that the productivity and quality of information-intensive work requiring the use of ict depends crucially on the user-friendliness of the new technology interface, both the hardware and software. communication and information technologies will change job contents, organization of work, working methods and competence demands substantially in all sectors of the economy in all countries. a number of new occupational health and safety hazards have already arisen or are foreseen, including problems with the ergonomics of video display units, and musculoskeletal disorders in shoulder-neck and arm-hand systems, information overload, psychological stress, and pressure to learn new skills. the challenge to occupational health and safety people is to provide health-based criteria for new technologies and new types of work organization. it is also important to contribute to the establishment of healthy and safe work environments for people. in the approved and draft standards of the international standardization organization, iso, there are altogether about different targets dealing with the standardization of eyesight-related aspects. vision is the most important channel of information in information-intensive work. from the point of view of seeing and eye fatigue, the commonly used visual display units (vdu) are not the most optimal solutions. stability of the image, poor lighting conditions, reflections and glare, as well as invisible flicker, are frequent problems affecting vision. the displays have, however, developed enormously in the s, and there is evidence that the so-called flat displays have gradually gained ground. information-intensive work may increasingly load the vision and sense of hearing, particularly of older workers. even relatively minor limitations in vision or hearing associated with ageing have a negative effect on receiving and comprehending messages. this affects the working capacity in information-intensive work. the growing haste in information-intensive work causes concern among workers and occupational health professionals. particularly older workers experience stress, learning difficulties and threat of exclusion. corrective measures are needed to adjust the technology to the worker. the most important extension of the man-technology interface has taken place in the interaction of two information-processing elements: the central nervous system and the microprocessor. the contact is transmitted visually and by the hands, but also by the software which has been developed during the s even more than the technology itself. many problems are still associated with the immaturity of the software, even though its user-friendliness has recently greatly improved. the logic and structure of the software and the user systems, visual ergonomics, information ergonomics, the speed needed, and the forgiving characteristics of programs, as well as the possibility to correct the commands at any stage of processing are the most important features of such improvements. also the user's skills and knowledge of information technology and the software have a direct effect on how the work is managed and how it causes workload. the user-friendliness and ergonomics of the technology, the disturbing factors in the environment, haste and time pressure, the work climate, and the age and professional skills of the individual user, even his or her physical fitness, all have an impact on the cognitive capacity of a person. this capacity can to a certain extent be improved by training, exercise and regulating the working conditions, as well as with expert support provided for the users when difficulties do occur. the use of new technologies has been found to be associated with high information overload and psychological stress. the problem is not only typical for older workers or those with less training, but also for the super-experts in ict who have shown an elevated risk of psychological exhaustion. there are four main types of ergonomic work loads: heavy dynamic work that may overload both the musculoskeletal and cardiovascular system; re-petitive tasks which may cause strain injuries; static work that may result in muscular pain; and lifting and moving heavy loads, which may result in overexertion, low back injury, or accidental injuries. visual ergonomics is gaining in importance in modern work life. the overload of the visual sensory system and unsatisfactory working conditions may strain the eye musculature, but can also cause muscle tension in the upper part of the body. this effect is aggravated if the worker is subjected to psychological stress or time pressure. in addition to being a biological threat, the risk of infections causes psychological stress to workers. the improved communication between health services and international organizations provides help in the early detection and control of previously unknown hazards. nevertheless, for example, the danger related to drug abusers continues to grow and present a serious risk to workers in, for example, health services and the police force. some new viral or re-emerging bacterial infections also affect health care staff in their work. the increase in the cases of drug-resistant tuberculosis is an example of such a hazard. the goal of preventive approaches is to exert control on the cause of unwelcome events, the course of such negative events or their outcome. in this context, one has to decide whether the harmful process is acute (an accident) or dependent on impact duration and stimulus (short-, medium-, and long-term). naturally, the prevention approaches depend on the phases of the harmful process, i.e. whether the harm is reversible, or whether it is possible only to maintain its status, or to slow down the process. it is assumed here that a stressful factor generates an inter-individual or intra-individual strain. thus the effects and consequences of stress are dependent on the situation, individual characteristics, capabilities, skills and the regulation of actions, and other factors. the overall consideration is related to work systems characterized by work contents, working conditions, activities, and actions. system performance is expected of this work system, and this system performance is characterized by a performance structure and its conditions and requirements (figure . ). the performance of the biopsychosocial unit, i.e. the human being, plays an important role within the human performance structure (see figures . and . ). the human being is characterized by external and internal features, which are closely related to stress compatibility, and thus to strain. in this respect, preventive measures serve to optimize and ensure performance, on the one hand, and to control stress and strain, on the other. preventive measures aim to prevent bionegative effects and to facilitate and promote biopositive responses. • the internal factors affecting performance are described by performance capacity and performance readiness. • performance capacity is determined by the individual's physiological and psychological capacity. • performance readiness is characterized by physiological fitness and psychological willingness. • the external factors affecting performance are described by organizational preconditions/requirements and technical preconditions/requirements. • regarding the organizational requirements, the organizational structure and organizational dynamics are of significance. • in the case of technical requirements, the difficulties of the task, characterized by machines, the entire plant and its constructions, task content, task design, technical and situation-related factors, such as work layout, anthropometrics, and quality of the environment, are decisive (table . ). mental stress plays an increasing role in the routine activities of enterprises. through interactive models of mental stress and strain, it is possible to represent the development of mental strain and its impairing effects (e.g. tension, fatigue, monotony, lack of mental satisfaction). it is important to distinguish the above-mentioned impairing effects from each other, since they can arise from different origins and can be prevented or eliminated by different means. activities that strain optimally enhance health and promote safe execution of work tasks. stress essentially results from the design parameters of the work system or workplace. these design parameters are characterized by, e.g.: • technology, such as work processes, work equipment, work materials, work objects; • anthropometric design; • work techniques, working hours, sharing of work, cycle dependence, job rotation; • physiological design that causes strain, fatigue; • psychological design that either motivates or frustrates; • information technology, e.g. information processing, cognitive ergonomic design; • sociological conditions; and • environmental conditions, e.g. noise, dust, heat, cold. the stress structure is very complex, and we therefore need to look at the individual parameters carefully, taking into account the interactions and links between the parameters at the conceptual level. the design parameters impact people as stress factors. as a result, they also turn into conditions affecting performance. such conditions can basically be classified into two types: a person's internal conditions, characterized in particular by predisposition and personality traits, and a person's external conditions, determined mainly by the design parameters. when we look at performance as resulting from regulated or reactive action, we find three essential approaches for prevention: • the first approach identifies strain. it is related to anatomical, biochemical, histological, physiological characteristic values, typical curves of organ systems, the degree of utilization of skills through stress, and thus the degree of utilization of the dynamics of physiological variables in the course of stress. • the second approach is related to the control of strain. the aim is to identify performance limits, the limits of training and practice, and to put them into positive use. adaptation and fatigue are the central elements here. • the third approach for prevention is related to reducing strain. the aim is to avoid harm, using known limits as guidelines (e.g. maximum workplace concentration limit values for harmful substances, maximum organspecific concentration values, biological tolerance values for substances, limit values for noise and physical loads). however, the use of guideline values can only be an auxiliary approach, because the stress-strain concept is characterized by highly complex connections between the exogenous stress and the resulting strain. an objectively identical stress will not always cause the same level of strain in an individual. due to action regulation and individual characteristic values and curves of the organ systems (properties and capabilities), differences in strain may occur. seemingly identical stress can cause differing strain due to the superposition of partial stress. combinations of partial stress can lead to compensatory differences (e.g. physiological stress can compensate for psychological stress) or accumulation effects. partial stress is determined by the intensity and duration of the stress, and can therefore appear in differing dimensions and have varying effects. in assessing overall stress, the composition of the partial stress according to type, intensity, course and time is decisive. partial stress can occur simultaneously and successively. in our considerations, the principle of homeostasis plays an important role. however, optimizing performance is only a means to an end in a prevention programme. the actual purpose is to avoid harm, and thus to control strain. harm is a bionegative effect of stress. the causative stress is part of complex conditions in a causal connection. causal relationships can act as dose-effect relationships or without any relation to the dose. in this respect, the causative stress condition can form a chain with a fixed or variable sequence; it can add up, multiply, intensify or have an effect in only specific combinations, and generate different effects (e.g. diseases). we are thus dealing with a multicausal model or a multi-factor genesis. low back pain is an example of a complex phenomenon. the incidence of musculoskeletal disorders, especially low back pain, is rapidly increasing. several occupational factors have been found to increase the risk for low back pain. some studies indicate that psychosocial and work-related conditions are far more accurate in the prognosis of disability than are physical conditions. chronic low back pain is perceived as a phenomenon which encompasses biological, social and psychological variables. according to the model of adaptation, the goal of reducing risks is to increase a person's physical abilities (i.e. flexibility, strength, endurance), the use of body mechanics, techniques to protect the back (following the rules of biomechanics), to improve positive coping skills and emotional control. the following unfavourable factors leading to back pain have been identified at workplaces: • the lifting of too heavy loads. • working in a twisted or bent-down position. • work causing whole-body vibration. • working predominantly in a sitting position. • carrying heavy loads on the shoulders. the prevention of acute back pain and the prevention of work disability must entail several features. one important element is work safety, which can be maximized by screening a worker's physical and intellectual capacities, by ensuring ergonomic performance of the work procedures, and by increasing awareness of proper working techniques that do not strain the back. the use of adaptation programmes makes it possible to attain a higher performance level and to be able to withstand more strain (figure . ) . research-based methods of training optimize and improve performance. they are a means for controlling stress and strain with the aim of preventing bionegative effects and facilitating and promoting biopositive responses. the stress (load) and strain model and human performance can be described as follows: • causative stress generates an inter-individual or intra-individual strain. • the effects and consequences depend on a person's properties, capabilities, skills and the regulation of actions, individual characteristics of the organ systems, and similar factors. • within the performance structure, the performance of the biopsychosocial unit, i.e. the human being, plays an important role. the human being is characterized by external and internal factors, which in turn are closely related to stress compatibility and thus to strain. the connection between stress and harm plays a significant role in the research on occupational health hazards. how should this connection be explored? different hypotheses exist in replying to this question, but none of them have been definitively proven. the three most common hypotheses today are: stress occurring in connection with a person's life events. the number and extent of such events is decisive. problem-coping behaviour and/or social conditions are variables explaining the connection between stress and harm. . the additive stress hypothesis. the ability to cope with problems and the social conditions has an effect on harm which is independent of the stress resulting from life events. when we refer to the complexity of risks in this context of occupational safety our focus shall be on the enterprise. there are different kinds of risks to be found in enterprises. many of them are of general importance, i.e. they are in principle rather independent of an enterprise's size or its type of activity. how to deal with such risks shall be outlined to some extent here. in order to treat those risks at work successfully resources are needed whose availability often depends on the enterprise's situation. the situation in enterprises usually is a determining indicator for available resources to control and develop safety and health and thus performance of the enterprise and its workers and employees through appropriate preventive measures. this situation has been described to some extent in chapter . big companies usually have well-developed safety and health resources, and they often transfer appropriate policies and practices to the less developed areas where they operate. even in big enterprises, however, there is fragmentation of local workplaces into ever smaller units. many of the formerly in-built activities of enterprises are outsourced. new types of work organizations are introduced, such as flat and lean organizations, increase of telework and call centres, many kinds of mobile jobs and network organizations. former in-company occupational health services are frequently transferred to service providers. this leads to the establishment of high numbers of micro-enterprises, small scale enterprises (sses), small and medium-sized enterprises (smes) and self-employed people. sses and smes are thus becoming the most important employers in the future. from a number of studies there is evidence that at least among a part of sses and smes awareness of osh risks is low. both managers and workers often do not see the need to improve occupational safety and health or ergonomic issues and their possibilities and benefits by reducing or eliminating risks at work. as these types of enterprises, even more the self-employed, do not have sufficient resources or expertise for implementing preventive measures, the need for external advisory support, services and incentives is evident and growing. interpersonal relations in sses and smes being generally very good provides a strong chance for effectively supporting them. other special features in the structure of small and medium-sized enterprises to be considered are: • direct participation of the management in the daily activities; • the management structure is designed to meet the requirements of the manager; • less formal and standardized work processes, organizational structures and decision processes; • no clear-cut division of work: -wide range of tasks; and -less specialization of the employees; • unsystematic ways of obtaining and processing information; • great importance of direct personal communication; • less interest in external cooperation and advice; • small range of internal, especially long-term and strategic planning; and • stronger inclination of individual staff members to represent their own interests. the role of occupational health services (ohs) in smes is an interdisciplinary task, consisting of: • risk assessment: -investigation of occupational health problems according to type of technology, organization, work environment, working conditions, social relationships. • surveillance of employees' health: -medical examinations to assess employees' state of health; and -offering advice, information, training. • advice, information, training: -measures to optimize safety and health protection; and -safe behaviour, safe working procedures, first aid preparedness. different kinds of risks are found in enterprises (see table . ). these different types of risks need to be handled by an interlinked system to control the risks and to find compromises between the solutions. figure . illustrates these linkages. the promotion of safety and health is linked to several areas and activities. all of these areas influence the risk management process. the results of risk treatment not only solve occupational health and safety problems, but they also give added value to the linked fields. specific risk management methods are needed to reach the set goal. one needs to know what a risk is. the definition of risk is essential: a risk is a combination of a probability -not frequency -of occurrence, and the associated unwelcome outcome or impact of a risk element (consequence). risk management is recognized as an integral part of good management practice. it is a recurring process consisting of steps which, when carried out in a sequence, allow decision making to be improved continuously. risk management is a logical and systematic method of identifying, analyzing, evaluating, treating, monitoring and communicating risks arising during any activity, function, or process in a manner enabling the organization to minimize losses and maximize productive opportunities. different methods are available for analyzing problems. each method is especially suited to respond to certain questions and less suited for others. a complex "thinking scheme" is necessary for arranging the different analyses correctly within the system review. such a scheme includes the following steps: . defining the unit under review: the actual tasks and boundaries of the system (a fictitious or a real system) must be specified: time, space and state. . problem analysis: all problems existing in the defined system, including problems which do not originate from the system itself, are detected and described. . causes of problems: all possible or probable causes of the problems are identified and listed. . identifying interaction: the dependencies of the effect mechanisms are described, and the links between the causes are determined. . establishing priorities and formulating targets: to carry out this step, it is necessary to evaluate the effects of the causes. . solutions to the problems: all measures needed for solving the individual problems are listed. the known lists usually include technical as well as non-technical measures. since several measures are often appropriate for solving one problem, a pre-selection of measures has to be done already at this stage. however, this can only be an approach to the solution; the actual selection of measures has to be completed in steps and . . clarifying inconsistencies and setting priorities: as the measures required for solving individual problems may be inconsistent in part, or may even have to be excluded as a whole, any inconsist-encies need to be clarified. a decision should then be made in favour or against a measure, or a compromise may be sought. . determining measures for the unit under review: the measures applicable to the defined overall system are now selected from the measures for the individual problems. . list of questions regarding solutions selected for the overall system: checking whether the selected measures are implementable and applicable for solving the problems of the overall system. . controlling for possible new problems: this step consists of checking whether new problems are created by the selected solution. the close link between cause and effect demands that the processes and sub-processes must be evaluated uniformly, and risks must be dealt with according to a coordinated procedure. the analysis is started by orientation to the problem. this is done in the following steps: . recognizing and analyzing the problem according to its causes and extent, by means of a diagnosis and prediction, and comparison with the goals aimed at. . description and division of the overall problem into individual problem areas, and specifying their dependencies. . defining the problem and structuring it according to the objectives, time relation, degree of difficulty, and relevance to the goal. . detailed analysis of the causes, and classification in accordance to the possible solution. the analysis of the problem should be integrated into the overall analytical process in accordance with the thinking schemes described earlier. the relevance and priorities related to the process determine the starting point for the remaining steps of the analysis. analyses are divided into quantitative and qualitative ones. quantitative analyses include risk analyses, that is, theoretical safety-related analysis methods and safety analyses, e.g. classical accident analyses. qualitative analyses include failure mode and effect analyses, hazard analyses, failure hazard analyses, operating hazard analyses, human error code and effect analyses, information error and effect analyses. the theoretical safety-related analysis methods include inductive and deductive analyses based on boolean models. inductive analyses are, e.g. fault process analyses. deductive analyses are fault tree analyses, analytical processes and simulation methods. theoretical safety-related analysis methods which are not based on boolean models are stochastic processes, such as markow's model, risk analyses and accident analyses which, as a rule, are statistical or probability-related analyses. a possible scheme to begin with is shown in figure . . since absolute safety, entailing freedom from all risks, does not exist in any sphere of life, the task of those dealing with safety issues is to avert hazards and to achieve a sustainable reduction of the residual risk, so that it does not exceed a tolerable limit. the extent of this rationally acceptable risk is also influenced by the level of risk which society intuitively considers as being acceptable. those who propose definitions of safety are neither authorized nor capable of evaluating the general benefit of technical products, processes and services. risk assessment is therefore focused at the potential harm caused by the use or non-use of the technology. the guidelines given in "a new approach to technical harmonization and standards" by the council resolution of may are valid in the european union. the legal system of a state describes the protective goals, such as protection of life, health, etc., in its constitution, as well as in individual laws and regulations. as a rule, these do not provide an exact limit as to what is still a tolerable risk. this limit can only be established indirectly and unclearly on the basis of the goals and conceptions set down by the authorities and laws of a state. in the european union, the limits are expressed primarily in the "basic safety and health requirements". these requirements are then put into more concrete terms in the safety-related definitions issued by the bodies responsible for preparing industrial standards. compliance with the standards is voluntary, but it is presumed that the basic requirements of the directives are met. the term which is opposite to "safety" is "hazard". both safe and hazardous situations are founded on the intended use of the technical products, processes and services. unintended use is taken into account only to the extent that it can be reasonably foreseen. the risks present in certain events are, in a more narrow sense, unwelcome and unwanted outcomes with negative effects (which exceed the range of acceptance). unwelcome events are • source conditions of processes and states; • processes and states themselves; and • effects of processes and states which can result in harm to persons or property. an unwelcome event can be defined as a single event or an event within a sequence of events. possible unwelcome events are identified for a unit under review. the causes may be inherent in the unit itself, or outside of it. in order to determine the risks involved in unwelcome events, it is necessary to identify probabilities and consequences. the question arises: are the extent and probability of the risk known? information is needed to answer this question. defining risk requires information concerning the probability of occurrence and the extent of the harm of the consequences. uncertainty is given if the effects are known but the probability is unknown. ignorance is given if both the effects and the probability are unknown. figure . shows the risk analysis procedure according to the type of information available. since risk analyses are not possible without practical, usable information, it is necessary to consider the nature of the information. the information is characterized by its content, truth and objectivity, degree of confirmation, the possibility of being tested, and the age of the information. the factors determining the content of the information are generality, precision and conditionality. the higher the conditionality, the smaller is the generality, and thus the smaller the information content of the statement. truth is understood as conformity of the statement with the real state of affairs. the closer that the information is to reality, the higher is its information content, and the smaller its logical margin. the degree of controllability is directly dependent on the information content: the bigger the logical margin, the smaller the information content, and thus the higher the probability that the information content will prove its worth. in this respect, probability plays a role in the information content: the greatest significance is attributed to the logical hypothetical probability and statistical probability of an event. objectivity and age are additional criteria for any information. the age and time relation of information play a particularly important role, because consideration of the time period is an important feature of analysis. as a rule, information and thus the data input in the risk analysis consist of figures and facts based on experience, materials, technical design, the organization and the environment. in this regard, most figures are based on statistics on incidents and their occurrences. factual information reveals something about the actual state of affairs. it consists of statements related to past conditions, incidents, etc. forecast-type predictions are related to real future conditions, foretelling that certain events will occur in the future. explanatory information replies to questions about the causes of phenomena, and provides explanations and reasons. it establishes links between different states based on presumed cause-effect relationships. subjunctive information expresses possibilities, implying that certain situations might occur at present or in the future, thus giving information about conceivable conditions, events and relationships. normative information expresses goals, standards, evaluations and similar matters; it formulates what is desirable or necessary. the main problem with risk analyses is incomplete information, in particular regarding the area of "uncertainty". in the eu commission's view, recourse to the so-called precautionary principle presupposes that potentially dangerous effects deriving from a phenomenon, product or process have been identified via objective scientific evaluation, and that scientific evaluation does not allow the risk to be determined with sufficient certainty. recourse to the precautionary principle thus takes place in the framework of general risk management that is concretely connected to the decision-making process. if application of the precautionary principle results in the decision that action is the appropriate response to a risk, and that further scientific information is not needed, it is still necessary to decide how to proceed. apart from adopting legal provisions which are subject to judicial control, a whole range of actions is available to the decision-makers (e.g. funding research, or deciding to inform the public about the possible adverse effects of a product or procedure). however, the measures may not be selected arbitrarily. in conclusion, the assessment of various risks and risk types which may be related to different types of hazards requires a variety of specific risk assessment methods. if one has dependable information about the probability and consequences of a serious risk or risky event, one should use the risk assessment procedure shown in figure . . • major industrial accidents; • damage caused by dangerous substances; • nuclear accidents; • major accidents at sea; • disasters due to forces of nature; and • acts of terrorism. • dangerous substances discharged (fire, explosion); • injury to people and damage to property; • immediate damage to the environment; • permanent or long-term damage to terrestrial habitats, to fresh water, to marine habitats, to aquifers or underground water supplies; and • cross-border damage. • technical failure: devices, mountings, containers, flanges, mechanical damage, corrosion of pipes, etc.; • human failure: operating error, organizational failure, during repair work; • chemical reaction; • physical reaction; and • environmental cause. system analysis is the basis of all hazard analyses, and thus needs to be done with special care. system analysis includes the examination of the system functions, particularly the performance goals and admissible deviations in the ambient conditions not influenced by the system, the auxiliary sources of the system (e.g. energy supply), the components of the system, and the organization and behaviour of the system. geographical arrangements, block diagrams, material flow charts, information flow charts, energy flow charts, etc. are used to depict technical systems. the objective is to ensure the safe behaviour of the technical systems by design methods, at least during the required service life and during intended use. qualitative analyses are particularly important in practice. as a rule, they are form sheet analyses and include failure mode and effect analyses, which examine and determine failure modes and their effects on systems. the preliminary hazard analysis looks for the hazard potentials of a system. the failure hazard analysis examines the causes of failures and their effects. the operating hazard analysis determines the hazards which may occur during operation, maintenance, repair, etc. the human error mode and effect analysis examines error modes and their effects which occur because of wrong behaviour of humans. the information error mode and effect analysis examines operating, maintenance and repair errors, fault elimination errors and the effects caused by errors in instructions and faulty information. theoretical analysis methods include the fault tree analysis, which is a deductive analysis. an unwelcome incident is provided to the system under review. then all logical links and/or failure combinations of components or partial system failures which might lead to this unwelcome incident are assembled, forming the fault tree. the fault tree analysis is suited for simple as well as for complex systems. the objective is to identify failures which might lead to an unwelcome incident. the prerequisite is exact knowledge about the functioning of the system under review. the process, the functioning of the components and partial systems therefore need to be present. it is possible to focus on the flow of force, of energy, of materials and of signals. the fault process analysis has a structure similar to that of the fault tree analysis. in this case, however, we are looking for all unwelcome incidents as well as their combinations which have the same fault trigger. analysis of the functioning of the system under review is also necessary for this. analyses can also be used to identify possible, probable and actual risk components. the phases of the analysis are the phases of design, including the preparation of a concept, project and construction, and the phases of use which are production, operation and maintenance. in order to identify the fault potential as completely as possible, different types of analyses are usually combined. documentation of the sufficient safety of a system can be achieved at a reasonable cost only for a small system. in the case of complex systems, it is therefore recommended to document individual unwelcome incidents. if solutions are sought and found for individual unwelcome incidents, care should be taken to ensure that no target conflicts arise with regard to other detail solutions. with the help of the fault tree, it is possible to analyse the causes of an unwelcome incident and the probability of its occurrence. decisions on whether and which redundancies are necessary can in most cases be reached by simple estimates. four results can be expected by using a fault tree: . the failure combination of inputs leading to the unwelcome event; . the probability of their occurrence; . the probability of occurrence of the unwelcome event; and . the critical path that this incident took from the failure combination through the fault tree. a systematic evaluation of the fault tree model can be done by an analytical evaluation (calculation) or by simulation of the model (monte-carlo method). a graphic analysis of the failure process is especially suited to prove the safety risk of previously defined failure combinations in the system. the failure mode and effect analysis and the preliminary hazard analysis as mentioned previously, no method can disclose all potential faults in a system with any degree of certainty. however, if one starts with the preliminary hazard analysis, then at least the essential components with hazard potential will be defined. the essential components are always similar, namely, kinetic energy, potential energy, source of thermal energy, radioactive material, biological material, chemically reactive substance. with the fault tree method, any possible failure combinations (causes), leading to an unwelcome outcome, can then be identified additionally. re- are especially suited for identifying failures in a system which pose a risk. liability parameters can be determined in the process, e.g. the frequency of occurrence of failure combinations, the frequency of occurrence of unwelcome events, non-availability of the system upon requests, etc. the failure effect analysis is a supplementary method. it is able to depict the effects of mistakes made by the operating personnel, e.g. when a task is not performed, or is performed according to inappropriate instructions, or performed too early, too late, unintentionally or with errors. it can pinpoint also effects resulting from operating conditions and errors in the functional process or its elements. an important aspect of all hazard analyses is that they are only valid for the respective case under review. every change in a parameter basically requires new analyses. this applies to changes in the personnel structure and the qualification of persons, as well as to technical specifications. for this reason, it is necessary to document the parameters on which each analysis is based. the results of the hazard analyses form the basis for the selection of protective measures and measures to combat the hazards. if the system is modified, the hazards inherent in the system may change, and the measures to combat the hazards may have to be changed as well. this may also mean that the protective measures or equipment which existed at time x for the system or partial system in certain operating conditions (e.g. normal operation, set-up operation, and maintenance phase) may no longer be compatible. different protective measures, equipment or strategies may then be needed. however, hazard analyses do not merely serve to detect and solve potential failures. they form the basis for the selection of protective measures and protective equipment, and they can also test the success of the safety strategies specified. a selection of methods used for hazard analysis is given in annex to section . . risk assessment is a series of logical steps enabling the systematic examination of the hazards associated with machinery. risk assessment is followed, whenever necessary, by actions to reduce the existing risks and by implementing safety measures. when this process is repeated, it eliminates hazards as far as possible. risk assessment includes: • risk analysis: -determining the limits of machinery; -identifying hazards; and -estimating risks. • risk evaluation. risk analysis provides the information required for evaluating risks, and this in turn allows judgements to be made on the safety of e.g. the machinery or plant under review. risk assessment relies on decisions based on judgement. these decisions are to be supported by qualitative methods, complemented, as far as possible, by quantitative methods. quantitative methods are particularly appropriate when the foreseeable harm is very severe or extensive. quantitative methods are useful for assessing alternative safety measures and for determining which measure gives best protection. the application of quantitative methods is restricted to the amount of useful data which is available, and in many cases only qualitative risk assessment will be possible. risk assessment should be conducted so that it is possible to document the used procedure and the results that have been achieved. risk assessment shall take into account: • the life cycle of machinery or the life span of the plant. • the limitations of the machinery or plant, including the intended use (correct use and operation of the machinery or plant, as well as the consequences of reasonably foreseeable misuse or malfunction). • the full range of foreseeable uses of the machinery (e.g. industrial, nonindustrial and domestic) by persons identified by sex, age, dominant hand usage, or limiting physical abilities (e.g. visual or hearing impairment, stature, strength). • the anticipated level of training, experience or ability of the anticipated users, such as: -operators including maintenance personnel or technicians; -trainees and juniors; and -general public. • exposure of other persons to the machine hazards, whenever they can be reasonably foreseen. having identified the various hazards that can originate from the machine (permanent hazards and ones that can appear unexpectedly), the machine designer shall estimate the risk for each hazard, as far as possible, on the basis of quantifiable factors. he must finally decide, based on the risk evaluation, whether risk reduction is required. for this purpose, the designer has to take into account the different operating modes and intervention procedures, as well as human interaction during the entire life cycle of the machine. the following aspects in particular must be considered: • construction; transport; • assembly, installation, commissioning; • adjusting settings, programming or process changeover; • instructions for users; • operating, cleaning, maintenance, servicing; and • checking for faults, de-commissioning, dismantling and safe disposal. malfunctioning of the machine due to, e.g. • variation in a characteristic or dimension of the processed material or workpiece; • failure of a part or function; • external disturbance (e.g. shock, vibration, electromagnetic interference); • design error or deficiency (e.g. software errors); • disturbance in power supply; and • flaw in surrounding conditions (e.g. damaged floor surface). unintentional behaviour of the operator or foreseeable misuse of the machine, e.g.: • loss of control of the machine by the operator (especially in the case of hand-held devices or moving parts); • automatic (reflexive) behaviour of a person in case of a machine malfunction or failure during operation; • the operator's carelessness or lack of concentration; • the operator taking the "line of least resistance" in carrying out a task; • behaviour resulting from pressure to keep the machine running in all circumstances; and • unexpected behaviour of certain persons (e.g. children, disabled persons). when carrying out a risk assessment, the risk of the most severe harm that is likely to occur from each identified hazard must be considered, but the greatest foreseeable severity must also be taken into account, even if the probability of such an occurrence is not high. this objective may be met by eliminating the hazards, or by reducing, separately or simultaneously, each of the two elements which determine the risk, i.e. the severity of the harm from the hazard in question, and the probability of occurrence of that harm. all protective measures intended to reach this goal shall be applied according to the following steps: this stage is the only one at which hazards can be eliminated, thus avoiding the need for additional protective measures, such as safeguarding machines or implementing complementary protective measures. . information about the residual risk. information for use on the residual risk is not to be a substitute for inherently safe design, or for safeguarding or complementary protective measures. risk estimation and evaluation must be carried out after each of the above three steps of risk reduction. adequate protective measures associated with each of the operating modes and intervention procedures prevent operators from being prone to use hazardous intervention techniques in case of technical difficulties. the aim is to achieve the lowest possible level of risk. the design process is an iterative cycle, and several successive applications may be necessary to reduce the risk, making the best use of available technology. four aspects should be considered, preferably in the following order: . the safety of the machine during all the phases of its life cycle; . the ability of the machine to perform its function; . the usability of the machine; and . the costs of manufacturing, operating and dismantling the machine. the following principles apply to technical design: service life, safe machine life, fail-safe and tamper-proof design. a design which ensures the safety of service life has to be chosen when neither the technical system nor any of its safety-relevant partial functions can be allowed to fail during the service life envisaged. this means that the components of the partial functions need to be exchanged at previously defined time intervals (preventive maintenance). in the case of a fail-safe design, the technical system or its partial functions allow for faults, but none of these faults, alone or in combination, may lead to a hazardous state. it is necessary to specify just which faults in one or several partial systems can be allowed to occur simultaneously without the overall system being transferred into a hazardous state (maximum admissible number of simultaneous faults). a failure or a reduction in the performance of the technical system is accepted in this case. tamper-proof means that it is impossible to intentionally induce a hazardous state of the system. this is often required of technical systems with a high hazard potential. strategies involving secrecy play a special role in this regard. in the safety principles described here, redundant design should also be mentioned. the probability of occurrence and the consequences of damage are reduced by multiple arrangements, allowing both for subsystems or elements to be arranged in a row or in parallel. it is possible to reduce the fault potential of a technical system by the diversification of principles: several different principles are used in redundant arrangements. the spatial distribution of the function carriers allows the possibilities to influence faults to be reduced to one function. in the redundant arrangements, important functions, e.g. information transmission, are therefore designed in a redundant manner at different locations. the measures to eliminate or avoid hazards have to meet the following basic requirements: their effect must be reliable and compulsory, and they cannot be circumvented. reliable effect means that the effect principle and construction design of the planned measure guarantee an unambiguous effect, that the components have been designed according to regulations, that production and assembly are performed in a controlled manner, and that the measure has been tested. compulsory effect includes the demand for a protective effect which is active at the start of a hazardous state and during it, and which is deactivated only when the hazardous state is no longer present, or stops when the protective effect is not active. technical systems are planned as determined systems. only predictable and intended system behaviour is taken into account when the system is designed. experience has shown, however, that technical systems also display stochastic behaviour. that is, external influences and/or internal modifications not taken into consideration in the design result in unintended changes in the system's behaviour and properties. the period of time until the unintended changes in behaviour and/or in properties occur, cannot be accurately determined; it is a random variable. we have to presume that there will be a fault in every technical system. we simply do not know in advance when it will take place. the same is true for repairs. we know that it is generally possible in systems requiring re-pair to complete a repair operation successfully, but we cannot determine the exact time in advance. using statistical evaluations, we can establish a timedependent probability at which a "fault event" or "completion of a repair operation" occurs. the frequency of these events determines the availability of the system requiring repair. technical systems are intended to perform numerous functions and, at the same time, to be safe. the influence of human action on safety has to be taken into account in safety considerations as well (i.e. human factor). a system is safe when there are no functions or action sequences resulting in hazardous effects for people and/or property. risks of unwelcome events (in the following called "risk of an event") are determined on the basis of the experience (e.g. catalogue of measures) with technical systems. in addition to this, safety analyses are used (e.g. failure mode and effect analysis, hazard analysis, failure hazard analysis, operating hazard analysis, information error analysis), as well as mathematical models (e.g. worst-case analysis, monte-carlo procedure, markow's models). unwelcome events are examined for their effects. this is followed by considerations about which design modifications or additional protective measures might provide sufficient safety against these unwelcome events. the explanations below present the basic procedure for developing safety-relevant arrangements and solutions, i.e. the thinking and decision-making processes, as well as selecting criteria that are significant for the identification of unwelcome events, the risk of an event, the acceptance limits and the adoption of measures. before preparing the final documentation, it is essential to verify that the limit risk has not been exceeded, and that no new unwelcome events have occurred. the sequence scheme describes the procedure for developing safety arrangements and for finding solutions aiming to avoid the occurrence of unwelcome events which exceed the acceptance limits, by selecting suitable measures. in this context, it is assumed that: • an unwelcome event is initially identified as a single event within a comprehensive event sequence (e.g. start-up of a plant), and the risk of an event and limit risk are determined. • the selection of technical and/or non-technical measures is subject to a review of the content and the system, and the decision regarding a solution is then made. • the number of applicable measures is limited, and therefore it may not be possible to immediately find a measure with an acceptable risk for a preliminary determination of the unwelcome event. • implementation of the selected solution can result in the occurrence of a new unwelcome event. • in the above cases, a more concrete, new determination of the unwelcome event and/or the unit under review, or the state of the unit under review, and another attempt at deciding upon measures may lead to the desired result, although this may have to be repeated several times before it is successful. in the case of complex event sequences, several unwelcome events may become apparent which have to be tackled by the respective set of measures. in accordance with the sequence scheme, the unit under review and its state have to be determined first. this determination includes information on, e.g., • product type, dimension, product parts/elements distinguished according to functional or construction aspects, if applicable; • intended use; • work system or field of application; • target group; • supply energy, transformed in the product, transmitted, distributed, output; • other parameters essential to safety assessment according to type and size of the product; • known or assumed effects on the product or its parts (e.g. due to transport, assembly, conditions at the assembly site, operation, maintenance); • weight, centre of gravity; • materials, consumables; • operating states (e.g. start-up, standstill, test run, normal operation); • condition (new, condition after a period in storage/shutdown, after repair, in case of modified operating conditions and/or other significant changes); and • known or suspected effects on humans. the next step is the identification of unwelcome events. they are source conditions of processes and states, or processes and states themselves. they can be the effects of processes and states which can cause harm to people or property. an unwelcome event can be a single event or part of a sequence of events. one should look for unwelcome events in sequences of processes and functions, in work activities and organizational procedures, or in the work environment. care has to be taken that the respective interfaces are included in the considerations. deviations and time-dependent changes in regard to the planned sequences and conditions have to be taken into account as well. the risk of an unwanted event results from the probability statement which takes into account both • the expected frequency of occurrence of the event; and • the anticipated extent of harm of the event. the expected frequency of occurrence of an event leading to harm is determined by, e.g., • the probability of the occurrence itself; • the duration and frequency of exposure of people (or of objects) in the danger zone, e.g. -extremely seldom (e.g. during repair), -seldom (e.g. during installation, maintenance and inspection), -frequently, and -very frequently (e.g. constant intervention during every work cycle); • the influence of users or third parties on the risk of an event. the extent of harm is determined by, e.g., • the type of harm (harm to people and/or property); • the severity of the harm (slight/severe/fatal injury of persons, or corresponding damage to property); and • number of people or objects affected. in principle, the safety requirements depend on the ratio of the risk of an event to the limit risk. criteria for determining the limit risk are, e.g., • personal and social acceptance of hazards; • people possibly affected (e.g. layman, trained person, specialized worker); • participation of those affected in the process; and • possibilities of averting hazards. the safety of various technical equipment with comparable risk can, for instance, be achieved • primarily by technical measures, in some cases; and • mainly by non-technical measures, in other cases. this means that several acceptable solutions with varying proportions of technical and non-technical measures may be found for a specific risk. in this context, the responsibility of those involved should be taken into consideration. technical measures are developed on the basis of e.g. the following principles: • avoiding hazardous interfaces (e.g. risk of crushing, shearing); hazard sources (e.g. radiation sources, flying parts, hazardous states and actions as well as inappropriate processes); • limiting hazardous energy (e.g. by rupture disks, temperature controllers, safety valves, rated break points); • using suitable construction and other materials (e.g. solid, sufficiently resistant against corrosion and ageing, glare-free, break-proof, non-toxic, non-inflammable, non-combustible, non-sliding); • designing equipment in accordance with its function, material, load, and ergonomics principles; • using fail-safe control devices employing technical means; • employing technical means of informing (e.g. danger signal); • protective equipment for separating, attaching, rejecting, catching, etc.; • suction equipment, exhaust hoods, when needed; • protection and emergency rooms; and • couplings or locks. technical measures refer to, e.g., • physical, chemical or biological processes; • energy, material and information flow in connection with the applied processes; • properties of materials and changes in the properties; and • function and design of technical products, parts and connections. the iterative (repeated) risk reduction process can be concluded after achieving adequate risk reduction and, if applicable, a favourable outcome of risk comparison. adequate risk reduction can be considered to have been achieved when one is able to answer each of the following questions positively: • have all operating conditions and all intervention procedures been taken into account? • have hazards been eliminated or their risks been reduced to the lowest practicable level? • is it certain that the measures undertaken do not generate new hazards? • are the users sufficiently informed and warned about the residual risks? • is it certain that the operator's working conditions are not jeopardized by the protective measures taken? • are the protective measures compatible with each other? • has sufficient consideration been given to the consequences that can arise from the use of a machine designed for professional/industrial use when it is used in a non-professional/non-industrial context? • is it certain that the measures undertaken do not excessively reduce the ability of the machine to perform its intended function? there are still many potential risks connected with hazardous substances about which more information is needed. because the knowledge about the relation between their dose and mode of action is not sufficient for controlling such risks, more research is needed. the following list highlights the themes of the numerous questions related to such risks: • potentially harmful organisms; • toxicants, carcinogens; • pesticides, pollutants, poisonous substances; • genetically engineered substances; • relation between chemical and structural properties and toxicity; • chemical structure and chemical properties and the relation to reactivity and reaction possibilities of organic compounds to metabolic reaction and living systems; • modes of action, genotoxicity, carcinogenicity, effects on humans/animals; • potentially harmful organisms in feedstuffs and animal faeces; • viruses and pathogens; • bacteria in feedstuffs and faeces; • parasites in feedstuffs and animal faeces; • pests in stored feedstuffs; • probiotics as feed additives; and • preservatives in feedstuffs. violent actions damaging society, property or people have increased, and they seem to spread both internationally as well as within countries. these new risks are difficult to predict and manage, as the very strategy of the actors is to create unexpected chaotic events. certain possibilities to predict the potential types of hazards do exist, and comprehensive predictive analyses have been done (meyerson, reaser ) . new methodologies are needed to predict the risk of terrorist actions, and also the strategies for risk management need to be developed. due to the numerous background factors, the preparedness of societies against these risks needs to be strengthened. table . lists important societal systems which are vulnerable to acts of terrorism. the situation in the developing countries needs to be tackled with specific methods. one has to answer the following questions: • what specific examples of prevention instruments can be offered? • what are the prerequisites for success? • how can industrialized countries assist the developing countries in carrying out preventive actions? • how should priorities be set according to the available resources? one possibility is to start a first-step programme, the goal of which is higher productivity and better workplaces. it can be carried out by improving • storage and handling of materials: -provide storage racks for tools, materials, etc.; -put stores, racks etc. on wheels, whenever possible; -use carts, conveyers or other aids when moving heavy loads; -use jigs, clamps or other fixtures to hold items in place. • work sites: -keep the working area clear of everything that is not in frequent use. • machine safety: -install proper guards to dangerous tools/machines; -use safety devices; -maintain machines properly. • control of hazardous substances: -substitute hazardous chemicals with less hazardous substances; -make sure that all organic solvents, paints, glues, etc., are kept in covered containers; -install or improve local exhaust ventilation; -provide adequate protective goggles, face shields, earplugs, safety footwear, gloves, etc.; -instruct and train workers; -make sure that workers wash their hands before eating and drinking, and change their clothes before going home. • lighting: -make sure that lighting is adequate. • social and sanitary facilities: -provide a supply of cool, safe drinking water; -have the sanitary facilities cleaned regularly; -provide a hygienic place for meals; -provide storage for clothing or other belongings; -provide first aid equipment and train a qualified first-aider. • premises: -increase natural ventilation by having more roof and wall openings, windows or open doorways; -move sources of heat, noise, fumes, arc welding, etc., out of the workshop, or install exhaust ventilation, noise barriers, or other solutions; -provide fire extinguishers and train the workers to use them; -clear passageways, provide signs and markings. • work organization: -keep the workers alert and reduce fatigue through frequent changes in tasks, opportunities to change work postures, short breaks, etc.; -have buffer stocks of materials to keep work flow constant; -use quality circles to improve productivity and quality. risk combination of the probability of an event and its consequences. the term "risk" is generally used only when there is at least a possibility of negative consequences. in some situation, risk arises from the possibility of deviation from the expected outcome or event. outcome of an event or a situation, expressed in quality and in quantity. it may result in a loss or in an injury or may be linked to it. the result can be a disadvantage or a gain. in this case the event or the situation is the source. in connection with every analysis it has to be checked whether the cause is given empirically, or follows a set pattern, and whether there is scientific agreement regarding these circumstances. note there can be more than one consequence from one event. note consequences can range from positive to negative. the consequences are always negative from the viewpoint of safety. extent to which an event is likely to occur. note iso - : gives the mathematical definition of probability as "a real number in the interval to attached to a random event. it can be related to a long-run relative frequency of occurrence or to a degree of belief that an event will occur. for a high degree of belief the probability is near ". note frequency rather than probability may be used in describing risk. degrees of belief about probability can be chosen as classes or ranks such as: rare/unlikely/moderate/likely/almost certain, or incredible/improbable/ remote/occasional/probable/frequent. remark: informal language often confuses frequency and probability. this can lead to wrong conclusions in safety technology. probability is the degree of coincidence of the time frequency of coincidental realization of a fact from a certain possibility. coincidence is an event which basically can happen, may be cause-related, but does not occur necessarily or following a set pattern. it may also not occur (yes-or-no-alternative). data for probability of occurring with specific kinds of occurrence and weight of consequences can be: in a statistical sense: empirical, retrospective, real in a prognostic sense: speculative, prospective, probabilistic occurrence of a particular set of circumstances regarding place and time. an event can be the source of certain consequences (empirically to be expected with certain regularity). the event can be certain or uncertain. the event can be a single occurrence or a series of occurrences. the probability associated with the event can be estimated for a given period of time. task range by which the significance of risk is assessed. note risk criteria can include associated costs and benefits, legal and statutory requirements, socio-economic and environmental aspects, the concerns of stakeholders, priorities and other inputs to the assessment. the way in which a stakeholder views a risk based on a set of values or concerns. note risk perception depends on the stakeholder's needs, issues and knowledge. note risk perception can differ from objective data. exchange or sharing of information about risk between the decision-makers and other stakeholders. overall process of risk analysis and risk evaluation. systematic use of information to identify sources and to estimate the risk. note risk analysis provides a basis for risk evaluation, risk treatment, and risk acceptance. note information can include historical data, theoretical analyses, informal opinions, and the concerns of stakeholders. process used to assign figures, values to the probability and consequences of a risk. note risk estimation can consider cost, benefits, the concerns of stakeholders, and other variables, as appropriate for risk evaluation. process of comparing the estimated risk against given risk criteria to determine the significance of a risk. process of selection and implementation of measures to modify risk. note risk treatment measures can include avoiding, optimizing, transferring or retaining risk. actions implementing risk management decisions. note risk control may involve monitoring, re-evaluation, and compliance with decisions. process, related to a risk, to minimize the negative and to maximize the positive consequences (and their respective probabilities). actions taken to lessen the probability, negative consequences or both, associated with a risk. limitation of any negative consequences of a particular event. decision not to become involved in, or action to withdraw from, a risk situation. sharing with another party the burden of loss or benefit of gain, for a risk. note legal or statutory requirements can limit, prohibit or mandate the transfer of a certain risk. note risk transfer can be carried out through insurance or other agreements. note risk transfer can create new risks or modify existing ones. note relocation of the source is not risk transfer. acceptance of the burden of loss, or benefit of gain, from a particular risk. note risk retention includes the acceptance of risks that have not been identified. note risk retention does not include means involving insurance, or transfer in other ways. this includes risk assessment, risk treatment, risk acceptance and risk communication. risk assessment is risk analysis, with identification of sources and risk estimation, and risk evaluation. risk treatment includes avoiding, optimizing, transferring and retaining risk. → risk acceptance → risk communication harm physical injury or damage to the health of people or damage to property or the environment [iso/iec guide ]. note harm includes any disadvantage which is causally related to the infringement of the object of legal protection brought about by the harmful event. note in the individual safety-relevant definitions, harm to people, property and the environment may be included separately, in combination, or it may be excluded. this has to be stated in the respective scope. potential source of harm [iso/iec guide ]. the term "hazard" can be supplemented to define its origin or the nature of the possible harm, e.g., hazard of electric shock, crushing, cutting, dangerous substances, fire, drowning. in every-day informal language, there is insufficient differentiation between source of harm, hazardous situation, hazardous event and risk. circumstance in which people, property or the environment are exposed to one or more hazards [iso/iec guide ]. note circumstance can last for a shorter or longer period of time. event that can cause harm [din en ] . the hazardous event can be preceded by a latent hazardous situation or by a critical event. combination of the probability of occurrence of harm and the severity of that harm [iso/iec guide ]. note in many cases, only a uniform extent of harm (e.g. leading to death) is taken into account, or the occurrence of harm may be independent of the extent of harm, as in a lottery game. in these cases, it is easier to make a probability statement; risk assessment by risk comparison [din en ] thus becomes much simpler. note risks can be grouped in relation to different variables, e.g. to all people or only those affected by the incident, to different periods of time, or to performance. the probabilistic expectation value of the extent of harm is suitable for combining the two probability variables. note risks which arise as a consequence of continuous emission, e.g. noise, vibration, pollutants, are affected by the duration and level of exposure of those affected. risk which is accepted in a given context based on the current values of society [iso/iec guide ]. the acceptable risk has to be taken into account in this context, too. note safety-relevant definitions are oriented to the maximum tolerable risk. this is also referred to as limit risk. note tolerability is also based on the assumption that the intended use in addition to a reasonably predictable misuse of the products, processes and services, is complied with. freedom from unacceptable risk [iso/iec guide ]. note safety is indivisible. it cannot be split into classes or levels. note safety is achieved by risk reduction, so that the residual risk in no case exceeds the maximum tolerable risk. existence of an unacceptable risk. note safety and danger exclude one another -a technical product, process or service cannot be safe and dangerous at the same time. means used to reduce risk [iso/iec guide ]. note protective measures at the product level have priority over protective measures at the workplace level. preventive measure means assumed, but not proven, to reduce risk. risk remaining after safety measures have been taken [din en ] . note residual risk may be related to the use of technical products, processes and services. systematic use of available information to identify hazards and to estimate their risks [iso/iec guide ]. determination of connected risk elements of all hazards as a basis for risk assessment. decision based on the analysis of whether the tolerable risk has been exceeded [iso/iec guide ]. overall process of risk analysis and risk evaluation [iso/iec guide ]. use of a product, process or service in accordance with information provided by the supplier [iso/iec guide ]. note information provided by the supplier also includes descriptions issued for advertising purposes. use of a product, process or service in a way not intended by the supplier, but which may result from readily predictable human behaviour [iso/iec guide ]. safety-related formulation of contents of a normative document in the form of a declaration, instructions, recommendations or requirements [compare en , safety related]. the information set down in technical rules is normally restricted to certain technical relations and situations; in this context, it is presumed that the general safety-relevant principles are followed. a procedure with the aim to reduce risk of a (technical) product, process or service according to the following steps serves to reach the safety goals in the design stage: • safety-related layout; • protective measures; • safety-related information for users. function inevitable to maintain safety. a function which, in case of failure, allows the tolerable risk to be immediately exceeded. depending on the situation, it is possible to use one method or a combination of several methods. intuitive hazard detection spontaneous, uncritical listing of possible hazards as a result of brainstorming by experts. group work which is as creative as possible. writing ideas down (on a flip chart) first, then evaluating them. technical documentation (instructions, requirements) is available for many industrial plant and work processes, describing the hazards and safety measures. this documentation has to be obtained before continuing with the risk analysis. the deviations between the set point and the actual situation of individual components are examined. information on the probability of failure of these elements may be found in technical literature. examining the safety aspects in unusual situations (emergency, repair, starting and stopping) when plans are made to modify the plant or processes. a systematic check of the processes and plant parts for effects in normal operation and in case of set point deviations, using selected question words (and -or -not -too much -too little?). this is used in particular for measurement, control units, programming of computer controls, robots. all possible causes and combinations of causes are identified for an unwanted operating state or an event, and represented in the graphic format of a tree. the probability of occurrence of an event can be estimated from the context. the fault tree analysis can also be used retrospectively to clarify the causes of events. additional methods may be: human reliability analysis a frequency analysis technique which deals with the behaviour of human beings affecting the performance of the system, and estimates the influence of human error on reliability. a hazard identification and frequency analysis technique which can be used at an early stage in the design phase to identify and critically evaluate hazards. operating safety block program a frequency analysis technique which utilizes a model of the system and its redundancies to evaluate the operating safety of the entire system. classifying risks into categories, to establish the main risk groups. all typical hazardous substances and/or possible accident sources which have to be taken into account are listed. the checklist may be used to evaluate the conformity with codes and standards. this method is used to estimate whether coincidental failures of an entire series of different parts or modules within a system are possible and what the probable effects would be. estimate the influence of an event on humans, property or the environment. simplified analytical approaches, as well as complex computer models can be used. a large circle of experts is questioned in several steps; the result of the previous step together with additional information is communicated to all participants. during the third or fourth step the anonymous questioning concentrates on aspects on which no agreement is reached so far. basically this technique is used for making predictions, but is also used for the development of new ideas. this method is particularly efficient due to its limitation to experts. a hazard identification and evaluation technique used to establish a ranking of the different system options and to identify the less hazardous options. a frequency analysis technique in which a model of the system is used to evaluate variations of the input conditions and assumptions. a means to estimate and list risk groups; reviews risk pairs and evaluates only one risk pair at a time. overview of data from the past a technique used to identify possible problem areas; can also be used for frequency analysis, based on accident and operation safety data, etc. a method to identify latent risks which can cause unforeseeable incidents. cssr differs from the other methods in that it is not conducted by a team, and can be conducted by a single person. the overview points out essential safety and health requirements related to a machine and simultaneously to all relevant (national, european, international) standards. this information ensures that the design of the machine complies with the issued "state of the art" for that particular type of machine. the "what-if" method is an inductive procedure. the design and operation of the machine in question are examined for fairly simple applications. at every step "what-if" questions are asked and answered to evaluate the effect of a failure of the machine elements or of process faults in view of the hazards caused by the machine. for more complex applications, the "what-if" method is most useful with the aid of a "checklist" and the corresponding work division to allocate specific features of the process to persons who have the greatest experience and practice in evaluating the respective feature. the operator's behaviour and professional knowledge are assessed. the suitability of the equipment and design of the machine, its control unit and protective devices are evaluated. the influence of the materials processed is examined, and the operating and maintenance records are checked. the checklist evaluation of the machine generally precedes the more detailed methods described below. fmea is an inductive method for evaluating the frequency and consequences of component failure. when operating procedures or operator errors are investigated, then other methods may be more suitable. fmea can be more time-consuming than the fault tree analysis, because every mode of failure is considered for every component. some failures have a very low probability of occurrence. if these failures are not analyzed in depth this decision should be recorded in the documentation. the method is specified in iec "analysis techniques for system reliability -procedure for failure mode and effects analysis (fmea)". in this inductive method, the test procedures are based on two criteria: technology and complexity of the control system. mainly, the following methods are applicable: • practical tests of the actual circuit and fault simulation on certain components, particularly in suspected areas of performance identified during the theoretical check and analysis. • simulation of control behaviour (e.g. by means of hardware and/or software models). whenever complex safety-related parts of control systems are tested, it may be necessary to divide the system into several functional sub-systems, and to exclusively submit the interface to fault simulation tests. this technique can also be applied to other parts of machinery. mosar is a complete approach in steps. the system to be analyzed (machinery, process, installation, etc.) is examined as a number of sub-systems which interact. a table is used to identify hazards and hazardous situations and events. the adequacy of the safety measures is studied with a second table, and a third table is used to look at their interdependency. a study, using known tools (e.g. fmea) underlines the possible dangerous failures. this leads to the elaboration of accident scenarios. by consensus, the scenarios are sorted in a severity table. a further table, again by consensus, links the severity with the targets of the safety measures, and specifies the performance levels of the technical and organizational measures. the safety measures are then incorporated into the logic trees and the residual risks are analyzed via an acceptability table defined by consensus. ilo ( ) , , , , , the risks to health at work are numerous and originate from several sources. their origins vary greatly and they cause vast numbers of diseases, injuries and other adverse conditions, such as symptoms of overexertion or overload. traditional occupational health risk factors and their approximate numbers are given in table . . the exposure of workers to hazards or other adverse conditions of work may lead to health problems, manifested in the workers' physical health, psysical workload, psychological disturbances or social aspects of life. workers may be exposed to various factors alone or in different types of combinations, which may or may not show interaction. the assessment of interacting risk factors is complex and may lead to substantial differences in the final risk estimates when compared with estimates of solitary factors. examples of interaction between different risk factors in the work environment are given in table . . the who estimate of the total number of occupational diseases among the billion workers of the world is million a year. this is likely to be an under-estimate due to the lack of diagnostic services, limited legislative coverage of both workers and diseases, and variation in diagnostic criteria between different parts of the world. the mortality from occupational diseases is substantial, comparable with other major diseases of the world population such as malaria or tuberculosis. the recent ilo estimate discloses . million deaths a year from work-related causes in the world including deaths from accidents, dangerous substances, and occupational diseases. eightyfive percent ( %) of these deaths take place in developing countries, where the diagnostic services, social security to families and compensation to workers are less developed. although the risk is decreasing in the industrialized world, the trend is increasing in the rapidly industrializing and transitory countries. a single hazard alone, such as asbestos exposure, is calculated to cause , cancers a year with a fatal outcome in less than two years after diagnosis (takala ). the incidence rates of occupational diseases in well registered industrialized countries are at the level of - cases/ , active employees/year, i.e., the incidence levels are comparable with major public health problems, such as cardiovascular diseases, respiratory disorders, etc. in the industrialized countries, the rate of morbidity from traditional occupational diseases, such as chemical poisonings, is declining, while musculoskeletal and allergic diseases are on the increase. about biological factors that are hazardous to workers' health have been identified in various work environments. some of the new diseases recognized are blood-borne infections, such as hepatitis c and hiv, and exotic bacterial or viral infections transmitted by increasing mobility, international travelling and migration of working people. also some hospital infections and, e.g., drug-resistant tuberculosis, are being contracted increasingly by health care personnel. in the developing countries the morbidity picture of occupational diseases is much less clear for several reasons: low recognition rates, rotation and turnover of workers, shorter life expectancy which hides morbidity with a long latency period, and the work-relatedness of several common epidemic diseases, such as malaria and hiv/aids (rantanen ). the estimation of so-called work-related diseases is even more difficult than that of occupational diseases. they may be about -fold more prevalent than the definite occupational diseases. several studies suggest that siegfried radandt, jorma rantanen and ortwin renn work-related allergies, musculoskeletal disorders and stress disorders are showing a growing trend at the moment. the prevention of work-related diseases is important in view of maintaining work ability and reducing economic loss from absenteeism and premature retirement. the proportion of work-relatedness out of the total morbidity figures has been estimated and found surprisingly high (nurminen and karjalainen , who ) (see table . ). the public health impact of work-related diseases is great, due to their high prevalence in the population. musculoskeletal disorders are among the three most common chronic diseases in every country, which implies that the attribution of work is very high. similarly, cardiovascular diseases in most industrialized countries contribute to % of the total mortality. even a small attributable fraction of work-relatedness implies high rates of morbidity and mortality related to work. the concept of disease is in general not a simple one. when discussing morbidity one has to recognize three different concepts: . illness = an individual's perception of a health problem resulting from either external or internal causes. . disease = an adverse health condition diagnosed by a doctor or other health professional. . sickness = a socially recognized disease which is related to, for example, social security actions or prescription of sick leave, etc. when dealing with occupational and work-related morbidity, one may need to consider any of the above three aspects of morbidity. a recognized occupational disease, however, belongs to group , i.e. it is a sickness defined by legal criteria. medical evidence is required to show that the condition meets the criteria of an occupational disease before recognition can be made. there are dozens of definitions for occupational disease. the content of the concept varies, depending on the context: a) the medical concept of occupational disease is based on a biomedical or other health-related etiological relationship between work and health, and is used in occupational health practice and clinical occupational medicine. b) the legal concept of occupational disease defines the disease or conditions which are legally recognized as conditions caused by work, and which lead to liabilities for recognition, compensation and often also prevention. the legal concept of occupational disease has a different background in different countries, often declared in the form of an official list of occupational diseases. there is universal discrepancy between the legal and medical concept, so that in nearly all countries the official list of recognized occupational diseases is shorter than the medically established list. this automatically implies that a substantial proportion of medically established occupational diseases remain unrecognized, unregistered, and consequently also uncompensated. the definition of occupational disease, as used in this chapter, summarizes various statements generated during the history of occupational medicine: an occupational disease is any disease contracted as a result of exposures at work or other conditions of work. the general criteria for the diagnosis and recognition of an occupational disease are derived from the core statements of various definitions: . evidence on exposure(s) or condition(s) in work or the work environment, which on the basis of scientific knowledge is (are) able to generate disease or some other adverse health condition. . evidence of symptoms and clinical findings which on the basis of scientific knowledge can be associated with the exposure(s) or condition(s) in concern. . exclusion of non-occupational factors or conditions as a main cause of the disease or adverse health condition. point often creates problems, as several occupationally generated clinical conditions can be caused also by non-occupational factors. on the other hand, several factors from different sources and environments are involved in virtually every disease. therefore the wordings "main cause" or "principal cause" are used. the practical solution in many countries is that the attribution of work needs to be more than %. usually the necessary generalizeable scientific evidence is obtained from epidemiological studies, but also other types of evidence, e.g. well documented clinical experience combined with information on working conditions may be acceptable. in some countries, like finland, any disease of the worker which meets the above criteria can be recognized as an occupational disease. in most other countries, however, there are official lists of occupational diseases which determine the conditions and criteria on which the disease is considered to be of occupational origin. in who launched a new concept: work-related disease (who ) . the concept is wider than that of an occupational disease. it includes: a) diseases in which the work or working conditions constitute the principal causal factor. b) diseases for which the occupational factor may be one of several causal agents, or the occupational factor may trigger, aggravate or worsen the disease. c) diseases for which the risk may be increased by work or work-determined lifestyles. the diseases in category (a) are typically recognized as legally determined occupational diseases. categories (b) and (c) are important regarding the morbidity of working populations, and they are often considered as important targets for prevention. in general, categories (b) and (c) cover greater numbers of people, as the diseases in question are often common noncommunicable diseases of the population, such as cardiovascular diseases, musculoskeletal disorders, and allergies and, to a growing extent, stressrelated disorders (see table . ). the concept of work-related disease is very important from the viewpoint of occupational health risk assessment and the use of its results for preventive purposes and for promoting health and safety at work. this is because preventive actions in occupational health practice cannot be limited only to legally recognized morbidity. the lists of occupational diseases contain great numbers of agents that show evidence on occupational morbidity. according to the ilo recommendation r ( ): list of occupational diseases, the occupational diseases are divided into four main categories: . diseases resulting from single causes following the categories listed in table . . the most common categories are physical factors, chemical agents, biological factors and physical work, including repetitive tasks, poor ergonomic conditions, and static and dynamic work. . diseases of the various organs: respiratory system, nervous system, sensory organs, internal organs, particularly liver and kidneys, musculoskeletal system, and the skin. . occupational cancers. . diseases caused by other conditions of work. research on risk perception shows differences in how different types of risks are viewed. instant, visible, dramatic risk events, particularly ones that cause numerous fatalities or severe visible injuries in a single event generally arouse much attention, and are given high priority. on the other hand, even great numbers of smaller events, such as fatal accidents of single workers, arouse less attention in both the media and among regulators, even though the total number of single fatal accidents in a year may exceed the number of fatalities in major events by several orders of magnitude. occupational diseases, with the exception of a few acute cases, are silent, develop slowly, and concern only one or a few individuals at a time. furthermore, the diseases take months or years to develop, in extreme cases even decades, after the exposure or as a consequence of accumulation of exposure during several years. as occupational health problems are difficult to detect and seriously under-diagnosed and under-reported, they tend to be given less priority than accidents. the perception of occupational disease risk remains low in spite of their severity and relatively high incidence. particularly in industrialized countries, the extent of occupational health problems is substantially greater than that of occupational accidents. on a global scale, the estimated number of fatalities due to occupational accidents is , and the respective estimate for fatalities due to work-related diseases is . million a year, giving a fatal accident/fatal disease ratio of to . the corresponding ratio in the eu- is to (takala ). the risk distribution of ods is principally determined by the nature of the work in question and the characteristics of the work environment. there is great variation in the risk of ods between the lowest and highest risk occupations. in the finnish workforce, the risk between the highest risk and the lowest risk occupations varies by a factor of . the highest risk occupations carry a risk which is - times higher than the average for all occupations. the risk of an occupational disease can be estimated on the basis of epidemiological studies, if they do exist in the case of the condition in question. on the other hand, various types of economic activity, work and occupations carry different types of risks, and each activity may have its own risk profile. by examining the available epidemiological evidence, we can recognize high-risk occupations and characterize the typical risks connected with them ( figure . , table . ). as an example, the risk of occupational asthma, dermatosis or musculoskeletal disorders is common in several occupations, but not in all. there may be huge differences in risks between different occupations. the occupations carrying the highest risk for occupational asthma, occupational skin diseases and work-related tenosynovitis, in - , are shown in table . . assessment of the risk of occupational diseases has an impact on research priorities. table . shows the priorities for research in four countries. the similarity of the priorities is striking, revealing that the problems related to the risks of occupational diseases are universal. the diagnosis of occupational diseases is important for the treatment of the disease, and for prevention, registration and compensation. the diagnosis is based on information obtained from: a) data on the work and the work environment usually provided by the employer, occupational health services, occupational safety committee, or expert bodies carrying out hygienic and other services for the workplace. b) information on the health examination of individual workers. the authorities in many countries have stipulated legal obligations for high-risk sectors to follow up the workers' health and promote early detection of changes in their health. occupational health services keep records on examinations. c) workers with special symptoms (for example, asthmatic reactions) are taken into the diagnostic process as early as possible. epidemiological evidence is a critical prerequisite for recognizing causal relationship between work and disease. epidemiology is dependent on three basic sources of information on work and the work environment: (a) exposure assessment that helps to define the "dose" of risk factor at work, (b) the outcome assumed to occur as a biological (or psychological) response to the exposures involved, and (c) time, which has a complex role in various aspects of epidemiology. all these sources are affected by the current dynamics of work life which has major impact on epidemiological research and its results. exposure assessment is the critical initial step in risk assessment. as discussed in this chapter, accurate exposure assessment will become more difficult and cumbersome than before in spite of remarkable achievements in measurement, analysis and monitoring methods in occupational hygiene, toxicology and ergonomics. great variations in working hours and individu-alization of exposures, growing fragmentation and mobility increase the uncertainties, which are multiplied. structural uncertainty, measurement uncertainty, modelling uncertainty, input data uncertainty and natural uncertainty amplify each other. as a rule, variation in any direction in exposure assessment tends to lead to underestimation of risk, and this has severe consequences to health. personal monitoring of exposures, considering variations in individual doses, and monitoring internal doses using biological monitoring methods help in the control of such variation. a monofactorial exposure situation in the past was ideal in the assessment because of its manageability. it also occurs usually as a constant determinant for long periods of time and can be regularly and continuously measured and monitored. this is very seldom the case today, and exposure assessment in modern work life is affected by discontinuities of the enterprise, of technologies and production methods, and turnover of the workforce, as well as the growing mobility and internationalization of both work and workers. company files that were earlier an important source of exposure and health data no longer necessarily fulfil that function. in addition, the standard -h time-weighted average for exposure assessment can no longer be taken as a standard, as working hours are becoming extremely heterogeneous. assessment of accurate exposure is thus more and more complex and cumbersome, and new strategies and methods for the quantification of exposure are needed. three challenges in particular can be recognized: a) the challenge arising from numerous discontinuities, fragmentation and changes in the company, employment and technology. although in the past company data were collected from all sources that were available, collective workroom measurements were the most valuable source of data. due to the high mobility of workers and variation in the work tasks, personal exposure monitoring is needed that follows the worker wherever he or she works. special smart cards for recording all personal exposures over years have been proposed, but so far no system-wide action has been possible. in radiation protection, however, such a personal monitoring system has long been a routine procedure. b) the complex nature of exposures where dozens of different factors may be involved (such as those in indoor air problems) and acting in combinations. table . gives a list of exposing factors in modern work life, many of which are difficult to monitor. c) new, rapidly spreading and often unexpected exposures that are not well characterized. often their mechanisms of action are not known, or the fast spread of problems calls for urgent action, as in the case of bovine spongiform encephalopathy (bse) in the s, sars outbreak in , and in the new epidemics of psychological stress or musculoskeletal disorders in modern manufacturing. the causes of occupational diseases are grouped into several categories by the type of factor (see table . ). a typical grouping is the one used in ilo recommendation no. . the lists of occupational diseases contain diseases caused by one single factor only, but also diseases which may have been caused by multifactorial exposures. exposure assessment is a crucial step in the overall risk assessment. the growing complexity of exposure situations has led to the development of new methods for assessing such complex exposure situations. these methods are based on construction of model matrices for jobs which have been studied thoroughly for their typical exposures. the exposure profiles are illustrated in job exposure matrices (jem) which are available for dozens of occupations (heikkilä et al. , guo . several factors can cause occupational diseases. the jem is a tool used to convert information on job titles into information on occupational risk factors. jem-based analysis is economical, systematic, and often the only reasonable choice in large retrospective studies in which exposure assessment at the individual level is not feasible. but the matrices can also be used in the practical work for getting information on typical exposure profiles of various jobs. the finnish national job-exposure matrix (finjem) is the first and so far the only general jem that is able to give a quantitative estimation of cumulative exposure. for example, finjem estimates were used for exposure profiling on chemical exposures and several other cancer-risk factors for occupational categories. the jem analysis has been further developed into task specific exposure matrices charting the exposure panorama of various tasks (benke et al. (benke et al. , . as the previous mono-causal, mono-mechanism, mono-outcome setting has shifted in the direction of multicausality, multiple mechanisms and multioutcomes, the assessment of risks has become more complex. some outcomes, as mentioned above, are difficult to define and measure with objective methods and some of them may be difficult to recognize by exposed groups themselves, or even by experts and researchers. for example, the objective measurement of stress reactions is still imprecise in spite of improvements in the analysis of some indicator hormones, such as adrenalin, noradrenalin, cortisol, prolactin, or in physical measurements, such as galvanic skin resistance and heart rate variability. questionnaires monitoring perceived stress symptoms are still the most common method for measuring stress outcomes. thanks to well organized registries, particularly in germany and the nordic countries, data on many of the relevant outcomes of exposure, such as cancer, pneumoconiosis, reproductive health disturbances and cardiovascular diseases can be accumulated, and long-term follow-up of outcomes at the group level is therefore possible. on the other hand, several common diseases, such as cardiovascular diseases, may have a work-related aetiology, but it may be difficult to show at individual level. the long-term data show that due to changes in the structure of economies, types of employment, occupational structures and conditions of work, many of the traditional occupational diseases, such as pneumoconiosis and acute intoxications have almost disappeared. several new outcomes have appeared, however, such as symptoms of physical or psychological overload, psychological stress, problems of adapting to a high pace of work, and uncertainty related to rapid organizational changes and risk of unemployment. in addition, age-related and work-related diseases among the ageing workforce are on the increase (kivimäki et al. , ilmarinen . these new outcomes may have a somatic, psychosomatic or psychosocial phenotype, and they often appear in the form of symptoms or groups of symptoms instead of well-defined diagnoses. practising physicians or clinics are not able to set an icd (international statistical classification of diseases and health-related conditions)-coded diagnosis for them. in spite of their diffuse nature, they are still problems for both the worker and the enterprise, and their consequences may be seen as sickness absenteeism, premature retirement, loss of job satisfaction, or lowered productivity. thus, they may have even a greater impact on the quality of work life and the economy than on clinical health. many such outcomes have been investigated by using questionnaire surveys among either representative samples of the whole workforce or by focusing the survey on a specific sector or occupational group. the combination of data from the surveys of "exposing factors", such as organizational changes, with questionnaire surveys of "outcomes", such as sickness absenteeism, provides epidemiological information on the association between the new exposures and the new outcomes. there are, however, major problems in both the accurate measurement of the exposures and outcomes, and also, the information available on the mechanisms of action is very scarce. epidemiology has expanded the focus of our observations from crosssectional descriptions to longitudinal perspectives, by focussing attention on the occurrence of diseases and finding associations between exposure and morbidity. such an extension of vision is both horizontal and vertical, looking at the causes of diseases. time is not only a temporal parameter in epidemiology, but has also been used for the quantification of exposure, measurement of latencies, and the detection of acceleration or slowing of the course of biological processes. as the time dimension in epidemiology is very important, the changes in temporal parameters of the new work life also affect the methods of epidemiological research. the time dimension is affected in several ways. first, the fragmentation and discontinuities of employment contracts, as described above, break the accumulation of exposure time into smaller fragments, and continuities are thus difficult to maintain. collecting data on cumulative exposures over time becomes more difficult. the time needed for exposure factors to cause an effect becomes more complex, as the discontinuities typical to modern work life allow time for biological repair and elimination processes, thus diluting the risk which would get manifested from continuous exposure. the dosage patterns become more pulse-type, rather than being continuous, stable level exposures. this may affect the multi-staged mechanisms of action in several biological processes. the breaking up of time also increases the likelihood of memory bias of respondents in questionnaire studies among exposed workers, and thus affects the estimation of total exposures. probably the most intensive effect, however, will be seen as a consequence of the variation in working hours. for example, instead of regular work of hours per day, hours per week and months per year, new time schedules and total time budgets are introduced for the majority of workers in the industrial society. the present distribution of weekly working hours in finland is less than hours per week for one third of workers, regular - hours per week for one third, and - hours per week for the remaining third. thus the real exposure times may vary substantially even among workers in the same jobs and same occupations, depending on the working hours and the employment contract (temporary, seasonal, part-time, full-time) (härmä , piirainen et al. . such variation in time distribution in "new work life" has numerous consequences for epidemiological studies, which in the past "industrial society" effectively utilized the constant time patterns at work for the assessment of exposures and outcomes and their interdependencies. the time dimension also has new structural aspects. as biological processes are highly deterministic in terms of time, the rapid changes in work life cannot wait for the maturation of results in longitudinal follow-up studies. the data are needed rapidly in order to be useful in the management of working conditions. this calls for the development of rapid epidemiological methods which enable rapid collection of the data and the making of analyses in a very short time, in order to provide information on the effects of potential causal factors before the emergence of a new change. often these methods imply the compromising of accuracy and reliability for the benefit of timeliness and actuality. as occupational epidemiology is not only interested in acute and short-term events, but looks at the health of workers over a - -year perspective, the introduction of such new quick methods should not jeopardize the interest and efforts to carry out long-term studies. epidemiology has traditionally been a key tool in making a reliable risk assessment of the likelihood of the adverse outcomes from certain levels of exposure. the new developments in work life bring numerous new challenges to risk assessment. as discussed above, the new developments in work life have eliminated a number of possibilities for risk assessment which prevailed in the stable industrial society. on the other hand, several new methods and new information technologies provide new opportunities for collection and analysis of data. traditionally, the relationship between exposure and outcome has been judged on the basis of the classical criteria set by hill ( ) . höfler ( ) crystallizes the criteria with their explanations as the following: . strength of association: a strong association is more likely to have a causal component than is a modest association. . consistency: a relationship is observed repeatedly. . specificity: a factor influences specifically a particular outcome or population. . temporality: the factor must precede the outcome it is assumed to affect. ing dose of exposure or according to a function predicted by a substantive theory. . plausibility: the observed association can be plausibly explained by substantive matter (e.g. biological) explanations. . coherence: a causal conclusion should not fundamentally contradict present substantive knowledge. . experiment: causation is more likely if evidence is based on randomized experiments. . analogy: for analogous exposures and outcomes an effect has already been shown. the hill criteria have been subjected to scrutiny, and sven hernberg has analyzed them in detail from the viewpoint of occupational health epidemiology. virtually all the hill criteria are affected by the changes in the new work life, and therefore methodological development is now needed. a few comments on causal inference are made here in view of the critiques by rothman ( ), hernberg ( ) and höfler ( ) : the strength of association will be more difficult to demonstrate due to the growing fragmentation that tends to diminish the sample sizes. the structural change that removes workers from high-level exposures to lower and shorterterm exposures may dilute the strength of effect, which may still prevail, but at a lower level. consistency of evidence may also be affected by the higher variation in conditions of work, study groups, multicultural and multiethnic composition of the workforce, etc. similarly, in the multifactorial, multi-mechanism, multi-outcome setting, the specificity criterion is not always relevant. the temporal dimension has already been discussed. in rapidly changing work life the follow-up times before the next change and before turnover in the workforce may be too short. the outcomes may also be defined by the exposures that have taken place long ago but have not been considered in the study design because historical data are not available. the biological gradient may be possible to demonstrate in a relatively simple exposure-outcome relationship. however, the more complex and multifactorial the setting becomes, the more difficult it may be to show the doseresponse relationship. the dose-response relationship may also be difficult to demonstrate in the cases of relatively ill-defined outcomes which are difficult to measure, but which can be detected as qualitative changes. biological plausibility is an important criterion which in a multimechanism setting may at least in part be difficult to demonstrate. on the other hand, the mechanisms of numerous psychological and psychosocial outcomes lack explanations, even though they undoubtedly are work-related. the missing knowledge of the mechanism of action did not prevent the establishment of causality between asbestos and cancer in a pleural sack or a lung. as many of the new outcomes may be context-dependent, the coherence criterion may be irrelevant. similarly, many of the psychosocial outcomes are difficult to put into an experimental setting, and it can be difficult to make inferences based on analogy. all of the foregoing implies that the new dynamic trends in work life challenge epidemiology in a new way, particularly in the establishment of causality. knowledge of causality is required for the prevention and management of problems. the hill criteria nevertheless need to be supplemented with new ones to meet the conditions of the new work life. similarly, more definitive and specific criteria and indicators need to be developed for the new exposures and outcomes. many of the challenges faced in the struggle to improve health and safety in modern work life can only be solved with the help of research. research on occupational health in the rapidly changing work life is needed more than ever. epidemiology is, and will remain, a key producer of information needed for prevention policies and for ensuring healthy and safe working conditions. the role of epidemiology is, however, expanding from the analysis of the occurrence of well-defined clinical diseases to studies on the occurrence of several other types of exposure and outcome, and their increasingly complex associations. as the baseline in modern work life is shifting in a more dynamic direction, and many parameters in work and the workers' situation are becoming more fragmented, incontinuous and complex, new approaches are needed to tackle the uncertainties in exposure assessment. the rapid pace of change in work life calls for the development of assessment methods to provide up-todate data quickly, so that they can be used to manage these changes and their consequences. many new outcomes which are not possible to register as clinical icd diagnoses constitute problems for today's work life. this is particularly true in the case of psychological, psychosocial and many musculoskeletal outcomes which need to be managed by occupational health physicians. methods for the identification and measurement of such outcomes need to be improved. the traditional hill criteria for causal inference are not always met even in cases where true association does exist. new criteria suitable for a new situation should be established without jeopardizing the original objective of ascertaining the true association. developing the bayesian inference further through utilization of a priori knowledge and a holistic approach may provide responses to new challenges. new neural network softwares may help in the management of the growing complexity. the glory of science does not lie in the perfection of a scientific method but rather in the recognition of its limitations. we must keep in mind the old saying: "absence of evidence is not evidence of absence". instead, it is merely a consequence of our ignorance that should be reduced through further efforts in systematic research, and particularly through epidemiology. and secondly, the ultimate value of occupational health research will be determined on the basis of its impact on practice in the improvement of the working conditions, safety and health of working people. changing conditions of work, new technologies, new substances, new work organizations and working practices are associated with new morbidity patterns and even with new occupational and work-related diseases. the new risk factors, such as rapidly transforming microbials and certain social and behavioural "exposures" may follow totally new dynamics when compared with the traditional industrial exposures (self-replicating nature of microbials and spreading of certain behaviours, such as terrorism) (smolinski et al. , loza . several social conditions, such as massive rural-urban migration, increased international mobility of working people, new work organizations and mobile work may cause totally new types of morbidity. examples of such development are, among others, the following: • mobile transboundary transportation work leading to the spread of hiv/aids. • increased risk of metabolic syndrome, diabetes and cardiovascular diseases aggravated by unconventional working hours. • increased risk of psychological burnout in jobs with a high level of longterm stress. • virtually a global epidemic of musculoskeletal disorders among vdu workers with high work load, psychological stress and poor ergonomics. the incidences of occupational diseases may not decline in the future, but the type of morbidity may change. the direction of trend in industrialized countries is the prominence of work-related morbidity and new diseases, while the traditional occupational diseases such as noise injury, pneumoconiosis, repetitive strain and chemical intoxications may continue to be prevalent in developing countries for long periods in the future. the new ergonomics problems are related to light physical work with a considerable proportion of static and repetitive workload. recent research points to an interesting interaction between unergonomic working conditions and psychological stress, leading to a combined risk of musculoskeletal disorders of the neck, shoulders and upper arms, including carpal tunnel syndrome in the wrist. the muscle tension in static work is amplified by the uncontrolled muscular tension caused by psychological stress. furthermore, there seems to be wide inter-individual variation in the tendency to respond with spasm, particularly in the trapezius muscle of neck, under psychological stress. about % of the health complaints of working-aged people are related to musculoskeletal disorders, of which a substantial part is work-related. the epidemics have been resistant against preventive measures. new regulatory and management strategies may be needed for effective prevention and control measures (westgaard et al. , paoli and merllié ) . the st century will be the era of the brain at work and consequently of psychological stress. between % and % of eu workers in certain occupations report psychological stress due to high time pressure at work (parent-thirion et al. ). the occurrence of work-related stress is most prevalent in occupations with tight deadlines, pressure from clients, or the high level of responsibility for productivity and quality given to the workers. undoubtedly, the threat of unemployment increases the perception of stress as well. as a consequence, for example, in finland some % of workers report symptoms of psychological overload and about % show clinical signs of burn out. these are not the problems of low-paid manual workers only, but also, for example, highly educated and well-paid computer super-experts have an elevated risk of burnout as a consequence of often self-committed workload (kalimo and toppinen ) . unconventional and ever longer working hours are causing similar problems. for example, one third of finns work over hours a week, and of these % work over hours, and % often work - hours per week. it is important to have flexibility in the work time schedules, but it is counterproductive if the biologically determined physiological time rhythms of the worker are seriously offended. over % have a sleep deficit of at least one hour each day, and % are tired and somnolent at work (härmä et al. ) . the toughening global competition, growing productivity demands and continuous changes of work, together with job insecurity, are associated with increased stress. up to - % of workers in different countries and different sectors of the economy report high time pressure and tight deadlines. this prevents them from doing their job as well as they would like to, and causes psychological stress. psychological stress is particularly likely to occur if the high demands are associated with a low degree of self-regulation by the workers (houtman ) . stress, if continuous, has been found to be detrimental to physical health (cardiovascular diseases), mental health (psychological burnout), safety (accident risks), and musculoskeletal disorders (particularly hand-arm and shoulder-neck disorders). it also has a negative impact on productivity, sickness absenteeism, and the quality of products and services. the resulting economic losses due to sickness absenteeism, work disability and lower quality of products and services are substantial. the prevention of stress consists not only of actions targeted at the individual worker. there is also a need for measures directed at the work organization, moderation of the total workload, competence building and collaboration within the workplace (theorell ). the support from foremen and supervisors is of crucial importance in stress management programmes. another type of psychological burden is the stress arising from the threat of physical violence or aggressive behaviour from the part of clients. in finland some % of workers have been subjected to insults or the threat of physical violence, % have experienced sexual harassment, and % mental violence or bullying at work. the risk is substantially higher for female workers than for men. stress has been found to be associated with somatic health, cardiovascular diseases, mental disorders and depression. one of the new and partly re-emerging challenges of occupational health services is associated with the new trends in microbial hazards. there are several reasons for these developments, for instance, the generation of new microbial strains, structural changes in human habitations with high population densities, growing international travel, and changes possibly in our microbiological environment as a consequence of global warming. of the to million species in the world, about million are microbes. the vast majority of them are not pathogenic to man, and we live in harmony and symbiosis with many of them. we also use bacteria in numerous ways to produce food, medicines, proteins, etc. the pathogenic bacteria have been well controlled in the th century; this control had an enormous positive impact on human health, including occupational health. but now the microbial world is challenging us in many ways. new or re-emerging biological hazards are possible due to the transformation of viruses, the increased resistance of some microbial strains (e.g. tuberculosis and some other bacterial agents) and the rapid spread of contaminants through extensive overseas travelling (smolinski et al. ) . the scenarios of health hazards from the use of genetically manipulated organisms have not been realized, but biotechnological products have brought along new risks of allergies. a major indoor air problem is caused by fungi, moulds and chemical emissions from contaminated construction materials. new allergies are encountered as a consequence of the increasingly allergic constitution of the population and of the introduction of new allergens into the work environment. health care personnel are increasingly exposed to new microbial hazards due to the growing mobility of people. evidence of high rates of hepatitis b antigen positivity has been shown among health care workers who are in contact with migrants from endemic areas. along with the growing international interactions and mobility, a number of viral and re-emerging bacterial infections also affect the health of people engaged in health care and the care of the elderly, as well as personnel in migrant and refugee services, in social services and other public services. this section applies the general framework for risk governance (chapter ) to the area of environmental risks. why should we include this topic in a book that is dominantly dealing with occupational health risks and safety issues? there are two major reasons for this decision: . most risks that impact health and safety of human beings are also affecting the natural environment. it is therefore necessary for risk managers to reflect the consequences of risk-taking activities with respect to workers, the public and the environment. these risk consequences are all interconnected. our approach to foster an integral approach to risk and risk management requires the integration of all risk consequences. . environmental risks are characterized by many features and properties that highlight exemplary issues for many generic risk assessment and management questions and challenges. for example, the question of how to balance benefits and risks becomes more accentuated, if not human life, but damage to environmental quality is at stake. while most people agree that saving human lives takes priority over economic benefits, it remains an open question of how much environmental change and potential damage one is willing to trade off against certain economic benefits. this section is divided into two major parts. part will introduce the essentials of environmental ethics and the application of ethical principles to judging the acceptability of human interventions into the environment. part addresses the procedures for an analytic-deliberative process of decision making when using the risk governance framework developed in chapter . it should be noted that this section draws from material that the author has compiled for the german scientific council for global environmental change and that has been partially published in german in a special report of the council (wbgu ). the last section on decision making has borrowed material from an unpublished background document on decision making and risk management that dr. warner north and the author had prepared for the us national academy of sciences. should people be allowed to do everything that they are capable of doing? this question is posed in connection with new technologies, such as nanotubes, or with human interventions in nature, such as the clearance of primaeval forests so that the land can be used for agriculture. intuitively everyone answers this question with a definitive "no": no way should people be allowed to everything that they are capable of doing. this also applies to everyday actions. many options in daily life, from lying to minor deception, from breaking a promise up to going behind a friend's back, are obviously actions that are seen by all well-intentioned observers as unacceptable. however, it is much more difficult to assess those actions where the valuation is not so obvious. is it justified to break a promise when keeping the promise could harm many other people? actions where there are conflicts between positive and negative consequences or where a judgement could be made one way or the other with equally good justification are especially common in risk management. there is hardly anyone who wilfully and without reason pollutes the environment, releases toxic pollutants or damages the health of individuals. people who pursue their own selfish goals on the cost and risk of others are obviously acting wrongly and every legislator will sanction this behaviour with the threat of punishment or a penalty. but there is a need for clarification where people bring about a benefit to society with the best intentions and for plausible reasons and, in the process, risk negative impacts on others. in ethics we talk about "conflicting values" here. most decisions involving risks to oneself or others are made for some reason: the actors who make such interventions want to secure goods or services to consumers, for example, to ensure long-term jobs and adequate incomes, to use natural resources for products and services or to use nature for recycling waste materials from production and consumption that are no longer needed. none of this is done for reasons of brotherly love, but to maintain social interests. even improving one's own financial resource is not immoral mere for this reason. the list of human activities that pose risks onto others perpetrated for existential or economic reasons could be carried on into infinity. human existence is bound to taking opportunities and risks. here are just a few figures: around , years ago about million people lived on the earth. under the production conditions those days (hunter-gatherer culture) this population level was the limit for the human species within the framework of an economic form that only interfered slightly with man's natural environment. the neolithic revolution brought a dramatic change: the carrying capacity of the world for human beings increased by a factor of and more. this agrarian pre-industrial cultural form was characterized by tightly limited carrying capacity, in around the earth was capable of feeding approx. million people. today the world supports billion people -and this figure is rising. the carrying capacity in comparison to the neolithic age has thus increased thousand-fold and continues to grow in parallel to new changes in production conditions (fritsch ; kesselring ; mohr ) . the five "promethean innovations" are behind this tremendous achievement of human culture: mastering fire, using the natural environment for agriculture, transforming fossil fuels into thermal and mechanical energy, industrial production and substituting material with information (renn ) . with today's settlement densities and the predominantly industrial way of life, the human race is therefore dependent on the technical remodelling of nature. without doubt, it needs this for survival, especially for the well-being of the innumerable people, goods and services that reduce the stock of natural resources. with regard to the question of the responsibility of human interventions in nature, the question cannot be about "whether" but -even better -about "how much", because it is an anthropological necessity to adapt and shape existing nature to human needs. for example, the philosopher klaus michael meyer-abich sees the situation as follows: ". . . we humans are not there to leave the world as though we had never been there. as with all other life forms, it is also part of our nature and our lives to bring about changes in the world. of course, this does not legitimise the destructive ways of life that we have fallen into. but only when we basically approve of the changes in the world can we turn to the decisive question of which changes are appropriate for human existence and which are not" (meyer-abich ). therefore, to be able to make a sensible judgement of the balance between necessary interventions into the environment and the risks posed by these interventions to human health and environmental quality, the range of products and services created by the consumption of nature has to be considered in relation to the losses that are inflicted on the environment and nature. with this comparison, it can be seen that even serious interventions in nature and the environment did not occur without reflection, but to provide the growing number of people with goods and services; these people need them to survive or as a prerequisite for a "good" life. however, at the same time it must be kept in mind that these interventions often inflict irreversible damage on the environment and destroy possible future usage potentials for future generations. above and beyond this, for the human race, nature is a cradle of social, cultural, aesthetic and religious values, the infringement of which, in turn, has a major influence on people's well-being. on both sides of the equation, there are therefore important goods that have to be appreciated when interventions in nature occur. but what form should such an appreciation take? if the pros and cons of the intervention in nature have to be weighed against each other, criteria are needed that can be used as yardsticks. who can and may draw up such criteria, according to which standards should the interventions be assessed and how can the various evaluative options for action be compared with each other for each criterion? taking risks always involves two major components: an assessment of what we can expect from an intervention into the environment (be it the use of resources or the use of environments as a sink for our waste). this is the risk and benefit assessment side of the risk analysis. secondly, we need to decide whether the assessed consequences are desirable. whereas the estimate of consequences broadly falls in the domain of scientific research and expertise, with uncertainties and ambiguities in particular having to be taken into account (irgc , klinke and renn ) , the question about the foundations for evaluating various options for action and about drawing up standards guiding action is a central function of ethics (taylor ) . ethics can provide an answer to the question posed in the beginning ("should people be allowed to do everything that they are capable of doing?") in a consistent and transparent manner. in section . . , environmental ethics will be briefly introduced. this review is inspired by the need for a pragmatic and policy-oriented approach. it is not a replacement for a comprehensive and theoretically driven compendium of environmental ethics. environmental ethics will then be applied to evaluate environmental assets. in this process, a simple distinction is made between categorical principles -that must under no circumstances be exceeded or violated -and compensatory principles, where compensation with other competing principles is allowed. this distinction consequently leads to a classification of environmental values, which, in turn, can be broken down into criteria to appreciate options for designing environmental policies. in section . . , these ideas of valuation will be taken up and used to translate the value categories into risk handling guidelines. at the heart of the considerations here is the issue of how the aims of ethically founded considerations can be used to support and implement risk-based balancing of costs and benefits. for this purpose, we will develop an integrative risk governance framework. the concept of risk governance comprises a broad picture of risk: not only does it include what has been termed "risk management" or "risk analysis", it also looks at how risk-related decision making unfolds when a range of actors is involved, requiring co-ordination and possibly reconciliation between a profusion of roles, perspectives, goals and activities. indeed, the problem-solving capacities of individual actors, be they government, the scientific community, business players, ngos or civil society as a whole, are limited and often unequal to the major challenges facing society today. then the ideas of the operational implementation of normative and factual valuations are continued and a procedure is described that is capable of integrating ethical, risk-based and work-related criteria into a proposed procedural orientation. this procedure is heavily inspired by decision analysis. answering the question about the right action is the field of practical philosophy, ethics. following the usual view in philosophy, ethics describes the theory of the justification of normative statements, i.e. those that guide action (gethmann , mittelstraß , nida-rümelin a , revermann . a system of normative statements is called "morals". ethical judgements therefore refer to the justifiability of moral instructions for action that may vary from individual to individual and from culture to culture (ott ) . basically, humans are purpose-oriented and self-determined beings who act not only instinctively, but also with foresight, and are subject to the moral standards to carry out only those actions that they can classify as good and justifiable (honnefelder ) . obviously, not all people act according to the standards that they themselves see as necessary, but they are capable of doing so. in this context, it is possible for people to act morally because, on the one hand, they are capable of distinguishing between moral and immoral action and, on the other, are largely free to choose between different options for action. whether pursuing a particular instruction for action should be considered as moral or immoral is based on whether the action concerned can be felt and justified to be "reasonable" in a particular situation. standards that cross over situations and that demand universal applicability are referred to as principles here. conflicts may arise between competing standards (in a specific situation), as well as between competing principles, the solution of which, in turn, needs justification (szejnwald-brown et al. ) . providing yardsticks for such justification or examining moral systems with respect to their justifiability is one of the key tasks of practical ethics (gethmann ) . in ethics a distinction is made between descriptive (experienced morality) and prescriptive approaches, i.e. justifiable principles of individual and collective behaviour (frankena , hansen . all descriptive approaches are, generally speaking, a "stock-taking" of actually experienced standards. initially, it is irrelevant whether these standards are justified or not. they gain their normative force solely from the fact that they exist and instigate human action (normative force of actual action). most ethicists agree that no conclusions about general validity can be drawn from the actual existence of standards. this would be a naturalistic fallacy (akademie der wissenschaften , ott ) . nevertheless, experienced morality can be an important indicator of different, equally justifiable moral systems, especially where guidance for cross-cultural behaviour is concerned. this means that the actual behaviour of many people with regard to their natural environment reveals which elements of this environment they value in particular and which they do not. however, in this case, too, the validity of the standards is not derived from their factuality, but merely used as a heurism in order to find an adequate (possibly culture-immanent) justification. but given the variety of cultures and beliefs, how can standards be justified inter-subjectively, i.e. in a way that is equally valid to all? is it not the case that science can only prove or disprove factual statements (and this only to a certain extent), but not normative statements? a brief discourse on the various approaches in ethics is needed to answer this question. first of all, ethics is concerned with two different target aspects: on the one hand, it is concerned with the question of the "success" of one's own "good life", i.e. with the standards and principles that enable a person to have a happy and fulfilled life. this is called eudemonistic ethics. on the other hand, it is concerned with the standards and principles of living together, i.e. with binding regulations that create the conditions for a happy life: the common good. this is called normative ethics (galert , ott . within normative ethics a distinction is made between deontological and teleological approaches when justifying normative statements (höffe ) . deontological approaches are principles and standards of behaviour that apply to the behaviour itself on the basis of an external valuation criterion. it is not the consequences of an action that are the yardstick of the valuation; rather, it is adhering to inherent yardsticks that can be used against the action itself. such external yardsticks of valuation are derived from religion, nature, intuition or common sense, depending on the basic philosophical direction. thus, protection of the biosphere can be seen as a divine order to protect creation (rock , schmitz , as an innate tendency for the emotional attachment of people to an environment with biodiversity (wilson ) , as a directly understandable source of inspiration and joy (ehrenfeld ) or as an educational means of practising responsibility and maintaining social stability (gowdy ) . by contrast, teleological approaches refer to the consequences of action. here, too, external standards of valuation are needed since the ethical quality of the consequences of action also have to be evaluated against a yardstick of some kind. with the most utilitarian approaches (a subset of the teleological approaches) this yardstick is defined as an increase in individual or social benefit. in other schools of ethics, intuition (can the consequence still be desirable?) or the aspect of reciprocity (the so-called "golden rule": "do as you would be done by") play a key role. in the approaches based on logical reasoning (especially in kant), the yardstick is derived from the logic of the ability to generalize or universalize. kant himself is in the tradition of deontological approaches ("good will is not good as a result of what it does or achieves, but just as a result of the intention"). according to kant, every principle that, if followed generally, makes it impossible for a happy life to be conducted is ethically impermissible. in this connection, it is not the desirability of the consequences that captures kant's mind, but the logical inconsistency that results from the fact that the conditions of the actions of individuals would be undermined if everyone were to act according to the same maxims (höffe ) . a number of contemporary ethicists have taken up kant's generalization formula, but do not judge the maxims according to their internal contradictions; rather, they judge them according to the desirability of the consequences to be feared from the generalization (jonas or zimmerli should be mentioned here). these approaches can be defined as a middle course between deontological and teleological forms of justification. in addition to deontological and teleological approaches, there is also the simple solution of consensual ethics, which, however, comprises more than just actually experienced morality. consensual ethics presupposes the explicit agreement of the people involved in an action. everything is allowed provided that all affected (for whatever reason) voluntarily agree. in sexual ethics at the moment a change from deontological ethics to a consensual moral code can be seen. the three forms of normative ethics are shown in figure . . the comparison of the basic justification paths for normative moral systems already clearly shows that professional ethicists cannot create any standards or des- ignate any as clearly right, even if they play a role in people's actual lives. much rather it is the prime task of ethics to ensure on the basis of generally recognized principles (for example, human rights) that all associated standards and behaviour regulations do not contradict each other or a higher order principle. above and beyond this, ethics can identify possible solutions that may occur with a conflict between standards and principles of equal standing. ethics may also reveal interconnections of justification that have proved themselves as examination criteria for moral action in the course of their disciplinary history. finally, many ethicists see their task as providing methods and procedures primarily of an intellectual nature by means of which the compatibility or incompatibility of standards within the framework of one or more moral systems can be completed. unlike the law, the wealth of standards of ethics is not bound to codified rules that can be used as a basis for such compatibility examinations. every normative discussion therefore starts with the general issues that are needed in order to allow individuals a "good life" and, at the same time, to give validity to the principles required to regulate the community life built on common good. but how can generally binding and inter-subjectively valid criteria be made for the valuation of "the common good"? in modern pluralistic societies, it is increasingly difficult for individuals and groups of society to draw up or recognize collectively binding principles that are perceived by all equally as justifiable and as self-obliging (hartwich and wewer , zilleßen ) . the variety of lifestyle options and subjectiernization. with increasing technical and organizational means of shaping the future, the range of behaviour options available to people also expands. with the increasing plurality of lifestyles, group-specific rationalities emerge that create their own worldviews and moral standards, which demand a binding nature and validity only within a social group or subculture. the fewer cross-society guiding principles or behaviour orientations are available, the more difficult is the process of agreement on collectively binding orientations for action. however, these are vital for the maintenance of economic cooperation, for the protection of the natural foundations of life and for the maintenance of cohesion in a society. no society can exist without the binding specification of minimum canons of principles and standards. but how can agreement be reached on such collectively binding principles and standards? what criteria can be used to judge standards? the answers to this question depend on whether the primary principles, in other words, the starting point of all moral systems, or secondary principles or standards, i.e. follow-on standards that can be derived from the primary principles, are subjected to an ethical examination. primary principles can be categorical or compensatory (capable of being compensated). categorical principles are those that must not be infringed under any circumstances, even if other prin- fication of meaning (individualization) are accompanying features of mod-ciples would be infringed as a result. the human right to the integrity of life could be named here as an example. compensatory principles are those where temporary or partial infringement is acceptable, provided that as a result the infringement of a principle of equal or higher ranking is avoided or can be avoided. in this way certain freedom rights can be restricted in times of emergency. in the literature on ethical rules, one can find more complex and sophisticated classifications of normative rules. for our purpose to provide a simple and pragmatic framework, the distinction in four categories (principles and standards; categorical and compensatory) may suffice. this distinction has been developed from a decision-analytical perspective. but how can primary principles be justified as equally valid for all people? although many philosophers have made proposals here, there is a broad consensus today that neither philosophy nor any other human facility is capable of stating binding metacriteria without any doubt and for all people, according to which such primary principles should be derived or examined (mittelstraß ) . a final justification of normative judgements cannot be achieved by logical means either, since all attempts of this kind automatically end either in a logical circle, in an unending regression (vicious cycle) or in a termination of the procedure and none of these alternatives is a satisfactory solution for final justification (albert ). the problem of not being able to derive finally valid principles definitively, however, seems to be less serious than would appear at first glance. because, regardless of whether the basic axioms of moral rules are taken from intuition, observations of nature, religion, tradition reasoning or common sense, they have broadly similar contents. thus, there is broad consensus that each human individual has a right to life, that human freedom is a high-value good and that social justice should be aimed at. but there are obviously many different opinions about what these principles mean in detail and how they should be implemented. in spite of this plurality, however, discerning and well-intentioned observers can usually quickly agree, whether one of the basic principles has clearly been infringed. it is more difficult to decide whether they have clearly been fulfilled or whether the behaviour to be judged should clearly be assigned to one or several principles. since there is no finally binding body in a secular society that can specify primary principles or standards ex cathedra, in this case consensus among equally defendable standards or principles can be used (or pragmatically under certain conditions also majority decisions). ethical considerations are still useful in this case as they allow the test of generalization and the enhancement of awareness raising capabilities. in particular, they help to reveal the implications of such primary principles and standards. provided that primary principles are not concerned (such as human rights), the ethical discussion largely consists of examining the compatibility of each of the available standards and options for action with the primary principles. in this connection, the main concerns are a lack of contradictions (consistency), logical consistency (deductive validity), coherence (agreement with other principles that have been recognized as correct) and other, broadly logical criteria (gethmann ) . as the result of such an examination it is entirely possible to reach completely different conclusions that all correspond to the laws of logic and thus justify new plurality. in order to reach binding statements or valuations here the evaluator can either conduct a discussion in his "mind" and let the arguments for various standards compete with each other (rather like a platonic dialogue) or conduct a real discussion with the people affected by the action. in both cases the main concern is to use the consensually agreed primary principles to derive secondary principles of general action and standards of specific action that should be preferred over alternatives that can be equally justified. a plurality of solutions should be expected especially because most of the concrete options for action comprise only a gradual fulfilment and infringement of primary principles and therefore also include conflicting values. for value conflicts at the same level of abstraction there are, by definition, no clear rules for solution. there are therefore frequently conflicts between conserving life through economic development and destroying life through environmental damage. since the principle of conserving life can be used for both options a conflict is unavoidable in this case. to solve the conflicts, ethical considerations, such as the avoidance of extremes, staggering priorities over time or the search for third solutions can help without, however, being able to convincingly solve this conflict in principle to the same degree for all (szejnwald-brown et al. ) . these considerations lead to some important conclusions for the matter of the application of ethical principles to the issue of human action with regard to the natural environment. first of all, it contradicts the way ethics sees itself to develop ethics of its own for different action contexts. just as there can be no different rules for the logic of deduction and induction in nomological science, depending on which object is concerned, it does not make any sense to postulate an independent set of ethics for the environment (galert ) . justifications for principles and moral systems have to satisfy universal validity (nida-rümelin b). furthermore, it is not very helpful to call for a special moral system for the environment since this -like every other moral system -has to be traceable to primary principles. instead, it makes sense to specify the generally valid principles that are also relevant with regard to the issue of how to deal with the natural environment. at the same time standards should be specified that are appropriate to environmental goods and that reflect those principles that are valid beyond their application to the environment. as implied above, it does not make much sense to talk about an independent set of environmental ethics. much rather, general ethics should be transferred to issues relating to the use of the environment (hargrove ) . three areas are usually dealt with within the context of environmental ethics (galert ): • environmental protection, i.e. the avoidance or alleviation of direct or indirect, current or future damage and pollution resulting from anthropogenic emissions, waste or changes to the landscape, including land use, as well as the long-term securing of the natural foundations of life for people and other living creatures (birnbacher a ). • animal protection, i.e. the search for reasonable and enforceable standards to avoid or reduce pain and suffering in sentient beings (krebs , vischer ). • nature conservation, i.e. the protection of nature against the transforming intervention of human use, especially all measures to conserve, care for, promote and recreate components of nature deemed to be valuable, including species of flora and fauna, biotic communities, landscapes and the foundations of life required there (birnbacher a) . regardless which of these three areas are addressed we need to explore which primary principles be applied to them. when dealing with the environment, the traditional basic and human rights, as well as the civil rights that have been derived from them, should be just as much a foundation of the consideration as other areas of application in ethics. however, with regard to the primary principles there is a special transfer problem when addressing human interventions into nature and the environment: does the basic postulate of conservation of life apply only to human beings, to all other creatures or to all elements of nature, too? this question does not lead to a new primary principle, as one may suspect at first glance. much rather, it is concerned with the delineation of the universally recognized principle of the conservation of life that has already been specified in the basic rights canon. are only people included in this principle (this is the codified version valid in most legal constitutions today) or other living creatures, too? and if yes, which ones? should non-living elements be included as well? when answering this question, two at first sight contradictory positions can be derived: anthropocentrism and physiocentrism (taylor , ott , galert . the anthropocentric view places humans and their needs at the fore. nature's own original demands are alien to this view. interventions in nature are allowed if they are useful to human society. a duty to make provisions for the future and to conserve nature exists in the anthropocentric world only to the extent that natural systems are classed as valuable to people today and subsequent generations and that nature can be classed as a means and guarantor of human life and survival (norton , birnbacher b . in the physiocentric concept, which forms an opposite pole to the anthropocentric view, the needs of human beings are not placed above those of nature. here, every living creature, whether humans, animals or plants, have intrinsic rights with regard to the chance to develop their own lives within the framework of a natural order. merit for protection is justified in the physiocentric view by an inner value that is unique to each living creature or the environment in general. nature has a value of its own that does not depend on the functions that it fulfils today or may fulfil later from a human society's point of view (devall and sessions , callicott , rolston , meyer-abich . each of these prevailing understandings of the human-nature relationship has implications that are decisive for the form and extent of nature use by humans (elliot , krebs . strictly speaking, it could be concluded from the physiocentric idea that all human interventions in nature have to be stopped so that the rights of other creatures are not endangered. yet, not even extreme representatives of a physiocentric view would go so far as to reject all human interventions in nature because animals, too, change the environment by their ways of life (e.g. the elephant prevents the greening of the savannah). the central postulate of a physiocentric view is the gradual minimization of the depth of interventions in human use of nature. the only interventions that are permitted are those that contribute to directly securing human existence and do not change the fundamental composition of the surrounding natural environment. if these two criteria were taken to the extreme, neither population development beyond the boundaries of biological carrying capacity nor a transformation of natural land into pure agricultural land would be allowed. such a strict interpretation of physiocentrism would lead to a radical reversal of human history so far and is not compatible with the values and expectations of most people. the same is true for the unlimited transfer of anthropocentrism to dealings with nature. in this view, the use of natural services is subjected solely to the individual cost-benefit calculation. this can lead to unscrupulous exploitation of nature by humans with the aim of expanding human civilization. both extremes quickly lead to counter-intuitive implications. when the issue of environmental design and policy is concerned, anthropocentric and physiocentric approaches in their pure form are found only rarely, much rather they occur in different mixtures and slants. the transitions between the concepts are fluid. moderate approaches certainly take on elements from the opposite position. it can thus be in line with a fundamentally physiocentric perspective if the priority of human interests is not questioned in the use of natural resources. it is also true that the conclusions of a moderate form of anthropocentrism can approach the implications of the physiocentric view. table . provides an overview of various types of anthropocentric and physiocentric perspectives. if we look at the behaviour patterns of people in different cultures, physiocentric or anthropocentric basic positions are rarely maintained consistently (bargatzky and kuschel ; on the convergence theory: birnbacher ) . in the strongly anthropocentric countries in the west, people spend more money on the welfare and health of their own pets than on saving human lives in other countries. in the countries of the far east that are characterized by physiocentrism, nature is frequently exploited even more radically than in the industrialized countries of the west. this inconsistent action is not a justification for one view or the other, it is just a warning for caution when laying down further rules for use so that no extreme -and thus untenable -demands be made. also from an ethical point of view, radical anthropocentrism should be rejected just as much as radical physiocentrism. if, to take up just one argument, the right to human integrity is largely justified by the fact that causing pain by others should be seen as something to avoid, this consideration without a doubt has to be applied to other creatures that are also capable of feeling pain (referred to as: pathocentrism). here, therefore, pure anthropocentrism cannot convince. in turn, with a purely physiocentric approach the primary principles of freedom, equality and human dignity could not be maintained at all if every part of living nature were equally entitled to use the natural environment. under these circumstances people would have to do without agriculture, the conversion of natural land into agricultural land and breeding farm animals and pets in line with human needs. as soon table . different perspectives on nature. adapted from renn and goble ( : ). as physiocentrism is related to species and not to individuals as is done in some biocentric perspectives human priority is automatically implied; because where human beings are concerned, nearly all schools of ethics share the fundamental moral principle of an individual right to life from birth. if this right is not granted to individual animals or plants, a superiority of the human race is implicitly assumed. moderate versions of physiocentrism acknowledge a gradual de-escalation with respect to the claim of individual table . different perspectives on nature (continued). adapted from renn and goble ( : ). life protection. the extreme forms of both physiocentrism and anthropocentrism are therefore not very convincing and are hardly capable of achieving a global consensus. this means that only moderate anthropocentrism or moderate biocentrism should be considered. the image of nature that is used as a basis for the considerations in this section emphasizes the uniqueness of human beings vis-à-vis physiocentric views, but does not imply carte blanche for wasteful and careless dealings with nature. this moderate concept derives society's duty to conserve nature -also for future generations -from the life-preserving and life-enhancing meaning of nature for society. this is not just concerned with the instrumental value of nature as a "store of resources", it is also a matter of the function of nature as a provider of inspiration, spiritual experience, beauty and peace (birnbacher and schicha ) . in this context it is important that human beings -as the addressees of the moral standard -do not regard nature merely as material and as a way towards their own self-realization, but can also assume responsibility for conservation of their cultural and so-cial function, as well as their existential value above and beyond the objective and technically available benefits (honnefelder ) . one of the first people to express this responsibility of human stewardship of nature in an almost poetic way was the american ecologist aldo leopold, who pointed out people's special responsibility for the existence of nature and land as early as the s with the essay "the conservation ethics". his most well-known work "a sand county almanac" is sustained by the attempt to observe and assess human activities from the viewpoint of the land (a mountain or an animal). this perspective was clearly physiocentric and revealed fundamental insights about the relationship between humans and nature on the basis of empathy and shifting perspectives. his point of view had a strong influence on american environmental ethics and the stance of conservationists. although this physiocentric perspective raises many concerns, the idea of stewardship has been one of the guiding ideas for the arguments used in this section (pickett et al. ) . we are morally required to exercise a sort of stewardship over living nature, because nature cannot claim any rights for itself, but nevertheless has exceptional value that is important to man above and beyond its economic utility value (hösle ) . since contemporary society and the generations to come certainly use, or will use, more natural resources than would be compatible with a lifestyle in harmony with the given natural conditions, the conversion of natural land into anthropogenically determined agricultural land cannot be avoided (mohr ) . many people criticized human interventions into natural cycles as infringements of the applicable moral standards of nature conservation (for example, fastened onto the postulate of sustainability). but we should avoid premature conclusions here, as can be seen with the example of species protection. for example, where natural objects or phenomena are concerned that turn out to be a risk to human or non-human living creatures, the general call for nature conservation is already thrown into doubt (gale and cordray ) . not many people would call the eradication of cholera bacteria, hiv viruses and other pathogens morally bad (mittelstraß ) if remaining samples were kept under lock and key in laboratories. also, combating highly evolved creatures, such as cockroaches or rats meets with broad support if we ignore the call for the complete eradication of these species for the time being. an environmental initiative to save cockroaches would not be likely to gain supporters. if we look at the situation carefully, the valuation of human behaviour in these examples results from a conflict. because the conservation of the species competes with the objective of maintaining human health or the objective of a hygienic place to live, two principles, possibly of equal ranking, come face to face. in this case the options for action, which may all involve a gradual infringement of one or more principles, would have to be weighed up against each other. a general ban on eradicating a species can thus not be justified ethically, in the sense of a categorical principle, unless the maintenance of human health were to be given lower priority than the conservation of a species. with regard to the issue of species conservation, therefore, different goods have to be weighed up against each other. nature itself cannot show society what it is essential to conserve and how much nature can be traded for valuable commodities. humans alone are responsible for a decision and the resulting conflicts between competing objectives. appreciation and negotiation processes are therefore the core of the considerations about the ethical justification of rules for interventions. but this does not mean that there is no room for categorical judgements along the lines of "this or that absolutely must be prohibited" in the matter of human interventions into the natural environment. it follows on from the basic principle of conserving human life that all human interventions that threaten the ability of the human race as a whole, or a significant number of individuals alive today or in the future, to exist should be categorically prohibited. this refers to intervention threats to the systemic functions of the biosphere. such threats are one of the guiding principles that must not be exceeded under any circumstances, even if this excess were to be associated with high benefits. in the language of ethics this is a categorical principle, in the language of economics a good that is not capable of being traded. the "club" of categorical prohibitions should, however, be used very sparingly because plausible trade-offs can be thought up for most principles, the partial exceeding of which appears intuitively. in the case of threats to existence, however, the categorical rejection of the behaviour that leads to this is obvious. but what does the adoption of categorical principles specifically mean for the political moulding of environmental protection? in the past, a number of authors have tried to specify the minimum requirements for an ethically responsible moral system with respect to biosphere use. these so-called "safe minimum standards" specify thresholds for the open-ended measurement scale of the consequences of human interventions that may not be ex-ceeded even if there is a prospect of great benefits (randall , randall and farmer ) . in order to be able to specify these thresholds in more detail the breakdown into three levels proposed by the german scientific council for global environmental change is helpful (wbgu ) . these levels are: • the global bio-geochemical cycles in which the biosphere is involved as one of the causes, modulator or "beneficiary"; • the diversity of ecosystems and landscapes that have key functions as bearers of diversity in the biosphere; and • the genetic diversity and the species diversity that are both "the modelling clay of evolution" and basic elements of ecosystem functions and dynamics. where the first level is concerned, in which the functioning of the global ecosystem is at stake, categorical principles are obviously necessary and sensible, provided that no one wants to shake the primary principle of the permanent preservation of the human race. accordingly, all interventions in which important substance or energy cycles are significantly influenced at a global level and where globally effective negative impacts are to be expected are categorically prohibited. usually no stringently causal evidence of the harmful nature of globally relevant information is needed; justified suspicion of such harmfulness should suffice. later in this chapter we will make a proposal for risk valuation and management how the problem of uncertainty in the event of possible catastrophic damage potential should be dealt with (risk type cassandra). on the second level, the protection of ecosystems and landscapes, it is much more difficult to draw up categorical rules. initially, it is obvious that all interventions in landscapes in which the global functions mentioned on the first level are endangered must be avoided. above and beyond this, it is wise from a precautionary point of view to maintain as much ecosystem diversity as possible in order to keep the degree of vulnerability to the unforeseen or even unforeseeable consequences of anthropogenic and nonanthropogenic interventions as low as possible. even though it is difficult to derive findings for human behaviour from observations of evolution, the empirically proven statement "he who places everything on one card, always loses in the long run" seems to demonstrate a universally valid insight into the functioning of systemically organized interactions. for this reason, the conservation of the natural diversity of ecosystems and landscape forms is a categorical principle, whereas the depth of intervention allowed should be specified on the basis of principles and standards capable of compensation. the same can be said for the third level, genetic and species protection. here too, initially the causal chain should be laid down: species conservation, landscape conservation, maintaining global functions. wherever this chain is unbroken, a categorical order of conservation should apply. these species could be termed primary key species. this includes such species that are not only essential for the specific landscape type in which they occur, but also for the global cycles above and beyond this specific landscape type thanks to their special position in the ecosystem. probably, it will not be possible to organize all species under this functional contribution to the surrounding ecosystem, but we could also think of groups of species, for example, humus-forming bacteria. in second place there are the species that characterize certain ecosystems or landscapes. here they are referred to as secondary key species. they, too, are under special protection that is not necessarily under categorical reservations. their function value, however, is worthy of special attention. below these two types of species there are the remaining species that perform ecosystem functions to a greater or lesser extent. what this means for the worthiness for protection of these species and the point at which the precise limit for permitted intervention should be drawn, is a question that can no longer be solved with categorical principles and standards, but with the help of compensatory principles and standards. generally, here, too, as with the issue of ecosystem and landscape protection, the conservation of diversity as a strategy of "reinsurance" against ignorance, global risks and unforeseeable surprises is recommended. it remains to be said that from a systemic point of view, a categorical ban has to apply to all human interventions where global closed loops are demonstrably at risk. above and beyond this, it makes sense to recognize the conservation of landscape variety (also of ecosystem diversity within landscapes) and of genetic variety and species diversity as basic principles, without being able to make categorical judgements about individual landscape or species types as a result. in order to evaluate partial infringements of compensatory principles or standards, which are referred to in the issue of environmental protection, we need rules for decision making that facilitate the balancing process necessary to resolve compensatory conflicts. in the current debate about rules for using the environment and nature, it is mainly teleological valuation methods that are proposed (hubig , ott . these methods are aimed at: • estimating the possible consequences of various options for action at all dimensions relevant to potentially affected people; • recording the infringements or fulfilments of these expected consequences in the light of the guiding standards and principles; and • then weighing them according to an internal key so that they can be weighed up in a balanced way. on the positive side of the equation, there are the economic benefits of an intervention and the cultural values created by use, for example, in the form of income, subsistence (self-sufficiency) or an aesthetically attractive landscape (parks, ornamental gardens, etc.); on the negative side, there are the destruction of current or future usage potentials, the loss of unknown natural resources that may be needed in the future and the violation of aesthetic, cultural or religious attributes associated with the environment and nature. there are therefore related categories on both sides of the equation: current uses vs. possible uses in the future, development potentials of current uses vs. option values for future use, shaping the environment by use vs. impairments to the environment as a result of alternative use, etc. with the same or similar categories on the credit and debit side of the balance sheet the decision is easy when there is one option that performs better or worse than all the other options for all categories. although such a dominant (the best for all categories) or sub-dominant option (the worst for all categories) is rare in reality, there are examples of dominant or sub-dominant solutions. thus, for example, the overfelling of the forests of kalimantan on the island of borneo in indonesia can be classed as a sub-dominant option since the short-term benefit, even with extremely high discount rates, is in no proportion to the long-term losses of benefits associated with a barren area covered in imperata grass. the recultivation of a barren area of this kind requires sums many times the income from the sale of the wood, including interest. apparently there are no cultural, aesthetic or religious reasons for conversion of primary or secondary woodland into grassland. this means that the option of deforestation should be classed as of less value than alternative options for all criteria, including economic and social criteria. at best, we can talk about a habit of leaving rainforests, as a "biotope not worthy of conservation", to short-term use. but habit is not a sound reason for the choice of any sub-optimum option. as mentioned at the start of this chapter, habit as experienced morality, does not have any normative force, especially when this is based on the illusion of the marginality of one's own behaviour or ignorance about sustainable usage forms. but if we disregard the dominant or sub-dominant solutions, an appreciation between options that violate or fulfil compensatory standards and principles depends on two preconditions: best possible knowledge of the consequences (what happens if i choose option a instead of option b?) and a transparent, consistent rationale for weighing up these consequences as part of a legitimate political decision process (are the foreseeable consequences of option a more desirable or bearable than the consequences of option b?) (akademie der wissenschaften ). adequate knowledge of the consequences is needed in order to reveal the systemic connections between resource use, ecosystem reactions to human interventions and socio-cultural condition factors (wolters ) . this requires interdisciplinary research and cooperation. the task of applied ecological research, for example, is to show the consequences of human intervention in the natural environment and how ecosystems are burdened by different interventions and practices. the economic approach provides a benefit-oriented valuation of natural and artificial resources within the context of production and consumption, as well as a valuation of transformation processes according to the criterion of efficiency. cultural and social sciences examine the feedback effects between use, social development and cultural self-perception. they illustrate the dynamic interactions between usage forms, socio-cultural lifestyles and control forms. interdisciplinary, problem-oriented and system-related research contribute to forming a basic stock of findings and insights about functional links in the relationship between human interventions and the environment and also in developing constructive proposals as to how the basic question of an ethically justified use of the natural environment can be answered in agreement with the actors concerned (wbgu ) . accordingly, in order to ensure sufficient environmental protection, scientific research, but especially transdisciplinary system research at the interface between natural sciences and social sciences is essential. bringing together the results of interdisciplinary research, the policy-relevant choice of knowledge banks and balanced interpretation in an environment of uncertainty and ambivalence are difficult tasks that primarily have to be performed by the science system itself. how this can happen in a way that is methodslogically sound, receptive to all reasonable aspects of interpretation and yet subjectively valid will be the subject of section . . . but knowledge alone does not suffice. in order to be able to act effectively and efficiently while observing ethical principles, it is necessary to shape the appreciation process between the various options for action according to rational criteria (gethmann ) . to do this it is, first of all, necessary to identify the dimensions that should be used for a valuation. the discussion about the value dimensions to be used as a basis for valuation is one of the most popular subjects within environmental ethics. to apply these criteria in risk evaluation and to combine the knowledge aspects about expected consequences of different behavioural options with the ethical principles is the task of what we have called risk governance. what contribution do ethics make towards clarifying the prospects and limits of human interventions into the natural environment? the use of environmental resources is an anthropological necessity. human consciousness works reflexively and humans have developed a causal recognition capacity that enables them to record cause and effect anticipatively and to productively incorporate assessed consequences in their own action. this knowledge is the motivating force behind the cultural evolution and the development of technologies, agriculture and urbanization. with power over an ever-increasing potential of design and intervention in nature and social affairs over the course of human history, the potential for abuse and exploitation has also grown. whereas this potential was reflected in philosophical considerations and legal standards at a very early stage with regard to moral standards between people, the issue of human responsibility towards nature and the environment has only become the subject of intensive considerations in recent times. ethical considerations are paramount in this respect. on the one hand, they offer concrete standards for human conduct on the bases of criteria that can be generalized, and, on the other hand, they provide procedural advice about a rational and decision-and policy-making process. a simple breakdown into categorical rules and prohibitions that are capable of being compensated can assist decision makers for the justification of principles and standards on environmental protection. as soon as human activities exceed the guidelines of the categorical principles, there is an urgent need for action. how can we detect whether such an excess has happened and how it can be prevented from the very outset that these inviolable standards and principles be exceeded? here are three strategies of environmental protection to be helpful for the implementation of categor-ical guidelines. the first strategy is that of complete protection with severe restrictions of all use by humans (protection priority). the second strategy provides for a balanced relationship between protection and use, where extensive resource use should go hand in hand with the conservation of the ecosystems concerned (equal weight). the third strategy is based on optimum use involving assurance of continuous reproduction. the guiding principle here would be an intensive and, at the same time, sustainable, i.e. with a view to the long term, use of natural resources (use priority). the following section will present a framework for applying these principles into environmental decision making under risk. the main line of argument is that risk management requires an analytic-deliberative approach for dealing effectively and prudently with environmental risks. assessing potential consequences of human interventions and evaluating their desirability on the basis of subsequent knowledge and transparent valuation criteria are two of the central tasks of a risk governance process. however, the plural values of a heterogeneous public and people's preferences have to be incorporated in this process. but how can this be done given the wealth of competing values and preferences? should we simply accept the results of opinion polls as the basis for making political decisions? can we rely on risk perception results to judge the seriousness of pending risks? or should we place all our faith in professional risk management? if we turn to professional help to deal with plural value input, economic theory might provide us an answer to this problem. if environmental goods are made individual and suitable for the market by means of property rights, the price that forms on the market ensures an appropriate valuation of the environmental good. every user of this good can then weigh up whether he is willing to pay the price or would rather not use the good. with many environmental goods, however, this valuation has to be made by collective action, because the environmental good concerned is a collective or open access good. in this case a process is needed that safeguards the valuation and justifies it to the collective. however, this valuation cannot be determined with the help of survey results. although surveys are needed to be able to estimate the breadth of preferences and people's willingness to pay, they are insufficient for a derivation of concrete decision-making criteria and yardsticks for evaluating the tolerability of risks to human health and the environment. • firstly, the individual values are so widely scattered that there is little sense in finding an average value here. • secondly, the preferences expressed in surveys change much within a short time, whereas ethical valuations have to be valid for a long time. • thirdly, as outlined in the subsection on risk perception, preferences are frequently based on flawed knowledge or ad hoc assumptions both of which should not be decisive according to rational considerations. what is needed, therefore, is a gradual process of assigning trade-offs in which existing empirical values are put into a coherent and logically consistent form. in political science and sociological literature reference is mostly made to three strategies of incorporating social values and preferences in rational decision-making processes (renn ) . firstly, a reference to social preferences is viewed solely as a question of legitimate procedure (luhmann , vollmer . the decision is made on the basis of formal decision-making process (such as majority voting). if all the rules have been kept, a decision is binding, regardless of whether the subject matter of the decision can be justified or whether the people affected by the decision can understand the justification. in this version, social consensus has to be found only about the structure of the procedures; the only people who are then involved in the decisions are those who are explicitly legitimated to do so within the framework of the procedure decided upon. the second strategy is to rely on the minimum consensuses that have developed in the political opinion-forming process (muddling through) (lindbloom (lindbloom , . in this process, only those decisions that cause the least resistance in society are considered to be legitimate. in this version of social pluralism groups in society have an influence on the process of the formation of will and decision making to the extent that they provide proposals capable of being absorbed, i.e. adapted to the processing style of the political system, and that they mobilize public pressure. the proposal that then establishes itself in politics is the one that stands up best in the competition of proposals, i.e. the one that entails the fewest losses of support for political decision makers by interest groups. the third strategy is based on the discussion between the groups involved (habermas , renn ). in the communicative exchange among the people involved in the discussion a form of communicative rationality that everyone can understand evolves that can serve as a justification for collectively binding decisions. at the same time, discursive methods claim to more appropriately reflect the holistic nature of human beings and also to provide fair access to designing and selecting solutions to problems. in principle, the justification of standards relevant to decisions is linked to two conditions: the agreement of all involved and substantial justification of the statements made in the discussion (habermas ) . all three strategies of political control are represented in modern societies to a different extent. legitimation conflicts mostly arise when the three versions are realized in their pure form. merely formally adhering to decisionmaking procedures without a justification of content encounters a lack of understanding and rejection among the groups affected especially when they have to endure negative side effects or risks. then acceptance is refused. if, however, we pursue the opposite path of least resistance and base ourselves on the route of muddling through we may be certain of the support of the influential groups, but, as in the first case, the disadvantaged groups will gradually withdraw their acceptance because of insufficient justification of the decision. at the same time, antipathy to politics without a line or guidance is growing, even the affected population. the consequence is political apathy. the third strategy of discursive control faces problems, too. although in an ideal situation it is suitable for providing transparent justifications for the decision-making methods and the decision itself, in real cases the conditions of ideal discourse can rarely be adhered to (wellmer ) . frequently, discussions among strategically operating players lead to a paralysis of practical politics by forcing endless marathon meetings with vast quantities of points of order and peripheral contributions to the discussion. the "dictatorship of patience" (weinrich ) ultimately determines which justifications are accepted by the participants. the public becomes uncertain and disappointed by such discussions that begin with major claims and end with trivial findings. in brief: none of the three ways out of the control dilemma can convince on its own; as so often in politics, everything depends on the right mixture. what should a mixture of the three elements (due process, pluralistic muddling through and discourse) look like so that a maximum degree of rationality can come about on the basis of social value priorities? a report by the american academy of sciences on the subject of "understanding environmental risks" (national research council ) comes to the conclusion that scientifically valid and ethically justified procedure for the collective valuation of options for risk handling can only be realized within the context of -what the authors coin -an analytic-deliberative process. analytic means that the best scientific findings about the possible consequences and conditions of collective action are incorporated in the negotiations; deliberative means that rationally and ethically transparent criteria for making trade-offs are used and documented externally. moreover, the authors consider that fair participation by all groups concerned is necessary to ensure that the different moral systems that can legitimately exist alongside each other should also be incorporated in the process. to illustrate the concept of analytic-deliberative decision making consider a set of alternative options or choices, from which follow consequences (see basic overview in dodgson et al. ) . the relationship between the choice made, and the consequences that follow from this choice, may be straightforward or complex. the science supporting environmental policy is often complicated, across many disciplines of science and engineering, and also involving human institutions and economic interactions. because of limitations in scientific understanding and predictive capabilities, the consequences following a choice are normally uncertain. finally, different individuals and groups within society may not agree on how to evaluate the consequences -which may involve a detailed characterization of what happens in ecological, economic, and human health terms. we shall describe consequences as ambiguous when there is this difficulty in getting agreement on how to interpret and evaluate them. this distinction has been further explained in chapter (see also klinke and renn ) . environmental assessment and environmental decision making inherently involve these difficulties of complexity, uncertainty, and ambiguity (klinke and renn ) . in some situations where there is lots of experience, these difficulties may be minimal. but in other situations these difficulties may constitute major impediments to the decision-making process. to understand how analysis and deliberation interact in an iterative process following the national research council (nrc) report, one must consider how these three areas of potential difficulty can be addressed. it is useful to separate questions of evidence with respect to the likelihood, magnitude of consequences and related characteristics (which can involve complexity and uncertainty) from valuation of the consequences (i.e. ambiguity). for each of the three areas there are analytical tools that can be helpful in identifying, characterizing and quantifying cause-effect relationships. some of these tools have been described in chapter . the integration of these tools of risk governance into a consistent procedure will be discussed in the next subsections. the possibility to reach closure on evaluating risks to human health or the environment rests on two conditions: first, all participants need to achieve closure on the underlying goal (often legally prescribed, such as prevention of health detriments or guarantee of an undisturbed environmental quality, for example, purity laws for drinking water); secondly, they need to agree with the implications derived from the present state of knowledge (whether and to what degree the identified hazard impacts the desired goal). dissent can result from conflicting values as well as conflicting evidence. it is crucial in environmental risk management to investigate both sides of the coin: the values that govern the selection of the goal and the evidence that governs the selection of cause-effect claims. strong differences in both areas can be expected in most environmental decision-making contexts but also in occupational health and safety and public health risks. so for all risk areas it is necessary to explore why people disagree about what to do -that is, which decision alternative should be selected. as pointed out before, differences of opinion may be focused on the evidence of what is at stake or which option has what kind of consequences. for example: what is the evidence that an environmental management initiative will lead to an improvement, such as reducing losses of agricultural crops to insect pests -and what is the evidence that the management initiative could lead to ecological damage -loss of insects we value, such as bees or butterflies, damage to birds and other predators that feed on insects -and health impacts from the level of pesticides and important nutrients in the food crops we eat? other differences of opinion may be about values -value of food crops that contain less pesticide residue compared to those that contain more, value of having more bees or butterflies, value of maintaining indigenous species of bees or butterflies compared to other varieties not native to the local ecosystem, value ascribed to good health and nutrition, and maybe, value ascribed to having food in what is perceived to be a "natural" state as opposed to containing manufactured chemical pesticides or altered genetic material. separating the science issues of what will happen from the value issues of how to make appropriate trade-offs between ecological, economic, and human health goals can become very difficult. the separation of facts and values in decision making is difficult to accomplish in practical decision situations, since what is regarded as facts includes a preference dependent process of cognitive framing (tversky and kahneman ) and what is regarded as value includes a prior knowledge about the factual implica-tions of different value preferences (fischhoff ) . furthermore, there are serious objections against a clear-cut division from a sociological view on science and knowledge generation (jasanoff ) . particularly when calculating risk estimates, value-based conventions may enter the assessment process. for example, conservative assumptions may be built into the assessment process, so that some adverse effects (such as human cancer from pesticide exposure) are much less likely to be underestimated than overestimated (national research council ) . at the same time, ignoring major sources of uncertainty can evoke a sense of security and overconfidence that is not justified from the quality or extent of the data base (einhorn and hogarth ) . perceptions and world views may be very important, and difficult to sort out from matters of science, especially with large uncertainties about the causes of environmental damage. a combination of analytic and deliberative processes can help explore these differences of opinions relating to complexity, uncertainty, and ambiguity in order to examine the appropriate basis for a decision before the decision is made. most environmental agencies go through an environmental assessment process and provide opportunities for public review and comment. many controversial environmental decisions become the focus of large analytical efforts, in which mathematical models are used to predict the environmental, economic, and health consequences of environmental management alternatives. analysis should be seen as an indispensable complement to deliberative processes, regardless whether this analysis is sophisticated or not. even simple questions need analytic input for making prudent decisions, especially in situations where there is controversy arising from complexity, uncertainty, and ambiguity. in many policy arenas in which problems of structuring human decisions are relevant, the tools of normative decision analysis (da) have been applied. especially in economics, sociology, philosophical ethics, and also many branches of engineering and science, these methods have been extended and refined during the past several decades. (edwards , howard , north , howard et al. , north and merkhofer , behn and vaupel , pinkau and renn , van asselt , jaeger et al. . da is a process for decomposing a decision problem into pieces, starting with the simple structure of alternatives, information, and prefer-ences. it provides a formal framework for quantitative evaluation of alternative choices in terms of what is known about the consequences and how the consequences are valued (hammond et al. , skinner . the procedures and analytical tools of da provide a number of possibilities to improve the precision and transparency of the decision procedure. however, they are subject to a number of limitations. the opportunities refer to: • different action alternatives can be quantitatively evaluated to allow selection of a best choice. such evaluation relies both on a description of uncertain consequences for each action alternative, with uncertainty in the consequences described using probabilities, and a description of the values and preferences assigned to consequences. (explicit characterization of uncertainty and values of consequences) • the opportunity to assure transparency, in that ( ) models and data summarizing complexity (e.g., applicable and available scientific evidence) ( ) probabilities characterizing judgement about uncertainty, and ( ) values (utilities) on the consequences are made explicit and available. so the evaluation of risk handling alternatives can be viewed and checked for accuracy by outside observers. (outside audit enabled of basis for decision) • a complex decision situation can be decomposed into smaller pieces in a formal analytical framework. the level of such composition can range from a decision tree of action alternatives and ensuing consequences that fits on a single piece of paper, to extremely large and complex computerimplemented models used in calculating environmental consequences and ascribing probabilities and values of the consequences. a more complex analysis is more expensive and is less transparent to observers. in principle, with sufficient effort any formal analytical framework can be checked to assure that calculations are made in the way that is intended. (decomposition possible to include extensive detail) on the other hand, there are important limitations: • placing value judgements (utilities) on consequences may be difficult, especially in a political context where loss of life, impairment of health, ecological damage, or similar social consequences are involved. utility theory is essentially an extension of cost-benefit methods from economics to include attitude toward risk. the basic trade-off judgements needed for cost-benefit analysis remain difficult and controversial, and often, inherently subjective. (difficulties in valuing consequences) • assessing uncertainty in the form of a numerical probability also poses difficulties, especially in situations when there is not a statistical data base on an agreed-on model as the basis for the assessment. (difficulty in quantifying uncertainty, assigning probabilities) • the analytical framework may not be complete. holistic or overarching considerations or important details may have been omitted. (analytical framework incomplete) • da is built upon an axiomatic structure, both for dealing with uncertainty (i.e., the axiomatic foundation of probability theory), and for valuing consequences (i.e., the axiomatic basis for von neumann-morgenstern utility theory). especially when the decision is to be made by a group rather than an individual decision maker, rational preferences for the group consistent with the axioms may not exist (the "impossibility" theorem of arrow, ) . so in cases of strong disagreements on objectives or unwillingness to use a rational process, decision analysis methods may not be helpful. decision analytical methods should not be regarded as inherently "mechanical" or "algorithmic", in which analysts obtain a set of "inputs" about uncertainty and valuing consequences, then feed these into a mathematical procedure (possibly implemented in a computer) that produces an "output" of the "best" decision. da can only offer coherent conclusions from the information which the decision maker provides by his/her preferences among consequences and his/her state of information on the occurrence of these consequences. where there is disagreement about the preferences or about the information, da may be used to implore the implications of such disagreement. so in application, there is often a great deal of iteration (sensitivity analysis) to explore how differences in judgement should affect the selection of the best action alternative. da thus merely offers a formal framework that can be effective in helping participants in a decision process to better understand the implications of differing information and judgement about complex and uncertain consequences from the choice among the available action alternatives. insight about which factors are most important in selecting among the alternatives is often the most important output of the process, and it is obtained through extensive and iterative exchange between analysts and the decision makers and stakeholders. the main advantage of the framework is that it is based on logic that is both explicit and checkable -usually facilitated by the use of mathematical models and probability calculations. research on human judgement supports the superiority of such procedures for decomposing complex decision problems and using logic to integrate the pieces, rather than relying on holistic judgement on which of the alternatives is best (this is not only true for individual decisions, see heap et al. : ff., jungermann ; but also for collective decisions, see heap et al. : ff., pettit . one should keep in mind, however, that "superior" is measured in accordance with indicator of instrumental rationality, i.e. measuring means-ends effectiveness. if this rationality is appropriate, the sequence suggested by da is intrinsically plausible and obvious. even at the level of qualitative discussion and debate, groups often explore the rationale for different action alternatives. decision analysis simply uses formal quantitative methods for this traditional and common-sense process of exploring the rationale -using models to describe complexity, probability to describe uncertainty, and to deal with ambiguity, explicit valuation of consequences via utility theory and other balancing procedures, such as cost-benefit or cost-effectiveness analyses. by decomposing the problem in logical steps, the analysis permits better understanding of differences in the participants' perspective on evidence and values. da offers methods to overcome these differences, such as resolving questions about underlying science through data collection and research, and encouraging tradeoffs, compromise, and rethinking of values. based on this review of opportunities and shortcomings we conclude that decision analysis provides a suitable structure for guiding discussion and problem formulation, and offers a set of quantitative analytical tools that can be useful for environmental decisions, especially in conjunction with deliberative processes. da can assist decision makers and others involved in, and potentially affected by, the decision (i.e., participants, stakeholders) to deal with complexity and many components of uncertainty, and to address issues of remaining uncertainties and ambiguities. using these methods promises consistency from one decision situation to another, assurance of an appropriate use of evidence from scientific studies related to the environment, and explicit accountability and transparency with respect to those institutionally responsible for the value judgements that drive the evaluation of the alternative choices. collectively the analytical tools provide a framework for a systematic process of exploring and evaluating the decision alternatives -assembling and validating the applicable scientific evidence relevant to what will happen as the result of each possible choice, and valuing how bad or how good these consequences are based on an agreement of common objectives. yet, it does not replace the need for additional methods and processes for including other objectives, such as finding common goals, defining preferences, revisiting assumptions, sharing visions and exploring common grounds for values and normative positions. the value judgements motivating decisions are made explicit and can then be criticized by those who were not involved in the process. to the extent that uncertainty becomes important, it will be helpful to deal with uncertainty in an orderly and consistent way (morgan and henrion ). those aspects of uncertainty that can be modelled by using probability theory (inter-target variation, systematic and random errors in applying inferential statistics, model and data uncertainties) will be spelled out and those that remain in forms of indeterminacies, system boundaries or plain ignorance will become visible and can then be fed into the deliberation process (van asselt , klinke and renn ) . the term deliberation refers to the style and procedure of decision making without specifying which participants are invited to deliberate (national research council (nrc) , rossi ) . for a discussion to be called deliberative it is essential that it relies on mutual exchange of arguments and reflections rather than decision making based on the status of the participants, sublime strategies of persuasion, or social-political pressure. deliberative processes should include a debate about the relative weight of each argument and a transparent procedure for balancing pros and cons (tuler and webler ) . in addition, deliberative processes should be governed by the established rules of a rational discourse. in the theory of communicative action developed by the german philosopher jürgen habermas, the term discourse denotes a special form of a dialogue, in which all affected parties have equal rights and duties to present claims and test their validity in a context free of social or political domination (habermas (habermas , b . a discourse is called rational if it meets the following specific requirements (see mccarthy , habermas a , kemp , webler . all participants are obliged: • to seek a consensus on the procedure that they want to employ in order to derive the final decision or compromise, such as voting, sorting of positions, consensual decision making or the involvement of a mediator or arbitrator; • to articulate and criticize factual claims on the basis of the "state of the art" of scientific knowledge and other forms of problem-adequate knowledge; (in the case of dissent all relevant camps have the right to be represented); • to interpret factual evidence in accordance with the laws of formal logic and analytical reasoning; • to disclose their relevant values and preferences, thus avoiding hidden agendas and strategic game playing; and • to process data, arguments and evaluations in a structured format (for example a decision-analytic procedure) so that norms of procedural rationality are met and transparency can be created. the rules of deliberation do not necessarily include the demand for stakeholder or public involvement. deliberation can be organized in closed circles (such as conferences of catholic bishops, where the term has indeed been used since the council of nicosia), as well as in public forums. it may be wise to use the term "deliberative democracy" when one refers to the combination of deliberation and public or stakeholder involvement (see also cohen , rossi . what needs to be deliberated? firstly, deliberative processes are needed to define the role and relevance of systematic and anecdotal knowledge for making far-reaching choices. secondly, deliberation is needed to find the most appropriate way to deal with uncertainty in environmental decision making and to set efficient and fair trade-offs between potential over-and under-protection. thirdly, deliberation needs to address the wider concerns of the affected groups and the public at large. why can one expect that deliberative processes are better suited to deal with environmental challenges than using expert judgement, political majority votes or relying on public survey data? • deliberation can produce common understanding of the issues or the problems based on the joint learning experience of the participants with respect to systematic and anecdotal knowledge (webler and renn , pidgeon ). • deliberation can produce a common understanding of each party's position and argumentation and thus assist in a mental reconstruction of each actor's argumentation (warren , tuler . the main driver for gaining mutual understanding is empathy. the theory of communicative action provides further insights in how to mobilize empathy and how to use the mechanisms of empathy and normative reasoning to explore and generate common moral grounds (webler ). • deliberation can produce new options and novel solutions to a problem. this creative process can either be mobilized by finding win-win solutions or by discovering identical moral grounds on which new options can grow (renn ) . • deliberation has the potential to show and document the full scope of ambiguity associated with environmental problems. deliberation helps to make a society aware of the options, interpretations, and potential actions that are connected with the issue under investigation (wynne , de marchi and ravetz ) . each position within a deliberative discourse can only survive the crossfire of arguments and counter-arguments if it demonstrates internal consistency, compatibility with the legitimate range of knowledge claims and correspondence with the widely accepted norms and values of society. deliberation clarifies the problem, makes people aware of framing effects, and determines the limits of what could be called reasonable within the plurality of interpretations (skillington ) . • deliberations can also produce agreements. the minimal agreement may be a consensus about dissent (raiffa ) . if all arguments are exchanged, participants know why they disagree. they may not be convinced that the arguments of the other side are true or morally strong enough to change their own position; but they understand the reasons why the opponents came to their conclusion. in the end, the deliberative process produces several consistent and -in their own domain -optimized positions that can be offered as package options to legal decision makers or the public. once these options have been subjected to public discourse and debate, political bodies, such as agencies or parliaments can make the final selection in accordance with the legitimate rules and institutional arrangements such as majority vote or executive order. final selections could also be performed by popular vote or referendum. • deliberation may result in consensus. often deliberative processes are used synonymously with consensus-seeking activities (coglianese ). this is a major misunderstanding. consensus is a possible outcome of deliberation, but not a mandatory requirement. if all participants find a new option that they all value more than the one option that they preferred when entering the deliberation, a "true" consensus is reached (renn ) . it is clear that finding such a consensus is the exception rather than the rule. consensus is either based on a win-win solution or a solution that serves the "common good" and each participant's interests and values better than any other solution. less stringent is the requirement of a tolerated consensus. such a consensus rests on the recognition that the selected decision option might serve the "common good" best, but on the expense of some interest violations or additional costs. in a tolerated consensus some participants voluntarily accept personal or group-specific losses in exchange for providing benefits to all of society. case studies have provided sufficient evidence that deliberation has produced a tolerated consensus solution, particularly in siting conflicts (one example in schneider et al. ) . consensus and tolerated consensus should be distinguished from compromise. a compromise is a product of bargaining where each side gradually reduces its claim to the opposing party until they reach an agreement (raiffa ) . all parties involved would rather choose the option that they preferred before starting deliberations, but since they cannot find a win-win situation or a morally superior alternative they look for a solution that they can "live with" knowing that it is the second or third best solution for them. compromising on an issue relies on full representation of all vested interests. in summary, many desirable products and accomplishments are associated with deliberation (chess et al. ) . depending on the structure of the discourse and the underlying rationale deliberative processes can: • enhance understanding; • generate new options; • decrease hostility and aggressive attitudes among the participants; • explore new problem framing; • enlighten legal policy-makers; • produce competent, fair and optimized solution packages; and • facilitate consensus, tolerated consensus and compromise. in a deliberative setting, participants exchange arguments, provide evidence for their claims and develop common criteria for balancing pros and cons. this task can be facilitated and often guided by using decision analytic tools (overview in merkhofer ) . decision theory provides a logical framework distinguishing action alternatives or options, consequences, likelihood of consequences, and value of consequences, where the valuation can be over multiple attributes that are weighted based on tradeoffs in multi-attribute utility analysis (edwards ) . a sequence of decisions and consequences may be considered, and use of mathematical models for predicting the environmental consequences of options may or may not be part of the process (humphreys , bardach , arvai et al. ): a) the structuring potential of decision analysis has been used in many participatory processes. it helps the facilitator of such processes to focus on one element during the deliberation, to sort out the central from the peripheral elements, provide a consistent reference structure for ordering arguments and observations and to synthesize multiple impressions, observations and arguments into a coherent framework. the structuring power of decision analysis has often been used without expanding the analysis into quantitative modelling. b) the second potential, agenda setting and sequencing, is also frequently applied in participatory settings. it often makes sense to start with problem definition, then develop the criteria for evaluation, generate options, assess consequences of options, and so on. c) the third potential, quantifying consequences, probabilities and relative weights and calculating expected utilities, is more controversial than the other two. whether the deliberative process should include a numerical analysis of utilities or engage the participants in a quantitative elicitation process is contested among participation practitioners . one side claims that quantifying helps participants to be more precise about their judgements and to be aware of the often painful trade-offs they are forced to make. in addition, quantification can make judgements more transparent to outside observers. the other side claims that quantification restricts the participants to the logic of numbers and reduces the complexity of argumentation into a mere trade-off game. many philosophers argue that quantification supports the illusion that all values can be traded off against other values and that complex problems can be reduced to simple linear combinations of utilities. one possible compromise between the two camps may be to have participants go through the quantification exercise as a means to help them clarify their thoughts and preferences, but make the final decisions on the basis of holistic judgements (renn ) . in this application of decision analytic procedures, the numerical results (i.e. for each option the sum over the utilities of each dimension multiplied by the weight of each dimension) of the decision process are not used as expression of the final judgement of the participant, but as a structuring aid to improve the participant's holistic, intuitive judgement. by pointing out potential discrepancies between the numerical model and the holistic judgements, the participants are forced to reflect upon their opinions and search for potential hidden motives or values that might explain the discrepancy. in a situation of major value conflicts, the deliberation process may involve soliciting a diverse set of viewpoints, and judgements need to be made on what sources of information are viewed as responsible and reliable. publication in scientific journals and peer review from scientists outside the government agency are the two most popular methods by which managers or organizers of deliberative processes try to limit what will be considered as acceptable evidence. other methods are to reach a consensus among the participants up front which expertise should be included in the deliberation or to appoint representatives of opposing science camps to explain their differences in public. in many cases, participants have strong reasons for questioning scientific orthodoxy and would like to have different science camps represented. many stakeholders in environmental decisions have access to expert scientists, and often such scientists will take leading roles in criticizing agency science. such discussions need to be managed so that disagreements among the scientific experts can be evaluated in terms of the validity of the evidence presented and the importance to the decision. it is essential in these situations to have a process in place that distinguishes between those evidence claims that all parties agree on, those where the factual base is shared but not its meaning for some quality criterion (such as "healthy" environment), and those where even the factual base is contested (foster ) . in the course of practical risk management different conflicts arise in deliberative settings that have to be dealt with in different ways. the main conflicts occur at the process level (how should the negotiations be conducted?), on the cognitive level (what is factually correct?), the interest level (what benefits me?), the value level (what is needed for a "good" life?) and the normative level (what can i expect of all involved?). these different conflict levels are addressed in this subsection. first of all, negotiations begin by specifying the method that structures the dialogue and the rights and duties of all participants. it is the task of the chairman or organizer to present and justify the implicit rules of the talks and negotiations. above and beyond this, the participants have to specify joint rules for decisions, the agenda, the role of the chairman, the order of hearings, etc. this should always be done according to the consensus principle. all partners in the negotiations have to be able to agree to the method. if no agreement is reached here the negotiations have to be interrupted or reorganized. once the negotiation method has been determined and, in a first stage, the values, standards and objectives needed for judgement have been agreed jointly, then follows the exchange of arguments and counter arguments. in accordance with decision theory, four stages of validation occur: • in a first stage, the values and standards accepted by the participants are translated into criteria and then into indicators (measurement instructions). this translation needs the consensual agreement of all participants. experts are asked to assess the available options with regard to each indicator according to the best of their knowledge (factual correctness). in this context it makes more sense to specify a joint methodological procedure or a consensus about the experts to be questioned than to give each group the freedom to have the indicators answered by their own experts. often many potential consequences remain disputed as a result of this process, especially if they are uncertain. however, the bandwidth of possible opinions is more or less restricted depending on the level of certainty and clarity associated with the issue in question. consensus on dissent is also of help here in separating contentious factual claims from undisputed ones and thus promotes further discussion. • in a second stage, all participating parties are required to interpret bandwidths of impacts to be expected for each criterion. interpretation means linking factual statements with values and interests to form a balanced overall judgement (conflicts of interests and values). this judgement can and should be made separately for each indicator. in this way, each of the chains of causes for judgements can be understood better and criticized in the course of the negotiations. for example, the question of trustworthiness of the respective risk management agencies may play an important role in the interpretation of an expected risk value. then it is the duty of the participating parties to scrutinize the previous performance of the authority concerned and propose institutional changes where appropriate. • third stage: even if there were a joint assessment and interpretation for every indicator, this would by no means signify that agreement is at hand. much rather, the participants' different judgements about decisionmaking options may be a result of different value weightings for the indicators that are used as a basis for the values and standards. for example, a committed environmentalist may give much more weight to the indicator for conservation than to the indicator of efficiency. in the literature on game theory, this conflict is considered to be insoluble unless one of the participants can persuade the other to change his preference by means of compensation payments (for example, in the form of special benefits), transfer services (for example, in the form of a special service) or swap transactions (do, ut des). in reality, however, it can be seen that participants in negotiations are definitely open to the arguments of the other participants (i.e. they may renounce their first preference) if the loss of benefit is still tolerable for them and, at the same time, the proposed solution is considered to be "conducive to the common good", i.e. is seen as socially desirable in public perception. if no consensus is reached, a compromise solution can and should be reached, in which a 'fair' distribution of burdens and profits is accomplished. • fourth stage: when weighing up options for action formal methods of balancing assessment can be used. of these methods, the cost-benefit analysis and the multi-attribute or multi-criteria decision have proved their worth. the first method is largely based on the approach of revealed "preferences", i.e. on people's preferences shown in the past expressed in relative prices, the second on the approach of "expressed preferences", i.e. the explicit indication of relative weightings between the various cost and benefit dimensions (fischhoff et al. ) . but both methods are only aids in weighing up and cannot replace an ethical reflection of the advantages and disadvantages. normative conflicts pose special problems because different evaluative criteria can always be classified as equally justifiable or unjustifiable as explained earlier. for this reason, most ethicists assume that different types and schools of ethical justification can claim parallel validity, it therefore remains up to the groups involved to choose the type of ethically legitimate justification that they want to use (ropohl , renn . nevertheless, the limits of particular justifications are trespassed wherever primary principles accepted by all are infringed (such as human rights). otherwise, standards should be classed as legitimate if they can be defended within the framework of ethical reasoning and if they do not contradict universal standards that are seen as binding for all. in this process conflicts can and will arise, e.g. that legitimate derivations of standards from the perspective of group a contradict the equally legitimate derivations of group b (shrader-frechette ). in order to reach a jointly supported selection of standards, either a portfolio of standards that can claim parallel validity should be drawn up or compensation solutions will have to be created in which one party compensates the other for giving up its legitimate options for action in favour of a common option. when choosing possible options for action or standards, options that infringe categorical principles, for example, to endangering the systematic ability of the natural environment to function for human use in the future and thus exceeding the limits of tolerability are not tolerable even if they imply major benefits to society. at the same time, all sub-dominant options have to be excluded. frequently sub-dominant solutions, i.e. those that perform worse than all other options with regard to all criteria at least in the long term, are so attractive because they promise benefits in the short term although they entail losses in the long term, even if high interest rates are assumed. often people or groups have no choice other than to choose the sub-dominant solution because all other options are closed to them due to a lack of resources. if large numbers of groups or many individuals act in this way, global risks become unmanageable (beck ) . to avoid these risks intermediate financing or compensation by third parties should be considered. the objective of this last section of chapter was to address and discuss the use of decision analytic tools and structuring aids for participatory processes in environmental management. organizing and structuring discourses goes beyond the good intention to have all relevant stakeholders involved in decision making. the mere desire to initiate a two-way communication process and the willingness to listen to stakeholder concerns are not sufficient. discursive processes need a structure that assures the integration of technical expertise, regulatory requirements, and public values. these different inputs should be combined in such a fashion that they contribute to the deliberation process the type of expertise and knowledge that can claim legitimacy within a rational decision-making procedure (von schomberg ). it does not make sense to replace technical expertise with vague public perceptions, nor is it justified to have the experts insert their own value judgements into what ought to be a democratic process. decision analytic tools can be of great value for structuring participatory processes. they can provide assistance in problem structuring, in dealing with complex scientific issues and uncertainty, and in helping a diverse group to understand disagreements and ambiguity with respect to values and preferences. decision analysis tools should be used with care. they do not provide an algorithm to reach an answer as to what is the best decision. rather, decision analysis is a formal framework that can be used for environmental assessment and risk handling to explore difficult issues, to focus debate and further analysis on the factors most important to the decision, and to provide for increased transparency and more effective exchange of information and opinions among the process participants. the basic concepts are relatively simple and can be implemented with a minimum of mathematics (hammond et al. ) . many participation organizers have restricted the use of decision analytic tools to assist participants in structuring problems and ordering concerns and evaluations, and have refrained from going further into quantitative trade-off analysis. others have advocated quantitative modelling as a clarification tool for making value conflicts more transparent to the participants. the full power of decision analysis for complex environmental problem may require mathematical models and probability assessment. experienced analysts may be needed to guide the implementation of these analytical tools for aiding decisions. skilled communicators and facilitators may be needed to achieve effective interaction between analysts and participants in the deliberative process whose exposure to advanced analytical decision aids is much less, so that understanding of both process and substance, and therefore transparency and trust, can be achieved. many risk management agencies are already making use of decision analysis tools. we urge them to use these tools in the context of an iterative, deliberative process with broad participation by the interested and affected parties to the decision in the context of the risk governance framework. the analytical methods, the data and judgement, and the 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Ökologie und ethik. ein versuch praktischer philosophie. ethik in den wissenschaften zur ethischen bewertung von biodiversität. externes gutachten für den wbgu. unveröffentlichtes manuskript decision theory and folk psychology towards a comprehensive conservation theory the limits to safety? culture, politics, learning and manmade disasters environmental standards. scientific foundations and rational procedures of regulation with emphasis on radiological risk management the art and science of negotiation what mainstream economists have to say about the value of biodiversity benefits, costs, and the safe minimum standard of conservation decision analytic tools for resolving uncertainty in the energy debate Ökologisch denken -sozial handeln: die realisierbarkeit einer nachhaltigen entwicklung und die rolle der sozial-und kulturwissenschaften ein diskursives verfahren zur bildung und begründung kollektiv verbindlicher bewertungskriterien a model for an analytic-deliberative process in 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nature. a theory of environmental ethics meanings, understandings, and interpersonal relationships in environmental policy discourse. doctoral dissertation designing an analytic deliberative process for environmental health policy making in the u.s. nuclear weapons complex the framing of decisions and the psychology of choice perspectives on uncertainty and risk akzeptanzbeschaffung: verfahren und verhandlungen the erosion of the valuespheres. the ways in which society copes with scientific, moral and ethical uncertainty can participatory democracy produce better selves? psychological dimensions of habermas discursive model of democracy welt im wandel: erhaltung und nachhaltige nutzung der biosphäre. jahresgutachten discourse in citizen participation. an evaluative yardstick the craft and theory of public participation: a dialectical process a brief primer on participation: philosophy and practice system, diskurs, didaktik und die diktatur des sitzfleisches konsens als telos der sprachlichen kommunikation? in: giegel biophilia: the human bond with other species rio" oder die moralische verpflichtung zum erhalt der natürlichen vielfalt risk and social learning: reification to engagement die modernisierung der demokratie im zeichen der umweltproblematik wandelt sich die verantwortung mit technischem wandel? key: cord- - plzyqd authors: krishnan, gokul s.; kamath, s. sowmya title: hybrid text feature modeling for disease group prediction using unstructured physician notes date: - - journal: computational science - iccs doi: . / - - - - _ sha: doc_id: cord_uid: plzyqd existing clinical decision support systems (cdsss) largely depend on the availability of structured patient data and electronic health records (ehrs) to aid caregivers. however, in case of hospitals in developing countries, structured patient data formats are not widely adopted, where medical professionals still rely on clinical notes in the form of unstructured text. such unstructured clinical notes recorded by medical personnel can also be a potential source of rich patient-specific information which can be leveraged to build cdsss, even for hospitals in developing countries. if such unstructured clinical text can be used, the manual and time-consuming process of ehr generation will no longer be required, with huge person-hours and cost savings. in this article, we propose a generic icd disease group prediction cdss built on unstructured physician notes modeled using hybrid word embeddings. these word embeddings are used to train a deep neural network for effectively predicting icd disease groups. experimental evaluation showed that the proposed approach outperformed the state-of-the-art disease group prediction model built on structured ehrs by % in terms of auroc and % in terms of auprc, thus proving our hypothesis and eliminating dependency on availability of structured patient data. electronic health records (ehrs) and predictive analytics have paved the way for clinical decision support systems (cdsss) development towards realizing intelligent healthcare systems. the huge amount of patient data generated in hospitals in the form of discharge summaries, clinical notes, diagnostic scans etc., present a wide array of opportunities to researchers and data scientists for developing effective methods to improve patient care. cdsss built on techniques like data mining and machine learning (ml) have been prevalent in healthcare systems, aiding medical personnel to make informed, rational decisions and execute timely interventions for managing critical patients. some existing ml based cdsss include -mortality prediction [ , , , , , ] , hospital readmission prediction [ , , ] , length of stay prediction [ , , ] , generic disease or icd group prediction [ , , ] and so on. however, it is significant to note that, most existing cdsss depend on structured patient data in the form of electronic health records (ehrs), which favors developed countries due to large scale adoption of ehrs. however, healthcare personnel in developing countries continue to rely on clinical notes in the form of free and unstructured text, as structured ehr adoption rate is extremely low in these countries. moreover, the conversion of patient information recorded in the form of text/clinical reports to a standard structured ehr is a labor-intensive, manual process that can result in loss of critical patient-specific information that might be present in the clinical notes. for this reason, there is a need for alternative approaches of developing cdsss that do not rely on structured ehrs. icd disease coding is an important task in a hospital, during which a trained medical coder with domain knowledge assigns disease-specific, standardized codes called icd codes to a patient's admission record. as hospital billing and insurance claims are based on the assigned icd codes, the coding task requires high precision, but is often prone to human error, which has resulted in an annual spending of $ billion in the us to improve coding efficacy [ , ] . hence, automated disease coding approaches have been developed as a solution to this problem. currently, this is an area of active research [ ] [ ] [ ] , , ] , but performance that has been recorded so far is below par indicating huge scope for improvement, thus making automated icd coding an open research problem. existing icd coding methods make use of only discharge summaries for coding, similar to the process adopted by human medical coders. however, as the records are digitized, other textual records under the same admission identifier might be available, which may provide additional patient-specific insights pertaining to the diagnosed diseases. further, for icd disease coding to be effective, the correct determination of generic disease categories or groups prior to specific coding is very crucial as information regarding generic disease groups can be informative for specific icd coding. with this in mind, we explore other textual sources of patient data, utilizing physician notes to predict the generic icd disease groups for the patient, following which actual icd disease code prediction can be achieved. in this article, we focus on effective prediction of icd disease groups and the code prediction will be considered in future work. while most existing works aim to predict icd codes rather than icd disease groups, purushotham et al. [ ] performed benchmarking of three cdss tasks -icu mortality prediction, icd group prediction and hospital readmission prediction on icu patients data using super learner and deep learning techniques. the icd group prediction task discussed in their work is a generic disease group prediction task that is a step prior to icd coding, which can also be a viable disease risk prediction cdss for caregivers. their work uses feature subsets extracted from structured patient data, where the best performing model uses multitude of numerical features such as input events (volume of input fluids through iv), output events (quantity of output events like urinary and rectal), prescriptions and other icu events like chart events (readings recorded in the icu) and lab events (lab test values). as deep learning models can effectively learn from numerous raw feature values quite well, their approach achieved good results. however, in a real world scenario, to measure all these values and then using them as inputs to a prediction model for an outcome, will cause an inevitable and significant time delay, during which the patient condition can worsen. cdss prediction models that use unstructured clinical text data can predict desired outcomes with lower latency and also help improve the prediction performance as these raw text sources contain more patient-specific information. some recent works [ , , ] focussed on investigating the effectiveness of modeling various unstructured text reports such as radiology and nursing notes of icu patients for disease group prediction. with the objective of continuing such analyses on other kinds of unstructrured clinical reports, we intend to focus on physician notes in this work. additionally, we also analyze how they can be used in predicting icd disease groups by effectively modeling these physician notes. in this article, a hybrid feature modeling approach that uses hybrid clinical word embeddings to generate quality features which are used to train and build a deep neural network model to predict icd disease groups is presented. we show the results of a benchmarking study of our proposed model (modeled on unstructured physician notes) against the current state-of-the-art model for disease group prediction (built on structured clinical data). the rest of this article is structured as follows: sect. describes the proposed approach in detail, followed by experimental results and discussion in sect. . we conclude the article with prospective future work and research directions. the proposed approach for disease group prediction is depicted in fig. . we used physicians' clinical notes in unstructured text form from the mimic-iii [ ] dataset for our experiments. mimic-iii contains data relating to , icu admissions of more than , patients admitted in beth israel hospital, new york, usa, during - . we extracted only the physician notes from the 'noteevents' table, which resulted in a total of , physician notes generated during , admissions. as per mimic documentation, physicians have reported some identified errors in notes present in the 'noteevents' table. as these notes can affect the training negatively, records with physician identified errors were removed from the cohort. additionally, those records with table . we observed that there were kinds of physician notes available in the mimic-iii dataset such as physician resident progress note, intensivist note, etc. the frequency statistics of the top ten kinds of physician notes are tabulated in table . a particular patient may suffer from multiple diagnoses during a particular admission and hence, it is necessary for the prediction to be a multilabel prediction task. therefore, for each physician note, all the diagnosed disease groups during that particular admission were considered as labels and given binary values - (if the disease was not diagnosed) and (if the disease was diagnosed). the physician notes corpus is first preprocessed using basic natural language processing (nlp) techniques such as tokenization and stop word removal. using [ ] , hence capturing relevant terms and concepts in the biomedical domain, which helps generate quality feature representation of the underlying corpus. the pre-trained model generates word embedding vectors of size × for each word and these vectors were averaged to generate a representation such that each preprocessed physician note is represented as a × vector. the preproccesed tokens are then used to train a word vec model [ ] , a neural network based word representation model that generates word embeddings based on co-occurrence of words. the skipgram model of word vec was used for training the physician notes tokens with a dimension size of (same as the pre-trained model) with an initial learning rate of . . the averaged word vector representation for each report tokens are extracted and then fed into the neural network model along with the vector representation extracted from the pre-trained model and the icd disease group labels. icu patients' diagnoses in terms of icd disease codes from the 'diag-noses icd' table of mimic-iii dataset were grouped as per standards (similar to the approach adopted by purushotham et al. [ ] ). a total of , unique icd disease codes thus obtained were grouped into icd disease groups, i.e., potential labels. as the icd group prediction task is considered as a binary classification of multiple labels, labels (disease groups) were considered with binary values: (negative diagnosis of the disease) and (positive diagnosis of the disease). the physician notes modeled into two feature matrices of shape × each, along with icd disease groups (labels) is now used to train the neural network model. the proposed deep neural network prediction model is illustrated in fig. . the neural network architecture is divided into parts -the first for determining the weights for the hybrid combination of features dynamically and the next for multi-label classification of icd disease codes. the process of dynamically modeling the weightage to be assigned for the combination of pre-trained word embeddings and the word embeddings generated using the physicians notes is fig. . the two feature set are fed as inputs into the neural network model, where both the input layers consists of neurons, equal to the number of features generated from both models. the addition layer merges the two sets of input features in a weighted combination which is dynamically determined through backpropagation of the overall neural network architecture thereby ensuring the optimal and effective combination that offers the best classification performance possible. this architecture also ensures that the weights for the hybrid combination of features is always determined dynamically and hence can be used for any clinical text corpus. the combined set of features, i.e., the hybrid features, are then fed on to the dense feed forward neural network model which performs the training for multi-label classification of icd disease code groups. the dynamically weighted feature matrix consisting of the hybrid word embedding features, along with icd group labels are next used for training a feed forward neural network (ffnn) used as the prediction model (depicted in fig. ). the input layer consists of neurons with input dimension as (number of input features); followed by three hidden layers with , and neurons respectively and finally an output layer with neurons, each representing an icd- disease group. to prevent overfitting, a dropout layer, with a dropout rate of % was also added to the ffnn model (see fig. ). as this is a binary classification for multiple labels, binary cross entropy was used as a loss function, while stochastic gradient descent (sgd) was used as the optimizer with a learning rate of . . rectified linear unit relu activation function was used as the input and hidden layer activation functions as the feature matrix values are standardized to the range − and . the major hyperparameters for the ffnn model -the optimizer, learning rate of the optimizer and the activation function, were tuned empirically over several experiments using the gridsearchcv function in python sklearn library. finally, the output layer activation function is a sigmoid, again as the classification is two-class for each of the labels. training was performed for epochs and then the model was applied to the validation set to predict disease groups after which the results were observed and analyzed. to evaluate the proposed approach, we performed several experiments using standard metrics like accuracy, precision, recall, f-score, area under receiver operating characteristic curve (auroc), area under precision recall curve (auprc) and matthew's correlation coefficient (mcc). we measured these metrics on a sample-wise basis, i.e., for each report, the predicted and actual disease groups were compared and analyzed. it can be observed from the table that, the proposed model achieved promising results: auprc of . and auroc of . . the accuracy, precision, mcc and f-score of . , . , . and . respectively also indicate a good performance. for evaluating the proposed hybrid text feature modeling approach, we designed experiments to compare its performance against that of baseline feature modeling approaches -tf-idf based bag-of-words approach, trained word embedding approach (only word vec model) and a pre-trained word embedding approach (using word embedding model trained on pubmed, pmc and wikipedia english articles). the bag-of-words approach is based on calculated term frequency and inverse document frequency (tf xidf ) scores determined from the physician notes corpus. the sklearn english stopword list was used to filter the stopwords and n-gram (n = , , ) features were considered. finally, the top features were extracted from the corpus and then fed into the neural network model for training. the other two baselines were kept the same as explained in sect. . it is to be noted that in the neural network configuration, only one input is present, i.e., there is no hybrid weighted addition layer. the results of comparison are tabulated in table . it can be observed that the proposed approach that involves a hybrid weighted combination of pre-trained and trained word embeddings is able to perform comparatively better in terms of all metrics. next, a comparative benchmarking against the state-of-the-art icd disease group prediction model developed by purushotham et al. [ ] was performed. for each hospital admission id (hadm id) considered by purushottam et al. [ ] , the physician notes (if present) were extracted from 'notevents' table. although the number of records under consideration for both the studies are different, it is to be noted that the labels are distributed similarly (statistics shown in fig. ) and is therefore considered a fair comparison. we consider this comparison in order to study the effect of the disease group prediction models built on structured patient data (state-of-the-art approach [ ] ) and unstructured patient data (physician notes in this case). the results of the benchmarking are tabulated in table , which clearly shows the proposed approach outperformed purushotham et al.'s model purushotham et al. [ ] by % in terms of auroc and % in terms of auprc. this shows that the predictive power of a model built on unstructured patient data exceeds that of those built on structured data, where some relevant information may be lost during the coding process. to encourage comparative studies that use physician notes in mimic-iii dataset, certain additional metrics were also considered. the recall & f-score performance as well as the mcc values of the proposed model over our easily reproducible patient cohort data subset are also observed and provided. discussion. from our experiments, we observed a significant potential in developing prediction based cdss using unstructured text reports directly, eliminating the dependency on the availability of structured patient data and ehrs. the proposed approach that involves a textual feature modeling and a neural network based prediction model was successful in capturing the rich and latent clinical information available in unstructured physician notes, and using it to effectively learn disease group characteristics for prediction. the word vec table and it is this combination that has further enabled the ffnn to generalize better and learn the textual feature representation, effectively improving prediction performance when compared to the state-of-the-art model built on structured data. it is interesting to note that the patient data was modeled using only textual features, without any ehrs, structured data or other processed information. the high auprc and auroc values obtained in comparison to the stateof-the-art's (based on structured data) performance is an indication that the unstructured text clinical notes (physician notes in this case) contain abundant patient-specific information that was beneficial for predictive analytics applications. moreover, the conversion process from unstructured patient text reports to structured data can be eliminated, thereby saving huge man hours, cost and other resources. the proposed approach also eliminates any dependency on structured ehrs, thus making it suitable for deployment in developing countries. we found that the data preparation pipeline adopted for this study could be improved, as it sometimes resulted in some conflicting cases during training. this is because of the structure of the mimic-iii dataset, in which, the physician notes do not have a direct assignment to icd disease codes. to overcome this problem, we designed an assignment method in which we extracted icd codes from the diagnoses icd table and assigned them to all patients with the same subject id and hadm id in the physician notes data subset. a sideeffect of this method is that, at times, the icd disease codes/groups assigned to the physician notes may not be related to that particular disease as a patient under same admission can have multiple physician notes. however, the model achieved promising results and the good performance indicates that prediction model was able to capture disease-specific features using the information present in the notes during the admission. in this article, a deep neural network based model for predicting icd disease groups from physician notes in the form of unstructured text is discussed. the proposed approach is built on a hybrid feature set consisting of word embedding features generated from a word vec skipgram model and also from a pre-trained word embedding model trained on biomedical articles from pubmed and pmc. the icd disease codes were categorized into standard groups and then used to train a binary classifier for multi-label prediction. the two sets of word embedding features were input into a neural network model as inputs and the weights of hybrid addition was determined dynamically using backpropagation. the combined features were further fed into a ffnn architecture to train the classifier and the prediction model was validated and benchmarked against stateof-the-art icd disease group prediction model. the experiments highlighted the promising results achieved by the proposed model, outperforming the state-ofthe-art model by % in terms of auroc and % in terms of auprc. the promising results achieved by the proposed model underscores its usefulness as an alternative to current cdss approaches, as it eliminates the dependency on structured clinical data, ensuring that hospitals in developing countries with low structured ehr adoption rate can also make use of effective cdss in their functioning. as part of future work, we plan to address the issues observed in the current data preparation strategy, and enhance it by sorting out the disease group assignment problems. we also intend to explore other techniques to further optimize topic and feature modeling of textual representations to study their effect on disease prediction. additionally, we intend to evaluate and compare other disease group prediction approaches on the patient cohort used in this study. furthermore, we also aim to perform a detailed efficiency analysis and comparison with respect to training times of the proposed approach and various baselines. finally, extensive benchmarking of approaches for modeling unstructured clinical notes on the physician notes, for further insights into the adapatability of the proposed approach in real-world scenarios will also be explored. prediction of length of stay for stroke patients tagging patient notes with icd- codes multi-label classification of patient notes: case study on icd code assignment automated diagnosis coding with combined text representations using electronic health record collected clinical variables to predict medical intensive care unit mortality doctor ai: predicting clinical events via recurrent neural networks automatic construction of rule-based icd- -cm coding systems farsight: long-term disease prediction using unstructured clinical nursing notes predicting icd- code groups with fuzzy similarity based supervised multi-label classification of unstructured clinical nursing notes. knowl.-based syst an interpretable icu mortality prediction model based on logistic regression and recurrent neural networks with lstm units multitask learning and benchmarking with clinical time series data an integrated machine learning framework for hospital readmission prediction. knowl.-based syst mimic-iii, a freely accessible critical care database risk prediction models for hospital readmission: a systematic review ontology-driven text feature modeling for disease prediction using unstructured radiological notes a novel ga-elm model for patient-specific mortality prediction over large-scale lab event data automated icd- coding via a deep learning approach efficient estimation of word representations in vector space deep patient: an unsupervised representation to predict the future of patients from the electronic health records overview of bionlp shared task benchmarking deep learning models on large healthcare datasets predicting hospital readmission for lupus patients: an rnn-lstm-based deep-learning methodology deepsofa: a continuous acuity score for critically ill patients using clinically interpretable deep learning optimizing intensive care capacity using individual length-of-stay prediction models a neural architecture for automated icd coding automatic icd- coding via deep transfer learning key: cord- -chwk bs authors: nan title: abstracts: poster session date: - - journal: ann neurol doi: . /ana. sha: doc_id: cord_uid: chwk bs nan an immune etiology has been postulated for acute cerebellar ataxia of childhood (acac) since it frequently follows viral infections. we analyzed serum and cerebrospinal fluid (csf) from acac patients for antibody cross-reacting with cerebellar neurons. serum and csf were obtained within days of onset of pancerebellar ataxia from subjects aged . to years. varicella infection preceded cases. results of enhanced cranial ct scans were normal; csf demonstrated - cells/mm with sterile cultures. serial dilutions from : of serum and undiluted csf were screened for antineuronal antibody by indirect immunofluorescence (iif) using frozen, unfixed normal human cerebellum. serum ( : ) was examined further for antineural antibody by western immunoblotting using purified cerebellar neuronal extracts as antigen. serum from age-matched, neurologically normal pediatric inpatients served as the control group for iif and immunoblot experiments. in acac patients, no antineuronal immunoreactivity was observed by iif. immunoblots demonstrated no consistent pattern of immunoreaction when comparing acac to controls, though patient exhibited distinct bands at kd (neurofilament protein) and kd. although antecedent infection suggests an immune etiology for acac, our preliminary results do not support a humoral mechanism for this disorder. ingrid taff; joseph zito, robert gould, and steven pavlakis, great neck and manhasset, ny in a -month period we studied patients between the ages of weeks and years with magnetic resonance angiography (mra). studies were performed on a . t magnet (siemens magnetom sp) with a circular polarized head coil. a three-dimensional time-of-flight technique was utilized. occasionally, images were obtained after gadopenetate dimeglumine infusion. two-dimensional projection images were calculated using a maximum intensity projection algorithm and recorded on laser film. sixty-seven patients also had routine mri. a sampling of vascular lesions was demonstrated. nineteen patients had clinical and mri evidence of stroke. mra revealed intracranial vascular occlusion in patients, diminished focal cerebral flow in the affected area in , and generalized ipsilateral underdeveloped cerebral circulation in . a moya-moya vascular pattern was found in and sickle-cell vasculopathy was found in patient. seven mras were normal. seventeen vascular hamartomas were demonstrated including vene of galen malformations, arteriovascular malformations, and venous angiomas. three aneurysms were found. thirty-one mras were normal. we find m u to be a valuable adjunct to routine mr imaging in the evaluation of pediatric patients with potential cerebrovascular disease. it demonstrates a spectrum of pathology, is noninvasive, and allows for serial follow-up examinations. angiography in pediatric cerebrovascular disease p . thalamic change in acute encephalopathy of adult rats. twelve weeks after grafting, clinical and histological studies were performed. we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. the survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin section. animals that received the transplants demonstrated qualitative and quantitative improvements in several parameters of locomotion. donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. we suggest embryonic rat spinal cord transplantation may be a useful treatment of spinal cord injury and a possible therapeutic strategy in human spinal cord injury and amyotrophic lateral sclerosis. the basic neuropathophysiology of hemineglect after unilateral cerebral lesions is still not clear. one theory holds that degraded perceptual processing occurs in the damaged hemisphere due to intrahemispheric deficits. another holds interhemispheric interaction at fault, with the intact hemisphere actively inhibiting spatial cognitive processes in the damaged one. we tested adult macaca fascicuhris with acute neglect on a task in which the whole visual surround was restricted to degrees from central fixation, and a second in which an opaque lens occluded the eye either ipsilateral (ipsi) or contralateral (contra) to the lesion. using paired t tests, in the first task there were no differences in reaction time to the ipsi and contralesional hemifields. in the second, there was no change in extent of the ipsilesional field (obtained with the contralesional eye occluded), as compared to its extent without occlusion. the contralesional field, however, improved significantly ( p < . ) with the ipsilesional eye occluded. since reducing sensory input to both hemispheres leads to no worsening of hemineglect, but reducing sensory input to the intact hemisphere alone leads to improvement of hemineglect, we conclude that adverse interhemispheric interactions play a major role in the pathophysiology of hemineglect. we assessed the sensitivity and applicability of a new, cornbined cognitive and mood screening battery for multiple sclerosis (ms). sixty consecutive, untreated clinically active ms patients, each relapsing-remitting and chronic progressive, underwent the battery and head mri upon entering concurrent treatment trials. the battery combines the faust-fogel brief cognitive screen and visual analogue dysphoria scale, both previously validated in other neurological diseases. cognitive domains tested were immediate and delayed sentence and word-pair recall, verbal fluency, and conflicting response suppression. patients marked ''usual mood" along a "happy-sad" cartoon continuum. relapsing patients were program and abstracts, american neurological association younger ( . vs . yr mean), with shorter ms durations ( . vs . yr), and had lower kurtzke disability scores ( . vs . ). modified qualitative mri grading (lesion burden, confluence, localization) was compared. half as many relapsing patients ( % vs %) scored ''abnormal'' cognitively, despite similar "sadness" rates ( % vs %). subjective dys-~ phoria in both groups correlated with denser periventricular lesion burdens. the battery was well tolerated and easily administered within minutes without special equipment. this combined cognitive and mood screening battery is sensitive and convenient for clinically active ms. alternate forms of the battery are needed for repeatability. questionnaires may be reliable and valid supplements to laboratory tests for brain-damaged patients, as they can be applied to situations for which laboratory testing is not possible. we investigated the usefulness of informant-based data in alzheimer's disease (ad) by comparing caregivers' subjective evaluations of probable a d patients' performance on an abbreviated version of the memory self-report questionnaire to objective evaluations derived from an extensive battery of neuropsychological tests and to clinicians' evaluations. similar information was obtained from healthy agematched controls. caregivers' subjective appraisals of patients' memory correlated significantly with objective measures of secondary memory, with all cognitive variables, measures of activities of daily living, and clinicians' evaluations of dementia staging. scores were independent of clinical indicators of depression. the abbreviated memory questionnaire showed good reliability, internal consistency, and external validity. its positive predictive value is . and its negative predictive value is close to %. results suggest that ( ) informant-based questionnaires may be useful for obtaining valid information on cognitive ability outside of laboratory settings; ( ) the scale reflected more than just memory functions; and ( ) the scale may be promising for screening cognitive difficulties in epidemiological or clinical settings. although neglect along the horizontal dimensions of extrapersonal space is well recognized, there are only a limited number of observations documenting neglect along the vertical and radial spatial dimensions. we report an investigation of neglect along the principal dimensions of extrapersonal space in a patient with bilateral mesial temporo-occipital infarctions. neglect was assessed by asking the patient and controls to bisect lines of lengths oriented in directions with respect to the body: horizontal, vertical, and radial. our patient showed significant neglect of upper vertical and far radial space, as well as neglect of left hemispace. his line bisection errors were consistently in a direction opposite the slight directional biases shown by controls for all line orientations ( p < . ). the magnitude of the patient's bisection errors increased by moving the lines toward the neglected sectors of -dimensional space. neglect of upper vertical and far radial space was also evident on line cancellation tasks. our results suggest that following focal brain injury, neglect may be observed along all dimensions of extrapersonal space. these findings provide further empirical support for functional specialization within inferior and mesial temporooccipital regions for attending to upper vertical and far visual space (previc, ) . p . posterior cortical atrophy: degenerative disease with primary visuospatial and visuosemantic deficits a. kertesz, m . polk, and a. kirk, london, ontario, and saskatoon, saskatchewan, canada posterior cortical atrophy is a recent, and heidenhahn's disease is an old, label for a miscellaneous group of patients with imaging or pathological and clinical evidence of visuocognitive deficits and cortical atrophy localized to the posterior cortex. the extent of this cortical localization and the nature of the pathological findings are not fully agreed upon, but spongiform degeneration and alzheimer pathology have been described. detailed examination of patients who are representative of the problem and have uniquely specific deficits is presented. one patient had visual associative agnosia, prosopagnosia, and transcortical sensory aphasia. lexicosemantic experiments of categorization, word retrieval, and comprehension of auditory and visual stimuli showed a specific impairment of visuoverbal semantics. a striking preservation of phonological, orthographic and visual structural input, and intercategory dissociations was demonstrated. consistency of errors argued for specific loss of semantic knowledge. another patient with apraxia, primary visuospatial deficit, agraphia, and amnesia at the beginning had predominantly right-sided posterior cortical atrophy, demonstrating further fractionation of the entity and the striking specificity of visuospatial function. the behavioral specification of degenerative disease is clinically and theoretically important. permanent neurological deficits after ischemic stroke are mainly determined by the location and size of the infarct. clinical recovery also depends on the functional state of adjacent brain tissue, where both neuronal loss and deactivation without gross morphological damage may affect flow and metabolism to a varying degree (g. mies et al, stroke ; - ) , and where the ability to respond to stimulation by appropriate neuronal recruitment may be impaired. therefore, degree of resting hypometabolism and of responsiveness to functional activation may provide a measure of prognosis. in patients (age . . yr) with aphasia consequent to ischemic stroke of the dominant hemisphere, regional cerebral metabolic rate of glucose (rcmrgi) was measured at rest and in of them also during spontaneous speech, using positron emission tomography (pet) of -(f )-fluoro- -deoxy-~-glucose (fdg). the pet study and a standardized neuropsychological test battery to assess the main aspects of language were performed around the fourteenth day after the stroke, and the language functions were assessed again to months later. performances in various dimensions of language weeks and to months after stroke were related to rcmrgl in topographically meaningful areas at rest and during activation using wilcoxon-rank program and abstracts, american neurological association sum test and multiple regression analysis. severity of aphasia was assessed by the token test, which showed a bimodal distribution to slight and severe, and a lower representation of moderate cases. global and all regional cmrgl at rest and during activation were significantly correlated to scores in token test at first and second examination, with the highest correlation coefficients ( - . to - . ) for broca's, wernicke's, and left temporoparietal regions. for performance after to months, the relationships were still significant with lower coefficients. verbal fluency also was correlated to kmrgi, but with lower coefficients that slightly increased for the recovery state. language performance at different stages in the course after ischemic stroke was significantly related (r = . for token test, r = . for verbal fluency). however, there exists a high variability in recovery that may be explained by stepwise regression of metabolic values. significant effects were observed only for cmrgl of the left hemisphere outside the infarct (partial r' = . ) at rest and for cmrgl within the infarct ( . ), the contralat-era mirror region ( . ), and broca's region ( . ) during activation, with a sum of all partial weight factors of . at rest and . during activation. our results furnish indicators for recovery of aphasia: the resting metabolism of the left hemisphere outside the infarct, and the activated metabolism in residual tissue within the infarct and in languagerelated areas. although the hemispheric metabolism at rest might be related to neuronal loss and thereby to the brain's reserve capacity, the extent of metabolic activation indicates neuronal recruitment and the capability of neuronal networks for functional recovery. heparin therapy for acute myocardial infarction: the timi-i pilot and randomized trial combined experience m . a. sloan, t . r. price, m . l. tevrin, and s. forman for the timi investigators, baltimore, m d of , myocardial infarction (mi) patients treated with rt-pa and heparin, ( . %) developed ischemic cerebral infarcts (ci). all ci patients had detailed neurological evaluations and ( %) had c t scans. age range was to years (mean yr), were male, and were caucasian. electrocardiographic location of mi was anterior in ( %) and nonanterior in ( %). six cis occurred within hours; between and hours; between and hours; between and hours; during the second week; and others distributed over the weeks after study entry. six of cis did not involve cerebral cortex; ( %) had multiple cis. of cis thought to be embolic in origin, had at least cardiac abnormality (mural clot, wall motion abnormality, aneurysm, or transient atrial fibrillation) known to be associated more specifically with embolism than just the diagnosis of myocardial infarction. eight of ( %) with ct scans had hemorrhagic conversion of varying degrees. the time of occurrence and sites of ci after rt-pa and heparin therapy for acute mi are similar to those reported in the prethrombolytic area. nancy futrell andjeanne m. riddle, detroit, m i photochemical irradiation of the carotid artery of rats has been used to induce endothelial damage, producing a nonocc h i v e thrombus (that apparently embolizes spontaneously) thrombi and emboli and multiple cerebral infarcts. evidence for embolism generally has a presumptive component. to document further that cerebral infarcts in this model are indeed due to embolism, we studied the ultrastructure of the carotid thrombi and the presumed cerebral emboli using scanning and transmission electron microscopy (sem, tem). the right carotid artery of wistar rats was irradiated with a laser ( nm, mw/cm , min) following the injection of the photosensitizing dye photofrin , . mgikg. rats were sacrificed from to hours later. endothelial damage with formation of a fragmenting thrombus, composed mainly of platelets and erythrocytes (with no fibrin in most areas), was present in the carotid arteries of all rats by sem. sem was done on cerebral vessels, containing peripheral blood elements, with single ( ) and aggregated ( ) platelets (causing occlusion in ), single ( ) and aggregated ( ) erythrocytes (without occlusion), and single ( ) and aggregated ( ) leukocytes (without occlusion). tem demonstrated that the platelet aggregates did not adhere to the cerebral endothelium. the endothelial surface of all cerebral vessels was normal, which provided additional evidence that the mechanism of cerebral infarction in this model is embolism. model of repetitive ischemia: this effect is significantly enhanced when combined with mild hypothermia ashfaq shuaib and elisabeth sechocka, saskatoon, saskatchewan. canada there is considerable evidence that glutamate release resulting in activation of postsynaptic receptors (especially nmethyhaspartate) is a major mechanism of ischemic neuronal injury. in vivo experiments have shown that a more severe release of glutamate may be responsible for the excessive damage seen with repeated ischemic insults. we have shown that in cell cultures the effect of brief repeated insults is more severe than a single insult of similar duration. in the present study, we tested the protective effects of cgs- in a cell culture model of single ischemic and multiple-insult paradigm. in the multiple-insult paradigm, in some cultures cgs- was combined with mild hypothermia to see if this would offer additional protection. cgs- offered a dose-dependent protection in cell cultures exposed to a single ischemic insult. cgs- was protective to cultures exposed to repeated ischemic insults. the protective effects were enhanced significantly when they were combined with hypothermia, resulting in almost complete protection of the cultures. the combination of therapies appears to be a valuable strategy in neuronal protection during cerebral ischemia. toby i. gropen, i . prohovnik, t . k. tatemirhi, z. sharif; and m. hirano, new york, n y although a rare syndrome, mitochondria encephalomyopathy, lactic acidosis, and stroke (melas) may offer a unique insight into stroke mechanisms. we report novel observations in a patient with melas studied with serial and quantitative cerebral perfusion after stroke using """tc-ceretec spect and ' ixe rcbf. a -year-old man with melas presented with left-sided headache, generalized seizures, fluent aphasia, and right hemianopia. serial ct and mri showed infarction of the posterior left hemisphere in a multiterritorial distribution. spect performed days after stroke showed to % greater flow in the infarct than in normal brain, which reversed days after stroke. quantitative rcbf (m isi, reflecting mostly gray matter), when corpatients died. we recommend ct evaluation in all patients who have a seizure or lose consciousness during the peripar-tum period. despite intensive management, mortality is high. seizures should not be attributed to eclampsia without careful neurological assessment. when prenatal care is sought, women should be counseled about the dangers of cocaine to themselves as well as to their babies. changes in circulating blood volume following stephan a. mayer, matthew e. fink, laura lenniban, louise m. klebanoff; auis beckford, lsak prohounik, william young, and robert a. solomon, new york, n y reduction of blood volume (bv) has been implicated as a risk factor for delayed cerebral ischemia (dci) due to vasospasm after aneurysmal subarachnoid hemorrhage (sah). volume expansion guided by target filling pressures has gained popularity as a means of preventing or reversing dci; however, the adequacy of central venous pressure (cvp) as a reflection of bv in this setting remains unclear. we measured bv and cvp concurrently in patients ( males, females; mean age yr) day after craniotomy (mean . days after sah) and an average of . days later. the mean bv (mllkg) measured using chromium '-labeled red blood cells (rbcs) fell from . to . (normal range - ), a reduction of % ( p = . , paired student's t test). despite this, mean cvp (mm hg) remained unchanged ( . vs . ). similar reductions of plasma volume ( %) and rbc volume ( %) accounted for no change in mean hematocrit ( . vs . ). bv fell . % among grade iiiliv patients (n = ) compared to . % among grade / patients (n = ). a moderate correlation between bv and cvp ( r = . , p = . ) was found only with the first set of measurements. time-related alterations in venous capacitance, myocardial contractility, or systemic vascular resistance may explain our findings. axel rosengart, louis r. caplan, michael s. pessin, atherostenosis of the extracranial vertebral artery (ec-va) has rarely been studied systematically in series of patients with acute vertebrobasilar strokes or transient ischemic attacks. we identified by conventional angiography and neuroimaging (ct, mri, ultrasound, mr angiography) patients with ec-va disease among patients with posterior circulation ischemia. patients with cardiac sources of emboli were excluded. the probable etiologic mechanisms were: group a: vertebral artery origin (vao) atherostenosis with embolism- patients; group b: vao atherostenosis with hemodynamic spells- patients; group c: patient with vao atherostenosis and both intra-arterial embolism and hernodynamic spells; group d: ec-va dissection- patients ( unilateral, bilateral); patient had perioperative compromise of the ec-va with presumed intra-arterial embolism; group e: vao disease in addition to other distal vascular lesions- patients ( with intracranial va and with basilar artery [baf occlusive disease). ec-va disease is not always benign. vao atherostenosis and dissection of the ec-va are sources of intra-arterial emboli. hemodynamicrelated ischemia occurs with bilateral and unilateral va disease but is often transient. vao atherostenosis is often accompanied by severe occlusive disease of the intracranial va and ba. program and abstracts, american neurological association sebastian e. ameriso, vicky l. y. wong, andvas gruber, hidemi ishii, and mark fisher, los angeles and la jolla, c a , and kanagawa, japan hemostasis abnormalities are associated with ischemic stroke. these changes typically are demonstrated in antecubital venous blood samples and may not necessarily represent changes within the vasculature of the brain. the purpose of this study was to identify potential differences in hemostatic profile from samples of cranial versus noncranial venous sites in patients with acute ischemic stroke. eight patients were studied within days of acute brain infarction. some patients were studied on separate days. blood was drawn from the external jugular vein and immediately thereafter from an antecubital vein without the use of tourniquet. we measured hematocrit, leukocyte count, platelet count, fibrin d-dimer (cross-linked fibrin fragment), plasminogen activator inhibitor- (pai- , an important antifibrinolytic protein), and anticoagulant proteins thrombomodulin and activated protein c. a jugular-to-antecubital ratio was calculated for each paired blood sampling. thirteen paired samples were obtained from the eight patients. external jugular-to-antecubital ratios (mean to-antecubitat ratio for pal was significantly different from ( p < . ), with higher concentrations in jugular samples. in conclusion, levels of hemostatic proteins measured from cranial venous blood may differ from antecubital samples in patients with acute ischemic stroke. in animal models of transient cerebral ischemia, the effects of repetitive insults are more severe than a single ischemic episode of similar duration. we used the cell culture model of ischemia to determine if the effects of repetitive ischemia are similarly more severe in this model of ischemia. for cell culture, we used fetal mice cortical astrocytic and postnatal cerebellar (glutamatergic) granular neurons and cerebral gamma-aminobutyric acid (gaba)ergic cells. lactic dehydrogenase (ldh) (activity per gram protein) release in the medium was used as a measure of cellular damage. compared to a single insult, there was a large increase in ldh release during repetitive ischemia in astrocytes ( vs , p < . ) and granular cells ( vs , p < . ) (highly significant) and a modest (but significant) increase in the cortical neurons ( . vs . p = . ). the demonstration that repetitive ischemia produces more severe damage in cell culture would suggest that the mechanisms are not predominantly vascular. cell culture could prove useful to study the mechanisms of neuronal damage with repetitive ischemia. we studied the spontaneous recovery of neurological function after acute ischemic stroke using a standardized stroke nih stroke scale scale ( n i h stroke scale) to assess the extent of improvement, differences in stroke types, and early predictors of later outcome. we performed serial neurological assessments on admission; , , and hours after admission; and to days and > days after admission. twenty-six patients had presumed embolic occlusion of the middle cerebral artery (mca) and had a clinical diagnosis of lacune. admission score was better in the lacune group compared to the mca group. the mean scores for all patients improved by the to -day and the > -day examination, but the degree of improvement was greater in the mca group than in the lacune group at > days ( p < . ). the degree of change at to days correlated with the change in score at hours ( r = . , p < . ) and hours ( r = . , p < . ). most patients improve after acute ischemic stroke, but to variable degrees and at different rates. david w. desmond, thomas k. tatemichi, miguel figueroa, dew'itt t . cross, and yaakov stern, new yovk, n y to investigate the effects of lacunar infarction (li) on cognitive function, we examined li patients months after stroke (age = . ? . yr; education = . . yr) and stroke-free nondemented control subjects (age = . k . yr; education = . k . yr) with a battery of neuropsychological tests. li was defined as a presenting infarct of cc and a mean volume of any additional subcortical infarctions of cc on ct scan. using multiple regression analyses, with significance set at p < . to minimize the risk of type i error, we considered the role of li as a correlate of performance in multiple cognitive domains. controlling for the effects of demographic factors, vascular risk factors, alcohol use, and depression within the multivariate models, li was a significant independent correlate of deficits in memory (( = -. , p = . ), verbal (p = -. , p = . ), visuospatial (p = -. , p < .oool), abstract reasoning (p = -. , p = . ), and attentional skills (p = -. , p < . ). we further investigated the effects of infarct number, volume, and location, as well as atrophy, on global cognitive function within the li group. the only significant independent correlate of global cognitive performance was a preponderance of left-hemisphere infarctions (p = -. , p = , ). these results suggest that li may produce dysfunction in multiple cognitive domains, particularly when the left hemisphere is differentially involved. p . increased intracranial atherosclerotic stroke in hispanics and blacks from northern manhattan ralph l. sacco, christina zamanillo, t . shi, andj. p. mobr, new york, ny intracranial atherosclerosis has been found to be more frequent in blacks compared to whites, whereas hispanics have rarely been characterized. among consecutive patients from northern manhattan over age hospitalized at the presbyterian hospital from to , cerebral infarction occurred in whites, blacks, and hispanics. all patients had at least one c t scan, % had duplex doppler, % transcranial doppler, and % angiography. strokes were classified as atherosclerotic (ath), cardioembolism, lacunar, and as infarcts of undetermined cause. ath was further subdivided into extracranial (eath) or intracranial (iath) . overall, the frequency of ath was similar in the three racelethnic groups (white , black ' , hispanic ) . the distribution of the atherosclerosis, however, was different in whites compared to blacks and hispanics. whites had more eath stroke than blacks and hispanics (white %, black % , hispanic lo%), while iath was similar in blacks and hispanics and greater than in whites (white , black , hispanic %). nonwhites have more iath stroke than whites. the similarity in the distribution of atherosclerosis between blacks and hispanics argues for shared environmental risk factors, rather than genetic differences. ethnic differences in stroke risk factors may help explain differences in infarct subtype. we studied mild to moderate alzheimer's disease (ad) patients with a series of " water bolus positron emission tomographic (pet) activation studies, and compared them to similar studies in age-matched normal controls. for each group, pet images were mapped onto the subjects' mri scan, and results of a particular activation condition were averaged across the group. naming a series of pictures (line drawings of animals) minus counting abstract designs as a baseline produced strong activation of the anterior cingulate gyrus only in the ad group. silent reading of words minus viewing a baseline series of "xs" similarly showed strong activation of the anterior cingulate gyms in the ad subjects but not the normals. naming block (activation condition) of % unnamed pictures, minus a second block (baseline) of easily named pictures, demonstrated much greater cingulate activation in the ad patients, for naming of the more difficult pictures. we conclude that this cingulate activation may reflect the greater involvement of an attentional network (of which the anterior cingulate is a part) in tasks requiring a higher degree of "mental work" on the part of ad patients. dementia in alzheimer's disease j. w. pettegrew, k. panchalingam, w. e. klunk, and r. j alzheimer's disease (ad) predominantly affects the brain, resulting in the loss of multiple cognitive abilities. some studies suggest the membranes of peripheral cells are involved in the disease. to investigate erythrocyte membrane molecular dynamics in ad patients and age-matched controls, we investigated erythrocyte membrane molecular motion at the surface (fluorescamine), aqueous-hydrocarbon interface (dppe-ans), and hydrocarbon core ( j-as; ppc-dph) by steady-state fluorescence anisotropy measurements of probable ad patients ( males; females) and ( males; females) age-matched controls. cognitive function was assessed by the mini-mental, mattis, and blessed scales. we found that intergroup comparisons revealed decreased motion at the surface ( p = . ) and aqueous-hydrocarbon interface ( p = . ) and increased motion in the hydrocarbon core ( p = . ) of the moderately to severely impaired ad patients compared to the controls. in the ad patients, there were significant correlations between decreasing membrane surface motion and worsening blessed scores (males p = . ; r = . ; femalesp = . ; r = . ). these findings suggest that molecules are being produced in the brain of ad patients that gain access to the circulation. these molecules insert into the erythrocyte membrane and secondarily alter erythrocyte membrane molecular motion. the production of these molecules correlates with the dementia and could contribute to the molecular pathophysiology of the disease. parkinson's disease (pd) and alzheimer's disease (ad) are common disorders of old age and may therefore coexist. the prognosis in demented pd patients is poor and early recognition of such cases is therefore desirable. the objective of this study was to identify characteristics that distinguish pd + ad from pd patients during early stage. all patients were clinically evaluated over a -year period . clinical diagnosis of dementia was made only when unequivocal clinical evidence of progressive decline in memory and cognitive function was documented, and pathological diagnosis of ad and pd was made using standard criteria. twentysix patients who had only pd or pd + ad were identified; had no dementia and at autopsy had pd. six patients had clinical evidence of parkinsonism and dementia and at autopsy had distinct pathological findings-pd and ad. these cases could be classified as having simultaneous or sequential evolution of pd + ad. those with sequential onset had pd before age years but were inexplicably functionally disabled early on, whereas those with simultaneous onset manifested pd after age years. pd + ad patients had rapid disease progression, shorter survival, poorer drug response, and more side effects of levodopa than pd patients. to study the prevalence ofwhite matter lesions in the general elderly population, and to investigate whether white matter lesions were relatively frequent in subjects with classic vascular risk factors and with hemostatic risk factors, magnetic resonance scans were obtained of participants, aged to years, of the rotterdam elderly study. the subjects for the imaging study were a random sample from the general population, stratified by age and gender. t -weighted images were obtained in the axial plane. white matter lesions were considered present when moderate or severe periventricular hyperintensities or when more than small focal lesions or focal confluent lesions were found. overall, % of subjects had white matter lesions. the prevalence and severity of le-program and abstracts, american neurological association sions increased with age. history of stroke or myocardial infarction, presence of peripheral arterial disease, factor viic activity, and fibrinogen level were each significantly and independently associated with the presence of white matter lesions. significant relations with actual systolic as well as diastolic blood pressure, with a history of hypertension, and with plasma cholesterol were observed only for subjects between and years. this study suggests that white matter lesions in the elderly may be related not only to the classic cardiovascular risk factors. but also to hemostatic factors. joan m. swearer, paula nelligan, hanno muelher, beatrice woodward, and david drachman, worcester, ma although behavioral disturbances occur frequently in alzheimer's disease and other dementing disorders, little is known about the factors that predict their development or predispose to their occurrence. in the present study we examined sets of possible predictive/predisposing factors retrospectively for behavioral disturbances in mildly to severely demented, community-dwelling patients. the factors examined included: individual distinguishing features (age, gender, age of onset, premorbid personality traits, prior psychiatric history) and dementia severity (dependence in activities of daily living [adls) and self-care, duration of dementia, global disease severity). spearman correlations and t tests were used to assess the relative influence of these factors on the occurrence of types of aberrant behaviors: aggressive behaviors, disordered ideation, and motor abnormalities. forty percent of the patients exhibited aggressive behaviors, % exhibited disordered ideation, and % had motor abnormalities. neither a prior history of psychiatric disorders nor premorbid personality traits were associated with the occurrence of the target behaviors. dependence in adls and self-care and greater global severity were associated ( p < . ) with the frequency and severity of aggressive behaviors, disordered ideation, and motor abnormalities. these results suggest that severity of dementia is a consistent and reliable factor in the development of aberrant behaviors, whereas preexisting personality traits are not. dementia of the alzheimer t y p e w. j. burke, a. ranno, w. h . roccafrte, s. p. wengel. b. l. bayer, and n . k. willcockson, omaha, ne l-deprenyl is an irreversible inhibitor of mao-b that has been reported to cause modest improvements in short-term memory and behavioral symptoms in persons with dementia of the alzheimer type (dat). thirty-eight subjects meeting research criteria for mild d a t were enrolled in a placebo-controlled, double-blind trial of r-deprenyl at a dose of mg twice a day. subjects underwent extensive clinical and neuropsychological assessments at entry, and at and months. after months, subjects taking both l-deprenyl and placebo showed a significant decline in their scores on the mini-mental state examination, the clinical dementia rating (cdr) scale, and the sum-of-boxes score derived from the cdr. when the change in scores on these clinical measures was examined across the groups, there was no significant difference. there were no significant differences within or between groups on several behavioral measures including the brief psychiatric rating scale and the cornell rating scale for depression in dementia. neuropsychological testing demonstrated no significant differences berween groups based on mean score change. l-deprenyl did not affect cognition or behavioral symptoms of dat in this -month study. k. marder, m-x. tang, r. ottman, l. cote, y. stern, and r. mayeux, new york, n y the etiology of dementia in parkinson's disease (pd) is probably multifactorial but there may be a shared susceptibility for p d and alzheimer's disease (ad). reliable risk factor interviews were conducted with informants of nondemented p d patients (pd-d) and demented p d patients (pd + d) enrolled in a longitudinal community study of pd. p d + d were older ( . yr) than pd-d ( . yr) and had later age at onset of motor signs ( . yr) than pd-d ( . yr) ( p < , ). the frequency of smoking, alcohol use, head injury, and family history (fh) of pd did not differ but fh of ad was significantly more frequent in the pd + d group (or . , ci . - . ). using stepwise logistic regression, only age of onset of motor signs (or . ), education < years (or . ), and the interaction of age of onset of motor signs and fh of a d (or . ) were independent predictors of dementia in pd. to address variable years at risk for development of dementia, life table analysis revealed the cumulative risk of a d to age in first-degree relatives of p d + d was . , and . in pd-d relatives ( p < . ). cox proportional hazards analysis controlling for the differences in ages of the relatives of both groups yielded a rate ratio of . (ci . - . ) for the development of a d among p d + d compared to pd-d relatives. we conclude that a genetic susceptibility to a d may raise the risk for dementia in patients with pd. differentiated from alzheimer's disease? john c. mowis, elizabeth grant, rita canfield, eugene rubin, and daniel mckeel, jr, st louis. mo vascular dementia (vd) is believed to account for to % of all us cases of dementia; however, pathologically confirmed cases are quite rare. this discrepancy suggests that current diagnostic criteria lead to the clinical overdiagnosis of vd. twenty v d subjects (mean age . yr; men, women) were diagnosed solely on the basis of the presence of dementia, a history of stroke(s), and a documented relationship of stroke to onset andlor course of dementia; ischemic scores (is) and neuroradiographic findings were not used for diagnosis. compared with subjects (mean age . yr; men, women) with dementia of the alzheimer type (dat), there were no significant group differences for comparable clinical dementia rating stages of dementia for measures of language, activities of daily living, or general cognition. the vd group scored significantly higher than the dat group on the modified is (f [ , ] = . , p < , ). all autopsied d a t subjects had verified alzheimer's disease (ad); also had cerebral infarctions. the autopsied v d subjects had , , and cc of brain tissue affected by stroke; ( cc) also satisfied histological criteria for ad. we conclude that ( ) the clinical features of vd and a d overlap considerably; ( ) diagnostic criteria based on the temporal association of stroke with dementia may have predictive value for vd; and ( ) the frequent coexistence of a d and strokes indicates that refinement of criteria is needed to distinguish "mixed" and "pure" vd. clinicopathological correlation remains essential for any study of putative vd. left-handedness has been proposed as a marker for decreased survival in the general population, but possible effects of handedness on longevity in alzheimer's disease (ad) have not been examined. we hypothesized that left-handed ad patients would evince more rapid deterioration and therefore die at an earlier age than right-handed patients. subjects were demented patients consecutively confirmed at autopsy to meet nincds-adrda criteria for "definite" ad. handedness was determined from structured interviews with primary caregivers and validated for most subjects with the edinburgh inventory of handedness. age at onset of dementia symptoms retrospectively determined by caregivers was used to calculate the duration of illness at the time of death. because of reported gender differences with regard to longevity, we first partialled out effects of gender before using hierarchical regression procedures to test the hypothesis. four of men and of women with definite ad were left-handed. the mean age at onset did not differ significantly between handedness groups (f [ l,loo] = . ), but the mean duration of symptoms ( alterations in the optical properties of brain can be used to detect pathological changes in patients with alzheimer's disease (ad). using time-resolved spectroscopy (trs) and phase-modulation spectroscopy (pms), we measured the absorption (ua) coefficient, scattering (us) coefficient, and mean photon pathlength (pl) of red light directed through the base of the frontal lobes of patients with ad and age-matched control subjects. the measured values and the asymmetry index (ai) (an indication of the symmetry of the measurements between the left and right side of the brain) were correlated with the severity of disease as determined by mini-mental state score. there were significant differences between the ad and control group for ua, us, pl, and the standard deviation of ai. there was no correlation between the mms score and ua, us, or pl. however, the highest asymmetry index values were seen in moderately impaired patients , which suggests that the asymmetrical nature of the pathological process detected by optical spectroscopy is most marked during this stage of the illness. this noninvasive technique may provide a convenient method to detect and monitor the pathological changes that occur in the brain of patients with ad. disease p . memory impairment in very mild alzheimer's a memory impairment is often the earliest indication of alzheimer's disease (ad). we investigated components of disease learning and recall to determine which aspect of memory function is impaired the earliest in incipient ad. using the mayo clinic alzheimer's disease patient registry, which is a longitudinal prospective project on ad and normal aging, we identified patients with very mild ad (i.e., with a mini-mental state score of or greater) and age-and sex-matched controls. we assessed performance on memory measures: the rey auditory verbal learning test and the buschke free and cued selective reminding test (fcsrt). the parameters evaluated included a measure of acquisition, total learning over trials (tl), and delayed recall (dr). on the fcsrt, an index of facilitation of performance with semantic cues (sc) was assessed. results indicated that all indices, tl., dr, and sc, were capable of separating the mild ad group from the controls ( p < , ). using a linear discriminant analysis with stepwise variable entry, the measure that assessed the patient's ability to use semantic cues (sc) was the most sensitive parameter for separating the groups ( f = . , p < . ), and the acquisition parameter (tl) was also useful at adding some additional predictive power (f = . , p < . ). the delayed recall measure, however, did not add anything to the previous measures. it appears that very early ad can be detected using appropriately structured memory tasks, and these procedures can be helpful in identifying at-risk individuals. alzheimer's disease (ad) has an insidious onset that is difficult to date reliably. we developed a standardized interview to provide objective criteria for dating the onset of different symptoms (memory complainr, performance problems, language deficits, disorientation, depression, behavior problems, and psychosis), yielding an estimated disease onset date. inrerrater reliability (icc = . ;p < , ) and interinformant reliability (icc = ; p < . ) for the onset of first symptom was high. interrater agreement for the order in which symptoms appeared was high (icc = . - . ) as was interinformant reliability for all symptoms except memory complaint. the interview was administered to patients with ad. mean estimate duration of illness was . years k . years and correlated significanrly with problems in instrumental activities of daily living. sixty-six percent had memory complaint and % had performance problems as their initial symptom. this technique provides a reliable characterization of disease onset. longitudinal studies will determine if particular onset symptoms differentially predict disease progression. the purpose of this study was to determine whether there is an excess of white matter disease (wmd) in alzheimer's disease (ad). brun and englund ( ) reported an excess of wmd in brains of patients with ad vs age-matched controls. there have been reports both confirming (bowen et al, ; fazekas et al, ) and refuting (leys, ) these findings using ct and mri in patients with clinically diagnosed ad. postmortem t -weighted mri scans and program and abstracts, american neurological association neuropathology were graded on brains of pathologically confirmed ad subjects and brains of age-matched neuropathologically normal controls. white matter lesions were scored on a to scale (none, mild, moderate, severe) separately for periventricular (pvl) and deep white matter (dwm) areas in mri scans and lux fast blue (lfb)-stained brain sections. correlations between mri and neuropathology were good ( r = . for pvl, r = . for dwm). pvl scores were higher in ad than in normal subjects on mri (ad: . l . vs controls: . rfr . ; p < . ). on pathology the difference in scores did not reach significance (ad: . . vs controls: . * . ; p = . ). similarly, dwm scores were higher in ad subjects than normals on mri, but not neuropathology. in conclusion, ad brains have a significant excess of wmd on mri compared to controls. although the pvl and dwm scores for pathological sections are not different in the groups, mri is much more sensitive than lfb-stained sections for wmd. thirteen autopsy cases of progressive supranuclear palsy (psp) were investigated for clinical-neuropathological correlations and heterogeneity. we reviewed clinical records of men and women aged to years (mean age yr) with disease duration ranging from to years. most patients had classic features of psp including ophthalmoplegia, postural instability, and extrapyramidal signs. dementia was eventually observed in of the patients ( %). six of patients ( %) on whom adequate initial documentation was available presented with memory loss or behavior change. five of the patients ( %), including with an initial presentation of memory loss, were diagnosed clinically as having alzheimer's disease (ad) rather than psp; neuropathological diagnoses in these cases varied: i had combined ad-psp; had ad-psp combined with parkinson's disease (pd) changes; had psp-pd; and had "pure" psp. the patients with concomitant pd changes showed lewy bodies in the substantia nigra, locus coeruleus, nucleus basalis, and neocortex. the remaining patients were clinically diagnosed as having psp; neuropathological diagnoses in these cases included with "pure" psp and with psp that also met neuropathological criteria for ad (psp-ad). these findings emphasize the clinical and neuropathological heterogeneity in psp. the neuropsychological battery developed for the consortium to establish a registry for alzheimer's disease (cerad) is currently used in many research studies to index the cognitive impairments of alzheimer's disease. in spite of its widespread use, normative informarion on the battery, important for interpretation of performance, has not been available. we report norms for the cerad battery based on a large sample of elderly control subjects (n = ; white men and women; ages - yr) enrolled in the national study of cerad. performance on the neuropsychological measures was examined separately for subjects with high ( yr) and low (< yr) education. distribution of scores and basic descriptive information (means and sd) for each measure were determined. significant age and sex effects were observed on most cognitive measures in the highly educated group. in contrast, no significant age effects were observed in the low education group. effect of sex was not explored in this group due to the limited sample size (n = ). further exploration of cerad performance in normal controls from underrepresented groups including minorities, residents of rural communities, and individuals with low education is in progress. intraneuronal inclusions of cytoskeletal proteins appear in several neurological diseases; for example, the neurofibrillary tangles of alzheimer's disease contain a cytoskeletal protein, tau. because the previously described slowing of axonal transport in aged animals might lead to accumulation of cytoskeletal proteins in nerve-cell bodies and axons, we assessed the abundance of major cytoskeletal proteins in brain tracts of rats at age months or months. immunoassay was performed with monoclonal antibodies to alpha and beta tubulin and to nf-l (the core neurofilament protein) by published methods. samples were dissected in a standardized fashion and -mrn pieces of the following tracts were assayed: optic nerve, corticospinal tract (medulla), superior cerebellar peduncle, l dorsal root, and l ventral root. between months and months, the nf-l content approximately doubled in each brain site. tubulin substantially increased at of aged rats all sites except the fimbria-fornix. in contrast, tubulin did not change in the spinal roots. nf-l increased slightly in the ventral but not the dorsal root. this tendency of senescent brain neurons to accumulate cytoskeletal proteins in their axoplasm may predispose them to formation of intraneuronal inclusions in various degenerative diseases. we performed a prospective study of preoperative magnetic resonance imaging (mri) in consecutive patients with intractable partial epilepsy who underwent a stereotactic resection of an extrahippocampal temporal lobe foreign-tissue lesion, "lesionectomy," between june and january . interpretation of the mri studies was performed by an investigator blinded to the presurgical evaluation, surgical outcome, and pathology. hippocampal formation (hf) atrophy was assessed using mri-based volumetry (n = ) and visual grading of the h f (n = ). mri-detected hf atrophy has been shown to be a reliable marker of moderate to severe mesial temporal sclerosis (mts) (cascino gd, et al, ann neurol ; : - evidence from experimental animals indicates that endogenously produced platelet-derived growth factor (pdgf) is an important regulator of glial proliferation and differentiation. because of the striking degree of glial proliferation in chronic epilepsy, we sought to determine whether cultured glia from human epilepsy tissue would be responsive to pdgf. the effects of pdgf on dna synthesis, proliferation, and relative distribution of a b (+) glia were studied in a cell culture derived from temporal lobe white matter of adult epilepsy lobectomy tissue. by immunocytochemistry, glial fibrillary acidic protein (gfap) was detectable in % of cells in untreated or -day pdgf-treated ( ng/ml) cultures, which confirmed their astrocytic nature. in contrast, a b ( +) cells increased from to % in untreated cultures to % after pdgf treatment, which suggested that type astrocytes (a b [ + , gfap[ +]) had been elicited. dna synthesis of cells resembling oligodendrocyte-type astrocyte ( - a) progenitors occurred within hours after program and abstracts, american neurological association pdgf treatment as evidenced by nuclear incorporation of brdu in bipolar a b ( +) cells. these studies demonstrate that expansion of adult human gfap( +) astrocyte populations is sensitive to regulation by pdgf. further, these data imply the existence of pdgf-responsive - a progenitor cells in astrocyte-rich cultures derived from human epilepsy tissue. ( we have recorded vagal and esophageal-evoked potentials after electrical (e) and balloon (b) stimulation in epileptic patients who had vagal stimulators for the control of intractable epilepsy and the results were compared with esophagealevoked potentials in healthy controls and diabetic patients. the vagal and esophageal-evoked potentials showed similar configurations with major positive and negative potentials. the amplitudes of the responses habituated rapidly over trials at per second up to per seconds. latencies were shorter from the upper esophagus ( cm above lower esophageal sphinctereles]) cf. lower esophagus ( cm above les) yielding conduction velocities of to meters per second but conduction was significantly slower in the diabetics. the vagal-evoked potentials have validated the use of esophageal-evoked potentials as a practical method of assessment of the integrity and speed of conduction in vagal afferent pathways in man. ronald e. kramer and neil l. rosenberg, englewood. co seizure disorders were analyzed in patients in whom toluene was the sole or major drug of abuse. toluene abuse is increasing; therefore, physicians should gain experience with its neuropathological and ciinical sequelae. a retrospective chart review found patients meeting criteria. the average patient age was years; abuse onset averaged . years; abuse averaged . years; and patients were male. ten were daily, were weekly, and were intermittent users. seizures occurred in . one suffered a single generalized tonic-clonic seizure without recurrence and without treatment. his we characterized the clinical dose-response curves for relief of parkinsonism and production of dyskinesias as a function of plasma levodopa and - -methyldopa levels in patients with parkinson's disease (pd) and fluctuating responses to oral levodopa/carbidopa. dose response to graded intravenous levodopa was measured after overnight drug withdrawal on occasions, first after chronic, intermittent oral levodopa/ carbidopa and second after to days of continuous intravenous levodopa. continuous intravenous levodopa shifted the dyskinesia dose-response curve to the right, and reduced maximum dyskinesia activity, but did not significantly alter dose response for relief of parkinsonism. improvement in dyskinesia was apparent by the second day of continuous levodopa, during which ratios of plasma dopa/ - -methyldopa remained constant. our results support the hypothesis that relief of parkinsonism and production of dyskinesias occur by separate mechanisms. continuous dopamine-mimetic therapy should be sought as a therapeutic goal for advanced pd. dopa-responsive dystonia (drd) is a distinct subset of idiopathic dystonia with diurnal fl uctuation and a dramatically beneficial response to l-dopa. it has hitherto been considered an autosomal-dominant disease with reduced penetration (mckusick no. ) . we studied an arabic family of members with d r d spanning generations. we examined members and of their spouses. l-dopa was withheld for hours from patients in treatment. five family members had generalized dystonia with diurnal auctuation ( i male, in an arabic family females). dystonia started between the age of and years with gait difficulty and involvement of the legs. mri, eeg, evoked potentials, and screening for wilson's disease were negative. an excellent response to l-dopa was noted in all patients with continued long-term clinical stability for as long as years. the patients were the products of consanguineous marriages, and their siblings were normal. the patients were descendants of the same great-great-grandparents. this pedigree suggests an autosomal-recessive type of inheritance. we believe this is the first report of d r d with an autosomal-recessive type of inheritance. a. achiron, m . gornish, h . goldberg, i . ziv, r. djaldetti, y. zoldan, h. smka, and e. mekzmed, petah tiqva, israel freezing gait is an incapacitating symptom that occurs often in advanced parkinson's disease and also in other neurological disorders, eg., multiinfarct state, multisystem atrophies, and normotensive hydrocephalus. we evaluated, videotaped, and rated patients ( men, age k , - yr) who developed pure progressive freezing gait during . . , . - years. severity was mild in with sudden motor blocks mainly when confronted with obstacles; moderate in with gait arrests upon any attempt to initiate walking and changing direction, requiring a walking stick or partial external assistance; and severe in with total inability to start walking, requiring a walker, massive assistance, or a wheelchair. in all, freezing was associated with postural instability. they could mimic normal gait when seated or lying prone and could overcome arrests by the "walking over lines" maneuver. neurological examination was otherwise normal with no signs of dementia, parkinsonism, or pseudobulbar palsy. ischemic risk factors including ischemic heart disease, hypertension, and diabetes occurred in and previous strokes in . brain c t and mri were normal or showed mild cortical atrophy in and putative lacunae in only patients. none responded to levodopa or dopamine agonists. progressive pure freezing gait should be recognized as a separate nonparkinsonian neurological entity. it may be due to degenerative or ischemic non-nigral brainstem lesions. we describe patients with causalgia and dystonia, triggered by peripheral injuries in patients and occurring spontaneously in patients. the injury was often trivial. the mean age at presentation was . years. the legs were affected in patients, and the arm was affected in the remaining patients. all had burning pain, allodynia, and hyperpathia, along with vasomotor, sudomotor, and trophic changes. all developed typical dystonic muscle spasms in the affected part. the spasms typically were sustained, producing a fixed dystonic posture, in contrast to the mobile spasms characteristic of idiopathic torsion dystonia. dystonia always followed the causalgia and was painful. there was spread of the causalgia and of the dystonia from its initial site both in the affected limb and to other extremities, the latter in a hemiplegic, transverse, and triplegic distribution. all forms of conventional treatment failed to relieve either the pain or the dystonia. we suggest that functional changes in the corticobasal ganglia-thalamic system are responsible for this painful dystonic syndrome. program and abstracts, american neurological association holiday for parkinson's disease: a controlled clinical trial r. kurlan, c . m . tanner, c. g. goetz, j . sutton, p. carvey, c. deeley, l. cui, c. itvine, and m . mcdemzott, rochester, ny, chicago, il, and san jose, c a the efficacy and mechanisms of levodopa (ld) drug holiday for parkinson's disease (pd) remain controversial. we performed a double-blind, randomized study with advanced pd patients ( men, women; aged - yr) with entry criteria of inadequate response to ld plus dose-limiting ld-induced side effects (dyskinesias, hallucinations, and confusion). subjects were assigned to: ( ) % placebo for ld (complete drug holiday) or ( ) % ld and % placebo for ld ( % drug reduction) for days. after subsequent open-label ld dose optimization, subjects were followed to end point (defined as the time when entry criteria were again satisfied or a maximum of year). median survival time to end point was not significantly different for the complete drug holiday ( days) and % drug reduction ( days) groups ( p = ) . aspiration pneumonia occurred in complete drug holiday patients and no significant morbidity occurred with drug reduction. after a -mg dose of ld, clinical and pharmacological responses were no different before and after drug holiday ( p = . ) or reduction ( p = ). subject to the limitations of our small sample size, we conclude that complete drug holiday is associated with greater morbidity and confers no major advantage over % drug reduction. we found no evidence of significant alterations of pharmacokinetic or pharmacodynamic properties of ld after drug holiday or reduction. ( (pc) of the substantia nigra on t -weighted images. the narrowing of the pc signal has been attributed either to atrophy of the pc or to increased deposition of iron in this region. we have studied details of iron distribution in the midbrain of formalin-fixed human brains by scanning pixe analysis. three p d brains, juvenile pd brain, and control (amyotrophic lateral sclerosis) brains were studied. in the controls, the iron content of the pars reticulata (pr) was almost equal to that of the red nucleus (rn), but that of the pc was less than % of the pr. in parkinsonian brains, the iron content in the pc was significantly higher than in the controls. the iron content ratios of pc/pr and pc/rn in parkinsonian brains were significantly higher than in the controls. these findings suggest that iron deposition increases in the pc of parkinsonian brains. the difference in the pattern of iron content corresponds to the intensity profile pattern noted on mri. mri findings in parkinsonian patients may reflect a change in the iron distribution pattern. (jankovic and brin, nejm, ) . such resistance within exposures is not likely due to toxin antibody formation. of cd patients evaluated per protocol receiving or more botox tx under multichannel emg monitoring, ( . %) showed no benefit after the first and second tx, despite mild neck weakness on static muscle testing and, in some instances, emg signs of denervation. the responders ( men, women) had younger age onset cd (mean yr vs yr), but similar duration (mean yr vs yr) compared to the male and female nonresponders (in contrast to jankovic and schwartz, arch neurol, ) . both groups had similar degrees of severity and tx dose (mean iu, range . - ). although disparate group size prohibits statistical analysis, some interesting comparisons include: nonresponders were more apt to have extranuchal dystonic sites ( % vs %), antecollis ( % vs %), dark eyes ( % vs %), women with elevated antinuclear antibody titer ( % vs %), history of intracranial operation ( % vs o%), significant for age focal mri abnormality ( of vs of ). history of cervical operation ( patients) did not limit responsiveness. rates of prior remission, perinatal stress, antecedent trauma, left-handedness, and family history of movement disorder were similar for both groups. antecollis presents problems for optimizing tx to affected muscles, but central mechanisms may play a role in why some patients with focal dystonia do not improve with botox tx from the outset. enrico fazzini, new york, n y with parkinson's disease does deprenyl have a symptomatic effect on patients with untreated and l-dopa-treated parkinson's disease (pd)? once deprenyl is started, how long is it before another medication is needed to control symptoms of continued disease progression? there has been controversy over whether deprenyl has effects on delaying disease progression (nejm ; : ) and/or in alleviating the symptoms of pd. one hundred seventy-five patients already taking l-dopa (group ) and patients who had never taken l-dopa (group ) were treated with deprenyl mg/day. unified pd rating scale (updrs) scores were measured before and after deprenyl. patients were followed until pd symptoms progressed to the point of requiring additional medication. one hundred eighteen of ( %) patients in group (reduced updrs mean activities of daily living [adl) to , motor [mtr] to ) and / ( %) patients in group (reduced updrs mean adl to , mtr to ) reported symptomatic benefit. an average of months' duration was found in both groups before further medication adjustments were needed. deprenyl provides symptomatic benefit for an average of months in the majority of patients with p d regardless of whether or not they are being treated with l-dopa. yasuo iwasaki, masao kinoshita, toshiya shojima, and ken ikeda, tokyo, japan, and cleveland, oh in parkinsonian patients we measured fasting plasma amino acids in parkinson's disease patients and controls matched for age and sex. all patients were receiving l-dopa and they were free of any medications other than l-dopa. normal controls were free of any medication. there were no differences in diets between patients and controls. fasting blood specimens were collected in heparinized tubes and immediately were centrifuged at , g for minutes. analysis of plasma amino acids was performed by automated ion-exchange chromatography with lithium-based buffer and an amino-acid analyzer. parkinsonian patients had significant elevations of aspartate, glutamate, and glycine. the other amino acids were not significantly different from those in controls. n o correlation between severity or activity and degree of abnormality in plasma level of amino acids in patients was established. we conclude that excitatory amino-acid metabolism is altered in patients with parkinson's disease. bonnie e. levin, rachel tomer, and william weiner, miami, fl disease there is evidence linking obsessive-compulsive symptoms (ocs) to basal ganglia dysfunction. we investigated the presence and severity of ocs in a sample of patients with an unequivocal diagnosis of idiopathic parkinson's disease (pd) using the leyton obsessional inventory. ocs was found in the majority of the patients, with ( %) scoring above the normative cutoff for the symptom score and ( %) scoring above the normative cutoff for the trait score. when severity of oc symptoms was correlated with a battery of neuropsychological measures, significant relationships were observed between ocs and a preponderance of tests associated with right-hemisphere functions. these findings were observed especially on those tests with a strong frontal lobe component (block design: r = -. ; embedded figures: r = -. ; set shifting: r = -. ; and perseverative responses: r = . ; p < . for all measures). in all cases, the more severe oc symptoms, the poorer the performance. a similar trend was observed between the leyton trait scores and the cognitive measures. these findings suggest that ocs is present in a subgroup of pd patients, which may reflect greater compromise of right-hemisphere basal ganglia-frontal lobe pathways. intraclass correlations (iccs) were calculated for the total motor score and for each individual sign. results indicated excellent agreement (icc > . ) for the total motor score, resting tremor, gait, arising from a chair, and speeded, repetitive movements; good agreement (icc > . ) for rigidity, action tremor, posture, postural stability, and bradykinesia; and poor agreement (icc < . ) for speech and facial mobility. a factor analysis was then performed on updrs motor scores for pd patients from a community-dwelling cohort. three factors were extracted by principal components analysis with subsequent varimax rotation, accounting for . % of the total variance: factor -balance and stability (posture, postural stability, gait, arising from a chair, and bradykinesia); factor -rigidity and motor speed (rigidity, speech, facial mobility, rapid alternating movements, leg agility, hand movements, and finger tapping); factor -tremor (resting and action). these results indicate that the updrs motor examination is reliable between raters and measures the cardinal signs of pd. an open pilot study was performed to evaluate the efficacy of botulinum a toxin (botox) injections for disabling hand tremors. a previous report on the use of botox for hand tremors suggested that it was helpful, but relied on subjective clinical rating scales. the extent of normal clinical fluctuations or a placebo response could not be determined. to investigate these issues more objectively, patients with parkinson's disease and with essential tremor with refractory hand tremors underwent electromyographically guided intramuscular injections of botox into wrist flexors and extensors. patients without great medication-related tremor fluctuations were selected. results before and after botox were determined by comparing ( ) patient perceptions of functional improvement, ( ) clinical assessments using the unified pd rating scale for tremor and the webster rating scales, and ( ) physiological measurements using accelerometric analysis of hand tremors of tremor frequency, amplitude, and waveform characteristics. all patients reported some improvement, ranging from mild to marked with a mean of . on a to ( = marked) global rating scale. however, only / patients showed a significant improvement in the clinical rating scales, confirmed by > % reduction in tremor amplitudes. these findings show that most patients reported improvement not confirmed by the clinical or physiological measures. efficacy of botox injections for tremors is implied, but controlled trials are needed before this procedure can be generally recommended. christopher g. goetz and glenn t . stebbins, chicago, i l we tested whether hallucinations, motor disability, and cognitive decline were risk factors for nursing home placement in advanced parkinson's disease (pd) and whether these effects were independent or synergistic. between and , we identified patients admitted to long-term nursing homes. using case control methodology, we matched each for age, pd duration, and sex with control pd patients remaining at home. parkinsonism was assessed by the motor and activities of daily living subscales of the unified p d rating scale (updrs); hallucinations and dementia were determined by scores on the thought disorder and intellectual impairment items of the updrs. tests of synergy were based on a mantel-hentel model. hallucinations were a significant risk factor with odds ratio = . , x = . , p < , . motor impairment alone and cognitive impairment alone were not significant risk factors for nursing home placement (x for motor severity = , , p > . , and x for cognitive impairment = . , p > . ). furthermore, combined odds ratios for hallucinationslmotor severity and hallucinations/cognitive impairment showed no synergy of effect (x < . for both,p > . ). of the variables studied, hallucinatory behavior is the most prominent and independent risk factor for nursing home placement in these patients; the data suggest that aggressive control of hallucinations may be warranted to prevent nursing home admission. temperature-sensitive paramyotonia congenita phenotype* louis j . ptacek, philip mcmanis, hzrbert kwiecinski, alfred george, robert barchi, launce gouw. and mark leppert, salt lake city, u t , rochester, mn, warsaw, poland, and philadelphia, pa the periodic paralyses are a group of autosomal-dominant muscle diseases sharing a common feature of episodic paralysis. in one form, paramyotonia congenita (pc), the paralysis is temperature-sensitive, usually occurring with muscle cooling. electrophysiological studies of muscle from patients with pc have revealed temperature-dependent alterations in sodium channel (nach) function. this observation led to the identification of distinct mutations in an s segment of a skeletal muscle nach in unrelated pc families. we describe the use of the single-strand conformation polymorphism (sscp) technique to define a third allele specific to pc patients in an additional family. this aberrant pattern, though distinct from the first , occurs in the same exon of this nach gene. sequencing is currently underway to define the molecular alteration causing this aberrant pattern. two additional families with the pc phenotype have been sampled and do not demonstrate these sscp variants. we are currently searching for new mutations in these families to define further the molecular heterogeneity of this temperature-sensitive pc phenotype. parag mehta and roger w. kukz, brooklyn, n y abnormal accumulation of calcium (ca) in myofibers is thought to play a role in pathogenic myonecrosis. attempts at reducing intracellular ca content with ca channel blockers in duchenne muscular dystrophy (dmd) have been clinically unsuccessful. dantrolene, however, which acts at the sarcoplasmic reticulum to inhibit ca release from intracellular stores, has produced dramatic reductions in serum creatine kinase (ck) in dystrophic mice and more recently in dmd. we investigated the effect of low-dose dantrolene in a group of patients with limb girdle dystrophy (lgd), dmd, and other myopathic disorders. all subjects received dantrolene in incrementing doses from to mg daily over a to -week period. mean baseline ck was compared to ck with dantrolene treatment. dramatic reductions in serum ck levels averaging % were seen at to -mg doses in lgd patients ( ). dmd patients ( ) and patients with other myopathies ( ) showed a similar but less dramatic reduction in ck. three of the weakest patients complained of increased fatigue while taking mg, which suggests that higher-dose dantrolene may confound longer-term trials assessing clinical muscle strength and function. dantrolene in dosages well below conventional antispastic doses has a dramatic effect on serum ck and possibly myofiber necrosis in lgd, other dystrophies, and other muscle disorders. p . antigen-specific therapy in myasthenia gravis: myasthenia gravis (mg) is mediated by anti-acetylcholine receptor (achr) antibodies, believed to be t-cell dependent, and antigen-specific therapy would be preferable to current nonspecific immunosuppression. exposing mouse t-cell clones to mhc class i molecules complexed with relevant antigen on planar membranes induced proliferative unresponsiveness (quill and schwartz, ) , and soluble mhc class i molecules complexed with myelin basic protein (mbp) peptide resulted in unresponsiveness of specific tlymphocyte clones in vitro (sharma et al, ) . we have used our well-defined dr -restricted t-cell clone (ong et a [, ) isolated from an mg patient and specific for p - of the achr alpha subunit. overnight incubation of these t cells with a soluble p - : dr complex substantially inhibited the subsequent response to challenge with soluble antigen and presenting cells. in contrast, antigen response after preincubation with dr complexed to an irrelevant peptide (mbp - ), soluble dr alone, or an experimental approach studied in vitro p - alone (at equimolar concentrations) did not differ appreciably from that in untreated cells. the p - :dr complex had no effect on other non-achrspecific cell lines/clones. these results suggest that the use of soluble mhc-peptide complexes may be an approach to selective immunotherapy in mg patients. p . high-dose intravenous immunoglobulin in the shawke a. soueidan and marinos c. dalakas, bethesda, md inclusion body myositis (ibm) is a severe disabling inflammatory myopathy with characteristic clinical and histological features. it is commonly suspected when a patient with presumed polymyositis does not respond to available immunotherapies. the need for an effective treatment in patients with ibm prompted the present pilot study using high-dose intravenous immunoglobulin (hd-ivig), an apparently effective immunomodulating agent in several autoimmune neuromuscular disorders. we treated patients with muscle biopsy-proven ibm with up to monthly infusions of gml kg ivig. after the first infusion, of the patients showed definite functional improvement consisting of independent ambulation, fewer falls, and increased ability to lift weights. the muscle strength of the proximal and less atrophic muscle groups improved by one grade mrc scale (from to ), whereas the distal and atrophic muscles remained unchanged. the improvement, sustained up to months, was greater in patients with the most severe endomysial inflammation. we conclude that hd-ivig may be the first promising agent that can improve the strength of certain muscle groups in patients with ibm. because ivig is prohibitively expensive, the present encouraging results warrant a large-scale controlled therapeutic study. hays, and n . lutov, new york, n y , and milan, italy anti-myelin-associated glycoprotein (mag) antibodies from patients with neuropathy cross-react with the glycolipid -sulfated glucuronyl paragloboside (sgpg). among patients tested by enzyme-linked immunosorbent assay and western blot, had highly elevated antibody titers ( , ) to both mag and sgpg, had highly elevated titers to mag alone, and had highly elevated titers to only sgpg. immunostaining of normal nerve myelin by the antibodies correlated better with anti-mag than anti-sgpg activity. twenty-one of the patients, including patients in all groups, had predominantly sensory or sensorimotor neuropathy, and biopsy specimens revealed deposits of igm and complement on affected myelin sheaths. three patients presented with motor syndromes, all with antibodies specific for sgpg; had a predominantly motor demyelinating neuropathy, had upper and lower motor neuron signs and peripheral neuropathy, and had amyotrophic lateral sclerosis confirmed post mortem. all had deposits of complement on peripheral nerve myelin sheaths. these studies suggest the following: ( ) that anti-mag or sgpg antibodies may differ in their fine specificities and biological activities, ( ) that anti-sgpg antibodies also may occur in motor neuron diseases, complicating the clinical presentation, and ( ) that both mag and sgpg should be used as antigens in testing for autoantibody activity in peripheral neuropathy. david b. williams, john steele, ulla-katrina craig, sandra bryant, peter o'brien, and leonard kurland, newcastle, new south wales, australia, mangilao, guam, and rochester, m n continuing surveillance of neurodegenerative diseases in the mariana islands reveals changes in frequency and clinical characteristics since the s that resemble those in other known western pacific foci (kii peninsula, japan, and irian jaya, new guinea). recent surveys of patients years and older were conducted on rota, tinian, and yigo, guam. possible cases of dementia, parkinsonism, and amyotrophic lateral sclerosis (als) were identified by local trained personnel using a questionnaire, world health organization neurology test, and cognitive screening. those who failed the screening were examined by a neurologist. in the small populations of rota and tinian, there were no definite cases of als compared to i to cases present in previous surveys. the high prevalence of parkinsonism-dementia complex (pdc) was unchanged and dementia was increased compared to earlier surveys. in yigo, als and pdc continue to be prevalent; however, the als patients are predominantly long-term survivors (> -year disease duration). in areas of previous high prevalence of als/pdc, dementia (as pdc) was associated with extrapyramidal signs, whereas in areas of previously low prevalence of als/pdc, dementia alone, possibly of alzheimer type, predominated. these observations help to confirm previous reports of changing clinical patterns, but suggest that the geographic distribution of the (presumed) environmental etiological agent for als/pdc remains stable after almost years. p. v . fragokz, . frongillo, m. michisanti, g. antonini. derangements of the cardiac conducting system are the most common features of heart involvement in myotonic dystrophy (md). in view of chis patients with various grades of md ( males and females, mean age yr) underwent -lead ecg and holter monitoring. in patients ( %), almost all with a severe grade of md, or more conduction defects were found: first-degree atrioventricular block (i-avb) in cases, second-degree avb in case, right bundle branch block in cases, left anterior hemiblock in cases, left bundle branch block in cases, and trifascicular block in case (pacemaker implanted). an -year-old boy had a chronic atrial fibrillation with slow ventricular rate; he died suddenly while awaiting electrophysiological study. thirty-seven patients were followed over a mean period of months (range - mo). a -year-old woman experienced a myocardial infarction and was excluded from subsequent considerations. conduction defects de novo appeared in patients: i-avb in and i-avb plus -avb (mobitz i and i type) in . nine patients, all with i-avb at initial evaluation, showed deterioration of their defects' conduction: a bifascicular block was observed in cases and a trifascicular block in cases (pacemaker implanted). conduction defects may run a malignant course in md, mainly in patients with more severe grades of the neuromuscular disease; thus, a close cardiological evaluation is mandatory for a proper therapeutic approach in single cases. hiroshi mitsumoto, surest kumar, kevy h. levin, robert w. shields, jr, michelle secic, asa j . wilbourn, and rajendra g . desai, cleveland, oh, and santa ana, ca twenty patients with amyotrophic lateral sclerosis (als) entered a pretreatment study with monthly quantitative isometric muscle strength tests ( muscles in each extremity) and quantitative tufts scales including vital capacity, bulbar diadochokinetic rate, timed water drinking, timed rising from a chair, and timed walking meters. after to months of pretreatment observation, the patients received mg/kg intravenous immunoglobulin (ivig) (gamimune-n, miles) every month for up to months. during the study period, patients died and patients withdrew from the study. the slope of each variable's changes over time before and after the ivig treatment were compared statistically. none of the quantitative scales showed significant change with ivig. however, the slope of the upper extremity muscle strength revealed improvement with ivig treatment ( p = . and . , right and left, respectively). when all extremities were combined, the slope was also significantly improved with ivig ( p = . ). lower extremity muscle strength alone showed similar trends but no statistical significance. electrophysiological and immunological data were also analyzed. our results warrant a double-blind, controlled study with ivig for the treatment of als. leber's hereditary optic neuropathy (lhon) is a mitochondrial disorder with predominantly optic nerve abnormality. it can be associated with dystonia, ataxia, encephalopathy, cardiac abnormalities, and other less well-characterized neurological syndromes. we describe members of a family with lhon with a slowly progressive motor polyneuropathy. a -year-old man and his -year-old sister have had mild motor impairment since early childhood. a gait disorder and distal muscle weakness became evident at puberty. the girl, but not the man, also has blindness, distal numbness, type i diabetes mellitus, and short stature. physical examination showed in both: bilateral foot drop, limb hyperreflexia but absent ankle reflexes, distal sensory loss, and a slight brownish scaly skin discoloration over the forearms. only the girl had clonus and optic nerve atrophy. the man had peripapillary telangiectasias. the jaw jerk was normal in both. motor nerve conductions in both showed absent tibia and peroneal responses, whereas other motor and sensory nerve conductions were normal. emg revealed denervation, more so distally. muscle biopsy findings showed recent denervation and previous denervation followed by reinnervation. n o raggedred fibers were observed with the modified trichrome and sdh stains. brain mri was normal in both. cerebrospinal fluid in the girl was normal. blood samples from both patients and maternally related family members revealed a mitochondrial dna point mutation at position (d. c. wallace). in this family, only male had lhon; females had lhon and others, including the patients' mother, were asymptomatic carriers. this association of chronic motor neuropathy and hyperreflexia with lhon appears to be a distinct syndrome. the pathogenic mechanism by which the mitochondrial dna defect causes the neuropathy (and other neurological deficits) requires analysis. duchennelbecker muscular dystrophy henry j . kaminski, mazen al-hakim, r. john leigh, bashar katirji, and robert l. ruff; cleveland, oh fast-twitch extremity muscle fibers are preferentially affected in duchenne/becker muscular dystrophy (dbmd). since saccades are thought to be mediated by fast-twitch fibers, saccadic velocities would be expected to be decreased among these patients. to investigate involvement of extraocular muscle (eom) by dbmd, we studied with infrared oculography patients who were wheelchair-bound and able to perform only minimal activities of daily living. saccades were slightly slowed but were within % confidence limits of normal. all patients showed square wave jerk movements (swj). in patients, the frequency of the swj exceeded that of normal subjects, which suggested central nervous system dysfunction. clinical neuroophrhalmological examination of other dbmd patients was normal. this investigation is the first study of ocular motility in dbmd and demonstrates that eom function is relatively preserved even in far advanced patients. eom is composed of a heterogenous mix of fiber types that differ in anatomical and physiological characteristics from extremity muscle. study of eom in dbmd may prove to be useful in understanding why some muscles are resistant to dbmd and in characterizing properties that limit muscle degeneration. (supported by nih grants ey , ey , the department of veterans affairs, and the evenor armington fund.) since patients with myotonic dystrophy (mtd) exhibit a marked resistance to insulin effect on glucose uptake and an impaired handling of the insulin-sensitive amino acids, it is possible that muscle wasting in mtd may reflect a derangement of insulin action on muscle protein metabolism. increased muscle protein breakdown in mtd would be expected if the normal inhibitory effect of insulin on protein catabolism is impaired. the forearm perfusion technique combined with measurements of -methylhistidine ( -mh) arteriovenous (a-v) differences by high-performance liquid chromatography provides a unique method to investigate skeletal muscle myofibrillar protein degradation in vivo. we studied -mh (a-v) and efflux from the forearm muscles in men moderately affected with mtd and normal men. efflux values (q) were calculated as the product of -mh (a-v) times forearm plasma flow measured by the indicator dilution technique. forearm -mh release (estimated as {a-v} or q) of mtd patients did not differ significantly from normal controls. we conclude that myofibrillar degradation is not increased in mtd even when measured in a muscle compartment selectively affected by wasting. the possibility of an impaired anabolic action of insulin in mtd has yet to be determined. to study the relationship between the ragged red fibers (rrf) and age, we have reviewed muscle biopsy specimens prospectively. patients with well-established mitochondrial myopathy syndrome and with myopathies known to produce secondary rrf were excluded. the number of ragged red fibers (rrf) was counted under x (lpf) magnification. rrf were identified by the modified trichrome and sdh stain. for the final analysis, the sdh staining was used. the frequency of rrf was analyzed in relation to patients' ages. the frequency of cases with more than rrf increased with aging: % in the first decade, % in the fourth decade, and % in the eighth decade. the frequency of cases with more than rrf also increased with aging: % in the first three decades, % in the fourth decade, and % in the eighth decade. of patients with well-established mitochondrial myopathy syndrome, had more than rrf under lpf. the number of rrf in muscle increases with aging, indicating that mitochondrial activity in muscle is affected by aging. this finding may complicate the diagnostic criteria of mitochondrial myopathy in older individuals. ten rrf under lpf seems to be a reasonable cutoff point for the diagnosis of mitochondrial myopathy. schwartz-jampel, a rare autosomal-recessive syndrome characterized by short stature, myotonia, skeletal abnormalities, and peculiar facies, was reported by aberfeld in . the same sibship was earlier reported by schwartz and jampel in with emphasis on blepharophimosis. as of this writing about cases have been reported in the literature. most of the features of this syndrome are believed to be secondary to primary muscle disease. several peripheral electrophysiological studies showing features of myotonia have been reported. we describe patients with schwartz-jampel syndrome showing evidence of central conduction disturbance documented by somatosensory-evoked potentials (seps). median nerve seps showed normal latencies to erb's point and n- in all. interpeak latencies between n and n were prolonged in with complete block in . emg showed typical myotonic discharges in all. motor nerve conduction velocities, visual and brainstem auditory-evoked potentials, ct, and mri were normal in all. seps in the parents were normal. we believe this is the first report documenting evidence of central nervous system (cns) involvement in schwartz-jampel syndrome. schwartz-jampel syndrome and myotonic dystrophy may have similar cns ''lesion'' as sep abnormalities also have been shown in myotonic dystrophy. epidemiological studies have associated consumption of certain batches of l-tryptophan (lt) with development of the eosinophilia-myalgia syndrome (ems). , '-ethyledenebisltryptophan) (ebt or peak e), a derivative of lt, is a trace contaminant associated with implicated batches of lt. three female lewis rats received ebt, mg per gm daily, by intraperitoneal injection. four control rats received unimplicated lt. n o peripheral eosinophilia, rash, or weakness were observed in either group. one rat from each group died during the experiment (control-bowel infarct; ebt-death under anesthetic). after days, forelimb and hindlimb muscles of the remaining animals were frozen and fixed for program and abstracts, american neurological association ultrastructural and histological studies. two ebt rats had a myopathy involving soleus with a perimysial infiltrate containing lymphocytes, macrophages and sparse eosinophils, and necrotic fibers; the other showed few necrotic fibers in gastrocnemius. occasional eosinophils were seen in fascia in both animals given ebt but not in controls. fiber-type specific quantitative analysis of the microvasculature showed no decrease in the capillary index. ultrastructural examination revealed an increase in the size of microvessels in ebt animals. no denervation or reinnervation were demonstrated. the perimysial inflammation replicates an important feature of human ems and supports the epidemiological evidence that ebt is the causative agent of the disease. britta ostermeyer-shoaib, bernard m. patten, and tetsuo ashizawa, houston, t x five women (patients - ) developed motor neuron disease (mnd) years (range - yr) after receiving silicone gel-filled breast implants. at explant in patients, had both and had the left implant ruptured with silicone spilled into tissue. one woman (patient ) developed amyotrophic lateral sclerosis (als) years after numerous injections of free silicone into her face. biceps muscle biopsy specimens in all showed neurogenic atrophy. patient developed als with bulbar involvement and died years later of respiratory failure. she had anti-gm antibodies and autopsy findings confirmed the diagnosis of typical als. patient developed als, but also fatigue, myalgia, arthralgia, and skin rash. she had anti-gm antibodies, antisilicone antibodies, positive antinuclear antibodies (ana), and decreased serum igg, iga, and c , but increased igm and creatine phosphokinase (cpk) and chronic inflammation was revealed in muscle biopsy specimens. patient developed als, but also had hair loss, skin rash, fatigue, headache, sjogren's syndrome, and positive ana. patient developed als, but also had myalgia and arthralgia. patient developed lower mnd, but also fevers, arthralgia, and joint stiffness. she had anti-gm antibodies, positive ana, antimyelin antibodies, and decreased serum igg and iga with chronic inflammation shown in nerve biopsy findings. patient developed a steroidresponsive and steroid-dependent als with bulbar involvement. she had a monoclonal gammopathy in the cerebrospinal fluid and increased cpk. we suggest that silicone acts as an adjuvant that damages motor neurons via an indirect autoimmune mechanism. implants and silicone injections into the face p . silicone adjuvant breast disease: more forty-five women developed mixed sensory-motor neuropathy ( ), motor neuron disease ( ), multiple sclerosis ( ), multiple sclerosis-like syndrome ( ), or myasthenia gravis ( ) years (range mo- yr) after receiving silicone-gel breast implants ( ), saline-filled silicone-covered breast implants ( ), or direct injections of silicone into the breast ( ). most patients had, in addition, severe fatigability, myalgia, arthralgia, morning stiffness, skin rash, lymphadenopathy, sjogren's syndrome, and short-term memory problems. laboratory results revealed in most of the women decreased or increased serum immunoglobulins, autoantibodies, a serum monoclonal gammopathy, or oligoclonal bands in cerebrospinal fluid. at explantation in , had both and had implant neurological cases ruptured. biopsy of the fibrous implant capsule in most patients showed foreign-body giant cells containing refractile material consistent with silicone whether or not the elastomer shell was ruptured, indicating silicone bleed. the major finding on surd nerve biopsy was loss of myelinated fibers, on biceps muscle biopsy was neurogenic atrophy, and on pectoralis muscle biopsy was myositis with vasculitis and free silicone in some. we suggest that silicone may provoke damage to nerve and muscle, probably indirectly promoting autoimmunity. james f. howard, jr, m . kathleen donovan. and m. susan tucker, chapel hill, nc urinary symptoms of urgency and incontinence have been reported only rarely in patients with myasthenia gravis (mg) and then most often in association with myasthenic crisis. we report the case of a -year-old woman who in december had the onset of chest pain and was found to have a lymphocytic thymoma. in june she developed urinary incontinence, was found to have an open bladder neck. and underwent a suspension procedure for stress incontinence in january . eight months later she developed exertional fatigue and a diagnosis of m g was made. in july there was a recurrence of urinary incontinence. these symptoms clustered toward the end of the day and at trough mestinon dose. neuro-urophysiological studies demonstrated her previous open bladder neck, the inability to sustain a pelvic floor contraction, and increased bladder wall contraction. singlefiber electromyography (sfemg) recordings from the anal sphincter demonstrated a mean consecutive difference (mcd) of ysec, and % of fiber pairs had impulse blocking while recordings in the extensor digitorium communis muscle were normal. following a course of plasma exchange, there was significant clinical improvement with a reduction in the frequency of urinary incontinence, and improvement in anal sphincter sfemg studies (mcd, ysec with no blocking). this case demonstrates that in those myasthenic patients with predisposing bladder outlet dysfunction, urinary incontinence may be a manifestation of worsening mg. diana m. escolar, mohamed eldaly, and jaime rich, boston, m a debate still exists as to the role of antibody versus celmediated factors in the pathogenesis of guillain-barre syndrome (gbs). we describe a patient with increased proportion of circulating t cells and a t-cell lymphoma who developed gbs and responded to intravenous immunoglobulin (ivig). a -year-old man with t-cell lymphoma drveloped gbs by clinical, nerve conduction, and cerebrospinal fluid criteria. he had an elevated proportion of t cells and markedly reduced b cells with a normal cd /cd ratio. he responded rapidly to ivig, with return of nearly normal motor function in week. three weeks later, he relapsed and his vital capacity dropped. ivig was again administered and within hours he nearly recovered. a third relapse, days later, again responded to ivig. he has remained asymptomatic with ivig maintenance. the role of t-cell lymphocytes in initiating experimental autoimmune neuritis has been shown by adoptive transfer experiments. this patient with t-cell neoplasia may represent an analogous model in hu-guillain-barre syndrome mans supporting the role of t-cell (cell-mediated) autoimmunity in the pathogenesis of gbs. ivig therapy may act primarily by inhibiting the t-cell-mediated attack on myelin. p . sympathetic skin response: age effect it is frequently stated that the sympathetic skin response (ssr) can be elicited in all normal subjects, but the age of the investigated population usually is not considered to be a significant factor. we have examined the ssr in the upper and lower limbs of normal subjects, aged to years ( of them males). the ssr was elicitable in the lower limbs in all subjects under the age of years and in the upper limbs in all subjects younger than years. in contrast, it could be elicited in the lower limbs in only % and in the upper limbs in % of octogenarians. the amplitude of the response, though highly variable, showed a remarkable decline with age, both in the upper ( p < . , r = . ) and in the lower ( p < . , r = . ) limbs. these results indicate that age affects both the elicitability and the amplitude of the ssr. this has to be taken into consideration when evaluating the autonomic function in the elderly. we reviewed records of patients appearing to have motor neuron disease (mnd) to whom we recommended immunosuppression over years ( of m n d patients). atypical findings engendered hope rhat they might have treatable neuropathy. electrophysiological studies were mainly consistent with mnd, but also showed conduction block or other evidence of relatively mild peripheral nerve disease in . sensory symptoms were present in ; or more reduced or absent deep tendon reflexes in ; elevated cerebrospinal fluid protein in ; and nonspecific abnormalities on sural nerve biopsies in of . anti-gm levels were measured in (including who improved), but none was significantly elevated. immunosuppression included cyclophosphamide ( patients); prednisone ( ); plasma exchange ( ); intravenous gammaglobulin ( ); cyclosporine ( ); and total lymphoid irradiation ( ). seven treated patients died, worsened, remained stable for years, and improved. two patients declined treatment. one died and the other did not worsen in years. we conclude that immunosuppression by our methods is, at best, rarely effective in atypical mnd. we studied serum antiglycolipid antibodies by enzymelinked immunosorbent assay in patients with typical miller fisher syndrome (mfs), patients with atypical mfs who were lacking in some of the cardinal signs, patients with guillain-barrc syndrome (gbs) with ophthalmoplegia, patients with gbs without ophthalmoplegia, patients with multiple sclerosis (ms), and patients with other immunological disorders (oid) including systemic lupus erythematosus, polymyositis, and mixed connective tissue disorder. all patients with typical mfs had increased activity of igg antibody against ganglioside g q l b in the early phase, and it reduced with time. such anti-gqlb igg activity also was detected in of the patients with atypical mfs and in of the patients with gbs with ophthalmoplegia. in atypical mfs, the only patient without increased anti-gq b igg activity demonstrated normal eye movement with ptosis, whereas eye movement was impaired in the other patients. no patients with gbs without ophthalmoplegia, ms, or oid had increased anti-gqlb igg activity. these findings suggest the close association between increased anti-gql b igg activity and impaired eye movement in mfs and gbs. serum anti-gqlb igg activity possibly plays a role in impaired eye movement in mfs. our goal is to develop an assay that can be used to monitor a relevant immune effect of interferon p (ifnp) in multiple sclerosis (ms) patients during the course of ifnp immuno-' therapy, since recombinant ifnp is being tested in multicenter clinical trials. this report extends our prior studies of the inhibitory effect of ifnp on t-cell activation. peripheral blood mononuclear cells (pbls) from healthy donors and clinically stable ms patients were studied. pbl cultures were stimulated with cona, mab to cd , or with the phorbol ester pma in the presence of the calcium ionophore ionomycin. parallel cultures were studied in the presence of ifnp, uiml. t-cell activation was monitored by determining the percent cells positive for il- receptor (il- r) using facs analysis, or with a sensitive enzyme-linked immunosorbent assay for ifny. ifnp markedly inhibited il- r expression induced by cona, by mab to cd , or by pma and ionomycin, which activate t cells via different pathways. the results suggest that ifnp inhibits t-cell activation by actions independent of membrane receptors. we observed significant inhibition of cona-induced t-cell il- r expression in both ms patients ( . % inhibition, p < . ) and controls ( . % inhibition, p < . ). there was no significant difference in percent inhibition between ms and controls, but there was more variance among the ms patients. variability of biological effects of ifnp on t cells may relate to differential therapeutic responses to exogenously administered ifnp in ms patients. preliminary experiments suggested that ifnp inhibited ifn gamma secretion by pbl stimulated with cona. ifnp inhibits a number of events associated with t-cell activation, in both normal and ms t cells. response to ifnp appears more variable in the ms cases. immunological monitoring of t-cell activation in patients receiving ifnp may assist in understanding the observed therapeutic responses and planning clinical protocols. myelin destruction is associated with many central nervous system disorders, such as multiple sclerosis, head trauma, and ischemic injury. inflammatory cells, monocytes/macrophages, and polymorphonuclear leukocytes (pmn) may me-program and abstracts, american neurological association diate myelin injury, and lipid peroxidation may be an important mechanism. inhibiting myelin oxidation could have substantial benefit, so we evaluated the ability of a -minosteroid, u a, to inhibit myelin oxidation by monocytes and pmn. fresh rat brain myelin was harvested by multiple sucrose gradient, ultracentrifugation steps, and the final product was confirmed to be pure myelin by sds-page electrophoresis. human monocytes or pmn were obtained from healthy, unmedicated volunteers by gradient separation techniques. monocytes ( . x lo cells/ml) and pmn ( . x lo cells/ml), myelin ( pg protein/ml), and lipopolysaccharide ( pg/ml) were incubated for hours with or without wm u a in -ml wells. myelin oxidation was evaluated by a thiobarbituric acid reactive substance assay for production of malondialdehyde (nmol/ ml). myelin oxidation by monocytes was . +- . (mean standard error of mean) without u a and was reduced to . ? . by pm u a ( p < . ). pmn-mediated myelin oxidation was . _t . without drug and . ? . with drug ( p < . ). these results demonstrate that u a markedly inhibits monocyte-and pmn-mediated myelin oxidation and suggest that the aminosteroids may help disorders associated with inflammatory cell-induced myelin injury. microglia cells participate in the pathological reactions of the cns to multiple insults including trauma, inflammation, and neuronal degeneration. functional roles for these cells could include mediating tissue in jury, promoting repair, or modulating immune responses. with regard to the latter, we have observed that the majority of adult human-derived microglia express major histocompatibility complex (mhc) class molecules under basal culture conditions, in contrast to astrocytes derived from the same surgical biopsy specimens. all morphological subtypes of the microglia (ameboid, bipolar, and ramified) expressed mhc class i molecules, indicating a discordance between morphology and mhc antigen expression as markers of microglia activation. the microglia actively ingest myelin constituents, as assessed using fluorescein-labeled myelin basic protein and laser confocal microscopy. autologous t cells (e') freshly isolated from the systemic blood and cocultured with candida antigen underwent active proliferation in the presence of to % microglia, indicating the functional capacity of the microglia to serve as antigen-presenting cells. y-interferon augmented both mhc class expression and functional antigen-presenting capacity. these results indicate the potential of the adult human microglia to promote immune reactivity within the cns. protein (mbp) neutralize anti-mbp purified from multiple sclerosis cerebrospinal fluid active phases of multiple sclerosis (ms) are associated with increased titers of intrathecally produced antimyelin basic protein (anti-mbp). anti-mbp can be purified by antigenspecific affinity chromatography from csf igg of patients with acute relapses of ms. eighteen synthetic peptides of human myelin basic protein (h-mbp) containing between and amino-acid residues and covering the entire length of the molecule were synthesized by the fmoc method. purified anti-mbp was reacted with increasing amounts of h-mbp as well as each of the peptides in an initial liquid phase assay, and subsequently titers of f anti-mbp in all resulting mixtures were measured by a solid-phase radioimmunoassay . purified anti-mbp was neutralized by h-mbp and of the synthetic peptides containing overall residues corresponding to to of h-mbp. the remaining synthetic peptides covering both the amino and carboxyl terminals of h-mbp did not significantly react with purified anti-mbp from these patients. in conclusion, anti-mbp purified from csf of ms patients has affinity for epitopes located between residues and of h-mbp. in a double-blind study involving patients, we recently demonstrated that -aminopyridine ( -ap) is superior to placebo in the treatment of multiple sclerosis (ms) (ann neurol, in press). the related agent , -diaminopyridine (dap) also appears to be effective. to enable a preliminary comparison, patients, who in our previous study had not benefitted from -ap, were now treated ( wk) with dap (up to . mg/kg/day) for weeks in an open-label fashion. instruments for assessment and registration of side effects were the same as in the previous trial. the optimal dose of dap was . mg/day compared to . mg/day for -ap. significant changes in the edss ( . point or more) were not found, whereas significant improvements in neurophysiological parameters were found (no difference between -ap and dap, allp > . ). subjective side effects during -ap ( patients) mainly suggested cns-function disturbance (dizziness and gait disturbance) and during dap ( patients) mainly suggested peripheral nervous system-function disturbance (paresthesias). systemic tolerability clearly was diminished for dap compared to -ap, with patients withdrawing because of severe gastric complaints and developing liver function abnormalities. these data suggest that -ap is more valuable than dap in the treatment of ms. cy clophosphamide/methylprednisolone therapy in multiple sclerosis multiple sclerosis (ms) is a presumed autoimmune disease in which various forms of immunotherapy have been attempted. mri studies show the disease to be more chronically active than is clinically evident, thus a single treatment is unlikely to provide lasting benefit. recently, the northeast cooperative treatment group found that pulse cyclophosphamide ( mg/m every other month for years) slows progressive ms. we initiated a pilot study to determine the effect of a more intensive and prolonged pulse therapy regimen in both progressive and earlier stages of the disease. pulse therapy was given after induction with either iv cyclophosphamide/corticotropin ( mg/m x over days) or iv methylprednisolone ( gm x over days). patients received a single iv dose of cyclophosphamide ( - , mg/m ) adjusted to produce leukopenia plus gram of iv methylprednisolone monthly for a year, every weeks for the next year, and every months in the third year. another group received pulse methylprednisolone without cyclophosphamide. as of this writing, patients have been treated, of which have completed years. interim analysis shows that patients treated with pulse methylprednisolone were more likely to become treatment failures than those treated with pulse cyclophosphamide/methylprednisolone ( % vs %) independent of induction therapy. withdrawal due to toxicity, however, was higher in the pulse cyclophospharnide group. current regimens involve methylprednisolone induction alone followed by pulse cyclophosphamide/methylprednisolone, analogous to lupus nephritis pulse therapy. this regimen can be given solely on an outpatient basis, does not cause alopecia, and is more amenable for use in earlier stages of the disease. to determine the incidence of pathologically confirmed malignancy in multiple sclerosis (ms) patients in funded clinical trials of cyclophosphamide (ctx) and of azathioprine (aza), data were collected longitudinally using telephone interviews, written questionnaires, physical examinations, and medical records for ctx and aza patients from a community in-hospital and out-patient ms clinic in fargo, nd. in the ctx study (goodkin et al, arch neurol ; : - ) , clinically definite (cd), chronic progressive ms patients were enrolled. twenty-four were controls and received a mean induction dose of . grams. fourteen of the induced patients then received boosters every other month for months resulting in a mean total dose of . grams. no malignancies were detected. in the aza study (goodkin et al, neurology ; : - ) , c d relapsing ms patients participated. twenty-five were controls and received mgtkg of aza daily by mouth adjusted to maintain a white blood count of greater than , o/cmm for years (mean dose . mg/kg). two of aza patients developed resectable skin cancers: basal cell (bcc) at months and i squarnous cell (scc) at months. the incidence of developing a bcc or scc was not significantly increased after initiating therapies as compared to the untreated ms controls (aza: fisher exact testp value = . ). no other malignancy has been detected during to months of clinical follow-up. allergic encephalomyelitis paula dore-dufly, ruth washington, and robert h. swanborg, detroit, m i postcapillary endothelium at sites of inflammation undergoes many changes referred to as activation. activated endothelial cells (ec) exhibit increased surface expression of immunorelevant proteins (icam- ; ncam, elam, and mhc class i and class i antigens [ags)). the sequence of events that characterizes ec activation may be important in susceptibility, induction, and perpetuation of experimental allergic encephalomyelitis (eae). in this study we examine expression of ec activation antigens in central nervous system (cns) microvessels in response to interferon gamma (ifn-y). cns microvessels from sjl and bio.s mice were incubated for hours in ifn-y ( u/ml), fixed, permeabilized, and then stained with an antibody that recognizes class i, class i mhc antigens, icam- , and factor viii. relative fluorescence intensity was determined using a laser cytometer. results indicate that microvessels from all strains tested expressed no detectable icam- and class i ags. little class i antigen and transferrin receptors were expressed. upon stimulation with ifn-y, sjl microvessels exhibited increased surface expression of all ec activation ags. b o.s microvessels exhibited icam- and class i mhc but mhc class i ags were not upregulated. results indicate that there are strain differences in the ec response to ifn-y. resistance of b o.s mouse ec to activation by ifn may be a factor in decreased susceptibility or induction of eae, or both. protein-t-cell line-mediated experimental to investigate a possible pathogenic role of interferongamma (ifn-y) in experimental allergic encephalomyelitis (eae), an immunocytochemical study was undertaken to localize this cytokine in the spinal cord of lewis rats in which eae was produced by adoptive transfer of myelin basic protein-specific t cells. one pm-thick cryosections of spinal cord were labeled with monoclonal antibodies (mab) db- and db- recognizing different epitopes of rat ifn-y. in the spinal cord of naive rats, mab db-i, but not db- , stained processes of astrocytes, suggesting that astrocytes contain a protein with an epitope cross-reacting with ifn-y. in rats with at-eae, numerous ifn-y-positive cells stained with both mab db- -and db- -positive cells were present from days to after cell transfer and had disappeared on day . at day they entered the spinal cords predominantly through subpial vessels. ifn-y-positive cells could be identified as w / + leukocytes as well as ed -positive macrophages. as in naive rats, astrocytes in at-eae were labeled only with mab db- , but not db- . we never observed labeling of motor neurons with these mab. the transient presence of ifn-y in the rat spinal cord at the onset of at-eae suggests a pathogenic role of this cytokine in acute immune-mediated demyelination of the cns probably as a local stimulus for expression of mhc class i antigens and adhesion molecules, as well as for the release of tnf-a and toxic oxygen radicals from macrophages and microglia. immune responses to stress or heat shock proteins are implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis (ms). we examined the hypothesis that antigens in myelin cross-reacted with stress protein antigens. two techniques were used: immunocytochemistry and western blotting. frozen histological sections were prepared from normal human central and peripheral nervous system tissues. sections were incubated with murine monoclonal antibodies to different mycobacterial stress proteins. antibody binding was determined using avidin-biotin complexed antimurine antibody linked to alkaline phosphatase. program and abstracts, american neurological association a monoclonal antibody to the stress protein sp from m . leprae strongly stained both central and peripheral nervous system myelin. no myelin staining was noted with antibodies to sp or sp . proteins from purified central and peripheral nervous system myelin were separated by sds-page. western blots were prepared using a monoclonal antibody to sp and a polyvalent rabbit antibody to myelin basic protein (mbp) as primary antibodies. antibody binding was determined using antimurine or antirabbit igg antibody coupled to alkaline phosphatase. strong staining of central but not peripheral mbp by the anti-sp antibody was observed. the rabbit anti-mbp antibody stained both central and peripheral nervous system mbp. the presence of antigenic epitopes shared by a stress protein and the potential autoantigen, mbp, supports the hypothesis that immune responses to stress proteins may be involved in the pathogenesis of presumed autoimmune diseases such as ms. ( ). of patients with borderline or positive csf lyme titer, received parenteral ceftriaxone. all had later relapses consistent with ms. one patient received a month of oral doxycycline. the fifth patient had a negative western blot and was not treated. we conclude that an incidental borderline or positive lyme serology in an ms patient is unlikely to indicate neurological lyme disease. borderline serologies should be documented to rise on later testing; positive serologies should be confirmed by retest in a different laboratory or by western blot. csf abnormalities suggestive of neurological lyme disease (pleocytosis, protein elevation, intrathecal lyme antibodies) are distinct from those suggestive of ms (ogb, elevated igg index, mbp). in such patients antibiotic treatment may be appropriate, but will not alter disease course. when designing and analyzing therapeutic trials for multiple sclerosis (ms), investigators commonly compare the proportion of patients in the experimental and control groups who worsen one or more steps on the disability status scale (dss) or expanded dss during the study (typically years' duration). however, the intervals between the scores in the dss may not be equal. it may be easier to change by one or more steps in the lower end of the scale (e.g., dss = - ) than in the midportion of the scale (e.g., dss = - ). to evaluate this possibility, we compared the proportion of patients who worsened by one or more steps in the years after entering our program with dss = or with dss = . fifty-one percent ( ) natural history data may be useful for designing therapeutic trials for multiple sclerosis (ms). since , we have collected such data in a standard format on patients in the ucla multiple sclerosis research and treatment program. t o describe the course in our group, we have performed survival analysis (kaplan-meier) of patients with or more assessments who entered the clinic with disability status scale (dss) scores of to (n = ). an increase of one or more steps in the dss score persisting for more than months defines worsening. median times to worsening for a dds at entry of to were approximately years (range . - . ); but were . years for dss and . years for dss . for those starting at dss (n = ), only % worsened by year, % by years, and % by years. the percent worsening when starting at dss (n = ) were %, %, and %, respectively. these variable rates of worsening (i.e., time spent at each starting level) influence therapeutic trial design. including patients with dss or will increase the sample size and study duration. for testing nontoxic agents, we recommend enrolling patients with dss to . for more toxic treatments, we suggest dss to . (partially supported by usphs grant ns , the conrad n. hilton foundation, and various donors.) pi . cholinergic antagonists and p-adrenergic agonists inhibit experimental allergic encephalomyelitis in an additive manner mark a. jensen, avertano noronha, and bawy g. w. amason, chicago, il lymphoid organs receive a sympathetic (sns) and possibly a parasympathetic innervation. lymphocytes express padrenergic and cholinergic receptors and thus are sensitive to regulation by these neurotransmitters. the severity of experimental allergic encephalomyelitis (eae) is increased in sns-ablated animals. local parasympathectomy decreases plaque-forming responses in submandibular nodes. p,-adrenergic receptors are upregulated on cds t cells in progressive multiple sclerosis, as are m,-muscarinic acetylcholine receptors on cd t cells. we examined the effect of isoproterenol, a p-adrenergic agonist, and scopolamine, a cholinergic antagonist, on the course of eae in lewis rats. eae was induced by injection of . ml of incomplete freund's ad juvant containing guinea pig spinal cord ( % wlv) and m. tubercdosis ( mgiml) in one hind footpad. scopolamine ( . mglkg, twice daily) and/or isoproterenol(o. mglkg, twice daily) or saline were injected subcutaneously starting on the day of immunization. scopolamine or isoproterenol alone reduced severity ( p < . , t test) and duration ( p < . , t test) of disease compared to controls. the combination of scopolamine and isoproterenol further reduced disease severity compared to either agent alone ( p < . , x test), suggesting an additive protective effect of cholinergic antagonists and p-adrenergic agonists in eae. antigen-presenting cells a . conrad, v. sanders, p. schmid, and w. w . tourtellotte, los angeles, c a tissue is cryopreserved by the method of tourtellotte; this procedure minimizes or eliminates ice artifacts and preserves surface protein markers. the dissected plaques are lightly fixed in % paraformaldehyde and then suspended in % sucrose. blocks are mounted in oct, cryosectioned at p,m, and picked up on gelatinized slides. the activity of plaques is determined by the presence or absence of myelin debris (antimyelin basic protein stain or lux fast blue) or presence or absence of neutral lipids indicating myelin digestion as seen by oil red (oro) staining and the presence or absence of a class i major histocompatibility complex antigen (evidence for an antigen-presenting cell) on macrophages or microglia as seen by immunocytochemically staining for hla-dr (hb atcc). the following is our classification of the activity of multiple sclerosis (ms) plaques. type i, the most active, is defined as an area of hypercellularity, positive for hla-dr with no o r staining or staining for myelin debris. type , or active, is defined as an area of hla-dr-positive cells that stain mildly with o r at the plaque edge but positive for myelin debris; evidence for demyelinating activity < hours (prineas; raine). there is an inner area of plump cells that are positive for hla-dr and oro. type , or modestly active, is defined as a "shelf" of plump hla-dr-positive cells at the edge loaded with program and abstracts, american neurological association oro-stained neutral lipid but a paucity of or no myelin debris. a region of hypocellularity is observed at the center of plaque. type iv, least active or inactive, is defined as scattered hla-dr-positive cells at plaque edge with little or no o r staining and no evidence of myelin debris. the frequency of plaque types was determined from a random sample of ms tissue blocks from patients who died of ms. ten percent of the plaques were type i; %: were type ; % were type . the majority of plaques ( %) were inactive (type iv). accordingly, it is necessary to classify the demyelinating activity of ms plaques for protocols designed to investigate etiopathogenesis. do these results suggest that whatever causes ms can be eradicated in half of the demyelinating areas by the time of death? p . localization of g d l b ganglioside antigen in the human peripheral nervous system susumu kusunoki, atsuro chiba, tadashi tai, and lchiro kanazawa, tokyo, japan serum antibodies against ganglioside gm and/or g d l b frequently are detected in autoimmune neuropathies such as rnultifocal motor neuropathy, igm paraproteinemic neuropathy, and guillain-barre syndrome. some of them bind to gm or g d l b monospecifically but the others cross-react with both of the antigens. to investigate respective localizations of gm and g d l b antigens in the human peripheral nervous system (pns), immunohistochemical study of dorsal root ganglia (drg), dorsal roots, ventral roots, and sympathetic ganglia (sg), which were obtained from human autopsy specimens, was performed by using mouse monoclonal antibodies, each monospecific to gm and gdlb. ggr , monospecific to gd b, immunostained nerve cell somas and axons of drg, sg, and dorsal and ventral roots. ggrl also recognized some myelin, including paranodal areas. however, gmb , monospecific to gm , did not bind to either neuron or myelin. thus, serum anti-gdlb antibody can bind to neurons and some myelin in the human pns. further study is necessary to identify the localization of gm antigen, because previous biochemical studies have shown that gm is also present in the human pns. the purpose of this study was to compare t and t lymphocytes in migraine patients versus controls, and a review of the literature was done. fifty-six migraine patients, aged to , were tested, as were controls. the t :t ratio in migraine patients was : , compared to : for controls. suppressors (t ) were reduced from in controls to in migraine patients. helpers (t ) decreased from , in controls to in migraine patients, and total ?' lymphocytes decreased from , to , . previous studies have revealed similar differences in t lymphocytes, with higher t : t ratios being found in both migraine and tension-type headache patients. in food-induced migraine, an increase in circulating immune complexes was noted, with increased t levels. differences in serotonin binding to mononuclear cells have been noted in migraine patients. a decreased sensitivity of the lymphocyte beta-adrenergic receptor in migraine patients was suggested by one study. after lymphocyte incubation with il- , the natural cytotoxic response is augmented in cluster patients. the percentage of lymphocytes expressing receptors for il- was decreased in cluster patients in a fur-review of the literature ther study. this il- receptor defect was independent of whether the clusters were present. there is a loss of highaffinity binding sites for serotonin on lymphocytes in both episodic tension and chronic tension headaches. we used positron emission tomography (pet) to measure local cerebral blood flow in volunteer subjects while they performed tasks of memory-guided saccades and a visual fixation control. tasks were performed continuously for seconds during emission scans, after bolus injection of h lso. eye movements were verified with electrooculography. areas of significant increase in regional blood flow between tasks were matched to three-dimensional reconstructions of brain magnetic resonance images of each subject. compared to the visual fixation control, saccade tasks evoked bilateral activation in the posterior superior parietal lobule with extension into the inferior parietal lobule. activation was also seen bilaterally in an area of frontal cortex immediately rostra to the precentral gyrus extending from the superior frontal gyrus to the inferior frontal sulcus. the increased blood flow in the posterior parietal cortex most likely corresponded to enhancement of visual attention required during saccades, while that in the frontal lobe, which included the frontal eye fields, indicated activation for saccade motor output. pamela blake, alexander s. mark, martin kolsky, and jorge kattah, washington, dc fifty patients with third cranial nerve (cn) palsy underwent precontrast and postcontrast mri to assess the utility of this study in this clinical context. mri demonstrated an appropriate lesion in cases. six patients had brainstem lesions ( infarcts, mass lesion, cryptic vascular malformation, hemorrhagic shearing injury, with compound q toxicity). lesions of the cisternal segment of the nerve were present in patients ( aneurysms, lymphomas, ophthalmoplegic migraine, viral meningitis, coccidiodomycosis, nerve avulsion), with enhancement of this segment in patients. fourteen patients had cavernous sinus lesions ( lymphomas, nasopharyngeal carcinoma, tolosa-hunt syndrome, cavernous carotid aneurysms, pituitary apoplexy, aspergillosis). eighteen patients, all with history of diabetes or vascular disease, had normal mri results, suggesting microvascular infarction of c n . in patients with cn i palsy, mri can detect the presence of brainstem or cavernous sinus lesions and often can suggest their cause. mri with contrast enhancement can demonstrate involvement of the cisternal segment of cn i in patients with inflammatory or infiltrative processes that previously could not be radiographically demonstrated. our study suggests microvascular infarction does not cause nerve enhancement on contrast-enhanced mri. we describe patients with acute hyperglycemic, hyperosmolal, nonketotic stupor who had ocular flutter or opsoclonus clinically. these are the fourth and fifth adult patients reported with the acute onset of stupor and opsoclonus; all patients had nonketotic hyperglycemia and hyperosmolality (rapid eye movement sleep also causes stupor and saccadic eye movements). three of the patients had myoclonic jerks in addition to opsoclonus. in the , opsoclonus began when glucose and osmolality acutely increased, and completely resolved when glucose and osmolality became normal, showing that opsoclonus is a specific and reversible effect of the metabolic disorder and implying that either acute hyperglycemia or hyperosmolality directly causes opsoclonus. since acute hyperosmolality caused by nacl or sucrose can cause a similar syndrome in experimental animals (trans am neurol assoc ; : - ) and infants, hyperosmolality is probably more important. opsoclonus is thought to be due to abnormal activity of saccadic "burst" cells in the pons. acute hyperosmolality may cause spontaneous saccades by disinhibiting burst cells from normal "pause" cell inhibition or by directly activating burst cells. the combination of acute deterioration in mental status and either ocular flutter or opsoclonus should suggest acute hyperosmolality, in particular, nonketotic hyperglycemia. neural cell adhesion molecule (n-cam) and the related cell adhesion molecule l play significant roles in axon outgrowth and mediation of cell-cell contact in development, and after peripheral nervous system injury. to understand the role of these molecules after injury in the adult cns, we studied alterations in n-cam and l in the rat brain's response to entorhinal cortex (erc) lesion. post lesion, reactive synaptogenesis and axonal sprouting follow a well-defined temporal course in restoring the synaptic density of the dederented outer two-thirds of the hippocampal dentate gyrus molecular layer (ml) to near prelesion levels. we found striking regionand lamina-specific staining of n-cam and l in the normal hippocampus and marked alterations in these molecules after injury. embryonic n-cam, present in high amounts during development but expressed at very low levels in the adult hippocampus, was massively re-expressed in the denervated zone; the embryonic form was still heavily expressed days later, when synapse number returned to >so% of prelesion levels. l staining, normally evenly distributed through the ml of the dentate, was completely lost in the outer ml, which is denervated by the erc lesion. this staining had not returned by days after lesion. neural cell adhesion molecules play a role in specificity of neural connectivity both in development and after injury. in reactive synaptogenesis and axonal sprouting after injury, both ontogenetic (the reexpression of embryonic epitopes) as well as uniquely adult sequences of repair are utilized. following hemispherectom y" a. pascual-leone, h . t. chugani, l. g. cohen, j . p . brasil-neto, e. m . wassermann, j . and m. hallett, betbesh, md, and los angeles, ca we studied subjects, aged months to years, who underwent hemispherectomy months to years earlier for intractable epilepsy. all had a spastic hemiparesis contralateral to the resected hemisphere, which was present presurgically. we used focal transcranial magnetic stimulation to map the areas of the preserved hemisphere targeting the abductor pollicis brevis (apb), the biceps, and the deltoid ipsilaterally and contralaterally. in subjects who had hemispherectomy after age , the same area targeted ipsilateral and contralateral muscles. in the remaining subjects, who were more functional, areas targeted ipsilateral muscles. one area coincided with the contralateral representation, but stimulation induced motor-evoked potentials (meps) of lower amplitude and longer latency in the ipsilateral muscles. the other area, to cm anterolaterally, targeted exclusively ipsilateral muscles and stimulation induced meps of normal amplitude and latency. this separate ipsilateral representation was more distinct in subjects studied a long time after the hemispherectomy and in those who were younger at the time of the operation. these results show evidence of motor reorganization after hemispherectomy. better motor function is associated with topographically differentiated ipsilat-era and contralateral representations, which may depend on age at the time of hemispherectomy and the time since then. cynthia l. comella, glenn t . stebbins, nancy brown-toms, and christopher g. goetz, chicago, il in a single-blind, crossover study, we evaluated the effect of an intensive outpatient physical rehabilitation program (re-hab) on the severity of parkinson's disease (pd). the re-hab program consisted of -hour sessions per week for weeks. sixteen patients completed phases, a rehab phase and a control phase, separated by months. the order of participation in each phase was randomized. all patients were evaluated using the unified pd rating scale with subscales for mentation (ment), activities of daily living (adl), and motor function (mot) by an investigator blinded to the rehab phase of the patient. all patients were evaluated immediately before and after each phase. pd medications were not changed during any phase. following the re-hab phase, there was significant improvement in adl score (pre-rehab , post-rehab , p = . wilcoxon) and in mot score (pre-rehab , post-rehab , p = , wilcoxon), but no change in ment score. after the con-trol phase, there was no significant change in any outcome measure. this is the first controlled, crossover study of an intensive rehab program in pd. it demonstrates that re-hab improves objective motor function and adl scores. sensitive and quantitative measurements of leg weakness, one of the most common deficits in multiple sclerosis (ms) patients, can be made using specialized extremity testing equipment. to determine whether such determinations could be used in clinical trials, ms patients with leg weakness that had been stable for at least months were evaluated every days over a -month period. each testing session was carried out at the same time of day and medication dosages and schedules were kept constant (only patient was taking baclofen and his dosing remained constant). quadriceps and hamstrings strengths were measured in isometric contraction in a mechanical testing apparatus (kincom). variability for each series of determinations was expressed as the standard deviation of the mean as a percent of the mean. the overall variability then was expressed as the mean of the individual variabilities. the mean variability for the strength determinations over months was . k . , and only one series of determinations out of had greater than % variability. these results suggest that quantitative strength determinations might be useful in clinical trials, and early experience in trials of aminopyridines will be discussed. polymyositis (pm) is an inflammatory myopathy of unknown cause, but the accumulating data strongly suggest an autoim-mune pathogenesis. the histological picture is of muscle fiber necrosis and inflammation, whereas in postviral fatigue syndrome (pfs), a disorder characterized by severe fatigue with myalgia and psychiatric symptoms, the histological picture of muscle is essentially normal. enteroviruses have been implicated on epidemiological and serological studies in both. we have used the polymerase chain reaction (pcr) and an enteroviral-specific probe and found persistent enteroviral genomic material in both pm and pfs muscle biopsy specimens. furthermore, we used a radiolabeled full-length cdna probe derived from coxsackie b in an in situ technique to look for viral d n a in pcr-positive cases. coxsackie genome was clearly identifiable in the muscle biopsy specimens of patients with pm but negative in pcr enteroviral-positive cases of pfs. the virus excites an inflammatory reaction only in pm. a murine animal model for pfs developed in our laboratory showed positive muscle pcr using enterovirus probes and a conspicuous increase in interleukin within the brain. these results provide major clues in the search for the etiology of these two puzzling disorders. human t-lymphotropic virus type i (htlv-i) is a cause of adult t-cell leukemia and tropical spastic paraparesis. in a specific population of iranian jews originating from the city of mashad, there is a high incidence of htlv-i infection ( . %) and associated t-cell leukemia. we evaluated the incidence of possible correlation between htlv-i infection and spastic paraparesis in israeli mashadi-born jews. we have examined mashadi-born immigrants in a mashadi community center ( men, women, mean age t_ . yr) and non-mashadi iranian-born jews. blood samples were tested for htlv-i antibodies by particle agglutination test. the polymerase chain reaction (pcr) was used to amplify htlv-i sequences of d n a from peripheral blood mononuclear cells. twelve mashadi-born immigrants ( %) were seropositive for htlv-i. in of those serologically positive for htlv-i ( %), neurological examination revealed spastic paraparesis of varying severity. none of the non-mashadi iranian jews were seropositive for htlv-i or had clinical signs of spastic paraparesis. these results support other studies of htlv-i-associated myelopathy. the high incidence of htlv-i-associated spastic paraparesis in the mashadi community might be related to their unique history of a high rate of intermarriage among members of this ethnically segregated group. further epidemiological studies are underway to evaluate the incidence of htlv-i in mashadi families as well as in mashadi-originating jews born in israel, to identify whether infection might be genetically transmitted. lymphotropic virus t y p e i-associated acute t-cell leukemia/lymphoma william j . harrington, jr, william a. sheremata, susan snodgrass, and mark raven, miami, fl human t-cell lymphotropic virus type i (htlv-i)-associated acute t-cell leukernia/lymphoma (atl) is thought to produce important c n s disease infrequently. we wish to correct this impression by presenting neurological findings in patients seen between and the present. all had positive western blots to htlv with polymerase chain reaction confirmation of htlv-i infection. only had a concomitant human immunodeficiency virus infection. all were black, aged to years, were men and women. all but americans came from the caribbean nations of haiti ( , dominican republic (l), jamaica ( ), and trinidad ( ). sexual transmission was the risk factor for htlv-i for all except for intravenous drug user. all patients were systemically ill. three had preceding neurological abnormality ( , months, and yr) and had major neurological disease concomitantly. two of these had tumor masses demonstrated in brain and in the spinal canal. one had a minor facial sensory abnormality; only had no deficits. we conclude that cns disease is commonly associated with atl, and that in the us atl occurs principally in caribbean natives. a. nath, v . hartloper, and m . furer, winni$eg, manitoba, canada microglia and astrocyte cultures were established from human fetal brain. microglia were infected with human immunodeficiency virus (hiv) strains, hiv,, or hiv,,,, resulting in a rising titer of p antigen in the supernatants. multinucleated giant-cell formation, vacuolar changes, and rising levels of lactate dehydrogenase in the supernatants were seen, indicating a cytopathic infection of microglia. astrocytes were infected with free virus or cocultivated with an hiv-infected lymphocyte cell line (hut- ). after a productive phase (rising titers of p antigen and detection of hiv antigens by immunocytochemistry), the cells went into a latent phase where hiv could be detected only by dna polymerase chain reaction. a -fold increase in astrocytes staining for hiv antigens was seen after cocultivation with hut- cells. lymphocytes adhered to astrocytes by hours of cocultivation. no adhesion was seen to microglia. fusion of plasma membranes was seen on electron microscopy. infected astrocytes did not show cytopathic or morphological changes. cell-to-cell contact may be important in viral transmission to astrocytes. hszao-huei chen, steven b. stein, wing kong, and raymond p. roos, chicago, i l one of our goals is tq delineate molecular determinants for disease phenotypes produced by theiler's virus (tv), a mouse picornavirus. the identification of these genes and gene products may clarify viral pathogenesis and also lead to the identification of genes that are important in normal cns function and nonviral cns disease. members of the gdvii subgroup of tv cause an acute, fatal neuronal infection, whereas members of the to subgroup are less neurovirulent and produce a demyelinating persistent infection. our studies of infectious tv cdna clones have demonstrated that the gdvii b(vp )- c segment is critical for neurovirulence. several other areas of the genome, including the ' untranslated region (s'utr), also affect tmev-induced disease. the 'utr of poliovirus, another picornavirus, has a critical role in paralysis; this effect on neurovirulence is believed to result from an altered translational efficiency related to bind-region ing of neural cell proteins. tv 'utr has an unusual predicted secondary structure, even for picornaviruses. our studies demonstrate that the tv 'utr affects translational efficiency and has a distinctive protein-binding pattern. investigations of the tv 'utr may clarify features of translational regulation of cns genes in general. p . coronaviruses infect primate brain from g a y f. cabirac, ronald s. murray, galen cai, kristen hoel, and kenneth soike, englewood, co, and covington, la recently, we described finding coronavirus (cv) rna and antigen in active demyelinating plaques of multiple sclerosis (ms) brain tissue (murray et al, ann neurol, in press). molecular analysis showed the cv rna to be more closely related to murine cvs than to human cvs. we then demonstrated that, following intracerebral inoculation, the murine cv jhm and the putative ms isolate cv-sd could infect and cause demyelination in primate brain (murray et al, virology, in press). we now have data showing that murine cv can infect primate brain following intranasal or intravenous routes of inoculation. standard virology, histopathology, and the molecular analysis of viral cytotropism will be presented. we conclude that cvs related to murine cvs can infect primate cns from peripheral routes and warrant consideration as potential human pathogens. probes (gag, pol, or enw). eleven were white and were black. a history of transfusion was obtained in , and sexual risk of transmission was present in but not in others. fulminant disease occurred in transfused men months to years later but such disease was only seen in woman (in month). in contrast, of women with multiple sexual partners had rapid progressive disease. the male-to-female ratio was : for transfusion association and : for those at sexual risk but : for unknown risk, transfusion is an important risk for tsp/ham and the diagnosis must be considered in all gait problems, regardless of the "diagnosis."transfusion association should decrease with regular testing of blood donors, but this will not affect the risk of sexual transmission. spastic paraparedhtlv-i-associated myelopathy (tsp/ ham). it is unclear why htlv-i infection causes atl in some individuals and tsplham in others. differences in the genome of viral isolates or immunological and host factors have been hypothesized to play a role in disease expression. at present there are no animal models of tsplham and no suitable animal models of htlv-i infection that can address these issues. we transplanted severe combined immunodeficient (scid) mice with peripheral blood mononuclear cells (pbm) from tsplham patients in an attempt to produce htlv-i disease. two weeks after transplantation of pbm from tsplham patients, we detected anti-htlv-i igg in serum samples of of scid mice by enzyme immunoassay using sonicated whole virus. five weeks after transplantation, we detected anti-htlv-i igg to p , p , gp , or gp in serum samples of of scid mice by immunoblot of disrupted virus. these findings suggest that scid mice may be valuable in the study of molecular and pathological determinants of htlv-i-induced disease. theiler's virus produces an encephalomyelitis in susceptible mice. during the course of the disease, specific regions in the cns become infected. compared to the immunocompetent mouse, the nude mouse provides a useful model where viral dissemination can be studied in the absence of functional t lymphocytes and antibodies. we investigated the distribution and spread of the da strain of theiler's virus in the cns of nude mice. by immunohistochemistry, the hippocampus, arnygdaloid nuclei, entorhinal cortex, cingulate cortex, thalamus (anteroventral nuclei), midbrain, and spinal cord all contained viral antigens by weeks after infection. in addition, the olfactory nuclei, mamillary body, hypothalamus, basal ganglia, and nucleus raphe dorsalis often were involved. in the brain, the limbic system was the site commonly infected by theiler's virus. the time course of virus dissemination varied depending on the site of initial virus infection, though the final distribution of virus was the same. olfactory bulb injection, which is a direct inoculation into the olfactory pathway, resulted in more rapid spread than did cortex injection. we demonstrated the constant presence of viral antigen in the limbic system and a different kinetics of viral dissemination between the two different routes of intracerebral inoculations. these results suggest that limbic structures and their connections are important to the dissemination of theiler's virus. -associated myelopathy in a northwest native indian d. foti, d. werke, g. dekaban, g. p. a . rice, andj. oger, vancouver, bc, and london, ontario, canada a -year-old indian of the oweekeno tribe was admitted for investigation of a myelopathy. initially symptomatic with midthoracic radicular pain, she developed progressive spastic paraparesis over year with mild sensory symptoms and urinary retention. mri of the cord and brain were normal. csf showed elevated protein ( mgll), lymphocytes, and weak oligoclonal banding with evidence of intrathecal igg synthesis. antibodies to human t-lymphotropic virus type i (htlv-i) were positive by enzyme-linked immunosorbent assay and western blot on both serum and csf. presence of htlv-i was demonstrated by polymerase chain reaction of blood lymphocytes. htlv-i-associated myelopathy, or tropical spastic paraparesis, is endemic in southern japan, the caribbean basin, and several tropical islands but has not been reported in natives of northwest canada. this patient's only risk factors for htlv-i infection were blood transfusions years previously. ongoing familial and epidemiological studies as well as virus sequencing should indicate if this case represents an indigenous or an imported infection. caused by herpes simplex virus type lawy blankensbz) and herbert . newton, columbus, oh myeloradiculitis occasionally occurs secondary to herpes simplex virus type (hsv ) infection, but rarely has been reported after herpes simplex virus type (hsv ) infection without encephalitis. we describe a -year-old man who developed cervical myelopathy and radiculitis, never developed symptoms of encephalitis, and had positive hsvl spinal fluid cultures. h e initially developed extremity weakness and incoordination, numbness, paresthesias, and neck pain. evaluation was negative except for mri results, which showed a high-signal lesion centrally within the cervical cord. the weakness, sensory loss, and radicular pain progressed over several months. subsequent mri showed extension of the high-signal abnormality and mild enlargement of the cervical cord. symptoms stabilized briefly with dexamethasone but soon worsened, and were accompanied by paroxysmal kinesiogenic dystonic episodes of his arms and right leg. repeat evaluation was unrevealing except for the spinal fluid, which grew out hsv . the patient was treated with dilantin and a i-month course of intravenous acyclovir, with slow improvement of neurological status and resolution of the dystonic episodes. this case illustrates that hsvl can cause a myelopathy with a subacute and protracted course, requiring serial was more frequent in the middle-aged group ( p < . , p = . ). history of hypertension, previous strokes, diabetes mellitus, and antithrombotic treatment was similar, as were sex ratio, qualifying event type (transient ischemic attack or nondisabling stroke), and angiographic features; stenosis severity in either side and presence of ulceration were all similar. in elderly patients, ecad is associated with symptomatic cardiac disease (scad or af), whereas in middleaged patients it is associated with precursors of generalized atherosclerosis (smoking and hyperlipidemia). to identify the anatomic factors correlating with dementia in patients with lacunar infarctions, we examined digitized ct data on elderly patients (mean age = . yr; education = yr) who presented with acute lacunar infarction. dementia was diagnosed in patients ( . %) based on neuropsychological tests given months after stroke onset. the following ct variables were assessed: infarct location and number, total infarct volume, brain parenchymal and csf areas at levels, and width of the frontal horn, third ventricle (tvw), and lateral ventricle plus their ratio to the intracranial width. atrophy and leukoaraiosis were rated semiquantitatively using a standard scoring method. in the group overall, mean infarct volume was . cc and mean infarct number was . . in univariate analyses, dementia was significantly related to infarct volume, number, tvw, bilaterality, and infarct predominance on the left side, but not to leukoaraiosis or atrophy. in a regression model adjusting for demographic factors, the ct variables correlating independently with dementia status were infarct number (p = . , p = . ) and the presence of left cerebral infarcts (p = . , p = . ). we conclude that the most important ct variables related to dementia in lacunar stroke are lesion multiplicity and the presence of lesions on the left side. brain ischemia results in potassium (k+)-induced voltageregulated presynaptic calcium (ca") accumulation, which may contribute directly to neuronal in jury presynaptically, and also promote excessive release of excitatory neurotransmitters leading to cell damage postsynaptically. k+-induced depolarization of brain synaptosomes may be used as an in vitro model to study the therapeutic potential of pharmacological agents to alter ischemia-induced presynaptic ca + accumulation. we preincubated gerbil cerebral cortical synaptosomes in r (janssen pharmaceutical) at concentrations of to lo-' m, subsequently depolarized the synaptosomes with k + , at concentrations of to mm, and measured intrasynaptosomal ca ' ([ca +]) with the fluorescent indicator fura . r had no effect on ecaz'i in nondepolarized synaptosomes, but significantly (<. , student t ) depressed depolarization-induced {ca +] at to lo-' m in a dose-dependent fashion. the greater the degree of depolarization employed, the greater degree of depression in [ca +] was seen at each dose of r . since r had no effect on [ca +] when utilizing ca +-free incubation media, it was demonstrated that r prevents depolaritation-induced [ca ' ] increase by blocking voltage-regulated influx. because r appears to block voltage-regulated presynaptic ca ' accumulation, it should be further evaluated as a potential therapeutic agent after cerebral ischemia. sneddon's syndrome (ss) is a focal and diffuse arthropathy affecting mainly the vascular wall of the skin and cerebral arteries. etiology is not determined. we studied patients with ss ( females, males), aged to years. clinical, radiological, and immunological studies were done. all patients developed cerebrovascular disorders: ischemic stroke in %, transient ischemic attack (tia) in %, and ischemic stroke and tia in %. cerebral scan showed small and medium (less than cm) ischemic lesions in %. these lesions were superficial in the cerebral cortex ( %); deeper in the centrum semiovalis, internal capsule, and basal ganglia ( %); and both superficial and deeper ( %). ultrasound and angiogram studies revealed obstruction of intracranial arteries in %, and of extracranial arteries in %. partial or complete improvement of cerebrovascular symptoms was observed in %. immunological studies showed increased content of b-cell lymphocytes ( p < . ), increased levels of igm ( p < o.oool), and circulating immunocomplexes ( p < . ). anticardiolipin antibodies were increased ( %) and lupus anticoagulant detected ( %). cerebrovascular disorders in ss that lead to small ischemic cortical lesions have a good prognosis. pathogenesis of this syndrome may be related to antiphospholipid antibodies. the cheiro-oral syndrome is characterized by pure sensory deficit limited to the hand and mouth. this syndrome has been described in diverse lesions of the parietal operculum and brainstem, but occurs most commonly due to lesions of the contralateral thalamus, and suggests a humuncular representation of sensation in the ventral posterior lateral (vpl) and ventral posterior medial (vpm) nuclei. we describe a -year-old hypertensive woman who presented with sudden onset of numbness of the left side of her body. examination revealed a left hemisensory deficit to all modalities that spared the hand and peri-oral regions. cat scan and mri demonstrated an acute infarction of the posterolateral right thalamus, with a rim of preservation adjacent to the internal capsule. pure hemisensory loss with sparing of the hand and mouth ("inverse cheiro-oral syndrome") has not been reported previously, and complements previously published studies of the cheiro-oral syndrome in demonstrating somatotopic sensory representation in the thalamus. to examine the relationship between depression and dementia after stroke, we administered the -item hamilton depression rating scale (hdrs) and neuropsychological tests to elderly patients months after ischemic stroke. using dsm- -r criteria, we found dementia in ( . %). hdrs score was . -t . overall, and higher in demented compared to nondemented patients ( . . vs . t . , p = . ). the frequency of depression (total hdrs score > ) was also higher with dementia ( . % vs . %, p = . ). however, demented patients did not differ from nondemented patients on ratings of depressed mood. instead, hdrs items for psychomotor retardation, reduced work activities, and impaired insight best distinguished the groups. in multiple regression analysis, stroke severity (p = . , p = . ) was the most important correlate of hdrs score; dementia status was not correlated independently. mean scores on mini-mental state examination and neuropsychological tests assessing memory, orientation, verbal, spatial, attentional, and abstract reasoning skills did not differ by depression status. although weakly related to intellectual impairment, hdrs score in our stroke sample was most importantly associated with stroke severity. higher scores on hdrs in demented stroke patients may be explained by physical and cognitive symptoms that are expected with dementia. these findings do not support a causal link between depression and dementia, and argue against the importance of "depressive pseudodementia" as an explanation for intellectual decline after stroke. to investigate the pathophysiological mechanism of the white matter lesions in progressive subcortical vascular encephalopathy (psve) of binswanger type, we measured regional water partition coefficient (pc) (reflecting water content) and effective p h (pht) (weighted average of intra-and extracellular ph) with dynamic positron emission tomographic technique using - co,, o,, and c- co,. si-multaneously, regional cerebral blood flow (rcbf) and regional cerebral metabolic rate of oxygen (rcmro,) were evaluated. eight subjects ( normal, psve, multiple infarction) were examined. in the white matter lesions in psve, corresponding to high-intensity areas in t -weighted images of mr, the pc increased and pht was unchanged or decreased, whereas both rcbf and rcmroz declined. the ratio of white matter pc to gray matter pc was . in psve and . in normals. in the frontal gray matter, the pc decreased in psve, whereas rcbf and rcmroz also decreased. these results suggest that tissue water content increases in the white matter lesions in psve reflecting the edematous status of the damaged regions. elevated pht with high pc may reflect the increase of extracellular water of the tissue. measurement of pc and pht provides useful information about the pathogenesis of psve. stroke has been the second most common cause of death in korea but its risk factors (rfs) have not been studied intensively, so the rfs or causes were investigated prospectively in , consecutive stroke patients who were admitted to the chungnam national university hospital, taejon, korea, between and . they included cases of cerebral ischemia (ci) ( . %), cases of intracerebral hemorrhage (ich) ( . %), and cases of subarachnoid hemorrhage (sah) ( . %). control data were obtained from healthy spouses of the patients. multivariate analyses showed that hypertension (ht) was the strongest rf for all stroke types and was followed by old age, diabetes mellitus (dm), smoking, high-density lipoprotein cholesterol, and fibrinogen. the rfs for atherothrombotic ci included old age, ht, dm, smoking, fibrinogen, and cholesterol. for lacunar infarcts, ht, dm, old age, fibrinogen, alcohol abuse, smoking, and female sex were the significant rfs. ht was the cause of ich in ( . %) and alcohol abuse ( , . %) and vascular anomalies ( , . %) followed. most alcoholassociated ichs occurred characteristically in the posterior fossa. frequencies of hospital admission of ich patients correlated positively with diurnal variation of temperature ( r = . , p < . ). aneurysmal rupture was the cause of sah in patients ( . %). three major sites of aneurysms identified on cerebral angiograms were the anterior communicating artery ( , . %), the middle cerebral artery ( , . %), and the posterior communicating artery ( , . %). thirty-nine patients ( . %) had no identifiable cause of sah. in korea curvilinear subinsular lesions have been noted on ct but the underlying pathology is unknown. we reviewed the cranial mr scans ( . tesla ge machine) of serial patients over the age of who had no cause for mr hyperintensities (hi) other than age or vascular risk factors. seven ( %) had linear subinsular h i (sihi). four of ( %) patients with sihi and of ( %) without sihi had marked periventricular h i compatible with subcortical arteriosclerotic encephalopathy (pvhi) ( p < . ). patients with sihi were older ( . yr) than patients without sihi ( . yr) ( p < . ). four of ( %) patients with sihi had hypertension program and abstracts, american neurological association or vascular risk factors. a further patient with diabetes, hypertension, and the opercular syndrome (bilateral facial, pharyngeal, and lingual weakness) had subinsular lesions on ct. the brain arteries were injected postmortem with a lead/ gelatin suspension, and mr of the whole brain and x-ray films of the brain slices were taken. bilateral sihi were seen on mr and corresponded with linear cavitary infarction pathologically, which on microangiograms was found to lie in the border-zone between cortical and basal penetrating arteries. we conclude that sihi are associated with older age and pvhi, and can be due to infarction in a deep watershed territory and can be associated with clinical deficits. hemodilution-treatment results in consecutive cases james l. frey, phoenix, az because precipitous neurological deterioration occurred during blood pressure reduction in a seminal case of lacunar infarction, subsequent patients with partial or evolving lacunar deficits were treated with hemodilution and blood pressure nonintervention to test the hypothesis that lacunar strokes represent perfusion failure. isovolemic hemodilution was performed using hetastarch with target hematocrit of to . results of pretreatment ct brain scans, carotid ultrasound, and echocardiograms were normal. nine patients recovered normal neurological function, and regained complete functional independence in close temporal correlation with hemodilution. mri brain scans demonstrated appropriate single white matter lesions in cases. no specific risk factor combination could be identified. n o patient has had recurrent stroke in follow-up from to months. response to hemodilution suggests a hemodynamic pathophysiology. successful treatment requires ( } blood pressure nonintervention and ( ) hemodilution prior to severe clinical deterioration. the hemodynamic classification for the carotid-cavernous sinus fistula (ccf) is important for the implication of prognosis and therapy, but satisfactory objective criteria for such differentiation is still lacking. retrospectively, we studied the application of extracranial duplex sonography in cases of ccf with emphasis on the hemodynamic parameters of resistivity index and flow volume. a correlation was made with the angiographic findings in an attempt to evolve an objective hemodynamic classification by this noninvasive method. the alterations in membrane metabolism and structure could be the primary etiological event in alzheimer's disease (ad) that results in the clinical and neuropathological findings. to investigate in vivo brain membrane phospholipid and highenergy phosphate metabolism in probable ad patients and control subjects, the building blocks (pme) and breakdown products (pde) of membranes and the high-energy phosphates pcr and atp were measured noninvasively by in vivo brain p mrs in probable ad patients ( males; females) and controls ( males; females). all subjects were assessed by mini-mental, mattis, and blessed scales. we found that, at clinical onset, ad females had elevated pme ( p = . ), decreased pcr ( p = . ), and decreased atp ( p = . ). similar changes were not seen in ad males at clinical onset, but the severely demented ad males had increased atp ( p = . ). correlation analysis for the ad patients revealed that increasing dementia was associated with decreasing pme ( p = . ; r = . ), increasing pde ( p = . ; r = . ), increasing pcr ( p = . ; r = . ), and increasing atp ( p = . ; y = . ). similar metaboliccognitive correlations were not seen in the controls. these results demonstrate alterations in membrane and energy metabolism at the earliest clinical stages of ad. increasing dementia correlates with markers of membrane degeneration and decreased utilization of high-energy phosphates. both of these findings suggest the dementia in ad is secondary to membrane changes resulting in synapse loss. alzheimer's disease: postmortem mri and histological correlates fen-lei f. chang, j. e. purisi, c. r. jack+ jr, and r. c. petersen, rochester, mn hippocampal atrophy, defined by mri-derived volumetric measurement, has been useful in differentiating alzheimer's disease (ad) patients from normals. since several studies have demonstrated anatomical and functional gradients along the rostral-caudal (r-c) axis of the hippocampus, it is tempting to speculate on the existence of a differential distribution of morphometric changes along this axis. the hippocampi from patients with clinically and pathologically confirmed ad were studied by postmortem mri and by reconstruction of serial histological sections. there was good correspondence between these two methods. the r-c length of the hippocampus in ad was preserved compared to normal. this length was longer on the left, both in normals and ad. the adassociated atrophy was due primarily to the reduction of the coronal cross-sectional area of the hippocampus. with increasing hippocampal atrophy, volume reduction was more prominent in the rostral area (pes hippocampus). this nonuniform reduction in volume may be associated with different connectivity patterns between rostral and caudal hippocampus. the purpose of this study was to determine the neuropathological validity of nincds-adrda criteria (nac) for probable and possible ad. mckhann et al ( ) provided clinical criteria for the categories probable ad and possible ad. the validity of these categories has not yet been reported. a retrospective, blinded evaluation of the complete neurological history, examination, neuroimaging, laboratory, and psychometric data was done for subjects from the state of florida brain bank. pathological classification, which was blind to clinical diagnoses, was in categories: pure ad, ad + , and other dementias. ninety-three percent of probable ad (n = ), whereas only % of possible ad (n = ) patients, had ad or a d + ( p = . ). pure ad was found in % of probable ad and % of possible ad patients ( p = . ). pathological evidence of coexisting parkinson's disease was present in % of all ad brains. these results suggest differential predictive power of nac for probable and possible ad, as suggested by the labels. nac are, therefore, usefui for research and clinical purposes. our prior study of falls in the elderly had shown significantly more white matter low attenuation (wmla) on ct scan among fallers than nonfallers, but no association between wmla and cognitive impairment among nondemented subjects. as these subjects were followed over time, it appeared that fallers were becoming demented at a more rapid rate than nonfallers. as a result, we analyzed the relationship between wmla and rate of change on the blessed test of information, memory, and concentration (bimc) in a combined cohort of subjects from the falls study and from a longitudinal study of dementia and normal aging. we selected of these subjects whose initial bimc score was less than (i.e., not already severely impaired) and who had at least yearly bimc evaluations. ct scans were scored on an -point ordinal scale for hemispheric wmla. linear regression was used to summarize the rate of change for each subjects' test scores. the rate of change on bimc was . points per year with standard error (se) of . among subjects who eventually became demented; nondemented subjects declined at . points per year with se of . . multiple regression analysis was performed with initial bimc score and wmla as the independent variables and rate of change of bimc as the dependent variable. wmla accounted for % of the variance (partial r = . , t = . , p < . ). these data suggest that elderly subjects with wmla may be at increased risk for rapid cognitive decline. such as g proteins and adenylate cyclase. the activities of adenylate cyclase, and of the g protein-associated enzyme activity, low-km glutamyl transpeptidase (gtpase), were assayed in membranes prepared from the postmortem brains of alzheimer-diseased and age-matched control subjects. both basal and fluoroaluminate-stimulated adenylate cyclase activities were significantly reduced in ad frontal cortex compared to control subjects ( p < . ; two-tailed student's t test). in addition, a significant, though smaller, reduction in basal gtpase activity also was detected in ad frontal cortex ( p < . ). in contrast, no significant change in the activity of either enzyme was detected in the hippocampus. the stimulation of gtpase activity by muscarinic and gababreceptor agonists was not altered significantly by the presence of alzheimer's disease. however, the degree of stimulation was much lower in human tissue compared to that observed using fresh rat brain, suggesting that the ability of receptors to activate g proteins declines post mortem. these results suggest that alzheimer's disease causes alterations in some key components involved in signal transduction. the association of lobar hemorrhage (lh) with cerebral amyloid angiopathy (caa) and that of caa with alzheimer's disease (ad) are well known. to determine how frequently lh and caa occur in ad, we reviewed patients with cerebral or cerebellar hemorrhage and caa. five patients were treated surgically, none was demented, died, and came to autopsy. eight patients died of lh and came to autopsy. of the autopsied patients, had ad, both clinically and neuropathologically. they comprised . % of cases of autopsy-confirmed ad in our laboratory. none of the other patients were known to be demented. five had senile plaques, with or without neurofibrillary tangles, in the hippocampus, and occasional senile plaques in the cortex, but none met the consortium for establishing a registry for alzheimer's disease neuropathological criteria for ad. ad patients were , , and years old and the ages of the nondemented patients ranged from to (mean = . yr). seven were younger than years of age. despite the frequent occurrence of caa in ad, we found that lh was uncommon. in addition, most of our patients with lh and caa were not demented and tended to be younger than ad patients with lh. these results indicate that the hf is involved primarily in acquisition or leaning processes and less so in retrieval of previously learned information. these findings relate to memory and structural brain changes found in normal aging. alzheimer's disease y . stern, l. stricks, g. alexander, . prohovnik, and there is an inverse relationship between parietotemporal cerebral blood flow and years of education in alzheimer's disease (ad) patients matched for clinical severity, which suggests delayed clinical manifestation of ad in patients with higher education (stern et al, soc neurosci abs ). we classified the lifetime primary occupations of ad patients using the dictionary of occupational titles of the us department of labor and derived factor scores describing intellectual, interpersonal, and physical job demands. after controlling for age, education, age at onset, illness duration, and dementia severity (mental status and activities of daily living), relative perfusion in the parietotemporal region (assessed using -xenon inhalation) showed significant correlations with job complexity (pl: r = -. , p < . ) and interpersonal (p : r = -. , p < . ) factor scores. in a stepwise multiple regression, job complexity and interpersonal skills increased explained parietotemporal flow variance by . % ( f = . , p < . ) over that explained by demo-graphic and severity indices; physical demands then accounted for another . % of the variance ( f = . , p < . ). we conclude that occupational demands, similar to but independent of education, may provide a reserve that delays the clinical expression of ad. richard mayeux and ming-xin tang, new york, n y risk factors for alzheimer's disease (ad) were collected from patients with ad and healthy elderly controls in an urban community population consisting of ethnic groups: black, hispanic, and white. advanced age (> yr) (or = . ; % ci . - . ) and head injury with loss of consciousness (or = . ; . - . ) were associated with ad, controlling for all known putative risk factors. factors such as low education (< yr) (or = . ; . - . ) and family history of ad (or = . ; . - . ) were not found to be significantly related to ad. head injury occurred in . % of the patients and .?% of controls. most ( %) head injuries in the patients with ad occurred after age , prior to disease onset. in controls with head injury, had experienced a head injury before age . the duration of unconsciousness was consistently longer in patients with ad than in the controls. the overall effect in each ethnic group was similar (or,, . ; . - . ). these results confirm and strengthen the previously described putative relationship between head injury and ad. we also conclude that both the severity and the timing of the head injury as well as the frequency of head injury in the population at risk may be important factors in understanding the causal relationship between head injury and ad. the purpose of this study was to determine how native language affects the cutoff scores in screening tests for dementia. there is a paucity of data o n this issue at present. screening tests used in the study were: folstein mini-mental state (mms); clockdrawing (clock); preparing a letter for mailing (mail); -item grocery list (list); and hamilton depression scale (ham). the subjects included ( demented) native english speakers (eng) and ( demented) native spanish speakers (spa) with memory complaints. diagnosis of dementia was determined by neurological, neuropsychological, and psychiatric evaluation. age and gender were unrelated to mms. in spa only, education was positively related to mms. ham scores were higher in de- and . (spa); c d and list did not add discriminative power to mms for eng but did so for list in spa. mms discriminates between demented and nondemented far better in english than in spanish speakers. only mail adds discriminative power to mms. a. heyman, g. fillenbaum, s. mirra, and participating cerad neuropathologists, durham, nc, and atlanta, ga the clinical diagnosis of alzheimer's disease (ad) has become more accurate in recent years due to the application of specific clinical criteria, wider use of neuroimaging procedures, and greater expertise among physicians. we report the frequency of clinical misdiagnosis of ad among patients who at autopsy were found to meet the rigorous clinical diagnostic criteria imposed by the consortium to establish a registry for alzheimer's disease (cerad) study. these patients included men and women (mean ages and yr, respectively) who were among the group of patients who died in cerad medical centers in the us between and . the clinical diagnosis of ad was neuropathologically confirmed in ( . %) of the cases. of these cases, varying degrees of concomitant cerebrovascular disease were present in % and coexisting parkinson's disease changes were found in %. in of the patients without neuropathological evidence of ad, the diagnoses were: lobar atrophy, diffuse lewy body disease, nonspecific neurodegenerative changes, and mesocorticolimbic dementia, respectively. the fifth patient showed no morphological abnormalities. on the basis of these results, it would appear that application of strict diagnostic criteria, as well as the use of brain scans and detailed clinical and neuropsychological tests by experienced clinicians, cannot yet distinguish some types of primary degenerative dementias from alzheimer's disease. we designed a scale that measures an underexplored facet of functional decline in alzheimer's disease (ad): the patient's dependence on others for supervising or performing activities. two hundred twenty-three informants for patients with mild ad (clinical dementia rating [cdr] = for ; cdr = for ) were interviewed and dependence was staged from to . interrater reliability was assessed by separate interviews of informants; agreement was % for dependence stage. dependence stage differed significantly at the cdr levels (chi square = . , p < . ) and correlated significantly with modified mini-mental state (mmms) ( r = -. , p < . ) and blessed dementia rating scale-part (bdrs) ( y = . , p < . ). seventy-eight percent of patients in a health-related facility were at stage or higher vs % of patients living at home. in a multiple regression model, both the bdrs and dependence scale accounted for unique portions of the variance in mmms, suggesting that they assess unique aspects of functional ability. dependence increased significantly in patients retested at year. we conclude that the dependence scale is reliable and relates to both disease severity and progression. formal assessment of dependence should prove useful for studies of the natural history of ad as well as for clinical trials. cortical-basal ganglionic degeneration (cbgd) is a disorder characterized by an asymmetrical akinetic-rigid syndrome and cortical signs such as apraxia, alien limb phenomena, and cortical sensory loss. dementia has been present in many cases, but always as a late manifestation. we report cases pathologically consistent with cbgd, presenting as primary degenerative dementia, fulfilling nincds-adrda criteria for probable alzheimer's disease (ad). the first patient presented with changes in memory and personality, language dysfunction, decreased verbal output, and shuffling gait. follow-up examinations over years showed progressive dementia, wide-based gait, and frequent falls. the second patient presented with complaints of memory loss. neuropsychological examinations showed progressive deficits in memory, attention, calculations, and visuospatial functioning. no movement disorder developed over years of follow-up. at autopsy, both patients had typical changes of cbgd and lacked pathological features of ad. definitive diagnosis of cbgd rests on both clinical and pathological criteria. cbgd should be considered in the differential diagnosis of patients with dementia resembling that found in ad, especially if extrapyramidal signs are present. a quick, easily administered and scored test for praxis is desirable in evaluation of neurological patients. during months in , each patient seen in the outpatient setting by the principal investigator (e. k.) received of praxis screening batteries. thirty-six patients were tested with a short battery. eleven had normal cognition based on neurological history, examination, and a short test of mental status. twenty-five had cognitive decline (cd). handedness, male/ female ratio, education, and mean age were similar in both groups. mean time of completion of the test was seconds in normals and * seconds in patients with cd. total scores (maximum ) were . * . in the normals and . in the cognitively declined patients. twenty-five other patients, with cd and with normal cognition, were tested with a longer battery containing oral/ facial, upper and lower limb, axial, sequential, and imitation subtests (maximum score ). normals completed the battery in i , and cognitively declined patients completed the battery in ? seconds. of the various subtests, tests of sequential praxis were performed most poorly by patients with cd: . * . in normals vs . * . in patients with cd. oral/facial praxis was least affected by cd: olivopontocerebellar atrophy (opca) is generally understood to be a nondementing neurodegenerative disorder affecting the cerebellum, lower brainstem, and spinal cord. one of us (s. k.) recently reported that postmortem cerebral cortex from patients with dominantly inherited opca shows a widespread reduction of cholinergic markers similar to that observed in alzheimer's disease (ad). we were interested to determine the status of other neurotransmitter systems in opca postmortem cerebral cortex. samples of frontal, parietal, temporal, and occipital cortex were dissected from confirmed cases of opca and age-matched controls. after processing, neuropeptide levels were measured by radioimmunoassay. concentrations of somatostatin were significantly reduced by to % in of the cortical areas of opca brain that were examined. the area that was spared was the inferior temporal gyrus, a region in which somatostatin levels are markedly reduced in ad. levels of neuropeptide y were normal in all areas, while concentrations of cholecystokinin, vasoactive intestinal polypeptide, and substance p were significantly increased in of the areas. these data show widespread neuropeptide changes in the cerebral cortex of opca postmortem brain. in contrast to cholinergic markers, the pattern of neuropeptide changes is different from what is observed in ad. it has been postulated that demise of the corticomotoneuron is the initial event in amyotrophic lateral sclerosis (als) and that the anterior horn cell dies as the result of antegrade glutamatergic excitotoxicity (muscle nerve ; : ). excitability of the corticomotoneuronal system can be tested by measuring threshold-to-cortical magnetic stimulation and the motor-evoked potential (mep)/compound muscle action potential (cmap) ratio, which estimates the number of corticornotoneurons stimulated. cortical threshold and mep/ cmap ratio were measured in patients early in the course of als. the mean time interval from onset of first symptoms was . months. mean threshold and mepicmap ratio measured . f . % and . rfr . %, respectively. in ( . %) patients, threshold was paradoxically low (< %, mean . +. . %) and in ( . %) patients there was no response. there was a significant ( r z = . ) inverse power relationship between cortical threshold and mep/cmap ratio given by . x mep/cmap-'j. six months later, / ( . %) patients still had low thresholds but the mean mep/ cmap ratio had dropped to . ? . % and in . % there was no response. we conclude that early in als the corticomotoneuronal pathways are abnormally excitable. this may explain early cramping and fasciculation, which characteristically diminishes as als progresses. one of the distinct clinical features in patients with amyotrophic lateral sclerosis (als) is loss of elasticity of skin. however, little is known concerning the biochemical nature of skin elastin in als. in our study, cross-links unique to elastin, desmosine and isodesmosine, were measured and compared in skin tissue (left upper arm) from patients with als and from age-matched controls. the contents of desmosine and isodesmosine were decreased significantly ( p < . and p < . , respectively) in patients with als (mean * sd, . ? . and . * . nmol/mg dry weight; range . - . and . - . nmol/mg dry weight, respectively) as compared with those of controls (mean sd, . * . and . . ;range . - . and . - . , respectively), and were negatively and significantly associated with duration of illness in patients with with amyotrophic lateral sclerosis als ( r = - . , p < . , and r = - . , p < . , respectively). the ratio of desmosine and isodesmosine was constant ( : ) in all samples analyzed. the decline in skin desmosine and isodesmosine is more rapid in als than in normal aging. thus, cross-linking of skin elastin is affected in als. (supported in part by n i h grants de , de , de , ar , ar , and nasa grant nag- - .) pl . natural history of amyotrophic ( ) collection of large numbers of twin pairs in disease of low prevalence is difficult. to circumvent this, we devised a new approach termed the "death discordant twin pair" method. eleven thousand deaths from motor neuron disease (mnd) were extracted from the office of population censuses and surveys during to . birth indexes from onward were searched for possible twins. for each twin so identified ( pairs), the national health service central registry located the relevant family practitioner committee and thence the co-twin's general practitioner. the search produced: ( ) living co-twins; ( ) embarked; ( ) dying as adults or infants; ( ) not mnd; and ( ) validity of the accuracy, sensitivity, and specificity of the world federation of neurology (wfn) subcommittee on motor neuron disease working group criteria for the clinical diagnosis of amyotrophic lateral sclerosis (als) has been tested against neuropathological criteria in autopsied patients (neurology, in press). integration of clinical and electrodiagnostic data to meet wfn criteria for possible, probable, and definite als was studied in this samegroup. patients received . ifr . (mean * standard deviation) electromyograms (emgs) per patient and included . . emg levels (bulbar, cervical, thoracic, lumbar) per patient. proportionately fewer emgs were performed as the level of diagnostic certainty at presentation increased: suspected ( . %), possible ( . %), probable ( . %), definite ( . %). in only . % of all patients studied did the first emg alone change the level of diagnostic certainty of the diagnosis of als. only . % of patients presenting with suspected als alone or with possible or probable als were associated with a change in level of diagnostic certainty following or more emgs. however, although increasing the number of emgs performed per patient may be associated with an increasing chance of increasing the level of diagnostic certainty ( emgdpatient = . %; emgdpatient = . %), selection of the level of emg analysis was more crucial. the "el escorial" criteria emphasize the importance of emg evidence of lower motor neuron involvement in a limb with clinical upper motor neuron signs. our analysis of emg studies in autopsy-confirmed als patients suggests that complete evaluation of bulbar and thoracic levels for lower motor neuron changes and complete evaluation of motor unit recruitment patterns are important for the integration of emg data with clinical data in the application of the "el escorial" criteria for the diagnosis of possible, probable, and definite als. sibships on guam annette grefe, john steele, linda flares, and stephen waving, birmingham, al, and umatac and mangikao, guam reports in the s indicated that % of guamanian chamorro patients with amyotrophic lateral sclerosis (als) gave october a positive family history. subsequent investigators have inferred that purely genetic factors are not responsible for guamanian als or its clinical variant, parkinsonism-dementia complex (pdc). we report the first chamorro sibships selected in an ongoing study of familial aggregations. the basis for the initial selection was that the youngest patient in the sibship had als. age of onset was to years (mean yr). fourteen of persons in these sibships were affected. others in the sibship developed pdc, progressive supranuclear palsy, or pure dementia later in life (mean yr, range - yr). these cases developed to years (mean yr) after onset of disease in the first sibling. the age at onset of the first case and the intervals between earliest and latest cases in such sibships may help to determine minimal and maximal latency (i.e., interval between exposure to an exogenous agent and onset of symptoms). our observations suggest that exposure may have occurred early (before age ) with varying and often long latency (up to yr). we also find that variability of clinical expression may be correlated with the age at onset. concentrating on the study of familial cases on guam may enhance the identification of the etiologic agent(s) of this prevalent and tragic disorder. the spinocerebellar ataxias are an uncommon group of genetic disorders that have been well characterized in north america and europe. information concerning these conditions in africa and other parts is scant. to address this problem, a large-scale survey has been undertaken in the cape province of south africa. in this investigation, more than persons in affected families have been appraised and investigated and phenotypic features have been analyzed in detail. linkage studies have been undertaken in families with similar phenotypes in which the condition was transmitted as an autosomal-dominant trait. human lymphocyte antigen (hla) typing was carried out on members of the families and linkage analysis was undertaken using the liped program to analyze the data with a correction factor for age of onset. maximum lod scores were: family a: . ( = . ); family b: . ( = . ); family c: . ( = . ); family d: . ( = . ). these results indicate linkage to hla in out of families. these findings provide support for the concept of genetic heterogeneity in these phenotypically homogeneous families. pcr typing with the reportedly more closely linked d s locus is now being undertaken in these south african families. h.-p. hartung, g. f. hoffmann, and g. becker, wunburg, heidelberg, germany recently, l- -hydroxyglutaric acidemia has been described as a novel metabolic disorder in children. we report the occurrence of this disease in adults. one of brothers developed at the age of an abnormal gait and dysarthria; in the other , clumsiness and walking delay were noted at age . symptoms progressed and at the time of admission, when the patients were and years, neurological examination revealed a spastic ataxic gait, limb ataxia, dysmetria, dysarthria, dystonic posturing, and mental retardation. ct and mr imaging revealed subcortical white matter changes with loss of arcuate fibers, folial atrophy, and leakage in the cerebellar vermis, as well as atrophic changes in the cerebellar hemi-acidemia program and abstracts, american neurological association spheres. on biochemical screening, highly elevated concentrations of l- -hydroxyglutaric acid were found in csf, plasma, and urine. the pathological accumulation of l- hydroxyglutaric acid in these adults, along with the clinical picture characterized by cerebellar, extrapyramidal, and pyramidal symptoms and oligophrenia, and the neuroradiological findings of severe loss of myelinated arcuate fibers in subcortical white matter, conform with what previously has been described in the few neuropediatric cases. the biochemical abnormality underlying accumulation of this organic acid remains elusive. this study was undertaken to differentiate primarily affected areas from functionally suppressed areas due to remote effect in aphasic patients with focal brain degeneration, using an activation method with - water, in comparison with [sf] -fluoro- -deoxy-~-g~ucose (fdg) positron emission tomographic examination at rest. the subjects were patients with slowly progressive aphasia showing contrasted clinical symptoms (nonfluent type vs fluent type). regional cerebral metabolic rate of glucose (cmrglu) was measured with intravenous injection of mbq of f- fdg at rest. regional cerebral blood flow (cbf) was measured with intravenous bolus injection of . gbq of - water in different conditions: at rest, under repetition tasks, and under naming tasks. changes of cbf were evaluated between a resting condition and task-performing conditions. regional cmrglu was decreased focally in both broca's and wernicke's areas similarly in these cases in spite of the difference in clinical symptoms, whereas the patterns of regional cbf changes were different. the fluent patient showed prominent activation in the bilateral frontal areas on repetition tasks. the nonfluent patient showed, whereas the fluent patient did not show, focal activation in the left occipitoparietal area on a naming task. evaluation with an activation method can provide detailed information about the pathophysiological process and the location of primary lesion. defective complex i activity has been linked to huntington's disease (hd) and parkinson's disease (pd). intrastriatal injection of inhibitors of complex i reproduces the pathological features of hd, and the neurotoxin mpp+ kills dopaminergic neurons by inhibiting complex i. defects in complex i have been reported in hd and pd, but the distribution of this enzyme in the brain is unknown. to map complex i in brain quantitatively, we developed an assay using e h)dihydrorotenone to label the enzyme in tissue sections. this high-affinity binding is saturable and is displaceable by rote-in brain none and mpp+. using pm rotenone to define nonspecific binding, more than % of binding is specific. highest levels of binding are found in kidney, followed by myocardium. moderate levels of complex i are seen in striated muscle and some brain regions. within the brain, binding varies more than -fold and is heaviest in the cerebellar molecular layer and dentate gyrus. lower levels of binding are found in cortex and striatum and very low levels are located in substantia nigra. this assay may help to clarify the role of complex i in neurodegenerative disorders. ( in , patients with medically intractable parkinson's disease underwent autologous adrenal medullary-to-caudate transplants at the university of california-los angeles (ucla). these persons had been followed for several years before operation at -or -month intervals. at each visit, their disability had been rated on the quantified ucla scale and the hoehn and yahr stage of disease. these provided a longitudinal assessment of the progression of disease in each patient, which could be compared to the rate of progression in the cohort of cases followed at ucla for years. in addition, the unified parkinson's disease rating scale provided supplementary data for the immediate preoperative and subsequent postoperative evaluations. the hours "off" also were recorded for the preoperative and postoperative periods. after operation, the same evaluations were performed by the neurologist who previously had cared for the patients. the longitudinal postoperative data revealed that at to months after operation, all patients improved. after years, continue to be less disabled than their preoperative baselines. the progression of their disease, while still evident, is nonetheless proceeding at a slower rate than before transplant. the fourth patient had brief improvement shortly after operation, but then rapidly worsened to his previous level. his disease has continued to progress at a rapid pace, unchanged from progression before operation. individuals at risk for huntington's disease h . p. h . kremer, w. shtybel, b. snow, c. clark, j . theilmann, m . r. hayden, and w. in huntington's disease (hd), caudate hypometabolism as demonstrated by positron emission tomography (pet) is a well-established feature in symptomatic patients. in individuals at risk, however, conflicting findings are reported. since we have performed pet scans in asymptomatic persons at risk for hd (age - yr) . linkage analysis with independent dna probes or subsequent evolution to clinical hd (in patients) allowed a risk estimate for subjects. twenty were considered to be at increased risk (? %), at decreased risk (< %), and in individual no modification could be given. nine subjects were not tested. a pet scan was considered abnormal if either the caudate/thalamus ratio or the caudate/whole-brain ratio of rcmrglu was more than these criteria, scans in individuals were abnormal. none had received a decreased risk. comparison of the ratios of increased risk, decreased risk, unmodified risk, and control subjects failed to show statistically significant differences (analysis of variance). follow-up of persons with an abnormal scan showed conversion to symptomatic status within years after the first abnormal scan. this result suggests that an abnormal pet scan in a person at risk for h d heralds the onset of choreic movements. robitaille, m . el-awar, b. clark, l. scbut, m. ball, l. young, r. currier, and k. sbannak, toronto, ontario, and montreal, quebec, canada; pittsburgh, pa, minneapolis, mn, portkmd, or, and jackson, ms we measured the levels of dopamine in striatum of patients with end-stage dominantly inherited olivopontocerebellar atrophy (opca). on average, dopamine levels were reduced in putamen ( - , as compared with controls), caudate ( - %), and nucleus accumbens ( - %). however, individual patient values showed a wide variation (normal to - ), indicating that striatal dopamine loss is a common, but not constant feature of opca. seven patients had marked putamen dopamine loss (- to - ) but without corresponding severe substantia nigra cell damage; this suggests a "dying-back'' phenomenon in which nerve terminal loss precedes cell-body degeneration. in this regard, opca may offer the possibility of examining nigrostriatal dopamine neuronal degeneration at an early stage. although patients were found to have severe nigral cell loss with near total ( - to - %) striatal dopamine loss, none had depression in parkinson's disease (pd) has been correlated with low cerebrospinal fluid (csf) levels of -hydroxyindoleacetic acid ( -hiaa). l-dopa may precipitate or exacerbate depression in % of pd patients. to determine if l-dopa affects -hydroxytryptamine ( ht) metabolism, patients were studied. four had pd. of these, were taking l-dopa and both were depressed. the diagnoses in the remaining were progressive supranuclear palsy (psp), striatonigral degeneration (snd), normal pressure hydrocephalus, and pseudoseizures with depression. neuropsychological examinations and lps of all patients were done. three patients were started on l-dopa (the previously untreated pd patients and the psp patient) and retested days later. one pd patient started on l-dopa became depressed. mood in the others was unchanged. csf was analyzed for ht and -hiaa. ht could not be detected in the csf of patients who were not taking l-dopa, but was easily detectable in all of the patients who were taking l-dopa. -hiaa levels were low in the untreated pd patients, and also in the patients with psp, snd, and pseudoseizures with depression. -hiaa levels were even lower in the l-dopa-treated patients. the ratio of -hiaa: ht (an index of ht turnover) was lowest in the l-dopa-treated pd patients who were depressed. low csf -hiaa in untreated pd may reflect depletion of brain ht. l-dopa may induce depression by inhibiting ht turnover in ht-depleted brain. p . ventroposterolateral medial pallidotomy in the e. fazzani, m. dogali, a. ben;, d. eidelberg, j. gianutsos, t. kay, b. newman, s. loftus, d. samehon, and l. laitinen, new york, n y , and stockholm, sweden in patients with parkinson's disease (pd), as a consequence of low dopamine there exists an increase in inhibitory output from the globus pallidus. ten patients ( men and women) with pd received unilateral ( right, left) ventroposterolateral medial globus pallidotomies (vplmp). the average patient age was years (range - yr), and the average duration of disease was years (range - yr). patients fluctuated between "on" chorea and "off" parkinsonism. hoehn and yahr stage "on" was i in , in ; and "off" was i in , iv in , and v in patients. unified pd rating scale (updrs) score averages hours off medicines ( hom) were activities of daily living (adl): and motor (mtr): preoperatively (preop). capit score averages preop hom were pronation-supination (ps): seconds (s), finger tap (ft): s, board (b): s for the most affected contralateral side, and gait: s ( patients could not walk). re-examination was done to days after pallidotomy. updrs scores hom decreased an average of %. three patients had major bilateral improvement in bradykinesia. rest tremor, prominent in patients, also was diminished. capit scores hom decreased to ps: s, ft: s, b: s; the average gait of the patients who could walk preop improved to s. there were no side effects. vplmp leads to an immediate overall significant improvement in patients with pd. s. kish, y . there is a growing interest in the genetic aspects of parkinson's disease and other basal ganglia disorders. we have studied families whose ancestors immigrated to north america from contiguous regions of northern germany and southern denmark. the pedigrees contain , , and individuals spanning , , and generations with , , and affected members, respectively. autosomal-dominant inheritance is clearly present in families and probable in the third. typical levodopa-responsive parkinsonism with bradykinesia, rigidity, resting tremor, and impaired postural reflexes uniformly develops in affected individuals from all families. n o downgaze impairment, pyramidal signs, sensory disturbances, cerebellar dysfunction, or orthostatic blood pressure changes have been observed. dementia, however, has developed in a few elderly individuals, especially in family. laboratory studies are normal. mri shows moderately enlarged ventricles and cortical atrophy. -fd positron emission tomography demonstrated reduced striatal uptake in examined patient and normal uptake in l individual at risk. autopsy of only subject has been performed (in ). brain weight was , grams and there were no obvious gross abnormali- program and abstracts, american neurological association tuesday, october ties, but microscopic examination was limited. further research on these families is planned. electrophysiological study s. maurri, m . cincotta, a. ragazzoni, g. descisciolo, and f. barontini, florence, ltah many neurophysiological examinations were conducted of a -year-old woman with familial mirror movements. n o other neurological abnormalities were detected. examination included voluntary electromyographic (emg) activity from various muscles, f-wave as well as short-and long-latency reflex responses of the thenar muscles from electrical stimulation of the median nerve, mapping of motor-evoked potentials (meps) to transcranial magnetic stimulation, movement-related cortical potentials (mrcps), and somatosensory-evoked potentials (seps). emg documented mirror activity in the upper limbs, most marked in the muscles of both hands. onset latency of emg activity in response to an auditory stimulus was identical in active and mirror muscle. long-latency responses from median nerve stimulation were recorded on contralateral as well as ipsilateral thenar muscles. short-latency reflexes and f wave were strictly ipsilateral. unilateral scalp magnetic stimulation evoked bilateral responses at similar latencies in the thenar muscles; midline scalp stimulation activated no responses. scalp distribution of median nerve seps and of mrcps associated with self-paced thumb abduction were normal. our findings suggest that congenital inherited mirror movements in otherwise normal subjects can be generated by corticospinal fibers pro jecting to ipsilateral motoneurons of the spinal cord. we have previously identified dysphagia and constipation (both slow transit and defecatory dysfunction types) as common gastrointestinal (gi) problems in parkinson's disease (pd). since apomorphine has been shown to be capable of terminating off-periods when injected subcutaneously, we have evaluated the effects of apomorphine injection on objective parameters of dysphagia and bowel dysfunction in pd patients. nine subjects underwent the following battery of studies to characterize their pd features, swallowing, and bowel function: gi assessment survey, unified pd rating scale, videoesophagram, colon transit study, defecography, and anorectal manometry. specific abnormalities on the studies were noted and the most abnormal study was repeated after subcutaneous administration of mg apomorphine. all individuals were pretreated with domperidone mg four times daily for days prior to apomorphine administration. improvement in both esophageal motility and deglutition was noted in the individual in whom videoesophagram was repeated. for the other patients, defecography or anorectal manometry was performed. significant improvement in specific parameters was demonstrated after apomorphine administration, but individuals experienced syncope during radiographic procedures. we conclude that subcutaneous apomorphine administration holds promise as a potential therapeutic approach to dysphagia and, especially, bowel dysfunction in pd, but that further investigation and refinement are necessary. asymmetrical effect of unilateral thalamotomy or subthalamotomy on tremor in parkinson's disease nico diedericb, christopber g. goetz, glenn t . stebbins, harold l. klawans, k. nittner, a. kozrlosakis, p. sanker, and v . strum, cologne, germany, and chicago, il in the past, stereotactic operation was a regular treatment for unilateral tremor in parkinson's disease (pd). however, follow-up studies were usually short term and always unblinded. we examined pd patients in long-term follow-up (mean . yr after operation) who underwent unilateral thalamotomy for parkinsonian tremor. we used videotapes and the unified parkinson's disease rating scale to blindly compare tremor ipsilateral and contralateral to the side of operation. since the patients were specifically selected for stereotactic operation because of asymmetric tremor, we reasoned that a sign of long-term efficacy would be current postoperative reversal of tremor side predominance. upper extremity tremor was significantly better contralateral to the side of operation compared to the ipsilateral side ( z = . ; p < . ). for the lower extremities the difference was not statistically significant. in chronic follow-up, stereotactic operation improved the absolute magnitude of arm tremor or ameliorated its rate of progression. since asymmetric bradykinesia and dyskinesia were not prerequisites for the choice of surgical side, we cannot make any conclusion about longterm impact of operation on these features. subtle extrapyramidal signs resembles that of patients with parkinson's disease m . richards, k. marder, l. cote, y . stern, and r. mayeux, new york, n y to investigate the relationship between extrapyramidal signs (eps) and cognition, eps severity and neuropsychological function were assessed in normal elderly individuals and nondemented patients with idiopathic parkinson's disease (pd) from a community-dwelling cohort in new york city. multivariate analysis of variance (manova) indicated poorer neuropsychological performance ( p = . ) in pd patients on verbal memory, orientation, verbal fluency, visuomotor construction, and psychomotor speed, but not naming, abstract reasoning, or matching. controlling for eps severity abolished these differences. one hundred fourteen ( %) of the normal individuals had subtle eps (mostly postural abnormality, bradykinesia, or rigidity) but no identifiable neurological disorder. manova indicated poorer neuropsychological test performance ( j = , ) in these individuals than in normals without eps on verbal memory, orientation, abstract reasoning, naming, verbal fluency, matching, and psychomotor speed but not visuomotor construction. we conclude that: ( ) cognitive impairment in pd is specifically associated with eps, and ( ) a similar association occurs in individuals with subtle eps but no neurological disorder. whether this represents a preclinical stage of pd or ad is yet to be determined. disease in olmsted county, minnesota emre kokmen, fatma sibel ozekmekci, c. mary beard, and peter c. o'brien, rochester, m n , and istanbd, turkey there have been many studies of prevalence of huntington's disease (hd) in diverse populations around the world. to study the incidence, we took advantage of the availability of detailed health care records for the population of olmsted county, mn, from mayo clinic, its affiliated hospitals, olmsted medical group, county hospital, state hospital, records of solo practitioners, nursing homes, death certificates, and autopsy records. we reviewed all records with a diagnosis of hd, huntington's chorea, chorea major, and chorea otherwise unidentified, and sought evidence for progressive chorea, progressive cognitive and/or behavioral dysfunction, and family history compatible with autosomal-dominant inheritance with onset of symptoms in the period between january , , and december , , while the patient lived in the geographic boundaries of olmsted county. we found males and females who met these criteria. average annual incidence rate (age/sex adjusted to us white population) for hd for this -year period was . cases/ year/ , population. we also estimated prevalence by taking account of in-migration, out-migration, and deaths. the agelsex adjusted ( ) prevalence for - - was . , and for - - it was . / , . the small number of cases caused the instability of the prevalence rates, but our rates are similar to rates reported in other populations. alton e . btyant, , l. breeden hollis, john a. hamjian, john d. wooten, ill, and francis . walker, nc we described patients with unusual episodic movement disorders and normal diagnostic work-ups: a -year-old woman who had recurrent episodes of tonic jaw deviation and forced right-eye closure; a -year-old woman who developed unexplained pain and subsequent spells of tonic inversion of the left leg; a -year-old man who presented with a bizarre episodic right-arm tremor; and a -year-old woman who experienced intermittent abdominal undulations. examination of the affected body part provoked or enhanced symptoms in all patients. using suggestion and placebo activation in the form of a medicated patch, intravenous saline, or cervical massage, we first induced and then aborted typical episodes of their abnormal movements. postinduction discussions of the procedure led to a marked reduction in the frequency of attacks in patients. activation procedures are useful in diagnosing psychogenic disorders because they demonstrate that situational, not medical, factors govern the expression of the abnormal behavior. we speculate that patients who are refractory to simple suggestion may respond to induction because it offers the potential of validating their symptoms. as in the case of psychogenic respiratory distress or pseudoseizures, positive induction can assist in counseling and symptom control. had alzheimer's disease with parkinsonism (adp), had essential tremor (et), had cerebellar tremor in multiple sclerosis (ms), and had tardive dyskinesia (td). clozapine was used either to treat psychosis ( pd, adp, dys, td) or tremor ( pd, et, ms). two pd patients were retrospective analysis of patients counted twice, who was treated for psychosis and then tremor and who was treated on separate occasions for psychosis with different responses. all dys patients improved, with complete resolution of their dystonia on changing antipsychotic drugs. the patients with et ( mg) and ms ( mg) improved mildly but sedation and clumsiness caused drug discontinuation in the ms patient. one adp patient ( . mg) responded well and the other became sedated and confused ( mg). the p d responses for psychosis at a dose range of . to mg daily were good ( ), very good ( ), and excellent ( ), whereas were intolerant. pd tremor responses were good ( , very good ( ), excellent ( ), and poor ( ) at doses of . to mg daily. one patient died of unrelated causes shortly after initiation of the drug. adverse effects included sedation, weight gain, hypersalivation, fainting, clumsiness, transient granulocytopenia, and "spasms" necessitating discontinuation in patients ( pd, l td, and l ms). tourette's syndrome and attention-deficit disorder patients s. m. silverstein, p. g. coma, d. palumbo, l. west, and r. kurlan, rochester, n y impaired attention is a common comorbid behavioral feature of tourette's syndrome (ts) and a key clinical feature of attention-deficit hyperactivity disorder (adhd). however, the pattern of attentional impairments reported in adhd has not been observed in ts. we therefore compared ts patients ( male, female; mean age ? yr), adhd patients ( male, female; * yr), and normal controls ( male, female; ? yr) on specific neuropsychological (np) and computer-administered tasks of attentional ability. adhd, but not ts, subjects performed significantly worse than controls on the n p tasks (digit symbol, perceptual speed) and had a trend toward poorer performance on a computerized measure of attention. however, both the adhd and ts groups had significantly greater test performance variability on some, but not all, tasks and had more subjects with deviant scores. among ts patients, higher scores on an obsessive-compulsive disorder (ocd) inventory and a greater number of adhd symptoms correlated significantly with poorer performance on the attentional tasks. moreover, ts patients with observed tics during testing had greater attentional impairment than those without tics. these results suggest that: ( ) many adult ts patients do not have impaired attention; ( ) attentional impairment in ts differs from that observed in adhd; and ( ) attentional impairment in ts is associated with the full neurobehavioral spectrum of ts (i.e., tics, ocd, and adhd). frank r. sharp, cathleen miller, thomas rando, and steven greenberg, san francisco and palo alto, c a six patients are described with choreoathetoid movements and marked proprioceptive sensory loss. one patient had a traumatic injury to the right parietal cortex that produced severe proprioceptive sensory loss and choreoathetosis in the left arm. another patient had a left thalamic infarction that resulted in profound proprioceptive sensory loss and chorea on the right side of the body. two patients had cervical spinal cord disease, proprioceptive sensory loss, and diffuse choreoathetosis. another patient had dorsal root ganglionitis associ-a hypothesis program and abstracts, american neurological association ated with small-cell lung carcinoma that produced diffuse loss of all sensory modalities and chorea. the last patient had an ulnar sensory neuropathy and choreic movements of the fifth finger. lesions anywhere along the pathway that transmits limb proprioception may cause pseudochoreoathetosis. furthermore, choreoathetosis without sensory loss caused by focal lesions of striatum may occur because of disruption of cortical proprioceptive inputs to striatum-perhaps explaining why most focal lesions of striatum do not produce chorea. sporadic inclusion-body myositis (s-ibm) and autosomalrecessive hereditary inclusion-bod y myositis (h-ibm) are of unknown cause and pathogenesis. in both there are muscle fibers with rimmed vacuoles containing to -nm cytoplasmic tubulofilaments (ctfs) and denervation atrophy; in s-ibm, but not h-ibm, there is a varying degree of inflammation. vacuolated fibers contain ubiquitinated inclusions (askanas ) and congo-red positivity indicating amyloid (mendell ). because immunoreactive p-amyloid precursor protein (app) and p-amyloid protein (p-ap) are constituents of ubiquitinated senile plaques in alzheimer's disease (ad) brain, we studied immunolocalization of app and p-ap fibers in ibm muscle using antibodies against: ( ) non-p-ap fragments of app, viz. (a) c-terminus (residue - courtesy d. selkoe) and (b) n-terminus (residue - courtesy b. frangione and d. levartovsky); ( ) p-ap (sequence - , courtesy g. glenner, and sequence - courtesy d. selkoe); ( ) ub (chemicon). in of ibm patients, including one h-ibm, % of the vacuolated muscle fibers contained large or several small app and p-ap immunoreactive (ir) inclusions, which by double-labeling fluorescence were closely colocalized with each other and with ub-ir. none of control muscle biopsy specimens (including polymyositis) contained app-ir, p-ap-ir, or ub-ir inclusions characteristic of ibm. control experiments utilizing omitted, replaced, or absorbed primary antisera were negative. p-ap, a product of proteolytic cleavage of app, is receiving attention regarding the pathogenesis of ad. our study provides ( ) the first demonstration of app and p-ap accumulations in abnormal human muscle, and ( ) raises the possibility that in ibm muscle and ad brain rhey may form from similar cellular events. jeffrrey d. rothstein, lin jin, and ralph kuncl, baltimore, m d the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (als) is unknown. accumulating evidence suggests that the disease is characterized neurochemically by a derangement in the control of neurotransmitter glutamate metabolism: csf levels of glutamate and aspartate are ele-of motor neuron degeneration vated and their high-affinity transporter is defective in brain and spinal cord. inefficient glutamate transport, and subsequent chronic increase in extracellular glutamate, could be responsible for selective motor neuron death. to test the hypothesis that chronic defects in glutamate uptake can produce motor neuron toxicity, we developed a tissue culture model employing organotypic rat spinal cord maintained under conditions of chronic glutamate uptake inhibition. slices ( pm) of lumbar spinal cord from -to -day-old rat pups were cultured on millicell membranes. chronic uptake inhibition was produced by culturing tissue in the presence of threohydroxyaspartate (tha) or pyrrolidine-dicarboxylic acid, both known to be specific inhibitors of glutamate transport. tha produced chronic elevation of glutamate in the medium and produced motor neuron toxicity after to days in culture using pm tha, and after days using pm tha, as determined by assay of tissue choline acetyltransferase (chat) activity and by histological analysis of -micron plastic sections. motor neuron toxicity was completely blocked by the non-n-methybaspartate (nmda) antagonists cnqx or nbqx, but not by the nmda antagonist mk- . this model demonstrates that the chronic loss of glutamate transport in als can produce motor neuron degeneration and that motor neurons appear to be susceptible to non-nmda-mediated glutamate toxicity. guillain last year, we described a distinct acute paralytic syndrome in children and young adults from northern china and differentiated it from guillain-barre syndrome (gbs) by epidemiological, clinical, and nerve conduction (nc) features. to distinguish chinese paralytic syndrome (cps) from gbs more clearly, we measured nc in cps patients (mean yr, range . - yr) and in gbs patients from johns hopkins (mean yr, range - yr). sensory nc was normal in all (n = ) but nerve of cps patients, whereas sensory nc was frequently abnormal in gbs patients: median nerve, %; ulnar nerve, %; and sural nerve, %. motor n c also differed between the groups. in all nerves, distal latency (dl) was significantly longer in gbs than in cps. for example, in the median nerve, mean dl was . ms (se ) in gbs and . ms ( . ) in cps ( p < . ). motor conduction velocity was significantly reduced in gbs median and ulnar nerves compared with cps nerves. f-wave latency was significantly longer in gbs median nerves than in cps nerves. these data support the distinction both clinically and electrodiagnostically between cps and north american gbs. the use of n c may be especially important in field epidemiological studies in separating the disorders. clinical manifestations and gene analysis of the first japanese kindred yoshihide sunada, teruo shimizu, lchiro kanazawa, and toru mannen, tokyo, japan familial amyloidotic polyneuropathy type iv (fap iv) has been clustered in the finnish population and only a few cases have been reported from the netherlands. denmark, and united states. we describe the first japanese family with fap iv. the family originates from nagano prefecture, a mountainous district in the middle part of japan, and has no relationship to the finnish population. this family has members in generations, and individuals are affected with slowly progressive cranial neuropathy and corneal lattice dystrophy. the genetic trait is autosomal-dominant. polarizing microscopy and immunohistochemistry show abundant amyloid deposits reactive to an anti-gelsolin monoclonal antibody. direct sequence analysis of a dna fragment spanning codon of the plasmagelsolin cdna from the propositus, and restriction analysis using a modified pcr from other family members demonstrate a single base substitution, g to a at the first base of codon , which is identical to the mutation of finnish fap iv. this suggests that the mutation causes the fap iv phenotype regardless of ethnic background. gene expression focal puncture injury has been used as a model to study degenerative and regenerative responses of skeletal muscle. previous studies have demonstrated the ultrastructural and metabolic effects of muscle injury. however, the early genomic response to focal injury is presently unknown. we asked whether the immediate early genes (iegs) or early response genes-~$ , c-jun, nur , and junb-are responsive to muscle injury. these iegs encode transcription factors and are expressed rapidly after cell-surface stimulation. we have previously shown that surgical denervation and neural stimulation of muscle induced differential patterns of ieg expression. in this study, we produced injury of mouse gastrocnemius muscle by injection of c . of normal saline. we used the contralateral (uninjected) muscle as a control and examined the milna levels of each of these iegs. we found that ~$ and junb levels were increased at and hours and returned to basal levels by hours. in contrast, mrna levels of nur and cjun remained unchanged. this pattern of ieg response is distinct from that seen after muscle stimulation or denervation. the selectivity of this pattern suggests that ieg expression may play a role in the response of muscle to injury. amyotrophic lateral sclerosis (als) is a degenerative disease that leads to the restricted loss of motor neurons (mn). the reason for the selective death of mn remains unknown. we hypothesize that mn-enriched or mn-specific genes are important for normal m n function and that their disturbance may play a role in the pathogenesis of als. we have produced clonal hybrid cells derived from embryonic and neonatal spinal cord m n for the study of m n gene properties. some of these hybrid mn clones express traits typical of mn, such as high levels of choline acetyltransferase enzyme activity and message, glycine receptor message, and neurofilament and neural cell adhesion molecule proteins. we are using molecular techniques to identify novel mn-enriched or mn-specific genes in these cells. with this strategy, we have identified several cdna clones preferentially expressed in mn hybrid cells but not in the parental neuroblastoma cells by differential hybridization of an embryonic mn hybrid cdna phage library. we are extending these observations by performing subtraction hybridization experiments. these results suggest that mn-enriched or mn-specific genes can be identified, and may lead to a greater understanding of the etiology of als. there is a syndrome of slowly progressive, mid-adult-onset fasciculating progressive muscular atrophy (pma) affecting upper more than lower limbs, without bulbar or corticospinal signs, more often in males, associated with igm monoclonal gammopathy, and no nerve conduction block. two such men, ages (a) years and (b) years, duration and and one-half years, csf protein and , had failed to achieve sustained improvement with: prednisone, cyclophosphamide, total-body irradiation, and multiple lymphoplasmaphereses in a; and interferon alpha a in b. intravenous immunoglobulin (ivig), . gm/kg/day, has provided dramatic benefit, sustained and increasing for > and > months to date. (there is a continuing base of depotestosterone, mg weekly, which initially alone provided very minimal improvement.) strength increase was evident at and days after the first course of daily ivig infusions. it further increased for to and one-half weeks after treatment, and then began to diminish. repeat -day treatment weeks after the first course resulted in summated improvement, now sustained and enhanced by an average of treatment per week. quantitated strength testing by a blinded observer has shown a -fold to >loo-fold gradually increasing muscle function in all limbs. patient a regained the ability to feed himself, get out of a chair, walk unaided, and go up steps; quantitated hip flexors increased -and -fold. patient b regained the ability to feed himself, take care of personal toilet needs, walk securely, and drive miles; quantitated hip flexors increased -fold, and biceps flexions increased from with no weight to > reps while holding -pound weights. bukhara jews: a new cluster with typical oculopharyngeal muscular dystrophy (opmd) is a rare, late-onset myopathy with autosomal-dominant inheritance. its ultrastructural hallmark is the finding in muscle fibers of intranuclear tubular filaments of . -nm outer diameter. most opmd cases were described among french canadians; in france, the homeland of their ancestors, the prevalence is / , (brunet et al, ) . in israel's central area live approximately , jews who have immigrated from the bukhara and samarkand regions in uzbekistan. they represent a homogeneous ethnic group with its own language and community life. among them we have identified opmd in families ( affected individuals). the inheritance, clinical, electrophysiological, and histological features of these pa-tients are similar to those described in other pdts of the world, with typical intranuclear inclusions seen on electron microscopy. the minimal estimated prevalence of opmd in this population is approximately : . this cluster of opmd among bukhara jews is the second largest in the world. because many bukharian families are large, they may be suitable for linkage genetic studies. human muscle during ontogenesis e . scarpini, g. conti, p. l. baron, and g. myoblast transfer has been proposed recently as a possible therapy for duchenne muscular dystrophy patients. because immune rejection can represent a major problem in myoblast implantation, immunological characteristics of human muscle should be investigated. previous studies showed that human muscle cells cultured in vitro can constitutively express human lymphocyte antigen (hla) class i, but not hla class . furthermore, human y-interferon induces the surface expression of hla class i on mononuclear myoblasts, but not on multinucleated myotubes. however, whether the cells produce and present the antigen by themselves or take this material from the environment, where it could be released by infiltrative cells, is not yet clear. in this study, we analyzed hla molecules at the protein level by immunocytochemistry with monoclonal antibodies against different hla-dr epitopes and hla-abc molecules on frozen serial sections of human muscle during development and at the adult stage. human muscle infiltration by macrophages and monocytesmacrophages also were studied with m and leum specific monoclonal antibodies at the same stages of development. our results show that during muscle development and maturation, hla-dr and hla-abc antibodies do not label muscle fibers but some m -and leum -positive cells within the muscle. these data can be useful to understand the role of infiltrating monocytes-macrophages in the muscle immune response. mounting evidence suggests that excitotoxicity, mediated via the glutamate receptor, is involved in the pathogenesis of amyotrophic lateral sclerosis (als), as well as in other neurological diseases. we therefore initiated an open label, phasen i trial of highdose dextromethorphan (dm), a noncompetitive, selective n-methybaspartate antagonist, in als. patients began with mg/kg/day, divided into doses, and incrementally escalated their medication to mg/kg/day or their maximum tolerable dose. thirteen patients, all extensive metabolizers of dm, were enrolled. total daily doses ranged from . to mg/kg. major side effects were lightheadedness ( ), slurred speech ( ), and fatigue ( ). no biochemical, hematological, or neuropsychiatric abnormalities occurred after up to months of maximal therapy, except for depression in patient. plasma kinetics of dextrorphan (dt) (the major metabolite of dm) were studied after an acute oral dose of . mg/kg dm. median elimination halflife was . hours. plasma dt concentration peaked at a median of hours, with a median cmax of . pm. median amyotrophic lateral sclerosis cerebrospinal fluid/plasma dt ratio was . . this study demonstrates the feasibility of long-term, high-dose dm therapy. we are now conducting a phase i study of highdose dm in als, designed to assess its efficacy. polyneuropathy: a chronic inflammatory demyelinating polyradiculoneuropathy variant? d. cros, k. h. chiappa, s. patel, and s. gominak, boston, ma, we describe patients ( men, woman) with a pure, adultonset sensory neuropathy. the course was chronic in all cases. three patients had a relapsing-remitting course over to years with several attacks every year; the onset was gradual and followed by a plateau in the fourth patient. all patients had positive and negative sensory symptoms, and had positive motor symptoms (fasciculations). in all patients, muscle power was normal at the time of peak deficit. all were areflexic and had large fiber sensory deficits, and patients had sensory ataxia. three patients had elevated csf protein, whereas the csf was normal in patient. mri demonstrated marked thickening of the lumbosacral spinal roots in patient. motor conduction studies were normal in all patients, and mild f-response abnormalities were noted in . neurophysiological investigations of the sensory pathways were abnormal in all. three patients had several studies over a -year period. sensory nerve action potentials were unobtainable in patients, and normal in the others. median and tibial somatosensory-evoked potentials showed conduction slowing consistent with demyelinating lesions affecting the peripheral sensory pathways, either globally or focally in the proximal segments. two patients appeared to respond to plasma exchange or intravenous immunoglobulin therapy, or both. glial fibrillary acidic protein cells in experimental motoneuron disease raul n . mandler, pam c. allgood, and james a. wallace, albuquerque, nm neuronal degeneration in human and animal motoneuron disease has been emphasized, but glial phenotype alterations have not been studied as extensively. we carried out a developmental and topographic study of astrocyte expression in the wobbler mouse model of motoneuron disease. wobbler mice and normal littermates were studied at , , , and weeks of postnatal development. anesthetized animals were perfused intracardially with paraformaldehyde. spinal cords were dissected and landmarks were identified carefully for systematic study. sections were stained with monoclonal antibodies against glial fibrillary acidic protein (gfap) neurofilament and neuron-specific enolase. cell quantitation was done with video-enhancing microscopy. in symptomatic animals, marked increases in gfap staining were found in rostral and caudal spinal cord areas. quantitation studies revealed a to -fold increase in gfap+ cells in the wobbler. we conclude that gfap+ cells are markedly increased in the wobbler mouse at cervical, thoracic, and lumbar areas. this cell may also be relevant in motoneuron disease pathogenesis. ( indirect evidence suggests that polio virus may persist in the human cns years after initial infection and may be a cause for the post-polio syndrome. to evaluate whether the polio virus genome can be detected in the cns of patients with previous polio infection, we identified patients who had died with autopsy findings and clinical history consistent with poliomyelitis. rna was extracted from paraffin-embedded sections of brain or spinal cord and subjected to reverse transcription followed by dna amplification by polymerase chain reaction (rt-pcr) using primers specific for heat shock protein (hsp ) and a conserved region of the polio viruses. hsp mrna could be detected in all specimens, indicating that amplifiable rna had been isolated. in no specimens could polio virus rna be detected. this study suggests that polio virus does not persist in the human cns in quantities detectable by the sensitive pcr method. with cmt type , seen at mayo clinic rochester between and , for the frequency of selective calf weakness in cmt type , the form of cmt most similar clinically to distal sma. anterior compartment weakness exceeded calf weakness in patients ( %); anterior and posterior involvement was equal in ( %). calfweakness exceeded anterior compartment weakness in patient ( %). selective calf weakness in distal sma thus helps distinguish this disorder from cmt type , and similarly from distal sma with weakness resembling cmt, in that we are unaware of the distributions in distal sma occurring in the same family. given the possibility of genetic heterogeneity, linkage studies of distal sma probably should include patient selection criteria such that the distribution of leg muscle weakness is homogeneous. p . conjugal amyotrophic lateral sclerosis: amyotrophic lateral sclerosis (als) is a sporadic neurodegenerative disorder of unknown cause. unusual cases may provide etiologic clues. we report a married couple, both of clue to etiology? whom developed als in year. the couple grew up in southeastern pennsylvania and attended the same schools. they married after high school and have healthy children. in september , a -year-old woman noted right-hand weakness and associated fasciculations that progressed to the entire right upper extremity. by january , the lower extremities were asymmetrically weak and fasciculating. she then developed left-arm weakness, d ysarthria, dysphagia, and emotional incontinence. she had hyperreflexia and bilateral extensor plantar responses. then, in may , her husband, aged , noted difficulty whistling, which progressed to frank dysarthria. later, he developed dysphagia, emotional incontinence, and weakness, wasting, and fasciculations in the upper extremities. hyperactive gag, jaw, and limb reflexes were present. in both, electrodiagnostic testing revealed widespread evidence of lower motor neuron degeneration. numerous laboratory tests were normal. although these cases may represent a chance association, the development of als in a young husband and wife suggests a possible environmental cause. the authors welcome suggestions about these cases from the neurological community. conduction block in demyelinating neuropathies usually is assessed from differences in the sizes of surface-recorded maximum m-potentials evoked by supramaximal stimulation at successively more proximal sites along the course of motor nerves. as the maximum m-potential is comprised of many bitriphasic surface-recorded motor unit action potentials (muaps), differences in the relative latencies between muaps may lead to phase cancellations, reducing the m-potential size and rendering any quantitative assessment of the extent of conduction block relative to phase cancellation difficult. cooling a muscle (not the nerve), however, by as much as °c increases the negative peak durations of muaps by as much as to times and moves the point at which maximum phase cancellation might occur to some theoretical point well proximal to the spinal roots. in cases of guillain-barre syndrome (gbs) studied to date, cooling produced little change in percent reductions in m-potential negative peak areas between successively more proximal sites of stimulation. this finding suggests that "true" conduction block rather than interpotential phase cancellation best explains reductions in m-potential size at successively more proximal sites of stimulation in gbs. associated with trimethoprim-sul famethoxazole rare cases of primarily motor polyneuropathy have been associated with the use of sulfonamides. the incidence of polyneuropathy has diminished substantially with the abandonment of earlier methylated compounds. we describe patients who developed allergic phenomena, including a skin rash and debilitating, painful sensory and autonomic polyneuropathy within days of receiving trimethoprimsulfamethoxazole. in patient, examination revealed resting tachycardia, marked blood pressure orthostasis and near-program and abstracts, american neurological association syncope, hyporeffexia, urinary incontinence, and reduced sensation distally in the lower extremities. his cerebrospinal fluid was acellular with a protein of mg/dl. the other patient showed a resting tachycardia, sluggish pupils, reduced distal vibration perception, and hyperpathia of hands and feet. conventional nerve conduction studies demonstrated normal motor results in both patients, absent or reduced sensory amplitudes in the first patient, and normal sensory results in the second. autonomic studies identified profound abnormalities in testing of sympathetic skin potentials, sinus arrhythmia, and valsalva's ratio. in both cases, nerve biopsy was not performed for fear of exacerbating the patient's hyperpathia. subsequent hemodynamic and electrophysiological testing showed improvement in autonomic function, paralleling the patients' clinical amelioration. although uncommon, a painful, sensory and autonomic, partially reversible polyneuropathy may develop after the use of trimethoprim-sulfamethoxazole. the remote effects of botulinum a toxin injections into vocalis muscles for treatment of focal laryngeal dystonia were investigated using single-fiber electromyography (sfemg). botulinum a toxin injections have been proven effective therapy for various dystonic disorders including focal laryngeal dystonia, blepharospasm, and torticollis. previous sfemg studies have demonstrated remote effects of the toxin in noninjected muscles after treatment for both blepharospasm and torticollis. these effects include an increase in fiber density, mean jitter (mcd), and percentage of fiber pairs with increased jitter. other researchers have postulated that the distant effects of this toxin may be related in part to the dose of botulinum toxin injected. to investigate this hypothesis we have studied patients treated for focal laryngeal dystonia because the amounts of toxin required are / th to / ooth of the doses used to treat other dystonias. using electromyographic (emg) guidance, bilateral injections of . or . mouse units of botulinum a toxin were injected into each vocalis muscle of patients. each patient had significant improvement in phonotory function within hours after injections and have been followed serially (usually within weeks and again at mo) after injections, with sfemg recordings of the left extensor digitorum communis and sternocleidomastoid muscles. five patients have had more than series of injections over the months since we began this study. sfemg studies have revealed no significant change in the fiber density, mean mcd, or percent of fiber pairs with normal jitter in either muscle. in conclusion, our studies support the hypothesis that the presence of remote effects of botulinum toxin may be related, in part, to the amount of toxin used. the c bl/ / a mouse exhibits the remarkable characteristic of prolonged survival of axons separated from their cell bodies (slow wallerian degeneration). previous work has demonstrated that the axon itself is responsible for the phenotype of prolonged survival. we investigated whether the lack of rapid axonal degeneration after axotomy in this substrain is due to an inability to break down cytoskeletal components, a process that is normally accomplished by activation of intrinsic calcium proteases. segments of desheathed sciatic nerves from normal and ola mice were incubated for hours under conditions that disrupt the axolemma (freeze/ thaw or in % triton x-loo), allowing external calcium free access to axoplasm. nerves were analyzed by western blot for neurofilament (nf) proteins and by electron microscopy. in high-calcium media ( mm caci,), nf immunoreactivity was lost and axoplasm was reduced to watery debris in both substrains, whereas in egta-buffered media, axoplasm was preserved. these results demonstrate that calcium-activated proteases are present and can be activated in ola nerves. the defect in these mice that allows for prolonged survival of transected axons is likely in the mechanism for calcium entry into the distal stump. the mechanism by which the analogue of adrenocorticotrophic hormone, acta - , prevents cisplatinum (cp) neurotoxicity is unknown. murine n e. neuroblastoma cells and neural crest-derived, squirrel fish erythrophore cells tuesday, have similar vesicular transport mechanisms to human neural cells. they were used to study the effects of cp and acth , on cellular transport. differentiated n e. cells were treated hour prior to observation with serum-free media (sfm, control); sfm/cp pg/ml; or sfm/cp pg/ml and ng/ml acth -,. organelle transport was studied ( neurites and - organelles per condition) using computer-enhanced video microscopy. mean fast anterograde ( . k . pmsec-' vs . * . pmsec-') and retrograde ( . . pmsec-' vs . +- . pmsec-') transport were decreased in cp-treated compared to control cells ( p < . ). in cp/acth ,-treated cells, mean anterograde ( . +_ . pmsec-') and retrograde ( . . pmsec-') velocities were greater than in cp cells ( p < . ). velocities in control and cp/acth , cells were not statistically different. erythrophore pigment granule transport was observed in a blinded study, using similar techniques. mean aggregation velocity was greater in control ( . k . msec-') and cp/acth , ( . f . msec-')-treated cells compared to cp ( . * . msec-') cells ( p < . ). incubation with cp for or hours affected velocities equally, but acute exposure was more easily reversed by control or acth,, containing media. there is striking inhibition by cp in cross-species models of organelle transport. this can be prevented by acth ,. erythrophores allow future study of individual transport components. neurotrophin to investigate signal transduction pathways involved in neurite growth, the cytoplasmic regions of p sngfr, the common neurotrophin receptor monomer, were searched for a motif analogous to the predicted secondary structure of the tetradecapeptide mastoparan. potential sequences were modeled using a semi-empirical molecular mechanical force field approach. the sequence rat ~ ~~~ - represents a highly conserved amphiphilic domain predicted to be involved in neurotrophin signal transduction via g-protein mechanisms. to test this prediction, peptides containing sequences homologous to p ngfr - were examined for effects on trophic factor-induced survival/differentiation responses of rat pc pheochromocytoma cells, chick embryo drg neurons, and chick embryo ciliary neurons. a peptide identical to ~ ~~~ - accelerated the neurite growth response to nerve growth factor (ngf) of pc cells and drg neurons in a time frame that paralleled uptake into cells, but mastoparan did not influence ngf-mediated neurite growth. millimolar mg+ + and benzalkonium chloride, known to block the actions of mastoparan, blocked the effect of the peptide on ngf-mediated neurite growth by pc cells. peptides mutated to alter cationic amino acid relationships or amphiphilicity were less effective than the peptide in accelerating ngf-mediated neurite growth. these observations complement and extend evidence suggesting a pivotal role of ~ ~~~ in ngf-mediated signal transduction. these studies complement and extend evidence suggesting a pivotal role of p sngfr in neurotrophin signal transduction and evidence that activation of intracellular signalling processes involving specific g-protein mechanisms are involved in neurotrophin-mediated neurite growth. crushing the hypoglossal nerve causes hypoglossal motor neurons to decrease expression of choline acetyltransferase (chat) and begin expressing p ngfr, the low-affinity ngf receptor. these changes are evident within days after the injury and continue for several weeks. inhibition of axonal transport by vincristine applied to uninjured nerves causes loss of chat expression without induction of ~ ~~" . we sought to determine if topical vincristine would alter p sngf' expression after nerve injury. hypoglossal nerves were surgically exposed unilaterally in anesthetized rats and crushed. one week later, rats were reanesthetized and the same nerves were re-exposed. vincristine or saline was applied at the crush sites by soaking a strip of cotronoid wrapped around the nerves. one week later, rats were anesthetized and perfused with aldehydes. frozen sections from the brainstems were stained by indirect immunoperoxidase to demonstrate chat and ~ ~~~' . saline-treated controls showed decreased chat and abundant ~ ~~" in hypoglossal motor neurons ipsilateral to the crush injury. vincristine-treated animals showed no chat and no p tngf'. we interpret these results as indicating that a signal originating from the injury site maintains ~ ~~" expression after nerve injury. catecholamines have been reported to be toxic to embryonic-derived rat neurons and glia via the formation of reactive oxygen species (rosenberg, ) . we tried to determine whether oligodendrocytes (ol) from adult -month-old rat brain are similarly susceptible. toxicity to ol was examined using light microscopy and galactocerebroside immunohistochemistry where the relative number of surviving ol and their extent of process formation were graded. five days of exposure to norepinephrine (ne) and epinephrine (epi) at and fm produced significant toxicity ( p < . , analysis of variance [anova)) to adult rat ol; this toxicity was evident by hours of exposure. treatment with catalase ( p,g/ml), a free-radical scavenger enzyme, completely prevented the toxicity of catecholamines. to ascertain whether astrocytes, which have free-radical scavenging capacity, could prevent the catecholamine-induced injury to ol, rat ol were seeded on neonatal rat astrocytes. under such conditions, the toxicity of n e and epi was reduced significantly ( p < . , anova). these findings suggest that impairment of this protective function of astrocytes may render ol and its myelin membrane susceptible to free-radical-mediated damage. lyme neuroborreliosis is an increasingly prevalent disorder, but the diagnosis generally has been indirect. thus, the pres-ence of other manifestations of the infection, consistent findings on neurological exam or lumbar puncture, or presence of csf antibody have been used rather than direct isolation or identification of the organism from the csf. we have previously developed polymerase chain reaction with hybridization (pcr/h) to identify borrelia burgdorferi in the blood and organs of infected mice, and found that the assay was equivalent or, in some cases, preferable to culture (ann neurol ; : ) . the assay used for primers oligos derived from a sequence of genomic b. burgdovfri d n a expressed on a plasmid by rosa and schwan. a two-stage nested pcr was performed on csf samples in which the d n a was isolated in a variety of ways. pcr products were subsequently hybridized with a digoxigenin-labeled internal probe by slotblot hybridization. the sensitivity of the assay was excellent, being <o. fg of purified b. burgdoorfevi dnaiml of csf, and to spirochetes per ml of csf when "spiked" csf was tested. the assay then was applied to a battery of csfs stored over the past years in patients with definite, probable, and possible lyme neuroborreliosis. the lack of pcr/h positivity in some patients with definite and probable disease signifies that the spirochete does not reside in the lumbar csf in all patients with lyme neuroborreliosis, and may be concentrated in the meninges or parenchyma. progenitor cell line on transplantation into the adult murine brain t . kitagwcbi, d.-h. chui, and t . tabira, tokyo, japan several multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into primary culture cells of the septum of embryonic murine brains (the retroviral vector, kindly provided by d r c. l. cepko, boston, ma). two of those cell lines, when transplanted back into to -week-old mice by stereotaxic operation, integrated into the septum in a nontumorigenic manner. the implanted cells, which had been labeled with pkh dye, could be identified in animals up to at least weeks after transplantation. immunocytochemical analysis demonstrated that the transplanted cells were seen assuming a round morphology and no processes. they did not stain with any glial or neuronal markers except for a b and hnk-i, in the same way as in vitro. interestingly, after weeks of implantation, the cells were located discretely in the transplanted site and displayed a small and round morphology with fine processes. most of the transplanted cells showed immunoreactivity with monoclonal antibodies directed against neuronal markers such as neurofilament and map . these data indicate that the immortalized cell lines might be useful in characterizing factors that regulate cell type differentiation in the mammalian nervous system. (supported by a grant from the science and technology agency of japan.) related to serum response factor in human brain and muscle dana leifer, dimitri krainc, racbael neve, roger bveitbavt, and stuart a. lipton, boston, m a we have identified cdna clones for a novel transcription factor that is expressed at high levels in human fetal brain and skeletal muscle, but not in a variety of other tissues, as demonstrated by northern blotting. sequence analysis indicates that the clones appear to have alternatively spliced forms and have extensive homology with human serum re-sponse factor (srf) and a family of related transcription factors that has representatives throughout eukaryotic evolution in species from yeast to man. srf is thought to have a role in regulation of immediate early genes and of muscle-specific genes. in gel mobility-shift assays, the srf-like proteins coded for by our clones bind specifically to the myocytespecific enhancer binding factor- (mef- ) regulatory element, a dna sequence that has so far been found to be functionally important in the promoter and enhancer regions of a variety of muscle-specific genes. moreover, our srf-like proteins specifically activate transcription of reporter genes containing the mef- element. given the abundant expression of our clones in human brain as well as in muscle, our results suggest that these proteins may have a significant role in development not only of muscle but also of brain. a host of quantitative eeg studies, most of which employed variations of the fast-fourier transform, have been made on isolated clinical entities such as the epilepsies, metabolic syndromes, and sleep with varying degrees of satisfactory correlations. using a time domain wave-by-wave computer program that simulated the logical approach of the clinical electroencephalographer, we have studied more than unselected routine neurological clinic and ward patients. the presenting problems ranged from headache to major cerebral involvement. employing the dichotomy of normal and abnormal, the computer-read eegs agreed with the routine eeg, the clinical manifestations (including imaging techniques), and neuropathological findings in % of all cases. this high level of correlation, which approached that achieved by welltrained eegers reading the same records, was obtained by solely utilizing the background eeg activity. such results became possible only when the computer program was so organized that the "raw" computed and original oscillograph records were made to match closely. the diagnosis, with topographically diagramed localization, was printed automatically as the final step of the program. our data strongly suggest that routine computer reading of the human eeg is feasible, reliable, and potentially useful. protein accumulation in damaged neurites and microglial cells abnormal accumulation or abnormal processing of amyloid precursor protein (app), or both, may be critical to the development of p-amyloid protein (bap) deposits. however, alzheimer's disease (ad) has another pathological hallmark, which is the appearance of neurofibrillary tangles. the question is, therefore, to clarify the possible relationship between altered app metabolism and neurofibrillary degeneration. one classic method for inducing neurofibrillary tangles is the administration of aluminum salts. although these tangles are morphologically different than those seen in ad brain, this phenomenon has given rise to the hypothesis that aluminum might be an environmental factor in the causation of ad. this theory is controversial, but the animal aluminum model remains one of the few ways that long-lasting neuroskeletal changes can be induced. to explore the effects of aluminum salts on app metabolism, we examined app immunodistribution in rat brain injected intraventricularly or intrastriatally with aici,. we found a long-lasting accumulation of app in affected neurites, as well as in activated microglia/macrophages. abnormal neurites also showed argentophilic changes, neurofilament accumulation, and alz- immunoreactivity. the results support the hypothesis that interruption of axoplasmic flow by aluminum salts, as by other means, can lead to both app accumulation and neuroskeletal alterations. p . nondemyelinating conduction slowing of myelinated fibers due to inactivation of n a channel takanori yokota, yukinobu saito, and tadashi miyatake, tokyo, japan in patients with acute or chronic inflammatory demyelinating polyradiculoneuropathy, the marked improvement of motor-nerve conduction velocity without change of amplitude or configuration of the compound muscle action potentials (cmaps) was observed in days. to investigate the mechanism of the rapid improvement of nerve conduction, the influence of na channel blockade on nerve conduction was studied. in healthy volunteers, the recovery of the cmap and sensory nerve action potential (snap) of median nerve stimulation were recorded after the intravenous infusion of mg of lidocaine. after the loading, cmap and snap were reduced rapidly in amplitude and were slowed in latency. after the recovery of amplitude of cmap and snap, the nerve conduction velocities were improved gradually in hours with the amplitudes and configurations of cmap and snap unchanged. this indicated that myelinated nerve conduction velocity could be slowed by the inactivation of na channels without demyelination or conduction block. the prolongation of rising time in depolarization of axonal membrane potential should be one of the mechanisms of this conduction slowing due to inactivation of na channels. paraneoplastic cerebellar degeneration (pcd) in gynecological and breast cancers frequently is accompanied by an autoantibody response (type i or "anti-yo") reacting with and -kd cytoplasmic antigens of cerebellar purkinje cells. sakai et al, using a cerebellar library (stratagene), recently have isolated a cdna clone expressing a -kd protein recognized by igg from a patient with pcd and uterine carcinoma (sakai et al, ann neurol ; : ) . this clone is believed to share some homology with the message encoding the -kd protein recognized by type i sera. because pcd was isolated using serum from a single patient, however, it is not known whether the expressed protein of pcd is recognized uniformly by all patients with type i antibody response. e. coli strain xl -blue transformed with pwr - containing the snabiihindiii fragment of pcd , pwrsbo-pcdl?sn, was induced using isopropylthiogalactoside. bacterial lysates were electrophoresed on % sds-page gels and analyzed by western immunoblot program and abstracts, american neurological association methods using sera and csf from patients with pcd associated with gynecological and breast malignancies who exhibited type i antibody response. the -kd expression product was labeled by sera and csf samples tested from antibody-positive patients but was not labeled by sera or csfs from controls. our studies demonstrate that the -kd protein expressed by pcd contains epitopes that are consistently recognized by both systemically and intrathecally produced antibodies from patients with pcd accompanying gynecological and breast malignancies. tax is a novel antitumor drug that has demonstrated impressive clinical activity in breast, ovarian, and lung cancers. although sensory neuropath y has been described secondary to both taxol alone and taxol-cisplatin combination, detailed neurophysiological and pathological studies have not been described. nineteen patients with solid tumors were treated with combinations of taxol ( - mg/m ), cisplatin ( - mg/m ), and granulocyte colony-stimulating factor (g-csf) ( pglkglday). sequential neurological examinations, nerve conduction studies (ncs), and quantitative vibration testing (qst) were performed during and after treatment. eighteen of patients developed sensorimotor neuropathy and developed myopathy. thirteen were symptomatic with numbness or weakness and had abnormal results on neuromuscular examination. ncs showed absent or prolonged h-reflexes ( ), reduced peroneal motor amplitude ( ), reduced sural sensory amplitude ( ), and myopathic motor unit potentials in proximal muscles ( ). qst results were abnormal in of tested patients. muscle biopsy specimen in a patient with myopathy showed features of a toxic myopathy. the neuropathy was worse with higher cumulative dosages of taxol (> mg), with higher single doses of taxol(> mg/m ), or with a preexisting neuropathy. we conclude that sensorimotor neuropathy and myopathy are dose-limiting neurotoxicities of combined cisplatin and taxol use, now that neutropenia can be controlled with neuropathy and myopathy g-csf. central nervous system lymphoma casilda balmacedz and lisa deangelis, new york, ny ally periventricular and may seed the csf by direct growth through the ependyma. we reviewed the csf profile of non-acquired immunodeficiency syndrome (aids) patients (pts) with pcnsl. all pts had lumbar puncture (lp) and had multiple samples from an ommaya reservoir. definite lm involvement was identified with a positive csf cytology, lymphomatous lm infiltration on a surgical specimen, or mri with gadolinium showing lm tumor. probable lm lymphoma was diagnosed in pts with suspicious or atypical csf cytology. there were women and men with a median age of (range - yr). at diagnosis, mean white blood cell count was /mm (range - , ); mean lumbar csf protein was mgldl (range - , ); and mean ventricular csf protein was mgldl (range - ). glucose was always normal. nineteen of pts sampled had oligoclonal bands, and / had elevated pz microglobulin. at diagnosis ( %) had an abnormal csf cytology: positive, suspicious, and atypical. one pt had pathological infiltration of the lm and had lm tumor on spine mri for a total of ( %) pts with definite or probable lm lymphoma. in pts with abnormal cytology, the abnormality was found in ( %) lps and / ( %) of the ommaya and ventricular specimens. in pts the lumbar cytology was the only abnormal specimen despite multiple ventricular samples, and in only the ventricular csf was abnormal. thirty-six of ( %) pts developed recurrent tumor afrer treatment. forty-two percent ( ) of all patients with relapse had lm recurrence. lm recurrence was accompanied by brain recurrence in pts, systemic in , ocular in , both systemic and ocular in , and isolated in . at diagnosis / patients received treatment directed against the lm. of these, ( %) had meningeal recurrence whereas of the ( %) patients who did not receive this treatment had lm recurrence. lm involvement by pcnsl is frequent, may be missed on a single csf sample, and requires specific therapy at diagnosis. sixty-three solid cancer patients with a single brain metastasis were prospectively randomized for neurosurgery and radiotherapy combined (arm ) or radiotherapy alone (arm ). they were stratified for lung or nonlung cancer and for active versus stable or absent extracranial disease. world health organization performance status was . age, sex, performance status, and location of brain metastasis were divided evenly over both groups. one-month mortality was % in arm and % in arm . median survival of months after combination therapy was significantly better compared to months after irradiation alone ( p < . ). it made no difference whether they had lung or nonlung cancer. the largest difference between both treatment arms was observed in patients with stable or absent extracranial disease ( vs mo, p < . ). when systemic disease activity was present, median survival was months irrespective of treatment arm. functional independent survival was to months shorter than overall survival and was significantly better for patients with stable extracranial disease after combined therapy. multivariate analysis showed that age was also an independent prognostic factor. patients older than years had a hazard ratio for dying of . (p. ). we will detail the type and pattern of neurological complications in t-cell non-hodgkin's lymphoma (nhl), and review how they differ from those associated with b-cell nhl, and the lymphomas in general. this study is the first step in a process to characterize these tumors to determine if special staging or cns prophylaxis are indicated in any of the subtypes of t-cell lymphoma. we recently have encountered women with breast cancer and an unusual sensorimotor neuropathy. the neuropathy was the major clinical problem. in women the initial symptom was severe itching, generalized and first localized to the involved breast and then generalized. all developed distal extremity numbness and burning that very slowly progressed proximally, and in became generalized. four complained of painful muscle cramps in the extremities ( ) and jaw ( ). all had mild extremity weakness, distal ( ) and proximal ( ) . three women developed symptoms up to months prior to cancer diagnosis, shortly after diagnosis, and years after diagnosis. four women had disease confined to the breast and regional lymph nodes, and had metastatic disease in remission. although annoying, symptoms were generally not disabling. three women stabilized or had slight improve-associated with breast cancer ment with cancer treatment, and continue to gradually progress while in cancer remission; required a cane to ambulate after years due to sensory ataxia. one who developed cancer relapse had concurrent neurological relapse. one woman treated with high-dose immunoglobulin did not improve. none had significant weight loss. laboratory abnormalities included elevated erythrocyte sedimentation rate ( - ) in , antinuclear antibody : in , csf with lymphocytic pleocytosis ( - white blood cellslmm) in / and elevated protein ( - mg/dl) in available. emgi ncv showed mild sensory-to-motor polyneuropathy in available. none had detectable antibodies against peripheral nerve or dorsal root ganglia. the etiology of sensorimotor neuropathy in these patients is unknown, but it may represent a distinct paraneoplastic syndrome that can herald the onset of, and parallel the course of breast cancer. our objective was to determine whether ceramide induces differentiation of anaplastic glioma cells. sphingomyelin hydrolysis resulting in ceramide production has been linked to differentiation of leukemia cells. t rat anaplastic glioma cells, seeded at x lo cells per well, were grown in serum-a glioma cell line program and abstracts, american neurological association free media. on day , cells were photographed, scored for processes longer than one cell body, and counted. c ceramide changed plump cells to flattened cells with many long processes: ceramide treatment increased the percentage of cells with processes from % (sd) to * % (n = , p < . ). control cells grew to . x lo cells/well . x lo cells; ceramide-treated cells grew to . x lo ? . x los (n = , p < . ). although one ceramide analog reproduced the c ceramide-associated changes, the optical isomer of this analog did not, demonstrating stereospecificity. cerarnide did not decrease cell viability by trypan blue. ceramide inhibits proliferation and induces process formation in a glioma cell line, causing it to assume a more differentiated phenotype. cerarnide or its analogs represent possible future therapeutic agents that would inhibit the growth and affect differentiation of anaplastic gliomas. [bashir, mod pathol ; ( ) : - )). this is similar to the pattern seen in ebv-infected human b cells and unlike the uniform latent infection seen in burkitt's lymphoma. we tested the hypothesis that long-term passaging of ebv-immortalized human b cells in immunodeficient mice leads to emergence of a uniform nonlytic pattern of ebv infection associated with appearance of the malignant profile. ebv-infected normal human b cells were serially passaged intracerebrally in severe combined immunodeficient cb mice ( x ' cells per mouse, mice per passage for a total of passages). frozen mouse brain sections from each passage were stained with vca antibody (ebv lytic cycle) and hybridized with biotinylated bamh -w sequence of ebv. all injected animals developed tumors as previously described (bashir, lab invest ; ( ): - ) . tumor cells continued to express vca and showed latent and lytic hybridization patterns with bamh,-w after passages despite exhibiting monoclonality (surface immunoglobulins) and random chromosomal changes. lytic infection of immortalized b cells with ebv is stable, resembling brain lymphomas in aids, and unlike the latent infection seen in burkitt's lymphoma. a previously healthy -year-old man developed diplopia and incapacitating, diffuse weakness over a period of weeks. examination showed a "one-and-a-half'' syndrome of horizontal gaze paresis, patchy severe weakness with atrophy and fasciculations, absent tendon reflexes in the legs and right biceps, and decreased vibration sense in the feet. csf contained a mild pleocytosis, elevated protein, and oligoclonal igg bands. electrophysiological testing indicated a generalized sensorimotor axonal neuropathy with diffuse denervation. small-cell lung carcinoma was diagnosed by bronchoscopy. prednisone produced mild subjective improvement. chemotherapy was begun but the patient developed fatal septicemia. serum was negative for anti-hu or anti-gm with paraneoplastic encephalomyeloneuritis antibodies. serum and csf contained high titers of igg antibodies reacting specifically with a protein antigen of approximately kd in immunoblots of human cerebral cortical neuronal nuclei or of human purkinje cells. this pattern of autoantibody reactivity was not present in sera from any of other patients with small-cell lung carcinoma, of whom had paraneoplastic encephalomyeloneuritis and anti-hu antibodies, nor was it present in many patients with other neurological disorders. the patient's serum has been used to probe a human cerebellum expression library and to isolate a cdna clone that is being characterized. acute encephalopathy is the problem in % of neurology consultations reported at mskcc. we studied patients ( prospectively and retrospectively) to determine clinical findings, causes, and outcome. fifty-five were women and were men, and the average age was years. all patients had cancer: lung ( %), gastrointestinal tract ( %), breast ( %), and others ( %), forty-two patients ( %) were delirious on admission and delirium developed an average of days later in %. encephalopathy occurred postoperatively in %. symptoms included confusion ( %), lethargy ( %), agitation ( %), hallucinations ( %), and seizures ( %). signs included deficits in attention ( %), memory ( %), language ( %), lateralizing signs ( %), and asterixis ( %). the average mini-mental status test (mms) score was ( = normal). a single cause for delirium was found in only % of patients with an average of etiologies per patient. metabolic abnormalities were found in % of patients, and were a primary cause in %; disseminated intravascular coagulation contributed to delirium in %. cns metastases were found in % and were a major cause of delirium in all. fifty percent of the patients had fever/ systemic infection, but sepsis was present in only %; only patient had cns infection. medication contributed to delirium in % but was a primary cause in only %. the -day mortality rate was % and delirium improved in % (average mms = ). patients with cancer have multiple, potentially treatable causes of delirium. delirium is associated with a high death rate, though patients generally improve. radiation therapy for brain tumors d. w. dodick, b. mokri, k. k. unni, g. m. miller, and e. g. shaw, rochester, m n osteosarcoma in a previously normal bone is a rare but recognized remote effect of radiation therapy. any bone in the field of radiation can be affected. involvement of cranial bones is exceedingly rare. we could identify only patients ( men and women) with postirradiation osteosarcoma of the calvarium seen at the mayo clinic over a -year period, from to . all had received radiation for brain tumor, osteosarcoma had appeared in the field of radiation in all, the interval from radiation therapy to the appearance of sarcoma ranged from to years, and diagnosis of sarcoma was confirmed histologically in all cases. the patients' age at the diagnosis of the brain tumor ranged from to years. the nature of the brain tumor was unverified in cases, was a low-grade ependymoma in the third case, and a pilocystic astrocytoma in the fourth case. one patient is still alive months after the diagnosis of the sarcoma. she received chemotherapy and subsequently underwent resection of the osteosarcoma. one patient died postoperatively after partial resection of the sarcoma. the other patients died months and months after the diagnosis of the osteosarcoma despite additional radiation therapy in the former and aggressive chemotherapy in the latter. element-la fusion gene as a potential marker of neural tumor differentiation lawrence recht, chiffon wu, and louis j. degennam, worcester, ma inducing cancers to differentiate into more benign differentiated tumors represents a novel oncological strategy. to establish a model that would permit assessment of this phenomenon at a molecular level, we created and have partially characterized a murine neuroblastoma line that has been stably transfected with a synthetic fusion gene containing the promoter element of the rodent synapsin i gene (synapsin i regulatory element isre)). in vivo, this promoter directs the neuron-specific expression of the synapsin i gene in normal adults. the gene also is expressed in varying amounts in neuronal tumors including neuroblastoma. in the synthetic fusion gene, the sre has been linked to the lacz gene that encodes bacterial p-galactosidase. a simple histochemical assay for p-galactosidase therefore provides a specific marker of the expression of the fusion gene. our preliminary experiments as of this writing have shown that it is possible to detect p-galactosidase activity in the transfected neuroblastoma cells both in vitro and in transplanted tumors. it appears possible therefore that this transfected neuroblastoma cell line can provide a useful model system with which to assess the effects of differentiation therapies. larger lesions (> cm ), and larger midline shifts (> mm). twenty-eight of ( %) patients with prior has had bt has compared to of ( %) patients without prior has. in patients, their bt h a was similar to their previous ha but was more frequent or severe. we conclude that has in bt patients are common but usually not severe. nausea, vomiting, an abnormal neurological examination, or a change in prior headaches warrant further investigation. cairncross and macdonald showed that procarbazine, lomustine, and vincristine (pcv) are effective for recurrent anaplastic oligodendrogliomas (ao) (ann neurol ; : - ) . pcv now has a major role in management of all forms of oligodendrogliomas ( ), but the biological basis for this response is unknown. to evaluate one subset of possibilities, we studied patients ( ao, ) with a ct method that permits measurement of blood-to-tissue transport (kj, tumor-to-blood transport (k ), and vascular volume (v,) (ann neurol ; : - ) . pcv was used to treat of the patients. k, (~ grr-' min-') values were highly variable for whole tumor, ranging from . to . (mean . k . ) with no difference between a and . k and v, were also highly variable. k, of tumor-free brain was . to . (mean . ? . ). in comparison to malignant astrocytomas, which have a mean k, in the range of . with some as high as . , a and appear to be much less permeable. this suggests that the efficacy of pcv may be due to factors other than capillary transport, such as tumor-cell sensitivity. frameless stereotactic localizer gene h. barnett, donald w. komos, and charles p . steiner, cleveland, oh extent of tumor resection has been shown to correlate with prognosis in malignant gliomas. although frame-based stereotactic techniques can provide information regarding tumor margin, they are often unwieldy and require expensive and elaborate computing systems. a frameless stereotactic neurosurgical localizing system was designed that overcomes these liabilities. this armless, frameless, stereotactic pointing device provides real-time three-dimensional localization information during operation. in addition to assisting in placement of a trephine craniotomy, it allows volumetric resection of the tumor with virtually complete excision of even large irregularly shaped tumors. mean error on localizing a point in space using this system has proven to be less than mm. a technical description of the system as well as surgical results are presented. to investigate the effect of age on response rate to chemotherapy and time to progression (ttp) and death ('itd) in patients with recurrent astrocytomas and malignant astrocytomas, we reviewed case records and scans of patients who received chemotherapy at the university of michigan with bischloroethylnitrosourea or procarbazine. three age groups were studied: ( ) < yr (n = ); ( ) - yr (n = ); ( ) > yr (n = ). tumors were grouped as grade r+ (recurrent grade plus grade , n = ) or grade (n = ). serial computed tomographic or magnetic resonance scans were analyzed in a blinded fashion and graded as progressive disease (pd), stable disease (sd), or partial response (pr, > % decrease in size). the pr rates for the age groups were %/ %/ % for grade r+ tumors ( p = . ) and %/ %/ % for grade tumors ( p = . ). median 'itp was weeks for grade r + and / weeks for grade . median ltd was / / weeks for grade r+ and / / weeks for grade . we conclude that age is an important prognostic factor with respect to likelihood of response to chemotherapy, duration of response, and survival irrespective of grade. cheryl p. harris and kurt a. jaeckle, salt lake city, ut intravascular malignant lymphomatosis (iml), a b-cell lymphoma confined to small venules and capillaries, often presents with neurological symptoms. this disease is uniformly fatal ( -month mean survival); no successful treatment has been identified. we observed marked reproducible neurological improvement after plasmapheresis in a -year-old woman with iml. presenting with a cauda equina syndrome, she progressed over year with neurological, hepatic, and hematological disease. persistent laboratory abnormalities included a high sedimentation rate ( mm/hr), coagulopathy, hemolytic anemia, and elevated liver enzymes. extensive evaluations for infectious, autoimmune, and neoplastic processes, including bone marrow examination, were inconclusive. because of neurological progression, empiric therapy with high-dose steroids followed by cyclophosphamide was initiated without response. plasmapheresis ( ml/kg in exchanges) effected resolution of encephalopathy and normalization of the coagulopathy and sedimentation rate. neurological progression recurred within weeks of pheresis; repetitive courses reproduced neurological response. finally, progressive dementia ensued, and a decision was made to cease pheresis; the patient died days later, months after presentation. autopsy disclosed diffuse intravascular cd- positive malignant lymphoma cells in small vessels of all organs. although the mechanism is unknown, the serendipitous discovery of response to plasmapheresis in this patient warrants further consideration. morphine is an effective analgesic in the rat after injection into a number of discrete brainstem regions, including the periaqueductal gray (pag), the locus coeruleus (lc), and the nucleus raphe magnus (nrm). early work with morphine gray and the nucleus raphe magnus established the existence of synergy between the brainstem and the spinal cord in rats. more recently, studies from our laboratory revealed synergy between two brainstem structures, the pag and the lc. in the current study, we explored the analgesic interactions between the pag and the nrm using indwelling cannulae. first, we established morphine dose-response curves and calculated the ed,, independently in the pag ( . pg) and the nrm ( . pg). we then simultaneously injected various morphine doses into both regions. injecting morphine at pg into either the pag or the nrm did not elevate tailflick latencies above baseline values. however, administered into both regions simultaneously, the -pg doses produced an % maximal response, corresponding to more than a threefold increase of baseline latencies. a fixed morphine dose of pg in the pag shifted the morphine dose-response curve fivefold in the nrm (ed,, . pg), whereas a fixed nrm dose of fg shifted morphine's dose-response in the pag approximately twofold. together, these results clearly show synergistic interactions for morphine between the pag and the nrm. the presence of synergistic interactions between brainstem nuclei as well as between the brainstem and the spinal cord underscores the complexity of opioid analgesic systems. p . the glutamate uptake inhibitor ~-trans- , -pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain john d. e. barks and faye s. siloerstein, ann arbor, m i important evidence of the neurotoxicity of endogenous glutamate (glu) in mammalian brain was provided by the observation that dl-threo- -hydroxyaspartate, a high-affinity glutamate uptake (hagu) inhibitor, was neurotoxic in adult rodent striatum (j neurochem ; : ) ; however, the absence of neurotoxicity in neonatal brain was interpreted as evidence that immaturity of glutamatergic innervation limited the potential role of endogenous glu as a neurotoxin in the immature brain. yet, considerable data provide indirect support for the hypothesis that glu can be neurotoxic at this stage. to resolve this issue, we assessed the neurotoxicity of a novel, selective hagu inhibitor, ~-trans- , -pyrrolidine dicarboxylate (l-pdc) (j med chem ; : ), in postnatal day (pnd) rats (n = ). l-pdc (ph . ) was stereotaxically injected into right anterior striatum (str) ( nrnol, n = ) or through dorsal hippocampus into posterior str ( nmol, n = ; nmol, n = ). animals were killed days later, and neuropathology was assessed in cresyl violet-stained sections. after anterior injections, focal neuronal necrosis was evident in dorsal str; high-dose posterior injections caused prominent hippocampal lesions with pyramidal layer thinning and focal necrosis in dorsal thalamus, while nmol produced small foci of pyramidal cell loss. in both groups, focal cortical necrosis and callosal cysts were apparent adjacent to the injection track. l-pdc-induced brain injury provides direct support for the hypothesis that endogenous glu may be neurotoxic in the developing brain. (sax et al, ann neurol ; : a) . the signs and symptoms of of these patients lessened for to months. furthermore, a patient with a severe oral-lingual biting dyskinesia improved when taking doses up to milligrams per day for months. we noted no-significant adverse reactions, although patients had mild but labile elevations in sgop and sgpt. one patient receiving opioid analgesics for pain inadvertently failed to discontinue the naltrexone but noted no reduction in the pain-alleviating effects of the analgesic. although hyperkinesia, especially of midline functions, as well as quality of life improved for the hd patients, their cognitive deficits remained unaffected. these observations suggest that chronic naltrexone is a safe and effective agent to treat chorea, dysphagia, and oral dyskinesia in hd for periods longer than a year. furthermore, they indicate that naltrexone can be effective in ameliorating oral-lingual biting tardive dyskinesia. these findings support our previous hypothesis that endogenous opioids play a role in rhe modulation of the dopamine system in hyperkinetic stereotypic movement disorders. a. jon stoessl, elizabeth szczutkmuski, and hanna fydvszak, london, ontario, canada we previously have demonstrated (psychopharmacology ; : - ) that intraperitoneally (ip) administered cholecystokinin (cck)- s suppresses vacuous chewing mouth movements (vcms, a putative mode of tardive dyskinesia) in rats exposed to chronic neuroleptics. as cck is not thought to cross the blood-brain barrier in significant amounts, its site of action in this paradigm is unclear. other behavioral and neurochemical effects of ip cck are blocked by vagotomy. male sprague-dawley rats were administered fluphenazine decanoate (flu; mg/kg im) or its vehicle every weeks for approximately weeks. cck ( , , or ng intracerebroventricularly) had no effect on neuroleptic-induced vcms. another group of neuroleptic-treated rats was subjected to bilateral subdiaphragmatic vagotomy or a sham procedure. cck- s ( , , or pg/kg intraperitoneally) suppressed neuroleptic-induced vcms in shamoperated animals, which confirmed our previous results. in vagotomited animals, chronic flu failed to induce vcms and cck was without effect in vehicle-or neuroleptictreated animals. these data suggest that the effects of cck on flu-induced vcms may be mediated peripherally, and that vagal pathways may be important for generating this response. (supported by the ontario mental health foundation and the ontario ministry of health.) p . excitotoxic amino acids are not involved in dopaminergic neurotoxicity of m p t p eldad mehmed, jutta rosenthal, and avinoam reches, petah tiqva, tel aviv, and jerusalem, israel the dopaminergic (da) neurotoxicity of -methyl- phenyl- , , , -tetrahydropyridine (mptp) is mediated via its oxidation in cns to mpp + , which enters da neurons and poisons mitochondrial complex i. da neuronal damage induced by direct nigral mpp+ injection is prevented by pretreatment with n-methyl-d-aspartate receptor antagonists, which suggests that excitatory amino acids are involved in mptp toxicity. since local mpp + application may produce nonselective nigral damage, we examined whether excitotoxins have a role in toxicity of systemically administered mptp. c black mice were injected intraperitoneally, once, with mptp.hci( mg/kg) and decapitated days later. groups of animals underwent the following pretreatments: (i) decortication week prior to mftp; ( ) intracerebroventricular injections of the excitatory amino acid receptor antagonists -amino-phosphonoheptanoate and d-glutamyl-glycine; and ( ) intraperitoneal injections of the calcium channel antagonists nimodipine, diltiazem, and flunarizine minutes prior to mptp. mptp produced marked striatal da depletions. decortication, destroying glutamatergic corticostriatal projections, intracerebral amino acid receptor antagonists, and systemic calcium channel antagonists did not protect mice against mptp toxicity; mptp-induced striatal da decreases were similar to those given the neurotoxin alone. this study suggests that excitotoxins are not involved in the mechanism of mptp toxicity. scopolamine-induced cognitive deficits k . j . meador, m . e. allen, p. franke, e. e. moore, and d. w. loring, augusta, ga a unique neurophysiological role for thiamine in cholinergic systems has been suggested. total thiamine content in cholinergic nerve terminals is comparable to that of acetylcholine, and the phosphorylation state of thiamine changes with release of acetylcholine. thiamine binds to nicotinic receptors and may exhibit anticholinesterase activity. based on these observations, we investigated the effects of pharmacological doses of thiamine on the cognitive deficits induced by the anticholinergic scopolamine in healthy young adults using a randomized, double-blind, placebo-conrrolled, double crossover design. cognitive tests included the p eventrelated potential and free recall memory for a verbal paragraph. conditions included baseline (bl), thiamine gm by mouth and scopolamine . mg/kg intramuscularly (b + scop), and lactose by mouth and scopolamine (plac + scop). testing was performed hours post thiamine or placebo, and . hours post scopolamine. thiamine significantly reduced the adverse effects of scopolamine on p latency (f [i, = . , p . ) and percent recall memory ( f el, ) = . , p . ). means (+sd) and p latency (ms) were bl = ( ), b + scop = ( ), and plac neurol ; : - ) . we previously reported that cff improved in of ms patients ( %) given el- orally in divided daily doses ranging from . to . mg (stefoski et al, neurology ; : - . subsequent analysis revealed that in of the patients cff improvements and reversals followed in a phase-locked trend the rising and falling serum concentrations of el- , including patients whose changes remained below the % increase needed to qualify for the improved category. these results resemble the phase-locked effects of temperature on neurological function in ms. the cff changes, because they so closely reflect variations in serum concentration, suggest that el- tissue levels closely follow those in serum and that el- rapidly crosses the blood-brain barrier. efficacy of el- in ms is also predicted to have a close relationship to serum levels. p . development of a n internal standard detectable by proton and phosphorus- nmr and hplc w. e. klunk, k. panchalingam, r. j . mcclure, and j . w. pettegrnu, pittsburgh, pa comparison of quantitative results from different analytical techniques can prove difficult due to the peculiarities of the particular techniques and the lack of a common standard applicable to all of the techniques. recently, both in vivo and in vitro nuclear magnetic resonance (nmr) have been applied to the quantification of a large variety of metabolites. although nmr can be applied to the study of living tissue, the question arises of how this technique compares with more traditional techniques such as high-pressure liquid chromatography (hplc). although this question can be addressed partly by studying perchloric acid (pca) extracts, it is difficult to directly compare this in vitro nmr data with results from hplc. to address this question, we have developed an internal standard that can be quantified directly by in vitro phosphorus- nmr, proton nmr, and by -fluorenylmethyl chloroformate (fmoc) derivatization followed by separation by hplc. a variety of aminophosphonic acids were studied by 'p nmr, 'h nmr, and hplc. promising compounds were added to pca extracts of human brain. the optimal compound was found to be -aminopropylphosphonic acid (app, ho p-ch -ch,-ch -nh ). app is easily detectable by all techniques, falls well outside of the region of interest in phosphorus- nmr, and is well resolved by proton nmr at mhz. it is easily separable from the phosphomonoesters and phosphodiesters observable by hplc and from the amino acids that occur in brain in significant concentrations. app appears to be a useful internal standard in the study of phosphorus and amino acid metabolites by in vitro p nmr, 'h nmr, and hplc. theories of sentence comprehension hypothesize at least a grammatical component that establishes the relationship among words in a sentence, and a semantic component that determines the meanings of these words. we used positron emission tomography (pet) to quantify regional cerebral blood flow (rcbf) in neurologically intact subjects during their detection of a letter target, a grammatical target, or a semantic target in the same written sentences. a mixedmodel analysis of variance (anova) revealed significant main effects for region (f e , ) = . ; p < . ), condition (f , = . ;~ < . ), and a significant region times condition interaction ( f {go, = . ; p < . ), but there were no differences between individual subjects. subsequent anovas revealed increased rcbf in a unique set of brain regions during the subjects' response to a grammatical probe when compared to their response to a letter probe of the same sentences. a unique distribution of rcbf also distinguished response to the semantic probe from response to the grammatical probe and the letter probe. other brain regions apparently contributed to performance for several activation conditions. these findings support the hypothesized dissociation of specific linguistic components based on their unique cerebral topographical representation, and that a distributed network of brain regions subserves sentence comprehension. cerebral music processing-a comparison study of musically trained and naive individuals louis s. russo, jr, jachonville, fl we performed topographical mapping of brain electrical activity in right-handed symphony musicians and righthanded, musically naive individuals during various musical tasks; namely, listening to solo piano music, silent singing of familiar music, and silent reading of unfamiliar music. fast-fourier transform ( f r ) of electrocortical activity was carried out during task performance and the eyes-open resting state. data were analyzed using a computer-assisted model. increases in regional beta activity of greater than standard deviations from the resting state were considered significant of activation. during audition, the musicians showed activation in the right posterior parietotemporal region; the naive showed no change from the resting state. during silent singing, the musicians showed bitemporal activation, r > l; the naive showed activation in the right mid-and posteriortemporal regions alone. during silent sight reading, the musicians showed a major activation in both temporal regions, l > > r, the naive showed only a marginal change in the posterior temporal-occipital regions. these data suggest that music processing is primarily a right cerebral function in untrained individuals and a bilateral function in musicians. musicians, in contrast to the naive, show progressively more left brain activation as task complexity increases. the present study analyzes language profiles in patients who presented with primary progressive aphasia (ppa) without global dementia for at least years. language and cognitive impairment were evaluated using the western aphasia battery (wab) and the mattis dementia rating scale (drs). expressive language disability with reduced speech fluency and anomia, but preserved language comprehension and nonverbal cognition, were typical features in early stages. spontaneous speech was significantly more impaired in ppa than in anomic aphasia after left-hemisphere stroke and in language impairment in probable alzheimer's disease (ad) ( p = . ). the profile of aphasia suggests that ppa tends to affect anterior parts of the language-dominant cortex first. neuroimaging generally showed mild to moderate brain atrophy. in patients atrophy involved especially the left frontal progressive aphasia cortex. follow-up examinations that were done in patients or several years after the first assessment revealed continuous, most often rapid deterioration of language impairment. two patients died and years after the onset of ppa. neuropathological examination showed ad in patient and pick's disease in the other patient. beverly clarke, adrian upton, markad kamath, and helene griff;., hamilton, ontario, canada eight patients implanted with a cyberonics neurocybernetic prosthesis model to stimulate the vagus nerve were assessed for changes in cognitive performance. the patients had complex partial seizures for more than years, with more than per month. patients were years * . sd old. cognitive evaluation included response time to a randomized light signal appearing on a switch box (test a); test b, in which the signal appeared bilaterally; and test c, in which a response to the signal was required while the patient simultaneously ignored a second signal. data were collected and analyzed using an apple i e computer and switch pad. all patients were taking therapeutic levels of anticonvulsant medications and dosages were constant. testing occurred times during a day preoperatively (day l), weeks postoperatively with the stimulator on (day ), and months after turn-on (day ). patients were randomized into high-and low-frequency stimulation groups (hfg and lfg). hfg parameters were hz, so msec pulse width (pw), and lfg hz, msec pw. examiners were blinded as to group. student's t-test analyses of mean differences between groups and individual measurements showed a significant difference between hfg and lfg for test c ( p < . ). lfg showed a significant improvement for tests a, b, c between day and , and for test c between day and . no group effect was seen between day and in the lfg. individual measurements showed improvement for test b ( p < . ) for the hfg between day and , test b ( p < . s) between day and , and tests a and b ( p < . ) and ( p < . ) day vs . the lfg group improved between days and and and . between day and day , the lfg showed improvement only for test b ( p < . ). chronic stimulation of the vagus nerve improves cognitive function in epileptic patients and this improvement is more marked with low-frequency stimulation. a. guidotti, and j. d. rothstein, bologna, itah, washington, dc, and baltimore, m d we reported idiopathic recurring stupor (irs) in a patient with stuporous episodes without known causes and reversed by flumazenil, a specific benzodiazepine (bz) antagonist. ictal plasmdcerebrospinal fluid (csf) showed increased bz-like activity (ann neurol, in press). recently, an endogenous bzreceptor ligand (endozepine [ez]) has been purified from mammalian brain with properties similar to diazepam. it acts like diazepam to potentiate gamma-aminobutyric acidmediated postsynaptic inhibition. we hypothesized that irs might be due to an excess of this substance. irs was diagnosed in patients, and years old, who had recurring stupor or coma episodes lasting hours to days. ictal brain ct/mri, kidney, liver, heart, blood glucose, ammonia, and osmolality were normal. eeg showed fast -hz background activity while the patients were unreactive to stimuli, reversed by flumazenil. ictal serum or csf revealed an enormous increase of the ez in both patients, with levels as high as nm, compared to to nm in control serum/csf. interictal csf or serum in irs contained ez levels similar to control csf and serum. irs may be due to excess ez. the cause for increased ez is unknown. parkinson's disease: evidence from word learning murray grossman, jenger mickanin, barbara schaefr, kris onishi, matthew b. stern, steven gollomp, and howard hurtig, philadelphia, pa several reports have suggested that patients with parkinson's disease (pd) have intellectual impairments in several domains such as memory, but few studies have explored difficulties in language processing. we investigated the ability of nondemented pd patients with mild motor impairments to learn about the grammatical and semantic information represented in a new verb. the new verb was presented to patients in a sentence-picture matching context, and we probed their recall of the verb minutes later. a sentence judgment task assessed grammatical knowledge by asking patients to judge the new verb, known verbs, and pseudowords used appropriately or incorrectly in a sentence. we found that % of pd patients were significantly impaired in their grammatical appreciation of the new verb (f [i, = . ;p < . ). this was not related to their motor disorder or neuropsychological performance. a picture classification task used pictures illustrating specific aspects of the new word's meaning to evaluate semantic knowledge. pd patients were as accurate as controls at deciding whether a picture illustrated the meaning of the new verb (f ( , = .lo;p > . ). only pd patient ( %) had difficulty sorting pictures. selective difficulty recalling only grammatical aspects of a new word suggests that the word learning impairment in pd cannot be entirely explained by poor memory. instead, in agreement with other recent findings, pd patients may be impaired in some aspect of grammatical processing in language. we discuss the hypothesis that defects in the frontocaudate axis in pd underlie this impairment. neuropsychological performance on both verbal and nonverbal tasks is reported to differ between healthy men and women. some of these cognitive differences are postulated to reflect differences in interhemispheric and intrahemispheric cerebral organization. our preliminary study indicated that women with alzheimer's disease (ad) performed worse than men on a composite neuropsychological battery, even after effects of potentially confounding variables were considered (buckwalter et al, j clin exp neuropsychol ; : ) . to explore further the nature of gender-associated differences in ad, we analyzed data from a verbal and a nonverbal task (the boston naming test and drawings from the spatial quantitative battery supplement to the boston diagnostic aphasia examination) for men and women who met nincds-adrda criteria for "probable" ad. prior to ana-grip strength kg [range . - kg]). the electrophysiological examination showed improvement with reversal of conduction block in patients and was unchanged in . an apparent response to the placebo was seen in patients. improvement after ivig therapy was maintained for variable durations ( - wk) and reoccurred with subsequent infusions. an equally effective response was documented after infusion of a single ivig dose of gm/kg. we conclude that ivig therapy is effective in some patients with cidp, even after long duration of illness. the best responses were observed in patients with recent relapse. a single high-dose treatment may be equally effective. the object of this study was to examine whether borrelia burgdorjeri antigens could be detected in csf in the absence of detectable antibodies to b. burgdorjeri (the etiological agent of lyme disease). osp a is a -kd antigen that is specific for . duvgdorferi osp a was probed using western (immuno) blot and specific mouse monoclonal antibodies. polyclonal lyme antibodies were detected in csf using standard micro enzyme-linked immunosorbent assay. seven patients had osp a in csf without detectable lyme antibodies. there were men and women aged to years (mean yr). disease duration ranged from weeks to years. neurological syndromes included confusion with acute flulike illness, optic neuritis, hemiparesis with inflammatory brain lesion, encephalitis, headache with erythema migrans, bilateral facial nerve palsies, and encephalomyeloradiculitis. three patients had csf abnormalities. in patients csf parameters were otherwise completely normal. possible explanations for undetectable csf lyme antibodies included early infection ( patients), prior antibiotics ( patients), and prior steroids plus antibiotics ( patient). in patient there was no obvious explanation. we conclude that osp a, a specific antigen of b. burgdorferi, may be present in csf without a detectable humoral response. the diagnosis of neurological infection with b. bwgdorjeri should not require a positive csf serology. mollaret's meningitis: demonstration by polymerase chain reaction a -year-old man was seen in september for his fourth episode of aseptic meningitis over a -year period. the episodes conformed to criteria for mollaret's meningitis as published by bruyn, straathof, and raymakers, and subsequently by others; they lasted about week, and were characterized by fever, headache, meningismus, lymphocytic pleocytosis, elevated protein in cerebrospinal fluid (csf), and spontaneous resolution without residua. extensive prior evaluations had failed to uncover a cause. the patient was otherwise well and neither he nor his wife had any history of sexually transmitted diseases. suspicion of herpes simplex virus (hsv) arose due to a single transient, raised skin rash several weeks earlier that failed to yield virus on culture. the patient was treated with acyclovir, which resulted in rapid resolution of symptoms. though culture and immunological studies of csf and blood again were unrevealing, polymerase chain reaction (pcr) studies of csf confirmed the presence of hsv type . we suspect that herpes simplex virus is a more common cause of recurrent aseptic meningitis than current culture and immunological techniques would suggest. pcr offers increased diagnostic sensitivity for neurotropic viruses and should be considered in patients with recurrent meningitis of cryptic etiology. differentiation by inorganic lead: a role for protein kinase c j. lutewa, j. p. bressler, r. r. indurti, l. belloni-olivi, and g. w . goldstein, baltimore, m d microvascular endothelial function in developing brain is altered by inorganic lead. this may result from changes in protein kinase c (pkc) modulation. we examined the effects of inorganic lead on an in vitro model of neural endothelial differentiation. astroglial-induced endothelial differentiation into capillary-like structures was inhibited by lead acetate with % maximal inhibition occurring at . pm lead. inhibition was independent of effects on cell viability or growth. we examined the effects of lead on cellular pkc pools under conditions that inhibited capillary-like structure formation. membranous pkc increased in c astroglial and neural endothelial cells after exposure to lead acetate. exposing c cells to p,m lead for hours increased membranous pkc by % as determined by immunoblotting. membranous pkc increased in response to as little as nm lead and saturated at pm. phorbol esters were used to determine if pkc modulation was mechanistically related to lead's inhibition of capillary-like structure formation. -myristate -acetate ( nm) inhibited endothelial differentiation by * %, whereas -alpha-phorbol , didecanoate was without effect. these findings demonstrate that inorganic lead may induce cerebral microvessel dysfunction by interfering with pkc modulation in microvascular endothelial or perivascular astroglial cells. w. f. brown, b. v . watson, j . garland, g . c. ebers, and n . desai, london, ontario, canada gait difficulties in multiple sclerosis (ms) are commonly accompanied by fatigue and dyspnea. possible explanations for the latter include weakness andlor dyssynergia of the respiratory muscles, including possible abnormalities in central pathways regulating respiration. this study examined central and peripheral motor conduction to the diaphragm in ms patients whose gait was notably labored and accompanied by breathlessness. peripheral conduction was assessed by measuring the latency and size of the surface-recorded diaphragmatic maximum m-potential responses to supramaximal stimulation of the phrenic nerve in the neck, and central motor conduction by comparable measurements in response to magnetoelectrical stimulation over the vertex. peripheral motor conduction was normal. the most striking abnormalities were in central motor conduction. cortical stimulus-evoked diaphragmatic responses were absent on both sides in patients, and unilaterally in l patient, whereas in others the latency of the cortical stimulus-evoked response was increased and the size clearly reduced and entirely normal in only patients. these studies show that central conduction program and abstracts, american neurological association to the diaphragm is commonly abnormal and may play a role in the fatigue and dyspnea experienced by ms patients. six men ( - yr) developed myelopathy that progressed slowly over several months and was characterized by asymmetrical, incomplete spinal cord syndrome manifested at the sensory level at the trunk, mild spastic paraparesis, and urinary incontinence. the spinal cord lesions at appropriate levels were recognized by mri as enhancing lesions in of the men. coxiella burnetii infection was confirmed in the blood of all patients by immunofluorescence microscopic assay (ifa) and transmission electron microscopy (tem). in patients, we detected c. burnetti by tem and ifa using csf of the patients inoculated onto fresh peripheral blood lymphocytes. four patients who were treated with appropriate antibiotics responded with either partial resolution of symptoms or arrest of further neurological progression. in patients the lesion was shown on mri to have decreased in size. in summary, we report cases of transverse myelopathy associated with c. barnetti infection. this is the first report, to our knowledge, of coxiella-related chronic myelopathy. we present a series of patients with different labyrinthine lesions diagnosed by mri. twelve patients with sensorineural hearing loss were studied by gadolinium-enhanced mri, including -mm contiguous t -weighted images through the labyrinth. ten patients had enhancement of the cochlea or vestibule, or both. all patients with cochlear enhancement had severe neural sensory hearing loss. all patients with vestibular enhancement had severe vestibular symptoms. the patients' final diagnosis included viral labyrinthitis ( patients), syphilitic labyrinthitis ( patients), bacterial labyrinthitis ( patient), and vestibular neuromas ( patients). one patient had an acoustic neuroma extending in the basal turn of the cochlea. the enhancement in patients with vestibular neuromas was brighter and there was slight mass effect in comparison with the patients with inflammatory labyrinthine lesions. one patient had hemorrhage within the vestibule from an adjacent temporal bone hemangioma. one patient with ct-proven cochlear otosclerosis had pericochlear areas of enhancement on gadolinium mri. mri can diagnose a variety of labyrinthine lesions that correlate very well with the patient's clinical symptoms. gadolinium should be used routinely in patients with suspected labyrinthine disease. the diagnosis of meniere's disease and endolymphatic hydrops remains a diagnosis of exclusion. few radiographic findings have been correlated with the clinical symptoms of this entity. we describe patients with symptoms of hearing loss or vertigo, or both, who demonstrated enhancement of the endolymphatic sac on gadolinium-enhanced mri. no enhancement was noted in a series of controls with no symptoms of hearing loss and vertigo. enhancement in the brain correlates with inflammatory or neoplastic conditions. we thus can speculate that enhancement of the endolymphatic sac reflects an inflammatory process in this location that may interfere with the normal resorption of indulin and secondary hydrops. in addition to excluding an acoustic neuroma and a labyrinthine schwannoma (which clinically may be confused with meniere's disease), contrast-enhanced mri may provide objective evidence in favor of labyrinthine hydrops. it was intermittent ( %) but progressive. the ha was mild to moderate in severity; it was the worst symptom in only ( %) and the first symptom in ( %) patients. has were worse in the morning in ( %) and interfered with sleep in ( %) patients. unlike true tension-type has, bt has were worse with bending over in ( %), with valsalva's maneuver in ( %), and nausea or vomiting were present in ( %) patients. an abnormal neurological exam was found in ( %) patients with has and ( %) patients without has lyzing the effects of gender, we used a hierarchical regression procedure to control for possible effects of subject age, education, age at onset of dementia symptoms, dementia duration, and family history of dementia. significant gender effects were found for the verbal task ( p < . ) (mean boston naming test score of . for women and . for men), but not for the drawing task. we conclude that verbal abilities are more severely affected in women than in men with ad, a difference that may in part reflect premorbid gender-associated differences in cerebral hemispheric organization. hemispatial placement is known to affect line bisection in patients with neglect. whereas placing stimuli in neglected space increases bisection error, placing stimuli in nonneglected space attenuates error. the effects of hemispatial placement on line bisection were examined in patients with chronic neglect (over months after stroke). all patients had large (frontotemporoparietal), unilateral, right-hemisphere lesions. each patient bisected lines of different lengths ( , , , and cm) in hemispatial conditions ( cm left of midline, midline, and cm right of midline). like previous reports, when patients bisected lines in left hemispace, a consistent ( / trials) left-sided neglect was observed ( . cm). however, when lines were bisected in center space, misbisections occurred on either side of the midline; and, unlike previous studies, when lines were bisected in right hemispace, a consistent ( / trials) right-sided neglect was observed ( . cm). the magnitude and directional consistency of line bisection errors were significant. neither visual field defects nor limitations in reaching accounted for the results. recovery in chronic neglect may involve a realignment of limited attentional resources favoring the body's midline. consequently, performance in both hemispatial fields can be biased toward midline, resulting in neglect of opposite directions. despite agreement that depression is the most common neuropsychiatric symptom associated with multiple sclerosis (ms), many aspects of this emotional change are unclear. one of the more controversial issues concerns the relationship between severity of ms and depression. this relationship is used to evaluate whether depression is an integral or reactive symptom of ms. examination of this relationship is complicated by the presumed overlap between somatic features of depressive and neurological symptoms in ms. to clarify this situation, we examined the relationship between severity of ms and categories of depressive symptoms using the beck depression inventory (bdi). eighty-nine patients and normal controls were examined. for certain comparisons, patients were classified as mild (extended disability status scale of - ) or moderatelsevere ( ) ( ) ( ) ( ) ( ) ( ) . results indicated that total bdi scores and the depressive symptom categories (mood, self-reproach, vegetative, and somatic features) were elevated in patients with ms, but the extent of these elevations was not related to severity of disease. these results suggest that depression in ms is not a simple reaction to physical disability. furthermore, clinical examination of depressive symptoms is straightforward and not confounded by severity of ms. neurological involvement in wegener's granulomatosis was studied in consecutive patients diagnosed at the mayo clinic. one hundred and nine patients ( %) had neurological involvement. peripheral neuropathy was seen in ( . %), cranial neuropathy in , external ophthalmoplegia in , cerebrovascular events in , seizures in , and miscellaneous involvement in . the mean age and sex ratio did not differ in those with or without neurological involvement. among the patients with peripheral neuropathy, had multiple mononeuropathy, had distal symmetric polyneuropathy, and had unclassified peripheral neuropathy. multiple mononeuropathy was one of the major presenting symptoms in patients. kidney involvement was significantly higher in the patients with peripheral neuropathy compared to those without it (p < . ). among the cranial nerves, the second, sixth, and seventh nerves were affected most frequently. multiple cranial nerves were affected in patients. unusual neurological manifestations among the miscellaneous group included spastic paraparesis, temporal arteritis, homer's syndrome, and papilledema. this is the first comprehensive study on the frequency and distribution of neurological involvement in wegener's granulomatosis. chronic inflammatory demyelinating polyneuropathy: a double-blind placebo-controlled crossover study treatment with high-dose intravenous human immunoglobulin (ivig) has been reported to be beneficial in some patients with chronic inflammatory demyelinating polyneuropathy (cidp), yet most observations have been nonblinded. we examined the effect of ivig therapy in patients ( men, women) with cidp in a double-blind, placebo-controlled crossover study. disease was chronic progressive (n = ) or chronic relapsing (n = ) and of variable duration ( mo to yr). the diagnosis was confirmed by electrophysiological ( ) and nerve biopsy ( ) examinations. the trial consisted of two -day periods each. patients were randomly treated with ivig ( . mg/kg/day) or placebo on consecutive days and followed. function was assessed by a quantitative neurological disability score, functional grade, grip strength measurement, and electrophysiological examinations at the beginning and end of each treatment period. with ivig therapy, significant improvement was documented in / patients (improvement in neurological disability score mean key: cord- -j riw ir authors: stikova, elisaveta; gjorgjev, dragan; karadzovski, zarko title: strengthening the early-warning function of the surveillance system: the macedonian experience date: - - journal: emerging and endemic pathogens doi: . / - - - - _ sha: doc_id: cord_uid: j riw ir epidemics and pandemics can place sudden and intense demands on health systems. the world requires a global system that can identify and contain public health emergencies rapidly and reduce panic and disruption of trade, travel, and society in general. strengthening public health preparedness requires establishing an integrated global alert and response system for epidemics and other public health emergencies along the lines of the world health organization’s international health regulations. the revised international health regulations provide a global framework to address these needs through a collective approach to the prevention, detection, and timely response to any public health emergency of international concern. a standardized approach for readiness and response to major epidemic-prone diseases should be developed. an early-warning and rapid-alert system is one of the possibilities to improve readiness at the local, regional, national, and international level to limit the spread of disease and to reduce health, economic, and social damage. the republic of macedonia, with world health organization support, has implemented an earlywarning system (alert) for priority communicable diseases to complement the routine surveillance system that reports individual confirmed cases. alert relies on reporting of eight syndromes by primary care facilities. data are analyzed weekly at the regional level and transmitted to national epidemiologists. it is perceived to be a simple and flexible tool for detecting and triggering timely investigation and control of outbreaks. alert was identified as a useful instrument for forecasting and detecting the start of the influenza season. at the beginning of the st century, the world still confronts: globally, from to december , the world health organization (who) identified , syndromes and diseases that were potential public health emergencies of international concern. of these events, subsequently were verified in the who european region [ ] . region reported cases of sars, including one death, from february through july . this figure corresponds to % of the cases reported worldwide over the same period. communicable diseases in the european region account for % of the disease burden measured in disability-adjusted life years. this is largely attributable to high rates of tuberculosis and growing rates of hiv infection, particularly in central and eastern european countries and in central asia, and to emerging and reemerging epidemic-prone diseases. some of the most prominent public health programs currently being undertaken are the eradication of smallpox, the ongoing efforts to eradicate poliomyelitis and to eliminate measles, the expanded programme on immunization, the stop tb partnership, the coordination of the global epidemic response to control sars, and the ongoing efforts to contain the spread of influenza a/h n virus (avian influenza) and to prepare for pandemic influenza. we must be aware, however, that widening development gaps, the collapse of public health infrastructure, poverty, urbanization, civil strife, environmental change and degradation, and the globalization of travel and trade can contribute to the new challenges posed by epidemic-prone and emerging communicable diseases worldwide. • the emergence of new or newly recognized pathogens such as nipah virus, ebola virus, marburg virus, severe acute respiratory syndrome (sars) corona virus, and influenza a/h n virus • the recurrence of well-characterized epidemic-prone diseases such as cholera, dengue, influenza, measles, meningitis, shigellosis, and yellow fever • the accidental release or deliberate use of biological agents such as anthrax [ ] in addition to the events described in table , member states in the european these are reasons for public health-capacity building at the local, national, and international level and strengthening of public health preparedness and response systems around the world [ , ] . avian influenza is a major challenge for the international community and a real public health threat. globally, as of june , , laboratory-confirmed human cases of influenza a/h n virus infection, including fatal cases (case-fatality rate about %), had been registered in member states. in the european region in , human cases including nine deaths were reported in turkey ( cases and four deaths) and azerbaijan (eight cases and five deaths) [ ] . many international organizations, including who, and experts are working together to coordinate activities regarding key actions, including controlling avian influenza in animals and reducing opportunities for human infection; strengthening the earlywarning system; containing or delaying the spread at the source; reducing morbidity, mortality, and social disruption; and conducting research to guide response measures. the challenges that epidemic-prone diseases, including avian influenza, pose to who are: the revised international health regulations (ihr) [ ] , which entered into force in june , provide a legal framework to assist countries in protecting the health of their populations against any potential public health emergency of international concern, implementing the necessary measures, and contributing to making the world more secure [ , ] . national and international partnerships will maximize the benefit of strengthening surveillance and response [ , ] . to ensure the timely detection of events that are potential public health emergencies of international concern, the who regional office for europe, aside from relying on official reports from national health authorities, systematically screens a wide range of formal and informal sources of information in several languages. the monitoring and control of communicable diseases are facilitated by wellfunctioning surveillance systems. surveillance systems provide information for early detection of potential outbreaks and help to identify disease trends, risk factors, and the need for interventions [ , ] . they provide information for priority setting, planning, implementation, resource allocation, and for evaluating preventive programs and control measures. surveillance systems are set up to detect and control communicable diseases in humans regardless of the cause and manner of transmission. their principal aim is to prevent further transmission of the disease to other persons by epidemiologic investigation [ , ] . the timely detection of outbreaks at the regional and national level is a priority function of communicable disease surveillance systems. in the process of implementing its ihr [ ] , who included the requirement for member states to maintain an adequate core capacity to detect and respond to significant public health threats. this requires that member states develop effective early-warning systems and strengthen their investigation and response capabilities [ , ] . • how to minimize the risk of international spread • how to assist countries in preparing for and controlling epidemics • how to coordinate and focus global resources when no single institution has the necessary capacity [ , ] since december , central and eastern europe and the baltic countries have worked together to strengthen surveillance and early-warning and response systems [ ] . to ensure a rapid and effective response to events (including emergencies) related to communicable diseases, an early-warning and response system has been put in place in macedonia. this is a web-based system linking the regional public health institutes (rphis) with the national public health institute (nphi) and the ministry of health. innovative electronic surveillance systems are being developed to improve early detection of outbreaks attributable to biologic and other causes of threats. a review of the rationale, goals, definitions, and realistic expectations for these surveillance systems is a crucial first step toward establishing a framework for further research and development in this area [ ] . syndromic surveillance has been used for early detection of outbreaks; to follow the size, spread, and tempo of outbreaks; to monitor disease trends; and to provide reassurance that an outbreak has not occurred [ ] . syndromic surveillance systems seek to use existing health data in real time to provide immediate analysis and feedback to those charged with the investigation and follow-up of potential outbreaks. optimal syndrome definitions for continuous monitoring and specific data sources best suited to outbreak surveillance for specific diseases have not been determined [ , ] . broadly applicable signal-detection methodologies and response protocols that would maximize detection while preserving scant resources are being sought [ , ] . stakeholders need to understand the advantages and limitations of syndromic surveillance systems. syndromic surveillance systems might enhance collaboration among public health agencies, health-care providers, information-systems professionals, academic investigators, and industry. however, syndromic surveillance does not replace traditional public health surveillance, nor does it substitute for direct physician reporting of unusual or suspect cases of public health importance [ , ] . specific definitions for syndromic surveillance are lacking, and the name itself is imprecise. diverse names used to describe public health surveillance systems for early outbreak detection include: however, syndromic surveillance is the term that has persisted. the fundamental objective of syndromic surveillance is to identify illness clusters • early-warning systems • prodrome surveillance • outbreak-detection systems • information system-based sentinel surveillance • biosurveillance systems • health-indicator surveillance • symptom-based surveillance early, before diagnoses are confirmed and reported to public health agencies, and to mobilize a rapid response, thereby reducing morbidity and mortality. syndromic surveillance aims to identify a threshold number of early symptomatic cases, allowing detection of an outbreak earlier than would conventional reporting of confirmed cases [ ] . the ability of syndromic surveillance to detect outbreaks earlier than conventional surveillance methods depends on such factors as the size of the outbreak, the population dispersion of those affected, the data sources and syndrome definitions used, the criteria for investigating threshold alerts, and health-care providers' ability to detect and report unusual cases [ ] . syndromic surveillance focuses on the early symptom (prodrome) period before clinical or laboratory confirmation of a particular disease and uses both clinical and alternative data sources. strictly defined, syndromic surveillance gathers information about patients' symptoms (e.g., cough, fever, shortness of breath). the analytic challenge in using syndromic surveillance for outbreak detection is to identify a signal corresponding to an outbreak or cluster amid substantial "background noise" in the data [ ] . however, signal-detection methods have not yet been standardized. temporal and spatio-temporal methods have been used to assess day-to-day and day and place variability of data from an expected baseline [ , ] . the new ihr [ ] entered into force on june , . the ihr are ( ) a legal framework for surveillance of international health threats, ( ) a procedure for who's recommendations to counteract public health emergencies of international concern, and ( ) a set of rules concerning routine measures against international disease spread. here we will briefly review the first of these features. in the globalized world, diseases can spread far and wide via international travel and trade. a health crisis in one country can affect livelihoods and economies in many parts of the world. such crises can result from emerging infections such as sars or a new human influenza pandemic. the ihr also can apply to other public health emergencies such as chemical spills, leaks, and dumping or nuclear accidents [ ] . the ihr aim to limit interference with international traffic and trade, ensuring public health through the prevention of disease spread. the ihr require countries to report certain disease outbreaks and public health events to who [ , , ] . building on the unique experience of who in global disease surveillance, alert, and response, the ihr define the rights and obligations of countries to report public health events and establish a number of procedures that who must follow in its work to uphold global public health security. within the framework of the ihr [ ] , seven areas of work have been identified to achieve the goals described above. the first area of work aims to strengthen global partnerships; the second and third address countries' capacities to meet ihr requirements; the fourth and fifth areas of work focus on surveillance, prevention, control, and response systems at the international level; and the sixth and seventh address awareness of rules and legal aspects and measuring progress ( table ) . the risk of international spread of disease is minimized through effective permanent public health measures and response capacity at designated airports, ports and ground crossings in all countries. prevent and respond to international public health emergencies . strengthen who global alert and response systems timely and effective coordinated response to international public health risks and public health emergencies of international concern. . strengthen the management of specific risks systematic international and national management of the risks known to threaten international health security, such as influenza, meningitis, yellow fever, sars, poliomyelitis, food contamination, chemical and radioactive substances. there are three groups of events that may constitute public health emergencies of international concern: group . a case of the following diseases is unusual or unexpected and may have serious public health effects and thus shall be reported: group . an event involving the following diseases shall always lead to use of the algorithm because these diseases have demonstrated the ability to have serious public health effects and to spread rapidly internationally: • smallpox • poliomyelitis due to wild-type poliovirus • human influenza caused by a new subtype • severe acute respiratory syndrome (sars) • cholera • pneumonic plague • yellow fever • viral haemorrhagic fevers (ebola, lassa, marburg) • west nile fever • other diseases that are of special national or regional concern, e.g., dengue fever, rift valley fever, and meningococcal disease group . any event of potential international public health concern, including those of unknown causes or sources and those involving events or diseases other than those listed above shall lead to use of the algorithm and criteria from the republic of macedonia has population of about two million people. the territory is divided into municipalities. in , in the framework of the new public health system, one national and rphis were established. they adopted a previously established system for routine surveillance for registration and notification of communicable diseases, which included diseases. in , new recommendations for protecting the population from communicable diseases were adopted, and a new obligatory list of diseases was introduced. past work has shown an absence of case definitions, a lack of laboratory confirmation, significant delays in reporting between surveillance levels, delayed and inadequate outbreak response, lack of feedback to reporting level, lack of training, lack of analysis at the peripheral level, under-reporting of unconfirmed cases or outbreaks, and poor motivation of healthcare staff. in , the nphi, with who support, started to develop a syndromic early-warning alert response system (ewars), called alert, with an ultimate goal of strengthening the early detection of outbreaks of epidemicprone and emerging infectious diseases. a panel of macedonian experts in the field of epidemiology and microbiology has assessed the needs and priorities for disease surveillance using a standardized questionnaire. the aim of this assessment was to define what the most important diseases are in republic of macedonia, from their point of view. the results of the assessment are shown in table . acute bloody diarrhea mucous stools containing (visible) blood in the previous h, with or without dehydratation, stomach pain, and cramps suspicion of acute infective hepatitis acute jaundice (yellow skin and sclera colour), weakness and exhaustion, dark urine, light stool, anorexia, nausea, pain below the right rib arch suspicion of acute hemorrhagic fever acute beginning of fever in a period shorter than weeks in a very ill patient and any two of the following signs/symptoms: petechial or purpural rash, nose bleeding, hematemesis, hemoptysis, oliguria or anuria, bloody stool, any other hemorrhagic manifestation without known cause in children younger than a list of eight health events was included in alert on the basis of the results of this study. to unify the reporting process for all participants in the system, a case definition is necessary. a case definition is a combination of symptoms and signs that have to be present in a patient for the patient to be placed in a certain category. the list case definitions for each. after the decision was made about the syndromic diseases that would be included in the ewars, the next challenge facing the expert panel was to make a decision about the threshold limits. two different approaches have been used for threshold definition. regarding the severity of the disease and expecting threats for three of the syndromic diseases, the fixed number of cases was used. for the other five syndromic diseases, threshold limits were established on the basis of previous epidemiologic data and already-registered cases. using these two methodologies, threshold limits were alert will go out automatically. for three groups of syndromic events -meningitis and meningoencefalitis, acute bloody diarrhea, and acute hemorrhagic fever -an alert will be declared after every registered case. for the other five syndromic events, an alert will be declared after the defined number of cases specific for each region of surveillance is exceeded. reporting units all are comprised of primary care physicians who work in different segments of the health system in the republic of macedonia. currently there are , primary health units, but only - % of them are included in ewars. through a written, standardized surveillance form, they report weekly the aggregated number of new cases in four age groups to the corresponding collecting units at the municipality level or directly to the local and regional surveillance units. they send aggregated data by mail or fax. there are ten regional surveillance units equipped and trained to process of eight syndromes that are part of the national ewars are presented in table with established for all ten surveillance units. they are shown in table . anytime the defined number of syndrome cases listed in table is exceeded, the data from the reporting units. the regional surveillance units are rphis and their epidemiologic departments. at the regional level, data are computerized and electronically transmitted to the nphi. the nphi prepares a report and sends it to the ministry of health and initiates and performs all requested interventions and additional activities. feedback is sent electronically from the nphi to the regional institutes. in addition, the rphis can send information to the reporting units. epidemiologists from the rphis and the nphi are responsible for data control and regularity, reporting any unusual changes and undertaking urgent activities. a program has been developed using public-domain software for relational data entry (epidata) and production of interactive reports (epiinfo). it includes features for data entry (with quality checks) at the rphi level and electronic transfer of records to the nphi. it provides links with excel and word. the application produces a weekly epidemiologic bulletin in word and allows interactive browsing of tables, charts, and maps in html format. the system generates alert reports based on disease-specific thresholds. the communicable diseases surveillance system in the republic of macedonia the system is considered simple and flexible. users emphasized that alert has improved communication between reporting and surveillance units and strengthened the surveillance network. the acceptability of the system is higher at the national level mainly because data from alert are received in a timely fashion, which allows the surveillance department at riph to monitor potential outbreaks at the national level. is shown in fig. . using syndromic case definitions allows remote areas that do not usually report, because of lack of confirmation capacity, to report, thereby providing valuable early-warning information. moreover, for some rare and serious diseases such as those targeted by the hemorrhagic fever syndrome, alert is used as zero reporting. syndromic surveillance is simple and often the only available surveillance tool at the primary health care level when laboratory confirmation of disease is not possible [ ] . it allows detection of potential outbreaks of targeted diseases earlier than with the diagnosis-based routine surveillance system and leads to field investigations for confirmation and control [ , ] . experience has shown that reporting units at the primary health care level are not the most appropriate source of notification for early detection of some epidemic-prone diseases. some specific syndromes may be seen first in emergency departments, private clinics, or pharmacies [ ] . syndromes such as hemorrhagic fever, as an indicator for hantavirus or crimean-congo hemorrhagic fever, are sensitive and specific enough to detect outbreaks. because it is a serious and uncommon syndrome, each individual case reported is an alert and triggers an action. for other diseases, such as influenza, targeted by acute respiratory illness, the alert for action is a rise in reported syndrome cases, indicating the onset of the influenza season. alert was able to detect this increase during the season. however, other categories of syndromes have not been sensitive or specific enough to detect outbreaks in a timely fashion. timely detection of public health threats relies on proper analysis of early-warning data at each level. alert software produces automated tables, charts, and maps highlighting increases. epidemiologists should use those resources to trigger actions when individual confirmed cases are reported. the evaluation of the effects of implementation of the pilot project for the ewars have shown us that sensitivity and usefulness should be increased. there are many possible ways to do this, such as adding emergency departments as notification sources for some syndromes, better defining the role of the laboratory to confirm the suspicion of outbreaks, revising the list and definition of syndromes to adjust their sensitivity and specificity for detecting the targeted diseases, and strengthening data analysis through training. our experience shows that the role of training should not be overlooked. it is a change of paradigm, which is impossible to induce by simply implementing new surveillance tools, difficult to induce by short training, and best induced by coaching programs such as field-epidemiology training programs. although the process for implementing the ewars was piloted by the ministry of health, the alert reporting procedures were not incorporated into public health laws. alert does not interrupt the continuity of the existing reporting system, regulated by law. all obligations and responsibilities prescribed by it still remain. the final goal is, by comparing the advantages and disadvantages of both systems, to enable the creation (establishment) of a new, combined system that would be more functional, safer, and more economically sustainable. on the basis of our experiences, the obligation for syndromic reporting through ewars will be laid down in our national law. some additional measures, such as financial copayment for reporting units, should be discussed. world health organization. the world health report enhancing health security: the challenges in the who european region and the health sector response. copenhagen: world health organization regional office for europe public health threats and disaster management world health organization. fifty-eighth world health assembly. resolution wha . . revision of the international health regulations. geneva: world health organization global public health surveillance under the new international health regulations public health emergencies of international concern and the revision of the international viewarticle.aspx? towards health security. a discussion paper on recent health crises in the who european region. copenhagen: world health organization regional catalogue risk reduction and emergency preparednes. who six-year strategy for the health sector and annual disaster statistical review: numbers and trends strengthening national public health preparedness and response to chemical, biological and radiological threats cumulative number of confirmed cases of avian influenza a roles and functions of european union public health centre for communicable diseases and other threats to health world health organization. fifty-fourth world health assembly. resolution wha . . global health security: epidemic alert and response. geneva: world health organization world health organization. fifty-ninth world health assembly. resolution wha . . application of //www implementing the international health article.aspx?articleid= framework for evaluating public health surveillance systems for early detection of outbreaks: recommendations from the cdc working group introduction to international disaster management geneva: world health organization european center for disease prevention and control. surveillance of communicable diseases in the activities/surveillance/pages/strategiesprinciples_long-termstrategy improvement of a national public health surveillance.org/viewarticle.aspx?articleid= notifiable disease surveillance and practicing physicians what is epidemic intelligence, org/viewarticle.aspx? if syndromic surveillance is the answer, what is the question? surveillance for early detection and monitoring of infectious disease outbreaks associated with bioterrorism the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health strengthening early warning function of surveillance in the republic of serbia: lessons learned after a year of articleid= the emerging science of very early detection of disease outbreaks syndromic surveillance: a local perspective microbial threats to health: emergence, detection, and response syndromic surveillance and bioterrorism-related epidemics enhanced drop-in syndromic surveillance epidemiological response to syndromic surveillance signals updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group epidemic intelligence: a new framework for strengthening disease surveillance in europe investigation of disease outbreaks detected by syndromic surveillance systems syndromic surveillance in public health practice hot spots in a wired world: who surveillance of emerging and reemerging infectious diseases retrospective validation of a surveillance system from unexplained illness and death syndrome definitions for diseases associated with critical bioterrorism-associated agents the early warning and response system for communicable diseases in the eu: an overview from epidemic alert and verification: summary report for ? additional reading biological and chemical terrorism: strategic plan for preparedness and response. recommendations of the cdc strategic planning workgroup syndromic surveillance for bioterrorism following the attacks on the world trade center the bioterrorism preparedness and response early aberration reporting system (ears) different approaches to gathering epidemic intelligence in europe the epidemic intelligence service in the united states innovative surveillance methods for rapid detection of disease outbreaks and bioterrorism: results of an interagency workshop on health indicator surveillance key: cord- -citynr c authors: p. shetty, nandini; s. shetty, prakash title: epidemiology of disease in the tropics date: - - journal: manson's tropical diseases doi: . /b - - - - . - sha: doc_id: cord_uid: citynr c nan the study of epidemiology in the tropics has undergone major changes since its infancy when it was largely a documentation of epidemics. it has now evolved into a dynamic phenomenon involving the ecology of the infectious agent, the host, reservoirs and vectors as well as the complex mechanisms concerned in the spread of infection and the extent to which this spread occurs. similar concepts in the study of epidemiology apply to communicable as well as non-communicable diseases. the understanding of epidemiological principles has its origins in the study of the great epidemics. arguably, the most powerful example of this is the study of that ancient scourge of mankind, the so-called black death or plague. a study of any of the plague epidemics throughout history has all the factors that govern current epidemiological analysis: infectious agent, host, vector, reservoir, complex population dynamics including migration, famine, fi re and war; resulting in spread followed by quarantine and control. the world health report : 'fighting disease, fostering development', states that infectious diseases are the world's leading cause of premature death. infectious diseases account for % of deaths in low-income countries (figure . ) and up to % of deaths in children under years of age worldwide. africa and south-east asia carry the highest mortality due to infectious diseases (figure . ). in addition, new and emerging infections pose a rising global threat (table . ). no more than six deadly infectious diseases: pneumonia, tuberculosis, diarrhoeal diseases, malaria, measles and more recently, hiv/aids, account for half of all premature deaths, killing mostly children and young adults (figure . ). acute respiratory infections (aris) are the leading cause of death of infectious aetiology, killing more than million people a year, . million of which constitute children under the age of fi ve. among the countries of the world that carry % of the child mortality burden, - % of the under- mortality is due to pneumonia and nearly % of this pneumonia mortality occurs in the africa and south and south-east asia regions. the majority of this burden is borne during early childhood, with the greatest risk from mortality occurring during the neonatal period. the global incidence of ari in children is estimated to be million cases per year. this range of infections, which includes pneumonia in its most serious form, accounts for more than % of the global burden of disease. pneumonia often affects children with low birth weight or those whose immune systems are weakened by malnutrition or other diseases. caused by different viruses or bacteria, ari is closely associated with poverty, overcrowding and unsanitary household conditions. several other factors seem to exacerbate the disease. exposure to tobacco smoke increases the risk of contracting these infections, and many studies implicate both indoor and outdoor air pollution. indoor air pollution has been the focus of particular concern: specifi cally, the soot and smoke associated with the burning of biomass fuels such as wood, coal, or dung. many people in the developing world, mostly in rural areas, rely on biomass fuels for heating or cooking. a cause-and-effect relationship between indoor air pollution and ari has been diffi cult to prove. even so, the world bank estimated in that switching to better fuels could halve the number of pneumonia deaths. approaches to the management of childhood pneumonia in the tropics are hampered by lack of diagnostic facilities to identify the aetiological agent. the who has devised a simple algorithm for use in fi eld situations, by primary healthcare workers, using clinical criteria such as respiratory rate and indrawing of ribs to decide whether a child needs hospitalization. proper implementation of this strategy has been shown to reduce the mortality from childhood pneumonias by - %. however, implementation of community ari treatment programmes remains patchy and current rates of children with ari being taken to a health provider are ~ % in africa and south asia. in nearly half of the countries with available data, less than % of the children with ari were taken to an appropriate healthcare provider. the aids pandemic has emerged as the single most defi ning occurrence in the history of infectious diseases of the late twentieth and early twenty-fi rst centuries. according to the aids epidemic update of december (unaids and who), the epidemiology of hiv in the tropics varies enormously from place to place (figure latest estimates show some . million people ( million adult women) were living with hiv in , including the . million people who became newly infected in the past year. aids claimed some lives in . these estimates are in line with known risk behaviour in this region, where men account for the majority of injecting drug users, and are responsible for sexual transmission of hiv, largely through commercial sex. commercial sex accounts for a large part of the estimated % of hiv infections in china that are due to unprotected heterosexual contact. it also features in the transmission of the virus among men who have sex with men: a recent survey among male sex workers in the southern city of shenzhen found that % of them were hivpositive. however, it is the potential overlap between commercial sex and injecting drug use that is likely to become the main driver of china's epidemic. diverse epidemics are underway in india, where, in , an estimated . million indians were living with hiv. although levels of hiv infection prevalence appear to have stabilized in some states (such as tamil nadu, andhra pradesh, karnataka and maharashtra), it is still increasing in at-risk population groups in several other states. as a result, overall hiv prevalence has continued to rise. a signifi cant proportion of new infections is occurring in women who are married and who have been infected by husbands who (either currently or in the past) frequented sex workers. commercial sex (along with injecting drug use, in the states of nagaland and tamil nadu) serves as a major driver of the epidemics in most parts of india. hiv surveillance in found % of commercial sex workers in karnataka ( % in the city of mysore) and % in andhra pradesh were infected with hiv. the wellknown achievements among sex workers of kolkata's sonagachi red-light area (in west bengal, india) have shown that safe sex programmes that empower sex workers can curb the spread of hiv. condom use in sonagachi has risen as high as % and hiv prevalence among commercial sex workers declined to fewer than the combination of high levels of risk behaviour and limited knowledge about aids among drug injectors and sex workers in pakistan favours the rapid spread of hiv, and new data suggest that the country could be on the verge of serious hiv epidemics. most countries in asia still have the opportunity to prevent major epidemics. bangladesh, where national adult hiv prevalence is well below %, began initiating hiv prevention programmes early in its epidemic. indonesia is on the brink of a rapidly worsening aids epidemic. with risk behaviour among injecting drug users common, a mainly drug-injection epidemic is already spreading into remote parts of this archipelago. in malaysia, approximately people were living with hiv in , the vast majority of them young men (aged - years), of whom approximately % were injecting drug users. after peaking at % in , national adult hiv prevalence in cambodia fell by one-third, to . % in . the reasons for this are two-fold: increasing mortality and a decline in hiv incidence due to changes in risk behaviour. thailand has been widely hailed as one of the success stories in the response to aids. by , estimated national adult hiv prevalence had dropped to its lowest level ever, approximately . %. however, thailand's epidemic is far from over; infection levels in the most at-risk populations are much higher: just over % of brothel-based female sex workers were hiv-infected in , as were % of injecting drug users who attended treatment clinics. while cambodia and thailand in the s were planning and introducing strategies to reverse the spread of hiv, another serious epidemic was gaining ground in neighbouring myanmar. there, limited prevention efforts led to hiv spreading freely. consequently, myanmar has one of the most serious aids epidemics in the region, with hiv prevalence among pregnant women estimated at . % in . the main hiv-related risk for many of the women now living with the virus was to have had unprotected sex with husbands or boyfriends who had been infected while injecting drugs or buying sex. in japan, the number of reported annual hiv cases has more than doubled since - , and reached in ; the highest number to date. much of this trend is due to increasing infections among men who have sex with men. prevalence of hiv remains low in the philippines and lao pdr. the advance of aids in the middle east and north africa has continued, with latest estimates showing that people became infected with hiv in . approximately people are living with hiv in this region. an estimated adults and children died of aids-related conditions in . although hiv surveillance remains weak in this region, more comprehensive information is available in some countries (including algeria, libya, morocco, somalia and sudan). available evidence reveals trends of increasing hiv infections (especially in younger age groups) in such countries as algeria, libya, morocco and somalia. the main mode of hiv transmission in this region is unprotected sexual contact, although injecting drug use is becoming an increasingly important factor (and is the predominant mode of infection in at least two countries: iran and libya). infections as a result of contaminated blood products, blood transfusions or a lack of infection control measures in healthcare settings are generally on the decline. by far the worst-affected country in this region is sudan. in a country with a long history of civil confl ict and forced displacement, internally displaced persons face higher rates of hiv infection. for instance, among displaced pregnant women seeking antenatal care in khartoum in , hiv prevalence of . % was found compared with under . % for other pregnant women. the epidemic in latin america is a complex mosaic of transmission patterns in which hiv continues to spread through male-tomale sex, sex between men and women, and injecting drug use. sub-saharan africa has just over % of the world's population, but is home to more than % of all people living with hiv - . the rights and status of women and young girls deserve special attention. around the world -from south of the sahara in africa and asia to europe, latin america and the pacifi c -an increasing number of women are being infected with hiv. it is often women with little or no income who are most at risk. widespread inequalities including political, social, cultural and human security factors also exacerbate the situation for women and girls. in several southern african countries, more than three quarters of all young people living with hiv are women, while in sub-saharan africa overall, young women between and years old are at least three times more likely to be hiv-positive than young men (figure . ). in many countries, marriage and women's own fi delity are not enough to protect them against hiv infection. among women surveyed in harare (zimbabwe), durban and soweto (south africa), % reported having one lifetime partner, % had abstained from sex at least until the age of (roughly the average age of fi rst sexual encounter in most countries in the world). yet, % of the young women were hiv-positive. many had been infected despite staying faithful to one partner. diarrhoea remains one of the most common diseases affl icting children under years of age and accounts for considerable mortality in childhood. estimates from studies published between and show that there was a median of . episodes of diarrhoea per child-year in developing countries. this indicates little change from previously described incidences. estimates of mortality revealed that . children per /year in these countries died as a result of diarrhoeal illness in the fi rst years of life, a decline from the previous estimates of . - . per /year. the decrease was most pronounced in children aged under one year. despite improving trends in mortality rates, diarrhoea accounted for a median of % of all deaths of children aged under years in developing countries, being responsible for . million deaths per year. there has not been a concurrent decrease in morbidity rates attributable to diarrhoea. as population growth is focused in the poorest areas, the total morbidity component of the disease burden is greater than previously. diarrhoea remains a disease of poverty affl icting malnourished children in crowded and contaminated environments. efforts to immunize children against measles, provide safe water and adequate sanitation facilities, and to encourage mothers to exclusively breast-feed infants through to months of age can blunt an increase in diarrhoea morbidity and mortality. preventive strategies to limit the transmission of diarrhoeal disease need to go hand in hand with national diarrhoea disease control programmes that concentrate on effective diarrhoea case management and the prevention of dehydration. the factors contributing to childhood mortality and morbidity due to diarrhoea are described in table . . studies in asia and africa have clearly shown that establishment of an oral rehydration therapy (ort) unit with training of hospital staff can signifi cantly reduce diarrhoea case fatality rates. for instance, at mama yemo hospital in kinshasa, zaire, there was a % decline in diarrhoea deaths after creation of an ort unit. in may , the world health organization and the united nations children's fund recommended that the formulation of oral rehydration solution (ors) for treatment of patients with diarrhoea be changed to one with a reduced osmolarity and that safety of the new formulation, particularly development of symptomatic hyponatremia, be monitored. a total of patients, including children younger than months, were monitored at the dhaka and matlab hospitals, bangladesh. the risk of symptoms associated with hyponatraemia in patients diarrhoeal disease treated with the reduced osmolarity ors was found to be minimal and did not increase with the change in formulation. changing patterns in the epidemiology of diarrhoea have been noted in many studies. in matlab, bangladesh, acute watery diarrhoea accounted for % of diarrhoea deaths in under-fi ves, while the remaining % were related to dysentery or persistent diarrhoea and malnutrition. this pattern was age dependent, with acute watery deaths being more important in infancy, being associated with % of deaths, and less important in later childhood, being associated with % of deaths. rotavirus is the most common cause of severe diarrhoeal disease in infants and young children all over the world, and an important public health problem, particularly in developing countries where deaths each year are associated with this infection. more than million cases of diarrhoea each year are attributed to rotavirus. in tropical developing countries, rotavirus disease occurs either throughout the year or in the cold dry season. almost all children are already infected by the age of - years. although the infection is usually mild, severe disease may rapidly result in life-threatening dehydration if not appropriately treated. natural infection protects children against subsequent severe disease. globally, four serotypes are responsible for the majority of rotaviral disease, but additional serotypes are prevalent in some countries. the only control measure likely to have a signifi cant impact on the incidence of severe disease is vaccination. since the withdrawal from the market of the tetravalent rhesus-human reassortant vaccine (rotashield, wyeth laboratories) because of an association with intussusception, ruling out such a risk has become critical for the licensure and universal use of any new rotavirus vaccine. recent studies have shown that two oral doses of the live attenuated g p [ ] human rotavirus vaccine were highly effi cacious in protecting infants against severe rotavirus gastroenteritis, signifi cantly reduced the rate of severe gastroenteritis from any cause, and were not associated with the increased risk of intussusception linked with the previous vaccine. man is both the reservoir and natural host of shigella, the commonest cause of dysentery in the tropics. the most severe infections are caused by the s. dysenteriae type (also known as shiga's bacillus); it is also the only serotype implicated in epidemics. infection is by the faecal-oral route and is usually spread by personto-person transmission. it takes only - shigella organisms to produce dysentery, a low infectious dose, whereas million to million organisms may need to be swallowed to cause cholera. during the late s, shiga's bacillus was responsible for a series of devastating epidemics of dysentery in latin america, asia and africa. in , it was detected in the mexican-guatemalan border area and spread into much of central america. an estimated half a million cases, with deaths, were reported in the region between and . in some villages the case fatality rate was as high as %; delayed diagnosis and incorrect treatment may have been responsible for this high death rate. one particularly disturbing feature was the resistance of the bacteria to the most commonly used antibacterial drugs: sulfonamides, tetracycline and chloramphenicol. serious epidemics due to the multiple-drug resistant s. dysenteriae type have occurred recently in bangladesh, somalia, south india, burma, sri lanka, nepal, bhutan, rwanda and zaire. west bengal in india has always been an endemic area for bacillary dysentery. preventive measures include boiling or chlorination of drinking water, covering faeces with soil, protecting food from fl ies, avoiding eating exposed raw vegetables and cut fruits, and washing hands with soap and water before eating and after using the latrine. however, such measures are not easy to implement in most areas. consequently epidemics take their own course and subside only gradually. tuberculosis tuberculosis (tb) is the leading cause of death associated with infectious diseases globally. the incidence of tb will continue to increase substantially worldwide because of the interaction between the tb and hiv epidemics. in many developing countries, tb is mainly a disease of young adults affecting carers and wage-earners in a household, thus placing a huge economic burden on society as a whole. chemotherapy, if properly used, can reduce the burden of tb in the community, but because of the fragile structure of treatment programmes in many countries tb cases are not completely cured and patients remain infectious for a much longer time. another important consequence of poor treatment compliance is development of drug resistance in many developing countries. resistance to tuberculosis drugs is probably present everywhere in the world. worldwide attention was focused on south africa, when in october a research project publicized a deadly outbreak of xdr-tb in the small town of tugela ferry in kwazulu-natal. xdr-tb is the abbreviation for extensively drug-resistant tuberculosis (tb). this strain of mycobacterium tuberculosis is resistant to fi rstand second-line drugs, and treatment options are seriously limited. of tb patients at the church of scotland hospital, which serves a rural area with high hiv rates, some were found to have multi-drug resistance and of these, were diagnosed with xdr-tb. some of these patients died, most within days of diagnosis. of the patients, had been tested for hiv and all were found to be hiv-positive. the patients were receiving antiretrovirals and responding well to hiv-related treatment, but they died of xdr-tb. since the study, more patients have been diagnosed with xdr-tb in kwazulu-natal. only three of them are still alive (see: http://www.who.int/tb/xdr/xdr_jan.pdf). directly observed treatment, short course (dots), is the most effective strategy available for controlling the tb epidemic today. dots uses sound technology and packages it with good management practices for widespread use through the existing primary healthcare network. it has proven to be a successful, innovative approach to tb control in countries such as china, bangladesh, vietnam, peru and countries of west africa. however, new challenges to the implementation of dots include health sector reforms, the worsening hiv epidemic, and the emergence of drugresistant strains of tb. the technical, logistical, operational and political aspects of dots work together to ensure its success and applicability in a wide variety of contexts. million africans who die from malaria each year, most are children under years of age. in addition to acute disease episodes and deaths in africa, malaria also contributes signifi cantly to anaemia in children and pregnant women, adverse birth outcomes such as spontaneous abortion, stillbirth, premature delivery and low birth weight, and overall child mortality. the disease is estimated to be responsible for an estimated average annual reduction of . % in economic growth for those countries with the highest burden. of the four species of plasmodium that infect humans: p. falciparum, p. vivax, p. malariae and p. ovale, p. falciparum causes most of the severe disease and deaths attributable to malaria and is most prevalent in africa south of the sahara and in certain areas of south-east asia and the western pacifi c (figure . ) . the second most common malaria species, p. vivax, is rarely fatal and is commonly found in most of asia, and in parts of the americas, europe and north africa. there are over species of anopheline mosquitoes that transmit human malaria, which differ in their transmission potential. the most competent and effi cient malaria vector, anopheles gambiae, occurs exclusively in africa and is also one of the most diffi cult to control. climatic conditions determine the presence or absence of anopheline vectors. tropical areas of the world have the best combination of adequate rainfall, temperature and humidity allowing for breeding and survival of anophelines. in areas of malaria transmission where sustained vector control is required, insecticide treated nets are the principal strategy for malaria prevention. all countries in africa south of the sahara, the majority of asian malaria-endemic countries and some american countries have adopted insecticide treated nets as a key malaria control strategy. one of the greatest challenges facing malaria control worldwide is the spread and intensifi cation of parasite resistance to antimalarial drugs. the limited number of such drugs has led to increasing diffi culties in the development of antimalarial drug policies and adequate disease management. resistance of p. falciparum to chloroquine is now common in practically all malariaendemic countries of africa (figure . ) , especially in east africa. resistance to sulfadoxine/pyrimethamine, the main alternative to chloroquine, is widespread in south-east asia and south america. mefl oquine resistance is now common in the border areas of thailand with cambodia and myanmar. parasite sensitivity to quinine is declining in several other countries of south-east asia and in the amazon region, where it has been used in combination with tetracycline for the treatment of uncomplicated malaria. in response to widespread resistance of p. falciparum to monotherapy with conventional antimalarial drugs such as chloroquine and sulfadoxine-pyrimethamine, who now recommends combination therapies as the treatment policy for falciparum malaria in all countries experiencing such resistance. the preferred combinations contain a derivative of the plant artemisia annua, which is presently cultivated mainly in china and vietnam. artemisininbased combination therapies (acts) are the most highly effi cacious treatment regimens now available. resistance of p. vivax to chloroquine has now been reported from indonesia (irian jaya), myanmar, papua new guinea and vanuatu. urban and periurban malaria are on the increase in south asia and in many areas of africa. military confl icts and civil unrest, along with unfavourable ecological changes, have greatly contributed to malaria epidemics, as large numbers of unprotected, non- immune and physically weakened refugees move into malarious areas. such population movements contribute to new malaria outbreaks and make epidemic-prone situations more explosive. another disquieting factor is the re-emergence of malaria in areas where it had been eradicated (e.g. democratic people's republic of korea, republic of korea and tadjikistan), or its increase in countries where it was nearly eradicated (e.g. azerbaijan, northern iraq and turkey). current malaria epidemics in a majority of these countries are the result of a rapid deterioration of malaria prevention and control operations. climatic changes have also been implicated in the re-emergence of malaria. in the past years, the worldwide incidence of malaria has quadrupled, infl uenced by changes in both land development and regional climate. in brazil, satellite images depict a 'fi sh bone' pattern where roads have opened the tropical forest to localized development. in these 'edge' areas malaria has resurged. temperature changes have encouraged a redistribution of the disease; malaria is now found at higher elevations in central africa and could threaten cities such as nairobi, kenya. this threat has been hypothesized to extend to temperate regions of the world that are now experiencing hotter summers year on year. although substantial progress has been made in reducing measles deaths globally, in measles was estimated to be the fi fth leading cause of mortality worldwide for children aged < years. measles deaths occur disproportionately in africa and south-east asia. in , the african region of who, with % of the world's population, accounted for % of estimated measles cases and % of measles deaths; the south-east asia region, with % of the world's population and % of measles cases, accounted for % of measles deaths. the burden of mortality in africa refl ects low routine vaccination coverage and high case-fatality ratios. in south-east asia, where vaccination coverage is slightly below average worldwide levels, the large population amplifi es the number of cases and deaths resulting from ongoing measles transmission. the overwhelming majority of measles deaths in occurred in countries eligible to receive fi nancial support from the global alliance for vaccines and immunization's vaccine fund (who, unpublished data ). the majority of measles deaths occur among young children living in poor countries with inadequate vaccination services. like human immunodefi ciency virus, malaria, and tuberculosis, measles can be considered a disease of poverty. however, unlike these diseases, measles can be prevented through vaccination. , in much of the world, particularly sub-saharan africa, south-east asia, china and the pacifi c basin, infection with hepatitis b virus (hbv) is very widespread. the carrier rate in some of these populations may be as high as - %. in developing countries most hepatitis b transmission occurs during the perinatal period. infection between children is another common route of infection; it is not uncommon to fi nd up to % of -year-olds have serological evidence of infection with hbv. intermediate levels of infection ( - %) are seen in parts of the former soviet union, south asia, central america and the northern zones of south america. these high rates of infection lead to a high burden of disease, mainly from the clinical consequences of long-term carriage of the virus, which may include chronic hepatitis, cirrhosis and liver cancer. it has been estimated that hbv infection is the second most common cause of cancer deaths in the world (after tobacco consumption). in india hepatitis b is linked to % of cases of hepatocellular carcinoma and % of cases of cirrhosis of the liver. on the basis of disease burden and the availability of safe and effective vaccines, the who recommended that by the end of the twentieth century, hepatitis b vaccine be incorporated into routine infant and childhood immunization programmes for all countries. the effi cacy of universal immunization has been shown in different countries, with striking reductions of the prevalence of hbv carriage in children. most important, hepatitis b vaccination can protect children against hepatocellular carcinoma and fulminant hepatitis, as has been shown in taiwan. nevertheless, the implementation of worldwide vaccination against hbv requires greater effort to overcome the social and economic hurdles. safe and effective antiviral treatments are available but are still far from ideal, a situation that, hopefully, will be improved soon. with hepatitis b immunization, the global control of hbv infection is possible by the end of the fi rst half of twenty-fi rst century. tetanus is a vaccine-preventable disease that causes a total of deaths annually. of particular concern is maternal and neonatal tetanus (mnt), which can be prevented through immunization of the mother in pregnancy. in , neonatal tetanus alone was responsible for an estimated deaths. in addition, an estimated - non-immunized women worldwide die each year from maternal tetanus that results from postpartum, postabortal or postsurgical wound infection with clostridium tetani. while the focus is on priority countries, % of the neonatal tetanus deaths occur in countries. unicef spearheaded the effort to eliminate mnt by the year , with the support of numerous partners. mnt elimination is defi ned as less than one case of neonatal tetanus per live births at district level. the main strategies consist of promotion of clean delivery practices, immunization of women with a tetanus toxoid (tt) containing vaccine, and surveillance. maternal tetanus immunization is, in most developing countries, implemented as part of the routine immunization programme. however, large areas remain underserved, due to logistical, cultural, economical or other reasons. in order to achieve the target of mnt elimination by , and to offer protection to women and children otherwise deprived from regular immunization services, countries are encouraged to adopt the high risk approach. this approach implies that, in addition to routine immunization of pregnant women, all women of child-bearing age living in high risk areas are targeted for immunization with three doses of a tetanus toxoid containing vaccine (tt or td). by the end of vaccination against a range of bacterial and viral diseases is an integral part of communicable disease control worldwide. vaccination against a specifi c disease not only reduces the incidence of that disease, but it also reduces the social and economic burden of the disease on communities. very high immunization coverage can lead to complete blocking of transmission for many vaccinepreventable diseases. the worldwide eradication of smallpox and the near-eradication of polio from many countries provide excellent examples of the role of immunization in disease control. despite these advances many of the world's poorest countries do not have access to vaccines and these infections remain among the leading global causes of death. the special programme for research and training in tropical diseases (tdr) of the world health organization has designated several infectious diseases as 'neglected tropical diseases' (ntds) that disproportionately affl ict the poor and marginalized populations in the developing regions of sub-saharan africa, asia and the americas. infectious diseases are considered as 'neglected' or 'orphan' diseases when there is a lack of effective, affordable, or easy to use drug treatments. as most patients with such diseases live in developing countries and are too poor to pay for drugs, the pharmaceutical industry has traditionally ignored these diseases. ntds cause an estimated to million deaths annually and cause a global disease burden equivalent to that of hiv-aids. who estimates that at least billion people, i.e. onesixth of the world's population suffers from one or more neglected tropical diseases, while other estimates suggest the number to be much higher. some diseases affect individuals throughout their lives, causing a high degree of morbidity and physical disability and, in certain cases, gross disfi gurement. others are acute infections, with transient, severe and sometimes fatal outcomes. patients can face social stigmatization and abuse, which only add to the already heavy health burden. neglected tropical diseases are contrasted with the 'big three' diseases (hiv/aids, tuberculosis and malaria) which receive much more attention and funding. the current neglected diseases portfolio includes parasitic diseases of protozoan origin like kala-azar (leishmaniasis), african sleeping sickness (african trypanosomiasis) and chagas' disease (american trypanosomiasis) as well as those caused by helminths such as schistosomiasis, lymphatic fi lariasis, onchocerciasis (river blindness) and dracunculiasis (guinea worm). infestations due to soil transmitted helminths such as ascariasis, trichuriais and hookworm also belong to the latter category. other neglected diseases include those of bacterial origin such as leprosy, buruli ulcer and trachoma as well as those of viral origin like dengue fever which are vector-borne. even cholera and yellow fever are considered by some as ntds, while some include cysticercosis, hydatidosis and food-borne trematode infections. it is now believed that ramped up efforts against the 'big three', will yield far bigger dividends if they are coupled with concerted attack on ntds . evidence now points to substantial geographical overlap between the neglected tropical diseases and the 'big three', suggesting that control of the neglected tropical diseases could become a powerful tool for effectively combating hiv/aids, tuberculosis, and malaria. since , resurgent and emerging infectious disease outbreaks have occurred worldwide. in addition, many diseases widely believed to be under control, such as cholera, dengue and diphtheria, have re-emerged in many areas or spread to new regions or populations throughout the world (figure . ) . a growing population and increasing urbanization contribute to emerging infectious disease problems. in many parts of the world, urban population growth has been accompanied by overcrowding, poor hygiene, inadequate sanitation and unclean drinking water. urban development has also caused ecological damage. in these circumstances, certain disease-causing organisms and some of the vectors that transmit them have thrived, making it more likely that people will be infected with new or re-emerging pathogens. the existing public health infrastructure is already overtaxed and ill prepared to deal with new health threats. breakdown of public health measures due to civil unrest, war and the movement of refugees has also contributed to the re-emergence of infectious diseases (table . ). international travel and commerce have made it possible for pathogens to be quickly transported from one side of the globe to the other (figure . ) . examples of new and resurgent infections include ebola, dengue fever, rift valley fever, diphtheria, cholera, nipah virus infection, west nile virus infection, severe acute respiratory syndrome (sars) and avian infl uenza. in ebola (named after the ebola river in zaire) fi rst emerged in sudan and the democratic republic of the congo (formerly zaire). ebola virus occurs as four distinct subtypes: zaïre, sudan, côte d'ivoire and reston. three subtypes, occurring in the democratic republic of the congo, sudan and côte d'ivoire, have been identifi ed as causing illness in humans. ebola haemorrhagic fever (ehf) is a febrile haemorrhagic illness which causes death in - % of all clinically ill cases. the natural reservoir of the ebola virus is unknown despite extensive studies, but seems to reside in the rain forests on the african continent and in the western pacifi c. through the global prevalence of dengue and dengue haemorrhagic fever (dhf) has grown dramatically in recent decades. the disease is now endemic in more than countries in africa, the americas, the eastern mediterranean, south-east asia and the western pacifi c. south-east asia and the western pacifi c are most seriously affected. some million people -two-fi fths of the world's population -are now at risk from dengue. who currently estimates there may be million cases of dengue infection worldwide every year. in alone, there were more than reported cases of dengue in the americas, of which cases were dhf. this is greater than double the number of dengue cases which were recorded in the same region in . not only is the number of cases increasing as the disease is spreading to new areas, but explosive outbreaks are occurring. in , brazil reported over cases including more than cases of dhf. during epidemics of dengue, attack rates among the susceptible are often - %, but may reach - %. an estimated cases of dhf require hospitalization each year, microbial adaptation changes in virulence and toxin production; development and change of drug resistance; microbes as co-factors in chronic diseases of whom a very large proportion are children. without proper treatment, dhf case fatality rates can exceed %. with modern intensive supportive therapy, such rates can be reduced to less than %. the spread of dengue is attributed to expanding geographical distribution of the four dengue viruses and of their mosquito vectors, the most important of which is the predominantly urban species aedes aegypti. a rapid rise in urban populations is bringing ever greater numbers of people into contact with this vector, especially in areas that are favourable for mosquito breeding, e.g. where household water storage is common and where solid waste disposal services are inadequate. rift valley fever (rvf) is a zoonotic disease typically affecting sheep and cattle in africa. mosquitoes are the principal means by which rvf virus is transmitted among animals and to humans. following abnormally heavy rainfall in kenya and somalia in late and early , rvf occurred over vast areas, producing disease in livestock and causing haemorrhagic fever and death among the human population. as of december , who fi gures indicate that the outbreak continues to affect the north western provinces of kenya. in september who documented the fi rst ever rvf outbreak outside africa, in yemen and the kingdom of saudi arabia (ksa). rna sequencing of the virus from ksa indicated that it was similar to the rvf viruses isolated from east africa in . a total of suspected cases were identifi ed, of which ( %) persons died. of the , ( %) cases reported exposure to sick animals, handling an abortus or slaughtering animals in the week before onset of illness. the vibrio responsible for the seventh pandemic, now in progress, is known as v. cholerae o , biotype el tor. according to the who, it continues to spread in angola and sudan; more than cases have been documented with over deaths: a case fatality rate of . - %. cholera (biotype el tor) broke out explosively in peru in , after an absence of years, and spread rapidly in central and south america, with recurrent epidemics in and . from the onset of the epidemic in january to september , a total of cases and deaths (overall case fatality rate . %) were reported from countries in the western hemisphere to the pan american health organization. in december , a large epidemic of a new strain of cholera v. cholerae began in south india, and spread rapidly through the subcontinent (figure . ) . this strain has changed its antigenic structure such that there is no existing immunity and all ages, even in endemic areas, are susceptible. the epidemic has continued to spread and v. cholerae o has been reported from countries in south asia. because humans are the only reservoirs, survival of the cholera vibrios during interepidemic periods probably depends on low-level undiagnosed cases and transiently infected, asymptomatic individuals. recent studies have suggested that cholera vibrios can persist for some time in shellfi sh, algae or plankton in coastal regions of emerging and resurgent infectious diseases infected areas and it has been claimed that they can exist in a viable but non-culturable state. in early , health offi cials in malaysia and singapore investigated reports of febrile encephalitis and respiratory illnesses among workers who had been exposed to pigs. a previously unrecognized paramyxovirus (formerly known as hendra-like virus), now called nipah virus, was implicated by laboratory testing in many of these cases. as of april , cases of febrile encephalitis were reported to the malaysian ministry of health, including deaths. laboratory results from patients who died suggested recent nipah virus infection. the apparent source of infection among most human cases continues to be exposure to pigs. human-tohuman transmission of nipah virus has not been documented. outbreak control in malaysia has focused on culling pigs; approximately pigs have been killed. other measures include a ban on transporting pigs within the country, education about contact with pigs, use of personal protective equipment among persons exposed to pigs, and a national surveillance and control system to detect and cull additional infected herds. nipah virus cases and deaths have also been reported from bangladesh. since then, no more human cases have been reported. sars is due to infection with a newly identifi ed coronavirus named as sars-associated coronavirus (sars-cov). the source of infection is likely to be a direct cross-species transmission from an animal reservoir. this is supported by the fact that the early sars cases in guangdong province had some history of exposure to live wild animals in markets serving the restaurant trade. animal traders working with animals in these markets had higher seroprevalence for sars coronavirus, though they did not report any illness compatible with sars. more importantly, sars-cov-like virus detected from some animal species had more than a % homology with human sars-cov. the clinical course of sars varies from a mild upper respiratory tract illness, usually seen in young children, to respiratory failure which occurred in around - % of mainly adult patients. as the disease progresses, patients start to develop shortness of breath. from the second week onwards, patients progress to respiratory failure and acute respiratory distress syndrome, often requiring intensive care. in may , a -year-old boy in hong kong contracted an infl uenza-like illness, was treated with salicylates, and died days later with complications consistent with reye's syndrome. laboratory diagnosis included the isolation in cell culture of a virus that was identifi ed locally as infl uenza type a but could not be further characterized with reagents distributed for diagnosis of human infl uenza viruses. by august, further investigation with serological and molecular techniques in the netherlands and in the usa had confi rmed that the isolate was a/hong kong/ / (h n ), which was very closely related to isolate a/chicken/hong kong/ / (h n ). the latter virus was considered representative of those responsible for severe outbreaks of disease on three rural chicken farms in hong kong during march , during which several thousand chickens had died. molecular analysis of the viral haemagglutinins showed a proteolytic cleavage site of the type found in highly pathogenic avian infl uenza viruses. by late december, the total number of confi rmed new human cases had climbed to , of which fi ve were fatal; the case fatality rates were % in children and % in adults older than years. almost all laboratory evidence of infection was in patients who had been near live chickens (e.g. in marketplaces) in the days before onset of illness, which suggested direct transmission of virus from chicken to human rather than person-to-person spread. in december , veterinary authorities began to slaughter all ( . million) chickens present in wholesale facilities or with vendors within hong kong, and importation of chickens from neighbouring areas was stopped. knowledge of how humans are infected, the real level of humanto-human transmission, the spectrum of disease presentation and the effectiveness of treatment remains scanty. human-to human transmission is known to have occurred, but there is no evidence that transmission has become more effi cient. all the human-tohuman infections with h n to date seem not to have transmitted on further. therefore, although the case fatality rate for human infection remains high (around % for cases reported to who), it seems that h n avian viruses remain poorly adapted to humans. global prevalence studies (figure . ) indicate that indonesia is currently the most active site of bird to human h n transmission in the asia pacifi c region, and a large number of human cases have been detected here in - . china and cambodia have also reported human cases in . in south asia (india and pakistan), there have only been sporadic reports of infection in poultry to date. in vietnam and thailand there have been offi cial reports of poultry outbreaks; these show a decline since . surveillance in africa is especially weak, and there is evidence of widespread infection in domestic poultry in parts of north, west and central africa. prospects of control are bleak here because of weaknesses in veterinary services, and a number of competing animal and human health problems. the outbreaks in egypt have been well described. these involved both commercial and backyard fl ocks, with considerable impact on economic life and food security. it is probable that large numbers of people in african countries are at risk of h n infection. if that virus had pandemic potential then a pandemic arising from africa must be considered a possibility. non-infectious diseases take an enormous toll on lives and health worldwide. non-communicable diseases (ncds) account for nearly % of deaths globally, mostly due to heart disease, stroke, cancer, diabetes and lung diseases. the rapid rise of ncds represents one of the major health challenges to global development in the twenty-fi rst century and threatens the economic and social development of nations as well as the lives and health of millions of their subjects. in alone, ncds were estimated to have contributed to . million deaths globally and % of the global burden of disease. until recently, it was believed that ncds were a minor or even non-existent problem in developing countries in the tropics. a recent analysis of mortality trends from ncds suggests that large increases in ncds have occurred in developing countries, particularly those in rapid transition like china and india (table . ). according to these estimates at least % of all deaths in the tropical developing countries are attributable to ncds, while in industrialized countries ncds account for % of all deaths. low-and middle-income countries suffer the greatest impact of ncds. the rapid increase in these diseases is seen disproportionately in poor and disadvantaged populations and is contributing to widening health gaps between and within countries. in , of the total number of deaths attributable to ncds % occurred in developing countries, and of the disease burden they represent % was borne by low-and middle-income countries. it has now been projected that, by , ncds will account for almost three-quarters of all deaths worldwide, and that % of deaths due to ischaemic heart disease (ihd), % of deaths due to stroke, and % of deaths due to diabetes will occur in developing countries and the number of people in the developing world with diabetes is expected to increase by more than . -fold, from million in to million in . on a global basis, % of the burden of ncds will occur in developing countries and the rate at which it is increasing annually is unprecedented. the public health and economic implications of this phenomenon are staggering, and are already becoming apparent. it is important to recognize that these trends, indicative of an increase in ncds, may be partly confounded by factors such as an increase in life expectancy, a progressive reduction in deaths due to communicable diseases in adulthood, and improvements in case detection and reporting in the tropics. however, increase in the incidence of these chronic degenerative diseases is real. the complex range of determinants (below) that interact to determine the nature and course of this epidemic needs to be understood in order to adopt preventive strategies to help developing societies in the tropics to deal with this burgeoning problem. the determinants of non-communicable diseases in developing societies are as follows: • demographic changes in population • epidemiological transition • urbanization and internal migration • changes in dietary and food consumption patterns • lifestyle changes (changes in physical activity patterns, sociocultural milieu and stress as well as increased tobacco consumption) • adult-onset effects of low birth weight and the effects of early life programming • infections and their associations with chronic disease risk • effect of malnutrition and nutrient defi ciencies • poverty, inequalities and social exclusion • deleterious effects of environmental degradation • impacts of globalization. four of the most prominent ncds: cardiovascular disease, cancer, chronic obstructive pulmonary disease and diabetes, are linked to common preventable risk factors related to diet and lifestyle. these factors are tobacco use, unhealthy diet and lack of physical activity. interventions to prevent these diseases should focus on controlling these risk factors in an integrated manner and at the family and community level since the causal risk factors are deeply entrenched in the social and cultural framework of society. developing countries in the tropics have to recognize that the emerging accelerated epidemic of ncds is a cause for concern and that it needs to be dealt with as a national priority. they have to learn from the experience of industrialized and affl uent countries to tackle the emerging crisis of chronic diseases that they are likely to face in the near future. the emerging health burden of chronic disease affecting mainly the economically productive adult population will consume scarce resources. it is important, however, to realize that the poorer countries will be burdened even more in the long run, if attempts are not made to evolve and implement interventions to address these emerging health issues on an urgent basis. ensuring that health policies are aimed at tackling the 'double burden' of the continued existence of the huge burden of infectious/communicable diseases alongside the emerging epidemic of non-communicable diseases in developing countries of the tropics becomes a priority. the world we live in is constantly changing. in the past years, we have witnessed signifi cant progress in sustainable and technological development. however, increases in mass population movements, continuing civil unrest and deforestation have helped carry diseases into areas where they have never been seen before. this has been aided by the massive growth in international travel. effective medicines and control strategies are available to dra-matically reduce the deaths and suffering caused by communicable and non-communicable diseases. despite reduced global military spending many governments are failing to ensure that these strategies receive enough funding to succeed. who priorities for the control of infectious diseases in developing countries include childhood immunization, integrated management of childhood illnesses, use of the dots strategy to control tb, a package of interventions to control malaria, a package of interventions to prevent hiv/aids, access to essential drugs, and the overall strengthening of surveillance and health service delivery systems. over % of all preventable ill-health today is due to poor environmental quality-conditions such as bad housing, overcrowding, indoor air pollution, poor sanitation and unsafe water. the challenge of disease in the tropics has continued into the new millennium -never before have we been so well equipped to deal with disease threats. it remains for humankind to summon the collective will to pursue these challenges and break the chain of infection and disease. national and international surveillance of communicable diseases health report: fighting disease fostering development. geneva: world health organization acute respiratory infections. geneva: world health organization indoor air pollution energy and health for the poor estimate of global incidence of clinical pneumonia in children under fi ve years the global burden of diarrhoeal disease number evl- - . a global review of diarrhoeal disease control new parameters for evaluating oral rehydration therapy: one year's experience in a major urban hospital in zaire symptomatic hyponatremia during treatment of dehydrating diarrheal disease with reduced osmolarity oral rehydration solution diarrhoea mortality in rural bangladeshi children for the human rotavirus vaccine study group. safety and effi cacy of an attenuated vaccine against severe rotavirus gastroenteritis guidelines for the control of epidemics due to shigella dysenteriae . publication no. who/cdr/ . . epidemiology of dysentery caused by shigella. geneva: world health organization global tuberculosis control-surveillance, planning financing, geneva: world health organization who/international union against tuberculosis and lung disease global project on anti-tuberculosis drug resistance surveillance. epidemiology of antituberculosis drug resistance (the global project on anti-tuberculosis drug resistance surveillance): an updated analysis geneva: world health organization climate, ecology and human health global burden of disease and risk factors. geneva: world health organization update: global measles control and mortality reduction -worldwide towards the elimination of hepatitis b: a guide to the implementation of national immunization programs in the developing world. the international task force on hepatitis b immunization. geneva: world health organization global control of hepatitis b virus infection tetanus in developing countries: an update on the maternal and neonatal tetanus elimination control of neglected tropical diseases (ntd) incorporating a rapid-impact package for neglected tropical diseases with programs for hiv/aids, tuberculosis, and malaria emerging infectious diseases review of state and federal diseases surveillance fact sheet: ebola haemorrhagic fever. fact sheet no. . geneva: world health organization report of the public health laboratories division. who collaborating centre for research and training in viral diagnostics national institute of health update: vibrio cholerae o -western hemisphere, - , and v. cholerae o -asia update: outbreak of nipah virus: malaysia and singapore sars and emerging infectious diseases: a challenge to place global solidarity above national sovereignty world avian infl uenza update: h n could become endemic in africa global strategy for the prevention and control of non-communicable diseases. geneva: world health organization global comparative assessments in the health sector. geneva: world health organization life in the st century: a vision for all. geneva: world health organization life course perspectives on coronary heart disease, stroke and diabetes: key issues and implications for policy and research diet and life-style and chronic non-communicable diseases: what determines the epidemic in developing societies? in: krishnaswami k, ed. nutrition research: current scenario and future trends the double burden of communicable and non-communicable diseases in developing countries key: cord- -jf xl vl authors: le duc, james w.; nathanson, neal title: emerging viral diseases: why we need to worry about bats, camels, and airplanes date: - - journal: viral pathogenesis doi: . /b - - - - . - sha: doc_id: cord_uid: jf xl vl the emergence of a new viral disease is one of the most dramatic aspects of virology, which often receives widespread attention from the scientific community and the lay public. considering that the discipline of animal virology was established over years ago, it may seem surprising that new virus diseases are still being discovered. how this happens is the subject of this chapter. one of the most dramatic aspects of virology is the emergence of new virus diseases, which often receives widespread attention from the scientific community and the lay public. considering that the discipline of animal virology was established over years ago, it may seem surprising that new virus diseases are still being discovered. how this happens is the subject of this chapter. there are many recent books and reviews (see further reading) that list the plethora of determinants that can lead to the emergence of infectious diseases (table ) . in this chapter, we concentrate on those determinants that relate to viral pathogenesis and deal only briefly with the many societal and environmental factors that can be instrumental in disease emergence. in some instances, the "emergence" of a viral disease represents the first identification of the cause of a well-recognized disease. an example is la crosse virus, a mosquitotransmitted bunyavirus that was first isolated from a fatal case of encephalitis in . the isolation of the causal agent and the development of serological tests made it possible to distinguish la crosse encephalitis from the rubric of "arbovirus encephalitis, etiology unknown." since that time, about cases have been reported annually, without any significant increase since the s. it appears that the emergence of this "new" disease reflected only the newfound ability to identify this etiologic entity, rather than any true change in its occurrence. hantavirus pulmonary syndrome is another example of the "emergence" of an existing but previously unrecognized disease. in , in the four corners area of the southwestern united states, there occurred a small outbreak of cases of acute pulmonary illness with a high mortality. epidemiologic and laboratory investigation rapidly identified the causal agent, a previously unknown hantavirus, now named sin nombre virus (snv). snv is an indigenous virus of deer mice (peromyscus maniculatus) that are persistently infected and excrete the virus. apparently deer mice produce virus-infected excreta and, when they infest human dwellings, aerosolized fomites can result in occasional human infections. the outbreak is thought to reflect a transient rise in deer mouse populations associated with an unusual crop of pine nuts, a major food source for these rodents. the recognition of snv soon led to the discovery of other heretofore unrecognized hantaviruses in north, central and south america, many of which also cause serious human disease. on occasion, a virus that is already widespread in a population can emerge as a cause of epidemic or endemic disease, due to an increase in the ratio of cases to infections. such an increase can be caused by either an increase in host susceptibility or enhancement of the virulence of the virus. although counterintuitive, there are some dramatic instances of such phenomena. increase in host susceptibility. poliomyelitis first appeared as a cause of summer outbreaks of acute infantile paralysis in sweden and the united states late in the nineteenth century (figure ). isolated cases of infantile paralysis had been recorded in prior centuries, and an eygptian tomb painting indicates that poliomyelitis probably occurred in early recorded history. why then did poliomyelitis emerge abruptly as an epidemic disease? when personal hygiene and public health were primitive, poliovirus circulated as a readily transmitted enterovirus, and most infants were infected while they still carried maternal antibodies (up to - months of age). under these circumstances, the virus produced immunizing infections of the enteric tract, but passively acquired circulating antibodies prevented invasion of the spinal cord. with the improvement of personal hygiene in the late-nineteenth century, infections were delayed until - years of age, after the waning of maternal antibodies. infections now occurred in susceptible children, resulting in outbreaks of infantile paralysis. this reconstruction is supported by seroepidemiological studies conducted in north africa in the s, when epidemic poliomyelitis first emerged in this region. increase in viral virulence. viruses may undergo sudden increases in virulence resulting in emergence of dramatic outbreaks. an outbreak of lethal avian influenza in pennsylvania in is one documented example. in eastern pennsylvania, avian influenza appeared in chicken farms early in , but the virus was relatively innocuous and most infections were mild. however, in the fall of that year a fatal influenza pandemic spread rapidly through the same farms. when viruses from the spring and fall were compared, it appeared that both isolates had almost identical genomes. the fall virus had acquired a single point mutation in the viral hemagglutinin that facilitated the cleavage of the hemagglutinin. the virus could now replicate outside the respiratory tract, markedly increasing its virulence (discussed in chapter , patterns of infection). this point mutation led to the emergence of an overwhelming epizootic, which was only controlled by a widespread slaughter program involving millions of birds. similar outbreaks of avian influenza have occurred subsequently in other countries. a virus that is endemic in a population may "fade out" and disappear, because the number of susceptibles has fallen below the critical level required for perpetuation in that population. if the population is somewhat isolated, the virus may remain absent for many years. during this interval, there will be an accumulation of birth cohorts of children who are susceptible. if the virus is then reintroduced, it can "reemerge" as an acute outbreak. in the years - , iceland had a population of about , , which was too small to maintain measles virus, and measles periodically disappeared. when travelers to iceland reintroduced the virus, measles reemerged in epidemic proportions. on occasion, a virus can enter and spread in a region where it had never previously circulated, leading to the emergence of a disease new to that locale. a dramatic example is afforded by the emergence of west nile virus (wnv) in the united states, beginning in ( figure ). wnv, like most arboviruses, is usually confined to a finite geographic area, based on the range of its vertebrate reservoir hosts and permissive vectors. in an unusual event, wnv was imported into new york city, probably by the introduction of infected vector mosquitoes that were inadvertent passengers on a flight from the middle east, where the virus is enzootic. this hypothesis was supported by the finding that the genomic sequence of the new york isolates was closely related to the sequence of contemporary isolates from israel. some american mosquito species were competent vectors for wnv, and certain avian species such as american crows were highly susceptible. as a result, west nile encephalitis emerged as a significant disease new to the united states. over a period of several years wnv spread across the continent, finally reaching the west coast and many areas in latin america. chikungunya virus (chikv), another mosquito-borne arbovirus, has been endemic for many years in regions of africa and asia where it periodically caused outbreaks of a febrile illness associated with severe arthritis and arthralgia. in - , chikv caused a large epidemic on the island of reunion in the indian ocean, apparently associated with a mutation that allowed the virus to be more efficiently transmitted by vector mosquitoes. chikungunya subsequently spread to india and elsewhere in asia, followed by the americas. as of november , transmission of chikv had been documented in countries or territories in the caribbean, central, south and north america, resulting in nearly one million suspected cases each year. it appears that chikv may become established in the new world, where virtually the entire human population currently lacks immunity. zoonotic infections of animals that can be transmitted to humans are a major cause of emerging virus diseases of humans. these viruses are transmitted by direct contact, by virus-laden droplets or aerosols, or by insect vectors. all zoonotic viruses have one or more animal reservoir hosts, which play an important role in the epidemiological dynamics of human infections. although many zoonotic viruses can be transmitted to humans on occasion, their relative ability to spread from human to human determines whether or not they emerge as significant new virus diseases of mankind (table ). most zoonotic viruses that are transmitted to humans cannot be spread directly from person to person, so humans are considered to be "dead-end hosts." one familiar example is rabies, which is enzootic in several animal hosts, such as dogs, skunks, foxes, raccoons, and bats. humans are infected by bite of a rabid animal or by aerosol exposure (in caves with roosting bats). several zoonotic arenaviruses, such as lassa, machupo (bolivian hemorrhagic fever), and junin (argentine hemorrhagic fever) viruses, are likely transmitted from the reservoir host (wild rodents) by inhalation of contaminated aerosols. there are more than viruses-belonging to several virus families-that are also classified as arboviruses (arthropod-borne viruses), based on a vertebrate-arthropod maintenance cycle in nature. arboviruses replicate in both the vertebrate host and the arthropod vector (mosquitoes, ticks, sandflies, and others), and transmission occurs when the vector takes a blood meal. typically, arboviruses have only a few vertebrate hosts and are transmitted by a narrow range of arthropods. humans are not the reservoir vertebrate hosts of most arboviruses, but can be infected by many of these viruses, if they happen to be bitten by an infected vector. in most instances, arbovirus-infected humans are dead-end hosts for several reasons. many arthropod vectors competent to transmit a zoonotic arbovirus prefer nonhuman hosts as a blood source, reducing the likelihood of transmission from human to vector. also, infected humans are usually not sufficiently permissive to experience a high titer viremia, so they cannot serve as effective links in the transmission cycle. there are only a few exceptions: in urban settings, dengue, urban yellow fever, oropouche, and chikungunya viruses can be maintained by an arthropod vector-human cycle. as table shows, a few zoonotic viruses can be transmitted directly from human to human, at least for a few passages, and can emerge as the cause of outbreaks involving a few to several hundred cases. since many viruses in this group cause a high mortality in humans, even a small outbreak constitutes a public health emergency. these viruses belong to many different virus families, and there is no obvious biological clue why they should be able to spread from human to human, in contrast to other closely related viruses. typically, infections are mainly limited to caregivers or family members who have intimate contact with patients, often in a hospital setting. however, transmission is marginal, so that most outbreaks end after fewer than - serial transmissions, either spontaneously or due to infection-control practices. in the history of modern virology (the last years) there are very few documented instances where zoonotic viruses have established themselves in the human population and emerged as new viral diseases of mankind (table ). most viruses have evolved to optimize their ability to be perpetuated within one or a few host species, and this creates what is sometimes called the "species barrier." in most instances, a virus must undergo some adaptive mutations to become established in a new species. sars coronavirus. in november, , an outbreak of severe acute respiratory disease (sars) began in guangdong province, in southeast china near the hong kong border. in retrospect, the first cases were concentrated in food handlers, who then spread the virus to the general population in that region. although not recognized immediately as a new disease, the outbreak continued to spread both locally and in other parts of china. in february, , a physician who had been treating likely sars patients, traveled to hong kong, where he transmitted sars to a large number of contacts in a hotel. these persons, in turn, spread the infection to singapore, taiwan, vietnam, and canada, initiating a global pandemic that eventually involved almost countries. from patient samples, several research groups isolated a novel coronavirus, which has been named the sars coronavirus (sars cov). clearly, this virus is new to the human population, and there is circumstantial evidence that it was contracted from exotic food animals that are raised for sale in restaurants in guangdong province. recent studies suggest that horseshoe bats (genus rhinolophus) may be the reservoir hosts and palm civets, consumed as food in china, may be the intermediary hosts for sars cov. sars cov went through a large number of human passages (perhaps ) before being contained by primary control measures, such as respiratory precautions, isolation, and quarantine. the virus has been eliminated from the human population, but the outbreak showed that it could be maintained by human-to-human transmission. from that perspective, it is potentially capable of becoming an indigenous virus of humans. since many coronaviruses infect the respiratory system and are transmitted by the respiratory route, the sars virus did not have to undergo any change in its pathogenesis. however, the virus did have to replicate efficiently in cells of the human respiratory tract and it is unknown whether this required some adaptive mutations from the virus that is enzootic in its reservoir hosts. middle eastern respiratory syndrome. mers is an acute respiratory disease of humans that was first recognized in , when a novel coronavirus (mers-cov) was isolated from two fatal human cases in saudi arabia and qatar. since that time, more than clinical cases of mers have been recognized, the great majority in saudi arabia but also in most of the other countries in the arabian peninsula and beyond ( figure ). hospitalized cases of mers are characterized by an acute respiratory disease syndrome (ards) with a fatality rate over %. however, evolving evidence suggests that there may be many additional human infections with little or no associated illness. what is the origin of this new disease? based on fragmentary data now available, it appears that mers-cov is likely to be an enzootic virus of one or more species of bats. camels are probably an intermediary host, and humans in close contact with camels can acquire infection. also, mers-cov can spread from human to human, particularly to caretakers or others in close contact with acutely ill patients. however, there are many unknowns in this speculative reconstruction: how do camels become infected? are camels the main source of human infections? what has caused this disease to emerge in , or had it preexisted but was just recognized at that time? is this an evolving outbreak, and if so, what is driving it? several relevant facts have been ascertained from basic studies. mers-cov replicates well in cell cultures obtained from many species, including bats and humans. the pantropic nature of this virus is explained in part by its cellular receptor, dipeptidyl peptidase (dppt ), a widely distributed cell surface molecule. this would facilitate the transmission of this zoonotic infection from bats to camels to humans. of interest, mers-cov infects type ii alveolar pneumocytes while sars cov uses a different cell receptor (angiotensin-converting enzyme , ace ) and infects type i alveolar pneumocytes. however, both viruses appear to cause a similar ards. type a influenza virus. genetic evidence strongly implicates avian and porcine type a influenza viruses as the source of some past pandemics of human influenza. it appears that new epidemic strains are often derived as reassortants between the hemagglutinin (and the neuraminidase in some cases) of avian influenza viruses with other genes of existing human influenza viruses. the new surface proteins provide a novel antigenic signature to which many humans are immunologically naïve. the human influenza virus genes contribute to the ability of the reassortant virus to replicate efficiently in human cells. it is thought that reassortment may take place in pigs that are dually infected with avian and human viruses. currently, there is concern that a new pandemic strain of type a influenza could emerge as a derivative of highly virulent avian h n or h n influenza viruses now causing epidemics in domestic chickens in southeast asia. there have been several hundred documented human infections with each of these avian viruses in recent years-mainly among poultry workers-with significant mortality. however, few if any of these infections have spread from human to human, perhaps because the infecting avian virus has not undergone reassortment with a human influenza virus. a critical determinant is that avian influenza viruses preferentially attach to α - sialic acid receptors while human viruses attach to α - sialic acid receptors. the pandemic of influenza is presumed to be an example of a zoonotic influenza virus that crossed the species barrier and became established in humans where it caused an excess mortality estimated at - million persons. recent viral molecular archaeology has recovered the sequences of the h n influenza virus from the tissues of patients who died during the epidemic. all of the genes of the reconstructed virus are avian in origin, but it is unknown whether the virus underwent mutations that enhanced its ability to be transmitted within the human population. the reconstructed hemagglutinin of the virus has been inserted into recombinant influenza viruses, and-in mice-markedly increases the virulence of primary human isolates of influenza virus (table ) , but the full virulence phenotype appears to require many of the avian influenza genes. several physiological factors play a role in disease enhancement, including increased replication in pulmonary tissues and an enhanced ability to stimulate macrophages to secrete pro-inflammatory cytokines which, in turn, cause severe pneumonitis. human immunodeficiency virus. hiv has emerged as the greatest pandemic in the recent history of medical science. modern methods have made it possible to reconstruct its history in great detail (see chapter , hiv/aids). a provisional reconstruction of the sequence of events is summarized in figure . the emergence of hiv may be divided into two phases: what was the zoonotic source of hiv and when did it cross into humans? and when did hiv spread from the first human cases to become a global plague? there have been several transmissions of simian immunodeficiency virus (siv) to humans (sharp and hahn, ) , and this account will focus on hiv- which appears to have been transmitted to humans in at least four separate instances, identified by individual hiv- lineages called groups (m, n, o, p). of these, the most important was the m group of hiv- , which has been responsible for the vast majority of human infections. furthermore, hiv- is most closely related to sivcpz, the siv strain infecting two subpopulations of chimpanzees. different segments of the sivcpz genome, in turn, are closely related to genome segments of two sivs of african monkeys, red-capped monkeys and cercopithecus monkeys. it is hypothesized that chimpanzees, which regularly kill and eat monkeys, were infected during consumption of their prey; and that a recombination event produced sivcpz, which was derived from parts of the genomes of the two acquired monkey viruses. it is speculated that a further transmission from chimpanzees may have occurred during the butchering of nonhuman primates, which occurs in rural africa ( figure ) . amazingly, genomic similarities tentatively map the substrain of sivcpz that is the ancestor of the m group of hiv- , to chimpanzees in the southeastern corner of cameroon, a small country in west africa. using a sequence analysis to compare diversity within current isolates, the common parent of the m group can be reconstructed. a molecular clock, derived from dated isolates, indicates that this virus was transmitted to humans during the period - . the period from to is a mystery (pepin, ) , but there are fragmentary data suggesting that the virus persisted as a rare and unrecognized infection in residents of jungle villages in west africa during this time. it has also been proposed that the reuse of unsterilized needles-a frequent practice during the period of colonial rule-could have inadvertently helped to spread the virus. starting about , the virus began to spread more rapidly. it appears that accelerated spread began in the region centered on kinshasa (previously leopoldville) in the democratic republic of the congo (previously the belgian congo, then zaire) and brazzaville, just across the congo river in congo. transmission was exacerbated by the chaos in postcolonial zaire. during the period - , hiv infection spread widely in africa as shown in figure . in the countries worst affected, the prevalence of infection among adults aged - years reached levels higher than %. the rapid spread was driven by many factors among which were: ( ) a high frequency of concurrent sexual contacts in some segments of the population and the hidden nature of sexual networks; ( ) the long asymptomatic incubation period during which infected individuals able to transmit the virus were sexually active; ( ) the spread along commercial routes of travel within africa; ( ) the failure of health systems to concomitantly with the spread of hiv in africa, the m group of hiv- evolved into nine different subtypes (a-d, f-h, j, k), based on sequence diversity. during the spread within africa, there were population bottlenecks that resulted in the predominance of different m group subtypes in different regions. subtype c is most frequent in southern africa, and subtypes a and d are most frequent in eastern africa. during the s, hiv also spread globally, although prevalence rates did not reach the levels seen in some african countries. subtype b is dominant in the western hemisphere and europe, while subtype c is most frequent in india and some other asian countries. this implies that each of these regional epidemics was initiated speculative reconstruction of events following the transmission of sivcpz to humans. this reconstruction is based on data in hahn ( ), pepin ( ) . bushmeat is part of the diet in rural africa. photograph courtesy of billy karesh ( ). by a small number of founder strains of hiv- , improbable though that may seem. thirty years after its appearance as a global disease, almost million persons have died, and there are more than million people living with hiv/aids. although the global incidence of hiv has fallen slightly since , there are still more than two million new infections each year ( ). ebola hemorrhagic fever. the ebola pandemic of - is the most recent emerging virus disease that has riveted the attention of the world. where did it come from? why did it cause a pandemic? why did the international health community fail to control it? where will it end? these questions are discussed below. ebola is a filovirus indigenous to africa that is maintained in one or more reservoir species of wild animals, among which bats are a likely host. the transmission cycle is not well documented but it is thought that humans get infected, either by direct contact with bats, or while slaughtering infected wild animals who may act as intermediate hosts. human-to-human spread can then occur. the pathogenesis of ebola virus infection may be briefly summarized. presumably ebola enters the human host via the mucous membranes or cuts in the skin. the virus infects mononuclear cells including macrophages and dendritic cells and traffics to lymph nodes, whence it spreads to target organs including the liver, spleen, and adrenal glands. it causes a high titer viremia and a dysregulation of the innate immune system. clinically, ebola patients undergo severe vomiting and diarrhea with massive fluid losses and become very dehydrated, with a mortality that varies from % to %. in fatal cases, the infection of the liver leads to disseminated intravascular coagulopathy, a shock syndrome, and multiorgan failure, although the sequential details are not well understood. infection is transmitted between humans by contact with bodily fluids of patients, but not by aerosols. therefore, the virus is spread most frequently to caregivers or others who are in intimate contact with patients and their fomites. rituals associated with funerals and burial practices often serve to transmit the virus. ebola virus was first isolated in in an outbreak near the ebola river in the democratic republic of the congo (then called zaire) and almost concurrently in a second outbreak in southern sudan. viruses recovered from these two outbreaks were subsequently shown to be different and are now known as ebola-zaire and ebola-sudan. since that time there have been more than individual outbreaks of ebola disease, mainly in central africa. past outbreaks have been controlled by use of protective garments by caregivers and quarantine of infected or potentially infected contacts. these controlled outbreaks have been limited to no more than ∼ serial human-to-human transmissions, and mainly ranged from about to cases. in december, , an ebola-zaire outbreak began in guinea, west africa. it appears that the initial case was in an infant who may have been infected by contact with bats. the outbreak then spread to two contiguous countries, liberia and sierra leone. by the fall, , the epidemic had become a catastrophe, and was raging out of control in several parts of these three countries. although the data are incomplete, it is estimated that there have been at least , cases (with at least % mortality) through february, (figure ) . the infection has spread to nigeria, senegal, mali, the united states, and a few european countries, but these invasions have so far been controlled, with limited secondary cases. a global effort to control this pandemic was initiated, with participation from doctors without borders, the red cross, other nongovernment organizations, the world health organization, and the u.s. centers for disease control and prevention. as of march, , it appeared that the epidemic was coming under control. aggressive border screening, both on exit from the affected countries and on arrival at destinations, has limited spread by air travelers, but the porous land borders remain areas of concern. from an epidemiological viewpoint, why did this outbreak of ebola explode into a massive pandemic, in contrast to the many prior outbreaks that were limited to no more than a few hundred cases at most? the outbreak began in guinea and was mainly confined to that country for about months before it spread to neighboring liberia and sierra leone (figure ). during this first months, incidence in guinea varied from a few to about cases a week, and cases were concentrated in rural areas. from a public health viewpoint, this represented a missed opportunity to contain the outbreak. contributing to this omission were a combination of factors: a weak health system fragmented by social disruption, a failure of local health authorities to recognize or respond to the outbreak, the failure of international health organizations such as who to take aggressive action, and local societal norms that brought family and friends into close contact with ebola victims, during their illness and at their funerals (washington post, october , ; cohen, ) . once ebola infections spread from rural villages to urban centers, the outbreak exploded. beginning in the fall of , it was recognized that the pandemic was a global threat. in response, a number of countries and international organizations provided resources to africa, including building facilities, sending equipment, and recruiting personnel to work in the pandemic areas. a major effort was made to get the local population to temporarily change some of their normal social responses to illness and death, to reduce the spread within the population. combined with the efforts of the affected countries, these initiatives started to take hold about january, . however, as of march, , the epidemic was not yet terminated. in retrospect, the international community has acknowledged that it lacks a contingency plan to respond to global outbreaks wherever they may occur. futhermore, because ebola was a "neglected" disease, the tools to combat it had not been developed. the ebola pandemic has spurred crash programs to develop a rapid diagnostic test, drugs and antibodies for treatment, and a vaccine for prevention (product, ; mire et al., ) . canine parvovirus. cpv is another example of a disease that emerged due to the appearance of a virus new to its host species. in the late s, a highly lethal pandemic disease appeared in the dog populations of the world. the etiologic agent was a parvovirus previously unknown in canines. the sequence of cpv is almost identical to that of feline panleukopenia virus (fpv), an established parvovirus of cats, which causes acutely fatal disease in kittens. cpv has a few point mutations that distinguish it from fpv, and these mutations permit cpv to bind to and infect canine cells, a property not possessed by fpv. it is presumed that these mutations permitted the emergence of a new virus disease of dogs. many mammalian viruses have evolved with their hosts so that different members of a virus family are associated with each host species. furthermore, under natural circumstances, each member of the virus family usually "respects" the species barrier and does not cross into other species, although it spreads readily between individual animals within its host species. diverse mechanisms contribute to the species barrier, including host defenses and viral genes. for a zoonotic virus to establish itself in humans or another new species, it is probable that mutations are needed for full adaptation. this requirement is likely part of the explanation for the rarity of such events. one of the best-studied examples is the transmission of sivcpz, a virus of chimpanzees, to hiv, a human virus. several recent studies have shown that apobec and tetherin are two very important gate keepers for transmission of lentiviruses between different primate species (reviewed in sharp and hahn, ) . apobec proteins represent a powerful first line of defense, believed to be responsible for preventing the transmission of sivs from monkeys to chimpanzees and from monkeys to humans (etienne et al., ) . however, apobec proteins can be counteracted by the hiv/siv viral infectivity factor (vif), albeit generally in a species-specific fashion. thus, mutations in vif were required for monkey sivs to be able to infect chimpanzees and then humans (letko et al., ) . tetherin is a second line of defense, and only hivs that have evolved an effective tetherin antagonism have spread widely in humans (kluge et al., ) . different lentiviruses use different viral proteins to antagonize tetherin: hiv- group m uses the viral protein u (vpu); hiv- group a uses the envelope glycoprotein (env), and hiv- group o uses the negative regulatory factor (nef). in contrast, hiv- groups n and p, whose spread in the human population has remained very limited, are unable to counteract tetherin efficiently. turning to another virus, recent studies using a mouseadapted strain of ebola virus to infect a panel of inbred mice provided by the collaborative cross (see chapter , animal models) found striking variation in the response to ebola virus infection, from complete resistance to severe hemorrhagic fever with % mortality. this underlines the role of host genetic background as a determinant of susceptibility. consistent with this are comments of clinicians that there are unpredictable differences in the outcome of individual ebola cases. these studies suggest a dynamic relationship between host and pathogen that may determine when a virus can cross the species barrier and create a new virus disease of humankind. although difficult to document in a rigorous manner, it does appear that new virus diseases of humans (and perhaps of other species) are emerging at an increased tempo. there are a number of reasons for this trend (table ) . the human population is growing inexorably, and is becoming urbanized even faster. as a result, there are an increasing number of large-crowded cities, which provide an optimal setting for the rapid spread of any newly emergent infectious agent. in the nineteenth century, it was noteworthy that someone could circumnavigate the globe in days, but now it can be done in less than h. however, the incubation period of viral infections (several days to several months) has stayed constant. someone can be infected in one location and-within a single incubation period-arrive at any other site on earth. this enhances the opportunity for a new human virus to spread as a global infection before it has even been recognized, markedly increasing the opportunity for the emergence of a new disease. the same dynamics also apply to viral diseases of animals and plants, which has important economic and social consequences for humankind. the sars pandemic of - , described above, is an example of how rapidly and widely a new virus disease can emerge and spread globally. in this instance, it is extraordinary that the disease was brought under control-and eradicated from the human population-by the simple methods of isolation, quarantine, and respiratory precautions. although conceptually simple, a heroic effort was required for success. another example is the emergence of a novel h n strain of influenza a virus that spread rapidly around the world before a vaccine could be produced. remote areas of the world are now being colonized at a high frequency, driven by population pressures and economic motives, such as the reclamation of land for agriculture or other uses, and the harvest of valuable trees and exotic animals. the construction of new dams, roads, and other alterations of the natural environment create new ecological niches. it is possible that this is the origin of urban yellow fever. yellow fever virus is an arbovirus maintained in a monkey-mosquito cycle in the jungles of south america and africa. humans who entered jungle areas became infected. when they returned to villages or urban centers, an urban cycle was initiated, where aedes aegypti mosquitoes-that are well adapted to the urban environment and preferentially feed on humansmaintained the virus. in the last years, agriculture has undergone a dramatic evolution with the development of "agribusiness." food and food animals are now raised on an unprecedented scale and under very artificial conditions, where the proximity of many members of a single plant or animal species permits an infection to spread like wildfire. furthermore, increasing numbers of plants, animals, and food products are rapidly transported over large distances; we enjoy fresh fruits and vegetables at any time, regardless of the season. international shipment of plants and animals can import viruses into new settings where they may lead to the emergence of unforeseen diseases. one example is the outbreak of monkeypox that caused about human cases in the united states. this was traced to the importation from africa of gambian giant rats as exotic pets; several rodent species in west africa appear to be reservoir hosts of this poxvirus. monkeypox spread from these animals to pet prairie dogs and from prairie dogs to their owners. because monkeypox causes infections in humans that resemble mild cases of smallpox, this outbreak was a major cause of public health concern. on occasion, a virus has been deliberately introduced into a susceptible population where it caused the emergence of a disease epidemic. for sport, rabbits were imported from europe into australia in the mid-nineteenth century. because of the absence of any natural predator the rabbits multiplied to biblical numbers, and threatened natural grasslands and agricultural crops over extensive areas of southern australia. to control this problem, myxomatosis virus was deliberately introduced in (fenner, ) . this poxvirus is transmitted mechanically by the bite of insects and is indigenous to wild rabbits in south america, in which it causes nonlethal skin tumors. however, myxomatosis virus causes an acutely lethal infection in european rabbits, and its introduction in australia resulted in a pandemic in the rabbit population. following the introduction of myxomatosis virus in , co-evolution of both virus and host were observed. the introduced strain was highly virulent and caused epizootics with very high mortality. however, with the passage of time field isolates exhibited reduced virulence, and there was a selection for rabbits that were genetically somewhat resistant to the virus. strains of moderate virulence probably became dominant because strains of lowest virulence were less transmissible and strains of maximum virulence killed rabbits very quickly. concern has also been raised about disease emergence due to the deliberate introduction of viruses into either human or animal populations, as acts of bioterrorism. because of the shortage of human organs for transplantation recipients, there is considerable research on the use of other species-particularly pigs-as organ donors. this has raised the question whether known or unknown latent or persistent viruses in donor organs might be transmitted to transplant recipients. since transplant recipients are immunosuppressed to reduce graft rejection, they could be particularly susceptible to infection with viruses from the donor species. in the worst scenario, this could enable a foreign virus to cross the species barrier and become established as a new human virus that might spread from the graft recipient to other persons. the impetus to identify a new pathogenic virus usually arises under one of two circumstances. first, a disease outbreak that cannot be attributed to a known pathogen may set off a race to identify a potentially new infectious agent. identification of the causal agent will aid in the control of the disease and in prevention or preparedness for potential future epidemics. sars coronavirus, west nile virus, and sin nombre virus are examples of emergent viruses that were identified in the wake of outbreaks, using both classical and modern methods. alternatively, an important new virus may be discovered as a serendipitous by-product of research directed to a different goal, as was the case with hepatitis b virus (hbv). in this instance, a search for alloantigens uncovered a new serum protein that turned out to be the surface antigen (hbsag) of hbv, leading to the discovery of the virus. when a disease outbreak cannot be attributed to a known pathogen, and where classical virus isolation, propagation, and identification fail, molecular virology is required. hepatitis c virus (hcv), sin nombre and other hantaviruses, certain rotaviruses, and kaposi's sarcoma herpesvirus (hhv ) , are examples of emergent viruses that were first discovered as a result of molecular technologies. below, we briefly describe some methods of viral detection and identification. more detailed information and technical specifics can be found in several current texts. the first question that confronts the investigator faced with a disease of unknown etiology is whether or not it has an infectious etiology? evidence that suggests an infectious etiology is an acute onset and short duration, clinical similarity to known infectious diseases, a grouping of similar illnesses in time and place, and a history of transmission between individuals presenting with the same clinical picture. for chronic illnesses, the infectious etiology may be much less apparent and a subject for debate. faced with a disease that appears to be infectious, the next question is whether it is caused by a virus. a classical example that predates modern virology is the etiology of yellow fever. in a set of experiments that would now be prohibited as unethical, the yellow fever commission, working with the us soldiers and other volunteers in cuba in , found that the blood of a patient with acute disease could transmit the infection to another person by intravenous injection. furthermore, it was shown that the infectious agent could pass through a bacteria-retaining filter and therefore could be considered a "filterable virus." virus isolation in cell culture and animals. the first step in identification of a putative virus is to establish a system in which the agent can be propagated. before the days of cell culture, experimental animals were used for this purpose. many viruses could be isolated by intracerebral injection of suckling mice, and some viruses that did not infect mice could be transmitted to other experimental animals. human polioviruses-because of their cellular receptor requirements-were restricted to old world monkeys and great apes; the virus was first isolated in by intracerebral injection of monkeys and was maintained by monkey-to-monkey passage until when it was shown to replicate in primary cultures of human fibroblasts. the modern era of virology (beginning about ) can be dated to the introduction of cultured cells as the standard method for the isolation, propagation, and quantification of viruses. there are now a vast range of cell culture lines that can be used for the isolation of viruses, and currently this is the first recourse in attempting to isolate a suspected novel virus. some viruses will replicate in a wide variety of cells but others are more fastidious and it can be hard to predict which cells will support their replication. it is also important to recognize that some viruses will replicate in cell culture without exhibiting a cytopathic effect. an important example is the identification of simian virus (sv ). poliovirus was usually grown in primary cell cultures obtained from the kidneys of rhesus monkeys, but sv had escaped detection because it replicated without causing a cytopathic effect. when poliovirus harvests were tested in similar cultures prepared from african green monkeys, a cytopathic effect (vacuolation) was observed, leading to the discovery of sv virus in . because inactivated poliovirus vaccine produced from to had been prepared from virus grown in rhesus monkey cultures, many lots were contaminated with this previously unknown virus, which inadvertently had been administered to humans. since that time, viral stocks and cell cultures have been screened to exclude sv and other potential virus contaminants. a number of methods are available to detect a noncytopathic virus that is growing in cell culture. these include visualization of the virus by electron microscopy, detection of viral antigens by immunological methods such as immunofluorescence or immunocytochemistry, the agglutination of erythrocytes of various animal species by virus bound on the cell surface (hemagglutination), the production of interferon or viral interference, and the detection of viral nucleic acids. detection of nonreplicating viruses. during the period from to , there was a concerted effort to identify the causes of acute infections of infants and children. in seeking the etiology of diarrheal diseases of infants, it was hypothesized that-in addition to bacteria, which accounted for less than half of the cases-one or more viruses might be responsible for some cases of infantile diarrhea. numerous unsuccessful attempts were made to grow viruses from stools of patients with acute diarrhea. it was conjectured that it might be possible to visualize a putative fastidious virus by electron microscopy of concentrated fecal specimens. when patients' convalescent serum was added to filtered and concentrated stool specimens, aggregates of nm virions were observed in stools from some infants with acute gastroenteritis. the ability of convalescent but not acute illness serum to mediate virion aggregation provided a temporal association of the immune response with an acute diarrheal illness. within years, rotavirus was recognized as the most common cause of diarrhea in infants and young children worldwide, accounting for approximately onethird of cases of severe diarrhea requiring hospitalization. once an emergent virus has been identified, it is necessary to classify it, in order to determine whether it is a known virus, a new member of a recognized virus group, or represents a novel virus taxon. this information provides clues relevant to diagnosis, prognosis, therapy, and prevention. in , an outbreak of acute hemorrhagic fever occurred in laboratory workers in marburg, germany, who were harvesting kidneys from african green monkeys (chlorocebus aethiops, formerly cercopithecus aethiops). in addition, the disease spread to hospital contacts of the index cases, with a total of over cases and % mortality. clinical and epidemiological observations immediately suggested a transmissible agent, but attempts to culture bacteria were unsuccessful. however, the agent was readily passed to guinea pigs which died with an acute illness that resembled hemorrhagic fever. after considerable effort, the agent was adapted to tissue culture and shown to be an rna virus. when concentrated tissue culture harvests were examined by electron microscopy, it was immediately recognized that this agent differed from known families of rna viruses, since the virions consisted of very long cylindrical filaments about nm in diameter. this was the discovery of marburg virus, the first recognized member of the filoviruses, which now include marburg and ebola viruses. isolation of a virus from patients suffering from an emergent disease provides an association, but not proof of a causal relationship. formal demonstration that an isolated virus is the causal agent involves several criteria formulated over the past years. these are often called the henle-koch postulates, after two nineteenth-century scientists who first attempted to enunciate the rules of evidence. sidebar summarizes these postulates, which have been modernized in view of current knowledge and experimental methods. the classic version of the henle-koch postulates required that the causal agent be grown in culture. however, as discussed below, a number of viruses that cannot be grown in culture have been convincingly associated with a specific disease. usually, this requires that many of the following criteria can be met: ( ) viral sequences can be found in the diseased tissue in many patients, and are absent in appropriate control subjects; ( ) comparison of acute and convalescent sera document induction of an immune response specific for the putative causal virus; ( ) the disease occurs in persons who lack a preexisting immune response to the putative virus, but not in those who are immune; ( ) the implicated virus or a homologous virus causes a similar disease in experimental animals; and ( ) epidemiological patterns of disease and infection are consistent with a causal relationship. some very important human diseases-such as hepatitis b and hepatitis c-are caused by viruses that cannot readily be grown in cell culture. experiences with these viruses have given credibility to the view that an infectious etiology can be inferred by clinical and epidemiological observations in the absence of a method for growing the causal agent. also, they have stimulated researchers to devise novel techniques that bypass the requirement for replication in cell culture. furthermore, the application of molecular biology, beginning about , has led to an array of new methods-such as the polymerase chain reaction (pcr), deep sequencing, and genomic databases-that can be applied to the search for unknown viruses. several case histories illustrate the inferences that lead to the hypothesis of a viral etiology, the strategy used to identify the putative causal agent, and the methods exploited by ingenious and tenacious researchers. sin nombre virus. hantavirus pulmonary syndrome was described above, as an example of an emerging virus disease. the disease was first reported in mid-may, , the henle-koch postulates were formulated in by henle, revised by koch in , and have undergone periodic updates to incorporate technical advances and the identification of fastidious agents with insidious disease pathogenesis. many of the following criteria should be met to establish a causal relationship between an infectious agent and a disease syndrome. . the putative causal agent should be isolated from patients with the disease; or the genome or other evidence of the causal agent should be found in patients' tissues or excreta; and less frequently from appropriate comparison subjects. temporally, the disease should follow exposure to the putative agent; if incubation periods can be documented they may exhibit a log-normal distribution. if an immune response to the putative agent can be measured, this response should correlate in time with the occurrence of the disease. subjects with evidence of immunity may be less susceptible than naïve individuals. . experimental reproduction of the disease should occur in higher incidence in animals or humans appropriately exposed to the putative cause than in those not so exposed. alternatively, a similar infectious agent may cause an analogous disease in experimental animals. . elimination or modification of the putative cause should decrease the incidence of the disease. if immunization or therapy is available, they should decrease or eliminate the disease. . the data should fit an internally consistent pattern that supports a causal association. and tissues and blood samples from these cases were tested extensively, but no virus was initially isolated in cell culture. however, when sera from recovered cases were tested, they were found to cross-react with a battery of antigens from known hantaviruses, providing the first lead (in june, ) . dna primers were then designed, based on conserved hantavirus sequences, and these were used in a pcr applied to dna transcribed from rna isolated from tissues of fatal cases. sequence of the resulting amplicon suggested that it was a fragment of a putative new hantavirus (july, ) , yielding a presumptive identification of the emerging virus within months after the report of the outbreak. an intense effort by three research teams led to the successful isolation of several strains of snv by november, . snv is a fastidious virus that replicates in vero e cells but not in many other cell lines. kaposi's sarcoma herpesvirus (hhv ). ks was described over years ago as a relatively uncommon sarcoma of the skin in older men in eastern europe and the mediterranean region. in the s, ks emerged at much higher frequency, as one of the diseases associated with aids. furthermore, ks exhibited an enigmatic epidemiological pattern, since its incidence in gay men was more than -fold greater than in other aids patients, such as injecting drug users and blood recipients. these observations led to the hypothesis that ks was caused by a previously undetected infectious agent that was more prevalent among gay men than among other hiv risk groups. however, researchers were unable to isolate a virus from ks tissues. searching for footprints of such a putative agent, chang and colleagues used the method of representational difference analysis to identify dna sequences specific for ks tumor tissue. several dna fragments were identified, and found to be homologous with sequences in known human and primate herpesviruses. in turn, these sequences were used to design primers to obtain the complete genome of a previously undescribed herpesvirus, since named hhv , human herpesvirus . to this date, hhv defies cultivation in tissue culture. is computer modeling a useful adjunct to the analysis or control of emerging viral diseases? the - ebola pandemic in west africa offers an interesting case study. as the epidemic unfolded, several groups attempted to project how it would evolve (meltzer et al., ; butler, ) . these projections had very wide confidence limits, and several of them had upper limits in the range of , or more cases. what the modelers could not foretell was that the infection did not spread across sub-saharan africa, and that several introductions (into countries such as nigeria and mali) were controlled by case isolation, contact tracing, and quarantine. however, modeling did contribute useful insights. dobson ( ) suggested that rapid quarantine (within - days) of contacts of ebola patients could be critical in epidemic control. one of the most exciting current issues in virology is the emergence of new viral diseases of humans, animals, and plants. even though the era of modern virology has been well established for more than years, virus diseases continue to appear or reemerge. the ebola pandemic of - highlights the associated dangers and obstacles to control. there are several explanations for emergence: ( ) discovery of the cause of a recognized disease; ( ) increase in disease due to changes in host susceptibility or in virus virulence; ( ) reintroduction of a virus that has disappeared from a specific population; ( ) crossing the barrier into a new species previously uninfected. many zoonotic viruses that are maintained in a nonhuman species can infect humans, but most cause dead-end infections that are not transmitted between humans. a few zoonotic viruses can be transmitted between humans but most fade out after a few person-to-person transmissions. rarely, as in the case of hiv, sars coronavirus, and ebola filovirus, a zoonotic virus becomes established in humans, causing a disease that is truly new to the human species. there are many reasons for the apparent increase in the frequency of emergence of new virus diseases, most of which can be traced to human intervention in global ecosystems. emergent viruses are identified using both classical methods of virology and newer genome-based technologies. once a candidate virus has been identified, a causal relationship to a disease requires several lines of evidence that have been encoded in the henle-koch postulates, guidelines that are periodically updated as the science of virology evolves. identifying, analyzing, and controlling emerging viruses involve many aspects of virological science. virus-host interactions play a key role, to explain persistence in zoonotic reservoirs, transmission across the species barrier, and establishment in human hosts. thus, the issues discussed in many other chapters contribute to our understanding of emerging viral diseases. hendra and nipah viruses: different and dangerous biological control as exemplified by smallpox eradication and myxomatosis sequence-based identification of microbial pathogens: a reconsideration of koch's postulates emerging virus diseases: can we ever expect the unexpected? emerging microbes and infection epidemiology and transmission dynamics of west nile virus disease emerging infections the emergence and evolution of canine parvovirus -an example of recent host range mutation social and environmental risk factors in the emergence of infectious diseases diseases of the central nervous system caused by lymphocytic choriomeningitis virus and other arenaviruses middle east respiratory syndrome coronavirus (mers-cov) evidence and speculation human monkeypox: an emerging zoonosis isolation of the causative agent of hantavirus pulmonary syndrome cultivation of the lansing strain of poliomyelitis virus in cultures of various human embryonic tissue. science mission to mers origin of hiv- in the chimpanzee pan troglodytes genomic surveillance elucidates ebola virus origin and transmission during the outbreak a decade after sars: strategies for controlling emerging coronaviruses chikungunya disease outbreak, reunion island enhanced virulence of influenza a viruses with the haemagglutinin of the pandemic virus mers surges again, but pandemic jitters ease origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia bats are natural reservoirs of sars-like coronavirus the many projected futures of dengue ks-associated herpesvirus, and the criteria for causality in the age of molecular biology genetic identification of a hantavirus associated with an outbreak of acute respiratory illness host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance recent researches concerning etiology, propagation and prevention of yellow fever, by the united states army commission recognition determinants of broadly neutralizing human antibodies against dengue viruses a single mutation in chikungunya virus affects vector specificity and epidemic potential characterization of the reconstructed spanish influenza pandemic virus molecular changes in a/chicken/ pennsylvania/ (h n ) influenza virus associated with acquisition of virulence ebola references models overestimate ebola cases breakdown of the year: ebola mathematical models for emerging disease mobilizing ebola survivors to curb the epidemic ebola hemorrhagic fever genomic surveillance elucidates ebola virus origin and transmission during the outbreak basic clinical and laboratory features of filoviral hemorrhagic fever ebola virus vaccines: an overview of current approaches. expert review of vaccines estimating the future number of cases in the ebola epidemic -liberia and sierra leone single-dose attenuated vesiculovax vaccines protect primates against ebola makona virus pathogenesis of the viral hemorrhagic fevers product: reebov™ antigen rapid test kit host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance who ebola response team. ebola virus disease in west africa-the first months of the epidemic and forward projections gene loss and adaptation to hominids underlie the ancient origin of hiv- sharp pm aids as a zoonosis: scientific and public health implications nef proteins of epidemic hiv- group o strains antagonize human tetherin vif proteins from diverse primate lentiviral lineages use the same binding site in apobec g the origins of aids origins of hiv and the aids pandemic key: cord- -z rc ubj authors: wilkins, pamela a. title: disorders of foals date: - - journal: equine internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: z rc ubj nan before the s, intensive management of the compromised neonate was unusual and little was known regarding many of the problems of this special patient population. although some specific conditions had been described by astute clinician-researchers, most notably the "dummy" foal syndrome and respiratory distress syndrome caused by primary surfactant deficiency, little information regarding the diagnosis and management of conditions of the foal during the neonatal period was available, although at least one active group was investigating fetal and neonatal physiology of the horse in great britain. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] when treatment of compromised foals was undertaken, the approach most commonly resembled treating them as small adults with little understanding of the different physiology of the equine neonate. the advent of improved management of reproductive efficiency of mares led naturally to increased interest in preservation of the conceptus to parturition and the foal thereafter. interested clinicians, taking their lessons from the field of human perinatology/neonatology and sometimes working hand-in-hand with their counterparts in the human field, pioneered investigations into these small patients and created the fields of equine perinatology and equine neonatal intensive care. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] because of the foresight and energy of these early investigators, the field of veterinary perinatology/neonatology exploded in the s, leading to the creation of equine neonatal intensive care units throughout the united sates and the world. from these units information about the normal and abnormal physiology of foals, the medical conditions affecting them, and methods for treatment and management of these problems has been developed through observational, retrospective, and prospective studies. this veritable explosion of information over the last years has improved greatly the ability of all practitioners to provide appropriate care for these patients, whether in the field or at an equine neonatal intensive care unit. the ability not only to save the lives of these patients but also to treat them in such a manner as to allow them to fulfill their purposes, whether as pleasure animals or racing athletes, has improved almost exponentially from those early days. [ ] [ ] [ ] [ ] this chapter aims to provide the clinician with some of the most current information regarding the management of these patients, recognizing that much still remains unknown and that advances will continue to be made in this dynamic field. the reader is cautioned that much of this chapter is flavored by the experiences of the author and that variation in approach and treatment of specific problems exists between neonatal intensive care units (nicus) and between clinicians in the same nicu and that each year results in change. in some cases, information that is presented has been gleaned from human nicu studies, essentially using the critically ill infant as the experimental model. many of the problems of the newborn foal have their genesis in utero. identification of high-risk pregnancies is an important component of prenatal care of the foal, and some of the most commonly encountered problems of the dam resulting in abnormal foals include previous or concurrent disease, poor reproductive history, poor perineal or pelvic conformation, poor general health, poor nutritional condition, prolonged transport, history of previous abnormal foals, placental abnormalities, and twins. some of the more common causes of abortion can result in the birth of severely compromised foals of variable gestation lengths (box - ). these include pa m e l a a . wi l k i n s infectious causes such as equine herpesvirus (ehv) types (most commonly) and (rarely), equine infectious anemia, equine arteritis virus, bacterial and fungal placentitis, leptospirosis, equine ehrlichiosis, and gram-negative septicemia/endotoxemia. [ ] [ ] [ ] noninfectious causes of abortion include twinning and noninfectious placental abnormalities such as extensive endometrial fibrosis, body pregnancy, and abnormal length (long or short) of the umbilical cord. , to the equine neonatologist opportunities for intervention may appear limited, and in the case of many of the aforementioned causes of fetal loss, this is true. however, one can do much in an attempt to preserve the pregnancy and in effect treat the fetus. when one is faced with a threatened pregnancy, one has various ways of evaluating the fetus and its environment and may use many potential therapies. once one identifies a pregnancy as high risk, one should evaluate the fetus for viability. evaluation should include as thorough an evaluation as possible of the reproductive tract, placenta, and fetal fluids. prepartum disorders in the mare usually are readily recognizable, but disorders of the fetus and placenta can be more subtle and difficult to determine. the first step is to take a thorough history of the mare. of particular interest is any history of previous abnormal foals, but the history taking should include questions regarding transportation; establishment of an accurate breeding date (sometimes more difficult than one would suspect); any pertinent medical history including any diagnostic testing performed for this pregnancy such as culture, endometrial biopsy, and cytologic results; and any rectal and ultrasound examination results. additionally, one should obtain information regarding possible ingestion of endophyte-infected fescue or exposure to potential infectious causes of abortion. , a complete vaccination and deworming history is requisite, as is a complete history of any medications and supplements administered during pregnancy. after obtaining a history, one examines the mare per rectum. this examination should include palpation of the cervix, uterus, fetus, and all palpable abdominal contents. one should note any abnormalities. the cervix should be tight throughout gestation; the late gestation uterus will be large and distended with fluid and usually pulled craniad in the abdomen. palpation of the fetus frequently results in some fetal movement; however, one should interpret lack of movement with caution, for some normal fetuses do not respond. ultrasonographic evaluation of the uterus and conceptus per rectum can provide valuable information, particularly regarding placental thickness if placentitis is a concern. one may evaluate fetal fluids and estimate fetal size from the size of the eye later in gestation. in the author's hospital the practitioners choose not to perform vaginal examinations or speculum examinations because of an association between these examinations and the subsequent development of placentitis. unless placentitis is recognized with ultrasonograhic evaluation per rectum and culture is desirable, these types of examinations are generally not necessary. following examination per rectum, one performs transabdominal ultrasonographic evaluation of the uterus and conceptus. one can generate a biophysical profile of the fetus from this examination in the late-term fetus and readily determine viability. , one also readily can determine the presence or absence of twins in the late pregnant mare in this manner. one performs the sonogram through the acoustic window from the udder to the xiphoid ventrally and laterally to the skinfolds of the flank. imaging of the fetus usually requires a lowfrequency ( . -mhz) probe, whereas examination of the placenta and endometrium requires a higher-frequency ( . -mhz) probe. a complete description of this examination is beyond the scope of this chapter, but the reader will find several complete descriptions of the technique and normal values for specific gestation lengths within the relevant veterinary literature. the utility of this examination lies in its repeatability and low risk to the dam and fetus. sequential examinations over time allow the clinician to follow the pregnancy and to identify changes as they occur. a companion to transabdominal ultrasongraphy is evaluation of the fetal electrocardiogram (ecg). one can measure fetal ecgs continuously using telemetry or can obtain them using more conventional techniques several times throughout the day. , , one places electrodes on the skin of the mare in locations aimed at maximizing the magnitude of the fetal ecg. because the fetus frequently changes position, multiple sites may be needed in any -hour period. to begin, one places an electrode dorsally in the area of the sacral prominence with two electrodes placed bilaterally in a transverse plane in the region of the flank. the fetal ecg maximal amplitude is low, usually . to . mv, and can be lost in artifact or background noise, so one commonly must move electrodes to new positions to maximize the appearance of the fetal ecg. the normal fetal heart rate during the last months of gestation ranges from to beats/min, a fairly wide distribution. the range of heart rate of an individual fetus can be narrow, however. bradycardia in the fetus is an adaptation to in utero stress, most commonly thought to be hypoxia. by slowing the heart rate, the fetus prolongs exposure of fetal blood to maternal blood, increasing the time for equilibration of dissolved gas across the placenta and improving the oxygen content of the fetal blood. the fetus also has altered the distribution of its cardiac output in response to hypoxia, centralizing blood distribution. , tachycardia in the fetus can be associated with fetal movement, and brief periods of tachycardia should occur in the fetus in any -hour period. persistent tachycardia is a sign of fetal distress and represents more severe fetal compromise than bradycardia. the author has recognized dysrhythmias in the challenged fetus, most commonly as atrial fibrillation but also apparent runs of ventricular tachycardia. the ability to monitor the fetus in a high-risk pregnancy inevitably has led to questions of whether, how, and when to intervene. most equine neonatologists would agree that removal of the fetus from the uterus before its attainment of readiness for birth is not desirable. one of the difficulties in determining fetal preparedness for birth is that prediction of parturition is difficult in these mares. many of the parameters used in normal mares are unreliable in the high-risk pregnant mare. one must have an accurate history of any previous gestation length in terms of days for the specific mare in question to allow a more accurate estimate of her usual gestational length. evaluation of the usual mammary gland parameters, including size, the presence of "wax," and alteration of electrolyte concentrations, is not generally predictive in the high-risk mare, for in the author's experience many of these mares have changes predictive of parturition for weeks before actual parturition. , this circumstance may be related to the observation that many high-risk pregnant mares, particularly those with placentitis, are presented for a primary complaint of early onset lactation. although pulmonary system maturity in human beings can be assessed with some degree of accuracy using measurement of lecithin/sphingomyelin ratios, this measurement-along with sphingomyelin, cortisol, and creatinine concentrations in the amnionic fluid-has proved to be of no benefit in the horse. [ ] [ ] [ ] amniocentesis carries a high risk of abortion in the horse, even with ultrasound guidance, and is not a clinically useful technique at this time. currently, no clear-cut guidelines are available as to when to intervene, but the presence of persistent fetal tachycardia or prolonged absence of fetal movements, including breathing movements, as determined by transabdominal ultrasound evaluation, should initiate discussion regarding the appropriateness of induction of parturition or elective cesarean section. the goal of induction or cesarean section is to remove a pregnancy that is threatening the survival of the dam with no thought to fetal survival or to remove the fetus from a threatening environment to improve its likelihood for survival. preterm induction is ill advised if fetal survival is desirable because of the limited ability to treat severely immature neonates. timing of intervention in these circumstances remains an art, not a science. the approach to management of the high-risk pregnancy is dictated to some degree by the exact cause for concern, but for many mares therapy is similar. many high-risk mares have placentitis, primarily caused by ascending bacterial or fungal infections originating in the region of the cervix. these infections can cause in utero sepsis or compromise the fetus by local elucidation of inflammatory mediators or altered placental function. , premature udder development and vaginal discharge are common clinical signs. treatment consists of administration of broad-spectrum antimicrobial agents and nonsteroidal antiinflammatory drugs (table - ). in the author's clinic, trimethoprim-sulfonamide drugs have been the antimicrobial of choice based on unpublished studies performed at the facility demonstrating increased concentration of these agents in the fetal fluids compared with penicillin and gentamicin. however, if culture and sensitivity results are available, one should institute directed therapy. nonsteroidal antiinflammatory agents such as flunixin meglumine are useful to combat alterations in prostaglandin balance that may be associated with infection and inflammation. although the efficacy of these agents is best when administered before the development of clinical signs, to date no detrimental effects have been reported in the fetus or dam when chronically used at low doses in well-hydrated patients. tocolytic agents and agents that promote uterine quiescence have been used and include altrenogest, isoxuprine, and clenbuterol. [ ] [ ] [ ] [ ] [ ] altrenogest usually is administered, although its need in late gestation has been challenged. the efficacy of isoxuprine as a tocolytic in the horse is unproven, and bioavailability of orally administered isoxuprine appears to be highly variable. the long-term use of clenbuterol is inadvisable because of receptor population changes associated with chronic use and its unknown effects on the fetus at this time. clenbuterol may be indicated during management of dystocia in preparation for assisted delivery or cesarean section. the intravenous form of clenbuterol is not currently available in the united states. one can use three additional strategies in managing high-risk pregnancy patients. in mares with evidence of placental dysfunction, with or without signs of fetal distress, the author provides intranasal oxygen supplementation in the hope of improving oxygen delivery to the fetus. intranasal oxygen insufflation of to l/min to the mare significantly increases pao and percent oxygen saturation of hemoglobin. because of the placental vessel arrangement of the horse, improvement of these two arterial blood gas parameters should result in improved oxygen delivery to the fetus. blood gas transport is largely independent of diffusion distance in the equine placenta, particularly in late gestation, and depends more on blood flow. information from other species cannot be extrapolated to the equine placenta because of its diffuse epitheliochorial nature and the arrangement of the maternal and fetal blood vessels within the microcotyledons. , umbilical venous po is to mm hg in the horse fetus, compared with to mm hg in the sheep, whereas the maternal uterine vein to umbilical vein po difference is near . also unlike the sheep, the umbilical venous po values decrease to mm hg in response to maternal hypoxemia and increase in response to maternal hyperoxia. [ ] [ ] [ ] vitamin e (tocopherol) is administered orally to some high-risk mares as an antioxidant. administration of large doses of vitamin e before traumatic brain injury improves neurologic outcome in experimental models and has been examined as possible prophylaxis for human neonatal encephalopathy. [ ] [ ] [ ] extrapolation of that information to the compromised equine fetus suggests that increased antioxidant concentrations in the fetus may mitigate some of the consequences of uterine and birth hypoxia, but no evidence is available to date demonstrating that protection occurs or that vitamin e accumulates in the fetus in response to supplementation of the mare. finally, many high-risk mares are anorectic or held off feed because of their medical condition. these mares are at particularly great risk for fetal loss because of their lack of feed intake, which alters prostaglandin metabolism. therefore one should administer . % to % dextrose in . % saline or water ( % dextrose) intravenously at maintenance fluid rates to these patients. perhaps the most important aspect of managing high-risk pregnancy mares is frequent observation and development of a plan. one should observe mares at least hourly for evidence of early-stage labor and should put them under constant video surveillance if possible. depending on the primary problem, the team managing the mare should develop a plan for handling the parturition once labor begins and for fetal resuscitation following delivery. any equipment that might be needed should be readily available stallside, and a call sheet, listing contact numbers for all involved, should be posted on or near the stall. the plan should include a decision as to how to handle a complicated dystocia, should it occur, with permission for general anesthesia and cesarean section obtained before the event so that time is not wasted. an important question to be posed to the owner at the outset is which is most important to the owner, the mare or the foal, for the answer may dictate the direction of the decision tree once labor begins. early recognition of abnormalities is of utmost importance for successful management of critically ill foals. to recognize the abnormal, one must know the normal. immediately following birth, foals effect several important physiologic and behavioral changes. chief among these changes is the adaptation of the cardiovascular and respiratory systems to extrauterine life. the normal transition of the respiratory tract involves opening closed part ii disorders of specific body systems alveoli and absorption of fluid from the airway, accomplished by a combination of breathing efforts, expiration against a closed glottis (grunting), and a change in sodium flux across the respiratory membrane from net secretion to net absorption. [ ] [ ] [ ] [ ] [ ] the transition from fetal to neonatal circulatory patterns requires resolution of the pulmonary hypertension present in the fetus, normally shunting blood flow through the lower resistance ductus arteriosus in the fetal state, to direct cardiac output to the pulmonary vasculature for participation in gas exchange. this change is achieved by the opening of alveoli, decreasing airway resistance and providing radial support for pulmonary vessels, functional closure of the ductus arteriosus, and increasing the oxygen tension in the lung, reversing pulmonary vasoconstriction mediated by hypoxia. , pulmonary tree vasodilators (prostacyclin, nitric oxide [no] ) and vasoconstrictors (endothelin- , leukotrienes) play apparently well-coordinated, but as yet not fully elucidated, roles. in the normal newborn this change is smooth and rapid. these critical events are undermined by factors such as inadequate lung development, surfactant deficiency (primary or secondary), viral or bacterial infection, placental abnormalities, in utero hypoxia, and meconium aspiration. spontaneous breathing should begin in the neonate within minute of birth, many foals attempt to breathe as their thorax clears the pelvic canal. during the first hour of life, the respiratory rate of a healthy foal can be as high as breaths per minute but should decrease to to breaths per minute within a few hours. similarly, the heart rate of a healthy newborn foal has a regular rhythm and should be at least beats/min at the first minute. , one usually can auscultate a continuous murmur over the left side of the heart, although its loudness may vary with position. this murmur is thought to be associated with some shunting through the ductus arteriosus. one may auscultate variable systolic murmurs, thought to be flow murmurs, during the first week of life. one should investigate more thoroughly murmurs that persist beyond the first week of life in an otherwise healthy foal, along with any murmur associated with persistent hypoxia. auscultation of the thorax shortly after birth reveals a cacophony of sounds as airways open and fluid is cleared. end-expiratory crackles are consistently audible in the dependent lung during and following lateral recumbency. for a normal newborn foal to appear slightly cyanotic during this initial adaptation period is not unusual, but this should resolve within minutes of birth. the equine fetus, as do all fetuses, exists in a moderately hypoxic environment, but the equine fetus has a greater partial pressure of oxygen, around mm hg. because the fetus is well adapted to low oxygen tensions, cyanosis is rarely present in newborn foals once adaption occurs, even those with low oxygen tensions. although in many species the fetal blood oxygen affinity is greater than the maternal blood, in the equine fetus the oxygen affinity of its hemoglobin is only about mm hg greater than the maternal blood because of decreased levels of , -diphosphoglycerate compared with other species. the result is enhanced oxygen unloading in the equine fetus compared with others. , -diphosphoglycerate concentration increases after birth in the foal and reaches mature levels by to days of age. the major blood adaptation of the equine fetus to chronic hypoxia is an increase in packed cell volume of up to %, increasing the oxygen content of the blood as compensation for decreased oxygen delivery at the placenta. a larger than expected packed cell volume in any newborn foal should alert the clinician for possible sequelae from chronic hypoxia. the presence of significant cyanosis that persists should prompt the clinician to evaluate the foal thoroughly for cardiac anomalies resulting in significant right-to-left shunting or separated circulations, such as transposition of the great vessels. the chest wall of the foal is compliant, facilitating passage through the pelvic canal during parturition. this compliance requires that the foal actively participate in inspiration and expiration with several potential consequences. first, restriction of the thorax or the abdomen can result in impaired ventilation, which can occur easily when one restrains a foal and may result in spuriously abnormal arterial blood gas values (see the discussion on arterial blood gas evaluation, respiratory diseases associated with hypoxemia in the neonate). second, foals with primary pulmonary parenchymal disease resulting in poorly compliant lungs develop paradoxical chest wall motion, with the thorax moving inward during inspiration. [ ] [ ] [ ] [ ] the work of breathing can increase greatly, resulting in respiratory failure because of respiratory muscle fatigue. a foal that appears suddenly to improve a previously abnormal respiratory rate and pattern may in fact be in greater respiratory difficulty because of fatigue. one can observe a reduction in respiratory rate or abnormal breathing pattern in premature/dysmature foals or foals subjected to peripartum hypoxia/asphxia. although the genesis of these patterns is not understood fully, cheyne-stokes (lengthy periods of apnea interrupted by short breaths that wax and wane in depth), cluster (short periods of apnea interspersed with long periods of breathing), and biot's breathing (periods of apnea and breathing with no discernible pattern) may occur in these cases. foals attempting to maintain an adequate lung volume expire against a partially closed glottis, called valsalva's maneuver, producing an audible grunt. foals are normally nonresponsive while in the birth canal but should respond to stimulation immediately after birth. the lack of responsiveness while in the birth canal has lead to presumption of fetal death during dystocia. because of this, one should attempt other tests before determining that a foal is dead intrapartum. one possibly may detect pulses in the tongue, neck, or any presented limbs or palpate the thorax for a heartbeat. in the author's facility, nasotracheal intubation of the foal combined with measurement of co tensions in the exhaled gas aids practitioners in cases where they can reach the nose. nasotracheal intubation of foals under these circumstances actually can be performed readily with minimal practice. having long endotracheal tubes available of several different diameters ( to mm outer diameter) with an inflatable cuff is important. one can pass the tube blindly using a finger in one nostril for guidance and can check the position frequently by palpation of the throatlatch region. one inflates the cuff and begins manual ventilation with % oxygen or room air using an ambu-bag or equivalent. one can obtain continuous measurement of co tension using a capnograph or single-use disposable end-tidal co monitor attached to the ambu-bag or the nasotracheal tube. in a dead foal the end-tidal co measurement will be negligible after the first to breaths. one must ensure tube placement and seal integrity and allow for multiple breaths. some co will "wash out" with the first few breaths and can result in false hope initially. end-tidal co varies in living intrapartum foals, depending on cardiac output and ventilation frequency, but should be consistently greater than mm hg and is usually closer to mm hg. once one establishes manual ventilation of a living foal, one must continue ventilation until the foal is delivered satisfactorily. the author has resuscitated and maintained many foals successfully in this manner throughout induction of general anesthesia in the mare and cesarean section delivery of the foal. the nasotracheal tube also provides a convenient site for administration of intratracheal medications such as epinephrine used for extrauterine intrapartum resuscitation of the foal. the reader is cautioned that intratracheal epinephrine increases endtidal co measurements transiently, even in a dead foal, because of local actions on tissues. one should allow a washout period after intratracheal administration of epinephrine. the righting reflex is present as the foal exits the birth canal, as is the withdrawal reflex. cranial nerve responses are intact at birth, but the menace response may take as long as weeks to develop fully. one should not consider lack of a menace reflex diagnostic of visual deficits in the newborn foal. within an hour of birth the normal foal will demonstrate auditory orientation with unilateral pinna control. the normal pupillary angle is ventromedial in the newborn foal; this angle gradually becomes dorsomedial over the first month of life. foals should begin attempting to stand shortly after birth and should be able to achieve this on their own within hours of birth. the normal newborn foal has a suck reflex shortly after birth and should be searching for an udder even before it stands. the expectation is that a normal foal will be sucking from the dam unaided by hours post partum; many foals are overachievers and will be sucking well before this time. the normal foal may defecate shortly after standing but may not attempt defecation until after it first successfully sucks from the dam. urination varies more, with filly foals usually urinating before colt foals, but both usually do not urinate for several hours following birth, up to hours for some colts. for colt foals to fail to drop their penises when urinating over the first few days of life is not unusual. the gait of the newborn foal is hypermetric and the stance is base wide. extreme hypermetria of the forelimbs, usually bilateral but occasionally unilateral, has been observed in some foals and is associated with perinatal hypoxic/ischemic insults, but this gait abnormality usually resolves without specific therapy within a few days. spinal reflexes tend to be exaggerated, whereas the crossed extensor reflex may not be fully present until weeks of age. foals also exhibit an exaggerated response to external stimuli (noise, sudden visual changes, touch) for the first few weeks of life. foals are not bonded strongly to their mother for the first few weeks of life and will follow any large moving object, including other horses and human beings. orphan foals bond with surrogate mothers until they are several months of age; their primary motivation appears to be appetite. conversely, mares strongly bond with their foals shortly after parturition; the process begins once the chorioallantois ruptures and is driven more by olfaction and taste than by vision or hearing. interference with this process, by medical intervention or excessive owner manipulation of the foal, can disrupt normal bonding and result in foal rejection by the dam. most newborn foals make the transition to extrauterine life easily. however, for those in difficulty, recognition of the condition immediately and institution of appropriate resuscitation is of utmost importance. a modified apgar scoring system has been developed as a guide for initiating resuscitation and assessing probable level of fetal compromise (table - ). one also must at least perform a cursory physical examination before initiating resuscitation, for issues of humaneness are associated with with serious problems such as severe limb contracture, microophthalmia, and hydrocephalus, among others. the initial assessment begins during presentation of the fetus. although the following applies primarily to attending the birth of a foal from a high-risk pregnancy, one can perform quiet and rapid evaluation during any attended birth. the goal in a normal birth with a normal foal is to disturb the bonding process minimally. this goal also applies to high-risk parturitions, but some disruption of normal bonding is inevitable. the lead clinician should control tightly the number of persons attending, and the degree of activity surrounding, the birth. one should evaluate the strength and rate of any palpable peripheral pulse and should evaluate the apical pulse as soon as the chest clears the birth canal. bradycardia (pulse < beats/min) is expected during forceful contractions, and the pulse rate should increase rapidly once the chest clears the birth canal. persistent bradycardia is an indication for rapid intervention. the fetus is normally hypoxemic compared with the newborn foal, and this hypoxemia is largely responsible for the maintenance of fetal circulation by generation of pulmonary hypertension. the fetus responds to conditions producing more severe in utero hypoxia by strengthening the fetal circulatory pattern, and the neonate responds to hypoxia by reverting to the fetal circulatory pattern. during a normal parturition, mild asphyxia occurs and results in fetal responses that pave the way for a successful transition to extrauterine life. if more than mild transient asphyxia occurs, the fetus is stimulated to breathe in utero; this is known as primary asphyxia. if the initial breathing effort resulting from the primary asphyxia does not correct the asphyxia, a second gasping period occurs in several minutes, known as the secondary asphyxia response. if no improvement in asphyxia occurs during this period, the foal enters secondary apnea, a state that is irreversible except with resuscitation. therefore the first priority of neonatal resuscitation is establishing an airway and breathing pattern. one should assume that foals not spontaneously breathing are in secondary apnea and should clear the airway of membranes as soon as the nose is presented. if meconium staining is present, one should suction the airway before delivery of the foal is completed and before the foal breathes spontaneously. one should continue to the trachea if aspiration of the nasopharynx is productive. overzealous suctioning worsens bradycardia as it worsens hypoxia. one should stop suctioning once the foal begins breathing spontaneously, as hypoxia will worsen with continued suction. if the foal does not breathe or move spontaneously within seconds of birth, one should begin tactile stimulation. if tactile stimulation fails to result in spontaneous breathing, one immediately should intubate the foal and manually ventilate the foal using an ambu-bag or equivalent. one can use mouth-to-nose ventilation if nasotracheal tubes and an ambu-bag are not available. the goal of this therapy is to reverse fetal circulation, and hyperventilation with % oxygen is the best choice for this purpose. however, recent evidence suggests that no clinical disadvantages are apparent in using room air for ventilation of asphyxiated human neonates rather than % oxygen. , human infants resuscitated with room air recovered more quickly than those resuscitated with % oxygen in one study as assessed by apgar scores, time to the first cry, and the sustained pattern of breathing. in addition, neonates resuscitated with % oxygen exhibited biochemical findings reflecting prolonged oxidative stress, present even after weeks of postnatal life, which did not appear in the group resuscitated with room air. thus the current accepted recommendations for using % oxygen in the resuscitation of asphyxiated neonates needs further discussion and investigation. , almost % of foals requiring resuscitation respond to hyperventilation alone and require no additional therapy. one can initiate nasotracheal intubation while the foal is in the birth canal if the foal will not be delivered rapidly, such as with a difficult dystocia. this technique is "blind" and requires some practice but may be beneficial and lifesaving. once spontaneous breathing is present, one apgar score in the foal should provide humidified oxygen via nasal insufflation at to l/min. one should initiate cardiovascular support in the form of chest compression if the foal remains bradycardic despite ventilation and a nonperfusing rhythm is present. one should make sure the foal is on a hard surface in right lateral recumbency with the topline against a wall or other support. approximately % of foals are born with fractured ribs and an assessment for the presence of rib fractures is in order before initiating chest compressions. palpation of the ribs identifies many of these fractures, which usually are multiple and consecutive on one side of the thorax and located in a relatively straight line along the part of the rib with the greatest curvature dorsal to the costochondral junction. unfortunately, ribs to frequently are involved, and their location over the heart can make chest compression a potentially fatal exercise. auscultation over the ribs during breathing results in a recognizable click, identifying rib fractures that may have escaped detection by palpation. one should initiate drug therapy if a nonperfusing rhythm persists for more than to seconds in the face of chest compression. epinephrine is the first drug of choice (table - ) . practitioners pose various arguments regarding the best dose and the best frequency of administration for resuscitation. however, most of the data are acquired from human cardiac arrest studies and are not strictly applicable to the equine neonate because the genesis of the cardiovascular failure is different. , vasopressin is gaining attention as a cardiovascular resuscitation drug, and although the author has used this drug in resuscitation and as a pressor, experience is limited at this time. the author does not use atropine in bradycardic newborn foals because the bradycardia usually is caused by hypoxia, and if the hypoxia is not corrected, atropine can increase myocardial oxygen debt. the author also does not use doxapram because it does not reverse secondary apnea, the most common apnea in newborns. because birthing areas are generally cold, one should dry the foal and place it on dry bedding once resuscitation is complete. the fetus has some homeothermic mechanisms, but its size in relation to its mother and its position within her body means that it is in effect a poikilotherm. the body temperature of the foal generally reflects that of its environment, namely its mother, although the human fetal temperature directly measured at cesarean section, induction of labor, or during labor is approximately . °c higher than the mothers. , adaptation from poikilothermy to homeothermy normally takes place rapidly following birth. the fetus is capable of nonshivering thermogenesis, primarily through the oxidation of brown fat reserves, but this type of thermogenesis is inhibited in utero, probably by placental prostaglandin e and adenosine. , immediately after birth the foal must adapt to independent thermoregulation. local physical factors, including ambient temperature and humidity, act to induce cold stress, and the newborn must produce heat by metabolic activity. in response to the catecholamine surge associated with birth, uncoupling of oxidative phosphorylation occurs within mitochondria, releasing energy as heat. this nonshivering thermogenesis is impaired in newborns undergoing hypoxia or asphyxiation and in those that are ill at birth. infants born to mothers sedated with benzodiazepines are affected similarly, a consideration in the choice of sedative and preanesthetic medications in mares suffering dystocia or part ii disorders of specific body systems undergoing cesarean section. [ ] [ ] [ ] heat losses by convection, radiation, and evaporation are high in most areas where foals are delivered, resuscitated ,and managed, and one must take care to minimize cold stress in the newborn and the critically ill foal. supplementary heat, in the form of radiant heat lamps or warm air circulating blankets, may be required. one should use fluid therapy conservatively during postpartum resuscitation, for the neonate is not volume depleted unless excessive bleeding has occurred. some compromised newborn foals are actually hypervolemic. fluid therapy of the neonate is discussed in more detail later in this chapter. because the renal function of the equine neonate is substantially different from the adult, one cannot simply scale down fluid therapy from adult therapy. [ ] [ ] [ ] if intravenous fluids are required for resuscitation and blood loss is identified, administration of ml/kg of a non-glucose-containing polyionic isotonic fluid over minutes (about l for a -kg foal) once intravenous access is established can be effective. the author stresses non-glucose-containing polyionic intravenous fluids because hyperglycemia, but not hypoglycemia, immediately after fetal or neonatal asphyxia interfered with the recovery of brain cell membrane function and energy metabolism in neonatal piglets in one recent study. these findings suggest that post-hypoxic-ischemic hyperglycemia is not beneficial and might even be harmful in neonatal hypoxic-ischemic encephalopathy. indications for this shock bolus therapy include poor mentation, poorly palpable peripheral pulses, and the development of cold distal extremities, compatible with hemorrhagic shock. one should reassess the patient after the initial bolus and administer additional boluses as necessary. ideally, one should follow up on blood pressures and ecg readings and initiate appropriate pressor therapy if needed. again, these procedures are discussed in detail later in the chapter. one can administer glucose-containing fluids after resuscitation at a rate of to mg/kg/min (about ml/hr of % dextrose or ml/hr of % dextrose) to the average -kg foal, particularly in the obviously compromised foal. this therapy is indicated to help resolve metabolic acidosis, to support cardiac output because myocardial glycogen stores likely have been depleted, and to prevent postasphyxial hypoglycemia. under normal conditions, the fetal-to-maternal blood glucose concentration gradient is % to % in the horse, and glucose is the predominant source of energy during fetal development. , glucose transport across the placenta is facilitated by carrier receptors (glucose transporter [glut] receptors), and a direct relationship exists between maternal and fetal blood glucose concentration when maternal glucose is in the normal range. the glut receptors in the placenta are stereospecific, saturable, and energy independent. although the enzyme kinetics for glut isoform suggest that they are not saturable under conditions of euglycemia, equine maternal hyperglycemia results in increased fetal glucose concentration to a plateau point, likely caused by glut saturation. at term, the net umbilical uptake of glucose is to mg/kg/min, with most of the glucose being used by the brain and skeletal muscle. [ ] [ ] [ ] the fetus only develops gluconeogenesis under conditions of severe maternal starvation. a certain percentage of the delivered glucose is used to develop large glycogen stores in the fetal liver and cardiac muscle in preparation for birth, and at birth the foal liver produces glucose at a rate of to mg/ kg/min by using these stores. fetal glycogen stores also are built using the substrates lactate, pyruvate, and alanine; fetal uptake of lactate across the placenta is about half that of glucose. , the transition to gluconeogenesis, stimulated by increased circulating catecholamine concentration from birth and by stimulation of glucagon release at the time the umbilical cord breaks takes to hours in the normal foal, and glycogenolysis supplies needed glucose until feeding and glucose production are accomplished. in the challenged foal, glycogen stores may have been depleted and gluconeogenesis delayed, so provision of glucose at rates similar to what the liver would normally produce during this period is requisite. persistent pulmonary hypertension (pph) also is known as reversion to fetal circulation or persistent fetal circulation, and its genesis lies in the failure of the fetus to make the respiratory and cardiac transition to extrauterine life successfully or reversion of the newborn to fetal circulatory patterns in response to hypoxia or acidosis. differentiating this problem from other causes of hypoxemia in the newborn requires some investigation, and multiple serial arterial blood gas analyses are necessary to confirm suspicion of this problem (see the section on arterial blood gas analysis, respiratory diseases associated with hypoxemia in the neonate). however, one should suspect the condition in any neonate with hypercapnic hypoxemia that persists and worsens; these foals are in hypoxemic respiratory failure. the fetal circulatory pattern, with pulmonary hypertension and right-to-left shunting of blood through the patent foramen ovale and ductus arteriosus, is maintained in these cases. pulmonary vascular resistance falls at delivery to about % of fetal values, while pulmonary blood flow increases accordingly. early in the postnatal period these two changes balance each other, and mean pulmonary and systolic pressures remain increased for several hours. systolic pulmonary pressures can remain equivalent to systemic pressure for up to hours of age in human infants, although diastolic pulmonary pressures are well below systemic diastolic pressures by hour. mean pulmonary artery pressures fall gradually over the first hours. the direct effects of lung expansion and increasing alveolar oxygen tension probably provide the initial stimulus for pulmonary arteriolar dilation and partly result from direct physical effects, but vasoactive substances are released in response to physical forces associated with ventilation, for example prostacyclin. other vasoactive mediators thought to play a role in regulating pulmonary arteriolar tone include no, prostaglandins d and e , bradykinin, histamine, endothelin- , angiotensin ii, and atrial natriuretic peptide. the increase in alveolar and arterial oxygen tensions at birth is required for completion of resolution of pulmonary hypertension. much of this increase is thought to be mediated by no, evidence for this being the parallel increase during gestation of the pulmonary vasodilation response to hyperoxia and the increase in no synthesis. however, inhibition of no synthesis does not eliminate the initial decrease in pulmonary artery resistance occurring because of opening of the airways. when these mechanisms fail, one can recognize pph. right-to-left shunting within the lungs and through patent fetal conduits occurs and can result from many factors, including asphyxia and meconium aspiration, but in many cases the precipitating trigger is unknown. inappropriately decreased levels of vasodilators (no) and inappropriately increased levels of vasoconstrictors (endothelin- ) currently are being examined as potential mechanisms. chronic in utero hypoxia and acidosis may result in hypertrophy of the pulmonary arteriolar smooth muscle. in these cases, reversal of pph can be difficult and cannot be achieved rapidly. treatment of pph is twofold: abolishment of hypoxia and correction of the acidosis, for both abnormalities only bolster the fetal circulatory pattern. initial therapy is provision of oxygen intranasally at to l/min. some foals respond to this therapy and establish neonatal circulatory patterns within a few hours. failure to improve or worsening of hypoxemic respiratory failure following intranasal oxygen administration should prompt intubation and mechanical ventilation with % oxygen. this serves two purposes, one diagnostic and one therapeutic. ventilation with % oxygen may resolve pph and, if intrapulmonary shunt and altered ventilation-perfusion relationships are causing the hypoxic respiratory failure, arterial oxygen tension (pao ) should exceed mm hg under these conditions. failure to improve pao suggests pph or large right-to-left extrapulmonary shunt caused by congenital cardiac anomaly. the vasodilators prostacyclin and telazoline (an α-blocking vasodilator) cause pulmonary vasodilation in human infants with pph, but the effects on oxygenation vary and the sideeffects (tachycardia, severe systemic hypotension) are unacceptable. recognition of no as a potent dilator of pulmonary vessels has created a significant step forward in the treatment of these patients, for inhaled no dilates vessels in ventilated portions of the lung while having minimal effects on the systemic circulation. based on evidence presently available, use of inhaled no in an initial concentration of about ppm in the ventilatory gas seems reasonable for term and near-term foals with hypoxic respiratory failure and pph that fails to respond to mechanical ventilation using % oxygen alone. , the author has used this approach in the clinic, administering a range of to ppm no with success. hypoxic ischemic encephalopathy (hie), currently referred to as neonatal encephalopathy in the human literature, is one systemic manifestation of a broader syndrome of perinatal asphyxia syndrome (pas), and management of foals with signs consistent with a diagnosis of hie requires the clinician to examine other body systems fully and to provide therapy directed at treating other involved systems. although pas primarily manifests as hie, the gastrointestinal tract and kidneys frequently are affected by peripartum hypoxia/ischemia/ asphyxia, and one should expect complications associated with these systems. hypoxic ischemic encephalopathy also may affect the cardiovascular and respiratory systems, and one also may encounter endocrine disorders in these patients. hypoxic ischemic encephalopathy has been recognized as one of the most common diseases of the equine neonate for generations. , , in the past hie has been known as dummy foal syndrome and as neonatal maladjustment syndrome. the designation hie, although not perfect, attempts to describe the syndrome in terms of the suspected underlying pathophysiology. a wide spectrum of clinical signs is associated with hie and can range from mild depression with loss of the suck reflex to grand mal seizure activity. typically, affected foals are normal at birth but show signs of central nervous system abnormalities within a few hours after birth. some foals are obviously abnormal at birth, and some do not show signs until hours of age. hypoxic ischemic encephalopathy commonly is associated with adverse peripartum events, including dystocia and premature placental separation, but a fair number of foals have no known peripartum period of hypoxia, suggesting that these foals result from unrecognized in utero hypoxia (box - ). severe maternal illness also may result in foals born with pas. in human beings, ascending placental infection now is suspected of being a major contributor to neonatal encephalopathy in infants, and the incidence of neonatal encephalopathy increases with the presence of maternal fever, suggesting a role for maternal inflammatory mediators. the underlying pathophysiologic details of hie in the foal are unknown, and to date accurate experimental models of hie and pas in the foal have not been described. however, a great deal of attention has been paid to peripartum hypoxia/asphyxia by human counterparts because the effects of adverse peripartum events in the human neonate have far ranging implications for the affected human neonate and for society. therefore equine neonatologists have long looked to human studies and models of the human disease for understanding of the syndrome in the equine neonate. perinatal brain damage in the mature fetus usually results from severe uterine asphyxia caused by an acute reduction of uterine or umbilical circulation. the fetus responds to this challenge by activation of the sympathetic adrenergic nervous system, causing a redistribution of cardiac output that favors the central organs: brain, heart, and adrenal glands. , if the hypoxic insult continues, the fetus reaches a point beyond which it cannot maintain this centralization of circulation, cardiac output falls, and cerebral circulation diminishes. the loss of oxygen results in a substantial decrease in oxidative phosphorylation in the brain with concomitant decreased energy production. the na + /k + pump at the cell membrane cannot maintain the ionic gradients, and the membrane potential is lost in the brain cells. in the absence of the membrane potential, calcium flows down its large extracellular/intracellular concentration gradient through voltage-dependent ion channels into the cell. this calcium overload of the neuron leads to cell damage by activation of calcium-dependent proteases, lipases, and endonucleases. protein biosynthesis is halted. calcium also enters the cells by glutamate-regulated ion channels as glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles following anoxic cellular depolarization. once the anoxic event is over, protein synthesis remains inhibited in specific areas of the brain and returns to normal in less vulnerable areas of the brain. loss of protein synthesis appears to be an early indicator of cell death caused by the primary hypoxic/anoxic event. a second wave of neuronal cell death occurs during the reperfusion phase and is thought to be similar to classically described postischemic reperfusion injury in that damage is caused by production of and release of oxygen radicals, synthesis of no, and inflammatory reactions. additionally, an imbalance between excitatory and inhibitory neurotransmitters occurs. part of the secondary cell death that occurs is thought to be caused by apoptosis, a type of programmed cell death termed cellular suicide. secondary cell death also is thought be caused by the neurotoxicity of glutamate and aspartate resulting again from increased intracellular calcium levels. , in human infants the distribution of lesions with hypoxic-ischemic brain damage following prenatal, perinatal, or postnatal asphyxia falls into distinct patterns depending on the type of hypoxia-ischemia rather than on postconceptual age at which the asphyxial event occurs. periventricular leukomalacia was associated with chronic hypoxia-ischemia, whereas the basal ganglia and thalamus were affected primarily in patients experiencing acute profound asphyxia, providing direct evidence that the nature of the event determines the severity and distribution of neurologic damage in human beings. these remarkably selective patterns of injury in children, with differential variability in the damage caused to regions anatomically located within millimeters of each other, resulted in the hypothesis that location within neurotransmitter-specific circuitry loops is important. this hypothesis has important implications in the design of neuroprotective strategies and therapies for neonates experiencing hypoxic-ischemic-asphyxial events. now the evidence is overwhelming that the excitotoxic cascade that evolves during hie extends over several days from the time of insult and is modifiable. , in brain injury, traumatic or hypoxic, the mechanisms underlying delayed tissue injury still are understood poorly. many believe that neurochemical changes, including excessive neurotransmitter release, are pivotal in the pathophysiology of secondary neuronal death. excitatory amino acid neurotransmitters and magnesium are known to play at least a minimal role in secondary cell death following brain injury; a fair body of literature regarding these factors has been generated over the last years. the activation of the n-methyl-d-aspartate (nmda) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury and is suspected to play a role in hie. [ ] [ ] [ ] mechanically injured neurons demonstrate a reduction of voltage-dependent mg + blockade of nmda current that can be restored partially by increasing extracellular mg + concentration or by pretreatment with calphostin c, a protein kinase c inhibitor. this finding suggested that administration of mg + to patients with brain injury could lead to improved outcome. subsequently, magnesium sulfate solution was shown to improve dramatically the immediate recovery of rats from hypoxia. however, although pretreatment with magnesium sulfate protected against hypoxic ischemic brain injury, postasphyxial treatment worsened brain damage in -day-old rats, suggesting an age-related response in the rat. delayed magnesium treatment of mature rats following severe traumatic axonal brain injury improved motor outcome when administered up to hours after injury, with early treatments providing the most benefit. maternal seizure in rats is associated with fetal histopathologic changes that are abolished by administration of magnesium sulfate to the mother, and magnesium sulfate has been demonstrated to protect the fetal brain from severe maternal hypoxia. clinical trials investigating the efficacy of magnesium treatment following hypoxia in infants are under way, with few reports currently in the medical literature. magnesium sulfate was used to treat nine infants after perinatal asphyxia in one study (no control group), and all children were neurologically normal at year of age. seizures did not occur in any of these children, nor were any adverse side effects noted. magnesium sulfate administration failed to delay the global impairment in energy metabolism after hypoxia ischemia, characteristic of severe brain damage, in newborn piglets; at hours after hypoxia ischemia, no difference could be found in the severity of injury in piglets treated with magnesium compared with piglets treated with placebo, suggesting magnesium may not be protective with severe acute injury. in developing countries, birth hypoxia frequently is associated with hie, and although this finding is attributed most frequently to inadequate obstetric care, poor nutrition also may play a role. red blood cell magnesium levels were measured in more than women in labor at a teaching hospital in south africa. fifty five of the women delivered infants with hie and had significantly lower levels of magnesium than controls; the infants with hie also had significantly lower magnesium levels than controls. the large majority ( of ) of the women giving birth to hie infants were from poor social circumstances, suggesting nutrition might play a role in some cases of hie, with maternal magnesium levels affecting outcome in the infants. the authors suggested an early pregnancy intervention study may help determine the role of magnesium in the pathogenesis of hie in human infants born to at-risk mothers. therapy for the various manifestations of hypoxiaischemia involves control of seizures, general cerebral support, correction of metabolic abnormalities, maintenance of normal arterial blood gas values, maintenance of tissue perfusion, maintenance of renal function, treatment of gastrointestinal dysfunction, prevention and recognition and early treatment of secondary infections, and general supportive care. control of seizures is important because cerebral oxygen consumption increases fivefold during seizures. one can use diazepam for emergency control of seizures (table - ). if diazepam does not stop seizures readily or one recognizes more than two seizures, then one should replace diazepam with phenobarbital given to effect. the half-life of phenobarbital can be long in the foal ( hours), and one should keep this in mind when monitoring neurologic function in these cases after phenobarbital administration (j.e. palmer, personal communication, ). earlystage, preseizure administration of phenobarbital has been advocated by some investigators for prevention of neonatal encephalopathy. however, one recent study in asphyxiated human infants demonstrated that early phenobarbital treatment was associated with a threefold increase in the incidence of subsequent seizures and consequently a trend toward increased mortality. seizures per se were associated with almost a twentyfold increase in mortality. their findings suggest that early phenobarbital administration may produce adverse rather than beneficial effects following asphyxia. because this was an observational study; the results need to be confirmed by appropriate randomized trials in similar clinical settings. if phenobarbital fails to control seizures, one may attempt phenytoin therapy. in cases of hie, one should avoid ketamine and xylazine because of their association with increased intracranial pressure. one must protect the foal from injury during a seizure and also ensure the patency of the airway to prevent the onset of negative pressure pulmonary edema or aspiration pneumonia. probably the most important therapeutic interventions are aimed at maintaining cerebral perfusion, which is achieved by careful titration of intravenous fluid support, neither too much nor too little (see fluid therapy in neonates) and judicious administration of inotropes and pressors to maintain adequate perfusion pressures (see pressor and inotrope therapy in neonates). cerebral interstitial edema is only truly present in the most severe cases , ; in most cases the lesion is intracellular edema and most of the classic agents used to treat cerebral interstitial edema (e.g., mannitol) are minimally effective treating cellular edema. occasionally the author uses thiamine supplementation in the intravenous fluids to support metabolic processes, specifically mitochondrial metabolism and membrane na + ,k + -atpases, involved in maintaining cellular fluid balance. , this therapy is rational and inexpensive but unproven in efficacy. only if cellular necrosis and vasogenic edema are present are drugs such as mannitol and dimethyl sulfoxide indicated, and again these cases are usually the most severely affected. in the author's clinic, practitioners rarely have used dimethyl sulfoxide in neonates for the last several years and have recognized no change in outcome by discontinuing its use. when the practitioners use intravenously administered dimethyl sulfoxide, they do so within the first hour after an acute asphyxial insult and use it primarily for its hydroxyl radical scavenging effects and its theoretical modulation of postischemic reperfusion injury. naloxone has been advocated for treating hie in human beings and in foals, [ ] [ ] [ ] perhaps based on a study suggesting that postasphyxia blood-brain barrier disruption was related causally to poor neurologic outcome in a lamb model of hie and that naloxone prevented disruption and neurologic dysfunction among those survivors with an intact blood-brain barrier. however, other studies have demonstrated that naloxone exacerbates hypoxic-ischemic brain injury in -day-old rats subjected to unilateral common carotid artery ligation and hypoxia. moreover, systemic acidosis and cellular edema were no different in naloxone-treated animals compared with animals treated with saline solution. the authors concluded that high doses of naloxone in fact may reduce the resistance of the fetus to hypoxic stress. the use of naloxone in human neonatal resuscitation remains controversial, for whether the contradictory effects are related to a reduction in acute neuronal swelling by osmotic effects or by a more direct receptor-mediated mechanism is currently unknown. naloxone is most effective in resuscitation of compromised human infants born to mothers addicted to drugs. some practitioners are using γ-aminobutyric acid adrenergic agonists to manage hie in foals, based on evidence showing neuroprotection when used in ischemia alone and combined with nmda antagonists. [ ] [ ] [ ] the author currently has no experience with these compounds and cannot comment regarding their efficacy in foals. regional hypothermia also is being investigated as a potential therapy for global hypoxia/ischemia; published data are consistent with the theory that cooling must be continued throughout the entire secondary phase of injury (about days) to be effective. experimentally, this approach has resulted in dramatic decreases in cellular edema and neuronal loss; its practical application remains to be demonstrated. despite a lack of consensus regarding the use of magnesium to treat infants with hie, the author has used magnesium sulfate infusion as part of the therapy for selected foals with hie for the past several years. the rationale is based primarily on the evidence demonstrating protection in some studies and a failure of any one study to demonstrate significant detrimental effects. the clinical impressions of the author to date suggest that the therapy is safe and may decrease the incidence of seizure in patients. the author administers magnesium sulfate as a constant rate infusion over hour after giving a loading dose. the author has continued the infusion for up to days without demonstrable negative effect beyond some possible trembling. given the current evidence, a -hour course of treatment may be effective and all that is necessary. postasphyxial treatment certainly may be beneficial in foals with hie, and maternal magnesium therapy may be beneficial in certain high-risk pregnancy patients. foals with pas often have a variety of metabolic problems including hypo-or hyperglycemia, hypo-or hypercalcemia, hypo-or hyperkalemia, hypo-or hyperchloremia, and varying degrees of metabolic acidosis. although one needs to address these problems, one should not forget the normal period of hypoglycemia that occurs postpartum and should not treat aggressively so as to avoid worsening the neurologic injury. foals suffering from pas also have frequent recurrent bouts of hypoxemia and occasional bouts of hypercapnia. intranasally administered oxygen is generally needed in these cases as a preventative therapy and as direct treatment, for the appearance of the abnormalities can be sporadic and unpredictable. additional respiratory support, particularly in those foals with centrally mediated hypoventilation and periods of apnea or abnormal breathing patterns, include caffeine (per os or per rectum) and positive pressure ventilation. caffeine is a central respiratory stimulant and has minimal side effects at the dosages used ( mg/kg loading dose; . mg/kg as needed). the author purchases whatever oral form of caffeine is available at the local convenience store or drug store and administers it dissolved in warm water per rectum. foals treated with caffeine have an increased level of arousal and are more reactive to the environment. adverse effects generally are limited to restlessness, hyperactivity, and mild to moderate tachycardia. mechanical ventilation of these patients can be rewarding and generally is required for less than hours. one must monitor and maintain blood ph within the normal range. metabolic alkalosis can develop in some of these foals and requires clinician tolerance of some degree of hypercapnia. ph is important in evaluation and consideration of alternatives for treatment. if the respiratory acidosis is not so severe as to affect the patient adversely (generally > mm hg), and the ph is within normal limits, the foal may tolerate hypercapnia. the goal is to normalize ph. foals with respiratory acidosis as compensation for metabolic alkalosis do not respond to caffeine. metabolic alkalosis in critically ill foals frequently is associated with electrolyte abnormalities, creating differences in strong ion balance. one handles this ph perturbation best by correcting the underlying electrolyte problem. maintaining tissue perfusion and oxygen delivery to tissues is a cornerstone of therapy for pas to avoid additional injury. one should maintain the oxygen-carrying capacity of the blood; some foals require transfusions to maintain a packed cell volume greater than %. adequate vascular volume is important, but one should take care to avoid fluid overload in the foal. early evidence of fluid overload is subtle accumulation of ventral edema between the front legs and over the distal limbs. fluid overload can result in cerebral edema, pulmonary edema, and edema of other tissues, including the gastrointestinal tract. this edema interferes with normal organ function and worsens the condition of the patient. one maintains perfusion by supporting cardiac output and blood pressure by judicious use of intravenous fluid support and inotrope/pressor support. the author does not target therapy to a specific systolic, mean, or diastolic pressure but monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for these patients to require pressor therapy is not unusual, but in some cases the hypoxic damage is sufficiently severe to blunt the response of the patient to the drugs. the kidney is a target for injury in patients with pph, and for renal compromise to play a significant role in the demise of these foals is not unusual. clinical signs of renal disease are generally referable to disruption of normal control of renal blood flow and tubular edema leading to tubular necrosis and renal failure. these foals have signs of fluid overload and generalized edema. one must balance urine output and fluid therapy in these cases to prevent additional organ dysfunction associated with edema. although evidence has accumulated that neither dopamine nor furosemide play a role in protecting the kidney or reversing acute renal failure, these agents can be useful in managing volume overload in these cases. [ ] [ ] [ ] the aim is not to drive oliguric renal failure into a highoutput condition but rather to enhance urine output. overzealous use of diuretics and pressors in these cases can result in diuresis requiring increased intravenous fluid support and can be counterproductive. the author's approach is more conservative. low doses of dopamine administered as a constant rate infusion of to µg/kg/min are usually effective in establishing diuresis by natriuresis. one should avoid large doses of dopamine (> µg/kg/min) because high doses can produce systemic and pulmonary vasoconstriction, potentially exacerbating pph. one can administer a bolus ( . to . mg/kg) or constant rate infusion ( . to . mg/kg/hr) of furosemide, but once furosemide diuresis is established, one must evaluate electrolyte concentrations and blood gas tensions frequently because potassium, chloride, and calcium losses can be considerable and because significant metabolic alkalosis can develop from strong ion imbalances. the author does not aim for urine production rates of ml/hr, as has been presented by other authors as a urine output goal for critically ill equine neonates. rather the author looks for urine output that is appropriate for fluid intake and does not attempt to drive urine output to an arbitrary goal by excessive fluid administration or pressor use. although the average urine output for a normal equine neonate is about ml/kg/hr (~ ml/hr for a -kg foal), these values were obtained from normal foals drinking a milk diet with a large free water component. [ ] [ ] [ ] the urine of normal newborn foals is dilute, reflecting the large free water load they incur by their diet. expecting critically ill foals to produce such large volumes of urine, particularly those on restricted diets or receiving total parenteral nutrition, is an exercise in futility, and manipulating fluid, pressor, or diuretic therapy in attempt to meet an artificial goal is inappropriate. fluid therapy in the critically ill neonate is discussed later in this chapter. one final caveat regarding renal dysfunction in pas is that one should perform therapeutic drug monitoring when it is available. many antimicrobial agents used to manage these cases, most notably the aminoglycosides, depend on renal clearance. aminoglycoside toxicity occurs in the equine neonate and exacerbates or complicates the management of renal failure originally resulting from primary hemodynamic causes. the author monitors aminoglycoside concentrations for -minute peak and -to -hour trough values in these cases and adjusts dosage and frequency of drug administration based on these results. the author considers a trough value of less than µg/dl as desirable for gentamicin and amikacin. foals with pas suffer from a variety of problems associated with abnormalities within the gastrointestinal tract. commonly they have ileus, recurrent excessive gastric reflux, and gas distention. these problems are exacerbated by constant feeding in the face of continued dysfunction and continued hypoxia. frequently, enteral feeding cannot meet their nutritional requirements, and partial or total parenteral nutrition is required. one must give special attention to passive transfer of immunity (see failure of passive transfer) and glucose homeostasis in these cases. although some practitioners use prokinetic agents as therapy for ileus in these cases, the author's approach is again more conservative. appearance of damage to the gastrointestinal tract can be subtle and lag behind other clinical abnormalities for days to weeks. low-grade colic, decreased gastrointestinal motility, decreased fecal output, and low weight gain are among the most common clinical signs of gastrointestinal dysfunction in these case, but more severe problems, including necrotizing enterocolitis and intussusception, have been associated with these cases. the return to enteral feeding must be slow in many of these cases. a currently debated topic is constant versus pulsed enteral feeding. [ ] [ ] [ ] the author uses pulsed feeding through an indwelling small-gauge feeding tube. in many foals these tubes stay in place for weeks and cause no problems as the foals are returned to their dams for sucking or are trained to drink from a bottle or bucket. foals with pas are also susceptible to secondary infection. treatment of recognized infection is covered under sepsis in this chapter. if infection is recognized in these patients after hospitalization, one should give attention to the likelihood of nosocomial infection and should direct antimicrobial therapy based on known nosocomial pathogens in the nicu and their susceptibility patterns until culture and sensitivity results become available. one should make repeat determinations of immunoglobulin g (igg) concentration; additional intravenous plasma therapy may be required. nosocomial infections are often rapidly overwhelming, and acute deterioration in the condition of a foal with pas should prompt a search for nosocomial infection. the prognosis for foals with pas is good to excellent when the condition is recognized early and aggressively treated in term foals. up to % of these neonates survive and go on to lead productive and useful athletic lives. [ ] [ ] [ ] [ ] the prognosis decreases with delayed or insufficient treatment and concurrent problems such as prematurity and sepsis. in human nicus the survival rates of low-gestationlength infants has increased dramatically since the s concurrent with improvements in obstetric and neonatal care. the now routine, well-validated use of antenatal steroid and artificial surfactant therapies has contributed greatly to the enhanced survival of this patient population, although the use of these particular therapies is not common or frequently indicated in the equine nicu. , however, with improved care, outcomes in the equine nicu population have improved also, with survival of premature patients in many nicus exceeding %. in the equine population, gestation length is much more flexible than in the human population; however, the definition of the term prematurity needs reexamination. traditionally, prematurity is defined as a preterm birth of less than days of gestation in the horse. given the variability of gestation length in the horse, ranging from days to more than days in some mares, a mare with a usual gestation length of days possibly could have a term foal at days, whereas a mare with a usual gestation length of days may have a premature foal at days, considered the normal gestation length. foals that are born postterm but are small are termed dysmature; a postmature foal is a postterm foal that has a normal axial skeletal size but is thin to emaciated. dysmature foals may have been classified in the past as small for gestational age and are thought to have suffered placental insufficiency, whereas postmature foals are usually normal foals that have been retained too long in utero, perhaps because of an abnormal signaling of readiness for birth, and have outgrown their somewhat aged placenta. postmature foals become more abnormal the longer they are maintained, also may suffer from placental insufficiency, and are represented best by the classic foal born to a mare ingesting endophyteinfested fescue. box - compares the characteristics of premature/dysmature foals with those of postmature foals. the causes of prematurity/dysmaturity/postmaturity include the causes of high-risk pregnancy presented in box - . additional causes include iatrogenic causes such as early elective induction of labor based on inaccurate breeding dates or misinterpretation of late-term colic or uterine bleeding as ineffective labor. most causes remain in the category of idiopathic, with no discernible precipitating factor. despite lack of an obvious cause, premature labor and delivery does not just happen, and even if undetermined, the cause may continue to affect the foal in the postparturient period. all body systems may be affected by prematurity, dysmaturity, and postmaturity, and thorough evaluation of all body systems is necessary. respiratory failure is common in these foals, although the cause usually is not surfactant deficiency. immaturity of the respiratory tract, poor control of respiratory vessel tone, and weak respiratory muscles combined with poorly compliant lungs and a greatly compliant chest wall contribute to respiratory failure in these cases. most require oxygen supplementation and positional support for optimal oxygenation and ventilation. one must extend effort to maintain these "floppy foals" in sternal recumbency. some foals may require mechanical ventilation. these foals also require cardiovascular support but are frequently unresponsive to commonly used pressors and inotropes: dopamine, dobutamine, epinephrine, and vasopressin. careful use of these drugs and judicious intravenous fluid therapy are necessary. the goal should not be one of achieving specific pressure values (e.g., mean arterial pressure of mm hg) but of adequate perfusion. renal function, reflected in low urine output, is frequently poor initially in these cases because of delay in making the transition from fetal to neonatal glomerular filtration rates. the delay can result from true failure of transition or from hypoxic/ischemic insult. one should approach fluid therapy cautiously in these cases; initial fluid restriction may be in order to avoid fluid overload. many premature/dysmature/postmature foals have suffered a hypoxic insult and have all of the disorders associated with pas, including hie. treatment is similar to that of term foals with these problems. these foals also are predisposed to secondary bacterial infection and must be examined frequently for signs consistent with early sepsis or nosocomial infection. the gastrointestinal system of these foals is not usually functionally mature, which may result from a primary lack of maturity or from hypoxia. dysmotility and varying degrees of necrotizing enterocolitis are common. one commonly encounters hyperglycemia and hypoglycemia. hyperglycemia generally is related to stress, increased levels of circulating catecholamines, and rapid progression to gluconeogenesis, whereas hypoglycemia is associated with diminished glycogen stores, inability to engage gluconeongenesis, sepsis, and hypoxic damage. immature endocrine function is present in many of these foals, particularly regarding the hypothalamic-pituitary-adrenal axis, and contributes to metabolic derangements. , one should delay enteral feeding when possible until the foal is stable regarding metabolic and cardiorespiratory parameters. on intiating enteral feeding, one should provide small volumes initially and slowly increase the volume over several days. one frequently encounters musculoskeletal problems, particularly in premature foals, that include significant flexor laxity and decreased muscle tone. postmature foals frequently are affected by flexure contracture deformities, most likely because of decreased intrauterine movement as they increase in size. premature foals frequently exhibit decreased cuboidal bone ossification that predisposes them to crush injury of the carpal and tarsal bones if weight bearing is not strictly controlled. physical therapy in the form of standing and exercise is indicated in the management of all these problems, but one should take care to ensure that the patient does not fatigue or stand in abnormal positions. bandaging of the limbs is contraindicated because this only increases laxity, although light bandages over the fetlock may be necessary to prevent injury to that area if flexor laxity is severe. the foals are predisposed to angular limb deformity and must be observed closely and frequently for this problem as they mature. the overall prognosis for premature/dysmature/ postmature foals remains good with intensive care and good attention to detail. many of these foals (up to %) survive and become productive athletes. complications associated with sepsis and musculoskeletal abnormalities are the most significant indicators of poor athletic outcome. the last years have seen an explosion of new therapeutic agents purportedly useful for treating sepsis. unfortunately, clinical trials investigating these new therapies have failed to demonstrate a positive effect, have shown negative results, or have resulted in diametrically opposed study results, one showing a benefit and another showing no benefit or a detrimental effect. on a positive note, the survival rate of foals being treated for sepsis has improved. work was done regarding foal diseases and their treatment in the s, but the field did not attract much serious attention until the s. since that time almost every major veterinary college and many large private referral practices have constructed nicus or their equivalent. next to hypoxic ischemic asphyxial syndromes, sepsis is the number one reason for presentation and treatment at these facilities. neonatal septicemia of the horse has been the subject of three international workshops, - and a perinatology lecture covering some aspect of neonatal sepsis has been presented at almost every large continuing education meeting attended by equine veterinarians. concensus criteria conferences in the early s defined sepsis and septic shock for human beings. , sepsis was defined as the systemic response to infection manifested by two or more of the following conditions as a result of infection: a) temperature > °c or < °; b) heart rate > beats/min; c) respiratory rate > breaths per minute or paco < torr; and d) white blood cell count > , cell/µl, < , cell/µl, or > % immature (band) forms. septic shock was defined as sepsis induced hypotension or the requirement for vasopressors/ionotropes to maintain blood pressure despite adequate fluid resuscitation along with the presence of perfusion abnormalities that may include lactic acidosis, oliguria, or acute alteration in mental status. these definitions are broadly acceptable and applicable to neonatal sepsis in foals, and many of the treatment modalities in human medicine have been applied in some manner to the equine neonatal patient. additional definitions that have come into vogue that are actually useful at times, include the following: sirs, the systemic inflammatory response system; mods, multiple organ system dysfuction; and mofs, multiple organ failure syndrome. (sirs is sick, mods is sicker, and mofs is dying.) the compensatory response syndrome (cars) ideally balances sirs and keeps it from becoming detrimental. if balance is achieved, recovery is possible. imbalance progresses to septic shock, mods, and mofs. in horses, mods is manifested most commonly as renal failure, hepatic failure, central nervous system dysfunction, and disseminated intravascular coagulation. managing the septic patient involves early recognition of all the potential alphabet combinations and supporting the patient or intervening in the face of multiple clinical consequences, termed chaos (cardiovacular compromise; homeostasis; apoptosis; organ dysfunction; suppression of the immune system). inflammatory mediators are involved in all these processes and can be beneficial or detrimental, depending on timing and opposing responses. neutrophils, platelets, lymphocytes, macrophages, and endothelial cells are involved, and the implicated inflammatory molecules grow daily in numbers. sepsis in the foal initially can be subtle, and the onset of clinical signs varies depending on the pathogen involved and the immune status of the foal. for the purposes here, the discussion is limited to bacterial sepsis, but the foal also is susceptible to viral and fungal sepsis, which can appear similar to bacterial sepsis. failure of passive transfer (fpt) of immunity can contribute to the development of sepsis in a foal at risk. , testing for and treating fpt has received attention in the veterinary literature. it remains true, however, that foals presented to nicus that have an ultimate diagnosis of sepsis have fpt. , the current recommendation is that foals have igg levels greater than or equal to mg/dl for passive transfer to be considered adequate. other risk factors for the development of sepsis include any adverse advents at the time of birth, maternal illness, or any abnormalities in the foal. although the umbilicus frequently is implicated as a major portal of entry for infectious organisms in the foal, the gastrointestinal tract may be the primary site of entry. other possible portals of entry include the respiratory tract and wounds. early signs of sepsis include depression, decreased suck reflex, increased recumbency, fever, hypothermia, weakness, dysphagia, failure to gain weight, increased respiratory rate, tachycardia, bradycardia, injected mucous membranes, decreased capillary refill time, shivering, lameness, aural petechia, and coronitis. if sepsis is recognized early, patients with sepsis may have a good outcome, depending on the pathogen involved. gram-negative sepsis remains the most commonly diagnosed, but increasingly gram-positive septicemia is being recognized. foals in intensive care units and at referral hospitals have an additional risk of nosocomial infection. an attempt to isolate the organim involved early in the course of the disease becomes important. if possible, one should obtain blood cultures, and if localizing signs are present, one should obtain samples as deemed appropriate. cultures should be aerobic and anaerobic. recently, work has been done evaluating real-time polymerase chain reaction technology in sepsis in the foal as a means of identifying causative organisms. , until one obtains antimicrobial sensitivity patterns for the pathogen involved, one should initiate broad-spectrum antimicrobial therapy (table - ). intravenously administered amikacin and penicillin are good first-line choices, but one should monitor renal function closely. other first-line antimicrobial choices might include high-dose ceftiofur sodium or ticarcillin/clavulanic acid. one should treat failure of passive transfer if present. one should provide intranasal oxygen insufflation at to l/min even if hypoxemia is not present to decrease the work of breathing and provide support for the increased oxygen demands associated with sepsis. should arterial blood gas analysis reveal significant hypoventilation, one may administer caffeine orally or per rectum to increase central respiratory drive. mechanical ventilation may be necessary in cases of severe respiratory involvement such as with acute lung injury or acute respiratory distress syndrome. if the foal is hypotensive, one may administer pressor agents or inotropes by constant rate infusion (table - ) . inotrope and pressor therapy generally is restricted to referral centers where these drugs can be given as constant rate infusions and blood pressure can be monitored closely. some practitioners use nonsteroidal antiinflammatory agents and, in specific circumstances, corticosteroids. use of these drugs should be judicious because they may have several negative consequences for the foal including renal failure and gastric/dunodenal ulceration. [ ] [ ] [ ] nursing care is one of the most important aspects of treating septic foals. foals should be kept warm and dry. they should be turned at -hour intervals if they are recumbent. feeding septic foals can be a challenge if gastrointestinal function is abnormal, and total parenteral nutrition may be needed. if at all possible, foals should be weighed daily and blood glucose levels monitored frequently. some foals become persistently hyperglycemic on small glucose infusion rates. these foals may benefit from constant rate low-dose insulin infusions (table - ) . recumbent foals must be examined frequently for decubital sore development, the appearance of corneal ulcers, and for heat and swelling associated with joints and physis. the prognosis for foals in the early stages of sepsis is fair to good. once the disease has progressed to septic shock the prognosis decreases, although short-term botulism is a neuromuscular disease of foals characterized by flaccid paralysis. although the disease is discussed in detail elsewhere in this text, the form most commonly observed in foals, the toxicoinfectious form, deserves some specific comments. the causative organism is clostridium botulinum, an anaerobic organism. although affected adults usually acquire the disease by ingestion of preformed toxin elucidated from the organism, in the foal less than months of age the organism can survive and multiply in the gastointestinal tract and produce necrotic foci within the liver, giving the foal constant exposure to newly formed toxin. the horse is exquisitely sensitive to the toxin, and only small quantities of toxin are required to produce clinical signs and death in affected animals. the ε-toxin of c. botulinum binds to the presynaptic membrane of motor neurons and prevents transmission of impulses by blocking the release of acetylcholine from the presynaptic vessicles. this block produces the clinical signs of muscle weakness, manifested in foals as trembling (shaker foals) or acute recumbency. pupillary dilation, dysphagia, tremors, recumbency, and terminal respiratory distress caused by respiratory muscle paralysis occur. foals can be found acutely dead. in endemic areas (the northeast and mid-atlantic regions of united states), for these foals to be evaluated first as having colic is not unusual. treatment aims to neutralize the toxin by administration of botulinum antitoxin and to provide antimicrobial treatment of the infection with penicillin, metronidazole, and/or oxytetracycline. , at a minimum, feeding of milk replacer via indwelling nasogastric tube at % of the body weight of the foal per day divided into every -hour meals is required. many of these foals require respiratory support (in the form of intranasal oxygen insufflation), because of respiratory muscle paralysis. respiratory acidosis is present on arterial blood gas analysis in most of these foals because of hypoventilation and lateral recumbency, but they can tolerate some degree of hypercapnia (paco ~ mm hg) if the ph is normal and oxygenation (pao > mm hg; percent oxygen saturation of hemoglobin, > %) is adequate. metabolic alkalosis can accompany the respiratory acidosis, but this is a compensatory change and resolves once gas exchange is normalized. some of these patients require mechanical ventilation, which may be lifesaving. one may discontinue mechanical ventilation as clinical signs resolve and the respiratory muscles gain strength. nursing care is important, and these foals should be turned every hours. they should be maintained in sternal recumbency if possible and kept warm and dry. with good nursing care, good nutritional support, and adequate respiratory support, the prognosis for these foals is good. the limiting factor in the prognosis for life is often financial. foals that recover from the acute stage of this disease eventually fully recover. botulism is an expensive disease to treat and is also an entirely preventable disease. , all pregnant mares in endemic areas should be vaccinated against c. botulinum. vaccination does not prevent all cases of botulism, particularly if the foal has failure of passive transfer or acquires the disease after maternal immunity wanes and before its own vaccination. nutritional muscular dystrophy or white muscle disease is a vitamin e/selenium-responsive muscle disease of horses of all ages probably caused by a dietary deficiency of selenium and vitamin e. the condition occurs most commonly in geographic areas with low selenium levels in the soil, generally the northeastern, northwestern, great lakes and mid-atlantic regions of the united states. two forms of the disease are described in foals: the fulminant form, in which the foal is found acutely dead, and the subacute form. in the fulminant form, death usually is attributed to myocardial lesions resulting in cardiovascular collapse. the subacute form is characterized by dysphagia and gait abnormalities primarily caused by stiffness of the muscles of locomotion. paralysis, if present, is not flaccid as in botulism. abnormal function of respiratory muscles may complicate the clinical situation. aspiration pneumonia may be present following problems associated with swallowing; the tongue and pharyngeal muscles frequently are affected in the early stages of disease. foals with severe disease may have widespread muscle necrosis leading to hyperkalemia, which can be severe and result in death of the foal. serum activities of the muscle enzymes creatine kinase and aspartate aminotransferase may be greatly increased. diagnosis is confirmed at necropsy or ante mortem by determination of decreased vitamin e, selenium, and glutathione peroxidase concentrations in the blood of the foal before supplementation. myoglobinuria and acute renal failure are not uncommon in these foals. treatment of foals with nutritional muscular dystrophy is primarily supportive. one should address all metabolic abnormalities. some foals require intranasal oxygen insufflation. affected foals are unable to suck effectively, and one should provide enteral (via an indwelling nasogastric tube) or parenteral nutritional support. because of the high likelihood of aspiration pneumonia, one should administer broad-spectrum antimicrobial therapy parenterally. the patient should be kept quiet and should be stimulated minimally. affected foals should receive parenteral (intramuscular) vitamin e and selenium supplementation. selenium is toxic in large doses. the prognosis for severely affected foals is guarded. for less severely affected foals the prognosis is good with appropriate treatment. the disease is preventable by ensuring that mares receive sufficient vitamin e and selenium while pregnant and by supplementing foals with parenteral injections of vitamin e and selenium at birth in endemic areas. a more complete discussion of the pathophysiology of this disease and the nutritional management is presented elsewhere in this text. primary liver disease is uncommon in the foal and occurs primarily as a sequela to sepsis. clinical signs of severe liver disease may include depression, ataxia, and seizures. in affected foals, increases in serum liver enzyme activities and concentrations of ammonia and bile acids frequently can be identified. the mechanism(s) underlying hepatoencephalopathy are not delineated clearly, although increased excitatory neurotransmitters, or compounds that mimic their activity, are implicated. hepatoencephalopathy is discussed in more detail elsewhere in this text. tyzzer's disease (clostridium piliformis infection) rarely causes primary liver disease in foals from to about days of age. this disease is almost uniformly fatal. the incubation period is short, and the mare is thought to be the carrier. [ ] [ ] [ ] [ ] [ ] clinical signs range from acute death to depression, fever, and pronounced icterus. the feces of affected foals may appear white to grey because of the lack of bile. clinicopathologic abnormalities include leukopenia, hyperfibrinogenemia, metabolic acidosis, and hypoglycemia. , liver lesions at postmortem are characterized microscopically by multiple foci of necrosis. one usually can demonstrate variable numbers of elongated, slender intracytoplasmic bacilli within hepatocytes bordering the necrotic foci. infiltration of the portal triads with inflammatory cells and biliary duct hyperplasia and degeneration are observable. the bacillus also occurs in association with myocardial lesions. lesions in the intestine are characterised by mucosal necrosis with inflammatory cell infiltration, increased mucus production, submucosal lymphoid hyperplasia, and submucosal hemorrhage. necrosis of lymphoid follicles, congestion, and hemorrhage can be present in the spleen and mesenteric lymph nodes. affected foals may have a profound metabolic acidosis that is unresponsive to treatment. the clinical course is short, and most affected foals die within a few hours of developing neurologic signs. primary liver disease has been reported in association with ferrous sulfate administration in a probiotic compound. the lesion was massive hepatocellular necrosis and liver failure. the product is no longer commercially available. portosystemic shunt is rare in the foal but has been reported in foals as young as months of age. [ ] [ ] [ ] most infectious causes of neurologic abnormalities in foals are associated with sepsis. although rarely reported, halicephalobus gingivalis (deletrix) infection has been reported in three foals; in one case the foal was weeks of age. , possibly transmission in these cases was transmammary; the dam in one case died year later with confirmed h. deletrix infestation of her udder. listeria monocytogenes has been reported as a cause of neurologic disease in foals. recently, sarcocystis neurona was identified as the causative agent of central nervous system disease in a foal, and equine herpes myeloencephalitis has been diagnosed in individual foals and in herd outbreaks involving foals. , neospora also was reported in one foal recently. rhodococcus equi abscesses can form in the central nervous system or cause neurologic signs associated with compression, as with vertebral body abscesses. [ ] [ ] [ ] cerebellar hypolasia, occipitoatlantoaxial malformation, and agenesis of the corpus callosum with cerebellar vermian hypoplasia have been reported in foals. [ ] [ ] [ ] [ ] [ ] [ ] ivermectin toxicity and moxidectin toxicity have been reported. , electrolyte abnormalities such as extreme hypo-or hypernatremia may result in neurologic manifestations of disease. , cervical stenotic myelopathy and degenerative myelopathy also have been reported in foals, although the age at onset is usually more than months. idiopathic epilepsy of arabian foals usually is associated with another infectious disease and is thought to be temporary and self-limiting. causes, diagnosis, and treatment of fpt of immunity are covered in detail elsewhere in this text. failure of passive transfer occurs when a foal fails to ingest a significant quantity of good-quality colostrum. failure of passive transfer may occur by several mechanisms: failure of the foal to suck from the dam for any reason and failure of the dam to produce sufficient quantity of quality colostrum. box - presents causes of fpt. several methods are available for measuring igg concentration in blood; the most reliable are enzyme-linked immunosorbent assay and single radial immunodiffusion technology-based tests. [ ] [ ] [ ] [ ] [ ] [ ] [ ] foals usually are tested at hours of age, but one may test the foal earlier if colostrum ingestion has occurred and a concern exists regarding the passive transfer of immunity status of the foal, recognizing that additional increases in igg concentration may occur with additional time. , the concentration of igg in the blood of the foal has been used as an indicator of the adequacy of passive transfer, but the actual blood concentration at which fpt is diagnosed has been challenged in recent years. [ ] [ ] [ ] foals with sepsis commonly have a serum igg concentration of less than mg/dl. , foals with fpt are more likely to die from sepsis. , , [ ] [ ] [ ] one should consider the igg concentration only as a marker for adequacy of colostral absorption. all the measured igg is unlikely to be directed against the specific pathogen affecting any particular neonate, and igg is not the only immune protection afforded the foal by colostrum. many factors that confer local and more general immunity to the newborn are present in colostrum; these include growth factors, cytokines, lactoferrins, cd , leukocytes, and other yet to be described proteins. [ ] [ ] [ ] [ ] [ ] by considering igg a marker of adequacy for passive transfer, similar to γ-glutamyltransferase in calves, the clinician can make choices for replacement that are more beneficial to the patient. after one identifies fpt in a foal, treatment depends on the current condition of the foal and its local environment. foals not presently ill and on well-managed farms with low population density and low prevalence of disease may not require treatment if their igg concentration is between and mg/dl. critically ill neonates with fpt in an equine nicu are by definition ill and in an environment with high disease prevalence. these patients require immediate treatment of fpt and frequent reassessment of their passive immunity status. critically ill foals often fail to demonstrate the expected increase in blood igg concentration based on grams of igg administered per kilogram of body mass compared with healthy, colostrum-deprived foals. , , sick foals also demonstrate a more rapid decline in igg concentration than do healthy foals because they use and catabolize available protein. one may treat foals with fpt by oral or intravenous administration of various products containing igg. one can attempt oral administration of additional colostrum or igg-containing products such as plasma, serum, or lyophilized colostrum in foals less than to hours of age. [ ] [ ] [ ] depending on the age of the foal and the maturity and function of the gastrointestinal tract, this treatment may be effective. many nicus and large breeding farms maintain colostrum banks for this purpose. one should administer plasma intravenously if the foal is not expected to absorb additional colostrum or if the enteral route is unavailable. commercially available hyperimmune plasma products designed for use in foals are available and can be stored frozen. plasma and banked colostrum should be stored in a non-frost-free freezer to minimize protein loss associated with freeze-thaw cycling. one should administer plasma through special tubing with an in-line filter and should monitor patients closely for transfusion reactions. one may use serum and concentrated igg products, but the practitioner should be aware that many of these products focus on igg retention and not on other factors associated with passive transfer of immunity. one should measure igg concentration after transfusion and provide additional plasma as necessary. administration of plasma to critically ill foals without fpt may be beneficial through provision of other factors present in the plasma. in these situations, fresh frozen plasma or fresh plasma may be best, particularly if transfusion of clotting proteins is desired. neonatal isoerythrolysis is a hemolytic syndrome in newborn foals caused by a blood group incompatibility between the foal and dam and is mediated by maternal antibodies against foal erythrocytes (alloantibodies) absorbed from the colostrum. the disease most often affects foals born to multiparous mares and should be suspected in foals less than days of age with clinical signs of icterus, weakness, and tachycardia. a primiparous mare can produce a foal with neonatal isoerythrolysis if she has received a prior sensitizing blood transfusion or has developed placental abnormalities in early gestation that allowed leakage of fetal red blood cells into her circulation. many are the causes of jaundice in newborn foals, including sepsis, meconium impaction, and liver failure, but these usually can be differentiated readily from neonatal isoerythrolysis by measuring the packed cell volume, which is usually less than % in foals with neonatal isoerythrolysis. foals with neonatal isoerythrolysis are born clinically normal then become depressed and weak and have a reduced suckle response within to hours of birth. the rapidity of onset and severity of disease are determined by the quantity and activity of absorbed alloantibodies. affected foals have tachycardia, tachypnea, and dyspnea. the oral mucosa is initially pale and then becomes icteric in foals that survive to hours. hemoglobinuria may occur. seizures caused by cerebral hypoxia are a preterminal event. the salient laboratory findings are anemia and hyperbilirubinemia. most of the increased bilirubin is unconjugated, although the absolute concentration of conjugated bilirubin generally is increased well above normal. urine may be red to brown and is positive for occult blood. the natural development of neonatal isoerythrolysis has several prerequisites. first, the foal must inherit from the sire and express an erythrocyte antigen (alloantigen) that is not possessed by the mare. blood group incompatibility between the foal and dam is not particularly uncommon, but most blood group factors are not strongly antigenic under the conditions of exposure through previous parturition or placental leakage. factor aa of the a system and factor qa of the q system are highly immunogenic, however, and nearly all cases of neonatal isoerythrolysis are caused by antibodies to these alloantigens. the exception is in the case of mule foals in which a specific donkey factor has been implicated. [ ] [ ] [ ] mares that are negative for aa or qa or both are considered to be at risk for producing a foal with neonatal isoerythrolysis. the risk involves approximately % and % of thoroughbred and standardbred mares, respectively. second, and perhaps most important, the mare must become sensitized to the incompatible alloantigen and produce antibodies to it. the mechanism for this is not known in many instances but generally is believed to result from transplacental hemorrhage during a previous pregnancy involving a foal with the same incompatible blood factor. sensitization via transplacental contamination with fetal erythrocytes earlier in the current pregnancy is possible, but an anamnestic response is generally necessary to induce a pathogenic quantity of alloantibodies. ten percent of thoroughbred mares and % of standardbred mares have antibodies to the ca blood group antigen without known exposure to erythrocytes. some common environmental antigen is postulated possibly to lead to production of anti-ca antibodies. data suggest that these natural antibodies may suppress an immune response to other blood group antigens because mares negative for aa that have anti-ca antibodies often do not produce antibodies to aa of the erythrocytes in their foals that also contain ca antigen. this antibodymediated immunosuppression is thought to result from the destruction of fetal cells before the dam mounts an immune response to other cell surface antigens. natural alloantibodies have not been associated with neonatal isoerythrolysis in horses. after the mare becomes sensitized to the erythrocytes of her foal, alloantibodies are concentrated in the colostrum during the last month of gestation. unlike the human neonate, which acquires alloantibodies in utero and thus is born with hemolytic disease, the foal is protected from these antibodies before birth by the complex epitheliochorial placentation of the mare. thus the final criterion for foal development of neonatal isoerythrolysis is ingestion in the first hours of life of colostrumcontaining alloantibodies specific for foal alloantigens. immunoglobulin-coated foal erythrocytes are removed prematurely from circulation by the mononuclear phagocyte system or are lysed intravascularly via complement. the rapidity of development and severity of clinical signs are determined by the amount of alloantibodies that was absorbed and their innate activity. alloantibodies against aa are potent hemolysins and generally are associated with a more severe clinical syndrome than antibodies against qa or other alloantigens. the highest alloantibody titers are likely to be produced by mares that were sensitized in a previous pregnancy and then subsequently reexposed to the same erythrocyte antigen during the last trimester of the current pregnancy. prior sensitization of a mare by blood transfusion or other exposure to equine blood products may predispose to neonatal isoerythrolysis. one can make a tentative diagnosis of neonatal isoerythrolysis in any foal that has lethargy, anemia, and icterus during the first days of life. blood loss anemia caused by birth trauma is attended by pallor. icterus caused by sepsis or liver dysfunction would not be associated with anemia. one must base the definitive diagnosis of neonatal isoerythrolysis on demonstration of alloantibodies in the serum or colostrum of the dam that are directed against foal erythrocytes. the most reliable serodiagnostic test for neonatal isoerythrolysis is the hemolytic cross-match using washed foal erythrocytes, mare serum, and an exogenous source of absorbed complement (usually from rabbits). although this test is impractical in a practice setting, a number of qualified laboratories routinely perform this diagnostic service. the direct antiglobulin test (coombs' test) may demonstrate the presence of antibodies on foal erythrocytes; however, false negatives occur frequently. most human or veterinary hematology laboratories can perform routine saline agglutination cross-match between mare serum and foal cells. because some equine alloantibodies act only as hemolysins, agglutination tests may be falsely negative. most field screening tests of colostrum have not proved to be reliable enough for practical use. if one recognizes neonatal isoerythrolysis when the foal is less than hours old, one must withhold the dam's milk and feed the foal an alternative source of milk during the first day of life. one can accomplish this by muzzling the foal and feeding it via nasogastric tube. the minimum necessary amount of milk is % of body mass every hours (e.g., a -kg foal should receive ml or pint of mare's milk or milk replacer every hours). the udder of the mare should be stripped regularly (at least every hours) and the milk discarded. in most instances, clinical signs are not apparent until after the foal is hours old, when colostral antibodies have been depleted or the absorptive capacity of the foal's intestine for immunoglobulin has diminished. withholding milk at this point is of minimal benefit. supportive care to ensure adequate warmth and hydration is paramount. the foal should not be stressed and exercise must be restricted. confining the mare and foal to a box stall is a best. intravenous fluids are indicated to promote and minimize the nephrotoxic effects of hemoglobin and to correct any fluid deficits and electrolyte and acid-base imbalances. antimicrobials may be necessary to prevent secondary infections. one should monitor foals carefully for the necessity of blood transfusion, although transfusion should be used only as a lifesaving measure. when the packed cell volume drops below %, blood transfusion is warranted to prevent life-threatening cerebral hypoxia. erythrocytes from the dam are perfect in terms of nonreactivity with the blood of the foal; however, the fluid portion of the blood of the mare has to be removed completely from the cells to prevent administration of additional harmful alloantibodies to the foal. one can pellet the erythrocytes of the dam from blood collected in acid-citrate-dextrose solution by centrifugation or gravity and then aseptically draw off the plasma by suction apparatus or syringe and replace it with sterile isotonic ( . %) saline. one thoroughly mixes the cells with the saline and then repeats the centrifugation or sedimentation, followed by aspiration and discarding of the saline. one should perform this washing process at least three times. one then can suspend the packed erythrocytes in an equal volume of isotonic saline for administration. erythrocyte washing by centrifugation is more desirable than gravity sedimentation because antibody removal is more complete and packed cell preparations can be prepared more quickly (each gravity sedimentation requires to hours). packed red blood cells are advantageous in overcoming the problem of volume overload. when equipment or conditions do not allow the safe use of dam erythrocytes, an alternative donor is necessary. because the alloantibodies absorbed by the foal generally are directed against aa or qa and because the latter are highly prevalent among most breeds of horses, a compatible blood donor is difficult to identify. the odds of finding a donor without aa or qa are higher in quarter horses, morgans, and standardbreds than in thoroughbreds and arabians. previously blood-typed individuals negative for aa and qa and free of alloantibodies are optimal. one should give to l of blood or to l of packed erythrocytes over to hours. these allogeneic cells have a short life span and represent a large burden to the neonatal mononuclear phagocyte system, which may cause increased susceptibility to infection. in addition, these cells sensitize the foal to future transfusion reactions. one must measure all potential harm against the benefit in each situation. if a mule foal is the patient, one should not use blood from a female previously bred to a donkey. in cases in which transfusion will be delayed, one cannot identify a compatible donor, or the packed cell volume is so low as to be life-threatening (hemoglobin < mg/dl), one may administer polymerized bovine hemoglobin products at a dose of to ml/kg. one may use dexamethasone ( . mg/kg) to treat peracute neonatal isoerythrolysis if the packed cell volume is less than % and transfusion may be delayed or is not fully compatible, but dexamethasone has detrimental effects on blood glucose regulation in the neonate, and because the antibody in question is of maternal origin, corticosteroid therapy in immunosuppressive doses probably is not indicated. intranasal oxygen insufflation ( to l/min) may be beneficial. most foals with neonatal isoerythrolysis have adequate passive transfer of immunity, but antimicrobial therapy is indicated to protect against secondary sepsis resulting from the compromised condition of the foal. supportive care and good nursing care, including keeping the foal warm and quiet are essential. one should expect the packed cell volume to decline again to days after transfusion. the prognosis for neonatal isoerythrolysis in foals depends on the quantity and activity of absorbed antibodies and is indirectly proportional to the rate of onset of signs. in peracute cases the foal may die before the problem is recognized, whereas foals with slowly progressive signs often live with appropriate supportive care. like most diseases, neonatal isoerythrolysis is much more effectively prevented than treated. any mare that has produced a foal with neonatal isoerythrolysis should be suspect for the production of another affected foal; thus one should provide all subsequent foals with an alternative colostrum source and discard the colostrum of the dam unless she is bred to a stallion with known blood type compatibility. mares negative for aa and qa alloantigens are most at risk of producing affected foals, thus they should be identified by blood-typing. subsequently, breeding of these mares may be restricted to aa-and qa-negative stallions, thus eliminating the possibility of producing an affected foal. in breeds with a high prevalence of aa or qa alloantigens (e.g., thoroughbreds and arabians), a stallion negative for these and suitable based on other criteria may be difficult to identify. if these "at risk" mares are bred as desired, their serum should be screened in the last month of pregnancy for the presence of erythrocyte alloantibodies. one must test mares with low or equivocal titers closer to the time of parturition. if one detects alloantibodies, the colostrum of the dam should be withheld and the foal then should be provided with an alternative colostrum source. maternal alloantibodies to ca do not appear to mediate neonatal isoerythrolysis in foals and actually may be preventive by removing potentially sensitizing cells from the circulation ; therefore one should not deprive foals of colostrum from mares possessing anti-ca antibodies, even when ca is present on their erythrocytes. rarely, the antigens de, ua, pa, and ab have been associated with neonatal isoerythrolysis in foals; however, to consider mares without these alloantigens to be at risk for neonatal isoerythrolysis is not practical. these syndromes recently have been recognized and described within the veterinary literature, although they have been recognized widely in human neonatology for many years. [ ] [ ] [ ] [ ] affected foals demonstrate these hematologic abnormalities within the first week of life, and the mechanism is similar to neonatal isoerythrolysis following ingestion of maternal antibody directed against the platelet or the neutrophil. in general, affected foals are healthy but may demonstrate bleeding tendencies if thrombocytopenia is severe or they may be more susceptible to sepsis. one confirms the diagnosis by appropriate testing for platelet-and neutrophil-associated antibody. one must rule out other causes of neonatal thrombocytopenia and neutropenia, particularly sepsis. foals born to the mare in the future seem likely to be at risk for developing similar problems, and one should treat future foals as one treats neonatal isoerythrolysis foals: prevent sucking from the dam and provide an alternate source of passive immunity in the form of banked colostrum or intravenous plasma. one should provide an alternative nutritional source, such as foal milk replacer, to the foal for the first hours of life and should muzzle the foal while it is in the company of its dam for that period of time. treatment is primarily supportive, but in the case of severe thrombocytopenia, transfusion of platelet-enriched fresh plasma may be indicated. granulocyte colony-stimulating factor has been used in foals with neutropenia, but substantial efficacy has yet to be demonstrated. broad-spectrum antimicrobial therapy may be prudent in cases of alloantibody-associated neutropenia. treatment with immunosuppressive doses of corticosteroids is probably unwarranted, given the increased risk of infection, because the antibody in question is of maternal origin. other specific diseases of the immune system of foals, severe combined immunodeficiency, selective igm deficiency, transient hypogammaglobulinemia, agammaglobulinemia, and other unclassified immunodeficincies are covered in detail elsewhere in this text. the neonate can experience respiratory distress immediately after birth because of several congenital respiratory tract or cardiac anomalies. chief among these causes are bilateral choanal atresia, stenotic nares, dorsal displacement of the soft palate caused by anatomic deformity or neurologic impairment, accessory or ectopic lung lobes, lung lobe hypertrophy, lung lobe dysplasia, cardiac anomalies with right-to-left shunting, and miscellaneous causes such as subepiglotic cysts and severe edema of the larynx. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] one must evaluate and treat these situations immediately and should consider them true emergencies. one readily can recognize foals with airway occlusion by the lack of airflow through the nostrils despite obvious attempts to breathe and by respiratory stridor. these foals may demonstrate open-mouth breathing and their cheeks may puff outward when they exhale. one foal with congenital bilateral choanal atresia was recognized during extrauterine intrapartum resusucitation because of an inability to pass a nasotrancheal tube. one can establish an effective airway by orotracheal intubation in these cases under most circumstances, but some foals require an emergency tracheostomy. one diagnoses the underlying problem by endoscopy or radiography in most cases. treatment of choanal atresia and cystic structures is surgical, whereas severe laryngeal edema and laryngeal paralysis frequently respond to medical management. until the underlying problem is resolved in these cases, one should administer broad-spectrum antimicrobial therapy and feed the foal by intubation or total parenteral nutrition. one can give colostrum, but these foals frequently develop aspiration pneumonia if allowed to suck from their dams, so intravenously administered plasma also may be necessary to provide sufficient passive immunity. arterial blood gas determinations are the most sensitive indicator of respiratory function readily available to the clinician. the most readily available arteries for sampling are the metatarsal arteries and the brachial arteries. portable arterial/venous blood gas analyzers now are making arterial blood gas analysis more practical in the field, and the technique is no longer reserved for large referral practices. managing a critically ill equine neonate without knowledge of arterial blood gas parameters is veritably impossible. pulse oximetry is useful, but these monitors only measure oxygen saturation of hemoglobin. desaturation can occur rapidly in critically ill neonates. the utility of these monitors in the foal has yet to be demonstrated clearly, particularly in cases of poor peripheral perfusion. the most common abnormalities recognized with arterial blood gas analysis are hypoxemia with normo-or hypocapnia and hypoxemia with hypercapnia. hypoxemia is defined as decreased oxygen tension of the arterial blood (decrease pao ), and hypoxia is defined as decreased oxygen concentration at the level of the tissue, with or without hypoxemia. hypoxia results from hypoxemia, decreased perfusion of the tissue bed in question, or decreased oxygen-carrying capacity of the blood resulting from anemia or hemoglobin alteration. five primary means by which hypoxemia may develop are ( ) low concentration of oxygen in the inspired air such as in high altitude or in an error mixing ventilator gas; ( ) hypoventilation; ( ) ventilation/perfusion mismatch; ( ) diffusion limitation; and ( ) intrapulmonary or intracardiac right-to-left shunting of blood. hypoxemia is not an uncommon finding in neonates but must be evaluated in terms of the current age of the foal and its position. , [ ] [ ] [ ] [ ] one also must consider the difficulty encountered in obtaining the sample because severe struggling can affect the arterial blood gas results. table - presents normal arterial blood gas parameters for varying ages of foals. the normal foal has a small shunt fraction (~ %) that persists for the first few days of life and contributes slightly to a blunted response to breathing % oxygen compared with the adult. hypoxemia frequently occurs in foals with prematurity, pas, and sepsis, although other conditions also result in hypoxemia in the neonate. in the early stage of sepsis associated hypoxemia, paco may be within normal limits or decreased if the foal is hyperventilating for any reason. if the lung is involved significantly in the underlying pathologic condition, such as with severe pneumonia, acute lung injury, or acute respiratory distress syndrome, increased paco may well be present, representing respiratory failure. hypoxemia usually is treated with intranasal humidified oxygen insufflation at to l/min. hypercapnia is not a simple matter to treat. one must try to distinguish between acute and chronic hypercapnia. acute hypercapnia usually is accompanied by a dramatic decrease in blood ph of . ph units for each mm hg increase in paco . this acidemia can promote circulatory collapse, particularly in the concurrently hypoxemic and/or hypovolemic patient. the effects of more chronic co retention are less obvious because the time course allows for adaptation. the ph change is less, about . ph units per mm hg increase in paco , because it is balanced by enhanced renal absorption of bicarbonate by the proximal renal tubule. most foals with acute respiratory distress are in the acute stages of respiratory failure, but chronic adaptation begins to occur within to hours and is maximal in to days. one will note an increase in bicarbonate, particularly if the acidemia is primarily respiratory in origin. intravenous administration of sodium bicarbonate to correct respiratory acidosis/ acidemia should be done cautiously in these foals because co retention may only be increased. also, one should remember that meq of sodium is administered with each meq of bicarbonate and hypernatremia has been seen in foals treated exuberantly with sodium bicarbonate. foals with hypercapnia of several days' duration also may develop a blunted respiratory drive to increased co . in these foals, oxygen administration, although essential to treat hypoxemia, may further depress ventilation and further decrease ph. this effect is caused by a loss of hypoxic drive following oxygen therapy. one should consider these foals candidates for mechanical ventilation if the paco is greater than mm hg or is contributing to the poor condition of the foal, such as causing significant ph changes. if hypercapnia is caused by central depression of ventilation, as frequently occurs in foals with pas, one can administer caffeine ( mg/kg loading dose; then . mg/kg as needed) per rectum or orally in foals with normal gastrointestinal function. other clinicians may recommend continuous rate infusions of doxapram hydrochoride (dopram; mg/total dose at . mg/ kg/min) for these foals. if this therapy fails, one should consider mechanical ventilation. mechanical ventilation of foals with central respiratory depression is rewarding and may be necessary only for a few hours to days. a special category is the foal with botulism exhibiting respiratory failure caused by respiratory muscle paralysis. these foals do well with mechanical ventilation, although the duration of mechanical ventilation is more prolonged, frequently more than week. foals with primary metabolic alkalosis usually have compensatory respiratory acidosis. treatment of hypercapnia is not necessary in these cases because it is in response to the metabolic condition. these foals do not respond to caffeine, and they should not be ventilated mechanically if this is the only disorder present. in the neonate, bacterial pneumonia usually results from sepsis or aspiration during sucking. foals with sepsis can develop acute lung injury or acute respiratory distress syndrome as part of the systemic response to sepsis, and this is frequently a contributor to the demise of foals in septic shock. the best way to diagnose bacterial pneumonia is by cytologic examination and culture of a transtracheal aspirate, but blood culture may aid in early identification of the causative organism and allow for early institution of directed antimicrobial therapy. a second frequent cause of bacterial pneumonia in the neonate is aspiration caused by a poor suck reflex or dysphagia associated with pas, sepsis, or weakness. one must take care to ensure that aspiration is not iatrogenic in foals being bottle fed. auscultation over the trachea while the foal is sucking helps identify occult aspiration. one should suspect occult aspiration pneumonia in any critically ill neonate that is being bottle fed or is sucking on its own that has unexplained fever, fails to gain weight, or has a persistently increased fibrinogen level. older foals develop bacterial pneumonia, frequently following an earlier viral infection. bacterial pneumonia is discussed in depth elsewhere in this text, but a few comments specific to the foal are necessary. one should auscultate and percuss the thorax of the foal, but results may not correlate closely with the severity of disease. the most commonly isolated bacterial organism in foal pneumonia is streptococcus zooepidemicus, and one may isolate it alone or as a component of a mixed infection. [ ] [ ] [ ] transtracheal aspirate for culture and cytologic examination is recommended because mixed gram-positive and gram-negative infections are common, and antimicrobial susceptibility patterns can be unpredictable. one should split the obtained aspirate and submit samples for bacterial culture, virus isolation, and cytologic examination. additional diagnostics include radiography, ultrasonography, and serial determination of white blood cell counts (with differential) and blood fibrinogen concentrations. treatment includes administration of appropriate antimicrobial therapy. some foals may benefit from nebulization with saline or other local products. ascarid larval migration through the lung can mimic bacterial pneumonia. in these cases the foal may not respond to antimicrobial therapy and should be dewormed with ivermectin. deworming the mare within month of parturition and frequent deworming of the foal prevent ascarid migration pneumonia in most foals. a special category of bacterial pneumonia in foals is rhodococcus equi bronchopneumonia. this pneumonia of young foals was described first in . the organism originally was known as corynebacterium equi and is a gram-positive pleomorphic coccobacillus usually less than µm in diameter and µm in length. the organisms frequently are associated in l-and v-shaped clusters that have been termed chinese character formations. r. equi has an acid-fast staining characteristic under some growing circumstances because of the presence of mycolic acid in its cell wall, similar to mycobacterium and nocardia species. mycolic acid promotes granuloma formation. the organism is able to multiply in and destroy macrophages as it prevents phagosome lysosome fusion. , much attention has been paid to this organism in recent years, given its propensity to produce enzootic and epizootic outbreaks of disease. the organism is thought to be primarily an opportunistic pathogen, and it lives in the soil of most geographic areas. foals are affected most frequently between the ages of and months, when maternally derived immunity has begun to wane. the disease is insidious, and foals may have significant pulmonary involvement before developing noticeable clinical signs. phagocytosis of r. equi by equine macrophages is not associated with a functional respiratory burst and, at least in human beings, the l-arginine-no pathway is not required for intracellular killing of this organism. , optimal binding of r. equi to mouse macrophages in vitro requires complement and is mediated by mac- , a leukocyte complement receptor type (cr , cd b/ cd ). opsonisation of r. equi with specific antibody is associated with increased phagosome-lysosome fusion and enhanced killing of r. equi, suggesting that the mechanism of cellular entry is important. neutrophils from foals and adult horses are fully bactericidal, and killing of r. equi is enhanced considerably by specific opsonizing antibody. the ability of r. equi to induce disease in foals likely depends on host and microbial factors. knowledge of the virulence mechanisms of r. equi was speculative until the discovery of the virulence plasmid. as opposed to most environmental r. equi organisms, isolates from clinically affected foals typically contain -to -kb plasmids encoding an immunogenic virulence-associated protein (vapa) that is expressed on the bacterial surface in a temperature-regulated manner. plasmid-cured bacteria lose their ability to replicate and survive in macrophages and are cleared from the lungs within weeks of intrabronchial challenge without producing pneumonia. however, expression of vapa alone is not sufficient to restore the virulence phenotype. six other genes have approximately % overall amino acid identity with vapa, and the identification of multiple genes with considerable homology suggests these genes constitute a virulence-associated gene family in r. equi. other candidates for virulence factors include capsular polysaccharides and cholesterol oxidase, choline phosphohydrolase, and phospholipase c exoenzymes ("equi factors"), but their roles have not been defined clearly. the primary manifestation of disease caused by r. equi infection is severe bronchopneumonia with granuloma, abscess formation, or both. up to % of foals diagnosed with bronchopneumonia also have extrapulmonary sites of infection. as the pneumonia progresses, clinical signs may include decreased appetite, lethargy, fever, tachypnea, and increased effort of breathing characterized by nostril flaring and increased abdominal effort. cough and bilateral nasal discharge are inconsistent findings. a smaller percentage of affected foals may have a more devastating, subacute form. these foals may be found dead or have acute respiratory distress with a high fever and no previous history of clinical respiratory disease. hyperfibrinogenemia is the most consistent laboratory abnormality in foals with r. equi pneumonia. neutrophilic leukocytosis (> , cells/µl), with or without monocytosis, is common. thoracic radiography is a useful diagnostic aid, frequently revealing a prominent alveolar pattern with poorly defined regional consolidation and/or abscessation. ultrasonography is a helpful diagnostic tool when the disease involves peripheral lung tissue. although a number of serologic tests have been described, serologic diagnosis of r. equi infections is controversial and difficult because exposure of foals to this organism at a young age leads to production of antibody without necessarily producing clinical disease. , serologic tests may be more useful at the farm level to detect overall exposure than at the individual level. bacteriologic culture combined with cytologic examination of a tracheobronchial aspirate remains the most definitive method for accurate diagnosis of r. equi pneumonia. however, foals without clinical disease exposed to contaminated environments may have r. equi in their tracheae from inhalation of contaminated dust; therefore one should interpret culture results in the context of the overall case presentation. culture results in one study were as sensitive as polymerase chain reaction-based assays and offered the advantage of allowing in vitro antimicrobial susceptibility testing. however, polymerase chain reaction is likely to be a useful tool, and results from a second trial suggest the assay is more sensitive and specific than culture of tracheobronchial aspirates for diagnosis. the combination of erythromycin and rifampin has become the treatment of choice for r. equi infections in foals, and the combination reduces the likelihood of resistance to either drug. the recommended dosage regimen for rifampin is mg/kg every hours or mg/kg every hours orally. the recommended dose of estolate or ethylsuccinate esters of erythromycin is mg/kg every or hours orally. recently, azithromycin has been recommended for treatment of r. equi infection at a dosage of mg/kg orally every hours for to days and then every other day. alternatively, clarithromycin at . mg/kg every hours orally, in combination with rifampin, may be therapeutically effective. severely affected foals may require intranasal oxygen insufflation, intravenous fluid support, and nutritional support. treatment generally continues for to weeks until all clinical and laboratory evidence of infection is resolved. although well tolerated by most foals, erythromycin can result in soft feces. this diarrhea is generally self-limiting and does not require cessation of therapy, but one should monitor affected foals carefully. an idiosyncratic reaction characterized by severe hyperthermia and tachypnea has been described in foals treated with erythromycin during periods of hot weather. affected foals should be moved to a colder environment and treated with antipyretic drugs and alcohol baths if necessary. clostridium difficile enterocolitis has been reported in the dams of nursing foals treated with erythromycin given orally. the dam is exposed to active erythromycin by coprophagy or by drinking from a communal water source where the foal has "rinsed" its mouth. prevention of r. equi pneumonia on farms with recurrent problems is problematic. the most clearly demonstrated prophylactic measure to date has been the administration of plasma that is hyperimmune to r. equi to foals within the first week of life and then again when maternal immunity begins to wane at around days of age. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] no effective vaccination protocols for the dam or foal have been described to date. farm management is important in preventing disease, and control measures include frequent manure removal, avoidance of overcrowded conditions, and planting of dusty or sandy soils. the prognosis for r. equi bronchopneumonia is fair to good in foals with the more chronic form of the disease. foals with acute respiratory distress have a more guarded prognosis, as do foals with sites of significant extrapulmonary infection. the long-term prognosis for survival for foals with r. equi bronchopneumonia is good, and many foals perform as expected as athletes. the most commonly identified causes of viral pneumonia in foals are equine herpesviruses and (ehv- and ehv- ), equine influenza, and equine arteritis virus (eva). equine herpesvirus is probably the most clinically important, but outbreaks of eva in neonates have occurred and are devastating. , [ ] [ ] [ ] [ ] [ ] [ ] adenovirus is reported sporadically and as a problem in arabian foals with severe combined immunodeficiency. [ ] [ ] [ ] in the neonate, infection with ehv- or eva is almost uniformly fatal and antemortem diagnosis is difficult, even once an outbreak on a particular farm is identified. several factors appear common to foals with ehv- , including icterus, leukopenia, neutropenia, and petechial hemorrhage, but these problems also are identified in foals with severe sepsis. , , the antiviral drug acyclovir ( to mg/kg orally or per rectum to times per day) has been used in cases of ehv- in neonates, with some evidence of efficacy in mildly affected foals or foals affected after birth. if viral pneumonia is a possibility, one should collect blood and tracheal aspirates at presentation for bacterial and virus isolation. the lungs of foals with ehv- or eva are noncompliant, and pulmonary edema may be present. mechanical ventilation of these cases may prolong life, but death is generally inevitable because of the magnitude of damage to the lungs. foals suspected of having ehv- or eva should be isolated because they may be shedding large quantities of virus and pose a threat to other neonates and pregnant mares. foals with eva generally are born to seronegative mares, and intravenous treatment with plasma with a high titer against eva may prove beneficial because passive immunity appears to have a large role in protection against this disease in neonates. , older foals and weanlings may be affected by herpesviruses. disease is usually mild, although a fatal pulmonary vasculotropic form of the disease has been described recently in young horses. , the clinical signs of disease are indistinguishable from influenza and include a dry cough, fever, and serous to mucopurulent nasal discharge, particularly if secondary bacterial infection occurs. rhinitis, pharyngitis, and tracheitis may be present. treatment of affected foals is primarily supportive. foals also may become infected with ehv- . the predominant clinical signs are fever and lymphoid hyperplasia with pharyngitis. , diagnosis is by virus isolation. rib fractures have been recognized in % to % of all neonatal foals and can be associated with respiratory distress. potential complications of rib fractures include fatal myocardial puncture, hemothorax, and pneumothorax. rib fractures frequently are found during physical examination by palpation of the ribs or by auscultation over the fracture sites. one can confirm the diagnosis by radiographic and ultrasonographic evaluation. often multiple ribs are affected on one side of the chest. specific treatment is generally unnecessary, but direct pressure on the thorax should be avoided in all cases. some specific patients may benefit from surgical stabilization of some fractures, particularly those fractures overlying the heart. pneumothorax can occur spontaneously or following excessive positive pressure ventilation or following tracheostomy surgery or trauma. any foal being ventilated mechanically that suddenly has respiratory distress and hypoxemia should be evaluated for pneumothorax. diagnosis is by auscultation and percussion of the thorax, but one can confirm the diagnosis with radiographic and ultrasonographic evaluation of the thorax. needle aspiration of air from the pleural space also confirms the diagnosis. treatment is required in cases in which clinical signs are moderate to severe or progressive and involves closed suction of the pleural space. subcutaneous emphysema can complicate treatment of this problem. idiopathic or transient tachypnea has been observed in clydesdale, thoroughbred, and arabian breed foals. in human infants, transient tachypnea can be related to delayed absorption of fluid from the lung, perhaps because of immature sodium channels. in foals, tachypnea generally occurs when conditions are warm and humid and is thought to result from immature or dysfunctional thermoregulatory mechanisms. clinical signs of increased respiratory rate and rectal temperature develop within a few days of birth and may persist for several weeks. treatment involves moving the foal to a cooler environment, body clipping, and provision of cool water or alcohol baths. these foals frequently are treated with broad-spectrum antimicrobial drugs until infectious pneumonia can be ruled out. a syndrome of bronchointerstitial pneumonia and acute respiratory distress has been described in older foals and appears to be a distinct entity from acute respiratory distress syndrome in neonatal foals in association with sepsis. the underlying cause has not been identified, but the genesis is probably multifactorial with several potential pathogens being implicated. affected foals have acute respiratory distress with significant tachypnea, dyspnea, nostril flare, and increased inspiratory and expiratory effort. auscultation reveals a cacophony of abnormal sounds including crackles and polyphonic wheezes in all lung fields. loud bronchial sounds are audible over central airways, and bronchovesicular sounds are lost peripherally. affected foals are cyanotic, febrile, and unwilling to move or eat. foals may be found acutely dead. laboratory abnormalities include leukocytosis, hyperfibrinogenemia, and hypoxemia with hypercapneic acidosis. foals can be dehydrated severely and have coagulation changes consistent with disseminated intravascular coagulation. hypoxic injury to other organs, primarily the kidneys and liver, can occur. chest radiographs reveal a prominent interstitial pattern overlying a bronchoalveolar pattern that is distributed diffusely throughout the lung. this syndrome is a respiratory emergency. treatment is broad-based and includes administration of oxygen, nonsteroidal antiinflammatory agents, broad-spectrum antimicrobial therapy, nebulization, judicious intravenous fluid therapy, nutritional support, and corticosteroid therapy. one must manage hyperthermia in the foal. corticosteroid therapy appears to have been lifesaving in most of the reported surviving foals. because this syndrome is associated with high environmental temperatures in some areas, prevention involves control of ambient temperatures, not transporting foals during hot weather, and keeping foals out of direct sun on hot days, particularly foals being treated with erythromycin for suspected or confirmed r. equi infection. uroperitoneum has been recognized as a syndrome in foals for more than years. , classically, affected foals are to hours old at the time clinical signs first are recognized. [ ] [ ] [ ] previous reports had a proportionately larger affected male than female population. , , the hypothesis was that colts were more at risk because their long, narrow, high-resistance urethra was less likely to allow bladder emptying, resulting in rupture of a full bladder during parturition when high pressures were applied focally or circumferentially around the bladder. more recent reports suggest that such extreme sex bias may have been an artifact of small case numbers in the early reports. rupture or disruption of any structure of the urinary tract can occur. the dorsal wall of the bladder has been reported to be a frequent disruption site, with the ventral wall less likely to be involved. the urachus appears to be the next most commonly affected structure. a few cases of ureteral and urethral defects have been reported. , sepsis does not appear to favor one site over the others. the pathophysiology of uroperitoneum is not yet understood fully. the high pressure exerted on a full bladder during parturition once was thought to be the main cause. full bladder and obstruction caused by a partial umbilical cord at parturition, strenuous exercise, and external trauma have been reported as causes. a few reports describe smooth and noninflamed edges of torn tissue, suggesting the possibility of congenital bladder wall defects. , , sepsis leading to urinary tract rupture and uroperitoneum may occur in foals hospitalized for a variety of unrelated problems. the onset of clinical signs of uroperitoneum may be insidious in these foals, and diagnosis may be less obvious. clinical signs associated with uroperitoneum in the neonatal foal typically include straining to urinate, dribbling urine, and a stretched-out stance. weakness, tachycardia, tachypnea, and not sucking well are also common. a distended abdomen may be evident, and one may feel a fluid wave on ballottement of the abdomen. occasionally, urine accumulates in the scrotum and should not be confused with hernia. foals also may show signs of sepsis, including fever, injected mucous membranes, diarrhea, and disease of other body systems. laboratory findings vary depending on the duration of the uroperitoneum and on the presence and severity of sepsis. classic findings include hyperkalemia, hyponatremia, and hypochloremia arising from equilibration of urine electrolytes and water with blood across the peritoneal membrane. [ ] [ ] [ ] the usual foal diet of milk, which is high in potassium and low in sodium, promotes the electrolyte abnormalities. foals that develop uroperitoneum while receiving intravenous fluids may not have classic electrolyte imbalances at the time clinical signs are recognized. increased serum creatinine concentration is often present, whereas blood urea nitrogen concentrations occasionally, but not consistently, are increased. [ ] [ ] [ ] metabolic acidosis and hypoxemia may be present. some patients also have serum hypoosmolality. one should test foals for failure of passive transfer. one of the most sensitive laboratory tests for uroperitoneum is the ratio of peritoneal to serum creatinine. a ratio greater than or equal to : is considered diagnostic of uroperitoneum. one should collect peritoneal fluid and test it for creatinine concentration, as well as for cytologic findings, culture, and sensitivity. cytologic evaluation of peritoneal fluid is necessary to identify concurrent peritonitis or other gastrointestinal compromise. one should perform an electrocardiogram on initial evaluation of a foal with suspected uroperitoneum because hyperkalemia may result in bradycardia, increased duration of the qrs complex, a shortened q-t interval, increased p-wave duration, prolonged p-r interval, or atrioventricular conduction disturbances. other possible cardiac sequelae to hyperkalemia include cardiac arrest, third-degree atrioventricular block, ventricular premature contractions, and ventricular fibrillation. , for any foal exhibiting signs of dypsnea, tachypnea, or hypoxemia, one should have thoracic radiographs taken before induction of anesthesia to rule out pleural effusion, pneumonia, or acute respiratory distress syndrome, which could complicate ventilation and oxygenation during anesthesia and the postoperative period. ultrasonography has become the tool of choice in the diagnosis of uroperitoneum and is a useful tool available to the practitioner. one can image free peritoneal fluid readily, and tears within the bladder are readily visible. the empty bladder with a significant defect, in a fluid-filled abdomen, will collapse on itself and often have a u shape. one also can visualize urachal and urethral lesions. six of eight foals in one study had urinary tract lesions identified sonographically, and all foals of another study underwent sonographic evaluation, and a significant correlation between ultrasonographic findings and location of the lesion at surgery existed. , initial treatment aims to stabilize the patient and correct any electrolyte and acid-base abnormalities and provide fluid volume replacement. one should use . % or . % saline with % dextrose until laboratory data are available. a potassium concentration of greater than . meq/l can be life threatening. one can manage hyperkalemia by peritoneal drainage to decrease whole-body potassium stores using teat cannulae, foley catheters, large-gauge ( or ) intravenous catheters, or human peritoneal dialysis catheters. fluid replacement at least should equal the amount of fluid removed from the abdomen to prevent acute hypotension caused by expansion of previously collapsed capillary beds. abdominal drainage also helps ventilation and decreases the work of breathing by decreasing pressure on the diaphragm. one may administer calcium gluconate, glucose, sodium bicarbonate, or insulin intravenously to decrease serum potassium concentrations. these maneuvers do not correct the whole-body potassium overload, however, and once therapy is discontinued, hyperkalemia can reappear until the urine is removed from the abdomen. one should correct hyponatremia slowly. because of the real possibility of concurrent sepsis, one should obtain blood cultures before preoperative administration of antimicrobials. broad-spectrum coverage (penicillin and amikacin or ceftiofur sodium) is recommended until culture results become available. one should perform therapeutic drug monitoring when using aminoglycoside therapy. however, the peak value may be depressed because of the increased volume of distribution represented by the volume of urine in the abdomen, so one should not make dose adjustment based on a low peak until obtaining a new peak after surgical correction of the uroperitoneum. one should treat foals with failure of passive transfer with adequate volumes of intravenously administered plasma. after one has addressed the metabolic abnormalities, one may consider surgical management. medical management using an indwelling foley catheter has been described. preoperative medical stabilization reduces anesthetic risk. safer inhalant agents such as isoflurane also have decreased risk. removal of the internal umbilical remnant at the time of surgery is usual. one should consider culturing any removed umbilical remnant and submitting the remnant for histopathologic evaluation. recurrence of urinary tract rupture can occur. sepsis, hypoxemia, pneumonia, peritonitis, and acute respiratory distress syndrome complicate the management of uroperitoneum. many affected foals are persistently oxygen dependent for several days following surgical correction, and one should perform serial arterial blood gas analyses before discontinuing intranasal oxygen supplementation. prognosis is associated closely with concurrent illness, especially septicemia. uncomplicated uroperitoneum from a defect in the bladder has a good prognosis. if the location of the lesion is other than the bladder, the prognosis is not as favorable. foals with septicemia have a much poorer prognosis. , acute renal failure most often occurs as a complication of prenatal asphyxial syndrome, sepsis, or aminoglycoside therapy. acute renal failure also has been reported following oxytetracycline administration in foals. the dose of oxytetracycline commonly used to treat flexural deformities in foals is approximately times the antimicrobial dose. many foals treated in this manner also have suffered some degree of perinatal asphxia, which also damages the kidney, because of prolonged parturition precipitated in part by the flexural deformity. evaluation of renal function in these foals before the administration of the first dose of oxytetracycline and continued monitoring of serum creatinine concentrations before administering subsequent doses of this nephrotoxic compound would seem reasonable. hemodialysis has been used as therapy in one of these cases, but prevention is important because these foals may fail to respond to usual therapy for oliguric renal failure and are euthanized. the most commonly reported congenital deformity of the kidney of the foal is renal hypoplasia and dysplasia, which may have a heritable component. , renal arteriovenous malformations have been reported also. ectopic ureters and fenestrated ureters have been described in the foal. [ ] [ ] [ ] congenital renal defects, among others, were reported in three weak, recumbent neonatal foals born to mares being treated for equine protozoal myeloencephalitis. mares received sulfadiazine or sulfamethoxazoletrimethoprim, pyrimethamine, folic acid, and vitamin e orally. the foals were anemic, leukopenic, azotemic, hyponatremic, and hyperkalemic. serum folate concentrations were lower than those reported in the literature for clinically normal brood mares. treatment was unsuccessful. necropsy revealed lobulated kidneys with thin cortices and a pale medulla. the authors postulated that oral administration of sulfonamides, , -diaminopyrimidines (pyrimethamine with or without trimethoprim), and folic acid to mares during pregnancy is related to congenital defects in newborn foals. the umbilicus serves as the conduit for nutrition and gas exchange between the dam and the fetal foal. the urine from the foal is expelled via this structure into the allantoic cavity. the author has recognized cases of in utero bladder distention in the fetus that were associated with multiple twists decreasing urine flow or focal stenosis creating the same effect. foals born with this condition did not have bladder rupture associated with parturition but did have other severe abnormalities that eventually resulted in their demise, primarily premature delivery with failure to adapt to extrauterine life (p.a. wilkins, j.e. palmer, and f.t. bain, unpublished data). at birth the umbilicus breaks, leaving a small external remnant and a large internal remnant. the umbilicus long has been regarded as the primary site of entry of pathogens into the neonate, although this has been challenged recently. treatment of the umbilicus after birth involves dipping it (preferably just the most distal component) with various caustic compounds. the most current recommendation is to treat the umbilicus with dilute chlorhexidine, povidone-iodine, or dilute iodine solutions for just a few times following birth. exhuberant treatment of the umbilical stump with caustic solutions can lead to scalding of the ventral abdomen and may promote patency of the urachus. the ultrasonographic appearance and measurements of the umbilical arteries, urachus, and umbilical vein of foals from hours to weeks of age have been described in detail. a . -mhz sector scanner transducer placed across the midline of the ventral portion of the abdominal wall of the foal works best because of the superficial location of these structures. the mean (± sd) diameter of the umbilical vein was . ± . cm immediately cranial to the umbilical stalk, . ± . cm midway between the umbilicus and liver, and . ± . cm at the liver. the urachus and umbilical arteries of normal foals have a mean total diameter of . ± . cm at the bladder apex. the umbilical arteries scanned along either side of the bladder have a mean diameter of . ± . cm. one can use these measurements and the ultrasonographic appearance of the internal umbilical structures from clinically normal foals as references to diagnose abnormalities of the umbilical structures in neonatal foals. , the most common abnormalities of these structures are focal abscess formation, hematoma, and urachal tear. herniae traditionally have been thought to develop from failure of closure at the umbilical stump after birth. however, the closure of the body wall defect at the umbilicus was studied in relation to the development of umbilical herniae in a large group of normal foals followed from birth until months of age or from birth until months of age. at birth, approximately half of these foals had a defect in the body wall at the umbilicus that was termed a palpable umbilical ring. in foals this defect disappeared within days, but in one foal the ring did not close and a hernial sac with abdominal contents was palpable. this foal was considered to be the only foal to have a truly congenital umbilical hernia. twelve foals developed an umbilical hernia between and weeks of age. the prevalence of umbilical herniae was much higher than in other studies, possibly because of the prospective nature of the study. based on this study, the large majority of umbilical herniae would appear not to result from failure of closure but rather to be acquired after birth. one should consider the palpable ring structure within the body wall at the umbilicus a variant of normal in the foal and should not call it a hernia until the foal is at least month of age. in one study of horses treated for umbilical herniae over a / -year period, only . % developed complications in association with umbilical defects. six horses had intestinal incarceration; the incarceration was reduced manually in horses before admission and resolved without treatment in others. the hernia was surgically reduced in horse. herniorrhaphy was performed on of the horses in which the incarceration did not require surgical reduction, and the fifth was managed conservatively. the study confirmed that complications of umbilical herniae are rare in horses; however, when they do develop, they may be one of various forms, some of which are insidious in onset. the primary differential diagnosis for an external swelling in the umbilical stump region is an external abdominal abscess, which will be firm, variably painful, warm, and nonreducible. ultrasonographic evaluation readily can confirm either possibility. one report describes a -day-old foal that died from intestinal strangulation caused by a remnant of vitelline vein that extended between the umbilicus and the portal vein. patent urachus frequently is recognized in the abnormal neonate, probably because of the increased recumbency and decreased movement of these patients. cauterization of a patent urachus is no longer recommended except in cases that persist for long periods of time (> month) after the foal becomes more active. surgical resection may provide relief in some foals, but most cases resolve without treatment if given enough time. foals with a patent urachus may posture and strain frequently to urinate, some of this may be associated with irritation or local infection of the urachus. one can alleviate this by administration of broad-spectrum antimicrobial therapy such that the drug has a high concentration in the urine (e.g., trimethoprim-sulfa drug combinations) and by oral administration of phenazopyridine hydrochloride (pyridium), a dye that anesthetizes the urinary tract epithelial surfaces (see table - ). this dye turns the urine orange and stains everything yellow-orange that it or the urine touches but can provide a great deal of relief to foals with this problem. the umbilicus has been considered the traditional point of entry of bacteria into the septic neonate, and septic foals have been referred to as having "navel ill" and "joint ill" in the past. although current thought suggests that the gastrointestinal tract may be the route of entry in most septic neonates, infection of the umbilicus-termed omphalitis, or omphalophlebitis if the vessels are involved-still occurs as a single focus of infection or along with more generalized infection. external signs, such as swelling, heat, pain, ventral edema, or purulent discharge may be present in some foals, but more usually external signs are minimal and one suspects infection because of infection in another site (e.g., an infected joint), fever, or otherwise unexplained increased blood fibrinogen concentration. one confirms the diagnosis by ultrasonographic evaluation of the internal umbilical remnant. any of the umbilical structures may be involved. a complete description of the evaluation is available within the relevant veterinary literature, but the examination is performed best with the foal standing using a . -mhz probe with a standoff. the usual finding is that the affected structure is larger than expected. a fluid-filled core and echogeneic shadows consistent with gas may be apparent in some cases. interpretation requires some experience, and the examiner should be familiar with variants of normal, such as gas shadows associated with a patent urachus and enlarged vessels caused by hematoma formation, so that treatment is not initiated inappropriately. two options for treatment are surgical and medical. medical treatment is preferable in cases in which the lesion is well localized and small and in foals with a medical condition that is not amenable to anesthesia and surgical intervention. one should institute broad-spectrum antimicrobial therapy, and one may need to continue therapy for to weeks. most affected foals respond to medical therapy. frequent reevaluation of the abnormality is necessary, every to days initially, and one should measure blood fibrinogen concentrations at reevaluation because they should stabilize and decrease with effective treatment. failure to respond to therapy within days to weeks suggests that an empiric change in the antimicrobial used may be necessary. in foals that are refractory to medical management or where the lesion is large, surgical excision of the entire umbilical remnant may be desirable. colic in the foal can be difficult to diagnose accurately because one cannot perform an examination per rectum. however, many diagnostic aids, most importantly ultrasonography, are available to help differentiate medical from surgical causes of abdominal discomfort in the foal. intestinal accidents of all types described in adult horses, with the possible exception of enteroliths, occur in foals. intussusception, volvulus, displacement, diaphragmatic hernia, and intra-and extraluminal obstruction have been reported in foals. abdominal ultrasonographic and radiographic evaluation greatly aids diagnosis. treatment is primarily surgical. foals with pas and intestinal dysmotility are at increased risk of intussusception and displacement, and miniature breed foals appear to be at increased risk for fecolith and enterolith formation. meconium retention or impaction is a common cause of abdominal discomfort in newborn foals. most foals defecate shortly after their first meal. the usual practice for most owners or veterinarians attending the birth of a foal is to administer an enema to aid this process. in the past, phosphate-based commercially available enemata (fleet) were used frequently, but if used excessively these types of enemata can create problems of their own, including rectal irritation and hyperphosphatemia. the best enema is warm soapy water made with a mild soap such as liquid ivory soap that can be administered through soft rubber tubing using gravity flow. foals with significant meconium retention become colicky within the first few hours of life as gas accumulates within their bowel. frequently, one can palpate the meconium through the abdominal wall. additional diagnostics can include abdominal ultrasonography and radiography, particularly if one must rule out other, more serious types of colic. these foals assume a classic stance with an arched back. one must differentiate this stance from the stance assumed by foals with uroperitoneum, which is more extended. foals with meconium retention have had simultaneous ruptured bladder, however, so the clinician must be sure to evaluate the foal fully for both problems. foals that do not respond rapidly to enema administration need additional treatment, which can include giving mineral oil ( to ounces) by nasogastric tube. one can treat persistent meconium retention resulting in significant abdominal distention by muzzling the foal to prevent further milk intake and administering intravenous fluids at an appropriate maintenance rate. if continuous rate infusion is possible, % to % dextrose is the preferred fluid to use to provide calories to the foal. one should not use dextrose as a bolus fluid. more aggressive treatment would include administration of retention enemata made using acetylcysteine, which serves as an irritant and increases secretion. extreme cases of meconium retention may require surgical intervention, but this is usually not necessary and most cases resolve with medical management alone within to hours. some foals require pain managment. one should avoid nonsteroidal antiinflammatory drugs in the neonate because of their effects on renal function and gastric mucosal blood flow (see gastric ulcers). many foals respond well to butorphanol administered intramuscularly at a dose of to mg to an average -kg foal. intranasal oxygen insufflation is beneficial in foals with significant abdominal distention. one should evaluate foals with meconium impaction/ retention for evidence of pas because intestinal dysmotility is common in pas. colostrum is a laxative, and these foals also may suffer from failure of passive transfer, with meconium retention resulting from the lack of adequate colostrum. these foals are also at risk of sepsis because the mucosal intestinal barrier probably has been disrupted and translocation of bacteria can occur. one should obtain blood cultures on these foals and should monitor them closely for signs of sepsis. atresia within the gastrointestinal system of the foal occurs infrequently, but clinical signs are characteristic. acute colic occurs within the first few hours and is accompanied by abdominal distention similar to meconium retention. three primary types of atresia are described in the foal: membrane atresia, cord atresia, and blind-end atresia. antemortem diagnosis of atresia, short of abdominal exploratory surgery, is aided by the lack of meconium staining of the rectum or any administered enema fluids. additional diagnostic tests may include administration of a barium enema for a radiographic study, colonoscopy, and abdominal ultrasonography. abdominocentesis is usually normal until bowel rupture is imminent or has occurred. one can make affected foals more comfortable by muzzling them to prevent further milk intake and by supplying them with fluids and nutrition intravenously. if one attempts surgical correction, one first should initiate broad-spectrum antimicrobial therapy and determine passive transfer status. frequently, these foals are hypoxemic because of the abdominal distention, and oxygen supplementation is desirable. solid white foals born to overo-overo matings of american paint horses may suffer from congential aganglionosis of the ileum, cecum, and colon. these foals present similarly to foals with meconium impaction or atresia in that colic develops shortly after birth and involves progressive abdominal distention with feeding. the inherited defect is in the endothelin receptor gene. [ ] [ ] [ ] [ ] no effective treatment exists, but the clinician should be aware that not all white foals of this mating are affected, and some simply may have meconium retention, so a short period of treatment may be warranted. necrotizing enterocolitis is considered the most common acquired gastrointestinal emergency of human infants. , the to infants that die every year from this disease in the united states and the large number of infants who develop short gut syndrome from this disease only represent the tip of the iceberg of the problems necrotizing enterocolitis causes. the widespread fear of necrotizing enterocolitis among neonatologists and pediatric surgeons has contributed in large part to the use of the intravenous route rather than the gastrointestinal tract for nourishing these infants for long periods. the pathogenesis of necrotizing enterocolitis is unknown but may result from a disturbance of the delicate balance among gastrointestinal perfusion, enteric organisms, and enteral feeding. risk factors for necrotizing enterocolitis in human infants include prematurity, hypoxic-ischemic insult, and formula or breast milk feedings. the clinical spectrum of necrotizing enterocolitis is multifactoral and ranges from temperature instability, apnea, lethargy, abdominal distention, bilious residuals, septic shock, disseminated intravascular coagulation, and death. medical management is usually adequate treatment for necrotizing enterocolitis. in the neonatal foal, necrotizing enterocolitis is probably one of the most underrecognized causes of gastrointestinal dysfunction and in the past has been attributed only to infection with anaerobic organisms including clostridium perfringens type c and c. difficile. although a specific form of enteritis is associated with intestinal infection by these organisms, most necrotizing enterocolitis is associated with prematurity or pas in the infant and the foal. one should suspect necrotizing enterocolitis in any foal that is having difficulty tolerating oral feeding, demonstrating signs of ileus, or having episodes of colic and in any foal with occult blood or frank blood in the stool. foals exhibiting any of these clinical signs should not be fed orally if possible and should receive parenteral nutrition until gastrointestinal function returns to near normal. the mucosal barrier of the intestine is unlikely to be fully intact, and these foals are at risk for sepsis from bacterial translocation. one should institute broadspectrum antimicrobial therapy in these foals and, if any evidence of coordinated gastrointestinal motility is apparent, should administer sucralfate orally as a protectant. gastric ulcer disease has been recognized in foals, and lesions vary in anatomic distribution, severity, and cause. in clinically normal neonatal foals (< days of age), gastric ulcers and mucosal desquamation have been documented. [ ] [ ] [ ] [ ] because of these reports and other early reports of death following ruptured clinically silent ulcers in neonatal foals, for years many clinicians felt it necessary to treat critically ill neonates with antiulcer medication prophylactically. [ ] [ ] [ ] recently, this paradigm has been challenged. the pathophysiology of gastric ulcer disease is described most reasonably as an imbalance in protective and aggressive factors. [ ] [ ] [ ] these protective factors are responsible for maintaining a healthy gastrointestinal tract by promoting adequate mucosal blood flow, adequate mucus and bicarbonate production, prostaglandin e production, epithelial growth factor production, gastric afferent innervation, epithelial cell restitution, and gastroduodenal motility. probably the most important factor is maintenance of mucosal blood flow. hypoxia, no, prostaglandins, and gastric afferent innervation influence mucosal blood flow. the aggressive factors include gastric acid, bile salts, pepsin, and enzymes. few specific causes have been found for gastric ulcer disease in foals. excessive administration of nonsteroidal antiinflammatory drugs can result in ulceration of the glandular and squamous epithelium because of an inhibition of prostaglandin production, which leads to a decrease in mucosal blood flow and an increase in acid production. nonsteroidal antiinflammatory drugs also can impair the healing of lesions and rarely are indicated in neonatal equine medicine. , in the critically ill neonate the suspected cause of gastric ulcers has shifted away from an excessive amount of intraluminal gastric acid toward gastric mucosal ischemia caused by hypoxia, low blood flow conditions, or both. perforating gastric ulcers are more likely a manifestation of necrotizing enterocolitis than of excessive gastric acid. shock, sepsis, or trauma can result in gastric mucosal ischemia, allowing for the disruption of epithelial cell integrity and permitting damage by aggressive factors or providing an environment suitable for the establishment of bacteria colonization. , impairment of mucosal blood flow also may result in reperfusion injury, allowing the formation of gastric ulcers. in the sick neonatal foal (< days of age) a wide variability in the intragastric ph has been documented depending on the type of disease, severity, and milk intake frequency and volume, suggesting that in the critically ill equine neonate, ulcer prophylaxis using histamine antagonists or proton pump inhibitors is not only unnecessary but unlikely to work. clinically significant gastric ulcers can occur in the squamous, glandular, or both portions of the stomach as a primary problem or resulting from another problem. clinical signs include diarrhea, abdominal pain, restlessness, rolling, lying in dorsal recumbency, excessive salivation, and bruxism. in the neonatal foal the only clinical signs present may be depression or partial anorexia until a more catastrophic event, such as perforation, occurs. some lesions in the gastric mucosa extend from the pylorus into the proximal duodenum and can result in stricture of the pylorus and proximal duodenum. these foals are usually older (> month of age) and have a greater volume of reflux. bruxism and ptyalism are also more prominent in these older foals. the most sensitive and specific method for diagnosing gastric ulcers is visualization by endoscopic examination. unfortunately, the use of gastric endoscopy has led to recognition of relative nonlesions and ulcers resulting from other problems and of clinically significant disease states. the clinician should not stop simply when ulceration of the stomach is recognized with endoscopy but should examine that patient fully for other potential sources of the clinical signs. other diagnostic tests may help in determining the severity of the ulcers, including fecal occult blood or gastric blood assessments, contrast radiography, abdominal ultrasound, and abdominocentesis. endoscopy of the foal stomach carries an additional risk of exacerbating colic in the short term, unless the examiner ensures that as much introduced air as possible is evacuated from the stomach at the end of the procedure. the presence of a brown gastric reflux fluid may indicate the presence of bleeding ulcers. blood in the feces of the neonate is more consistent with a diagnosis of necrotizing enterocolitis, which can be associated with gastric ulcers. contrast radiography is useful if one suspsects delayed gastric emptying or pyloric or duodenal stricture in older foals. if a stricture has occurred, one will note a delay in complete emptying of barium from the stomach (> hours). abdominal ultrasound may be useful to visualize free abdominal fluid and gastric or small intestinal distention if one suspects a perforation. one can visualize portions of the descending duodenum, and a thickened duodenum should increase the index of suspicion for duondenal stricture. abdominocentesis also may confirm perforation. traditional therapy for gastric ulceration includes mucosal adherents, histamine type receptor antagonists, proton pump inhibitors, and antacids. the most widely used mucosal adherent is sucralfate, which is a hydroxy aluminum salt of sucrose. the main therapeutic action of sucralfate is to bind to the negatively charged particles in the ulcer crater. , at a ph less than , sucralfate is converted to a sticky viscous gel, which adheres to the ulcer crater and remains adhered for hours, but at a higher ph, sucralfate remains in a suspension. sucralfate is still effective because it inhibits pepsin and buffers hydrogen ions. other important actions of sucralfate include stimulating production of prostaglandin e, which maintains mucosal blood flow; increasing bicarbonate secretion; stimulating mucous secretion; decreasing peptic activity; and binding epidermal growth factor. the histamine type receptor antagonists include cimetidine, ranitidine, and famotidine. these compounds block the interaction of histamine with the histamine type receptor on the parietal cell, resulting in inhibition of gastric acid secretion. clinically normal neonatal foals have a highly acidic gastric fluid that is influenced by sucking. intravenous and oral administration of ranitidine increases intragastric ph in normal foals but critically ill neonatal foals have a blunted response to ranitidine administration. , one possible conclusion reached from these studies is that in critically ill neonatal foals, gastric ulcers may not be caused by an increased intraluminal gastric acidity. the most commonly used proton pump inhibitor is omeprazole. this drug has not as yet been approved for use in foals under days of age. omeprazole inhibits the secretion of hydrogen ions at the parietal cell by irreversibly binding to the h + ,k + -atpase proton pump of the cell. most of the lesions in older foals were healed after daily administration of omeprazole for days according to one report. table - summarizes the therapeutic agents for treating gastric ulcers in foals. prophylactic treatment of critically ill neonates for gastric ulcers has been standard therapy for years because of the evidence of clinically silent ulcers. this approach may not be appropriate for several reasons. an increased incidence of nosocomial pneumonia and systemic sepsis is associated with high gastric ph in human patients in intensive care. [ ] [ ] [ ] patients in intensive care units treated prophylactically with histamine type receptor antagonists are more likely to develop pneumonia during ventilation therapy and gastric colonization with potentially pathogenic bacteria or yeast. , an acidic environment appears to protect against airway colonization by bacteria of intestinal origin and bacteria translocated across the gastrointestinal tract. pathogenesis of ulcers in the neonatal foal most likely does not involve increased intraluminal gastric acid but instead may be caused by decreased mucosal perfusion associated with shock, hypoxia, and hypoxic/ischemic insult to the gastric mucosa. a recent report revealed that gastric ulcer disease in equine nicu patients is independent of pharmacologic prophylaxis. in this study, despite decreased treatment, the incidence of gastric ulcers found in these foals at necropsy had decreased significantly. the decrease was attributed to overall improvement in management of these cases. similarly, in a human intensive care unit, the incidence of stress ulcers decreased independent of the use of prophylaxis. , early treatment of sepsis, sufficient oxygenation, improved monitoring, institution of enteral feedings, and improved nursing care may contribute to the reduction in gastric ulcers in the neonatal patient. use of histamine type receptor antagonist and proton pump inhibitors apparently may not be necessary; however, in some instances sucralfate may be useful. sucralfate reduced the rate of bacterial translocation in a rat model during hemorrhagic shock and also may prohibit the generation of acute gastric mucosal injury and progression to ulcer formation induced by ischemia-reperfusion. , in a human medical intensive care unit, airway colonization by new pathogens occurred more frequently in patients receiving agents that increased gastric ph than in those receiving sucralfate. , in the critically ill neonatal foal, risk factors for gastric ulceration have not been identified clearly, although foals treated routinely with nonsteroidal antiinflammatory drugs may be at increased risk for gastric lesions. prophylactic treatment for gastric ulcers in critically ill neonates may not be necessary, and one should consider carefully the pros and cons of their use before their administration. foal heat diarrhea is a mild, self-limiting form of diarrhea that occurs in foals between and days of age, about the time of the "foal heat" in the dam. the definitive cause of foal heat diarrhea has yet to be described, but the condition may be associated with dietary changes or changes in gastrointestinal function that occur around that time. this form of diarrhea is not caused by stongyloides westeri infestation as previously thought. foals with foal heat diarrhea are not systemically ill and should not require therapy. one should evaluate fully any foals with diarrhea at this time for other possible causes of diarrhea, particularly if they are unwell or exhibit anorexia or dehydration. viral diarrhea occurs most commonly in large groups of mares and foals that are housed together. rotavirus is an isolate from the feces of up to % of foals with diarrhea worldwide, alone or with another pathogen. , the virus infects and denudes the microvilli, resulting in increased secretion combined with decreased absorption. the virus interferes with disaccharidase function and alters the function of the intestinal sodium-glucose cotransport proteins. the initial clinical signs are anorexia and depression, with profuse watery diarrhea occurring shortly thereafter. severely affected foals may become significantly dehydrated and have electrolyte abnormalities, primarily hyponatremia and hypochloremia with metabolic acidosis. these foals generally require intravenous fluid support, whereas less severely affected foals may require only symptomatic therapy. definitive diagnosis is by detection of the virus in the feces of foals with diarrhea. however, none of the available tests are particularly sensitive, and the virus also may be found with other intestinal pathogens. recently, vaccination of pregnant mares has been suggested as a means of prevention, with preliminary results suggesting efficacy. , although a definitive role for adenovirus has not been established in the foal, adenovirus is a common co-isolate from foals with rotaviral diarrhea. a specific equine coronavirus recently has been identified from an immunocompetent foal with diarrhea, and a second report of cornavirus diarrhea was published recently. , one case report suggests a parvovirus caused diarrhea in the foal. treatment of viral diarrhea in foals is primarily supportive. intravenous fluid and parenteral nutritional support may be necessary in severe cases. very young foals may benefit from intravenous plasma administration and broad-spectrum antimicrobial coverage to limit bacterial translocation. one can administer sucralfate orally in these cases as a gastrointestinal protectant and to discourage bacterial translocation. foals with moderate to severe metabolic acidosis may benefit from sodium bicarbonate administration if their ventilatory function is normal. one administers sodium bicarbonate at half the calculated deficit ( . × standard base excess × body mass in kilograms) as an isotonic solution at the maintenance fluid rate. one should reevaluate sodium and bicarbonate (or standard base excess) concentrations regularly. nonspecific therapy of diarrhea is discussed elsewhere in this text. diarrhea is frequently the primary presenting complaint in foals with sepsis, so one should rule out this differential diagnosis in foals less than week of age. one should evaluate all neonatal foals with diarrhea for possible sepsis and should include a blood culture whenever possible. clostridium perfringens and c. difficile are recognized increasingly as serious pathogens of the foal. - foals with either pathogen generally have abdominal pain, dehydration, and profuse watery diarrhea. some foals may have red-tinged or frankly bloody feces, which carries a poorer prognosis. most foals with this type of diarrhea require intensive care or, at the minimum, intravenous fluid administration. outbreaks of this type of diarrhea on farms occasionally occur, and the suggestion is that the dam has a role in transmission of the bacteria. diagnosis is by recognition of the offending organism by gram stain of the feces, by bacterial isolation from the feces, and by detecting the presence of toxins associated with the organisms. specific treatment includes oral administration of metronidazole and broad-spectrum antimicrobial coverage as prophylaxis for bacterial translocation associated sepsis in younger foals. foals with severe blood loss in their feces may require transfusion of whole blood. salmonella spp., escherichia coli, bacteroides fragilis, and aeromonas hydrophila have been implicated in diarrhea in foals. salmonella generally is associated with septicemia in foals, and although some convincing evidence exists for a role for e. coli in foal diarrheal disease, the extent of e. coli as a pathogen of the gastrointestinal tract in foals has yet to be described fully. , [ ] [ ] [ ] [ ] proliferative enteropathy is a transmissible enteric disease caused by lawsonia intracellulare. , most foals have been weaned before the appearance of clinical signs of depression, rapid and significant weight loss, subcutaneous edema, diarrhea, and colic. poor body condition with a rough hair coat and a pot-bellied appearance are common in affected foals. clinicopathologic abnormalities included hypoproteinemia, leukocytosis, anemia, and increased serum creatine kinase concentration. postmortem reveals characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa. antemortem diagnosis of equine proliferative enteropathy is based on clinical signs, hypoproteinemia, and the exclusion of other common enteric pathogens. fecal polymerase chain reaction analysis may be positive for the presence of l. intracellulare, and affected foals develop antibodies against l. intracellulare. treatment with erythromycin estolate alone or combined with rifampin for a minimum of days is recommended with additional symptomatic treatment when indicated. cryptosporidium spp. cause gastroenteritis and diarrhea in many animal species and are not host-specific. cryptosporidium has been implicated as the casual agent of diarrhea in foals, but the organism is isolated from the feces of diarrheic foals and normal foals with the same frequency and concentration, making a clear role for the organism difficult to elucidate. - diarrhea caused by cryptosporidium in other species and that described for foals is generally self-limiting, with a clinical course of between to days. immunosuppressed patients, including foals compromised by concurrent disease, are thought to be at increased risk for complications resulting from infection with this organism. , treatment is symptomatic. cryptosporidiosis is a disease with zoonotic potential, and one should take appropriate precautions, including use of gloves and frequent hand washing, if organisms are identified in the feces of any patients so as to prevent spread to other patients and personnel. eimeria leukarti, trichomonas equi, and giardia equi have been identified in the feces of normal horses and horses with diarrhea. transmission studies have failed to produce reliable clinical signs, and the prevalence and significance of these organisms in the genesis of foal diarrhea remain unknown. strongyloides westeri is a common parasitic infection of foals. , transmission is transmammary, and patent infection is recognizable in the foal by to days of age. this nematode previously was associated anecdotally with foal heat diarrhea, but the association has not been demonstrated clearly. the diarrhea is generally mild and is treated effectively by deworming with benzimidazole or ivermectin anthelmintics. strongylus vulgaris fourth-stage larvae cause diarrhea in young foals during migration through the arterioles of the cecum and descending colon. clinical signs may resemble thromboembolic colic. the prepatent period is about months, and diagnosis is based on clinical examination, clinicopathologic changes, and farm deworming history. patients with diarrhea associated with this parasite may have peripheral leukocytosis, neutrophilia, eosinophilia, and hypoproteinemia. appropriate deworming with ivermectin (label dose), fenbendazole ( mg/kg/day orally for days), or thiabendazole ( mg/kg/day orally for days) is recommended, with the last two drug dosages being larger than the label dose. cyathostomiasis, or diarrhea resulting from the sudden emergence of encysted cyathostome larvae, is an unusual cause of diarrhea in the foal. the clinician managing critically ill neonates must recognize that intravenous fluid therapy simply cannot be scaled down from adult management approaches. fluid management of the ill neonate, particularly over the first few days of life, must take into consideration that the neonate is undergoing a large transition from the fetal to the neonatal state and that important physiologic changes are taking place. these transitions include shifts in renal handling of free water and sodium and increased insensible losses because of evaporation from the body surface area and the respiratory tract. the newborn kidney has a limited ability to excrete excess free water and sodium, and the barrier between the vascular and interstitial space is more porous than that of adults. water and sodium overload, particularly in the first few days of life, can have disastrous long-term consequences for the neonate. , in the equine neonate, excess fluid administration frequently manifests as generalized edema formation and excessive weight gain, frequently equivalent to the volume of excess fluid administered intravenously. in cases in which antidiuretic hormone secretion is inappropriate, as in some foals with pas, generalized edema may not form, but the excess free water is maintained in the vascular space. this syndrome of inappropriate antidiuretic hormone secretion is recognized in the foal that gains excessive weight not manifested as edema generally, with decreased urine output and electrolyte abnormalities such as hyponatremia and hypochloremia. the foal manifests neurologic abnormalities associated with hyponatremia. the serum creatinine concentration varies in these cases, but urine always is concentrated compared with the normally dilute, copious amounts of urine produced by foals more than hours of age on a milk diet. if measured, serum osmolarity is less than urine osmolarity. the treatment for this disorder is fluid restriction until weight loss occurs, electrolyte abnormalities normalize, and urine concentration decreases. if the clinician is unaware of this differential diagnosis, the neonate can be assumed mistakenly to be in renal failure, and the condition can be exacerbated by excessive intravenous fluid administration in an attempt to produce diuresis. the problem of appropriate fluid management in critically ill neonates has been recognized by medical physicians for years and has resulted in changes in fluid management of these patients. the approach taken has been one of fluid restriction, in particular sodium restriction but also free water restriction, and has resulted in improved outcome and fewer complications, such as patent ductus arteriosus and necrotizing enterocolitis. , the calculations used for maintenance intravenous fluid support in these patients takes into consideration the ratio of surface area to volume and partially compensates for insensible water losses. maintenance fluids are provided as % dextrose to limit sodium overload and provide sufficient free water to restore intracellular and interstitial requirements. the calculation for maintenance fluid administration is as follows: first kg body mass ml/kg/day second kg body mass ml/kg/day all additional kilograms of body mass ml/kg/day as an example, the average -kg foal would receive ml/day for the first kg of body mass, ml/day for the next kg of body mass, and ml/day for the remaining kg of body mass for a total of ml/day. this translates to an hourly fluid rate of about ml/hr. one should adjust the fluid and sodium requirements for ongoing losses exceeding the maintenance requirements. these losses can take the form of diarrheal losses and excessive urine output, such as those with glucose diuresis and renal damage resulting in an increased fractional excretion of sodium. the normal fractional excretion of sodium in neonatal foals is less than that of adult horses, usually less than % (j.e. palmer, unpublished data). in the critically ill foal the sodium requirement can be met with as little as meq of sodium per day, about that administered in a single liter of normal equine plasma. one can address sodium deficits by separate infusion of sodium-containing fluids, although this may not be necessary if one considers the sodium being administered in other forms, including drugs administered as sodium salts and any constant rate infusions (pressors, inotropes, etc.) that are being provided as solutions made with . % sodium chloride. the author has used this approach to fluid therapy in her nicu for the last few years and believes that the percentage of foals suffering from generalized edema and related problems has decreased. if one takes this approach to fluid therapy, one should take the weight of the patient once daily, or even twice daily, and monitor the fluid intake and output as closely as practical. one should evaluate any larger than anticipated weight gains or losses. one should not expect urine output to approach the reported normal of ml/hr for a -kg foal because the free water administered is limited, unless the patient is experiencing diuresis (glucosuria, resolution of the syndrome of inappropriate antidiuretic hormone secretion, resolution of previous edematous state, renal disease). one should obtain the urine specific gravity several times daily and should determine fractional excretion of sodium at regular intervals. if the volume of urine produced by the patient is measured accurately, one can determine sodium losses accurately and can obtain creatinine clearance values. one should obtain blood pressure measurements at regular intervals throughout the day because hypotension can be a problem in these patients, particularly in septic foals and foals suffering from pas, and one may need to increase fluid therapy to maintain adequate vascular volume. patients with hypotension may need inotrope and pressor support. inotrope and pressor therapy generally is restricted to referral centers where these drugs can be administered as constant rate infusions and blood pressure can be monitored closely. blood pressure can be monitored directly or indirectly by the use of cuffs placed on the base of the tail. both techniques have advantages and disadvantages. although direct blood pressure measurements are considered the gold standard and are generally more accurate, the difficulty in placing and maintaining arterial catheters and lines in these patients severely restricts the utility of this method. indirect techniques can be inaccurate and are affected by cuff size and placement. however, indirect techniques are easier to use in the nicu and can be useful if trained staff are using the equipment. in the author's nicu, once practitioners identify the appropriate cuff size, they dedicate that cuff to that patient for the duration of the hospitalization to decrease variability caused by using different cuffs. one should monitor the blood pressure of all recumbent patients at regular intervals, and trends upward or downward should prompt the clinician to make necessary adjustments. foals suffering from pas and sepsis are the patients most at risk for significant hypotension and perfusion abnormalities. perfusion is maintained by supporting cardiac output and blood pressure with judicious use of intravenous fluid support and inotrope/pressor support. the author does not aim for any specific target systolic, mean, or diastolic pressure. instead the author monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for nicu patients to require inotrope and pressor therapy is not unusual, but in some cases hypoxic and septic damage is sufficiently severe to blunt the response of the patient to the drugs. one must approach each patient as an individual, and no single inotrope/pressor protocol will suffice for all patients. dobutamine is a β-adrenergic inotrope that is frequently used as first choice therapy in nicu patients. its effects are β at the lower dose range. neonates have a limited ability to increase stroke volume in an effort to maintain cardiac output, and one may observe tachycardia in these patients as heart rate increases to maintain cardiac output and vascular pressure. dobutamine is useful after patients are volume replete for support of cardiac output. the dose range is between to µg/kg/min provided as a constant rate infusion. dopamine has dopaminergic activity at low doses, β and β activity at moderate doses, and α activity at high doses. dopamine causes norepinephrine release, which has lead to the suggestion that this is its major mode of action at higher doses. at doses greater than µg/kg/min, intrapulmonary shunting, pulmonary venous vasoconstriction, and reduced splanchic perfusion may occur. dopamine also produces natriuresis at lower doses through a direct effect on renal tubules. for these reasons, dopamine has fallen out of favor at some referral institutions. norepinephrine has α and β activity but variable β activity, resulting in potent vasopressor effects; it has inotropic and chronotropic effects, but its chronotropic effect usually is blunted by vagal reflexes slowing the heart rate induced by the increase in blood pressure. in many critical care units, norepinephrine has become a pressor of choice and frequently is used along with dobutamine. evidence suggests that splanchic perfusion is maintained better with norepinephrine than with some other pressors. the dose range is . to . µg/kg/min, although larger doses have been used when necessary in certain patients. epinephrine has α , α , β , and β activity; β activity predominates and results in increased cardiac output and decreased peripheral resistance at low doses. epinephrine has been associated with hyperglycemia, hypokalemia, lipolysis, increased lactate concentration, and increased platelet aggregation. the effect on renal function is controversial. use of epinephrine usually is limited to those patients not responding to other pressors. vasopressin (antidiuretic hormone) is a pressor gaining a great deal of attention in the critical care literature. vasopressin appears to be depleted from the neurohypophysis in septic shock, and short-term administration of vasopressin spares conventional vasopressor use, in addition to improving some measures of renal function. low-dose vasopressin infusion increases mean arterial pressure, systemic vascular resistance, and urine output in patients with vasodilatory septic shock that are hyporesponsive to catecholamines. these data indicate that low-dose vasopressin infusions may be useful in treating hypotension in patients with septic shock. the author has been using low-dose vasopressin in patients in her nicu for the past few years and has the clinical impression that blood pressure is defended more readily using this agent in concert with other management strategies. the author commonly uses low-dose vasopressin constant rate infusion with dobutamine constant rate infusion as the initial inotrope/pressor therapy in cases requiring pressure defense, although no prospective studies are yet available regarding this drug in veterinary medicine. for quality health care for animals, advances in medical science, and in some breeds the increasing value of the juvenile equine athlete. equine veterinarians that encounter pediatric orthopedic problems are only beginning to get the information needed to make appropriate treatment decisions. the equine neonate has specific differences in structure and physiology from adults that one must consider when designing an optimal therapeutic or management strategy. few investigations have focused on the equine neonatal musculoskeletal system, - but a large body of clinical information exists, and one can make cautious extrapolations from work in other species. neonatal equine bones have accelerated modeling and remodeling processes that result in accelerated fracture healing and an increased susceptibility to deformation caused by excessive loading. contralateral limb varus deformities of the growth centers (most commonly distal radius and metacarpus/ metatarsus) are common in overloaded limbs. the increased plasticity of the skeletal structure also is mirrored in the soft tissue support system, for these units become flaccid within weeks of immobilization. this laxity is important, because it further compromises the use of the fractured limb and can last as long as the coaptation was in place. additional divergences from adult physiology include musculoskeletal immaturity (generalized or focal) and immune system differences. finally, foals are lighter and can tolerate and will assume recumbency more readily than adults. the net results of these differences are that one must consider the use of external coaptation carefully, fractures heal quickly, one must consider damage to the contralateral limb from overstress, reducing weight bearing is possible, and infection is always lurking. stresses can affect the musculoskeletal system of the foal at any time, including in utero. although rare, reports describe in utero fractures (k. sprayberry, personal communication, ) that result in foal locomotor problems and even maternal uterine damage from sharp bone ends. the cause is presumably from vigorous muscular activity of the foal, but one cannot rule out direct trauma. the fractures result in foal lameness and can increase the likelihood of dystocia and caused colic in one mare when the broken bones damaged the uterus. treatment depends on how long the fracture has been present and on the fracture location and configuration, but if the fracture is repairable, internal fixation probably is necessary. fractures occurring during foaling result from aggressive obstetric manipulation (mandibles) or the advances in medical care of equine neonates in the last years have resulted in the survival of many foals that previously would have died from sepsis, asphyxia, and prematurity; and the successful management of their musculoskeletal system can be a major challenge. major factors adding to the challenge are the immaturity of components of the musculoskeletal system and the demands placed on them by a growing and active foal. additional pressures to treat orthopedic conditions in foals have come from an overall increase in the demand chest compression. one should stabilize unstable mandibular fractures. appendicular fractures usually do not occur during parturition because of the robust character of the bones of the foal. after birth, foals are susceptible to external trauma from many sources. the dilemma is that younger foals with fractures are more likely to heal but also are more likely to develop contralateral limb problems because of excessive weight bearing and affected limb flexor tendon laxity if the limb is immobilized fully. as a result, internal fixation is often the best choice for neonatal fractures to keep the fractured limb in use. proximal sesamoid bone fractures result from hyperextension of the fetlock joint. foals are lame after the fracture, but the lameness can be mild and often diminishes quickly. soft tissue swelling occurs over the sesamoids. fractures are usually simple, can occur uniaxially or biaxially, and can be apical, midbody, or basilar. fractures can occur in any joint and can affect multiple sesamoids in one foal. however, they most commonly are single forelimb fractures and in thoroughbreds are most frequent in the left front medial proximal sesamoid (j.p. morehead, personal communication, ). of particular interest to neonatologists is that proximal sesamoids fractures often occur in recovered neonatal patients that are allowed too much exercise too soon. foals from the nicu need a gradual introduction to pasture turnout to allow their musculoskeletal system to adjust. mares are often in need of turnout, but in the interest of their foals, they must wait. treatment of proximal sesamoid fractures in foals is stall confinement with support bandaging. healing occurs, albeit with some distortion of the shape of the sesamoid. severely displaced fragments result in large and misshapen sesamoids, and surgery may be considered for these foals, because restriction of fetlock flexion can occur after conservative therapy. third phalangeal fractures are also common in foals. these foals have a lameness that worsens with hoof compression. hoof abscesses are uncommon in young foals but should be considered. most commonly, radiographs reveal nonarticular small fractures on the wings on the third phalanx. the fractures are associated with hard ground and exercise. the fractures heal with stall confinement, and unlike adults, leave no discernable radiographic fibrous union. avulsion fractures of the proximal insertion of the peroneus tertius and the origin of the long digital extensor tendon have been reported. , both soft tissue structures attach to the extensor fossa of the distal femur. the two affected foals had lameness of a hindlimb associated with swelling, pain, and crepitation. radiographs revealed multiple avulsion fractures of the extensor fossa. because of the intraarticular fragments in the femoropatellar joint, and the fear of later degenerative joint disease, fragments were removed arthroscopically. both foals were juveniles at last follow-up; one foal was considered normal, and one had a mild residual lameness. tendon and ligament damage is uncommon in neonates probably because of their low body weight. extensor tendon damage following flexural deformities is the most common tendon problem and is discussed in congential flexural deformities of foals. gastrocnemius ruptures are one of the most devastating problems and have occurred after forced extraction because of a breech presentation, severe flexor tendon laxity, and tarsal contracture. loss of gastrocnemius function usually results in a non-weight-bearing limb, although an intact superficial digital flexor tendon may make some weight bearing possible. complete loss of support is difficult to treat successfully. coaptation of the limb is logical but difficult to obtain. schroeder-thomas splints have been used but are difficult to manage. tube casts also are used but must be changed frequently, and cast sores are inevitable (l.r. bramlage, personal communication, ) . the prognosis for athletic function is guarded. treatment for ligamentous injuries is usually some form of coaptation, although surgical repairs have been performed when coaptation was unworkable. coaptation in proper limb alignment allows the ligaments to heal and should be used if the injury will destabilize a joint and cause damage to growing epiphyses or cuboidal bones. one can achieve coaptation with casts or splints under a bandage. casts are initially a greater expense, and cast sores and their resulting white hairs are a risk, but the rigid immobilization and the lack of the requirement for daily adjustment makes them preferable. important to musculotendinous health is some measure of weight bearing to avoid laxity after coaptation removal, which one can achieve by using tube casts and splints that allow weight bearing. following coaptation, bandaging and a gradual return to exercise are recommended for ligamentous injuries. patellar luxation can affect foals in one or both hindlimbs, and the luxation can vary from a laxity in the medial attachments to complete luxations that cannot be replaced in the patellar groove of the distal femur. , medial luxations have not been reported. clinical signs vary from a slight discontinuous motion during stifle flexion to an inability to stand. many foals have a crouching stance on the affected limb because of an inability to extend the stifle. the pathophysiology of patellar luxations is unknown. congenital bilateral luxations are common in miniature horse foals and are believed to be genetic. luxations are rarer in other breeds and are occasionally traumatic. the affected limbs are usually not grossly abnormal except for effusion of the femoropatellar joint and the luxation. a shallow trochlear groove has been reported to be a cause of patellar luxation, but objective evidence is lacking. one should evaluate foals for the ability to stand. once the appropriate supportive care is provided, if a foal cannot stand, euthanasia is recommended. most bilateral luxations in horses fit in this category. however, miniature horse foals often can stand sufficiently to nurse despite bilateral luxations, and one may consider treatment. treatment consists of replacing and stabilizing the patella and sometimes surgically deepening the patellar groove. delaying surgical repair until the foal is approximately days old is recommended to avoid neonatal problems, allow the musculoskeletal system to mature, and provide good anchors for suture. some surgeons worry that delay may cause further femoropatellar developmental abnormalities, but in a small number of cases, this has not been an issue. the prognosis for miniature horse foals appears to be good because of their low body weights and modest performance expectations. too few reports about the correction of unilateral luxations in light horses exist to make a definitive statement about prognosis except that success and failure have been experienced. , congenital flexural deformities in foals can be classified as severe (rarely correctable), moderate (correctable with therapy), or mild (self-correctable). examples of severe flexural deformities include arthrogryposis (deformities of multiple limbs and often the head and neck), severe carpal deformities (flexor angle of the carpus less than degrees), and tarsal contractures (rare). extraordinary methods have been used to correct severe deformities but are often unsuccessful. mild flexural deformities are those that result in an upright conformation to the limb, but the foal can bear weight on the limb and load the flexor structures. these foals require no specific treatment and will self-correct with controlled exercise. moderate flexural deformities are those that make bearing weight on the limb and loading the flexor structures and ligaments difficult for the foal. when these deformities occur bilaterally (most common), the foals cannot rise to suckle or does so with great difficulty, and the lack of weight bearing worsens the flexural deformity. examples of moderate flexural deformities include carpal and forelimb fetlock flexural deformities that usually occur together, hindlimb fetlock flexural deformities with coronopedal flexion or hyperextension, and the uncommon coronopedal flexural deformity alone. treatment of moderate flexural deformities aims to place the solar surface of the foot on the ground so that the weight of the foal can stretch the flexor structures. splints are useful for restoring the limb to normal orientation but require attention to detail because the splints often exert an extreme amount of tension on the soft tissues, and the skin of the foal is thin. pressure sores are easy to create and at a minimum result in an extended convalescence. the first step in splint application is to apply a separate heavy bandage to the limb, which should be reapplied as necessary because the bandage can slip and cause focal constriction. commercial gauze over cotton bandage material works better than sheet cotton as a bandage. the splint is made of polyvinyl chloride pipe cut in half or thirds. using % of the diameter of the pipe results in less splint rotation but is bulkier and leaves more splint exposed to cause trauma. one cuts off the corners of the splint and pads the ends with gauze or roll cotton covered with tape. palmar or plantar placement of the splint is preferable, but severe deformities may require initial dorsal placement. as the limb straightens, one can bend the splint to tape the fetlock into the bend to extend it. one can tape the splint tightly to the limb over the bandage with nonelastic (white or duct) tape. this procedure requires at least two persons, one to extend the limb firmly and hold the limb and one to tape. one should leave the splint on for to hours and then remove it for to hours. one can reapply splints as necessary. in addition to splints, some medications are of value for treating flexural deformities. oxytetracycline ( to mg/kg) given intravenously appears to relax the soft tissues. the mechanism of action is unknown, and the drug is most efficacious when given in the first days of life. this dose is high but appears to be safe for healthy foals and can be repeated at -hour intervals. foals should be normovolemic during tetracycline administration. one should use the drug with caution in foals with renal impairment. foals should be urinating and have reasonable urinary parameters (serum urea nitrogen, creatinine, and urinalysis) before tetracycline use. diarrhea is an uncommon sequela to tetracycline use. one should monitor the unaffected limbs closely because all limbs experience a relaxation of the palmar/plantar support. , discontinuation of tetracycline therapy before affected limbs are normal but after they can bear weight is common because of worsening laxity in the "normal" limbs. one also can use phenylbutazone ( mg/kg) for a short time when the splints are used. some analgesia appears to help the foals use the limbs and stretch the soft tissues. one should not use phenylbutazone for long periods of time because of the potential of inducing gastric ulcers. surgical treatment of congenital flexural deformities rarely is indicated. severely affected foals rarely respond favorably to surgery, and mildly affected foals do not need it. surgery is most appropriate for foals with moderate flexural deformities that are neglected or have not responded to splinting and tetracycline. the most common surgical therapy performed for congenital flexural deformities is the inferior check ligament desmotomy for fetlock or coronopedal flexural deformities. ruptures of the extensor tendons commonly occur with congenital flexural deformities and result from the foal overloading the extensor tendons. no specific therapy for the ruptures is necessary. if the rupture is extensive, it can interfere with the ability to extend the fetlock and to place the foot flat. these foals then tend to knuckle over, even after correction of the flexural deformity. a firm fetlock bandage extends the digit and assists in foot placement until the extensor tendons heal. foals commonly are born with hyperextension deformities of the fetlock of varying degrees of severity. all but the worst deformities self-correct as muscle tone improves. a deeply bedded stall is all that is usually necessary to protect the soft tissues, but one can apply a light bandage to the coronary band and pastern if trauma is a problem. severe deformities are more problematic but rare, so therapeutic recommendations are not available. hyperextension of the carpus occasionally occurs and usually is treated conservatively. however, a tube cast to align the limb may be necessary to protect the dorsal surface of developing carpal bones. neonatal foals exhibit three categories of forelimb conformational deviations: angulation, rotation, and carpal offset. angular deviations most commonly are centered in the metaphysis and epiphysis, but their location is described by the closest joint, usually the carpus and fetlock. when the deviation of the distal limb is lateral to the long axis, the deviation is valgus, and when the deviation is medial, the deviation is varus. more than one joint can be affected, and although rare in neonates, valgus and varus can occur in different joints in one limb. rotational deformities appear to originate most commonly in the diaphysis or metaphysis of the radius or the metacarpus. in neonates the direction of rotation of the distal limb at both sites is almost exclusively outward. associated angular and rotational deviations occur. in neonates, limb deviations occur in foals with narrower chests and less developed pectoral muscles than in straight foals, and they appear to have an initial greater overall weakness in the musculoskeletal system because it first interacts with gravity, body mass, and ground reaction forces. however, after the first few days of life, the asymmetric loading of the growth centers does affect limb deviations. angulation results from a compressive load that is asymmetric in a frontal plane but is uniform in the sagittal plane, and rotation occurs when the compressive load is asymmetric in both planes and the limb develops around an overloaded axis point. considered this way, valgus and outward rotation deviations in young foals are coupled, as are varus and inward rotation in older foals. the loading asymmetry for valgus/outward rotation foals is accentuated as foals assume a base-wide posture that is more stable side-to-side but promotes a lateralization of the limb load. the specific effects of intermittent versus static loads, strain magnitude versus strain rate, and shear and hydrostatic stress on growing bones is only beginning to be understood. however, clinical experience supports the general observation that excessive cartilage compression is deleterious to bone growth. offset carpal conformation describes a joint that appears to deviate outwardly and then inwardly, all within the carpus. the deformity is thought to be centered at the radiocarpal joint, but the specific structural cause of offset has not been determined. this conformation is more common in older foals but occasionally occurs in neonates. the deviation is particularly common when incomplete ossification of the carpal bones is present. the causes of conformational deviations are a matter of some debate. as always, the major factors are genetics or environment. genetic influences include the assortment of alleles that controls bone form and growth and the assortment that modulates bone remodeling. many in the horse industry believe that genetics is a strong determiner of limb conformation. environmental influences are many and include the intrauterine environment, the postnatal limb load, nutrition, and bad luck. suffice to say, the situation is complex, but one must consider biologic and mechanicobiologic influences when evaluating the growth of long bones. several factors may contribute to the common occurrence of deviations in the carpus. first, the carpus is in the middle of the limb and is subject to the greatest bending forces. second, the carpal anatomy is complex and perhaps is not understood completely. the carpus has seven cuboidal bones, two long bones, and two epiphyses (distal radial and lateral styloid); and cartilage surrounds all. the ligamentous support includes collateral ligaments, innumerable intracarpal ligaments, and a palmar carpal soft tissue ligament. the distal radial physis is not flat transversely, but undulates in the frontal and sagittal planes. a separate center of ossification for the lateral styloid process is found at its palmar-lateral aspect. because of this separate center of ossification, more cartilage and less bone are in the lateral aspect of the distal radial growth center, suggesting it may be more susceptible to growth alterations from load. less common conformation deformities in young foals include hindlimb deformities, windswept conformation, diaphyseal deviations (usually of the metacarpus/ metatarsus), gross congenital malformations such as agenesis and polydactyly, and acquired varus deformities of the carpus and fetlock. hindlimb conformational deviations can manifest as tarsal and fetlock angular deformities and external limb rotation, usually centered above the tarsus. windswept foals have limbs (usually both forelimb or both hindlimbs) that are curved in the same direction in the frontal plane. diaphyseal deviations, agenesis, and polydactyly are rare and have various presentations. acquired varus deformities are caused by excessive loading, which appears to be focused medially on the growth plates. one should evaluate the limbs to determine the location, extent, and potential cause of the deviation. evaluation consists of observation and then palpation for heat, swelling, or ligament laxity. ligamentous laxity of the medial carpal ligaments is an important cause of carpal valgus and should be evaluated carefully. lameness is not a characteristic of uncomplicated angular limb deformities and suggests further evaluations are necessary. radiography is indicated for foals with severe deviations (all tarsal valgus), ligamentous laxity, lameness, or joint effusions. ultrasonography may be valuable for selected soft tissue evaluations. conservative therapy is by far the most commonly used therapy in foals less than days of age. mild to moderate carpal valgus and external rotation of the carpus and fetlock are common and normal in neonates, particularly light breed horses. most congenital limb deviations improve with age, if the developing musculoskeletal system is protected from overuse and abnormal loads. approximately % of thoroughbred foals with congenital carpal valgus self-correct. those foals that do not most often have abnormal bone (incomplete ossification) with normal stress or normal bone with abnormal stress (ligamentous laxity or contralateral limb lameness). correction continues for several months, and on average, foals reach their straightest conformation (regarding angulation) at approximately months of age (e.m. santschi, unpublished data). determination of the appropriate treatment for foals with angular limb deformities is based on the age of the foal, the severity and location of the deviation, and its causes. one must evaluate the entire foal and the affected limb. if the carpal collateral ligaments have no laxity and carpal incomplete ossification is not suspected, one may use an exercise program such as in table - , assuming that the foal has no contradicting additional problems. exercise is essential for the robust development of almost every body system for neonates, and fresh air and good ventilation reduce the occurrence of respiratory disease. appropriate limb loading along with growth and maturity is what straightens limbs, but excessive amounts of loading can be deleterious. for example, one should use exercise cautiously in foals with very asymmetric deviations. when one limb is much more deviated than the other, it appears to be loaded excessively and compromised more than if both limbs were affected similarly. and finally, limb deviations are additive. foals with external rotation and carpal valgus improve more slowly than those with one type of deviation. incomplete ossification of cuboidal bones and focal ligamentous laxity are complicating matters of great potential impact on adult conformation. they generally manifest as a moderate to severe limb deviation. physical examination indicates laxity because angular limb deviations are reducible. radiographs are the best way to evaluate the extent of carpal bone ossification. incomplete ossification of the cuboidal bones can be focal or widespread. focal immaturity is not common but can result in severe angulation. generalized immaturity is more frequent and initially often manifests as an offset conformation with valgus angulation. when the foal becomes heavier, assumes a base-wide stance, and is allowed exercise, crushing of the bones of the lateral carpus (usually the lateral styloid process of the radius, the ulnar, the fourth and the intermediate facet of the third carpal bone) results in a permanent intracarpal valgus deviation. the same result occurs when significant medial carpal ligament laxity goes untreated. in the forelimb, foals with collateral ligamentous laxity and moderate to severely immature cuboidal bones should have external coaptation placed on the affected limb to maintain axial orientation. tube casts that allow weight bearing on the digit are preferred to splints. ligamentous laxity in the carpus usually responds to tube casting for to days followed by bandaging and cautious exercise. the duration of similar coaptation necessary for immature carpal bones depends on the degree of immaturity and the speed with which the bones mature. because casts cannot be left on neonatal limbs for more than to days because of their fast growth, more than one cast may be necessary. treatment of tarsal valgus and rotational deformities is much less common than in the forelimbs because deviations are less common than in the forelimb, because some breeds prefer an outward position to the hindlimb, and perhaps because owners recognize it less frequently. hindlimbs generally are unaffected by ligamentous laxity, but tarsal incomplete ossification is common and often is associated with tarsal valgus. treatment of tarsal incomplete ossification is important because tarsal crushing results in an unfavorable prognosis for athletic performance. , hindlimbs require a slightly different approach to coaptation than forelimbs because of their anatomy. foals can rise to stand if their forelimbs are fixed in extension but cannot do so if their hindlimbs are extended. the multiple bony protuberances of the hock make cast sores more likely than in the forelimb, so casts are problematic. gutter splints are not useful because of the angle of the hock. severely limiting exercise is part of allowing the tarsus to mature without cartilage crushing, but foals cannot always be recumbent. extra small articulated anterior cruciate ligament splints for human beings (playmaker wraparound, dj orthopedics, vista, california) have given the best results. for small foals, a padded bandage is necessary under the splint, which is reversed to conform to the angle of the hock. the splints allow enough flexion in the hock for the foal to rise but appear sufficient when combined with stall rest to protect the cartilage from crushing. splints are left on the hocks until the cuboidal bones have ossified as shown by radiography. fetlock conformational deviations in neonates that are treated best conservatively are rare. outward rotation is the most common deviation but is thought to have minimal effect on the performance and improves with maturity. the only therapy used is to rasp the toe square to promote central breakover. severe outward rotation can promote a fetlock valgus conformation, so one can use a medial hoof wall extension of epoxy to bring the limb load medially. the most commonly treated fetlock deviations are inward but usually occur in foals older than days. however, if the deviation is noticed in neonates, one can use small lateral hoof wall extensions that generally are made of epoxy with fiberglass cloth embedded to prevent chipping. windswept foals are born with multiple deviations. evaluating the foal as a whole is best rather than focusing on individual joints. most of these foals become straight over time with conservative therapy. no surgical procedures are commonly accepted for direct treatment of rotational or carpal offset deviations, so angular deviations are described. surgical procedures to correct carpal and fetlock valgus include periosteal transection and elevation and transphyseal bridging. periosteal elevation is thought to accelerate growth on the concave side of the metaphysis, and transphyseal bridging is used to restrict the growth on the convex side of the physis. studies indicate an approximately % improvement of carpal valgus foals after periosteal transection and elevation, but unfortunately they do not compare foals that had surgery with controls that did not. , recently, some have suggested that most of the correction was unrelated to the surgery, and one experimental study supports that conclusion. as a result, at this time making firm recommendations about the indications for periosteal transection and elevation is difficult. however, periosteal transection and elevation has a low likelihood of complications and may be effective. the procedure is inexpensive and can be done in the field and therefore may be an option for clients with foals with carpal valgus in which a transphyseal bridging is undesirable or unnecessary. one indication is the very young foal born with a notably asymmetric epiphysis that results in a severe carpal valgus. this distal radial appearance is not particularly common, but the lack of ossification in the epiphysis can make a firm hold with a transphyseal bridging difficult to achieve. however, one can use distolateral radial periosteal elevation at an early age in an attempt to accelerate correction of the valgus and protect developing carpal bones. often a degree of anxiety exists about correction of fetlock angulations because of the much shorter time period for physeal growth. most fetlocks are in their final conformation by days of age, so correction is best accomplished with earlier treatment, usually by weeks of age. one can perform periosteal elevation on the medial (for varus deviations) or lateral (for valgus deviations) aspect of the distal metacarpus/metatarsus. the definitive treatment of limb angulation at a growth plate is transphyseal bridging. one should consider using the procedure at about weeks of age for all moderate to severe fetlock deviations, at about weeks for severe carpal deviations, and to weeks for mild fetlock deviations, moderate carpal deviations, and any worsening angular deformities. one must perform bridge removal when the limb straightens to prevent overcorrection. diaphyseal deviations are rare but can occur in varying degrees of severity. if the foal can bear weight on the limb, a conservative approach is indicated. one can consider periosteal elevation of the length of the concave surface of the long bone. if the foal cannot bear weight on the limb because of the severity of deviation, euthanasia is probably the best option. however, a revision osteotomy and internal fixation may be appropriate for selected foals. polydactyly is also rare and sometimes can be corrected surgically. the outcome is based on the degree of articular involvement. bacteria may invade the foal musculoskeletal system and cause orthopedic infection after delivery by the circulation, by direct extension from another system, or by direct inoculation. hematogenous delivery is by far the most common and results in infection of synovial structures (joints, tendon sheaths, bursae) and bone. extension from another site without hematogenous delivery is rare. direct inoculation almost exclusively results from traumatic rather than surgical wounds. much is still to be learned about the pathophysiology of orthopedic infection, including the source of the infecting bacteria. the umbilicus commonly is accepted as a possible source of bacteria, but many believe that the gastrointestinal and respiratory tracts are at least equally responsible. associated conditions in foals with septic arthritis include failure of passive transfer, pneumonia, and enteritis. the classification of orthopedic sepsis in foals into infection of bones and joints is probably irrelevant because most foals with septic arthritis also have infectious osteitis or osteomyelitis. , septic arthritis is more readily recognizable because the reactivity of the synovium to the bacteria causes joint effusion and lameness and because early radiographic signs of bone infection in foals are equivocal. also unclear are the reasons for the apparent site predilection for orthopedic infection in foals. the femoropatellar joint and the tarsocrural joint are affected most frequently, followed by the carpal and fetlock joints, and finally an assortment of miscellaneous joints such as the elbow, shoulder, and hip. the common association of osteomyelitis of the distal femoral, tibial, and metacarpal/metatarsal physes with a newly recognized septic arthritis suggests that the infection in that area started at the growth center (epiphysis, physis, or metaphysis). the localization of the apparent initial site of infection to the growth center has been suggested to result from "looping" metaphyseal vessels with sluggish blood flow that allow pathogens more time to escape the circulation. , however, transmission electron microscopy indicates that osteogenic cells and the vascular endothelium are a continuous network in developing embryos, indicating that the relationship between circulation and bone is more intimate than previously suspected. a possible association between osteomyelitis and thickened or traumatized cartilage exists. focal osteomyelitis lesions occur commonly at the bone cartilage junction , and particularly in areas where cartilage is attached at an angle to the long axis or where thickened. an association also exists between incomplete ossification of the central and third tarsal bones and osteomyelitis. trauma to the metaphysis is a known predisposing cause of osteomyelitis in young bacteremic rabbits. a trend exists for foals with more than one joint affected to be affected bilaterally in the same joint, rather than in random joints. this trend suggests that a "window" exists when a joint may be more susceptible to infection and that trauma to the developing cartilage may be a contributing factor. in neonates, cartilage is vascular, and possibly small traumatic cartilage lesions with associated hemorrhage and exposure of bacterial binding sites might be the inciting cause for the location of infection. the pathogens most commonly associated with septic arthritis in young foals are also those that frequently are implicated in neonatal sepsis. the most commonly isolated gram-negative organisms are escherichia coli and other enterobacteriaceae, actinobacillus equuli, and salmonella spp. frequently isolated gram-positive organisms include streptococcus spp., staphylococcus spp., and rhodococcus equi. anaerobic bacteria and fungi are rare but should be considered in refractory cases. the diagnosis of orthopedic sepsis can be challenging. the most common clinical sign is lameness, followed by swelling around a joint or metaphysis. joint effusion alone may cause the swelling, but edema is also common, especially if metaphyseal osteomyelitis is present. but effusion and edema can be difficult to detect because of the tissue surrounding the focus of infection in the shoulder, elbow, hip, and coffin joints. one should evaluate lame foals carefully by palpation to localize pain and swelling. if one can find no pain or swelling, one should obtain a complete blood count and fibrinogen level. although a complete blood count is not always abnormal in foals with septic arthritis, abnormalities should raise the index of suspicion of infection. elevations in fibrinogen are fairly common in septic arthritis, and fibrinogen almost always is elevated if the infection involves bone. if hematologic values are normal, the lameness could be caused by trauma, but the foal should be monitored closely for improvement, and closer evaluation is indicated if improvement is not rapid. an arthrocentesis is the diagnostic test of choice for confirmation of septic arthritis. one should perform joint puncture in a sterile fashion, and sedation is indicated to get an atraumatic tap. short-term anesthesia is preferable when joints have effusion because one may perform joint lavage at the same time. normal joint fluid should be clear to slightly yellow, should be viscous, and should contain less than nucleated cells per deciliter. the cell ratio should be roughly : polymorphonuclear and mononuclear. the total protein content should be less than . mg/dl. one should consider joints to be infected if the nucleated cell count is greater than , cells/dl. for joints falling between and , cells/dl, if the polymorphonuclear cell count is > %, one should consider the joints infected. cytologists are often reluctant to diagnose infection when nuclear degeneration or bacteria are not visible. this is overly conservative and results in delay in treating infections because bacteria and nuclear degeneration are rare in early cases of joint infection. out of an abundance of caution, one should treat lame foals with suspicious joints as infected unless they are clearly normal. one should always culture joint fluid in an attempt to identify the offending organisms, but because of difficulties in culturing pathogens from joint fluid samples, absence of growth does not mean absence of infection. one obtains the best culture results if the foal has not been treated with antimicrobial agents beforehand. one should obtain as much joint fluid as possible for culture and should incubate it overnight in blood culture media before plate inoculation. as always in potentially septic foals, blood culture may assist in the isolation of the organism. other orthopedic infections that do not involve the joint may be more difficult to detect. often these are not apparent until infection breeches the joint and causes lameness. however, astute caretakers may notice early clinical signs such as mild lameness, fever, or edema centered at a growth center. radiography and advanced imaging modalities such as magnetic resonance imaging are the best diagnostic tools for the localization of areas of osteitis and osteomyelitis. one should examine the area of concern carefully, giving particular attention to the growth centers and subchondral bone. interpretation of radiographs may be difficult because these areas are complex and normally have irregular bone margins in the growing foal. if a normal contralateral joint is available, comparison radiographs may be useful. because of the high metabolic turnover in growing foal bone, changes occur faster than with adults, so radiographs at the earliest sign of potential infection of bone and joint are recommended. if evidence of osteolysis is clear, aspiration of the area may yield material for culture. the goals of treatment are to eliminate infection immediately and then resolve inflammation. bacteria and products of inflammation elicited by infection are responsible for destruction of bone and cartilage. the ultimate aim of treatment is to protect the structures critical to athletic performance such as subchondral bone and cartilage in weight-bearing areas. advances in the treatment of sepsis have resulted in hospital discharge rates of % for foals with septic arthritis, but their rate of high performers is %, indicating a need for improvement. equine veterinarians cannot replace what has been destroyed, so early identification and aggressive therapies are presently the best methods to improve performance rates. one achieves the goals of treatment by physical removal of bacteria, products of inflammation, and debris and by medications to kill the bacteria and reduce inflammation. one should optimize the physiology and general health of the foal to assist this process; one should include other treatments and supportive therapies for septic foals, especially treatment of failure of passive transfer, in the therapeutic plan. intravenous administration of antimicrobials (see chapter ) is the cornerstone of treatment of orthopedic infection, and if the drug is administered early in the course of infection and bacteria are susceptible, intravenous administration may be sufficient to eliminate the organisms. however, treatment of many foals does not begin until disease is advanced. if treatment begins after bacteria have had a chance to establish themselves, one should bring all appropriate methods to bear to end the infection. additional therapies for septic arthritis include joint lavage, arthrotomy (for drainage), , debridement (arthroscopically or arthrotomy), intraarticular administration of antimicrobials, intravenous regional perfusion, and antimicrobial beads. , one can use any sterile isotonic solution to flush a joint, and additives do not appear to give significant additional benefit. if radiographs do not indicate osteomyelitis, lavage, intraarticular antibiotics, and if possible, regional perfusion are recommended. if osteitis or osteomyelitis is present, debridement is indicated arthroscopically or via arthrotomy (one should culture the debris if the pathogen is unknown). if the joint is closed, one may use antibiotics intraarticularly. if the joint is left open to drain, regional perfusion is useful. antimicrobial beads theoretically are best to use if the wound is closed, but they appear to give benefit even if the wound is open under a bandage. because of concerns about the use of beads in a joint, beads often are used in tissue defects and the surrounding tissues. the major goal is to remove material that is compromising healthy tissues and to obtain high concentrations of antimicrobials in infected tissues. high antimicrobial concentrations are necessary because adhered bacteria are difficult to kill and may require many times the in vitro bacterial minimum inhibitory concentration. intraarticular administration of antimicrobials has been used for many years and has great value. regional perfusion of diluted antimicrobials recently has come into use and may be administered intraosseously or intravenously. intravenous perfusion is preferable because no special equipment is needed, but intraosseous perfusion may be valuable where intravenous access is impossible. the concept behind both procedures is to fill the venous vasculature in the area of the infection with antimicrobials diluted by a sterile balanced electrolyte solution. one isolates the anatomic area of interest using one or two tourniquets. the perfusate diffuses into all tissues and achieves much higher concentrations than are possible using intravenous therapy. this technique has shown excellent results as an adjunct therapy for orthopedic infection. for foals, to ml total of perfusate containing mg amikacin is useful for most single joint sites. amikacin has given consistently good results without complication and is a good choice based on its concentration-dependent activity. one may use a higher volume for the stifle, but the thigh musculature makes an effective tourniquet difficult to achieve. because of concerns that perfusion might dislodge bacteria and renew systemic sepsis, high concentrations of systemic antimicrobials are recommended at the time of the perfusion. if joint lavage and intraarticular administration of antimicrobials are not sufficient to resolve infection, one may perform arthrotomy to assist the joint to drain. passive and active drains add foreign material and so are not useful. maintaining the joint under a sterile bandage is critical and can be difficult to do in proximal joints such as the stifle and elbow. tie-over bandages can be useful in this application. the best measure of success is the resolution of lameness and local inflammation. radiographs may be helpful, but the most common sign of success is a failure of the infection to progress, rather than radiographic healing. one should continue intravenously administered antimicrobials for at least week after the resolution of lameness. if an appropriate drug is available, one should give foals antimicrobials orally for at least weeks more. a total of at least weeks of antimicrobials is recommended for most foals with orthopedic infection. treatment failures usually result from an inability to kill bacteria adhered to isolated tissue (usually dead bone). sometimes this failure is caused by incomplete debridement or an inability to access a known site of infection, but more frequently it is because infection has flourished in an unknown site. for this reason, multiple imaging modalities (radiographs, ultrasound, computed tomography, and magnetic resonance imaging) used multiple times are recommended for all refractory cases of septic arthritis. osteomyelitis not associated with a joint still involves a growth center. the ideal treatment for these infections is surgical debridement, systemic antimicrobial therapy, and some form of local antibiotic delivery. , even in the face of large initial osseous defects, infection may resolve, the defect may heal, and the foal may regain normal limb anatomy and function with appropriate therapy. clinical studies on newborn throughbred foals suffering from convulsions with special reference to blood gas chemistry and pulmonary ventilation respiratory distress in a newborn foal with failure to form lung lining film clinical studies on the newborn thoroughbred foal. . heart rate, auscultation and electrocardiogram blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following h measurements of pulmonary ventilation in normal newborn thoroughbred foals during the first three days of life some parameters 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neonatal foals strategies for prevention of neonatal isoerythrolysis in horses and mules detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia neonatal alloimmune thrombocytopenia in a quarter horse foal neonatal thrombocytopenia: new insights into pathogenesis and implications for clinical management thrombocytopenia in horses detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia reef vb: cardiovascular disease in the equine neonate congenital polyalveolar lobe in three foals critical pulmonary stenosis in a newborn foal bilateral hypoplasia of the soft palate in a foal pulmonary lobar hypertrophy in a foal equine congenital defects congenital bilateral choanal atresia in a standardbred foal evaluation of pulse oximetry in anaesthetised foals using multiple combinations of transducer type and transducer attachment site electrocardiographic findings during parturition and blood gas tensions immediately after birth in thoroughbred foals blood gas and acid-base changes in the neonatal foal blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following h mixed venous blood gases in recumbent and upright positions in foals from birth to days of age ventilatory support of the neonatal foal report of foal pneumonia panel association of microbiologic flora with clinical, endoscopic, and pulmonary cytologic findings in foals with distal respiratory tract infection microbiologic changes during antimicrobial treatment and rate of relapse of distal respiratory tract infections in foals pathologic changes and pathogenesis of parascaris equorum infection in parasite-free pony foals spezifische infektiose pneumonie beim fohlen. ein neuer eiterreger beim pferd interaction of rhodococcus equi with phagocytic cells from rhodococcus equi-exposed and non-exposed foals electron microscopic investigation of intracellular events after ingestion of rhodococcus equi by foal alveolar macrophages influence of rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses rhodococcus equi infection of monocytes/macrophages from human immunodeficiency (hiv)-infected patients and healthy individuals: evaluation of intracellular killing and nitric oxide production the intracellular bacterium rhodococcus equi requires mac- to bind to mammalian cells rhodococcus (corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses identification of -to -kilodalton antigens associated with virulent rhodococcus equi virulence-associated -to kilodalton antigens in rhodococcus equi: temperature-dependent expression and location of the antigens role of the -kilobase plasmid and plasmid-encoded virulence-associated protein a in intracellular survival and virulence of rhodococcus equi characterization of avirulence-associated gene family in rhodococcus equi corynebacterium equi infections in horses, - : a review of cases clinical manifestations, diagnosis, treatment, and prevention of rhodococcus equi infections in foals detection of corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay rhodococcus equi pneumonia in foals: comparison of elisa and agid serology on a commercial thoroughbred breeding farm studies of naturally occuring and experimental rhodococcus equi (corynebacterium equi) pneumonia in foals detection of virulent rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of r. equi pneumonia in foals comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals use of erythromycin-rifampin combination in treatment of rhodococcus equi pneumonia pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals hyperthermia in foals treated with erythromycin alone or in combination with rifampin for respiratory disease during hot environmental conditions clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia strategies for the control of rhodococcus equi infections on enzootic farms immunoprophylaxis of rhodococcus equi pneumonia in foals failure of hyperimmune plasma to prevent pneumonia caused by rhodococcus equi in foals rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals protection against naturally acquired rhodococcus equi pneumonia in foals by administration of hyperimmune plasma methods of implementation of an immunoprophylaxis program for the prevention of rhodococcus equi pneumonia: results of a -year field study effect of prophylactic administration of hyperimmune plasma to prevent rhodococcus equi infection on foals from endemically affected farms valuation of equine immunoglobulin specific for rhodococcus equi virulenceassociated proteins a and c for use in protecting foals against rhodococcus equi-induced pneumonia associations between physical examination, laboratory, and radiographic findings and outcome and subsequent racing performance of foals with rhodococcus equi infection: cases ( - ) endoscopic and virological observations on respiratory disease in a group of young thoroughbred horses in training epidemiology of ehv- and ehv- in the mare and foal populations on a hunter valley stud farm: are mares the source of ehv- for unweaned foals? clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus- on a stud farm foetal and neonatal foal losses on equine herpesvirus type (ehv- ) infected farms before and after ehv- vaccination was introduced an outbreak of foal perinatal mortality due to equid herpesvirus type : pathological observations equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations involvement of adenovirus in pneumonia in a thoroughbred foal adenoviral infection of arab foals with respiratory tract disease isolation of an adenovirus from an arab foal with a combined immunodeficiency disease clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus- infection compared with septic and premature foals neonatal equine herpesvirus type infection on a thoroughbred breeding farm passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a standardbred herd with high seroprevalence fatal nonneurological ehv- infection in a yearling filly pulmonary vasculotropic ehv- infection in equids virological and molecular biological investigations into equine herpes virus type (ehv- ) experimental infections equine herpesvirus type : prevalence and seroepidemiology in foals subcutaneous emphysema in a neonatal foal immature epithelial na+ channel expression is one of the pathogenetic mechanisms leading to human neonatal respiratory distress syndrome bronchointerstitial pneumonia and respiratory distress in young horses: clinical, clinicopathologic, radiographic, and pathological findings in cases ( - ) risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: cases ( - ) disease of foals du plessis jl: rupture of the bladder in the newborn foal and its surgical correction metabolic abnormalities associated with rupture of the urinary bladder in neonatal foals exploratory celiotomy for suspected urinary tract disruption in neonatal foals: a review of cases uroperitoneum in the foal uroperitoneum in the hospitalized equine neonate: retrospective study of cases repair of a defect in the bladder of a foal rupture of the urinary bladder in neonatal foals bladder defects in newborn foals ultrasound of the urinary tract nonsurgical management of ruptured urinary bladder in a critically ill foal hemodialysis for treatment of oxytetracycline-induced acute renal failure in a neonatal foal bilateral renal hypoplasia in four young horses bilateral renal dysplasia and hypoplasia in a foal with an imperforate anus clinical vignette: renal arteriovenous malformation in a quarter horse foal percutaneous ultrasound-guided pyelography aided diagnosis of ectopic ureter and hydronephrosis in a -week-old filly bilateral ureteral tears in a foal multiple ureteral defects in a belgian foal congenital defects in newborn foals of mares treated for equine protozoal myeloencephalitis during pregnancy ultrasonography of umbilical structures in clinically normal foals clinical, ultrasonographic, and surgical findings in foals with umbilical remnant infections closure of the abdominal wall at the umbilicus and the development of umbilical hernias in a group of foals from birth to months of age complications of umbilical hernias in horses: cases persistent vitelline vein in a foal atresia coli in the foal: a review of six cases endothelin receptor b polymorphism associated with lethal white foal syndrome in horses a dinucleotide mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome (lwfs): a horse variant of hirschsprung disease a missense mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome: an equine version of hirschsprung disease incidence of the endothelin receptor b mutation that causes lethal white foal syndrome in white-patterned horses necrotizing enterocolitis presenting in the emergency department: case report and review of differential considerations for vomiting in the neonate new concepts in necrotizing enterocolitis hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals endoscopic appearance of gastric lesions in foals: cases endoscopic evaluation of changes in gastric lesions of thoroughbred foals gastroduodenal ulceration in foals prevalence of gastric lesions in foals without signs of gastric disease: an endoscopic survey gastric ulcers in foals exsanguination due to gastric ulceration in a foal gastrointestinal diseases of foals pathophysiology of peptic disorders in foals and horses: a review clinical syndromes of gastric ulceration in foals and mature horses gastroduodenal ulceration in foals stress ulcer: is routine prophylaxis necessary? peptic ulcer pathophysiology intragastric ph in critically ill neonatal foals and the effect of ranitidine a review of medical treatment for peptic ulcer disease effect of sucralfate on healing of subclinical gastric ulcers in foals effect of ranitidine on intragastric ph in clinically normal neonatal foals effects of omeprazole paste on healing of spontaneous gastric ulcers in horses and foals: a field trial incidence of pneumonia in mechanically ventilated patients treated with sucralfate or cimetidine as prophylaxis for stress bleeding: bacterial colonization of the stomach the protective role of gastric acidity in neonatal bacterial translocation upper gastrointestinal tract bleeding in critically ill pediatric patients gastric colonization as a consequence of stress ulcer prophylaxis: a prospective, randomized trial is prophylaxis for gastric ulcers necessary in critically ill equine neonates? a retrospective study of necropsy cases - stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration influence of stress ulcer prophylaxis on translocation of bacteria from the intestinal tract in rats effects of sucralfate on acute gastric mucosal injury and gastric ulcer induced by ischemiareperfusion in rats stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration efficacy of ivermectin in controlling strongyloides westeri infections in foals foal diarrhoea between and in the united kingdom associated with prevalence of and risk factors for fecal shedding of cryptosporidium parvum oocysts in horses overwhelming strongyloidosis in a foal verminous arteritis in a -month-old thoroughbred foal randomised trial of fluid restriction in ventilated very low birthweight infants restricted versus liberal water intake for preventing morbidity and mortality in preterm infants hyponatraemia in children with acute cns disease: siadh or cerebral salt wasting? comparison of norepinephrinedobutamine to dopamine alone for splanchnic perfusion in sheep with septic shock depletion of neurohypophyseal content of vasopressin in septic shock hemodynamic and metabolic effects of low-dose vasopressin infusions in vasodilatory septic shock references . firth ec, poulos pw: blood vessels in the developing growth plate of the equine distal radius and metacarpus microangiographic studies of metaphyseal vessels in young foals physeal form of the longbones of the foal comparison of splinting and casting on the degree of laxity induced in thoracic limbs in young horses histological features of the dorsal cortex of the third metacarpal bone mid-diaphysis during postnatal growth in thoroughbred horses effect of oxytetracycline on metacarpophalangeal and distal interphalageal joint angles in newborn foals the blood supply of the growth plate and the epiphysis: a comparative scanning electron microscopy and histological experimental study in growing sheep fractures of the proximal sesamoid bones in thoroughbred foals avulsion of the origin of the peroneus tertius tendon in a foal avulsion fracture of the origin of the extensor digitorum longus muscle in a foal surgical reconstruction of a ruptured medial collateral ligament in a foal correction of patellar luxation by recession sulcoplasty in three foals lateral patellar luxation in miniature horse foals anatomy and therapeutic resection of the peroneus tertius in a foal preliminary observations on oxytetracycline treatment of congenital flexural deformities in foals effect of oxytetracycline on metacarpophalangeal and distal interphalangal join angles in newborn foals torsion in quadrapeds and its impact on mammalian joints computer model of endochondral growth and ossification in long bones: biological and mechanobiological influences observations on the evaluation and selection of foal limb deformities for surgical treatment treatment response and athletic outcome of foals with tarsal valgus deformities: cases incomplete ossification of the tarsal bones in foals: cases periosteal transection and periosteal stripping for correction of angular limb deformities in foals periosteal transection and stripping for treatment of angular limb deformities in foals: clinical observations restricted exercise and transphyseal bridging for correction of angular limb deformities effect of hemicircumferential periosteal transection and elevation in foals with experimentally induced angular limb deformities diaphyseal angular limb deformities in three foals current concepts of infectious polyarthritis in foals factors associated with prognosis for survival and athletic use in foals with septic arthritis: cases ( - ) the site of focal osteomyelitis lesions in foals pathological features of multiple bone infection in the foal stromal cell culture and relationships in perimedullary spaces of chick embryo shaft bones tarsal osteomyelitis in foals cartilage canals in equine articular/epiphyseal growth cartilage and a possible association with dyschondroplasia a histological study of acute hematogenous osteomyelitis following physeal injuries in rabbits open drainage, intra-articular and systemic antibiotics in the treatment of septic arthritis/tenosynovitis in horses comparison of various treatments for experimentally induced equine infectious arthritis a retrospective study of horses affected with septic arthritis/tenosynovitis regional intravenous perfusion of the distal limbs of horses with amikacin sulfate treatment of sepsis in the small tarsal joints of horses with gentamicin-impregnated polymethylmethacrylate beads use of antibioticimpregnated polymethyl-methacrylate in horses with open or infected fractures or joints: cases ( - ) the effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse regional perfusion of the equine carpus for antibiotic delivery how to perform equine digital intravascular perfusion surgical management of rhodococcus equi metaphysitis in a foal intraosseous regional perfusion for treatment of septic physitis in a -week-old foal key: cord- -ig nwtmi authors: nan title: th european conference on rare diseases & orphan products (ecrd ) date: - - journal: orphanet j rare dis doi: . /s - - - sha: doc_id: cord_uid: ig nwtmi nan theme: when therapies meet the needs: enabling a patient-centric approach to therapeutic development. background: rare diseases (rd) often result in a wide spectrum of disabilities, on which information is lacking. there is a need for standardised, curated data on the functional impact of rd to facilitate the identification of relevant patient reported/patient centered outcome measures (proms/pcoms) as well as for the use of validated quality of life instruments based on functional outcomes. to address these issues, orphanet is partnering with mapi research trust (mrt) in order to connect orphanet to proqolid ™ , mrt's proms/pcoms database, through disease codes. visit orphanet at www.orpha .net. methods and materials: the orphanet functioning thesaurus (oft) is a multilingual controlled vocabulary derived from the icf-cy. a subset of rd present in the orphanet nomenclature is annotated with the oft, with the addition of attributes for each functional impact (frequency, severity, and temporality) for each specific rd. annotations result from structured interviews with clinical experts, medical-social sector care providers, and patient organisations. in order to link proqolid ™ data with orphanet disability data, the taxonomy used to qualify rds in proqolid ™ was reviewed and mapped to orphanet's. all proms developed for rds were identified, and all products approved by the fda and ema from to with an orphan drug designation (odd) and a pro claim were listed. results: the orphanet knowledge base contains over rd, of which rd have been assessed for their functional consequences, of which rd have been annotated: the remaining rd were annotated, after discussions with medical experts, as either being highly variable, non-applicable or resulting in early-death. of the most prevalent rare diseases, have been annotated according to their functional consequences. rds had a prom (n = ) in pro-qolid ™ and . % of odd included a pro claim. the rds with the most prom were sickle cell anemia, spinal cord injuries, cystic fibrosis, all forms of hemophilia a and b and duchenne muscular dystrophy. prom used in labels were primarily focusing on symptoms ( %), rarely on functioning ( %) or health-related quality of life ( %). conclusions: linking these two databases, and providing standardised, curated data, will enable the community to identify proms/ pcoms for rd, and is the first step towards validated quality of life instruments based on functional outcomes. and eurordis. some patient organisations distributed the survey too more common disease patients as well, e.g. hashimoto's disease, and these responses were excluded. the survey asked patients to give suggested topics (i.e. fertility, heritability, tiredness, daily medicine intake, sleep quality, physical discomfort, and ability to work, partake in social life, and sports) a priority score and to suggest their own topics for research in open fields. open field responses were analysed with topic modelling and klipp-analysis. results: after exclusion of responses from more common endocrine disease patients, survey responses were analysed. most responses were received from northern ( %) and western europeans ( %), while southern ( %) and eastern europe ( %) were underrepresented. of the suggested topics respondent were most interested in research concerning the ability to work and participate in social life, and on tiredness. when patients were open to suggest their own topics, common responses included long-term side effects of drugs and quality of life. however, priorities differed between disease groups. for example, adrenal, pituitary and thyroid patients were more interested in research concerning tiredness than others. conclusion: with this survey endo-ern is provided with a large sample of responses from european patients with a rare endocrine condition, and those patients experience unmet needs in research, though these needs differ between the disease groups. the results of this study should be incorporated by clinical experts in the design of future studies in the rare endocrine disease field. purpose: when developing a health technology that requires clinical studies, developers institute working relations with clinical investigators. patient representatives can also create and manage advisory boards with product developers. this was of high utility in the s, in the development of products to treat hiv infection. inspired by this model, the european organisation for rare diseases (eurordis) proposes the eurocab programme to facilitate a two-way dialogue between patient representatives and medicine developers. as of , disease-specific cabs exist of approximately members each and others are being formed. methods: eurordis invites developers to sign a charter for collaboration with patients in clinical research, and provides guidelines together with a mentoring and training programme for patient networks. cabs help set the agenda with the developer, work on topics as diverse as study design, feasibility, informed consent and site selection, qol and proms, and organize the meetings. discussions also cover compassionate use, pricing, relative efficacy, etc. meetings last for to days with sessions with different developers, all under confidentiality. there are regular between-meeting teleconferences for trainings and action plan updates, and some cabs have instituted working groups on access, psychological support, etc. the collaboration is evaluated via a post-meeting survey send to both cab members and medicine developers. in addition, cabs have recently started to monitor outcomes of the meeting and progress towards their goals with a tracker tool. results: the results of the first surveys from distinct cab meetings with companies show that this form of shared decision-making is valuable as well as ethical for both parties. we have seen that working relations always continue, even when discussions become heated. all involved show interest in the co-creation possibilities of such collaboration and we look forward to seeing progress and change via the tracker. conclusions: monitoring and evaluation are crucial to understand whether and how the cabs are making an impact on medicine development. demonstrating impact is challenging because of the contextualized nature and complexity inherent to patient engagement collaborations in research design. eurordis is working within para-digm on our monitoring and evaluation strategy, focusing on improving its comprehensiveness and including multi-stakeholder perspectives. our current experiences show that the eurocab programme, with collective thinking and exchange between patients and a collaborative mentality from both sides, ensures high-quality and constructive dialogue with researchers and developers and can eventually inform both hta and regulatory decision-making (fig. ) . we have started to work on the metrics of markers of success. purpose: the french national network for rare sensory diseases sensgene launched in a -min motion design video ( fig. ) aiming at guiding healthcare professionals to welcome visually impaired patients in the hospital. this educational video was created to address patients' expectations and improve their experience in the network's hospital. the european reference network for rare eye diseases (ern-eye) collaborated on the project and created an english version of the video in order to distribute it widely in europe. method: sensgene worked on this project with two big french associations of visually impaired people: fédération des aveugles et amblyopes de france and fédération des aveugles alsace lorraine grand est. more than french patients' associations actively contributed to the project through five focus groups (workshops) which collected testimonies and gathered the needs of visually impaired persons and health-care professionals (fig. ). an evaluation was made by the independent body ipso facto: health professionals answered to a survey before and after viewing the video. results: the video deals with common situations in the delivery of care activities for different types of visual impairment: reception in a hospital center, consultations, moves and orientation in a hospital room (fig. ) . this fits perfectly with the needs of the patients reported in the focus groups. besides, the evaluation showed that % of them improved their knowledge on the topic. background: traditional appraisal and reimbursement approaches such as cost/qaly are increasingly recognised as being potentially unsuitable for rare disease treatments (rdts). approaches to appraising rdts vary across countries, from the same processes used for all medicines, to those completely separate from the standard, to adapted standard processes with greater willingness to pay (wtp). this study examines the impacts of standard versus special appraisal processes for specific rdts in selected countries. methodology: a case study analysis was conducted in which countries with a variety of rdt appraisal processes were selected, along with two rdts representative of the following criteria: rare/ultra-rare treatment, affecting child/adult, cancer/non-cancer, life-threatening/disabling. public hta reports for each country's appraisal of the selected rdts were retrieved and used to extract information into predesigned templates, which allowed for systematic comparison of the rdt processes across countries to compare and exemplify the impact of the different processes. results: reports from belgium, england, france, germany, u.s., italy, lithuania, netherlands, poland, romania, scotland, slovakia, and sweden were selected for spinraza and voretigene. characteristics of each country's process were extracted, including special reimbursement for rdts, special rdt committees, economic evaluation modifications, greater wtp, quality of evidence flexibility, additional considerations, etc. special and standard processes seemed to have different impacts on the appraisal of rdts. special processes more consistently managed rdt issues such as evidential uncertainty and higher icers. standard processes sometimes informally applied some of the characteristics included in special processes, such as broader consideration of value. conclusions: comparing case study country examples of rdt appraisal exemplified the complexity of these processes. special processes were more consistent in managing the challenges in rdt appraisal than standard processes. practical application: findings suggest a need for adapted approaches for rdt appraisal, to facilitate management of associated challenges and more consistent decision-making. estimating the broader fiscal impact of rare diseases using a public economic framework: a case study applied to acute hepatic porphyria (ahp) mark p. connolly , background: the aim of this study was to apply a public economic framework to evaluate a rare disease, acute hepatic porphyria (ahp) taking into consideration a broad range of costs that are relevant to government in relation to social benefit payments and taxes paid by people with ahp. ahp is characterized by potentially life-threatening attacks and for many patients, chronic debilitating symptoms that negatively impact daily functioning and quality of life. the symptoms of ahp prevent many individuals from working and achieving lifetime work averages. we model the fiscal consequences for government based on reduced lifetime taxes paid and benefits payments for a person diagnosed aged experiencing attacks per year. materials & methods: a public economic framework was developed exploring lifetime costs for government attributed to an individual with ahp in sweden. work-activity and lifetime direct taxes paid, indirect consumption taxes and requirements for public benefits were estimated based on established clinical pathways for ahp and compared to the general population (gp). results: lifetime earnings are reduced in an individual with ahp by sek . million compared to the gp. this also translates to reduced lifetime taxes paid of sek . million for an ahp individual compared to the gp. we estimate increased lifetime disability benefits support of sek . million for an ahp individual compared to gp. we estimate average lifetime healthcare costs for ahp individual of sek . million compared to gp of sek . million. these estimates do not include other societal costs such as impact on caregiver costs. conclusions: due to severe disability during the period of constant attacks, public costs from disability are significant in the ahp patient. lifetime taxes paid are reduced as these attacks occur during peak earning and working years. the cross-sectorial public economic analysis is useful for illustrating the broader government consequences attributed to health conditions. ethics approval: the study results described here are based on a modeling study. no data on human subjects has been collected in relation to this research. the european cystic fibrosis (cf) society patient registry collects demographic and clinical data from consenting people with cf in europe. the registry's database contains data of over , patients from countries. high quality data is essential for use in annual reports, epidemiological research and postauthorisation studies. methods: a validation programme was introduced to quantify consistency and accuracy of data-input at source level and verify that the informed consent, required to include data in the registry, has been obtained in accordance with local and european legislation. accuracy is defined as the proportion of values in the software matching the medical record, consistency as definitions used by the centre matching those defined and required by the registry. data fields to verify: demographic, diagnostic, transplantation, anthropometric and lung function measurement, bacterial infections, medications and complications. the number of countries to validate: % of the total countries/year. in the selected country ≥ % of the centres should be visited and - % of the data validated. results: in , ten out of centres ( %) in countries (austria, portugal, slovakia, switzerland) with ≥ % of all patients in their countries were selected. in a day visit, the data of the registry were compared with the medical records, the outcomes and recommendations discussed, and a final report provided. demographic, diagnostic and transplant data were checked for patients ( %*), clinical data for patients ( %*) ( data). challenges were: informed consent, mutation information (genetic laboratory report missing), definitions interpretations. see fig. for the results. conclusion: the registry's data is highly accurate for most data verified. the validation visits proved to be essential to optimise data quality at source, raise awareness of the importance of correct informed consent and encourage dialogue to gain insight in how procedures, software, and support can be improved. *of the total patients in these countries. background: people with duchenne muscular dystrophy (dmd) adopt compensatory movement patterns to maintain independence as muscles get weaker. the duchenne video assessment (dva) tool provides a standardized way to document and assess quality of movement. caregivers video record patients doing specific movement tasks at home using a secure mobile application. physical therapists (pts) score the videos using scorecards with prespecified compensatory movement criteria. objective: to gather expert input on compensatory criteria indicative of clinically meaningful change in disease to include in scorecards for movement tasks. approach: we conducted rounds of a delphi panel, a method for building consensus among experts. we recruited pts who have evaluated ≥ dmd patients in clinic and participated in ≥ dmd clinical trials. in round , pts completed a preliminary questionnaire to evaluate compensatory criteria clarity and rate videos of dmd patients performing each movement task using scorecards. in round , pts participated in an in-person discussion to reach consensus (≥ % agreement) on all compensatory criteria with disagreement or scoring discrepancies during round . results: of the pts, % practiced physical therapy for ≥ years, % provided physical therapy to ≥ dmd patients, and % participated in ≥ dmd clinical trials. of version compensatory criteria, ( %) were revised in round . of version compensatory criteria, ( %) were revised in round . the pts reached % agreement on all changes made to scorecards during the in-person discussion except the run scorecard due to time restrictions. a subset of the panel ( pts) met after the in-person discussion and reached consensus on compensatory criteria to include in the run scorecard. conclusion: expert dmd pts confirmed that the compensatory criteria included in the dva scorecards were appropriate and indicative of clinically meaningful change in the disease. introduction: fifty percent of rare disease cases occur in childhood. despite this significant proportion of incidence, only % of adult medicines authorised by the european medicines agency (ema) completed paediatric trials [ , ] . as a result, many clinical needs are left unmet. various factors compound the development of treatments for paediatric rare diseases, including the need for new clinical outcome assessments (coas), as conventional endpoints such as the minute walking test ( mwt) have been shown to not be applicable in all paediatric age subsets, [ ] and therefore may not be useful in elucidating patient capabilities. coas are a well-defined and reliable assessment of concepts of interest, which can be used in adequate, well-controlled studies in a specified context. coas capture patient functionality and can be deployed through the use of wearable sensor technology; this feasibility study presents data obtained from patients with paediatric rare diseases who were assessed with this type of technology. methods: niemann pick-c (np-c) (n = ) and duchenne muscular dystrophy (dmd) (n = ) patients were asked to wear a wrist-worn wearable sensor at home for a minimum of weeks. feasibility was assessed qualitatively and quantitatively, with data captured in minute epochs, measuring the mean of epoch's with the most steps over a month (adm), average daily steps (ads), average steps per minute epoch (ade) (table ) and reasons for non-adherence (table ) . no restriction in the minimum number of epochs available for analysis were applied, and all patient data analysed. results: discrepancies in ambulatory capacity were observed between np-c and dmd patients overall, with np-c patients covering greater distances and taking more steps daily. qualitative assessment of both patient groups highlighted their relationships with the technology, which in turn detailed adherence. some patients exhibited behavioural issues which resulted in a loss of data and low engagement. conclusions: the wearable sensor technology was able to capture the ambulatory capacity for np-c/dmd patients. insights into disease specific parameters that differed were gained, which will be used for developing the technology further for use in future trials. additional work is required to correlate the wearable device data with other clinical markers, however the study displays the feasibility of wearable sensors/apps as potential outcome measures in clinical trials. background: neonatal surgery is decentralized in germany. in there were departments of pediatric surgery that treated % of the abdominal wall defects with an average case load of less than per unit [ ] . patient organizations stress the importance of quality measurements for the care of children with rare diseases. study plan: currently, there is no nationwide data collection regarding the short term and long term care of patients with congenital malformations, who often need surgery during the first weeks of life. the german society of pediatric surgery, which covers almost all of the german pediatric surgical units, has initiated the work of creating a national patient registry (kirafe) for the following congenital malformations: malformations of the gastrointestinal tract, the abdominal wall, the diaphragm, and meningomyelocele. the development of the registry involves three different patient organizations and health care professionals from all over germany. the registry will be set up in based on the open source registry system for rare diseases (osse). the primary objective of the registry is the measurement of quality attributes of rare congenital malformations. furthermore, the registry will facilitate recruitment of patients to clinical trials. it will also serve as a basis for policy making and planning of health and social services for people with rare disorders. informed consent will be obtained from the participants. the registry will include core data, mainly comprising information on the set of malformations of each patient. each malformation will then prompt further different modules for data collection. this modular structure offers the greatest possible flexibility for the documentation of patients with more than one congenital malformation. data will be collected by health care professionals. results: since the start of the preparation individuals, either working in one of hospitals or being member of one of the three patient organizations, have contributed in the ongoing activities. the registry is listed in the european directory of registries (erdri) [ ] . ethical approval was obtained, financial resources were secured. in , german hospitals and three non-german hospitals confirmed their intention to document their patients within the registry. conclusion: the registry is an example for a nationwide collaboration with the goal to optimize the quality of care for a patient group with rare diseases. is a collaboration between cf europe and five pharmaceutical companies (to date). through biannual meetings, we aim to institute a longterm educational collaboration with companies with an interest in cf. membership of industrial partners is dependent upon adherence to the cfrtoc code of conduct and a financial contribution for cf europe to fulfil its missions. common objectives include access to information. one strong example, applicable even beyond rare diseases, is the need for improved communication regarding clinical trials (cts) which has been inconsistent and often difficult to understand. from , the new european ct regulation / will oblige sponsors to share ct results through lay summaries. to help move this initiative forward, cf europe, with the active support of the cystic fibrosis trust, is collaborating with the european cystic fibrosis society-clinical trials network (ecfs-ctn) and cfrtoc members to establish a glossary of relevant cf terms. it will be freely available so that all stakeholders can systematically use it in patient-friendly scientific summaries and wider communication. in a pilot project, people with cf and patient associations, together with industrial partners will shortlist terms. these will be defined by lay members and subsequently subjected to the study and approval of the legal department of participating companies. provided this process is successful, we aim to create approved definitions by the end of . cf europe and ecfs-ctn intend to advertise the use of this glossary online and through communications at scientific events. national patient organisations will be further encouraged to provide translations in their national language. alkaptonuria (aku, ochronosis) is an inborn metabolic disease, resulting in the accumulation of the metabolic intermediate homogentisic acid (hga). oxidation of hga by air or within connective tissue causes darkening of the urine, pigmentation of eyes and ears, kidney-and prostrate-stones, aortic stenosis, but most severely an early onset of arthritis called ochronotic arthropathy (ochronosis) due to deposition in the cartilage. ochronosis is very painful, disabling and progresses rapidly. starting in the thirties with the spine and affecting large joints in the forties, patients frequently require joint replacements in their fifties and sixties [ , ] . like many of the rare diseases, aku-patients undergo a long odyssey of several years until their diagnosis. the german aku-society "deutschsprachige selbsthilfegruppe für alkaptonurie (dsaku) e.v. " was founded in and became subsequently registered as a non-profit patient organization. first of all, the dsaku identified aku-patients, set up a homepage [ ] and designed flyers with information for patients, their families, medical professionals and healthcare services. second, it offered workshops on aku-related issues and enabled personal exchange. third, it raised awareness of aku, both nationally and internationally by information booths, presentations and posters at scientific congresses as well as rare disease days (rdd). fourth, in response to the needs of patients, it established collaborations and built up national networks for a better health care accordingly. thus, patients were encouraged to visit the centers for metabolic diseases at the charité (berlin), hannover medical school (mhh), university of düsseldorf and institute of human genetics at the university of würzburg to bundle knowledge and expertise. the dsaku is member of achse e.v., nakos, eurordis and metabern and registered in the databases se-atlas, zipse and orphanet. finally, the dsaku is nationally and internationally active in health politics regarding training in drug safety and evidence-based medicine. introduction: autoinflammatory diseases are rare conditions characterized by recurrent episodes of inflammation with fever associated to elevation of acute phase reactants and symptoms affecting mainly the mucocutaneous, musculoskeletal or gastrointestinal system. these diseases affect the quality of life of patients and their families. objectives: aim of this project is to develop a tool able to ameliorate patients' management of the disease and to enhance patientphysician communication. to develop a tool based on real-life needs, we involved patients and caregivers since the initial phase of the project. a first workshop designed to capture their needs was organized. innovative co-design activities were performed through "legoserious-play ™ " (lsp) methodology [ ] [ ] [ ] . during a first phase of "divergence" patients (from teen-agers to adults) affected by different aids (fmf, trap, caps, mkd) and physicians where involved in the lsp activities. participants were asked to describe, through lego and metaphors: • the disease • themselves in comparison with the disease • solutions and supports which could help them in managing the disease after each step the participants presented their models, and everyone was engaged in the discussion. the ideas collected during the three phases allowed to make a list of functionalities identified as necessary for the app to be developed. due to the actual sars-cov- sanitary emergency the second phase of the project, aimed at presenting the participants the results of the first meeting and proceed with the app finalization was performed through web-based meeting and surveys in which the patients and caregivers actively participated. results: in the first phase patients and caregivers participated actively expressing various needs, that we subsequently summarized in main areas (table ) . participants were then further involved and their opinion taken into consideration for the user experience and interface definition for the development of the mobile app including the required functionalities (after a further activity of prioritization). introduction: gaps in communication and education are becoming one of the biggest key pain points for patients that are suffering rare diseases. due to the limited resources and the misleading information on the internet we wanted to test the poc systems to deliver more efficiently the information to our patients and their relatives. userfriendly information at the point of care should be well structured, rapidly accessible, and comprehensive. method: we implemented a specific poc channel using several touchpoints to deliver the right content at the right time. we created and selected the video content that will be most helpful to our patients. later on, we analyzed the patient journey and we decided to use a mobile app where the patients could search for information when they are at their home. at the medical practice, we use the waiting room and exam room as learning areas through monitors and tablets. moreover, healthcare professionals are prescribing content to their patients that they reviewed when they are home. results: thanks to the use of the poc channel and technologies related we were able to reduce the time needed to perform an explanation by %. furthermore, our healthcare professionals reported that their conversations with the patients improved % and patient satisfaction increased by %. conclusion: poc channel created a positive impact on our patient experience allowing us to be more efficient delivering the information to our patients and their relatives. [ , ] , realworld safety and efficacy data are limited -particularly for patients who receive > treatment. we report initial data from the restore registry, including cohort clinical characteristics, treatments received, and outcomes. materials and methods: restore is a prospective, multicenter, treatment-agnostic registry of sma patients. the primary objectives include assessment of contemporary sma treatments; secondary objectives include assessment of healthcare resource utilization, caregiver burden, and changes in patient functional independence over time. planned follow-up is years from enrollment. as of january , data were available for patients, all from de novo clinical sites in the united states; information on treatment regimens was available for patients (table ) . disease-modifying treatments were administered sequentially or in combination. % of treated patients showed symptoms at sma diagnosis, with the most common being hypotonia and limb weakness ( table ) . Ågrenska, a swedish national centre for rare diseases, has for thirty years arranged courses for families of children with rare diagnoses and has experienced that the conditions often have complex and varying consequences in the children ś everyday lives. knowledge of these consequences and of how to adapt the treatment, environment and activities to create the best possible conditions for participation and learning, is often lacking. many professionals also report lack of sources of knowledge. knowledge formation and dissemination are thus of outmost importance. in order to aid knowledge formation and dissemination Ågrenska has developed an observation instrument for children with rare diagnoses, identifying both abilities and difficulties on a group level. the instrument consists of quantitative and qualitative items and covers ten areas: social/communicative ability, emotions and behaviours, communication and language, ability to manage his/her disability and everyday life, activities of daily life, gross and fine motor skills, perception and worldview, prerequisites for learning and basic school abilities. observations are made during the children ś school and pre-school activities during the Ågrenska course. teachers and special educators, working with the children, are responsible observers. some school-related abilities are difficult to observe during the five-day stay. this information is instead collected through a telephone interview with the children ś home teacher. the instrument was content validated against a number of existing instruments. the items were considered relevant as they, with few exceptions, appear in well-known assessment tools. to test interrater reliability observations of six children were performed. each child was observed by two educators. interrater reliability was calculated for the quantitative items usually observed during the course. interrater reliability reached . %. background: sma is a neurodegenerative disease caused by survival motor neuron gene (smn ) deletion or mutation [ , ] . disease severity (sma type) correlates with smn copy number [ , ] . gene therapy with onasemnogene abeparvovec provides sustained, continuous production of smn protein, and is fda approved [ ] , with ongoing trials for sma type (sma ) and sma , and presymptomatic treatment for all sma types. with treatment options available, many states in the united states (us) are implementing newborn screening (nbs) to detect smn deletions and smn copies, providing early diagnosis and the option of pre-symptomatic treatment [ ] . we examine the economic consequences of implementing nbs for sma and pre-symptomatic treatment with onasemnogene abeparvovec gene therapy among newborns in the us. a decision-analytic model was built to assess the cost effectiveness of nbs in , hypothetical newborns from a us third-party payer perspective. the model included separate arms, each allowing for a different treatment strategy. model inputs for epidemiology, test characteristics, and screening and treatment costs were based on publicly available literature (table ) . inputs and assumptions of lifetime costs and utilities for sma types were obtained from the institute for clinical and economic review sma report [ ] ; other values were sourced from published literature. model outputs included total costs, quality-adjusted life years (qalys), and incremental cost-effectiveness ratios (icers). scenario and sensitivity analyses tested model robustness. park's programme, particularly across education and engagement and prioritisation and development of research. in addition to representation on governance structures, wales gene park (wgp) collaborates with patients and the public to involve them in rare disease and genetic research. wgp has co-produced a rare disease research gateway following consultation with patients and the public from its networks. the gateway hosts relevant studies in genetic and rare disease research on the wgp website. it promotes involvement opportunities in addition to signposting to studies that patients and other members of the public can participate in. it also links to training opportunities for ppi representatives. consultation with patients and the public regarding the usability, design and development of the gateway was undertaken. feedback has enhanced the user experience and it was launched in october . there are currently over studies featured, and the gateway is searchable according to condition or key word. impact will be monitored through online usage and website analytics. engagement with researchers through a professional network enables opportunities to be advertised from all areas of genetic and rare disease research and ensures that patient and public representatives are involved in the design and development of research from its inception. wgp were invited to present at the welsh health and care research wales conference in as the gateway was highlighted as an exemplar of good practice. specialist visit, medications) and non-medical resource use (lost productivity and homecare or caregiver's time). outcomes of interest for treatment options assessed the efficacy and safety of treatments for rett syndrome. results: the search on economic burden yielded articles; intervention type and costs were extracted from , representing studies. in the economic burden studies, enteral feeding and assisted walking increased the risk of respiratory-related hospital admissions, while length-of-stay was lower in younger patients. mean recovery-stay after scoliosis-correcting surgery was . days and . days in each of studies. care integration improved outcomes and reduced costs. the search on clinical trials yielded articles; efficacy and safety were extracted from , representing studies ( randomized controlled trials, single-arm; n = - ; follow-up - months). of these, focused on pharmacological symptom treatment; examined environmental enrichment effects; none targeted the underlying cause. the most common primary endpoints are stated in table . naltrexone, trofinetide, and mecasermin demonstrated clinical benefits versus placebo, but most treatments yielded no significant improvement ( table ) . the cml advocates network (cml an) is an active network specifically for leaders of chronic myeloid leukemia (cml) patient groups, connecting patient organisations in countries on all continents. it was set-up and is run by cml patients and carers. its aim is to facilitate and support best practice sharing among patient advocates across the world. the cml community advisory board (cml-cab) is a working group of the cml advocates network. since its inception the cml-cab has met on nineteen occasions with five sponsors. the cml-cab is comprised of two chairs and cab-members. cml-cab organisation, sustainability and follow-up is supported by a part-time cml-cab officer and the cml-an executive director. the principles of leaving no one behind are essential to the goals of world health organization (who) and united nations (un). in , an ambitious objective to ensure that billion more people will benefit from universal health coverage (uhc) until was entrenched in the who th general programme of work [ ] . all un member states have agreed to try to achieve universal health coverage by , as part of the sustainable development goals [ ] . however, it is essential, that rare disease (rd) patients are not left behind on our trip to uhc. in , un declared that rd are among the most vulnerable groups that are still on the fringes of uhc [ ] . the first step on a way to the full uhc cube [ ] for rd is an identification of root causes of health inequities. health determinants of rd fundamentally differ from those for common diseases. some of them are unavoidable: up to % of rd have a genetic basis (individual or genetic determinants). although socio-economic factors are highly important, in contrast to common diseases, they are a consequence rather than a cause of rd. meanwhile, one of the major root cause amenable to change are health system determinants: organization of services for rd requires unique solutions in our health systems that are mostly adapted for common diseases. political and legal determinants also play a key role: while rd is an explicit example of an area, loaded orphanet j rare dis , (suppl ): with needs for pan-european solutions, relative "weakness" of eu legal powers to regulate and have an impact on implementation of pan-european policies in health results in vast inequities among and inside member states and lack of engagement at a national level. health activism that includes strong advocacy and a loud voice of patient organizations has also been ascribed to health determinants and may have a crucial role in rd [ ] to improve the situation, we already have some powerful tools at hand including national plans for rd, european reference networks [ ] and european joint programme on rare diseases [ ] . however, to reach the full potential of these, multiple obstacles have to be removed and full implementation ensured. since march , there has been an explosion in digital health adoption as people look for remote ways to manage their health and wellbeing. national government covid- strategies, local authorities and consumers, have all turned to health apps, both as a potential means of slowing the spread of the virus, and a method of allowing people to self-manage their own health. in the first few weeks of the covid- pandemic, orcha worked with app developers to build a dedicated covid- app library full of evaluated apps. free to use for all, it included relevant, quality assured apps that had been through orcha's rigorous review process. to build such a tool in such a short space of time is testament to the speed of this market. more consumers have been using health and care apps. in just one week, orcha saw an increase of . % in app downloads from its app libraries, and a , % increase in app recommendations from health and care professionals. orcha can see from the data across its app libraries that the most popular search terms since the pandemic began have included: mental health, physiotherapy, fitness, anxiety, rehabilitation, diabetes, respiratory, and sleep. whereas 'covid' was initially the most searched term at the beginning of the outbreak, people have since searched for specific condition areas. this indicates a shift in focus to actively self-managing health and wellbeing, and a desire for knowledge about particular health areas. the recent increase in digital health adoption has highlighted that the challenge remains of helping consumers to understand which apps are potentially unsafe to use, and ensuring that consumers are armed with the full facts about the strengths and weaknesses of an app, before it is downloaded. while considerable progresses have been made in the last years in research on innovative medicinal products for adults, children have not benefited from progresses to the same extent as adults in terms of appropriate treatments and advanced tools. it is well known that the availability of drugs for paediatric use still represents a challenging issue, since research and development in this field is characterized by many that range from methodological, ethical and economic reasons, especially when neonates and rare diseases are involved. moreover, even when industry has the capacity to perform a paediatric drug development plan, there are many economic reasons limiting the commercial sponsors' interest (the paediatric population is a small population; paediatric diseases often concern rare disorders with unknown mechanism; it is very difficult to perform preclinical and clinical studies; ethical concerns are still relevant and additional regulatory requirements have to be considered). in this scenario, eptri can make the different in closing the gap between innovative technologies and paediatric drug development processes. it is a eu-funded project that arises from the need to find answers to the serious lack of medicines for children in eu and worldwide, and aimed to design the framework for a european paediatric translational research infrastructure dedicated to paediatric research. an high interest is tailored on rare diseases (rd) as they affect mainly children and genetic rd start early in the prenatal/childhood life with an high frequent use of medicines not specifically tested (off-label, unlicensed). eptri will work to accelerate the paediatric drug development processes from medicines discovery, biomarkers identification and preclinical research to developmental pharmacology, age tailored formulations and medical devices. this will allow is to facilitate the translation of the acquired new knowledge and scientific innovation into paediatric clinical studies phases and medical use. neonatal screening started in many countries around - after phenylketonuria turned out to be a treatable condition. if diagnosed early, a diet could help to avoid impaired brain development. public health programmes were developed to offer all newborn children the possibility to be tested. screening always has benefits and disadvantages, and only rarely pros outweigh cons at reasonable costs. the world health organization in published criteria to evaluate benefits and disadvantages, concerning amongst others ( ) important health problem ( ) treatment ( ) suitable test and ( ) appropriate use of resources. pku was mentioned as an example of an important health problem [ ] . neonatal screening is more than a test. information to parents, communication of results, ict infrastructure, follow-up of affected infants, reimbursement of test and treatment and governance all need adequate attention [ ] . around the number of diseases covered in european countries in neonatal screening programs was very diverse: from zero in albania to more than in austria, hungary, iceland, portugal and spain [ ] . many countries have seen an increase in the number of diseases covered because of new tests and treatments becoming available. health authorities were almost always involved in changes in the programmes, hta experts and parents organizations sometimes. half of the countries had laws on nbd, and half had a body overseeing nbs programs. less than half of the countries informed parents of the storage of dried blood spots [ ] . after the eu initiated "tender nbs" had provided advice to eu policy makers [ ] , little initiatives for harmonization were taken, because health is the mandate of member states. from the perspective of newborns this implies that early diagnosis and adequate treatment for nbs conditions may differ very much for children being born in one or another eu country. with more tests and more treatments becoming available, this makes it even more urgent to attune the perspectives of different eu stakeholders for the benefit of all newborns. background: as genome sequencing is rapidly moving from research to clinical practice, evidence is needed to understand the experience of patients with rare diseases and their families. in the presentation, we discuss families' experience of receiving, making sense of and living with genomic information. the presentation includes video-clips from two short films from families' narratives. specifically, families struggled with the lack of information on the course of the disease, the difficulties to access support and navigate health and social care services, and the challenges related to making sense of the implications of genomic information for other family members. despite these issues, families identified a wide range of benefits from taking part in genome sequencing, which were broader than the clinical utility of the diagnosis. the findings raise questions regarding how to talk about 'diagnosis' in a way that reflects families' experience, including their uncertainty but also their perceived benefits. they also have implications for the design and delivery of health services in the genomic era, pointing to the need to better support families after their search for a diagnosis. saluscoop [http://www.salus coop.org] is a non-profit data cooperative for health research that aims to make a greater amount and diversity of data available to a broader set of health researchers, and to help citizens to manage their data for the common good. data heals. health research is data-driven: the larger the universe, the greater the quantity, quality, and diversity of the data, the more potential the data has to cure. in our european context, it is clear: data belongs citizen. gdpr regulates ownership and our rights over data that include portability. data protection laws rightly consider that health data deserves the maximum protection. however, the only truth, we note every day: in practice citizen often cannot access their data or control its use. the future of our health depends significantly on the ability to combine, integrate and share personal health data from different sources. the only one who can integrate all your information (public, private, clinical, personal, habits, genetics) is the citizen himself. using data well, it is possible to obtain more and better health for all. we are a cooperative that works to facilitate the transformation process towards this goals doing: -dissemination, awareness, communication -studies, manifestos. -licenses to facilitate it -salus common good license - it is necessary to dissociate the provision of services, of the possession of the data. the accumulation and centralization of the data is not necessary. blockchain and the like allow the certification of transactions without the need for intermediaries. the need for the existence of new social institutions for the collective management of data for the common good is much clearer today: so that these citizens have the technological and legal tools effectively manage their data. so that health research can address the real problems of our societies. the abstract is being presented on behalf of a saluscoop management board group. the region of murcia, located on the southeast of spain, has . million inhabitants. in , approximately % of its population was identified with a rd, based on the regional rd information system, which showed a public health problem requiring an integral and coordinated approach. results: in , after years of participative work (interdepartmental government representatives, patients associations and professionals) the regional plan for rd integrated (holistic) care was approved, for a period of years ( - ) and a budget of millions euros; with the goal of improving health, education and social care through interdisciplinary coordination and placing patients and families in the center of the actions. the plan includes ten different strategic areas related to information, prevention and early detection, healthcare, therapeutic resources, social-health care, social services, education, training of professionals, research, monitoring and evaluation a regional rd coordination center, linked to the medical genetics unit in the tertiary reference hospital, is connected to the health areas, educational and social local services, through a case manager integrated in the multidisciplinary team. this was our building experience presented in the innovcare project, co-funded by the eu. to design a holistic care plan for rd we need to know the prevalence based on rd registries, available and needed resources and an interdisciplinary participative action approach with the appropriate government and financial support with periodic evaluation. case management has an important role. the recognition of clinical genetics as health specialty is also urgent in spain to provide equal access to rd patients and families all over the country. [ ] . these policies have served us well, but it is essential that the policies guiding us towards the future we wish to see are equipped to address the needs of the future rd population. the rare project [ ] is working towards precisely this goal, and has identified over a hundred future-facing trends likely to impact on the field. some of these trends concern demographic changes about which we can be reasonably certain: whilst overwhelmingly positive, changes such as ageing rd populations will bring new challenges in managing comorbidities. they will also create new opportunities as well as risks in areas such as reproductive choice; however, these choices incur major ethical, legal and social concerns, and it is unclear how many countries really have robust frameworks in place to cope with this. besides the fairly certain demographic changes, there are many topics -and many needs-for which the future is not clear. will there be easier access to expert multidisciplinary teams? what will be the role of technology in care delivery? these fundamental issues are here debated in interview format [ ] . adrenoleukodystrophy, or ald, is a complex x-linked genetic brain disorder which mainly affects males between the ages of four and -males who are previously perfectly healthy and 'normal' . ald damages the myelin in the brain and spinal cord, and those with cerebral symptoms become completely dependent on their loved ones or carers. this usually involves patients becoming wheelchair or bed bound, blind, unable to speak or communicate and tube fed. it is a difficult disorder to diagnose with behaviour problems usually the primary indicator. in males, cerebral ald is a terminal illness with most dying within one to years of symptoms developing. if diagnosed before symptoms become apparent, usually through identification of a family member, the condition can be successfully treated through bone marrow transplant. some adults (males and females) develop a related condition called adrenomyeloneuropathy, or amn. symptoms include difficulty walking, bladder and bowel incontinence and sexual dysfunction. tragically, around one third of males with amn go on to develop cerebral ald. initial behavioural symptoms often have an impact on the individual's professional and personal lives -their capacity to work, maintain relationships and family ties -over time, they can become isolated and socially unacceptable. commonly, those individuals without supportive family structures are missed or misdiagnosed. the presentation presents a personal case study detailing the impact of an ald diagnosis on the whole family, moving on to alex tlc's experience in applying to add ald to the uk's new born screening programme. the conclusion includes next steps following an initial negative response, and thoughts on the methods used to assess decisions on the prevention and treatment of rare disease. the rare foresight study gathers the input of a large group key opinion leaders through an iterative process to propose recommendations for a new policy framework for people living with rare diseases (rd) in europe. since the adoption of the council recommendation on european action in the field of rd in , the european union has fostered tremendous progress in improving the lives of people living with rd. rare will recommendations for the next ten years and beyond. the rare foresight study includes major stages (fig. ) . the european conference on rare diseases and orphan products (ecrd ) marked the occasion to present four proposed future scenarios (fig. the market-led approach first creates the technology innovation, then seeks out its market. deep understanding of needs as the starting point of the innovation process. with symptoms and being suspected of having a rare disease can be the longest in many steps to getting a diagnosis. this is something we have the power to change now by providing content tailored to medics, early in their careers that will equip them to #daretothinkrare. to prepare for delivery of gene therapies, companies typically focus on four key areas: patient identification & diagnostics; treatment centre qualification; manufacturing & supply and market access. timely diagnosis of patients is important as with progressive disorders, the earlier patients are treated, typically the better their long-term clinical outcomes will be. targeted tools and resources are used to educate clinical specialists on the early symptoms of the disease. improving access to the appropriate diagnostic tests is essential. if newborn screening is considered, validated assays and pilot studies are required. gene therapies have to be administered in qualified treatment centres. after regulatory approval, treatment centres are relatively few so patients may need to cross borders and work is required to expand the recognition of patient rights to be treated in another eu country (e.g. through the s mechanism). many companies partner with contract manufacturing organisations and are developing ways to preserve gene-corrected stem cells to enable their transportation from the manufacturing site to treatment centres. the final area is market access, whereby it is vital to evolve the way healthcare systems think about delivery, funding and value determination. manufacturers have the responsibility to generate health economic evidence. recent research [ ] in metachromatic leukodystrophy showed that caregivers (n = ) spend an average of hours a day caring for their child. % of parents were forced to miss work with % of this being unpaid leave. in addition, it is recommended to have the optionality of payment models that allow the sharing of risk between the healthcare system and manufacturer (e.g. annuity or outcomes-based payments). orchard has developed a holistic value framework as gene therapies are expected to benefit patients, families, communities, healthcare systems and society reference background: employment has always been one of the fundamental human rights. it is important for people with rare diseases, because it helps to stay connected to the community and to continue professional development. equal access to job employment can help to overcome the consequences of the condition and to gain financial independence. on the other hand unemployment can increase the social exclusion. in the last few years there is an improvement in the european policies about job employment. in spite of this, people with rare diseases still have to overcome discrimination in this field. as a proof of this statement is the recent online survey, conducted by eurordis. according to it, % of the respondents admitted they had to reduce their professional activities after they were diagnosed with rare condition. this means that more than half of the people with rare diseases in europe face employment challenges. the analysis of this survey was important input to the presentation of the epf youth group project -ways. results: this is the abbreviation of work and youth strategy and it is a two year project, disseminated among young patients with chronic conditions. the main purpose was to increase the awareness about positive and negative practices for young patients on the labour market and to develop recommendations to employers and decision makers. that is why epf youth group conducted an online survey and provided different deliverables like factsheet with recommendations to employers and video about young patients' rights on the work place. the results of both survey provided important insight about the challenges people with rare diseases face in job employment. it proved the fact that only if we work together as a community of patients, we will be able to provide better opportunities for national and international inclusion. paul rieger , eberhard scheuer centiva health ag, zug, switzerland correspondence: paul rieger -paul@centiva.health orphanet journal of rare diseases , (suppl ):s the lack of access to research participants is the number one reason why medical studies fail [ ] . real-world data is often difficult to get despite usd billion costs of patient data intermediation. therefore, a new model for patient access is necessary where patients get paid fairly for their data, retain control over their data, and drive citizencentered research. on the other hand, researchers and industry must be enabled to access patients directly without violating their privacy, while reducing time and costs of data access at the same time. current patient registries facilitate patient access and match patients with a centralized data flow while giving little to no incentives. whereas, a decentralized patient registry allows for direct and confidential matchmaking between patients and organizations looking for data through the use of blockchain technology. it lets the patient decide with whom they want to share their data. on such a platform, patients can receive incentives in the form of digital currency. currently, centiva health [ ] is used in the context of rare diseases and population health, i.e., outbreak monitoring. in the area of rare diseases centiva health cooperates with patient advocacy groups by enhancing existing registries with the ability to collect real-world data. the access to patient via a decentralized registry leads to aligned incentives, real-time access to data, improved disease visibility while preserving patient privacy. orphanet j rare dis , (suppl ): the united kingdom and in the czech republic, to co-design optimal methods/services for the communication of genomic results. methods and results: using a methodology called experience-based co-design (ebcd) , we supported families and health professionals to shared and discuss their experiences, identify priories for improvement and then work together to prototype and test out interventions to address these. the process involved observations of clinical appointments (), interviews with families () and health professionals and a series of workshops and remote consultations at both sites. results: five shared priorities for improvement were identified by participants at the two sites, and eight quality improvement interventions were prototyped/tested to address these ( table ) . discussion: the findings clearly indicate the need for improved follow-up care to support families in the short, medium term after the sharing of the results, including when a diagnosis is confirmed. different service models were prototyped, including follow up consultations with clinical geneticists and a dedicated role to facilitate co-ordinated care. the findings also demonstrate the need for continued workforce development on the psychosocial aspects of genomic and genetic communication, specifically on families' needs regarding genomic consent and the experience of guilt and (self-) blame. to use technology has been used in the home to provide objective seizure data prior to upcoming clinic appointments. the covid- pandemic has prompted an acceleration in telemedicine and epihunter has improved the effectiveness of virtual consultations bringing opportunities for both diagnostics and informed changes in treatment. epihunter is an example of technology repurposing to create a new normal for people with hidden disabilities such as those living with absence epilepsy. the rare disease patient community tried to get this well detailed plan to be transferred to regulation which usually means an adequate financial substitution of those expert services. the patients should benefit from a centralized expert treatment/care pathway. esophageal atresia (ea) is a rare congenital condition with an estimated prevalence of to in , live births. esophageal atresia patients require life-long attention. ernica has developed a 'patient journey' for ea patients, under the leadership of patient representatives from the international federation of ea support groups (eat). in germany, patients with congenital malformations which need surgery in early life are treated in hospitals with (very) low experience. how can we as patient representatives get the fruits of the erns into the national health system? we don't have public money. we have no official contract and no political support. keks e.v., the german ea support group together with other support groups (e.g. soma e.v.), and with surgical expert teams across germany, some of them members in erns, started to organize monthly virtual boards for those patients. a self-commitment on ethical and medical standards following the ern-criteria, and a collaborative attitude within the group, help us to get step by step the first ernica results to the bedside of ea patients. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. delays in completion and results reporting of clinical trials under the paediatric regulation in the european union: a cohort study new strategies for the conduct of clinical trials in pediatric pulmonary arterial hypertension: outcome of a multistakeholder meeting with held at the european medicines agency on monday decentralized rather than centralized pediatric surgery care in germany erdri.dor -european directory of registries natural history of alkaptonuria recent advances in management of alkaptonuria when you build in the world, you build in your mind white paper on lego serious play articulation of tacit and complex knowledge zolgensma (onasemnogene abeparvovec-xioi) childhood spinal muscular atrophy: controversies and challenges spinal muscular atrophy bannockburn, il indirect estimation of the prevalence of spinal muscular atrophy type i, ii, and iii in the united states pilot study of populationbased newborn screening for spinal muscular atrophy in new york state presymptomatic diagnosis of spinal muscular atrophy through newborn screening one year of newborn screening for sma -results of a german pilot project correlation between sma type and smn copy number revisited: an analysis of unrelated spanish patients and a compilation of reported cases available from: www.ibm.com/produ cts/micro medex -red-book available from: www.ibm.com/produ cts/micro medex -red-book references . united kingdom national health service international rett syndrome foundation presented at aacap's th annual meeting placebo-controlled crossover assessment of mecasermin for the treatment of rett syndrome cerebrolysin therapy in rett syndrome: clinical and eeg mapping study effects of acetyl-l-carnitine on cardiac dysautonomia in rett syndrome: prevention of sudden death? rett syndrome: controlled study of an oral opiate antagonist, naltrexone pharmacologic treatment of rett syndrome with glatiramer acetate safety, pharmacokinetics, and preliminary assessment of efficacy of mecasermin (recombinant human igf- ) for the treatment of rett syndrome effects of ω- pufas supplementation on myocardial function and oxidative stress markers in typical rett syndrome thirteenth general programme of work - . promote health -keep the world safe -serve the vulnerable principles and practice of screening for disease newborn screening programmes in europe; arguments and efforts regarding harmonization. part -from screening laboratory results to treatment, follow-up and quality assurance newborn screening programmes in europe; arguments and efforts regarding harmonization. part -from blood spot to screening result a framework to start the debate on neonatal screening policies in the eu: an expert opinion document communication from the commission to the european parliament, the council, the european economic and social committee and the committee of the regions on rare diseases: europe's challenges on an action in the field of rare diseases on the application of patients' rights in cross-border healthcare available from: https :// drive .googl e.com/file/d/ sfe xp deisc ogrbw swht uznx erj/ view?usp=shari ng . which scenarios are most preferred by the rd community? . which scenarios are most likely to happen? . how do we achieve the scenarios we prefer and avoid those we don factors associated with clinical trials that fail and opportunities for improving the likelihood of success: a review s background: to help inform cross-national development of genomic care pathways, we worked with families of patients with rare diseases and health professionals from two european genetic services bringing user experience to health care improvement: the concepts, methods and practices of experience-based design department of medical informatics correspondence: info-rdsgofair@go-fair.org (marco roos -m.roos@ lumc.nl, gülçin gümüş -gulcin.gumus@eurordis.org) in practice, it can take months of searching data, understanding the sources, mapping to consistent standards, and negotiating how one might use the data. many assume that for sharing and analysis, data need to be moved between sources. this can lead to sharing only minimal, non-sensitive data: a fraction of global rare disease data. alternatively, data elements and local access conditions can be described by globally agreed, computer understandable standards conform fair principles. this enables analysis at each source, while sharing only the analysis results. fair prepares data for rapid discovery, access, and analysis, also when data remain at source. projects such as the european joint programme for rare diseases work on the technical infrastructure to support this. adopting fair principles requires culture change. fair advocates working on rare diseases have organised the 'rare diseases global open fair implementation network rds go fair prioritizes patient representatives for their capacity to reshape current practices, welcoming them to organise their own network within rds go fair to foster fair for patient priorities (registration for follow-up meetings is possible via eucerd recommendations on quality criteria for centres of expertise for rare diseases in member states joint action rd-action (european union's health programme european commission website pdf s patient's view on disruptive innovations in clinical research elizabeth vroom we would like to thank the j-rare patient organization groups and the asrid research ethics committee. consent to publish: informed consent to publish has been obtained from patients. we thank all the families who took part in the interviews, the staff of the health services and charities who collaborated to advertise the study to eligible participants and the members of the family advisory groups who reviewed the interview schedule and provided invaluable feedback on the preliminary findings. the work has been presented on behalf of the study "improving the communication of genomic diagnosis results using experience based co-design (ebcd)", which is part of the solve rd project. the solve-rd project has received funding from the european union's horizon research and innovation programme under grant agreement no . we thank all the families who took part in the interviews, the staff of the health services and charities who collaborated to advertise the study to eligible participants and the members of the family advisory groups who reviewed the interview schedule and provided invaluable feedback on the preliminary findings. the work has been presented on behalf of the study "improving the communication of genomic diagnosis results using experience based co-design (ebcd)", which is part of the solve rd project. the solve-rd project has received funding from the european union's horizon research and innovation programme under grant agreement no . acknowledgements: we would like to thank all seed group members of the rare diseases global open fair implementation network, the go fair office, eurordis, the european union's horizon research and innovation program under the ejp rd cofund-ejp n° , the rd-connect community, the lumc biosemantics research group, simone louisse (guardheart epag), and the many patients and patient representatives that inspire us. recent advances in next-generation phenotyping (ngp) for syndromology, such as deepgestalt, have learned phenotype representations of multiple disorders by training on thousands of patient photos. however, many mendelian syndromes are still not represented by existing ngp tools, as only a handful of patients were diagnosed. moreover, the current architecture for syndrome classification, e.g., in deepgestalt, is trained "end-to-end", that is photos of molecularly confirmed cases are presented to the network and a node in the output layer, that will correspond to this syndrome, is maximized in its activity during training. this approach will not be applicable to any syndrome that was not part of the training set, and it cannot explain similarities among patients. therefore, we propose "gestaltmatch" as an extension of deepgestalt that utilizes the similarities among patients to identify syndromic patients by their facial gestalt to extend the coverage of ngp tools. methods: we compiled a dataset consisting of , patients with , different rare disorders. for each individual, a frontal photo and the molecularly confirmed diagnosis were available. we considered the deep convolutional neural network (dcnn) in deepgestalt as a composition of a feature encoder and a classifier. the last fully-connected layer in the feature encoder was taken as facial phenotypic descriptor (fpd). we trained the dcnn on the patients' frontal photos to optimize the fpd and to define a clinical face phenotype space (cfps). the similarities among each patient were quantified by cosine distance in cfps. results: patients with similar syndromic phenotypes were located in close proximity in the cfps. ranking syndromes by distance in cfps, we first showed that gestaltmatch provides a better generalization of syndromic features than a face recognition model that was only trained on healthy individuals. moreover, we achieved % top- accuracy in identifying rare mendelian diseases that were excluded from the training set. we further proved that the distinguishability of syndromic disorders does not correlate with its prevalence. conclusions: gestaltmatch enables matching novel phenotypes and thus complements related molecular approaches.an audience of over delegates voted on the rare scenarios and discussions throughout the sessions of ecrd indicated the following opinions:-if we continue as we are we will find ourselves in the "fast over fair" scenario which forecasts high collective responsibility but an emphasis on market-led innovation -the majority of the audience preferred a future scenario with continued high collective accountability but more of an emphasis on needs-led innovation, "investments for social justice" -a significant portion of the audience agreed that a balance must remain with the market led attractiveness of the "technology along will save you" scenario -a scenario where "it's up to you to get what you need" was least preferred by all the diagnostic pathway in rare disease has a number of bottlenecks that can result in the pathway becoming an odyssey. while some barriers are being removed through remarkable innovation, there is one story of diagnostic delay that is echoed by rare disease patients across the globe and across thousands of different rare diseases: doctors failed to suspect something rare. however we cannot expect doctors to suspect rare diseases when they haven't been trained to or, in some cases, have been trained to do the exact opposite with the mantra "common things are common". without appropriate training 'rare' can be mistaken for 'irrelevant' when in reality million european citizens live with a rare disease [ ] . medics rarediseases is driving an attitude change towards rare diseases in the medical profession. this begins with explaining that rare diseases are collectively common and all clinicians should expect to manage people with diagnosed and undiagnosed rare disease regularly during their careers. this attitude change is called #daretothinkrare. secondly m rd is suggesting a new approach to educating about rare disease for trainers and training institutes. this approach tackles rare disease as a collective and focuses on patient needs rather than details of individual diseases. this not only solves the impossible challenge of covering over rare diseases during medical training but also provides some equity between different diseases. lastly, m rd promotes the use of rare disease specific resources that will support both doctors and their patients. this includes the invaluable input from patient advocacy groups. the step between presenting quality assurance of rare disease (rd) centers of excellence (coe) through designation, accreditation, monitoring and constant improvement provides a means to ensure high quality, centralization of resources and expertise, and cost-efficiency. eucerd recommendations for quality criteria of coe, issued in , are still highly relevant [ ] . in the state of art resource, almost all european union (eu) member states (ms) claim, that their coes conform to eucerd recommendations [ ] . however, national quality assurance processes differ significantly: some ms apply robust procedures, while in other ms, many of them -but not exclusively -are eu- ms, processes of quality assurance are less developed. under the subsidiarity principle embedded into european treaties, the eu plays a limited role in many areas of healthcare, and coes quality assurance processes are a choice and responsibility of ms.with the establishment of erns, another layer of quality assurance has been developed by the european commission and the ms [ ] . this new quality assurance framework may be in line, or not, with national accreditation systems and involves i) assessment of coes when they apply for full membership of erns and ii) continuous monitoring afterwards [ ] . in every ern, members have to be "equal partners in the game" and share the same goals, rights and obligations. while the ern logo should eventually be a quality mark of the highest standards, strong links of ern members to national systems, including many more and less specialized healthcare providers, are essential to ensure proper care pathways for rd patients. importantly, erns themselves and patients/non-governmental organizations provide us with additional means of "informal quality assurance". many erns are implementing their own monitoring processes through the creation of registries to collect health outcomes that allow peer-benchmarking. meanwhile, patients provide their strong voice through european patient advocacy groups (epags) and help to signpost "the best" coes through information sharing. in both these processes, the power of open, transparent information on performance may finally lead to improved transparency and accountability at a national level and, presumably, may have an impact on the composition of erns in the future. in order to improve clinical research, patient preferences and outcome measures relevant to patients should become the core of drug development and be implemented from the earliest stage of drug development. from 'bedside to bench' instead of from 'bench to bedside' . at all levels the reuse of data could and should be enhanced. patient derived or provided data are not owned by those who collected them, and their reuse should be primarily controlled by the donors of these data. researchers and health professionals are custodians (gdpr). to enable the optimal reuse of real world data, the data needs to be findable, accessible, interoperable and reusable (fair) by medical professionals, patients and in particular also by machines. for this reason the world duchenne organization published a duchenne fair data declaration [ ] . reuse of placebo data and use of natural history data could speed up research especially in the field of rare diseases at this moment, in line with gdpr, patients are in a good position to decide about the reuse of their own data and should not only have access to these data but preferable also be in charge of their own data. background: drug repurposing for rare disease has brought more costeffective and timely treatment options to patients compared to traditional orphan drug development, however this approach focuses purely on medical interventions and requires extensive clinical trials prior to approval. in the case of refractory epilepsy, practical solutions are also required to better manage daily life. here we present an example of technology repurposing as a practical aid to managing absence epilepsy. methodology: existing research tells us that seizure control is not the only consideration of quality of life in children with epilepsy and that mental health and caregiver/peer support are of utmost importance. we explored the needs of stakeholders and determined that there was a delicate balance between the individual (and those that care for them) and those that have the power to change their lives. results: across all stakeholders there was a shared common need to obtain objective data on absence monitoring to relieve the burden on families/carers to retain manual seizure diaries whilst providing accurate and timely data to medical teams, researchers and social care. epihunter is an absence seizure tracking software using repurposed technology: a headset from wellness/leisure to collect electroencephalographic (eeg) data and an ai algorithm to detect and record absence seizures on a mobile phone application in real-time. both eeg and video recording of the seizure are automatically captured. this low cost, easy key: cord- -avn f w authors: nan title: symposium summaries date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: avn f w nan the creation of mouse models of cf has provided new opportunities to elucidate disease pathogenesis, correlate genotype with phenotype, and evaluate the safety and efficacy of novel cf therapies. epithelial tissues from cf mice exhibit the loss of camp-stimulated clsecretion characteristic of the human disease, and cf mouse models display a range of abnormal intestinal and pulmonary phenotypes dependent on the cftr genotype, independently segregating modifier genes, and environmental factors (diet, pathogens) ( ) . studies in cf mice may also be helpful in answering the question: how much cftr is enough? quantifying the level of gene and protein expression necessary for restoring lung and intestinal function is of paramount importance for the design of somatic gene therapy or protein therapy protocols. one strategy is to reduce the expression levels or activity of cftr in wild type mice by genetic or pharmacological approaches (e.g. insertional mutagenesis; sirna; specific cftr inhibitors) and determine the threshold level at which the disease symptoms become manifest. a typical example is the creation of the exon insertional cf mouse ( cftr tm hgu ) . unlike the cf mice generated by gene replacement (knockout null mice), the original mutant mouse with a mixed background showed residual low levels of normal cftr mrna (< % in the lungs) as a result of exon skipping and aberrant splicing ( ) , associated with a - % residual intestinal and nasal camp-mediated chloride secretion and only mild intestinal obstruction ( % preand post-weaning survival on solid diet); outbred mf / cftr tm hgu mice raised outside the isolator displayed signs of inflammation, reduced mucociliary clearance , and impaired airway clearance of aerosolized, cf associated bacteria ( ) , suggesting that a low residual expression of normal cftr may ameliorate intestinal obstruction but not cf lung pathophysiology. recently, the phenotypic evaluation of two cftr tm hgu inbred mutant strains (named cf/ -cftr tgh(neoim)hgu and cf/ -cftr tgh(neoim)hgu ) generated in hannover showed similarly low cftr transcript level in the intestine, a low residual amount ( - %) of mature cftr protein in the apical membrane of the enterocytes, but close to normal ( - %) chloride secretory responses to camp in the ussing chamber ( ) . these findings suggest that ~ % of wild-type cftr protein is sufficient to normalize transepithelial intestinal chloride secretion, survival and weight gain. in contrast to intestine, the level of cftr mrna, immunostained cftr protein, and camp-stimulated chloride secretion in the nasal epithelum of these mice was almost normal, but the amiloride-sensitive sodium absorption, previously found to be fold increased in cftr-null mice ( ), remained ~ -fold above normal values. this finding suggests that a complete correction of the hyperabsorption of sodium in the airways, a known hallmark of cf, is difficult to reach even at virtually normal cftr protein levels. as yet the consequence of this persistent abnormality in sodium transport for lung pathophysiology remains to be explored. an alternative strategy is to express human cftr (hcftr) in cftr-/-mice and to examine rescue of the gut defect by the transgene. for example, a transgenic mouse expressing hcftr cdna under control of the intestinal fatty-acid binding protein (fabp) promoter showed normal survival and functional correction of ileal goblet cell and crypt hyperplasia, and a ~ % rescue of transepithelial chloride current ( ) ; assuming that % chloride secretion corresponds with ~ % mature cftr (cf. cf/ mice), it follows that ~ % cftr protein is sufficient to eliminate intestinal pathology. the notion that only partial correction of cftr channel activity may have a significant clinical impact was confirmed by replicating the g c trafficking mutant in the murine cftr gene ( ): % of mature cftr protein corresponded with ~ % residual camp-stimulated jejunal chloride secretion and the complete absence of intestinal pathology. insight into the question: how much cftr is enough, is also relevant for attempts to ameriolate cf disease pharmacologically, e.g. by promoting the correct processing and trafficking of f del cftr by small molecules emerging from high-throughput screens, by altering chaperone function ( ) or by suppressing premature stop mutations ( ) . one approach is to treat cf mutant mice in vivo, or isolated tissues ex vivo with the rescue compounds/ approaches and compare the gain in cftr protein expression with the extent of functional rescue and the amelioration of disease symptoms. for example, ex vivo incubation of ileal mucosa from congenic fvb cftr tm eur mice homozygous for the f del mutation at low temperature ( h, c) resulted in a gain in mature cftr protein (band c on a western blot) from a starting value of ~ % to a maximal level of % of the amount in cftr +/+ ileum, concomittant with the full restoration of camp/genistein-stimulated transepithelial chloride current ( ) . assuming that the potentiator genistein fully corrects the ~ -fold lower open probability of f del cftr channels, this outcome confirms that ~ % of mutant cftr protein, in the presence of a potentiator, is sufficient to fully restore intestinal chloride transport. complete functional rescue could also be reached by exposing the ileal mucosa for h at c to the proteasome/calpain inhibitor alln, likewise resulting in a gain in steady state levels of apical cftr protein up tõ % of normal levels ( ) . importantly, even untreated rotterdam fvb cftr tm eur mice maintained on solid food (hope farms srm-a) did not show signs of intestinal or lung pathology despite a low level of mature f del cftr protein (~ %) in the intestine and a reduced level in the nasal epithelium (~ %). however a shift to a different solid diet (rm , special diet services) caused % mortality by intestinal obstruction after - days, whereas changing the background from fvb to c bl/ provoked clear symptoms of lung pathology, without altering the nasal bioelectrics. these findings not only define optimal conditions for testing the efficacy of f del cftr rescue approaches in these mice, but stress the importance of diet composition and genetic modifiers in cf pathology irrespective of the level of transepithelial chloride and sodium transport. what is termed a "genotype-driven" design. the second approach is to select patients with a similar level of disease severity and then determine their genotypes in a "phenotype-driven" study. both approaches have been utilized extensively in cf research, and each approach has yielded a number of important insights: ) a small amount of wild-type cftr rna transcript ameliorates disease severity. "leaky" splice-site mutations that permit production of some normally-spliced cftr transcript can moderate disease severity in the pancreas, lungs and vas deferens. correlating estimates of the amount of wild-type transcript associated with "leaky" splice-site mutations demonstrates that approximately - % wild-type transcript is associated with a less severe pancreatic disease, and approximately - % allows individual to escape from cf lung disease ( ; ). ) partially functional cftr can reduce disease severity. phenotype-driven studies searching for mutations occurring in patients with mild forms of cf revealed a disproportionate number of amino acid substitutions ( ; ) . analysis of cftr bearing these substitutions in cell-based systems has revealed that many retain chloride channel properties of the wild-type protein ( ) . in general, the degree of residual chloride channel function of cftr correlates with the severity of the cf phenotype, in particular pancreatic status and degree of sweat chloride abnormality ( ; ). correlation of chloride channel function and the severity of lung disease is less clear since there is a paucity of mutations that are convincingly associated with an improved pulmonary outcome. one exception is the a e mutation that confers a milder lung phenotype ( ) , cftr bearing this mutation has altered processing and chloride channel properties ( ) . ) increasing the amount of a partially functioning protein can avoid the life-limiting pulmonary complications of cystic fibrosis. the best example of this phenomenon is the r h mutation. this mutation occurred at least twice in human evolution in different cftr genes, one bearing an efficient splice site ( t) in intron , and the other bearing an inefficient splice site ( t) in intron ( ) . cftr bearing r h is properly folded and targeted to apical membranes however conduction of chloride via the cftr channel is reduced from wild-type cftr ( ) . genes with r h and t have reduced levels of full-length transcript leading to reduced production of partially functional cftr bearing r h. individuals bearing r h- t and a severe cf mutation present with cystic fibrosis manifesting as progressive lung disease, pancreatic sufficiency, absence of the vas deferens in males and elevated sweat chloride concentration. on the other hand, r h with the efficient t variant results in normal levels of full-length transcript bearing the r h mutation leading to normal levels of cftr protein bearing this mutation. males with r h- t and a severe cf mutation generally have congenital absence of the vas deferens, a form of male infertility seen in cf males, and females generally have no clinical phenotype. importantly, lung disease is usually absent in these individuals. thus, increasing the amount of a partially functional cftr mutant can alleviate the lifelimiting complication of cf. ) tissues affected in cf require different levels of cftr function to operate normally. an early revelation of genotype/phenotype studies was that the presence of one or two "mild" mutations was associated with a milder form of pancreatic disease but not milder lung disease or sweat gland abnormalities ( ) . from a large number of studies, it appears that the sinuses are most sensitive to reduction in cftr function, followed by the vas deferens, lungs, sweat gland, and pancreas. thus, the level of correction needed to reverse cf pathology will depend upon the target organ. studies of genotype/phenotype conducted in cf strongly suggest that correction of most features of the disease will occur when % of normal cftr function is achieved. however, individuals bearing genotypes that are predicted to generate this level of cftr function have minimal differences in biochemical measurements such as sweat chloride levels and nasal potential difference measurements. improvement in measurable cftr function in vivo may require % function based on what is observed in cbavd males. males who have mild mutations in one cftr gene and a severe mutation in the other cftr gene can present with normal or near normal lung function, borderline to elevated sweat chloride levels and nasal potential difference measurements that are abnormal, but can be distinguished from those who have life limiting forms of cf ( ) or are normal ( ) . the cf respiratory disease phenotype includes thick mucus secretion and colonization of the lung with bacteria, such as pseudomonas aeruginosa (psa). presumably, repeated bacterial infections compounded by an inability to clear the infecting organism results in a profound neutrophil migration into the airways and the secretion of a variety of inflammatory mediators, including neutrophil elastase, reactive oxygen species, and cytokines, such as the chemokine il- . the existence of an underlying defect in the airway immune response of cf patients remains controversial; however, increasing evidence suggests that the cf lung exhibits an exaggerated immune response even in the absence of bacterial infection ( ; ). moreover, increasing evidence suggests that the cystic fibrosis transmembrane conductance regulator (cftr) may directly or indirectly regulate cfassociated airway inflammation and bacterial clearance. in the cf community, the advance of gene therapy has precipitated the question of how much cftr expression and function is required for the correction of cftr-related functions, including the resolution of inflammation and bacterial clearance within the cf lung. studies examining the effect of cftr on these responses have been performed utilizing both in vitro and in vivo model systems. specifically, in vitro studies have focused upon the effect of cftr expression and / or function in airway epithelial cell models on inflammatory mediator expression, nf-b activation, and bacterial adherence. in these studies, a variety of non-cf, cf, and cftr corrected-cf primary and immortalized airway epithelial cell lines have been utilized; however, little consensus has been reached on the role that cftr plays in regulating inflammatory and bacterialrelated responses. for example, several reports suggest that mutant cftr expression and / or function correlates directly with enhanced il- and il- ( ; ) protein secretion, reduced rantes ( ), il- ( ), and inos ( ) production, exaggerated nf-b activation ( ) , and altered bacterial adherence ( ; ). in contrast, additional studies indicate that little or no correlation exists between mutant cftr expression and il- ( ; - ) or il- ( ; ) and il- ( ) production and nf-b activation ( ; ) . with regard to in vivo studies examining the role of cftr in the pathophysiology of the cf lung, there is a general consensus that, when challenged with psaembedded agarose beads, mice lacking functional cftr exhibit heightened pulmonary inflammatory responses as compared with wild-type controls ( ; ) ; such responses include increased production of tnf␣ and il- ␤. there is less agreement, however, between in vivo studies that examine the role of cftr in the bacterial clearance of an infected cf lung. a collection of recent studies indicate that correction of mutant cftr in the lungs of murine cf models enhances the clearance of burkolderia cepacia ( ) , but not of psa ( ; ) . interestingly, some studies have proposed that nutritional deficits, which result from cf-related intestinal defects, contribute to the exaggerated immune response in both cf patients and cf mouse models ( ) . in contrast, a recent report demonstrates that cftr knock-out (ko) and gut-corrected cftr ko mice displayed similar indices of inflammation when challenged with psa ( ) . this study also showed that cftr ko mice and wildtype control littermates exhibited similar lung inflammatory responses in the presence of a psa infection ( ) . such discrepancies between these collective in vitro and in vivo studies may be due to differences in the airway epithelial cell and mouse models utilized, tissue culture conditions, exogenous stimuli, bacterial strain, and type of bacterial infection. importantly, these discrepancies highlight the on-going controversies regarding the role of cftr in regulating inflammatory responses within the cf lung and, therefore, make it difficult to project the amount of cftr required to attenuate such responses. in conclusion, the relationship between the level of cftr expression and excessive inflammatory responses warrants further study. the earliest concept of cf pathophysiology was that disease arises because affected organs are blocked by abnormally thick secretions , . thick mucus was initially attributed to a defect in mucin molecules, but the discovery of electrolyte disturbances in sweat gradually led to a radically different concept: the basic defect is in ion transport, which leads to thickened secretions because of deficient water within mucus. it was long known that the first step in water secretion is often mediated by anion secretion. with the discovery that the defective cf gene codes for cftr, a channel for chloride and bicarbonate, it was immediately apparent that defective secretion of electrolytes and water could account for observed pathology in many cf-affected organs such as the vas deferens, pancreas, and intestines. an exception is the sweat duct, where cftr anion channels play an important role in electrolyte absorption, leaving water behind . what about airways? people with cf typically die from unremitting lung infections. both ciliated surface epithelia and submucosal gland serous cells express cftr, and decreased anion-driven fluid secretion from both of these cell types contributes to mucus stasis. in addition, cf airways have an additional defect: lack of cftr disinhibits enac, leading to increased sodium-dri-ven fluid absorption: if extreme enough, hyper-absorption alone can produce mucus stasis and a cf-like disease . we propose still a third defect: gland mucus contains a rich complement of proteins that act as anti-microbials, anti-oxidants, and inhibitors of serine proteases. we hypothesise that the bio-availability of these compounds is reduced in cf because of the altered mucus properties(a precedent for this concept has been established in cf mouse intestines . furthermore, the ability of mucus to inhibit bacterial growth wanes with time, so static mucus eventually becomes a bacterial growth medium . our laboratory has recently focused our efforts on gland secretion and serous cell models. glands are the major secretory organs of healthy lungs and gland serous cells express cftr . when provoked, glands produce copious amounts of mucus that traps pathogens and inhibits their growth while they are swept from the airways . mucus clearance normally keeps the airways sterile, and it has been proposed that altered gland function in cf airways impairs the mucus shield e.g. , . early studies of airway glands established many of their properties, but were carried out before it was understood that cftr is expressed in serous cells. since then, studies by ballard and colleagues, and more recently by our lab and the verkman lab, have expanded our understanding of glands and have established that cf glands have defective secretion. we developed an optical method for rapid and accurate assessment of single gland secretion rates and are using it to study detailed features of single gland secretion in sheep, pigs and humans. glands secrete to both vip and ach. gland secretions are hypotonic , and secretions to both mediators are slightly acidic relative to bath . we found that in humans with cf, the glands are completely refractory to vip or forskolin , and produce about half normal amounts of fluid in response to acetylcholine. secretions to ach have increased viscosity . to look more closely at gland secretion, we have developed methods to isolate single glands or portions of glands, so that responses of single tubules and single cells can be monitored with nomarski differential interference contrast microscopy. these studies reveal that mucus is a highly heterogeneous substance, and provide evidence for exocrine, apocrine and holocrine secretion within a single gland. surprisingly, vip, which produces zero mucus outflow from cf glands, produces clear evidence of secretion when individual cf gland tubules are monitored, suggesting that proteins are being released, but without sufficient water to exit from the gland duct. these findings accord well with earlier predictions made by ballard and colleagues using pharmacological block of fluid secretion . a complementary approach is to use cell culture models of serous cells. to help bridge the gap between secretion studies of glands and electrophysiological studies of cell sheets, we designed a 'virtual gland' that allows us to establish secretion rates (jv) and collect secreted fluid from secretory cells such as calu- cells . with that link established, we can more easily interpret electrophysiological studies of calu- cells now being carried out in many laboratories. recent work from our laboratory indicates that calu- cells express kir . and possess an apical k + channel with properties similar to kir . . they also possess an apical h-k atpase, resulting in the co-ordinate secretion of h + (via the h-k atpase) and hco -(via cftr). this system provides one method whereby an initially isotonic secretion can be rendered hypotonic by the subsequent conversion of h co into co and h o. the hallmark of cystic fibrosis (cf) lung disease is chronic colonization/infection of the airways by a variety of bacteria, including h. influenzae, s. aureus, and p. aeruginosa. in cf, bacteria grow in regions of the lung that are normally sterile. the clinical course of cf lung disease correlates with the acquisition of bacterial infection and its progression. while the pathogenic mechanisms linking cftr mutations to cf lung disease continue to be a subject of research and debate, it is accepted that the normal host defense barriers in the lung are fundamentally altered. whatever the cause of the underlying propensity for infection, it is lung specific. the result is a characteristic susceptibility to bacterial infection. these features indicate that cf impairs the innate defenses of the lung. prominent components of innate immunity include the peptides and proteins secreted into airway surface liquid (asl). the airway epithelium directly interacts with the environment in a dynamic fashion. because of this interplay between the host and environment, systems evolved to clear or inactivate the pathogens encountered (see fig) . these defense systems include components of both innate and adaptive immunity. innate immunity complements adaptive immunity by its "ever ready" nature, as many of its components are continuously present and require no previous encounter or memory for their activity ( ) . in the airways, the main cellular components of innate immunity are the epithelium and its products, and macrophages, dendritic cells, nk cells, cytotoxic t cells, and neutrophils. innate responses may be triggered via pathogens interacting with pattern recognition receptors such as the toll-like receptor (tlr) family ( , ). the airway epithelium senses and responds to microbial challenges and can vectorially secrete its specialized products in the apical or basolateral direction to mount the appropriate response. in addition to serving as an important physical barrier, two major functions of the epithelia lining the conducting airways and submucosal glands are the production and modification of asl and the secretion of factors that contribute to host defenses. these agents act by a variety of mechanisms including disruption of microbial cell walls, sequestration of nutrients, and acting as decoys of microbial attachment. presumably, asl composition provides an optimal microenvironment for the function of the microbicidal compounds. asl contains several products with bacteriostatic or bactericidal activities, forming an important line of defense ( , , ) . the specialized cells of submucosal glands (serous cells) also contribute to the secretion of airway surface fluid and are a major source of the production of macromolecules including antimicrobials and mucins ( , ). the antimicrobial peptides and proteins produced by surface and submucosal gland epithelia act in a broad-spectrum fashion, in some cases exerting antimicrobial effects against bacteria, fungi and viruses. major asl protein components in terms of concentration include lysozyme, lactoferrin, and secretory leukocyte proteinase inhibitor (slpi) ( , , , , ) . additional components are surfactant proteins a and d (sp-a, sp-d), collectin family members that are opsonins for bacteria and viruses ( , , ) , the alpha and betadefensins ( ), cathelicidins ( ) , ngal (lipocalin ) ( ), and members of the lipid transfer/lipopolysaccharide binding protein family ( ) . while the activities of many asl components have been defined, it is clearly a complex milieu, containing > proteins ( ), and the identity and function of many constituents are unknown. the pulmonary host defense defect in cf is complex. it is likely that the nature of the defect changes as the disease progresses. early lung disease is characterized by intermittent bacterial infection and the onset of inflammation. hallmarks of established lung disease include established bacterial infection with organisms living in biofilms, chronic neutrophilic inflammation, and progressive bronchiectasis. in the ~ yrs since the cftr gene was discovered, many ideas emerged to explain the link between mutations and lung disease. one hypothesis is that cftr mutations alter the processing of cell surface asialoglycoproteins such that the cf epithelia are more suitable for bacterial attachment ( ) . a second is that cftr at the cell surface functions as a bacterial receptor for clearance by epithelial phagocytosis; mutant cftr is unable to perform this function ( ). a third hypothesis is that cftr mutations cause increased nacl and liquid absorption across epithelia, diminishing the asl volume and impairing normal mucociliary clearance. as a secondary effect of impaired clearance, bacterial adherence and colonization of the airways begins ( , ) . a fourth theory is that mutations in cftr impair the normal ability of epithelia to modify the electrolyte composition of airway surface liquid (asl) by reabsorbing nacl ( ). as a result, asl ionic strength increases and this secondarily impairs the function of many endogenous antimicrobials ( , , ) . alterations in the composition of secretions from submucosal glands may contribute to dehydration of the asl layer and increased viscosity of cf secretions ( , ) . furthermore, the trapped mucus may create an environment that is well suited for bacteria such as pseudomonas to live as biofilms ( , ) . in addition to primary or secondary cftr-associated changes that lead to pulmonary disease manifestations, polymorphisms in other genetic loci may influence the cf phenotype ( , , ) . several candidate modifier genes have been proposed for cf including mannose binding protein ( ) , hbd- and hbd- ( ), alpha -antitrypsin enhancer ( ) , and hla class ii ( ) . these disease pathogenesis hypotheses are not necessarily mutually exclusive, and underscore controversies in our current understanding of cf lung disease. a theme common to all these hypotheses is that altered innate defenses predispose to an increased susceptibility to colonization with bacteria. the chronic infection and inflammation associated with cf causes epithelial injury and repair responses ( ) . this may also cause the epithelium to be impaired. for example, the production of the collectins sp-a and d progressively declines with disease progression ( ). proteolytic enzymes, arising from cf airway secretions or resident bacteria, also cleave and inactivate many proteins and receptors involved in innate and adaptive immune responses, further impairing host defense. for example, pseudomonas proteases cleave igg and destroy its opsonic activity ( ) . there is also evidence for host or pathogen derived enzymatic cleavage of transferrin, lactoferrin, and slpi ( , ) . recently, taggart et al demonstrated that the cysteine protease cathepsins b, l, and s, in concentrations that are present in cf secretions, degrade and inactivate hbd- ( ). in addition, the protease rich environment of the cf airways has been recently shown to cleave tlrs- and - that may further impair innate immune signaling ( ) . the exuberant production of mucus and dna released from host cells and bacteria creates a complex anionic matrix that may also bind cationic antimicrobial proteins and further impair their activity. in summary, the single gene defect in cf causes complex primary and secondary host defense defects that initiate and then perpetuate progressive chronic lung disease. understanding these defects may lead to improved treatment for cf lung disease. emerging evidence from in vitro and in vivo studies suggests that airway surface liquid (asl) volume depletion initiates the cascade of events that results in cf lung disease. although regional differences may exist in the relative contributions of the superficial epithelium and glands to asl volume depletion, a common sequence follows that is manifest by periciliary liquid volume depletion, concentration of mucins in the mucus layer, adhesion of the mucus layer to airway surfaces, delayed ciliary and cough-dependent clearance, inflammation perhaps without overt bacterial infection, and ultimately acquisition of bacterial infection and promotion of bacterial biofilms. four topics that pertain to this scenario will be reviewed. • mechanisms of autoregulation of asl volume by normal and cf airway epithelia: the focus will be on the signals that sense airway surface liquid volume and the mechanisms by which these signals coordinate the deactivation/activation of epithelial ion channels (enac, cftr, cacc) to control asl volume and hence surface mucus clearance. • regional differences in airway surface liquid volume regulation in normal and cf: data will be reviewed on the expression in the normal airway of cftr in the proximal versus distal regions of the lung and the superficial versus gland epithelia. in addition, studies on the volume regulatory capacity of the normal and cf bronchiolar region will be reviewed. • functional consequences of depleted asl volume: the focus will be on mucus adhesion as being an initiating lesion in the progression of the obstructive and infectious components of cf lung disease. data demonstrating adhesion both in vitro and in vivo will be reviewed, as well as inferences as to the nature of the adhesive interactions. • methods of restoring asl volume therapeutically: the actions of hypertonic saline in restoring asl volume in normal and cf airway epithelia in vitro will be reviewed, and correlates to recent in vivo studies of the effects of hypertonic saline on mucus clearance and lung function made. in addition, the actions of selected novel small molecule pharmaceutical compounds will be reviewed. in sum, it appears that studies focused on the hypothesis that asl volume depletion is the initiating event in cf lung disease have been fruitful in regard to generating follow-on studies investigating the details of the pathogenetic sequence of cf disease, including the importance of mucus adhesion to airway surfaces and the importance of concentrated mucins in promoting not only the acquisition of infection but the biofilm mode of bacterial growth that characterizes cf. the charge now is to assign in different lung regions the relative roles of superficial epithelia versus glands in order to design effective therapies for early and, it is hoped, established cf lung disease. mucociliary clearance (mc) represents the first line of innate defense against the environmental contaminants and diverse exogenous sources of injuries to which the airways are exposed ( ) . the ability of the airways to clear the mucus is depending on several factors including the number of ciliated cells and ciliary beat frequency (cbf), the volume of the airway surface liquid, the degree of hydration and the rheological properties of the periciliary (sol) and gel airway mucus. the airway secretory glandular cells may also markedly contribute to the hydration of the sol and gel mucus and therefore may directly influence the airway mucus transport ( ) . there is now increasing evidence that the airway surface liquid (asl) covering the surface of the normal airways is an isotonic liquid, characterized by an optimal height (near the length of the cilia), and ideal viscosity and surface properties. the asl volume must be adjusted continuously in relation to the environment of the airway epithelium. any mechanical stimulation such as excess mucus load or external injury will first stimulate cbf, activate surface effectors and regulate the volume of asl ( ) . different studies using freshly excised tissues and primary cell cultures have shown that the steady-state periciliary layer volume is maintained by a balance between na + absorption and clsecretion ( ). tarran ( ) has demonstrated that normal airway epithelium senses and autoregulates asl height by adjusting the rates of na + absorption and clsecretion to maintain mucus transport. it is well accepted that excess liquid at the periciliary layer level may be removed by a transepithelial enac transport. on the opposite, enac inhibition will induce clsecretion controlled by cftr activity. in these conditions, it can be easily speculated that in cf airways, the upregulation of na + and downregulation of clrelated to cftr defect will lead to a depleted asl associated with abnormal mucus transport. in cf patients with mild disease, compared with age-matched normal subjects, a reduced mucociliary clearance has been reported ( ). a progressive decrease in mc is generally associated with increasing disease severity ( ) . in patients with advanced cf disease, inflammation, infection and remodeling of the airway may be directly responsible of the decreased mc. whether mc is delayed early in cf infants before any infection is still unknown. most of the mc studies in cf mice have reported a large variability of the data ( ) . in the normal murine lower airways according to the mouse strain, the mucociliary clearance varies from to . mm/min, the lowest values being related to the low number of ciliated cells rather than to a decreased ciliary activity. in cf mice, the cbf is not decreased. although we and others ( ) have reported a decreased rate of mc in cf mice, other investigators failed to demonstrate a difference ( ) . interestingly, overexperession of enac in a recently published mouse model showed that the decreased volume of asl in the lower airways was associated with a large decrease (~ %) in mc ( ) . although the specific contribution of abnormal biochemical and rheological properties to the impairment of mucus transport at an advanced stage of the disease is accepted, the impairment of mc in early cf disease is still not clearly defined. secretory proteins, mucins and lipids are major components of airway mucus that contribute to the antibacterial defense of the airway epithelium but also contribute to the physical (adhesion), rheological (visco-elasticity) and transport capacity of the airway mucus. secreted mucins such as muc ac are reported to be enriched in the sol phase of cf mucus and mucins sulphation and sialylation are increased in cf secretions ( ) . we have also earlier shown in secretions from adult cf patients that the phospholipid content moves towards a profile of poorly lubricant and therefore more adhesive mucus characterized by a decreased content in surface-active molecules such as phosphatidylglycerol and phosphatidylcholine ( ) . whether these biochemical and rheological changes are constitutive or related to a possible defective intracellular acidification due to cftr mutation is unknown. in human fetal tracheal xenografts in the scid mice ( ), we could not demonstrate any dehydration nor increased viscosity of the airway liquid collected in the lumen of the cf xenografts despite the fact that we observed an increased number of amiloride-sensitive na + channel and an inefficient camp-dependent clchannel. in fetal airway mucosa, volume activated clchannels may also contribute to the fluid secretion as previously shown in the late stages of lung development and therefore could compensate the defective mucus fluid hydration related to cftr mutation. one important cause of impaired mucus transport in cf may be related to an abnormal glandular secretion. when the secretion response to glandular agonists is inhibited in pig airways, the mucus liquid becomes less hydrated and more viscous ( , ) and mc is markedly decreased, likely due to an uncoupling between mucin and liquid secretion. abnormal secretion by cf glands is expected on the basis that cftr is expressed in airway glandular serous cells not only at the apical membrane edith puchelle, sonia baconnais, jean-marie zahm inserm umr-s , reims, france surface but also at the level of membrane secretory granules ( ) . moreover, fluid secretions are reduced and viscosity is increased in cf gland cells and in pig tissue treated by cftr inhibitors ( ) . the implication of abnormal airway submucosal gland function in cf has been suggested. wine and joo ( ) hypothesized that exocytosed mucus may remain as condensed packets for a long time after secretion and that, as in cf intestinal crypts, the granule contents may be trapped as undispersed granules within the lumen. using phase contrast dynamic videomicroscopy, we could confirm in cf glandular cells that under basal conditions, mucus exocytosis and expansion of secretory granules is markedly delayed. in parallel, we could demonstrate by quantitative dark field imaging and x-ray microanalysis on freeze dried airway glandular cell cryosections, that the mucus hydration of the cf secretory granules is significantly decreased and the ion content (na + , mg + , p, s and cl -) is significantly higher compared with the non-cf secretory granule mucus content (baconnais et al., unpublished data). our data provide strong evidence that in cf cells, the alterations in ion composition and water content within the serous granules may partly explain the formation of thick mucus plugs lying over a dehydrated periciliary layer on airway surfaces. as proposed by j. wine ( ) "too little water, to late" could explain that the secretory granules of glandular cells cannot correctly expand and may remain anchored to the surface of the cells. in such conditions, the glandular mucus cannot correctly disperse in the airway surface liquid. taken altogether, these results suggest that the abnormal exocytosis associated with abnormal ion and water content of secretory granules inside the glandular cells may accentuate the dehydration of the periciliary layer and contribute to the deficient mucus transport in cf airways. other alternative pathways may partly compensate this glandular mucus and asl dehydration and more or less prevent a major early failure of mc. nevertheless, the inadequate hydration of epithelial fluid at the apical surface and in intracellular secretory granules level favour the hypothesis that in cf, decreased mucus transport is a reality. lung disease in patients with cystic fibrosis (cf) is associated with neutrophil-dominant lower airway inflammation and increased lower airway concentrations of pro-inflammatory cytokines and neutrophil elastase even among infants and young children with cf ( , ) . several studies report a robust inflammatory response in the airways of both bacterial culture-positive and culturenegative children; some studies show a greater inflammatory response in those patients with at least x colony forming units/milliliter of bacteria in their bronchoalveolar lavage (bal) fluid ( ) . lung disease in cf can have regional heterogeneity that complicates understanding the relationship between initial airway infection and the inflammatory response ( ). there is emerging computed tomography evidence for destructive regional inflammation/infection, i.e. bronchiectasis, in stable young children with cf ( ) ( ) ( ) . such data emphasize the need for more research on the pathogenesis and treatment of early lung disease in cf, and the further development of outcome measures to assess global and regional progression of early lung disease. currently, lower airway inflammation and infection in non-expectorating patients can only be assayed by bal. there is a growing interest in proteomic analysis of blood and exhaled breath condensate to identify non-invasive markers of lower airway inflammation and infection. patients with cf acquire a unique set of respiratory tract pathogens in an age-dependent sequence. early airway infections are most frequently caused by s. aureus and non-typeable h. influenzae ( ) . there is controversy on the role of s. aureus and h. influenzae in the progression of early lung disease. pseudomonas aeruginosa (pa) is a ubiquitous environmental organism and the most significant pathogen in cystic fibrosis (cf) lung disease. pa prevalence increases with age, with positive respiratory tract cultures in - % of infants, - % of children - y.o.,~ % of adolescents, and ~ % of adults with cf ( ). reported risk factors for early pa infection include prior s. aureus respiratory tract infection, chronic anti-staphylococcal antibiotic treatment, female gender, delta f homozygous genotype, and attendance at cf clinics without cohorting ( ) . there is currently no optimal method to assess lower airway pa infection in young children. sources of respiratory tract secretions for culture include expectorated or induced sputum, oropharyngeal (op) swabs, endolaryngeal suction, and bal fluid. sputum expectoration is the least invasive and most widely used method in older children, but children < years of age can rarely expectorate. sputum induction with hypertonic saline is generally not successful in children < years of age. op cultures are widely used as a surrogate for lower airway cultures in non-expectorating cf patients, but have poor sensitivity and positive predictive value for lower airway pa infection; op pa isolates can have different genotypes than bal isolates from the same patient ( ) . despite the limitations, the current u.s. standard of care is to collect quarterly op cultures. the host immune response to pa antigens appears to be the earliest marker of pa infection; this is controversial and likely depends on the assays performed ( , ) . recent u.s. studies found serologic responses to pa exotoxin a and whole-cell membrane proteins occurred prior to cultureevidence of pa infection in young children ( ) . the danish cf center uses precipitins against pa antigens as a marker of chronic infection ( ); they have reported reduced serologic responses to pa with intensive anti-pa treatment. limitations of serological markers include lack of commercially available standardized assays, and lack of specificity to the site of pa infection (i.e. upper or lower airway). generally, patients with cf are initially infected with unique environmental isolates of pa ( , ). regardless of treatment, patients commonly have a period of intermittent isolation of pa from the respiratory tract prior to becoming chronically infected ( ) . the duration of intermittent pa + cultures can vary widely, and this complicates the decision regarding aggressive early intervention. early pa isolates are generally non-mucoid in phenotype, highly antibiotic sensitive, and present at relatively low density ( ). these features suggest a "window of opportunity" for early intervention with antibiotics to possibly delay or prevent chronic pa infection and lung disease progression. early age at pa acquisition adversely affects lung disease and survival; yet, acquisition of pa does not appear to cause an immediate and rapid decline in lung function ( ) . the early clinical features of pa infection are subtle and variable, including slightly reduced lung function, and poorer chest radiograph and clinical scores ( ). by school age, the impact of early pa isolation is established. among patients from whom pa was isolated during the first years of life, there was a . times higher risk of death, on average a % predicted lower fev , and % lower weight percentile at year follow-up compared to patients without pa isolated early in life ( ) . danish investigators were the first advocates for aggressive treatment of early pa infection in cf ( ). subsequently, most published studies have shown a microbiologic effect of antibiotics for early pa infection based on transient eradication of pa from upper and lower respiratory cultures ( ) . there has been no randomized, placebo-controlled trial to evaluate clinical efficacy and safety, including drug toxicities and emergence of resistant pa or new pathogens. most studies suggest that recurrent pa infection is inevitable regardless of the initial treatment regimen ( ) . the potential sources for recurrent or persistent infection include the sinuses, undetected residual lower airway infection, or re-infection with an environmental isolate. current options for early anti-pa treatment include oral quinolones, inhaled tobramycin and colistin, and iv antibiotics ( - ). macrolides have not been investigated for this indication. attractive features of oral quinolones include ease of administration, evidence for efficacy in older patients, and accumulating safety data in pediatrics. quinolone monotherapy may result in rapid emergence of resistance. limited safety data exist for inhaled tobramycin in young children, but it has a well-established safety profile in patients > years of age ( ) . inhaled colistin at the time of pa acquisition is used extensively in some european centers, primarily in combination with oral ciprofloxacin ( ) . iv antibiotics are established treatment for pulmonary exacerbation in chronic pa infection ( ); their role in the therapy of initial pa infection deserves further investigation. an ultimate goal for treatment of early pa infection is to pre-scribe the least invasive and safest treatment for the shortest duration necessary to achieve both microbiologic and clinical benefit. the pathogenesis of cf lung disease is due to dysfunction of cftr in the airway epithelium and results in impaired chloride secretion and increased sodium absorption, leading to dehydrated hyperviscous mucus secretions and impaired mucus clearance. the hallmarks of cf lung disease are chronic bacterial infection, mostly notably pseudomonas aeruginosa, and exaggerated neutrophil-mediated inflammation, eventually leading to airway obstruction and death. although the role of bacteria in cf lung pathology is well established the consequences of respiratory viral infection in cf infants are less well understood. although viral infection of the lung can result in serious illness, viral infection of the cf lung may have more severe consequences. there is also the likelihood that viral infection can establish an environment that is favorable for subsequent bacterial infection, a process termed "bacterial superinfection". the upper respiratory tracts of healthy individuals often contain commensal bacteria such as haemophilus influenzae and streptococcus pneumoniae but are asymptomatic for infection. however, these pathogens can become a threat when concurrent with a respiratory viral infection. of the common viruses infecting the respiratory tract of humans, influenza, respiratory syncytial virus (rsv) and parainfluenza virus (piv) result in the highest morbidity and mortality especially amongst children. in particular, rsv is the most common respiratory virus associated with the development of otitis media in children and is often associated with the respiratory bacterial pathogen nontypable haemophilus influenzae (nthi). nthi is responsible for a significant proportion of bacterial conjunctivitis, otitis media, sinusitis, bronchitis and pneumonia all of which may also be associated with concurrent viral infection. many mechanisms have been proposed to result in bacterial superinfection including disruption of innate immune mechanisms, e.g., impaired cough reflex; reduced efficiency of mucociliary clearance; the accumulation of excess or altered airway secretions; and, reduced macrophage-mediated phagocytosis. substantial evidence also exists for viral-induced up-regulation of bacterial adherence receptors although whether these changes occur at the lumenal surface of the respiratory epithelium or as altered secretory products is unknown. although viral-induced pathology and bacterial superinfection can occur in otherwise normal individuals, patients with cf or other underlying lung diseases are considered 'high-risk' for developing a greater severity of disease. more rapid pulmonary deterioration in cf patients has been associated with respiratory viral infections ( ) and rsv infection produced more severe and prolonged disease in cf infants compared to infection of non-cf infants ( ) . however, respiratory viral infections were found to just as likely occur in cf infants as in non-cf infants but more likely to result in lower respiratory tract infection, impaired pulmonary function, and hospitalization for cf infants ( ) . in combination with these clinical data there also exists ample anecdotal evidence that cf patient lung disease gets much worse after viral infection. in terms of specific interactions with respiratory pathogens there is good clinical and experimental evidence that h. influenzae superinfection is associated with a concurrent airway viral infection and h. flu is recognized as an early bacterial pathogen in cf airways. however, data for an association of p. aeruginosa superinfection with airway viral infection is lacking. in the context of cf lung disease and the consequences of viral infection in cf individuals a number of important questions remain to be addressed: another rarely encountered species, b. ubonensis, should also be considered a member of the bcc based on phylogenetic assessment. all of these 'new' species have been identified among banked cf sputum isolates previously identified merely as 'b. cepacia,' indicating that all have caused infection in cf patients for many years. however, the distribution of these species in cf is quite disproportionate. studies from several countries indicate that b. cenocepacia (formerly 'b. cepacia genomovar iii') is the most prevalent bcc species recovered from cf patients. b. multivorans is the next most common species, with the remaining bcc species generally being recovered from relatively small numbers of patients. careful assessment of cf sputum isolates has also identified strains that, although clearly members of the bcc, can not be placed definitively into one of the ten currently described species; these 'species indeterminate' strains may represent still additional bcc species that await definition. genotyping (dna fingerprinting) analyses have demonstrated that in some cf care centers multiple patients are infected with the same bcc strain. some strains are even more broadly distributed. among these are strain et , prevalent in eastern canada and the uk, and strain phdc, which has been recovered from cf patients in us states and, most recently, in europe ( ). et and phdc, as well as several other common strains reported to date, reside in the species b. cenocepacia. shared strains belonging to other bcc species, including b. cepacia, b. multivorans, and b. dolosa, have been less frequently identified ( ) . the extent to which such common strains are inherently transmissible between patients is unclear and must be assessed in the context of the infection control measures employed to prevent their spread. further, available data suggest that bcc species differ with respect to their preferred niche(s) in the natural environment. a better understanding of the ecology of bcc species (and specific strains) is needed to assess the risk posed by natural reservoirs and to develop strategies to prevent acquisition from the environment. limited clinical outcomes data, including studies in lung transplant recipients, have shown an association between infection with b. cenocepacia and greater rates of morbidity and mortality ( , ) . these observations have led some to conclude that b. cenocepacia is the "bad cepacia," implying that all strains in this species are easily transmissible and cause particularly severe infection, while other bcc species present little risk. however, most studies have involved small numbers of patients infected with a limited set of common strain types, with results being extrapolated broadly to the entire species. in fact, many, if not most, cf patients infected with b. cenocepacia harbor seemingly 'unique' strains (i.e., not shared by other patients), and many patients have remained chronically infected with this species for prolonged periods with little apparent impact on progression of lung disease. furthermore, infection with bcc species other than b. cenocepacia has been associated with sepsis and death (so-called cepacia syndrome). rather than ascribing degrees of relative virulence to entire bcc species, it seems more likely that virulence and transmissibility vary among specific bcc strains. of course, as yet undefined human host factors almost certainly contribute to clinical outcome, and so broad conclusions regarding relative bcc virulence will be difficult. a number of recent investigations employing improved animal and in vitro models of infection have yielded important insight into potential bcc virulence factors and pathogenic mechanisms. the molecular events underlying the ability of bcc to adhere to, invade and elicit a robust pro-inflammatory response from human airway epithelia have been studied by using immortalized cell lines as well as primary well differentiated human cell cultures ( , ) . similar to p. aeruginosa, bcc are capable of expressing homoserine lactones likely involved in quorum sensing and biofilm formation ( ) . a number of genes required for bacterial survival in vivo have been recently identified ( ) , and provide an exciting opportunity to define potentially completely novel virulence factors. the availability of the complete genome sequence of b. cenocepacia strain j (a representative of the et lineage) provides a critical asset to investigations of bcc pathogenesis. the sequencing of several other bcc strains and the development of a burkholderia dna microarray are underway and will surely further enhance ongoing research efforts. in summary, during the past several years, a great deal has been learned regarding bcc taxonomy, clinical microbiology and epidemiology. clearly, there is now a need for a more complete understanding of bcc ecology and for large scale outcomes studies to better assess the relative risk of infection with specific bcc strains. ultimately, better definition of the bacterial virulence factors and human host factors that determine diseases progression is necessary to devise novel therapeutic and preventative strategies. increasingly, the emergence of cf respiratory pathogens, including bacteria with intrinsic or acquired antimicrobial resistance, non-tuberculous mycobacterium (ntm) species and fungi, challenge clinicians to better characterize their prevalence and epidemiology, pathogenecity in cf lung disease, optimize treatment of infection, and prevent acquisition in both healthcare and non-healthcare settings. staphylococcus aureus binds avidly to respiratory mucin and is often is the first pathogen to colonize the respiratory tract, with a prevelance of ~ % by one year of age. s. aureus may cause chronic inflammation of the cf respiratory tract by up-regulation of proinflammatory cytokines exotoxin-mediated immune dysregulation and tissue damage. (ref) currently, . % of cf patient s. aureus strains are resistant to methicillin (mrsa). mrsa strains may be transmitted via direct contact in and outside of healthcare settings, may cause transient or chronic respiratory colonization, and have been associated with severity of underlying disease and adverse outcomes in both cf and non-cf patients (cosgrove, others) although the impact among cf patients is limited to date, there is growing concern about genetically distinct community-acquired mrsa (ca-mrsa) strains that posses a unique resistance cassette and cause virulent soft tissue infections and necrotizing pneumonia mediated by the cytotoxin panton-valentine leukocidin (ref). stenotrophomonas maltophilia and achromobacter xylosoxidans are intrinsically multidrug-resistant gramnegative pathogens whose acquisition is promoted by use of broad-spectrum antimicrobials. they can cause healthcare-associated infections, including pulmonary exacerbations among cf patients (refs). among cf patients, current prevalence rates of . % for s. maltophilia and . % for a. xylosoxidans likely are underestimates because of the failure to use appropriate selective media and misidentification. although many patients have only transient colonization with these organisms and their pathogenecity remains poorly defined, longterm colonization s. maltophilia was associated with decreased lung function to years after acquisition in one study (ref) . studies have suggested that acquisition may occur both from environmental sources or from person to person. saphrophytic fungi, including aspergillus spp. and scedosporium apiospermum, are ubiquitous and may be found in soil and in the hospital environment associated with construction activities or water intrusion. aspergillus spp., predominantly aspergillus fumigatus, and can colonize the lungs of cf patients and can cause allergic bronchopulmonary aspergillosis (apba) in up to . % of patients and rarely aspergilloma and invasive aspergillosis ( ). registry data likely underestimates the prevalence of aspergillus. in the aerolized tobramycin trial, up to % of cf patients were colonized with aspergillus spp ( ) person-to-person transmission remains a rare but potential risk among patients with wound or tracheal infection (ref) . further studies are needed to assess the impact of use of selective media and infection control precautions on the prevalence of these emerging pathogens and to better define risk factors for acquisition in and outside of healthcare settings, the clinical impact and treatment strategies. it is pertinent to look at our evolving understanding of the clinical manifestations of cf in the context of key milestones that have occurred since the recognition of cf as a distinct clinical entity in ( ) . from initial recognition/description through , most patients with cf died in infancy or early childhood of pneumonia and severe malnutrition. the diagnosis was suggested by clinical features along with evidence of exocrine pancreatic deficiency on duodenal fluid analysis, and then confirmed at autopsy. recognition of the sweat gland defect in ( ) and development of the quantitative pilocarpine iontophoresis sweat test in ( ) led to the inclusion of a positive sweat test result as an essential criterion for the diagnosis of cf. recognition of physiologic abnormalities of sweat and serous-secreting glands led to a change in terminology from mucoviscidosis to cystic fibrosis. from to , the cf phenotype was better defined and it became clear that cf was characterized by significant phenotypic heterogeneity ( ). during this time it was recognized that ) there is an important subset of patients with chronic sinopulmonary disease typical of cf, pancreatic sufficiency and borderline or normal sweat electrolyte concentrations (variant, atypical or non-classic cf) ( ) and ) almost all males with cf are infertile secondary to bilateral absence of the vas deferens ( ) . there were two highly significant laboratory observations during this period; raised immunoreactive trypsin levels in dried blood spots could be used for newborn screening and a raised bioelectric potential difference across cf respiratory epithelia could be used as a diagnostic tool. the identification of the cftr gene in led to significant advances in our understanding of genotypephenotype correlations and also introduced the possibility of using mutation analysis for diagnostic (including prenatal) testing and carrier screening. it became recognized that there is a subset of "mild" mutations which are associated with pancreatic sufficiency and, in some instances (missense mutation a e) with a less severe pulmonary phenotype. also, it was recognized that there are a number of disorders (cbavd, idiopathic chronic pancreatitis, chronic rhinosinusitis, abpa, diffuse panbronchiolitis) in which cftr mutations (often associated with residual cftr function) are frequently identified, but in which a diagnosis of cf may not be appropriate. this led to the introduction of diagnostic terms such as cftr or cftr-related disease or cf-like disease. further complicating the issue of "what is a cf diagnosis?" were the observations that cf mutations cannot be identified in a subset of patients who have clinical features typical of cf ( ) , and that some individuals who carry two known cf mutations may remain asymptomatic over extended periods of time. also, it has become clear that differences in endorgan sensitivity to cftr protein deficiency and the genetic context in which a mutation exists can influence the cf phenotype. intron variants ( ) and modifier genes ( ) may play an important role in determining the severity of the pulmonary and gastrointestinal manifestations seen in patients with cf. in , there is still controversy regarding the diagnosis of cf. the overwhelming majority of patients fall into the category of "classic cf" in which there are typical clinical features in association with elevated sweat chloride concentrations and, in the majority of cases, two identified cf mutations. however, there remains a small but increasing subset of patients, often adults, who have atypical clinical features, a normal or borderline sweat test result and identification of one or no cf mutations, and a subset of asymptomatic newborns identified by newborn screening (two cf mutations and/or a positive sweat test result) but who lack clinical criteria to support a cf diagnosis. this has led to the somewhat controversial proposal ( ) that it might be appropriate to have a diagnostic category of "genetic pre-cf" for some individuals and raises the issue of how a disease such as cf is defined in the genomic era in which gene alterations are likely to be found in the absence of clinical features ( ) . it is clear that a genetic abnormality by itself does not equal a disease and that both clinical criteria and genetic abnormalities will need to be used to define a disease state. peadar g noone, m.d. the diagnosis of cystic fibrosis is straightforward in the majority of patients, particularly pancreatic insufficient (pi) patients. diagnostic criteria (set forth by a consensus statement, drawn up by a panel of experts in ) include clinical / phenotypic data, as well as data on cftr genetics and function; these can be applied in most clinical situations ( ). the criteria were designed to encompass as many of the potential clinical scenarios as possible; the potential pitfalls that may occur with the laboratory tests of cftr were discussed. an emphasis was placed that the guidelines are unlikely to cover every possible scenario (practicing clinicians will quickly agree). difficulties may be encountered in patients with atypical disease that do not conform to traditional clinical definitions, in conjunction with cftr data that may be difficult to interpret -establishing the line between proven cf and "cf-associated" diseases thus may not be easy for the clinician. as new knowledge is gained through clinical research and clinical practice, and with the potential for increasing presentation of atypical patients the guidelines will need to be refined and updated. the traditional laboratory investigation of cftr function is the sweat test. it is strongly recommended that this test be carried out in a center experienced in its performance, and that the only acceptable test is the pilocarpine iontophoresis sweat test. furthermore, clconcentrations rather than na + concentrations are regarded as more accurate for discriminating between cf and non-cf, though sweat na + may still be used in some laboratories. the literature in regard to sweat clconcentrations has varied somewhat. the older literature recommends a clconcentration of mmol/l as diagnostic of cf; these data are based on reference values obtained from pi cf patients during the 's, and do not include pancreatic sufficient (ps) patients, or any recently described phenotypes ( ) . in view of the increasing awareness of atypical and "variant" cf, a level > mmol/l is now regarded as diagnostic for cf ( ) . what is more difficult to interpret is a level less than mmol/l, although in the context of a compatible phenotype, the higher the clconcentration, the more suspicious of cf the clinician is likely to be. somewhat arbitrarily, a level of between and mmol/l is regarded as a gray zone (previously - mmol/l), often including ps / atypical cf patients. complementation with nasal potential difference (npd) measures can be useful. npd is now more widely available, has been standardized, and can be performed safely in all age groups, including neonates ( ). npd requires significant technical experience and validation for each center / operator, and across groups of sufficient patients and controls. the best evidence of cf is a raised basal pd consistent with increased na + absorption across airway epithelia. across different sites in the us, standaert et al found that patients with cf (n = ) had a mean basal pd (mv) of - . + . (vs - . + . for non-cf). similarly, a reduced or absent cftr-mediated clconductance (mv) was present in patients with cf (+ . + . as compared with - . + . for non-cf). despite these advances in npd measurement techniques and standardization, difficulties arise in atypical cf patients who may have normal or high normal na + transport (up to - mv), although usually such patients have reductions in cftr-mediated clconductance, intermediate between that of cf and non-cf ( ). as a technical note, inflammation of the nasal mucosa renders the test very difficult to perform and interpret (it may cause a falsely low basal pd and apparently absent clconductance because of loss of epithelial integrity). finally, molecular testing demonstrating two "cf-causing" mutations (i.e. associated with altered cftr synthesis from a premature termination signal, alternations in intron splice sites, or novel amino acid sequences) is strong evidence of cf ( ) . difficulties arise when faced with one cf-causing mutation, after completion of currently available commercial tests. then the issue arises as to how aggressive to be in searching for another cf-causing mutation on the opposite allele. exactly defining a cf-causing mutation can be a difficult process, even after consultation with a cf molecular geneticist or other experts in the field of "atypical cf" is necessary an example of clinical phenotypes described since the consensus statement include patients with chronic pancreatitis of unknown etiology, some of whom have what is now termed cftr-related pancreatitis ( ) . initial cftr genetic tests showed that some of these patients were carriers of a severe cf mutation, and only after extensive tests or gene sequencing did a subset turn out to have a mild or "variable" mutation in cftr on the opposite allele. sweat chloride values were less than mmol/l in some, with a mean value for all cftr compound heterozygotes of mmol/l (n = ). nasal pd tests showed values intermediate between that of normal and "classic" cf, supporting the concept of a subset of cftr mutations resulting in some residual cftr function and intermediate cftr physiology ( ) . the flip side of the coin are patients with a phenotype strongly resembling cf but with normal tests of cftr structure / function ( ) . such patients have some phenotypic features of cf, for example sino pulmonary disease, congenital absence of the vas deferens, gastrointestinal disease, or elevated sweat chlorides (> mmol/l). proportions of patients referred with these criteria (n = ) had two (n = ), one (n = ), or no (n = ) mutations in cftr. as with the patients with idiopathic pancreatitis, those patients referred already known to have one cf mutation were most likely to have another mutation (associated with reduced cftr function) on the opposite allele. the hypothesis was raised that a non-classic cf like phenotype may result from causes outside the cftr gene. finally, it used to be thought that isolation of mucoid pseudomonas aeruginosa in the airway secretions of someone with bronchiectasis was strongly suspicious of cf. however, other diseases such as primary ciliary dyskinesia may also be associated with such respiratory tract microbiology. thus, interpretation of the "clinical phenotype" portion of the current definition of a cf diagnosis is difficult when one considers these groups of patients. studies are underway looking at the issue of defining more precisely the diagnostic criteria of sweat / npd / molecular tests in the context of different phenotypes ranging from classic to mild cf to heterozygote and non-cf individuals. defects in the cftr gene cause a wide spectrum of manifestations, from classic cystic fibrosis (cf) diagnosed in childhood with multi-system involvement including sino-pulmonary disease and pancreatic insufficiency, to monosymptomatic conditions not diagnosed until much later in life (e.g. congenital absence of vas deferens (cbavd), recurrent pancreatitis). there is now confusion between what is considered cystic fibrosis and how to label other disorders associated with cftr mutations. there is a growing population of patients in whom a diagnosis of cf can neither be made nor ruled out. the lack of a diagnosis can be stressful for the individual and health care professionals currently have difficulty providing prognostic guidelines. the discovery of the cf gene has not been as helpful as hoped in these situations. the common cf-causing mutations detected on standard screening panels are usually seen in patients with classic disease, in whom the diagnosis can be made by a positive sweat test. patients who present with atypical manifestations are more likely to have less common mutations; however extensive genetic testing of the remaining , mutations or variants in cftr is not financially feasible or practical. the standard diagnostic sweat test has not been validated across this new clinical spectrum and so its role in assessing patients presenting with unusual manifestations is unclear. we know that patients presenting with atypical features may have negative or "borderline" sweat test results. newer investigations examining the function of cftr, such as measurement of nasal transepithelial potential difference, have not been standardized or validated at all. in an effort to refine the diagnostic criteria for cf, we have initiated the systematic recruitment of subjects in well-defined cohorts defined as follows: . typical cf . atypical cf (i.e. pancreatic sufficiency) . other conditions related to cftr(e.g. cbavd, idiopathic recurrent pancreatitis) . obligate cf heterogyzgotes . unaffected controls the cf patients have been clinically diagnosed and identified from the toronto cf clinics (pediatric and adult), with current patients who have been extensively genotyped, classified as pancreatic sufficient or insufficient and who are followed regularly with clinical information prospectively collected in an extensive database. patients with unusual manifestations or disorders associated with cftr mutations are recruited from three sources: a cohort of men with cbavd from a male infertility clinic; patients with idiopathic, recurrent pancreatitis after extensive investigations to rule out other etiologies of pancreatitis; and patients referred to the toronto cf clinics with bronchiectasis of unknown etiology. identification of subjects in the latter category has been enhanced by mailings to pulmonologists and general practitioners in the province and cf clinics in canada. presentations to the medical personnel in the toronto and regional cf clinics have been made to alert them to the opportunity to refer patients in whom cf has been queried but not established or excluded. obligate heterozygotes are the siblings or parents of patients with classic cf. control subjects included siblings of known cf patients who do not carry cf gene mutations, as well as unaffected volunteers. all subjects are fully investigated with clinical assessment; complete gene scanning of cftr with multiplex heteroduplex shift analysis and characterization of dna fragments by direct sequencing analysis; sweat chloride testing, using the gibson and cooke method, and nasal transepithelial potential difference measurement performed (using method of knowles) on the same day as the sweat test. these measurements are prospectively performed in these well-defined patient and control populations, and will form the basis for classification and regression tree analysis as described later in this symposium. this novel, multi-variable approach to defining the reference parameters of these diagnostic tests will help us to refine the diagnostic process. the ultimate goal is a redefinition of the diagnostic criteria for cystic fibrosis, and a more precise definition of related syndromes. according to the cf consensus statement [ ] , a diagnosis of cf is established when a cftr-associated phenotype is present in conjunction with either two abnormal sweat chloride measurements, or two disease causing cftr mutations, or an abnormal nasal potential difference (pd) result. although the sensitivity and specificity of these guidelines are high when applied to pancreatic insufficient patients and healthy controls, the growing number of patients identified with pancreatic sufficiency as well as cftr-associated clinical phenotypes has resulted in numerous diagnostic dilemmas for which current guidelines are frequently inadequate. the reference range for sweat chloride, established in , was based on assessments of primarily severe cf patients and healthy controls (who were not genotyped). similarly, the cf-causing mutations included in the consensus statement do not reflect current knowledge. furthermore, objectively defined guidelines and reference values for nasal pd as a diagnostic test have not been established. our group is currently evaluating a cohort of patients presenting with diverse phenotypes associated with mutations of the cftr gene to redefine the laboratory parameters that confirm or exclude a cf diagnosis. study subjects are recruited from among conventionally diagnosed cf patients, healthy controls, obligate heterozygotes, and query cf patients presenting with infertility, pancreatitis, or sino-pulmonary disease. patient evaluation includes comprehensive medical history, sweat chloride, nasal pd measurements on the same day, pulmonary function testing, serum trypsinogen, and extensive cftr mutation testing by multiplex hereroduplex analysis and dna sequencing. methods for characterizing this increasingly heterogeneous population are necessary to refine diagnostic criteria used for clinical and research purposes. in order to identify structure and "natural" grouping within the spectrum of cf disease observed in our data, cluster analysis [ ] , an exploratory technique, is used. the most commonly used hierarchical procedures, agglomerative methods, successively fuse similar individuals or groups together in clusters of increasing size and heterogeneity, ending with a single group containing all subjects. the resulting sequence of clusters is represented graphically using a dendogram, a tree diagram where individuals correspond to the 'leaves' whose 'stems' are grouped together at 'nodes'. the distances between clusters, a measure of the difference between the groups, is represented by the lengths of the stems. the dendogram thus provides a useful visual representation of the clustering algorithm that can assist in making decisions about the number of clusters present in the data. to gain further insight into the underlying factors defining the individual clusters, a figure is derived that borrows from the concept of microarray data analysis. the variables used in the cluster analysis are depicted in separate columns across the x-axis, with individual subjects each represented as a single row across the figure. shades of gray, ranging from white for values in the normal range to black for values representing greatest disease severity, are used to represent each data value. using this figure, researchers can simultaneously visualize all the variables included in the cluster analysis, revealing the underlying structure of the data and unusual observations within the clusters. classification analysis is then used to define a rule that can be used to assign new subjects to one of the clusters. unlike cluster analysis where no assumptions are made about the number of groups or their structure, in classification analysis the groups are known and are used as the basis for developing a diagnostic rule. the classification and regression tree (cart) method of breiman et al. [ ] is ideally suited for developing diagnostic rules ( figure ). this method uses recursive partitioning to segregate patients into one of two or more known groups. the decision tree representation incorporates complex interactions in a transparent and intuitive rule that can easily be applied to new cases. moreover, the method does not rely on normal distribution assumptions and is particularly effective for developing rules in the presence of a large number of variables. prior distributions and cost of misclassification can be incorporated into the model to optimize the diagnostic rule for use in a clinical or screening situation. cutoff values selected by cart can be further adjusted to optimize sensitivity and specificity evaluated by normal distribution assumptions, where appropriate. statistical analysis of the cystic fibrosis diagnosis cohort will identify homogeneous subgroups within the spectrum of cftr disease, providing alternative labels for subjects with an uncertain diagnosis of cf. longitudinal studies of these patient groups will refine models for prognosis and survival, assisting in frequency of follow-up and treatment decisions and providing patients and their families with more precise information. supported by nih dk - and ccff. figure : controls, obligate heterozygotes, and patients with cf were used as a training sample for cart analysis to establish a diagnostic rule based on sweat chloride and change in nasal pd from infusion to the final value after superfusion with isoproterenol (⌬am+clfr+iso). the first split based on a value of ⌬am+clfr+iso of . mv has perfect sensitivity and specificity for discriminating cf from non-cf. further splits based on sweat chloride at . and . mmol/l segregate the majority of controls and cfpi patients at the extremes, with overlapping groups in the intermediate range. finally, a split based on a ⌬am+clfr+iso of - . mv further discriminates between controls and an intermediate group of controls and heterozygotes. by basing the initial split on ⌬am+clfr+iso rather than sweat chloride, the cart rule correctly classifies cf-ps patients with sweat chloride values in the normal range. jane l. burns, m.d. antibiotic therapy is a mainstay of treatment for cf patients hospitalized with a pulmonary exacerbation. in order to use antimicrobial agents effectively, it is important to understand the individual patient's microbiology, the patterns of antibiotic resistance within your cf center, the activity of the drugs in cf, and the optimum mode of administration. using each patient's culture and susceptibility data as a guide, it is usually possible to identify a combination of two or three agents from different antimicrobial classes (e.g., β-lactams, aminoglycosides, quinolones) that demonstrates in vitro activity against the predominant pathogens. the use of agents from different classes with different mechanisms of action offers the potential for synergistic activity. it also decreases the likelihood of development of antibiotic resistance. even if susceptibility data are unavailable, knowing the organisms most recently cultured from a respiratory sample may help direct therapy. while pseudomonas aeruginosa is often susceptible to a combination of a β-lactam and aminoglycoside, burkholderia cepacia complex is uniformly resistant to the aminoglycosides and stenotrophomonas maltophilia is never susceptible to the carbapenem antibiotics. the patterns of antibiotic resistance within a given cf center may also influence the choice of antimicrobial therapy. for example, methicillin-resistant staphylococcus aureus is being seen increasingly at some cf centers ( ) and may be susceptible to clindamycin and trimethoprim/sulfamethoxazole, or perhaps only to vancomycin. knowledge of the local resistance patterns can help determine the best choice of agents. the pharmacokinetics of antimicrobial agents in cf patients may be distinct from those of other individuals. because many cf pathogens are relatively antibioticresistant, optimizing drug levels at the site of infection in the lung is critical. for drugs where serum monitoring is performed, such as the aminoglycosides, the use of a previously determined dose is optimum, unless renal function has changed. for other agents, where monitoring is not available, the use of cf-specific dosing is recommended ( ) . the route of administration is the final decision that must be made in identifying antibiotics for inpatient treatment. questions that often arise include the necessity for parenteral rather than oral administration of quinolones and the advisability of combined inhaled and parenteral tobramycin. these decisions may need to be individualized for each patient. once therapy has been initiated, clinical response is often adequate to determine hospital course and the advisability of continuing therapy as an outpatient. if a patient is not improving as expected, additional culture and susceptibility testing may be indicated. physiotherapy is one of the cornerstones of treatment in cf and is part of the multi-disciplinary treatment approach. p. aeruginosa may survive longer in airways abundantly filled with sputum, and by removing infected lung secretions the rate of proteolytic tissue damage can be decreased . combining intravenous anti-biotic therapy with intensified airway clearance physiotherapy has been shown to improve lung function more than iv anti-biotic treatment alone . higher sputum volumes are often associated with increased inflammation, lung obstruction and destruction. inflammatory processes increase metabolism and in turn minute ventilation and work of breathing . there are many reasons why patients with cf may be admitted to hospital. some of the most common reasons include acute flare up of cf lung disease, intestinal obstruction, preparation for elective surgery and for management of cf complications such and hemoptysis and pneumothorax. the objectives of hospital admissions vary depending on the reason for the admission. in the case of acute exacerbation, the main objective will be to restore optimal physical function as quickly as possible. broadly speaking, this is achieved through a multi-disciplinary approach including inhalation therapy, clearance of sputum and mucus plugs and pharmacotherapy to reduce infection and inflammation which in turn may decrease the work of breathing. further, a graduated exercise program to assist in sputum mobilization and increase physical working capacity and musculo-skeletal well-being (including attention to posture, muscle strength and mobility) should commence as soon as appropriate. nutritional and psychosocial support are also part of the care package. ongoing age appropriate education about effective and efficient airway clearance, inhalation therapy and the need to adhere to treatment regimens should be incorporated in treatment sessions. each treatment session should be seized as an opportunity for education. establishing good rapport, offering age appropriate treatment and gaining the patient and family's trust and confidence are crucial in the delivery of effective treatment. an inpatient admission should include: • relevant past history • recently recommended home physiotherapy program including inhalation therapy (agents, order and timing), airway clearance therapy (act) and physical exercise program and adherence • the possibility of gastroesophageal reflux in relation to physiotherapy • clinical status including subjective and objective measures of the following -amount, color, consistency and ease of expectoration of sputum -oximetry/pulmonary function tests/peak expiratory flow rate -breath sounds on auscultation, respiratory rate and pattern of breathing -exercise tolerance (current activity & incidental exercise/ exercise tolerance tests) -musculo-skeletal problems (posture, pain, muscle tightness/weakness, oedema) -urinary incontinence during coughing and forced expirations establishment of a treatment program incorporated in the multi-disciplinary care plan: . inhalation therapy: - broncho-dilator, mucolytic (pulmozyme, isotonic or hypertonic saline etc) inhaled anti-microbial and anti-inflammatory agents. the timing and order of these treatments in relation to act and exercise are important and need to be established. . choice of airway clearance therapy: [ ] [ ] [ ] [ ] [ ] positioning manual techniques effective forced expirations the active cycle (huffing) of breathing positive expiratory pressure autogenic therapy drainage oscillating positive expiratory non-invasive pressure therapy ventilation . establishment of an exercise program to: -increase aerobic capacity, improve muscle strength and joint mobility -o therapy if needed -promote normal postural alignment and physical function -motivate increased exercise participation in the short and long term . discharge planning occurs once the objectives of the admission have been achieved. a home treatment program needs to be negotiated with each patient individually. an early review of progress is usually organized in the cf outpatient department. the dosage and number of treatments per day need to be individualized to each patient's needs. acutely ill patients may need shorter treatments spread over the hour period which should be available days/week . treatments should be timed before meals or at least hours after to allow gastric emptying (which is delayed in some patients) in order to minimize gastroesophageal reflux during act. appropriate information needs to be recorded in each patient's unit record to meet communication and medico-legal requirements. the quality of treatment and adherence are crucial in achieving optimal outcomes short and longterm. noreen roth henig, m. d. the majority of icu admissions for cf occur in the setting of respiratory complications of cf including pulmonary exacerbations from chronic infection leading to respiratory failure, hemoptysis and pneumothoraces. admission to an intensive care unit (icu) for progressive respiratory failure has different implications than admission for hemoptysis or a pneumothorax, an extrapulmonary manifestation of cf, or for post-surgical care. critical care for patients with cystic fibrosis encompasses a large number and wide range of important issues. the critically ill cf patient has needs specific to the underlying disease that distinguishes them from other icu patients. the cf team is well positioned to guide the intensivists in these issues as well as in endof-life discussions. factors such as local cf center practices, icu norms, patient preferences, and the availability of lung transplantation predictably influence admissions and outcomes in the icu. in the largest published american series of adult cf patients in the icu, % of the admissions were for a cf specific respiratory event. [ ] extrapulmonary manifestations of cf account for additional icu admissions, although the exact number is not known. two such extrapulmonary indications for icu admission are heat stroke and hematemesis from esophageal varices secondary to cirrhosis. renal failure due to drug toxicity, narcotic overdoses, and anaphylaxis to antibiotics are unfortunate complications of usual cf care that may result in icu admissions. in the largest european experience published, post-surgical admissions make up the majority of icu admissions ( of admissions). [ ] in that series, of the admissions were for pleurodesis. in other centers, abdominal and pelvic surgeries are more common indications for icu admission. although published experience in this area is limited, it appears that cf patients admitted to an icu for any indication other than progressive respiratory failure are likely to survive to discharge from the hospital. although most physicians feel comfortable utilizing icu resources for potentially reversible problems in cf patients, progressive respiratory failure presents a unique challenge. an early multicenter study of patients who received endotracheal intubation with mechanical ventilation for respiratory failure showed abysmal outcomes: % of patients died within weeks of intubation, either on or off the ventilator. heavy sedation and immobilization of the patient often accompany endotracheal intubation and mechanical ventilation and compromise airway clearance. suctioning of secretions through an endotracheal tube, either blindly or with a bronchoscope, is less effective than a cough and expectoration in an awake patient. in addition, patients who cough while intubated are perceived as "bucking" the vent, uncomfortable, and at risk of self-extubation, and are commonly treated with heavier sedation and/or neuromuscular blockers. the combination of poor airway clearance and the potential for the development of myopathies from immobilization and drugs may actually contribute to poor outcomes in cf patients. non-invasive mechanical ventilation (niv) provides ventilatory support to patients with respiratory failure without the limitations of sedation and immobility imposed by endotracheal intubation. the advantage is that patients can cough and expectorate while using niv. it is increasingly used in patients with cf for a variety of indications and in both inpatient and outpatient settings. the uses of niv are beyond the scope of this discussion, but its increasing use and acceptance has changed how end of life care in cystic fibrosis has undergone a revolution in the past decade. prior to the s, end of life care for patients with advanced cf lung disease was based on the assumption that aggressive interventions were inappropriate; the advent of lung transplantation unraveled this consensus about the use of aggressive care at the end of life. there is anecdotal evidence that patients with end-stage cf lung disease may now see their choices as a stark dichotomy: either aggressive technologic intervention in the hopes of life extension, or acceptance of limited lifespan in order to receive high quality palliative care. this "either transplantation or palliation" model fails to recognize that the goals of pursuing restorative therapy and palliation are not by their nature in conflict, and that a mixed model of end of life care, incorporating the new realities of lung transplantation, is possible. although patients and physicians may not explicitly acknowledge it, opting for lung transplantation is certain to alter care in the final months of life. patients on the transplant list are more likely to opt for aggressive measures to sustain life in order to increase the chance of receiving a suitable organ. as a result, transplantation requires a reordering of priorities for patients and families, and a re-envisioning of the good death. it is unrealistic to assume that patients and their families have given no thought to the matter; autobiographical accounts of life with cf contain frank discussions about the circumstances of dying, both before and after transplantation. it is clear that the topic, while difficult, is important to patients, and it is clear that patients and families can both consider transplantation and make end of life plans. consideration of transplantation requires that the patient, family, and physician directly confront the lethal and where cf patients are treated. for instance, in the american study of cf patients in the icu, almost a third with infective exacerbations were treated with niv. in some hospitals, niv is used on the general medical ward and patients who fail niv do not opt for endotracheal intubation. all patients with cf in the icu require skilled attention to cf specific care, including attention to nutritional needs, enzyme supplementation, bowel care, drug clearance, hydration, and diabetes management. the nutritional needs of cf patients include overall high caloric needs, the decision between parenteral and enteral feeding, and the use of powdered enzyme supplements through feeding tubes vs. elemental formulas. distal intestinal obstructive syndrome is a potentially devastating complication of icu care and results from immobilization, dehydration, narcotic analgesics, and the difficulty in determining appropriate enzyme doses in enterally fed patients. hydration is a difficult management issue in cf patients who are prone to dehydration but who may have pulmonary edema as a contributor to the respiratory failure. drug metabolism, especially of aminoglycosides, presents unique challenges and often results in the need for dose alterations. a final issue is diabetes management. critical illness may worsen glucose control in cf, even in patients previous not recognized to have impaired glucose tolerance or cf-related diabetes. critically ill cf patients likely benefit from the same aggressive glucose control as other critically ill patients. [ ] inevitably, discussions of end-of-life occur in the icu. ideally, the cf center team is aware of a patient's preferences regarding end-of-life care including aggressive interventions such as icu admission, mechanical ventilation and lung transplantation, although when faced with imminent death, a patient's perspective on end-of-life issues may change. additionally, the availability of transplantation, both lung and liver, may change the decision making paradigm, and even the local practices of the transplant center may influence end-of-life care. ultimately, the guiding principle that there is no "one-sizefits-all" approach to cf and critical care allows flexibility and individualized care at a most challenging time. nature of cystic fibrosis. physicians, eager to provide hope for recovery, may downplay the struggle that transplantation can be for patients and families, yet this does patients and families a great disservice. discussion of transplantation needs to be initiated at a point that allows adequate time for the family and patient to come to terms with the threat of approaching death and the uncertain prospect of transplantation. since administration of the usual therapies for cf only rarely causes distressing symptoms for patients, caregivers may not face difficult decisions about discontinuing therapies. at the end of life, intravenous or inhaled antibiotics, pancreatic supplements, oxygen, and the rest of the usual daily therapies for cf can be continued in most cases without a great burden to patients both on and off the transplant waiting list. there is one notable example: if bipap has been instituted as a measure of symptom relief, its removal may be both psychologically and symbolically difficult for the patient, family and clinician; the decision to remove assisted ventilation may be a visible sentinel of the approaching end of life or of the "failure" of transplant listing. pain control. initiation of pain control therapies such as opiates may have also have powerful symbolic importance for the patient family and clinician. yet skilled pain control is simply the duty of compassionate care, and concerns over the symbolic nature of opiate use should be so great that the result is a patient in avoidable pain. the common fear of respiratory suppression following use of opiates in chronic lung disease is unfounded, and concerns over addiction in end of life care have been dispelled by years of experience with their use in a wide variety of palliative situations. retrospective studies suggest that the frequency of headache and chest pain in cf patients increases steeply in the final year of life, and opiates should be used in sufficient amounts to relieve both acute and chronic pain. hemoptysis. patients with a history of marked hemoptysis may have a major bleeding episode as the final event in life. this possibility should be discussed and plans made as to what actions will be taken. simple measures such as a supply of dark towels and plans for rapid control of the symptoms of dyspnea and choking can lessen the suffering of both the patient and the family in such an event. respiratory failure. for most patients, death will follow a gradual decline in ventilation, with the accompanying "narcosis" of elevated co levels. yet for many, there can be waxing and waning consciousness, as well as changes in work of breathing, and pharmacologic therapies for control of dyspnea will be needed. although there is no prospective research on the use of opiates to control the dyspnea of cf respiratory failure, the use of opiates in other deaths due to copd strongly suggests that moderate doses of opiate can provide excellent control of end-stage dyspnea. there may well be other family members with cf, or a previous death from cf in the family. these deaths may have formed a "script" for the family as an example of either a good or bad death, and an open exploration of the family's view of the previous death can be invaluable in uncovering unspoken expectations. in addition, there is often a tight circle of friendship among patients with cf, and the death of one member of the circle will have powerful and long lasting effects on the other patients, particularly if death occurs suddenly. clinicians too may have a "script" of how they expect a patient's dying to proceed, and should be aware that their expectations may conflict with those of the patient or family. clinicians should openly discuss their approaches to end of life care and transplantation with one another. consultation with palliative care teams within the hospital can bring a valuable perspective. until recently, most cf clinicians have operated on the assumption that the care they provided was uniformly excellent. however, it has become increasingly clear that there are differences in outcomes among different cf clinics, suggesting that there are more and less effective approaches for providing cf care. thus, while we await the expected advances in care that will be provided in the long run by breakthroughs in biomedical research, we have the opportunity to take better advantage of existing knowledge to rapidly improve the outcomes for people with cf. there are several key strategies that healthcare providers can use in order to make positive changes in the care they deliver. these include: . appreciate that changes must be made to the system of healthcare delivery. the first step, and one that is often the most difficult for physicians, is to understand that simply working harder within a non-supportive system will not yield the results desired. the model of the individual physician who by force of intellect and will establishes the correct diagnosis and prescribes the appropriate therapy to cure a patient is anachronistic and does not apply to the care of patients with cf. in caring for a chronic disease like cf, multiple caregivers must communicate and integrate a complex set of data and then prescribe therapy based on the appropriate use of that data. while it is incumbent on the system to ensure that providers are knowledgeable regarding best, or ideal practices, it further needs to support consistent application of those interventions that the provider knows to be optimal. variation in outcomes (when adjusted for variation in risk) is then due to variation in the system's ability to provide this support in a consistent manner. in general, cf care providers have successfully adopted a longitudinal approach to dealing with this chronic disease. in addition, it is useful to conceptualize and work towards instituting an idealized system of chronic care delivery that consists of various interdependent components inside and outside the local clinic setting. wagner's chronic illness care model provides a useful framework for consideration and may be summarized as follows (see also http://www.improvingchroniccare.org): a. community resources -medical center-based subspecialists should form partnerships with community organizations and primary care providers to support and develop interventions and fill gaps in needed services. furthermore, providers should publicly advocate for policies that improve patient care. b. overall health delivery system -organizations should create a culture that promotes safe, high quality care. all levels of the organization should visibly support improvement and provide incentives based on quality of care. there should be open and systematic handling of errors and quality problems and care should be coordinated within and across organizations. c. patient and family self-management -individual resources should be exploited by training and empowering patients and their families in self-assessment, goal-setting, action planning, problem-solving and follow-up. furthermore, patients' input should be sought in planning all aspects of care as well as delivery system design. d. delivery system design includes the structure and function of the clinic, from the telephone to the reception area to the examination room. it is essential to define roles and distribute tasks among team members, use planned interactions, ensure regular followup, and give care that patients understand. e. decision support comprises the promotion of evidence-based care at the provider-patient interface. this is accomplished via the use of clinical tools, guidelines and algorithms to ensure that intended care is actually prescribed, and reliance on rote memory is minimized. f. clinical information systems -at the individual patient level, the system might provide timely reminders for providers and patients, facilitate individual patient care planning, and allow data to be easily shared in order to coordinate care. at the clinicwide population level, the system can help to identify relevant subpopulations for proactive care, and allow providers to monitor performance of the practice team and care system. it is the lack of the latter data that keeps many providers in the dark regarding the true effectiveness of their care. an effective organizational change strategy is an essential component of improvement work. without a disciplined approach, practitioners who are newly aware of the extent of their system's deficiencies will attempt immediate, large dramatic changes which fail either in their planning stage because they get bogged down in endless preparatory meetings or self-destruct in their implementation phase because of the number of unanticipated problems encountered. one effective approach, is the langley et al's plan/do/study/act (pdsa) cycle. to implement the process, the first step is to plan the details of a small test of change [plan] . the planned change is then carried out [do] . once the change is attempted on a small scale, data on its effectiveness is gathered [study]. following discussion of what was learned by the initial endeavor, the change strategy is then modified and reattempted [act] . through repeated use of this cycle, small hunches, theories, and ideas gradually coalesce to into significant changes that consummate in significant improvement. the essential key to success is the use of small changes that are easily accomplished, followed by the analysis of data to evaluate the impact of the intervention. the use of data on performance is essential to recognize where opportunities for improvement exist, and also in order to garner feedback on what changes truly result in improved outcome. once an organization determines specific actions that must take place in order to improve outcomes, it needs to track the consistency with which those actions are taken. improved performance on these "process" measures can be measured as a preliminary step to improvement in the outcome measures that are the true goal of the work. feedback must be received promptly and on a regular basis, and data should be reported visually in a way that can be understood and used by members of the care team as well as interested outsiders. the synergy that derives from collaboration among workers investigating the same problem is well known to scientific researchers, the most successful of whom are typically embedded in networks of cooperating laboratories within and outside of their home institutions. this strategy is equally effective for the development and spread of innovations for improvement in the delivery of health services. the most commonly used cooperative model is one that recognizes "best practices" and attempts to copy and spread approaches used by providers whose outcomes are the best within their field. however, novel and effective ideas for how to accomplish certain specific goals exist even at centers whose overall performance is average, especially if they are actively striving to improve their outcomes. thus, collaboration among various centers and healthcare workers who are trying to accomplish the same or similar goals is an important and effective strategy to accelerate change. supported in part by cff schech c qi. the steady increase in median survival for those with cystic fibrosis (cf) is, in large part attributed to cf center-based care and the introduction of new therapies. however, variation in practice patterns and utilization of existing therapies, as well as patient outcomes, suggests less than optimal application of existing knowledge in caring for those with cf. epidemiologic data identifies "average" cf care patterns and outcomes, but does not inform cf care providers on strategies for benchmarking above average, or "going beyond good enough". improving cf center care using existing therapies and scientific knowledge requires thoughtful examination of current care patterns to identify potential areas for improvement. the national initiative for children's healthcare quality (nichq) recruited cf care centers of varying demographics across the united states to create a collaborative group. this group focused on two goals to improve cf center care: improving nutrition and reducing environmental tobacco smoke (ets) exposure. this collaborative group of centers spent eighteen months working towards these goals utilizing the care model for child health. the experience of one pediatric cf center within the collaborative is presented demonstrating successful strategies for improving outcomes in nutritional status and reducing ets exposure, through appropriate process changes, partnership with families, consistent aggressive nutritional interventions, and application of existing knowledge. practical application of elements within the care model for child health incorporated within the collaborative are shared including changes in clinical information systems, decision support, delivery system design, family education, and a prepared, proactive cf care team. the results of the collaborative efforts resulted in a steady increase in median weight percentile for all children, and an even greater increase in those children in nutritional failure at the first recorded encounter. key factors in the achievement of the collaborative goals included: nutrition focus and process changes, and engaging families and patients to teach about enteral supplementation options. . preview of patients prior to clinic, in addition to postclinic review. . improving self-management by providing specific nutrition education (monthly newsletters) and written self-management plan at each visit. targeted efforts to reduce environmental tobacco smoke (ets) exposure resulted in a % documented "no smoking policy" for all environments for children within the center, and a % "quit-rate" among parental smokers. a simple three-pronged strategy of: a) systematic assessment of ets, b) clinician education in tobacco cessation counseling, and c) identification and provision of appropriate support materials to families, allowed for significant success in the overall reduction of ets exposure. the experience of this center demonstrates the potential to improve the care and outcomes of children with cf through systematic application of existing knowledge and attention to basic tenants of the cf center-based interdisciplinary care model. identification of variation in practice allows for focused interventions in areas where changes can, and will drive cf care beyond "good enough". it is the goal of all health care practitioners to render optimal care to those who seek their assistance. to this end, the learning leadership collaborative of the cystic fibrosis foundation has begun the promotion of the education of our community in the processes of quality improvement. as the project for the mini-atp course at intermountain health care we developed a standardized approach to relate to our patients the importance of their nutritional status with the goal of having no patients in need of urgent nutritional rehabilitation by june of . noting the great variance in the rate of nutritional compromise among the many cf centers, and accepting the concept that a calorie will function similarly regardless of geography, it was our hypothesis that raising awareness of nutritional status in our patient population, when coupled with specific recommendations for nutritional intervention, would result in a significant improvement in the nutritional state of our patient population. in september of we stratified our pediatric and adult population into three categories (green, yellow, and red) based on weight/height percentile for patients less than years of age, bmi percentile for pediatric patients greater than years of age, and bmi in adult patients equal to or greater than years of age. the three categories of green, yellow, and red rendered visual reminders to institute category and age determined protocols for nutritional maintenance or rehabilitation. a flowchart was created to assist in accurately stratifying each patient at each visit. in october of , we began universal distribution of growth or bmi charts at all regular visits. we also began institution of a "calorie contest" designed to raise the awareness of patients and their families regarding the caloric content of varied food combinations. those found in need of nutritional rehabilitation were treated in accordance with established cf guidelines. we tracked our adherence to our own program by recording all of the above interventions in a flow sheet placed at the front of each of our patient's paper charts. our team rapidly adapted to our new formalized process and we were able to achieve greater than % adherence to all of our interventions by / / . to monitor the effect of our interventions we created graphical displays of the monthly total of patients within each category as % of total monthly attendees. these data were graphed within control limits calculated from retrospective analysis of monthly data for months prior to our interventions. upon reviewing our statistical process charts of our outcomes up until june , we have not achieved the lofty goal of remitting our entire patient population from nutritional compromise. however, there has been notable improvement in both the pediatric and adult program outcome data as manifested by more visits per month with patients classified as "green zone". the data indicate a more prominent effect in the adult population, which has prompted us to readdress our process to further augment our positive outcomes. this process has served to enhance the nutritional status of our cf patient population and has provided a mechanism by which we may continue to improve many aspects of patient care. in our lives, sickness comes to loved ones, accidents leave their cruel marks of remembrance, and tiny legs that once ran are imprisoned in a wheelchair. mothers and fathers who anxiously await the arrival of a precious child sometimes learn that all is not well with this tiny infant. a missing limb, sightless eyes, a damaged brain, or the term "cystic fibrosis" greets the parents, leaving them baffled and filled with sorrow. sixteen years ago i had visions of what parenting a child would be like. my visions were based on hopes and dreams, which didn't include a life threatening illness. i remember waiting in a tiny room with staunch white walls that seemed to enclose us with its smells of alcohol. my husband and i anxiously waited the arrival of the pulmonary doctor as we nervously passed our two month old back and forth between us. i remember the doctor's red, white and blue tie, his graying hair and the subtle way he cleared his throat as he pronounced, "your son has cystic fibrosis." i felt isolated, overwhelmed and numb as we left the hospital corridor. there followed the inevitable blaming, the condemnation, and the perennial questions: "why such a tragedy in our family?", "how did this happen?", "where was god?". why, where, how-those recurring words-did not bring back our lost dreams, our son's perfect body, and the plans we had as parents. i have learned that self-pity, personal withdrawal, or deep despair will not bring peace, assurance, or help, which i needed to courageously, continue on my parenting journey. rather, i had to find a way to go forward, look upward, and move onward. sixteen years later the cystic fibrosis parent-to-parent support network is proving to be just this type of help by offering comfort, hope and encouragement from parents who are reliable allies that immediately understand the situation because they have been through it themselves. imagine the atmosphere of a room filled with ninetyeight mothers of children with cystic fibrosis. each of these mothers was experiencing a different place on the cystic fibrosis journey; some were newly diagnosed, and others were more experienced dealing with various struggles of cf. each mother was given the opportunity to ask a question or give advice structured around the challenge of cystic fibrosis; "how do i get my threeyear-old to sit still while taking a breathing treatment?", "i feel like all i do is give breathing treatments, chest physical therapy and medicine all day. what can i do to manage my time better?", "how can i get my teenager to take his enzymes when he goes out with friends?". how did they get support that day? a connection was made between mothers; the loneliness and despair were replaced with friendship and companionship. the robert wood johnson pursuing perfection grant supporting healthcare quality improvement at cincinnati children's hospital encouraged focus on the viewpoint of patients and parents and perspective of their needs in an effort to improve care. a qualitative study of parent to parent self-help conducted by parents and researchers and published in the journal of pediatric psychology ( ) determined that an overwhelming majority of parents were positively helped by parent to parent support. this type of support was shown to increase parents' acceptance of their situations and their sense of coping. parents were also able to define their roles in the healthcare process, perfect their self-management skills and in turn feel more confident in caring for their children. with support from the cincinnati children's hospital and the pursuing perfection grant, and knowledge and experience from studies ( , ), a core group pf patients, parents and cf center staff was formed. the core group identified a void in support that the parents could fill. the next step was to promote a parent-to-parent support network with the goals of providing strategies for improved patient care, tips from other parents, coping skills, and hope and comfort. i don't want to make it sound like things just fell into place, because in actuality, it took a lot of hard work. as with any change, there were concerns and conflicts. we experienced challenges but we were able to work through them. the legal department wondered whether parents would be compliant with hippa. there were concerns with training and how well the staff and parents would work together. any of these concerns could have been used as an excuse to quit. but, as john f. kennedy said, "the problems of the world cannot possibly be solved by skeptics or cynics whose horizons are limited by the obvious realities. we need…to dream of things that never were." we have a great team of people who, despite the difficulties, are committed to succeeding and reaching a common goal. in fact look at what we have been able to accomplish so far. one-to-one support is a personalized type of support that is made available to parents particularly during periods of increased stress, such as at the time of diagnosis. parents are carefully matched with a mentoring parent who understands their situation. our team consists of tracey blackwelder cincinnati children's hospital, cincinnati, ohio twelve diverse mentoring parents who were selected by the healthcare team. these selected parents were trained in communication and listening skills. quarterly newsletter is a quarterly parent-to-parent support newsletter that is mailed to all families of patients of the cincinnati cystic fibrosis clinic. this newsletter is coordinated and edited by parents, and is written with the parent's perspective. it is an effective way to stay in touch with parents and it provides continuous uplifting stories, updates, self-management skills, and influential hints and tips that help parents feel motivated and successful. social functions offer an opportunity to share experiences, strengths and hope. these functions enable parents to connect with others and find their way out of isolation, frustration and hopelessness and turn their challenges into positive forces. socializing also provides a venue for information regarding successful care strategies. promoting awareness is an important component of parent-to-parent support to reach families. stewart henderson britt said, "doing business without advertising is like winking at a girl in the dark. you know what you're doing, but nobody else does." we connect with parents through brochures, voice mail, list-serve web service and e-mail. in summary, the parents and staff are working together as a team. just like two oxen yoked together can accomplish more than they can separately when working for a common goal, our combined team of parents and staff are accomplishing amazing things as we work together to "change the outcome" in our pursuit of perfect care. ainbinder,j., blanchard, l., singer, g.h.s., sullivan the us cystic fibrosis foundation patient registry has been collecting annual data from over , patients cared for at accredited cf care centers since . this data has been used to document the steady improvement in the survival of people with cf, and improvements in lung function and in nutritional status. how can we now use it to identify opportunities for change and to monitor progress in the care of people with cf? for the past years we have examined the variation in treatments and outcomes between care centers and given feedback reports from the registry to the centers on their processes and outcomes. the amount of variation at the center level is completely invisible to providers who care for patients one at a time in a busy clinic, so these reports are an important first step towards understanding each center's care and outcomes in the context of cf care in the us. clinicians want to know how they are doing; eventually patients and families are going to demand to know as well. there has been a steady improvement in those measures that have been highlighted in the reports. in the past five years, the rate of screening for cf related diabetes nationally has gone from % to %, and in six centers reported screening % of appropriate patients. the proportion of patients adhering annually to standards for numbers of pulmonary function tests, sputum cultures, and outpatient visits continues to rise. however, the center reports are limited to working within the framework of the clinical practice guidelines for cystic fibrosis. the guidelines summarize the best evidence for different aspects of cf management, but they also define those areas of clinical care that experts consider important. there is center level variation in every aspect of patient care: antibiotics are used chronically in over % of patients at some centers, zero percent at others (see figure on next page); tobi is used in to % of pseudomonas aeruginosa positive patients; pulmozyme is used in to % of patients; annual hospitalization rates vary from to % of adults, with median lengths of stay from to days; and home iv antibiotic use rates range from to %, with median courses between . and days. nutritional processes and outcomes are similarly variable. does all this variation matter? we have started the process of answering the question. "are some centers the 'best' and what does that mean for patient care?" by examining lung function and nutrition outcomes for children and adults annually from to , ranking centers and selecting those that were reliably in the top in several categories we identified centers with consistently good outcomes across patient groups. since the median age of death is . years for the "best" centers compared to a mean of . years for the rest of the country, and median survival is almost years longer (p< . ). this analysis is the basis for the cf foundation's vision statement: "we believe that the life expectancy of cf patients can be extended by to years through the consistent implementation of existing evidence-based care." what are these centers doing to be so successful? the "best" centers are diverse; small and large, academic, for profit, rural and urban. the registry can suggest areas in which to focus further study but it does not collect the level of information that is required to completely describe exemplary cf care. how could a clinically useful data instrument describe a center where there are fail safe systems in place, where every patient gets everything they need at every visit, where handoffs between care givers are smooth, where culture results are never lost, where patients and families are well informed, activated, responsible, and supported? the registry allows us to report what is currently achievable by some clinical centers and sets the performance possibilities for the present. the highest performing centers don't necessarily use more healthcare resources than other centers. benchmarking visits by clinic teams may help us to understand the fine detail of care that produces the greatest success. what can be done right now to accelerate the improvement in cf care that we have seen over the past decades? historically, the registry has been used for epidemiologic purposes; the conversion to a patient management and quality improvement tool requires that the clinical practice guidelines be written in such a way that adherence is quantifiable. we must keep the guidelines up to date for capturing and measuring exemplary cf care. already portcf helps track individual patient results, and has some important patient management and reminder tools. portcf is being enhanced so that center level reports will be available much more quickly, and eventually, the reports will be available as the data is collected. but data that is not examined is like a tree falling in the woods. the patient registry gives a broad view of cf care in the us and allows us to track progress and focus our attention on achievable goals. variation is a treasure, but it does not yield its secrets easily and to learn what creates success is going to require the concerted efforts of all the players -clinicians, patients and families, basic scientists, and the foundation. cystic fibrosis foundation patient registry annual report to the center directors, bethesda, maryland. the year was a one of dramatic change for the cf foundation patient registry. first, the implementation of new regulations pertaining to patient privacy necessitated that all care centers gain approval from their local institutional review boards (irbs) for submission of data to the registry. a formal consenting/ assenting process was required at all but care centers and affiliate centers. despite these logistical challenges, registry data was submitted from sites including care centers, adult programs with distinct registry center numbers, and affiliate centers. patient retention in the registry was excellent. we observed a modest (approximately %) drop in patient numbers as compared to , but fully expect the numbers to rebound in . the care center teams deserve accolades for their work on behalf of the patient registry. the transformation of the patient registry from the collection of year-end summary data to the web-enabled, encounter-based port cf application was the other major change in . the web-based application has opened up new challenges and opportunities. a number of resources can now be placed at the fingertips of our care teams including consensus documents and patient education materials. ongoing data entry throughout the year provides the potential for real time feedback of information to the care team. for example, the current iteration of port cf includes reporting features such as the patient roster, patients due for a visit, and the patient summary reports. the summary report displays complications, respiratory microbiology, and graphical trends of pulmonary function and nutritional status. the query tool provides the capability to identify subsets of patients, e.g., those with cf-related diabetes, which require special monitoring. our intent with these reports and query capabilities is to provide access to patient and center level information that will facilitate health care delivery. the future for port cf holds even greater promise. our quality improvement activities are focused on three core care areas in which suboptimal outcomes are associated with morbidity and mortality: ) growth and nutrition, ) pulmonary, maintenance and exacerbations, and ) cfrelated diabetes mellitus. the significant variability in these outcomes throughout the care center network suggests that they are high leverage areas for accelerating the rate of improvement in cf care. we are currently collecting data in port cf on the pertinent medical outcomes in these areas, but many of the associated practice patterns are not being captured. collecting this data throughout the year and making individual and aggregate information about these practices available to the care centers will be instrumental in supporting further improvement activities. decision support in the form of reminders and algorithms can also be incorporated for key care decisions. further enhancements to port cf will occur in concert with an updating of our clinical practice guidelines. our goal is to create a dynamic process with better integration of port cf, the practice guidelines and clinical care (see figure) . standing committees will be formed in the three core care areas. the growth and nutrition committee has recently been constituted and will be the first area for enhancements to port cf. one of the first tasks for the committees will be to develop a list of strong recommendations for care based on the scientific evidence and clinical experience. work done in our quality improvement collaboratives will also inform the process. the strong recommendations of the guidelines committees will serve as a basis for new questions and reports derived from the new data for port cf. while striving to incorporate value-added data fields into port cf, an important consideration will be the impact of data collection and data entry on care centers. in summary, port cf is a critical tool for supporting improvement activities and for tracking the outcomes of those efforts. further evolution of port cf will occur in concert with ongoing quality improvement activities and an updating of our clinical practice guidelines. the continued success of the registry is dependent upon the commitment and hard work of the care center teams and the willingness of patients and families to share their data. the response to recurrent injury, in liver and in other organs is one of wound healing. many different types of injury (i.e., chronic hepatitis, ethanol, biliary tract disease, cystic fibrosis, iron or copper overload, etc.) lead to injury and ultimately to hepatic fibrosis and cirrhosis. cirrhosis in turn leads to hepatocellular dysfunction and portal hypertension, each of which have grave clinical sequelae. cirrhosis in cystic fibrosis results in the same clinical complications as is found in patients with other forms of liver disease. extensive investigation over the past years has established that the effector in the liver wound healing process is the hepatic stellate cell (also known as a lipocyte or ito cell). a central feature of the wounding response to liver injury is the transformation of resident stellate cells from a "quiescent" (normal) to an "activated" (injured liver) state ( figure ). characteristics of this transition include morphologic and functional changes. morphologic changes include loss of vitamin a, acquisition of stress bundles, and development of prominent rough endoplasmic reticulum ( ) ( ) ( ) ( ) . one of the earliest described and most prominent effects of stellate cell activation is production of increased quantities of extracellular matrix, including types i, iii and iv collagens, fibronectin, laminin and proteoglycans, some of which are increased by greater than -fold ( ). further, the available evidence now indicates that the overall increase in extracellular matrix protein deposition typical of cirrhosis can largely be ascribed to excess production by stellate cells ( ) . thus, a great deal of emphasis has been placed on elucidating mechanisms underlying stimulation of fibrogenesis by stellate cells. a number of events, typically acting in concert, play a role in stimulating stellate cell fibrogenesis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . for example, stellate cells produce chemotactic factors ( ) , appear to be important in matrix remodeling ( ) , and produce a number of "vasoactive peptides" ( , ) , including the potent vasoconstrictor, endothelin- (et- ) ( , ). indeed, et- and vasoactive peptides have emerged as important components of the fibrogenic response. an important emerging theme is that current methods for precise diagnosis and quantitation of the effects of therapy are required. liver biopsy remains the gold standard for assessment of liver fibrosis. however, it is not without potential problems. therefore, a number of methods have been used to non-invasively assess fibrosis. these include imaging techniques, non-specific or indirect markers of liver fibrosis, and tests that more directly assess the fibrogenic biology present in the liver. such non-invasive tests are likely to emerge as critical components of the clinical armamentarium in management of hepatic fibrosis. a number of therapies (i.e., colchicines, ursodeoxycholic acid, ptu, vitamin e, etc…) have been tested in patients with fibrosing liver disease. for the most part, these approaches have proved to be ineffective. however, newer preclinical studies have highlighted a number of putative therapies to abrogate fibrogenesis. such therapies are targeted at reducing or removing the primary injury, modulation of stellate cell activation, stimulation of matrix degradation or stimulation of stellate cell death. for example, interferon gamma, a cytokine with potent immunomodulatory activity, has profound effects on multiple aspects of stellate cell activation. these data have led to clinical trials in humans in which interferon gamma is being examined in patients with fibrosis. other specific, mechanism based, "anti-fibrotic" therapies are rapidly emerging. liver disease is a major complication in many patients with cystic fibrosis (cf). the precise prevalence of cfassociated liver disease (cfld) is difficult to determine as no reliable method of diagnosis currently exists. traditional measures of liver function do not correlate with the severity of liver disease and there is no specific cf genetic association. thus the diagnosis is not usually made until cirrhosis or complications such as portal hypertension are established. while clinical and imaging studies may detect these late changes, and invasive liver biopsy, which is not always reliable, due to the focal nature of fibrotic changes might aid diagnosis, the challenge exists to develop a sensitive, reliable non-invasive assay to identify cf patients who are at risk of developing severe liver disease, which might also serve as a guide to monitoring progression or response to therapy. although the basic mechanisms remain unclear, abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cfld. we have documented a definitive role for hepatic stellate cells in the fibrogenesis associated with cfld, and suggested a potential mechanism for the induction of hsc collagen gene expression, through the production of the cytokine tgf-beta( ) by bile duct epithelial cells ( ) . accumulated bile acids have been proposed as potential mediators of cytokine production, and , indeed, we recently found a correlation between both cholic acid and cholic acid/chenodeoxycholic acid levels with histological liver injury and fibrosis progression ( ) . a potential monitoring role for serum bile acids and serum measures of components of hepatic matrix remodelling, which may serve as markers of early fibrosis is suggested by these studies. we thus have evaluated, by preliminary cross-sectional study, the utility of selected serum markers ie bile acids, hyaluronic acid (ha), collagen type iv (cl-iv), prolyl hydroxylase (ph), matrix metalloproteinases (mmps), and tissue inhibitors of mmps (timps), by their correlation with the degree of hepatic fibrosis in patients with cfld. we compared with serum levels from patients with cf and no liver disease (cfnold), and age matched control data. methods: bile acid composition was determined by gas-liquid chromatography/mass spectrometry in bile, urine, and serum samples from children with cfld, children with cf but without liver disease (cfnold), and controls. sera from cfld, cfnold and control subjects were assessed for timp- , cliv, mmp- , ha and ph by enzyme immunoassay. serum markers were correlated with blinded hepatic fibrosis score on liver biopsies from cfld subjects.results: there were significant correlations between serum cholic acid levels and the ratio of serum cholic acid/chenodeoxycholic acid with hepatic fibrosis score, the degree of inflammation, and limiting plate disruption. timp- , ph, and cl-iv in cfld, there was a negative correlation between fibrosis score and both timp- (r = - . , p = . ) and ph (r = - . , p = . ). there were no differences in timp- , cl-iv, ha or ph between cfnold and controls, nor in mmp- between the three groups. the correlation between both cholic acid and cholic acid/ chenodeoxycholic acid levels with histological liver injury and fibrosis progression suggests a potential monitoring role for these bile acids in cfld. elevated levels of serum timp- , cl-iv and ph in cfld suggests that these may be indicators of fibrogenesis in cf. higher levels of timp- and ph were associated with mild fibrosis, and these, in concert with serum bile acid measurements may be useful markers for the early detection of cfld. we suggest that these serum markers be subject to longitudinal study in cf as markers of progression of cfld, and/or response to future antifibrogenic therapies. clinical and biochemical parameters have been shown to be very insensitive in the identification of cystic fibrosis associated liver disease (cfld) ( ). thus, many centers have begun to investigate the use of radiologic screening for cfld. this is challenging in that the histologic findings with cfld can range from steatosis through early fibrosis to biliary cirrhosis ( ) . the primary therapy that is currently available, ursodeoxycholic acid, has been suggested to be most effective in early disease ( , ) , but may improve advanced cfld ( ) . thus, the challenge for treatment remains the identification of "early" cfld for potential treatment and the identification of "silent" advanced cfld to institute appropriate anticipatory management. to that end, radiologic studies offer noninvasive imaging to assess the liver. this summary will discuss ultrasonography, ct scanning, mr scanning and radioisotope excretion studies. ultrasonography (us) utilizes short pulses of high frequency sound that are reflected back at the interfaces of tissues of different acoustic properties. us is low in cost, portable and involves no radiation exposure. it is limited by the inability to penetrate bone or air which can prevent a complete examination of abdominal organs and occasionally by the depth of penetration required. us is the imaging modality of choice to screen for biliary disease in cf. cholelithiasis and biliary dilation can easily be determined. us has also been suggested as a tool to detect early changes in liver architecture in cfld ( ) ( ) ( ) . us has an excellent sensitivity and specificity for end stage cirrhosis with portal hypertension ( ) as the findings of a nodular liver, splenomegaly and varices are generally due to cirrhosis. however, the goal of screening for cfld would be to identify early liver disease prior to the progression to cirrhosis. two ultrasound classifications for cfld have been proposed ( , ) : in montreal, they have demonstrated minimal interobserver variability in the scoring and interpretation of us ( , ) . abnormal us findings were more common in patients with abnormal ast, alt or ggt. however, only about % of patients with abnormal ast, alt or ggt had an abnormal us and - % with of patients normal values had an abnormal us ( ) . no study has investigated inter-center variability using either scoring system or the correlation between early us findings and liver biopsy evidence of fibrosis in cf. us can be used to follow patients for the development of us abnormalities. in a longitudinal us study by leanerts, serial us was performed for an average of us per patient ( ) . of the normal patients at the start of the study, ( %) had any abnormalities identified during longitudinal screening. of these, developed and continued with a heterogeneous liver, developed a nodular liver and progressed to portal hypertension. kaplan meier risk analysis predicted that approximately % of subjects would develop a nodular liver on us by years of age. in this study, thirty subjects received ursodeoxycholic acid during the course of the study, clearly not all due to us abnormalities as - % had biochemical abnormalities despite a normal us. it is difficult to make a firm recommendation about the use of us in screening for cfld. the sensitivity and specificity of early us findings for fibrotic cfld have never been studied, making interpretation of the meaning of these findings difficult at present. it seems likely that us will increase the identification of "silent" cf associated liver imaging abnormalities, but the timing, frequency and cost effectiveness of the routine use of us in screening for cfld remains to be determined. a prospective study of the sensitivity of this modality would be helpful in our interpretation of these studies. computed tomography (ct) detects small differences in the attenuation of x-rays to identify structures not visible on standard radiographs ( ) . the addition of oral and iv contrast improves the identification of tissues and blood vessels and can detect differences in perfusion in organs such as the liver. much like us, ct is very effective at identifying end stage cirrhosis with portal hypertension. the ct findings in end stage cirrhosis from a variety of causes have been published ( ) . however, accurate ct imaging of the liver requires iv contrast and radiation exposure. in biliary cirrhosis, the primary end stage lesion of cf, nodularity of the liver is common and easily seen on ct. however, there are no studies of ct findings in early cfld. similar to us, there have been no studies correlating ct and early hepatic fibrosis in cf or other liver diseases. at present, ct cannot be recommended for screening for cfld given the need for contrast and the radiation exposure compared to us. however, ct is likely the most sensitive imaging modality for screening for hepatic cancer in patients with cirrhotic cfld. magnetic resonance imaging (mri) detects the density of protons in tissue water and lipids and their relaxation times ( ) . in mri, the magnetic environments of protons in fat, intracellular water and extracellular water have different relaxation properties leading to sharp contrast differentiation between tissues with differing contents of fat or water. mri is very valuable for non-invasive imaging the biliary and pancreatic tree (magnetic resonance cholangiopancreatography). mri findings in cf have been presented with excellent demonstration of biliary and pancreatic abnormalities ( ) . it is the imaging modality of choice for hemagiomas. the value of mri in diffuse parenchymal liver disease is still investigational ( ) . similar to us and ct, mri can detect the gross abnormalities of cirrhosis and portal hypertension. at present, the use of mri for screening for early cfld would require further investigation. hepatobiliary scintigraphy. after iv injection, several radioisotopes are extracted by the liver and excreted into the bile and eventually into the intestine. these are detected by a scintillation camera. in cf, abnormalities detected have included dilated intrahepatic and extrahepatic bile ducts and delayed excretion and intestinal appearance of the radioisotope ( ) ( ) ( ) . delayed intestinal appearance seems to predict a better response to ursodeoxycholic acid ( ) . however, the specificity and sensitivity of this imaging modality for cfld has never been studied. given the technical difficulties with the study, it has been superceded by mri, ct and us for routine hepatic imaging and is not suitable as a screening test for early cfld. liver disease is increasingly recognized as a major cause of morbidity and mortality in patients with cystic fibrosis (cf). the typical hepatic manifestation of cf is focal biliary cirrhosis and results from biliary obstruction and progressive periportal fibrosis; it is the most clinically relevant cf-associated hepatic problem, since extension of the initially focal lesions may lead to multilobular biliary cirrhosis, portal hypertension and related complications ( , ) . surveys on cf patients with liver disease have reported rapid progression of cirrhosis to portal hypertension and high mortality rates due to complications of cirrhosis and lung disease ( , ) . in a retrospective study spanning a -year period, of children with cf investigated for cirrhosis, esophageal varices developed in % and in half of these patients esophageal bleeding occurred early during the second decade of life; liver failure occurred in % of them at a mean age of years ( ). in another retrospective study, over a period of -year, / cf patients with multilobular biliary cirrhosis died as a result of respiratory ( cases) and hepatic ( cases) complications ( ). more recently information regarding natural history of liver disease in cf patients have been obtained through prospective studies in carefully monitored cf patients ( , ) . in a cohort of consecutive cf patients identified over a ten-year period and followed for a median time of years at the cf center of the university of milan, there were incident cases with no incidence peak in any age group ( ). we estimated that crude incidence of liver disease in our whole cf population was %, corresponding to . cases per patient-years. median age at time of diagnosis of liver disease was years (range: months to years) and in of the patients with liver disease ( %) this complication was diagnosed during the first five years of life. in our cohort, after the age of years, incidence declined and, by the age of , liver disease had developed in out of patient. liver disease should be therefore considered a relatively early complication of cf, which may be susceptible to prophylactic strategies. the patients with liver disease (treated with ursodeoxycholic acid since ) were followed for a median time of . years (range: month to years), corresponding to patient-years. cirrhosis was already present at time of diagnosis of liver disease in of these patients ( %) and developed in additional patients during follow-up after a median period of . years from time of diagnosis of liver disease (range: months to years). incidence rate of cirrhosis was . cases per liver disease patient-years ( % ci: . to . ). among the patients with liver cirrhosis who were altogether detected, ( %) developed portal hypertension (incidence: . cases per cirrhotic patient-years, % ci: . to . ), ( %) esophageal varices (incidence: . cases per cirrhotic patient-years, % ci: . to . ), ( %) developed liver decompensation (incidence: . cases per cirrhotic patient-years, % ci: to . ) and underwent liver transplantation at the age of yrs. at the end of follow-up, of the patients with liver disease who were still alive had not developed liver cirrhosis after a median time of . years (range: to years) from time of diagnosis of liver disease. present evidence concerning efficacy of ursodeoxycholic acid on patient survival in cf is still inconclusive ( ), but we cannot exclude that treatment with this bile acid also contributed to the favourable clinical course we have observed. in order to establish the impact of liver disease on the outcome of cf, we also compared the main clinical outcomes of patients who developed liver disease with those observed in the group of patients who did not ( ) . no significant differences between patients with or without liver disease were observed in the occurrence rates of clinically relevant end-points, like respiratory failure, need of oxygen therapy or number of hospitalisations during the last two years of follow-up. these data indicate that liver disease does not expose cf patients to higher risk of developing respiratory failure or other major outcome events. mortality from any cause also did not differ significantly among patients with different liver status (death rate ratio . ), indicating lack of association between liver disease and mortality in cf. other studies addressing cf-related liver disease prospectively ( ) have also provided evidence that this is a relatively early complication of cf, and that its clinical course may be milder than previously reported by retrospective studies ( , ) . in a cohort of carefully managed swedish cf patients who had been followed-up for more than a decade, liver disease developed before adolescence in % of cases and in none of the patients this occurred in adulthood. rate of progression was slow, as also indicated by histologic changes at repeat liver biopsy during the follow-up ( ) . in summary, liver disease associated with cf develops in a significant percentage of cf patients, usually before or at puberty, displays a slowly progressive course and until the most advanced stages are attained, it does not seem to influence nutritional status and severity of pulmonary involvement. only in a minority of patients, often in the pediatric age, liver disease may represent the main clinical problem and its progression may be unusually rapid. the role of genetic modifiers in determining this variability in terms of severity is presently under study ( ) . the most prevalent cf-causing mutation is a three base pair deletion that results in the loss of phenylalanine (f ) in the first nucleotide binding domain (nbd ) of cftr. over ninety percent of all cf patients carry at least one delta f allele ( ) . not surprisingly, the molecular and cellular phenotypes of this mutation have been the focus of intensive study over the past fifteen years. this work has established that the mutant cftr does not efficiently traffic to, nor accumulate at, the plasma membrane ( ) due to a disruption of nbd folding ( ; ) and destabilization and rapid turnover of the minor fraction that does reach the membrane ( ). the inefficiently folded delta f cftr is recognized by cellular quality control systems and marked for degradation by the proteasome ( ; ) . the lack of properly folded, native cftr at the apical membrane underlies the aberrant fluid and electrolyte homeostasis that define the cf pathology. delta f cftr that does fold and reach the plasma membrane retains some function as a chloride channel ( ; ). thus, promoting the folding of delta f cftr would be predicted to at least partially ameliorate the pathology ( ; ). in this regard, conditions that promote folding and stabilize native structures, such as reduced temperature ( ) and addition of osmolytes ( ) ( ) ( ) have been shown to improve mutant cftr folding and partially restore function. however, treatments such as these, as well as alterations of cellular chaperones that assist in folding, lack specificity and the consequences of chronic alterations of these parameters on cellular function remain unknown. more specificity could theoretically be achieved if ligands that bind tightly to the native, but not unfolded cftr could be identified ( ; ; ). the favourable binding energy for these ligands would thereby be coupled to the otherwise unfavourable folding of mutant cftr ( ) . ideal compounds would promote folding and potentiate cftr function although weak anatagonists could also have utility. detailed structural knowledge of cftr and its domains provides a context for understanding not only the functional mechanics of cftr, but also the role of f in folding and further suggest sites which could be targeted for ligand binding. the recent solution of the murine nbd at high resolution using x-ray diffraction methods provides the first such structural information ( ) . the structure reveals that f is exposed on the surface of the nbd in an alpha-helical sub-domain. replacement of the phenylalanine with any other amino acid has no measurable effect on the folding or final structure of the isolated nbd, indicating that it is the loss of the backbone that leads to inefficient nbd folding ( ) . in contrast, charged or bulky side chains at the position interfere with the the later steps in the folding of the full-length cftr. together with the surface exposure of the position, these results indicates that f may be at an interface between the nbd and other domains of cftr, most likely the tmds. these results have implications for understanding the function of cftr and the development of ligands to promote mutant cftr folding for therapeutic benefit. structural predictions suggest that cftr, a member of the abc superfamily of transport atpases, consists of two homologous halves, each comprised of six transmembrane (tm) helices and a nucleotide binding domain (nbd and nbd ) that are connected by the regulatory (r) domain ( ). this complex, multidomain structure, conceivably, renders the post-translational folding of cftr sensitive to point mutations, while ensures its regulation by phosphorylation and atp-binding/hydrolysis. depending on the expression system examined, - % of the newly synthesized wt cftr degrades at the endoplasmic reticulum (er), as coreglycosylated folding intermediate ( ) ( ) ( ) . the remaining - % of the cftr undergoes an atp-dependent posttranslational conformational maturation ( ) ( ) ( ) and attains an export competent, folded conformation that is incorporated into transport vesicles at the endoplasmic reticulum (er). during traversing the cis/medial golgi, the high mannose-type n-linked oligosaccharides of cftr (core-glycosylated form) are converted to complex-type oligosaccharides ( ) . it was proposed that the atp-dependent conformational maturation of the nascent, core-glycosylated cftr is a prerequisite for entering the distal stages of the secretory pathway from the er ( ). this conclusion was verified by examining the in vivo and in vitro protease susceptibility of the early folding intermediates and the fully mature cftr either in its core-or complex-glycosylated form ( , ) . the conformational maturation of cftr is facilitated by an array of chaperone interactions. accordingly, mutations perturbing the folding process of cftr alter the association/dissociation dynamics of these chaperones and vice versa; modulation of chaperone activity has an effect on cftr folding ( ) . in the cf gene more than mutations have been identified, leading to impaired biosynthesis, processing, activation and stability of cftr or a combination of these ( ) . missense mutations, including the most frequent one, deletion of phenylalanine at position (∆f ) in the nbd , are believed to interrupt the post-translational folding of cftr ( ) ( ) ( ) ) and target the core-glycosylated intermediate for degradation via the ubiquitin-proteasome pathway at the er ( , ). while exposure of er-retention signals, may also contribute to the inability of folding intermediate(s) to exit the er ( ), the consequence of ∆f at the molecular/structural level remains elusive. numerous model systems, represented by synthetic peptides and isolated nucleotide binding domains, harboring the wild-type sequence or the ∆f , have been applied to unravel the structural impact of the mutation. in our approach we used systematic amino acid substitution of the f residue in the context of the full-length cftr to investigate the role of the side-chain and back-bone structure of f on the biogenesis, stability and interdomain interaction of the channel. hydrophobic amino acids with side chain volumes comparable to, but not larger than phenylalanine supported the post-translational folding and stability of cftr. on the other hand, charged amino acids, as well as proline and glycine substitutions were non-permissive for both folding and stabilization of the channel. while deletion of f caused limited conformational defect in the nbd , similar to non-permissive amino acid substitutions, it substantially compromised the conformation of nbd and its interaction with nbd (unpublished data). these and other results, discussed in the symposium, strongly suggest that hydrophobic side chain interactions of f are indispensable to ensure normal folding nbd and thus to achieve global stabilization of the native conformation of cftr. reduced temperature or chemical chaperones can partially rescue the folding defect of ∆f cftr ( ) the majority of patients with cystic fibrosis (cf) express a mutant cystic fibrosis transmembrane conductance regulator (cftr) protein that is defective in folding ( ). the misfolded protein is retained in the endoplasmic reticulum (er) and is not trafficked to the cell surface (misprocessed mutants) ( ). these patients would benefit from therapy that can cause the mutant cftr protein to fold properly and be delivered to the cell surface in a functional form. recently, we discovered a strategy for correcting misfolded defects in p-glycoprotein (p-gp) that has direct application to cf. p-gp and cftr are both members of the atp-binding cassette (abc) family of proteins. the abc proteins generally have two transmembrane domains (tmds) and two nucleotide-binding domains (nbds). mutations in p-gp at equivalent positions to those in cftr that cause it to be misprocessed also caused the mutant p-gp to misfold and be retained in the er. the misprocessed p-gp mutants, however, could be induced to fold properly when they were expressed in the presence of a specific drug substrate ( ). the protein was then trafficked to the cell surface where it was fully active. therefore, p-gp is a useful model system for studying how processing mutations interfere with folding of abc proteins. the next objective was to understand how the misprocessed cftr and p-gp mutants were different from the mature wild-type proteins and how drug substrates the mutant channel to the cell surface. biochemical assays revealed that the ∆f mutation not only impairs the gating kinetics, but also decreases the channel stability by tenfold at the cell surface ( ) . to unravel the cellular machinery responsible for the recognition and elimination of the mutant from the plasma membrane, the internalization and recycling rates of cftr were determined. whereas native cftr recycled from sorting endosomes back to the cell surface, misfolding by ∆f and ∆ mutations prevented recycling and facilitated lysosomal targeting of cftr by promoting its ubiquitination. rescuing the peripheral folding defect or down-regulating the e ubiquitin-activating enzyme stabilized the mutant cftr without interfering with its internalization. these and other observations, in concert with the preferential association of the mutant cftr with ub-binding proteins (e.g. hrs and stam- ) and other components of the ub-dependent endosomal sorting machinery (e.g. tsg ), establish a functional link between ub-modification and lysosomal degradation of misfolded cftr from the cell surface ( ) . our data provide evidence for a novel cellular mechanism of cf pathogenesis in case of ∆ cftr. the results also imply that efficient relocation of the ∆f cftr from the er to the cell surface will require the conformational stabilization of the mutant. finally, these observations suggest a paradigm for the quality control of membrane proteins in general, involving the coordinated function of the ubiquitination and ub-dependent endosomal sorting machinery in post-golgi compartments. act as specific chemical chaperones to rescue the misprocessed mutants. initial studies suggested that processing mutations affect the structure of p-gp ( , ) or cftr ( ) by trapping the mutant proteins in the er in "loosely-folded" conformations that resemble the immature core-glycosylated form of the wild-type protein. these trapped proteins are inactive. one folding step that may be sensitive to the presence of processing mutations is the establishment of correct domain-domain interactions between the two tmds and the two nbds. to test whether processing mutations affect domain-domain interactions, we constructed a pgp/cftr(⌬f ) chimera in which the predicted third ␣-helix in nbd of p-gp was replaced with the equivalent region from cftr containing the ⌬f mutation. it was found that the ⌬f mutation caused misprocessing of the p-gp/cftr chimera and loss of activity by disrupting packing of the tm segments in the tmds ( ) . it also appeared that the ⌬f mutation disrupted interactions between nbd and the first cytoplasmic loop connecting tm segments and . more recently, we showed that processing mutations could also disrupt interactions between nbd and nbd (loo et al., , submitted) . these results indicate that processing mutations may interfere with folding of abc proteins by hindering the proper interactions between the domains. the drug substrates (specific chemical chaperones) may act by stabi-lizing proper domain-domain interactions during the folding process. mutations in cftr profoundly influence the function of respiratory epithelial cells, with resultant mucous accumulation, inflammation and infection. cftr plays numerous roles in cellular function, interacting directly and indirectly with cellular proteins and processes that regulate many aspects of epithelial cell biology. in order to identify pathways by which cftr influences cell function, and to explore the effects of cftr on cellular processes per se, we have generated a series of transgenic mice ( ) lacking cftr (fabp-cftr, cftr-/-), ( ) overexpressing human cftr in lung epithelial cells (sp-c-hcftr), and ( ) expressing the human ∆ cftr (sp-c-∆ cftr, cftr-/-, fabp-hcftr). all of the mice are viable and without overt pulmonary pathology. rna microarray analysis of the lung tissue was compared in lungs from adult mice of each genotype using the jeffrey a. whitsett, m.d. and yan xu, ph.d. affymetrix system. rnas differentially expressed were cross-compared. comparison of sp-c-∆ cftr, cftr-/-vs. cftr-/-demonstrated a strong, positive correlation (r = . ), and a weak, negative correlation (r = - . ) with the sp-c hcftr overexpression, indicating human cftr ∆ did not correct the abnormalities in gene expression characteristic of cftr deficiency. genes regulating inflammation, neutrophil chemotaxis, as well as fluid and electrolyte transport were induced in cftr-/-and sp-c-∆ cftr, cftr-/-mice, while those regulating cell growth, cell matrix and adhesion, and angiogenesis were significantly decreased. increased expression of human cftr (wild type) in the ∆ cftr induced genes in the heat shock protein family, indicating that this subset of genes was induced in response to cftr or misfolded ∆ cftr. dnajb and hsp rnas were increased by human cftr and human ∆ cftr, perhaps indicating a response to excess or misfolded cftr protein. genes involved in the endoplasmic reticulum response pathway (erad) were not induced by overexpression of cftr or ∆ cftr in vivo. while rnas in the ∆ and cftr deficient mice were quite similar, several genes involved in host defense including s a , s a , h -eb , h -dmb , h -t , and c-fos were induced in cftr-/-, but decreased in the ∆ mutant mice. several genes regulating lipid homeostasis were uniquely influenced by the ∆ cftr (scd- , lipocalin, and carboxyesterase- ). in summary, increased expression of cftr and ∆ cftr similarly induced genes in the heat shock protein (hsp) pathway, but did not induce erad transcriptional responses. genomic responses to cftr deficiency were, in general, not corrected by the ∆ cftr. cftr influences genes regulating host defense, inflammation, and fluid and electrolyte transport by processes likely mediated by its direct interaction with other cellular proteins and by influencing adaptive responses to the lack of cftr. the absence of cftr activity from the cf airway epithelium results in abnormal regulation of ion transport mechanisms leading to a reduced volume of airway surface liquid (asl) producing mucus dehydration and decreased mucociliary clearance, both hallmarks of cf lung disease. the consequences of these physiological alterations in the lung eventually result in chronic bacterial infection, mostly notably pseudomonas aeruginosa, and exaggerated neutrophil-mediated inflammation, eventually leading to airway obstruction and death. a logical therapeutic strategy for cf lung disease would restore cftr function to the airway epithelium and gene transfer strategies remain one approach towards this goal. at present, the regions of the cf lung that would require cftr expression for restoration of normal physiological function and reduction of disease symptoms are not well established. in normal human lung, the ciliated epithelial cells of the surface epithelium are considered to express cftr. however, restoration of normal ion transport functions to cf airway epithelium after delivery of cftr exclusively to ciliated cells has not yet been achieved to test this hypothesis. the possible involvement of the submucosal glands in the pathogenesis of cf lung disease suggests that cell-types other than ciliated cells may also require cftr expression for normal function ( ). however, the predominance of ciliated cells in the upper and lower airways especially in cf infants who have not developed chronic airway inflammation, and the accessibility of the ciliated surface epithelium to intralumenal-delivered therapeutics identifies ciliated cell-types as attractive targets for cf lung therapeutic strategies. a critical factor for cf gene transfer strategies has been overcoming the low transduction efficiency for intralumenal delivered vectors. there are several extracellular barriers to gene transfer vectors in human airway that may result in inefficiency of gene transfer, including: the mucociliary clearance system; the glycocalyceal barrier; the absence of most viral receptors from the airway lumen; and, the slow rate of lumenal endocytosis in airway epithelial cells. although strategies to circumvent these barriers with commonly available vectors are under investigation, i.e., retargeting vectors to new apical surface receptors, transiently opening tight junctions or using new serotypes of the common vectors, the results so far published have not shown significant improvement. novel vector-types are also being assessed for airway gene transfer that may be more efficient at breaching epithelial cell apical barriers. for example, sendai virus, human coronavirus e, and lentiviral vectors pseudotyped with ebola virus or sendai virus envelope proteins have shown improved gene transfer efficiency to airway epithelial cells in vitro and/or in vivo ( ) ( ) ( ) ( ) . however, the usefulness of these vectors remains to be determined. we have chosen to investigate the utility of the human parainfluenza viruses (piv) as gene transfer vectors since these viruses exclusively infect ciliated cells of the human airway epithelium after intralumenal delivery. to test these vectors, we used an in vitro model of human pseudostratified, mucociliary airway epithelium (hae) that recapitulates the morphologic and phenotypic characteristics of the in vivo human airway epithelium. recent studies have revealed that this model system displays the phenotypic differences that occur between cf and non-cf airway epithelium, i.e., reduced chloride ion transport, hyperabsorption of sodium ions, the failure to regulate the depth of the asl, and, the dehydration of secreted mucus that results in cilial dysfunction and mucostasis. we have previously shown that human recombinant parainfluenza virus (hpiv) efficiently infects human ciliated airway epithelial cells from the lumenal surface. to test our hypothesis that cftr expression in ciliated cells may restore normal physiological function to cf epithelia, we generated a hpiv that expressed cftr (hpiv-cftr) and systematically tested the effects of cftr expression in cf hae. western analyses performed hrs after apical surface inoculation of cf hae with hpiv-cftr, hpiv expressing gfp (hpiv -gfp) or vehicle control alone showed that only hpiv-cftr produced expression of fully glycosylated "band c" cftr. immunodetection of cftr protein in histological sections of cf hae inoculated with hpiv-cftr localized cftr to the apical surface of ciliated cells in regions corresponding to the microvilli of the ciliated cells. to determine whether cftr expression in cf ciliated cells resulted in chloride ion channel activity, cf hae were assayed in ussing chambers after viral inoculation. compared to control cf hae (vehicle or hpiv-gfp treated), inoculation with hpiv-cftr resulted in a significantly increased forskolin-mediated activation of cftr chloride channel activity [ . ± . _a/cm for control (n = ) and ± . _a/cm for hpiv-cftr (n = )]. to determine whether this level of cftr expression in ciliated cells was sufficient to restore the altered fluid transport of cf hae, we assessed the regulation of asl depth by measuring the rate of absorption of liquid across the apical surfaces of cf hae hrs after inoculation with vehicle control alone or hpiv-gfp or hpiv-cftr. in control cf hae, the asl stabilized at . ± . _m (n = ), a depth associated with decreased mucus transport. however, in cf hae inoculated with hpiv-cftr, liquid absorption ceased at a depth of . ± . _m (n = ), a depth similar to that maintained by normal hae ( . ± . _m, n = ). a hallmark of cf lung disease is mucostasis, a parameter that is recapitulated in cf hae. therefore, to determine if the restoration of fluid transport to cf hae by cftr expression in ciliated cells was sufficient to allow the transport of mucus secretions, we measured the capacity of cf hae to transport mucus after inoculation with hpiv-cftr. for control cf hae, rotational velocity of mucus transport was low with little or no mucus transport ( ± _m/sec, n = ) compared to significant mucus transport in cf hae inoculated with hpiv-cftr ( ± _m/sec, n = ). therefore, hpiv as a gene delivery vehicle has circumvented a major barrier to the effective use of gene transfer vectors, i.e., the restricted uptake of vectors from the apical surface. we have demonstrated that expression of cftr in ciliated cells of cf hae resulted in restoration of the ability of the epithelial cells to regulate the depth of the asl to levels that were sufficient for effective mucociliary transport. whether, expression of cftr in ciliated cells in the airway epithelium in vivo will be sufficient to restore these physiological indicators of cf lung disease remains to be determined. since hpiv-cftr is the most efficient and efficacious vector that we have so far tested for cftr gene transfer and results in correction of pathophysiological consequences of the cftr defect, efforts will be directed at generating derivatives of hpiv vectors that can be safely delivered to the human airway in vivo. the genetic etiology of human respiratory diseases is being increasingly emphasized as a means of better understanding disease pathogenesis, with the ultimate goal of improving preventive strategies, diagnostic tools and therapies. considerable effort and expense is currently being expended in attempts to detect genetic loci contributing to both 'simple' (single-gene) and 'complex' human diseases. association and linkage studies comprise the two dominant strategies, the former aiming to find disease predisposing alleles at the population level and the latter focusing on familial segregation. although both strategies have compelling strengths, association analyses are currently more widely conducted and likely to spread even further in the future, particularly in the pharmacogenetics domain. facilitating these studies are technical developments in molecular genetics and in the use of gene-specific variants derived from the human genome. in addition, extensive catalogues of anonymous dna sequence variants across the human genome have begun to be compiled. some large-scale, population-based human samples have been or are expected to be collected (e.g., epic, isis, million women study, mrc/wellcome trust biobank uk), and the use of dna variants in drug development is expanding. the coupling of high-throughput molecular technology, large numbers of genetic variants, and large samples offers some new opportunities for understanding the etiological basis of many diseases, including cystic fibrosis (cf). modifier genes are classically defined as genes that have small quantitative effects on the level of expression of another gene. studies of modifier genes therefore attempt to detect the sometimes subtle modulation of the main effects of a major locus regulating a disease (the cf transmembrane conductance regulator [cftr] gene in the case of cf). such effects may modify disease severity and progression. a broad spectrum of disease severity exists in cf, and it has become increasingly clear that cftr genotype does not account for the wide variance in the clinical spectrum of cf phenotype. many family-based and population-based study designs are possible to attempt detection of modifier genes in cf. this symposium will review the statistical concerns related to genetic studies of modifier genes and will discuss study design issues related to the investigation of modifier effects in gene discovery projects. university of western australia. phenotypic heterogeneity of cf lung disease is not explained by intragenic mutations in cftr, and likely reflects genetic heterogeneity at other loci, plus environmental effects. a multi-center study of gene modifiers for cf lung disease is underway to identify non-cftr genetic contributions to the variability of cf lung disease. the study is designed as a case-control association study in unrelated patients with the same cftr genetic background (homozygous ∆f ). the analysis focuses on comparing the prevalence of alleles and genotypes in patients who have "severe" (lowest th percentile for age) versus "mild" (highest th percentile) lung disease. we have enrolled subjects, who are segregated into three groups: ) young subjects with "mild" lung disease (age - yrs), ) older subjects with "mild" disease (>age yrs), and ) subjects with "severe" disease (age - yrs). longitudinal analysis of spirometric measures covering five years up to enrollment (average ( measures per patient) has been performed by a mixed model analysis with empirical bayes estimates of individual slopes and intercepts. the intercepts (at birth) for the fev (% pred.) for these three groups are , , and (% pred.), respectively. the rates of decline (%/year) for the two groups of mild subjects were similar (- . and - . ), whereas the severe cohort had a more rapid rate of decline (- . ). the mild versus severe groups also differed in body mass index (bmi). the bmi z-score was nearnormal (- . and - . ) for the younger and older mild lung disease cohorts, respectively, but much worse (- . ) for severe patients. the prevalence of pseudomonas aeruginosa during the years up to enrollment was similar for all three groups (- - %). thus, cf patients in this protocol are characterized as "mild" and "severe", and other key clinical variables are defined. genotypes in subjects have been completed for selected snps in genes previously studied as modifiers of cf lung disease, including alpha-antiprotease, ace, β-adrenergic receptor , gstm , gstm , gstp , il- , mbl , nos , tnfβ and tgfβ . results show a striking association of snps in tgfβ with severe lung disease (p = . ; odds ratio > ). snps flanking tgfβ show no association, which implies that tgfβ is the causative modifier gene, as has been reported for asthma and copd. ongoing studies are testing other genetic variants in several pathogenic cascades in cf related to infection and inflammation. *reporting for the gene modifier study group. supported by cff knowle a , nih r hl , and nih r . garry r. cutting, md institute of genetic medicine, johns hopkins university, baltimore, md usa genetic, environmental, and stochastic factors are the three major factors contributing to phenotype variation. for single gene disorders the nature of the defect in the disease-causing gene is a major contributor to variation. however, in most single gene disorders, including cf, the disease-causing gene is not the sole determinant of variation indicating an important role for the other major factors. this point is highlighted by the observation that cystic fibrosis patients with identical cftr genotypes have considerable variability in the severity of lung disease ( ). study of related affected individuals provides a method to dissect the contributions of genetic background, environmental and stochastic factors to phenotype variability ( ). this is achieved by comparing the degree to which monozygous (mz) twins, dizygous (dz) twins and siblings are similar to each other for selected traits. if pairs of mz twins are found to be highly correlated (e.g. if one member of the twin pair has a trait; the other twin also has the trait) then one could attribute this similarity to genes (since they share % of their genes) or the environment that they share (since they are together in utero and grow up together in the same household). if dz twins are examined for the same trait and found to have lower rates of correlation than mz twins, then genetic factors can be implicated since dz twins share only % of genes but have the same degree (or nearly so) of shared environment as mz twins. on the other hand, similar high levels of correlation among mz and dz twins implicates the environment that they share. finally, siblings share % of genes like dz twins but have a lower degree of shared environment since they are born at different times (although still raised in the same household). thus, siblings provide an opportunity to test the role of environment on genetically similar individuals. a trait that shows similarly high levels of correlation among twins but a lower level of correlation among siblings would implicate environmental rather than genetic factors for the trait under question. the european twin and sibling study has demonstrated the utility of this approach. this group reported that genetic factors independent of cftr can influence phenotype severity by altering chloride secretion ( ; ). to increase our understanding of the role of these major factors, a cf twin and sibling study is being conducted in the united states. over participating centers located throughout the united states have contributed patient information to the study. at the current time, pairs of monozygous twins, pairs of dizygous twins, and sibships have been enrolled into the study. several themes are emerging from the u.s. twin and sibling study. first, variation in pulmonary disease, as measured by fev % predicted is minimal among twins. in fact, mz and dz twins show similarly high levels of correlation for this parameter suggesting that genes other than cftr have a limited role in differences in lung disease severity. furthermore, lower levels of correlation for fev % predicted among siblings both by cross-sectional and longitudinal analysis supports the concept that genes are not a major factor, but that sharing of environment plays an important role. this conclusion is consistent with observations from cftr genotype-cf phenotype studies over the past decade. however, the small number of enrolled dz twins in both studies reveals a limitation in this approach. analysis of a larger collection of siblings will allow rigorous evaluation of the role of environment, and the specific factors therein, that influence pulmonary outcome. second, evaluation of gi manifestations of cf in twins and siblings has revealed an important role for genetic factors. previous studies have documented familial clustering of meconium ileus (mi) and a locus for this trait has been mapped on chromosome ( ) . mz twins display a high rate of concordance for mi ( %) while dz twins have a lower rate ( %) strongly implicating genetic factors, as has already been noted. liver disease and cf-related diabetes mellitus follow the same pattern suggesting that genetic factors also underlie these traits. third, some twins and siblings exhibit considerable difference in the age that they acquire bacterial infections. most impressive is the observation that mz twins can differ by several years in the initial age of acquisition of pseudomonas aeruginosa or burkholderia cepacia. since mz twins share % of genes and environment, the difference in acquisition of these bacteria suggest a substantial role for stochastic factors. in summary, study of affected twins and siblings permits an assessment of the contribution of genetic factors to trait variation. this is an important step prior to the investment of considerable effort to identify modifier genes for particular traits. genomic analysis is now widely used to probe the basis for disease and how disease may be expressed in individual patients. the attempt to identify specific disease-associated mutations, polymorphisms, or transcript patterns currently underpins much of our thinking about disease mechanisms, but the overall complexity of genebased data and the limited amount of functional annotation continue to present challenges in the interpretation of these experimental results. going forward, it will become increasingly important to look beyond single genes to genetic networks and ultimately to the integration of these data with quantitative measures of biological function at the system level to discover key molecules that can explain and classify clinically-relevant phenotypes. we have taken a systems biology approach to discovery that is based on the concurrent analysis of gene expression in combination with two other large-scale data streams: metabolomics and quantitative histomorphometry. metabolomics uses mass spectrometry to measure changes in the relative abundance of endogenous small molecular weight biochemicals (e.g. amino acids, sugars, lipids) present in cells, tissue and biofluids. quantitative histomorphometry uses computationally intensive image analysis of normal and pathological histology slides to quantify tissue structures and identify phenotypic changes in response to toxicity or progress of disease. the assessment of the integrated cellular state using biochemical profiles and quantitative tissue analysis is an important complement to the large-scale molecular state of a biological system. we are currently applying this approach to the study of drug-induced liver injury and to the discovery of novel drug targets and biomarkers for type ii diabetes. hepatotoxicity: acetaminophen (apap) overdose remains one of the most common causes of hospital admissions for acute liver toxicity. the initiating mechanism is believed to involve hepatic metabolism of apap to a reactive oxidative species, napqi, which subjects the liver to oxidative stress, resulting in depletion of glutathione. oxidative stress is also believed to play a pivotal role in the hepatotoxicities of a wide range of chemical and pharmaceutical agents. therefore, apap may serve as a useful model toxicant to elucidate the mechanism of liver injury. single oral doses of apap producing gross liver pathologies ranging from no histopathological change to frank necrosis were administered to rats. tissue and biofluid samples were analyzed by lc/ms (tof) with an esi source in either positive or negative mode. mass spectra at each retention time were matched to a library of known standards using proprietary software and linked to metabolic pathways. analysis of individual metabolites at the tissue level revealed major perturbations in pathways associated with known injury and repair mechanisms -e.g. depletion of glutathione and cystathionine (oxidative stress), decreases in nad and various nucleotides (nucleic acid repair), and a decrease in cdpcholine (phospholipid turnover). several of these decreases in liver metabolites were also reflected in urine, suggesting that they might serve as useful biomarkers for early detection of liver disease. time and dose-related changes were also observed in various other biochemicals that were not predicted by current knowledge. type ii diabetes: as a disease model, obesity, insulin resistance and hyperglycemia were induced by feeding c bl/ mice high-fat diets. responses were monitored over weeks. high-fat fed obese mice were chosen that demonstrate either hyperinsulinemia or symptoms of type ii diabetes and were then compared to control mice fed a normal diet and demonstrating normal weight gain, fasting plasma insulin levels and fasting blood glucose levels. the mice selected at each time point were sacrificed and tissues (liver, skeletal muscle, pancreas, white adipose tissue, heart, kidney, brain, testes, skin, tongue, stomach, intestine, spleen, and tongue) were harvested and frozen in liquid nitrogen until processed for rna isolation and metabolic profiling with adjacent sections fixed in formalin for histological processing. by combining data sets, we are able to identify novel and important drug targets and biomarkers that would be overlooked in a conventional target discovery processes. this unique approach to the discovery of novel drug targets and biomarkers is based upon the hypothesis that tissue structure, which is at the core of most diagnosis, holds the key to unlocking the link between genes, biochemicals, function, and disease. through correlations, differential analysis, and knowledge of gene function and pathways, we are able to identify biologically relevant drug targets and biomarkers that support this hypothesis. co-variant analysis of gene expression and tissue feature data identified different type of genes than were found using gene expression analysis alone. comparing the ontological classifications of the genes that were up or down regulated greater than -fold, and genes were closely correlated to liver features, fasting glucose, or insulin at sacrifice revealed, but there were only genes in common. comparing the molecular function and biological processes ontological classifications associated with these two sets also shows distinct differences in the classes of gene families represented in the individual groups. many of the genes identified through co-variant analysis were altered < -fold and therefore would have been lost in conventional analyses. in conclusion, these results indicate that a systems biology approach combining gene expression analysis with biochemical profiling and quantitative tissue analysis can help identify disease mechanisms, drug targets and biomarkers that other gene-only approaches often overlook. we are indebted to diathegen (athens, oh) who is a strategic research partner in the diabetes target discovery program. this research was funded in part by the national institute of standards and technology -advanced technology program (nist atp), award # nanb h . pseudomonas aeruginosa is an interesting organism since it provides a prototype for a nutritionally diverse, free living organism that is found in many microenvironments such as soil, water, plants, and animals, and because it is a common cause of hospital-acquired and cystic fibrosis (cf) patient infections, in part because of its high intrinsic resistance to many antibiotics. the most important recent development in p. aeruginosa research was the publication of the genome sequence in august (stover, kc, et al. nature : - ). however a genome sequence is only as good as the usage we put it to. to permit us to capitalize on and reach beyond the genome sequence with maximum efficiency, we and other laboratories have been developing genomic tools. these include bioinformatics, whole genome knockout and gene fusion libraries, and microarrays. a major bioinformatics resource has been provided by the pseudomonas genome web site (www.pseudomonas. com), which is an attractive, information rich resource, maintained with funding from the cystic fibrosis foundation. this site includes all known information about each of the genes in the prototype strain pao , permitting investigators to find genes of interest through key word searching or browsing through the genome. other resources are being continually developed, e.g. the recent development of psortb has permitted a prediction of the location of every gene in the genome and especially key secreted or surface genes that may be involved in pathogenesis. using the tactic of fusing fragments of the p. aeruginosa genome to an n-terminal signal sequence lacking alkaline phosphatase gene, we have confirmed several hundred of these predictions. whole genome knockout libraries are of tremendous value in pathogenesis. for example one can use such libraries to determine if a particular mutation attenuates virulence. in addition to the established library, we have made a knockout library that consists of random gene fusions to the promoter-less luxabcde cassette. this library is not as comprehensive as the library published by jacobs et al (pnas usa : - ; , but creates in addition to knockouts, a reporter system for expression of the particular gene into which the lux cassette, encoding light production, has been inserted. thus one can also study regulatory events related to pathogenesis. by far the most utilized genomic tool has been microarrays, which permit the analysis of the expression of every gene in the genome simultaneously. many of the known regulatory events in pseudomonas that are connected to pathogenesis are being probed through the use of microarrays. as the tool-kit for post-genomic studies grows, we are making rapid in-roads into understanding how this complex organism responds to life outside and in the host. funding through the functional pathogenomics of mucosal immunity program grant, provided through genome prairie and inimex pharmaceuticals is gratefully acknowledged. in the chronic airway infections that afflict cystic fibrosis patients p. aeruginosa live in matrix-encased groups known as biofilms ( , , ) . biofilms are often thought of as communities of bacteria because the cells occupy a defined territory, they can exhibit coordinated behavior facilitated by communication, and because key biofilm characteristics (like their marked resistance to killing) cannot be generated by the bacteria living as individuals ( ) . the functioning of many types of biological communities is enhanced by diversity. diversity is beneficial because the presence of diverse subpopulations increases the range of environmental conditions in which some community members will survive, or even thrive ( , , ) . we have observed that growing wild-type p. aeruginosa in biofilms for - days produced considerable colony morphology variation. planktonic (free-swimming) batch cultures grown to log, stationary, or late stationary phase produced no variants. the variant phenotypes we studied were heritable, suggesting genetic changes produced them. a prime candidate for mediating such variation is reca. this protein is involved in phase variation and adaptive mutation, both which can be triggered by specific environmental cues ( , ) . inactivation of reca dramatically reduced biofilm-induced colony variation and this defect was complemented with chromosomally inserted reca. the involvement of reca, which could mediate genetic change anywhere in the chromosome, led us to hypothesize that biofilm-induced diversity could extend to other functions. to test this, we examined the effect of biofilm growth on additional phenotypes, and found that biofilm growth produced heritable variation in several other traits. furthermore, some variants exhibit specialized functions in biofilms. isolates from cf airway infections have also been observed to undergo phenotypic diversification ( ) . our data suggest that shortterm biofilm growth can rapidly produce genetic diversification of p. aeruginosa. this diversity may increase the stability of biofilm communities and speed the evolution of the resident bacteria. despite advances in the assessment and management of pain in children, there are still subpopulations of pediatric patients for which pain and its management has not been well studied. children with cystic fibrosis (cf) are such a population. pain and discomfort associated with cf may be secondary to direct involvement of the organs (e.g., abdominal cramping and pleuritic chest pain), physiologic changes that may occur related to organ involvement (e.g., headaches from hypercarbia and sinusitis, musculoskeletal pain), or due to treatment such as procedural pain. historically, pain management has not been included in an overall treatment plan, and commonly used analgesics such as opioids have long been avoided in the cf population because of the fear they may compound respiratory difficulties. only a few studies have focused on assessment or management of acute or chronic pain symptoms in children and adolescents with cf. the first published report of chronic pain in a relatively older and sicker population with cf was conducted via a retrospective chart review of patients who died and patients who were referred to a pain treatment service. this study demonstrated that patients with cf primarily complained of chest pain and headaches but back pain, abdominal pain, and limb pain also occurred regularly. in the patients referred for treatment of chronic pain, safe and effective management with opioid analgesics was achieved. another study, focused on musculoskeletal complications experienced by pediatric patients with cf, found through clinical assessment, serology, and bone scan that of ( %) patients had joint pain. finally, pain and dyspnea have been shown to be a common symptom at the end of life in children with cf. opioids were effective in managing these symptoms in terminal patients. although these studies provide evidence of the type and nature of pain associated with cf, to our knowledge, there has not yet been any published report of the multidimensional experience of acute and chronic pain in a broad, un-referred population of children and adolescents with cf. unrecognized and untreated pain in children with cf may be associated with increased morbidity, poorer prognosis, and decrements in their overall quality of life. therefore, research to characterize the full spectrum of the pain experience from the patient's point of view may help to identify areas where pain management intervention is needed. our research group initiated a study, the dch pain in cf study, to characterize the experience of acute and chronic pain and its impact on the quality of life in children with cf. forty-six children ages - (m = . years; % male) with cf were recruited at one university-based and two community-based pediatric cf clinics. a previously validated survey instrument collected standardized data regarding the location, frequency, intensity, duration, associated emotional distress, and perceived functional limitations of chronic pain, and the intensity of procedural pain. findings demonstrated that chronic pain was a regular experience for children and adolescents with cf, occurring at least weekly in % of children. commonly reported locations of pain were: abdominal/pelvic region pain ( %); chest pain ( %); head pain ( %); leg pain ( %); and arm/hand pain ( %). most children ( %) reported the intensity of their pain as being mild and lasting for "less than hour" ( %). however, a small subgroup of patients ( %) reported pain lasting onehalf day or longer and % reported experiencing moderately intense pain. there were no significant differences in pain reports by age or gender. pain location and frequency were related to pulmonary function and perceived limitations. patients with chest pain had lower fev % (p<. ) than those patients with no chest pain. these patients also had more perceived functional limitations due to pain (p<. ) than patients without chest pain. patients with weekly pain had more perceived functional limitations (p<. ) than patients with less frequent pain. the majority of patients reported experiencing mild procedural pain. a small number of patients reported experiencing severe pain from blood draws ( %), picc line placement ( %), gastrostomy tube placement ( %), pfts ( %), throat cultures ( %), and chest physiotherapy ( %). patients who experienced higher intensity of pain from picc line placement were more likely to also experience higher intensity of chronic pain (p<. ). patients who experienced higher intensity of pain from patrick hubbard, r.n., b.s.n the purpose of this study was to develop a web-based education program tailored to patients with cf who may be experiencing pain, and to investigate, via the website, the pain experiences of those patients by studying their pain reports, disability, and coping strategies. this study was a cross-sectional survey of cf patients. recruitment efforts focused on informing patients of the website and research study. a sample of participants was recruited. three separate questionnaires were employed: a demographic questionnaire, the pain disability index, and the pain response inventory. approximately half of the sample reported experiencing pain episodes on a daily basis lasting two hours or less. the average intensity of a pain episode was reported to be in the moderate range. participants reported pain disability highest in areas of recreation, occupation, and social activities. the most commonly used coping strategies included active and accommodative coping techniques such as problem solving, acceptance, and self-encouragement. pain management is clearly a problem for some young adults with cf. this study provided information about the areas of disability caused by pain, as well as common coping strategies utilized by patients. the findings of this study provide direction for the future of pain management in cf patients. pfts and chest physiotherapy were more likely to experience chronic chest pain (p<. ). the majority of children reported that medication ( %), rest ( %), relaxation ( %), heat or cold ( %), distracting activities ( %), and family/friends ( %) provided some pain relief. twenty-four percent of patients reported taking no medication for pain. of those taking medication to treat pain, acetaminophen, nsaids, or a combination of the two was used. none of the patients reported taking opioids for pain management. it was interesting to note that all the patients who reported head pain were taking some medication, mostly nsaids. children with cf appear to experience pain, especially abdominal, chest, and head pain, on a regular basis. the finding of frequent chest and head pain is consistent with the work of ravilly et al. the majority of patients in our study described their pain as mild and of relatively short duration, however a significant number of patients seem to have pain of long enough duration to potentially have a negative impact on daily functioning. a good example of this is that children with chest pain have reduced fev % and more perceived functional limitations as a result of the pain. our study provides further insight into the experience of pain for patients with cf with mild to moderate disease who were seen as outpatients. it is important to note that even in a less sick population, problems with chronic pain were very common. evidence suggests that pain assessment should be a routine part of the clinical care of children and adolescents with cf. further research is clearly needed to better understand the sources of pain and how best to provide relief. pharmacological interventions were minimal in this population and possibly contribute to the under treatment of pain. effective pain management may serve an integral role in reducing morbidity and improving quality of life in children with cf. figure . relationships between pain, coping, and disability. assessment of health related quality of life (hrqol) in children and adolescents with cystic fibrosis (cf) is important to better understand disease and treatment-related factors that impact function and well-being, and to evaluate the effectiveness of therapies and methods of drug delivery. limited data exist concerning the hrqol of youth with cf, although the recent development and validation of cf specific measures of hrqol for children and adolescents will likely increase the use of qol measurement in clinical trials. measurement of how symptoms, such as pain, impact on hrqol is essential, but these data have not yet been reported in children and adolescents with cf. there are preliminary data that patients with cf do in fact experience and/or suffer from pain related to their disease or complications of their disease. in the only published study of chronic pain in cf, headaches, chest pain, back pain, abdominal pain, limb pain, arthritis, and neuropathic pain were experienced by the majority ( %) of an older sicker pediatric population with cf. it is well documented that chronic pain can have a significant influence on hrqol of youth by impacting a variety of normal daily activities, such as school attendance and social interactions as well as psychological functioning . in addition, pain may limit a child's ability to take deep breaths, cough, and tolerate pulmonary toilet exercises that are so impor-tant for patients with cf. therefore, our research group initiated a study, the dch pain in cf study, to examine the impact of pain on the hrqol of children and adolescents with cf. a convenience sample was recruited from the cf center at doernbecher children's hospital (dch). the study sample consisted of children and adolescents with cf ( males, females) and their parents; n = child subjects (ages - years) and n = teen subjects (ages - years). participants completed two survey instruments to measure hrqol and pain characteristics. the cystic fibrosis questionnaire (cfq) , is a disease-specific quality of life measure, with different versions for children, adolescents, and parents. the cfq teen version includes subscales (the child version includes subscales) to assess cf specific symptoms and perceptions of hrqol. higher scores indicate better quality of life. pain characteristics were measured using a self-report questionnaire assessing frequency, location, duration, intensity, and emotional upset due to pain. adequate reliability and validity have been reported in the investigators' previous research with this pain questionnaire. in addition, to assess disease severity, forced expiratory volume in second (fev percent) was obtained from the most recent clinic visit. as expected, findings demonstrated that having frequent pain (at least once/week) produced decrements in the hrqol of children and adolescents with cf. specifically, children with frequent pain reported increased treatment constraints compared to children with less frequent pain. adolescents with frequent pain, on the other hand, reported negative impact on their hrqol in many areas including physical and role functioning, vitality, eating disturbances, respiratory and digestive symptoms, as well as overall perception of their health, compared to adolescents with less frequent pain. cf specific symptoms, in particular, respiratory and digestive symptoms, were associated with more frequent, longer lasting, and more intense pain that is often in more than one location for both children and adolescents. several specific pain locations were related to decreased hrqol. in particular, having chest pain was related to decrements in physical, emotional, and role functioning, increased treatment constraints, respiratory symptoms, and poorer overall health perception. the presence of abdominal/pelvic pain was associated with more digestive symptoms. disease severity was also associated with hrqol; a significant positive association was found between fev percent and respiratory symptoms. children and adolescents with cf, who are seen in an outpatient setting, experience frequent pain that is associated with decrements in their hrqol. in the dch pain in cf study, adolescents experienced the greatest negative impact on their hrqol from frequent pain. moreover, patients experiencing chest or abdominal pain were particularly at risk for having more cf specific symptoms and reduced hrqol. our study highlights the need for future research to identify methods to reduce pain and improve quality of life, particularly in adolescent patients with cf. effective pain management is likely to include both pharmacological and psychological interventions. the role of biobehavioral or psychological interventions for cf has received limited research attention. a recent systematic review identified only eight studies of psychological interventions for cf, and none were focused on pain relief. however, the three studies that used biofeedback, music, and massage therapy to assist with physiotherapy found some effectiveness. such interventions may also play a potential role in providing pain relief to these children and adolescents. pain relief should constitute an important treatment goal for youth with cf, as adequate pain treatment is expected to lead to broad improvements in health-related quality of life. pain in patients with cystic fibrosis (cf) results from discomfort arising from the disease (e.g., chest pain, gastrointestinal complaints, headaches, renal stones, arthritis, and end-stage disease), and caused by therapies (e.g., chest physiotherapy, intravascular access, and surgery.) characterization of an individual's pain is necessary in order to treat it effectively. types of pain include acute, recurrent, and chronic. patients' experience of pain is dependent not only on its physical nature, but also on their emotional, psychological, and spiritual states. social factors usually contribute to the impact of pain in patients' lives. evaluation of pain involves review of physical and psychological factors. patients should be asked regarding the onset, duration, and frequency of the pain. the nature of the pain can be described by its location, distribution, quality, and intensity. psychological factors that impact the experience of pain include anxiety, depression, substance abuse, and certain behaviors, e.g., reinforcement by caregivers who attend to the patient when pain is reported. in order to gain insight into how patients perceive their pain, they should be queried about their beliefs regarding the reason for the pain, its meaning, when it is most bothersome, what would change when the pain improves, and their expectations of the therapy for pain. hypnosis is useful in the treatment of pain because it can affect perception of pain, and psychological interactions with the experience of pain. hypnosis can be defined as an altered state of consciousness, characterized by a heightened state of suggestibility, which allows instruction and alteration of perception based on patients' specific needs. hypnosis may block the pain signal by allowing the triggering of a substitute signal through focus on an alternative thought, which usually is pleasant. hypnosis can reduce acute pain, chronic pain, depression, and anxiety, and can decrease dependence on pain medications. it is more effective in controlling pain than placebo, relaxation, distraction, or alpha-feedback. self-hypnosis for the control of pain can be taught by a qualified hypnotherapist or members of the cf care team who have been trained in hypnotherapy, e.g. physicians, nurses, social workers, or psychologists. many patients can be instructed in or sessions, which require - minutes. some patients need several additional sessions, especially if there is a large psychological component to their presentation. steps for instruction in hypnosis can include a pre-hypnotic interview, induction, deepening, hypnotic suggestions, and trance termination. techniques that can be used in hypnosis to help improve patients' mastery of their pain experience include egostrengthening, dissociation, time distortion, amnesia, positive visualization, and validation. self-hypnosis can be used to reduce the intensity or develop complete anesthesia at the site of the pain. the pain sensation can be altered, e.g., changing a sharp pain to a dull, warm, or tingling sensation. patients also can learn to relocate pain to a part of the body where it is not as bothersome. the patient was an -year-old with cf who became agitated whenever he required phlebotomy, which typically required minutes, as he became very upset, cried, and struggled. the patient explained that he was frightened by the thought of a needle in his arm. use of a topical anesthetic was not helpful, probably because it did not alleviate his anxiety about the procedure. the patient was interested in learning how to use selfhypnosis to help himself. a single -minute instruction session was offered by his cystic fibrosis physician. hypnosis induction was achieved by rolling his eyes back as far as possible, inhaling deeply, and closing his eyes as he exhaled. deepening was achieved with the suggestion that the patient imagine being in a favorite place. the patient was coached to imagine what he might see, hear, smell, feel, and taste there. in order to promote dissociation, it was explained that the more he paid attention to his different senses the more relaxed he could become. to help with his comfort control during phlebotomy, the patient was given the hypnotic suggestion that he imagine his antecubital fossa was covered by a magic sleeve that reduced his perception of pain at that site. following hypnosis, the experience was validated when the patient was asked to pinch his antecubital fossa and reported no discomfort. he was congratulated for his success, thus providing ego-strengthening, and encouraged to use self-hypnosis as he saw fit, which reinforced his sense of self-mastery. subsequently, the patient was proud and almost eager to undergo phlebotomy with use of hypnoanalgesia alone. he reported feeling more confident. in later years the patient used self-hypnosis to reduce or eliminate headaches and abdominal pain, as well as to gain insight into how to cope better with stressful situations. hypnosis is best taught by those who have had specific training in hypnosis and hypnotherapy. instruction to uninterested patients is likely to be of little benefit because the efficacy of hypnosis is affected by the patients' desires. hypnosis sometimes can be used instead of medications for the treatment of pain, but oftentimes its optimal role is to augment the effectiveness of medical therapy. in conclusion, self-hypnosis for patients with cystic fibrosis can improve mastery of their response to the disease, cooperation with therapy, and comfort for patients, family members and the health care team. the primary defect in cfrd is insulin deficiency. abnormal glucose tolerance is present in the majority of adult cf patients because of fibrosis-related partial islet destruction. progression to overt diabetes, however, appears to require diminished function of the remaining beta cells, and this may be related to the same genetic defect that causes diminished beta cell function in type diabetes. insulin resistance must also play a role. in several different studies, insulin resistance has been reported to be decreased, normal, or increased. it is likely that these contrary observations have been related to different degrees of illness severity in the patient populations that were studied. why do we care if cf patients have diabetes? cfrd contributes to morbidity and mortality in cf. a prospective study followed pulmonary function for four years in patients separated at baseline by ogtt testing into three groups: normal glucose tolerance, impaired glucose tolerance and diabetes without fasting hyperglycemia. at baseline, the groups did not differ in age, weight, pulmonary function or bacterial colonization. the rate of decline in pulmonary function over the fouryear observation period was directly related to the severity of glucose tolerance abnormalities at baseline. pulmonary function deteriorated more rapidly in patients with igt than those with ngt, and patients with diabetes without fh had the greatest decline. when patients were grouped at baseline based on the amount of insulin secreted during the ogtt, the degree of pulmonary decline over the subsequent four years was directly related to the degree of insulin deficiency at baseline, suggesting a cause and effect relationship between insulin deficiency and cf clinical deterioration. insulin is a potent anabolic hormone that plays a pivotal role in carbohydrate, protein and lipid metabolism. insulin deficiency may contribute to morbidity and mortality in cf by promoting an overall catabolic state. mounting evidence supports the use of insulin to improve nutritional status and thus pulmonary function in cf. a retrospective survival analysis at the university of minnesota in found that while nearly % of cf patients without diabetes were still alive at years of age, less than % of those with diabetes reached this age. since , we have been aggressively treating all patients with fasting hyperglycemia with insulin. a new survival analysis was performed on patients followed at the university of minnesota over the last years. median survival time after a diagnosis of cfrd was . years, but after the first years the survival curve was less precipitous, suggesting that the patients dying in the first few years after diagnosis were probably sicker than the long-term survivors. median survival was . years for non-diabetic males, . years for diabetic males, . years for non-diabetic females, and . years for diabetic females. thus, the diagnosis of diabetes had a dramatic negative impact on survival in women but not men with diabetes. this did not appear to be related to bmi, genotype, pulmonary pathogens, steroid use, or pregnancy. women had worse pfts at the time of diagnosis. we hypothesize that gender-related hormonal factors may modulate the impact of diabetes on cf prognosis, with either a deleterious effect of estrogen or a beneficial anabolic effect of testosterone. holley f. allen, md department of pediatrics, baystate medical center children's hospital, springfield, ma cystic fibrosis related diabetes mellitus (cfrd) is different in etiology, course, and treatment than type or type diabetes. as life expectancy cf patients increases, so does the prevalence of cfrd. evidence to support standardized recommendations for screening and diagnosis of cfrd is just emerging; such evidence to support specific treatment modalities is nearly non-existent. cfrd occurs in cf subjects with pancreatic insufficiency, and increases with age, reaching a prevalence rate of over % in those over age ( ) . estimates of the prevalence of cf vary from center to center, likely related to the strategies used for screening and diagnosing cfrd. cfrd is caused by insulinopenia and is exacerbated by insulin resistance. initially it has an indolent course with absence or intermittent occurrence of the classic symptoms of hyperglycemia (polyuria and polydipsia) mostly during episodes of acute illness. ketoacidosis is extremely rare. clinical status has been shown to decline prior to the development of overt symptoms of diabetes ( ; ). to prevent the catabolic state which is present during insulinopenia, screening for abnormalities of glucose metabolism is advocated by most experts in the field. ogtt is the gold standard for detecting abnormal glucose metabolism. the issues around recommendation of universal ogtt screening for cf patients arise primarily because: )ogtt is felt to be a cumbersome test to perform, )intermediate results such as impaired glucose tolerance (igt) fasting< , -hr impaired ( - mg/dl) may normalize in subsequent tests, and ) no solid evidence shows that the asymptomatic cf patients who have diabetic glucose tolerance without fasting hyperglycemia (dgt), (fasting< , -hr> mg/dl) benefit from treatment. although other tests including hba c, fbg and actual bg are frequently used by physicians caring for cf patients ( ), none have consistently demonstrated the sensitivity required of a screening test. hba c has been in the normal range in %( ) to %( ; ) of subjects at the time of cfrd diagnosis. numerous groups have evaluated use of a combination of fbg and hba c and have reported that they are not reliable in identifying cfrd( ) ( ) ( ). annual ogtt screening for cf subjects over the age of has been advocated by several experts in the field ( ) . increasing numbers of u.s. centers are screening with ogtt. a cfrd consensus conference in ( ), however, did not recommend universal ogtt screening, but rather an algorithm based on random plasma glucose in asymptomatic subjects ongoing research showing benefit from early detection of cfrd is needed to justify universal ogtt screening. the goal of treatment of cfrd is to achieve optimal nutritional and clinical status by maintaining near normoglycemia, to avoid both short and long term complications. because cfrd is a disease characterized by insulin insufficiency, insulin is generally the treatment of choice. early in the development of cfrd, significant insulin secretory capacity remains. most problems (hyperglycemia, decreased protein synthesis) occur in the post-prandial state, suggesting a possible role for short acting insulin secretagogues. sulfonylureas have been used by some with success comparable to insulin early in the disease process ( ) . sulfonylureas bind with the sulfonylurea receptor (sur), which like cftr is a member of the atp-binding cassette superfamily of proteins. theoretical concern exists because of evidence that sulfonylurea like drugs bind to and inhibit the cftr. because of these concerns, sulfonlyureas should only be used by practitioners very aware of the risk benefit ratios. many insulin treatment regimes are available. the approach to insulin therapy needs to be tailored to the subject's preference, stage of disease, and ability to adhere to a particular regime. . maximum flexibility with best post-prandial glycemic control is achieved using rapid acting insulin administered at the time of food consump- dana s. hardin, m.d. cf related diabetes (cfrd) is a frequent clinical problem. the north american cf foundation has greatly improved the knowledge and management of this disease by providing research funding, hosting the cfrd consensus conference and providing educational opportunities and written materials for cf caregivers. although the current clinical initiatives are appropriate for many cf patients, particular patient subsets may have unique needs. our group has had experience with both research and clinical management of two such groups, hispanics with cf and pregnant women with cf, and will review what we have learned. a relatively unexplored method of treating patients with cfrd is the insulin pump. we have just completed a study of its use in cfrd and will share our findings as part of this seminar. we studied pregnant women and found that % developed gestational diabetes (gdm) by weeks gestation, % by weeks gestation and % by week gestation. our studies of the metabolic changes caused by pregnancy revealed that pregnant cf women have lower insulin levels, higher hepatic glucose production and more peripheral and hepatic insulin resistance than normal pregnant women. insulin secretion did not increase during pregnancy, hepatic glucose production increased and insulin resistance worsened. we believe that these defects contribute to earlier presentation and greater incidence of gdm in cf. we also measured protein turnover and learned that pregnant women with cf are more catabolic than normal pregnant women and that catabolism worsened during pregnancy. catabolism correlated with less gain in lean mass in the pregnant cf women. furthermore, despite markedly greater caloric intake, pregnant women with cf had significantly less weight gain than normal pregnant women. based on our findings, we recommend that women who are actively seeking pregnancy be screened for cfrd. during pregnancy, we recommend an oral glucose tolerance test (glucose load grams) be administered each trimester to any woman not yet diabetic. women who are diabetic should be treated with insulin to improve glycemic con-trol, improve weight gain and prevent muscle loss from on-going catabolism. although cystic fibrosis (cf) occurs less often in hispanics than in caucasians, clinicians have long recognized that the disease progresses more quickly, and the morbidity and mortality are higher, in this patient group. these observations have been supported by several research studies. we hypothesize that one major reason for increased morbidity and mortality in hispanic cf patients is increased glucose intolerance occurring at a younger age. we believe the earlier development of cfrd and glucose intolerance is secondary to underlying insulin resistance, a hallmark of type diabetes, which is worse in hispanics. our review of medical records supports our hypothesis and describes an incidence of normal random glucose levels in less than % of hispanic children and % of hispanic teens. data from moran et al collected at university of minnesota indicate normal glucose tolerance occurs in % of children and % of teens. to date we have performed measures of insulin secretion and insulin resistance using a frequently sampled ivgtt in hispanics with cf. we have found that insulin resistance is greater in these patients than in agematched caucasians. another possible factor affecting increased risk of cfrd in hispanics may be advanced clinical disease. these patients tend to be sicker. previous studies have identified a mechanistic role of cytokines, specifically tnf-_, in the development of insulin resistance, and increased hepatic glucose production. elevated levels of il- and tnf-_ have been associated with protein catabolism. we measured tnf-α in cf children and found higher levels in the hispanics (tnf-( = ± pg/ml) than in the caucasian children (tnf-_ = pg/ml). tnf-_ levels correlated with protein catabolism. furthermore we have found poorer longitudinal growth and weight gain in hispanic children, another indicator of worsened clinical status. the general recommendation for screening for cfrd is to wait until age . we recommend that all hispanic carol brunzell r.d., l.d., c.d.e. maintenance of a healthy body weight and optimal nutritional status are critical to survival for the patient with cf. with the additional diagnosis of cfrd, nearnormal blood sugar control is essential to normalize metabolism of macronutrients and to facilitate weight gain and weight maintenance ( ). these patients are at risk for diabetic microvascular disease, so optimal control of blood glucose is imperative for prevention ( , , ) . the risk of macrovascular disease necessitating the typical lowfat, low-sodium diet restrictions for type and type diabetes do not apply to the patient with cfrd as macrovascular complications appears to be non-existent at the present time ( , ) . however, one study showed that patients with cfrd had a % prevalence of elevated cholesterol and % prevalence of elevated triglycerides, suggesting that this recommendation may change as these patients live longer ( ) . the typical diet for cf, high-calorie, high-fat, and high-sodium remains essential to optimize weight and nutritional status. to meet the increased energy demands of cf many patients require some form of nutrition support in the form of oral or gastrostomy-delivered supplements. fluctuations in appetite make a rigid meal plan unrealistic. prescribing a complicated meal plan in addition to an already complicated medical regimen is not necessary today due to the dramatic improvements in insulin types and delivery systems. the need for simplicity is imperative. the cfrd consensus committee recommended matching insulin to carbohydrates for maximum flexibility as one approach to management ( ) the use of carbohydrate counting is a simplified way to superimpose some diet organization onto the traditional cf diet and works well with the use of rapid-acting insulin to optimize blood sugar control. the rationale for carbohydrate counting is based on the relative effects of carbohydrate, protein, and fat on blood glucose, with dietary carbohydrate having the most significant effect compared to protein and fat. ( , ) the advent of insulin pump therapy and the insulin lantus tm has made carbohydrate counting user-friendlier as many patients now administer rapid-acting insulin according to the amount of carbohydrates they plan on eating. one study showed excellent glycemic control in patients with type diabetes using unit of regular insulin for each grams of carbohydrate consumed. glycemic control was maintained over a wide range of carbohydrate ingested and was not affected by the glycemic index, fiber, caloric or lipid content of the meals ( ) . to confirm the approximate carbohydrate-to-insulin ratio, self-monitoring of blood glucose (smbg) data and food records for at least days are recommended, with pre-meal and frequent -hour post-prandial glucose values to verify the ratio. most patients require approximately . - . units rapid-acting insulin per grams carbohydrate ( ) . while using fixed doses of insulin may not be the best approach, patients receiving fixed doses of insulin need to be consistent with their carbohydrate in conjunction with the time action of the insulin injected ( ). currently, most patients who have cfrd without fasting hyperglycemia are not treated with insulin unless they are unable to maintain an appropriate weight or if pulmonary function is declining more rapidly than expected. at the university of minnesota, the strategy in diabetes not children ages and over receive an ogtt at least yearly, and random blood glucose levels should be checked during every acute illness. further study may indicate a need for pre-emptive treatment for these children who are at high-risk for cfrd. the insulin pump has been utilized for treatment of type diabetes, including pediatrics. the pump's benefits include adjustable basal coverage and the ability to give bolus insulin for all meals and snacks without giving an injection. cf patients require high caloric intake. constant carbohydrate intake (a method for regulating blood sugar when traditional insulin is given) is not recommended for these patients. for this reason, the insulin pump may provide ideal therapy for cfrd. we have just completed a study on the use of the insulin pump in cfrd patients. we found that as compared to multiple injections per day of subcutaneous insulin, the pump improved weight gain and lowered hemoglobin a c. reduced protein catabolism was also noted. all patients but one requested insulin pump therapy at the conclusion of the study. based on our findings we believe the pump is a beneficial treatment for cfrd. as part of this seminar, we will share our experience regarding basal and bolus settings for the pump. treated with insulin is to spread carbohydrates throughout the day to minimize large carbohydrate loads without reductions in total calories. patients obtain a blood glucose profile periodically at home to monitor status. patients with impaired glucose tolerance are at high risk of progressing to cfrd. the risks of igt in the general population with regards to cardiovascular disease do not appear to be of concern for the patient with cfrd ( , ). the risk of microvascular disease with igt is not known at present. unlike diet strategies used in the general population, which recommended weight loss and a low fat diet for people with igt, it is never appropriate to recommend weight loss or a restriction of fat and calories in cf patients. the only potential restriction may be to minimize the excessive consumption of regular soda or other sweetened beverages, and to try to maximize intake of more nutrient-dense foods to prevent weight loss. spreading carbohydrates throughout the day may also be beneficial. it is internationally recognised that urinary incontinence (ui) is a common problem for women with cf. there is a reported prevalence of between - % ( , , ) . this compares to an approximate - % prevalence of ui in healthy young women ( , ) . ui is also reported as a problem for male cf patients ( ) . there is an increased prevalence of ui described in children with cf ( ). a wide-ranging age at onset is reported, from to years of age ( , ) . whilst acknowledging the symptoms of urinary leakage in children and adults with cf, this report will focus on the possible mechanisms of ui in women with cf. the mechanism for preservation of continence is known to be a complex interaction of the muscles, fascia and ligaments of the abdomino-pelvic capsule and a competent urinary sphincter. in the general population risk factors for urinary leakage include female gender, obesity, genetic causes such as collagen weakness and conditions which increase intra-abdominal pressure (iap) such as constipation, vaginal delivery and persistent cough ( ) . leakage may be related to features of urge and/ or stress incontinence. urge incontinence occurs when leakage is accompanied or preceded by urgency, whilst stress incontinence occurs when the pressure within the bladder exceeds that of the maximum urethral closing pressure. it is necessary to perform urodynamic assessment in order to provide a diagnosis of genuine stress incontinence ( ) . the aetiology of ui in cf has not been fully evaluated. cough is recognised as the major cause of leakage in cf, where long spells of intense coughing are seen ( , , ) . normally in healthy subjects the pelvic floor muscle (pfm) contracts prior to voluntary abdominal muscle activity suggesting a pre-programmed response to iap ( ) . however in the presence of increased stress to the pelvic floor (pf), for example with chronic cough, the continence threshold may be exceeded. indeed chronic cough is recognised as a poor prognostic indicator for success of treatment of ui ( ) . coughing often occurs in a position of spinal flexion in which the urethral closing pressure is diminished. abnormal posture, which is increasingly reported in cf, may be implicated in the mechanism of urinary leakage ( , ) . increased kyphosis, over strengthened upper abdominals and a weaker transversus and multifidus are reported. it is recognised that the muscles of the abdomino-pelvic capsule play a role both as postural and respiratory muscles and therefore it is possible that dysfunction of any of these synergistic actions may contribute to the symptoms of urinary leakage ( ) . all of these factors may reduce the ability of the pf musculature to provide timely, co-ordinated and sufficiently strong contraction to maintain adequate urethral closing pressure in response to increased iap. invasive urodynamic studies are not routinely performed on cf women complaining of leakage as patients are surprisingly symptom tolerant and reluctant to be assessed ( , ) . in the absence of a definitive diagnosis it is therefore important to recognise other potential causes of ui in relation to both the muscular and neural aspects of continence. altered muscle metabolism in female athletes with cf is described and this may suggest differences in the quantity and quality of muscle in cf patients ( ) . autonomic neuropathy in cf is also reported and may disturb the autonomic control of bladder filling and voiding ( ) . the elevated inflammatory cytokines evident during pulmonary exacerbation may also have an effect on the muscle strength, as shown in-patients with copd ( ) . in summary, ui in cf is a widely reported problem and it may be considered over simplistic to suggest that chronic cough is solely responsible for the mechanism of leakage. multifactorial causes should be considered when posing the question of why ui occurs in cf. physiotherapist, spec. resp diseases, bsc, lund cf team, lund university hospital, lund, sweden many children, adolescents and young adults with cystic fibrosis (cf) are reported as having a hyperinflated chest, stiff intervertebral and costovertebral joints, bad posture, thoracic kyphosis and back pain [ ] [ ] [ ] [ ] [ ] [ ] . the reported wedging or compression of vertebrae [ , ] in adult cf populations may be caused by the ergonomic burden of the thoracic kyphosis in combination with osteoporosis [ ] . pulmonary hyperinflation is a response to the clogging and collapse of airways in an attempt to keep airways open and ventilated, i.e. to keep the functional residual capacity (frc) at the level of the closing vol-ume. most often the degree of hyperinflation follows the degree of obstruction. with hyperinflation, expansion of the chest is accomplished by contracted inspiratory muscles. expiratory flow and expired volume is controlled by eccentric contractions of inspiratory muscles and inspiration is accomplished by concentric contractions, but from an unfavourable point on the length/tension curve. the more hyperinflated the chest, the closer the inspiratory muscles get to active insufficiency. atelectasis occurs if airways are not kept open, resulting in decreased ventilation distribution, and further increased breathing frequency, minute ventilation and work of breathing. accessory muscles become involved in the process of hyperinflation. as hyperinflation increases and as ventilation becomes more insufficient these muscles are progressively more involved in the mechanics of breathing. accessory muscles originate from the chest and insert on the upper limbs, cervical region and skull. concentric contraction assists in the inspiratory movements required of the severely hyperinflated chest when upper limbs and head are fixed while sitting upright, leaning forward with elbows on a table and the head resting in the hands [ ] . all soft tissues shorten when not stretched to full range regularly. shortening of the musculoskeletal tissues in the hyperinflated chest results in the elevated and protracted shoulders, the thoracic kyphosis, cervical lordosis and the decreased mobility of the chest [ , ] that have been reported. a stiff, hyperinflated chest makes effective airway clearance therapy impossible. it has been shown that bad posture, thoracic kyphosis and back pain are partly reversible if properly treated [ , , , ] . but trying to regain what has been lost can be difficult. it is time-consuming, uncomfortable for the patient and an added burden to the cf care package. thoracic kyphosis due to wedged vertebrae cannot be rehabilitated, but rather increases the load on the adjacent vertebrae. therefore preventing a stiff chest and bad posture is of great importance. some simple physical exercises which maintains muscle length and strength and joint mobility as an incorporated part of the daily physiotherapy programme from the very beginning can make a big difference in the long run. physical loading in upright positions stimulates bone accretion, which may reduce the risk of osteopenia/osteoporosis and the risk of spontaneous fractures including wedging and compression of vertebrae. if physical exercise aiming to preserve physical function is included in the treatment, patients can maintain good posture and chest mobility, even if lung disease should progress. nowadays most cf centres recommend physical activity to their patients. however, children and adolescents with cf, despite having good lung function, have been shown to be engaged in less vigorous spontaneous physical activity than their non-cf peers [ ] . whether this is due to less spare time caused by time consuming therapy, to protective parents or health care system, or to the disease itself can be discussed. simply recommending patients to be physically active is obviously not enough. they probably need more active guidance and continuing encouragement to become and remain physically active. all children need physical activity to develop motor maturity and body awareness. if children with cf are given the opportunity to experience pleasure and satisfaction during physical activity, much may be gained for future outcomes. the different types of exercises that should be included in a physical exercise pro-gramme from the very beginning or as soon as possible are [ , ] : • chest mobility activities/exercises using movements around a vertical, sagittal and horizontal axis • shoulder mobility exercises, especially elevation and external rotation • muscle-strengthening activities/exercises, especially for postural muscles • working capacity training/activities/exercises exercises must never be uncomfortable. for infants, toddlers, children and adolescents, the activities/ exercises must be stimulating, enjoyable and age-appropriate. activities/exercises should always be individualized and provided at appropriate times in different settings. team sports/activities offer parts of what is to be included in the cf care and provide the added benefits of normal social interaction. good chest mobility allows effective airway clearance therapy. good posture probably reduces the risk of back pain and spinal complications. good posture contributes to positive body image and self-esteem. there is definitive evidence that nutritional status is the single most important factor that determines aerobic and anaerobic exercise performance in subjects with cystic fibrosis (cf) , , . however, diminished exercise capacity has been reported in subjects with normal nutritional status , . there are several factors that can limit skeletal muscle function in cf. this can be considered in the following categories: intrinsic abnormalities in the skeletal muscle cells of patients with cf have been demonstrated in the mitochondria of fibroblasts and leucocytes and include increased calcium concentration, lower nicotinamide adenosine dehydrogenase activity (respiratory chain enzyme complex) and a higher ph optimum of nicotinamide adenosine dehydrogenase . moser and colleagues have suggested that there is a muscle related abnormality in oxygen metabolism in patients with cf . studies utilizing -p magnetic resonance spectroscopy , during exercise have demonstrated inefficient and sub-optimal aerobic and anaerobic muscle metabolism in subjects with cf and good nutritional status. the mechanism of this remains unclear. the class of the cftr mutation may be a factor in determining muscle function . subjects with a cftr mutation belonging to either class i or ii have a lower peak aerobic capacity and anaerobic power compared to those with class iii, iv or v cftr mutations. in addition, compared to the dd angiotensin converting enzyme (ace) gene polymorphism, the ii polymorphism has been shown to be associated with less end organ damage in subjects with cf and better anabolic response to exercise training programs in healthy subjects . girls with cf have a lower peak aerobic capacity and anaerobic power . this gender difference is not unique to cf and have been attributed to differences in androgen levels, efficiency of fat metabolism, ratio of type i to type ii muscle fibres and total muscle mass. a significantly reduced breathing reserve is associated with reduced exercise capacity and habitual activity . while nutritional status is a major determinant of habitual activity , , increasing habitual activity as part of a training program can improve muscle . arthropathy occurs in up to % of patients with cf and limit activity. this can lead to deconditioning . although nutritional status is a primary determinant of muscle function, there are several other factors that can limit optimal performance. larry c. lands, m.d., ph.d. with increased life expectancy for cf patients, we are required to pay attention to organ systems beyond the respiratory and gastrointestinal systems, whose dysfunction can cause significant long-term morbidity. many authors have documented significant decreases in bone mineralization in cf patients - , with increased risk for fracture and pain. there are two distinct phases with respect to the skeletal system. in the growing child, especially in the critical peripubertal and pubertal periods, the goal is to achieve maximal bone accretion . maximal bone mass is achieved by the early 's and so a great emphasis must be made during the early growth phases. once adulthood is achieved, the goal is to maintain this bone bank. there are many influences on bone, including genetic, nutrition, body habitus, hormonal status, and exercise patterns . one of the most significant factors affecting ultimate bone mass is genetics. this is also true for patients with cf, where there is a significant relationship between a mother and child's bone mineral density . the other major way in which parents can influence bone mineralization is through the encouragement of a healthy diet. a variety of population-based studies have demonstrated the positive influence of vegetable and fruit intake and the supply of vitamins k and c, and minerals, such as magnesium . certainly, calcium and vitamin d intake are very important. sources of calcium include milk, tofu, and bok choy. these essential elements are particularly important during the phase of bone accretion in the first two decades of life . other factors that play important roles in bone accretion and maintenance include sex hormones and inflammatory factors. cf patients are at risk for delayed puberty and thus delay in bone maturation. at the other end of the spectrum, premature menopause and andropause can lead to loss of bone mineral . production and circulation of pro-inflammatory cytokines, such as il- and , and tnf-_, can affect the balance between osteoblastic bone formation and osteoclastic bone resorption in favor of bone resorption . activity, both the amount and type, has a major impact on both bone accretion and retention . a recent study from australia highlighted these factors . the australian population is particular in that there is lots of sun exposure, promoting vitamin d formation, and the population is oriented towards being physically active. in this population, adolescent patients maintained good calcium intakes and vitamin d levels and were as physically active as their contemporaries. yet their femoral neck bone densities were reduced, possibly due to the higher inflammatory level. in the adults, reductions in physical activity also played a significant role in reducing bone densities. in a healthy pediatric population, differences in the amount of time spent being physically active affects bone mineralization . in cf patients, both lung function and maximal exercise ability, which partially reflects the degree of physical activity, influence bone mineralization, such that higher lung function and exercise capacity are associated with greater bone density . since both lung function and peripheral skeletal muscle function influence exercise ability in both healthy individuals and cf patients , greater muscular ability will thus positively influence bone mineralization. of the exercises which most promote bone growth and maintenance, weight bearing exercises are primordial ; - . this is applicable to both children and adults. in summary, there are multiple influences on bone accretion and maintenance, with cf patients facing the additional challenges of chronic inflammation. it is important that a major effort be made to replete the bone reserves by early adulthood. this makes the peripubertal, pubertal, and post-pubertal periods the most critical. physical activity, especially impact activities and weight-bearing, plays an important role in achieving optimal bone reserves and maintaining these throughout life. phosphorylation of the cystic fibrosis transmembrane conductance regulator (cftr) by protein kinases is thought to be an absolute prerequisite for opening of cftr channels. in addition, nucleoside triphosphates were shown to regulate opening of phosphorylated cftr. cftr contains multiple sites of phosphorylation by camp-dependent protein kinase (pka) and protein kinase c (pkc) [ ] . it was also shown that the membrane-associated cgmp-dependent protein kinase isoform ii is able to phosphorylate cftr [ , ] . recently it was reported that in human sweat gland ducts endogenous cftr is activated by heterotrimeric g proteins via a camp-independent pathway [ ] . the question arose if alternative pathways exist to activate cftr, apart from protein phosphorylation. therefore we tested the effect of phosphatidylinositol , -bisphosphate (pip ), a known regulator of ion channels and transporters [ ] , on plasma membrane patches of oocytes, heterologously expressing human cftr. our study demonstrates that phospholipids, like pip , enable kinase-independent activation of cftr, resulting in atp-responsiveness of pip -treated cftr [ ] . pip alone is not sufficient to open cftr but atp opens nonphosphorylated cftr after application of pip . the effect of pip is independent of protein kinases, as pip activates cftr in the complete absence of mg. phosphatidylinositol (pi) and phosphatidylinositol monophosphate (pip) activate cftr less efficiently than pip . pip application to phosphorylated cftr may inhibit the cftr chloride current. we suggest that regulation of cftr by pip is a previously not recog-nized, alternative mechanism to control cftr-mediated chloride conductance. supported by the max-planck-society and the german research foundation(dfg). cftr channels help determine the rates of chloride and fluid transport across the epithelia affected in cystic fibrosis. early studies revealed two general modes of regulation, one mediated by secretagogues that elevate camp and activate cyclic-amp dependent protein kinase (pka), and another mediated by agonists that activate protein kinase c (pkc) and/or mobilize cell calcium. maneuvers that elevate camp induce sustained secretion across dog trachea and other preparations whereas global stimulation of pkc by phorbol esters leads to robust secretion lasting - min followed by profound inhibition . the stimulatory effect of pkc is due, in part, to activation of apical cftr channels whereas its antisecretory action probably reflects inhibition of basolateral potassium conductance, which is needed to maintain the driving force for apical chloride exit. a direct role of cftr in phorbol ester-stimulated secretion is implied by the presence of many consensus sequences for protein kinase c (pkc) phosphorylation on the r domain which, like the pka sites, are highly conserved across species. the physiological significance of the pkc sites is less obvious than for pka sites, however, since exposing inside-out membrane patches to pkc in combination with the diacylglycerol analog , dioctanoyl sn-glycerol (dic ) causes only a slight increase in channel activity (a few % compared to pka). this weak response can not be attributed to the use of dic as lipid activator because similar results are obtained when pkc is added in a cocktail containing phosphatidylserine and diacylglycerol under conditions that increase pkc activity by -fold. rather than acting alone, pkc phosphorylation seems to regulate cftr gating primarily by enhancing the rate and magnitude of its response to pka, effects which are most noticeable in patches after channel activity has been allowed to run down. pkc phosphorylation of cftr channels in vivo is undoubtedly regulated by hormones and/or transmitters but appears partially "constitutive" in cultured cells, perhaps a reflection of high pkc activity in cells soon after they are removed from medium containing serum and its growth factors. cftr regulation is often studied using potent activators (eg forskolin, cpt-camp, phorbol esters, etc), but to assess the physiological role of a pathway it is preferable to expose cells to a "first messenger" (eg vip, acetylcholine, neurokinin) and block the pathway of interest. pharmacologic inhibitors, antisense oligonucleotides, dominant-negative mutants and small interfering rnas have all been used in various preparations, although specificity is always a concern. chelerythrine and gö were both used to establish the role of pkc in stimulation of cftr by pka , but it is now clear that chelerythrine is less potent and less specific than gö or the other bisindoylmaleimides (eg bis or bis ; ) and should be avoided. interactions between signals could arise if an agonist activates more than one pathway or if multiple secretagogues stimulate the cell simultaneously. the role of such interactions in regulating cftr has not been studied but may be important in vivo since several transmitters are often co-released and cells also receive inputs from circulating hormones and locally-generated agonists such as atp, adenosine and no. sorting out the relative contributions and crosstalk between these signals remains a major challenge, however a key factor will probably lie in the subcellular localization of receptors and signaling molecules. the fact that phorbol esters have opposing actions at the apical and basolateral membranes itself implies that transport is controlled by multiple pkc isozymes having restricted distributions. pkc signaling involves activation by phospholipid and/or calcium, translocation to the site of action (eg plasma membrane), and binding to a receptor for activated c-kinase (rack) . racks are ideally suited to orchestrate localized signaling responses because they are located near pkc sub-strates and are specific for particular pkc isozymes. during secretion, basolateral uptake of chloride into human tracheal cells by sodium-potassium-chloride cotransporters is regulated by the pkc_ and pkc_ whereas apical chloride channels in the calu cell line are regulated by pkc_ ; . pkc_ binds to rack , which in turn binds the scaffolding protein ebp- (ezrin-radixin-moesin binding phosphoprotein of kd); ). regulation of these associations appears complex since phosphorylation of a pkc site on the pdz domain of ebp- can disrupt the pdz -cftr interaction . together these findings raise the possibility that when activated, pkc could potentially regulate its own targeting to cftr. finally, choosing the right preparation will be important for physiological studies of pkc signaling as cell lines may differ in their expression of receptors, anchoring proteins, pkc isozymes, and apical-basolateral polarization. using first messengers rather than artificial activators such as phorbol esters should minimize artifacts caused by hyperstimulation, and isozyme-specific translocation inhibitor and activator peptides are available which act specifically on pkc_ or other pkc isozymes. these tools in combination with mutant channels that lack pkc sites but are fully competent for gating (eg the " ca revertent" mutant ) should provide new insights into the regulation of cftr by pkc. cftr is a chloride channel in the atp-binding cassette (abc) transporter protein family and contains its defining features, two membrane-spanning domains and two nucleotide-binding domains (nbds) ( , ) . like other abc transporters cftr can bind and hydrolyze atp. earlier work has shown that enzymatic activity is required for normal channel gating ( , ) . but this has long seemed puzzling because ion flow is passive without a fixed stoichiometric relationship to atp hydrolysis. furthermore, no other ion channel is known to require the energy of atp hydrolysis for gating. therefore we asked whether cftr had another atpdependent enzymatic activity that released less energy. in previous work we found that a recombinant nbd polypeptide had atpase activity (atp ( adp + p i ) in the presence of atp alone. but when amp was also present, the nbd polypeptide functioned as an adenylate kinase (atp + amp ( adp + adp) and its atpase activity was suppressed ( ) . under physiologic conditions an adenylate kinase reaction is reversible and releases very little energy ( ) . we therefore tested the hypothesis that cftr has adenylate kinase activity that gates the channel. we did five sets of experiments. we first asked whether ap a (p ,p -di(adenosine- ') pentaphosphate), a well characterized inhibitor of adenylate kinases that does not inhibit atpases, would inhibit cftr. we found that ap a inhibited cftr currents by reducing the channel opening rate. our data with different di(nucleoside) polyphosphates indicate that the inhibition involved simultaneous binding to one of cftr's two atp binding-sites and to an amp binding-site. second we asked whether amp would influence cftr gating. we found that amp did not evoke currents in the absence of atp but could increase currents at non-saturating atp concentrations by increasing the channel opening rate. amp altered the relationship between atp concentration and current from one with no apparent cooperativity between the two atp-sites to one with positive two-site cooperativity for atp, indicating a different gating mechanism. third we asked whether the effect of amp involved atp:amp phosphotransfer. to address this question we tested the effect of amp-nh (adenosine 'monophosphoramidate), an amp analog that cannot act as a phosphate acceptor. we found that amp-nh did not mimic the effect of amp but inhibited gating produced by atp alone (i.e., atpase-dependent gating). amp, but not atp, reduced amp-nh inhibition, indicating that the inhibition occurred because amp-nh bound to the amp-site, which is distinct from the atp binding-site. fourth we asked whether adp would alter cftr gating through the readily reversible adenylate kinase reaction. we found that adp inhibited atp generated cftr currents. in addition, like amp, it induced positive twosite cooperativity for atp. adp-nh (adenylyl ´-phosphoramidate), an adp analog that does not allow phosphotransfer, and gdp, which does not bind to an adenylate kinase amp-site, failed to induce positive cooperativity. these results support the conclusion that phosphotransfer between two bound nucleotide diphosphates induced positive cooperativity. fifth we tested the nbd contribution to cftr adenylate kinase activity. we found that the mutations k a and d n in nbd and the homologous nbd mutations k a and d n reduced the potency of atp to stimulate cftr currents, indicating a reduction in atp binding and hydrolysis. the mutations k a and d n also abolished current stim-ulation by amp and current inhibition by ap a while k a and d n did not. these differential effects suggest that nbd , and not nbd , contains the adenylate kinase activity that gates the channel. in addition, we studied the effects of the cf-associated mutation n k in nbd . this mutation did not reduce the potency of atp to stimulate currents, but abolished amp-induced current stimulation and ap a inhibition, indicating that this mutation may be related to the amp-site. in summary, our data indicate that cftr has adenylate kinase activity that regulates channel gating. when functioning as an adenylate kinase, cftr showed positive cooperativity for atp suggesting its two nucleotide-binding domains may dimerize. thus, channel activity could be regulated by two different enzymatic reactions, atpase and adenylate kinase. both activities share a common atp binding-site in the second nucleotide-binding domain. our data indicate that it is the interaction of amp with the amp-site that induced adenylate kinase activity and suppressed atpase activity. the amp k m of ± µm for cftr gating suggests that cftr will function as an adenylate kinase in vivo. thus, at physiologic nucleotide concentrations adenylate kinase activity, rather than atpase activity may control gating, and therefore involve little energy consumption. this work was supported by the nhlbi (hl - and hl - ), the deutsche forschungsgemeinschaft (ra / - and ra / - ) and the hhmi. phosphorylation of cftr in the r domain by protein kinase a (pka) is essential for the channel to be active. however, after phosphorylation, atp is required to open phosphorylated cftr channels (gadsby and nairn, ; zou and hwang ) . atp-dependent gating of cftr is studied in inside-out membrane patches excised from nih t or cho cells heterologously expressing wild-type or mutant cftr. the opening rate of the channel follows a simple michaelis-menten function and the closed time histograms show a negative exponential component (zeltwanger et al., ) . both of these observations are consistent with the idea that cftr gating is not in microscopic equilibrium (i.e., injection of free energy from atp hydrolysis drives the gating transition). since the open time is drastically prolonged when lysine (k ) or glutamate (e ), both critical for atp hydrolysis, is mutated, it is reasoned that atp hydrolysis at nbd closes the channel (zou and hwang ) . several biochemical studies indicate that atp binding at nbd is extremely tight (szabo et al., ; alexandrov et al., ; basso et al., ) presumably because nbd does not hydrolyze atp (lewis et al., ) . if atp is occluded in nbd (i.e., the offrate is extremely slow), association and dissociation of atp at nbd cannot account for the electrophysiologically observed, millisecond-to-second gating transitions (vegani et al., ) . by default, atp opens the channel by binding to nbd . thus, nbd controls both opening and closing of cftr. although ample experimental results have supported the role of nbd in channel closing, kinetic evidence for its role in channel opening is lacking. since adp competitively inhibits atp-dependent opening of the channel, we reason that determining the site where adp binds to affect the channel opening rate will identify the nbd that controls channel opening. homology model of cftr's nbd was built by using the framework of the crystal structure of nbd from mouse cftr (lewis et al., ) . according to our homology model, tyrosine (y ) was identified as the aromatic amino acid that interacts with the adenine ring of atp at nbd . mutating y to glycine (y g) shifts the atp dose-response to the right with an apparent affinity > fold of that for wild-type channel. adp inhibition was also dramatically reduced with this mutant. these results are consistent with the idea that atp as well as adp binds at nbd to modulate channel opening. it thus appears that nbd can open and close the channel. does nbd play any role in channel gating? biochemical and crystallographic studies of nbds in bacterial abc transporters suggest that binding of two atp molecules at the interface of two nbds drives nbd dimerization smith et al., ; chen et al., ) . if an equivalent molecular motion happens in cftr during opening-closing transition, the free energy of atp binding at nbd should contribute to the overall energetics of association-dissociation of nbd dimer. we found that adp can increase the closing rate of the channel (also see weineich et al., ) , suggesting that adp may act at another binding site other than nbd . mutation of lysine at nbd also shortens the open time and the amp-pnp-locked open time (powe et al., ) , indicating that atp binding at nbd affects the energetic stability of the open (or the locked-open) state. based on these results, we propose a model that entails energetic coupling of atp binding (nbd ) and hydrolysis (nbd ) in controlling cftr gating. using automobile as an analogy, we picture that phosphorylation of the r domain serves as the ignition mechanism. atp binding at nbd acts as the clutch that controls the speed of the energy-consumption engine at nbd . . cftr null mice have more inflammation and higher mortality than normal mice after airway infection with pseudomonas aeruginosa . because airway epithelial cells (aec) express cftr and orchestrate inflammation, they may mediate dysregulated inflammation in cf. hyper-inflammation in cf could result from pro-inflammatory gene expression in aecs that is excessive, prolonged, and/or without stimulation. many in vitro models of aecs expressing a cf or non-cf phenotype demonstrate increased il- release by cf cells either spontaneously or after exposure to inflammatory stimuli. the models include aec lines from cf patients complemented with wildtype cftr and non-cf aec lines with decreased cftr function due to expression of the cftr regulatory domain or antisense oligonucleotides , . in one cf cell line, il- release was prolonged after removal of the inflammatory stimulus . increased constitutive or induced il- release has also been reported from primary cf aecs, including both surface cells cultured under submerged or airliquid interface conditions and gland cells [ ] [ ] [ ] . the observation that multiple cf model systems from different laboratories display greater pro-inflammatory gene expression provides strong support for the intrinsic hyper-inflammatory hypothesis. altered expression of inflammatory mediators other than il- has been demonstrated in cf aecs, including gm-csf, il- , il- , rantes, and nitric oxide synthase- , [ ] [ ] [ ] . altered regulation of multiple mediators suggests modulation of common signaling pathways by mutant cftr. the nf-b pathway appears to be an important target, likely via effects on upstream regulatory proteins [ ] [ ] [ ] . other signaling pathways may also function improperly in cf cells, including the tgf-␤ /smad and jak-stat cascades , . thus, altered regulation of diverse signaling pathways have been found in cf epithelial cell models by several groups, and these may provide unique therapeutic targets for controlling airway inflammation in cf. multiple studies do not clearly support the hypothesis that inflammation in intrinsically dysregulated in cf. for example, greater il- and neutrophils found in cf bal samples could easily be explained by defective mucus clearance rather than a hyper-inflammatory response. furthermore, many cftr-expressing tissues in patients with cf are not inflamed, suggesting that any hyper-inflammatory phenotype must be airway-specific. although multiple in vitro studies suggest hyper-inflammation in cf aecs, others show no change or even lower secretion of pro-inflammatory mediators , . most studies were done with immortalized cell lines, which are inherently unstable and acquire differences other than cftr status over time, making them difficult to control for experimentally. complementation with wildtype cftr inconsistently affects inflammatory responses , . primary aecs freshly removed from inflamed cf airways release more il- , but this phenotype is lost in culture suggesting an acquired, rather than intrinsic, response , . recent studies of well-differentiated primary aecs show significant person-to-person variation, but few differences in cf versus non-cf cells , , . taken together, it is clear that hyper-inflammation has stimulus-and condition-specificity that is highly variable between cell models , . another concern is that potential bias in the literature against negative studies may over-represent positive outcomes. finally, despite numerous investigations, one can argue that a solid mechanistic link between mutant cftr expression and altered regulation of inflammation has not yet been established. while cftr mutation links to disease pathogenesis and effects on the host response are hotly debated, there is essentially universal agreement that severe and sustained inflammation impairs pulmonary function and ultimately destroys the cf lung. anti-inflammatory therapy slows the progression of cf lung disease, confirming the importance of airway inflammation, but current therapeutic approaches are nonspecific and entail risks , . research directed towards understanding the regulation of ongoing airway inflammation in cf will suggest more selective treatments to mitigate lung damage without adversely affecting host defense and pathogen clearance. the authors acknowledge colleagues whose work could not be cited due to page limitations. cf airway inflammation is typified by the presence of neutrophils and their protein products, including elastase, myeloperoxidase and matrix metalloproteinases, in the airways. neutrophils are attracted to the airway by interleukin (il)- , a chemokine which is released from epithelial cells and macrophages as part of the innate immune response to infection. besides il- , epithelial cells and macrophages are capable of producing other pro-inflammatory cytokines such as tumor necrosis factor (tnf)-␣, il- ␤, and il- , each of which are increased in the airways of patients with cf. the gene expression of il- and other pro-inflammatory proteins is regulated largely by transcription factors of the nuclear factor (nf)-b and activator protein (ap- ) families. in the context of the il- promoter, the nf-b site serves as a cis-acting response element to diverse stimuli including tnf␣, il- ␤, pkc␦ and rhinovirus (unpublished data), whereas the ap- site serves as a basal level enhancer ( ). the basic nf-b complex is a dimer of two rel family members, p (nf-b ) and p (rel a). the ap- complex is a dimer of fos and jun family transcription factors. in unstimulated cells, nf-b is sequestered in the cytoplasm by ib proteins. phosphorylation and degradation of ib allows nf-b translocation to the nucleus, where it regulates gene transcription by binding to specific sequences of dna. the "classical" signaling pathway to nf-b activation includes successive phosphorylation and activation of nf-b activating kinase (nik) and ib kinase (ikk). ikk consists of two catalytic subunits (ikk-␣ and -␤) and a regulatory subunit (ikk␥). while ikk␣ and ikk␤ contain similar kinase domains with essentially identical activation loops, they are functionally distinct. recent studies suggest that ikk␤ serves as the target for pro-inflammatory signals, whereas ikk␣ plays a critical role in development ( , ) . the most potent ikk activator is the serine-threonine kinase nik. nik is required for activation of nf-b by non-typeable h. influenzae ( ) and micrococci ( ) . we have shown that successive activation of nik and ikk␤ is required for il- transcription induced by ligation of the asialogm ( ), the putative glycolipid receptor for p. aeruginosa. ikk␤ and the mitogen-activated protein (map) kinase kinases share structural elements, including the position of two activation loop serine phosphoaccepting sites. thus, it was found that map kinase/extracellular signal regulated kinase (erk) kinase kinase (mekk) phosphorylates and activates ikk ( ) . mekk is activated by tnf␣ and il- , and dominant negative mekk inhibits ikk␤ and nf-b activation ( , ) . the nf-b signaling pathway in cf could be activated either by cytokines, infection, or endoplasmic reticulum (er) stress. tnf␣ and il- ␤ are potent activators of nf-b-dependent gene expression. nf-b activation is required for maximal il- expression in response to p. aeruginosa ( ) ( ) ( ) . these studies also noted a requirement for the map kinases, which in turn function as upstream activators of ap- ( , ) . recent attention has focused on the mechanisms by which p. aeruginosa engagement of asialogm , a glycolipid which lacks transmembrane and intracellular domains, elicits host cell responses. asialogm ligation promotes atp release from the host cell, which is followed by successive activation of a nucleotide receptor, phospholipase c, ca + flux and erk, a map kinase family member ( ) . also, it has recently been shown that asialogm is associated with toll-like receptor (tlr)- , and that stimulation with p. aeruginosa flagella mobilizes tlr into a lipid raft receptor complex containing the tlr downstream effectors myeloid differentiation (myd)- , il- receptor-associated protein kinase (irak)- and tnf receptor associated factor (traf)- ( ). the pathway from traf- to nf-b activation has not been identified, but may involve transforming growth factor-␤-activated kinase (tak)- and nik ( , ) . infants with cf show airway inflammation without apparent infection, suggesting that inflammatory signaling pathways may be primarily upregulated in cf. consistent with this, tracheas from cf fetuses grafted into severe combined immunodeficient mice show increased intraluminal il- and leukocytic infiltration compared to those from normal fetuses ( ) . on the cellular level, airway epithelial cell lines derived from cf patients show increased basal and stimulated nf-b activation and il- expression vs. corrected cells ( , ) . similar observations have been made in cf airway gland cells ( ) . expression of ⌬f cftr, an incorrectly folded protein which partially accumulates in the er, but not g d cftr, which is trafficked normally to the epithelial cell surface, increases nf-b transcriptional activity in chinese hamster ovary cells ( ) , suggesting that cftr misfolding, with subsequent er stress, is responsible for dysregulated inflammatory signaling in cf. overload of the er with mis-or unfolded proteins leads to the unfolded protein response (upr), an integrated pathway leading to the expression of molecular marc b. hershenson, m.d. chaperones, attenuation of protein synthesis, and erassociated degradation ( ) . if these adaptive responses do not correct the protein folding defect, cells undergo apoptosis. in addition, a distinct but poorly-defined pathway leads to nf-b activation, perhaps via calcium mobilization or the generation of reactive oxygen species. the upr and er stress in cf cells have not been carefully studied. acute inflammation is a normal, protective response to tissue injury that, in the ideal circumstance, leads to removal of injurious stimuli and damaged cells or matrix and results in restoration of normal structure and function. in most forms of acute inflammation, neutrophil influx is an early and essential component of the process. after reaching a peak the influx ceases and the emigrated inflammatory cells are removed as a key component of the resolution process. emigration of monocytes into the inflamed lung is slower and appears critical to the neutrophil removal and resolution process. ultimately the excess macrophages are also removed. this simple scheme raises a number of important questions, including: ) what initiates the decline in neutrophil accumulation?. ) what causes the later influx of monocytes ( )? ) by what mechanisms are the neutrophils recognized and designated for removal? ) how are they removed? ) how are the excess mononuclear phagocytes cleared from the lung? in chronic, persistent inflammatory responses this normal, self-limited sequence is disrupted, perhaps because of persistent stimuli, initiation of new forms of stimulation, or abnormalities in the resolution. persistent and recurrent neutrophil influx in cf may encompass all three of these abnormal effects. neutrophils are short-lived cells and usually undergo spontaneous apoptosis within a few hours of release from the bone marrow. emigration into the lung can alter this apoptosis in complicated ways that can both delay or accelerate the process, but only by a few hours ( ) . the apoptotic neutrophils are recognized and cleared locally due to their ingestion by macrophages and tissue cells, including epithelial cells. this is normally a very efficient process; so efficient that detection of significant numbers of apoptotic cells within a tissue may be considered to suggest some defect in the clearance process. for example, only a few percent of the neutrophils being cleared during resolution of bacterial pneumonia or ards are seen to be apoptotic. by contrast, examination of cf patients revealed up to % apoptotic neutrophils ( ). apoptotic cell recognition and removal in vivo occurs quietly and unlike uptake by other phagocytic processes, is both non-inflammatory and actively anti-inflammatory ( ) . instillation of such cells into an ongoing inflammatory reaction in the lung enhances resolution ( ) . while a large number of molecules have been implicated in this recognition, a specific receptor for phosphatidylserine (the phosphatidylserine receptor, psr) appears to be critical for these anti-inflammatory and anti-immunogenic effects. in its absence, alternative receptors may in fact induce pro-inflammatory mediators and/or the lack of apoptotic cell clearance results in necrotic death with pro-inflammatory consequences. these observations led to the possibility that in cf, apoptotic cell clearance was ineffective, leading to persistence of the apoptotic neutrophils, increased necrosis and liberation of pro-inflammatory cell contents, as well as abrogation of the normal anti-inflammatory consequences of apoptotic cell recognition. all of these would lead to persistence of the neutrophilic phase of inflammation. in seeking the mechanism for defective apoptotic neutrophil clearance, the known presence of high levels of active elastase in cf airways suggested a likely possibility. apoptotic cell recognition receptors, particularly the psr, are highly susceptible to proteolysis. elastolytic cleavage of these in the airways would result in blockade of both apoptotic cell clearance as well as shutdown of the pro-inflammatory mediator generation ( ) . extending this concept more generally, the possibility arises that during the induction of acute inflammation, active neutrophil elastase liberated during the early phase of neutrophil influx, can cleave and inactivate the psr, thereby providing a window of time during which the acute inflammatory reaction can proceed. as vascular permeability increases, influx of protease inhibitors would inactive the elastase, now allowing incoming monocytes to mature into macrophages that are free to express their psr and initiate the anti-inflammatory resolution phase. in cf this puts a great deal of weight on the persistent presence of active elastase. to some extent, a positive feed-back loop from non-clearance of the apoptotic neutrophils, cytolytic release of their elastase content, with more suppression of apoptotic cell clearance, all in the presence of a high bacterial load, could drive a self-sustaining neutrophilic inflammation. this does not, how-ever, explain how it all got going in the first place, leading to questions about genetically driven alterations in apoptotic cell recognition and response as potential explanations for the very early post-natal infiltration of cf airways with neutrophils. although there may still be uncertainty about whether misfolded protein or other intracellular consequences of cftr defects initiate some inflammation in the lungs of cf patients, it seems clear that infection plays a major role in stimulating inflammation in the airways. there also seems to be a consensus that the response to infection in the cf airway is dysregulated and excessive ( ) . several studies in mice with cftr mutations have shown excessive responses to the prototypic inducer of inflammation, lipopolysaccharide (lps). most investigators have reported that huge quantities of pro-inflammatory cytokines, chemokines, and low molecular chemoattractants are found in lung secretions of cf patients, mice with mutations in cftr that have been challenged with lps or bacteria, and the supernatants of cultured cells with defective or blocked cftr. decreased production of anti-inflammatory mediators such as lipoxins ( ) and il- ( ) is also likely to be important in the dysregulation of the inflammatory response in the cf lung. there is considerable evidence that the nf-b dependent proteins, tnf, il- , il- and il- are over-produced in cf and contribute to both intrapulmonary and systemic morbidity (reviewed in ). the major inflammatory effector cell in the cf airway is the neutrophil (pmn). besides a role for il- and tnf in priming pmn, they induce expression of adherence molecules which facilitate migration of pmn into the airway; and il- is itself an important chemoattractant. additional chemoattractants important in cf are the lipoxygenase product, ltb , and the complement fragments c a and c a desarg ( , ) . the pmn themselves contribute additional ltb . more importantly, pmn products including active proteases, long stranded dna, and oxidants all have deleterious effects on airway function and structure which are out of proportion to their necessary role in controlling infection. these pmn products contribute to airway obstruction, induce nf-b activation and additional pro-inflammatory cytokine production, and impair phagocytic defense mechanisms. thus, the pmns initiate and perpetuate a vicious cycle of infection, inflammation and airway destruction which eventually claims the life of the patient ( , ). the paradigm above illustrates an array of targets for anti-inflammatory therapy. the role of infection as the major stimulus for the inflammatory process emphasizes the importance of controlling infection and reducing the burden of bacteria with antibiotics and airway clearance techniques/therapies. numerous studies have documented decreases in inflammatory mediators after "cleanouts", confirming the importance of limiting this stimulus. while we all hope that correction of the basic defect will terminate the cycle of infection and inflammation, if chronic infection and airway damage are already established, this may not be possible. inhibition of intracellular signaling pathways such as the i-b/nf-b pathway or correction of putative cf-related dysregulation of this pathway would reduce many of the proinflammatory cytokines and chemokines, and would thus hit many lung and systemic targets by reducing tnf and il- ( ). aspirin, nsaids, and corticosteroids all inhibit nf-b activation ( , ) , and ibuprofen and prednisone have shown beneficial effects in cf ( , ) . the pleotropic effects of cytokines might suggest that inhibiting any individual pro-inflammatory cytokine may not be very effective. however, inhibition of tnf alone has been very effective in rheumatoid arthritis (ra), which has several features in common with cf, especially the role of pmn in destruction of the end organ ( ) . interestingly, methotrexate, the mainstay of ra treatment, has shown promising effects in small studies in cf ( ) . inhibiting production of individual pathways like -lipoxygenase is also possible, and inhibitors of individual chemoattractant receptors are being studied. although preventing the pmn influx might seem to be a better strategy than attempting to neutralize their products one-by-one, the success of dnaase illustrates the efficacy of the latter approach. similar efforts could be directed at elastase and pmnderived oxidants. in evaluating anti-inflammatory therapy in cf, it is important to differentiate short-term improvement in symptoms from long-term preservation of lung function ( ) . a two-tiered approach is envisioned by the cff tdn aim program, in which preliminary day studies looking for efficacy in reducing inflammatory mediators in induced sputum will be used as a screening tool to select the best agents for larger, long-term studies of efficacy in preventing loss of lung function. this strategy offers our best chance of fairly evaluating new agents. any time one contemplates interfering with inflammatory mechanisms that also play important roles in host defenses, there is fear of allowing infection to get out of control. this has not occurred in trials of ibuprofen or corticosteroids in cf, but problems with infection, particularly activation of old tb in patients over , have been seen with the use of tnf inhibitors in ra ( ) . some of the concerns with these agents and other anti-inflammatories may be relevant to cf, but some may not. adverse effects frequently occur in organs other than the lung and may or may not be related to that mechanism of action of the drug which is believed to be important in cf. observation of the latter should lead to development of more specific agents in which the therapeutic effect is maintained while the action(s) causing the adverse effects is eliminated. specifically modifying the drug and/or route of delivery to decrease access to sites of toxicity while preserving effects on the therapeutic targets is the principle behind the use of inhaled corticosteroids for asthma, but these drugs seem to have only limited efficacy in cf. just as the long-term therapeutic effects must be considered, long-term adverse effects must also be weighed. it is particularly worrisome that "catch-up" growth has not occurred in cf patients who were given prednisone in earlier trials ( ) . although there are many reasons to believe that anti-inflammatory therapy should a part of every cf patient's regimen, recent cff surveys suggest that use of these agents, particularly ibuprofen, is actually decreasing. this seems ironic in the face of the confirmation of the efficacy of ibuprofen presented by schlucter et al at this meeting. development of newer strategies, realistic assessment of the risk of adverse effects, development of better targeted agents, and better understanding of the role of inflammation in lung damage are all necessary for determining the appropriate place for anti-inflammatory therapy in cf. a small-molecule discovery program was established to identify inhibitors of wildtype cftr and activators of ∆f -cftr ( ). primary high-throughput screening is done using automated instrumentation, a collection of > , diverse drug-like small molecules, and a cellbased assay utilizing a halide-sensitive green fluorescent protein ( ) . two interesting classes of small-molecule cftr inhibitors were identified. the compound cftr inh - contains a -thioxo- -thiazolidinone core ( ) . cftr inh - reversibly inhibits cftr clchannel function with k i in the range . - µm, depending on cell type/membrane potential. the mechanism of cftr inhibition by cftr inh - involves stabilization of the closed channel state with prolonged mean channel closed times by patch-clamp analysis ( ), probably as a consequence of cftr inh - binding to the first nucleotide binding domain of cftr. cftr inh - did not inhibit calciumor volume-activated chloride channels or the atp-binding cassette protein mdr- , and produced little toxicity in cell culture and mouse models. a single intraperitoneal dose of . - mg/kg cftr inh - inhibited cholera toxin-induced intestinal fluid secretion in closed-ileal loop models in mice and rats ( , ) . cftr inh - was also found in mice to reproduce the nasal epithelial ion transport defect in cystic fibrosis ( ) , and in pig and human airways to reproduce cystic fibrosis defects in submucosal gland fluid secretion ( ) . analysis of rodent pharmacology indicated slow renal elimination without metabolism, and efficient liver uptake with enterohepatic recirculation ( ) . the in vitro and in vivo results provide the rationale for further evaluation of thiazolidinone-type cftr inhibitors as antidiarrheals and agents for pharmacological creation of cystic fibrosis animal models. a second class of small-molecule cftr inhibitors, glycine hydrazides, was identified using a screen designed to identify compound that might act at the external cftr surface ( ) . the compound n-( -naphthalenyl)-[( , -dibromo- , -dihydroxyphenyl)methylene]glycine hydrazide (glyh- ) reversibly inhibited cftr clconductance in < min. whole-cell current measurements revealed voltage-dependent cftr block by glyh- with strong inward rectification, producing an increase in apparent inhibitory constant k i from . µm at + mv to . µm at - mv. apparent potency was reduced by lowering extracellular clconcentration. patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from to ms. glyh- inhibitory potency was independent of ph from . - . , where it exists predominantly as a monovalent anion with solubility ~ mm in water. topical glyh- in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. in a closed-loop model of cholera, intralu-minal . µg glyh- reduced by ~ % cholera toxin-induced intestinal fluid secretion. compared to cftr inh - , glyh- has much greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. glycine hydrazides may be useful as nonabsorbable antidiarrheals in enterotoxic-mediated secretory diarrheas. ∆f -cftr 'potentiators' (correctors of defective gating) and 'correctors' (correctors of cellular misprocessing) were identified using frt epithelial cells expressing human ∆f -cftr and an ultra-high halide sensitive green fluorescent protein. initial screening revealed a tetrahydrobenzothiophene class of ∆f -cftr potentiators that corrected defective ∆f -cftr chloride channel gating at concentrations down to nm ( ) . recent additional screening hasidentified novel classes of potentiators with good efficacy and medicinal profiles. after structure-activity analysis and optimization, one class of compounds corrected defective ∆f -cftr gating at concentrations down to nm. in cell attached patch-clamp experiments, the potentiators evoked increased channel open probability > -fold by reduction of interburst closed time. stimulation of chloride secretion was confirmed in low temperature-rescued human bronchial epithelial cells from ∆f cf subjects. two novel features of the new class of ∆f -cftr potentiators include amplifed response (synergy) with camp agonists, and correction of other cftr gating mutants including g d-cftr. screening of ∆f -cftr expressing cells cultured at o c for correctors of defective cellular processing produced classes of small-molecules with favorable medicinal properties that conferred greater chloride conductance to ∆f -cftr expressing cells than low temperature rescue. complex glycosylation was confirmed as measured by immunoblot analysis, as was plasma membrane expression as measured using an external epitope-tagged cftr. correction was seen within - hours after compound addition and persisted for up to hours after washout. a unique feature of some correctors was their ability to confer partial correction of defective ∆f -cftr gating, possibly by improved ∆f -cftr folding. the small-molecule potentiators and correctors maybe useful in therapy of cf caused by ∆f and possibly other mutations. cystic fibrosis (cf) is a fatal genetic disease caused by mutations in the cf transmembrane conductance regulator (cftr) protein, a protein kinase a-regulated anion channel in the apical membrane of many epithelial tissues, including the airway. approximately % of cf patients are homozygous for a single mutation that causes a deletion of phenylalanine at position (∆f -cftr). this mutation causes improper folding of the protein, deceasing both channel cell surface density and gating. either directly or indirectly, ∆f -cftr impairs ion transport, fluid secretion and mucociliary clearance, resulting in chronic infection, inflammation and eventual loss of lung function. our strategy for clinical treatment of cf is to discover drugs to increase cell surface expression and/or increase anion transport for mutant cftr, including ∆f -cftr. the hypothesis is that such pharmacological "rescue" of cftr would restore airway ion transport and ameliorate or slow the deterioration of lung function. agents such as -phenylbutyrate and genistein have been demonstrated to increase the density and gating of ∆f -cftr in recombinant cells, respectively. however, these agents have other biological effects and have not been chemically optimized for activity on cftr or for human pharmaco-kinetics. furthermore, these agents show little effect on chloride transport in primary airway cultures from cf lungs, and therefore likely lack either the potency or the efficacy to be effective drugs in humans. we have pursued a targeted approach to discover drugs that improve ∆f -cftr function. to identify starting points for chemistry optimization, we employed fluorescence-based assays of membrane potential in t cells expressing recombinant ∆f -cftr. approximately , compounds were screened to identify small molecules that either increased ∆f -cftr gating in the presence of pka stimulation (so-called "potentiators") or increased ∆f -cftr trafficking to the apical membrane (so-called "correctors"), or both. in addition to activity in the fluorescence assays, both potentiators and correctors were assayed for the ability to increase chloride transport in ussing chamber measurements using monolayers of primary human bronchiolar epithelial cell from the airway of ∆f -cftr patients (∆f -hbe). potentiators were prioritized if they exhibited drug-like chemical features and efficacy and potency greater than or equal to genistein. correctors were prioritized based on drug-like features and efficacy greater than or equal to temperature correction, which has been previously shown to partially reverse the defect in vitro. based on these criteria, distinct chemical series have been identified for correctors and potentiators. chemical optimization is aimed at improving both in potency and efficacy measured in both the fluorescence assay and (f -hbe chloride transport with the goal of achieving greater than % of ion transport observed in normal airway. several thousand molecules have been synthesized in the program to date. the presentation will provide an update on recent progress in lead optimization and describe some of the key challenges moving toward compounds suitable for clinical testing. the most common mutation associated with cf is ⌬f , which results in the in-frame deletion of the single codon encoding a phenylalanine residue at position . this mutation accounts for ~ % of all cf alleles, and ~ % of cf patients carry at least one copy of ∆f cftr. the protein encoded by the (f cftr allele has been classified as a processing mutant and as such, it is retained in the endoplasmic reticulum through interactions with elements of the er's quality control chaperone machinery and tar-geted for subsequent degradation in the proteasome. however, investigators have identified a number of in vitro manipulations that allow cohorts of newly synthesized ∆f cftr protein to depart the er, undergo post-synthetic processing in the golgi complex and be delivered to the cell surface. the resultant "rescued" surface population of ∆f cftr proteins are able to function as chloride channels with properties that are similar, but not identical, to those of the wild type polypeptides. in light of this behavior, numerous efforts aimed at developing small molecule pharmacotherapies for cf are focused on identifying compounds that can either stabilize the tertiary structure of the ∆f protein, or that can interfere with the interactions between the mutant protein and er chaperones. by preventing these interactions, the newly synthesized mis-folded but functional ∆f cftr protein might be allowed to escape recognition by the mechanisms that are responsible for its retention and ultimate degradation. we have previously shown that compounds that inhibit the endoplasmic reticulum ca-atpase (serca) can induce the release of er-retained _f cftr, which is delivered to the cell surface and able to function. many compounds that inhibit the ca-atpase are potentially toxic, and are thus not suitable as therapeutic candidates. curcumin is a non-toxic ca-atpase inhibitor that can be administered to humans safely in very large quantities. in preliminary studies the oral administration of curcumin to homozygous ∆f cftr mice in mg/kg doses com-parable to those well tolerated by humans resulted in alterations of these animals' characteristic nasal potential difference (npd) defect, such that there is a decrease in the baseline npd and the appearance of an isoproterenol response. furthermore, this treatment altered the electrical properties of gastrointestinal epithelia in these animals as demonstrated by the rectal potential difference assay. these effects are not observed in mice homozygous for a complete knockout of the cftr gene. curcumin also induces the functional appearance of _f cftr protein in the plasma membranes of transfected bhk cells. taken together, our initial studies suggest that curcumin is a nontoxic compound that can partially correct defects associated with the homozygous expression of ∆f cftr. however, in a number of follow up studies using different in vitro cell models and in vivo mouse models have not resulted in similar changes in ion transport properties. the difference in these in vivo and in vitro studies will be discussed. lastly, the findings from the first clinical study will be presented. an estimated one-third of mutations underlying human disorders result in premature termination of translation (nonsense or stop mutations) . nonsense mutations account for approximately % of the total mutant alleles in cystic fibrosis (cf) patients (cystic fibrosis mutation database), however, in certain populations the incidence is relatively high. among ashkenazi jews, nonsense mutations account for % of all cftr alleles , . these mutations are associated with a severe form of the disease due to no cftr chloride channel function. aminoglycosides, in addition to their antimicrobial activity, can promote read-through of nonsense mutations in eukaryotic cells by allowing synthesis of full-length proteins , . aminoglycosides were shown to restore the cftr function in cell lines and patients carrying the nonsense mutation w x - . in a recent randomized, double blind, placebo-controlled, crossover clinical trial we showed that the aminoglycoside gentamicin can increase the expression of fulllength cftr and correct the electrophysiological abnormalities, in cf patients carrying the w x nonsense mutation. however, in these studies several patients did not respond. here we studied the molecular basis for the variable response in cf patients carrying the w x mutation. a wide variability in the level of cftr nonsense transcripts was found among these patients (n = ). this variability correlated with the response of the patients to gentamicin treatment as demonstrated by nasal potential difference (npd) measurements. all the patients with relatively high transcript levels showed a significant reduction in basal potential difference and/or a significant response to chloride-free isoproterenol solution, indicating functional restoration of cftr following gentamicin treatment. in contrast, the patients with markedly reduced levels (n = , < %) of cftr transcripts did not correct the abnormal basal potential difference nor the chloride transport, indicating no response to the gentamicin treatment. we then analyzed the effect of the cftr transcript levels on the response to gentamicin treatment in two cell lines (cfp a and cfp b) from unrelated cf patients carrying the w x mutation. analysis of the cftr transcript levels revealed a significant difference between the cells, such that in cfp a the level was . fold higher than in cfp b. chloride efflux measurements in cfp a and cfp b cells revealed no forskolinstimulated chloride efflux, indicating that the cftr channels in both cell lines are inactive. following treatment with _g/ml gentamicin, restoration of the cftr function was demonstrated only in cfp a cells. following treatment with _g/ml gentamicin, chloride efflux was detected in both cell lines. these results indicate that the level of cftr nonsense transcripts correlates with the response to gentamicin, similarly to the findings in the patients. the level of nonsense transcripts is regulated by several factors, among which is the nonsense mediated decay (nmd) pathway. we hypothesized that inhibition of nmd would increase the level of nonsense transcripts available for read-through, and thus would improve the response to gentamicin. hence we treated the cfp a and cfp b cells with cycloheximide (chx), a potent inhibitor of nmd. this treatment significantly increased the level of nonsense transcripts. a direct inhibition of nmd by sirna directed against the nmd factors hupf and hupf , resulted in a similar increase in the level of cftr transcripts. we further analyzed the effect of nmd inhibition on the cftr chloride efflux. fro this we studied the effect of sirna against hupf and hupf on the cftr function. the results showed a significant higher chloride efflux in response to gentamicin, indicating that inhibition of nmd can improve the response to gentamicin. our results suggest that nmd plays a role as a genetic modifier of the response to aminoglycoside treatment in cf and many other inherited diseases. lung transplant needs to be considered for all cf patients once they develop end-stage lung disease. our goal should be that no cf patients should die without having had the option of considering lung transplantation. with still limited availability of lung grafts, up to - % of cf patients will die while awaiting transplant. thus timing of referral for transplant is the issue -bal-ancing risks of death of cf lung disease with risks of transplantation; identifying those ill enough to benefit from transplant but well enough to survive the procedure. selection of candidates at the appropriate level of illness is therefore the challenge for the transplant team. for the cf team, it is obviously better to err on the side of a premature referral. as it may take time for the patient to prepare for the idea of transplant, this time has to be factored into the equation as well. early referral to provide information for patient and family and to assess for transplant (but not necessarily listing) is likely the best option for patients. early referral should not mean premature transplantation and the system should have the flexibility to move patients on and off an active waiting list to "fine tune" or optimize the timing of transplant. the major difficulty is that cf is an unpredictable disease; the patients are young and can withstand severe dysfunction and treatment has a huge effect on disease stabilization. as patients become more ill, adherence to therapy can improve and intensification of treatment often occur. prediction of patients at high risk of dying has proven to be elusive -several models have shown we are better at predicting who will survive over who will die. mayer-hamblett et al used the cf foundation national patient registry to find predictors of mortality (fev , burkholderia cepacia, pseudomonas aeruginosa, hospitalizations, use of iv antibiotics) which were incorporated into a multivariate logistic regression model. this model did not significantly improve on the previous model developed in the toronto cf population where fev < % predicted alone was the most significant predictor of mortality (kerem et al ). liou et al. also used data from the cf foundation national patient registry and from unos (united network for organ sharing) to develop a multivariate logistic regression model to predict -year survival and they showed that patients with a -year survival of < % showed maximal survival benefit from transplantation. however, this still does not help predict the timing of referral to transplant. in addition to our inability to accurately predict at what stage a person should be referred and listed for transplant, we have to contend with other variables. predicted long term survival depends on survival post transplantation plus the waiting time to transplant. survival post transplant depends mainly on b. cepacia status (the species cenocepacia or genomar iii appears to result in higher immediate post operative mortality)positive or negative). the waiting time to transplant depends on country and centre. in the usa, the previous organ allocation policy depended on body size and blood type and accrued active waiting time on the national waiting list. in canada, waiting list time is affected by medical necessity giving much more flexibility for patients who suddenly deteriorate. a similar style of practice is present in many european countries. waiting list of less than year in toronto allows for later referral when patients are more ill (which can be easier to predict) and patients feel more "ready" at time of referral as they have a lower functional status. from a practical point of view, we must use the information and models that we have available, recognizing differences depending on our patient population, using predictors (fev , rate of decline in fev , bacteriology, co retention, hypoxia, clinical stability (hospitalizations, use of iv antibiotics), and functional status ( minute walk test)). all cf physicians will recognize older, clinically stable patients with slow decline in fev who may remain with fev below % predicted for decades. thus, it is imperiative for the cf clinician to communicate this information to the transplant center, because they have their finger on the pulse of each individual patient. the reality is that afterall, the transplant pulmonologists focus more on post transplant after care than on pre transplant-care. thenus, the goal of the cf centre is to maximize medical therapy and follow patient frequently to fine tune. rehabilitation programs improve functional condition and provide an early warning system for clinical deterioration. ongoing screening for cf complications which may impact the post transplant course (eg. cf related diabetes, osteoporosis) should not be overlooked. the concept of transplant is frightening and at the time of referral, patient may not feel that this is necessary for them. emotional and psychological preparation of the patient and family is required. linking the patient and family with another cf patient of similar age and life experience who has had a transplant is a helpful strategy. close links between cf centre and transplant centre lead to optimal care for patients with hopefully an enhanced ability to minimize deaths on the waiting list. we performed transplants in ventilator dependent patients from july to january . selected pretransplant characteristics of these patients are described in table . the pretransplant characteristics of the nonventilated patients and the stable ventilated patients were compared and no significant differences were found. the unstable ventilated patients were all acute retransplantations for early graft failure and represented such unique and unpredictable situations that statistical comparisons with stable patients did not make sense. the single re-transplant in the stable group was a woman who developed bronchiolitis obliterans syndrome after an en-bloc double lung transplant for copd and was relisted months after her first transplant. living lobar lung transplantation was developed as a procedure for patients considered too ill to await cadaveric transplantation. the procedure involves using a lower lobe from each of two donors for each recipient. living lobar lung transplants have been performed in patients between through june . patients were adults (mean age ) and were pediatric (mean age . ). the primary indication for transplantation was cystic fibrosis ( %). at the time of transplantation, . % of patients were hospitalized and . % were intubated. , , and year actuarial survival among living lobar recipients was , , and % respectively. there was no difference in actuarial survival between adult and pediatric living lobar recipients (p = . ). there were deaths among living lobar recipients, with infection being the predominant cause ( . %), followed by obliterative bronchiolitis ( . %), and primary graft dysfunction ( . %). the overall incidence of acute rejection was . episodes per patient. % of rejection episodes were unilateral. age, gender, indication, donor relationship, preoperative hospitalization status, use of preoperative steroids, and hla a, b, and dr typing did not influence survival or rejection. however, patients on ventilators preoperatively had significantly worse outcomes (odds ratio . , p = . ; kaplan-meier, p = . ), while those undergoing retransplants had an elevated risk of death (odds ratio . ). we examined the perioperative outcomes associated with the first donor lobectomies performed. there have been no perioperative or long-term deaths. . % of donors (n = ) had no perioperative complications, while fifty ( . %) had one or more complication. the incidence of intraoperative complications was . %. complications requiring reoperation occurred in . % of donors. . % of donors had other perioperative complications; the most serious were two donors who developed pulmonary artery thrombosis, while the most common was the need for an additional thoracostomy tube or a thoracostomy tube for ≥ days for persistent airleaks and/or drainage. right sided donors were more likely to have a perioperative complication than left sided donors (odd ratio . , p = . ), likely secondary to right lower and middle lobe anatomy. these results support the continued use of living lobar lung transplantation in patients deemed unable to await a cadaveric transplant. we consider retransplants and intubated patients to be at significantly high risk due to the poor outcomes in these populations. our experience has shown donor lobectomy to be associated with a relatively low morbidity and no mortality. this is important if this procedure is to be considered an option at more pulmonary transplant centers, given continued organ shortages and differences in philosophical and ethical acceptance of live organ donors. the postoperative length of intubation, intensive care unit stay, and overall hospital stay were significantly longer for the stable ventilated patients as compared to non-ventilated patients. when the stable ventilated transplant recipients were compared with the non-ventilated recipients, the difference between the survival curves failed to reach statistical significance with p = . . finally, the survival difference between stable and unstable ventilator dependent recipients was significant with p = . . acute lung retransplantation for severe graft dysfunction is a high risk strategy that has led to no long term survivors in our limited experience in five patients. in each case the recipient fulfilled two criteria: acute lung dysfunction of such severity that the surgeon and pulmonologist agreed that survival was unlikely, and generally intact physiologic systems without systemic infection or multisystems organ dysfunction. other strategies considered in this dire situation have been ecmo and profound hypothermia but the experience in these modalities is equally anecdotal and the results equally poor. lung transplantation for patients in stable respiratory failure is more promising. in our sixteen stable ventilated patients there were no postoperative hospital deaths and a long-term survival that is not statistically different from the general population of our program's transplant recipients. these patients do seem to be more prone to prolonged intubation and hospitalization after the transplant, but the results indicate that these problems are surmountable and that long term results are acceptable. our practice continues to be the same as described in this paper: we will consider transplantation in stable patients who have declined on the waitlist but we are skeptical of and generally discourage transplantation for acute lung dysfuction, regardless of the etiology. the medical consequences of delayed diagnosis in cf can be thought of as short term or long term. in the short term, infants with cystic fibrosis (cf) are subject to a wide range of complications if diagnosis is delayed. the most striking complications are related to severe nutritional deficiencies and to hypoelectolytemia. infants with cf can present with severe protein calorie malnutrition closely resembling kwashiorkor. these infants have low albumin levels, edema, anemia and hepatomegaly from steatosis. they are also developmentally delayed at diagnosis. trace metal deficiencies have also been described including severe zinc and selenium deficiencies. zinc deficiency is associated with severe, weeping rash and immune dysfunction. selenium deficiency can lead to congestive heart failure. deficiency of alpha tocopherol, fat soluble vitamin e, is associated with severe anemia. vitamin k deficiency can lead to intracerebral hemorrhage. ( ) linoleic acid deficiency likely coexists with other severe nutritional abnormalities and can cause rash. hypoelectrolytemia can lead to lethargy, poor intake and seizures. a death related to hypoelectolytemia and seizures in an infant with unsuspected cystic fibrosis has been reported. ( ) the causes of nutritional deficiency and hypoelectolytemia in infants with cf are well understood and help explain the short term consequences of delayed diagnosis. because of exocrine pancreatic insufficiency, fat and protein malabsorption occur in most infants by two months of age. ( , ) trace element metabolism is also abnormal by two months of age. albumin and fat soluble vitamin levels in young infants inversely correlate with the degree of nutrient malab- philip m. farrell, m.d., ph.d. during the past years, numerous investigations have identified medical and psychosocial benefits associated with early diagnosis of cf through neonatal screening. research has included both observational studies and a randomized controlled trial. the following list of potential benefits provides a compelling rationale for universal newborn screening using the trypsinogen/dna(cftr) method. . prevent deaths of undiagnosed patients-save lives! . improve access-avoid geographic and fiscal barriers . avoid disparities related to gender, race and ethnicity . prevent protein-energy malnutrition and stunted growth . prevent prolonged micronutrient deficiencies such as e and k . reduce risk for cognitive dysfunction due to malnutrition . preempt bronchopulmonary disease and pa infection* . provide genetic risk information ("counseling") to parents . reduce costs for diagnosis and possibly treatment . enhance clinical research opportunities while improving quality * create the opportunity to initiate respiratory therapy before irreversibility develops i.e., the "point of no return," which will eventually facilitate prevention of lung disease sorption. we also know that impaired weight gain after birth is correlated with malabsorption. hypoelectolytemia arises from sweat electrolyte loss. sweat electrolytes are abnormally high in cf within a week after birth. early diagnosis rather than delayed diagnosis along with treatment of pancreatic insufficiency and supplementation of salt intake might therefore avoid short term complications of cf in infancy. two lines of evidence support this notion. the most important evidence that early diagnosis and treatment can avoid complications in infants with cf comes from the wisconsin trial of newborn screening. this trial has clearly demonstrated that growth is improved in the short term (as well as the long term) in infants identified through newborn screening compared to those identified after symptoms appear. ( ) the second line of evidence stems from observational studies of infants identified through newborn screening showing that nutrient levels and growth improve with treatment. impaired growth is a long term consequence of that cf that is clearly related to delayed diagnosis. ( ) since improved pulmonary outcome in cf has been related to improved growth, ( , ) it might be expected that delayed diagnosis could lead to worse pulmonary outcome. the one study that has looked at this carefully however showed worse pulmonary outcome in patients identified through newborn screening compared to those with delayed diagnosis. ( ) there were special circumstances dealing with environmental pseudomonas exposure that made this study difficult to interpret, however. thus further studies of short term and long term medical consequences of delayed diagnosis are needed. to further explore the short and long term medical consequences of delayed diagnosis of cf we examined data from the cf foundation registry from , , and . short term consequences examined included presentation with hypoproteinemia or hypoelectrolytemia, as well as growth status pseudomonas culture positivity, and hospitalization in the year of diagnosis. long term medical consequences included growth status, pseudomonas culture positivity, pulmonary function and hospitalization. we specifically examined four modes of diagnosis (delayed diagnosis, newborn screening, meconium ileus and prenatal diagnosis). more than new diagnoses were studied. we found that stunting, wasting and hospitalizations in the year of diagnosis were three times as common in infants with delayed diagnosis compared to infants identified through newborn screening. occurrence of hypoproteinemia was significantly more common in infants with delayed diagnosis as was pseudomonas culture positivity. we examined long term consequences by age group ( - years, - years, and - years). delayed diagnosis was associated with stunting and wasting at essentially each age, higher percentage of mucoid pseduomonas at each age, and decreased fev (absolute and percent predicted) at ten years of age. hospitalization rate was higher in the delayed diagnosis group than the newborn screened group at two age ranges. examination of the cf registry therefore supports the notion that delayed diagnosis of cf is associated with significant short term and long term medical consequences. these data strongly support the need for early diagnosis and treatment to improve outcome in cf. cystic fibrosis (cf) is not readily diagnosed without newborn screening. it has few unique features, is very rare, and varies in its presentation.( ) in the united states, half of all persons with cf were diagnosed after six months of age. ( ) the median delay in diagnosis is well over one year in parts of the united states where newborn screening is not universal. the impact of delayed diagnosis of cf on families is poorly described. in the united states, most studies have been concerned with the clinical features of those with delayed diagnoses and have rarely explored the impact on the mother, father and siblings.(e.g., - ) a few bibliographic accounts of families with a child with cf have been written.(e.g., , ) outside the u.s., however, studies have found that misdiagnosis leads to increased anxiety, guilt and anger, and mistrust of the medical profession, and late diagnosis leads to more negative feelings about the pre-diagnostic period and less confidence in the medical profession.(e.g., , ) we aimed to describe the impact of delayed diagnosis on u.s. families with cf at different life stages. we requested diagnostic stories from individuals on the cf research, inc. e-mail list in september . we obtained diagnostic stories and used an additional stories found on the cystic-l listserv dating back to . we condensed the stories into that of a single family and qualitatively described the affected child's symptoms at different life stages and the medical and family responses to these symptoms. we provide likely impacts on health and family resulting from a diagnosis had it occurred at different ages. we found that the age at which a person with cf was diagnosed can have large, irreversible negative impacts on that person's health status, quality of life, longevity, compliance with medical regimens, self-image, family structure, and major life decisions. familial relationships were seriously impacted. the stress around not knowing what is wrong was damaging to the family. economic losses can be expected. strong views about incompetence in the medical care profession abound. these negative effects on families were likely to become compounded the later the diagnosis of cf was made. we found that long-term parental guilt around not finding out sooner was difficult to avert altogether or remove later. delayed diagnoses means that opportunities were lost to make informed decisions about health care, employment, housing, insurance, reproduction, and other matters. even if untrue, it was common for parents to feel like appropriate steps could have been taken to prevent their child's lung disease, for example, if only they had known sooner about the diagnosis. a few parents who had a first child diagnosed on account of a second child being diagnosed early in life via newborn screening indicated that it was far better knowing the diagnosis than being tormented by not knowing it. it was extremely hard to plan for the future when it was unclear what was wrong with the child. there was parental regret, anger and pity for their older child who was not able to benefit in the way the younger child can through prevention and focused cf medical care, which works best for persons who present early without damaged lungs or nutritional deprivation. in nearly all of the delayed diagnosis stories we reviewed, parents and persons with cf would end with the hope that in the future others could avoid the pain they suffered of not having known sooner. . how should we understand our collective use of "early" and "late" diagnosis of cf as we consider the ethics of newborn screening? in most medical communities, often repeated case reports become part of our explanatory narrative, such that they express the shared understanding of our work. these explanatory narratives form a common language by which we express normative assumptions about the appropriate roles of patients and doctors, and the proper approach to the treatment and diagnosis of cf. case reports about "late" diagnosis of cf are one such common explanatory narrative. among ourselves, we often tell the story of a child or young adult diagnosed with cf only after being seen in a specialist clinic. we all recognize this story immediately, and it has a kind of defining quality for those of us in the cf community: i doubt anyone who has come to a nacf conference could make it through the conference without either hearing or telling one version of this story. we tell it to each other even if we have just met, when the talk turns to our patients, and we commonly use this story, in one form or another, as a cautionary tale to our trainees. why do we tell each other this story so often? from a simple point of view, it's not so surprising, because the story makes us look smart-in particular, it confirms us as better clinicians than whoever saw the patient before us, and lets us comfortably and collectively shake our heads in acknowledging that another doctor could have missed the obvious diagnosis. perhaps the frequent telling of the late diagnosis story is a just a harmless form of self-congratulation among colleagues, much like a group of carpenters telling stories about a non-carpenter neighbor's inept attempt at building a tree house for his child. but perhaps there is more to it. in thinking about this story, we should first admit that the frequency of the story does not reflect the frequency of the event, that is, we are not simply telling the late diagnosis story so often because it is such a common one. the sort of late diagnosis reflected in the story happens far less often than, say, the first acquisition of pseudomonas, or the onset of cf related diabetes, or for that matter, the death of a patient from cf. like many narratives, the moral of the story of "late" diagnosis is more complicated than it first appears. in this case, the "late" diagnosis story stands in for a complex set of assumptions about life with cf, cf clinical care, and the role of the cf physician. our head-shaking reaction to the narrative of late diagnosis reflects our assumption that the patient's life would walter m. robinson, md, mph children's hospital, boston and harvard medical school analysis. this improves the detection sensitivity from % to approximately %. additionally, the direct confirmation of cf from the newborn screen by identification of two alleles increased from % with ∆f analysis only to % with multiple mutation analysis. . since the introduction of newborn screening for cf in wisconsin, the total number of annual sweat tests has decreased from in the year to in the year . concomitant with this decrease in the number of sweat tests has been a decrease in the number of institutions performing sweat tests from in the year to institutions in the year . the cost savings of a fewer number of sweat tests accounts for % of the cost to operate wisconsin's cf neonatal screening program. . in order to achieve excellent nutritional outcomes, we exceed the cff recommendation of quarterly clinic visits by utilizing every - week clinic visits in infants < year of age. newborn screening provides the opportunity for better outcomes, and excellent follow-up care is essential to achieve outstanding results. always be better if we had made the diagnosis sooner. that the story is much more complicated is evident in the outpatient clinic of every cf physician; the contemporary heterogeneity of the clinical course of cf, and the presence of a wide variety of clinical conditions at every age, simply belies the assumption that in any individual case a "late" diagnosis always means more suffering than an "early" diagnosis. and if the history of cf care has a cautionary tale for us, it is that our current therapies and models of care may well have unintended consequences, and that we cannot see ourselves as always wiser in our use of various therapies than our forebears. in short, it may be that more therapy at the youngest possible age is in every case a good idea, but then again it may not. the late diagnosis narrative also supports an historically inaccurate view of the process of diagnosis; it is the state of diagnostic technology, rather the presence or absence of symptoms, which is the strongest determinant of the timing of diagnosis. before the reliability of the sweat test was established, no child could be diagnosed with cf in the absence of characteristic symptoms; in this situation, there are no pre-clinical diagnoses, that is, there are no "early" diagnoses, and all "late" diagnoses are likely to be a reflection of unfamiliarity with the disease. as the diagnostic accuracy of the sweat test advances, we begin to see its use first to confirm a clinical suspicion based on signs and symptoms, then to establish the diagnosis in the face of a confusing set of signs and symptoms, and finally to screen for a diagnosis to detect the pre-symptomatic disease. in a very real sense, the screening aspect of the sweat test transforms the healthy into the diseased before the appearance of any symptoms, and in so doing redefines the boundary of "early" diagnosis. if neonatal screening is adopted, we can only expect further reconfiguring of the boundaries of early and late diagnosis, so that a child diagnosed at six months of age would then be seen as a "late" diagnosis and the asymptomatic only child with a cf genotype will be unable to live the quiet life of the undiagnosed. considerations of newborn screening often center on parental anxiety and its function as either a benefit or burden of screening programs. a variety of types and causes of anxiety are put forth as amenable by (or caused by) newborn screening: • anxiety about the presence of disease in an apparently healthy infant • anxiety about the lack of diagnosis for the symptoms seen in a sick infant • anxiety about the detection of life threatening illness in the apparently healthy, asymptomatic infant • anxiety about the detection of life threatening, as opposed to self-limited, illness in the sick infant • anxiety about future reproduction • anxiety about the blame for passing on a genetic illness • anxiety about the implications of the carrier state of the newborn it is perhaps peculiarly modern that we are debating the role of a public health program in promoting or alleviating anxiety. yet anxiety is a slippery diagnosis, and as in the late diagnosis story, our attribution of anxiety to others may reflect our own concerns rather than the concerns of others. how ought we as clinicians assess these claims and counterclaims about anxiety, and how ought we to weigh the relief or creation of anxiety when assessing the ethics of newborn screening? parts although cfrd is different than type or type diabetes, the development of diabetes-induced complications is similar. these complications include eye, kidney, circulation and nerve problems, and are caused by high blood glucose levels. thus the goals of cfrd management are similar to those of people with all forms of diabetes and help prevent development of complications from diabetes. insulin is an important anabolic hormone which prevents protein catabolism and studies have demonstrated catabolism in both adults and children with cf, and in both the fed and fasted state. protein catabolism is asso- appropriate nutrition is essential to management of patients with cystic fibrosis. many cannot maintain appropriate weight or growth without supplemental nutrition support. initiation of such support is facilitated by lifelong education of patients and their parents as to the importance of good nutrition in the management of cf. dieticians play a role in every cf visit and families are exposed to information about gastrostomy-tubes long before they become necessary. nutrition failure is identified using the guidelines established by the consensus report on nutrition for pediatric patients with cystic fibrosis and the cf adult care consensus conference. patients are initially evaluated for manageable causes of nutrition failure, including poor oral intake, uncontrolled malabsorption, and increased nutritional needs. poor oral intake can result from many causes, including chronic sinusitis, chronic pulmonary infections, onset of cf-related diabetes, and gastrointestinal causes (e.g., gastroesophageal reflux or distal intestinal obstructive syndrome). depression can also contribute to poor intake. malabsorption may result from inadequate or inappropriate enzyme use, cholestasis, short bowel syndrome, or small bowel overgrowth. the presence of pulmonary or sinus infections may lead to increased nutritional needs which the patient may not be able to meet orally. if oral supplementation and medical management fail, gastrostomy tube placement must be considered to restore normal nutritional status and normal growth. percutaneous endoscopic gastrostomy placement is generally successful. patients may then receive nocturnal drip feedings over - hours as supplement. aggressive control of reflux and monitoring for abnormal glucose tolerance is important. preoperatively, we counsel patients and families that gastrostomy tube placement is usually permanent in cf patients and will allow normal activity. we review their pulmonary status and may request an ng tube feeding trial to demonstrate efficacy. patients are admitted after placement to allow pain management, provide classes for parents and caregivers, and maintain good pulmonary function after the procedure. g-tube feedings are usually an elemental or semielemental formula, with enzymes used at bedtime and available if the patient arouses at night. am glucoses are monitored for a time after feeding introduction. good follow-up is essential to monitor progress and reduce complications. patients may experience pain or nausea during feedings, diminished daytime appetite, no weight gain despite gastrostomy tube feedings, onset of insulin requirement, or complications of the gastrostomy itself. malnutrition and nutritional growth failure in cf is a common and important issue in cf management because of its adverse effects on long term outcome. optimal standard of care in most cases requires meeting the excess energy, protein and vitamin needs of cf patients via overcoming malabsorption with appropriate pancreatic enzyme replacement therapy (pert), the use of oral supplements, sometimes helped by behavioral therapy, and /or, in some cases, enteral nutrition supplements (en). there are, however, some cf -related complications , where conventional nutritional support is not enough, may fail, or is impossible to maintain. these include neonatal meconium ileus with consequent short bowel syndrome, cf related liver disease with liver synthetic dysfunction, severe lung disease with frequent exacerbations, chronic lung disease or pulmonary failure, cfrelated diabetes, and some cases of poor intake and/or compliance, particularly if these are manifest during critical phases of growth such as infancy or adolescence. various adjuncts to nutritional therapy have been studied and may be considered in these circumstances ( ) . firstly, maximising pert with acid suppression or increasing duodenal bicarbonate may enhance absorption of nutrients ( , ) .however, there is insufficient evidence to indicate whether there is an improvement in nutritional status, lung function, quality of life, or survival ( ). secondly, enhancing en with specific supplements, eg trophic factors such as glutamine for short bowel syndrome ( ), and branched chain amino acids for liver disease may give added benefit ( ) . thirdly, improved protein turnover, and efficiency of energy/fuel use and consequent wight gain and growth may be achieved by adjunctive growth hormone therapy ( ) . other adjuncts, such as igf- , and megestrol acetate have been evaluated with more variable outcomes. .finally, where en is inadequate, or impossible, the use of parenteral nutrition may be necessary, and has documented benefits ( ) . the latter is particularly useful in pre-lung transplant patients, where improving/ maintaining nutrition may have an important bearing on outcomes. questions as to when and 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biopsies from the iliac crest of adults with cystic fibrosis the role of physiotherapy in the prevention and treatment of osteoporosis exercise, bone and nutrition bone mineral density and bone acquisition in children and young adults with cystic fibrosis: a follow-up study milk intake and bone mineral acquisition in adolescent girls: randomised, controlled intervention trial sex steroids and body composition in men with cystic fibrosis impact of lung inflammation on bone metabolism in adolescents with cystic fibrosis acquisition of optimal bone mass in childhood and adolescence bone mineral density in australian children, adolescents and adults with cystic fibrosis: a controlled cross sectional study a six-year longitudinal study of the relationship of physical activity to bone mineral accrual in growing children: the university of saskatchewan bone mineral accrual study decreased bone mineral density in normal-growing patients with cystic fibrosis analysis of factors limiting maximal 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in cho cells stably expressing the cystic fibrosis gene phosphorylation by protein kinase c is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase a specificity and mechanism of action of some commonly used protein kinase inhibitors the bisindolylmaleimide gf x is a potent and selective inhibitor of protein kinase c intracellular transport mechanisms of signal transducers the first nucleotide binding domain of cystic fibrosis transmembrane conductance regulator is a site of stable nucleotide interaction, whereas the second is a site of rapid turnover prolonged nonhydrolytic interaction of nucleotide with cftr's nh -terminal nucleotide binding domain and its role in channel gating a tweezers-like motion of the atp-binding cassette dimer in an abc transports cycle control of cftr channel gating by phosphorylation and nucleotide hydrolysis structure of nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator cooperative, atp-dependent association of the nucleotide binding cassettes during the catalytic cycle of atp-binding cassette transporters mutation of walker-a lysine in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide binding domains atp binding to the motor domain from an abc transporter drives formation of a nucleotide sandwich dimer nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. different patterns in the two nucleotide binding domains on the mechanism of mgatp-dependent gating of cftr cl -channels dual effects of adp and amp-pnp on cftr channel kinetics show binding to two different nucleotide binding sites gating of cftr by nucleoside triphosphates: quantitative analysis of a cyclic gating scheme gating of cftr by atp hydrolysis: structure and function early pulmonary inflammation in infants with cystic fibrosis the relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients inflammation and infection in naive human cystic fibrosis airway grafts excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with pseudomonas aeruginosa diverse pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin- overproduction of the cftr r domain leads to increased levels of asialogm and increased pseudomonas aeruginosa binding by epithelial cells proinflammatory cytokine responses to p. aeruginosa infection in human airway epithelial cell lines altered cytokine production by cystic fibrosis tracheal gland serous cells inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis cytokine secretion by cystic fibrosis airway epithelial cells altered respiratory epithelial cell cytokine production in cystic fibrosis chemokine expression in cf epithelia: implications for the role of cftr in rantes expression in vivo alterations of ifn regulatory factor- and pias protein levels in cystic fibrosis epithelium activation of nf-b by adherent pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells exaggerated activation of nf-b and altered ib-␤ processing in cystic fibrosis bronchial epithelial cells high susceptibility for cystic fibrosis human airway gland cells to produce il- through the ib kinase ␣ pathway in response to extracellular nacl content reduced smad protein expression and altered transforming growth factor-␤ -mediated signaling in cystic fibrosis epithelial cells reduced interleukin- production by cystic fibrosis airway epithelial cells interleukin- production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-␣ and respiratory syncycial virus stimulation alternate-day prednisone reduces morbidity and improves pulmonary function in cystic fibrosis effect of high-dose ibuprofen in patients with cystic fibrosis regulation of human airway epithelial cell il- expression by map kinases abnormal morphogenesis but intact ikk activation in mice lacking the ikk␣ subunit of the ib kinase ikk␣ provides an essential link between rank signaling and cyclin d expression during mammary gland development activation of nf-b by nontypeable hemophilus influenzae is mediated by tlr -tak -dependent nik-ikk␣/␤-ib␣ and mkk / -p map kinase signaling pathways in epithelial cells micrococci and peptidoglycan activate tlr -myd -irak-traf-nik-ikk-nf-b signaling pathway that induces il- transcription hershenson mb. signaling intermediates required for nf-b activation and il- expression in cf bronchial epithelial cells coordinate regulation of ib kinases by map kinase kinase kinase and nf-b inducing kinase ras and mekk- coregulate ap- and nf-bmediated gene expression in airway epithelial cells activation of nf-b via a src-dependent ras-mapk-pp rsk pathway is required for pseudomonas aeruginosa-induced mucin overproduction in epithelial cells pseudomonas pyocyanin increases interleukin- expression by human airway epithelial cells cf pathogens activate ca + -dependent map kinase signaling pathways in airway epithelial cells atp transduces signals from asgm , a glycolipid that functions as a bacterial receptor tlr is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells the kinase tak can activate the nik-ib as well as the map kinase cascade in the il- signalling pathway inflammation and infection in naive human cystic fibrosis airway grafts activation of nf-b in airway epithelial cells is dependent on cftr trafficking and cl (-) channel function selective upregulation of il- expression in cf bronchial gland cells in vivo and in vitro signal transduction from the endoplasmic reticulum to the cell nucleus monocyte retention and migration in pulmonary inflammation. requirement for neutrophils differential roles for alpha(m)beta( ) integrin clustering or activation in the control of apoptosis via regulation of akt and erk survival mechanisms elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/ paracrine mechanisms involving tgf-beta, pge , and paf phosphatidylserine-dependent ingestion of apoptotic cells promotes tgf-beta secretion and the resolution of inflammation cystic fibrosis in adults drug discovery in academia high-affinity activators of cftr chloride conductance identified by high-throughput screening thiazolidinone cftr inhibitor identified by highthroughput screening blocks cholera-toxin induced intestinal fluid secretion altered channel gating mechanism for cftr inhibition by a high-affinity thiazolidinone blocker prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule cftr inhibitor cftr involvement in nasal potential differences in mice and pigs studied using a thiazolidinone cftr inhibitor a small molecule cftr inhibitor produces cystic fibrosis-like submucosal gland fluid secretions in normal airways pharmacology and antidiarrheal efficacy of a thiazolidinone cftr inhibitor in rodents discovery of glycine hydrazide pore-occluding cftr inhibitors: mechanism, structure-activity analysis and in vivo efficacy nanomolar affinity small-molecule correctors of defective ∆f -cftr chloride channel gating nonsense-mediated mrna decay in health and disease highly variable incidence of cystic fibrosis and different mutation distribution among different jewish ethnic groups in israel cystic fibrosis in jews: frequency and mutation distribution suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics g- and paromomycin aminoglycoside suppression at uag, uaa and uga codons in escherichia coli and human tissue culture cells suppression of a cftr premature stop mutation in a bronchial epithelial cell line a pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis gentamicin-induced correction of cftr function in patients with cystic fibrosis and cftr stop mutation developing cystic fibrosis lung transplant referral criteria using predictors of -year mortality prediction of mortality in patients with cystic fibrosis survival effect of lung transplant among patients with cystic fibrosis living lobar transplantation lung transplantation of ventilator dependent patients recipient selection international guidelines for the selection of lung transplant candidates lung transplantation is warranted for stable, ventilator dependent recipients early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition and improves long-term growth longitudinal evaluation of bronchopulmonary disease in children with cystic fibrosis bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis cognitive function of children with cystic fibrosis: deleterious effect of malnutrition references . merelle me, griffioen rw, dankert-roelse je. cystic fibrosis presenting with intracerebral haemorrhage dehydration deaths in infants and young children pancreatic function in infants identified as having cystic fibrosis in a neonatal screening program pancreatic insufficiency, growth, and nutrition in infants identified by newborn screening as having cystic fibrosis early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition and improves long-term growth. wisconsin cystic fibrosis neonatal screening study group investigators and coordinators of the epidemiologic study of cystic fibrosis. growth and nutritional indexes in early life predict pulmonary function in cystic fibrosis stature as a prognostic factor in cystic fibrosis survival bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis the diagnosis of cystic fibrosis cystic fibrosis foundation cystic fibrosis diagnosed after age . twenty-five teenage and adult patients including three asymptomatic men diagnosis of cystic fibrosis during adolescence clinical features associated with a delayed diagnosis of cystic fibrosis delayed diagnosis of us females with cystic fibrosis the spirit of lo. an ordinary family's extraordinary journey dueling with cystic fibrosis: finding the chloride defect early versus late diagnosis: psychological impact on parents of children with cystic fibrosis effects of newborn screening of cystic fibrosis on reported maternal behaviour newborn screening for cystic fibrosis in wisconsin: nine years experience with routine trypsinogen/dna testing genetic counseling and neonatal screening for cystic fibrosis: an assessment of the risk communication process sweat chloride concentrations in infants homozygous or heterozygous for f cystic fibrosis analysis of the costs of diagnosing cystic fibrosis with a newborn screening program comprehensive analysis of risk factors for acquistion of pseudomonas aeruginosa in young children with cystic fibrosis pediatric clinics of north america managing cystic fibrois related diabetes (cfrd). a teaching manual for cystic fibrosis related diabetes. the cf foundation enteral tube feeding for cystic fibrosis consensus report on nutrition for pediatric patients with cystic fibrosis cystic fibrosis adult care: consensus conference report optimising nutrition in cystic fibrosis gastric acid suppression and treatment of severe exocrine pancreatic insufficiency omeprazole, a proton pump inhibitor, improves residual steatorrhoea in cystic fibrosis patients treated with high dose pancreatic enzymes drug therapies for reducing gastric acidity in people with cystic fibrosis.cochrane database syst rev trophic and cytoprotective nutrition for intestinal adaptation, mucosal repair, and barrier function nutritional support in children with end-stage liver disease: a randomized crossover trial of a branched-chain amino acid supplement growth problems and growth hormone treatment in children with cystic fibrosis effects of glutamine and recombinant human growth hormone on protein metabolism in prepubertal children with cystic fibrosis improved growth and clinical, nutritional, and respiratory changes in response to nutritional therapy in cystic fibrosis s . when enteral nutrition is not enough -adjuncts to nutrition support in cf newborn screening for cystic fibrosis (cf) has been performed as part of a randomized, controlled trial for patients born in wisconsin between april , and june , . in the randomized, controlled trial, the study investigators were informed of a positive neonatal screen (immunoreactive trypsinogen [irt] in the first six years of the study and irt/dna in the last three years of the study) for patients in the early diagnosis group. the study investigators informed the primary care physician of the positive newborn screen and the infant was brought to madison or milwaukee (the two study centers) for sweat testing at six weeks of age. in july , cf newborn screening was added to the panel of routine neonatal screening tests. over the past decade, we have learned the following lessons from the routine cf newborn screening program. . initially in the routine neonatal screening program, the newborn screen collection card had a check-off box for meconium ileus. if this box was checked, then dna analysis for the ∆f mutation was performed regardless of the trypsinogen level. the rationale for this was to detect cf patients who theoretically could have a low trypsinogen level after surgery for meconium ileus. however, in the first / years of the routine cf newborn screening program, there were a total of infants in which meconium ileus was checked on the newborn screening specimen card. the staff at newborn nurseries were possibly checking this box erroneously for meconium stained amniotic fluid. in confirmed cases of cf with meconium ileus, there were no infants in which the trypsinogen level was less than the cut-off value. therefore, the meconium ileus box on the newborn screening form was removed. . initially in the routine newborn screening program, the written recommendation for an infant with a positive cf newborn screen was to have a sweat test performed at four weeks of age. although the two cf centers in the state of wisconsin were listed on the report, it was not explicitly stated that sweat testing should occur at a cf center. this led to two difficulties: a. families were required to wait until the baby was four weeks of age for a sweat test. during this time, they encountered anxiety about the possibility that their child could have cf. b. a number of sweat tests were performed at hospitals that were not cf centers. this led to questions about the accuracy of the sweat testing procedure. additionally, families were not receiving genetic counseling in these non-cf center settings.these issues were addressed by revising the positive cf neonatal screening report. the report no longer states that the infant should be four weeks of age for a sweat test and, therefore, there is now less of a delay from the time that the primary care physician notifies the family of the positive newborn screening test until the time that a sweat test is performed. additionally, the newborn screening report now states that a quantitative pilocarpine iontophoresis sweat test should be performed at cf centers or affiliate centers listed on the report. as a result of these changes, in the calendar year , there was only one infant who had a sweat test outside of a cf core center or affiliate center. . we are aware that families are anxious when they learn that their infant has a positive neonatal screen (specifically, an elevated trypsinogen and one cf mutation).to help allay the parents' anxiety , a letter has been created that accompanies the positive cf newborn screening report to the primary care physician. this letter is intended to be given to the parents and emphasizes that there is only a possibility of cystic fibrosis. the letter briefly describes the sweat test and that no special preparation is needed. contact information for the cf core and affiliate centers and the state newborn screening coordinator is provided. additionally, the letter directs the parents to a website that we have created that provides useful information about cf newborn screening <http://www.pediatrics.wisc.edu/childrenshosp/cf/nbs.html>. within this website is a video entitled "understanding newborn screening for cystic fibrosis". all of these educational efforts have significantly decreased the number of phone calls that we receive from panicked parents. . from our experience in the cf newborn screening program, it is clear that the traditional decision levels for sweat chloride (< meq/l is normal, - meq/l is borderline, and > meq/l is diagnostic of cystic fibrosis) is not applicable to infants. we have demonstrated that sweat chloride levels of > meq/l in an infant is suspicious for cf. extended mutation analysis in infants with a sweat chloride of > meq/l almost invariably reveals two cf mutations. this agrees with previous data. . infection control measures are of utmost importance, as clinic exposures in a small waiting room ( sq. ft.) was a risk factor for acquisition of pseudomonas aeruginosa. . cf newborn screening has evolved from an algorithm of irt/∆f analysis to irt/multiple ( ) mutation ciated with insulin deficiency and resistance to insulin's anti-catabolic effects on whole body protein turnover. protein catabolism is an especially important consideration in the malnourished cf child and adult. malnourishment, principally loss of muscle mass, has been associated with worsened morbidity and mortality. insulin lowers blood sugar levels and functions as an anabolic agent by increasing cellular uptake of ingested nutrients and conversion of the nutrients to energy. insulin is the principal hormone preventing protein catabolism. the cf consensus conference recommended that insulin be the only medical therapy prescribed for cfrd with fasting hyperglycemia. the recommendations were based on the review of existing medical literature. the use of insulin may be especially important in a malnourished patient with cf, and several studies have documented weight gain and improvement of catabolism with insulin. insulin use can be made more convenient by using insulin injection devices and insulin pumps. there are many types of insulin and potential insulin regimens available for treating cfrd, and therapy should be individualized. however, there are several principles to guide the clinician in choice of therapy. . the regimen should as closely as possible fit the patient's life-style. . meal coverage should be provided to prevent postprandial hyperglycemia. . the regimen should confer as low a risk as possible for the development of hypoglycemia. . in most cases, good glycemic control can be obtained using of multiple injections. the broad classes of insulin are "rapid-acting," "short-acting", "longer-acting" and basal insulin. insulin action (when it peaks in activity, and how long it lasts) may vary from person to person. there is also some variability from one day to the next in the same person. when deciding on an insulin regimen, three types of coverage should be considered.• meal coverage (bolus insulin): a normal pancreas secretes insulin as a "bolus" to cover the meal (specifically the carbohydrates) ingested. rapid-acting or short-acting insulin is given before meals to mimic this extra insulin "bolus." generally, the best way to dose pre-meal insulin dose is to account for the carbohydrate content of the meal. the normal pancreas makes a small amount on insulin at all times. these low levels of insulin are called "background" or "basal" insulin. generally long-acting insulin such as ultralente (lasts approximately - hours and has a small peak) or lantus (lasts hours and has no peak) are used. most people with cf make some insulin, thus they often do not require as much basal insulin as people who have diabetes but do not have cf. however, protein catabolism is higher in malnourished patients; therefore basal insulin is an important part of the insulin regimen in these patients. in general a starting dose which minimizes the risk of hypoglycemia is . units per kilogram of weight per day. this dose should be increased as needed, but hypoglycemia should be avoided.• correction: when the blood glucose level is too high, rapid-acting insulin is given in addition to the usual insulin dose to "correct" the blood glucose level. correction is especially important during illness and to prevent hyperglycemia-related diabetes complications. in general, when a patient is normally insulin sensitive, one unit of rapid-acting insulin will lower the glucose level approximately mg/dl. night-time enteral feeding are particularly useful in the malnourished cf patient, and some may only have high blood sugar levels during these feedings. hyperglycemia at this time is best managed by a combination of longeracting (nph or lente) plus short-acting insulin (regular), at the start of the feeding. a starting dose which minimizes the risk of hypoglycemia is . units per kilogram per dose of regular and . units/kg/dose of nph. the cf consensus conference on diabetes did not recommend that oral agent be used to treat cfrd with fasting hyperglycemia (the only glucose tolerance category for they recommended mandatory treatment). there are multiple types of oral agents, and a few small studies have documented efficacy in cfrd. research is needed to test potential oral therapies. key: cord- -iz alys authors: francis, john g.; francis, leslie p. title: fairness in the use of information about carriers of resistant infections date: - - journal: ethics and drug resistance: collective responsibility for global public health doi: . / - - - - _ sha: doc_id: cord_uid: iz alys one standard menu of approaches to the prevalence of anti-microbial resistance diseases is to enhance surveillance, fund research to develop new antimicrobials, and educate providers and patients to reduce unnecessary antimicrobial use. the primarily utilitarian reasoning behind this menu is unstable, however, if it fails to take fairness into account. this chapter develops an account of the fair uses of information gained in public health surveillance. we begin by sketching information needs and gaps in surveillance. we then demonstrate how analysis of information uses is incomplete if viewed from the perspectives of likely vectors of disease who may be subjects of fear and stigma and likely victims who may be coerced into isolation or quarantine. next, we consider aspects of fairness in the use of information in non-ideal circumstances: inclusive participation in decisions about information use, resource plans for those needing services, and assurances of reciprocal support. fairness in information use recognizes the ineluctable twinning of victims and vectors in the face of serious pandemic disease. antimicrobials; and to intervene through education, treatment, and careful stewardship of the existing antimicrobials that retain some efficacy. this combination of approaches is founded primarily in utilitarian reasoning, attempting to achieve the best possible mitigation of the current crisis in the hopes that effective new treatment methods may soon become available. such utilitarian reasoning is not entirely stable in practice, however. on the one hand, when the prospects of exposure to untreatable and potentially fatal disease appear imminent, fear may become the overriding reaction to those who are identified as ill. the result may be forms of coercion against people suspected of being vectors of disease that appear prudential in the short term but that are insufficiently grounded in science and potentially counter-productive in the longer term. people may hide to avoid disclosure and deleterious consequences of over-regulation may lead to under-regulation. recent examples include demands to compel isolation of people believed to have been exposed to ebola or for banning travel from regions where outbreaks of conditions such as ebola or zika have been identified. on the other hand, concerns for victims may generate outpourings of resources for treatment, calls for investment in public health resources in underserved areas, and renewed emphasis on privacy protections. these too may be counterproductive if they result in confusion and waste of resources or multiple conflicting strategies. the upshot may be policies that oscillate between treating people as vectors and treating them as victims but without significant or coordinated progress against the problem of resistance. each of these perspectives-victim-hood and vector-hood-is morally important. but in our judgment analysis that is limited to these perspectives is incomplete in its failure to take certain considerations of fairness into account. our specific focus here is the use of information, but similar points could be made about other types of resources as well. collection, uses, and access to information, we contend in what follows, must be rooted in the effort to make progress against serious public health problems in a manner that is reasonably fair under the circumstances. this requires not only concern for people as victims and vectors but concerns about how the impact of policies are distributed and foster cooperative connections in both the shorter and the longer term. traditional public health surveillance methods are both individual and population based. where particular individuals are concerned, the role of information is primarily to enable strategies to interrupt disease transmission. case identification, case reporting, contact tracing, treatment if possible, and education and intervention if needed to prevent transmission come to the fore. at every stage, information is critical. if individuals with transmissible disease are unknown or cannot be located, efforts to interrupt transmission will fail. efforts will also fail if information is not transmitted to those who are capable of acting, whether they be authorities designated to enforce quarantine or isolation or health care personnel equipped to offer treatment or prophylaxis. education requires information, too, about where to direct educational efforts and what these efforts might contain. importantly, if people who might suffer exposures are insufficiently informed about the likelihood and seriousness of contagion and the need for precautions, they may unwittingly become infected vectors as well as victims themselves. such was the case for health care workers during the sars epidemic of and for many during the ebola epidemic of . information gleaned in population-level surveillance plays many additional important roles in addressing the problem of anti-microbial resistance. a longstanding recommendation of the who, codified in the world health regulations that entered into force in in article , is international cooperation in the development of surveillance capacities for the identification of potential global health emergencies of international concern (who ). surveillance can help to identify rates of incidence and prevalence of resistant disease. testing samples can yield information about histories and patterns of disease spread. samples also can be used to identify biological characteristics of resistant infectious agents that may be helpful in developing methods of treatment or identifying new anti-microbial agents. population level surveillance can be targeted to identifying the incidence and prevalence of resistant disease in particular geographical areas. gonorrhea is an example. there were . million estimated new cases of gonorrhea worldwide in ; the highest number occurred in low-income areas of the western pacific. resistant disease has become increasingly prevalent, especially in these areas and among groups such as sex workers and truck drivers (unemo et al. ) . extensively drug-resistant (xdr) gonorrhea cases also have appeared in spain and in france, although these strains do not appear to have spread, possibly because they are less hardy and so less likely to be passed on. however, significant resistance may not be detected because of "suboptimal antimicrobial resistance surveillance in many settings" (unemo et al. ) . a recent international panel reviewing resistant gonorrhea recommends strategies of case management, partner notification, screening (especially of sex workers and men having sex with men), and evidence-based treatment (unemo et al. ) ; these recommendations are based on surveillance data. population-level surveillance information may also be useful in identifying risks associated with providing humanitarian treatment. over , young people wounded in the libyan civil war that began in were evacuated elsewhere for treatment. concerns arose that many of these patients were recognized to carry with them resistant organisms-thus bringing along with their needs for treatment risks to other patients being treated in the host facilities (zorgani and ziglam ) . institutions accepting these patients were informed of this risk so that they could take appropriate precautions. libya itself was identified as a region with high prevalence of resistant organisms, despite the limited surveillance capacities in that conflict-torn nation. recommendations included improving surveillance in libyawhich lacks a national surveillance system-and implementation of infection prevention measures in libyan hospitals. surveillance is also used to identify practices that might contribute to the development of resistance. use of antimicrobials in agriculture is one area of inquiry, although its precise contribution to the problem is not easy to quantify (e.g. hoelzer et al. ). there have been many studies of problematic prescribing practices among physicians in the us (wigton et al. ) , europe (e.g. jørgensen et al. ) , asia (lam and lam ) , and elsewhere (trap and hansen ) , along with efforts to educate physicians about appropriate antimicrobial use. ever since the recognition grew that crowds celebrating the return of soldiers from world war i had created a ready opportunity for transmission of the spanish influenza, epidemiologists have observed the potential health risks of large gatherings that concentrate people together, even for brief periods of time. examples include music festivals, major sporting competitions, other large festivals, and religious gatherings such as the hajj or other pilgrimages. the largest estimated gathering is the periodic kumbh mela pilgrimage in which hindus come together to bathe in a sacred river such as the ganges; over million people, drawn largely from the indian subcontinent but increasingly international, attend the event (gautret and steffen ) . the largest annual gathering of pilgrims is the hajj at mecca which draws over two million people; the fifth pillar of islam is the obligation to undertake the once in a lifetime journey for those who can physically or financially afford to do so. with such great numbers of people together for sustained periods of time, there is a risk of disease outbreaks and the spread of resistant infections. such events may strain existing sanitation systems or health care facilities if people become ill. crowding and inadequate facilities contribute to the potential for disease outbreaks (gautret and steffen ) . these events draw people from around the globe and thus may result in the international spread of disease (gautret and steffen ) . at the same time, many of these events are of great cultural importance and suppression of them is neither a realistic nor a desirable option. there have been extensive discussions of how to address the public health needs of the great numbers of people who undertake pilgrimages or who attend other events that draw great numbers of people together. vaccination may create herd immunities that reduce risks of disease transmission; for example, for this year's hajj the saudi arabian government is requiring proof of a quadrivalent meningococcal vaccination in order to receive a visa (ministry of hajj ). nonetheless, risks may remain significant for conditions that cannot currently be addressed by vaccination or that are difficult to treat, such as middle east respiratory syndrome coronavirus (mers-cov) or resistant infections. information too is critical: such well-attended events require imaginative and thoughtful surveillance that informs short-term medical care. because saudi arabia has had the largest number of human cases of mers-cov-an estimated % (who b)-travelers for this year's hajj are being warned to take extra precautions with respect to sanitation and personal hygiene measures such as handwashing or avoiding direct contact with non-human animals (new zealand ministry of foreign affairs ). still other social factors may contribute to the development of resistant disease that can be identified through surveillance. given the difficulties for women in saudi arabia to see physicians without being escorted, it is understandable that in saudi arabia many community pharmacies will dispense antibiotics without a prescription. zowasi ( ) recommends addressing these issues by increased education especially through social media as to the best approach to respond to the risk of anti-microbial resistant organism. still other recommendations about information use involve research on the development of new forms of antimicrobials. according to the most recent review article (butler et al. ) , antibiotics "are dramatically undervalued by society, receiving a fraction of the yearly revenue per patient generated by next-generation anticancer drugs." they are in the judgment of these authors an "endangered species,"-but there is some faint encouraging news. who and a number of national governments have recently begun to direct attention to the potential threat of resistance and lack of new drugs. since , five new-in-class antibiotics have been marketed, but these unfortunately only target gram-positive organisms not the gram-negative organisms that are likely to be resistant. other compounds are also in various stages of the process of clinical trials, but these too are more likely to be active against gram-positive bacteria. in the judgment of the authors of this review article, "the acute positive trend of new approvals masks a chronic underlying malaise in antibiotic discovery and development." interest in antibiotic development is more likely to be present in smaller biotech companies and in biotech companies located in europe. the authors conclude: "the only light on the horizon is the continued increase in public and political awareness of the issue." they also observe that with the retrenchment in investment, "we potentially face a generational knowledge gap" and drug development "is now more important than ever." to address this perilous juncture in antimicrobial research, the pew charitable trust convened a scientific expert group in . the premise of the group was that regulatory challenges, scientific barriers, and diminishing economic returns have led drug companies largely to abandon antibiotic research-yet antimicrobial resistance is accelerating. no entirely new classes of antibiotics useful against resistant organisms have been brought to market that are not derivatives of classes developed before -over years ago. the pew report advances many explanations for this dismal situation, including importantly the lack of coordinated investment in the relevant basic and translational research. one aspect of the report detailed the major role played by information gaps. published research is out of date and out of print. moreover, in today's world of investment in drug discovery, "creating an environment in which data exchange and knowledge sharing are the status quo will be difficult given proprietary concerns and the variety of information types and formats, which may range from historical data to new findings produced as part of this research effort." the pew consensus is that the following forms of information sharing are needed: a review of what is known about compounds that effectively penetrate gram-negative bacteria, a searchable catalogue of chemical matter including an ongoing list of promising antibacterial compounds, information on screening assays and conditions tested, and an informational database of available biological and physicochemical data. mechanisms must also be developed for sharing drug discovery knowledge in the area (pew, . in line with pew, a european antimicrobial resistance project suggests that research is seriously underfunded (kelly et al. ) . this group argues that the bulk of the publicly funded research is in therapeutics ( %); among the remainder, % of the research was on transmission and only % specifically on surveillance. this group also concluded that research is not coordinated and there is little attention to data sharing or sharing of research results. funding is fragmented, too, with many smaller grants addressing smaller projects independently rather than in a way that builds. this group summarizes: "to conclude, investment at present might not correspond with the burden of antibacterial resistance and the looming health, social, and economic threat it poses on the treatment of infections and on medicine in general. antibacterial resistance clearly warrants increased and new investment from a range of sources, but improved coordination and collaboration with more informed resource allocation are needed to make a true impact. hopefully, this analysis will prompt nations to pay due consideration to the existing research landscape when considering future investments." additional recommendations from other groups include novel methods for management of resistant disease, such as addressing the intestinal microbiome (e.g. bassetti et al. ) ; these methods, too, may be furthered by surveillance information as well as information about individual patients. analysis of these uses of information from the perspective of vector or victim are, we now argue, incomplete. when contagious diseases are serious or highly likely to be fatal and treatments for them are limited at best, fear is understandable. fear may be magnified if the disease is poorly understood, especially until modes of transmission have been identified. fear may also be magnified if there are no known effective treatments for the disease, as may be the case for extremely drug resistant infections. it is therefore understandable that proposals may come to the fore that emphasize isolation of those who are known to be infected, quarantine of those who have been exposed, or travel bans from areas of known disease outbreaks. proposals may even include criminalization of those who knowingly or even negligently take risks of infecting others. all of these possibilities and more were features of the hiv epidemic. even as understanding of the disease grew and effective treatment became increasingly available, some of these remain. criminalization of hiv transmission has not waned, despite the many objections raised to it (e.g. francis and francis a, b) . although the us ended its immigration ban on hiv+ individuals in , concerns remain about the risks of undiagnosed infections among immigrant populations in the u.s. (winston and beckwith ) and some countries (for example, singapore) continue to ban entry for hiv+ travelers planning stays over thirty days (the global database ). as epidemic fears have waxed and waned over recent decades, so have imperatives for identifying vectors and constraining their activities. these patterns have been apparent for avian influenza, sars, ebola, and zika, among others. the us still bars entry by non-citizens with a list of conditions including active tb, infectious syphilis, gonorrhea, infectious leprosy, and other conditions designated by presidential executive order such as plague or hemorrhagic fevers (cdc ). indeed, resistant tb has been a frequent illustration of the vector perspective in operation. multi-drug resistant tuberculosis is transmissible, difficult to treat, and poses a significant public health problem. its presence can be identified by methods such as testing of sputum samples. when patients are identified with resistant disease, public health authorities may seek to compel treatment or isolation, especially for patients judged unreliable about compliance with treatment. to avoid transmission, public health authorities have proposed isolating patients who have been identified as infected. because a course of treatment for tb may take many months-and failure to complete the full course may increase the likelihood of resistant diseaseisolation may continue for long periods of time. controversially, during the early s public health officials in new york isolated over patients identified with mdr tb on roosevelt island for treatment out of concern that they would be noncompliant with treatment even when they were unlikely to infect others (coker ) . perhaps one of the most highly publicized events involving a single patient was the odyssey of andrew speaker, a lawyer believed to have extremely resistant tb who eluded authorities as he took airplane flights around the globe in the effort to return home. speaker's journey created an international scare and calls for travel restrictions. speaker's lawsuit against the centers for disease control and prevention alleging violations of the federal privacy act, he claimed by revealing more information than was necessary for public health purposes, was ultimately resolved on summary judgment for the government, largely because the challenged disclosures had been made by speaker himself. who travel guidelines provide that individuals known to be infected with resistant tb should not travel until sputum analysis confirms that they are not at risk of disease transmission (who ). evidence is limited, however, about the need for this policy. the most recent literature review suggests that risks of transmission during air travel are very low and that there is need for ongoing international collaboration in contact tracing and risk assessment (kotila et al. ) . blanket travel bans encouraging actions that elude detection may reduce, rather than enhance, this needed collaboration. more subtle policies tailored to need would be preferable, but the fears generated by a focus on fear of vectors may make them unlikely to be developed or implemented. at best, therefore, the vector perspective is incomplete. focus on it may be counter-productive, if people hide or try to avoid education. it may encourage expenditures on efforts to identify suspected vectors rather than on evidence based efforts to identify risks of transmission and effective modes of prevention. and, of course, it ignores the plight of victims, to which we now turn. people with resistant infections are not only vectors, they are also victims of disease and have ethical claims to be treated as such (battin et al. ) . indeed, it is likely that vectors will themselves be victims, unless they are carriers of the disease in a manner that does not affect them symptomatically. concern for victims may take the form of seeking to ease the burdens of constraints such as isolation. a good illustration of the victim perspective in operation is the who publication of a pamphlet on "psychological first aid" to those affected by ebola. the pamphlet is designed to provide comfort to and meet the basic needs of people infected by ebola and those who are close to them, while maintained the safety of aids workers (who ). the recommendations rest on the importance of respect for the dignity of those who are suffering amidst disease outbreaks. it also emphasizes the importance of respect for rights such as confidentiality and nondiscrimination. the pamphlet is provisional and designed to be updated as knowledge of safety measures improves; this provisional nature is a recognition of the importance of ongoing development of information about how victims' needs can be safely met. despite the concern for victims, foremost in the pamphlet's recommendations is safety, both of aid workers and of disease victims, so that no one is further harmed including victims themselves and others close to them. overall, the pamphlet attempts to counter impulses to come to the aid of victims that may increase transmission risks, such as unprotected contact with those who are ill. but unexplored tensions remain in the document's recommendations. for example: "respect privacy and keep personal details of the person's story confidential, if this is appropriate" (p. ). nowhere does the document discuss when confidentiality is appropriate or what personal details may be revealed and in what ways. its manifest and important concern for victims is countered by safety but without discussion of how these goals might be implemented together or reasonably reconciled in practice. the who's most recently-adopted strategy for dealing with health emergencies, the health emergencies programme, provides another illustration of concern for victims that may lie in unexplored tension with other values. the programme urges cooperative methods to meet the immediate health needs of threatened populations through humanitarian assistance while also addressing causes of vulnerability and recovery (who ) . it is a coordinated strategy for emergency response that will move far beyond merely technical help; who describes it as a "profound change for who, adding operational capabilities to our traditional technical and normative roles" (who ) . it is aimed to provide crisis help, such as to hurricane matthew in haiti or to areas affected by the zika virus. it requires a major increase in funding devoted to core emergency efforts. core funding will come from assessed contributions, flexible contributions that the director-general has discretion to allocate, and earmarked voluntary contributions. but it is clearly under-funded; who reported a % funding gap as of october , just to meet the program's core capabilities. moreover, who also reported that it has raised less than a third of the funding needed for the who contingency fund for emergencies, a fund deployed for the initial months of an emergency before donor funding becomes available (who ) . the health emergencies programme reflects reactions to the humanitarian disaster of the ebola epidemic and criticisms of the who level of response. the who - budget reflects this response as well (who ) . that budget "demonstrates three strategic shifts" (who , p. ). the first is application of the lessons from ebola especially the need to strengthen core capacities in preparedness, surveillance and response. the second strategic shift is a focus on universal health coverage, which includes enhancing contributions to maternal and child health, speeding progress towards elimination of malaria, and enhancing work on noncommunicable diseases, among other worthy goals. the final strategic shift is towards "emerging threats and priorities"; illustrations of these are "antimicrobial resistance, hepatitis, ageing, and dementia." these are not an obvious group to characterize as "emerging," to the extent that this suggests a developing threat that has not yet become urgent but that may be expected to become so in the near future. nor are they an obvious group to link together in the same category. this mixture of budgetary priorities suggests is responsiveness to issues raised through consultation with who member states, rather than proactive planning. who specific efforts directed to resistance can be characterized as primarily coordination. the who website devoted to resistance promotes information sharing and lists research questions and potential funding agencies (who a). who expresses no judgment about either funding agencies or which of the nearly listed research questions-ranging from research on resistance in day care centers to the biological price that microorganisms pay for resistance-might be fruitfully addressed first or how they might be interconnected. concern for victims is surely part of a response to a humanitarian emergency. responsiveness to urgent health needs is an important goal. including antimicrobial resistance in a list of "emerging" issues is at least recognition of the problem. but the who response to ebola and the who budget overall can be characterized as less than fully set into context in a reasoned way. thus, we contend, neither vector nor victim perspectives are adequate. one risks falling prey to fear while the other risks responses that are well-intentioned but that may be difficult to meet or compete with other values in ways that remain underexplored. these perspectives are inevitable and important, but they are each incomplete. in our judgment, a primary difficulty with both vector and victim perspectives is that neither are set into context or seen as interconnected. this section suggests how fairness considerations may help in focusing attention to the most pressing questions to ask about antimicrobial resistance and the directions for surveillance and information use to take. fairness entered the philosophical lexicon in discussions about justice as procedural, most famously in john rawls's "justice as fairness" (rawls ) . as rawls initially conceptualized his view, it involved a decision procedure for selecting basic principles of justice in which people were unable to gain unfair advantage. as the debates about rawlsian justice unfolded, a fundamental issue was whether people with radically different capacities and views of the good life could be expected to accept the results of the decision procedure as formulated. thus critics raised the concern that people with disabilities might be left out of the decision procedure as "non-contributors" to the practice of justice (nussbaum ; stark ) . critics also pressed the argument that people with radically illiberal conceptions of the good would ultimately destabilize the practice of justice in a rawlsian ideal society (e.g. williams a, b) . rawls ultimately accepted the point that proceduralism could not yield a universal theory of justice, pulling back his view to the claim that it only represented a vision of justice for a certain kind of liberal society (rawls ) . but fairness also entered the debates about justice in a more substantive way, especially in bioethics. norman daniels ( ) , for example, expanded a rawlsian approach to consider justice in health care. the british idea of a "fair innings," in which the opportunities of each to reasonable health over a normal life span are prioritized, was raised particularly with respect to the distribution of health care resources to the elderly (bognar ; farrant ; harris ; williams b) . like the metaphor of a level playing field, the fair innings argument comes from sports (francis ) . it reflects the idea of everyone having a chance to participate in a game that at least gives them a reasonable opportunity for success. there are four aspects of such opportunity: who plays and whether the rules are constructed to give each an opportunity to win that is reasonable are two. also important is the balance among opportunities to succeed, so that there aren't consistent tilts in one direction or another, as might be characterized by the further metaphor of leveling the playing field. finally, attention to the interaction between advantages and disadvantages matters, so that participants are encouraged to continue playing the game rather than dropping out. our invocation of fairness as a concept is rooted in the judgment that antimicrobial resistance-or other pressing global public health problems, for that matterexemplify multiple aspects of non-ideal and partial compliance circumstances. natural circumstances are less than forgiving; new health threats emerge on a regular basis. antimicrobial resistance is an ongoing natural challenge to effective therapy for deadly diseases. social circumstances are imperfect, too: overcrowding, poor sanitation, straitened resources for public health and health care, and cultural practices that increase potential for disease transmission all play roles in the development of resistance. alexander fleming, the discoverer of penicillin, warned that the development of resistance was likely, but his warning appears not to have been well heard. finally, efforts to address antimicrobial resistance are riven with noncompliance: over-prescribing by physicians, over-use of antimicrobials in agriculture, individual failures to take medications as prescribed, and concealment of disease out of fear of discovery and persecution. because the conditions that give rise to these problems of non-compliance may seem urgent-people seeking antimicrobials are in pain or ill, perhaps gravely; people in hiding from health authorities may fear stigmatization or death-they raise in particularly poignant form questions of the extent of obligations under circumstances in which others are not doing what arguably is their fair share (e.g. stemplowska ; murphy ) . fairness as an ethical concept is especially suited to such imperfect circumstances. it directs attention to how improvements are distributed. distributions can be more or less fair, if they distribute benefits and burdens in an increasingly inclusive manner (e.g. francis and francis a, b) . fairness thus construed is at the heart of perhaps the most influential set of recommendations for ethical pandemic planning, the canadian stand on guard for thee (toronto joint centre ) . although much of the discussion of fairness in this document emphasizes inclusive procedures, so that engagement may lead to acceptance of choices as fairly made (e.g., p. ), the recommendations also contain substantive dimensions. these include fair resource plans for those who fall ill providing necessary services during a pandemic (p. ) and assurance that people who are affected by choices are reciprocally supported in a way that they do not suffer "unfair economic penalties" (p. ). here, the links between fairness and reciprocity are explicit. these four aspects of fairness-who is included in the play, what opportunities they have, how these opportunities are balanced, and whether there are elements of reciprocity-can be used to set vector and victim perspectives into context in addressing the gathering and use of information about antimicrobial resistance. over-emphasizing vectors threatens their opportunities and even possible participation. overemphasizing victims tilts the field unidirectionally, understandably directing resources to immediate need but without consideration of longer-term consequences. reciprocity may be the most important of all, creating commitment to workable strategies for addressing resistance when there are difficult choices to be made. fear, understood as a threat personal health, is often an ally in persuading people to seek preventive care and to change life styles, or to persuade policy makers to create incentives or penalties for decisions that contribute to poor health. but great fear can also lead to immobility. the real threat posed by the rise of antimicrobial resistance does not seem to be easily addressed by a successful alternative in the view of victims or policy makers. medical personnel are fearful of not responding to the demands of patients for immediate reductions in pain or suffering at relatively low costs. the scale of the threat posed by rapid rise of antimicrobial resistance may be daunting to policy makers especially as funders of research. the cost of developing ever-new generations of antibiotics seems to suggest a great series of short-term solutions especially as pharmaceutical companies respond to incentives to generate near-term profits. in this context, it is worth recalling how the development of the first antimicrobials contributed to more generally shared benefits: when penicillin became known to people as a wonderful drug it actually helped to speed the adoption of the national health service in britain. the popular expectation was health care for all facilitated with the rise of a new generation of low cost wonder drugs and reinforced by low cost vaccinations (webster ) . but some of the advantages were short-lived, as the costs of pharmaceuticals grew exponentially and inadequate attention was paid to the risks of overprescribing-once again a cautionary reminder of the importance of emphasizing balance rather than one particular perspective such as victimhood. if a promise of sustaining production at lower costs of ever-new generations of antimicrobials from how information is used can offer benefits more widely, then it becomes easier to impose tougher regulations on antimicrobial use that may to some extent stave off the development of resistance. this approach in terms of fairness directs attention not only to vectors and to victims seen as separate entities. it also directs attention to how they are often, and unpredictably, twinned-given the epidemiology of resistance spread, it is likely to begin within interlaced communities where vectors are also victims. but it also directs our attention to these issues set in distributive context, raising questions such as these: who is most likely to be affected by resistance? who will suffer the most severe consequences from resistance? who is most likely to be disadvantaged by information gained to counter resistance? who will suffer the most severe disadvantage? who will benefit from efforts to counter resistance? how can these benefits be spread more inclusively? and, how are the benefits and burdens of addressing resistance intertwined? are some primarily beneficiaries, while others are primarily burdened? are there ways to increase reciprocal linkages in these benefits and burdens, so that efforts to counter resistance are accepted and supported more widely? these are the kinds of questions that need to guide how surveillance is deployed in the effort to counter resistance, not vague generalities about the importance of addressing health infrastructure or bromides about the need to increase resources. open access this chapter is licensed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. georgia: suit against disease centers is revived. the new york times antimicrobial resistance in the next years, humankind, bugs and drugs: a visionary approach the patient as 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individual countries have consequences for other countries. here, we review what economic analysis can offer in countering these problems through the design of interventions that modify the behaviour of institutions and nations in the direction of greatest global good. the past century has been marked by significant improvements in life expectancy, due to greater child survival and reductions in infectious disease. the greatest victories in global health have come through globally coordinated actions-the eradication of small pox in , the global polio eradication initiative (still ongoing), and the sharp reductions in malaria through the global malaria eradication program (gmep) in the s. just the first two of these initiatives resulted in roughly . m deaths averted each year (unicef, ; ehreth, ) , and the gmep was responsible for eliminating malaria in countries (kouznetsov, ) . largely as a consequence of these efforts and of improvements in wellbeing that have translated into better ability to prevent and treat infectious diseases, these conditions have diminished in importance as a source of ill health across much of the world. according to the global burden of disease estimates, the percentage of disability-adjusted life years (dalys) due to prominent infectious diseases (comprised of the following four cause groups: hiv/aids and tuberculosis; diarrhoea/lower respiratory infections/other infectious diseases; neglected tropical diseases and malaria; and other communicable diseases) decreased from . per cent in to . per cent in , while the percentage of deaths due to prominent infectious diseases decreased from to . per cent (ihme ) . nevertheless, infectious diseases continue to be a significant source of ill health globally and a number of the world's emerging global health threats involve infectious diseases that can easily cross boundaries. the emergence of a new infectious disease-ebola being the most recent example-poses a significant risk to other countries, no matter where it arises. the risk is not uniform: countries that are connected by geography or population movement with the country where the disease emerges, and those with weak health systems are particularly vulnerable. but there are counter-examples as well. new delhi metallo-β-lactamase (ndm) enzymes that cause drug resistance in bacteria, which were first reported in from one patient hospitalized in sweden, are now reported globally (nordmann et al., ) . multiple factors including human population growth, land-use changes, and infectious diseases originating from wildlife (also known as zoonoses) are accelerating the frequency with which infectious diseases emerge (jones et al., ) . even if the direct health toll from these emerging infections does not approach the levels that were observed during the global flu pandemic, when nearly m people died, these infections can nevertheless do serious damage to economies, health, and health systems by virtue of their speed of attack. ebola has killed roughly , people in the last year, mostly focused in the west african countries of guinea, liberia, and sierra leone, and resulted in a per cent loss of gdp in these countries (world bank, ) . a recent study projected that after - months of disruptions, the accumulation of a large connected cluster of children unvaccinated for measles across guinea, liberia, and sierra leone resulted in between , and , additional child deaths due to measles alone (takahashi et al., ) . the deaths of healthcare personnel may have ripple effects down the road and could even discourage people seeking to train to be tomorrow's healthcare workers. the overall damage to health systems due to the large numbers of health system professionals lost to ebola will only become apparent in coming years. drug resistance is now a global problem and threatens public health in nations regardless of economic status (laxminarayan et al., ) . antibiotic-resistant gonorrhoea emerged in vietnam in (holmes et al., ) , before spreading to the philippines and finally to the united states (rasnake et al., ) . ndm enzymes are now in nearly every country, as discussed earlier. in this paper, we discuss the global health threats that involve 'commons' problems. with such problems, the actions undertaken in one country have consequences for other countries, but these are 'externalities' that are not taken into consideration by decisionmakers. for instance, a country may not report a disease outbreak for fear that it would discourage tourism, but the failure to report the outbreak could put other countries at risk. other examples of country-level actions with global consequences include inadequate vaccination coverage; slow progress on disease elimination; failure to report and contain pandemic flu, antibiotic resistance, and counterfeit drugs; and climate-related health threats. we provide some examples and case studies of such negative externalities across borders. then, we discuss the need for international cooperation for tackling these global health threats. the remainder of the paper is organized as follows. section ii describes trans-boundary externalities in tackling infectious diseases. section iii deals with incentives for surveillance and reporting of disease outbreaks. section iv addresses incentives for disease elimination and eradication. section v addresses incentives and financing mechanisms for controlling drug-resistant pathogens. section vi concludes the paper. early examples of international medical cooperation in the modern age were based on the idea that because infectious diseases do not respect national boundaries, meaningful control necessarily transcends national programmes. the first international sanitary conference was convened in paris in to discuss the quarantine of ships to contain plague, yellow fever, and cholera; it predated the first geneva conventions on treatment of war casualties by years (stern and markel, ) . more recently, campaigns to eliminate smallpox and eradicate malaria have been built on the idea that infectious disease control depends not just on national priorities but also on the priorities of one's neighbours and trading partners. an understanding of transnational disease transmission was deeply rooted in the gmep, which was launched in . funding from the top contributors to the special account for malaria by member countries during - accounted for per cent of overall contributions over this period (table ) . of these contributors, only saudi arabia had any significant malaria. malariacontrol investments in the current era are also likely to be largely externally funded, but contributions are not likely to continue indefinitely. therefore, the gains made from control have to be sufficiently large not just in the focal country but also in neighbouring countries so that malaria control will continue to be a priority for national planners even after the donors have exited. malaria control benefits the country in which it occurs, of course, but in the longer term, its neighbours benefit as well because they face fewer cases of imported malaria. the spatial coordination problems introduced by trans-boundary malaria are also relevant for the problem of regional elimination within large countries, especially those with frequent in-country movement, such as india. in contrast, china has managed to eliminate malaria from most of the interior of the country, but imported malaria remains a problem on its southern border. the extent of the 'external' benefit (to a neighbour) depends on malaria prevalence in that neighbour and the frequency and direction of overland migration. if malaria is common, then the benefit of fewer imported cases is minimal. however, the benefits can be large if the neighbour has eliminated malaria but still has to deal with cases imported from the focal country. barrett describes four equilibria in interactions between two countries that share an infectious disease (barrett, ) . in the first equilibrium, neither country engages in control, irrespective of what the other country does. in the second, each country eliminates the disease, irrespective of what its neighbour decides to do. in the third, each country eliminates the disease only if the other can be relied upon to do so. in the fourth, one country does not eliminate the disease, irrespective of what the other does. when countries are not identical in either epidemiological conditions or economic prosperity, it may be in the interest of some countries to eliminate malaria but for others not to, even if all others have eliminated malaria. yet elimination may be the optimal outcome for the two countries as whole. this is the case in which richer adjacent countries have financed elimination in poorer countries, as we observe in the lubombo spatial development initiative (lsdi). lubombo spatial development initiative lsdi offers a recent example of trans-boundary control of infectious disease (sharp et al., ) . malaria control was seen as an essential element of economic development in the lubombo region of eastern swaziland, southern mozambique (maputo), and north-eastern kwazulu natal province in south africa. malaria prevalence in these three regions was closely intertwined because of the frequent migration of people (sharp and le sueur, ) . most malaria cases in swaziland and kwazulu natal were imported from mozambique: for instance, nearly per cent of the malaria cases in kwazulu were in the district adjoining mozambique. between november and february , indoor residual spraying with bendiocarb insecticide was carried out twice a year in mozambique. spraying started in zone ( figure ) and proceeded incrementally, eventually covering seven districts and a population of roughly , people. in swaziland, where there were no other changes in malaria control efforts over the same time period, new malaria cases declined by per cent (table ). malaria cases declined by per cent in mpumalanga province, probably because during this period, indoor residual spraying and artemisinin-combination treatment were introduced on the south africa side of the border. nevertheless, the sharp decline in malaria in swaziland and south africa was attributable at least in part to efforts in mozambique, which were largely paid for by south africa and, to a lesser extent, by the global fund to fight aids, tuberculosis and malaria. west african river blindness programme coordinated financing, specifically with reference to multi-lateral financing to more than one country, is essential to permit a coordinated approach to disease control. however, such coordination has rarely been accomplished outside of global disease eradication programmes. there are a few examples of regionally coordinated financing such as against river blindness. the onchocerciasis control programme (ocp), which was launched in , covered major portions of seven western african countries (burkina faso, benin, ghana, côte d'ivoire, mali, niger, and togo). because the initial set of countries did not cover the limits of the breeding sites of the main vector, the savannah blackfly, the programme was expanded in to also include guinea, guinea-bissau, senegal, and sierra leone. a rare example of a transnational disease control effort launched by the world bank (kim and benton, ) , ocp relied on regionally coordinated larvicide spraying along the niger river to control black fly populations, and, at its peak, the programme covered m people in countries. this coordinated funding was in recognition of the fact that controlling black fly populations in a single country would be infeasible and required the cooperation of all seven countries on the niger river. through the mectizan donation programme, which was initiated in , onchocerciasis was eliminated as a public health problem in west africa. over the period - , the programme prevented , cases of blindness, and brought about m hectares of arable land-enough to feed an additional m people a year-back into productive use. the earliest efforts in global cooperation in the context of sanitary conventions, which required countries to report cholera outbreaks, subsequently led to the establishment of the pan american health organization, a pre-cursor to the world health organization (who) in the twentieth century. despite the benefits of warnings and reports on infectious disease outbreaks, there are few incentives for countries to report disease outbreaks that occur within their borders. current international health regulations, which were first enacted in and most recently revised in , require countries to report disease outbreaks. however, as there are no penalties for non-reporting, reporting depends on the goodwill of nations (baker and fidler, ) . this may not be entirely true since 'the consequences of non-compliance may include a tarnished international image, increased morbidity/mortality of affected populations, unilateral travel and trade restrictions, economic and social disruption and public outrage' (who, ) . specifically, if countries do not report promptly, other countries may take actions to moderate their trade and travel relations with the target country for fear that a future outbreak may also not be reported. we have discussed this in detail below as ex ante sanctions that precede an actual future outbreak. from a practical standpoint, countries face conflicting incentives as to whether or not to report an outbreak. on the one hand, reporting brings the near certainty of trade sanctions that can impose large costs. for example, when peru reported an outbreak of cholera in , its south american neighbours imposed an immediate ban on peruvian food products. the $ m cost of these sanctions and the additional $ m lost from reduced tourist activity far exceeded the domestic health and productivity costs of the epidemic (panisset, ) . on the other hand, countries may report an outbreak in the belief that the information will be reported anyway through the media or informal channels. furthermore, reporting an outbreak may result in international assistance for containing the outbreak. for instance, in the same peruvian outbreak, foreign aid in the form of rehydration salts, saline solution, and antibiotics, while unable to prevent an epidemic, helped to significantly reduce the death rate (brooke, ; suárez and bradford, ) . the appearance of new infections is determined by a number of factors, but generally is mediated by large growing populations that have poor nutrition and lack access to medical care (woolhouse and gowtage-sequeria, ) . however, despite the regular appearance of novel infections (woolhouse et al., ) , few infections are able to spread effectively within a population. over the last century, although more than diseases are believed to have emerged, only five novel diseases have swept across the globe-three were novel strains of influenza, another was hiv/aids, and more recently we saw the spread of sars, which emerged in china in november and spread around the world infecting more than , people in countries and killing approximately before it was contained (zhong et al., ) . further delays in reporting sars by china could have resulted in catastrophic consequences worldwide if the pathogen had been more virulent (heymann and rodier, ) . there is evidence that countries respond to external incentives on whether or not they report infectious disease outbreaks. an outbreak of meningococcal meningitis during the hajj resulted in more vaccination requirements for travellers coming to saudi arabia (laxminarayan et al., ) . these requirements, which were introduced in , were associated with reduced reporting of meningitis outbreaks among countries in sub-saharan africa, especially among countries with relatively few cases reported between and . the announcement of a programme in to assist countries with immediate vaccines conditional on their reporting of outbreaks was associated with an increase in reporting among countries that had previously not reported meningitis outbreaks (laxminarayan et al., ) . incentives for surveillance and reporting lie at the heart of an effective strategy to respond to avian influenza . mathematical models have suggested that it may be possible to contain an emerging pandemic of avian influenza if detection and reporting of cases suggestive of increased human transmission occurs within approximately weeks of the initial case (ferguson et al., ; longini et al., ) . while the who is responsible for coordinating the global response to human cases of avian influenza, decisions on establishing surveillance networks and reporting of outbreaks are the province of national governments. incentives to report an outbreak once it has been detected are only one part of the story, since an outbreak must first be detected. incentives to invest in surveillance to detect an outbreak are likely to be endogenous, and depend on whether or not a country wishes to report an outbreak (malani and laxminarayan, ) . these incentives are driven in part by the 'private' value of early detection to the individual country, but also by the likely consequences of the availability of this information to the rest of the world, either through the act of formal reporting or by informal channels, such as news reports or rumours. the greater the anticipated sanctions, the less likely a country will be to invest in surveillance. conversely, the higher the perceived benefit of international assistance in preventing or ameliorating the cost of an outbreak, the greater the likely investment in surveillance. current international mechanisms to encourage better reporting of disease have, by and large, ignored the economic dilemma and strategic behaviour of countries with emergent outbreaks. investments in surveillance also depend on the likelihood that the detected outbreak will produce a significant epidemic. the more a country believes a disease will arise and spread, the more significant the incentive to invest in surveillance. however, this investment can be tempered by the likelihood of false positives-the detection of a disease when none exists (malani and laxminarayan, ) . thus, a trade-off exists between investing in increased surveillance and investing in more accurate surveillance. a government's decision to report an outbreak can be modelled as a signalling game in which a country has private but imperfect evidence of an outbreak (malani and laxminarayan, ). an important conclusion is that not all kinds of sanctions may discourage reporting. what does this mean? let us divide sanctions into two kinds. ex ante sanctions are imposed in the form of reduced trade and travel contact with countries that are perceived to be poor at reporting disease outbreaks promptly. it is for this reason that west africa is not a favoured tourist destination-even in the absence of ebola, one is never quite sure if the system is able to detect and report this and other diseases. in contrast, an ex post sanction is imposed following a disease outbreak. ex post sanctions discourage detection and reporting since they kick in only after an outbreak has been announced. however, ex ante sanctions do not deter reporting and if anything they encourage reporting so that countries can signal that they are on top of their disease surveillance programmes. furthermore, ex ante sanctions based on fears of an undetected outbreak can reduce reliance on ex post sanctions as ways of controlling outbreaks. second, improving the quality of surveillance networks to detect outbreaks may not promote the disclosure of an outbreak because the forgone trade from reporting truthfully is that much greater. in sum, obtaining accurate information about potential epidemics is as much about incentives for reporting as it is about the capability and accuracy of surveillance networks. solving trans-boundary disease problems requires coordinated financing solutions, as has been evident with global eradication programmes. eradication of a disease means that it is no longer prevalent in any country in the world and requires elimination in every country. elimination, however, requires only the absence of the disease from a single country. global small pox eradication was largely paid for by the united states, even though countries like india stood to gain from the reduction in the number of deaths but were unable to achieve elimination on their own. however, the united states continues to recoup its roughly $ m investment in small pox eradication every days through not having to vaccinate its citizens against the disease. the optimal coverage with a vaccination programme of a disease that can be eradicated is given by p c = /( −r ) where r is the reproductive number of the disease-the number of secondary infections generated by a single infected patient entering a completely susceptible population. note that this critical rate of vaccination coverage depends only on the reproductive number (an epidemiological variable) and not on the costs of vaccination averted or any other economic variables. eradication may not be optimal in the case of all diseases, however. for diseases like measles, where the pathogen can be easily engineered through artificial methods and re-introduced into the population, there is no option of stopping vaccination. indeed, the current cohort of immunized individuals represents a valuable stock that is not easily replaceable in the short term. the optimal level of vaccination coverage of a disease for which vaccination must continue even after the disease has been eliminated can be computed as below. total costs to society include the costs of the vaccination campaign (vaccination costs), which we assume to increase exponentially with coverage, and costs of infection (infection costs) that we use as an index of the severity of the disease. the assumption that costs are increasing exponentially with coverage is consistent with the idea that reaching the most difficult to access and geographically remote populations involves increasing marginal costs. the total infection costs are proportional to the total number of the infective individuals in the population. because there is little evidence for increasing or decreasing marginal costs of infection within a single population (the change in total costs that arises from having one additional infection in the population), we assume constant marginal cost and model the costs of infection as a linear function of the infected. the total cost of the vaccination plus infection is then, with per capita burden c i . the cost of coverage is c(p) = ae xp , where a is the cost of vaccinating the first child (the cost of setting up the programme), and x captures the increase in costs with the increasing coverage p. when there is no immigration, we can calculate the economic optimum by minimizing eq. to find the level of coverage that minimizes total costs, which is independent of transmission. if the economic optimum p i is above the critical elimination threshold, p c = − /  , the optimal strategy is to eliminate the infection locally: (details in appendix in klepac et al. ( ) ). local elimination can be optimal also in the case of very severe diseases. in fact, for large enough per capita burden c i , i.e. the economic optimum p i is always above p c , and optimal vaccination coverage p* is reduced to the critical elimination threshold determined by  (eq. ). the optimal level of vaccination coverage for a disease that cannot be eradicated is a function of only economic parameters. indeed, epidemiological parameters play no role at all. local elimination is optimal only for low  values that result in a critical elimination threshold p c that is smaller than p i . moreover, adding immigration of infection to a single population precludes elimination by local vaccination alone. drug resistance is a global commons problem and covers the full range of infectious disease-causing pathogens from viruses, bacteria, fungi, and parasites through to disease vectors including mosquitoes, blackflies, and sandflies. resistance can arise in any single country and move globally. in this section, we focus on bacterial resistance and parasite resistance in the context of malaria. the global burden of resistance is poorly quantified but is likely to be concentrated in three major categories: increasing costs of resistant infections, increasing costs of antibiotics, and inability to perform procedures that rely on effective antibiotics to prevent infection. a primary burden of resistance is that resistant infections are more expensive to treat, and patients infected with resistant strains of bacteria are more likely to require longer hospitalization and face higher treatment costs than patients infected with drug susceptible strains (holmberg et al., ; the genesis report, ). an estimated , people die each year in europe from antibiotic-resistant bacteria (ecdc/emea joint technical report, ). in the united states in , an estimated , invasive methicillin-resistant staphylococcus aureus, or mrsa, infections required hospitalization and were associated with , deaths (klevens et al., ) . these estimates are useful for indicating the order of magnitude, but are imprecise because resistant infections are more common in individuals on long courses of antibiotic treatment: it is difficult to ascertain whether resistance is the cause of death or a correlate of long antibiotic treatment, hospitalization, and underlying sickness. in low-and middle-income countries, where the ability to pay for second-line drugs is limited, worse health outcomes are common, particularly in newborn children. even with effective antibiotics, neonatal infections are the major cause of neonatal deaths, which in turn account for more than a third of the global burden of child mortality (zaidi et al., ) . over half of neonates with extended spectrum beta-lactamase (esbl) sepsis are likely to die (versus a quarter of neonates with non-esbl infections), and a half of neonates with mrsa die (versus per cent of neonates with methicillinsensitive staphylococcus aureus) (kayange et al., ) . at these rates of mortality, one can estimate roughly , neonatal deaths attributable to gram-negative organisms and s. aureus, and , neonatal deaths attributable to esbl resistance and mrsa in india alone. a further cost of resistance is that associated with the cost of introducing new, expensive, antimicrobials to replace old ineffective ones (office of technology assessment, ) . this represents forgone resources that society could deploy elsewhere (reed et al., ) . according to one estimate, between and , increases in drug resistance raised the cost of treating ear infections by about per cent in the united states ($ m) (howard and rask, ) . resistance can also render broader health system functions such as surgeries, transplantations, and chemotherapy ineffective (laxminarayan et al., ) . a recent study estimated that, without effective antibiotics, - per cent of patients undergoing total hip replacements would have a postoperative infection, with a case-fatality rate of roughly per cent (smith and coast, ) . this category of burden affects both low-and middle-income as well as highincome countries and is likely to be the predominant way in which resistance drives up health care costs. take the case of drugs to treat malaria. the use of antimalarials places selection pressure on parasites to evolve resistance to these drugs. moreover, resistance is bound to arise when these drugs are misused, and could have adverse consequences for all malaria-endemic countries. efforts to manage resistance across national borders would have to rely on international agreements and regulations (walker et al., ) or on tax or subsidy instruments (arrow et al., ) . in the absence of such agreements and regulation, countries are unable to commit themselves to an optimal use of antibiotics, which would be in all countries' interests. at the macroeconomic level, a too intensive use of antibiotics in the health sector results in excessive levels of resistance both for that country and to the rest of the world (cornes et al., ) . a supranational authority would have to consider both the externality benefits of antibiotic use, in terms of reducing infections, and the costs, in terms of resistance (rudholm, ) . whether antibiotic consumption should be taxed or subsidized to reach the first-best outcome then depends on the relative magnitude of the externalities. in practice, the consequences of antibiotic use in sectors such as to make livestock grow faster involve little by way of positive externalities but impose resistance costs on other sectors and should therefore be taxed. a new class of antimalarial drugs, called artemisinins, requires a different way of thinking about optimal subsidies to manage resistance. when chloroquine, a oncepowerful antimalarial drug, became obsolete, the public health world was left with the challenge of optimally deploying the last remaining effective drug class, artemisinins. the who has recommended that artemisinins be used in combination with a partner drug that is unrelated to artemisinin's mechanism of action and genetic bases of resistance, so that a single mutation cannot encode resistance to both components (who, ) . artemisinin combination treatments (acts), if used instead of monotherapies of either artemisinin or the partner drug on its own, should slow the emergence of antimalarial resistance. however, the who guidelines are routinely flouted because monotherapies are much less expensive than acts. in response to this problem, an institute of medicine report (arrow et al., ) recommended establishing an international fund to buy acts at producer cost and resell them at a small fraction of that cost. on economic efficiency grounds there is a second-best case for subsidizing acts, because the ideal policy-taxing monotherapies and other antimalarials according to the marginal external cost from the elevated risk of the evolution of resistance-is infeasible, given their widespread use in the informal sector. the efficiency argument is further strengthened by the positive externality, to the extent that effective treatment of one individual reduces the risk of infection transmission to other individuals. laxminarayan et al. ( ) show that it is possible to determine the optimal subsidy in a dynamic diseasemodelling framework. bioeconomic analysis has been helpful for determining whether the social benefit from the subsidy, in terms of delayed resistance and saved lives, exceeds the social cost of resistance because of increased use of acts (laxminarayan et al., ) . it was also instrumental in turning an idea into the affordable medicines facility for malaria (amfm), a global financing system launched in early . amfm was formally evaluated in . in the six pilots where the programme was implemented to a substantial degree, amfm met or exceeded benchmarks for availability, price, and market share of quality-assured acts. in private, for-profit pharmacies, the quality-assured act market share at baseline ranged from to per cent (tougher et al., ) . a drawback of this evaluation was that it did not attempt to measure the impact on malaria prevalence or artemisinin resistance, both of which would have been difficult to ascribe to the intervention in the timeframe of the evaluation. nevertheless, the global fund to fight aids, tuberculosis and malaria made a political decision to discontinue amfm based on political objections raised by some country delegations (arrow et al., ) . one way to improve the efficiency of amfm resources was possibly to target children, though it would avert significantly fewer deaths. however, the benefits of a child-targeted subsidy (i.e. deaths averted) are eroded as leakage increases (i.e. older individuals taking young child-targeted doses), with few of the benefits (i.e. reductions in overall prevalence) of a universal subsidy (klein et al., ) . although potentially more cost-effective, a child-targeted subsidy must contain measures to reduce the possibility of leakage. most global health problems are 'commons problems'. therefore, it is often essential to have cooperative financing mechanisms for global health interventions, whether to eradicate disease, encourage appropriate levels of disease surveillance and reporting, or to reduce the likelihood of drug resistance. innovative financing that takes into account cross-country spillovers can play a critical role in arriving at globally optimal outcomes. for instance, in the case of the amfm subsidy, a high-level financing mechanism that lowers the cost of quality acts to all countries, including those that were at highest risk of using monotherapies, both enabled access to effective treatment and also reduced the threat of resistance. no bilateral financing solution could have achieved the same impact because of potential leakage to other countries, as discussed earlier. a global mechanism that is able to provide resources that incentivize surveillance and reporting of disease outbreaks can successfully counter the disincentives faced by countries for prompt reporting. again, bilateral assistance that simply focuses on subsidizing surveillance but does not pay attention to the lack of incentives for reporting cannot solve the problem. the three exemplars of trans-boundary problems that we have discussed can be applied to other global health problems with a public goods nature. saving lives, buying time: economics of malaria drugs in an age of resistance, board on global health the affordable medicines facility-malaria: killing it slowly global public health surveillance under new international health regulations the smallpox eradication game peru's neighbors halt food imports', the new york times drugs and pests: intertemporal production externalities the bacterial challenge: time to react the global value of vaccination strategies for containing an emerging influenza pandemic in southeast asia the recent history of malaria control and eradication global surveillance, national surveillance, and sars health and economic impacts of antimicrobial resistance studies of venereal disease. i. probenecidprocaine penicillin g combination and tetracycline hydrochloride in the 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childhood infections post-ebola the real war on drugs: bacteria are winning effect of the affordable medicines facility-malaria (amfm) on the availability, price, and market share of quality-assured artemisinin-based combination therapies in seven countries: a before-and-after analysis of outlet survey data vaccines bring diseases under control', unicef, the progress of nations frequently asked questions about the international health regulations host range and emerging and reemerging pathogens temporal trends in the discovery of human viruses the economic impact of ebola on sub-saharan africa: updated estimates for hospital-acquired neonatal infections in developing countries epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china key: cord- -rij f authors: langfeld, kurt title: dynamics of epidemic diseases without guaranteed immunity date: - - journal: nan doi: nan sha: doc_id: cord_uid: rij f the global sars-cov- pandemic suggests a novel type of disease spread dynamics. who states that there is currently no evidence that people who have recovered from covid- and have antibodies are immune from a second infection [who]. conventional mathematical models consider cases for which a recovered individual either becomes susceptible again or develops an immunity. here, we study the case where infected agents recover and only develop immunity if they are continuously infected for some time. otherwise, they become susceptible again. we show that field theory bounds the peak of the infectious rate. consequently, the theory's phases characterise the disease dynamics: (i) a pandemic phase and (ii) a response regime. the model excellently describes the epidemic spread of the sars-cov- outbreak in the city of wuhan, china. we find that only % of the recovered agents have developed an immunity. we anticipate our paper to influence the decision making upon balancing the economic impact and the pandemic impact on society. as long as disease controlling measures keep the disease dynamics in the"response regime", a pandemic escalation ('second wave') is ruled out. demic escalation ('second wave') is ruled out. introduction the rapid spread of a disease across a particular region or regions (epidemic) or the global outbreak of a disease (pandemic) [ ] can have a detrimental effect on health systems, on local and global economies including the financial markets and the socio-economic interactions, ranging from the city to the international level. measures to reduce the pandemic spread include curtailing interactions between infected and uninfected parts of the population, reducing infectiousness or the susceptibility of members of the public [ ] . the two major strategies governments use to handle an outbreak are to slow down an outbreak (mitigation) or to interrupt the disease spread (suppression). since each of those interventions bears itself significant risks for the societal and economic well-being, it is crucial to understand the effectiveness of these strategies (or any hybrid of them). mathematical methods provide essential input for governmental decision making that aims at controlling the outbreak. among those are statistical methods [ , ] , deterministic statespace models [ ] with its prototype developed by kermack and mckendrick [ ] , and a variety of complex network models, e.g., [ , ] . the different mathematical approaches have different objectives: a significant application of the statistical methods frequently aims at the early detection of disease outbreaks [ ] , while modelling either tries to develop a model as realistic as possible for a given outbreak or to design a simplistic model, which, however, reveals some universal truth about the outbreak dynamics. in the simplest version, the so-called compartmental models [ , ] consider the fraction of the population which is either susceptible (s), infected (i) or removed (r) from the disease network. coupled differential equations capture the dynamics of the disease that determine the time dependence of s, i and r. extensions add more compartments to the sir model such as (e) exposed. for example, such an seir model was used in [ ] for a description of the ebola outbreak in the democratic republic of congo in . compartmental models have been applied to describe the recent sars-cov- outbreaks [ , , , , , ] . for example, the elaborate model from giordano at al. uses a total of compartments -susceptible (s), infected (i), diagnosed (d), ailing (a), recognized (r), threatened (t), healed (h) and extinct (e) -to describe the covid- epidemic in italy. compartmental models have been extended in order to capture stochastically unknown influences, such as changing behaviours [ ] . such models were recently used to analyse the covid- outbreak in wuhan [ ] . compartmental models address global quantities such as the fraction of susceptible individuals s and assume that heuristic rate equations can describe the disease dynamics. in cases of a strongly inhomogeneous (social) network, e.g. taking into account different population densities, the above assumption seems not always be justified. in these cases, spatial disease spread patterns can be described by a stochastic network model with monte-carlo simulations a common choice for the simulation. in this paper, we consider a disease dynamics for which the duration (severity) of the illness depends on the amount of exposure. using an elementary (social) network, we are looking for universal mechanisms describing a pandemic spread. we will reveal a connection to statistical field theory, enabling us to characterise an outbreak with the tools of critical phenomena. we will discuss the impact of the findings on policies to curb an outbreak and will draw conclusions from the recent covid outbreak in hubei, china. model basics: we assume that each individual has two states u with u = -susceptible and u = -infected. each individual interacts with four 'neighbours' of the social network. the disease spread is described as a stochastic process. at each time step (say 'day'), the probability that an individual gets infected (or recovers) depends on the status of the neighbours in the social network. here, we only study the simple case of a homogeneous network with four neighbours for each site. we also consider periodic boundary conditions to minimise edge effects. immunity: we study two closely related scenarios. (i) there is no immunity. every individual can be reinfected and can recover only to be susceptible again. (ii) individuals can be reinfected and recover. only if individuals stay infected for τ consecutive days, they are considered immune. in case (ii), the sites of immune individuals are removed from the disease network. disease dynamics: if x is a site of the disease network, at every time step the state u x ∈ { , } is randomly chosen with probability where xy is an elementary link on the lattice joining sites x and y and, hence, n is the number of infected neighbours, and n x = + exp { β n x + h} is the normalisation. the parameter β describes the contagiousness of the disease. the parameter h is linked to the probability to contract the disease from outside the network. in fact, if no-one of the network is infected (n x = , ∀x), the probability p that any individual contracts the diseases, is connected to h by if the lattice contains n individuals (i.e., sites), one time step is said to be completed if we have considered n randomly chosen sites for the update. the pandemic spread as a critical phenomenon: scenario (ii) show the typical time evolution of an epidemic with the infections rate approaching zero for large times due to agents for a vanishing external field h, the model shows a critical behaviour with a phase transition at β = β c = ln( + √ )/ ≈ . . in the ordered phase for β > β c , a small seed probability phase. for β < β c , the model is in the 'response' phase, i.e., the infection rate is in repose to the seed probability p, but no outbreak occurs. the asymptotic infection rate can be calculated using markov monte-carlo methods. we used a modified swendsen-wang cluster algorithm, which performs near the phase transition [ ] . our numerical findings are summarised in figure , left panel. curve clearly separates both phases -the pandemic phase and the response regime. note that the dynamics ( ) corresponds to the standard heat-bath update [ ] . starting from a healthy population (u x = , ∀x), it takes the 'thermalisation' time t th that the daily infection rate starts fluctuating around its asymptotic value. immunity: let us now study scenario (ii), where individuals can develop immunity if they are infected for τ consecutive days. for τ > t th , the peak infection rate is that of the asymptotic state of the corresponding model (i) and, hence, inherits the classification 'pandemic' or 'response' phase. this is illustrated in figure , right panel, for the pandemic phase for several values of τ . figure illustrates the vastly different behaviour of the disease spread in the pandemic phase (β = . , p = %) and in the response regime (β = . , p = %). results are for a n = × network and τ = . note that the curve for 'infected+immune' ('triangle' symbol) in the pandemic phase is not monotonically increasing with time since the infected individuals can return to 'susceptible' state, i.e., not every infected individual becomes immune. note that in the response regime ('circle' and 'square' symbol), the 'pandemic' peak is absent altogether. however, on the downside, the so-called 'herd immunity' slowly develops over time. comparison with data: we stress that the model assumption of a homogeneous (social) network with 'four neighbours' is unrealistic. the knowledge of the underlying disease network is essential to make quantitative predictions for e.g. the critical value β c of the contagiousness. here, we adopt a different approach: we assume that qualitative time evolution of bulk quantities such as the fraction of infected individuals is within the grasp of model scenario (ii) and use those as fit functions to determine the model parameters such as β, p and τ by comparison with actual data. for this study, we used data from the covid- outbreak in in the hubei province in china [ ] . the data of the number of infected individuals show a jump at day (on the arbitrary time scale) by %, which is due to a change in reporting. we assume that the same 'under-reporting' has occurred in the days before, and have corrected the data by multiplying the number of infected (and infected+recovered) by a factor . for times t ≤ . let d(t, τ, β, p) be the fraction of the population of infected individuals as a function of time t and depending on the parameters τ (time to develop immunity), β (contagiousness) and p seed probability to get infected. we have calculated d(t, τ, β, p) using a n = × lattice. we checked that the result is independent of the lattice size in the percentage range for the parameters relevant in this study. if d wuhei (t) quantifies the measured values for the number of infected in the hubei outbreak, we want to approximate these data, i.e., with a suitable choice of the parameter n pop , t s , β and p. since the offset of the time axis in the hubei data is arbitrary, we have chosen the shift t s such that the peaks of simulated data and measures data coincide. all other parameters are treated as fit parameters. altogether, we find a good agreement with the data for: the model data overshoot the data in the early days of the epidemic spread, which could be related to underreporting due to limited testing capabilities. it is interesting to observe that the curve of the infection rate is asymmetric: the slope of the raise at the beginning is larger than the slope of the decline after the maximum. also, the number of infected seem to level off at a nonzero value. in the present model, this is explained as follows: with more agents being immune, it is harder for susceptible agents to be continuously infected for time greater or equal τ and, thus, to develop immunity. we also find that only about % of the infected (and recovered) develop an immunity. spread corresponds to the ordered phase of the field theory, and the critical value for the contagiousness is that for the phase transition. the disease is in controllable response mode if the corresponding field theory is in the disordered phase. quantitative results, reported here, are derived with an unrealistic homogeneous disease network for which each agent interacts with four neighbours. nevertheless, we find that the covid data of the hubei outbreak are well represented. for this case, we find that only % of infected develop an immunity. the heavy tail of the decline of the number of infected, which levels off at non-zero values, is an inherent feature of the model and can be traced back to the fact that agents can be reinfected. in a network with a sizeable portion of immune agents, it is increasingly challenging to develop immunity. if these model assumptions were underpinned by medical investigations, achieving 'herd immunity' would be difficult. this should influence the decision to what extent efforts focus on developing a cure or a vaccine. strategies for containing an emerging influenza pandemic in southeast asia statistical methods for the prospective detection of infectious disease outbreaks: a review statistical studies of infectious disease incidence a contribution to the mathematical theory of epidemics thresholds for epidemic outbreaks in finite scale-free networks the impacts of network topology on disease spread the mathematics of infectious diseases statistical inference in a stochastic epidemic seir model with control intervention: ebola as a case study modelling the covid- epidemic and implementation of population-wide interventions in italy mathematical modelling on phase based transmissibility of coronavirus a conceptual model for the coronavirus disease (covid- ) outbreak in wuhan, china with individual reaction and governmental action data-based analysis, modelling and forecasting of the covid- outbreak estimating clinical severity of covid- from the transmission dynamics in wuhan, china capturing the time-varying drivers of an epidemic using stochastic dynamical systems early dynamics of transmission and control of covid- : a mathematical modelling study beitrag zur theorie des ferromagnetismus statistical mechanics of lattice systems: a concrete mathematical introduction nonuniversal critical dynamics in monte carlo simulations an r package with real-time data, historical data and shiny app i thank lorenz von smekal (giessen) and paul martin (leeds) for helpful discussions. key: cord- -szb jm authors: reza khorramizadeh, m.; saadat, farshid title: animal models for human disease date: - - journal: animal biotechnology doi: . /b - - - - . - sha: doc_id: cord_uid: szb jm this chapter introduces some types of animal models which are used for better understanding the disease mechanisms and its treatment. these experimental models fall into two categories: spontaneous models and induced models. among the diseases, rheumatoid arthritis (ra) as an autoimmune disease was considered. to study the pathogenesis of ra, we explained collagen-induced arthritis as an animal model that reflects a characteristic feature of ra patients. in addition, experimental allergic encephalomyelitis (eae) as an experimental model for multiple sclerosis (ms) was explained in detail to represent a standard method to investigate in its mechanism, finding the way for the amelioration of this incurable neurological disorder. the architecture of human body is comprised in such a manner that cells cannot be considered as a separate entity. physiologically, homeostasis is the reason that these components live and perform their functions within that environment. disruption of this process leads to fatal conditions and is considered a disease. to investigate the mechanism of disease and to find the means to reverse adverse conditions, various strategies are used including cell-based assays and tissue culture studies. although these models can provide useful information, they fail to address various physiological conditions and the complex interactions among different cell types of tissues and organs. ideally a useful animal model for any disease has to have pathology similar to the disease conditions in humans. use of animals in research has a long history that dates back to the fourth century b.c. in the s, william harvey used animals to describe the blood circulatory system. many scientists, such as louis pasteur and emil von behring, have used animal models for experimental purposes to prove their hypotheses. animal models are good for understanding disease mechanisms and treatment and for overcoming the limitations of clinical trials that use human subjects. for example, experimental animal models for diseases like rheumatoid arthritis or multiple sclerosis have been successfully employed to screen new bioengineered, chemical, or herbal therapeutics that might have the potential for the treatment of human patients. so far, more than , studies have been reported in the ncbi database; they use animal models for different diseases. animal model studies have been the main reason for a better understanding of disease mechanisms. animal models of disease can be divided into two categories (kurkó et al., ) : spontaneous disease models and ( van heemst et al., ) induced disease models. in the case of induced disease models, induction can occur by various agents, both chemical and biological. this chapter discusses some of the most important animal models. rheumatoid arthritis (ra) is an autoimmune disorder with progressive occurrence that preferentially affects peripheral joints. in spite of the fact that ra is severe and crippling and affects large numbers of people, very little knowledge about its etiology and pathogenesis is available in the literature. rheumatoid arthritis affects about % of the population. the ratio of the prevalence of ra in males and females is À . . ra can occur at any age, but it is mainly reported to affect the -to -year-old age group. no doubt the incidence has been reported to increase with age. the etiology of ra is unknown, but it has been predicted that genetic and environmental factors play an important role in the onset of ra. recent advances have identified genetic susceptibility markers both within and outside of the major histocompatibility complex (mhc). human leukocyte antigen (hla) genes located on chromosome p have been found to have a strong association with rheumatoid arthritis. the contribution of hla to heritability of ra has been estimated to be %À %. individuals carrying hla-dr and hla-dr alleles have been shown to have a higher risk of ra. apart from the known shared epitope alleles (hla-drb à , drb à ), other hla alleles, such as hla-drb à and drb à , have been linked to ra susceptibility (kurkó et al., ) . the hla class ii locus is the most important risk factor for anticitrullinated protein antibodies (acpa)-positive ra (acpa ra) (van heemst et al., ) . a positive correlation has been suggested for the role of hla in terms of the severity of ra rather than the onset of the disease. the most relevant non-hla gene single nucleotide polymorphisms (snps) associated with ra include ptpn , il r, traf , ctla , irf , stat , ccr , and padi (kurkó et al., ; suzuki and yamamoto, ; stanford and bottini, ) . although the data regarding this conclusion are inconsistent, some of the studies have shown associations between tumor necrosis factor (tnf) alleles and rheumatoid arthritis. other genes like those for corticotrophin-releasing hormone, interferon (ifn)-γ, and interleukin- (il- ) have also been implied for ra. it can be concluded that the role of genetic components in ra is modest at the best (viatte et al., ) . epigenetics is another important factor that contributes to ra. in the case of identical twins, ra has not been shown to have % concordance; therefore, the role of nongenetic factors has also been implicated in the etiology of ra (meda et al., ) . throughout the world, rheumatoid arthritis is more common in women than in men. this indicates that hormones may play an important role in the development of the disease. pregnancy has also been considered as a risk factor for rheumatoid arthritis. studies show that the onset of ra is rare during pregnancy, but the risk increases after delivery. smoking is associated with increased incidences of ra, especially in men. on the contrary, populations that consume a diet high in omega- fatty acids have been reported to be protected from rheumatoid arthritis. from experimental models in animals, a large number of infectious agents such as viruses and bacteria have also been suggested to trigger or contribute to the development of rheumatoid arthritis. however, no relationship between infectious agents and the development of ra has been found. tissue edema and fibrin deposition are prominent and can manifest clinically as joint swelling and pain. within a short period, the synovial lining becomes hyperplastic, commonly becoming ten or more cells deep and consisting of type a (macrophage-like) and type b (fibroblast-like) synoviocytes that produce glycosaminoglycans (e.g., hyaluronan, as reported to be present in synovial tissue and synovial fluid). the sublining also undergoes alterations for its cellularity, both in cell type and in cell numbers, with prominent infiltration of mononuclear cells, including t cells, b cells, macrophages, and plasma cells. the abundance and activation of macrophages at the inflamed synovial membrane correlates significantly with the severity of the disease. activated macrophages over-express major histocompatibility complex (mhc) class ii molecules and produce pro-inflammatory or regulatory cytokines and growth factors [il- , il- , il- , il- , il- , il- , tnf-α, and granulocyte macrophage colony stimulating factor (gm-csf)], chemokines [il- , macrophage inflammatory protein (mip- ), monocyte chemoattractant protein (mcp- )], metalloproteinases, and neopterin. these biomolecules are routinely detected in inflamed joints. most of the t cells infiltrating the rheumatoid synovium express cd ro and cd , which is an indication that the t-cell subset present in the synovium is memory helper t cells. surprisingly, %À % of the t cells present in the case of the synovium have granzymes a and perforins. this %À % of cells present in the synovium represents cytotoxic t-cell subsets. therefore, it can be concluded that cd -expressing cells are infrequent in the synovium. in the synovial fluid of rheumatoid arthritis patients, cd and cd t cells are equally represented. tcrα/tcrß is expressed on most of the t cells while only a minority of cells show tcrγ/tcrδ expression. it has, however, been found that the expression of tcrγ/tcrδ is increased in the synovium of patients with active ra. synovial-vessel endothelial cells transform into high endothelial venules early during the course of disease. high endothelial venules are specialized post-capillary venules usually present in secondary lymphoid tissue or inflamed nonlymphoid tissues; these venules facilitate the transit of leukocytes from the bloodstream into tissues. the cytokine-mediated events have conventionally been viewed in the milieu of the cd th /th paradigm. nowadays, newer cytokines of the il- /il- axis and others have changed investigations into the immunopathogenesis of arthritis. both the cd th and γδ-t cells secreted il- which is a chemotactic for neutrophils and its response inhibited by il- due to ifn-γ induction. the roles of other cytokines such as il- and il- in arthritis have been clarified further with inhibitors of them (veenbergen et al., ; palmer et al., ) . recent studies in arthritis models have revealed new aspects toward regulatory t cell (treg) activity. in the cia model, treatment with il- induced the regression of arthritis via expansion of regulatory t cells (kochetkova et al., ) . the formation of locally invasive synovial tissue (i.e. pannus) is a characteristic feature of rheumatoid arthritis. pannus is involved in the erosion of joints in rheumatoid arthritis. pannus is histologically distinct from other regions of the synovium and shows phases of progression. initially, there is penetration of cartilage by synovial pannus, which is composed of mononuclear cells and fibroblasts, with a high-level expression of matrix metalloproteinases (mmps) by synovial lining cells. in later phases of the disease, cellular pannus can be replaced by fibrous pannus comprised of a minimally vascularized layer of pannus cells and collagen overlying cartilage. the tissue derivation of pannus cells has not been fully elucidated, although they are thought to arise from fibroblast-like cells (type-b synoviocytes). in vitro work shows that these fibroblast-like synoviocytes have anchorageindependent proliferation and loss of contact inhibition, which a phenotype is usually found in transformed cells. however, the molecular pathogenic mechanisms driving pannus formation still remains poorly understood. the range of presentations of rheumatoid arthritis is broad, but the disease onset is insidious in most cases, and several months can elapse before a firm diagnosis can be ascertained. the predominant symptoms are pain, stiffness, and swelling of peripheral joints. although articular symptoms are often dominant, rheumatoid arthritis is a systemic disease. active rheumatoid arthritis is associated with a number of extraarticular manifestations, including fever, weight loss, malaise, anemia, osteoporosis, and lymphadenopathy. the clinical course of the disorder is extremely variable, ranging from mild, self-limiting arthritis to rapidly progressive multisystem inflammation with a profound morbidity and mortality. analyses of clinical course and laboratory and radiological abnormalities have been defined as negative prognostic factors for progressive joint destruction; unfortunately, none of these are reliable enough to allow therapeutic decisionmaking. frequent assessment of disease symptoms and responses to therapy is crucial for a successful and long-term management of rheumatoid arthritis. joint destruction from synovitis can occur rapidly and early in the course of the disorder; radiographic evidence is present in more than % of patients within the initial years. more sensitive techniques such as magnetic resonance imaging (mri) can identify substantial synovial hypertrophy, bone edema, and early erosive changes as early as months after the onset of disease. these radiographic changes predate misalignment and functional disability by years; by the time physical deformity is evident, substantial irreversible articular damage has commonly occurred. furthermore, the biopsy analysis of clinically symptomless knee joints in patients with early rheumatoid arthritis shows active synovitis, highlighting the poor correlation between clinical assessment and disease progression, and the rapid development of polyarticular synovitis. the main goal of ra treatment is to stop inflammation, relieve symptoms, prevent joint damage, and reduce long-term complications. the past decade has seen a major transformation in the treatment of rheumatoid arthritis in terms of approach and choice of drugs. the previous therapeutic approach generally involved initial conservative management with nonsteroidal antiinflammatory drugs (nsaids) for several years; disease-modifying antirheumatic drugs (dmards) were withheld until a clear evidence of erosion was seen. dmards were then added individually in slow succession as the disease progressed. this form of treatment has been supplanted by early initiation of dmards and combination dmard therapy in patients with the potential for progressive disease. the idea of early intervention with the conventional disease-modifying antirheumatic drugs (cdmard) has been validated in several randomized trials. cdmards contain medications from different classes of drugs including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards are often partly effective and poorly tolerated for long-term therapy. in metaanalyses of dropout rates from clinical trials, %À % of patients discontinued the use of dmards assessed as monotherapy during the duration of the trial; even in clinical practice, the median duration of dmard monotherapy was less than years for nonmethotrexate agents. although there are many reasons for the lack of long-term adherence to treatment, poor efficacy, delayed onset of action, and toxic effects are major limitations. additionally, dmards therapy requires patients to undergo frequent monitoring of blood and physical examinations for toxic effects of treatment protocol. results from clinical trials showed that dmard therapy decreased markers of inflammation such as erythrocyte sedimentation rate and swollen joint counts, and that improved symptoms in a selected subset of patients; however, most patients continued to show progression of irreversible joint destruction on radiography. cdmards is increasingly burdened by side effects or clinical inefficacy, so other immunosuppressive drugs such as tacrolimus that blocks t-cell activation by specifically inhibiting calcineurin pathway and leflunomide have been developed. a new synthetic dmard, iguratimod, which exerts its action by the inhibition of the inflammatory cytokines (tnf-α, interleukin (il)- β, il- , il- , and il- ), is recently developed. the findings illustrate the consequences of progressive disease and have shown the need for the development of new and more effective therapies based on the therapeutic principles used for oncology; it means that treatment protocols for ra patients require the use of several therapeutic agents from different classes to be used in combination. recent studies have shown that combination therapy of biological dmards like tnfα inhibitors with methotrexate has clear-cut benefits with tolerable toxic effects. treatment with agents that can block tnf-α function has proved to be highly effective against ra. further studies reported downregulation of synovial gm-csf, il- , and il- , suggesting that tnf-α supports the production of other pro-inflammatory cytokines. however, the mechanisms behind the clinical effect of the tnf-α-blocking treatment are not fully understood. in an animal model, tnf-α-blocking agents such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody) reduce the expression of vascular adhesion molecules and inhibit the spontaneous production of il- and il- . patients with a new onset of symptoms and those with diseases of several years' duration and who had failed previous dmard therapy all benefited. these results suggest that patients in many stages of disease progression can benefit from combination therapy (chiu et al., ) . with the approval of tnf-α inhibitors (infliximab, etanercept, adalimumab, certolizumab, and golimumab), non-tnf biologic agents (rituximab, abatacept, tocilizumab, and anakinra), and other biologic agents, determining advances in treatment options of ra were made. rituximab (chimeric monoclonal antibody targeted against cd ) is a selective b-cell depleting agent for treating refractory rheumatoid arthritis. abatacept selectively modulates t-cell co-stimulation and has shown efficacy in several clinical trials. tocilizumab, a humanized monoclonal antiinterleukin- receptor antibody, has proven to be efficacious in patients who did not respond to methotrexate or other synthetic dmards. recently, several clinical trials have focused on a new class of drug: the janus kinase (jak) inhibitors. jaks are a family of nonreceptor tyrosine kinases . animal models for human disease (jak , jak , jak , and tyk ) involved in the intracellular signal transduction of many cytokines. tofacitinib is a pan-jak inhibitor that primarily inhibits jak and jak . in addition to tofacitinib, other jak inhibitor molecules including baricitinib, peficitinib, and decernotinib have also been studied in rcts. finally, filgotinib is a selective jak inhibitor which is currently in clinical development for the treatment of ra (calabrò et al., ) . as mentioned before, pro-inflammatory/regulatory cytokines and growth factors play important roles in the pathogenesis of ra. therefore, each of them or their pathway represents an attractive therapeutic target for ra. tocilizumab, a humanized monoclonal antibody targeting il- receptor, has already been approved for the treatment of ra in patients who failed to achieve remission with cdmards. another cytokine that plays an important role in the pathogenesis of ra is il- in which ixekizumab and brodalumab as humanized monoclonal antibody were developed against il a and its receptor. a new possible therapeutic target for the treatment of ra is the gm-csf pathway. the efficacy and safety of mavrilimumab (an anti-gm-csf receptor monoclonal antibody) in patients with moderate-to-severe ra has been investigated (takeuchi et al., ) . as an increased activation of osteoclasts contributes to bone erosions in ra, the inhibition of rankl that is essential for the osteoclast activation by denosumab (human monoclonal antibody against rankl) can reduce joint destruction in ra patients. takeuchi et al. ( ) , finally, do not forget that certain nutritional components interfere in the pathological inflammatory process, so that they should be considered as coadjuvant in the treatment of ra. it has been mentioned that flavonoids reduce cytokine expression and secretion. in this regard, flavonoids may have a therapeutical potential in the treatment of inflammationrelated diseases as cytokine modulators (rosillo et al., ; leyva-lópez et al., ) . in order to study the pathogenesis of ra, one can use different animal models. there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease, there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate a particular aspect of the disease. despite the fact that ra is not a spontaneously developing disease, spontaneously developing models for arthritis may be useful to study the role of genetics in the development of the disease. an activated immune response was reported in models such as the human tumor necrosis factor-α transgenic (htnftg), interleukin receptor-α (il- ra) knockout, il- ractivating mutation knockin, or skg mouse which bears the primary inflammatory response in joints (keffer et al., ) . in addition, arthritis can be rapidly induced with an adoptive transfer of t-helper cells in the il- r knockin mouse. transgenic mice expressing a tcr specific for bovine pancreas ribonuclease develop spontaneous arthritis that is mediated by antibodies (korganow et al., ) . this model is particularly interesting because it demonstrates that t cells specific for a ubiquitous antigen may induce an organspecific autoimmune disease. the expression of the gene product causes an upregulation of several cytokines (il- , il- , tgf-β , ifn-γ, and il- ) and subsequent development of arthritis (iwakura et al., ) . there are some other spontaneous models for arthritis in nontransgenic mice (bouvet et al., ) . arthritis can be induced by complete freunds' adjuvant (cfa). pearson (pearson, ) described this model for the first time. subsequently, it was demonstrated that other adjuvants, such as ifa, pristane, or squalene, could also induce arthritis (carlson et al., ) . microbially derived products such as lipopolysaccharide (lps), muramyle dipeptide (mdp), and trehalosedimycolate (tdm) can also induce arthritis when given with mineral oil (lorentzen, ; kohashi et al., ) . collagen-induced arthritis (cia) is normally induced by the immunization of susceptible mouse (e.g., dba/ ) or rat (da, lewis) strains at the base of the tail. the inoculum used for immunization contains both adjuvant and collagen type ii. the adjuvant has to be sufficiently strong to cause tissue destruction as well as induction of a strong pro-inflammatory immune response (holmdahl and kvick, ; kleinau et al., ) . susceptibility to cia is dependent on both mhc (class ii region) and non-mhc genes (lorentzen and klareskog, ) . antibodies against collagen ii are essential for the development of cia. this fact has been demonstrated by the passive transfer of anti-cii antibodies, which results in synovitis (svensson et al., ) . t cells are also important for cia development during early stages of disease progression. the dependence of both t-and b-cell responses has also been demonstrated in the same model (seki et al., ) . in cia, an immune response is being directed against a joint collagen type ii (cii) antigen. inflamed joints in cia are infiltrated by inflammatory cells that accumulate in the synovial membrane and fluid, similar to ra. the most frequent cell type in the synovial fluid is granulocyte. there is also a great infiltration of leukocytes into the synovial membrane. these cells have signs of an activated phenotype of ra since mhc class ii molecules are expressed (klareskog and johnell, ). in addition, there is an intense production of macrophage-derived cytokines in inflamed joints (e.g., tnf-α and il- β) (mussener et al., ; ulfgren et al., ) . a small number of t cells are encountered, and some of these t cells have il- receptor α chain upregulated. the disease shows a thickened synovial membrane that subsequently forms a pannus on the cartilage surface (holmdahl et al., ; holmdahl et al., ) . in both cia and ra, cartilage and bone destruction occurs mainly at the carti-lageÀpannus junction. there are some features of the pathology of cia that differ from what is usually observed in ra (e.g., extra-articular manifestation). although the compatibility of the cia model to human ra has been argued, many pathological features of cia are similar to those of rheumatoid arthritis. currently, collagen type ii-induced arthritis in mice and rats is one of the most widely used arthritis models in academia and industry. experimental collagen-induced arthritis was initiated by injecting bovine collagen type ii at the base portion of the tail of the animal (saadat et al., ) . male lewis rats weighing about À g were used. after the induction of cia, animals were divided randomly into four or more groups based on the experimental design. at least four different groups were needed, including a control group without arthritis, animals with collagen-induced arthritis, cia animals with treatment, and cia animals treated with methotrexate as a positive control. sample preparation: bovine collagen type ii (cii) was dissolved in . m acetic acid at a concentration of mg/ml by stirring overnight at c (the dissolved cii can be stored at c if it has to be used at a later time). before injecting the animals, cii was emulsified with an equal volume of complete freund's adjuvant (cfa). for the induction of cia, on day rats were injected intradermally at the base of the tail with μl of emulsion (containing μg of cii). after À days, animals showed the development of inflammation at peripheral joints ( fig. . ). on day , a booster injection of cii in cfa was administered. this model was used to evaluate the anti-ra effect by giving intraperitoneally injections of test materials (e.g., chemical or herbal extracts). methotrexate was a control used to evaluate the effect of the test compound and to compare the efficacy of the new compound with methotrexate. in this model, the test compound was given from day , where the frequency, route of administration, and dose could be selected as needed. the end point and days for the evaluation of different parameters were selected; one of the most common points was day (flow chart . ). the paws and knees were then removed for the histopathological assay. the visual observation can be done by using the macroscopic system as given in table . . moreover, rats immunized with cfa should be checked for weight gain from the first to the end of experiment at least every other day. the decline in body weight that followed on the onset of arthritis was proportional to the disease severity and, hence, can be used as a measure of disease activity. the scaling to record the observation should be from to for each paw (szabó et al., ) . on day , animals were anesthetized with sodium pentobarbital ( mg/kg intraperitoneally) and euthanized. blood was collected by intracardiac puncture, and paws and knees were removed, trimmed, and fixed in % buffered formalin, decalcified, and then embedded in paraffin, sectioned at μm, and stained with hematoxylin and eosin for the histological examination. joint damage was assessed based on synovial hypertrophy, pannus formation, inflammatory cell infiltration, and cartilage and subchondral bone destruction. joint erosion was graded on a scale of À for each limb (table . ), according to the severity of damage ( fig. . ). radiological scoring was performed by an investigator who was blind to the treatment protocol (on day ). radiographical analysis of affected joints in control rats typically showed soft tissue swelling, joint space narrowing, reduced lucency due to demineralization, and areas of recalcification indicative of new bone formation. a score was assigned to each joint on the basis of the information as listed in table . . scores were À per joint ( , normal; , maximum joint destruction). multiple sclerosis (ms) is a chronic inflammatory demyelinating disorder of the central nervous system (cns) that affects over . million individuals worldwide. similar to the affected population in other autoimmune diseases, twice as many women as men have ms. multiple sclerosis, like many other diseases, has existed as long as human life. in the s, the first report by dr. jean-martin charcot certified ms as a disease. a patient of him who suffered an unusual symptom died. after dissection, brain lesions were discovered. he called the disease scleroseen plaques. myelin was subsequently discovered, although its exact role was not recognized. about one century of research resulted in the discovery multiple sclerosis is the most common inflammatory demyelinating disease of the cns in europe and north america. the prevalence of ms in north america and europe is b À per , people. however, the prevalence is not globally uniform, geographically decreases in latitudes, and has been observed in only b À per , individuals in africa and asia. the etiology of ms is unknown. both genetic involvement and environmental factors have been indicated in ms. the only consistent correlation of involvement of the mhc locus is the mhc class ii allele hla-dr , which reflects a linkage with ms. in addition to associations within the major histocompatibility complex (mhc) region, other non-mhc loci reached a genome-wide significance. they map to the genes l mbtl , maz, erg, and shmt . products of the genes l mbtl , maz, and erg play important roles in immune cell regulation. shmt encodes a serine hydroxymethyl transferase catalyzing the transfer of a carbon unit to the folate cycle, which is important for establishment and maintenance of epigenetic signatures (andlauer et al., ) . some other factors like dietary components (e.g., milk), pathogens like human herpes virus (hhv- ), measles virus, epsteinÀbarr virus, and chlamydia have been implied as etiological factors. however, the association between any of these agents with ms is debatable. the presence of cns inflammation is a hallmark of ms. this inflammatory process greatly increases in the cns by the activation and deregulation of different cell types of the immune system. activation and entry of myelin-specific lymphocytes into the cns cause damage to oligodendrocytes, leading to demyelination. most of the cells from the immune system can contribute toward demyelination, but the main process of demylation is mediated by antibody and complement. so far, it has been noticed that antibody and complement are responsible for lesions in %À % of ms patients. myeloid cells may cause axonal damage by releasing molecules, such as glutamate, reactive oxy-gen species, and reactive nitrogen species. besides, these cells decreased the expression of glutamate clearance. as a result of increased glutamate in the cerebrospinal fluid, ms patients would be vulnerable to degeneration (yandamuri and lane, ) . in addition, cd t cells play an important role in ms pathogenesis. cd t cells make up the largest percentage of lymphocytes found in the brain of ms patients. all neuroectodermal cells in ms lesions express mhc class i molecules, making them an excellent target for cd t cells. in addition to their proinflammatory properties, cd t cells can also suppress the immune system and down-regulate inflammation. however, experiments with perforin, an important regulator of cytotoxic damage to immune cells, have made it clear that cd t cells present at ms lesions cause cytotoxicity, which could be the main source for demyelination and axonal damage (sinha et al., ) . bystander cd t cells do not contribute to the demyelinating process, but once cd t cells move into the cns and become activated against myelin antigen, these cd t cells could be contributing directly toward the demyelination of cns (basdeo et al., ) . moreover, cd th effector t cells are postulated to play a crucial role in the pathogenesis of ms (bettelli et al., ) . in addition to autoreactive immune cells against myelin and nerves, a progressive loss of the structure and function of neurons occurs. it has been reported that the alterations in the expression of mirnas may play a crucial role in ms pathogenesis (huang et al., ) . after demyelination, remyelination is possible, which could further damage the cns. the ratio of demyelination to remyelination determines whether a patient will develop secondary progressive ms (spms) or relapse remitting ms (rrms). if remyelination occurs before axonal damage, irreversible physiological damage can be prevented. none of the fda-approved therapies target oligodendrocytes to stimulate remyelination, but it is a very interesting possibility for future therapeutic intervention (huang et al., ) . the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. clinical motor manifestations include weakness, stiffness, and/or pain in arms or legs, abnormal reflex activity, and spasticity. often, the earliest symptoms of ms are somatosensory, including numbness and tingling. in ms, cerebral involvement is often accompanied by symptoms such as ataxia and intention tremor. many individuals with ms complain of increased urinary frequency, urgency, and incontinence. bladder and bowel disturbances remain among the most disabling and embarrassing symptoms experienced by ms patients. sexual symptoms are also very common among both men and women. fatigue, sleep disturbances, depression, and deficits in cognitive functioning are also common. the clinical course of ms is often highly variable and is generally characterized by relapses or exacerbations and deterioration of neurologic function, which entitle relapsing remitting ms. the features of relapsing remitting ms are defined as "episodes of acute worsening of neurologic function followed by a variable degree of recovery, with a stable course between attacks." approximately %À % of patients are initially diagnosed with rrms that evolves from an isolated demyelinating attack, which is characterized by multifocal inflammation along with varying degrees of axonal injury. a patient may experience disease progression with or without relapses and minor remissions; that clinical condition is defined as secondary progressive ms. it has been seen that % of rrma patients will eventually develop the spms state. primary progressive ms (ppms) affects b %À % of ms patients. ppms is defined as "disease progression from onset, with occasional plateaus and temporary minor improvements in clinical condition." the duration of ms varies significantly among ms patients. some patients will live with ms for several decades, while about % will develop an acute, fulminant form of ms. patients with an acute and fulminant form of ms show a rapid deterioration in their clinical signs and symptoms that have fatal consequences; these patients usually die within À years after the onset of disease. in general, the clinical spectrums among ms patients represent a benign disease and a low relapse rate, and these may never develop into secondary progressive disease. the heterogeneity of the clinical course of ms is shown to have a similar variation in its pathology. multiple sclerosis lesions were recently segregated into four distinct subtypes. the general pathology of ms, the formation of demyelinating lesions in the cns associated with infiltrating cd t cells, activated macrophages, and microglia-containing myelin debris, and infiltrating b cells, is common to all forms of the disease. it is thought that ms lesions are mediated by soluble factors such as tnf-α and immunoglobulin deposition on the myelin sheath, and the local activation of the complement cascade. the diagnostic criteria for clinically definite ms (cdms) include factors such as clinical history, mri imaging, and csf abnormalities. at present, there are no identifiable biomarkers that can predict the clinical subtype of ms. similarly, there are no factors that can assist in predicting whether a patient diagnosed with ms will develop either a progressive or a benign version of the disease. the clinical and pathological heterogeneity in ms has made it important to either develop or identify reliable biomarkers. several cytokines, immunoglobulins, mmps, markers of axonal/neuronal injury, and apoptotic markers have been suggested to have potential as biomarkers, but these biomarkers need validation by rigorous durability trials. in ms, treatment strategies can be either acute or long term. during a relapse, the goal of acute treatment is to reverse neurological disability as well as to delay further neurological dysfunction, so that the normal function can be restored. this type of treatment for ms patients is in contrast to the goals of long-term treatments. the main objective of long-term treatment for ms patients is to decrease relapses (both severity and frequency), which could lend support to stopping the progression of disability. patients experiencing a relapse, such as optic neuritis or transverse myelitis, are often administered high-dose corticosteroid firstline therapy. during progressive phases of the disease, patients may be prescribed immunosuppressive agents such as cyclophosphamide or mitoxantrone because the progressive phase is often accompanied by worsening inflammatory demyelination and axonal degeneration (rommer et al., ) . in , ifn-β was the first agent to demonstrate the significant clinical efficacy among patients suffering with rrms. although the exact disease-modifying effects of ifn-β in ms are unknown, several immunomodulatory mechanisms have been suggested. presently, two forms of ifn-β, including ifn-βla (avonex and rebif) and ifn-βlb (betaseron and extavia), have been prescribed. glatiramer acetate (copaxone) is a synthetic mixture of polypeptides that has been approved to treat rrms. similar to ifn-β, glatiramer acetate is found to be not effective for progressive forms of ms. natalizumab (tysabri) is an alpha- integrin antagonist and is the first drug of an entirely new class of immune-directed therapies that has been approved by the fda to treat relapsing ms. natalizumab is a humanized recombinant monoclonal antibody that blocks leukocyte migration into the cns by binding to α- integrins; these are components of the very late antigen- (vla- ) complex constitutively expressed on the leukocyte surface. in monotherapy trials, natalizumab has been reported to reduce the risk for sustained progression of disability as well as decrease the frequency of relapses. based on the current literature, natalizumab appears to be one of the most effective agents to prevent relapses as well as to stop disease progression. other monoclonal antibodies administered by intravenous (iv) infusion include lemtrada (alemtuzumab) and novantrone (mitoxantrone). currently, numerous other monoclonal antibodies are under investigation as potential therapies for ms; for example, anti-cd (daclizumab), anti-cd (rituximab), and so on. a number of other agents are under investigation for possible future use in ms including secukinumab (a humanized monoclonal antibody to il- ), rtl (inhibitor of the activation of myelin-reactive t cells), firategrast (affect on the vla- system) and aimspro (neuropeptide stabilizer). moreover, stem cell-based therapy might be considered as another approach for attenuating ms through regulating the immune system, although several challenges should be resolved. more investigations and clinical trials should be designed to assess the effectiveness of several drugs and approaches that can target both inflammatory and degenerative components of ms. these kinds of approaches may offer hope for individuals who are suffering from this debilitating disease (mansoor et al., ; agrawal and yong, ; hart and bainbridge, ) . to gain ideas about ms mechanisms, a number of models have been developed. these experimental models fall into two categories: spontaneous models and induced models. each model reflects characteristic features of ms patients and has its own merits and demerits. myelin basic protein mutant (taiep rat), proteolipid protein mutants (rumpshaker and jimpy mice), as well as gene-knockout animals (the myelin-associated glycoprotein (mag) knockout, thy -eb -yfp mice, and thy -xfp mice) show dysmyelination, altered neurotransmission and, in some instances, clinical disease. these models have frequently been used to study myelination. with chemically induced lesions, viral and autoimmune models are developed to show some evidence of demyelination, which is considered a pathological hallmark of ms. direct injection of ethidium bromide or lysolecithin into the cns produces demyelination. these induced models are usually effectively repaired once macrophages clear the myelin debris. for this reason, these models are rarely used at the present time. besides, local administration of glutamate or nitric oxide donors induces axonopathy in mice and have also been used to understand mechanisms of axonal degeneration and regeneration (luchtman et al., ) . a number of viruses, including semliki forest virus, theiler's murine encephalomyelitis virus, and a murine coronavirus have been found to induce disease by neurotrophic infection of the cns, specifically oligodendrocytes (lane and hosking, ) . moreover, studies using immunodeficient rag / mice have indicated that cd and cd t lymphocytes as well as macrophages are key contributors to demyelination in coronavirus-infected mice (dandekar et al., ) . finally, experimental allergic encephalomyelitis (eae) has received the most attention as a model for ms; this animal model is routinely used for testing different therapeutic strategies. today, eae as the most commonly used preclinical murine model of ms induced actively by the injection of defined encephalitogenic myelin protein epitopes plus cfa, or passively by the transfer of encephalitogenic myelin-sensitized t lymphocytes. some of these eae models also require the administration of the microbial-based immunologic adjuvant pertussis toxin (pt) (yandamuri and lane, ) . eae exhibits many clinical and histological features of ms and is caused by autoimmunity induced against antigens that are expressed either naturally or artificially in cns (denic et al., ) . the method for eae induction and preparation of antigens to induce eae in c bl/ mice was adapted from the method described by kafami et al. ( ) . it is important for the successful induction of eae to follow standard precautions for the use of animals. female c bl/ mice that are -to -week old are used for the induction of eae. animals must adhere to the normal laboratory animal maintenance guide. animals were immunized with the hooke kits (hooke labs, ek- , lawrence, ma, usa). it is recommended to follow the manufacturer's instructions. a mesh was dampened in ether and put in a desiccator. the mouse was kept in the desiccator and observed until breathing slowed down to ascertain whether the mouse had been anesthetized. the mouse was removed from the anesthetic chamber and laid on its side. two syringes were filled with ml of myelin oligodendrocyte glycoprotein (mog) emulsion with complete freund's adjuvant. each animal was given an injection of μl. the needle was gently inserted into the subcutaneous space at the base of the tail, and μl of emulsion was injected into the site. since it was difficult to give the mouse a -μl injection, every mouse was given a -μl injection at two different sites on the same day. immediately, and after hours from the first injection, each mouse was given an intraperitoneal injection of pertussis toxin ( μl/ animal). the animal was observed until complete recovery, and it could move without a floppy gate. this procedure was repeated for all animals. after À days, the flanks were bulging in response to the subcutaneous injection (flow chart . ). one day before immunization, and from the th to the th day post-immunization, the animals were evaluated on a daily basis for signs of eae following the -point score system (table . three different clinical parameters were analyzed to compare the course of eae (fig. . ) : ( ) severity of disease as the cumulative disease index (cdi) was the mean of the clinical scores of the animals; ( ) disease onset, calculated as the mean of the first-day animals showed the signs of the disease in experimental animals; and ( ) peak of disease score, which represented the mean of the highest clinical score of disease for all animals in each group. tonicity of the tail and the distal part of the tail was ascertained by touching the tip of the tail. if the distal part of the tail was flaccid, the animal was removed from the base and observed to see if its tail remained erect or fell down (examined with the touch of the finger). after ascertaining tonicity of the tail, the gate of the animal was observed by keeping it in an open area (like a tabletop) and allowing it to walk. after checking the gate, the hind limb was observed by grabbing its tail. after that, the paralysis score was recorded for unilateral paralysis. by holding the animal in the palm of the hand, it was easy to evaluate the type of paralysis (unilateral or bilateral). it was noted whether the mouse rolled spontaneously in its cage or was dead with complete paralysis. after days, animals that had an eae score of and did not change for more days were euthanized by chloral hydrate injection ( . ml, ip). for histopathlogical evaluations, different tissues were harvested after dissecting the animals. animals were placed appropriately in the dissection tray. a midline incision was made on the abdomen; the diaphragm was opened while ribbons were cut to expose the beating heart. the needle was inserted into the left ventricle of the heart while a phosphate-buffered saline (pbs) tap was allowed to fill the heart for seconds. the right aorta was cut with small scissors to allow the pbs and pfa to circulate to exit. pbs allowed perfusion until the liver turned from red to yellow (b À minutes). the best sign was when the liquid flowed out of the incised left aorta and turned from red to clear. another indicator was when pbs entered the pulmonary system and emerged through the nose of the animal. then, the pbs tap was closed and the tap was turned on for % paraformaldehyde (pfa, ph . at c) to allow pfa to flow and perfuse the circulatory system for minutes. perfusion was evaluated by involuntary hind limb movement and tail shivering. when the mouse became stiff, it was time to stop pfa perfusion. after the perfusion was complete with pfa, the system was washed with pbs to remove residual pfa. after perfusion, the various tissues of interest were harvested and stored in fresh % pfa for days at c. then, these tissues were washed with pbs and the pfa-fixed tissue could be stored in pbs for a few months. these tissues were then available for sectioning and staining (fig. . ) . for immunohistochemistry, the three sections showing the highest infiltrations were studied. an area $ . μm from the brain/spinal cord was selected and analyzed under magnification to assess the average number of positive cells per millimeter square and to quantify it on a computerized imaging system [bx microscope (olympus, hamburg, germany) with analysis software (special sis docu; soft imagingsystem)] by planimetry. the inflammatory index had to be calculated as a percentage determined by dividing the number of visual fields with . cd t cells by the total number of visual fields examined. detection of amyloid precursor protein (app) was performed for acute axonal damage. to assess the content of circulating proinflammatory cytokines like il- , il- , il- , il- , tnf-α, and ifn-γ, enzyme-linked immunosorbent assay (elisa) was employed. to evaluate the levels of different cytokines, blood was collected into tubes by a retro-orbital plexus method. the collected blood was kept in the tube to clot. after the clotting of the blood serum, it was separated and stored at c. these serum samples were then used for the evaluation of different cytokines using the elisa. in order to quantify the mrna of different proinflammatory cytokines such as tnf-α and ifn-γ, antiinflammatory cytokines like il- , myelin-deteriorating matrix metalloproteinase mmp- , and the content of . animal models for human disease myelin basic protein (mbp À ), samples from animals had to be analyzed by real-time pcr. animals were sacrificed with lethal injection and perfused with cold pbs. then, the limbs and muscles were removed with scissors and the skin removed from these organs. a transverse cut was made at the base of the skull and vertebral column to separate them. the nasal bridge was broken with a small scalpel and the eyeballs removed. very thin forceps were used under the skull bones to break it into pieces from the frontal to occipital lobes. the bony connection under the cerebellum was broken to expose the cerebellum. the broken bones of the skull needed to be removed. the nerve root connection with the brain was cut. the brain was removed and stored in liquid nitrogen. for the removal of the spinal cord, an oblique cut was made from the lateral side of the spinal cord (started from the cervical part) to the furthest part of the vertebral column (both sides). the spinal cord was then exposed by cutting the boney flap. the steps above were repeated to get to the coda aquina. the spinal cord was taken out by cutting its adhesion to the base. it was then stored in liquid nitrogen. the frozen tissue sample was used for rna extraction. first, the sample was homogenized by pushing and rotating it with a sterile glass homogenizer. next, the homogenate sample was left on the bench top at room temperature ( cÀ c) for minutes to promote the dissociation of nucleoprotein complexes. then, μl of chloroform was added to the tube and the tube was shaken vigorously for seconds. the tube containing the homogenate was placed on the bench top at room temperature for À minutes and then centrifuged again at , rpm for minutes at c. after centrifugation, the sample separated into three phases: an upper, colorless, aqueous phase containing rna; a white interphase; and a lower, red, organic phase. the upper, aqueous phase was transferred to a new sterile eppendorf tube. one volume (usually μl) of % ethanol was added to the tube containing the aqueous phase and mixed thoroughly by vortexing. visible precipitates after the addition of ethanol could then be noticed. up to μl of the sample was processed for total rna extraction by using an rneasy mini spin column (roche germany) according to the kit instructions. after rna extraction, rna was quantified spectrophotometrically and the purity of rna was ascertained by taking out a ration between the od at and nm. a quantitative real-time reverse transcriptase pcr was performed to analyze the levels of mrna of different cytokines using cytokine-specific primers. the first step was to perform cdna synthesis by using a cdna synthesis kit (takara, japan), which was followed by a syber green i real-time pcr master mix kit (takara, japan). a house-keeping gene (like the β-actin gene) was included in the study to compare the results. the use of laboratory animals in research is of major ethical concern. much of the argument revolves around moral values. today, there is a wide spectrum of views on animal rights. this has prompted the establishment of guidelines on the care and use of experimental animal models. the guidelines endorse some essential principles for the care and use of animals for scientific projects. the basis of these principles is to replace animals with other methods such as mathematical models, computer simulations, and in vitro biological systems, thus reducing the number of animals used in order to obtain valid results without unnecessary duplication, and finally, refining projects by selecting appropriate species and techniques to minimize pain or distress to animals using appropriate sedation or anesthesia. as a researcher, one must always assume that procedures that cause pain to humans will cause pain in such situations in animals. surgical procedures should be performed on anaesthetized animals. it should be kept in mind that if the animal would suffer severe pain during a procedure, or if at the end point cannot be alleviated swiftly, the animals must be killed humanely. the transportation, housing, feeding, and handling of animals are also important. housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care. the place should be designed to facilitate control of environmental factors. cages should be comfortable and should fulfill behavioral requirements such as free movement and activity, bedding, contact with others of the same species, lighting, temperature, air quality, appropriate day/night cycles, and protection from excessive noise. the population density of animals within cages should also be considered from an ethical standpoint. this statement refers to the need for the reader to operate in accordance with the guidelines at her/his academy. the concept of translational research is to try to convert the results derived in animal models into a new understanding of disease mechanisms and therapeutics in human beings. it is a bridge from experimental models to clinical medicine. over recent years, the importance of this kind or research has progressively increased. consequently, translational research is considered a key component to finding practical applications, especially within medicine. with the improvement of technologies, significant progress has been made in producing various types of engineered experimental animal models based on a better understanding of the molecular and genetic principles of disease. as a result, any interventions in experimental models are more practical and repeatable when compared to patient-oriented research. various risk factors that are linked to, or even responsible for, differences in clinical results should also be considered as significant for the development of experimental models; this will enhance the translational value of experimental models. these risk factors can be categorized into genetic factors, acquired factors, and health conditions, which can be studied in models in a controlled manner. in medicine, the performance of successful translational research requires data from hospitals. as we mentioned before, rheumatoid arthritis as a progressive debilitating disease is characterized by hyperplasia of synoviocytes leading to joint destruction and permanent deformity. although the definite pathophysiology of ra is ambiguous, some evidence suggests that telomerase is also involved in the pathogenesis of this disease. nobel laureates in physiology/ medicine in , elizabeth blackburn, jack szostak and carol greider, have solved a major problem of the chromosomal protection against degradation during cell divisions. they identified telomerase and a unique dna sequence in the telomeres. telomerase is a ribonucleoprotein enzyme that adds repeated units of ttaggg to the ends of chromosomes. this enzyme is composed of an rna component, called htert which serves as a template for addition of telomeric repeats. although it is now known that the dna sequence in the telomere attracts proteins that form a protective cap around the fragile ends of the dna strands, a number of reports have mentioned a link between the increased telomerase activity of human tumor samples and degree of invasiveness. in patients with ra, an impaired telomerase enzyme and premature cellular ageing (senescence) of thymic naïve and memory t cells was reported. moreover, transfection of rheumatoid arthritis synovial fibroblasts with vectors expressing antisense oligonucleotide against the htert component of telomerase enzyme has led to cytolysis of these cells that exhibit high telomerase activity. taken together, their discoveries have shed light on disease mechanisms and stimulated the development of potential new therapies in experimental models. there are many methods to evaluate telomerase activity, but we measured it by telomere repeat amplification protocol using trapeze telomerase detection kit (intergen, inc., usa) in animals treated with camellia sinensis stew. in detailed, biopsies of synovial tissue were obtained aseptically from the knee joints of rat after the induction of cia. synovial tissue specimens were rinsed, minced, and digested with . % collagenase in high-glucose dmem containing % fbs and antibiotics. following overnight incubation at c, cells were collected, plated in culture flask, and allowed to reach confluency at c in a humidified atmosphere of % co . after the lysis of equal number of cells which harvested from synovial tissue with the chaps lysis buffer, the telomerase was first extended for minutes at % oc and then amplified by cycles of pcr. the products of pcr were detected by polyacrylamide gels and revealed by silver nitrate staining. telomerase activity was calculated as the ratio of the intensity of telomerase ladders to the intensity of the -bp internal standard. in conclusion, we show that c. sinensis stew effectively suppresses collagen arthritis and a potent inhibitory effect on telomerase activity. so, natural products should continue to provide innovative lead compounds currently entering clinical trials. recently, the circular plant peptide kalata b (cyclotide) was investigated by thell et al. using the ms mouse model experimental autoimmune encephalomyelitis. according to their findings, treatment of mice with the cyclotide resulted in a significant delay and diminished symptoms of eae by oral administration. taken together, natural product should be considered as a candidate for the future investigations to possible implication for human health. using these above-mentioned models associated with other experimental models gives us such an opportunity to accomplish many findings in human medicine. until now, many progresses in medical sciences have been achieved. the discovery of numerous types of antibiotics for controlling infectious disease and elimination some viral disease like smallpox might be considered as one of researcher and indeed experimental animals honor. also, blood transfusions, open heart surgery, and other life-saving techniques have all been developed. nevertheless, there are many unsolved subjects included cancer, aging, alzheimer's disease, and acquired immunodeficiency syndrome in front of the society. without no doubt, until to find another means for answering human beings dilemma, the use of living animals in scientific research would be the best and applicable procedure. with all those valuable function, it is pivotal to consider ethical concerns over the quality of life of animals when you as a young researcher start to write a proposal. medlineplus is the national institutes of health (nih) site for patients and their families and friends. produced by the national library of medicine, it brings you information about diseases, conditions, and wellness issues in easy-to-understand language. medlineplus offers reliable, up-to-date health information, anytime, anywhere, for free. http://www.ebi.ac.uk/ipd/imgt/hla/ the imgt/hla database provides a specialist database for sequences of the human major histocompatibility complex (hla) and includes the official sequences for the who nomenclature committee for factors of the hla system. the imgt/hla database is part of the international immunogenetics project. adjuvant a substance such as complete freund's adjuvant (cfa) that enhances t-and b-cell activation, mainly by promoting the accumulation and activation of antigen-presenting cells at the site of antigen exposure. adjuvants stimulate the expression of t-cellactivating co-stimulators and cytokines by antigen-presenting cells and may also prolong the expression of peptideÀmhc complexes on the surface of these cells. autoimmune disease a disease caused by a breakdown of selftolerance such that the adaptive immune system responds to selfantigens and mediates cell and tissue damage. autoimmune diseases can be organ specific (e.g., thyroiditis or diabetes) or systemic (e.g., systemic lupus erythematosus). cd molecules cell surface molecules expressed on various cell types in the immune system that are designated by the "cluster of differentiation (cd) number." disease-modifying antirheumatic drugs (dmards) they contain medications from different classes including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards were often only partly effective and poorly tolerated in long-term therapy of autoimmune diseases. enzyme-linked immunosorbent assay (elisa) a method of quantifying an antigen immobilized on a solid surface by use of a specific antibody with a covalently coupled enzyme. the amount of antibody that binds the antigen is proportional to the amount of antigen present and is determined by spectrophotometrically measuring the conversion of a clear substrate to a colored product by the coupled enzyme. experimental autoimmune encephalomyelitis (eae) this is an animal model of multiple sclerosis, an autoimmune demyelinating disease of the central nervous system. eae is induced in rodents by immunization with components of the myelin sheath (e.g., myelin basic protein) of nerves, mixed with an adjuvant. the disease is mediated in large part by cytokine-secreting cd t cells specific for the myelin sheath proteins. granulocyte-monocyte colony-stimulating factor (gm-csf) a cytokine made by activated t cells, macrophages, endothelial cells, and stromal fibroblasts that acts on bone marrow to increase the production of neutrophils and monocytes. gm-csf is also a macrophage-activating factor and promotes the differentiation of langerhans cells into mature dendritic cells. granuloma a nodule of inflammatory tissue composed of clusters of activated macrophages and t lymphocytes, often associated with necrosis and fibrosis. granulomatous inflammation is a form of chronic delayed-type hypersensitivity, often in response to persistent microbes or to particulate antigens that are not readily phagocytosed. granzyme a serine protease enzyme found in the granules of ctls and nk cells is released by exocytosis, enters target cells, and proteolytically cleaves and activates caspases and induces target cell apoptosis. homeostasis in the adaptive immune system, the maintenance of a constant number and diverse repertoire of lymphocytes, despite the emergence of new lymphocytes and the tremendous expansion of individual clones that may occur during responses to immunogenic antigens. homeostasis is achieved by several regulated pathways of lymphocyte death and inactivation. human leukocyte antigens (hla) mhc molecules expressed on the surface of human cells. human mhc molecules were first identified as alloantigens on the surface of white blood cells (leukocytes) that bound serum antibodies from individuals previously exposed to other individuals' cells. interferons a subgroup of cytokines originally named for their ability to interfere with viral infections, but that have other important immunomodulatory functions. type i interferons include interferon-α and interferon-β, whose main functions are antiviral; type ii interferon, also called interferon-γ, activates macrophages and various other cell types. interleukins any of a large number of cytokines named with a numerical suffix roughly sequentially in order of discovery or molecular characterization (e.g., interleukin- and interleukin- ). some cytokines were originally named for their biological activities and do not have an interleukin designation. lipopolysaccharide (lps) a component of the cell wall of gramnegative bacteria that is released from dying bacteria and stimulates many innate immune responses, including the secretion of cytokines, induction of microbicidal activities of macrophages, and expression of leukocyte adhesion molecules on endothelium. lps contains both lipid components and carbohydrate moieties. major histocompatibility complex (mhc) molecule a heterodimeric membrane protein encoded in the mhc locus that serves as a peptide display molecule for recognition by t lymphocytes. two structurally distinct types of mhc molecules exist. class i mhc molecules are present on most nucleated cells, bind peptides derived from cytosolic proteins, and are recognized by cd t cells. class ii mhc molecules are restricted largely to dendritic cells, macrophages, and b lymphocytes, bind peptides derived from endocytosed proteins, and are recognized by cd t cells. matrix metalloproteinase (mmp) mmps are a family of highly conserved endopeptidases dependent on zn ions for activity. mmps can collectively cleave most extracellular matrix. at present, vertebrate mmps and human homologs have been identified and characterized. mmps participate in many physiological processes, such as embryonic development, organ morphogenesis, blastocyst implantation, ovulation, nerve growth, cervical dilatation, postpartum uterine involution, mammary development, endometrial cycling, hair follicle cycling, angiogenesis, inflammatory cell function, apoptosis, tooth eruption, bone remodeling, and wound healing. myelin oligodendrocyte glycoprotein (mog) mog is a cnsspecific type i membrane glycoprotein of the immunoglobulin superfamily expressed mainly on the outermost layer of the myelin sheath, making it an ideal target for antibody-mediated demyelination. it is highly immunogenic, and unlike other myelin proteins used to induce eae, is unique in inducing both an encephalitogenic t-cell response and a demyelinating response in eae. multiple sclerosis (ms) a chronic inflammatory demyelinating disorder of the central nervous system. the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. nitric oxide (no) a biologic effector molecule with a broad range of activities that in macrophages functions as a potent microbicidal agent to kill ingested organisms. pannus formation of locally invasive synovial tissue is a characteristic feature of rheumatoid arthritis. perforin a protein that is homologous to the c complement protein and is present in the granules of ctls and nk cells. when perforin is released from the granules of activated ctls or nk cells, it promotes the entry of granzymes into the target cell, leading to apoptotic death of the cell. rheumatoid arthritis (ra) an autoimmune disease characterized primarily by inflammatory damage to joints and sometimes inflammation of blood vessels, lungs, and other tissues. cd t cells, activated b lymphocytes, and plasma cells are found in the inflamed joint lining (synovium), and numerous proinflammatory cytokines, including il- and tnf, are present in the synovial (joint) fluid. reverse transcriptase (rt) an enzyme encoded by retroviruses, such as hiv, that synthesizes a dna copy of the viral genome from the rna genomic template. purified reverse transcriptase is used widely in molecular biology research for purposes of cloning complementary dnas encoding a gene of interest from messenger rna. th cells subset of cd helper t cells whose principal function is to stimulate phagocyte-mediated defense against infections via secretion of a group of cytokines, including ifn-γ. th cells subset of cd helper t cells whose principal functions are to stimulate ige and eosinophil/mast cell-mediated immune reactions via a particular set of cytokines, including il- and il- . th cells subset of cd helper t cells that are protective against certain bacterial infections and also mediate pathogenic responses in autoimmune diseases. tumor necrosis factor (tnf) a cytokine produced mainly by activated mononuclear phagocytes that stimulates the recruitment of neutrophils to sites of inflammation. tnf-α blocking agents a group of biological disease-modifying antirheumatic drugs such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody). very late antigen (vla) the set of integrins that shares a common beta- chain. long-answer questions immunopathogenesis of multiple sclerosis novel multiple sclerosis susceptibility loci implicated in epigenetic regulation immunology increased expression of tbet in cd t cells from clinically isolated syndrome patients at high risk of conversion to clinically definite ms reciprocal developmental pathways for the generation of pathogenic effector th and regulatory t cells spontaneous rheumatoid-like arthritis in a line of mice sensitive to collagen-induced arthritis one year in review : novelties in the treatment of rheumatoid arthritis the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats access to the next wave of biologic therapies (abatacept and tocilizumab) for the treatment of rheumatoid arthritis in england and wales axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus the relevance of animal models in multiple sclerosis research current and emerging treatment of multiple sclerosis hla and rheumatoid arthritis: how do they connect? vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type ii collagen-induced arthritis in the same rat strain are different diseases early appearance of activated cd t lymphocytes and class ii antigen-expressing cells in joints of dba/ mice immunized with type ii collagen involvement of macrophages and dendritic cells in synovial inflammation of collagen induced arthritis in dba/ mice and spontaneous arthritis in mrl/lpr mice micrornas associated with the pathogenesis of multiple sclerosis autoimmunity induction by human t cell leukemia virus type in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in c bl/ mice transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis induced expression of class ii transplantation antigens in the cartilage-pannus junction in ra: chronic synovitis as a model system for aberrant t-lymphocyte activation role of adjuvants in turning autoimmunity into autoimmune disease il- stimulation of cd regulatory t cells confers protection against collagen ii-induced arthritis via the production ofil- arthritis-inducing ability of a synthetic adjuvant, n-acetylmuramyl peptides, and bacterial disaccharide peptides related to different oil vehicles and their composition from systemic t cell self-reactivity to organ-specific autoimmune disease via immunoglobulins genetics of rheumatoid arthritis -a comprehensive review the pathogenesis of murine coronavirus infection of the central nervous system flavonoids as cytokine modulators: a possible therapy for inflammation-related diseases identification of arthritogenic adjuvants of self and foreign origin susceptibility of da rats to arthritis induced with adjuvant oil or rat collagen is determined by genes both within and outside the major histocompatibility complex in vivo and in vitro effects of multiple sclerosis immunomodulatory ther-apeutics on glutamatergic excitotoxicity the potential use of mesenchymal stem cells for the treatment of multiple sclerosis the epigenetics of autoimmunity cytokine production in synovial tissue of mice with collagen-induced arthritis (cia) inhibition of interleukin- signaling attenuates the severity of experimental arthritis development of arthritis, periarthritis and periostitis in rats given adjuvants an update on dietary phenolic compounds in the prevention and management of rheumatoid arthritis effect of pyrimethamine in experimental rheumatoid arthritis type ii collagen-induced murine arthritis. i. induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity cd ( ) t-cells as immune regulators of multiple sclerosis ptpn : the archetypal non-hla autoimmunity gene from genetics to functional insights into rheumatoid arthritis b celldeficient mice do not develop type ii collagen-induced arthritis (cia) protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly (adpribose) synthetase efficacy and safety of mavrilimumab in japanese subjects with rheumatoid arthritis: findings from a phase iia study effect of denosumab on japanese patients with rheumatoid arthritis: a doseresponse study of amg (denosumab) in patients with rheumatoid arthritis on methotrexate to validate inhibitory effect on bone erosion (drive)-a -month, multicentre, randomized, double-blind, placebo-controlled, phase ii clinical trial interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment the natural soluble form of il- receptor beta exacerbates collagen-induced arthritis via modulation of t-cell immune responses genetics and epigenetics of rheumatoid arthritis imaging axonal degeneration and repair in preclinical animal models of multiple sclerosis use of animals in scientific research. indian council of medical research ministry of health & family welfare new delhi animal models of rheumatoid arthritis and their relevance to human disease animal models of multiple sclerosis-potentials and limitations the rights of animals animal models of multiple sclerosis. neuroinflammation À . glossary adhesion molecule a cell surface molecule (e.g., selectin, integrin, and member of the ig superfamily) whose function is to promote adhesive interactions with other cells or the extracellular matrix describe the significance of animal modeling in biotechnology? how cia is induced and how the ability of medications is evaluated in mice? discuss about different types of animal model to study the pathogenesis of rheumatoid arthritis? why the presence of inflammation in the cns is considered as a hallmark of multiple sclerosis? explain the various methods for evaluation of experimental models of multiple sclerosis? short answer questions what is the reason of reportedly experiencing different animal models for studying the pathogenesis of rheumatoid arthritis? give an example which shows the impact of the epigenetics in the initiation of ra? which types of evaluation should be performed after "collagen-induced arthritis" aroused? . what are the "intervening factors after the activation of the immune system, which type of lymphocytes enters into central nervous system answers to short answer questions . there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate particular aspect of the disease. . the epigenetics of ra have also been responsible in the initiation of ra. since the concordance of rheumatoid arthritis in identical twins is not % other nongenetic factors also play a role in the disease etiology. . daily clinical assessment according to a macroscopic scoring system, histological processing and assessment of arthritis damage, radiographic evaluation by an investigator blinded to the treatment protocol on day . . age, weight, and possible infectious disease in animals should be considered as the intervening factors. . following activation, myelin-specific lymphocytes enter into the cns and oligodendrocytes are damaged.yes/no type questions . natalizumab blocks leukocyte migration into the cns by binding to icam. . eae induced actively by injection of the microbialbased immunologic adjuvant pertussis toxin. . housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care.answers to yes/no type questions . yes-the most important risk factor for acpa ra is the hla class ii locus. key: cord- - rkemm authors: pascual, isaac; maisano, francesco; mestres, carlos a. title: mind the gap versus filling the gap. the heart beyond specialties date: - - journal: rev esp cardiol (engl ed) doi: . /j.rec. . . sha: doc_id: cord_uid: rkemm nan pre-and postgraduate medical education is a complex dynamic process, with a crucial influence on the future of our specialties and on the wellbeing of society. a critical aspect is the need to constantly maintain and update knowledge, aiming to preserve and increase professional capabilities to serve the patient. this is the goal of continuous medical education. the specialty training period involves facing daily clinical practice, generally in a teaching hospital. some important aspects are the hardness of training that can influence one's life experience, the stress of taking on-not always progressivelyresponsibilities, the possible shortfalls of the system and, as in the case of spain, poor remuneration in relation to effort and responsibilities. physicians live with this for long time, currently, even more intensely, if possible; an example is the current situation of physicians in training in different autonomous communities in spain. medical education therefore needs periodic review to selfadapt to changes according to the accumulated knowledge and technological development. in the field of cardiovascular medicine, this has been a constant since the beginning of cardiac surgery and of what is currently known as interventional cardiology. cardiovascular surgery and cardiology are of the oldest medical specialties in spain, as described in the law of medical specialties for a set of ; this law was later confirmed by the royal decree / of january , which regulated specialized medical training to obtain the title of medical specialist for a set of specialties. in these more than years, the paths traveled by both specialties have at some point diverged. the training programs have evolved in parallel, and to date those of for cardiology and of for cardiovascular surgery are still in force. regarding the latter, some updates to the training programs have not yet been approved by the general directorate of professional management of the spanish ministry of education, the regulatory body overseeing training programs in spain. for various reasons, it does not seem that the restructuring that would be appropriate is on the professional or political horizon. we are currently witnessing changes in the approach to disease and patients. multidisciplinary teams dedicated to cardiovascular disease have flourished in the last decade, following the example of cardiovascular infections. what is currently termed the ''heart team'' has been a logical development related to the progression of our knowledge and interdisciplinary cooperation over the years. coronary artery and structural heart diseases are assessed differently if discussion involves diverse professionals. these groups have for some time now been considered necessary and essential for the surgical treatment and transcatheter therapy of structural heart disease. , assuming the differences that have existed to date between cardiologists and surgeons, which still exist and will most likely exist in the future, simply because of the different views of the specialties on the same problem-for example in structural heart disease-past developments have revealed that different training itineraries end up with the performance of the same procedure. as mentioned, the teaching programs in cardiovascular surgery and cardiology in spain would need a review of these training itineraries, taking into account the need to converge on the final point that gives meaning to the entire process: the patient and the quality of the care delivered. discussion about who should provide patient care through a certain procedure has been and continues to be more of a dogmatic response, based more on each specialty's view of the problem than on a discussion with logical arguments to find a point of convergence. everyone has expressed the problem in different ways. , many will argue that since medicine is a dynamic entity, as we witness its evolution and progress, it is necessary to go back in time and understand that the current situation of structural heart disease treatment follows, like almost everything in life, the evolutionary model described by lamarck. the fundamental fact is always the ability to adapt to environmental changes. we are, therefore, in an evolutionary moment of cardiovascular care that confirms the need for a change in the therapeutic approach to structural heart disease, a term that is accepted by most, but is also controversial for others because it retains some degree of vagueness. various scientific societies have spoken out in this regard with a greater or lesser degree of protectionism, discussing training, quality, and interdisciplinary collaboration in structural heart disease , mainly in the management of aortic stenosis. regardless of the approach, recent literature seems to concur on the need for a future with shared responsibility and execution in the provision of the intervention. this is supported by the spanish society of cardiology. this is the question nobody in the setting of cardiovascular medicine wants to address, but it is repeatedly hinted at in the specialized literature. what model do we want? a model organized by cardiologists, by surgeons, by educational regulatory bodies at the national level, by scientific societies? perhaps the fundamental question is: are we willing to cede our individual viewpoints and become more receptive to those of are so-called opponents? cequier et al. discuss the suitability of medical and surgical programs for the provision of services in structural heart disease, specifically in aortic stenosis. however, as already mentioned, both teaching programs , need an update. both have a minimum -year validity. in a decade, cardiology and cardiac surgery have undergone profound changes that have revolutionized practice in our field. therefore, and regardless of individual or societal orientation, a reformulation of the care process is required, which is feasible without renouncing the essence of each specialty. since its origins, the study of the basis of cardiovascular disease has been directly related to the development of invasive treatment. the development of techniques and procedures has obliged interventional cardiologists to become structural interventionists and cardiac surgeons to become surgeons who incorporate structural intervention into their practice. as a result of this transformation, structural interventional cardiologists and hybrid surgeons have become very similar professional figures. this has generated some confusion and challenges, including a new reason to debate and fight for control of the field. the need and the obligation to offer the best individualized treatment by the best professional is what gives birth to the similarity. the border marked at this point does not separate, but must unite disciplines that are obliged to understand that this separation is artificial. nobody tries to ignore the existence of different specialties; they are different entities at least in their current conceptions. it is about understanding that there is a large common ground for diseases, for techniques, and above all for the ultimate goal of optimizing the care of structural heart disease in our patients. at a time when specialties are struggling, the educational model that we are currently testing and offering is a model of equality, coexistence and shared philosophy aiming to unify the interventional view of structural heart disease. the structure is based on knowledge of cardiovascular disease, and understanding of the entire spectrum of cardiologists, surgeons and all specialists involved in patient care. an example of such an educational model is the university of zurich's certificate of advanced studies that has congregated throughout its various editions an ecosystem of specialists (appendix ), united by seeking excellence in the treatment of structural heart disease. in addition to the theoretical aspects included in various programs, cross training with supervised and documented practice integrates simulation; a fundamental tool in the present and in the future of anyone interested in structural heart disease interventions. our own experience shows that it is necessary to deepen not only the manual aspects of interventional practice, but also cognitive and perceptive abilities. the use of simulation models with different degrees of fidelity has already allowed us to advance the understanding of complex models, such as valve repair, for example. innovation has allowed us to develop simulation models suitable for all those interested in structural heart disease, regardless of the origin of their knowledge and basic training. an innovation that is aimed at usability, haptic feedback and image solutions, configuring promising tools for simulation-based training, is incorporated in the current and future model. from these already validated practical solutions, a future model can be anticipated in which not only manual, but also visual control of the procedure will favor the learning of future specialists in the treatment of structural heart disease. recent experience, even in complex times such as those of the covid- pandemic, confirm that in addition to individual cross training, improving the overall view of the disease, the process and the patient, such a model will favor teamwork and networking in interdisciplinary action. cardiologists and surgeons agree to move forward together in training and clinical practice. a good example is what was expressed in the spanish society of cardiology's documentary to commemorate its th anniversary. we live in a decisive moment for changing the basic training model for the provision of services in structural heart disease. multidisciplinary teams have proven to be useful and important in reaching agreements to improve patient care and, therefore, clinical outcomes. the model of education in structural heart disease has already begun to change and it is our responsibility to lead and drive that change. the final objective is to focus the training process on acquiring skills to enable us to offer the most advanced treatment and not on the specialty of the care provider, for the benefit of future specialists, and therefore, of patients. el desencuentro salarial mantiene la huelga mir de madrid ley de de julio de sobre «enseñ anza, título y ejercicio de las especialidades mé dicas». «boe» nú m. , de de julio de por el que se regula la formació n mé dica especializada y la obtenció n del título de mé dico especialista. «boe» nú m. , de de enero de de de abril, por la que se aprueba y publica el programa formativo de la especialidad de cardiología. «boe» nú m. , de de mayo de the working group on infective endocarditis of the hospital clínic de barcelona. organization and functioning of a multidisciplinary team for the diagnosis and treatment of infective endocarditis: a -year perspective standards for heart valve surgery in a'heart valve centre of excellence scai/aats/acc/sts operator and institutional requirements for transcatheter valve repair and replacement. part ii mitral valve percutaneous cardiological intervention and cardiac surgery: patient-centered care. position statement of the spanish society of en respuesta al documento de posicionamiento de la sociedad españ ola de cardiología titulado: ''intervencionismo percutá neo cardioló gico y cirugía cardiaca: el paciente en el centro de los procesos philosophie zoologique flammarion: paris structural heart disease? aats/acc/scai/sts expert consensus systems of care document: operator and institutional recommendations and requirements for transcatheter aortic valve replacement: a joint report of the american association for thoracic surgery, the american college of cardiology, the society for cardiovascular angiography and interventions, and the society of thoracic surgeons visual behaviour strategies of operators during catheter-based cardiovascular interventions novel augmented physical simulator for the training of transcatheter cardiovascular interventions the certificate of advanced studies (cas) course adapted to a pandemic thomas key: cord- -cuvfy pj authors: baselga, eulalia; torrelo, antonio title: inflammatory and purpuric eruptions date: - - journal: neonatal dermatology doi: . /b - - - - . - sha: doc_id: cord_uid: cuvfy pj nan infl ammatory and purpuric eruptions this group of eruptions is composed of lesions of variable morphology and diverse etiology. however, all have erythema as a common feature, a refl ection of their infl ammatory nature. several disorders appear to represent hypersensitivity reactions, but for most the etiologic agents are unknown. the differential diagnosis of purpura is extensive in neonates, and includes hematological disorders, infections, trauma, and iatrogenic disorders. annular erythema is a descriptive term that encompasses several entities of unknown etiology characterized by circinate polycyclic lesions that extend peripherally from a central focus. , because of subtle differences in clinical features, age of onset, duration of individual lesions, and total duration of the eruptions, a variety of descriptive terms have been coined for these disorders (table - ) . for prognostic reasons, it is useful to subdivide annular erythemas into transient and persistent forms. transient forms include annular erythema of infancy and the less well-established entity erythema gyratum atrophicans transient neonatale. persistent annular erythemas include erythema annulare centrifugum, familial annular erythema, and erythema gyratum perstans. other annular erythemas known to be a manifestation of well-defi ned diseases (e.g. neonatal lupus) or with distinctive clinical or histologic features (e.g. erythema multiforme, erythema chronicum migrans, erythema marginatum rheumaticum, and erythema gyratum repens) are not considered under this heading. annular erythema of infancy is a benign disease of early infancy characterized by urticarial papules that enlarge peripherally, forming - cm rings or arcs with fi rm, raised, cord-like or urticarial borders. [ ] [ ] [ ] adjacent lesions become confl uent, forming arcuate and polycyclic lesions ( fig. - ) . neither vesiculation nor scaling is present at the border. the eruption is asymptomatic. individual lesions resolve spontaneously without a trace within several days, but new lesions continue to appear in a cyclical fashion until complete resolution within the fi rst year of life. a few cases lasting for years have been described. [ ] [ ] [ ] the cause of annular erythema is unknown, and there are no associated systemic fi ndings. histologic studies reveal a superfi cial and deep, dense, perivascular infi ltrate of mononuclear cells and eosinophils. no fl ame fi gures are observed. the epidermis is normal or mildly spongiotic. laboratory studies are normal. peripheral eosinophilia does not accompany tissue eosinophilia. immunoglobulin levels, including ige levels, are normal. the differential diagnosis should include other annular lesions of infancy (see the following discussion). no treatment is warranted because of the self-limited nature of the eruption. erythema gyratum atrophicans transiens neonatale is a less well-defi ned entity, characterized clinically by annular plaques with an erythematous border and an atrophic center. the lesions appear in the newborn period and resolve within the fi rst year of life. histologic fi ndings include epidermal atrophy and a mild perivascular mononuclear infi ltrate. immunofl uorescence studies reveal granular deposits of igg, c , and c at the dermoepidermal junction and around capillaries. erythema gyratum atrophicans transiens neonatale possibly represents a variant of neonatal lupus erythematosus. erythema annulare centrifugum is a more persistent type of annular erythema that usually affects adults, but may also occur in children and rarely in newborns. , [ ] [ ] [ ] [ ] the lesions are clinically somewhat similar to those of annular erythema of infancy, but scaling or vesiculation is seen at the border. the scales lag behind the advancing border, which, in contrast to annular erythema of infancy, is not indurated. individual lesions resolve spontaneously after a few weeks, but new plaques continue to develop for years, or may be a lifelong condition. there is no associated pruritus. erythema gyratum perstans falls within the spectrum of erythema annulare centrifugum. [ ] [ ] [ ] some authors defend the distinctness of erythema gyratum perstans and consider distinctive features of this disorder to be its early onset, a duration of more than years, the presence of slight to severe pruritus, and especially the presence of vesiculation. erythema annulare centrifugum is thought to represent a hypersensitivity reaction to several trigger factors, including infectious agents (candida, , epstein-barr virus, and ascaris ), drugs or foods, , and neoplasia, especially in adults. intradermal injection of candidin or trichophytin may reproduce the clinical lesions. histologic features consist of a dense, superfi cial, perivascular mononuclear infi ltrate. parakeratosis or epidermal spongiosis may be present. no therapy has been successful in all cases. treatment agents include oral nystatin, oral amphotericin b, topical antifungals, antihistamines, disodium cromoglycate, and interferon-α. familial cases of annular erythema with autosomal dominant inheritance have been described. , the onset is in early infancy. scaling, vesiculation, and pruritus may be more common than in erythema annular centrifugum. lesions resolve with residual hyperpigmentation. chronicity is the rule. geographic tongue may be associated. differential diagnosis includes other eruptions with ringlike lesions, such as neonatal lupus, erythema multiforme, urticaria, urticarial lesions of pemphigoid, fungal infections, erythema chronicum migrans, and congenital lyme disease. , , serum antibody determinations (antinuclear, ss-a, and ss-b) are recommended to exclude neonatal lupus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] neonatal lupus erythematosus (nle) is a disease of newborns caused by maternally transmitted autoantibodies. the major manifestations are dermatologic and cardiac. skin fi ndings are transient. cardiac disease, which is responsible for the morbidity and mortality of nle, begins in utero and affects the cardiac conduction system permanently. other fi ndings include hepatic and hematologic abnormalities. mothers of infants with neonatal lupus have anti-ro/ss-a autoantibodies in % of cases. anti-la/ss-b and anti-u rnp autoantibodies have also been implicated in the pathogenesis of nle in a minority of patients. , fifty per cent of infants with nle have skin lesions, and congenital heart block is present in about %. , lesions commonly develop at a few weeks of age but may be apparent at birth, which suggests that ultraviolet (uv) radiation is not essential for the development of skin lesions in nle. clinically, skin lesions are analogous to those of subacute cutaneous lupus in its two variants: papulosquamous and annular. papulosquamous lesions are more common and are characterized by erythematous, nonindurated scaly plaques ( fig. - ). sometimes the skin lesions have an atrophic appearance ( fig. - ). ulcerations may be present. in contrast to discoid lupus, scarring and follicular plugging are absent. the annular variant, which occurs almost exclusively in japan, consists of annular, more infl ammatory plaques. lupus profundus and generalized poikiloderma with erosions and patchy alopecia are rare manifestations. , nle lesions may be widespread but are most common on the face and scalp, predominantly affecting the periorbital and malar areas and often causing a 'raccoon eyes' appearance . the eruption is frequently precipitated or aggravated by sun exposure. sun exposure is not strictly required, however, as the lesions may occur in sun-protected areas such as the diaper region, palms, and soles. , , skin lesions are transient and cease to appear around the age of months, after the disappearance of maternal antibodies. transient hypopigmentation and epidermal atrophy may result ( fig. - ) . telangiectasia is a more permanent sequela. telangiectasia also may be an initial sign of nle, occurring without preceding identifi able infl ammatory lesions; features of cutis marmorata telangiectatica congenita have been observed. , a case of nle with a serological profi le consis-tent with drug-induced lupus has been described in a newborn whose mother was treated with α-interferon during pregnancy. the most signifi cant manifestation is isolated complete congenital heart block. more than % of such cases are due to nle. most patients have third-degree block, but progression from a second-degree block has been reported. heart block can often be detected as early as weeks of gestation. transient liver disease, manifesting as hepatomegaly (with a picture of cholestasis) or elevation of liver enzymes, , [ ] [ ] [ ] and thrombocytopenia or other isolated cytopenias, may occur. petechiae and purpura have been described as presenting signs of nle. less common fi ndings include thrombosis associated with anticardiolipin antibodies, hypocalcemia, spastic paraparesis, pneumonitis, and transient myasthenia gravis. [ ] [ ] [ ] central nervous system (cns) involvement has been reported in of consecutive infants with nle, in the form of ultrasound and ct scan abnormalities which did not result in clinical neurological manifestations. such cns involvement in nle is probably a transient phenomenon. between % and % of mothers of infants with nle have a connective tissue disease, most commonly sle or sjögren syndrome. most, however, are asymptomatic. the risk for developing overt connective tissue disease in these mothers is highly debated, with estimates ranging from % to more than %. , [ ] [ ] [ ] [ ] [ ] [ ] it is universally agreed that placentally transmitted maternal igg autoantibodies are necessary for the pathogenesis of nle, but not suffi cient. the most commonly implicated autoantibodies have been anti-ro/ss-a and anti-la/ss-b. more than % of nle infants have anti-ro antibody, and - % have anti-la antibodies. a small subset of affected infants do not have detectable anti-ro or anti-la antibodies, but instead have anti-u rnp. , mothers with high titers of anti-ro and anti-la antibodies are at greater risk of delivering an infant with nle. despite initial observations based on immunoblot or elisa testing that anti- kda ro antibodies conferred a higher risk of nle than anti- kda ro antibodies, more precise testing with line immunoassay has revealed that antibodies to kda ro are signifi cantly more sensitive than antibodies to kda ro and kda la. furthermore, comparing mothers of children with nle with rash alone or with congenital heart block, there is no signifi cant difference in the prevalence of any of the three antibodies between the two groups. however, signifi cantly more symptomatic mothers of children with congenital heart block have anti-la antibodies than do disease-matched mothers with unaffected children. moreover, the mean level of anti-la seems to be higher in mothers of infants with congenital heart block than in mothers of children with cutaneous nle. it is not clear why only less than % of mothers with anti-ro and anti-la antibodies give birth to affected children and why mothers of affected infants are often asymptomatic despite having the same antibodies. furthermore, fraternal twins are often discordant for nle, and nle does not occur in every pregnancy. genetic factors may be important for the development of nle in children with maternal lupus antibodies. a link has been suggested between nle rash and the allele hla-drb * , as well as a - a polymorphism in the tnf-α gene. alternatively, maternal and/or sibling microchimerism may play an additional role, as levels of microchimerism have been reported to correlate with nle disease activity. serologic studies for autoantibodies in the mother and infant demonstrate anti-ro, anti-la, and/or anti-u rnp antibodies. anti-ndna, anticardiolipin antibodies, antinuclear antibody, and rheumatoid factor may also be present. anti-sm antibody, highly specifi c for systemic lupus erythematosus, is not found in nle. the maternal antibody titer is usually higher than the infant titer, which may even be negative if only immunodiffusion techniques are used. more sensitive methods, such as elisa, immunoblotting, or line immunoassay, should be used in such instances. skin biopsy, which is usually not necessary for diagnosis, shows changes characteristic of lupus erythematosus, that is, epidermal atrophy and vacuolization of the basal layer with a sparse lymphohistiocytic infi ltrate at the dermoepidermal junction and in a periappendageal distribution. in many instances, histopathological features in children with nle rash are subtle. direct immunofl uorescence is positive in % of cases, demonstrating granular deposits of igg, c , and igm at the dermoepidermal junction. histopathologic examination of the heart shows replacement of the atrioventricular node by fi brosis or fatty tissue. endomyocardial fi broelastosis and patent ductus arteriosus may also be seen, , as well as deposits of igg and complement. the differential diagnosis includes congenital rubella, cytomegalovirus infection, annular erythema of infancy, tinea corporis, and seborrheic dermatitis. congenital syphilis should also be considered, but mucosal lesions are not a feature of nle. false positive serologic tests for syphilis may occur in nle. telangiectasia and photosensitivity may suggest bloom syndrome or rothmund-thomson syndrome. serologic studies for autoantibodies in both infant and mother help to confi rm the diagnosis. skin biopsy for histologic and direct immunofl uorescence studies is seldom necessary. neonates with suspected nle should receive a complete physical examination, electrocardiogram, complete blood count with platelet count, and liver function tests (box - ). skin lesions are transient. treatment of skin disease consists of sun protection and the application of topical steroids. pulsed dye laser therapy may be considered for residual telangiectasia. congenital heart block is permanent. half of newborns with complete congenital heart block require implantation of a pacemaker in the neonatal period. mortality from complete congenital heart block in the neonatal period is %; another - % die of pacemaker complications. , late-onset cardiomyopathy may develop in a few infants. [ ] [ ] [ ] for mothers with anti-ro or anti-la antibodies, the risk of delivering an infant with nle is - %, depending on whether they have asymptomatic or symptomatic sle. , the risk of recurrence of congenital heart block in subsequent pregnancies may be as high as %. such pregnancies should be closely monitored, with repeated fetal echocardiograms. if signs of intrauterine congestive heart failure are detected dexamethasone or plasmapheresis, or both, have been given. , [ ] [ ] [ ] [ ] although nle is usually self-limited, sle or other rheumatologic/autoimmune diseases may develop later in life in a small subset of patients. , , the exact risk is unknown. [ ] [ ] [ ] [ ] cutaneous drug reactions are extremely rare in the neonatal period because the ability to generate a drug-induced immune response appears to be lower in infants. [ ] [ ] [ ] furthermore, most drug reactions require time for sensitization, which may range from to weeks or more, as well as re-exposure to the causative drug. finally, newborns and young infants are less exposed to drugs than adults. cutaneous adverse reactions to drugs may be classifi ed according to the clinical characteristics of the eruption (box - ). whenever a suspect eruption is observed, a detailed history of medications should be obtained, including drugs administered to the mother, which may be present in breast milk. morbilliform ( fig. - ) or maculo-papular eruptions (fig. - ) are the most frequent type of drug reaction in neonates, and antibiotics are commonly implicated ( fig. - ) . distinguishing a drug eruption from a viral exanthem is often diffi cult. emla cream, a local anesthetic that may be used with great frequency in neonatal units, has been noted to produce a localized purpuric eruption. , this type of reaction is seen preferentially in neonates, and subsequent applications of the cream do not always reproduce the purpuric lesions. methemoglobinemia is another complication of emla use in this age group. emla cream should therefore be used with caution in infants who are taking methemoglobin-inducing medications such as sulfonamides, acetaminophen, nitroglycerin, nitroprusside, and phenytoin, and particularly in those with a history of methemoglobinemia. vancomycin, an antibiotic frequently administered to premature newborn infants for staphylococcus epidermidis nosocomial infections, may produce shock and rash in newborns (red-baby syndrome). [ ] [ ] [ ] this reaction is characterized by the appearance of an intense, macular, erythematous eruption on the neck, face, and upper trunk shortly after the infusion is completed. it may be accompanied by hypotension and shock. the reaction resolves rapidly in a matter of hours. it is frequently associated with rapid infusion; however, lengthening the infusion to more than hour does not completely eliminate the risk. newborns with aids have an increased susceptibility to drug reactions. , reactions to trimethroprim/sulfamethoxazole in patients with hiv infections can be severe and life-threatening. fixed drug reactions of the scrotum and penis, with erythema and edema resulting from hydroxyzine hydrochloride, have been described in early infancy. however, hydroxyzine hydrochloride is administered infrequently in the neonatal period because of the risk of antimuscarinic effects, such as restlessness and excitation. serum sickness-like reaction is a type of drug reaction that occurs predominantly in children and has been reported in infants months of age. it is characterized by fever, an urticarial eruption, and arthralgias. lymphadenopathy may be present. in contrast to true serum sickness, there are no immune complexes, vasculitis, or renal impairment. the most commonly implicated drug has been cefaclor. [ ] [ ] [ ] this type of reaction may be seen in infants with an unknown or presumably viral etiology ( fig. - ) hypersensitivity syndrome reaction is a serious drug reaction characterized by fever, skin rash, lymphadenopathy, and internal organ involvement, especially of the liver. , the most commonly implicated drugs are anticonvulsants, and therefore it is not rare in children. a fatal case in a -monthold infant has been reported, as well as a case in a premature infant. , toxic epidermal necrolysis (ten) is extremely rare in newborns. cases of ten in newborns have been related to antibiotics and phenobarbital. all cases described so far proved fatal. vegetant bromoderma is a reaction to bromides characterized by coalescing papules and pustules which form vegetant infl ammatory or pseudotumoral lesions. it usually affects the scalp, face, and legs. most cases of vegetant bromoderma have been described in infants after the ingestion of syrups and solutions containing bromide, which has sedative and expectorant properties, or the spasmolytic agent scopolamine bromide. the eruption ceases after withdrawal of bromide. the risk of systemic intoxication, known as bromism, makes it advisable to avoid bromide use in newborns and infants. other anectodal reports of toxicoderma in very young infants or newborns have been described, such as a papular eruption from g-csf for collection of stem cells (fig. - ) , a lichenoid reaction to ursodeoxycholic acid for neonatal hepatitis, and a maculopapular rash from diazoxide used for neonatal hyperglycemia (see fig. - ). urticaria (hives) occurs frequently in childhood but is uncommon in children younger than months and even rarer in the neonatal period. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] urticaria is usually sporadic; however, familial forms with autosomal dominant inheritance have been described for many of the physical urticarias, such as dermographism, heat urticaria, cold urticaria, and vibratory urticaria. urticaria can be divided into acute (lasting less than weeks) and chronic (lasting more than weeks) types. nevertheless, this arbitrary division has prognostic and etiopathogenic signifi cance. in infants, chronic urticaria is very rare. , physical urticarias represent a special subgroup of urticaria in which wheals are elicited by different types of physical stimuli. these include dermographism, cold, pressure, cholinergic, aquagenic, vibratory, and solar urticaria. urticaria is characterized by transient edematous pruritic wheals ( fig. - ). by defi nition, individual lesions last less than hours. hives may occur on the skin and mucous membranes. angioedema or giant urticaria is a closely related entity in which there is swelling of the deep subcutaneous tissues and diffuse swelling of the eyelids, genitalia, lips, and tongue. it may be seen alone, or more often in association with 'common' urticaria. urticaria in children has certain characteristic features. the hives tend to coalesce, forming bizarre polycyclic, serpiginous, or annular shapes (fi gurative urticaria, fig. - ; or annular urticaria, fig. - ), and may become hemorrhagic. , edema is often pronounced and painful. these features confer a dramatic appearance to the eruption. in children the itching may be absent. urticaria may be more common and recurrent in atopic patients. , acute urticaria may be accompanied by signs of anaphylactic shock. in cases of angioedema, abdominal pain, diarrhea, vomiting, respiratory compromise, and joint pain may occur. , urticaria develops as a result of an increased permeability of capillaries and small venules, which leads to leakage of fl uid into the extravascular space. mast cell activation and subsequent mediator release are responsible for these changes. histamine is the best-known mediator. many triggers (secretagogues) initiate mast cell degranulation through receptors on mast cell membranes, either via an ige-dependent mechanism or through complement activation (immunologic secretagogues) or by acting directly without the need for receptors (nonimmunologic secretagogues). the most common provocative agents in children are drugs, foods, and infections, which account for % of the cases of acute urticaria. , , antipyretics (primarily aspirin) and antibiotics (amoxicillin, macrolides, and oral cephalosporins) are the most frequently incriminated drugs. food-related urticaria is associated with atopy. cow's milk allergy is one of the main causes of urticaria in infants, being present in - % of cases of cow's milk intolerance. , , the diagnosis of urticaria is made on clinical grounds. histopathologic examination of a skin biopsy specimen shows vascular dilation, edema, and a perivascular infl ammatory infi ltrate composed of lymphohistiocytic cells, polymorphonuclear cells, and more specifi cally eosinophils. neutrophils may predominate. laboratory tests are not usually necessary for the evaluation of acute urticaria. ige levels can be elevated in some patients. an exhaustive search for an underlying cause not elicited by history alone is not warranted. an erythrocyte sedimentation rate may suffi ce as a screening test in cases of chronic urticaria because it is usually elevated in diseases associated with chronic urticaria (e.g. collagen vascular diseases). in - % of patients no cause is identifi ed. intradermal skin tests to discover suspected allergens are not reliable. urticaria in infants is often misdiagnosed as erythema multiforme, acute hemorrhagic edema, annular erythema of infancy, or kawasaki disease. in an infant with urticaria and dermographism the possibility of diffuse cutaneous mastocytosis without visible cutaneous lesions should also be considered. nomid/cinca (see below) should be considered in young infant with urticaria. the predominance of neutrophils in skin biopsies of children with nomid may help in the differential diagnosis, although it is not % specifi c. in case of doubt, genetic testic for nomid is now available. despite its alarming symptoms, urticaria in early infancy is usually benign. exceptions are chronic infantile neurological cutaneous and articular syndrome (cinca) (see below) and the inherited physical urticarias, which may have a lifelong course. if medication is required, antihistamines such as diphenhydramine or hydroxyzine are the mainstay of therapy. however, newborns have an increased susceptibility to antimuscarinic side effects, such as central nervous system (cns) excitation causing convulsions. systemic corticosteroids should be reserved for cases of intractable urticaria. autosomal dominant variants have been described for many of the physical urticarias. although rare, these familial cases begin early in life, even immediately after birth, and have a lifelong course, usually with increased severity. the exact pathogenic mechanism for many of the physical urticarias is unknown. familial cold urticaria (fcu) is an autosomal dominant disorder characterized by the development of burning wheals, and frequently pain and swelling of joints, stiffness, chills, and even fever after exposure to cold, especially in combination with damp and windy weather. [ ] [ ] [ ] [ ] the skin lesions appear on exposed areas and generalize afterwards. leukocytosis may be present during the attacks. the reaction may be delayed for up to hours after cold challenge. in contrast to acquired cold urticaria, the reaction cannot be elicited by an ice cube test: rather, the patient must be subjected to cold environmental temperatures or cold water immersion. on skin biopsy a neutrophilic infi ltrate predominates. the symptoms tend to improve with age. responses to h and h blockers and ketotifen are poor. stanozolol has been of limited benefi t. fcu has also been described along with amyloidosis and deafness as muckle-wells syndrome (mws). it has been recently demonstrated that both fcu and mws are due to mutations in the cias (cryopyrin) gene: in fact they are the same disorder and may share exactly the same genetic mutation. fcu and mws are also allelic diseases with cinca syndrome (see below), which is also due to cias gene mutations. familial dermographism (autosomal dominant) has been described in a single large family. in neonates dermographism can also be a manifestation of 'silent' diffuse cutaneous mastocytosis. , vibratory urticaria is an autosomal dominant physical urticaria in which wheals develop after repetitive vibratory stimulation or stretching. , the need for repetitive trauma differentiates it from dermographism. familial aquagenic urticaria and familial heat urticaria usually have onset in childhood. [ ] [ ] [ ] chronic infantile neurological cutaneous and articular syndrome (cinca) [ ] [ ] [ ] [ ] cinca syndrome, also known as neonatal onset multisystemic infl ammatory disease (nomid), is a chronic systemic infl ammatory disease of neonatal onset characterized by skin rash, arthropathy, and cns manifestations. cutaneous fi ndings are the presenting signs. the disease follows a chronic course with acute febrile exacerbations, lymph node enlargement, and hepatosplenomegaly. two-thirds of patients are born prematurely. a skin eruption is usually the fi rst manifestation of the disease and is present at birth or develops during the fi rst months of life. it is characterized by generalized, evanescent, urticarial macules and papules that migrate over the course of a single day and wax and wane in intensity ( fig. - ) . the rash is persistent, although recrudescence of the skin lesions is noted at fl are-ups. the lesions may be pruritic, especially after sun exposure, but are usually asymptomatic. , geographic tongue and oral ulcers have been noted in a single patient. symmetric or asymmetric arthropathy is another constant fi nding and is severe in half of patients. it is often absent in the fi rst few weeks of life, but usually develops during the fi rst year. , the severity of the arthropathy correlates with an early onset of joint symptoms. the knees are most frequently affected, followed by the ankles and feet, elbows, wrists, and hands. joint swelling and pain are more severe during febrile fl are-ups. on palpation, a bony consistency is characteristic as a result of epiphyseal and growth cartilage involvement and overgrowth of the patellae. joint contractures and severe deformities result. neurologic signs and symptoms such as headache, vomiting, and seizures develop at a variable age. intellectual impairment is also common. both spasticity and hypotonia have been described. eye involvement is an inconstant fi nding. papilledema with or without optic nerve atrophy is the most common feature. other ocular manifestations include uveitis, keratitis, conjunctivitis, and chorioretinitis. these changes may lead to complete blindness in adulthood. progressive sensorineural hearing loss and hoarseness are also common. affected children have a characteristic phenotype. there is progressive growth retardation and increased head circumference with frontal bossing. fontanel closure is retarded. icterus may be present in the neonatal period, especially in patients with severe arthropathy. mutations in the cias gene have been identifi ed in % of patients with cinca syndrome. , cias encodes a protein called cryopyrin, which is involved in the regulation of apoptosis and the infl ammatory signaling pathway. it is proposed that familial cold urticaria and cinca represent extreme groups of the same disease, defi ned by the magnitude of phenotypic expression. considerable clinical overlapping exists between these disorders. nonspecifi c fi ndings typical of a chronic infl ammatory process include microcytic anemia; leukocytosis with high neutrophil and eosinophil counts, elevated platelet counts, sedimentation rates, and acute-phase reactants; and polyclonal hyperglobulinemia g, a, or m. rheumatoid factor and antinuclear antibodies are usually absent. liver enzymes may be mildly elevated. csf examination shows pleocytosis and high protein levels. radiologic studies of the affected joints show irregularly enlarged, bizarre, spiculated epiphyses with a grossly coarsened trabecular appearance. , there is periosteal new bone formation, and growth cartilage abnormalities are frequent. with time, there is bowing deformity of long bones and shortening of diaphyseal length. ct scans of the head have demonstrated hydrocephalus and cerebral atrophy. histopathologic examination of the skin reveals interstitial and perivascular neutrophilia. , neutrophilic eccrine hidradenitis has been described. biopsies of lymph nodes, liver, and synovium show nonspecifi c signs of chronic infl ammation. nomid must be differentiated from systemic onset juvenile arthritis. the main differences are its neonatal onset, persistent rash, the short duration of bouts of fever, absence of morning stiffness, and central nervous system involvement. the arthropathy is more deforming, and the radiographic fi ndings of enlarged and disorganized epiphyses are distinctive. in addition, the response to nsaids is poor. urticaria should also be considered and the predominance of eosinophils in skin biopsy may be a relative clue. the disease follows a chronic course with acute febrile exacerbations. occasionally it causes death in the fi rst or second decade. nonsteroidal anti-infl ammatory drugs may be effective for pain relief but do not alter the course of the disease. prednisone has been palliative in doses ranging from . to . mg/kg/day. chlorambucil and penicillamine have been tried, with limited success. , thalidomide has shown benefi cial effects in a single patient. other choices include methotrexate, the recombinant human il- receptor antagonist anakinra, [ ] [ ] [ ] [ ] and the anti-tnf-α agent etanercept. erythema multiforme (em) is an acute, self-limited disorder of skin and mucous membranes. [ ] [ ] [ ] it has been considered a spectrum of disorders, designated em minor, consisting of skin involvement only or of both the skin and the mouth, and as em major (stevens-johnson syndrome; sjs), which involves at least two mucous membranes with variable cutaneous lesions. some authors include toxic epidermal necrolysis within this spectrum as a severe form of sjs. recent evidence suggests that em and sjs have distinct clinical features and different precipitating factors, so perhaps the terms em major and em minor are best avoided. , em is a common disease in children but extremely unusual in the neonatal period. , , [ ] [ ] [ ] [ ] toxic epidermal necrolysis is discussed in chapter . the prototypic lesion of em is a - cm erythematous, edematous papule that develops a dusky vesicular, purpuric, or necrotic center. a raised edematous ring of pallor surrounded by an erythematous outer ring is often present. these concentric color changes produce the typical target, or iris, lesion. in many cases only two zones are seen, with a single ring around the central papule (atypical target lesions). the lesions are distributed symmetrically and acrally on the extensor surface of the extremities. they may extend to the trunk, fl exural surfaces, palms, and soles. in children, lesions on the face and ears are common, but are rare on the scalp (fig. - ). in sjs, the lesions are more centrally located, predominating on the trunk. the targets are atypical and are usually fl at. individual lesions tend to coalesce in large patches. areas of epidermal detachment may occur, but usually affect less than % of the body surface area. mucosal lesions occur frequently in em and are requisite for a diagnosis of sjs. mucous membrane involvement is characterized by erythema or blisters that rapidly evolve to confl uent erosions with pseudomembrane formation. the oral mucosa and conjunctiva are most commonly involved, but genital, anal, pharyngeal, and upper respiratory tract involvement may be seen. the number of mucous membranes involved has been considered one of the main distinguishing features of em and sjs. mild, nonspecifi c, prodromal symptoms of cough, rhinitis, and low-grade fever are occasionally present in em. fever, arthralgias, and prostration are common in sjs. em has been considered a hypersensitivity phenomenon to multiple precipitating factors such as infectious agents or drugs. three etiologic factors have been well documented: herpes simplex for erythema multiforme, and mycoplasma infections and drugs for sjs. herpes simplex (hsv- or hsv- ) is considered to be responsible for more than % of em in children, even if clinical infection is inapparent. hsvassociated em follows the lesions of herpes by - weeks and is often recurrent. however, not every episode of recurrent herpes is followed by em. hsv-specifi c dna has been detected by polymerase chain reaction and in situ hybridization in lesional skin from a large number of children with em, whether 'idiopathic' or clearly hsv related. erythema multiforme fig. - target lesions of erythema multiforme in a newborn. cow's milk intolerance has been described as a cause of erythema multiforme in a neonate. drugs are the most common cause of sjs. sulfonamides, phenylbutazone, diphenylhydantoin, and penicillin derivatives are most frequently implicated. vaccinations were the only known possible causative agents in a newborn and two infants with erythema multiforme. , in cases of extensive involvement an elevated sedimentation rate, leukocytosis, and mild elevation of transaminases may be seen. electrolyte imbalance and hypoproteinemia may be encountered in sjs. eosinophilia may be seen in drug-related cases. histopathologic examination of early lesions reveals a lymphocytic band-like infi ltrate at the dermoepidermal junction, with exocytosis and individual necrotic keratinocytes in close proximity to lymphocytes ('satellite cell necrosis'). there is vacuolization of the basal layer with focal cleft formation at the dermoepidermal junction. the upper dermis is edematous. over time, more extensive confl uent necrosis of the epidermis supervenes, resulting in subepidermal blister formation. in em a lichenoid infi ltrate predominates, whereas in sjs epidermal necrosis predominates. in typical cases em or sjs is rarely confused with other entities. urticarial vasculitis may be considered in some cases. kawasaki disease may produce target-like lesions; however, associated fi ndings should allow differentiation. serum sickness-like reactions often associated with the use of cefaclor or other antibiotics, or even without any known etiology, can also produce targetoid lesions (see fig. - ). erythema multiforme is usually self-limited. individual lesions heal in - weeks, with residual hyperpigmentation. conservative supportive care is the preferred form of treatment. possible underlying causes should be sought. treatment of underlying infection and discontinuation of nonessential drugs are indicated. corticosteroids are unnecessary and may even worsen a concurrent infection. , in hsv-associated em, early intervention or even prophylactic treatment with oral aciclovir may be benefi cial. sjs has a less favorable prognosis, with a mortality rate of - % if left untreated. the use of corticosteroids in sjs is more controversial. , , , no controlled study has proved their effi cacy, and in some studies patients treated with corticosteroids have had a worse prognosis. corticosteroids may predispose to secondary infection while suppressing the signs of sepsis. supportive care is extremely important. sweet syndrome, or acute febrile neutrophilic dermatosis, is a benign disease characterized by tender, raised erythematous plaques, fever, peripheral leukocytosis, histologic fi ndings of a dense dermal infi ltrate of polymorphonuclear leukocytes, and a rapid response to systemic corticosteroids. [ ] [ ] [ ] [ ] [ ] only a few pediatric cases have been reported, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the youngest being weeks of age. two brothers with sweet syndrome starting at weeks of life have been reported. the lesions of sweet syndrome have an acute, explosive onset and are characterized by indurated, tender, erythematous plaques or nodules that vary in size from . to cm ( fig. - ). tiny pustules may appear at a later stage. the borders may be raised, mammillated, or even vesicular. some of the lesions may show central clearing, forming annular or gyrate plaques ( fig. - ) . the lesions are usually multiple and distributed over the face and extremities or, more rarely, the trunk. without treatment, they tend to heal spontaneously within a few months. in some patients, especially children, the lesions heal with areas of secondary cutis laxa, also known as marshall syndrome. , , a high, spiking fever is characteristic but may be absent in up to % of patients. arthralgias or asymmetric arthritis may be associated, and conjunctivitis or iridocyclitis may be seen in one-third of patients. renal involvement manifesting as proteinuria or hematuria, as well as lung involvement with infi ltrates visible on chest radiographs, has also been described. central nervous system involvement may occur in rare instances and manifest as headaches, convulsions, or disturbance of consciousness. cerebrospinal fl uid pleocytosis with lymphocyte predominance is usually found in such cases. the pathogenesis is unknown. many of the patients reported have had a preceding respiratory tract infection or elevated antistreptolysin o titers. ten per cent of the cases have been seen in the setting of a variety of hematologic malignancies, particularly acute myeloid and myelomonocytic leukemias. sweet syndrome has also been associated with solid tumors, infl ammatory bowel disease, connective tissue diseases, and chronic granulomatous disease, or it may occur as an adverse reaction to drugs, [ ] [ ] [ ] particularly granulocyte colonystimulating factor or after vaccination. because of these associations and the rapid response to systemic corticosteroids, sweet syndrome is thought to represent a hypersensitivity reaction to infectious agents or tumoral antigens. an elevated erythrocyte sedimentation rate and peripheral leukocytosis are frequent accompanying abnormalities. eosinophilia, microcytic anemia, mild elevation of liver enzymes, and urinalysis abnormalities may be present occasionally. antineutrophil cytoplasmic antibodies have been detected in some cases. α -antitrypsin defi ciency has been documented in one case of marshall syndrome. the histopathologic fi ndings are diagnostic. there is a dense perivascular infi ltrate composed almost entirely of neutrophils. the dermis appears edematous, and subepidermal blisters may form. spongiosis, exocytosis, and intraepidermal vesiculation may be seen. there is endothelial swelling and nuclear dust, but true vasculitis is characteristically absent. the lesions of sweet syndrome may initially resemble those of em or acute hemorrhagic edema. lesions on the lower extremities may resemble those of erythema nodosum, but lesions more characteristic of sweet syndrome are usually present in other locations. sweet syndrome is a benign disease but may be a marker of malignancy. if left untreated it resolves spontaneously over weeks to months. recurrences are common. marshall syndrome may have a poorer prognosis, with the development of elastolysis in the lungs or cardiovascular involvement. oral corticosteroids are the treatment of choice and usually elicit a prompt response. potassium iodide administration has been successful in a few cases, as have colchicine, dapsone, clofazimine, and intravenous immunoglobulin. kawasaki disease is an acute systemic vasculitis involving small and medium-sized muscular arteries, especially the coronary arteries, of young children. in the past, many cases were called infantile polyarteritis nodosa. the disease is characterized by fever lasting at least days, nonpurulent conjunctivitis, a polymorphous exanthem, erythema and swelling of the hands and feet, infl ammatory changes of the lips and oral cavity, and acute nonpurulent cervical adenopathy. [ ] [ ] [ ] [ ] coronary artery aneurysms or ectasia develop in - % of untreated children and may lead to ischemic heart disease or sudden death. kawasaki disease occurs predominantly in children under and has a peak incidence between and months. [ ] [ ] [ ] it is infrequent before months of age, although it has been reported in patients less than weeks of age [ ] [ ] [ ] boys are affected . times as often as girls. kawasaki disease is an endemic disease with epidemic and geographic clustering. there is seasonal predominance in late winter and spring, although this may differ in different countries. , it is most common in japan, with an annual incidence of cases per children under , and is steadily increasing. , familial cases in household contacts have been described. the recurrence rate is %, with some patients having two or more recurrences. , the skin is involved in % of patients. the fi rst sign often consists of diffuse erythema and painful induration of the hands and feet. between and weeks after disease onset the eruption characteristically begins to desquamate beneath the distal nail plates, and peeling may extend to involve the entire palm and sole. horizontal depressions in the nail plates (beau's lines) usually result. a polymorphous exanthem on the trunk and proximal extremities usually appears within days of onset of fever ( fig. - ). it is a nonspecifi c, diffuse maculopapular or kawasaki disease fig. - morbiliform eruption in an infant with kawasaki disease. morbilliform eruption, but may be urticarial, scarlatiniform, targetoid, or even pustular. bullous or vesicular eruptions have not been described. the rash is usually in the perineum, which is a distinctive feature at this early stage, and it desquamates within hours, preceding fi nger-tip and toe-tip desquamation (fig. - ) . plaque-type, guttate, and pustular psoriasis have been described, either during the acute or the convalescent phase of the disease, which supports a superantigenmediated etiology. [ ] [ ] [ ] [ ] changes in the lips and oral mucosa include erythema, swelling and fi ssuring of the lips, strawberry tongue, and erythema of the oropharynx. oral ulcerations and pharyngeal exudates are not seen. intermittent acrocyanosis has been observed in infants younger than months of age, as well as peripheral gangrene. infl ammatory changes with necrosis at the site of a previous bcg inoculation have been reported. [ ] [ ] [ ] [ ] extracutaneous findings , , , prolonged fever for at least days is the cardinal and initial feature of the disease. it begins abruptly and is high, with peak temperatures generally > °c ( °f) and in many cases > °c ( °f), with several spikes each day (remittent fever). in the absence of appropriate therapy, fever persists for a mean of days, but it may continue for - weeks and, rarely, even longer. bilateral nonexudative conjunctival injection, involving mainly the bulbar conjunctivae, begins shortly after disease onset and may already be resolved at time of fi rst consultation. anterior uveitis is frequently noted on slit-lamp examination but is rarely symptomatic. cervical lymphadenopathy is the least common diagnostic sign, with a prevalence of approximately %. it is usually unilateral, and confi ned to the anterior cervical triangle. the lymph nodes are often fi rm, nonfl uctuant, and only slightly tender. cardiac conditions are the main cause of long-term morbidity and mortality. the pericardium, myocardium, endocardium, and coronary arteries may all be involved. myocarditis may manifest in the acute phase, and arrhythmias due to ischemia, congestive heart failure, and valvular involvement, usually mitral, may occur. occasionally there may low cardiac output syndrome or shock. cardiac auscultation of the infant or child with kawasaki disease in the acute phase often reveals a hyperdynamic precordium, tachycardia, a gallop rhythm, and an innocent fl ow murmur. a pansystolic regurgitant murmur may be heard in children with signifi cant mitral regurgitation. electrocardiography may show arrhythmia, prolonged pr interval, or nonspecifi c st and t-wave changes. pericardial effusion may be detected by an echocardiogram in % of patients. without treatment, coronary artery aneurysms develop in % and are most commonly detected days to weeks after onset. risk factors for the development of coronary aneurysms include age younger than year, male gender, fever for more than weeks, recurrent fever, and delayed treatment. aneurysms may also develop in systemic medium-sized arteries and result in peripheral gangrene. polyarticular arthritis and arthralgias may occur in the fi rst weeks of the illness. it affects small as well as large joints. irritability is usually prominent. lethargy and other signs of aseptic meningitis may be present. abdominal symptoms such as vomiting, diarrhea, and pain are common. in rare instances acute abdominal pain, mimicking a surgical abdomen, may herald the onset of the disease. mild hepatitis occurs frequently, as does acute distension of the gallbladder (hydrops). transient unilateral peripheral facial nerve palsy occurs rarely. respiratory symptoms due to pulmonary nodules, infi ltrates, or pleural effusion may also be observed. [ ] [ ] [ ] rare fi ndings include testicular swelling, hemophagocytic syndrome, and transient high-frequency sensorineural hearing loss ( - db). [ ] [ ] [ ] epidemiologic and clinical data suggest that kawasaki disease has features of infectious disease in an immunologically susceptible host and of an immune-mediated vasculitis. many etiologic agents, ranging from bacteria such as propionibacterium, staphylococcus, streptocuccus, chlamydia and yersinia to viruses such as epstein-barr, parvovirus, adenoviruses, retroviruses, and a novel human coronavirus, have been linked to kawasaki disease in different geographic outbreaks, but none has been consistently demonstrated. much of the continuing debate in the literature concerns whether kawasaki disease is caused by a superantigen or a conventional antigen. , evidence of a superantigen-mediated disease process includes the identifi cation of superantigen-producing organisms, isolation of bacterial superantigens, or fi nding the hallmark of superantigen activation in affected children, such as selective expansion of vβ and vβ t-cell receptor families. however, the immune response in kawasaki disease is oligoclonal (antigen driven, i.e. similar to a response to a conventional antigen) rather than polyclonal (as found typically in superantigen-driven responses), and immunoglobulin a (iga) plasma cells play a central role. , regardless of the cause, evidence points to a generalized immune activation with production of various proinfl ammatory cytokines and endothelial cell activation which lead to coronary artery alteration. [ ] [ ] [ ] [ ] the most studied cytokine has been tnf-α, which is usually elevated in children with kawasaki disease. enzymes, including matrix metalloproteinases that are capable of damaging arterial wall integrity, may also be important in the development of aneurysmal dilatation. various chemotactins that attract neutrophils and monocytes to coronary arteries may also play an important role. host genetic determinants play a role in both susceptibility and coronary artery outcome in kawasaki disease. the fig. - early perineal desquamative eruption of kawasaki disease. incidence rate in siblings is times the population incidence. , , the risk of occurrence in twins is higher than in ordinary siblings. parents who had kawasaki disease in childhood are more likely to have affected children, and children with recurrent disease. in the acute phase, laboratory studies show leukocytosis (> /mm ) with a left shift, normochromic normocytic anemia, increased sedimentation rate and other acute-phase reactants, depressed albumin, and elevated igm and ige levels. the degree of elevation of esr and c-reactive protein may show discrepancy, and both should be measured. furthermore, elevation of esr can be caused by ivig therapy and therefore can not be the sole determinant of the degree of infl ammatory activity. plasma lipids are altered in the acute stage, with depressed plasma cholesterol and hdl. , there may be mild elevation of transaminases and polyclonal hypergammaglobulinemia. in the subacute stage, in the second and third weeks of illness, there is a marked and almost universal thrombocytosis, which returns to normal in - weeks. thrombocytopenia is rarely seen in the acute stage and may be a sign of disseminated intravascular coagulation. antineutrophil cytoplasmic antibodies may be detectable as a nonspecifi c epiphenomenon. there may be sterile pyuria with mild proteinuria. cerebrospinal fl uid shows a mononuclear pleocytosis with normal protein and glucose levels. skin biopsy fi ndings are not specifi c. there is edema in the papillary dermis, with a mild perivascular mononuclear cell infi ltrate. vasculitis of medium and large arteries is observed. there is no single diagnostic test for kawasaki disease and therefore clinical criteria have been established to guide treatment decisions (box - ) . the classic diagnosis has been based on the presence of days of fever and four of the fi ve principal clinical features. clinical features usually appear sequentially and are not all present at a single point in time, therefore watchful waiting is sometimes necessary before a diagnosis can be made. to avoid holding treatment until more than four clinical criteria are met, and the recognition that many patients with 'incomplete' kawasaki still develop coronary artery disease, one may diagnose and treat kawasaki on day of illness in the presence of four principal criteria. , also, the diagnosis can be made in patients with fever for days and fewer than four principal features when coronary artery disease is detected by two-dimensional echocardiography ( de) or coronary angiography. kawasaki disease should be considered in the differential diagnosis of a young child, specially under year of age, with unexplained fever for days that is associated with any of the principal clinical features of this disease, or even in the presence of other clinical and laboratory fi ndings that are not classic criteria but which are commonly encountered in this disease (box - ). for example, an elevated crp or esr and elevated platelet count after days of illness are uncommon in viral infections but are universally seen in children with kawasaki disease. echocardiography may be useful in evaluating 'incomplete kawasaki disease' and should be considered in any infant under months with fever of more than days' duration, laboratory evidence of systemic infl ammation, and no other explanation for the febrile illness. although aneurysms rarely form before day of illness there may be signs of coronary arteritis, decreased contractibility, mitral regurgitation, and pericardial effusion. with all these considerations a new algorithm has been proposed to help in deciding which patient with incomplete kawasaki disease should undergo echocardiography or receive ivig treatment (fig. - ) . the morbidity and mortality of kawasaki disease depend primarily on coronary artery lesions. [ ] [ ] [ ] [ ] [ ] coronary artery aneurysms or ectasia develop in - % of untreated children and may lead to ischemic heart disease or sudden death. with early treatment the risk is reduced to around - %. , small aneurysms resolve completely within the fi rst years after disease onset in - % of these patients. however, coronary aneurysms, especially if giant (> mm), may persist and be complicated by thrombotic occlusion or the development of stenosis at the outlet of the aneurysm. stenotic lesions as well as early coronary atherosclerosis may develop gradually over several years, so long-term follow-up is warranted. , , , several scoring systems have been developed to predict risk for coronary artery aneurisms. the harada score is one that is used in japan. sometimes kawasaki disease may be 'atypical,' presenting at onset with clinical features that are not generally seen, such as acute abdominal pain, renal impairment, meningeal irritation, pneumonia, or retropharyngeal abscess. many diseases mimic kawasaki disease, including viral infections, streptococcal infection, juvenile rheumatoid arthritis, erythema multiforme, staphylococcal scalded skin syndrome, toxic shock syndrome, drug hypersensitivity reactions, rocky mountain spotted fever, leptospirosis, mercury hypersensitivity reaction (acrodynia), and bacterial cervical adenitis. low white blood cell count, lymphocytosis and low platelet count may be useful in suggesting a viral infection instead of kawasaki disease. because the major sequelae of kawasaki disease are related to coronary artery systems, cardiac imaging is critical in the evaluation of all patients with suspected kawasaki disease, and serial echocardiograms are recommended. echocardiography should focus on coronary artery visualization and measurement, but also on left ventricle contractibility, valve function, the presence of pericardial effusion, and measurement of the aortic root. an initial examination should be performed as soon as the diagnosis is suspected and is useful as a baseline for follow-up. thereafter, for uncomplicated cases it should be repeated at weeks and at - weeks after disease onset. for those who develop coronary artery aneurysms or other cardiac abnormalities more frequent evaluation is recommended. other noninvasive imaging modalities, such as mri, mra, and ultrafast ct, as well as cardiac stress testing, are being evaluated in the management of kawasaki disease. until the aneurysm resolves a stress test may be needed to guide recommendations for physical activity and the need for angiography. treatment in the acute phase of the disease is directed to reducing infl ammation in the coronary artery wall and preventing coronary thrombosis, whereas long-term therapy in individuals who develop coronary aneurysms is aimed at preventing myocardial ischemia or infarction. intravenous γglobulin (ivig) combined with high-dose aspirin is the treatment of choice in the acute phase of the disease. aspirin alone does not appear to reduce the frequency of the development of coronary abnormalities, but together with ivig it has an additive anti-infl ammatory effect. in the acute stage aspirin is given in doses of - mg/kg divided into four. the duration of high-dose aspirin varies in different centers. in many institutions the dose is reduced after the child has been afebrile for - hours. others continue until day of illness and > - hours after fever cessation. following this acute phase low-dose aspirin ( - mg/kg) is given as an antiplatelet agent until there is no evidence of coronary changes at - weeks from disease onset. for patients who develop coronary abnormalities, low-dose aspirin is continued until regression of coronary artery aneurysms, but some clinicians continue indefi nitely. ibuprofen should be avoided in children taking aspirin for its antiplatelet effect because it antagonizes platelet inhibition. ivig has been shown to reduce the incidence of coronary artery aneurisms from % to - %. [ ] [ ] [ ] [ ] a single dose of mg/kg has been shown to be superior than lower doses for consecutive days. , ivig should be started early, within days of disease onset and preferably within days. treatment before day does not appear to prevent cardiac sequelae and may be associated with an increased need for ivig retreatment. , treatment after day should be considered if there are still signs of ongoing infl ammation (elevated esr or crp) or persistent fever. not all patients respond to a single dose of ivig and may have persistent or recrudescent fever hours after completion of the initial treatment, and require a second dose. it has been observed that those children who received ivig very early in the illness are more prone to require a second infusion. vaccination with live or other vaccines should be deferred for at least months after high-dose ivig treatment, both to ensure correct immunization and to avoid fl ares of kawasaki disease. the usefulness of steroids in combination with ivig in the initial therapy of kawasaki disease is not well established. steroids shorten the duration of fever and lower esr and crp, but do not seem to infl uence the coronary outcome. , steroids have also been used for ivig treatment failures, but their role in preventing or reversing coronary anomalies is uncertain. it has been recommended to restrict their use in children who have persistent fever after two infusions of ivig. the most common regimen is intravenous pulse methylprednisolone, mg/kg for - days. because of its inhibition of tnf-α messenger rna transcription, pentoxifylline may have a theoretical benefi t in the initial treatment of kawasaki disease, although there are only small clinical trials reported. the role of tnf-α antagonists such as infl iximab; abciximab, a platelet glycoprotein iib/iia receptor inhibitor; plasma exchange; and cytotoxic agents for patients with refractory kawasaki disease, remains uncertain. , acute hemorrhagic edema (ahe), purpura en cockade, or finkelstein disease, is an acute, benign leukocytoclastic vasculitis of limited skin involvement occurring in children under years of age. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ahe has been considered an infantile variant of henoch-schönlein purpura; however, because of clinical and prognostic differences it is sometimes regarded as a separate entity. the disease is characterized by the abrupt onset of fever; tender edema of the face, eyelids, ears, scrotum, and acral extremities; and ecchymotic purpura on the face and extremities. the trunk is usually spared. individual lesions often have a darker center and expand centrifugally, giving them a cockade or target-like confi guration. lesions range in size from . to . cm and may become confl uent, forming polycyclic, annular plaques ( fig. - ). necrotic , and bullous lesions may be seen. , petechiae in the mucous membranes have also been described. except for fever, there are no associated manifestations. in many patients there is a preceding upper respiratory tract infection. the dramatic cutaneous fi ndings contrast with the general wellbeing of the patient. acute hemorrhagic edema fig. - acute hemorrhagic edema. the cause of ahe is unknown. it is thought to represent an immune complex-mediated disease precipitated by a preceding infection, particularly an upper respiratory tract infection, drug intake, or immunization. staphylococci and streptococcus spp. and viruses (adenoviruses, rotavirus) have been implicated most commonly. leukocytosis (both lymphocytic and granulocytic), thrombocytosis, eosinophilia, and an elevated esr may be present. urinalysis, tests for occult blood in the stool, immunoglobulin, and complement levels are usually normal or negative. circulating immunocomplexes may occasionally be found. histopathologic examination of skin biopsy specimens demonstrates a small vessel leukocytoclastic vasculitis. direct immunofl uorescence shows deposition of c and fi brinogen in the vessel wall. igm, igg, iga, and ige deposition has also been noted in up to one-third of cases. , [ ] [ ] [ ] the differential diagnosis includes henoch-schönlein purpura, child abuse, meningococcemia and other infectious purpuras, erythema multiforme, kawasaki disease, and sweet syndrome. , distinction from henoch-schönlein purpura may be impossible (table - ). perivascular deposits of iga are not useful for differentiation because they may be present in both entities. the prognosis is excellent. the eruption resolves spontaneously without sequelae in - weeks. treatment with corticosteroids is not necessary and may lead to complications and worsen the prognosis. exacerbations may be observed during the clinical evolution, with new crops of lesions and fever, , but true recurrences weeks or months after the fi rst episode are rare. , there has been a single report of a fatal ileoileal intussusception in an infant with cutaneous lesions otherwise typical for ahe. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the porphyrias are a group of diseases characterized by abnormalities of porphyrin-heme metabolism. each type results from defi cient activity of one of the enzymes of the heme biosynthetic pathway, which leads to an accumulation of heme precursors within plasma, red blood cells, urine, and feces. the genes for these enzymes have been characterized. , , porphyrias are mainly inherited in an autosomal dominant manner with incomplete penetrance, but autosomal recessive and more complex patterns of inheritance are also possible. porphyrias are classifi ed as hepatic or erythropoietic, according to the organ site in which the underlying defect of heme synthesis is predominantly expressed (see table - ). cutaneous manifestations in porphyrias may be classifi ed as acute photosensitivity with burning pain, edema, and erythema shortly after sun exposure, or delayed photosensitivity manifesting as skin fragility, subepidermal blisters, milia, disorders of pigmentation, and sclerodermoid signs. hepatic porphyrias usually manifest acute neurovisceral attacks and delayed photosensitivity, and rarely present before puberty except from the homozygous variants. elevated porphyrins may be detected in the stool or urine. erythropoietic porphyrias are characterized by acute cutaneous photosensitivity from early childhood. the more delayed photosensitivity, although less characteristic of this type of porphyria, may be also present. erythrocyte and plasma porphyrin levels are elevated in erythropoietic porphyrias. photosensitivity in porphyrias is maximum for ultraviolet wavelengths between and nm ('soret band'), the spectrum of maximum absorption of porphyrins. the pathophysiologic mechanisms involved in the cutaneous manifestations of the porphyrias are multiple and involve the creation of reactive oxygen specimens. [ ] [ ] [ ] childhood porphyrias are relative uncommon and their exact incidence is unknown. only those porphyrias manifesting early in infancy are reviewed here. congenital erythropoietic porphyria (cep), also called günther disease, is a rare autosomal recessive disorder caused by deficient activity of uroporphyrinogen iii (urogen iii) synthase which leads to nonenzymatic conversion of hydroxymethylbilane to uroporphyrinogen i, a nonphysiologic substrate that is converted to coproporphyrinogen i; these porphyrinogen i isomers are then oxidized to uroporphyrin i (uro-i) and coproporphyrin i (copro-i), which are phototoxic compounds. elevated levels of uro-i and copro-i in erythrocytes result in massive hemolysis, and the released porphyrins accumulate in peripheral blood, skin, bone, and teeth and are excreted in large amounts in the urine and feces. , , cep presents with severe photosensitivity from birth or early infancy with formation of vesicles and bullae on areas exposed to sun, phototherapy devices, or even ambient lighting. there is also marked skin fragility. as a result of the phototoxic injury and the increased skin fragility, there are severe mutilations, mainly of the fi ngers, hands, and face, particularly the nose and ears, but also in sun-protected areas. hypertrichosis of the face and extremities, scarring alopecia of the scalp and eyebrows, and pigmentary changes (hyperpig- histologic examination of skin biopsy specimens from blisters reveals subepidermal cleavage (within the lamina lucida) and minimal infl ammatory infi ltrate. perivascular accumulation of pas-positive, diastase-resistant, homogeneous hyaline material (porphyrins) may be seen, which is best viewed with fl uorescence microscopy. see table - for porphyrin excretion profi le. measurement of uro iii synthase activity is available. prenatal diagnosis from amniotic fl uid is possible by either measurement of uroproporphyrin i or direct gene mutation analysis. [ ] [ ] [ ] [ ] other photosensitivity diseases presenting early in life (box - ) or diseases manifesting with blisters, such as epidermolysis bullosa and bullous pemphigoid, should be considered. determination of porphyrins is diagnostic and allows differentiation from other porphyrias presenting early in life with photosensitivity. the clinical severity of cep is highly variable, ranging from hydrops fetalis, hepatosplenomegaly, and severe anemia in utero to adult-onset disease with only cutaneous manifestations. in most cases, however, patients survive well into adulthood, albeit with severe mutilations or major disfi gurement. mentation and hypopigmentation) are also common. over time, severe facial mutilation results, with destruction of nasal and auricular cartilages, ectropion, and eclabium, as well as shortening and contraction of the fi ngers. milder phenotypes may have onset later in childhood. the accumulation of porphyrins in deciduous and permanent teeth produces red discoloration (erythrodontia) and reddish fl uorescence on wood's light examination, which is pathognomonic. the urine is also reddish, which causes pink discoloration of the diapers that fl uoresces -an early bed-side diagnostic sign. porphyrins accumulate in the amniotic fl uid and brownish amniotic fl uid may be observed. severe hemolytic anemia and secondary splenomegaly occur. anemia may be so severe as to lead to hydrops fetalis and death in utero. patients with late-onset disease may not develop hemolytic anemia but only thrombocytopenia and myelodysplasia. ocular changes include ectropion, photophobia, and keratoconjunctivitis. other manifestations include osteodystrophy with increased bone fragility, and porphyrin-rich gallstones. the gene for urogen iii synthase is localized on chromosome . several mutations have been identifi ed, protection from sun exposure is essential. chemical sunscreens do not achieve good protection against soret band radiation, so protective clothing and physical sunblocks are necessary. long-wavelength, uv-absorbing fi lms are encouraged on car windows and windows at home. children with the severe phenotypes and severe hemolysis benefi t from repeated erythrocyte transfusions and hydroxyurea to suppress erythropoiesis. hematocrits should be maintained above %, with appropriate iron chelation. the effi cacy or repeated erythrocyte transfusion may decrease at puberty. subsequent splenectomy is often needed to control hemolytic anemia. , activated charcoal, [ ] [ ] [ ] [ ] and β-carotene , have been used, with inconsistent results. bone marrow transplantation or stem cells from cord blood offer the possibility of correcting enzyme activity. [ ] [ ] [ ] [ ] [ ] replacement gene therapy has been accomplished in vitro. , erythropoietic protoporphyria , , erythropoietic protoporphyria (epp) is the most common form of cutaneous porphyria apart from porphyria cutanea tarda (pct). epp is caused by defi cient activity of ferrochelatase, leading to the accumulation of protoporphyrin in erythrocytes, plasma, and feces. clinical symptoms typically begin in infancy or early childhood, with a peak incidence between and years of age. epp is usually inherited as an autosomal dominant condition, but most individuals who are heterozygous for the inherited mutations are asymptomatic, because of halfnormal ferrochelatase activity for protoporphyrin to accu-mulate suffi ciently to cause photosensitivity, a reduction of enzymatic activity to below a critical threshold of about % of normal is required. some families may have autosomal recessive inheritance. [ ] [ ] [ ] clinical manifestations are those of an acute phototoxic reaction, which triggers an episode of crying within minutes of sunlight exposure due to burning pain or stinging sensations on exposed areas (the face and the dorsal aspect of hands). some patients are photosensitive to fl uorescent lighting. erythema, edema, and urticarial lesions occur, but vesicles and bullae are rare (fig. - ) . fine petechiae may occur on sunexposed areas after prolonged exposure. some patients have only subjective symptoms. with chronic exposure there is characteristic thickening and wrinkling of the knuckle pads, furrowing around the mouth (pseudorhagades), and shallow elliptical scars on the nose, cheeks, and forehead. hemolytic anemia is absent, but in some patients a mild hypochromic, microcytic anemia may occur. protoporphyrinrich gallstones may develop in childhood. fatal liver failure resulting from the progressive accumulation of protoporphyrin in hepatocytes is a possible outcome in about . % of patients, altering the prognosis for an otherwise clinically benign disorder. recessive inheritance may predispose to severe liver disease. families. , in most symptomatic patients inheritance of a second mutation is needed in order to reduce the enzymatic activity to a critical threshold where clinical symptoms are caused. autosomal recessive inheritance has been demonstrated in % of patients with epp. histopathologic examination of skin biopsy specimens of sunexposed areas shows marked concentric deposits of a hyaline material around dermal blood vessels. this material is pas positive and diastase resistant. patients with epp should undergo frequent liver function tests, and those with persistent abnormalities should have a liver biopsy. children with high erythrocyte protoporphyrins should have periodic determination of blood, urinary, and fecal porphyrins because increased excretion of copropophyrins, high erythrocyte protoporphyrins and reduced excretion of faecal protoporhyrins can predict liver failure. the diagnosis of epp is established by detecting elevated levels of protoporphyrin in erythrocytes, plasma, and feces. in addition, fecal and erythrocyte coproporphyrins may be increased. a rapid microfl uorometric assay for free erythrocyte protoporphyrins and examination of a blood smear for fl uorescent erythrocytes may also be used as screening tests. the differential diagnosis includes other types of porphyria, but causes of immediate photosensitivity such as pmle or solar urticaria do not occur in infants. the mainstay of treatment for erythropoietic protoporphyria is sun avoidance and the use of physical sunscreens. , topical dihydroxyacetone may be helpful in some patients by producing brown pigment. oral administration of βcarotene ( - mg/day for children) has been shown in uncontrolled studies to increase tolerance to sun exposure because it quenches the formation of free radicals. , , narrowband ultraviolet b phototherapy has been proposed, as this wavelength does not cause photosensitivity. desensitization with puva therapy has also been used. oral iron, intravenous hematin, transfusion therapy, and a highcarbohydrate diet have been used to prevent protoporphyrin accumulation in the liver by reducing protoporphyrin production, but their effi cacy is unproven. cholestyramine or activated charcoal have been used to interrupt the enterohepatic circulation of protoporphirins. avoidance of alcohol and drugs that interfere with hepatic excretory function is also essential. liver transplantation has been performed in a few patients with liver failure, although the enzymatic defect is not thereby corrected and hence the long-term outcome is poor. [ ] [ ] [ ] [ ] modifi cation of the lighting in the operating room is necessary to avoid photoxicity to exposed organs. hepatoerythropoietic porphyria (hep) is an extremely rare disorder caused by a marked defi ciency of uroporphyrinogen decarboxylase due to a homozygous state. [ ] [ ] [ ] [ ] clinical manifestations begin in infancy, or more commonly in early childhood, and resemble both porphyria cutanea tarda and günter disease. the disease usually presents with darkening of the urine and delayed-type cutaneous photosensitivity, with vesicles, skin fragility, milia, and scarring. with time, hypertrichosis, sclerodermoid changes, and mutilation similar to the manifestations of günter disease become apparent. anemia, hepatosplenomegaly, and abnormalities of liver function of varying degrees may also occur, but are less common than in congenital erythropoietic porphyria. the porphyrin excr etion pattern resembles that of porphyria cutanea tarda (pct), with elevated urinary uroporphyrins and -carboxylated porphyrins, and a smaller elevation of coproporphyrins, -and -carboxylated porphyrins. increased isocoproporphyrins in feces are characteristic. unlike in pct, erythrocyte proto is increased. treatment is directed to sun protection. hypertrichosis in hepatoerythropoietic porphyria has been treated successfully with high-intensity pulses of noncoherent light. other porphyrias with onset of symptoms in infancy or early childhood include homozygous variants of aminolevulinate dehydratase (alad) defi ciency, homozygous coproporphyria (harderoporphyria), homozygous variegate porphyria, and homozygous acute intermittent porphyria. alad defi ciency porphyria is rare (fewer than patients reported) and usually manifests later in childhood or adulthood, but neonatal onset has been reported. clinical manifestations from birth are recurrent attacks of pain, vomiting, hyponatremia, and symptoms of polyneuropathy affecting motor functions, including respiration. raised levels of aminolevulinic acid and coproporphyrin in urine are found. very low erythrocyte aminolevulinate dehydratase activity is diagnostic. liver transplantation in patients with neonatal onset has little effect. in harderoporphyria, neonatal jaundice, hemolytic anemia, and hepatosplenomegaly dominate the clinical picture. [ ] [ ] [ ] [ ] blisters may occur during phototherapy for neonatal jaundice. diagnosis depends on detecting very low coproporphyrinogen oxidase activity, elevated coproporphyrin in urine, markedly homozygous variegate porphyria may present shortly after birth with marked photosensitivity or, more commonly, with erosions, blisters, and milia following minor trauma in sunexposed areas. [ ] [ ] [ ] acute attacks are absent, but mental and growth retardation, seizures, nystagmus, and clinodactyly have been described. homozygous variant of acute intermittent porphyria may present early in life with ataxia, mental retardation, convulsions, cataracts, and hepatosplenomegaly, but acute attacks typical of acute intermittent porphyria do not occur in these children. [ ] [ ] [ ] [ ] there are no cutaneous manifestations. markedly increased porphobilinogen and alad in urine are found and are responsible for the orange urine. [ ] [ ] [ ] [ ] transient increases in porphyrin levels have been described in neonates with hemolytic disease of the newborn and in a neonate with severe liver failure due to tyrosinemia type . these infants develop erythema, violaceous discoloration, purpura, erosions, and blisters in areas exposed to phototherapy, with sharp demarcation at photoprotected sites. sensitivity to sunlight may occur. elevated levels of plasma/urine porphyrins (mainly coproporphyrin) and/or erythrocyte protoporphyrin are found, which normalize spontaneously during the fi rst few months. the cause of transient porphyrinemia is unclear but is probably due to cholestasis. other factors likely to be involved include blood transfusions and drug use. purpura in the neonate is almost always an emergency and should prompt an immediate search for an underlying disorder. apart from trauma, purpura in the newborn may be due to coagulation defects, platelet abnormalities, or infections (see . extramedullary erythropoiesis also causes purpuric lesions by a different mechanism. in the evaluation of a neonate with purpura it is important to obtain a maternal and familial history of bleeding diathesis and thromboembolic phenomena, drug intake, and symptoms of infectious diseases. a general physical examination and workup for sepsis is warranted. laboratory studies should include hemoglobin and hematocrit values, platelet count, white blood count, coagulation studies, and torch serologies. persistence of the erythropoietic activity of fetal dermal mesenchyme into the newborn period produces a characteristic purpuric eruption for which the term blueberry muffi n baby was coined. the eruption, fi rst observed in newborns with congenital rubella (fig. - ) , may be the result of other intrauterine infections (fig. - ) and hematologic dyscrasias. [ ] [ ] [ ] a blueberry muffi n-like eruption may also represent metastatic infi ltration of the dermis by congenital malignancies, without true extramedullary erythropoiesis (fig. - ). the cutaneous lesions of blueberry muffi n babies consist of dark blue or magenta, nonblanchable, round to oval papules ranging in size from to mm and have a generalized distribution, with emphasis on the head, neck, and trunk ( fig. - ). the papules are fi rm to palpation, with an infi ltrative quality that distinguishes them from petechiae and purpura, which often coexist in the same patient. these lesions evolve into dark purple to brown macules and involute spontaneously within - weeks. blueberry muffi n lesions caused by infi ltrative processes are usually larger, more nodular, less hemorrhagic, fewer in number, and fi rmer to palpation. accompanying abnormalities vary with the underlying cause. in the prevaccination era rubella was the most common cause of dermal erythropoiesis, but now congenital cytomegalovirus (cmv) infection is the major cause. , dermal erythropoiesis has been associated with other intrauterine infections, such as coxsackie b and parvovirus b , as well as hematologic dyscrasias such as rh incompatibility (fig. - ) , , maternofetal abo incompatibility, spherocytosis, and the twin transfusion syndrome , (box - ) . in rare instances it may occur in otherwise healthy newborns. , histopathologic examination demonstrates poorly circumscribed collections of nucleated and nonnucleated red blood cells, predominantly confi ned to the reticular dermis and extending to the subcutaneous tissue. [ ] [ ] [ ] occasionally a few myeloid precursors may be interspersed. laboratory fi ndings depend on the underlying cause. in the evaluation of a blueberry muffi n baby the following tests are indicated: peripheral blood count, hemoglobin level, torch serologies, viral cultures, and a coombs' test. skin biopsy is not always necessary for diagnosis, but may be helpful if an infi ltrative process is suspected. the differential diagnosis includes other causes of neonatal purpura, such as coagulation defects, platelet abnormalities, and infections. neoplastic diseases that produce infi ltrative metastases in the neonatal period, such as neuroblastomas, [ ] [ ] [ ] rhabdomyosarcomas, the lesions of true dermal erythropoiesis fade and resolve spontaneously - weeks after birth. treatment is directed at the underlying condition. neonatal purpura fulminans is a rare condition characterized by massive and progressive hemorrhagic necrosis of the skin accompanied by thrombosis of the cutaneous vasculature in the neonatal period. [ ] [ ] [ ] [ ] [ ] [ ] occasionally larger vessels and other organs are involved. the primary pathologic event is widespread thrombosis, which is responsible for a hematologic picture of disseminated intravascular coagulation (dic). in neonates, purpura fulminans is usually the result of inherited thrombophilic disorders that are attributable to protein c deficiency, protein s defi ciency, or resistance to activated protein c due to factor v mutations. neonatal purpura fulminans manifests - hours after birth. in rare instances, delayed onset of up to - months of age has been described. cutaneous lesions consist of extensive ecchymoses in a diffuse and often symmetric distribution that rapidly evolve into hemorrhagic bullae and purple-black necrotic skin lesions, which ultimately form a thick eschar ( fig. - ). the initial ecchymotic areas are sharply defi ned from the surrounding skin and usually have a red, advancing infl ammatory rim. they are most common at sites of trauma or pressure, the buttocks, extremities, trunk, and scalp. mucous membranes may rarely be involved. if treatment is instituted in the fi rst - hours, before necrosis ensues, the initial lesions may be reversible. other organs may be affected by the microvascular thrombosis, most commonly the cns and eye, but also the kidney and gastrointestinal tract. cavernous sinus involvement, which may occur in utero, can result in hydrocephalus, seizures, intracerebral hemorrhage, and mental retardation. , , microphthalmia, cataracts, and blindness due to vitreous or retinal hemorrhage may be seen. , deep venous thrombosis and pulmonary embolism have also been described. purpura fulminans in the neonatal period is almost always caused by inherited thrombophilic states such as homozygous protein c and s defi ciency or resistance to activated protein c. severe bacterial infection associated with dic can also induce purpura fulminans in the neonate, although it is more common in infancy or early childhood. , proteins c and s are vitamin k-dependent glycoproteins with antithrombotic properties. , protein c defi ciency is an autosomal dominant disease with incomplete penetrance. homozygous or compound heterozygous patients have a severe clinical phenotype and usually present with neonatal purpura fulminans, although they may be asymptomatic or present later in life with recurrent thrombosis. , protein s defi ciency is also transmitted as an autosomal dominant trait with incomplete penetrance. homozygous patients may develop neonatal purpura fulminans, although the risk is lower than in patients with homozygous protein c defi ciency. , neonatal purpura fulminans may also be caused by activated protein c resistance due to a mutation in the factor v gene. , resistance to activated protein c may coexist with protein s and protein c defi ciencies, becoming an additional genetic risk factor for purpura fulminans or thromboembolic complications, and explaining in part the incomplete clinical penetrance of inherited thrombophilic disorders. blood coagulation studies demonstrate evidence of dic, including prolonged prothrombin and partial thromboplastin times, increased fi brin split products, reduced fi brinogen, and reduced platelets. microangiopathic hemolytic anemia may occur. biopsy of the early skin lesions demonstrates occlusion of dermal blood vessels by microthrombi. hemorrhage and dermal necrosis are present in the more advanced stages. necrosis of the overlying epidermis with subepidermal hemorrhagic bullae occurs in later phases. secondary fi brinoid necrosis of dermal vessel walls may be present in the necrotic areas, but primary vasculitis is absent. , a defi nitive diagnosis of protein c and s defi ciency is established by measurements of protein c and s levels. protein c defi ciencies can be identifi ed by immunoenzymatic assays measuring the actual concentration of the protein in plasma, and two functional assays measuring the enzymatic activity and the anticoagulant activity. these tests distinguish two types of protein c defi ciency. in type i, which is the most common, reduced synthesis of the normal protein leads to a low plasma concentration in all three assays. in type ii, a qualitative defi ciency, levels are normal but functional assays are abnormal. for protein s defi ciency, functional and immunoenzymatic assays are available, and both the free form and the inactive form that circulates bound to c b-binding protein have to be measured. type i defi ciency is characterized by low total and free protein s, type ii by normal free protein s and low activity, and type iii by low free protein levels with normal total levels. interpreting the results of the assays may be diffi cult because protein c and s levels are physiologically reduced in the neonatal period, and may be undetectable in sick newborns with liver disease, respiratory distress syndrome, dic, or sepsis. , , , , a complete sepsis workup is therefore recommended in any case of neonatal purpura fulminans. serial determination of protein levels in patients and other family members is necessary to exclude a transient defi ciency and confi rm true congenital defi ciency. the cutaneous lesions of purpura fulminans are very characteristic and rarely mistaken for any other condition. other causes of purpuric eruptions in the newborn may be considered (see . without treatment, neonatal purpura fulminans is often fatal. if the diagnosis is suspected, therapy should be initiated immediately without waiting for the results of protein c and s measurements. prompt treatment may completely reverse early skin lesions. initial therapy consists of the administration of fresh frozen plasma ( - ml/kg/ h) or prothrombin complex concentrate, sources of protein c, protein s, and activated protein c. , a protein c concentrate has been developed that has the advantage of avoiding blood volume overload and does not carry the risk of transmission of viral fig. - neonatal purpura fulminans due to protein c defi ciency. ment therapy to avoid coumarin-induced skin necrosis. experience with long-term treatment using protein c infusions is limited. there are few case reports of a successful liver transplant for homozygous protein c defi ciency. , this benign, transient purpura in transfused neonates who undergo phototherapy is characterized by raspberry-colored, nonblanching lesions at exposed sites, sparing sites that are protected from lights (e.g. leads and temperature probes). , the eruption develops after - days of phototherapy and clears spontaneously after discontinuation of light therapy. histologically there is extravasation of red blood cells in the dermis without epidermal damage. the pathogenesis of this disease is unknown, although transient porphyrinemia has been detected in some patients. , the purpuric nature of the eruption and the absence of 'sunburn cells' differentiate this eruption from 'sunburn' caused by exposure to uva from fl uorescent lamps. congenital erythropoietic porphyria and transient elevated porphyrin levels in neonates with hemolytic disease may also cause photosensitivity. drug-induced phototoxicity in neonates who have received photosensitizing chemicals such as fl uorescein dye, furosemide or methyleneblue must be considered. [ ] [ ] [ ] [ ] diseases. , protein s concentrate is not yet available for clinical use. replacement therapy should be continued until all lesions have healed, usually after - weeks. long-term treatment involves careful administration of oral anticoagulants, starting at very low doses and with protective replace- the annular erythemas persistent' annular erythema of infancy an annular erythema of infancy annular erythema of infancy annular erythema of infancy congenital annular erythema persisting in a -year-old girl persistent annular erythema of infancy erythema annulare centrifugum: report of a case with neonatal onset erythema gyratum atrophicans transiens neonatale erythema gyratum atrophicans transiens neonatale: a variant of cutaneous neonatal lupus erythematosus reactive erythemas: erythema annulare centrifugum and erythema gyratum repens erytheme annulaire centrifuge de darier à debut neonatal avec ans de suivi. effi cacité de l'interféron et role de cytokines erythema 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analysis and review of the world literature with report of an additional case blueberry muffi n baby': hematopoiese extramedullaire neonatale? leucemie monoblastique congenitale involutive? ou histiocytose congenitale involutive? histiocytoses langerhansiennes congenitales cutanees. a propos de cas purpura fulminans. a cutaneous manifestation of severe protein c defi ciency homozygous protein s defi ciency in an infant with purpura fulminans neonatal purpura fulminans: a genetic disorder related to the absence of protein c in blood report of the working party on homozygous protein c defi ciency of the subcommittee on protein c and protein s, international committee on thrombosis and haemostasis neonatal purpura fulminans due to homozygous protein c or protein s defi ciencies purpura fulminans in a newborn baby homozygous protein c defi ciency with delayed onset of symptoms at to months neonatal purpura fulminans in association with factor v r q mutation homozygous protein c defi ciency manifested by massive venous thrombosis in the newborn severe homozygous protein c defi ciency severe acquired neonatal purpura fulminans causes of purpura fulminans protein c pathway in infants and children protein c and protein s defi ciencies late-onset homozygous protein c defi ciency asymptomatic homozygous protein c defi ciency homozygous protein s defi ciency due to a one base pair deletion that leads to a stop codon in exon iii of the protein s gene the discovery of activated protein c resistance blood coagulation and its regulation by anticoagulant pathways: genetic pathogenesis of bleeding and thrombotic diseases dermatopathology of skin necrosis associated with purpura fulminans neonatal purpura fulminans and transient protein c defi ciency homozygous protein c defi ciency -management with protein c concentrate replacement therapy with a monoclonal antibody purifi ed protein c concentrate in newborns with severe congenital protein c defi ciency en bloc heterotopic auxiliary liver and bilateral renal transplant in a patient with homozygous protein c defi ciency successful treatment of homozygous protein c defi ciency by hepatic transplantation transient erythroporphyria of infancy ultraviolet light burn: a cutaneous complication of visible light phototherapy of neonatal jaundice methylene-blue-induced hyperbilirubinemia and phototoxicity in a neonate methylene blue-induced phototoxicity: an unrecognized complication fluorescein phototoxicity in a premature infant phototoxic blisters from high frusemide dosage key: cord- - p yieqv authors: fan, chunyan; lei, xiujuan; tan, ying title: inferring candidate circrna-disease associations by bi-random walk based on circrna regulatory similarity date: - - journal: advances in swarm intelligence doi: . / - - - - _ sha: doc_id: cord_uid: p yieqv identification of associations between circular rnas (circrna) and diseases has become a hot topic, which is beneficial for researchers to understand the disease mechanism. however, traditional biological experiments are expensive and time-consuming. in this study, we proposed a novel method named bwhcda, which applied bi-random walk algorithm on the heterogeneous network for predicting circrna-disease associations. first, circrna regulatory similarity is measured based on circrna-mirna interactions, and circrna similarity is calculated by the average of circrna regulatory similarity and gaussian interaction profiles (gip) kernel similarity for circrnas. similarly, disease similarity is the mean of disease semantic similarity and gip kernel similarity for diseases. then, the heterogeneous network is constructed by integrating circrna network, disease network via circrna-disease associations. subsequently, the bi-random walk algorithm is implemented on the heterogeneous network to predict circrna-disease associations. finally, we utilize leave-one-out cross validation and -fold cross validation frameworks to evaluate the prediction performance of bwhcda method and obtain auc of . and . ± . , respectively. moreover, the predicted hsa_circ_ -gastric cancer association is analyzed. results show that bwhcda could be an effective resource for clinical experimental guidance. circular rnas (circrnas) are a type of non-coding rnas with closed loop structures formed by back splicing [ ] . recently, large number of circrnas are widely found in various livings [ ] , and they could regulate gene expression at transcriptional or posttranscriptional levels by titrating micrornas (mirnas) [ ] , regulating transcription and splicing [ , ] , even several circrnas could translate to produce polypeptides [ ] . increasing researches have demonstrated that the mis-regulation of circrnas may cause abnormal cellular functions and associated with various diseases [ ] . thus, diseaseassociated circrnas are becoming a class of promising biomarkers for disease diagnosis and treatment. however, it is costly and laborious to identify the disease-related circrnas with biomedical experiments. recently, several computational approaches have been developed. lei et al. [ ] firstly designed a path weighted approach named pwcda to predict circrna-disease associations. likewise, katzhcda [ ] is developed based on katz model to measure the probability for each pair of circrna-disease associations, in which the circrna expression similarity and disease phenotype similarity matrices are used as priori knowledge to establish the circrna-disease heterogeneous network. dwnn-rls [ ] is designed based on kronecker regularized least squares to predict the associations between circrnas and diseases. icircda-mf [ ] is developed based on non-negative matrix factorization by integrating the circrna-gene, gene-disease and circrna-disease relationships. wang et al. [ ] utilized a recommendation algorithm personalrank to measure the relevance between circrnas and diseases based on circrna expression profiles and functional similarity. although several methods have developed for the circrna-disease association prediction, it is still a challenge to obtain sufficiently accurate results. in this study, we developed a novel framework for forecasting circrna-disease associations named bwhcda, which integrated multiple similarity measures and implemented bi-random walk algorithm (fig. ) . first, circrna regulatory similarity is effective measured based on circrnas may play essential roles in regulating mirna function in disease occurrence and progression. moreover, combined with gaussian interaction profiles (gip) kernel similarity for circrnas, the integrated circrna similarity is effectively measured. similarly, disease similarity is denoted as the average of disease sematic similarity and gip kernel similarity for diseases. subsequently, the heterogeneous network is constructed by combing the circrna network, disease network and circrnadisease associations. then, circular bigraph (cbg) patterns are introduced in bi-random walk algorithm to predict the missing associations based on the heterogeneous network. the results show that bwhcda could be considered as a powerful tool for predicting circrna-disease associations. the experimentally validated human circrna-disease associations are extracted from the circr disease database [ ] . then, we choose the associations that circrnas have been recorded in circbase database [ ] and disease name recorded in disease ontology database [ ] . finally, we retained circrna-disease associations between cir-crnas and diseases as the gold standard dataset. the circrna-disease adjacency matrix a(i,j) is established, if there is an association between circrna and disease, a(i,j) is set as , otherwise . the mirna-circrna interactions are downloaded from the circbank database [ ] , and the interactions overlapped with disease-related circrnas are selected to measure the regulatory similarity of circrnas. it is measured as follows: where the set of m i have relationship with circrna c i and the set of mirna m j have relationship with circrna c j . the disease names are described as hierarchical directed acyclic graph (dag) based on the medical subject headings (mesh) descriptions for diseases. and disease semantic similarity is calculated by the dose [ ] tool with wang et al. method. based on the assumption that similar circrnas (diseases) are tend to have similar interaction or non-interaction pattern with diseases (circrnas) [ ] , the gip kernel similarity for circrnas and diseases are respectively calculated as follows: where c(i) (or d(i)) denotes the circrna (disease) interaction profiles, which is the i-th row (column) of the adjacency matrix a. the parameters γ c and γ d are used to control the kernel bandwidth. n c (or n d ) is the number of circrnas (diseases). the new circrna similarity scores (sc) are calculated with the average scores of the circrna regulatory similarity and gip kernel similarity for circrnas. similarly, the integrated disease similarity (sd) is denoted as the mean of the disease semantic similarity and gip kernel similarity for diseases. then, the integrated circrna similarity and integrated disease similarity are adjusted with the logistic function [ ] . where x is the value of element of matrix sc or sd. parameters c and d control the adjustment effects, and we set c as − and set d as log( ), respectively. according to the circrna similarity and disease similarity measures, the circrna network and disease network can be constructed. next, the weighted heterogeneous circrna-disease network is constructed based on the circrna network, disease network via gold standard circrna-disease associations. the heterogeneous network could be considered as a bipartite graph, the nodes represent circrnas or diseases, the edges represent three types of interactions of circrna-circrna, disease-disease and circrna-disease. based on the topology and structure characteristics of circrna network and disease network, the concept of cbg was introduced. a cbg is described as a subgraph of a circrna path {c , c , …, c n } and a disease path {d , d , …, d m }, in which the two ends connected by circrna-disease associations (c , d ) and (c n , d m ). the cbg indicates a vicinity relation between the two association (c , d ) and (c n , d m ), which is generalized by their distance to other associations in the circrna network and disease network. the length of cbg patterns (l, r) is determined by the longer length of circrna path and the disease path. in this study, we hypothesize that most potential associations tend to be covered by many shorter cbgs in the unknown circrna-disease network. if there are more cbg patterns between circrnas and diseases, the higher possibility of circrna-disease associations are. by iteratively adding the circrna path and disease path, we calculates the cbgs weighted by decay factor α that ranges from to . because of different structures and topologies in the circrna network and disease network, disparate optimal number of random walk steps are generated. therefore, parameters l and r are introduced to restrict the number of random steps in circrna similarity network and disease similarity network, respectively. the iterative process of bi-random walk is described as follows: on the circrna network: on the disease network: where α is the decay factor that controls the importance of cbg for different paths, sl l and sd l represent the normalized matrix by using laplace regularization. where dc(i,i) (or dd(i,i)) is the diagonal matrix of circrna similarity matrix s c (s d ). by combining the propagation scores of matrices cc and dd, the relevance scores of unknown circrna-disease associations could be obtained. the bwhcda algorithm is outlined as table . to assess the performance of bwhcda method, leave-one-out cross validation (loocv) and -fold cross validation ( -fold cv) framework are performed on the gold standard datasets. for loocv, each known circrna-disease association is removed in turn as testing sample, and the other associations are regarded as training samples. then, the unknown circrna-disease associations are considered as candidate associations, and the prediction performance is assessed by the predicted rank of test sample. in the framework of -fold cv, circrna-disease associations are randomly divided into ten subsets, and each subset is utilized in turn as test set and the remaining as the train set on each time. to decrease the sample division bias, we perform times repetitions of -fold cv. the receiver operating characteristic (roc) curves are plotted to show the prediction performance by calculating the true positive rate (tpr) and false positive rate (fpr). furthermore, the area under the curves (aucs) are calculated to evaluate the overall performance. there are three parameters in the bwhcda method, including α, i l , i r . to test the effects of the three parameters, we set α value as { . , . , . , . }, and i l , i r are set from to , respectively. then, we could calculate auc values based on loocv and the effects of these parameters are shown in tables , , and . the results indicate that α has little effects on prediction performance. when α = . , i l = and i r = , the auc value of loocv is the highest with step length less than five. when α = . , i l = , i r = , auc value of loocv is the highest within step length less than four. the auc value of loocv is the highest within step length than three steps when α = . , i l = , i r = . and when α = . , i l = , i r = , the auc value of loocv is the highest within two steps. finally, we set three parameters as α = . , i l = , i r = , respectively. to further evaluate the prediction performance of bwhcda, we compare it with other five methods including katzhcda [ ] , pagerank [ ] , ncp [ ] , bdsilp [ ] and table . when α is set as . , the effect of parameters i l and i r for loocv auc. table . when α is set as . , the effect of parameters i l and i r for loocv auc. table . when α is set as . , the effect of parameters i l and i r for loocv auc. table . when α is set as . , the effect of parameters i l and i r for loocv auc. hetesim [ ] . consequently, bwhcda method achieve the best performance among these six approaches based on auc values of loocv and -fold cv with the same datasets (figs. and ) . therefore, bwhcda method is better than other five methods. to further assess the prediction performance of bwhcda method, we analyze the predicted hsa_circ_ -gastric cancer association. as shown in fig. , hsa_circ_ may interact with mirnas including hsa-mir- , hsa-mir- , hsa-mir- , hsa-mir- b- p, hsa-mir- to play their biological roles. the mirna targets gene of these mirnas have been validated related with gastric cancer, including hsa-mir- target hpse, hsa-mir- b- p target per and irak , hsa-mir- target ercc , hsa-mir- - p target erbb . in addition, hsa-mir- has been validated associated with gastric cancer. therefore, hsa_circ_ may be a potential biomarker for gastric diagnosis and prognosis. prioritizing the potential associations between circrnas and diseases is benefit to the development of the understanding of the disease mechanism, diagnose and treatment for diseases. the reasons that why bwhcda method has better performance is shown as following aspects. first, bi-random explored the cbg patterns with iteratively implement random walk on the circrna similarity network and disease similarity network. in addition, bwhcda is a multi-task learning method that could forecast potential circrna-disease associations simultaneously rather than mine candidate circrnas for specific diseases. therefore, bwhcda could be an effective method for biomedical research. circular rna biogenesis can proceed through an exon-containing lariat precursor circular rnas are a large class of animal rnas with regulatory potency a cerna hypothesis: the rosetta stone of a hidden rna language? circular intronic long noncoding rnas a circrna from sepallata regulates splicing of its cognate mrna through r-loop formation circcode: a powerful tool for identifying circrna coding ability circrna: functions and properties of a novel potential biomarker for cancer pwcda: path weighted method for predicting circrnadisease associations prediction of circrna-disease associations using katz model based on heterogeneous networks dwnn-rls: regularized least squares method for predicting circrna-disease associations icircda-mf: identification of circrna-disease associations based on matrix factorization predicting circrna-disease associations based on circrna expression similarity and functional similarity circr disease: a manually curated database for experimentally supported circular rnas associated with various diseases circbase: a database for circular rnas disease ontology: a backbone for disease semantic integration circbank: a comprehensive database for circrna with standard nomenclature dose: an r/bioconductor package for disease ontology semantic and enrichment analysis gaussian interaction profile kernels for predicting drug-target interaction associating genes and protein complexes with disease via network propagation the pagerank citation ranking: bringing order to the web network consistency projection for human mirna-disease associations inference the bi-direction similarity integration method for predicting microbe-disease associations predicting the associations between microbes and diseases by integrating multiple data sources and path-based hetesim scores acknowledgements. this work was supported by the national natural science foundation of china ( , , ) and the fundamental research funds for the central universities, shaanxi normal university (gk ). key: cord- -xqj en authors: petras, robert e.; frankel, wendy l. title: large intestine (colon) date: - - journal: modern surgical pathology doi: . /b - - - - . - sha: doc_id: cord_uid: xqj en nan normal large bowel gross anatomy and microscopic anatomy - are outlined here. the large bowel extends from the ileocecal valve to the anus and measures cm to cm in adults. it can be clinically useful to divide the large bowel into regions. the cecum, the most proximal saccular part of the large bowel, lies inferior to a horizontal line defined by the ileocecal valve. the cecum is completely invested by peritoneum and contains the opening of the vermiform appendix. the right colon, cm to cm in length, extends to the hepatic flexure. the right colon lacks mesentery and lies mostly in the retroperitoneum except for its anterior and right lateral serosa. the transverse colon averages cm to cm in length, runs from the hepatic flexure to the splenic flexure, and has a mesentery. the descending colon begins at the splenic flexure, becomes retroperitoneal, and extends for cm to cm. at the distal portion of the descending colon, the large bowel once again acquires a mesentery to become the sigmoid colon, which measures approximately cm in length. the sigmoid colon arbitrarily becomes the rectum at approximately the level of the third sacral vertebra. the rectum, measuring cm to cm in length, ends at the anal canal. the upper one third of the rectum is covered by peritoneum; the lower two thirds lies in the retroperitoneum surrounded by the fatty mesorectum. beneath the mesothelium-covered serosa lies a subserosal layer of fibroadipose tissue. the muscularis externa of the large bowel is composed of an inner circular layer and an outer longitudinally running layer of smooth muscle that condenses into three longitudinally running taeniae coli, the mesocolic taenia and two antimesenteric taeniae. the taeniae unite at the base of the vermiform appendix. they flare at the rectum and incorporate into its external muscular layer. the inner and outer layers of muscularis externa are separated by the myenteric plexus of auerbach. appendages of subserosal fat typically hang from the large bowel to form the epiploic appendices. extending luminally from the muscularis externa lie the fibroadipose tissue, blood vessels, lymphatics, and nerves of the submucosa. the submucosa contains meissner's plexus, which is usually found closely juxtaposed to the muscularis mucosae. the inner surface of the large bowel is characterized by horizontally oriented folds (plicae semilunares) and fine innominate grooves of the mucosa. the proximal colon to the splenic flexure derives its blood supply from the superior mesenteric artery through the ileocolic, right colic, and middle colic branches. the remainder of the colon is supplied by the left colic and sigmoid branches of the inferior mesenteric artery. the inferior mesenteric artery and iliac vessels provide blood to the rectum. veins accompany the arteries and share their names. the large bowel venous drainage enters the portal circulation except for the distal rectum, which drains into the systemic circulation through the middle and inferior rectal veins. in portal hypertension, this area can serve as a portalsystemic shunt and can be a site of varices. the lymph node drainage is divided into those lymph nodes close to the bowel wall (e.g., pericolic, perirectal) and those that follow the blood vessels (e.g., mesenteric). the vagus nerves supply stimulatory nervous activity to the right colon and proximal transverse colon. the remainder of the large bowel is supplied by pelvic postganglionic parasympathetic nerves. the inhibitory nervous activity is derived from the superior and inferior mesenteric plexuses. the large bowel mucosa is composed of a single-cell layer of colorectal epithelium covering the lumen and lining the crypts, the supporting lamina propria and a small smooth muscle band referred to as the muscularis mucosae. the normal colorectal luminal surface of the mucosa is straight; the glands are made up of tubules (crypts) that are tightly packed, parallel, nonbranching, and closely approximating the muscularis mucosae ( fig. - ) . the appearance of the colonic tubules is similar to rows of test tubes placed in a rack. goblet cells interspersed between colorectal absorptive cells line the colonic tubules. scattered neu-roendocrine cells can be observed usually near the base of the crypt and contain basally oriented eosinophilic cytoplasmic granules. the lamina propria contains a modest amount of mixed inflammatory cells including plasma cells, lymphocytes, eosinophils, mast cells, and macrophages. intraepithelial lymphocytes are present normally but usually are fewer than per colorectal epithelial cells. the muscularis mucosae is arranged into two layers (an inner circular and outer longitudinally running layer) and is usually thin and regular. the submucosa is typically devoid of inflammatory cells. scattered mucosal and submucosal lymphoid follicles are normally encountered, especially in younger individuals. in areas of mucosal lymphoid follicles, the mucosal architecture may be distorted and the muscularis mucosae can be incomplete. flattened epithelial cells known as m cells overlie the mucosal lymphoid aggregates. the epithelium of the m-cell zone typically contains numerous intraepithelial lymphocytes. [ ] [ ] [ ] paneth cells with their basal nuclei and luminal cytoplasmic refractile red granules are seen in the base of colonic crypts but are considered normal only in the cecum and proximal right colon. , mucosal biopsy interpretation can be hampered by changes associated with bowel preparation and with the trauma of the biopsy procedure itself. changes ascribed to bowel preparation include decreased intraepithelial mucin, increased numbers of mitotic figures, surface apoptosis with karyorrhectic debris in the superficial lamina propria, and small numbers of neutrophils and eosinophils in surface or crypt epithelium. [ ] [ ] [ ] [ ] [ ] [ ] edema and recent hemorrhage into tissues not associated with other degenerative or inflammatory changes most likely represent biopsy-related trauma. muciphages (foamy macrophages containing faintly periodic acid-schiff [pas]-positive material) are often present in the lamina propria of the large bowel, especially the rectum, where they most likely represent a nonspecific response to mucosal injury (i.e., trauma). , muciphages should be distinguished from xanthelasma/xanthomatous polyp, which can also occur in the large intestine ( fig. - ). an adenoma, defined as a benign intraepithelial neoplasm composed of epithelial cells exhibiting cytologic dysplasia, is considered the precursor lesion of most colorectal carcinomas. , [ ] [ ] [ ] dysplasia is characterized by decreased intraepithelial mucin, epithelial nuclear enlargement with hyperchromasia, nuclear stratification, and increased numbers of mitotic figures. large bowel adenomas are highly prevalent in western societies. the frequency of these tumors markedly increases after age years and reaches a peak at age years. adenomas are usually asymptomatic but large ones may bleed. adenomas usually produce a raised endoscopically or grossly detectable abnormality, generally a protrusion or polyp that can often be further subclassified as sessile or pedunculated. some adenomas appear flat; some may cause mucosal depressions. adenomas occur singly or can be multiple. multiple (≥ ) adenomas may indicate a genetic syndrome such as familial adenomatous polyposis (fap), attenuated fap, or myh-associated polyposis syndrome. most adenomas are small, measuring less than mm. adenomas should be classified histologically based on the pattern of growth as tubular, villous, or tubulovillous following the world health organization (who) guidelines. , adenomas in which simple tubules make up more than % of the area are classified as tubular. adenomas with greater than % of their area showing a villiform configuration are called villous adenomas ( fig. - ) ; all others should be reported as tubulovillous adenomas. once discovered, adenomas are characteristically removed by endoscopy or surgery because they are an important precursor lesion to colorectal carcinoma. there-fore, it is not surprising that occasionally a resected polyp thought to be a benign adenoma may contain an area of carcinoma. the various nomenclatures applied to colorectal adenomas, dysplasia, and malignant polyps can be confusing. unfortunately, no unified accepted guidelines exist. [ ] [ ] [ ] most surgical pathologists use variations of the who terminology. in this system, the terms dysplasia, adenocarcinoma in situ, intramucosal adenocarcinoma, and invasive adenocarcinoma are accepted. each has a precise meaning when applied to colorectal polyps and appropriate patient care requires that the endoscopist, surgeon, and surgical pathologist understand the significance of each of these terms. all adenomas demonstrate at least low-grade epithelial dysplasia. without dysplasia, an adenoma cannot be recognized and distinguished from normal colonic mucosa. lowgrade dysplasia is characterized by a slight decrease in the amount of intracellular mucin, mild nuclear enlargement with hyperchromasia, some nuclear stratification, and an increased number of mitotic figures (fig. - ) . increasing degrees of dysplasia (low-grade to high-grade) show progressive loss of intracellular mucin, progressive increase in nuclear size with stratification, and a loss of nuclear polarity. adenocarcinoma in situ describes the next step in the dysplasia-carcinoma sequence. here, the atypical glands assume a complex cribriform or back-to-back gland configuration but the basement membrane remains intact (fig. . some experts consider adenocarcinoma in situ as part of the spectrum of high-grade glandular dysplasia and report both under the same term. when carcinoma cells infiltrate into the lamina propria or muscularis mucosae only, terms such as high-grade glandular dysplasia and adenocarcinoma in situ are technically no longer applicable because both require an intact basement membrane. there- fore, the term intramucosal adenocarcinoma is more accurate (fig. - ). , finally, when carcinoma cells have invaded the submucosa (or beyond) the lesion is labeled invasive adenocarcinoma. invasion is invariably associated with an infiltrative pattern to neoplastic glands associated with tumor desmoplasia (fig. - ) . this tumor desmoplasia is extremely helpful in recognizing invasion of at least the submucosa, especially in small biopsy specimens. the nomenclature controversy principally centers on the observation that in the colon and rectum, infiltrating carcinoma cells do not become clinically significant (i.e., able to metastasize) until they have invaded the submucosa. , , , only polyps containing invasive adenocarcinoma require a decision for additional treatment on the part of the clinician. adenoma, adenocarcinoma in situ, and even intramucosal adenocarcinoma lack metastatic capability and are considered adequately treated by polypectomy alone. , , , , as a result, some pathologists advocate modification of the nomenclature to account for clinical behavior and promulgate use of the term high-grade glandular dysplasia to encompass high-grade dysplasia, adenocarcinoma in situ, and even intramucosal adenocarcinoma. , although the who guidelines accepted and defined two (low-grade, high-grade) or three (mild, moderate, severe) grades of dysplasia, adenocarcinoma in situ, and intramucosal adenocarcinoma, the authors of those guidelines recommended a similar behavior-based modification for intramucosal carcinoma and stated that "… intramucosal adenocarcinoma of the colon has not been shown to metastasize, and for this reason 'carcinoma in situ' is more appropriate." the version of the who classification added little clarification and introduced new and even more confusing terms. the authors stated that the defining feature of colorectal adenocarcinoma is invasion through the muscularis mucosae into the submucosa. however, once defined, worrisome lesions not fulfilling this criterion become difficult to describe. for example, the who classification defines adenocarcinoma in situ and intramucosal adenocarcinoma as lesions with morphologic characteristics of "adenocarcinoma" confined to the epithelium or that "invade" the lamina propria alone and lack invasion through the muscularis mucosae. the who goes on to state that these lesions have virtually no risk of metastasis. according to the who, the term "… high-grade intraepithelial neoplasia is more appropriate than adenocarcinoma in situ and … intramucosal neoplasia is more appropriate than intramucosal adenocarcinoma." in the version, the who believes that use of these terms will help avoid overtreatment. the problems with this classification are many. the inaccurate use of the term invasion to describe lesions that are not by definition invasive carcinoma is confusing. the lesser lesion of high-grade intraepithelial neoplasia sounds worse than the term used to describe intramucosal adenocarcinoma (intramucosal neoplasia). furthermore, all adenomas, strictly speaking, are intraepithelial neoplasia. an effort to achieve consensus (largely between eastern [japanese] and western pathologists) [ ] [ ] [ ] [ ] resulted in the vienna classification of gastrointestinal (gi) neoplasia, presented in table - . problems with the vienna system include the following: ( ) inaccurate use of the word invasion; ( ) category "noninvasive" high-grade neoplasia including potentially dangerous lesions (e.g., suspicious for invasive adenocarcinoma); and ( ) category "invasive neoplasms" including intramucosal adenocarcinoma, which is widely accepted to be clinically benign in the colon and rectum. it is unlikely that this system of categories without clinical correlation will ever gain widespread acceptance. as modified from the who classification, low-grade dysplasia, high-grade dysplasia, adenocarcinoma in situ, and intramucosal adenocarcinoma exist and can be recognized by pathologists. , this nomenclature remains attractive because it can be applied throughout the gi tract. if one chooses to diagnose high-grade dysplasia, adenocarcinoma in situ, and intramucosal adenocarcinoma in colorectal biopsy specimens, specific mention in the report that these lesions lack metastatic potential is helpful to clinicians. because infiltrating carcinoma cells in a colorectal polyp do not become clinically significant (i.e., able to metastasize) until they have invaded the submucosa, , - , - only a polyp containing invasive adenocarcinoma (invasion of at least the submucosa) should be considered malignant. only invasive adenocarcinoma requires a decision regarding additional treatment. therefore, the presence or absence of invasive adenocarcinoma should be specifically mentioned in the pathology report. to comply with the american college of gastroenterology (acg), the u.s. multi-society task force on colorectal cancer, and the american cancer society guidelines, , , a villous component (villous or tubulovillous adenoma) and high-grade dysplasia should be reported because these features require more frequent surveillance. carcinoma in situ and intramucosal adenocarcinoma can be reported parenthetically as high-grade dysplasia. most mistakes that pathologists make in reporting colorectal adenomas, dysplasia, and malignant polyps occur in three major categories: ( ) the pathology report is not clear (nonspecific or noncommittal terms are used or the presence or absence of invasive adenocarcinoma is not clearly stated); ( ) mispositioned glands (pseudocarcinomatous invasion) are misinterpreted as invasive adenocarcinoma; and ( ) the margin of excision is either not identified or not discussed. a common problem concerns differentiating invasive carcinoma complicating a colorectal adenoma from pseudocarcinomatous invasion (pseudoinvasion). pseudoinvasion describes a situation in which neoplastic glands of the adenoma are mispositioned, presumably by trauma, into or beneath the muscularis mucosae. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] pseudoinvasion is relatively common, reported in % to % of resected colorectal polyps. , , distinguishing this epithelial mis- placement from invasive adenocarcinoma can be difficult. some reported series of "malignant polyps" have included and even illustrated polyps with pseudoinvasion as examples of invasive adenocarcinoma associated with adenoma. histologic features favoring pseudoinvasion include the lack of an infiltrative pattern, the lack of tumor desmoplasia, the presence of lamina propria around mispositioned glands, the lack of increased atypia in mispositioned epithelium as compared with the surface epithelium of the adenoma, and the presence of hemorrhage or hemosiderin deposits in nearby connective tissue ( fig. - ) . occasionally, the misplaced glands of pseudoinvasion can become cystic, can rupture, and can be associated with dissection of mucus into the connective tissues of the polyp. in this case, the distinction between mucinous adenocarcinoma and misplaced glands can be extremely difficult. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] table - illustrates histologic features that can aid in this differential diagnosis. examination of additional sections can help in difficult cases because almost all mucinous adenocarcinomas contain at least small foci of typical nonmucinous-type adenocarcinoma. a rational decision concerning management of a patient with an endoscopically removed malignant colorectal polyp (one containing invasive adenocarcinoma) requires weighing the chances of finding residual or metastatic cancer with a follow-up surgical excision (whom do i help?) against the risk of surgical mortality and morbidity (whom do i hurt?). some investigators have advocated surgical resection for all patients. currently, however, almost all surgeons and gastroenterologists embrace a more conservative approach and use certain gross and histologic features as indications for follow-up colectomy. , these features include sessile growth, , residual villous adenoma, a short stalk (< mm), stalk invasion, level invasion, , lymphatic or vascular permeation, lack of a residual adjacent adenoma (so-called polypoid carcinoma), poor differentiation, , , , and invasive carcinoma at or near a margin of resection. , , we and others , , , believe that two features identify patients likely to avoid an adverse outcome defined as residual or metastatic adenocarcinoma in a subsequent colectomy specimen or during clinical follow-up. patients with favorable histology (defined as well-or moderately differentiated adenocarcinoma with a -mm tumor-free margin of resection in the polypectomy specimen) experienced no adverse outcome and are considered adequately treated by polypectomy alone. similar, although not identical, therapeutic recommendations have been adopted by the acg. , these guidelines consider colonoscopic polypectomy definitive treatment for a patient with a malignant polyp if the following criteria are fulfilled: ( ) the polyp is considered completely excised at endoscopy, ( ) the specimen is properly processed by the pathology laboratory, ( ) the cancer is not poorly differentiated, ( ) no histologic evidence of vascular or lymphatic involvement exists, and ( ) the resection margin is not involved by carcinoma. lymphatic or venous invasion, proposed as an indication for follow-up colectomy, remains controversial. , , , , , , , only a few malignant polyps with these features have been reported and almost all have had positive margins, contained poorly differentiated invasive carcinoma, or both. we think that lymphatic or venous invasion is not a reliable criterion because the distinction from retraction artifact is frequently difficult. cooper and colleagues encountered significant interobserver variation in assessing this feature. furthermore, no guidelines exist that establish the extent to which a pathologist must go to diagnose lymphatic or venous invasion (e.g., number of sections or use of immunostains). although patients can be stratified into high-risk and low-risk groups based on margin status and grade of invasive adenocarcinoma, and lymphatic or and in deference to these guidelines, the presence or absence of angiolymphatic invasion should be reported. as a guide for therapy, the major studies of endoscopic polypectomy for malignant polyps have shown that the chance of finding residual or metastatic cancer in a subsequent colon resection specimen or during follow-up in the "favorable histology" group is less than %. weighing these odds against the published operative mortality rates for colectomy that range between % and %, [ ] [ ] [ ] it seems that subsequent major surgical resection should be avoided in the "favorable histology" subgroup. , if a decision for subsequent colorectal resection is made, a cancer operation is recommended rather than a more limited procedure because cancer was the indication for surgery. residual carcinoma in a follow-up resection specimen can be expected in only % of cases. these cases of residual or metastatic carcinoma that are discovered within this subset of pt lesions are overrepresented by cases containing poorly differentiated carcinoma. evaluation of the resection line is critical to proper patient management; therefore, correct handling of the polypec-tomy specimen is of utmost importance. , , , the entire polyp should be immediately placed into fixative. following adequate fixation, a polyp with a stalk should be trimmed on either side of the stalk as illustrated in figure - . the section of the polyp with stalk and margin can be embedded in a block, to maintain the correct anatomic relationship. the remainder of the polyp should be submitted in separate blocks. for polyps without stalks (sessile growths or those in which the stalk has retracted), look for the effect of cautery on the gross specimen. this will appear as a lightercolored area or a defect on the external surface of the polyp. carefully trim on either side of this defect (fig. - ) and place this tissue in a block. again, the remaining tissue should be submitted in separate blocks. routine examination of a minimum of three step-sections stained with hematoxylin-eosin (h&e) from each block is recommended. in the pathology report, the presence or absence of invasive carcinoma must clearly be stated. with malignant polyps, the grade of carcinoma must be noted, the resection line must be identified and assessed, and the status of that resection line must be clearly stated in the pathology report. a distance measurement of carcinoma-free margin should be included in the report. in deference to the acg, the presence or absence of angiolymphatic invasion should be investigated and reported. the treating physician must individualize the decision for follow-up colorectal excision by weighing the patient's wishes against the estimated cancer recurrence risk and the predicted operative morbidity and mortality. advances in laparoscopic resection of the colon and rectum could drastically reduce the morbidity and mortality of operative resection that now constitutes the major contraindication for surgery. these newer surgical techniques may require reassessment of the current management recommendations for malignant colorectal polyps. more than , new cases of colorectal carcinoma occur in the united states each year and account for approximately , deaths annually. the peak incidence occurs between the ages of and years; fewer than % of cancers occur in patients less than years of age. risk factors for carcinoma include diets rich in animal fat, sedentary lifestyle, and coexisting inflammatory bowel disease (ibd). at least five separate but overlapping molecular pathways to colorectal cancer may exist. approximately % of colorectal carcinomas occur sporadically, whereas % appear to have a genetic basis. , the genetic group includes the % of cases related to lynch's syndrome (hereditary nonpolyposis colon cancer syndrome [hnpcc] ) and the % associated with fap and its variants. the other % of cases show strong familial clustering but a specific genetic cause has yet to be found. colorectal carcinoma can also be viewed another way. approximately % of colorectal cancers are thought to originate through the chromosomal instability pathway. these tumors typically demonstrate dna aneuploidy and have abnormalities of chromosomes , , and and contain mutational changes in apc gene, k-ras proto-oncogene, dcc tumor suppressor gene, and p tumor suppressor gene. fap colorectal carcinomas arise through this pathway. approximately % of colorectal carcinoma appears to arise in the so-called mutator phenotype. these cancers tend to be dna diploid and are associated with microsatellite instability (msi). the cancers related to lynch's syndrome are associated with the mutator phenotype. dna integrity is essential for normal cell function. dna insults can result from the direct effects of chemicals or radiation and are usually corrected through the excision repair system. dna replication errors are of two types: ( ) simple mispairing of nucleotides, the most common type; and ( ) "slipping" errors, in which genes may contain too many or too few copies of repeat short dna nucleotide sequences known as microsatellites. normally, these errors are recognized, the cell cycle is arrested, and the mismatched segment is corrected. for those errors not immediately corrected by dna polymerase, the mismatch repair (mmr) system acts as a backup for additional proofreading of dna. failure to repair mismatches allows the error (mutation) to persist and to become the template for subsequent dna replication. the known mmr genes and their relative frequency in lynch's syndrome are presented in table - . msi is best viewed as an epiphenomenon found in colorectal tumor dna but not in non-neoplastic tissues. it indicates that extensive mutation exists in the nonencoding repetitive dna sequences that are particularly prone to replication error, the microsatellites. the majority of msi is linked to somatic inactivation of hmlh through hypermethylation inactivation of the promotor region, but it can also be detected in persons with germline mmr gene mutations, the definition of lynch's syndrome. msi is detected in % of colorectal cancers overall and is present in more than % of the cancers found in patients with lynch's syndrome. because patients with lynch's syndrome have a germline mutation of an mmr gene, they are at increased lifetime risk for colorectal (≈ %) and other cancers. , these cancers develop at significantly younger ages (e.g., average age for colorectal carcinoma ≤ years). other tumors related to lynch's syndrome include cancers of the endometrium, ovary, stomach, biliary tract, urinary tract, kidney, central nervous system, small bowel, and skin. patients and families with lynch's syndrome can sometimes be identified by taking a careful patient and family medical history, can be suggested from the pathologic findings of excised tumors, and can be detected by direct evaluation of the mmr system. pathologic features of colorectal cancer that suggest msi/lynch's syndrome include right-sided location, synchronous or metachronous large bowel cancers, large and bulky polypoid tumors with circumscribed pushing margins, tumors showing prominent lymphoid infiltrate, and cancers of poor differentiation (medullary or undifferentiated carcinoma) or mucinous and signet ring cell histologic pattern (figs. - and - ) . , , the diagnosis of lynch's syndrome is evolving. originally, the amsterdam criteria were used to identify hnpcc clinically including patients with lynch's syndrome. the original amsterdam criteria included ( ) three or more relatives with colorectal cancer with at least one first-degree relative, ( ) colorectal carcinoma in two generations, and ( ) one or more colorectal carcinomas occurring in a person less than years of age. to increase the sensitivity, the amsterdam criteria were modified (amsterdam ii criteria) to include ( ) three or more relatives with any carcinoma related to lynch's syndrome, ( ) colorectal carcinoma in two generations, and ( ) and one or more carcinomas related to lynch's syndrome in a person younger than years of age. detecting lynch's syndrome based on the amsterdam criteria alone poses many problems. patient histories are less useful now than in the past because of requires than just one of the following criteria be met: ( ) colorectal cancer before age years, ( ) synchronous or metachronous colorectal or other tumor related to lynch's syndrome regardless of the patient's age, ( ) colorectal cancer with msi-high pathology (msi-h) in a patient less than years old, ( ) person with colorectal cancer and a first-degree relative with colorectal adenoma or carcinoma or other lynch's syndrome-related tumor (cancer < years; adenoma < years), and ( ) colorectal cancer with two or more relatives with colorectal or other lynch's syndrome-related tumor regardless of age. the american gastroenterological association position states that genetic testing should be performed on ( ) families meeting amsterdam criteria, ( ) any affected person meeting the modified bethesda guidelines, and ( ) any firstdegree relative of those with known mutations of mmr genes. these guidelines suggest that after pretest genetic counseling and written informed consent, immunohistochemical analysis for mmr gene products or msi testing by pcr be performed on tumor tissue. the international guidelines for evaluation of msi by pcr recommend use of consensus markers: bat , bat , d s , d s , and d s . if two or more markers are abnormal, the carcinoma is considered msi-h. if one marker is abnormal, the tumor is classified as msi-low (msi-l). if no markers are abnormal, the cancer is referred to as msi-stable (mss). laboratories using more than five loci modify this classification with % to % or more abnormal defined as msi-h, less than % to % as msi-l, and none abnormal as mss. immunohistochemistry can be used to detect msi. almost all msi-h cancers can be identified if the antibody panel includes mlh , msh , pms , and msh (figs. - and - ) . , immunohistochemical analysis and msi analysis by pcr have specific advantages and limitations. pcr requires a molecular laboratory and usually requires normal tissue for comparison. immunohistochemistry is more widely available but can be limited by poor tissue fixation or poor technique rendering interpretation difficult. immunohistochemistry may be superior because the findings can direct gene sequencing and msi is not always seen in lynch's syndrome kindred with msh germline mutation. patients with msi-h cancer should undergo additional genetic testing including gene sequencing. mss and msi-l tumors require no further testing. additional genetic evaluation may be considered if the clinical history is compelling. the clinical significance of identifying lynch's syndrome is that affected individuals and at-risk persons are recognized and can be screened and treated with correct surgical procedures. subtotal colectomy is usually recommended to treat colon cancer related to lynch's syndrome because of the high likelihood of synchronous or metachronous cancers. partial colectomy with colonoscopy every to years is a reasonable alternative. furthermore, clinicians can institute proper screening such as colonoscopy at a young age (beginning at age or years younger than the youngest cancer history in the family), periodic endometrial sampling (every to years starting at age years), pelvic ultrasound, ca serum testing, and urine cytology or molecular testing for urinary tract carcinoma. many experts screen all resected colorectal cancers for msi initially by pcr. immunohistochemical analysis is a useful alternative and some prefer this as the initial test because an abnormality in protein expression correlates almost invariably with msi-h by pcr. in cases showing normal mmr proteins or equivocal staining by immunohistochemical analysis, msi testing by pcr should be done in clinically suspicious cases to exclude a germline mutation that can yield an antigenic protein that is biologically inactive. msi testing in sporadic colorectal carcinoma is a subject of considerable contemporary interest and debate. much like their counterparts in patients with lynch's syndrome, sporadic msi-h carcinomas have a predilection for the right colon, mucinous histologic features, and a prominent lymphoid infiltrate. strong arguments exist for routine testing for msi in all resected colorectal carcinomas including the lower mortality rate independent of tumor stage. sporadic msi-h cancer can also be associated with an increased rate of metachronous tumors with subsequent clinical implications for cancer surgery, surveillance, and follow-up. msi status may also have implications for chemotherapy. improved survival is reported in patients with mss and msi-l stage ii and stage iii cancers who are treated with fluorouracil-based regimens. , finally, routine msi testing could increase the detection of lynch's syndrome because % of probands were more than years old and up to % of patients with lynch's syndrome did not fulfill amsterdam or bethesda guidelines. colorectal serrated polyps and the serrated pathway to colorectal cancer hyperplastic polyps are the most common benign polyps of the large intestine. , these polyps are usually small (< mm), sessile, and are often about the same color as the surrounding colonic mucosa. histologically, evenly distributed absorptive and goblet cells line crypts that are elongate and dilated (fig. - ). inhibition of normal apoptosis is thought to be the underlying mechanism for polyp formation and because more epithelial cells are present per unit area than normal, the cells must pseudostratify, thus imparting a serrated or micropapillary appearance. characteristically, the basement membrane under the surface epithelium is thickened and hyalinized. regenerative epithelial changes, mitotic figures, and active inflammation can be quite prom- c d inent at the crypt bases. this regenerative area can occasionally cause diagnostic confusion with dysplasia and carcinoma, especially in a variant referred to as inverted hyperplastic polyp. , in this inverted variety, the regenerative epithelium of the crypt base is misplaced into or beneath the muscularis mucosae. most examples of inverted hyperplastic polyp are now probably best classified as sessile serrated polyp (see later) and are easily recognized if one is cognizant of its existence and also notes the overall architectural and cytologic similarity to hyperplastic polyp/sessile serrated polyp. the entity is distinguished from invasive adenocarcinoma by the lack of infiltration and tumor desmoplasia. the differential diagnosis between hyperplastic polyp and tubular adenoma can be difficult, especially in a diminutive polyp that has been treated by hot biopsy (so-called thermal polyp). useful features in the differential diagnosis are found in table - . when the choice between hyperplastic polyp and tubular adenoma is difficult, as long as an adenoma diagnosis is not going to result in surgical resection (e.g., right colonic adenoma incompletely excised), we err on the side of adenoma to ensure that the patient will receive more frequent surveillance. mixtures of hyperplastic polyp, sessile serrated polyp, and adenoma occur. , mixed polyps and serrated adenomas are considered in more detail later. rare examples of patients with colons carpeted by hyperplastic-like polyps (hyperplastic polyposis) have been described ( fig. - ). the who defines hyperplastic polyposis as ( ) or more hyperplastic polyps proximal to the sigmoid colon of which are larger than cm, ( ) any number of hyperplastic polyps proximal to the sigmoid colon if the person has a first-degree relative with hyperplastic polyposis, and ( ) more than hyperplastic polyps of any size and any molecular events involved in the serrated polyp family are now recognized. methylation-induced inactivation of mmr genes occurs in both hyperplastic polyps and carcinoma. as shown in table - , methylation inactivation of genes and certain gene mutations (especially braf) appear to be involved in the serrated pathway to carcinoma. , these molecular events have been verified. [ ] [ ] [ ] [ ] [ ] [ ] hyperplastic polyps associated with carcinoma have been unusually large and right-sided lesions. they have been reported using numerous synonyms, including giant hyperplastic polyp, sessile serrated adenoma, sessile serrated polyp, inverted hyperplastic polyp, and polyp with epithelial serrated proliferation. it is becoming clear that several different pathologic entities have been called hyperplastic polyps in the past. this serrated polyp family includes conventional hyperplastic polyp, mixed hyperplastic/sessile serrated polyp/adenoma ( fig. - ), serrated adenoma (epithelial dysplasia defined, usually pedunculated and left sided, having eosinophilic cytoplasm and showing gastric foveolar change and often referred to as the traditionally defined serrated adenoma) ( fig. - ) , and hyperplastic-like polyps with unusual features that have been referred to as sessile serrated polyps or sessile serrated adenomas. , , [ ] [ ] [ ] [ ] sessile serrated polyps appear related to serrated adenomas and mixed polyps and could be the specific precursor lesion to sporadic msi-h carcinoma. transitions from sessile serrated polyps to areas of dysplasia and carcinoma with loss of hmlh protein expression have been described . , sessile serrated polyps as the name implies are sessile, large (frequently ≥ cm), right sided, and often show poor endoscopic circumscription. numerous cytologic and architectural abnormalities have been reported in sessile serrated polyps, especially those associated with carcinoma. , , , the abnormal proliferation or dysmaturation features include persisting nuclear atypia with large nuclei and nucleoli high (upper third) in the crypts, high (upper third of the crypt) mitotic figures, and irregular distribution of dystrophic goblet cells. architectural abnormalities include basal crypt dilatation, horizontally oriented crypts, crypt branching, an increased epithelial-to-stromal ratio (> %), inverted crypts, prominent serration, increased surface villosity or papillations, and the lack of a surface basement membrane thickening typical of conventional hyperplastic polyps. some authors suggest that a diagnosis of sessile serrated polyp requires the presence of at least four of the architectural and abnormal proliferation features mentioned earlier (figs. - and - ) . once recognized, the sessile serrated polyp creates a patient management dilemma. calling them "sessile serrated adenomas" may not be an appropriate default. first and foremost, these lesions do not show the cytologic dysplasia that should be definitional for adenoma. sessile serrated adenoma is often confused by gastroenterologists and surgeons with serrated adenoma or villous adenoma. it is unknown whether colonic resection (which is typically done for incompletely excised adenomas) should be recommended for sessile serrated polyps that are incompletely excised at endoscopy. furthermore, endoscopic follow-up for serrated adenoma would typically be in years (if the clinician considers serrated adenoma or sessile serrated adenoma a variant villous adenoma) or in years. in a cohort of patients with sessile serrated polyps preceding msi-h carcinomas, predated the carcinomas by less than years. we think that these lesions should be diagnosed as sessile serrated polyps and that they should be treated by complete endoscopic excision if possible. until more is known, a shorter surveillance interval (e.g., to years) rather than resection seems prudent for these types of polyps that are incompletely excised or associated with additional, endoscopically similar polyps that have remained unsampled. [ ] [ ] [ ] [ ] colorectal carcinoma occurs more often in men (male-tofemale ratio, : ), with a median age of years. most patients present with rectal bleeding, anemia, change in bowel habits, bowel obstruction, or less often perforation. patients with right-sided colon carcinoma are more likely to present with anemia and fatigue, whereas left-sided car-cinoma is more likely to produce melena, constipation, and change in bowel habits. approximately half of all large bowel carcinomas occur in the rectum, % occur in the sigmoid colon, and the rest are evenly distributed throughout the remainder of the colon. that said, with increased use of colonoscopy with removal of adenomas, clinicians have seen a right-sided migration of carcinomas since the late s. carcinomas of the right colon tend to produce large, exophytic tumors (see fig. - ) . carcinomas of the descending colon are more likely to be stenotic and produce the so-called "napkin ring" tumor. carcinomas anywhere can be fungating, ulcerated, or necrotic masses; the most common macroscopic appearance is an ulcer with raised, indurated edges (fig. the following features adversely affect prognosis: advanced stage, extensive local spread, lymph node involvement, aggressive histologic type, high histologic grade, extramural venous invasion, and free mesothelial surface invasion. , , [ ] [ ] [ ] although useful information is gleaned through these classic grading and staging exercises, the process is not without problems and controversy. no general agreement exists on staging or grading and all current schemes have shortcomings. [ ] [ ] [ ] using current systems, most fall into a moderate-stage, moderate-grade category in which the probability of survival is roughly / . the cap considered and commented on the multitude of reputed prognostic factors in a consensus statement and concluded that certain factors have been definitively proved to be of prognostic import, including local extent of tumor (pt), regional lymph node metastases (pn), blood or lymphatic vessel invasion, and residual tumor following surgery with curative intent. other factors that have repeatedly been shown to be of prognostic importance include tumor grade, radial margin status, and residual tumor in specimens following neoadjuvant therapy. the cap recommends that these additional features should also be included in pathology reports. although customarily included in pathology reports, parameters such as tumor size and gross configuration have been well studied and are of no prognostic significance. that still leaves an incredibly large group of factors that may be considered prognostic but have not yet been sufficiently studied. this discussion focuses on controversial areas in classic staging and grading including methods of lymph node dissection and assessment of histologic grade and type. additionally, the role of flow cytometric analysis, markers of proliferative activity, and other ancillary testing will be examined. the single most important factor related to patient prognosis is the presence or absence of lymph node metastases. no doubt exists that searching for lymph nodes in a resection specimen is tedious. the lymph node yield per case is directly proportional to the dissector's enthusiasm and skill. as a general rule, a standard resection specimen for carcinoma of the sigmoid colon or rectum should contain to lymph nodes, although we all have had cases in which the dissector found far fewer. minimum numbers of lymph nodes harvested is increasingly considered a measure of quality. , therefore, the routine use of clearance techniques for lymph node dissection has been debated. certainly, clearance techniques have advantages. one is likely to find more lymph nodes in a specimen and the lymph node yield will no longer depend solely on the dissector's ability and enthusiasm. however, the clearance process is time consuming and it may delay reporting. , clearing is relatively expensive because of the large volumes of clearing agents used and the prolonged technologists' or pathologists' time. common clearing agents are often flammable and toxic. cawthorn and colleagues showed that clearance techniques increase the yield of lymph nodes per specimen when compared with routine dissection. however, the proportion of stage , , and cases did not change. the number of positive lymph nodes found was similar between cleared and noncleared groups. this finding was later confirmed. clearance techniques are considered unnecessary for routine cases. , additional controversy is added by consideration of nontraditional methods of lymph node examination such as the following: immunohistochemical analysis for carcinoembryonic antigen (cea), cytokeratins, and epithelial membrane antigen; pcr testing looking for tumor dna or rna; and sentinel lymph node examination. the biologic significance of these nontraditional methods lacks validation, , [ ] [ ] [ ] [ ] [ ] and "positive" nodes found by these techniques may have no effect on prognosis. currently, the cap recommends that all grossly identified lymph nodes be sectioned (without multiple levels) in a routine fashion. , pathologists should find as many lymph nodes as possible and should recognize that the rules of representative sampling and probability apply. as a general rule, negative lymph nodes usually correlate with true pn status. , , , extramural tumor nodules of any size with smooth con- tours are counted as replaced regional lymph nodes. sentinel lymph node examination does not accurately predict either conventionally defined nodal metastasis or micrometastasis and is not considered useful in the study of patients with colorectal carcinoma. pathologists admit that grading is more art than science. grading is subjective and prone to interobserver and intraobserver variation. one multicenter trial noted % welldifferentiated adenocarcinomas from one institution, whereas another hospital reported % well-differentiated cases. marked heterogeneity exists within a given tumor. some observers grade on the average, whereas others assign a grade corresponding to the least-differentiated area. many grading systems are used for colorectal carcinoma. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] all employ slightly different criteria that are poorly defined. some use three grades and others four. some exclude mucinous carcinoma altogether and others include it as grade iv or grade iii. criteria for mucinous carcinoma are hardly ever defined. we follow the guidelines of dukes and bussey and use a three-grade system. well-differentiated adenocarcinoma (grade i), which should account for % to % of cases, shows tubular differentiation; the nuclear polarity is easily discerned, and nuclei are generally uniform in size . approximately % of adenocarcinomas are moderately differentiated (grade ii) exhibiting a more complex and irregular tubular pattern, and the polarity of nuclei is lost or is only barely discernible ( fig. - ). the remaining poorly differentiated adenocarcinomas (grade iii) consist of highly irregular glands or may show an absence of glandular architecture. nuclear polarity is lost ( fig. - ). when variability exists within a given tumor, the grade is determined by the worst area no matter how small. mucinous carcinoma and signet ring cell carcinoma are considered poorly differentiated or grade iii. jass and colleagues investigated grading using a cox regression analysis model in resection specimens. the only grading parameters associated with prognosis were the amount of tubule configuration, the pattern of growth (expanding versus infiltrative), and the degree of lymphocytic infiltration. when stage-related parameters were added into the cox regression model, only three factors emerged as significant: ( ) lymph node involvement, ( ) local spread (i.e., the components of stage), and ( ) the amount of lymphocytic infiltration in the neoplasm (i.e., a reflection of msi status). this study provided the scientific verification of the original observation by dukes and bussey that grade was subservient to stage in prognosis and re-emphasizes the need for careful specimen dissection and examination to determine the amount of local spread and lymph node status. many clinicians believe that mucinous carcinoma and signet ring cell carcinoma are associated with significantly worse prognosis than nonmucinous adenocarcinoma. unfortu- nately, the definitions of mucinous and signet ring cell carcinoma vary. , , [ ] [ ] [ ] [ ] [ ] work by sasaki and colleagues and umpleby and associates verified that mucinous and signet ring cell carcinomas are associated with a worse prognosis, but these tumors manifest at high stage and are associated with extensive local spread. sasaki and colleagues scrutinized a large cohort of mucinous, signet ring cell, and nonmucinous carcinomas using a cox multiple regression model. according to this study, the only significant adverse prognosis-related independent variables were the presence of lymph node metastases and the extent of local spread (i.e., the components of stage), along with an infiltrative growth pattern and minimal lymphocytic infiltration. sasaki and colleagues and umpleby and associates concluded that mucinous carcinoma (> % to % by volume) and signet ring cell carcinoma (> % cells with signet ring morphology) ( fig. - ) are more aggressive. these histologic features were not, however, significantly associated with poor prognosis when they were controlled for stage. flow cytometry for examination of dna content in human tumors involves cells or isolated nuclei stained in suspension with a fluorescent dye that binds stoichiometrically with double-stranded dna. these stained cells or nuclei are then passed one by one through an excitor light source (laser). the amount of fluorescence produced by the bound dye is detectable by a photoelectric cell, and the information is stored electronically. with this technique, thousands of measurements can be made in seconds and displayed on a histogram. the position of peaks on the x-axis is proportional to the amount of dna per cell, and the height of the peaks on the y-axis is proportional to the number of cells demonstrating a particular dna content. using this method, diploid cell populations can be distinguished from nondiploid (including dna aneuploid) cell populations. studies of paraffin-embedded and fresh colorectal carcinoma specimens have demonstrated an inconsistent association between dna aneuploidy and survival. , in at least one of these studies, stage was retained as a strong independent variable associated with prognosis after mul-tiple regression analysis. other investigators showed no independent association between dna aneuploidy and prognosis in a large group of patients with colorectal carcinoma, with stage as the only independent variable associated with prognosis. dna content analysis by flow cytometry is of no proved clinical value. the technology and methods lack standardization, and the results among groups generally are not comparable. most published studies have employed paraffin-embedded material. this may not be optimal because dna fragments and partial nuclei tend to stick together, thus leading to increased yields of pseudoaneuploid histograms. the proportion of cases showing aneuploid peaks is lower when fresh intact cells are used. in terms of interpretation, control histograms are easy to read but tumor histograms are less clean and interpretations are subject to interobserver variation. in a cohort of patients with colorectal carcinoma prospectively studied, results of flow cytometric analysis showed no correlation between dna aneuploidy and any standard staging or grading parameter and had no independent association with prognosis. , the cap believes that dna analysis has not been adequately studied for determination of prognostic value and the data are insufficient to recommend a specific technologic method. various proliferation markers have been studied in colorectal carcinoma. for example, a cohort of patients with colorectal carcinoma were studied using an antibody that recognizes ki- , a nuclear antigen expressed in all phases of the cell cycle except g . no correlation was found between ki- scores and stage, grade, or prognosis. stage, growth pattern, and lymphocytic infiltration were the only factors independently associated with prognosis. the cap believes that data are insufficient to recommend inclusion of proliferation indices in pathology reports for prognostic information. early nonpolypoid colorectal carcinoma early invasive colorectal carcinoma (pt -invasive carcinoma limited to the submucosa) warrants special mention given the advent of endoscopic techniques allowing gastroenterologists and surgeons to resect some carcinomas locally either surgically or through the endoscope (e.g., endoscopic mucosal resection [emr]). because lymph node status is the strongest prognostic factor in colorectal carcinoma, the question asked, particularly by surgeons, is whether local excision or emr is enough or should definitive surgical resection be performed for pt lesions. the issue is further complicated by the low rate of lymph node metastases in pt colorectal carcinoma, estimated at % to %. , [ ] [ ] [ ] [ ] this dilemma prompted evaluation of histologic parameters and molecular markers that correlate with positive lymph node status in excised pt colorectal carcinoma. features that consistently correlate with positive lymph node status in pt colorectal carcinoma include angiolymphatic invasion, poor differentiation, tumor budding (fig. [ ] [ ] [ ] [ ] [ ] [ ] , and sm invasion (invasion of the deepest third of the submucosa). [ ] [ ] [ ] [ ] [ ] although various immunostains and molecular markers have not been significantly associated with lymph node status, gene expression profiling could improve the prediction of patients likely to have positive lymph nodes and could improve outcomes. cetuximab is a chimeric monoclonal antibody that binds to epidermal growth factor receptor (egfr). it has clinical significant activity when given alone or in combination with irinotecan in patients with advanced irinotecan-refractory colorectal carcinoma. [ ] [ ] [ ] approximately % of colorectal cancers express egfr on immunohistochemical analysis but that expression does not correlate with gene amplification. immunohistochemical analysis for egfr is sometimes used as a selection criterion for cetuximab. the threshold for positive staining results has been extraordinarily low ( + staining in > % of cancer cells) and neither the proportion of positive tumor cells nor the intensity of staining has correlated with clinical response. some patients who tested negative for egfr responded to cetuximab and many patients who tested positive did not. consequently, the national comprehensive cancer network guidelines for colorectal cancer management recommend against using egfr expression based on immunohistochemical results to select patients for cetuximab therapy. , patients with colorectal cancer bearing mutated k-ras do not benefit from cetuximab. a bevacizumab is a monoclonal antibody directed against vascular endothelial growth factor (vegf), which is critical in the regulation of angiogenesis. this monoclonal antibody added to fluorouracil-based chemotherapy regimens resulted in significant improvement in patients with advanced colorectal carcinoma. , vegf is expressed in approximately % of colorectal carcinomas. neither microvessel density determination, which is prone to significant methodologic variation, nor vegf determinations by immunohistochemical analysis were considered a selection criterion. before , fluorouracil, a thymidylate synthase inhibitor, was the only effective treatment for advanced colorectal cancer and because leucovorin (folinic acid) enhances the effect by stabilizing the bond between fluorouracil and thymidylate synthase, both agents are often given together. , , other cytotoxic drugs such as irinotecan, an inhibitor of topoisomerase i, and oxaliplatin, which distorts dna by cross-linking into adducts, are now approved for treatment for advanced colorectal carcinoma. , , irinotecan has efficacy as a first-line treatment for advanced colorectal cancer but lacks efficacy as adjunct therapy. irinotecan is hydrolyzed into an active metabolite (sn- ) by hepatic carboxyl esterase. sn- is converted into an inactive form by uridine diphosphate glucuronosyltransferase isoform a (ugt a ). in patients with polymorphisms of ugt a , the toxicities of irinotecan (diarrhea, nausea, vomiting, myelosuppression, alopecia) are more severe. although results indicate that ugt a * polymorphisms have some relevance to toxicity, especially hematologic toxicity with the first cycle of chemotherapy, determination of the polymorphism seems to have marginal clinical implications. the observed toxicities can be managed clinically, other ugt a enzymes may play a role as well, and data do not support dose reduction based on a molecular test. cisplatin and its analogues (oxaliplatin) are particularly toxic and molecular markers to identify patients likely to respond have been investigated. oxaliplatin adducts are repaired by the nucleotide excision repair complex. ercc (excision repair cross-complementation group ) is of genes that encode proteins of this complex. polymorphisms that reduce levels of ercc correlate with clinical sensitivity to oxaliplatin , and could be used for patient selection. the medullary variant of colorectal carcinoma occurs predominantly in women and usually occurs in the cecum and right colon. , histologically, it is composed of uniform polygonal cells arranged in a nesting or trabecular pattern with minimal gland formation. immunohistochemistry is often employed to rule out neuroendocrine carcinoma or melanoma. other characteristic features of medullary carcinoma include a prominent lymphoid component that can either be peritumoral, often described as crohn's diseaselike, or intratumoral with tumor-infiltrating lymphocytes (more than five per high-power field; see figs. - and - ) . medullary carcinoma is seen with increased frequency in msi-h colorectal carcinoma whether sporadic or in association with lynch's syndrome. adenosquamous carcinoma, defined by having a malignant glandular and squamous component, occurs rarely as a primary carcinoma in the colon and rectum (fig. - ). the possibility of metastasis must always be considered. adjacent adenoma can help to confirm a primary tumor. adenosquamous carcinoma has been described in patients with ulcerative colitis (uc), fap, schistosomiasis, and endometriosis. occasionally, squamous differentiation can be found in adenomas (fig. - ). pure squamous colorectal carcinoma outside of the anal canal is extremely rare. the possible diagnosis of metastasis must be excluded. squamous carcinoma has been reported in fistulas, in association with radiation, and in patients with ibd, tuberculosis, and schistosomiasis. , , occasional case reports of microglandular goblet cell adenocarcinoma (goblet cell carcinoid) identical to that seen in the vermiform appendix have been described in the large bowel. these tumors are composed of trabeculae and nests of well-differentiated adenocarcinoma cells showing differentiation toward goblet cells. scattered or no endocrine differentiation is characteristically seen by immunohistochemical analysis for chromogranin and synaptophysin. although cytologically bland, these carcinomas are often aggressive in the colon and rectum. carcinosarcoma carcinosarcoma is often referred to as spindle cell carcinoma or metaplastic carcinoma and can occur rarely in the large bowel. frequently, a high-grade squamous or glandular component is detected. the mesenchymal component can be undifferentiated or can show striated or smooth muscle differentiation or areas of cartilage or bone. these tumors are associated with a poor prognosis. , , giant cell carcinoma and choriocarcinoma can occur purely or as focal components of an otherwise typical high-grade adenocarcinoma or carcinosarcoma. in choriocarcinoma, the giant cells express beta human chorionic gonadotropin (hcg). carcinomas with giant cells that fail to stain for beta hcg are referred to as giant cell carcinomas. , , endocrine tumors involving the colon and rectum can be classified into three types based on morphology: ( ) carcinoid tumor (well-differentiated neuroendocrine tumor), ( ) intermediate-grade neuroendocrine carcinoma, and ( ) high-grade neuroendocrine carcinoma, which can be further subdivided into small cell and large cell types. , rightsided endocrine tumors are usually large, whereas rectal endocrine tumors manifest as small polyps, most being solitary and less than . cm in greatest cross dimension. rectal carcinoids are quite common. they most often exhibit hindgut patterns of growth with trabeculae, small acini, and tubular structures that may contain luminal mucin and that must not be confused with goblet cell carcinoid (microglandular goblet cell adenocarcinoma) . mitoses are rare. the cytoplasm of the carcinoid tumor cells may be clear or eosinophilic and can sometimes appear granular. nuclei are regular and ovoid and contain granular chromatin. neuroendocrine differentiation can be proved with immunohistochemical analysis for chromogranin and synaptophysin. hormonally inactive carcinoids that are less than . cm in greatest cross dimension without angiolymphatic invasion act in a clinically benign fashion. , intermediate-grade neuroendocrine carcinoma acts more aggressively and is often characterized by angiolymphatic invasion, more frequent mitotic figures, nuclear pleomorphism with open nuclear chromatin, increased apoptotic bodies, and areas of necrosis ( fig. - ). transitions from low-grade to intermediate-grade and high-grade neuroendocrine carcinoma can be observed in rare cases. high-grade neuroendocrine carcinoma is important to recognize because of its particularly poor prognosis and because of chemotherapeutic considerations. , the small cell variant is composed of sheets of densely packed small to intermediate-size cells with dark hyperchromatic nuclei, inconspicuous nucleoli, frequent mitoses, and very little cytoplasm with molding ( fig. - ). foci of squamous and glandular differentiation can be seen in high-grade neuroendocrine carcinoma. large cell neuroendocrine carci-noma shows an endocrinoid growth pattern but contains larger cells that are round or polygonal and contain more abundant cytoplasm. the nuclei show a coarse chromatin pattern and in contrast to small cell carcinoma are often nucleolated ( fig. - ). large cell carcinoma and small cell carcinoma of the colon and rectum have a poor prognosis. virtually any subtype of lymphoma or leukemia can involve the colon and rectum. the reader is directed to the section covering the hematolymphoid system for more detailed discussions of these entities. use of the who classification for tumors of the hematopoietic and lymphoid tissues is recommended. this discussion focuses on the most common lymphoproliferative disorders affecting the colon and rectum. approximately one half of extranodal lymphomas involve the gi tract. diffuse large b-cell lymphoma makes up the majority of these cases and is usually easily identified by the surgical pathologist as malignant. b-cell lineage should be confirmed with cd immunostaining. an immunohistochemical panel to rule out undifferentiated carcinoma, melanoma, and high-grade neuroendocrine carcinoma is helpful in practice. differentiation of germinal center type (better prognosis) from nongerminal center type of diffuse large b-cell lymphoma should be made using immunostains for cd , bcl- , and mum- . burkitt's lymphoma and burkitt-like lymphoma can involve the colon and rectum. , , these lymphomas are composed of relatively uniform medium-sized cells with coarse chromatin and prominent nucleoli. b-cell lineage should be confirmed with cd immunostain and, as described earlier, other high-grade neoplasms should be excluded. most investigators recommend the workup of burkitt's and burkitt-like lymphoma with mib- immuno- staining, which should highlight at least % of cells. burkitt's lymphoma cells also characteristically stain positive for cd and bcl- . lymphoid hyperplasia must be distinguished from follicular lymphoma involving the colon and rectum. this is especially true in the setting of low rectal or anorectal lymphoid hyperplasia (so-called anorectal tonsil) and in defunctioned bowel seen in the setting of primary ibd. localized lymphoid hyperplasia of the anorectal area can manifest with bleeding, prolapse, or anorectal discomfort. the endoscopic appearance can vary from a sessile polyp, which can measure up to . cm in greatest dimension, to mere thickening of the mucosa resembling cobblestoning. histologically, one sees lymphoid hyperplasia with germinal center formation and scattered interfollicular collections of pale-staining histiocytes. distinction from follicular lymphoma can usually be made with standard h&e-stained sections but immunostaining for bcl- , which is overexpressed in follicular lymphoma because of the t( ; ) translocation, can be an extremely helpful adjunct. positive bcl- staining is seen in neoplastic follicles but not in reactive germinal centers. up to % of cases of multiple lymphomatous polyposis are caused by follicular lymphoma (fig. - ). mantle cell lymphoma usually manifests with widespread lymphadenopathy and frequent bone marrow involvement. overt gi tract involvement occurs in % to % of patients and clinically occult disease can affect up to % of patients. colonic involvement can manifest as a mass, a polyp, diffuse mucosal thickening, or multiple lymphomatous polyposis. approximately one third of patients with lymphomatous polyposis have mantle cell lymphoma. the histologic pattern of mantle cell lymphoma typically shows mucosa and submucosal infiltrates by small atypical lymphocytes surrounding germinal centers with effacement. the lymphoma cells express the b-cell marker cd and often coexpress cd . the t( ; ) translocation causes overexpression of cyclin d- , which is now considered defining. , extranodal marginal zone b-cell lymphoma of the mucosa-associated lymphoid tissue (malt) type can involve the colon and rectum and is responsible for up to % of cases manifesting as multiple lymphomatous polyposis. , similar to its appearance in the stomach, colorectal marginal zone b-cell lymphoma of malt type creates destructive lymphoepithelial lesions and produces an expansive proliferation of marginal zone lymphocytes. an appropriate immunohistochemical profile includes positive staining for pan b-cell markers and negative staining for cd , cd , cd , cd , and cyclin d- . gene rearrangement studies by pcr can be helpful in difficult cases. , , mesenchymal tumors involving the colon and rectum are unusual and include gi stromal tumors (gists), true smooth muscle tumors, neural tumors, inflammatory fibroid polyp, fatty tumors, fibrohistiocytic tumors, tumors of skeletal muscle, and vascular lesions (see later). primary mesenchymal tumors of the colon and rectum must be distinguished from mesenchymal differentiation within a high-grade carcinoma. the reader is directed to the section on soft tissues for more detailed discussions of these entities. gists of the colon and rectum are rare and usually show high-grade features using the national institutes of health consensus approach for classification. approximately % of colon and rectum gists have shown aggressive clinical behavior. overexpression of c-kit using cd immunostaining must be ascertained, preferably as part of a panel of immunostains that includes cd , smooth muscle actin, desmin, cytokeratin, s- protein, and melan a to verify gist and to exclude other differential diagnostic possibilities. schwannoma in the colon and rectum is rare and is usually encountered in the absence of a predisposing condition. schwannomas are rarely seen in patients who have neurofibromatosis syndrome or multiple endocrine neoplasia type iib (men b) and must be distinguished from gists in that setting. , , neurofibromas can manifest as an isolated mass lesion or nodule but are more commonly seen as a diffuse infiltration of the bowel wall in the setting of neurofibromatosis. care must be taken to distinguish neurofibroma from schwannoma and gist, which also occur in neurofibromatosis. , , granular cell tumor can manifest as a polyp or nodule frequently in the distal rectum and anal canal where it can mimic a hemorrhoid. the tumor is composed of masses of histiocytic-like cells with granular eosinophilic or amphophilic cytoplasm, which can sometimes be spindled . the cytoplasmic granules stain positive on pas and stain intensely for s- protein. , true smooth muscle tumors are rare but leiomyomas of the muscularis mucosae are commonly encountered in biopsy specimens. these tumors are usually small, less than mm to mm in greatest cross dimension, and are composed of smooth muscle cells. atypical mitoses and necrosis typical of leiomyosarcoma are not seen and the reported behavior has been benign. primary glomus tumors of the colon and rectum are exceptionally rare , and can manifest as a polyp or mucosal thickening. microscopically, these tumors are composed of lobules of small round cells intimately related to vascular spaces and usually show positive immunoreactivity for smooth muscle actin and vimentin. the cases reported so far have acted in a benign fashion. lipohyperplasia of the ileocecal valve, which can be accentuated in obese persons, is the most common fatty tumor of the colon. , occasionally, the fat may produce a more localized tumor. patients are usually asymptomatic and the degree of lipohyperplasia generally correlates with body weight. lipomas are frequently encountered during colonoscopy. these tumors are composed of benign adipose tissue. most are small and asymptomatic but larger ones can bleed, can be subject to trauma or prolapse, cause abdominal pain, and may lead to resection ( fig. - ). lipomas are usually recognized at colonoscopy by their yellow color and pliability (pillow sign). atypical lipomas and even liposarcoma have rarely been described. rare examples of malignant fibrous histiocytoma, rhabdoid tumor, and rhabdomyosarcoma have been reported in the large bowel. numerous classifications for intestinal vascular lesions have been proposed. , we find it useful to divide the vascular lesions into two groups, those isolated to the gi tract and those associated with recognized clinical syndromes (tables - and - ). vascular ectasia of the right colon has numerous synonyms, including angiodysplasia, arteriovenous malformation, and the type i lesion of moore and colleagues. , , , vascular ectasia of the right colon is a well-defined clinicopathologic entity with a distinct type of lesion and pathogenesis. patients are typically elderly and present with episodes of recurrent low-grade lower gi bleeding. approximately % of patients present with massive gi hemorrhage. the lesions nearly always manifest in the cecum or right colon and are often multiple. vascular ectasia of the right colon is usually not associated with other conditions. reports linking vascular ectasia of the right colon to aortic valvular disease have been criticized. , however, bleeding vascular ectasias may be associated with aortic stenosis and associated abnormalities in von willebrand factor (deficiency of the largest multimers). colonoscopic and radiographic examination will show almost all lesions. endoscopically, vascular ectasias appear as erythematous mucosal macules or papules measuring . mm to mm in diameter ( fig. - ). they may be round, oval, or stellate. a "feeding" vessel may be visible. angiographic signs of vascular ectasia include the densely opacified, slowly emptying vein, an opacified vascular tuft, early opacification (filling) of a draining vein, or, rarely, extravasation of contrast media into the lumen. vascular ectasia is usually treated primarily by the endoscopist (heat probe or other endoscopic ablation technique) or by the radiologist (vasopressin infusion or transcatheter embolization) at the time of diagnosis. , the typical curative surgery, right hemicolectomy, is rarely performed and is often reserved for those cases in which endoscopy or radiography could not be performed or treatment was not successful or for perforative complications of endoscopic or angiographic treatment. vascular ectasia is notoriously difficult to identify by standard pathologic techniques in resection specimens. demonstration of the lesion usually requires some form of vascular injection. boley and brandt's technique employs injection of the fresh specimen vasculature with a silicon rubber compound. the specimen is then fixed in formalin, dehydrated in graded alcohols, and cleared with methylsalicylate. this technique produces a transparent specimen in which the vasculature can be examined by epi-illumination and transillumination under a dissecting microscope. using this technique, the vascular ectasias appear as "coral reef-like" arrangements measuring . cm to . cm in diameter contrasted against the honeycombed pattern of the normal colonic mucosal vasculature. histologic examination reveals abnormal thin-walled ectatic veins, venules, and capillaries localized to the mucosa and submucosa. the lesions are thought to result from repetitive partial obstruction to venous drainage caused by contraction of the muscularis propria. wall tension is directly proportional to lumen diameter, a finding that may explain the lesion's predilection for the right colon, where the diameter is the greatest. obviously, vascular injection techniques require preparation in terms of materials. if caught unprepared, one may still demonstrate larger ectasias by carefully and gently washing off the mucosal surface of the specimen with water. sometimes, a blood clot adheres to the bleeding point and this may direct the histologic sectioning. we have been disappointed with injection of contrast media and specimen radiography but have had success demonstrating right-sided vascular ectasia by injecting brightly colored ink into the ileocolic artery. some investigators have reported success by bluntly dissecting the mucosa from the muscular wall of the formalin-fixed bowel followed by dehydration and transillumination. hemangiomas many isolated hemangiomas may be classified on the basis of the predominant vessel type as capillary, cavernous, venous, arteriovenous, or mixed. capillary hemangiomas are typically small, rarely multiple, and composed of small, closely packed capillaries. these lesions may be found in the colon but are usually encountered in the small intestine, appendix, and perianal skin. some lesions reported as capillary hemangiomas may, in fact, be vascular ectasias (see earlier). capillary hemangiomas rarely cause clinical symptoms. cavernous, venous, arteriovenous, and other mixture hemangiomas occur in localized or diffuse forms [ ] [ ] [ ] [ ] and have the same clinical significance regardless of vessel type . cavernous hemangiomas are composed of blood-filled, sinus-like spaces supported by connective tissue. , the stroma occasionally contains smooth muscle. cavernous hemangiomas can occur in the small bowel but have a predilection for the colon and rectum. patients may present with symptoms of a mass or with rectal bleeding. localized cavernous hemangiomas can be treated by excision, fulguration, or sometimes radiation therapy, whereas diffuse forms of hemangiomas may be unresectable and treatment must be directed toward controlling complications. a lesion composed of abnormal proliferating veins and arteries with evidence of arteriovenous shunting is referred to as an arteriovenous hemangioma. venous hemangiomas are characterized by large, thick-walled vessels that resemble normal veins but occasionally have more disorganized smooth muscle in their walls. arteriovenous and venous hemangiomas have the same distribution and significance as cavernous hemangioma. rare reports have noted tumors composed of admixtures of dilated vascular chan- nels and lymphatics. these tumors can be referred to as hemangiolymphangiomas, lymphaticovenous malformations, or even generically as vascular malformations. , these lesions have the same clinical significance as cavernous hemangiomas. pure lymphangioma of the large bowel is extremely rare. depending on size and location, these lesions may be asymptomatic or they may cause pain, diarrhea, or bleeding. reported size has varied to up to cm. microscopically the lesions are composed of thin-walled, anastomosing endothelial-lined channels that are bloodless but usually contain eosinophilic material. the term phlebectasia defines a non-neoplastic venous varicosity. these lesions may occasionally cause severe gi bleeding. the most frequent site of bleeding caused by phlebectasias associated with portal hypertension is the lower esophagus. other sites have been reported, especially the rectosigmoid colon and rarely the small intestine. we have also seen cases of idiopathic colonic varices in children. because phlebectasias are encountered rarely, if ever, in surgical specimens, they are not considered further. hereditary hemorrhagic telangiectasia (hht) is inherited as an autosomal dominant trait. , two disease-causing genes have been identified, endoglin and alk- . vascular lesions occur in the skin, mucous membranes, and internal organs, including liver, genitourinary tract, meninges, eyes, and spinal cord. the mucocutaneous lesions appear in the second and third decades of life. many patients present with recurrent epistaxis in childhood. approximately % of patients with hht, usually older patients, have gi bleeding. the histopathologic pattern of the colorectal lesion is simple dilatation of normal vascular structures (i.e., telangiectasia) and in general resembles the lesions of right-sided vascular ectasia ( fig. - ). progressive systemic sclerosis can be associated with gi tract telangiectasias. , a predilection exists for the stomach, rectum, and colon. intestinal bleeding develops only rarely. the blue rubber bleb nevus syndrome can occur as an autosomal dominant trait or in a sporadic form. the characteristic histologic lesion is a cavernous hemangioma that can involve the skin, gi tract, and other viscera. [ ] [ ] [ ] the skin lesion is typically blue and tender and ranges in size from . cm to . cm. the lesion empties on digital pressure to leave a wrinkled blue sac that in time refills with blood. the preferred site of involvement in the gi tract is the small intestine but colorectal lesions have been reported. turner's syndrome is characterized by numerous somatic abnormalities associated with a xo karyotype. telangiectasias of the small and large intestine have been described in this disorder and may cause gi bleeding. klippel-trenaunay-weber syndrome occurs sporadically. patients demonstrate unilateral soft tissue and bony hypertrophy associated with a port-wine hemangioma. gi cavernous hemangiomas have been described in these patients. , pseudoxanthoma elasticum shows marked clinical and genetic variability. clinically, "plucked-chicken" skin and angiomatoid streaks in the retina have been described. bleeding in the gi tract may occur from ruptured blood vessels caused by the abnormalities in elastic structure. patients usually bleed from the stomach. arteriovenous hemangiomas have also been described. , the term ehlers-danlos syndrome describes more than subclasses of metabolic disorders associated with abnormal collagen synthesis. most patients demonstrate hyperelasticity of skin and joints (i.e., the circus rubber man). vascular ectasias or proliferative vascular lesions have not been described, but spontaneous arterial or intestinal rupture has been reported in some forms (especially type iv [vascular ehlers-danlos syndrome with mutations of col a gene ]) of ehlers-danlos syndrome. [ ] [ ] [ ] [ ] [ ] [ ] [ ] kaposi's sarcoma characteristically cause pigmented skin nodules predominantly on the lower extremities. currently, an aggressive variant of kaposi's sarcoma in patients with acquired immunodeficiency syndrome (aids) is most commonly seen [ ] [ ] [ ] and has been linked to infection with human herpesvirus . the gi tract of patients with aids is frequently involved by kaposi's sarcoma. investigators have estimated that about half of the patients with cutaneous or lymph node kaposi's sarcoma have concomitant gi tract involvement. [ ] [ ] [ ] rarely, the gi tract can be involved without skin lesions. gi tract kaposi's sarcoma is typically asymptomatic but occasional gi kaposi's sarcoma can cause bleeding, obstruction, perforation, or protein-losing enteropathy. , gi kaposi's sarcoma has numerous appearances grossly and endoscopically. although red macules have been described, kaposi's sarcoma more characteristically causes a red or purple nodule or plaque measuring mm or more. some patients with gi kaposi's sarcoma have large tumors. despite the vascular nature of the neoplasm, lesions can be sampled for biopsy and in most instances bleed surprisingly little. the diagnostic yield with biopsy has been notoriously low ; in one report, only % of upper gi specimens and % of sigmoidoscopic specimens tested positive. usually, the low yield is attributed to the preferential submucosal location of kaposi's sarcoma that may not be sampled in an endoscopic biopsy specimen. that said, examples of kaposi's sarcoma are easily overlooked by even experienced pathologists, because the sarcoma is subtle and can be dismissed as granulation tissue. in positive biopsy specimens, kaposi's sarcoma typically causes an expansion of lamina propria by a spindle cell proliferation that may not be very atypical. the spindle cells are usually not extremely mitotically active and at first glance may suggest granulation tissue (although macrophages and mononuclear inflammatory cells are typically absent) or the fibromuscular obliteration of the lamina propria associated with the solitary rectal ulcer syndrome (prolapse or trauma-related change). generally, gi kaposi's sarcoma is histologically similar to kaposi's sarcoma seen elsewhere. the histologic pattern shows spindle cells, vascular clefts, extravasated red blood cells frequently associated with hemosiderin deposits, and occasionally tumor necrosis ( fig. - ). kaposi's sarcoma cells usually stain with antibodies directed against factor viii-related antigen but more consistently stain positive with endothelial markers such as cd and cd . as mentioned earlier, the changes are subtle and diagnosis can be difficult. important clues to the proper diagnosis include ( ) knowledge of the clinical situation, ( ) knowledge of the endoscopic appearance, ( ) recognition of crowding and hyperchromasia in the spindle cell nuclei, ( ) recognition of slits and spaces containing red blood cells, and ( ) recognition of the tendency for kaposi's sarcoma cells to overrun or obliterate the muscularis mucosae. aggressive antiretroviral therapy is now considered the first-line treatment for gi kaposi's sarcoma. , other forms of treatment are reserved for symptomatic tumors. surgical procedures are performed for obstruction or severe bleeding. chemotherapy approaches have improved but the treatment is risky in an already immunocompromised patient. these therapies offer palliation at best. treatment with zidovudine (formerly azidothymidine [azt]) does not have an effect on gi kaposi's sarcoma. , immunomodulation has been tested but is not used. , , the addition of taxanes (paclitaxel and docetaxel) to aggressive antiretroviral therapy appears promising. , angiosarcoma angiosarcomas are only rarely encountered in the colon and rectum. the varied histologic spectrum ranges from wellformed vascular structures to sheets of malignant polygonal or spindle cells. grossly and endoscopically, angiosarcomas form red-gray polypoid masses. an immunohistochemical panel including cd and factor viii-antigen can be useful in diagnosis. the term dieulafoy's lesion refers to an inappropriately large artery located directly beneath the mucosa where it can erode and cause profuse bleeding. although typically described in the stomach, similar lesions are found in the colon and rectum. metastatic neoplasia to the colon and rectum come principally from the lung, breast, ovary, and skin (melanoma). advanced prostate carcinoma can cause confusion with primary rectal carcinoma or with neuroendocrine carcinoma. sometimes perirectal lymph node metastases in resections performed for colorectal carcinoma contain metastatic deposits from prostate carcinoma. fap is inherited as an autosomal dominant trait. bussey recognized that or more colorectal adenomas (recognized grossly) phenotypically identified patients with fap and distinguished them from patients with multiple adenomas in whom inheritance was not seen. , in typical fap, hundreds to thousands of adenomas develop within the colon ( fig. - ). the adenomas begin to appear in the second or third decades of life and are surprisingly asymptomatic considering their usually large numbers. symptom-atic patients present with signs and symptoms of increased bowel motility and the passage of blood or mucus, or both, which often heralds the onset of carcinoma. the average age of patients with colon cancer and fap is years. two thirds of patients with these so-called propositus cases present with carcinoma and nearly one half of them have more than one carcinoma in the colon. this high risk of invasive cancer in symptomatic patients forms the basis for polyposis registries and the extensive screening of asymptomatic kindred at risk for fap. screening recommendations have evolved with increased genetic information. genetic testing should be considered for fap, attenuated fap, and muty homologue (myh)-associated polyposis (map) when or more colorectal adenomas are found in a patient during a single examination or over time. screening of first-degree relatives of affected individuals should begin at the age of years. in the absence of genetic testing, endoscopic screening is still useful to detect fap. all affected patients have adenomas within the range of a flexible sigmoidoscope. it is therefore recommended that screening sigmoidoscopy begin at age years with re-examination every years. the diagnosis of fap must be confirmed with biopsy because lymphoid polyposis and hyperplastic polyposis can mimic fap grossly and endoscopically. once a diagnosis of fap has been established, prophylactic proctocolectomy is recommended. most investigators recommend sigmoidoscopy for mutation-negative kindred at age years just in case the genetic test is erroneous. thyroid examination for associated thyroid lesions (usually papillary carcinoma with cribriform pattern ) and determination of serum alpha fetoprotein (to screen for hepatoblastoma) are recommended. regular upper endoscopy should also be performed. gastric and duodenal polyps develop in % to % of patients with fap. the gastric lesions are usually fundic gland polyposis, whereas the duodenal polyps are usually adenomas. the fundic gland polyps can develop a peculiar surface epithelial atypia called foveolar dysplasia, but progression to carcinoma is extremely rare. the incidence of duodenal adenomas in fap increases with increasing age. duodenal adenomas have a propensity to develop in the periampullary region. adenomas anywhere in the gi tract can proceed through the dysplasia-carcinoma sequence. the relative risk of duodenal or periampullary carcinoma in patients with fap is approximately to times that seen in the general population, and duodenal or periampullary carcinoma has become the major cause of morbidity and mortality in patients with fap in the post-prophylactic colectomy era. the gene responsible for fap (apc gene) has been localized to the long arm of chromosome ( q -q ) and has been cloned. [ ] [ ] [ ] [ ] [ ] some apc gene mutation-negative cases may be caused by mutation of myh (see later). mutation in most patients with fap creates a stop codon resulting in a truncated protein product. the apc gene is a tumor suppressor gene and the apc protein is part of the wntsignaling pathway , involved in cell growth control. when the apc gene is mutated, beta-catenin accumulates, thus altering expression of certain genes affecting proliferation, differentiation, migration, and apoptosis. most patients are now diagnosed by dna sequencing, which has largely replaced the assay to detect the truncated apc protein (ptt). , monoallelic mutation analysis (mama) examines the two apc alleles independently and can detect more than % of patients with fap. another test, multiplex ligation-dependent probe amplification (mlpa), is useful in detecting large deletions. more than disease-causing apc gene mutations have been reported. localization of mutations within the apc gene locus correlates with phenotype. for example, germline mutations between codon and codon are associated with very large numbers of colonic adenomas, whereas mutations elsewhere, especially near the ′ end or the ′ end of the apc gene and an area of exon , yield lesser numbers of colonic adenomas (see the later discussion of attenuated fap). , [ ] [ ] [ ] in gardner's variant, in addition to colonic adenomas and upper gi polyps, patients can exhibit extraintestinal manifestations such as osteomas, epidermal inclusion cysts and other benign skin tumors, desmoid tumors of the abdomen or abdominal wall, fibrosis of mesentery, dental abnormalities, carcinoma of the periampullary region or duodenum, and carcinoma of the thyroid. patients with gardner's syndrome have apc gene mutations; however, no particular apc mutation distinguishes fap from gardner's variant. even within a "gardner's family," gardner's stigmata can be variably expressed and can skip generations. therefore, some unknown disease-modifying factors are required for phenotypic expression of the extraintestinal manifestations. turcot's syndrome has been the subject of some controversy. in many investigators' zeal to publish, the phenotypic spectrum has been unduly broad with colonic manifestations ranging from a single adenoma to a virtual carpeting of the colonic mucosa with polyps. furthermore, the brain tumors have comprised almost every histologic type. molecular studies done on families with turcot's syndrome have clarified the situation. families with turcot's syndrome and germline mutations of the apc gene have a typical fap colonic phenotype and develop medulloblastomas. other patients originally thought to have turcot's syndrome have mutations in the dna mmr genes that are characteristic of lynch's syndrome. the brain tumors in this group have varied, with many reported as glioblastoma. mutations of the apc gene near the ′ end and ′ end and in a particular region of exon result in fewer adenomas (< ; average, ), a tendency for the adenomas to be macroscopically flat, and a propensity for these adenomas to cluster in the right colon. originally reported as hereditary flat adenoma syndrome, this form is now more accurately referred to as attenuated fap (afap). , as in typical fap, these patients can develop fundic gland polyposis, duodenal adenomas, and periampullary carcinoma. the risk of colorectal carcinoma is increased in these patients albeit to a lesser degree than in the other form of fap and the cancers tend to occur later in life (average age, years). inherited variants of a base-excision repair gene myh have been associated with colorectal polyposis with an autosomal recessive mode of inheritance. , , some cases phenotypically resemble fap or attenuated fap and are referred to as myh polyposis or map. of those patients with a phenotype typical of fap or suspected afap in whom an apc gene mutation is not found, % to % will have mutation of the myh gene. approximately % of affected persons have one of two specific myh mutations (y c or g d). if one is found, then sequencing is done to find the mutation on the other allele because map is biallelic. these patients should be treated and followed similarly to patients with fap. juvenile polyps can occur in a sporadic form or can be part of juvenile polyposis syndrome. in the sporadic form, juvenile polyps have their peak prevalence in children between and years of age. some evidence indicates that juvenile polyps once formed can regress; they can certainly be seen in adults. sporadic juvenile polyps typically occur singly but patients can have up to five, usually located in the rectum. juvenile polyps typically range in size up to cm and can be associated with overt prolapse (fig. - ). because these polyps are often attached by a small pedicle, they are prone to autoamputation. histologically, typical juvenile polyps consist of a hamartomatous overgrowth of the lamina propria accompanied by elongation and cystic dilatation of colonic crypts lined by nondysplastic colonic epithelium ( fig. - ). osseous and cartilaginous stromal metaplasia can occur ( fig. - ). the inflammatory component of juvenile polyps can be quite prominent with neutrophils and lymphoid follicles within the lamina propria. frequently, the distinction between juvenile polyps and inflammatory polyps of primary ibd cannot be made on histologic grounds alone and requires clinical correlation. nonsyndromatic juvenile polyps appear to have no malignant potential. juvenile polyposis syndrome can be familial or nonfamilial and usually becomes clinically apparent within the first decade of life with painless rectal bleeding, prolapse, iron deficiency anemia, or passage of an autoamputated polyp. patients are considered to have juvenile polyposis syndrome if they have six or more juvenile polyps in the colon and rectum, have juvenile polyps throughout the gi tract, or have any number of juvenile polyps in association with a positive family history. , in the nonfamilial forms of juvenile polyposis syndrome (≈ % of the total), patients frequently have associated abnormalities, such as cardiac defects, hydrocephalus, gut malrotation, undescended testes, and skull abnormalities. patients with the familial forms usually lack these extraintestinal manifestations. inheritance has varied although almost all are considered autosomal dominant with variable penetrance. familial forms of juvenile polyposis syndrome appear to be associated with an increased risk of colorectal carcinoma. prophylactic colectomy may be prudent in juvenile polyposis syndrome. patients may also have an increased risk of gastric, small intestinal, and pancreatic carcinoma. juvenile polyposis syndrome coexisting with hht (osler-weber-rendu syndrome) is rarely reported. the number of polyps in juvenile polyposis syndrome typically ranges from a few dozen to several hundred ( fig. - ) . phenotypically, juvenile polyposis syndrome appears to occur in three varieties: ( ) polyps limited to the colon, ( ) polyps limited to the stomach, and ( ) polyps throughout the entire gi tract. [ ] [ ] [ ] the mucosal polyps found in the context of juvenile polyposis syndromes are often unusual histologically. in addition to the typical juvenile polyps described earlier, one can find juvenile polyps with atypical features such as more epithelium than lamina propria. in addition, mixture polyps (juvenile polyps with areas of adenoma or dysplasia) are quite frequent. , [ ] [ ] [ ] seen in % of cases. one should consider genetic testing for juvenile polyposis syndrome when three or more juvenile polyps have occurred in one individual or when juvenile polyps are found outside the colon. madh and bmpr a are both components of the signaling pathway for transforming growth factor-b and the bone morphogenetic proteins. patients with madh gene mutation are more likely to have gastric juvenile polyposis. , , juvenile polyps can be found in patients with other hamartomatous syndromes of the colon, such as intestinal ganglioneuromatosis or ganglioneurofibromatosis (see later), [ ] [ ] [ ] although some of these syndromes are now best classified as pten (phosphatase and tensin homologue) syndrome (see later). patients can sometimes be managed with endoscopy and polypectomy (every to years); however, colectomy must be considered for patients with large numbers of polyps or polyps with dysplasia or for patients with complications (e.g., bleeding, obstruction). screening colonoscopy every years should commence with symptoms or in the early teenage years in an asymptomatic patient. , upper endoscopy is also recommended in patients with juvenile polyposis syndrome. esophagogastroduodenoscopy and small bowel examination (every years) should begin at age years. , ruvalcaba-myhre-smith syndrome (bannayan-zonana syndrome, ruvalcaba-myhre-smith syndrome consists of macrocephaly, mental deficiency, unusual craniofacial appearance, pseudopapilledema, pigmented macules on the penis, and hamartomatous polyps in the gi tract. the syndrome appears to be passed on as an autosomal dominant condition. the gi polyps have been indistinguishable from juvenile polyps and in rare instances intestinal ganglioneuromatosis has also been described. the syndrome has been linked to mutations or deletions in the pten gene ( q . ) , , and with cowden's syndrome and can be considered as one of the pten polyposis syndromes. peutz-jeghers polyps can be found throughout the gi tract, either sporadically or as part of the peutz-jeghers syndrome. , , the polyp itself is characterized by fairly normal epithelium and lamina propria lining an abnormal arborizing network of smooth muscle that represents hamartomatous overgrowth of the muscularis mucosae ( fig. - ). , , peutz-jeghers syndrome, usually inherited as an autosomal dominant trait, is the combination of skin hyperpigmentation and peutz-jeghers polyps in the gi tract. the diagnosis of peutz-jeghers syndrome is considered definitive if the patient has a peutz-jeghers polyp and at least two of the following criteria: ( ) family history, ( ) mucocutaneous hyperpigmentation, and ( ) small bowel polyposis. , the pigmentation consists of clusters of brown or black freckles about the lips, buccal mucosa, and perianal and genital region. pigmented areas can occasionally be seen on the fingers and toes. the spots appear in the first year of life and tend to fade toward middle age. the polyps usually number only in the dozens and can be found throughout the gi tract. these polyps have a propensity to form in the small intestine where they often cause intussusception. in rare kindred, peutz-jeghers polyps have been limited to the large bowel. cases of complicating gi carcinoma have been reported. , approximately % of female patients with peutz-jeghers syndrome have a peculiar ovarian tumor, sex cord tumor with annular tubules (sctat). the rate of detection may increase if the ovaries are carefully examined, , and some tumors may be associated with sexual precocity. male patients with peutz-jeghers syndrome occasionally have unilateral or bilateral sertoli cell tumors of the testes. , adenoma malignum and pancreaticobiliary tract carcinomas are reported to occur at increased rates. the peutz-jeghers syndrome has been linked to the stk (serine/threonine-protein kinase , also known as lkb ) gene on chromosome p . [ ] [ ] [ ] [ ] and can be demonstrated in % of cases. this is a tumor suppressor gene involved in transduction of intracellular growth signals. investigators have suggested that genetic testing be considered for peutz-jeghers syndrome when any peutz-jeghers polyps or typical perioral pigmentations are found. meta-analysis of cancer risk in an evaluation of patients with known mutations of the stk gene have shown increased lifetime risk for cancer of the esophagus, stomach, small bowel, colon, pancreas, and breast. , putting this into perspective, the risk for breast cancer in peutz-jeghers syndrome is similar to the risk seen in individuals with germline mutations of brca and brca and peutz-jeghers syndrome is the strongest known risk factor for pancreatic carcinoma except for hereditary pancreatitis. screening at-risk individuals (first-degree relatives of a patient with peutz-jeghers syndrome) should begin at birth with an annual history and physical examination to look specifically for melanotic spots, precocious puberty, and testicular tumors. asymptomatic at-risk individuals without stigmata by age years should be tested for stk /lkb gene mutations. if mutation is not found in the family, small intestinal contrast radiography every years until age years is recommended. other investigators suggest that upper and lower endoscopy with small bowel series should be done at ages , , and years. esophagogastroduodenoscopy and upper gi radiographic series with small bowel follow through are recommended in patients with peutz-jeghers syndrome commencing at age years and repeated every years thereafter. , colonoscopy every years is recommended starting with symptoms or by age years if symptoms have not occurred. , testicular examination, pelvic examination by age years, mammographic examination by age years, and endoscopic ultrasound examination of the pancreas by age to years have been recommended. , annual transvaginal ultrasound examination and serum ca- determination are also recommended commencing at age years. intestinal ganglioneuromatosis is defined as proliferation of ganglion cells, neurites, and supporting cells that can affect any layer of the gi wall ( fig. - ). these proliferations often manifest as mucosal polyps in the colon. although these lesions most often occur as an isolated phenomenon, the importance of intestinal polypoid ganglioneuromatosis is in recognizing the other settings in which it can occur, such as von recklinghausen's disease (nf gene mutation), men b (ret gene mutation), cowden's syndrome (pten mutation), ruvalcaba-myhre-smith syndrome (pten mutation), and tuberous sclerosis (tsc [ q ] or tsc [ p ] mutation). [ ] [ ] [ ] [ ] [ ] intestinal ganglioneuromatosis can coexist with juvenile polyps although these patients may be better classified as having pten polyposis. [ ] [ ] [ ] cowden's syndrome describes an autosomal dominant multiple hamartoma syndrome in which patients have multiple orocutaneous hamartomas (e.g., facial trichilemmomas, mucosal papillomas, acral keratosis, subcutaneous lipomas), fibrocystic disease of the breast, an increased risk of breast carcinoma, thyroid abnormalities, and hamartomatous polyps in the stomach, small intestine, and colon. polyps of the gi tract, when described, often have shown an abnormal proliferation of the smooth muscle in the lamina propria and generally have resembled the polypoid variant of solitary rectal ulcer syndrome. some juvenile polyp-like proliferations have been described. intestinal ganglioneuromatosis has also been reported. other associated abnormalities include macrocephaly, high arched palate, hypoplastic mandible and maxilla, microstomia, supernumerary nipples, pectus excavatum, hemangiomas, ovarian cysts, and uterine leiomyomas. , the gene (pten) for cowden's disease has been mapped to chromosome ( q - ). , , cowden's syndrome and ruvalcaba-myhre-smith syndrome are sometimes referred to as the pten polyposis syndromes. genetic testing is suggested when features of this syndrome are present. esophagogastroduodenoscopy and small bowel examination every years beginning at age years is recommended. cronkhite-canada syndrome is an acquired nonfamilial syndrome characterized by intestinal polyposis, dystrophic changes of the fingernails, alopecia, and cutaneous hyperpigmentation. , patients first present with diarrhea, abdominal pain, and anorexia that progresses to weight loss and protein-losing enteropathy. many patients complain of loss of taste (hypogeusia) and loss of smell. as a rule, the ectodermal changes occur weeks to months after the other symptoms. the nail dystrophy consists of thinning, splitting, and separation from the nail bed (onycholysis). onychomadesis (complete loss of the nail) can also occur. the hair loss is rapid and may be seen in the scalp, eyebrow, face, axilla, or pubic region. the cutaneous hyperpigmentation ranges from small macules to confluent areas of hyperpigmentation that can be cm or more. histologically, the pigmented macules result from increased melanin in the basal layer. cronkhite-canada polyps are found throughout the gi tract but are most commonly seen in the stomach and large bowel. grossly, they are sessile; a few are pedunculated. the polyps tend to occur on a background of diffuse mucosal thickening ( fig. - ) . histologically, the polyps themselves are identical to juvenile polyps. however, the mucosa between polyps is abnormal and shows edema, congestion, and inflammation (chronic inflammation often with prominent eosinophils) of the lamina propria coupled with glandular ectasia (fig. - ) . carcinomas of the colon and stomach have been described rarely in patients with cronkhite-canada syndrome. the malabsorption in this syndrome is usually progressive and with no specific therapy available, the prognosis is generally poor. death results from anemia, septic shock, bleeding, or postoperative complications. treatment consists of supportive therapy, antibiotics, corticosteroids, and surgery. within the stomach, cronkhite-canada syndrome closely mimics ménétrier's disease. ménétrier's disease, however, is confined to the stomach and has no associated ectodermal changes. heterotopic gastric, pancreatic, sebaceous, and salivary gland tissues have been described in the colon and rectum. these ectopic tissues can be found throughout the gi tract but are most often seen in the rectum where they can cause a plaque, polyp, or mass ( fig. - ). [ ] [ ] [ ] inflammatory fibroid polyp is most commonly found in the stomach but can be encountered throughout the gi tract including the colon and rectum. , - symptoms include abdominal pain and bleeding. the polyp is usually solitary. it can be sessile or pedunculated and typically has a solid pale tan cut surface. microscopically one sees a loose myxoid fibrous tissue background containing regularly distributed blood vessels, some of which show hyalin change in their walls. the fibrous tissue can layer in a whorl-like fashion around these vessels in an onion-skin pattern. most lesions are rich in inflammatory cells including plasma cells and eosinophils ( fig. - ) . scattered macrophages and touton-type giant cells can also be seen. the stroma in most lesions is positive for cd but negative for cd . mutations in plateletderived growth factor receptor alpha gene have been described. a the mucosa overlying these typically submucosal tumors can be ulcerated, presumably by trauma, and can show areas of inflamed granulation tissue. the ulcerated surface can contain bizarre stromal cells, which are also seen in a variety of inflammatory polyps with chronic ulceration (e.g., ibd, trauma or prolapse, and radiation injury). the inflammatory fibroid polyp is benign and typically does not recur. malakoplakia, an abnormal immune response to gramnegative bacteria, can cause a tumor or polyp in any site of the gi tract, including the large bowel. histologically, it is characterized by xanthogranulomatous inflammation accompanied by the pathognomonic michaelis-gutmann body (fig. - ). the partially digested bacteria accumulate in macrophages and lead to deposition of calcium and iron on the residual bacterial glycolipids. an association between colorectal malakoplakia and colorectal neoplasia may exist. defined as the presence of endometrial glands or stroma usually with hemorrhage and hemosiderin deposits in an extrauterine location, endometriosis tends to affect sites closest to the female genital tract such as the sigmoid colon and rectum. symptoms include episodic abdominal pain. hematochezia can occur with mucosal involvement. endometriosis usually involves the serosa and muscularis externa and can cause smooth muscle proliferation and stricture. mucosal and submucosal involvement can cause mucosal polyps. endometriosis must be distinguished from müllerian adenosarcoma and endometrial stromal sarcoma. the glandular component can be confused with colitis cystica profunda and adenocarcinoma. immunohistochemical analysis for cd , which highlights endometrial stromal cells, can be helpful in the differential diagnosis as can recognition of ciliated epithelial cells. differential cytokeratin immunostaining can also help because endometriosis commonly stains positive for ck , whereas colorectal epithelium usually expresses ck . examples of malignant transformation (mostly endometrioid carcinoma and clear cell carcinoma) in endometriosis have been reported. injection of materials containing lipid bases into the lower rectum and anus can cause a mass or polyp referred to as an oleogranuloma. the lesion is composed of lipidcontaining cysts surrounded by a foreign body giant cell reaction. benign fibroblastic polyps and perineurioma have been described in the colon and rectum where they may represent the same or a similar lesion. - these mucosal polyps are usually solitary but can be multiple and have been reported throughout the gi tract, most commonly in the colon and rectum. histologically, these polyps contain proliferations of small tightly packed spindle cells within the lamina propria that often orient themselves parallel to the muscularis mucosae. this lesion frequently coexists with hyperplastic polyp-like epithelial proliferations, and indeed the polyp could represent a trauma-related change seen in the otherwise typical hyperplastic polyp or sessile serrated polyp ( fig. - ) . the spindle cells test negative for immunoreactive s- protein and other neuromarkers; they can express immunoreactive epithelial membrane antigen by immunohistochemical analysis. areas of increased elastin fibers in the submucosa and muscularis mucosae are referred to as elastosis or elastofibromatous change and can cause polyps in the colon and rectum. histologically, the elastosis appears as finely granular or fibrillar amphophilic material usually with a fibrous component and is often centered around prominent blood vessels (fig. - ) . the change could also be a manifestation of mucosal trauma or prolapse. elastosis can be confused with amyloid deposits but results of congo red stains have been negative. benign spindle cell proliferations that express immunoreactive s- protein can manifest as mucosal polyps in the colon and rectum and are usually termed mucosal neuromas or schwann cell hamartomas. care must be taken not to overlook ganglion cells, which would indicate a ganglioneuroma. these lesions can be seen in patients with neurofibromatosis but most occur sporadically and are unassociated with syndromes. with increased availability of total colonoscopy and flexible sigmoidoscopy, pathologists can expect an ever-increasing number of colorectal biopsy specimens. the pathologist plays a critical role in the diagnosis and management of patients with colitis and suspected colitis. when evaluating a colorectal biopsy specimen, it is useful to scan the tissue at low magnification and ask several questions. are abnormalities present? if present, are the changes diffuse or focal? is the luminal border of the specimen straight or irregular? is intraepithelial mucin (the goblet cell population) preserved or depleted? if intracellular mucin is depleted, is this change focal or diffuse? are inflammatory cells increased in the lamina propria or epithelium? is this increase diffuse or focal? what kind of inflammatory cells are they? is the lamina propria obliterated by fibrous or fibromuscular tissue? are crypt abscesses present? are colonic tubules straight? do the colonic tubules reach the muscularis mucosae or are they short, branched, or budded? with careful consideration of the features outlined earlier, one can often recognize a pattern of abnormality that when coupled with clinical and endoscopic information can lead to a fairly specific diagnosis in a large number of patients. the following patterns of inflammation can be identified in mucosal biopsy specimens: chronic colitis, diffuse active colitis, focal active colitis, ischemic-type injury, traumarelated change, apoptotic colopathy, and intraepithelial lymphocytosis. identification of an inflammation pattern can be helpful in assessing patients by creating a clinically relevant differential diagnosis. quiescent uc best typifies the chronic colitis pattern of injury. the predominant features are mucosal atrophy and architectural distortion (fig. - ) . [ ] [ ] [ ] [ ] the luminal border is often irregular and the number of crypts decreased. the remaining crypts typically appear short (i.e., they do not touch the muscularis mucosae), they lose their parallel arrangement, and they become branched and budded. the goblet cell population is usually preserved. chronic inflammatory cells including plasma cells are increased in the lamina propria. paneth cells may be present. the muscularis mucosae is usually hypertrophied. the foregoing changes, although consistent with a diagnosis of chronic uc, must be interpreted in light of the clinical, radiologic, and endoscopic findings because similar changes can be seen in focal, healed, or healing areas of other chronic colitides such as crohn's disease, ischemia, chronic irradiation injury, tuberculosis, and schistosomiasis. care must be taken when interpreting biopsy specimens obtained from the normal mucosa adjacent to lymphoid follicles, from normal mucosa containing the innominate groove, and from the lower portion of the rectum near the transition zone. these areas normally show some loss of crypt parallelism and should not be misinterpreted as evidence of chronic colitis. , conversely, histologically normal biopsy specimens must not be reported as showing "chronic nonspecific inflammation" consistent with uc. unless one or more of the features discussed in the preceding para- graph are also present, it is a good rule not to diagnose primary ibd based only on an evaluation of inflammatory cells within the lamina propria. the term active colitis describes inflammatory conditions in which neutrophils are present within the lamina propria, within epithelial cells (cryptitis), or within the lumen of crypts (crypt abscesses). included under this heading are uc in an active phase, most examples of crohn's disease, and infectious colitis or acute self-limited colitis. , uc in an active phase represents the prototype diffuse active colitis. biopsy specimens usually demonstrate diffuse abnormalities, meaning that changes are of approximately the same intensity in all areas of the tissue submitted from a particular region of the colon. the luminal border of the mucosa is irregular. [ ] [ ] [ ] [ ] [ ] [ ] [ ] increased numbers of chronic inflammatory cells are present in the lamina propria and may occasionally spill over into the superficial portion of the submucosa. intracellular mucin in goblet cells is diffusely depleted. cryptitis and crypt abscess formation are often prominent (fig. - ). it is surprising that even in uc of extremely short overt clinical duration, atrophy, branching, and budding of crypts are already apparent in many specimens. , [ ] [ ] [ ] [ ] [ ] [ ] this crypt distortion coupled with basal plasmacytosis, which is defined as increased plasma cells in the lower fifth of the mucosa, has been proposed as the most useful histologic criterion to differentiate primary ibd from infectious colitis or acute self-limited colitis. , [ ] [ ] [ ] remember, the most a pathologist can conclude from a biopsy specimen showing this pattern of injury is that the changes are consistent with uc in an active phase. the diffuse active colitis pattern of injury can also be seen in some examples of crohn's disease , and in some cases of documented infectious colitis, although we think it is likely that these cases represent an infectious exacerbation of an underlying, albeit clinically latent, primary ibd. the diffuse active colitis pattern of injury has been reported in a form of colitis associated with diverticular disease. however, this entity can be distinguished from classic uc by virtue of its rectal sparing and the presence of inflammation only in areas of diverticula. the focal active colitis pattern of injury refers to the patchy distribution of architectural or acute inflammatory change in mucosal biopsy specimens. chronic colitis showing diffuse chronic changes as described earlier, coupled with patchy acute inflammation, is not considered focal active colitis but is classified as chronic colitis showing mild activity. the focal active colitis pattern consists of limited areas of increased inflammatory cells sometimes coupled with focal minimal architectural distortion (fig. - ) . characteristically, some areas of the biopsy specimen from a region of the colon must maintain an essentially normal appearance. the focal active colitis pattern of injury is usually not seen with uc and when present suggests crohn's disease, , , injury related to nonsteroidal anti-inflammatory drugs (nsaids), or infectious colitis or acute self-limited colitis (see later). , , , , , the focal active colitis pattern of injury, however, can be seen in resolving uc under active medical management. , areas previously involved in the colon and rectum in uc can return to an almost normal histologic appearance with therapy. granulomas typically found in crohn's disease should be sought in all biopsy specimens, but especially those showing the focal active pattern. although serial sectioning of biopsy specimens is advocated by some investigators for the detection of granulomas, in our experience, granulomas are rarely missed. that said, germinal centers, tangential cuts of blood vessels, tangential cuts of the pericryptal fibroblastic sheath, and an inflammatory reaction to extravasated mucin (so-called mucin granulomas) are often misinterpreted as the granulomas of crohn's disease. in the absence of true granulomas, biopsy specimens from patients with crohn's disease often show the focal active colitis pattern of injury without neutrophils in the lamina propria. some examples of crohn's colitis can be indistinguishable from resolving mucosal uc in biopsy specimens. , a fibrinopurulent exudate in the specimen overlying but separate from the mucosa is always abnormal. the clinician should be informed that ulcers are likely to be present in a more proximal location in the bowel. on a practical note, all inflammatory exudates should be examined under high magnification for trophozoites of entamoeba histolytica, because this is their preferred location and these organisms are easy to overlook. the definitive classification of colonic inflammation depends on clinical pathologic correlation. in our opinion, the task of the pathologist is to convey the histologic pattern of injury to the clinician who then collates that information with the clinical history and data obtained from endoscopic, radiologic, and laboratory examination. antineutrophil cytoplasmic antibodies (ancas) have been identified in patients with ibd. [ ] [ ] [ ] the immunofluorescence pattern (perinuclear) differs from the diffuse cytoplasmic reaction detected in patients with wegener's granulomatosis. although initially thought to be specific for uc, ancas more likely reflect inflammatory conditions involving the colon. in one study, up to % of patients with presumed uc had anca positivity with a % prevalence in patients with colonic crohn's disease and a % prevalence in patients with infectious-type colitis. another antibody, anti-saccharomyces cerevisiae antibody (asca), has been closely linked to crohn's disease. ascas can be identified in up to % to % of patients with crohn's disease, as opposed to % to % in uc and % of controls. , some investigators have proposed the use of anca and asca together to increase specificity. these tests clinically have demonstrated low sensitivity (≈ % to %) but have been reported to be highly disease specific. these tests may have value in classification of indeterminate colitis but this remains to be proved. the following histologic appearances have been described in culture-proved or toxin-proved infectious colitis: normal colon, nonspecific increases in chronic inflammatory cells, the diffuse active colitis pattern of injury (although these likely represent infectious exacerbations of an underlying primary ibd), ischemic-like changes, , and the focal active colitis pattern of injury. although colonic mucosal biopsy appearance in infectious colitis can vary greatly, many specimens have the focal active colitis pattern. in general, invasive organisms cause greater changes in morphology than those producing their effects by toxins. histologic evaluation, although helpful in suggesting infection and ruling out primary ibd, can only rarely suggest a specific cause. the definitive diagnosis of infectious colitis requires laboratory identification of the serologic features of the offending organism. even after extensive microbiologic workup, a subset of patients presumed clinically to have infectious colitis will experience spontaneous recovery in less than months and will have biopsy specimens that demonstrate the focal active colitis pattern of injury without an identifiable infectious cause. the term acute self-limited colitis has been used to describe such patients. [ ] [ ] [ ] we prefer the term infectious-type colitis to acute self-limited colitis because some examples of acute self-limited colitis may not be self-limited. other investigators prefer the term nonrelapsing colitis. , the ischemic pattern the characteristic pattern of acute ischemic-type injury consists of hemorrhage into the lamina propria associated with superficial epithelial coagulative necrosis, with relative sparing of the deep portions of the crypts (fig. - ) . , , these changes can occasionally be associated with more extensive necrosis of epithelium with inflammatory pseudomembrane formation. surprisingly, acute and chronic inflammatory cells, especially plasma cells, are typically scant in ischemic-type damage and this feature can help differentiate ischemic-type damage from primary ibd. the differential diagnosis of ischemic-type damage is wide and includes all causes of true ischemia such as inadequate perfusion, narrowing of blood vessels, obstructing lesions of the bowel, and bowel distention. ischemictype change is also associated with a variety of drugs, including vasopressors, oral contraceptives, nsaids, cocaine, and glutaraldehyde, which is sometimes used to clean endoscopes. [ ] [ ] [ ] [ ] some infectious agents typically cause ischemic-type damage, including cytomegalovirus (cmv), clostridium difficile, clostridium septicum, and the enterohemorrhagic escherichia coli (ehec). trauma-type histologic changes frequently coexist clinically with mucosal ulcers or erosions. the characteristic traumatype histologic pattern is found in the mucosa adjacent to ulcers or in polypoid mucosa areas and consists of fibro- muscular proliferation within the lamina propria associated with mucosal architectural distortion and intramucosal capillary ectasia (fig. - ). [ ] [ ] [ ] [ ] the trauma-type histologic features can be seen in the solitary rectal ulcer syndrome, localized colitis cystica profunda, inflammatory cloacogenic polyp, the mucosa adjacent to orifices of colonic diverticula, and inflammatory cap polyposis and are frequent findings adjacent to neoplasia and in the vicinity of the ileocecal valve. surface colonic epithelial apoptosis and karyorrhectic debris within the superficial lamina propria are commonly seen in mucosal biopsy specimens and are widely attributed to bowel preparation. apoptotic bodies in the deep crypt are only rarely seen (< per crypts) outside of pathologic conditions. increased deep apoptotic bodies can be seen in ischemic-type damage, cmv infection, chemotherapy or radiation, and in association with mycophenolate mofetil (cellcept) (fig. - ). although associated with a variety of injurious agents, apoptosis is the characteristic form of cell death in cell-mediated immune cytotoxicity as demonstrated in graft-versus-host disease (gvhd), other immune deficiency syndromes , and thymic neoplasia. many patients with aids with diarrhea demonstrate deep apoptosis in the absence of pathogens, a finding suggesting that primary immune-mediated apoptotic colopathy may cause some of the diarrhea and wasting seen in aids. , intraepithelial lymphocytosis the term collagenous colitis, first coined by lindström in , describes a distinct clinicopathologic syndrome causing watery diarrhea, predominantly in middle-aged or older (mean age, years) women (male-to-female ratio, : . ). , results of colonoscopic and barium enema examination in these patients are usually normal. therefore, diagnosis depends on recognition of characteristic changes in biopsy specimens. the primary histologic change of collagenous colitis consists of a patchy increase in the thickness of the subepithelial collagen plate (fig. - ) . , the normal colonic epithelial collagen layer measures approximately µm in thickness, but in collagenous colitis it may increase to µm or more. we use µm as a morphologic cutoff point for collagenous colitis. a mild to moderate increase in chronic inflammatory cells including plasma cells and eosinophils expands the lamina propria. patchy injury to the surface epithelium characterized by increased numbers of intraepithelial lymphocytes, epithelial degeneration, and sloughing also occur. atrophy, mucosal architectural distortion, and acute inflammation are usually not present or are minimal. , , read and colleagues introduced the term microscopic colitis to describe patients with chronic watery diarrhea of unknown origin occurring in middle-aged (mean, years) patients. as in collagenous colitis, the colonoscopic appearance and the barium enema were usually described as normal. biopsy specimens demonstrated increased inflammatory cells in a pattern not specific for any established entity. since this initial report, investigators have refined the clinical and especially the histologic diagnostic criteria of lymphocytic colitis. , the histologic changes include increased chronic inflammatory cells in the lamina propria and surface epithelium, degenerative changes of the surface epithelium, minimal architectural change, and minimal acute inflammation (fig. - ) . the changes of lymphocytic colitis in general resemble the surface epithelial and lamina propria changes of collagenous colitis, and indeed changes identical to lymphocytic colitis are seen in biopsy specimens from patients with collagenous colitis when areas where the collagen plate is not well developed are sampled. the similarities between lymphocytic colitis and collagenous colitis are striking. they share similar symptoms and endoscopic findings. the histologic similarities include increased intraepithelial lymphocytes, surface epithelial damage, and increased chronic inflammatory cells within the lamina propria. , , both collagenous colitis and lymphocytic colitis can be associated with other autoimmune phenomena such as thyroid disease, enteropathic arthritis, rheumatoid arthritis, myasthenia gravis, and celiac sprue. , some cases of lymphocytic colitis demonstrate thickening of the subepithelial collagen plate, and distinguishing lymphocytic colitis from collagenous colitis may not be so clear-cut in all cases. it appears likely that lymphocytic colitis and collagenous colitis are either the same or very similar conditions, perhaps representing different morphologic phases of one disease process. , some minor differences between lymphocytic colitis and collagenous colitis deserve mention, however. collagenous colitis has been reported predominantly in women, whereas lymphocytic colitis affects men and women equally. , differences in human leukocyte antigen (hla) haplotypes between collagenous colitis and lymphocytic colitis have been described. finally, collagenous colitis and lymphocytic colitis differ histologically with the presence of the thickened collagen plate. lymphocytic colitis and collagenous colitis are associated with net fluid secretion in the colon that is responsible for the diarrhea. several investigators could find no association between the thickness and extent of the collagen plate and the amount of diarrhea. therefore, most investigators conclude the damage to the surface epithelial cells and the inflammation, rather than the collagen deposits, cause diarrhea. the presence and thickness of the subepithelial collagen plate appear unrelated to the patient's age or the duration of disease. some patients have also demonstrated evidence of small bowel dysfunction such as salt wasting, fatty acid malabsorption, small bowel net secretion, and rarely a small bowel villous lesion that resembles celiac sprue (see later). spontaneous resolution of collagenous and lymphocytic colitis has occurred, thus rendering evaluation of therapeutic regimens difficult. a few patients with these colitides have presented with relatively mild diarrhea and have achieved medical control with dietary restriction (elimination of caffeine and lactose-containing foods), bulking agents, and antimotility drugs (loperamide hydrochloride, diphenoxylate hydrochloride, atropine). a trial of bismuth compounds is worthwhile. many times, symptomatic therapy has failed, thus necessitating the addition of antiinflammatory agents. approximately % to % of patients eventually respond to therapy with sulfasalazine, prednisone, or budesonide. , , etiology and pathogenesis nsaids and ticlopidine have been linked to some occurrences of collagenous colitis, , and lymphocytic colitis has been reported in patients receiving ruscus extract (cyclo fort), ranitidine, ticlopidine, and flutamide. however, most cases cannot be linked to drug ingestion and the bulk of the evidence suggests that lymphocytic and collagenous colitis share common immune-mediated etiology and pathogenesis. both conditions have a striking histologic similarity to celiac sprue, a condition known to be autoimmune and possibly having a viral or infectious trigger. in addition, both lymphocytic and collagenous colitis have been linked to other conditions thought to have autoimmune pathogenesis such as crohn's disease, hypothyroidism, hyperthyroidism, inflammatory arthropathies, pernicious anemia, small bowel villous atrophy, iritis, diabetes mellitus, and myasthenia gravis. , , both conditions respond dramatically to anti-inflammatory agents. , the finding of "lymphocytic colitis-like" histologic features reported in an epidemic outbreak of brainerd diarrhea linked to a water tank aboard a cruise ship supports an infectious trigger for some cases of lymphocytic colitis (see later). the association between lymphocytic colitis or collagenous colitis and celiac sprue and spruelike lesions deserves special attention. in the experience of dubois and associates and wolber and colleagues, approximately % of patients with "celiac sprue" who had colonic biopsies also showed changes of lymphocytic colitis. colonic microscopic abnormalities in patients with celiac sprue occur after experimental exposure to wheat or gliadin enemas, a finding suggesting that the entire intestinal tract may be susceptible to gluten-induced injury. it is possible that in some patients with true celiac sprue (responsive to gluten withdrawal), occult dietary gluten reaches the colon and induces the histologic changes of lymphocytic colitis. however, approximately one half of the patients with spruelike small bowel lesions and lymphocytic colitis have not responded to gluten withdrawal. the term lymphocytic enterocolitis has been coined to describe this refractory spruelike condition associated with colonic intraepithelial lymphocytosis. as more cases are investigated, it is not unusual to see variations in histologic pattern commingled with the classic changes of lymphocytic and collagenous colitis. these variations include architectural change, cryptitis, paneth cell metaplasia, ulcers, and inflammatory membranes. [ ] [ ] [ ] [ ] approximately one third of patients demonstrate cryptitis. rarely, one encounters inflammatory pseudomembranes. neither histologic pattern correlates strongly with infection. approximately % of patients have ulcers, and architectural abnormalities have been reported in % of patients. paneth cell metaplasia is seen in % to % of patients, most often in collagenous colitis in which it may correlate with increased severity of diarrhea. acute inflammation is seen with increased frequency in patients taking antibiotics. ulcers have been linked with concomitant nsaid usage. some investigators have suggested that the "stiff" colon seen in collagenous colitis could be at increased risk for the development of ulcers and perforation during colonoscopy. other variants with subepithelial giant cells have been described (fig. - ). histologic features, aberrant or otherwise, do not seem to correlate with symptoms, results of medical treatment, or outcome. no patient with unusual histologic features, including architectural change and paneth cell metaplasia, has yet to develop primary ibd (e.g., crohn's disease or uc). the term brainerd diarrhea has been applied to outbreaks of diarrhea of unknown origin characterized by acute onset and prolonged duration. the disease was named after brainerd, minnesota, where in residents developed watery diarrhea after drinking unpasteurized milk. a second outbreak occurred in henderson county, illinois in , when people developed watery diarrhea associated with drinking contaminated well water at a roadside restaurant. in each outbreak, patients underwent extensive diagnostic evaluations including comprehensive microbiologic studies of their stool and the implicated exposure site. despite the workup, no causative agent was identified. the clinical and epidemiologic characteristics were typical of point-source epidemic infectious diarrhea. however, unlike in typical infectious diarrhea, these patients developed a chronic watery diarrhea syndrome with symptoms lasting longer than months and often lasting for years. in general, the small bowel biopsy specimens from patients with brainerd diarrhea were histologically normal. colonic biopsy specimens revealed surface epithelial lymphocytosis without distortion of mucosal architecture, surface degenerative changes, or thickened subepithelial collagen plate. the degree of surface epithelial lymphocytosis was similar to that seen in cases of collagenous and lymphocytic colitis. , , many experts believe that patients with the clinical syndrome of chronic watery diarrhea of unknown origin and patients reported as having lymphocytic colitis represent a heterogeneous group that may contain persons with unrecognized brainerd diarrhea. with long-term follow-up, cases of brainerd diarrhea appear to be self-limited, and patients recover in less than years. from a practical standpoint, surface epithelial lymphocyte counts should be performed on all colonic biopsy specimens from patients with chronic diarrhea, especially chronic watery diarrhea. currently, brainerd diarrhea cannot be recognized outside the setting of an epidemic. the solitary rectal ulcer syndrome, localized colitis cystica profunda, inflammatory cloacogenic polyp, prolapsing mucosal folds in areas of diverticular disease, and inflammatory cap polyposis are closely allied conditions that have been linked to large bowel mucosal prolapse and trauma. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] affected patients with solitary rectal ulcer, localized colitis cystica profunda, and inflammatory cloacogenic polyp often demonstrate abnormal function of the anal and pelvic floor musculature during defecation , , that leads to rectal mucosal prolapse or even intussusception. the resulting trauma is thought to cause the clinical symptoms and the pathologic changes. the term solitary rectal ulcer syndrome is quite a misnomer because the ulcers are often multiple, a preulcer polypoid phase is reported, and similar lesions occur in the anal canal and proximal colon. , , , the terms colitis cystica profunda and inflammatory cloacogenic polyp are also misnomers. because all three conditions share a common histologic appearance, clinical presentation, clinical course, and pathogenesis, we prefer to consider them together under the heading mucosal prolapse syndromes. patients with mucosal prolapse syndrome range in age from to years with the majority presenting in the third and fourth decades of life. the condition occurs more commonly in women. all patients report difficulties in defecation. other common symptoms include rectal bleeding, passage of mucus, anal and abdominal pain, and intermittent bowel habits in which periods of constipation interrupt bouts of loose stools. furthermore, surprising numbers of patients admit to the use of digital manipulation, spoons, or other instruments to assist in rectal evacuation. the diagnosis of mucosal prolapse syndromes can be difficult because the history often suggests primary inflammatory or ischemic bowel disease. in addition, mucosal prolapse is often covert requiring special techniques such as evacuation cine proctography and defecography to demonstrate abnormalities in these patients. , , , ulcers, when present, typically occur on the anterior or anterolateral wall of the rectum, are irregular in shape, and often appear well demarcated. in patients without ulceration, the mucosa appears polypoid, roughened, or erythematous. inflammatory cloacogenic polyp , involves the lower rectum and anal transition zone. the clinical impression in patients with mucosal prolapse syndromes is often incorrect and includes ulcer, crohn's disease, nonspecific proctitis, carcinoma, and villous adenoma. the characteristic histopathologic changes are found in the mucosa adjacent to the ulcers or in the polypoid areas and consist of fibromuscular obliteration of the lamina propria associated with mucosal architectural distortion often with a hyperplastic or villiform appearance (see fig. - ). [ ] [ ] [ ] [ ] inflammation is typically mild or absent and mucosal capillaries can be ectatic. superficial erosions occasionally occur and can be associated with acute inflammation and the formation of inflammatory pseudomembranes. on occasion, colonic glands may be misplaced into the muscularis mucosae or submucosa, a histologic pattern referred to as localized colitis cystica profunda. submucosal vessels may also be ectatic and hyalinized. the histologic features of specimens obtained from ulcerated areas appear nonspecific and usually show fibrinopurulent debris, fibrosis, and granulation tissue. differential diagnostic considerations include mucinous adenocarcinoma, chronic uc, cowden's disease, and ulcers resulting from ergotamine suppositories. the misplaced glands of mucosal prolapse syndrome (colitis cystica profunda) simulate pseudoinvasion seen in adenomas; these glands can be associated with dissecting mucous pools and easily can be mistaken for invasive mucinous adenocarcinoma. table - illustrates histologic features that aid in this differential diagnosis. the mucosal abnormalities of mucosal prolapse syndrome also closely mimic chronic uc. knowledge of the clinical picture and recognition of the characteristic fibromuscular obliteration of the lamina propria (not usually present in ibd) are helpful in this distinction. the histologic appearance of colorectal polyps from patients with cowden's disease appears identical to the histologic features seen in mucosal prolapse syndrome. mucosal prolapse syndrome and cowden's disease must be separated on clin-ical grounds. clinical history is also required to separate the rectal ulcers associated with the use of ergotamine suppositories from mucosal prolapse. these lesions can be grossly and microscopically identical to those seen in mucosal prolapse syndrome. the few studies with long-term follow-up report a chronic, stable appearance to the lesions. , , most patients with mucosal prolapse syndrome tolerate their symptoms following reassurance that they do not have cancer, the addition of fiber to the diet, and instruction to reduce straining and to avoid digital manipulation. the rare patient with severe bleeding or obstruction requires excisional therapy. medical remedies such as sulfasalazine, local or systemic corticosteroids, and antibiotics are useless. , , unusual patients with severe, incapacitating symptoms have been treated by resection, diverting colostomy, or rectal prolapse repair. results of these operations have varied and because approximately different operations have been described for mucosal prolapse syndrome, comparisons are difficult. inflammatory conditions of the colon can be caused by specific agents or conditions, such as bacteria or ischemia, or can be nonspecific. after specific causes of colitis have been ruled out, one is left with a group of diseases referred to as nonspecific or primary ibd. these include uc, crohn's disease, and ibd type indeterminate (unclassified colitis). in most cases, careful examination of gross and microscopic features will allow categorization of these nonspecific ibds into either uc or crohn's disease. approximately % of cases will cause significant differential diagnostic problems by illustrating ambiguous features. these cases are best classified as ibd type indeterminate (see later). evolving surgical techniques have changed the pathologist's role in the analysis of ibd. patients with uc have several surgical options that either create continence through ileostomy (kock ileostomy) or preserve anal sphincter function and restore continuity to the bowel (ileal pouch-anal anastomosis). [ ] [ ] [ ] these operations have in common the creation of a pouch or reservoir formed by interconnecting loops of terminal ilium. in general, these pouch procedures are contraindicated in patients with crohn's disease because of increased morbidity such as fistula and abscess. furthermore, complications requiring pouch removal can result in loss of considerable lengths of small bowel, sometimes enough to cause short-bowel syndrome. in our experience, there is nothing quite like a pouch to bring out the crohn's disease in someone. these cases highlight the limitations of the current pathologic classification especially in the setting of fulminant clinical disease. accurate pathologic diagnosis of colonic ibd contributes greatly to patient care. in patients who have had subtotal colectomy, relaxed and thorough pathologic examination of the colectomy specimen to rule out crohn's disease is pos-sible. a staged resection of the large bowel is still the safest and most advisable approach for patients with severe or fulminant colitis, even though it requires an additional operation to remove the rectum and a period with a temporary ileostomy. although pathologic assessment and interpretation are more difficult in fulminant disease, the two-stage procedure still allows for a complete examination of the colectomy specimen before any contemplated pouch construction with ileal-anal anastomosis. some surgeons do not consider two-stage proctocolectomy necessary for selected patients with colonic ibd. the one-stage operative approach therefore requires accurate preoperative diagnosis and emphasizes the importance of clinical evaluation, radiologic examination, endoscopic analysis, and proper interpretation of mucosal biopsy specimens (see earlier). pouch complications include inflammation, fistula, obstruction, incontinence, and anastomotic leaks. , although many complications result from surgical and mechanical difficulties, and others relate to the development of "primary" inflammation in the pouch (see "pouchitis," later), some of these complications represent pouch recurrence of initially unrecognized crohn's disease. these cases illustrate the inability to differentiate uc from crohn's disease reliably in severe colitis, even after thorough examination of the colectomy specimen. all reports of surgical experience with ileal pouch-anal anastomosis for presumed uc contain approximately % to % of patients in whom the final diagnosis proves to be crohn's disease. , [ ] [ ] [ ] [ ] we recommend use of a three-tiered classification system for primary ibd in colon resection specimens: uc, crohn's disease, and ibd of indeterminate type. the definitive diagnosis of uc requires all the following features: diffuse disease limited to the large intestine, involvement of the rectum, more proximal colonic disease occurring in continuity with an involved rectum (i.e., no gross or histologic skip lesions), the absence of deep fissural ulcers, the absence of mural sinuses, and the absence of transmural lymphoid aggregates or granulomas (fig. - ) . , , the definitive diagnosis of crohn's disease requires histologic verification with the demonstration of transmural lymphoid aggregates in an area not deeply ulcerated or the presence of non-necrotizing granulomas (figs. - and - ). , [ ] [ ] [ ] [ ] when the gross and clinical features suggest crohn's disease by the presence of skip lesions, linear ulcers, cobblestoning, fat wrapping, or terminal ileal inflammation, extensive histologic sampling to find the definitive histologic features of crohn's disease is suggested (fig. - ) . the term primary ibd of an indeterminate type is used for cases of idiopathic colonic ibd that have ambiguous pathologic features in the resection specimen and are inconclusive for a diagnosis of uc or crohn's disease. , , [ ] [ ] [ ] [ ] [ ] [ ] the classification system has prognostic significance. the pouch failure rate in indeterminate colitis (≈ %) is intermediate between that seen with crohn's disease (≈ %) and that reported in uc (≈ %). even so, patients without clinical, endoscopic, or radiologic evidence suggestive of crohn's disease in whom the final pathologic results are indeterminate generally are considered suitable for ileal pouch-anal anastomosis. , , [ ] [ ] [ ] [ ] [ ] [ ] colon and rectum surgically placed out of circuit acquire histologic changes associated with defunctioning alone, regardless of the original reason for diversion. [ ] [ ] [ ] [ ] the changes reflect a physiologic response to stasis and the loss of trophic factors in the feces, most notably short-chain fatty acids. most patients are asymptomatic. the mucosa of the diverted segment appears erythematous, granular, and friable ( fig. - ). histologic changes include marked lymphoid hyperplasia with germinal center formation, usually accompanied by mild colitis with crypt abscess formation (fig. - ) . these changes may be indistinguishable from follicular proctitis (ulcerative proctitis or localized uc). with time, the muscularis mucosae hypertrophies, the submucosa shows fatty and fibrous tissue infiltration, the muscularis propria thickens, and the lumen becomes progressively smaller. the mucosal lymphoid hyperplasia may be accompanied by lymphoid aggregates scattered in the deep submucosa and muscularis externa and may occur in diverted segments in patients without ibd. care must be taken not to base a diagnosis of primary ibd, especially crohn's disease, solely on the histologic changes seen in diverted specimens. in many patients, the rectum is placed out of circuit during an operation for primary ibd. in these instances, the rectum can show changes of both ibd and diversion. the histologic changes in a defunctioned rectum do not correlate with the original diagnosis or clinical outcome. a late complication of kock pouch and ileal pouch-anal anastomosis is the development of "primary" inflammation in the pouch with its associated clinical syndrome termed pouchitis. , the reported incidence ranges from % to %. this wide range may be the result of the lack of an accepted case definition of pouchitis. patients generally experience nausea, vomiting, malaise, fever, and abdominal cramping. the increased effluent/stool from the pouch may be watery, foul smelling, or grossly bloody. patients frequently become incontinent. pouch bacterial ecology is often altered and many patients respond to antibiotics such as metronidazole and ciprofloxacin, a finding that suggests a bacterial cause in some cases. some patients have required -aminosalicylates, sulfasalazine, corticosteroids, immunomodulators, or even pouch excision to manage symptoms. pouch biopsy may be performed to confirm the presence of inflammation or to evaluate the possibility of crohn's disease. pouch biopsy specimens from nondysfunctional pouches can show mild villus shortening and increased chronic inflammation with augmented crypt proliferation. most specimens from pouches that are functioning well resemble normal terminal ileum. a few neutrophils within surface epithelium and within the lamina propria are commonly seen in pouches that are functioning well. [ ] [ ] [ ] [ ] [ ] in contrast, pouches with classic pouchitis show decreased epithelial mucin and decreased or absent lymphoid follicles. the most consistent finding in this classic form of pouchitis has been ulcers with granulation tissue and patchy accumulations of neutrophils within crypt epithelium and within the lamina propria with deep crypt abscess formation. , , , , to define pouchitis more precisely, sandborn proposed the pouchitis disease activity index. , in this system, up to points are awarded for various clinical and endoscopic findings. the amount of neutrophilic infiltration and the degree of ulceration are graded on a scale of to points each. histologic findings are weighted in this system because pouchitis is diagnosed in patients who have or more points. many investigators report an inconsistent relationship between endoscopic and histologic changes in the pouch and patients' symptoms. therefore, many clinicians diagnose pouchitis on clinical criteria and reserve endoscopic examination with biopsy for those patients with refractory pouchitis or possible crohn's disease. , , , unfortunately, no reliable endoscopic or histologic criteria are available to differentiate most examples of pouchitis from a new onset or recurrence of crohn's disease in the pouch. confusion over pouchitis is easily understood when one considers that the clinical syndrome of pouchitis probably represents at least six different conditions (table - ) . , , the two variants of antibiotic-responsive pouchitis syndromes are classic pouchitis and jejunal bacterial overgrowth. , , classic pouchitis is associated with endoscopic and histologic findings that support the pouch as the source of clinical symptoms. [ ] [ ] [ ] [ ] , patients with classic pouchitis usually respond to antibiotics such as metronidazole and ciprofloxacin. occasional patients with clinical symptoms of pouchitis have had endoscopically and histologically negative pouches but have responded to antibiotics. , although rare, some of these patients have had proximal jejunal bacterial overgrowth, probably as a result of pouch distention causing an ileal brake phenomenon that decreases motility in the proximal intestine. , this decreased motility may predispose some individuals to proximal small bowel bacterial overgrowth. pouchitis syndromes refractory to antibiotic therapy include irritable pouch syndrome, short-strip pouchitis ("cuffitis"), crohn's disease, and chronic primary refractory pouchitis. , patients with irritable pouch syndrome may have severe clinical symptoms but demonstrate normal pouch endoscopy and histology. some of these patients respond to dietary fiber supplements and antidepressant therapy. to obtain a better-functioning pouch, many surgeons have abandoned rectal mucosectomy in the ileal pouch-anal anastomosis procedure. , in short-strip pouch itis, clinical symptoms may be caused by exacerbation of uc in the small retained rectal segment. , many patients with this form of pouchitis have responded to topical corticosteroids or mesalamine. although debated, missed crohn's disease is likely to manifest as a late pouch fistula or abscess rather than as refractory pouchitis. in occasional reported cases of refractory pouchitis, pouch biopsy specimens contained granulomas or the excised pouch showed histologic criteria for crohn's disease. , , invariably, the original colectomy showed either missed crohn's disease or colitis of an indeterminate type. these cases are best classified as crohn's disease. , , patients with chronic pouchitis should be investigated with endoscopy and biopsy to rule out crohn's disease or cmv infection, which has been linked to some cases of refractory pouchitis. , routine biopsy specimens of the pouch should be examined as well as biopsy specimens of the afferent limb. afferent limb ulcers tend to correlate with crohn's disease and, in patients who do not have crohn's disease, with the use of nsaids. we have seen cases of refractory pouchitis requiring surgical removal of the pouch. after careful pathologic evaluation, no specific infection and no criteria for crohn's disease can be found in either the excised pouch or the original colectomy specimen. , , these cases are best categorized as primary refractory pouchitis. the causes of classic pouchitis and primary refractory pouchitis are unknown but are probably related to a combination of stasis, bacterial overgrowth, the abnormal immune response of patients with ibd, or perhaps associated colonic-type metaplasia, which is reported to occur in a minority of pouches (see later). , , some investigators have identified histologic patterns of mucosal adaptation in pouches. [ ] [ ] [ ] approximately % to % of patients exhibit what has been called type a mucosa with normal small bowel biopsy histology or only mild mucosal atrophy with no or minimal inflammation. type b mucosa, characterized by transient atrophy with temporary moderate to severe inflammation followed by normalization of intestinal mucosa, is seen in % to % of patients. type c mucosa with permanent persistent atrophy and severe inflammation occurs in up to % of pouches. colonic-type features have been reported at least focally in pouches of all types by routine morphology, mucin histochemistry, immunohistochemistry, lectin binding, or electron microscopy. this colonic-type change is best developed in type c mucosa but is not complete. all pouches seem to retain mostly small bowel properties regardless of mucosal type or the duration of the pouch. with long-term follow-up, it appears that epithelial dysplasia can rarely develop in the pouch. this extremely rare complication seems to be limited to the subgroup of patients (< %) in whom refractory pouchitis and colonic-type metaplasia develop (type c mucosa). [ ] [ ] [ ] most investigators suggest yearly pouch surveillance once type c mucosa is established. however, until further information on cancer risk is available, it would seem prudent to survey types a and b mucosa as well, perhaps every other year. , dysplasia can also develop in the small retained rectal segment (often incorrectly referred to as the anal transition zone) in patients who have had restorative proctocolectomy and stapled ileal pouch-anal anastomosis. annual endoscopic surveillance with biopsy for these retained rectal segments seems prudent. the incidence of dysplasia is rare (< % of patients), correlates with the original colectomy specimen containing dysplasia or cancer, and can be safely treated by completion mucosectomy. [ ] [ ] [ ] colostomy changes specimens from colostomy revisions usually show changes associated with trauma and mucosal prolapse such as erosions, ulcers, hemorrhage, acute and chronic inflammation, and fibromuscular obliteration of the lamina propria. one often encounters peri-intestinal fibrosis and suture granulomas. inflammatory polyps can be seen as well. infiltration of the large intestine by large numbers of eosinophils can be seen in eosinophilic gastroenteritis. these patients usually show peripheral blood eosinophilia and have a clinical history of atopy. [ ] [ ] [ ] [ ] the entity that has been termed allergic proctitis is in our opinion a form of uc. whenever large numbers of eosinophils are encountered in a colorectal biopsy specimen, this finding should prompt thorough search for parasites, especially strongyloides species. the most common form of allergic proctitis or colitis occurs in infants as a result of dietary protein-related allergy. these children present typically with rectal bleeding, sometimes with diarrhea. , colorectal biopsy specimens show increased numbers of eosinophils within the lamina propria, occasionally with focal active colitis. biopsy specimens that contain more than eosinophils per high-power fields are suggestive of this disease. allogeneic bone marrow transplantation can be complicated by acute or chronic gvhd. , [ ] [ ] [ ] [ ] the changes of acute gvhd (apoptotic colopathy) in mucosal biopsy have been described and various grading systems for acute gvhd are used. , , grade acute gvhd correlates with increased apoptosis alone. grade changes include crypt abscesses as well as apoptotic bodies (see fig. - ) . total necrosis of crypts is classified as grade , whereas denudation of areas of bowel is considered grade . biopsy specimens should be carefully examined for cmv infection. cmv may also cause apoptotic change that can mimic gvhd and occasionally both conditions can coexist. apoptosis is seen with damage associated with chemotherapy and irradiation. patients receiving bone marrow transplantation for malignant conditions are prepared with chemotherapy and irradiation; therefore, distinguishing the effects of therapy from gvhd without a clinical history is impossible. histologic changes associated with chemotherapy and irradiation typically resolve in to days. , apoptotic colopathy identical to grade gvhd can be seen in immunodeficiency syndromes, in patients receiving mycophenolate mofetil, and in primary aids-related colitis. grade gvhd cannot be distinguished from ischemic damage without a clinical history. chronic gvhd usually spares the colon but a few examples have been described. the pathologic changes include submucosal fibrosis, mucosal calcification, and fibrosis of the lamina propria. , , chronic colitis-like changes have been seen in a subset of patients with gvhd; the relationship between this condition and chronic gvhd requires more study. a localized form of uc with a better prognosis has been described as mucosal proctosigmoiditis, follicular proctitis, or ulcerative proctitis. [ ] [ ] [ ] the gross and endoscopic mucosal appearance resembles that seen with more extensive forms of uc. mucosal biopsy specimens typically show changes of uc but some have shown prominent mucosal lymphoid aggregates (follicular proctitis) (fig. - ) . most patients respond to the local administration of corticosteroids or -aminosalicylates; approximately % of patients develop more extensive colitis. , some evidence suggests that patients with prominent lymphoid follicles are less likely to respond to treatment. inflammatory changes are relatively common in patients with diverticular disease and are often related to trauma or prolapse. ibd and diverticular disease are both common and occasionally coexist. uc-like inflammation in association with diverticular disease has been described. crohn's disease and diverticular disease share similar features such as focal mucosal inflammation, stricture, and fistula. frequently, the pathologist will ascribe all the changes to one disease (usually diverticular disease) and overlook the concurrent ibd (usually crohn's disease). features that suggest crohn's disease in this setting are the presence of fissural ulcers, patchy active mucosal inflammation outside diverticula, granulomatous inflammation, and internal fistulas other than colovesical or colovaginal fistula. approximately % to % of patients develop other bowel lesions of ibd on follow-up. [ ] [ ] [ ] [ ] [ ] [ ] systemic mastocytosis is a rare disorder characterized by the infiltration of mast cells and eosinophils in the skin, bones, lymph nodes, and other organs such as the gi tract. [ ] [ ] [ ] one group of investigators suggested that colorectal biopsy specimens showing more than mast cells per high-magnification field as highlighted by cd or mast cell tryptase immunostain should be diagnosed as mastocytic enterocolitis, a disease best treated with antihistamines or mast cell stabilizers. this controversial study did not control for thickness of immunohistochemically stained sections and lacked a treatment control group, thus making such conclusions premature. mast cells coexpressing cd usually indicate neoplastic mast cells. patients with long-standing uc are at increased risk for colorectal carcinoma. the prevalence of colorectal cancer in patients with uc is estimated to be . % overall and . % for those with pancolitis. those at greatest risk are patients with extensive colitis , who have been afflicted for more than to years. the cumulative risk of cancer in a patient with uc is approximately % at years, % at years, and % at years. , early age at onset of colitis, family history of colorectal cancer, histologic evidence of active inflammation, and sclerosing cholangitis may also increase the risk. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] colitis-associated carcinoma makes up only a small proportion of total colorectal cancer cases, but these tumors are particularly bothersome to the medical community. they frequently occur in young patients and often develop insidiously even while the patient is under active medical care. many times the carcinomas are not clinically apparent until distant spread has occurred. , colitis-associated carcinomas are often flat and infiltrative and difficult to visualize with standard radiographic and endoscopic techniques. because the symptoms associated with carcinoma mimic those of colitis, diagnosis is often delayed. the estimated risk of carcinoma complicating uc varies considerably. however, epidemiologic data suggest that patients with uc have to times the risk of developing carcinoma when compared with the general population, and looking at the subgroup of patients with uc who have extensive colitis, the risk is -to -fold. , this increased risk poses a management dilemma for physicians caring for patients not sick enough to require colectomy. several management options are available. first, the physician can ignore the risk. many physicians prefer this approach when dealing with an older patient or a patient who is not otherwise a surgical candidate, especially if the actual cancer risk is believed to be low. a second management approach is prophylactic colectomy in patients with colitis for longer than to years. this may be the best approach when dealing with a young patient because of the expected long duration of cancer risk and the possibility that surveillance can fail. furthermore, the results of ileal pouch-anal anastomosis are better in young patients. although prophylactic colectomy theoretically eliminates the cancer risk, several factors have made this approach generally unacceptable. patients with extensive colitis for more than to years are often asymptomatic or have only mild disease. such patients often find it difficult to accept the risks associated with major surgery, the possible morbidity associated with mobilizing the rectum, or the social implications of a permanent ileostomy (in the event of a pelvic pouch failure). besides, most studies suggest that prophylactic colectomy would have been unnecessary in the majority of patients because they would not have developed carcinoma. , a third option is colonoscopic surveillance with biopsy. the strategy of such surveillance is to identify a marker that signals a subgroup of patients with colitis who are at increased risk for carcinoma or the detection of carcinoma in an early, curable phase. , , currently, recognition of dysplasia in surveillance biopsy specimens is used as such a marker. dysplasia, the presumed precancerous epithelial change, has been regularly recognized in colectomy specimens both adjacent to and distant from colitis-associated carcinomas. , circumstantial evidence suggests that dysplasia may not only be the marker for carcinoma but also may itself be the carcinoma in a preinvasive phase. , dysplastic epithelium can occur in grossly flat mucosa, in mucosa with a villous or plaquelike configuration, or in a nodular growth resembling adenoma (fig. - ). dysplasia is recognized by histologic examination of biopsy specimens using well-defined cytologic criteria that include nuclear enlargement with hyperchromasia, increased mitotic figures, and decreased intracellular mucin. , most colitisassociated dysplasia resembles adenomas seen in patients without colitis. the pathologist should use the term dysplasia only as a synonym for intraepithelial neoplasia and not in reference to reactive or reparative changes seen with active inflammation. the distinction between repair and dysplasia can be difficult, requires experience, and in certain instances can be impossible. in general, cytologic abnormalities seen in the presence of active inflammation must be interpreted with caution. , similar features can be seen in repair and dysplasia and no one histologic feature is absolute in making the distinction. both conditions are associated with nuclear enlargement and hyperchromasia, increased mitotic figures, and decreased intracellular mucin. some features favor repair over dysplasia. although cells undergoing repair demonstrate nuclear enlargement with hyperchromasia, these nuclei are often evenly spaced and not crowded, round to oval with a smooth external nuclear contour, contain granular chromatin with a single or multiple chromocenter or nucleoli, and are remarkably similar to one another in size and appearance. in contrast to dysplasia, the nuclear-tocytoplasmic size ratio of regenerative cells is often actually decreased, especially in cells adjacent to ulcerated areas. during this phase, the cell cytoplasm is often eosinophilic. nearby crypt abscesses or neutrophils within epithelium (cryptitis) help to confirm the diagnosis of repair. as a general rule, reparative epithelial change is limited to or accentuated in the crypt base and will not extend onto the surface of the crypt. features that favor dysplasia over repair are variable nuclear hyperchromasia, nuclear pleomorphism, irregular external nuclear contours, nuclear stratification with irregular nuclear crowding and overlap, and a loss of nuclear polarity. the changes of dysplasia usually extend onto the surface of the crypt. major distortions of mucosal architecture also favor dysplasia. the inflammatory bowel disease-dysplasia morphology study group proposed a three-tiered classification for biopsy interpretation in ibd: positive, negative, and indefinite for dysplasia. our experience has shown that this classification is useful and reasonably reproducible. , biopsy specimens negative for dysplasia include normal colon and those biopsy specimens showing changes of active or quiescent uc. positive biopsy specimens are reported as showing high-grade dysplasia or low-grade dysplasia. most dysplasias histologically resemble adenomas seen in patients without colitis, although some do not. dysplasias in this latter group are easy to miss. in low-grade dysplasia, nuclear changes such as crowding, pleomorphism, hyperchromasia, and increased mitotic figures are present, but in general, the nuclei are limited to the basal half of the cell (fig. - ) . usually, intracellular mucin is decreased, but on occasion intracellular mucin can be increased. dystrophic or signet ring-type goblet cells are common in dysplasia although not pathognomonic. the general mucosal architecture should not be greatly disturbed. the distinction between low-grade dysplasia and highgrade dysplasia is made largely on the degree of cytologic and architectural change. in high-grade dysplasia, hyperchromasia and pleomorphism are marked (fig. - ) . stratification with loss of nuclear polarity is often present. nuclei may be found in the luminal portions of the cells. high-grade dysplasia is often associated with a distortion of mucosal architecture, most often taking the form of a villous growth similar to villous adenomas seen in patients without colitis. biopsy specimens are classified as showing changes indefinite for dysplasia when cytologic abnormalities are seen but these changes are of insufficient degree to warrant a diagnosis of true dysplasia. indefinite changes are usually encountered in a background of active inflammation in which atypical epithelial changes could represent repair or regeneration rather than dysplasia. the indefinite category also includes odd mucosal patterns of growth that have not yet been observed to give rise to carcinoma (e.g., sessile serrated polyp-like change [ fig. - ], epithelial changes that resemble gastric foveolar epithelium). the category, indefinite for dysplasia, is a legitimate diagnosis alerting the treating physician that worrisome changes are present that may place a patient in a higher risk category that requires more frequent surveillance. the inflammatory bowel disease-dysplasia morphology group originally subdivided indefinite dysplasia into three groups: probably inflamma-tory, probably dysplastic, and unknown. obviously, in a category that is already uncertain, subcategorization is cumbersome, subjective, and associated with marked interobserver and intraobserver variation. therefore, subdivision of biopsy specimens in the indefinite category is not recommended. the use of -aminosalicylate compounds decreases the risk of cancer by % to %, , as does seeing the physician regularly and having more than one colonoscopy. most patients and physicians opt for lifelong annual or biannual colonoscopic surveillance. , current practice guidelines recommend two to four biopsy specimens obtained every cm throughout the diseased bowel in addition to special sampling of macroscopic lesions. compliance with this recommendation has been inadequate. [ ] [ ] [ ] [ ] patients whose biopsy specimens remain negative for dysplasia can probably safely continue regular surveillance. most authorities recommend annual total colonoscopic examination for patients with extensive uc who have had their disease for more than to years. , , , the surveillance interval can be increased to to years for patients with negative initial colonoscopy because in only % to % of these patients has disease been shown to progress. patients demonstrating epithelial changes indefinite for dysplasia should have shorter follow-up (e.g., months to year). , they must not be ignored because % of patients in whom the initial biopsy results were indefinite progressed to high-grade dysplasia and % progressed to cancer. management recommendations for patients with low-grade dysplasia in a flat mucosa are difficult because of the paucity of long-term follow-up information. some clinicians consider it safe to continue short-term follow-up (e. g., to months) for patients with low-grade glandular dysplasia; , if dysplasia persists , , , or is associated with any suspicious gross lesion or stricture (dysplasiaassociated lesion or mass [dalm]), colectomy should be considered. , , , forty-three percent of reported patients with dalm already had carcinoma in immediate colectomy specimens. , increasing numbers of experts have advo- cated immediate colectomy for low-grade dysplasia. [ ] [ ] [ ] these experts have stated that % of patients with lowgrade dysplasia already had complicating carcinomas, and they also have cited a very high rate of progression to highgrade dysplasia or carcinoma with follow-up ( %, st. marks, london; %, mt. sinai, new york; %, mayo clinic, rochester, minn). these data are questioned because of case selection bias. if high-grade dysplasia is encountered, then colectomy should be recommended because the risk of concurrent carcinoma is estimated to be as high as %. , experience with surveillance biopsy interpretation has shown that truly negative results are rarely interpreted as dysplasia, and dysplasia, especially high-grade dysplasia, is rarely missed. , that said, variations in interpretation do occur, , , , - and in general confirmation of a biopsy diagnosis of dysplasia is desirable before colectomy. , any one or more of the following may be considered adequate confirmation: finding of dysplasia in a repeat biopsy from the same site, finding of dysplasia in one or more additional sites during the same endoscopic examination, or review and confirmation of the original dysplasia interpretation by another pathologist experienced with the classification system. , , surveillance endoscopy with biopsy has limitations. no compelling evidence proves that surveillance benefits the patient. , , , , participation in a surveillance program does not guarantee that lethal cancer will not develop. , , , , , , dysplasia is unusual and it is difficult for any one pathologist to acquire extensive experience. therefore, errors in histologic interpretation can be expected. dysplasia can be focal and is subject to tremendous sampling error. , , because most authorities consider dysplasia a neoplastic change, it is unlikely that it ever resolves spontaneously. thus, a clinician should not be lulled into a false sense of security by negative follow-up biopsy results once true dysplasia has been identified. although not proved scientifically, it seems likely that patients benefit from surveillance programs because many carcinomas are detected in a curable phase. , , , , , in addition, the incidence of carcinoma in patients whose biopsy specimen results have remained negative is low. , , it is unclear whether this benefit justifies the large cost of surveillance endoscopy. , finally, surveillance programs are pointless unless the patient complies with regular surveillance and agrees to colectomy when the end point (dysplasia) has been reached. many groups have searched for other markers for precancer in uc. sialomucins predominate in cancer and dysplasia in uc but the role of mucin histochemistry findings is debated. results of lectin binding, cea immunohistochemical study, and immunohistochemical analysis for products of c-myc and ras oncogenes have produced variable results and could not reliably differentiate dysplasia from repair. several investigators described a correlation between dna aneuploidy and dysplasia or carcinoma in uc. [ ] [ ] [ ] however, only % to % of invasive carcinomas and % to % of dysplasia demonstrated dna aneuploidy. this finding indicates that dna analysis by flow cytometry could not be used alone in a cancer surveillance program. many specimens (≥ %) interpreted as negative or indefinite for dysplasia showed dna aneuploidy. , this finding can be interpreted in several ways. it could indicate technical problems linked to false-positive results including failure to disaggregate nuclear clumps, prolonged exposure of the sample to higher temperatures, and debris in the sample. , alternatively, this test could identify a subgroup of patients different from the group identified by dysplasia that is showing objective chromosomal abnormalities in the absence of recognizable dysplasia. it is tempting to speculate that this dna aneuploidy could be used as a marker of some carcinomas complicating uc, although other investigators concluded that the detection of dna aneuploidy was not useful as a predictor of the presence of concurrent carcinoma in uc. [ ] [ ] [ ] until large prospective studies determine the usefulness of dna aneuploidy in uc, histologic dysplasia is still the only reliable marker for cancer in a surveillance program. , , dna analysis could benefit some patients. patients with diploid dna content and no signs of dysplasia probably can be examined at longer surveillance intervals. , approximately % of patients would fall into this category and a longer interval surveillance could save considerable amounts of money and time. a special problem concerns the occurrence of adenoma in patients with colitis. colorectal adenomas and ibd are both relatively common. therefore, there is probably no reason that both conditions could not coexist. because most ibdassociated dysplasias resemble adenomas, the distinction in practice is often impossible. management of patients with uc and adenoma has been controversial. does the lesion represent a harmless sporadic adenoma or is it a dangerous dalm? in general, a pathologic diagnosis of "adenoma" in a patient with ibd must be carefully evaluated by the treating physician and should at least prompt further consultation with the pathologist because the lesion could represent ibd-associated dysplasia, an ominous change that implies a substantial risk for the development of carcinoma or for coexisting cancer. , adenoma versus dysplasia-associated lesion or mass dysplasia in ibd can be broadly classified endoscopically as flat or raised. raised lesions can be further subdivided into adenoma-like (i.e., discrete, well-circumscribed sessile or pedunculated lesions that resemble sporadic adenomas [see fig. - ] ) or non-adenoma-like (i.e., irregular plaque or masslike) lesions (figs. - and - ). , most experts recommend colectomy for flat high-grade dysplasia and endoscopically raised non-adenoma-like dysplasia lesions. correct management of patients with uc who have endoscopically adenoma-like dysplasia is not clear. the differential diagnosis between conventional adenoma and dysplastic lesions in ibd has been the focus of several publications. distinction based on pathomorphologic features has been proposed. , although lesions can be classified histologically using this system, to date this approach has not been clinically validated. the study of torres and colleagues suggested that an admixture of normal and dysplastic glands at the surface of the lesion could be used to distinguish ibd-associated dysplasia from conventional adenoma. several drawbacks to this study limit its clinical usefulness. an association with carcinoma or flat dysplasia was definitional for ibd-associated dysplasia in this study. furthermore, the study had limited clinical follow-up to determine whether the distinction based on this feature (admixture of normal and dysplasia at the surface) was clinically relevant in patients not yet known to have other areas of dysplasia or carcinoma. many authorities believe that admixtures of normal and dysplastic glands are so highly prevalent in sporadic adenomas that this criterion is not helpful in practice. molecular analysis has been applied in an attempt to resolve this diagnostic problem. odze and associates reported a similar prevalence of p, apc, and p muta-tions in adenoma-like dysplasia lesions in uc and sporadic adenomas. however, non-adenoma-like dysplasia in uc showed significantly higher proportions of p and p mutation, a finding indicating different timing of molecular events in these lesions. although promising, these molecular approaches are technically difficult, are not generally available, and remain to be validated clinically. immunohistochemistry has also been used in an attempt to discriminate between conventional adenoma and ibdassociated dysplasia. , p overexpression by immunohistochemical analysis that generally correlates with mutation of the corresponding tumor suppressor gene appears to be the most promising. p gene mutations reportedly occur earlier in ibd-associated dysplasia than in conventional adenoma or carcinoma. therefore, strong p overexpression tends to correlate with ibd-associated dysplasia. unfortunately, only % of ibd-associated dysplasias overexpress p (versus % of sporadic adenomas). , this p testing is probably not sensitive enough for routine practice. combining p immunoreactivity with betacatenin expression (which exploits the relatively low prevalence of apc gene mutation in ibd-associated dysplasia) or bcl- immunohistochemistry (which is more often overexpressed in sporadic adenomas) does not appear to improve the sensitivity or specificity of this approach. , immunohistochemistry is clearly technically easier and more widely available. however, as with the molecular approaches outlined earlier, diagnoses based on immunohistochemistry have not been clinically validated. practical guide to patient management pathomorphologic, molecular, or immunohistochemical testing could eventually separate harmless sporadic adenomas from the more ominous dalms in patients with ibd but this remains to be proved. in the meantime, follow-up information is sufficient to guide a careful physician facing this dilemma. the most important criterion for stratification is the topographic relation of the adenoma-like lesion to areas of colitis. adenoma-like lesions occurring in areas not affected by colitis histologically or endoscopically have not been associated with a high risk of concurrent or subsequent carcinoma. these lesions should be considered conventional adenomas and treated by polypectomy alone. , , , , management of adenoma-like lesions occurring in areas involved by colitis is more challenging. as many as two thirds of patients have had concurrent or subsequent invasive carcinoma at follow-up. , , , from a practical standpoint, we believe that it is prudent to consider all adenoma-like lesions occurring in areas involved by colitis as potentially ibd-associated dysplasia lesions. these lesions must be excised endoscopically and not merely sampled with biopsy. the diagnosis of ibd-associated dysplasia alone in this unique setting (adenoma-like dysplasia in a macroscopic polyp) may not necessarily be an indication for immediate colectomy. , , , [ ] [ ] [ ] , endoscopic polypectomy alone may be adequate treatment provided that careful patient selection criteria are applied, including the following: ( ) the patient is in an adenoma age group (> years of age), ( ) the adenoma-like lesion is discretely defined macroscopically and can be excised in its entirety, ( ) excision of the lesion appears complete to the endosco- pist, ( ) no flat dysplasia is identified in the colon, and ( ) the colon is easy to survey (i.e., compliant patient without inflammatory polyposis). current data suggest that the majority of patients managed in this fashion will follow a clinically benign course. , , , [ ] [ ] [ ] however, these patients must receive careful short-term surveillance. a -to month initial surveillance interval can be increased to months to year following negative results of colonoscopy. colectomy should be recommended for patients not fulfilling these selection criteria. increasing epidemiologic and pathologic evidence suggests that patients with crohn's disease are also at increased risk for the development of carcinoma. , - those patients who are younger than years of age at the onset of crohn's disease may be at even higher risk. colonic carcinomas in patients with crohn's disease have in general developed after about years of disease. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the diagnosis is usually made clinically because a gross intraluminal lesion can often be visualized (fig. - ) . the colonic carcinomas have been better differentiated than their small bowel counterparts in crohn's disease, with an increased prevalence of mucinous histologic features. , , , reports using current histologic criteria almost invariably note dysplasia in the epithelium adjacent to small bowel and colonic carcinomas in crohn's disease. , [ ] [ ] [ ] dysplasia distant from carcinomas has also been encountered in specimens exhibiting both colonic carcinoma and crohn's disease. these features suggest that the dysplasiacarcinoma sequence similar to that proposed for uc also occurs in crohn's disease. , , a practice-based surveillance study of patients with crohn's colitis affecting at least one third of the colon for more than years found that % of patients developed dysplasia or cancer at years. opinions vary regarding cancer surveillance in patients with long-standing crohn's disease. , [ ] [ ] [ ] , , it is possible that surveillance could benefit an occasional patient with colonic crohn's disease by detecting dysplasia or early carcinoma, but the safety, necessity, and benefit of regular cancer surveillance are questioned. colonoscopy may be technically impossible or dangerous in some patients with crohn's disease. retained rectal stumps are often strictured and inaccessible. the absolute number of colon carcinomas reported in crohn's disease is still low, so again the expected yield of positive cases in a surveillance program would be quite small. some experts advocate a uc-like surveillance strategy for patients with at least years of crohn's colitis involving at least one third of the colon. rather than ignoring the risk in crohn's disease, options are available for a cautious clinician. , , a patient with recrudescence of colitis-like symptoms and a background of long-standing inactive crohn's colitis should be thoroughly investigated for carcinoma. yearly surveillance of an out-of-circuit rectum seems reasonable considering that approximately % of the reported cases of large bowel cancer in crohn's disease have occurred in such segments. , it may, however, be better to advise removal of the defunctioned rectum, especially if reanastomosis is not planned, not possible, or contraindicated. the presence of dysplasia in a biopsy specimen from a patient with crohn's disease must alert the physician to the possibility of coexistent invasive carcinoma. in this situation, management approaches similar to those proposed for dysplasia in uc are recommended. , , clinicians should also recognize that chronic fistula or anal stricture in crohn's disease may be complicated by squamous carcinoma or adenocarcinoma. , any abrupt change in the volume or nature of a fistula discharge or the development of an area of induration or mass near a fistula should be investigated with biopsy. so-called left-sided colonic diverticular disease commonly affects middle-aged and older individuals and shows a characteristic muscular abnormality of the bowel wall. the abnormality is characterized by marked thickening of the inner circular layer of the muscularis externa that results in a narrowed lumen and a mucosa thrown up into prominent accordion-like folds. the mucosal abnormality and associated diverticula are most prominent within the sigmoid colon but they may extend proximally for a variable distance and may even involve the entire colon. diverticula are not seen in the rectum. the diverticula are usually still enveloped by an intact longitudinal muscle layer. diverticula usually manifest in two longitudinally running rows situated between the mesenteric and two anti-mesenteric taeniae. in severe cases of diverticular disease, diverticula can also be found between the antimesenteric taeniae. similar clinical signs and symptoms occur regardless of whether objective inflammatory changes are present; therefore, the term diverticular disease is preferred rather than routinely making the distinction between diverticulosis and diverticulitis. diverticula are sometimes difficult to demonstrate in freshly resected specimens because of lost muscle tone and intraluminal pressure. as a result the diverticula often evert and are mistaken for polyps. the best way to demonstrate diverticula is to fix an intact specimen with formalin under pressure for at least day before dissection (fig. - ) . complications of diverticular disease include inflammation within the diverticula (diverticulitis), perforation of diverticula, adhesion, fistula formation, pericolonic abscess with inflammatory mass, hemorrhage, and obstruction. although colovesical and colovaginal fistula can occur in diverticular disease, other fistula combinations must raise the suspicion of coexisting crohn's disease. any granularity or ulceration of the luminal mucosa that is accompanied by microscopic findings of crypt abscesses or deep lymphoid aggregates away from inflamed diverticula must also raise the possibility of coexisting crohn's disease (see earlier). likewise, features of colitis distal to the zone of diverticula (e.g., at the distal resection margin) must also raise the question of coexisting crohn's disease. right-sided diverticular disease is often isolated and usually unassociated with diverticula in the rest of the colon. right-sided diverticula occur in younger patients and are frequently seen in asians. [ ] [ ] [ ] [ ] clinically, right-sided diverticular disease manifests with symptoms that mimic acute appendicitis. some authors classify these diverticula as congenital if the outpouching contains all layers of the bowel wall or acquired if the diverticula resemble those seen in the left-sided diverticular disease. complications include inflammation, hemorrhage, pericolonic abscess, and peritonitis. these diverticula have been implicated in some solitary nonspecific ulcers of the cecum and right colon. the clinical diagnosis of ischemic bowel disease can be difficult. patients usually present with nonspecific symptoms and routine radiographic examinations such as plain abdominal films or barium enemas are rarely of use. even arteriography may not be helpful because of the poor correlation between angiographic findings and the ultimate diagnosis or clinical outcome. angiographic demonstration of patent mesenteric blood vessels does not exclude ischemia. conversely, stenosis or occlusion of mesenteric arteries (even two of the three major vessels) does not establish a diagnosis of ischemia because these are frequent findings in asymptomatic patients. therefore, the diagnosis of ischemic bowel disease requires a high index of clinical suspicion complemented by continued monitoring of clinical, laboratory, and radiographic information. the pathologist can play an important role in the diagnosis and management of ischemic bowel disease by recognizing the patterns of injury that suggest ischemia in biopsy and resection specimens. at first glance, intestinal ischemia seems simple, but in reality the topic is almost impossibly complex; the ultimate outcome of the patient depends on complicated interrelationships among temporal, anatomic, and physiologic factors. for example, gradual occlusion of large vessels rarely causes ischemic damage because adequate collateral circulation (e.g., arc of riolan, marginal artery of drummond) will develop. in contrast, acute arterial occlusion is likely to result in infarct. considering anatomic factors, occlusion of a large vessel does not often result in ischemic damage because collateral circulation is usually adequate to protect the bowel. however, occlusion of smaller end arteries frequently results in segmental ischemic damage and infarct. finally, the mere existence of an entity referred to as nonocclusive intestinal ischemia emphasizes the importance of physiologic considerations. systemic hypotension and bowel distention lead to marked decreases in splanchnic blood flow caused by vasoconstriction and arteriovenous shunting in the bowel wall. , this phenomenon can result in widespread bowel infarct even in the presence of patent blood vessels. ischemic injury to the gi tract occurs whenever the oxygen or vascular supply cannot meet the metabolic demands of the tissue. gi ischemia has many causes, including inadequate perfusion, narrowing of blood vessels from any cause, bowel obstruction and distention, drug effects, and infections that can mimic ischemic damage. the list of differential diagnostic possibilities often overwhelms the pathologist and clinician. keeping the diagnostic considerations in their proper perspective, almost all cases of mesenteric ischemia result from systemic hypotension or occlusion of a major blood vessel. knowledge of these relative frequencies can be invaluable in decreasing the pathologist's anxiety and frustration when dealing with these types of specimens and when interacting with the concerned clinician, patient, and family. most ischemic episodes result from nonocclusive ischemic bowel disease (low-flow states) and in these cases no vascular lesion or specific cause for ischemia can be demonstrated on pathologic examination. superior mesenteric artery embolism or thrombosis is usually diagnosed by radiography or at operation. almost all significant superior mesenteric artery occlusions occur in the proximal cm to cm of the artery, an area not likely to be present in a resection specimen; such occlusive plaques, thrombi, or emboli are either bypassed surgically or removed and submitted separately to the pathology laboratory. mesenteric venous thrombosis is also diagnosed and identified at laparotomy and treated with thrombectomy. low-flow states, superior mesenteric artery occlusion, and mesenteric venous thrombosis account for more than % of major ischemic events. ischemia can result in a wide range of pathologic changes depending on the cause, severity, and duration of the hypoxia. thus, ischemia can cause little or no change, mucosal plaques or hemorrhages with erosion, discrete or serpiginous ulcers that mimic crohn's disease, strictures, or at the severe end of the spectrum, hemorrhagic infarct with perforation. any part or length of bowel can be affected depending on the cause and duration of hypoxia and the state of the collateral circulation. in general, those cases of ischemic bowel disease associated with narrowing of a blood vessel show pathologic changes in the bowel in the corresponding distribution. in contrast, nonocclusive intestinal ischemia causes patchy and irregular distribution of damage not necessarily corresponding to an area supplied by a specific blood vessel (fig. - ) . ischemic damage associated with reduced blood flow shows a propensity to affect "watershed zones" such as the splenic flexure, which is the boundary between the superior mesenteric and inferior mesenteric artery distributions. , acute, organizing, and healed phases of ischemia can be recognized histologically. acute ischemic damage can be focal or diffuse. the characteristic pattern of acute injury consists of hemorrhage into the lamina propria associated with superficial epithelial coagulative necrosis, often with sparing of the deep portion of the intestinal crypt (see fig. - ) . , the mucosal distribution of these changes can be explained by the existence of a countercurrent exchange mechanism that has been suggested for intestinal mucosa. in the mucosa, a central arteriole carries blood luminally in a direction parallel to but opposite to the direction of blood flow in the draining mucosal venule. this situation causes a gradient in oxygen tension to form, with the luminal aspect of the bowel relatively hypoxic when compared with the crypt base. , the changes of acute ischemia can occasionally be associated with more extensive necrosis of the epithelium, often associated with an attached inflammatory pseudomembrane. surprisingly, acute and chronic inflammatory cells are typically minimal in acute ischemic damage. this finding helps to differentiate ischemic damage from changes seen in primary ibd. depending on the severity and duration of the hypoxia, the acute change can be reversible or can proceed to mucosal ulcers, at which point the histologic features become relatively nonspecific. with increasing severity and duration, ischemia can lead to full-thickness infarct with hemorrhage and coagulative necrosis involving all bowel layers. granulation tissue and fibrosis with plump fibroblasts predominate in the organizing phase of ischemic damage. the histologic change becomes nonspecific. ischemia should be suspected when an ulcer or area of organizing inflammation in the bowel appears quite bland and is not associated with a marked increase in chronic inflammatory cells with plasma cells and lymphoid aggregates so characteristic of primary ibd. often, the mucosa of the ulcer edge shows changes similar to those seen in acute ischemic damage, and extensive samplings of these areas can help in diagnostic problem cases. the presence of hemorrhage and hemosiderin deposits in the connective tissues, although not pathognomonic, strongly suggests an ischemic origin. during the healing phase, the colonic mucosa often assumes an architectural appearance similar (if not identical) to chronic uc with atrophy, shortened crypts, and branched and budded glands. , these changes usually have a patchy distribution in healed ischemic damage but are diffuse in uc. ischemia usually does not show the basal plasmocytosis of primary ibd. healed phases of ischemia are often complicated by fibrous stricture in which fibrosis can affect all the layers of the bowel wall including the muscularis externa. fibrosis of the muscularis externa is distinctly unusual in primary ibd and its presence should raise the suspicion of ischemic bowel disease. again, hemosiderin deposits can be a helpful clue in recognizing the ischemic nature of the injury. most ischemic bowel lesions result from a combination of atherosclerosis and low-flow states and are referred to as nonocclusive ischemic bowel disease. specimens showing ischemic damage or infarct are usually resected on an emergency basis and include relatively short segments of mes- entery. large segments of infarct or ischemic damage are usually caused by an occlusive lesion in a proximal segment of a large artery or vein. in other words, the offending vessel is not likely to be present in the resection specimen. although one is unlikely to see specific vascular abnormality in the resection specimen, the blood vessels should be carefully dissected and many cross sections examined microscopically to detect vascular lesions such as atherosclerosis, embolus, thrombosis, or inflammation (vasculitis). the significance of vascular inflammation in areas of active ulcer and infarct is questionable because this "vasculitis" may be secondary. however, acute inflammation in vascular walls associated with fibrinoid necrosis found in apparently viable areas separated from an ulcerated and necrotic area by a margin of normal tissue may indicate systemic vasculitis. polyarteritis nodosa is a multisystem disease characterized by random necrotizing inflammation involving small and medium-sized arteries. because the distribution and severity of the vascular lesions are haphazard, polyarteritis nodosa produces protean clinical manifestations without pathognomonic signs or symptoms. abdominal pain, fever, leukocytosis, hypertension, and neuropathy are common in patients with polyarteritis nodosa. according to the american college of rheumatology, the presence of of the following criteria are considered diagnostic of the disease : weight loss, livedo reticularis, testicular pain or tenderness, myalgia or myopathy, neuropathy, hypertension, renal impairment, hepatitis b infection, abnormal arteriogram, and positive biopsy results. the diagnostic histopathologic change of polyarteritis nodosa is necrotizing panarteritis with inflammation involving intima, media, and adventitia. the inflammatory infiltrate is composed of neutrophils, eosinophils, plasma cells, and lymphocytes and is often associated with a deposition of fibrin. , the vascular lesions are usually seen in various stages of development and healing. disruption of the internal elastic lamina weakens the vascular wall and can lead to aneurysm formation, an important sign used in radiographic diagnosis. of patients with polyarteritis nodosa, % to % have gi tract involvement such as duodenal or gastric ulcer, melena, hematemesis, or small bowel infarcts. gi tract involvement is usually a manifestation of systemic polyarteritis nodosa. however, a growing body of literature has described polyarteritis nodosa apparently limited to one organ, most commonly the skin, gallbladder, or vermiform appendix. [ ] [ ] [ ] [ ] [ ] only a rare patient with polyarteritis nodosa that is initially diagnosed in the gallbladder or appendix develops systemic vasculitis , , , and those who do frequently have elevated serum rheumatoid factor or antinuclear antibodies. phlebitis, churg-strauss angiitis, small vessel vasculitis, buerger's disease, and giant cell arteritis can manifest initially in the gi tract and cause ischemic injury. , phlebitis and small vessel vasculitis are frequently associated with drugs and medications and are often self-limited. , special consideration should be given to an entity referred to as intra-abdominal (lymphocytic) phlebitis. rare cases of ischemic damage to the colon have been linked to a curious form of phlebitis reported under numerous synonyms including lymphocytic phlebitis, necrotizing and giant cell granulomatous phlebitis, idiopathic myointimal hyperplasia of mesenteric veins, mesenteric inflammatory veno-occlusive disease, intramural mesenteric venulitis, and idiopathic colonic phlebitis. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] since the original descriptions, approximately additional cases have been described. the patients have ranged in age from to years, and no gender predilection is apparent. most cases involve the small intestine and colon but involvement of other organs such as the gallbladder, omentum, and stomach have been described. , four patients received treatment with the drug rutoside. microscopically, specimens show phlebitis and thrombophlebitis mostly involving the submucosal veins and venules ( fig. - ) . the most prominent lesion is infiltrative lymphocytic phlebitis. in approximately half of these patients, the phlebitis has been necrotizing, and in about one third a granulomatous component has been visible. in approximately half of the patients, myointimal proliferation has been described. some investigators have proposed that myointimal hyperplasia represents an end stage of lymphocytic phlebitis. immunohistochemical analysis has been performed on a few cases and demonstrates a mixture of t cells and b cells with a few macrophages. , some of the t cells express the cytotoxic granule-associated protein tia- and granzyme b, findings supporting the contention that the lymphocyte-mediated vascular damage is a central event in the pathogenesis. so far, cases in the literature have had a favorable clinical outcome. the disease usually has been diagnosed and treated by resection in these patients. no patient to date has required repeat resection, nor has any patient developed systemic vasculitis. some patients con- tinue to have abdominal pain, and at least one report noted recurrent phlebitis documented by biopsy. other entities considered part of the spectrum of ischemic bowel disease include necrotizing enterocolitis of the neonate, neutropenic enterocolitis (typhlitis), intestinal behçet's disease, late irradiation enterocolitis, uremic colitis, stercoral ulcer associated with bowel obstruction, potassium chloride-induced ulcer, stress ulcer and ulcers associated with drugs (e.g., nsaids and chemicals), certain infections such as clostridium difficile-associated colitis, and ehec infection. the pathologic features of these disorders resemble ischemic bowel disease and are generally indistinguishable without knowledge of clinical and laboratory information. necrotizing enterocolitis is a serious form of intestinal injury that resembles ischemic bowel disease. although many investigators think it part of the spectrum of ischemia, increasing evidence supports a role for infectious agents in the pathogenesis of the lesion. necrotizing enterocolitis of the neonate generally affects low-birth-weight or premature infants. the disease usually occurs in babies younger than days of age and symptoms usually develop after feeding has begun. the lesions resemble acute ischemic damage. , ulcers that can be patchy or diffuse occur and in general may affect any part of the gi tract. preferred sites of involvement include the terminal ileum, right colon, and stomach. histologically bland transmural necrosis occurs and may be associated with gas cysts within the bowel wall (pneumatosis cystoides intestinalis [pci]) ( fig. - ) . babies surviving the acute episode may later develop fibrous strictures. neutropenic enterocolitis goes by numerous synonyms, including hemorrhagic necrosis of the gi tract, necrotizing enterocolitis, agranulocytic colitis, typhlitis, and the ileocecal syndrome. [ ] [ ] [ ] [ ] the condition is fulminant segmental colitis that is often seen in patients with lymphoma or leukemia receiving immunosuppressive drug therapy, but it has also been seen in association with other chemotherapyinduced or non-neoplastic-induced neutropenias such as cyclic neutropenia. many investigators consider this disorder a form of ischemic bowel disease, but increasing evidence implicates bacteria, especially clostridium septicum, as causative agents. [ ] [ ] [ ] [ ] the pathogenesis is unclear but is likely to be multifactorial with neutropenia, altered mucosal integrity, ischemia, and infection all playing a role. the lesion can occur anywhere in the gi tract, but the preferred sites are cecum, right colon, and terminal ileum. the histologic picture combines infarct necrosis with profound secondary bacterial or fungal overgrowth and is often associated with a paucity of acute inflammatory cells. the prognosis is grave; however, reports of successful surgical and medical management have been published. , , , , nonspecific ulcers of colon investigators have reported benign cecal ulcers and segmental necrosis of the right colon in which the pathologic features are nonspecific or resemble ischemia. [ ] [ ] [ ] the cause of many of these ulcers remains unclear. some may represent inflammatory changes or bleeding vascular lesions in right-sided diverticula. , many are associated with endstage renal disease, hemodialysis, renal or cardiac transplantation, and corticosteroid therapy. some are clearly related to ischemia caused by low-flow states, which are known to occur in patients undergoing hemodialysis. however, a strong correlation exists among cecal and terminal ileal ulcers, renal or cardiac transplantation, immunosuppression, and cmv infection. the possible role of cmv in these ulcers is unknown. the exact cause of many colonic ulcers cannot be determined at present. nsaid use should also be investigated. the term behçet's disease describes the clinical triad of aphthous stomatitis, genital ulcers, and relapsing iritis. behçet's disease is seen worldwide, but most cases have been reported from the mediterranean basin, the middle east, and japan. male patients are twice as commonly affected as female patients and the mean age of onset is in the third decade. , behçet's disease is a systemic disorder; only % to % of patients demonstrate gi tract involvement, [ ] [ ] [ ] usually in the form of intestinal ulcers. the ulcers of intestinal behçet's are described as multiple, deep, and punched-out with a preferential localization in the ileum and cecum. the ulcers often perforate. [ ] [ ] [ ] [ ] the primary clinical differential diagnosis is crohn's disease. intestinal behçet's disease differs from crohn's disease in several ways. behçet's disease is usually not associated with strictures or granulomas. free perforation, common in behçet's disease, occurs rarely in crohn's disease. details of histologic pattern in intestinal behçet's are difficult to find. , the inflammatory infiltrate of behçet's ulcer appears nonspecific. the lesions of so-called acute behçet's ulcer closely resemble acute ischemic bowel disease. the chronic lesion of behçet's disease resembles crohn's disease except the transmural lymphoid aggregates in behçet's disease contain more germinal centers, the bowel wall can be fibrosed, and granulomas are not usually encountered. some authors have reported mononuclear cell infiltrates around capillaries and venules with some areas of fibrinoid necrosis. , indeed, large vessel inflammation with occlusion and aneurysm formation have been described in % to % of patients with behçet's disease. , vasculitis may be the common underlying factor producing the gi lesions of behçet's disease. late complications of irradiation can occur weeks to years after therapy and include colitis, stricture, ulcer, and fistula ( fig. - ) . , many of these abnormalities may be related to ischemia as a result of the effect of radiation on blood vessels. histologically, the mucosa appears atrophic and similar (often identical) to that seen in chronic uc. having said that, the basal plasmacytosis in the lamina propria characteristic of primary ibd is usually but not always absent. ectatic mucosal capillaries with thrombosis and hyalinization are important histologic signs that can point to irradiation effect ( fig. - ). irradiation may be associated with fibrosis of any of the layers of the bowel wall. the fibrosis can appear dense and hyalinized and may contain large, atypical radiation fibroblasts. vascular changes are often prominent. blood vessels may be ectatic in the mucosa and submucosa. more commonly, however, one encounters marked intimal fibroplasia with hyaline thickening of the blood vessel walls that leads to luminal stenosis. sharply demarcated ulcers sometimes with secondary bacterial overgrowth can often be seen in the colon and rectum proximal to obstructing lesions such as invasive carcinoma, or they can be seen in patients with intractable constipation and fecal impaction. , [ ] [ ] [ ] the ulcer, bland in appearance, is thought to result from ischemia caused by a combination of pressure injury related to the hard fecal mass and the physiologic effects of bowel distention. , many drugs or chemicals have been associated with ischemic-type changes in the colon, including hydrogen peroxide and glutaraldehyde (used to clean endo scopes), nsaids, , , cocaine, , , oral contraceptives, and estrogen compounds. colitis can be caused by a host of bacteria including campylobacter species, shigella species, salmonella species, neisseria gonorrhoeae, yersinia species, mycobacterium species, and aeromonas species. although the histologic features of colonic mucosal biopsy specimens can vary greatly in these infections from essentially normal to lesions mimicking idiopathic ibd, large numbers of specimens show active colitis, as outlined earlier in the chapter, that when seen should suggest infectious-type colitis. , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the histologic finding usually consists of the focal active colitis pattern of injury with relative preservation of mucosal architecture except for edema. the active inflammation is associated with little or no mucin depletion. neutrophils can be present within the lamina propria and scattered cryptitis and crypt abscess formation, sometimes with luminal accentuation, can be seen. the definitive diagnosis of infectious colitis requires laboratory documentation by culture, pcr on the paraffin block, or serologic examination. histologic evaluation, although helpful in suggesting infection, can only rarely point to a specific agent. true granulomas can be seen in tuberculosis, syp hilis, chlamydia species infection, and yersinia pseudotuberculosis infection. microgranulomas are described in infection with salmonella species, campylobacter species, and yersinia enterocolitica. isolated mucosal giant cells are nonspecific but have been described in chlamydia trachomatis infection. , some bacterial infections cause little inflammation but can be diagnosed by observation of adherent organisms such as with intestinal spirochetosis and adherent e. coli species. although intestinal spirochetosis (infection with brachyspira aalborgi or brachyspira pilosicoli) can be seen in patients with human immunodeficiency virus (hiv) infection or aids, , it is more frequently encountered in immunocompetent patients in whom its significance remains controversial. , [ ] [ ] [ ] [ ] the microscopic appearance is subtle, consisting of a thickening or accentuation of the colonic brush border that stains deeply with hematoxylin. this effect is caused by adherent spirochetes that align in parallel and embed themselves in the absorptive cells. identification can be enhanced by use of a warthin-starry (or other silver) stain. the organism also cross-reacts with treponema species immunostains. adherent e. coli infections, described in hiv/aids under the term diarrheogenic bacterial enterocolitis, are discussed later. gi involvement with mycobacterium avium-intracellulare complex (maic) is seen in hiv/aids usually as part of disseminated infection. the organism is easily cultured from stool and even blood. maic can affect any part of the gi tract, including the large bowel. , the characteristic histologic feature is infiltration of the lamina propria by foamy macrophages that can mimic muciphages. in addition, maic stains positive with pas, a finding further adding to the possibility of misdiagnosis. maic is easily diagnosed with an acid-fast stain. cryptosporidium parvum infection can occur in immunocompetent persons but is more often seen in hiv/aids , and occasionally can be seen in the colon (fig. - ) . in biopsy specimens, cryptosporidial organisms are identified as basophilic dots measuring approximately µm attached to the surface of the colonocyte. immunocytochemical methods for identification have been described. amebiasis (infection with e. histolytica) is quite common worldwide and is seen in hiv/aids. clinical disease varies widely from asymptomatic conditions to fulminant colitis. early lesions consist of a slight mucosal depression and erosion with adherent exudate containing trophozoites of e. histolytica (fig. - ). focal active colitis can also be seen. more severe disease can cause ulcers that have been described as flask-shaped because they undermine the adjacent mucosa. e. histolytica is easily distinguished from the much larger balantidium coli, which is only rarely seen in humans. in addition, the trophozoite of b. coli is ciliated, the nucleus is much larger and kidney-shaped, and the cytoplasm contains vacuoles. schistosoma species infestation is a major cause of colitis worldwide but is rarely seen in north america. the adult worms elicit no inflammatory response. the colitis and the symptoms are caused by the marked inflammatory response to the eggs that can lead to mucosal ulcers ( fig. - ) . chronic colitis and inflammatory polyps can be seen in areas of endemic infection. strongyloidiasis is usually a self-limiting infection with adult worms residing in the proximal small bowel; the eggs develop into rhabdoid larvae in the lumen that pass in the stool. in immunocompromised patients, the rhabdoid larvae may develop into the infective filiform larvae. these infective larvae may invade intact colonic mucosa or perianal skin and set up a cycle of infection referred to as autoinfection. many such infections are severe and often fatal. in colorectal biopsy specimens the infective larvae can be seen usually associated with marked infiltration of mucosa with the eosinophils (fig. - ) . sometimes an eosinophilic and granulomatous reaction can be seen in biopsy and resection specimens. occasionally intraluminal worms are encountered in the colonic lumen and are removed at colonoscopy. these parasites are usually identified as trichuris (whipworm) or enterobius (pinworm) by examination of the worm and egg morphology. common viral pathogens include adenovirus, rotavirus, coronavirus, echovirus, enterovirus, astrovirus, and norwalk virus. colorectal biopsy is rarely performed in the setting of viral gastroenteritis; therefore, histologic changes are not well documented. norwalk virus and rotavirus are not known to cause morphologic changes in the colon. adenovirus can cause diarrhea in hiv/aids. histologic changes affecting epithelial cells include cellular disorder with loss of orientation and degeneration. eosinophilic viral intranuclear inclusions can be seen in surface goblet cells but are quite subtle. immunohistochemical analysis can be performed as an aid to diagnosis. cmv infection is extraordinarily common in patients with hiv/aids and in other immunosuppressed patients such as those receiving transplants. the characteristic inclusions are seen in endothelial cells, fibroblasts, and smooth muscle cells and are only rarely encountered in epithelial cells. severe infection can lead to vascular thrombosis and ischemic-type damage with ulcers (figs. - and - ) . herpes simplex virus infection is associated with painful ulcers in the distal rectum and perianal skin. , the pathologic changes (ulcer, neutrophils in the lamina propria, cryptitis, and crypt abscess formation) may suggest infec- invasive candidiasis can be seen in severely debilitated patients and in patients with hiv/aids. this disorder is rarely encountered in colorectal biopsy specimens. colorectal histoplasmosis can be seen in immunocompetent patients and in immunocompromised patients such as those with hiv/aids. , in disseminated forms one can see collections of foamy macrophages. some patients demonstrate granulomatous inflammation, which is sometimes suppurative. diagnosis can be made using gomori methenamine silver or other fungal stains. administration of any antibiotic that favors the growth of toxin-producing c. difficile can lead to pseudomembranous colitis. , early studies linked clindamycin and lincomycin to pseudomembranous colitis, but in terms of absolute numbers, most cases are linked to ampicillin, penicillin, the cephalosporins, and the fluoroquinolones (especially the c- -methoxyfluoroquinolones gatifloxacin, and moxifloxacin) because of their far more prevalent use. , currently, the second-and third-generation cephalosporins and the fluoroquinolones are the leading instigators of c. difficileassociated colitis. [ ] [ ] [ ] pseudomembranous colitis has even been associated with antineoplastic chemotherapeutic agents that have antimicrobial activity. in many cases, the source of clostridium difficile infection is the patient's own gut flora. alterations of the gut flora allow the patient's own c. difficile organisms to multiply. however, the spore-forming organism c. difficile is widely distributed in nature. although cases may be acquired by these routes, increasing evidence indicates that c. difficile is often acquired in hospitals. , , indeed, reports of a previously uncommon strain of c. difficile, the nap strain, showed that it is responsible for severe hospital outbreaks and serious disease in otherwise healthy individuals. , [ ] [ ] [ ] [ ] these reports of close-contact transmission, high recurrence rate, young age, bloody diarrhea, and lack of antibiotic exposure suggest changing epidemiologic features. advanced age is a risk factor; patients who are older than years old have an approximately times greater risk of developing c. difficile infection. statistically, c. difficile is associated with only a minority of cases of antibioticassociated diarrhea. in gi diseases other than primary ibd, isolation rates are about the same as in healthy adults. c. difficile is more frequently identified in patients with ibd and may be linked to exacerbations of ibd and fulminant colitis. , symptoms of pseudomembranous colitis usually develop during the administration of antibiotics but in up to one third of patients, the onset of symptoms can be delayed for up to to weeks; occasionally, no antibiotic exposure has occurred. , , , the characteristic endoscopic and histologic lesion is found early in the course of the disease and only in some individuals. the surface of the mucosa is covered by a plaquelike, cream-colored to yellow pseudomembrane ( fig. - ) . the intervening mucosa frequently appears normal but can be hyperemic or edematous. with increasing severity, the membranes can become confluent and linear ulcers can develop. usually the pseudomembranes are evenly distributed throughout the colon but in up to one third of patients, the pseudomembranes can be confined to the right colon. this finding emphasizes the need for total colonoscopy to make an endoscopic diagnosis. histologically, patchy necrosis of the superficial colonic crypts is evident that is not unlike that seen in ischemia. the affected crypts become dilated near the surface and an inflammatory exudate erupts from the surface aspect of the degenerating crypts in an explosive or mushroom-like configuration. the pseudomembrane may cover adjacent virtually normal colonic mucosa (fig. - ). the karyorrhectic debris and neutrophils in the pseudomembranes tend to orient in a curious linear fashion within the fibrin and mucus. early lesions of c. difficile and occasionally the mucosa between diagnostic pseudomembranes can show the focal active colitis pattern of injury frequently described in acute infectious-type colitis (acute self-limited colitis). , , left untreated, some cases of c. difficileassociated colitis progress and can become indistinguishable from ischemic bowel disease. toxic megacolon and perforation can occur. treatment falls into three major categories: ( ) nonspecific therapy, ( ) specific therapy, and ( ) therapies aimed at altering the gut flora. nonspecific treatments include discontinuance of the offending antibiotic, supportive measures, enteric precautions to retard the spread of c. difficile outbreaks, and surgery. surgical excision may occasionally be indicated for patients too sick to take oral antibiotics (the specific therapy), as well as for some patients with relapses and for nonresponders. toxic megacolon and perforation also require surgical treatment. specific therapies include the oral administration of antibiotics such as vancomycin, metronidazole, bacitracin, and the toxin binder cholestyramine. vancomycin, the preferred treatment, is relatively expensive. bacitracin and metronidazole appear to be as effective as vancomycin and are less expensive. theoretical disadvantages to metronidazole therapy include stool levels of the drug that appear far lower than those of vancomycin and the finding that metronidazole has occasionally been implicated as a cause of pseudomembranous colitis. treatment or prevention of pseudomembranous colitis by altering the gut flora with the introduction of such agents as lactobacillus and saccharomyces boulardii , , has been studied. saccharomyces boulardii was shown to decrease the incidence of antibiotic-associated diarrhea but did not change the rate of c. difficile colonization. another provocative approach to prevention is to restrict the use of newer antibiotics such as third-generation cephalosporins and fluoroquinolones in the hospital setting. , patients with c. difficile-associated pseudomembranous colitis who are treated specifically show a % to % response rate usually with defervescence in to days and resolution of diarrhea in days. however, % to % of patients have a relapse and relapse treatment can be challenging. most investigators recommend a second course of the same antibiotic (metronidazole or vancomycin) for days. for a second recurrence, tapered-pulsed vancomycin is advised. for a third or subsequent recurrence, a probiotic or toxin binder is added to tapered-pulsed vancomycin therapy. newer treatments for recurrences include passive immunoglobulin therapy, toxin receptor decoys (e.g., tolevamer), and active immunization against toxin a. stool culture for c. difficile is usually not recommended but some investigators report a significantly higher yield of positive results based on fecal culture followed by toxin assay on positive colonies. cell culture assay for toxin b is not routinely performed. results of latex agglutination tests have been disappointing. enzyme-linked immunoassays (eias) that detect toxin a (meridian diagnostic, vitec, bd, and cambridge) or toxin a and toxin b are widely used. these assays have a % to % positive correlation with cell culture assay. commercially available tests that detect toxin a and toxin b are preferred because % to % of c. difficile strains produce only toxin b. , an alternative and more sensitive but slower approach is to perform an eia for the detection of common antigen (a highly sensitive marker for c. difficile) followed by a cytotoxic assay if the results of the eia are positive. various gene probes and pcr techniques that detect toxin at the dna level are available for research purposes. enterohemorrhagic escherichia coli-associated colitis the clinical syndrome of hemorrhagic colitis is characterized by abdominal cramping, bloody diarrhea, and either no fever or low-grade fever. , patients typically demonstrate right-sided colonic edema, erosion, and hemorrhage and the absence of conventional enteric pathogens. in , investigation of hemorrhagic colitis outbreaks occurring in oregon and michigan implicated a then-rare serotype of e. coli, o :h , as the cause of the syndrome. subsequently, investigations of several additional outbreaks confirmed the association between hemorrhagic colitis and the verocytotoxin-producing e. coli, the most important of which is e. coli o :h . patients with hemorrhagic colitis typically present with the sudden onset of crampy abdominal pain occurring to days after ingestion of contaminated food, usually undercooked hamburger. outbreaks have also been linked to other foods, drinking water, and swimming pools. watery diarrhea, nausea, and vomiting follow within hours. one to days later, grossly bloody diarrhea replaces the watery diarrhea. in almost all patients, the disease resolves spontaneously, usually within days. investigation of the epidemic outbreaks of e. coli o :h infection revealed that not all patients acquire the full syndrome of hemorrhagic colitis. rather, a clinical spectrum exists ranging from asymptomatic carrier or self-limited nonbloody diarrhea to severe cases complicated by hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. , , e. coli o :h produces several toxins active against vero cells (verocytotoxins) and hela cells that have been termed shiga-like toxins because their mode of action is similar to that of the toxin produced by shigella dysenteriae type i. the toxins interact with the membrane receptor globotriosyl ceramide, are apparently absorbed into epithelial and endothelial cells, and cause damage or cell death by interfering with protein synthesis. , colonic histologic features in ehec infection are the best documented of the diarrheogenic e. coli organisms because ehec infection can lead to hospitalization and it clinically mimics ischemia and primary ibd, thus prompting colonoscopy with biopsy. , , colonoscopy typically demonstrates patchy erythema, edema, and surface ulceration of the colon (fig. - ) . the cecum and right colon are usually described as markedly abnormal, whereas the descending colon typically has mild or no changes. histologically, specimens usually show hemorrhage and edema within the lamina propria. specimens most often show focal necrosis associated with hemorrhage and acute inflammation within the superficial mucosa with preservation of the deep colonic crypts, similar to the pattern of injury described in acute ischemic colitis. specimens from many patients show neutrophils infiltrating the lamina propria and crypts resembling the focal active colitis pattern of injury seen in infectious colitis or acute self-limited colitis ( fig. - ). rarely patients also demonstrate inflammatory pseudomembranes. this combination of ischemic-like and infectious-like injury with capillary thrombi should at least suggest ehec-associated colitis in biopsy specimens. routine stool culture media do not enable one to distinguish e. coli o :h from other strains of e. coli normally found in the stool. physicians suspecting hemorrhagic colitis caused by e. coli o :h should specifically request that stools be screened for this organism. specimens may be examined using sorbitol fermentation as a strain marker; unlike most e. coli organisms, e. coli o :h strains test negative for sorbitol or have delayed positive results. colonies that test negative for sorbitol at hours can be screened with commercial o antisera. additional biochemical tests and h-antigen determinations can be performed later. dna hybridization techniques, pcr, direct immunofluorescence, and latex agglutination techniques have also been described for identification. procedures for detection of free fecal verocytotoxin and more sensitive methods for screening stool cultures for verotoxinproducing e. coli using polymyxin b on colony sweeps have been reported. , other escherichia coli pathogens e. coli organisms are the predominant components of the gut microflora. although most of these organisms are harmless or even beneficial, at least six categories of e. coli intestinal pathogens are recognized (table - ) . , much is known about the microbiology, pathogenic mechanisms, virulence factors, molecular genetics, and epidemiology of intestinal e. coli infections. however, surprisingly little is known about the histopathologic features of the human gut infection. information remains scant because most infectious diarrheas, being self-limited, do not require specific treatment even when an infectious organism is identified. therefore, sophisticated diagnostic tests such as organism identification, virulence factor determination, and endoscopy with biopsy are reserved for outbreaks or for cases with unusual features (e.g., severe or protracted diarrhea, systemic symptoms, need for hospitalization, or a differential diagnosis including serious diseases such as primary ibd or ischemia). analysis of the information available yields limited numbers of reaction patterns in e. coli-associated infection that have been described in the human gut or inferred from in vitro models or from human infection with other organisms having similar virulence factors. these include the following: no histologic change ( etec organisms are thought to adhere to and colonize the surface of the small bowel where they elaborate their toxins. it is possible that a serendipitously obtained small bowel biopsy specimen may contain adherent surface bacteria. histologic appearances of the small bowel and colon are inferred from etec's close relationship with vibrio cholerae, which does not cause histologically recognizable lesions in the small bowel or the colon. therefore, normal colon would be expected in etec-associated diarrhea. it is possible that etec could cause some acute self-limited colitis. recognizing the difficulty in identifying pathogenic e. coli in routine stool culture, it is possible that eiec and ehec could otherwise be responsible for some cases of acute self-limited colitis. as stated earlier, diffuse active colitis was also seen in some examples of documented cases of infectious colitis. these infectious colitis cases were associated with an epidemic outbreak of shigella dysentery. because the pathogenic features of shigella species are virtually identical to those of eiec, it is possible that eiec may on rare occasions cause the diffuse active colitis pattern of injury. that said, eiec infection with this pattern of injury may represent an infectious exacerbation of underlying primary ibd. the ability to adhere to and colonize host enterocytes or colonocytes is requisite for human infection for all six recognized categories of diarrheogenic e. coli. however, epec, eaec, and daec, which are traditionally referred to as the enteroadherent e. coli, are best differentiated on the basis of their adherence patterns to hep- cells in culture. these patterns include localized adherence (epec), aggregative adherence (eaec), and diffuse adherence (daec). the human gut histopathologic features in epec in vivo have been described , and are similar to those seen in experimental animal models. jejunal biopsy specimens have demonstrated variable villous abnormalities without acute inflammation. , adherent bacteria could be identified on routine h&e-stained sections on the luminal surface. adherent surface bacteria have also been seen in colorectal biopsy specimens. the characteristic attaching and effacing lesion can be recognized only by the use of electron microscopy. , , the bacteria intimately adhere to the host cell by an attachment pedestal and cause effacement of the adjacent microvilli. human histopathologic features associated with eaec and daec infection can be inferred only from animal models and in vitro studies. , [ ] [ ] [ ] presumably, bacteria adhere to the surface epithelium of the small intestine and the colon. eaec and daec infection may be associated with variable villous abnormalities. electron microscopic study has shown adherent bacteria in cell culture with a normal microvillous structure. in the colon, eaec produces cytotoxic effects ultrastructurally (microvillous vesic- ulation, enlarged crypt openings, creation of intercrypt crevices, and mucosal epithelial cell extrusion). the most detailed description of human ileal and colorectal infection with epec, eaec, and possibly daec probably is found in a published review of patients with aids. unfortunately, the exquisite light and electron microscopic descriptions of these diarrheogenic bacterial enterocolitides were not complemented by microbiologic studies. therefore, identification of these organisms as e. coli rests on the pathologic similarity to other reported cases and preliminary isolation studies that have shown that at least some of these cases were caused by eaec and daec. the colonic histologic pattern showed surface epithelial degeneration with adherent bacteria (some extremely subtle) without colonic architectural distortion or significant inflammation. electron microscopic examination demonstrated three patterns: ( ) typical adhering and effacing lesions (as with epec), ( ) a loosely adherent pattern with effacement (as with eaec), and ( ) an intercalated pattern with effacement in which vertically oriented bacteria were seen burrowing between intact microvilli. the intercalated pattern could be daec in vivo because it is similar to the pictures and descriptions of daec in other in vitro and animal models. the term intestinal pseudo-obstruction describes a disorder in which patients present with signs and symptoms of intestinal obstruction and in whom no mechanical obstructive lesion can be demonstrated. intestinal pseudoobstruction can be associated with a heterogeneous group of conditions some of which can affect the colon. [ ] [ ] [ ] systemic lupus erythematosus, dermatomyositis, and scleroderma can cause fibrosis of the muscularis externa. , amyloid deposits can also affect colonic motility. visceral myopathies can occasionally be identified in the colon. recognized variants of familial visceral myopathy demonstrate differences in the mode of inheritance (autosomal dominant versus recessive), site of gut involvement, clinical symptoms, and extraintestinal manifestations. visceral myopathies also occur in sporadic form. the intestinal pathologic changes of many familial and sporadic visceral myopathies are identical and consist of muscle cell degeneration, muscle cell loss, and fibrosis of the muscularis externa. the degenerative fibers appear swollen and rarified. collagen may encircle the residual muscle fibers in areas of muscle fiber dropout and impart a vacuolated appearance. , , these changes are often limited to, or are more severe in, the external layer of the muscularis externa. the differential diagnosis of these forms of visceral myopathy includes other entities that cause fibrosis of the muscularis externa and encompasses ischemia, tuberculosis, and scleroderma. ischemia usually is associated with a fibrous stricture and hemosiderin deposits. tuberculosis typically causes strictures and granulomas, often with central necrosis. progressive systemic sclerosis is associated with more patchy bowel involvement than are the visceral myopathies. the fibrosis is often denser and replaces all the muscle layers of the muscularis externa or is accentuated in the inner layer ( fig. - ). vacuolar change is usually not seen with progressive systemic sclerosis. the visceral neuropathies - form a complex group of unusual entities that vary in their pattern of inheritance, the extent of intestinal and extraintestinal involvement, and the nature of the histologic changes in the neural plexuses of the gut. many of the neuronal and axonal changes are subtle; with the exception of inflammatory neuropathies and some neuropathies associated with intranuclear or intracytoplasmic inclusions (e.g., red cytoplasmic inclusions in ganglion cells of mitochondrial neurogastrointestinal encephalomyopathy), they cannot be recognized in routine h&e-stained sections. difficult and unusual cases should probably be referred for consultation to pathology departments with particular expertise in the evaluation of visceral neuropathies. that said, referral is rarely indicated clinically. some sporadic cases demonstrate mononuclear inflammation in the myenteric plexuses, and these cases can be identified by routine light microscopy. , some of these cases are manifestations of paraneoplastic syndromes often linked to small cell carcinoma of the lung. others are postinfectious. the inflammatory neuropathies can sometimes cause acquired aganglionosis and can be associated with circulating antibodies, such as anti-hu, (anna- , antineuronal nuclear antibody- ), anti-ri, and anti-yo (purkinje cell cytoplasmic autoantibody). , an acquired form of hypoganglionosis associated with buserelin-induced formation of anti-gnrh (anti-gonadotropin-releasing hormone) antibodies has been reported. investigators now recognize the role interstitial cells of cajal (iccs) play as gut pacemakers and as mediators of neurotransmission. , iccs stain specifically with the tyrosine kinase receptor c-kit. immunohistochemical analysis for c-kit (cd ) and for cd (which reacts with many c-kit receptors) represents a relatively easy way to study severe constipation and intestinal pseudoobstruction. streutker and colleagues described completely absent or markedly reduced numbers of iccs in some cases of intestinal pseudo-obstruction. reduced volumes of iccs have also been described in some patients with slow-transit constipation. [ ] [ ] [ ] [ ] [ ] although the observations could be an epiphenomenon, they could form the basis of an alternate classification system for these cases. severe intestinal malabsorption for whatever reason (e.g., celiac sprue, cystic fibrosis) can be associated with dark brown or orange-brown discoloration of the bowel wall, , owing to deposits of a granular material that has the characteristics of lipofuscin in the smooth muscle of the muscularis externa and to a lesser degree the muscularis mucosae ( fig. - ). this excessive accumulation of lipofuscin is termed ceroidosis or the brown bowel syndrome. whether this pigment deposition adversely affects muscle function is debated; however, damage to smooth muscle mitochondria has been described, and several reports have linked ceroidosis to intestinal pseudo-obstruction. because of the name brown bowel syndrome, ceroidosis may be confused with melanosis coli (discussed in the next section). melanosis coli is a condition in which macrophages filled with lipofuscin-like pigment are found within the lamina propria or deeper in the wall of the colon (fig. - ) . , these macrophages may be of such numbers as to impart a brown or black color to the colonic mucosa ( fig. - ) . melanosis coli has been associated with increased apoptosis, which is often linked to ingestion of purgatives of the anthracene group (cascara sagrada, aloe, rhubarb, senna, frangula). [ ] [ ] [ ] one group of patients has severe and persistent constipation with no apparent cause (idiopathic constipation or morbid obstipation). these patients, usually women, can have as little as one bowel movement every to weeks, and many of these patients are so uncomfortable that they require colectomy for relief. [ ] [ ] [ ] [ ] because these patients almost invariably take laxatives, melanosis coli is usually found. what is so frustrating to the pathologist is that by h&e staining, the neuronal plexus and smooth muscle in these patients appear normal. distinctive abnormalities of the myenteric plexuses (e.g., loss of "argyrophilic" neurons) have been reported using special silver staining techniques. , , , these tests are best performed by pathology departments with expertise in this area. reduced volumes of iccs have been described in some patients with severe constipation. , cathartic colon is the name given to end-stage idiopathic constipation in which the bowel can no longer contract effectively. the mucosa has the gross appearance of snakeskin and histologically shows melanosis coli. the muscularis propria is thin and atrophic, and neurons are decreased in auerbach's plexus. hirschsprung's disease (aganglionic megacolon) has a predilection for boys and men. approximately % of patients present in infancy with constipation, abdominal distention, vomiting, and delay of meconium stool; diarrhea may occur and some patients may even be affected by lifethreatening enterocolitis. reports suggest that many cases of hirschsprung's disease have a genetic basis. both hirschsprung's disease and men are associated with mutations of the ret proto-oncogene. [ ] [ ] [ ] in cases of men , the mutations are activating and enhance the function of the coded protein, whereas in hirschsprung's disease the mutations are inactivating. several cases of familial hirschsprung's disease have been linked to mutations of the endothelin receptor-b (endrb) gene. at least nine additional genes have been implicated in the pathogenesis of hirschsprung's disease: ntn (seen only with ret mutations), gdnf, edn , ece- , sox , phox b, zfhx b, l cam, and dhcr. despite this progress, mutations of one or more of the known genes are detected in only half the patients with hirschsprung's disease. in the typical clinical picture of hirschsprung's disease, the anus is normal. the anal canal and rectum are usually small and devoid of stool. in classic cases, these physical findings are confirmed by barium enema study. the contrast material flows into an unexpanded distal segment, then passes through a cone-shaped area, and finally passes into the dilated proximal bowel (fig. - ) . the pathologic change is aganglionosis. the narrowed distal segment shows loss of ganglion cells in both the submucosa and myenteric plexuses, usually accompanied by hypertrophy of the muscularis mucosae and muscularis externa and increased numbers of nerve fibers in the submucosa and between the muscle layers of the muscularis externa. hypertrophied nerve fibers (> µm) derived from the extrinsic and sensory fibers are observed in many but not all cases of hirschsprung's disease. in the tapered or cone-shaped region, the number of ganglion cells may be decreased (hypoganglionosis). historically, histologic diagnosis was based on fullthickness rectal biopsy specimens. however, this procedure requires general anesthesia and risks the development of stricture and perforation. because the submucosal and myenteric plexuses stop at about the same level in hirschsprung's disease, , suction biopsy that samples the mucosa and submucosa is now considered the method of choice for the diagnosis. all rectal biopsy specimens for suspected hirschsprung's disease should be serially sectioned throughout the block and each section examined. , if no ganglion cells are found, then some comment should be made concerning the adequacy of the specimen. biopsy specimens devoid of ganglion cells, but in which the amount of submucosa is less than the thickness of the mucosa, should be considered insufficient to diagnose hirschsprung's disease. if the biopsy specimen contains epithelium of the anal canal, this specimen should also be considered inadequate, because the anal canal and distal cm of rectum are normally hypoganglionated or aganglionated. many pathologists prefer to examine frozen section slides stained for acetylcholinesterase in addition to standard h&e-stained sections. in hirschsprung's disease, examination using the acetylcholinesterase stain demonstrates increased acetylcholinesterase-positive nerve fibers in the lamina propria and muscularis mucosae. the utility of this technique as an adjunct to diagnosis is debated. false-positive and false-negative reactions have been reported, and the use of this method is a matter of personal preference. [ ] [ ] [ ] occasionally, ganglion cells may be difficult to identify using light microscopy alone, especially in the neonate. in such cases, a positive immunohistochemical reaction for neuron-specific enolase can be valuable in documenting ganglion cells. we have been successful in performing immunohistochemical analysis for neuron-specific enolase on sections after they have been stained with h&e and examined. other immunostains such as cathepsin d, pgp . (protein gene product . ), ret, bmpr a (bone morphogenetic protein receptor type a), and bcl- decorate ganglion cells. , frozen section is often used as an adjunct to visual inspection to select the site for colostomy. however, use of the frozen section to establish a primary diagnosis of hirschsprung's disease is best avoided because of the high rate of incorrect interpretations. in % of patients with hirschsprung's disease, the aganglionic segment of colon is less than cm in length. the remaining patients have longer aganglionic segments that may extend even into the small intestine. microscopically, the hypertrophied nerve trunks of short-segment hirschsprung's disease are absent, but increased numbers of acetylcholinesterase-positive mucosal nerve fibers are usually but not always seen. ultrashort-segment hirschsprung's disease (segments < cm) reportedly exists but is impossible for a pathologist to document by routine h&e stains of the rectal mucosa and submucosa alone because this segment of the colon is relatively hypoganglionated or aganglionated even in physiologically normal individuals. rectal manometry may be used in the diagnosis of this lesion. recognition of acetylcholinesterase nerve abnormalities similar to those seen in hirschsprung's disease may complement that study. some patients may have internal sphincter achalasia with abnormalities of nitric oxide-induced sphincter relaxation. regardless of pathogenesis, some patients benefit from sphincterotomy. hypoganglionosis is regularly observed in the cone-shaped transition zone between normal and aganglionic bowel in hirschsprung's disease. some authors believe that diffuse hypoganglionosis of the colon may give rise to megacolon similar to that observed in hirschsprung's disease. , no accepted definition of hypoganglionosis exists; however, guidelines were offered by meier-ruge. this study suggested that a decrease by a factor of in the number of ganglion cells per centimeter of bowel as compared with normal ( to myenteric plexus neurons/cm bowel) is diagnostic of hypoganglionosis. other investigators have accepted much higher numbers of ganglion cells as evidence of hypoganglionosis. in general, the condition has not been well characterized and many reports lack quantitation. diverse abnormalities have been described by special silver staining in cases that would have been called hypoganglionosis by h&e staining results, and some cases of "hypoganglionosis" may be similar to those cases reported as severe idiopathic constipation or cathartic colon. intestinal neuronal dysplasia is characterized by hyperplasia of the myenteric plexuses, increased acetylcholinesterase activity in nerves of the lamina propria and submucosa, and increased numbers of ganglion cells with the formation of giant ganglia. , [ ] [ ] [ ] these giant ganglia, typically containing more than to neurons (normal ganglia contain to ), make up only % to % of all ganglia seen in a given case and are usually not seen in the distal cm of the rectum. occasionally, ganglion cells may be found within the lamina propria but this feature should not be considered diagnostic for intestinal neuronal dysplasia because it can be seen in physiologically normal individuals. , the condition may give rise to signs and symptoms similar to those seen in hirschsprung's disease. intestinal neuronal dysplasia may occur in a localized or disseminated form. similar lesions sometimes referred to as ganglioneuromatosis can be observed in patients with von recklinghausen's disease or men b. , , although some clinicians diagnose intestinal neuronal dysplasia based on abnormal acetylcholinesterase staining in specimens containing ganglion cells, others believe that one cannot rely on acetylcholinesterase staining alone for the diagnosis. diagnostic criteria for intestinal neuronal dysplasia and even its very existence are challenged , because % of infants so diagnosed experience normalization of gut motility within year. therefore, many of the observed "abnormalities" could be within normal range and in general the diagnosis should be reserved for florid pathologic cases. in zonal aganglionosis or skip-segment hirschsprung's disease, ganglion cells are found distal to one or more aganglionic segments. [ ] [ ] [ ] evidence suggests that zonal aganglionosis is rare and is likely to be acquired by ischemia (e.g., necrotizing enterocolitis), viral infection, or some other (e.g., immunologic) injury. the problem is that a rectal biopsy specimen may yield ganglion cells in spite of an authentic hirschsprung's disease-like aganglionic lesion. immaturity of ganglion cells , and hypogenesis of myenteric plexuses , have been reported to cause signs and symptoms similar to those seen in hirschsprung's disease. immunostains for bcl- may be helpful in detecting immature ganglion cells. the gi tract is a common site for amyloid deposits; such deposition has been documented in % of primary amyloidosis cases and in half of secondary cases. , , occasionally the deposition is symptomatic and causes ischemic manifestations such as bleeding diarrhea or infarct, sometimes referred to as amyloid colitis. on rare occasions, the deposits can be localized and form a tumor. the aa (secondary) type of amyloid primarily deposits in the lamina propria and walls of blood vessels; the al (primary or myeloma-associated) type tends to accumulate preferentially in blood vessel walls and in the muscularis externa. , amyloid can be subtyped using immunohistochemical analysis. although subcutaneous biopsy or aspiration is an easy way to obtain tissue for diagnosis, rectal biopsy is still widely used to diagnose amyloidosis. , the term pneumatosis cystoides intestinalis (pci) describes the occurrence of gas-filled cysts within the bowel wall. the large intestine is a common site of involvement; benign and fulminant forms have been described. , the fulminant form of pci is most often seen in infants as a complication of ischemia. pci in this setting is caused by mural invasion by gas-forming bacteria and subsequent formation of cysts (see fig. - ) . the fulminant form can be seen in adults sometimes in association with drugs such as antineoplastic chemotherapeutic agents or in pseudomembranous colitis. histologically, one sees ischemic change, bacterial overgrowth, and gas cysts predominantly within the submucosa. it is easy to dismiss the gas cysts as cutting artifact because an endothelial or histiocytic lining is rarely seen in this form of pci. the benign form of pci is most often seen in adults and is usually asymptomatic, although diarrhea, constipation, and rectal bleeding have been documented. , benign pci is often seen with comorbid conditions that either increase intraluminal pressure or provide a breach in mucosal integrity by which the gas can enter the bowel wall. these conditions include chronic obstructive pulmonary disease, emphysema, diverticular disease, appendicitis, cholelithiasis, peptic ulcer disease, abdominal trauma, crohn's disease, and gi tract surgery. , at endoscopy, mucosal broadbased elevations that are sometimes semitranslucent have been described. resection specimens often show crepitance. microscopic analysis demonstrates occasional tears in the submucosal connective tissues, but more often one sees dilated spaces lined totally or partially by endothelium, inflammatory cells, histiocytes, and marked foreign body giant cell reaction (fig. - ). the principal differential diagnostic consideration is the entity referred to as pseudolipomatosis. , pseudolipomatosis resembles fatty infiltration of the lamina propria but ultrastructural study convincingly demonstrates that the spaces are, in fact, gas cysts ( fig. - ). pseudolipoma-tosis has been associated with air inflation used to distend the bowel during colonoscopy. some investigators have implicated cleaning agents used to disinfect colonoscopes. the term fibrosing colopathy has been applied to colonic strictures seen in some patients with cystic fibrosis. , the pathologic change is submucosal fibrosis, which can sometimes extend into the muscularis externa. fibrosing colopathy has been linked to administration of high-dose pancreatic replacement therapy. large bowel agenesis and atresia are extremely rare. , congenital atresia and stenosis are associated with failure to pass meconium, abdominal distention, and vomiting. these conditions are often seen on a background of other congenital anomalies. pathologically, one can see an imperforate septum, a portion of the colon replaced by fibrous cord, or the absence of a segment of colon and associated mesentery. colonic malrotation occurs with malrotation of the small bowel and is associated with abnormal anatomic relationships and fibrous bands; it may predispose patients to volvulus. , mispositioning of the cecum and appendix may lead to delayed diagnosis of acute appendicitis. congenital duplications and diverticula are located within the mesentery and often occur in combination with other congenital malformations. , sometimes, the duplications are tubular, represent doubling of the bowel, and run parallel to the colon and rectum. other duplications can become cystic and are often referred to as enterogenous cysts. patients may have associated spine abnormalities. we classify tubular duplications that communicate with the lumen at one end as congenital diverticula. small duplications and diverticula are usually asymptomatic. larger ones may cause mass lesions, abdominal pain, constipation, or bleeding. hindgut duplications may be associated with complex genital and urinary tract abnormalities. duplications and congenital diverticula usually have organized layers of smooth muscle sometimes with a nerve plexus within their walls. mucosal linings if present resemble colon, respiratory epithelium, or gastric mucosa. as lesions enlarge to become cystic, the lining and the wall can become atrophic. the retrorectal space is a relatively common location for developmental cysts that can become symptomatic in adults. [ ] [ ] [ ] epidermoid or dermoid cysts are unilocular, are lined by squamous epithelium, and may contain adnexal structures (dermoid cyst) and lack smooth muscle in the wall. rectal duplications can become cystic. rectal duplications are also unilocular, are lined by colonic, gastric, or respiratory epithelium, and have an organized muscular wall that recapitulates the muscularis externa. the retrorectal cystic hamartoma is often referred to as a tailgut cyst. this lesion manifests as a multilocular cystic and solid tumor ( fig. - ) . the variably sized cysts can be lined by squamous, transitional, or glandular epithelium. disorganized bundles of smooth muscle are found within the wall. inflammatory changes such as a foreign body giant cell reaction are quite common. developmental cysts in the retrorectal space are susceptible to infection and fistula, and associated malignancy has been reported. therefore, total excision is recommended. the histologic changes associated with acute chemotherapyor radiation-induced colitis are similar. necrosis, ulcer, and inflammation occur usually within weeks of the cessation of therapy. , , epithelium lining the colonic tubules often shows marked enlargement with large atypical nuclei and loss of intracellular mucin (fig. - ). apoptotic bodies similar to those seen with grade gvhd may be prominent. typically, the acute changes subside in to months. , the epithelial atypia seen as a result of acute irradiation and chemotherapy can be alarming and may mimic the appearance of glandular dysplasia and carcinoma. features favoring chemotherapy or radiation effect over dysplasia or carcinoma include ( ) overall preservation of mucosal architecture, ( ) bizarre atypia, ( ) maintenance of a relatively low nuclear-to-cytoplasmic size ratio despite cellular enlargement and nuclear atypia, ( ) a paucity of mitotic figures, ( ) recognition of similar atypia in nearby fibroblast and endothelial cells, and ( ) lack of an infiltration pattern in tumor desmoplasia. the taxanes can cause epithelial changes that can mimic high-grade glandular dysplasia similar to the changes seen with colchicine toxicity. the histologic changes include increased apoptosis, increased mitotic figures, and "ringed" mitotic figures that correlate with metaphase arrest (fig - ). the nuclear stratification and loss of polarity can mimic dysplasia. , late complications of radiation-induced change are better documented than are antineoplastic chemotherapy effects and may occur weeks to years after therapy. these late complications of radiation include chronic colitis, stricture, ulcers, and fistula. the histologic pattern resembles that of ischemic damage with mucosal atrophy and architectural change. mucosal and submucosal blood vessels can become ectatic. fibrosis occurs and often resembles hyalin. fibrosis can affect any bowel layer, including the muscularis externa. atypical radiation-type fibroblasts can persist for years. other vascular changes include intimal fibroplasia, accumulation of foamy macrophages in blood vessel walls, and luminal stenosis. epiploic appendices are pedunculated, serosa-covered accumulations of adipose tissue seen on the lateral aspects of the colon. they can become very large in obese individuals. the pedicles of epiploic appendices are thin and prone to torsion, a feature that can cause infarct and even amputation. infarcted epiploic appendices can appear as fine, graywhite nodules attached to the bowel or even can be found loose within the abdomen at surgery. these appearances can mimic metastatic carcinoma or foreign body. the subsequent biopsy specimen can be confusing to the pathologist who is asked to identify the frozen section. microscopically, these lesions show a central zone of infarct with mummification of the adipose tissue surrounded by a variable amount of fat necrosis and calcification. the outermost portion usually shows fibrosis. pacinian corpuscles occur in many areas of the body. 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different significance acute irradiation in proctitis in man: development of eosinophilic crypt abscesses gastric epithelial atypia associated with hepatic arterial infusion chemotherapy: its distinction from early gastric carcinoma gastrointestinal tract epithelial changes associated with taxanes: marker of drug toxicity versus effect colchicine toxicity: distinct morphologic findings in gastrointestinal biopsies pseudotumoral organization of a twisted epiploic fringe or "hard-boiled egg" in the peritoneal cavity enlarged intra-abdominal pacinian corpuscles simulating tumor implants intra-abdominal pacinian neuroma: a rare lesion in an unusual location intramural extravasation of barium simulating carcinoma of the rectum we wish to acknowledge and thank michele dover for her tenacity and help in transcribing this manuscript. we also thank drs. jeff kneile and amber petrolla for their contributions. key: cord- -f khcjdy authors: lópez, alfonso; martinson, shannon a. title: respiratory system, mediastinum, and pleurae date: - - journal: pathologic basis of veterinary disease doi: . /b - - - - . - sha: doc_id: cord_uid: f khcjdy nan diseases of the respiratory system (respiratory apparatus) are some of the leading causes of morbidity and mortality in animals and a major source of economic losses. thus veterinarians are routinely called to diagnose, treat, and implement health management practices to reduce the impact of these diseases. in companion animals, diseases of the respiratory tract are also common and, although of little economic significance, are important to the health of the animals and thus to clinicians and pet owners. in the past few years, animal shelters have been recognized as a major risk factor for respiratory diseases in dogs and cats, a comparable situation to what is reported in human beings with nosocomial infections. to facilitate the understanding of the structure and function, it is convenient to arbitrarily divide the respiratory system into conducting, transitional, and gas exchange systems ( fig. - ). the conducting system includes nostrils, nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, and extrapulmonary and intrapulmonary bronchi, all of which are largely lined by pseudostratified, ciliated columnar cells, plus a variable proportion of secretory goblet (mucous) and serous cells (figs. - and - and e- fig. - ). the transitional system of the respiratory tract is composed of bronchioles, which are microscopic structures that serve as a transition zone between the conducting system (ciliated) and the gas exchange (alveolar) system (see fig. - ). the disappearance of cilia in the transitional system is not abrupt; the ciliated cells in the proximal bronchiolar region become scarce and progressively attenuated, until the point where distal bronchioles no longer have ciliated cells. normal bronchioles also lack goblet cells but instead have other types of secretory cells, notably club cells (formerly clara cells) and neuroendocrine cells. club cells, also referred to as secretory bronchiolar cells, contain numerous biosynthetic organelles that play an active role in detoxification of xenobiotics (foreign substances), similar to the role of hepatocytes ( fig. - ) . club cells are also critical stem cells in the repair and remodeling of not only the bronchioles but also of most of the respiratory tract. in addition, club cells contribute to the innate immunity of the lung by secreting protective proteins (collectins) and pulmonary surfactant (see b) . in carnivores and monkeys, and to a much lesser extent in horses and human beings, the terminal portions of bronchioles are lined not only by cuboidal epithelium but also by segments of alveolar capillaries. these unique bronchioloalveolar structures are known as respiratory bronchioles ; also see fig. - ). the gas exchange system of the respiratory tract in all mammals is formed by alveolar ducts and millions of alveoli ( fig. - ; also see fig. - ). the surface of the alveoli is lined by two distinct types of epithelial cells known as type i (membranous) pneumonocytes and type ii (granular) pneumonocytes ( fig. - ) . all three-the conducting, transitional, and exchange systems of the respiratory system-are vulnerable to injury because of constant exposure to a myriad of microbes, particles and fibers, and toxic gases and vapors present in the air. vulnerability of the respiratory system to aerogenous (airborne) injury is primarily because of ( ) the extensive area of the alveoli, which are the interface between the blood in alveolar capillaries and inspired air; ( ) the large volume of air passing continuously into the lungs; and ( ) the high concentration of noxious elements that can be present in the air (table - ). for human beings, it has been estimated that the surface of the pulmonary alveoli is approximately m , roughly the area animal and cultured for microbes, yeasts, and fungi, many species of bacteria are recovered, such as mannheimia (pasteurella) haemolytica in cattle; pasteurella multocida in cats, cattle, and pigs; and bordetella bronchiseptica in dogs and pigs. the organisms that constitute the normal flora of the respiratory tract are restricted to the most proximal (rostral) region of the conducting system (nasal cavity, pharynx, and larynx). the thoracic portions of the trachea, bronchi, and lungs are considered to be essentially sterile. the types of bacteria present in the nasal flora vary considerably among animal species and in different geographic regions of the world. some present in the nasal flora are pathogens that can cause important respiratory infections under some circumstances. for instance, mannheimia (pasteurella) haemolytica is part of the bovine nasal flora, yet this bacterium causes a devastating disease in cattle-pneumonic mannheimiosis (shipping fever). experimental studies have established that microorganisms from the nasal flora are continuously carried into the lungs via tracheal air. despite this constant bacterial bombardment from the nasal flora and from contaminated air, normal lungs remain sterile because of their remarkably effective defense mechanisms. the conducting portion of the respiratory system is lined by pseudostratified columnar ciliated epithelium (most of the nasal cavity, paranasal sinuses, part of the larynx, and all of the trachea and bronchi), olfactory epithelium (part of the nasal cavity, particularly ethmoidal conchae), and squamous epithelium (nasal vestibulum and parts of the larynx). the pattern of injury, inflammation, and host response (wound healing) are characteristic for each of these three types of epithelium independent of its anatomic location. pseudostratified ciliated epithelium, which lines most of the nasal cavity and nasopharynx, part of the larynx, and all of the trachea and bronchi, is exquisitely sensitive to injury. when these cells are irreversibly injured, whether caused by viral infection, trauma, or inhalation of toxic gases, the ciliated cells swell, typically lose their attachment to underlying basement membrane, and rapidly exfoliate ( fig. - ) . a transient and mild exudate of fluid, plasma proteins, and neutrophils covers the ulcer. in the absence of of a tennis court. the alveolar surface of the equine lung is estimated to be approximately m . it has also been estimated that the volume of air reaching the human lung every day is approximately l. lungs are also susceptible to blood-borne (hematogenous) microbes, toxins, and emboli. this fact is not surprising because the entire cardiac output of the right ventricle goes into the lungs, and approximately % of the total blood volume is within the pulmonary vasculature. the pulmonary capillary bed is the largest in the body, with a surface area of m in the adult human; this area is equivalent to a length of km of capillaries, with ml of blood occupying up to km of capillary bed. the respiratory system has its own normal flora (microbiota), as does any other body system in contact with the external environment. if a sterile swab is passed deep into the nasal cavity of any healthy (rhinoviruses), infectious bovine rhinotracheitis (bovine herpesvirus ), feline rhinotracheitis (felid herpesvirus ), and viruses of the canine infectious respiratory disease (cird) group such as canine adenovirus (cav- ) and canine parainfluenza virus (cpiv) . if damage to the mucociliary blanket becomes chronic, goblet cell hyperplasia takes place, leading to excessive mucus production (hypersecretion) and reduced mucociliary clearance, and when there is loss of basement membrane, repair is by fibrosis and granulation tissue (scarring). in the most severe cases, prolonged injury causes squamous metaplasia, which together with scarring causes airway obstruction and an impediment to mucociliary clearance. in laboratory rodents, hyperplastic and metaplastic changes, such as those seen in nasal polyps and squamous metaplasia, are considered a prelude to neoplasia. the second type of epithelium lining the conducting system is the sensory olfactory epithelium, present in parts of the nasal mucosa, notably in the ethmoidal conchae. the patterns of degeneration, exfoliation, and inflammation in the olfactory epithelium are similar to those of the ciliated epithelium, except that olfactory complications or secondary bacterial infections, a specific type of progenitor cells known as basal cells or nonciliated secretory cells (preciliated cells) , which are normally present in the mucosa, migrate to cover the denuded basement membrane and undergoes mitosis, eventually differentiating into new ciliated epithelial cells (see fig. - ). cellular migration, proliferation, and attachment are regulated by locally released interleukins (il- β, il- , il- , and il- ), growth factors, integrins and extracellular matrix (ecm) proteins such as collagen, and fibronectin. the capacity of ciliated epithelium to repair itself is remarkably effective. for example, epithelial healing in an uncomplicated ulcer of the tracheal mucosa can be completed in only days. this sequence of cell degeneration, exfoliation, ulceration, mitosis, and repair is typically present in many viral infections in which viruses replicate in nasal, tracheal, and bronchial epithelium, causing extensive mucosal ulceration. examples of transient infections of this type include human colds epithelium. neurons in the olfactory mucosa have the unique ability to regenerate, a fact that is being explored as a potential source of new neurons in the treatment of spinal cord injury. squamous epithelium, located in the vestibular region of the nose (mucocutaneous junction), is the third type of epithelium present in the nasal passages. compared with ciliated and olfactory epithelia, nasal squamous epithelium is quite resistant to all forms of injury. the pharyngeal mucosa, composed of squamous epithelium, has similar patterns of necrosis and inflammation as the oral mucosa (see chapter ). the patterns of necrosis, inflammation, and repair in intrapulmonary bronchi are similar to those previously described for the nasal and tracheal epithelium. in brief, injury to ciliated bronchial epithelium may result in degeneration, detachment, and exfoliation of necrotic cells. under normal circumstances, cellular exfoliation is promptly followed by inflammation, mitosis, cell proliferation, cell differentiation, and finally by repair ( fig. - and see . depending on the type of exudate, bronchitis can be fibrinous, catarrhal, purulent, fibrinonecrotic (diphtheritic), and sometimes granulomatous. when epithelial injury becomes chronic, production of mucus is increased via goblet cell hyperplasia (chronic catarrhal inflammation). this form of chronic bronchitis is well illustrated in habitual smokers who continually need to cough out excessive mucus secretions (sputum). unfortunately, in some cases, excessive mucus cannot be effectively cleared from airways, which of the bronchial wall or cylindrical when destruction involves a large segment of a bronchus. grossly, bronchiectasis is manifested by prominent lumps in the lungs (bosselated appearance or having rounded eminences) resulting from distention of bronchi with exudate, which results in a concurrent obstructive atelectasis of surrounding parenchyma ( fig. - ). the cut surfaces of dilated bronchi are filled with purulent exudates; for this reason, bronchiectasis is often mistaken for pulmonary abscesses. careful inspection, usually requiring microscopic examination, confirms that exudate is contained and surrounded by remnants of a bronchial wall lined by squamous epithelium and not by a pyogenic membrane (connective tissue) as it is in the case of a pulmonary abscess. the squamous metaplasia further interferes with the normal function of the mucociliary escalator. the epithelial lining of the bronchiolar region (transitional zone) is exquisitely susceptible to injury, particularly to that caused by some respiratory viruses (bovine parainfluenza virus , bovine respiratory syncytial virus, adenovirus, or canine distemper virus), oxidant gases (nitrogen dioxide [no ], sulfur dioxide [so ], or ozone [o ]), and toxic substances ( -methylindole or paraquat). the precise explanation as to why bronchiolar epithelium is so prone to injury is still not clear, but it is presumably due in part to ( ) its high vulnerability to oxidants and free radicals; ( ) the presence of leads to chronic obstructive bronchitis and emphysema (see . chronic bronchial irritation causes squamous metaplasia of highly functional but vulnerable ciliated epithelium to nonfunctional, but more resistant, squamous epithelium. squamous metaplasia has a calamitous effect on pulmonary clearance because it causes a structural loss and functional breakdown of portions of the mucociliary escalator. hyperplasia of bronchial glands occurs frequently in chronic bronchitis, which translates to an increase of the reid index (bronchial-gland to bronchial-wall ratio) (e- fig. - ). this index is less than % in the healthy human lung and in the lungs of most domestic species, except for cats, which generally have an index higher than %. the term airway remodeling encompasses all the structural changes that accompany chronic bronchitis such as hypertrophy and hyperplasia of smooth muscle, submucosal glands, and goblet cells; fibrosis; and increased bronchial vascularity. bronchiectasis is one of the most devastating sequelae to chronic remodeling of the bronchi. it consists of a pathologic and permanent dilation of a bronchus with rupture of the bronchial wall as a result of obstruction or chronic inflammation. destruction of walls occurs in part when proteolytic enzymes and oxygen radicals released from phagocytic cells during chronic inflammation degrade and weaken the smooth muscle and cartilage (chondromalacia) that help to maintain normal bronchial diameter ( fig. - ). bronchiectasis may be saccular when destruction affects only a small localized portion . this same type of lesion is seen in viral or mechanical injury to the mucosa of the conducting system. two days after exposure, the basement membrane is lined by rapidly dividing preciliated cells, some of which exhibit mitotic activity (inset). ten days after injury, the nasal epithelium is completely repaired. h&e stain. b, schematic representation of the events of injury and repair in the respiratory mucosa of the conducting system. blue cell, ciliated mucosal epithelial cell; pink cell, goblet cell; red cell, neutrophil. (a from lópez a, prior m, yong s, et al: am j vet res : - , into well-organized, microscopic polyps inside the bronchiolar lumen. the external surface of the exudate eventually becomes covered by ciliated cells. this lesion is referred to as bronchiolitis obliterans, and the polyps may become so large as to cause airflow impairment ( fig. - and see fig. - ). in mild but persistent bronchiolar injury, goblet cells normally absent from bronchioles proliferate from basal cells, resulting in goblet cell metaplasia and causing a profound alteration in the physicochemical properties of bronchiolar secretions ( fig. - ). the normally serous bronchiolar fluid released by club (clara) cells becomes a tenacious material when mucus produced by goblet cells is added. as a result of increased viscoelasticity of the mucus, bronchiolar secretions cannot be removed effectively by ciliary action, leading to plugging and obstruction of distal airways. under such conditions, often grouped as chronic obstructive pulmonary disease, coughing is required to clear mucus from obstructed bronchioles. pulmonary emphysema and atelectasis are further sequelae to bronchiolar metaplasia and mucous hypersecretion blocking or partially blocking the lumens of these bronchioles. these two inflation abnormalities are characteristically present in chronic obstructive pulmonary disease (copd), which is called "recurrent airway obstruction (rao or "heaves") in horses (see recurrent airway obstruction, under disorders of horses). peribronchiolar club (clara) cells rich in mixed function oxidases, which locally generate toxic metabolites (see fig. - ); and ( ) the tendency for pulmonary alveolar macrophages and leukocytes to accumulate in this region of the lungs. depending on the types of injury and inflammatory response, bronchiolitis is classified as necrotizing, suppurative, catarrhal (mucous metaplasia), or granulomatous. once injury to bronchiolar ciliated cells becomes irreversible, the cells degenerate and exfoliate into the bronchiolar lumen, leaving a denuded basement membrane. repair in the bronchiolar region is similar to, but less effective than, that in the tracheal or nasal mucosa. under normal circumstances, recruited phagocytic cells remove exudate and cell debris from the lumina of affected bronchioles, thus preparing the basement membrane to be repopulated with new, undifferentiated cells originating from a rapidly dividing pool of club (clara) cells. after several days, these proliferating cells fully differentiate into normal bronchiolar cells. in severe acute injury, such as that caused by aspiration pneumonia or by highly pathogenic microorganisms, exudate attaches and cannot be removed from the basement membrane of bronchioles. the exudate becomes infiltrated by fibroblasts, which form small nodular masses of fibrovascular tissue that develop postviral bronchiolitis is associated with increased expression of tlrs and unusual susceptibility to inhaled endotoxin. hyperreactive animals typically have an increased number of mast cells, eosinophils, and t lymphocytes in the airway mucosa. clinically, airway hyperresponsiveness is characterized by an exaggerated bronchoconstriction after natural exposure to mild stimuli, such as cold air, or after animals are experimentally exposed to aerosols of histamine or methacholine. because of their extremely delicate structure, alveoli are quite vulnerable to injury once the local defense mechanisms have been overwhelmed. the alveolar wall is a thin membrane formed by a core of interstitium supporting an extensive network of alveolar capillaries. fibroblasts (septal cells), myofibroblasts, collagen, elastic fibers, and few interstitial macrophages and mast cells constitute the alveolar interstitium. the wall of the alveolar capillaries facing the airspace is remarkably thin and has three layers composed of vascular endothelium, basal lamina, and alveolar epithelium. these three layers of the alveolar capillaries constitute what is customarily referred to as the blood-air barrier (see fig. - ). the epithelial side of the alveolus is primarily lined by rather thin type i proliferation of lymphocytes (balt hyperplasia) is also a common microscopic lesion seen in chronic bronchiolitis. airway hyperresponsiveness, or hyperreactive airway disease, is another sequela of bronchiolar injury arising from gene-environment interactions. it develops in human beings and animals (experimentally) after a transient and often innocuous viral infection of the lower respiratory tract or from exposure to certain allergens. experimental work has shown that airway hyperreactivity in club (clara) cell edema. alveolar repair is possible as long as the basement membrane remains intact and lesions are not complicated by further injury or infection. within days, cuboidal type ii (granular) pneumonocytes, which are the precursor cells and more resistant to injury, undergo mitosis and provide a large pool of new undifferentiated cells . these new cells repave the denuded alveolar basement membrane and finally differentiate into type i pneumonocytes. when alveolar injury is diffuse, proliferation pneumonocytes, which are arranged as a very delicate continuous membrane extending along the alveolar surface (see fig. - ). type i pneumonocytes are particularly susceptible to noxious agents that reach the alveolar region either aerogenously or hematogenously. injury to type i pneumonocytes rapidly causes swelling and vacuolation of these cells . when cellular damage has become irreversible, type i cells detach, resulting in denudation of the basement membrane, increased alveolar permeability, and alveolar figure - hyperplasia of type ii pneumonocytes. a, acute alveolar injury, crude oil aspiration, cow. note proliferation of cuboidal epithelial cells (type ii pneumonocytes) (arrows) along the luminal surface of the alveolar wall. during alveolar repair, type ii pneumonocytes are the precursor cell for necrotic and lost type i pneumonocytes. h&e stain. b, chronic alveolar injury, interstitial pneumonia, horse. note entire alveolar membrane lined with cuboidal type ii pneumonocytes (arrowheads). the alveolar interstitium is expanded with inflammatory cells, and the alveolar lumens contain cell debris mixed with leukocytes. h&e stain. ( ( ). necrosis of these cells leads to transient alveolar edema (area that is pink) ( ), which is followed by hyperplasia of type ii pneumonocytes ( ), stem cells that differentiate ( ) into type i pneumonocytes as part of alveolar repair and healing ( ). (courtesy dr. a. lópez, atlantic veterinary college.) more information on postmortem examination of the lung can be found at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. microbes, toxins, and pneumotoxicants can gain access into the respiratory system by the following routes (table - ; also see table - ): aerogenous, hematogenous, direct extension, and by local production of free radicals and toxic metabolites. pathogens, such as bacteria, mycoplasmas, and viruses, along with toxic gases and foreign particles, including food, can gain access to the respiratory system via inspired air. this is the most common route in the transmission of most respiratory infections in domestic animals. some viruses, bacteria, parasites, and toxins can enter the respiratory system via the circulating blood. this portal of entry is commonly seen in septicemias, bacteremias, and with protozoa and viruses that target endothelial cells. also, circulating leukocytes may release infectious organisms such as retroviruses and listeria monocytogenes while traveling through the lungs. of type ii pneumonocytes becomes so spectacular that the microscopic appearance of the alveolus resembles that of a gland or fetal lung; this lesion has been termed epithelialization or fetalization. although it is part of the normal alveolar repair, hyperplasia of type ii pneumonocytes can interfere in gas exchange and cause hypoxemia. in uncomplicated cases, type ii pneumonocytes eventually differentiate into type i pneumonocytes, thus completing the last stage of alveolar repair (see fig. - ). in some forms of chronic interstitial lung injury, the surface of the alveolar basement membrane could become populated with migrating bronchiolar cells, a process known as alveolar bronchiolization or lambertosis. in severe cases, lambertosis, a metaplastic change, can be mistaken microscopically with alveolar adenomas. type i pneumonocytes are one of the three structural components of the blood-air barrier, so when these epithelial cells are damaged, there is an increase in alveolar capillary permeability and transient leakage of plasma fluid, proteins, and fibrin into the alveolar lumen (see fig. - ) . under normal circumstances, these fluids are rapidly cleared from the alveolus by alveolar and lymphatic absorption, and necrotic pneumonocytes (type i) and fibrin strands are phagocytosed and removed by pulmonary alveolar macrophages. when there is persistent and severe injury, fibroblasts and myofibroblasts may proliferate in the alveolar walls (alveolar interstitium), causing alveolar septal fibrosis, whereas in other forms of severe injury, fibroblasts and myofibroblasts actively migrate from the interstitium into the alveolar spaces, causing intraalveolar fibrosis. these two types of alveolar fibrosis are most commonly seen in toxic and allergic pulmonary diseases and have a devastating effect on lung function. endothelial cells are also major players in the normal and abnormal physiology of the alveolus (see . these cells trap and share circulating antigens with intravascular and interstitial macrophages. the junction between alveolar endothelial cells is not as tight as that of the type i pneumonocytes, allowing some movement of fluid and small-size molecular weight proteins into the alveolar interstitium. endothelial cells maintain an intimate cell contact with erythrocytes and leukocytes passing through the lung, since the lumen of alveolar capillaries is slightly smaller ( . µm) than the diameter of red and white blood cells. erythrocytes are easily deformable, so their transit time through the alveolar capillaries is shorter than that of leukocytes, which are less deformable cells. this longer transit time of leukocytes and their close cellular contact with alveolar endothelial cells have major impacts in lung inflammation and acute respiratory distress syndrome (ards). on a minute-to-minute basis, the pulmonary defense mechanisms deal effectively with noxious stimuli and mild tissue injury without the need for an inflammatory response. however, if normal defense mechanisms are ineffective or insufficient (overwhelmed), the inflammatory process is rapidly turned on as a second line of defense. postmortem examination of the respiratory tract should always be conducted in a thorough and systematic manner and include the conducting system (trachea, bronchi, and bronchioles), the lungs, and the thoracic cavity and pleura. detailed record keeping and photographic documentation are essential elements of a thorough examination. normal lungs typically have a homogeneous pink color and are slightly deflated from loss of negative intrathoracic pressure. the e-sections that follow describe a systematic approach to this process. .e chapter respiratory system, mediastinum, and pleurae the respiratory tract should always be examined in a systematic manner. to determine whether negative pressure is present in the thoracic cavity, the diaphragm is punctured through the abdominal cavity before the thoracic cavity has been opened. when the diaphragm is punctured in a fresh carcass, the loss of negative pressure in the thorax causes the diaphragmatic cupola to drop back caudally toward the abdominal cavity, and at the same time, there is an audible sound caused by the inrush of air into the thorax. lack of this movement may be an indication of advanced pneumothorax, pleural effusion, or the presence of uncollapsed lungs caused by pulmonary edema, pneumonia, fibrosis, or emphysema. in carcasses that have been dead for a long time, pulmonary air and gas produced by saprophytic bacteria leak into the pleural cavity, reducing the negative thoracic pressure and collapsing the lung. the rib cage must be removed by cutting along the costosternal joints and along the neck of the ribs (close to the costovertebral joints) in such a way that pleural adhesions and abnormal thoracic contents can be observed and grossly quantified (e.g., ml of clear, yellow fluid). the tongue, pharynx, esophagus, larynx, trachea, and thoracic viscera (lungs, heart, and thymus) should be removed as a unit (often called the pluck) and placed on the necropsy table. the pharynx and esophagus are opened starting at the pharynx by a single cut with scissors along the dorsal midline and are inspected for ulcers, foreign bodies, and neoplasms. the larynx and trachea must be examined by opening both along the dorsal midline from cranial to caudal ends and then extending the incision into the large bronchi of the caudal lung lobes. normal tracheobronchial mucosa has a smooth and glistening pearl-colored surface with empty lumina in airways. the presence of foamy fluid in airways indicates pulmonary edema. feed particles may suggest aspiration; however, careful examination of the mucosa is required because aspiration of ingesta from stomach or rumen into the lungs commonly takes place agonally or can be displaced into these areas when the carcass is moved. the lungs should be examined before incision. normal lungs typically have a homogeneous pink color (see fig. - ). external changes include the presence of rib imprints on the pleural surface when lungs fail to collapse. in addition, the lungs should be inspected for changes in color and texture and distribution of lesions. color changes can be various shades of red, indicating hypostatic congestion, hyperemia (acute pneumonia), and hemorrhage; dark blue collapsed lobules or areas are indicative of atelectasis; pale pink to white lungs indicate notable anemia, fibrosis, or emphysema; and uniformly or patchy yellow-brown lungs indicate chronic passive congestion and pulmonary fibrosis likely secondary to chronic heart failure. lungs from exsanguinated animals are generally paler than the normal pink color because of reduced blood in the pulmonary tissue. lungs with postmortem autolysis show green discoloration, a change that is also seen in other organs (e- fig. - ). a covering of yellowish material on the pleural surface indicates accumulation of fibrin. because it is impossible to describe the texture of normal lungs, experience in palpation is required to appreciate the actual texture of a normal lung. texture is determined by gently palpating the surface and parenchyma of the lungs. normal texture can change to firm, hard, elastic (rubbery), or crepitus (with a crackling sound or feeling). for a detailed description of lung texture, see the section on classification of pneumonias in domestic animals. palpation of the lungs, which should be gentle, also permits detection of nonvisible nodules or abscesses in the parenchyma. knowing the distribution of a lesion in the lungs also facilitates diagnosis because particular etiologic agents cause lesions with specific distribution. distribution of lesions is generally described as focal, multifocal, locally extensive, or diffuse. according to their topography, pulmo-nary lesions can also be classified as cranioventral, dorsocaudal, and so on. necropsy reports must also contain an estimate of the extent of the pulmonary lesions, preferably expressed as a percentage of the volume of the lungs affected. for instance, a report may read "cranioventral consolidation involving % of the lungs." if the lungs have focal lesions, a rough estimate of the number should also be included in the report. for instance, "numerous (approximately ), small ( to cm in diameter), hard nodules were randomly distributed in all lung lobes." two methods are used to examine the nasal structures. the first is making a midsagittal cut through the head and removing the nasal septum; the second is making several transverse sections of the nose at the level of the second premolar teeth. this latter method is preferred when examining pigs suspected of having atrophic rhinitis or animals suspected of having nasal neoplasms. microscopic examination of pulmonary tissue is routinely done in diagnostic laboratories. samples of normal and abnormal lungs, along with other appropriate tissue, should always be submitted in % buffered-neutral formalin for histopathologic evaluation. a minimum of four lung samples (left cranial, left caudal, right cranial, and right caudal) should be taken for histopathologic examination in animals with a history of respiratory signs. to improve fixation, a paper towel can be placed over the samples of lung floating in fixative. when detailed evaluation of the alveolar walls is required, lungs can be fixed by a gentle intratracheal injection of fixative; however, this technique displaces transudates and exudates and can artificially cause distention of the perivascular and peribronchial spaces. lung biopsy specimens are taken only sporadically because complications often outweigh the diagnostic value. however, the use of new techniques, such as endoscopic-directed biopsies, has notably reduced some of these complications. biopsies of the lungs are recommended in cases of chronic persistent pulmonary disease unresponsive to treatment or intrathoracic masses of undetermined origin. endoscopic-directed biopsies of the nasal and bronchial mucosa are routinely used in clinical practice and generally have a much better diagnostic value. two valuable diagnostic tools in human medicine, bronchoalveolar lavage (bal) and transtracheal wash (ttw), have in recent years become more widely used in veterinary clinical diagnosis of respiratory ailments, particularly in horses, dogs, and cats. the basis of bal and ttw is sampling the lung or trachea of a living animal by infusing sterile fluid into the trachea or deep lung (respectively) and retrieving it to determine the cellular and biochemical composition of this fluid. in other words, the composition of the fluid reflects what is present in the bronchioloalveolar spaces and trachea. these procedures are performed by inserting a tube directly through the larynx into the trachea or bronchus, or transtracheally by inserting a tube through a needle percutaneously into the cervical trachea. microscopic examination of properly collected, stored, and processed samples may reveal many erythrocytes and siderophages in pulmonary hemorrhage or left-sided heart failure; inclusion bodies or syncytial cells in viral pneumonias; increased number of leukocytes in pulmonary inflammation; abundant mucus in asthma or equine recurrent airway obstruction (rao); the presence of pulmonary pathogens, such as parasites, fungi, and bacteria; or tumor cells in cases of pulmonary neoplasia. in the healthy animal, % to % of the bal cells are pulmonary alveolar macrophages (see fig. - , a) . clearance, or a combination of both is the underlying pathogenetic mechanism in many pulmonary diseases ( fig. - ) . the anatomic configuration of the nasal cavity and bronchi plays a unique role in preventing or reducing the penetration of noxious material into the lungs, especially into the alveoli, which is the most vulnerable portion of the respiratory system. the narrow nasal meatuses and the coiled arrangement of the nasal conchae generate enormous turbulence of airflow, and as a result, physical forces are created that forcefully impact particles larger than µm onto the surface of the nasal mucosa ( fig. - ). although particles smaller than µm could escape trapping in the nasal cavity, these mediumsized particles meet a second barrier at the tracheal and bronchial in some instances, pathogenic organisms can also reach the pleura and lungs through penetrating injuries, such as gunshot wounds, migrating awns, or bites, or by direct extension from a ruptured esophagus or perforated diaphragm. the lungs, particularly the bronchioles and alveoli, are vulnerable to endogenous injury caused by the local generation of free radicals during inflammation or by toxic metabolites generated by club (clara) cells (see fig. - , b). inflammatory processes in the respiratory system, particularly those caused by infectious organisms, can spread to contiguous or distant tissues. for instance, rhinitis may spread into the sinuses causing rhinosinusitis. similarly, laryngeal inflammation may spread into the lungs when exudate in the larynx is aspirated. lung disease can have profound systemic effects when cytokines, produced locally during necrosis or inflammation, are released into circulation. as a result of the enormous vascular bed present in the lung, sepsis and septic shock often develop when proinflammatory molecules overwhelm the antiinflammatory response during the so-called "cytokine storm." it is axiomatic that a particle, microbe, or toxic gas must first gain entry to a vulnerable region of the respiratory system before it can induce an adaptive immune response or have a pathologic effect. the characteristics of size, shape, dispersal, and deposition of particles present in inspired air are studied in aerobiology. it is important to recognize the difference between deposition, clearance, and retention of inhaled particles. deposition is the process by which particles of various sizes and shapes are trapped within specific regions of the respiratory tract. clearance is the process by which deposited particles are destroyed, neutralized, or removed from the mucosal surfaces. the difference between what is deposited and what is cleared from the respiratory tract is referred to as retention. the main mechanisms involved in clearance are sneezing, coughing, mucociliary transport, and phagocytosis (table - ). abnormal retention of particles resulting from increased deposition, decreased same rate in all levels of a conducting system, a "bottleneck" effect would be created in major airways as the minor but more numerous airways enter the bronchi. for this reason, the mucociliary transport in proximal (rostral) airways is physiologically faster than that of the distal (caudal) ones. ciliary activity and mucus transport increase notably in response to stimuli such as in respiratory infections. the mucociliary blanket of the nasal cavity, trachea, and bronchi also plays an important role in preventing injury from toxic gases. if a soluble gas contacts the mucociliary blanket, it mixes with the mucus, thus reducing the concentration of gas reaching deep into the alveoli. in other words, mucus acts as a "scavenger system," whereby gases are solubilized and subsequently cleared from the respiratory tract via mucociliary transport. if ciliary transport is reduced (loss of cilia) or mucus production is excessive, coughing becomes an important mechanism for clearing the airways. in addition to the mechanical barrier and physical transport provided by the mucociliary escalator, other cells closely associated with ciliated epithelium contribute to the defense mechanism of the conducting and transitional systems. among the most notable are the microfold (m) cells, which are modified epithelial cells covering the bronchial-associated lymphoid tissue (balt), both of which are strategically situated at the corner of the bifurcation of bronchi and bronchioles, where inhaled particles often collide with the mucosa because of inertial forces. from here, inhaled particles and soluble antigens are phagocytosed and transported by macrophages, dendritic cells, and other professional antigen-presenting cells (apcs) into the balt, thus providing a unique opportunity for b and t lymphocytes to enter into close contact with inhaled pathogenic substances. pulmonary lymphocytes are not quiescent in the balt but are in continual traffic to other organs and contribute to both cellular (cytotoxic, helper, and suppressor t lymphocytes) and humoral immune responses. immunoglobulin a (iga), produced by mucosal plasma cells, and, to a lesser extent, immunoglobulin g (igg) and m (igm) play important roles in the local immunity of the conducting and transitional systems, especially with regard to preventing attachment of pathogens to the cilia. chronic airway diseases, especially those caused by infectious agents such as mycoplasmas or retroviruses, are often accompanied by severe hyperplasia of the balt. the mucociliary clearance terminates at the pharynx, where mucus, propelled caudally from the nasal cavity and cranially from the tracheobronchial tree, is eventually swallowed and thus eliminated from the conducting system of the respiratory tract. some respiratory pathogens, such as rhodococcus equi, can infect the intestines after having been removed and swallowed from the respiratory tract into the alimentary system. alveoli lack ciliated and mucus-producing cells; thus the defense mechanism against inhaled particles in the alveolar region cannot be provided by mucociliary clearance. instead, the main defense mechanisms of alveoli (exchange system) are phagocytosis provided by the pulmonary alveolar macrophages and antimicrobial molecules of the alveolar lining fluid ( fig. - ). pulmonary alveolar macrophages are highly phagocytic cells, which are not to be confused with pulmonary intravascular macrophages, and are derived largely from blood monocytes and, to a much lesser extent, from a slowly dividing population of interstitial macrophages. after a temporary adaptive stage within alveolar interstitium, blood monocytes reduce their glycolytic metabolism and increase their oxidative metabolism to function in an aerobic rather than an anaerobic environment. pulmonary alveolar macrophages contribute to the bifurcations. abrupt changes in the direction of air (inertia), which occurs at the branching of major airways, cause particles in the -to -µm size range to collide with the surface of bronchial mucosa (see fig. - ). because the velocity of inspired air at the level of the small bronchi and bronchioles has become rather slow, inertial and centrifugal forces no longer play a significant role in the trapping of inhaled particles. here, in the transitional (bronchiolar) and exchange (alveolar) regions, particles µm or smaller may come into contact with the mucosa by means of sedimentation because of gravitation or by diffusion as a result of brownian movement. infective aerosols containing bacteria and viruses are within the size range ( . to µm) that can gain access to the bronchiolar and alveolar regions. in addition to size, other factors, such as shape, length, electrical charge, and humidity, play an important role in mucosal deposition, retention, and pathogenicity of inhaled particles. for example, particles longer than µm may also reach the lower respiratory tract provided their mean aerodynamic diameter is less than µm. asbestos is a good example of a large but slender fiber that can bypass the filtrating mechanisms by traveling parallel to the airstream. once in the terminal bronchioles and alveoli, asbestos fibers cause asbestosis, a serious pulmonary disease in human beings. in summary, the anatomic features of the nasal cavity and airways provide an effective barrier, preventing the penetration of most large particles into the lungs. once larger particles are trapped in the mucosa of conducting airways and small particles are deposited on the surface of the nasal, tracheal, or bronchoalveolar mucosa, it is crucial that these exogenous materials be promptly removed to prevent or minimize injury to the respiratory system. for these purposes, the respiratory system is equipped with several defense mechanisms, all of which are provided by specialized cells operating in a remarkably well-coordinated manner. conducting system (nose, trachea, and bronchi) mucociliary clearance is the physical unidirectional movement and removal of deposited particles and gases dissolved in the mucus from the respiratory tract. mucociliary clearance, also referred to as the waste disposal system, is provided by the mucociliary blanket (mucociliary escalator) and is the main defense mechanism of the conducting system (nasal cavity, trachea, and bronchi) (see figs. - and - ). mucus acts primarily as a barrier and a vehicle, and it is a complex mixture of water, glycoproteins, immunoglobulins, lipids, and electrolytes. these substances are produced by goblet (mucous) cells, serous cells, submucosal glands, and fluid from transepithelial ion and water transport. once serous fluid and mucus are secreted onto the surface of the respiratory mucosa, a thin, double-layer film of mucus is formed on top of the cells. the outer layer of this film is in a viscous gel phase, whereas the inner layer, which is in a fluid or sol phase, is directly in contact with cilia (see fig. - and see e- fig. - ). the respiratory system of a healthy human produces approximately ml of mucus per day. each ciliated cell in the conducting system has approximately to motile and chemosensory cilia ( µm long), beating metachronously (forming a wave) at a ciliary beat frequency of approximately strokes per minute, and in a horse, for example, mucus moves longitudinally at a rate of up to mm per minute. rapid and powerful movement of cilia creates a series of waves that, in a continuous and synchronized manner, propel the mucus, exfoliated cells, and entrapped particles out of the respiratory tract to the pharynx. the mucus is finally swallowed or, when present in large amounts, is coughed up out of the conducting system. if mucus flow were to move at the activated alveolar macrophages. similarly, inhaled particles, such as dust, pollen, spores, carbon, or erythrocytes from intraalveolar hemorrhage, are all phagocytosed and eventually removed from alveoli by pulmonary alveolar macrophages. most alveolar macrophages leave the alveoli by migrating toward the bronchiolar (transitional) region until the mucociliary blanket is reached. once there, pulmonary macrophages are removed in the same way as any other particle: along the mucociliary flow to the pharynx and swallowed. in the cat, as many as million macrophages per hour move out from the alveoli into the conducting system and pharynx. destruction and removal of inhaled microbes and particles by alveolar macrophages is a well-orchestrated mechanism that engages many cells, receptors (i.e., toll-like receptors [tlrs]), and pulmonary secretions in the lung. the cell-to-cell interactions are complex and involve pulmonary alveolar macrophages, pneumonocytes, endothelial cells, lymphocytes, plasma cells, natural killer (nk) cells, and dendritic cells. antibodies are also important in the protection (acquired immune response) of the respiratory tract against inhaled pathogens. iga is the most abundant antibody in the nasal and tracheal secretions and prevents the attachment and absorption of antigens (immune exclusion). igg and, to a lesser extent, ige and igm promote the uptake and destruction of inhaled pathogens by phagocytic cells (immune elimination). igg is the most abundant antibody in the alveolar surface and acts primarily as an opsonizing antibody for alveolar macrophages and neutrophils. in addition to antibodies, there are several secretory molecules locally released into the alveoli that constitute the alveolar lining material and contribute to the pulmonary defense mechanisms. the most important of these antimicrobial products are transferrin, anionic peptides, and pulmonary surfactant (table - ). to facilitate phagocytosis and discriminate between "self" and "foreign" antigens, pulmonary alveolar macrophages are furnished with a wide variety of specific receptors on their cell surfaces. among the most important ones are fc receptors for antibodies; complement receptors (for c b, c a, and c a); tumor necrosis factor (tnf) receptor; and cd receptors, which facilitate phagocytosis and destruction of opsonized particles. toll-like receptors (tlrs) recognize microbial components, and apoptosis stimulating fragment (fas) receptors are involved in apoptosis and in the phagocytosis of apoptotic cells in the lung. "scavenger receptors," which are responsible for the recognition and uptake of foreign particulates, such as dust and fibers, are also present on pulmonary alveolar macrophages. lungs are also susceptible to hematogenously borne microbes, toxins, or emboli. the hepatic (kupffer cells) and splenic macrophages are the primary phagocytic cells responsible for removing circulating bacteria and other particles from the blood of dogs, some rodents, and human beings. in contrast, the cell responsible for the removal of circulating particles, bacteria, and endotoxin from the blood of ruminants, cats, pigs, and horses is mainly the pulmonary intravascular macrophage, a distinct population of phagocytes normally residing within the pulmonary capillaries (see fig. - ). in pigs, % of the pulmonary capillary surface is lined by pulmonary intravascular macrophages. in ruminants, % of intravenously injected tracer particles or bacteria are rapidly phagocytosed by these intravascular macrophages. studies have shown that an abnormally reduced number of kupffer cells in diseased liver results in a compensatory increase in pulmonary intravascular macrophages, even in animal species in which these phagocytic cells are normally absent from the lung. in some abnormal conditions, such as sepsis, pulmonary innate and adaptive immune response by rapidly attaching and phagocytosing bacteria and any other particles reaching the alveolar lumens. the number of free macrophages in the alveolar space is closely related to the number of inhaled particles reaching the lungs. this ability to increase, within hours, the number of available phagocytic cells is vital in protecting the distal lungs against foreign material, particularly when the inhaled particle load is high. unlike that of tissue macrophages, the life span of alveolar macrophages in the alveoli is notably short, only a few days, and thus they are continuously being replaced by newly migrated blood monocytes. alveolar phagocytosis plays a prominent role in the innate defense mechanism against inhaled bacteria without the need of an inflammatory reaction. bacteria reaching the alveoli are rapidly phagocytosed, and bactericidal enzymes present in lysosomes are discharged into the phagosome containing the bacteria (see b) . except for some facultative pathogens that are resistant to intracellular killing (e.g., mycobacterium tuberculosis, listeria monocytogenes, brucella abortus, rhodococcus equi, and some salmonella spp.), most bacteria reaching the lungs are rapidly destroyed by (ros) not only induce extensive pulmonary injury but also impair the defense and repair mechanisms in the lung. oxygen and free radical scavengers, such as catalase, superoxide dismutase, ubiquinone, and vitamins e and c, are largely responsible for protecting pulmonary cells against peroxidation. these scavengers are present in alveolar and bronchiolar epithelial cells and in the extracellular spaces of the pulmonary interstitium. in summary, the defense mechanisms are so effective in trapping, destroying, and removing bacteria that, under normal conditions, animals can be exposed to aerosols containing massive numbers of bacteria without any ill effects. if defense mechanisms are impaired, inhaled bacteria colonize and multiply in bronchi, bronchioles, and alveoli, and they produce infection, which can result in fatal pneumonia. similarly, when blood-borne pathogens, inhaled toxicants, or free radicals overwhelm the protective defense mechanisms, cells of the respiratory system are likely to be injured, often causing serious respiratory diseases. for many years, factors such as viral infections, toxic gases, stress, and pulmonary edema have been implicated in predisposing human beings and animals to secondary bacterial pneumonia. there are many pathways by which the defense mechanisms can be impaired; only those relevant to veterinary species are discussed. viral agents are notorious in predisposing human beings and animals to secondary bacterial pneumonias by what is known as viral-bacterial synergism. a good example of the synergistic effect of combined virus-bacterial infections is documented from epidemics of human beings with influenza virus in which the mortality rate has been significantly increased from secondary bacterial pneumonia. the most common viruses incriminated in predisposing animals to secondary bacterial pneumonia include influenza virus in pigs and horses; bovine herpesvirus (bohv- ), bovine parainfluenza virus (bpiv- ), and bovine respiratory syncytial virus (brsv) in cattle; canine distemper virus (cdv) in dogs; and felid herpesvirus (fehv- ) and feline calicivirus (fcv) in cats. the mechanism of the synergistic effect of viral-bacterial infections was previously believed to be the destruction of the mucociliary blanket and a concurrent reduction of mucociliary clearance, but in experimental studies, viral infections did not significantly reduce the physical removal of particles or bacteria out of the lungs. now, it is known that to days after a viral infection, the phagocytic function of pulmonary alveolar macrophages and, to a lesser extent, the mucociliary clearance are notably impaired (see fig. - ). other mechanisms by which viruses impair defense mechanisms are multiple and remain poorly understood (box - ). immunization against viral infections in many cases prevents or reduces the synergistic effect of viruses and thus the incidence of secondary bacterial pneumonia. certain gases also impair respiratory defense mechanisms, rendering animals more susceptible to secondary bacterial infections. for instance, hydrogen sulfide and ammonia, frequently encountered on farms, especially in buildings with poor ventilation, can impair pulmonary defense mechanisms and increase susceptibility to bacterial pneumonia. the effects of environmental pollutants on the defense mechanisms of human beings and animals living in crowded and polluted cities remain to be determined. excessive release of cytokines by pulmonary intravascular macrophages may result in acute lung injury. existing in an oxygen-rich environment and being the site of numerous metabolic reactions, the lungs also require an efficient defense mechanism against oxidant-induced cellular damage (oxidative stress). this form of damage is caused by inhaled oxidant gases (e.g., nitrogen dioxide, ozone, sulfur dioxide, or tobacco smoke), by xenobiotic toxic metabolites produced locally, by toxins reaching the lungs via the bloodstream (e.g., -methylindole and paraquat), or by free radicals (reactive oxygen species) released by phagocytic cells during inflammation. free radicals and reactive oxygen species anomalies localized congenital anomalies of the nasal cavity are rare in domestic animals and are often merely part of a more extensive craniofacial deformity (e.g., cyclops) or a component of generalized malformation (e.g., chondrodysplasia). congenital anomalies involving the nasal cavity and sinuses, such as choanal atresia (lack of communication between the nasal cavity and pharynx), some types of chondrodysplasia, and osteopetrosis, are incompatible with life. examples of nonfatal congenital anomalies include cystic nasal conchae, deviation of the nasal septum, cleft upper lip (harelip and cheiloschisis), hypoplastic turbinates, and cleft palate (palatoschisis) (see fig. - ). bronchoaspiration and aspiration pneumonia are common sequelae to cleft palate. nasal and paranasal sinus cysts are slowly growing and expansive lesions that mimic neoplasia and cause severe cranial deformation in horses. as in other organs or systems, it is extremely difficult to determine the actual cause (genetic vs. congenital) of anomalies based on pathologic evaluation. metabolic disturbances affecting the nasal cavity and sinuses are rare in domestic animals. immunodeficiency disorders, whether acquired or congenital, are often associated with increased susceptibility to viral, bacterial, and protozoal pneumonias. for example, human beings with acquired immunodeficiency syndrome (aids) are notably susceptible to pneumonia caused by proliferation of pneumocystis (carinii) jirovecii. a similar ubiquitous organism, which under normal circumstances is not pathogenic, is also found in the pneumonic lungs of immunosuppressed pigs, foals, dogs, and rodents. pigs infected with the porcine reproductive and respiratory syndrome (prrs) virus frequently develop pneumocystis carinii infection ( fig. - ) . arabian foals born with combined immunodeficiency disease easily succumb to infectious diseases, particularly adenoviral pneumonia. combined infections with two respiratory viruses, such as canine distemper virus (cdv) and canine adenovirus (cav- ), are sporadically reported in immunosuppressed puppies. also, large doses of chemotherapeutic agents, such as steroids and alkylating agents, cause immunosuppression in dogs, cats, and other animals, increasing susceptibility to secondary viral and bacterial infections. stress, uremia, endotoxemia, dehydration, starvation, hypoxia, acidosis, pulmonary edema, anesthesia, and ciliary dyskinesia are only some of the many conditions that have been implicated in impairing respiratory defense mechanisms and consequently predisposing animals to develop secondary bacterial pneumonia. the mechanisms by which each of these factors suppresses pulmonary defenses are diverse and sometimes not well understood. for example, hypoxia and pulmonary edema decrease phagocytic function of pulmonary alveolar macrophages and alter the production of surfactant (abnormal head tilt and abnormal gait), which in severe cases may lead to emaciation. based on the nature of exudate, rhinitis can be classified as serous, fibrinous, catarrhal, purulent, or granulomatous. these types of inflammatory reactions can progress from one to another in the course of the disease (i.e., serous to catarrhal to purulent), or in some instances exudates can be mixed, such as those seen in mucopurulent, fibrinohemorrhagic, or pyogranulomatous rhinitis. microscopic examination of impression smears or nasal biopsy, and bacterial or fungal cultures are generally required in establishing the cause of inflammation. common sequelae of rhinitis are hemorrhage, ulcers, and, in some cases, nasopharyngeal polyps (hyperplasia) arising from inflamed mucosa. rhinitis also can be classified according to the age of the lesions as acute, subacute, or chronic; to the severity of the insult as mild, moderate, or severe; and to the etiologic agent as viral, allergic, bacterial, mycotic, parasitic, traumatic, or toxic. serous rhinitis. serous rhinitis is the mildest form of inflammation and is characterized by hyperemia and increased production of a clear fluid locally manufactured by serous glands present in the nasal submucosa. serous rhinitis is of clinical interest only. it is caused by mild irritants or cold air, and it occurs during the early stages of viral infections, such as the common cold in human beings, upper respiratory tract infections in animals, or in mild allergic reactions. catarrhal rhinitis. catarrhal rhinitis is a slightly more severe process and has, in addition to serous secretions, a substantial increase in mucus production by hypersecretion of goblet cells and mucous glands. a mucous exudate is a thick, translucent, or slightly turbid viscous fluid, sometimes containing a few exfoliated cells, leukocytes, and cellular debris. in chronic cases, catarrhal rhinitis is characterized microscopically by notable hyperplasia of goblet cells. as the inflammation becomes more severe, the mucus is infiltrated with neutrophils, giving the exudate a cloudy appearance. this exudate is referred to as mucopurulent. purulent (suppurative) rhinitis. purulent (suppurative) rhinitis is characterized by a neutrophilic exudate, which occurs when the nasal mucosa suffers a more severe injury that generally is accompanied by mucosal necrosis and secondary bacterial infection. cytokines, leukotrienes, complement activation, and bacterial products cause exudation of leukocytes, especially neutrophils, which mix with nasal secretions, including mucus. grossly, the exudate in suppurative rhinitis is thick and opaque, but it can vary from white to green to brown, depending on the types of bacteria and type of leukocytes (neutrophils or eosinophils) present in the exudate . in severe cases, the nasal passages are completely blocked by the exudate. microscopically, neutrophils can be seen in the submucosa and mucosa and form plaques of exudate on the mucosal surface. neutrophils are commonly found marginated in vessels, in the lamina propria, and in between epithelial cells in their migration to the surface of the mucosa. fibrinous rhinitis. fibrinous rhinitis is a reaction that occurs when nasal injury causes a severe increase in vascular permeability, resulting in abundant exudation of plasma fibrinogen, which coagulates into fibrin. grossly, fibrin appears as a yellow, tan, or gray rubbery mat on nasal mucosa. fibrin accumulates on the surface and forms a distinct film of exudate sometimes referred to as pseudomembrane ( fig. - ). if this fibrinous exudate can be removed, leaving an intact underlying mucosa, it is termed a croupous or pseudodiphtheritic rhinitis. conversely, if the pseudomembrane is difficult to remove and leaves an ulcerated mucosa, it is referred to as diphtheritic or fibrinonecrotic rhinitis. the term diphtheritic was derived from human diphtheria, which causes a severe and destructive inflammatory process of the nasal, tonsillar, pharyngeal, and laryngeal mucosa. nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses and human beings (see nasal amyloidosis, in disorders of horses). congestion and hyperemia. the nasal mucosa is well vascularized and is capable of rather dramatic variation in blood flow, whether passively as a result of interference with venous return (congestion) or actively because of vasodilation (hyperemia). congestion of the mucosal vessels is a nonspecific lesion commonly found at necropsy and presumably associated with the circulatory failure preceding death (e.g., heart failure, bloat in ruminants in which the increased intraabdominal pressure causes increased intrathoracic pressure impeding the venous return from the head and neck). hyperemia of the nasal mucosa is seen in early stages of inflammation, whether caused by irritation (e.g., ammonia and regurgitated feed), viral infections, secondary bacterial infections, toxemia, allergy, or trauma. hemorrhage. epistaxis is the clinical term used to denote blood flow from the nose (nosebleed) regardless of whether the blood originates from the nasal mucosa or from deep in the lungs, such as in horses with "exercise-induced pulmonary hemorrhage." unlike blood in the digestive tract, where the approximate anatomic location of the bleeding can be estimated by the color the blood imparts to fecal material, blood in the respiratory tract is always red. this fact is due to the rapid transport of blood out of the respiratory tract by the mucociliary blanket and during breathing. hemorrhages into the nasal cavity can be the result of local trauma, can originate from erosions of submucosal vessels by inflammation (e.g., guttural pouch mycosis), or can be caused by neoplasms. hemoptysis refers to the presence of blood in sputum or saliva (coughing or spitting blood) and is most commonly the result of pneumonia, lung abscesses, ulcerative bronchitis, pulmonary thromboembolisms or hemorrhage, and pulmonary neoplasia. inflammation of the nasal mucosa is called rhinitis, and inflammation of the sinuses is called sinusitis. these conditions usually occur together, although mild sinusitis can be undetected. clinically, rhinosinusitis is characterized by nasal discharge. rhinitis. the occurrence of infectious rhinitis presupposes an upset in the balance of the normal microbial flora of the nasal cavity. innocuous bacteria present normally protect the host through a process called competitive exclusion, whereby potential pathogens are kept at a harmless level. disruption of this protective mechanism can be caused by respiratory viruses, pathogenic bacteria, fungi, irritant gases, environmental changes, immunosuppression, local trauma, stress, or prolonged antibacterial therapy. inflammatory processes in the nasal cavity are not life-threatening and usually resolve completely. however, some adverse sequelae in cases of infectious rhinitis include bronchoaspiration of exudate leading to bronchopneumonia. chronic rhinitis often leads to destruction of the nasal conchae (turbinates), deviation of the septum, and, eventually, craniofacial deformation. also, nasal inflammation may extend into the sinuses causing sinusitis; into facial bones causing osteomyelitis; through the cribriform plate causing meningitis; into the eustachian tubes causing otitis media or guttural pouch empyema (eustachitis) in horses; and even into the inner ear causing otitis interna and vestibular syndrome the nasal septum has been removed to expose nasal conchae. the nasal mucosa is hyperemic and covered by yellow-white purulent exudate (arrows). inset, histological section showing submucosal congestion and edema and also large aggregates of neutrophils on the superficial mucosa (asterisk). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) microscopically, the lesions include a perivascular edema with fibrin, a few neutrophils infiltrating the mucosa, and superficial plaques of exudate consisting of fibrin strands mixed with leukocytes and cellular debris covering a necrotic and ulcerated epithelium. fungal infections, such as aspergillosis, can cause a severe fibrinonecrotizing rhinitis. granulomatous rhinitis. granulomatous rhinitis is a reaction in the nasal mucosa and submucosa that is characterized by infiltration of numerous activated macrophages mixed with a few lymphocytes and plasma cells (figs. - and - ). in some cases, chronic inflammation leads to the formation of polypoid nodules that in severe cases are large enough to cause obstruction of the nasal passages ( fig. - ). granulomatous rhinitis is generally associated with chronic allergic inflammation or infection with specific organisms, such as fungi (see fig. - ), tuberculosis, systemic mycosis (see section on granulomatous pneumonia), and rhinosporidiosis ; also see fig. - ). in some cases, the cause of granulomatous rhinitis cannot be determined. sinusitis. sinusitis occurs sporadically in domestic animals and is frequently combined with rhinitis (rhinosinusitis), or it occurs as extend into the adjacent bone (osteomyelitis) or through the ethmoidal conchae into the meninges and brain (meningitis and encephalitis). nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses. unlike amyloidoses in other organs of domestic animals where amyloid is generally of the reactive type (amyloid aa), equine nasal amyloidosis appears to be of the immunocytic type (amyloid al). affected horses with large amyloid masses have difficulty breathing because of nasal obstruction and may exhibit epistaxis and reduced athletic performance; on clinical examination, large, firm nodules resembling neoplasms (amyloidoma) can be observed in the alar folds, rostral nasal septum, and floor of nasal cavity. microscopic lesions are similar to those seen in other organs and consist of a deposition of hyaline amyloid material in nasal mucosa that is confirmed by a histochemical stain, such as congo red. progressive ethmoidal hematoma. progressive ethmoidal hematoma (peh) is important in older horses and is characterized clinically by chronic, progressive, often unilateral nasal bleeding. grossly or endoscopically, an ethmoidal hematoma appears as a single, soft, tumor-like, pedunculated, expansive, dark red mass arising from the mucosa of the ethmoidal conchae ( fig. - ) . microscopic examination reveals a capsule lined by epithelium and hemorrhagic stromal tissue infiltrated with abundant macrophages, most of which are siderophages. viral infections. viruses, such as equine viral rhinopneumonitis virus, influenza virus, adenovirus, and equine picornavirus, cause mild and generally transient respiratory infections in horses. the route of infection for these respiratory viruses is typically aerogenous. all of these infections are indistinguishable clinically; signs consist mainly of malaise, fever, coughing, conjunctivitis, and nasal a sequela to penetrating or septic wounds of the nasal, frontal, maxillary, or palatine bones; improper dehorning in young cattle, which exposes the frontal sinus; or maxillary tooth infection in horses and dogs (maxillary sinus). based on the type of exudate, sinusitis is classified as serous, catarrhal, fibrinous (rare), purulent, or granulomatous. paranasal sinuses have poor drainage; therefore exudate tends to accumulate, causing mucocele (accumulation of mucus) or empyema (accumulation of pus) ( fig. - ). chronic sinusitis may promised horses, particularly in arabian foals with inherited combined immunodeficiency disease. bacterial infections. strangles, glanders, and melioidosis of horses are all systemic bacterial diseases that cause purulent rhinitis and suppuration in various organs. these diseases are grouped as upper respiratory diseases because nasal discharge is often the most notable clinical sign. strangles. strangles is an infectious and highly contagious disease of equidae that is caused by streptococcus equi ssp. equi (streptococcus equi) . it is characterized by suppurative rhinitis and lymphadenitis (mandibular and retropharyngeal lymph nodes) with occasional hematogenous dissemination to internal organs. unlike streptococcus equi ssp. zooepidemicus (streptococcus zooepidemicus) and streptococcus dysgalactiae ssp. equisimilis (streptococcus equisimilis), streptococcus equi is not part of the normal nasal flora. infection occurs when susceptible horses come into contact with feed, exudate, or air droplets containing the bacterium. after penetrating through the nasopharyngeal mucosa, streptococcus equi drains to the regional lymph nodes-mandibular and retropharyngeal lymph nodes-via lymphatic vessels. the gross lesions in horses with strangles (mucopurulent rhinitis) correlate with clinical findings and consist of copious amounts of mucopurulent exudate in the nasal passages with notable hyperemia of the nasal mucosa. affected lymph nodes are enlarged and may contain abscesses filled with thick purulent exudate (purulent lymphadenitis). the term bastard strangles is used in cases in which hematogenous dissemination of streptococcus equi results in metastatic abscesses in such organs as the lungs, liver, spleen, kidneys, or brain or in the joints. this form of strangles is often fatal. common sequelae to strangles include bronchopneumonia caused by aspiration of nasopharyngeal exudate; laryngeal hemiplegia ("roaring"), resulting from compression of the recurrent laryngeal nerves by enlarged retropharyngeal lymph nodes; facial paralysis and horner syndrome caused by compression of sympathetic nerves that run dorsal to the medial retropharyngeal lymph node; and purpura hemorrhagica as a result of vasculitis caused by deposition of streptococcus equi antigen-antibody complexes in arterioles, venules, and capillaries of the skin and mucosal membranes. in severe cases, nasal infection extends directly into the paranasal sinuses or to the guttural pouches via the eustachian tubes, causing inflammation and accumulation of pus (guttural pouch empyema). rupture of abscesses in the mandibular and retropharyngeal lymph nodes leads to suppurative inflammation of adjacent subcutaneous tissue (cellulitis), and in severe cases the exudate escapes through cutaneous fistulas. strangles can affect horses of all ages, but it is most commonly seen in foals and young horses. it is clinically characterized by cough, nasal discharge, conjunctivitis, and painful swelling of regional lymph nodes. some horses become carriers and a source of infection to other horses. glanders. glanders is an infectious world organization for animal health (oie)-notifiable disease of equidae caused by burkholderia mallei (pseudomonas mallei) that can be transmitted to carnivores by consumption of infected horsemeat. human beings are also susceptible, and untreated infection is often fatal. this gramnegative bacterium has been listed as a potential agent for biologic warfare and bioterrorism. in the past, burkholderia mallei was found throughout the world, but today, glanders has been eradicated from most countries, except for some areas in north africa, asia, and eastern europe. there also have been sporadic outbreaks reported in brazil. the pathogenesis of glanders is not fully understood. results from experimental infections suggest that infection occurs discharge varying from serous to purulent. viral respiratory infections are common medical problems in adult horses. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr) is caused by two ubiquitous equine herpesviruses (ehv- and ehv- ) and may be manifested as a mild respiratory disease in weanling foals and young racehorses, as a neurologic disease (myeloencephalopathy), or as abortion in mares. the portal of entry for the respiratory form is typically aerogenous, and the disease is generally transient; thus the primary viral-induced lesions in the nasal mucosa and lungs are rarely seen at necropsy unless complicated by secondary bacterial rhinitis, pharyngitis, or bronchopneumonia. studies with polymerase chain reaction (pcr) techniques have demonstrated that, like other herpesviruses, ehv- and ehv- persist in the trigeminal ganglia for long periods of time (latency). reactivation because of stress or immunosuppression and subsequent shedding of the virus are the typical source of infection for susceptible animals on the farm. equine influenza. equine influenza is a common, highly contagious, and self-limiting upper respiratory infection of horses caused by aerogenous exposure to type a strains of influenza virus (h n [a/equi- ] and h n [a/equi- ]). equine influenza has high morbidity (outbreaks) but low mortality, and it is clinically characterized by fever, conjunctivitis, and serous nasal discharge. it occurs mainly in -to -year-old horses at the racetrack. as with human influenza, equine influenza is usually a mild disease, but occasionally it can cause severe bronchointerstitial pneumonia with pulmonary edema. in some horses, impaired defense mechanisms caused by the viral infection are complicated by a secondary bacterial bronchopneumonia caused by opportunistic organisms (streptococcus zooepidemicus, staphylococcus aureus, or bacteroides sp.) found in the normal flora of the upper respiratory tract. uncomplicated cases of equine influenza are rarely seen in the postmortem room. equine influenza virus (h n ) recently did an equine to canine "host-jump" causing extensive outbreaks of respiratory disease in dogs (see pneumonias of dogs). other equine respiratory viruses. equine picornavirus, adenovirus, and parainfluenza virus produce mild and transient upper respiratory infections (nasopharynx and trachea) in horses, unless complicated by secondary pathogens. in addition to reduced athletic performance, infected horses may have a temporary suppression of cell-mediated immunity leading to opportunistic infections such as pneumocystis carinii pneumonia. fatal adenoviral infections with severe pneumonia or enteritis occur commonly in immunocom- disorders of ruminants (cattle, sheep, and goats) infectious bovine rhinotracheitis. infectious bovine rhinotracheitis (ibr), or "rednose," occurs worldwide and is a disease of great importance to the cattle industry because of the synergism of the ibr virus with mannheimia haemolytica in producing pneumonia. the causative agent, bovine herpesvirus (bohv- ), has probably existed as a mild venereal disease in cattle in europe since at least the mid- s, but the respiratory form was not reported until intensive management feedlot systems were first introduced in north america around the s. typically, the disease is manifested as a transient, acute, febrile illness, which results in inspiratory dyspnea caused by obstruction of the airways by exudate only in very severe cases. other forms of bohv- infection include ulcerative rumenitis; enteritis; multifocal hepatitis in neonatal calves; nonsuppurative meningoencephalitis; infertility; and in experimental infections, mastitis, mammillitis, and ovarian necrosis. except for the encephalitic form, the type of disease caused by bohv- depends more on the site of entry than the viral strain. like other herpesviruses, bohv- also can remain latent in nerve ganglia, with recrudescence after stress or immunosuppression. this virus also causes bovine abortion, systemic infections of calves, and genital infections such as infectious pustular vulvovaginitis (ipv) and infectious balanoposthitis (ibp). the respiratory form of ibr is characterized by severe hyperemia and multifocal necrosis of nasal, pharyngeal, laryngeal, tracheal, and sometimes bronchial mucosa ( fig. - and see fig. - ). as in other respiratory viral infections, ibr lesions are microscopically characterized by necrosis and exfoliation of ciliated cells followed by repair. secondary bacterial infections of these areas of necrosis result in the formation of a thick layer of fibrinonecrotic material (diphtheritic) in the nasal, tracheal, and bronchial mucosa (see fig. - ). intranuclear inclusion bodies, commonly seen in herpesvirus infections, are rarely seen in field cases because inclusion bodies occur only during the early stages of the disease. the most important sequela to ibr is bronchopneumonia, which is caused either by direct aspiration of exudate from airways or as a result of an impairment in pulmonary defense mechanisms, thus predisposing the animal to secondary bacterial infection, most frequently mannheimia haemolytica (see pneumonic mannheimiosis discussion). postmortem diagnosis of ibr is confirmed by isolation of the virus or its identification by immunohistochemistry or pcr in affected tissues. other causes of rhinitis. nasal granulomas occur in cattle presumably as a result of repeated exposure to an unidentified inhaled antigen. nasal granulomas (atopic rhinitis) are reported mainly in cattle in australia, south africa, and the united kingdom, where affected cattle develop multiple, small, pink or red, polypoid nodules, starting in the nasal vestibule that in time extend into the caudal aspect of the nasal septum (see fig. - ). these nodules are composed of fibrovascular tissue mixed with lymphocytes (granulation tissue) superficially lined by hyperplastic epithelium with abundant mast cells and eosinophils in the lamina propria (nasal eosinophilia). the microscopic features suggest that hypersensitivity type i (immediate), type iii (immune complex), and type iv (delayed) may be involved in nasal granulomas of cattle. bovine (idiopathic) nasal granuloma must be differentiated from nasal mycetomas, nasal rhinosporidiosis, and nasal schistosomiasis, which also cause the formation of nodules in the nasal mucosa of cattle. an eosinophilic material consistent with the splendore-hoeppli phenomenon is occasionally observed in bovine mycotic granulomas. this phenomenon seen in some mycotic or bacterial infections is microscopically via the ingestion of contaminated feed and water and, very rarely, via inhalation of infectious droplets. the portals of entry are presumed to be the oropharynx or intestine, in which bacteria penetrate the mucosa and spread via lymph vessels to regional lymph nodes, then to the bloodstream, and thus hematogenously to the internal organs, particularly the lungs. lesions in the nasal cavity start as pyogranulomatous nodules in the submucosa; these lesions subsequently ulcerate, releasing copious amounts of burkholderia mallei-containing exudate into the nasal cavity (see fig. - , a) . finally, ulcerative lesions in conchal mucosa heal and are replaced by typical stellate (star-shaped), fibrous scars. in some cases, the lungs also contain numerous gray, hard, small ( to mm), miliary nodules (resembling millet seeds) randomly distributed in one or more pulmonary lobes because of the hematogenous route. microscopically, these nodules are typical chronic granulomas composed of a necrotic center, with or without calcification, surrounded by a layer of macrophages enclosed by a thick band of connective tissue infiltrated with macrophages, fewer giant cells, lymphocytes, and plasma cells. cutaneous lesions, often referred to as equine farcy, are the result of severe suppurative lymphangitis characterized by nodular thickening of extended segments of lymph vessels in the subcutaneous tissue of the legs and ventral abdomen (see fig. - , c). eventually, affected lymph vessels rupture and release large amounts of purulent exudate through sinuses to the surface of the skin. melioidosis (pseudoglanders). melioidosis (pseudoglanders) is an important, life-threatening disease of human beings, horses, cattle, sheep, goats, pigs, dogs, cats, and rodents caused by burkholderia pseudomallei (pseudomonas pseudomallei). this disease in horses is clinically and pathologically similar to glanders, hence the name pseudoglanders. in human beings, this infection can cause severe sepsis and septic shock and has also been considered to have potential for biologic welfare. melioidosis is currently present in southeast asia and, to a much lesser extent, in northern australia and some european countries where the causative organism is frequently found in rodents, feces, soil, and water. ingestion of contaminated feed and water appears to be the main route of infection; direct transmission between infected animals and insect bites has also been postulated as a possible mechanism of infection. after gaining entrance to the animal, burkholderia pseudomallei is disseminated by the bloodstream and causes suppuration and abscesses in most internal organs, such as nasal mucosa, joints, brain and spinal cord, lungs, liver, kidneys, spleen, and lymph nodes. the exudate is creamy or caseous and yellow to green. the pulmonary lesions in melioidosis are those of an embolic bacterial infection with the formation of pulmonary abscesses, which can become confluent. focal adhesive pleuritis develops where abscesses rupture through the pleura and heal. rhinosporidiosis. the protistan parasite, rhinosporidium seeberi, causes nasal infection in human beings, horses, mules, cattle, dogs, and cats. gross lesions vary from barely visible granulomas to large expansive polypoid nodules that may be mistaken as tumors. these granulomatous nodules are detected by direct observation when present in the nasal mucosa close to the nares or by rhinoscopy when located in the deep nasal cavity. the offending organism, rhinosporidium seeberi, is readily visible in histologic preparations and in impression smears, appearing as a large ( µm), oval sporangium containing thousands of endospores (see fig. - ). rhinosporidium seeberi was once considered a mycotic agent, but recent phylogenetic investigations suggest that it is an aquatic protistan parasite of the class mesomycetozoea. giant, basophilic, intranuclear inclusion bodies in the nasal epithelium, particularly in the nasal glands ( fig. - ). immunosuppressed piglets can develop a systemic cytomegalovirus infection characterized by necrosis of the liver, lungs, adrenal glands, and brain with intralesional inclusion bodies. inclusion body rhinitis is clinically a characterized by a deeply eosinophilic homogeneous material surrounded by bacteria or mycelia. it is thought to result from a localized antigen-antibody response in tissue. oestrus ovis. oestrus ovis (diptera: oestridae; nasal bot) is a brownish fly about the size of a honeybee that deposits its first-stage larvae in the nostrils of sheep in most areas of the world. microscopic larvae mature into large bots (maggots), which spend most of their larval stages in nasal passages and sinuses, causing irritation, inflammation, and obstruction of airways. mature larvae drop to the ground and pupate into flies. this type of parasitism in which living tissues are invaded by larvae of flies is known as myiasis ( fig. - ). although oestrus ovis is a nasal myiasis primarily of sheep, it sporadically affects goats, dogs, and sometimes human beings (shepherds). the presence of the larvae in nasal passages and sinuses causes chronic irritation and erosive mucopurulent rhinitis and sinusitis; bots of oestrus ovis can be found easily if the head is cut to expose the nasal passages and paranasal sinuses. rarely, larvae of oestrus ovis penetrate the cranial vault through the ethmoidal plate, causing direct or secondary bacterial meningitis. other causes of rhinitis. infectious rhinitis is only sporadically reported in goats, and most of these cases are caused by pasteurella multocida or mannheimia haemolytica. the lesions range from a mild serous to catarrhal or mucopurulent inflammation. foreign body rhinitis caused by plant material is sporadically seen cattle, sheep, and goats ( fig. - ). inclusion body rhinitis. inclusion body rhinitis is a disease of young pigs with high morbidity and low mortality caused by a porcine cytomegalovirus (suid herpesvirus- ) and characterized by a mild rhinitis. this virus commonly infects the nasal epithelium of piglets younger than weeks and causes a transient viremia. because this disease is seldom fatal, lesions are seen only incidentally or in euthanized animals. in uncomplicated cases, the gross lesion is hyperemia of the nasal mucosa, but with secondary bacterial infections, mucopurulent exudate can be abundant. microscopic lesions are typical and consist of a necrotizing, nonsuppurative rhinitis with toxigenic strains of pasteurella multocida. the only lesion associated with infection with bordetella bronchiseptica alone is a mild to moderate turbinate atrophy (nonprogressive atrophic rhinitis), but this bacterium actively promotes the colonization of the nasal cavity by pasteurella multocida. the toxigenic strains of pasteurella multocida produce potent cytotoxins that inhibit osteoblastic activity and promote osteoclastic reabsorption in nasal bones, particularly in the ventral nasal conchae, where abnormal bone remodeling results in progressive atrophy of conchae. the degree of conchal atrophy in pigs with atrophic rhinitis varies considerably, and in most pigs, the severity of the lesions does not correspond to the severity of the clinical signs. the best diagnostic method of evaluating this disease at necropsy is to make a transverse section of the snout between the first and second premolar teeth. in normal pigs, conchae are symmetric and fill most of the cavity, leaving only narrow airspaces (meatuses) between coiled conchae. the normal nasal septum is straight and divides the cavity into two mirror-image cavities. in contrast, the septum in pigs with atrophic rhinitis is generally deviated and the conchae appear smaller and asymmetric ( fig. - ). conchal atrophy causes dorsal and ventral meatuses to appear rather enlarged, and in the most advanced cases, the entire nasal conchae may be missing, leaving a large, empty space. it may seem logical to assume that after loss of conchae in an obligate nasal breather, such as the pig, the filtration defense mechanism of the nasal cavity would be impaired, thus enhancing the chances of aerogenous infections in the lung. however, the relationship between atrophic rhinitis, pneumonia, and growth rates in pigs is still controversial. osteoclastic hyperplasia and osteopenia of the conchae are the key microscopic lesions in atrophic rhinitis. depending on the stage of the disease, mucopurulent exudate may be found on the surface of the conchae. hyperplastic or metaplastic changes can occur in the nasal epithelium and glands, and infiltrates of lymphoplasmacytic cells can be present in the lamina propria. in summary, atrophic rhinitis is an important disease in pigs worldwide; morphologic characterized by a mild and transient rhinitis, causing sneezing, nasal discharge, and excessive lacrimation. atrophic rhinitis. a common worldwide disease of pigs, atrophic rhinitis (progressive atrophic rhinitis) is characterized by inflammation and atrophy of nasal conchae (turbinates). in severe cases, atrophy of the conchae may cause a striking facial deformity in growing pigs because of deviation of the nasal septum and nasal bones. the etiopathogenesis of atrophic rhinitis is complex and has been a matter of controversy for many years. pathogens historically associated with atrophic rhinitis include bordetella bronchiseptica, pasteurella multocida, haemophilus parasuis, and viral infections such as porcine cytomegalovirus (inclusion body rhinitis). in addition, predisposing factors have included genetic makeup, environment, and nutritional deficiencies. the cause of atrophic rhinitis is currently believed to be a combined infection by specific strains of bordetella bronchiseptica producing dermonecrotic toxin and linguatula serrata. linguatula serrata is a rare but highly specialized pentastomid parasite that shares some morphologic features with arthropods and annelids and causes infection when dogs consume uncooked ruminant meat containing infective larvae. it occurs primarily in carnivores, although sheep and goats may become aberrant hosts. human beings can also acquire the infection by ingesting raw ovine or caprine meat. the adult parasite is found throughout the nasal passages and sometimes can reach the sinuses and middle ear by moving through the exudate in the eustachian tubes. in common with other nasal parasites, linguatula serrata acts as an irritant, causing sneezing, catarrhal inflammation, and epistaxis. the eggs of this parasite leave the host in the exudate, which is coughed up or swallowed and eliminated in the feces. the nasal cavity and paranasal sinuses of dogs can occasionally be infested with other parasites, including mites (pneumonyssus caninum) and rhinosporidium seeberi (see figs. - and - ). allergic rhinitis. allergic rhinitis (hay fever; nasolacrimal urticaria), which is so common in human beings sensitized and reexposed to inhaled pollens or allergens, has been reported only sporadically in dogs and cats. hay fever in human beings and animals is a type i hypersensitivity reaction in which an ige-mediated degranulation of mast cells results in an acute rhinitis and conjunctivitis. microscopically, the nasal mucosa is edematous and infiltrated with numerous eosinophils, neutrophils, and some macrophages. clinically, allergic rhinitis is characterized by profuse serous nasal discharge and lacrimation. other causes of rhinitis. a nonspecific (idiopathic) chronic lymphoplasmacytic rhinitis is occasionally seen in dogs. immotile cilia syndrome (ciliary dyskinesia), a congenital disease, reduces mucociliary clearance and is an important factor in recurrent canine rhinosinusitis, bronchitis, bronchiectasis, and pneumonia. feline viral rhinotracheitis. feline viral rhinotracheitis (fvr) is a common, worldwide respiratory disease of cats caused by felid herpesvirus (fehv- ). the disease causes an impairment of pulmonary defense mechanisms predisposing cats to secondary bacterial pneumonia or to a coinfection with feline calicivirus. the virus also can remain latent in ganglia. the vast majority of cats that recover from fvr become carriers and shed fehv- , either spontaneously or following stress. susceptible animals, particularly kittens with low maternal immunity, become infected after exposure to a diseased or carrier cat. replication of fehv- in the nasal, conjunctival, pharyngeal, and, to a lesser extent, tracheal epithelium causes degeneration and exfoliation of cells. lesions caused by fehv- are fully reversible, but secondary infections with bacteria, such as pasteurella multocida, bordetella bronchiseptica, streptococcus spp., and mycoplasma felis, can cause a chronic, severe suppurative rhinitis and also conjunctivitis. intranuclear inclusion bodies are rarely seen in cats with fvr because inclusions are only present during the early stages of infection and have already disappeared by the time the cat is presented for diagnosis. respiratory sequelae to fvr can include chronic bacterial rhinitis and sinusitis with persistent purulent discharge; lysis of nasal bones, which can lead to conchal atrophy; permanent damage to the olfactory epithelium; and secondary bacterial pneumonia. in addition to rhinitis and interstitial pneumonia, fvr also causes ulcerative keratitis, hepatic necrosis, emaciation, abortion, and diagnosis is simple, but additional understanding of the pathogenesis will be necessary before effective preventive measures can be established. atrophic rhinitis is clinically characterized by sneezing, coughing, and nasal discharge. obstruction of the nasolacrimal duct is common and results in accumulation of dust and dried lacrimal secretions on the skin inferior to the medial canthus of the eye. viral infections. dogs have no specific viral infections affecting exclusively the nasal cavity or sinuses. acute rhinitis and sinusitis occurs as part of the canine infectious respiratory disease (cird) group caused by several distinct viruses, such as canine distemper virus, cav- and - , canine parainfluenza virus, reovirus, and canine herpesvirus. the viral lesions in the respiratory tract are generally transient, but the effect of the virus on other tissues and cells can be fatal, as in distemper encephalitis in dogs. bacterial infections. as in other species, secondary bacterial rhinitis, sinusitis, and pneumonia are possible sequelae of respiratory viral infections; bordetella bronchiseptica, escherichia coli, and pasteurella multocida are the most common isolates in dogs with bacterial rhinitis. mycotic infections. aspergillus spp. and penicillium spp. cause mycotic rhinitis and sinusitis in dogs (canine nasal aspergillosis) ( fig. - ). nasal biopsies reveal extensive necrosis of the nasal epithelium and thick plaques of fibrinopurulent exudate mixed with many fungal hyphae. cryptococcus neoformans and blastomyces dermatitides infections of the nasal cavity occur sporadically in dogs ( fig. - ). lesions are characterized by mucosal granulomas containing periodic acid-schiff (pas)-positive fungal organisms, and the infection is clinically characterized by mucopurulent nasal discharge. fvr; these two viral infections account for % of all cases of feline respiratory diseases. a febrile systemic hemorrhagic syndrome with high mortality (up to %) has been reported in cats infected with virulent strains of fcv. feline chlamydiosis. feline chlamydiosis is a persistent respiratory infection of cats caused by chlamydophila felis. infection results in a conjunctivitis (similar to the conjunctivitis seen in human trachoma caused by chlamydia trachomatis) and serous or mucopurulent rhinitis. in the past, chlamydophila felis was incriminated as the agent responsible for "feline pneumonitis," but its role in causing bronchointerstitial pneumonia in cats has been seriously challenged in recent years (see pneumonias of cats). mycotic infections. the most common mycotic infection in the feline nasal cavity is caused by cryptococcus neoformans and cryptococcus gatti, but not all animals exposed to these fungi necessarily develop cryptococcosis unless they are immunosuppressed. stillbirths. clinical signs of fvr infection are characterized by lethargy, oculonasal discharge, severe rhinitis, and conjunctivitis. feline calicivirus. feline rhinitis can be caused by different strains of feline calicivirus (fcv). it is an important infection of the respiratory tract of cats, and depending on the virulence of the strain, lesions vary from a mild oculonasal discharge to severe rhinitis, mucopurulent conjunctivitis, and ulcerative gingivitis and stomatitis. the lesions, in addition to rhinitis and conjunctivitis, include acute, diffuse interstitial pneumonia with necrotizing bronchiolitis (see pneumonias of cats) and in some cases prominent ulcers of the tongue and hard palate. primary viral lesions are generally transient, but secondary bacterial infections (bordetella bronchiseptica, pasteurella multocida, or escherichia coli) are a common complication. some kittens develop lameness after infection or vaccination with calicivirus because of an acute and self-limiting arthritis ("limping kitten syndrome"). carrier state and virus shedding from oronasal secretions and feces are natural sequelae after recovery from the acute phase of the disease. clinical and pathologic features of fcv disease are strikingly similar but not identical to those of hemorrhage, increased lacrimation as a result of obstruction of nasolacrimal ducts, and sneezing. in some instances, it is not possible to clinically or grossly differentiate neoplasms from hyperplastic nodules or granulomatous rhinitis. some neoplasms may infiltrate adjacent bone structures and produce notable facial deformities, loss of teeth, exophthalmus, and nervous signs. large neoplasms also project into the meatuses, narrow the lumen, and interfere with airflow, causing stertorous breathing (see . biopsies, as well as brush and imprint cytology, have proven effective in the antemortem diagnosis of nasal neoplasms, particularly in those of epithelial lineage. a unique group of nasal carcinomas (enzootic nasal tumors, enzootic intranasal tumors, and enzootic nasal carcinoma) of sheep and goats arise from the surface epithelium and glands of the ethmoidal conchae. these types of carcinomas are caused by betaretroviruses in sheep (entv- ) and goats (entv- ). the enzootic nasal tumor has been successfully transmitted to susceptible animals by the lesions vary from discrete nasal granulomas to large confluent masses of mucopurulent exudate filling the entire nasal cavity and paranasal sinuses. microscopic examination of the exudate reveals the typical thick-walled pas-positive organisms (see fig. - ). mycoplasma felis can also cause mucopurulent conjunctivitis and a mild upper respiratory infection, with clinical signs and lesions overlapping those seen with chlamydiosis, fvr, and fcr infections. respiratory infections and bronchopneumonia in cats may also be associated with the immunosuppressive effects of feline retroviruses such as feline leukemia virus (felv) and feline immunodeficiency virus (fiv). nasal aspergillosis and allergic rhinosinusitis are sporadically reported in cats (see disorders of the conducting system: species-specific diseases of the nasal cavity and paranasal sinuses: disorders of dogs: mycotic infections). neoplasms of the nasal cavity and paranasal sinuses may arise from any of the tissues forming these structures, including bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), connective tissue (fibroma or fibrosarcoma, myxoma or myxosarcoma), and blood vessels (hemangioma or hemangiosarcoma), and from all the different types of cells of glands and lining epithelium (adenoma, carcinoma, or adenocarcinoma). nasal tumors originating from stromal tissues, such as bone, cartilage, and connective tissue, are morphologically indistinguishable from those seen in other sites. in general, nasal neoplasms are rare in domestic animals, except for enzootic ethmoidal tumor (retroviral) in sheep and goats, which can occur in several animals in a herd (see the next section). in companion animals, nasal neoplasms are most common in dogs, particularly in medium to large breed dogs such as the collie, airedale terrier, basset hound, and german shepherd. the cat and the horse are less frequently affected. the main sites in order of frequency are the nasal passages and sinuses for dogs, the tip of the nose and nasal passages for cats, and the maxillary sinus and nasal passages for horses. the majority of neoplasms in the nasal cavity are malignant. benign nasal neoplasms (papilloma and adenoma) are rare and generally are either solitary or multiple, well-delineated nodules. in contrast, nasal carcinomas and nasal sarcomas are generally larger but vary in size and are often pale and multilobulated masses composed of fleshy to friable tissue (figs. - and - ). malignant neoplasms are locally invasive and tend to infiltrate sinuses, meninges, frontal brain, olfactory nerves, and vessels resulting in epistaxis. carcinomas vary from anaplastic (poorly differentiated) to well differentiated, in which cell and tissue morphology retains some glandular (adenocarcinoma) or squamous cell patterns. because nasal tumors in dogs and cats are usually large and invasive at the time of diagnosis, prognosis is usually poor and survival times are short. sarcomas originating in the nasal cavity and paranasal sinuses are less common than carcinomas. mesenchymal tumors can arise from bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), blood vessels (hemangioma or hemangiosarcoma), and connective tissue (fibroma or fibrosarcoma). overall, benign epithelial and mesenchymal tumors are less common than their malignant counterparts. secondary tumors in the nasal cavity are rare, with lymphoma being the most common secondary tumor in the nasal cavity of domestic animals ( fig. - ). nasal neoplasms become secondarily infected by bacteria, and clinical signs often overlap with those of infectious rhinitis and include catarrhal or mucopurulent nasal discharge, periodic anomalies congenital anomalies of the pharynx, guttural pouches, larynx, and trachea are rare in all species. depending on their location and severity, they may be inconsistent with postnatal life, pose little or no problem, interfere with quality of life, or manifest themselves in later life. if clinical signs of respiratory distress, such as stridor, coughing, dyspnea, or gagging, do occur, they are usually exacerbated by excitement, heat, stress, or exercise. brachycephalic airway syndrome. see disorders of the conducting system: species-specific diseases of the pharynx, guttural inoculation of cell-free tumor filtrates. enzootic nasal tumors are typically invasive but do not metastasize ( fig. - ). in some regions of the world, ethmoid tumors have been reported in horses and pigs, particularly in those farms where the endemic nasal tumors of ruminants are known to occur. nonneoplastic exophytic masses that resemble neoplasms are commonly found in horses, cats, and, to a lesser extent, other species. in horses, polyps tend to form in the ethmoidal region, whereas in cats, polyps are most frequently found in the nasopharynx and eustachian tubes. the pathogenesis of these benign growths is uncertain, although in many cases they follow chronic rhinitis or sinusitis. most recently, lymphatic obstruction secondary to inflammation has been postulated as the main culprit. grossly, polyps appear as firm, pedunculated nodules of various sizes protruding from the nasal mucosa into the nasal passages or nasopharynx ( fig. - ); the surface may be smooth, ulcerated, secondarily infected, and hemorrhagic. microscopically, polyps are characterized by a core of wellvascularized stromal tissue that contains inflammatory cells and are covered by pseudostratified or squamous epithelium (see fig. - ). nasal and paranasal sinus cysts are common idiopathic lesions in horses and are medically important because they clinically mimic table - for potential, suspected, or known genetic disorders. tracheal collapse and tracheal stenosis. tracheal collapse with reduction in tracheal patency occurs in toy, miniature, and brachycephalic breeds of dogs, in which the condition is also called tracheobronchial collapse or central airway collapse. the defect also occurs in horses, cattle, and goats. by radiographic, endoscopic, or gross examination, there is dorsoventral flattening of the trachea with concomitant widening of the dorsal tracheal membrane, which may then prolapse ventrally into the lumen ( fig. - ). most commonly, the defect extends the entire length of the trachea and only rarely affects the cervical portion alone. affected segments with a reduced lumen contain froth and even are covered by a diphtheritic membrane. in horses, the so-called scabbard trachea is characterized allergic reactions. grossly, the mucosa of the epiglottis and vocal cords is thickened and swollen, often protrudes dorsally onto the epiglottic orifice, and has a gelatinous appearance ( fig. - ). laryngeal and tracheal hemorrhage. hemorrhages in these sites occur as mucosal petechiae and are most commonly seen in coagulopathies; inflammation; septicemia and sepsis, particularly in pigs with classical swine fever (hog cholera); african swine fever or salmonellosis; and horses with equine infectious anemia. severe dyspnea and asphyxia before death can cause congestion, ecchymosis, and petechiae in the laryngeal and tracheal mucosa; this lesion must be differentiated from postmortem imbibition of hemoglobin in autolyzed carcasses (see chapter ). by lateral flattening so that the tracheal lumen is reduced to a narrow vertical slit. segmental tracheal collapse causing stenosis has been associated with congenital and acquired abnormalities. in severe cases, abnormal cartilaginous glycoproteins and loss of elasticity of tracheal rings causes the trachea to collapse. in some other cases, it is an acquired tracheal lesion that follows trauma, compression caused by extraluminal masses, peritracheal inflammation, and flawed tracheotomy or transtracheal aspirate techniques. other tracheal anomalies include tracheoesophageal fistula, which is most commonly found in human beings and sporadically in dogs and cattle. congenital fistulas can occur at any site of the cervical or thoracic segments of the trachea. acquired tracheoesophageal fistula can be a complication of improper intubation, tracheotomy, or esophageal foreign body. laryngeal hemiplegia. laryngeal hemiplegia (paralysis), sometimes called roaring in horses, is a common but obscure disease characterized by atrophy of the dorsal and lateral cricoarytenoid muscles (abductor and adductor of the arytenoid cartilage), particularly on the left side. muscular atrophy is most commonly caused by a primary denervation (recurrent laryngeal neuropathy) of unknown cause (idiopathic axonopathy) and, to a much lesser extent, secondary nerve damage (see the section on denervation atrophy in chapters and ). idiopathic laryngeal hemiplegia is an incurable axonal disease (axonopathy) of the cranial laryngeal nerve that affects mostly larger horses. secondary laryngeal hemiplegia is rare and occurs after nerve damage caused by other pathologic processes such as compression or inflammation of the left recurrent laryngeal nerve. the medial retropharyngeal lymph nodes are located immediately ventral to the floor of the guttural pouches. as a result of this close anatomic relationship, swelling or inflammation of the guttural pouches or retropharyngeal lymph nodes often results in secondary damage to the laryngeal nerve. common causes of secondary nerve damage (wallerian degeneration) include guttural pouch mycosis, retropharyngeal abscesses, inflammation because of iatrogenic injection into the nerves, neck injury, and metastatic neoplasms involving the retropharyngeal lymph nodes (e.g., lymphosarcoma). grossly, the affected laryngeal muscle in a horse with laryngeal hemiplegia is pale and smaller than normal (muscle atrophy) . microscopically, muscle fibers have lesions of denervation atrophy (see chapters and ). atrophy of laryngeal muscles also occurs in dogs as an inherited condition (siberian husky and bouvier des flanders), as a degenerative neuropathy in older dogs, secondary to laryngeal trauma in all species (e.g., choke chain damage), or secondary to hepatic encephalopathy in horses. the abnormal inspiratory sounds (roaring) during exercise in horses with laryngeal hemiplegia are caused by paralysis of the left dorsal and lateral cricoarytenoid muscles, which cause incomplete dilation of the larynx, obstruction of airflow, and vibration of vocal cords. laryngeal edema. laryngeal edema is a common feature of acute inflammation, but it is particularly important because swelling of the epiglottis and vocal cords can obstruct the laryngeal orifice, resulting in asphyxiation. laryngeal edema occurs in pigs with edema disease; in horses with purpura hemorrhagica; in cattle with acute interstitial pneumonia; in cats with systemic anaphylaxis; and in all species as a result of trauma, improper endotracheal tubing, inhalation of irritant gases (e.g., smoke), local inflammation, and animal species is classified as fibrinous, catarrhal, purulent, or granulomatous (figs. - and - ). chronic polypoid tracheitis occurs in dogs and cats, probably secondary to chronic infection. the most common causes of tracheitis are viral infections, such as those causing infectious bovine rhinotracheitis (see fig. - ), equine viral rhinopneumonitis, canine distemper, and feline rhinotracheitis. viral lesions are generally mild and transient but often become complicated with secondary bacterial infections. at the early stages, the mucosa is notably hyperemic and can show white foci of necrosis. in the most severe cases, the affected mucosa detaches from the underlying basement membrane, causing extensive tracheal ulceration. chemical tracheitis is also commonly seen after aspiration (see fig. - ). also, inhalation of fumes during barn fires can cause extensive injury and necrosis of the tracheal mucosa. in forensic cases, the presence of carbon pigment in the mucosal surface of trachea, bronchi, and bronchioles indicates that the burned animal was alive during the fire. parasitic infections of the larynx and trachea can cause obstruction with dramatic consequences, but burdens sufficient to cause such effects are not commonly seen in veterinary practice. besnoitiosis (besnoitia spp.) . besnoitiosis (besnoitia spp.) is caused by several species of this apicomplexan coccidian parasite, whose life cycle is still unknown. this parasite can cause pedunculated lesions on the skin, sclera, mucosa of the nasal cavity, and larynx of horses and donkeys, cattle, goats, and wild animals. besnoitiosis has been reported from africa, central and south america, north america, and europe. grossly, pale, round, exophytic nodules up to cm in diameter can be observed protruding from mucosal surfaces. microscopically, these nodules consist of finger-like projections covered by hyperplastic and sometimes ulcerated epithelium containing numerous thick-walled parasitic cysts with little inflammatory response. cattle. see disorders of cattle. inflammation of the pharynx, larynx, and trachea are important because of their potential to obstruct airflow and to lead to aspiration pneumonia. the pharynx is commonly affected by infectious diseases of the upper respiratory and upper digestive tracts, and the trachea can be involved by extension from both the lungs and larynx. pharyngeal obstruction and perforation. intraluminal foreign bodies in the pharynx, such as medicament boluses, apples, or potatoes, can move down and obstruct the larynx and trachea. also, pharyngeal obstruction can be caused by masses in the surrounding tissue, such as neoplasms of the thyroid gland, thymus, and parathyroid glands. a number of nonspecific insults can cause lesions and clinical signs. trauma may take the form of penetrating wounds in any species: perforation of the caudodorsal wall of the pharynx from the improper use of drenching or balling guns in sheep, cattle, and pigs; choking injury because of the use of collars in dogs and cats; and the shearing forces of bite wounds. the results of the trauma may be minimal (local edema and inflammation) or as serious as complete luminal obstruction by exudate. foreign bodies may be lodged anywhere in the pharyngeal region; the location and size determine the occurrence of dysphagia, regurgitation, dyspnea, or asphyxiation. pigs have a unique structure known as the pharyngeal diverticulum ( cm long in adult pigs), which is located in the pharyngeal wall rostral and dorsal to the esophageal entrance. it is important because barley awns may lodge in the diverticulum, causing an inflammatory swelling that affects swallowing. the diverticular wall may be perforated by awns or drenching syringes, which results in an exudate that can extend down the tissue planes between muscles of the neck and even into the mediastinum. the pharynx of the dog may also be damaged by trauma from chicken bones, sticks, and needles, resulting in the formation of a pharyngeal abscess. equine inflammation of the trachea (tracheitis). the types of injury and host inflammatory responses in the trachea are essentially the same as those described for the nasal mucosa. although tracheal mucosa is prone to aerogenous injury and necrosis, it has a remarkable capacity for repair. according to the exudate, tracheitis in all palate, are important causes of respiratory problems and reduced athletic performance in horses. an undersized epiglottis is prone to being entrapped below the arytenoepiglottic fold, causing an equine syndrome known as epiglottic entrapment. this syndrome also occurs in horses with lateral deviation and deformity of epiglottis, epiglottic cysts, or necrosis of the tip of the epiglottis. hypoplastic epiglottis also occurs in pigs. dorsal displacement of the soft palate, particularly during exercise, narrows the lumen of the nasopharynx and creates abnormal air turbulence in the conducting system of horses. epiglottic entrapment is clinically characterized by airway obstruction, exercise intolerance, respiratory noise, and cough. subepiglottic and pharyngeal cysts. anomalous lesions, such as subepiglottic and pharyngeal cysts, are occasionally seen in horses, particularly in standardbred and thoroughbred racehorses. these cysts vary in size ( to cm) and occur most commonly in the subepiglottic area and to a lesser extent in the dorsal pharynx, larynx, and soft palate. cysts are lined by squamous or pseudostratified epithelium and contain thick mucus. large cysts cause airway obstruction, reduced exercise tolerance, or dysphagia and predispose to bronchoaspiration of food. equine pharyngeal lymphoid hyperplasia. equine pharyngeal lymphoid hyperplasia, or pharyngitis with lymphoid follicular hyperplasia, is a common cause of partial upper airway obstruction in horses, particularly in -and -year-old racehorses. lymphoid hyperplasia is also seen in healthy horses as part of a response to mild chronic pharyngitis, which in many instances tends to regress with age in older animals. the cause is undetermined, but chronic bacterial infection combined with environmental factors may cause excessive antigenic stimulation and lymphoid hyperplasia. the gross lesions, visible endoscopically or at necropsy, consist of variably sized ( to mm) white foci located on the dorsolateral walls of the pharynx and extending into the openings of the guttural pouches and onto the soft palate. in severe cases, lesions may appear as pharyngeal polyps. microscopically, the lesions consist of large aggregates of lymphocytes and plasma cells in the pharyngeal mucosa. clinical signs consist of stertorous inspiration, expiration, or both. inflammation of guttural pouches. the guttural pouches of horses are large diverticula ( to ml) of the ventral portion of the auditory (eustachian) tubes. these diverticula are therefore exposed to the same pathogens as the pharynx and have drainage problems similar to the sinuses. although it is probable that various pathogens, including viruses, can infect them, the most common pathogens are fungi, which cause guttural pouch mycosis and guttural pouch empyema in the horse. eustachitis is the term used for inflammatory processes involving the eustachian (pharyngotympanic) tube. because of the close anatomic proximity of guttural pouches to the internal carotid arteries, cranial nerves (vii, ix, x, xi, and xii), and atlantooccipital joint, disease of these diverticula may involve these structures and cause a variety of clinical signs in horses. guttural pouch mycosis occurs primarily in stabled horses and is caused by aspergillus fumigatus and other aspergillus spp. infection is usually unilateral and presumably starts with the inhalation of spores from moldy hay. grossly, the mucosal surfaces of the dorsal and lateral walls of the guttural pouch mucosa are first covered by focal, rounded, raised plaques of diphtheritic (fibrinonecrotic) exudate, which with time can become confluent and grow into a large fibrinonecrotic mass ( fig. - ) . microscopically, the lesions are severe necrotic inflammation of the mucosa and submucosa with widespread vasculitis and intralesional fungal hyphae. necrosis of the mammomonogamus (syngamus) spp. mammomonogamus (syngamus) laryngeus is a nematode that is seen attached to the laryngeal mucosa of cattle in tropical asia and south america, and cats (gapeworm: mammomonogamus ierei) in the caribbean and southern united states. occasionally, human beings with a persistent cough or asthma-like symptoms have the parasite in the larynx or bronchi. oslerus (filaroides) osleri. see disorders of dogs. b a c empyema of guttural pouches is a sequela to suppurative inflammation of the nasal cavities, most commonly from streptococcus equi infection (strangles). in severe cases, the entire guttural pouch can be filled with purulent exudate ( fig. - ). the sequelae are similar to those of guttural pouch mycosis except that there is no erosion of the internal carotid artery. it is clinically characterized by nasal discharge, enlarged retropharyngeal lymph nodes, painful swelling of the parotid region, dysphagia, and respiratory distress. guttural pouch tympany develops sporadically in young horses when excessive air accumulates in the pouch from the one-way valve effect caused by inflammation or malformation of the eustachian tube. arabian and german warm-blooded horses are particularly susceptible to develop guttural pouch tympany. it is generally unilateral and characterized by nonpainful swelling of the parotid region. cattle. tracheal edema and hemorrhage syndrome of feeder cattle, also known as the honker syndrome or tracheal stenosis of feedlot cattle, is a poorly documented acute disease of unknown cause, most often seen during the summer months. severe edema and a few hemorrhages are present in the mucosa and submucosa of the dorsal surface of the trachea, extending caudally from the midcervical area as far as the tracheal bifurcation. on section, the tracheal mucosa is diffusely thickened and gelatinous. clinical signs include inspiratory wall of the guttural pouches can extend into the wall of the adjacent internal carotid artery causing hemorrhage into the lumen of the guttural pouch and recurrent epistaxis. invasion of the internal carotid artery causes arteritis, which can also lead to formation of an aneurysm and fatal bleeding into the guttural pouches. in other cases, the fungi may be angioinvasive, leading to the release of mycotic emboli into the internal carotid artery, generally resulting in multiple cerebral infarcts. dysphagia, another clinical sign seen in guttural pouch mycosis, is associated with damage to the pharyngeal branches of the vagus and glossopharyngeal nerves, which lie on the ventral aspect of the pouches. horner's syndrome results from damage to the cranial cervical ganglion and sympathetic fibers located in the caudodorsal aspect of the pouches. finally, equine laryngeal paralysis (hemiplegia) can result from damage to the laryngeal nerves as previously described in the section on laryngeal hemiplegia. pekingese, and others. the defects are a result of a mismatch of the ratio of soft tissue to cranial bone and the obstruction of airflow by excessive length of the palatine soft tissue. secondary changes, such as nasal and laryngeal edema caused by forceful inspiration, eventually lead to severe upper airway obstruction, respiratory distress, and exercise intolerance. tracheal hypoplasia. tracheal hypoplasia occurs most often in english bulldogs and boston terriers; the tracheal lumen is decreased in diameter throughout its length. canine infectious respiratory disease. canine infectious respiratory disease (cird), formerly called canine tracheobronchitis or kennel cough, is a highly contagious group of infectious diseases characterized clinically by an acute onset of coughing notably exacerbated by exercise. the term is nonspecific, much like the dyspnea that can progress to oral breathing, recumbency, and death by asphyxiation in less than hours. necrotic laryngitis. necrotic laryngitis (calf diphtheria, laryngeal necrobacillosis) is a common disease of feedlot cattle and cattle affected with other diseases, with nutritional deficiencies, or housed under unsanitary conditions. it also occurs sporadically in sheep and pigs. necrotic laryngitis, caused by fusobacterium necrophorum, is part of the syndrome termed necrotic stomatitis or laryngeal necrobacillosis, which can include lesions of the tongue, cheeks, palate, and pharynx. an opportunistic pathogen, fusobacterium necrophorum produces potent exotoxins and endotoxins after gaining entry either through lesions of viral infections, such as ibr and vesicular stomatitis in cattle, or after traumatic injury produced by feed or careless use of specula or balling guns. the gross lesions, regardless of location in the mouth or larynx (most common in the mucosa overlying the laryngeal cartilages), consist of well-demarcated, dry, yellow-gray, thick-crusted, and fibrinonecrotic exudate ( fig. - ) that in the early stages is bounded by a zone of active hyperemia. deep ulceration develops, and if the lesion does not result in death, healing is by granulation tissue formation. microscopically, the necrotic foci are first surrounded by congested borders, then by a band of leukocytes, and finally the ulcers heal by granulation tissue and collagen (fibrosis). the lesions can extend deep into the submucosal tissue. numerous bacteria are evident at the advancing edge. there are numerous and important sequelae to calf diphtheria; the most serious is death from severe toxemia or overwhelming fusobacteremia. sometimes, the exudate may be copious enough to cause laryngeal obstruction and asphyxiation or be aspirated and cause bronchopneumonia. the clinical signs of necrotic laryngitis are fever, anorexia, depression, halitosis, moist painful cough, dysphagia, and inspiratory dyspnea and ventilatory failure because of fatigue of the respiratory muscles (diaphragmatic and intercostal). laryngeal contact ulcers. ulcerative lesions in the larynx are commonly found in feedlot cattle. grossly, the laryngeal mucosa reveals circular ulcers (up to cm in diameter), which may be unilateral or bilateral and sometimes deep enough to expose the underlying arytenoid cartilages. the cause has not been established, but causal agents, such as viral, bacterial, and traumatic, have been proposed, along with increased frequency and rate of closure of the larynx (excessive swallowing and vocalization) when cattle are exposed to market and feedlot stresses such as dust, pathogens, and interruption of feeding. contact ulcers predispose a calf to diphtheria (fusobacterium necrophorum) and laryngeal papillomas. ulceration of the mucosa and necrosis of the laryngeal cartilages have also been described in calves, sheep, and horses under the term laryngeal chondritis. laryngeal abscesses involving the mucosa and underlying cartilage occur as a herd or flock problem in calves and sheep, presumably caused by a secondary infection with trueperella (arcanobacterium) pyogenes. anomalies brachycephalic airway syndrome. brachycephalic airway syndrome is a clinical term that refers to increased airflow resistance caused by stenotic nostrils and nasal meatuses and an excessively long soft palate. these abnormalities are present in brachycephalic canine breeds such as bulldogs, boxers, boston terriers, pugs, a "common cold" in human beings or bovine respiratory disease complex (brdc) in cattle. the infection occurs commonly as a result of mixing dogs from different origins such as occurs at commercial kennels, animal shelters, and veterinary clinics. between bouts of coughing, most animals appear normal, although some have rhinitis, pharyngitis, tonsillitis, or conjunctivitis; some with secondary pneumonia become quite ill. the pathogenesis of cird is complex, and many pathogens and environmental factors have been incriminated. bordetella bronchiseptica, canine adenovirus- (cav- ), and canine parainfluenza virus- (cpiv- ) are most commonly implicated. the severity of the disease is increased when more than one agent is involved or if there are extreme environmental conditions (e.g., poor ventilation). for example, dogs asymptomatically infected with bordetella bronchiseptica are more severely affected by superinfection with cav- than those not carrying the bacterium. other agents are sometimes isolated but of lesser significance and include canine adenovirus- (cav- : infectious canine hepatitis virus), reovirus type , canid herpesvirus- (cahv- ), canine respiratory coronavirus (crcov), and mycoplasma species. depending on the agents involved, gross and microscopic lesions are completely absent or they vary from catarrhal to mucopurulent tracheobronchitis, with enlargement of the tonsils and retropharyngeal and tracheobronchial lymph nodes. in dogs with bordetella bronchiseptica infection, the lesions are suppurative or mucopurulent rhinitis and tracheobronchitis, and suppurative bronchiolitis. in contrast, when lesions are purely viral, microscopic changes are focal necrosis of the tracheobronchial epithelium. sequelae can include spread either proximally or distally in the respiratory tract, the latter sometimes inducing chronic bronchitis and bronchopneumonia. oslerus (filaroides) osleri. oslerus (filaroides) osleri is a nematode parasite of dogs and other canidae that causes characteristic protruding nodules into the lumen at the tracheal bifurcation. they are readily seen on endoscopic examination or at necropsy. in severe cases, these nodules can extend cm cranially or caudally from the tracheal bifurcation and even into primary and secondary bronchi. the disease occurs worldwide, and oslerus osleri is considered the most common respiratory nematode of dogs. the gross lesions are variably sized, up to cm, submucosal nodules that extend up to cm into the tracheal lumen ( fig. - , a). microscopically, nodules contain adult parasites with a mild mononuclear cell reaction; with the death of the parasite, an intense foreign body reaction develops with neutrophils and giant cells b) . clinically, it can be asymptomatic, although it most often causes a chronic cough that can be exacerbated by exercise or excitement. severe infestations can result in dyspnea, exercise intolerance, cyanosis, emaciation, and even death in young dogs. neoplasms of the guttural pouches occur rarely in horses and are usually squamous cell carcinomas. laryngeal neoplasms are rare in dogs and extremely so in other species, although they have been reported in cats and horses. the most common laryngeal neoplasms in dogs are papillomas and squamous cell carcinomas. other less common tumors are laryngeal rhabdomyoma, previously referred to as laryngeal oncocytoma, and chondromas and osteochondromas. lymphoma involving the laryngeal tissue is sporadically seen in cats. when large enough to be obstructive, neoplasms may cause a change or loss of voice, cough, or respiratory distress with cyanosis, collapse, and syncope. other signs include dysphagia, anorexia, and exercise intolerance. the neoplasm is sometimes visible from the oral cavity and causes swelling of the neck. the prognosis is poor because most lesions recur after excision. tracheal neoplasms are even more uncommon than those of the larynx. the tracheal cartilage or mucosa can be the site of an osteochondroma, leiomyoma, osteosarcoma, mast cell tumor, and carcinoma. lymphoma in cats can extend from the mediastinum to involve the trachea. each lung is subdivided into various numbers of pulmonary lobes (see fig. - ). in the past, these were defined by anatomic fissures. however, in current anatomy, lobes are defined by the ramification of the bronchial tree. following this criterion, the left lung of all domestic species is composed of cranial and caudal lobes, whereas the right lung, depending on species, is composed of cranial, middle (absent in horse), caudal, and accessory lobes. each pulmonary lobe is further subdivided by connective tissue into pulmonary lobules, which in some species (cattle and pigs) are rather prominent and in others are much less conspicuous. from a practical standpoint, identification of the lungs among different species could be achieved by carefully observing the degree of lobation (external fissures) and the degree of lobulation (connective tissue between lobules). cattle and pigs have well-lobated and well-lobulated lungs; sheep and goats have well-lobated but poorly lobulated lungs; horses have both poorly lobated and poorly lobulated lungs and resemble human lungs; finally, dogs and cats have well-lobated but not well-lobulated lungs. the degree of lobulation determines the degree of air movement between the lobules. in pigs and cattle, movement of air between lobules is practically absent because of the thick connective tissue of the interlobular septa separating individual lobules. this movement of air between lobules and between adjacent alveoli (via the pores of kohn) constitutes what is referred to as collateral ventilation. this collateral ventilation is poor in cattle and pigs and good in dogs. the functional implications of collateral ventilation are discussed in the section on pulmonary emphysema. the lungs have an interconnecting network of interstitial stromal tissue supporting the blood and lymphatic vessels, nerves, bronchi, bronchioles, and alveoli. for purposes of simplicity, the pulmonary interstitium can be anatomically divided into three contiguous compartments: ( ) bronchovascular interstitium, where main bronchi and pulmonary vessels are situated; ( ) interlobular interstitium separating pulmonary lobules and supporting small blood and lymph vessels; and ( ) alveolar interstitium supporting the alveolar walls that contain pulmonary capillaries and alveolar epithelial cells (no lymphatic vessels here) (see discussion on the blood-air barrier in the section on alveoli). pulmonary changes, such as edema, emphysema, and inflammation, may affect one or more of these interstitial compartments. anomalies congenital anomalies of the lungs are rare in all species but are most commonly reported in cattle and sheep. compatibility with life largely depends on the type of structures involved and the proportion of functional tissue present at birth. accessory lungs are one of the most common anomalies and consist of distinctively lobulated masses of incompletely differentiated pulmonary tissue present in the thorax, abdominal cavity, or subcutaneous tissue virtually anywhere in the trunk. large accessory lungs can cause dystocia. ciliary dyskinesia (immotile cilia syndrome, kartagener's syndrome) is characterized by defective ciliary movement, which results in reduced mucociliary clearance because of a defect in the microtubules of all ciliated cells and, most important, in the ciliated respiratory epithelium and spermatozoa. primary ciliary dyskinesia often associated with situs inversus has been reported in dogs, which as a result usually have chronic recurrent rhinosinusitis, pneumonia, and infertility. pulmonary agenesis, pulmonary hypoplasia, abnormal lobulation, congenital emphysema, lung hamartoma, and congenital bronchiectasis are occasionally seen in domestic animals. congenital melanosis is a common incidental finding in pigs and ruminants and is usually seen at slaughter (fig. - ). it is characterized by black spots, often a few centimeters in diameter, in various organs, mainly the lungs, meninges, intima of the aorta, and caruncles of the uterus. melanosis has no clinical significance, and the texture of pigmented lungs remains unchanged. congenital emphysema is sporadically seen in dogs (e- fig. - ). pulmonary calcification ("calcinosis"). calcification of the lungs occurs in some hypercalcemic states, generally secondary to hypervitaminosis d or from ingestion of toxic (hypercalcemic) plants, such as solanum malacoxylon (manchester wasting disease), that contain vitamin d analogs. it is also a common sequela to uremia and hyperadrenocorticism in dogs and to pulmonary necrosis (dystrophic calcification) in most species. calcified lungs may fail to collapse when the thoracic cavity is opened and have a characteristic "gritty" texture ( fig. - ) . microscopically, lesions vary from calcification of the alveolar basement membranes (see by pathologists. recent investigations suggest that excessive lipid originates from the breakdown products of neoplastic cells. bronchial and bronchiolar obstructions such as those caused by lungworms can also cause alveolar lipidosis. the pathogenesis relates to the inability of alveolar macrophages that normally remove part of the surfactant lipids to exit the lung via the mucociliary escalator. exogenous lipid pneumonia. another form of lipid pneumonia occurs accidentally in cats or horses given mineral oil by their owners in an attempt to remove hairballs or treat colic (aspiration pneumonia). to achieve gaseous exchange, a balanced ratio of the volumes of air to capillary blood must be present in the lungs (ventilation/perfusion ratio), and the air and capillary blood must be in close proximity across the alveolar wall. a ventilation-perfusion mismatch occurs if pulmonary tissue is either collapsed (atelectasis) or overinflated (hyperinflation and emphysema). the term atelectasis means incomplete distention of alveoli and is used to describe lungs that have failed to expand with air at the time of birth (congenital or neonatal atelectasis) or lungs that have collapsed after inflation has taken place (acquired atelectasis or alveolar collapse) (figs. - and - ). during fetal life, lungs are not fully distended, contain no air, and are partially filled with a locally produced fluid known as fetal lung fluid. not surprisingly, lungs of aborted and stillborn fetuses sink when placed in water, whereas those from animals that have breathed float. at the time of birth, fetal lung fluid is rapidly reabsorbed and replaced by inspired air, leading to the normal distention of alveoli. congenital atelectasis occurs in newborns that fail to inflate their lungs after taking their first few breaths of air; it is caused by obstruction of airways, often as a result of aspiration of amniotic fluid and meconium (described in the section on meconium aspiration syndrome) (see fig. - ). congenital atelectasis also develops when alveoli cannot remain distended after initial aeration because of an alteration in quality and quantity of pulmonary surfactant produced by type ii pneumonocytes and club (clara) cells. this infant form of congenital atelectasis is referred to in human neonatology as infant respiratory distress syndrome (irds) or as hyaline membrane disease because of the clinical and microscopic features of the disease. it commonly occurs in babies who are premature or born to diabetic or alcoholic mothers and is occasionally found in animals, particularly foals and piglets. the pathetic, gasping attempts of affected foals and pigs to breathe have prompted the use of the name "barkers"; foals that survive may have brain damage from cerebral hypoxia (see chapter ) and are referred to as "wanderers" due to their aimless behavior and lack of a normal sense of fear. acquired atelectasis is much more common and occurs in two main forms: compressive and obstructive (see fig. - ). compressive atelectasis has two main causes: space-occupying masses in the pleural cavity, such as abscesses and tumors, or transferred pressures, such as that caused by bloat, hydrothorax, hemothorax, chylothorax, and empyema ( fig. - ). another form of compressive atelectasis occurs when the negative pressure in the thoracic cavity is lost because of pneumothorax. this form generally has massive atelectasis and thus is also referred to as lung collapse. obstructive (absorption) atelectasis occurs when there is a reduction in the diameter of the airways caused by mucosal edema and inflammation, or when the lumen of the airway is blocked by fig. - ) to heterotopic ossification of the lungs (e- fig. - ). in most cases, pulmonary calcification in itself has little clinical significance, although its cause (e.g., uremia or vitamin d toxicosis) may be very important. alveolar filling disorders are a heterogeneous group of lung diseases characterized by accumulation of various chemical compounds in the alveolar lumens. the most common are alveolar proteinosis, in which the alveoli are filled with finely granular eosinophilic material; pulmonary lipidosis, in which alveoli are filled with macrophages containing endogenous or exogenous lipid; and alveolar microlithiasis, in which the alveoli contain numerous concentric calcified "microliths" or "calcospherites." a similar but distinct concretion is known as corpora amylacea, which is an accumulation of laminated bodies composed of cellular debris, lipids, proteins, and possibly amyloid. for most alveolar filling disorders, there is little host response, and in many cases, it is an incidental finding. most of the alveolar filling disorders originate from inherited metabolic defects in which alveolar cells (epithelial or macrophages) cannot properly metabolize or remove lipids or proteins, whereas others result from an excessive synthesis of these substances in the lung. endogenous lipid (lipoid) pneumonia. endogenous lipid pneumonia is an obscure, subclinical pulmonary disease of cats and occasionally of dogs, which is unrelated to aspiration of foreign material. although the pathogenesis is not understood, it is presumed that lipids from pulmonary surfactant and from degenerated cells accumulate within alveolar macrophages. accumulation of surfactant lipids can occur in metabolic abnormalities of alveolar macrophages or in bronchial obstruction where surfactant-laden macrophages cannot exit the lungs via the mucociliary escalator. the gross lesions are multifocal, white, firm nodules scattered throughout the lungs (e- fig. - ) . microscopically, the alveoli are filled with foamy lipid-laden macrophages accompanied by interstitial infiltration of lymphocytes and plasma cells, fibrosis, alveolar epithelialization, and, in some cases, cholesterol clefts and lipid granulomas. lipid (lipoid) pneumonia occurs frequently in the vicinity of cancerous lung lesions in human beings, cats, and dogs. the reason for this association remains unknown and frequently unrecognized appearance of atelectasis is more common in species with poor collateral ventilation, such as cattle and pigs. the extent and location of obstructive atelectasis depends largely on the size of the affected airway (large vs. small) and on the degree of obstruction (partial vs. complete). atelectasis also occurs when large animals are kept recumbent for prolonged periods, such as during anesthesia (hypostatic atelectasis). the factors contributing to hypostatic atelectasis are a combination of blood-air imbalance, shallow breathing, airway obstruction because of mucus and fluid that has not been drained from bronchioles and alveoli, and from inadequate local production of surfactant. atelectasis can also be a sequel to paralysis of respiratory muscles and prolonged use of mechanical ventilation or general anesthesia in intensive care. in general, the lungs with atelectasis appear depressed below the surface of the normally inflated lung. the color is generally dark blue, and the texture is flabby or firm; they are firm if there is concurrent edema or other processes, such as can occur in ards or "shock" lungs (see the section on pulmonary edema). distribution and extent vary with the process, being patchy (multifocal) in congenital atelectasis, lobular in the obstructive type, and of various degrees in between in the compressive type. microscopically, the alveoli are collapsed or slitlike and the alveolar walls appear parallel and close together, giving prominence to the interstitial tissue even without any superimposed inflammation. pulmonary emphysema. pulmonary emphysema, often simply referred to as emphysema, is an extremely important primary disease in human beings, whereas in animals, it is always a secondary condition resulting from a variety of pulmonary lesions. in human medicine, emphysema is strictly defined as an abnormal permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of alveolar walls (alveolar emphysema). this definition separates it from simple airspace enlargement or hyperinflation, in which there is no destruction of alveolar walls and which can occur congenitally (down syndrome) or be acquired with age (aging lung, sometimes misnamed "senile emphysema"). the pathogenesis of emphysema in human beings is still controversial, but current thinking overwhelmingly suggests that destruction of mucus plugs, exudate, aspirated foreign material, or lungworms (see fig. - ). when the obstruction is complete, trapped air in the lung eventually becomes reabsorbed. unlike the compression type, obstructive atelectasis often has a lobular pattern as a result of blockage of the airway supplying that lobule. this lobular lungs are extremely well-vascularized organs with a dual circulation provided by pulmonary and bronchial arteries. disturbances in pulmonary circulation have a notable effect on gaseous exchange, which may result in life-threatening hypoxemia and acidosis. in addition, circulatory disturbances in the lungs can have an impact on other organs, such as the heart and liver. for example, impeded blood flow in the lungs because of chronic pulmonary disease results in cor pulmonale, which is caused by unremitting pulmonary hypertension followed by cardiac dilation, right heart failure, chronic passive congestion of the liver (nutmeg liver), and generalized edema (anasarca). hyperemia is an active process that is part of acute inflammation, whereas congestion is the passive process resulting from decreased outflow of venous blood, as occurs in congestive heart failure ( fig. - ). in the early acute stages of pneumonia, the lungs appear notably red, and microscopically, blood vessels and alveolar capillaries are engorged with blood from alveolar walls is largely the result of an imbalance between proteases released by phagocytes and antiproteases produced in the lung as a defense mechanism (the protease-antiprotease theory). the destructive process in human beings is markedly accelerated by defects in the synthesis of antiproteases or any factor, such as cigarette smoking or pollution, that increases the recruitment of macrophages and leukocytes in the lungs. this theory originated when it was found that human beings with homozygous α -antitrypsin deficiency were remarkably susceptible to emphysema and that proteases (elastase) inoculated intratracheally into the lungs of laboratory animals produced lesions similar to those found in the disease. more than % of the problem relates to cigarette smoking, and airway obstruction is no longer considered to play a major role in the pathogenesis of emphysema in human beings. primary emphysema does not occur in animals, and thus no animal disease should be called simply emphysema. in animals, this lesion is always secondary to obstruction of outflow of air or is agonal at slaughter. secondary pulmonary emphysema occurs frequently in animals with bronchopneumonia, in which exudate plugging bronchi and bronchioles causes an airflow imbalance where the volume of air entering exceeds the volume leaving the lung. this airflow imbalance is often promoted by the so-called one-way valve effect caused by the exudate, which allows air into the lung during inspiration but prevents movement of air out of the lung during expiration. depending on the localization in the lung, emphysema can be classified as alveolar or interstitial. alveolar emphysema characterized by distention and rupture of the alveolar walls, forming variably sized air bubbles in pulmonary parenchyma, occurs in all species. interstitial emphysema occurs mainly in cattle, presumably because of their wide interlobular septa, and lack of collateral ventilation in these species does not permit air to move freely into adjacent pulmonary lobules. as a result, accumulated air penetrates the alveolar and bronchiolar walls and forces its way into the interlobular connective tissue, causing notable distention of the interlobular septa. it is also suspected that forced respiratory movements predispose to interstitial emphysema when air at high pressure breaks into the loose connective tissue of the interlobular septa ( fig. - ). sometimes these bubbles of trapped air in alveolar or interstitial emphysema become confluent, forming large (several centimeters in diameter) pockets of air that are referred to as bullae (singular: bulla) (see e- fig. - ); the lesion is then called bullous emphysema. this lesion is not a specific type of emphysema and does not indicate a different disease process but, rather, is a larger accumulation of air at one focus. in the most severe cases, air moves from the interlobular septa into the connective tissue surrounding the main stem bronchi and major vessels (bronchovascular bundles), and from here it leaks into the mediastinum, causing pneumomediastinum first, and eventually exits via the thoracic inlet into the cervical and thoracic subcutaneous tissue causing subcutaneous emphysema. note that mild and even moderate alveolar emphysema is difficult to judge at necropsy and by light microscopy unless special techniques are used to prevent collapse of the lung when the thorax is opened. these techniques include plugging of the trachea or intratracheal perfusion of fixative ( % neutral-buffered formalin) before the thorax is opened to prevent collapse of the lungs. important diseases that cause secondary pulmonary emphysema in animals include small airway obstruction (e.g., heaves) in horses and pulmonary edema and emphysema (fog fever) in cattle (see fig. - ) and exudates in bronchopneumonia. congenital emphysema occurring secondary to bronchial cartilage hypoplasia with subsequent bronchial collapse is occasionally reported in dogs. severe and persistent cases of heart failure, the lungs fail to collapse because of edema and pulmonary fibrosis. terminal pulmonary congestion (acute) is frequently seen in animals euthanized with barbiturates and should not be mistaken for an antemortem lesion. hypostatic congestion is another form of pulmonary congestion that results from the effects of gravity and poor circulation on a highly vascularized tissue, such as the lung. this type of gravitational congestion is characterized by the increase of blood in the lower side of the lung, particularly the lower lung of animals in lateral recumbency, and is most notable in horses and cattle. the affected portions of the lung appear dark red and can have a firmer texture. in animals and human beings who have been prostrated for extended periods of time, hypostatic congestion may be followed by hypostatic edema, and hypostatic pneumonia as edema interferes locally with the bacterial defense mechanisms. pulmonary hemorrhage. pulmonary hemorrhages can occur as a result of trauma, coagulopathies, and disseminated intravascular coagulation (dic), vasculitis, sepsis, and pulmonary thromboembolism from jugular thrombosis or from embolism of exudate from a hepatic abscess that has eroded the wall and ruptured into the caudal vena cava (cattle). a gross finding often confused with intravital pulmonary hemorrhage is the result of severing both the trachea and the carotid arteries simultaneously at slaughter. blood is aspirated from the transected trachea into the lungs, forming a random pattern of irregular red foci ( to mm) in one or more lobes. these red foci are readily visible on both the pleural and the cut surfaces of the lung, and free blood is visible in the lumens of bronchi and bronchioles. rupture of a major pulmonary vessel with resulting massive hemorrhage occurs occasionally in cattle when a growing abscess in a lung invades and disrupts the wall of a major pulmonary artery or vein ( fig. - ). in most cases, animals die rapidly, often with spectacular hemoptysis, and on postmortem examination, bronchi are filled with blood (see fig. - ). pulmonary edema. in normal lungs, fluid from the vascular space slowly but continuously passes into the interstitial tissue, where it is rapidly drained by the pulmonary and pleural lymphatic vessels. clearance of alveolar fluid across the alveolar epithelium is also a major mechanism of fluid removal from the lung. edema develops when the rate of fluid transudation from pulmonary vessels into the interstitium or alveoli exceeds that of lymphatic and alveolar removal ( fig. - ). pulmonary edema can be physiologically classified as cardiogenic (hydrostatic; hemodynamic) and noncardiogenic (permeability) types. hydrostatic (cardiogenic) pulmonary edema develops when there is an elevated rate of fluid transudation because of increased hydrostatic pressure in the vascular compartment or decreased osmotic pressure in the blood. once the lymph drainage has been overwhelmed, fluid accumulates in the perivascular spaces, causing distention of the bronchovascular bundles and alveolar interstitium, and eventually leaks into the alveolar spaces. causes of hemodynamic pulmonary edema include congestive heart failure (increased hydrostatic pressure); iatrogenic fluid overload; and disorders in which blood osmotic pressure is reduced, such as with hypoalbuminemia seen in some hepatic diseases, nephrotic syndrome, and protein-losing enteropathy. hemodynamic pulmonary edema also occurs when lymph drainage is impaired, generally secondary to neoplastic invasion of lymphatic vessels. permeability edema (inflammatory) occurs when there is excessive opening of endothelial gaps or damage to the cells that constitute the blood-air barrier (endothelial cells or type i pneumonocytes). hyperemia. pulmonary congestion is most frequently caused by heart failure, which results in stagnation of blood in pulmonary vessels, leading to edema and egression of erythrocytes into the alveolar spaces. as with any other foreign particle, erythrocytes in alveolar spaces are rapidly phagocytosed (erythrophagocytosis) by pulmonary alveolar macrophages. when extravasation of erythrocytes is severe, large numbers of macrophages with brown cytoplasm may accumulate in the bronchoalveolar spaces. the brown cytoplasm is the result of accumulation of considerable amounts of hemosiderin; these macrophages filled with iron pigment (siderophages) are generally referred to as heart failure cells ( fig. - ). the lungs of animals with chronic heart failure usually have a patchy red appearance with foci of brown discoloration because of accumulated hemosiderin. in this type of edema is an integral and early part of the inflammatory response, primarily because of the effect of inflammatory mediators, such as leukotrienes, platelet-activating factor (paf), cytokines, and vasoactive amines released by neutrophils, macrophages, mast cells, lymphocytes, endothelial cells, and type ii pneumonocytes. these inflammatory mediators increase the permeability of the blood-air barrier. in other cases, permeability edema results from direct damage to the endothelium or type i pneumonocytes, allowing plasma fluids to move freely from the vascular space into the alveolar lumen ( fig. - and see fig. - ). because type i pneumonocytes are highly vulnerable to some pneumotropic viruses (influenza and brsv), toxicants (nitrogen dioxide [no ], sulfur dioxide [so ], hydrogen sulfide [h s], and -methylindole), and particularly to free radicals, it is not surprising that permeability edema commonly accompanies many viral or toxic pulmonary diseases. a permeability edema also occurs when endothelial cells in the lung are injured by bacterial toxins, sepsis, ards, dic, anaphylactic shock, milk allergy, paraquat toxicity, adverse drug reactions, and smoke inhalation (e- fig. - ) . the concentration of protein in edematous fluid is greater in permeability edema (exudate) than in hemodynamic edema (transudate); this difference has been used clinically in human medicine to differentiate one type of pulmonary edema from another. microscopically, because of the higher concentration of protein, edema fluid in lungs with inflammation or damage to the blood-air barrier tends to stain more intensely eosinophilic than that of the hydrostatic edema from heart failure. grossly, the edematous lungs-independent of the cause-are wet and heavy. the color varies, depending on the degree of congestion or hemorrhage, and fluid may be present in the pleural cavity. if edema is severe, the bronchi and trachea contain considerable amounts of foamy fluid, which originates from the mixing of edema fluid and air ( fig. - ). on cut surfaces, the lung parenchyma oozes fluid like a wet sponge. in cattle and pigs that have distinct lobules, the lobular pattern becomes rather accentuated because of edematous distention of lymphatic vessels in the interlobular septa and the edematous interlobular septum itself ( fig. - ). severe pulmonary edema may be impossible to differentiate from peracute pneumonia; (h&e)-stained sections (see fig. - ), particularly if a fixative such as zenker's solution, which precipitates protein, is used. acute respiratory distress syndrome. acute (adult) respiratory distress syndrome (ards; shock lung) is an important condition in human beings and animals characterized by pulmonary hypertension, intravascular aggregation of neutrophils in the lungs, acute lung injury, diffuse alveolar damage, permeability edema, and formation of hyaline membranes ( fig. - ) . these membranes are a mixture of plasma proteins, fibrin, surfactant, and cellular debris from necrotic pneumonocytes (see fig. - , b) . the pathogenesis of ards is complex and multifactorial but in general terms can be defined as diffuse alveolar damage that results from lesions in distant organs, from generalized systemic diseases, or from direct injury to the lung. sepsis, major trauma, aspiration of gastric contents, extensive burns, and pancreatitis are some of the disease entities known to trigger ards. all these conditions provoke "hyperreactive macrophages" to directly or indirectly generate overwhelming amounts of cytokines causing what is known as a "cytokine storm." the main cytokines that trigger ards are tnf-α, interleukin (il)- , il- , and il- , which prime neutrophils previously recruited in the lung capillaries and alveoli to release cytotoxic enzymes and free radicals. these substances cause severe and diffuse endothelial and alveolar damage that culminates in a fulminating pulmonary edema (see fig. - ). ards occurs in domestic animals and explains why pulmonary edema is one of the most common lesions found in many animals dying of sepsis, toxemia, aspiration of gastric contents, and pancreatitis, for example. a familial form of ards has been reported in dalmatians. the pulmonary lesions in this syndrome are further discussed in the sections on interstitial pneumonia and aspiration pneumonia in dogs. neurogenic pulmonary edema is another distinctive but poorly understood form of life-threatening lung edema in human beings that follows cns injury and increased intracranial pressure (i.e., head injury, brain edema, brain tumors, or cerebral hemorrhage). this type of pulmonary edema can be experimentally reproduced in laboratory animals by injecting fibrin into the fourth ventricle. it involves both hemodynamic and permeability pathways presumably from massive sympathetic stimulation and overwhelming release of catecholamines. neurogenic pulmonary edema has sporadically been reported in animals with brain injury or severe seizures or after severe stress and excitement. pulmonary embolism. with its vast vascular bed and position in the circulation, the lung acts as a safety net to catch emboli before they reach the brain and other tissues. however, this positioning is often to its own detriment. the most common pulmonary emboli in domestic animals are thromboemboli, septic (bacterial) emboli, fat emboli, and tumor cell emboli. pulmonary thromboembolism (pte) refers to both local thrombus formation and translocation of a thrombus present elsewhere in the venous circulation ( fig. - ). fragments released inevitably reach the lungs and become trapped in the pulmonary vasculature ( fig. - and see fig. - ). small sterile thromboemboli are generally of little clinical or pathologic significance because they can be rapidly degraded and disposed of by the fibrinolytic system. larger thromboemboli may cause small airway constriction, reduced surfactant production, pulmonary edema, and atelectasis resulting in hypoxemia, hyperventilation, and dyspnea. parasites (e.g., dirofilaria immitis and angiostrongylus vasorum), endocrinopathies (e.g., hyperadrenocorticism and hypothyroidism), glomerulopathies, and hypercoagulable states can be responsible for pulmonary arterial thrombosis and pulmonary thromboembolism in dogs (e- fig. - ) . pieces of this fact is not surprising because pulmonary edema occurs in the very early stages of inflammation (see e- fig. - ) . careful observation of the lungs at the time of necropsy is critical because diagnosis of pulmonary edema cannot be reliably performed microscopically. this is due in part to the loss of the edema fluid from the lungs during fixation with % neutral-buffered formalin and in part to the fact that the fluid itself stains very poorly or not at all with eosin because of its low protein content (hemodynamic edema). a protein-rich (permeability) edema is easier to visualize microscopically because it is deeply eosinophilic in hematoxylin and eosin fig. - ) . , normal alveolar capillary externally covered by type i and type ii pneumonocytes and internally by vascular endothelium (see fig. - for more detail). , at the early stages of sepsis, proinflammatory cytokines (interleukin [il- ] and tumor necrosis factor [tnf]) cause circulating neutrophils to adhere to the endothelial surface. following a "cytokine storm," the marginated neutrophils further activated by inflammatory mediators suddenly release their cytoplasmic granules (proteolytic enzymes and elastases myeloperoxidase) into the surrounding milieu (arrows). , enzymes released by these neutrophils cause injury to type i pneumonocytes (arrows) and endothelial cells (arrowheads), disrupting the blood-air barrier and causing permeability edema (curved arrows), alveolar hemorrhage (double-headed arrow), and exocytosis of neutrophils into the alveolar space (double-headed arrow). , extravasated plasma proteins admixed with surfactant and cell debris form thick hyaline membranes along the alveolar wall. , in the unlikely event that the animal survives, the healing process starts with alveolar macrophages removing cellular debris, reabsorption of edema, and hyperplasia of type ii pneumonocytes (double-headed curved arrow) that subsequently differentiate into type i pneumonocytes (see fig. b a recognized in the bovine lung after strong pneumatic stunning at slaughter (captive bolt) ( fig. - , a) . although obviously not important as an antemortem pulmonary lesion, brain emboli are intriguing as a potential risk for public health control of bovine spongiform encephalopathy (bse). fragments of hair can also embolize to the lung following intravenous injections (see fig. - , b). hepatic emboli formed by circulating pieces of fragmented liver occasionally become trapped in the pulmonary vasculature after severe abdominal trauma and hepatic rupture (see fig. - , c) . megakaryocytes trapped in alveolar capillaries are a common but incidental microscopic finding in the lungs of all species, particularly dogs (see fig. - , d) . tumor emboli (e.g., osteosarcoma and hemangiosarcoma in dogs and uterine carcinoma in cattle) can be numerous and striking and the ultimate cause of death in malignant neoplasia. in experimental studies, cytokines released during pulmonary inflammation are chemotactic for tumor cells and promote pulmonary metastasis. pulmonary infarcts. because of a dual arterial supply to the lung, pulmonary infarction is rare and generally asymptomatic. however, pulmonary infarcts can be readily caused when pulmonary thrombosis and embolism are superimposed on an already compromised pulmonary circulation such as occurs in congestive heart c d thrombi breaking free from a jugular, femoral, or uterine vein can cause pulmonary thromboembolism. pulmonary thromboembolisms occur in heavy horses after prolonged anesthesia (deep vein thrombosis), recumbent cows ("downer cow syndrome"), or in any animal undergoing long-term intravenous catheterization in which thrombi build up in the catheter and then break off (see fig. - ). septic emboli, pieces of thrombi contaminated with bacteria or fungi and broken free from infected mural or valvular thrombi in the heart and vessels, eventually become entrapped in the pulmonary circulation. pulmonary emboli originate most commonly from bacterial endocarditis (right side) and jugular thrombophlebitis in all species, hepatic abscesses that have eroded and discharged their contents into the caudal vena cava in cattle, and septic arthritis and omphalitis in farm animals (see fig. - ). when present in large numbers, septic emboli may cause unexpected death because of massive pulmonary edema; survivors generally develop pulmonary arteritis and thrombosis and embolic (suppurative) pneumonia, which may lead to pulmonary abscesses. bone marrow and bone emboli can form after bone fractures or surgical interventions of bone. these are not as significant a problem in domestic animals as they are in human beings. brain emboli (i.e., pieces of brain tissue) in the pulmonary vasculature reported in severe cases of head injury in human beings have recently been pulmonary macrophages (alveolar, intravascular, and interstitial), which have an immense biologic armamentarium, are the single most important effector cell and source of cytokines for all stages of pulmonary inflammation. these all-purpose phagocytic cells modulate the recruitment and trafficking of blood-borne leukocytes in the lung through the secretion of chemokines (see e- table - ). before reviewing how inflammatory cells are recruited in the lungs, three significant features in pulmonary injury must be remembered: ( ) leukocytes can exit the vascular system through the alveolar capillaries, unlike in other tissues, where postcapillary venules are the sites of leukocytic diapedesis (extravasation); ( ) the intact lung contains within alveolar capillaries a large pool of resident leukocytes (marginated pool); and ( ) additional neutrophils are sequestered within alveolar capillaries within minutes of a local or systemic inflammatory response. these three pulmonary idiosyncrasies, along with the enormous length of the capillary network in the lung, explain why recruitment and migration of leukocytes into alveolar spaces develops so rapidly. experimental studies with aerosols of endotoxin or gram-negative bacteria have shown that within minutes of exposure, there is a significant increase in capillary leukocytes, and by hours the alveolar lumen is filled with neutrophils. not surprisingly, the bal fluid collected from patients with acute pneumonia contains large amounts of inflammatory mediators such as tnf-α, il- , and il- . also, the capillary endothelium of patients with acute pneumonia has increased "expression" of adhesion molecules, which facilitate the migration of leukocytes from capillaries into the alveolar interstitium and from there into the alveolar lumen. in allergic pulmonary diseases, eotaxin and il- are primarily responsible for recruitment and trafficking of eosinophils in the lung. movement of plasma proteins into the pulmonary interstitium and alveolar lumen is a common but poorly understood phenomenon in pulmonary inflammation. leakage of fibrinogen and plasma proteins into the alveolar space occurs when there is structural damage to the blood-air barrier. this leakage is also promoted by some types of cytokines that enhance procoagulant activity, whereas others reduce fibrinolytic activity. excessive exudation of fibrin into the alveoli is particularly common in ruminants and pigs. the fibrinolytic system plays a major role in the resolution of pulmonary inflammatory diseases. in some cases, excessive plasma proteins leaked into alveoli mix with necrotic type i pneumonocytes and pulmonary surfactant, forming microscopic eosinophilic bands (membranes) along the lining of alveolar septa. these membranes, known as hyaline membranes, are found in specific types of pulmonary diseases, particularly in ards, and in cattle with acute interstitial pneumonias such as bovine pulmonary edema and emphysema and extrinsic allergic alveolitis (see pneumonias of cattle). in the past few years, nitric oxide has been identified as a major regulatory molecule of inflammation in a variety of tissues, including the lung. produced locally by macrophages, pulmonary endothelium, and pneumonocytes, nitric oxide regulates the vascular and bronchial tone, modulates the production of cytokines, controls the recruitment and trafficking of neutrophils in the lung, and switches on/off genes involved in inflammation and immunity. experimental work has also shown that pulmonary surfactant upregulates the production of nitric oxide in the lung, supporting the current view that pneumonocytes are also pivotal in amplifying and downregulating the inflammatory and immune responses in the lung (see e- table - ). as the inflammatory process becomes chronic, the types of cells making up cellular infiltrates in the lung change from mainly neutrophils to largely mononuclear cells. this shift in cellular composition is accompanied by an increase in specific cytokines, such as failure. it also occurs in dogs with torsion of a lung lobe (fig. - ) . the gross features of infarcts vary considerably, depending on the stage, and they can be red to black, swollen, firm, and cone or wedge shaped, particularly at the lung margins. in the early acute stage, microscopic lesions are severely hemorrhagic, and this is followed by necrosis. in or days, a border of inflammatory cells develops, and a few days later, a large number of siderophages are present in the necrotic lung. if sterile, pulmonary infarcts heal as fibrotic scars; if septic, an abscess may form surrounded by a thick fibrous capsule. in the past three decades, an information explosion has increased the overall understanding of pulmonary inflammation, with so many proinflammatory and antiinflammatory mediators described to date that it would be impossible to review them all here (see chapters and ). pulmonary inflammation is a highly regulated process that involves a complex interaction between cells imported from the blood (platelets, neutrophils, eosinophils, mast cells, and lymphocytes) and pulmonary cells (type i and ii pneumonocytes; endothelial and club [clara] cells; alveolar and intravascular macrophages; and stromal interstitial cells, such as mast cells, interstitial macrophages, fibroblasts, and myofibroblasts). blood-borne leukocytes, platelets, and plasma proteins are brought into the areas of inflammation by an elaborate network of chemical signals emitted by pulmonary cells and resident leukocytes. long-distance communication between pulmonary cells and blood cells is largely done by soluble cytokines; once in the lung, imported leukocytes communicate with pulmonary and vascular cells through adhesion and other inflammatory molecules. the best known inflammatory mediators are the complement system (c a, c b, and c a), coagulation factors (factors v and vii), arachidonic acid metabolites (leukotrienes and prostaglandins), cytokines (interleukins, monokines, and chemokines), adhesion molecules (icam and vcam), neuropeptides (substance p, tachykinins, and neurokinins), enzymes and enzyme inhibitors (elastase and antitrypsin), oxygen metabolites (o •, oh•, and h o ), antioxidants (glutathione), and nitric oxide (e -table - ). acting in concert, these and many other molecules send positive or negative signals to initiate, maintain, and, it is hoped, resolve the inflammatory process without causing injury to the lung. chapter respiratory system, mediastinum, and pleurae e- with several episodes of hemorrhage are characterized by large areas of dark brown discoloration, largely in the caudal lung lobes. microscopically, lesions are alveolar hemorrhages, abundant alveolar macrophages containing hemosiderin (siderophages), mild alveolar fibrosis, and occlusive remodeling of pulmonary veins. recurrent airway obstruction. recurrent airway obstruction (rao) of horses, also referred to as copd, heaves, chronic bronchiolitis-emphysema complex, chronic small airway disease, alveolar emphysema, and "broken wind," is a common clinically asthma-like syndrome of horses and ponies. rao is characterized by recurrent respiratory distress, chronic cough, poor athletic performance, airway neutrophilia, bronchoconstriction, mucus hypersecretion, and airway obstruction. the pathogenesis is still obscure, but genetic predisposition, t h (allergic) immune response, and the exceptional sensitivity of airways to environmental allergens (hyperreactive airway disease) have been postulated as the basic underlying mechanisms. what makes small airways hyperreactive to allergens is still a matter of controversy. epidemiologic and experimental studies suggest that it could be the result of preceding bronchiolar damage caused by viral infections; ingestion of pneumotoxicants ( -methylindole); or prolonged exposure to organic dust, endotoxin, and environmental allergens (molds). it has been postulated that sustained inhalation of dust particles, whether antigenic or not, upregulates the production of cytokines (tnf-α, il- , and monokine-inducible protein ) and neuropeptides (neurokinin a [nka], neurokinin b [nkb], and substance p), attracting neutrophils into the bronchioloalveolar region and promoting leukocyte-induced bronchiolar injury. summer pasture-associated obstructive pulmonary disease (spaopd) is a seasonal airway disease also reported in horses with similar clinical and pathologic findings. more recently, the term inflammatory airway disease (iad) has been introduced in equine medicine to describe rao-like syndrome in young horses to years old. the lungs of horses with heaves are grossly unremarkable, except for extreme cases in which alveolar emphysema may be present. microscopically, the lesions are often remarkable and include goblet cell metaplasia in bronchioles; plugging of bronchioles with mucus mixed with few eosinophils and neutrophils (see fig. - ); peribronchiolar infiltration with lymphocytes, plasma cells, and variable numbers of eosinophils; and hypertrophy of smooth muscle in bronchi and bronchioles. in severe cases, accumulation of mucus leads to the complete obstruction of bronchioles and alveoli and resultant alveolar emphysema characterized by enlarged "alveoli" from the destruction of alveolar walls. feline asthma syndrome. feline asthma syndrome, also known as feline allergic bronchitis, is a clinical syndrome in cats of any age characterized by recurrent episodes of bronchoconstriction, cough, or dyspnea. the pathogenesis is not well understood but is presumed to originate, as in human asthma, as a type i hypersensitivity (igemast cell reaction) to inhaled allergens. dust, cigarette smoke, plant and household materials, and parasitic proteins have been incriminated as possible allergens. this self-limited allergic disease responds well to steroid therapy; thus it is rarely implicated as a primary cause of death except when suppressed defense mechanisms allow a secondary bacterial pneumonia. bronchial biopsies from affected cats at the early stages reveal mild to moderate inflammation characterized by mucosal edema and infiltration of leukocytes, particularly eosinophils. increased numbers of circulating eosinophils (blood eosinophilia) are present in some but not all cats with feline asthma. il- , interferon-γ (ifn-γ), and interferon-inducible protein (ip- ), which are chemotactic for lymphocytes and macrophages. under appropriate conditions, these cytokines activate t lymphocytes, regulate granulomatous inflammation, and induce the formation of multinucleated giant cells such as in mycobacterial infections. inflammatory mediators locally released from inflamed lungs also have a biologic effect in other tissue. for example, pulmonary hypertension and right-sided heart failure (cor pulmonale) often follows chronic alveolar inflammation, not only as a result of increased pulmonary blood pressure but also from the effect of inflammatory mediators on the contractibility of smooth muscle of the pulmonary and systemic vasculature. cytokines, particularly tnf-α, that are released during inflammation are associated, both as cause and as effect, with the systemic inflammatory response syndrome (sirs), sepsis, severe sepsis with multiple organ dysfunction, and septic shock (cardiopulmonary collapse). as it occurs in any other sentinel system where many biologic promoters and inhibitors are involved (coagulation, the complement and immune systems), the inflammatory cascade could go into an "out-of-control" state, causing severe damage to the lungs. acute lung injury (ali), extrinsic allergic alveolitis, ards, pulmonary fibrosis, and asthma are archetypical diseases that ensue from an uncontrolled production and release of cytokines (cytokine storm). as long as acute alveolar injury is transient and there is no interference with the normal host response, the entire process of injury, degeneration, necrosis, inflammation, and repair can occur in less than days. on the other hand, when acute alveolar injury becomes persistent or when the capacity of the host for repair is impaired, lesions can progress to an irreversible stage in which restoration of alveolar structure is no longer possible. in diseases, such as extrinsic allergic alveolitis, the constant release of proteolytic enzymes and free radicals by phagocytic cells perpetuates alveolar damage in a vicious circle. in other cases, such as in paraquat toxicity, the magnitude of alveolar injury can be so severe that type ii pneumonocytes, basement membranes, and alveolar interstitium are so disrupted that the capacity for alveolar repair is lost. fibronectins and transforming growth factors (tgfs) released from macrophages and other mononuclear cells at the site of chronic inflammation regulate the recruitment, attachment, and proliferation of fibroblasts. in turn, these cells synthesize and release considerable amounts of ecm (collagen, elastic fibers, or proteoglycans), eventually leading to fibrosis and total obliteration of normal alveolar architecture. in summary, in diseases in which there is chronic and irreversible alveolar damage, lesions invariably progress to a stage of terminal alveolar and interstitial fibrosis. for pneumonia, see section species-specific pneumonia of domestic animals. exercise-induced pulmonary hemorrhage. exercise-induced pulmonary hemorrhage (eiph) is a specific form of pulmonary hemorrhage in racehorses that occurs after exercise and clinically is characterized by epistaxis. because only a small percentage of horses with bronchoscopic evidence of hemorrhage have clinical epistaxis, it is likely that eiph goes undetected in many cases. the pathogenesis is still controversial, but current literature suggests laryngeal paralysis, bronchiolitis, and extremely high pulmonary vascular and alveolar pressures during exercise, alveolar hypoxia, and preexisting pulmonary injury as possible causes. eiph is seldom fatal; postmortem lesions in the lungs of horses that have been affected disease may be known by different names. in pigs, for instance, enzootic pneumonia and mycoplasma pneumonia refer to the same disease caused by mycoplasma hyopneumoniae. the word pneumonitis has been used by some as a synonym for pneumonia; however, others have restricted this term to chronic proliferative inflammation generally involving the alveolar interstitium and with little or no evidence of exudate. in this chapter, the word pneumonia is used for any inflammatory lesion in the lungs, regardless of whether it is exudative or proliferative, alveolar, or interstitial. on the basis of texture, distribution, appearance, and exudation, pneumonias can be grossly diagnosed into four morphologically distinct types: bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia. by using this classification, it is possible at the time of a necropsy to predict with some degree of certainty the likely cause (virus, bacteria, fungi, or parasites), routes of entry (aerogenous vs. hematogenous), and possible sequelae. these four morphologic types allow the clinician or pathologists to predict the most likely etiology and therefore facilitate the decision as to what samples need to be taken and which tests should be requested to the diagnostic laboratory (i.e., histopathology, bacteriology, virology, or toxicology). however, overlapping of these four types of pneumonias is possible, and sometimes two morphologic types may be present in the same lung. the criteria used to classify pneumonias grossly into bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia are based on morphologic changes, including distribution, texture, color, and general appearance of the affected lungs (table - ). distribution of the inflammatory lesions in the lungs can be ( ) cranioventral, as in most bronchopneumonias; ( ) multifocal, as in embolic pneumonias; ( ) diffuse, as in interstitial pneumonias; or ( ) locally extensive, as in granulomatous in the most advanced cases, chronic bronchoconstriction and excess mucus production may result in smooth muscle hyperplasia and obstruction of the bronchi and bronchioles and infiltration of the airway mucosa by eosinophils. a syndrome known as canine asthma has been reported in dogs but is not as well characterized as the feline counterpart. few subjects in veterinary pathology have caused so much debate as the classification of pneumonias. historically, pneumonias in animals have been classified or named based on the following: . presumed cause, with names such as viral pneumonia, pasteurella pneumonia, distemper pneumonia, verminous pneumonia, chemical pneumonia, and hypersensitivity pneumonitis . type of exudation, with names such as suppurative pneumonia, fibrinous pneumonia, and pyogranulomatous pneumonia . morphologic features, with names such as gangrenous pneumonia, proliferative pneumonia, and embolic pneumonia . distribution of lesions, with names such as focal pneumonia, cranioventral pneumonia, diffuse pneumonia, and lobar pneumonia . epidemiologic attributes, with names such as enzootic pneumonia, contagious bovine pleuropneumonia, and "shipping fever" . geographic regions, with names such as montana progressive pneumonia . miscellaneous attributes, with names such as atypical pneumonia, cuffing pneumonia, progressive pneumonia, aspiration pneumonia, pneumonitis, farmer's lung, and extrinsic allergic alveolitis until a universal and systematic nomenclature for animal pneumonias is established, veterinarians should be acquainted with this heterogeneous list of names and should be well aware that one the parts of the face with the tip of your finger has been advocated by some pathologists. the texture of a normal lung is comparable to the texture of the center of the cheek. firm consolidation is comparable to the texture of the tip of the nose, and hard consolidation is comparable to the texture of the forehead. the term consolidation is frequently used to describe a firm or hard lung filled with exudate. pneumonias ( fig. - ) . texture of pneumonic lungs can be firmer or harder (bronchopneumonias), more elastic (rubbery) than normal lungs (interstitial pneumonias), or have a nodular feeling (granulomatous pneumonias). describing in words the palpable difference between the texture of a normal lung compared with the firm or hard texture of a consolidated lung can be a difficult undertaking. an analogy illustrating this difference based on touching changes in the gross appearance of pneumonic lungs include abnormal color, the presence of nodules or exudate, fibrinous or fibrous adhesions, and the presence of rib imprints on serosal surfaces (see fig. - ). on cut surfaces, pneumonic lungs may have exudate, hemorrhage, edema, necrosis, abscesses, bronchiectasis, granulomas or pyogranulomas, and fibrosis, depending on the stage. palpation and careful observation of the lungs are essential in the diagnosis of pneumonia. (for details, see the section on examination of the respiratory tract.) bronchopneumonia refers to a particular morphologic type of pneumonia in which injury and the inflammatory process take place primarily in the bronchial, bronchiolar, and alveolar lumens. bronchopneumonia is undoubtedly the most common type of pneumonia seen in domestic animals and is with few exceptions characterized grossly by cranioventral consolidation of the lungs (fig. - and see fig. - ). the reason why bronchopneumonias in animals are almost always restricted to the cranioventral portions of the lungs is not well understood. possible factors contributing to this topographic selectivity within the lungs include ( ) gravitational sedimentation of the exudate, ( ) greater deposition of infectious organisms, ( ) inadequate defense mechanisms, ( ) reduced vascular perfusion, ( ) shortness and abrupt branching of airways, and ( ) regional differences in ventilation. the term cranioventral in veterinary anatomy is the equivalent of "anterosuperior" in human anatomy. the latter is defined as "in front (ventral) and above (cranial)." thus, applied to the lung of animals, "cranioventral" means the ventral portion of the cranial lobe. however, by common usage in veterinary pathology, the term cranioventral used to describe the location of lesions in pneumonias has come to mean "cranial and ventral." thus it includes pneumonias affecting not only the ventral portion of the cranial lobe (true cranioventral) but also those cases in which the pneumonia has involved the ventral portions of adjacent lung lobes-initially the middle and then caudal on the right and the caudal lobe on the left side. bronchopneumonias are generally caused by bacteria and mycoplasmas, by bronchoaspiration of feed or gastric contents, or by improper tubing. as a rule, the pathogens causing bronchopneumonias arrive in the lungs via inspired air (aerogenous), either from infected aerosols or from the nasal flora. before establishing infection, pathogens must overwhelm or evade the pulmonary defense mechanisms. the initial injury in bronchopneumonias is centered on the mucosa of bronchioles; from there, the inflammatory process can spread downward to distal portions of the alveoli and upward to the bronchi. typically, for bronchopneumonias, the inflammatory exudates collect in the bronchial, bronchiolar, and alveolar lumina leaving the alveolar interstitium relatively unchanged, except for hyperemia and possibly edema. through the pores of kohn, the exudate can spread to adjacent alveoli until most or all of the alveoli in an individual lobule are involved. if the inflammatory process cannot control the inciting cause of injury, the lesions spread rapidly from lobule to lobule through alveolar pores and destroyed alveolar walls until an entire lobe or large portion of a lung is involved. the lesion tends to spread centrifugally, with the older lesions in the center, and exudate can be coughed up and then aspirated into other lobules, where the inflammatory process starts again. at the early stages of bronchopneumonia, the pulmonary vessels are engorged with blood (active hyperemia), and the bronchi, bronchioles, and alveoli contain some fluid (permeability edema). in cases in which pulmonary injury is mild to moderate, cytokines locally released in the lung cause rapid recruitment of neutrophils and alveolar macrophages into bronchioles and alveoli ( fig. - and see fig. - ). when pulmonary injury is much more severe, proinflammatory cytokines induce more pronounced vascular changes by further opening endothelial gaps, thus increasing vascular permeability resulting in leakage of plasma fibrinogen (fibrinous exudates) and sometimes hemorrhage in the alveoli. alterations in permeability can be further exacerbated by structural damage to pulmonary capillaries and vessels directly caused by microbial toxins. filling of alveoli, bronchioles, and small bronchi with inflammatory exudate progressively obliterates airspaces, and as a consequence of this process, portions of severely affected (consolidated) lungs sink to the bottom of the container when placed in fixative. the replacement of air by exudate also changes the texture of the lungs, and depending on the severity of bronchopneumonia, the texture varies from firmer to harder than normal. the term consolidation is used at gross examination when the texture of pneumonic lung becomes firmer or harder than normal as a result of loss of airspaces because of exudation and atelectasis. (for details, see the discussion of lung texture in the section on classification of pneumonias in domestic animals). inflammatory consolidation of the lungs has been referred to in the past as hepatization because the affected lung had the appearance and texture of liver. the process was referred to as red hepatization in acute cases in which ( ) congestion, ( ) red hepatization (liver texture), ( ) gray hepatization, and ( ) resolution. because of the use of effective antibiotics and prevention, pneumococcal pneumonia and its four classic stages are rarely seen; thus this terminology has been largely abandoned. currently, the term bronchopneumonia is widely used for both suppurative and fibrinous consolidation of the lungs because both forms of inflammation have essentially the same pathogenesis in which the pathogens reach the lung by the aerogenous route, injury occurs initially in the bronchial and bronchiolar regions, and the inflammatory process extends centrifugally deep into the alveoli. it must be emphasized that it is the severity of pulmonary injury that largely determines whether bronchopneumonia becomes suppurative or fibrinous. in some instances, however, it is difficult to discriminate between suppurative and fibrinous bronchopneumonia because both types can coexist (fibrinosuppurative bronchopneumonia), and one type can progress to the other. suppurative bronchopneumonia. suppurative bronchopneumonia is characterized by cranioventral consolidation of lungs (see figs. - and - ), with typically purulent or mucopurulent exudate present in the airways. this exudate can be best demonstrated by expressing intrapulmonary bronchi, thus forcing exudate out of the bronchi (see fig. - ). the inflammatory process in suppurative bronchopneumonia is generally confined to individual lobules, and as a result of this distribution, the lobular pattern of the lung becomes notably emphasized. this pattern is particularly obvious in cattle and pigs because these species have prominent lobulation of the lungs. the gross appearance often resembles an irregular checkerboard because of an admixture of normal and abnormal (consolidated) lobules (see fig. - ). because of this typical lobular distribution, suppurative bronchopneumonias are also referred to as lobular pneumonias. different inflammatory phases occur in suppurative bronchopneumonia where the color and appearance of consolidated lungs varies considerably, depending on the virulence of offending organisms and chronicity of the lesion. the typical phases of suppurative bronchopneumonia can be summarized as follows: . during the first hours when bacteria are rapidly multiplying, the lungs become hyperemic and edematous. . soon after, neutrophils start filling the airways, and by hours the parenchyma starts to consolidate and becomes firm in texture. . three to days later, hyperemic changes are less obvious, but the bronchial, bronchiolar, and alveolar spaces continue to fill with neutrophils and macrophages, and the affected lung sinks when placed in formalin. at this stage, the affected lung has a gray-pink color, and on cut surface, purulent exudate can be expressed from bronchi. . in favorable conditions where the infection is under control of the host defense mechanisms, the inflammatory processes begin to regress, a phase known as resolution. complete resolution in favorable conditions could take to weeks. . in animals in which the lung infection cannot be rapidly contained, inflammatory lesions can progress into a chronic phase. approximately to days after infection, the lungs become pale gray and take a "fish flesh" appearance. this appearance is the result of purulent and catarrhal inflammation, obstructive atelectasis, mononuclear cell infiltration, peribronchial and peribronchiolar lymphoid hyperplasia, and early alveolar fibrosis. complete resolution is unusual in chronic bronchopneumonia, and lung scars, such as pleural and pulmonary fibrosis; bronchiectasis as a consequence of chronic destructive bronchitis (see bronchiectasis [dysfunction/responses to injury and patterns of injury]); atelectasis; pleural adhesions; and lung abscesses may remain unresolved there was notable active hyperemia with little exudation of neutrophils; conversely, the process was referred to as gray hepatization in those chronic cases in which hyperemia was no longer present, but there was abundant exudation of neutrophils and macrophages. this terminology, although used for and applicable to human pneumonias, is rarely used in veterinary medicine primarily because the evolution of pneumonic processes in animals does not necessarily follow the red-to-gray hepatization pattern. bronchopneumonia can be subdivided into suppurative bronchopneumonia if the exudates are predominantly composed of neutrophils and fibrinous bronchopneumonia if fibrin is the predominant component of the exudates (see table - ). it is important to note that some veterinarians use the term fibrinous pneumonia or lobar pneumonia as a synonym for fibrinous bronchopneumonia, and bronchopneumonia or lobular pneumonia as a synonym for suppurative bronchopneumonia. human pneumonias for many years have been classified based on their etiology and morphology, which explains why pneumococcal pneumonia (streptococcus pneumoniae) has been synonymous with lobar pneumonia. in the old literature, four distinct stages of pneumococcal pneumonia were described as lumen of bronchiole capillary pulmonary defense mechanisms to allow them to colonize the lungs and establish an infection. suppurative bronchopneumonia can also result from aspiration of bland material (e.g., milk). pulmonary gangrene may ensue when the bronchopneumonic lung is invaded by saprophytic bacteria (aspiration pneumonia). fibrinous bronchopneumonia. fibrinous bronchopneumonia is similar to suppurative bronchopneumonia except that the predominant exudate is fibrinous rather than neutrophilic. with only a few exceptions, fibrinous bronchopneumonias also have a cranioventral distribution (fig. - and see fig. - ) . however, exudation is not restricted to the boundaries of individual pulmonary lobules, as is the case in suppurative bronchopneumonias. instead, the inflammatory process in fibrinous pneumonias involves numerous contiguous lobules and the exudate moves quickly through pulmonary tissue until the entire pulmonary lobe is rapidly affected. because of the involvement of the entire lobe and pleural surface, fibrinous bronchopneumonias are also referred to as lobar pneumonias or pleuropneumonias. in general terms, fibrinous bronchopneumonias are the result of more severe pulmonary injury and thus cause death earlier in the sequence of the inflammatory process than suppurative bronchopneumonias. even in cases in which fibrinous bronchopneumonia involves % or less of the total area, clinical signs and death can occur as a result of severe toxemia and sepsis. the gross appearance of fibrinous bronchopneumonia depends on the age and severity of the lesion and on whether the pleural surface or the cut surface of the lung is viewed. externally, early stages of fibrinous bronchopneumonias are characterized by severe congestion and hemorrhage, giving the affected lungs a characteristically intense red discoloration. a few hours later, fibrin starts to permeate and accumulate on the pleural surface, giving the pleura a ground glass appearance and eventually forming plaques of fibrinous exudate over a red, dark lung (see fig. - ). at this stage, a yellow fluid starts to accumulate in the thoracic cavity. the color of fibrin deposited over the pleural surface is also variable. it can be bright yellow when the exudate is formed primarily by fibrin, tan when fibrin is mixed with blood, and gray when a large number of leukocytes and fibroblasts are part of the fibrinous plaque in more chronic cases. because of the tendency of fibrin to deposit on the pleural surface, some pathologists use the term pleuropneumonia as a synonym for fibrinous bronchopneumonia. on the cut surface, early stages of fibrinous bronchopneumonia appear as simple red consolidation. in more advanced cases ( hours), fibrinous bronchopneumonia is generally accompanied by notable dilation and thrombosis of lymph vessels and edema of interlobular septa (see fig. - , b) . this distention of the interlobular septa gives affected lungs a typical marbled appearance. distinct focal areas of coagulative necrosis in the pulmonary parenchyma are also common in fibrinous bronchopneumonia such as in shipping fever pneumonia and contagious bovine pleuropneumonia. in animals that survive the early stage of fibrinous bronchopneumonia, pulmonary necrosis often develops into pulmonary "sequestra," which are isolated pieces of necrotic lung encapsulated by connective tissue. pulmonary sequestra result from extensive necrosis of lung tissue either from severe ischemia (infarct) caused by thrombosis of a major pulmonary vessel such as in contagious bovine pleuropneumonia or from the effect of necrotizing toxins released by pathogenic bacteria such as mannheimia haemolytica. sequestra in veterinary pathology should not be confused with "bronchopulmonary sequestration," a term used in human pathology to describe a congenital malformation in which whole lobes or parts of the lung develop without normal connections to the airway or vascular systems. for a long time. "enzootic pneumonias" of ruminants and pigs are typical examples of chronic suppurative bronchopneumonias. microscopically, acute suppurative bronchopneumonias are characterized by hyperemia, abundant neutrophils, macrophages, and cellular debris within the lumen of bronchi, bronchioles, and alveoli (see fig. - ). recruitment of leukocytes is promoted by cytokines, complement, and other chemotactic factors that are released in response to alveolar injury or by the chemotactic effect of bacterial toxins, particularly endotoxin. in most severe cases, purulent or mucopurulent exudates completely obliterate the entire lumen of bronchi, bronchioles, and alveoli. if suppurative bronchopneumonia is merely the response to a transient pulmonary injury or a mild infection, lesions resolve uneventfully. within to days, cellular exudate can be removed from the lungs via the mucociliary escalator, and complete resolution may take place within weeks. in other cases, if injury or infection is persistent, suppurative bronchopneumonia can become chronic with goblet cell hyperplasia, an important component of the inflammatory process. depending on the proportion of pus and mucus, the exudate in chronic suppurative bronchopneumonia varies from mucopurulent to mucoid. a mucoid exudate is found in the more chronic stages when the consolidated lung has a "fish flesh" appearance. hyperplasia of balt is another change commonly seen in chronic suppurative bronchopneumonias; it appears grossly as conspicuous white nodules (cuffs) around bronchial walls (cuffing pneumonia). this hyperplastic change merely indicates a normal reaction of lymphoid tissue to infection. further sequelae of chronic suppurative bronchopneumonia include bronchiectasis (see figs. - and - ), pulmonary abscesses, pleural adhesions (from pleuritis) ( fig. - ) , and atelectasis and emphysema from completely or partially obstructed bronchi or bronchioles (e.g., bronchiectasis). clinically, suppurative bronchopneumonias can be acute and fulminating but are often chronic, depending on the etiologic agent, stressors affecting the host, and immune status. the most common pathogens causing suppurative bronchopneumonia in domestic animals include pasteurella multocida, bordetella bronchiseptica, trueperella (arcanobacterium) pyogenes, streptococcus spp., escherichia coli, and several species of mycoplasmas. most of these organisms are secondary pathogens requiring a preceding impairment of the fulminating hemorrhagic bronchopneumonia can be caused by highly pathogenic bacteria such as bacillus anthracis. although the lesions in anthrax are primarily related to a severe septicemia and sepsis, anthrax should always be suspected in animals with sudden death and exhibiting severe acute fibrinohemorrhagic pneumonia, splenomegaly, and multisystemic hemorrhages. animals are considered good sentinels for anthrax in cases of bioterrorism. interstitial pneumonia refers to that type of pneumonia in which injury and the inflammatory process take place primarily in any microscopically, in the initial stage of fibrinous bronchopneumonia, there is massive exudation of plasma proteins into the bronchioles and alveoli, and as a result, most of the airspaces become obliterated by fluid and fibrin. leakage of fibrin and fluid into alveolar lumina is due to extensive disruption of the integrity and increased permeability of the blood-air barrier. fibrinous exudates can move from alveolus to alveolus through the pores of kohn. because fibrin is chemotactic for neutrophils, these types of leukocytes are always present a few hours after the onset of fibrinous inflammation. as inflammation progresses ( to days), fluid exudate is gradually replaced by fibrinocellular exudates composed of fibrin, neutrophils, macrophages, and necrotic debris ( fig. - ). in chronic cases (after days), there is notable fibrosis of the interlobular septa and pleura. in contrast to suppurative bronchopneumonia, fibrinous bronchopneumonia rarely resolves completely, thus leaving noticeable scars in the form of pulmonary fibrosis and pleural adhesions. the most common sequelae found in animals surviving an acute episode of fibrinous bronchopneumonia include alveolar fibrosis and bronchiolitis obliterans, in which organized exudate becomes attached to the bronchiolar lumen (see fig. - ) . these changes are collectively referred to as bronchiolitis obliterans organizing pneumonia (boop), a common microscopic finding in animals with unresolved bronchopneumonia. other important sequelae include pulmonary gangrene, when saprophytic bacteria colonize necrotic lung; pulmonary sequestra; pulmonary fibrosis; abscesses; and chronic pleuritis with pleural adhesions. in some cases, pleuritis can be so extensive that fibrous adhesions extend onto the pericardial sac. pathogens causing fibrinous bronchopneumonias in domestic animals include mannheimia (pasteurella) haemolytica (pneumonic mannheimiosis), histophilus somni (formerly haemophilus somnus), actinobacillus pleuropneumoniae (porcine pleuropneumonia), mycoplasma bovis, and mycoplasma mycoides ssp. mycoides small colony type (contagious bovine pleuropneumonia). fibrinous broncho- a b n alveolar epithelium. inhaled antigens, such as fungal spores, combine with circulating antibodies and form deposits of antigen-antibody complexes (type iii hypersensitivity) in the alveolar wall, which initiate a cascade of inflammatory responses and injury (allergic alveolitis). hematogenous injury to the vascular endothelium occurs in septicemias, sepsis, dic, larva migrans (ascaris suum), toxins absorbed in the alimentary tract (endotoxin) or toxic metabolites locally generated in the lungs ( -methylindole and paraquat), release of free radicals in alveolar capillaries (ards), and infections with endotheliotropic viruses (canine adenovirus and classical swine fever [hog cholera]). interstitial pneumonias in domestic animals and human beings are subdivided based on morphologic features into acute and chronic. it should be kept in mind, however, that not all acute interstitial pneumonias are fatal and that they do not necessarily progress to the chronic form. acute interstitial pneumonias. acute interstitial pneumonias begin with injury to either type i pneumonocytes or alveolar capillary endothelium, which provokes a disruption of the blood-air barrier and a subsequent exudation of plasma proteins into the alveolar space (see fig. - ) . this leakage of proteinaceous fluid into the alveolar lumen constitutes the exudative phase of acute interstitial pneumonia. in some cases of diffuse alveolar damage, exuded plasma proteins mix with lipids and other components of pulmonary surfactant and form elongated membranes that become partially attached to the alveolar basement membrane and bronchiolar walls. these membranes are referred to as hyaline membranes because of their hyaline appearance (eosinophilic, homogeneous, and amorphous) microscopically (see figs. - and - ). in addition to intraalveolar exudation of fluid, inflammatory edema and neutrophils accumulate in the alveolar interstitium and cause thickening of the alveolar walls. this acute exudative phase is generally followed a few days later by the proliferative phase of acute interstitial pneumonia, which is characterized by hyperplasia of type ii pneumonocytes to replace the lost type i pneumonocytes (see fig. - ). type ii pneumonocytes are in fact progenitor cells that differentiate and replace necrotic type i pneumonocytes (see fig. - ) . as a consequence, the alveolar walls become increasingly thickened. this process is in part the reason why lungs become rubbery on palpation, what prevents their normal collapse after the thorax is opened, and why the cut surface of the lung has a "meaty" appearance (see fig. - ). of the three layers of the alveolar walls (endothelium, basement membrane, and alveolar epithelium) and the contiguous bronchiolar interstitium (see fig. - ) . this morphologic type of pneumonia is the most difficult to diagnose at necropsy and requires microscopic confirmation because it is easily mistaken in the lung showing congestion, edema, hyperinflation, or emphysema. in contrast to bronchopneumonias, in which distribution of lesions is generally cranioventral, in interstitial pneumonias, lesions are more diffusely distributed and generally involve all pulmonary lobes, or in some cases, they appear to be more pronounced in the dorsocaudal aspects of the lungs (see fig. - ). three important gross features of interstitial pneumonia are ( ) the failure of lungs to collapse when the thoracic cavity is opened, ( ) the occasional presence of rib impressions on the pleural surface of the lung indicating poor deflation, and ( ) the lack of visible exudates in airways unless complicated with secondary bacterial pneumonia. the color of affected lungs varies from diffusely red in acute cases to diffusely pale gray to a mottled red, pale appearance in chronic ones. pale lungs are caused by severe obliteration of alveolar capillaries (reduced blood-tissue ratio), especially evident when there is fibrosis of the alveolar walls. the texture of lungs with uncomplicated interstitial pneumonia is typically elastic or rubbery, but definitive diagnosis based on texture alone is difficult and requires histopathologic examination. on a cut surface, the lungs may appear and feel more "meaty" (having the texture of raw meat) and have no evidence of exudate in the bronchi or pleura (fig. - ). in acute interstitial pneumonias, particularly in cattle, there is frequently pulmonary edema (exudative phase) and interstitial emphysema secondary to partial obstruction of bronchioles by edema fluid and strenuous air gasping before death. because edema tends to gravitate into the cranioventral portions of the lungs, and emphysema is often more obvious in the dorsocaudal aspects, acute interstitial pneumonias in cattle occasionally have a gross cranioventral-like pattern that may resemble bronchopneumonia, although the texture is different. lungs are notably heavy because of the edema and the infiltrative and proliferative changes. the pathogenesis of interstitial pneumonia is complex and can result from aerogenous injury to the alveolar epithelium (type i and ii pneumonocytes) or from hematogenous injury to the alveolar capillary endothelium or alveolar basement membrane. aerogenous inhalation of toxic gases (i.e., ozone and no ) or toxic fumes (smoke inhalation) and infection with pneumotropic viruses (influenza, herpesviruses, or canine distemper virus) can damage the figure - interstitial pneumonia, lung, feeder pig. a, the lung is heavy, pale, and rubbery in texture. it also has prominent costal (rib) imprints (arrows), a result of hypercellularity of the interstitium and the failure of the lungs to collapse when the thorax was opened. b, transverse section. the pulmonary parenchyma has a "meaty" appearance and some edema, but no exudate is present in airways or on the pleural surface. this type of lung change in pigs is highly suggestive of a viral pneumonia. (courtesy dr. a. lópez, atlantic veterinary college.) pneumonia are centered in the alveolar wall and its interstitium, a mixture of desquamated epithelial cells, macrophages, and mononuclear cells are usually present in the lumens of bronchioles and alveoli. ovine progressive pneumonia, hypersensitivity pneumonitis in cattle and dogs, and silicosis in horses are good veterinary examples of chronic interstitial pneumonia. pneumoconioses (silicosis and asbestosis), paraquat toxicity, pneumotoxic antineoplastic drugs (bleomycin), and extrinsic allergic alveolitis (farmer's lung) are well-known examples of diseases that lead to chronic interstitial pneumonias in human beings. massive pulmonary migration of ascaris larvae in pigs also causes interstitial pneumonia ( fig. - ). there is an insidious and poorly understood group of chronic idiopathic interstitial diseases, both in human beings and in animals, that eventually progress to terminal interstitial fibrosis. these were originally thought to be the result of repeated cycles of alveolar injury, inflammation, and fibroblastic/myoblastic response to an unknown agent. however, aggressive antiinflammatory therapy generally fails to prevent or reduce the severity of fibrosis. now, it is acute interstitial pneumonias are often mild and transient, especially those caused by some respiratory viruses, such as those responsible for equine and porcine influenza. these mild forms of pneumonia are rarely seen in the postmortem room because they are not fatal and do not leave significant sequelae (see the section on defense mechanisms/barrier systems). in severe cases of acute interstitial pneumonias, animals may die of respiratory failure, usually as a result of diffuse alveolar damage, a profuse exudative phase (leakage of proteinaceous fluid) leading to a fatal pulmonary edema. examples of this type of fatal acute interstitial pneumonia are bovine pulmonary edema and emphysema, and ards in all species. chronic interstitial pneumonia. when the source of alveolar injury persists, the exudative and proliferative lesions of acute interstitial pneumonia can progress into a morphologic stage referred to as chronic interstitial pneumonia. the hallmark of chronic interstitial pneumonia is fibrosis of the alveolar walls (with or without intraalveolar fibrosis) and the presence of lymphocytes, macrophages, fibroblasts, and myofibroblasts in the alveolar interstitium (figs. - and - ). in other cases, these chronic changes are accompanied by hyperplasia and persistence of type ii pneumonocytes, squamous metaplasia of the alveolar epithelium, microscopic granulomas, and hyperplasia of smooth muscle in bronchioles and pulmonary arterioles. it should be emphasized that although the lesions in interstitial the term bronchointerstitial pneumonia is used in veterinary pathology to describe cases in which microscopic lesions share some histologic features of both bronchopneumonia and interstitial pneumonia (e- fig. - ). this combined type of pneumonia is in fact frequently seen in many viral infections in which viruses replicate and cause necrosis in bronchial, bronchiolar, and alveolar cells. damage to the bronchial and bronchiolar epithelium causes an influx of neutrophils similar to that in bronchopneumonias, and damage to alveolar walls causes proliferation of type ii pneumonocytes, similar to that which takes place in the proliferative phase of acute interstitial pneumonias. it is important to emphasize that bronchointerstitial pneumonia is a microscopic not a gross diagnosis. examples include uncomplicated cases of respiratory syncytial virus infections in cattle and lambs, canine distemper, and influenza in pigs and horses. embolic pneumonia refers to a particular type of pneumonia in which gross and microscopic lesions are multifocally distributed in all pulmonary lobes. by definition, lung injury is hematogenous, and the inflammatory response is typically centered in pulmonary arterioles and alveolar capillaries. lungs act as a biologic filter for circulating particulate matter. sterile thromboemboli, unless extremely large, are rapidly dissolved and removed from the pulmonary vasculature by fibrinolysis, causing little, if any, ill effects. experimental studies have confirmed that most types of bacteria when injected intravenously (bacteremia) are phagocytosed by pulmonary intravascular macrophages, or they bypass the lungs and are finally trapped by macrophages in the liver, spleen, joints, or other organs. to cause pulmonary infection, circulating bacteria must first attach to the pulmonary endothelium with specific binding proteins or simply attach to intravascular fibrin and then evade phagocytosis by intravascular macrophages or leukocytes. septic thrombi facilitate entrapment of bacteria in the pulmonary vessels and provide a favorable environment to escape phagocytosis. once trapped in the pulmonary vasculature, usually in small arterioles or alveolar capillaries, offending bacteria disrupt endothelium and basement membranes, spread from the vessels to the interstitium and then to the surrounding lung, finally forming a new nidus of infection. embolic pneumonia is characterized by multifocal lesions randomly distributed in all pulmonary lobes (see fig. - and e-figs. - and - ). early lesions in embolic pneumonia are characterized grossly by the presence of very small ( to mm), white foci surrounded by discrete, red, hemorrhagic halos ( fig. - ). unless emboli arrive in massive numbers, causing fatal pulmonary edema, embolic pneumonia is seldom fatal; therefore these acute lesions are rarely seen at postmortem examination. in most instances, if unresolved, acute lesions rapidly progress to pulmonary abscesses. these are randomly distributed in all pulmonary lobes and are not restricted to the cranioventral aspects of the lungs, as is the case of abscesses developing from suppurative bronchopneumonia. the early microscopic lesions in embolic pneumonias are always focal or multifocal ( fig. - ) ; thus they differ from those of endotoxemia or septicemia, in which endothelial damage and interstitial reactions (interstitial pneumonia) are diffusely distributed in the lungs. when embolic pneumonia or its sequela (abscesses) is diagnosed at necropsy, an attempt should be made to locate the source of septic emboli. the most common sources are hepatic abscesses that have ruptured into the caudal vena cava in cattle, omphalophlebitis in farm animals, chronic bacterial skin or hoof infections, and a contaminated catheter in all species (see fig. - ) . valvular or mural endocarditis in the right heart is a common source of septic emboli and embolic pneumonia in all species. most frequently, bacterial proposed that a genetic mutation alters the cell-cell communication between epithelial and mesenchymal cells in the lung. this aberrant cellular communication leads to an overexpression of inflammatory and repair molecules (i.e., il- , il- , tgf-β , and caveolin), leading to increased apoptosis and interstitial deposition of extracellular matrix (ecm). the chronic interstitial (restrictive) diseases in human medicine include "idiopathic pulmonary fibrosis," "nonspecific interstitial pneumonia," "unusual interstitial pneumonia," and "cryptogenic organizing pneumonia," also referred to as idiopathic bronchiolitis obliterans organizing pneumonia (idiopathic boop). feline idiopathic pulmonary fibrosis is an example of this type of progressive interstitial disease in veterinary medicine. it has been reported that in rare cases, chronic alveolar remodeling and interstitial fibrosis can progress to lung cancer. the lung has numerous circular areas of hemorrhage distributed randomly throughout all lung lobes (embolic pattern [see fig. - ]). these foci arise from injury to the microvasculature in alveolar septa and the visceral pleura secondary to lodgment of bacterial or fungal emboli (septic emboli) from valvular or mural endocarditis in the right heart or from other bacterial or fungal diseases where the bacterium or fungus gains access to the circulatory system as occurs in many bacterial and fungal enteritides or pneumonias caused by salmonella spp., e. coli, or aspergillus spp. the pathogenesis of granulomatous pneumonia shares some similarities with that of interstitial and embolic pneumonias. not surprisingly, some pathologists group granulomatous pneumonias within one of these types of pneumonias (e.g., granulomatous interstitial pneumonia). what makes granulomatous pneumonia a distinctive type is not so much the portal of entry or site of initial injury in the lungs but, rather, the unique type of inflammatory response that results in the formation of granulomas, which can be easily recognized at gross and microscopic examination. as a rule, agents causing granulomatous pneumonias are resistant to intracellular killing by phagocytic cells and to the acute inflammatory response, allowing prolonged persistence of these agents in tissues. the most common causes of granulomatous pneumonia in animals include systemic fungal diseases, such as cryptococcosis (cryptococcus neoformans and cryptococcus gatti), coccidioidomycosis (coccidioides immitis), histoplasmosis (histoplasma capsulatum), and blastomycosis (blastomyces dermatitidis) (see fig. - ). in most of these fungal diseases, the port of entry is aerogenous, and from the lungs the fungi disseminate systemically to other organs, particularly the lymph nodes, liver, and spleen. filamentous fungi such as aspergillus spp. or mucor spp. can also reach the lung by the hematogenous route. granulomatous pneumonia is also seen in some bacterial diseases, such as tuberculosis (mycobacterium bovis) in all species and rhodococcus equi in horses. sporadically, aberrant parasites such as fasciola hepatica in cattle and aspiration of foreign bodies can also cause granulomatous pneumonia (e- fig. - granulomatous pneumonia is characterized by the presence of variable numbers of caseous or noncaseous granulomas randomly distributed in the lungs (see fig. - ). on palpation, lungs have a typical nodular character given by well-circumscribed, variably sized nodules that generally have a firm texture, especially if calcification has occurred ( fig. - ) . during postmortem examination, granulomas in the lungs occasionally can be mistaken for neoplasms. microscopically, pulmonary granulomas are composed of a center of necrotic tissue, surrounded by a rim of macrophages (epithelioid cells) and giant cells and an outer delineated layer of connective tissue commonly infiltrated by lymphocytes and plasma cells ( fig. - ). unlike other types of pneumonias, the causative agent in granulomatous pneumonia can, in many cases, be identified isolates from septic pulmonary emboli in domestic animals are trueperella (arcanobacterium) pyogenes (cattle), fusobacterium necrophorum (cattle, pigs, and human beings), erysipelothrix rhusiopathiae (pigs, cattle, dogs, and human beings), streptococcus suis (pigs), staphylococcus aureus (dogs and human beings), and streptococcus equi (horses). granulomatous pneumonia refers to a particular type of pneumonia in which aerogenous or hematogenous injury is caused by organisms or particles that cannot normally be eliminated by phagocytosis and that evoke a local inflammatory reaction with numerous alveolar and interstitial macrophages, lymphocytes, a few neutrophils, and sometimes giant cells. the term granulomatous is used here to describe an anatomic pattern of pneumonia typically characterized by the presence of granulomas. g g but yet unproven that viral infections may also predispose horses to airway hyperresponsiveness and recurrent airway obstruction (rao). equine influenza. equine influenza is an important and highly contagious flulike respiratory disease of horses characterized by high morbidity and low mortality and explosive outbreaks in susceptible populations. it is an oie-notifiable disease. two antigenically unrelated subtypes of equine influenza virus have been identified (h n [a/equi- ] and h n [a/equi- ]). the course of the disease is generally mild and transient, and its importance is primarily because of its economic impact on horse racing. the types of injury and host response in the conducting system are described in the section on disorders of the nasal cavity and paranasal sinuses of horses. uncomplicated lesions in the lungs are mild and self-limiting bronchointerstitial pneumonia. in fatal cases, the lungs are hyperinflated with coalescing areas of dark red discoloration. microscopically, there is a bronchointerstitial pneumonia characterized by necrotizing bronchiolitis that is followed by hyperplastic bronchiolitis, hyperplasia of type ii pneumonocytes, hyaline membranes in alveoli, and sporadic multinucleated giant cells. the microscopic changes are ards in severe and fatal cases. the influenza virus antigen can be readily demonstrated in ciliated cells and alveolar macrophages. clinical signs are characterized by fever, cough, abnormal lung sounds (crackles and wheezes), anorexia, and depression. secondary bacterial infections (streptococcus equi, streptococcus zooepidemicus, staphylococcus aureus, and escherichia coli) commonly complicate equine influenza. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr), or equine herpesvirus infection, is a respiratory disease of young horses that is particularly important in weanlings between and months of age and to a much lesser extent in young foals and adult horses. the causative agents are ubiquitous equine herpesviruses (ehv- and ehv- ) that in addition to respiratory disease can cause abortion in pregnant mares and neurologic disease (equine herpes myeloencephalopathy) (see the section on disorders of the nasal cavity and paranasal sinuses of horses). the respiratory form of evr is a mild and a transient bronchointerstitial pneumonia seen only by pathologists when complications with secondary bacterial infections cause a fatal bronchopneumonia microscopically in sections by pas reaction or grocott-gomori's methenamine silver (gms) stain for fungi or by an acid-fast stain for mycobacteria. viral infections of the respiratory tract, particularly equine viral rhinopneumonitis and equine influenza, are important diseases of horses throughout the world. the effects of these and other respiratory viruses on the horse can be manifested in three distinct ways. first, as pure viral infections, their severity may range from mild to severe, making them a frequent interfering factor in training and athletic performance. second, superimposed infections by opportunistic bacteria, such as streptococcus spp., escherichia coli, klebsiella pneumoniae, rhodococcus equi, and various anaerobes, can cause fibrinous or suppurative bronchopneumonias. third, it is possible equine henipavirus (hendra virus). fatal cases of a novel respiratory disease in horses and human beings suddenly appeared in approximately in hendra, a suburb of brisbane, australia. this outbreak was attributed to a newly recognized zoonotic virus that was tentatively named equine morbillivirus. now called hendra virus (hev), this emerging viral pathogen is currently classified as a member of the genus henipavirus (includes hendra virus and nipah virus), in the family paramyxoviridae. fruit bats (flying foxes) act as natural reservoirs and are involved in the transmission by poorly understood mechanisms. the lungs of affected horses are severely edematous with gelatinous distention of pleura and subpleural lymph vessels. microscopically, the lungs have diffuse alveolar edema associated with vasculitis, thrombosis, and the presence of multinucleated syncytial cells in the endothelium of small pulmonary blood vessels and alveolar capillaries. the lymphatic vessels are notably distended with fluid. the characteristic inclusion bodies seen in other paramyxovirus infections are not seen in horses; however, the virus can be easily detected by immunohistochemistry in pulmonary endothelial cells and alveolar epithelial cells (pneumonocytes). clinical signs are nonspecific and include fever, anorexia, respiratory distress, and nasal discharge. equine multinodular pulmonary fibrosis. equine multinodular pulmonary fibrosis is a lung disease characterized by well-demarcated fibrotic nodular lesions in the lung (e- fig. - ). until recently, the pathogenesis was unclear, but recent studies proposed equine herpesvirus (ehv- ) as the putative etiology. grossly, the lungs show multifocal to coalescing, firm tan nodules scattered in all pulmonary lobes, which resemble pulmonary neoplasia. microscopically, alveolar walls are thickened due to collagen deposition, infiltration of lymphocytes and macrophages, and cuboidal cells lining the alveolar walls. the alveolar lumens contain neutrophils and macrophages, some of which may contain a large eosinophilic intranuclear inclusion body. typical clinical signs include weight loss, low-grade fever, and progressive exercise intolerance. this condition has a poor prognosis. rhodococcus equi. rhodococcus equi is an important cause of morbidity and mortality in foals throughout the world. this facultative intracellular gram-positive bacterium causes two major forms of disease: the first involves the intestine, causing ulcerative enterocolitis, and the second severe and often fatal bronchopneumonia. although half of foals with pneumonia have ulcerative enterocolitis, it is rare to find animals with intestinal lesions alone. occasionally, infection disseminates to lymph nodes, joints, bones, the genital tract, and other organs. because rhodococcus equi is present in soil and feces of herbivores (particularly foals), it is common for the disease to become enzootic on farms ("hot spots") where the organism has been shed earlier by infected foals. serologic evidence of infection in horses is widespread, yet clinical disease is sporadic and largely restricted to young foals or to adult horses with severe immunosuppression. virulence factors encoded by plasmids (virulenceassociated protein a [vapa gene]) are responsible for the survival and replication of rhodococcus equi in macrophages, thus determining the evolution of the disease. this bacterium has also been sporadically incriminated with infections in cattle, goats, pigs, dogs, and cats, and quite often in immunocompromised human beings, for example, those infected with the aids virus, after organ transplantation, or undergoing chemotherapy. it is still debatable whether natural infection starts as a bronchopneumonia (aerogenous route) from which rhodococcus equi reaches the intestine via swallowed sputum or whether infection starts as an enteritis (oral route) with a subsequent bacteremia into the lungs. (streptococcus equi, streptococcus zooepidemicus, or staphylococcus aureus) . uncomplicated lesions in evr are seen only in aborted fetuses or in foals that die within the first few days of life. they consist of focal areas of necrosis ( . to mm) in various organs, including liver, adrenal glands, and lungs. in some cases, intranuclear inclusion bodies are microscopically observed in these organs. outbreaks of interstitial pneumonia in donkeys have been attributed to multiple strains of asinine herpesviruses (ahv- and - ). clinically, horses and donkeys affected with the respiratory form of evr exhibit fever, anorexia, conjunctivitis, cough, and nasal discharge. equine viral arteritis. equine viral arteritis (eva), a pansystemic disease of horses, donkeys, and mules caused by an arterivirus (equine arteritis virus [eav]), occurs sporadically throughout the world, sometimes as an outbreak. this virus infects and causes severe injury to macrophages and endothelial cells. gross lesions are hemorrhage and edema in many sites, including lungs, intestine, scrotum, and periorbital tissues and voluminous hydrothorax and hydroperitoneum. the basic lesion is fibrinoid necrosis and inflammation of the vessel walls (vasculitis), particularly the small muscular arteries (lymphocytic arteritis), which is responsible for the edema and hemorrhage that explain most of the clinical features. pulmonary lesions are those of interstitial pneumonia with hyperplasia of type ii pneumonocytes and vasculitis with abundant edema in the bronchoalveolar spaces and distended pulmonary lymphatic vessels. viral antigen can be detected by immunoperoxidase techniques in the walls and endothelial cells of affected pulmonary vessels and in alveolar macrophages. clinical signs are respiratory distress, fever, abortion, diarrhea, colic, and edema of the limbs and ventral abdomen. respiratory signs are frequent and consist of serous or mucopurulent rhinitis and conjunctivitis with palpebral edema. like most viral respiratory infections, eva can predispose horses to opportunistic bacterial pneumonias. african horse sickness. african horse sickness (ahs) is an arthropod-borne, oie-notifiable disease of horses, mules, donkeys, and zebras that is caused by an orbivirus (family reoviridae) and characterized by respiratory distress or cardiovascular failure. ahs has a high mortality rate-up to % in the native population of horses in africa, the middle east, india, pakistan, and, most recently, spain and portugal. although the ahs virus is transmitted primarily by insects (culicoides) to horses, other animals, such as dogs, can be infected by eating infected equine flesh. the pathogenesis of african horse sickness remains unclear, but this equine orbivirus has an obvious tropism for pulmonary and cardiac endothelial cells and, to a lesser extent, mononuclear cells. based on clinical signs (not pathogenesis), african horse sickness is arbitrarily divided into four different forms: pulmonary, cardiac, mixed, and mild. the pulmonary form is characterized by severe respiratory distress and rapid death because of massive pulmonary edema, presumably from viral injury to the pulmonary endothelial cells. grossly, large amounts of froth are present in the airways, lungs fail to collapse, subpleural lymph vessels are distended, and the ventral parts of the lungs are notably edematous (see fig. - ) . in the cardiac form, recurrent fever is detected, and heart failure results in subcutaneous and interfascial edema, most notably in the neck and supraorbital region. the mixed form is a combination of the respiratory and cardiac forms. finally, the mild form, rarely seen in postmortem rooms, is characterized by fever and clinical signs resembling those of equine influenza; it is in most cases transient and followed by a complete recovery. this mild form is most frequently seen in donkeys, mules, and zebras and in horses with some degree of immunity. detection of viral antigen for diagnostic purposes can be done by immunohistochemistry in paraffin-embedded tissues. chapter respiratory system, mediastinum, and pleurae clinically, rhodococcus equi infection can be acute, with rapid death caused by severe bronchopneumonia, or chronic, with depression, cough, weight loss, and respiratory distress. in either form, there may be diarrhea, arthritis, osteomyelitis, or subcutaneous abscess formation. parascaris equorum. parascaris equorum is a large nematode (roundworm) of the small intestine of horses; the larval stages migrate through the lungs as ascarid larvae do in pigs. it is still unclear whether migration of parascaris equorum larvae can cause significant pulmonary lesions under natural conditions. experimentally, migration of larvae results in coughing, anorexia, weight loss, and small necrotic foci and petechial hemorrhages in the liver, hepatic and tracheobronchial lymph nodes, and lungs. microscopically, eosinophils are prominent in the interstitium and airway mucosa during the parasitic migration and in focal granulomas caused by dead larvae in the lung. dictyocaulus arnfieldi. dictyocaulus arnfieldi is not a very pathogenic nematode, but it should be considered if there are signs of coughing in horses that are pastured together with donkeys. donkeys are considered the natural hosts and can tolerate large numbers of parasites without ill effects. dictyocaulus arnfieldi does not usually become patent in horses, so examination of fecal samples is not useful; bal is only occasionally diagnostic because eosinophils (but not parasites) are typically found in the lavage fluid. mature parasites (up to cm in length) cause obstructive bronchitis, edema, and atelectasis, particularly along the dorsocaudal lung. the microscopic lesion is an eosinophilic bronchitis similar to the less acute infestations seen in cattle and sheep with their dictyocaulus species. the results of experimental studies suggest that natural infection likely starts from inhalation of infected dust or aerosols. once in the lung, rhodococcus equi is rapidly phagocytosed by alveolar macrophages, but because of defective phagosome-lysosome fusion and premature lysosomal degranulation, bacteria survive and multiply intracellularly, eventually leading to the destruction of the macrophage. interestingly, rhodococcus equi appears to be easily killed by neutrophils but not macrophages. released cytokines and lysosomal enzymes and bacterial toxins are responsible for extensive caseous necrosis of the lungs and the recruitment of large numbers of neutrophils, macrophages, and giant cells containing intracellular gram-positive organisms in their cytoplasm. depending on the stage of infection and the immune status and age of affected horses, pulmonary lesions induced by rhodococcus equi can vary from pyogranulomatous to granulomatous pneumonia. in young foals, the infection starts as a suppurative cranioventral bronchopneumonia, which progresses within a few days into small variable-size pulmonary abscesses. these abscesses rapidly transform into pyogranulomatous nodules, some of which become confluent and form large masses of caseous exudate ( fig. - ). microscopically, the early lesion starts with neutrophilic infiltration, followed by an intense influx of alveolar macrophages into the bronchoalveolar spaces. this type of histiocytic inflammation persists for a long period of time because rhodococcus equi is a facultative intracellular organism that survives the bactericidal effects of equine alveolar macrophages. in the most chronic cases, the pulmonary lesions culminate with the formation of large caseonecrotic masses with extensive fibrosis of the surrounding pulmonary parenchyma. pcr analysis of tracheobronchial aspirates has successfully been used as an alternative to bacteriologic culture in the diagnosis of rhodococcus equi infection in live foals. b a rhinotracheitis (ibr)/bovine herpes virus (bohv- ), bovine parainfluenza virus (bpiv- ), and bovine respiratory syncytial virus (brsv); and noninfectious interstitial pneumonias, such as bovine pulmonary edema and emphysema, reinfection syndrome, and many others. bovine enzootic pneumonia. enzootic pneumonia, sometimes simply referred to as calf pneumonia, is a multifactorial disease caused by a variety of etiologic agents that produces an assortment of lung lesions in young, intensively housed calves. the hostmicrobial-environmental triad is central in the pathogenesis of this disease. morbidity is often high (up to %), but fatalities are uncommon (> %) unless management is poor or unless new, virulent pathogens are introduced by additions to the herd. enzootic pneumonia is also called viral pneumonia because it often begins with an acute respiratory infection with bpiv- , brsv, or possibly with one or more of several other viruses (adenovirus, bohv- , reovirus, bovine coronavirus [bcov] , and bovine rhinitis virus). mycoplasmas, notably mycoplasma dispar, mycoplasma bovis, ureaplasma, and possibly chlamydophila, may also be primary agents. following infection with any of these agents, opportunistic bacteria, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, histophilus somni, mannheimia haemolytica, and escherichia coli, can cause a secondary suppurative bronchopneumonia, the most serious stage of enzootic pneumonia. the pathogenesis of the primary invasion and how it predisposes the host to invasion by the opportunists are poorly understood, but it is likely that there is impairment of pulmonary defense mechanisms. environmental factors, including air quality (poor ventilation), high relative humidity, and animal crowding, have been strongly incriminated. the immune status of the calf also plays an important role in the development and severity of enzootic pneumonia. calves with bovine leukocyte adhesion deficiency (blad), which prevents the migration of neutrophils from the capillaries, are highly susceptible to bronchopneumonia. lesions are variable and depend largely on the agents involved and on the duration of the inflammatory process. in the acute phases, lesions caused by viruses are those of bronchointerstitial pneumonia, which are generally mild and transient, and therefore are seen only sporadically at necropsy. microscopically, the lesions are necrotizing bronchiolitis, necrosis of type i pneumonocytes with hyperplasia of type ii pneumonocytes, and mild interstitial and alveolar edema. in the case of bpiv- and brsv infection, intracytoplasmic inclusion bodies and the formation of large multinucleated syncytia, resulting from the fusion of infected bronchiolar and alveolar epithelial cells, can also be observed in the lungs (fig. - ) . airway hyperreactivity has been described in calves after brsv infection; however, the significance of this syndrome in relation to enzootic pneumonia of calves is still under investigation. the mycoplasmas also can cause bronchiolitis, bronchiolar and alveolar necrosis, and an interstitial reaction, but in contrast to viral-induced pneumonias, mycoplasmal lesions tend to progress to a chronic stage characterized by striking peribronchiolar lymphoid hyperplasia (cuffing pneumonia). when complicated by secondary bacterial infections (e.g., pasteurella multocida and trueperella pyogenes), viral or mycoplasmal lesions change from a pure bronchointerstitial to a suppurative bronchopneumonia (fig. - ) . in late stages of bronchopneumonia, the lungs contain a creamy-mucoid exudate in the airways and later often have pulmonary abscesses and bronchiectasis (see fig. - ) . note that the same viruses and mycoplasmas involved in the enzootic pneumonia complex can also predispose cattle to other diseases, such as pneumonic mannheimiosis (mannheimia aspiration pneumonia. aspiration pneumonia is often a devastating sequela to improper gastric tubing of horses, particularly exogenous lipid pneumonia from mineral oil delivered into the trachea in treatment of colic. gross and microscopic lesions are described in detail in the section on aspiration pneumonias of cattle. opportunistic infections. chlamydophila (chlamydia) spp., obligatory intracellular zoonotic pathogens, can cause systemic infection in many mammalian and avian species; in horses, they can also cause keratoconjunctivitis, rhinitis, pneumonia, abortion, polyarthritis, enteritis, hepatitis, and encephalitis. serologic studies suggest that infection without apparent disease is common in horses. horses experimentally infected with chlamydophila psittaci develop mild and transient bronchointerstitial pneumonia. there are unconfirmed reports suggesting a possible association between these organisms and recurrent airway obstruction in horses. detection of chlamydial organisms in affected tissue is not easy and requires special laboratory techniques such as pcr, immunohistochemistry, and fluorescent antibody tests. horses are only sporadically affected with mycobacteriosis (mycobacterium avium complex, mycobacterium tuberculosis, and mycobacterium bovis). the intestinal tract and associated lymph nodes are generally affected, suggesting an oral route of infection with subsequent hematogenous dissemination to the lungs. the tubercles (granulomas) differ from those in ruminants and pigs, being smooth, gray, solid, sarcoma-like nodules without grossly visible caseous necrosis or calcification (e- fig. - ) . microscopically, the tubercles are composed of macrophages, epithelioid cells, and multinucleated giant cells. fibrosis increases with time, accounting in part for the sarcomatous appearance. adenovirus infections occur commonly in arabian foals with combined immunodeficiency (cid), a hereditary lack of b and t lymphocytes. in cases of adenoviral infection, large basophilic or amphophilic inclusions are present in the nuclei of tracheal, bronchial, bronchiolar, alveolar, renal, and intestinal epithelial cells. as it occurs in other species, infection with a unique fungal pathogen known as pneumocystis carinii typically occurs in immunosuppressed or immunoincompetent individuals such as arabian foals with cid (see fig. - ). diagnosis of pneumocystis carinii requires microscopic examination of lungs and special stains. idiopathic interstitial pneumonia. interstitial and bronchointerstitial pneumonias of undetermined cause that can progress to severe pulmonary fibrosis have been reported in foals and young horses. the gross and microscopic lesions are reminiscent of those of bovine pulmonary edema and emphysema or ards. the lungs are notably congested and edematous and microscopically are characterized by necrosis of the bronchiolar epithelium, alveolar edema, hyperplasia of type ii pneumonocytes, and hyaline membranes. the cause of this form of equine interstitial pneumonia is not known, but toxic and particularly viral causes have been proposed. bovine respiratory disease complex (brdc) and acute undifferentiated respiratory disease are general terms often used by clinicians to describe acute and severe bovine respiratory illness of clinically undetermined cause. these terms do not imply any particular type of pneumonia and therefore should not be used in pathology reports. clinically, the brd complex includes bovine enzootic pneumonia (multifactorial etiology); pneumonic mannheimiosis (mannheimia haemolytica); respiratory histophilosis (histophilus somni), previously known as respiratory hemophilosis (haemophilus somnus); mycoplasma bovis; respiratory viral infections, such as infectious bovine .e chapter respiratory system, mediastinum, and pleurae pneumonic mannheimiosis (shipping fever) is the most important respiratory disease of cattle in north america, particularly in feedlot animals that have been through the stressful marketing and assembly processes. mannheimia haemolytica biotype a, serotype is the etiologic agent most commonly responsible for the severe pulmonary lesions. a few investigators still consider that pasteurella multocida and other serotypes of mannheimia haemolytica are also causes of this disease. even after many years of intense investigation, from the gross lesions to the molecular aspects of the disease, the pathogenesis of pneumonic mannheimiosis remains incompletely understood. experiments have established that mannheimia haemolytica a alone is usually incapable of causing disease because it is rapidly cleared by pulmonary defense mechanisms. these findings may explain why mannheimia haemolytica, despite being present in the nasal cavity of healthy animals, only sporadically causes disease. for mannheimia haemolytica to be established as a pulmonary infection, it is first required that stressors impair the defense mechanisms and allow the bacteria to colonize the lung (see section on impairment of defense haemolytica). clinically, enzootic pneumonia is usually mild, but fatal cases are occasionally seen even in farms with optimal health management. pneumonic mannheimiosis (shipping fever). shipping fever (transit fever) is a vague clinical term used to denote acute respiratory diseases that occur in cattle several days or weeks after shipment. the disease is characterized by a severe fibrinous bronchopneumonia, reflecting the fact that death generally occurs early or at an acute stage. because mannheimia haemolytica (formerly pasteurella haemolytica) is most frequently isolated from affected lungs, the names pneumonic mannheimiosis and pneumonic pasteurellosis have been used synonymously. it is known that pneumonic mannheimiosis can occur in animals that have not been shipped and that organisms other than mannheimia haemolytica can cause similar lesions. therefore the term shipping fever should be relinquished in favor of more specific names, such as pneumonic mannheimiosis or respiratory histophilosis. irregular areas of coagulative necrosis are typically bordered by a rim of elongated cells often referred to as oat-shaped cells or oat cells that are degenerating neutrophils mixed with a few alveolar macrophages (see fig. - ). in the early stages of necrosis, there is no evidence of vascular thrombosis, suggesting that necrosis is primarily caused by the cytotoxin of mannheimia haemolytica and is not the result of an ischemic change. the interlobular septa become distended with protein-rich edematous fluid, and the lymphatic vessels contain fibrin thrombi. the trachea and bronchi can have considerable amounts of blood and exudate, which are transported by the mucociliary escalator or coughed up from deep within the lungs, but the walls of the trachea and major bronchi may or may not be involved. because of the necrotizing process, sequelae to pneumonic mannheimiosis can be serious and can include abscesses, encapsulated sequestra (isolated pieces of necrotic lung), chronic pleuritis, fibrous pleural adhesions, and bronchiectasis. clinically, pneumonic mannheimiosis is characterized by a severe toxemia that can kill animals even when considerable parts of the lungs remain functionally and structurally normal. cattle usually become depressed, febrile ( ° to ° f [ ° to ° c]), and anorexic and have a productive cough, encrusted nose, mucopurulent nasal exudate, shallow respiration, or an expiratory grunt. hemorrhagic septicemia. pneumonic mannheimiosis should not be confused with hemorrhagic septicemia (septicemic pasteurellosis) of cattle and water buffalo (bubalus bubalis) caused by inhalation or ingestion of serotypes :b and :e of pasteurella multocida. this oie-notifiable disease does not occur in north america and currently is reported only from some countries in asia, africa, and recently in germany. in contrast to pneumonic mannheimiosis, in which lesions are always confined to the lower respiratory tract, the bacteria of hemorrhagic septicemia always disseminates hematogenously to other organs. at necropsy, typically, generalized petechiae are present on the serosal surfaces of the intestine, heart, and lungs and in skeletal muscles. superficial and visceral lymph nodes are swollen and hemorrhagic. variable lesions include edematous and hemorrhagic lungs with or without consolidation; hemorrhagic enteritis; blood-tinged fluid in the thorax and abdomen; and subcutaneous edema of the head, neck, and ventral abdomen. bacteria can be cultured from blood, and animals have high fever and die rapidly ( % case fatality). respiratory histophilosis (haemophilosis). respiratory histophilosis is part of the histophilus somni (haemophilus somnus) disease complex, which has at least eight different clinicopathologic forms, each one involving different organs. this complex includes septicemia, encephalitis (known as thrombotic meningoencephalitis [tme]), pneumonia (respiratory histophilosis), pleuritis, myocarditis, arthritis, ophthalmitis, conjunctivitis, otitis, and abortion. the portals of entry for the different forms of histophilosis have not been properly established. the respiratory form of bovine histophilosis is the result of the capacity of the bacterium to induce both suppurative and fibrinous bronchopneumonia (e- fig. - ). the latter is in some cases indistinguishable from that of pneumonic mannheimiosis. the pathogenesis of respiratory histophilosis is still poorly understood, and the disease cannot be reproduced consistently by administration of histophilus somni alone. like mannheimia haemolytica, it requires predisposing factors such as stress or a preceding viral infection. histophilus somni is often isolated from the lungs of calves with enzootic pneumonia. the capacity of histophilus somni to cause septicemia and localized infections in the lungs, brain, eyes, ear, heart, mammary gland, male and female genital organs, or placenta is perhaps attributable to specific virulence factors, such as immunoglobulin-binding proteins (igbps) and lipooligosaccharide (los). also, histophilus mechanisms). these stressors include weaning, transport, fatigue, crowding, mixing of cattle from various sources, inclement weather, temporary starvation, and viral infections. horizontal transmission of viruses and mannheimia haemolytica occurs during crowding and transportation of cattle. viruses that most commonly predispose cattle to pneumonic mannheimiosis include bohv- , bpiv- , and brsv. once established in the lungs, mannheimia haemolytica causes lesions by means of different virulence factors, which include endotoxin, lipopolysaccharide, adhesins, and outer membrane proteins; however, the most important is probably the production of a leukotoxin (exotoxin), which binds and kills bovine macrophages and neutrophils. the fact that this toxin exclusively affects ruminant leukocytes probably explains why mannheimia haemolytica is a respiratory pathogen in cattle and sheep but not in other species. during mannheimia haemolytica infection, alveolar macrophages, neutrophils, and mast cells release maximum amounts of proinflammatory cytokines, particularly tnf-α, il- , il- , adhesion molecules, histamine, and leukotrienes. by locally releasing enzymes and free radicals, leukocytes further contribute to the injury and necrosis of bronchiolar and alveolar cells. the gross lesions of acute and subacute pneumonic mannheimiosis are the prototypic fibrinous bronchopneumonia, with prominent fibrinous pleuritis ( fig. - and see fig. - ) and pleural effusion. lesions are always cranioventral and usually ventral to a horizontal line through the tracheal bifurcation. the interlobular septa are distended by yellow, gelatinous edema and fibrin. the "marbling" of lobules is the result of intermixing areas of coagulation necrosis, interlobular interstitial edema, and congestion ( fig. - ) . microscopically, lung lesions are evident hours after experimental infection in which neutrophils fill the bronchial, bronchiolar, and alveolar spaces. within to hours, the cytotoxic effect of mannheimia haemolytica is manifested by necrosis of individual alveolar cells and fibrin begins to exude into the alveoli from increased permeability of the air-blood barrier. these changes are exacerbated by endothelial swelling, altered platelet function, increased procoagulant activity, and diminished profibrinolytic activity in the lungs. by hours, alveolar macrophages start to appear in the bronchoalveolar space. at this time, large and the pulmonary defense mechanisms. lung lesions are typically those of a chronic bronchopneumonia with numerous well-delineated caseonecrotic nodules (fig. - and e-fig. - ) . microscopically, lesions are quite characteristic and consist of distinct areas of pulmonary necrosis centered on bronchi or bronchioles. the lesion is formed by a core of fine eosinophilic granular debris surrounded by a rim of neutrophils, macrophages, and fibroblasts (see fig. - ) . although the origin of the caseonecrotic lesions is under investigation, recent studies incriminate reactive oxygen species (ros) and reactive nitrogen species (rns) as the major contributors for cell injury in the lung. the diagnosis is confirmed by isolation or somni has the ability to undergo structural and antigenic variation, evade phagocytosis by promoting leukocytic apoptosis, inhibit intracellular killing, reduce transferrin concentrations, and induce endothelial apoptosis in the lungs of affected calves. mixed pulmonary infections of histophilus somni, mannheimia haemolytica, pasteurella multocida, trueperella pyogenes, and mycoplasmas are fairly common in calves. mycoplasma bovis pneumonia. mycoplasma bovis is the most common mycoplasma sp. isolated from pneumonic lungs of cattle in europe and north america. pulmonary infection is exacerbated by stress or any other adverse factor (e.g., viral infection) that depresses n control programs for infectious disease. it was eradicated from north america in and from australia in the s, but it is still enzootic in large areas of africa, asia, and eastern europe. the etiologic agent, mycoplasma mycoides ssp. mycoides small colony type, was the first mycoplasma isolated and is one of the most pathogenic of those that infect domestic animals. natural infection occurs in cattle and asian buffalo. the portal of entry is aerogenous, and infections occur when a susceptible animal inhales infected droplets. the pathogenic mechanisms are still inadequately understood but are suspected to involve toxin and galactan production, unregulated production of tnf-α, ciliary dysfunction, immunosuppression, and immune-mediated vasculitis. vasculitis and thrombosis of pulmonary arteries, arterioles, veins, and lymphatic vessels lead to lobular infarction. the name of the disease is a good indication of the gross lesions. it is a severe, fibrinous bronchopneumonia (pleuropneumonia) similar to that of pneumonic mannheimiosis (see figs. - and - ) but having a more pronounced "marbling" of the lobules because of extensive interlobular edema and lymphatic thrombosis. typically, % to % of lesions are in the caudal lobes (not cranioventrally), and pulmonary sequestra (necrotic lung encapsulated by connective tissue) are more frequent and larger than pneumonic mannheimiosis. unilateral lesions are common in this disease. microscopically, the appearance again is like that of pneumonic mannheimiosis, except that vasculitis and thrombosis of pulmonary arteries, arterioles, and capillaries are much more obvious and are clearly the major cause of the infarction and thrombosis of lymphatic vessels in interlobular septa. mycoplasma mycoides ssp. mycoides small colony type remains viable in the sequestra for many years, and under stress (e.g., starvation), the fibrous capsule may break down releasing mycoplasma into the airways, thus becoming a source of infection for other animals. clinical signs are those of severe sepsis, including fever, depression, and anorexia followed by severe respiratory signs such as opened-mouth breathing, dyspnea and coughing, and crepitation and pleural friction on thoracic auscultation. vaccination is highly effective in preventing the disease. bovine tuberculosis. tuberculosis is an ancient, communicable, worldwide, chronic disease of human beings and domestic animals. it continues to be a major problem in human beings in underdeveloped countries, and it is on the rise in some industrialized nations, largely because of the immunosuppressive effects of aids, immigration, and movement of infected animals across borders. the world health organization (who) estimates that more than million people die of tuberculosis and million new cases appear each year, mostly in developing countries. mycobacterium tuberculosis is transmitted between human beings, but where unpasteurized milk is consumed, mycobacterium bovis from the milk of cattle with mammary tuberculosis is also an important cause of human tuberculosis. mycobacterium bovis infections have also been reported in a number of domestic and wild mammalian species; in some countries, wildlife reservoirs exist and may act as a source of infection for cattle. bovine tuberculosis is primarily caused by mycobacterium bovis, but infection with mycobacterium tuberculosis, the pathogen of human tuberculosis, and mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) can occur sporadically. tuberculosis can be acquired by several routes, but infection of the lungs by inhalation of mycobacterium bovis is the most common in adult cattle, whereas ingestion of infected milk is more predominant in young animals. organisms belonging to the mycobacterium avium complex can also infect cattle, but for infection caused by these organisms, the term atypical mycobacteriosis (not tuberculosis) is currently preferred. immunohistochemical labeling of tissue sections for mycoplasma antigens. mycoplasma bovis is also incriminated in arthritis, otitis, mastitis, abortion, and keratoconjunctivitis. contagious bovine pleuropneumonia. contagious bovine pleuropneumonia is an oie-notifiable disease of historic interest in veterinary medicine because it was the object of early national a b c than % of bovine cases, a chronic, moist cough can progress to dyspnea. enlarged tracheobronchial lymph nodes can contribute to the dyspnea by impinging on airways, and the enlargement of caudal mediastinal nodes can compress the caudal thoracic esophagus and cause bloating. interstitial pneumonias. atypical interstitial pneumonia (aip) is a vague clinical term well entrenched in veterinary literature but one that has led to enormous confusion among veterinarians. it was first used to describe acute or chronic forms of bovine pneumonia that did not fit in any of the "classic" forms because of the lack of exudate and lack of productive cough. microscopically, the criteria for diagnosis of aip in cattle were based on the absence of obvious exudate and the presence of edema, interstitial emphysema (see the section on pulmonary emphysema), hyaline membranes, hyperplasia of type ii pneumonocytes, and alveolar fibrosis with interstitial cellular infiltrates. at that time, any pulmonary disease or pulmonary syndrome that had a few of the previously mentioned lesions was traditionally diagnosed as aip, and grouping all these different syndromes together was inconsequential because their etiopathogenesis were then unknown. field and laboratory investigations have demonstrated that most of the bovine syndromes previously grouped under aip have rather different causes and pathogeneses ( fig. - ) . furthermore, what was "atypical" in the past has become so common that it is fairly routine nowadays to find "typical cases" of aip. for all these reasons, investigators, largely from britain, proposed that all these syndromes previously clustered into aip should be named according to their specific cause or pathogenesis. the most common bovine syndromes characterized by edema, emphysema, hyaline membranes, and hyperplasia of type ii pneumonocytes include bovine pulmonary edema and emphysema (fog fever), "extrinsic allergic alveolitis" (hypersensitivity pneumonitis), "reinfection syndromes" (hypersensitivity to dictyocaulus sp. or brsv), milk allergy, ingestion of moldy potatoes, paraquat toxicity, toxic silo gases, mycotoxins, and others. acute bovine pulmonary edema and emphysema (fog fever). acute bovine pulmonary edema and emphysema (abpee), known in britain as fog fever (no association with atmospheric conditions), occurs in cattle usually grazing "fog" pastures (i.e., aftermath or foggage, regrowth after a hay or silage has been cut). epidemiologically, abpee usually occurs in adult beef cattle in the fall when there is a change in pasture from a short, dry grass to a lush, green grass. it is generally accepted that l-tryptophan present in the pasture is metabolized in the rumen to -methylindole, which in turn is absorbed into the bloodstream and carried to the lungs. mixed function oxidases present in the nonciliated bronchiolar epithelial (club) cells metabolize -methylindole into a highly pneumotoxic compound that causes extensive and selective necrosis of bronchiolar cells and type i pneumonocytes (fig. - and see fig. - ) and increases alveolar permeability, leading to edema, thickening of the alveolar interstitium, and alveolar and interstitial emphysema. -methylindole also interferes with the lipid metabolism of type ii pneumonocytes. the gross lesions are those of a diffuse interstitial pneumonia with severe alveolar and interstitial edema and interlobular emphysema (see fig. - , a) . the lungs are expanded, pale, and rubbery in texture, and the lesions are most notable in the caudal lobes. microscopically, the lesions are alveolar and interstitial edema and emphysema, formation of characteristic hyaline membranes within alveoli (see fig. - , b) , and in those animals that survive for several days, hyperplasia of type ii pneumonocytes and alveolar interstitial fibrosis. respiratory infection usually starts when inhaled bacilli reach the alveoli and are phagocytosed by pulmonary alveolar macrophages. if these cells are successful in destroying the bacteria, infection is averted. however, mycobacterium bovis, being a facultative pathogen of the monocytic-macrophage system, may multiply intracellularly, kill the macrophage, and initiate infection. from this first nidus of infection, bacilli spread aerogenously via airways within the lungs and eventually via the lymph vessels to tracheobronchial and mediastinal lymph nodes. the initial focus of infection at the portal of entry (lungs) plus the involvement of regional lymph nodes is termed the primary (ghon) complex of tuberculosis. if the infection is not contained within this primary complex, bacilli disseminate via the lymph vessels to distant organs and other lymph nodes by the migration of infected macrophages. hematogenous dissemination occurs sporadically when a granuloma containing mycobacteria erodes the wall of a blood vessel, causes vasculitis, and allows the granuloma to discharge mycobacteria into the alveolar circulation. if dissemination is sudden and massive, mycobacteria are widely disseminated and numerous small foci of infection develop in many tissues and organs and the process is referred to as miliary tuberculosis (like millet seeds). the host becomes hypersensitive to the mycobacterium, which enhances the cell-mediated immune defenses in early or mild infections but can result in host-tissue destruction in the form of caseous necrosis. the evolution and dissemination of the pulmonary infection are closely regulated by cytokines and tnf-α production by alveolar macrophages. unlike abscesses that tend to grow rather fast, granulomas evolve slowly at the site of infection. the lesion starts with few macrophages and neutrophils ingesting the offending organism, but because mycobacterium organisms are resistant to phagocytosis, infected macrophages eventually die, releasing viable bacteria, lipids, and cell debris. cell debris accumulates in the center of the lesion, whereas viable bacteria and bacterial lipids attract additional macrophages and a few lymphocytes at the periphery of the lesion. some of these newly recruited macrophages are activated by local lymphocytes and become large phagocytic cells with abundant cytoplasm resembling epithelial cells, thus the term epithelioid macrophages. multinucleated giant cells (also macrophages) appear at the edges of the lesion, and finally the entire focus of inflammatory process becomes surrounded by fibroblasts and connective tissue (see fig. - ). it may take weeks or months for a granuloma to be grossly visible. bovine tuberculosis, the prototype for granulomatous pneumonia, is characterized by the presence of a few or many caseated granulomas (see fig. - ). the early gross changes are small foci (tubercles) most frequently seen in the dorsocaudal, subpleural areas. with progression, the lesions enlarge and become confluent with the formation of large areas of caseous necrosis. calcification of the granulomas is a typical finding in bovine tuberculosis. single nodules or clusters occur on the pleura and peritoneum, and this presentation has been termed pearl disease. microscopically, the tubercle is composed of mononuclear cells of various types. in young tubercles, which are noncaseous, epithelioid and langhans' giant cells are at the center, surrounded by lymphocytes, plasma cells, and macrophages. later, caseous necrosis develops at the center, secondary to the effects of cell-mediated hypersensitivity and enclosed by fibrosis at the periphery. acid-fast organisms may be numerous but more often are difficult to find in histologic section or smears. clinically, the signs of tuberculosis relate to the dysfunction of a particular organ system or to general debilitation, reduced milk production, and emaciation. in the pulmonary form, which is more grossly, the postmortem lesions vary from subtle, gray, subpleural foci (granulomatous inflammation) to severe lesions, in which the lungs are firm and heavy and have a "meaty appearance" because of interstitial pneumonia (e- fig. - ) with type ii pneumonocyte hyperplasia, lymphocytic infiltration, and interstitial fibrosis. characteristically, discrete noncaseous granulomas formed in response to the deposition of antigen-antibody complexes are scattered throughout the lungs. chronic cases of extrinsic allergic alveolitis can eventually progress to diffuse fibrosing alveolitis. clinically, it can be acute or chronic; the latter has a cyclical pattern of exacerbation during winter months. weight loss, coughing, and poor exercise tolerance are clinical features. full recovery can occur if the disease is recognized and treated early. reinfection syndrome. hypersensitivity to reinfection with larvae of dictyocaulus viviparus is another allergic syndrome manifested in the lungs that causes signs and lesions indistinguishable from abpee, with the exception of eosinophils and possibly larvae in the alveolar exudate. the hypersensitivity reaction in the lung causes diffuse alveolar damage and edema, necrosis of type i pneumonocytes, and hyperplasia of type ii pneumonocytes. in the later stages of the disease, there is formation of small granulomas with interstitial infiltrates of mononuclear cells. it has been suggested but not confirmed that emphysema with diffuse proliferative alveolitis and formation of hyaline membranes can also occur sporadically in the late stages of brsv infection in cattle. presumably, this disease shares many similarities with "atypical" infections occasionally seen in children with respiratory syncytial virus (rsv human strain), in which a hypersensitivity to the virus or virus-induced augmentation of the immune response results in hypersensitivity pneumonitis (see fig. - ). brsv infection is also known to enhance hypersensitivity to environmental allergens in cattle. other forms of bovine interstitial pneumonia. inhalation of manure ("pit") gases, such as nitrogen dioxide (no ), hydrogen interstitial cell infiltrates, fibrosis, emphysema acute proliferation phase hyperplasia of type ii pneumonocytes clinically, severe respiratory distress develops within days of the abrupt pasture change, and cattle develop expiratory dyspnea, oral breathing, and evidence of emphysema within the lungs and even subcutaneously along the back. experimentally, reducing ruminal conversion of l-tryptophan to -methylindole prevents the development of abpee. a number of other agents cause virtually the same clinical and pathologic syndrome as is seen in abpee. the pathogenesis is assumed to be similar, although presumably other toxic factors are specific for each syndrome. one of these pneumotoxic factors is -ipomeanol, which is found in moldy sweet potatoes contaminated with the fungus fusarium solani. mixed function oxidases in the lungs activate -ipomeanol into a potent pneumotoxicant capable of producing irreversible oxidative injury to type i pneumonocytes and bronchiolar epithelial cells, presumably through lipoperoxidation of cell membranes. similarly, purple mint (perilla frutescens), stinkwood (zieria arborescens), and rapeseed and kale (brassica species) also cause pulmonary edema, emphysema, and interstitial pneumonia. extrinsic allergic alveolitis. extrinsic allergic alveolitis (hypersensitivity pneumonitis), one of the most common allergic diseases in cattle, is seen mainly in housed adult dairy cows in the winter. this disease shares many similarities with its human counterpart known as farmer's lung, which results from a type iii hypersensitivity reaction to inhaled organic antigens, most commonly microbial spores, mainly of the thermophilic actinomycete, saccharopolyspora rectivirgula (micropolyspora faeni), commonly found in moldy hay. this is followed by an antibody response to inhaled spores and local deposition of antigen-antibody complexes (arthus reaction) in the lungs (see fig. - ). because it affects only a few animals of the herd or the sporadic person working in a farm, it is presumed that intrinsic host factors, such as dysregulation of dendritic cells, t lymphocytes, igg, interleukins, ifn-γ, and surfactant, are involved in the pathogenesis of the disease. chapter respiratory system, mediastinum, and pleurae e- figure - interstitial pneumonia, adult cow. note meaty appearance of the pulmonary parenchyma and mild edematous distention of the interlobular septa. inset, thick hyaline membranes (arrows) lining hypercellular alveolar walls. hypersensitivity pneumonia was suspected. (courtesy dr. a. lópez, atlantic veterinary college.) gases, inhalation of no (silo gas) also causes bronchiolitis, edema, and interstitial pneumonia and, in survivors, bronchiolitis obliterans ("silo filler's disease"). smoke inhalation resulting from barn or house fires is sporadically seen by veterinarians and pathologists. in addition to skin burns, animals involved in fire accidents suffer extensive thermal injury produced by the heat on the nasal and laryngeal mucosa, and severe chemical irritation caused by inhalation of combustion gases and particles in the lung. animals that survive or are rescued from fires frequently develop nasal, laryngeal, and tracheal edema, and pulmonary hemorrhage and alveolar edema, which are caused by chemical injury to the blood-air barrier or by ards caused by the excessive production of free radicals during the pulmonary inflammatory response (see e- fig. - ) . microscopic examination of the lungs often reveals carbon particles (soot) on mucosal surfaces of the conducting system. verminous pneumonia (dictyocaulus viviparus). pulmonary lesions in parasitic pneumonias vary from interstitial pneumonia caused by migrating larvae to chronic bronchitis from intrabronchial adult parasites, to granulomatous pneumonia, which is caused by dead larvae, aberrant parasites, or eggs of parasites. in many cases, an "eosinophilic syndrome" in the lungs is characterized by infiltrates of eosinophils in the pulmonary interstitium and bronchoalveolar spaces and by blood eosinophilia. atelectasis and emphysema secondary to the obstruction of airways by parasites and mucous secretions are also common findings in parasitic pneumonias. the severity of these lesions relates to the numbers and size of the parasites and the nature of the host reaction, which sometimes includes hypersensitivity reactions (see section on reinfection syndrome). a common general term for all of these diseases is verminous pneumonia, and the adult nematodes are often visible grossly in the airways ( fig. - ) . dictyocaulus viviparus is an important pulmonary nematode (lungworm) responsible for a disease in cattle referred to as verminous pneumonia or verminous bronchitis. adult parasites live in the bronchi of cattle, mainly in the caudal lobes, and cause severe bronchial irritation, bronchitis, and pulmonary edema, which in turn are responsible for lobular atelectasis and interstitial emphysema. atelectasis is confined to the lobules of the lungs ventilated by the obstructed bronchi (dorsocaudal). interstitial emphysema (interlobular) is caused by forced expiratory movements against a partially obstructed single bronchus. in addition to the inflammation of bronchial mucosa, bronchoaspiration of larvae and eggs also causes an influx of leukocytes into the bronchoalveolar space (alveolitis). verminous pneumonia is most commonly seen in calves during their first summer grazing pastures that are repeatedly used from year to year, particularly in regions of europe that have a moist cool climate. the parasite can overwinter in pastures, even in climates as cold as canada's, and older animals may be carriers for a considerable length of time. at necropsy, lesions appear as dark or gray, depressed, wedgeshaped areas of atelectasis involving few or many lobules usually along the dorsocaudal aspect of the lungs. on cut surface, edematous foam and mucus mixed with white, slender (up to -mm long) nematodes are visible in the bronchi (see fig. - ). in the most severe cases, massive numbers of nematodes fill the bronchial tree. microscopically, the bronchial lumens are filled with parasites admixed with mucus because of goblet cell hyperplasia, and there is squamous metaplasia of the bronchial and bronchiolar epithelium because of chronic irritation. there are also inflammatory infiltrates in the bronchial mucosa; alveolar edema; hyperplasia of balt sulfide (h s), and ammonia (nh ), from silos or sewage can be a serious hazard to animals and human beings. at toxic concentrations, these gases cause necrosis of bronchiolar cells and type i pneumonocytes and fulminating pulmonary edema that causes asphyxiation and rapid death (see fig. - ) . like other oxidant secretory granules released by club cells contain several proteins, such as surfactantlike protein, antiinflammatory protein (cc ), and bronchiolar lining proteins. b, ros produced by club cells are also absorbed into capillaries within the lamina propria and are transferred by the circulatory system to pulmonary capillaries where they disrupt the air-blood barrier, causing degeneration and necrosis of type i pneumonocytes. this process leads to leakage of plasma fluid (alveolar edema [pink color]) and extravasation of erythrocytes (alveolar hemorrhage) and neutrophils (inflammation). ingested pneumotoxicants can be metabolized by the liver, leading to release of ros into the circulatory system that then disrupts the air-blood barrier in a similar manner. fig. - ). microscopically, there are focal intraalveolar hemorrhages caused by larvae migrating through the alveolar walls. some larvae admixed with edematous fluid and cellular exudate (including eosinophils) may be visible in bronchioles and alveoli. the alveolar walls are thickened because of edema and a few inflammatory cells. clinical signs include cough and expiratory dyspnea to the point of oral breathing. hydatid cysts, the intermediate stage of echinococcus granulosus, can be found in the lungs and liver and other viscera of sheep and to a lesser extent in cattle, pigs, goats, horses, and human beings. the adult stage is a tapeworm that parasitizes the intestine of canidae. hydatidosis is still an important zoonosis in some countries, and perpetuation of the parasite life cycle results from animals being fed uncooked offal from infected sheep and consumption of uninspected meat. hydatid cysts are generally to cm in diameter, and numerous cysts can be found in the viscera of affected animals ( fig. - ). each parasitic cyst is filled with clear fluid; numerous daughter cysts attach to the wall, each containing several "brood capsules" with protoscolices inside. hydatid cysts have little clinical significance in animals but are economically important because of carcass condemnation. aspiration pneumonias. the inhalation of regurgitated ruminal contents or iatrogenic deposition of medicines or milk into the trachea can cause severe and often fatal aspiration pneumonia. bland substances, such as mineral oil, may incite only a mild suppurative or histiocytic bronchopneumonia, whereas some "home remedies" or ruminal contents are highly irritating and cause a fibrinous, necrotizing bronchopneumonia. the right cranial lung lobe tends to be more severely affected because the right cranial bronchus is the most cranial branch and enters the ventrolateral aspect of the trachea. however, the distribution may vary when animals aspirate while in lateral recumbency. in some severe cases, pulmonary necrosis can be complicated by infection with saprophytic organisms present in ruminal contents, causing fatal gangrenous pneumonia. aspiration pneumonia should always be considered in animals whose swallowing has been compromised-for example, those with cleft palate or hypocalcemia (milk fever). on the other hand, neurological diseases such as encephalitis (e.g., rabies) or encephalopathy (e.g., lead poisoning) should be investigated in animals in which the cause of aspiration pneumonia could not be caused by persistent immunologic stimuli; hypertrophy and hyperplasia of bronchiolar smooth muscle because of increased contraction and decreased muscle relaxation; and a few eosinophilic granulomas around the eggs and dead larvae. these granulomas, grossly, are gray, noncaseated nodules ( to mm in diameter) and may be confused with those seen at the early stages of tuberculosis. the clinical signs (coughing) vary with the severity of infection, and severe cases can be confused clinically with interstitial pneumonias. expiratory dyspnea and death can occur with heavy parasitic infestations when there is massive obstruction of airways. a different form of bovine pneumonia, an acute allergic reaction known as reinfection syndrome, occurs when previously sensitized adult cattle are exposed to large numbers of larvae (dictyocaulus viviparus). lesions in this syndrome are those of a hypersensitivity pneumonia as previously described. other lung parasites. ascaris suum is the common intestinal roundworm of pigs; larvae cannot complete their life cycle in calves, but the larvae can migrate through the lungs and cause severe pneumonia and death of calves within weeks of infection. infection is usually acquired from the soil on which infested pigs were previously kept. the gross lesions are a diffuse interstitial pneumonia with hemorrhagic foci, atelectasis, and interlobular edema and it also occurs in canada, europe, australia, and probably elsewhere. this disease has two major clinicopathologic forms: one involves the central nervous system of goat kids and young goats and is characterized by a nonsuppurative leukoencephalomyelitis; the other form involves the joints of adult goats and is characterized by a chronic, nonsuppurative arthritis-synovitis. in addition, infection with cae virus can cause chronic lymphocytic interstitial pneumonia. the lentivirus of cae, caprine arthritis and encephalitis virus (caev), is closely related to visna/maedi virus and, in fact, cross infection with cae virus in sheep has been achieved experimentally. similar to maedi, cae infection presumably occurs during the first weeks of life when the doe transmits the virus to her offspring through infected colostrum or milk. horizontal transmission between infected and susceptible goats via the respiratory route has also been described. after coming into contact with mucosal cells at the portal of entry, the virus is phagocytized by macrophages, which migrate to the regional lymph nodes. infected macrophages are disseminated hematogenously to the central nervous system, joints, lungs, and mammary glands. like maedi, there is some evidence that the recruitment of lymphocytic cells results from dysregulation of cytokine production by infected macrophages and lymphocytes in affected tissues. it can take several months before serum antibodies can be detected in infected goats. grossly, the interstitial pneumonia is diffuse and tends to be most severe in the caudal lobes. the lungs are gray-pink and firm in texture with numerous, -to -mm, gray-white foci on the cut surface. the tracheobronchial lymph nodes are consistently enlarged. microscopically, the alveolar walls are thickened by lymphocytes and conspicuous hyperplasia of type ii pneumonocytes ( fig. - ). one important difference between the pneumonias of cae and maedi is that in cae the alveoli are filled with proteinaceous eosinophilic material (alveolar proteinosis), which in electron micrographs has structural features of pulmonary surfactant. the pulmonary form of cae can be mistaken for parasitic pneumonia (muellerius capillaris) because these two diseases have lymphocytic interstitial pneumonia and can coexist in the same goat. explained otherwise. depending on the nature of the aspirated material, histopathologic evaluation generally reveals foreign particles such as vegetable cells, milk droplets, and large numbers of bacteria in bronchi, bronchioles, and alveoli (e- fig. - ). vegetable cells and milk typically induce an early neutrophilic response followed by a histiocytic reaction with "foreign body" multinucleated giant cells (see e- fig. - ). special stains are used for the microscopic confirmation of aspirated particles in the lung (e.g., pas for vegetable cells and oil red-o for oil or milk droplets). maedi (visna/maedi). maedi is an important, lifelong, and persistent viral disease of sheep and occurs in most countries, except australia and new zealand. maedi means "shortness of breath" in the icelandic language, and it is known as graaff-reinet disease in south africa, zwoegerziekte in the netherlands, la bouhite in france, and ovine progressive pneumonia (opp) in the united states. more recently, the disease has also been referred to as ovine lentivirusinduced lymphoid interstitial pneumonia or simply lymphoid interstitial pneumonia (lip). maedi is caused by visna/maedi virus (vmv), a nononcogenic small ruminant lentivirus (srlv) of the family retroviridae that is antigenically related to the lentivirus causing caprine arthritisencephalitis (cae). seroepidemiologic studies indicate that infection is widespread in the sheep population, yet the clinical disease seems to be rare. the pathogenesis is incompletely understood, but it is known that transmission occurs largely vertically, through ingestion of infected colostrum, and horizontally, via inhalation of infected respiratory secretions. once in the body, the ovine lentivirus causes lifelong infections within monocytes and macrophages, including alveolar and pulmonary intravascular macrophages; clinical signs do not develop until after a long incubation period of years or more. pulmonary lesions at the time of death are severe interstitial pneumonia and failure of the lungs to collapse when the thorax is opened. notable rib imprints, indicators of uncollapsed lungs, are often present on the pleural surface ( fig. - ). the lungs are pale, mottled, and typically heavy (two or three times normal weight), and the tracheobronchial lymph nodes are enlarged. microscopically, the interstitial pneumonia is characterized by balt hyperplasia and thickening of alveolar walls and peribronchial interstitial tissue by heavy infiltration of lymphocytes, largely t lymphocytes (see fig. - ). recruitment of mononuclear cells into the pulmonary interstitium is presumably the result of sustainable production of cytokines by retrovirus-infected pulmonary macrophages and lymphocytes. hyperplasia of type ii pneumonocytes is not a prominent feature of maedi, likely because in this disease there is no injury to type i pneumonocytes, but there is some alveolar fibrosis and smooth muscle hypertrophy in bronchioles. secondary bacterial infections often cause concomitant bronchopneumonia. enlargement of regional lymph nodes (tracheobronchial) is due to severe lymphoid hyperplasia, primarily of b lymphocytes. the virus can also infect many other tissues, causing nonsuppurative encephalitis (visna), lymphocytic arthritis, lymphofollicular mastitis, and vasculitis. maedi is clinically characterized by dyspnea and an insidious, slowly progressive emaciation despite good appetite. death is inevitable once clinical signs are present, but it may take many months. caprine arthritis-encephalitis. caprine arthritis-encephalitis (cae) is a retroviral disease of goats (small ruminant lentivirus) that has a pathogenesis remarkably similar to that of visna/maedi in sheep. it was first described in the united states in the s, but such as pasteurella multocida, pneumonia may progress to fibrinous or suppurative bronchopneumonia. one might expect some specific evidence pointing to the infectious agents (e.g., large intranuclear inclusion bodies in epithelial cells with adenoviral infection), but this is often not the case, either because examination is seldom done at the acute stage when the lesions are still present or because secondary bacterial infections mask the primary lesions. in the late stages, chronic enzootic pneumonia is characterized by hyperplastic bronchitis, atelectasis, alveolar and peribronchiolar fibrosis, and marked peribronchial lymphoid hyperplasia (cuffing pneumonia). ovine pneumonic mannheimiosis. ovine pneumonic mannheimiosis is one of the most common and economically significant diseases in most areas where sheep are raised. it is caused by mannheimia haemolytica and has a pathogenesis and lesions similar to those of pneumonic mannheimiosis of cattle. colonization and infection of lungs are facilitated by stressors such as changes in weather; handling; deworming; dipping; viral infections such as parainfluenza virus (piv ), respiratory syncytial virus (rsv), and adenovirus; and probably chlamydiae and bordetella parapertussis infections. lesions are characterized by a severe fibrinous bronchopneumonia (cranioventral) with pleuritis ( fig. - and e-fig. - ). subacute to chronic cases progress to purulent bronchopneumonia, and sequelae include abscesses and fibrous pleural adhesions. a similar form of pneumonic mannheimiosis has been reported with increased frequency in bighorn sheep. septicemic pasteurellosis. septicemic pasteurellosis, a common ovine disease, is caused by bibersteinia trehalosi (formerly pasteurella trehalosi or mannheimia haemolytica biotype t) in lambs months of age or older or by mannheimia haemolytica (biotype a) in lambs younger than months of age. both organisms are carried in the tonsils and oropharynx of clinically healthy sheep, and under abnormal circumstances (particularly under stress from dietary or environmental changes) bacteria can invade adjacent tissues, enter the bloodstream, and cause septicemia. gross lesions include a distinctive necrotizing pharyngitis and tonsillitis; ulcerative esophagitis (e- fig. - ) ; severe congestion and edema of the lungs; focal hepatic necrosis; and petechiae in the mucosa of the tongue, esophagus, and intestine and particularly in the lungs and pleura. clinically, goats are active and afebrile but progressively lose weight despite normal appetite. the encephalitic or arthritic signs tend to obscure the respiratory signs, which are only evident on exertion. secondary bacterial bronchopneumonia is common in affected animals. bacterial pneumonias. in the past, pasteurella haemolytica was incriminated in four major ovine diseases known as ( ) acute ovine pneumonic pasteurellosis (shipping fever), ( ) enzootic pneumonia (nonprogressive chronic pneumonia), ( ) fulminating septicemia, and ( ) mastitis. under the new nomenclature, mannheimia haemolytica is responsible for ovine pneumonia resembling shipping fever in cattle (ovine pneumonic mannheimiosis), septicemia in young lambs (younger than months of age), and ovine enzootic pneumonia and sporadic severe gangrenous mastitis in ewes. bibersteinia (pasteurella) trehalosi (formerly pasteurella haemolytica biotype t) is the agent incriminated in septicemia in lambs to months old. chronic enzootic pneumonia. in sheep, this entity is a multifactorial disease complex that, in contrast to ovine pneumonic mannheimiosis, causes only a mild to moderate pneumonia and it is rarely fatal. it generally affects animals younger than year of age. significant costs associated with chronic enzootic pneumonia include reduction of weight gain, labor costs, veterinary fees, and slaughterhouse waste. the modifier "chronic" is used here to avoid any confusion with pneumonic mannheimiosis ("acute enzootic pneumonia"). it is also sometimes called atypical pneumonia, chronic nonprogressive pneumonia, proliferative pneumonia, or other names. chronic enzootic pneumonia is a clinical epidemiologic term and does not imply a single causal agent but is the result of a combination of infectious, environmental, and managerial factors. the list of infectious agents involved in ovine enzootic pneumonia includes mannheimia haemolytica, pasteurella multocida, parainfluenza virus (pi- ), adenovirus, reovirus, respiratory syncytial virus (rsv), chlamydiae, and mycoplasmas (mycoplasma ovipneumoniae). in the early stages of enzootic pneumonia, a cranioventral bronchointerstitial pneumonia is characterized by moderate thickening of alveolar walls because of hyperplasia of type ii pneumonocytes. in some cases, when lungs are infected with secondary pathogens, viviparus of cattle. as seen in cattle with dictyocaulus viviparus, areas of atelectasis secondary to bronchiolar obstruction are present, particularly along the dorsal caudal aspects of the caudal lung lobes. microscopically, affected lungs are characterized by a catarrhal, eosinophilic bronchitis, with peribronchial lymphoid hyperplasia and smooth muscle hyperplasia of bronchi and bronchioles. bronchioles and alveoli can contain edematous fluid, eosinophils, and parasitic larvae and eggs. microscopic granulomas caused by aspirated eggs can be observed in the distal lung. the clinical signs (cough, moderate dyspnea, and loss of condition) and lesions relate mainly to obstruction of the small bronchi by adult worms and filaria. anemia of undetermined pathogenesis and secondary bacterial pneumonia are common in small ruminants with this parasitic disease. muellerius capillaris. muellerius capillaris, also called the nodular lungworm, occurs in sheep and goats in most areas of the world and is the most common lung parasite of sheep in europe and northern africa. it requires slugs or snails as intermediate hosts. the lesions in sheep are typically multifocal, subpleural nodules that tend to be most numerous in the dorsal areas of the caudal lung lobes ( fig. - , a) . these nodules are soft and hemorrhagic in the early stages but later become gray-green and hard or even calcified. microscopically, a focal, eosinophilic, and granulomatous reaction occurs in the microscopically, the hallmark lesion is a disseminated intravascular thrombosis often with bacterial colonies in the capillaries of affected tissues. the alveolar capillaries contain bacteria and microthrombi, and the alveolar lumens have fibrin and red blood cells. mannheimia haemolytica and bibersteinia trehalosi are readily isolated from many organs. affected animals usually die within a few hours of infection, and these animals only rarely have clinical signs such as dullness, recumbency, and dyspnea. contagious caprine pleuropneumonia. a number of mycoplasma spp., often referred to as the "mycoides cluster," can produce respiratory tract infections in goats; however, only mycoplasma capricolum ssp. capripneumoniae is considered to cause contagious caprine pleuropneumonia. this disease is the goat counterpart of contagious bovine pleuropneumonia in cattle; sheep do not have a corresponding disease. this oie-notifiable disease is important in africa, the middle east, and areas of asia, but it is also seen elsewhere. the gross lesions caused by mycoplasma capricolum ssp. capripneumoniae are similar to those of the bovine disease and consist of a severe, often unilateral fibrinous bronchopneumonia and pleuritis; however, distention of the interlobular septa (which are normally not as well developed in goats as in cattle) and formation of pulmonary sequestra are less obvious than in the bovine disease. clinically, contagious caprine pleuropneumonia is similar to contagious bovine pleuropneumonia, with high morbidity and mortality, fever, cough, dyspnea, and increasing distress and weakness. other small ruminant mycoplasmas. pneumonia, fibrinous polyarthritis, septicemia, meningitis, mastitis, peritonitis, and abortion are possible manifestations of disease caused by mycoplasma mycoides ssp. mycoides large colony type and mycoplasma mycoides ssp. capri. the pathogenicity of other mycoplasmas, such as mycoplasma ovipneumoniae, mycoplasma arginini, and mycoplasma capricolum ssp. capricolum, in sheep and goats is still being defined and specific description of the lesions would be premature. these organisms probably cause disease only in circumstances similar to those for enzootic pneumonia, where host, infectious, and environmental factors create a complex interaction in the pathogenesis of the disease. it has been suggested that igg antibodies directed against ovine mycoplasmal antigens cross-react with ciliary proteins, causing inflammation and ciliary dysfunction, a condition in lambs referred to as coughing syndrome. tuberculosis. although tuberculosis has generally been considered uncommon in sheep and goats, caprine tuberculosis has become a significant disease in areas of spain and europe. mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) is the most common cause, but infection with mycobacterium bovis or with the mycobacterium avium complex does occur when the disease is prevalent in other species in the locality. the pulmonary form, similar to that seen in cattle, is characterized by a granulomatous pneumonia with multiple, large, caseous, calcified, and well-encapsulated granulomas scattered throughout the lungs. intralesional acid-fast organisms within macrophages are not as abundant as in bovine tuberculosis. staphylococcus aureus. young sheep ( to weeks old) are susceptible to staphylococcus aureus septicemia (tick pyemia). this bacterium causes disseminated inflammation and abscesses in the joints, heart, liver, kidneys, and cns, and in the lung it can also produce bronchopneumonia and pulmonary abscesses (e- fig. - ). dictyocaulus filaria. dictyocaulus filaria, also called the large lungworm, is a serious, worldwide, parasitic disease of the lungs, most commonly of lambs and goat kids but occurring in adults as well. the life cycle and lesions are similar to those of dictyocaulus epithelial cells spreads rapidly throughout the nasal, tracheal, and bronchial mucosa, with the more severe outbreaks reflecting more involvement of intrapulmonary airways and secondary infection with pasteurella multocida, trueperella (arcanobacterium) pyogenes, or haemophilus spp. although uncommon, human beings infected with swine influenza (h n ) can transmit the virus to pigs; therefore it is important that veterinarians or workers with influenza-like illness stay away from pig farms. natural transmission of h n and h n from human beings to ferrets (mustela putorius furo) and from human beings to cats and dogs has also been reported. pulmonary lesions caused by influenza virus alone are rarely seen in the postmortem room because this disease has a very low mortality rate unless complicated with secondary bacterial infections. grossly, a copious catarrhal to mucopurulent inflammation extends from the nasal passages to the bronchioles, with the volume of mucus being sufficient to plug small airways and cause a lobular or multilobular atelectasis in the cranioventral regions of the lungs. the appearance can be similar grossly, although not microscopically, to that of mycoplasma hyopneumoniae. fatal cases have severe alveolar and interstitial pulmonary edema. microscopically, the lesions in uncomplicated cases are typical of a virus-induced, necrotizing bronchitis-bronchiolitis, which in severe cases extends into the alveoli as bronchointerstitial pneumonia. it is characterized by necrosis of the bronchial/bronchiolar epithelium, thickening and infiltration of the alveolar wall with mononuclear cells and aggregates of macrophages, neutrophils, mucus, and some necrotic cells within the alveolar lumen. if these changes are extensive enough, the lumen of bronchioles can be occluded by exudate, causing lobular atelectasis. viral antigen can be demonstrated in infected epithelial cells by immunoperoxidase techniques. in the later stages of alveolar inflammation, neutrophils are progressively replaced by intraalveolar macrophages, unless the pneumonia is complicated by secondary bacterial infections. recent serologic surveys indicate that infection is also prevalent in wild pigs. clinically, a sudden onset of fever, nasal discharge, stiffness, labored breathing, weakness or even prostration, followed by painful and often paroxysmal coughing, is seen in animals of all age groups and may affect most of the herd. the outbreak subsides virtually without mortality within or weeks; the clinical appearance is much more alarming than the pathologic changes, unless the pigs have secondary infection with bacteria. infection can be confirmed using pcr in secretions collected with nasal swabs. the most important effect of most outbreaks of influenza is severe weight loss, but pregnant sows may abort or give birth to weak piglets. porcine reproductive and respiratory syndrome. a disease originally named mystery swine disease was first recognized in the united states in . in , it was seen in europe, and the disease now occurs worldwide in most major pig-raising countries. in , dutch investigators isolated a virus as the etiologic agent; porcine reproductive and respiratory syndrome virus (prrsv) is currently classified in the genus arterivirus of the family arteriviridae. as its name implies, prrs is characterized by late-term abortions and stillbirths and respiratory problems. the respiratory form is generally seen in nursery and grow/finish pigs. the pathogenesis has not been completely elucidated, but it is presumed that there is a mucosal portal of entry with virus replication in macrophages of the lymphoid tissue, followed by viremia and finally dissemination of infected macrophages to the lungs and other organs, such as the thymus, liver (kupffer cells), spleen, lymph nodes, and intestine. the pulmonary alveolar and intravascular macrophages are the major targets for prrs virus, which induces apoptosis of these cells. the virus also downregulates the innate immune response by subpleural alveoli where the adults, eggs, and coiled larvae reside ( fig. - , b) . clinical signs are usually not apparent. goats differ from sheep by having diffuse interstitial rather than focal lesions, and the reaction to the parasites seen microscopically varies from almost no lesions to a severe interstitial pneumonia with heavy infiltrates of mononuclear cells in alveolar walls resembling cae or mycoplasmal infections. secondary effects of muellerius capillaris infection in sheep and goats include decreased weight gain and possibly secondary bacterial infections. protostrongylus rufescens. protostrongylus rufescens is a worldwide parasite of sheep, goats, and wild ruminants. it requires an intermediate snail as a host. infection is usually subclinical, but protostrongylus rufescens can be pathogenic for lambs and goat kids and can cause anorexia, diarrhea, weight loss, and mucopurulent nasal discharge. the adult parasite lives in bronchioles as dictyocaulus spp., but it causes pulmonary nodules similar to those of muellerius capillaris. porcine pneumonias are unequivocally a major obstacle for the contemporary swine industry. the incidence, prevalence, and mortality rates of pneumonias in pigs depend on a series of complex, multifactorial interactions. among the most commonly recognized elements linked to porcine pneumonias are the following: • host (age, genetic makeup, immune status) • infectious agents (viruses, bacteria) • environmental determinants (humidity, temperature, ammonia concentrations) • management practices (crowding, mixing of animals, air quality, nutrition, stress) because of the nature of these multifactorial interactions, it will become obvious in the following paragraphs that more often than not a specific type of pneumonia frequently progresses to or coexists with another. the term porcine respiratory disease complex (prdc) has been introduced in clinical practice to describe pigs with signs of respiratory infection involving combined bacterial and viral infections. commonly implicated microbes include porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), porcine circovirus (pcv ), porcine respiratory coronavirus (prcov), mycoplasma hyopneumoniae, and pasteurella multocida. swine influenza (swine flu). swine influenza is a highly contagious acute respiratory viral disease of swine that is caused by swine influenza virus (siv), a type a influenza virus of the family orthomyxoviridae. it is generally accepted that swine influenza resulted from adaptation of the type a influenza virus that caused the human influenza pandemic during world war i. the most common subtypes of siv currently circulating in pigs are h n , h n , and h n . swine influenza is enzootic worldwide and is known to infect human beings who are in close contact with sick pigs. in , an outbreak of swine-human influenza (h n ), presumably transmitted from pigs to human beings, emerged in mexico and rapidly spread to many countries throughout the world. this new "pandemic" was attributed to a triple-reassortant of influenza a virus containing gene segments of swine, eurasian avian, and human strains. human infection with this novel strain affected mainly children and young adults, as well as individuals of any age with an underlying debilitating condition. transmission between influenza-infected and susceptible pigs occurs mainly by aerosol or oral route. siv attaches to and replicates within epithelial cells of the upper respiratory tract; the infection of similar inclusions are occasionally seen in bronchial glandular and renal epithelial cells. the lungs show thickening of the alveolar walls because of hyperplasia of type ii pneumonocytes and interstitial infiltrates of mononuclear cells, peribronchiolar fibrous hyperplasia, and necrotizing bronchitis/bronchiolitis. circovirus can be confirmed in affected tissue by immunohistochemical or pcr techniques. dual infections with pcv and prrsv frequently occur in pigs, and secondary infections with pneumocystis carinii are commonly seen in pigs with this coinfection. characteristically, alveoli are filled with a distinctive foamy exudate that contains the organism, which is not visible in h&e-stained sections but is easily demonstrated with gomori's methenamine silver stain (see fig. - ) . in human beings, pneumocystis (carinii) jirovecii pneumonia (pneumocystosis) is one of the most common and often fatal complications in aids patients. as in aids patients, abnormal populations of cd + and cd + t lymphocytes have been incriminated as the underlying mechanism leading to pneumocystosis in foals and pigs. nipah virus. nipah virus belongs to the paramyxoviridae family and shares a genus (henipavirus) with the closely related hendra virus (see section on pneumonias of horses). another emerging zoonotic disease, nipah virus caused a major epidemic with significant human mortality in southeast asia in and . people handling pigs were primarily affected. similar to hendra virus, fruit bats (flying foxes) act as natural reservoir and are involved in the transmission to pigs by poorly understood mechanisms. in pigs, this virus infects the respiratory system resulting in pneumonia with syncytial cells occurring in the vascular endothelium and in the respiratory epithelium at all levels of the lung. disease is spread to human beings via the respiratory route. human-to-human transmission of this virus has been reported in more recent outbreaks. other viral pneumonias of pigs. porcine respiratory coronavirus (prcov) is sporadically incriminated in pneumonia in pigs. this viral pneumonia is generally mild, and most pigs fully recover if the pneumonia is not complicated with other infections. lesions in the lung are those of bronchointerstitial pneumonia with necrotizing bronchiolitis. interestingly, infections with porcine and other respiratory coronaviruses have been used to investigate the pathogenesis of severe acute respiratory syndrome (sars), an emerging and highly contagious condition in human beings that is attributed to a novel human coronavirus (sars-cov). the relationship between sars-cov and animal coronavirus is still under investigation. other viruses rarely incriminated in porcine respiratory disease complex (prdc) include paramyxovirus, encephalomyocarditis virus, hemagglutinating encephalomyocarditis virus, and adenovirus. petechial hemorrhages in the lung and pulmonary edema may be seen with african swine fever, classical swine fever, and pseudorabies virus infections. porcine enzootic pneumonia. porcine enzootic pneumonia, a highly contagious disease of pigs caused by mycoplasma hyopneumoniae, is grossly characterized by suppurative or catarrhal bronchopneumonia ( fig. - and e- fig. - ). when its worldwide prevalence and deleterious effect on feed conversion are taken into account, this disease is probably the most economically significant respiratory disease of pigs. although an infectious disease, it is very much influenced by immune status and management factors, such as crowding (airspace and floor space), ventilation (air exchange rate), concentrations of noxious gases in the air (ammonia and hydrogen sulfide), relative humidity, temperature fluctuations, and mixing of stock from various sources. it has been demonstrated with inhibiting interferons and deregulates the adaptive immune response, thus interfering with the normal defense mechanisms predisposing pigs to septicemia and bacterial pneumonia. the most common opportunistic organisms are streptococcus suis, salmonella choleraesuis, mycoplasma hyopneumoniae, haemophilus parasuis, bordetella bronchiseptica, pasteurella multocida, and pneumocystis carinii. dual viral infections with prrsv and porcine circovirus (pcv ), siv, and porcine respiratory coronavirus (prcov) are commonly found in pigs, and such coinfections increase the severity of disease. on postmortem examination, pulmonary lesions vary from very mild changes characterized by failure of the lung to collapse when the thorax is opened and the presence of rib imprints (see fig. - ) to severe changes manifested by consolidation of the lung in cases that have been complicated with bacterial pneumonia. tracheobronchial and mediastinal lymph nodes are typically enlarged. microscopically, pulmonary changes are those of interstitial pneumonia characterized by thickening of alveolar walls by infiltrating macrophages and lymphocytes and mild hyperplasia of type ii pneumonocytes. necrotic cells are scattered in the alveolar lumens. unlike some other viral infections, bronchiolar epithelium does not appear to be affected. diagnosis of prrs in tissue collected at necropsy can be confirmed by immunohistochemistry and pcr techniques. infected pigs may become carriers and transmit the infection through body fluids and semen. clinically, prrs in nursery and young growing animals is characterized by sneezing, fever, anorexia, dyspnea, cough, and occasional death. some piglets develop severe cyanosis of the abdomen and ears, which explains why this syndrome was named blue ear disease when first described in europe. porcine circovirus-associated disease. another emerging porcine syndrome, characterized clinically by progressive emaciation in weaned pigs, was originally described in the s in canada, the united states, and europe. since then, it has disseminated to many countries, causing economic devastation in pig farms worldwide. because of the clinical signs and lesions in many organs, this syndrome was named postweaning multisystemic wasting syndrome (pmws). porcine circovirus (pcv ) has been incriminated as the etiologic agent and is a member of the circoviridae family. pcv has been associated with a number of syndromes in pigs, including systemic pcv infection (the preferred term for pmws because it may also affect mature pigs), pcv -associated pneumonia, pcv -associated enteritis, porcine dermatitis and nephropathy syndrome (pdns), pcv -associated reproductive failure, and, most recently, pcv -associated cerebellar vasculitis. the diseases caused by pcv are now collectively known as porcine circovirus-associated disease (pcvad); the most common manifestations are systemic pcv infection (pmws) and pcv -associated pneumonia as part of the porcine respiratory disease complex. all of these manifestations affect more than one organ, and there is substantial overlap between the syndromes. at necropsy, pigs with systemic pcv infection (pmws) and pcv -associated pneumonia are often in poor body condition, and the most remarkable changes, not considering other possible secondary infections, are enlargement of the superficial and visceral lymph nodes and a mild interstitial pneumonia characterized by failure of the lungs to collapse when the thorax is opened. jaundice is occasionally observed. microscopically, the lymphoid tissues show lymphoid depletion, histiocytic replacement of follicles, and notable proliferation of parafollicular histiocytes, some of which fuse and form syncytial cells (granulomatous lymphadenitis); necrosis of the lymphoid follicles is seen less often. in some cases, large basophilic inclusion bodies are present singly or as grapelike clusters (botryoid inclusions) within the cytoplasm of macrophages, particularly in peyer's patches, spleen, and lymph nodes (e- fig. - ). chapter respiratory system, mediastinum, and pleurae peribronchial, bronchiolar, and alveolar interstitium. additional virulence factors include the ability of mycoplasma hyopneumoniae to cause immunosuppression, reduce the phagocytic activity of neutrophils in the lung, and change the chemical composition of mucus. all of these functional alterations can predispose the lung to secondary bacterial infections. the lesions caused by mycoplasma hyopneumoniae start as a bronchointerstitial pneumonia and progress to a suppurative or mucopurulent bronchopneumonia once secondary pathogens are involved (commonly seen at necropsy). in most pigs, gross lesions affect only portions of the cranial lobes, but in more severely affected pigs, lesions involve % or more of the cranioventral portions of the lungs (see fig. - ). the affected lungs are dark red in the early stages but have a homogeneous pale-gray ("fish flesh") appearance in the more chronic stages of the disease. on cut surface, exudate can easily be expressed from airways, and depending on the stage of the lesions and secondary infections, the exudate varies from purulent to mucopurulent to mucoid. microscopic lesions are characterized by an influx of macrophages and neutrophils into the bronchi, bronchioles, and alveoli, and with time there is also notable balt hyperplasia (see fig. - , b) . in some cases, accumulation of exudate can be severe enough to cause occlusion of bronchioles and atelectasis of the corresponding lobules. the suppurative bronchopneumonia may be accompanied by a mild fibrinous pleuritis, which is often more severe if other organisms, such as mycoplasma hyorhinis, pasteurella multocida, or actinobacillus pleuropneumoniae, are also involved. abscesses and fibrous pleural adhesions are sequelae of chronic complicated infections. clinically, enzootic pneumonia occurs as a herd problem in two disease forms. a newly acquired infection of a previously clean herd causes disease in all age groups, resulting in acute respiratory distress and low mortality. in a chronically infected herd, the mature animals are immune and clinical signs are usually apparent only in growing pigs at times of particular stress such as at weaning. in such herds, coughing and reduced rate of weight gain are the most notable signs. porcine pasteurellosis. porcine pasteurellosis is an infectious disease complex with unclear pathogenesis that includes primary infections by pasteurella multocida alone (primary pasteurellosis) or, more frequently, after the defense mechanisms are impaired and a secondary bacterium colonizes the lung (porcine pneumonic pasteurellosis). in rare cases, pasteurella multocida causes acutely fatal septicemias in pigs (primary septicemic pasteurellosis). it is important to remember that pasteurella multocida serotypes a and d are both part of the normal nasal flora and are also causative agents of bronchopneumonia, pleuritis, and atrophic rhinitis in pigs. pasteurella multocida is one of the most common secondary pathogens isolated from the lungs of pigs with swine influenza virus (siv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus (pcv ), pseudorabies (suhv- ), classical swine fever (hog cholera), enzootic pneumonia, and porcine pleuropneumonia. secondary infections with pasteurella multocida notably change the early and mild bronchointerstitial reaction of enzootic and viral pneumonias into a severe suppurative bronchopneumonia with multiple abscesses and sometimes pleuritis. the other important role of pasteurella multocida in porcine pneumonias is as a cause of a fulminating, cranioventral, fibrinous bronchopneumonia (pleuropneumonia) after influenza virus infection or stress from inadequate ventilation resulting in high levels of ammonia in the air. the nature of the lesion and the predisposing factors of poor management or coexisting viral infections suggest that fulminating porcine pasteurellosis has a pathogenesis similar to that of pneumonic mannheimiosis of cattle. pharyngitis with subcutaneous cervical edema, fibrinohemorrhagic polyarthritis, and focal lymphocytic pcr that mycoplasma hyopneumoniae is present in the air of infected farms. the causative agent, mycoplasma hyopneumoniae, is a fastidious organism and very difficult to grow; thus the final diagnosis is frequently based on interpretation of lesions alone or supported by ancillary tests to detect this mycoplasma in affected lungs by immunohistochemistry, immunofluorescence, or pcr. the bronchopneumonic lesions of porcine enzootic pneumonia are in most cases mild to moderate, and thus mortality is low unless complicated with secondary pathogens, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, bordetella bronchiseptica, haemophilus spp., mycoplasma hyorhinis, and other mycoplasmas and ureaplasmas. although the pathogenesis of porcine enzootic pneumonia is not completely elucidated, it is known that mycoplasma hyopneumoniae first adheres to the cilia of the bronchi by means of a unique adhesive protein, produces ciliostasis, and finally colonizes the respiratory system by firmly attaching to the ciliated epithelial cells of the trachea and the bronchi of the cranioventral regions of the lungs. once attached to the respiratory epithelium, it provokes an influx of neutrophils into the tracheobronchial mucosa; causes extensive loss of cilia (deciliation); stimulates an intense hyperplasia of lymphocytes in the balt; and attracts mononuclear cells into the factors have been identified. these factors allow actinobacillus pleuropneumoniae to attach to cells; produce pores in cell membranes; damage capillaries and alveolar walls, resulting in vascular leakage and thrombosis; impair phagocytic function; and elicit failure of clearance mechanisms. the gross lesions in the acute form consist of a fibrinous bronchopneumonia characterized by severe consolidation and a fibrinous exudate on the pleural surface. although all lobes can be affected, a common site is the dorsal area of the caudal lobes. in fact, a large area of fibrinous pleuropneumonia involving the caudal lobe of a pig's lung is considered almost diagnostic for this disease (fig. - ) . on cut surface, consolidated lungs have notably dilated interlobular septa and irregular but well-circumscribed areas of necrosis caused by potent cytotoxins produced by actinobacillus pleuropneumoniae. except for the distribution, pulmonary lesions of porcine pleuropneumonia are identical to those of pneumonic mannheimiosis of cattle. the microscopic lesions are also very similar and include areas of coagulative necrosis surrounded by a thick cluster of "streaming (oat-shaped/oat cell) leukocytes" and notable distention of the interlobular septa because of severe edema and lymphatic thrombosis. bronchioles and alveoli are filled with edematous fluid, fibrin, neutrophils, and few macrophages (see fig. - ). pigs with the chronic form have multiple pulmonary abscesses and large ( to cm) pieces of necrotic lung encapsulated by connective tissue (sequestra)-changes frequently seen in slaughterhouses. interstitial nephritis are also present in porcine pneumonic pasteurellosis. sequelae of porcine pneumonic pasteurellosis include fibrous pleuritis and pericarditis, pulmonary abscesses, so-called sequestra, and usually death. in contrast to ruminants, mannheimia haemolytica is not a respiratory pathogen for pigs, but in some instances, it can cause abortion in sows. porcine pleuropneumonia. porcine pleuropneumonia is a highly contagious, worldwide disease of pigs caused by actinobacillus (haemophilus) pleuropneumoniae (app), which is characterized by a severe, often fatal, fibrinous bronchopneumonia with extensive pleuritis (pleuropneumonia). survivors generally develop notable residual lesions and become carriers of the organisms. porcine pleuropneumonia is an increasingly important cause of acute and chronic pneumonias, particularly in intensively raised pigs ( to months old). transmission of actinobacillus pleuropneumoniae occurs by the respiratory route, and the disease can be reproduced experimentally by intranasal inoculation of the bacterium. considered a primary pathogen, actinobacillus pleuropneumoniae can sporadically produce septicemia in young pigs and otitis media and otitis interna with vestibular syndrome in weaned pigs. two biovars and serotypes of the organism have been identified; all serotypes can cause the disease, but differences in virulence exist. the pathogenesis is not yet well understood, but specific virulence factors, such as rtx toxins (hemolytic/cytolytic toxins apx i to apx iv), capsular factors, fimbriae and adhesins, lipopolysaccharide, and permeability tuberculosis. tuberculosis is an important disease in domestic and wild pigs that has a much greater prevalence in pigs than in cattle or other domestic mammals in many countries. porcine tuberculosis is attributed to infection with mycobacterium bovis and porcine mycobacteriosis to infection with mycobacterium avium complex. a common scenario in small mixed-farming operations is the diagnosis of avian tuberculosis at the time that pigs are slaughtered, and the source is ingestion of tuberculous chickens or contaminated litter. as would be expected, granulomas are found in the mesenteric, mandibular, and retropharyngeal lymph nodes; to a lesser extent in the intestine, liver, and spleen; and only in rare cases in the lung. the route of infection in pulmonary tuberculosis and mycobacteriosis of pigs is most often hematogenous after oral exposure and intestinal infection. lung lesions are those of a granulomatous pneumonia. the microscopic lesions are basically those of tubercles (granulomas), but the degree of encapsulation, caseation, and calcification varies with the type of mycobacterium, age of the lesion, and host immune response. other bacterial pneumonias of pigs. septicemias in pigs often cause petechial hemorrhages in the lung and pulmonary edema. salmonellae, escherichia coli, and listeria monocytogenes can cause severe interstitial pneumonia in very young animals. salmonella choleraesuis causes a necrotizing fibrinous pneumonia similar to porcine pleuropneumonia, and salmonella typhisuis causes a chronic suppurative bronchopneumonia. in high health herds, actinobacillus suis may cause fibrinohemorrhagic pleuropneumonia and is easily confused with porcine pleuropneumonia. metastrongylosis. metastrongylus apri (elongatus), metastrongylus salmi, and metastrongylus pudendotectus (lungworms) of domestic and feral pigs occur throughout most of the world and require earthworms as intermediate hosts for transmission. the incidence of disease has therefore decreased with development of confinement housing. the importance of pig lungworms is mainly because infection results in growth retardation of the host. clinical signs include coughing because of parasitic bronchitis. the gross lesions, when noticeable, consist of small gray nodules, particularly along the ventral borders of the caudal lobes. the adult worms are grossly visible in bronchi, and microscopically, the parasites cause a catarrhal bronchitis with infiltration of eosinophils and lobular atelectasis ( fig. - ) . ascaris suum. the larvae of ascaris suum can cause edema, focal subpleural hemorrhages, and interstitial inflammation (see fig. - ). along their larval migration tracts, hemorrhages also occur in the liver and, after fibrosis, become the large white "milk spots" seen so frequently as incidental findings at necropsy. it has been reported that ascaris suum may cause immunosuppression in severely affected pigs. pigs can be killed if exposed to an overwhelming larval migration. other causes of pneumonia. foreign body granulomatous pneumonia occurs frequently in pigs after inhalation of vegetable material (starch pneumonia), presumably from dusty (nonpelleted) feed. lesions are clinically silent but are often mistaken for other pneumonic processes during inspection at slaughterhouses. microscopically, pulmonary changes are typical of foreign body granulomatous inflammation in which variably sized feed particles are surrounded by macrophages and neutrophils, and often have been phagocytosed by multinucleated giant cells. feed (vegetable) particles appear as thick-walled polygonal cells that stain positive with pas because of their rich carbohydrate (starch) content (see e- fig. - ) . clinically, porcine pleuropneumonia can vary from an acute form with unexpected death and blood-stained froth at the nostrils and mouth to a subacute form characterized by coughing and dyspnea accompanied by clinical signs of sepsis such as high fever, hypoxemia, anorexia, and lethargy (e- fig. - ) . a chronic form is characterized by decreased growth rate and persistent cough. animals that survive often carry the organism in the tonsils, shed the organism, and infect susceptible pigs. haemophilus pneumonia. in addition to glasser's disease characterized by polyserositis (pericarditis, pleuritis, peritonitis, polyarthritis, and meningitis) (e- fig. - ) , some serotypes of haemophilus parasuis (originally haemophilus influenzae suis) can also cause suppurative bronchopneumonia that in severe cases can be fatal. the causal organism, haemophilus parasuis, is usually carried in the nasopharynx of normal pigs and requires abnormal circumstances such as those following stress (weaning and cold weather) or viral infections (swine influenza or pcv ). specific pathogen-free (spf) pigs seem to be particularly susceptible to glasser's disease (arthritis and serositis) but not to pulmonary infection (bronchopneumonia). streptococcal pneumonia. streptococcus suis is a common cause of porcine disease worldwide and a serious zoonosis capable of causing death by septic shock or meningitis and residual deafness in butchers, veterinarians, and pig farmers. typically, streptococcus suis gains entrance to the susceptible young pig through the oropharyngeal mucosa and is carried in the tonsils, nasal mucosa, and mandibular lymph nodes of healthy animals, particularly in survivors of an outbreak. infected sows can abort or vertically transmit the infection to their offspring. some serotypes of streptococcus suis cause neonatal septicemia, and this can result in suppurative meningitis, otitis, arthritis, polyserositis, myocarditis, valvular endocarditis, and embolic pneumonia ( fig. - ). other serotypes of streptococcus suis can reach the lung by the aerogenous route and cause a suppurative bronchopneumonia, in combination with pasteurella multocida, escherichia coli, or mycoplasma hyopneumoniae, or in combination with actinobacillus pleuropneumoniae, which causes a fibrinous bronchopneumonia. coinfections of streptococcus suis with pcv and prrsv are also frequently seen in some farms. gross lesions in the acute stages include serous to catarrhal to mucopurulent nasopharyngitis and conjunctivitis. the lungs are edematous and have a diffuse interstitial pneumonia ( fig. - ) microscopically characterized by necrotizing bronchiolitis, necrosis and exfoliation of pneumonocytes, mild alveolar edema, and, several hours later, thickening of the alveolar walls because of interstitial mononuclear cell infiltrates and hyperplasia of type ii pneumonocytes. secondary infections with bordetella bronchiseptica and mycoplasmas are common and induce life-threatening suppurative bronchopneumonia. the thymus may be small relative to the age of the animal because of viral-induced lymphocytolysis. microscopically, eosinophilic inclusions are present in the epithelial cells of many tissues, in the nuclei or cytoplasm, or in both (see fig. - ). they appear early in the bronchiolar epithelium but are most prominent in the epithelium of the lung, stomach, renal pelvis, and urinary bladder, making these tissues good choices for diagnostic examination. viral inclusions are rarely seen in the later stages of this disease. the suppurative secondary bronchopneumonias often hinder the detection of viral lesions in the lung, particularly because bronchiolar cells containing inclusion bodies exfoliate and mix with the neutrophils recruited by the bacterial infection. distemper virus antigens can be readily demonstrated in infected cells by the immunoperoxidase technique (see fig. - ), which can also be used in skin biopsies for the antemortem diagnosis of canine distemper. distemper virus also has a tendency to affect developing tooth buds and ameloblasts, causing enamel hypoplasia in dogs that recover from infection. of all distemper lesions, demyelinating encephalomyelitis, which develops late, is the most devastating (see chapter ). sequelae to distemper include the nervous and pneumonic complications mentioned previously and various systemic infections, such as toxoplasmosis and sarcocystosis, because of depressed immunity. persistent viral infection occurs in some dogs that survive the disease, and they may become carriers and the source of infection for other susceptible animals. clinical signs consist of biphasic fever, diarrhea, vomiting, weight loss, mucopurulent oculonasal discharge, coughing, respiratory distress, and possible loss of vision. weeks later, hyperkeratosis of the foot pads ("hard pad") and the nose are observed, along with nervous signs, including ataxia, paralysis, convulsions, or residual myoclonus (muscle twitches, tremors, and "tics"). in general, inflammatory diseases of the lungs are less of a problem in dogs than in food-producing species and can be subdivided in two major groups, infectious and noninfectious pneumonias. "canine infectious respiratory disease" (cird) is the term currently used by clinicians to describe a heterogeneous group of respiratory infections in dogs; these diseases were previously clustered under the name of infectious tracheobronchitis or "kennel cough." cird is the canine counterpart of brd and prd complexes in cattle and pigs, respectively. the most common viruses in cird include canine parainfluenza virus (cpiv), canid herpesvirus (cahv- ), canine adenovirus- (cav- ), canine respiratory coronavirus (crcov), canine distemper virus (cdv), and canine influenza virus (civ). bordetella bronchiseptica, streptococcus equi ssp. zooepidemicus, and mycoplasma spp. are the most frequent bacterial isolates in cird. it has been recently recognized that animal shelters are an important source of viral and bacterial infections for dogs and cats. uremia and paraquat toxicity are perhaps the two most notable noninfectious causes of canine respiratory disease. canine distemper. canine distemper is an important and ubiquitous infectious disease of dogs, other canidae, wild felidae, mustelidae, and marine mammals throughout the world. it is caused by a morbillivirus that is antigenically related to the human measles, rinderpest (officially eradicated in ), "peste de petit ruminants," and phocine distemper viruses. canine distemper virus (cdv) is transmitted to susceptible puppies through infected body fluids. the virus invades through the upper respiratory tract and conjunctiva, proliferates in regional lymph nodes, becomes viremic, and in dogs with an inadequate antibody response, infects nearly all body tissues (pantropic), particularly the epithelial cells. distemper virus hampers the immune response, downregulates cytokine production, and persists for a long time in some tissues. cdv can target the lungs either directly as a viral pneumonia or indirectly by its immunosuppressive effects rendering the lungs susceptible to secondary bacterial and protozoal infections, or as a coinfection with other viruses such as canine adenovirus- and canid herpesvirus . : - , .) inclusion bodies occur within epithelial cells in early lesions. cahv- has also been identified as a cause of ulcerative keratoconjunctivitis in older dogs. canine influenza (canine flu). canine influenza is an emerging contagious respiratory infection of dogs that was first described in the united states and subsequently in other countries. it has a high morbidity (close to %), but the mortality, as with most other influenza infections, is relatively low (less than %). this disease, first diagnosed in greyhounds, is caused by a novel influenza-a virus (canine influenza virus or civ), a mutation from a previously recognized h n strain of equine influenza virus. dog-to-dog transmission does occur and therefore this infection must be distinguished from other viruses of the canine infectious respiratory disease (cird) group. pulmonary lesions are generally mild and transient, but infected dogs are susceptible to secondary bacterial bronchopneumonia. the most relevant lesions in dogs dying unexpectedly from canine influenza are pleural and pulmonary hemorrhages. microscopically, there is necrotizing tracheitis, bronchitis, and bronchiolitis with exudation of neutrophils and macrophages. in severe cases, hemorrhagic interstitial or bronchointerstitial pneumonia may be accompanied by vasculitis and thrombosis. influenza antigen can be demonstrated by immunohistochemistry in airway epithelium and alveolar macrophages. clinically, dogs with canine influenza are lethargic, inappetent, and hyperthermic and frequently cough and show nasal discharge. these signs resemble those seen in dogs with kennel cough or secondary bacterial pneumonia. in addition, there are confirmed cases of canine influenza caused by the porcine h n presumably transmitted from infected pet owners. bacterial pneumonias. dogs generally develop bacterial pneumonias when the pulmonary defense mechanisms have been impaired. pasteurella multocida, streptococcus spp., escherichia coli, klebsiella pneumoniae, and bordetella bronchiseptica can be involved in pneumonia secondary to distemper or after aspiration of gastric contents ( fig. - and e- fig. - ). streptococcus zooepidemicus can cause acute and fatal hemorrhagic pleuropneumonia with canine adenovirus type infection. cav- infection is a common but transient contagious disease of the respiratory tract of dogs, causing mild fever, oculonasal discharge, coughing, and poor weight gain. the portal of entry is generally by inhalation of infected aerosols followed by viral replication in the surface cells of the upper respiratory tract, mucous cells of the trachea and bronchi, nonciliated bronchiolar epithelial cells, and type ii pneumonocytes. pulmonary lesions are initially those of bronchointerstitial pneumonia, with necrosis and exfoliation of bronchiolar and alveolar epithelium, edema, and, a few days later, proliferation of type ii pneumonocytes, mild infiltration of neutrophils and lymphocytes in the alveolar interstitium, and hyperplastic bronchitis and bronchiolitis. large basophilic intranuclear viral inclusions are typically seen in bronchiolar and alveolar cells ( fig. - ) . infection with cav- is clinically mild unless complicated with a secondary bacterial infection or coinfections with other viruses such as distemper virus. experimental work suggests cav- reinfection may lead to hyperreactive airways, a nonspecific condition in which the bronchial mucosa becomes highly "responsive" to irritation such as that caused by cold air, gases, or cigarette smoke. however, it is not clear if this outcome is true in natural infections. canid herpesvirus . canid herpesvirus (cahv- ) can cause fatal systemic disease in newborn puppies and is probably a contributing factor in "fading puppy syndrome." hypothermia has been suggested as a pivotal component in the pathogenesis of fatal infections in puppies. many dogs are seropositive, suggesting that transient or subclinical infections are more common than realized; the virus remains latent in the trigeminal and other ganglia and can be reactivated after stress, resulting in asymptomatic transmission of cahv- virus to offspring via the placenta, thus resulting in abortion or stillbirths. in puppies, cahv- causes ulcerative tracheitis, interstitial pneumonia (e- fig. - ) , and focal necrosis and inflammation in the kidneys, liver, and brain. eosinophilic intranuclear aspiration pneumonia starts as an acute necrotizing bronchitis and bronchiolitis caused by aspiration of irritant materials such as gastric acid or a caustic material administered by mouth. the aspirate also contains potentially pathogenic bacteria, and because the mucociliary apparatus is damaged and these bacteria are not removed, they settle into the ventral portions of the lung (from gravity) and provoke a fibrinosuppurative and necrotizing bronchopneumonia. b, bronchoalveolar spaces are filled with neutrophils, macrophages, and bacteria (arrows). h&e stain. inset, large colonies of bacteria (arrows). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) a b infection; thus it most frequently affects outdoor and hunting dogs. from the lung, infection is disseminated hematogenously to other organs, mainly bone, skin, brain, and eyes. pulmonary lesions are characterized by multifocal to coalescing pyogranulomatous pneumonia, generally with firm nodules scattered throughout the lungs (fig. - ) . microscopically, nodules are pyogranulomas with numerous macrophages (epithelioid cells), some neutrophils, multinucleated giant cells, and thick-walled yeasts (see fig. - , c). yeasts are to µm in diameter and are much better visualized when they are stained with pas reaction or gomori's methenamine silver stain. nodules can also be present in other tissues, chiefly lymph nodes, skin, spleen, liver, kidneys, bones, testes, prostate, and eyes. this fungus can be easily identified in properly prepared and stained transtracheal washes or lymph node aspirates. clinical signs can reflect involvement of virtually any body tissue; pulmonary effects include cough, decreased exercise tolerance, and terminal respiratory distress. coccidioidomycosis. coccidioidomycosis (san joaquin valley fever), caused by the dimorphic fungus coccidioides immitis, occurs mainly in animals living in arid regions of the southwestern united states, mexico, and central and south america. it is a primary respiratory tract (aerogenous) infection commonly seen at slaughterhouses in clinically normal feedlot cattle. in dogs, coccidioidomycosis also has an aerogenous portal of entry and then a b hemorrhagic pleural effusion in dogs. death is generally a consequence of severe sepsis and septic shock or from β-hemolytic streptococcal bacteremia causing emboli in the lungs, liver, brain, and lymph nodes. the primary source of the infection cannot be determined in most cases. dental disease in dogs may be a source of systemic and pulmonary infection, a concept wellrecognized in human medicine for many years. the role of mycoplasmas in canine pneumonia is still uncertain because these organisms are frequently isolated from normal nasopharyngeal flora. tuberculosis is uncommon in dogs because these animals appear to be quite resistant to infection; most cases occur in immunocompromised dogs or in dogs living with infected human beings. dogs are susceptible to the infection with mycobacterium tuberculosis, mycobacterium bovis, and mycobacterium avium complex, and therefore canine infection presupposes contact with human or animal tuberculosis. the clinicopathologic manifestation is pulmonary after inhalation or alimentary after oral exposure, but in most cases infection is disseminated to lymph nodes and visceral organs. the gross lesions are multifocal, firm nodules with necrotic centers, most often seen in the lungs, lymph nodes, kidneys, and liver. diffuse granulomatous pleuritis and pericarditis with copious serofibrinous or sanguineous effusion are common. microscopically, granulomas are formed by closely packed macrophages but with very little connective tissue. mycotic pneumonias. mycotic pneumonias are serious diseases seen commonly in animals in some regions. there are two main types: those caused by opportunistic fungi and those caused by a group of fungi associated with systemic "deep" mycoses. all of these fungi affect human beings and most domestic animals but are probably not transmitted between species. aspergillosis. opportunistic fungi, such as aspergillus spp. (particularly aspergillus fumigatus), are important in birds, but in domestic animals, they mainly affect immunosuppressed individuals or those on prolonged antibiotic therapy. the pulmonary lesion is a multifocal, nodular, pyogranulomatous, or granulomatous pneumonia. microscopically, there is necrosis and infiltrates of neutrophils, macrophages, and lymphocytes, with proliferation of fibroblasts eventually leading to encapsulation of the granuloma. fungal hyphae are generally visible in the core of the lesion and in the walls of blood vessels. systemic mycoses (dimorphic fungal infections) . systemic (deep) mycoses are caused by blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans/ cryptococcus gatti (see fig. - ). blastomycosis mainly affects dogs and is discussed here, whereas cryptococcosis is discussed in the section on pneumonias of cats. in contrast to other fungi, such as aspergillus spp., organisms of the systemic mycosis group are all primary pathogens of human beings and animals and thus do not necessarily require a preceding immunosuppression to cause disease. these fungi have virulence factors that favor hematogenous dissemination and evasion of immune and phagocytic responses. systemic dissemination is often exacerbated by the administration of immunosuppressant drugs such as corticosteroids. these fungi are usually detected by cytological evaluation of affected tissues. blastomycosis. blastomycosis occurs in many countries of the north american continent, africa, the middle east, and occasionally in europe. in the united states, it is most prevalent in the atlantic, st. lawrence, and ohio-mississippi river valley states, compared with the mountain-pacific region. blastomyces dermatitidis is a dimorphic fungus (mycelia-yeast) seen mainly in young dogs and occasionally in cats and horses. this fungus is present in the soil, and inhalation of spores is considered the principal route of from the alveolar interstitium associated with larvae or dead worms because little reaction develops to the live adults. crenosoma vulpis. crenosoma vulpis is a lungworm seen commonly in foxes and sporadically in dogs with access to the intermediate hosts-slugs and snails. the adult lungworms live in small bronchi and bronchioles in the caudal lobes, causing eosinophilic and catarrhal bronchitis manifested grossly as gray areas of inflammation and atelectasis. in some animals, crenosoma vulpis causes bronchiolar goblet cell metaplasia and mucous obstruction, resulting in lobular atelectasis due to the valve effect of the mucous plug. eucoleus aerophilus. eucoleus aerophilus (capillaria aerophila) is a nematode parasite typically found in the trachea and bronchi of wild and domestic carnivores. in some cases, this parasite may also involve the nasal passages and sinuses. although generally asymptomatic, some dogs cough because of the local irritation caused by the parasites on the tracheal or bronchial mucosa. paragonimus spp. paragonimus kellicotti in north america and paragonimus westermani in asia are generally asymptomatic fluke infections in fish-eating species. the life cycle involves two intermediate hosts, the first a freshwater snail and the second a freshwater crab or crayfish; in north america, cats and dogs acquire infection by eating crayfish. gross lesions include pleural hemorrhages where the metacercariae migrate into the lungs. later, multifocal eosinophilic pleuritis, and subpleural cysts up to mm long containing pairs of adult flukes, are found along with eosinophilic granulomas around clusters of eggs. like many other parasitic pneumonias, lesions and scars are more frequent in the caudal lobes. pneumothorax can occur if a cyst that communicates with an airway ruptures to the pleural surface. other parasitic infections. angiostrongylus vasorum and dirofilaria immitis are parasites of the pulmonary arteries and right ventricle and, depending on the stage, can produce different forms of pulmonary lesions. adult parasites can cause chronic arteritis that leads to pulmonary hypertension, pulmonary arterial thrombosis, interstitial (eosinophilic) granulomatous pneumonia, pulmonary interstitial fibrosis, congestive right-sided cardiac failure, and eventually caudal vena caval syndrome. other lesions include pleural petechial hemorrhages and, in later stages, diffuse pulmonary hemosiderosis and multifocal pulmonary infarcts. larvae and eggs also cause alveolar injury, thickening of the alveolar walls with eosinophils and lymphocytes (interstitial pneumonia), and multifocal or coalescing granulomas with giant cells (parasitic granulomas). pneumocystis carinii has been reported as a sporadic cause of chronic interstitial pneumonia in dogs with a compromised immune system (see pneumonias of horses; also see fig. - ). aspiration pneumonia. aspiration pneumonia is an important form of pneumonia that occurs in dogs when vomit or regurgitated materials are aspirated into the lungs, or when drugs or radiographic contrast media are accidentally introduced into the airways (e- fig. - ) . as in other animal species, aspiration pneumonia may be unilateral or may more often affect the right cranial lobe ( fig. - ). the severity of lesions depends very much on the chemical and microbiologic composition of the aspirated material. in general, aspiration in monogastric animals, particularly in dogs and cats, is more severe because of the low ph of the gastric contents (chemical pneumonitis). in severe cases, dogs and cats die rapidly from septic shock and ards (see fig. - ), which is microscopically characterized by diffuse alveolar damage, protein-rich pulmonary edema, neutrophilic alveolitis, and formation of typical hyaline membranes along the alveolar walls (see fig. - ). in animals that survive the acute stages of aspiration, pulmonary lesions progress to bronchopneumonia. aspiration pneumonia is a common sequela to disseminates systemically to other organs. clinical signs relate to the location of lesions, so there can be respiratory distress, lameness, generalized lymphadenopathy, or cutaneous lesions, among others. the lesions caused by coccidioides immitis consist of focal granulomas or pyogranulomas that can have suppurative or caseated centers. the fungal organisms are readily seen in histologic or cytologic preparation as large ( to µm in diameter), double-walled, and highly refractile spherules containing numerous endospores (see fig. - , d) . histoplasmosis. histoplasmosis is a systemic infection that results from inhalation and, in dogs, possibly ingestion of another dimorphic fungus, histoplasma capsulatum. histoplasmosis occurs sporadically in dogs and human beings and, to a lesser extent, in cats and horses. bats often eliminate histoplasma capsulatum in the feces, and droppings from bats and birds, particularly pigeons, heavily promote the growth and survival of this fungus in the soil of enzootic areas. pulmonary lesions are grossly characterized by variably sized, firm, poorly encapsulated granulomas and, sometimes, more diffuse involvement of the lungs. microscopically, granulomatous lesions typically have many macrophages filled with small ( to µm), punctiform, intracytoplasmic, dark oval bodies (yeasts) (see fig. - , a) that are best demonstrated with pas reaction or gomori's methenamine silver stain. similar nodules or diffuse involvement can be present in other tissues, chiefly lymph nodes, spleen, intestine, and liver. toxoplasmosis. toxoplasmosis is a worldwide disease caused by the obligate intracellular, protozoal parasite toxoplasma gondii. cats and other felidae are the definitive hosts in which the mature parasite divides sexually in the intestinal mucosa. human beings, dogs, cats, and many wild mammals can become intermediate hosts after accidental ingestion of fertile oocysts shed in cat feces or ingestion of undercooked or raw meat containing tissue cysts, and fetuses can be infected transplacentally from an infected dam. in most instances, the parasite infects many cells of different tissues and induces an antibody response (seropositive animals) but does not cause clinical disease. toxoplasmosis is often triggered by immunosuppression, such as that caused by canine distemper virus. toxoplasmosis is characterized by focal necrosis around the protozoan. pulmonary lesions are severe, multifocal necrotizing interstitial pneumonia with notable proliferation of type ii pneumonocytes and infiltrates of macrophages and neutrophils. other lesions in disseminated toxoplasmosis include multifocal necrotizing hepatitis, myocarditis, splenitis, myositis, encephalitis, and ophthalmitis. the parasites appear microscopically as small ( to µm) basophilic cysts that can be found free in affected tissues or within the cytoplasm of many epithelial cells and macrophages (see e- fig. - ) . similar findings can be seen sporadically in dogs infected with neospora caninum and sarcocystis canis, and immunohistochemistry would be required to differentiate those protozoal organisms from toxoplasma gondii. filaroides hirthi. filaroides hirthi, a lungworm of the alveoli and bronchioles of dogs, has long been known as a cause of mild subclinical infection in large colonies of beagle dogs in the united states. however, it can on occasion cause severe and even fatal disease in individual pets, presumably as a result of immunosuppression. clinical signs may include coughing and terminal respiratory distress. grossly, the lesions are multifocal subpleural nodules, often with a green hue because of eosinophils, scattered throughout the lungs. microscopically, these nodules are eosinophilic granulomas arising .e chapter respiratory system, mediastinum, and pleurae other pneumonias. idiopathic pulmonary fibrosis is a rare condition of uncertain etiology reported in the west highland white terrier breed that shares similarities with human and feline idiopathic pulmonary fibrosis. microscopically, there is diffuse interstitial pneumonia and progressive alveolar fibrosis with capillary obliteration, hyperplasia of type ii cells, some of which exhibit cellular atypia, and finally hypertrophy and hyperplasia of smooth muscle. the interstitial fibrosis eventually spills over alveolar spaces causing conspicuous intraalveolar fibrosis. although upper respiratory tract infections are common and important in cats, pneumonias are uncommon except when there is immunosuppression or aspiration of gastric contents. viral infections such as feline rhinotracheitis and calicivirus may cause lesions in the lungs, but unless there is secondary invasion by bacteria, they do not usually cause a fatal pneumonia. feline rhinotracheitis. feline rhinotracheitis is an important viral disease of cats caused by the ubiquitous felid herpesvirus (fehv- ). this infection affects primarily young or debilitated cats causing inflammation in the nasal, ocular, and tracheal mucosa and, to a much lesser extent, the lung (see species-specific diseases of the nasal cavity and paranasal sinuses). when lungs are affected, fehv- causes bronchointerstitial pneumonia with necrosis of bronchiolar and alveolar epithelium, thickening of the alveolar walls, and extensive permeability edema. eosinophilic intranuclear inclusion bodies may be seen in infected epithelial cells early in infection. feline calicivirus. feline calicivirus (fcv) causes upper respiratory disease, stomatitis, conjunctivitis, and, to a lesser extent, interstitial pneumonia. microscopically, affected lungs exhibit the typical pattern of bronchointerstitial pneumonia with necrotizing bronchiolitis, thickening of alveolar walls, occasionally hyaline membranes, hyperplasia of type ii pneumonocytes, and macrophages admixed with cellular debris in the alveolar lumens. because pulmonary lesions are similar to those caused by fehv- , isolation or in situ detection is required for final diagnosis. feline infectious peritonitis. feline infectious peritonitis (fip) is caused by fip virus (fipv), a mutated form of feline enteric cleft palate, and in dogs with megaesophagus secondary to either myasthenia gravis or persistent right aortic arch. it is also an important complication of general anesthesia or neurologic diseases affecting laryngeal function. paraquat. paraquat, a broad-spectrum herbicide widely used in gardening and agriculture, can cause severe and often fatal toxic interstitial pneumonia (pneumonitis) in dogs, cats, human beings, and other species. after ingestion or inhalation, this herbicide selectively accumulates in the lung where paraquat toxic metabolites are produced by club (clara) cells. these metabolites promote local release of free radicals in the lung, which causes extensive injury to club cells and to the blood-air barrier, presumably through lipid peroxidation of type i and ii pneumonocytes and alveolar endothelial cells (see fig. - ). paraquat toxicity has been used experimentally as a model of oxidant-induced alveolar injury and pulmonary fibrosis. soon after poisoning, the lungs are heavy, edematous, and hemorrhagic because of extensive necrosis of epithelial and endothelial cells in the alveolar walls. the lungs of animals that survive acute paraquat toxicosis are pale, fail to collapse when the thorax is opened, and have interstitial emphysema, bullous emphysema, and occasionally pneumomediastinum. microscopic findings in the acute and subacute phases include necrosis of type i pneumonocytes, interstitial and alveolar edema, intraalveolar hemorrhages, and proliferation of type ii pneumonocytes. in the chronic stages ( to weeks later), the lesions are typically characterized by severe interstitial and intraalveolar fibrosis. uremic pneumopathy. uremic pneumonopathy (pneumonitis) is one of the many extrarenal lesions seen in dogs with chronic uremia. lesions are characterized by a combination of pulmonary edema and calcification of vascular smooth muscle and alveolar basement membranes. in severe cases, alveolar calcification prevents lung collapse when the thorax is opened. in the more advanced cases, the lungs appear diffusely distended, pale red or brown in color, and show a rough pleural surface with rib imprints (see fig. - ). on palpation, the pulmonary parenchyma has a typical "gritty" texture because of mineralization of the alveolar and vascular walls, which are best visualized microscopically by using special stains such as von kossa (see fig. - ). because this is not primarily an inflammatory lesion, the term pneumonitis should not be used. fig. - ) . a, note that the lungs did not collapse when the thorax was opened (loss of negative pressure) and as a result fill almost the entire thoracic cavity. the cranioventral aspects of the lung are consolidated with hemorrhage. b, alveolar capillary congestion, thick hyaline membranes along the alveolar septa (arrows), and intraalveolar hemorrhage. these microscopic changes are typical of the diffuse alveolar damage seen in lungs with ards. h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) feline calicivirus has removed chlamydophila felis from its previously overstated importance as a lung pathogen. tuberculosis. cats are susceptible to three types of mycobacterial infections: classic tuberculosis, feline leprosy, and atypical mycobacteriosis. classic tuberculosis in cats is rare and generally caused by mycobacterium bovis and mycobacterium microti but also, to a lesser extent, by mycobacterium tuberculosis. nosocomial tuberculosis (mycobacterium bovis) in cats has been reported with increased frequency. the usual route of infection for feline tuberculosis is oral, through infected rodents/meat or unpasteurized milk, so the granulomatous lesions are mainly in the intestine and mesenteric lymph nodes where they may disseminate through infected phagocytes to other organs. the solid and noncaseated appearance of tuberculous nodules is grossly similar to that of neoplasms, so they must be differentiated from pulmonary neoplasms (e.g., lymphoma). classic tuberculosis with dermal lesions in cats should be differentiated from feline leprosy (localized skin granulomas) caused by mycobacterium lepraemurium and other nonculturable species of acid-fast bacilli. atypical mycobacteriosis is caused by contamination of a skin wound with saprophytic and nonsaprophytic mycobacteria such as those of the mycobacterium avium complex. advances in pcr techniques have notably reduced the time required for etiologic diagnosis of mycobacteriosis in veterinary diagnostic laboratories. cryptococcosis. cryptococcosis (pulmonary cryptococcus neoformans or cryptococcus gatti) is the most frequent systemic mycosis in cats, and lesions are akin to those discussed in the section on mycotic pneumonias of dogs. it occurs worldwide in all species but is diagnosed most frequently in cats, horses, dogs, and human beings. some healthy dogs and cats harbor cryptococcus in the nasal cavity and become asymptomatic carriers. clinical infection may occur in immunocompetent cats and in cats that are immunologically compromised, such as by felv, fiv, malnutrition, or corticosteroid treatment. lesions can occur in nearly any tissue, resulting in a wide c coronavirus (fecv), and is one of a few viral infections of domestic animals that result in pyogranulomatous pneumonia. this disease is microscopically characterized by a vasculitis affecting many tissues and organs ( fig. - ) . other viral pneumonias. other viruses sporadically incriminated in feline interstitial pneumonia are cowpox virus (cpxv) and influenza a h n . pasteurellae. bacteria from the nasal flora such as pasteurella multocida and pasteurella-like organisms are occasionally associated with secondary bronchopneumonia in cats ( fig. - ) . pasteurella multocida also causes otitis media and meningitis, but its role as a respiratory pathogen is mainly associated with pyothorax. interestingly, there are reports of pasteurella multocida pneumonia in older or immunosuppressed human beings acquired through contact with domestic cats. mycoplasmas. mycoplasmas are often isolated from the lungs of cats with pulmonary lesions but are not definitively established as primary pathogens in feline pneumonias. feline pneumonitis. the term feline pneumonitis is a misnomer because the major lesions caused by chlamydophila felis (formerly chlamydia psittaci) are severe conjunctivitis and rhinitis (see species-specific diseases of the nasal cavity and paranasal sinuses). the elucidation of the importance of feline viral rhinotracheitis and organism infects erythrocytes in the erythrocytic stage of disease and multiplies in intravascular macrophages/monocytes, including those in the alveolar capillaries (e- fig. - ) , during the leukocytic stage of disease. aspiration pneumonia. aspiration pneumonias are common in cats as a result of vomiting, regurgitation, dysphagia, or anesthetic complication or after accidental administration of food, oral medicaments, or contrast media into the trachea (iatrogenic). pulmonary lesions are similar to those described for dogs, and the type of lung lesion depends on the chemical and bacterial composition of the aspirated material (see the section on aspiration pneumonia of dogs). feline idiopathic pulmonary fibrosis. feline idiopathic pulmonary fibrosis is a rare, progressive, and fatal disease of cats of uncertain etiology characterized by multifocal fibrotic nodules subpleurally and randomly in the lung making the pleural surface resemble nodular cirrhosis of the liver ( fig. - ) . microscopically, the affected alveolar and peribronchiolar interstitium is thickened by excessive fibrosis, abundant deposition of extracellular matrix, and hypertrophy of smooth muscle. some investigators suggest an intrinsic cellular defect in type ii pneumonocytes as the underlying cause. the alveolar walls are diffusely lined by cuboidal hyperplastic type ii pneumonocytes, and the alveolar lumens often contain exfoliated cells and necrotic debris. this feline condition has morphologic features similar to "equine multinodular pulmonary fibrosis" and "cryptogenic pulmonary fibrosis" in human beings. fetal pneumonias. pneumonia is one of the most frequent lesions found in fetuses submitted for postmortem examination, particularly in foals and food-producing animals. because of autolysis, lack of inflation, and the lungs being at various stages of development, fetal lesions are often missed or misdiagnosed. in the nonaerated fetal lung, the bronchoalveolar spaces are filled with a viscous, locally produced fluid known as lung fluid or lung liquid. it has been estimated that an ovine fetus produces approximately . ml of "lung fluid" per kilogram of body weight per hour. in the variety of clinical signs. however, granulomatous rhinitis, sinusitis, otitis media and interna, pneumonia, ulcerative dermatitis, and meningoencephalitis are most common. the pulmonary lesion in cryptococcosis is a multifocal granulomatous pneumonia and, like those occurring in other internal organs, they are small, gelatinous, white foci. the gelatinous appearance is due to the broad mucous capsule around the yeast (see fig. - , b) . microscopically, lesions contain great numbers of fungal organisms ( to µm in diameter without the capsule) and only a few macrophages, lymphocytes, and multinucleated giant cells. this thick polysaccharide capsule does not stain well with h&e, and thus there is a large empty space or halo around the yeast. feline lungworm. aelurostrongylus abstrusus, known as feline lungworm, is a parasite that occurs in cats wherever the necessary slug and snail intermediate hosts are found. it can cause chronic respiratory disease with coughing and weight loss and, sometimes, severe dyspnea and death, particularly if there are secondary bacterial infections. the gross lesions are multifocal, amber, and subpleural granulomatous nodules up to cm in diameter throughout the lungs. on incision, these nodules may contain viscous exudate. microscopically, the adult parasites, eggs, and coiled larvae are in the bronchioles and alveoli, where they cause catarrhal bronchiolitis, hyperplasia of submucosal glands, and, later, granulomatous alveolitis, alveolar fibrosis, and fibromuscular hyperplasia ( fig. - ) . during routine examination of feline lungs, it is quite common to find fibromuscular hyperplasia in bronchioles and arterioles in otherwise healthy cats. it was alleged in the past that this fibromuscular hyperplasia was a long-term sequela of subclinical infection with aelurostrongylus abstrusus. however, this view has been challenged; thus the pathogenesis and significance of pulmonary fibromuscular hyperplasia in healthy cats remains uncertain. in severe cases, fibromuscular hyperplasia is grossly visible in the lungs as white subpleural nodules. other parasitic pneumonias. toxoplasma gondii, paragonimus kellicotti, and dirofilaria immitis can also affect cats (see the section on parasitic pneumonias of dogs). cytauxzoon felis is an apicomplexan hemoparasite that affects domestic and wild felidae. the diseases that cause fetal pneumonia in farm animals. gross lesions in the lungs are generally undetected, but microscopic lesions include focal necrotizing interstitial pneumonia and focal necrosis in the liver, spleen, or brain. fetal bronchointerstitial pneumonia also occurs in some viral abortions, such as those caused by infectious bovine rhinotracheitis (ibr) virus and bovine parainfluenza virus (bpiv- ) in cattle and equine viral rhinopneumonitis (evr) in horses. fetal pneumonias in dogs and cats are infrequently described, perhaps because aborted puppies and kittens are rarely submitted for postmortem examination. with advancements in molecular biology techniques, the etiologic diagnosis of abortions and their association with pulmonary fetal lesions is rapidly improving. neonatal pneumonias and septicemias. these entities are rather common in newborn animals lacking passive immunity because of the lack of either ingestion or absorption of maternal colostrum (failure of passive transfer or hypogammaglobulinemia). in addition to septicemias causing interstitial pneumonia, farm animals with hypogammaglobulinemia can develop bronchopneumonia by inhalation of bacterial pathogens. these include histophilus somni and pasteurella multocida in calves; streptococcus spp. in foals; and escherichia coli, listeria monocytogenes, and streptococcus suis in pigs. meconium aspiration syndrome. meconium aspiration syndrome (mas) is an important but preventable condition in human babies that originates when amniotic fluid contaminated with meconium is aspirated during labor or immediately after birth. the pathogenesis of mas is basically the same as in those of fetal bronchopneumonia (see fig. - ) . fetal hypoxia, a common event during dystocia or prolonged parturition, causes the fetus to relax the anal sphincter and release meconium into the amniotic fluid. aspiration of meconium can occur directly from aspirating contaminated amniotic fluid before delivery (respiratory movements with an open glottis) or immediately after delivery when the meconium lodged in the nasopharynx is carried into the lung with the first breath of air. this latter form of aspiration is prevented in delivery rooms by routine suction of the nasopharynx in meconium-stained babies. mas is well known in human babies, but the occurrence and significance in animals remains largely unknown. mas has been reported in calves, foals, piglets, and puppies. although pulmonary lesions are generally mild and transient, aspiration of meconium can be life-threatening for newborn babies and animals because it typically occurs in compromised neonates already suffering from intrauterine hypoxia and acidosis. neonatal acidosis is known to impair colostrum absorption in calves. common mas sequelae are lobular atelectasis, pulmonary hypertension, and possibly airway hyperreactivity. in the most severe cases of mas, focal (patchy) atelectasis can be observed grossly in the lung, indicating failure of the lungs to be fully aerated because of the mechanical obstruction and the chemical effect of meconium on pulmonary surfactant (see fig. - ). microscopically, meconium and keratin exfoliated from skin of the fetus into the amniotic fluid are present in bronchi, bronchioles, and alveoli and accompanied by mild alveolitis characterized by infiltration of leukocytes followed by alveolar macrophages and occasional giant cells (e- fig. - ) . lung cancer in animals is rare, unlike in human beings, in which the incidence is alarming and continues to be the number one cause of death due to cancer in canada, the united states, and europe. interestingly, prostatic and breast cancers, so much feared by men fetus, this fluid normally moves along the tracheobronchial tree, reaching the oropharynx, where a fraction is swallowed into the gastrointestinal tract, and a small portion is released into the amniotic fluid. at the time of birth, the lung fluid is rapidly reabsorbed from the lungs by alveolar absorption and lymphatic drainage. aspiration of amniotic fluid contaminated with meconium and bacteria from placentitis is the most common route by which microbial pathogens reach the fetal lungs. this form of pneumonia is secondary to fetal hypoxia and acidosis ("fetal distress"), which cause the fetus to relax the anal sphincter, release meconium into the amniotic fluid, and, in the terminal stages, inspire deeply with open glottis, resulting in the aspiration of contaminated fluid ( fig. - ). gross lesions are only occasionally recognized, but microscopic changes are similar to those of a bronchopneumonia. microscopically, bronchoalveolar spaces contain variable numbers of neutrophils, macrophages, epidermal squames, and pieces of meconium that appear as bright yellow material because of its bile content. in contrast to postnatal bronchopneumonia, lesions in fetuses are not restricted to the cranioventral aspects of the lungs but typically involve all pulmonary lobes. in cattle, brucella abortus and trueperella (arcanobacterium) pyogenes are two of the most common bacteria isolated from the lungs of aborted fetuses. these bacteria are usually present in large numbers in the amniotic fluid of cows with bacterial placentitis. inflammation of the placenta interferes with oxygen exchange between fetal and maternal tissue, and the resultant fetal hypoxia induces the fetus to "breathe" with an open glottis and aspirate the amniotic fluid. aspergillus spp. (mycotic abortion) and ureaplasma diversum cause sporadic cases of placentitis, which results in fetal pneumonia and abortion. in addition to the respiratory route (aspiration), pathogens, such as bacteria and viruses, can also reach the lungs via fetal blood and cause interstitial pneumonia. listeriosis (listeria monocytogenes), salmonellosis (salmonella spp.), and chlamydiosis (chlamydophila abortus [c. psittaci]) are the best known examples of blood-borne primary benign neoplasms of the lungs, such as pulmonary adenomas, are highly unusual in domestic animals. most primary neoplasms are malignant and appear as solitary masses of variable size that, with time, can metastasize to other areas of the lungs and to distant organs. it is sometimes difficult on gross and microscopic examination to differentiate primary lung cancer from pulmonary metastasis resulting from malignant neoplasms elsewhere in the body. it is often difficult to determine the precise topographic origin of a neoplasm within the lungs-for example, whether it originates in the conducting system (bronchogenic carcinoma), transitional system (bronchiolar carcinoma), exchange system (alveolar carcinoma), or bronchial glands (bronchial gland carcinoma). according to the literature, pulmonary carcinomas in animals arise generally from club (clara) cells or type ii pneumonocytes of the bronchioloalveolar region, in contrast to those in human beings, which are mostly bronchogenic. tumors located at the hilus generally arise from major bronchi and tend to be a solitary large mass with occasional small metastasis to the periphery of the lung. in contrast, tumors arising from the bronchioloalveolar region are often multicentric with numerous peripheral metastases in the lung parenchyma. because of histologic architecture and irrespective of the site of origin, many malignant epithelial neoplasms are classified by the all-encompassing term of pulmonary adenocarcinomas. dogs and cats are the species most frequently affected with primary pulmonary neoplasms, largely carcinomas, generally in older animals. the mean age for primary lung tumors is years for dogs and years for cats. pulmonary carcinomas in other domestic animals, except for retrovirus-induced pulmonary carcinoma in sheep, are less common, possibly because fewer farm animals are allowed to reach their natural life span. these neoplasms can be invasive or expansive, vary in color (white, tan, or gray) and texture (soft or firm), and often have areas of necrosis and hemorrhage, which result in a "craterous" or "umbilicate" appearance. this umbilicate appearance is frequently seen in rapidly growing carcinomas in which the center of the tumoral mass undergoes necrosis as a result of ischemia. some lung neoplasms resemble pulmonary consolidation or large granulomas. cats with moderately differentiated neoplasms had significantly longer survival time (median, days) than cats with poorly differentiated neoplasms (median, days). dogs with primary lung neoplasms, grades i, ii, and iii, had survival times of , , and days, respectively. ovine pulmonary adenocarcinoma (ovine pulmonary carcinoma). ovine pulmonary adenocarcinoma, also known as pulmonary adenomatosis and jaagsiekte (from the south african afrikaans word for "driving sickness"), is a transmissible, retrovirus-induced neoplasia of ovine lungs caused by jaagsiekte sheep retrovirus (jsrv). it occurs in sheep throughout the world, with the notable exception of australia and new zealand; its incidence is high in scotland, south africa, and peru and unknown but probably low in north america. this pulmonary carcinoma behaves very much like a chronic pneumonia, and jsrv shares many epidemiologic similarities with the ovine lentivirus responsible for maedi and the retrovirus responsible for enzootic nasal carcinoma in small ruminants. pulmonary adenomatosis has been transmitted to goats experimentally but is not known to be a spontaneous disease in that species. ovine pulmonary adenocarcinoma affects mainly mature sheep but can occasionally affect young stock. intensive husbandry probably facilitates horizontal transmission by the copious nasal discharge and explains why the disease occurs as devastating epizootics with % to % mortality when first introduced into a flock. differential diagnosis between maedi and pulmonary adenomatosis can prove difficult because both diseases often coexist in the same flock and women, are a distant second. to say that cigarette smoking is responsible for this epidemic of lung cancer is unnecessary. although dogs have been proposed as valuable "sentinels" for environmental hazards, such as exposure to passive smoking, asbestos, dyes, and insecticides, it is not known if the prevalence of canine lung tumors has increased in geographical areas with high contamination. alterations in genes (oncogenes) and chromosomes and changes in biologically active molecules have been linked to lung cancer in recent years. as with many other forms of cancer, epidemiologic studies indicate that the incidence of pulmonary neoplasms increases with age, but there are still insufficient data to confirm that particular canine or feline breeds have a higher predisposition to spontaneous lung neoplasms. a standard nomenclature of pulmonary neoplasms in domestic animals is lacking, and as a consequence, multiplicity of names and synonyms occur in the veterinary literature. some classifications are based on the primary site, whereas others emphasize more the histomorphologic type. the most common types of benign and malignant pulmonary neoplasms in domestic mammals are listed in box - . clinically, the signs of pulmonary neoplasia vary with the degree of invasiveness, the amount of parenchyma involved, and locations of metastases. signs may be vague, such as cough, lethargy, anorexia, weight loss, and perhaps dyspnea. in addition, paraneoplastic syndromes, such as hypercalcemia, endocrinopathies, and pulmonary hypertrophic osteoarthropathy, have been associated with pulmonary neoplasms. primary neoplasms of the lungs. primary neoplasms of the lungs arise from cells normally present in the pulmonary tissue and can be epithelial or mesenchymal, although the latter are rare. any malignant tumor metastatic from another body location (e.g., osteosarcoma in dogs, uterine carcinoma in cows, and malignant melanoma in horses) box - classification of pulmonary neoplasms .e chapter respiratory system, mediastinum, and pleurae abundant cytoplasm containing numerous acidophilic granules, which are positive for pas and for s- protein using immunohistochemistry. although this tumor can cause bronchial obstruction and respiratory signs, in most cases, it is an incidental finding in older horses submitted for postmortem examination. lymphomatoid granulomatosis. lymphomatoid granulomatosis is a rare but interesting pulmonary disease of human beings, dogs, cats, and possibly horses and donkeys characterized by nodules or large solid masses in one or more lung lobes. these frequently metastasize to lymph nodes, kidneys, and liver. microscopically, tumors are formed by large pleomorphic mononuclear (lymphomatoid) cells with a high mitotic rate and frequent formation of binucleated or multinucleated cells. tumor cells have a distinct tendency to grow around blood vessels and invade and destroy the vascular walls. lymphomatoid granulomatosis has some resemblance to lymphoma and is therefore also referred to as angiocentric lymphoma; phenotypic marking confirms that neoplastic cells are a mixed population of plasma cells, b and t lymphocytes, and histiocytes. cerebral and cutaneous forms of lymphomatoid granulomatosis have also reported in human beings, dogs, and cats. secondary neoplasms of the lungs. secondary neoplasms of the lungs are all malignant by definition because they are the result of metastasis to the lungs from malignant neoplasms elsewhere. because the pulmonary capillaries are the first filter met by tumor emboli released into the vena cava or pulmonary arteries, secondary neoplasms in the lung are relatively common in comparison to primary ones. also, secondary tumors can be epithelial or mesenchymal in origin. common metastatic tumors of epithelial origin are mammary, thyroid ( fig. - ) , and uterine carcinomas. tumors of mesenchymal origin are osteosarcoma ( fig. - , a) ; hemangiosarcoma ( fig. - , b) ; malignant melanoma in dogs; lymphoma in cows, pigs, dogs, and cats ( fig. - ) ; and vaccineassociated sarcoma in cats. usually, secondary pulmonary neoplasms are multiple; scattered throughout all pulmonary lobes (hematogenous dissemination); of variable size; and, according to the growth pattern, can be nodular, diffuse, or radiating (e- fig. - ) . the appearance of metastatic neoplasms differs according to the type of neoplasm. for example, dark red cystic nodules containing blood indicate hemangiosarcoma, dark black solid nodules indicate melanoma, and hard solid nodules (white, yellow, or tan color) with bone spicules indicate osteosarcoma. the gross appearances of or in the same animal. death is inevitable after several months of the initial onset of respiratory signs, and a specific humoral immune response to jsrv is undetectable in affected sheep. during the early stages of ovine pulmonary carcinoma, the lungs are enlarged, heavy, and wet and have several firm, gray, variably sized nodules that in some cases can be located in the cranioventral lobes mimicking a bronchopneumonic lesion ( fig. - , a) . in the later stages, the nodules become confluent, and large segments of both lungs are diffusely, but not symmetrically, infiltrated by neoplastic cells. on cross section, edematous fluid and a copious mucoid secretion are present in the trachea and bronchi ( fig. - , b) . microscopically, the nodules consist of cuboidal or columnar epithelial cells lining airways and alveoli and forming papillary or acinar (glandlike) structures (see fig. - , a) . because the cells have been identified ultrastructurally as originating from both type ii alveolar epithelial cells and club (clara) cells, the neoplasm is considered a "bronchioloalveolar" carcinoma. sequelae often include secondary bronchopneumonia, abscesses, and fibrous pleural adhesions. metastases occur to tracheobronchial and mediastinal lymph nodes and, to a lesser extent, to other tissues such as pleura, muscle, liver, and kidneys. neoplastic cells stain strongly positive for jsrv using immunohistochemistry. clinically, ovine pulmonary adenocarcinoma is characterized by a gradual loss of condition, coughing, and respiratory distress, especially after exercise (e.g., herding or "driving"). appetite and temperature are normal, unless there are secondary bacterial infections. an important differentiating feature from maedi (interstitial pneumonia) can be observed if animals with pulmonary adenomatosis are raised by their hind limbs; copious, thin, mucoid fluid, produced by neoplastic cells in the lungs, pours from the nostrils of some animals. carcinoid (neuroendocrine) tumor of the lungs. carcinoid tumor of the lungs is a neoplasm presumably arising from neuroendocrine cells and is sporadically seen in dogs as multiple, large, firm pulmonary masses close to the mainstem bronchi. it has also been reported in the nasal cavity of horses. tumor cells are generally polygonal with finely granular, pale, or slightly eosinophilic cytoplasm. nuclei are small, and mitotic figures are absent or rare. granular cell tumor. granular cell tumor is a rare and locally invasive tumor that has been reported mainly in human beings and older horses. the cell origin of this tumor was thought to be the myoblast, but it is currently presumed to be schwann cells, which are normally present in the bronchovascular bundles of the lung. microscopically, neoplastic cells are large, polyhedron-shaped with metastatic carcinomas are generally similar to the primary neoplasm and sometimes have umbilicated centers. proper diagnoses of pulmonary neoplasms in live animals require history, clinical signs, radiographs, cytologic analysis of bal fluid, and, when necessary, a lung biopsy. identification of a specific lineage of neoplastic cells in biopsy or postmortem specimens is often difficult and requires electron microscopy or immunohistochemical techniques. electron microscopy allows identification of distinctive cellular components such as osmiophilic lamellar phospholipid nephritic bodies in alveolar type ii epithelial cells or melanosomes in melanomas. immunohistochemical staining is also helpful in identifying tumor cells. the thoracic wall, diaphragm, and mediastinum are lined by the parietal pleura, which reflects onto the lungs at the hilum and continues as the visceral pleura, covering the entire surface of the lungs, except at the hilus where the bronchi and blood vessels enter. the space between the parietal and visceral pleura (pleural space) is only minimal and under normal conditions contains only traces of clear fluid, which is a lubricant, and a few exfoliated cells. samples of this fluid are obtained by thoracocentesis, a simple procedure in which a needle is passed into the pleural cavity. volumetric, biochemical, and cytologic changes in this fluid are routinely used in veterinary diagnostics. anomalies congenital defects are rare and generally of little clinical significance. cysts within the mediastinum of dogs and, less often, cats in severe cases, the amount of fluid present in the thoracic cavity can be considerable. for instance, a medium-size dog can have l of fluid, and a cow may accumulate l or more. excessive fluid in the thorax causes compressive atelectasis resulting in respiratory distress (see fig. - ). hydrothorax is most commonly seen in cattle with right-sided heart failure or cor pulmonale (hydrostatic) (e- fig. - ); dogs with congestive heart failure (hydrostatic), chronic hepatic disease (hepatic hydrothorax) ( fig. - ) , or nephrotic syndrome (hypoproteinemia); pigs with mulberry heart disease (increased vascular permeability); and horses with african horse sickness (increased vascular permeability). hemothorax. blood in the thoracic cavity is called hemothorax, but the term has been used for exudate with a sanguineous component. causes include rupture of a major blood vessel as a result of severe thoracic trauma (e.g., hit by car); erosion of a vascular wall by malignant cells or inflammation (e.g., aortitis caused by spirocerca lupi); ruptured aortic aneurysms; clotting defects, including coagulopathies; warfarin toxicity; disseminated intravascular coagulation (consumption coagulopathy); and thrombocytopenia. hemothorax is generally acute and fatal. on gross examination, the thoracic cavity can be filled with blood, and the lungs are partially or completely atelectatic ( fig. - ). chylothorax. the accumulation of chyle (lymph rich in triglycerides) in the thoracic cavity ( fig. - ) is a result of the rupture of major lymph vessels, usually the thoracic duct or the right lymphatic duct. the clinical and pathologic effects of chylothorax are similar to those of the other pleural effusions. causes include thoracic neoplasia (the most common cause in human beings but a distant second to idiopathic cases in dogs), trauma, congenital lymph vessel anomalies, lymphangitis, dirofilariasis, and iatrogenic rupture of the thoracic duct during surgery. the source of the leakage of chyle is rarely found at necropsy. when the leakage of chyle occurs in the abdominal cavity, the condition is referred to as chyloabdomen. cytologic and biochemical examination of fluid collected by thoracocentesis typically reveals large numbers of lymphocytes, lipid droplets, few neutrophils in chronic cases, and high triglyceride content. can be large enough to compromise pulmonary function or mimic neoplasia in thoracic radiographs. these cysts may arise from the thymus (thymic branchial cysts), bronchi (bronchogenic cysts), ectopic thyroid tissue (thyroglossal duct cysts), or from remnants of the branchial pouches, and they are generally lined by epithelium and surrounded by a capsule of stromal tissue. anomalies of the thoracic duct cause some cases of chylothorax. pleural calcification. pleural calcification is commonly found in dogs and less often in cats with chronic uremia. lesions appear as linear white streaks in parietal pleura, mainly over the intercostal muscles of the cranial part of the thoracic cavity. the lesions are not functionally significant but indicate a severe underlying renal problem. vitamin d toxicity (hypervitaminosis d) and ingestion of hypercalcemic substances, such as vitamin d analogs, can also cause calcification of the pleura and other organs. pneumothorax. pneumothorax is the presence of air in the thoracic cavity where there should normally be negative pressure to facilitate inspiration. human beings have a complete and strong mediastinum so that pneumothorax is generally unilateral and thus not a serious problem. in dogs, the barrier varies, but in general it is not complete, so often some communication exists between left and right sides. there are two main forms of pneumothorax. in spontaneous (idiopathic) pneumothorax, air leaking into the pleural cavity from the lungs occurs without any known underlying disease or trauma. in secondary pneumothorax, movement of air into the pleural cavity results from underlying pulmonary or thoracic wall disease. the most common causes of secondary pneumothorax in veterinary medicine are penetrating wounds to the thoracic wall, perforated esophagus, iatrogenic trauma to the thorax and lung during a transthoracic lung biopsy or thoracoscopy, tracheal rupture from improper intubation, and rupture of emphysematous bullae or parasitic pulmonary cysts (paragonimus spp.) that communicate with the thoracic cavity. pneumothorax and pneumomediastinum caused by high air pressure (barotrauma) are also well documented in cats after equipment failure during anesthesia. clinical signs of pneumothorax include respiratory distress, and the lesion is simply a collapsed, atelectatic lung. the air is readily reabsorbed from the cavity if the site of entry is sealed. pleural effusion. pleural effusion is a general term used to describe accumulation of any fluid (transudate, modified transudate, exudate, blood, lymph, or chyle) in the thoracic cavity. cytologic and biochemical evaluations of pleural effusions taken by thoracocentesis are helpful in determining the type of effusion and possible pathogenesis. based on protein concentration and total numbers of nucleated cells, pleural effusions are cytologically divided into transudates, modified transudates, and exudates. hydrothorax. when the fluid is serous, clear, and odorless and fails to coagulate when exposed to air, the condition is referred to as hydrothorax (transudate). causes of hydrothorax are the same as those involved in edema formation in other organs: increased hydrostatic pressure (heart failure), decreased oncotic pressure (hypoproteinemia, as in liver disease), alterations in vascular permeability (inflammation), or obstruction of lymph drainage (neoplasia). in cases in which the leakage is corrected, if the fluid is a transudate, it is rapidly reabsorbed. if the fluid persists, it irritates the pleura and causes mesothelial hyperplasia and fibrosis, which thickens the pleura. from a perforated esophagus. chronic injury typically results in serosal fibrosis and tight adhesions between visceral and parietal pleurae (see fig. - ). when extensive, these adhesions can obliterate the pleural space. pleuritis or pleurisy. inflammation of the visceral or parietal pleurae is called pleuritis, and according to the type of exudate, it can be fibrinous, suppurative, granulomatous, hemorrhagic, or a combination of exudates. acute fibrinous pleuritis can progress with time to pleural fibrosis ( fig. - ). when suppurative pleuritis results in accumulation of purulent exudate in the cavity, the lesion is called pyothorax or thoracic empyema ( fig. - ) . clinically, pleuritis causes considerable pain, and in addition, empyema can result in severe toxemia. pleural fibrous adhesions (between parietal and visceral pleura) and fibrosis are the most common sequelae of chronic pleuritis and can significantly interfere with inflation of the lungs. pleuritis can occur as an extension of pneumonia, particularly in fibrinous bronchopneumonias (pleuropneumonia), or it can occur alone, without pulmonary involvement ( fig. - ). bovine and ovine pneumonic mannheimiosis and porcine and bovine pleuropneumonia are good examples of pleuritis associated with fibrinous bronchopneumonias. polyserositis in pigs and pleural empyema, particularly in cats and horses, are examples of pleural inflammation in pleural tissue is readily susceptible to injury caused by direct implantation of an organism through a penetrating thoracic or diaphragmatic wound; by hematogenous dissemination of infectious organisms in septicemias; or by direct extension from an adjacent inflammatory process, such as in fibrinous bronchopneumonia or in contrast to those with the effusive ("wet") form, in which thoracic involvement is primarily that of a pleural effusion. cytologic evaluation of the effusion typically shows a low to moderate cellularity with degenerated leukocytes, lymphocytes, macrophages, and mesothelial cells, and a pink granular background as a result of the high protein content. pleuritis is also an important problem in horses. nocardia spp. can cause fibrinopurulent pneumonia and pyothorax with characteristic sulfur granules. although mycoplasma felis can be isolated from the respiratory tract of normal horses, it is also isolated from horses with pleuritis and pleural effusion, particularly during the early stages of infection. the portal of entry of this infection is presumably aerogenous, first to the lung and subsequently to the pleura. the pleural surface of the lung is often involved in neoplasms that have metastasized from other organs to the pulmonary parenchyma and ruptured the visceral pleura to seed the pleural cavity. mesothelioma is the only primary neoplasm of the pleura. which involvement of the lungs may not accompany the pleuritis. pleural inflammation is most frequently caused by bacteria, which cause polyserositis reaching the pleura hematogenously. these bacteria include haemophilus parasuis (glasser's disease) (see , streptococcus suis, and some strains of pasteurella multocida in pigs; streptococcus equi ssp. equi and streptococcus equi ssp. zooepidemicus in horses; escherichia coli in calves; and mycoplasma spp. and haemophilus spp. in sheep and goats. contamination of pleural surfaces can be the result of extension of a septic process (e.g., puncture wounds of the thoracic wall and, in cattle, traumatic reticulopericarditis) and ruptured pulmonary abscesses (e.g., trueperella pyogenes). in dogs and cats, bacteria (e.g., nocardia, actinomyces, and bacteroides) can cause pyogranulomatous pleuritis, characterized by accumulation of blood-stained pus ("tomato soup") in the thoracic cavity. this exudate usually contains yellowish flecks called sulfur granules ( fig. - ), although these are less common in nocardial empyema in cats. many species of bacteria, such as escherichia coli, trueperella pyogenes, pasteurella multocida, and fusobacterium necrophorum, can be present in pyothorax of dogs and cats. these bacteria occur alone or in mixed infections. the pathogenesis of pleural empyema in cats is still debatable, but bite wounds or penetration of foreign material (migrating grass awns) are likely. pyogranulomatous pleuritis with empyema occurs occasionally in dogs, presumably associated with inhaled small plant material and penetrating (migrating) grass awns. because of their physical shape (barbed) and assisted by the respiratory movement, aspirated grass awns can penetrate airways, move through the pulmonary parenchyma, and eventually perforate the visceral pleura causing pyogranulomatous pleuritis. cats with the noneffusive ("dry") form of feline infectious peritonitis (fip) frequently have focal pyogranulomatous pleuritis, mesothelioma is a rare neoplasm of the thoracic, pericardial, and peritoneal mesothelium of human beings that is seen most commonly in calves, in which it can be congenital. in human beings, it has long been associated with inhalation of certain types of asbestos fibers (asbestos mining and ship building) alone or with cigarette smoking as a probable cocarcinogen; no convincing association between the incidence of mesothelioma and exposure to asbestos has been made in domestic animals. in animals, there may be pleural effusion with resulting respiratory distress, cough, and weight loss. mesothelioma initially causes a thoracic effusion, but cytologic diagnosis can be difficult because of the morphologic resemblance of malignant and reactive mesothelial cells. during inflammation, mesothelial cells become reactive and not only increase in number but also become pleomorphic and form multinucleated cells that may be cytologically mistaken for those of a carcinoma. grossly, mesothelioma appears as multiple, discrete nodules or arborescent, spreading growths on the pleural surface ( fig. - ) . microscopically, either the mesothelial covering cells or the supporting tissue can be the predominant malignant component, so the neoplasm can microscopically resemble a carcinoma or a sarcoma. figure - nocardiosis. a, chronic pleuritis (nocardia asteroides), pleural cavity, cat. the pleural cavity is covered with abundant red-brown ("tomato soup") exudate" (syringe). once considered to be pathognomonic of nocardia spp. infection, it is no longer regarded as being diagnostic of nocardiosis. the fluid contains abundant protein, erythrocytes, granulomatous inflammatory cells, and sulfur granules. b, chronic pleuritis (nocardia asteroides), visceral pleura, dog. the thickened pleura has a granular pink-gray appearance because of granulomatous inflammation and the proliferation of fibrovascular tissue of the pleura. c, chronic pleuritis (nocardia asteroides), dog. the pleura has been thrown up into villous-like projections composed of abundant fibrovascular tissue and granulomatous inflammation. leakage from the neocapillaries of the fibrovascular tissue is responsible for the hemorrhagic appearance of the pleural exudate. h&e stain. d, chronic pleuritis (nocardia asteroides), thoracic cage, parietal pleura, cat. large pieces of exudate, which contain yellow sulfur granules, are present on the thickened pleura. although considered malignant, mesotheliomas rarely metastasize to distant organs. secondary neoplasms of the pleura. secondary tumors may also spread into the visceral and parietal pleura. thymomas are rare neoplasms that grow in the cranial mediastinum of adult or aged dogs, cats, pigs, cattle, and sheep. thymomas are composed of thymic epithelium and lymphocytes (see chapter ). old age, both in human beings and in animals, is known to be a risk factor for pulmonary infections, but the precise mechanisms involved in this increased susceptibility are still under investigation. some studies have shown that in aged individuals the antibacterial properties provided by surfactant proteins, proinflammatory cytokines, and complement are altered. pulmonary hyperinflation (often referred to as senile emphysema) has been reported as an age-related change in human and canine lungs. other age-related changes described in canine lungs include mineralization of bronchial cartilage, pleural and alveolar fibrosis, and heterotopic bone formation (so-called "pulmonary osteomas"). we thank all pathologists at the atlantic veterinary college, university of prince edward island for providing case material. suggested readings are available at www.expertconsult.com. lung section showing a distended and partially occluded blood vessel (center of figure) containing large granular cells. these large cells are macrophages, and their cytoplasm is filled with myriad merozoites isolation of porcine circoviruslike viruses from pigs with a wasting disease in the usa and europe exercise-induced pulmonary hemorrhage effect of mucociliary transport relies on efficient regulation of ciliary beating epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats canine h n influenza virus infection in dogs and mice failure of respiratory defenses in the pathogenesis of bacterial pneumonia in cattle the respiratory system advances in diagnosis of respiratory diseases of small ruminants canine nasal disease transmission of equine influenza virus to dogs dear jd: bacterial pneumonia in dogs and cats acute respiratory distress syndrome in dogs and cats: a review of clinical findings and pathophysiology inflammatory response to infectious pulmonary injury laryngeal paralysis: a study of cases in a mixed-breed population of horses stem cells of the respiratory tract exudative pleural disease in small animals bovine respiratory disease research pulmonary thromboembolism coccidioidomycosis in dogs and cats: a review cousens c: pathology and pathogenesis of ovine pulmonary adenocarcinoma prognosis factors for survival in cats after removal of a primary lung tumor: cases ( - ) the acute respiratory distress syndrome: from mechanism to translation endogenous lipid pneumonia in cats: cases ( - ) retroviral infections in sheep and goats: small ruminant lentiviruses and host interaction canine and feline nasal neoplasia the acute respiratory distress syndrome equine respiratory medicine and surgery canine pleural and mediastinal effusions: a retrospective study of cases a review of histiocytic diseases of dogs and cats estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in australian live export cattle polymicrobial respiratory disease in pigs current state of knowledge on porcine circovirus type -associated lesions common and emerging infectious diseases in the animal shelter chronic rhinitis in the cat advances in the understanding of pathogenesis, and diagnosis and therapeutics of feline allergic asthma mannheimia haemolytica and bovine respiratory disease detection of respiratory viruses and bordetella bronchiseptica in dogs with acute respiratory tract infections current perspectives on the diagnosis and epidemiology of mycoplasma hyopneumoniae infection mannheimia haemolytica: bacterialhost interactions in bovine pneumonia acute lung injury review rhodococcus equi: the many facets of a pathogenic actinomycete tumors of the respiratory system key: cord- -l jcw h authors: tang, claire title: discovering unknown diseases with explainable automated medical imaging date: - - journal: medical image understanding and analysis doi: . / - - - - _ sha: doc_id: cord_uid: l jcw h deep neural network (dnn) classifiers have attained remarkable performance in diagnosing known diseases when the models are trained on a large amount of data from known diseases. however, dnn classifiers trained on known diseases usually fail when they confront new diseases such as covid- . in this paper, we propose a new deep learning framework and pipeline for explainable medical imaging that can classify known diseases as well as detect new/unknown diseases when the models are only trained on known disease images. we first provide in-depth mathematical analysis to explain the overconfidence phenomena and present the calibrated confidence that can mitigate the overconfidence. using calibrated confidence, we design a decision engine to determine if a medical image belongs to some known diseases or a new disease. at last, we introduce a new visual explanation to further reveal the suspected region inside each image. using both skin lesion and chest x-ray datasets, we validate that our framework significantly improves the accuracy of new disease discovery, i.e., distinguish covid- from pneumonia without seeing any covid- data during training. we also qualitatively show that our visual explanations are highly consistent with doctors’ ground truth. while our work was not designed to target covid- , our experimental validation using the real world covid- cases/data demonstrates the general applicability of our pipeline for different diseases based on medical imaging. extensive ai-based research and attempts have been made on automated medical imaging. recent researches have witnessed remarkable progress in diagnosing known diseases when dnn classifier models are trained on a large number of images on known diseases [ ] . however, in real world, unknown/new diseases continuously emerge, i.e., covid- . unfortunately, since no training data for the new/unknown diseases are available at training time, existing dnn classifiers trained only on the known disease (in-domain data) oftentimes fail on the new/unknown disease (out-of-domain data) in open-world practice. this problem is challenging even for a human. when doctors see a new disease, they could wrongly diagnose such a new disease as some other known diseases. in fact, at the beginning of the covid- outbreak, doctors mistook the new covid- disease as pneumonia/sars/mers which are known diseases from the past. the detection of out-of-domain unknown diseases is currently a challenging open research problem. unknown diseases are theoretically unlimited. for each unknown disease, again there are theoretically infinite variations. to make it even harder, none of these data is available at model training and learning time. recent work [ ] has shown that dnn classifiers oftentimes suffer from overfitting and overconfidence issues, i.e., prediction accuracy is much lower than average confidence scores for predictions. as a consequence, dnn classifiers mistake unknown out-of-domain diseases as one of the known in-domain diseases. on the other hand, deep learning models are black boxes. it is not clear why it works when it works, and why it does not work when it fails. blindly accepting the decision from computer-aided diagnosis based on dnn classifiers can have serious consequences on patients in practice. thus, it is highly desirable for models to provide explanations that can assist doctors to think and make the right decisions. to explain deep networks, several methods have been proposed based on internal states of the network [ ] [ ] [ ] . recently, selvaraju [ ] proposed grad-cam to compute neuron importance as part of a visual explanation. however, these approaches are only designed to explain the decision for existing diseases and cannot be applied to explain the decision when an unknown/new disease is detected. in this paper, we aim to develop a high-quality explainable automatic medical imaging system that can accurately detect new/unknown diseases as well as provide reliable visual explanations to doctors. our contributions can be summarized as follows: -we provide in-depth mathematical analysis to explain the overconfidence phenomena that leads to misdiagnosis of new/unknown diseases and present the calibrated confidence that can mitigate the overconfidence; we develop an automatic unknown disease discovery capability via confidence calibration for dnn classifiers trained only on known diseases data. -we develop an automatic visual explanation into deep learning models to reveal suspected evidence in medical images for potential unknown diseases. -we propose a novel explainable deep learning framework and pipeline that incorporates the above two automatic modules. -based on our proposed new pipeline, we conduct comprehensive experimental evaluations showing that our system achieves significant performance improvement on both quantitatively (unknown disease detection) and qualitatively (visual explanation) on skin lesion and chest x-ray datasets. in this section, we propose a novel framework and pipeline for explainable automated medical imaging. figure shows the whole framework including both in-domain known disease diagnosis and out-of-domain unknown disease discovery. next, we will present both training and testing processes with the focus of out-of-domain unknown disease discovery. training process: the components inside the dotted box in fig. indicate the training process. that said, dnn classifier and confidence calibration for unknown disease discovery will learn their parameters during training and later be used during testing. in the training, a dnn classifier is first trained only on known disease training images with class labels. then, our confidence calibration component is to further adjust the confidence scores from dnn classifier output. this will largely mitigate the dnn overconfidence and avoid misdiagnosing a new disease as some known diseases. to make our setting practical, our training process only takes the images of known diseases as inputs. we assume that new/unknown disease images are not available during model training time. in addition, our visual explanation component can automatically generate visual explanations only using the trained dnn classifier without needing to train a separate image segmentation model. the trained components in the dotted box are used in the testing process. given an input image, it first goes through dnn classifier and confidence calibration components to generate the calibrated confidences. next, we compare the calibrated confidence of the input image with a given threshold. if it is smaller than the threshold, we decide that this is a new/unknown disease; and we use our new visual explanation to show the potential suspected regions that have led to our detection of "new/unknown". otherwise, we directly use the trained dnn classifier model to automatically diagnose to be one of the known diseases and provide its visual explanation [ ] for doctors to review and confirm. in the rest of our paper, we will focus on introducing our novel designs for the two blue components in fig. . overconfidence phenomenon has been observed empirically in literature [ ] . in this section, we propose a mathematical explanation of why overconfidence happens in deep neural networks (dnn) classifiers that lead to misdiagnosis of new/unknown diseases. motivated by our mathematical logic, we shall present calibration solutions. dnn classifiers implicitly assume all data are in-domain. thus, they model: in open world settings, one needs to learn: since unknown data is not available during training, we can only model the following based on training data: then, we hope to indirectly model out-of-domain probability: thus, the combination of p(y in |x) and p(y out |x) forms a probability distribution. in open world (since y in is a label for in-domain samples). thus, eq. can be rewritten as follows: thus, we reorganize the formula: hypothesis . let f c be the unnormalized probability p(y in |x) and f d be the unnormalized probability . we call the unnormalized probability "logits". we hypothesize the following: mathematical explanation of overconfidence observation [ ] : assuming that hypothesis holds, we show the explanation via eq. . given hypothesis , we can rewrite eq. as follows: then, we use the following "softmax function" [ ] to normalize the logits to be a probability distribution: we illustrate our overconfidence explanation in fig. using an example: assuming there are two indomain classes in our classifier. for an out-of-domain x, it is expected that f c (x) (the unnormalized p(y in |x)) (blue points in fig. ) for both classes are small, e.g., . and . . the normalization function maps f c (x) to probabilities % and %. however, for an out-of-domain , the final model logits (red points in fig. ) for both classes become and . the softmax normalization maps them to probabilities % and %. with that, the model will conclude that x is classified into class # with a confidence level of %. this shows how a wrong decision can be made with overconfidence for out-of-domain images. based on the above mathematical explanation of overconfidence, an intuitive solution to mitigate overconfidence is temperature scaling [ ] , i.e., scale f d (x) with a large temperature t to compute the calibrated confidence score. where s c is calibrated confidence score for each class c. unfortunately, since this temperature t is not trainable, it is hard to determine the right temperature for any case. in our experiments, t is simply set as a large number. thus, we present another confidence calibration approach using mahalanobis distance based on a generative classifier layer to replace with the softmax layer [ ] . according to a simple theoretical connection, the pretrained softmax classifier is likely to follow class-conditional gaussian distribution. that said, we can parameterize the class-conditional gaussian distribution with class mean μ i and covariance matrix Σ as follows: during testing, given an input image, we can compute its confidence score based on mahalanobis distance (distance between a point and a probability distribution) as follows: where s c is the same for each class c. f (x) represents the output features at the penultimate layer of dnn classifier models. since all s c does not have to form a probability distribution, we will introduce how these scores are matched to the final decision engine in the next section. in this section, our goal is to use the confidence scores to derive the final probability distribution, i.e., both p(y in |x) and p(y out |x). consider the calibrated confidences s c for any class c. accordingly, since y out has only one unknown class, i.e., y out equivalent to d = . we consider the following threshold-based function to derive the probability of out-of-domain probability: where s = max c s c meaning the largest confidence scores among all in-domain classes. δ is the threshold based on the true positive rate requirement. note that the in-domain images (s > δ) will be further diagnosed as one of the unknown diseases using the conventional softmax layer in dnn classifiers. when an image x is detected as in-domain, we directly compute its classification probability as: p(y in |x) = p(y in |d = , x) existing work tried to provide visual explanations for the in-domain classification decision [ ] . it produces heat maps to visualize the most indicative regions in the image regarding the diagnosed disease using class activation mappings (cam). however, cam cannot be directly used to discover unknown regions since none of the in-domain disease classes is diagnosed. thus, we devise the discovery cam (discam) based on the original cam. we use the calibrated confidence to combine the weights in the final classification layer as follows: at last, we follow cam to generate a heat map based on the neuron importance weights m by upscaling m to the dimensions of the image and overlaying the image for each pixel. we have conducted experimental evaluations based on our proposed new deep learning pipeline. we use two medical datasets in our experiment. for each dataset, we discuss its in-domain and out-of-domain data respectively. for in-domain images, we use the latest isic skin lesion challenge dataset [ ]. it contains , training images and each image is labeled as one of categories/classes, including different diseases and benign. the task is to classify an image into one of these eight classes. since the class ground truth of testing images are not available, we evaluate our approaches via -fold crossvalidation on training data and report the average results. for out-of-domain images, we download the images in the "unknown" category from gallery in isic archive website [ ] . dermatologists have determined these images do not belong to any of the above categories. in addition, each image is provided with segmentation ground truth by dermatologists. for in-domain images, we use the chest x-ray dataset [ ] from kaggle. it contains , training images and validation images. each image is labeled . since the in-domain data only have frontal chest x-ray, we only keep frontal x-ray out-of-domain covid- images for our testing purpose. each covid- data sample consists of a chest x-ray image, a patient's basic information, and clinical notes from doctors. figure shows a sample chest x-ray images including in-domain normal and pneumonia as well as a new outof-domain covid- image. covid- started in late and is caused by a new virus, a.k.a. severe acute respiratory syndrome coronavirus , or sars-cov- . the infection may result in severe pneumonia with clusters of illness onsets. its impacts on public health make it paramount to clarify the clinical features with other pneumonia. thus, the computer-aided discovery of covid- is challenging but in the meanwhile practically very useful. we implement our code using the pytorch . . framework. the experiment is run on nvidia gpus (tesla v gb gpu). our first step is to train state-of-the-art based cnn models for both datasets. we first normalized both datasets using the mean and standard deviation calculated on the statistics of all training images. the skin lesion dataset has mean ( . , . , . ) and standard deviation ( . , . , . ), and chest xray dataset has mean ( . , . , . ) and standard deviation ( . , . , . ). note that the gray-scale chest x-ray images have the same values for all rgb channels. for each image, we first resize it to be × . we performed dynamic in-memory augmentation by randomly cropping to × , horizontal & vertical flips, and zooming by appropriate transformations in the pytorch data loader. following the previous work [ ] , we conduct transfer learning with resnet- , resnet- and resnet- pre-trained on the imagenet [ ] . we also use batch size and use the same approach in [ ] to choose the optimal learning rate. using this learning rate, we continue following the two-step model training in [ ] . to validate our model training, we first evaluate the performance of indomain classification on all trained models. we use top- accuracy and auc table shows the in-domain performance. since our out-of-domain detection will not retrain the model, the in-domain classification performance will not be impacted in our new deep learning pipeline. we follow the evaluation metrics in the literature [ ] . let tp, tn, fp, and fn denote true positive, true negative, false positive, and false negative, respectively. we use the following out-of-domain evaluation metrics: -tnrktpr (high) (true negative rate (tnr) at k% true positive rate (tpr)): can be interpreted as the probability that an out-of-domain image is classified correctly when the true positive rate (tpr) for in-domain data is as high as k%, where tpr = tp/(tp + fn). in our experiment, we choose k to be . -detection error (low): measures the misclassification probability when tpr is k%. detection error is defined as follows: where s is a confidence score. we follow the same assumption that both fn) ) by varying a threshold. table and table show the unknown disease detection results on skin lesion and chest x-ray diseases respectively. as one can see, the baseline suffers from failure due to overconfidence. temperature scaling (ts) improves the performance but still not satisfactory due to the untrainable temperature t . generative classifier (gc), after removing the source of overconfidence by replacing the softmax layer, achieves significant performance improvement for all metrics. the gc performance on the skin lesion dataset is slightly lower since it contains colorful images and there are many varieties of noises on the images such as color, illumination, skin hair, etc. gc improved baseline performance by over times on chest x-ray to detect new out-of-domain covid- disease using the model trained on known pneumonia and normal images. in fact, we achieved almost perfect detection of covid- when tested on a small dataset. next, we conduct some case study of visual explanation on new/unknown diseases to ( ) qualitatively validate our visual explanation method by comparing with ground truth doctor explanation, and ( ) visually elaborate the underlying reason why our unknown disease detection method works well. each skin lesion unknown image has a doctor provided segmentation ground truth (green lines). each chest x-ray covid- image has clinic notes which explain the suspected regions in x-ray that indicate covid- diagnosis. it is important to note that the left and right sides are flipped over in conventional x-ray images. figure , fig. and fig. show three different types of wrong regions baseline cam method looks at, which leads to all wrong decisions. in fig. , cam looks at completely wrong regions which no doubt leads to wrong predictions. figure and fig. are more interesting. in fig. , although the regions cam looks at include the correct region, it also looks at other distracting regions. for example, the hair on the skin and white abdomen area in chest x-ray possibly confused the decision engine. on the other hand, fig. shows that cases where cam looks at too narrow regions and missed the holistic view of the disease. meanwhile, our discam looks at correct regions in all these three cases and also correctly detects all these unknown disease images. figure shows another interesting visual explanation in which our discam method shows no particular suspected region in the image. that said, our trained model does not discover any suspected regions based on the learned knowledge of known diseases and therefore also concludes this is a new/unknown disease. in contrast, cam identifies completely wrong regions and mistakes the unknown disease as a known disease. we proposed a framework for explainable automatic medical imaging that can discover unknown diseases and provide a visual explanation for that decision. we first mathematically analyzed and explained why existing models oftentimes fail to classify new/unknown data correctly. we then showed calibration methods that can mitigate the overconfidence. we validated the new calibration method with multiple datasets and demonstrated its effectiveness for unknown data detection via quantitative evaluations. we successfully detected covid- with our new deep learning pipeline trained with only known pneumonia data. we provided visual explanations of our new/unknown detection decisions based on the calibrated confidence methods. our explanations are consistent with doctors' ground truth and clinical notes. for future work, we will continue to validate our work by evaluating more and larger datasets. as a natural next step, we also plan to continue working on few-shot learning using a small amount of new disease data to efficiently learn the new diseases for future predictions/classifications. deep learning. adaptive computation and machine learning on calibration of modern neural networks. in: icml a baseline for detecting misclassified and out-ofdistribution examples in neural networks a simple unified framework for detecting out-ofdistribution samples and adversarial attacks enhancing the reliability of out-of-distribution image detection in neural networks interpreting fine-grained dermatological classification by deep learning imagenet large scale visual recognition challenge grad-cam: visual explanations from deep networks via gradient-based localization visualizing and understanding convolutional networks object detectors emerge in deep scene cnns learning deep features for discriminative localization acknowledgement. i would like to express my sincere gratitude to professor jiang du and his staff from the department of radiology at the university of california at san diego who introduced me to the wonderful medical imaging space, especially on mris and image segmentation. i would also like to express my deep appreciation to dr. jeremy shen from samsung for educating me on related work and the pitfalls. i am very grateful to ai all for awarding me a travel grant to attend the neurips workshop in vancouver, canada for "machine learning health". without their support, this work would have been impossible. key: cord- -syirijql authors: adiga, aniruddha; chen, jiangzhuo; marathe, madhav; mortveit, henning; venkatramanan, srinivasan; vullikanti, anil title: data-driven modeling for different stages of pandemic response date: - - journal: arxiv doi: nan sha: doc_id: cord_uid: syirijql some of the key questions of interest during the covid- pandemic (and all outbreaks) include: where did the disease start, how is it spreading, who is at risk, and how to control the spread. there are a large number of complex factors driving the spread of pandemics, and, as a result, multiple modeling techniques play an increasingly important role in shaping public policy and decision making. as different countries and regions go through phases of the pandemic, the questions and data availability also changes. especially of interest is aligning model development and data collection to support response efforts at each stage of the pandemic. the covid- pandemic has been unprecedented in terms of real-time collection and dissemination of a number of diverse datasets, ranging from disease outcomes, to mobility, behaviors, and socio-economic factors. the data sets have been critical from the perspective of disease modeling and analytics to support policymakers in real-time. in this overview article, we survey the data landscape around covid- , with a focus on how such datasets have aided modeling and response through different stages so far in the pandemic. we also discuss some of the current challenges and the needs that will arise as we plan our way out of the pandemic. as the sars-cov- pandemic has demonstrated, the spread of a highly infectious disease is a complex dynamical process. a large number of factors are at play as infectious diseases spread, including variable individual susceptibility to the pathogen (e.g., by age and health conditions), variable individual behaviors (e.g., compliance with social distancing and the use of masks), differing response strategies implemented by governments (e.g., school and workplace closure policies and criteria for testing), and potential availability of pharmaceutical interventions. governments have been forced to respond to the rapidly changing dynamics of the pandemic, and are becoming increasingly reliant on different modeling and analytical techniques to understand, forecast, plan and respond; this includes statistical methods and decision support methods using multi-agent models, such as: (i) forecasting epidemic outcomes (e.g., case counts, mortality and hospital demands), using a diverse set of data-driven methods e.g., arima type time series forecasting, bayesian techniques and deep learning, e.g., [ ] [ ] [ ] [ ] [ ] , (ii) disease surveillance, e.g., [ , ] , and (iii) counter-factual analysis of epidemics using multi-agent models, e.g., [ ] [ ] [ ] [ ] [ ] [ ] ; indeed, the results of [ , ] were very influential in the early decisions for lockdowns in a number of countries. the specific questions of interest change with the stage of the pandemic. in the pre-pandemic stage, the focus was on understanding how the outbreak started, epidemic parameters, and the risk of importation to different regions. once outbreaks started-the acceleration stage, the focus is on determining the growth rates, the differences in spatio-temporal characteristics, and testing bias. in the mitigation stage, the questions are focused on non-prophylactic interventions, such as school and work place closures and other social-distancing strategies, determining the demand for healthcare resources, and testing and tracing. in the suppression stage, the focus shifts to using prophylactic interventions, combined with better tracing. these phases are not linear, and overlap with each other. for instance, the acceleration and mitigation stages of the pandemic might overlap spatially, temporally as well as within certain social groups. different kinds of models are appropriate at different stages, and for addressing different kinds of questions. for instance, statistical and machine learning models are very useful in forecasting and short term projections. however, they are not very effective for longer-term projections, understanding the effects of different kinds of interventions, and counter-factual analysis. mechanistic models are very useful for such questions. simple compartmental type models, and their extensions, namely, structured metapopulation models are useful for several population level questions. however, once the outbreak has spread, and complex individual and community level behaviors are at play, multi-agent models are most effective, since they allow for a more systematic representation of complex social interactions, individual and collective behavioral adaptation and public policies. as with any mathematical modeling effort, data plays a big role in the utility of such models. till recently, data on infectious diseases was very hard to obtain due to various issues, such as privacy and sensitivity of the data (since it is information about individual health), and logistics of collecting such data. the data landscape during the sars-cov- pandemic has been very different: a large number of datasets are becoming available, ranging from disease outcomes (e.g., time series of the number of confirmed cases, deaths, and hospitalizations), some characteristics of their locations and demographics, healthcare infrastructure capacity (e.g., number of icu beds, number of healthcare personnel, and ventilators), and various kinds of behaviors (e.g., level of social distancing, usage of ppes); see [ ] [ ] [ ] for comprehensive surveys on available datasets. however, using these datasets for developing good models, and addressing important public health questions remains challenging. the goal of this article is to use the widely accepted stages of a pandemic as a guiding framework to highlight a few important problems that require attention in each of these stages. we will aim to provide a succinct model-agnostic formulation while identifying the key datasets needed, how they can be used, and the challenges arising in that process. we will also use sars-cov- as a case study unfolding in real-time, and highlight some interesting peer-reviewed and preprint literature that pertains to each of these problems. an important point to note is the necessity of randomly sampled data, e.g. data needed to assess the number of active cases and various demographics of individuals that were affected. census provides an excellent rationale. it is the only way one can develop rigorous estimates of various epidemiologically relevant quantities. there have been numerous surveys on the different types of datasets available for sars-cov- , e.g., [ ] [ ] [ ] [ ] , as well as different kinds of modeling approaches. however, they do not describe how these models become relevant through the phases of pandemic response. an earlier similar attempt to summarize such responsedriven modeling efforts can be found in [ ] , based on the -h n experience, this paper builds on their work and discusses these phases in the present context and the sars-cov- pandemic. although the paper touches upon different aspects of model-based decision making, we refer the readers to a companion article in the same special issue [ ] for a focused review of models used for projection and forecasting. multiple organizations including cdc and who have their frameworks for preparing and planning response to a pandemic. for instance, the pandemic intervals framework from cdc describes the stages in the context of an influenza pandemic; these are illustrated in figure . these six stages span investigation, recognition and initiation in the early phase, followed by most of the disease spread occurring during the acceleration and deceleration stages. they also provide indicators for identifying when the pandemic has progressed from one stage to the next [ ] . as envisioned, risk evaluation (i.e., using tools like influenza risk assessment tool (irat) and pandemic severity assessment framework (psaf)) and early case identification characterize the first three stages, while non-pharmaceutical interventions (npis) and available figure : cdc pandemic intervals framework and who phases for influenza pandemic therapeutics become central to the acceleration stage. the deceleration is facilitated by mass vaccination programs, exhaustion of susceptible population, or unsuitability of environmental conditions (such as weather). a similar framework is laid out in who's pandemic continuum and phases of pandemic alert . while such frameworks aid in streamlining the response efforts of these organizations, they also enable effective messaging. to the best of our knowledge, there has not been a similar characterization of mathematical modeling efforts that go hand in hand with supporting the response. for summarizing the key models, we consider four of the stages of pandemic response mentioned in section : pre-pandemic, acceleration, mitigation and suppression. here we provide the key problems in each stage, the datasets needed, the main tools and techniques used, and pertinent challenges. we structure our discussion based on our experience with modeling the spread of covid- in the us, done in collaboration with local and federal agencies. • acceleration (section ): this stage is relevant once the epidemic takes root within a country. there is usually a big lag in surveillance and response efforts, and the key questions are to model spread patterns at different spatio-temporal scales, and to derive short-term forecasts and projections. a broad class of datasets is used for developing models, including mobility, populations, land-use, and activities. these are combined with various kinds of time series data and covariates such as weather for forecasting. • mitigation (section ): in this stage, different interventions, which are mostly non-pharmaceutical in the case of a novel pathogen, are implemented by government agencies, once the outbreak has taken hold within the population. this stage involves understanding the impact of interventions on case counts and health infrastructure demands, taking individual behaviors into account. the additional datasets needed in this stage include those on behavioral changes and hospital capacities. • suppression (section ): this stage involves designing methods to control the outbreak by contact tracing & isolation and vaccination. data on contact tracing, associated biases, vaccine production schedules, and compliance & hesitancy are needed in this stage. figure gives an overview of this framework and summarizes the data needs in these stages. these stages also align well with the focus of the various modeling working groups organized by cdc which include epidemic parameter estimation, international spread risk, sub-national spread forecasting, impact of interventions, healthcare systems, and university modeling. in reality, one should note that these stages may overlap, and may vary based on geographical factors and response efforts. moreover, specific problems can be approached prospectively in earlier stages, or retrospectively during later stages. this framework is thus meant to be more conceptual than interpreted along a linear timeline. results from such stages are very useful for policymakers to guide real-time response. consider a novel pathogen emerging in human populations that is detected through early cases involving unusual symptoms or unknown etiology. such outbreaks are characterized by some kind of spillover event, mostly through zoonotic means, like in the case of covid- or past influenza pandemics (e.g., swine flu and avian flu). a similar scenario can occur when an incidence of a well-documented disease with no known vaccine or therapeutics emerges in some part of the world, causing severe outcomes or fatalities (e.g., ebola and zika.) regardless of the development status of the country where the pathogen emerged, such outbreaks now contains the risk of causing a worldwide pandemic due to the global connectivity induced by human travel. two questions become relevant at this stage: what are the epidemiological attributes of this disease, and what are the risks of importation to a different country? while the first question involves biological and clinical investigations, the latter is more related with societal and environmental factors. one of the crucial tasks during early disease investigation is to ascertain the transmission and severity of the disease. these are important dimensions along which the pandemic potential is characterized because together they determine the overall disease burden, as demonstrated within the pandemic severity assessment framework [ ] . in addition to risk assessment for right-sizing response, they are integral to developing meaningful disease models. formulation let Θ = {θ t , θ s } represent the transmission and severity parameters of interest. they can be further subdivided into sojourn time parameters θ δ · and transition probability parameters θ p · . here Θ corresponds to a continuous time markov chain (ctmc) on the disease states. the problem formulation can be represented as follows: given Π(Θ), the prior distribution on the disease parameters and a dataset d, estimate the posterior distribution p(Θ|d) over all possible values of Θ. in a model-specific form, this can be expressed as p(Θ|d, m) where m is a statistical, compartmental or agent-based disease model. in order to estimate the disease parameters sufficiently, line lists for individual confirmed cases is ideal. such datasets contain, for each record, the date of confirmation, possible date of onset, severity (hospitalization/icu) status, and date of recovery/discharge/death. furthermore, age-and demographic/comorbidity information allow development of models that are age-and risk group stratified. one such crowdsourced line list was compiled during the early stages of covid- [ ] and later released by cdc for us cases [ ] . data from detailed clinical investigations from other countries such as china, south korea, and singapore was also used to parameterize these models [ ] . in the absence of such datasets, past parameter estimates of similar diseases (e.g., sars, mers) were used for early analyses. modeling approaches for a model agnostic approach, the delays and probabilities are obtained by various techniques, including bayesian and ordinary least squares fitting to various delay distributions. for a particular disease model, these are estimated through model calibration techniques such as mcmc and particle filtering approaches. a summary of community estimates of various disease parameters is provided at https://github.com/midas-network/covid- . further such estimates allow the design of pandemic planning scenarios varying in levels of impact, as seen in the cdc scenarios page . see [ ] [ ] [ ] for methods and results related to estimating covid- disease parameters from real data. current models use a large set of disease parameters for modeling covid- dynamics; they can be broadly classified as transmission parameters and hospital resource parameters. for instance in our work, we currently use parameters (with explanations) shown in table . challenges often these parameters are model specific, and hence one needs to be careful when reusing parameter estimates from literature. they are related but not identifiable with respect to population level measures such as basic reproductive number r (or effective reproductive number r eff ) and doubling time which allow tracking the rate of epidemic growth. also the estimation is hindered by inherent biases in case ascertainment rate, reporting delays and other gaps in the surveillance system. aligning different data streams (e.g., outpatient surveillance, hospitalization rates, mortality records) is in itself challenging. when a disease outbreak occurs in some part of the world, it is imperative for most countries to estimate their risk of importation through spatial proximity or international travel. such measures are incredibly valuable in setting a timeline for preparation efforts, and initiating health checks at the borders. over centuries, pandemics have spread faster and faster across the globe, making it all the more important to characterize this risk as early as possible. formulation let c be the set of countries, and g = {c, e} an international network, where edges (often weighted and directed) in e represent some notion of connectivity. the importation risk problem can be formulated as below: given c o ∈ c the country of origin with an initial case at time , and c i the country of interest, using g, estimate the expected time taken t i for the first cases to arrive in country c i . in its probabilistic form, the same can be expressed as estimating the probability p i (t) of seeing the first case in country c i by time t. data needs assuming we have initial case reports from the origin country, the first data needed is a network that connects the countries of the world to represent human travel. the most common source of such information is the airline network datasets, from sources such as iata, oag, and openflights; [ ] provides a systematic review of how airline passenger data has been used for infectious disease modeling. these datasets could either capture static measures such as number of seats available or flight schedules, or a dynamic count of passengers per month along each itinerary. since the latter has intrinsic delays in collection and reporting, for an ongoing pandemic they may not be representative. during such times, data on ongoing travel restrictions [ ] become important to incorporate. multi-modal traffic will also be important to incorporate for countries that share land borders or have heavy maritime traffic. for diseases such as zika, where establishment risk is more relevant, data on vector abundance or prevailing weather conditions are appropriate. modeling approaches simple structural measures on networks (such as degree, pagerank) could provide static indicators of vulnerability of countries. by transforming the weighted, directed edges into probabilities, one can use simple contagion models (e.g., independent cascades) to simulate disease spread and empirically estimate expected time of arrival. global metapopulation models (gleam) that combine seir type dynamics with an airline network have also been used in the past for estimating importation risk. brockmann and helbing [ ] used a similar framework to quantify effective distance on the network which seemed to be well correlated with time of arrival for multiple pandemics in the past; this has been extended to covid- [ , ] . in [ ] , the authors employ air travel volume obtained through iata from ten major cities across china to rank various countries along with the idvi to convey their vulnerability. [ ] consider the task of forecasting international and domestic spread of covid- and employ official airline group (oag) data for determining air traffic to various countries, and [ ] fit a generalized linear model for observed number of cases in various countries as a function of air traffic volume obtained from oag data to determine countries with potential risk of under-detection. also, [ ] provide africa-specific case-study of vulnerability and preparedness using data from civil aviation administration of china. challenges note that arrival of an infected traveler will precede a local transmission event in a country. hence the former is more appropriate to quantify in early stages. also, the formulation is agnostic to whether it is the first infected arrival or first detected case. however, in real world, the former is difficult to observe, while the latter is influenced by security measures at ports of entry (land, sea, air) and the ease of identification for the pathogen. for instance, in the case of covid- , the long incubation period and the high likelihood of asymptomaticity could have resulted in many infected travelers being missed by health checks at poes. we also noticed potential administrative delays in reporting by multiple countries fearing travel restrictions. as the epidemic takes root within a country, it may enter the acceleration phase. depending on the testing infrastructure and agility of surveillance system, response efforts might lag or lead the rapid growth in case rate. under such a scenario, two crucial questions emerge that pertain to how the disease may spread spatially/socially and how the case rate may grow over time. within the country, there is need to model the spatial spread of the disease at different scales: state, county, and community levels. similar to the importation risk, such models may provide an estimate of when cases may emerge in different parts of the country. when coupled with vulnerability indicators (socioeconomic, demographic, co-morbidities) they provide a framework for assessing the heterogeneous impact the disease may have across the country. detailed agent-based models for urban centers may help identify hotspots and potential case clusters that may emerge (e.g., correctional facilities, nursing homes, food processing plants, etc. in the case of covid- ). formulation given a population representation p at appropriate scale and a disease model m per entity (individual or sub-region), model the disease spread under different assumptions of underlying connectivity c and disease parameters Θ. the result will be a spatio-temporal spread model that results in z s,t , the time series of disease states over time for region s. data needs some of the common datasets needed by most modeling approaches include: ( ) social and spatial representation, which includes census, and population data, which are available from census departments (see, e.g., [ ] ), and landscan [ ] , ( ) connectivity between regions (commuter, airline, road/rail/river), e.g., [ , ] , ( ) data on locations, including points of interest, e.g., openstreetmap [ ] , and ( ) activity data, e.g., the american time use survey [ ] . these datasets help capture where people reside and how they move around, and come in contact with each other. while some of these are static, more dynamic measures, such as from gps traces, become relevant as individuals change their behavior during a pandemic. modeling approaches different kinds of structured metapopulation models [ , [ ] [ ] [ ] [ ] , and agent based models [ ] [ ] [ ] [ ] [ ] have been used in the past to model the sub-national spread; we refer to [ , , ] for surveys on different modeling approaches. these models incorporate typical mixing patterns, which result from detailed activities and co-location (in the case of agent based models), and different modes of travel and commuting (in the case of metapopulation models). challenges while metapopulation models can be built relatively rapidly, agent based models are much harder-the datasets need to be assembled at a large scale, with detailed construction pipelines, see, e.g., [ ] [ ] [ ] [ ] [ ] . since detailed individual activities drive the dynamics in agent based models, schools and workplaces have to be modeled, in order to make predictions meaningful. such models will get reused at different stages of the outbreak, so they need to be generic enough to incorporate dynamically evolving disease information. finally, a common challenge across modeling paradigms is the ability to calibrate to the dynamically evolving spatio-temporal data from the outbreak-this is especially challenging in the presence of reporting biases and data insufficiency issues. given the early growth of cases within the country (or sub-region), there is need for quantifying the rate of increase in comparable terms across the duration of the outbreak (accounting for the exponential nature of such processes). these estimates also serve as references, when evaluating the impact of various interventions. as an extension, such methods and more sophisticated time series methods can be used to produce short-term forecasts for disease evolution. formulation given the disease time series data within the country z s,t until data horizon t , provide scale-independent growth rate measures g s (t ), and forecastsẐ s,u for u ∈ [t, t + ∆t ], where ∆t is the forecast horizon. data needs models at this stage require datasets such as ( ) time series data on different kinds of disease outcomes, including case counts, mortality, hospitalizations, along with attributes, such as age, gender and location, e.g., [ ] [ ] [ ] [ ] [ ] , ( ) any associated data for reporting bias (total tests, test positivity rate) [ ] , which need to be incorporated into the models, as these biases can have a significant impact on the dynamics, and ( ) exogenous regressors (mobility, weather), which have been shown to have a significant impact on other diseases, such as influenza, e.g., [ ] . modeling approaches even before building statistical or mechanistic time series forecasting methods, one can derive insights through analytical measures of the time series data. for instance, the effective reproductive number, estimated from the time series [ ] can serve as a scale-independent metric to compare the outbreaks across space and time. additionally multiple statistical methods ranging from autoregressive models to deep learning techniques can be applied to the time series data, with additional exogenous variables as input. while such methods perform reasonably for short-term targets, mechanistic approaches as described earlier can provide better long-term projections. various ensembling techniques have also been developed in the recent past to combine such multi-model forecasts to provide a single robust forecast with better uncertainty quantification. one such effort that combines more than methods for covid- can be found at the covid forecasting hub . we also point to the companion paper for more details on projection and forecasting models. challenges data on epidemic outcomes usually has a lot of uncertainties and errors, including missing data, collection bias, and backfill. for forecasting tasks, these time series data need to be near real-time, else one needs to do both nowcasting, as well as forecasting. other exogenous regressors can provide valuable lead time, due to inherent delays in disease dynamics from exposure to case identification. such frameworks need to be generalized to accommodate qualitative inputs on future policies (shutdowns, mask mandates, etc.), as well as behaviors, as we discuss in the next section. once the outbreak has taken hold within the population, local, state and national governments attempt to mitigate and control its spread by considering different kinds of interventions. unfortunately, as the covid- pandemic has shown, there is a significant delay in the time taken by governments to respond. as a result, this has caused a large number of cases, a fraction of which lead to hospitalizations. two key questions in this stage are: ( ) how to evaluate different kinds of interventions, and choose the most effective ones, and ( ) how to estimate the healthcare infrastructure demand, and how to mitigate it. the effectiveness of an intervention (e.g., social distancing) depends on how individuals respond to them, and the level of compliance. the health resource demand depends on the specific interventions which are implemented. as a result, both these questions are connected, and require models which incorporate appropriate behavioral responses. in the initial stages, only non-prophylactic interventions are available, such as: social distancing, school and workplace closures, and use of ppes, since no vaccinations and anti-virals are available. as mentioned above, such analyses are almost entirely model based, and the specific model depends on the nature of the intervention and the population being studied. formulation given a model, denoted abstractly as m, the general goals are ( ) to evaluate the impact of an intervention (e.g., school and workplace closure, and other social distancing strategies) on different epidemic outcomes (e.g., average outbreak size, peak size, and time to peak), and ( ) find the most effective intervention from a suite of interventions, with given resource constraints. the specific formulation depends crucially on the model and type of intervention. even for a single intervention, evaluating its impact is quite challenging, since there are a number of sources of uncertainty, and a number of parameters associated with the intervention (e.g., when to start school closure, how long, and how to restart). therefore, finding uncertainty bounds is a key part of the problem. data needs while all the data needs from the previous stages for developing a model are still there, representation of different kinds of behaviors is a crucial component of the models in this stage; this includes: use of ppes, compliance to social distancing measures, and level of mobility. statistics on such behaviors are available at a fairly detailed level (e.g., counties and daily) from multiple sources, such as ( ) the covid- impact analysis platform from the university of maryland [ ] , which gives metrics related to social distancing activities, including level of staying home, outside county trips, outside state trips, ( ) changes in mobility associated with different kinds of activities from google [ ] , and other sources, ( ) survey data on different kinds of behaviors, such as usage of masks [ ] . modeling approaches as mentioned above, such analyses are almost entirely model based, including structured metapopulation models [ , [ ] [ ] [ ] [ ] , and agent based models [ ] [ ] [ ] [ ] [ ] . different kinds of behaviors relevant to such interventions, including compliance with using ppes and compliance to social distancing guidelines, need to be incorporated into these models. since there is a great deal of heterogeneity in such behaviors, it is conceptually easiest to incorporate them into agent based models, since individual agents are represented. however, calibration, simulation and analysis of such models pose significant computational challenges. on the other hand, the simulation of metapopulation models is much easier, but such behaviors cannot be directly represented-instead, modelers have to estimate the effect of different behaviors on the disease model parameters, which can pose modeling challenges. challenges there are a number of challenges in using data on behaviors, which depends on the specific datasets. much of the data available for covid- is estimated through indirect sources, e.g., through cell phone and online activities, and crowd-sourced platforms. this can provide large spatio-temporal datasets, but have unknown biases and uncertainties. on the other hand, survey data is often more reliable, and provides several covariates, but is typically very sparse. handling such uncertainties, rigorous sensitivity analysis, and incorporating the uncertainties into the analysis of the simulation outputs are important steps for modelers. the covid- pandemic has led to a significant increase in hospitalizations. hospitals are typically optimized to run near capacity, so there have been fears that the hospital capacities would not be adequate, especially in several countries in asia, but also in some regions in the us. nosocomial transmission could further increase this burden. formulation the overall problem is to estimate the demand for hospital resources within a populationthis includes the number of hospitalizations, and more refined types of resources, such as icus, ccus, medical personnel and equipment, such as ventilators. an important issue is whether the capacity of hospitals within the region would be overrun by the demand, when this is expected to happen, and how to design strategies to meet the demand-this could be through augmenting the capacities at existing hospitals, or building new facilities. timing is of essence, and projections of when the demands exceed capacity are important for governments to plan. the demands for hospitalization and other health resources can be estimated from the epidemic models mentioned earlier, by incorporating suitable health states, e.g., [ , ] ; in addition to the inputs needed for setting up the models for case counts, datasets are needed for hospitalization rates and durations of hospital stay, icu care, and ventilation. the other important inputs for this component are hospital capacity, and the referral regions (which represent where patients travel for hospitalization). different public and commercial datasets provide such information, e.g., [ , ] . modeling approaches demand for health resources is typically incorporated into both metapopulation and agent based models, by having a fraction of the infectious individuals transition into a hospitalization state. an important issue to consider is what happens if there is a shortage of hospital capacity. studying this requires modeling the hospital infrastructure, i.e., different kinds of hospitals within the region, and which hospital a patient goes to. there is typically limited data on this, and data on hospital referral regions, or voronoi tesselation can be used. understanding the regimes in which hospital demand exceeds capacity is an important question to study. nosocomial transmission is typically much harder to study, since it requires more detailed modeling of processes within hospitals. challenges there is a lot of uncertainty and variability in all the datasets involved in this process, making its modeling difficult. for instance, forecasts of the number of cases and hospitalizations have huge uncertainty bounds for medium or long term horizon, which is the kind of input necessary for understanding hospital demands, and whether there would be any deficits. the suppression stage involves methods to control the outbreak, including reducing the incidence rate and potentially leading to the eradication of the disease in the end. eradication in case of covid- appears unlikely as of now, what is more likely is that this will become part of seasonal human coronaviruses that will mutate continuously much like the influenza virus. contact tracing problem refers to the ability to trace the neighbors of an infected individual. ideally, if one is successful, each neighbor of an infected neighbor would be identified and isolated from the larger population to reduce the growth of a pandemic. in some cases, each such neighbor could be tested to see if the individual has contracted the disease. contact tracing is the workhorse in epidemiology and has been immensely successful in controlling slow moving diseases. when combined with vaccination and other pharmaceutical interventions, it provides the best way to control and suppress an epidemic. formulation the basic contact tracing problem is stated as follows: given a social contact network g(v, e) and subset of nodes s ⊂ v that are infected and a subset s ⊂ s of nodes identified as infected, find all neighbors of s. here a neighbor means an individual who is likely to have a substantial contact with the infected person. one then tests them (if tests are available), and following that, isolates these neighbors, or vaccinates them or administers anti-viral. the measures of effectiveness for the problem include: (i) maximizing the size of s , (ii) maximizing the size of set n (s ) ⊆ n (s), i.e. the potential number of neighbors of set s , (iii) doing this within a short period of time so that these neighbors either do not become infectious, or they minimize the number of days that they are infectious, while they are still interacting in the community in a normal manner, (iv) the eventual goal is to try and reduce the incidence rate in the community-thus if all the neighbors of s cannot be identified, one aims to identify those individuals who when isolated/treated lead to a large impact; (v) and finally verifying that these individuals indeed came in contact with the infected individuals and thus can be asked to isolate or be treated. data needs data needed for the contact tracing problem includes: (i) a line list of individuals who are currently known to be infected (this is needed in case of human based contact tracing). in the real world, when carrying out human contact tracers based deployment, one interviews all the individuals who are known to be infectious and reaches out to their contacts. modeling approaches human contact tracing is routinely done in epidemiology. most states in the us have hired such contact tracers. they obtain the daily incidence report from the state health departments and then proceed to contact the individuals who are confirmed to be infected. earlier, human contact tracers used to go from house to house and identify the potential neighbors through a well defined interview process. although very effective it is very time consuming and labor intensive. phones were used extensively in the last - years as they allow the contact tracers to reach individuals. they are helpful but have the downside that it might be hard to reach all individuals. during covid- outbreak, for the first time, societies and governments have considered and deployed digital contact tracing tools [ ] [ ] [ ] [ ] [ ] . these can be quite effective but also have certain weaknesses, including, privacy, accuracy, and limited market penetration of the digital apps. challenges these include: (i) inability to identify everyone who is infectious (the set s) -this is virtually impossible for covid- like disease unless the incidence rate has come down drastically and for the reason that many individuals are infected but asymptomatic; (ii) identifying all contacts of s (or s ) -this is hard since individuals cannot recall everyone they met, certain folks that they were in close proximity might have been in stores or social events and thus not known to individuals in the set s. furthermore, even if a person is able to identify the contacts, it is often hard to reach all the individuals due to resource constraints (each human tracer can only contact a small number of individuals. the overall goal of the vaccine allocation problem is to allocate vaccine efficiently and in a timely manner to reduce the overall burden of the pandemic. formulation the basic version of the problem can be cast in a very simple manner (for networked models): given a graph g(v, e) and a budget b on the number of vaccines available, find a set s of size b to vaccinate so as to optimize certain measure of effectiveness. the measure of effectiveness can be (i) minimizing the total number of individuals infected (or maximizing the total number of uninfected individuals); (ii) minimizing the total number of deaths (or maximizing the total number of deaths averted); (iii) optimizing the above quantities but keeping in mind certain equity and fairness criteria (across socio-demographic groups, e.g. age, race, income); (iv) taking into account vaccine hesitancy of individuals; (v) taking into account the fact that all vaccines are not available at the start of the pandemic, and when they become available, one gets limited number of doses each month; (vi) deciding how to share the stockpile between countries, state, and other organizations; (vii) taking into account efficacy of the vaccine. data needs as in other problems, vaccine allocation problems need as input a good representation of the system; network based, meta-population based and compartmental mass action models can be used. one other key input is the vaccine budget, i.e., the production schedule and timeline, which serves as the constraint for the allocation problem. additional data on prevailing vaccine sentiment and past compliance to seasonal/neonatal vaccinations are useful to estimate coverage. modeling approaches the problem has been studied actively in the literature; network science community has focused on optimal allocation schemes, while public health community has focused on using meta-population models and assessing certain fixed allocation schemes based on socio-economic and demographic considerations. game theoretic approaches that try and understand strategic behavior of individuals and organization has also been studied. challenges the problem is computationally challenging and thus most of the time simulation based optimization techniques are used. challenge to the optimization approach comes from the fact that the optimal allocation scheme might be hard to compute or hard to implement. other challenges include fairness criteria (e.g. the optimal set might be a specific group) and also multiple objectives that one needs to balance. while the above sections provide an overview of salient modeling questions that arise during the key stages of a pandemic, mathematical and computational model development is equally if not more important as we approach the post-pandemic (or more appropriately inter-pandemic) phase. often referred to as peace time efforts, this phase allows modelers to retrospectively assess individual and collective models on how they performed during the pandemic. in order to encourage continued development and identifying data gaps, synthetic forecasting challenge exercises [ ] may be conducted where multiple modeling groups are invited to forecast synthetic scenarios with varying levels of data availability. another set of models that are quite relevant for policymakers during the winding down stages, are those that help assess overall health burden and economic costs of the pandemic. epideep: exploiting embeddings for epidemic forecasting an arima model to forecast the spread and the final size of covid- epidemic in italy (first version on ssrn march) real-time epidemic forecasting: challenges and opportunities accuracy of real-time multi-model ensemble forecasts for seasonal influenza in the u.s realtime forecasting of infectious disease dynamics with a stochastic semi-mechanistic model healthmap the use of social media in public health surveillance. western pacific surveillance and response journal : wpsar the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak basic prediction methodology for covid- : estimation and sensitivity considerations. medrxiv covid- outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand. imperial college technical report modelling disease outbreaks in realistic urban social networks computational epidemiology forecasting covid- impact on hospital bed-days, icudays, ventilator-days and deaths by us state in the next months open data resources for fighting covid- data-driven methods to monitor, model, forecast and control covid- pandemic: leveraging data science, epidemiology and control theory covid- datasets: a survey and future challenges. medrxiv mathematical modeling of epidemic diseases the use of mathematical models to inform influenza pandemic preparedness and response mathematical models for covid- pandemic: a comparative analysis updated preparedness and response framework for influenza pandemics novel framework for assessing epidemiologic effects of influenza epidemics and pandemics covid- pandemic planning scenarios epidemiological data from the covid- outbreak, real-time case information covid- case surveillance public use data -data -centers for disease control and prevention covid- patients' clinical characteristics, discharge rate, and fatality rate of meta-analysis estimating the generation interval for coronavirus disease (covid- ) based on symptom onset data the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application estimating clinical severity of covid- from the transmission dynamics in wuhan, china the use and reporting of airline passenger data for infectious disease modelling: a systematic review flight cancellations related to -ncov (covid- ) the hidden geometry of complex, network-driven contagion phenomena potential for global spread of a novel coronavirus from china forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study. the lancet using predicted imports of -ncov cases to determine locations that may not be identifying all imported cases. medrxiv preparedness and vulnerability of african countries against introductions of -ncov. medrxiv creating synthetic baseline populations openstreetmap american time use survey multiscale mobility networks and the spatial spreading of infectious diseases optimizing spatial allocation of seasonal influenza vaccine under temporal constraints assessing the international spreading risk associated with the west african ebola outbreak spread of zika virus in the americas structure of social contact networks and their impact on epidemics generation and analysis of large synthetic social contact networks modelling disease outbreaks in realistic urban social networks containing pandemic influenza at the source report : impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand the structure and function of complex networks a public data lake for analysis of covid- data midas network. midas novel coronavirus repository covid- ) data in the united states covid- impact analysis platform covid- surveillance dashboard the covid tracking project absolute humidity and the seasonal onset of influenza in the continental united states epiestim: a package to estimate time varying reproduction numbers from epidemic curves. r package version google covid- community mobility reports mask-wearing survey data impact of social distancing measures on coronavirus disease healthcare demand, central texas, usa current hospital capacity estimates -snapshot total hospital bed occupancy quantifying the effects of contact tracing, testing, and containment covid- epidemic in switzerland: on the importance of testing, contact tracing and isolation quantifying sars-cov- transmission suggests epidemic control with digital contact tracing isolation and contact tracing can tip the scale to containment of covid- in populations with social distancing. available at ssrn privacy sensitive protocols and mechanisms for mobile contact tracing the rapidd ebola forecasting challenge: synthesis and lessons learnt acknowledgments. the authors would like to thank members of the biocomplexity covid- response team and network systems science and advanced computing (nssac) division for their thoughtful comments and suggestions related to epidemic modeling and response support. we thank members of the biocomplexity institute and initiative, university of virginia for useful discussion and suggestions. this key: cord- - qawjquv authors: lara, r.j.; islam, m.s.; yamasaki, s.; neogi, s.b.; nair, g.b. title: aquatic ecosystems, human health, and ecohydrology date: - - journal: treatise on estuarine and coastal science doi: . /b - - - - . - sha: doc_id: cord_uid: qawjquv this chapter treats two main topics: the relationship between human health, aquatic ecosystems, and water use; and the necessity of interdisciplinary approaches for the development of water management policies and disease control. main waterborne diseases, mostly affecting developing countries and relevant in terms of water management and changes in land use, such as malaria, schistosomiasis, or cholera, are discussed stressing links to the global water crisis. also, the role of artificial and natural wetlands in influenza epidemics is treated. the effects of increasing water use and scarcity on human health are discussed considering historical and contemporary incidence of diarrheal diseases in european and south asian megacities, relationships between dams and on waterborne diseases in asia and africa, and intensive agri- and aquaculture resulting in man-made ecotones, fragmented aquatic ecosystems, and pathogen mutations. it is emphasized that the comprehension of the multiple interactions among changes in environmental settings, land use, and human health requires a new synthesis of ecohydrology, biomedical sciences, and water management for surveillance and control of waterborne diseases in basin-based, transboundary health systems. surveillance systems should monitor changes in water management, ecotones, and hydrological cycles and shifts in, for example, the outbreak timing of strongly seasonal diseases. these indicators would provide criteria for the development of innovative water management policies, combining methods of vector control and the safe creation of water reservoirs, irrigation systems, and wetland habitats. this chapter is not intended to provide an extensive review of water-borne diseases, since there are already excellent examples of these in the literature. rather, the intention is to call atten tion to particular aspects of the principal water-borne diseases and related water management issues. the emphasis is often on controversial aspects which it is hoped will stimulate an open, undogmatic, and fruitful discussion on possibilities and cur rent and future challenges for ecohydrology in relation to human health. ecological integrity is central to health (epstein, ) . pollution, disturbed environments, habitat loss, and climate change promote disease emergence in a number of ways. threats to human health arising from man's interaction with aquatic ecosystems can originate from multiple factors, which can be broadly grouped into the following major categories: • natural biological cycles in which humans can act as hosts of pathogenic microorganisms (protozoans, bacteria, etc.); • consequences of the management of aquatic resources (e.g., wetlands drainage or creation, aquaculture, and dam construction); • effects of water pollution (chemical, microbiological, radio active, and thermal) on man and on the physiology of individual organisms; and • the impact of global changes affecting climate and hydrolo gical cycles (e.g., habitat degradation, warming, increased rainfall, and storms). clearly, these are interlinked and specific case studies are likely to present evidence of more than one causal factor. thus, an effort has been made to integrate these different topics into sections covering the most relevant factors related to diseases that can be considered indicators of determined hydrological processes. globally, approximately % of deaths due to infectious dis eases are water related. this amounts to . million deaths per year, % of which are caused by diarrhea, a disease killing around million people every year, mostly children in devel oping countries (wwd, ) . thus, it is crucial to develop an integrated water and health management system, as well as the tools required to identify and predict interconnected trends in the evolution of aquatic ecosystems and diseases. although the importance of integrating natural sciences with socioeconomic research is stressed and recommended in every forum dealing with sustainable use of natural resources, human health, one of the most valuable public goods, is seldom included as a significant issue in coastal or basin management programs. a frequent goal of cooperation between socioeconomic and natural sciences research is the evaluation of the sustainability of resource use and the vulnerability of the coastal zone. the seven step assessment framework of the international panel on climate change (ipcc, ) is the customary tool used for this purpose (figure ) . similarly, the health map program from the world health organization (who) provides criteria and a software platform for linking epidemiological data with layers of geographic information (who and unicef, ) . however, these two types of research programs mostly lack any substantial interaction. ecohydrology can provide the conceptual framework for establishing such links. this involves a scale (the river basin and the coastal zone), an integrative process (flooding dynamics), and an integrative tool such as digital elevation models (dem) of the study area (figure ) . high-resolution topography is essential for data integra tion in low-lying areas. for this purpose, a closer interaction of socioeconomic studies, climate, hydrology, ecology, micro biology, biogeochemistry, molecular biology, and medicine is required, as exemplified in the case of cholera (discussed in section . . . . ). further, it is necessary to generate a common language to facilitate and promote inter-and transdisciplinary communication. several concepts related to disease dynamics, such as outbreak, epidemics, and pan demics, are commonly used in a qualitative way. yet, the creation of interdisciplinary databanks or the compilation of information from diverse sources (e.g., hydrological events and outbreak intensity) including historical ones would ben efit from objective category definitions. in the following sections, some examples are dealt with, which will appear in this chapter and in other related literature. it is considered that an epidemic occurs when new cases in a given human population, during a given period, sub stantially exceed what is expected, based on recent experience (the number of new cases in the population during a speci fied period of time is called the 'incidence rate'). an epidemic may be restricted to one locality (an outbreak), be more general (the usual 'epidemic'), or even global (pandemic). common diseases that occur at a constant but relatively low rate in the population are said to be 'endemic'. an example of an endemic disease is malaria in some parts of africa, where a large portion of the population is expected to contract malaria at some point in their lifetime. these somewhat subjective definitions require more precisionand a wider diffusion among the populationwhen, for aquatic ecosystems, human health, and ecohydrology figure integration of socioeconomic, ecological, and medical research within the ecohydrology framework. example, public funds have to be allocated for prevention, alleviation, or fight against determined diseases or for insur ance policies. an increasing number of direct or indirect cases of water-borne diseases are due to global warming, increasing frequency and intensity of storms, flood control, and environmental management (dam construction, irriga tion, use of fertilizers, etc.), and this has greatly widened the spectrum of stakeholders with an interest in the use of water and its impact on health. this underlines the need for agree ment on and diffusion of definitions and criteria between users and managers to facilitate the quantification of impacts of disease, ecological damage, and poor sanitation, as well as for cost-benefit analysis of proposed prevention or remedia tion measures. the global burden of disease analysis (who, ) pro vides a comprehensive and comparable appraisal of mortality and loss of health due to diseases and injuries, and risk factors for all regions of the world. the overall burden of disease is assessed using the disability-adjusted life year (daly), a timebased measure that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health. worldwide, about million dalys per year are due to water-borne diseases (pruess et al., ) . a relevant specific example is the definition and evaluation of the impact of disease related to drinking water quality. this concerns the presence of chemicals or pathogenic microorganisms that are transmitted when contaminated drinking water is directly con sumed. if contaminated drinking water is used in the preparation of food, it can be the source of food-borne disease through consumption of the same microorganisms, often in the form of gastrointestinal illnesses. according to the who (who, a) , diarrheal disease accounts for an estimated . % of the total daly global burden of disease and is respon sible for the deaths of . million people every year. it is estimated that % of that burden is attributable to unsafe water supply, sanitation, and hygiene. at the national level, surveillance systems are the primary source of data concerning the scope and effects of water-borne diseases on persons. these data can be organized to provide information for political and administrative units such as counties or federal states, and/or geomorphological units, that is, basins. in the united states, since , the centers of disease control and prevention (cdc), environmental protection agency, and the council of state and territorial epidemiologists have maintained a collaborative surveillance system which monitors the occurrence and causes of water borne-disease outbreaks (wbdos) (calderon et al., ). the surveillance system includes data for outbreaks associated with drinking water and recreational water. state, territorial, and local public health departments are primarily responsible for detecting and investigating wbdos and voluntarily reporting them to cdc on a standard form. the unit of analysis for the wbdo surveillance system is an outbreak, not an individual case of a water-borne disease. two criteria must be met for an event to be defined as a wbdo. first, more than two persons must have experienced a similar illness after either ingestion of drinking water or exposure to water encountered in recreational or occupational settings. this cri terion is waived for single cases of laboratory-confirmed primary amebic meningoencephalitis and for single cases of chemical poisoning if water-quality data indicate contamina tion by the chemical. second, epidemiologic evidence must implicate water as the probable source of the illness. the integration of national information on water-borne disease incidence into collaborative regional, transboundary basin databank networks of open access is crucial to allow a fair and efficient implementation of water management regula tions related to, for example, dam operation and international irrigation agreements. this is particularly relevant in tropical countries where large river basins (e.g., amazon, nile, mekong, indus, ganges, and yarlong-brahmaputra) represent the main source of income, water, and food for millions of people. the rivers originating in the tibetan plateau ( figure ) are particu larly relevant as they flow through several, densely populated tropical and subtropical countries with quite different degrees of development. in general, water-borne disease can be caused by protozoa, viruses, or bacteria, many of which are intestinal parasites. in the following tables, some relevant examples of various disease types are grouped and summarized, including those which mostly affect developing countries, in many cases in tropical and subtropical regions. in the following sections, diseases have been selected that are relevant in terms of numerical incidence, water management, and changes in land use, parti cularly under the ecohydrological approach. these are malaria, schistosomiasis, lymphatic filariasis, onchocerciasis, cholera, spotted fever, and those illnesses induced by toxins produced during cyanobacteria blooms due to increased nutrient loads. we will provide a general overview of these diseases, stressing the links to the global water crisis, possible contributions of ecohydrology, and challenges to its classical approach. parasitic infections, particularly malaria and schistosomiasis, represent some of the most universal health problems. the who estimates that million people are infected and another million people are at risk of infection (who, ) . in fact, only malaria accounts for more cases of disease than schis tosomiasis, and both are closely related to water management (who, ) and will be extensively treated in this chapter. water scarcity and uncoordinated water management go hand in hand with poverty and disease. russel ( ) explains the links clearly: bilharziasis and malaria are both debilitating diseases and debility is not conducive to good farming. throughout history, malaria and bilharziasis have interfered with the use of arid land and indeed, calcified eggs of bilharzia worms were found in the kidneys of two mummies of the th dynasty - - b.c. there is a crucial chain reaction that in some instances has contributed to the virtual abandonment of irrigation systems: the more debility, the less canal maintenance, therefore the more anopheles mosquitoes and the more snails transmitting more malaria and more bilharziasis, thus leading to more debility, and so on…. (russel, : ) . table provides a summary of relevant water-borne diseases produced by protozoan parasites. in this section, we will con centrate on malaria, currently still the world's most important parasitic disease. today, malaria is one of the world's deadliest diseases and occurs mostly in tropical and subtropical countries, being transmitted from one person to another through the bite of female anopheles mosquitoes. the who estimates that there are - million cases of malaria, with over million deaths each year (who, a) . t he main burden of malaria (more than %) is in africa, south of the sahara. two-thirds of the remaining burden affects six countries: brazil, colombia, india, solomon islands, sri lanka, and vietnam. the ecology of the disease is closely associated with the availability of water, as the larval stage of mosquitoes develops in different kinds of water bodies. the mosquito species vary considerably in their water-ecological require ments (sunlit or shaded, with or without aquatic vegetation, stagnant or slowly streaming, and fresh or brackish; discussed in section . . . . . ), and this, to a great extent, determines the ecology of the disease. in many places, the nat ural habitat sustains intense malaria transmission; in others, the development of water resources (irrigation, dams, and urban water supply) has exacerbated the intensity of transmission and caused the disease to spread. in yet others, for example, the central asian republics of the commonwealth of independent states, malaria has returned as a result of a breakdown in water management and maintenance problems of local irrigation systems (who, ) . climate change (global warming) appears to be moving the altitudinal limits of malaria to higher elevations, for example, in the east african highlands and madagascar. further, in some of the tropical regions of the developing world, the incidence of malaria has increased in recent years as the mosquito and the malaria parasite it transmits have evolved more resistance to sewage, nontreated drinking water, flies in water supply (hand-to-mouth) untreated water, poor disinfection, pipe leaks, groundwater pollution, sharing of water source by humans and wildlife. beavers and muskrats create ponds that act as reservoirs for giardia (oral-fecal, hand-to-mouth) encephalitozoon intestinalis has been detected in groundwater chills, period fever attacks; debility, spleen and liver enlargement; anemia, jaundice; clogging of brain vessels can lead to death flu-like symptoms, watery diarrhea, loss of appetite, substantial loss of weight, bloating, increased gas, and nausea abdominal pain, fatigue, weight loss, diarrhea, bloating, and fever diarrhea, bloating abdominal discomfort, and flatulence diarrhea and wasting in immunocompromised individuals alternatives to dichlorodiphenyltrichloroethane (ddt) and to the medications used to prevent or treat the disease. in some regions (e.g., south africa), ddt is again increasingly used to control mosquitoes (thurow, ) . in general, land reclama tion for agriculture, deforestation, and changes in land use are probably the principal causes of the climatic and habitat changes responsible for these developments. yet, it should be borne in mind that it was the drainage of wetlands for agricul tural purposes in the nineteenth century which contributed most significantly to the eradication of malaria in europe (reiter, ) . these are crucial aspects to be carefully evaluated when wetland creation and management policy are being considered as an ecohydrological tool, for example, for sequestration of nutri ents in estuaries and for preventing toxic algal blooms. these issues will be discussed extensively in the following section (also discussed in section . . . ). it is often erroneously thought that larvae of malariatransmitting mosquitoes can only develop in freshwater. it is also frequently forgotten that malaria is not restricted to the tropics, and that only in did the who declare that europe was free of malaria. about years ago, malaria was a leading cause of death in many marshland commu nities along the coast of southern england. there, extensive salt marshes provided high-quality grazing for sheep and cattle, but were also a favored habitat for anopheles atroparvus, a highly effective malaria vector, which prefers to breed in brackish water along river estuaries and in the presence of abundant algae. until the nineteenth century, malaria was a major mortality factor in the netherlands. however, by the end of that century transmission had dropped precipitously in the more prosperous countries of north europe. a major factor contributing to this decline was that the mosquito habitat had been eliminated by improved drainage and extensive land reclamation. major epidemics still occurred in russia and poland in the s, with high death rates reaching regions near the arctic circle (wolanski et al., and references therein) . today, malaria is again common in many parts of central america, northern south america, tropical and subtropical asia, some mediterranean countries, and many of the republics of the former ussr. this spread of the disease has been attrib uted to, among other factors, forest clearance, irrigation, ecological change, population increase, deterioration of public health services, resistance of mosquitoes to insecticides, and resistance of the malarial parasite to antimalarial drugs (reiter, ) . thus, policies on wetland creation or restoration must take account of not only the benefits of the reestablishment of lost ecological services, but also the potential consequences of increased areas of slow-flowing or stagnant waters on disease vector proliferation, particularly under a scenario of increasing temperatures. the related ecohydrological concepts of system robustness and flushing dynamics can make a major contribu tion to the integrated analysis and handling of this issue, seeking equilibrium between the necessary water residence time, for example, efficient nutrient sequestration, and the minimization of mosquito reproduction. the choice cannot be formulated as 'mosquitoes in the basin' or 'toxic algal blooms in the estuary'. there are techniques such as 'runneling' that have been used with varying success to control mosquito proliferation in cre ated or natural wetlands. as mosquitoes can be vectors for several other diseases besides malaria, this will be treated in a separate section (discussed below in section . . . . . ). table provides a summary of relevant water-borne diseases produced by worm infections. in this section, we will concen trate on schistosomiasis, lymphatic filariasis, and river table main water-borne diseases produced by worms blindness because of their strong links to water management measures, such as irrigation and dam operation schemes. schistosomiasis is considered the second most important para sitic infection after malaria in terms of public health and economic impact. it is a chronic debilitating disease that is estimated to affect between and million people in countries. as many as million live in endemic areas. also known as bilharzia, bilharziosis, or snail fever, it is caused by several species of fluke (trematode) of the genus schistosoma. (gryseels et al., ; figure ) . infection with any of the five species of schistosome worms is rarely fatal. although it has a low mortality rate, schistosomiasis is often a chronic illness that can damage internal organs and, in children, impair growth and cognitive development. the urinary form of schis tosomiasis is associated with increased risks for bladder cancer in adults (hodder et al., ) . human infections are most common in asia, africa, south america, or the middle east, especially in areas where the water contains numerous fresh water snails, which are intermediate hosts, that is, may carry the parasite. however, trematodes can be found anywhere human waste is used as fertilizer. the disease affects many people in developing countries, particularly children who may acquire the disease by swim ming or playing in infected water, and field workers in arid or semiarid regions where agriculture depends heavily on irrigation. first infection with schistosomiasis usually occurs during the early school years and is a frequent cause of absen teeism. it is not uncommon for % of a school's student population to be infected in some highly endemic areas in africa. in addition to its effect on children, schistosomiasis has a major impact on the agricultural workforce and on national economic productivity. in egypt, where % of the people are infected, economic losses due to lost work are estimated to exceed $ million a year (anonymous, a) . development, both planned and unplanned, has resulted in a number of changes in the epidemiology of the disease that threaten to increase its spread and the number of people infected, reduce economic productivity, and compromise development gains. water development schemes, including dam building and irrigation systems, with slow water flows and aquatic vegetation, have created new breeding sites for snails. intensive agriculture has encouraged people to migrate to urban and peri-urban areas that are ill-prepared to meet their needs for sanitation and water. in these areas, snail-infested streams and canals are often the most convenient water sources. new agricultural systems that emphasize irrigation, double cropping, and other intensive cultivation practices have increased farmers' exposure to infection. the emergence or reemergence of schistosomiasis as a result of large-scale hydroprojects has been reported from the egyptian aswan high dam (khalil, ; strickland, ) , the sudanese gezira-managil dam (amin, ; omer, (teklehaimanot and fletcher, ) . in china, the danling dam in the province of sichuan and the huangshi dam in the province of hunan have all had adverse effects by increasing local schistosome transmis sion (zhang and guo, ) . however, not all dam regions suffer from schistosomiasis. for example, when the ertan dam in the province of sichuan became operational, local schistoso miasis control centers collaborated with dam management offices and government ministries to actively monitor and pre vent the spreading of schistosome worms. as a result of these efforts, potential schistosome transmissions in the ertan region were successfully averted (gu et al., ) . schistosomiasis infection in humans, the definitive hosts, is caused mainly by three species of flatworm, namely schistosoma haematobium, s. japonicum, and s. mansoni. infection occurs when free-swimming larvae penetrate human skin. the larvae develop in freshwater snails, which serve as intermediate hosts for the parasite. humans are infected when they enter larvaeinfested water for domestic, occupational, and recreational purposes and the larvae of the parasite penetrate the unbroken skin. the life cycle is continued when people infected with schistosome worms deposit urine or fecally borne eggs into the water. the disease particularly affects children who may acquire the disease by swimming or playing in infected water (hodder et al., ) . the life cycle of s. mansoni provides a simplified example for all species of schistosomes and helps understand the potential contribution of water management to snail proliferation. after the eggs of the human-dwelling parasite are emitted in the feces, into the water, the ripe miracidium hatches out of the egg. the miracidium searches for a suitable freshwater snail to act as an intermediate host and penetrates it. following this, the parasite develops, via a so-called mother-sporocyst and daughter-sporocyst generation, into the cercaria. the purpose of the growth in the snail is the numerical multiplication of the parasite. a single miracidium can produce several thousand cercaria, each one of which is capable of infecting humans. the cercaria propel themselves in water with the aid of their bifurcated tail and actively seek out their final host. when they recognize human skin, they penetrate it within a very short time. following a migration through the body within the bloodstream, they develop into sexually mature adults. the larvae enter through the skin, migrate via the blood vessels, and mature in the lungs. from there they travel to the veins of the upper or lower intestine or bladder and, if they find a partner of the opposite sex, they reproduce. there are specific host/parasite combinations that lead to typical forms of bilhar ziasis in endemic areas in different regions of the world, frequently associated with a particular economic activity. there are also region-specific control measures with positive and negative consequences or side effects (mannesmann and fuchs, ) as discussed in section . . . . . . in these regions, the snail bulinus truncatus, b. globosus, and b. forskali can be hosts of s. haematobium, which produces uro genital bilharziasis (lang, ) . the two clearest examples of large-scale irrigation systems spreading schistosomiasis are found in africa, especially in the nile valley. in upper egypt south of cairo, it has been known for a long time that the shift from basin irrigation by the floodwaters of the nile to perennial irrigation results in a dramatic increase in schistosomiasis. basin irrigation allows the land to dry out seasonally, killing almost all snails. under perennial irrigation, the land is wet all year round. soon after the construction of aswan low dam, four locations that changed irrigation methods in the mid- s experienced a surge in s. haematobium infections (khalil and abdel azim, ; khalil, ) . infection rates increased from - % in to - % in . urinary schistosomiasis had a pre valence at % in areas of perennial irrigation in upper egypt and only % in areas with basin irrigation (scott, ) . in the s, prevalence ranged from % to % in three districts with perennial irrigation, while in two districts with basin irriga tion, it was % and % (wright, ) . in the s, after the construction of the aswan high dam, the nile delta became a major breeding habitat for the snail hosts of both urinary and intestinal schistosomiasis. irrigation canals and drains ( figure ) harbored stable populations of these snails throughout the year. this resulted from the elimina tion in these canals of the so-called 'winter closure'. before the construction of the dam, the closure was enforced for about days, during which the canals were closed and dried up, and the silt deposited on their beds during the nile flood was dredged out together with the snails and aquatic weeds (malek, ) . the other large-scale nile valley irrigation system impli cated in schistosomiasis transmission is the gezira irrigation scheme in the sudan. the major increase in prevalence of schistosomiasis in gezira came after , when the cropping rotation changed to include 'winter' wheat. farmers kept the canals filled with water from march to may, when they were previously dry (fenwick, ) . another factor was the creation of the adjoining managil extension irrigation system which left tenants without adequate water supplies or sanitation facilities. also, there was an influx of migrant laborers in the original gezira scheme, who lived near irrigation canals, under very poor sanitary conditions, leading to the propagation of further water-borne diseases (tameim et al., ) . in north africa and the middle east, research has demon strated the association of even small-scale irrigation plans, decentralized at the village level, with increases in schistoso miasis transmission. malek ( ) reports that in sudanese villages along the nile, north of khartoum, prevalence of urin ary schistosomiasis in children was - %, compared to an average of less than % in other areas. wright ( ) reports that in the rural area around baghdad, iraq, prevalence of schistosomiasis increased from % to % following the installation of lift pumps. . . . . . asia in east and southeast asia, intensification and expansion of irrigated rice production systems over the past decades have increased the habitats for snail and schistosoma. the host/para site combinations tricula aperta/s. mekongi and oncomelania hupensi/s. japonicum produce intestinal bilharziasis predomi nantly in rural communities living close to irrigation ditches. the association of asian schistosomiasis with rice-growing areas has been reported by different authors. however, in many cases, rice fields themselves did not seem to be breeding habitats; rather snails found in the rice fields appeared to have spread from nearby irrigation canals. cattle and water buffalo can also be important reservoir hosts. various methods have been applied to substantially reduce schistosomiasis in rice agriculture, which will be discussed later in section . . . . . in this part of the world, it is mainly the combination of s. mansoni with biomphalaria glabrata snails that causes bilhar ziasis. brazil, with million people living in the endemic areas and million infected, is the most affected country in the americas (chitsulo et al., ) . however, it is not obvious to what extent hydrological factors, including large dams or irrigation systems, contribute to spreading schistosomiasis in brazil. the african slave trade was probably responsible for the introduction of schistosomiasis in brazil soon after the country was discovered by european explorers, and internal migration was responsible for spreading it from seashore to interior. nowadays, schistosomiasis transmission occurs over a vast endemic region, from maranhão to espírito santo, and minas gerais, and there are further areas with a high risk of endemic expansion (araujo, ; paraense, ) . there are also iso lated foci in the federal district and in the states of pará, goiás, rio de janeiro, são paulo, paraná, and rio grande do sul. some examples from the literature point to patterns of labor and household migration as significant factors in the incidence of disease in brazil, probably more than irrigation schemes. cases of disease importation from endemic areas have been registered over almost the entire country, mainly in the states that are considered migration destination areas, such as rondônia (coura and amaral, ) . a study involving irrigation projects in the semiarid region of five northeastern states of brazil (coutinho et al., ) found that schistoso miasis transmission was not a major problem in the areas studied. socioeconomic-sanitary analysis identified the pre sence of migrant farm workers coming from endemic areas of schistosomiasis and living in poor sanitary conditions in the irrigation areas as a main factor of epidemiological importance. however, continued epidemiological surveillance is essential in all irrigated areas of northeastern brazil if schistosomiasis control is to be maintained, as well as improvement in the water supply and sanitation measures for migrant workers (coutinho et al., ) . the potential association between irrigation levels and the occurrence and spread of s. mansoni infection was investigated (martins and barreto, ) in the state of bahia, where two forms of irrigation have been developed. the first is capital intensive and mechanized, requiring little manual labor. the second is labor intensive and characterized by limited mechan ization. according to the study, the municipalities with the largest irrigated areas are not the ones with the highest s. mansoni infection rates. in most of these counties, irrigation is capital-and technology intensive. according to these find ings, unlike africa, irrigation in the state of bahia has had little impact on the spatial profile of the schistosomiasis endemism. regarding the impact of schistosomiasis on labor-intensive irrigation schemes, some research points to the importance of the household in disease transmission, as a result of the cluster ing of domestic activities associated with water collection, storage, and usage. such activities can result in the sharing of water-contact sites and water-contact behavior, which expose all members of the household to an increased risk of infection. in previous studies in brazil (bethony et al., ) , it was deter mined that shared residence accounted for % of the variance in schistosoma fecal egg excretion rates. further, shared residence accounted for % of the variation in total water contacts per week. it also accounted for a large proportion of the variation in individual water-contact behavior: for example, agricultural con tacts ( %), washing limbs ( %), or bathing ( %). these results implicate the household as an important composite measure of the complex relationships between socioeconomic, environmental, and behavioral factors that influence water-con tact behavior and, therefore, the transmission of schistosomiasis. these results also support the idea of focusing on safe water supply and household density in the implementation of schis tosomiasis prevention and control measures. the role of aquatic animals in maintaining the schistosome life cycle in brazil requires further clarification. the influence of s. mansoni on a population of the water rat, nectomys squamipes, was studied at sumidouro, rio de janeiro, and brazil (d' andrea et al., ) . the population dynamics of parasites was studied. water contamination (i.e., the source of miraci dia), abundance of the intermediate host, and rodent migration were found to be related to schistosome prevalence. the n. squamipes population was not obviously influenced by the infection, as shown by the high number of reproductive infected females, high longevity of infected individuals, and the absence of a relationship between recruitment or survivorship rates and the intensity of schistosome infection. the data indicate that n. squamipes can increase transmission of s. mansoni in endemic areas and carry it to noninfected areas. furthermore, this rodent can be used as an indicator of trans mission foci. in the caribbean, b. glabrata can be found in shallow ponds with abundant vegetation or with fallen banana leaves. it has been also found in drains around banana plantations on the west indian island of st. lucia (sturrock, ) . this is a mosquito-borne parasitic worm infection, a debilitating parasitic disease, which affects million people in the tropical and subtropical areas of southeast asia, south america, africa, and the islands of the pacific. while filariasis is rarely fatal, it is the second leading cause of permanent and long-term disability in the world. a person with the disease tends to have more bacterial infections in the skin and lymph system. this causes hardening and thickening of the skin, which in its most dramatic form is expressed in the symptoms of elephantiasis, the accumu lation of lymph, usually in legs. it is not a killer disease, but causes severe debilitation and social stigma. the who has named filariasis one of only six 'potentially eradicable' infectious diseases and has embarked upon a -year campaign to eradi cate the disease. in addition to consistent long-term treatment by oral medicines, eradication efforts focus on controlling the pro liferation of mosquitoes in aquatic environments, which will help to reduce the transmission of lymphatic filariasis, as well as that of malaria, which is prevalent in many of the same communities in africa (cdc, ) . onchocerciasis or river blindness is the world's second leading infectious cause of blindness. it is found in countries in africa as well as in guatemala, southern mexico, some areas of venezuela, small areas in brazil, colombia, and ecuador, and in the arabian peninsula. a total of million people are affected worldwide. the disease is caused by onchocerca volvulus, a parasitic worm that breeds in water and that can live for up to years in the human body. controlling insect breeding sites in rivers is one of the pillars of prevention. the disease is trans mitted person to person by bite of a blackfly (simulium), which breeds solely in fast-flowing waters. symptoms of the disease in a person usually begin to show - years after infection. each adult female worm, which can be more than half a meter in length, produces millions of microscopic young worms (micro filaria). the microfilaria migrate through the skin and, upon death, cause intense itching and depigmentation of the skin (leopard skin), lymphadenitis, resulting in hanging groins and elephantiasis of the genitals, serious visual impairment, and blindness when they reach the eye. the disease blinds between % and % of its victims and seriously undermines the economic productivity of communities in endemic areas (who, b; figure ). figure onchocerciasis also affects the development of exposed children. their school performance is affected by the unrelenting itching associated with this disease. many youth are deprived of their childhood as they are often forced to guide and look after elderly relatives blinded by the disease. photo credit: bill vanderdecker in: http://www.pqmd.org/cms/node/ unlike malaria and schistosomiasis, transmission of river blindness is usually found along fast rivers or streams with white-water rapids and cascades. the species of blackflies, which transmit this blinding parasite, require well-aerated, high-velocity flow to deposit their eggs, usually on rocks or overhanging vegetation. the larval stages are filter feeders and need large flows passing their habitat to obtain sufficient food and oxygen for development. in many parts of africa, people living near rivers migrate out of the fertile river valleys because of the painful bites of the flies and the eventual blindness resulting from this parasite (kim and merritt, ) . river blindness has historically plagued the fertile valleys of west africa, but it was the arrival of europeans that unleashed the full force of the disease upon the region's inhabitants. traditional taboos had kept people from settling along river banks or visiting streams in broad daylight, when blackflies are most active. white colonists, however, insisted on recreating the riverside towns they remembered from home. by removing longstanding cultural prohibitions, they made onchocerciasis more prevalent than it had been before. by the s, several hundred thousand people had been blinded by the disease. also tragically for the region, the most fertile farmlandan area roughly the size of michiganwas abandoned due to the risk of contracting the disease (wong, ) . currently, river blindness has been eliminated as a major public health problem in west african countries. despite success in west africa, million people remain at risk of contracting river blindness in the countries of central, eastern, and southern africa (anonymous, ) . due to the tight connec tions between the dynamics of this disease and changes in flow velocity, we will return to it in section . . . in the discus sion of the effect of dams on human health. table provides a summary of relevant water-borne diseases produced by bacteria. we will concentrate on cholera and other vibrio illnesses for the reasons explained below. the case of cholera has been considered paradigmatic of the links between global climate change and infectious diseases. it offers an excellent example of how information about environ mental factors permits better understanding of disease virulence, transmission, and epidemiology. therefore, and due to the links to wetlands and other coastal ecosystems, aquaculture, and water management in megacities, this disease will be discussed in detail in this and the following sections, in relation to the dynamics of its causative agent in aquatic ecosystems. cholera is still an important cause of morbidity and mor tality in many countries in asia, africa, and latin america due to lack of safe water supply and poor hygienic practices (colwell, ) . cholera is endemic in the ganges and brahmaputra deltas. it was originally endemic to the indian subcontinent but spread worldwide along the trade routes, and mostly affects developing countries, particularly in coastal zones. in the last ∼ years, there have been seven pandemics and there is evidence of accelerated change of vibrio and disease dynamics as consequence of environmental changes and increased global connectivity (faruque et al., ) . the cur rent pandemic, which started in , is the most extensive in geographic spread and duration. cholera epidemics were reported from over countries during , the largest scale ever recorded in human history (who, ) . vibrio cholerae, a gram-negative comma-shaped gammapro teobacterium, is the causative agent of cholera ( figure ). vibrios are aquatic bacteria of marine and estuarine origin but can survive and be pathogenic in freshwater ecosystems. apart from v. cholerae, some other vibrios (v. parahaemolyticus, v. vulnificus, etc.) are also responsible for incidences of diarrhea, gastroenteritis, necrotizing fasciitis, and septicemia, which afflict human populations (table ) all over the world (chakraborty et al., ) . besides, many vibrios can also cause diseases in fish, shrimp, corals, and other aquatic organisms (thompson et al., ) . there are convergent approaches to the investigation of coral and human diseases. these include the research into the figure the causative agent of cholera, vibrio cholerae, is a faculta tively anaerobic bacterium, . - . µm to . - . µm, and a natural inhabitant of temperate/tropical estuaries, salt marshes, mangroves, coastal waters, and reefs. photo credit: moredun animal health, ltd/ science photo library/photo researchers, inc. same bacteria genus (vibrio), responsible for coral bleaching and for cholera, the use of remote sensing of ocean-surface temperature, climate research, and an emerging common cur rent of epidemiological thinking. traditionally, the association between water temperature and coral bleaching has been stressed, and only recently it has been discovered that this is probably triggered by a vibrio bacterium, and that it could be transmitted by a coral-feeding worm, acting as 'vector'. it is the first time a vector has been found for a coral disease (rosenberg and falkovitz, ) . table main water-borne illnesses produced by bacteria at the same time, the study of cholera disease has begun to encompass marine and estuarine research. the association of v. cholerae with plankton was established only recently, allow ing analysis of patterns of cholera epidemics, especially in those regions where it is endemic. the sporadic and erratic nature of cholera epidemics can now be related to climate-ocean coupling events, such as el niño (colwell, ) . since zoo plankton has been shown to harbor the bacterium and zooplankton blooms follow phytoplankton blooms, remote sensing can be employed to determine the relationship between cases of cholera and ocean chlorophyll concentration, as well as sea-surface temperature, ocean height, nutrient con centrations, salinity, and turbidity. during each pandemic, cholera has struck coastal regions before spreading inland. the principal agents responsible for cholera epidemics are the o and o serogroups of v. cholerae, of more than serogroups so far identified. within the o serogroup, the classical biotype caused the pre vious six pandemics, while the el tor biotype is associated with the present seventh pandemic. a coupling of vibrio dynamics to that of aquatic, brackish ecosystems is strongly suggested by the marked cholera seasonality in coastal villages of the bay of bengal (colwell, ) . the role of coastal areas in maintain ing endemicity is clearly a significant feature of cholera ecology. however, it was mainly laboratory research that provided evi dence for the existence of aquatic environmental reservoirs where v. cholerae survives for long periods of time and from which a toxigenic form may emerge to support epidemic con ditions (miller et al., (miller et al., , (miller et al., , barua and greenough, ) . the o serogroup emerged in the coastal zone in through a natural genetic alteration of the o el tor biotype. a survey in bangladesh established that this strain was mainly found in southern coastal marshes (faruque et al., ) . however, systematic, interdisciplinary field studies on the relationship between habitat characteristics and vibrio diversity and virulence are scarce. little has been done to widely monitor v. cholerae and study its ecology from a basin perspec tive in different related coastal environments such as mangroves, marshes, and estuaries, despite clear evidence that this could be one significant step forward for early warning and understanding human vulnerability to the disease (collins, ) . as early as , robert koch suggested that the bay of bengal, especially the sundarban mangrove forest, was the main source of cholera, noting that the combination of a brackish, organic-matter-rich environment, with a high den sity of human population represented the ideal conditions for the proliferation of v. cholerae (koch, ) . however, since then, there have been no extensive surveys of this transbound ary ecosystem (bangladesh and india), which represents the largest unitary mangrove and marsh system worldwide (akhtaruzzam, ) . difficult access, cyclones, floods, and the difficulty of obtaining funding for long-term research have precluded the systematic spatiotemporal monitoring of these habitats. most of the few available works are based on one-site samplings and do not provide information on seasonal trends. plankton is a significant marine reservoir of v. (huq and colwell, ) . in most cases, phytoplankton and zooplank ton are spatially and temporally associated (kiorboe and nielsen, ) and their abundance can be estimated by remote sensing, which has been related to cholera incidence in the bay of bengal region by lobitz et al. ( ) . living zooplankton can be a reservoir for vibrios, which attach them selves to the zooplankton's chitinaceous exoskeleton (kaneko and colwell, ) . nevertheless, watkins and cabelli ( ) found that growth and survival of v. parahaemolyticus was more stimulated by addition of pulverized chitin, than by living zooplankton, which however had a greater effect than the addition of sewage or other nutrients. in addition to salinity, nutrients, and plankton, suspended particle load and sediment resuspension also influence the vibrio amount in the estuary. recent investigations (lara, unpublished results) on seston size fractionation in sunderbans waters showed that the largest amounts of chitin and cultivable vibrio spp. were generally present in the fractions correspond ing to micro-, nanoplankton, microdetritus, and silt/clay particles, and not in zooplankton. it is still an open question whether the vibrio serotypes o and o , as well as the viable but not cultivable, and nonviable forms are preferen tially associated with determined seston size classes or wetland ecosystem compartments. better understanding of vibrio diversity in aquatic environ ments such as estuaries, marshes, and mangroves can lead to new insights into their genetic expression and co-regulation in the environment where they interact with each other. however, most vibrio studies treat species of pathogenic importance (e.g., v. cholerae, v. parahaemolyticus, and v. vulnificus) and not the diversity of the genus itself. a further key issue is the compre hension of the seasonality, life cycle, and dormant phases of bacterial population in nature. the environmental persistence of v. cholerae may be facilitated by entering a dormant state in which it remains viable but becomes nonculturable (vbnc) in conventional laboratory media (colwell et al., ) . the v. cholerae cells attached to plankton enter into the vbnc state as survival strategy (colwell and huq, ) . therefore, the detection of the nonculturable state is crucial to understanding v. cholerae ecology. however, the mechanisms that cause the organism to associate with plankton or other particles, to form biofilms, or to enter dormant or free-swimming phases are not yet completely understood. in the karnaphuli estuary, bangladesh, recent studies (lara, unpublished) showed that suspended particulate matter (spm) other than zooplankton contained significant amounts of chitin, especially in the size class < µm. the quantitative contribution of respectively nano-or bacterioplankton, microdetritus of biological origin, or resuspended sediment particles to the chitin pool in that size class is still unknown. particle load together with salinity significantly influenced estuarine vibrio distribution. we compared the microbial landscape dur ing a pre-monsoon situation and after a strong cyclone: the amount of cultivable vibrio, and its relative contribution to total aerobic bacteria, increased dramatically after the cyclone. amounts of spm also increased and there were higher salinities along the estuary. sediment resuspension and salt intrusion can thus strongly influence the abundance and distribution of estuarine vibrio population. the above findings call attention on essential questions relating estuarine dynamics and human health: are vibrios in sediment part of a benthic community with its own character istics or do they basically consist of a fraction of a pelagic population reaching the sediment after sedimentation of the particles to which they are attached? nair et al. ( ) also addressed the role of sediments as a possible vibrio reservoir in freshwater environments in calcutta. in florida, there was a predominance of non-o v. cholerae infections at the time the organisms flourished in the sediment (williams and larock, ) , which was detected down to -cm depth. higher sedi ment vibrio concentrations were associated with organic matter flocs occurring after the seasonal phytoplankton productivity maximum and during the zooplankton decline. this suggested that the flourishing of vibrio in the sediments was related to the presence of organic matter input from plankton detritus. thus, although plankton itself is an important aquatic vibrio reser voir, its relevance for fueling benthic vibrio seasonal cycles has probably been overlooked. the relevance of seasonally driven sediment resuspension in relation to annual cholera cycles in endemic regions deserves more attention. the incorporation of particle-bound vibrios and porewater nutrients into the water column could favor a sharp vibrio increase, even at otherwise unfavorable salinities (singleton et al., ) . future studies should investigate the links between the spatiotemporal estuarine variability and the ecology, diversity, and spreading mechanisms of vibrios including v. cholerae. a key question is how and to what extent these microorganisms persist in the transition from a brackish to a freshwater envir onment, involving strong gradients in salinity, ph, inorganic nutrients, dissolved and particulate organic matter, turbidity, plankton, and wetland vegetation. further, an ecohydrological research approach should focus on how the distribution of the above factors influences the relative abundance of v. cholerae compared to the total vibrio population and other bacterial groups in aquatic environments of west bengal, including water and sediment compartments. there have been efforts to predict cholera outbreaks through models relating disease incidence and environmental variables such as seawater temperature, chlorophyll content, height of the ocean surface, and rainfall, as well as by remote sensing (e.g., lobitz et al., ) . recently, an empirical model relating multiyear data of the number of cholera cases, rainfall, and chlorophyll was able to successfully reproduce outbreaks of cholera in kolkata and matlab over the time span of the data set (magny et al., ). yet, these authors stated that a finer temporal resolution (submonthly) in environmental data col lection was needed to improve mechanistic models and account for short-term variability, especially for the kolkata region. there is also evidence that cholera cases increased following a rise in ocean-surface temperature. however, a direct correla tion exists only for the spring peak, while during the rest of the year there are lags and even an inverse correlation between the two variables (colwell, ) . simple (lag) correlations between the seasonality of cholera and that of climate variables such as monsoon rainfall merely confirm that cholera is seaso nal (bouma and pascual, ) . these authors hypothesized that there would be two different aquatic habitats: the marineestuary type and the inland water bodies, with potentially different driving factors. although much has been speculated about possible ocean/ land interactions based on empirical correlations, the causal links between ocean parameters and cholera incidence in ripar ian inland villages are tenuous, as discussed in the following. zooplankton in freshwater has been considered a main trans mission means of v. cholerae to humans . although tidal transport of vibrio-carrying marine zooplankton toward the inland is likely and has been proposed by these authors as a possible infection source, until now there have been no studies demonstrating that marine copepods survive long enough to represent an infection source in freshwater, which is what people drink finally, and not seawater. simultaneous variation of two parameters does not imply causality and, although the existing mechanistic models can be useful as predictive tools, they have not significantly contributed to explaining the reasons for cholera endemicity or the existence of bimodal and unimodal occurrence patterns in the indian subcontinent. for example, sea-surface tempera ture in the bay of bengal shows a bimodal cycle similar to the seasonal pattern of cholera in dhaka, bangladesh (colwell, ; figure ) , and is therefore often used, directly or through covarying parameters, with success for such models. however, this seems to mask the fact that strong unimodal patterns are observed elsewhere in india (e.g., north bengal and vellore; jesudason et al., ; bouma and pascual, ) without any obvious relationship to the marine environment or even to temperature and rainfall. several studies found that the effect of rainfall on cholera incidence does not show any univocal pattern (glass et al., ; bouma and pascual, ; ruiz-moreno et al., ) . even in dhaka, which has a strong bimodal seasonality, there is no clear relationship between rainfall and cholera. the number of cases peaks before the monsoon (high rainfall period) and at its end, with a strong decrease in the middle of the monsoon. recently, hashizume et al. ( ) showed that the weekly number of cholera cases in the period - in dhaka did not show any direct relationship to rainfall, and suggested that river levelbelow flooding levelalso plays a role. river discharge is controlled not only by rainfall but also by snow melting in the himalayas and can control the survival of the bacterium through determining salinity and ph levels (bouma and pascual, ) . hashizume et al. ( ) stated that, because of the observed and potential effects of chemical changes in surface waters due to rainfall on vibrio survival and toxicity, it is necessary to quantify the level of v. cholerae in aquatic environments at the same time as measuring rainfall and river levels ( table ) . this section deals with examples of diseases derived from or influenced by high nutrient loads, particularly those derived from toxic algal blooms, as well as with the general effect of global changes in nutrient cycles on parasitic infectious diseases according to toxin type and exposure: skin irritation, stomach cramps, vomiting, nausea, diarrhea, fever, sore throat, headache, muscle and joint pain, liver damage, and kidney disease in the body nitrate is reduced to nitrite, which reacts with hemoglobin forming methemoglobin, and reducing blood ability to carry oxygen; affects mostly infants and old persons (pids). although arsenicosis affects millions of people, parti cularly in bangladesh, the problem arises from using water from wells reaching layers where groundwater naturally con tains high as levels and there is, to date, no obvious way that ecohydrological methods could help solve this problem. for this reason, we will not discuss this disease further in this chapter. eutrophication in water reservoirs or semi-enclosed water bodies leads to the formation of intensive phytoplankton blooms. during recent years, both the incidence and intensity of such blooms appear to be increasing, when examined at the global scale. this increasing severity of algal blooms may be a consequence of increasing levels of nutrient enrichment as a result of sewage disposal, increased agricultural runoff, and changes in hydrological regimes potentially related to climate change (unep, ) . blooms caused by toxic cyanobacteria (harmful algal blooms) lead to outbreaks of disease and dete riorated recreational and aesthetic values, causing both economic losses and illness and death of both humans and animals. in evolutionary terms, cyanobacteria are one of the oldest organisms on earth, dating back to more than ma (schopf, ) . they are oxygenic photosynthetic prokaryotes possessing the ability to synthesize chlorophyll a as their photosynthetic pigment. the nitrogen-to-phosphorus ratio (n:p) has been frequently used as a key indicator in predicting algal biomass and compositions, and its seasonal succession in lentic systems (tilman, ; kilham, ) . smith ( ) pointed out that bloom-forming cyanobacteria had a tendency to dominate in a lake when the n:p ratio was less than . the significance of the n:p ratio as a critical factor, however, is still controversial, due to the variability of the other chemical char acteristics and phytoplankton composition within a geographic region. for example, cyanobacteria blooms can be induced by increases in the phosphorus concentration instead of by a decrease in the n:p ratio (trimbee and prepas, ; sheffer et al., ) . cyanobacteria ( figure ) produce a variety of toxic com pounds known as cyanotoxins. the impacts of cyanotoxins on human health have been of increasing concern, as the impacts of cyanobacterial blooms on water supplies as well as nearshore marine ecosystems have become better understood. outbreaks of poisoning from cyanobacterial blooms can be catastrophic, such as the death of dialysis patients following exposure to inadequately treated water from the tabocas reservoir in brazil (pouria et al., ) or the hospitalization of children and adults who drank cyanobacterially contaminated water impounded by the solomon dam in australia (hawkins et al., ) . cyanobacterial toxins have also been implicated in mortality in wild fisheries and terres trial mammals, and can accumulate in the ecosystem . toxins are classified by how they affect the human body (who, c): hepatotoxins (which affect the liver) are produced by some strains of the cyanobacteria microcystis, anabaena, oscillatoria, nodularia, nostoc, cylindrospermopsis, and umezakia. neurotoxins (which affect the nervous system) are produced by some strains of aphanizomenon and oscilatoria. cyanobacteria from the species cylindroapermopsis raciborski may also produce toxic alkaloids, causing gastrointestinal symptoms or kidney disease in humans. not all cyanobacteria of these species form toxins and it is likely that there are other as-yet unrecognized toxins. further, cyanobacteria produce a wide range of secondary metabolites, among which new cya notoxins continue to be found. besides the recognized effects of the toxins mentioned above, a recent study opened up a discussion about possible new causes of known neurodegenerative illnesses. cyanobacterial strains were found to produce the neurotoxic nonprotein amino acid, β-n-methylamino-l-alanine (bmaa) (cox et al., (cox et al., , . the bmaa is considered a possible causative agent of human motor neuron disease, amyotrophic lateral sclerosis parkinsonism/dementia complex (als/pdc), and has been found to accumulate in brain tissues of patients with progressive neurodegenerative illness (murch et al., ) . this was first detected in humans as a consequence of dietary habits and biomagnification in terrestrial ecosystems in guam (cox et al., ) . however, sometime later, bmaa was detected in aquatic ecosystems, such as in seawater in the area of a trichodesmium bloom and in freshwater and brackish waterbodies (metcalf et al., ) . cox et al. ( ) isolated bmaa in several species of free-living cyanobacteria. possible implications of these findings (cox et al., ; ince and codd, ) are that bmaa of cyanobacterial origin might occur in diverse natural and controlled environments where massive cyanobacterial populations occur. because these environments can include waterbodies used for drinking and recreational use, bmaa in drinking water may contribute to chronic intoxication. this may involve incorporation of the neurotoxin into an endogenous protein reservoir with slow release (murch et al., ) and a possible lag between expo sure and effect of years or decades. since protein-associated bmaa can accumulate within food chains, it is possible that biomagnification of bmaa occurs in marine ecosystems in a way similar to that in terrestrial ecosystems (banack et al., ) . thus, production of bmaa by marine cyanobacteria may represent another route of human exposure to this neuro toxin. cox stated that the bmaa produced by the algae may act as a slow toxin. for these reasons, it would be advisable to monitor bmaa concentrations in drinking waters contami nated by cyanobacterial blooms and in fish and animals that may be ingesting the microbes, even at low environmental densities. this can be particularly relevant in semiarid or arid regions that are highly dependent for their drinking water supply on reservoirs, in which cyanobacteria can regularly occur on a seasonal basis, normally in summer. fertilizer use, widespread cultivation of leguminous crops, and fossil fuel burning have produced strong changes in the global n cycle (galloway et al., ) . more than half of all n fertili zers ever used in earth's history has been applied in the past two decades (howarth et al., ) . moreover, increased n loading in the environment often occurs together with that of phosphorus. in freshwater ecosystems in particular, a com bined increase in both n and p is highly likely to drive eutrophication and associated significant ecological change. mckenzie and townsend ( ) reviewed direct and indirect evidence that changing global nutrient cycles are influencing the dynamics and incidence of pids ( figure ). some of the disease agents discussed earlier in this chapter could react particularly sensitively to increased n and p loads. although generalizations are difficult, the evidence presented by these authors, together with past reviews by lafferty ( ) and townsend et al. ( ) , suggests a trend in which most asso ciations between increased nutrients and disease are positive. a parasite or pathogen may respond to nutrients directly, that is, without requiring an intermediate or vector host; or an inter mediate or vector host may respond to nutrients, mediating the overall disease response. there can be an increase in vector host population density as a result of increased primary productivity, or a decrease in population due to some disturbance related to nutrient addition, in both cases resulting in changes in transmis sion success of the parasite or pathogen (anderson and may, ; dobson, ; arneberg et al., ) . besides the above-mentioned increase in cyanobacterial blooms, increased nutrients could particularly affect ecological processes associated with, for example, malaria, schistomiasis, cholera, and filariasis, leading to a higher occurrence of these diseases. for example, vector-borne pathogens that cause malaria and schistosomiasis involve intermediate hosts (mos quitoes and snails) whose growth and reproduction depend on algae or weed abundance in their respective environments. where n additions cause greater plant growth and/or changes in plant species composition, the density of these intermediate hosts is also likely to be affected, with cascading consequences for the risk of disease. lafferty ( ) reviewed parasitic responses to a suite of environmental changes and found that, in most cases, eutrophi cation caused an increase in parasite abundance. johnson and carpenter ( ) suggested that planorbid snails in nutrient-rich environments grow faster. larger individuals are able to produce significantly more trematode cercariae at a faster rate. the avail able evidence suggests that, in general, nutrient additions should favor trematode development and increase disease risks. in the case of cholera, the bacteria display a strong associa tion with marine plankton, and, therefore, factors that cause increases in plankton primary productivity can also increase the prevalence of v. cholerae. thus, nitrogen-based eutrophication of coastal regions (nrc, ) has been linked to increased cholera risks (epstein, ; colwell and huq, ) , prob ably as an indirect response to plankton dynamics. changes in the n cycle are not globally uniform (galloway et al., ) . the largest changes have occurred in industria lized countries but the focus of global change is shifting to tropical and subtropical countries, where greatest relative changes in the n cycle over the next years are expected (galloway et al., ; dentener, ) . for example, the booming soybean agriculture in brazil in the last decade is leading to rapid increases in regional n deposition and nutrient loading to aquatic ecosystems (martinelli et al., ) . similar increases are occurring throughout tropical and subtropical portions of asia and central america. these same regions harbor the greatest diversity of human pids (guernier et al., ) , including those that currently cause the majority of pidrelated human deaths (who, b) . thus, the next decades will probably bring an increase in the potential for nutrients, especially n, to affect parasitic and infectious diseases in these regions. the changes in temperature and rainfall regime in temperate regions and the changes in land use resultant from them or from market shifts can exacerbate that potential. diseases produced by viral infections can emerge, reemerge, and be transmitted by several different, but often interlinked, mechanisms involving tight interactions between man and domestic and wild animals sharing artificial or natural aquatic systems. table summarizes some viral diseases of worldwide relevance. in the following section, we will focus on avian influenza due its multiple and complex connections with the dynamics and use of wetlands by man and animals. . . . . influenza: human and animal links between artificial and natural wetlands nutrient reduction in wastewaters or surface waters is a prior ity for reducing risks to human health. nevertheless, from the point of aqua-and agriculture, the nutrients contained in wastewater are a valuable resource, in particular in arid and semiarid regions. wastewater form has been used for aquaculture, for example, in duck ponds, in several countries, mainly in asia, for centuries to produce human food. organizations such as who, food and agriculture organization (fao), etc., have developed guidelines for the safe use of wastewater, excreta, and gray water in agriculture and aquaculture in order to provide a basis for the develop ment and implementation of health risk assessment and management approaches, including standards and regula tions, to address hazards associated with human waste-fed aquaculture (edwards, ) . however, wastes and fecally polluted surface water are often used without any pretreat ment or assessment of the presence of pathogens. various hazards are associated with waste-fed aquaculture: excretarelated pathogens (bacteria, helminths, protozoans, and viruses), skin irritants, vectors that transmit pathogens, and toxic chemicals. wastewater systems were also developed independently in india from the s, in china from the s, and in vietnam from the s onward, but they were designed primarily for aquaculture, not to treat wastewater. few engineered waste water-fed aquaculture systems have been developed recently. systems primarily engineered to treat wastewater that incorpo rated aquaculture were developed in germany from the end of the nineteenth century, but only the munich system remains, and only for tertiary wastewater treatment and currently as a bird sanctuary (edwards, ) . in this, the following aspects in the dynamics of bird popu lations are of ecohydrological and biomedical relevance. during migratory movements, birds carry pathogens that can be transmitted between species at breeding, wintering, and stopover places where numerous birds of various species are concentrated, such as wetlands. a study by jourdain et al. ( ) focused on bird migration routes to the camargue in relation to risk of pathogen dispersion into western mediterranean wetlands. they considered two pathogens clo sely associated with wild birds: avian influenza (ai) virus and west nile virus (wnv). the ai viruses have a water-borne transmission, and ducks are their main natural reservoirs (easterday et al., ; alexander, ) ; wnv has a vectorborne transmission, and passerines are believed to play a major role in the amplification cycle (hurlbut, ; malkinson and banet, ) . despite different transmission cycles and ecology, both viruses are known to be carried by reservoir birds during migration and have been associated with emer ging disease transmission risk for humans and domestic animals (rappole et al., ; reed et al., ; olsen et al., ) . environmental conditions, avifauna abundance, and diversity, as well as the interactions among birds from different species and departure sites in stopover wetlands, may be of key importance in terms of virus communication (hudson et al., ) . for water-transmitted pathogens such as ai viruses, the risk of transmission may be associated with the number of ducks congregated on the same water body, particularly in autumn and winter. this crowding of wintering species, in addition to the permanent presence of a transient population of birds using wetlands to stop off during migration, could provide the conditions for the circulation and rapid dissemi nation of ai viruses. for vector-transmitted pathogens such as wnv, transmission possibilities depend both on reservoir bird density and on the dispersion capability and activity periods of the arthropod vectors. the risk for interspecific transmission of disease is particularly problematic when wild and domestic species are involved. ducks are the aquatic birds most likely to come in contact with free-range poultry, especially because the presence of congeners can induce migrating wild ducks to make a stopover (jourdain et al., ) . the study by jourdain et al. ( ) showed that western mediterranean wetlands are a hub for birds from several differ ent origins in central asia, siberia, northern and eastern europe, western africa, and the mediterranean basin. as exam ple for the potential of wetlands for introduction or reemersion of these viruses, they state that wnv dispersion by birds migrat ing from sub-saharan africa might explain why an outbreak occurred in in the camargue, even though the virus had not been observed there since the s. besides migration, breeding ducks in the aquaculture ponds can increase virus circulation between ducks, water, sediment, fish, and man. in a study (markwell and shortridge, ) of the occurrence and persistence of influ enza viruses (hong kong type) within domestic duck communities, the virus was isolated throughout the year from feces or pond water or both, indicating a cycle of water-borne transmission. infection was asymptomatic and virus persistence in the duck community appeared to be dependent upon the continual introduction of ducklings sus ceptible to infection onto virus-contaminated water, since the feces of ducks - days old were generally virus-free despite the ducks' exposure to the virus in pond water. the normal practice of raising ducks of different ages on the same farm, where the water supplies are shared (figure ) , appears to be instrumental in maintaining a large reservoir of influenza viruses in the duck communities. domestic ducks may act as a silent reservoir for the h n ai virus. the concern is greatest in rural areas of affected countries, where traditional free-range ducks, chickens, and wildlife frequently share the same water source. domestic ducks can harbor the virus for long periods and without showing any sign of illness. an altered role for domestic ducks is further supported by evidence that the h n virus circulating in parts of asia has increased its virulence in chick ens and mice (a laboratory model for mammals), and has expanded its host range to include larger mammals (e.g., cats and tigers), not previously considered susceptible to infection (fao, ) . the assessment of respective roles of routes and timing of wild waterbird migration and poultry imports is of utmost importance in order to objectively identify the origin and pos sible evolution of an outbreak in a determined country. for example, the avian flu outbreak in nigeria in may have been caused by the supply of infected live poultry including day-old chicks from different sources, including east asia and turkey, and not by wild waterbirds. this is supported by sam plings of wild waterbirds in african wetlands, in which no evidence of the h n virus was found, indicting that wild birds probably played a relatively minor role in the spread of ai in that region. northward migration of wild birds from africa to europe in the northern spring of did not cause any major outbreaks. nor do wild birds seem to play a role in indonesia, where h n has been present for some years and several cases of human infections have been recorded. however, although not many major outbreaks took place in europe in , there is evidence to suggest that wild birds did play a significant role in spreading the disease on the european continent (aiweb, ). useand scarcityon human health: some examples from aquaculture, megacities, dams, and intensive agriculture aquaculture in or close to wetlands is increasing worldwide. besides being responsible for massive wetland destruction, aqua culture itself faces serious problems, arising from several diseases that can affect shrimp ponds. among the groups of microorgan isms that cause serious losses in shrimp culture, the best known are bacteria because of the devastating economic effects they have on the affected farms. as mentioned before, vibrio organ isms attach themselves preferentially to chitin surfaces, such as in zooplankton and shrimp exoskeletons. bacterial diseases, mainly due to vibrio, have been frequently reported in penaeid shrimp culture systems. at least vibrio species are implicated in disease outbreaks in shrimp, including vibrio harveyi, v. alginolyticus, v. vulnificus, v. parahaemolyticus, and v. cholerae (non-o ) (venkateswara, ; figure ). vibrios can produce different chitinases to degrade various chitin types (svitil et al., ) in marine, estuarine, and figure example of vibrio disease affecting shrimp. in such severe cases, extensively melanized black blisters can be seen on the carapace/ abdomen of the infected animals. copyright national institute of oceanography, dona paula, goa, india, . pond and irrigation canal next to live poultry markets. in http://www.eastwestcenter.org/index.php?id= &print= freshwater environments. further, meibom et al. ( ) found that v. cholerae can acquire new genetic material by natural transformation during growth on chitin. thus, natural compe tence occurring in chitin-attached bacterial communities can act as a powerful driver of v. cholerae evolution, which could be accelerated by environmental events such as high nutrient input, giving rise to copepod blooms. it is still unclear whether growth on a determined type of chitin substrateand the production of the corresponding specific chitinasespromotes the capture of external genetic material by vibrios. this empha sizes the need for biogeochemical characterization of different aquatic microhabitats, such as different types of chitin-contain ing particulate matter, besides living zooplankton, in environmental studies of vibrio diversity and virulence. as organic-matter-rich aquatic environments contain multiple microbial strains and species and high concentrations of bac teriophage and free dna, horizontal gene transfer (hgt) provides the most likely explanation for why vibrionaceae have developed high levels of genomic diversity (meibom et al., ) . interestingly, the cholera toxin gene (ctx) is a phage-mediated mobile genetic element that is transferrable to genetically closely related bacterial strains. in the aquatic environment, vibriophages can regulate seasonal disease out breaks, for example, seasonal cholera epidemics in dhaka were inversely correlated with the prevalence of environmental cho lera phages (faruque et al., ) . the v. cholerae (non-o ) is frequently isolated from sewage, estuarine waters, and seafood in cholera-endemic and noncholera-endemic countries. it has been associated with sporadic episodes of diarrhea worldwide, but has not caused pandemics. interestingly, in , there was the first report of an epidemic of diarrhea caused by v. cholerae non-o that produces heatstable toxin, affecting khmers in a camp in thailand. in con trast to the v. cholerae o isolated from the same camp, in % of the cases, the non-o were resistant to three or more anti biotics (bagchi et al., ) . this calls attention to several important aspects in relation to threats to human health. the fact that the toxin is heat stable could imply a higher diarrhea risk for humans consuming cultured seafood as cooking would not completely eliminate toxin activity. its higher resistance to antibiotics compared with v. cholerae o potentially increases the disease hazards associated with these non-o strains. further, as mentioned previously, the abundance of chitinac eous substrate can favor mutations. thus, just as v. cholerae o evolved from a nonpathogenic to a pandemic-causing form (faruque et al., ) , it cannot be excluded that the high individual density of aquaculturein the same way as megacitiescould be favoring the emergence of new and highly pathogenic vibrio types. in many of the world's cities, water management and sanitation are in crisis and will dramatically worsen with the continuing growth of cities and slums. sewage pollution is the largest and most common type of pollution and one of the most common causes of illnesses. illnesses caused by sewage pollution are estimated to affect the health of more than million people at any one time. contaminated water, inadequate sanitation, and poor hygiene cause over % of all disease in developing countries; diarrhea is the world's second most serious killer of children, but paradoxically in % of cases it could be easily prevented or treated. pollution of water sources by sewage contributes to billion cases of diarrhea in the world each year, killing some . million children under the age of . poor sanitation currently affects . billion people, % of the world's population, who lack access to even the most minimal toilet facilities. the number of people without sanita tion will double to almost billion in , as the world becomes more urbanized (anonymous, ) . some people are moving to cities from the country side every day. at least million people in africa, asia, and latin america now live in squatter settlements without any sanitation whatsoever. the pollution of rivers and groundwater by sewage spreads disease and causes environmental degrada tion. in latin america, as a whole, only % of sewage receives any treatment. in asia, the level of sewage in rivers is times higher than the united nations (un) guidelines. levels of suspended solids in asia's rivers almost quadrupled since the late s. every minute . million liters of raw sewage are dumped into the ganges river (vidal, ) . most megacities are in asia. dhaka's population rose from in the early s to ∼ million today, including the metropolitan area, and will probably reach million in . in the same time, kolkata's population grew from to million; including the metropolitan region, its population is million and is predicted to reach million in . pakistan experienced one of the highest growth rates of popu lation worldwide: it quadrupled in only years to over million in (prb, ) . according to the world bank, karachi is one of the fastest-growing megacities of the world and is expected to rank seventh by the year (kamal, ) . the country faces a serious situation in terms of water availability, depletion, and pollution of its water bodies and irrigation systems as well as a severe degradation of its coastal ecosystems. this complex and multifaceted setting will be clo sely analyzed in the following sections. it has been reported that at least of the towns of the city are supplied with water unfit for human consumption, in most cases infected with escherichia coli. only around % of the total sewerage generated by karachi at present is treated. the e. coli, found in human feces, and other bacteria found in drinking water could cause life-threatening diseases, including diarrhea and cholera. the bulk of the drinking water concerned is taken from the indus river. bacteria easily enter the drinking water as the pipelines are rusted and leaking (irin, ) . lowintensity seismic activity, though normally not felt by people, probably further damages worn-out pipelines. additionally, due to the water shortages, the pipelines remain empty for a considerable amount of time daily, during which time they develop negative pressure and absorb moisture and sewage that has leaked from the nearby, similarly worn-out sewers. additionally, after rains, rainwater mixes with sewage and gar bage can enter the pipelines through the leaks to contaminate the drinking water supply, making people vulnerable to numer ous health hazards (hasan, ) . karachi is situated in a desert. however, in a study by sheikh et al. ( ) the analysis of microbiological data for the period - showed the permanent presence of cholerawith seasonal periodicityin karachi. cholera cases peak each year between may and august in both epidemic and nonepidemic years. both v. cholera o and o serogroups were involved in the outbreaks in and ; o disappeared in . the role of rain in disease seasonality and incidence is not clear. rainfall is scarce and sporadic, usually only between june and august. in , the city could have been flooded, with wide spread overflowing sewers, whereas, in , there was virtually no rain at all. cholera appears each year before the rains, and epidemic years in the - data set appear to have occurred independently of rainfall. like most enteric diseases in an endemic setting, cholera in karachi is a disease of young children. the mean age of patients with acute cholera in karachi closely resembles that in the rest of the indian subcontinent where social conditions are com parable. despite this association with poverty, % of the patients in the study were admitted to expensive private rooms in good hospitals. this shows that, in a city with a sanitary infrastructure like that of karachi, personal wealth affords no protection (sheikh et al.. ) . visitors and tourists are also at risk. in the same context, despite the common perception that bottled water was safe and pure, microbiologi cal tests showed that about % of samples of water supplied in many bottled brands tested all over the city was unfit for human consumption (hasan, ) . according to the same above-mentioned study, in , the new strain of v. cholerae, serogroup o , established itself in karachi and during and karachi experienced over lapping, but distinct epidemics of both strains. the serogroup o never wholly replaced serogroup o , and by it had disappeared. at that time, it was still unclear whether the dis appearance would be permanent, or whether o would reemerge in subsequent epidemic years. karachi is in a semi desert area, and the strain may not be able to maintain itself outside the human host (sheikh et al., ) . this strain has also shown diminished ability to maintain its epidemic potential in bangladesh, and it has been suggested that one reason for this may be that it is less able to persist long term in the aquatic environment (faruque et al., ) . however, years later, in july and june , cholera outbreaks were detected by a diarrhea surveillance system in a fisher village near karachi (siddiqui et al., ) . the first outbreak was caused by v. cholerae o and the second one by serotype o . it would be erroneous to conclude that, because of the relatively small number of persons affected, these cases are not relevant in terms of public health. on the contrary, they should be considered extremely valuable indicators of environmental change, especially of aquatic systems, which provide an early warning of possible future trends for policymakers and sanitar ians. water source was a risk factor only in the first outbreak: a reservoir in the village containing brackish water was only used for washing utensils and clothes and for bathing. only illness caused by v. cholerae o was associated with the use of reservoir water, while o cases were not. washing the clothes of infected persons may have introduced the pathogen into the reservoir. this implies that v. cholerae o was probably able to survive for a time outside the body and in water long enough to infect other people. this may be partly because the water was salty and that v. cholerae is a salt-loving bacterium. in summary, fecal pollution, increased nutrients, turbidity, and sodium content create favorable conditions for the propagation of v. cholerae in pakistan's coastal zone, megacities, and irrigation systems. when the increasing pollu tantchemical and microbiologicalload reaches the coastal region, it encounters a disturbed wetland ecosystem (discussed in section . . . . ), where vibrios could potentially multi ply and mutate to new pathogenic types. a close regular surveillance of vibrios at basin level, including in the coastal region, rivers, and channels, and in humans is essential in this region, which has the potential to become an epidemic center. further, restoration of riparian forests and wetlands habitat should be a priority to avoid further habitat loss, and potential host shifts, although salinization would probably preclude the reinstallation of the same species existent before dam building and population explosion. the hoogly river is the most important source for the water supply of kolkata. through an agreement with bangladesh, only a determined amount of water from the ganges can be diverted into the hooghly river during the dry season. although this does not increase the amount of pollution in the river, it does increase its concentration. during the mon soon, rubbish and feces are washed out from the city into the ground and into the river (karthe, ) . around % of the water supply is lost through leakages in the obsolete distribu tion network, reservoirs, and public water tap connections. however, the disposal of sewage is an even greater threat to human health. reduced capacity of the inadequate sewer net work, aggravated by obstruction caused by mud or garbage, as well as flooding during the monsoon produces pollution of surface and groundwater with enteric bacteria. as in karachi, interruptions or shortages in the water supply produce a negative pressure in the pipelines, which then absorb polluted water that had leaked from sewers and the surface, especially during the monsoon season. before and after the monsoon, water quality increases, that is, bacterial load decreases. following several cholera epidemics, the chlor ination of unfiltered water supply began in . this reduced cholera incidence, but supply of potable water to many parts of the city is still insufficient. people in shanties frequently get untreated water from hydrants, or from the river (hensgens, ) . cholera seasonality in kolkata is further discussed in section . . . . . the situation in london years ago resembled current conditions in many megacities in developing countries. by , half of the population of britain was living in townsthe first society in human history to do so. over the previous years, britain's population had risen at an unpre cedented rate. large towns were desperately unhealthy, with death from sickness at a level not seen since the black death (daunton, ) . london had a large scavenger class living off the refuse of the citya group so numerous that it could have formed the fifth largest city in england. new epidemics affected the citiescholera and typhoid were carried by polluted water, typhus was spread by lice, and 'summer diarrhea' was caused by swarms of flies feeding on horse manure and human waste. london suffered from recurring epidemics of cholera and in - more than londoners were killed by the disease (johnson, ) . the frequent occurrence of cholera in london gave impetus to legislation, enabling the metropolitan board to begin work on sewers and street improvements. by most of london was connected to a sewer network brilliantly devised by joseph bazalgette (bbc, ) . the flow of foul water from old sewers and underground rivers was intercepted and diverted along new, low-level sewers, built behind embankments on the river front and taken to new treatment works. by , both the albert and the victoria embankments had been opened. the victoria embankment protected bazalgette's low-level sewer from the hydraulic pressure from the thames estuary. the chelsea embankment was completed in . the public health act of required local authorities to implement building regulations, or bylaws, which insisted that each house should be self-contained, with its own sanitation and water. this change in the design of housing complemented the public investment in sewers and water supply. cholera never reappeared in london after that. london was the largest city on the planet in , but now it is on the small side, in comparison to, for example, mexico city, são paulo, or mumbai. massive shantytowns have exploded at the margins of today's megacities. in such places, the water-borne diseasesincluding cholerathat plagued victorian london are still widespread, thanks to insufficient public health and sanitation resources. worldwide, up to a billion people live in shantytowns and according to some projections this will increase to a quarter of the world's popula tion by (johnson, ) . despite enormous progress in the molecular biology of v. cholera, still little is known about basic forces, such as spatial biogeochemical gradients, seasonal rainfall variations, or cyclones, driving its abundance, diversity, and virulence in the basins of rivers and estuaries of the indian subcontinent. the most recent detailed studies on seasonal variations of estuarine salinity and related urban cholera incidence are from the s (chatterjee and gupta, ) . these compare the river systems kolkata-hoogly (a main branch of the ganges) and london-thames (data from the nineteenth century), and are discussed in miller et al. ( ) . an important aspect of the s data is that minute but clearly delimited salinity oscillations (e.g., . - . and . - ppt) in the hoogly river tightly correlate with cholera incidence in kolkata. several authors reported significantly higher salinity ranges for the growth and persistence of v. cholerae in the environ ment, for example, . - ppt (miller et al., ) or - ppt (singleton et al., ; louis et al., ; randa et al., ) . salinities < ppt were considered suboptimal (miller et al., ) . however, kolkata's data clearly indicate cholera out breaks at much lower salinities. such oscillations could also be a proxy of other processes occurring at basin level, which were responsible for triggering the cholera outbreaks. vibrio survival at low salinities can be facilitated by adsorption onto algae, zooplankton, or by high nutrient concentrations. unfortunately, this information mostly originated from stag nant water bodies or short-term investigations (e.g., islam et al., , and references therein) , but no studies are available about the seasonality of hydrology, biogeochemistry, and vibrio dynamics in flowing waters of the large rivers in this region through which vibrios most likely spread from the coastal zone toward inland habitats. since the s, seven large dams for irrigation purposes have been constructed in india. the farakka barrage, complete in , diverts the ganges river water into the hooghly river during the dry season to flush out the accumulating silt in the port of calcutta. it cuts off bangladesh's water supply, elevating salinity, and has affected fisheries, caused desertification, and hindered navigation, and poses a threat to water quality and public health (wolf, ) . there is evidence of changes in cholera seasonality due to hydrologic disturbances. before the s, the peak cholera season in dhaka was november-february; now it is september-november. in kolkata, season ality has changed twice since the mid- s (niced, . these shifts may be related to changes in salinity, particle load, and associated estuarine biogeochemistry due to, for example, the construction of the farakka barrage on the river ganges (mirza, ) or increased melting of himalayan glaciers (unep, ) . more that years ago, miller et al. ( ) postulated that dam construction in india could influence vibrio dynamics by salt intrusion. these aspects deserve further investigation; apart from the s data, there are no other published data series systematically relating these parameters in the rivers of the region with cholera incidence. this informa tion is essential in order to evaluate the transboundary effects of dam construction and water management. dam construc tion in india has reduced riverine discharge in bangladesh, inducing desertification in its northern sector and facilitating salt intrusion into its estuaries, particularly in the southwestern region (wolf, ; adel, ) . this resulted in changes in land use from rice cultivation to shrimp farming in the southern bay of bengal (gebauer, ) (discussed in section . . . ). salinization of inland water bodies can facilitate the spreading of the halophilic vibrio organisms and affect drinking water availability. on the other hand, global warming increases glacier melting and associated riverine runoff. both factors, in a frame of increasing intensity and frequency of cyclones and flooding events, create an extremely complex situation in the coastal zone that could result in shifts in seasonal cholera patterns. despite the different overall climatic setting of pakistan as com pared to india and bangladesh, increasing water needs in pakistan are leading to a similar situation in the coastal zone, including increased cholera incidence. the common factors are the halophilic character of vibrio cholerae and other vibrios and the salinization of estuaries and inland waters. water needs for irrigation of desertic and semidesertic areas, as well as for drinking water supplies, mainly for karachi and islamabad, have led to the construction of several dams along the indus river. within the sindh province, there are three major barrages on the indus-guddu, sukkur, and kotri. severe reduction of water flow below the kotri barrage started affecting environments in the area from s onward, with the following consequences in the river basin: ( ) drying up and death of riparian forests, figure the indus delta faces major degradation threats, whose major cause is the reduction in the flow of freshwater from the indus river. as the delta dries up and the mangrove forests decline, the sea is slowly sweeping in. which occurred soon after ; ( ) reduction of the area under fruit and vegetable crops; ( ) destruction of natural pastures causing a reduction in animal populations; and ( ) desertifica tion leading to a shifting of human settlements. in the coastal region, intrusion of seawater in the river bed to a distance of km upstream from the shore, with percolation of saline water from the riverine areas into groundwater of adjoining irrigated areas, has turned shallow water lenses brackish ( figure ) . another reason for accelerated salinization in the indus river is the saline water discharges from the salinity control and reclamation program in the north-west frontier province, punjab, and india (hasan, ) . along the coast, the increase in salinity of seawater along the whole coastline of sindh has resulted in damage to man groves, colonization by other halophilic species, and the abandonment of ∼ ha of land reserved for shrimp farming by the government of sindh. sea shrimp can survive within salinity range of - of water, but in the space of years , salinity rose beyond the tolerance limit. water salinity in sea creeks and estuaries increased from ∼ to over ppt, making estuaries inhabitable for some shrimp and other species of commercial interest. in conditions of low salinity, shrimp farming could have been established all along km of seacoast. sea fish and prawn catch has declined considerably and severe erosion due to reduced sediment load occurs along the coast. in the coastal region, the livelihood of fishing communities and the fishing industry as a whole depends on ecosystem integrity. however, this has already been devastated by reclamation of former marine areas, and of mangrove marshes and mudflats. it desperately needs to be protected, for no city that destroys the ecology of the region where it is situated can be sustainable. the south asian tsunami gave ample proof of this and so did the flooding of karachi, much of which is the result of reclama tion from mangrove marshes, creeks, and natural drainage channels for elite real estate (hasan, ) . in addition to the deeply disturbed aquatic ecosystems described above, only % of cities with a population of over have wastewater-treatment facilities. of the wastewater generated daily, % is used in agriculture and % is disposed of into rivers or the arabian sea (iwmi, ) . directly or indirectly, % of the people of sindh, in rural or urban regions, drink water from the indus. salt content at kotri reaches . ppt in winter months, and supplies to karachi range from . to . ppt (panhwar, ) . the combina tion of a riverine environment with increasing salinity, growing populations, lack of sanitation, input of untreated sewage, drinking water contaminated with enteric bacteria, and rela tively high sodium content is very similar to that in london during the cholera epidemics in the nineteenth century (see section . . . . ). recently, a cholera outbreak in mirpur khas, a district of sindh province, has been reported (anonymous, ) . scientists at the university of health sciences found out that around % of the patients were affected by cholera instead, as was previously thought, by gastroenteritis. the authorities have been requested to pay particular attention to this outbreak of cholera, which could spread to the other areas of the province. mirpur khas has a population of ∼ , has successful agriculture, and is con nected to the indus via irrigation canals. the irrigation system of pakistan has been considered "one of the largest contagious systems of the world" by gachal et al. ( ) . the reservoir formed by the three gorges dam (tgd) is the largest in the world at over km in length. studies about the impact of parasite dynamics have been restricted to medically important species such as schistosoma japonica (zheng et al., ) . these studies have focused on the distribution of the intermediate host oncomelania hupensis within the tgd area and associated downstream water bodies. before construction of the dam, neither o. hupensis nor s. japonica occurred in the reservoir region. however, it is widely predicted that the dam will lead to the introduction of s. japonica into the tgd region, while downstream both positive and negative effects on schistosomiasis transmission will occur (zheng et al., ) . nevertheless, a survey of the recently filled reservoir (jobin, ) concluded that because of the very steep shoreline along most of its length and subsequent narrow photic zone, which provides only a limited area for the growth of plants, there was little chance of o. hupensis, principally a marshland snail, becoming established. however, these unfavorable topo graphic conditions do not preclude other mollusk species colonizing the shoreline (morley, ) . for example, former gravel quarries converted into lakes have a steep-sided profile with a narrow discontinuous zone of plant life but support a diverse molluskan-trematode community (adam and lewis, ; morley et al., ) . river blindness is an important parasitic disease around tropi cal dams in africa, along the red sea in arabia, in central america, and in parts of south america. the rapids of the upper nile river used to be a classic focus of this blinding parasite, spread by the bite of a species of blackfly, which breeds in white-water habitats and on dam spillways. in uganda (jobin, ) , the history of river blindness can be traced to the owen falls dam in the upper nile river. this dam flooded out owen falls and also ripon falls near the outlet of lake victoria. the history of the impact from the owen falls dam on onchocerciasis over the last years shows the importance of optimal water current velocity regulation for the avoidance of reduction of this and other water-borne dis eases such as schistosomiasis. prior to construction of the owen falls dam (figure ), river blindness was endemic among the buganda people, downstream along the nile. in , the prevalence of the parasite was %. to protect the workers, weekly applications of ddt were made at the outlet of lake victoria, treating the entire flow during the construction phase, and eliminating the blackflies for at least km downstream. after dam completion in and discontinuation of ddt applications, the black flies did not return to their former habitats downstream of the dam. by , the prevalence of the parasite had decreased to . % among the populations along the river. the fact that blackfly populations did not return to former levels indicates that there must have been a change in the basic habitat condi tions in the river. the dam had two major hydraulic effects on the river: one effect of turbine operation is a reduction of velocities downstream. the preferred range of water velocity for breeding of the east african species of blackfly involved in river blindness is between . and m s − . at present, the mean velocities downstream of owen falls dam are between . and . m s − , slightly less than the required velocities. prior to dam construction, the mean velocity was roughly twice the present, and thus highly suitable for blackfly breeding. yet, the major ecohydrologic effect of the dam reservoir was the complete submergence of owen falls and ripon falls upstream of the dam. these falls were preferential sites for blackfly breeding, and their permanent submergence eliminated the breeding completely. in west africa, dam construction has led to a significant switch in dominant water-borne diseases. the volta river basin extends over six west african countries ( % in burkina faso, % in ghana, and % in togo, benin, cote d'ivoire, and mali) and covers an area of about km . the volta lake is the largest man-made lake in the world, created after the construction of the akosombo dam in . the primary purpose of the project was to supply cheap electricity to smelt aluminum, other significant uses being transportation, fishery, water supply (commercial and domestic purposes), tourism, and irrigation. construction of the volta lake led to the resettlement of about people from several hundreds of villages. in the other riparian countries of the basin, small and larges dams have been built by governments, nongovernmental organizations (ngos), and local people to secure food produc tion after the severe droughts that occurred in the s and s. in the nakambe sub-basin (burkina faso) alone, more that small dams have been built, mostly during that period (barry et al., ) . there have been serious health issues associated with the volta lake, in particular with two major diseases: schistosomia sis and river blindness. before the creation of the volta lake, schistosomiasis was endemic in ghana; but endemicity was low along the volta river. according to an epidemiological survey done in - before the lake was formed, infection rates of schistosomiasis in the area had been - %, mostly affecting children. in the asukwakwaw area, north of the akosombo dam, the prevalence of onchocerciasis is now almost %. principal public health impacts of the formation of the lake have included reduced prevalence of river blindness, but increased incidence of urinary schistosomiasis and a mas sive increase in malaria, as discussed in the following paragraphs. the dam virtually halted the rate of flow in the volta river, increasing stagnant water conditions and consequently creating ideal breeding grounds for carriers of water-borne diseases. in the period following the construction of the dam at akosombo, there has been a steady decline in agricultural productivity along the lake and the associated tributaries (gyau-boakye, ). the land surrounding lake volta is not nearly as fertile as the for merly cultivated land residing underneath the lake, and intensive agricultural activity has quickly exhausted the already inade quate soils. without the periodic floodings that brought nutrients to the soil, before the natural river flow was halted by the dam, upstream agricultural systems are also losing soil ferti lity (van de giesen et al., ) . the growth of commercially intensive agriculture has produced a rise in fertilizer runoff into the river. this, along with runoff from nearby cattle stocks and sewage pollution, has caused eutrophication of the river waters (gyau-boakye, ) . this nutrient enrichment, in combination with the low water movement, has allowed for the invasion of aquatic weeds (cerratophyllum) (fobil and attaquayefio, ) . these weeds, associated with the aquatic snail, the 'intermediate host', together with mass migration into the fishing commu nities from regions in which the disease was endemic, have led to a great increase in the prevalence of schistosomiasis in many localities around the lake. the presence of aquatic weeds along the lake and within tributaries has resulted in even greater devastation to local human health as they provide an excellent habitat not only for snails but also for mosquitoes (gyau-boakye, ) . before the construction of the akosombo and kpong dams, malaria was not much of a problem along the swift-flowing volta river, but, when it became a stagnant lake, it became a greater public health problem in lakeside villages. by , urinary schisto somiasis had increased to become the most prevalent disease in the area, affecting some % of lakeside residents (gitlitz, ) and reaching a prevalence rate of % among children in certain localities. the problem of schistosomiasis in the lake basin must be seen as embracing both the lake and the volta delta. the migratory habits of the fishermen ensure the spread of the disease from endemic areas to other areas. in particular, resettlement villages have showed an increase in disease pre valence since the establishment of lake volta, and a village's likelihood of infection corresponds to its proximity to the lake. previously, the population in the basin generally lived away from the main watercourses because of the threat from water borne diseases. children and fishermen have been especially hard hit by this rise of disease prevalence (zakhary, ) . additionally, the degradation of aquatic habitat has resulted in the decline of shrimp and clam populations. the physical health of local communities has declined as a result of this loss of shellfish populations, as they provided an essential source of dietary protein (fobil and attaquayefio, ) . conversely, while leading to a dramatic increase in schisto somiasis, the lake has flooded out the riparian forests which constituted a breeding place for a species of tsetse fly, glossina spp. from the palpalis group, the vector of protozoan trypanosoma brucei gambiense, which causes the western african trypanosomiases (sleeping sickness) in people. the lake has also inundated and eliminated the major breeding sites of the onchocerciasis blackfly in rapidly flowing streams and rivers north of the akosombo dam. the construction of the second dam at kpong also eliminated the breeding sites downstream of akosombo and therefore stopped the transmis sion of river blindness in the vicinity. the main benefit to health because of the construction of the akosombo dam in and the kpong dam in is undoubtedly the reduc tion of the incidence of onchocerciasis in the volta basin. about fishermen living mostly in isolated villages around the lake were exposed to the riverine disease and did not have access to health facilities (jobin, ) . increased global demand for biofuels has incentivized sugar cane plantation in brazil, giving rise to several social and environmental modifications (martinelli and filoso, ) . the widespread occurrence of generalist animal species in sugarcane areas has been associated with public health pro blems. for instance, the population increase of the semiaquatic rodent, capybara (hydrochoerus hydrochaeris), in the piracicaba river basin has led to the spread of brazilian spotted fever (bsf) (labruna et al., ) . the bsf is the most important tick-borne disease in brazil and is caused by the bacterium, rickettsia rickettsii, and transmitted by the tick, amblyomma cajennense, its main vector, and capybaras serve as host for the ticks (estrada et al., ) . r. rickettsii infections can cause a wide range of clinical manifestations, ranging from asympto matic or mild febrile illness to overwhelming and fatal disease. failure in diagnosis and delayed therapy have contributed to hidden mortality, frequently a result of atypical fulminant forms of the disease (gonçalves da costa et al., ) and physician's lack of knowledge about the disease, which is exa cerbated by the difficulty of adequate confirmatory laboratory tests during its acute phase. mortality can reach % of the infection cases. the bsf has been known in brazil since . during the period between and the s there was a marked drop in the number of reported cases of bsf in brazil, as well as in the united states (angerami et al., ) . however, since the s an apparent reemergence of the disease has been observed with an increase in the number of reported cases in the southeast of brazil (angerami et al., ) . rickettsial diseases have been considered emerging zoo noses worldwide (raoult and roux, ) and should no longer be classified as rare diseases in brazil. despite the still moderate number of bsf cases, its increasing trend, together with high mortality rates, reflects and calls atten tion to deep changes in land use and habitat structure in brazil. increased fertilizer use, pollution, and soil erosion have caused deterioration of aquatic systems. as colluvium sediments are transported downhill across the landscape from sugarcane fields, they are deposited onto wetlands, and into small streams, rivers, and reservoirs. deposition affects water quality, and ecosystem biodiversity (politano and pissarra, ) and functions. high rates of n export into rivers draining watersheds heavily culti vated with sugarcane in brazil, such as the piracicaba and mogi river basins, have been reported (filoso et al., ) . the indus trial processing of sugarcane for production of sugar and ethanol is another source of pollution for aquatic systems with poten tially harmful effects for human health. waste products (vinasse) are rich in organic matter, and increase the biochemical oxygen demand (bod) of waters receiving these effluents, often causing anoxia (ballester et al., ) . with the boom of ethanol pro duction in brazil in the early s, new legislation was enacted to ban the direct discharge of vinasse into surface waters. since then, nutrient and carbon-rich vinasse has been mixed with wastewater from washing sugarcane and is recycled back to sugarcane fields as organic fertilizer (gunkel et al., ) , although this practice is still far from generalized. as a conse quence, high nutrient concentrations in these effluents also contribute to the problem by enhancing algal blooms and pro moting eutrophication of surface waters (matsumura-tundisi and tundisi, ) . the increase and dispersion of the capybara population and the associated health risk is most likely due to a strong anthro pogenic habitat modification caused by extensive monoculture. paradoxically, this situation could be aggravated by current efforts to restore aquatic ecosystems. typical capybara habitat is com posed of two main components: water and a patch of forest or woodland. in são paulo state, capybaras and ticks share a habitat component, the riparian vegetation called gallery forest. these forests form as corridors along rivers or wetlands and project into landscapes that are otherwise only sparsely wooded, such as savannas, grasslands, or deserts. the boundary between gallery forest and the surrounding woodland or grassland is usually very abrupt, with the ecotone being only a few meters wide. in são paulo state, capybaras shelter in the gallery forest and feed in the sugarcane fields adjacent to the ecotone ( figure ) . thus, the abundant food and lack of predators in this new habitat have led to a strong population increase (labruna, ). in the s, the capybara was considered in danger of extinction in são paulo state, where population can reach densities times higher than in natural environments in some areas, such as the extended wetlands of pantanal (verdade and ferraz, ) . this offers optimum conditions for the increase in the tick and rickettsia population. further, the capybara is a protected species and is gradually adapting to aquatic urban habitats with a larger spectrum of possible vertebrate hosts for ticks ( figure ). capybaras have been observed in the outskirts of são paulo city along the highly polluted pinheiros river (labruna, ) , which flows through the fourth largest metropolitan area worldwide, with ∼ million inhabitants. presently, a substantial cleanup pro gram for this river is underway and an increase of secondary vegetation and fragmented, regenerated systems is expected. with this, a further expansion of capybaras, ticks, and bsf toward anthropogenically modified habitats, including urban and suburban areas close to water sources such as rivers and lakes, is likely. in addition, it has recently been reported (meireles et al., ) that capybaras in the são paulo state were infected by cryptosporidium parvum, which is a protozoan pathogen that causes a diarrheal illness called cryptosporidiosis, an acute short-term infection that can become severe and chronic in children and immunocompromised individuals. despite not being identified until (meisel et al., ; nime et al., ) , it is one of the most common water-borne diseases and is found worldwide, being spread by direct ingestion of con taminated water or food and through recreational water activities. the finding of zoonotic c. parvum infection in this figure aerial photo of a sugarcane plantation and a small water body with highly fragmented remains of riparian forest, brazil. photo credit: geraldo arruda, jr. semiaquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies in brazil may be contaminated with cryptosporidium oocysts from wildlife. cryptosporidiosis is the most significant water-borne dis ease associated with the public water supply in western europe. when contamination occurs, it has the potential to infect very large numbers of people. some notable outbreaks of cryptos poridiosis have been associated with heavy rainfall events. in this chapter and elsewhere (despommier et al., and references therein) , there are indications that the boundaries between ecological systems play a role in some of the most important emerging infectious diseases, with a correspondence between ecotonal processes and the ecological and evolution ary processes responsible for zoonotic and vector-borne infections. terrestrial ecotones include forest-edge habitats, fragmented forest landscapes, and forest-grassland interfaces. terrestrial-aquatic ecotones are found in riparian habitats, riverine landscapes, freshwater and estuarine wetlands, and in the coastal zone. anthropogenic ecotones can include crop land/pasture-natural habitat and settlement-natural habitat, and a combination of these. processes in ecotones can contribute to the shifts or changes in hosts, vectors, or pathogens that produce disease emergence. these dynamics are associated with changes in land cover/use and with the changing nature of the land-water interface. anthropogenic influences can intensify ecotonal processes by increasing their geographic extent and overlap. various ecotone features can contribute to disease emergence. animals congre gate in ecotones. populations of species that normally are members of distinct ecological communities from different habitats or ecosystems overlap in ecotones, facilitating patho gen spillover. host-vector hyperabundance increases the potential for pathogens to achieve critical threshold density. enhanced dispersal conditions facilitate dispersal at a higher rate or over longer distances, along linear habitats defined by habitat edges, such as riverine or gallery forest, and flowing water in streams or rivers themselves. cropland-forest-river transitions appear particularly relevant as sensitive indicators of change in this context. for example, the transition between fragmented riparian habitats such as degraded gallery forests and, for example, sugarcane fields seems to be a priority sector for the control of rickettsia-related diseases since capybaras must cross the ecotone to feed on sugarcane, as described else where in this article. further, nutrient pollution, degradation of riparian habitat, and the loss of ecological functions involving assimilation of nutrients and pathogens, combined with high concentrations of domestic fowl and their waste, are commonly associated with human settlement/aquatic-terrestrial ecotones. the emer gence of ai involved the mixing of three different communities: wild migratory waterfowl (wetlands), wild local birds, and domestic fowl (ponds), and, later, also pigs. like influenza, the emergence of japanese encephalitis has involved transmis sion in the spatial area of overlap of human settlements, agriculture, and natural habitat (despommier et al., ) . while waterbirds and wetland habitat are implicated in influ enza, nonaquatic wild birds and irrigation systems provide the vector habitat for encephalitis. the intensification and expan sion of irrigated rice production systems in southeast asia over the past years have made an important contribution to the spread of this disease, which is produced by a mosquito-borne virus (see table ). the flooding of the fields at the start of each cropping cycle leads to a sizable increase in the mosquito population. domestic pigs and wild birds are reservoirs of the virus and transmission to humans may cause severe symptoms. japanese encephalitis is a leading cause of viral encephalitis in asia with - clinical cases reported annually (who, d) . in general, research in ecotoneparticularly terrestrialaquatic dynamicscan provide vital information about changes in climate, river hydrology, sea level (cohen and lara, ) , and land use (lara et al., ) . a strengthened integration of ecological and biomedical monitoring is essen tial for successfully restoring ecological functions and enhancing environmental and human health. globally, the surveillance of locally and regionally relevant ecotones could provide evidence of disease emergence related to environmen tal change. in this context, restoration of lost ecological functions, such as nutrient sequestration by wetland creation or regeneration of riparian vegetation, must be accompanied by careful monitoring of other changes in the surrounding land scape and in the connectivity between basin processes and land use. created or regenerated aquatic systemsindependently of their purposewill increment existing ecotonal processes or generate new ones. thus, surveillance of the created/recovered/ enhanced system ecological functions should include at least those disease agents (host, vectors, or pathogens) , which according to the present knowledge would have a higher prob ability of proliferation under modified or changing conditions (e.g., mosquito larvae in temperate wetlands and snails in tropical regions). floods and droughts will intensify with climate change and affect health through the spread of disease resulting from habi tat modification, with high risk of rapid increase in diarrheal and other diseases (lipp et al., ; ipcc, ) . there are many pathways through which hydrologically relevant events can affect health; notably when a river or stream bursts its banks producing changes in mosquito abundance (malaria, and dengue), or contamination of surface water with human or animal waste such as, for example, rodent urine (leptospiro sis). flooding may become more intense with climate change and can result in the spread of disease. conversely, droughts can produce changes in vector abundance if, for example, a vector breeds in ponds left in dried-up riverbeds (noji, ; menne et al., ) . coastal ecosystems and their basins are rapidly changing due to anthropogenic pressure and global warming, also inducing changes in patterns of resource use (lara et al., ) . integrative, comparative approaches are needed for the understanding of functional links between basin structure; morphology of different estuaries, marshes, and mangroves; flooding and biogeochemical regimes (lara and cohen, ) ; pathogen life cycles; and disease incidence (wolanski et al., ; lara et al., ) . for improved prediction of the dispersal of inundation waters, formation of drought ponds, or preventive identifica tion of vulnerable locations or sectors that could be used as drainage areas, it is crucial to have a detailed knowledge of the regional and local topography. the elaboration of highresolution topographic models (dem) of basins in connection with hydrology, meteorology, and biogeochemistry data will also allow the assessment of vulnerability descriptors such as soil moisture potential, salinity, or organic matter content, which can be crucial state parameters for the development of microorganisms and/or disease vectors. however, precisely in tropical coastal areas, where the impact of climate change on vector-transmitted diseases is of high concern, there is frequently a lack of topographic informa tion with an adequate resolution for low-lying sectors. in vulnerable regions, the combination of risks to both food and water can exacerbate the impact of even minor weather extremes (floods and droughts) on the households affected (webb and iskandarani, ) . a methodological approach including wetland basin microtopography and its relation with inundation dynamics and estuarine biogeochemistry is necessary for vulnerability assess ment and risk management. the use of geographic information systems (giss) provides an excellent basis for network coopera tion at the interfaces between environmental and biomedical research, adding a critical componenthuman healthto coastal management research. this is a major concern for the who, which also has set a priority on the link between gis and disease surveillance (who, ) . through the joint who/united nations children's fund (unicef) program health map, specific gis software was developed for that purpose, combining a standar dized geographic database, a data manager, and a mapping interface. however, although these concerns are closely linked to the subject matter of ecohydrology, they are not usually included in interdisciplinary research projects dealing with, for example, coastal wetlands. clearly, the only way to concretely reduce vulnerability is to ensure that infrastructure is in place for the removal of solid waste and wastewater and the supply of potable water. no sanitation technology is safe when covered by floodwaters, as fecal matter mixes with floodwaters and is spread wherever the floodwaters run (lara et al., ) . consideration should also be given to the deterioration of groundwater quality caused by salinity intrusion due to climate change and rising sea levels (e.g., sherif and singh, ) . thus, as stated in section . . . , such health issues clearly require a basin approach, that is, considering basins as a natural unit of territorial management. however, a usual shortcoming of gis-derived vulnerability studies is that data sources from official institutions are most frequently based on municipalities or counties as an administrative unit, whose limits do not necessarily coincide with basin boundaries. thus, this vision of the relationship between climate and sealevel change and effect on human health converges with zalewski's ( ) statement that: as a consequence, the issue of water quality at the basin scale cannot be resolved without a profound understanding of the effects of hydrology on biotic processes and of biota on hydrology. the frame work for developing the principles of ecohydrology is logically the water basin scale. (zalewski, : ) creation of wetlands for nutrient sequestration from surface waters requires the inclusion of measures for control of locally major and regionally relevant disease vectors such as snails or mosquitoes. the latter are relevant for disease transmission in several climatic zones besides the tropics, and global warming is widening their habitats with severe consequences for human health (e.g., the dengue outbreak in argentina in ) and will therefore be treated with some detail. early methods of managing salt-marsh mosquitoes have primarily focused on maximizing the reduction of mosquito populations, with mini mizing environmental impact as a secondary consideration. however, in the last few decades, there have been attempts to apply diverse water management models to marsh systems, especially in terms of vector control and habitat modification (dale and hulsman, ; wolfe, ) using programs with minimal environmental impacts. the success of these programs requires a thorough knowledge of mosquito developmental conditions, as well as potential impacts on adjoining ecosys tems. a deep knowledge of the local microtopography and tidal regimes is critical. marsh drainage and hydrological linkage to the tidal source are essential in the determination of what type of wetland occurs where, and for the development of appro priate wetland management measures. elevation of a few centimeters is more critical in the coastal wetlands than that of hundreds of meters in the mountains. in this section we cover not only well-established methods for mosquito control, but a series of successful methods for snail control derived from good agriculture practice, basically from techniques for rice cultivation. ecohydrology can contri bute to and learn from these experiences. water management, molluskicides, and chemotherapy have been the main instruments for preventing or treating schisto somiasis. biological control of snails through predators such as ducks, fish, turtles, crustaceans, water rats, leeches, and aquatic insects have been also used, as yet with very limited success. an interesting lesson on the potential for improved water management to reduce vector proliferation from the s can be drawn from the experience in the baluchi irrigation scheme in then tanganyika (sturrock, ) . this system was remark ably clear of snails, although several species occurred in small numbers. several factors may account for this. first, the water flow in the canal system was very rapid when it was in use, but the canals were completely dried out in the dry season. second, silt and vegetation were dug out of the canals twice a year. third, a complex system of rice husbandry was used in which the rice fields were ploughed, manured, and subjected to a program of alternate drying and flooding. consequently, neither the canal system nor the rice fields contained much in the way of snail habitats. snails were confined to temporary pool sites, filled with rain or seepage water. all these schemes were built on land with an appreciable slope and with relatively porous soils. even when irrigation was in progress, the field canals were not always in use and dried out rapidly. while snails are often able to withstand limited periods of drought, it is unlikely that any large snail populations could develop under these conditions in any one season. furthermore, on five of these schemes, field canals were often re-routed from season to season so that the establishment of suitable snail habitats was rendered even more unlikely. mobarak ( ) reported that after prevalence of urinary schistosomiasis in upper egypt reversed a previous downward trend and began to increase again because of the shift from basin to perennial irrigation. however, in any case, increased use of parenteral antischistosomal therapy (pat, injections of antimony-based drugs) was bringing schistoso miasis under control in egypt. in the northern part of upper egypt, prevalence dropped from . % in to . % in , while further south, prevalence reportedly fell from . % in % in to % in % in (who, . most experts agreed that applying the proper combination of sanitary engi neering, water control management, snail control, infection surveillance, and treatment drugs can avert adverse effects of irrigation on schistosomiasis. moreover, there was an impres sion that even without water control measures and environmental sanitation, chemotherapy with or without treat ing water to kill snails would adequately control schistosomiasis transmission. fenwick ( ) noted that in the newer rahad irrigation scheme, east of the gezira plain in the sudan, because of the use of drugs and snail control, the incidence of schistosomiasis remained very low, despite very poor sanitary conditions. it was predicted, however, that relax ing control measures would cause a surge in schistosomiasis. although oral drugs started to be used in egypt in the s, pat use continued into the mid- s. praziquantel, which also has a high cure rate for s. mansoni, became available in egypt in and has been the treatment of choice there since the late s. although the massive pat campaigns were successful in strongly reducing disease incidence, a study by frank et al. ( ) concludes that the intensity, widespread geographical coverage, and duration of the campaigns, together with unsafe injection practices (inappropriate sterilization pro cedures), have been responsible for the nationwide spread of hepatitis c in egypt in recent decades. the authors state that the enormous dimension of egypt's schistosomiasis problem and the sheer size of the antischistosomiasis effort, combined with the characteristics of pat, provided an effective mechanism for a massive increase and establishment of hepatitis c virus in the egyptian population. according to these authors, this is "the world's largest iatrogenic transmission of blood-borne patho gens known to date." moreover, it is probable that the heavy reliance on an effective chemical treatment also allowed the continuation of water management schemes that were contri buting to the maintenance of large numbers of snails in aquatic environments. the evolution of the schistosomiasis problem in egypt and sudan described above highlights the importance of developing water management programs able to keep vector proliferation under control. this lesson is also highly relevant to the construction of large-scale reservoirs and irrigation facil ities as in the case of the three gorges dam. in israel, biomphalaria alexandrina was eradicated through a combination of factors including chemical applications. as in all cases, snails return some time after the application of mol luskicides. some combined measures used for the control of snail vectors have been successful, such as increasing water currents to over cm s − , rapid emptying and drying up of water reservoirs, and weekly deflection of infested water courses along different routes (saliternik, ) . as stated previously, the success of chemotherapy in treating this disease and of molluskicides for eradicating snails does not imply that preventive measures should not be taken for avoid ing vector proliferation based on knowledge of their ecohydrological setting. all vector snails require water, at least for breeding. the management of water bodies is therefore a potentially powerful control method. for example, in the case of rice cultivation there are opportunities for vector control by changing the aquatic habitat of the snail in a way compatible with maximum crop production. infection of humans mostly takes place not in the rice field itself, but in irrigation canals and surrounding living quarters. different hydrological and rice husbandry approaches have been used in various countries. however, generalizations should be made with care because each snail species has its own preferences and tolerances regard ing shade, water velocity, the steepness of canal walls, and drought tolerance. rice cultivation by itself can be used as an environmental method of snail control in that it brings about ecological changes that can reduce snail habitat. the philippines has promoted more intensive scientific methods of rice cultivation to control schistosomiasis (hairston and santos, ) . snail control is achieved at different stages of rice growing in a number of different ways, for example, by deep plowing, which turns over the soil and buries the snails; or by draining the ricefield at harvest and keeping it dry until the next crop, which kills the snails and prevents them from breeding. in japan, s. japonicum eradication was accomplished by treatment, sanitation, control of animal reservoir host, educa tion, and elimination of most of the snail colonies (garcia, ) . the steepness of the walls of irrigation channels was increased and later they were lined with concrete and main tained clear of silt, vegetation, and debris to supplement the eradication of snails in the ricefields, which resulted from intensive cultivation. extensive rice-growing areas in china's mainland have been cleared of oncomelania snails (garcia, ) . the measures taken have included digging new water channels parallel to the existing snail-infested streams and using the excavated soil to fill the old ones, clearing streambeds, and removing vegetation. where the soil structure permits, the banks have been made steeper. in the philippines, similar measures have been taken as in china; in addition, converting undrainable swampy areas into fishponds and improving agricultural prac tices have successfully controlled snails in limited areas. thus, an integrated approach to drainage problems can result in increased production while reducing health risks. in the ecohydrology approach, it is essential to consider the whole basin in management policies, especially when a disease agent can be transmitted by different species of the same host (in this case a snail) that is differentially distributed along altitude gradients in river basins. both b. truncatus and planorbarius metidjensis are intermediate hosts of s. haematobium in southwestern morocco. a basin investigation (yacoubi et al., ) in five rivers identified sites colonized by these species and compared the habitats in which they were found. the p. metidjensis was observed in the upper valleys of three rivers, whereas b. truncatus was found in sites of lower altitude. a component analysis demonstrated that altitude (from to m), water ph (from . to . ), and electric conductivity (from to μs cm − ) were the main descriptors of environment. the p. metidjensis was associated to high altitude and low electric conductivity. however, b. truncatus was asso ciated to being found in lower altitude sites with medium electric conductivity in water. it has been mentioned above that periodic canal or field drying had been used successfully for snail control. nevertheless, before a control plan is adopted and implemen ted, the effects of drying out have to be monitored and thoroughly understood for each snail species: snails that are capable of undergoing diapause can circumvent unfavorable environmental conditions, including long periods of drought. cooper et al. ( ) found that diapause influenced the sus ceptibility of biomphalaria glabrata snails to s. mansoni infection. juvenile snails exposed just prior to diapause, or immediately following a diapause period of weeks, were highly susceptible to infection by s. mansoni miracidia. however, snails that underwent diapause produced compar able or only slightly fewer cercariae than did nondiapausing snails. these studies indicate that diapause in b. glabrata does little to decrease a snail's ability to act as an intermediate host for s. mansoni or to interrupt the development of the parasite. for these reasons, great attention should be given to diapausing snail populations when planning programs for mollusk control. thus, for snail control, it is important that not only the agricultural field but also irrigation and drainage channels, as well as the water source, be considered part of the agroecosys tem. this parallels the ecohydrology approach that considers the whole basin, from the river source to the wetlands, estuar ine, and coastal zone as an integrated management unit. both agricultural and ecohydrological models should converge in a new synthesis integrating their own tools with practices based on traditional knowledge, socioeconomical cost-benefit analysis of vector eradication, and agri-and/or aquacultural production. the type of wetland management approach, particularly when restoration or creation is planned, will require previous surveys of the mosquito species and their habitat types, locally and at basin level. in the following sections, we present a summary of main species/habitat combinations for different hydrological settings extracted from anonymous ( b) . relatively few mosquito species breed in running waters, such as streams. larvae can be flushed out when stream volume increases, and to remain in the stream requires a large amount of energy. the tropical genus, chagasia, and some anopheles species are stream breeders. stream breeders will find vegeta tion along banks with which to anchor themselves or attempt to remain away from the main flow of the stream by seeking isolated eddies. transient water sources, such as flooded areas and ditches, are used as breeding grounds for species whose eggs can with stand desiccation and whose life cycles require alternating periods of wet and dry, such as aedes and psorophora. the quality of transient water changes with time, which can result in a succession of different species using the same pool. transient waters include woodland pools created by spring rains (aedes stimulans), fresh floodwater (aedes canadensis), and tidal floodwater (aedes sollicitans). permanent or semipermanent waters support characteristic aquatic vegetation. cattail, rushes, and sedges are typical fresh water swamp vegetation. genera associated with permanent water are anopheles, culex, culiseta, coquillettidia, and uranotaenia, whose eggs are not desiccant resistant and must be laid directly on the water. aedes adults will oviposit near the edge of the swamp, or within tussocks of vegetation, requiring later flooding of the eggs for hatching. as with transient waters, there are seasonal changes in the vegetation, water quality, and mosquito species present. permanent waters can include fresh water swamps, such as, for example, tussock (aedes abserratus) or cattail swamps (coquillettidia perturbans), as well as brackish water swamps with salt marshes (culex salinarius). besides nat ural environments, polluted water with floating debris can be a habitat for species such as culex pipiens. container water habitat can be found in both natural set tings, such as water held by plants to artificial settings and water found in tires. container water is characteristically clear and many container species now also use artificial sites as they provide insulation against the weather and are more numerous (aedes aegypti and aedes albopictus). increasing dengue incidence in not only tropical but also subtropical countries requires a thorough elimination of such urban, man-made microhabitats. there are various techniques for mosquito control based on different principles. all involve modification of the hydrologi cal setting, ranging from total wetland drainage to an increase in their tidal flooding. a summary of these methods, including parallel grid ditching, open marsh water management, and runneling, is presented in the following paragraphs. parallel grid ditching consists in the physical removal of water from intertidal marshes and was one of the first largescale forms of mosquito control (lesser ( ) ; figure ). parallel grid ditches were dug in salt marshes, spacing these ditches about m apart to remove standing surface water where mosquitoes might breed. extensive ditching programs for mosquito control in north america were only moderately effective, since many breeding potholes were not drained dry, and there were long-term nega tive effects on wildlife and salt-marsh ecosystems. many nonmosquito breeding wetlands that provided wildlife habitat were unnecessarily drained and salt-marsh vegetation commu nities were changed into fragmented wetlands. birds were particularly affected through the draining of larger natural ponds. by the early s, there was an increasing awareness of wetland values and functions, and the value of parallel grid ditching was questioned. understanding of the drawbacks of the parallel grid-ditching technique led to the development of a new mosquito control source-reduction technique called open marsh water manage ment (omwm). it started in the late s and was further optimized until the early s. the goals of omwm are: ( ) control of salt-marsh mosquitoes; ( ) reduction of insecti cide applications; and ( ) habitat enhancement for salt-marsh fish and wildlife (ferrigno and jobbins, ; hruby et al., ) . the omwm method involves the selective installation of small, shallow ponds and interconnecting ditches superim posed on known mosquito-breeding habitats ( figure ). this aims to eliminate wet-dry-wet cycles necessary for determined species and any newly created permanent water habitats are unattractive for mosquito egg deposition. this simultaneously improves habitats for mosquito-eating larvivorous fishes which can quickly invade, via tidal flooding, any newly created omwm pond or ditch. scattered mosquito breeding depressions and sheetwater habitats are connected through pond and ditch excavations to allow unimpeded water flow and predatory fish movement, while isolated potholes are often filled with natural soils to eliminate these smaller-sized breeding depressions (lesser, ; figure ). the increase of tidal inundation frequency and predation by fish significantly reduce mosquito density. runneling is an effective method for controlling mosqui toes that breed in intertidal salt marshes through a type of habitat modification using shallow channels. this technique is based on omwm principles (wolfe, ) . it increases tidal frequency to a marsh and removes surface sheet water from low-lying areas high on the marsh. runnels are linked to the tidal source, promoting tidal exchange between graded regions of the marsh. they are conceived to allow transport of lowamplitude tides to areas of salt marsh in a way so that pools do not form, even after spring tides. runnels are shallow figure the omwm system, involving the selective installation of small, shallow ponds and interconnecting ditches superimposed on known mosquito-breeding habitats. (< cm deep) spoon-shaped channels constructed along nat ural drainage lines on the salt marsh ( figure ) to a maximum gradient of : (hulsman et al., ) . due to the slight slope, runnels enable slow water movement even during lowamplitude tides. the net result is a reduction in mosquito breeding areas, the modification of pools and edges for egg conditioning (the process involving flooding and drying events that prepares mosquito eggs for hatching), and larval development. there are few apparent negative impacts at the modified site (hulsman et al., ; dale and hulsman, ; dale et al., ; latchford, ) . further, like omwm, they allow water to drain from trap pools and permit predatory fish to gain access to the mosquito larvae, at least during high tide. in comparison to grid ditching and omwm, runneling is an environment-oriented approach to salt-marsh management for mosquito control that aims to alter the salt marsh as little as possible, while causing significant reductions in mosquito numbers. the main difference between the three approaches lies in the magnitude of the habitat modification. ditching involves the greatest alteration to the marsh, and runneling the least. runneling has a lesser effect on the estuarine environ ment as a whole than does either ditching or omwm. . . conclusions . . . some reflections on dams, water scarcity, ecohydrology, and health although the construction of reservoirs is controversial, the rising demand for water by an increasing human population makes more dams inevitable (jobin, ) . in a review, morley ( ) calls attention of the fact that most parasitological studies in relation to reservoir construction have been focused on schistosomiasis and other tropical diseases of humans (stanley and alpers, ; jobin, ) . in comparison, the impact of reservoir construction on indigenous aquatic parasite fauna of wildlife has been a subject largely ignored by the scientific community. however, reservoir formation can have profound effects on the parasite fauna of fish, birds in the reservoir, as well as up-and downstream of it. changes in host-parasite relationships and switches between animal and human hosts can occur (morley, and references therein) . the role of parasites in environmental monitoring is increas ingly recognized (lafferty, ; lafferty and kuris, ) . thus, the surveillance of the effects of reservoirs on parasite fauna of aquatic wildlife may provide important general infor mation on both short-and long-term changes that occur within and downstream of new reservoirs during the maturation pro cess of the reservoir. the above-described cases that show the effect of dam con struction on onchocerciacis and schistosomiasis call attention to the need to evaluate changes in flow power and velocity downstream of proposed dams, in order to assess their likely health impact. ecohydrological measures are required to improve the operation of existing dams to provide more effective control of disease vectors (see section . . . . ). the modeling of the effect of controlled flooding pulses on the plankton dynamics in the guadiana estuary (wolanski et al., ) is an outstanding example of the potential of the appli cation of ecohydrological principles for the control of toxic algal blooms. in arid and semiarid regions, the quality of water in artificial reservoirs is essential to human health, particularly in climati cally unstable regions. for example, recent el niño/la niña-southern oscillation teleconnections have produced a pro longed drought of ∼ years in south argentina. this has interrupted a period of about years of rainfall significantly above the historical average and produced a decline to critical levels in the reserves of drinking water reservoirs, which like other water bodies in the region, have suffered from recurrent summer algal blooms (kopprio et al., ) . however, the perception that cyanobacteria can represent a threat to human health only in connection with acute events such as blooms should be widened to consider the possibility of neurologic damage as consequence of chronic exposition to toxins such as bmaa via long-term ingestion of water or aquatic fauna. this should be taken into account when developing schemes for preventing cyanobacterial blooms by manipulating natural predator communities. the reduction of the nutrient load input to the water reservoir probably remains the best preven tive measure to prevent harmful algal blooms. water treatment should be regularly checked and improved to remove the organisms and their toxins from drinking-water supplies, where appropriate. water treatment by flocculation and sedi mentation, followed by sand filtration, is supposed to remove live cyanobacterial cells and debris. however, there is evidence that plants that are not working properly can actually increase cell counts of algae with potential toxicity (in this case, anabaena circinalis and microcystis aeruginosa) in the treated water (echenique et al., ) . throughout this chapter, examples have been referred to of actual and potential conflicts of interest between alternative uses of aquatic systems, for example, between the desire to restore degraded wetlands and the need to protect health of the human population living nearby. a relevant example of policy conflicts in an industrialized country is the experience of the tennessee valley authority (tva), where health concerns in the past gave rise to management measures that conflict with modern recreational interests (bos, ) . the meeting report of the ninth meeting of the who/fao/united nations environment programme (unep) panel of experts on environmental management for vector control in includes the following passage: as national and regional priorities change, so do policies, and there must therefore always be a provision for their reconsideration and modification. sometimes, though, the acute problems that led to the original priority setting might have become latent rather than have disappeared completely, and while public awareness and political pressure favour a policy change, the original goals of such policies should not be ignored. this was well illustrated by the water man agement policies including mosquito control established by the tva in the s. the standards of mosquito control maintained by tva equalled those maintained in privately owned river impoundments under prevailing public health regulations. the measures included the programmed fluctuation of water levels in the reservoirs, a practice that played a key role in reducing anopheles populations and eradicating malaria transmission from the valley. new uses of the reservoirs, including recreation and the promotion of nature conservation had led to a conflict of interests. for recreation, stable water levels during summer and early autumn were required; con servation of certain fish species and of waterfowl required higher water levels in spring to promote fish spawning and the rapid growth of aquatic vegetation for the fowl. such changes in water manage ment regimes would without doubt result in increased mosquito populations, yet the potential risk for the reintroduction of vectorborne disease was not appreciated after three generations of malaria free experience. in recent years much interest has been directed towards the protection and establishment of wetlands, without pay ing sufficient attention to their mosquito breeding potential. consequently, tva has been faced with a conflict of new policy directives concerning wetlands, existing mosquito control policies and state regulations for impounded water. the use of constructed (artificial) wetlands for the treatment of domestic waste water and its processing for reuse is of particular concern, since these could pro duce large quantities of potential disease vectors and they are often sited close to populated areas (peem/who, : - ). this stresses the necessity of harmonizing policies of reservoir management for vector control with other policies concerned with land use patterns, to ensure that areas of potential risk, such as artificial wetlands, are planned away from areas of human habitation. presently we face a complex environmental situation that seems to be changing at a much faster rate than our current capacity to revise and renew our intellectual schemes or to generate integrated management structures capable to enhance both ecosystem and human health. take, for example, the case of influenza virus. the loss of wetlands around the globe may force many wild birds onto alternative sites like farm ponds and paddy fields, bringing them into direct contact with domestic fowl and humans and providing greater opportu nities for the spread of the h n virus (anonymous, ) . poor planning in response to development pressures has led to the increasing loss or degradation of wild ecosystems which are the natural habitats for wild birds. the displaced wild birds increasingly seek to feed and live in areas populated by domestic poultry and humans. thus, wetland creation must also consider these factors, since they will attract migratory birds if located near their routes. despite this being considered an enrichment of landscape ecology in terms of diversity, recreational value, landscape beauty, etc., the risk of increasing transmission of emerging diseases must be dispassionately considered. an increase in biodiversity does include not only those species humans like, but also the diversity of vectors capable of transmitting diseases. the distance of the wetlands to be created from populated centers, the direction of prevail ing winds, and other transport mechanisms should be carefully taken into account. this issue of 'ecohealth' highlights the interplay between agriculture, animal (domestic and wildlife) and human health, the integral health of aquatic ecosystems, and sociocultural factors. nutrient reduction in wastewaters or surface waters is a priority for reducing risks to human 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and control of schistosomiasis (bilharziasis) overview of disease fact sheets habitats of bulinus truncatus and planorbarius metidjensis, the intermediate hosts of urinary schistosomiasis, under a semiarid or an arid climate factors affecting the prevalence of schistosomiasis in the volta region of ghana ecohydrology -the use of ecological and hydrological processes for sustainable management of water resources impact of the three gorges dam construction on transmission of schistosomiasis in the reservoir area relationship between the transmission of schistosomiasis japonica and the construction of the three gorge reservoir key: cord- -j kg qf authors: jones, samuel l.; blikslager, anthony t. title: disorders of the gastrointestinal system date: - - journal: equine internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: j kg qf nan however, the abdominal wall is too rigid to allow effective palpation of intraabdominal structures. abdominal auscultation is particularly useful for assessing the motility of the large intestine. progressive motility of the small intestine, conversely, is difficult to distinguish by auscultation from nonprogressive motility. the distinct character of the borborygmi produced during propulsive contractions of the cecum and ascending colon allow evaluation of the frequency and strength of retropulsion and propulsion. propulsive contractions of the cecum and ventral colon occur every to minutes and give rise to prolonged rushing sounds heard over long segments of intestine. retropulsive sounds presumably are similar to propulsive sounds, but they occur less frequently. the distinction of propulsion from retropulsion is not important clinically because both types of contractions signify normal motility. inter-and intrahaustral mixing contractions produce nonspecific sounds of fluid and ingesta movement that are difficult to distinguish from other borborygmi, such as small intestinal contractions or spasmodic contractions. auscultation over the right flank and proceeding along the caudal edge of the costal margin toward the xiphoid allows evaluation of the cecal borborygmi. auscultation over a similar area on the left side allows evaluation of the pelvic flexure and ascending colon. typical progressive borborygmi heard every to minutes on both sides of the abdomen indicate normal motility of the cecum and ascending colon. less frequent progressive sounds may indicate a pathologic condition of the large intestine or may result from anorexia, nervousness (sympathetic tone), or pharmacologic inhibition of examination of patients with disease of the gastrointestinal tract must include evaluation of the metabolic and cardiovascular status of the patient, because acute conditions of the proximal or distal intestinal tract have the potential to lead to endotoxemia and sepsis. examination of the cardiovascular system (heart, peripheral pulse, and mucous membranes), lungs, and abdomen is essential to detect clinical signs of systemic inflammation from endotoxemia, coagulation disorders, dehydration, ileus, shock, and other abnormalities resulting from injury to the small or large intestine. chapter . covers clinical signs of systemic inflammation from endotoxemia and sepsis. one performs the physical examination of the abdomen primarily by auscultation, transabdominal ballottement, and transrectal palpation. abdominal distention often indicates distention of the large intestine; however, small intestinal distention also can cause visible abdominal distention if a large proportion of the small intestine is involved. one can perform abdominal palpation in neonatal foals; after several weeks of age, motility (i.e., α -adrenergic agonists such as xylazine). [ ] [ ] [ ] absolute absence of any auscultable borborygmi suggests abnormal motility and indicates ileus resulting from a serious pathologic condition but is not specific to any segment of the intestine. , if borborygmi are audible but progressive sounds are not detectable, determining whether a significant abnormality exists is difficult. borborygmi heard more frequently than normal may result from increased motility following feeding; from excessive stimulation from irritation, distention, or inflammation; or after administration of parasympathomimetic drugs such as neostigmine. large intestinal motility increases in the early stages of intestinal distention regardless of the site. mild inflammation or irritation of the large intestinal mucosa also can stimulate motility. parasympathomimetic drugs stimulate contractions and auscultable borborygmi in the large intestine; however, an increase in parasympathetic tone may result in segmental contractions, which actually inhibit progressive motility. one can detect sand or gravel in the large intestinal ingesta by auscultation behind the xiphoid process. one can hear sand or gravel particles grinding together during progressive contractions of the ascending colon. the presence of sand in the ingesta becomes clinically detectable by auscultation or fecal sedimentation before the amount of sand is enough to produce clinical signs of pain or irritation (diarrhea). if progressive contractions are audible without hearing sand sounds, clinically important quantities of sand likely are not present. if the frequency of progressive contractions is low or absent, detecting sand by auscultation is difficult. percussion of the abdomen during auscultation can reveal gas in the large intestine. the characteristic ping produced by simultaneous digital percussion and auscultation over a gas-filled viscus often is associated with abnormal accumulation of gas under pressure. this technique is particularly useful in foals, ponies, and miniature horses because of the limitations of palpation per rectum. one can use transabdominal ballottement to detect large, firm masses or an abnormal volume of peritoneal fluid. the usefulness of this technique is usually limited to animals too small to palpate per rectum. one can detect soft tissue masses or fetuses by bumping the structures with a hand or fist. if excessive peritoneal fluid is present, one can generate a fluid wave by ballottement; however, this technique is not as useful in horses older than weeks because the abdominal wall is rigid. transrectal palpation is the most specific physical examination technique for investigation of intestinal disease and is particularly valuable when evaluating obstructive diseases. , the primary objectives of transrectal palpation are to assess the size, consistency, and position of the segments of the large intestine; to determine the presence of any small intestinal distention; and to detect intraabdominal masses. evaluation of the wall thickness and texture and the mesenteric structures (blood and lymphatic vessels and lymph nodes) also may aid in diagnosis of large intestinal disease. the interpretation of transrectal palpation findings in light of clinical signs and laboratory results is an important diagnostic aid for developing appropriate treatment strategies for intestinal diseases manifested by abdominal pain. enlargement of one or more segments of large intestine detected by transrectal palpation provides evidence of obstruction at or distal to the enlarged segment. by systematically evaluating each segment, one may determine the site of obstruction. obstruction of the pelvic flexure, for instance, results in enlargement of the pelvic flexure and ventral colon, but the dorsal and descending colons are of normal size. enlargement of a segment of the large intestine usually is accompanied by abnormal consistency of the contents. one may distinguish accumulation of gas, fluid, or ingesta and may detect foreign bodies in palpable segments. accumulation of gas and fluid infers complete and acute obstruction, whereas accumulation of ingesta infers chronic and incomplete obstruction. accumulation of fluid usually indicates ileus. one must evaluate the consistency of the contents in light of the size of the segment; ingesta in the ventral colon of a dehydrated patient may be firm, but the size of the ventral colon will be normal. conversely, if the ingesta is firm because of a distal obstruction, the ventral colon will be enlarged. displacement of a segment of the large intestine may create an obstruction detectable by enlargement of the segment and accumulation of gas and fluid, even if the site of obstruction is not palpable. torsion of the ascending colon at the sternal and diaphragmatic flexures results in acute accumulation of gas and fluid proximal to the torsion, causing distention of the left dorsal and ventral colons. depending on the degree of torsion, the position of the ventral and dorsal colons may not be significantly abnormal. displacement of a segment of large intestine often results in incomplete obstruction, and the diagnosis relies solely on detection of the displaced segment in an abnormal position. the position of the displaced segment may not be palpable, and the diagnosis then relies on the inability to find the segment in a normal position. one must take care to ensure that the segment that appears to be displaced is not in a normal position but has become too small to palpate from a decrease in the volume of ingesta. the cecum, right dorsal and ventral colons, pelvic flexure, and descending colon are palpable in most horses. one should palpate the nephrosplenic space to detect the presence of intestine, usually pelvic flexure, entrapped within the ligament. small intestine is not normally palpable in the horse. distention is an indicator of ileus with gas or fluid retention, usually following a strangulating or nonstrangulating obstruction. strangulating obstructions result from conditions such as volvulus or torsion, lipoma, or entrapments. such conditions often are accompanied by severe pain, dehydration, peritoneal fluid changes, and a varying degree of gastric fluid accumulation. the small intestine in these cases is turgid and firm on palpation. one should assess the mesentery and wall thickness as for large intestinal disorders. careful palpation of the inguinal rings in stallions with small intestinal distention is crucial for determining inguinal herniation. evaluation of the wall thickness and mesenteric vessels can reveal venous congestion (mural edema and enlarged blood and lymphatic vessels) or inflammation (mural edema with normal vessels). disruption of arterial blood flow does not cause venous congestion, but the arterial pulse is not detectable. mesenteric tears may not be palpable, but the entrapped ischemic intestinal segment may be thickened with edema. one may detect acute or chronic inflammation with cellular infiltration of the intestinal wall as thickening of the wall without edema and also may note enlargement of mesenteric lymph nodes. one should interpret abnormalities in the wall or vessels in light of the size, consistency, and position of the segment of intestine and the clinical signs. several conditions involving small intestinal strangulating lesions do not necessarily cause abnormal rectal examination findings until the disease has been present for an extended time. these conditions include diaphragmatic herniae and epiploic foramen entrapments. peritoneal fluid analysis may be normal in these cases as well because the fluid is trapped in the thorax or in the cranial abdomen. surgery is usually necessary for diagnosis. nonstrangulating causes of small intestinal distention can be divided further into intraluminal and extraluminal obstructions. ileal impactions are probably the most common cause of intraluminal obstructions, and on rare occasions one can palpate the impaction in the upper right quadrant, near the ileocecal opening. intraluminal masses caused by lymphoma, eosinophilic enteritis, foreign bodies or ascarid impactions often lead to small intestinal distention and are usually indistinguishable from one another based on palpation alone. small intestine in these cases can be moderately to severely distended, depending on the degree of obstruction. extraluminal obstructions include abdominal masses, abscesses or tumors, and large colon displacement. one always should palpate the rest of the abdomen carefully to help rule out these causes. some cases of small intestinal distention result from a physiologic rather than a mechanical obstruction. ileus may result postoperatively or following inflammatory diseases of the bowel (proximal enteritis) or peritoneal cavity (peritonitis). the bowel is usually mildly to moderately distended and almost always is accompanied by significant amounts of accumulated gastric fluid. the small colon is easily distinguishable by the presence of normal fecal balls and an antimesenteric band. in cases of impaction of the small colon, a long, hard, tubelike structure is present in the caudal abdomen, and the band is palpable along the length. fluid stool is often present in the rectum in these cases, as is tenesmus. one can detect and carefully evaluate rectal tears by palpation. one can detect mural masses in palpable segments of intestine or mesentery; however, if a mass causes obstruction, one can detect the result of the obstruction in proximal segments of intestine even if the mass is unreachable. palpation of the mesenteric vessels may reveal thickening and thrombosis, which can lead to ischemia or infarction. one can perform visual inspection of the mucosa of the rectum and descending colon with a speculum or flexible endoscope and also can evaluate rectal tears or perforations, mural masses, strictures, or mucosal inflammation. one also can perform guided biopsy of the mucosa or masses. the obvious limitations are the amount of fecal material interfering with the examination and the distance of the lesion of interest from the anus. these techniques offer little advantage over palpation in many cases unless the patient is too small to palpate. examination of the oral cavity in cases of dysphagia or weight loss is a necessary part of the physical examination. one should adequately sedate the horse and should use a full-mouth speculum to allow palpation and visualization of all parts of the oral cavity. one should examine the area for abnormal dentition, foreign bodies, fractures, abscesses, and ulceration. the presence of fluid accumulation in the stomach indicates a decrease or absence in propulsive motions of the small intestine or obstruction of gastric outflow. decreased small intestinal motility may result from a functional or mechanical blockage. masses, feed impactions, or strictures in the pylorus or in the proximal duodenum may obstruct gastric outflow. one routinely assesses fluid accumulation in the stomach by siphoning off the gastric contents with a nasogastric tube and examining the fluid for amount, color, and any particular odor. normal fluid is green and may contain foamy saliva. the volume obtained by gastric lavage is usually less than l. large volumes of fluid (> to l) accumulate in the stomach of horses with proximal enteritis, and the fluid is foul smelling and often has an orange to yellow discoloration. if one suspects proximal enteritis, one can submit the fluid for culture and gram staining. salmonella sp. and clostridium sp. have been cultured in some cases. patients with postoperative ileus also frequently accumulate large amounts of gastric fluid. horses with section . examination for disorders of the gastrointestinal tract strangulating obstructions or luminal obstructions often accumulate moderate amounts of gastric fluid, but the amount is generally less than in horses with proximal enteritis or postoperative ileus. hemorrhage in the gastric fluid usually indicates devitalized small intestine, stomach wall, or severe gastric ulceration. fluid with large amounts of food material often indicates a gastric impaction, and one should lavage the stomach until obtaining no more ingesta. horses and foals with chronic gastric ulceration in the glandular mucosa of the stomach or in the duodenum may develop strictures and have fluid accumulate in the stomach. endoscopy or contrast radiography aids in diagnosing gastric outflow obstruction. evaluation of the hemogram is essential when one assesses conditions of the gastrointestinal tract. however, hematologic alterations associated with diseases of the gastrointestinal tract are often nonspecific, reflecting systemic response to inflammation, endotoxemia, or sepsis. neutrophilic leukocytosis and normochromic, normocytic anemia with or without hyperfibrinogenemia commonly are associated with chronic inflammatory conditions of the intestine. anemia from chronic blood loss occurs infrequently in adult horses because of the large iron stores and high concentrations of iron in their diet; usually anemia follows chronic inflammation, as do alterations in the leukon and plasma fibrinogen concentrations. plasma protein concentrations vary depending on gastrointestinal losses of albumin and globulin and elevation of globulin concentration from antigenic stimulation. protein-losing enteropathies may manifest predominantly as a hypoalbuminemia or may have a concurrent hypoglobulinemia. immunoglobulin quantification can be useful in selected cases; immunosuppression with low immunoglobulin m concentration has been shown to occur in some cases of lymphosarcoma. parasitic infections, especially strongylosis, may be characterized by elevated serum immunoglobulin g(t) concentration. significant alterations of the hemogram do not accompany acute disease of the intestine unless severe inflammation, dehydration, endotoxemia, or sepsis is present. during the early stages of endotoxemia, elevations in circulating concentrations of inflammatory mediators, epinephrine, and cortisol produce characteristic changes in the hemogram. leukopenia, with neutropenia and a left shift, toxic changes in the neutrophil cytoplasm, and lymphopenia occur commonly. hemoconcentration and hyperfibrinogenemia are also common. thrombocytopenia and other coagulopathies are also features of endotoxemia. indeed, thrombocytopenia may be the earliest indicator of sepsis. endotoxemia and circulating mediators of inflammation activate the coagulation cascade, causing a hypercoagulable state that can lead to consumption of coagulation factors and coagulation defects manifested as elevated prothrombin time, partial thromboplastin time, fibrin degradation products, and bleeding time, and reduced activity of antithrombin iii. [ ] [ ] [ ] neutrophilic leukocytosis occurs during the later stages of endotoxemia. the most common serum biochemical abnormalities with diseases of the large or small intestine are electrolyte imbalances. serum calcium concentrations are often low with strangulating obstructions and acute inflammatory diseases. inflammation of the mucosa can disrupt electrolyte fluxes severely. diarrhea or gastric reflux greatly exacerbates the loss of sodium, potassium, calcium, magnesium and bicarbonate. ischemia of the intestine causing hypoxia and cellular damage may be reflected by an elevated serum phosphate concentration resulting from phosphate leakage from damaged cells. ischemia and cellular hypoxia in any segment of the intestine also causes a shift in energy metabolism to anaerobic glycolysis, resulting in increased production of lactate and elevated serum lactate concentration. reduced perfusion of peripheral tissues from hypotensive shock and intestinal ischemia can cause elevations in serum lactate. however, obstruction of the intestine during ischemia may result in absorption of lactate from the lumen. , anion gap is an indirect measurement of organic acid production during states of tissue hypoxia and is a reasonable estimate of serum lactate concentration. metabolic acidosis may accompany lactic acidemia, but an inconsistent association exists between the two, especially when mixed acid-base imbalances are present. , elevations of hepatic enzymes, specifically γ-glutamyltransferase, may occur with large colon displacements, duodenal strictures, or anterior enteritis. relative polycythemia from hemoconcentration or splenic contraction and changes in red blood cell deformability from hypoxia or hypocalcemia may increase blood viscosity. blood viscosity increases in patients with acute obstructive disease. hyperviscosity reduces perfusion of capillary beds, thereby exacerbating ischemia and tissue hypoxia. hyperviscosity is one manifestation (along with lactic acidemia, coagulopathies, and clinical signs of shock) of the pathophysiologic events that take place during acute inflammatory or vascular injury to the large intestine. laboratory tests designed to reflect the systemic effects of endotoxemia, ischemia, sepsis, and shock are important to design therapeutic strategies, and monitor response to therapy. abdominocentesis and analysis of peritoneal fluid (pf) is a diagnostic technique performed on many patients with disease of the gastrointestinal tract. one can quantitate cytologic examination of pf; white blood cell and red blood cell counts; protein, fibrinogen, lactate, phosphate, and glucose concentrations; lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity; and ph. the results of pf analysis may help establish a specific diagnosis but more importantly may reflect inflammatory, vascular, or ischemic injury to the intestine requiring surgical intervention. pf reflects a sequence of events that takes place during acute vascular injury to the intestine. the pf protein concentration first increases, followed by an increase in the red blood cell count and fibrinogen concentration. a transudative process resulting from vascular congestion and increased endothelial permeability allows small macromolecules (albumin) to escape into the pf, followed by larger macromolecules (globulin and fibrinogen), and finally diapedesis of cells (red blood cells, then white blood cells). , if ischemic inflammation of the intestine and visceral peritonitis occur, an exudative process ensues. severe inflammation of the intestine and visceral peritoneum causes large quantities of protein and white blood cells, primarily neutrophils, to escape into the pf. as damage to the bowel progresses, the protein concentration and red blood cell and white blood cell counts continue to rise. as the degree of irreversible damage to the intestine increases, the pf characteristics become more exudative. , eventually, bacteria begin to translocate across the intestinal wall and appear in the pf as the mucosal barrier breaks down. neutrophils predominate, and their cytoplasm becomes granulated, and döhle bodies often are visible. if perforation occurs, bacteria and particles of ingesta appear in the pf, and the neutrophils become degenerate, that is, pyknotic, with karyorrhexis, karyolysis, and smudge cells. elevated pf protein concentration is a sensitive indicator of early inflammation, whereas elevated red blood cell counts in the presence of normal white blood cell counts suggest vascular damage without significant tissue ischemia. elevation of the white blood cell count usually indicates severe tissue inflammation or intestinal injury. the gross color of the pf can be helpful in detecting injury and necrosis of the intestine. a serosanguinous appearance indicates vascular injury, whereas orange or brown-red indicates necrosis with the release of pigments such as hemosiderin. serial samples of pf are most useful in determining the nature and extent of damage to the intestine, but in many cases of ischemia, irreversible tissue damage has occurred by the time pf abnormalities appear. tissue hypoxia and ischemia cause a rapid elevation of pf lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity and lactate concentration. , , phosphate concentration increases when cellular disruption occurs. pf enzyme activities, phosphate, and lactate concentration increase faster and higher than serum activities. [ ] [ ] [ ] pf ph and glucose concentration tend to decrease during intestinal ischemia, but not as low as in septic peritonitis. although biochemical alterations may provide early indicators of intestinal ischemia and necrosis, they are nonspecific and offer no advantage over conventional methods of pf analysis in many cases. pf alkaline phosphatase has been shown to arise predominantly from degenerating white blood cells, and elevations of other enzyme activities may occur with many inflammatory diseases. thus the specificity of many tests run on pf is questionable. however, in selected cases in which conventional pf analysis and physical examination do not provide sufficient information to develop a treatment plan, biochemical analysis of the pf may be useful. cytologically examined cells of the pf may reflect chronic inflammatory conditions of the large intestine, especially eosinophilic or lymphocytic processes. infectious and inflammatory conditions often cause increases in the neutrophil count and may be indistinguishable unless bacteria are visible. one also may detect neoplastic diseases by pf examination. chronic infection and inflammation may be associated with elevated pf protein and fibrinogen concentrations. culture of pf usually is required to distinguish bacterial infections from noninfectious inflammation unless bacteria are visible on cytologic examination. however, culture of pf is often unrewarding because factors that are found in inflammatory pf inhibit bacterial growth, and leukocytes phagocytose many bacteria in the pf. decreases in pf glucose concentrations (< mg/dl) and ph (< . ) are early indicators of a septic process. the glucose concentration and ph in the pf should approximately equal the blood glucose concentration and ph. a pf fibrinogen concentration greater than mg/dl also indicates bacterial infection. gross examination of the feces can provide information about digestion and transit time in the large intestine. large fiber particles in the feces represent poor mastication or poor digestion in the large intestine. small, mucuscovered, hard fecal balls indicate prolonged transit through the descending colon, whereas increased fluidity implies decreased transit time. feces containing sand or gravel are not necessarily abnormal. however, a significant amount of sand implies that large quantities are present in the colon. frank blood indicates substantial bleeding into the distal colon (right dorsal colon and/or small colon) from mucosal damage. laboratory analysis of the feces is performed frequently in cases of diarrhea. fecal cytologic examination and tests for occult blood detect mucosal inflammation, section . examination for disorders of the gastrointestinal tract erosion, or ulceration. severe inflammatory diseases in human beings, invasive bacterial infections in particular, have been shown to increase the shedding of leukocytes in the feces. a higher percentage of horses with salmonellosis and diarrhea have fecal leukocyte counts greater than cells per high power field than horses with negative fecal cultures for salmonella. these results suggest that high fecal leukocyte counts indicate salmonellosis in horses with diarrhea. however, the specificity of this test is probably low. low fecal leukocyte counts do not rule out salmonellosis. fecal occult blood tests detect blood in the feces, presumably from erosion or ulceration of the mucosa, but do not distinguish the source of the blood. large volumes of blood ( to l) given by nasogastric tube were required to produce a positive test for occult blood in the feces, but the amount of blood originating from the large intestine required to produce a positive test is unknown. a positive test implies significant hemorrhage into the gastrointestinal tract. newer, more sensitive tests detect not only occult blood but also degraded blood and may be useful to determine the site and quantity of blood loss. a positive test implies significant hemorrhage into the gastrointestinal tract. bacteriologic examination of the fecal flora has been used to quantitate specific bacterial species in the feces of horses with diarrhea. quantitation of clostridial species may be beneficial in diagnosing clostridial infection of the large intestine. tests to detect clostridial toxins in intestinal contents or feces are important to determine whether clostridia cultured from the feces are causing disease. the most common bacterial pathogens isolated from the feces of horses are salmonella and clostridium. the number of salmonella organisms isolated from the feces of horses with clinical salmonellosis is usually higher than from horses with asymptomatic infections. however, the volume of feces in many cases of acute diarrhea is high, and the concentration of salmonella organisms may be lower than would be expected, accounting for many false-negative fecal cultures. the sensitivity of fecal cultures for detecting salmonella infection may be as low as %. culture of five consecutive daily fecal samples is recommended to increase the sensitivity of the test. because salmonellae are intracellular organisms, culture of rectal scrapings or a rectal biopsy sample, along with fecal material, may increase the sensitivity of culture for detecting salmonella infection to %. one can perform a polymerase chain reaction assay on fecal samples to detect dna from salmonella sp. the polymerase chain reaction test is more sensitive than culture and is frequently positive in clinically normal horses that continuously shed small amounts of bacteria. polymerase chain reaction or immunologic tests also may detect clostridium perfringens and c. difficile exotoxins in the feces. qualitative fecal examination is a technique to detect nematode and cestode ova, protozoan oocysts, parasitic larvae, and protozoan trophozoites. a direct smear of fecal material is a rapid method to screen feces for ova and oocysts, to detect parasite larvae and trophozoites, and to observe motility of ciliates and parasite larvae. fecal flotation is a more sensitive technique for isolating and detecting ova and oocysts because the eggs are concentrated from the sample. zinc sulfate and sucrose solutions are often used to concentrate less dense ova and oocysts. zinc sulfate produces less distortion of trophozoites and larvae than sucrose solutions. fecal sedimentation is particularly appropriate for ciliates, giardia, and trichomonads. quantitative techniques such as the cornell-mcmaster method allow one to estimate the number of eggs per gram of feces and are most appropriate in monitoring parasite control programs. survey radiography of the normal esophagus is usually unrewarding but may be useful in horses with esophageal obstructions to determine the extent and location of the obstruction. one may detect foreign bodies or soft tissue masses, and in cases of esophageal rupture, one may see free air and ingesta in the tissues surrounding the esophagus and may observe pneumomediastinum. thoracic radiographs may be necessary to detect intrathoracic esophageal obstructions, megaesophagus, or cranial mediastinal masses causing extraluminal obstruction. one may use barium swallows or double-contrast esophagrams after resolution of the obstruction to determine whether a stricture or diverticulum or other underlying disorder is present. barium sulfate is the usual contrast medium and can be administered orally via a dose syringe or by nasogastric tube ( to ml of a % barium sulfate suspension or barium paste). oral administration is preferred for evaluation of swallowing and lesions in the proximal esophagus. administration of contrast using a nasogastric tube (preferably cuffed) allows for delivery of larger volumes of barium (up to ml) but should be performed without sedation if possible. one can follow administration of contrast material with air insufflation to create a double-contrast effect. if one suspects a rupture of the esophagus or if the likelihood of aspiration of the contrast material is high, one should use iodinated organic compounds in an aqueous solution as contrast material. contrast radiography may be the most definitive method for the diagnosis of primary megaesophagus or other functional disorders such as autonomic dysautonomia (grass sickness) affecting the esophagus. one should take care when interpreting esophageal radiographs if the horse is sedated. acepromazine or detomidine administration causes esophageal dilation in normal horses, especially after passage of a nasogastric tube. radiography of the adult equine abdomen is an effective technique in detecting radiodense material in the large intestine, such as enteroliths, sand, and metallic objects. , one survey demonstrated that radiography has . % sensitivity and . % specificity for diagnosing enterolithiasis. radiography also can be a useful tool for detecting sand accumulation in the colon that causes diarrhea or impactions ( figure . - ) and for monitoring resolution in medically treated horses. the large size and density of the adult abdomen precludes evaluation of soft tissue structures because the detail and contrast of the radiographs are usually poor. one is more likely to obtain diagnostically useful abdominal radiographs from small ponies and miniature horses than from full-size adult horses. accumulation of gas is visible on radiographs of adult horses, but distinguishing normal intestinal gas from obstruction is often difficult. horses should be fasted for to hours to reduce the amount of ingesta in the large intestine before radiography. abdominal radiography is more useful in foals than in adult horses. radiographs are more detailed and contrast can be good. radiographic evidence of gas distention in the large intestine may indicate large intestinal obstruction, and radiographic signs of displacement are often diagnostic. one may diagnose impactions, intussusceptions, foreign bodies, and other disorders with the aid of radiography. functional ileus may be difficult to distinguish from mechanical obstruction. , administration of contrast (barium sulfate % at ml/kg) via nasogastric tube increases the diagnostic capabilities of radiography. gastric ulceration also is recognizable with contrast radiography in the foal, although this is not as accurate a method as endoscopy. contrast administered retrograde via a -f foley catheter inserted into the rectum at a dose of up to ml/kg has excellent potential for diagnosing disorders of the small colon, transverse colon, and large colon in foals. ultrasonographic evaluation of the abdomen can add valuable information in cases of acute or chronic gastrointestinal disease. examination of the adult horse requires a . -to . -mhz transducer at minimum. one may use sector, linear, or curved linear transducers. clipping of the hair over the area to be examined, along with the application of isopropyl alcohol and ultrasound coupling gel, enhances evaluation. to evaluate the abdomen adequately, one must know the anatomic location and normal appearance of the individual organs. in the left cranial abdomen, one can assess the greater curvature of the stomach between the eleventh and thirteenth intercostal space, and one can use the spleen and the large splenic vein as landmarks. cases of gastric dilation from gas or impaction appear as an enlargement of the viewing area to cover greater than five rib spaces. one also can evaluate the stomach for intramural or extramural masses such as abscesses or for squamous cell carcinoma. the lesser curvature is not routinely visible. assessment of the small intestine should include evaluation for changes in thickness, motility, location, and visibility. one may find small intestinal loops easily in the left lower quadrant of the abdomen, but these normally are visible in other locations. one can visualize the duodenum consistently on the right side of the abdomen deep to the liver in the tenth to twelfth intercostal space or deep to the right kidney at the fifteenth to sixteenth intercostal space. mural thickening (> mm) may occur in cases of infiltrative or proliferative diseases, postoperative cases, enteritis, and paralytic or mechanical ileus. thickening of the small intestinal wall in foals, with or without the presence of gas shadows within the wall, should raise suspicions of clostridial enteritis. one can assess motility by monitoring a specific area for contractions over time. ultrasonography is an accurate method of distinguishing strangulating versus nonstrangulating disorders of the small intestine. strangulated small intestine has thicker small intestinal walls and larger intestinal diameter than in nonstrangulating disorders. strangulating lesions have decreased motility in the incarcerated segments with normal motility elsewhere. cases of paralytic ileus or nonstrangulating obstruction have a diffusely decreased peristalsis, but not to the degree observed with strangulating lesions. , one may diagnose some specific lesions of the small intestinal tract using ultrasonography. one may see ascarids in foals in cases of ascarid impaction and epiploic foramen entrapments as edematous loops of small intestine found in the right cranial abdomen. one may note small intestinal intussusceptions as targetlike lesions when viewed in cross sections. the presence of bowel loops, stomach, or liver in the thoracic cavity indicates the presence of herniation through the diaphragm and should be confirmed using radiography or surgical exploration. evaluation of the large intestine may be difficult because of the large amounts of gas within the lumen. however, certain disorders are readily identifiable via ultrasonography. one can assess the nephrosplenic ligament area for bowel entrapment in the left paralumbar fossa. in cases of entrapment, the spleen will be pulled away from the body wall and fluid or gas shadows will be observable dorsal to the spleen, obscuring the kidney, which is normally adjacent and abaxial to the spleen. small colon, small intestine, or pneumoperitoneum also may produce a gas shadow and obscure the kidney from view. sand impactions may appear as hyperechoic bands on the ventral abdominal wall. one may see ileocecal and cecocolic intussusceptions in the upper right paralumbar fossa. in cases of colitis, large, fluid-filled colons may be visible with or without intramural edema. one can find the right dorsal colon consistently abaxial to the liver, within the right thirteenth to fifteenth intercostal space and may be thickened (> mm) in cases of right dorsal colitis. evaluation of the abdomen always should include assessment of the peritoneal space for any evidence of an increased amount of pf or increased cellularity of the fluid as indicated by an increase in echogenicity. ultrasonography also can be useful in determining the ideal location for abdominocentesis. one also should evaluate the liver, kidneys, and spleen. one may detect choleliths, nephroliths, masses, abscesses, or enlargement of any of these organs. abscesses or tumors not associated with visceral organs may be difficult to visualize and interpret via ultrasonography. although more commonly used to diagnose lameness and musculoskeletal problems, nuclear scintigraphy has several uses for evaluation of the gastrointestinal tract. scintigraphy is now available at most universities and many private referral hospitals. one must use proper isolation protocols and waste disposal techniques strictly. the procedure requires special gamma cameras and the injection of radioactive materials into the bloodstream. one can use one of two methods: injection of technetium- m methylene diphosphonate ( m tc-mdp) directly into the blood or injection of m tc-labeled leukocytes. labeling of leukocytes involves aseptically collecting heparinized blood samples, isolating the buffy coat, and mixing those leukocytes with a radioactive dye ( m tc hexamethylpropyleneamine oxime, or m tc-hmpao) in vitro. one then reinjects the labeled leukocytes and obtains images. the principle of nuclear scintigraphy then lies in increased uptake of the dye or the white blood cells into areas of inflammation. one of the most common uses of nuclear scintigraphy in evaluating the gastrointestinal tract is diagnosis of dental disease. scintigraphy using m tc-mdp proved to be more sensitive in cases of dental disease than was radiography. scintigraphy was slightly less specific, however, and therefore should be used with radiography or computed tomography for ultimate accuracy. scintigraphy using radiolabeled white blood cells can support a diagnosis of right dorsal colitis in the horse. images taken of the abdomen hours after injection showed an increased linear uptake of leukocytes in the region of the right dorsal colon in horses with right dorsal colitis compared with normal horses. this technique also may prove useful for diagnosing intraabdominal abscesses in the horse. other uses of nuclear scintigraphy include evaluation of metastasis of abdominal tumors to bony areas, assessment of biliary kinetics, and determination of gastric emptying times. [ ] [ ] [ ] endoscopy endoscopic examination of the gastrointestinal tract begins with evaluation of the pharyngeal area by examination for any signs of collapse or dysfunction. one should evaluate the ability of the horse to swallow. the floor of the pharynx should be clean and free of feed material and foreign bodies. one can examine the oral cavity with the horse under heavy sedation or anesthesia and with the help of a full-mouth speculum. one can examine the teeth for any irregularities, obvious cavities, sharp points, or hooks and the hard and soft palpate for completeness and any evidence of ulceration, masses, or foreign bodies. one should use a -m flexible fiberoptic endoscope to examine the esophagus, which is accomplished best by passing the endoscope into the stomach and viewing the esophagus as one withdraws the endoscope while dilating the lumen with air. the esophageal mucosa normally should be a glistening, light pink color. ulceration can occur with cases of choke, reflux esophagitis or in horses that have had an indwelling nasogastric tube. erosions may be punctate, linear, or circumferential. one should evaluate carefully for any ulcers to ensure that no areas of perforation through the entire thickness of the esophageal wall exist. distinguishing normal peristaltic contractions from areas of stricture requires observation of the area and its motility over time. one also may note diverticula as outpouchings of the mucosa, sometimes associated with a stricture distally. megaesophagus, although rare, appears as a generalized dilation of the esophagus. one may detect food or foreign body impactions of the esophagus via endoscopy. one always should reevaluate the esophagus after removing any obstruction to detect the presence of complications (ulceration, rupture) or initiating causes (strictures, diverticula, and masses). a -m flexible endoscope also allows examination of the stomach. the horse should be fasted for at least hours before endoscopy. one can examine the cardia and fundus easily, as well as the margo plicatus. the squamous mucosa should resemble the esophageal mucosa. the glandular mucosa should be glistening red and may have a reticulated pattern. one should carefully examine for evidence of ulceration or masses. one can obtain transendoscopic biopsy material easily from esophageal, pharyngeal, or gastric masses, and because the biopsy size will be small, one should take several samples for histopathologic examination. pharmacologic agents (bethanechol) to empty the stomach and provide complete visualization of the entire fundic region, the pylorus, and the duodenum may be useful. for a complete description of gastroscopy and evaluation of gastric and gastroduodenal ulceration, please refer to chapter . . d-glucose or d-xylose absorption tests are useful in determining malabsorption of carbohydrates from the small intestine in horses. the protocol for absorption tests using either carbohydrate is similar. the horse should be fasted for to hours before testing. increased periods of fasting actually have been shown to decrease absorption of d-xylose and interfere with results. one administers a dosage of . to g/kg of d-glucose or d-xylose via a nasogastric tube. administration of sedatives may increase the blood glucose levels falsely and interfere with gastrointestinal transit times. one then collects blood samples to measure glucose or xylose concentrations at , , , , , , , , and minutes after administration. one may take additional samples up to hours after dosing if the results are questionable. one should measure glucose in blood samples collected with sodium fluoride as an anticoagulant and measure xylose in samples collected in heparinized plasma. a normal d-glucose absorption test, also known as an oral glucose tolerance test, should have a peak between and minutes, and this peak should be greater than % above the resting glucose value. complete malabsorption is defined as a peak less than % above the resting levels, and partial malabsorption is defined as a peak between % and % above the resting level. one must keep in mind that gastric emptying, gastrointestinal transit time, length of fasting, cellular uptake and metabolism, and endocrine function influence glucose absorption curves. malabsorption demonstrated by the oral glucose tolerance test is sensitive but not specific. diseases that may cause a lowered or delayed peak include infiltrative lymphosarcoma, inflammatory bowel disease (lymphocytic-plasmacytic or eosinophilic), cyathostomiasis, chronic colitis (salmonella sp.), multisystemic eosinophilic epitheliotropic disease, food allergies, and small intestinal bacterial overgrowth. d-xylose absorption tests have some advantages over the oral glucose tolerance test because xylose is not metabolized in the small intestinal mucosa and insulin does not influence its absorption. gastric and intestinal motility, intraluminal bacterial overgrowth, and renal function still influence xylose absorption, because the kidneys clear xylose. the other main drawback to d-xylose is that it is generally available only in research settings. however, xylose measurements are available at most major universities. a normal d-xylose absorption curve should peak between and mg/dl at to minutes after dosing. decreased xylose absorption can occur in horses with inflammatory bowel disease, lymphosarcoma, multisystemic eosinophilic epitheliotropic disease, cyathostomiasis, extensive small intestinal resections, and any cause of villous atrophy. maldigestion is a common occurrence in foals with diarrhea. bacteria (especially clostridium sp.) and viruses (especially rotavirus or coronavirus) may invade and destroy the villous epithelial cells that manufacture lactase and other disaccharidases, resulting in an inability to digest lactose. in this case, continued ingestion of the mare's milk may cause an osmotic diarrhea, which may exacerbate the underlying enterocolitis. one can perform lactose tolerance testing to assess the degree of maldigestion. one administers d-lactose at g/kg as a % solution via nasogastric tube and measures glucose concentrations in the blood at , , , , , , , , and minutes. a normal curve shows doubling of glucose levels compared with baseline by minutes after administration. accurate measurement of gastric emptying can be difficult to assess. several methods are currently available. multiple diagnostic imaging techniques have been used to study gastric emptying times. one can use contrast radiography to assess gastric emptying in foals. in the normal foal, barium remains in the stomach for varying amounts of time, but a significant amount should be gone within hours. gastric emptying of solid, nondigestible, radiopaque markers also has been used in adult horses and ponies, but the results were variable and unpredictable even in the normal horse. nuclear scintigraphy is used commonly in human beings to measure gastric emptying and can be used in horses where available. this technique requires oral administration of m tc pentenate ( mci), and serial images taken of the cranial abdomen. the tracer is usually not visible minutes after administration in normal horses. alternatively, if nuclear scintigraphy is not available, one can use acetaminophen absorption testing as an indirect determination of gastric emptying. , one performs this test by administering mg/kg of acetaminophen orally and measuring subsequent blood values and calculating the time to reach maximum serum concentrations and the absorption constant. in human beings, the proximal small intestine absorbs almost all of the acetaminophen. the median time to reach peak plasma levels using acetaminophen absorption in horses was . minutes. one often requires histopathologic examination of tissues from the intestine to diagnose chronic inflammatory, infiltrative, or neoplastic conditions, and such examination can be useful in evaluating the extent of injury after obstruction or ischemia. rectal mucosal biopsies are easy to collect with few complications. however, to collect a full-thickness biopsy of the intestine requires a surgical approach (flank or ventral midline approach). laparoscopy offers a safer technique to observe the large intestine and other abdominal structures. one can obtain biopsies of masses, lymph nodes, mesentery, or intestinal serosa via laparoscopy and mucosal biopsies of the upper gastrointestinal tract via endoscopy. other diagnostics, specifically laparoscopy and computed tomography, are available but require specialized equipment and personnel with specific training. flexible or rigid endoscopes used for laparoscopic evaluation of the abdomen allow for visualization of visceral organs and potentially for collection of biopsy material from masses or organs. full-thickness biopsies of the intestines are not routinely possible through the laparoscope and usually require flank or ventral midline laparotomy. the laparoscopic procedure can be done in the standing or recumbent horse. advantages of this technique over a flank or ventral midline celiotomy include smaller incisions, less healing time, and less procedure time. disadvantages include the large amount of equipment needed, skill involved, and the limitation as a diagnostic modality, rather than a treatment. clinical applications of diagnostic laparoscopy include rectal tears, percutaneous abscess drainage, assessment of adhesions, displacements, and integrity of the serosa of various bowel segments, and biopsy of abdominal masses. computed tomography scans are available at several universities across the country. they have been used frequently to evaluate dental disease and may be useful in evaluating tumors and masses of the head, larynx, pharynx, and proximal esophagus. computed tomography also has promise for evaluating abdominal disorders in foals. most equipment can accommodate up to lb. restrictions to computed tomography as a diagnostic aid include expense, availability, expertise, and weight and size limitation. permeability increases, cells are recruited rapidly into the tissue, plasma protein cascades are activated, and a myriad of soluble products are released that coordinate the response, trigger innate and adaptive immunity, and mobilize reparative elements. although the cellular and vascular response and the secreted mediators of inflammation are important for killing pathogens and limiting invasion of injured tissues by commensal organisms, they can be damaging to host cells and proteins if not tightly regulated. thus if the inciting stimulus is not eliminated quickly, the inflammatory response itself causes significant tissue injury. the mechanism regulating inflammation has been the focus of much research to identify therapeutic targets to modulate the damage to host tissues during many gastrointestinal diseases. recent work has provided some of the molecular and cellular details of this complex physiology and has led to novel therapeutic strategies for treating inflammation. the gastrointestinal epithelium interfaces with a luminal environment inhabited by potentially hostile microbial organisms. the epithelium presents a physical barrier to invasion by the flora of the gastrointestinal tract, consisting of the apical cellular membrane, intercellular tight junctions the permeability of which is highly regulated, and a secreted layer of mucus. breaching of the mucosal barrier by invading pathogens generates potent soluble and neural signals that initiate an inflammatory response. conceptually, the epithelium can be thought of as a sensory organ detecting pathogen invasion to trigger an appropriate host defense and reparative response. noninfectious mucosal injury or invasion of epithelial cells by pathogenic organisms such as salmonella activates synthesis of proinflammatory chemokines (chemoattractants) by epithelial cells and triggers a robust influx of neutrophils into the tissue within hours of the damage. of the chemoattractants produced by epithelium, interleukin- (il- ) has a particularly important role in initiating inflammation by recruiting neutrophils from blood [ ] [ ] [ ] and regulating neutrophil migration through tissue matrix adjacent to epithelium. , bacteriaderived formylated chemotactic peptides also act as potent chemoattractants that are fully capable of stimulating a robust inflammatory response in the intestine if the epithelial barrier permits the diffusion of the peptides across the mucosa. epithelial cells activated during infection produce cytokines such as tumor necrosis factor α (tnf-α), arachidonic acid metabolites, and other proinflammatory mediators that activate recruited leukocytes. bacterial products, particularly lipopolysaccharide and other cell wall components, are potent activators of leukocytes recruited into the tissue. once the inflammatory response has been initiated, tnf-α, il- β, and other proinflammatory products of neutrophils, monocytes, mast cells, and epithelial cells amplify the inflammatory response. the enteric nervous system has a key role in sensing and regulating inflammatory responses in the intestine. for example, clostridium difficile toxin a activates a neural pathway that triggers mast cell degranulation and neutrophil influx into the tissue. , blockade of this neural pathway is sufficient to abolish the profound inflammatory response induced by toxin a and many of the effects of toxin a on enterocyte secretion. other pathogens and immune-mediated hypersensitivity reactions similarly stimulate inflammation by mechanisms that involve the enteric nervous system. thus the epithelium interacts in a highly complex manner with the intestinal milieu, the enteric nervous system, and inflammatory cells to regulate the tissue response to injury and infection. resident macrophages located in the lamina propria, submucosa, and intestinal lymphoid organs are among the first cells beyond the epithelium to respond to infection or injury. macrophages are activated by bacterial products via pattern recognition receptors and begin to produce proinflammatory molecules important for recruiting and activating neutrophils and monocytes. pattern recognition receptors recognize microbial products ranging from lipopolysaccharide to peptidoglycan and even cpg-containing bacterial dna and signal the invasion by pathogens. of the pattern recognition receptors, the lipopolysaccharide receptor complex is perhaps the best defined. lipopolysaccharide activates macrophages via the cd -toll-like receptor complex to initiate transcription of the inflammatory cytokines tnf-α and il- β, which synergize with lipopolysaccharide to amplify the macrophage response. lipopolysaccharide, particularly in concert with inflammatory cytokines, stimulates macrophages to produce copious amounts of nitric oxide, which is microbicidal and vasoactive. nitric oxide and other nitrogen radicals react with reactive oxygen intermediates (rois) to produce some of the most toxic molecules of the host defense system: the peroxynitrites. il- is produced as well to recruit neutrophils. as the response progresses, other inflammatory mediators, particularly the arachidonic acid-derived lipids, are produced. these lipids have potent vasoactive effects and are important stimuli of endothelial cells, neutrophils, and platelets. four important changes occur in the intestinal vasculature during inflammation: . alteration of blood flow . increased vascular permeability . increased adhesiveness of endothelial cells, leukocytes, and platelets . exposure of the basement membrane and activation of the complement, contact, and coagulation cascades a wide range of mediators alters blood flow during intestinal tract inflammation, from gasses such as nitric oxide (a major vasodilator of the intestinal vasculature) to lipids (prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor), cytokines, bradykinin, histamine, and others. the major sources for these mediators include activated leukocytes, endothelial cells, epithelial cells, and fibroblasts. the primary determinant of blood flow early in inflammation is vascular caliber, which initially decreases in arterioles, but then quickly changes to vasodilation coincident with opening of new capillary beds, increasing net blood flow. the increase in blood flow is short lived, for the viscosity of the blood increases because of fluid loss and tissue edema through leaky capillaries. leukocyte margination, platelet adhesion to endothelial cells and exposed matrix, and areas of coagulation protein accumulation further decrease local circulation. inflammatory mediator actions on the endothelial cells initially increase vascular permeability. histamine, leukotrienes, platelet-activating factor, prostaglandins, bradykinin, and other mediators stimulate endothelial cell contraction, and interendothelial gaps form. , this stage of increased vascular permeability is readily reversible. concurrently, mediators such as the cytokines tnf-α and il- β induce a structural reorganization of the interendothelial junctions, resulting in frank discontinuities in the endothelial monolayer. cytokines also stimulate endothelial cells to express adhesion molecules that support adhesion of leukocytes and platelets, leading to the next and perhaps most devastating event. leukocytes (primarily neutrophils) and platelets adhere to exposed basement membranes and activated endothelial cells. adherent neutrophils and platelets then are exposed to the mediators of inflammation present in the surrounding milieu, which activates the cells to release oxidants and proteases (particularly elastase) that injure the endothelium and have the potential to cause irreparable harm to the microvasculature. [ ] [ ] [ ] marginated neutrophils begin to transmigrate between endothelial cells (as described in later sections), and if their numbers section . pathophysiology of gastrointestinal inflammation are large enough, they disrupt the integrity of the interendothelial junctions, worsening the vascular leakage. conceptually, these stages of enhanced vascular permeability can be thought of as a mechanism to allow plasma proteins to enter the tissues and to potentiate the critical influx of leukocytes into tissues. however, if not regulated precisely, alterations in hydrostatic and oncotic forces and irreversible damage to the vascular bed may have devastating consequences. moreover, inappropriate activation of plasma protein cascades and leukocytes by activated endothelium and exposed matrix proteins can contribute to systemic inflammation (systemic inflammatory response syndrome; see chapter . for more information) characterized by hypotension, generalized vascular leak syndrome, and multiorgan dysfunction, which may be fatal. phosphodiesterase inhibitors reduce endothelial permeability in ischemia-reperfusion injury and other models of inflammation-induced vascular leakage , by increasing endothelial tight junction integrity and thus may be a viable therapeutic strategy to prevent or reduce the permeability alterations associated with inflammation. endothelial cells respond to products of activated epithelial cells and macrophages in the intestinal tissue to recruit cells and humoral mediators of inflammation into the tissue. activated endothelial cells display a range of molecules critical for neutrophil and platelet adhesion. intercellular permeability increases to expose basement membrane proteins that trigger humoral defense systems (complement, coagulation, and contact system cascades) and to provide access for these macromolecules to the tissue. endothelial cells are an important source of inflammatory mediators that amplify the response and vasoactive substances (particularly nitric oxide) that alter blood flow. infection or injury to the gastrointestinal mucosa causes an influx of leukocytes from the blood that lay the foundation of the inflammatory response. neutrophils, being the first to arrive during inflammation, have a dominant role in the acute response. within minutes, neutrophils are recruited into the tissue where they are activated to release products that not only are proinflammatory and lethal to pathogens but also may damage host cells and tissues. not surprisingly, much attention has been paid to the role of neutrophils in the pathophysiology of many inflammatory conditions. neutrophil depletion is protective in many models of gastrointestinal inflammatory disease. of interest to clinicians, blockade of neutrophil migration into inflamed tissues prevents many of the pathophysiologic events associated with infectious enteritis, ischemia-reperfusion injury, and other gastrointestinal diseases. , [ ] [ ] [ ] [ ] [ ] neutrophil transendothelial migration is a multistep process that is temporally and spatially regulated and has a degree of cell type specificity ( figure . - ) . the predominant sites of neutrophil transendothelial migration are in the postcapillary venules and, in some tissues, the capillaries. endothelial cells in these vessels respond to cytokines and other soluble signals by expressing molecules that promote neutrophil adhesion and transmigration, including selectins and counterreceptors for integrins. as neutrophils flow through these vessels, they are first tethered to activated endothelium. tethering is mediated by selectin molecules expressed on neutrophils (l-selectin) and on activated endothelial cells (p-and e-selectins) that bind to p-selectin glycoprotein ligand- (psgl- ), e-selectin ligand- (esl- ), and other mucin counterreceptors. , tethering functions to increase the exposure of the neutrophil to activating chemokines presented on the surface of the endothelial cells. stimulation of neutrophils by il- and other chemokines activates the second step of transendothelial migration. chemokine binding to their receptors on the neutrophil generates signals that activate the binding of integrin adhesion receptors to their ligands, called intracellular adhesion molecules or vascular cell adhesion molecules expressed on endothelial cells in inflamed mucosa. integrin ligation to cellular adhesion molecules arrests the tethered neutrophils, resulting in firm adhesion to the endothelium. of the integrins expressed on neutrophils, the β integrins have a particularly important role in transendothelial migration. calves and human beings with a disorder known as leukocyte adhesion deficiency illustrate the requirement for β integrinmediated adhesion in neutrophil function. leukocyte adhesion deficiency results from an autosomal recessive trait causing the lack of the β integrin expression. the neutrophils from these individuals cannot migrate into most tissues and do not function normally, resulting in poor tissue healing and profound susceptibility to infection, especially at epithelial barriers. , other integrins also have a role in transendothelial migration. β integrins mediate transendothelial migration in some cells and seem to be particularly important for mediating emigration of monocytes into many tissues. following this firm adhesion step, neutrophils migrate through the endothelium along a chemotactic gradient of il- and other chemoattractants such as leukotriene b . , , neutrophils migrate across the endothelial monolayer at intercellular junctions via a mechanism involving a series of integrin-ligand interactions mediated by β and β integrins and other adhesion molecules that is generally capable of maintaining the integrity of the endothelial barrier. however, massive flux of neutrophils through the endothelium alters endothelial tight junctions and injures the basement membrane, resulting in increased endothelial permeability to molecules as large as plasma proteins and even endothelial cell detachment from the basement membrane. , nonintegrin molecules such as platelet/endothelial cell adhesion molecules (pecams) also are involved in transendothelial migration of neutrophils. homotypic binding of pecams on adjacent endothelial cells form part of the intercellular junction. neutrophils express an integrin of the β family that can bind pecam, and via sequential binding of β integrins to pecam, the neutrophil can , and other proinflammatory mediators. endothelial cells stimulated by inflammatory mediators produce chemoattractants (such as il- ) and display adhesion molecules that promote neutrophil emigration. the three steps of neutrophil (polymorphonuclear [pmn]) emigrationcapture/rolling (mediated by selectins), adhesion (mediated by β integrins), and transendothelial migration (mediated by integrins and platelet/endothelial cellular adhesion molecule [pecam])-occur on activated endothelium. chemoattractant molecules, such as il- trigger neutrophil emigration. in inflamed tissues, cytokines (il- β and tnf-α) and a variety of other proinflammatory mediators stimulate the neutrophil oxidase complex to produce reactive oxygen intermediates (rois; o and h o and their derivatives). activated neutrophils degranulate to release proteases and other hydrolases, cationic peptides (defensins), myeloperoxidase, and other products into the tissue. activated neutrophils synthesize a variety of inflammatory mediators, including prostaglandins (pge ) that modulate the inflammatory response. the products of activated neutrophils (rois, proteases, and mediators) stimulate epithelial secretion and alter tight junction permeability, promoting diarrhea. neutrophils eventually migrate across the infected epithelium by a mechanism that involves integrins, disrupting tight junction integrity and increasing permeability to bacterial products, thus exacerbating the inflammatory response. "unzip" the intercellular junction and migrate through, closing it behind itself. a key feature of neutrophils and other leukocytes is the requirement for integrin-mediated adhesion to extracellular matrix proteins (ecms) or other cells to achieve an optimal effector phenotype. critical components of the ecms in inflamed tissues include fibronectin, fibrinogen, and vitronectin, deposited in tissues as a result of plasma leakage and by synthesis of new proteins by stromal cells and resident macrophages in response to inflammatory mediator activation. the changing composition of the matrix proteins deposited in tissues during inflammation serves as a clue as to the nature of the tissue environment for recruited inflammatory cells as they become activated. individual gene expression studies have demonstrated that adhesion to matrix proteins induces the expression of cytokines and chemokines and their receptors, arachidonic acid-derived lipid mediator synthases, metalloproteinases, growth factors, transcription factors, and other genes that influence the differentiation and activation of inflammatory cells. roi production, phagocytosis, degranulation, and other effector functions stimulated by inflammatory mediators and bacterial products are optimal only when neutrophils adhere to the ecms. adhesion to distinct ecm proteins selectively activates signaling pathways and gene expression of neutrophils, monocytes, and other leukocytes with differing abilities to promote certain functions such that the composition of ecms in many ways controls the development of the ultimate effector phenotype. thus integrin-mediated adhesion provides a mechanism by which neutrophils and other leukocytes can sense the complex tissue environment and respond appropriately. of the activators of neutrophils at sites of inflammation, complement (c -opsonized particles), cytokines (tnf-α and il- β), platelet-activating factor, immune complexes, and bacterial products are among the most potent stimuli. other mediators produced during inflammation may modify neutrophil activity, particularly formylated bacterial peptides, chemokines, complement fragments (c a), leukotriene b , and prostaglandins. activated neutrophils are highly phagocytic; produce large amounts of rois; degranulate to release myeloperoxidase, cationic antimicrobial peptides (defensins), serine proteases (mainly elastase), and metalloproteinases; and secrete inflammatory mediators (tnf-α, il- β, prostaglandins, leukotrienes, and others) (see figure . - ). mast cells strategically reside in mucosal tissues, including the submucosa and lamina propria of the gastrointestinal tract, and constitute a crucial first line of defense at epithelial barriers. however, they are also important effector cells of the pathophysiology of inflammatory gastrointestinal diseases. experimental depletion of mast cells, genetic deficiency in the development of mast cells, or pharmacologic stabilization of mast cells to prevent degranulation have a protective effect in a variety of models of gastrointestinal inflammatory disease, including dextran-or trinitrobenzenesulfonic acid-induced colitis, , ischemia-reperfusion injury, , and immediate hypersensitivity responses. mast cells are activated by a wide variety of microbial products and host-derived mediators. among the activators of mast cells the so-called anaphylatoxins (complement fragments c a, c a, and c a), are potent stimuli causing release of mediators of inflammation. in addition, mast cells are the primary effector cells of immunoglobulin e-mediated anaphylaxis (type i hypersensitivity reactions) by virtue of their high affinity receptors for immunoglobulin e. cross-linking of receptor-bound immunoglobulin e on mast cell surface by antigens (i.e., food antigens) causes rapid degranulation, resulting in the explosive release of granule contents. neural pathways in the intestine also regulate mast cells. mast cells respond to enteric pathogen invasion via neural reflexes that stimulate the release of inflammatory mediators. activated mast cells release preformed histamine, -hydroxytryptamine, proteases, heparin, and cytokines from granules. activation also stimulates de novo synthesis of a range of inflammatory mediators, including prostaglandins, platelet-activating factor, and leukotrienes. transcription of a number of peptide mediators, such as the cytokines tnf-α and il- β among many others, also increases on stimulation of mast cells. mast cell products have profound effects on the vasculature, increasing endothelial permeability and causing vasodilation. moreover, mast cell-derived mediators greatly enhance epithelial secretion by a mechanism that activates neural pathways of epithelial secretion and directly stimulates epithelial cells. mast cell products significantly alter intestinal motility, generally increasing transit and expulsion of intestinal contents. mast cell-derived leukotrienes and tnf-α also have a crucial role in host defense against bacterial pathogens, acting to recruit and activate neutrophils. , mast cells have a newly identified role in host defense and inflammatory responses to bacterial pathogens, which in part is caused by the release of proinflammatory mediators during bacterial infection, which is critical for recruiting and activating other innate host defense cells such as neutrophils. however, mast cells are also phagocytic, have microbicidal properties, and can act as antigen-presenting cells to the adaptive immune system. although accumulating evidence was establishing the role of mast cells in innate immunity, a seminal study that unconditionally identified the importance of mast cells in host defense demonstrated that mast cell-deficient w/ w v mice have impaired responses to gram-negative bacterial peritonitis, resulting in a significant increase in mortality. the role for mast cells in host protective responses appears to be as a sensor of bacterial invasion. unlike immunoglobulin e-mediated responses, bacterial products seem to elicit a highly regulated and selective response from mast cells. the complement cascade is a fundamental part of the inflammatory response. activation of the complement cascade by immune complexes (classical pathway) or by bacteria or bacterial products, polysaccharides, viruses, fungi, or host cells (alternative pathway) results in the deposition of complement proteins on the activating surface and the release of soluble proteolytic fragments of several complement components. in particular, activation of either pathway results in the deposition of various fragments of the complement protein c , which are potent activators of neutrophils and monocytes. opsonization of particles with c fragments constitutes a major mechanism of target recognition and phagocyte activation. during the activation of the complement cascade culminating in deposition of c , soluble fragments of c (c a), c (c a), and c (c a) are liberated. these fragments, termed anaphylatoxins, have potent effects on tissues and cells during inflammation. perhaps most notably, anaphylatoxins are chemotactic for neutrophils (particularly c a), activate neutrophil and mast cell degranulation, and stimulate roi release from neutrophils. the termination of the complement cascade results in the formation of a membrane attack complex in membranes at the site of complement activation, and if this occurs on host cells such as endothelium, it may cause irreversible cell injury. although the primary source of complement is plasma, epithelial cells of the gastrointestinal tract also produce c , suggesting that local production and activation of the complement cascade during inflammation occurs in intestinal tissues. clearly, if the regulatory mechanisms of the complement cascade fail, then the inflammatory response may be inappropriate and tissue injury can occur. the role of complement in gastrointestinal inflammation has been most studied extensively in models of ischemia-reperfusion injury. activation of the complement cascades has a major role in altered endothelial and epithelial permeability in these models. several lines of evidence support the importance of complement in intestinal injury. mice deficient in c or c are protected against ischemia-reperfusion injury. moreover, administration of monoclonal antibodies against c reduced local and remote injury and inflammation during intestinal reperfusion injury in a rat model. administration of a soluble form of complement receptor , a regulatory protein that halts the complement cascade by dissociating c and c on host cell membranes, reduced mucosal permeability, neutrophil influx, and leukotriene b production during ischemia-reperfusion injury in rats and mice. , although neutrophils and mast cells mediate many of the pathophysiologic effects of the complement cascade, the membrane attack complex may have a primary role in altered vascular permeability during ischemia-reperfusion injury. four components initiate the contact system of coagulation: hageman factor (hf), prekallikrein, factor xi, and high-molecular-weight kininogen. hf is a large plasma glycoprotein that binds avidly to negatively charged surfaces. bacterial cell walls, vascular basement membranes, heparin, glycosaminoglycans, and other negatively charged surfaces in the intestine capture hf and the other three important initiators of the contact system in a large multimolecular complex. of the surfaces that bind hf, the extracellular matrix is a potent activator of the contact system. once bound, hf is converted to hf-α, which cleaves prekallikrein to kallikrein and factor xi to factor xia. the ultimate result is further cleavage of hf by kallikrein and triggering of the contact system cascade, activation of intrinsic coagulation by factor xia, activation of the alternative pathway by hf, and proteolytic cleavage of high-molecular-weight kininogen by kallikrein, releasing biologically active kinins. the products of the contact system, particularly bradykinin, have several important biologic properties that drive many of the vascular and leukocytic responses during inflammation. bradykinin induces endothelial cell contracture and intracellular tight junction alterations that increase vascular permeability to fluid and macromolecules. bradykinin also affects vascular smooth muscle contracture, resulting in vasoconstriction or vasodilation depending on the location. bradykinin also increases intestinal motility, enhances chloride secretion by the intestinal mucosa, and intensifies gastrointestinal pain. in neutrophils, kinins stimulate the release of many inflammatory mediators, including cytokines, prostaglandins, leukotrienes, and rois. kallikrein cleaves c to release c a, a potent chemotactic factor for neutrophils, and thus has a role in recruiting and activating inflammatory leukocytes. the plasma kallikrein-bradykinin system is activated in a variety of acute and chronic inflammatory diseases of the gastrointestinal tract. , recent evidence has demonstrated that blockade of the pathophysiologic effects of bradykinin has clinical applications. oral or intravenous administration of the bradykinin receptor antagonist icatibant reduces the clinical signs, onset of diarrhea, and many of the histopathologic changes in experimental models of colitis in mice. inhibition of kallikrein by oral administration of p attenuated the intestinal inflammation, clinical score, and systemic manifestations in a model of chronic granulomatous enterocolitis. thus the contact system is a viable therapeutic target for inflammatory diseases of the intestine. changes in blood flow to the mucosa and other regions of the intestine that reduce perfusion of the tissues can potentiate the initial damage caused by infection or injury. for example, reperfusion of ischemic tissues is associated with platelet and neutrophil clumping in the small vessels of the mucosa, which can impede blood flow. platelets are activated and adhere to exposed basement membrane and activated endothelial cells and provide a surface for leukocyte adhesion. the accumulation of platelets and leukocytes can reduce vessel diameter and blood flow significantly while potentiating local coagulation and thrombus formation. soluble mediators released by activated leukocytes and endothelial cells also affect blood flow. histamine and the vasoactive lipids derived from arachidonic acid (leukotrienes, prostaglandins, thromboxane, prostacycline, and platelet-activating factor) have a prominent role in regulating local perfusion during inflammation and also may have systemic effects on blood flow. procoagulant mediators released by inflammatory cells in response to the inflammatory process (i.e., tissue factor produced by macrophages or endothelial cells), exposed basement membrane proteins, and bacterial components can trigger the contact system and the coagulation and complement cascades, the products of which affect blood flow. nitric oxide, whether produced by endothelial cells or leukocytes (macrophages), is a potent regulator of blood flow and has a significant role in the control of perfusion during inflammation. many of the mediators that affect perfusion also affect endothelial permeability, altering osmotic and hydrostatic balance and tissue edema. in extreme cases, local and systemic coagulopathies initiated by vascular injury and absorption of microbial products and inflammatory mediators induce a hypercoagulable state, leading to microthrombus formation, which can reduce blood flow, or macrothrombus formation, which causes tissue infarction. the cellular mediators of inflammation have the potential to inflict severe injury to intestinal tissues. neutrophils have an important role in the pathophysiology of many intestinal diseases, including ischemia-reperfusion injury, , infectious enterocolitis, [ ] [ ] [ ] nonsteroidal antiinflammatory drug-induced mucosal ulceration, and others. depletion of neutrophils, blockade of their emigration into tissues, or inhibition of neutrophil activation reduce the severity of these and other inflammatory diseases. thus many antiinflammatory therapies are emerging that specifically target neutrophil adhesion, migration, and activation. migration of neutrophils through endothelium during emigration into inflamed tissues is remarkable in that the permeability of the endothelial monolayer is preserved under most circumstances. however, a limit exists above which neutrophil migration alters the permeability characteristics of the endothelium. the effect is in part physical in that mere movement of large numbers of neutrophils through the endothelium is sufficient to disrupt the tight junctions mechanically and is caused in part by toxic products of neutrophils that damage endothelial cells and basement membranes. , serine proteases (particularly elastase) and metalloproteinases released by degranulating neutrophils destroy tissue matrix proteins and cell-surface proteins that make up endothelial intercellular junctions. these degradative enzymes are particularly damaging to basement membranes and the cellular barriers of the endothelium, thus contributing to vascular permeability (and local tissue edema) and thrombosis. the permeability may be affected to the extent that not only water but macromolecules (albumin, matrix proteins, complement, etc.) leak into the interstitium. blockade of neutrophil adhesion to endothelium with anti-β integrin antibodies has a sparing effect on the microvasculature in experimental intestinal ischemiareperfusion injury, reducing the alterations in vascular permeability and histopathologic evidence of microvascular damage. similar to the endothelium of inflamed tissues, massive neutrophil transmigration occurs across the epithelium in response to infection or injury. neutrophil transepithelial migration increases epithelial permeability by disrupting tight junctions. like the endothelium, neutrophils disrupt the epithelial barrier mechanically as they migrate through (see figure . - ). proteases, particularly elastase, degrade basement membrane components and tight junction proteins. products of activated neutrophils (tnf-α and interferon-γ) increase tight junction permeability by direct effects on the enterocytes. prostaglandins released by activated neutrophils stimulate epithelial secretion, thus contributing to diarrhea. subepithelial accumulation of neutrophils can lead to deadhesion of the epithelial cells from the basement membrane and mild to severe ulceration. the physiologic results of the effects of neutrophils and their products on the epithelial barrier include protein-losing enteropathy and absorption of bacterial cell wall constituents, which potentiates the local and systemic inflammatory responses. neutrophils in inflamed tissues stimulated by potent host-derived activators (such as il- β and tnf-α) and bacterial products (lipopolysaccharide) release copious amounts of rois (see figure . - ). although these oxygen and oxyhalide radicals are important for killing pathogens, they are also potentially toxic to epithelial and endothelial cells and matrix proteins. reactive nitrogen intermediates produced primarily by macrophages during inflammation combine with rois to form peroxynitrites, which are particularly toxic. in addition to injury to mucosal tissues, rois also have an as yet ill-defined role in recruiting and activating neutrophils, thereby potentiating the inflammatory response. in support of the role of rois in inflammatory diseases of the gastrointestinal tract, administration of inhibitors of roi production or pharmacologic roi scavengers can be protective in many models of reperfusion injury or enterocolitis. many therapies are aimed at inhibiting neutrophil activation and effector functions in tissues have been evaluated for use in intestinal diseases. phosphodiesterase inhibitors, by causing cyclic adenosine monophosphate accumulation in neutrophils, are antiinflammatory by virtue of their ability to suppress neutrophil activation and roi production. new phosphodiesterase inhibitors selective for the predominant neutrophil isoform of phosphodiesterase hold promise for use in many inflammatory diseases. subepithelial mast cells also have an important role in altering epithelial permeability in inflamed intestine. during the intestinal hypersensitivity response, subepithelial mast cell release of mast cell protease ii by degranulation increases epithelial permeability via an effect on tight junctions. , , this alteration in tight junction permeability results in enhanced transepithelial flux of macromolecules, including proteins and bacterial products. cytokines released by mast cells and phagocytes also regulate tight junction permeability. interleukin- , a product of mast cells and macrophages, increases epithelial permeability. moreover, tnf-α and interferon-γ, products of many inflammatory cells, synergistically increase tight junction permeability. acute equine colitis causes rapid, severe debilitation and death in horses (more than % of untreated horses die or require euthanasia). since , several reports have described a number of different acute diarrheal conditions in the horse that appear to share a common characteristic clinical presentation. - diarrhea associated with acute equine colitis occurs sporadically, is highly fatal, and is characterized by intraluminal sequestration of fluid, moderate to severe colic (abdominal pain), and profuse watery diarrhea with resultant endotoxemia, leukopenia, and hypovolemia. , , the condition can affect adult horses of all ages but usually occurs in horses between and years of age. disease onset is sudden with a rapid progression and often is preceded by a stressful event. a definitive diagnosis is made in only about % to % of cases. , most ante-and postmortem diagnostic tests remain speculative. treatment of the condition in horses is costly because of the massive fluid therapy required. currently, no curative treatment is available for acute colitis in horses, human beings, or other mammals. treatment regimens provide rehydration, electrolyte and plasma protein replacement, mitigation of the effects of circulating endotoxin, and antimicrobial therapy when indicated. attempts during the past years to develop appropriate treatments (i.e., vaccines or pharmacologic agents) have been hampered by the unavailability of acceptable equine models and have been unsuccessful because of the complex pathophysiology of the intestinal epithelium. although the mechanisms responsible for the fluid losses are not known, inflammatory cells may play an integral role because this condition is characterized by large numbers of granulocytes infiltrating the large intestinal mucosa. [ ] [ ] [ ] [ ] [ ] equine cecal and colonic tissues collected during the acute stages of experimentally induced acute equine colitis (equine ehrlichial colitis, lincomycin with and without clostridium spp. inoculation, nonsteroidal antiinflammatory drug administration) reveal the presence of numerous neutrophils and eosinophils in the lamina propria and submucosa. , , , granulocytederived reactive oxygen intermediates are crucial to antimicrobial defenses in the gut and stimulate chloride and water secretion by interactions with enterocytes. , normal equine intestinal tissue is unique compared with that in most other mammalian species for a preponderance of eosinophils located in the intestinal mucosa and submucosa. , production of reactive oxygen intermediates by stimulated phagocytic granulocytes following mucosal barrier disruption may be responsible for the massive fluid secretory response that occurs during the early stages of acute equine colitis. colitis refers to inflammation and mucosal injury of the colon and cecum (typhlocolitis) that may occur in response to a number of causes. , the cause of the colonic injury may be well-defined such as in naturally occurring infectious or experimentally induced colitis. however, many cases of human and animal diarrhea have a speculative or unknown diagnosis or no diagnosis. , , irrespective of the underlying or initiating cause of colonic injury, the colon apparently has a limited repertoire of responses to damage because most forms of colitis demonstrate similarities in histopathologic appearance and clinical presentation. various degrees of mucosal erosion and ulceration, submucosal/mucosal edema, goblet cell depletion, and presence of an inflammatory cellular infiltrate within the mucosa and submucosa are common to many types of human and animal colitis. , [ ] [ ] [ ] characteristic clinical manifestations include intraluminal fluid sequestration, abdominal discomfort, hypovolemia, and most often profuse, watery diarrhea. large bowel diarrhea results from abnormal fluid and ion transport by cecal and colonic mucosa. loss of fluid by the large intestine can result from malabsorptive or hypersecretory processes and is often a combination of the two. colonic secretory processes are a function of the crypt epithelium, whereas absorptive processes are limited to surface epithelial cells. under normal baseline conditions, an underlying secretion by crypt epithelium is masked by a greater rate of surface epithelial cell absorption. abnormal forces influencing the rates of secretion and absorption can result in massive, uncontrolled secretion and malabsorption by large intestinal mucosal epithelial cells, leading to rapid dehydration and death. , two intracellular processes control colonic secretion: the cyclic nucleotide (cyclic adenosine monophosphate [camp] and cyclic guanosine monophosphate [cgmp]) and the calcium system. , agents may activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e [pge ]) or guanyl cyclase (bacterial enterotoxins) and induce increases in camp or cgmp, respectively. this reaction causes phosphorylation of specific protein kinases that induce the actual apical and basolateral membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotide-dependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. , calcium may act through calmodulin, which then can activate membranephosphorylating protein kinases. at least four central systems control intestinal secretion: ( ) the hormonal system, ( ) the enteric nervous system, ( ) bacterial enterotoxins, and ( ) the immune system. , hormonal control of colonic electrolyte transport is exerted primarily through the renin-angiotensinaldosterone axis. , the enteric nervous system controls transport through three separate components: ( ) extrinsic nerves of the parasympathetic and sympathetic pathways; ( ) intrinsic ganglia and nerves, secreting a variety of neurotransmitters including peptides; and ( ) neuroendocrine cells (intraepithelial lymphocytes) that reside in the epithelium and release messengers onto the epithelial cells in a paracrine manner. , [ ] [ ] [ ] [ ] many bacterial enterotoxins can induce intestinal secretion by camp or cgmp signal transduction. bacterial enterotoxins can stimulate enteric neurons, providing evidence for interaction between two controlling systems. preformed inflammatory mediators such as histamine, serotonin, or adenosine and newly synthesized mediators such as prostaglandins, leukotrienes, platelet-activating factor, various cytokines, the inducible form of nitric oxide, and reactive oxygen metabolites can initiate intestinal secretion by directly stimulating the enterocyte and by acting on enteric nerves indirectly to induce neurotransmitter-mediator intestinal secretion. for instance, when added to the t colonic cell line, the known mast cell mediators histamine, adenosine, and pgd induce chloride secretion. [ ] [ ] [ ] prostaglandins of the e and f series can cause an increase in chloride secretion in intact tissue and isolated colonic cells. [ ] [ ] [ ] leukotrienes, platelet-activating factor, and a number of cytokines have been shown to have no effect on t cell secretion but have a significant effect on electrolyte transport in intact tissue, suggesting that intermediate cell types may be involved in these secretory responses. [ ] [ ] [ ] the epithelial cell chloride secretory response occurs via prostaglandin-and adenosine-mediated increases in cellular camp, whereas histamine acts by h receptor induction of phosphatidylinositol turnover, production of inositol triphosphate, and mobilization of intracellular calcium stores. , lipoxygenase products (leukotrienes) are capable of activating a colonic secretory response and do not appear to involve the cyclic nucleotides or calcium ions. phagocyte-derived reactive oxygen mediators (roms) can induce colonic electrolyte secretion in vitro, suggesting that oxidants may contribute directly to the diarrhea associated with colitis. [ ] [ ] [ ] [ ] [ ] reactive oxygen species initiate the secretory response by increasing cellular camp or stimulating mesenchymal release of pge or prostacyclin, which in turn stimulates the epithelial cell or enteric neuron, respectively. [ ] [ ] [ ] [ ] [ ] [ ] sodium nitroprusside, an exogenous source of nitric oxide, stimulated an increase in chloride secretion in rat colon that was mediated by cyclooxygenase products and enteric neurons. table . - summarizes inflammatory mediator-induced epithelial cell chloride secretion. acute colitis rarely develops by a simple cause or effect phenomenon but is influenced by many extrinsic and intrinsic host and microorganism factors. inflammatory mediators released from mast cells and monocytic or granulocytic phagocytes cause intestinal chloride and water secretion and inhibit neutral sodium and chloride absorption. , , inflammatory cells, particularly the phagocytic granulocytes, play an important role in mucosal pathophysiology in cases of colitis. , large numbers of these cells are observed on histopathologic examination of tissues from human and animal cases of colitis. products of cell activation stimulate direct and indirect secretory responses in intestinal cells and tissues. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] products of phagocyte secretion may amplify the inflammatory signal or have effects on other target cells in intestine such as enterocytes and smooth muscle cells (table . - ). the nadph-oxidase system of phagocytes (neutrophils, eosinophils, monocytes/macrophages) is a potent inducer of superoxide radicals used as a host defense mechanism to kill invading microorganisms. , during inappropriate stimulation such as inflammation, trauma, or ischemia followed by reperfusion, increased levels of toxic oxygen species are produced, causing damage to host tissues. engagement of any of several receptor and nonreceptor types including phagocytosis mediators, chemotactic agents, various cytokines, and microbial products can stimulate phagocytes. resident phagocytes or those recruited to colonic mucosa early in the disease process are considered to augment mechanisms causing fluid and electrolyte secretory processes, a so-called amplification process. , activation of the respiratory burst results in the production and release of large amounts of superoxide anion (o − ) and h o . , in addition to these roms, activated phagocytes secrete peroxidase enzyme (myeloperoxidase from neutrophils and eosinophil peroxidase from eosinophils) into the extracellular space. the peroxidases catalyze the oxidation of clby h o to yield hocl, the active ingredient in household bleach products. the peroxidase-h o -halide system is the most cytotoxic system of the phagocytes; hocl is to times more toxic than o or h o . hocl is a nonspecific oxidizing and chlorinating agent that reacts rapidly with a variety of biologic compounds including dna, sulfhydryls, nucleotides, amino acids, and other nitrogen-containing compounds. hocl reacts rapidly with primary amines (rnh ) to produce the cytotoxic n-chloramines (rnhcl). the mechanisms by which hocl and rnhcl damage cells and tissue remain speculative, but possibilities include direct sulfhydryl oxidation, hemoprotein inactivation, protein and amino acid degradation, and inactivation of metabolic cofactors of dna. peroxidase-derived oxidants have been shown to degrade hyaluronic acid and collagen. in addition, luminal perfusion of specific roms increased mucosal permeability and serosal application caused increases in clsecretion in vitro. tissue myeloperoxidase activity, an index of tissue granulocyte infiltration, is used clinically and experimentally to assess degree of intestinal inflammation. , myeloperoxidase activity is elevated in acute flare-ups of human inflammatory bowel disease and various animal models of acute colitis. [ ] [ ] [ ] [ ] [ ] the acute inflammatory response in these conditions is characterized predominantly by neutrophils, the predominant source of myeloperoxidase activity. however, this assay measures total hemoprotein peroxidase, which includes monocyte and eosinophil peroxidase in addition to neutrophils. moreover, levels of peroxidase activity in equine circulating eosinophils are greater than in circulating neutrophils, and this may apply to resident tissue eosinophils as well. arachidonic acid metabolites are thought to play a role in intestinal inflammation in diarrheal disease. , , elevated levels of these intermediate metabolites have been demonstrated in natural disease and experimental models of colitis and appear to parallel increases in roms in inflamed intestine. addition of h o or hocl to rat colonic tissue in ussing chambers has been shown to induce pge release and active clsecretion. , prostaglandins can stimulate increases in clsecretion in intact intestinal tissue , , and in isolated colonic t cells. , interactions between roms and mesenchymal release of pge /pgi may be relevant to the mechanisms producing the diarrheic condition. fibroblasts co-cultured or juxtaposed to colonic t cells greatly increased the clsecretory response to h o in vitro through the release of pge . in addition, equine colonic mucosa has an increased sensitivity to endogenously released prostaglandin by exhibiting a significant secretory response under in vitro conditions. endotoxin, the lipopolysaccharide component of the outer cell wall of gram-negative bacteria, is present in large quantities in the large intestine of healthy horses. , endotoxins are released into the immediate surroundings when gram-negative bacteria undergo rapid proliferation or die. , the intact bowel forms an effective barrier to the transport of significant amounts of these highly antigenic toxins, but the diseased gut absorbs these macromolecules in large amounts, causing the subsequent adverse systemic effects that are often life threatening. disruption of the intestinal barrier (i.e., ischemia, trauma, inflammatory conditions) overwhelms the capacity of the liver to clear endotoxins, and systemic endotoxemia ensues. endotoxins have been shown to be potent activators of the inflammatory process, stimulating the production and release of numerous cytokines by activated macrophages and other immunocytes. in vitro studies suggest that endotoxin activates phagocytic granulocytes to secrete roms, increase release of lysozymes, and enhance the migratory response to chemotactic stimuli. prostacyclin and thromboxane a mediate hemodynamic dysfunction, and lipoxygenase products may induce tissue ischemia. the cytokine interleukin- causes a febrile response and initiates the acute phase response. tumor necrosis factor contributes to many of the abnormal physiologic responses, particularly hemostatic functions that potentiate coagulopathy. additional mediators include interleukin- , platelet-activating factor, procoagulant mediators, and various other speculated substances. endotoxins trigger mucosal immune cells and subsequent release of inflammatory mediators in cases of colitis. the first report of experimentally induced endotoxemia described clinical signs and hematologic findings that closely paralleled those reported for severe colitis in horses. studies in which endotoxin was administered intravenously in human beings and laboratory animals caused significant dose-related gastrointestinal changes, ranging from mild diarrhea to bloody, watery diarrhea. , in vitro studies on the effects of endotoxin on intestinal water and electrolyte transport in adult male rats showed a significant decrease in net colonic sodium absorption and increased colonic permeability. in animal models of protein energy deficiency, endotoxin-induced mortality increased compared with that of well-nourished control animals. endotoxin depresses lymphocyte responses to specific mitogens. thus the adverse effects of malnutrition and endotoxin are mutually aggravating. the importance of a normal immune system to the defense of the mucosal surface of the gastrointestinal tract is evident in the immunosuppressed state. primary immunodeficiencies affecting the gastrointestinal tract are well documented. [ ] [ ] [ ] common agammaglobulinemia is the most frequently reported gastrointestinal disease and causes b cell deficiency-associated giardiasis. interestingly, selective immunoglobulin a (iga) deficiency rarely results in intestinal disease because of a speculated increase in mucosal igm response. however, combined iga and igm deficiencies with a higher incidence of intestinal disease occur. a selective deficiency of secretory iga has been associated with intestinal candidiasis. certain mucosal pathogens may enhance their pathogenicity by producing iga proteases. defects in cell-mediated immunity are associated most commonly with intractable diarrhea, and organisms frequently involved include salmonella spp., escherichia coli, and shigella spp. acquired immunodeficiency or immunosuppression in adults can result from infectious diseases (particularly viral), nutritional deficiencies, aging phenomenon, and drugs (corticosteroids, azathioprine, cyclophosphamide). nutrition is a critical determinant of immunocompetence and risk of illness. , impaired systemic and mucosal immunity contribute to an increased frequency and severity of intestinal infections observed in cases of undernourishment. abnormalities occur in cell-mediated immunity, complement system, phagocytic function, mucosal secretory antibody response, and antibody affinity. morbidity caused by diarrheal disease is increased particularly among individuals with stunted growth rate because of malnourishment. the critical role of several vitamins and minerals in immunocompetence has been substantiated in animals deprived of one dietary element and findings in human patients with single-nutrient deficiency. tables . - and . - summarize nutritional-related immune system abnormalities. in summary, nutritional deficiency can cause increased colonization of the intestine with microorganisms, alter the symbiotic characteristics of resident intestinal bacterial populations, and impair defenses of the gastrointestinal tract, allowing increased risk of systemic spread of infection and absorption of macromolecules (in particular, endotoxin). indigenous microflora greatly impede colonization of the gastrointestinal mucosa by pathogenic organisms. the ability of a potential pathogen to initiate an infection depends on its ability to breach the mucosal epithelial barrier. one mechanism by which the normal flora inhibit establishment of pathogens is by preventing adherence of the pathogen to mucosal cells by occupying the site or by stearic hindrance. [ ] [ ] [ ] [ ] resident microbes also produce byproducts such as antibacterial factors that allow symbiosis rather than competition between them. another hindrance mechanism is production of volatile fatty acids by normal microbial digestive processes to create an environment that is toxic to many bacterial populations, particularly the enterobacteriaceae. disturbances in motility patterns occur during inflammatory diseases of the colon, but the role of motility alterations in the pathogenesis of diarrhea remains unclear. invasive bacteria cause characteristic motor patterns in the colon consisting of rapid bursts of motor activity that appear to decrease transit time through the large intestine. the result is reduced clearance of bacteria from the large intestine, which may contribute to the virulence of the organism. absorption of endotoxin and the release of inflammatory mediators such as prostaglandins disrupts the motility patterns of the large intestine, resulting in less coordinated contractions, and may contribute to the alterations in motility seen with invasive bacteria. although the effect of endotoxin and prostaglandins on transit time is not profound, the disruption of coordinated activity may play a role in causing diarrhea. thorough mixing and prolonged retention time of ingesta are important not only in microbial digestion of nutrients but also in absorption of microbial byproducts and fluid. , the ingesta is viscous and therefore must be mixed to bring luminal ingesta in contact with the mucosa for absorption. in addition, poor mixing increases the thickness of the unstirred layer, decreasing contact of ingesta with the mucosa and decreasing absorption. , progressive motility must be present, however, if a diarrheal state is to occur. , ileus may be accompanied by increased fluid in the lumen of the large intestine, pyridoxine, folic acid, impairs cell-mediated immunity and vitamin c, vitamin e reduces antibody response. vitamin b decreases lymphocyte stimulation response to specific mitogens. zinc deficiency affects humoral and cell-mediated immunity. iron inhibits bacterial multiplication. copper depresses antibody response. needed by neutrophils and lymphocytes for optimal function, which may be related to myeloperoxidase and ribonucleotidyl reductase deficiencies. but without progressive motility the fluid is not passed. frequently, acute colitis causes a period of ileus characterized by scant stool. diarrhea is apparent only when motility returns and the ingesta is passed. increased progressive motility has been suggested to cause diarrhea by decreasing transit time and is thought to play a role in irritant catharsis and in the mechanism of action of some laxatives. irritation and distention increase motility and may well decrease transit time, but increased secretion also is thought to contribute to diarrhea caused by these substances. one should direct the clinical investigation of malabsorption at ascertaining and trying to localize the source of the abnormality. in medical practice, impairment of one or more phases of the digestion and absorption of dietary constituents may precipitate clinical signs that are associated primarily with carbohydrate, protein, or fat malabsorption. this level of differentiation is not possible in the horse because of the herbivorous diet and the contribution of large intestinal functions. in human and small animal medicine, disturbances in digestive processes especially from exocrine pancreatic insufficiency or reduced intestinal bile salt concentration are principal determinants of many clinical malabsorption syndromes. the rarity of pancreatic problems in horses and the herbivorous diet makes maldigestion less of a concern and difficult to pursue diagnostically. nevertheless, maldigestion undoubtedly contributes to chronic weight loss conditions in horses, which may be significant with severe infiltrative disease of the small intestine with partial to total villous atrophy and flattened mucosa. impairment of digestive processes may exacerbate diarrhea in the suckling foal through reduced intestinal bile salt concentrations from hepatic or ileal dysfunction. malabsorption is not synonymous with diarrhea, although diarrhea may be a feature. adult horses rarely exhibit diarrhea with small intestinal problems unless large intestinal involvement is concomitant. chronic diarrhea is predominantly a large intestinal disorder that reflects an overload of water and electrolytes and thus may be considered a state of impaired absorption. primary small intestinal disease is more likely to occur in neonates and young foals. for example, acquired small intestinal brush border lactase deficiency may result in increased lactose fermentation in the large intestine and induction of osmotic diarrhea. box . - lists conditions that have been or potentially could be associated with malabsorption syndromes and maldigestion in the horse. malabsorption in horses has no pathognomonic clinical syndrome. case recognition derives from the robust investigation of horses with chronic wasting. prevalence is unknown. no strict case definition exists, even for chronic wasting. interest generated by unusual clinical test results and their related pathologic findings has stimulated publication of reports of cases considered as malabsorption in horses. pathologic description of the predominant cellular infiltrate and the pattern of intestinal distribution have resulted in the classification of many conditions as representing examples of chronic inflammatory bowel disease (cibd) (see box . - ), drawing analogies from human medicine. in the affected animal, coexistent enteric protein loss reflecting changes in mucosal integrity from extensive infiltration and inflammation in the intestinal tract is likely to be more debilitating than the malabsorption. the principal concern of the owner is the weight loss and poor condition of the horse. many clinical examination findings, except for body condition, may appear within normal limits. investigation of the weight loss, together with the clinical pathologic findings, helps to eliminate other more commonly encountered causes of wasting. box . - lists clinical signs that may be associated with malabsorption syndromes. no characteristic clinical pathologic profile of malabsorption exists. findings relate to the stage of the underlying disease process and intercurrent problems. the syndrome tends to cause anemia (normocytic, normochromic) and neutrophilia. hemolytic or macrocytic anemia and thrombocytopenia have been observed in alimentary lymphosarcoma. lymphocytosis (leukemia) rarely is encountered. eosinophilia is uncommon even with suspected immune-mediated conditions and widespread tissue eosinophilia. many animals are hypoalbuminemic and hypoproteinemic; horses with alimentary lymphosarcoma may exhibit hyperproteinemia and hypergammaglobulinemia. serum or plasma may be lipemic. the clinician may find elevated hepatic and biliary tract enzymes (γ-glutamyltransferase and alkaline phosphatase) in multisystemic conditions; for example, eosinophilic granulomatosis (multisystemic eosinophilic epitheliotropic disease; eg [meed]). abdominocentesis has been of diagnostic value in several alimentary lymphosarcoma cases, but rarely in the granulomatous conditions. ultrasonographic examination of the abdomen can yield infomation on intestinal distention, wall thickness, and unexplained masses detected on rectal palpation. rectal biopsy is easy to perform and may provide an indication of cellular infiltration that could be present at more proximal locations. however, pathologists examine few equine rectal samples, and the interpretation is frequently equivocal. adoption of standardized grading or classification would improve the diagnostic value. a proposed classification system was based on a retrospective study of rectal biopsies from horses ages to years with clinical signs of intestinal disease. necropsy results were studied from horses. biopsy specimens ( horses) and necropsy rectal tissue ( horses) from horses ages to years served as controls. simple proctitis, the presence of neutrophils in the crypt or surface epithelium, was an abnormal finding compared with mild scattered neutrophil infiltration in controls. simple proctitis was found in association with malignant lymphoma and other inflammatory disorders. inflammatory bowel disease was diagnosed from rectal biopsy specimens in of eg (meed) cases and of granulomatous enteritis cases confirmed at necropsy. rectal biopsy aided diagnosis for of horses in a series of lymphocytic-plasmacytic enterocolitis cases. eosinophils were demonstrated on impression smears of rectal mucosal biopsies from of horses with eosinophilic enterocolitis. skin biopsies or ultrasound-guided biopsies of liver, lymph node, or lung may reveal evidence of multisystemic disease. one can obtain intestinal and lymph node biopsies via a standing laparotomy. exploratory laparotomy facilitates rigorous inspection of the gastrointestinal tract and associated organs to obtain multiple biopsies from intestinal sites and lymph nodes. the procedure may provide a diagnosis, enabling one to make decisions on potential treatment and management options. cost and potential postoperative complications may limit surgical procedures for diagnosis. laparoscopy may provide an alternative means to facilitate biopsy of certain tissues. however, one should consider surgical exploration as an option early in the process rather than as a last resort. the noninvasive breath hydrogen test used to assess carbohydrate malabsorption in human beings has not proved reliable in equine studies. intestinal function tests can provide a practical and inexpensive means to assess the absorptive capability of the small intestine. for clinical practice purposes this is limited to carbohydrate absorption. abnormality of carbohydrate absorption has become an important precept on which to base a diagnosis of malabsorption in the horse. however, results of the oral glucose tolerance test (ogtt) or d-xylose absorption test require cautious interpretation. pathologic changes in the mucosa and submucosa must be extensive and widely distributed to greatly affect the peak plasma concentration and shape of the curve. the tests are easy to perform in practice and require a baseline blood sample predosing and further samples for up to hours after administration of the solution. many commercial laboratories conduct glucose and xylose assays. the immediate dietary history, gastric emptying rate, intestinal transit, age, and hormonal effects of the horse influence glucose peak and curve shape. higher glucose peaks are recorded from healthy animals eating grass or hay than from those eating concentrates. recent appetite or the level of cachexia may affect test results. maximum plasma glucose level (> % baseline) is reached by minutes in healthy animals given g glucose per kilogram body mass as a % solution. , break points below which the probability increases of carbohydrate malabsorption associated with intestinal morphology changes have been proposed. a referral population of mature horses with chronic weight loss was divided into three groups based on ogtt results to determine if any concurrence with the morphologic diagnoses existed. group (n = ) had a normal ogtt (peak glucose concentration at minutes > % baseline) and contained animals that had normal small intestinal morphology, and a few with large intestinal lesions. group (n = ) had partial malabsorption and included horses with small intestinal infiltrative disease that allowed some glucose uptake. diagnoses included lymphosarcoma, villous atrophy, granulomatous enteritis and eg (meed). seven horses had normal small intestinal histologic findings. peak glucose concentrations were less than % and greater than % of baseline at minutes. seventeen horses in the group had large intestinal pathologic conditions. group horses (n = ) had total malabsorption; the peak concentration at minutes was less than % above baseline. these horses had severe infiltration throughout most of the small intestine that was attributed predominantly to lymphosarcoma or granulomatous enteritis. however, the test is far from definitive; one cannot assume a flat curve indicates malabsorption and a poor prognosis. two horses with chronic weight loss initially diagnosed with malabsorption based on flat ogtt curves subsequently showed more normal ogtt responses. full-thickness intestinal biopsies were unremarkable. one horse had an elevated serum immunoglobulin e to oat allergen. oats and oat straw were removed from access. dexamethasone was given on a tapered protocol, and a repeat ogtt was normal at months. the other horse received oral probiotics to counter suspected small intestinal bacterial overgrowth, was clinically normal in months, and had an improved ogtt with a minute peak. therefore malabsorption, as defined by an absorption test and weight loss, may occur in the horse without significant morphologic changes in the intestine, and the condition may be transient. demonstration of carbohydrate malabsorption in of horses with chronic diarrhea showed poor diagnostic sensitivity for small intestinal involvement. impaired glucose absorption was recorded in horses with predominantly large intestinal problems, cyathostomiasis, chronic colitis, alimentary lymphosarcoma, and meed. although prior dietary history influences peak plasma xylose concentration, xylose is not confounded by hormonal effects or mucosal metabolism. gastric emptying rate, intestinal motility, intraluminal bacterial overgrowth, and renal clearance do affect curve shape. healthy mares not fed for up to hours had flatter curves and a slower decrease in plasma xylose than when deprived for to hours. hence recent appetite or the level of cachexia may influence test results. abnormal d-xylose absorption represented by a flat curve or delayed absorption is considered indicative of significant jejunal disease and has been observed with most examples of cibd, parasitism, and idiopathic villous atrophy. , ponies may have lower peak d-xylose concentrations at and minutes than horses, although the range of peak values at the test dosage ( . g d-xylose per kilogram body mass in a % solution) is wide. potentially diagnostic discriminatory cut off points for peak plasma xylose concentrations have not been determined. abnormal absorption curves have been detected in the absence of small intestinal histologic changes, and interpretation is clouded further by findings from small intestinal resection studies in healthy ponies. nine ponies with % distal small intestinal resection and four shamoperated controls were placed on interval feeding for weeks and then turned out to pasture until months after surgery. grazing was supplemented by twice daily (meal feeding) concentrate rations. all ponies gained weight and were clinically normal, and none developed diarrhea. however, the mean peak xylose concentration at minutes declined progressively (at monthly intervals) over the study period in the resection group to % of that of controls. lack of clinical malabsorption was attributed to adaptation of the residual % of healthy small intestine and of large intestinal function. bacterial overgrowth in the small bowel remnant from refluxed cecal contents (resected ponies had ileocecal valve bypass) may have contributed to the abnormal xylose assimilation. by contrast, xylose absorption decreased over months, associated with substantial weight loss, lethargy, and diarrhea, in an earlier study of extensive (≥ %) small intestinal resection in ponies. an important difference was the feeding pattern; those ponies received pelleted feed twice daily for the entire month follow-up period. consequently, horses with suspected malabsorption may adapt to an interval feeding regimen. the critical factor could be the availability of sufficient unaffected or minimally affected small intestine and large intestinal functional capacity. the outcome for animals with small intestinal disease and some unknown degree of large intestinal pathologic dysfunction may be less successful than shown in the experimental study. abnormal xylose absorption reverted to normal following days of corticosteroid therapy in an adult thoroughbred gelding with a -week history of weight loss and diarrhea for weeks; peak xylose concentration at minutes was within normal limits and the horse had gained weight. d-xylose absorption was abnormal in an adult standardbred gelding with a -month history of poor performance, weight loss, intermittent fever, mesenteric lymphadenopathy, elevated fibrinogen, and decreased albumin and globulin levels. multiple fullthickness small intestinal biopsies revealed evidence of granulomatous enteritis. the horse received antibiotics postoperatively and then corticosteroids parenterally for to months. after weeks, peak plasma xylose had increased, although absorption was delayed. five months after cessation of corticosteroid therapy, the horse had regained weight and was bright and alert, and d-xylose absorption was normal. diagnostic predictions were made retrospectively by examining d-xylose absorption in horses with granulomatous enteritis compared with those with eg. peak xylose concentrations were much lower in horses with granulomatous enteritis than those with eg, whereas in eg the absorption curve shifted to the right with the peak occurring at minutes. the small intestine is affected predominantly in granulomatous enteritis with extensive villous atrophy and more diffuse lesions in the large intestine, whereas in eg (meed) the large intestine is more involved. hence, the extent and distribution of pathologic changes in the small and large intestines may influence xylose absorption test results. the chronic wasting horse with suspected malabsorption and probable enteric protein loss has at best a guarded to poor prognosis. prognosis may be improved through early and aggressive investigation to achieve a diagnosis, and perhaps assess the stage in the natural progression of the disorder. the owner may elect euthanasia of the animal or may be willing to determine whether the condition can be improved. in the short term, intravenous infusion of plasma or colloids, with or without fluids and electrolytes, may be necessary to stabilize the condition. prognosis is much worse for the horse that is inappetent. prolonged intensive total parenteral nutrition and/or oral alimentation may not be a realistic course of action. the overall therapeutic and management plan can prove to be expensive. the owner must be cognizant from the start that the outcome may not be altered, even after protracted therapy. one cannot make predictions for outcome of therapy without meaningful data because only a few case reports of successful responses with long-term follow up exist. some level of digestive and absorptive capability remains in the diseased small intestine. interval feeding of small quantities of food may be beneficial if the horse continues to eat, and particularly for animals with ravenous appetites that seem able to maintain their reduced state of body condition without further losses. diet should include feeds with a high fiber content to favor large intestinal fermentation, including grass hay and access to pasture complemented by commercial high-fiber rations based on beet pulp and soybean hulls. energy intake can be increased through feeding high-energy dense fats that provide . times more calories than carbohydrates. most affected horses should tolerate high fat ( % to %) processed feeds containing vegetable oils or rice bran (up to % of the concentrate mix, equivalent to % vegetable oil) to achieve the higher-fat composition. changeover to a higher-fat concentrate should be gradual. even in healthy animals that can eat up to % added fat, appetite may decrease as the percentage increases, and fecal consistency may change. clearly, the objective for the horse with suspected malabsorption is to sustain, and preferably increase, dietary intake, value, and efficiency. the owner of an affected horse must be prepared to experiment with feeds, must be patient, and must keep records. no standard procedure exists. exposure to a feed component may contribute to the problem as an allergen eliciting a hypersensitivity reaction. identifying the potential allergen through immunologic testing or by stepwise removal and outcome assessment over a longer period may be difficult. the clinician should give immunosuppressive drugs early in the process. immunosuppressive agents have produced the most promising responses to ameliorate the effects of conditions associated with malabsorption, particularly cibd. short-duration, and in some cases more prolonged and sustained, improvements in body condition, weight gain, demeanor, energy and activity levels have occurred following corticosteroid administration. one should start treatment as early as possible. one should follow initial parenteral (intramuscular or intravenous) loading doses of dexamethasone (sodium phosphate) with a series of depot injections, or orally administered prednisolone or prednisone, on a tapered dose protocol over a period of months. interval low-dose therapy may be necessary if clinical signs return after treatment ends. one uses the lowest dose to control the clinical signs for alternate-day therapy. clinical benefits far outweigh concerns over potential adverse effects. chemotherapeutic agents such as vincristine, cytosine, cyclophosphamide, and hydroxyurea have been tried in a few cases of cibd or lymphosarcoma with no apparent success, probably related to the advanced stage of the disease when treatment was initiated and the dose selected. resection of a segment of intestine that is edematous, hemorrhagic, or constricted is an option in localized forms of cibd, , particularly if gross changes are not discernible in adjacent or distant parts of the intestinal tract, that is, malabsorption is not a feature. long-term outcome has been favorable. removal of a substantial proportion of the diseased small intestine may be indicated in a horse with malabsorption, considering that resection of % distal small intestine was performed in healthy animals without inducing adverse effects. however, because pathologic changes may exist in normalappearing small or large intestine that is not resected or biopsied, the prognosis remains guarded. two young horses with granulomatous enteritis had the thickened terminal small intestine resected with positive outcomes; one survived months, the other has a follow up extending more than years. anthony t. blikslager to gain an appreciation of the mechanisms whereby the mucosa is injured and subsequently repaired, one must understand how the integrity of the mucosa is regulated physiologically. regulation of mucosal integrity is referred to as mucosal barrier function, which is vital because it prevents bacteria and associated toxins from gaining access to subepithelial tissues and the circulation. however, the mucosa has two conflicting functions: it must serve as a protective barrier and continue to absorb solutes necessary to maintain well-being of the host. this conflict is most notable at the intercellular (paracellular) space, which allows passage of select solutes and water, - but which does not admit large molecules, including bacterial toxins. the paracellular space is regulated almost exclusively by the tight junction, which is the interepithelial junction at the apical-most aspect of the paracellular space. although these tight junctions originally were viewed as inert cellular adhesion sites, what has become clear in recent years is that tight junction permeability depends on tissue-specific molecular structure and is regulated by a complex array of intracellular proteins and the cytoskeleton. tight junctions consist of a group of transmembrane proteins that interdigitate from adjacent cells. although occludin originally was thought to be the predominant tight junction transmembrane protein, a group of proteins termed claudins appear to be more critical. these transmembrane proteins interact with the cytoskeleton via a series of intracellular proteins, including zonula occludens , , and ; cingulin; and others. in addition, local regulatory proteins such as the small guanosine triphosphatase-rho are also critical to tight junction function. in general the relative contractile state of the actin cytoskeleton determines the degree to which tight junctions are open or closed, but the complexities of regulation of this process are understood poorly. , the most sensitive measure of mucosal barrier function is transepithelial electric resistance, which is measured by mounting mucosa in an ex vivo system called an ussing chamber, because this measurement is largely a reflection of the permeability of mucosa to ions. , ions may follow one of two routes when traversing epithelium: transcellular and paracellular. because cell membranes have a resistance to passive flow of ions . to log units greater than that of the epithelium as a whole, measurements of transepithelial resistance largely reflect the resistance of the paracellular space, and in particular the tight junctions that regulate paracellular flow of ions. because tight junctions differ in structure from different portions of the mucosa, measurements of transepithelial resistance reflect the net resistance of epithelium of variable permeability within a given tissue. for example, tight junctions in the intestinal glandular structures called crypts are leakier than those in the surface epithelium because of fewer and less organized tight junction strands. , conversely, surface epithelium has a greater number of well-organized tight junction strands that result in epithelium with a high resistance. this correlates well with the absorptive function of epithelium located on the mucosal surface and the secretory function of crypt epithelium. structure of tight junctions also varies with the segment of intestine. for example, tight junctions have more strands in the ileum than in the jejunum, which is reflected by a higher transepithelial resistance in the ileum. the stomach has four regions based on the type of mucosal lining (in an orad to aborad order): nonglandular stratified squamous epithelium, cardiac epithelium, proper gastric mucosa, and pyloric mucosa. stratified squamous epithelium has distinct differences in terms of barrier function compared with the remainder of the gastrointestinal tract. this epithelium has baseline transepithelial resistance measurements of approximately to Ω/cm , which is an order of magnitude higher than the adjacent cardiac mucosa. , thus the stratified squamous mucosa is exceptionally impermeable. this in effect is the only mechanism this mucosa has to defend itself against injury. the stratified squamous epithelium consists of four layers: the outer stratum corneum, stratum transitionale, stratum spinosum, and the basal stratum germinativum. however, not all layers contribute equally to barrier function, the barrier being composed mostly of interepithelial tight junctions in the stratum corneum and mucosubstances secreted by the stratum spinosum. , the relative impermeability of stratified squamous mucosa can be demonstrated by the effects of hcl on this type of epithelium in vitro, which has little effect until it reaches a ph of . or lower. thus although most of the literature on equine ulceration pertains to the effects of hcl and inhibitors of hcl secretion, [ ] [ ] [ ] [ ] other factors may be critical to the development of gastric ulcer disease. the site of hcl secretion (proper gastric mucosa) also is protected from so-called back-diffusion of h + by a high transepithelial electric resistance (compared with cardiac mucosa), but a number of other critical mechanisms also exist to prevent acid injury. the gastric mucosa secretes mucus and bicarbonate, which together form a hco --containing gel that titrates acid before it reaches the lumen. , the mucus layer is formed principally by glycoproteins (mucins) secreted by goblet cells but also includes other gastric secretions and sloughed epithelial cells. mucins consist of core peptides with a series of densely packed o-linked polysaccharide side chains that, once secreted, become hydrated and form a viscoelastic gel. however, the mucus layer does not form an absolute barrier to back-diffusion of acid. thus for acid that does back-diffuse into the gastric mucosa, epithelial na + /h + exchangers are capable of expelling h + once the cell reaches a critical ph. recent studies have renewed interest in the protective mechanisms of mucus because of the discovery of a group of compounds secreted by goblet cells called trefoil peptides. the name of these peptides is derived from a highly conserved cloverleaf structural motif, which confers substantial resistance to degradation by proteases including pepsin. three members of this group are known, ps , sp, and intestinal trefoil factor, the latter of which is secreted solely by goblet cells in the small and large intestine. ps and sp are secreted by goblet cells within the stomach and are believed to intercalate with mucus glycoproteins, possibly contributing to the barrier properties of mucus. these peptides also play a critical role in repair of injured mucosa. an additional mucosal function that serves to reduce the level of injury is adaptive cytoprotection, wherein application of topical irritants to gastric mucosa results in subsequent protection of mucosa in response to repeated exposure to damaging agents. for example, pretreatment with % ethanol protected against mucosal damage in response to subsequent application of absolute ethanol, and this effect was abolished by treatment with the cyclooxygenase inhibitor indomethacin. the cytoprotective effect of prostaglandins has been demonstrated directly in studies in which preadministration of prostaglandins protected gastric mucosa from damage by agents such as concentrated hcl and hypertonic saline. prostaglandins appear to be cytoprotective in the stomach at doses less than those used to inhibit gastric acid secretion, ruling out a simple antacid mechanism. although not fully characterized, cytoprotection has been attributed in part to prostaglandin-stimulated mucus production. an associated beneficial effect of prostaglandins is the increased production of bicarbonate, which is trapped within mucus on the surface of the mucosa. , interestingly, prostaglandin e (pge ) appears to lose its cytoprotective activity in the presence of the mucolytic agent n-acetylcysteine. attention also has been directed at enhanced mucosal blood flow as a potential mechanism for prostaglandin-mediated cytoprotection. for example, pretreatment with pgi protected against ethanol-induced mucosal damage as a result of increased mucosal blood flow. although pge , which is also cytoprotective, does not increase blood flow, it may prevent vascular stasis associated with irritant-induced vascular damage resulting from inhibition of neutrophil adherence to damaged endothelium. sensory nerves also have been implicated in cytoprotective mechanisms. these nerves are distributed throughout gastrointestinal mucosa. as an example of their importance in mucosal cytoprotection, pretreatment of newborn rats with capsaicin (to which sensory nerves are sensitive) renders the mature rats more susceptible to gastric injury. alternatively, use of a low dose of capsaicin, which stimulates rather than destroys sensory nerves, protects gastric mucosa against injurious agents. , sensory nerves contain neuropeptides such as calcitonin-gene-related peptide (cgrp) and substance p, which may play a protective role via vascular mechanisms. for instance, cgrp stimulates increased gastric blood flow, which is theorized to reduce injury in much the same way as prostaglandins do. in fact, recent studies suggest that the roles of prostaglandins and cgrp in gastric cytoprotection are intertwined intimately. in particular, pgi is believed to sensitize sensory nerves following treatment with a mild irritant, with resultant increases in cgrp release and mucosal flow. similar studies have shown that antagonists of cgrp inhibit the cytoprotective action of pge . another neural mediator, nitric oxide, also has been implicated in adaptive cytoprotection. interestingly, nitric oxide has a number of actions that are similar to those of prostaglandins, including maintenance of mucosal blood flow. regulation of barrier function in the intestine is not as well characterized as that of the stomach, although mechanisms of barrier function, including secretion of mucus and regulation of mucosal blood flow, are presumed to be similar. the proximal duodenum also has to protect itself from acid damage as it receives gastric contents, and this involves secretion of mucus and bicarbonate in much the same way as the stomach. one other mechanism that helps the stomach and the intestine to maintain mucosal barrier function is the speed with which the mucosa repairs. thus for a defect to develop in the mucosal barrier, injurious factors have to outpace mucosal recovery. such recovery initially involves epithelial migration across denuded regions of basement membrane (restitution), a process so rapid that epithelial defects may be resurfaced within minutes. for example, in bile salt-injured colon, denuded surface mucosa was covered completely by restitution. in the small intestine, villi greatly amplify the surface area of the mucosal luminal surface, which in turn takes far longer to resurface with restituting epithelium once it has become denuded. however, intestinal villi are able to reduce the denuded surface area considerably by extensively contracting. these mechanisms are described in detail under mechanisms of gastrointestinal mucosal repair. although the stratified squamous epithelium is relatively impermeable to hcl, a number of factors can enhance the damaging effects of hcl in this epithelium. in particular, bile salts and short-chain fatty acids are capable of breaking down the squamous epithelial barrier at an acid ph, thereby exposing deep layers to hcl, with subsequent development of ulceration. , high concentrations of short-chain fatty acids normally exist within the equine stomach because of microbial fermentation. these weak acids penetrate squamous mucosa and appear to damage na + transport activity principally located in the stratum germinativum. bile salts also may be present in the proximal stomach because of reflux from the duodenum. although such reflux has a high ph, bile salts appear to adhere to stratified squamous epithelium, becoming lipid soluble and triggering damage once the ph falls below . diet and management (e.g., periods of fasting) also play crucial roles in the development of conditions conducive to gastric ulceration. typically, a ph gradation in horses exists from proximal to distal compartments of the stomach, with the lowest ph values in the distal stomach. however, fasting disrupts this stratification such that low ph values may be recorded in the proximal stomach. fasting conditions also increase the concentration of duodenal contents within the proximal stomach, particularly bile. proper gastric mucosa is exposed to injurious agents, including pepsin, bile, and acid. parietal cells in the horse secrete acid constantly as an adaptation to near-continuous intake of roughage, but the enterochromaffin-like cells within the proper gastric mucosa and g and d cells within the pyloric mucosa tightly regulate acid secretion. histamine released by enterochromaffin-like cells amplifies acid secretion and interacts with h receptors on parietal cells and g cells, which release the prosecretory hormone gastrin. a combination of histamine and gastrin can have a synergistic effect on parietal cell gastric secretion, because these mediators have distinct receptors and second messengers. however, d cells are sensitive to an acidic environment and release somatostatin, which inhibits acid secretion. nonetheless, gastric mucosa may be exposed to acid for prolonged periods of time, particularly in horses that are extensively meal fed and that do not have the benefit of roughage, which tends to buffer stomach contents. , aside from peptic ulceration induced by combinations of acid and pepsin, research in human medicine has revealed the tremendous importance of helicobacter pylori in inducing ulceration. infection with this organism has the effect of raising gastric ph because of disruption of gastric glands and also induces an inflammatory reaction that causes damage. however, little evidence to date indicates that this organism is involved in gastric ulcers in horses. in the absence of a known role for infectious agents in gastric ulceration in animals, ulceration likely develops from injurious factors similar to those found in the proximal stomach, including gastric acid and bile. however, some factors that are important to induction of squamous epithelial ulceration may not be important in development of proper gastric mucosal ulceration. for example, feed deprivation and intensive training reproducibly induce squamous epithelial ulceration in horses but have little effect on proper gastric mucosa in horses. gastric acid likely plays a key role, whereas other factors such as nonsteroidal antiinflammatory drugs (nsaids) serve to reduce gastric defense mechanisms. in particular, inhibition of prostaglandin production reduces mucus and bicarbonate secretion while also reducing gastric mucosal blood flow. some of the nsaids also have a topical irritant effect, although this appears to be of minor significance because the route of administration (oral or parenteral) seems to have little influence on development of ulceration. the source of prostaglandins responsible for gastric protection originally was assumed to be cyclooxygenase (cox- ), because this isoform is expressed constitutively in gastric mucosa, whereas cox- is not expressed in the stomach unless induced by inflammatory mediators. however, mice in which the cox- gene has been knocked out fail to develop spontaneous gastric lesions, possibly because of compensatory increases in prostaglandin production by cox- . this concept agrees with recent data indicating that inhibition of both cox isoforms is required to induce gastric ulceration. from a clinical perspective this data indicate that drugs selective for cox- or cox- may be less ulcerogenic in the horse. because cox- elaborates prostaglandins induced by inflammatory stimuli, selective inhibitors of cox- may be particularly useful because of their ability to serve as antiinflammatory agents that are less ulcerogenic. the most notable cause of intestinal mucosal injury in horses, particularly those suffering from colic, is ischemia. initially, that a reduction in gastrointestinal blood supply leads to mucosal injury seems intuitive. however, the anatomy of the gastrointestinal tract and the differing structure of the intestinal mucosa at various anatomic locations have a significant influence on the extent of mucosal injury. furthermore, ischemic injury may be induced by several different mechanisms, including occlusion of arterial supply by a thrombus, strangulation of intestinal vasculature, and generalized reduction in blood flow associated with various shock states. in addition, a number of seemingly distinct mechanisms of intestinal injury, such as intestinal distention, also trigger mucosal injury via an ischemic mechanism. finally, reperfusion injury also may influence the extent of mucosal injury following an ischemic episode and has been proposed as a potential site of therapeutic intervention. , thus understanding the mechanisms of ischemia-reperfusion injury is critical to developing an understanding of the severity of various clinical conditions and beginning to formulate a therapeutic approach to diseases characterized by this devastating form of injury. the intestinal circulation is capable of closely regulating blood flow during periods of low systemic perfusion pressure. , in particular, local regulation of resistance vessels within the microvasculature is particularly prominent, whereby metabolic end products of adenosine triphosphate (atp) result in continued dilation of resistance vessels despite reductions in systemic arterial pressure. dilation results in continued perfusion of gastrointestinal tissues during the early stages of shock, while other organs such as skeletal muscle undergo massive shunting of blood resulting from increased constriction of resistance vessels. the reasons for these differences in regulation are not entirely clear but may relate to the high level of energy required to fuel the intestinal mucosa and the serious systemic effects of breaches in the mucosal barrier. however, as blood flow falls below a critical level, regulatory systems are no longer effective and oxygen uptake by the gastrointestinal tissue decreases, culminating in tissue damage. the tip of the villus is the most susceptible region affected by hypoxia in the equine small intestine, largely because of the countercurrent exchange mechanism of blood flow in the small intestinal villus. this countercurrent exchange mechanism is attributable to the vascular architecture, which consists of a central arteriole that courses up the core of the villus, arborizes at the tip, and is drained by venules coursing down the periphery of the villus. as oxygenated blood flows into the central arteriole, oxygen tends to diffuse across to the adjacent venules, which flow in the opposite direction. this series of events takes place along the length of the villus, resulting in a tip of the villus that is hypoxic even under normal conditions. furthermore, reduced blood flow as occurs in shock exacerbates the countercurrent exchange of oxygen, and the tip becomes absolutely hypoxic. this mechanism might explain why the small intestinal mucosa is more susceptible to ischemic injury, compared with the colon, which has no villi. for example, the duration required to produce severe morphologic damage to the equine colon is approximately % longer than in the small intestine. intestinal mucosal epithelium is susceptible to hypoxia because of the high level of energy required to fuel the na + /k + -atpase that directly or indirectly regulates ion and nutrient flux. the first biochemical event to occur during hypoxia is a loss of oxidative phosphorylation. the resulting diminished atp concentration causes failure of the energy-dependent na + /k + -atpase resulting in accumulation of sodium, and subsequently intracellular water. the ph of the cytosol drops as lactic acid and inorganic phosphates accumulate from anaerobic glycolysis. the falling ph damages cell membranes, including lysosomal membranes, resulting in the release and activation of lysosomal enzymes into the cytosol, further damaging cellular membranes. damage to the cell membrane allows the accumulation of high concentrations of calcium in the cytosol, which activates calciumdependent degradative enzymes. these events result in cytoplasmic blebbing of the basal membrane with subsequent detachment of cells from the underlying basement membrane. recent studies on epithelial injury during ischemia suggest that most epithelial cells undergo programmed cell death (apoptosis) during ischemia and reperfusion rather than necrosis, allowing retention of reusable components of irreversibly injured cells. in one study, % of detached epithelium during small intestinal ischemia and reperfusion underwent apoptosis. although the most obvious result of apoptosis is loss of surface epithelium, a number of cells on the lower portion of the villus (in the small intestine) and cells within the crypts also may undergo apoptosis that only may become evident up to hours following reperfusion of ischemic tissue. morphologic changes observed in ischemic-injured small intestinal mucosa follow a similar sequence regardless of whether ischemia alone or ischemia and reperfusion induce injury (table . - ). initially, epithelium separates from the underlying basement membrane, forming a fluid-filled space termed grüenhagen's space ( figure . - ). the mechanism of fluid accumulation in this space is not understood entirely but may result from continued epithelial absorption of nacl and water before it has detached fully from neighboring epithelial cells. this fluid accumulation likely exacerbates epithelial separation from the basement membrane. subsequently, epithelium progressively sloughs from the tip of the villus toward the crypts, which are the last component of the intestinal mucosa to become injured. [ ] [ ] [ ] injury of crypts likely relates to the vascular architecture, because crypts receive a blood supply separate from the vasculature involved in the villous countercurrent exchange mechanism. the early morphologic changes observed in the equine large colon during ischemia are different from those described in the equine small intestine because of the lack of intestinal villi. however, as might be expected, the more superficially located surface cells are sloughed before those in crypts. , the orderly progression of tissue injury has been used by one group of investigators to predict accurately the survival of horses with large colon volvulus. the researchers took biopsies from the pelvic flexure, which has been shown previously to reflect mucosal changes along the length of the colon accurately, and examined them histologically for the width of the crypts and intercrypt interstitial space. they expressed the latter measurements as a ratio of interstitium to crypt width (i:c) and defined nonviable colon as that which has greater than % loss of crypt and an i:c ratio greater than . using this methodology, researchers correctly predicted survival in % of horses. because of the dramatic decline in strongylus vulgarisinduced colic, which was associated frequently with infarction of intestinal arterial blood supply, most ischemic lesions are associated with strangulating obstruction. therefore considering mechanisms of ischemic injury in horses with naturally occurring strangulating lesions is important. the majority of experimental work has assessed complete ischemia (complete occlusion of the arterial blood supply) or low-flow ischemia (during which arterial blood flow is reduced). , however, during intestinal strangulation, a disparity between the degree of occlusion of the veins and arteries occurs whereby veins are occluded before arteries because of differences in compliance of vascular walls. thus strangulating lesions are typically hemorrhagic (hemorrhagic strangulating obstruction) as the arteries continue to supply blood to tissues that have little or no venous drainage. the result is ischemic injury, as previously outlined, but also a tremendous congestion of the tissues. such hemorrhagic congestion has two opposing effects: it disrupts tissue architecture, including the mucosa and its epithelium, and continues to provide oxygenated blood to the tissues during much of the ischemic episode. in contrast, when strangulation results in sudden cessation of arterial blood flow (ischemic strangulating obstruction), tissues appear pale, and the mucosa rapidly degenerates because of a complete lack of oxygenated blood. because intestine that may look nonviable (dark red) may in fact have less mucosal injury than that of ischemic strangulated intestine. an additional consideration in clinical strangulating obstruction is the degree of ischemia that intestinal distention may induce. for example, experimental distention ( cm of h o for hours) and decompression ( hours) of jejunum resulted in a significant increase in microvascular permeability and a significant decrease in tissue oxygenation similar to that which would be expected with low-flow ischemia. , in particular, microscopic evaluation of vasculature revealed capillary endothelial cell damage and local edema formation. this data suggest that distended intestine proximal to an obstruction may undergo mucosal injury despite its normal appearance. indeed, in one study, intraluminal pressures greater than cm h o in naturally occurring cases of colic correlated with a poor prognosis for survival. although that reperfusion of ischemic tissues results in exacerbation of mucosal injury recently has been taken for granted, one should remember that mechanisms underlying intestinal reperfusion injury have been defined largely in laboratory animals under specific conditions. [ ] [ ] [ ] [ ] [ ] however, studies on reperfusion injury in horses have had some conflicting results. , , the conflict may be attributable to the way in which the studies have been performed. in particular, the type of ischemia used in most laboratory animal studies has been low-flow ischemia (in which the blood flow typically is reduced to % of baseline flow), whereas studies in horses have used a number of different ischemic models, including various types of strangulating obstruction. although strangulating obstruction is of great clinical relevance, this type of ischemic insult is less likely to develop reperfusion injury. , , conversely, low-flow ischemia appears to prime tissues for subsequent injury once the tissue is reperfused, and considerable evidence supports the presence of reperfusion injury in horses following low-flow ischemia. , , , nonetheless, lowflow ischemia may not be a common clinical entity. in addition to the type of ischemia, other factors are involved in priming tissues for reperfusion injury, including species and anatomic-specific variation in oxidant enzyme and neutrophil levels ( table . - ). for example, the foal appears to have low levels of small intestinal xanthine oxidase, an enzyme that has been shown to play a critical role in triggering reperfusion injury in laboratory animals, , , whereas adult levels are much greater, particularly in the proximal small intestine. in addition, horses appear to have low numbers of resident neutrophils in the intestinal mucosa, and this population of neutrophils (rather than those recruited from the circulation) appears to be most critical for induction of reperfusion injury. however, studies demonstrating reperfusion injury in the equine colon following low-flow ischemia have shown significant accumulation of neutrophils within the mucosa. therefore a complete understanding of mechanisms of neutrophilic infiltration and the mechanisms whereby they damage tissue requires further study. reperfusion injury is initiated during ischemia when the enzyme xanthine dehydrogenase is converted to xanthine oxidase and when its substrate, hypoxanthine, accumulates simultaneously because of atp use ( figure . - ). , however, little xanthine oxidase activity occurs during ischemia, because oxygen is required as an electron acceptor. during reperfusion, xanthine oxidase rapidly degrades hypoxanthine in the presence of oxygen, producing the superoxide radical as a by-product. the superoxide radical contributes to oxidative tissue damage and, most importantly, activates neutrophil chemoattractants. , thus inhibition of xanthine oxidase in feline studies of intestinal ischemiareperfusion injury prevents infiltration of neutrophils and subsequent mucosal injury. , however, inhibition of xanthine oxidase has had no effect on ischemiareperfusion injury in equine small intestine and colon, suggesting that reperfusion injury is simply a continuation of injury initiated during ischemia, as suggested in some equine studies, or that the classic reperfusion injury pathway is activated by alternate sources of reactive oxygen metabolites. the latter has been suggested by studies in feline models of ischemia-reperfusion injury in which the source of a significant proportion of reactive oxygen metabolites is unknown and is independent of xanthine oxidase and neutrophils. a veterinary review of the pathogenesis of intestinal reperfusion injury in the horse suggested the concept of a therapeutic window wherein treatment of reperfusion injury would be beneficial. the basis of this concept is that certain conditions exist under which ischemic injury is minimal and that tissues are damaged severely during reperfusion. thus under conditions of low-flow ischemia, little injury is demonstrated during hours of ischemia, but remarkable injury occurs during hour of reperfusion. [ ] [ ] [ ] however, a therapeutic window may not exist under conditions of strangulating obstruction in which severe injury occurs during ischemia and minimal injury occurs during reperfusion. this in turn greatly reduces clinicians' ability to ameliorate ischemiareperfusion injury with treatments such as antioxidants at the time of reperfusion. mechanisms of gastric repair depend greatly on the extent of injury. for instance, superficial erosions can be covered rapidly by migration of epithelium adjacent to the wound; a process termed epithelial restitution. however, ulceration (full-thickness disruption of mucosa and penetration of the muscularis mucosa) requires repair of submucosal vasculature and extracellular matrix. the formation of granulation tissue initiates repair and supplies connective tissue elements and microvasculature necessary for mucosal reconstruction. connective tissue elements include proliferating fibroblasts that accompany newly produced capillaries that form from proliferating endothelium. recent studies indicate that nitric oxide is critical to both processes, , which likely explains the reparative properties of nitric oxide in the stomach. once an adequate granulation bed has formed, newly proliferated epithelium at the edge of the wound begins to migrate across the wound. in addition, gastric glands at the base of the ulcer begin to bud and migrate across the granulation bed in a tubular fashion. repairing epithelium expresses epidermal growth factor, which appears to facilitate these processes. in addition, a mucoid cap facilitates these events and retains reparative factors and serum adjacent to the wound bed. once the ulcer crater has been filled with granulation tissue and the wound has been reepithelialized, the subepithelial tissue remodels by altering the type and amount of collagen. despite the remodeling process, ulcers tend to recur at sites of previous ulceration, and the concern is that this remodeling can result in excessive deposition of collagen and fibrosis. reparative mechanisms are similar in the intestine, except that in the small intestine, mucosal villi contribute to mucosal repair. once intestinal epithelium is disrupted, two events occur almost immediately to reduce the size of the denuded portion of the villus: contraction of the villus and epithelial restitution ( figure . - ). for example, in porcine ileum subjected to hours of ischemia, villi were % of their former height and % of the denuded villous surface area was covered in flattened epithelium within hours. enteric nerves appear to regulate villous contraction, because inhibition of enteric nerve conduction prevents villous shortening following injury. the contractile component of the villus is a network of myofibroblasts distributed throughout the lamina propria of the villus and along the central lacteal. inhibition of villous contraction results in retarded epithelial repair because of the larger denuded surface that remains to be covered by migrating epithelium compared with similarly injured villi that have contracted. pge also has been implicated in regulating villous contraction, because application of pge resulted in villous contraction when perfused through normal rat ileum. as villi contract, assuming the basement membrane is intact, epithelium from the margins of the wound migrates centripetally to resurface toward the tip of the villus. the process of restitution is similar in denuded colonic mucosa, except that it may proceed more rapidly because of the lack of villi. epithelial restitution is solely a migratory event that does not depend on provision of new enterocytes by proliferation. cellular migration is initiated by extension of cellular lamellipodia that receive signals from the basement membrane via integrins. intracellular signaling converges on the actin cytoskeleton, which is responsible for movement of lamellipodia. specific components of the basement membrane appear to be critical to the migratory process. for example, application of antibodies to collagen types iii and iv, which are important components of intestinal mucosal basement membrane, impeded epithelial restitution. , other elements of the basement membrane, including proteoglycans, hyaluronic acid and noncollagenous proteins such as fibronectin and laminin also may provide important signals. these subepithelial matrix components that facilitate restitution may form the basis for clinical treatments designed to speed up the repair process, analogous to administration of matrix components to horses with articular cartilage damage. although epithelial restitution results in gross closure of previously denuded regions of gastrointestinal mucosa, closure of interepithelial spaces ultimately is required to restore normal epithelial barrier resistance. because the tight junction is principally responsible for regulating the permeability of the interepithelial space, repair and closure of this structure likely is critical to restore intestinal barrier function. recent research indicates that prostaglandins play a vital role in recovery of tight junction resistance, indicating that administration of nonselective cox inhibitors to horses with colic, particularly those recovering from strangulating obstruction, may be deleterious. therefore judicious use of nsaids is appropriate until more selective drugs that allow continued production of reparative prostaglandins are available for use in horse. after restoration of the epithelial barrier, the epithelium must reestablish normal mucosal architecture to allow normal gut absorptive and digestive function. in porcine ileum subjected to hours of ischemia, the epithelial barrier was restored within hours, but villi were contracted and covered in epithelium with a squamous appearance. restoration of normal villous architecture required another days. newly proliferated crypt epithelium replaces the flattened villous epithelium that characterizes restitution. under normal circumstances the dividing stem cells, of which the base of each mucosal crypt has approximately four, form new enterocytes. newly divided enterocytes migrate from the crypt onto the villus. during migration, enterocytes differentiate and acquire specific absorptive and digestive functions. fully differentiated enterocytes reside on the upper third of the villus for to days and then are sloughed into the intestinal lumen. this process accelerates during mucosal repair and requires increased proliferative rates. a variety of locally available gut-derived factors, including luminal nutrients, polyamines, and growth factors, may stimulate increased proliferation within to hours. the return of the normal leaflike shape of the villus occurs following the appearance of normal columnar epithelium. although prostaglandins have been implicated in mucosal cytoprotective function, few studies have assessed their importance in mucosal repair. one study implicated prostaglandins in growth factor-stimulated restitution, but a more prominent role of prostaglandins in mucosal repair is their ability to close interepithelial tight junctions. , , for instance, ischemic-injured small intestine rapidly recovers barrier function (as measured in vitro as transepithelial resistance) in the presence of pgi and pge , despite the fact that these prostanoids had little effect on villous contraction and epithelial restitution. however, electron microscopic examination of tissues reveals dilation of tight junctions in tissues treated with nsaids, whereas those additionally treated with prostaglandins have closely apposed tight junctions (figures . - and . - ). prostaglandins stimulate closure of tight junctions via the second messengers cyclic adenosine monophosphate and ca + , which interestingly were among the first mediators found to modulate tight junction permeability. , such tight junction closure is of importance to patients with intestinal injury that are treated with nsaids, because reduced prostaglandin levels may result in increased intestinal permeability. for example, in a study on ischemic-injured porcine ileum, treatment with the nsaid indomethacin resulted in a significant increase in intestinal permeability to inulin and lipopolysaccharide compared with tissues that were treated additionally with pgi and pge . section . pathophysiology of mucosal injury and repair a b figure . - ultrastructural appearance of repairing ischemic-injured mucosa. a, restituting epithelium hours following a -hour ischemic episode in the presence of the nonselective cyclooxygenase inhibitor indomethacin. dilation of the interepithelial space and the apical tight junction (arrows) correlates with a leaky intestinal barrier, b, similar restituting epithelium had been treated additionally with prostaglandins e and i . the close apposition of the tight junction (arrows) and the interepithelial space correlate with normalization of intestinal barrier function. -cm bar = µm. the process of restitution absolutely depends on a group of compounds called polyamines. , the rate-limiting enzyme in the formation of the polyamines spermine, spermidine, and putrescine is ornithine decarboxylase. in rats with stress-induced duodenal ulcers, systemic administration of the ornithine decarboxylase inhibitor α-difluoromethylornithine significantly reduced polyamine levels and greatly reduced epithelial restitution. furthermore, intragastric treatment of these same rats with putrescine, spermidine, and spermine prevented the delayed mucosal repair induced by α-difluoromethylornithine. interestingly, gastric tissue levels of ornithine decarboxylase increased in rats with stress-induced gastric ulcers, suggesting that tissue injury enhances polyamine production, which may contribute to the normal rapid rate of epithelial restitution. the mechanisms whereby polyamines stimulate epithelial restitution are not clear. mccormack, wang, viar, et al. hypothesized that polyamines increase transglutaminase activity, an enzyme that catalyzes the cross-linking of cytoskeletal and basement membrane proteins. further investigation of the role of polyamines in iec- cell migration showed that depletion of polyamines resulted in disruption of the cytoskeleton and reduced the physical extension of lamellipodia. more recent studies have clarified this pathway. in particular, polyamines have been shown to regulate cytoskeletal cellular migration via activation of the small guanosine triphosphatase-rho-a by elevating intracellular ca + levels. these elevations in ca + result from polyamine regulation of expression of voltagegated k + channels and altered membrane electric potential. polyamines also play a role in the normal physiologic regulation of crypt cell proliferation and differentiation. , polyamines are produced by fully differentiated enterocytes at the tip of the villus and may reach the crypt within sloughed luminal epithelium or via local villous circulation. following intestinal injury, polyamines appear to stimulate enhanced proliferation by increasing the expression of protooncogenes, which control the cell cycle. the mechanism whereby polyamines influence gene expression likely relates to the cationic nature of these compounds, which may influence the tertiary structure of negatively charged dna and rna. locally produced growth factors-including epidermal growth factor (egf), transforming growth factor α (tgf-α), tgf-β, and hepatocyte growth factor-have the ability to modulate mucosal recovery. the most important of these growth factors in early mucosal repair events is tgf-β, which is a potent stimulus of epithelial restitution and modulator of the extracellular matrix. neutralization of tgf-β retards epithelial migration in vitro, and tgf-β apparently may serve as a point of convergence for mediators of restitution, because neutralizing tgf-β also inhibits the effects of other peptides. however, tgf-β paradoxically inhibits epithelial proliferation, thereby reducing the supply of new enterocytes for mucosal repair. conversely, egf, produced by the salivary glands and duodenal brunner's glands, and the related tgf-α, produced by small intestinal enterocytes, are potent stimulants of enterocyte proliferation. these growth factors share approximately % of their amino acid structure, bind to the same receptor on the basolateral surface of enterocytes, and are not related to tgf-β. the physiologic role of egf is difficult to discern because it is present in the intestinal lumen, with no apparent access to its basally located receptor. however, egf has been proposed to act as a "surveillance agent" that gains access to its receptor during epithelial injury (when the egf receptor likely would be exposed) to stimulate proliferation. tgf-α presumably has a similar role, but it is present in greater concentrations in the small intestine because it is produced by differentiated villous enterocytes. the mature peptide is cleaved from the extracellular component of the transmembrane tgf-α precursor and released into the lumen. another group of proreparative peptides produced within the gastrointestinal tract are the trefoil peptides. under physiologic conditions, trefoil peptides are secreted by mucus-producing cells at distinct anatomic sites. for example, gastric epithelium produces the trefoil peptide ps , whereas the small and large intestine mucosa produce intestinal trefoil peptide. however, any of the trefoil peptides may be upregulated within repairing epithelium regardless of anatomic site. , in addition, trefoil peptides have the ability to induce their own expression, amplifying the level of these reparative factors at sites of mucosal repair. trefoil peptides are the most potent stimulants of epithelial migration in vitro, and their effects are independent of growth factors, including tgf-β. however, recent evidence suggests that egf receptor activation is required for induction of ps and another of the trefoil peptides, termed spasmolytic peptide, in gastric epithelium in vitro. the importance of trefoil peptides to the mucosal repair response in vivo is illustrated by gene knockout studies, in which mice deficient in intestinal trefoil factor have greatly reduced ability to repair intestinal injury. in fact, detergent-induced mucosal injury was lethal because of a lack of restitution compared with wild-type mice that fully recovered from similar mucosal injury. the fact that administration of intestinal trefoil factor restored restitution has important therapeutic implications. the mechanism whereby trefoil peptides stimulate epithelial migration is yet to be characterized fully but appears to involve translocation of the adherens junction protein e-cadherin, thereby allowing cells to become untethered from neighboring cells. the principal metabolic fuel of enterocytes is glutamine and of colonocytes, butyrate. however, recent studies suggest that glutamine and butyrate have more specific proliferative actions aside from their role as nutrients. for example, in the piglet ipec-j enterocyte cell line, glutamine enhanced gene transcription by increasing mitogen-activated protein kinase activity. , similarly, butyrate stimulated mucosal growth following colonic infusion in the rat. because of such growth-promoting actions, glutamine was shown to prevent intestinal mucosal atrophy and dysfunction that accompanies starvation , and long-term total parental nutrition. , additionally, glutamine improves function of transplanted small intestine , and protects intestinal mucosa from injury if administered before chemotherapy and radiation therapy. , intestinal nutrients also may synergize with other proliferative agents. for example, administration of glutamine and tgf-α to porcine ileum that had been subjected to hours of ischemia resulted in a synergistic increase in mitogen-activated protein kinase activity, enterocyte proliferation, and villous surface area. although concern has arisen that such early return to normal surface area may result in dysfunctional mucosal digestive and absorptive function because of resurfacing denuded mucosa with immature epithelium, nutrients and growth factors also appear to promote early differentiation. in the case of glutamine and tgf-α restoration of postischemic small intestine, rapid recovery of digestive enzymes also was documented. effective gastrointestinal motility involves a complex interaction between the enteric nervous system, muscular wall, and luminal contents. additional factors that influence the net transit of digesta include gravity, the volume and viscosity of the contents, and pressure gradients created by simultaneous contraction and relaxation of adjacent segments of bowel. casual use of the term intestinal motility in veterinary medicine often underestimates the complexity of the processes involved in the transit of intestinal contents. this is particularly true when the term is used to describe the frequency and or intensity of intestinal sounds, or borborygmi. the existence of borborygmi does not always equate with progressive movement of intestinal contents. disruption to normal motility occurs commonly in horses for a variety of reasons. examples of diseases in which altered motility may be present include gastroduodenal ulceration, intraluminal obstruction or impaction, excessive wall distention, strangulating obstructions, peritonitis, and inflammatory bowel diseases such as duodenitis proximal jejunits or colitis. ineffective intestinal motility is also a feature of several neonatal diseases, including prematurity, systemic sepsis, and perinatal asphyxia. certain parasitic infections, electrolyte derangements, and endotoxemia can modify digesta transit in horses of all ages. general anesthesia and specific sedatives, such as xylazine, romifidine, or detomidine, also disturb motility. the inhibition of propulsive bowel activity usually is referred to as ileus. ileus is ascribed most frequently to the condition that occurs after laparotomy and is termed simple or uncomplicated postoperative ileus (poi). the term complicated or paralytic ileus describes intestinal motility disturbed for longer periods after surgery. poi in horses is associated most commonly with surgery of the small intestine, particularly after resection and anastomosis, , is a common complication of small intestinal surgery, and can have a negative effect on short-term postoperative survival. , motility dysfunction likely is present in all horses after laparotomy, but many are affected subclinically and require minimal or no specific intervention. in symptomatic animals, clinical signs are apparent shortly after recovery and include colic, tachycardia, dehydration, decreased borborygmi and fecal output, and sequestration of fluid within the stomach. rectal examination and ultrasound reveal small intestinal distention with rare or absent wall movement. the severity and duration of intestinal stasis varies, lasting from minutes to days. a specific motility disorder involving the cecum or ileocecocolic region occurs sporadically in horses. [ ] [ ] [ ] the condition most commonly occurs after general anesthesia and extraabdominal surgery, particularly orthopedic and upper airway procedures, and therefore often is categorized as a form of poi. anecdotally, horses at greatest risk are young male performance animals. other cases occur spontaneously, often in animals with painful primary conditions such as uveitis or septic tenosynovitis. the syndrome is frustrating in that clinical signs are often subtle unless cecal perforation has occurred. in horses with a cecal emptying defect after anesthesia, overt signs are usually apparent to days after the procedure. the earliest detectable signs include depression and a reduction in feed intake and fecal output. ineffective emptying results in overfilling of the cecum with moist contents, which is manifest by signs of mild to moderate colic. if the condition is recognized late or untreated, the cecum may rupture and result in fatal peritonitis. the inherent rhythmicity of electric activity in the intestine is controlled by the interstitial cells of cajal, specialized cells that are electrically coupled to myocytes via gap junctions. these cells are responsible for generating and propagating slow-wave activity and may be critically involved in a range of motility disorders. the enteric nervous system primarily controls and coordinates intestinal contraction. a combination of central and autonomic innervation influences events, but contraction does not require external neural input. the parasympathetic supply to the gastrointestinal tract is via the vagus and pelvic nerves, and the sympathetic supply is through postganglionic fibers of the cranial and caudal mesenteric plexuses. a complex network of interneurons within each plexus integrates and amplifies neural input; the intensity and frequency of resultant smooth muscle contractions are proportional to the amount of sympathetic and parasympathetic input. additional binding sites for a number of other endogenous chemicals, including dopamine, motilin, and serotonin exist within the enteric nervous system and on smooth muscle cells. acetylcholine is the dominant excitatory neurotransmitter in the gastrointestinal tract and exerts its action through muscurinic type receptors on smooth muscle cells. sympathetic fibers innervating the gastrointestinal tract are adrenergic, postganglionic fibers with cell bodies located in the prevertebral ganglia. activation of α adrenergic receptors on cholinergic neurons within enteric ganglia inhibits the release of acetylcholine and therefore reduces intestinal contraction. β -, β -, and β-atypical receptors are directly inhibitory to the intestinal smooth muscle. inhibitory nonadrenergic, noncholinergic neurotransmitters include adenosine triphosphate, vasoactive intestinal peptide, and nitric oxide. , these neurotransmitters are critical for mediating descending inhibition during peristalsis and receptive relaxation. substance p is a nonadrenergic, noncholinergic neurotransmitter that may be involved in contraction of the large colon. , the rate and force of intestinal contractions along the small intestine and large colon of the horse are important determinants of intestinal motility; of even greater importance to the net propulsion of digesta are the cyclical patterns of contractile activity. these patterns are known as the small intestinal and colonic migrating motility (or myoelectric) complexes (mmcs). , the colonic complex usually originates in the right ventral colon and variably traverses the ascending and descending colons. many of these complexes are related temporally to a specialized motility event of the ileum, the migrating action potential complex. local inflammation within the intestinal muscularis and inhibitory neural events are important initiators of intestinal ileus. , intestinal inflammation not only is important in primary intestinal diseases in horses, such as duodenitis-proximal jejunitis and colitis but also is induced after simple intestinal handling during laparotomy. experimental data from other species suggests that handling of the small or large intestine at the time of surgery activates resident macrophages with resultant increased expression of p-selectin and intercellular adhesion molecule on endothelial cells within the vasculature of the muscularis. the upregulation of associated ligands on leukocytes leads to sequential "sticking and rolling," followed by neutrophil migration into the interstitium. the subsequent release of neutrophil products interferes with cell signaling and results in reduced intensity of smooth muscle contraction. furthermore, the inflamed intestine fails to contract normally in response to putative prokinetic agents. another key factor in the development of intestinal stasis after inflammation is the local overproduction of nitric oxide caused by the upregulation of inducible nitric oxide synthase (inos) by resident macrophages. nitric oxide is a key inhibitory neurotransmitter of the nonadrenergic, noncholinergic system. nitric oxide synthase inhibition has been a pharmacologic target in the treatment of experimental ileus. the inhibitory effects of α -agonists such as xylazine and detomidine on cecal and large colon motility are well described. [ ] [ ] [ ] [ ] [ ] [ ] intravenously administered xylazine inhibits cecal and large colon motility for to minutes without seriously disrupting small intestinal myoelectric activity, and detomidine can reduce large intestinal myoelectric activity for up to hours. the α -antagonist yohimbine has a weak but positive effect on cecal emptying in normal ponies, suggesting that normal motility is under constant α -adrenergic tone. atropine is a postganglionic blocking agent that binds to muscarinic receptors. when administered at . mg/kg, atropine inhibits individual small intestinal, cecal, and colonic contractions for about minutes but supresses small intestinal and colonic migrating complexes for up to hours. neural reflexes also may mediate inhibition of motility associated with peritoneal inflammation. , the afferent segment is composed partly of capsaicin-sensitive visceral afferent c fibers that terminate in the dorsal horn of the spinal cord, where they can activate inhibitory sympathetic fibers or synapse directly on the sympathetic ganglia. consequently, the efferent limb of the reflex expresses increased sympathetic outflow, primarily mediated through stimulation of α -adrenoreceptors, and inhibition of acetylcholine release, which provides the rationale for α -blockade in treating ileus. intraluminal infusion of capsaicin before abdominal surgery ameliorated the severity of poi in experimental rats. this finding highlights the importance of visceral afferent fibers in the development of poi. ileus also can occur in association with intestinal obstruction or displacement. mild to moderate distention of the bowel, such as that occurring in the early stages of an intraluminal obstruction, evokes an increase in local contractile activity. , excessive distention results in inhibition of motility within the distended segment of bowel. intestinal stasis is not always detrimental and under certain conditions may be protective. endotoxemia is a clinical feature of many diseases of the equine gastrointestinal tract, and endotoxins independently can exert a negative effect on intestinal motility and transit. a variety of mediators likely are involved, but activation of α -adrenoreceptors and production of prostanoids appear to be important, for pretreatment with yohimbine or nonsteroidal antiinflammatory drugs (nsaids; phenylbutazone or flunixin), respectively, ameliorates the inhibitory effects of experimental endotoxin infusion. , endotoxin infusion induced an inflammatory response in the intestine of rats that mimicked the response induced by handling during laparotomy. the similarity of the responses were highlighted in a recent study that demonstrated that prior exposure of the muscularis to endotoxin protected the intestine from the effects of manipulation. the pathophysiology of cecal emptying defect is not known. this syndrome may best mimic poi in human beings and generally is considered a large intestinal disorder. an important difference in horses is that laparotomy is a rare predisposing factor, and most cases occur in horses undergoing routine extraabdominal surgical procedures. general anesthesia itself is a potent inhibitor of gastrointestinal motility in horses, but these effects are short-lived and reversible within hours of anesthetic withdrawal. the return of normal motility in horses after experimental ileus was most delayed in the cecum, suggesting that this may be a common site of ileus in horses. a link between routine postoperative medications, such as phenylbutazone and aminoglycoside antibiotics, has been suspected but not established. an inhibitory effect of nsaids on large colon contractility has been demonstrated using in vitro techniques. primary sympathetic overstimulation could be involved, for many of the affected animals are young, male horses or animals with painful diseases. the duration of surgery influences the development of small intestinal poi, but not cecal emptying dysfunction. , technique may have a weak influence on small intestinal poi after jejunojejunostomy. the duration of intestinal ileus was shorter in animals that received a sideto-side stapled anastomosis than those that had a hand sewn end-to-end procedure. the duration of ileus after stapled end-to-end anastomosis was not different from that after either procedure. reported risk factors for the development of poi in horses include age (> years), small intestinal resection and anastomosis, breed (arabians had a greatest risk than other breeds), and duration of surgery. interestingly, performing a pelvic flexure enterotomy and emptying the colon had a protective effect against poi. the diagnosis of ileus is based on history and physical examination findings. important tests include determination of pulse rate and rhythm, auscultation and percussion of the abdomen, rectal palpation, and passage of a nasogastric tube. a complete blood count with fibrinogen estimation and cytologic analysis of peritoneal fluid may improve the accuracy of diagnosis. affected animals may be colicky because of accumulation of fluid in the upper gastrointestinal tract (classical poi) or cecal contents (cecal emptying defect). decompression of the stomach is important diagnostically and therapeutically in horses with poi after small intestinal surgery. failure to relieve pain with gastric decompression could point toward mechanical obstruction, severe inflammation of the intestine, or peritonitis. most animals with ileus are depressed and have reduced fecal output and intestinal borborygmi. one should interpret intestinal sounds with caution, however, because the presence of borborygmi does not always equate to progressive intestinal motility and merely may reflect local, nonpropagated contractions. rectal palpation findings in cases of persistent poi or duodenitis-proximal jejunitis are usually nonspecific but may reveal dilated, fluid-filled loops of small intestine. the clinician occasionally can palpate roughened peritoneal surfaces if peritonitis is present. one can palpate cecal distention with digesta in horses with advanced cecal dysfunction. distinguishing functional ileus from mechanical obstruction is important and can be difficult, but horses with mechanical obstruction typically have sustained high volumes of gastric reflux that vary little over time. the management of intestinal ileus depends on the segment of gastrointestinal tract involved. therapy for ileus of the proximal gastrointestinal tract involves a combination of gastric decompression, fluid and electrolyte therapy, and antiinflammatory drug therapy. electrolyte therapy is critical, particularly for maintaining adequate extracellular concentrations of potassium, calcium, and magnesium. calculation of the volume of fluid to be administered should include maintenance requirements ( to ml/kg/day) plus an estimate of losses, especially those lost through gastric decompression. one should consider parenteral provision of calories when feed has been withheld for more than hours, particularly after the horse has had surgery. a combination of amino acids and dextrose with or without lipids effectively provides these calories. hand walking also may provide some benefit to these animals but is not likely to have a direct effect on intestinal motility. one should avoid drugs that may impair normal intestinal motility, including the anticholinergics such as atropine and opiate receptor agonists such as morphine and meperidine. butorphanol appears to have little or no adverse effect on small or large intestinal motility. , one should use α -agonists sparingly because of their inhibitory effects on large intestinal motility. fluid therapy is the key component in managing cecal emptying defect, usually in combination with lubricants or laxatives, such as mineral oil or magnesium sulfate, and with careful use of antiinflammatory drugs. horses with primary cecal impaction or impaction caused by an emptying defect frequently require surgery to prevent fatal rupture. the surgical management of these cases is controversial and may include typhlotomy alone, typhlotomy with a bypass procedure such as ileocolic or jejunocolic anastomosis, or a bypass without typhlotomy. most horses that undergo simple typhlotomy have an uneventful recovery, although a small number experience impaction again and require a second laparotomy. experimental and anecdotal evidence provides a strong rationale for using antiinflammatory drugs to prevent and treat gastrointestinal ileus, particularly in animals that may have endotoxemia. flunixin meglumine is used widely in equine practice as an analgesic and antiinflammatory agent, and it ameliorates many of the adverse systemic effects of endotoxin, particularly those on the cardiovascular system. a potential negative effect of nsaids on large intestinal contractility has been suggested. broad-spectrum antimicrobials are indicated when one suspects sepsis or for the compromised immune system, as in cases of moderate to severe endotoxemia. theoretical concerns have been raised regarding the use of aminoglycoside antibiotics in animals with ileus. high concentrations of aminoglycoside antimicrobials inhibited intestinal contractions in exposed sections of intestine in vitro, but this inhibitory effect is unlikely to occur at clinically relevant doses. motility-enhancing drugs have been advocated to treat gastrointestinal ileus. unfortunately, information directly pertinent to horses is limited and must be extrapolated cautiously from that of other species because of the differences in intestinal anatomy and physiology. prokinetic drugs potentially can shorten the length of hospitalization, thereby reducing the cost of treatment and the number of potential complications such as weight loss, thrombophlebitis, and laminitis. experimental evidence indicates that prokinetic drugs can minimize the development of postoperative abdominal adhesions. most prokinetic drugs require a healthy gut wall to enhance intestinal contraction. therefore one should not assume that many of these drugs would be effective in the presence of an inflammatory injury such as that which can occur after intestinal manipulation at surgery or that associated with duodenitis-proximal jejunitis. bethanechol is a parasympathomimetic agent that acts at the level of the myenteric plexus and directly on intestinal smooth cells through muscarinic receptors. bethanechol is a synthetic ester of acetylcholine and is not degraded by anticholinesterase. bethanechol has cholinergic side effects, including abdominal discomfort, sweating, and salivation, although these are minimal when the drug is administered at . mg/kg body mass subcutaneously or orally. bethanechol has efficacy in diseases that involve abnormal gastric emptying and delayed small intestinal transit and has been shown to increase gastric contractility and hasten the emptying of liquid and solid phase markers from the stomach of normal horses. [ ] [ ] bethanechol also increases the strength and duration of wall contractions in the cecum and right ventral colon and consequently speeds up cecal emptying. neostigmine increases receptor levels of acetylcholine by inhibiting cholinesterase. the drug ( . to . mg/kg intravenously) promotes cecal and colonic contractile activity and hastens the emptying of radiolabeled markers from the cecum. neostigmine has been used to manage small intestinal ileus, but it significantly delayed the emptying of -mm beads from the stomach of normal adult horses. metoclopramide acts principally as a -hydroxytryptamine -receptor ( ht- ) agonist and ht- -receptor antagonist. in contrast to newer generation benzamides, metoclopramide is also an antagonist at dopamine (da ) and (da ) receptors. antagonism of prejunctional da receptors facilitates acetylcholine release and smooth muscle contraction. metoclopramide crosses the blood-brain barrier, where its antagonist properties on central da receptors can result in extrapyramidal signs, including seizure. these signs were responsible for poor acceptance of the drug in equine practice. most investigators have failed to demonstrate significant effects of metoclopramide in experimental animals, but constant intravenous infusion ( . mg/kg/hr) in a population of postoperative horses significantly decreased the volume and duration of gastric reflux over control and intermittent drug infusion groups. infusion was well tolerated and appeared to be superior to intermittent infusion or no treatment at all. cisapride is a second-generation benzamide that acts as a ht- agonist and ht- receptor antagonist but is without antidopaminergic action. stimulation of ht- receptors within the enteric nervous system enhances release of acetylcholine from the myenteric plexus. several reports suggest the efficacy of cisapride in managing intestinal disease in horses, including the resolution of persistent large colon impaction, treatment of equine grass sickness, and as a preventative for poi in horses after small intestinal surgery ( . mg/kg body mass intramuscularly during the postoperative period). [ ] [ ] [ ] [ ] the horse erratically absorbs tablets administered rectally, but a method for preparing a parenteral form of the drug from tablets has been described. cisapride has the potential to cause adverse cardiac side effects mediated through blockage of the rapid component of the delayed rectifier potassium current that include lengthening of the qt interval and development of torsades de pointes, a potentially fatal arrhythmia. these adverse effects have resulted in withdrawal of the drug in the united states. domperidone acts as a competitive antagonist at peripheral da receptors. the drug is a therapeutic agent ( . mg/kg/day) for mares grazing endophyte-infected tall fescue, principally because of drug-enhanced prolactin release. the potential prokinetic effects of domperidone have not been studied extensively in horses, but a modest efficacy of domperidone ( . mg/kg intravenously) has been demonstrated in experimental ileus in two ponies. erythromycin is a direct motilin receptor agonist on smooth muscle cells and also may act within the enteric nervous system to facilitate the release of acetylcholine and motilin. erythromycin enhances gastric emptying in normal horses but has a more pronounced effect on the hindgut. , erythromycin lactobionate ( . mg/kg intravenously) hastens cecal emptying in normal animals and induces colonic mmc-like activity across the colon. administration often is associated with defecation and abdominal discomfort. the drug may help prevent cecal impaction in horses after anesthesia, although its effectiveness on cecal motility in the immediate postoperative period may be reduced. high doses, constant infusion, or prolonged use of erythromycin induces receptor downregulation and inhibition of activity. erythromycin can induce diarrhea in adults, therefore one should avoid dosing over many days. naloxone ( . mg/kg intravenously) induces contractile activity in the cecum and left colon. defecation commonly follows administration of naloxone within to minutes. α -adrenoreceptor antagonists such as yohimbine or tolazoline counteract increased sympathetic outflow in response to nociceptive stimulation. yohimbine infusion ( µg/kg) also may attenuate the negative effects of endotoxin on motility. intravenous infusion of lidocaine may suppress primary afferent neurons, thereby limiting reflex efferent inhibition of motility. an infusion dose of to mg/min over to hours has been recommended for horses. lidocaine infusion is associated with reversible side effects that include muscle fasciculations, ataxia, and seizure. consequently, the rate of infusion requires close monitoring. katharina l. lohmann, michelle henry barton endotoxemia is defined as the presence of endotoxin in the bloodstream. most often, however, the term is used to refer to the associated clinical manifestations caused by an overshooting inflammatory reaction. in its pathophysiologic consequences the innate immune response to endotoxin (lipopolysaccharide) is similar to the response to other stimuli; for example, overwhelming bacterial infection, viral infection, or severe trauma. the term systemic inflammatory response syndrome therefore was introduced to describe a general systemic inflammatory process independent of cause. sepsis is defined as the "systemic inflammatory response to infection," and septic shock as "sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction." according to these definitions the diagnosis of sepsis requires documentation of infection by culture in addition to two or more of the following findings: hypo-or hyperthermia, tachycardia, tachypnea or hypocapnia, and leukocytosis, leukopenia, or an increased proportion of immature leukocyte forms. organ failure is a common sequela of endotoxic or septic shock, and the term multiple organ dysfunction syndrome describes insufficiency of two or more organ systems, as evident by clinical or clinicopathologic changes. in horses one should include the laminae of the feet in the list of organs susceptible to failure. german scientist richard pfeiffer ( - ), in working with vibrio cholerae, first described endotoxin as a toxin "closely attached to, and probably integral of, the bacterial body." he observed this toxin to be distinct from the actively secreted, heat-labile, and proteinaceous bacterial exotoxins. endotoxin later was found to be a heat-stable lipopolysaccharide structure, and the terms endotoxin and lipopolysaccharide now are used interchangeably. lipopolysaccharide is a major structural cell wall component of all gram-negative bacteria, including noninfectious species (figure . - ). with to × molecules per cell, lipopolysaccharide makes up about % of the outer layer of the outer cell membrane and is a key functional molecule for the bacterial outer membrane, serving as a permeability barrier against external noxious agents. the lipopolysaccharide molecule consists of four domains, which are essential for the virulence of gram-negative bacteria. three of the domains (inner core, outer core and o-specific chain) represent the hydrophilic polysaccharide portion of the molecule, whereas the lipid a portion represents the hydrophobic lipid portion ( figure . - ). combined, these domains confer the overall amphiphilic properties of the molecule that lead to the formation of micellar aggregates in aqueous solutions. o-specific chains (also called o-antigen polysaccharides or o-chains) are characteristic of any given type of lipopolysaccharide and show enormous structural variability between bacterial serotypes. o-chains are synthesized by addition of preformed oligosaccharide blocks to a growing polymer chain and therefore have a repetitive structure. o-specific chains determine part of the immunospecificity of bacterial cells and, on interaction with the host immune system, serve as antigens for the production of species-specific antibodies. o-specific chains are further responsible for the smooth appearance of gram-negative bacterial colonies on culture plates, and lipopolysaccharide molecules containing an o-chain are termed smooth lipopolysaccharide. the inner (lipid a-proximal) and outer (o-chainproximal) core oligosaccharide portion is more conserved between different strains of gram-negative bacteria than the o-specific chain. the core of all lipopolysaccharide molecules contains the unusual sugar kdo ( -deoxy-dmanno-oct- -ulopyranosonic acid), which links the core region to the lipid a molecule. synthesis of a minimal core is essential for the survival of bacteria, and the smallest naturally occurring lipopolysaccharide structure consists of lipid a and kdo. in contrast to the s-form colonies, colonies of gram-negative bacteria with lipopolysaccharide molecules that lack the o-specific chain but contain a core region show a rough appearance on culture plates. rough lipopolysaccharide molecules are denoted further as ra, rb, etc. to indicate the length of the core region. in re-lipopolysaccharide (also called deep rough lipopolysaccharide), the core region is reduced to a kdo residue. remutants often are used to raise antibodies against the core region in an attempt to provide cross-protection against a variety of bacterial species. the lipid a portion serves to anchor the lipopolysaccharide molecule in the bacterial outer membrane and has been identified as the toxic principle of lipopolysaccharide, and its structure is highly conserved among gramnegative bacteria. the common structure shared by lipid a molecules is a , '-bisphosphorylated β , -linked d-glucosamine disaccharide backbone (lipid a backbone), which is acylated by up to six fatty acids. figure . - shows the acylation pattern for escherichia coli lipopolysaccharide. variation in the lipid a structure between gramnegative bacteria affects the number, length, and position of fatty acids and the backbone structure and the substitution of phosphate by other polar groups. according to its nature as a structural cell wall component, the presence of endotoxin implies the presence of gram-negative bacteria as a source. depending on the nature of the underlying disease, these bacteria may circulate in the bloodstream in their intact form (i.e., bacteremia), may be confined to a localized infectious process, or may be part of the endogenous bacterial flora colonizing the gastrointestinal tract. in any of these scenarios, endotoxin molecules are released as a by-product of bacterial growth and in large numbers on bacterial cell death. common infectious conditions associated with endotoxemia in horses include neonatal gram-negative sepsis, bacterial pneumonia and pleuropneumonia, endometritis, peritonitis, and infectious colitis with bacteria such as salmonella spp., that are not part of the normal intestinal flora. in one study, for example, endotoxin was detectable in plasma of % of foals evaluated for presumed sepsis. the term translocation describes entry of endogenous bacteria and bacterial products from the gastrointestinal tract into tissues and the systemic circulation. the natural intestinal flora of horses consists mainly of gramnegative, anaerobic bacteria, and thus large amounts of endotoxin normally exist in the lumen of the equine intestinal tract. even in health, small amounts of endotoxin cross the intact mucosal barrier and reach the portal circulation and the liver. these molecules are cleared, however, by the mononuclear phagocytic system in the liver and only lead to a localized and restricted activation of the host immune system. for endotoxin translocation to become detrimental, excessive amounts have to cross the intestinal barrier and overwhelm the mononuclear phagocytic system or the capacity of the liver to detoxify lipopolysaccharide must be compromised. the latter may be a concern in conditions such as hepatitis, cholangiohepatitis, or portosystemic shunting of blood. permeability of the intestinal mucosal barrier frequently increases in cases of acute gastrointestinal disease. colic patients are prime candidates to development endotoxemia, and plasma endotoxin was detectable in % to % of colic patients on admission. , a higher percentage of horses tested positive for endotoxin when only patients presented for surgical intervention were evaluated. aside from gastrointestinal rupture, increased permeability to intact bacteria or free endotoxin molecules is thought to be associated most commonly with ischemic insults such as strangulating obstruction and bowel infarction, severe inflammation as in proximal enteritis and colitis, bacterial overgrowth, and intraluminal acidosis, which occurs with grain overload. , one study, however, found no difference in plasma endotoxin detection between disease groups, therefore emphasizing the fact that any disease of the abdominal cavity can induce endotoxemia in horses. in the same study, endotoxin was approximately times more likely to be detected in peritoneal fluid as opposed to plasma samples. similarly, higher cytokine concentrations have been measured in peritoneal fluid than in plasma. the likely explanation for these findings is a local inflammatory response in the peritoneal cavity elicited by translocated bacteria and/or lipopolysaccharide molecules before their absorption into the systemic circulation. although certainly the most important factor in horses, conditions other than gastrointestinal disease may result in translocation of endotoxin and bacteria. in experimental studies using laboratory animals, entry of gutassociated bacteria into the lymphatic system was demonstrated after hypovolemic shock, burn injuries, trauma, malnutrition, and starvation. [ ] [ ] [ ] furthermore, endotoxin itself caused bacterial translocation into mesenteric lymph nodes after intraperitoneal administration to mice. these findings have received much attention in the literature concerning human patients because they serve to explain cases of endotoxic shock in the absence of demonstrable bacterial infection. one should keep in mind the possibility of translocation when evaluating cases of presumed systemic inflammatory response syndrome in horses, in which one cannot demonstrate bacterial infection or gastrointestinal disease. endotoxin translocation also may be associated with strenuous exercise, which results in reduced splanchnic blood flow, hypoxemia, and a higher body temperature. in fit racehorses a significantly increased mean plasma lipopolysaccharide concentration was found after racing, whereas antilipopolysaccharide immunoglobulin g levels were decreased. fit horses showed significantly higher antilipopolysaccharide immunoglobulin g concentrations at rest than sedentary controls, suggesting leakage of small amounts of endotoxin from the intestinal lumen during training and racing. the clinical significance of these findings requires further investigation. the initiating event in the pathophysiology of endotoxemia is the activation of lipopolysaccharideresponsive cells by endotoxin, resulting in altered cellular functions and increased expression of inflammatory mediators. immune cells such as macrophages, which are the first to encounter endotoxin, respond to minute amounts of lipopolysaccharide, which usually allows them to eliminate gram-negative bacteria and free lipopolysaccharide molecules efficiently. an important factor in the exquisite sensitivity to lipopolysaccharide is the presence of lipopolysaccharide-binding protein (lbp). lbp is an approximately -kd plasma glycoprotein synthesized by hepatocytes and belongs to the group of acute phase proteins. under the control of inflammatory agents and cytokines, lbp concentration in plasma increases approximately -fold within hours of an inflammatory stimulus. the main function of lbp is to transfer lipopolysaccharide to endotoxin-responsive cells, which include mononuclear phagocytes, neutrophils, lymphocytes, and endothelial cells. the importance of a highly sensitive response to lipopolysaccharide for protection against gram-negative bacterial infection is demonstrated in experiments using lbp "knock-out" mice (mice that lack the lbp gene and are therefore unable to synthesize lbp). although these animals are resistant to the effects of isolated lipopolysaccharide, they are unable to control bacterial infection and rapidly succumb. despite its crucial importance for an effective host defense, lbp is not essential for lipopolysaccharide-receptor interaction per se, because high concentrations of lipopolysaccharide can activate cells in the absence of lbp. aside from its role as a catalyst of cellular activation by lipopolysaccharide, lbp has opsonizing activity and participates in the phagocytosis of lipopolysaccharide by macrophages and neutrophils. , although phagocytosis of lipopolysaccharide is receptor dependent, it appears to be uncoupled from intracellular signaling events and occurs in the absence of cell activation. lbp further catalyzes transfer of lipopolysaccharide to lipoproteins such as high-density lipoprotein, which neutralizes lipopolysaccharide activity. this detoxifying effect may become important when large amounts of lipopolysaccharide are present. a protective effect of lbp against lipopolysaccharide challenge and infection has been demonstrated in a murine model. the most important lipopolysaccharide receptors known to date are cluster differentiation antigen (cd ) and toll-like receptor (tlr ). both are classified as pattern recognition receptors, which means that they recognize lipopolysaccharide as a pattern common to all gram-negative bacteria. cd is a -kd protein that in its membrane-bound form (mcd ) is inserted into the cell membrane via a glycosylphosphatidyl-inositol anchor. cd is expressed primarily on monocytes and tissue macrophages and to a lesser extent on neutrophils. cd also is found in a free, or soluble, form (scd ) that can bind to cell types lacking cd , such as endothelial cells, and make them lipopolysaccharide-responsive. in addition to this proinflammatory effect, high concentrations of scd can sequester and neutralize lipopolysaccharide. the amount of circulating scd greatly increases during inflammation, which makes it a useful marker of acute and chronic inflammation. although cd is known to be crucial for cellular activation, it cannot transmit signals to the inside of the cell because it lacks a transmembrane domain. the missing link between cd and the cytosolic environment is a toll-like receptor in association with a molecule named md- . the name toll-like receptor stems from the homology of the mammalian receptor with a receptor type in drosophila (toll) that is important for dorsoventral orientation and immune responses in the fly. a number of toll-like receptors have been identified in mammalian species so far, but tlr appears to be the receptor subtype most important for lipopolysaccharide signaling. the importance of cd and tlr in the cellular response to lipopolysaccharide has been demonstrated in a number of experiments. mice deficient in cd are incapable of mounting a normal inflammatory response to lipopolysaccharide, whereas mutation or deletion of the gene encoding for tlr causes lipopolysaccharide hyporesponsiveness. [ ] [ ] [ ] after binding of lipopolysaccharide to cellular receptors, a multitude of signaling events takes place within the cell and results in the alterations of cellular metabolism known as cell activation. signaling pathways are characterized by sequential phosphorylation and thereby activation of enzymatic activities. a typical end result of intracellular signaling is the activation of transcription factors; for example, proteins that bind to dna and promote gene transcription. translational mechanisms are activated in a similar manner. among the bestcharacterized pathways in endotoxin-induced cell signaling are the mitogen-activated protein kinase (mapk) pathways and the activation of transcription factor nuclear factor κb (nfκb) (figure . - ) . , in the nfκb pathway the intracellular domain of tlr associates with the adapter protein myeloid differentiation factor and recruits interleukin- receptor-associated kinase to the complex. activation of interleukin- receptor-associated kinase, tumor necrosis factor receptor-associated factor, nfκb-inducing kinase, and iκb-kinase follow, and lastly, iκb is phosphorylated. iκb is an inhibitor protein complex that sequesters and inactivates nfκb in the cytoplasm. on phosphorylation, iκb is ubiquinated and degraded, and nfκb is translocated to the nucleus where it unfolds its activity. nfκb is a dimeric protein complex with several isoforms of which the p /p heterodimer is the most important inducible complex in mammals. proteins of importance for the pathogenesis of septic shock, the genes of which contain promoter elements for nfκb, include cytokines, inducible nitric oxide synthase, and cyclooxygenase (cox- ). three groups of mapks known to be crucially important for lipopolysaccharide-induced signal transduction are extracellular signal-regulated kinase, c-jun-terminal kinase, and p . final effects of signaling through mapk pathways include the activation of several transcription factors, translation initiation factors, and cytosolic enzymes such as phospholipase a , as well as an increase in the expression of adhesion molecules on the cell surface. despite the characterization of seemingly separate pathways, one should recognize that interaction and synergy between pathways is likely to occur. for example, simultaneous activation of p , c-jun-terminal kinase, and extracellular signal-regulated kinase results in much higher levels of tumor necrosis factor (tnf) reporter expression than activation of a single pathway. , aside from the mechanisms described here, pathways involving atypical protein kinase c , and receptor-independent integration of lipopolysaccharide into the cell membrane and ceramide-like second messenger activity of lipopolysaccharide have been proposed. additional pathways are likely to be uncovered in the ongoing investigation of intracellular signaling mechanisms and their in vivo significance. although endotoxin can exert some direct effects, cytokines are a primary mediator of lipopolysaccharide effects. cytokines are glycoprotein molecules that regulate inflammatory and immune responses by acting as a signal between cells. cytokines of major interest in the pathogenesis of endotoxemia include tnf, the interleukins, chemokines, and growth factors such as granulocyte-monocyte colony-stimulating factor. tnf is thought of as the most "proximal" cytokine released in response to lipopolysaccharide. studies corroborate this by showing that administration of recombinant tnf mimics the effects of lipopolysaccharide, and that antibodies directed against tnf protect against the lethal effects of endotoxin. increased plasma activity of tnf is associated with increased mortality in equine patients with acute gastrointestinal disease and in septic neonates. despite being a structurally diverse group of proteins, cytokines share several characteristics that allow them to execute their complex functions in the inflammatory response. any individual cytokine generally is produced by several different cell types, can act on different cell types, and has multiple effects on any given cell. furthermore, cytokine effects are redundant, meaning that different cytokines can share the same effect. in endotoxemia, this is particularly true for the effects of interleukin- (il- ) and tnf. many of the biologic activities of cytokines in vivo result from synergistic or antagonistic actions involving two or more cytokines. within itself the cytokine response is highly regulated: cytokines induce or suppress synthesis of other cytokines including their own (feedback regulation), regulate expression of cytokine receptors, and regulate cytokine activities. additional regulatory mechanisms include the release of specific cytokine inhibitors such as soluble il- and tnf-α receptors, cytokine receptor antagonists such as il- receptor antagonist, and antiinflammatory cytokines including il- , il- , il- , and transforming growth factor β. glucocorticoids also are produced increasingly in response to endotoxin and inhibit the production of cytokines. during a controlled inflammatory response, therefore, cytokine secretion is a selflimited event, whereas excessive stimulation of cytokine release can lead to the perpetuation of the inflammatory response even after the initial stimulus has been removed. conversely, the compensatory antiinflammatory reaction can become severe enough to cause anergy of the immune system and increased susceptibility to infection, which has been termed the compensatory antiinflammatory response syndrome. overall, excessive and unbalanced stimulation of the immune system therefore may result in predominantly proinflammatory (systemic inflammatory response syndrome), antiinflammatory (compensatory antiinflammatory response syndrome), or combined (mixed antagonist response syndrome) responses. interestingly, tolerance to endotoxin develops after repeated exposure to lipopolysaccharide. tolerance can be demonstrated in vitro and in vivo and encompasses decreased production of cytokines and a diminished clinical response. , mechanisms that likely are responsible for the development of endotoxin tolerance include receptor downregulation and inhibition of intracellular signaling pathways. , cytokines such as tnf are important mediators in the development of endotoxin tolerance. the development of endotoxin tolerance in horses has been reported. , aside from cytokines, a number of other molecules function as inflammatory mediators in the pathogenesis of endotoxemia, the synthesis and release of which are stimulated by endotoxin and by cytokines. these mediators include the arachidonic acid metabolites or prostanoids, platelet-activating factor (paf), oxygenderived free radicals, nitric oxide (no), histamine, kinins, and complement components. table . - summarizes the origins, targets, and effects of the most important inflammatory mediators involved in the pathogenesis of endotoxemia. figure . - shows the pathways of arachidonic acid metabolism by cox and lipoxygenase. cox products are the prostaglandins (pgs), prostacyclin (pgi ) and thromboxanes, and the lipoxygenase produces the leukotrienes. the innate immune response to lipopolysaccharide is an efficient defense mechanism that provides maintenance of homeostasis and therefore health in the face of an almost continuous exposure to microorganisms and their products. detrimental consequences of this immune response only occur if excessive and uncontrolled mediator output results in endothelial damage, neutrophil-mediated tissue damage, and uncontrolled activation of the coagulation and fibrinolytic cascades and the complement system. ultimately, the combination of these events culminates in cardiovascular instability, impaired hemostasis, organ failure, shock, and death. the following discussion addresses the various pathophysiologic events in the development of endotoxemia and shock and the role of inflammatory mediators. normal endothelium plays an important role in regulating blood pressure and regional tissue perfusion and provides an anticoagulant surface. endothelial dysfunction and damage result in a decreased responsiveness to vasoactive agents (vasoplegia), increased vascular permeability, and a tendency for clot formation in the microvasculature. if the basement membrane and underlying matrix are compromised, further microvascular hemorrhage can occur. endothelial cell damage is primarily neutrophilmediated. more specifically, damage is caused by oxygen-derived free radicals, which are produced within endothelial cells via reactions involving neutrophil-derived elastase and hydrogen peroxide molecules, endothelial cell enzymes such as xanthine oxidase, and endothelial cytosolic iron. the hypochloric anion radical (ho˙) is thought to be responsible most directly for endothelial cell cytotoxicity. no˙produced by constitutively expressed nitric oxide synthase in endothelial cells may afford protection from oxygen radical-induced endothelial cell damage. no is able to scavenge superoxide radicals and react with them to form peroxynitrite. variations in the ability to produce no may explain why vascular beds in different organs vary in their susceptibility to neutrophilmediated damage. excessive production of no by an inducible form of nitric oxide synthase (i nos), however, contributes to tissue damage, and increased peroxynitrite concentrations may be responsible in part for paf-induced increases in vascular permeability. in addition to oxygen-derived free radicals, activated neutrophils release matrix metalloproteinases, which contribute to tissue damage. vascular endothelial cells are further susceptible to direct effects of various cytokines, most prominently tnf-α and il- . these cytokines are thought to cause damage via the induction of cox activity and production of prostanoids and through generation of free radicals. neutrophil activation by lipopolysaccharide and cytokines results in stimulation of phagocytosis and the respiratory burst, release of lysosomal enzymes and inflammatory mediators, and expression of adhesion molecules. perhaps the single most specific clinicopathologic indicator of endotoxemia is pronounced neutropenia, which temporally correlates with peak plasma concentrations of tnf. neutropenia is caused primarily by margination of neutrophils in the vasculature, whereas significant loss through active migration into peripheral tissues likely is limited to the presence of a localized source of infection. margination is made possible by adhesion molecules on endothelial cells and leukocytes that interact and allow sticking of leukocytes to the endothelial lining of blood vessels. endotoxin or cytokines such as tnf and il- can initiate expression of adhesion molecules. subsequent transmigration of cells into tissue spaces is aided by the production of leukocyte collagenase, which allows enzymatic destruction of the vascular basement membrane. margination and transmigration of neutrophils occurs in three phases. , in the first phase of tethering and rolling, endothelial cells are stimulated to express p-selectin and e-selectin, which bind to p-selectin glycoprotein ligand- and sialylated lewis-x-like structures on leukocytes, respectively. whereas p-selectin is stored preformed in weibel-palade bodies of endothelial cells, e-selectin is expressed only following stimulation by cytokines. additionally, constitutively expressed l-selectin on neutrophils can bind to endothelial glycoproteins and glycolipids. during the second phase, firm adhesion is mediated by binding of neutrophil integrins (lfa- and mac- , also known as cd a/cd and cd b/cd ) to intercellular adhesion molecule (an immunoglobulin structure) on endothelial cells. although integrins are expressed constitutively on the leukocyte surface, activation signals are necessary to induce a high-affinity state and interaction with the endothelial surface. transmigration finally requires the expression of yet another adhesion molecule, namely platelet/endothelial cell adhesion molecule , which is located at the intercellular junction of endothelial cells. chemotactic factors including activated complement factor c a and the cxc chemokines control transmigration. the latter group includes il- , which is expressed by endothelial cells in response to activation. rebound neutrophilia, which is observed frequently following episodes of endotoxemia, is caused by neutrophil release from the bone marrow reserve pool and by stimulation of myeloid cell proliferation via granulocyte-macrophage colony-stimulating factor and is mediated primarily by tnf and il- . in health, coagulation and fibrinolysis underlie stringent control mechanisms that allow appropriate clot formation and their resolution. coagulopathies frequently are observed in horses with colic , , and foals with sepsis and are likely attributable to endotoxemia. disseminated intravascular coagulation (dic) results from a widespread activation of the coagulation and fibrinolytic systems and failure of their control mechanisms. ultimately, this leads to disseminated fibrin deposition in the microvasculature, consumption of platelets and clotting factors, and accumulation of fibrin degradation products (fdps). depending on the underlying disease process and the impairment of the systems, dic can manifest as a diffuse thrombotic syndrome leading to ischemic organ failure, a fibrinolytic syndrome with uncontrolled hemorrhage, or a combination of both. a procoagulant state in which one can detect clinicopathologic abnormalities precedes dic. activation of the coagulation cascade culminates in the cleavage of fibrinogen to fibrin by the serine protease thrombin. thrombin deposition on endothelial cell surfaces leads to platelet adherence and their activation by surface-bound paf. the intrinsic and extrinsic arms of the coagulation cascade are activated in endotoxemia. the intrinsic pathway is initiated by activation of coagulation factor xii (hageman factor), prekallikrein, and highmolecular-weight kininogen, which compose the contact system. although direct activation of coagulation factor xii by endotoxin has been demonstrated, the extrinsic pathway likely is more important for the development of coagulopathy in endotoxemia and sepsis. activation of the extrinsic pathway depends on the interaction of coagulation factor vii with tissue factor, which is the only coagulation factor not constitutively present in blood. tissue factor is present in subendothelial tissues and is exposed on vascular injury but also is expressed on endothelial cells and mononuclear phagocytes in response to lipopolysaccharide. , increased expression of monocyte tissue factor (also described as increased procoagulant activity) was found to be associated significantly with coagulopathy and poor prognosis in horses with colic. furthermore, lipopolysaccharide-induced tissue factor expression by equine peritoneal macrophages may be associated with the development of intraabdominal adhesions. regulatory mechanisms of the coagulation cascade include tissue factor pathway inhibitor, antithrombin iii (at iii), and the protein c system. protein c acts as an anticoagulant by inactivating clotting factors v and viii and promotes fibrinolysis by inactivating plasminogen activator inhibitor (pai). protein c activation by thrombin-thrombomodulin complexes is important for the anticoagulative properties of normal endothelium, and downregulation of endothelial thrombomodulin expression by tnf and il- and decreased expression of at iii and tissue factor pathway inhibitor by damaged endothelial cells contribute to the procoagulant state in endotoxemia and sepsis. [ ] [ ] [ ] in addition, activation of vascular endothelial cells leads to a loss of prostacyclin and no production and an increased release of thromboxane a (txa ). as a result, platelets are stimulated to aggregate and release txa and paf, thereby further promoting clot formation. the crucial step in the fibrinolytic cascade is the conversion of plasminogen to plasmin, a fibrin-degrading enzyme. tissue-type (tpa) and urokinase-type (upa) plasminogen activator are the major initiators of fibrinolysis, whereas pai and α -antiplasmin are the main regulatory components. , tnf and il- have been shown to induce the release of upa and tpa and the synthesis of pai. activation of fibrinolysis leads to consumption of α -antiplasmin and accumulation of fdps, which if present in high concentrations can interfere with platelet aggregation, fibrin polymerization, and thrombin formation and can promote bleeding. additionally, fdps mediate an increase in vascular permeability. lipopolysaccharide infusion in rabbits and human beings resulted in an early increase in plasma tpa activity, followed by a later profound rise in pai activity and fall in tpa activity. increased plasma pai concentrations also were found in horses with colic compared with controls. , thus although fibrinolysis may compensate initially for accelerated coagulation, its subsequent inhibition contributes to clot formation. activation of the complement system in endotoxemia occurs via the alternative pathway through interaction with lipopolysaccharide. increased concentrations of plasmin and kallikrein (caused by activation of the fibrinolytic and contact system) further promote this pathway by directly activating complement factors c a and c a. aside from being key molecules in the complement cascade, c a and c a are anaphylatoxins and cause an increase in vascular permeability via mast cell degranulation. c a further activates the lipoxygenase pathway in neutrophils and monocytes, acts as a chemotaxin for leukocytes and monocytes, and promotes neutrophil adhesion to endothelial cells. in response to acute inflammation, synthesis and secretion of a number of proteins called the acute phase proteins increases in hepatocytes, whereas synthesis of albumin decreases. the primary function of this acute phase response may be to suppress and contain inflammatory responses. il- and il- are the most important cytokines that induce the acute phase response, which typically begins within a few hours of the insult and subsides within to hours, unless the initiating cause persists. in horses, fibrinogen (the most commonly evaluated), haptoglobin, transferrin, ferritin, ceruloplasmin, coagulation factor viii:c, serum amyloid a protein, c-reactive protein, α -acid glycoprotein, and phospholipase a are considered part of the acute phase response. one must consider the effect of acute inflammation on the serum concentration of several coagulation factors when evaluating coagulation profiles. serum fibrinogen concentration is determined primarily by the acute phase response, although fibrinogen is consumed increasingly on activation of the clotting cascade. shock is characterized by a loss of homeostasis attributable to breakdown of hemodynamic control mechanisms, decreases in cardiac output and the effective circulating volume, and inadequate perfusion of vital organs. shock caused by endotoxemia is classified as distributive shock and is largely initiated by vascular dysfunction in the periphery. peripheral vascular beds are of major importance for the regulation of local tissue perfusion and affect systemic blood pressure by regulating total peripheral resistance. normally, vascular smooth muscle tone is regulated by endothelin- (vasoconstriction), no, and prostacyclin (vasodilation) released from vascular endothelial cells. as mentioned before, detrimental effects of no are attributable to induction of i nos in macrophages and other cell types, rather than endothelialderived no. peripheral vasomotor effects of endotoxin manifest as vasodilation and vasoplegia and are mediated by pgi , no, and mediators such as bradykinin. widespread vasodilation leads to vascular blood pooling, decentralization of blood flow, decreased venous return, and in effect decreased effective circulating volume and cardiac output. compensatory responses in the form of an initial hyperdynamic phase include tachycardia, increased cardiac output and central venous pressure, pulmonary hypertension, peripheral vasoconstriction, and increased peripheral vascular resistance. , , the early vasoconstrictive phase corresponds to an increased serum concentration of txa , but additional vasoconstrictors such as arginine vasopressin, angiotensin ii, serotonin, endothelin, and norepinephrine likely are implicated in the pathogenesis of shock and organ failure. with progression of disease, the animal enters a stage of decompensated shock and progressive systemic hypotension, which correspond to increased plasma concentrations of prostacyclin, pge and bradykinin. , inadequate blood flow and oxygen delivery to tissues caused by hypotension is confounded by direct myocardial suppression via no, increased vascular permeability, intravascular microthrombosis, and impaired tissue oxygen extraction and results in progressive metabolic acidosis and inhibition of normal cellular metabolism. quantification of endotoxin in plasma samples is possible. the limulus amebocyte lysate assay is an activity assay based on the endotoxin-sensitive hemolymph coagulation cascade in the horseshoe crab limulus polyphemus. in limulus this reaction is thought to be a defense mechanism against gram-negative infection. although frequently used as a research tool, the assay is not convenient enough to become a routine clinical test. the clinician therefore must appreciate the primary disease processes associated with a high risk of endotoxemia and rely on clinical signs and clinicopathologic data to achieve a diagnosis. in some cases, endotoxemia may be the first indication of disease or may be the most overt of otherwise subtle clinical manifestations. with colitis or proximal enteritis, for example, one may detect signs of endotoxemia before the development of colic, diarrhea, or gastric reflux, which more specifically indicate the nature of the primary illness. diseases such as peritonitis or pleuritis, however, may show nonspecific clinical findings including fever, anorexia, and depression. findings such as neutropenia, which indicate endotoxemia, should urge the clinician to search for a septic process. in vivo experiments in horses clearly show that many of the clinical signs associated with acute gastrointestinal disease and sepsis are attributable to the activities of lipopolysaccharide and cytokines such as tnf. on administration of sublethal doses of lipopolysaccharide the clinical response can be divided into the early hyperdynamic and the later hypodynamic or shock phases. clinical signs during the first phase, which begins within to minutes after lipopolysaccharide administration, include anorexia, yawning, sweating, depression, evidence of abdominal discomfort, muscle fasciculation, and recumbency. heart and respiratory rates increase, and decreased borborygmi suggest ileus. hyperemia of the mucous membranes and an accelerated capillary refill time indicate the hyperdynamic state. if one administers large amounts of lipopolysaccharide or if exposure is ongoing, depression worsens progressively, anorexia persists, and feces develop diarrheic character. signs of colic typically abate after the initial stage. fever develops as a result of direct action of tnf on the thermoregulatory center and il- -induced local production of pge in or near the hypothalamus. , because of compromised peripheral perfusion, mucous membrane color changes to brick red or purple, a dark "toxic" line appears, and capillary refill time is prolonged. inadequate peripheral perfusion and compromised organ function finally characterize the hypodynamic shock phase. body temperature may become subnormal and the skin, especially on extremities, is cool to the touch. the arterial pulse weakens and venous fill is decreased. vascular endothelial damage and increased capillary permeability result in a muddy mucous membrane color and diffuse scleral reddening. hemostatic abnormalities can manifest in the form of thrombosis, such as of the jugular vein, or increased bleeding tendency with mucosal petechiation or ecchymoses and prolonged bleeding from venipuncture sites. bleeding also may occur in spontaneous epistaxis or prolonged hemorrhage after nasogastric intubation. additional clinical signs typically reflect the development of organ failure. renal failure and laminitis appear to be common complications of endotoxemia in horses. renal failure results from ischemic cortical necrosis and acute tubular necrosis caused by coagulopathy-induced afferent arteriolar obstruction. clinical signs may include oliguria, anuria, or hematuria caused by renal infarction. laminitis may lead to lameness, increased digital arterial pulsation, increased warmth of the hoof wall, and sensitivity to hoof tester pressure. other signs of organ failure include icterus, anorexia and depression caused by liver failure, tachypnea and dyspnea caused by pulmonary failure, colic and bleeding caused by ischemia-induced gastrointestinal ulceration and abnormal motility patterns, and persistent tachycardia or cardiac arrhythmia in cases of cardiac failure. in pregnant mares, fetal death and abortion can occur because of increased production of pgf α and decreased serum progesterone concentrations. , alterations in the hemogram and serum biochemical profile are nonspecific and mainly reflect the underlying disease process and the occurrence of organ failure. leukopenia caused by neutropenia may be the most specific indicator of acute bacterial sepsis or endotoxemia. in prolonged cases, an increased proportion of immature neutrophil forms (bands) and toxic changes are observable. toxic changes resulting from neutrophil activation include vacuolation, cytoplasmic granulation, basophilic cytoplasm, and döhle's bodies. because neutropenia occurs early in the development of endotoxemia, it also may be a useful parameter for monitoring horses at risk. on recovery, neutropenia typically is followed by a pronounced rebound neutrophilia. an elevated hematocrit and total serum protein concentration are evidence of dehydration; however, splenic contraction caused by increased sympathetic stimulation and protein losses also influence these parameters. a normal or only slightly decreased serum protein and albumin concentration in the face of an elevated hematocrit and clinical signs of dehydration should alert the clinician to the possibility of protein loss. hypoproteinemia and hypoalbuminemia can occur because of loss via the gastrointestinal or urinary tract or with pleural or peritoneal cavity effusion. increased vascular permeability and edema formation contribute to hypoproteinemia. serum electrolyte abnormalities primarily depend on the nature and duration of underlying disease processes and need to be evaluated individually. common sources of electrolyte loss are gastrointestinal secretions, urine, and sweat; however, severe effusion into body cavities may contribute. in anorexic patients, lack of dietary intake is a confounding factor that warrants consideration. in human patients, gram-negative sepsis frequently is associated with hypocalcemia, more specifically a decrease in serum ionized calcium concentration. endotoxin is thought to be a causative factor, and proposed mechanisms include acquired parathyroid gland insufficiency, dietary vitamin d deficiency, impaired calcium mobilization, and renal α-hydroxylase insufficiency leading to decreased , -hydroxylation of vitamin d. hypocalcemia in septic human patients was found to be associated with hypotension and poor outcome. in horses with surgically managed gastrointestinal disease, decreased serum ionized calcium concentration was a common finding and was most severe in patients with strangulating or nonstrangulating infarctions. in some horses, ionized calcium concentration decreased further throughout surgery. treatment with calcium gluconate resulted in normalization of serum ionized calcium concentrations in all cases. septic neonatal patients are frequently hypoglycemic. aside from decreased oral intake and generally increased metabolism, glucose use by the infecting bacteria, inhibition of gluconeogenesis by endotoxin, and insulinlike activity produced by macrophages are responsible for hypoglycemia. interestingly, experimental endotoxin administration results in transient hyperglycemia in adult horses, whereas profound hypoglycemia occurs in foals. one should evaluate coagulation parameters if coagulopathy is suspected. the most significant changes can be expected with severe inflammatory disease such as colitis, , devitalized intestine as with strangulating obstruction, , and with increased duration of disease. in horses with acute gastrointestinal disease, coagulation profiles were considered normal in only horses. although coagulation times may be shortened during the procoagulant state, commonly observed abnormalities with developing dic include an increased concentration of fdps and soluble fibrin monomer, prolonged prothrombin time indicative of factor vii consumption, prolonged activated partial thromboplastin time indicative of factor viii:c and ix consumption, prolonged thrombin time, decreased at iii activity, thrombocytopenia, and decreased protein c and plasminogen activities. fibrinogen concentration frequently increases and reflects the acute phase response rather than coagulation abnormalities. some clinicians make a diagnosis of dic if three or more coagulation parameters (specifically at iii, fdps, platelet count, prothrombin time, and activated partial thromboplastin time) are abnormal, whereas others require overt clinical signs of hemorrhage and concomitant thrombosis in addition to classic laboratory findings. the prognostic value of coagulation parameters has been evaluated. , , overall, persistence or worsening of abnormalities in the face of treatment appears to be more indicative of poor outcome than alterations in any specific parameter. in one study, decreased serum at iii concentration was the parameter most commonly associated with fatal outcome in mature horses with colic. one should further evaluate the serum biochemical profile regarding compromise or failure of specific organ systems. increases in serum creatinine and urea nitrogen concentration can have prerenal, renal, or postrenal causes. in cases of endotoxemia and sepsis, prerenal azotemia caused by dehydration and decreased renal blood flow and renal azotemia caused by organ failure are most likely to occur. one can use urine specific gravity and the response to fluid therapy to differentiate renal from prerenal causes of azotemia. although ideally one should perform urinalysis before initiating fluid therapy, one should never delay treatment to obtain a sample and instead should use the first available urine sample. with prerenal azotemia, urine specific gravity is increased, urinalysis is normal in other respects, and azotemia resolves with adequate fluid therapy. azotemia in the face of normal or decreased urine specific gravity, however, indicates compromised renal function. depending on the extent of renal damage, proteinuria and hematuria also may be present. bacteriuria and an elevated urine leukocyte count may occur if urinary tract infection is the underlying cause for the development of endotoxemia. in these cases, urine culture and sensitivity testing are indicated to aid appropriate antimicrobial therapy. increased serum activity of liver enzymes (aspartate aminotransferase, γ-glutamyltransferase, sorbitol dehydrogenase, alkaline phosphatase) are common in endotoxemic patients; however, liver failure caused by endotoxemia is rare. sorbitol dehydrogenase is the most liver-specific of the enzymes and a sensitive indicator of acute hepatocellular necrosis; however, sorbitol is unstable and routine assays may not be available. one should evaluate liver enzymes and function tests (serum indirect and direct bilirubin concentration, serum bile acids and blood ammonia) in cases of prolonged and profound depression to rule out hepatoencephalopathy. one should evaluate arterial blood gases in patients with primary respiratory disease or with clinical evidence of respiratory failure and in profoundly depressed, recumbent patients, especially neonates. hypoxemia observed in response to endotoxin infusion is thought to be caused by an increase in ventilation-perfusion mismatch rather than pulmonary edema as occurs in human patients with acute respiratory distress syndrome. the lung is not a major shock organ in horses; however, pulmonary edema may occur in patients with associated sepsis or complications such as dic. the ideal treatment for endotoxemia is prevention. if one possibly can recognize and closely monitor patients at risk, one can provide treatment proactively and may reverse the effects of endotoxin before the inflammatory response has developed a dynamic of its own. unfortunately, endotoxemia can develop rapidly, and horses are exquisitely sensitive to the effects of endotoxin; therefore, many equine patients are not evaluated until reaching more severe stages of endotoxemia or shock. prognosis and patient outcome then frequently depend on the severity of complications associated with endotoxemia. treatment of endotoxemia involves multiple aspects, and the following strategies have been proposed : • inhibition of endotoxin release into the circulation • scavenging of lipopolysaccharide molecules to prevent direct effects and interaction with inflammatory cells • inhibition of cellular activation by lipopolysaccharide • inhibition of mediator synthesis • interference with the effects of inflammatory mediators • general supportive care in addition, complications such as renal failure, one also must address liver failure, cardiac failure, laminitis, and abortion in pregnant mares. when evaluating reports concerning the efficacy of any one treatment, one should keep in mind differences in underlying disease processes and the complexity of the inflammatory cascade. a "one for all" treatment most likely will not be found, and similarly, any one treatment can only address one or few pathophysiologic aspects of endotoxemia. understanding the rationale for different treatment strategies is important to be able to tailor treatment to the needs of the patient. inhibition of endotoxin release requires identification and removal of its source. therefore whenever endotoxemia is evident, the clinician should strive to reach a diagnosis of the underlying disease and ascertain whether ischemic or inflamed bowel or a gram-negative septic process is present. aside from history, physical examination, and routine laboratory tests, evaluation may include exploratory laparotomy in colic patients, roentgenologic and ultrasonographic evaluation of the pleural and peritoneal cavity and organs, ultrasonographic evaluation of umbilical remnants in neonatal foals, evaluation of passive transfer and calculation of a sepsis score in foals, and repeated culturing of blood or other specimens. if one suspects an infectious process, one should pursue identification of the responsible microorganisms and their antimicrobial sensitivity spectrum; however, one should not delay treatment to obtain culture results. specimen containers with removal devices for antimicrobials are available and are useful in cases for which one has initiated treatment before specimen collection. once one reaches a diagnosis, one must take appropriate measures to correct the primary disease process. examples are removal of devitalized sections of bowel or infected umbilical remnants, drainage of infected pleural or peritoneal fluid, and gastric lavage followed by administration of mineral oil and/or activated charcoal in cases of grain overload to prevent further absorption of endotoxin. one must address any septic process with appropriate antimicrobial therapy. initially, broad-spectrum coverage of the most likely organisms is recommended; one then should specify therapy according to results of culture and sensitivity testing. sepsis in foals is caused predominantly by gram-negative organisms, of which e. coli, actinobacillus spp., klebsiella spp., salmonella spp. and pasteurella spp. frequently are identified. the reader is referred to other texts for review of general principles of antimicrobial therapy. regarding endotoxemia specifically, antimicrobial therapy has been suggested to increase the amount of circulating endotoxin by inducing endotoxin release on cell death of gram-negative bacteria. a recent in vitro study compared endotoxin release and inflammatory mediator activity between antimicrobials commonly used to treat e. coli septicemia in foals and specifically evaluated amikacin, ampicillin, amikacin plus ampicillin, ceftiofur, and imipenem. although these antimicrobials showed no difference in the ability to kill bacteria, amikacin and the amikacin/ampicillin combination resulted in the lowest and ceftiofur in the greatest release of endotoxin. endotoxin release appeared to be dose-dependent in that lesser amounts were released at higher antimicrobial concentrations. based on these results and clinical experience, combining antimicrobial therapy with endotoxin-binding agents such as polymyxin b may be beneficial, especially when using β-lactam antimicrobials. antimicrobials frequently are given perioperatively to colic patients to lower the risk of peritonitis, incisional infection, and generalized sepsis and endotoxemia. because antimicrobial therapy has been implicated in the development of colitis, the duration of treatment should be minimal. unless evidence of sepsis, such as fever or changes in the leukogram, is present, perioperative administration of antimicrobials should not exceed a to hours. conversely, antimicrobial therapy frequently is used in cases of infectious colitis to treat the inciting cause and to prevent sepsis from translocation of bacteria. endotoxin typically has a short plasma half-life and is removed rapidly by mononuclear phagocytes or neutralized by binding to serum proteins and lipoproteins. many conditions responsible for the development of endotoxemia in horses, however, are associated with an ongoing release of endotoxin. examples include severe gastrointestinal inflammation as in proximal enteritis or colitis, grain overload, or uncontrolled sepsis. therapy directed against endotoxin itself may be able to interrupt the continuous activation of the inflammatory cascade in these cases. further benefits of antiendotoxin treatment may be derived if large amounts of endotoxin have been released before the inciting cause can be addressed. an important consideration regarding the efficacy of immunotherapy is the region of the lipopolysaccharide molecule against which antibodies are raised. the o-chain of lipopolysaccharide acts as a potent antigen on infection with gram-negative bacteria ; however, antibodies directed against the o-chain are serotype specific and cannot afford significant cross-protection against heterologous bacterial strains. the core and lipid a region, both of which show a much higher degree of homology between lipopolysaccharide derived from different bacterial strains, offer a more promising target for immunotherapy. active immunization against endotoxin has been reported for horses. vaccination with a bacterin/ toxoid vaccine prepared from rough mutants of salmonella typhimurium or s. enteritidis protected horses against homologous and heterologous endotoxin challenge , and carbohydrate overload. despite these encouraging results and the current availability of a vaccine for use in horses (endovac-equi, immvac inc., columbus, missouri), active immunization against endotoxin does not appear to be a common practice. in comparison, passive immunization with antilipopolysaccharide antibodies is used widely. rough bacterial mutants, most commonly j of e. coli o :b and s. minnesota re , are used to immunize donor horses and subsequently prepare serum or plasma products. proposed mechanisms of action after binding of the antibodies to lipopolysaccharide include steric blockade of lipid a interaction with cellular receptors and enhanced bacterial clearance by opsonization. [ ] [ ] [ ] studies concerning the efficacy of antibody administration in equine patients vary in their results. beneficial effects have been described in experimental models of endotoxemia, acute gastrointestinal disease, and neonates with sepsis, , [ ] [ ] [ ] whereas in other studies, antibodies failed to protect foals and horses against endotoxin effects. - furthermore, administration of a s. typhimurium antiserum to foals was associated with an increased respiratory rate and higher serum activities of il- and tnf. various equine serum and plasma products are currently commercially available. an antiserum raised against the lipopolysaccharide-core of s. typhimurium (endoserum) is available for administration to endotoxemic horses at a recommended dose of . ml/kg body mass. diluting the serum ten-to twentyfold in crystalloid intravenous solutions, administering it slowly over to hours, and monitoring the patient for adverse reactions is advisable. although the product is marketed for use in foals with failure of passive transfer, adverse effects have been reported, and one should use caution when administering it to neonates. plasma from donors inoculated with j (e. coli) and s. typhimurium (re mutant) is available under a restricted license (polymune-j, vet dynamics, inc., san louis obispo, california). the manufacturer recommends administration of at least to l in cases of endotoxemia. in addition, hyperimmune plasma, which has a guaranteed minimum immunoglobulin g content but does not contain specific antiendotoxin antibodies (hi-gamm equi, lake immunogenics, inc., ontario, new york; polymune and polymune-plus, vet dynamics, inc.), is marketed for treatment of failure of passive transfer, and many clinicians use it to treat endotoxemia and sepsis. in addition to antibodies and protein, plasma contains active constituents such as complement components, fibronectin, clotting factors, and at iii and therefore may be particularly useful in patients with endotoxemia-induced coagulopathy. volumes of to ml/kg body mass of hyperimmune plasma have been recommended for use in endotoxemic patients. , polymyxin b polymyxin b is a cationic polypeptide antibiotic that binds to the anionic lipid a portion of lipopolysaccharide and neutralizes its endotoxin capacity. at dosages required for antimicrobial activity, polymyxin b carries the risk of respiratory paralysis and ototoxic, nephrotoxic, and neurotoxic side effects; however, a much lower dose is required for endotoxin-binding activity. the effects of polymyxin b in horses have been evaluated in different experimental models. , , in an in vivo study in foals, treatment with polymyxin b at a dosage of u/kg body mass before infusion with s. typhimurium lipopolysaccharide resulted in significantly less severe elevations of body temperature, respiratory rate, and serum activities of tnf and il- compared with untreated controls. similarly, polymyxin b treatment of adult horses given endotoxin significantly ameliorated clinical signs and decreased plasma tnf activity. in the latter study, benefits of treatment were also evident at lower dosages of polymyxin b ( and u/kg body mass) and administration of polymyxin b hour after the start of endotoxin infusion. conversely, polymyxin b failed to ameliorate clinical signs of endotoxemia or prevent the development of coagulopathy, acidosis, lameness, and shock in experimental carbohydrate overload. side effects suggestive of neurotoxicity appeared after repeated administration of mg/kg body mass ( , u/kg) and in milder form, . mg/kg body mass ( , u/kg) polymyxin b. nephrotoxicity was not observed. currently, use of polymyxin b in equine patients is recommended at dosages of to u/kg body mass every to hours. one should initiate treatment as early in the disease process as possible, because the beneficial effects of lipopolysaccharide scavenging are limited to the first to hours after the onset of endotoxemia, before tolerance develops. side effects in the form of neuromuscular blockade and apnea, which necessitate slow infusion of the drug in human patients, have not been observed in horses. one therefore can administer the entire dose as a slow bolus. if one uses polymyxin b in horses with hypovolemia, dehydration, or azotemia, one should attempt to improve peripheral tissue perfusion, minimize the polymyxin b dose, and closely monitor patients for nephrotoxicity. close monitoring is also important if one administers medications such as aminoglycoside antibiotics, which share a similar spectrum of potential side effects, concurrently. azotemic neonates have been reported to be more susceptible to the nephrotoxic effects of polymyxin b than adult horses. in an attempt to decrease the risk for adverse effects while preserving lipopolysaccharide-neutralizing ability, a conjugate of polymyxin b with dextran has been developed. in conjugated form, polymyxin b is prevented from extravasation into tissues, where it exerts toxic effects by interaction with cell membranes. in addition, conjugation increases the residence time of polymyxin b in the circulation and therefore should prolong the antiendotoxic effect. the polymyxin b-dextran combination was evaluated at a total dose of mg/kg body mass of polymyxin b in . g/kg body mass dextran given minutes before administration of endotoxin in horses. treatment was found to block the development of tachycardia, tachypnea, fever, and neutropenia completely and to prevent increases in serum concentrations of tnf, il- , txb (a txa metabolite), and the prostacyclin metabolite -keto-pgf α . although mild adverse effects in the form of tachypnea, sweating, and increased systolic blood pressure were observed, these were transient and could be prevented by pretreatment with ketoprofen. the polymyxin b-dextran combination is not commercially available at this time. natural endotoxin-binding proteins such as lbp, lipoproteins, and scd have been evaluated experimentally. results of these studies are controversial, and detrimental effects occurred in some cases. a protein receiving much attention regarding potential therapeutic efficacy is the bactericidal permeability-increasing protein (bpi). this protein is structurally similar to lbp but is expressed exclusively in myeloid precursors of polymorphonuclear leukocytes. bpi is stored in primary granules of mature neutrophils and during inflammation is expressed on their cell membranes and secreted into the extracellular environment. bpi has an even higher affinity for lipopolysaccharide than lbp and shows antibacterial activity specific for gram-negative bacteria. binding of bpi to the gram-negative bacterial membrane results in growth arrest and is an important factor in the antibacterial activity of intact neutrophils. furthermore, bpi binding disrupts normal membrane organization and makes bacteria more susceptible to hydrophobic substances, including antimicrobials. experimentally, recombinant bpi has been shown to protect against the toxic and lethal effects of isolated lipopolysaccharide and intact gram-negative bacteria, and clinical trials in human patients show promising results concerning its therapeutic use. the biology and potential use of bpi in horses has not been evaluated. treatments aimed at inhibiting lipopolysaccharide interaction with cells or turning off intracellular signaling pathways are under investigation. nontoxic lipopolysaccharide or lipid a structures can act as endotoxin antagonists, if they competitively inhibit binding to lbp or cellular receptors or inhibit cellular activation by other mechanisms. of the potential antagonists that have been evaluated experimentally, lipopolysaccharide and lipid a from the phototrophic bacterium rhodobacter sphaeroides, and a synthetic compound (e ) the structure of which is based on r. sphaeroides lipopolysaccharide, have been most promising. [ ] [ ] [ ] [ ] [ ] unfortunately, species differences exist regarding cellular response to these structures, and r. sphaeroides lipopolysaccharide acts as a potent inducer of cytokine expression in equine cells. based on results of receptor transfection studies in other species, , tlr is likely responsible for this phenotypic variation. additional compounds including lipopolysaccharide derived from nitrogen-fixing plant bacteria of the species rhizobium are being evaluated to reveal further insight into structural requirements for endotoxin antagonists in horses. nonsteroidal antiinflammatory drugs (nsaids) are probably the most commonly used drugs to treat endotoxemia. the rationale for their use is inhibition of prostaglandin endoperoxide h synthase, that is, cox, and thereby inhibition of prostanoid production (see figure . - ). additional beneficial effects may include scavenging of oxygen-derived free radicals and iron chelation; however, side effects may occur at dosages required to achieve these effects. prostanoids have been identified as important mediators in the inflammatory response in a number of studies, and inhibition of their synthesis is associated with beneficial effects. two cox isoforms are recognized: constitutively expressed cox- and inducible cox- . upregulation of cox- expression results from various proinflammatory stimuli, including lipopolysaccharide, tnf-α, and il- . constitutively expressed cox products are likely important for maintenance of homeostasis, whereas increased production of prostanoids by cox- is thought to be responsible for detrimental effects during inflammation and shock. research has focused on the development of selective cox- inhibitors; however, none of these products are currently available for use in horses. in horses the most commonly used nsaid to treat endotoxemia is flunixin meglumine. beneficial effects of flunixin meglumine have been described in experimental models of endotoxemia [ ] [ ] [ ] and in clinical cases. in equine colic patients, treatment with flunixin meglumine before exploratory surgery resulted in reduced plasma concentrations of txb and pge and had a favorable effect on cardiovascular parameters. flunixin meglumine was shown further to maintain cardiac output and systemic arterial blood pressure, improve blood flow to vital organs, reduce pulmonary endothelial damage, and improve survival on endotoxin challenge. [ ] [ ] [ ] [ ] nsaid use in horses carries the risk of side effects, most importantly the development of gastrointestinal ulceration and renal papillary necrosis (renal crest necrosis). in a study comparing the side effects of flunixin meglumine ( . mg/kg body mass), phenylbutazone ( . mg/kg body mass), and ketoprofen ( . mg/kg body mass) given times daily for days, lesions of the gastric glandular mucosa occurred most commonly. phenylbutazone resulted in the most severe side effects, which included small intestinal edema, erosions, and ulcers in the large colon and decreased serum albumin concentration. renal crest necrosis occurred more frequently in horses treated with phenylbutazone but also occurred with flunixin meglumine treatment. despite the higher risk of side effects, use of phenylbutazone has been suggested for certain cases. in colic patients, phenylbutazone may provide analgesia and ameliorate endotoxin-induced ileus without masking cardiovascular effects of endotoxin, which are used to determine the necessity of surgical exploration. for similar reasons and to minimize side effects a reduced dose of flunixin meglumine ( . mg/kg body mass) has been suggested and is used widely in horses. at this dosage, flunixin meglumine was shown to inhibit eicosanoid synthesis efficiently in an in vivo model of endotoxemia. reduction of clinical signs, however, was dose dependent, and therefore one should choose the appropriate dose based on the circumstances of each case. ketoprofen has been suggested to have superior effects because of a proposed dual inhibitory effect on cox and lipoxygenase and may carry a decreased risk of side effects compared with flunixin meglumine and phenylbutazone. a comparison of cytokine and eicosanoid production by lipopolysaccharide-stimulated isolated monocytes in vitro, however, showed no significant difference between horses pretreated with flunixin meglumine ( . mg/kg body mass) or ketoprofen ( . mg/kg body mass), respectively. eltenac has been evaluated in an experimental endotoxemia model in horses. given minutes before lipopolysaccharide infusion, eltenac at a dose of . mg/kg protected against changes in clinical, hemodynamic, and hematologic parameters and blunted the lipopolysaccharide-induced rise in plasma cytokine concentrations in comparison with controls. some parameters, however, including heart rate, leukocyte count, lactate concentration, and plasma tnf activity, were not improved. ibuprofen may have beneficial effects superior to the other nsaids, because it may be possible to achieve tissue concentrations safely that allow iron chelation to occur. according to a study in healthy foals, dosages of ibuprofen up to mg/kg every hours can be given safely for up to days. the use of corticosteroids for antiinflammatory therapy in sepsis and endotoxemia has been controversial in human and equine patients, and beneficial effects superior to the ones achieved by nsaids have not been demonstrated consistently overall. corticosteroids inhibit the activity of phospholipase a and the release of arachidonic acid from cell membrane phospholipids, as well as the production of tnf, il- , and il- in response to a lipopolysaccharide stimulus. experimentally, beneficial effects of dexamethasone in equine endotoxemia have been demonstrated. , to inhibit tnf production by equine peritoneal macrophages, however, the required concentration of dexamethasone was high and corresponded to an in vivo dosage (approximately mg/kg body mass) greatly exceeding current recommendations. although single doses of corticosteroids are unlikely to carry a disproportionate risk of side effects, one should consider the suggested association of laminitis with corticosteroid use in horses. in cases of sepsis, further immunosuppressive effects could be detrimental. in human patients with certain types of septic shock, dysfunction of the hypothalamic-pituitary-adrenal axis has been recognized and successfully treated with hydrocortisone replacement therapy. use of corticosteroids for this indication has not been evaluated in horses. pentoxifylline, a methylxanthine derivative and phosphodiesterase inhibitor, has been suggested for use in endotoxemia because of its effects on neutrophil function and its ability to inhibit the production of various cytokines, interferons, and thromboplastin. decreased production of tnf, il- , txb , and thromboplastin in response to endotoxin was shown in an equine ex vivo model. in horses given endotoxin followed by treatment with pentoxifylline ( . mg/kg body mass followed by continuous infusion of mg/kg/hr for hours), however, only minimal beneficial effects were observed. treatment significantly improved body temperature, respiratory rate, and whole blood recalcification time, but no effect was observed regarding heart rate, blood pressure, leukocyte count, plasma fibrinogen concentration, and serum cytokine concentrations. the conclusion was that benefits of treatment with pentoxifylline might be restricted to administration of high bolus doses or continuous infusion early in the pathophysiologic process. in an in vivo endotoxemia model in horses, combination of pentoxifylline ( mg/kg body mass) and flunixin meglumine ( . mg/kg body mass) was found to have greater benefit than each treatment on its own. the currently recommended dosage for oral administration of pentoxifylline is mg/kg every hours. because of its rheologic properties, that is, the ability to increase erythrocyte deformability and microvascular blood flow, pentoxifylline may be particularly useful in endotoxemic patients showing evidence of laminitis. an intravenous preparation of pentoxifylline is not commercially available. dimethyl sulfoxide (dmso) is used frequently in an attempt to scavenge oxygen-derived radicals. the treatment may be most appropriate in cases of ischemia-induced intestinal damage and associated reperfusion injury. however, dmso failed to show beneficial effects in an experimental model of intestinal ischemia when administered on reperfusion of the ischemic intestine. dmso at the commonly used dose of g/kg body mass was shown to increase mucosal loss after ischemia and reperfusion of the large colon, and hence a reduced dose of . g/kg body mass has been proposed for cases of intestinal ischemia. for intravenous administration, dmso needs to be diluted in polyionic solutions to a concentration not exceeding %. oral administration of a % to % solution via nasogastric intubation is also possible. aside from dmso the xanthine oxidase inhibitor allopurinol has been suggested as a treatment to prevent oxygen radical-induced tissue damage. during periods of ischemia, tissue xanthine dehydrogenase is converted to xanthine oxidase, which on reperfusion catalyzes the generation of superoxide radicals. , evaluation in horses showed beneficial effects of mg allopurinol per kilogram body mass administered hours before endotoxin challenge. in another study, mucosal damage attributable to oxygen-derived free radicals was not attenuated by allopurinol in an experimental ischemia-reperfusion model. lidocaine given intravenously has been suggested as an antiinflammatory, analgesic, and prokinetic agent, and some clinicians use it to treat colic and laminitis in horses. in an experimental endotoxemia model in rabbits, lidocaine was found to inhibit hemodynamic and cytokine responses to endotoxin profoundly if given immediately following lipopolysaccharide infusion. use of lidocaine therefore may have additional merit in endotoxemic patients. a common regimen for lidocaine use in horses is administration of an initial bolus ( . mg/kg body mass) section . endotoxemia followed by continuous infusion at a rate of . mg/ kg/min. one should monitor patients for toxic neurologic effects associated with a lidocaine overdose. high concentrations of ω- fatty acids can alter the phospholipid composition of cellular membranes toward a decreased ratio of ω- to ω- and thereby can affect membrane functions such as phagocytosis, receptor binding, and activities of membrane-bound enzymes. most importantly for the treatment of endotoxemia, ω- fatty acid incorporation into cell membranes decreases the availability of arachidonic acid (an ω- fatty acid) for eicosanoid synthesis and provides alternative substrates. metabolism of ω- fatty acids via the cox and lipoxygenase pathway leads to the production of -series prostaglandins and -series leukotrienes, which have less biologic activity than their -series and -series counterparts derived from arachidonic acid. aside from these mechanisms, ω- fatty acids prevent lipopolysaccharideinduced upregulation of cd in monocytic cells and therefore may be able to block transmembrane signaling of lipopolysaccharide. cells from horses given linseed oil (high in ω- fatty acids) for weeks before blood collection showed significantly decreased expression of procoagulant activity, txb , and tnf in response to lipopolysaccharide stimulation. , in an in vivo experimental model of endotoxemia in horses, treatment resulted in prolonged activated partial thromboplastin time and whole blood recalcification time, suggesting an anticoagulant effect; however, a significant beneficial effect on clinical response and serum eicosanoid concentrations was not observed. because dietary addition of ω- fatty acids requires several weeks of treatment, intravenous infusion was evaluated and shown to alter the composition of cell membrane phospholipids rapidly. further evaluation of this treatment for use in horses is necessary before dosage recommendations can be made. monoclonal and polyclonal antibodies against equine tnf have been evaluated. [ ] [ ] [ ] administration of a monoclonal antibody preparation before lipopolysaccharide infusion resulted in significantly reduced plasma tnf-activity, improved clinical abnormality scores, lower heart rate, and higher leukocyte count compared with controls. furthermore, plasma concentrations of lactate and -keto-pgf α were reduced significantly, whereas txa production was not affected. in another study, administration of a rabbit polyclonal antibody against recombinant human tnf was unable to improve clinical and hematologic parameters when given shortly ( minutes) after lipopolysaccharide infusion, although inhibition of tnf activity was present in vitro. , findings in horses are in agreement with studies in other species and suggest that beneficial effects of tnf inhibition may be limited to administration before lipopolysaccharide exposure. widespread clinical use therefore is unlikely to become feasible. clinical trials in human patients have not shown significant benefits of tnf antibody treatment. , the effects of selective paf receptor antagonists have been evaluated. paf is implicated in the development of systemic hypotension, lipopolysaccharide-induced platelet aggregation, ileus, and increased vascular permeability and may mediate recruitment of leukocytes to inflamed tissues. , a study in horses using the paf receptor antagonist sri - before lipopolysaccharide infusion showed significant decreases in heart rate and shorter elevation of lactate concentrations in response to the treatment. although not statistically significant, additional beneficial effects included delayed onset of fever, a shortened period of neutropenia, and reduced maximal platelet aggregation. whenever possible, the clinician should correct volume and electrolyte deficits, or at least improve them, before anesthetizing a patient for a surgical procedure. for initial resuscitation, polyionic solutions such as lactated ringer's solution given at rates of to ml/kg/hr are appropriate. patients with severe hypovolemia and shock may require higher fluid volumes. a viable alternative to large-volume resuscitation with isotonic fluids is the use of small volumes of hypertonic solutions, which transiently raise plasma osmolality, thereby causing a fluid shift from the interstitial space into the vasculature and rapidly restoring circulating volume. hypertonic saline solution ( . % sodium chloride) is the most commonly used hypertonic solution and has been shown to have beneficial effects in endotoxemic horses. a dose of ml/kg is recommended, which one should give as a bolus infusion over to minutes, followed by administration of an isotonic solution to restore total body fluid volume. one should use hypertonic saline with caution in patients with sodium and/or chloride derangements and should monitor serum electrolyte concentrations in the case of repeated administration. improvement of the cardiovascular status in response to fluid therapy is indicated by normalization of heart rate, mucous membrane color, and capillary refill time. failure of urination to occur despite appropriate fluid resuscitation should result in critical evaluation of renal function. once one has stabilized the patient, one should choose a maintenance fluid rate to maintain adequate hydration and plasma volume. for adult horses, the maintenance fluid rate is approximately ml/kg/hr, whereas neonatal foals that are not nursing may require larger volumes ( ml/kg/hr). one should monitor fluid administration carefully in endotoxemic patients, because lowered plasma oncotic pressure caused by hypoproteinemia along with an increased vascular permeability increase the risk of tissue edema formation. furthermore, a rapid increase in total body fluid volume may be detrimental in patients with compromised cardiac and peripheral vasomotor function and may increase the severity of vascular pooling in peripheral organs. in these patients, hypertonic saline or colloids may be more appropriate means of stabilization than large volumes of crystalloid solutions. plasma is an ideal colloid and should be administered to maintain a serum total protein concentration above . g/dl. to raise plasma protein concentration and colloid osmotic pressure significantly, however, horses often require large volumes of plasma ( to l or more in a -kg horse), and one should consider alternative colloids. furthermore, high-molecular-weight polymers are thought to provide superior oncotic effects in cases of sepsis and endotoxemia, when vascular permeability is increased. hetastarch, or hydroxyethyl starch, (hespan) is commercially available as a % solution in . % sodium chloride. hetastarch molecules have very high molecular weight, and degradation must occur before renal excretion. these properties result in a longer plasma half-life and prolonged oncotic effects compared with other colloids; persistence of the oncotic effect for hours was found in hypoproteinemic horses. a dosage of to ml/kg given by slow intravenous infusion along with an equal or greater volume of crystalloid fluids is recommended. , in human patients, prolonged activated partial thromboplastin time, decreased factor viii activity, and decreased serum fibrinogen concentration have been described in association with hetastarch use. in the limited number of equine studies, bleeding times were not affected , ; however, one should monitor patients treated with hetastarch for coagulopathy. one should base correction of serum electrolyte concentrations on the results of laboratory evaluation. ideally, one should evaluate serum electrolyte concentrations of patients receiving fluid therapy daily. one should take ongoing losses and lack of dietary intake into account, especially when serum concentrations, as in the case of potassium, poorly reflect total body electrolyte stores. potassium supplementation is recommended in patients experiencing prolonged (greater than hours) periods of anorexia. one can add calcium in the form of calcium gluconate, which is available as a % solution. based on a study in healthy horses, rates of administration for calcium gluconate in the range of . to . mg/kg/min are recommended, and as a guideline, one should administer . to ml/kg body mass per day of a % solution. one can add potassium in the form of potassium chloride or potassium gluconate to intravenous solutions at a dose of to meq/l given at a maintenance rate. administration of potassium should not exceed a rate of . to meq/kg/hr. metabolic acidosis in endotoxic shock is attributable to lactic acidemia and inadequate tissue perfusion. acid-base balance often improves considerably after fluid resuscitation (preferably with alkalinizing solutions such as lactated ringer's solution) alone; however, additional sodium bicarbonate may be required in cases in which serum bicarbonate concentration remains below meq/l. for adult horses, the bicarbonate deficit (in meq hco ) is calculated as . × body mass (kg) × base deficit, whereas for foals one should use a factor of . . as a general rule, one should administer half the required amount as a bolus followed by the remaining half over to hours. because endotoxemia is a dynamic process and losses are ongoing, one should reevaluate acid-base status at least once daily. foals with sepsis are frequently hypoglycemic, and % dextrose solutions are useful as initial resuscitation fluids. one should reduce the glucose concentration of intravenous solutions according to the blood glucose concentration to avoid prolonged hyperglycemia. administration of hyperimmune plasma ( to ml/kg body mass) is highly recommended in foals with evidence of partial or complete failure of passive transfer. one should consider positive inotropic and vasomotor agents in patients with persistently inadequate tissue perfusion. lower dosages of dopamine ( . to µg/kg/min) result in vasodilation of the renal, mesenteric, coronary, and intracerebral vasculature via dopaminergic effects, whereas higher dosages (up to µg/kg/min) also exert stimulation of β -adrenergic receptors, resulting in increased myocardial contractility and heart rate. dobutamine is a direct β -adrenergic agonist and does not appear to have significant vasodilator properties. dosages for dobutamine of to µg/kg/min as continuous intravenous infusion have been recommended for use in horses. in addition, norepinephrine was evaluated in hypotensive critically ill foals that were refractory to the effects of dopamine and dobutamine. at dosages up to . µg/kg/min administered concurrently with dobutamine, six out of seven foals showed an increase in mean arterial pressure, and all foals had increased urine output. because of the risk of cardiac side effects, close monitoring of heart rate and rhythm should accompany infusion of inotropes. indirect blood pressure measurements using a tail cuff may be used to monitor the effects of treatment. more frequently than overt thrombosis or bleeding attributable to dic, hemostatic abnormalities occur in the form of alterations in the coagulation profile. a procoagulant state with shortened bleeding times or prolonged bleeding times caused by consumption of clotting factors may be evident. one should address abnormalities in the coagulation profile as early as possible but especially if they persist more than hours after initiation of therapy. because of the complex interactions of coagulation and fibrinolysis during endotoxemia, one should combine anticoagulant therapy with the administration of fresh frozen plasma to replace clotting and fibrinolytic factors. heparin acts as an anticoagulant by activation of at iii and subsequent inhibition of thrombin, release of tissue factor pathway inhibitor from endothelial cells, and inhibition of platelet aggregation. because endogenous at iii levels frequently are decreased in patients with coagulopathy, addition of heparin to fresh frozen plasma may be the most effective route of administration. an initial dose of iu/kg body mass followed by to iu/kg body mass times daily has been recommended. anemia caused by erythrocyte agglutination occurs in some patients during therapy with unfractionated heparin , but typically resolves within hours if therapy is discontinued. because of the risk of microthrombosis associated with erythrocyte agglutination, use of low-molecular-weight heparin ( iu/kg body mass subcutaneously every hours) has been recommended but may be cost-prohibitive. one may give aspirin orally ( to mg/kg body mass, every hours), which irreversibly inhibits platelet cox activity, to inhibit platelet aggregation and microthrombosis. platelet hyperaggregability has been implicated in the pathogenesis of carbohydrate-induced laminitis, and heparin and aspirin have been recommended to prevent development of laminitis. in an in vitro study, however, aspirin did not inhibit endotoxin-induced platelet aggregation. luteolysis caused by increased concentrations of pgf α leads to pregnancy loss in endotoxemic mares before day of pregnancy. daily administration of altrenogest (regu-mate, hoechst-roussel agri-vet, somerville, new jersey) at a dose of mg orally consistently prevented fetal loss in mares if administered until day of pregnancy. treatment with flunixin meglumine, by blockade of pgf α release, also may contribute to the maintenance of pregnancy in endotoxemic mares. the pathogenesis of fetal loss and abortion caused by endotoxemia, surgery, or systemic disease later in gestation is not understood completely. proposed mechanisms include direct effects on the fetus, placental function, or placental progesterone production. the pathophysiology of laminitis caused by endotoxemia is understood incompletely; however, decreased digital blood flow , and intravascular microthrombosis have been implicated. decreased no production by vascular endothelial cells in response to endotoxin has been suggested as a mechanism for vasoconstriction and decreased blood flow ; however, use of no donors remains controversial. maintenance of adequate peripheral perfusion and anticoagulant and antiinflammatory therapy may be helpful in preventing and treating laminitis caused by endotoxemia. although the innate immune response to endotoxin (lipopolysaccharide) is crucially important for the preservation of homeostasis and health, large amounts of endotoxin can evoke an excessive and uncontrolled inflammatory response and result in a dysfunction of hemostatic and circulatory control mechanisms, loss of vascular integrity, and finally tissue damage. conditions commonly associated with the development of endotoxemia in horses are acute gastrointestinal diseases, especially of ischemic and severe inflammatory nature, and localized or generalized infections. although measuring endotoxin concentrations in equine plasma is possible, this is not feasible in a clinical setting, and one typically reaches a diagnosis of endotoxemia based on clinical signs and clinicopathologic data. successful treatment of endotoxemia requires resolution of the primary disease process in addition to neutralization of circulating endotoxin, interference with the activities of inflammatory mediators, and general supportive care. newer treatments, such as blockade of endotoxin-interaction with cells or interruption of cell signaling pathways, are under investigation. possible sequelae of endotoxemia include dic, multiple organ failure, circulatory failure, and death. frequently, the outcome of conditions associated with endotoxemia in horses depends on the severity of associated complications; for example, renal compromise, laminitis, and abortion. resting state the lateral margins of the vesicle, that is, the buccal mucosa, are in close contact with the cheek teeth. caudally, the external space communicates with the pharynx through the aditus pharyngis. the mucous membrane of the mouth is continuous at the margin of the lips with the skin and during life is chiefly pink but can be more or less pigmented, depending on the skin color and the breed type. the lips are two muscular membranous folds that unite at angles close to the first cheek teeth. each lip presents an outer and an inner surface. the upper lip has a shallow median furrow (philtrum); the lower lip has a rounded prominence or chin (mentum). the internal surface is covered with a thick mucous membrane that contains small, pitted surfaces that are the openings of the ducts of the labial glands. small folds of the mucous membrane called the frenula labii pass from the lips to the gum. the free border of the lip is dense and bears short, stiff hairs. the arteries of the mouth are derived from the maxillary, mandibular, labial, and sphenopalatine arteries of the major palatine artery. the veins drain chiefly to the lingual facial vein. sensory nerves originate from the trigeminal nerve (cranial nerve v) and the motor nerves from the facial nerve (vii). the cheeks spread back from the lips and form both sides of the mouth and are attached to the alveolar borders of the bones of the jaws. the cheeks are composed of skin and muscular and glandular layers and then the internal mucous membrane. the skin is thin and pliable. in contrast, the oral mucous membrane is dense and in many areas of the oral cavity is attached firmly to the periosteum so that construction of oral mucosal flaps can be achieved only by horizontal division of the periosteal attachment. such a feature is important in reconstructive techniques applied to the oral cavity. the blood supply to the cheeks comes from the facial and buccal arteries and the sensory nerves from the trigeminal and motor nerves from the facial nerve. the hard palate (palatum durum) is bounded rostrally and laterally by the alveolar arches and is continuous with the soft palate caudally. the hard palate has a central raphe that divides the surface into two equal portions. the word mouth is used commonly to signify the first part of the alimentary canal or the entrance to it. the mouth is bounded laterally by the cheeks, dorsally by the palate, and ventrally by the body of the mandible and by the mylohyoideus muscles. the caudal margin is the soft palate. the mouth of the horse is long and cylindric, and when the lips are closed, the contained structures almost fill the cavity. a small space remains between the root of the tongue and the epiglottis and is termed the oropharynx. the cavity of the mouth is subdivided into sections by the teeth. the space external to the teeth and enclosed by the lips is termed the vesicle of the mouth, and in the enter the nose from the glands of the vomeronasal duct. to what extent these secretions aid in pheromone reception is not known. that portion of the palatine mucosa immediately behind the incisor teeth frequently is swollen (lampas) during eruption of the permanent teeth. this swelling is physiologic and not pathologic. the tongue is situated on the floor of the mouth between the bodies of the mandible and is supported by the sling formed by the mylohyoideus muscles. the root of the tongue is attached to the hyoid bone, soft palate, and pharynx. the upper surface and the rostral portion of the tongue are free; the body of the tongue has three surfaces. the apex of the tongue is spatulate and has a rounded border. the mucous membrane adheres intimately to the adjacent structure and on the dorsum is dense and thick. the lingual and sublingual arteries supply the tongue from the linguofacial trunk and matching veins. the linguofacial trunk drains into the linguofacial vein. the lingual muscles are innervated by the hypoglossal nerve (xii) and the sensory supply is from the lingual and glossopharyngeal (ix) nerves. the formula for the deciduous teeth of the horse is times i - c - p - for a total of . the permanent dental formula is times i - c - p - or p - m - for a total of or . in the mare the canine teeth are usually small or do not erupt, hence reducing the number to or . the first premolar tooth (wolf tooth) is often absent and has been reported as occurring in only % of the upper dentition of thoroughbred horses. the teeth of the horse are complex in shape and are compounded of different materials (dentin, cementum, and enamel). they function as grinding blades to masticate and macerate cellulose food in the important first stage of the digestive process. the cheek teeth in the horse are a well-documented feature of the evolution of equus caballus. the first incisor is present at birth or the first week of life. the second incisor erupts at to weeks of age; the third incisor, at to months of age; the first and second premolars, at birth to weeks of age; and the third premolar, months of age. the eruption times for the permanent teeth are as follows: first incisor, / years of age; second incisor, / years of age; third incisor, / years of age; the canine tooth, to years of age; the first premolar (wolf tooth), to months of age; the second premolar, / years of age; the third premolar, years of age; the fourth premolar, years of age; the first molar, to months of age; the second molar, years of age; and the third molar, / to years of age. this eruption sequence clearly indicates that the eruption of the second and third permanent premolar teeth give the potential for dental impaction. the modern horse has six incisor teeth in each jaw that are placed close together so that the labile edges form a semicircle. the occlusal surface has a deep enamel invagination (infundibulum) that is filled only partially with cementum. as the incisor teeth wear, a characteristic pattern forms in which the infundibulum is surrounded by rings of enamel, dentin, enamel, and crown cementum in a concentric pattern. each incisor tooth tapers from a broad crown to a narrow root so that as the midportion of the incisor is exposed to wear, the cross-sectional diameters are about equal; that is, at years of age, the central incisor tooth of the horse has an occlusal surface that is an equilateral triangle. observations on the state of eruption, the angles of incidence of the incisor teeth, and the pattern of the occlusal surfaces are used as guides for aging of horses. the canine teeth are simple teeth without complex crowns and are curved. the crown is compressed and is smooth on its labial aspect but carries two ridges on its lingual aspect. no occlusal contact occurs between the upper and lower canine teeth. when erupted, the six cheek teeth of the horse function as a single unit in the mastication of food. each arcade consists of three premolar and three molar teeth. the maxillary arcade is slightly curved, and the teeth have a square occlusal surface. the occlusal surfaces of the mandibular teeth are more oblong, and each arcade is straighter. the horse is anisognathic, that is, the distance between the mandibular teeth is narrower (one-third) than the distance between the upper cheek teeth. this anatomic arrangement affects the inclination of the dental arcade as the jaws slide across each other in the food preparation process. the unworn upper cheek tooth presents a surface with two undulating and narrow ridges, one of which is lateral and the other medial. on the rostral and lingual side of the medial style is an extra hillock. the central portion of these surfaces is indented by two depressions that are comparable with, but much deeper than, the infundibula of the incisor teeth. when the teeth have been subjected to wear, the enamel that closed the ridges is worn through and the underlying dentin appears on the surface. thus after a time the chewing surface displays a complicated pattern that may be likened to the outline of an ornate letter b, the upright stroke of the b being on the lingual aspect. dentin supports the enamel internally, cementum supports the enamel lakes, and the peripheral cementum fills in the spaces between the teeth so that all six teeth may function as a single unit, that is, the dental arcade. transverse ridges cross each tooth so that the whole maxillary arcade consists of a serrated edge. the serrations are formed so that a valley is present at the area of contact with adjacent teeth. these serrations match fitting serrations on the mandibular arcade. the true roots of the cheek teeth are short compared with the total length of the tooth. cheek teeth have three roots: two small lateral roots and one large medial root. by custom, that portion of the crown embedded within the dental alveolus is referred to as the reserve crown, and the term root is confined to that area of the tooth that is comparatively short and enamel free. wear on the tooth gradually exposes the reserve crown, and the roots lengthen. in an adult -lb horse the maxillary cheek teeth are between . and . cm in length. dental wear accounts for erosion and loss of tooth substance at a rate of mm/yr. the pulp chambers of the teeth are also complex. the incisors and canines have a single pulp chamber. the mandibular cheek teeth have two roots and two separate pulp chambers. the maxillary cheek teeth, although they have three roots, have in fact five pulp chambers. as occlusal wear proceeds, deposition of secondary dentin within the pulp chambers protects the chambers (e.g., the dental star, medial to the infundibulum on the incisor teeth). in the mandibular cheek teeth the transverse folding of the enamel anlage (during morphogenesis of the tooth) does not take place, and the occlusal surface is a simple surface of central dentin surrounded by enamel. each tooth then is conformed to a single arcade by the presence of peripheral crown cementum. the oral cavity and oropharynx are subject to a variety of diseases. however, many conditions affecting the first portion of the alimentary system produce the same clinical signs, regardless of their cause. the clinical signs may include inappetance or reluctance to eat, pain on eating or swallowing, oral swelling, oral discharge, and fetid breath. affected animals may show some interest in food but hesitate to eat it. salivation may be excessive and may be contaminated with purulent exudate or blood. the occurrence of bruxism (i.e., grinding of teeth) can indicate discomfort in other areas of the alimentary tract; for example, bruxism and frothing oral saliva are characteristic features of gastric ulceration in the horse. the clinician needs to be aware that considerable weight loss can occur rapidly with inability to feed and swallow. diseases that result in denervation of the pharynx and inappropriate swallowing can have the complication of inhalation pneumonia. after a complete physical examination and ascertaining the history, the clinician should approach examination of the mouth systematically in all cases. one can examine a considerable portion of the mouth and teeth from the outside by palpation of the structures through the folds of the cheek. most horses allow an oral examination without sedation or the use of an oral speculum. in many cases, however, one best achieves the detailed oral examination by sedation and the use of an oral speculum and a light source. one should irrigate the mouth to wash out retained food material so as to be able to inspect and palpate the lips, cheeks, teeth, and gums. the classic signs of dental disease in the horse include difficulty and slowness in feeding, together with a progressive unthriftiness and loss of body condition. in some instances, the horse may quid, that is, it may drop poorly masticated food boluses from the mouth, and halitosis may be obvious. additional problems reported by owners include bitting and riding problems and headshaking or head shyness. facial or mandibular swelling may occur. nasal discharge can result from dental disease associated with maxillary sinus empyema. mandibular fistulae frequently are caused by lower cheek tooth apical infections. some correlation exists between the age of the animal and clinical signs (table . - ). ancillary aids for a complete examination of the oral cavity of the horse may include radiology, endoscopic examination, fluoroscopy, biopsy, and culture. one should take care always during endoscopic evaluation of the oral cavity using a flexible endoscope. the author recommends sedation and the use of an oral speculum to prevent inadvertent mastication of the endoscope. if one uses general anesthesia as part of the diagnostic workup, then endoscopic evaluation of the oral cavity is much easier. in selected cases, advanced imaging technologies such as computed tomography, magnetic resonance imaging, or nuclear scintigraphy may be beneficial. the lips of the horse are mobile and prehensile. in many ways they function like the tip of the elephant's trunk in that they test, manipulate, and sample the environment for potential nutritive value. consequently, loss of motor function (e.g., facial palsy) affects the efficiency of the prehensile system. the lips grasp food in grazing or browsing, and the incisor teeth section the food. with mastication and lubrication with saliva, the bolus of food forms and is manipulated from side to side across the mouth, assisted by the tight cheeks of the horse and the palatine ridges. swallowing begins as the food bolus contacts the base of the tongue and the pharyngeal walls. during swallowing, the soft palate elevates to close the nasopharynx, the base of the tongue elevates, and the hyoid bone and the larynx move rostrally following contraction of the hyoid muscles. during this process, the rima glottidis closes and the epiglottis tilts dorsally and caudally to protect the airway so that food is swept through lateral food channels around the sides of the larynx into the laryngoesophagus. fluoroscopic studies in nursing foals in the dorsoventral view showed that contact occurs between the lateral food channels in the midline so that in outline the food bolus achieves a bow tie shape. dysphagia is defined as a difficulty or inability to swallow. anatomic classifications for dysphagia include prepharyngeal, pharyngeal, and esophageal (postpharyngeal) dysphagias. the site of the cause for dysphagia influences the clinical signs. prepharyngeal dysphagia is characterized by dropping food (quidding) or water from the mouth, reluctance to chew, hypersalivation, or abnormalities in prehension. pharyngeal and esophageal dysphagias are characterized by coughing; nasal discharge containing saliva, water, or food material; gagging; anxiousness; and neck extension during attempts to swallow. the following section describes esophageal dysphagia in more detail. causes of dysphagia can be divided into four types: painful, muscular, neurologic, or obstructive (table . - ). pain and obstruction cause dysphagia by interfering with the mechanics of prehension, bolus formation and transfer to the pharynx, and deglutition. muscular and neurologic causes of dysphagia impede prehension and swallowing by affecting the motor function of the lingual or buccal musculature, muscles of mastication (temporal and masseters), and pharyngeal and cranial esophageal muscles. sensory loss to the lips, buccal mucous membranes, pharynx, or tongue also may cause dysphagia. neurologic causes of dysphagia may affect the forebrain, brainstem, or peripheral nerves that control prehension (cranial nerves vm, vs, vii, and xii), transfer of the food bolus to the pharynx (cranial nerves vs and xii) and swallowing (cranial nerves ix and x). diagnosis of the cause of dysphagia is based on physical examination including a careful oral examination, neurologic examination, clinical signs, and endoscopy of the pharynx, esophagus, and guttural pouches. radiology may be useful to assess the bony structures of the head and throat. ultrasonography is valuable for examining the retropharyngeal space and esophagus to detect and evaluate masses. one may detect pharyngeal or esophageal causes of dysphagia with routine endoscopic examination or with contrast radiography. although one also can use endoscopy to assess deglutition, one must remember that sedation adversely affects the deglutition mechanism. one may assess deglutition using fluoroscopy or manometry, but these techniques require specialized equipment. specific diagnostic procedures for nonalimentary causes of dysphagia are covered elsewhere in this text (see chapter ). specific treatments aimed at resolving the underlying disorder causing dysphagia are discussed in detail elsewhere. one should avoid feeding roughage with long fiber length (hay or grass) to most horses with dysphagia. dietary modifications that promote swallowing such as feeding slurries made from complete pelleted feeds may be sufficient to manage some cases of partial dysphagia. one must take care to prevent or avoid aspiration pneumonia in horses with pharyngeal or esophageal dysphagia. one can manage foals by feeding mare's milk or a suitable substitute through a nasogastric tube. one also may administer pellet slurries or formulated liquid diets via nasogastric tubes to older horses. prolonged nutritional management of dysphagic horses may require extraoral feeding using a tube placed through an esophagostomy. formulated pelleted diets are often easy to administer through a tube as slurry and are balanced to meet the nutritional requirements for healthy horses. one must feed sufficient quantities to deliver adequate calories ( to mcal/day for a -kg horse). adjustments may be necessary for horses that are cachectic or have extra metabolic demand (such as pregnancy). adding corn oil to the ration ( cup every or hours) is a common method of increasing fed calories. liquid diets also have been used for enteral feeding but may not be tolerated as well as pelleted diets. regardless of the method of nutritional management, one must monitor and replace salivary losses of electrolytes. saliva contains high concentrations of na, k, and cl. a group of ponies with experimental esophagostomies and a horse with esophageal squamous cell carcinoma were fed a complete pelleted diet through esophagostomy tubes but developed metabolic acidosis, hyponatremia, and hypochloremia apparently because of salivary losses. surprisingly, salivary losses of potassium did not result in hypokalemia in these cases, presumably because of replacement in the diet. however, if the diet is deficient in potassium, hypokalemia may result. one often can accomplish electrolyte replacement by adding nacl and kcl to the diet. one can maintain horses for months with frequent feedings through an esophagostomy tube. parenteral nutrition (total or partial) may be useful in the short term but is not often feasible for long-term management. tooth eruption is a complex phenomenon involving the interplay of dental morphogenesis and those vascular forces responsible for creating the eruption pathway. these changes are responsible for osteitis and bone remodeling within the maxilla and mandible. young horses frequently show symmetric bony swelling resulting from these eruption cysts. in some cases, additional clinical signs of nasal obstruction with respiratory stridor or nasal discharges may be apparent. pathologic problems associated with maleruption include a variety of dental diseases. oral trauma can displace or damage erupting teeth or the permanent tooth buds. as a result, teeth may be displaced and erupt in abnormal positions or may have abnormal shapes. supernumerary teeth, incisors and molars, can develop, as well as palatal displacement of impacted teeth (maxillary p - , or third cheek tooth). in almost all of these conditions some form of surgical treatment is necessary. significant evidence from the location of apical osteitis in diseased teeth (table . - ) confirms that dental impaction is a major cause of dental disease in the horse. in a series of extracted teeth, were p - or p - (cheek tooth or , respectively). early observations had indicated that the first molar (m , or cheek tooth ) was the most commonly diseased tooth, and an "open infundibulum" in this tooth has been suggested as the cause. studies on cementogenesis of the maxillary cheek teeth have shown, however, that in fact most maxillary cheek teeth have a greater or lesser degree of hypoplasia of cementum within the enamel lakes and that this "lesion" rarely expands into the pulp. the central infundibular hole is the site of its vascular supply to the unerupted cement lake. on those occasions in which cases one can use apicoectomy and retrograde endodontic techniques to save the diseased tooth. one must take care, however, in selection of patients. in most cases of apical osteitis in the horse that result from dental impaction, immature root structures make achieving an apical seal of the exposed pulp difficult. gingival hyperemia and inflammation occur during the eruption of the permanent teeth and are common causes of a sore mouth in young horses (particularly -year-olds as the first dental caps loosen). such periodontal changes usually resolve as the permanent dental arcade is established. during normal mastication, the shearing forces generated by the occlusal contact of the cheek teeth essentially clean the teeth of plaque and effectively inhibit deposition of dental calculus. wherever occlusal contact is ineffective, periodontal changes and calculus buildup occur; for example, the deposition of calculus on the canine teeth of mature geldings and stallions is common. routine dental prophylaxis forms an important component of maintaining normal occlusal contact, and for this reason one should remove arcade irregularities that result in enamel point formation on the buccal edges of the maxillary cheek teeth and the lingual edges of the mandibular cheek teeth. one should remove these edges annually in horses that are at grass and twice yearly in young horses, aged horses, and stabled horses. horses at grass have been shown to have a greater range of occlusal contact and therefore better periodontal hygiene than stabled horses. in stabled horses the range of occlusal contact is narrower and the formation of enamel points occurs more frequently with subsequent buccal ulceration and the initiation of a cycle of altered occlusal contact and hence irregular arcade formation. this process leads to severe forms of periodontal disease and wave mouth formation. periodontal disease occurs with abnormal occlusal contact and initiation of the cycle of irregular wear and abnormal contact. such changes progress to loss of alveolar bone, gross periodontal sepsis, and loss of tooth support. in this sense periodontal disease truly is the scourge of the equine mouth and results in tooth loss. palatine clefts may result from an inherited defect and are caused by failure of the transverse palatal folds to fuse in the oral cavity. harelip accompanies few palatine clefts in the horse. the degree of palatine clefting depends on the stage at which interruption in the fusion of the part ii disorders of specific body systems caries of cementum occurs, that is, secondary inflammatory disease and acid necrosis of the cementum, apical osteitis may develop. pulpitis is key to the pathogenesis of dental decay in the horse. the initiation of inflammatory pulp changes may be a sequela to dental impaction or dental caries or may result from fracture of a tooth. if the onset of the inflammatory process is slow, then formation of secondary dentin within the pulp chambers may protect the pulp and the tooth. secondary dentin formation occurs from stimulation of odontoblasts within the pulp chamber. such changes are the normal process of protection during dental wear and attrition as crown substances wear away and the reserve crown comes into wear. in acute disease, however, this defense mechanism is ineffective, and the changes that occur and that are sequelae to pulpitis reflect the location of each affected tooth. for example, pulpitis and apical osteitis of the third mandibular cheek tooth most commonly results in the development of a mandibular dental fistula. pulpitis of the third maxillary cheek tooth, however, results in an inflammatory disease within the rostral maxillary sinus and in development of chronic maxillary sinus empyema (figure . - ) . oblique radiographs greatly assist the diagnosis of dental decay by demonstrating sinus tract formation, sequestration of bone, mandibular osteitis, hyperplasia of cementum, and new bone formation (so-called alveolar periosteitis). the management of dental decay in the horse usually involves surgical extraction of the diseased tooth. in some palatopalatal folds occurs. toxic or teratogenic effects are documented in other species, but little data are available in the horse. in recent years, treatment for repair of uncomplicated palatine defects has been recommended but prognosis is generally poor because of the considerable nursing care required and the high incidence of surgical failures. one should emphasize early surgery and the use of mandibular symphysiotomy in affording surgical exposure. the combination of mandibular symphysiotomy and transhyoid pharyngotomy to approach the caudal margins of the soft palate affords surgical access, and one can construct mucosal flaps to repair the defects. however, the incidence of surgical breakdown is high, and healing by first intention is the exception rather than the rule. a recent surgical report documented the successful closure of a median cleft of the lower lip and mandible in a donkey. foals born with a severely deviated premaxilla and palate have a wry nose. one can achieve surgical correction of the deviated premaxilla by submucosal division of the premaxilla across the nose at the line of the first cheek tooth. circumstantial evidence indicates that such a defect has a genetic cause, and the defect occurs most frequently in the arabian breed. other developmental abnormalities are subepiglottic cysts resulting from cystic distortion of remnants of the thyroglossal duct, which may cause dyspnea and choking in foals. surgical removal of these cysts results in normal function. the most significant developmental defect of dental origin is a maxilla that is longer than the mandible, that is, the horse is parrot-mouthed. an overbite of cm in the incisor arcade may be present in a horse with a mismatch of less than cm between the first upper and lower cheek teeth. parrot mouth and monkey or sow mouth are thought to be inherited conditions. some correction of minor incisor malocclusion occurs up to years of age. recognition and detection of parrot mouth are important in the examination of potential breeding stock. surgical attempts to inhibit overgrowth of the premaxilla by wiring or by the application of dental bite plate procedures have been documented in recent years. as has been indicated, the horse is by nature a curious animal and uses its lips as a means of exploring a variety of objects. wounds of the lips, incisive bone, and the mandibular incisor area occur commonly in the horse and usually result from the horse getting the lips, jaw, or teeth caught in feeding buckets, in fence posts, or in halters or having a segment of tongue encircled with hair in tail chewing. as the horse panics and pulls away from its oral entrapment, considerable trauma can occur to the lips, teeth, and gums. most wounds repair satisfactorily, provided one finds them early and observes the basic principles of wound hygiene, excision of necrotic tissue, and wound closure. one must ensure that oral mucosal defects are closed and that effective oral seals are made before external wounds are closed. in some cases, offering specially constructed diets or even feeding the horse by nasogastric tube or esophagostomy during the healing processes may be necessary. foreign body penetration of the tongue, cheek, or palate has been reported in grazing and browsing horses and in particular in horses that have certain hay sources that contain desiccated barley awns or yellow bristle grass. other plant material and grass awns also occasionally may penetrate the tongue, gingiva, or cheek, causing inflammation or abscesses. ulcerative stomatitis also results from the toxicity of phenylbutazone therapy. vesicular stomatitis is a highly contagious viral blistering disease described in more detail elsewhere. treatment of glossitis and stomatitis primarily aims at removing the inciting cause. actinobacillus lignieresii, the causative agent of actinobacillosis, has been isolated and identified from ulcers on the free border of the soft palate and oral and laryngeal granulomata. the bacterium also was reported in a sublingual caruncle in a horse with a greatly swollen tongue. therapy with ml of % sodium iodide and g of ampicillin every to hours effected a clinical cure. saliva is important for lubricating and softening food material. the horse has paired parotid, mandibular, and polystomatic sublingual salivary glands. the parotid gland is the largest of the salivary glands in the horse and is situated in the space between the ramus of the mandible and the wing of the atlas. the parotid duct is formed at the ventral part of the gland near the facial crest by the union of three or four smaller ducts. the duct leaves the gland above the linguofacial vein, crosses the tendon of the sternocephalicus muscle, and enters the mouth obliquely in the cheek opposite the third upper cheek tooth. the parotic duct orifice is small, but some dilation of the duct and a circular mucous fold (the parotid papillae) exist at this point. the mandibular gland is smaller than the parotid gland and extends from the atlantal fossa to the basihyoid bone. for the most part, the mandibular gland is covered by the parotid gland and by the lower jaw. the mandibular duct is formed by union of a number of small duct radicles that emerge along the concave edge of the gland and run rostral to the border of the mouth opposite the canine tooth. the orifice is at the end of a sublingual caruncle. the mandibular gland possesses serous, mucous, and mixed alveolar glandular components. the parotid gland is a compound alveolar serous gland. the parotid salivary gland can secrete saliva to yield rates of ml/min, and a total daily parotid secretion can be as much as l in a -kg horse. parotid secretion only occurs during mastication, and administration of atropine or anesthesia of the oral mucosa can block secretion. parotid saliva is hypotonic compared with plasma, but at high rates of flow, concentrations of sodium, chloride, and bicarbonate ions increase. parotid saliva of the horse has a high concentration of calcium, and occasionally calculi (sialoliths) form within the duct radicles of the parotid salivary gland. congenital parotid duct atresia, acquired stricture from trauma to the duct, or obstruction by plant material (sticks or foxtails and other seeds) also may occur. the clinical signs of sialolithiasis or other forms of ductule obstruction include a fluid swelling in the form of a mucocele proximal to the stone and occasionally inflammation of the parotid gland. ultrasonography is useful to diagnose salivary mucoceles and to detect foreign bodies or sialoliths. measurement of electrolyte concentrations in aspirates from suspected mucoceles might be helpful to distinguish them from hematomas. salivary potassium and calcium concentrations are higher than plasma. treatment may require surgical removal of the stone or plant material in the case of sialolithiasis or foreign body obstructions. other causes of obstruction may require resection of the affected portion of the duct or chemical ablation of the gland. primary sialoadenitis is unusual but can occur in one or both glands. the condition is painful and may be associated with a fever and anorexia. secondary sialoadenitis is more common and usually is associated with trauma. infectious sialoadenitis from corynebacterium pseudotuberculosis or other bacterial pathogens also may occur. diagnosis is by physical examination and by finding an enlarged edematous parotid gland tissue on ultrasonographic examination. culture and cytologic examination of aspirates may be useful for diagnostic purposes. treatment in usually palliative, consisting of nonsteroidal antiinflammatory drugs. appropriate antibiotic therapy is indicated as directed by culture and sensitivity results. chemical irritation, glossitis, stomatitis, or other causes of prepharyngeal dysphagia cause ptyalism or excessive salivation in horses. specific therapy for the ptyalism usually is not required as long as salivary losses are not excessive, resulting in dehydration and electrolyte imbalances. ingestion of the fungal toxin slaframine also causes hypersalivation in horses. the fungus rhizoctonia leguminicola, which produces slaframine, causes black patch disease in red clover. slaframine is a parasympathomimetic compound that stimulates exocrine secretion in the parotid gland. slaframine toxicosis most commonly occurs in the spring or early summer and rarely requires treatment other than removal from the pasture. mowing removes the source in most cases because regrowth in pastures often has less fungal contamination. . the esophagus has no digestive or absorptive functions and serves as a conduit to the stomach for food, water, and salivary secretions. the esophageal mucosa is a keratinized stratified squamous epithelium. the submucosa contains elastic fibers that contribute to the longitudinal folds of the esophagus and confer elasticity to the esophageal wall. a transition occurs in the muscle type composing the tunica muscularis from striated skeletal muscle in the proximal two thirds of the esophagus to smooth muscle in the distal third. in the proximal esophagus the skeletal muscle layers spiral across one another at angles. within the smooth muscle layers of the distal esophagus the outer layer becomes more longitudinal, whereas the inner layer thickens and becomes circular. the wall of the terminal esophagus can be to cm thick. deep cervical fascia, pleura, and peritoneum contribute to the thin fibrous tunica adventitia of the esophagus. motor innervation to the striated skeletal muscle of the esophagus includes the pharyngeal and esophageal branches of the vagus nerve, which originate in the nucleus ambiguus of the medulla oblongata. parasympathetic fibers of the vagus nerve supply the smooth muscle of the distal esophagus. sympathetic innervation of the esophagus is minimal. passage of ingesta through the esophagus can be considered part of the swallowing process, which consists of oral, pharyngeal, and esophageal stages. the oral stage is voluntary and involves transport of the food bolus from the mouth into the oropharynx. during the involuntary pharyngeal stage the food bolus is forced through the momentarily relaxed upper esophageal sphincter by simultaneous contractions of the pharyngeal muscles. in the esophageal phase of swallowing the upper esophageal sphincter closes immediately, the lower esophageal sphincter opens, and esophageal peristalsis propels the bolus into the stomach. unlike a food bolus, liquids do not require peristalsis to reach the lower esophageal sphincter and may precede the food bolus during swallowing. the upper esophageal sphincter prevents esophagopharyngeal reflux during swallowing and air distention of the esophagus during inspiration. upper esophageal pressure increases in response to pressure from a food bolus and to increased intraluminal acidity, as would occur with gastroesophageal reflux. the lower esophageal sphincter is a smooth muscle located at the gastroesophageal junction that is morphologically ill defined but forms an effective functional barrier. normally the lower esophageal sphincter is closed in response to gastric distention to restrict gastroesophageal reflux. relaxation of the lower esophageal sphincter permits passage of ingested material from the esophagus to the stomach. distention of the stomach with ingesta mechanically constricts the lower esophageal sphincter. gastric distention also triggers a the esophagus is a musculomembranous tube that originates from the pharynx dorsal to the larynx and terminates at the cardia of the stomach. in adult thoroughbred horses the esophagus is approximately cm long. the cervical portion is approximately cm long; the thoracic portion, approximately cm long; and the short abdominal portion, only approximately cm long. the cervical esophagus generally lies dorsal and to the left of the trachea in the cervical region. in the thorax the esophagus courses through the mediastinum lying dorsal to the trachea and crosses to the right of the aortic arch dorsal to the heart base. impactions. intramural causes of esophageal obstruction include tumors (squamous cell carcinoma), strictures, diverticula, and cysts. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mediastinal or cervical masses (tumors or abscesses) may cause extramural obstructions. congenital anomalies are covered in detail later. the clinician must perform a thorough physical examination, including complete oral and neurologic examination, to help rule out causes of dysphagia and nasal discharge other than esophageal obstruction. the clinical signs associated with esophageal obstructions are related primarily to regurgitation of food, water, and saliva caused by esophageal (postpharyngeal) dysphagia. horses with esophageal obstruction are often anxious and stand with their neck extended. one may note gagging or retching, particularly with acute proximal obstructions. bilateral frothy nasal discharge containing saliva, water, and food material; coughing; odynophagia; and ptyalism are characteristic clinical signs, the severity of which varies with the degree and location of the obstruction. distention in the jugular furrow may be evident at the site of obstruction. one may observe other clinical signs related to regurgitation of saliva, water, and food material, such as dehydration, electrolyte or acid-base imbalances, weight loss, and aspiration pneumonia. in extreme cases, pressure necrosis from the impaction or trauma to the esophagus may cause esophageal rupture. if the rupture is in the cervical esophagus, crepitus or cellulitis may be evident along with signs of systemic inflammation. thoracic auscultation is important to determine whether aspiration pneumonia is present. intrathoracic esophageal rupture may result in pleuritis and its associated clinical signs. passage of a nasogastric tube is an effective way to detect and localize an obstruction but provides little information about the nature of the obstruction or the condition of the esophagus. the most direct method for diagnosis of esophageal obstructions is endoscopic examination. most cases of esophageal obstruction occur at sites of natural narrowing of the esophageal lumen, such as the cervical esophagus, the thoracic inlet, base of the heart, or the terminal esophagus, thus one may need an endoscope longer than m for complete evaluation. endoscopic evaluation is useful before relief of an impaction to localize the obstruction and to investigate the nature of the impaction if one suspects a foreign body. foreign bodies may be retrievable via transendoscopic tethering. one can obtain critical diagnostic and prognostic information following resolution of the impaction. assessing the affected esophagus for mucosal ulceration, rupture, masses, strictures, diverticula, and signs of functional abnormalities is important (figure . - ) . ultrasonography of the cervical region is useful not only to confirm a cervical esophageal impaction but also part ii disorders of specific body systems vagal reflex that increases lower esophageal sphincter tone, a safety mechanism against gastroesophageal reflux. the mechanical and vagal mechanisms that promote lower esophageal sphincter tone prevent spontaneous decompression of the stomach, which along with a lack of a vomiting reflex in the horse, increases the risk of gastric rupture during episodes of severe distention. esophageal obstruction has many causes (table . - ) and most often is manifested clinically by impaction of food material and resulting esophageal dysphagia. esophageal obstruction may be caused by primary impactions (simple choke) of roughage, particularly leafy alfalfa hay, coarse grass hay, bedding, and even grass. prior esophageal trauma or poor mastication caused by dental abnormalities may predispose horses to primary esophageal impaction. wolfing or gulping food may precipitate primary impactions, particularly if the horse is exhausted or mildly dehydrated after a long ride or is weakened from chronic debilitation. impactions also may result from disorders that physically impede the passage of food material and fluid by narrowing the luminal diameter, reduce the compliance of the esophageal wall, or alter the conformation of the esophageal wall such that food material accumulates in a pocket or diverticulum. foreign bodies, intra-or extramural masses, or acquired or congenital anomalies cause these so-called secondary to provide critical information about the location and extent of the impaction and esophageal wall thickness and integrity. ultrasonography may provide information about the cause. radiographic assessment of the esophagus can confirm the presence of esophageal obstruction in cases in which one cannot view the affected area adequately using endoscopy. one can detect impacted food material in the esophagus by a typical granular pattern and often can observe gas accumulation proximal to the obstruction. air or barium contrast radiographic studies are most useful for evaluating the esophagus following relief of the impaction if one suspects a stricture. one often can detect esophageal dilation, diverticula, rupture, functional disorder (megaesophagus), or luminal narrowing caused by extraluminal compression more easily using contrast radiographic studies instead of endoscopy ( figure . - ). - one should take care when interpreting radiographic studies in sedated horses, particularly after passage of a nasogastric tube or other esophageal manipulations that may contribute to esophageal dilation. the primary goal of treatment for esophageal impaction is to relieve the obstruction. parenteral administration of acepromazine ( . mg/kg intravenously), xylazine ( . to . mg/kg intravenously) or detomidine ( . to . mg/kg intravenously), oxytocin ( . to . iu/kg intramuscularly), and/or esophageal instillation of lidocaine ( to ml of % lidocaine) may reduce esophageal spasms caused by pain or may decrease esophageal tone. [ ] [ ] [ ] [ ] some clinicians advocate parasympatholytic drugs such as atropine ( . mg/kg intravenously) to reduce salivary secretions and lessen the risk of aspiration. however, undesirable effects of atropine including excessive drying of the impaction and inhibition of distal gastrointestinal motility may preclude its use. resolution of an impaction may require physical dispersal of the material. one can use a nasogastric tube to displace the impacted material along with external massage if the obstruction is in the cervical region. often, carefully lavaging the esophagus with water via an uncuffed or a cuffed nasogastric tube while the head is lowered is necessary to aid in breaking up the impaction. some clinicians advocate a dual tube method whereby a tube is placed through each nasal passage into the esophagus for ingress and egress of the lavage fluid. because of the risk of aspiration of water and food material, esophageal lavage sometimes is done under general anesthesia with a cuffed nasotracheal tube. in refractory cases, intravenous administration of isotonic fluid containing . % nacl and kcl ( to meq/l) for hours at a rate of to ml/kg/ day along with esophageal relaxants such as oxytocin may promote hydration and softening of the impaction and help prevent or alleviate any electrolyte or acid-base imbalances resulting from salivary losses of chloride, sodium, and potassium. one should note that the effects of oxytocin on esophageal tone occur in the proximal two thirds of the esophagus and may not be effective for mucosa has recovered as assessed by endoscopy, one can feed the horse soft food (moistened pellets and bran mashes). one can return the patient gradually to a highquality roughage diet over to days, depending on the degree of esophageal damage induced by the impaction and the nature of any underlying disease. the prognosis for survival is good ( %), but some horses may require permanent dietary modification if persistent chronic obstruction is a problem. aspiration pneumonia and perforation are potential complications of severe or prolonged esophageal obstructions. if aspiration is suspected, administration of broad-spectrum antibiotics that are effective against gram-positive and gram-negative organisms, including metronidazole ( mg/kg orally every hours) for anaerobes is advisable. a subsequent section describes treatment of esophageal perforation or rupture. esophagitis refers to a clinical syndrome of esophageal inflammation that may or may not be ulcerative. the major protective mechanisms of the esophageal mucosa include salivary and food material buffers, normal peristaltic motility, and the barrier formed by the gastroesophageal sphincter. reflux esophagitis is caused by repeated episodes of gastric fluid regurgitation into the distal esophagus and subsequent chemical injury to the mucosa ( figure distal obstructions. , rarely, esophageal obstruction ultimately may require esophagotomy to relieve the impaction. one must enforce strict restriction of food and water, including access to bedding material, until the obstruction is resolved and the esophagus has regained function. systemic effects of dysphagia associated with esophageal impaction include dehydration, hyponatremia, hypochloremia, and metabolic alkalosis from prolonged loss of salivary free water and electrolytes. if the duration of a complete esophageal obstruction is hours or longer, one should correct dehydration and electrolyte and acid-base imbalances. one can restore fluid and electrolyte balance with oral electrolyte solutions if the patient is less than % to % dehydrated and the esophageal obstruction is resolved. horses that are greater than % to % dehydrated or those that have a refractory obstruction or moderate to severe electrolyte imbalances may require intravenous fluid therapy with solutions containing . % nacl and kcl ( to meq/l). one should perform esophageal endoscopy after relief of the impaction to determine whether any complications of the impaction have developed or if a primary cause of the obstruction is present. endoscopic examination is critical to determine the postobstruction treatment plan and for follow-up evaluation of esophageal healing. one should reevaluate the horse every to weeks following resolution of the impaction if one notes esophageal dilation or mucosal injury. additional evaluation via radiography may be warranted to assess motility and transit times. dilation proximal to the site of obstruction, mucosal injury from trauma, stricture formation, formation of a diverticulum, megaesophagus, and esophagitis are sequelae to esophageal obstruction that predispose patients to reobstruction. the rate of reobstruction may be as high as %. depending on the duration of the obstruction and the degree of trauma or dilation, the risk of reobstruction is high for to hours or longer, thus one should withhold food for at least to hours after resolution of the obstruction. sucralfate ( mg/kg orally every hours) may hasten healing if esophageal ulceration is evident, but the efficacy of sucralfate for this purpose is not established. some clinicians suggest that administration of a nonsteroidal antiinflammatory drug (nsaid) such as flunixin meglumine ( mg/kg orally or intravenously every hours) or phenylbutazone ( to mg/kg orally or intravenously every to hours) for to weeks after resolution of the impaction may reduce the development of strictures. judicious use of nsaids is recommended to prevent nsaid-induced worsening of esophageal mucosal injury. one should avoid orally administered nsaids if esophagitis is present. after to hours or when the esophageal duodenal strictures caused by chronic ulceration commonly have reflux esophagitis. diagnosis requires endoscopic examination of the esophagus. one may note diffuse, patchy, linear, or coalescing erosion or ulcerations (see figures . - and . - ). one also may observe significant edema or hyperemia. determining whether an underlying disease, such as infection, neoplasia, esophageal strictures, or diverticula, is present is important. in addition, one must examine the stomach to determine whether the esophagitis is associated with gastritis, gastric obstruction, or gastric ulcer disease. contrast radiography may be helpful to detect esophageal ulceration and is useful to assess esophageal motility and transit time. the principles of therapy for reflux esophagitis include control of gastric acidity, mucosal protection, and correction of any underlying disorder contributing to gastroesophageal reflux. reduction of gastric acid production with h histamine receptor blockers such as ranitidine or proton pump antagonists such as omeprazole is critical for resolution of the esophagitis. some clinicians advocate using sucralfate to promote healing of ulcerated esophageal mucosa. however, the ability of sucralfate to bind ulcerated esophageal mucosa is not proven, nor is the efficacy of sucralfate for hastening esophageal ulcer healing. horses with reflux esophagitis following delayed gastric outflow caused by gastroduodenal ulcer disease, gastric paresis, or proximal enteritis may benefit from prokinetic drugs that act on the proximal gastrointestinal tract. metoclopramide ( . to . mg/kg subcutaneously every to hours) reduces gastroesophageal reflux by increasing lower esophageal sphincter tone, gastric emptying, and gastroduodenal coordination. one should exercise caution when giving metoclopramide to horses because they are prone to extrapyramidal neurologic side effects of the drug. cholinergic drugs such as bethanechol ( . to . mg/kg subcutaneously every to hours or . to . mg/kg orally every to hours) may improve gastric emptying and are effective for treating reflux esophagitis. for esophagitis from trauma or pressure injury after esophageal impaction, judicious use of nsaids may be warranted to reduce esophageal inflammation and pain. dietary modification may be necessary for patients with esophagitis, depending on the degree of ulceration or if motility is impaired. one should feed horses with mild esophagitis frequent small meals of moistened pellets and fresh grass. severe esophagitis may necessitate withholding food and complete esophageal rest for several days. although the prognosis for esophagitis is good in the absence of underlying disease, the risk of esophagus is delayed, such as in functional disorders of the esophagus. like ulceration of the squamous portion of the stomach in horses, gastric acid and bile salt chemical injury is a major mechanism of esophageal squamous epithelial ulceration. , reflux esophagitis may occur along with gastric ulcer disease, motility disorders, increased gastric volume from gastric outflow obstructions, gastric paresis, intestinal ileus, or impaired lower esophageal sphincter function. , other causes of esophagitis in horses include trauma (foreign bodies, food impactions, nasogastric tubes), infection (mural abscesses), or chemical injury (pharmaceuticals, cantharidin) ( figure . - ). [ ] [ ] [ ] [ ] the clinical signs of esophagitis are nonspecific and similar to esophageal obstruction and gastric ulcer diseases. gagging or discomfort when swallowing may be evident, and hypersalivation and bruxism are signs of esophageal pain. esophageal (postpharyngeal) dysphagia may be evident. one may note partial or complete anorexia such that horses with chronic esophagitis may have significant weight loss. esophageal hypomotility dysfunction caused by the inflammatory process may result in esophageal impaction. clinical signs of underlying diseases that predispose to esophagitis may predominate or mask the signs of esophagitis. horses with gastrointestinal motility disorders such as proximal enteritis or gastric outflow obstruction are at a high risk of developing reflux esophagitis because of the presence of gastric acid and bile salts in the fluid reflux. foals with gastric, pyloric, or stricture formation is high if severe circumferential or coalescing ulcerations are present. esophagitis from severe trauma or infection may be prone to stricture formation. motility dysfunction of the equine esophagus is caused most commonly by hypomotility resulting in esophageal dilation (ectasia) or megaesophagus. although megaesophagus in horses most commonly is acquired, reports indicate idiopathic megaesophagus in young horses may be congenital. [ ] [ ] [ ] [ ] acquired megaesophagus in horses may be a consequence of chronic or recurrent esophageal obstruction. , esophageal impactions of a short duration cause a proximal dilation of the esophagus that is generally reversible. however, if the duration of the obstruction is long enough, the motility of the esophagus proximal to the site of obstruction may be impaired permanently. other causes of acquired megaesophagus include extraesophageal obstruction by tumors or abscesses, pleuropneumonia, and vascular ring anomalies. , acquired megaesophagus also may result from neurologic, neuromuscular, and muscular disorders. neurologic diseases that cause vagal neuropathy-such as equine protozoal myeloencephalitis, equine herpesvirus myeloencephalitis, and idiopathic vagal neuropathy-have been associated with megaesophagus in horses. pleuropneumonia may be associated with a vagal neuropathy resulting in megaesophagus. megaesophagus is an early sign of equine dysautonomia and may be observable in patients with botulism. myasthenia gravis is a well-known cause of megaesophagus in nonequine species but has not been reported in horses. also in other species, electrolyte disorders, cachexia, primary myopathies, myositis, and addison's disease may affect esophageal motility but have not been associated with megaesophagus in horses. one can induce iatrogenic megaesophagus by the α adrenergic agonist detomidine, but this is transient and reversible. , nonetheless, the use of this drug may complicate clinical evaluation of esophageal motility. esophageal inflammation, particularly reflux esophagitis, may affect motility and cause megaesophagus. however, because esophageal hypomotility affects the tone and function of the lower esophageal sphincter, reflux esophagitis also may be a complication of a primary functional disorder. thus assessing esophageal motility in horses with esophagitis that is not responding appropriately to treatment is important. along with a complete physical examination one should include a careful neurologic examination to help rule out primary neurologic causes of megaesophagus. because esophageal hypomotility is a functional obstruction, the clinical signs of esophageal hypomotility or megaesophagus are similar to esophageal obstruction. unlike mechanical obstruction the onset of clinical signs is insidious rather than acute. the clinical signs include those associated with esophageal dysphagia. , , [ ] [ ] [ ] [ ] the cervical esophagus may be dilated enough to be evident externally. weight loss is a common sign. signs attributable to an underlying disease may be evident. diagnosis of esophageal hypomotility requires transit studies. one can measure the transit time of a bolus from the cervical esophagus to the stomach by fluoroscopy or contrast radiography. , other signs of esophageal hypomotility and megaesophagus include pooling of contrast material and an absence of peristaltic constrictions. , , , endoscopy may reveal a dilated esophagus and an absence of peristaltic waves. , one may observe evidence of underlying disease causing obstruction or esophageal dilation. , one should evaluate the esophagus for evidence of esophagitis that is causing esophageal motility dysfunction or is a result of impaired esophageal clearance of gastric fluid. esophageal manometry may be useful to document abnormal postdeglutition contraction pressures, contraction time, and propagation times but is not often available for routine clinical application. , one should perform other diagnostic tests such as a complete blood count and chemistry to help determine a possible underlying cause. cerebral spinal fluid analysis may be indicated to rule out neurologic disorders. specialized testing such as electromyography to detect neuromuscular disorders may also be indicated. treatment of esophageal hypomotility or megaesophagus should aim at treating the underlying cause. dietary modification should aim at improving esophageal transit of food. one should feed the horse slurries of pellets, and feeding from an elevated position to promote transit may be beneficial. metoclopramide or bethanechol may benefit patients with reflux esophagitis associated with megaesophagus by increasing lower esophageal tone, gastric emptying, and reducing gastroesophageal reflux. the prognosis depends on the underlying cause and the degree of dilation. although many cases of megaesophagus associated with reflux esophagitis respond well to treatment, many other forms of megaesophagus including congenital megaesophagus have a poor prognosis. strictures most commonly are caused by pressure necrosis from esophageal impactions that induce circumferential erosion or ulceration of the esophageal mucosa, although esophageal injury caused by oral administration of corrosive medicinal agents and trauma to the neck may also result in stricture formation. congenital strictures also have been reported. strictures caused by mucosal and submucosal trauma are termed esophageal webs or rings. strictures may also originate in the muscular layers and adventitia of the esophagus (mural strictures) or in all of the layers of the esophagus (annular stenosis). , horses with these lesions have a presentation similar to those with simple obstructions, because strictures result in partial obstruction and impaction of food material in the lumen. one can detect esophageal webs or rings with endoscopy (see figure . - ), whereas identification of mural strictures or annular stenosis may require a double-contrast esophogram (see figure . - ). in a retrospective study of horses with esophageal stricture following simple obstruction, maximal reduction in esophageal lumen diameter occurred within days of the esophageal obstruction. although surgery has been used to relieve such strictures, initial medical management is warranted because strictures may resolve with conservative therapy, and the esophagus continues to remodel for up to days following ulceration. in one report, seven horses with esophageal obstruction-induced stricture were treated conservatively by feeding a slurry diet and administering antiinflammatory and antimicrobial medications, and five of seven were clinically normal within days. one of the five successfully treated horses had a -cm area of circumferential ulceration, suggesting that the potential exists for extensive mucosal injury to resolve without permanent stricture formation. if resolution of strictures within days is insufficient, one should investigate other methods to increase esophageal diameter. bougienage has been used successfully in small animal patients and human beings. the technique involves passage of a tubular dilatable instrument down the esophagus and stretching of the stricture. one may perform the technique by passing a nasogastric tube with an inflatable cuff. however, one has to perform the procedure frequently to have any success, and horses do not tolerate it well. alternatively, a number of surgical techniques have been used to resolve strictures, including resection and anastomosis, , temporary esophagostomy with fenestration of the stricture, esophagomyotomy for strictures of the muscularis and adventitia, , or patch grafting with local musculature. however, such surgeries are fraught with complications, largely because of the propensity of the traumatized esophagus to restricture. , the esophagus lacks a serosal layer and does not rapidly form a fibrin seal as does the remainder of the intestinal tract, so anastomoses tend to leak. in addition, tension on the esophagus during swallowing and movement of the neck impairs healing of anastomoses. , in spite of these difficulties, the long-term prognosis for horses with chronic esophageal strictures treated surgically is better than for those treated nonsurgically. two types of diverticula are traction (true) diverticula and pulsion (false) diverticula. traction diverticula result from wounding and subsequent contraction of periesophageal tissues, with resultant tenting of the wall of the esophagus. pulsion diverticula arise from protrusion of esophageal mucosa through defects in the muscular wall of the esophagus and usually result from trauma or acute changes in intraluminal pressure. traction diverticula appear as a dilation with a broad neck on contrast esophagography, whereas pulsion diverticula typically have a flask shape with a small neck on an esophagram (see figure . - ). , although traction diverticula are usually asymptomatic and of little clinical significance, pulsion diverticula may fill with feed material, ultimately leading to esophageal obstruction. [ ] [ ] [ ] a movable mass in the midcervical region may be noticeable before onset of complete obstruction. pulsion diverticula may be corrected surgically by inverting or resecting prolapsed mucosa and closing the defect in the wall of the esophagus. , , inversion of excessive mucosa may reduce the diameter of the esophageal lumen and predispose horses to esophageal obstruction and therefore should be reserved for small diverticula. congenital disorders of the esophagus are rare. reported congenital abnormalities include congenital stenosis, persistent right aortic arch, esophageal duplication cysts, [ ] [ ] [ ] intramural inclusion cysts, , and idiopathic megaesophagus. , , in the one report of congenital stenosis, double-contrast radiography revealed concentric narrowing of the thoracic esophagus in the absence of any vascular abnormalities at the base of the heart. successful treatment included having the foal stand with the forelimbs elevated off the ground following each feeding. persistent right aortic arch is a congenital anomaly in which the right fourth aortic arch becomes the definitive aorta instead of the left aortic arch, which results in constriction of the esophagus by the ligamentum arteriosum as it extends between the anomalous right aorta and the left pulmonary artery. clinical signs may include those associated with esophageal (postpharyngeal) dysphagia, drooling, and distention of the cervical esophagus resulting from partial obstruction of the thoracic esophagus. , endoscopic examination typically reveals dilation of the esophagus cranial to the obstruction section . esophageal diseases with evidence of diffuse esophagitis. successful surgical treatment of persistent right aortic arch has been reported in one foal. esophageal duplication cysts and intramural inclusion cysts cause typical signs of esophageal obstruction, including salivation, esophageal dysphagia, and swelling of the cervical esophagus as the cysts enlarge. , , such signs can make them difficult to differentiate from other forms of esophageal obstruction (choke). endoscopic examination may reveal compression of the esophageal lumen and communication with the esophageal lumen if it exists. ultrasonographic examination may be the most useful method of antemortem diagnosis if the cyst is in the cervical esophagus. examination of an aspirate of the mass may aid in the diagnosis by revealing the presence of keratinized squamous cells. , surgical treatments have included complete surgical resection and surgical marsupialization. , , the latter appears to be more successful and results in fewer complications. , complications of surgical resection have included laryngeal hemiplegia following surgical trauma to the recurrent laryngeal nerve in the region of the esophagus and esophageal fistula formation. perforation typically occurs in the cervical region in response to external trauma, necrosis of the esophageal wall caused by a food impaction, or rupture of an esophageal lesion such as an impacted diverticulum. the esophagus is particularly vulnerable to external trauma in the distal third of the neck because only a thin layer of muscle covers it at this point. iatrogenic perforation may occur in response to excessive force with a stomach tube against an obstruction or a compromised region of the esophagus. esophageal perforations may be open or closed and tend to cause extensive cellulitis and necrosis of tissues surrounding the wound because of drainage of saliva and feed material within fascial planes. systemic inflammation associated with endotoxemia from septic cellulitis may occur. closed perforations of the esophagus are particularly troublesome because food material, water, saliva, and air may migrate to the mediastinum and pleural space via fascial planes. , because of the leakage of air into the tissues surrounding the rupture, extensive subcutaneous and fascial emphysema frequently develops and is usually evident clinically and on cervical radiographs. pneumomediastinum and pneumothorax are potentially fatal complications of esophageal ruptures. treatment should include converting closed perforations to open perforations if possible, extensive debridement and lavage of affected tissues, broadspectrum antibiotics, tetanus prophylaxis, and esophageal rest. the clinician may achieve the latter by placing a feeding tube into the esophagus via the wound. alternatively, one may place a nasogastric tube using a small tube ( -f diameter) . for open perforations, once the wound has granulated and contracted to a small size, one may attempt peroral feeding. extensive loss of saliva via esophageal wounds may lead to hyponatremia and hypochloremia. in addition, transient metabolic acidosis occurs because of salivary bicarbonate loss, followed by progressive metabolic alkalosis. although reports of esophageal wounds healing well by second intention exist, healing takes a prolonged time. in addition, some perforations never completely heal and form permanent esophagocutaneous fistulae that may require surgical correction. the development of esophageal strictures is not common because wounds are usually linear and not circumferential. however, traction diverticula may develop. other complications of esophageal wounds include horner's syndrome and left laryngeal hemiplegia. in a retrospective study on esophageal disorders, only of horses with esophageal perforations survived long-term ; in a report of esophageal trauma following nasogastric intubation, of horses were euthanized. the prognosis is therefore poor in horses with esophageal perforations, largely because of the extent of cellulitis, tissue necrosis, shock, and local wound complications. specialized endoscopic equipment allowing visual inspection of the entire adult equine stomach has become increasingly available to veterinarians in academia and private practice. thus gastric disease in horses recently section . diseases of the stomach has gained increasing awareness among veterinarians, owners, and trainers. peptic ulcer disease is defined as erosions or ulcers of any portion of the gastrointestinal tract normally exposed to acid. mucosal damage can include inflammation, erosion (disruption of the superficial mucosa), or ulceration (penetration of the submucosa). in severe cases, fullthickness ulceration can occur, resulting in perforation. the proximal (orad) portion of the equine stomach is lined by stratified squamous mucosa similar to the esophageal lining. the distal (aborad) portion of the stomach is lined with glandular mucosa, and the distinct junction between the two regions is deemed the margo plicatus. ulceration can occur in either or both gastric regions, although different clinical syndromes and pathophysiologic mechanisms apply. as a result, the broad term equine gastric ulcer syndrome (egus) has been used to encompass the wide array of associated clinical syndromes. egus develops in horses of all ages and continues to be of major clinical and economical importance. the prevalence of gastric ulceration has been reported for a variety of breeds and types of horses; however, most current data involve thoroughbreds in race training. the prevalence of squamous ulceration in horses in race training varies from % to % - and can be as high as % when limited to animals actively racing. in a survey of active show horses, % had gastric ulceration, with only horse having ulceration of the glandular fundus. in one large retrospective study ( adult horses from to ) evaluating incidence of gastric ulceration identified at necropsy, an overall prevalence of . % was found. the highest prevalence was found in thoroughbreds (including arabians) and standardbred trotters, and cold-blooded horses were affected significantly less. lesions were located most commonly in the squamous mucosa along the margo plicatus, followed by the glandular body, proximal squamous mucosa, and antrum. many studies investigating prevalence of gastric ulceration do not differentiate between squamous and glandular lesions or evaluate only squamous disease. in a recent study in which the gastric antrum and pylorus were evaluated in horses in a hospital setting, % had antral or pyloric erosions or ulcerations, % had squamous mucosal lesions, and % had lesions involving the glandular body. a correlation between the presence or severity of squamous disease and antral/pyloric disease was not identified. the reported prevalence of gastric ulceration in foals varies from % to %. [ ] [ ] [ ] an imbalance between inciting and protective factors in the mucosal environment can result in ulcer formation. , the major intrinsic factors promoting ulcer formation include hydrochloric acid, bile acids, and pepsin, with hydrochloric acid being the predominant factor. various intrinsic factors protect against ulcer formation such as the mucus-bicarbonate layer, maintenance of adequate mucosal blood flow, mucosal prostaglandin e and epidermal growth factor production, and gastroduodenal motility. in human beings, extrinsic ulcerogenic factors include nonsteroidal antiinflammatory drugs, helicobacter pylori, stress, changes in diet, or gastrointestinal disorders, especially those resulting in delayed gastric emptying. in human neonates, physiologic stress associated with a major primary illness seems to be associated strongly with gastric ulcers. many of the other factors mentioned previously are believed to be important in horses, but clear evidence of an infectious agent has not yet been identified in horses or foals with egus. , recently, the possibility of helicobacter infection in horses has reemerged with the identification of polymerase chain reaction products from urel, a protongated urea channel unique to gastric-dwelling helicobacter species, in the squamous epithelium of three horses, two of which had squamous erosions. the specific factors involved in injury and the protective mechanisms vary between regions of the proximal gastrointestinal tract. the pathophysiology of squamous mucosal ulceration in the horse appears similar to that in gastroesophageal reflux disease in human beings and ulceration of the nonglandular mucosa in pigs. excess acid exposure is the predominant mechanism responsible for squamous mucosal ulceration, although many details remain unclear. hydrochloric acid is secreted by parietal cells in the gastric glands via a hydrogen-potassium adenosine triphosphatase (h + ,k + -atpase) pump on the luminal side. horses secrete acid continuously, and measured ph of equine gastric contents varies from less than to greater than depending on the dietary state of the horse (fed or fasted). , a protocol of repeated -hour periods of fasting and feeding has been shown to induce squamous erosion and ulceration. because this protocol results in periods of prolonged gastric acidity (ph < . ) and because concurrent administration of the histamine (h ) receptor antagonist ranitidine reduces lesion severity, the protocol supports the role of acid exposure in the pathogenesis of squamous ulcer disease. several peptides can stimulate or inhibit parietal cell secretion of acid. the predominant stimuli for hydrochloric acid secretion are gastrin, histamine, and acetylcholine via the vagus nerve. g cells release gastrin within the antral mucosa, whereas mast cells and enterochromaffin-like cells release histamine in the gastric gland. histamine binds to type receptors on the parietal cell membrane, causing an increase in cyclic adenosine monophosphate and resulting in phosphorylation of enzymes that activate the proton pump. gastrin and acetylcholine can act via calcium-mediated intracellular pathways and also stimulate histamine release directly. isolated equine parietal cells respond maximally to histamine stimulation and only minimally to carbachol and pentagastrin. gastrin release is controlled primarily by gastrin-releasing peptide, which is stimulated by gastric distention and increased luminal ph, but the interaction between gastrin and histamine has not been elucidated fully in the horse. somatostatin, released by fundic and antral d cells, is the primary inhibitor of gastric acid secretion by parietal cells. the inhibitory effect of somatostatin is primarily paracrine, but plasma levels of somatostatin negatively correlate with gastric luminal acidity. epidermal growth factor, a peptide produced in saliva, also inhibits gastric acid secretion. foals can produce significant amounts of gastric acid by the second day of life, with consistent periods of acidity (ph < . ) in clinically normal animals. , in one study, foals tended to have a high gastric ph at day of age, but in a study of critically ill foals, some foals demonstrated periods of gastric acidity on the first day of life. suckling was associated with an immediate rise in gastric ph, whereas periods of rest in which foals did not suck for more than minutes were associated with prolonged periods of acidity. whereas premature human infants are capable of gastric acid production at weeks of gestation, only of premature foals demonstrated an acidic ph recording in a study of gastric ph profiles in critically ill foals. however, multiple factors likely were involved in critically ill foals of this study, and the true ontogeny of gastric acid production in foals is currently unknown. equine squamous mucosa is thin at birth but becomes hyperplastic and parakeratotic within days. the parallel between decreasing ph and proliferation of squamous epithelium correlates with that observed in other species. the combination of a thin gastric epithelium with a high acid output may leave neonatal foals susceptible to ulcer formation at a young age. in addition, one must remember the difference in normal appearance of the squamous mucosa when interpreting gastric endoscopy in a neonatal population. in esophageal squamous mucosa, intercellular tight junctions and bicarbonate secretion are the major factors involved in protection against acid injury in other species, although squamous bicarbonate secretion had not been documented in the horse. [ ] [ ] [ ] the principal barrier is a glycoconjugate substance secreted by cells in the stratum spinosum, with a contribution from the tight junctions in the stratum corneum. this barrier function is considered weak at best, and thus a functioning lower esophageal sphincter, normal salivary flow, and salivary mucins contribute to the prevention of acid injury in human gastroesophageal reflux disease. in horses a mechanical barrier like the lower esophageal sphincter is not available to protect the gastric squamous mucosa from acid exposure. the normal gastric fill line rests just below the cardia, so only the squamous mucosa along the lesser curvature adjacent to the margo plicatus should receive exposure to acidic gastric contents regularly. not surprisingly, this correlates with the most common location of squamous mucosal ulceration. bile salts and pepsin have been implicated as contributing factors to ulcer disease in many species. in rabbit esophageal mucosa, bile salt absorption occurs and is correlated directly with mucosal barrier disruption. the unconjugated bile salts cholate and deoxycholate have a pk a (negative logarithm of the ionization constant of an acid) of and . , respectively, and therefore cannot remain in solution and cause mucosal damage in the presence of acid. alternatively, the conjugated bile salt taurocholate (pk a . ) can cause mucosal injury in the ionized salt form at ph or the un-ionized acid form at ph to . in the pig, bile salts or acid alone cause squamous mucosal damage, whereas a combination of the two result in extensive damage in vitro. in the horse a similar synergistically damaging effect was found with the addition of bile salts and acid (ph . ) to stratified squamous mucosa in vitro in one study. in addition, the investigators were able to document levels of bile salts and acid sufficient to cause mucosal damage in gastric contents within hours of feed deprivation. this is not surprising, given that duodenogastric reflux occurs normally in the horse. in a separate in vitro study of equine squamous mucosa, prolonged exposure to acid alone (ph . ) had a damaging effect, and synergism with exposure to a combination of acid and pepsin or taurocholate was not found. the lack of synergism likely is caused by the lower ph used in this study and stresses the importance of acid exposure in squamous ulcer disease. pepsinogens are secreted primarily by chief cells, although secretion by neck cells, cardiac glands, and antral pyloric glands also occurs. in an acidic environment (ph < . ), pepsinogen is converted to the active pepsin. although the proteolytic activity of pepsin normally is directed toward dietary protein, it also can act on the gastric mucosa. thus acid remains the major contributing factor to squamous mucosal damage, although other factors such as pepsin and bile salts may play an important role as well in the initiation or perpetuation of disease. several mechanisms help protect the glandular mucosa from acid injury. the mucus-bicarbonate layer serves to titrate h + ion from the gastric lumen to co and h o. cellular restitution and prostaglandins of the e series, which enhance mucosal blood flow and secretion of mucus and bicarbonate in the glandular mucosa have not been documented in squamous epithelium. , of these mechanisms, mucosal blood flow is likely the most important contributor to overall gastric mucosal health. nitric oxide is a key regulator of mucosal blood flow and prostaglandin synthesis and thus may play a role in mucosal protection. dietary factors also have been implicated in ulcer disease. horses in race training have a high incidence of gastric ulceration and frequently are fed high-concentrate, low-roughage diets. in one study, higher volatile fatty acid (acetic, propionic, and isovaleric acid) concentrations, higher gastric juice ph, and lower number and severity of nonglandular ulceration were documented after feeding an alfalfa hay-grain diet compared with a bromegrass hay diet. however, many factors differed between the diets, such as digestible energy, bulk, crude protein, and mineral content (especially calcium). thus dietary factors represent an important area of further investigation in the pathophysiology of egus, particularly squamous ulceration. the pathophysiologic correlation between exercise and squamous ulcer disease has not yet been defined despite the high prevalence of ulceration in performance horses. preliminary work suggests that gastric compression occurs during treadmill exercise, presumably because of an increase in intraabdominal pressure. such contracture could result in increased acid exposure to the squamous mucosa by raising the fill line of gastric contents. further studies in this laboratory have provided support for this theory by demonstrating a high ph in the proximal stomach, immediately distal to the lower esophageal sphincter, during resting conditions that decreases during treadmill exercise (m. lorenzo-figueras and a.m. merritt, personal communication, ) . risk factors associated with gastric ulceration include gender and age, and the reported prevalence of gastric ulcers has increased over time. in one study, ulcers were found more commonly in stallions, and the prevalence of gastric ulceration decreased with age, independent of gender, although this trend was only significant in the population of standardbred trotters. interestingly, the frequency of gastric ulceration increased from less than % before to approximately % after . in a study of thoroughbred horses in race training, an increase in squamous ulcer severity was noted in horses years old or older and in those horses that had raced. in the same study, severity of glandular lesions did not change between examinations, and age (> years) was the only factor associated with glandular lesion severity. several studies have failed to document a correlation between nonsteroidal antiinflammatory drug (nsaid) administration and naturally occurring ulcer disease. , , , , however, nsaid administration is a well-known cause of gastric ulceration under experimental conditions. [ ] [ ] [ ] [ ] [ ] nsaid-related ulceration typically is described as predominantly glandular, although nonglandular ulceration also can occur by a mechanism that has not yet been characterized fully. nsaids cause a decrease in prostaglandin e synthesis because of inhibition of the cyclooxygenase pathway. therefore a resultant decrease in glandular mucosal protection, most notably via decreased mucosal blood flow and mucus production, is the most likely mechanism of action. in one study, however, phenylbutazone administration resulted in ulceration of the glandular mucosa at the pyloric antrum but did not alter mucosal prostaglandin e concentration significantly. clinical signs typically associated with gastric ulceration in foals include poor appetite, diarrhea, and colic. many foals probably never exhibit clinical signs, and some do not exhibit clinical signs until ulceration is severe or fatal perforation has occurred. glandular ulceration typically is considered the most clinically significant type of disease in this population. the physiologic stress of a concurrent illness has been associated with gastric ulceration in foals. retrospectively, ( %) of foals up to days of age with a clinical disorder were found to have lesions in the gastric glandular mucosa, and prospectively ( %) of foals up to days of age with a clinical disorder had glandular ulceration. by contrast, only % to % of clinically normal foals examined in endoscopic surveys had lesions observed in the gastric glandular mucosa. , critically ill neonatal foals can have a greatly different ph profile compared with that in clinically normal foals, potentially because of alterations in gastric motility and acid secretion. gastric ulceration was not identified in any animals at necropsy in that study; however, ulceration has been documented in a similar population. thus factors other than acid exposure, most notably mucosal blood flow, may play an important role in the stressrelated ulceration in neonates. subjectively, gastric ulceration and rupture in the hospitalized neonatal population occurs less commonly now than in previous reports. advances in overall neonatal care, especially supportive care, likely have contributed to this decline. in suckling foals less than days old, lesions typically originate in the squamous mucosa adjacent to the margo plicatus along the greater curvature. such lesions can occur in foals as young as days of age and have been observed in % of foals less than days old. histologic examination of these lesions has revealed disruption of the epithelial layers of the mucosa and a neutrophilic infiltration. another phenomenon that occurs in young foals is the shedding, or desquamation, of squamous epithelium, which appears as flakes or sheets of epithelium. desquamation occurs without ulceration in up to % of foals less than days of age, and this process typically is not associated with clinical signs. , , in older foals, lesions become more prevalent in the squamous mucosa, particularly along the lesser curvature. lesions also are found in the squamous mucosa of the fundus and adjacent to the margo plicatus. these lesions can be severe and often are associated with clinical signs such as diarrhea, poor appetite, and poor growth and body condition. diarrhea is the most frequent sign in symptomatic foals with squamous mucosal lesions and is associated with more diffuse erosion or ulceration of the squamous mucosa than that which occurs in asymptomatic foals. in some foals, poor growth, rough hair coat, a potbelly appearance, or all of those occur along with moderate to severe squamous mucosal ulceration. in horses with severe or diffuse squamous ulceration, bruxism or colic may occur. gastroduodenal ulcer disease occurs almost exclusively in suckling and early weanling foals. clinical signs of duodenal ulceration are similar to those described for gastric ulceration (bruxism, colic, salivation, diarrhea), but the consequences are often more severe. lesions occur primarily in the proximal duodenum and range from diffuse inflammation to severe ulceration. foals with duodenal ulceration often have delayed gastric emptying and may have gastroesophageal reflux. complications can include gastric or duodenal rupture, duodenal stricture, and ascending cholangitis. severe squamous and esophageal ulceration and aspiration pneumonia can occur following gastroesophageal reflux. , [ ] [ ] [ ] [ ] the gastroduodenal ulcer disease syndrome can occur in outbreaks and most commonly is identified in intensive breeding operations. the cause of duodenal lesions in foals is not known. one theory is that the problem begins with diffuse duodenal inflammation that can coalesce down to a focal area of ulceration (g.d. lester and a.m. merritt, personal communication, ) . a temporal relationship between gastroduodenal ulcer disease and rotaviral diarrhea has been suggested, but an infectious cause remains unproven. although lesion location and severity associated with rotaviral infection varies among species, duodenal ulceration has not been reported. clinical signs attributable to egus in older horses vary and classically include anorexia and chronic or intermittent colic of varying severity. many horses with endoscopic evidence of disease may appear to be clinically normal or have vague signs that include decreased consumption of concentrates, postprandial episodes of colic, poor performance or failure to train up to expectations, poorquality hair coat, and decreased condition or failure to thrive. diarrhea typically is not associated with gastric ulceration in adult horses, although ulceration can occur concurrently with other causes of diarrhea. horses actively racing are more likely to have squamous ulceration than those solely in training. lesions occur predominantly in the squamous mucosa, particularly adjacent to the margo plicatus ( figure . - ) . in more severe cases, lesions can extend dorsally into the squamous fundus. clinically relevant lesions typically affect a greater portion of the squamous mucosa and can be deep enough to cause bleeding. however, bleeding from ulcers in the gastric squamous mucosa typically is not associated with anemia or hypoproteinemia. according to a recent study, the incidence of glandular lesions, particularly within the pyloric region, may be higher than previously reported, which emphasizes the importance of a thorough endoscopic examination and proper documentation of lesion location when reporting or discussing egus, especially the differentiation between squamous and glandular disease. although one may suspect a diagnosis of egus based on clinical signs and response to treatment, the only current method of confirmation is via gastroendoscopy, which one can perform easily in the standing horse or foal with mild sedation. in adult horses a -m endoscope allows for visual inspection of the entire stomach, pylorus, and proximal duodenum. shorter scopes permit examination of the gastric body and fundus, but not the pyloric antrum in most cases. one should use an endoscope with a maximum external diameter of mm for neonatal foals. numerous scoring systems for lesion severity have been described, but a recent consensus has been published by the equine gastric ulcer council (table . - ). duodenal ulceration can be difficult to confirm. duodenoscopy is the most specific means of diagnosis, although the procedure is more difficult than gastroscopy. additionally, an endoscope at least cm in length is needed for foals up to to months old, and a longer endoscope usually is required for older animals. diffuse reddening or inflammation may be the only recognizable lesion in cases of early duodenal disease. excessive enterogastric reflux of bile through the pylorus suggests duodenal dysfunction. however, the pylorus frequently appears open, and some degree of enterogastric reflux is common under normal conditions. ulceration at the pylorus or pyloric antrum also suggests the presence of a duodenal lesion. if one can perform gastroendoscopy, but not duodenoscopy, the severity of lesions, particularly in the glandular mucosa and in the squamous mucosa of the lesser curvature dorsal to the pyloric antrum, usually will be severe when duodenal ulcers are present. multiple pharmacologic treatments have been suggested for treating egus. because acid has been implicated as the most important pathophysiologic component of squamous ulcer disease, most antiulcer therapy centers on suppression or neutralization of gastric acid. severity and location of gastric lesions and severity and duration of clinical signs, as well as medication cost, can play a role in the therapeutic management of egus (table . - ). if gastroendoscopy is unavailable, some guidelines to therapy can be used, but the efficacy of the treatment is based on clinical signs, which are often vague or nonspecific. signs of colic or diarrhea that result from gastric ulcers often resolve within hours. one can note improvements in appetite, bodily condition, and attitude within to weeks. if one does not observe improvement in clinical signs, treatment has not been effective or gastric ulceration was not the primary problem. the principal therapeutic options for ulcer treatment include h antagonists (cimetidine, ranitidine, famotidine, nizatidine), proton pump blockers (omeprazole, pantoprazole, rabeprazole, esomeprazole), the mucosal adherent sucralfate, and antacids. the h antagonists suppress hydrochloric acid secretion through competitive inhibition of the parietal cell histamine receptor that can be overcome partially with exogenous pentagastrin. use of h antagonists has been successful in raising gastric ph and resolving gastric lesions in foals and adult horses. , , clinical and experimental evidence has demonstrated greater individual variability with lower dosages of h antagonists. thus dosage recommendations are based on levels necessary to increase gastric ph and promote ulcer healing in a majority of horses. commonly recommended dosages are to mg/kg orally every hours or . mg/kg intravenously every hours for cimetidine and . mg/kg orally every hours or . to mg/kg intravenously every hours for ranitidine. famotidine has been used less extensively in the horse, but a dose of to mg/kg/day has been recommended. because gastric perforation caused by glandular ulcer disease has been reported in hospitalized neonates, many clinicians routinely use prophylactic antiulcer therapy in this population. although clinically normal foals respond predictably to ranitidine, sick neonates have shown variability in ph response to intravenously administered ranitidine, with a much shorter duration of action and in some cases no noticeable response. thus currently used dosing schedules for hospitalized foals may be inadequate. because some critically ill foals have a predominantly alkaline gastric ph profile and because gastric acidity may be protective against bacterial translocation in neonates, the need for prophylactic ulcer therapy is controversial. in critically ill human neonates, intravenous administration of ranitidine raises gastric ph and gastric bacterial colonization but does not increase the risk of sepsis. in a retrospective study of hospitalized foals less than days of age, no difference in the frequency of gastric ulceration at necropsy was found between those foals that received prophylactic treatment for gastric ulcers and those that did not. because the study was retrospective, specific details regarding lesion location and severity were not available; however, none of the foals in the study died because of gastric ulcer disease. h antagonist therapy should continue for to days, but complete ulcer healing may take to days. if an animal is kept in race training during therapy, clinical signs may resolve but the lesions may not. currently, cimetidine and ranitidine are available in injectable, tablet, and liquid forms. famotidine and nizatidine are available in tablets. proton pump inhibitors block secretion of h + at the parietal cell membrane by irreversibly binding to the h + ,k + -atpase proton pump of the cell. these agents have a prolonged antisecretory effect, which allows for once-daily dosing. omeprazole, the first proton pump inhibitor to be developed, is the only currently approved agent for the treatment of egus. several studies have documented the safety of orally administered omeprazole in foals and adult horses. , omeprazole has demonstrated efficacy in the healing of nsaid-induced ulcers in horses and in naturally occurring cases of egus. , more importantly, omeprazole has been shown to eliminate or reduce the severity of gastric ulcers in thoroughbreds maintained in race training. the available equine preparation of omeprazole (gastrogard, merial, ltd., duluth, georgia) is recommended at a dose of mg/kg orally every hours. initial reports suggested that to days of omeprazole therapy were necessary to achieve maximum acid suppression; however, an increase in gastric ph and a decrease in acid output are evident to hours after omeprazole paste administration. after initial treatment ( days), treatment with or mg/kg every hours has been shown to decrease or prevent the recurrence of disease in animals maintained in training. the powder form of omeprazole degrades rapidly in an acidic environment, thus one must use an enteric-coated capsule (as used in the human preparation) or a specially formulated paste (such as gastrogard) to allow delivery of the active drug to the small intestine for absorption. many compounding pharmacies prepare omeprazole in liquid or paste formulation for use in horses, but their efficacy has not been evaluated to date. other proton pump inhibitors have been developed recently for use in human beings, including rabeprazole, lansoprazole, esomeprazole, and pantoprazole. in gastroesophageal reflux disease treatment in human beings, esomeprazole has demonstrated a higher rate of healing at and weeks compared with omeprazole, but rabeprazole, lansoprazole, and pantoprazole have similar efficacy. an intravenous formulation of pantoprazole recently became available commercially and may prove beneficial for patients in need of antiulcer therapy that cannot be treated orally. research regarding the pharmacokinetics and efficacy of other proton pump inhibitors in horses is not currently available. sucralfate is effective in treating peptic ulcers and preventing stress-induced ulcers in human beings. the mechanism of action likely involves adherence to ulcerated mucosa, stimulation of mucus secretion, enhanced prostaglandin e synthesis, and increased concentration of growth factor at the site of ulceration, although the prostaglandin effects may not play an important role in ulcer healing. these are factors relevant to glandular mucosa, and the efficacy of sucralfate in treating ulcers in the equine gastric squamous mucosa remains undetermined. sucralfate may be effective in preventing stress-induced ulcers in neonatal foals, because these occur in the glandular mucosa, although no clinical evidence directly supports this concept. in human beings, sucralfate provides protection against stress-induced ulcers with a decreased risk of pathogenic gastric colonization. one should give sucralfate at a dosage of to mg/kg every to hours. the efficacy of sucralfate in an alkaline ph is controversial but appears likely. [ ] [ ] [ ] moreover, at the time of administration of an section . diseases of the stomach h antagonist, the gastric ph likely will have returned to an acidic ph since the last dosage and will remain so for to minutes depending on the route of administration; thus one likely can administer the agents simultaneously if so desired. the use of antacids to treat gastric ulcers has not been examined critically in the horse. research in horses has shown that g aluminum hydroxide per g magnesium hydroxide results in an increase in gastric ph above for approximately hours. thus although antacids may be useful for treating ulcers in horses, a dose of approximately to ml at least every hours is necessary for a standard adult horse. the use of synthetic prostaglandin e analogs, such as misoprostol, has been effective in treating gastric and duodenal ulcers in human beings, and the proposed mechanism of action involves inhibition of gastric acid secretion and mucosal cytoprotection. frequently reported adverse effects of intestinal cramping and diarrhea in human beings have precluded the use of misoprostol in horses. one should consider prokinetic drugs in foals with duodenal disease and gastroesophageal reflux and when one suspects delayed gastric emptying without a physical obstruction. the cholinergic drug bethanechol has been shown to increase the rate of gastric emptying in horses. in cases of acute gastric atony, bethanechol . to . mg/kg administered subcutaneously every to hours has been effective in promoting gastric motility and emptying, followed by oral maintenance dosages of . to . mg/kg to times daily. adverse effects can include diarrhea, inappetance, salivation, and colic, but at the dosages stated, adverse effects have been infrequent and mild. a complete review of ileus and prokinetic therapy is available in chapter . . for foals with severe gastroduodenal ulcer disease that have developed duodenal stricture, surgical therapy is necessary. , these animals require a serious financial commitment because intensive perioperative medical therapy is critical for a successful outcome. even with surgical therapy, these foals often warrant a guarded prognosis. pyloric stenosis is a structural resistance to gastric outflow. congenital pyloric stenosis has been reported in foals and one yearling and results from hypertrophy of the pyloric musculature. [ ] [ ] [ ] acquired pyloric stenosis can result from neoplasia or duodenal ulceration. [ ] [ ] [ ] [ ] clinical signs depend on the degree of obstruction and include abdominal pain, salivation, and teeth grinding. complete or near complete obstruction can result in gastric reflux and reflux esophagitis. in foals with congenital pyloric hypertrophy, clinical signs may begin with the consumption of solid feed. in foals one can make a presumptive diagnosis via gastric endoscopy and radiography (plain and contrast studies). depending on the cause and severity of disease, gastric endoscopy may provide a presumptive diagnosis in the adult horse. measurement of gastric emptying can aid the diagnosis. several methods of measurement are currently available, including nuclear scintigraphy, acetaminophen absorption, and postconsumption [ c] octanoic acid blood or breath testing. , , exploratory laparotomy shows a distended stomach and thickened pylorus accompanied by a relatively empty intestinal tract. if complete obstruction is not present, medical therapy with a prokinetic such as bethanechol can increase the rate of gastric emptying. phenylbutazone and cisapride also have been shown to attenuate the delay in gastric emptying caused by endotoxin administration. , surgical repair is necessary for definitive treatment of complete or near-complete obstruction and consists of gastroenterostomy or pyloroplasty. , gastric dilation can be classified as primary, secondary, or idiopathic. causes of primary gastric dilation include gastric impaction, grain engorgement, excessive water intake after exercise, aerophagia, and parasitism. , secondary gastric dilation occurs more commonly and can result from primary intestinal ileus or small or large intestinal obstruction. time to development of gastric reflux is proportional to the distance to the intestinal segment involved, with duodenal obstruction resulting in reflux within hours. clinical signs of gastric dilation include those associated with acute colic and in severe cases, ingesta appearing at the nares. associated laboratory abnormalities include hemoconcentration, hypokalemia, and hypochloremia. the most common reported cause of gastric rupture in horses varies between reports. in a retrospective study of horses, gastric rupture occurred most commonly as a secondary phenomenon ( %), usually because of small intestinal obstruction, with primary gastric dilation and idiopathic rupture occurring almost equally ( % and %, respectively). in another retrospective study of horses in combination with a search of the veterinary medical database (vmdb), % of the gastric rupture cases were classified as idiopathic. risk factors for gastric rupture include feeding grass hay, not feeding grain, gelding, and a nonautomatic water source. , nasogastric intubation does not preclude the possibility of gastric rupture, and the amount of reflux obtained before rupture varies greatly. because these reports were retrospective, one cannot rule out confounding factors with certainty. regardless of the initiating cause, gastric rupture usually occurs along the greater curvature. in horses with rupture caused by gastric dilation, tears in the seromuscular layer are frequently larger than the corresponding tears in the mucosal layer, indicating that the seromuscularis likely weakens and tears before the mucosa. , in contrast, horses with gastric rupture following gastric ulceration usually demonstrate full-thickness tears of equal size in all layers. gastric rupture is usually fatal because of widespread contamination of the peritoneal cavity, septic peritonitis, and septic shock. initial clinical signs vary with the primary disease; however, when rupture occurs, a previously painful animal can exhibit signs of relief. subsequent signs are consistent with peritonitis and shock, including tachypnea, tachycardia, sweating, and muscle fasciculations. surgical repair is thus limited but has been reported for partial-thickness tears, and in one case of a combined tear of the mucosa and muscularis with only a focal serosal tear, a full-thickness repair was performed with a favorable outcome. gastric impaction can result in acute or chronic signs of colic in the horse. although a specific cause is not always evident, ingestion of coarse roughage (straw bedding, poor-quality forage), foreign objects (rubber fencing material), and feed that may swell after ingestion or improper mastication (persimmon seeds, mesquite beans, wheat, barley, sugar beet pulp) have been implicated. possible predisposing factors include poor dentition, poor mastication and rapid consumption of feedstuffs, and inadequate water consumption. clinical signs can vary from anorexia and weight loss to those consistent with severe abdominal pain. in severe cases, spontaneous reflux may occur, with gastric contents visible at the nares. in cases of acute severe abdominal pain, one often makes a diagnosis during exploratory celiotomy. in animals not exhibiting signs of colic warranting surgical intervention, an endoscopic finding of a full stomach after a normally adequate fast ( to hours) often can confirm the diagnosis. abdominal radiographs are reserved for smaller horses and ponies. in addition to pain management, specific treatment consists of gastric lavage via nasogastric intubation or massage and injection of fluid to soften the impaction during laparotomy. [ ] [ ] [ ] nonulcerative gastritis rarely occurs in the horse; however, a single case of emphysematous gastritis caused by clostridium perfringens has been reported. mimic that of a strangulating or nonstrangulating small intestinal obstruction, so distinguishing between the two syndromes is important because appropriate treatment of small intestinal obstruction usually requires surgical intervention. studies suggest that the survival rate for horses with dpj that endured surgical exploratory laparotomy was poor compared with those treated medically, although differences in disease severity may have accounted for the results in these early reports. , the clinical syndrome of dpj was well described in the s, and although recognized by its classical presentation, varying degrees of focal intestinal and systemic illness may occur. - dpj usually occurs alone but can occur along with gastritis, ileitis, typhlitis, and or colitis. typical pathologic findings in horses with dpj include involvement of the duodenum and usually the proximal jejunum. the ileum and large colon usually are determined to be grossly normal. gastric distention is a common finding and is thought to be caused by hypersecretory mechanisms in the proximal small intestine and a functional ileus of affected enteric segments. the small intestine may be to cm in diameter because of fluid distention with malodorous, red to brown-red intralumenal fluid accumulation. duodenal (and jejunal) serosal surfaces may have varying degrees and distribution of bright-red to dark-red petechial and ecchymotic hemorrhages and yellow to white streaks. the enteric mucosal surfaces are usually hyperemic and have varying degrees of petechiation and ulceration. microscopically, the most severe lesions have been located in the duodenum and proximal jejunum but may extend proximally to the gastric mucosa and aborally to the large intestinal mucosa and submucosa. microscopic lesions consist of varying degrees of mucosal and submucosal hyperemia and edema. more severe lesions include villus degeneration with necrosis and more severely, sloughing of villous epithelium. the lamina propria, mucosa, and submucosa may have varying degrees of granulocyte infiltration (predominantly neutrophils), and the muscular layers and serosal surfaces contain small hemorrhages. proximal small intestinal serosal fibrinopurulent exudate is a common finding in the more severe cases; therefore the term hemorrhagic fibrinonecrotic duodenitis-proximal jejunitis has been suggested as a more descriptive name for this syndrome. horses with dpj often have evidence of multiple organ involvement such as hepatic changes including congestion and varying degrees of biliary duct hyperplasia. additional systemic involvement likely is caused by endotoxin absorption, metabolic imbalances such as acidemia, and circulatory changes. the cause of this syndrome remains an enigma (much like the cause of other inflammatory conditions affecting the intestinal tract). several microorganisms have been implicated as playing a role in triggering dpj, including clostridium spp., salmonella spp., and some mycotoxins, but efforts to reproduce the syndrome experimentally have been futile. a recent dietary change with an abrupt increase in dietary concentrate level has been suggested to predispose a horse to developing dpj because of intraluminal microbial imbalances. two intracellular processes control intestinal secretion, the cyclic nucleotide (cyclic adenosine monophosphate and cyclic guanosine monophosphate) and the calcium systems. agents (inflammatory mediators, microorganisms, toxic agents) can activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e ) or guanyl cyclase (bacterial enterotoxins) and induce increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate, respectively. this reaction causes phosphorylation of specific protein kinases, which induce the actual mucosal membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotidedependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. calcium may act through calmodulin, which then can activate membrane-phosphorylating protein kinases. the net effect is increased movement of sodium and chloride into the mucosal cell from the interstitium, with secretion of sodium and chloride into the intestinal lumen. water follows the directional flux of sodium and chloride through highly permeable intercellular spaces. several bacterial toxins and endogenous mediators can cause active secretion and contribute to a synergistic mucosal secretory response. passive secretion of protein-rich fluid into the lumen occurs following damage to the mucosal epithelium, capillary endothelium, and submucosal inflammation in the proximal small intestine. the clinically relevant events that result from active and passive fluid secretion are proximal small intestinal distention and nasogastric reflux, dehydration, and circulatory shock. the concentration of protein in the peritoneal fluid from horses with dpj is usually higher than in horses with small intestinal obstruction. a disproportionate increase in total protein concentration relative to nucleated cell count occurs probably by leakage of blood or plasma into the peritoneal cavity without a significant stimulus for leukocyte chemotaxis. suggested mechanisms for increased abdominal fluid protein concentration include serositis associated with inflamed intestine and small intestinal distention causing passive congestion and increased capillary hydrostatic pressure of visceral peritoneal vessels. small intestinal ileus is another hallmark sign of dpj and the pathophysiology is complicated, involving primary and secondary dysfunction of the central, autonomic, and enteric nervous systems and their purported roles in governing intestinal motility. primary role-players in dpj-associated ileus include peritoneal inflammation, inflammatory cell migration/activation within the muscularis, small intestinal mechanical distention, and effects of endotoxin absorption. the use of prokinetic agents for treating ileus and gastric/small intestinal distention in horses with dpj is becoming more common, but veterinarians should realize that a potential restriction on their use is the need for normal intestinal integrity. in spite of that, one may use motility modifiers judiciously. the veterinarian has the challenge of differentiating horses with dpj from horses with small intestinal obstructive lesions so as to avoid surgical intervention (table . - ). horses with dpj typically show signs of acute abdominal pain initially, and then after gastric decompression, volume replacement, and analgesic therapy, the colic signs subside, but signs of lethargy and malaise become more apparent. in contrast, horses with obstructive lesions of the small intestine usually show signs of abdominal pain until the affected viscus is repaired via surgical intervention or the viscus ruptures. another differentiating characteristic is the large volume (> to l with each decompressive effort) of nasogastric reflux that is often malodorous and orange-brown or red-brown. dpj-affected horses have moderate to severe small intestinal distention palpated on rectal examination, temperature of . °to . °c ( . °to . °f), dehydration, brick-red mucous membranes, lethargy and absent borborygmi, prolonged capillary refill time, tachycardia (> beats/min), and tachypnea. although the signs of abdominal pain usually resolve after gastric decompression, most horses remain severely lethargic. without periodic removal of the fluid that accumulates in the proximal intestinal tract, signs of abdominal pain usually recur. horses with dpj often require gastric decompression at -hour intervals, with to l of fluid recovered each time. nasogastric tubes left in place for long periods of time cause varying degrees of pharyngitis, laryngitis, and esophagitis. typical clinical laboratory findings include an increased packed cell volume and total plasma protein reflective of volume depletion, a metabolic acidosis (with elevated anion gap) in longstanding or severe cases, an increased peritoneal fluid protein concentration (often > . g/dl), and a mild to moderate elevation of the peritoneal white blood cell count, although the count usually is less than , cells per microliter. , the peritoneal fluid is usually yellow and turbid, but in severe cases diapedesis occurs resulting in a serosanguinous color. the white blood cell count in the peripheral blood may be normal, decreased, or increased. in addition, hyponatremia, hypochloremia, hypokalemia, and acid-base alterations (elevated anion gap) are often evident. the loss of enteric bicarbonate through evacuation of enterogastric reflux and poor tissue perfusion from hypovolemia can lead to metabolic acidosis. one makes a definitive diagnosis of dpj in most cases by gross examination of the duodenum and proximal jejunum at surgery or at necropsy. some equine practitioners have observed an apparent geographic relationship in the incidence and severity of the syndrome, with more cases occurring in the southeastern united states. horses with dpj appear to share a common characteristic clinical presentation, and the mechanisms leading to electrolyte imbalances, fluid loss, ileus, and endotoxemia and septicemia are similar. treatment regimens are supportive and aim at plasma volume replacement (usually in the form of crystalloid fluid replacement), analgesia and antiinflammatory therapy, gastric decompression, antiendotoxin therapy, antimicrobial therapy if indicated, nutritional support, and nursing care. one should institute aggressive intravenous polyionic fluid therapy immediately in a horse with dpj. one should calculate the total fluid deficit based on clinical assessment of dehydration (e.g., for % or moderate dehydration, . × kg body mass = l) and should administer replacement fluids rapidly (up to to l per hour per -kg adult horse). administering intravenous hypertonic saline ( %) may be useful to treat hypovolemic shock in horses with severe circulatory shock. the use of to l of hypertonic saline ( % nacl) improved systemic blood pressure and cardiac output in horses with hemorrhagic shock and in a model of equine endotoxemia. if one chooses this treatment option, intravenous administration of replacement isotonic fluids must follow immediately to maintain tissue integrity. one should not allow horses with significant volumes of gastric reflux to ingest foodstuffs or liquids orally. once one has administered replacement fluids and the horse is well hydrated, one should administer maintenance fluid amounts, which may be as high as ml/kg/day. unfortunately, the intravenous fluid therapy itself may accelerate the flux of fluid from the vasculature into the intestinal lumen because of a reduction in intravascular oncotic pressure and an increased capillary perfusion pressure, which can result in an increased volume of gastrointestinal reflux. however, the veterinarian should not consider reducing the volume of intravenous fluid therapy because excessive fluid losses continue to occur. one should monitor plasma protein concentration, overall hydration, and the volume of reflux and then determine the rate of intravenous fluid administration. during the initial hours of therapy, even aggressive intravenous fluid administration results in only moderate clinical improvement. the clinical response, as evidenced by improved hydration status, decreased nasogastric reflux, improved attitude, and improvement in values reflecting kidney function (decreased blood urea nitrogen and creatinine), correlates with improvement of intestinal damage. horses with dpj that continue to reflux large volumes of enterogastric fluid frequently for more than to hours most likely will experience protein loss from the inflamed and disrupted intestinal mucosal barrier and from systemic protein catabolism. decreased colloid oncotic pressure leads to decreased effective circulating fluid volume and edema. total plasma protein may decline to below g/dl and the albumin may decrease to below . g/dl. fresh or thawed frozen plasma is ideal for replacement of functional proteins. one should consider treatment with intravenous plasma therapy or a combination of plasma and synthetic colloid (e.g., synthetic amylopectin) as soon as one sees evidence of a consistent decline in total plasma protein or albumin (< . g/dl) or if the horse is developing dependent edema. fresh plasma (preferred) or fresh frozen plasma is the treatment of choice if coagulation disorders accompany protein loss. an average-size horse ( kg) requires to l of plasma (albumin . g/dl) or synthetic colloid to improve plasma oncotic pressure. administration of additional aliquots of to l of a balanced colloidal solution may be necessary if the dpj crisis continues. in addition to albumin (the major colloid component), plasma contains other components that provide overall systemic support (e.g., fibronectins, complement inhibitors, elastase and proteinase inhibitors, antithrombin iii). one may administer a % solution of hydroxyethyl starch (hetastarch ( %), abbott laboratories, north chicago, illinois), a synthetic colloid, at to ml/kg. because of the large size of the starch molecules, this solution is an effective plasma volume expander, resulting in sustained dosedependent decreases in packed cell volume and plasma protein concentration with increased oncotic pressure. the cost of an appropriate amount of commercial plasma or synthetic colloid solution for treatment of adult horses with dpj may be prohibitive but can be life-saving. horses with enteritis frequently absorb large amounts of endotoxin from the disrupted intestinal mucosal barrier, therefore putting these horses at a high risk for laminitis. one should monitor digital pulses every to hours until systemic signs of enteritis have abated (fever, leukopenia, etc.). treatment to combat endotoxemia is critical, and several therapeutic approaches are available. choice of treatment options is based on severity of disease, renal function, hydration status, and economics. the reader is referred to chapter . for a thorough discussion of endotoxemia pathophysiology, treatment, and prevention. nonsteroidal antiinflammatory drugs are the most frequently used group of drugs for treatment of abdominal pain in horses (flunixin meglumine . mg/kg intravenously every hours or phenylbutazone . mg/kg orally or intravenously every hours). the clinician must weigh the benefit of the analgesic effect of nonsteroidal antiinflammatory drugs with the possibility of further damage to the intestine by potentially blocking the protective effects of intestinal mucosal prostaglandins. one should consider other classes of drugs for treating colic associated with dpj. butorphanol (torbugesic; an opioid analgesic) at . to . mg/kg with detomidine (dormosedan; an α-agonist) at . to . mg/kg given intramuscularly every to hours is a useful combination that has minimal effects on gastrointestinal motility. because clostridium spp. are suspected as a causative agent of dpj, penicillin often is administered to affected horses. however, one should consider broad-spectrum antimicrobial coverage for horses with dpj. one can add an aminoglycoside (gentamicin, amikacin) or thirdgeneration cephalosporin (ceftiofur [naxcel], upjohn co., kalamazoo, michigan) to the penicillin therapy, keeping in mind the potential adverse effects of these drugs on renal function. effective antisecretory medications targeting the equine small intestine have not been identified. one should consider the nutritional needs of horses with dpj. most horses have a total body protein loss from cachexia and a protein-losing enteropathy. total parenteral nutrition may be indicated in horses that remain anorectic for more than to days. parenterally administered solutions containing glucose, balanced amino acid solutions, lipid emulsions, balanced electrolyte and trace minerals, and vitamins have been administered to adult horses with small intestinal ileus or enterocolitis. based on a small number of horses, this therapy has proved promising in terms of minimizing protein losses and decreasing the duration of illness. providing for part of the nutritional requirements of the horse ( to , kcal/day) is possible with glucose-amino acid solutions, which are of moderate cost. one may suppose reasonably that providing nutritional support to an anorectic, severely ill horse will facilitate the healing process and even shorten the duration of illness. thus the overall cost of providing parenteral nutritional supplementation to horses with dpj may well be offset by quicker recovery and diminished requirements for other, expensive treatments. normal (healthy) intestine is necessary for optimum performance of prokinetic agents in horses. many motilitymodifying agents likely are ineffective in cases of dpj. however, some benefit may come of the judicious use of prokinetic agents in inflammatory conditions of the equine intestine, particularly if the agent provides additional effects such as analgesia. for example, lidocaine infusion has several actions that may be beneficial in the treatment of ileus, including suppression of primary afferent firing, antiinflammatory properties, an observed analgesic effect, and direct stimulation of smooth muscle. an infusion dose of to mg/min over to hours has been recommended. the reader is referred to chapters . and . for a complete description of motility modifying agents. medical therapy is sufficient in most cases of dpj, but in those cases in which the horse continues to produce copious enterogastric reflux, one may consider surgery as an option. refractory cases have been observed to improve with surgical intervention; however, some horses with refractory dpj have been observed to recover with supportive medical care alone even after days of refluxing large amounts of fluid every to hours (personal observation). the decision of when to intervene surgically often is difficult. one may elect surgery to determine the extent of gross pathologic condition and intestinal distention and to perform intestinal bypass so as to direct enterogastric reflux toward the cecum and colon, where the fluid can be reabsorbed. allen and clark have described two approaches for surgical therapy in such cases. a standing right flank laparotomy with resection of the last rib has been used to approach the duodenum and cecal base. using this approach, one makes a small stoma between the duodenum and cecum using a handsewn . -to . -cm side-to-side anastomosis. the stoma may act as a shunt to decompress the proximal small intestine and deliver the small intestinal fluid to the cecum for reabsorption. following recovery, the stoma likely will close. when a veterinarian is confronted with a horse exhibiting abdominal discomfort, with small intestinal distention palpable per rectum, and greater than l of gastric reflux, the veterinarian should recommend referral of the horse to a facility capable of performing abdominal surgery. the chance that such a horse has an intestinal obstruction is too great to decide to treat it as if it may have dpj. surgery on such horses is not unusual, even though dpj is possible, to rule out an obstruction. at present, the survival of horses with dpj that undergo surgery is much greater than previously described, and certainly greater than that of horses with small intestinal obstruction that do not have surgery. horses with dpj that receive appropriate therapy have a reasonably good chance of making a full recovery. horses that continue to have frequent episodes of voluminous nasogastric reflux and systemic signs of endotoxemia and septicemia have a poorer prognosis for recovery. frequent complications of dpj include laminitis, thrombophlebitis, and weight loss. the clinical signs of chronic wasting and poor body condition, although nonspecific for a diagnosis of malabsorption antemortem, can be attributed to proliferative or inflammatory intestinal disorders, often collectively referred to as chronic inflammatory bowel diseases. clinical signs include alimentary lymphosarcoma, granulomatous enteritis, multisystemic eosinophilic epitheliotropic disease (meed), and lymphocyticplasmacytic enterocolitis-conditions affecting young and adult horses. proliferative enterocolitis, a transmissible disease of foals to months of age characterized by significant small intestinal pathologic changes, will be included in this group. however, several other primarily small intestinal conditions described from a morphologic perspective, such as chronic postinfarctive inflammation and mycobacterial infections, will not be discussed. in addition, a single case of aa amyloid-associated gastroenteropathy in an -year-old morgan stallion that had evidence of severe malabsorption based on poor d-xylose absorption is included. for comparative purposes, table . - lists the clinical and clinicopathologic features of the diseases, and tables . - and . - present the gross morphologic and histopathologic findings, respectively. the extent of small intestinal disease is the key to determine whether one can demonstrate malabsorption based on abnormal carbohydrate absorption. as described in chapter . , this is not an all-or-nothing situation. in the same animal the staging of the pathologic changes differs in different regions of the small and large intestines, thus influencing severity of clinical signs and absorption findings. furthermore, the extent of pathologic changes in different animals with ultimately the same morphologic diagnosis affects absorption studies and progress of the disease. early diagnosis remains a challenge, and even multiple intestinal biopsies taken at exploratory laparotomy may prove unhelpful. by contrast, intestinal infiltration with the predominant cell types can be found in grossly normal appearing intestinal tissue. alimentary lymphosarcoma of the horse may represent a primary neoplasia of the gut associated lymphoid tissue with significant cellular infiltration of the small intestine and associated lymph nodes with minimal large intestinal or systemic involvement. case series and pathology reports indicate that young horses to years of age primarily are affected, although the age range can be broad. [ ] [ ] [ ] no breed or sex predilection exists. prevalence is unknown. despite the progressive nature of lymphomata, onset of clinical signs can be rapid and the animal may become acutely ill. as with all adult cases of chronic inflammatory bowel disease, antemortem diagnosis is by a process of exclusion and usually is confirmed post mortem. frequently, the horse has anemia, thrombocytopenia, neutrophilia or neutropenia, hypoalbuminemia, normal serum protein or hyperproteinemia, and hypergammaglobulinemia. lymphocytosis is rare. one may palpate intraabdominal masses, mainly enlarged mesenteric lymph nodes, rectally. abdominocentesis has been of diagnostic value. carbohydrate absorption tests usually reveal partial to total malabsorption indicative of the severely reduced surface area resulting from significant villous atrophy and the extensive mucosal or transmural infiltration. rectal biopsy has aided diagnosis. early confirmation of a suspected diagnosis necessitates exploratory laparotomy to obtain multiple intestinal and lymph node biopsies. in the future, markers of cancer cells may become available and may be cost-effective to aid diagnosis. prognosis is poor. natural progress of the disease is unknown. most horses are presented in an advanced state of disease. immunosuppressive drugs or chemotherapy may afford temporary improvement. however, outcome is unaffected. the chronic wasting condition granulomatous enteritis was first described in ; of horses were young standardbreds. most affected horses are to years of age. case reports from many countries revealed a predominance of standardbred over thoroughbred horses by three to one. , some of the standardbreds were related, implicating a genetic predisposition. prevalence is low. the condition is sporadic and has an insidious onset, and the course can be protracted. significant diagnostic features include anemia, slight increases or decreases in white blood cell counts, hypoalbuminemia, normal serum protein or hypoproteinemia, occasional increases in serum alkaline phosphatase activity, normal serum γ-glutamyltransferase activity, and enlarged mesenteric lymph nodes on rectal palpation. reduced carbohydrate absorption to the level of partial to total malabsorption is reported frequently, consistent with the severe morphologic changes throughout the small intestine. one can attribute the low proportion of horses exhibiting diarrhea , to the preferential distribution of inflammatory infiltration in the small intestine, with lesser involvement of the large intestine. rectal biopsy can be a useful aid to diagnosis. treatment of horses with granulomatous enteritis with a variety of drugs, particularly corticosteroids, has not affected the outcome except in the short term. one successful response has been reported. prolonged corticosteroid administration produced clinical remission in a -year-old standardbred gelding based on improvement in clinical signs and in d-xylose absorption. five months after cessation of approximately months therapy, d-xylose absorption was normal and the horse was bright, alert, and resumed a level of athletic performance. parenteral administration of dexamethasone sodium phosphate was tapered to achieve a minimal effective dose to reduce intestinal inflammation and abolish clinical signs. adverse effects were not reported. the outcome of this single case is encouraging. surgery may be indicated if the disease is localized. two young horses underwent resection of the thickened terminal small intestine to confirm a diagnosis and provide a means of treatment; one horse died months after surgery, and the other has remained clinically normal for at least years. the cause of granulomatous enteritis is unknown. several infectious agents have been implicated, including mycobacterium avium. the condition may represent a granulomatous hypersensitivity reaction. immunemediated responses to dietary, parasitic, or bacterial antigens may be important initiating factors. recently, six cases purported to represent granulomatous enteritis were linked to environmental contamination with aluminum. although the case definition was flawed and problems existed with the data and interpretation, the report nevertheless raised the possibility that a toxicologic basis may exist for some equine inflammatory bowel disorders. lumen from parasitic, bacterial, or dietary sources. infectious agents have not been identified. , widespread use of the avermectins has tended to reduce parasite loads and composition to favor small strongyles (cyathostomes). eosinophilia is a feature of parasitism in the equine intestinal tract, although nematodes rarely have been identified in any lesions of meed. , however, failure to detect larval structures in these lesions may be attributable to chronicity of the disease and destruction of the parasites in tissue. biopsies of the rectal mucosa or of the skin, liver, intestinal tract, and lymph nodes may assist in diagnosis. treatment has been attempted with a variety of drugs, including antibiotics, corticosteroids, and anthelmintics with larvicidal activity. immediate improvement has not been borne out in the long term. prognosis is poor. the clinical objective is to reach a tentative diagnosis early in the course of the disease for intervention to be more than transient. unlike the other conditions (see table . - ), meed has definitive liver and pancreatic involvement, and thus maldigestion may make a significant contribution to the wasting disease. for example, the lowered albumin and protein could result in part from impaired pancreatic enzyme digestion, and the effects of inflammatory lesions in the liver and ileum may decrease bile salt concentrations. the morphologic findings in lymphocytic-plasmacytic enterocolitis reflect the predominant infiltrative cellular elements of this rarely encountered condition. a retrospective study of horses provided the information presented in the tables. no specific clinical or clinicopathologic features differentiate this condition antemortem from other inflammatory diseases of adult horses. carbohydrate absorption was abnormal or delayed in of horses, consistent with the predominance of small intestinal pathologic changes. rectal biopsies were abnormal in of horses, two of which were reported as having lymphocytic-plasmacytic proctitis. prognosis is poor. treatment has been unsuccessful, probably because of the advanced nature of the condition at the beginning of treatment. proliferative enteropathy has not been associated with abnormal carbohydrate absorption based on three horses subjected to carbohydrate absorption tests. however, the florid mucosal lesions in the jejunum and ileum undoubtedly contribute to impaired digestive function and potential malabsorption of vitamins, minerals, and amino acids in the distal small intestine. the condition meed encompasses disorders characterized by a predominant eosinophilic infiltrate in the gastrointestinal tract, associated lymph nodes, liver, pancreas, skin, and other structures and accompanied by some degree of malabsorption and enteric protein loss. the disorders include chronic eosinophilic gastroenteritis, eosinophilic granulomatosis, chronic eosinophilic dermatitis, and probably basophilic enterocolitis. the condition differs from idiopathic eosinophilic enterocolitis, in which segmental lesions in the small or large intestine induce signs of colic requiring surgical intervention , without evidence of malabsorption or multisystem involvement. although prevalence is low, meed appears to be more common than granulomatous enteritis based on the accumulated published reports and personal experience in australia and the united states. most affected horses are to years of age, and standardbreds and thoroughbred are reported to predominate. the condition is sporadic and has an insidious onset, and the course is protracted with a duration of to months. diarrhea is common in contrast to granulomatous enteritis. severe skin lesions with exudative dermatitis and ulcerative coronitis are prominent and frequently are the principal reason for veterinary attention being sought. despite extensive tissue eosinophilia, systemic eosinophilia is rare. hematologic values are usually unremarkable. notable features include hypoalbuminemia and normal serum protein or hypoproteinemia, and because of liver involvement, serum γ-glutamyltransferase and alkaline phosphatase activities may be increased. most reports of carbohydrate absorption test findings (glucose or d-xylose) indicate retarded absorption and a reduced or normal peak concentration delayed to at least minutes. one can interpret this pattern as the existence of sufficient small intestinal absorptive capacity to enable moderate absorption with possibly delayed gastric emptying or ileocecal ejection. morphologic changes are less pronounced in the small intestine than in the large intestine, and small intestinal lesions predominate segmentally in the proximal duodenum and distal ileum. furthermore, significant hyperkeratosis of the fundic region may contribute to gastric muscle contractile disruption. diarrhea can be a consequence of the severe segmental or multifocal granulomatous lesions in the large intestine with mucosal and transmural thickening and extensive ulceration. abundant fibrosis is a feature of all affected tissues (see table . - ). the cause of meed is unknown and could represent a chronic ongoing immediate hypersensitivity reaction against undefined antigens ingested or excreted into the affects foals to months of age, particularly those that have been weaned recently. the disease is caused by lawsonia intracellulare, an obligate intracellular bacterium found in the cytoplasm of proliferative crypt epithelial cells of the intestine. the condition in a foal was described first as intestinal adenomatosis, because of similarity to the swine disorder of the same name. later, molecular studies showed that intestines from an affected foal contained l. intracellulare sequences as determined by polymerase chain reaction analysis and confirmed by southern blot hybridization. recently, studies of a cluster of affected foals on three breeding farms in canada provided much information on the clinical syndrome, laboratory investigations, and response to treatment. two of the three farms bred arabians, hence a demographic predominance of arabian foals exists. clinical signs included depression, rapid and significant weight loss, edema, diarrhea, and colic. poor body condition, a rough hair coat, and potbelly appearance were common findings. other problems often were concurrent, including respiratory tract infection, dermatitis, intestinal parasitism, and gastric ulceration. significant laboratory findings were anemia, transient leucocytosis, hypoalbuminemia, hypoproteinemia, and elevated serum creatine kinase concentrations. diagnosis was confirmed by identifying characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells using silver stains and by results of polymerase chain reaction analysis and immunohistochemical testing. antemortem diagnosis relied on clinical signs, hypoproteinemia, and exclusion of common enteric infections. one can confirm diagnosis in live animals by fecal polymerase chain reaction analysis (positive in of foals tested) and serologic testing; foals with proliferative enteropathy were evaluated serologically and had antibodies against lawsonia intracellulare. treatment is effective. most foals received erythromycin estolate ( to mg/kg per os every to hours), alone or with rifampin ( to mg/kg per os every hours) for to weeks. foals frequently needed supportive therapy at the outset for stabilization. response to therapy has been excellent. rapid improvement in clinical signs even within hours preceded the rise in plasma protein concentration. the source of the infection was undetermined. no apparent link existed between the three farms and a swine operation or solid and liquid waste disposal on pasture. however, one cannot exclude airborne spread of dried fecal material over distances. comparisons of epidemiologic findings from the swine disease indicated that overcrowding, feed changes, antibiotic usage, and mixing and transportation were potential risk factors at two of the farms. recent weaning appeared to be a key element in the pathogenesis. samuel l. jones acute diarrhea caused by colitis in adult or young horses is a potentially life-threatening disorder of a variety of causes (table . - ) characterized by hypersecretion of fluid, motility disturbances, altered microbial flora in the colon, and an impaired mucosal barrier caused by direct injury or inflammation. many of the clinical and clinicopathologic features are similar regardless of the cause. severe dehydration with profound electrolyte abnormalities is common, as is systemic inflammation from absorption of endotoxin or other bacterial products through the compromised mucosa. gastrointestinal protein loss may result in reduced colloid oncotic pressure from hypoproteinemia, leading to tissue edema. colitis is a highly catabolic disorder, and weight loss may be rapid and severe. some cases of colitis may be complicated by extensive mucosal ulceration, serosal inflammation, or mural ischemia/infarction extending from the inflammation or resulting from coagulopathies. thus diagnostic measures aimed at determining the cause necessarily must be accompanied by clinical and laboratory assessment of hydration, electrolyte and acid-base balance, plasma protein concentration and colloid oncotic pressure, organ function, and evaluation of the degree of systemic inflammation and of the integrity of the intestinal wall. although therapeutic strategies are similar for many causes of colitis, consisting primarily of control of local and systemic inflammation, maintenance of fluid and electrolyte balance, promotion of tissue perfusion, replacement of plasma protein, preservation of colloid oncotic pressure, promotion of mucosal repair, restoration of the microbial ecology of the colon, and nutritional management, some causes of acute colitis have specific therapies aimed at eliminating the cause. a variety of infectious organisms has been identified as causes of acute colitis in adult horses. the clinical syndromes associated with these infections are indistinguishable in most horses. however, appropriate diagnostic tests including fecal bacterial culture, fecal bacterial toxin analysis, pcr, and/or serology may identify specific infectious organisms. salmonella is a genus of gram-negative facultatively anaerobic bacteria that are common gastrointestinal pathogens in horses. many serotypes of salmonella have been reported to infect horses, but those classified in group b appear to be associated more commonly with disease than those in other groups. group b includes s. typhimurium and s. agona, two of the species most frequently isolated from horses. - s. typhimurium is the most pathogenic serotype in horses and is associated with a higher case fatality rate than other species of salmonella. the number of horses that are infected inapparently with and actively shed salmonella in their feces has been reported to be as high as % to %, but actual prevalence of salmonella-shedding in the general horse population is likely to be much lower, less than % to %. horses shedding salmonellae are a potential source of infection to susceptible horses, , as are environmental reservoirs. [ ] [ ] [ ] for these reasons, salmonellosis is one of the most common nosocomial diseases in horses. nosocomial salmonellosis significantly affects morbidity and mortality in hospitalized horses. the emergence of multidrug resistance in equine salmonella isolates has been a cause of concern because of the importance of salmonellosis as a nosocomial disease and because salmonella represents a significant zoonotic pathogen. , [ ] [ ] [ ] the virulence of the bacteria varies tremendously with serotype and even among strains of the same serotype in part because of the important role of host susceptibility in the pathogenicity of particular organisms. the infective dose is generally millions of organisms inoculated orally, but various environmental and host factors can reduce the infective dose to a few thousand or even hundreds of organisms. [ ] [ ] [ ] environmental factors or stresses that increase susceptibility to salmonella infection are not well defined, but high ambient temperature, for example, is known to increase the prevalence of salmonellosis in horses greatly. indeed, the peak incidence of salmonellosis in horses occurs in late summer and fall. , , other environmental and host factors that increase the risk of salmonella infection include transportation, antibiotic administration, gastrointestinal surgery, general anesthesia, preexisting gastrointestinal disease, change in diet, and immunosuppression. , , host factors that restrict gastrointestinal colonization and invasion by pathogens include gastric ph, commensal gastrointestinal flora, gastrointestinal motility, the mucosal barrier and mucosal immunity. , gastric acidity is an important defense mechanism preventing live organisms from reaching the intestine. altering the gastric ph with histamine receptor antagonists, for example, may increase susceptibility to infection. gastrointestinal flora inhibits the proliferation and colonization of salmonella by secreting bacteriocins, short-chain fatty acids, and other substances that are toxic to salmonella. in addition, elements of the normal flora compete for nutrients and space, especially on the mucosa. being predominantly anaerobic, the normal flora maintain a low oxidationreduction potential in the environment of the large intestine, which inhibits the growth of many bacterial pathogens. the importance of normal host gastrointestinal ecology is illustrated by the fact that disturbances of the colonic flora with antibiotics, changes in feed, ileus, or other underlying gastrointestinal disease greatly increase the susceptibility of the host to infection by salmonella, often resulting in serious disease. the immune status of the host may be one of the most important factors determining not only the susceptibility to salmonella infections but also the degree of invasion and subsequent outcome of the infection. local immunity, such as mucosal antibody secretion and enterocyte-derived cationic peptides, prevents colonization of the mucosa. , , opsonizing antibodies and activation of the complement cascade are important in fighting systemic invasion by salmonella by increasing the efficiency of phagocytosis and by direct bactericidal activity. humoral immunity, however, is often ineffective in preventing disease and dissemination once invasion occurs and salmonella has established in its intracellular niche. following invasion, salmonella is capable of surviving and multiplying within macrophages, rendering humoral (noncellular) immune systems ineffective. , specific cellular immunity may be the most effective defense mechanism in the host arsenal against dissemination and systemic infection by salmonella. , oral inoculation with small numbers of virulent organisms may induce protective immunity in horses and calves, but the duration of the immunity is not known. , oral and parenteral vaccines using killed or attenuated organisms and bacterial products have been promising but are effective only against homologous organisms and are usually not cross-protective among different serogroups. [ ] [ ] [ ] in adult horses, salmonella primarily infects the cecum and proximal colon, causing enterocolitis, and the ability to disseminate beyond the intestine and cause enteric fever is limited. in foals, however, salmonellosis often is associated with septicemia. the ability of salmonella to cause enterocolitis depends on the ability of the bacteria to invade the gastrointestinal mucosa. , invasion of the gastrointestinal mucosa occurs preferentially through specialized enterocytes called m cells that overlay intestinal lymphoid tissues such as peyer's patches in nonequine species. a variety of enteric pathogens exploit m cells during infection of intestinal tissue. invasion of the epithelium occurs by self-induced uptake via the apical membrane of the m cell, often killing the cell in the process. salmonellae then invade neighboring cells via the basolateral membrane, eventually spreading the destruction of the epithelium beyond the principle area of attack. virulent salmonellae have a well-developed invasion mechanism involving generation of an apparatus called a type iii secretory system that enables virulence gene products to be injected directly into enterocytes. virulence proteins injected by salmonellae into enterocytes engage the cellular machinery and induce the cell to engulf the bacteria by macropinocytosis. salmonella virulence gene products also induce enterocyte chloride and fluid secretion and upregulate enterocyte transcription of inflammatory cytokines (tumor necrosis factor α and interleukin- β) and chemokines that trigger a mucosal inflammatory response. , , after salmonellae invade the mucosa, they are phagocytosed quickly by macrophages and dendritic cells in the lamina propria and lymphoid tissues. the ability of salmonellae to disseminate systemically and cause enteric fever is associated with the ability to survive and proliferate in macrophages. indeed, phagocytes have an important role in dissemination of the pathogen to blood, lymph nodes, liver, and spleen. most salmonellae in the blood and tissues of infected animals that are competent to cause enteric fever are within phagocytic cells. in adult horses with salmonellosis, dissemination appears to be limited to the intestine and mesenteric lymph nodes, and salmonella rarely is cultured from blood. however, in foals and in some adults, salmonella causes an enteric feverlike disease with dissemination to mesenteric lymph nodes, liver, spleen, and blood. salmonella organisms require specific virulence gene clusters encoded on the chromosome or on plasmids for intracellular survival in macrophages. some of these genes are sensors that signal the bacteria that it has entered an intracellular environment and turn on genes required for intracellular survival. others, like invasion genes, are transported from the bacteria and injected into macrophage cytosol by a type iii secretory system to prevent phagosome/lysosome fusion and subvert other essential macrophage killing mechanisms. salmonellae also possess multiple genes that confer resistance to reactive oxygen and nitrogen metabolites, perhaps the most lethal antimicrobial mechanisms of macrophages. diarrhea associated with salmonellosis has multiple causes. a salmonella cytotoxin inhibits protein synthesis in mucosal cells, causing morphologic damage and altered permeability. virulent salmonellae also produce an enterotoxin similar to the heat-labile toxin (lt) produced by escherichia coli. , the enterotoxin contributes to but is not required in the pathogenesis of diarrhea. , salmonella enterotoxin increases secretion of chloride and water by colonic mucosal cells in many species, including horses, by increasing intracellular cyclic adenosine monophosphate concentrations. the ability of virulent salmonellae to cause diarrhea appears to be associated most closely with the ability to invade enterocytes and to trigger an inflammatory reaction in the intestinal tissue. , gene products injected into enterocyte cytosol by the type iii secretory system of invading salmonellae stimulate chloride and fluid secretion. salmonella invasion of enterocytes is also a potent activator of inflammatory chemokine and cytokine production, resulting in the recruitment of leukocytes, particularly neutrophils, and activation of resident macrophages and mast cells. products of these activated leukocytes, including prostaglandins, leukotrienes, reactive oxygen metabolites, and histamine, are potent stimulators of chloride secretion in the colon of many species. , [ ] [ ] [ ] the enteric nervous system integrates the diverse processes of pathogen recognition, triggering of the inflammatory response, and induction of enterocyte fluid secretion. many of the inflammatory mediators studied stimulate colonic secretion by prostaglandin-dependent mechanisms, resulting in increased intracellular cyclic adenosine monophosphate or calcium concentrations or both in mucosal cells. in addition, these mediators and the enteric nervous system may stimulate secretion by prostaglandin-independent mechanisms, inhibit sodium and water absorption, cause motility disturbances, and potentiate tissue injury, all of which enhance the pathogenicity and dissemination of salmonella and contribute to the pathogenesis of diarrhea. , neutrophils recruited to the mucosa by signals generated by the infected enterocytes physically contribute to mucosal injury by producing a variety of products that are lethal to pathogens but are also toxic to host cells. , moreover, neutrophils attracted to infected epithelial cells accumulate beneath the monolayer, lifting it off the basement membrane in sheets. neutrophils also migrate across the epithelial monolayer in potentially massive numbers. transepithelial migration of neutrophils increases the permeability to macromolecules, bacterial products, and even bacteria. potentially massive losses of electrolytes, water, and protein can occur depending on bacterial and host factors. perhaps most devastatingly, mucosal injury and altered permeability allow systemic absorption of bacterial products and dissemination of bacteria, resulting in systemic inflammatory responses such as occur with endotoxemia and septicemia. four syndromes of salmonella infection have been described clinically and reproduced experimentally in horses: ( ) inapparent infections with latent or active carrier states; ( ) depression, fever, anorexia, and neutropenia without diarrhea or colic; ( ) fulminant or peracute enterocolitis with diarrhea; and ( ) septicemia (enteric fever) with or without diarrhea. inapparent infections can be activated to clinical disease in compromised horses, such as horses with colic or horses being treated with antibiotics, causing mild to severe enterocolitis. in addition, latent infections (nonshedding) can become active infections (shedding) under certain conditions, such as transportation stress and antibiotic treatment. horses with depression, anorexia, fever, and neutropenia without diarrhea generally have a good prognosis and recover in several days without specific treatment. the septicemic form is restricted mostly to neonatal foals and is uncommon in adult horses. this discussion focuses on acute enterocolitis. acute enterocolitis is characterized by severe fibrinonecrotic typhlocolitis, with interstitial edema and variable degrees of intramural vascular thrombosis that may progress to infarction. severe ulceration of the large intestinal mucosa may occur with serosal ecchymoses and congestion. the earliest signs of enterocolitis are usually fever and anorexia. , signs of colic may be apparent early in the course of the disease, especially if ileus is present. clinical signs of endotoxemia are common and range from fever, elevated heart and respiratory rates, poor peripheral perfusion, and ileus to fulminant and rapidly progressive signs of endotoxemic shock. oral mucous membranes are often pale with perigingival hyperemia (a toxic rim) but may be brick red or cyanotic, with prolonged capillary refill time. one may note weakness, muscle fasciculations, cold extremities, and other signs suggestive of hypotensive shock; synchronous diaphragmatic flutter; abdominal pain; and significant metabolic and electrolyte abnormalities in severe cases of enterocolitis. one also may note signs of mild dehydration before diarrhea is apparent. once diarrhea is evident, dehydration may become severe rapidly. occasionally, horses die peracutely, without developing diarrhea. diarrhea may not be apparent for several days but usually occurs by to hours after the fever begins. , the duration of the diarrhea may be days to weeks. the character of the first diarrheal feces is usually watery with particles of roughage but may become fluid rapidly without solid material. finding frank blood and fibrin in the feces is unusual. the volume of feces is often large, with frequent defecation. one may note straining or signs of colic when the patient is defecating, and rectal prolapse may occur occasionally. persistent straining and rectal prolapse may be a sign of colonic infarction. abdominal borborygmi are often absent early in the course of the disease because of ileus but become evident later, usually when diarrhea begins. fluid and gas sounds are commonly audible, but normal progressive motility is less frequently audible than normally. transrectal palpation may reveal edematous rectal mucosa and colon and fluid-filled colon and cecum. one may obtain gastric reflux, especially early in the course when ileus is evident. hematologic abnormalities early in the course of the disease include moderate to severe neutropenia, lymphopenia, and leukopenia, a mild to moderate left shift, and toxic changes in the neutrophils. , thrombocytopenia, moderate to severe hemoconcentration, and hyperfibrinogenemia are also common. neutropenia is an early but nonspecific indicator of salmonellosis, often occurring concurrently with the onset of fever. later in the course of disease, one may see neutrophilic leukocytosis, indicating recovery. a degenerative left shift, with metamyelocytes and myelocytes in the peripheral blood, is a poor prognostic sign. serum biochemical analysis may reveal azotemia, elevations in serum sorbitol dehydrogenase and γglutamine aminotransferase activity, and elevated serum lactic acid concentration. azotemia is often prerenal, but acute hemodynamic renal failure may occur in severely dehydrated, endotoxemic, or septicemic patients. indeed, elevation of creatinine concentration is a poor prognostic indicator in horses with acute colitis. hemodynamic renal disease may be complicated by toxic injury caused by administration of nephrotoxic drugs. hyponatremia may also contribute to prerenal azotemia. elevations in hepatocellular enzymes are usually mild and reflect damage to the hepatocytes from absorbed toxins such as endotoxin and from poor perfusion caused by hypotensive shock or dehydration. lactic acidemia may be present, reflecting poor tissue perfusion. plasma protein rapidly drops as protein is lost in the gastrointestinal tract, causing moderate to severe hypoalbuminemia and hypoglobulinemia. peripheral or organ edema (vascular leak syndrome) may occur if hypoproteinemia is severe, coupled with increases in endothelial permeability induced by systemic inflammation. hypokalemia, hyponatremia, hypochloridemia, and hypocalcemia are common electrolyte abnormalities in patients with enterocolitis. metabolic acidosis also may be present. coagulopathies, such as decreased antithrombin iii activity and disseminated intravascular coagulation, may occur. urinalysis may reveal isosthenuria, proteinuria, hematuria, cylindruria, and glucosuria if hemodynamic or toxic renal injury is present. the number of leukocytes in the feces usually is elevated, and occult blood may be detectable. peritoneal fluid is usually normal except when severe mural inflammation or colonic infarction occurs. routine detection of salmonellae in feces involves five daily cultures of large samples ( to g) of feces using enrichment techniques. , , however, the sensitivity of fecal culture can be as low as % to %, even if one cultures several fecal samples collected daily. concurrent culture of rectal biopsy specimens and feces increases the sensitivity of culture techniques to % to %. currently, the polymerase chain reaction (pcr) is the most sensitive and rapid test for detecting salmonella in feces. a single pcr test applied early in the course of disease is a more sensitive test for the presence of salmonella than repeated fecal cultures. , detection of salmonellae in feces does not prove a diagnosis of salmonellosis, but the positive predictive value of a positive pcr or culture results is high in horses with compatible clinical signs. culture of peripheral blood may allow isolation of the organism if bacteremia or septicemia is present, but blood cultures are not a sensitive test for salmonellosis in adult horses. although foals are more likely than adults to become septicemic, culture of blood is recommended in all cases with signs suggestive of septicemia. increased numbers of fecal leukocytes suggest an invasive process in the colon but are not specific for salmonellosis. early in the course of the disease, dehydration, electrolyte and acid-base imbalances, endotoxemia, and sepsis may be life threatening. aggressive treatment during the acute stages to replace fluids lost in the diarrhea and to control sepsis and endotoxemia is often effective in controlling the primary disease. weight loss and hypoproteinemia are often severe. complications such as multiorgan dysfunction, vascular leak syndrome with peripheral and organ edema, laminitis, acute renal failure, venous thrombosis and septic phlebitis, irreversible protein-losing enteropathy or chronic malabsorption, pulmonary aspergillosis, and gastrointestinal infarction can occur. in many instances, horses recover from acute salmonellosis with aggressive treatment, only to succumb to complications of the disease, partially explaining the high fatality rate of equine salmonellosis compared with human salmonellosis. chronic, mild to moderate diarrhea occasionally occurs in horses after a bout of severe salmonellosis, usually with protein-losing enteropathy. if the chronic diarrhea persists beyond to weeks after the onset of signs, the prognosis for recovery is poor. potomac horse fever (equine monocytic ehrlichiosis) is caused by the obligate intracellular rickettsial organism neorickettsia risticii. [ ] [ ] [ ] [ ] the disease is most common from late summer to early fall, with a peak incidence in july and august. potomac horse fever was described first in the northeast but since has been diagnosed in most areas of the continental united states, with a particularly high prevalence in the northeast and midwest. the geographic distribution is characterized by a significantly higher percentage of cases found along waterways and rivers. , the disease occurs sporadically, temporally and geographically, and can affect any age group of horses. the case fatality rate is % to %. transmission of n. risticii has been reproduced experimentally by oral, intramuscular, intradermal, subcutaneous, and intravenous routes. , however, the natural route of infection has remained a mystery until recently. the epidemiologic data, the fact that many other rickettsial diseases are transmitted by insect vectors, and the finding that the disease can be transmitted via whole blood have implicated insect vectors in the natural transmission of the organism. attempts to transmit the disease experimentally with ticks (dermacentor variablis) or biting flies (stomoxys calcitrans) have been unsuccessful. , recently, n. risticii has been found to infect virgulate cercariae larval stages of trematodes that use operculate freshwater snails (juga spp.) as part of their life cycle in northern california. infected virgulate cercariae have been identified in aquatic snails collected in other parts of the country as well. virgulate cercariae are part of the life cycle of trematodes that are common parasites of many species and use freshwater snails and aquatic insects as intermediate hosts. although the trematode species infected with n. risticii remains to be identified definitively, at least two species are considered potential vectors. during the trematode life cycle, aquatic snails release large numbers of infected cercariae into water, where they seek their next intermediate host. infected metacercaria have been identified in a variety of aquatic insects. preliminary studies suggest that n. risticii in fact may be transmitted via ingestion of mature caddis flies containing infected metacercariae. possibly horses are infected by drinking water containing infected cercaria released from snails or by ingesting infected metacercariae in other aquatic insects. the number of pcr-positive snails in endemic regions corresponds to the seasonal incidence of potomac horse fever and may be as high as %. the pathogenesis of n. risticii is not understood completely. the organism infects and survives in monocytes and monocyte-derived leukocytes and can be found in blood monocytes during natural infections, but the sequence of events resulting in enterocolitis remains speculative. the organism appears first to infect blood monocytes in experimentally infected horses, which may be the vehicle of organ infection. , however, in naturally infected horses, whether leukocytes of monocytic lineage or epithelial cells are infected first is unclear. the target organ is the gastrointestinal mucosa, with the most severe lesions found in the large intestine. , infection of human colonic cells in vitro does not cause major cytopathologic effects for several days. disruption of the microvilli in the region of the plasma membrane where sodium chloride channels are located has been observed in human colonic cell cultures. infection in horses is associated with variable degrees of morphologic damage. , mild morphologic damage and mononuclear cell infiltration of the lamina propria occur early during the infection, but fibrinous, necrotizing typhlocolitis with severe mucosal ulceration and inflammation of the lamina propria may occur later in the disease. vasculitis and intravascular coagulation are consistent features in the large intestine, with perivascular edema. one can observe n. risticii in mucosal cells and macrophages and mast cells of the lamina propria. , n. risticii can survive and multiply in macrophages by inhibiting the production of reactive oxygen intermediates and by avoiding lysosomal digestion by blocking phagosome-lysosome fusion. [ ] [ ] [ ] evidence of impaired sodium chloride absorption in the colon has been suggested to contribute to diarrhea in infected horses and may be related to destruction of the enterocyte membrane structure in the region of sodium chloride channels. , direct injury to the mucosa by n. risticii and colonic inflammation are likely to be prominent features leading to diarrhea, especially later in the disease. fluid, protein, and electrolyte loss likely is caused by mucosal injury and effects on enterocyte fluid secretion caused by the inflammatory response. like other inflammatory conditions of the colon, systemic inflammation caused by absorption of bacteria and bacterial products is a potential complication of n. risticii infections if mucosal injury is severe, which contributes to the clinical signs seen during the disease. n. risticii infection is clinically similar to other forms of enterocolitis and is characterized by anorexia, depression, and fever. , , experimental infections produce a biphasic fever occurring to days apart. decreased gastrointestinal motility, manifested as reduced borborygmi, occurs during the early stages before the onset of diarrhea. diarrhea occurs in % of cases and occurs days after the second fever episode during experimental infections. , the diarrhea can be moderate to severe and dehydrating. ileus can develop at any stage of the disease and can cause signs of moderate to severe colic. systemic signs of endotoxemia, shock, and peripheral edema may occur and are similar to those described for salmonellosis. experimental and natural infection with n. risticii can cause abortion of infected fetuses in pregnant mares. , laminitis is a complication in % to % of naturally occurring cases and is often severe. other complications include protein-losing enteropathy, thrombosis, and renal failure, as described for salmonellosis. hematologic abnormalities reflect endotoxemia, dehydration, and sepsis and are essentially identical to those described for salmonellosis. neutropenia with a left shift is a consistent feature and occurs concurrently with or soon after the onset of diarrhea. thrombocytopenia is common and often severe. neutrophilic leukocytosis occurs later in the course of the disease. hyperfibrinogenemia is usually more pronounced than that with salmonellosis. serum electrolyte, acid-base, and biochemical abnormalities are also similar to those described for salmonellosis. coagulopathies are common during n. risticii infection and reflect activation of coagulation pathways. disseminated intravascular coagulation is common and may be related to the high frequency of laminitis associated with n. risticii infection. one cannot base diagnosis of n. risticii infection solely on clinical signs because the disease is clinically similar to other forms of enterocolitis. however, in endemic areas, acute colitis is likely to be caused by n. risiticii, and thus the clinical signs of acute inflammatory colitis in fact may have a high predictive value. serologic evidence of infection, such as rising antibody titers to n. risticii detected by indirect immunofluorescence or enzymelinked immunosorbent assay in paired serum samples may be helpful in establishing a diagnosis. , one should take care when interpreting the indirect immunofluorescence serologic test for n. risticii because the test appears to have a high false-positive rate. culture of the organism from blood is possible but is difficult and is generally useful only in the research laboratory. recently developed polymerase chain pcr tests for n. risticii dna are rapid, highly sensitive (as sensitive as culture), and specific for n. risticii infection and can be applied to blood or feces. [ ] [ ] [ ] prevention prevention of the disease by reducing exposure to the causative organism is difficult because the mode of transmission is not known. a killed vaccine has been developed that is effective in preventing clinical illness other than fever in % of experimentally challenged horses using the vaccine strain. however, field studies suggest the vaccine has limited benefit for prevention or decreasing the severity of natural infection. vaccine failures have been attributed to strain differences in antigenicity or to poor antibody responses to the vaccine. , clostridiosis is an important cause of acute enterocolitis in foals and adult horses. clostridium perfringens and c. difficile are associated most commonly with intestinal clostridiosis in horses, but other clostridial species, including c. septicum, c. cadaveris, and c. sordellii also have been isolated from horses with enterocolitis. [ ] [ ] [ ] [ ] [ ] [ ] in horses of all ages, clostridial enterocolitis appears to be a common antibiotic-associated and nosocomial cause of enterocolitis. , , however, clostridiosis in neonatal foals is a distinct clinical entity and is discussed in more detail elsewhere. this chapter focuses on adult intestinal clostridiosis. clostridia are obligate anaerobic to aerotolerant spore-forming gram-positive rods that are ubiquitous in the environment in the spore form. clostridia are elements of the normal flora of horses of all ages and are among the first bacteria acquired after birth. however, clostridia inhabiting the gastrointestinal tract normally are found in low numbers and do not produce enterotoxins. clostridiosis is associated with an increase in the number of a particular species of clostridia in the gastrointestinal tract and, perhaps most importantly, exotoxin production. although the conditions resulting in exotoxin production are not understood fully, several factors increase clostridial numbers in the gastrointestinal tract. dietary factors are known to affect the numbers of clostridium species shed in the horse feces. experimental induction of colic increases fecal shedding of clostridium species in the absence of diarrhea. antibiotics, particularly administered orally or recycled via the enterohepatic system, are well documented to increase the recovery of clostridia colony-forming units (cfus) in equine feces and may result in clinical clostridiosis. , , [ ] [ ] [ ] [ ] indeed, clostridiosis associated with c. perfringens or c. difficile is likely to be the most important cause of antibiotic-induced enterocolitis in the horse. clostridium perfringens c. perfringens is made up of many genetically distinct strains of variable virulence that produce one or more of a large group of exotoxins. the pattern of exotoxin production is used to classify c. perfringens into five types, a, b, c, d, and e. c. perfringens type a is the most common clostridium isolate from healthy horses of all ages and adults and foals with diarrhea worldwide. c. perfringens types a, b, c, and d have been associated with hemorrhagic enteritis in foals less than days of age, with type c being the most common cause in north america. the primary toxin produced by c. perfringens type a is α-toxin (phospholipase c), which interferes with glucose uptake and energy production and activates arachidonic acid metabolism and signaling pathways in enterocytes. oral administration of α-toxin does not cause tissue necrosis but causes increased secretion by small intestinal mucosal cells in human beings. , the β-toxin of types b and c is a cytotoxin that causes enterocyte necrosis, ulceration, and ultimately severe intestinal inflammation and hemorrhage. a novel toxin designated β may also have a role in c. perfringens enterocolitis. the biologic activity of the β -toxin is similar to β-toxin, but β -toxin is not related to β-toxin in sequence. the β -toxin was prevalent in two groups of horses with acute enterocolitis but not in healthy horses. the β -toxin appears to be associated predominantly with c. perfringens that would have been classified otherwise as type a but that in fact may represent a previously undescribed type. virulent strains of c. perfringens type a and to a lesser extent type c also may produce enterotoxin. enterotoxin is a cytotoxin that inserts into cell membranes to form pores, which alter permeability to water and macromolecules and ultimately lead to cellular necrosis. massive desquamation of the intestinal mucosa resulting from enterotoxin cytotoxicity triggers an inflammatory response, intestinal edema, mural hemorrhage, and systemic inflammation. enterotoxin also alters tight junction integrity, resulting in increased paracellular permeability by a noncytotoxic mechanism. clostridium difficile c. difficile produces several toxins, but only two, toxin a and toxin b have been studied in detail. toxin b is a potent cytotoxin in vitro, but its role in enterocolitis is less clear than the role of toxin a. toxin b does not induce fluid secretion, inflammation, or characteristic alterations in intestinal morphology. c. difficile induces an inflammatory response with hypersecretory diarrhea that is induced in large part by the enterotoxin toxin a. toxin a induces neutrophil influx into intestinal tissue, mast cell degranulation, and secretion of prostaglandins, histamine, cytokines, and -hydroxytryptamine by these activated leukocytes. , the products of neutrophils and mast cells have a prominent role in the vasodilatory and secretory responses in the intestine during c. difficile infection. the enteric nervous system is central to the induction of intestinal inflammation and mucosal secretion by toxin a. a model for toxin a-induced secretory diarrhea has emerged in which toxin a stimulates substance p-containing afferent sensory nerve fibers, which in turn stimulate mast cell degranulation, recruitment and activation of polymorphonuclear leukocytes, and vasodilation. [ ] [ ] [ ] toxin a-induced stimulation of enterocyte secretion can occur via secretomotor neuronal stimulation by substance p-containing sensory neurons or products of mast cells and polymorphonuclear leukocytes. neural blockade or depletion of substance p abolishes mast cell degranulation, polymorphonuclear leukocyte influx, and enterocyte secretion. how toxin a triggers the sensory component of the enteric nervous system is not known yet, but toxin a-induced necrosis of enterocytes likely exposes afferent neurons to the noxious milieu of the intestinal contents. equine intestinal clostridiosis is clinically similar to other forms of acute enterocolitis in horses. , although the clinical course is usually acute, peracute colitis with rapid death may occur. occasionally, a milder, more prolonged clinical course occurs. one may note fever, anorexia, and depression before the onset of gastrointestinal signs, but more commonly no prodromal signs are apparent. signs of endotoxemia and shock may accompany acute signs of colic and severe, dehydrating diarrhea. diarrhea may not be profuse but is usually dark and foul smelling. like the clinical signs, hematologic and serum biochemical abnormalities are similar to those associated with other forms of enterocolitis and reflect fluid, protein, and electrolyte loss and systemic inflammation from endotoxemia. neutropenia, leukopenia, and hemoconcentration are common. hypoproteinemia may be profound. one often may note hyponatremia, hypokalemia, hypochloremia, hypocalcemia, and a mixed prerenal/renal azotemia, as well as metabolic acidosis and coagulopathies. serum concentrations of hepatocellular enzymes such as sorbitol dehydrogenase may be elevated, and liver function may be reduced. preliminary diagnosis of equine intestinal clostridiosis caused by c. perfringens is based on the isolation of greater than cfus of c. perfringens type a per gram of feces from patients with diarrhea and signs suggestive of toxemia. similar criteria are used to screen human patients for c. perfringens type a infection. normal horses shed fewer than cfus/g of feces, and usually horses with intestinal clostridiosis shed greater than × cfus/g. , however, identification of increased numbers of clostridium organisms in the feces does not prove infection. detection of c. perfringens toxins in feces or intestinal contents in horses with increased numbers of fecal cfus and clinical signs of enterocolitis is more conclusive evidence of an enterotoxigenic infection than culture alone. immunoassays are available that are primarily designed to detect c. perfringens enterotoxin. however, the reliability (specificity) of some immunoassays for diagnosis of c. perfringens infection has come into question. recently, pcr multiplex and gene probe assays have been developed to detect the major lethal toxins in isolates or fecal samples to determine the pattern or toxin production and are currently the preferred methods of detection. [ ] [ ] [ ] as for c. perfringens, diagnosis of c. difficile infection consists of culture of the organism from feces and identification of toxins in the feces. bacterial culture of c. difficile may be difficult and may be an insensitive test in horses. [ ] [ ] detection may require enrichment techniques and culture of multiple fecal samples. , detection of toxins a and b in feces is regarded as the preferred test for diagnosis of c. difficile infection in human beings. commercial enzyme-linked immunosorbent assays are available for toxins a and b that are specific and appear to be more sensitive than a single culture for identifying c. difficile infection in adult horses. , one also may induce toxin production by c. difficile isolates. sensitive pcr methods also have been developed for application to fecal samples and isolates to detect the genes for toxins a and b. proliferative enteropathy is a chronic hyperplastic disorder of the small intestine and has been described in a variety of mammalian and avian species. , the only causative agent identified to date that induces proliferative enteropathy is the obligate intracellular pathogen lawsonia intracellulare. , the pig is the most frequently naturally affected species. however, the reports of equine proliferative enteropathy associated with l. intracellulare have increased in recent years. [ ] [ ] [ ] [ ] the relatedness of the strains of l. intracellulare causing proliferative enteropathy in pigs and horses or even among other affected species is not known. no host restriction is apparent because hamsters and other rodents can be infected with porcine strains of l. intracellulare. before the year , reports of proliferative enteropathy in the literature describing isolated cases were sporadic. [ ] [ ] [ ] however, since , outbreaks on breeding farms have been documented on farms in canada, suggesting that a new strain has emerged. the mode of infection is fecal-oral, and infected animals can shed large numbers of organisms in feces. affected animals shedding the organism in the feces serve as a source of infection for herdmates. possibly nonequine species serve as reservoirs contributing to outbreaks on horse farms. factors that increase the risk of proliferative enteropathy in pigs include overcrowding, ration changes, transport, and weaning. , like pigs, horses are affected as weanlings. factors associated with weaning and other stresses may affect immunity and increase susceptibility to infection. the incubation period is to weeks in nonequine species and is presumed to be similar in horses. experimental l. intracellulare infection produces characteristic pathologic lesions in pigs and hamsters that are identical to lesions in horses with proliferative enteropathy. , a profound hyperplasia of the mucosa predominantly caused by proliferation of crypt epithelium and crypt hyperplasia is induced locally in infected islands of tissue and eventually extends to the entire distal jejunum and ileum. l. intracellulare preferentially infects proliferating cells, thus the tropism for the crypt epithelium. infected cells proliferate far more rapidly than uninfected cells, suggesting that l. intracellulare directly induced the proliferative response. however, the molecular basis for the enhanced proliferation is not known. l. intracellulare penetrates epithelial cells in a membrane-bound vesicle but eventually escapes the vacuole and is found free in the cytoplasm concentrated at the apical pole of the cell. the gross pathologic lesions found in equine proliferative enteropathy are characteristic. [ ] [ ] [ ] [ ] lesions may be segmental and typically are found in the ileum and terminal jejunum in horses. however, lesions also may affect the duodenum. severe mucosal hypertrophy often occurs but may wane during the chronic stages of the disease. the mucosa may become corrugated with focal erosions or ulcers. one often can identify submucosal edema easily on cut sections of affected segments. moderate to severe crypt hyperplasia with atrophy of the intestinal villi is a consistent feature. the hyperplastic crypts are branched and may herniate into the submucosa. necrosis, edema of the submucosal and lamina propria, hemorrhage, mononuclear inflammation and muscular hypertrophy have been reported in affected intestinal segments but are not consistent. special stains such as silver stain are required to detect intracellular organisms. the organisms are curved or comma-shaped rods found clustered in the apical cytoplasm of hyperplastic crypt epithelium. the proliferative response of the intestinal mucosa alters absorption of nutrients and fluid secretion by disrupting the architecture of the villi and by altering the maturation of epithelial cells into absorptive cells, accounting for the secretory diarrhea and often severe weight loss. , the combined effects of the inflammatory part ii disorders of specific body systems response and malabsorption may account for the protein-losing enteropathy. proliferative enteritis most commonly affects weanling foals ages to months. the clinical signs of proliferative enteropathy include ill thrift, weight loss, peripheral edema, diarrhea, and colic. [ ] [ ] [ ] [ ] the diarrhea is usually in the form of soft feces but may be profuse and watery. some foals with mild diarrhea have black, tarry feces. secondary complications such as gastric ulceration, bronchopneumonia, or parasitism may occur concurrently with the proliferative enteropathy. clinicopathologic features include mild to moderate anemia, moderate to severe hypoalbuminemia (often < g/dl), hypoglobulinemia, neutrophilic leukocytosis, and hyperfibrinogenemia. creatine kinase activities may be elevated in affected foals. prerenal azotemia and electrolyte imbalances such as hyponatremia may be associated with diarrhea. peritoneal fluid analysis is usually unremarkable. ultrasonographic examination of the small intestine often reveals significant thickening of the intestinal wall ( figure . - ). intestinal edema may be evident as a hypoechoic appearance to one or more layers of the intestinal wall. methods for antemortem diagnosis include serologic analysis of l. intracellulare antibodies and pcr analysis of feces. serologic analysis using an indirect immunofluorescent antibody test may be the most useful single test available. the pcr test is specific but the sensitivity may be low. by the time clinical signs appear, % of pigs are serologically positive for anti-lawsonia intracellulare immunoglobulin g (igg). in contrast, only % of pigs had positive fecal pcr tests. of the seven foals tested in an outbreak of equine proliferative enteropathy, four foals with confirmed disease and three with suspected proliferative enteropathy had serologic titers against l. intracellulare of : or greater. in contrast, serum samples collected from foals before the outbreak were negative for l. intracellulare antibodies. fecal pcr for l. intracellulare was positive in of foals tested, and half of the serologically positive foals had negative fecal pcr tests. many clinicians combine serologic analysis with fecal pcr testing to increase the sensitivity and specificity of these diagnostic methods. isolation and culture of the organism requires cell culture techniques that are not widely available. thus no practical method exists for culturing the organism from feces or tissues that is available for clinical use. definitive diagnosis requires histopathologic examination of affected tissues. diagnosis is based on typical histopathologic findings of mucosal hypertrophy and submucosal edema and identification of small, curved, rod-shaped intracellular bacteria at the apical pole of epithelial cells in affected segments of intestine. special stains such as warthin-starry silver stain are required to detect the bacteria in histopathologic specimens. pcr analysis of affected intestinal tissue is a specific test for the presence of l. intracellulare and, unlike fecal pcr analysis, appears to be sensitive. strongyle infections in horses are caused by two groups of nematodes: large and small strongyles (see the section cyathostomiasis). large strongyles that are pathogenic in horses include strongylus vulgaris, s. edentatus, and s. equinus. of these species, s. vulgaris is by far the most important cause of disease in the large intestine and in fact is the most pathogenic parasitic infection in horses. s. vulgaris infection in horses is manifested clinically by two forms: acute and chronic disease. the age and resistance of the host, the infective dose, and the size and function of the affected arteries influence the type and degree of disease that occurs. sudden ingestion of large numbers of infective larvae by a naïve host causes acute strongylosis, whereas ingestion of fewer infective larvae over a long period of time by an older, more resistant host causes chronic strongylosis. acute strongylosis is more likely to cause colic than diarrhea and may be rapidly fatal. chronic strongylosis tends to cause debilitation and signs of colic but may also cause diarrhea. diarrhea associated with acute strongylosis occurs within several days of infection and is likely to be caused by migration of the larvae through the intestinal wall. fourth-stage larvae migrate through the mucosa and submucosa into the arterioles of the intestine, causing mural edema, hemorrhage, and infiltration with inflammatory cells into the intestinal wall. , increased secretion and decreased absorption of fluid and electrolytes, stimulated by inflammatory mediators such as prostaglandins and histamine, may play a role in the diarrhea induced by s. vulgaris. interstitial edema and damage to the interstitial matrix and mucosa may result from inflammation and migration of the parasites, causing increased secretion of fluid and albumin loss. abnormal gastrointestinal motility also may play a role in the development of diarrhea. migration of larvae through the intestinal wall early in the course of infection affects myoelectric activity and motility in the large intestine and may affect retention of ingesta and absorption of fluid. , the cause of death in acute strongylosis has not been addressed but may be related to massive migration through the vasculature, causing thrombosis with ischemia and infarction of the intestine. chronic strongylosis causes typical verminous arteritis and is associated more commonly with natural infections in horses than with acute strongylosis. lesions of the large intestinal vasculature caused by migration of larvae through the intima are characterized by thrombus formation, narrowing of the arterial lumen, fibrosis, and thickening of the arterial wall. , embolization may occur, causing acute segmental infarction of the large intestine, but more commonly reduced blood flow without embolization causes ischemia and occasionally infarction. , postmortem examination of horses with colonic infarction failed to reveal embolization as the cause in most cases. reduced blood flow in the tissues of the intestine usually results from narrowing of the arterial lumen by the thrombus and formation of microthrombi at sites independent of the parasites. release of vasoconstrictive inflammatory mediators such as leukotrienes from platelets, neutrophils, and eosinophils and elaboration of parasitic antigens or toxins may cause vasoconstriction and ischemia. horses with experimental strongylosis were found to have a % reduction of blood flow in the colonic vasculature. clearly, reduced blood flow is an important effect of chronic strongylosis, but the relationship between blood flow and diarrhea is unclear. disrupted motility resulting from ischemia may lead to diarrhea by reducing the retention of ingesta and absorption of fluid. acute infarction and mucosal ulceration have been found to cause severe chronic diarrhea in naturally infected horses. release of inflammatory mediators such as prostaglandins, histamine, and kinins from inflammatory cells associated with thrombi and inflamed intestine also may affect secretion, absorption, and motility, leading to diarrhea. the clinical signs of acute strongylosis caused by s. vulgaris infection are characterized by depression, moderate to severe colic, and fever. diarrhea is less often a feature of acute strongylosis than is colic. most cases of acute strongylosis occur in young, naïve horses that are introduced to an infested environment or are inoculated experimentally with infective larvae. this form of strongylosis often is not recognized naturally. chronic strongylosis, however, occurs most commonly as a natural syndrome. weight loss or poor weight gain; chronic, intermittent colic; fever; poor appetite; and diarrhea are frequent signs. , diarrhea may be profuse and watery, or the feces may be soft but of normal volume. transrectal palpation may reveal thickening and fremitus in the cranial mesenteric artery. young horses are most commonly affected, but older horses also may be affected. horses with acute infarction or large intestinal ulceration following chronic strongylosis may have signs of severe abdominal pain, sepsis, and endotoxemia, and profuse, watery diarrhea is common. hematologic abnormalities associated with strongylosis include neutrophilic leukocytosis and eosinophilia. [ ] [ ] [ ] neutrophilia appears to be an early event during the course of the disease, and eosinophilia tends to appear later. , hyperfibrinogenemia also may occur, especially later in the course of the disease. serum αand β-globulin and igg(t) concentrations are characteristically elevated. [ ] [ ] [ ] horses with chronic ulcerative colitis following strongylosis may exhibit severe hypoalbuminemia. peritoneal fluid analysis may reveal an elevated protein concentration and eosinophilia. , tentative diagnosis is based on clinical signs, hematologic abnormalities, and peritoneal fluid analysis. elevated serum αand β-globulin concentrations and igg(t) concentration support the diagnosis. fecal analysis may reveal strongyle eggs, but fecal egg counts are often unreliable because nonpatent larvae cause the disease. appropriate preventive measures are important in controlling this disease, including management procedures such as preventing overcrowding, reducing exposure of susceptible individuals, and instituting proper deworming schedules. ivermectin is the preferred anthelmintic used to control strongylosis in horses. monitoring fecal egg counts as a means of evaluating the efficacy of parasite control measures is recommended. infection with small strongyles (cyathostomiasis) is well recognized as a cause of diarrhea and large intestinal disease in horses of all ages. [ ] [ ] [ ] [ ] [ ] [ ] clinical disease is caused by intramural larval stages. the cyathostome life cycle requires migration by fourth-stage larvae through the mucosa of the large intestine and may include a period of hypobiosis during which the larvae remain encysted within the mucosal layer of the large intestine. after a period of hypobiosis the larvae emerge in response to a largely unknown stimulus. most cases occur when larval emergence takes place, classically in the late winter and spring in the northern temperate zones when larvae are expected to emerge and in the late fall or winter months in the southeastern united states and subtropical regions. sudden emergence of encysted larvae causes the mucosal injury, ulceration, and inflammatory reaction responsible in large part for the clinical disease. , however, migration of the larvae as they penetrate the mucosa affects motility patterns and can cause inflammation that may contribute to diarrhea. chronic, eosinophilic, granulomatous colitis and diarrhea with histopathologic evidence of hypobiotic cyathostome larvae in the large intestine have been reported in two horses during a period in which emergence of larvae would not be expected to occur (early winter). natural emergence of cyathostome larvae causes fibrinous inflammation of the large intestine, focal necrosis, mural hemorrhage, and ulceration of the large intestinal mucosa, which even may result in bleeding into the lumen. mild to moderate eosinophilic and mononuclear inflammation of the lamina propria occurs, and moderate to severe interstitial edema frequently occurs. , colonic inflammation and interstitial edema may contribute to the diarrhea, along with the loss of the mucosal barrier, by causing increased active and passive secretion of fluid, electrolytes, and protein. protein loss is often significant, resulting in profound hypoalbuminemia and interstitial edema of skin and other organs. chronic, granulomatous colitis has been reported to occur in response to encysted larvae and may cause diarrhea by increased secretion following granulomatous inflammation or disruption of the interstitium by granulomatous infiltration. administration of an anthelmintic to horses with a heavy load of encysted larvae also may cause rapid larval death and acute and often severe inflammation similar to natural emergence. cyathostomiasis may be the most commonly identified cause of chronic diarrhea in the horse. [ ] [ ] [ ] however, an acute syndrome also has been associated with cyathostomiasis. clinical signs of cyathostomiasis are characterized by moderate to severe weight loss or poor weight gain, ill thrift, ventral edema, intermittent fever, and intermittent, mild colic. acute onset of diarrhea is typically profuse and progresses to chronic diarrhea that is often mild, is the consistency of bovine feces, and may be intermittent. [ ] [ ] [ ] [ ] [ ] [ ] appetite is usually normal, but affected horses occasionally have a ravenous appetite. transrectal palpation usually does not reveal any abnormalities. cyathostomiasis may affect any age of horse, and clinical signs are more common during periods of emergence of larvae, corresponding to late winter and spring in northern temperate zones. the deworming history may appear to be adequate. neutrophilic leukocytosis is typically evident, but the white blood cell count may be normal. [ ] [ ] [ ] [ ] [ ] [ ] profound hypoalbuminemia is a characteristic feature of cyathostomiasis, manifested clinically by ventral edema. plasma αand β-globulin concentrations may be elevated, which can result in a normal total plasma protein concentration in spite of hypoalbuminemia. [ ] [ ] [ ] the serum igg(t) concentration, however, has been reported to be normal, which may help distinguish cyathostomiasis from s. vulgaris infection. , , in addition, peritoneal fluid analysis does not usually reveal any abnormalities, in contrast to horses with s. vulgaris infection. fecal analysis may be unrewarding because the infection is often not patent when clinical signs are apparent. measurement of plasma fructosamine may provide a measure of protein catabolism or protein loss in the absence of hypoalbuminemia. plasma fructosamine concentrations are significantly lower in horses with experimental cyathostomiasis than in normal controls, , suggesting that this test may be a useful diagnostic tool. however, the test has not yet been validated in naturally occurring cases, and neither the specificity nor the sensitivity is known. rectal scrapings or rectal mucosal biopsies may reveal evidence of cyathostome larvae. , definitive diagnosis usually requires microscopic examination of biopsy specimens of the cecum and ascending colon, collected by laparotomy. examination of biopsy specimens collected from the small intestine is recommended to rule out other causes of weight loss and diarrhea. one should include appropriate diagnostic tests, such as culture of feces for pathogenic bacteria, in the workup to rule out other causes. preventive measures are appropriate for other horses on premises known to have a problem with cyathostomiasis, particularly frequent deworming (every weeks) during times of high infectivity (spring and summer in the north and fall, winter, and early spring in the south) to eliminate parasites before they become patent. because of high levels of resistance to benzimidazoles, avermectins (ivermectin or moxidectin) are the drugs of choice. [ ] [ ] [ ] resistance to ivermectin has been demonstrated, but the prevalence of ivermectin resistance appears to remain low. although daily pyrantel pamoate administration also has been reported to reduce worm burdens and pasture infectivity in young and mature horses effectively, cyathostome resistance has been reported and is a concern for the use of this drug as a routine preventive anthelmintic. , diarrhea in adult horses may also occur secondary to administration of antimicrobial or antiinflammatory medications or after ingestion of toxic compounds. affected horses exhibit clinical signs that may be indistinguishable from signs exhibited by horses with diarrhea of infectious etiology. antibiotic-associated diarrhea has been reported in many species, including horses. certain antibiotics-such as trimethoprim-sulfonamide combinations, erythromycin, penicillins, tetracyclines, clindamycin, and lincomycinare associated with naturally occurring and experimental enterocolitis syndromes in horses. , [ ] [ ] [ ] [ ] in some cases, such as with trimethoprim-sulfonamide combinations, the geographic incidence of antibiotic-associated diarrhea appears to differ considerably. clostridium perfringens, c. difficile, and salmonella spp. are apparently the most common causes of antibioticassociated diarrhea in horses. outbreaks of c. difficile have been reported in hospitalized horses being treated with antibiotics. , in sweden, accidental erythromycin ingestion has been associated with c. difficile enterocolitis in mares in which their foals were being treated for rhodococcus equi. , , interestingly, this phenomenon has not been reported in other areas of the world. foals being treated with erythromycin are at a higher risk for diarrhea than foals being treated with other antibiotics. tetracycline administration has been shown to be associated with an increase in the numbers of gram-negative enteric bacteria and c. perfringens in the feces of horses and reactivation of salmonellosis and prolongation of fecal shedding of salmonella. , the most common mechanism by which antibiotics cause diarrhea is by disrupting the gastrointestinal flora. the normal large intestinal flora, comprised of mainly obligate anaerobes and streptococci, protects the host from pathogenic bacteria by colonization resistance. ecologic factors play an important role in colonization resistance. for example, surface bacteria in the large intestine interact with receptors on the mucosal cells, facilitating adherence to the mucosa. , in doing so, the normal organisms compete more successfully for this important niche. competition for space and nutrients is an important means of preventing colonization and proliferation of pathogenic bacteria. in addition, anaerobic bacteria produce short-chain fatty acids (scfas) and other metabolites that are toxic to facultative anaerobic bacteria, especially in the conditions of the large intestine. , , organisms of the normal flora also produce bacteriocins that inhibit growth of potential pathogens. antibiotics that deplete the population of obligate anaerobes and streptococci efficiently decrease colonization resistance. production of fatty acids diminishes, thus reducing competition for space and nutrients. as a result, gram-negative enteric bacteria such as salmonella are able to proliferate. in addition, pathogenic anaerobes normally found in low numbers can proliferate. antibiotic-resistant strains of bacteria, especially gram-negative enteric bacteria and possibly clostridia, may be selected by antibiotic administration, allowing proliferation of pathogenic bacteria resistant to many antibiotics. obligate anaerobic commensal organisms, perhaps the most critical group of microbes for maintaining colonization resistance, are usually susceptible to macrolides, tetracyclines, β-lactams, and lincosamides, perhaps explaining the high incidence of diarrhea associated with the administration of these antibiotics. in addition to reduction of colonization resistance, depletion of the normal anaerobic microbial population in the intestine decreases carbohydrate fermentation and production of scfas, which contributes to the pathogenesis of antibiotic-associated diarrhea by decreasing absorption of sodium and water by the colonic mucosa. ampicillin decreases colonic fermentation of carbohydrates in human beings. human patients with antibioticassociated diarrhea have greatly impaired colonic fermentation and low production of scfas. erythromycin, ampicillin, or metronidazole treatment is associated with decreased production of scfas in patients with and without diarrhea. absorption of sodium and water is stimulated by absorption of scfas in the equine colon, suggesting that reduction of colonic scfa content by antibiotic-induced depletion of anaerobic flora has similar effects in horses as in human beings. broad-spectrum antibiotics exert a more profound effect on the gastrointestinal flora than narrow-spectrum antibiotics. antibiotics administered orally, especially those that are poorly absorbed, are more likely to cause diarrhea than are parenterally administered antibiotics. for instance, clindamycin is less likely to cause diarrhea in human beings when administered intravenously than when administered orally. antibiotics with extensive enterohepatic circulation, such as tetracyclines and erythromycin, are excreted in high concentrations in the bile and are associated more commonly with diarrhea than antibiotics that do not undergo enterohepatic circulation. antibiotics may cause diarrhea by means other than by disrupting the normal flora. direct toxic effects may play a role in producing irritation, increasing secretion, and disrupting motility patterns. tetracyclines are irritating to the gastrointestinal mucosa and may cause inflammation and increase secretion. erythromycin has been shown to interact with smooth muscle cells, stimulating gastrointestinal motility. , normal peristalsis plays an important role in suppressing the population size of potentially pathogenic bacteria. normally, bacteria that are prevented from adhering to the mucosa by colonization resistance are swept aborally by peristalsis and are excreted in the feces. disruption of normal motility patterns may prevent clearance of pathogenic bacteria, contributing to the colonization of mucosal surfaces. diarrhea induced by antibiotics usually occurs within days of antibiotic administration or can occur several days after cessation of antibiotic treatment. the clinical syndrome of antibiotic-associated diarrhea can vary from mild diarrhea to fulminant enterocolitis with severe diarrhea. mild cases of diarrhea are common, especially in foals receiving erythromycin, trimethoprim-sulfonamide combinations, or rifampin. , mild cases of diarrhea are usually not clinically significant. however, acute, severe enterocolitis can occur in all ages of horses receiving antibiotics and can be life threatening. clinical signs are identical to other causes of acute enterocolitis. severe, dehydrating diarrhea, endotoxemia, sepsis, and shock may occur. hemoconcentration, neutropenia, hypoproteinemia, and electrolyte and acid-base imbalances are common. severe hyponatremia may occur in foals with antibiotic-associated diarrhea, especially if trimethoprimsulfonamide and rifampin combinations are the cause. more detailed descriptions of the clinical and laboratory findings were given previously. diagnosis is presumptive, because definitive diagnosis of antibiotic-associated diarrhea is impossible. fecal culture and pcr testing may reveal salmonella or clostridium infection. toxicity resulting from nonsteroidal antiinflammatory drug (nsaid) administration has been well documented in several species, including horses. [ ] [ ] [ ] [ ] [ ] [ ] [ ] in horses and human beings, nsaid toxicity is manifested by renal and gastrointestinal disease. elderly human patients are more susceptible to nsaid toxicity, but the effects of age on nsaid toxicity in horses are less well defined. foals are considered to be more susceptible than adult horses to gastrointestinal disease following nsaid administration, and ponies may be more susceptible than horses. all nsaids are capable of inducing gastrointestinal and renal damage at toxic concentrations, and the toxicity is not significantly different among products. aspirin is potentially more toxic than other nsaids because it irreversibly inactivates cyclooxygenase by acetylation, whereas other nsaids reversibly inhibit cyclooxygenase. however, phenylbutazone is the drug most commonly reported to cause toxicity in horses, perhaps because of its widespread use by veterinarians and horse owners. phenylbutazone toxicity in horses is characterized by mucosal ulceration throughout the gastrointestinal tract, oral ulceration, renal papillary necrosis, vasculopathy, thrombosis, and protein-losing enteropathy with hypoalbuminemia. [ ] [ ] [ ] this discussion focuses on the toxic effects of nsaids on the large intestine but necessarily includes elements of upper gastrointestinal and renal disease. horses with large intestinal disease resulting from nsaid toxicity generally are receiving inappropriately large doses. the dosage regimen recommended for phenylbutazone ( . mg/kg twice in day and then . mg/kg twice daily) is considered to be safe. experimental studies in horses, however, have shown toxicity to occur when greater than the recommended dosage ( . mg/kg/day) is administered for several days. , in most reported cases of phenylbutazone toxicosis horses were receiving higher than recommended dosages. , , regardless, administration of phenylbutazone at the recommended dosage has been reported to cause a significant decrease in plasma protein concentration and gastrointestinal disease. , moreover, signs of nsaid toxicity have been reported in normovolemic horses treated with appropriate doses of phenylbutazone. , dehydration, sepsis, and endotoxemia exacerbate the renal and gastrointestinal toxicity of nsaids. clearly, the margin of safety is narrow for phenylbutazone and probably for other nsaids used in horses as well. gastrointestinal disease induced by nsaids is manifested by mucosal ulceration, inflammation, bleeding, and protein-losing enteropathy. , , , in addition to direct effects on the mucosal barrier, nsaid administration has been shown to cause an acute relapse of preexisting colonic inflammatory disease and worsen colonic inflammation in human beings. , , whether this occurs in horses is not clear. the mechanism by which nsaids induce mucosal damage is probably multifactorial. direct irritation may play a role in oral and gastric irritation and ulceration; however, parenteral administration of nsaids produces oral and gastric ulceration as well. inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (the constitutive cox) and cyclooxygenase (the inducible cox) appears to be the most important mechanism of mucosal injury. prostaglandins, particularly pge and pgi , are critical for mucosal health. , pge has been shown to increase mucosal blood flow; increase secretion of mucus, water, and bicarbonate; increase mucosal cell turnover rate and migration; stimulate adenyl cyclase activity; and exert other protective effects in the gastric mucosa of several species. , , perhaps most importantly, pge and pgi have a role in maintaining epithelial tight junction integrity, which is indispensable for mucosal barrier function and repair after mucosal injury. in spite of the overwhelming amount of information about the role of prostaglandins in maintaining the mucosal barrier in other species and clear clinical and experimental evidence that nsaids injure the equine colonic mucosa, the role of prostaglandins in mucosal protection in the equine colon is not yet well defined. inhibition of cox- and cox- in equine colonic mucosa with flunixin meglumine resulted in reduced electric resistance of the mucosa and increased permeability to macromolecules in vitro (a.t. blikslager and s.l. jones, ) , suggesting that flunixin treatment disrupts the epithelial tight junctions in the equine colon. mucosal changes were correlated with a profound inhibition of pge and pgi concentrations in the treated tissues. in other studies, administration of a pge analog prevented the gastrointestinal manifestations of phenylbutazone toxicosis in ponies. recent development of nsaids specific for cox- have greatly reduced the frequency and severity of gastrointestinal side effects in human beings taking nsaids for chronic musculoskeletal conditions. thus cox- -specific nsaids hold promise for use in horses to treat arthritis and reduce the incidence of toxicity. for example, the cox- -specific inhibitor etodolac was less harmful to equine colonic mucosa than flunixin meglumine in vitro (a.t. blikslager and s.l. jones, ) . moreover, etodolac was significantly more permissive than flunixin for recovery of the mucosa in equine ischemic-injured intestinal tissues, and in fact, recovery was no different than control tissues. however, their use is at present limited because the specificity of the so-called cox- selective inhibitors and their efficacy as analgesics have not been demonstrated in the horse. nsaid-induced mucosal injury is associated with a significant inflammatory response to microbial products exposed to the lamina propria. this inflammation exacerbates mucosal dysfunction and injury associated with nsaid toxicity. for example, depletion of neutrophils or blockade of neutrophil influx into gastrointestinal tissues or inhibition of neutrophil activation and release of toxic products prevents many of the pathophysiologic effects of nsaid toxicity in the gastrointestinal tract. [ ] [ ] [ ] [ ] the inflammatory response alone may result in moderate to severe gastrointestinal ulceration, mural vascular thrombosis and edema, fluid secretion, protein-losing enteropathy, and mucosal hemorrhage. nsaid colitis manifests as two clinical syndromes: right dorsal colitis (rdc) and generalized nsaid toxicity. rdc is an ulcerative disorder isolated to the right dorsal segment of the large intestine. , , the most prominent clinical signs of rdc are anorexia, lethargy, and colic. anorexia, depression, diarrhea, fever, and signs of section . inflammatory diseases of the gastrointestinal tract causing diarrhea endotoxemia also may be features. if the rdc is chronic, weight loss, intermittent colic, lethargy, anorexia, and ventral edema are common clinical signs, along with soft and unformed feces. severe ulceration of the right dorsal colonic mucosa results in proteinlosing enteropathy and significant hypoproteinemia attributable mainly to hypoalbuminemia. hypoproteinemia may be severe enough to cause peripheral (usually ventral) edema.in some cases, one may note dehydration, electrolyte abnormalities, neutropenia or anemia, azotemia, and biochemical abnormalities if the ulceration and diarrhea are severe or if systemic inflammation is present. clinical signs of generalized nsaid toxicity may vary from mild diarrhea with no systemic signs to severe dehydrating diarrhea with anorexia, fever, depression, peripheral edema, oral ulceration, and colic. , , clinical signs of systemic inflammation caused by endotoxemia may occur, manifested as poor peripheral perfusion, tachycardia, tachypnea, weakness, trembling, and cyanotic or hyperemic oral mucous membranes. hematuria or oliguria may be present if renal involvement is present. complications associated with other forms of severe enterocolitis, such as laminitis, thrombophlebitis, and severe weight loss, may occur. hematologic abnormalities of generalized nsaid toxicity are nonspecific and include neutropenia with a left shift or leukocytosis and hemoconcentration. serum biochemical analysis is characterized by profound hypoproteinemia, hyponatremia, and metabolic acidosis. , hypocalcemia, hypokalemia, hypochloremia, and elevated hepatocellular enzyme activities also may occur. hypoproteinemia may occur without signs of diarrhea. azotemia may be prerenal from dehydration but frequently is caused by renal failure resulting from a combination of hemodynamic and toxic renal injury. urinalysis frequently reveals hematuria, proteinuria, cylindruria, and isosthenuria. fecal occult blood is frequently detectable. diagnosis of either form of nsaid colitis is often presumptive, with a history of overdose of nsaids being strong evidence of nsaid toxicity. but as discussed earlier, toxicity may occur with dosage regimens that are not considered inappropriate, particularly if the horse experiences a concurrent period of dehydration. one can use ultrasonographic examination of the right dorsal colon to confirm a diagnosis of rdc, but the sensitivity of this method is questionable. ultrasonography ( . -to -mhz transducer at the right twelfth to fifteenth intercostal spaces below the margin of the lung axial to the liver) may reveal a thickened right dorsal colon (> . cm) and evidence of colonic edema in horses with rdc. however, the sensitivity of this method of diagnosis is questionable. one can use nuclear scintigraphy of horses after infusion with technetium -labeled white blood cells to document inflammation of the right dorsal colon. diagnosis of rdc may require one to perform laparotomy or laparoscopic examination of the right dorsal colon. one must rule out other causes of enterocolitis, such as salmonellosis, potomac horse fever, clostridiosis, and antibiotic-associated diarrhea. cantharidin is the toxic principle found in beetles of the genus epicauta, commonly known as blister beetles. [ ] [ ] [ ] ingestion of the beetles in contaminated alfalfa hay causes release of the toxin from the tissues of the beetle and absorption through the gastrointestinal tract. transcutaneous absorption may occur but appears to be rare in horses. blister beetles feed on the flowers of alfalfa and may be incorporated into processed alfalfa hay if the hay is cut and processed simultaneously, as by crimping. [ ] [ ] [ ] the beetles often swarm, and one may find large numbers of beetles in small portions of hay. the lethal dose of cantharidin is less than mg/kg, but the concentration of cantharidin varies from species to species of blister beetles and between sexes. , as many as to as few as to beetles may be lethal. usually, only one or a few horses fed contaminated hay ingest beetles because the beetles are concentrated in a small portion of the hay. however, outbreaks involving many horses on a farm have occurred. most cases occur in texas and oklahoma, but horses in other states may be affected as well, especially if hay is imported from states where blister beetles are common. peak incidence is in late summer and fall. the fatality rate may be % or greater, , but if the patient survives several days, recovery is probable. cantharidin is absorbed from the gastrointestinal tract and excreted via the kidney. cantharidin is a potent irritant, causing acantholysis and vesicle formation when applied topically. , , the chemical is thought to disrupt oxidative metabolism in the mitochondria, causing mitochondrial swelling, plasma membrane damage, and changes in membrane permeability. the mucosa of the gastrointestinal tract is affected most commonly in horses because they ingest the toxin. cell swelling and necrosis occur, resulting in mucosal ulceration. oral, esophageal, gastric, and small and large intestinal ulceration have been observed in natural and experimental canthariasis. , , severe fibrinous to pseudomembranous inflammation and submucosal edema of the intestine also have been reported. diarrhea probably results from the severe ulceration and inflammation of the large intestine, causing increased secretion of water, electrolytes, and protein and decreased absorption of fluid. large volumes of fluid and protein are lost in the gastrointestinal tract, causing hemoconcentration and profound hypoalbuminemia in some cases. , , cystitis and myocarditis occur in natural and experimentally produced cases of cantharidin toxicity. , , cystitis occurs because renal excretion of cantharidin results in high concentrations in urine. occasionally, hemorrhagic cystitis may occur, with hematuria or frank hemorrhage into the bladder. the cause of the myocarditis and myocardial necrosis is unknown but also may be a direct effect of the toxin on the myocardium. elevated plasma creatine kinase activity often occurs and has been postulated to arise from the damaged myocardium. , horses have a characteristically stiff gait, but histopathologic evidence of skeletal muscle injury that explains the elevated plasma creatine kinase activity has not been observed. the kidneys are often pale, swollen, and moist, with occasional infarcts. hypocalcemia and hypomagnesemia are biochemical features of cantharidin toxicity in horses that have not been explained. , , hypocalcemia may occur from hypoalbuminemia, but the ionized calcium concentration often is decreased along with the total calcium concentration, indicating that hypoalbuminemia is not responsible for the hypocalcemia. in addition, clinical signs of hypocalcemia, such as synchronous diaphragmatic flutter, are often associated with hypocalcemia from cantharidin toxicity. hypocalcemia associated with hypoalbuminemia alone does not produce clinical signs. cantharidin toxicity can cause a range of clinical signs from mild depression and abdominal discomfort to fulminant signs of toxemia and rapid death, depending on the ingested dose of toxin. most commonly, clinical signs include depression, sweating, irritability, abdominal pain, elevated heart and respiratory rates, fever, polyuria, polydypsia, and profuse diarrhea. , , blood is rarely visible in the feces. affected horses frequently posture to urinate; indeed, stranguria and pollakiuria are characteristic of cantharidin toxicity. signs of hypocalcemia include synchronous diaphragmatic flutter and tremors. a stiff and stilted gait may be evident. one may note neurologic signs such as head pressing, swaying, and disorientation. signs of systemic inflammation from endotoxemia may be apparent in severe cases. some horses develop severe depression and toxemia and may die within hours after ingestion of cantharidin without developing diarrhea. , hematologic abnormalities include hemoconcentration and neutrophilic leukocytosis. occasionally, neutropenia and leukopenia may accompany endotoxemia. serum biochemical analysis usually reveals elevated creatine kinase activity, hypocalcemia, and hypoalbuminemia. , biochemical abnormalities include hypocalcemia (ionized and total calcium concentrations), hypomagnesemia, and azotemia. , , urine specific gravity is characteristically in the hyposthenuric range. , microscopic hematuria and mild proteinuria may be evident. fecal occult blood is often present, but hematochezia is unusual. one can make a tentative diagnosis based on clinical signs and the finding of blister beetles in the hay. determining the species of the insects may be necessary to estimate the amount of cantharidin ingested. all species of epicauta contain cantharidin, but some have small amounts. definitive diagnosis requires the measurement of the cantharidin concentration in gastric or intestinal contents and urine. , measurement of cantharidin concentration in the beetles is often done but is not necessary. arsenic toxicosis is an unusual cause of diarrhea in horses, resulting from ingestion of arsenic-containing herbicides, insecticides, and other pest control products contaminating water or roughage used as a food source. the toxicity of arsenic depends on the valence of the element. , arsenate may be reduced to arsenite in mammalian systems, and arsenite is thought to be more toxic than arsenate and less rapidly excreted in urine. arsenate and arsenite uncouple oxidative phosphorylation, leading to breakdown of energy metabolism in the cells of many tissues. widespread cellular injury and death occur rapidly during acute arsenic toxicosis. multiorgan failure usually results. in fact, cardiomyopathy and pulmonary disease are common causes of death in human beings. damage to the large intestine is probably caused in part by direct cellular toxicity and corrosion by the compound. however, vasculitis is a hallmark of the disease in human beings and horses and is thought to be the most important mechanism of large intestinal disease in human beings. , acute hemorrhagic colitis is a feature of arsenic toxicosis, with severe mural edema and mucosal ulceration. profuse, hemorrhagic diarrhea and abdominal pain result. chronic arsenic toxicity can occur but appears to be rare in horses. acute depression, weakness, abdominal pain, hemorrhagic diarrhea, and shock are characteristic of acute arsenic toxicosis in horses. death may occur before diarrhea is evident. initial clinical signs may be difficult to distinguish from other peracute forms of colitis and are related to endotoxic shock, metabolic disturbances, and dehydration. later, cardiac arrhythmias, pulmonary edema, acute renal failure, and neurologic deficits (ataxia and stupor) may develop. one may observe anuria or polyuria. hemolytic anemia caused by preferential binding of arsenic compounds to red blood cells is a feature of arsenic poisoning in human beings. hematologic abnormalities may be apparent after the peracute stages from injury to bone marrow cells and ongoing hemolysis. leukopenia and thrombocytopenia have been described in human patients. serum biochemical analysis may reveal azotemia, hepatocellular enzyme activities higher than generally attributed to endotoxemia, and elevated creatine kinase activity. urine specific gravity may be in the isosthenuric range, with hematuria, cylindruria, and proteinuria evident by urinalysis. diagnosis may be possible by measuring blood and urine arsenic concentration, but these tests may not be diagnostic. postmortem diagnosis is by measuring the arsenic concentration in liver and kidney samples. history of exposure and clinical signs remain the primary means of diagnosis. other disorders associated with diarrhea in adult horses include anaphylaxis, carbohydrate overload, and sand enteropathy. careful evaluation of history, environment, and management will assist the clinician in arriving at an accurate diagnosis. severe intestinal anaphylaxis is a syndrome in horses characterized by peracute, rapidly fatal colitis. the severe syndrome is clinically and pathologically similar to other known causes of peracute colitis, such as salmonellosis, clostridiosis, and antibiotic-associated diarrhea. some cases are less severe and manifest as mild to moderate diarrhea or colic. an ige-mediated type i hypersensitivity or an ige-independent anaphylactoid reaction can produce the syndrome of intestinal anaphylaxis. , local gastrointestinal exposure to a food, environmental contaminant, drug, or other allergen usually induces intestinal anaphylaxis, , but anaphylaxis also may occur with systemic exposure to an allergen. [ ] [ ] [ ] [ ] massive mast cell degranulation, secretion of inflammatory mediators, and activation of enteric neural reflexes in the intestine causes profound alterations in blood flow, increased vascular permeability and interstitial edema, recruitment of neutrophils, altered motility, mucosal injury, absorption of microbial products, and mucosal hypersecretion. [ ] [ ] [ ] [ ] [ ] systemic signs may be caused by the anaphylactic reaction or may be associated with systemic inflammation triggered by microbial products (endotoxin) absorbed through the injured and hyperpermeable mucosa. intestinal anaphylaxis in horses may be a peracute, fulminant enterocolitis with endotoxemia that may be fatal. , this form is characterized by severe intramural edema and hemorrhagic inflammation of the large intestine, often producing submucosal thickening on the order of many centimeters. vascular thrombosis may be widespread with mucosal and serosal petechia and ecchymoses. less severe forms of intestinal anaphylaxis may manifest as patchy areas of intestinal edema and congestion. diarrhea results from intestinal inflammation initiated by the type i hypersensitivity response. many of the mediators of type i hypersensitivity, such as histamine and -hydroxytryptamine, have well-documented stimulatory effects on mucosal secretory activity, vascular and epithelial permeability, and motility [ ] [ ] [ ] in the intestine. systemic inflammation from endotoxemia may be overwhelming once the mucosal barrier breaks down. infarction of intestinal segments and other organs may occur from intravascular coagulation. ileus, abdominal distention, and moderate to severe abdominal pain may result from motility disturbances and infarction of the large intestine. the clinical signs are similar to those described for other forms of peracute colitis. however, the severity may vary, manifesting as colic or moderate diarrhea. characteristically, severe shock, signs of systemic inflammation from endotoxemia, and severe metabolic disturbances are observable. , heart and respiratory rates may be elevated greatly, with other signs of cardiovascular collapse such as weak and thready peripheral pulses and peripheral vasoconstriction. however, peripheral vasodilation may occur later in the course of disease. dark red, muddy, or cyanotic mucous membranes with a prolonged capillary refill time signify sepsis. borborygmi are usually absent, and abdominal tympany may be heard on percussion, following ileus. moderate to severe colic may accompany ileus. severe diarrhea may occur, but death may occur before diarrhea is evident. multiorgan failure from disseminated intravascular coagulation is not unusual. rapid onset of weakness, staggering, and trembling commonly precedes death. the syndrome may cause death in to hours. hematologic abnormalities include severe neutropenia and leukopenia, thrombocytopenia, and hemoconcentration. serum biochemical alterations include hyponatremia, hypokalemia, hypocalcemia, and severe metabolic acidosis. blood urea nitrogen and creatinine may be elevated from prerenal or renal azotemia. if acute renal failure accompanies the colitis, hyperkalemia may result. hepatocellular enzyme activity may be elevated in the serum from endotoxemia. severe coagulopathies are common, resulting in prolonged coagulation times, elevated fibrinogen, decreased antithrombin iii activity, and elevated plasma concentration of fibrin degradation products. analysis of peritoneal fluid may be valuable because infarction of the large intestine is not unusual. protein concentration and the white blood cell count may be elevated. red blood cell counts are less likely to be elevated, because infarction and not strangulation of the intestine occurs. diagnosis is based on clinical signs, postmortem findings, and exclusion of other causes. cultures and toxicologic analysis of fecal samples and gastrointestinal tissues fail to demonstrate a clear cause. other diagnostic tests are also inconclusive. if an antigen is suspected as the trigger of the anaphylaxis, a prausnitz-küstner passive cutaneous anaphylaxis sensitization test can confirm the presence of antigen-specific ige in the patient serum. overeating of soluble carbohydrates, especially so-called hot grains such as corn, overwhelms the digestive capability of the small intestine, resulting in a high percentage of the soluble carbohydrates entering the large intestine. the amount of soluble carbohydrates that produce diarrhea varies according to the previous dietary history of the individual. horses fed diets higher in soluble carbohydrates are more resistant to the deleterious effects of carbohydrate overload. gradual accommodation to a diet high in carbohydrates can be accomplished over several weeks. however, horses fed an unusually large amount of grains or other form of soluble carbohydrates often develop diarrhea and may, depending on the amount ingested, develop severe colitis, systemic inflammation from endotoxemia, metabolic acidosis, and laminitis. [ ] [ ] [ ] [ ] the pathogenesis of colitis from carbohydrate overload is caused primarily by the toxic effects on the microbial flora in the large intestine. a sudden delivery of soluble carbohydrates to the large intestine causes rapid fermentation by gram-positive lactic acid-producing bacteria and a sudden increase in organic acid production. the cecal ph rapidly decreases, and the lactic acid concentration rapidly increases. rapid organic acid production overwhelms the buffering capacity of the large intestine not only by directly depleting the buffers found in the contents but also by reducing the efficiency of buffer secretion. bicarbonate secretion is linked to absorption of volatile fatty acids, which are produced in low amounts by fermentation of soluble carbohydrates. the contents of the large intestine become profoundly acidic, resulting in unfavorable conditions for the microbial flora. lactic acid-producing bacteria flourish, while the gram-negative bacteria, especially the enterobacteriaceae, are killed in large numbers by the acids. large quantities of endotoxin are released from the dying bacteria. the osmotic load from the lactic acid produced in the large intestine is an important factor in the development of diarrhea because organic acids such as lactic acid are absorbed poorly. mild cases of carbohydrate overload may result purely from osmotic diarrhea. in more severe cases, the acidic contents of the large intestine are toxic to the mucosa, causing necrosis of the mucosal tissues, similar to that occurring in ruminal acidosis. mucosal ulceration allows absorption of large quantities of endotoxin and lactic acid produced by the massive die-off of acid-intolerant microbes and fermentation of soluble carbohydrates, normally poorly absorbed by intact mucosa. systemic inflammation from endotoxemia may be overwhelming, and profound metabolic acidosis may occur. secretory diarrhea caused by the direct effects of acid luminal contents on the mucosa, as well as the effects of inflammatory mediators on enterocyte secretion, worsens the acidosis and dehydration. systemic inflammation from endotoxemia, along with intestinal inflammation, adversely affects intestinal motility, and ileus develops. ileus and gas production from fermentation of the carbohydrates may cause severe distention of the large intestine and signs of abdominal pain. laminitis is a frequent complication of endotoxemia and lactic acidosis. in fact, carbohydrate overload is used to induce laminitis as an experimental model because of the consistency of the laminitis produced. [ ] [ ] [ ] clinical signs of colitis from carbohydrate overload can vary according to the amount of carbohydrates ingested and accommodation of the flora to a high-carbohydrate diet. mild cases may result in a transient osmotic diarrhea with no systemic effects. more severe cases are characterized by signs similar to those described for other forms of colitis, including abdominal pain, moderate to severe diarrhea, and dehydration. signs of endotoxemia and sepsis are frequently present in severe cases. elevated heart and respiratory rates are common, with peripheral vasoconstriction early in the disease, followed by peripheral vasodilation as the disease progresses. depression may be profound from metabolic acidosis and endotoxemia. abdominal auscultation and percussion may reveal ileus and intestinal tympany. nasogastric intubation may yield significant gastric acidic reflux. one may note particles of grain in the gastric reflux and the feces, if grain overload is the source of the carbohydrate overload. laminitis may complicate mild and severe cases of carbohydrate overload, especially if the animal has had previous bouts of laminitis. hematologic abnormalities include neutropenia and leukopenia. severe dehydration may result in profound hemoconcentration. protein loss later in the course of disease may result in hypoproteinemia. serum biochemical abnormalities include azotemia, elevated hepatocellular enzyme activity, hyponatremia, and hypokalemia. severe hypocalcemia and metabolic acidosis are characteristic of the disease. serum lactate concentrations are elevated in the absence of evidence of intestinal strangulation or infarction. peritoneal fluid analysis often reveals no abnormalities. sand enteropathy is described in more detail under the heading of obstructive diseases, because acute obstruction is often associated with abnormally large amounts of sand in the large intestine. however, chronic sand-induced diarrhea is a distinct syndrome that can occur at any age from abnormal accumulation of sand in the large intestine. , chronic diarrhea and signs of colic may occur without obstruction. the pathogenesis of sand accumulation in individual horses, other than simple ingestion of large quantities, is unclear. presumably the sand causes irritation and may disrupt motility, leading to diarrhea. the diarrhea is usually not severe and dehydrating and may be intermittent. weight loss is characteristic and can be severe in some cases. complications may occur such as peritonitis and acute obstruction. diagnosis usually is based on finding abnormal amounts of sand in the feces. because sand-induced chronic diarrhea is associated primarily with sand accumulation in the ventral colon, auscultation of the ventral abdomen immediately behind the xiphoid process may reveal characteristic sand sounds. this technique is only sensitive if peristalsis is present. ultrasonography also may be useful to identify sand in the ventral colon but is not useful to quantitate the amount of sand. occasionally, radiography may be required to detect sand in the colon. the principles of therapy of acute diarrhea from colitis are similar regardless of the cause and include replacement of fluid and electrolyte losses, control of colonic inflammation and reduction of fluid secretion, promotion of mucosal repair, control of endotoxemia and sepsis, and reestablishment of normal flora. this section focuses on a review the principles of therapy with references to specific therapies for particular causes as they arise. replacement of fluid and electrolyte losses is of primary concern in treating horses with salmonellosis. depending on the severity of the disease, fluid losses may be minimal or massive. one can administer fluid and electrolytes orally or intravenously. some horses with mild to moderate diarrhea may maintain hydration and electrolyte balance by consuming water and electrolytes voluntarily. freshwater and water containing electrolytes should be available in all cases. in many instances, periodic nasogastric intubation and administration of water and electrolytes via the tube may be sufficient to maintain hydration. in more severe cases, one can maintain indwelling nasogastric tubes and can administer up to to l of fluid by the tube every to minutes, if ileus is not evident. however, intravenous administration of fluids is preferred in most cases, requiring significant quantities of fluid to replace and maintain hydration and electrolyte balance. for patients with severe diarrhea to require large volumes ( to l/day) of intravenous fluids to maintain hydration is not unusual. frequent monitoring of packed cell volume, serum electrolyte concentration, venous blood gases or total serum carbon dioxide, blood urea nitrogen and creatinine, urine protein and cytologic findings, and body weight is important to monitor hydration, electrolyte and acid-base balance, and renal function. isotonic sodium chloride or lactated ringer's solution frequently is used to restore and maintain fluid and electrolyte balance. one can add potassium chloride to the fluids and administer it at a rate up to . to . meq/ kg/hr. generally, a rate of less than . meq/kg/hr is used. hypertonic nacl solutions ( to l of % to % nacl) have been used in horses that are severely hyponatremic (< meq/dl). one should not administer hypertonic solutions to severely dehydrated horses, but such solutions have been used clinically without complication and with considerable beneficial effect in patients with endotoxemia. the beneficial effects of hypertonic nacl are short-lived ( to minutes). one should administer isotonic solutions concurrently or immediately following administration of hypertonic nacl solutions. isotonic ( . %) or hypertonic ( . %) sodium bicarbonate solutions are used to correct metabolic acidosis. prolonged administration of sodium-containing fluids may promote diuresis and renal water loss or accumulation of peripheral edema and should be used conservatively when one notes a free water loss. administration of isotonic dextrose ( %) or . % dextrose/ . % nacl solutions may be beneficial when free water loss (sodium excess) is evident. many horses with acute colitis are concurrently hypoproteinemic because of gastrointestinal losses and are absorbing bacterial products that induce a systemic inflammatory response. thus plasma oncotic pressures are abnormally low in the face of increased vascular permeability. interstitial edema formation is a clinical problem in these patients and contributes to organ dysfunction. crystalloid fluids, although critical for replacing water and electrolyte losses from diarrhea, actually may contribute to a drop in plasma oncotic pressure because of hemodilution. , administration of colloid solutions are important for volume expansion and to maintain plasma oncotic pressures, which improve tissue perfusion and oxygenation and organ function in hypovolemic, hypotensive, and hypoproteinemic patients with or without systemic inflammatory response syndrome. colloids are more effective than crystalloid fluids at expanding plasma volume and thus require smaller volumes. moreover, the effect of colloid volume expansion is longer lasting than crystalloid fluid volume expansion, because colloids are retained in the vasculature better. , natural colloids, such as plasma and purified albumin are used commonly. in addition to its beneficial colloidal properties, plasma harvested from donor horses immunized with rough mutants of escherichia coli (j ) or salmonella typhimurium may have other benefits for treatment of endotoxemia from gastrointestinal disease. , the horse may require large volumes ( to l/day) to increase and maintain plasma protein concentration significantly. synthetic colloids such as dextrans, starches, or polymerized hemoglobin are also available for use in the horse. hetastarch ( to ml/kg of a % solution) increases colloidal oncotic pressures for up to hours in hypoproteinemic horses and has beneficial effects on cardiac output and other cardiorespiratory parameters, vascular permeability, interstitial fluid content, and tissue perfusion in models of hypoproteinemia and systemic inflammatory response syndrome. when one administers synthetic or even natural colloids, monitoring plasma oncotic pressure may be more relevant than monitoring plasma protein concentrations as a means of assessing the need for plasma or other colloid administration. hetastarch may prolong bleeding times by altering von willebrand's factor function; thus one should use this synthetic colloid cautiously in horses with suspected coagulopathies, active hemorrhage, or other bleeding problems. control of colonic inflammation and secretion is a difficult and poorly studied aspect of equine acute colitis. the role of inflammation and mediators such as prostaglandins as causes of fluid loss is well known for salmonella and clostridium infections. cox inhibitors (nsaids) have antisecretory effects in the equine colon and in models of salmonellosis that appear to extend to clinical management of salmonellosis. , , [ ] [ ] [ ] indeed, nsaids commonly are administered to horses with salmonellosis. however, prostaglandins such as pge and pgi are also cytoprotective to gastrointestinal mucosa and critical for mucosal repair. the doses of nsaids used pharmacologically to inhibit colonic inflammation and secretion in fact may be detrimental to the mucosa if not used judiciously. nsaids have been shown to exacerbate colonic inflammation in human beings with inflammatory colitis, impede mucosal healing in several models of mucosal injury, and have well-documented detrimental effects on colonic mucosa in horses. , , in addition to toxicity to the colonic mucosa, gastric ulceration is not unusual in horses with enterocolitis and may be related to treatment with nsaids. in addition to nsaids, other drugs occasionally are used as antiinflammatory or antisecretory therapy. metronidazole has beneficial effects in experimental models of gastrointestinal inflammation, including nsaid toxicity and may be useful for treating horses with colitis, but evidence supporting its use is lacking. bismuth subsalicylate solutions administered orally often are used to decrease inflammation and secretion in the colon. in adult horses the volume of solution necessary to be beneficial is large ( to l every to hours). often the solution is administered twice daily instead of to times daily. if one does not achieve a beneficial effect within to days of treatment, one should discontinue administration of bismuth subsalicylate solution. one can administer the treatment more frequently in foals, and clinical improvement occurs more often in foals than in adult horses. in light of the role of reactive oxygen metabolites in colonic inflammation, free radical scavengers have been advocated to reduce the effects of these molecules. sulfasalazine metabolites have been shown to reduce reactive oxygen metabolite-induced colonic inflammation in other species, and sulfasalazine has been used to treat chronic inflammatory disease in horses but has not been used to treat acute colitis. the only free radical scavenger used commonly in horses with colitis is dimethyl sulfoxide, which at a dosage of . to . g/kg intravenously every to hours in a % solution has been used in clinical cases of colitis, but evidence of efficacy has not been established. systemic inflammatory response syndrome associated with endotoxemia frequently occurs in patients with salmonellosis. the principles of therapy for endotoxemia are covered in detail elsewhere in this chapter. oral administration of activated charcoal and mineral oil is used commonly to reduce absorption of endotoxin in horses with colitis. low doses of nsaids (such as flunixin meglumine at . to . mg/kg intravenously every to hours) inhibit eicosanoid synthesis induced by endotoxin. in addition, administration of nsaids prevents laminitis from endotoxemia, a devastating complication of salmonellosis. one must remember that prostaglandins are important for mucosal healing and may worsen mucosal injury in colitis. although the benefits of low doses of nsaids administered to horses with systemic inflammatory response syndrome are believed to outweigh the risks of worsening gastrointestinal damage, judicious use is recommended. sucralfate ( mg/kg orally every hours) has been advocated to aid in healing the colonic mucosa, but the efficacy in the large intestine is questionable. misoprostol ( µg/kg orally to times daily) and other synthetic pge analogs have been shown in several species including horses to enhance mucosal healing in the intestine and promote recovery in experimental models of colitis. misoprostol may be particularly useful for treating nsaid toxicity, the generalized form or rdc. however, the efficacy of misoprostil for hastening mucosal healing is clinically unproven in equine colitis. the primary drawbacks of prostaglandin analogs such as misoprostol are the side effects of the drug, including abdominal cramping, diarrhea, sweating, and abortion in pregnant mares. one can add psyllium mucilloid to the diet ( tablespoons once or twice daily) to increase the production of scfas in the colon. amylase-resistant fermentable fiber such as psyllium is hydrolyzed by colonic bacteria to scfas such as butyrate, which represent a major energy source for colonocytes. butyrate and other scfas hasten epithelial maturation and stimulate salt (and thus fluid) absorption in the colon, improve the clinical course of ulcerative colitis, and hasten colon healing. psyllium is itself a source of butyrate in the colon and also promotes the movement of amylase sensitive carbohydrates into the distal colon, which then are fermented to scfas. thus psyllium is thought to be clinically useful for promoting mucosal healing in colitis. many horses with salmonellosis or other forms of colitis have mild to severe signs of abdominal pain from gas and fluid distention of the colon, colonic ischemia, or infarction. one can accomplish analgesia with nsaids such as flunixin, but the potential for worsening mucosal injury or nephrotoxicity may prevent the use of analgesic doses, especially in horses with suspected nsaid toxicity. newer nsaids that specifically target cox- (the inducible cox) but have little activity against cox- (the constitutive cox) may be useful analgesics that spare the gastrointestinal mucosa. for example, etodolac ( to mg/kg intravenously or orally once daily) has analgesic properties in horses and may spare the intestinal mucosa from the detrimental effects associated with nonselective cox inhibitors (a.t. blikslager, personal communication, ) . however, the specificity for cox- in horses is unproven. thus avoiding the use of any nsaids in horses with rdc or other forms of nsaid toxicity is advisable. xylazine or detomidine may provide temporary relief of pain. butorphanol is a useful analgesic that one can administer intramuscularly ( . mg/kg every hours) or as a continuous infusion. an infusion of . µg/kg/hr in isotonic crystalloid fluid such as lactated ringer's solution has been suggested. continuous lidocaine infusions ( . mg/kg intravenous loading dose administered slowly over minutes and followed by mg/kg/hr infusion in isotonic crystalloid fluids) can provide profound visceral analgesia and may have added prokinetic benefits if ileus is present. broad-spectrum antibiotic treatment often is recommended in neutropenic horses or horses with signs of septicemia. neutropenia is associated with an increased risk of septicemia and septic complications such as septic phlebitis and infection of surgical site. septicemia is a potentially life-threatening complication of enterocolitis and may be caused directly by salmonella, clostridium, other invasive enteric bacteria, or indirectly by toxic injury to the colonic mucosa that breaks down the barrier to luminal microbes. neutropenia possibly may weaken host defenses enough to render horses susceptible to organisms that breach the mucosal barrier. although most attempts to culture bacteria from the blood of adult horses with colitis fail to isolate organisms, no detailed studies have been undertaken to determine the prevalence of bacteremia or septicemia in these patients. disseminated aspergillosis has been reported in horses as a complication of acute colitis, demonstrating the potential for systemic infections with rarely pathogenic organisms stemming from colonic mucosal injury in the face of potential immunosuppression from neutropenia. , broad-spectrum antibiotics lessen septic complications in human patients. however, evidence supporting this principle in horses with colitis is lacking. treatment with antibiotics is controversial in horses with salmonellosis and is not thought to alter the course of the enterocolitis. antibiotics directly targeted at the salmonella are reserved for patients with the enteric fever (septicemia) form of salmonellosis, documented with positive blood cultures. lipid-soluble antibiotics are suited ideally for salmonella infections, because the bacteria persist intracellularly. trimethoprim-sulfadiazine or other potentiated sulfa drugs, enrofloxacin, and chloramphenicol are preferred antibiotics for the enteric fever form of salmonellosis for this reason. as with other causes of enterocolitis, the use of antibiotics for equine monocytic ehrlichiosis is controversial. fear of inducing salmonellosis or other forms of antibiotic-induced diarrhea and the difficulty of diagnosing the disease early have caused most authors to recommend judicious use of antibiotics. however, in patients with a high suspicion of neorickettsia risticii infection, treatment with antibiotics often is indicated before definitive diagnosis. lipid-soluble drugs are desirable because the organism can live within cells. oxytetracycline ( . mg/kg intravenously every hours), doxycycline ( mg/kg orally every hours), trimethoprim-sulfadiazine ( mg/kg trimethoprim orally or intravenously every to hours and mg/kg sulfadiazine every to hours), or erythromycinrifampin ( mg/kg and mg/kg, respectively, orally every hours) have been used effectively to treat clinical cases. , [ ] [ ] [ ] the tetracyclines appear to be the most effective antibiotics for treatment of potomac horse fever. treatment is most successful if initiated before the onset of diarrhea. , clostridiosis if one has administered antibiotics since the onset of enterocolitis, one should discontinue administration as soon as possible. specific treatment with metronidazole ( to mg/kg orally every hours) is effective for treating clostridiosis in human beings and appears to be effective in horses. , metronidazole resistance in clinical isolates of clostridium difficile has been reported in one outbreak but appears to be rare in most human and equine cases. metronidazole-resistant isolates were sensitive to vancomycin, which may be effective for treating clinical cases if one suspects metronidazole resistance. however, metronidazole remains the treatment of choice. some authors describe the off-label use of c. perfringens type c antitoxin in cases of neonatal clostridiosis, described in more detail elsewhere. antitoxin preparations generally are not advocated for use in adult horses with clostridiosis. lawsonia intracellulare is susceptible to a variety of antibiotics in vitro, including chlortetracycline, erythromycin, penicillin, difloxacin, and ampicillin. lipid-soluble antibiotics with a large volume of distribution usually are chosen to treat proliferative enteropathy because l. intracellulare is an intracellular organism. erythromycin estolate ( to mg/kg orally every to hours) alone or with rifampin ( mg/kg orally every hours) is the most commonly reported efficacious treatment for proliferative enteropathy. chloramphenicol ( mg/kg orally every hours) has also been reported to be effective if erythromycin worsens the diarrhea. anecdotal reports suggest that oxytetracycline and doxycycline also may be effective. supportive care including maintenance of hydration and electrolyte balance and plasma or colloid administration to increase colloid oncotic pressure in hypoalbuminemic patients is also indicated. one should treat affected foals until clinical signs, hypoproteinemia, and ultrasonographic evidence of intestinal thickening resolve. the prognosis depends on the duration of the disease and the degree of fibrosis and destruction of the intestinal architecture. hypercoagulability is a common complication of enterocolitis, associated with systemic inflammation from endotoxemia. administration of heparin ( to iu/kg subcutaneously or intravenously every to hours) may prevent thrombosis in these patients, provided antithrombin iii concentrations are adequate in the plasma. concentrated sources of antithrombin iii are not available for use in horses, but whole plasma may provide an important source. treatment with heparin is thought to decrease thrombosis, especially of the jugular vein, a serious complication of salmonellosis. low-dose aspirin treatment ( mg/kg orally every to hours) along with heparin treatment may provide added benefit by irreversibly inhibiting platelet function. heparin and aspirin may have protective effects on the digital lamina. , heparin also may enhance the phagocytic activity of the reticuloendothelial system by enhancing the efficiency of opsonins such as fibronectin and immunoglobulin, thereby stimulating phagocytosis of products of coagulation and possibly other particles, including bacteria. , maintenance of the bacterial flora and antagonism of pathogenic bacteria such as salmonella in the gastrointestinal tract are important defense mechanisms preventing colonization by pathogenic bacteria. the use of probiotic preparations containing beneficial bacteria has been shown to prevent colonization of pathogenic bacteria, including salmonella, in poultry. little work has been done to investigate the efficacy of these products in preventing salmonellosis in horses, but ongoing studies may provide important information. probiotic and other preparations designed to restore normal flora to the gastrointestinal tract, such as fecal suspensions, sour milk, and yogurt, have been used clinically to shorten the course of salmonellosis, with variable results. therefore prevention of infection by using probiotic agents and other means is important. exposure of susceptible horses to salmonella should be avoided, but the task is difficult, especially because asymptomatic infections are common and the bacteria are ubiquitous in the environment. prophylactic use of probiotic preparations, judicious use of antibiotics in susceptible horses, control of environmental conditions such as temperature, and restricted exposure to pathogenic bacteria are important for control of salmonellosis. because altered large intestinal flora appears to play an important role in the pathogenesis of equine intestinal clostridiosis or any antibiotic-associated diarrhea, probiotic preparations have been advocated to treat affected horses. sour milk, a product containing lactose-producing streptococcus species, appears to improve the clinical course greatly in horses suspected of having clostridium perfringens type a infection. sour milk may benefit the patient by altering the flora and antagonizing enterotoxigenic c. perfringens type a but also is reported to be bactericidal against c. perfringens type a. preparations of saccharomyces boulardi are effective for reducing diarrhea and the frequency of c. difficile recurrence in human beings. however, whether relapse is a problem in horses with c. difficile colitis is not clear. lactobacillus preparations have a protective effect in human beings and decrease the severity and duration of antibioticassociated diarrhea. , however, evidence of their clinical usefulness in horses is lacking. good nursing care and adequate nutrition are vital to the treatment of horses with salmonellosis. salmonellosis is a severely catabolic disease, increasing caloric requirements greatly. normal intake of roughage to provide energy may be inadequate; however, one should avoid feeding of grains to prevent carbohydrate overload. dietary management usually consists of restricting or eliminating long-stem roughage (hay) from the diet and feeding exclusively a complete pelleted diet (at least % dietary fiber). the rationale behind this recommendation is to reduce the mechanical load on the colon. frequent meals ( to times a day) are recommended. one can add corn oil ( cup every to hours) to the pellets to increase the caloric intake without adding roughage or grain. one should note that if a horse with colitis refuses to eat pelleted feed, then one should feed good-quality grass hay. in anorectic or severely catabolic patients, enteral and parenteral nutrition (total and partial) has been used successfully to provide calories and nutritional support. strongylus vulgaris infection requires treatment of the migrating parasite larvae and the lesions produced by the parasite. fenbendazole ( mg/kg orally every hours for days or mg/kg orally every hours for days) and ivermectin ( mg/kg orally) are effective in killing fourth-stage larvae. other anthelmintics also may be part ii disorders of specific body systems effective when given at higher doses than those required to kill adult worms. the efficacy of these anthelmintics against larvae within thrombi is not known. thrombolytic and antithrombotic therapy has been advocated in horses with suspected strongylosis. , heparin ( to iu intravenously or subcutaneously every to hours) is often administered as an anticoagulant. aspirin ( to mg/kg orally every to hours) is usually combined with heparin to inhibit platelet adhesion. aspirin also may inhibit release of platelet products such as thromboxane that affect the motility of the large intestine. low-molecular-weight dextrans have been advocated as antithrombotics that act by inhibiting platelet function and coagulation. , the clinical efficacy of dextran administration appears to be good, but no controlled studies have been performed. anthelmintic administration is usually the only treatment necessary for mild to moderate cases of cyathostomiasis treated early in the course of the disease (within to weeks of onset). fenbendazole is effective against many larval stages, but resistance is increasing. although the reported efficacy of ivermectin varies against certain stages, one study reported an overall efficacy of %. currently, fenbendazole ( . to mg/kg orally every hours for days) followed on day by ivermectin ( mg/kg orally) is the most commonly advocated treatment regimen. , moxidectin ( µg/kg orally once daily) also may be effective against adults and l and l larval stages and may be useful for treating cyathostomiasis. antiinflammatory therapy also may be beneficial, especially in severe or refractory cases. nsaid administration may have limited value, but dexamethasone appears to be efficacious in refractory cases when used with larvicidal anthelmintics. , pretreatment with dexamethasone or prednisolone is indicated before anthelmintic administration if heavy larval loads are suspected to prevent an acute exacerbation of the disease by rapid death of encysted larvae. bismuth subsalicylate often is administered orally as an antisecretory agent in young animals. supportive care may be necessary in severe cases, particularly if hypoproteinemia is severe. horses occasionally require administration of intravenous crystalloid fluids and plasma or other colloids. proper nutritional support is also important. supportive care is the most important principle of therapy for cantharidin toxicity. intravenous fluid administration; maintenance of electrolyte balance, especially calcium; and prevention of further renal and urinary tract damage is important. , diuresis by intravenous fluid administration is often sufficient to prevent renal failure. furosemide often is administered after rehydration of the patient to further promote diuresis and to decrease the concentration of the toxin in the urine, which may ameliorate some of the effects on the urinary tract mucosa. diuresis also has been suggested to increase clearance of the toxin, but no evidence for this has been found. judicious use of nsaids may be necessary to control abdominal pain but should be reserved until the patient is rehydrated and renal failure has been ruled out. cantharidin is lipid-soluble; therefore oral administration of mineral oil may prevent further absorption of the toxin. activated charcoal often is administered with the mineral oil. to reduce arsenic absorption, one should initiate administration of cathartics such as mineral oil and magnesium sulfate slurries and activated charcoal by nasogastric tube immediately. chelation therapy with sodium thiosulfate to g in ml of water orally and dimercaprol (bal) mg/kg intramuscularly every hours is indicated. dimercaprol is a specific antidote for trivalent arsenicals, but its efficacy in horses is questionable. intravenous fluid administration may help treat shock, replace fluid lost in feces, and promote diuresis but should be monitored carefully because pulmonary edema is a frequent complication. the horse may require more specific treatment of renal, cardiac, pulmonary, or neurologic disease. treatment of intestinal anaphylaxis is in principle similar to treatment of other forms of colitis but is often unsuccessful because of the rapidly progressive nature of the syndrome. inclusion of heparin in intravenous fluids ( to iu/kg intravenously every to hours) may help prevent vascular thrombosis. administration of hypertonic saline solutions or colloids may prove to be useful during initial periods of shock. early treatment with prednisolone succinate ( to mg/kg intravenously) or dexamethasone ( . to . mg/kg intravenously) may be essential for successful treatment. mild cases of carbohydrate overload may not require treatment other than exclusion of grains from the diet for several days to weeks and gradual reintroduction of grain into the diet later if the horse needs the extra energy. patients showing signs of colic or diarrhea without other systemic signs may benefit from administration of mineral oil, charcoal, and fluids via nasogastric tube. one also may lavage residual carbohydrates from the stomach with the nasogastric tube. nsaids such as phenylbutazone ( . to . mg/kg/day intravenously) or flunixin meglumine ( mg/kg intravenously every hours) often are administered to prevent laminitis. phenoxybenzamine and heparin given before the onset of laminitis may prevent or decrease the severity of laminitis. , more severe cases with dehydrating diarrhea, systemic signs of endotoxemia, or metabolic acidosis require intravenous fluid support to maintain water, electrolyte, and acid-base balance in addition to the previously mentioned treatments. large amounts of bicarbonatecontaining solutions may be required. one should take care when administering hypertonic bicarbonate solutions, because many patients already may be hyperosmotic from lactic acidemia. isotonic sodium bicarbonate . % may be useful in the hyperosmotic patient. careful attention to calcium balance is also important, because severe hypocalcemia may occur. one should institute aggressive therapy for systemic inflammation from endotoxemia. one should administer broad-spectrum antibiotics intravenously to combat bacteremia and septicemia, which frequently complicate colitis induced by carbohydrate overload. in extreme cases, especially if the patient has ingested a large quantity of grain, surgical removal of the grain from the large intestine may be indicated, especially if one can accomplish surgery before the onset of severe clinical signs. however, administration of oral cathartics, such as magnesium sulfate slurries or mineral oil, or a combination of these, is often sufficient to clear the carbohydrates from the large intestine before fermentation, mucosal damage, and absorption of endotoxin and lactic acid occur. oral administration of activated charcoal may prevent absorption of endotoxin by binding the molecules in the lumen of the bowel. in any case, one should discontinue feeding of the source of the soluble carbohydrates, such as grains. one should feed the horse low-carbohydrate and low-protein roughage such as grass or oat hays until the microbial flora recovers. oral administration of probiotic preparations containing lactobacillus is contraindicated; however, other sources of normal equine large intestinal microbial flora, such as fecal extracts from normal feces, may be useful to reintroduce appropriate microorganisms. complications from laminitis and sepsis are common and often cause death. treatment of sand enteropathy requires removal of the sand from the gastrointestinal tract using psyllium products and magnesium sulfate slurries administered orally. analgesics may be required initially to relieve pain and stimulate appetite. a diet high in roughage often stimulates further passage of sand. treatment may require several weeks to remove as much sand as possible. prevention of the disease is important, and recurrence is not unusual. lumen results in clinical signs similar to those of simple obstruction, occlusion of the blood supply results in a more rapid deterioration of the intestinal mucosa and subsequent onset of endotoxemic shock. although a great deal of interest in the relevance and treatment of intestinal reperfusion injury has arisen recently, - the lesion that develops during strangulation is often severe, leaving little viable bowel for further injury during reperfusion. although extensive lengths of strangulated small intestine may be resected, strangulation of the large colon presents a much greater treatment dilemma because strangulated intestine usually extends beyond the limits of surgical resection. therefore horses with large intestinal strangulation often recover with extensive intestinal injury left in place. thus subtle degrees of reperfusion injury may be important in horses with large colon disease, warranting further work in this area in an attempt to reduce mortality. strangulating obstruction may be divided into hemorrhagic and ischemic forms. , hemorrhagic strangulating obstruction, which is most common, involves initial occlusion of veins before occlusion of arteries because of the greater stiffness of arterial walls. this lesion is characterized by a darkened appearance to affected bowel and increased thickness as blood is pumped into the lesion. ischemic strangulating obstruction occurs if the intestine is twisted tightly enough to occlude arteries and veins simultaneously. in the case of the colon, such strangulation has been suggested to be determined by how much ingesta is in the colon, because intestinal contents may prevent the intestine from twisting tightly. tissue involved in ischemic strangulating obstruction appears pale and of normal or reduced thickness because of a complete lack of blood flow ( figure . - ). bowel peripheral to strangulating lesions also may become injured because of distention, which reduces mural blood flow once it reaches critical levels. furthermore, as this intestine is decompressed, it also may undergo reperfusion injury. [ ] [ ] [ ] small intestinal strangulation horses with small intestinal strangulating obstruction typically have moderate to severe signs of abdominal pain that are only intermittently responsive to analgesic medications. during the latter stages of the disease process, horses may become profoundly depressed rather than painful as affected intestine necroses. horses have progressive signs of endotoxemia, including congested mucous membranes, delayed capillary refill time, and an elevated heart rate (> beats/min in most cases). in addition, one typically obtains reflux following passage of a stomach tube, and one usually can detect loops of distended small intestine on rectal palpation of the abdomen. however, these latter findings vary depending on the duration and location of the obstruction. for example, horses with ileal obstructions tend to reflux later in the course of the disease process than horses with a jejunal obstruction. furthermore, a horse that has an entrapment of small intestine in the epiploic foramen may not have palpable loops of small intestine because of the cranial location of these structures. abdominocentesis can provide critical information on the integrity of the intestine and is indicated in horses in which one suspects strangulation of the small intestine. a horse that has signs compatible with a small intestinal obstruction and additionally has serosanguinous abdominal fluid with an elevated protein level (> . mg/dl) is likely to require surgery, although one must differentiate these a b figure . - ischemic strangulating obstruction of the small colon by a mesenteric lipoma. a, the lipoma (arrow) has encircled a segment of small colon tightly. b, following resection of the lipoma, a pale area of strangulated small colon clearly is demarcated (arrows), the appearance of which is consistent with ischemic strangulating obstruction. cases from proximal enteritis. in general, horses with small intestinal strangulation show continued signs of abdominal pain, whereas horses with proximal enteritis tend to be depressed after initial episodes of mild abdominal pain. in addition, horses with small intestinal strangulation continue to deteriorate clinically despite appropriate medical therapy and will likely begin to show an increased white blood cell count (> , cells/µl) in the abdominal fluid as the duration of strangulation increases. however, cases occur in which the differentiation between small intestinal strangulation and proximal enteritis is not clear, at which point one may elect surgery rather than risking delay of abdominal exploration of a horse with a potential strangulating lesion. the prognosis for survival in horses with small intestinal strangulating lesions is generally lower than for most forms of colic. however, recent studies indicate that in excess of % of horses with small intestinal strangulating lesions are discharged from the hospital. nonetheless, veterinarians should warn owners that the long-term survival rates are reduced substantially to below %, in part because of long-term complications such as adhesions. , in addition, the prognosis is particularly low for some forms of strangulation, including entrapment of small intestine within a mesenteric rent. the epiploic foramen is a potential opening (because the walls of the foramen are usually in contact) to the omental bursa located within the right cranial quadrant of the abdomen. the foramen thus is bounded dorsally by the caudate process of the liver and caudal vena cava and ventrally by the pancreas, hepatoduodenal ligament, and portal vein. intestine may enter the foramen from the visceral surface of the liver toward the right body wall or the opposite direction. studies differ as to which is the most common form. , in the case of entrapments that enter the foramen in a left-to-right direction, the omental bursa ruptures as the intestine migrates through the epiploic foramen, which may contribute to intraabdominal hemorrhage often seen with this condition. clinical signs include acute onset of severe colic with examination findings compatible with small intestinal obstruction. the condition tends to be more prevalent in older horses, possibly because of enlargement of the epiploic foramen as the right lobe of the liver undergoes ageassociated atrophy. however, the disease also has been recognized in foals as young as months of age. one makes a definitive diagnosis at surgery, although ultrasonographic findings of distended loops of edematous small intestine adjacent to the right middle body wall suggest epiploic foramen entrapment. in general, thickened, amotile intestine on ultrasonographic examination is highly predictive for small intestinal strangulating obstruction. small intestine entrapped in the epiploic foramen may be limited to a portion of the intestinal wall (parietal hernia), and the large colon may become entrapped within the epiploic foramen. in treating epiploic foramen entrapment, one must not enlarge the epiploic foramen by blunt force or with a sharp instrument, because rupture of the vena cava or portal vein and fatal hemorrhage may occur. prognosis has improved substantially over the last decade, with current short-term survival rates (discharge from the hospital) ranging from % to %. preoperative abdominocentesis has been found consistently to be the most predictive test of postoperative survival. , lipomata form between the leaves of the mesentery as horses age and develop mesenteric stalks as the weight of the lipoma tugs on the mesentery. the stalk of the lipoma subsequently may wrap around a loop of small intestine or small colon causing strangulation. one should suspect strangulating lipomata in aged (> years old) geldings with acute colic referable to the small intestinal tract. , ponies also appear to be at risk of developing disease, suggesting alterations in fat metabolism may predispose certain horses to development of mesenteric lipomata. one usually makes the diagnosis at surgery, although on rare occasions one can palpate a lipoma per rectum. treatment involves surgical resection of the lipoma and strangulated bowel, although strangulated intestine is not always nonviable. studies indicate that approximately % to % of horses are discharged from the hospital following surgical treatment. a volvulus is a twist along the axis of the mesentery, whereas torsion is a twist along the longitudinal axis of the intestine. small intestinal volvulus theoretically is initiated by a change in local peristalsis or the occurrence of a lesion around which the intestine and its mesentery may twist (such as an ascarid impaction). volvulus is reportedly one of the most commonly diagnosed causes of small intestinal obstruction in foals. , the theory is that young foals may be at risk of small intestinal volvulus because of changing feed habits and adaptation to a bulkier adult diet. onset of acute, severe colic, a distended abdomen, and radiographic evidence of multiple loops of distended small intestine in a young foal suggest small intestinal volvulus. however, one cannot differentiate volvulus from other causes of small intestinal obstruction preoperatively. in adult horses, volvulus frequently occurs in association with another disease process, during which small intestinal obstruction results in distention and subsequent rotation of the small intestine around the root of the mesentery. although any segment of the small intestine may be involved, the distal jejunum and ileum are affected most frequently because of their longer mesenteries. one makes the diagnosis at surgery by palpating a twist at the origin of the cranial mesenteric artery. treatment includes resection of devitalized bowel, which may not be an option because of the extent of small intestinal involvement (similar to large colon volvulus). prognosis is based on the extent of small intestine involved and its appearance following surgical correction of the lesion. in general, horses with greater than % of the small intestine devitalized are considered to have a grave prognosis. a number of structures, when torn, may incarcerate a segment of intestine (typically the small intestine), including intestinal mesentery, the gastrosplenic ligament, the broad ligament, and the cecocolic ligament. horses with such incarcerations have signs typical of a horse with strangulating small intestine, including moderate to severe signs of abdominal pain, endotoxemia, absent gastrointestinal sounds, distended small intestine on per rectal palpation, nasogastric reflux, and serosanguinous abdominal fluid. however, the prognosis for many of these horses appears to be lower than for horses with other types of small intestinal strangulations. for example, in horses with small intestine entrapped in a mesenteric rent, only of horses were discharged from the hospital, and only of horses for which follow-up information was available survived long term (> months). poor outcome may result from the difficulty in unentrapping incarcerated intestine, the degree of hemorrhage, and the length of intestine affected. inguinal herniae are more common in standardbred and tennessee walking horses that tend to have congenitally large inguinal canals. inguinal herniae also may occur in neonatal foals but differ from herniae in mature horses in that they are typically nonstrangulating. the nature of the hernia (direct versus indirect) is based on the integrity of the parietal vaginal tunic. in horses in which the bowel remains within the parietal vaginal tunic, the hernia is referred to as indirect, because strictly speaking the bowel remains within the peritoneal cavity. direct herniae are those in which strangulated bowel ruptures through the parietal vaginal tunic and occupies a subcutaneous location. these direct herniae most commonly occur in foals and should be suspected when a congenital inguinal hernia is associated with colic, swelling that extends from the inguinal region or the prepuce, and intestine that may be palpated subcutaneously. , although most congenital indirect inguinal herniae resolve with repeated manual reduction or application of a diaper, surgical intervention is recommended for congenial direct herniae. historical findings in horses with strangulating inguinal herniae include acute onset of colic in a stallion that recently had been used for breeding. a cardinal sign of inguinal herniation is a cool, enlarged testicle on one side of the scrotum (figure . - ). , however, inguinal herniae also have been reported in geldings. one also can detect inguinal herniae on rectal palpation, and one can use manipulation of herniated bowel per rectum to reduce a hernia, but this is generally not recommended because of the risk of rectal tears. in many cases, the short segment of herniated intestine greatly improves in appearance after reduction and in some cases can be left unresected. the affected testicle will be congested because of vascular compromise within the spermatic cord, and although the testicle may remain viable, resection generally is recommended. the prognosis in adult horses is good, with up to % of horses surviving to months. horses that have been treated for inguinal herniae may be used for breeding. in these horses, the remaining testicle will have increased sperm production, although an increased number of sperm abnormalities will be noticeable following surgery because of edema and increased temperature of the scrotum. although umbilical herniae are common in foals, strangulation of herniated bowel is rare. in one study, of ( %) horses with umbilical herniae had incarcerated intestine. clinical signs include a warm, swollen, firm, and painful hernia sac associated with signs of colic. the affected segment of bowel is usually small intestine, but herniation of cecum or large colon also has been reported. in rare cases, one may find a hernia that involves only part of the intestinal wall, called a richter's hernia. in foals that have a richter's hernia, an enterocutaneous fistula may develop. in one study, of foals with strangulating umbilical herniae survived to discharge, although at least died of long-term complications. an intussusception involves a segment of bowel (intussusceptum) that invaginates into an adjacent aboral segment of bowel (intussuscipiens). the reason for such invagination is not always clear but may involve a lesion at the leading edge of the intussusception, including small masses, foreign bodies, or parasites. in particular, tapeworms (anoplocephala perfoliata) have been implicated. ileocecal intussusceptions are the most common intestinal intussusceptions in the horse and typically affect young animals. in one study evaluating cases of ileocecal intussusception, the median age of the horses was year old. acute ileocecal intussusceptions are those in which the horses has a duration of colic of less than hours and involve variable lengths of intestine that ranged in one study from to cm long. in acute cases the involved segment of ileum typically has a compromised blood supply. chronic ileocecal intussusceptions typically involve short segments of ileum (up to cm long), and the ileal blood supply is frequently intact. abdominocentesis results vary because strangulated bowel is contained within the adjacent bowel. obstruction of the small intestine often is evident, including nasogastric reflux and multiple distended loops of small intestine on rectal palpation. horses with chronic ileocecal intussusceptions have mild, intermittent colic, often without evidence of small intestinal obstruction. in one study, a mass was palpated in the region of the cecal base in approximately % of cases. transabdominal ultrasound may be helpful in discerning the nature of the mass. the intussusception has a characteristic target appearance on cross section. other segments of the small intestine also may be intussuscepted, including the jejunum (figure . - ) . in one study of jejunojejunal intussusceptions, the length of bowel involved ranged from . to . m. attempts to reduce intussusceptions at surgery are usually futile because of intramural swelling of affected bowel. one should resect jejunojejunal intussusceptions. for acute ileocecal intussusceptions, one should transect the small intestine as far distally as possible and perform a jejunocecal anastomosis. in horses with particularly long intussusceptions (up to m has been reported), one may attempt an intracecal resection. for horses with chronic ileocecal intussusceptions, one should perform a jejunocecal bypass without small intestinal transection. the prognosis is good for horses with chronic ileocecal intussusceptions and guarded to poor for horses with acute ileocecal intussusceptions, depending on the length of bowel involved. herniation of intestine through a rent in the diaphragm is rare in the horse and may involve any segment of bowel, although small intestine is herniated most frequently. diaphragmatic rents may be congenital or acquired, but acquired herniae are more common. congenital rents may result from incomplete fusion of any of the four embryonic components of the diaphragm: pleuroperitoneal membranes, transverse septum, and esophageal mesentery. in addition, abdominal compression of the foal at parturition may result in a congenital hernia. acquired herniae are presumed to result from trauma to the chest or a sudden increase in intraabdominal pressure, such as might occur during parturition, distention of the abdomen, a sudden fall, or strenuous exercise. herniae have been found in a number of different locations, although large congenital herniae are typically present at the ventral most aspect of the diaphragm, and most acquired herniae are located at the junction of the muscular and tendinous portions of the diaphragm. a peritoneopericardial hernia has been documented in at least one horse. figure . - jejunojejunal intussusception in a horse presented for colic. the intussusceptum has become ischemic because of invagination of intestine and its mesenteric blood supply into the intussuscipiens. clinical signs usually are associated with intestinal obstruction rather than respiratory embarrassment. however, careful auscultation may reveal an area of decreased lung sounds associated with obstructed intestine and increased fluid within the chest cavity. such signs may prompt thoracic radiography or ultrasound, both of which one can use to make a diagnosis. auscultation also may reveal thoracic intestinal sounds, but differentiating these from sounds referred from the abdomen typically is not possible. in one report, two of three horses diagnosed with small intestinal strangulation by diaphragmatic hernia had respiratory acidemia attributable to decreased ventilation. treatment of horses with diaphragmatic hernia is fraught with complications because of the need to reduce and resect strangulated bowel and the need to repair the defect in the diaphragm. , because dorsal defects in the diaphragm are among the common forms of diaphragmatic defect, closing the diaphragmatic hernia via the approach used for abdominal exploration may not be possible. however, because herniation is likely to recur, scheduling a second surgery using an appropriate approach to resolve the diaphragmatic defect is appropriate. horses with large colon volvulus have rapid onset of severe, unrelenting abdominal pain, most often in postpartum broodmares. once the large colon strangulates (≥ -degree volvulus), gas distention is significant, leading to gross distention of the abdomen, compromised respiration as the distended bowel presses up against the diaphragm, and visceral pooling of blood as the caudal vena cava is compressed. horses with this condition are frequently refractory even to the most potent of analgesics. these horses may prefer to lie in dorsal recumbency, presumably to take weight off the strangulated colon. an abbreviated physical examination is warranted in these cases, because the time elapsed from the onset of strangulation to surgical correction is critical. under experimental conditions, the colon is irreversibly damaged within to hours of a -degree volvulus of the entire colon. despite severe pain and hypovolemia, horses may have a paradoxically low heart rate, possibly related to increased vagal tone. in addition, results of abdominocentesis often do not indicate the degree of colonic compromise , and in many cases are not worth attempting because of extreme colonic distention. palpation per rectum reveals severe gas distention of the large colon, often restricting access to the abdomen beyond the pelvic brim. one may make the diagnosis tentatively based on signalment, severity of pain, and degree of distention. at surgery, the volvulus typically is located at the mesenteric attachment of the colon to the dorsal body wall and the most common direction of the twist is dorsomedial using the right ventral colon as a reference point. however, the colon may twist in the opposite direction, twist greater than degrees (up to degrees has been reported) or twist at the level of the diaphragmatic and sternal flexures. in all cases, one should decompress the colon as much as possible, and in many cases a colonic evacuation via a pelvic flexure enterotomy greatly aids correction of the volvulus. one must determine after correction of the volvulus whether the colon has been injured irreversibly and should base the determination on mucosal color and bleeding (if an enterotomy has been performed), palpation of a pulse in the colonic arteries, serosal color, and appearance of muscular motility. if one judges the colon to be damaged irreversibly, one can consider the feasibility of a large colon resection. although % of the colon can be resected (that part of the colon distal to the level of the cecocolic fold), damage from the volvulus usually exceeds that which can be resected. in these cases, surgeons may elect to resect as much damaged bowel as possible or may advise euthanasia. the prognosis is guarded to poor because of the rapid onset of this disease. in one study the survival rate was %. in a more recent report the survival rate was % for horses with -degree volvulus of the large colon compared with % for horses with -degree volvulus. however, one study in central kentucky documented a high success rate, possibly because of early recognition of the disease and the proximity of the hospital to the surgical caseload. postoperative complications include hypovolemic and endotoxic shock, extensive loss of circulating protein, disseminated intravascular coagulation, and laminitis. in addition, large colon volvulus has a propensity to recur. although one study documented a recurrence rate of less than %, some authors believe recurrence may be as high as %. therefore one should consider methods to prevent recurrence in patients at risk of recurrence, particularly broodmares that tend to suffer from the disease recurrently during the foaling season. , the most common intussusceptions of the large intestine are cecocecal and cecocolic intussusceptions. , both are likely attributable to the same disease process, with variable inversion of the cecum. these conditions doughnut-shaped prolapse of rectal mucosa and submucosa. type ii prolapses involve full-thickness rectal tissue, whereas type iii prolapses additionally have invagination of small colon into the rectum. type iv prolapses involve intussusception of proximal rectum or small colon through the anus in the absence of prolapse of tissue at the mucocutaneous junction at the anus. one can differentiate type iv from other forms of prolapse by their appearance and a palpable trench between prolapsed tissue and the anus. type i prolapses occur most frequently in horses with diarrhea, in which the rectal mucosa becomes irritated and protrudes intermittently during episodes of tenesmus. if tenesmus persists, rectal mucosa can remain prolapsed. rectal mucosa rapidly becomes congested and edematous under these conditions, which one should treat with osmotic agents such as glycerin or magnesium sulfate and by massaging and reducing the prolapse. a purse-string suture may be required to keep the mucosa inside the rectum. topical application of lidocaine solution or jelly, epidural anesthesia, and sedation may help reduce tenesmus that incites and exacerbates rectal prolapse. one can apply similar treatments to type ii rectal prolapses. however, these more severe prolapses may not be reducible without surgical resection of mucosa and submucosa from the prolapsed bowel. , type iii and iv rectal prolapses are more serious injuries because of involvement of small colon. in horses with type iii prolapses, one should perform an abdominocentesis to determine if injured small colon has resulted in peritonitis. one should reduce the small colon component manually if possible, although prolapsed rectal tissue typically requires mucosal/ submucosal resection. one should perform surgical exploration of the abdomen to determine the status of the small colon, although one can use serial abdominocenteses in lieu of surgery to detect progressive necrosis of bowel. type iv prolapses occur most commonly in horses with dystocia. these prolapses are almost always fatal because of stretching and tearing of mesenteric vasculature, with subsequent infarction of affected bowel. therefore euthanasia usually is warranted tend to occur in young horses ( % were less than years old in one study) and may be associated with intestinal tapeworms. horses show highly variable clinical signs, including acute severe colic, intermittent pain over a number of days, or chronic weight loss. these variable presentations likely relate to the degree to which the cecum has intussuscepted. initially, the cecal tip inverts, creating a cecocecal intussusception, which does not obstruct flow of ingesta. as the intussusception progresses, the cecum inverts into the right ventral colon (cecocolic intussusception), obstructs flow of ingesta, and often causes severe colic. the cause of abdominal pain is often difficult to differentiate in these cases, although detecting a mass on the right side of the abdomen by per rectal palpation or ultrasound examination sometimes is possible. , treatment involves manual surgical reduction by retracting the intussusceptum directly or via an enterotomy in the right ventral colon. however, a number of cases occur in which one cannot reduce the cecum readily because of severe thickening or in which surgical procedures result in fatal contamination. for example, one report stated that of horses were euthanized in the perioperative period because of complications, and another report stated that of horses were euthanized before or during surgery. the latter included all of the horses with chronic disease because of irreversible changes to the cecum. however, one recent report on cecocolic intussusceptions indicated that seven of eight horses that underwent right ventral colon enterotomy and cecal resection survived long-term, suggesting that continued improvements in surgical techniques may improve the prognosis. colocolic intussusceptions are rare but have been reported to affect the pelvic flexure and the left colons. [ ] [ ] [ ] [ ] although the condition is reported to be more common in young horses, - the condition may affect older horses. clinical findings may include a palpable mass on the left side of the abdomen. ultrasonography also may be useful. treatment requires manual reduction of the intussusception at surgery, , or resection of affected bowel. because the left colons may be exteriorized extensively and manipulated at surgery, - the prognosis is fair. rectal prolapse may occur following any disease that causes tenesmus, including diarrhea, rectal neoplasia, and parasitism, or prolapse can occur following elevations in intraabdominal pressure during parturition or episodes of coughing. , rectal prolapses are classified into four categories (table . - ) based on the extent of tissue prolapsed and the severity. type i rectal prolapse is most common and is characterized by a intussusception of rectum and poor small colon through the anus based on physical examination findings. however, confirmation of severe small colonic injury requires abdominal exploration via a midline approach or laparoscopy. a horse with compromised small colon conceivably could undergo a colostomy of the proximal small colon, but the compromised small colon typically necroses beyond that which can be resected via a midline abdominal approach. nonstrangulating infarction occurs following cranial mesenteric arteritis caused by migration of strongylus vulgaris and has become a rare disorder since the advent of broad-spectrum anthelmintics. although thromboemboli have been implicated in the pathogenesis of this disease, careful dissection of naturally occurring lesions has not revealed the presence of thrombi at the site of intestinal infarctions in most cases. these findings suggest that vasospasm plays an important role in this disease. clinical signs vary greatly depending on the extent to which arterial flow is reduced and the segment of intestine affected. any segment of intestine supplied by the cranial mesenteric artery or one of its major branches may be affected, but the distal small intestine and large colon are more commonly involved. no clinical variables exist that one can use to differentiate this disease from strangulating obstruction reliably. in some cases, massive infarction results in acute, severe colic. occasionally, one may detect an abnormal mass and fremitus on palpation of the root of the cranial mesenteric artery per rectum. one should consider this disease a differential diagnosis in horses with a history of inadequate anthelmintic treatment and the presence of intermittent colic that is difficult to localize. although one should perform fecal parasite egg counts, they are not indicative of the degree of parasitic infestation. in addition to routine treatment of colic, dehydration, and endotoxemia, medical treatment may include aspirin ( mg/kg every hours) to decrease thrombosis. definitive diagnosis requires surgical exploration. however, these cases are difficult to treat because of the patchy distribution of the lesions and the possibility of lesions extending beyond the limits of surgical resection. in addition, further infarction may occur following surgery. the prognosis is fair for horses with intermittent mild episodes of colic that may be amenable to medical therapy but is poor in horses that require surgical intervention. , surgical exploration of a horse with on-going intestinal injury exacerbates shock induced largely by endotoxin traversing damaged mucosa, and this in turn correlates with mortality. the initial clinical step in the workup of horses with colic is taking a thorough history. however, one may have to delay taking a complete history until after the physical examination and initial treatment, because management of abdominal pain may take precedence. if possible, one should obtain the vital components of the history before examination and treatment: the duration and severity of colic symptoms, analgesics already administered, and a history of any adverse drug reactions. the two most critical factors from a history that would support a decision to explore a horse with colic surgically are the duration of signs and the extent of pain. one deduces the latter from asking the owner about the presence and frequency of pawing, looking at the flanks, rolling, repeatedly going down and getting back up, posturing as if to lie down or urinate, among other clinical evidence of pain. table . - lists other important components of the history one should obtain to try to ascertain why colic has occurred. just as the history necessarily may need to be brief to allow rapid treatment of colic, so the clinician must be able to alter the extent of the physical examination to treat the horse in a timely fashion. the most critical examination finding is the heart rate of the horse, because it provides an excellent assessment of the cardiovascular status of the horse. the heart rate is likely the single most reliable predictor of the need for surgery and survival. , because analgesics can alter the heart rate dramatically, if possible, one should obtain the heart rate before administering analgesics. other components of the examination are designed specifically to gather information about the cardiopulmonary status of the horse (quality of the pulse, mucous membrane color, capillary refill time, respiratory rate, and full auscultation of the chest), and the nature of the intestinal obstruction (auscultation of gastrointestinal sounds, per rectal palpation of the abdomen, and presence of nasogastric reflux). although classic presentations exist for horses with obstructions of the small or large intestine (table . clinical management of colic is distinctly different from management of many other clinical syndromes because the initial focus is often not on defining the definitive diagnosis but rather on deciding whether a horse requires surgical exploration. therefore the clinician must collect historical, physical examination, and clinicopathologic information and make a decision whether these findings warrant medical management or whether to perform surgical exploration of the abdomen because of a suspected obstructive or ischemic lesion. for example, one may examine a horse with signs of severe abdominal pain, poor cardiovascular status, and abdominal distention that may be compatible with an extensive list of differential diagnoses but that more importantly indicate the need for abdominal exploration to minimize the extent of intestinal injury. the speed with which one can make this clinical decision has a tremendous effect on the well-being of the patient, , because delaying gastric fluid accumulation because of direct compression of the small intestine by distended colon or via tension on the duodenocolic ligament. the most useful diagnostic test for determining the type of intestinal obstruction is rectal palpation of the abdomen. however, one can reach only approximately one third of the abdomen via the rectum, and this percentage may be substantially less in large horses or heavily pregnant horses. nonetheless, attempting to determine the type of obstruction present (small intestine versus large intestine, and simple obstruction versus strangulating obstruction) is worthwhile; this information directly affects prognosis. in one study, interns and residents at a veterinary teaching hospital were able to predict the type of lesion with a specificity exceeding %. findings from palpation are helpful in educating the client about the potential findings in surgery and the likelihood of survival for the horse. before considering how to manage signs of colic, one should remember that such signs are poorly localized. therefore although colic is most frequently associated with intestinal disease, one should consider dysfunction of other organ systems, including urinary obstruction, , biliary obstruction, uterine torsion or tears, , ovarian artery hemorrhage, and neurologic disease as differential diagnoses. however, the duration and severity of colic the most immediately useful clinicopathologic information in horses with colic are the packed cell volume and total protein, because one can use them to substantiate clinical estimates of dehydration and they correlate strongly with prognosis. , a serum biochemical profile is useful for assessing electrolyte imbalances, tissue perfusion (anion gap or lactate), and kidney and liver function. one can use serum biochemical or blood gas analysis to assess acid-base status. horses with colic most frequently show evidence of metabolic acidosis associated with poor tissue perfusion caused by hypovolemia or endotoxemia, but one may note other abnormalities such as metabolic alkalosis in association with extensive loss or sequestration of stomach chloride. metabolic acidosis has been investigated further in horses with colic by measuring blood lactate, although this test is not offered routinely in many laboratories. lactate levels also have been inferred from measurement of the anion gap, although one study noted that lactate in horses with colic did not account for the entire anion gap. lactate levels and anion gap closely correlate with prognosis for survival. , , other key components of assessment of the horse with colic are abdominocentesis and complete blood count. the total white blood cell count and differential can provide crucial evidence of systemic inflammation associated with endotoxemia stemming from colic attributable to colitis (leukopenia, neutropenia, and a left shift) rather than an obstruction (highly variable complete blood count findings). peritoneal fluid may be helpful in determining the integrity of the intestine. specifically, as the intestine becomes progressively devitalized, the peritoneal fluid becomes serosanguinous as red blood cells leak into the abdomen, followed by an elevation in the total protein (> . g/dl) and progressive increases in total nucleated cell count (> , cells/µl). however, these findings do not always correlate well with the condition of the intestine, particularly in horses with large colon volvulus. for example, in a study of horses with large colon volvulus, the average total protein ( . g/dl) and total nucleated cell count ( cells/µl) were normal despite the fact that only % with a -degree volvulus survived. , these measures may appear normal because the development of severe mucosal injury following large colon volvulus is rapid and may not allow enough time for protein and leukocytes to equilibrate with the abdominal fluid. investigators have taken all the variables routinely assessed during evaluation of horses for colic and have attempted to develop models to predict accurately the need for surgery and the prognosis for life. [ ] [ ] [ ] [ ] none of these predictor models has taken the place of clinical decision making, although these studies have added part ii disorders of specific body systems signs are excellent predictors of whether a horse requires surgical exploration of the abdomen. in fact, refractory pain supersedes all other predictors of the need for surgery in the colic patient. once signs of colic have been recognized and categorized as to their severity, rapidly and effectively relieving the pain is critical for the well-being of the horse and to reduce the owner's anxiety. in addition, pain is best managed before it becomes severe. several classes of analgesics are readily available to treat horses with colic (table . - ), including α -agonists (xylazine, detomidine), opiates (butorphanol), and nonsteroidal antiinflammatory drugs (nsaids, such as flunixin meglumine). although much of this information is familiar to most practitioners, several principles deserve emphasis. the short-duration drugs xylazine and butorphanol, which provide analgesia for to minutes, allow the veterinarian to determine if pain is recurrent within the time period of the typical examination. in contrast, flunixin meglumine is not as potent as an analgesic but has a much longer duration of action. to avoid deleterious effects on gastrointestinal mucosa and the kidneys, one should not administer flunixin meglumine more frequently than recommended. , the recent discovery of two isoforms of cyclooxygenase (cox), the enzyme inhibited by nsaids, has resulted in discovery of drugs that can more selectively inhibit proinflammatory cox- while permitting continued constitutive production of prostanoids. such specificity may be advantageous in horses with colic, particularly when one considers recent evidence of reduced intestinal recovery from an ischemic event with flunixin compared with a drug that is more selective for cox- . one should reserve the α agonist detomidine for horses with severe, unrelenting pain because of its tremendous potency. in addition, one should remember that α -agonists reduce the heart rate associated with a transient increase in blood pressure, , thereby reducing the predictive value of the heart rate and pulse pressure. tremendously to understanding of the importance of some prognostic factors, particularly those reflecting cardiovascular function. simple obstruction involves intestinal obstruction of the lumen without obstruction of vascular flow. however, because a tremendous volume of fluid enters the small intestinal lumen daily, , the obstructed intestine tends to become distended, which in turn may reduce mural blood flow. ultimately, such distention may result in necrosis of tissues, particularly in the immediate vicinity of the obstruction. few are the causes of simple obstruction in the small intestine, and the incidence of these obstructions is low (approximately % of all referred horses in one large hospital-based study). however, in some geographic regions, this type of obstruction is prevalent. for example, in the southeastern united states, ileal impactions are common. ileal impactions most commonly occur in adult horses in the southeastern united states. although feeding of coastal bermuda hay has been implicated in the regional distribution of the disease, separating geographic location from regional hay sources as risk factors has been difficult. nonetheless, feeding coastal bermuda hay likely places horses at risk of ileal impaction, particularly if the coarse fiber content of the hay is high. furthermore, sudden changes in feed from an alternate type of hay to coastal bermuda hay likely places a horse at risk of ileal impaction. studies in england have revealed tapeworm infection as another important risk factor for ileal impaction. based on risk analysis, the data suggested that in excess of % of the ileal impaction cases studied were associated with serologic or fecal evidence of tapeworm infection. because of the poor sensitivity of fecal analysis for tapeworms, proudman and trees have developed a serologic test (enzyme-linked immunosorbent assay) with a sensitivity of approximately % and a specificity of %. clinical signs of horses with ileal impaction are typical for a horse with small intestinal obstruction, including onset of moderate to severe colic and loops of distended small intestine palpable per rectum as the condition progresses. because the ileum is the distal most aspect of the small intestinal tract, nasogastric reflux may take a considerable time to develop and is found in approximately % of horses requiring surgical correction of impacted ileum. , one usually makes the diagnosis at surgery, although on occasion one may palpate an impacted ileum per rectum. multiple loops of distended small intestine frequently make the impaction difficult to palpate. ileal impactions may resolve with medical treatment but frequently require surgical intervention ( figure . - ) . at surgery, one can infuse fluids directly figure . - appearance of roundworms that have been retrieved within the nasogastric reflux from a foal with an ascarid impaction. the large size of these ascarids (bar = cm) contributes to the risk of impaction following sudden kills of these parasites by broad-spectrum anthelmintics. into the mass, allowing the surgeon to breakdown the impaction. the surgeon may include dioctyl sodium sulfosuccinate in the infused fluid to aid in disruption of the mass. extensive small intestinal distention and intraoperative manipulation of the ileum may lead to postoperative ileus, but recent studies indicate that this complication is less frequent as the duration of disease before admission decreases. recent studies indicate that the prognosis for survival is good. , ileal hypertrophy is a disorder in which the muscular layers (circular and longitudinal) of the ileum hypertrophy for unknown reasons (idiopathic) or following an incomplete or functional obstruction. for idiopathic cases, proposed mechanisms include parasympathetic neural dysfunction resulting in chronically increased muscle tone and subsequent hypertrophy of the muscular layers of the ileal wall. such neural dysfunction possibly could result from parasite migration. alternative hypotheses include chronic increases in the muscular tone of the ileocecal valve, leading to muscular hypertrophy of the ileum as it contracts against a partially occluded ileocecal valve. the jejunum also may be hypertrophied, alone or with the ileum. clinical signs include chronic intermittent colic as the ileum hypertrophies and gradually narrows the lumen diameter. in one study, partial anorexia and chronic weight loss ( to months) were documented in % of the horses, most likely because of intermittent colic and reduced appetite. because hypertrophy does not affect the ileal mucosa, no reason exists to believe that these horses experience malabsorption of nutrients. one usually makes the diagnosis at surgery, although one may palpate the hypertophied ileum per rectum in some cases. for treatment, one performs an ileocecal or jejunocecal anastomosis to bypass the hypertrophied ileum. without surgical bypass, intermittent colic persists and the thickened ileum ultimately may rupture. the prognosis is fair with surgical treatment. secondary ileal hypertrophy is most commonly notable in horses that previously have had colic surgery and that may have a partial or functional obstruction at an anastomotic site. for example, in one case report, a horse developed ileal hypertrophy after surgical correction of an ileocecal intussusception. ileal hypertrophy also was noted in a horse with cecal impaction in which an ileocolic anastomosis was oriented incorrectly. horses are typically re-presented for recurrence of colic in these cases. surgical therapy is directed at addressing the cause of small intestinal obstruction and resecting hypertrophied intestine. meckel's diverticulum is an embryonic remnant of the vitelloumbilical duct, which fails to atrophy completely and becomes a blind pouch projecting from the antimesenteric border of the ileum. , however, similar diverticula also have been noted in the jejunum. these diverticula may become impacted, resulting in partial luminal obstruction, or may wrap around an adjacent segment of intestine, causing strangulation. occasionally, an associated mesodiverticular band may course from the diverticulum to the umbilical remnant and serve as a point around which small intestine may become strangulated. mesodiverticular bands also may originate from the embryonic ventral mesentery and attach to the antimesenteric surface of the bowel, thereby forming a potential space within which intestine may become entrapped. clinical signs range from chronic colic for an impacted meckel's diverticulum to acute severe colic for intestine strangulated by a mesodiverticular band. one makes the diagnosis at surgery, and treatment requires resection of the diverticulum and any associated bands. the prognosis is good for horses with simple impaction of a meckel's diverticulum and is guarded for horses with an associated small intestinal strangulation. adhesions of one segment of bowel to another or of a segment of intestine to other organs and the body wall most typically occur following abdominal surgery and may be clinically silent, cause chronic colic attributable to partial obstruction, or result in acute obstruction. these differing clinical syndromes are attributable to the type of adhesions that develop. for example, a fibrous adhesion that does not by itself obstruct the intestinal lumen might serve as the pivot point for a volvulus, whereas an adhesion between adjacent segments of the intestinal tract may create a hairpin turn that causes chronic partial obstruction. the number of adhesions that develop also may vary greatly from horse to horse. some horses may develop a single adhesion adjacent to an anastomotic site or a discrete segment of injured intestine, whereas other horses may develop diffuse adhesions involving multiple segments of intestine, likely because of widespread inflammation of the peritoneum at the time of the original surgery. the mechanism whereby adhesions develop is complex but likely involves initial injury to the serosa initiated by intestinal ischemia, reperfusion injury, and luminal distention. importantly, such injury involves infiltration of neutrophils into the serosa accompanied by loss of mesothelial cells. in one study assessing the margins of resected small intestine, extensive neutrophil infiltration was documented in the serosa, particularly in the proximal resection margin that had been distended before correction of a variety of strangulating lesions. regions of serosal injury and inflammation subsequently undergo reparative events similar to any wound, including local production of fibrin, de novo synthesis of collagen by infiltrating fibroblasts, and ultimately maturation and remodeling of fibrous tissue. unfortunately, during this process, fibrin may result in injured intestinal surfaces adhering to adjacent injured bowel or an adjacent organ. once a fibrinous adhesion has developed, new collagen synthesis may result in a permanent fibrous adhesion. alternatively, proteases released by local phagocytes may lyse fibrinous exudate, thereby reversing the adhesive process. thus one can view formation of adhesions as an imbalance of fibrin deposition and fibrinolysis. prevention of adhesions depends on inhibition of the mechanisms involved in adhesion formation, including reduction of serosal injury with early intervention and good surgical technique, reduction of inflammation by administration of antiinflammatory medications, physical separation of inflamed serosal surfaces (e.g., carboxymethylcellulose and hyaluronan), [ ] [ ] [ ] and pharmacologic modulation of fibrinous adhesion formation (e.g., heparin). in addition, early return of motility in the small intestine after surgery may reduce contact time between inflamed surfaces of intestine, thereby reducing the chances of adhesion formation. horses at greatest risk of developing adhesions after colic surgery appear to be those that have small intestinal disease. , in one study of horses undergoing surgical correction of small intestinal obstruction, % developed a surgical lesion associated with adhesions. foals appear to have an increased incidence of adhesions compared with mature horses regardless of the nature of the abdominal surgery. one study indicated that % of foals developed lesions attributable to adhesions regardless of the type of initial surgery. studies conflict as to whether the degree of surgical intervention influences adhesion formation, but in one study, horses that require enterotomy or resection and anastomosis were at greatest risk of developing adhesions. as an indication of the importance of postoperative adhesion formation, adhesions were among the most common reasons for repeat laparotomy in postoperative colic patients. , clinical signs of horses with adhesions vary greatly depending on whether the adhesion is causing partial obstruction or complete luminal obstruction or involves intestinal vasculature. adhesions would be an important differential diagnosis for intermittent colic in the postoperative period, particularly if such colic was not relieved by nasogastric decompression of the stomach. continued intermittent colic should prompt abdominocentesis to determine if septic peritonitis is present, which may contribute to adhesion formation. placement of a large bore drain and peritoneal lavage ( figure surgery should prompt immediate nasogastric intubation to decompress the stomach. treatment should include attempts at obtaining reflux from the horse at frequent intervals rather than relying on passive flow of reflux. in addition, administration of intravenous fluids should account for the maintenance requirement ( ml/kg/day, about l/hr in the average horse) and fluid losses via reflux. in practice, this requires frequent monitoring of packed cell volume and total protein to ensure that the horse remains well hydrated. although concerns have arisen that overhydrating horses may contribute to increased nasogastric reflux, keeping horses well-hydrated to avoid hypovolemic shock is critical. additionally, one should monitor electrolytes frequently, particularly considering their potential role in smooth muscle contraction and nerve excitability. because of the important role of inflammation in postoperative ileus, including elaboration of cox- -produced prostanoids, administration of nsaids is indicated. nsaid administration is particularly necessary if postoperative ileus is associated with endotoxemia, because lipopolysaccharide-induced prostanoid production disrupts propulsive motility in horses. , interestingly, phenylbutazone is more effective than flunixin meglumine at reducing the deleterious actions of lipopolysaccharide on intestinal motility. however, one should use caution when administering nsaids to patients with postoperative ileus in light of research suggesting that complete inhibition of prostanoid production can alter motility patterns in normal equine intestine. the advent of selective cox- inhibitors may provide optimal antiinflammatory treatment in the future. other treatments aimed at specifically modulating intestinal motility include lidocaine (bolus of . mg/kg followed by . mg/kg/min for hours), erythromycin ( . to . mg/kg slow intravenous infusion in l saline every hours), and metoclopramide ( . mg/kg/hr). , , the mechanism of lidocaine is presumed to be inhibition of sensory nerve activity within the wall of the intestine, thereby reducing reflex sympathetic inhibitory activity. in addition, intravenously administered lidocaine appears to be an effective analgesic. thus an important feature of intravenous lidocaine therapy may be to control postoperative pain-induced reduction of gastrointestinal motility and mucosal secretory activity. metoclopramide may stimulate intestinal motility by several mechanisms, including dopamine receptor blockade, cholinergic stimulation, and adrenergic blockade. although metoclopramide has been shown to be beneficial for reversing postoperative ileus in clinical patients and research animals, it has central nervous system excitatory side effects in the horse that make its use difficult. nonetheless, administration of metoclopramide to horses with postoperative ileus resulted in elect repeat laparotomy or laparoscopy. in one study of adhesions, % of repeat laparotomies were performed within days, suggesting that surgical colic attributable to adhesions typically occurs within months of an initial surgical procedure. unfortunately, the prognosis for horses with colic attributable to adhesions is low, with only % of horses in one study surviving from adhesion-induced colic. the definition of ileus is intestinal obstruction, including physical and functional obstructions. however, in veterinary medicine, the term typically is used to designate a lack of progressive aboral propulsion of ingesta resulting in functional obstruction. one typically bases the diagnosis of postoperative ileus on the presence of excessive gastric fluid accumulation (reflected as excessive nasogastric reflux). postoperative ileus may occur following any abdominal exploratory procedure. however, horses undergoing surgery for strangulating small intestinal lesions or small intestinal obstructive lesions such as an ileal impaction are at greatest risk. recently, the syndrome of postoperative ileus in horses has been broadened to include those horses that may have delayed transit of ingesta through the large intestine following surgery. this large intestinal ileus may follow any type of surgery, particularly horses that have had orthopedic surgery, and is characterized by reduced fecal output (fewer than three piles of manure per day) rather than excessive nasogastric reflux. however, horses with excessive nasogastric reflux are unlikely to have normal fecal output, so the distinction between these two manifestations of ileus is not absolute. mechanisms involved in precipitating postoperative ileus characterized by small intestinal dysfunction likely involve local inflammation, reduced coordination of progressive motility, and increased sympathetic tone. a recent series of studies in the rat has shown that surgical manipulation of intestine results in delayed transit time associated with infiltration of neutrophils into intestinal longitudinal muscle [ ] [ ] [ ] and upregulation of inducible nitric oxide synthase and cox- . the mechanisms in the horse may be similar in that extensive manipulation of the intestine resulted in abnormal intestinal motility in ponies, and prostanoids and nitric oxide alter or reduce intestinal motility in horses. [ ] [ ] [ ] clinical signs of postoperative ileus following colic surgery include evidence of abdominal pain, increased heart rate, reduced gastrointestinal sounds, and reflux of gastric fluid via a nasogastric tube. of these signs, heart rate is critical because it appears to be a more sensitive indicator of pain in the postoperative period than overt evidence of colic. therefore a sudden increase in the heart rate of a postoperative patient following colic a significantly reduced duration of reflux and shorter postoperative hospital stays compared with horses not receiving this drug. in the same study, constant infusion of metoclopramide was superior to intermittent infusion. recent in vitro studies indicate that metoclopramide effectively increases smooth muscle contractile activity throughout the small intestine. similarly, the motilin agonist erythromycin had stimulatory effects on equine small intestine, although the results were not uniform throughout the small intestine. erythromycin stimulates contractile activity in the longitudinal muscle of the pyloric antrum but inhibits contractile activity in circular smooth muscle in this segment of the gastrointestinal tract. the latter may be attributable to activation of motilin receptors on inhibitory nerves and may result in enhanced gastric emptying. in vivo studies on erythromycin confirmed the stimulatory action of this drug on the distal small intestine and indicated this drug also stimulates contractile activity in the cecum and pelvic flexure. however, the stimulation depends on the temporal association with surgery. erythromycin stimulated contractile activity in the postoperative period in the ileum and pelvic flexure but not the cecum, suggesting this drug may be useful for treating select cases of postoperative ileus. for horses with presumed ileus of the large colon, signs included reduced fecal output (fewer than three piles of manure per day), reduced gastrointestinal sounds, variable presence of colic, and on occasion a palpable impaction of the cecum or large colon. risk factors for this syndrome include orthopedic surgery, length of the operative period, and most importantly inadequate treatment with phenylbutazone, presumably resulting from insufficient control of postoperative pain. although treatment of large colon impaction in the postoperative period typically is uncomplicated, onset of cecal impaction is fatal in many cases because of the difficulty in recognizing horses that have cecal dysfunction. therefore one should pay close attention to fecal production and optimal analgesic treatment in any horse following an orthopedic procedure. other painful procedures, including ophthalmologic procedures, also likely place horses at risk of developing ileus of the large intestine. simple obstructions of the large intestine such as impaction tend to have a more gradual onset than those of the small intestine, although horses may become acutely and severely painful with some forms of colon displacement. in fact, some of these cases mimic and may progress toward large colon volvulus. medical therapy is frequently successful in correcting large colon impactions. however, cecal impactions present much more of a dilemma because of the greater propensity of this organ to rupture, the relative difficulty of surgically manipulating the cecum, and the onset of cecal dysfunction that may prevent the cecum from emptying following surgical resolution of impaction. cecal impaction may be divided into two syndromes: primary cecal impactions that result from excessive accumulation of ingesta in the cecum and secondary cecal impactions that develop while a horse is being treated for a separate problem. , although primary impactions typically consist of impacted, relatively dry fecal material and secondary cecal impactions tend to have fluid contents, considerable overlap exists between the two syndromes, and one must approach each case carefully. in horses with primary cecal impactions, onset of abdominal pain occurs over a number of days, reminiscent of the development of a large colon impaction. one should differentiate cecal impactions from large colon impactions on the basis of rectal palpation findings. cecal impactions have a propensity to rupture before the development of severe abdominal pain or systemic deterioration and therefore must be monitored closely. secondary cecal impactions typically develop following unrelated surgical procedures that result in postoperative pain (particularly orthopedic surgeries). secondary cecal impactions may be even more difficult to detect because one may attribute postoperative depression and decreased fecal output to the operative procedure rather than to colic. by the time horses with secondary cecal impactions show noticeable signs of colic, the cecum may be close to rupture. in many cases, no signs of impending rupture are evident. therefore all horses that undergo surgeries in which considerable postoperative pain may develop should have feed intake and manure production closely monitored. a recent study indicated that horses that produce fewer than three piles of manure daily in the postoperative period are at risk of developing a large intestinal impaction. furthermore, horses that underwent prolonged (> hour) orthopedic surgery that received inadequate treatment with phenylbutazone were at considerable risk of reduced postoperative fecal output. these results are in contrast to statements indicating that nsaids may place horses at risk of impaction, statements that appear to be based largely on clinical impressions rather than on risk analysis. the diagnosis of primary cecal impaction is based on palpation of a firm, impacted cecum per rectum. in some cases, cecal impactions may be difficult to differentiate from large colon impactions. however, careful palpation reveals the inability to move the hand completely dorsal to the impacted viscus because of the attachment of the cecum to the dorsal body wall. treatment for horses with primary cecal impactions may include initial medical therapy, including aggressive administration of intravenous fluids and judicious use of analgesics. however, if the cecum is distended grossly or if medical therapy hasno effect within a reasonable period of time, surgical evacuation of the cecum via a typhlotomy is indicated. in addition, performing an ileocolostomy to bypass the cecum is advisable, because postoperative cecal motility dysfunction with recurrence of the impaction is common. , in horses that develop secondary cecal impactions, diagnosis is based on palpation of a greatly distended cecum filled with semifluid intestinal contents. the nature of the contents likely is related to the more rapid progression of this disease compared with primary cecal impaction. one should not delay surgery because of the risk of cecal rupture. however, if the cecum appears healthy following typhlotomy and evacuation, bypass of the cecum is not as critical as it is for primary impactions as long as one can control the inciting cause of the impaction (such as orthopedic pain). the prognosis is guarded for surgical treatment of all cecal impactions because of the potential for the cecum to rupture during prolonged medical treatment or during surgical manipulation, the possibility of abdominal contamination during surgery, and the extensive surgical procedures required. in a recent report, seven of nine horses for which cecal impaction was treated by typhlotomy and ileocolostomy or jejunocolostomy lived long term. however, a separate report indicated that all horses with cecal impaction following another disease process had cecal rupture without any signs of impending rupture. ingesta impactions of the large colon occur at sites of anatomic reductions in luminal diameter, particularly the pelvic flexure and the right dorsal colon. although a number of risk factors have been reported, most have not been proved. however, a sudden restriction in exercise associated with musculoskeletal injury appears frequently to be associated with onset of impaction. another consideration is equine feeding regimens, which usually entail twice daily feeding of concentrate. such regimens are associated with large fluxes of fluid into and out of the colon, associated with readily fermentable carbohydrate in the colon and subsequent increases in serum aldosterone, respectively. one may prevent these fluid fluxes, which may cause dehydration of ingesta during aldosterone-stimulated net fluid flux out of the colon, with frequent small feedings. amitraz, an acaricide associated with clinical cases of colon impaction, can induce impaction of the ascending colon. , this effect may provide some clues as to the pathogenesis of large colon impaction. in particular, amitraz appears to alter pelvic flexure pacemaker activity, resulting in uncoordinated motility patterns between the left ventral and left dorsal colon and excessive retention of ingesta. absorption of water from the ingesta increases with retention time, dehydrates the contents of the colon, and results in impaction. conceivably, parasite migration in the region of a pacemaker may have a similar action. other factors implicated in large colon impaction include limited exercise, poor dentition, coarse roughage, or dehydration. clinical signs of large colon impaction include slow onset of mild to moderate colic. fecal production decreases, and the feces are often hard, dry, and mucuscovered because of delayed transit time. the heart rate may be elevated mildly during episodes of pain but is often normal. signs of abdominal pain are typically well controlled with administration of analgesics but become increasingly more severe and refractory if the impaction does not resolve. the diagnosis is based on palpation of a firm mass in the large colon per rectum. however, one may underestimate the extent of the impaction by rectal palpation alone because much of the colon is out of reach. adjacent colon may be distended if the impaction has resulted in complete obstruction. one should attempt initial medical treatment. administration of analgesics (e.g., flunixin meglumine at . to . mg/kg intravenously every to hours; butorphanol at . to . mg/kg intramuscularly every to hours; or xylazine at . to . mg/kg intravenously as needed) controls intermittent abdominal pain. administration of oral laxatives such as mineral oil ( to l by nasogastric tube every to hours) and the anionic surfactant dioctyl sodium sulfosuccinate ( to g/ kg diluted in to l of water by nasogastric tube every to hours) are used commonly to soften the impaction. saline cathartics such as magnesium sulfate ( . mg/kg in to l by nasogastric tube) also may be useful. one should not permit access to feed. for impactions that persist, one should institute aggressive oral and intravenous fluid therapy ( to times the maintenance fluid requirement). if the impaction remains unresolved, the horse becomes uncontrollably painful, or extensive gas distention of the colon occurs, surgery is indicated. in addition, one can monitor abdominal fluid serially to determine the onset of intestinal compromise. at surgery, one evacuates the contents of the colon via a pelvic flexure enterotomy. the prognosis is good for those horses in which impactions resolve medically ( % long-term survival in one study) and fair in horses that require surgical intervention ( % long-term survival in the same study). enteroliths are mineralized masses typically composed of magnesium ammonium phosphate (struvite). however, magnesium vivianite also has been identified in enteroliths, along with variable quantities of sodium, sulfur, potassium, and calcium. the formation of magnesiumbased minerals is puzzling because of the relative abundance of calcium in colonic fluids, which would favor the formation of calcium phosphates (apatite) rather than struvite. however, elevated dietary intake of magnesium and protein may play a role. many horses that develop enteroliths are located in california and are fed a diet consisting mainly of alfalfa hay. analysis of this hay has revealed a concentration of magnesium approximately times the daily requirements of the horse. furthermore, the high protein concentration in alfalfa hay may contribute to calculi formation by increasing the ammonia nitrogen load in the large intestine. enteroliths most commonly form around a nucleus of silicon dioxide (a flintlike stone), but nidi have included ingested nails, rope, and hair. enteroliths usually are found in the right dorsal and transverse colons. although enterolithiasis has a wide geographic distribution, horses in california have the highest incidence. in one california study, horses with enterolithiasis represented % of the surgical colic population, and arabians, morgans, american saddlebreds, and donkeys were at greatest risk of this disease. in a study of enterolithiasis in texas, risk factors also included feeding of alfalfa hay and arabian breed. however, in that study, miniature horses were also at risk. horses with enteroliths are rarely under years old, although an enterolith in an -month-old miniature horse has been reported recently. enterolithiasis is characterized by episodic, mild to moderate, intermittent abdominal pain. progressive anorexia and depression may develop. the amount of pain depends on the degree of obstruction and amount of distention. partial luminal obstruction allows the passage of scant, pasty feces. heart rate varies and depends on the degree of pain. in some cases, an enterolith is forced into the small colon, where it causes acute small colon obstruction. one may diagnose enteroliths by abdominal radiography or at surgery. on rare occasions, one may palpate an enterolith per rectum, particularly if it is present in the distal small colon. in general, these cases require surgery, although enteroliths being retrieved per rectum have been reported. in fact in one study, % of horses presented for treatment of enterolithiasis had a history of passing an enterolith in the feces. however, enteroliths typically are located in the right dorsal colon, transverse colon, or small colon. at surgery, one gently pushes the enterolith toward a pelvic flexure enterotomy, but removal frequently requires a separate right dorsal colon enterotomy to prevent rupture of the colon. following removal of an enterolith, one must conduct further exploration to determine if other enteroliths are present. solitary enteroliths are usually round, whereas multiple enteroliths have flat sides. the prognosis is good ( % -year survival in one study of cases), unless the colon ruptures during removal of an enterolith. in one recent study, rupture occurred in % of cases. sand impactions are common in horses with access to sandy soils, particularly horses eating feed placed on the ground. some horses, especially foals, deliberately eat sand. fine sand tends to accumulate in the ventral colon, whereas coarse sand may accumulate in the dorsal colon. , however, individual differences in colonic function may contribute to accumulation of sand, because some horses can clear consumed sand, whereas others cannot. distention from the impaction itself, or gas proximal to the impaction, causes abdominal pain. in addition, sand may trigger diarrhea, presumably because of irritation of the colonic mucosa. in horses with sand impactions, clinical signs are similar to those of horses with large colon impactions. one may find sand in the feces, and auscultation of the ventral abdomen may reveal sounds of sand moving within the large colon. however, unlike sand-induced diarrhea, one may not hear sand impactions easily because of the lack of colonic motility. to determine the presence of fecal sand, one places several fecal balls in a rectal palpation sleeve or other container, which subsequently is filled with water. if sand is present, it accumulates at the bottom of the container. in addition, one may detect mineral opacity within the colon on abdominal radiographs, particularly in foals, ponies, and small horses. abdominal paracentesis typically yields normal fluid and poses some risk because large quantities of sand in the ventral colon make inadvertent perforation of the colon more likely. peritoneal fluid is often normal but may have an elevated protein concentration. initially, medical therapy is warranted. administration of psyllium hydrophilic mucilloid ( . to . kg/ kg in to l of water by stomach tube) may facilitate passage of sand. one should administer the solution rapidly because it will form a viscous gel. an alternative method of administration is to mix psyllium with l of mineral oil, which will not form a gel and can be pumped through a nasogastric tube easily. one then pumps to l of water through the tube. the psyllium separates from the oil phase and mixes with the water, forming a gel within the gastrointestinal tract. psyllium is thought to act by stimulating motility or by agglutinating the sand. however, a recent experimental study failed to show a benefit of this treatment for clearing sand from the colons of otherwise normal horses. if a severe impaction is present, one should not give the psyllium until softening the impaction by administrating intravenous or oral fluids and other laxatives. perforation is a potential complication in horses with sand impactions because the sand stretches and irritates the intestinal wall and causes inflammation. therefore if colic becomes intractable, one should perform surgical evacuation of the large colon. the prognosis is generally good. , displacement of the ascending colon is a common cause of large intestinal obstruction. the ascending colon is freely movable except for the right dorsal and ventral colons. contact with adjacent viscera and the abdominal wall tends to inhibit movement of the ascending colon from a normal position; however, accumulation of gas and fluid or ingesta may cause the colon to migrate. feeding practices, including feeding of large concentrate meals, likely plays a role in initiating displacement of the large colon. large concentrate meals increase the rate of passage of ingesta, allowing a greater percentage of soluble carbohydrates to reach the large intestine, which in turn increases the rate of fermentation and the amount of gas and volatile fatty acids produced. the production of large amounts of volatile fatty acids stimulates the secretion of large volumes of fluid into the colon. the association between feeding concentrate and development of displacements of the large colon is illustrated by studies indicating that ascending colon displacement is more prevalent in horses fed a highconcentrate, low-roughage diet. abnormal motility patterns of the ascending colon also have been suggested to contribute to the development of colonic displacement. feeding stimulates colonic motility via the gastrocolic reflex, but large meals may alter normal motility patterns and concurrently allow rapid accumulation of gas and fluid from fermentation. , migration of parasite larvae (strongyles) through the intestinal wall also has been shown to alter colonic motility patterns. other experimental studies also have shown that strongylus vulgaris infection results in reduced blood flow to segments of the large intestine without necessarily causing infarction. electric activity of the colon and cecocolic junction increases after infection with s. vulgaris and cyathostome larvae, probably reflecting a direct effect of migration through the intestine and an early response to reduced blood flow. displacements of the ascending colon generally are divided into three types: left dorsal displacement, right dorsal displacement, and retroflexion. left dorsal displacement is characterized by entrapment of the ascending colon in the renosplenic space. the colon often is twisted degrees such that the left ventral colon is situated in a dorsal position relative to the left dorsal colon. the entrapped portion may be only the pelvic flexure or may involve a large portion of the ascending colon, with the pelvic flexure situated near the diaphragm. the colon may become entrapped by migrating dorsally between the left abdominal wall and the spleen or may migrate in a caudodorsal direction over the nephrosplenic ligament. occasionally, one can palpate the ascending colon between the spleen and abdominal wall, lending support to the first mechanism of displacement. gastric distention is thought to predispose horses to left dorsal displacement of the ascending colon by displacing the spleen medially, allowing the colon room to migrate along the abdominal wall. right dorsal displacement begins by movement of the colon cranially, medial (medial flexion) or lateral (lateral flexion) to the cecum. according to one author, the proportion of right dorsal displacements with medial versus lateral flexion is approximately : . in either case the pelvic flexure ends up adjacent to the diaphragm. retroflexion of the ascending colon occurs by movement of the pelvic flexure cranially without movement of the sternal or diaphragmatic flexures. displacement of the ascending colon partially obstructs the lumen, resulting in accumulation of gas or ingesta and causing distention. secretion of fluid in response may exacerbate the distention. tension and stretch of the visceral wall is an important source of the pain associated with colonic displacement. tension on mesenteric attachments and the root of the mesentery by the enlarged colon also may cause pain. ischemia rarely is associated with nonstrangulating displacement of the colon. however, vascular congestion and edema often occur in the displaced segments of colon, resulting from increased hydrostatic pressure from reduced venous outflow. morphologic damage to the tissues is usually minor. clinically, displacement of the ascending colon is characterized by intermittent signs of mild to moderate abdominal pain of acute onset. however, one also may note an insidious onset of colic. one may note dehydration if the duration of the displacement is prolonged. the heart rate may be elevated during periods of abdominal pain but is often normal. abdominal distention may be present if the colon is enlarged by gas, fluid, or ingesta. fecal production is reduced because progressive motility of the large intestine is absent. one often diagnoses left dorsal displacements by palpation per rectum. one can feel the left ventral colon in a dorsal position; it often is filled with gas. one can trace the ascending colon to the nephrosplenic space, and the spleen may be displaced medially. alternatively, one can reach a tentative diagnosis using abdominal ultrasonography. the spleen is visible on the left side of the abdomen, but the gasdistended bowel obscures the left kidney. evaluation of this technique indicates that false positives occur in few instances, although false negatives occasionally may occur. a definitive diagnosis therefore may require surgery. right dorsal displacements are characterized by the presence of the distended ventral colon running across the pelvic inlet and may be felt between the cecum and the body wall if a lateral flexion is present. the pelvic flexure is usually not palpable. retroflexion of the ascending colon may produce a palpable kink in the colon. if the displaced colons are not distended by gas in the instance of right dorsal displacement and retroflexion, the ascending colon may not be palpable and is conspicuous by its absence from a normal position. peritoneal fluid may increase in amount, but the color, protein concentration, and white blood cell count are usually normal. however, as the displaced segment becomes edematous, fluid leaking through the serosa into the peritoneal fluid increases the protein concentration. surgical correction of colon displacement is the most effective means of resolving this disorder. however, nonsurgical intervention has been successful in select cases of nephrosplenic entrapment of the large colon. [ ] [ ] [ ] before attempting such manipulations, the clinician must be certain of a diagnosis. one anesthetizes the horse and places it in right lateral recumbency, rotates the horse up to dorsal recumbency, rocking it back and forth for to minutes, and then rolls the horse down into left lateral recumbency. one should palpate the nephrosplenic space per rectum to determine whether the entrapment has been relieved before recovering the horse from anesthesia. one may administer phenylephrine ( - µg/kg/min over minutes) to decrease the size of the spleen. more recently, phenylephrine has been used successfully with to minutes of exercise to reduce nephrosplenic entrapments in four of six horses. the authors suggested that the technique be used on horses with mild to moderate colonic distention, particularly when financial constraints are severe. a number of cases occur in which nonsurgical interventions do not correct the problem and others in which nonsurgical manipulations correct the entrapment but result in large colon volvulus or displacement. one should take horses in such condition to surgery promptly. the prognosis for horses with large colon displacement is good. in one study on horses with nephrosplenic entrapment of the large colon, survival exceeded %. the horse, particularly young horses, may ingest foreign material that can cause obstruction, such as bedding, rope, plastic, fence material, and feedbags. these foreign bodies may result in impaction with ingesta and distention of the intestine, typically in the transverse or descending colon. young horses usually are affected. in one study the obstructing mass could be palpated per rectum in three of six horses. fecaliths are common in ponies, miniature horses, and foals. older horses with poor dentition also may be predisposed to fecaliths because of the inability to masticate fibrous feed material fully. fecaliths commonly cause obstruction in the descending colon and may cause tenesmus. other clinical signs are similar to those of enterolithiasis. abdominal radiography may be useful in smaller patients to identify the obstruction, especially if gas distention around the foreign body or fecalith provides contrast. the horse usually requires surgical treatment. mural masses such as abscesses, tumors (adenocarcinoma, lymphosarcoma), granulomata, and hematomas ( figure . - ) can cause luminal obstruction and impaction, typically in older horses. impaction may result from obstruction of the lumen or impaired motility in the segment of intestine with the mass. abscesses may originate from the lumen of the intestine or may extend from the mesentery or mesenteric lymph nodes. intramural hematomas form most commonly in the descending colon and cause acute abdominal pain. once the acute pain from the hematoma subsides, impaction proximal to the hematoma develops because of impaired motility through the affected portion of the colon. trauma, ulceration of the mucosa, and parasitic damage are speculated causes of intramural hematomas. , stricture of the large intestine occurs when fibrous tissue forms in a circular pattern around or within the intestine, reducing the luminal diameter and the ability of the wall to stretch. strictures may be congenital or may follow peritonitis, previous abdominal surgery, or inflammatory bowel disease. in a report of horses with inflammatory bowel disease, horses had strictures, four of which were in the small intestine and two of which were in the large colon. clinical signs vary according to the degree of luminal obstruction. partial obstruction and impaction tend to produce mild to moderate abdominal pain of insidious onset. mural hematomas tend to produce signs of acute abdominal pain. , per rectal palpation of the abdomen may reveal the presence of a mass or simply the impacted segment but not the mass itself. one may note fever, weight loss, and anorexia if an abscess or tumor is the cause. an elevated white blood cell count; hyperfibrinogenemia; hyperglobulinemia; or normocytic, normochromic anemia may occur with abscesses or tumors. peritoneal fluid may reflect the cause of the mass. tumor cells may occur infrequently. one may note evidence of inflammation with bacteria if the cause of colic is an abscess or granuloma, in which case one should culture the fluid. hematomas may cause hemorrhage into the peritoneal fluid. treatment usually requires surgical resection of the mass. one may treat abscesses with appropriate antibiotics if the impaction can be resolved medically with oral or intravenous analgesics and laxatives. streptococcus spp, actinomyces pyogenes, corynebacterium pseudotuberculosis, rhodococcus equi, anaerobic bacteria, and gram-negative enteric organisms commonly are involved in abscesses. atresia of a segment of the colon is a rare congenital abnormality in horses. the heritability and causes of the condition are unknown. one potential mechanism for development of the lesion is intestinal ischemia during fetal life, which results in necrosis of a segment of intestine. clinical signs include a failure to pass meconium and colic within the first to hours of life. secondary abdominal distention results from complete intestinal obstruction, and abdominal radiographs may reveal gas-distended colon. one makes the diagnosis at surgery. any portion of the colon may be absent, but the distal segment of the large colon or the proximal small colon usually is affected most severely. if sufficient tissue is present, one may attempt anastomosis to the proximal blind end of the colon. the prognosis depends on which segment of the colon is absent but is usually poor because of an absence of distal colon. neoplasia in the alimentary tract of the horse is uncommon. primary and metastatic neoplasia can affect multiple locations within the oral cavity and gastrointestinal tract (boxes . - and . - ). neoplasia is not limited to older horses. the average age of horses with squamous cell carcinoma is . to . years. , the alimentary form of lymphoma occurs most commonly in horses less than years of age. identification of benign versus malignant tumors is imperative to justify treatment and predict survival. clinical signs associated with alimentary neoplasia are related to the tumor location. clinical signs of oral neoplasia can include enlargement or ulceration of the mandible or maxilla. neoplasia of the tongue results in weight loss, quidding, prepharyngeal dysphagia, halitosis, and nasal discharge containing feed material if the oropharynx is involved. [ ] [ ] [ ] tumors of the esophagus cause signs typical of esophageal dysphagia, ptyalism, choke, intermittent colic, fever, weight loss, and halitosis. , , gastric neoplasia can be associated with abnormal chewing and swallowing behavior, anorexia, weight loss, chronic intermittent colic, abdominal distention, and intermittent fever. abdominal neoplasia has been implicated in % of horses with intermittent or chronic colic. , altered stool character, weight loss, ventral edema, and recurrent fever have been associated with intestinal neoplasia. acute signs of abdominal discomfort can occur in intestinal obstructions from malignant and benign neoplastic disease. paraneoplastic syndromes may occur in the horse. the most common syndromes are cancer cachexia, ectopic hormone production, anemia, leukocytosis, thrombocytopenia, hypergammaglobulinemia, fever, and neurologic abnormalities. horses with cancer cachexia have profound weight loss despite adequate consumption of calories. diagnosis of alimentary neoplasia can be challenging. data collected from a complete blood count, biochemistry panel, and urinalysis may support the diagnosis of neoplasia but rarely confirms it. normocytic normochromic anemia indicates chronic disease and is the most likely cause of anemia associated with neoplasia. blood loss anemia (via gastrointestinal tract) and immune-mediated hemolytic anemia (lymphoma) are less frequent causes of anemia associated with abdominal neoplasia. peripheral eosinophilia has been reported in association with multisystemic eosinophilic, epitheliotropic disease with lymphoma. leukocytosis and hyperfibrinogenemia are common findings. serum chemistry can confirm hypoalbuminemia caused by inflammation of the bowel wall. hyperglobulinemia can be characterized with serum electrophoresis, which is nonspecific and can reveal chronic inflammation. a few cases of lymphoma have been identified with monoclonal hypergammaglobulinemia. ectopic hormone production may result in hypercalcemia (calcium > mg/dl), which is associated with alimentary neoplasia such as lymphoma, multiple myeloma, carcinomata, and ameloblastoma. , hypoglycemia (blood glucose < mg/dl) can occur with neoplasia of the pancreas or liver. rectal examination may detect an abdominal mass, thickening of the intestinal wall, lymph node enlargement, or a gritty texture in horses with carcinomatosis. rectal biopsy can reveal lymphoma in some cases. fecal occult blood test is nonspecific for neoplastic disease but can reveal blood loss through the gastrointestinal tract. occasionally, abdominocentesis can identify neoplasia if the tumor exfoliates cells into the abdomen. one can diagnose squamous cell carcinoma, adenocarcinoma, and mesothelioma from peritoneal fluid. , , , characterization of peritoneal fluid as an inflammatory exudate or modified transudate without any neoplastic cells present is common. cytologic analysis of peritoneal fluid samples collected by abdominocentesis accurately predicted the presence of neoplasia in of cases in one study. cytologic examination of two or more peritoneal fluid samples increases the sensitivity of this test for detecting abdominal neoplasia. measurement of peritoneal fluid glucose concentration and ph is valuable to differentiate inflammation in the peritoneum caused by neoplasia from bacterial peritonitis. abdominal neoplasia typically is associated with peritoneal glucose concentrations similar to blood and ph higher than . . d-xylose absorption tests can reveal malabsorptive diseases that include lymphoma. , immunoglobulin m deficiency is associated with lymphoma in some young adult horses, but the prevalence of immunoglobulin m deficiency in horses with lymphoma and the value of measuring serum immunoglobulin m concentrations for the diagnosis of lymphoma have not been evaluated. dna cell cycle analysis of suspect neoplastic cells has been used to detect lymphoma in equine patients confirmed with the disease. this method of evaluating fluid or tissues aspirates may increase the accuracy for diagnosing neoplasia in the future. a complete evaluation of the oral cavity may include using a full-mouth speculum, radiographs, and endoscopy of the pharynx. evaluation of the esophagus and stomach with a -m endoscope can reveal intralumenal masses. pleuroscopy has been used to obtain biopsy samples of extralumenal masses surrounding the esophagus. contrast radiography can assist in the diagnosis of neoplasia within the wall or outside of the esophagus. , ultrasonography of the stomach, small intestine, cecum, and large colon is useful in detecting intestinal wall thickness, abdominal masses, and excessive peritoneal fluid. identification of neoplasia in the liver, kidney or spleen may support metastasis to other parts of the gastrointestinal tract or lymph nodes. laparoscopy and exploratory laparotomy often are required to obtain a final diagnosis. lymphoma is the most common neoplasia in the horse and has been divided into four categories. this section covers only the intestinal/alimentary form. in the past, lymphoma has been called lymphosarcoma, but the preferred term by oncologists is lymphoma because no benign form of this disease exists. lymphoma originates from lymphoid tissue and predominantly affects the small intestine and intestinal lymph nodes. chronic weight loss from malabsorption, intermittent colic, and fever are the most common clinical findings. , chronic diarrhea has been reported in some cases. paraneoplastic pruritus and alopecia have been identified in one case of diffuse lymphoma. one generally does not note peripheral lymphadenopathy, but one may palpate enlargement of the intestinal lymph nodes on rectal examination. large colon resection for treatment of lymphoma in horses has increased short-term survival in two horses. chemotherapy in two mares that were pregnant extended their lives long enough to foal normally. long-term prognosis for intestinal lymphoma is poor. squamous cell carcinoma (scc) is a malignant tumor of the gastrointestinal epithelium. scc is the second most common neoplasia in the horse and is the most common oral neoplasia. however, the incidence of scc is rare. , , in the oral cavity scc may affect the lips, tongue, hard palate, pharynx, and mucosa. , treatment for scc in the oral cavity may involve surgical resection, iridium- brachytherapy, -fluorouracil, or intralesional cisplatin. , [ ] [ ] [ ] the prognosis for survival is good if complete removal of the tumor is possible. metastasis beyond the regional lymph nodes is rare for oral scc. squamous cell carcinoma is the most common tumor of the stomach and esophagus , and can invade these areas and metastasize to the lymph nodes and lungs. abnormal masses were palpated on rectal examination in four of five cases of gastric scc. treatment by surgical resection is not possible in most cases and the horses die or are euthanized. adenocarcinoma is a malignant tumor that can occur in the small intestine, cecum, and large colon. the tumor arises from the glandular crypts of the gastrointestinal tract and has been reported in middle-aged and older horses. metastasis to the lymph nodes, liver, and lungs can occur. intestinal adenocarcinoma has been reported to metastasize to the bone and was diagnosed using nuclear scintigraphy following injection of technetium- m hydroxymethylene diphosphate. , no reports of successful surgical resection have been published. leiomyosarcoma is a malignant tumor of the smooth muscle lining the gastrointestinal tract and has been reported in the stomach, small intestine, and rectum. , , , , in one case report, gastroscopy could not identify the mural mass in the stomach that was found during exploratory surgery. another report describes a favorable outcome for surgical resection of a leiomyosarcoma that was protruding from the anal sphincter in a -year-old quarter horse. prognosis for survival is favorable if surgical resection is possible. leiomyoma is a benign tumor of the smooth muscle of the gastrointestinal tract that can occur in the stomach, small intestine, and small colon. , clinical signs are consistent with intestinal obstruction. surgical resection and anastomosis of the affected portion of the intestine have been performed without complications. lipoma is a benign tumor that occurs in older horses ( to years) and arises from mesenteric adipocytes. the tumor grows on a stalk that wraps around the intestine, causing a strangulating lesion manifested clinically by acute obstructive colic. intestinal injury caused by pedunculated lipomata may occur in the small intestine, small colon, and rectum. long-term survival with surgical resection and anastomosis of the affected segment has been reported to be % to %. , oral neoplasia oral cavity neoplasia may involve the dental tissue (odontogenic tumors), bone (osteogenic tumors), or soft tissues (see box . - ). ameloblastoma occurs in horses greater than years old and mainly affects the mandible. ameloblastic odontoma affects younger horses and usually involves the maxilla. both are benign but locally invasive. radiographs may distinguish the difference between an ameloblastoma (radiolucent lesion) and ameloblastic odontoma (radiolucent lesion with partially mineralized density). the best treatment option is surgical resection and radiation therapy regardless of the type. juvenile mandibular ossifying fibroma occurs in the rostral mandible of young horses between the ages of months and years. the fibroma may cause significant distortion of the bone. with early diagnosis and surgical excision of the mass, the horse has a good prognosis. melanomas, sarcoids, and oral papilloma occur on the mouth and lips. melanomas rarely metastasize, but they commonly are found in the parotid salivary glands and lymph nodes. sarcoids are the most common skin tumor that can involve the mouth. ulcerations of the buccal mucosa are difficult to treat. intralesional cisplatin, cryosurgery, radiation, and laser excision have been tried with limited success. equine papilloma virus is responsible for the common skin wart found on the lips and muzzle of young horses. these lesions are usually selflimiting but may be removed successfully by cryosurgery or excision. a number of detailed and informative reviews are available describing the anatomy, physiology, and pathophysiology of the equine peritoneum. [ ] [ ] [ ] [ ] [ ] the peritoneum consists of a single layer of mesothelial cells lining the peritoneal cavity and serosal surfaces of the intraabdominal viscera. the mesothelial lining of the diaphragm, abdominal walls, and pelvic cavity is termed parietal peritoneum. the visceral peritoneum includes the serosal surfaces of the intraabdominal organs. the parietal and visceral portions of the peritoneum are contiguous with each other through the omentum, mesenteries, and ligaments. caudally, the peritoneum reflects over the surfaces of the pelvic organs (portions of the urogenital tract and rectum), excluding them from the peritoneal space, and thus much of the pelvic cavity and contents are described as retroperitoneal. the peritoneal space communicates with the lumen of the uterus (and thus the external environment) via the fallopian tubes in females. in males the peritoneum forms a true blind sac. the vascular supply and nervous innervation of the visceral peritoneum are supplied by the splanchnic vessels and visceral autonomic nerves, respectively. branches of the intercostal, lumbar, and iliac arteries supply the parietal peritoneum, and the phrenic and intercostal nerves provide nervous innervation. the clinical relevance is that inflammation of the parietal peritoneum is perceived as somatic pain, resulting in a splinted abdominal wall, pain on external palpation, and reluctance to move. visceral pain is mediated by small type c sensory fibers, which are believed to be stimulated by bowel distention, smooth muscle spasms, tension on the mesentery, and ischemia. the peritoneal lining functions as a semipermeable barrier to the diffusion of water and low-molecular weight solutes between the blood and the abdominal cavity. the peritoneum secretes a serous fluid that lubricates the abdominal cavity, inhibits adhesion formation, and has minor antibacterial properties. , macrophages, mast cells, mesothelial cells, and lymphocytes provide immune function within the peritoneum. , peritoneal macrophages impart antibacterial activity via complement receptors, phagocytic activity, interaction with t lymphocytes, neutrophil chemotaxis, and fibroblast activation. the peritoneal surface maintains a high level of fibrinolytic activity through the production of plasminogen activators by mesothelial cells. this function, together with the lubricant properties of the peritoneal fluid, helps to maintain gliding surfaces within the peritoneum and prevent adhesion formation. in quadrupeds, peritoneal fluid produced by the mesothelium tends to move ventrally and cranially, aided largely by diaphragmatic movement. peritoneal fluid, waste products, and foreign material (including bacteria) exit the peritoneal cavity to enter the lymphatic system through diffusely distributed subendothelial pores or via the large diaphragmatic stomata, depending on particle size. large molecules and particles greater than approximately , mw (such as bacteria) exit through the diaphragmatic stomata and ultimately enter the thoracic duct. peritonitis is inflammation of the mesothelial lining of the peritoneal cavity and is characterized by desquamation and transformation of mesothelial cells; chemotaxis of neutrophils; release of several soluble mediators of inflammation; exudation of serum, fibrin, and protein into the peritoneal cavity; and depression of fibrinolytic activity. peritonitis occurs in association with a variety of disorders that result in mechanical, chemical, or infectious insult to the peritoneal lining. [ ] [ ] [ ] [ ] any process resulting in disruption or irritation of the peritoneal lining, inflammation or infection of abdominal organs, or compromise of the intestinal wall can result in peritonitis (box . - ). common mechanical injuries include blunt or perforating trauma to the abdominal wall, breeding and foaling accidents, and abdominal surgery. a variety of traumatic insults of iatrogenic origin can cause peritonitis, such as the pathogenesis of peritonitis as a series of stages, as reviewed and described by trent, is useful. the contamination stage, lasting to hours, involves introduction of bacteria into the peritoneum and initiation of the acute inflammatory response previously described. if the organisms are not eliminated and infection is established, the process evolves to the stage of acute diffuse peritonitis. although the overall movement of contaminants is toward the diaphragmatic stomata and into the thoracic duct, the nature of the peritoneal circulation is such that regardless of the location of the initial contamination, bacteria spread throughout the peritoneum within several hours. the stage of acute diffuse peritonitis lasts up to days. the inflammatory response persists and escalates with continued exudation of proteinaceous fluid and influx of inflammatory cells. offending organisms are delivered to the lymphatic system and may be eliminated by the immune system. organisms, however, may gain access to the systemic circulation in sufficient numbers to result in clinically relevant septicemia. in human beings and laboratory animals having undergone polymicrobial contamination of the peritoneum, the organisms causing septicemia at this stage are usually coliforms, e. coli in particular. this stage of the disease process has the highest mortality because of the effects of severe peritoneal inflammation, endotoxemia, and septicemia. if the animal survives this stage but fails to eliminate the infection in the peritoneal cavity, the disease enters a transitional phase referred to as the acute adhesive (or localizing) stage. this stage occupies a time frame of perhaps to days after the initial insult. neutrophils are still active, macrophages are increasing in numbers, and fibrin aggregates are being organized or lysed. in human beings and laboratory animals, selective reduction and synergism continue such that anaerobes and gram-negative aerobes predominate. if infection persists beyond this point, organization of fibrin proceeds and organisms become isolated from host defenses. at this point, the disease process enters the stage of chronic abscessation. this stage can begin as early as days after inoculation and persists indefinitely. clinical signs of peritonitis depend on the primary disease process, the duration of the problem, and the extent of peritoneal inflammation. localized peritonitis may have few or no systemic manifestations, whereas severe localized or generalized peritonitis often is accompanied by severe toxemia or septicemia or both. septic peritonitis usually causes more severe clinical signs because of the inflammatory mediators released in response to bacterial toxins and because of the presence of endotoxin when gram-negative organisms are involved. most clinical signs are nonspecific and include fever, depression, inappetance, decreased borborygmi, and dehydration. additional signs, reported in horses (ages months to years) with peritonitis, were colic, ileus, weight loss, and diarrhea. horses with peracute peritonitis, as occurs with rupture of the bowel or rectal tear, have severe toxemia, weakness, depression or severe colic, tachycardia, tachypnea, and circulatory failure. fever may not be present depending on the degree of shock. typical clinical findings include sweating, pawing, muscle fasciculations, weak peripheral pulses, red to purple mucous membranes, prolonged capillary refill time, and decreased skin elasticity. parietal pain, characterized by reluctance to move, splinting of the abdominal wall, and sensitivity to external abdominal pressure occur in some acute cases. urination or defecation may be painful for the horse, and urine and fecal retention may be evident on rectal examination. palpation of the abdomen externally may elicit flinching, aversion movements, or groaning. with extensive abdominal fecal contamination, rectal examination may reveal a gritty feeling of the serosal and parietal surface of the peritoneum because of fibrin deposition and a dry texture of the peritoneum. in horses with more chronic peritonitis, rectal examination findings can include pain on palpation of fibrinous or fibrous adhesions, intestinal impaction or distention following ileus and dehydration, an abdominal mass (abscess or neoplasia), or an impression of bowel floating in fluid. in many cases, one can detect no abnormalities on rectal examination. horses with localized, subacute, or chronic peritonitis may have signs of chronic or intermittent colic, depression, anorexia, weight loss, intermittent fever, ventral edema, exercise intolerance, decreased or absent intestinal sounds, and mild dehydration. heart rate and respiratory rate may be normal. fecal output may be normal; however, horses with chronic diarrhea and weight loss have been reported. foals with peritonitis usually exhibit signs of colic (acute or chronic) and are febrile, depressed, and inappetant. in some foals with primary peritonitis, pleural effusion occurs. in young foals, peritonitis can cause rapid metabolic deterioration, and determination and correction of the primary problem requires immediate attention. in older foals, peritonitis may occur insidiously, as occurs following s. equi or r. equi infections. clinicopathologic abnormalities vary depending on the time of onset and severity of peritonitis. horses with acute, septic peritonitis can have leukopenia, hemoconcentration, metabolic acidemia, azotemia, and electrolyte imbalances reflective of systemic inflammation from endotoxemia and hypovolemia. horses with peritonitis of a few days' duration may have leukocytosis and hyperfibrinogenemia. plasma protein levels vary depending on the hydration status, degree of exudation into the peritoneum, and type of underlying problem. in chronic peritonitis, hyperproteinemia with hyperglobulinemia may be present. neonates with uroperitoneum caused by urinary bladder rupture or urachal disease tend to develop azotemia, hyponatremia, hypochloremia, hyperkalemia, and acidosis. foals with peritonitis following septicemia, severe enterocolitis, severe meconium impaction, intussusception, small intestinal volvulus, gastric or duodenal rupture, or ascarid impactions usually have clinicopathologic findings reflective of systemic inflammation, such as inflammatory leukogram or leukopenia, hemoconcentration, and acidosis. chronic abscessation, as occurs in foals with r. equi and streptococcal infections, results in clinicopathologic findings reflecting chronic inflammation (anemia, hyperfibrinogenemia, hyperglobulinemia). abnormalities in the composition of peritoneal fluid occur with peritoneal inflammation, and peritoneal fluid analysis is principal to the diagnosis of peritonitis. one collects peritoneal fluid through puncture of the abdomen on the ventral midline. one should clip and prepare an area aseptically. usually, the lowest point of the abdomen, to cm caudal to the xiphoid cartilage, is prepared for puncture; although in some cases one may perform paracentesis more caudally, particularly when one suspects a specific area of sequestered fluid or abscessation. in addition, one may choose a site to the right of midline in an attempt to avoid the spleen. one can perform peritoneal puncture using a / -inch, -gauge needle or, following local anesthesia and a stab incision with a no. scalpel blade, using a sterile cannula. one collects fluid by gravity flow and should collect fluid in a tube containing anticoagulant, preferably edta for cytologic examination, and in a sterile tube without anticoagulant for visual inspection and, if desired, for culture. one should fill the edta tube to half its volume, because the edta will alter the refractive index of the fluid, resulting in a falsely elevated value for total solids when one collects only a small volume and tests it with a refractometer. one should evaluate peritoneal fluid routinely as to color, turbidity, total protein, white blood cell (wbc) count and differential, and the presence of bacteria as determined by gram stain. normal peritoneal fluid is clear and straw-colored and does not coagulate spontaneously. peritoneal fluid becomes turbid when increased numbers of white blood cells and concentration of protein are present. pink or red fluid indicates free hemoglobin or hemorrhage. blood introduced into the peritoneal fluid iatrogenically in some cases may be differentiated from blood from internal hemorrhage based on the presence of platelets and hematocrit. fluid with iatrogenic blood contamination contains platelets, whereas fluid with blood following internal hemorrhage or diapedesis often does not have platelets. blood contamination resulting from splenic puncture often results in the packed cell volume of the sample being greater than that of the blood. large volumes of dark brown or green fluid with a fetid odor obtained from several sites strongly suggest bowel rupture, but one should perform cytologic examination for confirmation. the distribution of polymorphonuclear and mononuclear cells varies widely, and one should interpret the results of cell counts and differentials as supporting a number of disorders rather than a specific diagnosis. normal equine peritoneal fluid contains fewer than nucleated cells per microliter. , wbc counts in acute peritonitis (> , /µl) are reported to be higher than those in chronic peritonitis ( , to , /µl) [ ] [ ] [ ] ; however, this is not always the case, and the wbc count depends most on the cause of the peritonitis. the wbc level does not always correlate with severity of peritonitis or the prognosis. the peritoneal fluid wbc count can be greater than , /µl following enterocentesis, with no clinical signs or problem. conversely, peritoneal wbc counts of fewer than , /µl may be found in foals or horses with intraabdominal abscesses. the peritoneal wbc count can increase to greater than , /µl following celiotomy and can be higher if an enterotomy is done. postoperatively, the wbc count normally continues to decline and returns to near normal by to days. failure of the wbc count to decrease suggests peritonitis resulting from a postoperative complication. finally, peritoneal fluid wbc counts greater than , /µl indicate severe focal or generalized peritoneal sepsis. the distribution of polymorphonuclear and mononuclear cells varies in normal peritoneal fluid, , but polymorphonuclear cells usually predominate. with acute peritonitis, polymorphonuclear cells typically increase to a greater degree than mononuclear cells, but this depends on the cause. in horses that have bowel disease accompanied by endotoxemia, the number of peritoneal mononuclear cells increases, as does transformation of mesothelial cells to macrophages. in chronic cases, one easily may mistake transforming mesothelial cells for neoplastic cells, which can make diagnosis difficult, particularly when the presenting problem is compatible with a neoplastic process. in such cases, consultation with a clinical pathologist regarding cytologic findings is prudent. factor, and interleukin- have been measured in the peritoneal fluid of horses with abdominal disorders, but the diagnostic and prognostic implications of the presence or absence of these enzymes and analytes is limited. [ ] [ ] [ ] one should submit peritoneal fluid samples in appropriate media (port-a-cul vial, bbl microbiology system) for aerobic and anaerobic cultures in an attempt to identify the pathogenic organism(s). obligate anaerobic bacteria such as bacteroides are difficult to culture, because one must collect, transport, and culture the sample under strict anaerobic conditions. frequently, bacterial cultures are negative when bacteria are present in peritoneal fluid. to enhance recovery of bacteria, one can inoculate peritoneal fluid into blood culture medium (septi-chek columbia, hoffmann-laroche inc., nutley, new jersey), and if the horse has received antimicrobial treatment, one first should pass fluid through an antimicrobial removal device (a.r.d., becton dickinson & co., cockeysville, maryland). early and aggressive therapy is required if treatment of peritonitis is to be successful. the goals of treatment are to resolve the primary problem, minimize the inflammatory response, and prevent long-term complications. in the acute phase, one gives primary consideration to the arrest of endotoxic, septic, or hypovolemic shock; correction of metabolic and electrolyte abnormalities and dehydration; and management of pain. in the absence of blood gas and electrolyte determinations, adequate volumes of a balanced electrolyte solution are required to correct dehydration and support the cardiovascular system. if the plasma protein concentration of the horse is less than . g/dl, one should consider administration of plasma or synthetic colloids. one should administer flunixin meglumine (banamine) for its local and systemic antiinflammatory effects. dosages vary depending on the severity of peritonitis, degree of toxemia, severity of pain, and hydration status of the horse and range from . mg/kg intramuscularly or intravenously every to hours to . mg/kg intramuscularly or intravenously every hours. the higher dosage provides greater visceral analgesia, whereas the lower dosage is effective in modifying the effects of experimental endotoxemia. in addition to analgesic and general antiinflammatory effect, flunixin meglumine may be effective in retarding adhesion formation when administered early in the acute, diffuse stage of septic peritonitis. heparin therapy has been recommended to prevent adhesion formation and to render bacteria more susceptible to cellular and noncellular clearing mechanisms. in experimental models using laboratory animals, normal peritoneal fluid protein concentration is less than . g/dl. protein levels between . g/dl and . g/dl can be difficult to interpret, but one should consider levels greater than . g/dl to be elevated abnormally. fibrinogen concentration increases with inflammation, and levels greater than mg/dl in the peritoneal fluid suggest that an acute inflammatory process is present. fibrinogen content will also increase from blood contamination. the presence of free and phagocytosed bacteria in peritoneal fluid indicates generalized suppuration, abscessation, or compromised bowel. if one observes numerous microorganisms of mixed types free in the peritoneal fluid or if one observes plant material, massive bacterial contamination of the abdomen following bowel rupture likely has occurred. the presence of toxic or degenerate neutrophils and bacteria within polymorphonuclear cells helps to distinguish peritoneal fluid from intestinal contents in such cases. enterocentesis yields discolored fluid containing mixed microorganisms and plant material and that is largely devoid of white blood cells. bacterial contamination of a sample can occur during collection of the sample, and iatrogenic contamination of a sample can result in free and intracellular bacteria in peritoneal fluid, particularly if processing is delayed. in such cases the bacterial numbers are few and the neutrophils appear healthy. in some cases of gastrointestinal perforation the luminal material, inflammatory cells, and protein may be sequestered by the omentum and further contained by fibrinous adhesions. abdominal fluid obtained via standard ventral paracentesis may have low cellularity and protein content but large numbers of mixed bacteria indicating bowel rupture. examples include gastric rupture along the greater curvature of the stomach between the omental layers (omental bursa) and perforated gastric or duodenal ulcers in foals. correlating all cytologic findings with clinical and clinicopathologic findings is important for interpreting the results of peritoneal fluid cytologic examination. biochemical analysis of peritoneal fluid may be useful in detecting sepsis when cytologic examination and culture are negative or otherwise unavailable. in a prospective study by van hoogmoed, rodger, spier, et al., peritoneal fluid ph and glucose concentrations from horses with septic peritonitis were significantly lower than horses with nonseptic peritonitis and healthy horses. peritoneal fluid ph less than . , glucose less than mg/dl, and fibrinogen concentration greater than mg/dl were considered highly predictive of septic peritonitis. serum to peritoneal glucose concentration differences of greater than mg/dl was considered the most diagnostically useful test for septic peritonitis in the study. increased activities of alkaline phosphatase, lactic dehydrogenase, creatine kinase, aspartate aminotransferase, tumor necrosis heparin therapy was associated with decreased adhesions in septic peritonitis. heparin has not yet been demonstrated clearly to have similar efficacy in horses, although it may. suggested dosages range from to iu subcutaneously every hours for hours to to iu/kg subcutaneously every hours. one should note that heparin induces red blood cell aggregation in horses, which may adversely affect capillary blood flow. one should initiate antimicrobial therapy after making a diagnosis of peritonitis and before the results of peritoneal culture are available, because isolating an organism may take several days and often culture fails to isolate the organism(s). intravenous administration of antimicrobials is preferred over oral or intramuscular routes in acute, diffuse, septic peritonitis because more reliable levels of drug are achieved in the tissues and peritoneal fluid than otherwise would be obtained in horses with hypovolemia or decreased intestinal motility. the combination of a β-lactam antibiotic with an aminoglycoside is appropriate in most circumstances and certainly in the acute diffuse stage of septic peritonitis. these drugs act synergistically to provide a broad spectrum of activity against a variety of gram-positive and gramnegative aerobic and anaerobic bacteria. potassium penicillin ( , to , iu/kg intravenously every hours) combined with gentamicin ( . mg/kg every hours) is an appropriate regimen. in most cases, peritonitis will have resulted from bowel contamination, and thus one should presume a mixed infection with gram-negative aerobic bacteria and gram-positive and gram-negative anaerobic bacteria. one also should presume the same in many cases of traumatic peritonitis, as occurs with foreign body puncture, breeding trauma, or foaling trauma. therefore a strong possibility exists of infection involving penicillin resistant bacteroides fragilis, so that adding metronidazole ( mg/kg orally every to hours) to the regimen is prudent. combination therapy with β-lactam and aminoglycoside antibiotics (and metronidazole when indicated) is a standard and generally effective protocol. one can modify this antimicrobial regimen when culture and antimicrobial sensitivity results become available. aminoglycosides and nonsteroidal antiinflammatory drugs have the potential to induce acute renal tubular damage, particularly when dehydration and decreased renal perfusion are present. therefore adequately rehydrating the patient and ensuring that renal function is intact before initiating treatment with these drugs are important. furthermore, maintaining hydration and monitoring renal function during the course of treatment are important. monitoring serum creatinine concentration; performing serial uninalysis observing for pigment, red blood cells; and casts; determining the ratio of γ-glutamyltransferase to creatinine in the urine; and therapeutic drug monitoring of aminoglycoside levels are useful in this regard. sodium ampicillin and ceftiofur sodium are β-lactam antibiotics that may be useful in combination therapy. these drugs have an extended gram-negative spectrum compared with penicillin. however, as a third-generation cephalosporin, ceftiofur is less effective against anaerobes than penicillin. one also may consider ceftiofur, trimethoprim-potentiated sulfonamides, amikacin, and enrofloxacin for treatment of gram-negative infection based on culture and sensitivity results. enrofloxacin is a quinolone drug with excellent activity against gramnegative pathogens, including pseudomonas, and also can be effective against resistant staphylococci (personal observation). such staphylococci may be involved in infections caused by traumatic puncture of the abdominal wall. enrofloxacin has a variety of potential toxic effects, including cartilage damage in young growing animals. however, a recent study concluded the drug was safe when administered to adult horses intravenously at mg/kg every hours for weeks. one probably should avoid using the drug in young, growing animals until the issue of cartilage damage is resolved. administration of enrofloxacin to horses constitutes off-label usage. one should treat horses with acute, diffuse, septic peritonitis with antibiotics until the white blood cell count, plasma fibrinogen, and abdominal fluid analysis are normal. in horses that respond to therapy, this process takes a variable amount of time depending on the offending organisms and stage of disease at the time treatment is initiated. horses with abdominal abscessation resulting from polymicrobial infection may require many months of antibiotic treatment. abdominal abscesses caused by streptococci and corynebacterium pseudotuberculosis also may require long-term treatment (weeks to months). long-term antibiotic treatment generally necessitates the use of oral antibiotics, and the options are limited. trimethoprim-potentiated sulfonamides are administered orally and are effective against a variety of gram-positive and gram-negative organisms, although some streptococci are resistant. metronidazole is an orally administered drug effective against anaerobic bacteria, as previously discussed. other orally administered antimicrobials one may consider for long-term use include doxycycline (broad spectrum), erythromycin (gram-positive spectrum), and enrofloxacin (mostly gram-negative spectrum). importantly, rifampin, when used with other drugs, can be effective in penetrating and resolving abscesses. combination therapy with erythromycin and rifampin is the standard treatment for rhodococcus equi infection in foals. peritonitis caused by actinobacillus equuli usually is manifested as a diffuse, supporative peritoneal exudate. the same is true for some cases involving streptococci (personal observation). these infections generally respond well to combination therapy with penicillin and gentamicin. if streptococci are involved as the sole pathogen, then penicillin alone should be effective. streptococci potentially can be involved in mixed, synergistic peritoneal infections in horses. drainage or lavage of the peritoneal cavity may be of benefit in removing toxic bacterial by-products and products of inflammatory cells. high numbers of inflammatory cells and release of their mediators can persist even after the primary stimulus of the inflammatory response has resolved. infusing large volumes of isotonic, warmed fluid into the peritoneal cavity also dilutes the inflammatory mediators, possibly reducing their deleterious effects. when successful, peritoneal lavage decreases the peritoneal fluid wbc count and total protein, potentially reflecting a decrease in diffuse inflammation. the benefits of peritoneal lavage are controversial, and a positive effect may be more likely during the acute, diffuse stage of disease. , some studies suggest peritoneal lavage, along with heparin therapy, may reduce the incidence of adhesions. one should perform peritoneal drainage and lavage using a drain of no less than f diameter. foley-type catheters can be used, but "mushroom" drains provide a larger area for fluid to enter the drain. two approaches to peritoneal lavage are ( ) retrograde irrigation through a ventrally placed ingress-egress drain and ( ) placement of ingress catheter(s) in the paralumbar fossa(e) for infusion of fluids, with a drain placed ventrally for removal of infused fluid. one must recognize that thorough peritoneal lavage can be achieved only via ventral midline laparotomy. complications associated with the use of abdominal drains or repeated peritoneal penetration to drain fluid include retrograde infection, local irritation, pneumoperitoneum, and subcutaneous seepage around the drain and resultant cellulitis. if the patient is hypovolemic or hypoproteinemic, one should consider volume replacement and administration of plasma before removing large quantities of fluid from the abdomen. in horses with suspected parasite involvement, such as verminous arteritis, one should give larvicidal doses of an anthelmintic once the condition of the horse is stabilized. ivermectin, fenbendazole, and thiabendazole have been recommended as larvacidal therapies. visualization of auscultation sounds of the large intestine small intestinal betagalactosidase activity in the horse rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases equine lymphocyticplasmacytic enterocolitis: a retrospective study of cases eosinophilic enterocolitis and dermatitis in two 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microbiological culture for detection of salmonellae in equine feces and environmental samples rapid pcr detection of salmonella in horse faecal samples potomac horse fever equine monocytic ehrlichiosis (potomac horse fever): a review experimental reproduction of potomac horse fever in horses with a newly isolated ehrlichia organism causative ehrlichial organisms in potomac horse fever ehrlichial diseases attempted ehrlichia risticii transmission with dermacentor variabilis (acari: ixodidae) attempted transmission of ehrlichia risticii (rickettsiaceae) with stomoxys calcitrans (diptera: muscidae) production and characterization of ehrlichia risticii, the agent of potomac horse fever, from snails (pleuroceridae: juga spp.) in aquarium culture and genetic comparison to equine strains infection of aquatic insects with trematode metacercariae carrying ehrlichia risticii, the cause of potomac horse fever transmission of ehrlichia risticii, the agent of potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report infection rate of ehrlichia risticii, the agent of potomac horse fever, in freshwater stream snails (juga yrekaensis) from northern california ultrastructural study of ehrlichial organisms in the large colons of ponies infected with potomac horse fever enterocolitis caused by ehrlichia sp. in the horse (potomac horse fever) growth of ehrlichia risticii in human colonic epithelial cells respiratory burst activity associated with phagocytosis of ehrlichia risticii by mouse peritoneal macrophages in vitro killing of ehrlichia risticii by activated and immune mouse peritoneal macrophages lack of lysosomal fusion with phagosomes containing ehrlichia risticii in p d cells: abrogation of inhibition with oxytetracycline pathophysiological changes in the large colon of horses infected with ehrlichia risticii disease features in horses with induced equine monocytic ehrlichiosis (potomac horse fever) clinical and hematologic variables in ponies with experimentally induced equine ehrlichial colitis (potomac horse fever) evaluation of fetal infection and abortion in pregnant ponies experimentally infected with ehrlichia risticii identification of ehrlichia risticii as the causative agent of two equine abortions following natural maternal infection effect of equine ehrlichial colitis on the hemostatic system in ponies detection of serum antibodies against ehrlichia risticii in potomac horse fever by enzyme-linked immunosorbent assay evidence for a high rate of false-positive results with the indirect fluorescent antibody test for ehrlichia risticii antibody in horses detection and quantitation of ehrlichia risticii genomic dna in infected horses and snails by real-time pcr comparison of pcr and culture to the indirect fluorescent-antibody test for diagnosis of potomac horse fever diagnostic application of polymerase chain reaction for detection of ehrlichia risticii in equine monocytic ehrlichiosis (potomac horse fever) evaluation of vaccination of horses as a strategy to control equine monocytic ehrlichiosis association of deficiency in antibody response to vaccine and heterogeneity of ehrlichia risticii strains with potomac horse fever vaccine failure in horses equine intestinal clostridiosis: an acute disease in horses associated with high intestinal counts of clostridium perfringens type a a method for reproducing fatal idiopathic colitis (colitis x) in ponies and isolation of a clostridium as a possible agent hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals apparent outbreaks of clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital a prospective study of the roles of clostridium difficile and enterotoxigenic clostridium perfringens in equine diarrhoea clostridial enterocolitis prevalence of clostridium perfringens enterotoxin and clostridium difficile toxin a in feces of horses with diarrhea and colic clostridium difficile associated with acute colitis in mature horses treated with antibiotics the effect of colic on the microbial activity of the equine intestine comparative effects of oral administration of trimethoprim/sulphadiazine or oxytetracycline on the faecal flora of horses colonic fermentation to short-chain fatty acids is decreased in antibiotic-associated diarrhea clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia lincomycin-induced severe colitis in ponies: association with clostridium cadaveris an investigation into clostridium perfringens enterotoxin-associated diarrhoea enterotoxigenic clostridium perfringens esophageal duplication cyst as a cause of choke in the horse beta toxin, a novel toxin produced by clostridium perfringens prevalence of beta -toxigenic clostridium perfringens in horses with intestinal disorders an overview of clostridium perfringens enterotoxin the effects of clostridium perfringens type a enterotoxin in shetland ponies: clinical, morphologic and clinicopathologic changes an update on clostridium perfringens enterotoxin clostridium difficile infection direct evidence of mast cell involvement in clostridium difficile toxin a-induced enteritis in mice neutrophil recruitment in clostridium difficile toxin a enteritis in the rabbit neuronal involvement in the intestinal effects of clostridium difficile toxin a and vibrio cholerae enterotoxin in rat ileum increased substance p responses in dorsal root ganglia and intestinal macrophages during clostridium difficile toxin a enteritis in rats cp- , , a substance p antagonist, inhibits rat intestinal responses to intestinal infarction associated with mesenteric vascular thrombotic disease in the horse the role of parasites in colic retropulsion-propulsion in equine large colon diarrhoea in horses associated with ulceration of the colon and caecum resulting from s. vulgaris larval migration clinical response of pony foals experimentally infected with strongylus vulgaris host response to experimentally induced infections of strongylus vulgaris in parasite-free and naturally infected ponies morphologic and clinicopathologic changes following strongylus vulgaris infections of immune and nonimmune ponies increase of immunoglobulin t concentration in ponies as a response to experimental infection with the nematode strongylus vulgaris larval cyathostomiasis immature stages of trichonema spp as a cause of diarrhoea in adult horses in spring larval cyathostomiasis (immature trichonema-induced enteropathy): a report of clinical cases diagnosis and successful treatment of diarrhoea in horses caused by immature small strongyles apparently insusceptible to anthelmintics recurrent diarrhoea in aged ponies associated with larval cyathostomiasis outbreak of larval cyathostomiasis among a group of yearling and two-year-old horses pathogenicity of cyathostome infection chronic diarrhoea in adult horses: a review of referred cases diarrhoea in adult horses: a survey of clinical cases and an assessment of some prognostic indices the pathogenic effects of experimental cyathostome infections in ponies fructosamine measurement in ponies: validation and response following experimental cyathostome infection re-evaluation of ivermectin efficacy against equine gastrointestinal parasites prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses equine cyathostome infection: suppression of faecal egg output with moxidectin experimental cyathostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: comparison of parasite burdens, immunity and colonic pathology identification and characterization of a pyrantel pamoate resistant cyathostome population lethal complications following administration of oxytetracycline in the horse lincomycin-associated colitis in horses risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: cases ( - ) case control and historical cohort study of diarrhea associated with administration of trimethoprim-potentiated sulphonamides to horses and ponies the association of erythromycin ethylsuccinate with acute colitis in horses in sweden effects of transportation, surgery, and antibiotic therapy in ponies infected with salmonella the influence of the normal flora on clostridium difficile colonisation of the gut studies on experimental enteric salmonellosis in ponies physiology of diarrhea: large intestine impaired colonic fermentation of carbohydrate after ampicillin the relationship of absorption characteristics and gastrointestinal side effects of oral antimicrobial agents prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period hypovolemic hyponatremia and signs of neurologic disease associated with diarrhea in a foal toxicology of nonsteroidal antiinflammatory drugs colitis induced by nonsteroidal anti-inflammatory drugs: report of four cases and review of the literature the effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study experimentally induced phenylbutazone toxicosis in ponies: description of the syndrome and its prevention with synthetic prostaglandin e phenylbutazone toxicosis in the horse: a clinical study ulceration and stricture of the right dorsal colon after phenylbutazone administration in four horses phenylbutazone toxicity in a horse biochemical and haematological effects of phenylbutazone in horses medical management of right dorsal colitis in horses: a retrospective study mechanisms of intestinal mucosal repair prostaglandins in the gut and their relationship to non-steroidal anti-inflammatory drugs the future of antiinflammatory therapy the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse mechanisms of acute and chronic intestinal inflammation induced by indomethacin mechanisms of nsaid-induced gastrointestinal injury defined using mutant mice pathogenesis of nsaid gastropathy: are neutrophils the culprits? icam- and p-selectin expression in a model of nsaid-induced gastropathy gastric ulceration induced by nonsteroidal anti-inflammatory drugs is a neutrophil-dependent process the recognition and medical management of right dorsal colitis in horses the application of technetium- m hexamethylpropyleneamine oxime ( mtc-hmpao) labeled white blood cells for the diagnosis of right dorsal ulcerative colitis in two horses cantharidin toxicosis in horses experimental cantharidiasis in the horse pathology of blister beetle (epicauta) poisoning in horses etiologic agents, incidence, and improved diagnostic methods of cantharidin toxicosis in horses clinical features of blister beetle poisoning in equids: cases clinical and diagnostic veterinary toxicology environmental biochemistry of arsenic trace metal poisoning gee, honey, why does the iced tea have a garlic taste? arsenic intoxication acute diarrheal disease in the horse the mucosal barrier, ige-mediated gastrointestinal events, and eosinophilic gastroenteritis ige-mediated (and food-induced) intestinal disease induction of anaphylaxis in mouse intestine by orally administered antigen and its prevention with soluble high affinity receptor for ige vaccine-associated anaphylactic-like reaction in a horse equine anaphylaxis equine anaphylaxis pathologic changes in experimental equine anaphylaxis role of intestinal mast cells in modulating gastrointestinal pathophysiology role of -hydroxytryptamine in intestinal water and electrolyte movement during gut anaphylaxis intestinal anaphylaxis in the rat: mediators responsible for the ion transport abnormalities motility effects of intestinal anaphylaxis in the rat intestinal anaphylaxis: rapid changes in mucosal ion transport and morphology exhaustion shock in the horse lactic acidosis: a factor associated with equine laminitis changes in the caecal flora associated with the onset of laminitis intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report plasma endotoxin levels in horses subjected to carbohydrate induced laminitis surgical treatment of sand colic: results in horses diarrhea associated with sand in the gastrointestinal tract of horses sand-induced diarrhea in a foal abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation nasogastric electrolyte replacement in horses acute colitis in horses. . initial management effect of hydroxyethyl starch infusion on colloid oncotic pressure in hypoproteinemic horses oncotic, hemodilutional, and hemostatic effects of isotonic saline and hydroxyethyl starch solutions in clinically normal ponies colloid volume expanders: problems, pitfalls and possibilities immunity targeting common core antigens of gram-negative bacteria protection against clinical endotoxemia in horses by using plasma containing antibody to an rc mutant e. coli (j ) enterotoxin activity of a salmonella typhimurium of equine origin in vivo in rabbits and the effect of salmonella culture lysates and cholera toxin on equine colonic mucosa in vitro enterotoxin-induced fluid accumulation during experimental salmonellosis and cholera: involvement of prostaglandin synthesis by intestinal cells nacl transport across equine proximal colon and the effect of endogenous prostanoids nonsteroidal anti-inflammatory drugs activate quiescent inflammatory bowel disease misoprostol provides a colonic mucosal protective effect during acetic acidinduced colitis in rats rationale for the luminal provision of butyrate in intestinal diseases pharmacokinetics and adverse effects of butorphanol administered by single intravenous injection or continuous intravenous infusion in horses necrotizing mycotic vasculitis with cerebral infarction caused by aspergillus niger in a horse with acute typholocolitis pulmonary aspergillosis in horses: cases ( - ) effect of antibiotics on clinical, pathologic and immunologic responses in murine potomac horse fever: protective effects of doxycycline effect of treatment with oxytetracycline during the acute stages of experimentally induced equine ehrlichial colitis in ponie effect of treatment with erythromycin and rifampin during the acute stages of experimentally induced equine ehrlichial colitis in ponies use of metronidazole in equine acute idiopathic toxaemic colitis antimicrobial susceptibilities of equine isolates of clostridium difficile and molecular characterization of metronidazole-resistant strains equine neonatal clostridiosis: treatment and prevention antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy antithrombotic actions of aspirin in the horse evaluation of heparin for prophylaxis of equine laminitis: cases ( - ) phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin cold insoluble globulin and heparin interactions in phagocytosis by macrophage monolayers: mechanism of heparin enhancement probiotics in man and animals double-blind report on the efficacy of lactic acid-producing enterococcus sf in the prevention of antibiotic-associated diarrhoea and in the treatment of acute diarrhoea effect of lactobacillus gg yoghurt in prevention of antibiotic associated diarrhoea comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites efficacy of oral ivermectin paste against mucosal stages of cyathostomes elimination of mucosal cyathostome larvae by five daily treatments with fenbendazole moxidectin: spectrum of activity and uses in an equine anthelmintic program references . laws eg, freeman de: significance of reperfusion injury after venous strangulation obstruction of equine jejunum how important is intestinal reperfusion injury in horses? clinical relevance of intestinal reperfusion injury in horses strangulating volvulus of the ascending colon in horses mucosal alterations in experimentally induced small intestinal strangulation obstruction in ponies histologic findings in the gastrointestinal tract of horses with colic large colon resection evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal diseases of the small intestine small intestinal herniation through the epiploic foramen: cases ( - ) determining the diagnosis and prognosis of the acute abdomen duodenitis-proximal jejunitis risk factors and clinical signs associated with cases of equine colic short-term survival and prevalence of postoperative ileus after small intestinal surgery in horses short-and longterm survival and prevalence of postoperative ileus after small intestinal surgery in the horse survival after small intestine resection and anastomosis in horses abdominal adhesions after small intestinal surgery in the horse mesenteric rents as a source of small intestinal strangulation in horses: cases ( - ) small intestine incarceration through the epiploic foramen of the horse right hepatic lobe atrophy in horses: cases incarceration of the jejunum in the epiploic foramen of a four month old foal use of diagnostic ultrasonography in horses with signs of acute abdominal pain parietal hernia of the small intestine into the epiploic foramen of a horse transection of the pelvic flexure to reduce incarceration of the large colon through the epiploic foramen in a horse incarceration of the small intestine in the epiploic foramen: report of cases pedunculated lipomas as a cause of intestinal obstruction in horses: cases ( - ) an analysis of cases of intestinal obstruction caused by pedunculated lipomas strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma abdominal surgery in foals intestinal surgery in the foal effects of extensive resection of the small intestine in the pony incarceration of small intestine through rents in the gastrosplenic ligament in the horse jejunal displacement through the mesometrium in a pregnant mare strangulating obstruction caused by intestinal herniation through the proximal aspect of the cecocolic fold in horses congenital inguinal hernias associated with a rent in the common vaginal tunic in five foals ruptured inguinal hernia in new-born colt foals: a review of cases acquired inguinal hernia in the horse: a review of cases surgical treatment of acquired inguinal hernia in the horse: a review of cases different types of inguinal herniation in two stallions and a gelding surgery of the small intestine complications of umbilical hernias in horses: cases strangulated umbilical hernias in horses: cases ( - ) surgical management of intussusception in the horse ileocecal intussusception in horses: cases ultrasonographic diagnosis of small-intestinal intussusception in three foals jejunal intussusception in adult horses: cases ileocecal intussusception corrected by resection within the cecum in two horses diaphragmatic hernias in horses and cattle diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases peritoneopericardial hernia in a horse diaphragmatic herniation as a cause of lethargy and exercise intolerance in a mare diaphragmatic hernia repair in three young horses surgical repair of a diaphragmatic hernia in a racehorse diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases morphologic alterations observed during experimental ischemia of the equine large colon equine large intestinal volvulus: a review of cases diseases and surgery of the large colon large colon volvulus: surgical treatment of horses colopexy in broodmares: cases cecocolic intussusception in horses: cases ( - ) cecocolic and cecocecal intussusception in horses: cases ( - ) cecal amputation via a right ventral colon enterotomy for correction of nonreducible cecocolic intussusception in horses resection of intussuscepted large colon in a horse intussusception of the large colon in a horse intussusception of the left dorsal colon in a horse intussusception of the colon in a filly rectal prolapse in the horse rectal prolapse and cystic calculus in a burro rectal prolapse in a foaling mare management of rectal injuries rectum and anus disruption to the blood supply to the small colon following rectal prolapse and small colon intussusception in a mare laparoscopic diagnosis of ischemic necrosis of the descending colon after rectal prolapse and rupture of the mesocolon in two postpartum mares intestinal infarction associated with mesenteric vascular thrombotic disease in the horse diseases of the large colon diagnostic and prognostic procedures for equine colic surgery examination of the horse with colic detection of endotoxin in cases of equine colic determining the diagnosis and prognosis of the acute abdomen risk factors and clinical signs associated with cases of equine colic accuracy of clinicians in predicting site and type of lesion as well as outcome in horses with colic struvite urethral calculus in a three-month-old thoroughbred colt urolithiasis in horses cholelithiasis in horses: ten cases review of cases of colic in the pregnant mare surgical management of uterine torsion in the mare: a review of cases rabies in horses: cases mechanisms of pain and their therapeutic implications gastrointestinal pharmacology nonsteroidal anti-inflammatory drugs the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse alpha adrenoceptor agonists in the horse: a review cardiovascular effects of medetomidine, detomidine and xylazine in horses cardiovascular effects of xylazine and detomidine in horses prognosis in equine colic: a study of individual variables used in case assessment a: study of variables commonly used in examination of equine colic cases to assess prognostic value clinical evaluation of blood lactate levels in equine colic use of clinical pathology in evaluation of horses with colic the anion gap as a prognostic indicator in horses with abdominal pain strangulating volvulus of the ascending colon in horses diseases and surgery of the large colon multivariable prediction model for the need for surgery in horses with colic prognosis in equine colic patients using multivariable analysis prognosis in equine colic: a comparative study of variables used to assess individual cases prognostic index for acute abdominal crisis (colic) in horses feeding and digestive problems in horses: physiologic responses to a concentrated meal effect of meal feeding on plasma volume and urinary electrolyte clearance in ponies evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression morphologic effects of experimental distention of equine small intestine surgical reduction of ileal impactions in the horse: cases medical treatment of horses with ileal impactions: cases ascarids: recent advances the purported role of coastal bermuda hay in the etiology of ileal impactions: results of a questionnaire (abstract) tapeworm infection is a significant risk factor for spasmodic colic and ileal impaction colic in the horse use of excretory/secretory antigens for the serodiagnosis of anoplocephala perfoliata cestodosis ileal impaction in the horse: cases evaluation of factors associated with postoperative ileus in horses: cases idiopathic muscular hypertrophy of the equine small intestine: cases ( - ) obstruction of the ileum in the horse: a report of clinical cases ileal muscular hypertrophy and rupture in a pony three years after surgery for ileocaecal intussusception jejunocolostomy or ileocolostomy for treatment of cecal impaction in horses: nine cases ( - ) small intestinal strangulation caused by meckel's diverticulum in a horse volvulus associated with meckel's diverticulum in the horse congenital jejunal diverticulum in a foal mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse abdominal adhesions after small intestinal surgery in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal the characteristics of intestinal injury peripheral to strangulating obstruction lesions in the equine small intestine current concepts in management of abdominal adhesions one percent sodium carboxymethylcellulose prevents experimentally induced abdominal adhesions in horses effect of carboxymethylcellulose and a hyaluronate-carboxymethylcellulose membrane on healing of intestinal anastomoses in horses intraperitoneal use of sodium carboxymethylcellulose in horses undergoing exploratory celiotomy prevention of intraabdominal adhesions in ponies by low-dose heparin therapy retrospective analysis of the results of exploratory laparotomies in horses with gastrointestinal disease surgical treatment for colic in the foal retrospective evaluation of repeat celiotomy in horses with acute gastrointestinal disease risk factors for reduced postoperative fecal output in horses: cases ( - ) role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model the effect of prostaglandin e on motility of the equine gut in vitro investigation of the effect of prostaglandins and nonsteroidal antiinflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure evaluation of nitric oxide as an inhibitory neurotransmitter in the equine ventral colon prostanoid production via cox- as a causative mechanism of rodent postoperative ileus the action of low dose endotoxin on equine bowel motility sir frederick hobday memorial lecture: all wind and water-some progress in the study of equine gut motility antagonism of endotoxin-induced disruption of equine bowel motility by flunixin and phenylbutazone cyclooxygenase inhibitors in equine practice in vitro effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: cases ( - ) role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period cecal impaction in the horse caecal disease in equids ileocolostomy: a technique for surgical management of equine cecal impaction diseases and surgery of the cecum treatment of impaction colics large colon impaction in horses: cases ( - ) effects of amitraz, several opiate derivatives and anticholinergic agents on intestinal transit in ponies experimental studies of druginduced impaction colic in the horse retropulsion-propulsion in equine large colon clinical and structural features of equine enteroliths petrographic and geochemic evaluation of equine enteroliths enteroliths in horses evaluation of enterolithiasis in equids: cases risk factors for enterolithiasis among horses in texas obstructive enterolith in an -month-old miniature horse surgical treatment of sand colic in equids: cases ( - ) surgical treatment of sand colic: results in horses diarrhea associated with sand in the gastrointestinal tract of horses abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation failure of psyllium mucilloid to hasten evaluation of sand from the equine large intestine nonstrangulated colonic displacement in horses functions of the equine large intestine and their interrelationship in disease comparisons of age, breed, history and management in horses with colic motor functions of the intestine the effect of strongylus vulgaris larvae on equine intestinal myoelectrical activity displacement of the large colon reversal of colonic net absorption to net secretion with increased intraluminal pressure use of ultrasound in horses for diagnosis of left dorsal displacement of the large colon and monitoring its nonsurgical correction renosplenic entrapment of the large colon in horses: cases renosplenic entrapment of the large colon in horses: cases further experiences with non-surgical correction of nephrosplenic entrapment of the left colon in the horse effect of phenylephrine on hemodynamics and splenic dimensions in horses displacement of the large colon associated with nonsurgical correction of large-colon entrapment in the renosplenic space in a mare foreign body obstruction of the small colon in six horses fecalith impaction in four miniature foals obstruction of the small colon by intramural haematoma in three horses submucosal haematoma as a cause of obstruction of the small colon in the horse: a review of four cases inflammatory bowel disease in horses: cases intestinal atresia in horses clinical survey of tumours and tumourlike lesions in horses in south east queensland abdominal neoplasia (excluding urogenital tract) clinical manifestation of squamous cell carcinoma in horses lymphoma (lymphosarcoma) in horses oral and dental tumors in equine denistry t cell-rich b cell lymphosarcoma in the tongue of a horse multiple myeloma in a horse rhabdomyosarcoma of the tongue in a horse paraneoplastic bullous stomatitis in a horse gastric squamous cell carcinoma in three horses use of esophagoscopy in the diagnosis of esophageal squamous cell carcinoma in a horse gastric hyperplastic polyp in a horse gastric leiomyosarcoma in a horse multisystemic, eosinophilic, epitheliotropic disease with intestinal lymphosarcoma in a horse an immunohistochemical study of equine b-cell lymphoma squamous cell carcinoma of the equine stomach: a report of five cases six cases of squamous cell carcinoma of the stomach of the horse small intestinal adenocarcinoma in a horse ganglioneuroma as a cause of small intestinal obstruction in the horse: a case report intestinal carcinoid in a mare: an etiologic consideration for chronic colic in horses leiomyoma of the small intestine in a horse jejunal intussusception associated with leiomyoma in an aged horse duodenal leiomyoma associated with colic in a two-year-old horse leiomyosarcoma of the duodenum in two horses pedunculated lipomas as a cause of intestinal obstruction in horses: cases ( - ) clinical aspects of lymphosarcoma in the horse: a clinical report of cases multiple peripheral nerve sheath tumors in the small intestine of a horse equine adenocarcinomas of the large intestine with osseous metaplasia intestinal myxosarcoma in a thoroughbred mare gastrointestinal stromal tumors of the equine cecum colonic adenocarcinoma with osseous metaplasia in a horse intestinal adenocarcinoma causing recurrent colic in the horse equine colonic lipomatosis large colon resection for treatment of lymphosarcoma in two horses colic in a mare caused by a colonic neurofibroma small colon intussusception associated with an intralumenal leiomyoma in a pony leiomyoma of the small colon in a horse an analysis of cases of intestinal obstruction caused by pedunculated lipomas rectal leiomyosarcoma in a horse strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases standing rectal and tail surgery disseminated peritoneal leiomyomatosis in a horse ascites as a result of peritoneal mesotheliomas in a horse a case of peritoneal mesothelioma in a thoroughbred mare omental fibrosarcoma in a horse neoplasia of the mouth and surrounding structure pleuroscopic diagnosis of gastroesophageal squamous cell carcinoma in a horse recurrent esophageal obstruction due to squamous cell carcinoma in a horse chronic colic in the mature horse: a retrospective review of cases recurrent colic in the mature horse: a retrospective review of cases paraneoplastic syndromes lymphosarcoma and associated immune-mediated hemolytic anemia and thrombocytopenia in horses differentiation of chronic lymphocytic leukemia in the horse: a report of two cases hypercalcemia associated with malignancy in a horse adenocarcinoma of intestinal origin in a horse: diagnosis by abdominocentesis and laparoscopy differentiation between intra-abdominal neoplasms and abscesses in horses, using clinical and laboratory data: cases ( - ) malabsorption in the horse associated with alimentary lymphosarcoma immunodeficiency associated with lymphosarcoma in a horse flow cytometric methods to diagnose selected equine immune-mediated disorders pleural effusion associated with squamous cell carcinoma of the stomach of a horse use of diagnostic ultrasonography in horses with signs of acute abdominal pain the indications for equine laparotomy: an analysis of cases equine lymphosarcoma diarrhoea in the horse as a result of alimentary lymphosarcoma paraneoplastic pruritus and alopecia in a horse with diffuse lymphoma lymphoma in the horse. proceedings of the twelfth annual meeting of the american college of veterinary internal medicine squamous cell carcinoma of the pharyngeal wall in a horse squamous cell carcinoma of the oral, pharyngeal and nasal mucosa in the horse treatment of superficial ulcerative squamous cell carcinoma in three horses with topical -fluorouracil intratumoral chemotherapy with cisplatin in oily emulsion in horses excision of oral squamous cell carcinoma in a horse extensive resection and anastomosis of the descending (small) colon in a mare following strangulation by a mesenteric lipoma section . peritonitis references . hosgood g: peritonitis. . a review of the pathophysiology and diagnosis the peritoneum and peritoneal cavity large animal internal medicine diseases of the peritoneum and mesentery equine internal medicine peritonitis associated with actinobacillus equuli in horses: cases veterinary gastroenterology peritonitis and other intra-abdominal infection antibiotic susceptibility of bacterial pathogens from horses peritonitis in horses: cases biologic reactions to endotoxin effect of dietary alpha-linoleic acid on equine monocyte procoagulant activity and eicosanoid synthesis review of cases of peritonitis in the horse reference values for equine peritoneal fluid diagnostic cytology in the equine species: overview of effusions (peritoneal, pleural, and synovial joint) and transtracheal wash effects of enterocentesis on peritoneal fluid constituents in the horse internal abdominal abscesses in the horse: a study of cases equine peritoneal fluid analysis following celiotomy analysis of equine peritoneal fluid evaluation of peritoneal fluid ph, glucose concentration, and lactate dehydrogenase activity for detection of septic peritonitis in horses tumor necrosis factor and interleukin- activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease low dose flunixin meglumine: effects on eicosanoid production and clinical signs induced by experimental endotoxemia in horses heparin in the treatment of experimental peritonitis erythrocyte agglutination associated with heparin treatment in three horses therapeutic strategies involving antimicrobial treatment of large animals with peritonitis effect of long-term administration of injectable enrofloxacin solution on physical and musculoskeletal variables in adult horses peritoneal lavage in the horse pharmacologic principles therapeutic drug monitoring: a tool for rational drug therapy. proceedings of the seventh american college of veterinary internal medicine forum toxicity of quinolones although a number of potential causes of peritonitis exist, sepsis is a common and serious complication, and the identification and control of bacterial sepsis is critical for a successful outcome. bowel leakage (as well as external trauma) results in contamination of the peritoneum with large numbers of many types of bacteria. the intestinal tract contains a mixed population of bacteria, and the quantity of bacteria and prevalence of anaerobic species increase in the distal segments. [ ] [ ] [ ] [ ] [ ] [ ] [ ] there are approximately × anaerobic and × aerobic bacteria per milliliter of cecal and colonic fluid, thus the potential for bacterial contamination of the peritoneum is great. high mortality is associated with contamination from the lower bowel because of the large numbers of bacteria present. hirsch and jang reported isolation of an infective agent from equine peritoneal fluid in approximately % of attempts. obligate anaerobic bacteria were cultured most frequently, followed by members of the enterobacteriaceae family (escherichia coli). penicillin-resistant bacteroides fragilis was isolated from % to % of cases. in another study in which bacteria were identified in equine abdominal fluid by cytologic examination or culture, e. coli was the organism most commonly isolated. in human beings and laboratory animals the well-established fact is that despite the variety of organisms initially introduced subsequent to these events, established infections are characterized by only a few types of bacteria, which are often gram-negative aerobes and anaerobic bacteria. this selectivity occurs through the processes of selective reduction of bacterial populations and bacterial synergism. a well-known example of synergism in human beings and laboratory animals is peritonitis involving e. coli and b. fragilus. the presence of each organism is beneficial to the survival of the other, and each is important in the overall pathogenesis of the disease. e. coli is associated with septicemia and early mortality, whereas b. fragilis infection tends to result in chronic abscessation with delayed morbidity and mortality. some evidence suggests that in horses, in addition to coliforms and anaerobes, streptococci and perhaps c. psuedotuberculosis may survive selective reduction and participate in synergistic infection following polymicrobial contamination.biologic events resulting from contamination of the abdomen or injury to the mesothelial cells have been described [ ] [ ] [ ] [ ] and include release of catecholamines, histamine, and serotonin from peritoneal mast cells; vasodilation and hyperemia; increase in peritoneal vascular permeability; secretion of protein-rich fluid into the peritoneum; transformation of mesothelial cells into macrophages; and influx of polymorphonuclear cells, humoral opsonins, natural antibodies, and serum complement into the peritoneal cavity. additionally, depression of the peritoneal fibrinolytic activity, fibrin deposits on the peritoneal surface, and sympathetic-mediated ileus of the gastrointestinal tract can occur.these processes benefit the animal by confining contamination and infection, and indeed, with clean, minimally invasive procedures such as enterocentesis or trocharization, this is effective. however, with greater severity of peritoneal contamination or irritation, these processes are magnified and become deleterious, resulting in problems such as hypovolemia, hypoproteinemia, ileus with resultant bowel distention, ischemia of the bowel wall with subsequent absorption of bacteria and toxins, and ultimately adhesion and abscess formation. additionally, systemic responses to bacterial toxins, particularly lipopolysaccharide, , can compromise the metabolic condition of the patient further. equine peritoneal macrophages release several mediators when exposed to bacterial lipopolysaccharide, undoubtedly an important component of septic peritonitis.pathologic description of peritonitis includes origin (primary or secondary), onset (peracute, acute, chronic), distribution (localized versus diffuse), and presence of bacteria (septic versus nonseptic). , clinically, viewing key: cord- -yaxawqhj authors: bucknall, r.a. title: the continuing search for antiviral drugs date: - - journal: adv pharmacol doi: . /s - ( ) - sha: doc_id: cord_uid: yaxawqhj this chapter discusses the continuing search for antiviral drugs. many virus diseases, both of humans and animals, have been successfully controlled by vaccines. these successes have naturally led to improvements in the spectrum and duration of protection offered by vaccines until, at present it is difficult to see how antiviral drugs could compete with vaccines in the control of many virus diseases. one may cite smallpox, yellow fever, polio, and recently measles among human diseases, newcastle disease, marek's disease, and infectious bronchitis among poultry diseases—an area of veterinary disease control where vaccines have been particularly important. research into the treatment of virus diseases by drugs is at present directed toward three general areas: ( ) attempts to stimulate the defense mechanism of the host animal, ( ) large screening programs to find drugs which directly block some virus-specific process, and ( ) alleviation of the symptoms of the disease. the treatment of the symptoms, rather than the cause of a disease, has been the mainstay of medical practice from time immemorial, and this is still the case with most virus disease. the short incubation period of many virus diseases will inevitably restrict the therapeutic use of antiviral drugs and in cases where symptoms have already appeared. although research into the mechanisms of virus infection is carried out by many sections of the scientific community, the search for antiviral drugs is almost exclusively the province of pharmaceutical manufacturers. the reasons for this are partly historical, but chiefly it is because the facilities for running large-scale screening programs are expensive and can only be met by commercial and, occasionally, governmental resources. because of the need to protect their discoveries from unauthorized exploitation, a good deal of secrecy inevitably surrounds the work being carried out in commercial organizations. this secrecy is regrettable but necessary, since the survival of such organizations depends largely then on getting a fair financial return on the money invested by them in research. the security aspects of commercial research naturally restrict frank and constructive discussions between competitors as well as third parties, and this has been particularly true in antiviral research. perhaps it was a revolt against this enforced isolationism which led, at least in part, to the first conference on antiviral substance's held by the new york academy of sciences in , in which manufacturers disclosed many of the details of their antiviral research and had a chance to discuss their failures and comparative successes (whipple, ) . since then there have been a number of reviews of progress in the field of antiviral chemothrrapy, and in most of these there have appeared comments and recommendations ivhich indicate that a radical rethinking is taking place of the prospects for antiviral drugs (osdene, ; mcfadzean, ; goz and prusoff, ; swallow, ). it was natural in the early s to espect that antiviral drugs would be discoverrd which would be analogous to the antibacterial antibiotics, the darlings of the prrvious decade. the experience up to date has proved that this was not to br the case, and despite prodigious efforts by the drug houses, only three clinically useful antiviral agents, which are far from perfect, have emerged. i believe thcre are lessons to be learned from this disappointing record, and i hope that the following remarks may help to continue further the critical reappraisal of this field, so that future progress may be faster. before embarking on a search for antiviral drugs, a number of points must be considered in order to assess thr technical feasibility of treating or preventing a virus disrase u-ith a drug. too often in the past massive screens have bccn set up against many viruses in the hope that a drug would turn up, and it would then find a natural place in human or veterinary medicine. although it is true that random screening still offers the best chance of discovering new drugs, unless a realistic appraisal of the whole project is made a t the outset, the products of random screens could well be useless as potential medicines. somr of the more important considcrations are listed below. a. diseases of economic importance i n ordcr to he commercially viable a drug must sell in sufficient quantities to pay for its devclopmcnt and manufacture as well as for future research. because of this, and because antiviral chemicals tend to inhibit specific viruses. diseases of low incidence or loneconomic importance are ruled out as primary targets for drug devclopment. this may seem inhumane, particularly in the field of human virus diseases, but the fact remains that it is no easier to find a drug against rabies than against influenza, and the sales from a specific antirabies drug would never cover its own developmrnt costs. diseases of loiv incidence or low cconomic importance, no matter how serious the outcome may be for the infected individual, inevitably must remain the subjects of sponsored research. nevertheless, treatments for some of these diseases may emerge as drugs are developed against major diseases. b. immunological control many virus diseases, both of humans and animals, have been successfully controlled by vaccines. these successes have naturally led to improvements in the spectrum and duration of protection offered by vaccines until, today, it is difficult to see how antiviral drugs could compete with vaccines in the control of many virus diseases. as examples one may cite smallpox, yellow fever, polio, and, more recently, measles among human diseases, and newcastle disease, marek's disease, and infectious bronchitis among poultry diseases-an area of veterinary disease control where vaccines have been particularly important. despite these triumphs, vaccines are not, and probably never will be, the complete answer to the control of certain virus diseases. it is in these areas where drugs would be useful, and it is on these diseases that efforts should be concentrated. the two most important human diseases in this class are influenza and the common cold. when a novel strain of influenza appears among the human population, as happened in , , and , existing immunity to the previous current influenza strain is not effective, and widespread epidemics of disease occur. the disease spreads so rapidly after it first appears that it is not possible to develop, distribute, and administer a vaccine based on the new strain soon enough to protect useful numbers of the population. even after the initial overwhelming pandemic, successive epidemics will occur as the disease penetrates into pockets of the community that 'had escaped infection. the disease is then maintained partly by the continuing appearance of susceptible juveniles, partly by spontaneous antigenic modifications in the virus enabling it to overcome previous immunity, and partly by the general decline in immunity of other individuals with the passage of time. after the initial pandemic, it is theoretically possible to control the disease with widespread vaccination, but in practice this is not done. consequently the disease smoulders on in the community, appearing as isolated cases and occasional outbreaks and epidemics. these are the conditions under which the disease normally exists, and it is this situation, rather than the much publicized pandemics, which causes the greatest economic loss to industrialized countries. the overall loss in - in great britain due to the asian influenza pandemic was estimated at x million, but the continuing loss, from that time on has been at least x million each year. losses in europe, north america, japan, and similar industrialized communities must be romparablr, and if only a fraction of the disease could be prevented by a drug, the economic benefits would be enormous. losses due to the common cold are comparable. the careful study of lidwell and williams ( ) showed that approximately x lo working days arc lost cach year in great britain alone from the common cold. scvertheless, the prospects for a common cold vaccine are poor because of the large number of viruses which are known to cause the disease. there are known rhinoviruses (kapikian, ) , probably a comparable number of as yct unclassified rhinoviruses, a growing catalog of coronaviruses (kapikian, ) , and a selection of other viruses including myxoviruses, adenoviruses, and herpcsviruses (tyrrell, ) , all of which have been isolated from clinical colds. it is the serological diversity of these etiological agents which makes the prospects for a vaccine so poor, and the common cold must, therefore, be considered as a target, even though a difficult target, for antiviral drugs. besides the problems of antigenic variation, exemplified by influenza, and thc multiplicity of serotypes, exemplified by the common cold, there are two further problems associated with the control of respiratory diseases by parmterally administered vaccines. the first is that circulating antibodies appear in only small amounts in respiratory mucus, and, consequently, the degree of protection afforded to the respiratory tract is less and of shorter duration than might be expected. the second problem has been brought to light by the use of experimental vaccines against respiratory syncytial virus disease in infants. when infants who had been vaccinated parenterally against this disease contracted the natural disease, they were more ill than infants who had not received the vaccine. the reason seems to be that the circulating antibodies resulting from parenteral vaccinations not only offer little protection to the respiratory tract, but when a natural infection occurs, these antibodies combine with the virus antigens at the surface of the respiratory epithelial cclls causing an inflammatory response with a corresponding increase in the severity of clinical symptoms kim et al., ; kapikian et al., ) . chanock et a . ( ) have pointed out that if an immunopathological process involving serum antibodies occurs during respiratory syncytial virus infection, then stimulation of local, respiratory tract, secretory antibody by intranasal instillation of live or inactivated virus may give adequate protection without unwanted hyperreactivity. a similar allergic reaction between virus antigen and preexisting antibody is a factor, possibly a major factor, in the pathological processes initiated by herpesviruses (jones and patterson, ) and marks the herpes diseases as possible candidates for antiviral drug development. one factor that deserves more careful consideration than it usually receives is the way in which a potential antiviral drug would be administered to the animal or patient requiring protection. clearly a common cold treatment would be unacceptable if it had to be given intravenously times a day. but there are less obvious, but no less real, difficulties in dosing large herds of cattle or sheep so that effective protection is maintained. with freeranging animals the duration of protection from a single dose would need to be prolonged to offset the labor of administering the dose. also, it is easier to dose herds by injection than by mouth, so that certain veterinary antiviral drugs may not be required in an orally active formulation. conversely, oral dosing, preferably by an addition to food or drinking water, is the most convenient way of dosing poultry. the onset of symptoms in most virus diseases is acute and may be the first indication that the host has contracted an infection. because of this, it is usually assumed that antiviral drugs will only be of value in preventing and not in curing virus diseases. nevertheless, although the periods of virus growth in infected individuals may be short, they may well be long enough to allow useful therapy. for example, pate et al. ( ) have shown in volunteers infected intranasally with coxsackie a virus that maximum virus growth precedes the onset of symptoms by hours. but from their work it may be seen that a considerable amount of virus growth is concurrent with the period of overt symptoms, and application of antiviral drugs during this period of - days might well prevent the full development of the disease. the work of douglas et al. ( ) with volunteers shows that a similar period exists in acute rhinovirus infections, and dawkins et al. ( ) and wingfield et al. ( ) have shown that the course of influenza in humans can be modified, even after the onset of symptoms, by treatment with -aminoadamantane. it hardly seems necessary to point out that before undertaking a search for a drug against a particular disease, the etiological agent of the disease should be unequivocally identified. there are a number of important virus diseases for which the causative agent is either unknown or is in doubt, for example, bovine pneumonia and human epidemic viral gasteroenteritis. it would be a mistake to set up screens against agents that were only suspected of being implicated in these diseases in case subsequent work should demonstrate that these were not in fact the causative agents. although it is difficult to predict changes in governmental legislation toward public or animal health, nevertheless, the existing and prospective legal position in various countries should be considered since these may affect the prospects for potential drugs. for example, in great britain, foot and mouth disease is controlled by the policy of slaughter and compensation, but in other countries the disease is controlled by vaccination. in the lattrr countries, a drug may find a ready market, but if legislation should change, then that market would be lost. in summary, we may say that before embarking on a search for antiviral drugs, the target disease must be carefully selected by a consideration of all the relevant factors. the more important of thesc a r t : . there must be an adequate market for the drug. . there should be no effective immunological control, and no prospects . due regard should be given to the practicability and the timing of . the etiology of the disease should be clearly established. other relevant factors, e.g., medical or veterinary legislation, should be takcn into consideration. in table i , a number of virus diseases of economic importance are listed together with some comments to illustrate how many seemingly attractive drug-target diseases are in fact precluded by other factors. for such control. dosing the patients or animals at risk. it is easy to list the properties of the ideal antiviral drug-wide spectrum of activity, nontoxic, accessible to the target organ, etc. what is not so easy is to predict what sort of properties one might expect from antiviral leads detected by screening programs. all the same, it is only by intelligent attempts to do just this that screens may be designed to detect compounds that one day may lead to the development of useful medicines. the scientific literature abounds with reports of antiviral chemicals discovered by screening random compounds in tissue culture systems, but all too frequently these compounds turn out to be false positives or active only against some relatively unimportant virus. for the products of a tissue culture screen to be of potential value, the screen must meet the requirements outlined below. first, the screen should be able to process large numbers of chemical compounds, or fermentation products, since the more that are tested, the greater the chance of success in finding active leads. perhaps the time will come when new drugs can be designed and synthesized on entirely rational grounds, but at present most active leads are chance discoveries. buthala ( ) quotes % of all compounds tested in a tissue screen as showing some antiviral activity, but in my experience the rate varies from to o.ol$!&, the higher rate referring to influenza a viruses, and the lower rate to picornaviruses. bauer ( ) has suggested that the larger the virus, the more susceptible it is to inhibition, since, for any given intracellular concentration of drug, a large virus will encompass, both physically and in terms of synthetic requirements, more drug molecules than a small one. undoubtedly, this principle will contribute to our observed high rate of inhibition of influenza virus, but another important factor seems to be that the adsorption of influenza viruses to cellular receptors is particularly vulnerable to interference by extraneous substances. given that only a fraction of a percent of all compounds tested in tissue culture will show activity and that of these only a small proportion will show activity in animal models, a realistic screening rate would be not less than compounds a year. at rates less than this, the chances of finding useful compounds become so small as to make the whole project not worthwhile. in many of the published screening procedures, the virus with which the screens are run seem to have been chosen more for the ease with which they can be handled rather than for their relevance to any virus disease target. for example, ehrlich et al. ( ) describes a screen where the primary test viruses include parainfluenza type , measles, and poliovirus, and johnson ( ) describes a screen that includes pseudorabies, adenovirus , and mouse hepatitis virus. of course, if wide-spectrum leads appear, the choice of test virus may be irrelevant, but the antiviral compounds (as distinct from interferon inducers) known at present are characterized by their relatively limited spectrum of activity, e.g., methisazone is active only against poxviruses (bauer and sadler, ) and possibly adenoviruses (bauer and apostolov, ) ; l-aminoadamantane is active only against influenza a and as and not against other myxo-or paramyxoviruses (davies et al., ) ; guanidine and a-hydroxybenzyl benzimidazole are active only against picornaviruses and not against other small ribonucleic acid (rna) viruses (eggers and tamm, ) . thus, whenever possible, the viruses used in routine screens should be those that are responsible for the clinical large-scale screens use large numbers of tissue culture cells, and there has been an inevitable trend toward the use of continuous cell lines in screening procedures. such cells have many attractive features. they grow rapidly, they can easily be obtained in large quantities, they remain "the same" year after year, they can be madr to prrform useful technical tricks such as rapidly changing the ph of their medium and surviving for long periods under agar, and, perhaps most important, they will support the growth of a wide range of viruses. continuous cell lines seem to be the natural choice for running routine screens. xevertheless, it is worthwhile remembering that many of the desirable technical properties exhibited by these cells may be a direct result of their neoplastic nature, and to use a continuous rather than a primary or diploid cell in a tissue culture system is to take yet another step away from the natural disease. it is truc that antiviral agents, such as l-aminoadamantane and methisazone, which can be shown to protect humans against virus diseascs, also exert their antiviral action in neoplastic cells in tissue culture, for example hela and kb cells, but we have striking evidence that this may not always follow. we have recently discovered a family of chemical compounds that have high activity against rhinoviruses when grown in human diploid lung cells, but virtually no activity against the same viruscs growing in monkey kidney cells, hela cells, or kb cells (bucknall, unpublished results) . these compounds and any others that may exhibit this property would have been missed in tests carried out in continuous cell lines. in summary, a tissue culture screen should be able to proccss large numbers of tcst compounds, using viruses as relevant as possible to the diseases for which a drug is required, and should employ normal rather than neoplastic cells. unfortunately, in most of the published screening procedures the last two requirements have been sacrificed to technical considerations designed to increase the number of compounds tested, as the following descriptions will show. in its simplest form, a test for antiviral activity involves treating cultures of cells with a range of concentrations of a test compound. first the maximum concentration tolerated by the cclls is assessed; then, second, the growth of virus at lowcr concentrations of compound that are not cytotoxic is measured. several ingenious methods have been devised for measuring these two responses-cytotoxicity and virus growth-all designed to facilitate the screening of large numbers of compounds. for example, herrmann et al. ( ) devised a zone-inhibition test in which large flat dishes of chicken cells were infected with test virus, overlaid with agar containing a vital stain, and paper discs impregnated with test compounds placed on the surface of the agar. compounds with antiviral activity showed two concentric zones around the paper disc, the innermost being pale in color due to the destruction of host cells by cytotoxic concentrations of compound diffusing from the disc. outside this was a deeply staining zone where cells were exposed to nontoxic concentrations of compound which also protected them from the destructive effects of the virus with which they had been infected. beyond this, where the concentration of compound was too low to protect the cells, the cell sheet was destroyed by virus and stained poorly. thus, by visual inspection of the dishes after to days, active compounds could be quickly detected. rada et al. ( ) devised a similar agar diffusion test in which test compounds were applied to the virus-infected cell sheets in circular wells in the agar overlay. although agar diffusion tests are capable of processing large numbers of test compounds, they suffer from two drawbacks. first, they are of comparatively low .sensitivity in detecting both the cytotoxic and antiviral levels of compounds, and second, they are limited to viruses that produce plaques under agar. rightsel et al. ( ) devised a system based on the fact that if cells were damaged either by the toxic effects of a chemical compound or by virus growth, they would not swing the ph of their medium. thus, by incubating virus-infected cells in a series of concentrations of a compound and then looking for the cultures that had changed the color of the phenol red indicator in their medium from pink to yellow, active compounds could be detected. this technique has also been used to assay neutralizing antibody and interferon action (pauker, ). finter ( ) described a system whereby the cytotoxic effects of test compounds could be assayed by the reduction in the amount of neutral red taken up by treated cells, and, similarly, the cytopathic effects of virus growth could be quantitated by measuring the reduction in the uptake of neutral red by infected cells. the system can readily be adapted to the screening of test compounds for antiviral activity. if myxoviruses are used in this system, because their cytopathic effects may not be pronounced, their growth is best monitored, not by a reduction in neutral red uptake, but by a quantitative hemadsorption method which matches the neutral red uptake method in its accuracy and sensitivity (finter, ) . in contrast to the eone-inhibition and ph-swing tests, the neutral red uptake test is precise in operation and may be used to demonstrate fine differences in the relative toxicity and activity of test compounds; it is probably no more timeconsuming than the former tests. a system of testing for antiviral agents based on the inhibition of nucleic acid synthesis was dcscribcd by lliller et al. ( ) and has been used to screen compounds and mold metabolites for antiviral activity (miller et ul., ) . for the test, hela cells were suspended in a medium containing uridine- h. if a test compound has toxic effects on the hela cells, then the cellular r s a synthesis, as measured by u r i d i n~-~h fixation, will be reduced. similarly, if cells are infected with an r s a virus and treated with sctinomycin d. then r s a synthesis will be due to virus growth only. thus, if test compounds reduce this virus-directed rxa synthesis at concentrations that do not affect cellular r s a synthesis, then the compound is exerting a specific effect on virus growth. the test can also be used for deoxyribonucleic acid (dsa) viruses, the cellular and virus dxa synthesis being monitored by including th~midine-~h in the medium. virus dsa synthesis is distinguished from cellular dsa synthesis by disrupting the cells at the end of the test and treating them with dcoxyribonuclease when encapsulated virus dsa is resistant to digestion and the unprotected host cell dsa is not. thus, the selective effect of test compounds on the synthesis of virus dsa can be measured. the authors claim that the system operates satisfactorily ivith a range of viruses-some of them important disease organisms-and is simple, reliable, and rapid. the chief criticism of this method is that, because nucleic acid synthesis is used as the sole measure of virus growth, test compounds that might act on subsequent stages in the virus replicative cycle may not be detected. for example, any disturbances in the sequencing of virus nucleic acid, inhibition of structural protein synthesis, or failure of assembly or release of mature virions, would provide a sound basis for a useful drug, but these phenomena may not be detected in this type of test. also, since high infecting doses of virus are used to give satisfactory operation of this test (up to virus particles per cell), the test may be rather insensitive in detecting antiviral activity. all antiviral activity is, in the broadest sense, competitive, either a t the level of cellular membrane receptors or at an cnzymic or template level. thus, the more virus is used to initiate infection, the less effective an antiviral compound is likely to be. although this factor will not turn a highly active compound into an inactive one, it may well obscure low levels of activity which might be useful starting points for chemical exploitation. the real value of miller's test is the use of the important biochemical system of nucleic acid synthesis to monitor the toxic manifestations of test compounds. this subject is discussed furthrr in the following section. reference has already been made to four methods for measuring the toxicity of chemical compounds in tissue culture cells: direct cytopathic ef-fects, vital dye uptake, metabolic activity (ph-swing), and nucleic acid inhibition, and there is no shortage of other methods. nevertheless, the inadequate assessment of compound toxicity in antiviral testing probably gives rise to more false leads than any other single cause, before discussing how compound toxicity might be measured, it must first be defined. strictly, any interference with cellular metabolism by an extraneous compound is a toxic effect, and the most stringent tissue culture test of lack of toxicity is the continued normal division and growth of cells in the presence of an extraneous compound. however, this test is too cumbersome for use in rapid screening procedures, and simpler, but less critical, tests are invariably used in primary screens. undoubtedly, the simplest method of assessing compound toxicity is by direct microscopic examination of cells for cytopathic effects or more subtle morphological changes. it is necessary for the observer to be trained to detect such changes, and an arbitrary scale must be devised to record the observations, but if these simple requirements are met, the method is generally successful. it may be objected that this system would be unworkable where large numbers of compounds are being screened because of the correspondingly large numbers of microscopic examinations required; but given a good low-power microscope, an experienced reader, and the fact that most random compounds tested will show no antiviral activity and will, therefore, not require more than a cursory examination, the system is reliable, fast, and economical. in the author's laboratory a system of this kind has been in use for over years, and it is possible for one worker to screen a hundred compounds against three viruses each week. we have found that with this system, compounds appear to be toxic a t lower concentrations than with either the zone diffusion or the dye uptake method. we conclude, therefore, that our method is more sensitive than the others mentioned in detecting the toxic effects of compounds. the direct microscopic assessment of toxicity is not without its deficiencies, but if the method is seen only as a preliminary determination of toxicity, these deficiencies are not serious. chief among these (and this applies even more to indirect methods) is the occasional failure to detect certain types of toxicity. for example, from time to time we have had compounds which appeared to prevent virus growth and to show no toxicity to confluent sheets of tissue culture cells, these cultures looked normal for several days in the presence of the compound, but viruses would not grow in these cells. nevertheless, further studies (see below) have shown that the compounds were exerting an inhibitory effect on some aspect of the cellular metabolism and it was this which prevented virus growth. we have investigated this effect with (a) inhibitors of nucleic acid synthe-sis and ( b ) uncouplers of oxidative phosphorylation, two classes of compound that are particularly prone to giving misleading results. nucleic acid inhibitors are often slow to produce cytopathic effects in confluent monolayers of cultured cells. the d s a synthrsis of such cells is low, and sufficient r s a synthesis is often maintained in the presence of partially effective concentrations of an inhibitor, enabling the cellular structural integrity to be sustained. all the same, an invading virus is unable to replicate in a cell under these reduced circumstances, and this will lead to an apparent antiviral specificity. this is the mechanism by which the chlorinated ribofuranosylbenzimidazoles exert their antiviral effects (bucknall, ) . these compounds were extensively studied as antiviral agents before their "activity" was found not to be specific for the virus (tamm et al., ; tamm and srmes, ; tamm and overman, ) . uncouplers of oxidative phosphorylation also often appear to be antiviral agents becausc concentrations that greatly reduce the energy-generating systems of cells in confluent monolayers are often slow to produce morphological changes. in this half-poisoned state, the cultures appear normal, but do not support virus growth, and thus another false "lead compound" is generated. as mentioned earlier, these remarks apply to all tissue culture systems to a greater or lesser extent, and tissue culture tests for antiviral activity must always be regarded as strictly preliminary. active leads from such tests must always be subjected to the closest scrutiny to determine whether the activity is truly specific for a virus-coded process or simply results from a subtle toxic effect on the host cell. the margin betwen the maximum nontoxic concentration and the minimum antiviral concentration of a test compound is conveniently expressed as thrrapcutie ratio = max. nontoxic concentration/min. antiviral concentration and will vary according to how these two concentrations are determined. the simplest and most stringent test of the maximum nontoxic concentration of a compound in zdtro is to grow cells in the presence of the compound and determine the maximum concentration a t which division and growth will proceed normally. if this concentration, and lower ones, protect the cells from virus attack, then this is an unequivocal demonstration that the compound is exerting a specific effect on some aspect of virus replicat ion. like miller et al. ( ) , we have found the inhibition of cellular nucleic acid synthesis, particularly r s a synthesis, to be a useful system for detecting the toxic effects of test compounds. cells are treated with a range of concentrations of a compound, then the uptake of ~r i d i n e -~h into acidinsoluble material is measured and compared with that of normal cells (bucknall, ) . the test is simple to run, and since the nucleic acid metabolism is a cardinal area in the cellular metabolism, even if a compound has no direct effect on the nucleic acid synthesis, disturbances of other synthetic or homeostatic mechanisms are quickly reflected in changes in the synthesis of rna or dna or both. in fig. , the dose-response curves of one experimental compound are determined in human diploid lung cells by the three methods outlined above-direct cytopathic effect in confluent monolayers, inhibition of rna synthesis, and inhibition of cell growth in newly seeded cultures. although this compound is not a specific inhibitor of rna synthesis, the inhibition of rna synthesis and the production of cytopathic effects run close together. at concentrations that indirectly affect the nucleic acid synthesis, sufficient disturbance is caused in other areas of the cellular metabolism to lead to a general cytopathic effect. cell division is affected a t lower concentrations and in this particular case, inhibitory (and therefore toxic) effects can be detected with concentrations times lower than those that cause cell destruction, nevertheless, even with cell growth as a measure of toxicity, and effects on the cellular growth rate (&-a) are higher than those that suppress virus growth, indicating that ici , is exerting a specific effect on virus growth. ( % end points: cpe, pg/ml; rna synthesis, pglml; cell growth, . pg/ml; virus growth, . pg/ml.) there is a clear margin between the toxic and antiviral effects, as may be seen from the virus yield curve. fusidic acid, which has been reported to show specific antiviral activity in tissue culture (acornley et al., ) , was also tested in the same way (fig. ) . in this case, the concentration causing % cytopathic effect after hours was pg/ml, whereas virus yield was depressed to % by only pg/lml, giving an apparent therapeutic ratio of . but when the effects of fusidic acid on cellular synthesis were studied, it was clear that cellular rka synthesis was drastically reduced by pg,!ml. thus, fusidic acid probably reduces virus growth by inhibiting cellular, rather than virus, synthetic processes, and probably accounts for the fact that this compound showed no clinically useful effects in virus-infected volunteers, despite good levels of drug in blood and nasal secretions (acornley et al., ) . since the toxic effects of compounds in vitro usually increase with time, it is important when comparing toxicity and antiviral activity, to ensure that the compound has been in contact xvith cells for the same length of time in each case. in the above experiment, the cytopathic effect, rna synthesis, cell growth, and virus inhibition were all measured in cells that had been exposed to the compound for hours. when a virus disease is limited to a particular target organ, such as the respiratory tract, it is of great value to be able to culture a portion of the organ for in vitro studies. the culture of portibns of trachea has been extensively used to study the growth of respiratory viruses (hoorn and tyrell, , ; mcintosh et al., ; craighead and brennan, ; herbst-laier, ) , and recently organ cultures of human embryonic gut have been used to study the agents of human "virus" gastroenteritis ( d o h et al., ) . the technique could presumably be extended to the study of viruses, such as polio, rabies, smallpox, and herpes, which localize in specific organs of infected individuals. we have found the use of human embryo and animal tracheal pieces of value in studying the toxicity and the antiviral activity of leads produced by tissue culture screening programs. our technique is to excise a trachea and cut it transversely into rings - mm thick. these are placed in x in. tubes with ml of eagle's medium and rolled exactly as conventional tissue cultures. with a low-power microscope the ciliary activity of the respiratory epithelium is assessed on an arbitrary scale of to . in the presence of a test compound, the reduction of ciliary action is a highly sensitive measure of compound toxicity, and it is easy to determine the concentration at which full ciliary activity can be maintained (fig. ) . at lower concentrations the effects on virus growth may be measured by harvesting the culture fluid at intervals and titrating for infectious virus. by using this technique, we have found that almost % of the so-called active compounds produced by a tissue culture screen against influenza a appear negative when tested in ferret tracheal cultures. in almost all cases, compounds were toxic at lower concentrations in tracheal cultures than in conventional tissue culture monolayers, as judged by a cessation of ciliary activity. in a proportion of these compounds, some data concerning their biochemical action were available, and in most cases these drugs were uncouplers of oxidative phosphorylation, nucleic acid inhibitors, or general antimetabolites. the inhibition of ciliary action in tracheal cultures is, therefore; a much more sensitive index of toxic effects than morphological changes in monolayers. in fig. , the % cilia-inhibitory concentration of ici , is . pg/ ml. this effect is detected at a concentration times lower than is necessary to cause morphological changes in conventional tissue culture cells (fig. l) , presumably because the metabolic patterns of the ciliated cells are more complex than those of static cells in culture and are, therefore, more readily disturbed. in general, the concentrations of compounds that suppress ciliary activity arc comparable to those that prevent cell growth, except in the case of specific inhibitors of dsa synthesis for which cell division and growth are usually more sensitive than ciliary activity. any conventional technique may be used to measure virus growth in antiviral tests-cytopathic effect, plaque reduction, yield of infectious virus, and ht.madsorption, bring thc most common. because of thcir simplicity, cytopathic effect and hcmadsorption are widdy used, hut these techniques must be used with some precautions if certain types of antiviral activity are not to be missed. in order to speed up the rate of antiviral testing, the quantity of challenge virus is often increased to a theoretical maximum of virus particle per cell. the whole cell culture then behaves synchronously, and results are obtained in whatever time the virus takes to complete its replicative cycle-usually between and hours. with this procedure, however. a test compound that prevented the formation of infectious virus but was unable to protect the infected cell from destruction would not be detected. for example, in a relatively complex virus, such as influenza, it is not difficult to imagine that rna synthesis could be interrupted while hemagglutinin production continued, much as it does in the van magnus effect. the result would be a monolayer showing full hemadsorption and yet no transmissible virus would have been formed. test compounds that produce this effect would be of great interest as potential drugs but could be missed in tests where the dose of challenge virus is too high. wherever possible, tests should permit several cycles of virus growth to occur so that compounds that interrupt any part of the cycle may be detected. there is a school of thought that tissue culture testing is so artificial as to be of little value in detecting useful antiviral substances. it is argued that by testing for antiviral effects directly in animals, the activity that is detected is likely to be more valid and more useful than that detected in tissue culture. it is true that the majority of active compounds detected in tissue culture screens are not active in animals, even after full authentication of the antiviral activity by tests such as those discussed above. the reason for this is usually that the compounds do not reach the target organs in sufficient amounts to show activity rather than because of some intrinsic defects in the antiviral activity of the compounds. also, apart from interferon inducers, unless a compound manifests some activity in vitro, it is highly unlikely to do so in vivo. on the one hand, it is argued that a virus growing in a tissue culture cell is of little relevance to the processes by which that virus causes disease in the whole animal. on the other hand, it can be said that, since virus growth is the basis of the pathological processes, if virus growth can be halted, then the disease can be stopped. furthermore, the literature discloses that some highly irrelevant viruses are being used in animal test systems and that, even when human pathogens are used, e.g., influenza, they require extensive adaption to their animal host and the course of the disease is usually very different from that in humans. finally, there are no convenient animal models for studying the growth of human rhino-and coronaviruses and, unless tissue culture tests are used, there could be no screening for antiviral compounds against these important pathogens. there is, therefore, no convincing theoretical advantage in using animals for routine antiviral screens. this, together with the cumbersome nature of animal tests and the difficulties of "scaling-up" to test large numbers of compounds makes tissue culture testing a more attractive proposition for the initial screening program. the foregoing remarks apply, of course, only to the detection of compounds that have a direct effect on specific virus processes, e.g., absorption, penetration, and replication. in the field of interferon inducers, immune enhancers, and other stimulators of thr host defense mechanisms, obviously one has no choice but to use test animals; but here one is concerned to detect any overall rffcct on the course of a disease rathrr than accurately t o model a particular human or veterinary infection. accordingly, the choice of test system is less critical provided it fulfills certain requirements. for instancc, (a) thr virus and test compound should be administered a t separate sites to avoid any possible local destruction of the challenge virus by test compound; ( ) the test compound should be given parenterally to give the best chance of absorption, and (c) the dose of challenge virus should be sufficiently small to allow a useful incubation period before symptoms develop. the following system has been used successfully by us. groups of mice are dosed intraperitoneally with test compounds a t , . , and . mg/ kg on four successive days. twenty-four hours after the first dose, they are challenged intramuscularly with jild,, of semliki forest virus, and after the last dose they are observed for symptoms twice daily. the mcan rcciprocal day of death (mrdd) for each test group is calculated after days and comparcd with that of a similar undosed group as w l l as with that of a group givcn four daily injections of a protective agent such as polyinsinie-polyrytidilir acid (poly ic) . figure shows the typical response of mice treated nith poly ic and untreated controls. under these particular conditions, the t~ response curves overlap. by selecting an mrdd of . as the criterion of "active" or ' nactive," then theoretically % of all inactive compounds tested will appear as spurious actives and would require to be retested to establish their true status. some caution is needed in interpreting the converse overlap. given known active compounds, or a single active compound tested times, then tests in every would miss such a compound. but in practice, the vast majority of compounds passing through the test will be inactive, and the chance that the occasional true active compound will by chance fall into the " % missed" category is correspondingly reduced. even with an in vivo test reduced to such a minimum as this, it still requires a large effort in terms of manpower and facilities to test realistic numbers of compounds. although many human viruses will grow in animal hosts it is often difficult to assess the potential value of an antiviral compound for human use by using animal models. there are five main reasons for this. first, a relatively benign human virus infection will often follow a very different, and often severe course in an animal. for example, influenza virus, herpes simplex, and coxsackie viruses may cause much more serious diseases in laboratory animals than they do in man. second, human viruses often must be adapted by multiple passaging before they will grow satisfactorily in animal hosts, and, therefore, the challenge virus in the animal model may be a very different creature from the original human pathogen. third, the quantity of virus administered to an animal in order to produce some measurable effect, e.g., symptoms, virus isolation, and seroconversion, is usually vastly greater than would ordinarily be encountered by the natural host, and this can have a profound bearing on the efficacy of any curative agent. for example, finter ( ) has shown that the protection offered to mice by doses of interferon is greatly increased as the quantity of challenge virus is reduced. fourth, the fate of a drug when administered t o an animal may be very different from that seen in man. and last, a compound may show toxio effects in man which it did not show in animals. the last two points are probably the most important in determining how far the results obtained with animal models are relevant to man. in theory the above considerations should operate in both directions; that is, a compound that shows a positive result in an animal model may be positive or negative in man, and a compound that is negative in an animal may be negative or positive in man. but, since many animal models offer a greater challenge to the therapeutic potential of a drug than the natural disease in man, a positive result in an animal model always gives great hopes that a positive result might be achieved in man. this consideration often encourages the evaluation of potential antiviral drugs in man on the very slimmest of grounds. an example of the dilemma that an animal model may pose is afforded by the ork of boyle and his colleagues ( ) on the compound skf . this compound was shown to be active against a number of viruses, including a wide range of human rhinoviruses, in tissue culture. the problem arose of how to evaluate this compound in vivo. there are no smallanimal modrls of human rhinovirus infections, and the authors, therefore, decided to test the compound in chimpanzees, which are one of the few primates susceptible to human rhinoviruses. because of lack of knowledge on infectivity of human rhinoviruses for chimpanzees, the authors gave up to , ooo tcd,, of challenge virus to each animal to ensurc infcction. the animals were given the drug orally times a day, and the course of the disease was monitored by virus shedding from the nose. the rate of antibody rise was also measured. the numbers of animals in each experiment were necessarily small-usually or treated with drug and or controls. the final results were tantalizingly inconclusive: not clearly negative, nor convincingly positive that the drug had produced a curative effect. the investigators admit that this system is far from satisfactory but conclude from their results that the compound is n-orthwhile studying further in human subjects. the same conclusion, however, would probably have been reached if the compound had been clearly inactive in the chimpanzees, on the grounds that the excessive doses of challenge virus and unknown factors in the chimpanzee metabolism could have led to this result. animal models of human virus disease must at best be regarded as poor imitations of the natural condition, and results obtained with animal models, whether they are positive or negative, encouraging or discouraging, should be interprcted cautiously and never be used as the sole basis for predicting the outcome in man. research into the treatment of virus diseases by drugs is a t present directed toward three general areas: ( ) attempts to stimulate the defense mechanism of the host animal; ( ) large screening programs to find drugs that directly block some virus-specific process; and ( ) alleviation of the symptoms of the disease. the first approach is exemplified by the variety of interferon inducers which are a t present under intensive study. a disappointing feature of these is their uniformly low activity in man, despite highly promising results in laboratory animals, such as mice, rats, and rabbits. perhaps the interferon response in man and primates is less important in defense against virus disease than it is in other taxonomic groups. searches are being made for more general stimulants of host defense mechanisms, for example, stimulators of phagocytosis and the immune response, but very little progress has been reported so far. the search for drugs that will directly inhibit virus replication, by stopping a virus-coded synthetic process, or the absorption, penetration, uncoating, assembly, or release of virions, has been intensive and is still continuing. nevertheless, the products of this effort will find application only in a relatively small number of virus diseases for the reasons outlined above. only for those diseases of high economic importance, and for which no effective vaccines are available, will specific antiviral drugs ever be a commercial reality. the present paucity of such drugs is undoubtedly due largely to the intimate association of viruses at the molecular level with their host cells. for this reason, disease targets must be carefully defined, screening procedures made as meaningful as possible, and the limitations of animal models be clearly recognized. only by attention to these details can the maximum effort be brought to this difficult problem. again, the lack of clinically useful drugs allows no more than speculation on the possibilities of drug-resistant viruses emerging when antiviral drugs are eventually in widespread use. the phenomenon of drug resistance in viruses is well established in the laboratory (melnick et al., ; tamm and eggers, ; renis and buthala, ) , and, unless potential antiviral drugs are free of this serious defect, their commercial life will be embarrassingly short. the treatment of the symptoms, rather than the cause of a disease, has been the mainstay of medical practice from time immemorial, and this is still the case with most virus disease. the short incubation period of many virus diseases will inevitably restrict the therapeutic use of antiviral drugs, and in cases where symptoms have already appeared, the physician and layman alike will have recourse to the extensive armamentanurn of palliatives available for alleviating the symptoms of virus disease. all the same, this is clearly an unsatisfactory state of affairs, and the ultimate goal of antiviral research is the prevention of virus disease. as the understanding of viruses increases, so a more rational approach to the chemotherapy of virus diseases will become feasible. also, random discoveries of antiviral activity in novel chemical compounds will shed further light on those areas of virus metabolism that are susceptible to chemical attack. with increasing con-tributions from both these approaches, virus chemotherapy should soon emerge from a theoretical possibility to a practical reality, and antiviral drugs ivill make their long awaited contributions to clinical medicine. modern trends in medical virology zn "virus-induced immunopathology arch. gesanlfe virusforsch. zn "diagnostic procedures for virus and rickettsia infections amer zn "topics in medicinal chemistry ezperientia , virology , . tamm, j., and overman antiviral substances key: cord- -mc zruyx authors: toksvang, linea natalie; schmidt, magnus strøh; arup, sofie; larsen, rikke hebo; frandsen, thomas leth; schmiegelow, kjeld; rank, cecilie utke title: hepatotoxicity during -thioguanine treatment in inflammatory bowel disease and childhood acute lymphoblastic leukaemia: a systematic review date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: mc zruyx background the recently established association between higher levels of dna-incorporated thioguanine nucleotides and lower relapse risk in childhood acute lymphoblastic leukaemia (all) calls for reassessment of prolonged -thioguanine ( tg) treatment, while avoiding the risk of hepatotoxicity. objectives to assess the incidence of hepatotoxicity in patients treated with tg, and to explore if a safe dose of continuous tg can be established. data sources databases, conference proceedings, and reference lists of included studies were systematically searched for tg and synonyms from – . methods we included studies of patients with all or inflammatory bowel disorder (ibd) treated with tg, excluding studies with tg as part of an intensive chemotherapy regimen. we uploaded a protocol to prospero (registration number crd ). database and manual searches yielded unique records. of these, full-texts were screened for eligibility. finally, reports representing studies were included. results and conclusions we included data from studies of all and ibd patients; four randomised controlled trials (rcts) including , patients, observational studies including patients, and case reports including patients. hepatotoxicity in the form of sinusoidal obstruction syndrome (sos) occurred in – % of the all patients in two of the four included rcts using tg doses of – mg/m /day, and long-term hepatotoxicity in the form of nodular regenerative hyperplasia (nrh) was reported in . %. in ibd patients treated with tg doses of approximately mg/m /day, nrh occurred in % of patients; sos has not been reported. at a tg dose of approximately mg/m /day, nrh was reported in % of ibd patients, which is similar to the background incidence. according to this review, doses at or below mg/m /day are rarely associated with notable hepatotoxicity and can probably be considered safe. two review authors independently evaluated risk of bias of the included reports using the cochrane collaboration tool for rcts. [ ] the study quality assessment tools of the national heart, lung, and blood institute of the national institutes of health (nih) for quality assessment of observational cohort and cross-sectional studies and controlled intervention studies [ ] were used to assess the risk of bias of observational studies. a graphic illustration of potential bias of observational studies ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and were case reports ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . no similar reviews were found in the database search or on prospero. the included rcts were generally well executed with low risk of bias, except in the areas of blinding participants and outcome assessment, which may lead to performance and detection bias. risk of bias assessments in the included observational studies did not result in any suspicion of specific biases. the confidence in the cumulative estimate of the incidence of hepatotoxicity was graded as moderate, and the use of diagnostic methods as moderate, primarily because of the inconsistencies in reporting within these subjects. the confidence in the cumulative estimate of dose reduction or truncation of tg was graded as high. the findings of the present systematic review indicate that tg-induced hepatotoxicity in the form of sos or nrh is highly dose-dependent, and that it rarely occurs at daily doses of less than mg/m /day. furthermore, tg-induced hepatotoxicity appears to be largely reversible, except at high doses exceeding mg/m /day. the three rcts of childhood all, the coall- trial, the ccg- trial, and the uk mrc all / trial, respectively, have previously been included in a meta-analysis, which estimated the increase in sos between treatment arms to be a factor . (or, % ci . - . ). [ ] sos was highlighted as a dose-related toxicity with a multifactorial aetiology. no sos was reported in the coall- trial, albeit a high frequency of discordant thrombocytopenia, which may reflect a minor degree of hepatotoxicity. in that trial, vincristine and corticosteroids were not co- administered with tg -as was the case in the other two rcts. [ ] this difference in the co- medication has been proposed to be an explanation for the differing incidence of sos between the three rcts. [ ] the theory that nrh is tg dose or ery-tgn level-dependent was first presented in . [ ] this is supported by a mouse model, in which sos arose from high peak concentrations of tg, lnt created and pilot tested the data extraction form. sa, mss and lnt extracted the data, made assessments of risk of bias and confidence in cumulative evidence. lnt drafted the manuscript. the manuscript was read, revised, and approved by all authors. setting, duration, inclusion and exclusion criteria, source of funding, conflict of interest, ethical approvals, key conclusions), patient characteristics (number of patients, age, sex, ethnicity, disease, comorbidity, concomitant therapy), details of the interventions (duration of tg, dose of tg, cumulative dose of tg, maximum dose of tg, route of administration, ery-tgn levels), comparator (comparator drug, duration of mp or other standard of care drug, dose of mp or other standard of care drug, cumulative dose of mp or other standard of care drug, maximum dose of mp or other drug incidence of any hepatotoxicity reported as sos, veno-occlusive disease (vod) drug- induced liver injury or non-specified hepatotoxicity. due to the lack of standardised definitions of hepatotoxicity, authors of the included studies may not have used the above-mentioned terms. to assess additional hepatotoxicity we therefore report any pathological findings of liver biopsies and use the ponte di legno (pdl) toxicity working group consensus criteria for sos, which entail fulfilment of at least three out of the following five criteria: (i) hyperbilirubinaemia; (ii) hepatomegaly furthermore, we considered an increase in alanine transaminase, aspartate transaminase, alkaline phosphatase, conjugated bilirubin or total bilirubin of more than two times upper normal limit as evidence of hepatotoxicity secondary outcomes: diagnostic methods (number of patients who had a liver biopsy, indication for liver biopsy, other diagnostic methods, study conclusions about diagnostic methods); dose reduction or truncation of tg due to hepatotoxicity (how many patients had tg truncated or the dose reduced if protocols were unavailable, we compared outcomes reported in the methods and results sections. we did not quantitate the impact of meta ) consistency, and ( ) directness. the evidence on each outcome was graded as 'very low', 'low', 'moderate' or 'high'. higher in studies on tg for adult ibd and childhood all. tg-related hepatotoxicity persists in the form of nrh in % of the patients. however, the use of tg doses of approximately mg/m /day or less leads to hepatotoxicity in only % of the adult patients, corresponding to the incidence in the background population veno-occlusive disease of the liver after chemotherapy of acute leukemia. report of two cases toxicity and efficacy of -thioguanine versus -mercaptopurine in childhood lymphoblastic leukaemia: a randomised trial oral -mercaptopurine versus oral -thioguanine and veno-occlusive disease in children with standard-risk acute lymphoblastic leukemia: report of the children's oncology group ccg- clinical trial hepatic sinusoidal obstruction syndrome during maintenance therapy of childhood acute lymphoblastic leukemia is associated with continuous asparaginase therapy and mercaptopurine metabolites. pediatr blood cancer review article: the association between nodular regenerative hyperplasia, inflammatory bowel disease and thiopurine therapy long-term risk of portal hypertension and related complications in a novel mouse model of venoocclusive disease provides strategies to prevent thioguanine-induced hepatic toxicity nodular regenerative hyperplasia and thiopurines: the case for level-dependent toxicity meta-analysis of randomised trials comparing thiopurines in childhood acute lymphoblastic leukaemia novel therapy for childhood acute lymphoblastic leukemia relapsed childhood acute lymphoblastic leukemia in the nordic countries: prognostic factors, treatment and outcome the cytotoxicity of thioguanine vs mercaptopurine in acute lymphoblastic leukemia drug insight: pharmacology and toxicity of thiopurine therapy in patients with ibd dna-thioguanine nucleotide concentration and relapse-free survival during maintenance therapy of childhood acute lymphoblastic leukaemia (nopho all ): a prospective substudy of a phase trial cochrane handbook for systematic reviews of interventions version . . [updated preferred reporting items for systematic reviews and meta-analyses: the prisma statement the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration consensus definitions of severe acute toxic effects for childhood lymphoblastic leukaemia treatment: a delphi consensus thiopurine-induced liver injury in patients with inflammatory bowel disease: a systematic review the study quality assessment tools of the national heart, lung, and blood institute of the national institutes of health (nih) for quality assessment of observational cohort and cross-sectional studies and con grading quality of evidence and strength of recommendations thioguanine offers no advantage over mercaptopurine in maintenance treatment of childhood all: results of the randomized trial coall- long-term outcome in children with relapsed all by risk-stratified salvage therapy: results of trial acute lymphoblastic leukemia-relapse study of the berlin-frankfurt-munster group substitution of oral and intravenous thioguanine for mercaptopurine in a treatment regimen for children with standard risk acute lymphoblastic leukemia: a collaborative children's oncology group/national cancer institute pilot trial (ccg- ). pediatr blood cancer -thioguanine: a new old drug to procure remission in inflammatory bowel disease high variation of tioguanine absorption in patients with chronic active crohn's disease -thioguanine can cause serious liver injury in inflammatory bowel disease patients -thioguanine associated nodular regenerative hyperplasia in patients with inflammatory bowel disease may induce portal hypertension treatment of inflammatory bowel disease with -thioguanine ( -tg): retrospective case series from a tertiary care center efficacy of -thioguanine in patients with crohn's disease intolerant to azathioprine biotransformation of -thioguanine in inflammatory bowel disease patients: a comparison of oral and intravenous administration of -thioguanine lower" -thioguanine ( -tg) levels may be as effective as "higher" -tg levels in ibd patients treated with thioguanine -thioguanine in children with acute lymphoblastic leukaemia: influence of food on parent drug pharmacokinetics and -thioguanine nucleotide concentrations -thioguanine as an alternative therapy in inflammatory bowel disease-experience in a london district general hospital splitting a therapeutic dose of thioguanine may avoid liver toxicity and be an efficacious treatment for severe inflammatory bowel disease: a -center observational cohort study efficacy and safety of -thioguanine in the management of inflammatory bowel disease to evaluate the efficacy and safety of -thioguanine therapy in patients with inflammatory bowel disease-a london dgh experience a systematic survey evaluating -thioguanine-related hepatotoxicity in patients with inflammatory bowel disease toxicity and efficacy of intensive chemotherapy for children with acute lymphoblastic leukemia (all) after first bone marrow or extramedullary relapse the prevalence of nodular regenerative hyperplasia in inflammatory bowel disease patients treated with thioguanine is not associated with clinically significant liver disease thioguanine in inflammatory bowel disease: long-term efficacy and safety. united tpmt and mthfr genotype is not associated with altered risk of thioguanine-related sinusoidal obstruction syndrome in pediatric acute lymphoblastic leukemia: a report from the children's oncology group. pediatr blood cancer portal hypertension develops in a subset of children with standard risk acute lymphoblastic leukemia treated with oral -thioguanine during maintenance therapy. pediatr blood cancer psychosomatic complications during treatment for ulcerative colitis long-term follow-up of children with -thioguanine-related chronic hepatoxicity following treatment for acute lymphoblastic leukaemia variceal hemorrhage in a patient with ulcerative colitis treated with -thioguanine acute sinusoidal obstruction syndrome after -thioguanine therapy for crohn's disease thioguanine treatment-related sinusoidal obstruction syndrome in children mri patterns in a case of -thioguanine-related hepatic sinusoidal obstruction syndrome safe -thioguanine therapy of a tpmt deficient crohn's disease patient by using therapeutic drug monitoring veno-occlusive disease of the liver associated with thiopurines in a child with acute lymphoblastic leukemia pharmacokinetics of -thioguanine and -mercaptopurine combination maintenance therapy of childhood all: hypothesis and case report the case of colitis ulcerosa -diagnostic and therapeutic difficulties hepatotoxicity associated with -thioguanine therapy for crohn's disease liver venoocclusive disease (vod) in a patient given -thioguanine for crohn's disease early nodular hyperplasia of the liver occurring with inflammatory bowel diseases in association with thioguanine therapy safety of tioguanine during pregnancy in inflammatory bowel disease thioguanine-induced symptomatic thrombocytopenia on the limitation of -tioguaninenucleotide monitoring during tioguanine treatment thioguanine use in inflammatory bowel disease: year experience in a tertiary centre toxicity of -thioguanine: no hepatotoxicity in a series of ibd patients treated with longterm, low dose -thioguanine. some evidence for dose or metabolite level dependent effects? dig liver dis transient elastography to assess liver stiffness in patients with inflammatory bowel disease early hepatic nodular hyperplasia and submicroscopic fibrosis associated with -thioguanine therapy in inflammatory bowel disease histopathology of liver biopsies from a thiopurine-naïve inflammatory bowel disease cohort: prevalence of nodular regenerative hyperplasia micronodular transpormation (nodular regenerative hyperplasia) of the liver: a report of cases among , autopsies and a new classification of benign hepatocellular nodules -thioguanine treatment in inflammatory bowel disease: a critical appraisal by a european -tg working party a multicenter assessment of liver toxicity by mri and biopsy in ibd patients on -thioguanine -thioguanine-related chronic hepatotoxicity and variceal haemorrhage in children treated for acute lymphoblastic leukaemia -a dual-centre experience nodular regenerative hyperplasia: evolving concepts on underdiagnosed cause of portal hypertension nodular regenerative hyperplasia rarely leads to liver transplantation: a -year cohort study in all dutch liver transplant units. united eur gastroenterol j key: cord- -t mbsr authors: weyand, cornelia m.; goronzy, jörg j. title: vasculitides date: journal: primer on the rheumatic diseases doi: . / - - - - _ sha: doc_id: cord_uid: t mbsr despite the spatial closeness of blood vessels and inflammatory cells, blood vessel walls are infrequently targeted by inflammation. giant cell arteritis (gca) and takayasu’s arteritis (ta) are characterized by inflammation directed against the vessel wall. gca and ta display stringent tissue tropism and affect defined vascular territories in a preferential manner. gca predominantly affects the second- to fifth-order aortic branches, often in the extracranial arteries of the head. the aorta itself may also be affected in gca, albeit less often than other regions. in contrast, in ta, the aorta and its major branches are the prime disease targets. despite the spatial closeness of blood vessels and infl ammatory cells, blood vessel walls are infrequently targeted by infl ammation. giant cell arteritis (gca) and takayasu's arteritis (ta) are characterized by infl ammation directed against the vessel wall. gca and ta display stringent tissue tropism and affect defi ned vascular territories in a preferential manner. gca predominantly affects the second-to fi fth-order aortic branches, often in the extracranial arteries of the head. the aorta itself may also be affected in gca, albeit less often than other regions. in contrast, in ta, the aorta and its major branches are the prime disease targets. in both gca and ta, clinical symptoms of vascular infl ammation and vascular insuffi ciency are usually accompanied or preceded by a systemic infl ammatory process not localizable to a single tissue or organ. systemic infl ammation is also characteristic of polymyalgia rheumatica (pmr), a syndrome of muscle pain and stiffness in the neck, shoulders, and hips. pmr can accompany, precede, or follow gca, but it also occurs independently. in a subset of pmr patients, gca is present but not clinically evident. giant cell arteritis is the most common primary form of vasculitis among adults in the united states and europe. the disease occurs almost exclusively in individuals aged years and older, and its incidence increases progressively with age ( ) . women are more likely to be affected than men. the prevalence is highest in scandi-navian countries and in regions settled by people of northern european descent, with incidence rates reaching to cases per , persons aged years and older. gca occurs much less frequently in southern europeans ( cases per , individuals) and is rare in blacks and hispanics ( - cases per , individuals). the histological hallmark of gca is a mononuclear cell infi ltrate dominated by t lymphocytes and macrophages. the infl ammatory infi ltrate penetrates all layers of the arterial wall ( figure a- ) . the infi ltrates can be granulomatous with the accumulation of histiocytes and multinucleated giant cells. granuloma formation is most likely to be observed in the media. although the presence of multinucleated giant cells inspired the name of the disease, they are often absent, and the mononuclear infi ltrates lack a complex organization. if present, giant cells lie in close proximity to the fragmented internal elastic lamina. their presence correlates with increased risk for ischemic complications. gca can also present with perivascular cuffi ng of vasa vasorum or t cell-macrophage infi ltrates in the adventitia, sometimes arranged along the external elastic lamina. this fi nding is consistent with recent studies suggesting that the adventitia is a critical site in the disease process. the infl ammation causes a series of structural changes to the arterial wall. among the fi rst pathologic changes observed is the fi nding of a lymphoplasmacytic infi ltrate in the adventitia. with progress of the infl ammatory process, the media of the arterial wall becomes thinner. as the medial smooth muscle cell layer loses thickness, the intima becomes hyperplastic, compromising or occluding the arterial lumen. although the vessel lumen may become critically narrowed, thrombosis is not the central event. hyperplasia of the intimal layer with scarring in the media and fragmentation of the elastic lamina are irreversible changes that persist beyond the stages of active arterial infl ammation. fibrinoid necrosis is rare and should raise the suspicion for other forms of vasculitis. polyarteritis nodosa, microscopic polyangiitis, and wegener's granulomatosis, for example, are known to affect the temporal artery as well as other more typical vascular beds. when these forms of vasculitis affect the temporal artery, their fi rst pathological manifestations may be lymphoplasmacytic infi ltrates within the adventitia, indistinguishable at an early stage from gca. the immune response in the arterial wall experimental evidence supports a t-cell-mediated immunopathology of gca ( ) . humoral immunity does not appear to be important: b cells are not found within the arterial wall; no pathognomic antibodies have been identifi ed; and hypergammaglobulinemia is absent. t cells enter the vessel wall from the vasa vasorum in the adventitia, not from the macroendothelium. recruitment and activation of tissue-invading t cells is controlled by dendritic cells (dcs) in the adventitia. dcs are an indigenous cell population in normal mediumsized and large vessels. in the adventitia, they are typically localized at the outside of the external elastic lamina, close to the adventitia-media junction. evidence suggests that these vascular dcs utilize toll-like receptors (tlrs) to scan their environment for signs of infection, specifi cally for pathogen-related molecules. in gca and pmr, such adventitial dcs are strongly activated, produce chemokines, and express t-cell stimulatory ligands. this model is supported by experiments in human artery mouse chimeras. in these experiments, human temporal arteries from gca patients are implanted into severe combined immunodefi ciency mice. depletion of either t cells or dcs from the implanted vascular lesions terminates the infl ammatory response, with subsequent clearing of the infl ammatory infi ltrate. in contrast, administration of tlr ligands to chimeras implanted with normal temporal arteries followed by the adoptive transfer of t cells is suffi cient to induce the initial steps of vasculitis ( ) . based on these studies, it has been proposed that the vessel wall, in its physiologic state, is an immunoprivileged site. in gca, activation of vascular dcs by microbial products can break this immunoprivilege and lead to the recruitment and stimulation of t cells. the nature of the peptide antigens recognized by these t cells is histomorphology of giant cell arteritis. a typical temporal artery biopsy specimen is shown. characteristic changes include a panmural mononuclear infi ltrate, destruction of the internal and external elastic laminae, and concentric intimal hyperplasia. undetermined, but it may be that common self-antigens are suffi ciently immunogenic when dcs are activated. macrophage function in gca is known to be multifaceted, with specifi c commitments of these cells linked closely to their topographical arrangements within the cell wall ( ) . interferon (ifn) gamma, a t-cell cytokine, regulates both macrophages and giant cells. macrophages in the adventitia, intermingling with activated t cells, produce interleukin (il)- , il- , and transforming growth factor (tgf) beta. macrophages in the medial layer are specialized in the production of metalloproteinases, and also contribute to oxidative damage. end products of lipid peroxidation, a cell injury mechanism driven by oxygen radicals, are typically found on medial smooth muscle cells ( ) . macrophages recruited to the intimal layer are committed to the production of nitric oxide synthase- . nitric oxide is suspected to be involved in tissue injury, cellular activation, and vascular remodeling. multinucleated giant cells, once assumed to function in the removal of indigestible debris, are actually active secretory cells, producing molecular mediators relevant in structurally changing the arterial wall. the presence of giant cells in gca corresponds with the presence of high adventitial levels of ifn-gamma ( ). the assumption that all pathogenic mechanisms in infl amed arteries are mediated by tissue-infi ltrating immune cells is simplistic. t cells and macrophages in the arterial wall do not live and function in isolation, but rather closely interact with stromal components of the blood vessel in a bidirectional pattern ( ) . the major vascular abnormality leading to clinical disease is a nonthrombotic luminal occlusion, caused by rapid and concentric growth of the intima. these structural alterations result from the response to injury elicited in arterial cells ( figure a - ). intimal hyperplasia is generated by the mobilization of smooth muscle cells, their directed migration towards the lumen, and their proliferation and matrix deposition. this process is under the control of growth factors. platelet-derived growth factor (pdgf), a factor with the capability of supporting the outgrowth of the hyperplastic intima, is present in infl amed arteries. pdgf derives from macrophages and multinucleated giant cells. patients with low pdgf production have no or minimal lumen-occlusive intimal proliferation. in contrast, those with excessive pdgf production are at a risk for ischemic complications ( ) . a second pathway of the arterial injury-response program relates to the formation of new capillaries. the media and intima of normal arteries are avascular, but intense neoangiogenesis is induced in gca ( ) . vascular endothelial growth factor (vegf) is critical in driving the generation of neovessels in the media and intima. vegf, like pdgf, originates from macrophages and multinucleated giant cells. the arterial response pattern initiated by the production of pdgf and vegf leads to profound structural arterial abnormalities with subsequent stenosis and tissue ischemia, emphasizing that the immune system coerces the artery toward a counterproductive pattern of reaction. however, the infl ammation also leads to the induction of protective response patterns that are aimed at healing and tissue repair. an example is the upregulation of the enzyme, aldose reductase ( ) , which metabolizes and detoxifi es end products of oxidative damage. the activation of vascular dcs as an early step in the pathogenesis of gca has two major implications: ( ) infl ammation and immune activation are not limited to vascular lesions; and ( ) a systemic component of gca is an independent dimension of the disease process and not simply a spillover from vessel wall infl ammation. further evidence for this systemic component is the activation in gca patients of circulating monocytes, which produce il- and il- . elevated levels of il- , a potent inducer of acute phase responses, are characteristic of gca. in this model, gca is a systemic infl ammatory disease, with vasculitis of medium and large arteries as a consequence of the disease process. age is the major risk factor for gca. no other environmental risk factors, including a variety of infectious agents, have been demonstrated convincingly to play important roles in this disease. the high incidence rates of gca in all geographical regions settled by people of scandinavian ethnicity strongly suggest inherited risk factors ( ) . the best available information is for human leukocyte antigen (hla) genes. hla-dr haplotypes are associated with increased disease risk. several allelic variants of hla-dr are enriched among patients. selective binding of antigenic peptides has been proposed as the mechanism underlying this genetic association. in contrast to other hla-dr -associated diseases such as rheumatoid arthritis, hla polymorphisms do not correlate with clinical phenotypes and disease severity. many other genetic risk factors have been suggested, but none have been proven to date. the diagnostic category of gca encompasses multiple variants ( figure a- ) . each of these subtypes has characteristic clinical features ( ) ; however, the clinical manifestations of the different subtypes overlap substantially, and none of the clinical symptoms is unique for any one of the variants. increased awareness of gca, a growing population of individuals older than years of age, and improvement in diagnostic procedures (e.g., the availability of magnetic resonance angiography to image the aorta and its branches) have led to increased detection of cases formerly considered to be atypical presentations. giant cell arteritis presents with two major symptomatic complexes, signs of vascular insuffi ciency resulting from impaired blood fl ow and signs of systemic infl ammation. in general, vascular changes are those of occlusion; arterial wall dilatation only occurs when the aorta is involved. giant cell arteritis, also known as temporal arteritis, has a predilection for involving the extracranial branches of the carotid arteries. the temporal artery, which courses just below the skin over the temple region, is the vessel most amenable to biopsy. in % to % of patients, histopathology of vasculitis is detected in the extracranial arterial tree, most often in the superfi cial temporal artery; the vertebral, ophthalmic, and posterior ciliary arteries; and, less frequently, the internal and external carotid and central retinal arteries. patients complain of throbbing, sharp, or dull headaches that are usually severe enough to prompt clinical evaluation. the headaches may or may not be associated with scalp tenderness. in classic cases, the patients notice temporal tenderness when wearing glasses, grooming, or lying on a pillow. on physical examination, involved vessels may be thickened, tender, and nodular. pulses may be reduced or absent. abnormalities are most frequent in the temporal arterial branches, but they can also be detected in the occipital arteries or other superfi cial scalp vessels. in one third of the patients with biopsy-proven gca, the temporal arteries are normal on physical examination. focal arteritic lesions in the ophthalmic artery produce the most feared complication of gca: vision loss. the disease is an ophthalmological emergency, because prompt recognition and treatment can prevent blindness. ischemia anywhere along the visual pathway can lead to visual loss, but anterior ischemic optic neuropathy is the most common cause. visual loss is sudden, painless, and usually permanent. amaurosis fugax, reported as fl eeting visual blurring with heat or exercise or posture-related visual blurring and diplopia, may precede partial or complete blindness. on ophthalmologic examination, anterior ischemic optic neuropathy is recognized by optic disc edema, eventually followed by sectoral or generalized optic atrophy with optic disc cupping. besides optic neuropathy, the spectrum of ophthalmic complications is wide, ranging from pupillary defects to orbital ischemia and from ocular motor ischemia to anterior and posterior segment ischemia. a relatively disease-specifi c manifestation of gca that is present in about half of the patients is jaw claudication: pain in the masseter or temporalis muscles caused by compromised blood fl ow in the extracranial branches of the carotid artery. prolonged talking and chewing produce pain in the muscles of mastication. the onset of jaw claudication following the initiation of chewing is surprisingly swift. cases of trismus have been described. claudication of the tongue is less frequent, but tongue infarctions have been reported. vasoocclusive disease of the carotid and vertebrobasilar arteries results in ischemia of the central nervous system (cns), manifesting as transient ischemic attacks or infarcts. neurologic manifestations are increasingly being recognized and can be expected in % to % of patients. true intraparenchymal cns vasculitis in gca is rare, but reported. occult presentations of gca are common. gca is the cause of fever of unknown origin in up to % of elderly individuals, for example. nonspecifi c symptoms of pain in the face, neck, or throat are other warning signs of possible gca. chronic nonproductive cough can be an initial presentation of gca. the involvement of cough receptors (present throughout the repiratory tree) by the vasculitic process is believed to be the cause of cough in gca. symptoms related to systemic infl ammation are frequently present. laboratory abnormalities are detectable in more than % of patients. in a subset of patients, the disease process is dominated by a systemic infl ammatory syndrome. fever of unknown origin with spiking temperatures and chills usually leads to diagnostic evaluations designed to exclude infections and malignancies. in less dramatic cases, malaise, anorexia, weight loss, low-grade fever, and fatigue eventually become severe enough to prompt medical attention. physical examination of the scalp arteries is often negative, and symptoms of vascular insuffi ciency can be absent. temporal artery biopsy, even if the artery is normal on clinical examination, remains the diagnostic procedure of choice. clinical spectrum of the giant cell arteritis/ polymyalgia rheumatica syndrome. in at least % to % of patients, gca involves the large arteries in a clinically evident manner. (the percentage of cases with subclinical large vessel disease may be substantially higher.) preferred vascular beds are the carotid, subclavian, and axillary arteries. vasculitis of the femoral arteries is infrequent. the major clinical presentation is that of aortic arch syndrome, producing claudication of the arms, absent or asymmetrical pulses, paresthesias, and (rarely) symptoms of digital ischemia. patients with the large vessel variant of gca often lack evidence of cranial involvement; they do not complain about headaches, have normal temporal arteries on examination, and almost % of temporal artery biopsies are negative for vasculitis ( ) . aortitis in gca can coexist with cranial arteritis. whether the patient subset with subclavian-axillary gca is distinct from the subset progressing to aortic involvement is not known. overall, the risk of patients with gca to develop thoracic aortic aneurysm is increased -fold ( ) . the elastic membranes supporting the aortic wall are destroyed and replaced by fi brotic tissue. the resulting histopathology can be indistinguishable from that of ta. most cases of aortitis have been diagnosed several years after the initial diagnosis of gca, raising the possibility that smoldering aortitis is more common than previously expected ( ) . the spectrum of clinical manifestations ranges from silent aneurysm to aortic dissection and fatal rupture. in , the american college of rheumatology (acr) formulated classifi cation criteria for gca. these criteria, not intended for the purposes of establishing a clinical diagnosis of gca, are shown in appendix i. the diagnosis of gca should be considered in patients aged years and older with recent onset of unexplained headache, signs of tissue ischemia in the extracranial vascular territory, loss of vision, symptoms of limb or jaw claudication, or polymyalgia rheumatica. laboratory evidence of an acute phase response heightens concern about gca. the diagnostic procedure of choice is the histological verifi cation with the superfi cial temporal artery. in a recent meta-analysis, positive clinical predictors of a positive biopsy were jaw claudication, diplopia, and abnormalities of the temporal artery biopsy on physical examination ( ) . all other symptoms, including vision loss, elevated sedimentation rate, headaches, and constitutional symptoms, were not particularly helpful in predicting the results of temporal artery biopsies (i.e., in diagnosing gca). the presence of synovitis was a negative predictor of gca, indicative of the fact that most patients with true arthritis have another diagnosis, such as rheumatoid arthritis. even the most specifi c fi ndings for history, physical examination, and routine laboratory testing have sensitivities of only (at best) %. in view of the fact that rendering the diagnosis of gca commits a patient to long-term course of glucocorticoid therapy, confi rmation of the diagnosis by temporal artery biopsy is essential whenever possible. true negative results are expected in more than % to % of all patients undergoing biopsies at most institutions. false-negative biopsies, which occur as frequently as % of the time, can be minimized by taking a suffi cient length of biopsy, by examining serial sections, and by removing the contralateral temporal artery when the fi rst biopsy is free of arteritis. short-term glucocorticoid treatment (up to weeks, or even signifi cantly longer) is unlikely to interfere with the results of a temporal artery biopsy. prednisone should therefore not be withheld if a biopsy cannot be performed immediately. a pathognomic laboratory test for gca does not exist. specifi c autoantibodies have not been identifi ed. highly elevated acute phase responses are typical for gca but are not present in all patients. although a high erythrocyte sedimentation rate (esr) is usually considered a hallmark of gca, in a recent study % of all patients with positive temporal artery biopsies had normal esrs before the initiation of glucocorticoid therapy ( ) . other markers of acute phase response, particularly creactive protein (crp), may be more sensitive than esr in some patients, but studies have not demonstrated this consistently. some evidence indicates that the most sensitive serum marker for ongoing systemic infl ammation in gca (both before and after glucocorticoid therapy) is il- . il- , a strong inducer of acute phase reactants, probably functions upstream in the disease process. unfortunately, reliable il- measurements are not widely available, and knowledge about how (or if) to adjust therapy in the context of changing il- levels remains incomplete. there is currently no evidence that treatment decisions should be predicated upon the results of laboratory tests-esr, crp, or il- -in the absence of clinical symptoms. other laboratory abnormalities in gca include mild-to-moderate normochromic or hypochromic anemia. elevated platelet counts are common. liver function tests, particularly alkaline phosphatase, can be abnormal. precise mapping of the vaso-occlusive process still requires angiography. angiography is also essential for patients with signifi cant stenoses in vessels to all four extremities, for the purpose of measuring central aortic pressure directly. alternatives to conventional angiography, however, have made great strides in recent years ( ) . magnetic resonance angiography (mra) permits evaluation of vessel wall thickness and perivascular edema-signifi cant advantages over conventional angiograms, which evaluate only the vascular lumen. in the proper clinical context, therefore, certain mra fi ndings may be diagnostic of large vessel vasculitis. the noninvasive nature of mra also lends important advantages in serial monitoring. unfortunately, the appropriate interpretations of some mra fi ndings, for example, enhancement of the vessel wall following gadolinium administration and the presence of vessel wall edema, remain uncertain and require additional longitudinal studies. computed tomography angiography, another promising technique, has not been evaluated thoroughly in large vessel vasculitis. position emission tomography (pet) with f-fl uorodeoxyglucose also holds promise for the assessment of the degree of disease activity in large arteries, but has not yet been validated for general clinical use. other noninvasive vascular studies, including fl uorescein angiography, transcranial doppler fl ow studies, and doppler ultrasonography, are useful in assessing certain vascular beds, for example, the retinal, vertebral, or subclavian arteries. these techniques only identify vascular insuffi ciency in cases with pronounced, lumen-stenosing disease, however, and do not provide specifi c information useful from the standpoint of diagnosis. although doppler ultrasound was once hypothesized to be useful in the diagnosis of gca, the value of identifying a "hyperechoic halo" on ultrasound in the temporal artery has not been confi rmed in subsequent studies ( ) . glucocorticoids are explicitly effective in suppressing clinical manifestations of gca. since the introduction of glucocorticoids, the rate of gca-related blindness has declined, documenting the effectiveness of this immunosuppressive approach. in almost all patients, glucocorticoids induce relief within to hours. the excellent response of the disease to this therapy has been suggested by some as a diagnostic criterion. in view of the severity of gca-related morbidity, initial doses of mg prednisone or equivalent have been recommended. glucocorticoids cannot reverse intimal hyperplasia but may help attenuate the ischemic insult by reducing tissue edema. in ophthalmologic emergencies (e.g., amaurosis fugax occurring in suspected gca), pulse glucocorticoids may be appropriate ( ) . initial doses should be maintained until reversible manifestations of the disease have responded and the systemic infl ammatory syndrome is suppressed. subsequently, under close monitoring for clinical signs of disease reactivation, the dose of prednisone generally can be tapered by % every to weeks. so far, the use of glucocorticoid-sparing agents to allow a more rapid taper has been unsuccessful ( ) . initial positive results with methotrexate could not be confi rmed in a subsequent study ( ) . a recent randomized, controlled trial of tumor necrosing factor (tnf) alpha blockade found this therapy to be ineffective as a glucocorticoid-sparing medication ( ) . a recent study suggests that a more aggressive induction therapy at the onset of the disease, including three daily pulses of gram of methylprednisolone, may allow for a rapid tapering of glucocorticoids and, in particular, a discontinuation of glucocorticoids in the second year of disease ( ) . aspirin is an important adjunctive treatment for gca patients without contraindications. retrospective studies have indicated strong reductions in the risks of visual loss and central nervous system ischemic events among patients taking aspirin for other reasons at the time their gca was diagnosed ( ) . the mechanism of aspirin's effi cacy in this setting is not entirely clear, but the medication may exert its effect through the selective suppression of interferon gamma production ( ) . although the optimal dose of aspirin has not been established, doses ranging from mg/day to mg/day may be benefi cial. the most signifi cant morbidity of gca relates to reduced blood fl ow to the eye and optic nerve as well as hypoperfusion of the brain ( ) . if diagnosed and treated promptly, progression of the downstream effects of arterial wall infl ammation, in particular lumen occlusion with tissue ischemia, can be prevented. side effects of high doses of glucocorticoids given over a prolonged period of time can be serious, especially in patients older than years, and treatment should therefore only be initiated if the diagnosis is confi rmed. in the majority of patients, gca does not enter remissions that are sustained indefi nitely after discontinuation of glucocorticoids. in a prospective study of patients with biopsyproven gca, all of the patients responded to mg prednisone with disappearance of clinical signs of the disease ( ) . however, % of patients had disease relapses that occurred throughout the course of treatment. typically, reactivation of the disease produced symptoms of systemic infl ammation or polymyalgic symptoms, but no vascular complications were seen. polymyalgia rheumatica is diagnosed in patients presenting with pain and stiffness in the muscles of the neck, shoulder girdle, and pelvic girdle of at least weeks' duration ( ) . the myalgias are combined with signs of systemic infl ammation manifesting clinically as malaise, weight loss, sweats, and low-grade fever. most patients have laboratory abnormalities such as elevated esr, elevated crp, and anemia, which are indicative of a systemic infl ammatory syndrome. upregulation of acute phase reactants is helpful in distinguishing pmr from other pain syndromes, yet (as in gca and ta) not all patients with active disease have elevated markers of infl ammation within their serum. no pathognomic test for pmr is available; exclusion of other diseases with similar clinical presentations is essential. the systemic infl ammatory syndrome associated with pmr is exquisitely sensitive to glucocorticoid therapy, such that prompt improvement of clinical symptoms with glucocorticoid therapy has been proposed as a diagnostic criterion. the pathophysiology of pmr is related closely to those of gca. pmr is now often considered a form of gca that lacks fully developed vasculitis. because pmr remains a clinical diagnosis, epidemiological studies are diffi cult. pmr affects the same patient population as gca, but occurs approximately two to three times more frequently ( ) . women are affected more often than men, and the diagnosis is extremely unlikely in individuals younger than years of age. in high-risk populations, such as scandinavians and other peoples of northern european descent, annual incidence rates have been estimated at to per , persons over the age of years. in low-risk populations, such as italians, the annual incidence rates for individuals aged years and older are only cases per , . although the sudden onset of intense infl ammation is suspicious for an infectious etiology, no causative agent has been identifi ed. most pathogenic abnormalities in pmr patients are reminiscent of those in gca, supporting the concept that pmr is a variant of gca characterized by the dominance of the systemic infl ammatory over the vascular component. human leukocyte antigen polymorphisms that are genetic risk factors for gca are also associated with pmr. there is no evidence that the hla has a role in determining whether the disease process will remain limited to pmr or progress to fully developed gca. polymyalgia rheumatica appears to be associated with a global activation of the innate immune system, including circulating monocytes that produce il- and il- . activated dcs render arteries susceptible to vasculitis. in many patients with pmr, in situ cytokine production can be demonstrated in biopsy specimens, although in lower quantities than in gca. of note, ifngamma is absent in pmr, but abundant in gca ( ) . a subset of pmr patients develops infl ammation of periarticular structures, for example, bursae. whether these patients comprise a different subset from those susceptible to developing frank vasculitis remains unclear. patients complain about aching and pain in the muscles of the neck, shoulders, lower back, hips, thighs, and occasionally the trunk. in typical cases, the onset is abrupt and the myalgias symmetrical; they usually affect the shoulders fi rst. often the patients have pain during the night and have diffi culties rising and dressing themselves. weight loss, anorexia, malaise, and depression are common. fever and chills should raise the suspicion of fully developed gca. pmr is frequently diffi cult to distinguish from forms of seronegative polyarthritis. in particular, male patients can present with proximal aching and diffuse edema of the hands and feet that is highly glucocorticoid-responsive. polymyalgia rheumatica includes patients with mild disease that is promptly responsive to therapy and remits within a few months ( ) . in many patients, however, reactivation of myalgias occurs when glucocorticoid doses are tapered. some patients require higher initial doses of glucocorticoids than are usually considered to be effective in pmr. patients with pmr must be carefully evaluated for possible gca. a negative temporal artery biopsy does not exclude the possibility of large vessel vasculitis targeting primarily the subclavian and axillary arteries and the aorta. signs of vascular insuffi ciency, including claudication in the extremities, bruits over arteries, and discrepant blood pressure readings should alert the physician to the possibility of gca ( ) . mra can be helpful in confi rming the concomitant diagnosis of large vessel vasculitis. in pmr patients with infl ammation of periarticular structures, the most prominent fi ndings are subdeltoid and subacromial bursitis ( ) . biceps tendonitis and glenohumeral synovitis may also be present. ultrasonography reveals fl uid accumulation in the bursae; t -weighted mri shows thickening and edema. these involved areas show increased uptake on pet scans. the clinical symptoms of pmr can be mimicked by a number of arthropathies, shoulder disorders, infl ammatory myopathies, hypothyroidism, and parkinson's disease. the differential diagnosis also includes malignancies and infections. no clear guidelines have been developed to determine whether patients with pmr should be screened for occult malignancies. lack of the typical and impressive improvement upon initiation of therapy can provide a clue towards reevaluating the diagnosis of pmr. polymyalgia rheumatica is dramatically responsive to glucocorticoid therapy. currently there are no data documenting glucocorticoid-sparing effects of other medications. however, almost all patients with pmr can be safely managed with glucocorticoids; doses for long-term treatment are low and unlikely to cause serious side effects. a critical decision in treating pmr is the dose of glucocorticoids required for successful suppression of symptoms and infl ammation. the glucocorticoid requirements may differ quite markedly among patients. two thirds of patients can be expected to respond with remission of pain and stiffness when started on mg/day or less prednisone ( ) . some patients will need doses as high as mg/day for complete clinical control. such patients may be at higher risk of full-blown gca. patients initially controlled on mg/day of prednisone can usually taper the dose by . mg every to days. more protracted tapering may be necessary once daily doses of to mg prednisone are attained. dose adjustments should be based mainly on clinical evaluation, not exclusively on laboratory abnormalities. in many patients, pmr can go into long-term remission, and prednisone can be discontinued. occasionally, successful suppression of recurrent myalgias and stiffness may only be achieved by giving very low doses of prednisone over an extended period. patients should be warned about the potential of pmr progressing to gca and should be monitored for vascular complications, particularly when discontinuing glucocorticoid therapy. the prognosis of patients with pmr is good. in the majority of patients, the condition is self-limited. a proportion of patients will eventually present with typical symmetrical polyarthritis, fulfi lling the criteria for the diagnosis of seronegative rheumatoid arthritis. such patients may require disease-modifying antirheumatic drug (dmard) therapy. takayasu's arteritis is a vasculitis of the large elastic arteries, specifi cally the aorta and its main branches. the disease may also affect the coronary and pulmonary arteries ( ) . infl ammatory injury to the vessel wall leads to patchy disappearance of the elastica and smooth muscle layer and subsequent intimal hyperplasia, resulting in vascular stenosis in virtually all patients and dila-tation and aneurysm in about %. complete occlusion of upper extremity arteries results in the loss of palpable pulses, which is why ta is also termed the pulseless disease. the preference for the aorta and its primary branches is signifi ed in another alternative name, aortic arch syndrome. the acr has developed a set of criteria to distinguish ta from other vasculitic syndromes (see appendix i). takayasu's arteritis is a rare disease that primarily affects adolescent girls and young women. the diagnostic criteria include an age of less than years at disease onset; however, ta can start later in life, particularly in asians ( ) . (in addition, the diagnosis is often not made until the patient is older than , but symptoms may have begun years before the diagnosis.) incidence rates are highest in asia (japan, korea, china, india, and thailand), with estimates of approximately case per million persons annually. ta can occur in all races and geographic regions, but south american countries have recently been recognized as additional areas of relatively high incidence. an international survey among countries has indicated differences in the clinical spectrum of ta in different ethnic groups. takayasu's arteritis is a granulomatous polyarteritis. the adventitia is characterized by striking thickening, often with intense perivascular infi ltrates around the vasa vasorum. granuloma formation and giant cells are predominantly found in the media of the large elastic arteries. the medial elastic smooth muscle cell layer is destroyed in a centripetal direction and replaced by fi brotic tissue, leading (in the aorta) to vessel wall dilatation and aneurysm formation. smooth tapering, narrowing, or complete occlusion of the vascular lumen results from proliferation of the intima, occasionally with thrombosis. the etiology of ta remains unknown. in view of the systemic features of the syndrome, microbial infections have been implicated, but no conclusive evidence for infectious organisms has been provided. cd t cells are a major component of vascular infi ltrates, setting ta apart from gca. cytotoxic activities of tissue infi ltrating cd t cells, mediated by the release of the poreforming enzymes perforin and granzyme b, have been suspected of contributing to smooth muscle cell damage ( ) . support for a role of cd t-cell-mediated cytolytic tissue injury has come from the observation that selected hla class i molecules, specifi cally hla-b , are overrepresented among ta patients ( ) . cd t cells recognize antigens when bound to hla class i molecules. the role of cd t-cell responses and the contribution of macrophage effector functions in the vascular lesions are not understood. the focus of lymphocytic infi ltrates on the adventitia and accumulation of t cells around vasa vasorum makes it less likely that the macroendothelium has major involvement in the pathogenesis of ta. a generalized infl ammatory syndrome with fever, night sweats, malaise, anorexia, weight loss, and diffuse myalgias often dominates initial manifestations of ta. these symptoms are frequently misdiagnosed as infection. the clinical pattern of ischemic complications that emergeoften years later-directly refl ect the vascular territory targeted by the disease (figure a- ) . involvement of the carotid and vertebral arteries leads to neurologic and ophthalmologic symptoms, including dizziness, tinnitus, headaches, syncope, stroke, and visual disturbances. atrophy of facial muscles and jaw claudication are mostly late manifestations. occlu-sions of the brachiocephalic and subclavian arteries impair blood fl ow to the upper extremities, presenting as arm claudication, pulselessness, and discrepant blood pressures. the detection of bruits can be helpful in making the diagnosis. cardiac disease, including ischemic coronary disease, arrhythmia, and congestive heart failure, can be related to aortitis of the ascending aorta or severe hypertension. aortic regurgitation, a serious complication requiring prompt clinical attention, is a consequence of aortic dilatation. coronary arteries can be involved directly or indirectly, producing classical symptoms of myocardial ischemia. progressively enlarging aneurysms and possible rupture are a major concern in patients with ta of the aortic arch and the descending thoracic aorta. patients from india, china, and korea often have lesions in the abdominal aorta and its branches (particularly the renal arteries, causing renovascular hypertension). the proximal ends of mesenteric arteries are less frequently affected, but gastrointestinal symptoms, such as nausea, vomiting, and ischemic bowel disease can be seen in patients with ta. patients presenting (%) clinical spectrum of takayasu's arteritis in relationship to vascular bed involvement. a combination of vaso-occlusive disease and systemic infl ammation in a young patient should immediately raise suspicion for ta. typically, the diagnosis is made by characteristic fi ndings on vascular imaging ( ) . tissue is rarely available. the fi ndings on conventional angiography can be diagnostic for ta in the proper clinical setting. angiography reveals long, smooth taperings of involved vessels, with a remarkable web of collateral blood vessels in advanced cases. as in gca, conventional angiography is essential in many patients with ta in order to measure accurately the central aortic blood pressure. several noninvasive imaging techniques are informative for assessing progression of occlusive disease, but currently lack standardization and are subject to investigator bias and experience. far more problematic than assessing the degree of stenosis within a given blood vessel is the reliable assessment of infl ammatory activity by imaging. mri/a has largely replaced conventional angiography for serial assessments of the distribution and degree of vessel involvement, and also permits evaluations of the vessel wall as well as the lumen. mri/a is particularly important in the longitudinal monitoring of ta although, as noted, the correct interpretation of all mri/a fi ndings is not always clear. mri/a has clear utility in monitoring the progress or stability of vascular stenoses, provided that serial studies are compared carefully for changes. doppler ultrasound provides a good assessment of cervical vessels. computed tomography angiography can be used to survey the aorta and proximal vessels, but rigorous serial studies of its use in ta remain to be performed. the role of pet scanning in gauging the degree of ongoing infl ammation (as opposed to uptake that might be related to a process of healing or fi brosis) has not been established. although some patients with ta have disease that appears to "burn out," becoming quiescent after years of active disease, most patients have progressive or relapsing/remitting disease and require long-term immunosuppressive treatment ( ) . glucocorticoids are the therapy of choice for management of ta. recommendations of initial doses have varied, but to mg of prednisone may be necessary to control vascular as well as systemic infl ammation. monitoring of acute phase reactants (esr, crp) is only helpful in a subset of patients. in a national institutes of health (nih) cohort, % of patients had active progressing disease despite nonelevated acute phase reactants ( ) . prednisone doses are tapered as clinically indicated and tolerated, usually by mg/day every weeks until a maintenance dose of mg/day is reached. further dose reductions must be tailored to the individual patient. low-dose aspirin or other antiplatelet agents should complement glucocorticoid therapy. methotrexate, given in weekly doses of up to mg, has shown promise in improving remission rates and sparing glucocorticoids ( ) , but has never been tested in a randomized trial (the same is true for all other potential steroid-sparing agents). azathioprine, mycophenolate mofetil, cyclosporine, and tnfalpha blockers have been used with reported success in individual patients, but controlled studies are required. contrary to other vasculitides, cyclophosphamide does not play a major role in this disease because of its toxicity and uncertain effi cacy. stenotic lesions are irreversible. surgical management and angioplasty or stent placement have a role in selected patients, but for most patients revascularization attempts of vessels to the extremities are not necessary because of the exuberant collateralization that develops in ta. when revascularization is necessary, bypass grafts are generally successful, while stenting appears to have a high rate of reocclusion ( ) . angioplasty is reserved for short stenotic segments. treatment of hypertension secondary to renal artery stenosis may or may not benefi t from revascularization, depending on the location of the lesion leading to renovascular hypertension. decisions about whether or not to attempt revascularization should be undertaken in consultation with experts accustomed to the management of complex hypertension cases. for much of the past several decades, ta has been viewed as an inevitably devastating disease. the diagnosis was seldom made before damage from prolonged vascular infl ammation was already extensive. more recently, the potential for earlier diagnosis, effective immunosuppressive therapy, and astute surgical management have led to an improved prognosis for many patients. long-term follow up of almost japanese patients found stable clinical conditions in two thirds of the patients and serious complications occurring in only % of affected individuals. cardiac complications, including congestive heart failure and ischemic heart disease, have become the most common cause of death in japanese patients with ta. acceleration of atherosclerotic disease emerges as a critical factor in long-term outcome. polyarteritis nodosa (pan) primarily affects mediumsized arteries that supply the skin, gut, nerve, and kidney, but may involve multiple organs. microaneurysms of arteries to or within the kidneys, liver, or gastrointestinal tract are highly characteristic of pan. polyarteritis nodosa is not associated with antineutrophil cytoplasmic antibodies (anca) directed against either proteinase- or myeloperoxidase. mononeuritis multiplex, an asymmetric sensory and motor neuropathy due to ischemia and infarction of peripheral nerves, occurs frequently in pan. in mononeuritis multiplex, nerve conduction studies of peripheral nerves reveal a distal, asymmetric, axonal neuropathy involving both motor and sensory nerves. polyarteritis nodosa is characterized pathologically by patchy, transmural infl ammation in medium-and small-sized muscular arteries, sparing large arteries, capillaries, and the venous system. the infl ammation leads to fi brinoid necrosis, but is not associated with granulomatous features. high-dose glucocorticoids are the mainstay of therapy in pan. in cases that are rapidly progressive or life-or organ-threatening, however, cyclophosphamide is added to glucocorticoid treatment. most cases of idiopathic pan do not recur once a sound remission has been achieved with to months of therapy. a minority of pan cases (now > %) are associated with acute hepatitis b infection. cases associated with hepatitis b are treated with regimens emphasizing antiviral therapy and only short courses of immunosuppression and plasmapheresis. polyarteritis nodosa (pan) is a vasculitis affecting predominantly medium-sized arteries. clinically, pan often presents insidiously with nonspecifi c, constitutional symptoms. the disease has a predilection for mediumsized arteries supplying skin, gut, nerve, and kidney, but may involve multiple organs. the majority of pan cases have no known cause, but cases secondary to hepatitis b virus infection have been reported. described by kussmaul and maier in ( , ), pan is often regarded as the fi rst reported form of systemic vasculitis. in fact, earlier descriptions of behcet's disease, takayasu's arteritis, henoch-schönlein purpura, and even pan itself exist in the medical literature. for nearly a century after the case reported by kussmaul and maier, however, most forms of systemic vasculitis were termed periarteritis nodosa, and forms of vasculitis recognized later were contrasted and classifi ed in comparison to pan. the patient described by kussmaul and maier was a -year-old male with fever, weight loss, abdominal pain, and a polyneuropathy that progressed over the period of month to paralysis. an autopsy revealed microaneurysms ("whitish small tumors up to the size of poppy and hemp seeds") throughout medium-sized arteries, conspicuously sparing both the venous circulation and the lungs. before the delineation of vasculitis subsets and the formulation of defi nitions based principally on vessel size ( ), pan was used to describe two now-distinct forms of vasculitis: the classic pan described by kussmaul and maier, and "microscopic pan" (now called microscopic polyangiitis). according to current convention, and as described in this text, pan is a vasculitis affecting medium-sized arteries. pan is also not associated with antineutrophil cytoplasmic antibodies (anca), at least not those directed against proteinase- (pr ) or myeloperoxidase (mpo) that are such a distinctive feature of the majority of cases of wegener's granulomatosis, microscopic polyangiitis, and, to a lesser extent, the churg-strauss syndrome. although patients may be p-anca-positive on immunofl uorescence testing, enzyme immunoassays for pr -and mpo-anca are negative in pan. further, in contrast to the anca-associated vasculitides (see chapter c), pan does not involve either the lungs or blood vessels as small as the renal glomeruli (which are essentially capillaries). polyarteritis nodosa affects men and women approximately equally and has a broad age range of patients. although the incidence of pan varies according to the population studied, it is rare in all populations, with annual incidence rates generally ranging from to cases per million. higher rates have been reported in populations with a high burden of hepatitis b virus infection, but with the availability of a vaccine, hepatitis b virus infection now accounts for less than % of pan cases in developed countries ( ) . pan has also been reported to occur in conjunction with hairy cell leukemia. polyarteritis nodosa can present with nonspecifi c constitutional symptoms such as fever, fatigue, malaise, myalgias, and arthralgias. this phase of the illness can last weeks or months. the more specifi c clinical manifestations of pan are the direct results of infl ammation in medium-and small-sized muscular arteries. pan often has cutaneous involvement, a feature it shares with small vessel vasculitides such as the ancaassociated disorders. this differentiates it from large vessel vasculitis (e.g., giant cell arteritis and takayasu's arteritis), in which skin disease is very rare. however, unlike the anca-associated vasculitides, pan is not associated with glomerulonephritis or pulmonary involvement. common clinical features of pan and their frequency are listed in table b - ( ) . in autopsy studies, the most frequently involved organ in pan is the kidney. involvement of the mediumsized arteries supplying the renal parenchyma can result in hypertension due to renal ischemia; hypertension is mediated by the renin-angiotensin system. renal insuffi ciency, another common manifestation of pan, is due to ischemia resulting from the involvement of arteries the size of renal arteries and smaller. microaneurysms, detectable by angiography [ figure b - (a,b)], are a hallmark of pan. the gastrointestinal (gi) tract is involved in up to % of patients. postprandial periumbilical pain, or intestinal angina, is the result of mesenteric ischemia. more severe disease can result in bowel infarction and perforation. other gi symptoms can include nausea, vomiting, diarrhea, and bleeding. while the small intestine is most commonly involved, rare presentations involving ischemia of the gallbladder or appendix have been described ( ) . moderately elevated hepatic transaminases often betray liver involvement. asymptomatic mircoaneurysms within the liver are common; these occasionally rupture. involvement of the peripheral nervous system is seen in % to % of patients, usually as an asymmetric sensory and motor neuropathy due to ischemia of peripheral nerves ( ) . infarction of named nerves results in mononeuritis multiplex ( figure b - ). progressive sensory neuropathy has been described less frequently. central nervous system involvement is much less common, but has been reported in the form of cerebrovascular accidents. polyarteritis nodosa can have multiple cutaneous manifestations: livedo reticularis, nodules, ulcerations ( figure b - ), and frank ischemia of digits ( ) . a small group of patients have a form of disease termed cutaneous pan, a variant limited ostensibly to the skin. these patients develop nodules and ulcerations, primarily of the lower legs, which can occur in crops and can be very painful. however, as with any other vasculitis, cutaneous manifestations should prompt a thorough evaluation for evidence of systemic disease. as with kawasaki's disease, the other medium vessel vasculitis described in this text, pan can also involve the coronary arteries. clinical proof of coronary disease during life is diffi cult. myocardial infarction is uncom-mon, and coronary involvement is usually only seen at autopsy. contraction band necrosis, indicative of segmental ischemia, is a common fi nding in the myocardium at autopsy, attesting to the presence of vasculitis below the resolution of conventional angiography. pan can involve other organs, such as the testicle, ovary, breast, and eye. polyarteritis nodosa is characterized pathologically by patchy, transmural infl ammation in medium-and smallsized muscular arteries, sparing large arteries, capillaries, and the venous system. there is a pleomorphic cellular infi ltrate and fi brinoid necrosis in the vessel, but no features of granulomatous infl ammation. disruption of the elastic laminae of the vessel wall can lead to aneurysmal dilatation at the site of the lesion. pan has a predilection for certain organs: arteries to the kidney are estimated to be involved % to % of the time, the gi tract is involved in % of cases, the peripheral nerves are involved in % of cases, and the central nervous system is involved in % of cases ( ) . polyarteritis nodosa is diagnosed based on characteristic symptoms, physical examination fi ndings, and compatible laboratory, angiographic, and pathologic data. because pan is a rare disease and its treatment can result in serious adverse events, the diagnosis should be supported with either abdominal angiography or biopsy whenever possible. pan must be differentiated from other forms of vasculitis, such as the anca-associated disorders, cryoglobulinemia, and buerger's disease. common vasculitis mimics, such as viral hepatitis, bacterial endocarditis, or other embolic diseases, should be excluded. undiagnosed connective tissue diseases, such as systemic lupus erythematosus, rheumatoid arthritis, or systemic sclerosis, must be ruled out, as such diseases can be associated with systemic vasculitis or widespread vascular dysfunction that involves multiple organs. atrophie blanche, a thrombotic disorder that may lead to lower extremity ulcerations, must be differentiated from pan by skin biopsy. the american college of rheumatology criteria for the classifi cation of pan are listed in table b - ( ). the criteria were developed through the selection of clinical fi ndings that identify pan and distinguish it from other forms of vasculitis. although the criteria are useful for classifying patients in clinical studies, they were not intended for use in diagnosing individual patients ( ). routine laboratory studies are often abnormal but nonspecifi c, such as elevated infl ammatory markers (erythrocyte sedimentation rate or c-reactive protein), anemia, and thrombocytosis. the patient may have mild renal insuffi ciency, with an elevated blood urea nitrogen and creatinine. non-nephrotic range proteinuria and mild hematuria are also seen, but active urine sediments are not a feature of pan. as not, pan is not associated with anca. indeed, there is no characteristic autoantibody for pan-a fact that creates one of the diagnostic challenges in this disease. electromyography/nerve conduction velocity (emg/ncv) studies may be very useful in confi rming patterns of nerve dysfunction consistent with mononeuritis multiplex; namely, a distal, asymmetric, axonal neuropathy involving both motor and sensory nerves. imaging in a patient with suspected pan should be guided by symptoms. in patients with abdominal pain, abdominal arteriography often reveals characteristic strictures and aneurysms (beading) of the mesenteric vessels [see figure b - (b)]. similar fi ndings can be seen in the renal vasculature. as with imaging, biopsy should be guided by organ involvement. blind biopsy of an asymptomatic organ, such as muscle or testicle, is not recommended. skin biopsy is often the easiest way to confi rm this diagnosis, with a biopsy from the center of a nodule or the edge of a vasculitic ulcer. routine punch biopsy of involved skin reveals leukocytoclastic vasculitis and fi brinoid necrosis within the blood vessel wall. because punch biopsy samples include only epidermis and superfi cial dermis, they do not capture medium-sized, muscular-walled arteries whose infl ammation is characteristic of pan. when pan is suspected and skin biopsy is indicated, a full thickness skin biopsy that includes some subcutaneous fat should be performed (arteries within the fat lobules of subcutaneous tissue are often involved.) another option for confi rming the diagnosis of pan is a peripheral nerve biopsy. the sural nerve is biopsied most often because it does not mediate motor function. whenever the sural nerve is biopsied, a muscle biopsy (of the gastrocnemius) should be performed simultaneously. because of the highly vascular nature of muscle, biopsies of this organ may yield proof of vasculitis even in the absence of clinical indications of muscle involvement ( figure b - ). muscle biopsy showing fi brinoid necrosis within the wall of a medium-sized muscular artery. although the patient had clinical symptoms of a neuropathy and nerve conduction studies were consistent with a mononeuritis multiplex, the nerve biopsy was negative. the diagnosis of polyarteritis was confi rmed by the muscle biopsy. (courtesy of dr. john stone.) untreated pan has a high mortality, with an estimated -year survival of % prior to the introduction of glucocorticoids. with current treatment, survival is greatly improved, approximately % at years. in a population of patients enrolled in prospective trials for pan, mpa, and churg-strauss syndrome, approximately % of the deaths occurred during the fi rst months after the diagnosis was made and treatment initiated. of the patients who died, % died from progression of their vasculitis, while % died of infectious complications related to treatment. no major differences were seen among the three vasculitides studied ( ) . not surprisingly, more severe disease is associated with increased mortality. a five factor score has been used to classify disease severity ( ) . the fi ve factors are ( ) proteinuria > g/day, ( ) renal insuffi ciency (cr > . mg/dl), ( ) cardiomyopathy, ( ) gastrointestinal symptoms, and ( ) cns involvement. a five factor score of is associated with a -year mortality of only % (with not all deaths caused directly by pan). five factor scores of and or more are associated with mortalities of % and %, respectively ( ) . the treatment of pan is guided by both the etiology of the disease (if known) and its severity. pan cases associated with hepatitis b are treated with a short course of prednisone ( mg/kg/day) to suppress the infl ammation. patients begin -week courses of plasma exchange (approximately three exchanges per week) simultaneously with the start of glucocorticoids. the dose of prednisone is tapered rapidly (over approximately weeks), followed by the initiation of antiviral therapy (e.g., lamivudine mg/day). for idiopathic pan, the mainstay of treatment is glucocorticoids, with an initial dose of approximately mg/ kg daily of prednisone. intravenous glucocorticoids can be used in patients with diffi culty taking oral medications due to gi involvement. pulse doses (e.g., methylprednisolone g intravenously each day times three) may be used in severe disease. glucocorticoids alone may be enough to treat milder cases. approximately half of all patients with pan may be cured with glucocorticoids alone. in cases of pan that are rapidly progressive or lifeor organ-threatening, cyclophosphamide is added to glucocorticoid treatment. cyclophosphamide should be considered for any patient with a five factor score of or greater. in addition, severe peripheral neuropathy or mononeuritis multiplex is also a strong indication for cyclophosphamide. although many clinicians still prefer daily oral cyclophosphamide to monthly pulsed intravenous cyclophosphamide, a meta-analysis comparing the two regimens in anca-associated vasculitis showed little difference ( ) . therapy should be tailored to the individual patient's circumstances. most cases of idiopathic pan do not recur after remission has been achieved and the patient has received to months of cyclophosphamide. current regimens generally emphasize shorter courses of cyclophosphamide, with durations of therapy closer to months than to . after treatment with months of cyclophosphamide, patients in remission-the great majority-should be switched to another immunosuppressive agent for remission maintenance. as with the anca-associated vasculitides, azathioprine or methotrexate is often used. after a total treatment length of approximately months, the remission maintenance agent can often be stopped, with a low relapse rate. patients should continue to be monitored for evidence of recurrence. much potential morbidity in pan relates to adverse events from inappropriate (or overly aggressive) treatment. conversely, poor outcomes also result from undertreatment, for example, failure to employ cyclophosphamide in a patient clearly failing highdose glucocorticoids. an important aspect of treatment is avoiding known side effects of agents used. this includes the use of calcium and vitamin d supplementation in all patients on glucocorticoids, along with use of a bisphosphonate in those at high risk for bone loss and monitoring of bone density. patients on cyclophosphamide should have routine monitoring for cytopenias and hematuria and receive trimethoprim/ sulfamethoxazole for prevention of pneumocystis jiroveci (formerly carinii) pneumonia. patients receiving pulsed intravenous cyclophosphamide are also candidates for mesna (sodium- -sulfanyl ethanesulfonate) for prevention of hemorrhagic cystitis. premenopausal females on cyclophosphamide are candidates for leuprolide to suppress the gnrh axis and prevent premature ovarian failure; males may opt to bank sperm. finally, as a teratogen, patients should not become pregnant or father children on cyclophosphamide. multiple antibodies may lead to positive immunofl uorescence testing for anca in either perinuclear (p-anca) or cytoplasmic (c-anca) patterns. however, only antibodies to myeloperoxidase (mpo) and proteinase- (pr ) are associated with the aav. wegener's granulomatosis may be associated with destructive upper respiratory tract disease, including saddle-nose deformity, erosive sinusitis, and subglottic stenosis. the css is often associated with allergic rhinitis, nasal polyposis, or sinusitis, but is rarely associated with destructive lesions. a host of ocular lesions may occur in the aav, including episcleritis, scleritis, peripheral ulcerative keratitis, and orbital pseudotumor. lung disease is common in the aav and ranges from asthma (in css) to nodular lesions with a tendency to cavitate (in wg) to interstitial lung disease (mpa) to alveolar hemorrhage (all forms of aav). segmental, necrotizing glomerulonephritis commonly accompanies the aav, particularly wg and mpa. eosinophilia is the sine qua non of css. in , godman and churg observed that wegener's granulomatosis (wg), microscopic polyangiitis (mpa), and the churg-strauss syndrome (css) share certain pathological similarities despite their clinical distinctions ( ). these diseases, godman and churg noted, "group themselves into a compass, (ranging from) necrotizing and granulomatous processes with angiitis . . . to vasculitis without granulomata." validation of the pathological links between these disorders became clear three decades later with the discovery of antineutrophil cytoplasmic antibodies (anca) and the fi nding that most patients with wg, mpa, and (to a lesser extent) css have anca in their serum. these diseases are commonly termed anca-associated vasculitides (aav), even though not all patients with these diseases have detectable anca (table c- ) . anticytoplasmic antibodies directed against neutrophils (i.e., anca) were reported in association with segmental necrotizing glomerulonephritis in the early s. in , the presence of diffuse cytoplasmic staining of neutrophils was reported in patients with wg ( ) . in studies of patients with wg, mpa, or renal-limited vasculitis, falk and jennette ( ) noted another pattern of immunostaining-perinuclear fl uorescence of alcohol-fi xed neutrophils. the two types of antibodies associated with aav are those directed against ( ) proteinase- (pr ) and ( ) myeloperoxidase (mpo). pr and mpo, both serine proteases, are constituents of the primary granules of neutrophils and monocytes. antibodies directed against these antigens are known, respectively, as pr -anca and mpo-anca. the american college of rheumatology classification criteria for wg and the css (table c- ) ( , ) were developed to ensure the inclusion of uniform disease populations in research studies ( ) . these criteria did not address the utility of anca for classifi cation or the difference between polyarteritis nodosa and mpa. these limitations were addressed by the chapel hill consensus conference (table c - ) ( ). to date, widely accepted diagnostic criteria for these diseases have not been developed. a population-based study from norfolk, england, reported incidences of . cases per million for wg, . cases per million for mpa, and . cases per million for the css ( ). in two large u.s. cohorts of patients with wg ( , ), whites comprised more than % of all cases, whereas african americans, hispanics, and asians together represented % to % of cases. the mean age at diagnosis is about years, but cases involving octogenarians are not unusual. wegener's granulomatosis granulomatous infl ammation involving the respiratory tract, and necrotizing vasculitis affecting small-to medium-sized vessels (e.g., capillaries, venules, arterioles, and arteries). necrotizing glomerulonephritis is common. microscopic polyangiitis necrotizing vasculitis, with few or no immune deposits, affecting small vessels (i.e., capillaries, venules, or arterioles). necrotizing arteritis involving small-and medium-sized arteries may be present. necrotizing glomerulonephritis is very common. pulmonary capillaritis often occurs. churg-strauss syndrome eosinophil-rich and granulomatous infl ammation involving the respiratory tract, and necrotizing vasculitis affecting small-to medium-sized vessels, associated with asthma and eosinophilia. there is substantial overlap in many of the clinical features of the aavs. in some cases, distinguishing among two or more of these diseases on the basis of clinical features alone is diffi cult (table c- ) . although patients with the css or mpa may experience substantial ear, nose, or sinus disease, this pattern of involvement is most characteristic of wg. more than % of patients with wg eventually develop upper airway or ear abnormalities. the nasal symptoms of wg include nasal pain and stuffi ness, rhinitis, epistaxis, and brown or bloody crusts. nasal infl ammation may lead to septal erosions, septal perforation, or, in many cases, nasal bridge collapse-the "saddle-nose deformity" (figure c - ). the distinction between active wg in the sinuses and secondary infections in the sinuses may be challenging (see nonmedical interventions section). in % to % of patients with the css, allergic rhinitis is the earliest disease manifestation, typically appearing years before the development of full-blown saddle-nose deformity in wegener's granulomatosis. systemic vasculitis. rhinitis may be severe and may require serial polypectomies to relieve obstruction and sinusitis. nasal crusting and conductive hearing loss (due to serous otitis or granulomatous middle ear infl ammation) may also occur in the css. two principal categories of ear disease-conductive and sensorineural hearing loss-are typical of wg. the most common cause of conductive hearing loss may be eustachian tube dysfunction due to nasopharyngeal disease. inner ear disease in wg may be associated with sensorineural hearing loss, vestibular dysfunction, or both. in contrast to middle ear disease, the mechanism of inner ear disturbances in wg is poorly understood. subglottic stenosis and stenotic lesions of the bronchi are potentially serious complications of wg. subglottic involvement, often asymptomatic initially, becomes apparent as hoarseness, pain, cough, wheezing, or stridor. thin-cut computed tomographic scans and often direct laryngoscopy are useful in assessing these airway narrowings. scleritis may lead to necrotizing anterior scleritis (scleromalacia perforans) and blindness. peripheral ulcerative keratitis may cause the corneal melt syndrome. other ocular manifestations of aav include conjunctivitis, episcleritis, and anterior uveitis. in wg, orbital masses termed pseudotumors occur in a retrobulbar location in % to % of patients, causing proptosis, diplopia, or visual loss. nasolacrimal duct obstruction is most typical of wg. in wg, the pulmonary manifestations range from asymptomatic lung nodules and fl eeting (or fi xed) pulmonary infi ltrates to fulminant alveolar hemorrhage. the nodules are usually multiple, bilateral ( figure c- ) , and often cavitary. infi ltrates are often misdiagnosed initially as pneumonia. pulmonary capillaritis, equally likely to occur in wg and mpa, may lead to lung hemorrhage, hemoptysis, and rapidly changing alveolar infi ltrates ( figure c- ) . patients with mpa may also develop interstitial fi brosis of the lungs. obstructive airway disease and fl eeting pulmonary infi ltrates are the hallmarks of the css. the majority of patients report the new onset of asthma months to years before the appearance of overt vasculitis. following resolution of the vasculitic phase with treatment, many patients with css suffer from steroid-dependent asthma. the most feared clinical presentation of renal disease among the aavs is rapidly progressive glomerulonephritis. more than % of patients with wg will eventually develop renal involvement. the progression of the disease often appears to accelerate once kidney involvement is apparent. in mpa, renal disease may have a more indolent course, and renal biopsies typically demonstrate more sclerosis and fi brosis than do specimens from patients with wg. severe renal disease in css is very rare. "renal-limited" vasculitis is pauci-immune glomerulonephritis (see pathology section) associated with anca, usually directed against mpo, without evidence of disease in other organs. anca-associated renal disease may lead to fi brotic crescents and other scarring within the kidney. subsequent disease fl ares and progression of renal dysfunction through hyperfi ltration may lead to end-stage renal disease. infl ammatory joint complaints, often migratory and oligoarticular in nature, occur in at least % of patients with aav. joint problems are frequently the presenting complaint, but the diagnosis is seldom made until other symptoms are manifest. the combination of joint complaints, cutaneous nodules (frequently mistaken for rheumatoid nodules), and the high frequency of rheumatoid factor positivity among patients with aav (approximately one third are rheumatoid factor positive) often lead to the misdiagnosis of rheumatoid arthritis early in the disease course. arthralgias are more common than frank arthritis. the recurrence of musculoskeletal complaints in a patient in remission often marks the start of a disease fl are. in both the css and wg, cutaneous nodules may occur at sites that are also common locations for rheumatoid nodules, particularly the olecranon region ( figure c- ) . skin fi ndings in the aavs also include all of the potential manifestations of cutaneous vasculitis: palpable purpura, vesiculobullous lesions, papules, ulcers, digital infarctions, and splinter hemorrhages. vasculitic neuropathy may lead to a devastating mononeuritis multiplex or a disabling sensory polyneuropathy. mononeuritis multiplex occurs more commonly in the css [up to % of patients ( ) ] and mpa (up to %) than in wg. central nervous system abnormalities occur in approximately % of patients with wg, usually in the form of cranial neuropathies, mass lesions, or pachymeningitis. the frequency of parenchymal brain involvement in aav, though not yet known with certainty and generally regarded as rare, has been reported. central nervous system disease generally occurs only when more typical disease manifestations are present elsewhere. the css is the type of aav that is most likely to involve the heart, usually in the form of rapid-onset heart failure. cardiac complications in wg and mpa are both less common and more diffi cult to attribute with certainty to the underlying disease. focal cardiac valvular lesions, valvular insuffi ciency, pericarditis, and coronary arteritis have been described in wg. eosinophilic gastroenteritis often precedes the frank vasculitic phase of the css. among patients with either the css or mpa, unexplained abdominal pain occurs in up to one third of patients and may lead to ischemic bowel. gastrointestinal involvement is less common in wg. eosinophilia (before treatment) is a sine qua non of the css. eosinophil counts are usually sensitive markers of disease fl ares, but respond very quickly (within hours) to treatment with high doses of glucocorticoids. tissue infi ltration by eosinophils, however, may remain. mild eosinophilia (rarely more than % of the total white blood cell count) may also occur in wg. most patients with css also have elevated serum immunoglobulin e levels. in addition to anca, nonspecifi c autoantibodies, such as antinuclear antibodies and rheumatoid factor, also occur in high percentages of patients with aav. antineutrophil cytoplasmic antibodies-associated vasculitides rarely affect the parotid gland, pulmonary artery, breast, or genitourinary organs. involvement of these organs by aav is usually an unexpected fi nding on biopsies performed to exclude other diseases, particularly cancer and infection. churg-strauss granulomas, that is, cutaneous extravascular necrotizing granulomas, occurring over the elbow. these lesions may occur in both the churg-strauss syndrome and wegener's granulomatosis, mimicking rheumatoid nodules. fibrinoid necrosis, a pathological hallmark of aav, may be found in a variety of vasculitic (and nonvasculitic) conditions, such as polyarteritis nodosa, scleroderma renal crisis, systemic lupus erythematosus, and malignant hypertension. both vasculitic and necrotizing granulomatous features, which do not invariably coexist, may be confi rmed in lung biopsy specimens. in addition, pulmonary wg frequently demonstrates an extensive, nonspecifi c infl ammatory background. coalescence of such neutrophilic microabscesses leads to extensive regions of "geographic" necrosis. palisading granulomas, scattered giant cells, and poorly formed granulomas may also be found in wg. churg-strauss syndrome typically evolves through three phases, with corresponding pathological fi ndings. in the fi rst phase, allergy, asthma, and other atopic symptoms predominate. in the second, eosinophilic infi ltration occurs in the lung and other organs (eosinophilic pneumonia, eosinophilic gastroenteritis; figure c - ). in the third phase, vasculitis ensues. curiously, at the time the vasculitic phase begins, patients' asthma often improves signifi cantly. the histopathological fi ndings in css in the lung include eosinophilic infi ltrates; extensive areas of necrosis (reminiscent of the geographic necrosis in wg); a granulomatous vasculitis of small arteries and veins, associated with striking eosinophilic infi ltration. in contrast to wg and mpa, lymphadenopathy (with overwhelming eosinophilic infi ltration into the lymph nodes), is frequently found in css. the interstitial lung disease of mpa resembles usual interstitial pneumonitis (uip), with the exception that necrosis of the alveolar septae and areas of hemorrhage can occur. more characteristic fi ndings in mpa, however, reveal nonspecifi c infi ltrates or alveolar hemorrhage. vasculitis of the pulmonary capillaries may be diffi cult to prove. renal disease in the aavs is associated with focal, segmental lysis of glomerular tufts, disruption of the basement membrane, and accumulation of fi brinoid material (i.e., fi brinoid necrosis). crescents in bowman's space develop as a result of spillage of infl ammatory mediators across the ruptured glomerular capillaries, accumulation of macrophages, and epithelial cell proliferation. thrombotic changes in the glomerular capillary loops are among the earliest histologic changes. acute tubular necrosis and tubulointerstitial nephritis are also seen commonly. immunofl uorescence studies of renal biopsy specimens demonstrate scant deposition of immunoglobulin and complement, hence the term pauci-immune glomerulonephritis. tissue samples from involved areas of the upper respiratory tract (nose, sinuses, and subglottic region) in wg often reveal only acute and chronic infl ammation. nevertheless, these biopsies are easier to obtain than are biopsies of the lung and kidney. moreover, the combination of these pathological fi ndings (nondiagnostic in and of themselves) and compatible clinical features (e.g., pulmonary nodules and pr -anca) may yield the diagnosis in some cases. upper respiratory tract biopsies are therefore worth undertaking in patients with significant upper respiratory involvement. eosinophilic infi ltration of a salivary gland in a patient with the churg-strauss syndrome. the arrows in panel b indicate the formation of a churg-strauss granuloma, with multinucleated giant cells, palisading histiocytes, and scattered eosinophils. proteinase- , a -kda serine protease, is found in the azurophilic granules of neutrophils and peroxidase-positive lysosomes of monocytes. mpo, which constitutes nearly % of the total protein content of the neutrophil, is localized to the same cellular compartments as pr . the protein is a covalently linked dimer with a molecular weight of kda. the autoantibodies directed against pr and mpo are directed against multiple epitopes. sera from different patients may recognize different epitopes. all anca, however, recognize restricted epitopes of pr involving its catalytic site. two types of assays for anca-immunofl uorescence and enzyme immunoassay-are now in common use. capture enzyme immunoassays may offer some advantages over the more widely available tests, but are currently performed only in specialty centers. with immunofl uorescence, three principal patterns of fl uorescence are recognized: the cytoplasmic (c-anca), perinuclear (p-anca), and "atypical" patterns. in patients with vasculitis, the c-anca pattern usually corresponds to the detection of pr -anca by enzyme immunoassay. the combination of a c-anca pattern on immunofl uorescence testing and pr -anca is associated most strongly with wg. the p-anca pattern, which usually corresponds to the presence of mpo-anca in vasculitis patients, occurs in approximately % of patients with wg, but is more typical of mpa, the css, and renal-limited vasculitis. the great majority of patients with drug-induced aavs are p-anca positive, often with very high titers of mpo-anca. regardless of the immunofl uorescence pattern, positive immunofl uorescence assays should be confi rmed by the performance of enzyme immunoassays for the specifi c antibodies associated with vasculitis: pr -and mpo-anca. even for c-anca, the positive predictive value for wg is only in the range of % to % ( , ) . despite advances in anca testing techniques, the cornerstone of diagnosis in wg remains the rigorous interpretation of histopathological specimens within the overall clinical context. when biopsy specimens are nondiagnostic, anca assays provide an important adjunct to diagnosis (table c- ). in the proper clinical setting, a positive anca assay greatly increases the likelihood that a form of aav is present. most series indicate that up to % to % of patients with active, untreated wg are anca negative. for patients with limited wg, % or more of patients lack anca. approximately % of patients with mpa and % of those with css (higher in some series) have anca. positive anca serologies are extremely useful in suggesting the diagnosis in the proper clinical setting. positive immunofl uorescence assays without confi rmatory enzyme immunoassays for anti-pr or anti-mpo antibodies are of limited utility. histopathology remains the gold standard for diagnosis in most cases. negative anca assays do not exclude anca-associated vasculitis because between % and % of patients with anca-associated vasculitis (depending on the particular disease) may be anca-negative. persistence of anca in the absence of clinical indications of active disease does not indicate a need for continued treatment. in a patient who was anca-positive during active disease, persistent anca-negativity provides reassurance-but no guarantee-that the disease is not active. if disease fl ares occur in such patients, they are usually limited. a patient who becomes anca-positive again following a period of clinical quiescence associated with negative anca assays may be at an increased risk for a disease fl are. the temporal correlation between the return of anca and a disease fl are, however, is poor. treatment of anca-associated vasculitis should never be predicated upon anca serologies or titers alone. abbreviations: anca, antineutrophil cytoplasmic antibody; mpo, myeloperoxidase; pr , proteinase . in general, anca titers have imperfect correlations with disease activity. in one study, the positive predictive value of a rise in anca titers as measured by immunofl uorescence was only % in a prospective study, compared with % for enzyme immunoassay ( ) . moreover, among patients with an elevation of anca titers measured by enzyme immunoassay, only % suffered disease fl ares within months. a more recent prospective study ( ) showed that increases in pr -anca levels did not predict disease relapses. the proportion of patients who relapsed within year following an increase in anca levels was only %. although some studies suggest that a rise in anca titer is a risk factor for a fl are, the temporal relationship between a rise in anca titers and the development of disease activity requiring treatment is very poor, with months to years between these two events. thus, the adjustment of immunosuppressive medications based solely on the rise or fall of anca titers is never justifi ed. the aavs are complex disorders mediated by the immune system in which tissue injury results from the interplay between an initiating infl ammatory event and a highly specifi c pathogenic immune response (i.e., the production of anca) to previously shielded epitopes of neutrophil granule proteins. ancas produce tissue damage via interactions with primed neutrophils and endothelial cells. the hypothesis, supported strongly by in vitro evidence, is that the antibodies induce a necrotizing vasculitis by inciting a respiratory burst and degranulation of leukocytes (neutrophils and monocytes), leading to endothelial injury. the initial events in the process require the priming of leukocytes by cytokines and perhaps other stimuli, leading to the expression of pr and mpo on the cell surface. the effects of ancas are determined by the state of neutrophil activation. ancas may constitutively activate primed neutrophils and promote binding of the primed neutrophils to the vascular endothelium, degranulation, and the release of neutrophil chemoattractants, hence creating an autoamplifying loop. there is now substantial evidence that ancas are directly involved in the widespread tissue damage that is the hallmark of the aavs. recombinant activating gene (rag- )-defi cient mice that receive anti-mpo antibodies develop clinical features consistent with aav, including crescentic glomerulonephritis and sys-temic necrotizing vasculitis ( ) . in humans, the evidence is indirect. propylthiouracil is known to accumulate within neutrophil granules and may lead to a druginduced aav ( ) , possibly by increasing the immunogenicity of mpo (leading to the characteristically high titers of mpo-anca seen in this disease). in addition to anca, multiple other elements of the immune system participate in the pathophysiology of these diseases. if the autoantibody response leading to anca production follows the exposure of a cryptic epitope, epitope spreading may then generalize the antibody response to the rest of the molecule. this hypothesis implies a prominent role for the t cell in the pathogenesis of the aavs. moreover, most patients with aavs produce isotype-switched igg anca, implying a secondary immune response driven by t cells. growing evidence, particularly data from clinical studies ( ) , now also implicates b cells as important participants in the infl ammation of aav. as the precursors of plasma cells (which produce anca), b cells now seem a logical therapeutic target in aav. in addition to disrupting anca production, however, interference with b-cell function may also ameliorate aav by disabling critical b cell/t cell interactions, by removing the antigen presenting function of b cells, and perhaps other mechanisms. b-cell depletion is the focus of ongoing randomized trials involving patients with wg and mpa. the pathophysiology of css likely bears many similarities to that of wg and mpa, albeit anca are less common in css. in the css, however, relatively little is currently understood about the special role played by the eosinophil in that disease. because of their multiorgan system nature, the differential diagnosis of aav is lengthy. one frequently challenging task is the differentiation of these diseases from other forms of vasculitis. indeed, clear distinctions are often impossible between wg and mpa, because granulomatous infl ammation is not detected on all biopsy specimens from patients with wg. distinguishing the aavs from other forms of vasculitis is often more critical because the specifi c treatments differ according to diagnosis. in addition, the aavs must be distinguished from a host of other disorders associated with infl ammation and multiorgan system dysfunction. the differential diagnosis of aav is shown in table c- . churg-strauss syndrome has an additional major branch of its differential diagnosis because of the eosinophilia associated with the disease. allergic bronchopulmonary aspergillosis, chronic eosinophilic pneumonia, eosinophilic gastroenteritis, eosinophilic fasciitis, the hypereosinophil syndrome, and eosinophilic leukemias must all be excluded. current choices for treatment in wg are based on the classifi cation of patients into the categories of either severe or limited disease ( ) . severe wg constitutes an immediate threat either to the function of a vital organ or to the patient's life. conversely, limited wg consists of disease manifestations that do not pose such threats. the practical distinction between severe and limited disease is that under the current standard of care the diagnosis of severe wg mandates the use of cyclophosphamide, whereas milder therapeutic approaches may be appropriate and should be considered for limited disease. the treatment of mpa parallels very closely that of severe wg. because of the propensity of mpa to involve major organs (lungs, kidneys, peripheral nerves) in a severe fashion, limited forms of the disease are recognized less often. some cases of css can be treated with glucocorticoids alone, but those with vasculitic neuropathy, life-threatening pulmonary involvement, and other sobering organ involvement require cyclophosphamide from the outset of therapy. in treating severe aav, the standard approach now is to employ cyclophosphamide [e.g., mg/kg/day, adjusted for renal dysfunction ( ) ] for to months. some experts prefer intermittent (intravenous) regimens of cyclophosphamide (e.g., - mg/m every month). no data yet available strongly endorse one cyclophosphamide regimen over another. remission induction with cyclophosphamide is usually followed by longer periods of remission maintenance therapy with either azathioprine ( ) or methotrexate ( ) . for patients who demonstrate propensities to fl are, long-term use of the least toxic drug for the maintenance of remission may be appropriate. this may include methotrexate or azathioprine and, for patients with recurrent disease fl ares, low doses of prednisone (e.g., mg/day). the optimal length of treatment with methotrexate or azathioprine is not clear, but continuation of these medications for at least year after remission is reasonable in most patients. tumor necrosis factor inhibition (or at least etanercept) does not appear to be effective in wg ( ) , and its use in combination with cyclophosphamide may heighten the risk of solid malignancies substantially ( ) . among patients with limited wg, remission is induced in approximately three fourths of patients with the use of methotrexate (up to mg/week) and glucocorticoids alone. patients treated with methotrexate and glucocorticoids must be observed carefully for breakthrough disease, particularly glomerulonephritis. the use of trimethoprim-sulfamethoxazole as a treatment for wg has fallen increasingly into disfavour. the use of trimethoprim-sulfamethoxazole alone for the treatment of active wg is not appropriate. the medication may have some role in remission maintenance for patients with upper respiratory tract disease, however, its effi cacy is uncertain and its mechanism of action unclear. a wide array of other therapies, such as plasmapheresis, intravenous immunoglobulin, mycophenolate mofetil, and lefl unomide, have been employed in small numbers of patients, but so far there are insuffi cient data to judge their effi cacy. rituximab and other strategies for b-cell depletion are now being tested. interferonalpha has been reported to have some benefi t for css. once scarring and fi brosis are well established in the subglottic region, airway narrowing may be due to the progression of scar tissue rather than to wg-related infl ammation. in such cases, subglottic stenosis responds poorly to immunosuppressive therapy, and the most effective therapeutic approach to this problem is laryngoscopic dilatations of the airway, augmented by intralesional corticosteroid injections ( ) . serial procedures are often required. if severe subglottic stenosis precludes a safe dilatation procedure, a patent airway should fi rst be secured by a tracheostomy. wg often leads to chronic nasosinus dysfunction. regardless of disease activity, most patients require multiple daily saline irrigations to minimize the accumulation of secretions and crusts, and to reduce the incidence of secondary infections. persistent or recurrent infections may require surgical drainage. distinguishing between worsening sinus disease caused by active wg and superinfection may be diffi cult. in the absence of a prompt response to antibiotics, surgical drainage and biopsy are often required for a more defi nitive diagnosis. in contrast to the situation in the fi rst years following the descriptions of the aavs, these diseases are now highly treatable. unfortunately, disease relapses are a major threat. mpa and the css are somewhat less likely than wg to fl are after the achievement of remission. the percentages of patients with those diseases who suffer disease fl ares after appropriate courses of treatment have been estimated to be about % to %. even with therapy, mortality and morbidity are substantial. in a cohort of patients followed at the national institutes of health (nih) from the late s through the early s ( ), % of deaths were due to either the disease or complications of treatment, and % suffered permanent disease-related morbidity, including chronic renal insuffi ciency ( %), end-stage renal disease requiring dialysis ( %), hearing loss ( %), nasal deformity ( %), tracheal stenosis ( %), and visual loss ( %). many patients incurred more than one type of permanent morbidity. in a more recent retrospective study of patients with anca-associated renal vasculitis ( ), cumulative patient survival at years was %. there was an % mortality rate at year, however, with infections as a major cause of death. in this cohort, mortality was associated with age older than years, the development of end-stage renal failure, and an initial serum creatinine level greater than . mg/dl. in the wegener's granulomatosis etanercept trial (wget) ( ) , although > % of all patients achieved at least a transient disease activity score of zero, fewer than % of patients achieved and maintained these disease remissions over the mean -month course of follow-up on treatment. thus, although remissions are achieved in most patients with wg, relapse remains a major threat. furthermore, % of the patients enrolled in the wget had sustained at least one item of damage from the disease or treatment within year of enrollment ( ) . the most common items of damage recorded were hearing loss ( %) and proteinuria ( %). much of the morbidity in the aavs relates to prolonged courses of immunosuppression, particularly the need to re-treat patients who suffer multiple relapses. in the patient-years of follow-up in the nih series, only % of these years were spent in remission. serious infections occurred in %. other morbidities included drug-induced cystitis caused by cyclophosphamide ( %), increased risk of malignancy (particularly bladder cancer, leukemia, and lymphoma), infertility ( % of women with childbearing potential), and a host of side effects related to the use of glucocorticoids. pathogenic immune complexes formed between antigen and antibodies tend to occur during periods of antigen excess. when the immune complexes precipitate into the tissues, they fi x complement, leading to an intense immune reaction. in immune complex vasculitis, immune complexes deposit in the vascular endothelium or in capillary beds, such as those found in the skin, kidneys, or lungs. the most common skin manifestation of small vessel vasculitis is palpable purpura. hypersensitivity vasculitis is characterized by immune complex deposition in capillaries, postcapillary venules, and arterioles. the usual causes are either medications (e.g., penicillins, sulfonamides, or cephalosporins) or infections. cryoglobulinemic vasculitis is usually caused by hepatitis c infections. the antigens involved in cryoglobulinemic vasculitis are portions of the hepatitis c virus (hcv) virion. the relevant antibodies are both igg and igm, leading to the designation "mixed" cryoglobulinemia. henoch-schönlein purpura is associated strongly with iga deposition within blood vessel walls. hypocomplementemic urticarial vasculitis has many features that overlap with systemic lupus erythematosus. exposure to a foreign antigen activates an adaptive immune response that leads to the production of antigen-specifi c antibodies. the combination of antibody with antigen creates immune complexes that neutralize the foreign antigen, and allow it to be cleared safely by the reticuloendothelial system. this complex system, however, contains within it the potential for failure. if the antibody response is just right, these immune complexes may escape early detection, and instead imbed themselves into joints and blood vessels. these immune complexes can then activate complement, leading to local infl ammation. immune complexes deposited in the kidneys, for example, create glomerulonephritis ( ). those deposited in the synovium lead to arthritis. if the immune complexes are found in the blood vessels, vasculitis is the result. in pathology, the word vasculitis describes infl ammation within blood vessel walls. this process frequently leads to cellular destruction, damage to the vascular structures, and compromise of blood fl ow to organs supplied by the involved vessels, resulting in organ compromise. several forms of vasculitis are the direct result of immune complex deposition. this chapter outlines several examples of immune complex-mediated vasculitis and highlights some common themes. in , maurice arthus noted that intradermal injection of a rabbit with horse serum resulted in a cutaneous infl ammatory reaction that evolved into localized tissue necrosis ( ). the reaction was faster, he observed, if the animal had been previously exposed to horse serum. this response, now known as the arthus reaction, forms the basis of our understanding of immune complexmediated diseases. in the arthus model, injection of the horse serum leads to immune complex formation that initiates complement activation and an infl ux of infl ammatory cells. in the areas of most intense infl ammation, in situ thrombosis formation can lead to tissue ischemia and hemorrhagic infarction. in general, immune complexes are not pathogenic. their immunogenicity is governed by a large number of factors, including antigen load, antibody response, the effi ciency of the reticuloendothelial system in the clear-ance of immune complexes, physical properties of the blood vessels (including fl ow dynamics and previous endothelial damage), and the solubility of the immune complexes themselves. immune complex solubility is determined by the ratio of antibody to antigen. when antibody and antigen are present in equal proportion, large immune complexes are formed, which are identifi ed easily and removed by the reticuloendothelial system. when there is an excess of antibody, small immune complexes are formed, which remain in solution, and do not elicit an immune response. when there is a slight excess of antigen, however, the immune complexes precipitate from solution, and become trapped in characteristic areas-either the capillary beds (such as those found in the skin, kidneys, or lungs), or the endothelium of medium-sized blood vessels previously damaged by turbulent blood fl ow. when the immune complexes precipitate into the tissues, they fi x complement, leading to an intense immune reaction. complement fi xation and local infl ammation recruit neutrophils, which attempt to engulf the immune complexes. during this process, the neutrophils degranulate, releasing lysosomal enzymes and oxygen free radicals that cause tissue necrosis. the inciting antigen can be from numerous sources. in infective endocarditis, the antibody response, formed against bacterial antigens, can lead to painful cutaneous lesions known as osler nodes. in systemic lupus erythematosus (sle), antibodies form against nuclear components (e.g., dna and histones) that are released during tissue injury. certain forms of malignancy can be associated with immune complex formation, with antibodies directed against tumor-associated antigens. immune complexes may also form in response to a large number of drugs, including penicillin and sulfonamides. hypersensitivity reactions were fi rst noted in patients who were treated with antitoxin derived from horse serum. such patients developed an antibody response to the horse antigens, which led to a characteristic syndrome of fever, joint pain, and rash now known as serum sickness. hypersensitivity vasculitis refers to a heterogeneous group of syndromes (including serum sickness and druginduced vasculitis) characterized by immune complex deposition in capillaries, postcapillary venules, and arterioles. this is the most common form of vasculitis. although multiple agents have been implicated, including penicillins, sulfonamides, and cephalosporins, the inciting agent cannot always be identifi ed. the presence of three or more criteria has a sensitivity of % and specifi city of % for the diagnosis of hypersensitivity vasculitis. hypersensitivity vasculitis that occurs without systemic manifestations is sometimes referred to as cutaneous vasculitis or cutaneous leukocytoclastic angiitis. the term serum sickness, on the other hand, is reserved to describe a systemic illness, including rash and arthralgias, which occurs to weeks after exposure to a drug or foreign antigen. most patients with hypersensitivity vasculitis will develop cutaneous manifestations, the most common of which is purpura ( figure d- ) . these lesions are distributed usually in a symmetric fashion over dependent regions of the body, particularly the lower legs (and, in recumbent patients, over the buttocks) because of the increased hydrostatic pressure in such areas. purpuric lesions are not always palpable to the touch. the term palpable purpura is essentially synonymous with small vessel vasculitis, but does not necessarily imply an immune complex-mediated pathophysiology; pauciimmune forms of vasculitis, such as wegener's granulomatosis, microscopic polyangiitis, and the churg-strauss syndrome, for example, may present with identical skin fi ndings (see chapter c). biopsy is the diagnostic method of choice. cutaneous biopsies are associated with low morbidity, and are generally suffi cient to confi rm one's clinical suspicion. light microscopy examination of a hematoxylin and eosin (h&e) preparation will demonstrate an infl ammatory infi ltrate primarily composed of mononuclear or polymorphonuclear cells, as well as telltale signs of small vessel vasculitis: leukocyte diapedesis, karyorrhexis, and leukocytoclasis. although h&e preparations are adequate for confi rming the presence of vasculitis, they often provide insuffi cient data for a precise diagnosis. in cases of immune complex-mediated vasculitis, direct immunofl uorescence (dif) studies on the biopsy demonstrate the types of immunoreactants (i.e., immunoglobulin and complement proteins) present at the site of disease. although direct immunofl uorescence requires a biopsy of a second cutaneous site, this procedure is critical in many cases to differentiating the array of conditions associated with cutaneous vasculitis. biopsy is especially useful to exclude other causes of vascular injury, including embolism and hypercoagulability states, which may demonstrate evidence of erythrocyte extravasation but not immune complex deposition. of course, the presence of small vessel vasculitis does not always confi rm the presence of a primary autoimmune disease; malignancy, for example, can also be associated with leukocytoclastic vasculitis. all biopsies, therefore, must be evaluated in the appropriate clinical context. removal of the inciting agent is the only reliable therapy. in patients who have been exposed to multiple medications, determining the inciting agent may be diffi cult, and may require withdrawal of multiple agents simultaneously until the syndrome clears, typically in to weeks. immunosuppression with glucocorticoids should be reserved for patients with particularly fulminant disease, and may be discontinued usually within several weeks. the prognosis for patients with hypersensitivity vasculitis depends on the nature of the inciting agent. in the case of drug-induced vasculitis, multiple agents may need to be discontinued and re-introduced gradually. approximately half of cases of isolated cutaneous angiitis do not have an obvious cause. many such cases are associated with a relapsing and remitting course, but remain restricted to the skin and do not mandate aggressive immunosuppressive therapy. in , wintrobe and buell noted that when serum from a patient with a hyperviscosity syndrome due to multiple myeloma was held at temperatures less than °c, a protein precipitate formed ( ). in , this "cold precipitable serum globulin" was referred to for the fi rst time as a "cryoglobulin." cryoglobulins are immune complexes that are characterized by their tendency to precipitate from serum under conditions of cold. cryoglobulins, detectable to a varying degree in a wide array of infl ammatory conditions, are not invariably pathogenic. in some patients, however, cryoglobulins deposit in the small-and medium-sized blood vessels and activate complement, leading to cryoglobulinemic vasculitis ( ) . three major types of cryoglobulinemia are recognized, defi ned by the specifi c kinds of immunoglobulins with which they are associated. type i cryoglobulinemia, characterized by a monoclonal gammopathy (generally igg or igm), can be associated with waldenström's macroglobulinemia or, less frequently, multiple myeloma. type i iga cryoglobulinemia has also been described, although this is quite rare. in contrast to the monoclonal nature of type i cryoglobulinemia, type ii and type iii cryoglobulinemias are known as "mixed" cryoglobulinemias because they are comprised of both igg and igm. in most cases of type ii cryoglobulinemia, more than % of which are caused by hepatitis c infections, the cryoproteins consist of monoclonal igm and polyclonal igg. cases of type ii cryoglobulinemia not associated with hepatitis c infections are sometimes termed "mixed essential" cryoglobulinemia. such cases may be associated with a still undefi ned viral infection. type iii cryoglobulinemia, typically associated with polyclonal igg and polyclonal igm, is associated with many forms of chronic infl ammation, including infection and autoimmune disease. not every form of cryoglobulinemia fi ts neatly into this classifi cation system; cryoglobulins can, for example, have an oligoclonal antibody component. type i cryoglobulinemia rarely presents with signs and symptoms of vasculitis. when symptomatic, type i cryoglobulinemia may be associated with a hyperviscosity syndrome that can lead to serious neurologic manifestations, including dizziness, confusion, headache, and stroke. type i cryoglobulinemia can also be associated with other evidence of vascular stasis, including livedo reticularis, acrocyanosis, and digital gangrene ( figure d- ) . type ii and type iii cryoglobulinemias often present with a triad of signs and symptoms: purpura, arthralgias, and myalgias. the purpura may be extensive and confl uent, and sometimes involves the trunk, upper extremities, and even the face (albeit in most cases the rash is confi ned to the lower extremities). other common organ system involvement, more common in type ii than in type iii cryoglobulinemia, includes membranoproliferative glomerulonephritis ( figure d- ) , peripheral neuropathy, and cutaneous ulcerations (the result of medium vessel vasculitis, in contrast to the smallsized vessel involvement that leads to purpura). biopsy of an affected organ (such as the skin or kidney) may be the most straightforward method of confi rming the diagnosis. light microscopy of purpuric lesions demonstrates leukocytoclastic vasculitis. direct immuno fl uorescence studies reveal various types of immunoglobulin and complement deposition, depending on cryoglobulinemia type. in type ii cryoglobulinemia, for example, direct immunofl uorescence reveals igg and igm deposition, as well as complement components. renal biopsies from patients with glomerulonephritis caused by cryoglobulinemia reveal a membranoproliferative glomerulonephritis that must be distinguished from lupus nephritis (another disorder in which immune complexes play a major role). if biopsy is impractical or impossible, serologic testing may be helpful. the most directly relevant test is a serum cryoglobulin assay, which can give a direct sense of the burden of disease. the serum is allowed to remain at °c for several days before interpretation. the percentage of the serum occupied by the cryoprecipitate is commonly referred to as the cryocrit, and may be useful for following such patients longitudinally. serum cryoglobulin tests are, unfortunately, notoriously diffi cult to obtain, largely because the collection apparatus must be prewarmed and the blood must be allowed to clot at °c before processing. falsenegative assays for cryoglobulins are common, particularly in laboratories not experienced in the collection and processing of samples. for patients with type ii and type iii cryoglobulinemia, nonspecifi c serologic testing may also heighten suspicion. first, a strong clue to the presence of mixed cryoglobulinemia is an extremely low (to almost undetectable) level of c . for reasons that are not entirely clear, c levels are lowered out of proportion to the decrease observed in c . second, the monoclonal component of type ii cryoglobulins almost invariably has rheumatoid factor activity (i.e., binds to the fc portion of igg). thus, essentially all patients with type ii cryoglobulinemia are rheumatoid factor-positive-usually strikingly so. in many cases, rheumatoid factor tests are a more reliable screening test for type ii cryoglobulinemia than are assays for cryoglobulins themselves. although rheumatoid factor titers correlate with immunoglobulin burden in such patients and can be used in longitudinal follow-up in a manner similar to the cryocrit, the level of clinical symptomatology often correlates poorly with both rheumatoid factor level and cryocrit. once patients have achieved a state of disease quiescence following treatment, both rheumatoid factor levels and cryocrit often remain elevated. neither should be used as a gauge of therapy for most patients. finally, acute-phase reactants such as the erythrocyte sedimentation rate and the c-reactive protein are elevated in many patients with cryoglobulinemia. in patients with type i cryoglobulinemia, for example, the erythrocyte sedimentation rate may be even higher than anticipated because of the excess circulating immunoglobulin. treatment of the underlying cause of the cryoglobulins is the only approach that may lead to long-term response. immunosuppression alone will not be suffi cient to treat a cryoglobulinemic vasculitis that is driven by malignancy or chronic infection. in the case of hepatitis cassociated cryoglobulinemic vasculitis, for example, the optimal therapy consists of the effective control of the underlying viral infection (typically with interferonalpha and ribavirin). in patients who experience severe consequences of cryoglobulinemia (such as mononeuritis multiplex, glomerulonephritis, or other forms of tissue necrosis), immunosuppression with high dose glucocorticoids and cyclophosphamide may be necessary to prevent further damage. in patients with rampant systemic vasculitis, anecdotal evidence suggests that the systemic infl ammation should be controlled fi rst with glucocorticoids and other immunosuppressive agents, as appropriate, before the institution of antiviral therapy. in some cases of fl agrantly active vasculitis, the introduction of antiviral therapy fi rst is believed to have triggered disease exacerbation through an unfavorable alteration of the antigen/antibody ratio. the prognosis of patients with cryoglobulinemia generally depends on the underlying cause. the outcome of type i cryoglobulinemia relates closely to the effi cacy in treating the cause of the cryoglobulin. type ii cryoglobulinemia secondary to hepatitis c may be treated effectively if the viral infection is responsive to therapy. if patients do not tolerate antiviral therapy well or if the treatment is ineffective, however, some patients may require low-to-moderate doses of prednisone to control the disease. type iii cryoglobulinemia is frequently a response to an infl ammatory event (such as infection), and may not require specifi c therapy. henoch-schönlein purpura (hsp) is an immune complex-mediated form of small vessel vasculitis that is associated strongly with iga deposition within blood vessel walls. many cases of hsp are reported to occur after upper respiratory tract infections. group a streptococci, mycoplasma, epstein-barr virus, varicella, and other infectious agents have all been implicated in the pathogenesis of this disease, but the true etiology remains unknown. in , the american college of rheumatology proposed the following criteria for the classifi cation of hsp ( ): • palpable purpura • age ≤ years at disease onset • bowel angina • granulocytes in arteriole or venule walls on biopsy meeting two of four criteria is associated with a sensitivity of % and a specifi city of % for this diagnosis. the hallmarks of hsp include an upper respiratory tract infection followed by a syndrome characterized by a purpuric rash, arthralgias, abdominal pain, and renal disease. hsp is usually viewed as a disease of childhood and, indeed, the majority of cases affect children younger than years of age. adults, however, can also be affected by hsp, and have a greater tendency toward prolonged disease courses (with recurrent bouts of purpura) than do children ( ) . colicky abdominal pain, presumably secondary to gastrointestinal vasculitis, is a common characteristic of hsp, and frequently occurs within a week after the onset of rash. occasionally the abdominal pain in hsp occurs before the onset of purpura, and may be diffi cult to distinguish from an acute abdomen. endoscopy may demonstrate purpura in the upper or lower intestinal tract. mild glomerulonephritis is common and generally self-limited, although some patients will develop end-stage renal disease. in children with mild manifestations, the clinical history alone may be suffi cient to confi rm the diagnosis. in more serious cases or when there is suffi cient doubt about the diagnosis, biopsy of an involved organ is essential. unlike other forms of immune complex-mediated disease, however, direct immunofl uorescence will reveal fl orid iga deposition. in the proper clinical setting, this fi nding is diagnostic of hsp. other forms of small vessel vasculitis may have small quantities of iga within blood vessels, but iga is not the predominant immunoreactant in such cases. in mild cases of hsp, no specifi c therapy is necessary. even for patients with glomerulonephritis, it has been diffi cult to demonstrate that treatment with glucocorticoids or immunosuppressive agents signifi cantly alters outcomes. despite this, it may be prudent to treat aggressive renal involvement with an immunosuppressive regimen, including high-dose glucocorticoids and another immunosuppressive agent such as cyclophosphamide, azathioprine, or mycophenolate mofetil, depending on disease severity ( ) . in patients with renal involvement, anecdotal evidence suggests that plasmapheresis and intravenous immunoglobulin may also be benefi cial, particularly in patients who are refractory to standard immunosuppressive regimens. recurrences of skin disease, often comprised of multiple episodes occurring over many months, are not unusual. generally, however, even in patients with recurrent disease, the rule is for the disorder to subside and to resolve completely over a few months to a year. in a minority of patients, some evidence of permanent renal damage persists in the form of proteinuria and hematuria. only a small minority, probably well under %, develop renal failure as a result of hsp. the study of urticaria is hampered by multiple terms that sound similar but describe different types of diseases. the word urticaria is most frequently used to describe acute urticaria, an ige-mediated hypersensitivity reaction to a variety of stimuli, including medications, infection, and other triggers. acute urticaria manifests as pruritic wheals that resolve days after the allergen is removed. chronic urticaria is an autoimmune condition that is probably driven by an autoantigen. this form of urticaria may require immunosuppressive therapy in addition to antihistamines to prevent recurrence ( ) . urticarial vasculitis describes a form of small vessel vasculitis that is characterized by the appearance of urticarial wheals. normocomplementemic urticarial vasculitis is most often simply an example of hypersensitiv-ity vasculitis in which the principal skin manifestation is urticaria. normocomplementemic urticarial vasculitis tends to be self-limited, as is the case with hypersensitivity vasculitis. in contrast, urticarial vasculitis associated with hypocomplementemia is much more likely to constitute a signifi cant and persistent clinical problem. two categories of urticarial vasculitis associated with hypocomplementemia are recognized. the distinctions between these two categories are not always sharp, and both categories also share a number of features with systemic lupus erythematosus. the fi rst category, known simply as hypocomplementemic urticarial vasculitis, refers to cutaneous vasculitis associated with low levels of serum complement (c and c ). the diagnosis of hypocomplementemic urticarial vasculitis is predicated upon the exclusion of other disorders that may present in a similar fashion, particularly cryoglobulinemia and sle. the second category-a more specifi c but still loosely defi ned entity known as the hypocomplementemic urticarial vasculitis syndrome (huvs)-consists of the constellation of low complement levels and urticaria for a period of at least months, as well as some or all of the following: arthritis, glomerulonephritis, uveitis, angioedema, chronic obstructive pulmonary disease, pleurisy, or pericarditis. although the lesions associated with chronic urticaria and urticarial vasculitis are similar in appearance, certain differences help differentiate these two conditions. in urticarial vasculitis, the urticaria typically have a purpuric quality, indicative of small blood vessel damage and red blood cell extravasation ( figure d- ) . unlike common urticaria, the lesions of urticarial vasculitis are frequently associated with moderate pain, burning, and tenderness, in addition to pruritus. whereas common urticaria typically resolve completely within to hours, the lesions of urticarial vasculitis may take days to resolve completely and often worsen without therapy. arthralgias and myalgias are common in urticarial vasculitis. as noted above, patients with huvs may also develop glomerulonephritis, pulmonary manifestations (particularly obstructive airway disease), and other fi ndings. gastrointestinal, cardiovascular, and neurologic manifestations are uncommon, but have been reported. there is striking overlap between huvs and sle, and patients will frequently have characteristics of both, although angioedema and copd are more common in huvs. biopsy of an urticarial wheal in uv will demonstrate evidence of leukocytoclastic vasculitis, including injury to the endothelial cells of the postcapillary venules, erythrocyte extravasation, leukocytoclasis, fi brin deposition, and a perivascular neutrophilic (or less commonly, lymphocytic) infi ltrate. direct immunofl uorescence demonstrates immune complex deposition around blood vessels in the superfi cial dermis and striking deposition of immunoglobulins and complement along the dermal-epidermal junction ( figure d- ) . the "interface dermatitis" is identical to that observed in lupus-a histopathological fi nding termed the lupus band test. in the proper setting, these fi ndings (interface dermatitis as well as immunoreactant deposition within blood vessels) are diagnostic of hypocomplementemic urticarial vasculitis. huvs, in contrast, is a clinical diag-nosis based not only on the presence of urticarial vasculitis but also the occurrence of typical features in extracutaneous organ systems. some cases of hypocomplementemic urticarial vasculitis respond to therapies commonly used for the treatment of sle, including low-dose prednisone, hydroxychloroquine, dapsone, or other immunomodulatory agents. there is anecdotal evidence that antihistamines, calcium channel antagonists, doxepin, methotrexate, indomethacin, colchicine, and pentoxifylline are effective in some cases. serious cases, particularly those presenting with glomerulonephritis or other forms of serious organ involvement, may require treatment with high doses of glucocorticoids and cytotoxic agents. both chronic obstructive pulmonary disease (copd) and cardiac valvular abnormalities are associated with huvs, and may require specifi c treatment as well. the prognosis of huvs is frequently linked to the disorder with which it is associated. sle, copd, angioedema, and valvular abnormalities are all known to occur in association with this disorder, and in such cases, may strongly infl uence both quality and quantity of life. the immune complex-mediated vasculitides are a clinically heterogeneous group of disorders linked by ineffi cient, defective, or dysregulated clearance of immune complexes by the reticuloendothelial system. biopsy of an involved organ is frequently helpful in establishing the diagnosis. direct immunofl uorescence studies of involved blood vessels demonstrate characteristic patterns of immunoglobulin and complement deposition, which may be particularly useful in distinguishing these diseases. the prognosis of patients with immune complex-mediated vasculitis is tied closely to the ability to identify and to treat the underlying cause of the immune response. the prevalence of behçet's disease is highest in countries of the eastern mediterranean, the middle east, and east asia. aphthous oral ulcers are usually the fi rst and most persistent clinical feature of behçet's disease. aphthous ulcers also occur frequently on the genitals (e.g., the scrotum or vulva). uveitis-either anterior or posterior-is common in behçet's disease and a source of major morbidity. many forms of central nervous system disease may occur in behçet's disease. these include aseptic meningitis and white matter lesions in the brainstem. human leukocyte antigen (hla)-b is a strong risk factor for behçet's disease. the diagnosis of primary angiitis of the central nervous system is predicated upon either biopsy evidence of vasculitis or angiographic fi ndings suggestive of vasculitis in the setting of other compelling features, for example, strokes demonstrated by magnetic resonance imaging or the fi ndings of a cerebrospinal fl uid pleocytosis. the diagnosis of primary angiitis of the central nervous system should never be made on the basis of an angiogram alone. patients with benign angiopathy of the central nervous system are predominantly female, tend to present acutely with headache (with or without focal symptoms), and have normal or near normal cerebrospinal fl uid. cogan's syndrome refers to the association of infl ammation in both the eyes and ears: specifi cally, the occurrence of nonsyphilitic interstitial keratitis and immune-mediated inner ear disease, resulting in audiovestibular dysfunction. any type of ocular infl ammation may occur in cogan's syndrome (e.g., scleritis, uveitis, orbital pseudotumor). the inner ear disease associated with this condition often leads to deafness. in erythema elevatum diutinum, skin lesions consist of purple, red, or brown plaques and often have an annular or nodular appearance. the skin lesions have a predilection for the extensor surfaces of the distal extremities and often overlie joints, but may be generalized. behçet's disease (bd) is a chronic infl ammatory disorder of unknown cause. its manifestations are thought to be caused by an underlying vasculitis. although this disease is recognized worldwide, the prevalence is highest in countries of the eastern mediterranean, the middle east, and east asia, thus the name silk road disease. the disease tends to be more severe in areas where it is more common. prevalence rates in all areas of the world are increasing, probably because of improved recognition and reporting. behçet's disease occurs primarily in young adults. the mean age at onset is between and years. the incidence of disease in males and females with the disease is approximately equal along the silk road, but in japan, korea, and western countries the disease occurs more frequently in women. familial aggregation and juvenile cases are not common. case confi rmation can be challenging because many patients labeled as having behçet's disease have oral ulcers as the primary or sole manifestation. aphthous oral ulcers are usually the fi rst and most persistent clinical feature of bd. lesions occur in crops and some patients may have them during most of the course of the disease. aphthae occur as ulcers that are to mm or larger. these are discrete, painful, round or oval red-rimmed lesions that affect mainly the nonkeratinized mucosa of the cheeks, the border of the tongue, the soft palate, and the pharynx ( figure e- ) . oral ulcers are identical to the lesions of recurrent aphthous stomatitis. the severity and behavior of the oral ulcers in bd often fi t the description of complex aphthosis, in which multiple, recurrent, or persisting lesions result in a severe syndrome that may include perianal or genital ulceration. genital ulcers resemble oral aphthae but occur less frequently. they occur as single or multiple lesions of the vulva and in the vagina, or on the scrotum or penile shaft ( figure e- ) . genital lesions are usually painful and may result in scarring, but vaginal ulcers may be asymptomatic or only produce a discharge. perianal ulcers may occur. skin lesions are common in bd. the international study group (isg) criteria for the diagnosis of bd (table e- bd. a neutrophilic infi ltrate is typical of other dermatoses occasionally seen with the disorder, including pyoderma gangrenosum and sweet's syndrome. pathergy is an excessive skin response to trauma, refl ecting neutrophil hyperreactivity, and is highly specifi c for bd. pathergy is suggested if there is a history of red papules, pustules, or sterile abscesses after therapeutic injections, at intravenous catheter sites, or after minor skin trauma. testing for pathergy can be done with a sterile -gauge needle, used to penetrate the cleansed skin perpendicularly to a depth of approximately one quarter of an inch, rotated briefl y on its axis, and then removed. after hours, the appearance of an erythematous papule or pustule at the puncture site constitutes a positive test. the volar forearm is usually chosen for the test and sensitivity is greater when three needle punctures are made. the positivity of the test may vary during the course of the disease and is more likely to be positive at times of active disease. the sensitivity of the test is lower in western countries than in silk road countries, but a positive test adds great support for the diagnosis of bd. ocular infl ammation typically follows mucocutaneous symptoms by a few years, but it often progresses with a chronic, relapsing course affecting both eyes. the ocular fi nding in behçet's original patients was anterior uveitis associated with a hypopyon [the accumulation of an infl ammatory cell infi ltrate-essentially pus-in the anterior chamber ( figure e- ) ]. anterior uveitis that is not treated promptly with a mydriatic agent may result in synechiae formation between the iris and the lens and permanent papillary distortion. the ocular manifestations of behçet's disease may also include a panuveitis, however, with posterior chamber involvement and retinal vasculitis, the complications of which may extend to visual loss. retinal vasculitis, which leads to episodes of retinal occlusion and areas of ischemia, may be followed by neovascularization, vitreous hemorrhage and contraction, glaucoma, and retinal detachment. the earliest fi ndings of retinal vasculitis may be detected with fl uorescein angiography. isolated optic disk edema in bd suggests cerebral venous thrombosis rather than ocular disease, but papillitis may occur with ocular infl ammation and central nervous system disease. cranial nerve palsies may result from brain stem lesions, and visual fi eld defects may also be caused by intracranial disease involving the optic pathways. with cerebral venous thrombosis, patients usually present with symptoms of intracranial pressure: headache, visual obscurations, and papilledema. magnetic resonance imaging may be used to demonstrate acute or recent clot in the larger dural sinuses, but magnetic resonance venography is more reliable for recognizing clot in the cerebral venous system, especially in the smaller veins and older thromboses. central nervous system symptoms in bd may be due to aseptic meningitis or parenchymal lesions, resulting in focal or diffuse brain dysfunction. an increased protein concentration and lymphocytic pleocytosis in the cerebrospinal fl uid (csf) is supportive of the diagnosis. the clinical combination of stroke, aseptic meningitis with csf pleocytosis, and mucocutaneous lesions can be diagnostic of bd. focal or multifocal nervous system involvement refl ects the predilection of the disease for diencephalon, midbrain, and brainstem ( figure e- ) . in contrast to multiple sclerosis, there is hypopyon in the anterior chamber of a patient with behçet's disease, caused by anterior uveitis. brainstem involvement in a patient with behçet's disease. no preference for the periventricular structures ( ) . isolated headaches in bd are common but the cause is not well understood. these may represent secondary migraine or may not be related to the disease. large vessel involvement, which occurs in about one fourth of patients with bd, is a major cause of morbidity and mortality ( ) . patients with vascular disease often have multiple lesions and involvement of both the arterial and venous systems ( ) . deep venous thrombosis (dvt) is the most common large vascular lesion. patients with recurrent dvts are at risk for chronic stasis changes in the legs. occlusions of the vena cava, hepatic, and portal veins, other recognized thrombotic complications in bd, are associated with an increased risk of mortality. chest wall, abdominal, and esophageal varices may occur from deep-seated venous thrombosis. right ventricular thrombi have been reported, usually in association with pulmonary vasculitis. no primary abnormality of the coagulation, anticoagulation, or fi brinolytic system explaining the thrombotic tendency in bd has been identifi ed consistently. arterial complications occur in up to % of patients with bd ( ). stenoses, occlusions, and aneurysms occur in the systemic circulation or the pulmonary arterial bed. arterial aneurysms, caused by vasculitis of the vasa vasorum, involve the aorta or its branches. the risk of rupture is high. pulmonary artery aneurysms ( ) may lead to fi stulae between the pulmonary artery and bronchi, presenting with hemoptysis. anticoagulant treatment for presumed pulmonary emboli can result in massive hemorrhage and death. clinically apparent cardiac vascular involvement is unusual, but may result in myocardial infarction. gastrointestinal symptoms in bd include melena and abdominal pain. colonoscopic lesions appear as single or multiple ulcerations involving primarily the distal ileum and cecum. gastrointestinal lesions have a tendency to perforate or to bleed. the lesions in bd should be differentiated from those of crohn's disease and those due to the use of nonsteroidal antiinfl ammatory drugs. an intermittent, symmetric oligoarthritis of the knees, ankles, hands, or wrists affects one half of the patients with bd; arthralgia is also common. an erosive or destructive arthropathy is unusual. infl ammatory cells of the synovium and synovial fl uid are primarily polymorphonuclear leukocytes. epididymitis occurs in about % of affected patients. glomerulonephritis and peripheral neuropathy occur much less frequently in bd than in other forms of systemic vasculitis. aa-type amyloidosis, presenting as nephrotic syndrome, can accompany bd. the occasional association of the disorder with ankylosing spondylitis [in human leukocyte antigen (hla)-b -positive patients] or relapsing polychondritis (magic syn-drome [mouth and genital ulcers with infl amed cartilage]) likely represents the simultaneous occurrence of two disorders. no laboratory abnormality is diagnostic of bd. acute-phase reactants may be increased, especially in patients with large vessel vasculitis, but they may be normal in other patients, even those with active eye disease. the histocompatibility antigen hla-b is associated with bd in areas of high prevalence and in patients with ocular disease. the multiple manifestations of bd in the same patient may be separated in time, occasionally by several years. for defi nitive diagnosis, the manifestations must be documented or witnessed by a physician. the isg criteria for the classifi cation of bd (table e- infl ammatory bowel disease, sprue, cyclic neutropenia or other hematologic disorders, herpes simplex infection, and acquired immune defi ciency syndrome may cause similar lesions. other disorders responsible for orogenital/ocular syndromes include erythema multiforme, mucous membrane pemphigoid, and the vulvovaginal-gingival form of erosive lichen planus. the differential diagnosis can be clarifi ed with the aid of an experienced dermatologist and biopsy fi ndings. in reiter's disease, mucocutaneous lesions are nonulcerative and painless, and the uveitis is usually limited to the anterior chamber. similarities between bd and crohn's disease include gastrointestinal lesions, fever, anemia, oral ulcers, uveitis, arthritis, thrombophlebitis, and erythema nodosum. granuloma formation in intestinal lesions is not typical in bd, and in crohn's disease the iritis is typically confi ned to the anterior chamber. genital ulcerations and central nervous system disease are rare in crohn's disease. frequent ophthalmologic examinations are essential for patients with ocular disease, and periodic monitoring of the eyes is recommended for all patients. a careful history and examination, with attention to the vascular and neurologic systems, should be part of the physician's assessment. standardized forms for scoring disease activity and ocular infl ammation have been developed for use in clinical trials and the care of individual patients ( ) . aphthous lesions are treated with topical or intralesional corticosteroids. an empiric trial of dapsone or methotrexate may be appropriate in diffi cult cases. colchicine is used in the treatment of mucocutaneous manifestations and as an adjunct in the treatment of more serious manifestations ( . mg three times daily may be required to achieve a therapeutic effect; many patients suffer gastrointestinal intolerance of the drug at that dose). the effectiveness of colchicine has been demonstrated for genital ulcers and erythema nodosum in females and for arthritis in both sexes ( ) . thalidomide has been used for the treatment of mucosal and follicular lesions, but toxicity is a major concern. short courses of prednisone are useful in the management of mucocutaneous disease in some patients. in others, low-dose prednisone as maintenance therapy is required. cyclosporine can be effective for the control of uveitis. a controlled study has demonstrated the value of azathioprine at a dose of . mg/kg per day in limiting the progression of ocular disease and preventing new eye disease in males. combination treatment with cyclosporine and azathioprine can been used when singleagent treatment has failed. azathioprine can have a benefi cial effect on mucosal ulcers, arthritis, deep venous thrombosis, and long-term prognosis ( ) . because young males are at the greatest risk for severe disease, especially uveitis, aggressive treatment is warranted in this disease subset. in open trials, interferon-alpha has been found useful for treating mucocutaneous lesions and arthritis and is emerging as an effective treatment for ocular disease ( ) . etanercept, a tumor necrosis factor inhibitor, was shown to be benefi cial for mucocutaneous manifestations in a controlled study ( ) . reports of uncontrolled experience with infl iximab for eye infl ammation have been positive, but controlled data are lacking. immunosuppression with chlorambucil or cyclophosphamide is used for uncontrolled ocular disease, central nervous system disease, and large vessel vasculitis, including recurrent deep venous thrombosis. glucocorticoids are useful in suppression of infl ammation in acute phases of the disease, but these agents are insuffi cient by themselves to treat such severe disease manifestations as posterior uveitis or parenchymal brain disease. because of the high risk of rupture, surgical treatment is indicated for systemic arterial aneurysms. glucocorticoids and alkylating agents should also be used to minimize the high risk of anastomotic recurrences or continued disease. pulmonary arterial aneurysms may respond to these same medications, but uncontrolled bleeding requires percutaneous embolization or surgical treatment. cerebral venous thrombosis is treated with anticoagulation and corticosteroids. the treatment of budd-chiari syndrome has included anticoagulants or antiaggregants, colchicine, and glucocorticoids. portocaval shunting should be considered if the inferior vena cava is patent. in many geographic areas, genetic studies have shown a strong association with hla-b , but the exact role of this gene in the development of bd is uncertain. neutrophilic hyperfunction is recognized in bd, in normal subjects with hla-b , and in hla-b transgenic mice ( ) . evidence also exists for antigen-driven immune mechanisms in the pathogenesis of bd. cytokine analysis and cellular characterization suggest a thelper cell (th ) response by lymphocytes in bd. molecular techniques have identifi ed herpes simplex viral rna and dna in cells from patients with bd, and streptococcal antigens have been proposed as triggers of active disease. activated gamma-delta t cells are increased in the circulation and in mucosal lesions, but the precise role of these cells in the pathogenesis of bd is uncertain. peptides from mycobacterial heat shock protein (hsp) and homologous human peptides have been found to stimulate gd+ t cells from patients with bd in a specifi c fashion ( ) . cross-reactivity and molecular mimicry between peptides from streptococcal or viral hsp, homologous human hsp, and mucosal antigens may result in selection of autoreactive t cells ( ) . more recently, similarities between bd and infl ammatory disorders associated with autoimmunity have been recognized ( ) . primary angiitis of the central nervous system (pacns) is a rare form of vasculitis limited to the brain and spinal cord. the term granulomatosis angiitis of the central nervous system was previously applied because of the histopathologic fi ndings observed in arteries from early reported cases. however, an analysis of a larger number of cases supports varied mononuclear cell infi ltrates, with fewer than % of cases showing granulomatous infl ammation ( ) . anatomically, the angiitis is multifocal and segmental in distribution and involves the small leptomeningeal and intracerebral arteries. in general, the arteries are involved much more frequently than the veins. the disease predominantly affects males. most patients are young or middle-aged, although patients of a broad age range are affected. cases of pacns in children have also been described. the clinical manifestations of pacns are not distinctive. the most common symptom is headache. because virtually every anatomic area of the central nervous system may be affected by the vasculitis, a wide range of neurologic presentations and defi cits may be seen, including transient ischemic attacks (tias), cerebral infarction, paraparesis, quadriparesis, hemiparesis, ataxia, seizures, aphasia, and visual fi eld defects, among others. decreased cognitive function or fl uctuating levels of consciousness are not uncommon. progressive multifocal symptoms over time in a younger patient should suggest the possibility of pacns, particularly in the absence of other risk factors. the spinal cord may occasionally be involved. presentations with subarachnoid or intracerebral hemorrhage are rare. timely diagnosis of pacns is critical, before the occurrence of massive brain damage. preliminary diagnostic criteria for pacns have been proposed ( ) but never validated. the diagnosis of pacns is usually predicated upon either biopsy evidence of vasculitis or angiographic fi ndings suggestive of vasculitis in the setting of other compelling features, for example, strokes demonstrated by magnetic resonance imaging or the fi ndings of a cerebrospinal fl uid pleocytosis ( ) . histologic confi rmation remains the most specifi c diagnostic procedure for pacns, but the sensitivity of brain biopsy is limited because of the focal segmental distribution of the disease. a negative biopsy does not exclude the diagnosis of pacns, but may be essential to excluding other disorders that mimic pacns clinically. in the absence of histologic confi rmation, a cerebral angiogram typical of vasculitis in the appropriate clinical settings is frequently used to establish the diagnosis of pacns. suggestive angiographic fi ndings include segmental narrowing, dilatation, or occlusion affecting multiple cerebral arteries in the absence of proximal atherosclerotic changes ( figure e- ) . the fi ndings of narrowing are, however, highly nonspecifi c, and can be caused by a host of nonvasculitic causes. angiographic fi ndings compatible with vasculitis are commonly encountered in conditions such as vasospasm, central nervous system infection, cerebral arterial emboli, intravascular lymphomatosis, and atherosclerosis. furthermore, the sensitivity of angiography is limited if small vessels beyond its resolution are primarily involved. cerebral angiography has been normal in some biopsy-proven cases. general laboratory tests, including acute-phase reactants such as c-reactive protein and the erythrocyte sedimentation rate, are not useful in the diagnosis of pacns. in addition to being nonspecifi c, in fact, acutephase reactants are known often to be normal even in the setting of biopsy-proven active disease. cerebrospinal fl uid (csf) analysis, however, is an essential part of the diagnostic workup of pacns. the csf fi ndings are abnormal in % to % of cases documented pathologically. csf fi ndings are characterized by a modest pleocytosis and elevated protein levels. csf analysis should include appropriate stains, cultures, and serologic tests to exclude for cns infections. magnetic resonance imaging (mri) is the most sensitive imaging study in the evaluation of pacns. only rare cases have no mri abnormalities. the most common fi ndings are multiple, bilateral, supratentorial infarcts distributed in the cortex, deep white matter, and/or leptomeninges, but the fi ndings lack specifi city. magnetic resonance angiography (mra) is limited in sensitivity in most cases of pacns. angiographically demonstrable lesions are often beyond the resolution of current mra technology. thus, a normal mra does not rule out the disorder. primary angiitis of the central nervous system is considered a progressive disorder with a fatal course unless treated vigorously with a combination of high-dose glucocorticoids and a cytotoxic agent (usually cyclophos- cerebral angiogram in a patient with central nervous system vasculitis. the angiogram reveals multiple segmental stenoses of the a and a segments of the anterior cerebral artery and the distal segments of the middle cerebral artery. phamide). there are no controlled treatment trials on which to base this standard. the optimal duration of therapy is unknown, but in view of the substantial side effects associated with cyclophosphamide and the successful use of shorter courses of therapy in other forms of vasculitis, a -month course of cyclophosphamide followed by an additional year of azathioprine appears reasonable. prednisone should be discontinued in a tapering fashion over to months. the presence of a subset of patients with some features suggesting pacns but demonstrating a more benign course has been suggested ( ) . this subset of patients is considered to have a disease entity known as benign angiopathy of the central nervous system (bacns). bacns patients are predominantly female, primarily present acutely with headache, with or without focal symptoms, and have normal or near normal csf analysis. the diagnosis in these cases has been established angiographically and appeared to have a monophasic course with a favorable neurologic outcome. most of these patients recover after only short-term glucocorticoid treatment, often supplemented by a calcium channel blocker to mitigate against vasospasm. cytotoxic agents are not required for patients with bacns. the etiology of the vascular disease in this benign form has not been defi ned clearly, but could be the result of arteritis or reversible vasospasm. the existence of a benign, angiographically defi ned subset, however, remains controversial ( ) . recently, we identifi ed a subset of patients presenting with evidence of prominent leptomeningeal enhancement on mri ( ) . these patients were characterized by normal cerebral angiography, brain biopsy evidence of vasculitis predominantly affecting the small leptomeningeal vessels, and a good response to corticosteroids and/or immunosuppressive therapy with a favorable neurologic course. cogan's syndrome refers to the association of nonsyphilitic interstitial keratitis ( figure e- ) and immunemediated inner ear disease, resulting in audiovestibular dysfunction. the disorder affects men and women equally at any age, but typically in their third and fourth decade. presenting manifestations include sudden hearing loss, ménière's-like vertigo and tinnitus, and ocular infl ammation, alone or in any combination ( ) . other features of the disease, if not present initially, usually follow within several months. hearing loss is bilateral, shows a downsloping pattern on audiograms, and is often progressive and profound. vestibular testing shows bilateral cochlear dysfunction, helping to distinguish this disorder from ménière's syndrome. in comparison to ménière's syndrome, the damage to hearing from cogan's syndrome is typically more unremitting. scleritis, uveitis, or other infl ammatory conditions of the eye ( figure e - ) may be present initially but often patients will subsequently develop interstitial keratitis if not present initially. systemic manifestations include headache, fever, arthralgia, and vasculitis, with or without aortitis. the critical evaluation and monitoring of cogan's patients requires the expertise and collaboration of the treating rheumatologist, otolaryngologist, and ophthalmologist ( ) . there are no controlled studies on the treatment of cogan's syndrome. glucocorticoids are used topically for anterior eye disease and systemically for audiovestibular manifestations, unremitting ocular disease, or when the disorder is complicated by vasculitis or significant systemic manifestations. these agents should be started as soon as the disorder is recognized, in adequate doses (at least mg/kg/day), and for a suffi cient duration to initially control the disease or for relapse. documented improvement in to weeks supports a therapeutic response and can be followed by gradual tapering of the dose and use of immunosuppressive agents if necessary for maintenance. the prognosis for hearing in these patients has been poor ( ) , but cochlear implants are used successfully in these patients with bilateral deafness. erythema elevatum diutinum (eed) is an extremely rare, chronic, recurrent vasculitis with distinctive clinical and histopathologic features ( , ). the disorder affects both men and women in middle age. individual lesions consist of purple, red, or brown plaques that often have an annular or nodular appearance. the skin lesions of eed have a predilection for the extensor surfaces of the distal extremities and often overlie joints, but may be generalized. older lesions may be dense and coalesce ( figure e- ) . erupting lesions may be associated with stinging, burning, or tenderness, and may be accompanied by systemic symptoms. in early lesions, the pathology of eed is one of a leukocytoclastic vasculitis with a perivascular neutrophilic infi ltrate. more mature lesions demonstrate perivascular or onion-skinlike fi brosis. capillary proliferation and cholesterolcontaining histiocytes may also be seen. the differential diagnosis includes other neutrophilic dermatoses, primarily sweet's syndrome. erythema elevatum diutinum has been recognized to occur in association with infections diseases, including human immunodecifi ency virus infection, hematologic disorders (particularly iga gammopathies), and several immune-mediated infl ammatory diseases, including rheumatoid arthritis. treatment of any associated disorder may benefi t eed. dapsone ( mg/day) has been reported to be successful in some patients. kawasaki's disease (kd), once known as mucocutaneous lymph node syndrome, is a systemic infl ammatory disorder occurring in children that is accompanied by vasculitis and a risk of coronary artery aneurysms. other typical features of kd include spiking fevers, cervical lymphadenopathy, conjunctivitis, erythematous changes on the lips and in the oral cavity, dryness and cracking of the lips, a strawberry appearance to the tongue, and a polymorphous rash. eighty percent of kd cases occur in children less than years of age. attempts to link kd defi nitively to some types of infection, particularly ones associated with superantigens, have thus far been unsuccessful. high dose aspirin and intravenous immune globulin (ivig) are the cornerstones of therapy in kd. ivig is essential to the prevention of coronary aneurysms. years after kd has occurred during childhood years, some cases of myocardial infarction caused by thrombosis of coronary aneurysms have been reported. kawasaki's disease (kd) is a form of systemic vasculitis that occurs in young children and may be associated with the development of coronary arteritis and aneurysm formation ( figure f- ) . kd is the leading cause of acquired heart disease of children in the united states. this illness, fi rst recognized to be a new entity by tomasaku kawasaki in japan in ( , ), was termed for mucocutaneous lymph node syndrome (mclns) until kd became the accepted designation for this disorder ( ). although the disorder was named after kawasaki, at least one previous case exists in the medical literature ( ) . this case, recounted in detail below, is classic in its clinical features of kd. a -year-old girl presented with a sore throat, a fever to °f, and an erythematous rash over her trunk, appearing "desperately and acutely ill" ( ). oropharyngeal lesions included an aphthous stomatitis, erythematous lesions of the hard palate, and prominent lingual papillae. on the fi fth hospital day the hectic fevers ceased, but low-grade fevers and tachycardia to beats per minute persisted. the skin of her fi ngers desquamated, but over the ensuing weeks she improved steadily. one month after admission, however, she developed acute chest pain, shortness of breath, and expired. a postmortem examination revealed blood and clots in the pericardial space, and several large aneurysms along the epicardial vessels. one aneurysm, the size of a large ripe cherry in the left coronary artery, was the site of hemorrhage into the pericardium. although the microscopic appearance of the disease was typical of periarteritis nodosa (i.e., pan; see chapter b), no hepatic or renal infarctions were present. indeed, among the internal organs the heart alone was involved. the child's death was attributed to an atypical case of "infantile periarteritis nodosa," now recognized as kd. kawasaki's disease strikes quickly, runs a furious course over a few weeks, and then apparently resolves. in all of the patients described initially by kawasaki, the symptoms resolved without sequelae within month. in subsequent years, however, mortality from cardiac complications (usually coronary artery thrombosis) was reported ( , ) . cardiac complications of kd result from a severe panvasculitis, leading to narrowing of the coronary lumina by the migration of myointimal cells from the media through the fragmented internal elastic lamina. although catastrophic heart complications occur in only a small minority of patients (< %), the preponderance of patients with kd appear to have at least some cardiac involvement. heart lesions may include myocarditis, pericarditis, aneurysmal dilatation and thrombosis of the coronary arteries ( figure f- ) , and myocardial infarction. the tropism of the vascular infl ammation for coronary arteries and its unusual propensity to cause aneurysm formation remain unexplained. in addition to the cardiac fi ndings, kd is associated with a number of other dramatic clinical fi ndings (table f- ) . spiking fevers may last for days or more. the conjunctivae, generally infl amed in a nonpurulent manner, are accompanied by erythematous changes on the lips and in the oral cavity [ figure f- (a) ]. the lips become dry and cracked [ figure f- (b) ], with a diffuse reddening of the oropharyngeal area and a strawberry appearance to the tongue ( figure f- ) . a polymorphous rash typically involves the trunk [ figure f - (a)], and there may be extensive lymphadenopathy in the neck region. the palms and soles become erythematous and indurated, followed by desquamation in the skin of these areas during the healing phase ( - ). the term atypical kd has been used to describe both older children and young infants presenting outside the typical age range of to years, as well as those presenting with features other than the classical criteria. incom- coronary and peripheral aneurysms in kawasaki's disease (kd). magnetic resonance angiogram in an infant with kd, revealing irregularities of the subclavian, axillary, and proximal brachial arteries, as well as fusiform dilatation of the right common iliac and right proximal internal iliac arteries. there is also a focal aneurysm of the left internal iliac artery. plete kd has been applied to any patient felt to have kd but who did not fulfi ll classical criteria. these are often diagnosed by echocardiogram fi ndings of coronary aneurysms and often occur in the older children or young infants ( , ) . coronary aneurysms, in fact, are most likely to occur in infants < months of age. because the epidemiology of kd is consistent with an infectious cause: clinical features that resemble infection (fever, lymphadenopathy), time/space clusters, epidemic occurrences, and alleged proximity of case foci to bodies of water. to date, however, no infectious etiology has been proven. there has been no culture or serologic evidence for conventional viral agents, mycoplasmae, rickettsiae, or bacterial agents (streptococcus, staphylococcus). molecular biologic techniques have provided support, however, for a propionibacterium acnes variant, retroviruses, rickettsiae, parvovirus b , epstein-barr virus, and coronavirus, as well as for the participation of the s. aureus toxin tsst- and other superantigens (e.g., yersinia pseudotuberulosis). support exists for a superantigen-mediated process both from clinical studies ( ) ( ) ( ) ( ) ( ) and from a murine model for coronary arteritis stimulated by lactobacillus casei cell wall extracts ( ) . this hypothesis proposes that the etiologic agents-which may differ across geographic sites throughout the world-are capable of evoking immunologic responses via t-cell receptor v beta restriction. an oligoclonal response is supported by the discovery of iga-secreting plasma cells within gia and arthritis, aseptic meningitis, diarrhea, abdominal pain, pericardial effusion, obstructive jaundice, and hydrops of the gallbladder. intravenous immune globulin (ivig), a critical medication in the treatment of kd, is a limited resource in many parts of the world because of its expense. the american heart association (aha), concerned about both the potential for overuse of ivig as well as the failure to employ this medication in a timely manner in appropriate patients, issued guidelines on the diagnosis and treatment of kd (tables f- through f- ) ( , ) . in these guidelines, the epidemiologic case defi nition of kd included fever of at least days and four or more principal criteria (table f - ) without other explanation; or fever and less than four principal criteria if coronary artery abnormalities are detected by echocardiogram or coronary angiography. in japan, the illness appears in late winter and spring. the peak age is to months, with % of cases occurring in patients younger than years of age. the male: female ratio is . : . except for three major pandemics ( , , / ) , the cases have reached a plateau of to per year. the endemic annual incidence is / , children < years old, with a recurrence rate of %. in the united states, there is also a seasonal variation in most places. the peak age is to months, and the illness accounts for hospitalizations/year. the recurrence rate is % to %. data from hawaii from - show ethnic incidence rate/ , children < years old per year of . in japanese, . in chinese, . in hawaiians, . in filipinos, . in caucasians. in los angeles from - , rates per , children the walls of the affected arteries. this fi nding lends credence to the hypothesis that the respiratory or gastrointestinal tract may be the portal of entry for the inciting organism, and that the process is antigen-driven ( , ) . the pathogenesis is characterized by immune activation. a host of immunologic irregularities have been described in kd, not all of which have been confi rmed consistently: endothelial cell activation [particularly human leukocyte antigen (hla)-dr expression on coronary endothelial cells]; autoantibody formation (e.g., anti-endothelial cell antibodies); complement activation and immune complex formation; abnormalities of immunoregulation (lymphocyte infi ltration, activated cd + and b cells, activated monocyte/macrophages, t lymphopenia, polyclonal b-cell activation); adhesion molecule upregulation (soluble p-, e-, and l-selectins); increased vascular endothelial growth factor; and marked cytokine production with high levels of interferon-gamma, interleukins- , - , - , and - , and tumor necrosis factor (tnf)-alpha ( , ( ) ( ) ( ) . in severe cases, this "cytokine storm" results in a macrophage activation syndrome (mas). following the initial recognition of kd, this illness was treated with salicylates, using the same doses of aspirin employed in the treatment of rheumatic fever. because of the potential for impedance of aspirin absorption caused by vasculitic involvement of the gastrointestinal tract, however, the use of aspirin must be monitored carefully in this setting. if aspirin doses are too high (e.g., - mg/kg/day), improvement of intestinal absorption with therapy may lead to symptoms of toxicity. in japan, doses of to mg/kg/day have been employed because of the high incidence of the slowacetylator gene in the japanese population. a combined us and japanese multicenter study demonstrated that to mg/kg of aspirin plus ivig (see below) was effective at preventing aneurysm formation in most cases ( ) . current aha guidelines, however, endorse aspirin doses of to mg/kg/day, in four divided doses (table f- ) . furusho studied the use of aspirin alone versus the combination of aspirin plus ivig ( . mg/kg/day × days), using a protocol then in use for immune thrombocytopenic purpura ( ) . a multicenter study demonstrated a decrease in the incidence of coronary artery abnormalities: only % ( of ) in the ivig group, compared with % ( of ) in the aspirin-only arm ( ) . no patients in the ivig arm developed giant coronary artery aneurysms. in contrast, % of the aspirinonly group suffered this occurrence. this study established ivig as the standard of care. several years later, a follow-up trial compared a single dose of ivig ( g/kg) to the traditional . mg/kg/day × schedule, confi rming the superiority (a further lowering of the coronary aneurysm rate) of the single-dose regimen ( ) . thereafter, the single-dose regimen became the standard of care recommendation by the aha (table f - ) ( , ). the use of glucocorticoids in kd is, surprisingly, controversial. one retrospective study assessed the outcomes of fi ve different treatment regimens, including aspirin alone, aspirin plus prednisolone, prednisolone alone, prednisolone plus warfarin, and no treatment aside from background antibiotic therapy (which all other treatment groups received, as well). although aspirin alone reduced the aneurysm rate from % to % compared with the no-treatment group, treatment with prednisolone was associated with an increase in the percentages of patients who developed aneurysm to % ( ) . of note, the seven patients treated with aspirin plus prednisolone-none of whom developed aneurysm-were not emphasized in the discussion. in addition, the patients in the prednisolone-only group were perhaps the most ill at baseline (and hence were treated with glucocorticoids, presumed empirically to be the most powerful therapy). after the publication of this study's results, glucocorticoids for the treatment of kd fell into disfavor among pediatricians and in fact were viewed as contraindicated for this disease. more recent case series, evaluating the use of pulse methylprednisolone as rescue therapy for ivig nonresponders, have been more encouraging with regard to the potential for a benefi cial effect of glucocorticoids ( ) ( ) ( ) . initial results from a multicenter trial ( ) indicate no worsening in the coronary aneurysm rate among patients treated with glucocorticoids, and a decrease in fever, infl ammatory markers, length of hospital stay, and ivig side effects. a consensus conference at the national institutes of health ( ) was prompted by the recognition of an ongoing immune activation at microvascular levels in patients treated adequately by the current therapies. outcome data from japan with long-term ( - year) follow-up demonstrated persistence of disease in some cases, with intravascular ultrasound and ultrafast computed tomography studies demonstrating lingering coronary aneurysms and/or wall fi brosis. of greatest alarm was the fi nding of such abnormalities in areas of the vasculature previously documented as normal by echocardiogram and even coronary angiography. electron microscopy studies of endomyocardial biopsies up to years after the kd episode showed ongoing microaneurysms and small vessel coagulopathy. in a small number of young adults who have experienced myocardial infarctions in the absence of known cardiac risk factors, angiograms have revealed giant coronary artery aneurysms compatible with old kd. the extent of active kd in such patients, if any, as opposed to the clinical sequelae occurring in arteries damaged years before, is not clear. newer treatment modalities have been utilized in selected patients and patient populations. small studies and anecdotal reports of treatment with the antiglycoprotein iib/iiia monoclonal antibody (abciximab) or with low-molecular-weight heparin have suggested more rapid regression of aneurysms and perhaps endothelial cell remodeling. noninvasive imaging modalities, such as magnetic resonance imaging studies of the chest and abdomen, have identifi ed the extracardiac arterial aneurysms and dilatation ( figure f- ) . the knowledge of the more widespread nature of the vasculitic involvement has prompted more aggressive and combination therapies ( ) . pentoxifylline, a phosphodiesterase inhibitor, has antiplatelet activity, vasodilatory effects, effects on red blood cell rheology, and the ability to inhibit tnf synthesis. a regimen of mg/kg/day of pentoxifylline in three divided doses demonstrated an improvement in clinical features and the rate of aneurysm formation in kd ( ) . further pharmacokinetic studies of a commercial liquid preparation of pentoxifylline demonstrated safety and a reduction in tnf levels in kd patients of % with doses up to mg/kg/day ( ) . anecdotal reports indicate tolerability of doses of to mg/kg/day in infants with kd. a multicenter trial of infl 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- journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: wetqqt i disease ecology is an interdisciplinary field that has recently rapidly grown in size and influence. we described the composition and educational experiences of disease ecology practitioners and identified changes in research foci. we combined a global survey with a literature synthesis involving machine-learning topic detection. disease ecology practitioners have diversified in the last decade in terms of gender identity and institution, with weaker diversification in terms of race and ethnicity. topic detection analysis of over , research articles revealed research foci that have declined (e.g., hiv), increased (e.g., infectious disease in bats), and have remained common (e.g., malaria ecology, influenza). the steady increase in topics such as climate change, and emerging infectious diseases, superspreaders indicate that disease ecology as a field of research will continue advancing our understanding of complex host-pathogen interactions and forms a critical and adaptable component of the global response to emergent health and environmental threats. among these is the urgency to understand and address novel disease threats, which are rooted in natural systems but are often exacerbated by societal inequalities (carlson & mendenhall ) . for example, the impacts of habitat degradation on pathogen spillover is an expanding area of research that can be used to guide risk assessments and environmental policy (patz et al. ). at the same time, infrastructure has developed around disease ecology, such as a specialized national science foundation and national institutes of health funding program and conference series (scheiner & rosenthal ) , which have helped to direct research effort and create networks amongst researchers. still, many questions remain as to the composition of disease ecology practitioners, core research foci, and how research trends have changed to meet societal needs. answering these questions will help to improve training pathways and prioritize research emphases for future research. however, understanding these complex and interrelated factors as they apply to an interdisciplinary research topic requires diverse and innovative approaches. here we characterize both the practitioners and field of disease ecology by addressing the following questions: ( ) who comprises the field in terms of education, demographics, and research foci? ( ) which are the most influential scientific articles and journals? ( ) and significantly, how have research trends emerged and changed over time? for example, do they follow global health priorities such as disease outbreaks? to answer these questions, we surveyed self-declared disease ecologists globally and conducted a literature synthesis with machine-learning topic detection ( had to meet specific criteria using boolean filters, including a focus on studying a pathogen or parasite, host infections (to distinguish from solely environmental persistence of microorganisms), and individual-level or higher-order dynamics (e.g. not cellular processes, with the exception of those analyzed as a population-level process). the full list of search terms is provided in the supporting information, alongside a set of exclusionary terms to remove similar but non-disease ecology articles. web of science categories were used to narrow our search and also reduce false-positive inclusions. finally, articles with fewer than four citations were removed as a form of quality control. to evaluate false-positives, two authors (djb and kmf) independently evaluated the same randomly selected articles and classified them as 'disease ecology' or 'outside the field'. papers that fell outside the field predominantly described bacterial communities, within-host behavior or adaptations, or genetics/genomics. over % of the articles in the final corpus were classified as disease ecology, and consensus was strong among evaluators ( % agreement, cohen's κ= . ). within-host studies were accepted if they focused on population-level processes (e.g., to evaluate false-negatives, we cross-validated our corpus using our survey data. specifically, we assessed whether articles that were identified by at least two respondents as influential were present in our corpus. we calculated the proportion of papers that were included in our corpus out of the list of such articles, with the requirement that at least % of papers had to be included. of the influential articles identified by survey respondents (written ≥ times) restricted to journals used in building the corpus, approximately % ( / ) were present in the corpus. yet the 'most influential' articles had a higher probability of being included: the corpus included % of articles written four or more times, % of articles written three times, and % of articles written twice. we adjusted the search and exclusion terms twice using the workflow described in figure small or too large, we were unable to detect temporal variation in that topic. if j was too small or too large, the topics were not clearly defined. for example, a topic with only five words may not be interpretable; similarly, a topic with words may be too broad to assign meaning. we used i = and j = , so our corpus was analyzed for topics with words each. we used k-means clustering from the nltk python library to construct topics, where each topic comprised commonly co-occuring words. we assigned a name to each topic to describe its theme. for example, we named a topic containing immunodeficiency, hiv, patient, therapy, drug, aids, background, treatment, and risk, as an hiv topic. we gave each topic name a 'confidence' measurement of - , from high to low confidence in identifying the topic. in addition to topics that emerged from the literature, we also generated and assessed our own topic lists based on key research areas, such as climate change, dilution effect, superspreaders, network analysis, eids, bovine tuberculosis, infectious diseases in bats and rodents, and chytrid fungus ( fig. ) . to ensure topic trends were not confounded by an increase in the total number of published articles through time, we constructed a baseline topic using neutral words that should be in all disease ecology articles: analysis, study, and paper. we evaluated temporal trends in publications for each theme using generalized additive models (gams) fit using the mgcv package in r (wood ) . the proportion of words in each topic relative to all words was modeled as a binomial response using thin plate splines with shrinkage for publication year. lastly, to assess covariation among topics, we estimated spearman's rank correlation coefficients (ρ) at the zero-year lag. survey a total of self-declared disease ecologists participated in the survey. the average respondent was . years old ( - , median: ; n= ). . % of participants (n= ) considered at least half of their research to fall within disease ecology. participants that considered ≥ % of their research to be disease ecology were concentrated from ages - , and most self-identified as women ( %) (n= ). more broadly, . % of participants identified as women (n= ), . % as men (n= ), . % as other (n= ), and . % preferred not to say (n= ). we report on participants that chose to disclose a gender identity for results regarding gender. most respondents identifying as women were younger (age ≤ - ) than most respondents identifying as men (age - ). the youngest age category (≤ years) was . % women (n= ), and the oldest age category ( + years) was . % men (n= ). current positions held by survey participants were: undergraduate student ( . %, n= ), master's student ( . %; n= ), phd student ( . %; n= ), post-doctoral researcher ( . %; n= ), faculty ( . %; n= ), researcher ( . %; n= ), and other ( . %; n= ). respondents identifying as women comprised most of each academic position except master's student and faculty (table s ). in general, most phd students and post-doctoral researchers were young and identified as women. most masters' students were young and identified as men, and most faculty were middle-aged and identified as men (tables s -s , fig. s c ). participants that did not identify with a strict gender binary were distributed across age (≤ - ) and position categories. important' areas were parasitology, immunology, field/laboratory techniques, microbiology/virology, and genetics/genomics/bioinformatics. ecology was also listed as least important, suggesting that most participants considered ecology as either the most or least important area of research; however, the number of responses for the former was nearly three times greater than for the latter. survey respondents were asked to write in scientific journals and articles that they believed were the most influential in disease ecology (table ) we compiled a list of journals that at least four survey participants said were the most important in disease ecology, plus science and nature. we searched these journals for relevant articles in the field using the algorithm described below, and our final corpus many of the topics that emerged from the disease ecology literature, such as malaria, influenza, and vaccination, have remained constant over time (fig. b) . others, such as hiv and serology, have declined over time, and host-pathogen coevolution has instead steadily increased. these emergent topics comprised a notable portion of the disease ecology literature and were more prominent than author-selected topics. we constructed a neutral topic for comparison, which was constant through time (fig. b, gray line in panels) , thus validating the observed temporal changes in these topics. using key term searches, we next explored select topic trends: climate change, emerging infectious diseases (eids), the dilution effect, superspreaders, network analysis, pathogens in rodents and bats, bovine tuberculosis, and chytrid fungus in amphibians (fig. b) . as with emergent topics, our topic detection was sensitive to detecting changes in frequency over time, have remained prominent foci of disease ecology, whereas an increase in a priori selected topics such as emerging infectious diseases, climate change, and effects of biodiversity loss emphasize how this expanding field has grown to meet global health crises and priorities. further, addressing diversity and allocating resources toward these growing topics could promote equity within disease ecology, improve training programs, designate funding opportunities, and provide the infrastructure for concentrated advancements. self-declared disease ecology practitioners are becoming more diverse in terms of country of education, gender identity, and institution (fig. ) . this echoes similar trends in conservation in general, research on epidemics tended to be responsive rather than anticipatory, such that we observed an immediate spike in publications on high-profile pathogens followed by a decline or plateau (e.g., bovine tuberculosis and chytrid fungus). emergent topics were remarkably stable through time, with the exception of hiv and host-pathogen coevolution, which have respectively decreased and increased. research focusing on concepts (e.g., dilution effect, superspreaders, coevolution) or approaches (e.g., network analyses) rather than specific hosts or pathogens tended to rise more gradually and remain a notable proportion of the literature. on the other hand, mosquito-borne pathogens and influenza have been defining topics over the entire time series, which we expect to persist for the foreseeable future. although our analysis of cross-correlation between the topic time series is associative, we observed several especially interesting relationships. in particular, publications on bat disease, chytrid fungus, climate change, the dilution effect, superspreaders, and emerging infectious diseases were all positively economic burden of livestock disease and drought in northern tanzania climate change and infectious diseases: from evidence to a predictive framework the proximal origin of sars-cov- infectious diseases of humans: dynamics and control population biology of infectious diseases: part i quantifying the burden of vampire bat rabies in peruvian livestock natural language processing with python: analyzing text with the natural language toolkit probabilistic topic models women and science careers: leaky pipeline or gender filter? gender global trends in 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and management effects of species diversity on disease risk the rise of disease ecology and its implications for parasitology-a review achieving synthesis with meta-analysis by combining and comparing all available studies facilitating systematic reviews, data extraction and meta-analysis with the metagear package for r plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques the changing face of pathogen discovery and surveillance superspreading and the effect of individual variation on disease emergence nltk: the natural language toolkit biological invasions: a field synopsis, systematic review, and database of the literature population biology of infectious diseases: part ii the educational benefits of diversity: evidence from multiple sectors national science foundation. . women, minorities, and persons with disabilities in automated content analysis: addressing the big literature challenge in ecology and evolution a guide to conducting a standalone systematic literature review host and viral traits predict zoonotic spillover from mammals unhealthy landscapes: policy recommendations on land use change and infectious disease emergence ecological responses to altered flow regimes: a literature review to inform the science and management of environmental flows recognizing the benefits of diversity: when and how does diversity increase group performance? global expansion and redistribution of aedes-borne virus transmission risk with climate change amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity ecology of infectious disease: forging an alliance leaks in the pipeline: separating demographic inertia from ongoing gender differences in academia ecological interventions to prevent and manage zoonotic pathogen spillover the influence of feeding behaviour and temperature on the capacity of mosquitoes to transmit malaria interactions between hiv/aids and the environment: toward a syndemic framework press release: infectious diseases kill over million people a year: who warns of global crisis the top causes of death unesco institute for statistics (uis) higher education. heterogeneity in pathogen transmission: mechanisms and methodology quantifying the impact of human mobility on malaria wildlife disease ecology: linking theory to data and application generalized additive models: an introduction with r evaluating ecological restoration success: a review of the literature key: cord- -yn pvb authors: nan title: full issue pdf date: - - journal: jacc case rep doi: . /s - ( ) -x sha: doc_id: cord_uid: yn pvb nan t he coronavirus disease- (covid- ) pandemic has revolutionized clinical practice in recent months unlike any other health emergency in recent years. jacc: case reports has taken this challenge very seriously, dedicating to it an entire issue. we have received excellent case reports from across the world. our goal is to provide a comprehensive report of representative cardiovascular involvement in covid- (central illustration). besides the concern regarding qt interval prolongation with hydroxychloroquine and azithromycin treatment, covid- is strongly associated with the occurrence of sudden unexpected arrhythmias. given the general ignorance of the pathophysiological mechanisms of the virus at the time of submission of these case reports, it is hard to state with certainty that covid- has been the cause of these arrhythmias, but they surely offer an interesting direction for future research in the field. included are cases of brugada type i pattern positivization ( ) in the context of fever, one of the most common presenting symptoms of the disease ( ); electrical ventricular storm ( ); transient atrioventricular block in the absence of myocarditis ( ); sinus node dysfunction requiring pacemaker implantation ( ) ; and finally a provocative report on the use of amiodarone as a possible treatment for covid- ( ) . overall, the cytokine storm has proved to have a strong impact on the conduction system. the clinical presentation of patients with covid- has ranged from asymptomatic to acute respiratory distress syndrome requiring mechanical ventilation. one cause of sudden respiratory deterioration is the increased risk for venous thromboembolic disease in these patients ( ) (figure ). these events have been noted both early and in the recovery phase of covid- . furthermore, thromboembolic events have occurred despite the use of prophylactic anticoagulation or even full anticoagulation ( ) . a majority of patients have elevated levels of d-dimer and laboratory findings consistent with sepsis-induced diffuse intravascular coagulation, suggesting a coagulopathic process, yet no prospective studies have demonstrated the predictive nature of these markers for the occurrence of a thromboembolic event, only a higher risk for mortality ( ) . the early recognition and treatment of venous thromboembolic disease has therefore been a dilemma. we therefore provide commentary and suggest treatment algorithms ( ) . we received case reports in general categories detailing an increased risk or arterial thrombosis during the covid- pandemic. causing stemi in infected patients ( , ) . second, there were a number of cases of stemi in patients younger than years without risk factors for atherosclerosis ( ) . this phenomenon occasionally occurred before covid- , but the increased incidence reminds us to always include myocardial infarction in the differential diagnosis of a younger patient with chest pain and to always consider performing screening electrocardiography. third, a number of cases of stemi mimics were submitted, including patients with st-segment elevation due to myopericarditis, brugada pattern, takotsubo cardiomyopathy, or endothelial dysfunction due to infection or cytokine storm. as many as % of patients with covid- and st-segment elevation may have normal coronary arteries on angiography, complicating the decision whether to administer fibrinolytic therapy to patients without early access to primary percutaneous coronary intervention. finally, there were reports of spontaneous thrombosis ( ) involving the ascending and descending aorta, the cerebrovascular arteries, the mesenteric and renal arteries, and the peripheral arteries, underscoring an increased systemic risk for arterial thrombosis in patients with covid- . the development of heart failure in patients infected with severe acute respiratory syndrome coronavirus- has been described to involve different, and overlapping, mechanisms. one is cytokine release resulting in myocardial inflammation (figure ) , and affected patients has demonstrated both inflammatory infiltrates and viral particles. the heart failure cases presented in this special issue, however, describe more varied presentations of patients, some with pre-existing heart failure and others with no known cardiac disease prior to becoming ill with infection. in addition to cases of direct myocardial injury, some with pathological evidence, we also present cases of takotsubo cardiomyopathy ( , ) two cases highlight the special circumstances faced by patients with left ventricular assist devices ( , ) , which include the inability to tolerate prone positioning to augment respiratory support because of the mechanical equipment and the hypothesis that mechanical circulatory support may provide a type of protection against the most serious hemodynamic consequences of severe acute respiratory syndrome coronavirus- infection. included also is a case series of pediatric patients who had hemodynamic collapse and cardiac dysfunction ( ) , a presentation that has been rare in this younger age group. the now well-recognized thromboembolic disorders encountered with this disease include a case in this issue of massive pulmonary embolism and resultant severe right heart failure ( ) . and separate reports describe patients with concomitant noncardiac disease, with human immunodeficiency virus infection ( ) and with influenza ( ) . all the cases of heart failure represent the investigators' best attempts at providing supportive and emerging therapies at a time when they had no established guidelines and no best practices to follow. from the beginning of the pandemic, we believed that it was very important to publish the "voices" of our frontline colleagues across the world, to help cardiologists to get acquainted with mechanical ventilation ( ) , to describe the first evidence of sex differences in covid- ( ) , and to discuss the significant changes in health care with telemedicine and virtual clinics. the impact of covid- on african americans has also recently been described in jacc (table ) revealed leukopenia, normocytic anemia, thrombocytopenia, and significant increase in c-reactive protein levels. a nasopharyngeal swab sample tested positive for severe acute respiratory syndrome-coronavirus- (sars-cov- ) using real time-reverse transcriptionpolymerase chain reaction. the patient had a medical history of bipolar disorder and hypothyroidism. he reported having fever for week. given the patient's presentation of prolonged asystole, differential diagnosis included metabolic disorders, pharmacologic agents and extracardiac diseases with special attention to thyroid hormone levels. to recognize cardiovascular complications among covid- patients. to demonstrate arrhythmic risk related to covid- disease. to manage sick sinus dysfunction in covid- disease. during the following days, the patient had episodes of asystole associated with hypotension but without loss of cardiac output ( figure ). moreover, alternating episodes of bradycardia and tachycardia note bilateral ground glass opacities. once reversible causes were excluded, symptoms were related to dysfunction of the intrinsic sinus node, and the patient underwent dual-chamber ratemodulated implantation of a pacemaker (pm) ( figure ). because of evidence of sinus tachycardia, bisoprolol was administrated. further and later pm control showed only this episode. transthoracic echocardiography was repeated before the patient was discharged and showed no differences from the previous one. in consideration of his progressive clinical improvement, the endotracheal tube was removed, and the patient began to breath spontaneously. on psychiatric indication, therapy for bipolar disorder was reintroduced. this could be due to hypoxia and electrolyte abnormalities, which could lead to episodes of cardiac arrhythmia, or due to central nervous system alterations caused by sars-cov- disease. increasing evidence shows that covs are not always confined to the respiratory tract but may also invade the central nervous system, inducing neurological diseases ( , ) , and some covs have been shown to be able to spread to the medullary cardiorespiratory center through chemoreceptors and mechanoreceptors in the lung and lower respiratory airways through a synapse-connected route ( ) . considering that most covs share a similar viral structure and infection pathway ( ) , the infection mechanisms previously found for other covs may also be applicable to sars-cov- . furthermore, the transsynaptic transfer also has been reported for avian bronchitis virus ( ) in fact, the intrinsic cardiac nervous system has regional control over different cardiac functions, such as sinus node electrical activation and propagation, as well as atrioventricular nodal conduction, and consists of ganglia composed of afferent, efferent, and interconnecting neurons to other cardiac ganglia. these ganglia coordinate the sympathetic and parasympathetic inputs received from the rest of the cardiac autonomic nervous system. generally, autonomic dysfunction refers to a disorder of an autonomic nervous system that may arise from intrinsic or extrinsic mechanisms. intrinsic autonomic dysfunction arises from diseases that directly affect the autonomic nerves, such as diabetes mellitus and the various syndromes of primary autonomic failure. extrinsic autonomic dysfunction often is secondarily induced by cardiac or other disease ( ) . patients with autonomic dysfunction commonly have poor long-term prognosis, and death can occur from pneumonia, acute respiratory failure, sudden cardiopulmonary arrest, or fatal arrhythmias related, for example, to ssd. specifically, ssd includes a spectrum of heart rhythm disturbances related to abnormal sinus impulse formation or propagation ( ) and has different presentations, such as bradycardia, alternating episodes of bradycardia and tachycardia and sinoatrial block. in some cases, ssd presents with sinus node arrest and prolonged asystole, such as in the present patient. symptoms related to ssd are generally fatigue and syncope or presyncope, but patients can be asymptomatic in the early phase of the disease. when symptoms are related to dysfunction of sinus node, pm implantation is required. currently, data regarding the neuroinvasive potential of sars-cov- with subsequent autonomic dysfunction are less described. furthermore, to these authors' knowledge, this is the first case in medical literature of ssd related to covid- infection. an improved understanding is crucial primarily for guiding the need for additional arrhythmia monitoring during hospitalization and after discharge ( ) . the present authors believe that recognition by the scientific community of these risks related to covid- disease may be helpful for strict monitoring of affected patients and also for furthering knowledge of such complications for global public health. diagnostic coronary angiography performed through the right radial approach revealed angiographically normal coronary arteries (figures and ) . ventriculography confirmed the globally mildly reduced ejection fraction. the patient was admitted to a dedicated coronavirus disease- (covid- ) intensive care unit. the covid- results became available within h and were positive. his condition continued to improve, and he required minimal supplemental oxygen to maintain arterial saturation. all serial troponin values were negative. two days later he developed a brief episode of supraventricular tachycardia that was successfully terminated with intravenous adenosine ( figure ). four days after the initial presentation, he was doing well without fever. the c-reactive protein level had decreased to . mg/l, and the ecg demonstrated nearly complete resolution of the initial brugada-like ecg pattern ( figure ). the patient was discharged to home after the -week hospital stay. diagnosis and treatment of st-segment elevation myocardial infarction during the covid- pandemic present multiple diagnostic and logistic challenges ( ) . myocardial injury, myocarditis, acute coronary syndromes, and arrhythmias have all been described in the setting of covid- infection ( ) . st-segment elevation in the right precordial leads and brugada-like ecg patterns have previously been associated with various conditions (e.g., fever, myocarditis toxicity, metabolic disorders, certain drugs). these brugada-like patterns usually disappear once the inciting event is removed ( ) . a brugada-like ecg pattern presents an additional diagnostic and therapeutic challenge because it may be seen in patients presenting with chest pain, thus mimicking st-segment elevation. atrioventricular nodal re-entrant tachycardia, such as developed in our patient, has similarly been associated with brugada syndrome ( ) . most recently, covid- infection has been described as unmasking brugada syndrome in a patient who presented with syncope ( ). our case is important because it demonstrates the need to differentiate between the brugada syndrome and the brugada-like ecg configuration. given that our patient had a covid- -associated brugada ecg pattern with no history of syncope, observation therapy was recommended because the risk of major adverse cardiac events is low ( ) . covid- manifests mainly as a respiratory syndrome that includes pneumonia and, in the worst case scenario, acute respiratory distress syndrome ( ) . we have also learned that, in a not negligible number of cases, the virus can provoke myocardial ischemia and/or inflammation, with or without an associated respiratory syndrome ( ) . there are already numerous cases of covid- manifesting as st-segment elevation myocardial infarction that have triggered activation of primary percutaneous coronary intervention protocols. the cause of this stsegment elevation is unknown: it has been linked to traditional plaque rupture in those patients who have required coronary angioplasty, but it has been suggested that myocarditis or microvascular thrombosis could be the cause when no obvious thrombus or coronary flow interruption is detected. if all this were not sufficient, here comes brugada type i pattern, interfering with and complicating the lives of interventional cardiologists. indeed, in the case reported by vidovich ( ) , the patient presented with shortness of breath, substernal chest pain, and fever. the electrocardiogram showed a brugada type i pattern in the right precordial leads with no reciprocal changes; the presence of chest pain, shortness of breath, and reduction of systolic left ventricular function, assessed with a -dimensional echocardiogram, led to urgent coronary angiography, which excluded an ongoing acute coronary syndrome. no significant electrolyte imbalance was found. vidovich's ( ) conclusion was that the brugada type i pattern, completely unknown to the patient until this admis- a link between fever and a brugada type i pattern is very well known and has been described extensively ( ) ( ) ( ) . in fact, the international guidelines on sudden cardiac death recommend lowering body temperature as soon as possible in those patients with an established diagnosis of bs, as well as in carriers of the mutations with a proved inducible brugada type i pattern ( ) . the increase in body temperature has indeed been proven to cause a higher degree of inactivation of sodium channels, both mutated and wild ones: in the subjects who are genetically predisposed, this reduced sodium flow can result in a dangerous transmural heterogeneity that is the basis for phase re-entry ventricular arrhythmias and sudden death ( , ) . it would also be of interest understand whether the virus itself could interact directly with the myocardial ion channels and provoke the electrocardiographic modification typical of bs. the take-home message is therefore that patients with bs and concomitant covid- infection should be monitored in the intensive care unit or in the telemetry ward until the fever is resolved, regardless of their respiratory conditions. further research will be needed to help clinicians to navigate this uncharted sea. a -year-old man presented to the emergency department with acute-onset high-grade fevers accompanied by dry cough and shortness of breath that had been ongoing for a week before presentation. he denied any associated nausea or vomiting, diarrhea, sore throat, congestion, or skin rash. of note, he had recently returned from a high-prevalence area for coronavirus disease- (covid- ) within the united states and was in self-quarantine. he was monitoring his symptoms; however, when his shortness of breath was not improving with his asthma medications (albuterol inhaler and cetirizine), he presented to the emergency department. on arrival, he was noted to be febrile at . f, he was tachypneic to breaths/min, he was normotensive at / mm hg, his heart rate was beats/min, and he was saturating % oxygen on room air. physical examination was remarkable for decreased breath sounds bilaterally. his past medical history was significant for mild intermittent asthma. our patient's clinical presentation was concerning for viral or bacterial lower respiratory tract infection. an electrocardiogram revealed normal sinus rhythm with normal pr ( ms) and qrs ( ms) intervals to anticipate and diagnose conduction disturbances associated with the novel coronavirus. to understand the mechanism responsible for high-degree av block associated with covid- without evidence of overt myocarditis. ( figure ). no acute st-t wave changes were noted. a single-view chest radiograph showed blunted costophrenic angles bilaterally with concern for right middle lobe opacity ( figure inflammatory markers were mildly elevated; the ferritin level was mg/ml (normal range to ng/ml), and c-reactive protein was elevated at . mg/dl (normal range to . mg/dl). the procalcitonin level was negative at . ng/ml (normal range to . ng/ml), and thyroid hormone levels were within normal limits. his nasopharyngeal swab tested positive for sars-cov- ribonucleic acid. given the patient's underlying asthma, which predisposed him to an increased risk for pulmonary patients were noted to have cardiac arrhythmias ( ) . covid- involvement of the heart has ranged from asymptomatic myocardial injury to acute coronary syndrome, mild to fulminant myocarditis, stress cardiomyopathy, and cardiogenic shock; however, the mechanism of cardiac involvement is not exactly clear ( ) . furthermore, underlying cardiovascular disease or risk factors and myocardial injury have been shown to portend poor prognosis in these patients ( ) . in this case, we present a patient with moderate covid- infection who showed evidence of transient conduction disturbances with highdegree atrioventricular (av) block. high-degree av block is known to be an uncommon presentation of acute myocarditis in adults, more commonly seen in cardiac sarcoidosis and giant cell myocarditis ( ) . however, because our patient did not have any other overt evidence of myocardial involvement, with normal cardiac biomarkers and a normal echocardiogram, his presentation is unusual and interesting. it is possible that covid- may have caused subclinical myocarditis leading to high-degree av block in this case. ace receptors are abundant in the heart and are present in multiple cell types, including macrophages, endothelial cells, smooth muscle cells, and cardiomyocytes ( ) . further, animal models have shown the presence of ace receptors in sinoatrial nodal cells in rats ( ) , and conduction disturbances and ventricular fibrillation have been noted with overexpression of the ace receptor in experimental mice models ( ) . hence, another possibility is that isolated involvement of the av node and infra-hisian conduction system by sars-cov- may have caused transient high-grade av block. whether this block is secondary to direct viral involvement or is an autoimmune response is unknown at this time. our patient did not have a recurrence of these conduction disturbances after he was started on supportive her medical history included mild asthma, heart failure with preserved ejection fraction, coronary artery disease (percutaneous coronary intervention ), paroxysmal atrial fibrillation (af), hypertension, obesity, and total hip arthroplasty -month prior. her differential diagnosis included bacterial/viral pneumonia, acute on chronic heart failure with preserved ejection fraction, pulmonary embolism, and coronavirus disease- (covid- ) . on arrival she was hemodynamically stable: heart rate beats/min, blood pressure / mm hg, respiratory rate of , and oxygen saturation % on l nasal canula. physical examination noted bilateral rales. chest x-ray showed patchy bilateral consolidations with mild interstitial edema ( figure ) . a computed tomography pulmonary angiogram revealed no pulmonary embolism but bilateral interlobular septal thickening and peripheral ground glass opacities most prominent in the posterior and lower zones. laboratory tests were remarkable for a white blood cell count of . k/ml, absolute lymphocyte count of /ml, the differential for electrical storm in covid- remains broad. myocarditis and cytokine storm may not be universal drivers of cardiac sequelae in covid- . management of these arrhythmias requires consultation with expert, multidisciplinary teams. hemoglobin . mg/dl ( . mg/dl month prior), hyponatremia to mmol/l, ferritin of , ng/ml, nterminal pro-brain natriuretic peptide of pg/ml, and c-reactive protein of . mg/dl (figure a ). remaining laboratory tests were within normal limits. a nasopharyngeal swab was sent for severe acute respiratory syndrome-coronavirus- (sars-cov- ) and respiratory viruses, blood cultures were collected, vancomycin and cefepime were started, and the patient was admitted to a negative-pressure room. were mildly elevated at admission and continued to rise with ast, reaching a peak on day of admission. similarly, alt peaked on day of admission. by discharge, the transaminase levels were downtrending ( figure ). an abdominal ultrasound showed normal liver size ( . cm) with increased liver echogenicity and a nodular contour suggestive of liver fibrosis, likely due to fontan associated liver disease (fald). his cbc was trended with normalization of his platelet count by day of hospitalization. in the emergency room, the patient was hypoxic with oxygen saturations of % on right atrium with accompanying dyspnea. he was placed on to l of oxygen via a nasal cannula, which led to an improvement in the oxygen saturations to low s. ggt (g-glutamyl transferase) being the most common ( ) . this patient had modest transaminitis likely due to the effects of covid- on his underlying fald ( ) . the patient was discharged after a -day hospitali- over the -month period preceding the sars-cov- -related stay-at-home order, a total of patients followed by the achd service at the university of washington medical center with defects of various severities died in an acute setting. in this report we unexpected mortality among patients with achd appears to have acutely increased at a single academic achd center during the sars-cov- pandemic. the ongoing sars-cov- pandemic appears to be contributing to increased achd mortality by delaying patient contact with health care. routine follow-up care for high-risk groups, including those with congenital heart disease, during the sars-cov- pandemic is critical to ensure appropriate triage and care for vulnerable populations. even with prior clinical stability, these populations remain at risk for acute cardiovascular complications and increased mortality. describe a series of acute-setting achd deaths that occurred within a single week at the time of the effective stay-at-home order. acute-onset death was defined as death that occurred either out of the hospital or within h of presentation to a medical facility. with the exception of patient (case ) who was called but did not answer the phone days prior to being found dead at home, none of the patients had a missed clinic appointment since the initiation of the stay-at-home order, and none had made contact with the medical system to report concerning symptoms prior to their deaths. a after the procedure, the impella cp catheter was removed without complications, and the femoral access was closed with the use of proglide closure systems (abbott vascular, santa clara, california). the staff allowed to enter the catheterization lab since its outbreak in italy in mid-february, covid- has spread rapidly, with over , cases and more than , deaths to date. epidemiologic analysis shows that the presence of comorbidities significantly increases mortality: . % in patients with cardiovascular diseases; . % in patients with diabetics; . % in patients with chronic respiratory diseases; % in patients with hypertension; and . % in oncologic patients ( ) . given the high number of infected patients, we often diagnose cardiovascular diseases at different stages during the viral pathology. in this perspective, some selected patients could benefit from treatments that deviate from current guidelines. we report the first case of a patient with covid- and acute coronary syndromes treated in italy for unprotected lmca stenosis with protected percutaneous coronary intervention. the use of the impella cp cardiac assist system to provide left ventricular support during high-risk percutaneous coronary interventions is recommended in such settings, because its efficacy is supported by randomizedcontrolled trials ( ) and large registries ( , ). nevertheless, this case also highlights the importance the patient had no significant medical history. he never smoked. there was no family history of cardiovascular disease (cvd). an early viral panel polymerase chain reaction was negative for multiple respiratory viruses. a severe acute respiratory syndrome-coronavirus- (sars-cov- ) nucleic acid amplification test early and rapid testing is critically necessary in patients with suspected covid- to prevent severe evolution. ecg st-segment elevations in inferior leads have been described in several covid- patients, with variable clinical significance. an accurate evaluation of the true incidence of acute myocardial injury related to covid- requires a standardized definition, which should include a combination of ecg changes, biochemical markers, and imaging abnormalities. given the very low pre-test probability for coronary artery disease and the absence of coronary calcifications on the chest ct scan, a coronary ct angiogram was not indicated, and the patient was not referred for invasive coronary angiography. the patient was treated conservatively, without thrombolytic agents or initiation of the acute coronary syndrome management protocol. the covid- pandemic represents the largest worldwide health care challenge to date. limited but rapidly emerging data have documented the role of cvd in increasing both the risk of infection and the severity of its clinical presentation ( ) ( ) ( ) ( ) . in particular, cvd is associated with a sharp increase in overall mortality, which reaches almost % of patients hospitalized ( ) . however, although such an association can be anticipated to a certain degree (on the basis of existing data from previous outbreaks of influenza and severe acute respiratory syndrome), the incidence of myocardial injury in covid- infection appears to be higher ( ) . furthermore, the definition of covid- -associated "myocardial injury" lacks standardization and is based primarily on elevated (and highly variable) serum levels of cardiac-specific troponins as the single most common defining markers. this myocardial injury has been associated with possibilities. logistically, we now understand that the decision to proceed with angiography carries a significant risk for nosocomial spread of the virus endangering hospital staff. we are also learning that acute kidney injury is quite prevalent and highly associated with mortality in covid- patients ( ). one should think twice before administering intravenous contrast medium in these patients. consensus documents from our professional societies that are based on early covid- observations have resurrected considering the use of fibrinolytic therapy for stemi ( ) . in a setting of limited staffing and resources, and where time to treatment is expected to be significantly delayed, fibrinolytic therapy provides a more rapid and logistically easier approach to reperfusion therapy while reducing staff exposure to infection. however, contraindications to fibrinolytic therapy have to be absent, and stemi mimics have to be excluded. the fibrinolytic strategy is probably most reasonable for hospitals without pci capability or immediate availability. at pci-capable hospitals with adequate staffing, primary pci is still preferred ( , ) . until there is universal availability of rapid testing (< min) for both the virus and the antibodies, our approach to stemi will have to be modified. this is primarily the result of new infection control considerations that will have to be included in our daily workflow. the current door-to-balloon time quality metric should be suspended by hospital quality improvement committees as a measure of system performance because of the current diagnostic and logistical challenges in delivering stemi care. in the american college of cardiology national cardiovascular data registry cathpci registry reporting form, noting a "system delay" as a reason for a prolonged door-to-balloon time will avoid any external quality of care penalties. we now work in the era of covid- stemi care. the patient remained asymptomatic, and no confirmatory tests were performed for the same reason as in the first patient. very late lad artery stent thrombosis was found, and a new des was implanted. the patient was asymptomatic, but because the covid- pandemic had reached its peak, a pre-admission polymerase chain reaction test was performed, with a positive result. the patient had a favorable course and was discharged days later. as antiplatelet therapy. ten days later, prasugrel was replaced by clopidogrel (after antiviral treatment was completed), and the patient was discharged. the covid- pandemic has significantly decreased worldwide interventional cardiology activity. in spain, cardiac catheterization procedures have been reduced by %, with a reduction of % for primary angioplasty ( ) . similar data have been reported in we present case of acute stent thrombosis and very late stent thrombosis cases ( table ) . despite no initial covid- testing in cases, symptoms and subsequent testing ( figures a to d ) supported that the patients were infected at the time of stent thrombosis ( table ) . the patient reported an active lifestyle with a history of playing competitive football and had a body mass st-segment elevation myocardial infarction with a high thrombus burden can appear as the first and only onset of covid- symptoms. plaque rupture in predisposed patients with cardiovascular risk factors can be enhanced by severe inflammation and worsened by prothrombotic characteristics of this new infectious disease. in the course of this covid- pandemic, sars-cov- infection should probably be ruled out in patients with unusual or severe thrombotic and ischemic events, even when there are no symptoms of covid- disease. index of kg/m . he had no significant medical history except for past smoking ( pack-years), which he had quit years before. he had no family history of cardiovascular disease and was taking no medication or drugs. no diagnosis other than a stemi was possible. the initial electrocardiogram displayed a sinus rhythm with an anterior st-segment elevation and q waves with negative t waves in the inferior leads the patient did not develop heart failure, or heart rhythm disturbances, or other complications of myocardial infarction. furthermore, he remained free of covid- disease symptoms. in the context of the covid- pandemic, unusual myocardial infarction presentations, such as in young individuals at low cardiovascular risk, should lead to with t-wave inversion in diii and avf ( figure ). the patient had no past medical history and was not on any medications. to describe the management of patients with refractory ards requiring coronary angiography. to describe the feasibility of coronary angiography with the patient in prone position. the differential diagnosis included acute myocardial infarction, myocarditis, and takotsubo syndrome. there was no evidence of obstructive coronary disease, and the final diagnosis was myocarditis, although we were not able to perform cardiac magnetic resonance in this highly unstable patient. with a rapid spread worldwide, covid- has become a public health emergency of international concern ( ). the clinical course of sars-cov- infection is mostly characterized by respiratory tract symptoms, including fever, cough, pharyngodynia, fatigue, and complications related to pneumonia and ards, often in a patient in prone position, the geometry and orientation of the heart as well as the coronary anatomy do not allow to obtain perfectly symmetric pictures of the coronary arteries using usual views. consequently, the interpretation of coronary angiography was simply done following the heart's shape. with unchanged cranial/caudal tilts ( , ) or using the double-inversion technique to normalize all angiographic pictures such as in a left-located heart ( ) is usually enough to perform and analyze coronary angiography in such patients. finally, even though we did not perform percutaneous coronary intervention, performing percutaneous coronary intervention with a patient in prone position would not be a critical issue for an experienced operator. pandemic, the most plausible diagnosis seemed to be a severe acute respiratory syndrome-coronavirus- (sars-cov- ) infection. tables and summarize the biochemical tests and atrial blood gas analysis before, during, and after amiodarone therapy. figure shows the illness clin- no adverse events were reported. the patient was discharged on day . infection in cell cultures and mouse models ( , ) . amiodarone is a widely available, low-cost antiarrhythmic drug that in the past has been considered as a possible antiviral medication ( ) . amiodarone and its main metabolite (mono-n-desethyl amiodarone) were shown to inhibit the entry of filoviruses (a family of single-stranded, negative-sense rna viruses that includes ebola virus) at the same serum concentration found in patients treated for arrhythmias ( , ) . amiodarone also proved able to remain to be investigated, and drug interaction with other treatments (e.g., hydroxychloroquine, lopinavir/ritonavir, atazanavir, and darunavir/cobicistat) are major concerns ( ) . notably, amiodarone toxicity at follow-up visit, days after discharge, the patient was asymptomatic. in this case, amiodarone was given for research purposes in a hospital setting. this is an approach still under investigation. do not try this at home. to diagnose acp in patients with sars-cov- -related ards. to appreciate potential role of almitrine in improving oxygenation and rv function. to understand sars-cov- -related atypical type of ards. medical history included only an overweight with a body mass index of . kg/m . the differential diagnosis included pulmo- and septal dyskinesia disappeared ( figure c ). moreover, rv global longitudinal strain improved from - . % to - . % ( figure d ). twelve hours following almitrine infusion, rvswi decreased from . to . g/m/beat/m . the clinical spectrum of sars-cov- -related cardiovascular complication includes myocarditis, pericarditis, vasoplegia, rv failure, and acute coronary syndromes ( , ) . in this case, we highlight the rv dysfunction another explanation is the impact of ards and mechanical ventilation on the rv. acp is a well-known complication of ards despite a protective ventilation, with an incidence of %. hence, acp may be related to a high driving pressure, leading to an increased rv afterload ( ) . moreover, the patient was on norepinephrine, which may increase rv afterload. another explanation is that hypoxia could lead to rv dysfunction in its own right ( ). ventilation-to-perfusion ratio ( ) . moreover, previous studies in the s showed that at a low dose, the deleterious effect on pulmonary vascular resistance was negligible, especially when associated with nitric oxide ( ) . hence, we hypothesized that almitrine use in the case of sars-cov- atypical ards might be useful. in the present case, almitrine infusion was associated with rv function improvement and decrease in pulmonary vascular resistance. this is probably due not only to a better oxygenation, but also to a better distribution of pulmonary vascular flow to aerated lung areas. before almitrine infusion, we observed a high rvswi with a normal-to-low range of ci, suggesting a hemodynamic disconnection between the rv and left ventricle. the reduction in rv afterload by almitrine infusion resulted in an improvement in this disconnection (decreased rvswi and improved ci). as almitrine infusion could induce reversible lactic acidosis and hepatic dysfunction ( ) to describe high-risk clinical features in a patient on durable lvad support who developed covid- . to illustrate potential complications and clinical dilemmas in managing covid- in a patient supported with a durable lvad. reverse transcription polymerase chain reaction results for severe acute respiratory syndrome-coronavirus- (sar-cov- ) was positive at the initial emergency department visit and at the authors' institution. serial laboratory and imaging tests are detailed in table . several markers of disease severity were abnormal including absolute lymphocyte count, c-reactive protein level, and cardiac enzymes. chest radiographs showed bilateral infiltrates concerning for atypical pneumonia (figure ). the patient was quarantined in a negative-pressure intensive care room. the host response to covid- infection is often localized in the lung parenchyma, but a surge in proinflammatory cytokines can occur ( , ) . known as a "cytokine storm," this phenomenon is described in dynamics. we should closely monitor for: ) rv failure and need for inotropic support; ) drops in lvad speed or suction events, low flow, or pulsatility index events due to vasoplegia associated with infection. to limit health care workers' exposure to covid- , nonessential testing such as echocardiograms, the antimalarial medication hydroxychloroquine, which was chosen as the initial treatment agent for our patient, was shown to reduce in vitro sar-cov- cell entry, and a retrospective study suggested its clinical benefit in covid- ( , ) . a major side effect is qtc prolongation, so the present protocol provides monitoring guidance of this complication. immunomodulatory biological agents such as tocilizumab are reserved for severe covid- , defined by the values in bold are in-hospital values that were consistent with baseline values. *last visit values were the latest values obtained within the previous months. baseline ldh, wbc, platelet, absolute polymorphonuclear leukocytes, and absolute lymphocytes were recorded as an average of the previous values measured within year. †this patient was placed on ventilator support on the night of hod and was given tocilizumab on the evening of hod . ‡this patient experienced pulseless electrical activity arrest after the return of spontaneous circulation. krelative change is the percentage of increase or decrease from baseline value. alt ¼ alanine aminotransferase; ards ¼ acute respiratory distress syndrome; ast ¼ aspartate aminotransferase; bnp ¼ b-type natriuretic peptide; ck-mb ¼ creatine kinase mb; cvp ¼ central venous pressure (obtained from right heart catherization at baseline, and from central venous line in the hospital); egfr ¼ estimated glomerular filtration rate; fio ¼ fraction of inspired oxygen; hod ¼ hospital day; ldh ¼ lactate dehydrogenase; lvad ¼ left ventricular assist device; map ¼ mean arterial pressure (obtained from doppler or arterial line); pao ¼ arterial partial pressure of oxygen; wbc ¼ white blood cell. presence of both worsening respiratory failure and a cytokine storm as shown by increasing inflammatory markers. still, caution is warranted as major adverse effects of tocilizumab include infection, infusion reactions, dyslipidemia, neutropenia, and potential malignancy ( ) . patients on lvad support are particularly vulnerable to infectious complications due to the inherent presence of hardware and driveline finally, prone ventilation is beneficial in cases of severe ards. the maneuver has been effective in improving lung mechanics and gas exchanges, and in some cases, it may prevent the need to escalate to venous-venous extracorporeal membrane oxygenation ( , ) . although there are no published outcomes, early experience in wuhan, china indicates that prone position was widely used in patients with covid- -related severe ards with possible benefits ( ) . nonetheless, it may be prohibitive in heart failure patients on lvad support. a -year-old woman presented with productive cough, fatigue, fever, and diarrhea for the previous days. physical abbreviations as in figure . ( ) . stress cardiomyopathy has also been reported with viral infections ( ) . histological studies have shown mild inflammatory infiltration ( , ) , and it is possible that heightened inflammation with viral infections, particularly that seen with covid- , may contribute to development of stress cardiomyopathy. overall, the prognosis of stress cardiomyopathy is favorable, with the majority of patients fully recovering lv function by months ( ). twitter: @ferrasdabbagh . he had a history of hypertension and was taking lisinopril. the differential diagnosis included sars-cov- causing severe ards and acute cardiac injury from direct viral toxicity (i.e., myocarditis), acute coronary syndrome (acs), demand ischemia, and stress cardiomyopathy. prevalence and prognostic implications of cardiac injury (defined as troponin elevation > th percentile upper reference limit) in covid- . considerations for differentiating causes of cardiac injury in covid- . management strategies for myocarditis and severe ards in covid- . and mortality benefit in animal models ( ) . improved airway inflammation has also been observed in animals treated with aris ( ) . other viral infections such as influenza and respiratory syncytial virus were considered, but the pre-test probability for covid- was high because other residents at the facility had been diagnosed with covid- recently. in the emergency department, the patient was tachypneic with an initial oxygen (o ) saturation %. table lists the results of his initial laboratory testing including normal levels of ferritin, procalcitonin, interleukin (il)- , and il- . levels of c-reactive protein, lactate dehydrogenase, and troponin were elevated. there was a reduced white blood cell count without lymphopenia. a chest radiograph had no air space or interstitial infiltrates (figure ). there was a single low-flow lvad alarm noted days prior to presentation. based upon the adequate room air saturation, absence of pulmonary infiltrates, and minimally abnormal inflammatory markers, the patient was classified as having a mild case of covid- . due to persistent breathlessness, hydroxychloroquine was initiated on day with qtc monitoring. to the best of our knowledge, this is the first re- in the current covid- pandemic, lopinavir/ritonavir was studied in a randomized, controlled trial in table ) . the patient was discharged to his nursing facility on hospital day once a room was available where he could be quarantined, and he continued to feel well days later. mahmood et al. to make a diagnosis of fulminant myocarditis concomitant with covid- pneumonia. to understand the value of serial cardiac magnetic resonance after myocarditis due to covid- . sinus tachycardia ( beats/min) with negative t waves from v to v . to be able to arrive at the differential diagnosis of acute cardiac dysfunction in the setting of severe covid- disease early after heart transplantation. to understand the need for individualized management, balancing risks of infection and rejection in heart transplantation recipients with severe infections early after transplantation. was discharged to isolation at home with pulse oximetry and plans for daily telemedicine assessments. three days later the patient presented to the emergency department with rapidly progressive respiratory distress and hypoxia. the patient had a history of end-stage non- figure . the patient reported continued gradual recovery by weekly telemedicine assessments after discharge. he had a history of pityriasis lichenoides chronica. he had no personal or family history of congenital heart disease, immunodeficiency, or autoimmune disease. the differential diagnosis included viralinduced myocarditis or underlying cardiomyopathy unmasked by an acute viral illness. table ) . the patient has had no recurrent episodes of chb since day of admission, and a repeat echocardiogram on day of admission demonstrated lownormal biventricular systolic function. and conduction abnormalities appears to be a rare manifestation of sars-cov- infection in children ( ) ( ) ( ) . nonetheless, evaluation for myocardial injury may be warranted in pediatric patients with symptomatic sars-cov- infection, particularly in patients whose clinical symptoms (e.g., dyspnea, hypoxia) seem out of proportion to chest imaging findings. on autopsy, histopathologic examination of the heart showed mild to moderate myocyte hypertrophy with mild to moderate diffuse interstitial and perivascular fibrosis (figures a and b) . the impact of influenza co-infection in this patient with covid- must also be considered because this virus is known to contribute to cardiovascular morbidity and mortality secondary to up-regulation of the inflammatory response and endothelial dysfunction ( ) . as such, influenza a likely had significant effects on her cardiac functioning. coinfection with sars-cov- is of great concern, with limited data delineating the the patient's medical history showed arterial hypertension, dyslipidemia, and impaired fasting blood sugar. covid- has extrapulmonary and cardiovascular manifestations. covid- may be associated with exaggerated inflammatory response with an abnormal activation of coagulation, so a screening of coagulation setup may be indicated. covid- may show up with takotsubo syndrome. the differential diagnosis included acute myocardial infarction, takotsubo syndrome, myocarditis, and coronary embolism. the patient was transferred to our center for an urgent coronary angiography, which revealed nonsignificant coronary atherosclerosis. figures and , videos and ). our priority was to treat the patient with enoxaparin , iu twice daily as per the patient's weight. during the first days of hospitalization, and taking into consideration that the patient was hypotensive (systolic blood pressure: mm hg; mean blood pressure: < mm hg), we treated the patient with chest radiography was repeated in the following days and showed progressive reduction of interstitial pneumonia. also, blood test results revealed an improvement of inflammation indexes ( table ) . on day of hospitalization, the nasopharyngeal swab was repeated, with a positive result. the first negative result was registered on day . on the th day, we performed another transthoracic echocardiography, which showed the resolution of the thrombi ( figure ) and a complete restoration of lvef ( %) (video ). his past medical history was notable for type diabetes mellitus, remote prostate cancer, and ventricular tachycardia. covid- and concern over prolonged separation from his family. our service is modeled after previously published "e-consultation" workflow recommendations in "peacetime" prior to the sars-cov- outbreak ( ). in phase , we implemented these processes, and all emergent cases were treated as puis. as we approach phase , our processes continue to undergo iterative improvements and all cases coming to the ccl will be considered puis. our approach to stat and routine tee is outlined in remote monitoring is used for electrophysiology clinic device checks, with patients triaged to present for evaluation if they develop concerning arrhythmias, heart failure alerts, or device-related issues. *vt storm that has failed medical treatment including at least antiarrhythmic drugs (including propranolol), treatment of underlying reversible condition if present (qtc prolongation due to ischemia, medications, or metabolic/electrolyte imbalance), general anesthesia, and left stellate ganglion block (if available). †not reversible or fails to respond to chronotropic drugs such as isoproterenol, dopamine, and/or scopolamine, and temporary pacing cannot be safely maintained in an intensive care setting. in this scenario a screw-in active fixation lead connected to an externalized generator or an active fixation temporary pacing lead may be considered depending on the patient's clinical condition and could be performed in the intensive care setting under fluoroscopic guidance (if available) or in an operating room with negative airflow capabilities and fluoroscopy. cied ¼ cardiac implantable electronic devices; eos ¼ end of service; eri ¼ elective replacement indicator; vt ¼ ventricular tachycardia. in the wake of this pandemic, formal medical student and trainee didactics were disbanded to allow for social distancing. additionally, several states expe- her past medical history included hypertension and diabetes mellitus. the differential diagnosis included communityacquired pneumonia, atypical pneumonia, and coro- figures a and b ). the patient was started on therapeutic enoxaparin and was closely monitored for hemodynamic instability. she declined to take hydroxychloroquine, recommended by some experts for management for covid- . she remained hemodynamically stable and was transitioned to oral anticoagulant therapy (apixaban) with plans to continue anticoagulation for months. the covid- outbreak is an unprecedented global public health challenge. since the end of december , when the first cases of sars-cov- infection were detected in wuhan, china, the disease has spread exponentially ( ). on january , , the world health organization declared covid- , the disease caused by the novel coronavirus, a public health emergency of international concern and later officially upgraded it to a global pandemic. as of april , , more than , , confirmed cases from more than countries and more than , deaths have been documented worldwide. the projected u.s. death toll is > , , with an estimated total burden of more than million covid- cases. in approximately % of cases, fever is the most common presentation, followed by cough ( %), vomiting ( %), and diarrhea ( . %) ( ) . in up to % of patients, the natural course of the disease is complicated by severe interstitial pneumonia, which can lead to acute respiratory distress syndrome, multiorgan failure including acute kidney injury, dissemi- to suspect pe early in the disease process in confirmed or suspected covid- patients. to identify high-risk patients early and to offer appropriate therapies while mitigating patient and provider risk. the patient was managed with intravenous unfractionated heparin (ufh) and dobutamine; infection. cdt is associated with early improvement in rv function and hemodynamics in deteriorating patients with lower doses of tpa ( , ) ; however, pui approximately times less frequent ( , ) . importantly, a substantial proportion of the thrombotic events were diagnosed very early during the hospital stay, suggesting that they had already occurred before admission ( ) . in view of the previously mentioned (preliminary) findings, and although it cannot yet be concluded with safety that the thrombosis risk among patients with severe covid- is substantially higher than that of patients with severe infection caused by other bacterial or viral pathogens ( , ) , thrombotic events are very likely to be a key aspect of covid- -associated morbidity and mortality ( ) . it is therefore now necessary to make the patient's medical history was notable only for obesity (body mass index kg/m ) and type diabetes. the primary differential diagnosis for the patient's the usual risk stratification schema for acute pe rely on a combination of hemodynamic clinical parameters, such as hypoxemia, tachycardia, and hypotension along with serum biomarkers, such as troponin or brain natriuretic peptide, followed by confirmatory imaging tests ( ) . severe covid- -related ards may present with many similar hemodynamic and biomarker derangements masking underlying vte. illness. figure ). an axillobifemoral bypass was performed, followed by therapeutic anticoagulation with good initial results. the patient died days after surgery from a major hemorrhage. case . the third patient was a -year-old male with history of hypertension, diabetes, and coronary artery disease ( table ) who was admitted for hypoxic ct angiography of patient shows a nonobstructive thrombus formation of descending aorta (arrow) in an axial view (a) and a sagittal view (b). disease. a recent paper attributes this state "to excessive inflammation, platelet activation, endothelial dysfunction, and stasis" ( ) . others have suggested that formation and polymerization of fibrin are responsible for this hypercoagulability ( ) . therefore, recent recommendations insist on thromboprophylactic measures to prevent thromboembolism ( , , ) . a recent publication found evidence of the presence of virus in endothelial cells ( ) . one explanation is that the angiotensin-converting enzyme receptor that the virus uses to infect cells is widely expressed in endothelial cells. this causes endotheliitis, which could explain why covid- patients seem prone to venous and arterial thrombosis. this paper ( ) blockade may also be considered ( ) . it should be noted that mild ards may be managed with noninvasive forms of ventilation. however, during the present pandemic, modifications to usual critical care may be necessary. given concern for viral transmission, current recommendations advise it is also important to monitor the patient's driving pressure, or difference between the peep and plateau pressure, as increased driving pressures have been associated with higher mortality in ards ( ) . with a basic understanding of these fundamentals, it is possible for all cardiologists to provide safe and effective care for our patients with covid- . as many of us prepare to use skill sets long forgotten, it will be important to remember to ask for help when needed. one of the few bright spots in this pandemic has been the resurgence of interdisciplinary team- and is thought to protect against lung injury. these functions may be due to differences in the location of the ace proteins, transmembrane or in the plasma ( ) . the ace gene is located on the x chromosome, which suggests that women might have higher ace levels and thus be protected against more severe disease compared to men ( ) . there has been recent concern in the cardiology community about the possible negative effect of italy, as it has done in the past, will improve its health and economic systems after this tragedy. probably nothing will be like before, and this catastrophe will be a great opportunity to further improve an efficient and effective national universal health system. heroes, we look like prey in heroes' capes. that kind of bravery, that work integrity, is not boundless. no one is so fearless or short-sighted as to discount all risks. when i try to figure out how i feel in this moment, the italian motto "andrà tutto bene" ("everything will be alright") that has been viral since the onset of the the ccl nursing staff was reinforced to speed up procedures. all noncritical equipment or supplies were removed from the ccl to facilitate cleaning and disinfection procedures. availability of ppe is a concern, so we created sets of ppe to best manage available resources: a mid-level kit and a fullprotection kit for suspected and confirmed cases. despite the concern of the medical community, we believe society has largely adhered to the social isolation recommendations, as we are looking at a constant drop in admissions to intensive care units and an increase in patients successfully discharged. the availability of masks for everyone is still not a reality as we are conceiving the first draft of a plan to reduce restriction measures. subsequently, as we are receiving more papers, we have decided to divide jacc: case reports publications into sections: acute coronary syndromes, heart failure, arrhythmias, thromboembolic events, and stories from the front line, in the format of "voices of cardiology" papers. all these papers have been highlighted in this issue ( ) . recognizing the value of not overloading our audience with publications, and in an effort to keep the quality high and up to the standards of jacc journals, we accepted approximately % to % of the manuscripts submitted. we selected the best of the best cases and brought together world-renowned specialists to write editorials. although we understand that clinical cases have been of crucial importance for our understanding of covid- , it is of equal importance that they cannot substitute for large studies and pharmacological trials. therefore, unless we were dealing with an impressive side effect of a medicine, we have been very cautious in publishing pharmacological evidence, as large trials would prove the benefits and side effects of these medicines currently under trial. another important task of jacc: case reports is that to ease navigation on the acc covid- hub, its content was organized into sections on clinical guidance, practice considerations, and frontline perspectives. given the novelty and rapidity of the covid- pandemic, most of the initial content was based on analyses of frontline experiences and expert opinion. the hub executive team and sqc worked to ensure that the content struck a balance between reasonable, actionable suggestions and acknowledgment that more rigorous research was needed to better inform the best approach to covid- management. going forward, the hub will continue to generate content but now turn its attention to highlighting the growing peer-reviewed research on covid- and cv disease. the acc has commissioned a task force to promote research in this area, and the hub will serve as a primary dissemination platform, in conjunction with jacc and other cardiology-focused journals. in addition, the hub will highlight best practices and frontline experiences from its membership on "reopening" protocols. with projections that covid- will ebb and flow worldwide over the next several years, our membership will need to navigate the best way to continue to treat cv disease during this time. the acc covid- hub has proved to be a useful resource to assembling and distributing information broadly during a rapidly evolving pandemic. lessons learned include the need to build a nimble process to commission, organize, and distribute content, an ability to engage with experts to generate content, a method to closely monitor of member and community needs to inform content development, and an emphasis on highlighting rigorously conducted research and expert consensus over mere opinion and speculation. sars-cov- infection in children available at: https:// picsociety.uk/news/pics-statement-regardingnovel-presentation-of-multi-system-inflammatorydisease covid- and the heart sars-cov- and viral sepsis: observations and hypotheses myocardial localization of coronavirus in covid- cardiogenic shock: covid- does not spare the heart cardiac involvement in a patient with coronavirus disease (covid- ) the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality epidemiology of covid- among children in china cardiac 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italy: changing perspectives on preparation and mitigation covid- and italy: what next? italy's health performance, - : findings from the global burden of disease study coronavirus infections-more than just the common cold the obelisk press-seurat editions, ; new address for correspondence: dr e-mail: george.collins@ucl.ac.uk. twitter: @drgeorgecollins clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease (covid- ): the experience of clinical immunologists from china exploring personal protection during high-risk pci in a covid- patient: impella cp mechanical support during ulmca bifurcation stenting for the american college of cardiology's interventional council and the society for cardiovascular angiography and interventions. catheterization laboratory considerations during the coronavirus (covid- ) pandemic: from the acc's interventional council and scai reacquainting cardiology with mechanical ventilation in response to the covid- pandemic the covid- pandemic and cardiovascular complications: what have we key: cord- - ydczid authors: funkhouser, william k. title: pathology: the clinical description of human disease date: - - journal: essential concepts in molecular pathology doi: . /b - - - - . - sha: doc_id: cord_uid: ydczid pathology is that field of science and medicine concerned with the study of diseases, specifically their initial causes (etiologies), their step-wise progressions (pathogenesis), and their effects on normal structure and function. this chapter will consider the history of relevant discoveries and technologies that have led to our current understanding of diseases, as well as the pathologist’s current role in the diagnosis, prognosis, and prediction of response of human diseases. this chapter will discuss the fundamental concepts, terminology, and practice of pathology as the discipline dedicated to the understanding of causes, mechanisms, and effects of diseases. a section on key terms, definitions, and concepts is followed by sections on historical human approaches to diseases, an overview of current diagnostic practice, and a vision for new interface with applied molecular biology. pathology (from the greek word pathologı´a, meaning the study of suffering) refers to the specialty of medical science concerned with the cause, development, structural/functional changes, and natural history associated with diseases. disease refers to a definable deviation from a normal phenotype (observable characteristics due to genome and environment), evident via patient complaints (symptoms), and/or the measurements of a careful observer (signs). the cause of the disease is referred to as its etiology (from the greek word meaning the study of cause). one disease entity can have more than one etiology, and one etiology can lead to more than one disease. each disease entity develops through a series of mechanistic chemical and cellular steps. this stepwise process of disease development is referred to as its pathogenesis (from the greek word meaning generation of suffering). pathogenesis can refer to the changes in the structure or function of an organism at the gross/clinical level, and it can refer to the stepwise molecular abnormalities leading to changes in cellular and tissue function. the presentation of a disease to a clinician is in the form of a human patient with variably specific complaints (symptoms), to which the examining physicians can add diagnostic sensitivity and specificity by making observations (screening for signs of diseases). these phenotypic (measurable characteristic) abnormalities reflect the interaction of the genotype (cytogenetic and nucleic acid sequence/expression) of the patient and his/her environment. patient workup uses present illness history with reference to past medical history, review of other organ systems for other abnormalities, review of family history, physical examination, radiographic studies, clinical laboratory studies (for example, peripheral blood or csf specimens), and anatomic pathology laboratory studies (for example, tissue biopsy or pleural fluid cytology specimens). as you will see from other chapters in this book, the ability to rapidly and inexpensively screen for chromosomal translocations, copy number variation, genetic variation, and abundance of mrna and mirna is adding substantial molecular correlative information to the workup of diseases. the differential diagnosis represents the set of possible diagnoses that could account for symptoms and signs associated with the condition of the patient. the conclusion of the workup generally results in a specific diagnosis which meets a set of diagnostic criteria, and which explains the patient's symptoms and phenotypic abnormalities. obviously, arrival at the correct diagnosis is a function of the examining physician and pathologist (fund of knowledge, experience, alertness), the prevalence of the disease in question in the particular patient (age, race, sex, site), and the sensitivity/ specificity of the screening tests used (physical exam, vital signs, blood solutes, tissue stains, genetic assays). the pathologic diagnosis represents the best estimate currently possible of the disease entity affecting the patient, and is the basis for downstream follow-up and treatment decisions. the diagnosis implies a natural history (course of disease, including chronicity, functional impairment, and survival) that most patients with this disease are expected to follow. be aware that not all patients with a given disease will naturally follow the same disease course, so differences in patient outcome do not necessarily correspond to incorrect diagnosis. variables that independently correlate with clinical outcome differences are called independent prognostic variables , and are routinely assessed in an effort to predict the natural history of the disease in the patient. it is also important to note that medical therapies for specific diseases do not always work. variables that independently correlate with (predict) responses to therapy are called independent predictive variables. diagnosis of a disease and development of an effective therapy for that disease do not require knowledge of the underlying etiology or pathogenesis. for example, granulomatous polyangiitis (née wegener's granulomatosis) was understood by morphology and outcomes to be a lethal disease without treatment, yet responsive to cyclophosphamide and corticosteroids, before it was found to be an autoimmune disease targeting neutrophil cytoplasmic protein pr ( fig. . ). however, understanding the molecular and cellular pathogenesis of a disease allows development of screening methods to determine risk for clinically unaffected individuals, as well as mechanistic approaches to specific therapy. the pathologist is that physician or clinical scientist who specializes in the art and science of medical risk estimation and disease diagnosis, using observations at the clinical, gross, body fluid, light microscopic, immunophenotypic, ultrastructural, cytogenetic, and molecular levels. clearly, the pathologist has a duty to master any new concepts, factual knowledge, and technology that can aid in the estimation of risk for unaffected individuals, the statement of accurate and timely diagnosis, accurate prognosis, and accurate prediction of response to therapy for affected individuals. the ability of h. sapiens to adapt and thrive has been due in part to the ability of humans to remember the past, respect tradition, recognize the value of new observations, develop tools/symbols, manipulate the environment, anticipate the future, and role-specialize in a social structure. the history of human understanding of diseases has progressed at variable rates, depending on the good and bad aspects of these human characteristics. our understanding of ancient attitudes toward diseases is limited by the historical written record. thus, the start point for written medical history corresponds to around bce for mesopotamian rules in the code of hammurabi, and around bce for the analogous egyptian rules in the ebers papyrus. by definition, these philosophers, theologians, and physicians had access and assets to allow a written record, and materials and storage sufficient for the written records to survive. the mesopotamian records indicate a deity-driven, demon-driven theory and empirical practice by recognized professional physicians. in this context, the prevailing thought was that "disease was caused by sprit invasion, sorcery, malice, or the breaking of taboos; sickness was both judgment and punishment . . ." the greek medical community evolved a theory of disease related to natural causes and effects, with less emphasis on deity-driven theory. the hippocratic corpus includes "on the sacred disease" (circa bce), which rejected a divine origin for diseases, and postulated a natural rather than supernatural basis for disease etiology (". . . nowise more divine nor more sacred than other diseases, but has a natural cause . . . like other affections. . ."). aristotle ( À bce) wrote broadly on topics including logic, biology, physics, metaphysics, and psychology. to aristotle, observations led to a description of causes, or first principles, which in turn could be used logically in syllogisms to predict future observations. we would agree with these basic notions of induction and deduction. however, there was a different background philosophical construct regarding the nature of matter and causality (four elements, four humors, and four causes, including a final or teleological purpose). we would recognize aristotle's "efficient" cause of a disease as its etiology. alexander the great's conquest of egypt in the th century bc led to greek (ptolemaic) leadership of egypt from bce to bce, with development of the alexandrian library and university. faculty such as euclid developed geometric models of vision (optica), and herophilus described human anatomy by direct dissection and observation (greek medicine apparently allowed dissection in alexandria, including vivisection of the condemned). during the roman imperial era, galen ( À ce) used dissection and observation of other animals such as the macaque (human dissection was illegal) to extrapolate to human anatomy and physiology. like aristotle's approach, galen's approach to patients, diseases, and treatments was guided by philosophical constructs of four humors (blood, phlegm, yellow bile, and black bile) and the resulting temperaments (buoyant, sluggish, quick-tempered, and melancholic) due to humoral imbalances. it is thought that many of galen's texts were destroyed with the alexandrian library before the th century ad, but a subset was preserved and translated by middle eastern scholars. these ancient classic texts were then retranslated into latin and greek when printing houses developed in the th century (for instance, hippocrates'de natura hominis, circa ce, and galen's therapeutica, circa ce). the historical picture of the greco-roman understanding of disease is one of empirical approaches to diseases based on inaccurate understanding of anatomy, physiology, and organ/cellular pathology. greek medicine became less superstitious and more natural cause-and-effect oriented, yet philosophy still trumped direct observation, such that evidence was constrained to fit the classic philosophical constructs. some of the concepts sound familiar; for example, normal represents equilibrium and disease represents disequilibrium. however, we would differ on what variables are in disequilibrium (the historical humors, numbers, and opposites versus contemporary chemical and kinetic equilibria). following the collapse of the western roman empire in ce, the classic texts of aristotle, hippocrates, and galen were protected, translated, and built upon in the byzantine and arab societies of the near east, and in spain during the muslim/moorish period through the th century. during these middle ages for western europe outside spain, there was apparently a retreat to pre-hellenistic beliefs in supernatural forces that intervened in human affairs, with protecting saints and relics for disease prevention and therapy. centers of medical learning following the spanish muslim model developed in montpellier, france, and in salerno, italy, beginning in the th century. aristotle's concept of induction from particulars to general first principles, then use of syllogistic logic to predict particulars, evolved into the scientific method during the renaissance. ibn alhazen's (al-haitham's) work on the physics of optics in the th century challenged euclid's concepts of vision from the alexandrian era. (euclid thought that the eye generated the image, rather than light reflected from the object being received by the eye). in the th century, roger bacon reinforced this use of observation, hypothesis, and experimentation. the printing press (gutenberg, ce) allowed document standardization and reproduction, such that multiple parallel university and city libraries could afford to have similar collections of critical texts, facilitating scholarly publications in journals. access to publications in libraries and universities led to a system of review, demonstration, discussion, and consensus regarding new scientific findings. the concept of the body as an elegant machine was captured by not only th and th century renaissance artists like da vinci and michelangelo, but also by anatomists and pathologists interested in the structure/function of health and disease. the ancient models of aristotle and galen had become sacrosanct, and newer, evidence-based models were considered heretical to some degree. so it was somewhat revolutionary when vesalius dissected corpses, compared them with galenic descriptions, and published de humani corporis fabrica libri septem ( ce; "seven books on the structure of the human body"; the fabrica), challenging and correcting th century understanding of normal human anatomy. vesalius's successors (colombo, fallopius, and eustachius) further improved the accuracy of human anatomic detail. thus, correction of galen's anatomical inaccuracies (including the rete mirabile at the base of the brain, blood vasculature, the five lobed liver, and curved humerus) required at least centuries for challenge, scientific disproof, and eventual medical community acceptance. the scientific revolution describes the progressive change in attitude of scientists and physicians toward understanding of the natural world, health, and disease. this revolution began circa ce, when copernicus published arguments for a heliocentric universe and vesalius published the fabrica series on human anatomy. by the th century, galileo, kepler, newton, harvey, and others had used this observation-based, matter-based, and mathematical law-based perspective to develop a scientific approach similar to our own modern approach of testing hypotheses with experimental data and statistics. in human biology, the investigation of structure led to studies of function, initially of human cardiovascular physiology, for example, harvey's anatomical exercise concerning the motion of the heart and blood in animals ( ). whereas galen conceived of parallel but unconnected arteries and veins, with continuous blood production in the liver and continuous blood consumption in the periphery, harvey demonstrated that blood was pumped by the heart through arteries, through tissue capillaries, to veins, and then back to the heart in a circle (circulation). correction of these and other galenic physiological inaccuracies (such as nasal secretions representing the filtrate of cerebral ventricle fluid) thus required at least centuries before challenge, scientific disproof, and eventual medical community acceptance. the scientific method facilitates empirical, rational, and skeptical approaches to observational data, and minimizes human dependence on non-evidence-based traditional models. in spite of the scientific method, physicians are still human, and the medical community still shows an inertial reluctance to adapt to new information when it disrupts traditional paradigms. recent examples would include reluctance to accept an etiologic role for the h. pylori bacterium in peptic ulcer disease, and reluctance to offer less than radical mastectomy for primary breast carcinoma. before the use of lenses to magnify objects, it was physically impossible to make observations on objects smaller than the resolving limit of the human eye (about . À . mm). thus, prokaryotic and eukaryotic cells, tissue architecture, and comparisons of normal and disease microanatomy were philosophical speculation until description of the mathematics of optics and lens design. in a real sense, optical technology was rate-limiting for the development of the fields of tissue anatomy, cellular biology, and microbiology. concepts of optics were written as early as bce in alexandria (optica, euclid). clear glass (crystallo) was developed in venice in the th century. a compound microscope was invented by janssen in ce. microanatomy and structural terminology was begun by malpighi ( ce), who examined capillaries in frog lung, trachea tubes for airflow in silkworms, and stomata in plant leaves. robert hooke used a compound microscope to describe common objects in micrographia ( ce). antony van leeuwenhoek used self-made simple magnifying lenses to count the threads in cloth in a dutch dry-goods store, then later published descriptions of bacteria (termed animalcules), yeast, and algae, beginning in ce. yet the relevance of these observations in microanatomy and microbiology to human diseases required changes in conceptual understanding of the etiology and pathogenesis of diseases. for example, it was years after van leeuwenhoek that a streptococcus sp. was recognized by koch and pasteur as the likely etiologic agent of puerperal fever in post-partum women. the relevance of microanatomy and microbiology to human disease required expansion of conceptual understanding to include morphologic changes in diseased cells and tissues, as well as recognition of an etiologic role for microorganisms. rokitansky's gross correlates with clinical disease (a manual of pathological anatomy, ), paget's surgical perspective on gross pathology, and virchow's morphologic correlates with clinical disease were critical to the development of clinico-pathologic correlation, and served to create a role for pathologists to specialize in autopsy and tissue diagnosis. virchow's description of necrotizing granulomatous inflammation, the morphologic correlate of infections caused by mycobacteria such as tb and leprosy, preceded the discovery of the etiologic agents years later by hansen (m. leprae, ) and koch (m. tuberculosis, ). the causal relationship between microorganisms and clinical disease required scientific demonstration and logical proof before medical community acceptance. for example, identification of the cause-and-effect relationship between streptococcus sp. and puerperal fever required an initial recognition of unusual clinical outcomes (clusters of postpartum deaths), then correlation of puerperal fever clusters with obstetrician habits, then semmelweiss's experimental demonstration in that hand-washing reduced the incidence of puerperal fever, then the demonstration that particular bacteria (streptococci) are regularly associated with the clinical disease (koch, circa ), and finally by culture of the organism from the blood of patients with the disease (pasteur, ) . 'germ theory' articulates this causal relationship between microorganisms and clinical diseases in animals and humans. technical improvements in microscopes (abbe condenser, apochromatic lenses, oil immersion lenses), the development of culture media, and the development of histochemical stains no doubt made it possible for koch to identify m. tuberculosis in . to be emphasized is the process of recognition, first of all of the variables associated with the clinical disease, then the scientific demonstration of a causal relationship between one or more of these variables with the clinical disease. this latter step was enunciated as koch's postulates ( ): (i) the bacteria must be present in every case of the disease, (ii) the bacteria must be isolated from a diseased individual and grown in pure culture, (iii) the specific disease must be reproduced when a pure culture is inoculated into a healthy susceptible host, and (iv) the same bacteria must be recoverable from the experimentally infected host. viruses, like bacteria, were understood and manipulated clinically prior to their isolation. manipulation of viruses as vaccines is traced to description of smallpox variolation in turkey by at least (described by lady montagu). smallpox variolation was used in the us continental army in the s. local availability of cowpox, a non-lethal poxvirus, allowed jenner to demonstrate (cross-) vaccination against smallpox ( ). vaccination against smallpox was used in napolean's army in , and was mandated for massachusetts schoolchildren by . the success of these vaccination programs prompted development of vaccines against other human viruses through the th and th centuries, including rabies ( prior to , organic (carbon-containing) compounds were thought to derive from living organisms, and it was thought that they could never be synthesized from nonliving (inorganic) material. this concept ('vitalism') was disproven by the in vitro synthesis of urea by f. wohler in . work from this era initiated the field of organic chemistry. predictable rules for in vitro and in vivo organic reactions, structural theory, modeling, separation technologies, and accurate measurement subsequently allowed the chemical description of natural products, and the chemical synthesis of both natural products and synthetic compounds. in addition to setting the stage for a systematic understanding of cellular biochemistry and physiology, organic chemistry set the stage for laboratory synthesis of natural products, such as dyes, vitamins, hormones, proteins, and nucleic acids. in that era, the textile industry was the main consumer of dyes. access to imported natural product dyes from plants was predictable for seafaring nations, but not for landlocked nations. in parallel with natural product extraction and purification was the recognition that aniline from coal (perkin, ) could be modified to generate a spectrum of colors, catalyzing the development of the german dye industry in the last half of the th century. some of these synthetic dyes were found useful for histochemical staining. morphologic diagnosis requires thin ( À μm), contrast-rich (requiring dyes) sections of chemically fixed (cross-linked or precipitated) tissue. thin sections allow the passage of light through the tissue, but reduce overlapping of cells in the light path. thus, technologies had to develop for cutting and staining of thin fixed tissue sections. work leading to our current technique for tissue solidification in paraffin wax was first described by klebs in . prototypes of our current mechanical microtome for making thin (~ μm thick) tissue sections was developed by minot in . precursor work leading to our current technique for tissue fixation with diluted formalin was first described by blum in . histochemical stains developed in parallel with dye technology for the textile industry. botanists used carmine as a stain in , and subsequently gerlack applied carmine to stain brain tissue in . the current hematoxylin dye used in tissue histochemistry was originally extracted from logwood trees from central america for the dye industry (to compete with indigo). metallic ion mordants made oxidized hematoxylin (hematein) colorfast in textiles, and a protocol for tissue staining was published by boehmer in . similar to the hematoxylin story, semisynthetic analine dye technology was adapted by histochemists from to . many of these dyes are still routinely used for recognition of tissue structure, peripheral blood cells, and microorganisms, including hematoxylin, eosin, methylene blue, ziehl-neelsen, gram, van gieson, mallory trichrome, and congo red. the most commonly used stains for general tissue diagnosis are the hematoxylin and eosin (h&e) stains, which provide a wealth of nuclear and cytoplasmic detail not visible in an unstained section. supplemental histochemical stains demonstrate specific structures and organisms: collagen and muscle (trichome), elastin (verhoff-von giessen), glycogen/mucin (periodic acid-schiff, pas), mucin (pas diastase, mucicarmine, alcian blue), fungi (gemori methenamine silver, gms), mycobacteria (ziehl-neelsen, fite), and bacteria (gram, warthin-starry). each of these stains is inexpensive ($ À$ ), fast (minutes to hours), and automatable, making them extremely valuable for service diagnostic pathology use. light microscopy lens technology matured during the last half of the th century. critical were abbe's introductions of the apochromatic lens system to eliminate chromatic aberration (different focal lengths for different wavelengths of visible light) in , a novel condenser for compound microscopes (to provide better illumination at high magnification) in , and an oil immersion lens in . by , maturation of tissue fixation chemistry, histochemical stain protocols, and light microscope technology had evolved into the workhorse technique for evaluation of morphologic abnormalities in tissue examination in anatomic pathology labs, and for the evaluation of morphologic features of microorganisms in microbiology labs. the scope of this chapter is limited to pathology, but it should be clear to the reader that the momentous discoveries of deep general anesthetics (long, ; morton, ), commercial electricity (edison, ), and radiography (roentgen, ) also contributed to the development of the modern medical specialties of diagnostic pathology and laboratory medicine. the development of antisera in the th century for therapeutic purposes (for instance, treatment of diptheria) led to progressive understanding of the antibody, the efferent arm of the humoral immune response. similarly, tissue transplantation experiments led to the recognition of cellular rejection due to thymus-derived t-cells. antibodies and t-cells cooperate to react to foreign (non-self) molecules, common examples being allergic responses, viral infections, and organ transplants. antibodies were found to be made by b-cells and plasma cells, and were found to be exquisitely specific for binding to their particular antigens (ligands) either in solid phase or in solution. the analogous t-cell receptor (tcr) recognizes a ligand made up of a À mer peptide presented by self mhc (hla in human) molecules on the surface of antigen-presenting cells (macrophages, dendritic cells, activated b-cells). b-cell and t-cells activate and proliferate when exposed to non-self proteins, but not to self proteins, attesting to tolerance to self. when b-cell and t-cell self-tolerance breaks down, autoimmune diseases can result (including myasthenia gravis, grave's disease, and lupus erythematosus). antibodies (immunoglobulins) were found to be heterodimers of kd heavy chains and kd light chains, folded together so that a highly variable portion defines the antigen-binding site, and a constant portion defines the isotype (igm, igd, igg, ige, or iga). similarly, t-cell receptors were found to be heterodimers of immunoglobulinlike molecules with a highly variable portion for ligand binding, and a constant portion, but without different isotypes. the range of antigen-binding specificities in a normal mammal is extensive, perhaps infinite, subtracting out only those self proteins to which the animal is tolerant. the genes encoding immunoglobulins and t-cell receptors were sequenced and, surprisingly, the extensive variation of specificities was due to a unique system of gene rearrangements of polymorphic v, d, and j gene segments with random nucleotide addition at the junctions. this system allows generation of literally billions of different ig and tcr binding specificities. although the ig and tcr molecules define the specificity of antigen (ligand) binding, we now understand that the probability of t lymphocyte activation is adjustable, based on activating (e.g. cd :b ) or inhibitory (e.g. pd- :pd-l ) receptor:ligand interactions. polyclonal antibodies raised in other species (goat, mouse, rabbit) against an antigen can be used to detect the antigen in diffusion gels (ochterlony, western blot), in solution (elisa), and in tissue sections. use of fluorescent-tagged antibodies for frozen section immunohistochemistry was developed first, and immunofluorescence (if) is still routinely used in renal pathology and dermatopathology. peroxidase-tagged secondary antibodies and dab chemistry were developed to generate a stable chromogen in the tissue, and this is now the primary method for detecting antigens in formalin-fixed tissue sections. improved antibody binding specificity and industrial production required monoclonal antibodies, which in turn required the development of mouse plasmacytomas/myelomas and cell fusion protocols. the net result is that commercial antibodies are now available for antigens of both clinical and research interest, including antibodies specific enough to distinguish minimally modified variant antigens (for instance, phosphorylated proteins or proteins with single amino acid substitutions). natural product chemistry and the rise of clinical laboratories diseases due to dietary deficiencies (like scurvy) and hormonal imbalances (like diabetes mellitus) were described clinically long before they were understood pathologically. dietary deficiency diseases prompted searches for the critical metabolic cofactors, so-called 'vital amines' (vitamins). xerophthalmia was linked to retinol (vitamin a) deficiency in (mccollum). rickets was linked to calcitriol (vitamin d) deficiency in . beri-beri was linked to a deficiency of thiamine (vitamin b ) in . scurvy was linked to ascorbic acid (vitamin c) deficiency in . pellagra in the united states was linked to niacin deficiency in . pernicious anemia was linked to cobalamin (vitamin b ) deficiency in . it is currently unusual to see morphologic features of these diseases in this country. diseases due to non-dietary physiologic imbalances prompted isolation of circulating molecules with systemic effects, i.e. the hormones. parathyroid hormone was isolated (berman, collip) in the s. thyroxine and cortisone were isolated (kendall) in , and table salt was iodized starting in . insulin was isolated (banting, best) in , non-human insulin was industrially purified and marketed soon thereafter, and recombinant human insulin was marketed starting in . these examples highlight the ability of th century chemists to fractionate, purify, synthesize, measure bioactivity, and manufacture these compounds for safe use by humans. study of diseases due to deficiencies and excesses of single molecule function led to a mechanistic understanding of biochemistry and physiology, with resultant interconnected reaction pathways of byzantine complexity (now referred to as systems biology). clinical demand for body fluid levels of ions (such as sodium, potassium chloride, and bicarbonate), glucose, creatinine, hormones (such as thyroxine and parathyroid hormone), albumin, enzymes (related to liver and cardiac function), and antibodies (reactive to aso, rh, abo, and hla antigens) led to the development of clinical laboratories in chemistry, endocrinology, immunopathology, and blood banking. functional assays for coagulation cascade status were developed, as were methods for estimating blood cell concentration and differential, leading to coagulation and hematology laboratories. serologic and cell activation assays to define hla haplotype led to hla laboratories screening donors and recipients in anticipation of bone marrow and solid organ transplants. culture medium-based screening for infectious agents led to dedicated clinical microbiology laboratories, which are beginning to incorporate nucleic acid screening technologies for speciation and prediction of treatment response. the clinical laboratories now play a critical, specialized role in inpatient and outpatient management, and their high test volumes (a -bed hospital may perform million tests per year) have catalyzed computer databases for central record-keeping of results. the previous vignettes indicate a scientific approach to natural products of the steroid and protein types, but do not indicate how proteins are encoded, what accounts for variation in the same protein in the population, or how inherited diseases are inherited. it turns out that the instruction set for protein sequence is defined by dna sequence. the role of nucleoproteins as a genetic substance was alluded to by miescher in , and was shown by avery to be the pneumococcal transforming principle in . the discovery of x-ray crystallography in made it possible for franklin, wilkins, and gosling to study dna crystal structure, and led to the description of the antiparallel double helix of dna by watson and crick in . this seminal event in history facilitated dissection of the instruction set for an organism, with recognition that -base codons specified amino acids in , and description of the particular codons encoding each amino acid in . demonstration of in situ hybridization in made it possible to localize specific dna or rna sequences within the cells of interest. recognition and purification of restriction endonucleases and dna ligases, and the development of cloning vectors, made it possible to clone individual sequences, leading to methods for synthesis of natural products (such as recombinant human insulin in ). chemical methods were developed for sequencing dna, initially with radioisotope-tagged nucleotide detection in plate gels, then with fluorescent nucleotide detection in . subsequent conversion to capillary electrophoresis and computer scoring of sequence output allowed high throughput protocols which generated the human genome sequence by . the development of polymerase chain reaction (pcr) chemistry in has made it possible to quickly screen for length polymorphisms of microsatellite (identity testing, donor:recipient ratio after bone marrow transplant, and microsatellite instability), coding sequence abnormalities in genes (translocations, rearrangements, insertions, deletions, substitutions), and epigenetic control of transcription (promoter methylation) in a targeted fashion. quantitative pcr methods using fluorescent detection of amplicons has made it possible to study dna and rna copy number, and to mimic northern blots/oligo microarrays in estimating rna transcript abundance for cluster analysis. massively parallel solid-state ("next-generation") sequencing chemistries now allow rapid alignment of hundreds of independent sequencing reactions, facilitating rapid comparison of germline and somatic sequence, as well as identification of low-abundance mutations. diseases can be distinguished from each other based on differences at the molecular, cellular, tissue, fluid chemistry, and/or individual organism level. one hundred and sixty years of attention to the morphologic and clinical correlates of diseases has led to sets of diagnostic criteria for the recognized diseases, as well as a reproducible nomenclature for rapid description of the changes associated with newly discovered diseases. sets of genotypic and phenotypic abnormalities in the patient are used to determine a diagnosis, which then implies a predictable natural history and can be used to optimize therapy by comparison of outcomes among similarly afflicted individuals. the disease diagnosis becomes the management variable in clinical medicine, and management of the clinical manifestations of diseases is the basis for day-to-day activities in clinics and hospitals nationwide. the pathologist is responsible for integration of the data obtained at the clinical, gross, morphologic, and molecular levels, and for issuing a clear and logical statement of diagnosis. clinically, diseases present to front-line physicians as patients with sets of signs and symptoms. symptoms are the patient's complaints of perceived abnormalities. signs are detected by examination of the patient. the clinical team, including the pathologist, will work up the patient based on the possible causes of the signs and symptoms (the differential diagnosis). depending on the differential diagnosis, the workup typically involves history-taking, physical examination, radiographic examination, fluid tests (blood, urine, sputum, stool), and possibly tissue biopsy. radiographically, abnormalities in abundance, density or chemical microenvironment of tissues allow distinction from surrounding normal tissues. traditionally, the absorption of electromagnetic waves by tissues led to summation differences in exposure of silver salt photographic film. tomographic approaches such as computerized tomography (ct, ) and nuclear magnetic resonance (nmr, mri, ) complemented summation radiology, allowing finely detailed visualization of internal anatomy in any plane of section. in the same era, ultrasound technology allowed visualization of tissue with density differences, such as a developing fetus or gallbladder stones. more recently, physiology of neoplasms can be screened with positron emission tomography (pet, ) for decay of short half-life isotopes such as fluorodeoxyglucose. neoplasms with high metabolism can be distinguished physiologically from adjacent lowmetabolism tissues, and can be localized with respect to normal tissues by pairing pet with standard ct. the result is an astonishingly useful means of identifying and localizing new space-occupying masses, assigning a risk for malignant behavior and, if malignant, screening for metastases in distant sites. this technique is revolutionizing the preoperative decision-making of clinical teams, and improves the likelihood that patients undergo resections of new mass lesions only when at risk for morbidity from malignant behavior or interference with normal function. pathologically, disease is diagnosed by determining whether the morphologic features match the set of diagnostic criteria previously described for each disease. multivolume texts are devoted to the gross and microscopic diagnostic criteria used for diagnosis, prognosis, and prediction of response to therapy. pathologists diagnose disease by generating a differential diagnosis, then finding the best fit for the clinical presentation, the radiographic appearance, and the pathologic (both clinical lab and morphologic) findings. logically, the venn diagram of the clinical, radiologic, and pathologic differential diagnoses should overlap. unexpected features expand the differential diagnosis and may raise the possibility of previously undescribed diseases. for example, legionnaire's disease, human immunodeficiency virus (hiv), hantavirus pneumonia, and severe acute respiratory syndrome (sars) are examples of newly described diagnoses during the last years. the mental construct of etiology (cause), pathogenesis (progression), natural history (clinical outcome), and response to therapy is the standard approach for pathologists thinking about a disease. a disease may have one or more etiologies (initial causes, including agents, toxins, mutagens, drugs, allergens, trauma, or genetic mutations). a disease is expected to follow a particular series of events in its development (pathogenesis), and to follow a particular clinical course (natural history). disease can result in a temporary or lasting change in normal function, including patient death. multiple diseases of different etiologies can affect a single organ, for example, infectious and neoplastic diseases involving the lung. different diseases can derive from a single etiology, for example, emphysema, chronic bronchitis, and small cell lung carcinoma in long-term smokers. the same disease (for instance, emphysema of the lung) can derive from different etiologies (emphysema from α- -antitrypsin deficiency or cigarette smoke). modern surgical pathology practice hinges on morphologic diagnosis, supplemented by special stains, immunohistochemical stains, cytogenetic/molecular data, and other clinical laboratory findings, as well as on the clinical and radiographic findings. findings that meet all of these criteria are diagnostic for the disease. if some, but not all, of the criteria are present to make a definitive diagnosis, the pathologist must either equivocate or make an alternate diagnosis. thus, a firm grasp of the diagnostic criteria, and the instincts to rapidly create and sort through the differential diagnosis, must be possessed by the service pathologist. the tissue diagnosis has to make sense, not only from the morphologic perspective, but from the clinical and radiographic vantage points as well. it is both legally risky and professionally erosive to make a clinically and pathologically impossible diagnosis. in the recent past, limited computer networking meant numerous phone calls to gather the relevant clinical and radiographic information to make an informed morphologic diagnosis. for example, certain diseases such as squamous and small cell carcinomas of the lung are extremely rare in non-smokers. thus, a small cell carcinoma in the lung of a non-smoker merits screening for a non-pulmonary primary site. fortunately for pathologists, computing and networking technologies now allow access to preoperative clinical workups, radiographs/reports, clinical laboratory data, and prior pathology reports. all of these data protect pathologists by providing them with the relevant clinical and radiographic information, and protect patients by improving diagnostic accuracy. just as research scientists ". . . ignore the literature at their peril . . .", diagnostic pathologists ". . . ignore the presentation, past history, workup, prior biopsies, and radiographs at their peril. . ." there are limitations to morphologic diagnosis by h&e stains. first, lineage of certain classes of neoplasms (including small round blue cell tumors, clear cell neoplasms, spindle cell neoplasms, and undifferentiated malignant neoplasms) is usually clarified by immunohistochemistry, frequently by cytogenetics (when performed), and sometimes by electron microscopy. second, there are limitations inherent in a snapshot biopsy or resection. thus, the etiology and pathogenesis can be obscure or indeterminate, and rates of growth, invasion, or timing of metastasis cannot be inferred. third, the morphologic changes may not be specific for the underlying molecular abnormalities, particularly the ratelimiting (therapeutic target) step in the pathogenesis of a neoplasm. for example, ret gain of function mutations in a medullary thyroid carcinoma will require dna level screening to determine germline involvement, familial risk, and presence or absence of a therapeutic target. fourth, the same morphologic appearance may be identical for two different diseases, each of which would be treated differently. for example, there is no morphologic evidence by h&e stain alone to distinguish host lymphoid response to hepatitis c viral (hcv) antigens from host lymphoid response to allo-hla antigens in a liver allograft. this is obviously a major diagnostic challenge when the transplant was done for hcv-related cirrhosis, and when the probability of recurrent hcv infection in the liver allograft is high. paraffin section immunohistochemistry has proven invaluable in neoplasm diagnosis for clarifying lineage, improving diagnostic accuracy, and guiding customized therapy. if neoplasms are poorly differentiated or undifferentiated, the lineage of the neoplasm may not be clear. for example, sheets of undifferentiated malignant neoplasm with prominent nucleoli could represent carcinoma, lymphoma, or melanoma. to clarify lineage, a panel of immunostains is performed for proteins that are expressed in some of the neoplasms, but not in others. relative probabilities are then used to lend support (rule in) or exclude (rule out) particular diagnoses in the differential diagnosis of these several morphologically similar undifferentiated neoplasms. the second role is to make critical distinctions in diagnosis that cannot be accurately made by h&e alone. examples of this would include demonstration of myoepithelial cell loss in invasive breast carcinoma but not in its mimic, sclerosing adenosis ( fig. . prostatic adenocarcinoma (fig. . ) . the third role of immunohistochemistry is to identify particular proteins, such as nuclear estrogen receptor (er) (fig. . ) or the plasma membrane her proteins (fig. . ), both of which can be targeted with inhibitors rather than generalized systemic chemotherapy. morphology remains the gold standard in this diagnostic process, such that immunohistochemical data support or fail to support the h&e findings, not vice versa. probability and statistics are regular considerations in immunohistochemical interpretation, since very few antigens are tissue-specific or lineage-specific. cytokeratin is positive in carcinomas, but also in synovial and epithelioid sarcomas. this example may imply aspects of the lineage of these two sarcomas that may be helpful in our categorization of these neoplasms. another example would be the diagnosis of small cell carcinoma in the lung of a nonsmoker. because lung primary small cell carcinoma is extremely uncommon, in non-smokers, this diagnosis would prompt the pathologist to inquire about screening results for other, non-pulmonary, sites. likewise, immunohistochemistry results are always put into the context of the morphologic, clinical, and radiographic findings. for example, an undifferentiated cd + neoplasm of the testis supports embryonal carcinoma primary in the testis, whereas a lymph node effaced by sclerotic bands with admixed cd + reed-sternberg cells supports nodular sclerosing hodgkin's disease. demand for both diagnostic accuracy and report promptness has increased as hospitals come under increasing financial pressure to minimize length of patient stay. hospitals now manage all but the sickest patients as outpatients. minimally invasive approaches for the acquisition of tissue samples for diagnosis use flexible endoscopic biotomes or hollow needles that sample À mm diameter tissue specimens. multidisciplinary conferences function almost realtime with respect to the initial biopsies. together, these changes have forced modern pathologists to make critical diagnoses on progressively smaller biopsy specimens, sometimes bordering on the amounts seen in cytopathology aspirates, and to do this in a timely fashion. this requires a clear understanding of the limitations to development of an accurate diagnosis, and a willingness on the part of the pathologist to request repeat biopsy for additional tissue when it is necessary for accurate diagnosis. diagnostic criteria involving electron microscopic ultrastructure found relevance for the evaluation of neoplasms described as small round blue cell tumors, spindle cell tumors, melanocytic tumors, and neuroendocrine/neuroblastic tumors, as well as delineation of ciliary ultrastructural abnormalities in primary ciliary dyskinesia. current approaches to these neoplasms are now generally approached using paraffin section immunohistochemistry. electron microscopy is now currently used mainly for nephropathology, platelet morphology, ciliary axoneme morphology, and for rare cases where immunohistochemistry is not diagnostic and where demonstration of premelanosomes, neuroendocrine granules, or amyloid is diagnostic. adequate sampling of a lesion is critical to making an accurate diagnosis. undercall diagnostic discrepancies are frequently due to sampling of a small portion of a large lesion that is not representative of the most abnormal portion of the lesion. insufficient sampling can result in an equivocal diagnosis or, worse, an inaccurate diagnosis. empirical rules have been adopted over the decades to ensure statistically adequate sampling of masses and organs, e.g. transurethral resections of prostate, soft tissue sarcomas, and heart allograft biopsies. in spite of the limitations and statistical uncertainties relating to morphologic diagnosis, a wealth of information is conveyed to a service pathologist in a tried-and-true h&e section. analogous to the fact that a plain chest x-ray is the sum total of all densities in the beam path, the morphologic changes in diseased cells and tissues are the morphologic sum total of all of the disequilibria in the abnormal cells. for most neoplastic diseases, morphologic criteria are sufficient to predict the risk of invasion and metastasis (the malignant potential), the pattern of metastases, and the likely clinical outcomes. for example, the etiology and pathogenesis in small cell lung carcinoma can be inferred (cigarette smoking, with carcinogen-induced genetic mutations) and the outcome predicted (early metastasis to regional nodes and distant organs, with high probability of death within years of diagnosis). new molecular data for both neoplastic and non-neoplastic diseases will most likely benefit unaffected individuals by estimating disease risk, and will most likely benefit patients by defining the molecular subset for morphologically defined diagnostic entities, thus guiding individualized therapy. diagnostic pathology will continue to use morphology and complementary data from protein (immunohistochemical) and nucleic acid (cytogenetic, in situ hybridization, dna sequence, epigenetic, and rna abundance) screening assays. improvements in current technologies should improve current test performance, reduce test cost, and shorten turnaround time. development of new technologies will lead to better diagnostic algorithms, improved diagnostic accuracy and prognosis, improved patient assignment to prospective randomized clinical trials, improved prediction of response to customized therapy, and improved sensitivity for measurement of residual subclinical disease. for transplant candidates, major histocompatability complex (mhc, hla in human) screening is evolving from cellular assays and serology toward sequencing of the alleles of the class i and ii hla loci. rapid sequencing of these alleles in newborn cord blood would allow creation of a database of the population's haplotypes, facilitating perfect matches for required bone marrow or solid organ transplants. current uses of in situ hybridization to screen for viruses (such as ebv), light chain restriction (in b lymphomas), and copy number variation (for instance, her gene amplification) demonstrate the benefit of in situ nucleic acid hybridization assays. interphase in situ hybridization with fluorescent probes (fish) is now being used in the initial diagnostic workup for certain diseases, e.g. sarcoma-specific translocations, ploidy analysis in hydatidiform moles, and copy number variation analysis for detection of locus amplification and deletion. rapid dna sequence, rna sequence, and rna abundance screening current uses of dna screening for bcr-abl translocation, donor:recipient ratios after allogeneic bone marrow transplant, microsatellite instability, quantitative viral load (e.g. ebv, bk, cmv), single gene mutations (e.g. cftr, factor , serpina (α- -antitrypsin), hfe), and promoter methylation (e.g. mlh , mgmt) demonstrate the benefit of nucleic acid screening in diagnosis and management. it is likely that each new malignant neoplasm will be promptly inventoried for chromosomal ploidy, gene translocations, gene copy number variation, dna base substitutions/insertions/deletions, annotation of non-synonymous coding sequence and splice-site changes as either benign polymorphisms or pathogenic mutations, local and global promoter methylation status, and rna expression cluster subset. this inventory at the time of initial diagnosis, prior to therapy, should allow molecular subgrouping, individualized therapy, and targeted residual disease screening. as the cost of genome sequencing approaches the $ mark, sequencing of newborns for germline mutations that predispose to subsequent developmental or neoplastic diseases now seems practical for the purposes of early diagnosis, prompt management, and routine follow-up. current uses of morphology, immunohistochemistry, and molecular pathology demonstrate their benefit through improved diagnostic accuracy. however, diagnosis, extent of disease, and molecular subsets are currently imperfect estimators of prognosis and response to therapy. relational databases which correlate an individual's demographic data, family history, and concurrent diseases with the neoplasm's morphologic features, immunophenotype, and molecular subset, and which integrate disease prevalence by age, sex, and ethnicity using bayesian probabilities, should improve accuracy of prognosis and prediction of response to therapy. as risk correlates are developed, it is possible that healthy individuals can be screened and given risk estimates for development of different diseases, as desired. it is worth noting here that unaffected patients in inherited-disease kindreds such as huntington's disease may prefer not to know what the future holds with respect to the disease that runs in their extended family, so germline screening should hinge on pre test patient consent. current uses of normal ranges for serum chemistry assume similar bell-curve distributions across ages, sexes, and ethnicity. this may not be true for all analytes, so normal ranges should be measured and compared, with publication of analytes whose normal ranges differ by age, sex, or ethnic group. computer reference databases facilitate this sort of normal range database, stratified by age/sex/ethnicity of individual patients. similarly, familial risk for an inherited disease may vary by ethnic group, and this variation may be used in bayesian calculations to define risk for unaffected atrisk family members. current uses of liver and renal impairment to guide drug dosage demonstrate the benefit of using patient physiology to customize therapy. we are now starting to use gene haplotype data for specific genes that encode specific drugmetabolizing enzymes, so as to guide starting doses for particular drugs (e.g. warfarin, tamoxifen, or clopidogrel/ plavix) prior to treatment initiation. current uses of prostate specific antigen (psa) to screen for prostate carcinoma and its recurrence demonstrate the benefit of serum biomarkers in common neoplasms. it is likely that high-sensitivity screening for single and clustered serum analytes using proteomic and metabolomic technologies will lead to improved methods for early detection of neoplasms, autoimmune diseases, and infections. residual disease testing for malignancies with clonal mutations or translocations can now be performed via assays based on circulating tumor cells or circulating cell-free tumor dna (so-called liquid biopsies). similar high-sensitivity testing for disease-specific analytes may also improve diagnosis, treatment, and residual disease testing for autoimmune diseases, and infections. treatment by pathway, rather than by tissue diagnosis traditional medical oncology chemotherapy strategies for malignant neoplasms hinged on the tissue diagnosis and the organ of origin. this is changing, as current knowledge of signaling pathway disruption by gain-of-function mutations, e.g. egfr or braf, points to specific treatment targets. the pharmaceutical industry is able to create small-molecule inhibitors and humanized antibodies that allow specific treatment of specific mutant enzyme isoforms. current data suggests that many, if not most, patients with driver gain-of-function mutations treated with specific inhibitors will show an initial brisk response, although later recurrence is possible due to outgrowth of subclones with different driver mutations. the challenge for the next generation of scientists and oncologists is how to rescue homozygous loss-of-function mutations that are etiologic in human diseases, e.g. cftr or p . pathologists consider each disease to have a natural, mechanical, physicochemical basis. each disease has an etiology (initial cause), a pathogenesis (stepwise progression), and a natural history with effects on normal function (clinical outcome). pathologists collect the data needed to answer patients' and clinicians' questions, simply phrased as "what is it?" (diagnosis), "how it going to behave?" (prognosis), and "how do i treat it?" (prediction of response to therapy). instincts and diagnostic criteria, as well as the optical, mechanical, chemical, and computing technologies described previously, are the basis for modern service pathology. as the human genome is deciphered, and as the complex interactions of cellular biochemistry are refined, risk of disease in unaffected individuals will be calculable, disease diagnosis will be increasingly accurate and prognostic, and molecular subsets of morphologically defined disease entities will be used to guide customized therapy for individual patients. it is a great time in history to be a pathologist. g clinically, diseases present to front-line physicians as patients with sets of signs and symptoms. symptoms are the patient's complaints of perceived abnormalities. signs are detected by examination of the patient. the clinical team (including the pathologist) evaluate the patient based on the possible causes of the signs and symptoms (the differential diagnosis). g pathologically, disease is diagnosed by determining whether the morphologic features match the g set of diagnostic criteria previously described for each disease. pathologists diagnose disease by generating a differential diagnosis, then finding the best fit for the clinical presentation, the radiographic appearance, and the pathologic (both clinical lab and morphologic) findings. g etiology describes the causes of a disease. one disease entity can have more than one etiology, and a single etiology can lead to more than one disease. for example, emphysema, chronic bronchitis, and small cell lung carcinoma can all occur in long-term smokers (different diseases derived from a single etiology). likewise, the same disease (for instance, emphysema of the lung) can derive from different etiologies (emphysema from a- -antitrypsin deficiency or cigarette smoke). g the pathogenesis of a disease describes its stepwise progression after initiation in response to a specific etiologic factor (or factors). pathogenesis can refer to the changes in the structure or function of an organism at the gross/clinical level, and it can refer to the stepwise molecular abnormalities leading to changes in cellular and tissue function. g the natural history of a disease describes the expected course of disease, including chronicity, functional impairment, and survival. however, not all patients with a given disease will naturally follow the same disease course, so differences in patient outcome do not necessarily correspond to incorrect diagnosis. variables that independently correlate with clinical outcome differences are called independent prognostic variables, and are assessed routinely in an effort to predict the natural history of the disease in the patient. g variables that independently correlate with response to therapy are called independent predictive variables, and are assessed routinely in an effort to optimize therapeutic response for each patient. suggested readings the greatest benefit to mankind: a medical history of humanity unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration the legacy of karl rokitansky lectures on surgical pathology. london: brown, green, and longmans cellular pathology investigations concerning the etiology of leprosy die Ä etiologie der tuberkulose (the etiology of tuberculosis) rudolf virchow-father of cellular pathology contagiousness of puerperal fever oliver wendell holmes ( À ) and his essay on puerperal fever ignac semmelweis and the etiology of fetal and neonatal sepsis on the germ theory ueber kunstliche bildung des harnstoffs in search of the origins of modern surgical pathology the immunology of transplantation an immunoglobulin heavy chain variable region gene is generated from three segments of dna: vh, d and jh recombination events that activate, diversify, and delete immunoglobulin genes emerging paradigms of t-cell co-stimulation localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody continuous cultures of fused cells secreting antibody of predefined specificity molecular structure of deoxypentose nucleic acids molecular configuration in sodium thymonucleate molecular structure of nucleic acids; a structure for deoxyribose nucleic acid general nature of the genetic code for proteins the rna code and protein synthesis formation and detection of rna-dna hybrid molecules in cytological preparations molecular hybridization of radioactive dna to the dna of cytological preparations expression in escherichia coli of chemically synthesized genes for human insulin dna sequencing with chain-terminating inhibitors a new method for sequencing dna fluorescence detection in automated dna sequence analysis initial sequencing and analysis of the human genome the sequence of the human genome specific enzymatic amplification of dna in vitro: the polymerase chain reaction sternberg's diagnostic surgical pathology. philadelphia: lippincott diagnostic histopathology of tumors. churchill livingstone the continuing role of morphology in the molecular age key: cord- -xj hn authors: moya, alfonso; mínguez, juan j.; martorell, jaime; gallinato, maría j.; recio, alfredo title: congenital peripheral vestibular syndrome in a domestic ferret (mustela putorius furo) date: - - journal: j exot pet med doi: . /j.jepm. . . sha: doc_id: cord_uid: xj hn a -month-old intact female ferret (mustela putorius furo) was presented with a -month history of ataxia. on physical examination, the ferret had difficulty standing upright. during the neurologic examination, the patient had a left head tilt and positional strabismus, circled to the left, and was ataxic. results of the complete blood count were consistent with a mild normocytic normochromic anemia. initial treatment was supportive. magnetic resonance imaging was performed and revealed an asymmetry of the inner ears. a brainstem auditory evoked response test was also performed. history, clinical signs, and diagnostic test results indicated that the ferret was suffering from congenital peripheral vestibular syndrome and left-sided deafness. congenital disease should be considered in the differential diagnosis of young ferrets with peripheral vestibular syndrome. supportive care and physiotherapy can improve balance and motor function, leading to an acceptable quality of life. a -month-old intact female ferret (mustela putorius furo) was presented with a -month history of ataxia, circling, and left head tilt. the condition was obvious at weeks of life, when the ferret's littermates started normal ambulation. the ferret was unable to maintain balance and often would fall on its left side. the ferret was not able to eat without help; therefore, it was fed a handmade chicken puree diet. the rest of the litter showed no clinical signs of illness. on physical examination, the ferret presented with a poor hair coat, weighed . kg with a body condition score of / , and showed difficulty in maintaining equilibrium. no other abnormalities were observed. during the neurologic examination, the patient had a left head tilt, circled to the left, vestibular ataxia, and positional strabismus (fig. ) . no deficits in proprioception were noted. spinal reflexes were present and normal in all limbs. cranial nerves evaluation was normal, except for left vestibulocochlear nerve (cranial nerve viii) deficits. nevertheless, imbalance made further neurologic evaluation difficult. motor activity of the limbs was apparently normal. no other abnormalities were noted during the neurologic examination. the initial diagnostic workup included blood analysis. a complete blood count revealed a mild normocytic normochromic anemia. serum chemistry results were within normal limits. at that time, a differential diagnosis was determined based on the peripheral location of the lesion (receptors in the membranous labyrinth and vestibulocochlear nerve), and included congenital defect, trauma, inflammatory/infectious disease, toxicity, vascular defect, neoplasia, and idiopathic disease. the ferret's owners declined further diagnostic tests during the initial visit owing to financial concerns. supportive treatment was initiated. nutrition was improved by switching to a complete nutritional hand-feeding product for carnivores (carnivore care; oxbow enterprises, inc., murdock, ne usa). medical treatment included citicoline ( mg/kg orally, every hours, somazina; ferrer internacional, barcelona, spain) for its neuroprotective and nootropic effects. homeopathic antivertiginous plant-based medication ( . ml/ferret orally, every hours, vertigoheel gotas; laboratorios heel españa, madrid, spain) and a vitamin and mineral supplement (nutri-plus gel; virbac laboratories, carros, france) were also added to the treatment. physiotherapy exercises and swimming were also recommended to improve limb musculature. the ferret was examined weeks following the initial visit and appeared more alert, weighed . kg, and showed an improvement in body condition (score / ). neurologic examination revealed a left head tilt, but circling was no longer present. no other abnormalities were noted. diagnostic tests were once again recommended, and the owners consented to pursue magnetic resonance imaging (mri). the ferret was premedicated with midazolam ( . mg/kg, intramuscular injection, dormicum; roche farma, madrid, spain) and butorphanol ( . mg/kg, intramuscular injection, torbugesic; fort dodge, ia usa). anesthesia was induced and maintained with . % to % isoflurane (isoflo; abbott laboratories, north chicago, il usa) and -l flow of oxygen through a -mm (outer diameter) endotracheal tube. crystalloid fluids (lactated ringer's solution mixed with % glucose) were administered at the rate of ml/kg/hour through a -g catheter located in the left cephalic vein. capnography and pulse oximetry were used to monitor the patient during the procedure. an mri study of the cranium was performed on the ferret using a . -t system (esaote vetmr). it included sequences corresponding to sagittal, transverse, and dorsal t -weighted (t w); dorsal and transverse t -weighted; transverse fluid attenuated inversion recovery; dorsal dynamic contrast enhanced; and transverse and sagittal multiplanar reconstruction in dynamic contrast enhanced. there were no signs of ischemic, demyelinating, or space-occupying lesions. enlargement or asymmetry of ventricles was not observed. bullae appeared air filled, and fluid or mass lesions were not observed within the tympanic bullae. the only abnormal mri finding was a slight asymmetry in the morphology of inner ears (labyrinth and cochlea). this mild asymmetry was assessed cautiously, as it could have been related to patient positioning during the mri procedure. t w sequences after contrastenhancement paramagnetic gadolinium-based medium ( . ml/kg, intravenously, multihance; laboratorio farmacéutico rovi, madrid, spain) did not reveal any enhancement (fig. ). an otoscopic examination of both ears was performed under anesthesia using a portable otoscope. physical abnormalities such as inflammation, stenosis, or masses were not observed in the external ear canals. microscopic evaluation of samples from the ear canals was unremarkable. the ferret recovered uneventfully from anesthesia and was released from the hospital later that same day. to evaluate the auditory system, a brainstem auditory evoked response (baer) test was recommended. the baer test was performed while the ferret was under general anesthesia. again, the ferret was premedicated with midazolam ( . mg/kg, intramuscularly) and butorphanol ( . mg/kg, intramuscularly). anesthesia was induced and maintained with . % to % isoflurane via a facemask. the patient was then placed in ventral recumbency, and pulse oximetry was used to monitor oxygen saturation and heart rate. subdermal electrodes were inserted using adapted needles. a recording electrode was applied to the vertex (dorsal aspect of the skull), reference electrodes in the base of the external ear canal, and a ground electrode in the nuchal crest. electroacoustic transducers were inserted into the external auditory canals. owing to small patient size, eppendorf tubes were adapted as earphones for the transducers, modified to fit securely over the ferret's external ears (fig. ) . alternating wave patterns were used for the test and had an increasing range of to db normal hearing level. click stimuli were employed, with a frequency range of to khz and -ms length as well as a masking noise in the nonstimulated ear and db under the stimulated ear. an average of click stimuli were generated in each ear to get the tracing. right ear baer test recorded all the main waves (p , p , p , p , and p ), with p to p latencies of . , . , . , and . ms. right ear results were considered normal for a -month-old ferret, as minor prolonged p and p latencies could be attributed to the use of adapted earphones. the left ear baer test was unable to record any main wave (fig. ) . these results were consistent with a sensorineural lesion causing left-sided deafness. the ferret recovered uneventfully from anesthesia, and it was released from the hospital later that same day. the clinical signs and results of complementary tests were consistent with a diagnosis of peripheral vestibular syndrome, presumably of congenital origin considering the young age of the ferret and early onset of clinical signs, and the presence of left-sided deafness. owing to the assumed congenital etiology of the lesion and mild improvement in clinical signs, no other diagnostic tests were indicated. initial treatment and physiotherapy exercises were continued to improve muscle tone of the limbs. the ferret showed a slow progressive improvement of balance, and adapted by leaning on vertical surfaces to facilitate movement. owing to difficulty of mastication, the ferret continued to be fed a liquid diet. quality of life was judged adequate based on activity level, food and water consumption, and absence of pain or other disease conditions. at last report, the ferret was alive with no increase of clinical disease signs months after diagnosis. to the authors' knowledge, this is the first case report describing congenital peripheral vestibular syndrome in a ferret. in ferrets, several causes of vestibular diseases have been described, including toxins, infectious diseases (e.g., canine distemper virus, aleutian disease, rabies, toxoplasmosis, and ferret systemic coronavirus), neoplasia (e.g., choroid plexus papilloma and granular cell tumor), trauma, and systemic disease (e.g., hypoglycemia). [ ] [ ] [ ] [ ] [ ] [ ] causes of vestibular disease in dogs and cats include neoplasia, trauma, toxin exposure, infectious disease, inflammatory disease, vascular disease, nutritional disorders, idiopathic disease, and congenital disorders. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for congenital causes, a hereditary origin is assumed as some breeds appear to be predisposed to vestibular disease. nonetheless, pathogenesis of the disease is not well known. onset of vestibular clinical signs in these animals can vary, ranging from birth to several weeks of age, with both unilateral or bilateral deafness being present. , vestibular disease can be classified as either peripheral or central, depending on the location of the lesion. , lesions located in receptors of the membranous labyrinth and vestibulocochlear nerve deficits result in peripheral vestibular disease. central vestibular disease results from lesions to the vestibular nuclei in the medulla or the vestibular projections to the cerebellum (cerebellar nuclei). both peripheral and central vestibular disease can usually be distinguished by the presence of specific neurologic signs and diagnostic testing. [ ] [ ] [ ] common clinical disease signs of vestibular disease include ataxia, head tilt, nystagmus, and strabismus. lesions affecting the central vestibular system typically produce clinical signs suggesting involvement of the medulla, including altered mentation, ipsilateral proprioceptive deficits, vertical nystagmus, and involvement of multiple cranial nerves. the anatomic diagnosis of peripheral vestibular disease is made in the absence of clinical disease signs associated with the medulla. problems of balance resulting in incoordination are prominent in animals with both peripheral and central vestibular diseases. [ ] [ ] [ ] [ ] [ ] [ ] animals with both peripheral and central vestibular diseases often have decreased extensor muscle tone in the forelimbs and rear limbs on the side of the lesion and increased extensor tone in the limbs on the contralateral side. these abnormalities result in a tendency for the animal to fall or roll toward the side of the lesion. however, animals with peripheral vestibular disease have no loss of general proprioception, which is assessed by performing postural reaction tests. owing to impaired balance, postural reaction tests can be difficult to perform in affected animals. ferrets with cochlear agenesia or hypoplasia show various degrees of imbalance associated with vestibular syndrome. in addition to this, these animals generally have poor muscle development, aggravating the inability to correctly stand. there is evidence that the development of the vestibular system is essential for correct neuromuscular development of the ferret during the initial neonatal period (first month of life). , ferrets undergoing labyrinthectomies before complete development of mechanoreceptors showed delays in motor development and shorter and less contractile muscles. antemortem diagnosis of congenital vestibular syndromes can be difficult. compatible history and clinical disease signs are strongly suggestive. however, complementary tests are needed to rule out other common causes of vestibular ataxia, such as otitis media/interna. mri is the diagnostic imaging modality of choice for the central nervous system, as it allows the clinician to evaluate soft tissue, especially those surrounded by bone. specific additional mri sequences can be useful to detect hemorrhage (gradient echo sequences), ischemic disease (diffusion weighted imaging), peri-lesional edema and the presence of pure fluid (fluid attenuated inversion recovery sequences), and muscle, bone and nerve root abnormalities (short ti inversion recovery). computed tomographic imaging provides excellent images of bony structures and is indicated where osseous changes are of greatest diagnostic importance. computed tomography and mri are complementary imaging studies of the middle ear, labyrinth, internal auditory canal, and their contents, but mri is superior in imaging soft tissue components including intralabyrinthine fluid. [ ] [ ] [ ] [ ] [ ] in this case, only one of the tests was available for economic reasons, and mri was considered the imaging test of choice for better evaluation of the central nervous system and tympanic bullae. peripheral deafness is due to disorders of the peripheral auditory organ (middle/inner ear and cochlea). peripheral deafness can be classified as sensorineural or conductive. conductive deafness results when sound is blocked from reaching the internal ear because of outer or middle ear pathologies. sensorineural deafness occurs when there is damage to the hair cells or afferent neurons of the cochlea. in dogs and cats, pigmentassociated deafness is both the most common type of deafness and form of congenital deafness. , the white, deaf phenotype has been reported in multiple species, including the ferret, cat, dog, mouse, mink, horse, rat, syrian hamster, alpaca, and human. a hereditary basis is described, and the genes responsible for white or lightened pigmentation in skin and hair suppress melanocyte function. suppression of melanocytes in the stria vascularis disrupts their regulation of the endolymph composition, and deafness ensues as a secondary effect. , vestibular disease is not a finding in these animals. in ferrets, the pigmentassociated hereditary defect has been described as waardenburg syndrome. albino ferrets usually retain hearing for the very high frequencies, which may be of limited practical benefit. these animals demonstrate an abnormally small hearing mechanism in the middle and inner ear. congenital peripheral vestibular syndrome accompanied by nonpigment-associated deafness has been documented in dogs. these animals have cochlear and vestibular hair cell degeneration. although the mechanisms for this type of deafness is not known in dogs, human and mouse models frequently show channelopathies (mutations in genes responsible for neuron membrane ion channels, especially potassium). a similar condition unrelated to coat color was suspected in the ferret in this case report, owing to the presence of vestibular disease. other nonhereditary congenital causes of both peripheral vestibular syndrome and deafness have been described, and these include perinatal anoxia, dystocia, and intrauterine ototoxin exposure. , neither dystocia nor medical treatments administered during gestation were reported by the owners. the baer test is used for hearing assessment of people, dogs, and cats presenting for congenital deafness screening. [ ] [ ] [ ] the baer test allows for assessment of the cochlear division of vestibulocochlear nerve, the receptors, and various brainstem nuclei. , in puppies, baer studies report both high sensitivity ( %) and specificity ( %) for diagnosing deafness. in ferrets, baer is a noninvasive test that can be performed in a similar way. sedation or anesthesia is recommended. ferrets have a relatively late onset of maturity of the auditory system, and adultlike baer thresholds are observed by postnatal day . the normal baer in adult ferret consists of four prominent positive peaks (p to p ) and a fifth peak of smaller amplitude and more variable latency. published mean reference ranges for latencies in ferrets for p to p are . , . , . and . ms. the absence of baer waves in the left ear of this ferret indicated a sensorineural disease (deafness). the baer test should be considered when a ferret presents with vestibular disease, and evaluation of inner ear function is required, or to confirm deafness. citicoline (cytidine diphosphate-choline) is a neurostimulant, which has neuroprotective and nootropic properties. its mechanism of action is through biosynthesis promotion of neuronal membrane phospholipids and inhibiting apoptosis. , recent studies suggest the neuroregenerative potential of citicoline. [ ] [ ] [ ] the homeopathic antivertiginous plant-based medication vertigoheel gotas contains the herbs conium maculatum, ambra grisea, petroleum rectificatum, and anamirta cocculus. indications for the use of this compound in humans include nonspecific causes of vertigo and other related imbalance disorders, and related symptoms such as nausea. , although the exact mechanism of action of vertigoheel gotas is not fully understood, studies suggest that it activates the vestibular system in the brainstem area, which may facilitate more accurate communication between the peripheral vestibular system and the brain. , prognosis for congenital vestibular disease in mammals depends on location and the extension of the lesions. - peripheral vestibular disease generally carries a better prognosis when compared with central vestibular disease. in the authors' experience, ferrets suffering from congenital peripheral syndrome can progressively improve balance and thus, improve their ability to walk. ferrets will likely benefit from supportive care and physiotherapy, including assisted exercise and swimming. the use of antivertiginous, neuroprotective, and nootropic drugs could help to improve balance and promote better quality of life in cases of peripheral vestibular diseases in ferrets. musculoskeletal and neurological diseases neurological syndrome in the ferret (mustela putorius furo) diagnostic exercise: ataxia and incoordination in ferrets vestibular syndrome due to a choroid plexus papilloma in a ferret granular cell tumor in the central nervous system of a ferret (mustela putorius furo) ferret coronavirusassociated diseases small animal internal medicine vestibular dysfunction diseases of the middle and inner ear vestibular system: special proprioception ataxia of the head and the limbs the neurology of balance: function and dysfunction of the vestibular system in dogs and cats congenital peripheral vestibular disease attributed to lymphocytic labyrinthitis in two related litters of doberman pinscher pups congenital vestibular disease in a german shepherd dog congenital deafness and vestibular deficit in the doberman canine deafness a critical period for the impact of vestibular sensation on ferret motor development development of banded afferent compartments in the inferior colliculus before onset of hearing in ferrets analysis of the -dimensional fluid-attenuated inversion-recovery ( d-flair) sequence in idiopathic sudden sensorineural hearing loss magnetic resonance imaging after translabyrinthine complete excision of vestibular schwannomas magnetic resonance imaging of the normal and diseased feline middle ear review of diagnostic imaging of ear diseases in the dog and cat inner ear disorders feline deafness another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear diseases of the ferret ear, eye and nose physiology of the ferret kv -type channel currents in spiral ganglion neurons. involvement in sensorineural hearing loss newborn hearing screening and strategy for early detection of hearing loss in infants transient evoked otoacoustic emissions testing for screening of sensorineural deafness in puppies unilateral and bilateral congenital sensorineural deafness in client-owned pure-breed white cats auditory brainstem of the ferret: maturation of the brainstem auditory evoked response bilateral deafness in a maltese terrier and a great pyrenean puppy: inner ear morphology brainstem auditory evoked response in the ferret (mustela putorius) citicoline: update on a promising and widely available agent for neuroprotection and neurorepair neuroprotection and recovery: recent data at the bench on citicoline citicoline and postconditioning provides neuroprotection in a rat model of ischemic spinal cord injury citicoline enhances neuroregenerative processes after experimental stroke in rats cpd-choline and its endogenous metabolites, cytidine and choline, promote the nerve regeneration and improve the functional recovery of injured rat sciatic nerves the clinical efficacy of vertigoheel in the treatment of vertigo of various etiology homeopathic vs conventional treatment of vertigo: a randomized double-blind controlled clinical study the low-dose combination preparation vertigoheel activates cyclic nucleotide pathways and stimulates vasorelaxation myogenic vestibular evoked potentials used to objective estimation of effectiveness of central action drugs the authors would like to thank rina maguire, bvsc, for her useful comments and text review on the manuscript, and maría páez for her help with english grammar. key: cord- -aowe kyl authors: chen, li; ghanbarnejad, fakhteh; brockmann, dirk title: phase transitions and hysteresis of cooperative contagion processes date: - - journal: nan doi: . / - /aa bd sha: doc_id: cord_uid: aowe kyl we investigate the effects of cooperation between two interacting infectious diseases that spread and stabilize in a host population. we propose a model in which individuals that are infected with one disease are more likely to acquire the second disease, both diseases following the susceptible-infected-susceptible reaction scheme. we analyze cooperative coinfection in stochastic network models as well as the idealized, well-mixed mean field system and show that cooperative mechanisms dramatically change the nature of phase transitions compared to single disease dynamics. we show that, generically, cooperative coinfection exhibits discontinuous transitions from the disease free to high prevalence state when a critical transmission rate is crossed. furthermore, cooperative coinfection exhibits two distinct critical points, one for outbreaks the second one for eradication that can be substantially lower. this implies that cooperative coinfection exhibits hysteresis in its response to changing effective transmission rates or equivalently the basic reproduction number. we compute these critical parameters as a function of a cooperativity coefficient in the well-mixed mean field system. we finally investigate a spatially extended version of the model and show that cooperative interactions between diseases change the general wave propagation properties of conventional spreading phenomena of single diseases. the presented work may serve as a starting and reference point for a more comprehensive understanding of interacting diseases that spread in populations. we investigate the effects of cooperation between two interacting infectious diseases that spread and stabilize in a host population. we propose a model in which individuals that are infected with one disease are more likely to acquire the second disease, both diseases following the susceptibleinfected-susceptible reaction scheme. we analyze cooperative coinfection in stochastic network models as well as the idealized, well-mixed mean field system and show that cooperative mechanisms dramatically change the nature of phase transitions compared to single disease dynamics. we show that, generically, cooperative coinfection exhibits discontinuous transitions from the disease free to high prevalence state when a critical transmission rate is crossed. furthermore, cooperative coinfection exhibits two distinct critical points, one for outbreaks the second one for eradication that can be substantially lower. this implies that cooperative coinfection exhibits hysteresis in its response to changing effective transmission rates or equivalently the basic reproduction number. we compute these critical parameters as a function of a cooperativity coefficient in the well-mixed mean field system. we finally investigate a spatially extended version of the model and show that cooperative interactions between diseases change the general wave propagation properties of conventional spreading phenomena of single diseases. the presented work may serve as a starting and reference point for a more comprehensive understanding of interacting diseases that spread in populations. the proliferation and prevalence of infectious diseases continue to pose a significant threat to human society [ ] . the combination of increasing population densities and global mobility facilitates the emergence of new pathogens that can quickly spread on a global scale, exemplified by outbreaks in the last decade, e.g. the sars epidemic [ , ] , the h n pandemic [ , ] , the emergence of the novel mers-cov in [ ] , and the ebola crisis [ ] . mathematical, dynamical, statistical, and computational models have become an essential tool for understanding disease dynamics and for designing effective mitigation strategies [ ] . dynamical models cover a broad scope in terms of complexity, ranging from parsimonious and conceptual models [ , ] , network models that account for population structure and/or host mobility patterns [ ] [ ] [ ] [ ] [ ] [ ] [ ] , to sophisticated, large-scale computational models [ , ] that incorporate high resolution data on multi-scale transportation, demographics, epidemiological factors, and behavioral response rules. state-of-the-art computational models have become remarkably successful in reproducing observed patterns * electronic address: chenl@rki.de † electronic address: fakhteh@pks.mpg.de ‡ electronic address: dirk.brockmann@hu-berlin.de and predicting the trend of ongoing epidemics. the majority of epidemic models are designed to capture epidemiological dynamics of single pathogen species in a host population, characterized by epidemiological parameters associated with a single virus or bacterium. however, bacterial and viral pathogens generically interact, either directly or indirectly by means of their effects on the host. prominent examples are hiv infections that through immune suppression increase the host's susceptibility towards other infections [ ] , including hepatitis [ ] , malaria [ ] , syphilis, herpes virus, tuberculosis [ , ] and more. this phenomenon, i.e. being infected with more than one pathogen is known as syndemics or coinfection [ ] . although, coinfections have been well documented and received considerable attention by epidemiologist, the dynamical implication of disease interactions in epidemic progression is unclear and the generic dynamical behavior is poorly understood. although some models have been developed recently to describe coinfection dynamics, the majority of them consider competitive interactions between diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] , i.e. infection with one disease induces a host's cross immunity towards another infection. in these systems different phases such as extinction, coexistence, and onestrain dominance have been identified. in one class of models coinfection is captured by a multiplex network approach [ ] [ ] [ ] , where each infectious agent is transmitted along a different sets of links in the same population [ ] [ ] [ ] . recently, researchers also considered cooperative pathogen interactions [ ] [ ] [ ] ] tion with one disease facilitating infection with another pathogen and vice versa. in ref. [ ] , a simple coinfection model of susceptible-infected-recovered (sir) type was investigated within the framework of cooperative bond percolation. this model exhibits avalanche-like outbreak scenarios, depending on the level of cooperation and the structure of the underlying transmission network. furthermore, it has been found that highly clustered structures in population aid the proliferation of coinfections, contrary to the effect observed in single disease dynamics [ ] . nevertheless, some fundamental issues remain elusive: what basic dynamical features can we expect in multiple-epidemic spreading with disease-disease interactions? to what extent does coinfection change the classic outbreak scenario of single diseases? how does coinfection dynamics shape the generic features of spatial propagation? here we introduce and investigate a straightforward network model for the contagion process of two interacting epidemics (labeled a and b) in which the interaction is explicitly incorporated. the kinetics of both diseases follows the generic susceptible-infected-susceptible (sis) reaction scheme, in which infected individuals transmit the disease to susceptibles at a specified rate. infected individuals remain infectious for a typical infectious period before they become susceptible again. based on this model, we show that cooperative coinfections possess a variety of important features that are absent in single disease dynamics. for sufficiently strong cooperation, increasing the infection rate of both diseases yields abrupt, discontinuous outbreak transitions. furthermore, cooperative coinfection generically exhibits dynamical hysteresis, a consequence of different threshold for disease outbreak and disease eradication. finally we show that in spatially extended systems, wavefront velocities are considerably accelerated in certain parameter regimes, while the propagation wave can be considerably slowed down when the system approaches the eradication threshold, even progress in a backwards direction. we consider a stochastic network in which each node represents a host individual, e.g. a person, in a population and links are potential transmission channels. in the conventional sis system each node is either susceptible or infected. if the transmission probability across a link is p, a susceptible node can acquire an infection from one of its m infected neighbors with probability −( −p) m . an infected node recovers from infection and becomes susceptible again with probability r. the coinfection scenario is modeled analogously. having two types of infections, we denote the infected states by one of the diseases by a and b, respectively and coinfection by ab (see fig. ). we assume that susceptibles are infected at probability p, whereas individuals in class a and b are infected with the complementary disease at probability q. cooperative coinfection corresponds to a scenario with q > p, i.e. being infected by one pathogen increases the infection probability with the other. in contrast, the condition q < p corresponds to antagonistic effects. when q = p the system corresponds to two non-interacting infections. for simplicity we consider identical recovery probabilities (r = . ) for both infections [ ] . to understand the general behavior of the system and identify the nature of phase transitions, we investigated the dynamics on d square lattices and random networks while monitoring the fraction of non-susceptible nodes, − ρ s in equilibrium, i.e. the fraction of nodes that are either infected by a or b or both, as a function of transmission rate parameters p and q. −ρ s can be considered the order parameter of the system. at fixed q, we distinguish forward (i.e. increasing p) and backward (i.e. decreasing p) direction, yielding an outbreak phase transition (opt) and an eradication phase transition (ept), respectively. these transitions are identical in the ordinary sis model. to initiate an epidemic, two nodes are randomly chosen and seeded with a and b, respectively. we first study the system on d square lattice. in the limit of strong cooperation [ ] p q = , we find a discontinuous opt. when p increases beyond the critical point p o c ≈ . , the system exhibits a discontinuous jump to a large endemic state, see fig. a , contrary to the single disease case (conventional sis dynamics with a threshold p c ). however, the critical point is identical in both systems (i.e., p o c ≈ p c ). this is understandable, as for a and b seeds to generate cooperative effects, each single disease must be sustained for some time in order for single disease fronts to meet and initiate cooperative effects. once the two wave fronts touch, the amplified coinfection establishes and acquires access to a consider-able fraction of the population yielding a discontinuous phase transition [ ] . this type of behavior was also observed in cooperative bond percolation [ ] . in addition to the discontinuous opt we find that eradication of an endemic state occurs at a substantially reduced threshold p e c (p e c < p o c ) in the cooperative regime [ ] . this means that even a very small basic transmission probability p is sufficient to sustain the coinfection in the population. note that although the transition is very steep, it is continuous in the d-lattice. cooperative coinfection therefore exhibits a clear hysteresis in its order parameter. once a coinfection outbreak has established an endemic state in the population, a large effort is required to eradicate the cooperating diseases. the magnitude of this hysteresis effect increases with increasing degree of cooperation, i.e. increasing q, see fig. c . the hysteresis loop disappears if q < q c ≈ . , where these two thresholds coincide. qualitatively, we observe the same behavior in erdős-réyni (er) random networks (figure b and d) . a prominent difference, however, is that here the eradication phase transition is also discontinuous. this implies that the infection suddenly disappears as the eradication threshold is crossed from above. this different behavior at the ept in the er-network is due to the net- work's lack of short loops that are present in the lattice and that are required to sustain coinfection. in the ernetwork, single infection pertrusions have to move along much longer paths to meet and incite a coinfection spark. fig. a and b show typical endemic patterns in the dlattice system for independent, non-cooperative (p = q) and cooperative (q = ) parameter values, both being slightly beyond the critical point. in the first case (fig. a) , the pattern is loosely distributed without any significant spatial structure, with a small fraction of ab infected sites. in contrast, in the cooperative case (fig. b) , the infected clusters exhibit spatial structure, in the sense that the backbones of the infected islands always consist of essential ab individuals that sustain the coinfection. this morphological difference is further evidence that in the cooperative case, it is the disease interaction and a significant fraction of coinfected ab individuals that support the survival in the regime p < p c (see fig. ). this is evident in fig. c that depicts the fraction individuals that are infected by both diseases normalized by the fraction of infected individuals of all types, i.e. ρ ab /(ρ a +ρ b +ρ ab ). without cooperation this fraction continuously decreases until the critical points is reached. when cooperation is acting, the fraction of ab individuals remains almost constant until the eradication threshold is reached below which the endemic state col-lapses. to better understand the nature of phase transitions, we provide the full phase diagrams for the two topologies in fig. a and b . in either case, the outbreak transition point is at p o c = p c , irrespective to q. tricritical points [ ] are identified, which separate the discontinuous transition (for q > q c ) and the continuous transition (for q < q c ). coincidentally, this tricritical point of outbreak transition is also the tricritical point of the eradication phase transition for er networks, but not for the d lattice, since its eradication phase transitions are all continuous. apart from the outbreak size a characteristic feature of the stochastic dynamics is the outbreak probability p , i.e. the chance that an outbreak occurs in response to a small infected seed. in the coinfection dynamical system we distinguish two outbreak probabilities. first we define p (a ∪ b) as the probability that an initial seed yields an endemic state for either single infection a, b or coinfection ab. we also define p (a ∩ b) as for generating coinfection endemic where both diseases must survive. the results are shown in fig. c and d . although the outbreak size transition is discontinuous, the two probability transitions are continuous and occur at the same threshold p c . note that, er network and dlattice exhibit clearly different p (a ∩ b) profiles. in the random network p (a ∩ b) increases more slowly with p although both topologies have identical average degree. again, a plausible explanation here is that the lattice system exhibits a higher likelihood of short loops that facilitate the outbreak of coinfection. the similarities as well as differences in the nature of phase transitions in the lattice system on one hand and the er-network on the other hand pose the question which properties of the phase transitions are observed in a well mixed particle kinetic system that is unaffected by network structure and fluctuations in the system. to address this question we analyzed the mean field dynamical system that corresponds to cooperative sis dynamics. the basic reaction scheme underlying the dynamical process is given by as a function of infection rate α for different levels of cooperativity c. solid and dashed lines correspond to stable and unstable stationary states, respectively. (b) typical bifurcation types for continuous (c = ), tri-critical (c = ), and discontinuous (c = ) transitions. all show transcritical bifurcation at αc except for tri-critical case, which is of pitchfork type instead. furthermore, a saddle-node bifurcation precedes at α e c in the discontinuous case. as a consequence, a bistable region is found for the case with c > , which is bounded by αc and α e c , corresponding to critical outbreak and eradication transition rate, respectively. where s, i a , i b , i ab correspond to the fraction of s, a, b and ab individuals in the population. as in the previous section we assume for simplicity that the diseases are characterized by identical parameters. this assumption can be relaxed without changing the qualitative behavior but eases the analysis. we can simplify the system by assuming that i a (t) = i b (t) which follows for symmetric initial conditions. however, even for nonsymmetric conditions the system approaches this symmetric state. defining new variables p = i a = i b and x = i a + i ab = i b + i ab , the above system simplifies tȯ where we have absorbed the recovery rate γ into time, other rates are expressed in units of γ. we can conveniently define c = β/α as the cooperativity coefficient. the epidemic interaction is cooperative if c > and antagonistic for c < . the special cases with c = corresponds to non-interacting diseases (equivalent to p = q in sec. ii). figure depicts the asymptotic behavior of the system for different levels of cooperativity c. a bifurcation analysis shows that for weak cooperation (c < ), the system exhibits a continuous transition, at α c = , the same threshold as the single sis case. for c > , a bistable region emerges, with a different eradication threshold α e c = √ c − /c. hence, a hysteresis loop is formed on the interval (α e c , α c ). both bifurcations at α c are transcritical. the key bifurcation is the additional saddle-node bifurcation at α e c that generates a bistable region. the case of c = has the critical level of cooperation which connects continuous and discontinuous bifurcations yielding a pitchfork bifurcation. this analysis also implies that in the bistable regime, an unstable stationary state exists between both stable states and that only for sufficiently substantial perturbations above this unstable stationary state will invoke a non-trivial endemic state. overall, these results qualitatively are in good agreement with what we observed in numerical experiments in sec. ii, especially in the er-network system, which is naturally more similar to the well-mixed population structure as required by the mean field treatment. the defining feature of the bistable regime in cooperative coinfection is the fact that two stable stationary states co-exist: i) the disease free state and ii) the macroscopic endemic state, separated by an unstable intermediate stationary state. because of this, a non-infinitesimal perturbation of the disease free state is required to push the system into the attractor of the endemic state. therefore, it is reasonable to assume that the standard properties of wave propagations of pulled-fronts (diffusion into an unstable state) in a spatially extended system is dif- ferent for cooperative coinfection. in order to address these question we investigate the generic generalization of the above model to include spatial coordinates and allow individuals to diffuse in space. for simplicity, we first consider the corresponding reaction-diffusion system in one dimension: where d in last terms is the diffusion coefficient. we assume that diffusion is constant and independent of the state of a host individual. here we use also the symmetric parameters for the two diseases and set γ = . for sufficiently localized initial conditions (and only one disease) the above system exhibits a propagating wave asymptotically with a wave front velocity of v = αd( − /α) for α > , these types of wave fronts are usually referred to as "pulled fronts" [ ] . interestingly, very different propagation modes arise in the cooperative coinfection system (red solid line in fig. a ). the first observation is that the velocity is larger than the single disease case for a given α, which can be explained by cooperative effect and is less surprising. however, a negative wavefront velocity v is found for values of α e c < α < α st , equivalent to a wave that is propagating backwards and shrinking the coinfection bulk. coinfec-tion cannot be sustained in this range of α. note however, that at these values of α the diffusion free system possesses a stable coinfection state. therefore, the diffusion in this regime dilutes the coinfected individuals to a level where the endemic state vanishes. this can only occur, however, in a system that requires a finite threshold (in coinfection the unstable stationary state) above which a concentration needs to grow in order to reach the stable endemic state. the effect becomes more prominent when α → α e c , because the attractive basin of the non-outbreak branch expands to substantial fraction of phase space. a marginal scenario is observed when diffusion and reaction forces balance and yield a stable "standing wave" (v = ) that we also observe in numerical experiments. we denote this point by α st , which marks the separation of the two moving directions. figure illustrating the three typical propagation modes in d space are depicted ( )). bottom panels (i-l): backward propagation (α = . ). the infected fraction ia+ib +iab is color coded. the sequence (from left to right) of panels depicts the time course of the infected regions at time t = , , , , respectively. here αst is slightly different from the value in d space, up to the dimension correction. initial conditions start from some randomly infected round regions with random radius as shown in the first column. other parameters: c = , d = . (see the movies in the supplemental material [ ] ). one situation of particularly practical interest is when a disease invades a region that has already been infected by the other one. this happens when the two diseases enter a region at different time so that the secondary infection acts in an established region by the first infection wave. when this happens, the spread of the secondary disease (blue dashed-dotted) is found not only much faster than its usual speed of single disease (black dashed), but is also faster than the wave of coinfection propagation (red solid). we presented a class of structurally parsimonious models of two cooperating epidemics that spread within the same structured population. we have shown that new dynamical properties emerge as a consequence of cooperative coinfection that are absent in single disease dynamics or antagonistic coinfection dynamics. the most prominent property is the existence of discontin-uous transitions to an endemic state when the disease specific outbreak threshold is crossed and that outbreak and eradication transitions occur at different critical parameters. this is an important insight for designing eradication protocols for pathogens that are known to interact with other diseases in a cooperative way. because of the universality of this property of intrinsic hysteresis, we believe that with increasing data on interactions between pathogens in traditional epidemiology the simple single threshold concept may have to be revised and extended. the proposed model can serve as a valuable reference point for dynamical systems in which not only two but more contagion processes cooperate, potentially with a variety of disease specific parameters. for example, an entire network of diseases with different degrees of cooperation or conflict may yield a complex system of possible stationary state and much more complex transitions as parameter thresholds are crossed. furthermore, because the simple sis dynamical system finds applications outside of epidemiology, for example in adoption dynamics, our results may also find applications in situations when technologies are adopted in social systems. infectious diseases of humans: dynamics and control forecast and control of epidemics in a globalized world predictability and epidemic pathways in global outbreaks of infectious diseases: the sars case study pandemic potential of a strain of influenza a (h n ): early findings the hidden geometry of complex, network-driven contagion phenomena middle east respiratory syndrome world health organization modeling infectious diseases: in humans and animals a contribution to the mathematical theory of epidemics the mathematics of infectious diseases dynamical processes on complex networks epidemic spreading in scale-free networks the role of the airline transportation network in the prediction and predictability of global epidemics the scaling laws of human travel understanding individual human mobility patterns multiscale mobility networks and the spatial spreading of infectious diseases natural human mobility patterns and spatial spread of infectious diseases modeling disease outbreaks in realistic urban social networks the gleamviz computational tool, a publicly available software to explore realistic epidemic spreading scenarios at the global scale hiv and co-infections epidemiology of viral hepatitis and hiv co-infection dual infection with hiv and malaria fuels the spread of both diseases in sub-saharan africa tuberculosis and hiv co-infection world health organization introduction to syndemics: a critical systems approach to public and community health interacting epidemics on overlay networks modeling the dynamical interaction between epidemics on overlay networks competitive epidemic spreading over arbitrary multilayer networks threshold effects for two pathogens spreading on a network epidemic dynamics of two species of interacting particles on scale-free networks competing epidemics on complex networks host mobility drives pathogen competition in spatially structured populations catastrophic cascade of failures in interdependent networks mathematical formulation of multilayer networks dynamics of interacting diseases interacting epidemics and coinfection on contact networks outbreaks of coinfections: the critical role of cooperativity avalanche-outbreaks emerging in cooperative contagions phase transitions in cooperative coinfections: simulation results for networks and lattices complex dynamics of synergistic coinfections on realistically clustered networks we have qualitatively the same observations for other values of the recovery probability r . here, we choose the value of r = . being a bit smaller than unity (often chosen in previous studies of single disease), just because a smaller r leads to a longer stay in a or b state the choice of q with other large values showing strong cooperativity doesn't change our observations quanlitatively, as one can see from fig see supplemental material in the attached files for movies illustrating the typical outbreak and eradication processes. also an online interactive d webpage is available for playing at generalized epidemic process and tricritical dynamic percolation continuation and bifurcation software for ordinary differential equations (with hom-cont) see supplemental material in the attached files for movies illustrating the three wave propagation modes key: cord- -nwbmxepi authors: margină, denisa; ungurianu, anca; purdel, carmen; tsoukalas, dimitris; sarandi, evangelia; thanasoula, maria; tekos, fotios; mesnage, robin; kouretas, demetrios; tsatsakis, aristidis title: chronic inflammation in the context of everyday life: dietary changes as mitigating factors date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: nwbmxepi the lifestyle adopted by most people in western societies has an important impact on the propensity to metabolic disorders (e.g., diabetes, cancer, cardiovascular disease, neurodegenerative diseases). this is often accompanied by chronic low-grade inflammation, driven by the activation of various molecular pathways such as stat (signal transducer and activator of transcription ), ikk (iκb kinase), mmp (matrix metallopeptidase ), mapk (mitogen-activated protein kinases), cox (cyclooxigenase ), and nf-kβ (nuclear factor kappa-light-chain-enhancer of activated b cells). multiple intervention studies have demonstrated that lifestyle changes can lead to reduced inflammation and improved health. this can be linked to the concept of real-life risk simulation, since humans are continuously exposed to dietary factors in small doses and complex combinations (e.g., polyphenols, fibers, polyunsaturated fatty acids, etc.). inflammation biomarkers improve in patients who consume a certain amount of fiber per day; some even losing weight. fasting in combination with calorie restriction modulates molecular mechanisms such as m-tor, foxo, nrf , ampk, and sirtuins, ultimately leads to significantly reduced inflammatory marker levels, as well as improved metabolic markers. moving toward healthier dietary habits at the individual level and in publicly-funded institutions, such as schools or hospitals, could help improving public health, reducing healthcare costs and improving community resilience to epidemics (such as covid- ), which predominantly affects individuals with metabolic diseases. chronic inflammation is a central process involved in a high number of metabolic disorders (e.g., obesity, metabolic syndrome, diabetes, dyslipidemia, etc.), including neurodegenerative (alzheimer), malignant diseases, and autoimmune diseases. in most if not all chronic inflammatory conditions, there is an extensively failed resolution of inflammation with high influx of leukocytes, which in their effort to resolve inflammation stimulate the synthesis of pro-inflammatory molecules and establish a highly inflammatory micro-environment, leading to extensive fibrosis and tissue damage [ ] . chronic low-grade inflammation has been shown to either induce or aggravate metabolic disturbances, including insulin resistance and dyslipidemia, which contributes to the development of other complications [ ] . there is accumulating evidence that, in the case of autoimmune diseases, when the immune system loses self-tolerance and attacks the body's cells and tissues, metabolic disturbances are key contributors to disease progression. results from the type diabetes mellitus (t dm) prediction and prevention studies on t dm showed that metabolic disturbances preceded the seroconversion to positive autoantibodies by several months or years in type diabetes mellitus [ , ] . many chronic inflammatory diseases originate or have their development promoted by an unbalanced diet. although the exact mechanism remains unclear, de rosa et al. suggest that metabolic pressure, as a result of increased caloric intake, leads to an altered adipose tissue homeostasis. this results in the synthesis of adipokines and facilitates the overactivation of nutrient-sensing mechanisms, altering the balance between pro-inflammatory and regulatory t-cells, ultimately resulting in the loss of immunotolerance [ ] [ ] [ ] . in addition, dietary components have the ability to influence the immune response through the modulation of gut bacteria metabolism, impacting the risk of developing chronic diseases either directly in the gastrointestinal tract, or in other more distant organs that impact general metabolism [ ] [ ] [ ] [ ] . recent studies have investigated long term exposure to low doses of chemical mixtures that can be a part of modern lifestyles, such as pesticides, food additives, or additives contained in food coating materials, proving that different disturbances appeared from minor biochemical disturbances. these early alterations are generally followed by oxidative stress induction and organ damage depending on the period of exposure [ ] [ ] [ ] [ ] [ ] [ ] . recently, it has been shown that long term exposure to stressors might also have a positive association with increased vulnerability of the population to the microbial and viral infections [ ] . metabolomics are an emerging biological field that allow for the identification and simultaneous measurement of a large number of small molecules called metabolites in biological matrixes. it has become the most accurate method to detect metabolic imbalances and is useful for prevention and early detection of diseases. moreover, metabolomics have vast applications in clinical practice [ ] . targeted metabolomic analysis provides insights regarding the normal function of endogenous metabolism, dietary intake, microbiota, drug metabolism, and nutrient adequacy [ ] . the challenge of chronic inflammatory diseases with respect to early diagnosis can be tackled with metabolomics through the identification of biomarkers that can discriminate high-risk populations. in a group of autoimmune patients, it was found that their fatty acid-based metabolic profile and lifestyle factors including physical activity and alcohol consumption were valuable predictive markers of autoimmune diseases [ ] . humans are exposed to a large number of substances from food, water, cosmetics, air, and so forth, each at low levels of exposure, and are able to induce cumulative/synergistic effects. many studies have focused on the effects induced by administering a single substance at medium-high doses to laboratory animals. recently, the concept of real-life risk simulation has emerged, since there is growing evidence that the effects of chemical mixtures at concentrations for which individual components failed to elicit have adverse effects when tested individually [ ] . the concept of real-life risk simulation can also incorporate dietary interventions because, in our diets, we expose the human body to myriad substances in diverse doses [ , , ] . the discovery of inflammation regulators opened a new window in therapeutics to clear low-grade chronic inflammation. a large number of physiological processes promote the physiological process of regulating inflammation. the development of such an approach targets the stimulation of endogenous processes that naturally occur during inflammation, which are hampered mainly by the lack of suitable human models and the heterogeneity of inflammatory disorders. another limitation includes the lack of sensitive measurements able to capture the different stages of inflammation and metabolites [ ] [ ] [ ] [ ] [ ] . the present paper aims to evaluate the impact diet might have on immune response, with special attention as to how lifestyle changes can help mitigate low-grade inflammation. real-life risk simulation (rlrs) concept. this analysis can be highly relevant in the context of the present viral spread of sars-cov- , since the inflammation is once again in the front line of an acute pathological response. identifying strategies to modulate the immune response might prove useful for reducing the virus's impact on the respiratory tract and thus diminishing its impact on each patient, as well as on the general medical system. the majority of studies that assess dietary habits, metabolism, and nutrient intake are based on food frequency questionnaires. however, food frequency questionnaires (ffq) have several limitations, including inconsistent responses on food choices, mostly because answers depend on responders' memory. moreover, ffq that are filled out by the responder instead of a trained healthcare professional only provide an overview of the macronutrients' intake while not fully capturing the micronutrient status of the person [ ] . micronutrient deficiencies are common in both developing and affluent countries, affecting two billion people worldwide, according to the world health organization (who) [ ] . the primary cause of micronutrient deficiencies or "hidden hunger" is poor dietary intake of micronutrients while other socioeconomic factors play an important role as well [ ] . several diet and nutrients assessment tools have been developed to evaluate inflammation status. the dietary inflammation index (dii) is based on literature data and aims to evaluate if a responder follows a proor anti-inflammatory diet. since its development, there has been an increasing interest in dii, although other indexes are being developed with similar efficacy [ ] . an important limitation of these indexes is the lack of causality and direct association to a person's symptoms. thus, the application of these indexes in clinical practice is hampered. a novel empirical, close-ended, and self-administered questionnaire developed by the european institute of nutritional medicine provides an inflammation status score that captures the interaction between the autonomic nervous system and inflammation [ ] . there is growing evidence that imbalances in the autonomic nervous system reflect local or systemic inflammation found in various diseases and that diet and lifestyle factors can act as regulators of sympathetic and parasympathetic activity [ ] [ ] [ ] [ ] . through a -question series, responders provide data on the presence/absence and status of inflammatory response in different body systems. overall, the nutritional medicine exam (numex) consists of questions and assesses the nutritional deficiencies status in seven categories: inflammation, nutrition, perceived stress, oxidation, sugar metabolism, amino acids metabolism, and gut microbiome. designed by medical doctors and nutritionists, the aim of this empirical test is to assist the individual and the healthcare professional to evaluate the overall inflammatory status based on autonomic nervous system changes and track its progression after targeted lifestyle changes. at a molecular level, metabolomics is the only method that can capture small, time-dependent fluctuations in the metabolism, thus indicating ncd-related metabolic imbalances [ ] . overall, traditional and well-established diet and nutrient assessment methods including ffq and dii have provided valuable information on the role of specific foods on health and disease, as discussed in the present review. with the advent of advanced tools, metabolomics is complementary to the standard approach to provide tailor-made recommendations depending on an individual's specific needs at a given time. recent literature considers bmi cut-off values to not fully depict metabolic disturbances associated with obesity. the bmi is a mathematical approximation and does not reflect the percentage of total body fat between body fat and total body muscle or bone mass. as such, bmi does not reflect cardiometabolic risk. a more comprehensive classification describes four phenotypes for obese individuals: normal weight obese (nwo), metabolically obese normal weight (monw), metabolically healthy obese (mho), and metabolically unhealthy obese (muo), or "at risk" obese with ms. this classification takes into account bmi, fat mass, and waist circumference, but also general biochemical parameters (e.g., fasting plasma glucose, total cholesterol, ldl, hdl, triglycerides) [ ] [ ] [ ] [ ] [ ] [ ] . all four classes are characterized by impairments of different severity of inflammatory pathways [ , ] . lifestyle and nutrition are modifiable factors that interact with genetics in regulating chronic inflammation, leading to aforementioned complications. the changes in nutritional patterns in western societies-caused by a high intake of fat and energy-dense, processed foods, as well as a low intake of fibers, fruits, and vegetables-are associated with a rising prevalence of asthma, allergies, and autoimmune diseases involving inflammatory mechanisms [ , ] . high fat diets determine, among other things: intestinal inflammation, favoring lipopolysaccharides (lps) absorption from gram-negative gut bacteria, and increasing lipoperoxidation that induces insulin resistance and inflammation. saturated fatty acids and lps activate toll-like receptor (tlr ) signaling pathways further contribute to promoting systemic inflammation and consequent metabolic disorders [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( figure ). lifestyle-and diet-induced inflammation affects several cellular pathways, which stimulates the synthesis and secretion of various pro-inflammatory molecules. this ultimately maintains the low-grade inflammation state. interestingly, populations that consume a diet rich in fruits, vegetables, and fibers have lower incidences of inflammatory diseases compared to western populations [ , ] . the mediterranean diet-based on olive oil, fish, vegetables, and fruits, in addition to incorporating myriad beneficial phytochemicals-discourages cardiovascular diseases [ ] [ ] [ ] [ ] . sourcing food from organic agriculture could further improve the beneficial health effects of a mediterranean diet, as suggested in a study comparing an organic and nonorganic mediterranean diet on male patients with chronic kidney disease [ ] . this was hypothesized to be due to a decreased exposure to pesticides, since animal studies have repeatedly found that exposure to pesticide mixtures can be a source of toxicity [ ] . however, although most studies have found that organic food consumers are healthier, it is not clear whether health benefits can be attributed to a decreased exposure to synthetic pesticides [ ] . dietary changes that include specific metabolites can modulate gene expression via epigenetic modifications, such as dna methylation or chromatin remodeling (e.g., histone acetylation or deacetylation). for example, a diet rich in folate and methionine can shape the host epigenome with a direct impact on molecular pathways associated with obesity-related inflammation. moreover, global dna hypermethylation in adipocytes derived from obese subjects is correlated with the expression of genes involved in proinflammatory interactions [ , ] . for example, hypermethylation at kb upstream of the adiponectin gene's promoter site was observed in adipocytes of obese mice fed a high-fat diet, but also in human adipocytes. dna methyltransferase (dnmt ) expression is correlated with the methylation of the adiponectin gene, resulting in decreased expression of adiponectin in obese mice and increased expression in healthy mice. studies on human adipocytes show a correlation between dnmt expression and bmi, suggesting that obesity is a cause or cofactor of hypermethylation of adiponectin gene [ , ] . another factor inducing epigenetic changes is ros overproduction in expanded adipose tissue, influencing histone acetylation/deacetylation equilibrium, thus inducing nfκb activation [ ] [ ] [ ] . on the other hand, nutrient restriction decreases akt (protein kinase b) activity and stimulates foxo (a forkhead box o transcription factor) activity, thus stimulating the expression of proteins involved in cell metabolism, autophagy, and stress-response, contributing to the resolution of inflammation [ , ] . fasting regimens are correlated with increased insulin sensitivity, improvement of blood pressure, and inflammatory status, regardless if they are associated with weight reduction. for example, days of intermittent fasting induced an increase of glucose uptake rates and a significant increase of anti-inflammatory adiponectin in lean young men (bmi of kg/m ) without a significant decrease in body weight. these results were consistent with data from animal studies [ , ] . an important causal factor for low grade inflammation influenced mainly by lifestyle is the impairment of gut microbiota. bacteroidetes and firmicutes constitute approximately % of the intestinal population, but the equilibrium is fundamentally changing with ageing and depending on diet composition. a decline of microbiota diversity occurs during ageing and obese individuals. gut dysbiosis has been found in several inflammatory pathologies such as obesity, diabetes, cardiovascular, and neurodegenerative diseases. this can be connected to the induction of chronic low-grade inflammation since the gut microbiome is intimately connected to innate immune responses [ ] [ ] [ ] . the relationship between gut microbiome and the host immune system are influenced by lifestyle interventions. for instance, secretory iga levels increase after periodic fasting. this can be linked to changes in gut microbiome composition [ , ] , with proteobacteria modulating the adaptive humoral local response. some studies showed that microbiota composition and diversity has a great impact on a population's general health status. for example, when comparing the fecal microbiota of european and rural african children (burkina faso), a higher proportion of prevotella and xylanibacter (involved in the digestion of fibers and generation of short chain fatty acids (scfas)) was found in the latter group, which lacked european subjects. these observations could be correlated with the higher prevalence of inflammatory diseases in european populations compared to rural african ones [ , ] . chronic exposure to environmental pollutants or food additives could also predispose one to chronic pathologies, which promotes inflammation [ ] . xenobiotics promotes chronic inflammation, which is thought to be the generation of lipotoxic conditions, i.e., in the development non-alcoholic fatty liver disease [ ] . this can be mitigated by lifestyle interventions such as periodic fasting [ ] . the exposure to xenobiotics such as heavy metals, pesticides, nanoparticles, polycyclic aromatic hydrocarbons, dioxins, furans, polychlorinated biphenyls, or non-caloric artificial sweeteners can also promote chronic inflammation by disturbing the gut microbiota [ , , ] . decreasing inflammatory burden is more important than ever during the covid- pandemic. this can be accomplished through everyday actions (e.g., lifestyle, diet, smoking cessation, weight decrease, sport, etc.). there is a lot of information available in the scientific community regarding the risk of covid- complications; even the likelihood of death is highly increased by some chronic diseases, mostly associated with an impaired inflammatory profile (e.g., obesity, type ii diabetes, hypertension, chronic pulmonary disease, etc.) [ , ] . the literature data shows that people without comorbidities have a much lower risk of severe symptoms as a result of the sars-cov- infection [ ] . on the other hand, increased levels of inflammatory markers cytokines with pro-inflammatory outcomes constitute predictors of adverse outcome in covid- patients [ ] . evidence proves that some dietary elements such as zinc or vitamin d might provide protective effects against viral load [ ] . as such, this reduces the inflammatory burden through a healthy diet, associating (based on rlrs principles) several protective components (e.g., fiber, polyphenols, pufas, vitamins, etc.) that constantly increase our chance of being better protected against different immune challenges. fermentable dietary fiber are not enzymatically digested in the small intestine; they pass into the colon and are transformed by gut bacteria into scfas [ , ] . the systemic distribution and generation of scfas-acetate, propionate, and butyrate (the most abundant)-in the distal colon is important to inhibit inflammatory signals. germ-free animal models were characterized by inflammatory flairs, due to the absence of tissue/blood scfas [ ] [ ] [ ] . butyrate is a representative member of scfas and has a high affinity for different g-protein-coupled receptors (gpcrs) found throughout the body: gpr is found in adipose tissues and immune cells and gpr is found in immune cells. however, gpr a is present in colonic cells and gpr and gpr are activated by butyrate, which favors the production of peptide yy (pyy). this contributes to gastric emptying and intestinal transit inhibition, which thereby reduces appetite and promotes glucagon-like peptide (glp- ). these outcomes indirectly stimulate insulin secretion. gpr a activates the inflammation-associated pathway in colonic macrophages and dendritic cells, inducing the differentiation of il- -producing t-cells and release of il- from intestinal epithelial cells [ , , ] . the presence of fiber in the diet is extremely important ( table ) as it generates scfas and promotes the proliferation of commensal bacteria, which limits the access of pathogenic bacteria to the gut epithelium. moreover, scfas favor epithelial mucus secretion that increases the protective effect on the intestinal surface and the proper maintenance of the barrier function [ , ] . scfas have anti-inflammatory effects that bind to the nuclear transcription factor pparγ (peroxisome proliferator-activated receptor γ) and, consequently, inhibit the nf-kb pathway [ , ] . this ultimately lowers the expression of vcam- (vascular adhesion molecule ) and icam- (intracellular adhesion molecule ), as well as the synthesis of tnfα, il- , and ifn-γ (interferon γ) [ ] . the main physiological role of histones is to interact with dna and stabilize its structure. when they are acetylated, this loosens the contact between histones and dna, uncoiling the dna structure that thus becomes transcriptionally active. the histone acetylation process is a result of the balance between the induction of histone acetyl transferases (hats) and the inhibition of histone deacetylase (hdacs). the same acetylation process causes dna to bind to transcription factors, such as stat (signal transducer and activator of transcription ), nf-kβ (nuclear factor kappa-light-chain-enhancer of activated b cells, and foxp . consequently, this regulates gene expression, including inflammation proteins [ , ] . scfas (with butyrate and acetate being the most and least effective, respectively) act as inhibitors for histone deacetylase (hdacs), thus contributing to the inhibition of the transcription for inflammatory proteins [ , ] . fasting is a process that has been known for thousands of years. it was quite frequent in ancient times, because access to food was difficult and as a result, individuals were obliged to survive without food until it was available again [ ] . fasting is a survival mechanism in both animals [ , ] and humans, especially in countries where food conservation is not widespread [ ] . in the rest of the world, fasting has been employed either due to religious convictions or in wellness centers. since , one of the methods used to address morbid obesity and related diseases has been the "zero calorie diet", thus translating into clinical practice the scientific data generated by centuries of fasting. fasting can be divided into three broad categories: • periodic fasting, which lasts from days to a few weeks; • intermittent fasting, which lasts from to h and can be done daily or every second day or twice a week, and • fasting-mimicking diet, the diet that mimics fasting to achieve its beneficial effects, in which restriction of calories and specific foods is necessary (e.g., fat) [ ] [ ] [ ] . there is a lot of research that shows the beneficial effects of fasting on health and also on different pathological conditions. fasting increased lifespan in prokaryotic organisms such as yeast s. cerevisiae and nematode c. elegans [ , [ ] [ ] [ ] [ ] , but also on animal models that performed fasting for long periods (e.g., the royal penguin) [ ] . other models have shown better brain function [ , ] , increased lifespan and longevity [ ] [ ] [ ] , and improved maintenance of muscle mass after fasting [ ] . studies on animal models reveal the beneficial effect fasting has on cancer as a complementary disease management strategy in concert with drug treatments [ ] [ ] [ ] [ ] . studies on animal models show an improvement in neurodegenerative diseases after fasting, while other studies prove that intermittent fasting diets boost the levels of antioxidant defense, neurotrophic factors (bdnfs, h- and fgf ), proteins involved in adaptive response (hsp- and grp- ), and reduce pro-inflammatory cytokines levels (tnfa, il- β, and il- ) [ ] [ ] [ ] [ ] [ ] . it has been found that intermittent fasting can prevent and reverse all aspects of metabolic syndrome in rodents: body fat, inflammation, and blood pressure are reduced; insulin sensitivity is increased; and the functional capacity of the neuromuscular and cardiovascular systems are improved [ ] [ ] [ ] . an intermittent fasting diet has also been found to improve hyper-glycaemia in diabetic rodent models [ ] and in myocardial infarction models, as the heart is protected from ischemic damage by this type of regimen [ ] . elevated leptin levels usually predict a pre-inflammatory condition, while adiponectin and ghrelin may suppress inflammation and increase insulin sensitivity [ , ] . fasting can reverse every major abnormality caused by metabolic syndrome, by increasing insulin and leptin sensitivity, suppressing inflammation and stimulating autophagy [ , ] . there are several studies that show an increased use of fat and ketone bodies for energy [ , ] , as well as an increase of growth hormone and glucagon secretion [ ] [ ] [ ] , with a decrease in blood sugar, insulin, and igf- levels. after intermittent fasting, total fat, abdominal fat, and blood pressure are decreased, while glucose metabolism is improved in obese individuals [ , [ ] [ ] [ ] [ ] . in addition, periodic fasting significantly changes the composition of the human gut microbiota [ ] . finally, studies of utmost importance show the effect of intermittent fasting mainly in the fight against cancer as a supplement along with the classic treatment, with promising results [ , ] . below, we analyze the effects of the interrelation between fasting and inflammation as well as the relevant molecular mechanisms. fasting not only results in weight loss but it is a survival mechanism that impacts many metabolic pathways [ , , ] . fasting's many benefits are related to the regulation of key molecular pathways. initially, during fasting, the downregulation of insulin-like growth-factor- (igf- ) and mammalian target of rapamycin (mtor) occur. these pathways are upregulated in the presence of food excess as they sense nutrients and therefore activate anabolic metabolism. when there is a lack of food for several hours, catabolic processes are activated. aging appears modulated by changes in the insulin-like growth-factor- receptor signaling system, as longevity is enhanced by a decrease in igf- signaling [ ] [ ] [ ] . the igf- signal induces mtor activation. reduced mtor activity is related to extended lifespan in different organisms [ ] , as mtor induces activation of foxo proteins. foxo proteins are transported to the nucleus and activate genes associated with autophagy [ ] , which emphasizes the link between autophagy and foxo proteins. when the amp/atp ratio is high, the ampk path is activated [ ] ; this results in increased energy production and reduced atp utilization. in addition, the mitochondrial biogenesis and mitophagy repair and replace damaged mitochondria. as a result, the cells have "younger" and more efficient mitochondria. in addition to the aforementioned condition, activation of this path has been associated with increased lifespan in various studies in both c. elegans and drosophila melanogaster [ , ] . in mammals, fasting does not appear to affect ampk activation, but further studies are needed to be able to draw surer conclusions [ ] . like ampk, sirtuins are associated with life [ ] and autophagy [ ] . some sirtuins are found in the cytoplasm (sirt ), some (sirt ) in the nucleus having dna repair action, and others in mitochondria. in general, sirtuins are associated with mitochondrial biogenesis and mitophagy for damaged mitochondria, thereby enhancing mitochondrial cells without problems and are thus more efficient in energy production [ , ] . sirt is modulated by nad + level and is increased in energy depletion states (such as fasting or exercise) for which nad + is a sensor, which contributes to the reduction of inflammation through nf-kβ down-regulation and related transcription factors [ , , ] . a study on rats showed that inflammation decreased with fasting [ ] . other work has shown nf-kβ inhibition and the modulation of nrf , sirtuins, sod , and increased lifespan [ ] [ ] [ ] [ ] . in a study, intermittent fasting appears to significantly reduce corticosterone (cort), interleukin (il- ), and tumor necrosis factor-alpha (tnf-α) levels [ ] . nrf plays a key role in oxidative stress and toxicity; the right balance in ros levels is very important so that mitochondrial and all other pathways can function properly. the absence of ros, however, does not activate nrf , which in turn does not activate are (antioxidant response). thus, a critical amount of ros ("hormesis hypothesis") is necessary for the upregulation of are, which allows cells and mitochondria to be able to deal with oxidative stress and different kinds of toxins [ ] , which consequently increases their lifespan [ ] (figure ). during the last decades, many studies have been conducted on the effect of fasting on several markers related to metabolism. most of them determine the effect of fasting on weight. however, there are several studies that identify changes in lipid and carbohydrate metabolism as well as key hormones that affect the above (e.g., insulin). recently, some studies have focused on fasting's effect on inflammatory markers such as tnf-α, interleukins, crp, and bdnf, as well as the hormones adiponectin and leptin. the largest study on fasting's effects is an observational study including subjects that describes metabolic changes after a -to -day fasting period [ , ] . all the participants fasted according to the buchinger wilhelmi fasting guidelines, which include a daily caloric intake of - kcal together with a variety of lifestyle changes (e.g., dietary advices, physical exercise). a beneficial modulating effect of fasting was observed on blood lipids, glucoregulation, and altogether general health-related blood parameters. additionally, it was associated with a reduction in weight, abdominal circumference, and blood pressure. in another study, which used the same fasting guidelines, improved metabolic markers were observed after periodic fasting, including a decrease in blood glucose levels associated with changes in gut microbiome composition [ ] . in this study, the analysis of the gut microbiome after days of periodic fasting showed that fasting caused a decrease in the abundance of bacteria known to degrade dietary polysaccharides such as lachnospiraceae and ruminococcaceae, concomitant to an increase in bacteroidetes and proteobacteria known to use host-derived energy substrates. a study of eight healthy non-obese men discovered that in days of fasting every other day, with h fasting on fasting days, adiponectin increased and leptin decreased, while no changes in il- or tnf-α were observed. protocol allowed them to maintain normal exercise but also to consume food in order to keep their weight stable [ ] . redman et al., in a two-year study of people who followed a reduced calorie intake diet ( %) observed a reduction in leptin. these individuals lost an average of . kg while the control group gained an average of . kg in the same period [ ] . another study including eight women and two men, all overweight with asthma, showed that fasting every other day and reducing calories to less than % of their normal intake on the days of fasting, for eight weeks, resulted in a reduction in tnf-α and bdnf, but no change in crp. in this study, patients lost % of their initial weight during the study. asthma symptoms also improved as well as some indicators of oxidative stress ( -protein carbonyls, isoprostane, nitrotyrosine, and -hydroxynonenal adducts) [ ] . in , another study [ ] found that weeks of reduced calorie intake every other day resulted in reduced crp levels, increased adiponectin levels, and reduced leptin levels in adults who were either overweight or normal weight. on fasting days, they consumed only % of the calories they normally consumed each day. in addition, their weight was significantly reduced by . ± . kg and the coronary heart disease risk was improved as the concentration of tg reduced. another study [ ] found reduced high sensitivity-c-reactive protein (hs-crp) of women with polycystic ovary syndrome (pcos) during the ramadan period in iran and in which participants aged to years old with an average of age . , followed everyday . h fasting, isocaloric diet, for days. an important study involving men (resistance-trained) who fasted every day h, followed an isocaloric diet for eight weeks, and consumed % of their energy needs in the -h eating window, showed that there was an increase in adiponectin and a reduction of leptin of il- and il- β [ ] . faris' study on healthy volunteers ( men and women) fasting for - h each day for days, showed a reduction in il- , il- β, tnf-α, total leukocytes, granulocytes, lymphocytes, and monocytes [ ] . another study [ ] involving patients aged to years old with nonalcoholic fatty liver disease, following ramadan fasting (every day -h fasting for days), showed a reduction in il- and hs-crp, compared to volunteers who did not fast. these and other studies are presented in detail in table . collectively, an increasing number of studies show that fasting has numerous health benefits and could be used to prevent or manage the development of cardiometabolic disorders, metabolic diseases, and immune diseases. although extended periods of fasting can be challenging without medical advices, recent studies also showed that time-restricted eating can be practiced safely as a routine. for instance, a recent study showed that h of time-restricted eating for weeks improved cardiometabolic health for patients with metabolic syndrome [ ] . [ ] ns-non-significant; hs-crp-highly sensitive crp; ldl-low density lipoproteins; hdl-high density lipoproteins; gsh-glutathione; tac-total antioxidant capacity; t -triiodotironine; tg-triglycerides; bmi-body mass index; bf-body fat; sbp-systolic blood pressure; dbp-diastolic blood pressure; , -dinor-ipf( α)-iii - , -dinor- -iso prostaglandin f α; hba c-glycated hemoglobin; igf- -insulin growth factor . given that a large part of the global population suffers from various metabolic disorders, it is important to look for non-pharmacological ways to deal with these conditions. targeted changes in lifestyle and especially diet can be economical tools to mitigate the development of metabolic disorders when they are at an early stage. these changes include increased fiber and polyphenol intake compared to the current western diets, but also well-structured, personalized fasting protocols, which can reduce the risk of metabolic disorders (figure ) . this could be implemented in various 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risk factors during an intermittent fasting/calorie restriction regimen: relationship to adipokine modulations key: cord- -s vzf q authors: fang, evandro f.; xie, chenglong; schenkel, joseph a.; wu, chenkai; long, qian; cui, honghua; aman, yahyah; frank, johannes; liao, jing; zou, huachun; wang, ninie y.; wu, jing; liu, xiaoting; li, tao; fang, yuan; niu, zhangming; yang, guang; hong, jiangshui; wang, qian; chen, guobing; li, jun; chen, hou-zao; kang, lin; su, huanxing; gilmour, brian c.; zhu, xinqiang; jiang, hong; he, na; tao, jun; leng, sean xiao; tong, tanjun; woo, jean title: a research agenda for ageing in china in the st century ( nd edition): focusing on basic and translational research, long-term care, policy and social networks date: - - journal: ageing res rev doi: . /j.arr. . sha: doc_id: cord_uid: s vzf q one of the key issues facing public healthcare is the global trend of an increasingly ageing society which continues to present policy makers and caregivers with formidable healthcare and socio-economic challenges. ageing is the primary contributor to a broad spectrum of chronic disorders all associated with a lower quality of life in the elderly. in , the chinese population constituted % of the world population, with . million chinese citizens aged and above ( +), and million aged or above ( +). china has become an ageing society, and as it continues to age it will continue to exacerbate the burden borne by current family and public healthcare systems. major healthcare challenges involved with caring for the elderly in china include the management of chronic non-communicable diseases (cncds), physical frailty, neurodegenerative diseases, cardiovascular diseases, with emerging challenges such as providing sufficient dental care, combating the rising prevalence of sexually transmitted diseases among nursing home communities, providing support for increased incidences of immune diseases, and the growing necessity to provide palliative care for the elderly. at the governmental level, it is necessary to make long-term strategic plans to respond to the pressures of an ageing society, especially to establish a nationwide, affordable, annual health check system to facilitate early diagnosis and provide access to affordable treatments. china has begun work on several activities to address these issues including the recent completion of the of the ten-year health-care reform project, the implementation of the healthy china action plan, and the opening of the national clinical research center for geriatric disorders. there are also societal challenges, namely the shift from an extended family system in which the younger provide home care for their elderly family members, to the current trend in which young people are increasingly migrating towards major cities for work, increasing reliance on nursing homes to compensate, especially following the outcomes of the ‘one child policy’ and the ‘empty-nest elderly’ phenomenon. at the individual level, it is important to provide avenues for people to seek and improve their own knowledge of health and disease, to encourage them to seek medical check-ups to prevent/manage illness, and to find ways to promote modifiable health-related behaviors (social activity, exercise, healthy diets, reasonable diet supplements) to enable healthier, happier, longer, and more productive lives in the elderly. finally, at the technological or treatment level, there is a focus on modern technologies to counteract the negative effects of ageing. researchers are striving to produce drugs that can mimic the effects of ‘exercising more, eating less’, while other anti-ageing molecules from molecular gerontologists could help to improve ‘healthspan’ in the elderly. machine learning, ‘big data’, and other novel technologies can also be used to monitor disease patterns at the population level and may be used to inform policy design in the future. collectively, synergies across disciplines on policies, geriatric care, drug development, personal awareness, the use of big data, machine learning and personalized medicine will transform china into a country that enables the most for its elderly, maximizing and celebrating their longevity in the coming decades. this is the nd edition of the review paper (fang ef et al., ageing re. rev. ). the research agenda in response to rapid population ageing in china has been broad, covering areas including the study of the ageing process itself in laboratory and animal studies, to clinical-level studies of drugs or other treatments for common chronic diseases, and finally policy-level research for the care of the elderly in hospital, community and residential care settings, and its influence on health and social care policies . chinese population statistics taken between - show a reduction in crude death rate (cdr) and total fertility rate (tfr), accompanied by an increase in life expectancy at birth and an expansion of the population aged and above ( +, termed the elderly) (fig. a) . as of , the population of mainland china constitutes % of global total, with . million chinese citizens aged +, million of whom are +. by , it is expected that there will be . billion chinese, with million aged +, a number representing . % of the country's total population (fig. b) . furthermore, among this ageing population, million are expected to reach an age of at least and . million are expected to become centenarians (fig. b) . when compared with their counterparts born a decade earlier, the current + generation has reduced annual mortality and disability rates, but has increased cognitive impairment and reduced objective physical performance capacity (zeng et al., b) . to achieve what may be considered a 'healthy ageing society', it is first important to address and prepare for the challenges and issues that are associated with rapidly ageing populations. ageing is the primary driver of most, if not all, chronic diseases, including cancer, cardiovascular diseases, diabetes, and neurodegenerative diseases, particularly alzheimer's disease (ad) and parkinson's disease (pd) (kerr et al., ; lautrup et al., ; lopez-otin et al., ) . the most predominant diseases affecting the elderly in china ( +, data from ) include sensory diseases, other non-communicable diseases, digestive diseases, respiratory infections and tuberculosis, skin and subcutaneous diseases, neurological diseases, and musculoskeletal disorders, among others (fig. c) . from to , there were dramatic increases in prevalence of all diseases, excluding a very minor reduction in 'neglected tropical diseases and malaria' (fig. c) . the major diseases responsible for death of the elderly in china are cardiovascular diseases, neoplasms, chronic respiratory diseases, and neurological diseases, among others (figs. d and ). compared to their younger counterparts, the elderly population are more fragile, and susceptible to conditions such as cardiovascular diseases, chronic respiratory diseases, diabetes, kidney diseases, unintentional injuries, hiv/aids and sexually transmitted diseases (stds), among others (fig. b ). in comparison with data from , new patterns of disease mortality characterize the modern elderly, such as a dramatic reduction in the percentage of death contributed by 'neurological disorders' with an increase in deaths due to hiv/aids and stds (fig. b, d) . recognition of the current disease demographics in the elderly in china, and accurate prediction of future trends will enable us to be best prepared for different healthcare needs at different times. in wake of the expanding ageing society in china, and the formidable socio-economic and healthcare challenges, we offer the nd edition of our previously published review . here, we aim to provide an update regarding the situation of the elderly in china using a range of expertise and suggestions from multiple fields which may further propel the exciting and ongoing reforms to china's healthcare system. we hope to explore different ageing care models that can be used to best produce a healthy ageing society (chen, ; yip et al., ; zhan et al., ) . the following sections highlight recent developments in the above areas as well as areas for future research. based on ageing phenotypes and the major disease demographics in the elderly in china (figs. and ), we chose to focus on frailty (including sarcopenia as an independent subsection), cncds (including cardiovascular disease as an independent subsection), mental health disorders, dental health challenges, elderly infections and immune diseases, as well as hiv, syphilis, and other stds. in view of recent reviews on other grand challenges, including cancer tsoi et al., ) , chronic respiratory diseases (zhu et al., ) , diabetes and kidney diseases (hu and jia, ; wei et al., ) , these areas will not explored here. frailty is a biologic syndrome characterized by deteriorating function across a broad spectrum of physiological symptoms (fried et al., ) . it can be thought of as a state of vulnerability. some have proposed an index approach to categorize different degrees of frailty; however, these attempts are complicated by the multidimensionality of the underlying causes of frailty, thus creating a dynamic, ever-changing value that is difficult to index (rockwood et al., ) . the term physical frailty has been applied to age-related loss of muscle mass and function, that is sarcopenia (detailed in the next section). in recent years, frailty research has increased rapidly in china as a strategy to prevent disability in response to an ageing population (chhetri et al., ) . research projects were showcased in the following scientific conferences: the st and nd international china conference on frailty in china, jointly organized by the who collaborating centre on frailty, clinical research and geriatric training at the gerontopole, toulouse, france, the chinese embassy in paris, and the national clinical centre for geriatric diseases, china, and the th asian conference for frailty and sarcopenia in dalian in october , organized by the chinese geriatrics society, beijing institute of geriatrics and gerontology and the chinese health promotion foundation. wide-ranging topics included basic science, epidemiology, definitions and measurements, management, as well as service models. such conferences greatly accelerate basic and clinical research on as well as clinical treatment for frailty. frailty may be used as a population indicator of ageing, and be a useful indicator of a need for treatment. research into prevalence, risk factors, prevention, and incorporation into service delivery models in community, hospital and residential care settings are an important part of the ageing research agenda for china. the importance of recognizing frailty in communitydwelling older people in china has been highlighted in a systematic review and meta-analysis by he et al. (he et al., a) . risk factors for a worsening in frailty among community-living older adults include hospitalizations, older age, previous stroke, lower cognitive function, diabetes and osteoarthritis, while higher socioeconomic status and neighborhood green space were protective factors (lee et al., ; yu et al., c) . a comparison of prevalence and incidence of frailty between populations may stimulate further research into prevention strategies and inform government policies. using data from a nationally representative study, wu et al. found that % of community-dwelling adults aged years or above were frail in mainland china and the prevalence increased dramatically with age, reaching . % for those aged years or above ). substantial regional disparities exist in the prevalence as well as incidence of frailty in mainland china. for example, the incidence rate of frailty in the northeast was more than double than that in the southeast . furthermore, a comparison of frailty and its contributory factors across three chinese populations (hong kong, urban and rural populations of taiwan) using the ratio of frailty index (fi) to life expectancy (le) as an indicator of compression of morbidity showed higher fi/le in taiwan compared with hong kong. risk factors include low physical activity and living alone . the importance of protein intake to slow the decline in muscle mass and physical function over four years supports the importance of nutrition as an underlying factor for physical frailty .the role of inflammatory cytokines in the pathophysiology of sarcopenia is supported by the finding of slower decline in grip strength for those in the highest quartile of telomere length (woo et al., b) . simple tools for frailty and sarcopenia may be used in a community setting as case finding, without the need for professionally trained personnel (woo et al., a; woo et al., b) . this may represent the first step in the approach to community-based intervention such as group exercises with or without nutritional supplementation for frailty and sarcopenia (yu et al., in press; . how community services may be developed to make frailty as a cornerstone of health and social care systems (woo, ) depends on the development of existing community infrastructures. two examples have been described previously: the tai po cadenza hub, and the jockey club e health project, where screening data based on the who integrated care for older people toolkit (who) were collected via ipad, followed by action algorithms for items where action is indicated: e.g., frailty, sarcopenia. this model emphasizes the empowerment of older people and their care-givers, societal-level behavioral changes, and the use of technology in the absence of a low cost primary care system orientated to meeting the needs of older people (woo, ) . in the hospital setting, detection of frailty may inform choice of therapies and prognosis, such as mortality and hospitalization in chronic heart failure . closely related to the concept of frailty, sarcopenia is an age-related gradual loss of mass and strength of skeletal muscles resulting in reduced physical performance. major pathological features include a loss of satellite cells and motor neurons, as well as less active neuromuscular junctions (cruz-jentoft et al., ) . following the publication of the european consensus group on sarcopenia (cruz-jentoft et al., ; cruz-jentoft et al., ) , an asian group including chinese researchers formed a panel to arrive at a consensus on the definition of sarcopenia, published in , and recently updated in . the asian criteria differed from the european consensus definitions. an individual international statistical classification of diseases and related health problems code (m . ) was assigned to 'sarcopenia' which has stimulated both diagnostic and therapeutic trials worldwide. in china, sarcopenia diagnosis requires some special considerations, including anthropometric and cultural differences. the guideline of asia working group of sarcopenia (awgs) provides updated guidelines on epidemiology, case-finding, the diagnostic algorithm, measurements of muscle mass, muscle strength and physical performance, and intervention and treatment. the prevalence of sarcopenia is estimated to be between . - . % among the general older population and was over % in the oldest populations ( +) wang et al., a; woo et al., a; xu et al., , in press; yu et al., ) . while old age is the primary risk factor for sarcopenia, other risk factors in the chinese population include household status, lifestyle, physical inactivity, poor nutritional and dental status, and some diseases (osteoporosis, metabolic diseases, etc.). in terms of longer-term clinical outcomes, awgs-defined sarcopenia was significantly associated with increased risks of physical limitations at years, slowness at years, and -year mortality, but not of hospitalization wang et al., a; woo et al., a; yu et al., ) . interventional strategies for the elderly of china have been the subject of recent research. for instance, an intervention for community-dwelling older adults yielded significant improvements in muscle function based on which when protein was offered as an oral nutritional supplement in combination with resistance exercises (kang et al., ) . similar findings were reported in j o u r n a l p r e -p r o o f major behavioral risk factors that are responsible for cncds are prevalent among the elderly in china. nearly % of deaths are attributable to unhealthy diet, high blood pressure, smoking, high glucose, air pollution (indoor and outdoor), and physical inactivity (who, ) . in china, . % of adults aged + have insufficient dietary balance (daily intake of < g fruit and vegetables), . % are current smokers, % use unclean fuel for cooking, % are physically inactive, and . % have harmful alcohol use (who, ) . risk factors for major cncds, particularly smoking and alcohol use, are unevenly distributed among older men and women. the prevalence of cigarette smoking is substantially higher among men ( . %) than women ( . %). the prevalence of harmful alcohol use among men is more than three times as much as that among women ( . % vs. %). substantial rural-urban disparities in the distribution of risk factors exist among older chinese adults. rural residents have a higher prevalence of smoking ( . % vs. . %), harmful alcohol use ( . % vs. . %), insufficient dietary intake ( % vs. . %), and unclean fuel use ( % vs. . %) than those in urban areas, while residents of urban areas have a substantially higher prevalence of physical inactivity than their rural counterparts. , cvd was ranked first in mortality rates, higher than the mortality rates attributed to tumors and other prevalent diseases. studies have shown that in , age-standardized cvd mortality in china was % lower than in (gbd, ; zhou et al., ) . although the age-standardized cvd mortality rate has declined, the absolute number of cvd deaths is still rising rapidly, and increased by % between and . cvd is a large burden for the chinese healthcare system. with the development of medical technology, and the government's focus on chronic disease management, the problem of cvd in china has improved. however, due to the problems arising from population ageing, cvd still has a great impact on national health. major factors for cvd include hypertension, dyslipidemia, diabetes, air pollution, and excess weight (overweight and obesity). some risk factors are specific to china compared to other countries, however, this is changing as china's economy develops and the population ages. hypertension is an important public health problem in china. the prevalence of hypertension in china among those over the age of is . %, and the number of patients with hypertension in china is estimated to be million . in , . million deaths were attributed to hypertension in china, accounting for . % of all causes of death (trammell et al., ) . with the rapid development of the economy and the ageing population, problems with blood lipid levels in china have gradually increased, and the prevalence of dyslipidemia has increased significantly. the main symptoms of dyslipidemia seen in china are low levels of low-density lipoprotein cholesterol (ldl-c) and hypertriglyceridemia (pan et al., ) , while dyslipidemia in the west is characterized by hypercholesterolemia and high levels of ldl-c (toth et al., ) . with the change of lifestyle following china's economic development, the number of chinese diabetic patients is growing. overall, % of adults in china have diabetes or pre-diabetes, which is slightly lower than the - % in the united states . in recent years, there has been a significant increase in the prevalence of excess weight (bmi: . - . kg/m ) and obesity (bmi≥ . kg/m ) in chinese residents, as noted over a five-year study period (he et al., b) . the prevalence of combined overweight and obesity among men was . %. air pollution is another important factor leading to cvd. among different particles, pm . (an aerodynamic diameter of . μm or less ) is most closely related to cvd (brook et al., ) . a follow-up study of cohorts of elderly people + in hong kong showed that for every μg /m increase in pm . concentration, the risk of total cvd death increased by % (wong et al., ) . air pollution is also associated with increased blood pressure. for each μg/m increase in pm . concentration, the per capita systolic blood pressure level increased by . mmhg, the per capita diastolic blood pressure level increased by . mmhg, and the risk of hypertension increases by % . coronary heart disease, atrial fibrillation (af), heart failure, and atherosclerosis are common forms of cvd. technological developments have allowed for an increase in treatment options and testing methods, including percutaneous coronary intervention (pci), radiofrequency ablation, implantable cardiac defibrillator (icd) and pacemaker implantation. since elderly patients are often associated with more complications, treatment decisions for cvd in elderly patients need to be adjusted individually based on an overall scoring of health. coronary heart disease is a common fatal cvd. for the treatment of coronary heart disease, the number of pci cases has steadily increased in china (zhao et al., b) . the creative study explored antiplatelet treatment options for patients after pci in china, and studies have shown that for patients with low response to antiplatelet drugs after pci, a triple antiplatelet intensive therapy combined with cilostazol is safe and effective (tang et al., ) . bleeding events should be paid special attention when administrating dual antiplatelet treatment to acs patients aged and older receiving pci (zhao et al., a) . the risk of all-cause, cardiovascular, and stroke deaths in patients with af is significantly higher than in patients with sinus rhythm . the proportion of chinese patients receiving anticoagulation treatments is low. only . % of patients with af and a chads score of or more received anticoagulation treatment . patients with af aged + tend to higher chads scores but receive less anticoagulation therapy. the risk of one-year follow-up deaths and adverse events in the elderly is more than doubled compared to other populations yang et al., ) . cases of af ablation procedures and icd implantation have steadily increased in china. however, european danish studies suggest that primary prevention through icd implantation has limited benefits in elderly patients with non-ischemic cardiac diseases (kober et al., ) . therefore, it is necessary to pay attention to the indications when expanding the population eligible for icd implantation in china. in recent years, the etiology of heart failure in china has changed significantly. the proportion of valvular disease (especially rheumatic valvular disease) has decreased. as china is becoming an ageing society, the number of elderly patients with heart failure has increased. at present, most studies suggest that coronary heart disease is a common cause of heart failure in the elderly, and the proportion of hypertension and pulmonary heart disease in elderly patients with heart failure increases with age. in recent years, the use of diuretics in hospitalized heart failure patients in china has not changed significantly, while the usage rate of digoxin has shown a downward trend. the use of acei, arb, aldosterone receptor antagonists and beta-blockers have shown a significant upward trend . lower extremity atherosclerotic disease (lead) is a common disease in the elderly and an important starting point for systemic atherosclerosis. early detection of lead is of great value in the diagnosis and treatment of systemic atherosclerosis (hiramoto et al., ) . to reduce the burden of cvd in china, we recommend interventions directed at altering lifestyles and programs dedicated to the detection and management of risk factors, especially for elderly people. research modeling has shown that if dyslipidemia and hypertension are effectively managed, medical expenses to the tone of $ billion us from - (stevens et al., ) . controlling blood lipids and blood pressure of elderly people over years of age represents the most cost-effective strategy (stevens et al., ) . mental health disorders, particularly dementia and depression, are major diseases in the elderly of china. alzheimer's disease international (adi) estimates that over million people worldwide were living with dementia in , and that this figure will rise to million by ; the current annual cost of dementia is estimated at trillion us dollars which will be doubled by (adi, ). it is estimated that the number of patients with dementia in china constitutes % of the dementia population worldwide, with the prevalence of dementia ranging from . % ( % ci . - . , in ) to . % ( % ci . - . , in ) for individuals aged + jia et al., ; jia et al., ) . the patterns and spread of dementia in china vary geographically and between genders. women are . times more susceptible than men. western china has a higher prevalence at . %, while central and northern china are lower at . and . %, respectively, southern china has the lowest prevalence at . %, this variation is possibly due to a variety of reasons including diet, exercise, social networks, healthcare, etc. (chan et al., ; jia et al., ; wu et al., b) . the incidence of dementia in individuals aged + ranged from . to . per person-years using / dementia research group criteria, while it was . per person-years using dsm-iv criteria (jia et al., ; prince et al., ; yuan et al., ) . while health conditions such as depression, diabetes mellitus, and insomnia correlate with dementia in a global fashion, epidemiological evidence from different regions in china also suggests smoking and heavy alcohol consumption as high risk factors (fan et al., ; pei et al., ; xue et al., ) . depression, a risk factor for dementia, is a common but often neglected disease in the elderly in china . data from a cross-sectional study suggest a prevalence of depression of % in the elderly which increases to % in the most elderly (yu et al., ) . in view of the stigma of mental illness in some areas of china coupled with inadequate health services in rural areas, depression is likely underdiagnosed suggesting the real prevalence may be higher. in addition to its contribution to dementia, depression aggravates the quality of life of the elderly and of their family members, brings the risk of death caused by different reasons, and accordingly is a heavy burden on the society and the healthcare system (zhang and li, ) . much effort should be made to address mental health disorders in china, including increasing government investment, the training of more geriatric care professionals with specialties in mental disorders, and raising public awareness, especially in conjunction with more active social activities and exercises. although there have been increased care facilities for citizens + and improved access to health services, the diagnosis and management of dementia and depression are still inadequate, especially in rural areas (jia et al., ) . the inclusion of steps to manage dementia in the th five-year plan of the central chinese government marked a major step forwards, and such efforts need to be continued. in view of the insufficiency of medical professionals in regards to mental disorders, especially in rural areas, we recommend increased training to such professionals, and the development of policies to encourage health professionals to work (at least for a short period) in rural areas . in recent years, the public awareness of mental disorders, especially ad, has greatly improved thanks to efforts from social media (e.g., drama shows on ad) and dementia organizations. professional interventions, comprising medicine and combined cognitivepsychological-physical intervention (e.g., family and community support plus playing mahjong and practicing taichi) can mitigate subclinical depression and improve overall mental health (kong et al., ; wang et al., c; wong et al., ) . although no drug at present is available to cure ad, recent progress on the understanding of ad etiology, such as the involvement of impaired mitophagy and reduced grid-cell-like representations in the human ad brain, along with the development of novel stem cell models, and the use of artificial intelligence (ai), will undoubtedly propel the development of novel drugs (fang, ; fang et al., ; gilmour et al., ; kunz et al., ; lin et al., ) . the china brain project, covering studies on basic neuroscience, brain diseases, and brain-inspired computing, will greatly benefit the development of novel drugs for different neurological diseases (poo et al., ) . while oral health is an important part of the whole body, the prevalence of oral disease is high in the elderly in china, but is largely ignored, while here we focus on dental health. dental caries (tooth decay), periodontal disease and tooth loss in the elderly are issues of global health concern. the burden on healthcare cost and the quality of life of these dental diseases in the elderly remain high . maintaining good dental health is an integral part of healthy ageing. as such, developing effective preventive and therapeutic interventions are needed to protect and enhance dental health and well-being tonetti et al., ) . dental caries and periodontal diseases are common oral diseases in the elderly and often lead to tooth loss, edentulism (toothlessness), impaired masticatory function and poor nutrition. according to the th national oral health epidemiological survey (fnohes, (fnohes, - covering the whole of mainland china, caries and periodontal diseases are highly prevalent in the elderly in china; while the prevalence of caries was above % in all age groups ( - , - , - , - , and - years) , the rate was % in the - years groups (lu et al., ; si et al., ) . in adults aged - years, . % had periodontal diseases, including gingival bleeding ( . %), dental calculus ( . %) and a deep periodontal pocket ( . %) (lu et al., ; si et al., ) . human oral tissues naturally and gradually degrade with age; a fact also exacerbated by modern lifestyle choices, including the prevalence of sugary diets and a lack of oral hygiene (belibasakis, ; lamster et al., ) . more specifically, age-dependent changes include a reduction in periodontal support, loss of elastic fibers, and thickening and disorganization of collagen bundles in the connective tissue of the oral mucosa (belibasakis, ; lamster et al., ; wu et al., b) . severe dental health challenges can cause loss of self-esteem, social difficulties, while also being drivers of common diseases, such as ad, pd, diabetes, and hypertension (belibasakis, ; bollero et al., ; dominy et al., ; lamster et al., ) . major risk factors of the high prevalence of dental diseases in the elderly in china include the scarcity of dental health knowledge in the general population, low frequency of daily oral hygiene practices, insufficiency of dental care services, and unhealthy diet habits. daily oral hygiene practices are effective for removing plaque and preventing gingivitis. the average awareness rate of dental health in the chinese elderly was . %, only . % of the elderly brush their teeth twice daily, and a mere . % used dental floss (lu et al., ; si et al., ; . increased attention to the dental health needs of an ageing population urgently requires combined efforts by relevant stakeholders (lu et al., ; si et al., ; tonetti et al., ; . specifically in the case of older adults, knowledge and competence in oral care, awareness of medical comorbidities and of medications relevant to oral care should all be strengthened. epidemiological surveillance and monitoring of oral diseases and oral healthrelated quality of life in the elderly is needed. oral self-care, access to treatments and preventive services and assuring the affordability of dental care are critical for oral health. looking after teeth and gums by brushing twice a day with fluoride toothpaste and cleaning with dental floss are effective in achieving a good oral health status. likewise, the control of risk factors, such as refraining from the frequent consumption of foods and drink high in sugar, and refraining from smoking, are also important. provisions to expand services to older adults, to meet increasing oral healthcare needs in the ageing population, and to ensure the affordability of dental care should all be emphasized by policymakers. we suggest programmes that promote general oral health education as well as public outreach programmes directed towards the elderly via understandable brochures, and the use of television and other social medias. additionally, it is important to improve the country's dental care infrastructure by training more dentists and oral specialists and ensuring the provision of affordable dental healthcare. it has been well documented that altered immune system components and function are characteristic of ageing and form part of the causes of age-related diseases (nikolich-zugich, ) . in ageing, a significant decline in the homeostatic, defensive, and surveillance functions of the immune system is noted. prominent features of the ageing immune system include thymus involution, a decrease in naïve lymphocytes, and an accumulation of memory and senescent lymphocytes; more recently, the concept of 'inflammageing' has been developed (ferrucci and fabbri, ) . functionally, impaired immune defense, especially against new antigens for which no memory exists, makes older adults increasingly vulnerable to incident and more severe infections. in addition, a decline in immune surveillance hampers the elimination of premalignant cells, leading to cancer development. older adults also manifest a chronic low-grade inflammatory phenotype (clip), a manifestation of the inflammageing concept, that likely results from uncompensated inhibitory immune regulation and/or an inability to eliminate senescent cells (chen and yung, ; chen et al., b) . as such, immune dysregulation is a general feature of ageing. here we provide an update on infectious diseases in the elderly in china. we carried out a comprehensive review on infections in china based on the following public databases: the chinese center for disease control and prevention (ccdc), the data-center of china public health science (ccdc, ), and the national bureau of statistics of china (nbsc, ). the three most common infectious diseases in were viral hepatitis, pulmonary tuberculosis (tb) and syphilis (detailed in section . ) while the three with the highest mortality rate were aids, tb, and viral hepatitis ( fig. a-d) . of note, pulmonary tb was more prevalent than the other two in older adults over years of age (fig. c ). generally speaking, infectious diseases are more frequent and deadly in older adults, as seen with the recent -ncov epidemic worldwide huang et al., ) ; thus, infectious diseases deserve more attention. viral hepatitis is caused by the hepatitis viruses a, b, c, d, and e (hav, hbv, hcv, hdv, hev) and is prevalent throughout the world, posing a significant threat to human health. china is a highly epidemic area of viral hepatitis with . million people infected with hbv and . million infected with hcvas of (who, . in , there were . million new cases and deaths among the chinese population (nbsc, ) . according to the chinese statutory infectious disease report, viral hepatitis mainly occurred in adults between to years old ( . %, fig. e ). its morbidity in older adults was estimated to be . % in ( fig. e ). however, compared with the morbidity, the mortality of viral hepatitis was higher ( . %) in the aged population ( fig. e ). from the survey, the morbidity and mortality rate of viral hepatitis have ranked in the top five for many years. the morbidity of viral hepatitis was stable in last decade, which is likely due to the wide usage of the hepatitis vaccine ( fig. g , h). however, the morbidity of hepatitis in the elderly continues to increase yearly. since most cases of viral hepatitis developed into chronic hepatitis, the lifespan extension seen in china has contributed to a higher number of elderly hepatitis cases. luckily, the mortality of hepatitis has declined in both the aged population and the population at large (fig. h ). among the five hepatitis viruses, hdv is rarely detected and is not discussed here. hcv ( . %) and hev ( . %) demonstrated high morbidity in the aged population (fig. f) . however, the highest mortality is caused by two acute types, hav ( %) and hev ( . %) (fig. f) , indicating a weakened immune responses against acute infection in the elderly. significantly, both the morbidity and mortality of hbv in the aged population were lowest among the four types ( fig. f ), further indicating the benefit of the hbv vaccine. however, prophylactic vaccines for hcv and other types of viral hepatitis are still lacking. for patients who have been infected, current treatments are still limited, especially for the elderly patients. one of the reasons for this is the lack of a long-term infection model for use in laboratory conditions (winer et al., ) . developing an elderly-representative model would be a useful tool for screening treatment options for those affected by hepatitis diseases. mycobacterium tuberculosis and is typically transmitted through coughs and sneezes. the lack of global tb control is the result of several factors, including hiv coinfection, limited vaccine efficacy, a lack of highly specific and sensitive diagnostic tests, and the rise of multidrug-resistant (mdr) and extensively drug-resistant (xdr) tb strains (venketaraman et al., ) . according to the who's global tuberculosis report, china is ranked third in terms of tb burden when compared with other countries (who, a). pulmonary tuberculosis (p-tb) is the second highest ranked cause of morbidity and mortality among the infectious diseases ranked in (fig. a, b) . however, it is the most frequent infectious disease in the elderly (fig. c ). the elderly occupied more than half ( . %) of all deaths from p-tb (fig. e ). in the last decade, the incidence of p-tb has decreased year to year, however, the incidence and mortality rates of p-tb in the elderly remains high in china (fig. g , f). there are several reasons for the high incidence and mortality rates of p-tb in the elderly: i) an increasingly ageing population; ii) immune decline; iii) delay of diagnosis and treatment. with the increase of the number of elderly patients, p-tb is rapidly becoming a new public health challenge. several risk factors, such as immune decline, smoking, malnutrition, hiv infection and other chronic diseases, make the elderly susceptible to tb . compared with p-tb in the young, p-tb in the elderly has its own characteristics. elderly patients with p-tb are more contagious than the young, and elderly men are more likely to suffer from tuberculosis than elderly women (lee et al., ) . in the elderly, early symptoms of tb are atypical and insidious, and can result in misdiagnosis (rajagopalan, ) . furthermore, chronic fibrous cavitation and hematogenous disseminated tb are more common in the elderly population. most elderly patients with p-tb get tb in their youth at which time it is better controlled but, as they age, p-tb can result as immune function declines. moreover, elderly tb patients usually present with several complications, which further complicates diagnosis and treatment (nagu et al., ) . all these characteristics have brought special focus on the treatment and diagnosis of tb in the elderly. at present, there are several tb guidelines for high-risk groups (who, b), but few for the elderly. previous studies in the elderly have also focused less on the evaluation of targeted strategies for control and prevention. thus it is necessary to pay more attention in the future to the production of control programs and evaluation of targeted interventions for tb in the elderly. aids is a chronic, potentially life-threatening infectious disease caused by hiv, which was first detected in the united states in ( barré-sinoussi et al., ) . in the last decade, the morbidity and mortality of hiv/aids has increased yearly ( fig. g, h) , and it has become the top cause of death by infectious disease in china, including in the elderly (fig. b, d) . the morbidity and mortality of hiv/aids in the elderly population is also rising significantly, and notably the mortality in the elderly is much higher than that seen in the young (ccdc, ). furthermore, because elderly people have many basic diseases and low awareness of selftesting after hiv infection, the elderly are more likely to already be aids patient at the time of diagnosis of their hiv infection (xing et al., ) . a study has shown that . % of newly diagnosed elderly hiv infectors had already developed into the aids stage (liu et al., ) . with increasing use and efficacy of antiretroviral therapy for hiv infection, the lifespan of hiv/aids patients has been greatly extended, and more and more hiv/aids patients will enter old age (nizami et al., ) . the problem of hiv/aids in the elderly will become increasingly serious in the future. firstly, hiv infection is not commonly checked in the elderly in china upon visit to the hospital, which may lead to uncontrolled disease progression and infection to others. second, the treatment of aged hiv/aids patients may cause more adverse effects, such as cardiovascular disease (hanna et al., ; kramer et al., ) , ad (brousseau et al., ) , and diabetes (guaraldi et al., ) . furthermore, cognitive disorders, loneliness, shame and depression may increase the likelihood that they fail to follow their drug regimen, or refuse treatment altogether (greene et al., ; vincent et al., ) . interestingly, hiv infection is also likely a driver of early ageing, as aids patients age more rapidly than the general healthy population (he et al., b; lin et al., ) . to address these problems, the diagnostic process in the aged population should be addressed more cautiously; therapeutic drugs and technologies suitable for the elderly patients should be developed. special attention should also be paid to psychological problems of elderly patients. the hiv epidemic as a sexually transmitted disease will be discussed further below. influenza is an acute viral infection caused by the influenza virus. at present, a total of four types of influenza viruses have been identified, including influenza a, b, c, and d (iav, ibv, icv and idv) (petrova and russell, ) . among them, only iav and ibv are able to cause seasonal epidemics and clinical disease. yearly, the extent of the influenza pandemic varies around the world, which causes high morbidity and mortality. because elderly individuals above years of age are immunocompromised and may have preexisting conditions, they are more susceptible to influenza infection and its complications. data accumulated in the last decade showed that the morbidity of influenza has increased in both the general and aged populations (fig. g ). like other acute infections, the mortality of influenza in aged patients was higher than in younger population (fig. h ). during january , to september , , a total of severe influenza cases were reported in hong kong, among which patients ( . %) were over years old (chp, b). in , a total of influenza cases were reported in macau, among which there were cases were over years (hbgm, a) . however, only a small number of influenza cases acquires laboratory confirmation, as patients usually die of other related illnesses brought on by influenza. thus, the influenza-related mortality rate is greatly underestimated. in , the ccdc estimated that the death rate caused by influenza was / in northern china and . / in southern china, and most of the deaths occurred among people aged over years ( . % in southern cities and . % in the northern) (feng et al., ) . the excess mortality of respiratory and circulatory diseases caused by influenza was . / and . / , respectively, among which % occurred in people aged over years (feng et al., ) . pneumonia is an acute respiratory infection that affects the lungs, which is especially deadly in children under years and in the elderly ( +). pneumonia has become one of the major causes of death for the elderly over years. the harm and mortality of pneumonia increases with age. the " china health statistics yearbook" reported that the mortality rate (/ ) of urban residents aged - , - , - , - , and over with pneumonia was . , . , . , . and . , respectively; and that of rural residents was . , . , . , . and . , respectively (nbsc, ) . since , pneumonia has been one of the top three causes of death in hong kong (chp, a) . according to statists by the hong kong centre for health protection, the mortality rate of pneumonia was / in , with a total of pneumonia-related deaths. of these cases, . % occurred in people aged over years (chp, c) . in macau, pneumonia also has been cited as one of the top three causes of death for many years (hbgm, b). in summary, old age is known to affect the immune system negatively. immunocompromised elderly adults are more susceptible to common diseases such as influenza and pneumonia, both of which were responsible for many deaths in this age group. in some cases, these infections may lead to complications that then lead to death, and this likely contributes to underreporting, hiding the true effects of influenza and pneumonia. there are multiple methods for improving and maintaining healthy immune function in the elderly: physical activity and exercise are known to enhance the immune system, however effective ranges still need to be established and disseminated (venjatraman and fernandes, ) . additionally, the development of vaccines must be prioritized, although challenges exist such as finding suitable mass production methods. perhaps surprisingly, sexually transmitted diseases (stds) are becoming an increasing problem among older age groups. many people aged years or older in china remain sexually active, and the shift towards nursing homes has led to an increase in exposure to possible sexual partners (yang and yan, ) . unfortunately, many older adults do not take precautions in their sex life, due to reasons such as a decreased worry about pregnancy (tht_uk, ) . high-risk sexual behaviors render them vulnerable to the transmission of hiv and other sexually transmitted diseases (stds), likewise low awareness of the potential risks and low use of sexual health services can result in late diagnosis and treatment of stds among older adults. we here describe the current situation of hiv/aids and other stds in older adults in china, and propose potential preventative measures. as mentioned before the incidence and proportion of older adults in the total number of reported hiv/aids cases is on the rise in china (fig. a-d) . the rise in both the number of absolute cases and the proportion of std infections was observed in both genders. the vast majority of cases in older adults resulted from heterosexual copulation, and has brought about an alarming increase in the rate of new infections. for example, in chongqing, the proportion of hiv infections reported in those aged years and older increased dramatically from to . % between and (chinanews, )at the same time, the overall number of male cases quadrupled, and the female cases tripled between - (wu, ) . among women newly diagnosed with hiv in china between - , the proportion of those aged years and older increased from . % in ( / ) to . % in ( / ) . this proportion is even higher in regions with larger rural populations. in guangxi, % of newly reported hiv cases in were men aged + (hu et al., a) . in addition to the increase in newly reported infection among older adults, people infected with hiv can now survive to an older age, increasing the proportion of advanced-age hiv cases. in addition to hiv other stds are increasing in prevalence among the elderly in china. from to , the incidence of syphilis in people over years of age increased by over %. the proportion of people aged years and older among all syphilis cases was also on the rise, from . % in to . % in . between - , the incidence of condyloma acuminate in china showed a downward trend, with an average annual decline of . %. however, the incidence rate among people aged and over increased by . % annually (yue et al., ) . gonorrhea is not common in the elderly, and china saw an average annual decline of . % in the incidence of gonorrhea. this trend was also seen in older adults ( . %- . %) (gong et al., ) . this phenomenon may be related to the short incubation period of gonorrhea, the high self-medication rate of patients, the sensitivity of gonococcal bacteria to antibiotics, and the insignificant clinical symptoms of female patients (wang and ni, ) . there are several contributing factors behind hiv/stds transmission in older adults. ageing is associated with various physiological changes in the human body collectively known as frailty. however, physiological changes in sexual function often fail to attract societal attention. male sexual dysfunction and disorders often manifest in the slowing of penile erection, prolonged ejaculation, the dampening of sexual desire, impotence, etc. as women age, their vaginal tissue becomes thinner, drier, and less likely to become fertile. for the above reasons, the use of condoms in the elderly seems to be less important. older women may have less interest in or need for sexual intercourse; however, their male counterparts may continue to be sexually active for a long period of time. cravings for sex combined with loneliness may push men to resort to commercial sex to quench their desire for sex. in rural areas, the hiv prevalence is high among street-based female sex workers and female sex workers working at sex-on-premise venues with low quality of hygiene, such as hairdressing shops. use of condoms and other precautions in these scenarios is likely to be lacking . sexual education in older adults is nearly absent, and it is generally assumed that "age is a condom". embarrassment may discourage older adults from obtaining condoms and other precautions. in a survey in guangxi, although . % of respondents were willing to accept condoms issued free of charge by healthcare services, . % of the respondents were unwilling to take them of their own due to embarrassment (qi and pang, ) . despite the growing importance of sexual health among older adults, many of them do not seek health services for sexual problems. in china, data on sexual health in older adults are scarce. existing research focuses mostly on males (jiang, ) . few actions have been taken to accommodate older adults' sexual health needs in china. engaging older adults in health program development and policy changes is particularly challenging due to concurrent incidences of disability, frailty, and other comorbidities. conventional top-down strategies are often unappealing and less trusted by the target audience. innovative solutions are needed to develop contextualized sexual health services and ensure that they are inclusive, trusted, and reliable. collectively, hiv/stds are becoming an increasing problem in the elderly in china due to diminished precautions in their sex life, a lack of condom usage, and insufficient sexual education, among other issues. future research focuses should include a) routine sexual healthcare and screening for hiv/stds among older adults, especially those who have highrisk sexual behaviors; b) sexual health education and hiv/stds prevention among older adults; c) late diagnosis of hiv/stds among older adults; and d) healthcare providers' attitude on the sexual health of older adults. modifiable health-related behaviors (hrbs) are key contributors to chronic diseases and early mortality, such that by maintaining a vigorous lifestyle, the processes of frailty, disability, and dementia can be postponed or even prevented (lafortune et al., ; rizzuto and fratiglioni, ; who, c) . similar public health recommendations for hrbs have been promoted worldwide, namely, refraining from smoking and excessive alcohol consumption, consuming a balanced diet, partaking in regular physical exercise, and maintaining frequent social engagements (who, d ). an international comparison study revealed a large degree of consistency in hrb clustering across six nationally-representative ageing cohorts in the east and west, alongside considerable gender-and country-specific variations (liao et al., b) . particularly, older chinese males were characterized by a much higher probability of being smokers ( %) than their counterparts in japan ( %), korea ( %), usa ( %), uk ( %), and in other european countries ( %~ %) (liao et al., b) . comparable findings have been reported in the who's report on the global tobacco epidemic, which further indicates that the progress of smoking reduction tends to be noticeably slower in china than the global average (who, d). nevertheless, positive developments of china's concerted tobacco control efforts, such as smoke-free public places, a strengthened ban on tobacco advertising, etc., should be acknowledged (li and galea, ) . these smoke-free movements have challenged and hope to gradually change social norms regarding smoking, though they may be less effective among older generations with poor health literacy (hu et al., ) . the implementation of the healthy china action plan provides an opportunity to increase tobacco control (li and galea, ) , as well as to address a range of risk factors via a population-based multi-sectoral approach (nhcprc, a) . aiming to enhance the overall health of the chinese population, the plan prioritizes major actions, including the promotion of health literacy, the improvement of nutrition, a new national exercise campaign, more tobacco control measures, the promotion of mental health and environmental health; and specific actions dedicated to four target populations (i.e. women and children, teenagers, older adults, and those undertaking special occupations) and five categories of diseases, i.e. cardiovascular and cerebrovascular diseases, cancer, respiratory diseases (e.g. copd), diabetes, and infectious diseases. besides health-related targets for the health promotion actions for older adults, the importance of building an elderly-friendly and engaging environment is highlighted, which embodies "ageing in place" with humane, equitable and sustainable health and social care resources. social engagement is a key determinant of active ageing (world health organization, ) , especially within china's collective cultural background (liao et al., b; liao et al., ) . in tandem with physical exercise, social activities may generate health benefits not only for the body but also for the soul. chinese square dancing is a social group-based exercise performed to music in public squares or parks. this low-cost and easy-participation activity is highly popular among middle-aged and retired chinese women, estimated at million participants in (fang, ) . square dancers can meet as often as every day, usually in the early morning or evening after dinner, and sometimes both, upon meeting they organize themselves into rank and file, and exercise for nearly two hours, led by the most proficient dancer (liao et al., a) . as an aerobic exercise accompanied by a dance rhythm, square dancing mobilizes the participants' whole body, improving their balance and cardiopulmonary function (liu and guo, ) . it is also cognitively challenging, requiring participants to listen to and process the music, focus on movement and balance, and dance to the rhythm with coordinated body movements (kattenstroth et al., ) . moreover, square dancing creates a socially enriched environment for participants to interact with peers, keeping them socially engaged and dispelling loneliness (liao et al., a; liao et al., ) . square dancing is a typical example of a grassroots group activity that may serve as inspiration for the design of culturally appropriate health promotion programs for older adults. one possibility is developing similar programs that can be implemented throughout the country, and possibly tailoring them to the local needs and/or cultures. in the past five years, central and local governments in china have made enormous efforts in establishing a multi-dimensional geriatric care system to support healthy ageing in chinese society. more than national policies have been issued to drive the development of this care system, including cross-ministerial policy measures for promoting the growth of elderly services and the integrated development of medical, health and elderly care, through the guiding opinions on advancing the development of age-friendly livable environment (ndrc, ) , and the state council opinions on promoting the development of elderly care services (nhcprc, b, c) . following the strategies of the national -year plan, provincial and municipal governments have all issued local implementation plans. in places such as shanghai, shandong, jiangsu, zhejiang and guangdong, political will has been accompanied by strong financial support (cnca, ). as compared to q , in q there was an additional . million beds added in public and private nursing homes across china, resulting in a total national supply of . million beds (mcaprc, a, b) . in , the ministry of civil affairs allocated rmb . billion (usd million) to support the local expansion of care beds in nursing homes as well as the development of community and home care services. in terms of service utilization, the occupancy rate of nursing home beds is at around %, i.e. at any time, there are less than . million residents in these facilities. , elderly benefited from nursing care subsidies while . million benefited from social care subsidies (mcaprc, a, b) . in july , the first national pilot of a long-term care insurance (ltci) program was announced in cities across different regions of china (mhrssprc, ) . identification of elderly people with severe care dependency was carried out, and local models of financing care for them in nursing homes, community centers as well as at home were implemented. by june this pilot program covered a total of . million people, funding services for , beneficiaries at rmb , per year per person (nhsaprc, ). while geriatric care system development has attracted strong attention from stakeholders and become a major theme for policy, research and investment, the following challenges need to be understood and addressed before meaningful progress can be made to prepare the country for its rapid entrance into an ageing society. the first challenge is that care needs must be assessed comprehensively and should be subject to regular reassessment in order to develop personalized care plans and identify goals that are aligned among care recipients, providers and payers (who, ) . generally, there are currently two types of assessments in use in china: one conducted before admission into nursing homes, the other for entry into the ltci programs. the first type can be quite comprehensive but is often used to decide the charge levels associated with the care service. the second type uses a simple -item adl questionnaire and links its results to the funding schemes, e.g. maximum hours of care per month. as the assessment of care needs tends to be one-off and disconnected to care plans or goals (hua, ; ma, ) , it is difficult to allocate resources dynamically and to analyze care performance or economics. the second challenge involves problems with service capacity. on the one hand, % of nursing home beds are left unoccupied and, contrary to international best practice, for the beds that are occupied, only less than % are actually utilized by people with severe dependency; on the other hand, according to the national health commission, nearly million seniors have chronic diseases, and million have various levels of disabilities (nhcprc, b, c) . among the over million people with different degrees of care dependency and care needs, under % have been served by community and home, and the majority have yet to be cared for (mcaprc, a, b) . some policies have been put in place to attempt to fill the huge gap in caregivers, stating that million more caregivers are needed just to care for the existing group of dependent seniors. however, if the current mainstream model of "replacive care" is not changed, growing care service capacity will only lead to an accelerated rate of care dependency among the high-risk population. additionally, such a model of care is highly unattractive to potential workforce candidates. as a corrective move, the central government has now set a goal to train million more caregivers by (mcaprc, a, b) . the third challenge is distorted allocation of resources. up until the end of , despite plans to establish a home care-dominant, community-backed and nursing home-supplemented system, investment has remained predominantly in heavy assets, i.e. the development of nursing homes as well as senior-living property projects, resulting in the above-mentioned "oversupply" of care beds (qiao, ) . since the th five-year plan, the central government has committed to an annual funding of rmb billion to support innovative pilots of home-or community-based care models (mcaprc, (mcaprc, - . however, for many local governments, the first and foremost priority when developing local care capacity is to specify land for elderly care use and invest in care facilities construction before or while looking for operators of such facilities. in addition to resistance and reluctance from nursing homes and preexisting policymakers, difficulty in understanding senior population's care needs and evaluating care competency among community and home care providers have prevented financial support schemes from materializing in most parts of china. typical examples of the insufficient support for community and home care service development can be seen in the number of government purchase tenders that fell through without enough qualified bidders. while there is no lack of political will and resources to be invested in further developing the care system, there is an urgent need to pay for access and quality. a value-based resource allocation model focusing on improving population health rather than the current fee-for-service care model would provide china a rare opportunity to benefit from a healthily ageing society (gyurmey and kwiatkowski, ; mandal et al., ) . to address the above-mentioned challenges and seize the opportunity associated with them, pilots should be designed based on local evidence and should be established in four dimensions. firstly, development of care plans should be focused on individually centered goal based on comprehensive assessments. as highlighted in the latest who icope (integrated care for older persons) package, it is essential for countries and health systems to align the efforts of different stakeholders with a shared care plan that is customized to serve the individuals' priorities and goals. secondly, health and social care resources should be integrated to support the realization of personalized goals of care and, at the population level, to delay and reduce care dependency. rather than further developing passive care capacity to compensate for the increasing need for other fragmented services, devoting resources to the reaching of a consensus among care providers and receivers will serve to empower the population itself, and maximize the pooling of financial and human resources, decreasing the need for an expansion of passive services (who, b). thirdly, the education and training of "integrated care managers" should be developed, whose job would be to work actively in primary care settings to identify care needs and coordinate care resources crucial to achieving societal and individual care goals. mobilizing talents with various backgrounds to understand and operate under the comprehensiveness of geriatric health needs, developing their capability to better communicate and coordinate care efforts across public and private sectors would not only facility the integration of various care services, but make the care work more attractive for those seeking long-term career opportunities (wang and song, ) . fourthly, a reform of the payment model used in elderly care services should be carried out, focusing on value rather than volume of care for populations at risk of care dependency. healthcare payments have long been moving from an inefficient, fragmented, fee-for-service model to a value-based capitation or bundled payment model. for geriatric care financing, this reform is likely to develop faster than the reform of payments for healthcare services. setting sustainable goals for care and allocating resources accordingly will be a viable realistic solution to caring for the millions of chinese citizens in need . we recognize the complexity of establishing such a health-oriented care system. for the four dimensions of an integrated care system to be aligned around common goals as discussed above, a pre-requisite should be the interconnectivity of data: linking results across personal health records, assessments of geriatric care needs, and total costs of care, including: social and commercial health insurance payment, out-of-pocket private payment, social welfare payment, as well as other sources of funding for elderly care (threapleton et al., ) . palliative care is emerging as a new alternative for hospitalized elderly with life-threatening illness. the who defines palliative care as the prevention and relief of suffering of adult and pediatric patients and their families facing the problems associated with life-threatening illness (including malignant and non-malignant diseases). these problems include physical, psychological, social and spiritual suffering of both patients and their family members the aim of palliative care is to enhance the quality of life, promote dignity and comfort, and may also positively influence the course of illness (who, a). palliative care is the basic skill of medical staff in departments where medical care is provided to end-stage patients (e.g. icus, emergency rooms, geriatric and oncology departments) (ning, ) . the quality of death index survey showed that the death quality of mainland china ranked st out of countries, while taiwan and hong kong ranked th and nd , respectively (eiu, ) . while palliative care is widely available in western countries, it is limited in mainland china. according to a report in , only . % ( / , ) of hospitals offered palliative care services. in china, the proportion of course in palliative medicine at medical schools is relatively low and, often only available as electives for undergraduates or postgraduates (liu and yuan, ) . questionnaire data of th year medical students in and of geriatric nurses in , showed that . % of medical students and . % of nurses had no training or education regarding death or terminal care, and % of medical students and . % of nurses had not received any education about hospice and palliative care. thus the need for course education in hospice and palliative care at chinese medical schools is extremely urgent. palliative care is recommended to be introduced early in curative treatments when patients are diagnosed with a life-limiting disease or when the palliative care needs of patients are identified. current palliative care in mainland china is still mainly focused on patients with cancer, with only a few palliative care resources available for other chronic conditions such as copd, hiv and renal failure . therefore, in the future, palliative care should be extended to both patients with cancer, with other life-limiting diseases, and their families. many palliative care guidelines have emphasized that the discussion of advanced decisionmaking among patients and their families should be initiated when patients still possess decision-making capacity (cheng, ) . patients in mainland china sometimes fail to grasp or accept the truth of a diagnosis and limited survival time (cheng, ) . moreover, according to questionnaire reports from , patients in , awareness of the concept of advance care planning or advance directives in china is still low (kang et al., ) . in mainland china, family members are often held responsible for making decisions for the elderly in their care, despite a lack of knowledge or training, and thus may resort to homeopathic remedies. healthcare professionals generally have to ''respect'' any decision made by the families and try their best to ''save'' patients' lives using many life-sustaining treatments, although they generally hold negative attitudes to useless treatments. such an approach is regarded as an appropriate measure in terms of protecting themselves from medical conflicts. misunderstanding of palliative care as 'giving up on treatment and waiting for the death of the patients' by family members of the patients as well as even by some doctors, should be corrected (hu et al., b; ning, ; xiao et al., ) . there is an urgent need for the development of hospice and palliative care in china. in recent years, hospice and palliative care have witnessed rapid development. more and more patients, families, and health-care professionals come into contact with the concept and realize the benefit of hospice and palliative care, while more and more educators, organizations, government and other intermediary leaders have paid more attention to the promotion and development of hospice and palliative care. the current trend towards an ageing society poses difficulties due to the additional challenges seen in diseases in the elderly, including longer disease durations, more complications, underwhelming responses to treatment, and poor prognosis, thus in response to this trend, china has established the national clinical research center for geriatric disorders (ncrcgd) (o'meara, ; yu et al., a) . funded by the central government, the ncrcgd aims to provide innovative models for the diagnosis, management and further research into geriatric diseases at a national level. the ncrcgd focuses mainly on comprehensive and systematic research into pathogenesis, prevention, diagnosis and treatment of age-related diseases such as ad, pd, and cerebrovascular disease (xwhosp, b, ). at the same time, it is committed to building a national elderly medical service network and scientific innovation system by integrating resources of clinical and basic research. for instance, a health data management platform for the elderly could provide a scientific basis for management and decision making. furthermore, through the education and promotion of new theories and technologies of geriatrics to grassroots hospitals, the ncrcgd can build a better medical service system, improving the health of elderly people. the ncrcgd strives to promote the combination of academic and clinical research (xwhosp, b, ). research on agerelated diseases has been carried on such various aspects as diagnosis, treatment, and prevention in fields such as immunology and molecular biology (o'meara, ). the characterization of the gene pool, a series of research findings and new technologies have been applied at the clinic, which promotes the development of gerontology in the direction of precision medicine for early diagnosis, early prevention, and early treatment. the ncrcgd also serves as an educational harbor to foster the training of geriatricians and promote academic exchange (frailty-china, ; xwhosp, b, ). the organization also undertakes other social responsibilities, including partnerships with hundreds of institutions across the country, relying on their collaborative research network to successfully carry out a comprehensive assessment of multiple systems for the elderly (xwhosp, a). through a comprehensive assessment of the multiple aspects of the elderly's diseases, fitness, cognition, psychology and society, it may be possible to develop a system for early identification of health imbalances in the elderly that characterize certain diseases, aiding in their early prevention, and helping to reduce the burden of the ageing chinese society, as well as improving the elderly health service system. prospectively, the ncrcgd will also play its essential role in guiding the research and clinical guidelines for elderly people care as well as making more contributions to improve elderly people's quality of life in china. in order to deal with the ongoing boom in the elderly population, the chinese government has put more effort into funding research on ageing and its related diseases in recent decades. during the recent ( ) outbreak of coronavirus in china, older patients with preexisting ageing-related diseases were found to have a much higher casualty rates than younger patients (chen n et al, ) , again highlighting the importance of preventing ageing-related diseases. in response to this, along with the growing need for improving the quality of life of the elderly in china, more attention has been placed on the development of pharmacological strategies against ageing, organ degeneration and major ageing-related diseases. in this section, we will discuss recent world-wide progress in pharmacological attempts to improve healthspan, and the significant contributions that chinese researchers have made. calorie restriction (cr) was first demonstrated as an effective way to extend lifespan in rodents (de cabo and mattson, ), however the physiological mechanisms behind its anti-ageing effectiveness were not fully understood at the time, and remain uncertain. later studies have suggested that cr might extend lifespan by regulating insulin-like growth factor (igf) and mammalian target of rapamycin (mtor) pathways. metformin is primarily known for treating type diabetes, with its underlying molecular mechanisms leading to the to down-regulation of igf- signaling, and the inhibition of cellular proliferation, mitochondrial biogenesis, ros production, dna damage, activity of the mtor pathway, etc. . the anti-ageing effect of metformin is under investigation by the tame (targeting ageing with metformin) trial in the usa. acarbose, an antidiabetic drug, could also disrupt the igf pathway. acarbose has been shown to partially mimic the effects of cr and extend lifespan in mice by controlling blood sugar and slowing carbohydrate digestion (harrison et al., ) . a clinical trial on acarbose (clinicaltrials.gov identifier: nct ), named study of acarbose in longevity (sail), is in phase , and will hopefully shed some light on its pro-longevity effect in humans. mtor is a pivotal nutrition sensor that links cellular metabolism with proliferation, growth and survival by regulating amino acid metabolism, proteostasis, mitochondria dynamics, cellular senescence, etc. (liu and sabatini, ) . rapamycin, a well-known inhibitor of mtor, has shown life-extending effects in all model organisms and postpones the onset of age-associated diseases harrison et al., ; liu and sabatini, ) . despite the promising pro-longevity outcome of using rapamycin in animals, its clinical application in human has been obstructed by growing concern of potential side effects from immunosuppression and hyperglycemia (pallet and legendre, ) . whether the dosage can be fine-tuned to avoid these side effects will be the determining factor in whether or not rapamycin becomes a future pro-longevity drug. the application of induced pluripotent stem cells (ipscs) from healthy and pathological ageing individuals (liu et al., ) is also propelling further mechanistic studies and translational applications for cr. nicotinamide adenine dinucleotide (nad + ) is a fundamental molecule in human life and health; while there is an age-dependent reduction of nad + , nad + augmentation extends lifespan and improves healthspan in different animal models as well as shows potential to treat different neurodegenerative diseases based on phase i clinical trials (gilmour et al., ; lautrup et al., ; yoshino et al., ) . nad + precursors such as nicotinamide riboside (nr) and nicotinamide mononucleotide (nmn) have emerged as promising approaches for intervention against ageing phenotypes and age-related diseases. supplementation via these precursors can elevate nad + level in vivo and improve glucose metabolism, mitochondria biogenesis, dna repair, neovascularization and neuroprotection . additionally, more than five phase i clinical trials indicate that orally taking nr is well tolerated and able to elevate nad + in the blood (gilmour et al., ; lautrup et al., ) . several clinical trials are currently operating in parallel, investigating nr's effects on metabolic function in bones (nct ), in immunity (nct ), and nmn's effect in cardiometabolic function (nct ), with others also ongoing. in china, although nad + precursors have become widely available commercially as supplements, clinical trials exploring their disease-treating ability in humans are still lacking. senescent cells accumulate in aged tissues and this accumulation is considered one of the driving forces of ageing. senolytics are a class of molecules specifically designed to induce apoptosis of these senescent cells. clearing senescent cells in mice has been shown to substantially alleviate ageing phenotypes, producing potent therapeutic effects in ageingrelated diseases such as ad (bussian et al., ; zhang et al., b) , atherosclerosis (childs et al., ) and osteoarthritis (jeon et al., ) . in , a joint research team of chinese and american researchers found that the molecule abt reduced irradiationinduced senescent bone marrow hematopoietic stem cells (hscs) and muscle stem cells (muscs) in mice . abt (a bcl- family inhibitor), together with dasatinib (an anticancer drug) and quercetin (an apoptosis inducer) are the most commonly used senolytic drugs. the senolytic cocktail of dasatinib plus quercetin (dq) decreased naturally occurring senescent cells, improved mobility and reduced the risk of mortality . however, a small pilot clinical study using the same dq cocktail in patients with idiopathic pulmonary fibrosis (ipf) reported no change in pulmonary function, frailty index, clinical chemistries and reported health, though the beneficial effects on mobility were still noted (justice et al., ) . while clinical trials on senolytic drugs are mainly conducted in the usa, the concept of reducing senescent cells to delay the ageing progress has attracted interest from all over the world. since , the national natural science foundation of china (nsfc) has set up special programs, providing millions of rmb to support research on cellular senescence and organ degeneration. as such, it is recommended that china further expand its investment in senolytics research. targeting the microbiota may also improve age-related diseases, including ad. in , china approved the first domestically invented ad drug, oligomannate (gv- ) (wang et al., d) . considering there has been no new approved anti-ad drugs in the past years, this has been exciting news. despite the potential of these advances, more work is necessary to understand how gv- works. additionally, due to the relatively short clinical trial period, further investigation with longer lasting trials is highly recommended. most human trials for potential anti-ageing drug candidates are conducted in patients with certain age-related diseases. despite partial overlap of the pathologies of these diseases, the knowledge from these trials cannot be interpreted as treating ageing itself. therefore, to reach the goal of identifying anti-ageing compounds, a more comprehensive study on disease-free, healthily ageing groups with no obvious health issues is in immediate need. china has the advantage of a large and diverse population, providing an ideal subject pool for this type of study. the knowledge gained from such studies would likely open new avenues to better understand the fundamental aspects of ageing mechanisms, facilitating their treatment. from a public health and policy perspective, it can be seen that continuing research into prevention and management strategies will be important for both non-communicable diseases as well as geriatric syndromes, to ensure that it is not only life expectancy that is increased, but also the quality of life, by promoting independence and reducing reliance on elderly care services. regular monitoring of trends in incidence and case fatalities of common chronic diseases would enable estimates of future disease burdens and guide preventive health policies (chau et al., a; chau et al., b) . in addition, solutions to trends in the occurrence of disability and frailty are also needed (yu et al., b) . such data would inform the design of elder-friendly service delivery models across the whole spectrum, from prevention to primary care, hospital and residential care settings (woo et al., ; yu et al., ) . currently, hong kong, a special administrative region (sar) of china, has the longest life expectancies in the world for both men and women, such that the need to redesign service models is particularly pressing. by , it is predicted that % of the population in hong kong will be aged years and over; % will have at least one chronic condition, with an increasing prevalence of disability also predicted (yeoh and lai, ) . while the health and social care systems are well developed, there is a mismatch of needs as those with chronic conditions are predominantly managed in the public hospital systems, whereas primary care is predominantly in the private sector. a recent review concluded that better integration of health and social care systems with a primary emphasis on the community could be the best way forward for the ageing population in hong kong (threapleton et al., ) , exemplified by the formation of nurse-led district health centers in (fhb, ). other community models with an emphasis on promoting group activities to prevent frailty and aid selfmanagement of chronic diseases have also been developed (cadenza). such developments have the potential to enhance the role of primary healthcare professionals in preventing functional decline (morley et al., ) , so that many can retain independence even as life expectancy increases. the who's integrated care for older people (icope), formally launched in october , will form a useful blueprint for policymakers to build on their existing health and social care infrastructure (who, c). experiences of elderly healthcare in the european union (eu) may provide useful tips for the situation in china (table ). in the eu, elderly care is provided in each country based on its own social security system and cultural norms. in most european countries, the family and the state are the main providers of support to older people both in activities of daily living (adl) and in instrumental activities of daily living (iadl) (schmid et al., ) . europe is characterized by three types of care provision: ) 'crowding out', whereby the state largely replaces family care; ) 'crowding in', whereby the state promotes family care; ) 'mixed responsibility', whereby both the state and the family take a joint responsibility for care, yet have separate functions (brandt et al., ) . in china, family is still the traditional provider for elderly care (wu et al., a) . under current national and social developmental conditions of china, the chinese government encourages a ' / / ' pattern of eldercare system, namely: % of all older people are cared for at home, % are cared for in communities, and % are cared for in institutions (mayston et al., ) . a 'crowd out' system dominates in the nordic countries (denmark, finland, norway, sweden, iceland) , where the government strives to create a comprehensive system of care services in order to reduce the care obligation of the family. in continental european countries such as austria, belgium, france, germany and the netherlands, systems are more mixed in their provision of elderly care, though tend towards a 'crowd out' approach (kasearu and kutsar, ) . in the island countries, i.e. the uk and ireland, the system is more mixed, and the private market is the dominant welfare provider, with the government providing two main social care services to older people, one being old age pensions and the other being healthcare . southern european countries (e.g. greece, italy, portugal, spain) have a 'crowd in' system whereby families have more responsibilities for care services to older people (kasearu and kutsar, ; wu et al., ) . eastern european countries have undergone dramatic political, social and economic changes after the soviet era and experienced a rapid change from 'crowd out' to a 'crowd in' system where family is the main care provider and the government provides basic formal care services (kasearu and kutsar, ; wu et al., ) . in china, owing to confucian culture and its emphasis on the family, it is taken for granted that the family, most notably adult children, has the responsibility to care for older parents, especially in the rural areas of china, thus older people rely mainly on their children or family for support (chen and silverstein, ; wu et al., a) . rapid demographic ageing increases the demand for care in all ageing societies. currently, european countries face the enormous challenge of implementing major reforms to elderly care in order to ensure that the needs of older people can be continuously met in the future (brandt et al., ; broese van groenou and de boer, ) . to this end, european governments have increasingly relied on informal care in addition to regular and traditional formal care providers from professional home care services, day care units and nursing homes (broese van groenou and de boer, ; verbakel et al., ) . informal care for older people is generally provided by caregivers from both kin and non-kin groups, including spouses, children, relatives, neighbors, friends, etc. (swinkels et al., ) . in europe, around a third of people aged years or older provide informal care to older people. however, shrinking family sizes, the increasing participation of females in the workplace, and rising retirement ages, may pose a drastic challenge to informal care in the future (verbakel et al., ) . china is currently facing challenges in its family-based elderly care model due to new family formation, the spread of individualistic values, and frequent internal migration from rural to urban areas encouraged by rapid economic development (wu et al., a) . moreover, china's one-child policy has sped up the process of population ageing by accelerating the change of the fertility rate and, in turn, has weakened the family-based elderly care model in china . in europe, new elderly care arrangements have been gradually developing based on a new combination of family obligations, market provision and public support. in nordic countries, the state, family and market have been changing with regards to their roles in the provision of elderly care, specifically by increasing the provision of publicly funded care services in a forprofit capacity (marketization of elderly care) and increasing the importance of family care (szebehely and meagher, ) . in estonia, the idea of community-based support for older people has been increasingly set forth in order to postpone the need for institutional eldercare (tulva et al., ) (tulva et al., ) . when it comes to the current trend of eldercare in china, marketization has also been discussed to a large extent both at academic and policy levels. the 'public-private-partnership' (ppp) model may improve the efficiency of familybased eldercare. in the th five-year plan for national economic and social development ( - ) , the opening-up of the market for elderly care services (e.g. purchase of services by the government) was clearly stated (du and wang, ) . elderly care reforms might create new challenges for both europe and china, an important challenge being an increase in inequality in eldercare service utilization among different social groups of older people. older people with higher socioeconomic status will be able purchase private care services whereas those with less social capital will have to rely on more family-based care. in addition, for the chinese government, there is a need to take into account larger inequalities derived from immense resource variations across regions during the development and reform of elderly care services. while mainland china can learn many successful experiences from hong kong, the eu, etc., there remains many unique features that demand the creation of an elderly care system tailored to mainland china. in addition to responding to changes and preparing to adapt to an ageing society at the societal and individual levels, understanding of the mystery of ageing at a molecular level will aid the development of novel strategies to slow ageing and to promote healthy longevity. in the below sub-sections, we will focus on how to use centenarians, the china national genebank database (cngbdb), and ai to further propel ageing research. in china, the numbers of the oldest-old individuals (those aged +), near-centenarians ( +), and centenarians are increasing at roughly % yearly (fig. a-b) , providing unique resources for both basic research and clinical studies. there were , centenarians in , with the number rising to , by (abida and gu, ) . based on the un's medium variant projection, by over a quarter of the global oldest-old population will live in china. as the numbers of the most elderly have expanded, the gender structure of centenarians, the proportion from urban and rural areas, and differences in geographical distribution have formed "three-high" trends in china (data from china's population census, excluding hong kong, macau and taiwan) . first, there is a gender difference, with % of centenarians being female (peng, ) (fig. a) . data from the th population census of china ( ) reported , ( . %) female and , ( . %) male centenarians. this could be due to both physiological (e.g., female hormone estrogen) and cultural differences (women often do more housework, pay more attention to healthcare) (austad and fischer, ; peng, ) . second, urban and rural disparities were clear, wherein more centenarians ( %) live in rural areas, possibly due to a healthier living environment, diet and lifestyle in these regions (cai, ; peng, ; zeng et al., a) (fig. c ). and third, there was geographical difference in the distribution of centenarians. the distribution of longevity areas in china presents several significant characteristics, including province-specific: being majorly in hainan, guangxi, sichuan, yunnan, guangdong and xinjiang, and mostly distributed along river basins, with more centenarians along the pearl and yangtze rivers and the lancang river basins. these characteristics of the area distribution of centenarian suggest that areas beneficial to longevity can be divided into two types: 'natural' and 'economically developed' longevity areas (he et al., ; zeng et al., a) (fig. d) . studies of centenarians can provide valuable information for early prevention of major diseases, premature ageing, and early death, thus providing the scientific support necessary to cope with the quickly approaching arrival of an ageing society in china. centenarians may represent a prototype of successful ageing. a longitudinal study of a danish cohort suggests exceptional longevity does not result in excessive levels of disability (christensen et al., ) . in fact, some centenarians experience a delayed onset of age-related illnesses (delayers), whereas others did not succumb to any age-related illnesses (escapers) (christensen et al., ; hitt et al., ) . in addition, one case-control study showed that older individuals had a delayed age of onset of cancer, cardiovascular disease, diabetes mellitus, hypertension and osteoporosis than their respective younger reference groups (ismail et al., ) . the china hainan centenarian cohort study (chccs) on , centenarians is now in progress, focusing primarily on examining biological indicators and medical aspects, and extensively examining psychological and sociological factors (he et al., ) . all in all, the study of centenarians is a topic of immense importance for population and health policymakers, as well as for the larger aim to achieve long, healthy lives. state-of-the-art technologies enable the 'big data'-based investigation of the molecular mechanisms of human ageing and its associated diseases, providing unique information for therapies and interventions. the cngbdb is a centralized 'big data' hub of biological data, providing data sharing, knowledge search, computational analysis, management authorization, and visualization services to the global research community. built and maintained by the china national genebank (cngb), cngbdb draws from "banks": the living biobank, the biorepository, and the bioinformatics center, and from "platforms": the digitization platform and the synthesis and editing platform. the research data system of cngbdb integrates molecular data from internal and external sources into nine sub-databases including literature, gene, variation, protein, sequence, project, sample, experiment, and assembly (https://db.cngb.org/news/ /). comparative analyses of species and tissues can identify the molecular causes of ageing phenotypes, corroborate or disprove theories on ageing, and help to understand differences in j o u r n a l p r e -p r o o f the mechanisms of ageing across species. genotype comparisons within a species at the level of individuals and populations can help identify genetic reason for differences in lifespan. this approach may be used to compare populations from different regions of china, or chinese and foreign populations, such as ashkenazi jews and okinawan centenarians in japan, two populations well-known for their longevity, facilitating the discovery of chinese-specific agemodifying genes. the identification of potential life-extending genes eases the design of therapeutics that can mimic the effect of these genes in people without those genes. likewise, treatments can be designed for age-related diseases that result from mutated or nonfunctional genes in specific populations. it is also possible to comparatively analyze gene expression at the tissue level, as tissues age at different speeds. since many age-related diseases, such as ad, occur within a specific tissue, understanding the speed at which tissues age can help chinese gerontologists assess the risk of and help to prevent tissue-specific age-related diseases (wieser et al., ) . the advent of 'big data' and machine learning have eased the collection and identification of biomarkers associated with biological age and may allow for the development of personalized clinical diagnostic tools for physicians in the near future (aman et al., ) . in the field of medicine, biomarkers refer to measurable indices capable of identifying a condition, state of being, disease, or environmental marker whose presence may reflect a pathophysiological state (naylor, ) . the use of biomarkers has been applied to the field of anti-ageing technologies, including the prevention and treatment of age-related disease, and has been used to explore methods to delay or offset the ageing process altogether, and will likely serve as key components to advances in the field (campisi et al., ) . in some cases biomarkers may more accurately represent a patient's 'biological age', as opposed to a patient's simple 'chronological age', the former of which is thought to be more clinically relevant (lopez-otin et al., ) . the following sections will review three applications of biomarkers at the molecular, individual and societal levels, including current findings as well as potential research directions. as stated above, there are multiple tests that can be used to obtain molecular biomarkers, and several have been validated to some degree by current research. molecular biomarker studies can be roughly separated into classes: smaller scale studies attempting to determine the utility of a given biomarker, and machine learning studies involving thousands of samples with the intention of developing clinical assessment tools. as an example of this, a recent study involving elderly hypertensive patients was used to investigate a wide library of potentially useful biomarkers . here, elderly chinese participants were matched with subjects from a pool of , normal volunteers. after adjusting for confounding covariate factors, the researchers found that only elevated triglyceride levels were strongly linked to high blood pressure (hong et al., ) . while these cross-sectional studies are certainly important for determining which biomarkers should be considered for clinical evaluation, one of the limitations, at least in comparison to machine learning studies, is that they have a low number of samples. in the above examples, most participant groups contained less than people. while this is a surplus number in other medical contexts, one of the advantages of machine learning is its ability to process thousands of samples granting increased accuracy. fittingly, one of its primary uses is to draw meaningful conclusions from mass, simple, cheap, and non-invasive tests. another key limitation is that biomarker assessment studies using these 'smaller' cohorts tend to lack any external validation. perhaps one of the most easily accessible tests that comes to mind is a standard blood test, here the usefulness of machine learning has been demonstrated by putin and colleagues, who designed a modular ensemble of deep neural networks (dnns) of varying depth, structure and optimization for the prediction of human chronological age using a basic blood test (putin et al., ) . the team trained the dnns using a collection of over , samples from routine blood biochemistry and cell counting assays. the researchers reported that the accuracy of their results provided evidence to suggest that machine learning algorithms could be used to design minimally invasive biomarker tracking methods for ageing that would only improve with greater access to training samples (putin et al., ) . another study examined serum biomarkers, and its results were externally validated using a separate data set from the framingham heart study (sebastiani et al., ) . such studies highlight the ability of machine learning techniques to infer conclusions from basic samples, and to externally validate such conclusions. still, this was one of the very few studies with this type of external validation and more are needed for clinical application. big data can also be used to assist geriatricians for personalized medicine, defined as a medical approach in which treatment is customized on an individual basis based upon disease subtype, genetics, risk, prognosis, or treatment response using specialized diagnostic tests (frohlich et al., ) . for instance, predictive biomarkers for the early detection of certain diseases, may help both patients and doctors to decide on appropriate treatment pathways. in addition, the 'internet of things' refers to the ability of technology to send and receive data via the internet. as wearable/compact technologies become more prevalent (i.e., phone pedometers, pacemakers, insulin trackers) and their data becomes easier to store and share, so too does it become easier to use life-logging data to track individual's wellbeing. unfortunately, while there is great potential for this type of technical approach, there are currently very few cases of applications within clinical practice (frohlich et al., ) , with many studies still in an exploratory phase, requiring further research. for example, one study shows that machine learning techniques have significant potential in developing personalized decision support for chronic disease tele-monitoring systems; however, it was noted that the system would be improved with a larger library of comprehensive predictive markers (finkelstein and jeong, ) . the use of radiomics, the high-throughput mining of quantitative image features from standard-of-care medical imaging that enables data to be extracted and applied within clinical-decision support systems, has also been proposed, especially within the realm of dementia prevention and detection . these studies have benefited from large sample sizes (> , images) using machine learning. this brings us to the issue of noise reduction, which is crucial for effective use of big data and will enable a more robust extraction of features. given the immense amount of data expected to be handled in future projects, finding ways to store, process, and analyze this data also presents a challenge for future research. at the societal level biomarkers have numerous applications. monitoring population-level biomarkers will likely provide an accurate, real-time view of the health state of a given area. this will allow for targeted interventions catered to suit the specific needs of a population. as stated earlier in this piece, modern medicine has provided for major increases in both quality of life at old age, and life expectancy, however, this can also be considered a potential societal burden. earlier the use of federated systems with respect to online medical records and data sharing was discussed as a potential hurdle to some countries and medical systems in the world. china has recently embraced a centralized health informatics scheme, with over % of medical organizations above the county/district level, % of town level hospitals and all cdc above the county/district level capable of transmitting real-time reporting on the status of epidemics via the public health information system (zhang et al., ) . in the future, the data provided by a centralized medical record system has the capacity to train numerous machine learning algorithms for use with biomarkers. another challenge for population-level biomarker implementation is to select low-cost, minimally invasive testing that can be used at a large scale. with respect to china, great advances have been made in the use of medical informatics within the past years. however one of the hurdles going forward for the country is that much of this investment has been driven by industry and the private sector, and a major priority for the country's future should be to divert resources to academic research (liang et al., ) . this is especially true for the poorer members of society, or those without ready access to healthcare, as biomarker are an asset in devising appropriate healthcare plans for populations in need . addressing rural areas may be a challenge both in terms of healthcare delivery and biomarker testing, as these regions may lack sufficient infrastructure for both, posing a challenge for the future . we recommend the use of mobile-equipped information technology services to reach more remote regions. in summary, biomarkers have a great deal of potential for how doctors can prevent, diagnose, and treat illness associated with ageing. while there are many hurdles going forward, the application of machine learning and big data to biomarker research will provide new opportunities to understand ageing at the molecular level, deliver personalized treatment at the individual level, and design influential and effective policy at the societal and population level. since the beginning of the st century, china started to enter a period where it may be classified as an ageing society. at the same time, the compulsory healthcare insurance systems in china has undergone a comprehensive and rapid development, while still emphasizing the ideologies of health equity and social justice . three major health insurance schemes have been launched, achieving near-universal coverage in a short time, which gained early appraisal by emulating the goal of the provision of affordable and equitable basic healthcare for all by (yip et al., ) . after the establishment of the urban employee basic medical insurance (uebmi) in , the chinese government implemented the new rural cooperative medical scheme (ncms) for rural residents in , and the urban residents basic medical insurance (urbmi) for urban residents without employment in . as a result, social health insurance coverage increased from . to . % between and , and further to . % by , and has been stable since (meng et al., ) . in order to further reform the fragmented health insurance system, the latter two of these schemes were combined into the basic medical insurance for rural and urban residents in early , with a target of making the system less complicated, but more equitable for various social groups. in the past years of the new round of healthcare reform beginning in , the chinese government dramatically increased financial investment, with half of all investment in the form of funded premium subsidies for residents to be covered by the social health insurance system (yip et al., ) . universal coverage has since led to improved access to and utilization of healthcare (meng et al., ) , decreased the prevalence of catastrophic health expenditure (yip et al., ) , and reduced out-of-pocket expenditure as a proportion of total health expenditure, especially for vulnerable groups, including older adults (xu and mills, ) . however, the social health insurance system in china still faces the dual challenge of population ageing (demand) and inefficient delivery on the side of the healthcare system (supply), raising both health expenditures and individual disease burden. out-of-pocket expenditure as a proportion of disposable personal income increased from . % in urban regions and . % in rural areas in to . % in (xu and mills, ) . concretely speaking, population ageing addressed the increasing health and social care needs of older people. according to the report on the fifth national health services survey, the prevalence of non-communicable disease had increased more than % between - , from . to . %, while the inpatient rate rose from . to . % in between - . the outpatient rate also increased to . % in (nhfpc, ) . the reimbursement of social health insurance improved rapidly and accounted for % of total health expenditure in , though while out-of-pocket payments dropped from % in to % to , financial protection and services packages were insufficient for the elderly (meng et al., ) , especially for those in rural regions. reported a three times-higher risk of catastrophic health expenditure among the old population in rural regions . in , expenditures on hospitalization for older people in urban areas were reimbursed % by social health insurance and % were covered for their rural counterparts (who, b). regional disparity in health benefits for the elderly with insurance aside, a problem of inequity among different health insurance schemes on health outcomes for older adults is still a great challenge. uebmi recipients were found to have better physical and psychological health outcomes compared to those with urbmi or ncms insurance. this demonstrates a transformation in health insurance reform from an emphasis on the opportunity-oriented health equity measured by coverage and healthcare accessibility to stressing outcome-based equity composed of health outcomes for the elderly, namely "outcome-based health equity", giving priority to disadvantaged groups . in terms of supply-side deficiency and unsatisfied progress in the past years, gaps in the public hospital and pharmaceutical reform have tremendously limited the effectiveness of social health insurance reform, even though the public hospital has removed mark-ups for drug sales, adjusted pricing mechanism, reformed provider payment systems and changed governance structures at the county level (yip et al., ) . the hospital-centered health delivery system has induced the growth of both health expenditure and health insurance expenses, which worsened the control of non-communicable diseases and health outcome improvement in ageing society. unexpectedly, the usage of outpatient and inpatient services in primary health facilities declined from % and % in to % and % in . due to the lack of qualified long-term care facilities, the length of hospitalization was longer for the old population aged and over (who, b), demanding higher expenditure input to cope while wasting health resources. we advocate the immediate application of an integrated health and social care-oriented, particularly in community settings, with the objective of increasing affordability and improving the quality of care for older people. population ageing, family structure shift, and migration, were three major challenges limiting the efficacy of traditional informal care provided by families and their networks. a large proportion of older people with functional disability or dementia will continue to create enormous challenges for an immature long-term care system in china . it was estimated about million ( . % of the elderly) older people had some sort of functional disability by , among which . million ( . % of old population) had a serious status of disability (nhfpc, ) . at the same time, china has become the largest country in the world to have over . million people with dementia (jia et al., ) . in response to the increasing need for social care of older people with disability, the central government of china has implemented a pilot practice of ltci policies in cities, while some local governments were also encouraged by the central government to initiate county-level pilot experiments on ltci since , in hopes of stimulating the growth of long-term care providers (luo and zhan, ) . most ltci schemes were based on the social health insurance system, though these pilots had distinct and diverse eligibility conditions, premium contributions, need assessment instruments, and benefit packages. the reason for carrying out a pilot practice rather than fully implementing a uniform nation-wide scheme reflected the complexity of ltci, and a worry about cost escalation noted ltci introduction in the more mature ageing societies of japan and germany. after two years of practice, a few evaluations were conducted to estimate the outcome of these pilot practices, identifying a host of problems. there are several characteristics and unique features present in the chinese ltci scheme. at first, coverage was narrow and limited only to older people with the most serious degrees of disability, and excluded older adults with dementia due to security issues and lower quality of care skills. by the end of , less than % of the older population in the pilot cities were covered by ltci plans in qingdao (zhu and osterle, ) , which was the first city to launch the ltci scheme in china, a higher proportion of those years and older was achieved in the ltci practice of the mature ageing populations in germany ( . %) and japan ( . %) (oecd, ) . secondly, the need assessment tools used by each pilot city were fragmented and biased. some pilot cities or counties only employed the barthel index to measure physically functional disability, but did not measure cognitive function with any scales, thus leading to the exclusion of older people with dementia from ltci coverage. more seriously, the results of need assessment were not applied to long-term care service provision, but only used as a "gate keeper" for receiving the fixed benefit package. assessment tools should be transformed from simple to comprehensive, from a physically oriented test to a multi-dimension health status one, even from health assessment to service assessment. thirdly, in most pilot plans, long-term care was provided by designated institutions through a contract, and a homeand-community-based caregiver was paid by the insurance scheme, however reimbursements were limited such that a large proportion of costs was still paid out-of-pocket by service users themselves, and unmet needs were still high among the disabled elderly . in addition, the inequality in access to long-term care services between advantaged and vulnerable elderly was enlarged. in most pilot schemes, higher numbers of benefit packages were allocated to insured groups living in nursing homes or receiving formal care than to those living at home receiving informal care. retired people with uebmi had higher affordability and preferred to live in the institutions and received higher reimbursements from insurance. rural residents could not access good quality long-term care facilities, and received fewer benefits. the inequality that remains in ltci practices highlights how policy reform ought to reevaluate and reconstruct the currently fragmented schemes and direct more attention to the disabled elderly with lower socio-economic status and without financial or family support. although china attempted the ltci scheme, its most urgent priority was to establish a unified meanstest public budget system to cover the most vulnerable social groups regardless of their living locations. through lu et al. ( ) 's projection, an investment as small as . % of gdp (equivalent to about . % of fiscal revenue) would greatly benefit the frail elderly and those with serious problems of functional disability and/or poor financial status . in , the central committee of the communist party of china and the state council issued the healthy china plan. corresponding with the health-related sustainable development goal (sdg), this is a national mid-term and long-term strategic plan for moving towards universal health coverage and improving health equity, with emphases on health coverage for the whole life circle, including healthy ageing (prc, ). in , china made a major restructuring of national healthcare governance. the national health commission (originally called the ministry of health) administers and regulates the healthcare delivery system and include two new areas of responsibility: elderly care and tobacco control (yip et al., ) . in addition, the national healthcare security administration was established. it is in charge of administering essential health insurances (urban employee basic health insurance, urban-rural resident basic health insurance, which integrated the original urban resident basic health insurance and new cooperative medical scheme) and medical assistance for the poor and vulnerable groups as well as deciding on pricing and drug procurement. rapid ageing and an alarming increase in non-communicable diseases (ncds) have arisen as major health concerns in china marten et al., ) . in , the national basic public health service program was established, which included health management for elderly people, patients with major ncds (hypertension and diabetes), among others (nhfpc, ) . the program is financed by government funds, and the government's per capita allocation increased from to rmb between (nhfpc, . china's ongoing healthcare system reform prioritizes transforming hospital-centered treatment care to integrated and continued care through a tiered healthcare delivery system (meng et al., ) . a tiered healthcare delivery model defines the functions at each health facility level, and coordinates care across levels. a common model is that hospitals lead medical alliances to deliver integrated care, and provide support and training to strengthen primary health services wang et al., b; wbwho, ) . in addition, residents are able to register with a family doctor team who provide preventive and basic healthcare as well as referral services. the government target is universal registration by (nhc, ) . china has made good progress in improving equal access to healthcare and financial risk protection for socially vulnerable people over the past decade (fu et al., ; meng et al., ; yip et al., ) , but challenges remain. there is a lack of qualified primary healthcare providers who are able to serve as gatekeepers, and the quality of primary healthcare is poorly characterized meng et al., ) . previous studies reported very low proportions of blood pressure and blood glucose control among patients with hypertension and diabetes seeking care from primary health facilities, and common over-prescription of antibiotics su et al., ; wang et al., ) . patients persistently bypass primary health facilities and seek perceived good quality of care in high level hospitals, despite many patients complaining of high medical costs and long wait times . on the other hand, most hospitals still largely rely on fee-for-service payment and tie doctors' salary to the hospital revenue generation, which gives hospitals an incentive to attract and retain patients rather than shifting them primary healthcare. overuse and overprovision of health services are common in china (meng et al., ; yip et al., ) . consequently, health expenditure has continued to escalate, a trend which threatens the long-term financial sustainability of basic health insurance schemes. the efficiency in using health resources is low (meng et al., ; yip et al., ) . as china continues to progress as an ageing society, strengthening primary healthcare system to provide integrated care will be fundamental to meet growing health needs and obtain the best value from existent health resources. it is difficult to shift from treatment-based intensive care to population-based preventive care and health management while perverse financial incentive for hospitals are not controlled or eliminated. this requires effective collaboration across related sectors led by a strong coordinating authority and needs to bridge policy dialogue to ensure health in all policies workable and achievable. china's prolonged demographic shift has led to decreased fertility, elevated sex ratios, rapid ageing, fast urbanization and major geographic redistributions (peng, ) , an interdisciplinary collaborative approach is necessary to prepare and face the challenges as society continues to age. we present a summary on ways to achieve a healthy ageing society in china at societal, individual, and molecular levels (fig. ) . breaking knowledge gaps and eliminating boundaries among different sectors to further integrate and synergize different healthcarerelated parties at societal, individual, and molecular levels will optimize the outputs of the chinese healthcare system. the chinese government has adopted a positive stance to investment across the whole spectrum of ageing policies, medical education and training, basic ageing and geriatric research, prevention, primary care, and hospital and residential care. it is necessary to establish updated ageing policies on retirement age, to incentivize employment of the elderly, to encourage lifelong learning, and to invest in senior volunteer programmes (yeoh and lai, ) . the education and practice of geriatric medicine has been and will continue to be enhanced, including to further increase the teaching of geriatrics-related subjects in medical school, to design high-quality residency and fellowship programs, and to further integrate geriatric principles into general clinical practice (yu et al., a) . the establishment of the national alliance of ncrcgd has been highly appreciated and welcomed and it will continue to serve as a national platform to educate and train geriatricians. while the achievement of healthy ageing and longevity is emerging as an important task for china as in many other countries, this may also be accompanied by socio-economic challenges. one of the major concern is that creating a society where healthy and active ageing and longevity are taken for granted may lead to a swelling of the elderly in the workforce, leading to limitations in job availability for the young, and proving to be a potent economic issue. countries like china and south korea are entering a society of population ageing, showing low birth rates and increased life expectancy, which changes the whole economy korea, ) . population ageing will likely have several macro-economic effects, touching various domains such as overall industrial structure, current account and inflation, output growth, household finance, labor markets, consumption, and even fiscal and monetary policy (korea, ) . it is therefore imperative to give a comprehensive assessment of population ageing in view of its effects on society and the economy in the long-term, to provide evidence to inform future policies. while a challenging task, some suggested responses to the early-emerging changes that could be taken to offset the effects of the ageing society include promoting the production of larger families in the young, finding ways to ensure jobs remain for the young should the elderly be able to continue longer in their positions, along with more general preparations on transform to an elderly-friendly society. it is delightful to witness the progress of basic and translational ageing research in china, as supported by increases in the number of grants and funding opportunities, as well as by rising numbers of high profile publications and discoveries (he et al., c) . joint efforts from the government and stakeholders of each and every sector should be encouraged to nurture an elderly-friendly society, of most import are reforming the social support system to support china's ageing society, and the introduction of health service/investment interventions aimed at reducing inequalities in health among older people in china. we suggest current research focus on basic and translational gerontology to improve healthy longevity in the elderly, and on developing an integrated and affordable health and social care delivery system to meet the complex needs of a growing elderly population, and to finally transform china into an ageenabling country where well-being and healthy longevity can be celebrated for decades to come. in response to the ageing society and in order to improve the quality of life of the elderly in china, strategies at societal, individual, and cellular levels are presented, detailed in 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(per people) of viral hepatitis, pulmonary tuberculosis (p.tb), influenza, hiv infection and aids from to were extracted and presented as general (open) or aged ( years or older, closed) population ), the th population census of china ( ) and projection ( - ) of china's national bureau of statistics. c. women account for % of the total number of centenarians in china and the proportion of rural centenarians is far higher than that of urban area. data source: china's population census, excluding hong kong, macau and taiwan area. the map was made by r. d. geographical distribution of the relative number of centenarians. the proportion of centenarians in china's total population (centenarian ratio) has a significant regional imbalance the authors acknowledge the valuable work of the many investigators whose published articles they were unable to cite owing to space limitations. the authors thank dr. vilhelm bohr at the nia and university of copenhagen for reading of the manuscript. e.f. key: cord- -pnjhi cu authors: foreman, stephen; kilsdonk, joseph; boggs, kelly; mouradian, wendy e.; boulter, suzanne; casamassimo, paul; powell, valerie j. h.; piraino, beth; shoemaker, wells; kovarik, jessica; waxman, evan(jake); cheriyan, biju; hood, henry; farman, allan g.; holder, matthew; torres-urquidy, miguel humberto; walji, muhammad f.; acharya, amit; mahnke, andrea; chyou, po-huang; din, franklin m.; schrodi, steven j. title: broader considerations of medical and dental data integration date: - - journal: integration of medical and dental care and patient data doi: . / - - - - _ sha: doc_id: cord_uid: pnjhi cu dental health insurance coverage in the united states is either nonexistent (medicare and the uninsured), spotty (medicaid) and limited (most employer-based private benefit plans). perhaps as a result, dental health in the united states is not good. what public policy makers may not appreciate is that this may well be impacting medical care costs in a way that improved dental benefits would produce a substantial return to investment in expanded dental insurance coverage. have been rising at double digit rates. most employers have been dropping health care coverage rather than expanding it ( kaiser family foundation ) . medicare trust funds are bankrupt (social security and medicare boards of trustees ). adding coverage would exacerbate an already alarming problem. medicaid funding is a major source of state government defi cits. many states are slashing medicaid coverage during this time of crisis (wolf ). improving medicaid dental coverage during times of budget crisis would meet substantial political resistance. strikingly, strong and increasing evidence suggests relationships between oral health and a range of chronic illnesses. for example, recent fi ndings show relationships between periodontal infl ammatory conditions and diabetes, myocardial infarction, coronary artery disease, stroke, preeclampsia and rheumatoid arthritis. this suggests that improved oral health may well have the potential to reduce the incidence of chronic diseases as well as their complications. if chronic disease incidence is reduced it may be possible to avoid medical care costs related to treating them. it would be important to know more about the extent to which improved oral health could reduce health care costs and improve lives. there are few, if any, studies of the costs of providing medicare dental benefi ts, the costs of improving the medicaid dental benefi t or the cost of providing dental insurance to the uninsured. there are a few studies that indicate that periodontitis increases medical care costs, perhaps by as much as % (ide et al. ; albert et al. ) . ideally there should be a controlled study to assess the benefi t of providing dental coverage through a government payer system. for a preliminary inquiry we can consider work already done and using some cost and benefi t estimates, determine whether it is possible that benefi ts of extending dental coverage may outweigh costs. the failure of medicare to cover dental care has engendered some (albeit not much) public debate. in , congress enacted the medicare prescription drug, improvement, and modernization act (medicare part d). by medicare provided $ . billion in benefi t payments for outpatient prescription drugs and medicaid paid . billion for outpatient prescription drugs (center for medicare and medicaid services ) . benefi ciaries provided billions more in the form of monthly part d premiums. the expense of the medicare prescription drug program and the controversy surrounding its enactment may well have eroded public support for increased medicare coverage. so while there has been no shortage of effort paid to improving medicare, the one common theme in all of the recent initiatives is that dental care has been conspicuously a new study by hedlund, jeffcoat, genco and tanna funded by cigna of patients with type ii diabetes and periodontal disease found that medical costs of patients who received maintenance therapy were $ . per year lower than patients who did not. cigna, research from cigna supports potential association between treated gum disease and reduced medical costs for people with diabetes, http://newsroom.cigna.com/newsreleases/research-from-cigna-supports-potential-association-between-treated-gum-disease-and-reduced-medical-costs-for-people-with-diabetes. omitted. as a result, million medicare recipients in (us census bureau ) continue to have no dental insurance coverage through medicare. medicaid dental coverage is an optional benefi t that states may or may not elect to provide. in medicaid, both the state and the federal government provide funds to cover healthcare services to eligible patients. the bulk of the money comes from the federal government. because the medicaid dollars are limited and coverage for systemic diseases has precedence, medicaid coverage of dental care has been spotty. even where it has been provided, payments to dental providers have been so low as to make it diffi cult or impossible for medicaid benefi ciaries to obtain adequate dental care (broadwater ) . the recession increased the number of medicaid eligible individuals nationwide. further, the federal budget defi cits of the past few years have reduced the federal contribution to state medicaid programs. the combination of increases in the number of benefi ciaries and diminished revenues has caused a number of states to eliminate or curtail medicaid dental coverage (ehow ; mullins et al. ) . the result, million medicaid benefi ciaries in the us (us census bureau ) in either had no dental insurance coverage or inadequate coverage. approximately million people in the united states do not have health insurance (kaiser family foundation ) . presumably, they have no dental insurance either. further, not every employer provides dental insurance. a cdc survey found that . % of adults do not have dental insurance coverage (centers for disease control ) . a montana survey found that % of employers who offer health insurance do not offer dental insurance coverage (montana business journal ) . in there were approximately million people enrolled in health insurance plans (us census bureau ) . if half (a rough combination of the cdc and montana percentages) of them do not have dental insurance it is likely that an additional million (nonelderly, non-poor) people in the us do not have dental insurance coverage. finally, the term "dental insurance" is actually a misnomer. dental policies cover routine treatments, offer discounts for more complex treatment and impose a low yearly on total payments. in fact, it has been called "part insurance, part prepayment and part large volume discount" (manski ) . effectively, many (if not most) people who have dental insurance fi nd it coverage to be quite restrictive. for example, many impose a small yearly cap ($ , is common) or large coinsurance amounts ( % for orthodontia, for example) (rubenstein ) . even with discounts it is easy for many people to exceed the annual limit. given the lack of dental insurance coverage it is not surprising that the status of oral health in the us is not particularly good. in approximately . % of adults between the ages of and had untreated caries, % had decayed, missing and fi lled tooth surfaces and more than one-half of adults had gingival bleeding (dental, oral and craniofacial data resource center of the national institute of dental and craniofacial research ) . three fourths of adults in the us have gingivitis and % have periodontitis (mealey and rose ) . if these levels of untreated disease were applied to most systemic diseases, there would be public outcry. over the past decade evidence has been building that there is a relationship between dental disease, particularly periodontal disease, and chronic illnesses. mealey and rose note that there is strong evidence that "diabetes is a risk factor for gingivitis and periodontitis and that the level of glycemic control appears to be an important determinant in this relationship" (mealey and rose ) . moreover, diabetics have a six times greater risk for worsening of glycemic control over time compared to those without periodontitis and, periodontitis is associated with an increased risk for diabetic complications. for example, in one study more than % of diabetics with periodontitis experienced one or more major cardiovascular, cerebrovascular or peripheral vascular events compared to % of the diabetic subjects without periodontitis (thorstensson et al. ) . also, a longitudinal study of type diabetics found that the death rate from ischemic heart disease was . times higher in subjects with severe periodontitis and the death rate from diabetic nephropathy was . times higher (saremi et al. ) . clinical trials have demonstrated that treatment of periodontal disease improved glycemic control in diabetics (miller et al. ) . moreover, investigations have found an association between periodontal disease and the development of glucose intolerance in non-diabetics (saito et al. ) . while it is diffi cult to establish causality and it is possible that other factors infl uence periodontal disease and medical complications, these studies suggest that treatment of periodontitis substantially improves health and greatly reduces medical complications related to diabetes. similarly, periodontitis is associated with cardiovascular disease and its complications including ischemia, atherosclerosis, myocardial infarction and stroke. a study by slade and colleagues found both a relationship between periodontitis and elevated serum c-reactive protein levels (systemic marker of infl ammation and documented risk factor for cardiovascular disease) as well as a relationship among body mass index, periodontitis and crp concentrations (slade et al. ) . hung and colleagues evaluated the association between baseline number of teeth and incident tooth loss and peripheral arterial disease. they determined that incident tooth loss was signifi cantly associated with pad, particularly among men with periodontal disease potentially implying an oral infection-infl ammation pathway (hund et al. ) . the same group of researchers used the population enrolled in the health professionals' follow-up study ( , men free of cardiovascular disease and diabetes at baseline) to assess the relationship between tooth loss and periodontal disease and ischemic stroke. controlling for a wide range of factors including smoking, obesity, and dietary factors, the researchers found a "modest" association between baseline periodontal disease history and ischemic stroke . as early as destefano and colleagues found that among subjects, those with periodontitis had a % increased risk of coronary heart disease relative to those without. the association was particularly high among young men. the authors questioned whether the association was causal or not, suggesting that it might be a more general indicator of personal hygiene and possibly health care practices (destefano et al. ) . in wu and colleagues used data from the first national health and nutrition examination survey and its epidemiologic follow-up study to examine the association between periodontal disease and cerebrovascular accidents. the study found that periodontitis was a signifi cant risk factor for total cva, in particular, for non-hemorrhagic stroke (wu et al. ) . in addition to diabetes and coronary artery disease, associations have been found between periodontal disease and rheumatoid arthritis and respiratory disease. this is not surprising given the role of periodontal disease in the production of infl ammation related proteins. dissick and colleagues conducted a pilot study of the associate ion between periodontitis and rheumatoid arthritis using multivariate regression and chi square tests. they found that periodontitis was more prevalent in patients with rheumatoid arthritis than in the control group and that patients who were seropositive for rheumatoid factor were more likely to have moderate to severe periodontitis than patients who were rf negative and also that patients who were positive for anti-cyclic citrullinated peptide antibodies were more likely to have moderate to severe periodontitis (redman et al. ) . paju and scannapeico investigated the association among oral biofi lms, periodontitis and pulmonary infections. they noted that periodontitis seems to infl uence the incidence of pulmonary infections, particularly nosocomial pneumonia in high-risk subjects and that improved oral hygiene has been shown to reduce the occurrence of nosocomial pneumonia. they found that oral colonization by potential respiratory pathogens, for possibly fostered by periodontitis and possibly by bacteria specifi c to the oral cavity contribute to pulmonary infections (paju and scannapeico ) . the implications for these fi ndings are profound. professionally, they suggest that managing patients with chronic illness and periodontal disease will require teamwork and a deeper knowledge base for dentists and for physicians (mealey and rose ) . dentists will need to be alert for early signs of chronic illness among their patients and physicians will need to be alert for signs of dental disease. both will need to consider wider treatment options than their specialty indicates. dentistry and medicine have operated as professional silos in the past. the relationship between dental disease and chronic medical conditions suggests that continued separation is detrimental to patient centered care. beyond treatment implications, there are extremely important health policy concerns. if treatment of periodontitis and other dental problems leads to reduced incidence of chronic illness, fewer complications from chronic diseases and reduced morbidity among chronically ill patients, increased access to dental services could signifi cantly reduce health care costs. the diseases associated with periodontitis are among the most common illnesses, the fastest growing and the most expensive diseases that we treat. a recent robert wood johnson report notes that approximately million americans have one or more chronic conditions, that the number of people with chronic conditions is expected to increase by % per year for the foreseeable future and that the most common chronic conditions include hypertension, disorders of lipid metabolism, upper respiratory disease, joint disorders, heart disease, diabetes, cardiovascular disorders, asthma and chronic respiratory infections (anderson ) (see fig . . ). one in four americans has multiple chronic conditions. ninety-one percent of adults aged and older have at least one chronic condition and % have two or more of them (anderson ) . people with chronic conditions account for % of all healthcare spending. seventy eight percent of private health insurance spending is attributable to the % of privately insured persons with chronic conditions. seventy three percent of healthcare spending for the uninsured is for care received by the one third of uninsured people who have chronic conditions. seventy nine percent of medicaid spending goes to care for the % of non-institutionalized benefi ciaries who have chronic conditions (anderson ) (see fig. . ). further, health care spending increases with the number of chronic conditions (anderson ) (see fig. . ). more than three fi fths of healthcare spending (two thirds of medicare spending) goes to care for people with multiple chronic conditions. those with multiple chronic conditions are more likely to be hospitalized, fi ll more prescriptions, and have more physician visits (anderson ) . in the american diabetes association estimated direct medical expenditures for diabetes at $ . billion: $ . billion for diabetes care, $ . billion for chronic complications and $ . billion for excess prevalence of general medical conditions. approximately % of direct medical expenditures were incurred by people over . indirect expenditures included lost workdays, restricted productivity mortality and permanent disability -a total of $ . billion. all told, diabetes was found to be responsible for $ billion of $ billion in total expenditures. per capita medical expenditures totaled $ , annually for people with diabetes and $ for people without diabetes (hogan et al. ) . more recently, dall and colleagues estimated that the us national economic burden of prediabetes and diabetes had reached $ billion in , $ million in higher medical costs and $ billion in reduced productivity. annual cost per case was estimated at $ , for undiagnosed diabetes and , for type diabetes (dall et al. ) . the costs of caring for people with diabetes have risen both because the numbers of diabetics has been increasing and because the per capita costs of care have increased. the number of diabetics increased from . million on to . million in (ashkenazy and abrahamson ) . a recent report by the unitedhealth group center for health reform & modernization provides a dire estimation -that more than % of adult americans could have diabetes ( %) or prediabetes ( %) by at a cost of $ . trillion over the decade. this compares with current estimates of % of the population with diabetes and % with prediabetes, or %. these estimates conclude that diabetes and prediabetes will account for % of total healthcare spending in at an annual cost of $ billion, up from an estimated $ billion in (unitedhealth center for health reform and modernization ) . average annual spending over the next decade by payer type is $ billion for private health insurance, $ billion for medicare, $ billion for medicaid and $ . billion for the uninsured. what about cardiovascular disease and rheumatoid arthritis? among the top ten health conditions requiring treatment for medicare benefi ciaries in approximately % of benefi ciaries suffered from hypertension, % from heart conditions, % had hyperlipidemia % had copd, % had osteoarthritis and % had diabetes (thorpe et al. ) . the american heart association estimates the cost of cardiovascular disease and stroke to be $ billion in direct expenditures and $ . billion for productivity losses due to morbidity and $ . billion in lost productivity due to mortality (present value of lost wages at %) (lloyd- ) . the centers for disease control estimates that during - million americans had selfreported doctor diagnosed arthritis, million of them with activity limitations (cheng et al. ) . cisternas and colleagues estimated that total expenditures by us adults with arthritis increased from $ billion in to $ billion in . most of the increase was attributable to people who had co-occurring chronic conditions (cisternas et al. ) . the cisternas study appears to aggregate all medical care expenditures by people with arthritis (which would include expenditures to treat diabetes and cardiovascular disease). an earlier cdc study focused on the direct and indirect costs in attributable to arthritis that estimated $ . billion in direct costs (medical expenditures) and $ billion in indirect costs (lost earnings) (yelin et al. ) . in short, current cost estimates for direct health care expenditures (excluding productivity losses) related to diabetes are approximately $ billion, for cardiovascular treatment, $ billion, and for rheumatoid arthritis, approximately $ billion (estimating that the $ . billion in costs have grown approximately % per year), a total of $ billion of the $ . trillion that will be spent in the us in . moreover, given current growth in the prevalence of diabetes, the unitedhealth estimate of $ million in spending for diabetes alone is not unreasonable. if health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis only increase by % over the next decade (even given added demand produced by the aging baby boomer population), annual costs of these chronic diseases will exceed $ . trillion in . if we use the unitedhealth estimates for the proportions of diabetes costs paid by private insurance ( %), medicare ( %), medicaid ( %) and the uninsured ( %) and estimate total costs based on the studies projecting a % increase in years and a % increase in years we can obtain an estimate of future costs for treating diabetes, cardiovascular disease and arthritis. table . set forth below, summarizes these cost estimates. by medicare costs for these chronic illnesses would be approximately $ billion. the estimated costs to medicaid will be approximately $ billion. the costs for the uninsured will be approximately $ billion. any intervention that has the potential to substantially reduce these costs will produce meaningful results. unfortunately, even though there had been a substantial numbers of studies that show relationships between dental disease and chronic illness that are have been very few studies that actually test whether improved dental treatment reduces the incidence of chronic illness and complications due to chronic illness. the potential for large health care cost savings through an active and aggressive program of dental care is so large that such studies are clearly indicated. suppose, for example, that % of all medical care costs required to treat diabetes, cardiovascular disease and arthritis could be avoided through an active aggressive program of dental care. what this would mean is that in private health insurers could see a $ billion reduction in healthcare costs, medicare would see a $ . billion reduction and medicaid pay $ . billion reduction. recent health reform has provided for the issuance of health insurance to the uninsured by state exchanges. aggressive dental care that saved % of costs attributable to diabetes, cardiovascular disease and arthritis could save the exchanges $ billion per year. and, if greater proportions of costs can be saved or if the estimates of costs are low, potential benefi ts will be even larger. once again, it would be important to know whether aggressive dental care could produce such savings and how much. ide and colleagues found that people who were treated for periodontitis incurred % higher health care costs than those who were free of periodontal disease (ide et al. ) . similarly, albert, et al., found medical costs associated with diabetes, cardiovascular disease and cerebrovascular disease were signifi cantly higher for enrollees who were treated for periodontitis than for other dental conditions (albert et al. ) . additional studies of this nature would be important to support a measured approach to expanding dental coverage. so what do we mean by an aggressive dental treatment plan? suppose we were to provide dental insurance to all medicare benefi ciaries at the level of current private dental insurance coverage and strongly encourage benefi ciaries to receive dental treatment. suppose we were to provide for medicaid payment for all benefi ciaries at the level of current private dental insurance coverage. suppose health care insurers provided dental coverage in order to reduce their costs and that such coverage was consistent with current private dental insurance coverage. suppose health insurance companies, understanding the benefi ts from dental care, were to require their private employer customers to cover the costs of dental care. how much would all of this cost? how would it compare to the benefi ts that may be available? in order to estimate the potential costs of providing enhanced coverage for dental care we start use the cms estimates of national health care spending for dental services and statistical abstract of the us estimates for medicare enrollment, medicaid enrollment, private health insurance enrollment and uninsured persons. based on the estimate that half of private employers with health insurance provided dental insurance coverage we estimate that of the private health insurance enrollment one half would have dental insurance coverage and one half would not. table . sets forth the national health care expenditures for dental services in millions and enrollment in private dental plans, medicare, medicaid, the uninsured without health insurance and dental insurance, the uninsured with health insurance and dual eligibles. from this we derive a cost per enrollee for private dental insurance, medicare dental benefi ts and medicaid dental benefi ts. in order to estimate the annual cost of providing full dental coverage to medicare benefi ciaries we subtracted dual eligibles (who receive some dental insurance) from total medicare enrollees to determine the number of persons who would need coverage. in our example there were million medicare benefi ciaries including million dual eligibles. accordingly, the estimates would cover the million medicare benefi ciaries that are not dual eligible at a cost equal to the per capita cost of private dental insurance ($ . ) less amounts that medicare is already paying for dental services ($ . per person). the result provides an estimate of the cost of covering all medicare benefi ciaries for dental services at a level equivalent to private health insurance. using the example the cost of providing full dental insurance coverage to medicare benefi ciaries would have been $ . billion. in addition, we used the cms national health expenditure fi gures to determine administrative costs for private health insurance, medicare and medicaid as a percentage of program expenditures for medical care. we found that the administrative costs of the medicare program were . % on average for - . in order to fully estimate the cost of medicare dental coverage we added . % to the cost health insurers will be in the same position as medicare and medicaid regarding dental coverage. if quality dental coverage saves health care costs attributable to diabetes, cardiovascular disease and rheumatoid arthritis then the exchanges will have an incentive to provide quality dental coverage to reduce costs. accordingly, we estimated the cost of providing dental coverage equivalent to private dental insurance coverage through the exchanges. again we assume that the costs of such coverage will be equivalent to the number of uninsured persons multiplied by the annual per capita cost of coverage. for the example, this would refl ect coverage for million people at $ . per person, a total of $ . billion. with administrative costs, the cost of providing dental insurance coverage to the uninsured at a level equivalent to private dental coverage would be $ . billion. finally, given the evidence that improved dental care has the potential to reduce health care costs private health insurers may wish to expand health insurance to cover dental care. here, we estimate the cost of providing dental insurance to the % of the workforce whose employers currently do not provide dental insurance benefi ts. once again, we multiply the number of covered lives by the estimated annual per capita cost. for the example we estimate million adults will receive dental coverage at $ per person: $ billion for dental services and $ . billion for administrative costs or a total of $ . billion. of course, as noted a number of times above, these estimates are based on providing full "universal" dental insurance coverage at levels equivalent to current benefi t levels for private dental insurance. it may be that an appropriate package of dental services that deals specifi cally with periodontitis can be provided for less than the full cost of private dental insurance. once again, further research should provide better information. the health reform law does not attempt to provide coverage to all million people without health insurance. estimates are that only million people will be covered by the bill. even though this is the case we prepare our estimates using all million uninsured americans. indeed, the failure of % of employers to cover dental services may well constitute a classic externality in the market for health insurance. internalizing this externality may well provide better effi ciency. it is also possible that dental care for persons with greater incidence of chronic illness as is the case with medicare benefi ciaries may require even higher levels of spending per benefi ciary. again, it would be good to know scientifi cally if this is the case. as noted in sect. above, costs for diabetes, cardiovascular disease and arthritis will be $ billion for private health insurance, $ billion for medicare, $ billion for medicaid and $ billion for the uninsured. costs of providing "full" dental coverage will be $ . billion for medicare, $ . billion for medicaid, $ . billion for the uninsured and $ . billion for private health insurance. given this, if . % or more of the medicare costs can be "saved" through improved dental care, medicaid dental insurance will pay for itself and will provide a positive return on investment. see table . . similarly, private health insurers could justify providing dental insurance coverage to employees who do not have it so long as they spend . % or more of their chronic care costs for diabetes, cardiovascular disease and arthritis. on the other hand, it would appear that medicaid expansion would require cost savings of approximately % and that health care insurance coverage of the uninsured would require savings of approximately % in order to justify coverage. while it is possible, it may not be likely that full dental coverage would be justifi ed for these programs. of course, these estimates do not consider indirect costs in the form of lost wages or premature death. these costs are externalities to the health insurance programs. to the extent that they represent a social benefi t that a national dental insurance program might internalize, it would be appropriate to consider their impact in the cost-benefi t analysis. in any event, better understanding of the potential for deriving savings in health insurance costs related to chronic diseases like diabetes, cardiovascular disease and arthritis would be crucial to any determination whether to expand insurance coverage for dental care. heretofore the case for expanding medicare coverage to include dental care has taken the form of "benefi t" to patients rather than benefi t to health insurance programs and society and has been cast in emotional and political terms. for example, oral health america grades "america's commitment to providing oral health access to the elderly" (oral health america ) . in truth, there is no american commitment to providing oral health access to any age group, much less the elderly. rubenstein notes that "at least one commentator has suggested that the dental profession should join with senior citizen groups when the time is right to ask congress to expand medicare to cover oral health" (rubenstein ) . rubenstein emphasizes that "calls for action" are "mere words" unless they are accompanied by political actions that health policy professionals and the dental profession must help promote (rubenstein ) . another commentator has suggested that "as soon as the debate over medicare prescription drug coverage and, the debate to provide dental care coverage for the elderly may soon begin" (manski ) . rubenstein, again suggests that "the dental community must convince americans, and particularly aging boomers, that oral health is integral to all health, and for that reason, retiree dental benefi ts are an important issue". in truth, a decade of defi cit spending and public distaste for out of control program costs in the medicare and medicaid programs as well as the unpopularity of the process that was used to provide medicare prescription drug coverage (with perceived abuses by the health insurance and drug lobbies) and national health reform makes it unlikely that the public would be willing to approve expansions in insurance coverage for dental care "for its own sake" or "as the right thing" or to "benefi t seniors." what this political climate has produced is an arena in which a good idea that could provide appropriate return on investment for society might well be rejected out of hand based on political history of health insurance coverage. as a result, it is incumbent on policymakers, medical and dental research scientists and health economists to investigate and confi rm the potential savings that expansion of dental insurance coverage has the potential to produce and to develop hard evidence regarding potential costs of the expansion prior to, not as a part of, political efforts aimed at dental coverage expansion. a responsible, well informed effort to expand dental coverage may well go far to restore public confi dence in the health policy process. joseph kilsdonk and kelly boggs the adage of "putting your money where your mouth is" is often referenced when being challenged about public statements or claims. in this instance, we use it literally. in health care costs in us were $ . trillion. there have been numerous reports on health disparities, the burden of chronic diseases, increasing healthcare costs and the need for change. long-term economic benefi ts associated with the cost of care are dependent upon integrating oral health with medicine. this is particularly true as it relates to the management of those conditions which impact the economics of healthcare the most. as examples, % of medicare costs and % of medicaid costs are in managing chronic health conditions (partnership for solutions national program offi ce ) . more than % of the u.s. population has one or more chronic condition (cartwright-smith ) and in , % of medicare spending was on patients with fi ve or more chronic diseases (swartz ) . effective management of health care resources and information are critical to the economic well-being of our healthcare system. we can no longer afford to manage care in isolation. integration of care between medicine and dentistry holds much promise in terms of reducing the cost of care and an integrated medical-dental electronic healthcare record (iehr) is the vehicle that will lead to downstream cost savings. in the united states the center for medicare & medicaid services (cms) has conducted demonstration projects around chronic disease management. section of the benefi ts improvement and protection act of mandated cms to conduct a disease management demonstration project. april , , as an effort to reduce the cost of care and improve quality associated with chronic diseases, cms partnered with ten premier health systems to effectively manage chronic diseases in a medicare physician group practice demonstration (pgp). it was the fi rst pay-for-performance initiative for physicians under the medicare program (center for medicare and medicaid services ) . it involved giving additional payments to providers based on practice effi ciency and improved management of chronically ill patients. participants included ten multispecialty group practices nationwide, with a total of more than , physicians, who care for more than , medicare benefi ciaries (frieden ) . the chronic diseases that were targeted were based on occurrence in the population and included diabetes, heart failure, coronary artery disease, and hypertension (frieden under the pgp, physician groups continued to be paid under regular medicare fee schedules and had the opportunity to share in savings from enhancements in patient care management. physician groups could earn performance payments which were divided between cost effi ciency for generating savings and performance on quality measures phased in during the demonstration as follows: year , measures, year , measures and years and having quality measures. for each of the years only the university of michigan faculty group practice and marshfi eld clinic, earned performance payments for improving the quality and cost effi ciency of care. a large part of the success of this project was attributed to being able to extract, evaluate, and monitor key clinical data associated with the specifi c disease and to manage that data through an electronic health record (table . ). during the third year of the demonstration project marshfi eld clinic, using a robust electronic health record succeeded in saving cms $ million dollars; that's one clinic system in year. as a result of such demonstration projects and as of this writing, cms is looking to establish accountable care organization's as the medical front runners to new care delivery methods for quality and cost control. accountable care organization (aco) is a term used to describe partnerships between healthcare providers to establish accountability and improved outcomes for the patients. in a cms workshop on october , , don berwick, the administrator of cms, stated "an aco will put the patient and family at the center of all its activities…" an emerging model of an aco is the patient-centered medical home (pcmh). pcmh is at the center of many demonstration projects. acos were derived from studies piloted by cms. since funds provided by cms, do not cover routine dental care as part of the patient management or quality and cost objectives cms aco studies are limited if they become models for the pcmh, due to the exclusion of dental. more recently, organizations representing the major primary care specialtiesthe american academy of family practice, the american academy of pediatrics, the american osteopathic association, and the american college of physicianshave worked together to develop and endorse the concept of the "patient-centered medical home," a practice model that would more effectively support the core functions of primary care and the management of chronic disease (fisher ) . in geisinger health system, kaiser permanente, mayo clinic, intermountain healthcare and group health cooperative announced they will be creating a project called the care connectivity consortium. this project is intended to exchange patient information. although progressive in their approach their project does not include dental. these benefi ts however, are yet to be adapted in the arena of oral health. as of this writing, dentistry remains largely separate from medical reimbursement mechanisms such as shared billing, integrated consults, diagnosis, shared problem lists, and government coverage. for example, cms does not cover routine dental care. dentistry is also working to establish its own "dental home" with patients. however to reap the economic benefi ts of integrated care, a primary care "medical-dental" home is what needs to be created. according to an institute of oral health report ( ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships that create treatment solutions. the case for medical and dental professionals' comanaging patients has been suggested for almost the past century, in william gies reported that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies ) . as described by dr. richard nagelberg, dds "the convergence of dental and medical care is underway. our patients will be the benefi ciaries of this trend. for too long, we have provided dental care in a bubble, practicing -to a large degree -apart from other health-care providers. even when we consulted with our medical colleagues, it was to fi nd out if premedication was necessary, get clearance for treatment of a medically compromised patient, or fi nd out the hba c level of a diabetic individual, rather than providing true patient co-management. we have made diagnoses and provided treatments without the benefi t of tests, reports, metrics, and other information that predict the likelihood of disease development and progression, as well as favorable treatment outcomes. we have practiced in this manner not due to negligence, but because of the limitations of tools that were available to us" (nagelberg ) . integrated medical/dental records need to be a tool in a providers' toolbox. in the case of marshfi eld clinic, dental was not included in their past cms demonstration project as dental is not a cms covered benefi t, and thus not part of the demonstration. however, as a leader in healthcare, the marshfi eld clinic recognizes the importance of data integration for both increased quality and cost savings. "marshfi eld clinic believes the best health care comes from an integrated dental/medical approach," said michael murphy, director, business development for cattails software. integration enhances communication between providers and can ultimately lead to better management of complex diseases with oral-systemic connection, avoidance of medical errors, and improved public health. while the cms pgp and other demonstration projects along with independent studies have shown to improve quality and reduce costs through integration, greater results may be afforded if studies are not done in isolation from dental data. in fact, if healthcare does not fi nd a way to manage the systemic nature of the pathogens known to the oral cavity the economic impact and cost savings around chronic disease management will hit a ceiling. the economic opportunity of having clinical data for integrated decision making is readily identifi ed by the insurance industry. the effective management of clinical data around chronic and systemic oral and medical disease as part of an iehr is the greatest healthcare cost savings opportunity associated with such a tool. the insurance industry sustains itself through risk management [obtaining best outcomes] using actuarial analysis [data] and controlling costs [reduction of costs] in order to ensure coverage [profi tability]. as such they have pursued the economic and outcome benefi ts of integrated medical -dental clinical decision making. as an example, in there was a study conducted by the university of michigan, commissioned by the blue cross blue shield of michigan foundation ( ) , the study included , blue cross blue shield of michigan members diagnosed with diabetes who had access to dental care, and had continuous coverage for at least year. with regular periodontal care, it was observed diabetes related medical costs were reduced by %. when compounding chronic health complications were also examined, the study showed a % reduction in cost related to the treatment of cardiovascular disease in patients with diabetes and heart disease. a % reduction in cost related to treatment of kidney disease for patients with diabetes and kidney disease. and a % reduction in costs related to treating congestive heart failure for patients with diabetes and congestive heart failure. according to a joint statement by lead researchers, and blue cross blue shield of michigan executives, "our results are consistent with an emerging body of evidence that periodontal disease…it addresses quality of care and health care costs for all michigan residents." also, at the institute for oral health conference in november joseph errante, d.d.s., vice president, blue cross blue shield of ma reported that blue cross blue shield of massachusetts claims data showed medical costs for diabetics who accessed dental care for prevention and periodontal services averaged $ /month, while medical costs for diabetics who didn't get dental care were about $ /month (errante ) . similarly insured individuals with cardiovascular diseases who accessed dental care had lower medical costs, $ /month lower than people who did not seek dental treatment (errante ) . the cost is $ less per visit for those diabetics who accessed prevention and periodontal services. those savings could be translated into access to care or additional benefi ts for more individuals. in the case of neonatal health there is similar research. over % of all births in the u.s. are delivered preterm, with many infants at risk of birth defects ( martin et al. ) . according to a january statement issued by cigna, announcing their cigna oral health maternity program, "the program was launched in response to mounting research indicating an increased probability of preterm birth for those with gum disease. these research-based, value-added programs are designed to help improve outcomes and reduce expense" (cigna ) . the program was initially designed to offer extended dental benefi ts free of charge to members who were expecting mothers, citing "research supporting the negative and costly impact periodontal disease has on both mother and baby." according to research cited by cigna, expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before th week), and the costs associated with treating premature newborns is an average of times more during their fi rst year, and premature newborns have dramatically more healthcare challenges throughout their life. cigna also cited the correlation between periodontal disease and low birth weight, pre-eclampsia, gestational diabetes as additional rationale to support extended dental benefi ts to expecting mothers. six months later cigna initiated well aware for better health, an extended benefi ts free of charge program for diabetic and cardiovascular disease patients aimed at "turning evidence into action by enhancing dental benefi ts for participants in disease management" programs. it is interesting to note, not only does cigna offer extended dental benefi t to targeted groups, they also reimburse members for any out-of-pocket expenses associated to their dental care (co-pays, etc.) in , columbia university researchers conducted a -year retrospective study of , aetna ppo members with continuous medical and dental insurance, exhibiting one of three chronic conditions (diabetes mellitus, coronary artery disease, and cerebrovascular disease) (aetna ) . researchers found members who received periodontal treatments incurred higher initial per member per month medical costs, but ultimately achieved signifi cantly lower health screening (episode risk group/erg) risk scores than peers receiving little or no dental care. convinced by the data and understanding lower risk scores ultimately leads to healthier people and cost savings, aetna initiated the dental/medical integration (dmi) program in . aetna's dmi program offers enhanced benefi ts in the form of free-of-charge extended benefi t dental care to aetna's . million indemnity, ppo and managed choice medical plan members, specifi cally targeting members deemed at-risk, including those who are pregnant, diabetic, and/or have cardiovascular disease and have not been to a dentist in year as a result of various outreach methods during the pilot, % of at-risk members who had not been to a dentist in the previous months, sought dental care (aetna ) . "the fi ndings from this latest study we conducted continue to show that members with certain conditions who are engaged in seeking preventive care, such as regular dental visits, can improve their overall health and quality of life," said alan hirschberg, head of aetna dental (aetna ) . delta dental of wisconsin understands the connection between oral and systemic health and has created a program that is designed to offer members with certain chronic health conditions the opportunity to gain additional benefi ts. more than , groups now offer delta dental of wisconsin's evidence-based integrated care plan (ebicp) option (delta dental of wisconsin ) . ebicp provides expanded benefi ts for persons with diseases and medical conditions that have oral health implications. these benefi ts include increased frequency of cleanings and/or applications of topical fl uoride. they address the unique oral health challenges faced by persons with these conditions, and can also play an important role in the management of an individual's medical condition. ebicp offers additional cleanings and topical fl uoride application for persons who are undergoing cancer treatment involving radiation and/or chemotherapy, persons with prior surgical or nonsurgical treatment of periodontal disease and persons with suppressed immune systems. the ebic offers additional cleanings for persons with diabetes and those with risk factors for ie, persons with kidney failure or who are on dialysis and for women who are pregnant. the iehr provides the insurance industry in partnership with the healthcare industry an integrated tool to facilitate these health and subsequently economic outcomes across medicine and dentistry. in addition to the anticipated savings through better outcomes using integrated clinical data, an example of a positive economic outcome associated with an integrated record as related to increased effi ciency and patient safety is found in the united states veterans administration (va) hospitals and clinics. the va is one of the few institutions that have implemented the shared electronic medical-dental record successfully. the va has the ability to be the "one stop shop" for their patients. an april press release published on the department of veterans affairs website highlighted the success of va's health information technology in terms of cost reductions and "improvements in quality, safety, and patient satisfaction" (department of veterans affairs ) . the press release spotlighted a recent study conducted by the public health journal, health affairs, which focused on va's health it investment from to . the study confi rmed that while va has spent $ billion on their technology initiative, a conservative estimate of cost savings was more than $ billion. after subtracting the expense of the it investment, there was a net savings of $ billion for the va during the years covered by the study (mcbride ) . furthermore, the study estimated that "more than percent of the savings were due to eliminating duplicated tests and reducing medical errors. the rest of the savings came from lower operating expenses and reduced workload." independent studies show that the va system does better on many measures, especially preventive services and chronic care, than the private sector and medicare. va offi cials say "its [integrated] technology has helped cut down hospitalizations and helped patients live longer" (zhang ) . recently, the journal of obstetrics and gynecology reported on a tragic loss of life due to the systemic nature of oral health. a study found oral bacteria called fusobacterium nucleatum was the likely culprit in infecting a -year-old woman's fetus through her bloodstream (carroll ) . the doctors determined that the same strain of oral bacteria found in the woman's mouth was in the deceased baby's stomach and lungs. integrated records would provide critical data to the obstetrician including oral health issues and when the patient had her last dental exam. how does one measure the economic impact of a life not lived and another derailed by such tragedy? in a randomized controlled study, lopez et al. ( ) determined that periodontal therapy provided during pregnancy to women with periodontitis or gingivitis reduced the incidence of preterm and of low birth weight. the institute of medicine and national academies estimate that preterm births cost society at least $ billion annually . data integration of the iehr enables the effective management between the dentist and obstetrician to ensure proper periodontal therapy has been provided during pregnancy. such management based on the lopez et al. study, will have direct impact in reducing the prevalence per preterm births leading to reduced health care costs. there have also been studies indicating a correlation between poorer oral hygiene or defi cient denture hygiene and pneumonia or respiratory tract infection among elderly people in nursing homes or hospitals (rosenblum ; ghezzi and ship ; scannapieco ) . one such study of elderly persons in two nursing homes in japan (adachi et al. ) concluded that "the number of bacteria silently aspirating into the lower respiratory tract was lower in the group who received professional oral care, which resulted in less fatal aspiration pneumonia in that group." over the month period of the study, of the patients receiving professional oral care, % died of pneumonia versus . % of the patients that died of the same cause who maintained their own oral hygiene. lack of access is certainly a key factor to consider. however, lack of available data respective to the interrelationship between oral health and systemic health also contributed to the apathy in these cases. as identifi ed above, complications are correlated to cost. as conditions compound, costs go up. marshfi eld clinic, as part of their iehr is creating a shared problem list that identifi es both oral and medical conditions and history to recent visits and medication lists for monitoring at point of care [be it a medical or dental visit], such cross access to clinical data and care management milestones serves as a tool to prevent conditions from compounding and escalating costs such as those described above. several other areas of economic impact will be seen as iehr's become broadly deployed. some of these are listed as follows: medication management. a great deal of provider and allied support time is • spent obtaining medication information between dentistry and medicine [and vice versa] including current medications, contraindications, tolerances, etc. marshfi eld clinic cattails software has created a dashboard that readily identifi es this for both the medical and dental providers. not is time saved but chances for complications or escalation of conditions is reduced [both of which impact cost]. for example an integrated record allows medical providers treating respiratory infections to include or exclude oral fl ora as the possible source of the infection which would lead to more knowledgeable prescribing decision on the antibiotic used. coordination of care has a direct impact on cost for the system and the patient. • for example, in . % of the us population aged years and older that was diagnosed diabetes had been to the dentist in the past year (healthy people ( )). the us government's program healthy people includes an initiative to increase the proportion of people with diagnosed diabetes who have at least an annual dental examination. the american diabetes association recommends that diabetic patients be seen semi-annually and more if bleeding gums or other oral issues are present. the american diabetes association also recommends the consultation between the dentist and doctor to decide about possible adjustments to diabetes medicines, or to decide if an antibiotic is needed before surgery to prevent infection. the target from the healthy people is a % improvement at . %. integrated medical/dental records could allow for the coordination of efforts between providers to include communication of treatment plan and services leading to quicker resolution, increased patient compliance, and less patient time away from work or home and potentially less travel. similarly, integrated records also create a platform to integrate clinical appointing • between medicine and dentistry. as such, combative patients or severely disabled patients needing anesthesia in order for care to be delivered can be treated with one hospital sedation vs. multiple sedations. family health center of marshfi eld, inc. (fhc) dental clinics shares an iehr with marshfi eld clinic and uses it integrated scheduling feature to complete dental care, lab work, ent care, woman's health, preventive studies, all in one visit. follow up care management can be more focused and coordinated. for example, • without the knowledge or dental conditions, medical providers could spend months attempting to control diabetes with periodontal disease. however, with access to an iehr, the practitioner or allied care manager can determine patient's oral health status immediately to determine possible infl uence of periodontal disease. similarly an iehr with a shared patient data dashboard brings to light history • and physical examination data without having to have patients be the historian to their physician on their last dental visit or for the dentist to have to rely on the patient's recall of medications or medical diagnosis. for example, if an integrated record saved providers min per hour of patient care, that would be min per day. imagine giving a physician or dentist min more a day. in a capitated system, this allows for more patients to be seen in a day for roughly the same amount of expenditure. in a production based clinic this allows more patients to be seen and more charges per day. in either case, the investment into informatics is covered. in an underserved area, more patients get care quicker, which creates the opportunity for quicker resolution, which can lead to a healthier society, which in turn may lead them back to a productive livelihood sooner. an iehr results in one system for acquisition, orientation, training and support. • pc based owners who also own a mac and mac owners who also have to operate a pc can relate. need we say more? imagine if your pc function just like a mac [or your mac function just a pc]. no cross learning of software quirks. not having to purchase two separate units to begin with. reduced costs, increased space. not having to jump from one computer to the other computer to get data from one data from another to create a report. not having to call two separate computer companies for service or updates. third party coordination. having an iehr creates a platform for interfacing • with third party payers. a common system and language for timely reimbursement. in part, the result of an iehr is driving the diagnostic coding for dentistry. such an integrated interface provides a tool to bridge with healthcare payors that historically kept payment as segregated as the oral and medical health professions. the iehr overcomes that limitation. timely payment, consolidation of payment, expansion of covered patient and provider benefi ts based on clinical integration, and a viable system for interfacing are all potential economic benefi ts of iehr clinical data. the iehr creates new horizons for research that will lead to cost saving discov-• eries. as example, knowing the benefi ts of research, marshfi eld clinic research foundation (mcrf) has created an oral and systemic health research project (oshrp). the creation of oshrp, led by dr. murray brilliant, will allow mcrf to capitalize on its existing and growing strengths in the areas of complex disease interactions and personalized health care (phc) to advance oral health and the health of the rest of the body. the oshrp has three specifi c goals: understand the connections between oral and systemic health (diabetes, heart disease, pre-term births) understand the causes of oral diseases and determine the effect of genetics, diet, water source (well/city + fl uoridation) and microbiome. understand how improving oral health aids systemic health (comparative effectiveness) and bring personalized health care (phc) to the dental arena. the oshrp research resource will be unique in the nation. as mcrf has done • with other projects, it will share this resource with qualifi ed investigators at other academic institutions both within and outside of wisconsin. oshrp will advance scientifi c knowledge, improve healthcare and prevention, reduce the cost of oral healthcare, and create new economic opportunities. such knowledge will have a direct economic impact on the cost of care and care management. the iehr creates an ability to have an integrated patient portal to comprehen-• sively maintain their health. portals are becoming more and more popular in the healthcare industry as a means to helping maintain compliance with care management recommendations and preventative procedures. portals provide patents a tool to stay up to date on their care and recommendations. portals can take iehr clinical data, adapt it through programming, and provide creative visual reinforcement for patients as they monitor their health status. the more patients engage in owning their health status, the more preventative services are followed through with. the more medicine and dentistry can leverage the prevention potential [which insurance companies have come to realize] the more likely costly conditions can be avoided. the link between oral health and systemic health is well documented. the separation of dental and medical is not a sustainable model in modern healthcare delivery. a new model of integrated care is necessary. aristotle said, "the whole is greater than the sum of its parts." increased access to combined medical and dental histories and diagnosis at the providers' fi ngertips makes vital information available. shared diagnosis between physicians and dentists could aid in formulating interventions and to accelerate decision making abilities by allowing for prioritizing of medical/ dental procedures. clinical management and treatment of the patient would be expedited with immediate access to both records. quality could be improved through a complete picture of the patient through the dashboard. all of which have a direct or indirect economic benefi t. the iehr will be the tool that facilitates such delivery and the studies and scenarios described in these pages point to signifi cant economic benefi ts to patients, payors, and providers. if increased access, multi-provider monitoring, shared problems lists with enhanced decision making abilities from iehr could reduce healthcare costs. the greatest cost reduction will be with using the iehr to manage chronic disease. a combined dental-medical electronic record with a shared data informatics platform is most likely to yield the best long-term economic solution while maintaining or enhancing positive patient outcomes. this section reveals viewpoints from a variety of medical and dental providers. one section focuses on optimal use of ophthalmic imaging, which should show how that the challenges of clinical data integration go beyond those encountered in the effort to bring oral health and systemic health together. wendy e. mouradian , suzanne boulter , paul casamassimo , and valerie j. harvey powell oral health is an important but often neglected part of overall health. historically separate systems of education, financing and practice in medicine and dentistry fuel this neglect, contributing to poorer health outcomes for vulnerable populations such as children, while increasing costs and chances for medical error for all patients. advances in understanding the impact of oral health on children's overall health, changing disease patterns and demographic trends strengthen the mandate for greater integration of oral and overall healthcare, as reviewed in two recent institute of medicine reports (iom a, b ) . the pediatric population could realize substantial benefit from oral disease prevention strategies under a coordinated system of care enhanced by integrated electronic health records (ehr). this approach would benefit all children but especially young children and those from low socioeconomic, minority and other disadvantaged groups who are at higher risk for oral disease and difficulties accessing dental care. this section focuses on the pediatric population and the need for close collaboration of pediatric medical and dental providers. first we consider how a child's developmental position and their parents' level of understanding might affect oral health outcomes. next we address the importance of children's oral health and the urgency of seizing missed opportunities to prevent disease. we then briefl y outlines some steps to preventing early childhood oral disease utilizing some of the many health providers that interact with families. finally we examine one pediatric hospital's approach to choosing an integrated ehr technology. children have unique characteristics which distinguish their needs from those of adults. children's developmental immaturities may increase their risks for poor oral health outcomes ( fig. all children, but especially young children, are limited in their ability to care for their own health and must depend upon adults. a child's parent/caregiver may also lack basic oral health knowledge and an awareness of their child's oral health needs, and/or suffer from poor oral health themselves. low oral health literacy is prevalent among patients and health professionals alike in america; individuals of low socioeconomic status or from ethnically diverse backgrounds may be at particular risk for low oral health literacy (iom a ) . without appropriate education, a parent…. may not correctly interpret a child's symptoms or signs of oral disease • may not know that caries is an infectious disease that can be spread to a child by • sharing spoons, for example, may not know the potential value of chewing gum with xylitol, • may not fully grasp the importance of good oral health hygiene habits, • may not grasp the consequences of a child consuming quantities of sugared • foods or beverages, may have diffi culty controlling the child's consumption of sugared foods or bev-• erages in or out of the home, may not realize the consequences of chronic use of sugared medications, • may not know the potential for systemic spread of disease from a toothache, or • for liver damage due to overuse of acetaminophen or other analgesics, may not grasp the long-term consequences of early childhood caries, • may live in a community without fl uoride in the tap water and not know about • alternative sources of fl uoride, may overlook oral health due to the stress of living in poverty, • may be fearful of dentists or oral health care due to their own experiences, • may have diffi culty locating a dental provider accepting public insurance, or • have other problems navigating the health care system. parents in turn depend on access to medical and dental providers with current understanding of the most effective ways to prevent caries and promote the child's oral and overall health. an important element in helping families is the provision of culturally-sensitive care to a diverse population. children are the most diverse segment of the population with % from minority backgrounds compared with % of the overall population (us census bureau ) . the separation of medical and dental systems and the lack of shared information can create additional barriers for families, especially for those with low health literacy or facing linguistic or cultural barriers. all pediatric health professionals have increased ethical and legal responsibilities to promote children's health, including advocacy for them at the system level (mouradian ) . although many factors can infl uence children's oral health outcomes, caries is largely a preventable disease. despite this, national trends and other data on broader considerations of medical and dental data integration children's oral health attest to this persistent national problem ) . some important facts include the following…. caries is the most prevalent chronic disease of childhood, • caries is a preventable disease unlike many chronic diseases of childhood, • yet according to (nicdr ) % of children - have had dental caries in • their primary teeth; % of children - have untreated dental caries. further, " % of children - have had dental caries in their permanent teeth; % of children - have untreated decay." overall "[c]hildren - have an average of . decayed primary teeth and . decayed primary surfaces," the latest epidemiologic evidence shows increasing rates of caries for young-• est children, reverse from the healthy people goal of decreasing caries. according to (nicdr ), overall "dental caries in the baby teeth of children - declined from the early s until the mid s. from the mid s until the most recent ( ) ( ) ( ) ( ) ( ) ( ) ) national health and nutrition examination survey, this trend has reversed: a small but signifi cant increase in primary decay was found. this trend reversal was more severe in younger children." disparities in children's oral health and access to care persist by age, income • level, race and ethnicity, and parental education level (edelstein and chinn ) . of concern, the latest increase was actually in a traditionally low-risk group of young children (dye and thornton-evans ) . the human and economic costs of early childhood caries are substantial • (casamassimo et al. ) . according to catalanotto ( ) , health consequences include… extreme pain, spread of infection/facial cellulitis, even death (otto - ) diffi culty chewing, poor weight gain falling off the growth curve (acs et al. - ) risk of dental decay in adult teeth (broadbent et al. - ; li and wang ) crooked bite (malocclusion) -children with special health care needs (cshcn) may be at higher risk for oral • disease and diffi culties accessing care. analyzing data from the national survey of children with special health care needs, (lewis ) found that "cshcn are more likely to be insured and to receive preventive dental care at equal or higher rates than children without special health care needs. nevertheless, cshcn, particularly lower income and severely affected, are more likely to report unmet dental care need compared with unaffected children." children who were both low-income and severely affected had . times the likelihood of unmet dental care needs, dental care is the highest unmet health care need of children; . million children • had unmet dental care needs because families could not afford care compared with . million with unmet medical needs for the same reasons (cdc ) , according to the national survey of children's health, children are . times as • likely to lack dental as medical insurance (lewis et al. ) , there is evidence that children who get referred to a dentist early may have lower • costs of care and disease. savage et al. ( ) reported that children "who had their fi rst preventive visit by age were more likely to have subsequent preventive visits but were not more likely to have subsequent restorative or emergency visits" and concluded that preschool "children who used early preventive dental care incurred fewer dentally related costs," ramos- gomez and shepherd ( • ) , in their "cost-effectiveness model for prevention of early childhood caries," conclude that preventive ecc interventions could reduce ecc by - % for a particularly vulnerable population of children, and that part of the costs of interventions will be offset by savings in treatment costs. as these facts convey, and the deaths of more than one child from consequences of untreated caries make painfully clear, there is an urgent need for more attention to the oral health needs of children. a more coordinated system for oral health care including integrated ehr would be an important advance. a glance at table . , an ideal model, reveals that intervention should begin before birth and that a range of medical and oral health professionals can contribute to the child's oral health. early intervention is necessary because of the transmissibility of cariogenic bacteria from mother/caregiver to infant, and importance of oral health practice in preventing disease. the following professionals may be involved: • pediatric medical provider family physician pediatrician pediatric nurse nurse practitioner in pediatric/family practice physician assistant in pediatric /family practice - other appropriate allied health professionals • the availability of some of these professionals can be affected socioeconomic status, health insurance, place of residence, or by a child's special health care need. one obvious limitation on developing a "relay" as in table . , with a "hand-off" from family care to obstetric care to pediatric care is the education of the medical providers. as part of pre-conception and perinatal healthcare, providers should address oral health, but may lack the knowledge to do so. additionally, as noted by ressler-maerlaender et al. ( ) , "some women may believe that they or their table . timeline of some oral health interventions to prevent early childhood caries (ecc) -birth to years age (marrs et al. ; lannon et al. ; han et al. ; ezer et al. ; aap ; mouradian et al. ) child's age intervening professional(s) planning conception, prenatal and perinatal family physician the physician and/or obstetric provider educates mother-to-be about good maternal oral hygiene and infant oral health issues, including transmissibility of caries. mother's dentist assesses and treats caries, gingivitis or other oral health problems and educates the mother-to-be obstetrician/nurse midwife obstetric nurse general dentist obstetric nurse obstetric nurse advises new mother to chew xylitol gum, limit salivary contact between mother and infant, and help child avoid sugar intake (exposure) while asleep and from common sugar sources (medicines, sugared water, bottle feeding on demand at night with fl uid other than water -following tooth eruption, certain foods), and to schedule dental exam at year age months pediatric medical provider first dental examination recommended by aapd when the fi rst tooth comes in, usually between to months pediatric/general dentist educate mother about optimal fl uoride levels . assess the woman's oral health status, oral health practices, and access to a dental home; . discuss with the woman how oral health affects general health; . offer referrals to oral health professionals for treatment; . educate the woman about oral health during pregnancy, including expected physiological changes in the mouth and interventions to prevent and relieve discomfort; and . educate the woman about diet and oral hygiene for infants and children and encourage breastfeeding a combination of anticipatory guidance, with continuity from prenatal and perinatal care to pediatric care, can help move infant oral health from "missed opportunities" to "seized opportunities." others who may be of assistance to families in closing these gaps are professionals at the women, infants and children's (wic) supplemental nutrition program, early head start/head start and neurodevelopmental/birth to three programs. together medical, dental and community professionals can help create a system of care to improve maternal and child oral health. for the envisioned model in table . to be realized, the mother requires access to a general dentist with accurate information on her oral health during pregnancy and on her infant's oral health, including the need for an early dental visit. the mother and child then need access to a pediatric medical provider who will provide oral health screening/counseling, and who will guide the family to establishing the child's dental home by age . success in dental referral requires access to a pediatric or general dentist willing and able to provide infant oral health. (dela cruz et al. ) , in a discussion of the referral process mentioned that among the factors in assessing the likelihood of a dental referral were the medical providers' "level of oral health knowledge, and their opinions about the importance of oral health and preventive dental care." since young children are much more likely to access medical than dental care, the medical provider plays an important role in promoting children's oral health. (catalanotto ) recommends, as part of a pediatric well child checkup: an oral screening examination, • a risk assessment, including assessment of the mother's/caregiver's oral health, • application of fl uoride varnish • anticipatory guidance (parental education) including dietary and oral hygiene • information, attempted referral to a dental home. • the aap recommends that child healthcare providers be trained to perform an oral health risk assessment and triage all infants and children beginning by months of age to identify known risk factors for early childhood caries (ecc). the oral health component of pediatric care is integrated into the aap's "recommendations for preventive pediatric health care (periodicity schedule)" (aap ) . to what extent are medical and dental and providers aware of recommendations for a fi rst dental visit for a child by age one, as recommended by the aap, the american academy of pediatric dentistry (aapd), and the american dental association? (wolfe et al. ) reported that % of licensed general dentists in iowa were familiar with the aapd age dental visit recommendation and that most obtained the information through continuing education; % believed that the fi rst dental visit should occur between and months of age. however, according to (caspary et al. ) , when pediatric medical residents were asked the age for the fi rst dental visit, the average response was . years, while % reported received no oral health training during residency. in a national survey of pediatricians ) reported that less than % of had received oral health education in medical school, residency, or continuing education. finally (ferullo et al. ) surveyed allopathic and osteopathic schools of medicine and found that . % reported offering less than h of oral health curriculum, while . % offered no curriculum at all. other workforce considerations relevant to preventing early childhood caries include the training of dentists in pediatric oral health (seale et al. ) , the number and diversity of the dental workforce, the number of pediatric dentists, and the use of alternative providers such as dental therapists, expanded function dental assistants and dental hygienists (mertz and mouradian ; nash ) . examples of integrated care models do exist, such as that presented by (heuer ) involving school-linked and school-based clinics with an "innovative health infrastructure." according to heuer, "neighborhood outreach action for health (noah)" is staffed by two nurse practitioners and a part-time physician to provide "primary medical services to more than , uninsured patients each year" in scottsdale, arizona. heuer counts caries among the "top ten" diagnoses every year. mabry and mosca ( ) described community public health training of dental hygiene students for children with neurodevelopmental/intellectual disabilities. they mentioned that the dental hygiene students had worked together with school nurses and "felt they had impacted the school nurses' knowledge of oral disease and care." the decision to acquire an integrated ehr as pediatric clinicians (both medical and dental) work more closely together, they require appropriate ehr systems that integrate a patient's medical and dental records. following is a set of local "best practices" from nationwide children's hospital in columbus, ohio, which may help other children's hospitals in planning acquisition of an integrated pediatric ehr system. integrated (medical-dental) ehr technologies are becoming more widely available outside the federal government sector (see integrated models e and e in fig. . ). nationwide children's 'drivers' for the acquisition process were, in : . minimize registration and dual databases . patient registration takes time and requiring both a stand-alone dental and a medical patient registration inhibits cost-effective fl ow of services. integration allows for the use of single demographics information for all clinics in the comprehensive care system serving the patient. clinicians always have an updated health history on patients, if they have been a patient of record. if not, and for a dental clinic that sees walk-ins, a brief "critical" dental health history can be completed on paper by a parent and scanned into the emr. in designing an integrated medical-dental record for patients of record, the system can sort essential health history elements into a brief focused dental history without the detail needed by other medical specialty clinics. kioskdriven electronic health histories for those children who are new to clinic similar to those used in airline travel could be considered if feasible in busy clinics. . for charting, no more key/mouse strokes than with paper . some commercial dental record products try to accomplish too much. moving from paper to electronics should be driven in part by effi ciencies. the tooth chart, which is an essential part of any dental record, must be such that examination fi ndings can be transferred quickly and accurately to either paper or electronic capture. a helpful exercise is visualization of the functionality of the charting process, including both the different types of entries (caries, existing restorations and pathology) and how these are entered in the paper world. if charting will be able to be used for research the system should be able to translate pictures to numerical values, often a complex programming function. dental practitioners and faculty may want to use drawings of teeth or graphics of surfaces because that is their current comfort level. a true digital charting is possible with no images of teeth, but some habits are hard to change. . maximizing drop downs with drop down building possible . duplication of paper chart entries using drop downs which can be upgraded as more clinical entities are found is a staple of an emr. the paper process usually relies on a clinician's wealth of medical-dental terms since inclusion of every possible, or even the most common fi ndings, is prohibitive on a paper chart. the emr drop down requires front-end loading of the most common clinical fi ndings with opportunity for free-hand additions. being able to add terms to any drop down is a needed capability. . don't design a system for uncommon contingencies, but for your bulk of work . a pediatric dental record should be primarily designed around dental caries, with secondary emphases on oral-facial development (orthodontics) and a lesser capability to record traumatic injuries and periodontal fi ndings. these second and third level characteristics can be hot-buttoned and should not drive the design of the basic system which is caries charting for % of our patients. sadly in most dental schools, the chart is slave to every teaching form, few of which ever exit with the dds into practice! these forms may have little relationship to patient care and only create "signature black holes" that need to be addressed, usually after treatment is completed. . progress notes should be designed for the routine entries with free-hand modification possible . student learners tend to write too much and a carefully crafted progress note format with standard entries in required fi elds helps patient fl ow and record completion. in federally funded clinics and residencies, attending reconciliation of student/resident service delivery is a compliance requirement. a well-designed emr system can "stack" required co-signing tasks on a computer screen, offer standard entries as well as free-hand options, and create a process far faster than paper records for an attending's validation (same as reconciliation?). . tie examination results to treatment planning and treatment planning into billing . a good system allows easy transfer of clinical fi ndings needing treatment into some problem "basket" and ideally in a tabulated format. an alternative is a split screen that allows a clinician to visualize clinical fi ndings, radiographic fi ndings while compiling a treatment plan. again, in clinical settings where compliance to medicaid/medicare regulations is required, the design of the record should give attention to auditing principles and security. a good emr system allows portals of entry for billing and compliance personnel. . plan for users of different skill levels and different periods of exposure . the teaching hospital or dental school environment often involves learners and attendings with varying skill levels and computer experience who may be there for brief periods of time. this reality adds signifi cant security and userfriendliness issues. some medical record systems are far too complex for shortterm or casual users. a well-integrated medical-dental emr allows navigation of the depths of the medical side should a user want to explore, but should focus on the dental portion. some suggestions in design: initial opening or logging into the dental portion for dental users, rather than • opening into the medical portion, clearly indicated options for exploration of medical portions, • orientation of major dental component (examination, radiographs, treatment • plan) in a logical dental treatment fl ow to replicate the way dentistry works rather than trying to reshape dentistry's normal fl ow to the record, minimization of seldom-used functions on the main dental screen, such as • specialty medical clinics, old laboratory tests and hyperfunctionalities like letter writing, clear identifi cation of existing non-caries dental portions like orthodontics or • trauma, so a novice user need not randomly search to see if a patient has any of these records. unfortunately, many pediatric hospitals do not yet have an ehr system that supports convenient communication among a pediatric patient's medical and dental providers. evidence of this state of affairs was provided unintentionally by (fiks et al. ) . some pediatric hospitals may have an awkward mix of systems serving physicians, dentists, and orthodontists and their shared patients. this section demonstrates how closely medical and dental professionals must collaborate to deliver appropriate oral health care for infants and children. such collaboration is especially important given the developmental vulnerabilities of children and the urgency of the oral health needs of many children, especially those from underserved populations. collaboration is made more diffi cult by the long-standing separation of medical and dental systems and poor oral health literacy of parents and medical professionals alike. teamwork in the delivery of pediatric care requires appropriate electronic patient record technology to facilitate sharing of patient information, to avoid patient record discrepancies between systems, and to create effi ciencies by maintaining only a single repository for patient demographics. only comparatively recently have appropriate integrated systems become available to support a range of clinical sites from pediatric special needs clinics to the largest children's hospitals. nationwide children's has given practical examples of effi cient decision-making in identifying an integrated system to acquire. much more work will be needed to develop the means to move towards integrating offi ce and community-based care for children through the sharing of electronic health records. oral health is an oft neglected area in the care of patients who have chronic kidney disease. furthermore, the provision of care by dentists and physicians to the same patient is fragmented as communication between the two health care providers is scant. emerging data suggesting the periodontal disease is closely linked to chronic kidney disease highlights the importance of proper oral health and the importance of communication between dentists and physicians in the care of the patient. investigators used data from nhanes iii, including information on , adults who had an oral examination by a dentist who categorized each patient as having no periodontal disease, periodontal disease or edentulous to examine the relationship between numerous risk factors for moderate to severe chronic kidney disease, as determined by calculation of estimated gfr through use of the mdrd formula (fisher et al. ) . no chronic kidney disease was defi ned as an estimated gfr of ml per min per . m . three percent of the patients had ckd, . % were hypertension and . % had diabetes ( . % with glycated hemoglobin of % or higher). four models were constructed to examine the potential relationship between periodontal disease and ckd. in model one adults with either periodontal disease or edentulous had an adjusted odds ratio of . (with % confi dence intervals of . - . ) of having ckd, independent of the other risk factors for ckd including of age above years, ethnicity, hypertension, smoking status, female gender and c-reactive protein elevation. the fourth model contained potential risk factors including the periodontal disease score and for every -unit increase in the score, the risk of having ckd increased by % controlling for the other risk factors. the authors hypothesized from their results that the relationship between periodontal disease and ckd was bidirectional in that ckd may increase the risk of periodontal disease which in turn increases the risk of ckd. grubbs et al. ( ) also used nhanes data to look more closely at the relationship between periodontal disease and ckd, using dental examinations obtained from to (n = , adults, - years) (grubbs v, et al. ) . in this analysis edentulous subjects were excluded and those with albuminuria were included in the defi nition of ckd. in the entire population ckd was present in . %, but in those with moderate to severe periodontal disease this increased to . %. other associations with moderate to severe periodontal disease were being older, male, nonwhite, less educated and poor. there was a strong relationship between periodontal disease and ckd ( . unadjusted odds ratio). when adjusted for age, gender, tobacco use, hypertension, diabetes, ethnicity, poverty and educational attainment, the odds ratio for the association of periodontal disease and ckd was still signifi cant ( . ). in some groups (mexican american, poor, and poorly educated) dental care was not received on an annual basis in the majority of this segment of the population. periodontal disease has been associated with an increased risk of death in hemodialysis patients (kshirsagar et al. ). this relationship has been poorly studied in peritoneal dialysis patients. this requires further study but it appears possible that periodontal disease might hasten loss of residual kidney function and perhaps contribute to atherosclerosis in dialysis patients and therefore, contribute to the high mortality in this population. patients who desire a kidney transplant are required to undergo a thorough evaluation beforehand including an oral examination by a dentist. some patients on dialysis have inadequate insurance which does not cover dental care, leading to a situation in which a kidney transplant is denied because the patient cannot afford the dental examination. communications between dentists and physicians in the care of the patient is scant. if oral surgery is required in a dialysis patient, the surgeon generally requires a brief summary from the nephrologist with recommendations. these might include suggestions for prophylactic antibiotics, avoidance of vasoconstrictor agents to an excess locally (which can elevate blood pressure) and the increased risk of bleeding of a dialysis patient. for more routine dental examinations no information is requested which could potentially lead to drug interactions or a dangerous situation. most nephrologists and health care providers in the dialysis unit do not inquire of the patient concerning dental health and examination of the mouth is quite uncommon. although the dialysis patient is seen monthly at a minimum, there is little conversation or documentation of oral health. connecting the electronic health records of in-patient care, the out-patient dialysis unit and the dentists' offi ce could potentially have a large impact in improving the care of those with end stage kidney disease. integrating medical and dental records in ehr's may or may not be the "golden ring." first, we need to integrate the clinical thinking…something we both realize is important, but not likely to be solved by an inert computer. i also think that integrated records will be very cumbersome, given the fact that the language used by the separate disciplines is so different, and the kind of detail required to support good decisions and good work is so different. it could be done…but for many professionals on either "side," they would never open the other module. to me, a more sensible solution may be to have a condensed "nugget" of information that could cross populate. "moderate periodontal disease" may be what the medical doctor needs to know, plus know what a treatment plan may include. she won't need to know the number of the teeth with the deepest pockets and erosions but will need to support the patient's determination to follow through. on the other hand, if the patient has shown remarkable initiative in gum care and has successfully migrated to a lower severity index, that would be important for congratulation and reinforcement…and also to encourage similar diligence in managing, let's say, the hypertension that is not optimally controlled. in the other direction, the dentist should know that a patient has been erratic in clinical follow up, does not self-test blood glucose, uses hypoglycemic drugs only intermittently, and has failed several appointments for eye exams. this would lead to a rather different set of approaches from a highly motivated grandmother who is enrolled in a community cultural center's senior exercise club, and is learning to become a lay community teacher for diabetes. right now, i don't think even this superfi cial degree of information is exchanged. we need to support each other's efforts, but we probably do not need to share minute details. the benefi ts of an electronic health record are well described. ehrs allow for legible standardized documentation and easier sharing of patient data between providers at multiple locations. they are less prone to loss and require much less space to store. they have the potential to result in a reduction in the cost of health care. a distinct disadvantage of the ehr, in its current confi guration, is the problem of information overload. simply put, there is often too much information presented in a way that is diffi cult to review and digest. the ehr equivalent of thumbing through a chart quickly is not yet available. as a result we frequently see practitioners look only at the last note or two as they review a patient's history. we require a way to communicate information directly relevant to patient diagnosis, treatment and prognosis among subspecialists and primary care providers. we require a way to identify subclinical cerebrovascular disease in a patient, independent of blood pressure and other traditional risk factors. we require a way to recognize which patients with cerebrovascular disease are two to four times more likely than average to develop a stroke in the next years. we have a way -retinal imaging. the eye is the one place in the body we can directly observe arteries, veins and a cranial nerve in a noninvasive manner. routine imaging of the retina and optic nerve could allow primary care providers to assess retinal, and by proxy systemic, end organ damage from atherosclerosis in an effi cient manner. the key to optimal use of the medical record and effi cient yet effective communication among providers may lie with the familiar adage; a picture is worth a , words. traditionally, when ophthalmologists communicate with primary care providers they send brief letters regarding the fi ndings seen during a yearly dilated examination and the presence, absence or progression of diabetic retinopathy. these letters end by exhorting the virtues of improved blood sugar, blood pressure and lipid control, a sentiment that the primary care provider likely shares. this system of communication does not provide particularly useful information for the primary care provider, except to serve as a notice that the standard of care screening guidelines have been met. the box has been checked. if primary care providers, cardiologists, nephrologists had access to routine ophthalmic imaging, they would be able to directly visualize the effect that suboptimal blood sugar control is having on their diabetic patients. as importantly, they would be equipped with information directly predictive of congestive heart failure, stroke, and cardiovascular mortality for their patient with hypertension, hyperlipidemia and for those who smoke. large clinical studies have shown that assessment of retinal vascular changes such as retinal hemorrhages, microaneurysms and cotton wool spots provides important information for vasculopathy risk stratifi cation. as an example, wong et al. showed that the presence of retinopathy indicates susceptibility to and onset of preclinical systemic vascular disease, independent of and qualitatively different from measuring blood pressure or lipids (wong and mcintosh ) . in the atherosclerosis risk in communities (aric) study, individuals with hypertensive retinopathy signs such as cotton wool spots, retinal hemorrhages and microaneurysms were two to four times more likely to develop a stroke within years, even when controlling for the effects of blood pressure, hyperlipidemia, cigarette smoking and other risk factors (wong et al. ) . in a recent study by werther et al., patients with retinal vein occlusions were found to have a two-fold increased risk of stroke compared to controls (werther et al. ) . in addition, the aric study group reported that individuals with retinopathy were twice as likely to develop congestive heart failure as individuals without retinopathy, even after controlling for pre-existing risk factors (wong et al. a ) . interestingly, even among individuals without pre-existing coronary artery disease, diabetes or hypertension, the presence of hypertensive retinopathy was associated with a three-fold increased risk of congestive heart failure events (wong et al. a ) . in the beaver dam eye study, cardiovascular mortality was almost twice as high among individuals with retinal microaneurysms and retinal hemorrhages as those without these signs ( wong et al. a, b ) . the aric and beaver dam eye studies have also shown that, independent of other risk factors, generalized retinal arteriolar narrowing predicts the incidence of type ii diabetes among individuals initially free of the disease (wong et al. a (wong et al. , b . a primary care provider with access to patients' retinal photographs may therefore have the evidence needed to suggest which patient with either established systemic vascular disease or preclinical systemic vascular disease requires a more aggressive treatment and risk factor modifi cation. they could do this without wading through the electronic equivalent of piles of records. one photograph could refl ect both acute changes in blood pressure (retinal hemorrhages, microaneurysms and cotton wool spots) and chronic changes resulting from cumulative damage from hypertension (av nicking and generalized arteriolar narrowing) (sharrett et al. ; wong et al. a ; leung et al. ) . in brown et al. out of patients, excluding those with known diabetes, that presented with a single cotton wool spot or a predominance of cotton wool spots on examination of the retina were found to have underlying systemic disease (brown et al. ) . systemic work-up revealed diagnoses including previously undiagnosed diabetes, hypertension, cardiac valvular disease, severe carotid artery obstruction, leukemia, metastatic carcinoma, systemic lupus erythematosus, aids and giant cell arteritis (brown et al. ) . these fi ndings illustrate the importance of retinal fi ndings on a systemic level. the utilization and integration of ophthalmic imaging may serve to achieve more effective communication among subspecialists and primary care providers and ultimately to provide improved diagnosis and treatment for delivery of optimal quality of patient care. moreover, the improved integration and maximal use of resources may serve to reduce overall health care cost and perhaps decrease provider frustration with the electronic health record (fig. . ). there are cotton wool spots, exudates, intraretinal dot-blot hemorrhages and microaneurysms. av nicking is also present especially along the superior arcade just as the vessel leaves the optic nerve ( fig. . ) . av nicking, tortuosity of vessels, intraretinal hemorrhages and dry exudates are seen ( fig. . ) . there is edema of the optic nerve head, with cotton wool spots and fl ame shaped hemorrhage along the disc margin. there are several cotton wool spots along the vascular arcades and scattered dot hemorrhages throughout the posterior pole and periphery ( fig. . ) . notice the cholesterol plaque in the vessel just as it exits the optic nerve head and the pallor in the superior macula corresponding to retinal ischemia and edema ( fig. . ). the cholesterol embolus has resulted in lack of blood fl ow to the superior arcade ( fig. . ) . there is pooling of subretinal blood just superior to the optic disc with a central fi brin clot and associated vitreous hemorrhage (fig. . ) . optic disc edema, fl ame hemorrhages and venous congestion are seen in a patient with severe hypertension. biju cheriyan in clinical practice, an otolaryngologist often needs a dental consult not only because of the topographically adjacent nature of the structures but also because most structures are supplied by the same neurovascular bundle and therefore there is overlapping of symptoms. the converse scenario can also apply. apart from this, there are many systemic medical conditions (for example: bleeding diatheses, diabetes) a hypertensive optic neuropathy dentist encounters throughout his or her practice which can determine the outcome of a successful treatment. sometimes, providers may observe a cluster of diagnostic criteria which may have to a single source. in the sections below, i will explore a few of these scenarios and conditions, and indicate where and how an integrated electronic health record (ehr) could optimize delivery of health care by dentists and otolaryngologists. cleft palate/cleft lip : cleft lip and cleft palate (cl/cp) are congenital conditions that require multidisciplinary management by dentists, oral and maxillofacial surgeons, orthodontists, otolaryngologists, speech pathologists and plastic surgeons a number of studies report that a multidisciplinary approach is essential for better treatment outcomes (wangsrimongkol and jansawang ) and for post operative rehabilitation (furr et al. ). these multidisciplinary approaches may lead to new ways to manage and treat cl/cp patients (salyer et al. ). hutchinson's teeth : notching of the upper two incisors is typically seen in individuals infl icted with congenital syphilis. macroglossia refers to enlarged tongue in relation to oral cavity. macroglossia is an important sign. it can indicate important systemic diseases like systemic amyloidosis, congenital hypothyroidism, acromegaly, or down syndrome. a common complaint that dentists and otolaryngologists encounter in their practice is the common headache. because of the special nature of the neurovascular bundle of the head and neck this symptom can be presented to both dentists and otolaryngologists (ram et al. ). any sinus pathology can present as a headache to an otolaryngology practice. since the maxillary sinus fl oor is in close proximity to the maxillary premolars and molars, it is imperative to obtain a dental evaluation in persistent cases of headache. there are a number of causes for headache from the dental and otolaryngology perspective. a mal-aligned denture patient with chronic headache, whom i saw in my practice was shuttled between departments and an array of investigations only to fi nd at the end that an ill-fi tting denture caused the intractable headache. in these cases, an integration of fi ndings is extremely important in providing quality treatment to the patient and also saves money and time for the whole health care system. hence it is important to have an integrated patient record for this particular symptom alone. trigeminal neuralgia is facial pain of neurogenic origin experienced along the distribution of the trigeminal nerve(fi fth cranial nerve). it can present as a dental pain and can also be triggered by brushing teeth among other trigger factors. as a result, patients with dental pain without obvious causes are required to have a physicians' consultation to rule out this obscure condition. sometimes it is diagnosed by omission (aggarwal et al. ; rodriguez-lozano et al. ; spencer et al. ). any tumor of the nasal sinuses (specifi cally maxillary and ethmoids) can erode the lower bony wall and present in the oral cavity (usually the maxillary arch) as dental pain, loose tooth, etc. therefore, these are areas of interest to both dentists and otolaryngologists. such tumors most commonly present fi rst to a dentist or could also be an accidental fi nding. cancers of the naso/oro/laryngo pharynx can also present as toothache to a dentist as these structures have a common nerve supply from cranial nerves , and . therefore, an integration of the patient record may even help in early diagnosis of the tumor. the same principle applies to all oral tumors, tumors of the nasopharynx, the oropharynx etc. this is especially true of malignant lesions of the oral cavity as these may help in early detection and treatment of cancer. in these cases, an early biopsy and histopathology can save the life of the patient. therefore, it is imperative to say that a collaborative patient record can save patients' lives. ulcers of the oral cavity from aphthous ulcers to carcinomas can present both to a dentist and an otolaryngologist. oral ulcers can be of dental origin. contact ulcers from sharp edges of a mal-aligned tooth can result in intractable ulcers, where a simple smoothing of sharp edges may eradicate the ulcer and terminate it as a chronic condition and can even prevent the ulcer turning into a malignancy. if you have an integrated electronic health record (ehr) these problems are immediately addressed and managed. otherwise, the condition will consume valuable time of both the patient and the physician concerned. in addition to this, there are a few conditions which require special attention: aphthous stomatitis (canker sore), which may indicate oral manifestation of defi ciencies of iron, vitamin b , folate deficiency and oral candidiasis, which can be a sign of diabetes mellitus or of an immunocompromised patient (e.g. aids). temperomandibular joint (tmj) disorders can present in a variety of symptoms to both dentists and otolaryngologists. they can present as a headache, earache, toothache, or as facial pain. there can be a number of causes for this including osteoarthritis of the tmj, recurrent dislocation, bruxism, or even an ill fi tting denture. there have been cases where patients have been subjected to removal of teeth for chronic toothache only to discover at the end that the symptom was a referred pain from tmj! therefore, an integrated ehr can prevent misdiagnoses and resulting impairment or disability to patients. trismus (lock jaw) can indicate important diagnoses such as tetanus and rabies.it is due to a spasm of muscles of mastication, which is an important oral manifestation of widespread muscle spasm. apart from these conditions, other causes of trismus are peritonsillar abscesses, and scleroderma. other problems dentists and otolaryngologists encounter in clinical practice are concurrent systemic diseases (patients with multiple problems): patients with bleeding diatheses, diabetes mellitus and a hidden primary malignancy. a non-healing ulcer in the oral cavity may hide a primary malignancy behind it. in these cases, you have to look for it specifi cally. similarly, one has to be aware of oral manifestations of internal pathology. some of them are crohn's disease, ulcerative colitis and gastro-intestinal tract malignancies. often dentists see patients after a tooth extraction with intractable bleeding to fi nd that they have a bleeding diathesis. so, this may be the fi rst presentation of these patients' bleeding disorder. when this patient undergoes any elective procedure in future, it will be a great help to surgeons to be aware of this information to prevent any inadvertent complications. therefore an integrated ehr can prevent unwanted complications where a patient's life may be in jeopardy. the source of otalgia or earache can be from a number of sites other than ear itself. technically ear lobe and ear canal are supplied by four different cranial nerve branches ( th, th, th, th). therefore, an area with a common nerve supply can present as earache. common dental problems which present as referred otalgia are ( ) dental caries ( ) oro-dental diseases or abscesses ( ) an impacted molar tooth (which is a common cause) ( ) malocclusion ( ) benign and malignant lesions of oral cavity and tongue (kim et al. ) . therefore, it is essential these two departments collaborate with each other in diagnosing and treating these diseases, and one way of facilitating it is through an integrated ehr system. there is a lot of overlap between dentists and otolaryngologists in the diagnosis and treatment of patients with halitosis (delanghe et al. ; bollen et al. ) . poor oral hygiene is the most common cause for this common complaint. oral causes include tooth caries, oral ulcers, periodontal diseases, unhealthy mucosa of the oral cavity. it is interesting to note that a simple oral ulcer can form an abcess eroding the fl oor of mouth and becoming a life-threatening oral cellulitis (ludwig angina). once the cellulitis has developed, it becomes a medical emergency. therefore, it is essential to prevent it before it can progress into a life-threatening condition, which of course is possible. causes pertaining to otolaryngologists include: chronic sinusitis or mucociliary disorder, chronic laryngitis or pharyngitis, pharyngeal pouches-related pathology, tumors or ulcers of naso/oro/laryngopharynx, diseases or conditions that impair normal fl ow of saliva such as salivary gland diseases or stones preventing fl ow of saliva, medications which cause dryness of mouth: antihistamines, antidepressants; local manifestation of systemic disorders: auto immune disorders, sjögren syndrome, dehydration from any cause, diabetes mellitus and gastro esophageal refl ux disorder (gerd). gerd is caused by improper neuro-autonomy of the lower esophageal sphincter (les). the les does not close tightly after food intake which causes gastric content to enter the esophagus. over time this can erode mucosa and cause various diseases even becoming cancerous (friedenberg et al. ). this disorder is attributed to life style. fast food consumption habits (oily fried foods) and eating habits (swallowing food without properly chewing) are partly responsible for this disorder (lukic et al. ; al-humayed et al. ) . here again an early diagnosis can manage the disease process before it is fully developed. at present there are no integrated ehr systems serving these specialties (dentistry and otolaryngology). an integrated ehr would facilitate effi cient communication between a dentist and an otolaryngologist who are providing care to the same patient and addressing a problem with a shared focus between the two disciplines. such integrated communication, may only require consulting the available medical or dental record of the patient, based on the particular circumstance. even enabling this simple communication would avoid duplication of effort, clarify the context of certain symptoms and reduce stress endured by the patient. it also has the potential to reduce healthcare delivery costs, and in some cases, even contribute to saving the patient's life. henry hood, allan g. farman, and matthew holder in this chapter, the authors attempt to put forth a justifi cation for precisely this kind of collaborative approach through a summary and discussion of a series of actual clinical cases. the protocols discussed in the management of each of these clinical cases illustrate the value in providing whole-person, interdisciplinary health care to this complex patient population. there is arguably no single patient population for whom the provision of collaborative, interdisciplinary health care is more challenging than for patients with neurodevelopmental disorders and intellectual disabilities (nd/id). in planning and delivering the generally-accepted standard of health care to this unique population, myriad biomedical, psychosocial and sociopolitical realities converge to create a landscape that is, at best, daunting for patients with these disorders, and for the clinicians who are charged with their care. anecdotal and scientifi c evidence suggest that this landscape has produced a paucity of physicians and dentists who are willing and able to provide care to patients with nd/id, and that american medical and dental schools are providing little training focused on their care (holder et al. ; wolff et al. ) . in february of , th surgeon general david satcher issued a report, which documented that americans with nd/id experience great diffi culty accessing quality health care (thompson ) . in that same report, former health and human services secretary tommy thompson said, "americans with mental retardation and their families face enormous obstacles in seeking the kind of basic health care that many of us take for granted." (thompson ) the disparities identifi ed by dr. satcher and secretary thompson require that physicians and dentists approach this population in a spirit of collaboration, compassion, and teamwork in order to produce positive health outcomes for them. perhaps, an even greater imperative driving the need for collaboration between medicine and dentistry in this arena is the fact that many patients with intellectual disabilities have developed this cognitive impairment as the result of an underlying neurodevelopmental disorder that is often undiagnosed. and it is this neurodevelopmental illness and the constellation of potentially devastating complications associated with that illness that create a biomedical fragility and a vulnerability that neither begins nor ends at the oral cavity, and that leaves these patients at risk in almost every aspect of their daily lives. when, for example, patients with nd/id are dependent upon publicly-funded programs for their health care, and when these systems fail to provide the health services that biomedically complex cases require because they fail to account for and accommodate the link between medical and dental pathologies, the risk of a negative outcome is greatly enhanced. such was the case for an intellectually disabled woman in michigan who, in october of , was unable to access dental services through the state's public medical assistance program, and who fatally succumbed to a systemic bacteremia resulting from an untreated periodontal disease (mich. dent. assoc. ). the american academy of developmental medicine and dentistry (aadmd) defi nes a neurodevelopmental disorder as a disorder involving injury to the brain that occurs at some point between the time of conception and neurological maturationapproximately age or (zelenski et al. ). examples of frequently-encountered neurodevelopmental disorders would include fragile x syndrome, a genetically acquired neurodevelopmental disorder caused by a mutation at the distal end of the long arm of the x chromosome (see fig. . ), trisomy , another genetic disorder, which features extra genetic material at the chromosome site (see fig. . ), and cerebral palsy, a prenatal or perinatal, acquired neurodevelopmental disorder (see fig. . ). patients with neurodevelopmental disorders tend to present clinically with one or more of fi ve frequently-encountered, objective symptom complexes or primary complications. these fi ve, classic primary complications include intellectual disability (aka: mental retardation), neuromotor impairment, seizure disorders, behavioral disturbances, and sensory impairment (aadmd). additionally, multiple secondary health consequences can derive from the fi ve primary complications; and any one of these secondary health consequences, or a combination of them, can produce profound morbidity. an example of a common secondary health consequence seen in patients with nd/id, which is derived from intellectual disability and / or neuromotor impairment, is the patient who is unable to care for his or her own mouth, and who develops ubiquitous caries and advanced periodontal disease as a result (see: fig. . ). another example would be the patient who suffers from the secondary health consequence of gastroesophageal refl ux disease (gerd) as a result of the neuromotor impairment associated with multiple neurodevelopmental disorders; and whose tooth enamel and dentinal tissues become chemically eroded as a result of the chronic intraoral acidity produced by gerd (see: fig. . ) . the diagnosis and management of these secondary health consequences provide dentists and physicians with a unique opportunity to work together to improve the quality of health and quality of life for their patients by implementing a team approach, which crosses the traditional interdisciplinary lines of communication, and which expands each clinician's ability to make meaningful treatment options available. indeed, it is often the case that quality primary care provided in one discipline will provide potentially valuable information to an attending clinician from another discipline. such is the case with the patients featured in figs. . and . . the patient whose intraoral photograph is featured in fig. . is a year-old male patient who presented to a special needs dental clinic accompanied by his mother. the mother indicated that her son was exhibiting hand-mouthing behaviors that she believed suggested he was experiencing mouth pain. a comprehensive radiographic and intraoral exam revealed, among other maladies, notched incisors, multiple diastemas, grossly decayed mulberry molars, and advanced periodontal disease. the patient also exhibited moderate to severe intellectual disability. these fi ndings were all consistent with a diagnosis of congenital syphilis. however, in developing the medical history with the mother, it was learned that no previous diagnosis of syphilis had been discussed with the mother, nor was it included in the health history. in cases like this, a comprehensive dental treatment plan should always include consultation with the primary care physician for purposes of moving forward with confi rmation of the clinical diagnosis by serologic testing, and consultation with a cardiologist to assist in the management of potential cardiovascular sequelae. as the dental treatment plan is being developed, consideration should also be given to human immunodefi ciency virus (hiv) testing for this patient, as coinfection is a common fi nding . this issue could easily be attended to by a primary care physician, an internist or an infectious disease specialist. in the absence of any of these team members, the dentist should feel entirely comfortable ordering hiv testing. the primary care physician and the developmental dentist should continue to advise each other and their respective consultant specialists of any signifi cant developments or new information, which could in any way impact either the medical or the dental treatment plan. as treatment progresses, both the physician and the dentist should expect improvement in the patient's periodontal status, which will likely be refl ected in a decrease in the frequency of immune-related illnesses, and in the maladaptive behaviors produced by chronic oral pain. it is quite often the case in this patient population that, with a reduction in maladaptive behaviors, comes a reduction of the use of psychotropic medications prescribed in a frequently futile attempt to manage behaviors that were born of an undiagnosed medical or dental illness. gerd is defi ned as the refl ux of gastric contents into the esophagus. gerd is primarily associated with incompetence of the lower esophageal sphincter; however there are numerous co-contributors, which may predispose a patient to gerd or exacerbate an existing refl ux problem. these co-contributors include a diet high in fat, neuromotor impairment associated with functional abnormalities such as dysphagia, neuromotor impairment associated with impaired ambulation and prolonged periods of recumbence, and the use of multiple medications including anxiolytics, calcium channel blockers, and anticholinergics. gerd is thought to affect approximately - % of the general us population. it has been established in the literature that the incidence of gerd in patients with intellectual disabilities is signifi cantly higher than in the neurotypical population, and that the relative number of unreported cases of gerd is much higher in patients with a neurodevelopmental diagnosis, as well. patients who have gastric refl ux as a function of a neurodevelopmentally-derived neuromotor impairment and a coexisting intellectual disability are impaired in their ability to voice the complaint that would, in the neurotypical patient, commonly lead to an encounter with either a family physician or a gastroenterologist and, ultimately, to a diagnosis. this inability to voice a complaint can be problematic in that, left untreated, gerd can produce maladaptive and sometimes aggressive behaviors in this population. and, of even greater concern, is the fact that undiagnosed esophageal refl ux can lead to more complex conditions that can produce signifi cant morbidity or even mortality -maladies such as barrett's esophagus or adenocarcinoma of the esophagus. chronic gerd can also produce an acidic intraoral environment, which can lead to the chemical erosion of the enamel and dentinal tissues of the teeth. ali et al. have established a link between erosion of the enamel and dentinal tissues of the teeth and gerd. there is additional anecdotal evidence suggesting a link between tooth enamel erosion and gerd, and related maladies. a special needs dental clinic in the eastern united states serving , patients with nd/id, has reported that, of nine patients referred to gastroenterology who presented for dental exam with a fi nding of either tooth enamel erosion or ubiquitous caries, two cases were diagnosed with gerd, two with barrett's esophagus, three with gastritis, and one with duodenitis. in all cases, medical treatment was required. in light of all that is known about the incidence of gerd and of the gerdrelated risks unique to this patient population; and in light of the link between tooth enamel erosion and gerd, it is incumbent upon any dentist encountering tooth enamel erosion in a patient with an intellectual disability to immediately refer that patient to gastroenterology for a work up, which should include esophagogastroduodenoscopy (egd) and ph monitoring. a dentist encountering gerd in a patient with an intellectual disability must be aware that he or she may be the fi rst and only link between that patient and the diagnosis of a potentially life-threatening illness. phenytoin-induced gingival enlargement can appear as either an infl ammatory lesion or a more dense, fi brotic hyperplastic lesion. the infl ammatory lesion is one in which the gingival tissues are swollen and bleeding, and in which pain is often a component. this type of gingival enlargement is the more acute lesion, frequently seen in patients who are currently taking phenytoin. in advanced cases of infl ammatory gingival enlargement, the tissues can appear botryoid, with a characteristic grape-cluster appearance. in advanced cases of phenytoin-induced gingival enlargement, the lesion can sometimes shroud entire sections of the dentition. phenytoin has long been a common medication used to treat seizure disorders in patients with neurodevelopmental disorders and intellectual disabilities. however, the gingival enlargement it produces, and the obstacle this lesion can pose to effective oral hygiene -especially in a population in which oral hygiene is typically compromised -can, over time, lead to periodontal disease, edentulism, and in advanced cases, systemic bacteremias. gingivectomy performed to reduce phenytoin-induced gingival enlargement will typically fail unless the patient is weaned off the offending medication, and another anti-seizure medication is titrated to effect. multiple alternative anti-seizure medications are currently available, which do not have the side effect profi le of phenytoin, and most patients who are weaned off phenytoin will demonstrate a virtual % resolution of the infl ammatory lesion within a matter of or months. the image in fig. . is of a year-old, microcephalic african-american male with intellectual disability, neuromotor impairment, and a seizure disorder. figure . illustrates the appearance of this patient's gingival tissues while he was currently on phenytoin. figure . features the same patient months after being weaned off phenytoin and placed on topiramate. these images illustrate the dramatic result that can be achieved when a dentist and a physician work in collaboration in the best interests of the patient. it is worth noting that this particular collaboration required only one intervention to achieve this result: the patient was weaned off phenytoin and was placed on a safer alternate anti-seizure medication. any dentist caring for a patient with an intellectual disability who presents with phenytoin induced gingival enlargement should immediately contact either the primary care physician or neurologist managing the patient's seizure disorder, and strongly urge that the patient be weaned off phenytoin and placed on a safer alternative anti-seizure medication. edentulism and bacteremia need not be a side-effect of a seizure management protocol. the patient seen in fig. . is a year old male patient with idiopathic intellectual disability who presented to an outpatient dental clinic for comprehensive dental evaluation and treatment. he was accompanied by his father. his father was referred to the clinic by the staff at his son's day program workshop. the day program staff had observed hand-mouthing behaviors, and they had voiced concern that the patient may be in pain. in the waiting room, the patient exhibited behaviors consistent with neurodevelopmental dysfunction. he was non-communicative, and his gaze aversion and tactile defensiveness were suggestive of autism. he was resistant and somewhat combative when directed to the dental chair, and effective behavior management in both the waiting room and operatory required the combined efforts of his father and two staff fig. . the adult patient suspected of having fragile x syndrome members. the patient's health history was positive for attention defi cit hyperactivity disorder (adhd), and there was no history of seizure or neuromotor impairment. the father indicated that, at age ten, the patient was admitted to an inpatient psychiatric unit for evaluation of his uncontrollable behavior. the following day, the parents were told that managing the patient's behavior was beyond the ability of the psychiatric unit staff, and the parents were asked to take the child home. the father also indicated that the psychiatric unit staff described the child's behavior as overwhelming. the patient was last seen by a dentist years prior to presentation; examination and treatment at that time were carried out in the operating room under general anesthesia. effective oral examination of this patient required utilization of papoose board and molt mouth prop. multiple options for behavior management, including utilization of general anesthesia in the operating room, were discussed with the father, and informed consent to utilize medical immobilization techniques for purposes of this examination was obtained and documented prior to taking the patient into the operatory. in the operatory a dental examination was performed, and a baseline panel of digital radiographs was obtained. the head and facial features of this patient were suggestive of fragile x syndrome (see: fig. . ) . the body of the mandible was somewhat elongated; the nose was prominent; the head had somewhat of a triangular shape, and the patient readily averted his gaze. upon further inquiry, the father reported that the patient also exhibited macroorchidism, although he indicated that no physician or dentist had ever suggested a work up for fragile x. fragile x syndrome is a disorder with which many clinicians are unfamiliar. yet it is the second leading genetic cause of intellectual disability in the united states, and it is the leading known cause of autism in the u.s. in addition to the phenotypic fi ndings noted in this case, there are other frequently-encountered physical characteristics consistent with fragile x that may move a clinician toward this diagnosis. they include pectus excavatum or funnel chest (see fig. . ) and joint laxity (see fig. . ) . gaze aversion, as previously mentioned, is a typical fi nding in autism and in fragile x syndrome. indeed, in conjunction with non-verbal behaviors, gaze aversion is often the fi nding that initially alerts the clinician to the possibility of a neurodevelopmental diagnosis featuring autism as a complication. figure . features a photograph of fi ve children at a school for children with special needs. four of the children have been diagnosed with autism, and a fi fth child is a neurotypical child who was visiting his brother on the day the photograph was taken. the reader is left to decide which child is the neurotypical child. any physician or dentist who encounters a patient with an obvious intellectual disability, who does not have an established underlying neurodevelopmental diagnosis, and who presents with additional fi ndings, which may include gaze aversion, shyness, a prominent chin, pectus excavatum, a large nose or large ears, should suspect a possible fragile x diagnosis. the primary care clinician -physician or dentist -should discuss with the guardian or family member the importance of establishing a neurodevelopmental diagnosis. the family member or guardian should be informed that genetic counseling should be made available to all members of the extended family, since fragile x syndrome is a genetic disorder that can be passed from parents to offspring. once this discussion has taken place, a referral to a geneticist for a complete genetic work up is indicated. both the dentist and physician should feel entirely comfortable making this referral. in remote areas where the services of a geneticist may not be available, the attending physician or dentist may order a high resolution chromosomal analysis and a fragile x dna test, and have those results sent to a remote location for interpretation by a geneticist. consultation with a psychiatrist or a clinical psychologist may also be advisable, as patients with fragile x can sometimes experience enhanced social integration as a benefi t of behavioral therapy. the healthcare access problem for americans with neurodevelopmental disorders and intellectual disabilities is, at its core, a healthcare education problem -an education problem resulting from a long-standing defi ciency in professional training focused on the care of this patient population. and it is clear that the medical and dental professions share equally in responsibility for these defi ciencies. eighty-one percent of america's medical students will graduate without ever having rendered clinical care to a single patient with a neurodevelopmental disorder or intellectual disability; and the graduates of % of america's medical residency programs will graduate from those residencies having had no formal training whatsoever -didactic or clinical -in the care of this patient population. additionally, % of graduating dentists have never treated a single patient with a disability. it is no wonder that patients like those whose cases were discussed in earlier sections of this chapter have such diffi culty accessing quality health care. as robert uchin, dean of nova southeastern university college of dental medicine observed in a speech in to his faculty, "not only do we not have enough doctors to care for these patients; we don't have enough teachers to teach them how to care for them." as a result of these defi ciencies in professional education, few clinicians with any expertise in developmental medicine or developmental dentistry are to be found in communities across america. the experts in developmental medicine and dentistry, for the most part, tend to be physicians and dentists who work at the few remaining intermediate care facilities, and at special needs outpatient clinics, psychiatric hospitals, and nursing homes. these physicians and dentists possess the knowledge and expertise in these disciplines because they are the physicians and dentists with the clinical experience. unfortunately for the patients with neurodevelopmental disorders who are clamoring for quality care, there are too few of these clinicians. national experts in developmental medicine and dentistry, however, have begun to collaborate in the creation of patient care protocols; and they have produced multidisciplinary curricula in both dvd and online format. the aadmd has made available hours of online curriculum in developmental medicine, developmental dentistry, and developmental psychiatry (see: list of urls). the curriculum program is entitled, the continuum of quality care , and it teaches collaborative patient care in three disciplines through an interdisciplinary format. the aadmd, through a grant from the wal mart foundation and the north carolina developmental disabilities council, and in collaboration with the north carolina mountain area health education center and the family medicine education consortium, has also established the national curriculum initiative in developmental medicine. this initiative, which is scheduled for completion in , will develop curriculum standards for physicians in the primary care of adults with nd/id. the curriculum stresses the importance of a collaborative approach, which includes medicine, dentistry, podiatry, optometry, and multiple ancillary health professions. if the disparities in access to healthcare for americans with nd/id are to be resolved, physicians and dentists must be willing to cross professional boundaries and work together to plan and deliver whole-person healthcare to their patients with nd/id. interdisciplinary protocols in the diagnosis of neurodevelopmental disorders and in the management of the secondary health consequences associated with these disorders must be established. additionally, clinicians with expertise in these arenas must be willing to work and teach in our nation's medical and dental schools. the clinicians with expertise must be willing to develop predoctoral and postdoctoral curricula, and the deans of america's professional schools must be willing to include these curricula as part of their larger programs in primary and specialized care. the clinicians with expertise in developmental medicine and dentistry must also be willing to conduct patient-focused, interdisciplinary, clinical research in an effort to solve the myriad problems that create obstacles to the delivery of the standard of care for patients with nd/id. they must be willing to obtain institutional review board approval for this research, and they must be willing to make this research available to their colleagues through publication in peer-reviewed journals and text books, and in professional lecture forums. the patient featured in figs. . and . is a man named james. he is a year old patient with idiopathic intellectual disability who presented to a dental clinic for evaluation of a painful facial swelling. a comprehensive intraoral exam revealed a cellulitis resulting from multiple grossly decayed teeth, and a generalized advanced periodontitis. no fewer than fi ve clinicians became involved in this patient's care. they included a general dentist, two oral surgeons, a family practice physician, and a geneticist. over the course of several months, as the treatment plan was completed, and as the chronic dental and periodontal infections were eliminated, james experienced signifi cant improvement in his overall state of health. a comparison of these two photographs reveals not only signifi cant improvement in his aesthetic appearance, but also in his skin turgor and color. these improvements in the patient's health translated to improvements in his daily life. he found gainful employment, and his caregivers now report that he smiles constantly -at work and at home. these photographs were entered into evidence in before a congressional subcommittee investigating the death of a young african-american boy who died as a result of an untreated dental abscess. the photographs were intended to make the point that patients with intellectual disabilities need not die as a result of medical illnesses derived from untreated dental disease. this patient's case illustrates that, when physicians and dentists are willing to work together toward a common goal of whole-person health for their patients, profoundly positive outcomes can be achieved. in a larger context, if our nation's medical and dental professions are willing to commit to a shared agenda, one which promotes the idea of collaborative, interdisciplinary care as a foundational concept, signifi cant improvements in quality of health and quality of life can be realized, not just for americans with neurodevelopmental disorders, but for every patient seeking quality care. in light of the events of , bioterrorism has become subject of increased attention from all members of society. government agencies, professional associations, academia, etc. have expressed their determination to wage war on such threats by all means available. dentists can also participate in this effort by providing assistance at interested groups and the general public (flores et al. ) . in this chapter we will examine the elements and components that may play a role in the establishment of an electronic network for the dental profession for supporting the fi ght against bioterrorism. in this section we review the threats, the public health system, current electronic surveillance systems, regulations and ethical issues, the computerization of dentistry, and how dentistry can serve in improving biosurveillance efforts. the aftermath of september and the anthrax incidents in october ( lane and fauci ) , made the us government reorganize its priorities and reform its current structure (white house offi ce of the press secretary ) . in response to these incidents, president bush proposed the "health security initiative" (white house letter ) in february nd of . this effort labeled the "bioshield initiative," (white house letter ) has the purpose to stimulate research and development of medical countermeasures against bioterrorism attacks. however, despite all these efforts, terrorist attacks are likely to happen in the future and even the best work from intelligence and security agencies will be unable to prevent such events (betts and richard ; council on foreign relations ; baker and koplan ) . to cope with this threat, a report published by an independent task force sponsored by the council on foreign relations "america-still unprepared, still in danger" (council on foreign relations ) , suggested a series of steps to assist the government in preparing to better protect the country. one of these suggestions is the bolstering of the "public health systems". baker et al. defi ne the u.s. public health system as a system that consists of a broad range of organizations and partnerships needed to carry out the essential public health services, such as hospitals, voluntary health organizations, other non-governmental organizations and the business community (baker and koplan ) which can collaborate with local, state and federal public health entities. after the unfortunate incidents in the public health system was revisited and the realization that "the nation's public health infrastructure is not fully prepared to meet this growing challenge" (frist ) became clear. to address this need, congress and president bush enacted the public law (p.l.) - titled "public health security and bioterrorism preparedness and response act of " (frist ; th congress ) . the main purpose of this law was to improve the public health capacity by means of increasing funding and fostering other measures. frist ( ) , described the law as a "good start" and that "to be prepared for bioterrorism, it is imperative that we develop a cohesive and comprehensive system of ongoing surveillance and case investigations for early detection". in this way, several early detection systems have been implemented with different levels of success among different geographic regions in the us. one of the most important initiatives over the years has been the establishment of the national electronic disease surveillance system (nedss) (baker and koplan ; nedss ) . the national electronic disease surveillance working group establishes that the "nedss is a broad initiative focused on the use of data and information systems standards to advance the development of effi cient, integrated, and interoperable surveillance systems at the state and local levels. the long-term objectives for nedss are the ongoing automatic capture and analyses of data needed for public health surveillance". the purpose of this system is to take into consideration and integrate the information of current public health systems implemented at different health department levels: county, state and fi nally at the centers for disease control and prevention (cdc). another initiative spearheaded by the cdc is biosense (looks ) . the purpose of this program is to develop advance detection capabilities of health related events including disease outbreaks. in addition, its emphasis is to improve situational awareness by integrating advanced analytics to process data generated by different health providers and other entities in the us. now that we have examined the general aspects, we will continue our background review focusing on the aspects that pertain to the specifi cs of the dental profession. this section will provide some perspective of the structure of the dental profession in comparison with its medical counterpart. "there are approximately , active dentists in the united states" (mertz and o'neil ) . in the dentistto-population ratio was of - , . and it is expected that by the year the ratio will be . , which translates into one dentist for every , people. " in contrast, the physician-to-population ratio has been increasing for the past years and now stands at per , , about one physician for every people." eighty percent of the dentists are in general practice. during march and of , the american dental association and the us public health service sponsored the conference "dentistry's role in responding to bioterrorism and other catastrophic events" (palmer ; national institute of dental and craniofacial research ) . this meeting reviewed several aspects of bioterrorism and the dental profession: the nature of biological pathogens and its oral manifestations, what needed to be communicated, how dentists should participate, etc. dr. michael c. alfano described the diffi culties that biological pathogens create for clinicians because "they are so insidious." while discussing the anthrax mailings after september th he pointed out that: "… early symptoms appeared so they resembled the aches, fever, and malaise of fl u so those affected delayed seeking treatment, a delay that has proven fatal in some cases". lieutenant colonel ross h. pastel of the us army medical research institute of infectious disease (usamriid) listed the "category a" pathogens as defi ned by the centers for disease control and prevention, and those are: smallpox, anthrax, plague, botulinum toxin, tularemia and viral hemorrhagic fever. he also described an outbreak of smallpox in yugoslavia in and the measure that had to be taken to control it. dr. michael glick described the oral manifestations of smallpox showing "signs hours before skin rash. these oral signs include tongue swelling, multiple mucosa vesicles, ulceration, and mucosal hemorrhaging. oral signs are also evident in inhalation and gastro-intestinal anthrax. in oropharyngeal anthrax the mucosa appears edematous and congested; there may be neck swelling, fever, and sore throat" . dr. ed thompson, deputy director of the centers for disease control and prevention mentioned that "none of the new counter-bioterrorism measures can be effective unless local health practitioners are vigilant in observing and reporting a possible disease outbreak. such surveillance-knowing what to look for and whom to report to-is critical and applies not only to suspected bioterrorist agents, but to a list of reportable diseases which has grown to include such entities as west nile virus and sever acute respiratory syndrome (sars)." dr. sigurs o. krolls presented the response at the local level and he "stressed the importance of communication and the need for redundant systems", "to keep all the parties informed". he also posed the question "can dentists recognize signs and systems of contagious diseases?", and emphasized that education can be essential. dr. louis depaola made several connotations that can be key in the scope of this paper by saying "dentists can contribute to bioterrorism surveillance by being alert to clues that might indicate a bioterrorism attack. such surveillance would note if there is an infl ux of people seeking medical attention with non-traumatic conditions and fl ulike or possibly neurological or paralytic symptoms… or even specifi c signs of a bioterrorist agent. patterns of school of work absence, appointment cancellations or failures to appear, could also be indicators." dr. depaola made clear that in cases of limited release of bioterrorist agents, dentists "have little to offer" but "a widespread attack can certainly tap into dental professional skills in recognition, isolation and management". in addition, dr. guay ( ) lists all the possible roles in which dentists can participate including "education, risk communication, diagnosis, surveillance and notifi cation, treatment, distribution of medications, decontamination, sample collection and forensic dentistry." dental informatics must pay attention to these and other recommendations, in order to develop integrated systems that take these recommendations into consideration. it is also important to understand that informatics has to work with technologies already in place like the computer-based oral health record and current standards. the fi nal recommendation from the meeting stated that to play an important role in biodefense, a serious amount of coordination and preparation will be required, not only from dentists but from other groups, most likely requiring medical and dental data integration. the cohr as described by rhodes ( ) "can provide a structure for documentation that goes beyond the concept of a blank form on a page, it includes a glossary of dental terminology for the entire content of the form as well as knowledge bases and expert systems that can enhance the practitioner's diagnostic and treatment planning decisions". he also acknowledges that one of the advantages of this type of documentation is that it "is much more transportable". he also recognizes the need for standardized methods for collecting information from dentists. schleyer and eisner ( ) defi ned several scenarios where the cohr is used in a "shared" environment where several healthcare providers interact and information is seamless communicated, improving the decisions made by clinicians. delrose and steinberg ( ) discuss how the "digital patient record" enhances clinical practice by providing "better quality information" to the clinician. although all of these benefi ts sound promising and encouraging some still express concern of the lack of standards among different information systems, which translates in communication breakdowns (schleyer ) . on the other hand, heid and colleagues ( ) mention a list all the steps that are currently being taken by different organizations such as the ada in order to produce a standardized cohr. other examples of standardization can be found in a paper presented by narcisi ( ) where ada's participation as a voting member in the american national standards institute has allowed edi or the cohr to be discussed and improved at a national level. additional infl uences in the standardization of the cohr are the security regulations mandated by hipaa, the health insurance portability and accountability act of . dentists are required to "adopt practices necessary for compliance" (sfi kas ; chasteen et al. ) . these and other regulations (szekely et al. ) will encourage the homogeny among different system vendors. computer ownership, on the other hand, has increased steadily during the last years. according to schleyer et al. ( ) in only % of dental professionals used computers in their practices compared to % in the year . additionally similar trends in internet connectivity where described. the issues mentioned above describe the issues that have to be considered in order to create surveillance system against bioterrorism for the dental profession. this review has tried to be inclusive by covering different aspects starting with the current state of affairs and environment, treats, technology, law, etc. next we present a blueprint for developing a biosurveillance system. the purpose of developing an electronic health surveillance system is to gather information from patients directly ( wagner et al. ) by detecting signs and/or symptoms, or indirectly by obtaining other types of information such as over the counter medication sales, patients' no-shows, usage of internet search engines keywords, etc. in this particular case, the proximity of contact between the dentist and the patient is equivalent to a medical inspection in terms of immediacy and/or closeness. such signs and symptoms can be easily detected if the dentist is properly prompted to search for them. this is just one example of ways how a system could provide assistance in the detection of a bioterrorism incident. but, before describing our proposed system, it would be important to address the fact that current syndromic surveillance systems have certain advantages in terms of its particular technological implementation . the rods laboratory obtains data directly from chief complains in the emergency departments from hospitals. the advantage of this surveillance system is that the implementation has to be made with only a limited number of parties (hospitals, clinics, health systems, etc.). on the other hand, our system would have to deal with thousands of different implementations (one in each dental offi ce). this and other challenges have to be considered when designing the proposed system: the proposed system should work at multiple levels: the system would have to provide a mechanism to alert the dentist if there is • suspicion that a bioterrorist attack may be happening. the mechanism would increase the dentist's awareness in case of fi nding suspicious signs or symptoms in a patient. this can be triggered by the patient's characteristics such as geographic location of residence, etc. automated collection of information from the patient's oral health record. the • system would report to a central database signs or symptoms of interest. the aggregation of this data could generate information that would eventually identify the presence of patterns that may lead to the early detection of such events. collection of additional information, which combined with other sources, can be • useful in terms of detecting or tracing some incident. patients' "no-shows" is the primary example, that, if combined with others such as work or school absenteeism can provide a relevant pattern for public health offi cials. dr. x, who practices in a community min away from capitol city, installed a new clinical management system months ago. among the features that were included in this new clinical management system (cms), a bioterrorism detection module was added. she felt curious because of recent news she read in the newspaper about possible attacks against the us and decided to install such feature. he read about how the module would work in combination with the cms she just bought. the educational information provided with the software instructed dr. x, that in case that a patient victim of a bioterrorism attack happens to be seen in her practice, the software would collect information and would send it to public health offi cials. when installing the software, dr. x was asked if she agreed to share such information with authorities. she was provided the option to receive notifi cation in case some information was sent but she decided not to enforce it. during the last week a patient walked into dr. x's dental offi ce. the patient presented some signs that indicated the presence of a disease; still its origin was not clear. an epidemiologic study later would show that the patient was present at the football stadium when an infectious agent was released (fig. . ) . although, at that time his medical history showed no indication of a systemic disease, the presence of multiple oral vesicles prompted the dentist to make an annotation into the cohr. the system, by using a natural language processing engine, detected such sign and sent this information to a central database. the patient was discharged and instructed to take some support medication to treat the oral ulcers. the next day, the central database pinpointed the presence of an out of the ordinary increase in the number of cases with the same signs and symptoms around that region. when the presence of this peak was detected, the central server sent a request to the dentist computer for additional information. one of the requested elements was if there was any use of medication for treating oral ulcers. fortunately this information was available. the central database crossed this with the information of other surveillance systems together with the information from other patients that happen to have similar clinical signs and/or symptoms. dr. x received an email from a public health offi cial asking her to communicate to the local health department to discuss information about one her patients. the case depicted above simulates the release of smallpox during a football game. in the case of smallpox oral symptoms include tongue swelling, multiple oral mucosal vesicles, ulceration, and mucosal hemorrhaging (national institute of dental and craniofacial research ) . dentists could be alerted by an electronic system to search for such signs or they can be detected automatically. in case of a high incidence within a group of patients, in a confi ned area, public health offi cials get to be notifi ed. in our hypothetical case there are issues that need to be addressed in order to make such detection system feasible: as described by schleyer et al. ( ) , % of dentists in the us use a computer in their practices. this fi gure would generate an estimate of , computers in dental practices. this prevalence of computers represents an opportunity for public health data collection. the creation of a software application for surveillance purposes must rely on existing technology. currently there are approximately major clinical management software packages in the market (dentistry today ) . out of these , clearly permit direct database manipulation. this characteristic can easily allow the creation of a "querying" application that would look for specifi c information within the data stored by those packages. additionally, a natural language processing engine could be embedded into the application in order to detect variations in data input on the computer oral health record. nevertheless, it is necessary to obtain a detailed list of the oral manifestations of diseases that are likely to be found on patients. successful implementations of similar systems have been shown to work successfully (chapman et al. ; ivanov et al. ) and using the same approach for our system seems technically feasible. this collected information later would be send to a central server in order to be analyzed and interpreted. the components of our system would be as follows (fig. . ) : thin client: a software application distributed for data collection. it would be • conformed of a "querying" mechanism, combined with a natural language processing engine and a communication module. this software client should be as thin as possible to reduce the work load on the dentist's equipment and should be embedded as a plug-in for current clinical management systems. vendors should be contacted to ask for their collaboration in the development of such application to ensure maximum compatibility and integrity of data collection. central servers: server software in charge of integrating all the data collected • from dental offi ces. it has to be capable of handling simultaneous requests from multiple users. this server would integrate all the data and would perform an analysis with the intention of detecting anomalies. it would be recommended that redundant servers should be located in different data centers with mirroring capabilities to guarantee their survivability in case of technical diffi culties. communication network: the transmission of information should be done using • the internet. this, of course, would essentially depend on the practitioner's current connectivity. if that is not available, backup connection to the central servers should be established. dentistry uses several standards for transmission of health related information. clinical management systems use standard-based technology to transmit information (narcisi ; chasteen et al. ; szekely et al. ; dentrix dental systems ) . dentists are aware of these standards and use them in a day-to-day basis to transmit information to insurers. additionally, in order to interact with other surveillance systems such as the nedss, our application should rely on the same standards. the software both client and server should be thoroughly verifi ed to be secure in terms of being safe against hacker attacks. on the server side, redundancy should be provided so downtime is reduced from design. the system should be developed so mirrored servers are always up and running. data integrity mechanism should also be considered. privacy and confi dentiality are important issues that need to be incorporated as part of a robust biosurveillance system and distinct regulations such as hipaa require protecting patient information (frist ; chasteen et al. ; bayer and colgrove ; etzioni ; ivanov et al. ) . in our hypothetical case we describe the use of several sources of information for detecting a bioterrorist attack. we described how syndromic information is transmitted to a central database which initially should be de-identifi ed. later, after the suspicion a bioterrorist attack more information is requested (medications) and more inferences are made. this, although technically possible, would require changing our processes and also the will to share clinical information. this leads to the discussion mentioned in the background section about "individual rights" vs. "common good". although hipaa addresses public health , some other implications may arise and the health professionals including dentists, physicians, public health offi cials and patients should discuss and address such issues. as discussed earlier, legislators face a diffi cult task in terms of determining what is best on behalf of the individuals they were asked to represent. legislation may have to be passed in order to guarantee the functioning of such a system. individual freedom and privacy are important values which may pose a confl ict when collecting individuals' information even for their own good. in any case, careful consideration has to be given to which information is required to detect a bioterrorist attack and also, by keeping in mind that it is always important to reduce, as much as possible, the collection and transmission of patients' information over the internet or any other network. a detection algorithm has to be created or adapted in order to determine the presence of a bioterrorist attack. some algorithms have proven their effectiveness (wong et al. a, b ) and it is likely that from these, a new analysis should be done in order to select or create one that addresses the particular needs of our system. a study was conducted to assess the feasibility of using oral manifestations in order to detect disease outbreaks (torres-urquidy et al. ) . it was found that for diseases such as botulism and smallpox it would be feasible to gather data that contains oral manifestations that would allow creating a detection signal using natural language processing followed by the use of statistical methods such as moving average to serve as part of a detection algorithm. the system should also be thoroughly evaluated, before and after implementation. to perform the evaluation before the system implementation computer simulation can be used to assess the effectiveness and likelihood of detection. simulation and modeling techniques (reshetin and regens ) have been used to estimate the effects of a bioterrorist attack. the same techniques can be used to evaluate our system. in case of the study by torres-urquidy ( ) , the investigators utilized synthetic outbreaks to test the performance of different signals. from their evaluation process, they learned, for instance, how many cases would be necessary to occur for the system to reach certain detection thresholds. several dental organizations have shown publicly their support of measures against bioterrorism. the american dental association and the national institute of dental and craniofacial research are two organizations who could play an important role in the development, deployment and ongoing support for our system. local dental societies also would also play an important role in the deployment of the proposed system. similarly, local, state and federal public health agencies should engage in activities that could make these mechanisms for health surveillance feasible. if dentists want to play an active role in the fi ght against bioterrorism, they should commit to collaborate with public health entities as well as to seek a way to integrate their information with the rest of electronic biosurveillance systems. professional organizations such as the american dental association can also participate by endorsing such efforts and by collaborating in the educational process of the dental professionals and their patients. as mentioned by dr. depaola (national institute of dental and craniofacial research ) dentists "have little to offer" in the current biosurveillance state of affairs. however, the integration of different technologies can change this perception. goldenberg et al. ( ) described over-the-counter medication sales as a technique for discovering disease outbreaks and stated that their approach may be "more timely" than traditional medical or public health approaches. medical cases that result from bioterrorism attacks do not produce symptoms until they have fully developed, so it is likely that different patterns can be detected before the patients start reaching the emergency department. as stated earlier (torres-urquidy et al. ) , it may be possible to have dentists participating of biosurveillance efforts, if we solve the proper organizational and technical challenges. dr. john r. lumpkin ( ) states that "hippocrates noted the health of the community was dependent on characteristics of a community and the habits of the people who lived there." dr. krolls (nidcr ) in his fi nal remarks during his presentation at the dentistry's role conference against bioterrorism, said, "dentists may pick up telltale information about what is happening in the community. after all, dentists spend more time with their patients than any other health specialty". kass-hout t, zhang x. biosurveillance: methods and case studies. muhammad f. walji maintaining patient records are essential for both clinical care and research. clinical research often occurs in the context of also providing patient care, yet the systems that are used for each are different and often cannot exchange data. the lack of data exchange between systems pose signifi cant barriers to effi ciently treating patient and conducting clinical research in dentistry. the purpose of this section is to review the benefi ts and challenges of integrating electronic health record (ehr) used for patient care and electronic data capture (edc) which is used for clinical research such as clinical trials. an increasing number of dentists routinely use ehrs (schleyer et al. ) . most dental schools in north america also use ehrs. benefi ts of using ehrs include increased legibility, portability, and improved patient safety (buntin et al. ) . recent federal incentives, although not directly benefi cial to dentists, will also likely spur the adoption of ehr (blumenthal and tavenner ) . clinical researchers, especially those conducting clinical trials, are also discovering benefi ts of using electronic data capture compared to paper. a clinical trial is a process in which new treatments, medications and other innovations are tested to evaluate safety and effi cacy. a standard part of health care, clinical trials are often lengthy and costly due to myriads of regulatory oversight. recent estimates set the cost of drug development in excess of $ million (grabowski et al. ) . accurately documenting data with suffi cient detail is critical for providing patient care and conducting clinical research. while the medical record is the foundation for patient care, the case report form is the foundation in a clinical trial. not all clinical research is clinical trials. clinical trials whose data will be submitted to the fda as a new therapy or device have additional requirements relating to the collection and transmission of the data. similarly for patient care data, ehrs need to meet the privacy and security requirements of hipaa. case report forms (crf) are a medium in which research study sites collect subject data in pre-defi ned formats for communication with clinical trial sponsors (rondel and webb ) many clinical trials data are collected on paper (rondel and webb ) . data measurement, collection, and recording are considered the "most crucial stage" in the data management process (hosking et al. ) . traditionally, study coordinators often record information in a case report form and subsequently mail or fax the crf to the centralized coordinating center. there, data entry staff, sometimes with the aid of optical character recognition systems, input crf data into a computer. errors made during this second data entry process are diffi cult to detect and correct (hosking et al. ) . lengthy guidelines in literature discuss methods for developing paper case report forms to reduce data entry mistakes (hosking ) . a well-designed crf may allow a user to effi ciently collect and record pertinent data. however, forms are often revised and redesigned during a clinical trial due to changes in protocol, unforeseen outcomes, or oversight (singer and meinert ) . there has been a recent drive to use electronic case report forms (ecrf). direct data entry at a study site shortens time to analysis and provides opportunities to audit data at time of entry. this could reduce data errors that might otherwise be caught weeks after submission. for quality control purposes, some studies require double data entry using computers and paper (day et al. ) , though alternative solutions have been explored including the use of data sampling (king and lashley ) and probability statistics to select only those forms likely to contain errors (kleinman ) . ecrfs may also facilitate data collection from existing electronic information systems such as lab systems. however, ecrfs are almost always reside in a separate system that is not linked to a patients record. although many clinical research studies are still being conducted using paper, an increasing number of studies are using ecrfs and electronic data capture (edc). for example, a review of canadian clinical trials found that % use edc (el emam et al. ). studies that are sponsored by a pharmaceutical company and are multicenter appear to use edc at a higher rate than those sponsored by government or a university. the cost of a commercial edc is substantial. recently a freely available edc has become popular amongst universities called redcap. a tool originally developed at vanderbilt university, it is now being used at over a institutions worldwide (harris et al. ). however, such tools are generally not integrated with the institutions ehr. although moving from paper to electronic will afford benefi ts there is a great need to allow data exchange between the patient care and clinical research components of information systems. although ehr and edc are similar, several challenges remain unresolved that prevent integration. one of the major barriers is likely to be different workfl ows for patient care purposes and to collect data for research. research is needed in defi ning an optimal workfl ow that can streamline the tasks associated with patient care and research, while at the same time providing a unifi ed information system that support these activities. also, the data that are collected for care and research are likely to differ. a researcher may require far more granularity of an oral health measurement than a clinician seeking to provide care. in cases when conducting a double blind placebo controlled clinical trial, the investigator may not even know the type of treatment that has been delivered to the patient. due to complexities of each domain, and large differences in goals, to date mutually exclusive workfl ows have arisen. a clinician investigator who sees a patient for both care and research, will likely need to enter data on this same patient twice; once in the ehr and once in the edc system. despite the availability of electronic systems, a major barrier is the integration and compatibility of disparate health information systems to converse with one another. the languages are important because they can help data sharing. clinical trials are not usually conducted in isolation, but are part of conventional medical care. therefore sharing data by clinical trials, patient care and laboratory systems becomes especially important with the adoption of ehrs in dentistry. in biomedical informatics, standardized terminologies are recognized as a critically important area to help better represent and share data for use in electronic systems (cimino ) . the systematized nomenclature of medicine clinical terms (snomed-ct), developed by the college of american pathologists, is the most comprehensive medical terminology (strang et al. ; chute et al. ) and is used in a number of health informatics applications. the us department of health and human services ( ) has also licensed snomed-ct, allowing access throughout the us at no charge. therefore snomed-ct is even more likely to be the vocabulary used in electronic formats of patient records in the future. the medical dictionary for regulatory activities (meddra) is terminology used by the fda and drug development industry to classify, retrieve, present, and communicate medical information throughout the medical product regulatory cycle (brown et al. ) . in particular it is used to record and report adverse drug event data. therefore standard languages are essential in sharing clinical trials data between sites, and also with regulatory agencies. no one single terminology is suited for all tasks. snomed-ct is likely to be more comprehensive to code clinical encounters, while meddra is more suited to help adverse event reporting. however, it is important that terminologies are widely adopted and used for similar purposes. even when standard terminologies are agreed upon, such information needs to be interchanged in standard formats. health level (hl ) is an important organization whose standards are widely adopted in healthcare to exchange information between computer systems. the clinical data interchange standards consortium (cdisc) is also an important group that helps to defi ne different data standards specifi cally for clinical trials research, such as clinical trials or regulatory submissions. one particular challenge in oral health has been the lack of a standardized terminology to describe diagnoses. although icd contains oral health concepts, they are often not granular enough to be useful for some patient care or research purposes. recently a dental diagnostic terminology has been developed by a group of dental schools, and has already been adopted by several institutions and used within dental ehrs (kalenderian et al. ) . the american dental association (ada) has also been developing snodent, but is not yet publically available for clinical use (goldberg et al. ). another link between ehr data and clinical research is the potential to fi nd human subjects. recruiting suffi cient numbers of patients that meet eligibility requirements within an allotted time frame for clinical trials is challenging. as ehrs contain detailed information about patients, they can be used to fi nd patients that meet specifi c inclusion and exclusion criteria. informatics for integrating biology and the bedside (i b ), an open source data warehousing platform, has been found to be a useful tool for cohort selection especially if the source data from an ehr is represented in a structured format (deshmukh et al. ). further, with health information increasingly available to patients through the internet, it is possible interested patients will be more effective in fi nding clinical trials than investigators looking for patients. many clinical trial registers are now available online. the national institutes of health (nih) have made available their database of nih funded research (mccray ) . there is currently no single repository for patients to fi nd all trials studying a health condition. a recent study assessed the comprehensiveness of online trial databases concerning prostate and colon cancer and found that online trial registries are incomplete, especially for industry-sponsored trials (manheimer and anderson ) . a more collaborative effort between government and industry-sponsored research groups to compile and standardize information may be a mutually benefi cial effort. it is not clear how many patients now enroll in clinical trials through online discovery. ehr data originally collected for patient purposes can be potentially used for research. aggregating data from multiple sources can provide a large dataset that could otherwise not be available. electronic health records (ehr) contain a wealth of information and are a promising source to conduct research. data extracted from ehrs differ from other sources such as population surveys or data obtained from payers, as they provide a more detailed and longitudinal view of patients, symptoms, diseases, treatments, outcomes, and differences among providers. therefore ehr data in dentistry can potentially provide valuable insight into oral health diseases, and treatments performed on a large cohort of subjects. ehrs also play an important role in enhancing evidence-based decision-making in dentistry (ebd) and improving clinical effectiveness through decision support (atkinson et al. ; walji et al. ; valenza and walji ; taylor et al. ; spence et al. ; chambers et al. ; langabeer nd et al. ; walji mf et al. ). the consortium of oral health related informatics (cohri) provides an example of how dental ehrs are used for research purposes (schleyer et al. ; stark et al. ) . cohri was formed in by a group of dental schools who used the same ehr platform and who are interested in sharing clinical and education data. through funding from the national library of medicine, four dental schools are participating in a pilot project to develop an inter-university oral health research database by extracting and integrating data from ehrs. one promising area where data repositories derived from ehr data can be used for new discoveries is in the area of comparative effectiveness research. comparative effectiveness research is defi ned as "a rigorous evaluation of the impact of different options that are available for treating a given medical condition for a particular set of patients." (congressional budget offi ce ) further, such research includes focusing on the clinical benefi ts and risks of each option (clinical effectiveness), and an analysis on the costs and benefi ts (cost effectiveness analysis). comparative effectiveness research (cer) is also likely to reduce costs of dental care and increase access to the majority of the population who currently receive no dental care. unfortunately many recent systematic reviews focusing on cer questions in dentistry have been inconclusive due to the lack of existing evidence in the scientifi c literature. secondary analysis of the data that reside in dental electronic health records (ehr) is a particularly appealing approach to facilitate cer and generate new knowledge. ehr data has the potential to provide a comprehensive picture of patients' histories, treatments, and outcomes, and if integrated with similar data from other dental clinics can include a large and diverse set of patients. however, numerous challenges must be solved before ehrs can be used for cer. first, data suitable for cer must actually be collected from ehr systems. second, this data, which often resides in proprietary systems, must be accessible and retrievable. and lastly, this data should be structured in a format that can be integrated with data from other sources or institutions. practice-based research networks (pbrn) are groups of primary care clinicians and practices working together to answer community-based health care questions and translate research fi ndings into practice. pbrns engage clinicians in quality improvement activities and an evidence-based culture in primary care practice to improve the health of all americans. in , the national institute of dental craniofacial research funded three such research networks. the dental pbrn's to date have been conducting both prospective and retrospective research. for example, barasch et al. conducted a case controlled study to investigate risk factors associated with osteonecrosis of the jaws . many prospective studies conducted as part of pbrns still require separate data collection systems for the research data. ehr data contained in practices as part of pbrns are beginning to be used for secondary purposes. for example fellows et al. conducted a retrospective analysis of data contained in electronic health records to estimate incidence rates of osteonecrosis of the jaws ( fellows et al. ) . pbrns provide great promise of how ehr and clinical research data can be used effectively to promote both patient care and new discoveries. another area that intersects both the patient care and research realm are patient registries. patient registries are ways to track groups of patients who have had specifi c diseases or have had certain treatments. while ehr data would contain information on all types of patients, their diseases, and treatments, registries would allow focus on specifi c diseases or treatments of interest. registries would not be as costly in terms of resource requirements like a traditional clinical trial, but would require specifi c eligibility criteria, informed consents, and collection addition to that collected as part of routing care. dentistry has lagged far behind in forming data registries, primarily because dentistry is practiced in small offi ces and not in large hospitals making the process of integrating data very diffi culty. however, dental schools which themselves house large clinical operations are ideally positioned to create disease specifi c registries that can potentially use data collected for patient care and extend for research purposes. there is great potential for providing new insight in oral health by the integration of patient records and clinical research from both a workfl ow and information systems perspective. the technology challenges of developing systems that can exchange data, and use standardized terminologies appear solvable. however, the socio-technical issues such as determining how to incorporate optimal workfl ows for conducing both patient care and research with minimal additional overhead appear to be the greatest challenge before widespread adoption. similarly, there appears to be great potential in using ehr data originally collected for patient care for the secondary use of research and discovery. this will require collaboration between patients, providers and researchers from all healthcare disciples, and institutions with friendly policies for sharing data to improve both patient care and drive new discoveries. amit acharya , andrea mahnke , po-huang chyou , and franklin m. din more recently there has been a strong push from the united states federal government for the adoption of the electronic health record (ehr) within the healthcare industry. as a result, $ . billion is made available to incentivize the physicians, dentists and hospitals for the adoption of the ehr through the health information technology for economic and clinical health (hitech) act. as the nation head towards adoption of the ehrs, there has also been a growing interest with the majority of the u.s. dental schools to implement ehrs within the educational setting. fifty of the fi fty-six u.s. dental schools, as well as dental schools in canada and europe, are either using or in the process of adopting some aspects of a common dental ehr framework (white et al. ) . a group of dental schools known as consortium for oral health-related informatics (cohri) was formed in which used this common dental ehr framework -axium (stark et al. ) . currently there are about dental schools within cohri. the ehr will not only support clinical care, but will also result in training the next generation dental students and to conduct innovative research that was not possible earlier. however, not much is known about how many of these dental schools' electronic dental records are integrated with their respective university's electronic medical record. a common medical-dental ehr model at healthcare universities would enable a holistic approach to providing patient care and provide the much needed electronic infrastructure to study interrelationship between the various oral-systemic diseases. recently a group of researchers from marshfi eld clinic in wisconsin, us conducted a survey to investigate the current states of health information technology and informatics within the dental school in the us. list of us dental schools were identifi ed through the american dental education association (adea) web site. dental schools were contacted to determine who the most appropriate person to take the survey would be. once the list of contact was developed from each dental school, an email was sent to us dental schools with a link to a survey created in surveymonkey. the survey was administered on tuesday march , . reminder survey emails were sent to all recipients on march and march . the survey was closed on march . the anonymous survey was at most questions, depending on how questions were answered. the survey focused on topics such as presence of dental informaticians within the dental schools, use of fi nancial and clinical information systems, interest in federal stimulus support for ehr adoption provided through american recovery and reinvestment act and meaningful use of ehr, relationships with health care entities and bidirectional nature of the dental and medical ehrs. the study was approved as exempt from the marshfi eld clinic institutional review board under section cfr . (b) and waived requirement for an authorization. thirty out of the fi fty fi ve dental schools responded to the survey (response rate of %). however, fi ve of the thirty dental schools representative did not complete the survey and hence their response was not included in the analysis. regarding the question about the presence of a dedicated department or a center for information technology (it) or informatics within the dental school in us, % (n = ) of the responding dental schools had a dedicated it/informatics department or center (p-value of . ). the it or the informatics department size (in terms of the number of personnel) at the dental schools is illustrated in fig. . . thirty fi ve percent (n = ) of the us dental schools that housed an it / informatics departments had personnel with not only it training but also dental informatics training. while % (n = ) of the dental schools were considering integration of dental informatics personnel within their department or center. twenty fi ve percent (n = ) of the dental schools did not have any plans of integrating personnel with dental informatics personnel within their department or center (see fig. . ). partial responses to additional questions in the section of the survey is provided under table . . the majority of the responding dental schools were currently using financial electronic systems (fes) (p-value of < . ) and electronic dental records (edr) (p-value of . ). the use of fes outnumbered the use of edrs in the dental schools (see fig . ) . about % of the dental schools that were currently utilizing the edrs used it in all the clinical modules (p-value of . ), while % of the dental schools used the edrs in some of the clinical modules. when asked about the commercial edr system that the dental schools were using, axium (exan group, canada) was by far the most implemented edr system. two dental schools had salud (two-ten health limited, ireland) implemented and two dental schools had gsd academic (general systems design group, iowa, us) implemented. combinations of two ehr systems (home grown and dentrix) were implemented at two dental schools. one school had a dentrix only implementation, while another had developed its own edr system (home grown) (see fig. . ). there were dental schools which had implemented an edr fi ve or more years ago, dental schools - years ago, dental schools - years ago and dental schools less than a year ago (see fig. . ) (p value of . ). when the dental schools were asked the question as to whether they were expecting to apply for the medicaid meaningful use incentive program, majority ( %) of the dental schools did not know and only % of the dental schools were expecting to apply within the next years (fig. . ) (p-value of . ). challenges or barriers identifi ed by some of the dental schools in complying with the meaningful use objectives were (a). lack of certifi ed edr and information regarding it, (b). issues with getting auxium certifi ed and (c). qualifi cations of the edr as many of the meaningful use objectives do not apply to dentistry and lack of specifi c information about it. only % of the responded dental schools were part of a health system. fifty two percent (n = ) of the responded dental schools had a formal relationship with other health care delivery entities in terms of sharing facilities, patient transfer, training programs. some of the types of relationship mentioned by the dental schools that had a formal relationship with other health care delivery entities included: (a). a gpr program and an emergency dental unit in the hospital, (b). affi liated hospital, (c). affi liation agreements, (d). oral and maxillofacial surgery (omfs), anesthesia and pedodontics all have some portion of education in medical health center, (e). omfs residents are also residents of medical health center, (f). residents providing care under contract with area hospitals, (g). sharing patients wand facilities with the health center, (h). students rotating in the community health centers and (i) collaborative grand programs. eighty fi ve percent of the dental schools that had a formal relationship with the health care delivery entities had routine interaction with them because of their existing relationship (p-value of . ). their usual method for exchanging information was through informal medium such as phones, emails and faxes and formal medium such as memorandums, letters and contracts. when the dental schools were asked about the communication between the health systems' emr and the school's edr, majority of the dental schools did not have any communication ( %) or did not know is such a communication existed ( %) (p-value of < . ) (see fig. . ). out of the % (n = ) of the responded dental schools who's edr did not communicate with the health system's emr, % (n = ) of the dental schools stated that they did not need to exchange patient information electronically as a reason for the non-communication, while % (n = ) dental schools states that they would like to exchange patient information electronically but there were barriers that prevent them from doing so. some of the barriers identifi ed by these dental schools were (a). the hospitals and the dental school are not part of the same medial system and hipaa concerns prevent sharing data, (b). the dental school currently neither did have an edr nor the infrastructure to support one and (c). hospital is not interested and has high and perhaps unrealistic security standards. the remaining % (n = ) of the dental schools expected to exchange patient information electronically in the near future (next years). some of the information categories that were shared between the edr and emr in the small number of dental schools are illustrated in fig . . finally when asked about any research projects under way in their dental school to investigate discrepancies between medical and dental records for the same patient, only ( %) dental school was currently undertaking such project. in all common diseases, including those that affect the oral cavity, both the environment and genetics are pathogenic conspirators. unfortunately, we currently know little about the specifi c mechanisms underlying any common disease; and oral diseases are among the least understood. elucidating the etiology of chronic oral diseases will involve a synthesis of results from careful experiments of environmental exposures such as diet and tobacco use, the oral microbiome, co-morbidities, largescale, well-designed genetic studies, and the various interaction effects. with regard to genetics, the past few decades have witnessed transformative developments in our ability to interrogate the entire genome for genes that contribute to disease. while dramatic advances in experimental designs, statistical approaches, and clinical insights have greatly aided this scientifi c campaign, the central driver of this progress has been the development of high-throughput, inexpensive genetic technologies. following initial molecular studies using variant forms of enzymes, or allozymes, a major breakthrough was the use of highly informative dna-based markers throughout the genome (botstein et al. ) . this idea of directly assaying existing dna variation to conduct linkage and association studies in genetics began a revolution in disease gene mapping. recent interest from commercial entities has produced a feverish pace of technological innovation, markedly reducing cost and expanding the depth of inquiry. previously unfathomable, the testing of over one million single nucleotide polymorphisms (snps) in thousands of patients and controls is now commonplace (wellcome trust case control consortium ; schaefer et al. ) ; and very recently, next generation sequencing technologies have progressed to the point where sequencing of the entire protein-coding portion of the genome (exome) or even the entire genome is a costeffective method to examine disease traits across the entire spectrum of genetic variants in small numbers of affected individuals (ng et al. ) . there is little doubt that soon whole genome sequencing will be applied to nuclear family-based designs, studies among distantly-related affected individuals in extended pedigrees, and case/control studies involving thousands of individuals. this unprecedented scope of inquiry made possible by large-scale genetics, has begun to yield fascinating resulting into predisposition to oral cancers, caries, and periodontal disease that will molecularly redefi ne these pathologies, explicate unique biological connections with related diseases, give impetus to the development of directed therapeutics, and indeed personalize medicine. still, much more genetic focus on oral disease phenotypes is required if we are to realize this medical impact in a timely fashion. as genetic technologies have allowed the progression of interrogating single protein variants to single dna markers to entire genes to markers across the genome, and now to the entire genome sequence, the promises of these large-scale genetic studies have understandably undergone monumental expansion. it may be reasonable to expect the results from whole genome sequencing to decidedly revolutionize medicine within the next two decades. however, this new scientifi c capacity comes at a cost. as genetics, and biology in general, transitions to a data-rich science, practitioners have found themselves woefully unprepared to store and analyze the volume of data generated. once analyzed, interpretation and integration of these abundant and multifaceted results into medical practice will also be an appreciable challenge. insuffi cient assimilation of genetic fi ndings into merged dental and medical records will severely limit the ability of clinicians to appropriately treat patients. inadequately addressing these informatics issues will severely derail efforts in the basic sciences efforts as well as the translational and clinical sciences. this chapter explores the current state of genomics studies, what we have learned from genetic investigations into oral diseases, and where we may be headed. genetic studies have much to offer investigations of disease etiology. why do some acquire diseases and others do not? for those affected, why do some progress more rapidly than others? what causes some patients to respond to therapies, while others suffer from adverse reactions? these are all fundamental questions in both biology and medicine, whether the focus is on the gastrointestinal tract, the hippocampus, the lymphatic system, metabolic disorders, or oral diseases. speaking generally across disease areas, a portion of the answers to these questions often lies in described environmental effects. in numerous chronic diseases, infectious agents are likely contributors to the disease process -periodontitis, for example, is initiated by gram negative anaerobes in susceptible individuals (holt and ebersole ) . surely, unique and latent environmental exposures provide a random component to common disease susceptibility and progression. through twin studies, studies of risk in close relatives, and quantitative traits experiments, it is well-understood that heritable factors, including but not limited to dna variation, are typically responsible for - % of the phenotype variability for common diseases. this section will attempt to cover, at least at a cursory level, the major salient developments affecting genetic insights into chronic and aggressive periodontitis, with some comment on genetic factors infl uencing susceptibility to caries and oral cancers. while it would be extremely naïve to view genetic studies as an immediate panacea for our ills, the discovery of disease-causing genes does illuminate hitherto unknown biological pathways and molecular mechanisms, draws unforeseen connections with other traits, may improve prognostic models applicable for individuals, and suggests specifi c therapeutics. industrialization has brought forth increased lifespan and wellness through vaccination, modern sanitation practices, public health policies, and advances in medical science translated into practice. however, the accompanying physical inactivity coupled with a high calorie diet are probable contributors to an extremely common, chronically infl amed metabolic syndrome (hotamisligil ) that is thought to give rise to a multitude of intimately related disease traits: insulin resistance, compromised insulin signaling, hyperglycemia, obesity, dyslipidemia, hypertension, impaired kidney function, elevated liver enzymes and steatohepatitis, poor wound healing, neurodegeneration, vascular disease, pregnancy complications, accelerated immunosenescence, and periodontal disease (ford et al. ; ferrannini et al. ; eaton et al. ; holvoet et al. ; speliotes et al. ; eckel et al. ; d'aiuto et al. ) . these diseases often co-occur within the same patient and could be considered variable expression complications arising from a state of aberrant caloric fl ux that induces metabolic dysfunction and chronic, systemic infl ammation. these features constitute a disruption in a fundamental homeostatic mechanism with intensifying pathogenic consequences. the rapidly increasing incidence and decreasing age of onset for this pathophysiological state have generated a major source of mortality and morbidity in modern cultures (ford et al. ; ferrannini et al. ; weiss et al. ) . it is becoming increasing clear that many chronic diseases have an infectious component. there is relatively convincing evidence that many systemic, t-cell mediated autoimmune disorders may be initiated by infections. for example, from archaeological data, it is believed that an infectious agent -currently unknown -is necessary for rheumatoid arthritis (firestein ) , and both guillain-barre syndrome and rheumatic fever have well-described pathogeneses triggered by specifi c infections in susceptible individuals (bach ) . in many instances, oncogenesis and tumor progression can be traced to pro-infl ammatory responses at the site of chronic infection (coussens and werb ) , although it is not known whether these effects are mediated through the actions of the immune system, the infectious agents, or a combination thereof. several cancers fall into this category including gastric adenocarcinoma (uemura et al. ) , cervical cancer (walboomers et al. ) , hepatocellular carcinoma (saito et al. ) , and kaposi's sarcoma (dictor ) , all having unequivocal infectious agent etiologies. recent fi ndings of antiinfl ammatory pharmaceuticals, particularly those that inhibit cox- and cox- , reduce the incidence of certain classes of cancers are consistent with this view (dannenberg and subbaramaiah , rothwell rothwell et al. ) . in addition, there is moderate evidence that several bacteria -the most studied is chlamydia pneumoniae -play a role in atherosclerosis and myocardial infarction (saikku et al. ; watson and alp ) , however the studies are not conclusive and antibiotic treatment does not appear to be effective (andraws et al. ) . chronic periodontal disease is fi rmly footed at the intersection of infection, chronic infl ammation, and metabolic dysfunction. chronic periodontitis is characterized by infl ammation of the periodontal membrane, slowly causing gingival recession and eventual bone loss. the proximate cause of periodontitis is the virulent oral microbiome. the involvement of gram negative anaerobes has been fi rmly established for the disease. aside from the known oral pathogenic species p. gingivalis , t. denticola , and t. forsythensis , the so-called "red complex" (holt and ebersole ) , new bacterial species associated with chronic periodontitis have also been described (kumar et al. ) . the advent of an extensive database covering the oral microbiome will surely propel such investigations . numerous studies have shown that periodontal disease covaries with many diseases, presumably due to overlapping molecular etiologies. compelling meta-analyses demonstrate a highly signifi cant synchronicity of obesity and periodontal disease (chaffee and weston ) . in addition, the correlation between periodontal diseases/ alveolar bone loss and frank metabolic syndrome is repetitively observed (nesbitt et al. ; andriankaja et al. ) . extensive work has also shown a strong role for both infl ammation-related genes and circulating infl ammatory markers in periodontal disease (nikolopoulos et al. ; bretz et al. a, b ) . treatment studies further support the link between periodontal disease and immuno-metabolic syndrome. these experiments have demonstrated a signifi cant improvement in intermediate molecular markers of infl ammation when chronic periodontitis in the presence of metabolic syndrome (acharya et al. ) or type diabetes (iwamoto et al. ) was treated. conversely, treatment of periodontal disease with reduction of bacterial load leads to greater glycemic control among diabetic patients (simpson et al. ; stewart et al. ) . given the high prevalence of periodontitis and the co-morbidity of metabolic syndrome with periodontal disease, these treatment experiments appear to suggest that the virulent oral microbiome could play an important role in the pathogenesis of systemic infl ammatory metabolic syndrome, and is exacerbated by the syndrome. certainly, further studies are needed to defi nitively answer this question. as chronic periodontal disease seems to be a critical feature of sustained, systemic dysfunction of both metabolic and infl ammatory networks, uncovering the genetic variants carried by susceptible individuals would not only provide much needed insight into the molecular pathogenesis of chronic periodontal disease, but would also markedly aid our understanding of the infl ammatory metabolic syndrome and how it drives related co-morbidities. such genetic studies may also shed light on the specifi c mechanisms that appear to improve cardiovascular, infl ammatory, and diabetic outcomes when periodontal disease is treated, potentially leading to therapies and medical/dental intervention with greater effectiveness. such studies may also provide clues to which subsets of individuals respond more effectively than others and why they do so. periodontal disease can also present in a rapid manner with aggressive bone loss and early-onset. this is termed aggressive periodontitis (lang et al. ) . in contrast to chronic periodontitis, there is often a greater degree of familial aggregation with aggressive periodontitis, and it is hypothesized that most aggressive cases may affl ict individuals with one or more defective immune genes (zhang et al. ; amer et al. ; machulla et al. ; carvalho et al. ; toomes et al. ; hart et al. ; hewitt et al. ) . mutations in the lysosomal protease, cathepsin c, have been shown to be responsible for some forms of aggressive periodontitis, along with complications associated with other infl ammatory diseases (laine and busch-petersen ) . the specifi c hla variants thought to play a role in aggressive periodontitis, are also involved in infectious disease susceptibility and autoimmunity; and, interestingly, two of the non-mhc-linked regions, fam c and a locus on chromosome p , have been implicated in myocardial infarction (connelly et al. ) and may have action as a tumor suppressor in tongue squamous cell carcinoma (kuroiwa et al. ) . as with chronic periodontal disease, an infectious microbiome is heavily involved. however, in general, microbiome differences could not explain the presence of chronic versus aggressive forms of the disease, although in some aggressive periodontitis patients, a highly leukotoxic a. actinomycetemcomitans strain may contribute to the disease process (mombelli et al. ) . we currently do not fully know the differences between the genetic susceptibility factors for the chronic and aggressive forms of the disease. the most prevalent chronic disease in both children and adults is dental caries (national institute of dental and craniofacial research) . caries formation is a complex disease with several interacting components form the environment and host genetics. similar to gingivitis and periodontitis, caries have an infection-initiating etiology with acidifi cation leading to localized demineralization. epidemiological studies have long shown that diet is a strong predictor of caries formation; and the reduction in ph is exacerbated by high consumption of carbohydrates. the principal pathobacterial species are streptococcus mutans and lactobacillus ( van houte ) . there are also several reports of positive correlations of caries with infl ammatory diseases, although the association is not always repeatable. it is also not clear what proportion of the putative association with infl ammatory disease is due to innate upregulation of immune networks in contrast to the immuno-modulating pharmaceuticals prescribed to those with infl ammatory disease (steinbacher and glick ) . much of the effect is reported to result from lack of saliva volume (steinbacher and glick ) . interestingly, the presence of epilepsy may be associated with higher caries rates (anjomshoaa et al. ) . fluoride is an effective antimicrobial agent that interferes with bacterial growth and metabolism (wiegand et al. ) . hence, topical fl uoride administration as well as ingestion of fl uoridated water inhibits cariogenesis and caries progression (ripa ) . amelogenesis is a key process involved in modifying the rate of caries formation. both common variation and rare mutations in enamel formation genes such as amelogenin and enamelin are involved in caries rates (patir et al. ; kim et al. ; crawford et al. ) , the molecular actions of which are beginning to be revealed (lakshminarayanan et al. ) . over , new cases of cancers affecting the oral cavity and pharynx were expected in the united states for , with deaths numbering , (jemal et al. ) . the majority of these malignancies involved solid tumors originating from cancerous changes in squamous cells of the mouth. again, oral cancers have a complex etiology existing of entangled genetic, epigenetic, infectious, and dietary causes, further modifi ed by tobacco, alcohol and other environmental exposures. as with most cancers, it is reasonable to expect that both germline and somatic genetic changes will be involved in carcinogenesis, tumor growth, and metastasis. promoter hypermethylation of genes central to cellular growth, differentiation, dna fi delity, apoptosis, and metabolic stability is an important facet of these cancers (poage et al. ) . indeed, methylation-mediated silencing of genes involved in tumor suppression (e.g. the cyclin-dependent kinase inhibitor a), detoxifi cation (e.g. mgmt ), and apoptosis (e.g. the death-associated protein kinase- ) are commonly found in oral squamous cell carcinoma samples (ha and califano ) . to quantify the proportion of the variance in a phenotypic trait that is due to variance in genetic factors, population geneticists defi ned the concept of heritability (visscher et al. ; falconer and mackay ) . researchers subsequently developed several methods for estimating heritabilities using the measure of a trait (e.g. occurrence of disease/not-disease) in combinations of relatives (e.g. parentoffspring, or monozygotic-dizygotic twins). in general, the higher the measured heritability of a variable phenotype, the larger the contribution of genetic factors is in comparison to environmental effects. it is fallacious to assume that the heritable variation is composed entirely of alleles residing in the dna sequence, for heritability studies simply examine the covariance between relatives without comment on specifi c molecular mechanisms. hence, any heritable variation such as methylation patterns, vertically-transmitted infectious agents, as well as dna variation can contribute to the heritability measure. heritability results are important because they not only give a rough estimate of the collective effects of heritable factors, but also can provide a measure to quantify how much of the total genetic effect is accounted for by specifi c loci examined. for periodontal disease, four twin-based studies of heritability have been performed (michalowicz et al. ; corey et al. ; michalowicz et al. ; mucci et al. ) . although varying in sample size and methodological details, all four arrived at consistent results, with - % of the variance in periodontal disease being attributed to genetic variability for chronic periodontitis. given the segregation patterns described in the literature, it is reasonable to assume that aggressive periodontal disease exhibits a higher heritability. therefore, given the prevalence of periodontal disease, heritable factors within the population at large are likely appreciable. using twin pairs, bretz and colleagues reported substantial heritability values for multiple traits related to caries ranging from % to % (bretz et al. a, b ) . lastly, mutagen sensitivity studies of head and neck cancer patients suggest a signifi cant effect of genetic factors for the carcinogenesis of oral cancers (cloos et al. ) . hence, there is every reason to believe that a sizable pool of genetic and/or epigenetic factors await discovery for oral diseases. once the development of pcr (saiki et al. ) was applied to the idea of using naturally-occurring dna variation (botstein et al. ) , large-scale dna-based studies of disease underwent a substantial acceleration (schlotterer ) . genotyping of short, tandem repeated sequences (weber and may ) -microsatellites -spurred on a wave of genome-wide linkage studies, which evaluate the co-segregation of disease state with microsatellite markers, for both rare mendelian disorders as well as more common diseases with complex inheritance patterns. while the rarer traits with more coherent transmission patterns generally relinquished their genetic secrets to linkage analysis, more common diseases did not. in the mid-to late s, several theoretical studies had shown that the power to detect disease-causing alleles is higher with association-based designs such as a case/control experiment or association in the presence of a linkage signal as in the transmission/disequilibrium test if the frequency of those alleles is high and the effects are moderate (kaplan et al. ; risch and merikangas ; jones ; long and langley ) . however, to conduct genomewide association studies presented an ominous obstacle for the genetic technologies at the time. the number of markers required to effectively cover the genome was prohibitively large as the chromosomal blocks in population-based samples used in association designs were expected to be small. even within large extended families, the limited number of recombination events generates substantial chromosomal blocks passed through the pedigree, but researchers had both theoretical and empirical evidence that the blocks in population-based samples were on the order of k base pairs for most large human populations. as the reader can imagine, the mean length of blocks that are shared by descent is inversely related to the product of recombination rate, the number of affected individuals and the number of meioses separating the affected individuals. in practice, even very large extended families segregate regions shared by affected members on the order of several million base pairs in length. however, once geneticists seriously considered large-scale studies using a case/control design where individuals are separated by say , meioses, it became clear that to adequately cover the much smaller shared regions across the entire genome, hundreds of thousands of markers would be required (kruglyak ) . utilizing the human genome sequence (venter et al. ; lander et al. ) , a number of studies at celera diagnostics provided an intermediate solution, where approximately , putative functional snps primarily located in genes were assayed through allelespecifi c pcr in a number of common diseases using a staged case/control design. these studies were successful in identifying several gene-centric polymorphisms associated with common diseases (begovich et al. ; cargill et al. ) (fig. . ) . concurrently, several groups had performed extensive sequencing and genotyping across the genome to produce a genome-wide map of haplotype structure (hinds et al. ) , useful in linkage disequilibrium mapping. within years, technology for snp hybridization arrays had advanced so as to enable genome-wide association studies capable of capturing most of the common genetic variation in the genome either through direct genotyping or indirect interrogation using linkage disequilibrium -the term linkage disequilibrium is a measure of the correlation of alleles at closely-linked sites (see fig. . ). these investigations were met with numerous successes (klein et al. ; kathiresan et al. ; graham et al. ; gudmundsson et al. ) . inexpensive genotyping platforms and urging from theoreticians ensured that these genome-wide association studies were, in general, highly powered to detect all but very mild effects from high frequency alleles. these efforts, led by large academic consortia such as the wellcome trust, the international multiple sclerosis genetics consortium, and the broad institute and commercial entities such as decode genetics and perlegen have greatly expanded our understanding of the basic biology of common diseases: we now know, for example, that (i) autophagy-related genes are involved in crohn's disease (rioux et al. ) , (ii) there are a number of genes such as the protein tyrosine phosphatase, ptpn and the interleukin- receptor, il r , that exhibit ample pleiotropic effects among autoimmune conditions (lopez-escamez ; safrany and melegh ) , (iii) in the case of age-related macular degeneration, predictive models using the genetic results enable fairly accurate prognosis of individuals who are at high risk of disease (seddon et al. transcription factor tcf l plays a role in type diabetes (grant et al. ) , and (v) aberrant il- signaling likely contributes to multiple sclerosis susceptibility (gregory et al. ) . the plot shows the tremendous progress in genotyping technology where, a decade ago, very little of the genome was accessible for disease studies using association designs through the current wave of viable sequencing-based whole exome studies ( ) ( ) and whole genome studies ( ) ( ) . in fig. . , the average distance between adjacent genetic markers is plotted as a function of year of introduction to the disease mapping community. impressively, the total number of genetic markers has increased a million-fold over the past decade. although successful in uncovering numerous pathogenic pathways for common diseases, results from the current wave of genome-wide association studies, with a few exceptions, explain little of existing disease heritability. the reasons for this are cryptic and the subject of heavy debate (manolio et al. ) . multiple rare sequence variants generating high levels of allelic heterogeneity, functional de novo mutations, structural mutations such as copy number variants and large deletions, and epigenetic effects constitute four of several possible disease models that could account for the heritability discrepancy. the answer will almost certainly consist of a conglomeration of these and other effects. bringing forth the new genome-wide technologies that illuminate these previously non-or under-interrogated properties of the genome to bear on this enigma is a reasonable next step for all complex traits including oral diseases. the most commonly used measure of ld in a sample of chromosomes is linkage disequilibrium (ld) is a measure of the correlation between alleles at two sites in a sample of chromosomes. for two biallelic sites, if the a allele is always paired with the b allele, and the a allele is always on the same haplotype as the b allele, then the two sites are said to be in perfect ld. successive recombination diminishes ld. interrogating one site for disease association allows investigators to indirectly interrogate other sites in sufficiently high ld with the interrogated site. a key feature explicitly studied in molecular population genetics and implicitly used in disease gene mapping studies is the site frequency spectrum; that is, the distribution of allele frequencies at single sites in the genome that vary in the human population studied. from both diffusion models (kimura ) and coalescent theory (hudson ) in theoretical population genetics, we know that the vast majority of realistic models generate many more rare variants compared to common polymorphisms. this is particularly true for expanding populations. are these rare variants the source of much of the missing heritability? recently, with the application of high-throughput sequencing technology to human studies over the past decade, empirical studies have clearly verifi ed these predictions -the large majority of variants have low frequencies (the international hapmap consortium ) . the distribution of deletions appears to be skewed toward more rare frequencies, presumably due to the deleterious effects of such variants. individual mutations appearing de novo typically are extremely rare events per locus, but collectively are numerous. other types of genetic variability, such as copy number repeats, span both ends of the frequency spectrum with the preponderance of the markers being rare. thus, there is a sizable pool of low-frequency variants in human populations that have yet to be thoroughly investigated. over the past few years it has become increasingly clear that structural variants exist in the human genome at a far higher rate than previously thought. structural variants can exist in a multitude of forms including deletions, copy number variants, and inversions among others. due to the nature of these genetic changes, many are considered to be highly disruptive of molecular function if they lie in functional motifs. indeed, there are several mendelian diseases are caused by fully-penetrant structural variants impacting a chromosomal region (lupski ) . numerous structural variants have recently been reported to be associated with common diseases, particularly in the neurological fi eld (sebat et al. ; stefansson et al. ; elia et al. ) , infectious disease susceptibility (gonzalez et al. ) , and drug metabolism (zackrisson et al. ) . although they have improved dramatically over the past few years, algorithms using snp-based data from hybridization arrays to infer copy number variants have had high error rates, perhaps explaining the rather low rates of replication of structural variation association results for common diseases. nevertheless, given the high frequency of structural variants, their pathogenic potential, and that we are on the precipice of a sequencing revolution in genome-wide studies, examination of these variants should be a high priority for new sequencing-based studies in oral disease susceptibility, progression, and related pharmacogenetic applications. as different technologies examine different portions of the site frequency spectrum (i.e. genome-wide snp scans interrogate variation that is common in the hapmap populations, whereas sequencing-based studies typically interrogate the entire frequency spectrum), where one believes genetic causation is harbored should infl uence the selection of genotyping technology. if common genetic variation contains the vast majority of heritable effects on disease phenotypes, then an investigator would be wise to employ a snp-based experimental design. if, however, there is reason to believe that a signifi cant portion of the genetic load of the disease studied exists in the highly populated portion of the distribution -the rare variants -then a sequencing-based study may be better suited to unravel causative alleles. the studies of heritability discussed previously show that there is heritable variation underlying a substantial portion of the variance observed in oral diseases. as discussed above, sequencing technologies may address many aspects of dna variation including copy number loci, rare haplotypes, inversions, and insertions/deletions, but it is also worthwhile to repeat that the molecular mechanisms for disease heritability are not necessarily limited to variation at the dna level. for a disease state, the covariance between relatives could be driven by co-inherited chromosomal regions or other phenomena. chief alternative heritable mechanisms include dna methylation (hammoud et al. ) , modifi cations to the histones (bestor ) , complex rna zygotic transfer (rassoulzadegan et al. ) , and vertical transmission of infectious agents. additionally, transgenerational effects offer an intriguing class of epigenetic mechanisms (nadeau ) . in a thorough review on epigenetics and periodontitis, gomez et al. make a strong argument for consideration of both cpg dinucleotide methylation and deacetylation actions on cytokine expression as a credible avenue for further investigation in periodontal disease etiology (gomez et al. ) . genome-wide epigenetic studies have been successfully conducted for oral cancers (poage et al. ) . the scale of this study on head and neck squamous cell carcinomas allowed these researchers to show a global pattern of tumor copy number changes signifi cantly correlated with methylation profi les that was not detectable at the individual gene promoter level. with advanced chromatin immunoprecipitation and new methods to study dna methylation, efforts to apply highthroughput epigenetic methods to oral diseases should be accelerated. numerous studies have been conducted in oral disease traits using a candidate gene approach. there are two large reviews of the existing candidate gene results (nikolopoulos et al. ; ) . laine and colleagues have recently put together a comprehensive review article covering gene polymorphisms. there are some suggestive fi ndings for cyclooxygenase- gene, cox- , the cytokineencoding genes, il and il b , the vitamin d receptor, vdr , a polymorphism immediately upstream of cd , and the matrix metalloproteinase- gene, mmp . however, these initial results will require further confi rmation, for the association patterns are inconsistent across independent studies, the statistical signifi cance is moderate, and the posterior probability of disease is decidedly bland. the striking pattern that emerges from the laine et al. summary data is the lack of coherent replication of genetic association for the vast majority of polymorphisms examined. the situation is reminiscent of genetic association studies prior to large-scale snp studies where poor repeatability of results plagued the fi eld. in a pivotal study from , hirschhorn and colleagues (hirschhorn et al. ) examined the state of genetic association studies, fi nding that "of the putative associations that had been studied three or more times, only six have been consistently replicated." the dearth of robust results was largely remedied when large-scale genetic studies were applied to very substantial numbers of well-characterized patients and geneticallymatched controls and stringent statistical criteria enforced. one can only suspect that a similar state of affairs is operating in genetic studies of chronic periodontitis. perhaps efforts to ( ) reduce the heterogeneity of the disease state through detailed clinical and laboratory assessments, ( ) drastically increase sample sizes, and ( ) expand the scope of inquiry to larger numbers of genes/regions, and examine a more comprehensive set of variants/epigenetic effects will improve the current situation. the second large study is a meta-analysis of studies, where nikolopoulos and colleagues analyzed six cytokine polymorphisms linked to il a , il b , il , and tnf-alpha (nikolopoulos et al. ) . two of these, an upstream snp in il a and a snp in il b , exhibited signifi cant association with chronic periodontal disease risk. although the results were not particularly strong, as is typical with complex diseases, the results do suggest the importance of infl ammation-response variability in chronic periodontitis predisposition. perhaps the strongest, most replicable genetic association fi nding with coronary heart disease and myocardial infarction is centered on the short arm of chromosome ( p . ) (mcpherson et al. ; helgadottir et al. ) . two studies of periodontal disease showed that the same alleles at the p . locus confer risk for aggressive periodontitis (schaefer et al. ; ernst et al. ) . the discovery of such a pleiotropic locus may explain a portion of the aggregation of periodontal disease with other co-morbid conditions. further studies investigating overlapping genetic susceptibility factors between periodontitis and cardiovascular disease, diabetes mellitus, metabolic syndrome, rheumatoid arthritis, and other related diseases may be a fruitful strategy for honing in on shared genes affecting these immuno-metabolic disorders. using patients from families from the philippines, the fi rst genome-wide linkage study for caries was completed in (vieira et al. ) . the study identifi ed fi ve loci which exhibit suggestive statistical evidence (lod scores exceeding . ): q . , q . , xq . , q . , and q . . the latter of which overlapped with a quantitative trait locus discovered from mapping work in the mouse. further work is necessary to refi ne these signals and localize the variants that may be driving these linkage signals. aggressive periodontal disease and rarer dental diseases have also been subjected to linkage analysis. results from linkage studies for dentinogenesis imperfecta type i, for example, have gone on to produce the novel gene fi ndings of the dentin sialophosphoprotein-encoding gene on q . being responsible (song et al. ; crosby et al. ) . a linkage study in african american families examining localized aggressive periodontitis found a strong linkage signal in a region covering approximately megabases on chromosome (li et al. ) . several interesting genes are in this region. in a study earlier this year further mapping from carvalho et al. in brazilian families identifi ed haplotypes in this region on q in fam c which were associated with aggressive periodontitis (carvalho et al. ) . the function of the fam c protein is not fully understood. fam c is localized in the mitochondria and it appears to play a role in vascular plaque dynamics and risk of myocardial infarction (laass et al. ) . it should also be noted here that other types of mapping analyses such as homozygosity mapping to identify have yielded gene discoveries. for example, the lysosomal protease cathepsin c gene for the recessively-inherited papillon-lefevre syndrome which is characterized by aggressive and progressive periodontitis was effectively mapped using homozygosity mapping (fischer et al. ; connelly et al. ) . cathepsin c is highly expressed in leukocytes and macrophages and is a key coordinating molecule in natural killer cells (rao et al. ; meade et al. ) . although sparse, these linkage results are undoubtedly encouraging. employing very large extended families subjected to genome-wide genotyping or sequencing will surely shed much needed light on chromosomal regions and genes relevant to oral disease research (fig. . ). for periodontitis, a single study has employed a genome-wide association design in an effort to uncover aggressive periodontal variants (schaefer et al. ) . this study by schaefer and colleagues discovered and replicated an intronic snp, rs , in the glycosyltransferase glt d which is signifi cantly correlated with aggressive periodontal disease in both german and dutch samples. often, seemingly signifi cant results from large studies are due to the effect of reporting the top result from a great many statistical tests -this is called the multiple testing problem. in this situation, the strength of the fi nding, along with the replication across three case/control studies, argues for true association with aggressive periodontal susceptibility. the snp may modulate the binding affi nity of gata- . the association with glt d is currently one of strongest genetic associations for aggressive periodontal disease, testifying to the power of genome-wide studies to generate novel, relevant molecular pathophysiology for complex diseases. it seems unlikely that glt d would be extremely high on a candidate gene list, and it was only through a genome-wide scan that it appeared. like many excellent studies, the fi nding by schaefer et al. raises more questions than it answers and will undoubtedly provide fertile ground for ensuing molecular work. after a somewhat sluggish start, due to a lack of critical mass of investigators aiming to collect large numbers of patient samples and bring high throughput genetic technologies to caries susceptibility, gingivitis, and periodontal disease traits, the future of genetic studies in oral health is bright. scientifi c progress in revealing the molecular pathogenesis of oral diseases is dependent on genome-wide genetic studies; and i have argued that progress in related immuno-metabolic diseases is also dependent on these large-scale genetic studies in periodontal disease. to study sporadic disease, substantial patient collection efforts are required for the application of these technologies. this may involve a combination of new recruitment and consortiumrelationships with existing collections. the beginning of such a collection for sporadic aggressive periodontitis in europe has shown extremely intriguing initial results, but more patients are needed to examine rare variants of moderate effect. both the german/dutch collection of aggressive periodontitis and the brazilian collection have begun to revolutionize the study of periodontal disease susceptibility with the discovery of glt d snps and fam c -linked haplotypes. there is little doubt that subsequent molecular work on these two genes will uncover novel mechanisms for the predisposition to aggressive periodontal disease. focus should also be placed on the collection of extended families segregating these diseases. applying sequencing technologies to large pedigrees can be an effective method of identifying rare variants and structural variants in a highly-refi ned phenotype. furthermore, applying these methods to the entire genome would make for a comprehensive genetic study. several trends in large-scale genomics science hold promise to signifi cantly advance our understanding of oral disease pathogenesis: the sociology of biological sciences has changed over the past years so as to • become more collaborative. essential for association-based designs, consortiumbased genetic research has blossomed over that time period, increasing sample sizes and therefore the power to detect disease-causing variants. there currently is consortium-based research in periodontal disease and oral cancer. further expanding these efforts will enhance subsequent studies, particularly those investigating rare alleles and/or rare epigenetic effects. through over a century of laboratory work, the collective knowledge of bio-• chemical pathways, signal transduction, cell physiology, regulatory mechanisms, and structural biochemistry is weighty. incorporation of this information into etiological models may substantially advance oral disease work as well as the fi eld of complex disease genetics in general. sophisticated analysis techniques are needed to perform this task. recent advances merging results from network science with probability theory within the context of computer science have produced the fi eld of machine learning. this rigorous framework can be used to identify those factors responsible for disease status and can also be used to develop robust predictive models using known biological networks and genetic data. the output from such models, typically the probability of disease, an estimate of disease progression rate, or a probability of adverse reaction, can be used by physicians and dentists to personalize medical care. until relatively recently, population genetics did not contribute a great deal to • human genetics research. that has changed in the past decade where effort spent on association studies surpassed that spent on family-based studies. those investigating disease gene mapping began to collaborate with population geneticists and population geneticists took up a wide-spread interest in fi nding disease alleles. incorporation of population genetics theory into such studies markedly improved association studies on several levels: confounding by population stratifi cation was effectively treated using population genetics, linkage disequilibrium patterns. use of population genetics theory in large-scale oral disease mapping studies may accelerate discoveries. sequencing technology has rapidly progressed over the past decade. currently, • sequencing studies across the exome can be accomplished at reasonable cost and yield data for all known genes in the genome. within the next few years, sequencing costs will depreciate to a point where whole-genome sequencing studies will be commonplace, using both family-based and population designs. application of these technologies to oral disease studies is imperative for comprehensive studies of etiology. high-throughput dna methylation and chromatin immunoprecipitation studies • will enable large-scale epigenetic studies in oral diseases (meade et al. ; ehrich et al. ; bibikova et al. ; ren et al. ; pokholok et al. ) . these have already started to play an important role in delineating mechanisms responsible for oral cancers (poage et al. ) . additional application of these techniques to studies of gingivitis, caries, and periodontal diseases may generate novel fi ndings. molecular biologists and pharmacologists have increasingly become able to • develop and evaluate highly targeted pharmaceuticals based on genetic discoveries. the use of such genetic information may improve the chances of developing effi cacious therapies. geneticists and disease researchers are beginning to realize that oral diseases • both impact and are intrinsically tied to susceptibility and progression of other common diseases. a synthesis of genetic fi ndings from immuno-metaboliclinked disorders would seem to greatly increase the knowledge of these diseases and better pinpoint their respective etiologies. as the new high-throughput genomics and epigenomics technologies become • implemented in oral disease research, the storage, management, analysis, and interpretation of the ensuing colossal amounts of data will be critical to enable clinicians to use these results in daily practice. advances in dental and medical informatics will facilitate these steps. we are in exciting times where advances in genetic technologies will uncover the genetic causes of diseases, including those that affect the oral cavity. with more focus in the area of oral disease genomics and the harnessing of new high-throughput sequencing and epigenetic technologies, novel insights into the pathways driving these diseases are imminent. these discoveries will, in turn, motivate directed therapies, aid in illuminating the molecular etiology of related disorders such as diabetes, and increase the level of personalized medicine. joseph kilsdonk the title of this section reinforces a institute of medicine (iom) report titled "dental education: at the crossroads." to quote yogi berra, a baseball sage: "when you come to a fork in the road, take it." the implication being that dental education must take action and move beyond its crossroads. these crossroads are described in the fi rst third of the section. it includes a summary and recommendations of the iom report and three transitional reports that followed: the surgeon general's report identifying oral health as a silent epidemic, the josiah macy foundation report, and a "pipeline" study funded by both the robert wood johnson and the california foundations. having been at the crossroads for a decade or so, the middle portion of the section highlights educational models that may lead to a more promising future. the later third of this section describes an alternative path of action for dental education which emphasizes the central roles of clinic-based education and dental informatics in dental education curriculum. it is unknown how traditional dental educators may view this model; however, it is effectively a logical conclusion and responsive to the reports. in the institute of medicine (iom) published "dental education at the crossroads" (field ) . the title was apropos as the authors' analysis concluded: ( ) economics surrounding dental education were unsustainable ; ( ) student service learning opportunities and access to care for patients were limited; and ( ) new dental schools were not replacing those forced to close due to the economic climate. the iom report additionally proposed key recommendations to reform dental education and service delivery. fifteen years later, we remain at "the crossroads" as these issues remain largely unresolved. furthermore, these recommendations have retained their validity. their implementation would directly impact structures and services for contemporary models of dental education in the future. the following iom recommendations (field ) are intrinsic to the proposed dental education reform: recommendation : to increase access to care and improve the oral health status of underserved populations… recommendation : to improve the availability of dental care in underserved areas and to limit the negative effects of high student debt… recommendation : to prepare future practitioners for more medically based modes of oral health care and more medically complicated patients, dental educators should work with their colleagues in medical schools and academic health centers to: move toward integrated basic science education for dental and medical • students; require and provide for dental students at least one rotation, clerkship or • equivalent experience in relevant areas of medicine and offer opportunities for additional elective experience in hospitals, nursing homes, ambulatory care clinics and other settings; continue and expand experiments with combined md-dds programs and • similar programs for interested students and residents; increase the experience of dental faculty in clinical medicine so that they, • and not just physicians, can impart medical knowledge to dental students and serve as role models for them. recommendation : to prepare students and faculty for an environment that will demand increasing effi ciency, accountability, and evidence of effectiveness, the committee recommends that dental students and faculty participate in effi ciently managed clinics and faculty practices in which the following occurs: patient-centered, comprehensive care is the norm; • patients' preferences and their social, economic, and emotional circumstances • are sensitively considered; teamwork and cost-effective use of well-trained allied dental personnel are • stressed; evaluations of practice patterns and of the outcomes of care guide actions to • improve both the quality and the effi ciency of such care; general dentists serve as role models in the appropriate treatment and referral • of patients needing advanced therapies; larger numbers of patients, including those with more diverse characteristics • and clinical problems, are served. recommendation : because no single fi nancing strategy exists, the committee recommends that dental schools individually and, when appropriate collectively evaluate and implement a mix of actions to reduce costs and increase revenues. potential strategies, each of which needs to be guided by solid fi nancial information and projections as well as educational and other considerations, include the following: increasing the productivity, quality, effi ciency, and profi tability of faculty • practice plans, student clinics, and other patient care activities; pursuing fi nancial support at the federal, state, and local levels for patient-• centered predoctoral and postdoctoral dental education, including adequate reimbursement of services for medicaid and indigent populations and contractual or other arrangements for states without dental schools to support the education of some of their students in states with dental schools; rethinking basic models of dental education and experimenting with less • costly alternatives; raising tuition for in or out-of-state students if current tuition and fees are low • compared to similar schools; developing high-quality, competitive research and continuing education • programs; consolidating or merging courses, departments, programs, and even entire • schools. in summary, the iom report identifi ed that: ( ) an outdated curriculum continues to be retained which refl ects past dental practice rather than current and emerging practice and knowledge; ( ) clinical education does not suffi ciently incorporate the goal of comprehensive care, with instruction focusing too heavily on procedures; ( ) medical care and dentistry are not integrated; and ( ) the curriculum is crowded with redundant material, often taught in disciplinary silos. the iom's report was followed by the surgeon general's report on oral health in and a subsequent supplement by the surgeon general in called "the national call to action" (u.s. department of health and human services ) . five signifi cant fi ndings and recommendations from the surgeon general's report(s) that have implications pertaining to the envisioned structure and services of new models for dental education include: changing the perception of oral health so that it will no longer be considered • separate from general health; improving oral health care delivery by reducing disparities associated with popu-• lations whose access to dental treatment is compromised by poverty, limited education or language skills, geographic isolation, age, gender, disability, or an existing medical condition; encouraging oral health research, expanding preventive and early detection pro-• grams, and facilitating the transfer of knowledge about them to the general population; increasing oral health workforce diversity, capacity, and fl exibility to overcome • the underrepresentation of specifi c racial and ethnic groups in the dental profession. in this regard, the national call to action urged the development of dental school recruitment programs to correct these disparities and to encourage parttime dental service in community clinics in areas of oral health shortage; increasing collaboration between the private sector and the public sector to cre-• ate the kind of cross-disciplinary, culturally sensitive, community-based, and community-wide efforts to expand initiatives for oral health promotion and dental disease prevention. spurred by the iom report and the surgeon general's report, the josiah macy foundation ( ) conducted a study entitled "new models of dental education." the study was prompted by concerns about declines in dental school budgets and the diffi culties experienced by schools in meeting their educational, research, and service missions. the macy study concluded that: financial problems of dental schools are real and certain to increase. • current responses of schools to these economic challenges are not adequate. • most promising solutions require new models of clinical dental education. • macy study lead researcher dr howard bailit, and his team recently concluded in reference to points one and two above, that: "if current trends (to aforementioned) continue for the next years, there is little doubt that the term crisis will describe the situation faced by dental schools. further, assuming that it will take at least ten or even more years to address and resolve these fi nancial problems, now is the time for dental educators, practitioners, and other interested parties from the private and public sectors to come to a consensus on how to deal with the coming crisis. clearly, these fi nancial problems will not be solved by minor adjustments to the curriculum, modest improvements in the clinical productivity of students or faculty, or even signifi cant increases in contributions from alumni. the solutions 'must involve basic structural changes in the way dental education is fi nanced and organized' (bailit et al. ) ." this statement is supported by the fact that in the past years more dental schools have closed than opened. specifi cally eight schools have closed, whereas to date a couple has opened and a handful is pending. curriculum relevance was also a focus of the study. findings concluded that "changing the curriculums in dental schools to allow students to spend more time in community venues would be highly benefi cial to both society and student. society benefi ted from having underserved patients cared for while students were assessed as being fi ve to ten times more productive, more profi cient, more confi dent, more technically skilled and more competent in treating and interacting with minority patients" (brodeur ) . macy study (formicola et al. ) outcomes represented signifi cant and foundational guideposts for assessing and planning any future models for dental education. their report led to the robert wood johnson foundation pipeline study ( ) , a major research study funded by the robert wood johnson foundation and the california endowment (tce). the goal of the dental pipeline program was to reduce disparities in access to dental care. the pipeline study provided over $ million for the start up or expansion of schools and student clinical programs that incorporated services to underserved extramural clinical settings (primarily community health centers). the following recommendations from the surgeon general's report structured • the goals of the pipeline's initiative: increase the number of under-represented minority and low-income students enrolled in the dental schools participating in the pipeline program so that there would be a voice of minority and low-income students at all the funded schools. provide dental students with courses and clinical experience that would prepare • them for treating disadvantaged patients in community sites. have senior dental students spend an average of days in community clinics • and practices treating underserved patients. increasing the community experience of dental students was expected to have an immediate impact on increasing care to underserved patients (brodeur ) . this third point is pivotal to future success of dental curricula and dental education economics. recently published in a supplemental volume to the journal of dental education , february, , the pipeline study reported the following outcomes: minority recruitment of low-income students increased by %; • the rate of recruitment for under-represented populations was almost twice that • of non-pipeline schools; the length of time dental students spent in extramural rotations increased from a • mean of days to a mean of days over a period of years. procedural profi ciency increased compared to that of their non-extramural peers. of the pipeline-funded programs, only four schools achieved the goal of • days of extramural rotations; through extra funding from tce, the four schools extended extramural rotations to an average of days; based on this publication, it appears that only a handful of pipeline schools defi -• nitely plan to sustain their extended extramural rotations. financial concerns were highlighted as the major problem in sustaining future recruitment and placement of students beyond the timeframe of the study; a survey of program seniors indicated a mean of % [range of - % by • school] were planning to devote ³ % of their practice to serving minority patients. only % [range of - % by school] were planning to practice at community clinics. in the context of these outcomes, discussion indicated that the unwillingness of students to practice in underserved settings was based on several factors: students that participated were already enrolled in traditional programs and were • not necessarily seeking a pipeline experience or a future in community service. concern over future reimbursement as a provider in a community setting; • limited time spent in underserved settings; • limited loan forgiveness scholarship opportunities. • the fact that the large majority of pipelines were unsustainable was attributed to lack of productivity in the school clinics while the students were on rotation at community based clinics. schools generate meager, yet necessary revenue streams on intramural student clinical activity to support the costly clinical and faculty infrastructure. currently, similar economic constraints involved with outsourcing students to serving rural and underserved populations impacts the ability of tradition dental schools to participate in sustained outreach programs. most recently, the pew center on the states national academy for health policy ( ) released "help wanted: a policy makers guide to new dental providers". this report provided an excellent summary outlining workforce needs, access issues, and strategies for dental-related services to help states and institutions develop creative ways to solve oral health access and care issues. the guide proposes the following relevant components and trends for consideration in development of future sustainable school models: dental colleges are willing to bear a large and disproportionate share of the burden • in terms of access to care, particularly during a time of incredibly scarce resources. expanded, extensive, and/or creative extramural rotations have been developed • in recent years under the conceptual umbrella of service-learning. these often involve clinics providing direct or indirect payment to dental schools or clinics managed in some way by dental schools. dental education has certain obligations. first, education must adhere to accreditation standards with the goal of producing competent practitioners. second, education must remain responsive and impact the societal need for care. lastly, the delivery of dental education must be economically sustainable. the macy, rwjf, and iom reports note that improved oral health, sustainable dental education economic models, and competent workforce pipelines converge around community health centers (chc). university of michigan researchers fitzgerald and piskorowski ( ) reaffi rm this conclusion in an evaluation of an ongoing -year program, stating that: (the chc model) is self-sustaining and can be used to increase service to the underserved and increase the value of students' clinical educational experiences without requiring grant or school funding, thus improving the value of dental education without increased cost. self-sustaining contracts with seven federally qualifi ed health centers (fqhcs) have resulted in win-win-win-win outcomes: win for the underserved communities, which experienced increased access to care; win for the fqhcs, which experienced increased and more consistent productivity; win for the students, who increased their clinical skills and broadened their experience base; and win for the school in the form of predictable and continuing full coverage of all program costs (fitzgerald and piskorowski ) (fig. . ). however, unlike medicine that outsources their students to clinical sites, dental education programs retain the majority of the student time within their own "clinical laboratories" as documented by the aforementioned studies, this limits students' exposure to extramural experiences. costs to operate such intramural clinical programs are ever increasing and many schools' clinical operations run defi cits. if that component can be outsourced to community-based resources such as a chc, then the burden of cost is shifted away from the school. an example would be a.t. still university's arizona school of dentistry and oral health (asdoh) which matriculated its fi rst class in . at the prompting of the state's community based clinics, asdoh designed a program that placed students into community-based settings for up to months, an unprecedented length of time for an extramural rotation. they also saw this as an opportunity to use an adjunct centric faculty that signifi cantly reduced traditional education overhead. through this innovation, the school was able to develop a program that was sustained by "fair market" value tuition and trained students where community needs were greatest for up to months (which was then unprecedented). conversely, if the chc can rely on student service-learning to care for patients, the cost of care is reduced. other schools are also advancing with innovative education and care delivery. adea's charting progress (valachovic ) fig. . the synergy between access to care, student competency, and fi nancially sustainable dental education converge around chc/fqhcs little rock, arkansas; and the university of southern nevada in south jordan, utah. western university is planning placement of % of their fourth year class in community health centers, while east carolina is seeking to set up rural clinical campuses as well as clinical partnerships with the state's fqhc. at the time of this publication, several existing schools are expanding or looking to expand including the university of north carolina, marquette university, midwestern university in downer's grove, il. such expansions will contribute to solving the existent access supply and demand issues. however, it was observed even with all the start ups and expansions, graduation numbers will not approach the output of schools in the late s and early s. these creative models establish the foundation for a sustainable clinic structure by generating self-sustaining revenue through student service-learning, which, unlike medical student services, are billable. simultaneously these new models provide access to care for the needy while student exposure to clinical experiences that are often not available in academic patient pools. these models also shift some of the cost of providing clinical education from the dental college to community-based clinics. however, this innovation is not without criticism. schools are dependent on the success of their clinics and clinic partnerships. one author cautions: "however, these creative models also may present potential political strategic risk or confl ict: private practitioners may organize and protest higher than normal reimbursement schemes. potentially, such protests could even jeopardize the very existence of such models (dunning et al. ) ." notably, community health centers have historically received strong bipartisan support. for example, during the bush administration, fqhc funding was doubled and most recently expanded through health reform legislation by the obama administration. according to the institute for oral health, "the group practice of the future is the dentist working with the physician" (ryan ) . the ada reported "multidisciplinary education must become the norm and represent the meaning and purposes of primary care as it applies to dentistry. educational sequences should include rotation strategies across discipline specialties in medicine and dentistry, clerkships and hospital rotations, and experience in faculty and residency clinics." (barnett and brown ) the models alluded to, were school-based attempts at improving educational outcomes. perhaps the proverbial fork in the road regarding the future of dental education leaves two paths for consideration. is it better to travel down a road that leads a school to develop and operate a clinic? or is the road less traveled, where a clinic becomes a school, the better of the two options? the answer, perhaps, is that a combination of both will accomplish the desired outcome. for example, didactic knowledge is measured by examination whereas competency as a practitioner is measured by clinical demonstration. at a minimum, the result must achieve learner competency, quality, and sustainability. however, the road less traveled has not been taken yet. william gies, in his revered report written years ago on the state of american dental education, wrote "dental faculties should show the need…. for integrated instruction in the general principles of clinical dentistry and in its correlations with clinical medicine" (gies ) . basic sciences aside, could a clinicalbased educational training center have an advantage over a school-based clinical center? soon-to-be-implemented new commission on dental accreditation (coda) standards will require schools to demonstrate competency in patient-centered care (valachovic ) . might an enterprise profi cient at running a successful clinical business model have an advantage running a professional, patient-centered clinical training program as compared to a pedagogical business model attempting to run a clinical training model? these questions should challenge us to reexamine why our thinking about educational models should be limited to schools being the starting point for the development of a profession that demands clinical competency, patientcenteredness, and integration as outcomes. the clinic based model may serve as an equivalent starting point and, have some distinct advantages for achieving responsiveness to recommendations and directions cited in this section. beginning in november through august , the family health center (fhc) of marshfi eld, inc, marshfi eld, wisconsin, launched of a broad network of developing dental clinics, targeting dental professional shortage areas with the provision of dental services to the underserved communities whose dental needs were not being adequately met by the existing infrastructure. fhc-marshfi eld is a federally qualifi ed health center (fqhc). as an fqhc, fhc receives cost-based reimbursement for its dental services to medicaid populations. along with the cost-based reimbursement, fqhcs are obligated to provide care to anyone regardless of their ability to pay. presently, fhc is the nation's largest federally qualifi ed dental health center. to date, this network of dental clinics has served over , unique patients, % of whom were under % of poverty. notably, service was provided to a signifi cant number of cognitively and developmentally disabled patients in special stations developed for serving patients with special needs. these patients frequently travel the furthest to get to our dental centers for care. beginning in , fhc stepped up the pace of dental clinic expansion, constructing two new dental centers in , two in , and two more are slated to open in . when fully operational, this will establish capacity to serve , patients annually. each site has proactively included dedicated clinical and classroom training space for dental residents or students, thus laying the framework for clinic-based training of new dental professionals. the plan is to continue to stand up new dental centers until they have the capacity to serve , patients annually or approximately % of the , underserved patients in the rural service area. in addition to the capacity for training residents and students, a dental post-baccalaureate program is being considered in partnership with regional year under graduate campuses. the post-baccalaureate program is aimed at preparing students from rural and underserved areas who desire to practice in rural and underserved areas for acceptance and success in dental schools. presently fhc in partnership with marshfi eld clinic is moving forward with plans to develop dental residencies at these sites and a dental post baccalaureate training program to better prepare pre-doctoral students from rural and/or underserved backgrounds to be successful in dental school as a means to create a dental academic infrastructure responsive to rural environments which have been classically underserved. marshfi eld clinic has a long-standing history in medical student education and multiple medical residency programs. creating access for the underserved population was the major motivational force driving the establishment of the dental clinic network back in . the fi ndings of the iom, macy, and rwjf reports became the foundational framework for developing the vision of a dental education model that would realize the major recommendations found in the reports. by establishing clinical campuses in regional underserved dental health professional shortage areas, access to care where care is needed most was provided. sustainment of a work force for provision of care across the dental clinic network is accomplished by schools contracting with fqhc's for service learning, thus circumventing challenges associated with releasing dental students at traditional dental schools to distant extramural training sites as discussed previously. this model is however not without its own set of challenges including calibration of faculty, supervision and evaluation of students in training, and achieving accreditation acceptance. however, through video connectivity and iehr technology curriculum, learning plans, competency assessment, progression, performance, faculty development, and learner evaluations can be centrally calibrated. additionally, this dental service-learning model based in a community health center setting offers students unique state-of-the-art exposures to alternative access models, cutting-edge informatics (including access to a combined dental-medical record) and a quality-based outcomes-driven practice. given the novelty of such an extended extramural dental clinical training model, there is limited data on the success of rural placement leading to retention to practice in a rural setting. the pipeline study piloted a model for getting students into underserved communities. however, that experiment was limited to -day rotations. outcome driven programs may provide a predictive surrogate for purposes of comparative analysis. for example, the rural medical education "rmed" program of the university of illinois medical school at rockford, has sustained a longstanding program in illinois. over years in duration with over student participants of whom % have been retained as primary care medicine practitioners in rural illinois. rabinowitz et al. ( a ) further reinforced that medical school rural programs have been highly successful in increasing the supply of rural physicians, with an average of - % of graduates choosing to practice in rural areas. they also noted rural retention rates of - % among the programs (rabinowitz et al. a) . recently, the university of wisconsin school of medicine and public health (uwsmph) launched the wisconsin academy for rural medicine (warm program). the warm program places medical students in rural academic medical centers during their third and fourth years in medical school. marshfi eld clinic is one of those sites. warm students affi liating with marshfi eld clinic's system would ultimately share learning experiences with dental students, clinical rotations, team-based rounding, lectures, and exposure to a combined medical-dental patient record. in an analogous manner, the marshfi eld clinic dental education model will incorporate a curriculum that embeds students in rural clinical practice for up to years. a secondary but not insignifi cant outcome of placing residents and students in clinical campuses focused on developing competency and providing care where needs are often greatest is the cost savings to taxpayers associated with the public care of patients. these savings are accomplished through the "service-learning" of the student. for example, in the model described where clinical training is embedded within the fhc clinics, the stipend resident or unpaid student learner provides the patient care as part of their service learning training while requiring oversight from one paid faculty per four to six learners. as a result, an academic based clinical partnership creates a model that reduces the cost for care provided to underserved patients. an additional benefi t to the community based clinic might be realized through tuition assistance by the academic program to help support patient procedures that develop learner competencies. in educational quality and infl uence, dental schools should equal medical schools, for their responsibilities are similar and their tasks analogous (william gies ) . the commission on dental accreditation (coda) notes that one of the learning objectives of an advanced education general dentistry (aegd) residency is to have the graduate function as a "primary care provider". to function competently in this role, the graduate needs to have a strong academic linkage to primary care medicine. at a dental deans forum, years after the gies report, dr polverini made the statement "dentistry has never been linked to the medical network but unless dentistry becomes part of the solution to the challenge of providing comprehensive patient care, it will be looked on as part of the problem, and ultimately, all dental schools will be called into question." (polverini ) the use of dental informatics and an integrated record are elements essential to this competency. on april , , fhc and marshfi eld clinic successfully transitioned all of their dental centers to a new practice management and electronic health record system that fully integrates medical and dental; one of the fi rst such systems in the nation. along with the benefi ts derived in fig. . , chc placement also exposes students to an integrated medical-dental care setting where learners can develop skills in system-based practice to include the interdependence of health professionals, systems, and the coordination of care. on the administrative side, dental and medical appointments can be coordinated to enhance convenience for patients and improve compliance with preventive dental visits. in , marshfi eld clinic's research foundation biomedical informatics research center hired their fi rst dental informatician, dr. amit acharya, bds, ms, phd. with dedicated biomedical informatics and research resource centers, the marshfi eld clinic has laid the groundwork for true medical/dental integration with appropriate electronic health record decision support and is positioned to develop a dental education curriculum capable of implementing the iom recommendations. downstream benefi ts of using such a curriculum are the ability of future practitioners to use informatics to improve quality of care and reduce the burden of disease. according to an institute of oral health report ( ) it is widely accepted across the dental profession that oral health has a direct impact on systemic health, and increasingly, medical and dental care providers are building to bridge relationships to create treatment solutions. as early as , william gies recognized that "the frequency of periodic examination gives dentists exceptional opportunity to note early signs of many types of illnesses outside the domain of dentistry" (gies ) . the following examples show how integration of dental and medical care can impact patient outcomes, underlining the importance of this concept in dental curriculum design. a study of , blue cross blue shield of michigan (bcbs) members with diabetes, who had access to dental care lead researchers, and bcbs executives to conclude that treatment of periodontal disease signifi cantly impacts outcomes related to diabetes care and related costs (blue cross blue shield of michigan ) . another example is found in the context of preterm delivery and miscarriage. according to research cited by cigna ( ) , expecting mothers with chronic periodontal disease during the second trimester are seven times more likely to deliver preterm (before th week), and have dramatically more healthcare challenges throughout their life. cigna also cites the correlation between periodontal disease and low birth weights, pre-eclampsia, gestational diabetes. equally important is the opportunity to develop and implement the team-based curriculum that trains future dentists and physicians in the management of chronic disease as an accountable care organization (aco) in a patient-centered environment. as an example, joseph errante, d.d.s., vice president, blue cross blue shield of ma, reported that medical costs for diabetics who accessed dental care for prevention and periodontal services were signifi cantly lower than those who didn't get dental care (errante ) . these data suggest that team based case management of prevalent chronic health conditions have considerable cost savings opportunities for government payers, third party payers, employers and employees (errante ) . these economic benefi ts to integration as it relates to the iehr are discussed elsewhere in this book, but begin with the ability of providers to function in a team based environment and as such, underscore the importance of training in such an environment. dentists trained in a fqhc iehr integrated educational model will be well positioned to function successfully within an aco model. an aco is a system where providers are accountable for the outcomes and expenditures of the insured population of patients they serve. the providers within the system are charged with collectively improving care around cost and quality targets set by the payor. within this system, care must be delivered in a patient-centered environment. the patient-centered environment according to the national committee for quality assurance (ncqa), is a health care setting that cultivates partnerships between individual patients and their personal physicians and, when appropriate, the patient's family. care is facilitated by registries, information technology, health information exchange and other means to assure that patients receive defi ned, timely and appropriate care while remaining cognizant of cultural, linguistic and literacy needs of the patient being served. the model includes the opportunity to deliver patient care that is patient-centric, incorporates the patient in the care planning, considers the patient's beliefs and views, and incorporates the patient's families as needed. the model allows providers to deliver care that is inclusive of needs, attentive, and accessible. the model equips payers to purchase high quality and coordinated care among teams of providers across healthcare settings. while this describes the medical home, most dental practices also follow this process. many dental practices function in this regard with insured populations and refl ect elements of the model that medicine is creating. william gies would be proud. training in the delivery of accountable and patient-centered medical-dental care must be done purposefully. commenting on the inadequate training relative to the integration of medical and dental education, baum ( ) stated that "we need to design new curricula with meaningful core competencies for the next generation of dentists rather than apply patches to our existing ones." while this statement was made in reference to the basic sciences, the same holds true for patient-centered system-based practice competencies. utilizing state-of-the-art electronic medical records as a tool and the fhc infrastructure as the service venue, meaningful patient-centered system-based practice core competencies achievement becomes possible in a manner highly responsive to societal needs. by defi nition, fqhcs must provide primary medical care, dental care, and behavioral health. fqhc have also historically been utilized as healthcare workforce training centers and the affordable care act of reinforced their role as healthcare training centers. specifi cally, this legislation serves to promote fqhcs as the entity through which the primary care workforce (including dental) will be developed and expanded. in combination, fqhcs and primary care centers are positioned to be the front runners in a medical/-dental home training model which will be essential to preparing future practitioners for practice in an aco. critical to this success is the ability to train these practitioners on an integrated medical-dental record and informatics platform. use of this platform imprints most strongly during the learner's formative years of training; instructing and guiding disease management, decision making, patient care coordination, prevention, and both outcome-based and comprehensive care. training in this hybrid academically orientated clinically integrated setting moves dental education off its crossroads and creates the highway to its future. concerns with the new models extend to their ability to integrate medical and dental disciplines at the clinical and informatics level. while the iom report identifi ed the need to integrate medical and dental curriculum, success at the curricular and technological level within schools, has been limited. three major factors have contributed to the limited progress: access priorities. creating access to care has outranked the need to integrate • care. in part, this refl ects societal need for care and public demand to reduce the burden of the "silent epidemic." schools play an important role as a safety net to care for the uninsured and underinsured through intramural clinical service learning. even though "dental colleges seem to be willing to bearing a large and disproportionate share of the burden in terms of access to care" (dunning et al. ) , schools were challenged as part of iom, surgeon general, and macy reports, to expand that role. while these reports have prompted creative educational solutions to increase access, the reports understate the tremendous opportunity, quality and cost benefi ts that could result from an integration of medicine and dentistry. it is diffi cult to change the culture and structure of existing schools. this is not • unique to dentistry. however, the iom report specifi cally recommended that schools "eliminate marginally useful and redundant courses and design an integrated basic and clinical science curriculum". the challenges with this are many. examples include: some schools may not have other disciplines to draw from to create an inte-grated curriculum; a number of schools use a faculty senate to determine curriculum. this can result in curriculum that preserves the current faculty structure; changing curriculum is associated with expense and can be fi nancially pro-hibitive to some schools physical changes may be needed and represent an expense and/or may, in some instances, may not be practicable based on structure of existing facilities. public school programs may direct the fi nal curriculum, as boards or regent's one or two steps removed from the curriculum often have fi nal authority conversely, private schools may specify business or mission objectives that determine fi nal design. perhaps most germane to this text is the lack of a common technology plat-• form between disciplines in a learning environment. an integrated curriculum requires an integrated platform to accomplish delivery and evaluation. this is particularly essential to clinical management of the patient by professionals in training as part of a healthcare delivery team. some progress in establishing shared basic sciences curricula has been documented in the literature. to date, no single integrated electronic health (medical-dental) record has been meaningfully adapted for educational purposes, including incorporation of assessment of the learner relative to integrated competencies, integrated case-based and problem-based curriculum, and integrated evaluation and assessment. another concern with new educational programs emerging in response to these reports and relative to creating a transformational integrated curriculum is that some of the programs are focusing primarily on creating clinicians with no value or emphasis on integrating training with research and/or scholarly activity. integrated training models counter such concerns. research will be fundamental to measuring the relative benefi ts and outcomes associated with treatment of patients in a shared curriculum setting and will be the catalyst for the development of integrated medical-dental informatics incorporating educational capabilities. additionally, accreditation will also need to evaluate its response to such models. presently it is unclear how accrediting bodies will view an integrated crossdisciplinary curriculum. further, due to its integrated nature, such a curriculum would lie outside of the expertise of a single traditional accrediting body focused on one particular discipline. it has yet to be determined how accrediting bodies will review and appraise such cross-disciplinary competencies. lastly, it is important to recognize that a successful education model with innovative informatics is only successful if its focus is patient care. graduating learners with competency only in the use of informatics will be limited unless adapted to training and delivery programs that result in patient centric care. research and reports over the past years support the need to reform dental education. first steps have been taken and lead the way for continued innovation around clinic-based education and integrated curriculum. the models identifi ed point to a strong partnership and interrelationship with chcs for creative, cost saving, effective and sustainable delivery methods. moreover, chc's must be more involved in a training curriculum integrated with informatics. chcs, in turn, benefi t from residents and students through service-learning to help meet a societal and workforce need, while the learners benefi t from increased competency. in order to train an evidence-based, patient-centered, medical-dental workforce, it is imperative that medical and dental data and record accessibility be incorporated into these training and care delivery initiatives. in order to keep moving away from the crossroads, such integration must become the pathway on which curriculum is developed and implemented. public law - 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environ health perspect doi: nan sha: doc_id: cord_uid: pdnkabwj balkan endemic nephropathy (ben) has attracted increasing attention as a possible environmental disease, and a significant amount of research from complementary scientific fields has been dedicated to its etiology. there are two actual competing theories attempting to explain the cause of this kidney disease: ) the mycotoxin hypothesis, which considers that ben is produced by ochratoxin a ingested intermittently in small amounts by the individuals in the endemic regions, and ) the pliocene lignite hypothesis, which proposes that the disease is caused by long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds leaching into the well drinking water from low rank coals underlying or proximal to the endemic settlements. we outline the current developments and future prospects in the study of ben and differentiate possible factors and cofactors in disease etiology. balkan endemic nephropathy (ben) has attracted increasing attention as a possible environmental disease, and a significant mount of research from complementary scientific fields has been dedicated to its etiology. there are two actual competing theories attempting to explain the cause of this kidney disease: ) the mycotoxin hypothesis, which considers that ben is produced by ochratoxin a ingested intermittently in small amounts by the indivriduals in the endemic regions, and ) the pliocene lignite hypothesis, which proposes that the disease is caused by long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds leaching into the well drinking water from low rank coals underlying or proximal to the enidemic settdements. we outline the current developments and future prospects in the study of ben and differentiate possible factors and cofactors in disease etiology. key workr. aromatic compounds, balkan endemic nephropathy, chronic interstitial nephropathy, environmental disease, ochatoxin, pliocene lignite, urinary tract tumors. environ health perspect : - ( ) . [online october htp:/ellehpnetl.niehs.nih.gov/docs/ / p - tat abstract.html a matter of debate for more than four decades ( ), balkan endemic nephropathy (ben) is a chronic tubulointerstitial nephropathy (cin) described so far in several rural regions of bulgaria, romania, and the former yugoslavia (serbia, croatia, and bosnia). most of the endemic villages are located on alluvial valleys of streams that are tributaries of the danube river, and the area where the disease occurs does not exceed , km ( ) . the topography of the disease sites varies from the flood plains in the former yugoslavia to the undulating hills around drobeta turnu severin in romania and the low montane region of the vratza district in bulgaria ( ) (fig. ). the geographical distribution of the disease has not changed significantly since it was first described in the s. villages afflicted in the past continue to be afflicted today, while nonendemic villages and towns, sometimes located in close proximity to afflicted villages, have remained free of ben. at least , individuals may suffer from ben or are suspected of having the disease, while the total number of people at risk in the three countries may exceed , ( , ) . some of the intriguing features of ben are ) focal occurrence of the clinical cases; ) familial aggregation without an obvious pattern of mendelian genetic inheritance; ) long "incubation" period before the clinical onset of the disease (people coming from outside must reside in the endemic region for more than - years to acquire the disease); ) occurrence only in adults (ages years are most heavily affected, while almost no children or individuals over years of age develop the disease); ) a slight sex distribution, with a female:male sex ratio of about . : ; ) a similar incidence in different ethnic and religious groups; and ) restriction to a rural farming population. of particular interest are the upper urinary tract tumors (utts) seen in ben patients at frequencies much higher than in individuals not suffering from ben from endemic regions or in the general population in the countries with ben. the distribution of these tumors generally follows the geographical distribution of ben, with a steep decrease in incidence in nonendemic villages located even - miles away from the endemic ones. utts are frequently multiple and bilateral and rarely occur in the general population. their high incidence in ben patients led to speculation that the two diseases could be causally related ( ) or that the urinary tract tumors should be seen as a symptom, although inconstant, of ben. there have been several attempts to trace the history of endemic nephropathy to the beginnings of this century or to the period between the two world wars. because ben becomes manifest at older ages and the average life expectancy in the endemic settlements was less than - years before world war ii (some of the major causes of earlier death were parasitic or infectious diseases such as malaria and tuberculosis), no rigorous description of the disease or clue it existed is available from that period ( ) . this, however, does not preclude the possibility that the etiological factor(s) responsible for ben has been present in endemic areas for centuries or millennia. the increase in the average life expectancy after world war ii may have allowed ben to become manifest and thereafter to be described as a distinct nosological entity. ben was almost simultaneously recognized for the first time in the three countries involved as well as in each major affected region of these countries by tanchev et al. in bulgaria in ( ), by danilovic and others in in yugoslavia ( ) , and by fortza and negoescu in romania in ( . in spite of considerable research performed in the three afflicted countries, the etiology of ben and of its accompanying uroepithelial tumors still remains a medical enigma. multidisciplinary studies involving environmental and occupational medicine, microbiology, oncology, toxicology, epidemiology, cytogenetics, and more recently, geology, hydrogeology, and biogeochemistry have added some pieces to the puzzle, but have also raised new questions ( ) . among these, the most central remains: what is the true cause of the disease? clinical presentation and laboratory findings in ben ben is a progressive kidney disease inexorably leading to end stage renal failure. although some symptoms seem to be more specific, there is no clear-cut clinical or pathological definition that is solely sufficient for disease diagnosis. the onset is without any acute episode, and the anecdotal cases of acute ben described in the literature seem to be related to kidney diseases other than ben (or going along with ben) that may occur in the endemic regions. the disease progresses slowly with the occurrence of nonspecific signs (lassitude, fatigue, headache, weight loss and reduced appetite, xanthochromic or pale skin) and specific signs of kidney damage (reduced tubular transport, intermittent proteinuria with low molecular weight proteins such as [- microglobulin, and a gradual rise in blood nitrogen) ( ) . usually, no increased blood pressure is found, even in the more advanced phases of the disease; this was explained by dammin ( ) as the outcome of the early impairment of the juxtaglomerular apparatus. anemia (normocytic, normochromic, or in rare cases, hypochromic or even aplastic) is one of the major symptoms, and it seems to develop well before other signs of ben become manifest. although in the advanced stages of the disease anemia is proportional to the degree of kidney insufficiency and, thus, could be attributed to the increased blood urea level and to the reduction of erythropoietin synthesis (as a result of the destruction of the erythropoietin-secreting peritubular endothelium of the kidney by the etiological agent), it usually appears before significant kidney damage takes place. in this regard, it was presumed that the nephrotoxic factor(s) responsible for ben could also be hematotoxic, inducing anemia and other possible hematological disturbances ( , ) . flow cytometric investigation of ben patients and area control individuals (people born and living in the endemic area and clinically asymptomatic, but having ben and a ben death-related family history) revealed in many cases a decreased b-cell count, eosinophiia, and an increased proportion of cd +cd +cd +cd + cytotoxic lymphocytes ( , ) . because similar modifications can be induced by certain toxic compounds [halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons (pahs)] in experimental animals, the authors linked these alterations to the same presumed toxic agent that caused the kidney alterations. the immunoglobulin (igm, igg) levels are normal and no signs of humoral or cellular immunodeficiency were seen, while a decreased phagocytic activity was observed in certain cases (s. gotia, personal communication). the serum levels of inflammatory cytokines [interleukin- (il- ), tumor necrosis factor-a (tnf-a), interleukin- (il- ), and interferon-y (ifn-y) are normal in most of the cases [drugarin et al., tatu et al., unpublished observations); ( ) ], stressing again the fact that ben is not an inflammatory kidney disease. the serum levels of albumin, c reactive protein, ceruloplasmin, fibrinogen, and a-l-antitrypsin are normal except for the advanced phases of the disease, when their lower levels could be attributed to uremia and to the reduced liver synthesis. morphopathologically, ben is a tubulointerstitial nephropathy with a relatively nonspecific aspect. in the advanced stages, kidneys are reduced in size symmetrically and can weigh as little as g or less. histological examination reveals pronounced fibrotic changes of the interstitium without cellular infiltrate or signs of inflammation. the initial lesion seems to be confined to the proximal convoluted tubules, leading to tubular atrophy and pronounced basilar membranes. in the incipient stages of the disease, glomeruli do not seem to be affected, although later periglomerular fibrosis and cysts in the cortical areas can be seen. atherosclerosis may also occur in kidney vessels ( ) . a peculiar feature of ben is its frequent association with malignant tumors of the upper urinary tract (renal pelvis and ureters). these tumors develop from the urothelium and usually have an aggressive behavior. there are many unknown aspects concerning ben as a disease, but its etiology is the central topic of dispute. perhaps no other human disease has generated so many different hypotheses and ideas in an attempt to explain its causal factors. for ben, which was classified as a disease only in the second half of the century, there have even been rumors among the villagers that the illness is caused by cosmic and astrological influences beyond human control and comprehension. to protect themselves against these dreadful forces, people in certain endemic regions (e.g., from former yugoslavia, romania, and probably bulgaria) still wear amulets or pendants and perform ritual ceremonies to be spared from becoming sick. the profusion of factors considered to cause ben reflects, at least in part, the limited knowledge of the etiology of cin in general. in comparison with other forms of cin, the main peculiarity of ben is its restricted geographical distribution, which became more conspicuous after descriptions of the nephropathy from the involved countries became available. considering only the geography of the disease, an environmental etiological agent seems plausible, presumably acting on a predisposing genetic background of the exposed population. other factors that make ben etiology research difficult indude poor epidemiological data gathered from the endemic region; confusing and often contradictory results due to the lack of communication and cooperation of ben researchers from the afflicted countries; the lack of standardized investigation methods; the communist mentality (e.g., the former communist government from bulgaria relocated whole endemic villages and never recognized the disease; thus, important medical data was lost) ( ); a shortage of funds for specialized teams to thoroughly investigate the endemic regions ( ) ; and social events (such as the war in former yugoslavia, which in cut a fruitful line of research related to the role that low-rank coals may play in ben). despite these difficulties, major research efforts have been made over several decades in an attempt to understand the causes of this still confusing disease. ben is a complex medical condition, and its apparent multifactorial etiology makes it a challenging research topic. because the symptoms and pathology of ben in all the endemic zones are the same regardless of the country where it occurs, there is probably a common etiological factor(s) for the disease. one of the drawbacks of many of the investigations of ben is that studies performed in one of the three countries were not reproduced by similar studies made in the other two countries, posing questions about their reliability. for instance, the proposal of a viral etiology of the disease was first advanced by georgescu in romania ( ) and apostolov in yugoslavia ( ) , but was not confirmed by bulgarian studies. similarly, in cytogenetic studies, bulgarian groups reported the presence of a chromosomal marker ( q ) in ben ( , ) , but this has not been demonstrated so far in romania or yugoslavia. other factors inconsistently incriminated in the etiopathogeny of ben include heavy metals claimed to be present at higher (lead, cadmium, chromium) ( ) or lower (manganese, cobalt) than normal concentrations in water or foodstuffs from endemic areas; selenium deficiency ( , ) ; x ray-emitting compounds; and industrial pollution. other studies incriminate low molecular weight protein (lmwp) excretion in patients with ben as a factor in disease pathogeny ( ) . microproteinuria with - microglobulin has been found in many ben patients and area controls. although these proteins are suspected to be involved in renal diseases in humans and experimental animals, they do not explain the geographical restriction of ben. in this regard, lmwp involvement is probably secondary, after the etiopathogenic agent has triggered the kidney damage. immunologic and hematologic alterations (thought not to be of primary role in ben etiopathogenesis) may also influence the evolution and outcome of the ongoing (established) disease. anemia and the phenotypic changes in lymphocyte subpopulations (e.g., the increase of a cytotoxic cd + t-cell subset and a decrease in the b lymphocyte subpopulation) are considered to be consequential to the action of the same agent(s) inducing kidney pathology ( , ) rather than to the nitrogen retention and uremia. it is likely that such modifications, although not marked, could result in an impaired antiinfectious and antitumoral defense, mainly in the advanced stages of ben when the risk to develop utts is highest. another possible cofactor or risk factor in ben and utt is selenium deficiency, which is well documented in rocks, soils, water, and foodstuff from serbia, as well as in serum samples collected from endemic and nonendemic regions (fig. ) . in certain endemic locations, levels of se deficiency approach those encountered in the keshan province in china, where a severe cardiomyopathy linked to se deficiency is endemic ( ) . environmental health perspectives * volume , number , november reviews a tatu et al. although no known human kidney disease has been associated with a low se intake, the chemoprotective effects make this metal an essential oligoelement in the defense against chemicals inducing oxidative damage and cancer. the immune antitumor response is also boosted by se ( ) and, in this regard, se deficiency cannot be completely ruled out, given the high incidence of utts in the ben endemic territories. genetic polymorphisms in environmentally regulated genes (such as those involved in the distribution, metabolism, and disposal of toxic compounds, and dna repair pathways and their regulators) ( ) may also account for differential susceptibility to ben in the endemic region population and particularly to upper urinary tract oncogenesis. the present paradigm is that ben is an environmentally acquired disease. currently, the two most plausible environmental agents supposedly involved in its etiology are mycotoxins produced by fungi in moldy cereals and various food commodities (grains, meats, beans, etc.) from endemic areas, and aromatic compounds present in the drinking water from the endemic areas ( ) . the mycotoxin cause for ben gained support more than years ago ( - ) when a striking morphopathological and epidemiological analogy between porcine mycotoxic nephropathy, caused by ochratoxin a (ota), and ben was observed. ota is produced in temperate and subtropical regions mainly by fungi of genus penicillium (i.e., p. verrucosum, p. cyclopium, p. chrysogenum, etc.) and aspergillus (a. ochraceus, a alliaceus, a. elegans, etc.) ( , ) . subsequent investigations, which revealed a high frequency of ota contamination of foodstuffs from yugoslavian and bulgarian endemic regions, higher levels of ota in foodstuffs from endemic versus nonendemic villages and in the blood of ben patients versus individuals from nonendemic regions, and the detection of several characteristic ota-dna adducts in urinary tract tumors from bulgarian individuals living in ben endemic regions ( , ) , substantiated this hypothesis. the recent description of a ben-like nephropathy in tunisia, africa ( , ) , which is allegedly produced by ota and of a possible causal link between ota and the karyomegalic interstitial nephropathy ( ), a form of nephropathy that resembles ben in certain morphopathological and clinical aspects, also strengthens the idea of an ota etiology of ben. ota, however, is a relatively common contaminant of various foodstuffs in many countries with temperate continental dimate and can be sporadically found in the blood of healthy individuals. this hypothesis also leaves unclear the fact whether ota is the principal etiologic factor in ben, or only a cofactor that, although required, is not enough to produce ben itself. a major caveat in studies of ota related to ben is that the higher levels of ota found in the serum of ben patients compared to controls may be the consequence of secondary accumulation of ota, following the impairment of kidney transport and excretion functions by the original etiological agent rather than reflecting the natural exposure to ota. also, if ota were the only etiological factor for ben, one would expect to find cases of ben or ben-like disease in many other places around the world where food contamination with ota has been reported (e.g., in the united kingdom or italy where the highest levels of ota contamination of certain food commodities, moldy wheat flour and moldy bread, respectively, have been observed) or in countries where a high prevalence of human sera contamination with ota was detected (for instance in france, denmark, and germany, where ota was found in %, . %, and . - . % of serum samples, respectively) [reviewed by godin et al. ( ) ]. ben, however, seems to be unique in its geographical distribution, evolution, manifestation, and pathology. although several studies daim that ben could be a disease "beyond the balkans" ( ), more research will be needed to darify this. an alternative hypothesis was formulated in based on the geographical correspondence between the location of pliocene lignite deposits in the balkans and the location of endemic areas, as well as preliminary geochemical analyses of well water from endemic villages in yugoslavia, which showed the presence of organic compounds not observed in well water from nonendemic villages ( , ) . all endemic regions except one are in close proximity to known deposits of low rank coals of tertiary age (pliocene lignites) (fig. ). the reason for the one exception is undear. this endemic area is in central serbia and does have some nearby pliocene lignite deposits, although not in as direct proximity as found in other endemic areas. this area of exception may have uncharacterized lignite deposits or be hydrologically connected to nearby lignite beds. further work will be needed to delineate this. the hypothesis emerging from these observations is that groundwater leaches toxic organic compounds from these low rank coals, with subsequent transportation of these organics to wells in the alluvial valleys below the lignite deposits ( , ) (fig. ) . the inhabitants of the rural villages who rely on well water for drinking, cooking, and other purposes are thus exposed to these toxic organics for long periods of time. although the concentrations of these organic molecules in the well water may be low, long exposure and/or accumulation in body tissues over time may lead to kidney lesions, the development of urothelial carcinomas in some individuals, and ultimately to fill-blown ben. pliocene lignites are some of the youngest coals in the balkans and are relatively unmetamorphosed. as such, they retain many of the complex organic compounds contained in their decaying plant precursors, and many kinds of potentially toxic organic compounds may be leached from them ( ) . semiquantitative analysis of shallow well water from endemic villages revealed the presence of pahs (pyrene, anthracene) and aromatic amines (aniline, naphtylamine). some pahs (e.g., benzopyrene, benzanthracene, benzofluoranthene) are known or suspected carcinogens, and aromatic amines have been linked to urinary tract cancer and tubulointerstitial nephropathies. thus, long exposure to these and/or similar compounds in well water in a population dependent on this source ofwater could be linked to ben etiology and the high incidence of urothelial cancer in individuals with ben. this hypothesis is appealing in that it accounts for the geographical restriction as well as for the epidemiological and clinical features of ben. based on the present status of knowledge in nephrotoxicology, however, it should be noted that the currently observed pahs and aromatic amines in the well water from endemic villages are not sufficient to explain the tissue (kidney)confined toxicity in ben. more specific (kidney-oriented) toxic molecules present in well water and derived from pliocene lignites also will need to be identified to further validate this hypothesis. many such compounds may indeed be present in pliocene lignites, given the complex organic structure of coal. on the other hand, ben could be a unique experiment of nature, the concentration, the route of entry, the association with other nephrotoxic compounds (such as mycotoxins), and the intermittent intake pattern of the toxicant(s) characterized by seasonal variations, causing some simple molecules such as pahs to be specific in their target tissue action, with chronic nephrotoxicity and slow carcinogenic activity as their most salient biological effects. besides the balkans, pliocene lignite deposits are also found in italy, turkey, greece, and myanmar. also, low rank, highly unaltered coals such as the pliocene lignites from the balkans are distributed worldwide (e.g., australian brown coals, north dakota and gulf coast lignites, chinese lignites, etc.). however, in the pliocene lignite hypothesis of ben etiology, other factors besides the presence of low rank coals must also be in play. the hypothesis also implies many or all of the following circumstances: the right hydrologic conditions for leaching and transport of the toxic organic compounds from the coal to the wells; a rural population largely dependent on untreated well water; a population with a relatively long lifespan (ben commonly becomes manifest in people in their s and s); a relatively settled population for long exposure to the source of nephrotoxic/carcinogenic substances; and a former yugoslavia and bulgaria. results were presented as fluorescence excitation/emission matrix spectral plots, providing gross chemical characterization of the organic matter present in the water samples. water samples from ben households in endemic villages had distinctive eem matrix spectra, different from those of water samples from nonendemic villages. for example, water samples from endemic villages had distinctive teardrop-shaped eem matrix spectra, with excitation peaks at nm and nm and an emission peak at nm. in contrast, water samples from the nonendemic areas had eem matrix spectra that lacked the teardrop shape and the -nm excitation maximum characteristic of endemic villages, and had litde fluorophoric activity in general. the eem matrix spectra of well water samples from endemic villages but from households not affected by ben had characteristics similar to those of ben-afflicted households, but fluorophoric activity in the non-ben households was considerably lower (fig. ) . these results showed significant differences between the dissolved organic constituents in well water from endemic and nonendemic areas of the balkans, with endemic areas exhibiting far greater fluorophoric activity. also, well water from ben households appeared to have higher concentrations of these fluorophoric compounds compared to non-ben households from endemic villages. based on these data, one could hypothesize that toxic dissolved organic compounds present in the well water contribute to the onset and progression of ben. well water from households from nonendemic villages appeared to lack the specific types of organic fluorophoric compounds observed in endemic villages, and water samples from non-ben households from endemic villages had these fluorophoric compounds at much lower concentrations compared to ben households. the specific nature of the fluorophoric dissolved organic compounds present in the well water samples from endemic villages is beyond the scope of this study, but aromatic compounds of less than daltons molecular weight are consistent with the data. additional work was performed on the pliocene lignite hypothesis in (orem et al., unpublished data) to examine ) the chemical structure of pliocene lignites from the endemic areas in yugoslavia; ) to conduct laboratory leaching experiments on these coals; and ) to determine the concentrations of aromatic compounds in well water from endemic villages and a nonendemic (control) village. analysis of the pliocene lignites from endemic areas in the former yugoslavia using c nudear magnetic resonance (nmr) spectroscopy showed that these coals are of very low rank (highly unaltered) and retain a very high degree of organic functionality (e.g., methoxyl, phenolic, and -bonded aliphatic hydrocarbons), as shown in figure . the high degree of organic oxygen functionality probably imparts a high degree of aqueous solubiity to these coals compared to more altered coal. this high degree of water solubiity was also observed in leaching experiments carried out on the coal samples. pliocene lignites from the endemic areas were leached with distilled water in the laboratory to simulate groundwater leaching. the resulting aqueous extract had a distinctive yellow tint, indicating the presence of dissolved organic matter. the yield of dissolved organic matter from the leaching experiments was - mg from g of starting coal. the c nmr spectra of the dissolved organic matter from the leaching experiments are shown in figure . the spectra show a large peak in the - ppm region, suggesting a high degree of condensed aromatic character in the dissolved organic matter leachate. this is consistent with the presence of polycyclic aromatic structures. the leachates also contain aliphatic hydrocarbon structures (peak at - ppm in the spectra) and a high the l degree of carboxyl (organic acid) functionali-ben etic ty (peak at ppm). tion of p well water samples from two endemic water fr villages and one nonendemic (control) village endemic in yugoslavia were analyzed for the presence performe of dissolved aromatic organic matter using et al. ( gas chromatography/mass spectrometry to rats an (gc/ms). a total of compounds were observed identified in the samples (table ) , with conin the cc centrations in the low microgram per liter tions of range. compounds , , , , and table were higher in well water from the endemic villages. comparing the concentrations of pahs in well water from the hyperendemic petka village to the control village, the petka village had times higher fluoranthene, times higher phenanthrene and pyrene, times higher anthracene, and - times higher concentrations of all other pahs. the higher concentrations of pahs in the endemic petka village may be linked to the high incidence of urothelial cancer associated with ben. potential nephrotoxic agents such as aminophenols or aromatic amines were not analyzed in this study, although previous work has indicated the presence of aromatic amines in well water from endemic villages ( ) . more additional work induding analyses of more well water samples and screening for more potentially carcinogenic and nephrotoxic organic substances is needed to validate the pliocene lignite role in ben etiology. link between the low-rank coals and ology is also reinforced by the detechenolic compounds in the drinking rom wells in several romanian villages and by animal experiments ed in the s in romania. bordas l fed endemic region water extracts nd mice for months to year; they renal lesions that were not found rntrol group. preliminary investigathe geological environment of the an ben locations also revealed the ity of endemic villages to coal .hence, endemic villages near the jiu, and cerna rivers adjoin the pliocene oltenian lignite deposits, e villages south of the carasul river roximity with smaller pliocene ligosits from the carasova and oravita ). field investigations in several villages in romania also showed presence of only one or a few well water supplies used by all or almost all of the village is apparently associated with a hyperendemic character of ben. that is, a higher incidence of the disease and more families (living in different households) were affected compared to the normoendemic or hypoendemic regions. other studies conducted in the s in yugoslavia also support a hydrogeological origin of the disease. field studies performed in a village (glogovac) located at the border between the endemic and the nonend.emic regions in bosnia revealed a striking association between ben incidence, mortality due to ben, and altitude ( ) . glogovac is made up of two parts: lower glogovac (an average m above sea level) and upper glogovac (an average m above sea level). while there is no difference between the two parts in population origin, housing conditions, diet, customs, occupations, or economical, social, or ecological conditions, ben occurs more frequently, and more households are affected in the lower glogovac than in the higher glogovac. the only notable differences between the two parts are altitude ( - m) and the depths of the wells used as a water supply. in lower glogovac, the depth of the wells is - m, whereas wells in upper glogovac are m or deeper. no cases of nephropathy have occurred in families using water from wells deeper than m ( ) . although the hydrology of the region is not very rigorously characterized, gaon ( ) hypothesized that the wells of upper glogovac derive water mainly from the mountains, whereas those from lower glogovac derive water primarily from the river drina. the pliocene lignite theory for ben etiology was also used in a predictive mode. a previously unknown endemic area in southeastern serbia (kosovo region) was described based on the proximity of the area to a previously identified pliocene coal field (coal deposit # in figure ) ( , . a summary of the findings that sustain and disprove the implication of the pliocene lignites in ben and the future research needed to reinforce the theory is presented in table . as long as the ben etiological agent(s) and its origins remain unproved, it is hard to predict if the exposure to the nephrotoxicant is continuous or discontinuous. however, irrespective of the continuity pattern, there seems to be an aggravation of the disease symptoms and an increased mortality from ben in certain seasons, usually following a prolonged rainy period. both the mycotoxin (ochratoxin) and the pliocene lignite hypotheses can explain this seasonal variation, the former by an increased growth of fungi and ota production in foodstuffs in the humid, high water activity periods, and the latter by an accelerated weathering of the coal deposits during the rainy season, increasing the concentration of the weathered compounds into the ground and well water. an acute exposure to higher concentrations of the nephrotoxic molecules will exhaust the functional cj * ¶~j~'c .' xr .>v r ...c. .y mc,~~~~~~~~f igure . solid state c nuclear magnetic resonance (ni distilled water leachates of two pliocene ignites from e of the former yugoslavia and a north dakota lignite. the characterized by a high degree of aliphatic hydrocarb and aromatic ( - ppm) and carboxyl ( ppm) stru reserve of the kidney, causing the disease to evolve from the asymptomatic to the symptomatic phase of the renal failure. the initial description of a high incidence of utt in ben patients soon led to the suspicion that the nephropathy and urinary tract oncoge-:.ii, nesis were causally related. , rare patients over - years of age (on average, years earlier than utts diagnosed in the general population from the nonendemic regions) ( ) . moreover, compared to the general population in which utts are encountered three to five times more frequently in males, the sex ratio of utt occurrence in ben equals or is slightly reversed, with women being more frequently affected ( ) . this could reflect the overall higher incidence of ben in women, but also a particular (hormonal, etc.) susceptibility not encountered in men. the sporadic association between ben and utts could be explained by idiosyncratic susceptibility, ethnic behavioral differences (e.g., hydration patterns and food preparation habits), and/or the action of environmental cofactors (e.g., selenium deficiency in serbia, which is unevenly distributed in the endemic regions). phenotypic and genotypic differences in a number of activating and detoxifying enzymes have been frequendy linked to individualized susceptibility to certain forms of cancer ( , ) . related to this constitutional susceptibility, interindividual variations in the genes encoding cytochromes p , such as cypiaj, cyp d , cyp e , and polymorphisms in glutathione swtransferases and n-acetyl transferases and are well described. a clinically useful marker of the interindividual variation in susceptibility to xenobiotics is the efficiency of debrisoquine metabolism. administered in healthy individuals, debrisoquine is converted into a hydroxy derivative by debrisoquine hydroxylase (cyp d ). polymorphisms in cyp d genes resulting in impaired debrisoquine metabolism occur in less than % of the caucasian populations and have been associated with variable susceptibility to chemical carcinogenesis (bladder cancer, leukemias, and possibly melanomas), pesticide-induced parkinson disease, and iatrogenic reactions to drugs ( ) . the association between the efficiency of oxidative metabolism of debrisoquine and the risk for developing ben and/or utt was examined in bulgaria ( ) . this study included individuals with ben and/or utt (both categories from the endemic region), individuals suspected of having ben, healthy controls from the endemic regions, and healthy controls from nonendemic regions. the extensive metabolizer phenotype was encountered in most of the ben cases and subjects with ben and utt. the mean metabolic ratio (unmodified debrisoquine:hydroxylated debrisoquine) decreased progressively as follows: controls from nonendemic regions > controls from endemic villages > suspected ben patients > ben patients. the following condusion can be drawn from this study whatever the etiological factor is, it will induce ben and/or volume , number i , november * environmental health perspectives utt only after metabolical activation in susceptible individuals (i.e., extensive debrisoquine metabolizers). moreover, the development of utts in less than half of the ben patients may reflect differences in type or degree of enzymatic efficiency in these subjects ( ) . it is not known, however, if the supposed ben causing toxicant(s) is activated specifically in the kidney, or if it is first metabolized to the active form in the liver and subsequently transported to the kidney where it induces the progressive tissue damage. on the other hand, experiments in animals showed that the nephrotoxicity and carcinogenicity of ota is not altered significantly by metabolism. one of the major metabolites that preserves almost the full toxicity of ota is (r)-hydroxyochratoxin a, generated by the mixed function oxidase system ( ) . this suggests, once again, that it is unlikely that ota is the (sole) causative factor for ben and utt. ochratoxin a also seems to be metabolized by a different hydroxylase than for debrisoquine, as only the former is inducible by methylcholanthrene or phenobarbital ( ) . nevertheless, in this case, interspecies metabolization and enzymatic differences make extrapolation from laboratory animals to humans difficult. it cannot be ruled out that the suspected aromatic compounds from the endemic drinking water, besides being nephrotoxic, contribute additionally to the disease etiopathogenicity by inducing enzymes that alter ota and extend in this way its pattern of toxicity. the higher incidence of uits, with no increase in the incidence of bladder cancer, in non-ben interstitial nephropathies (e.g., analgesic nephropathy, interstitial nephropathy induced by renal lithiasis) contradicted the carcinogenicity of ben agent(s), suggesting in the extreme case that ben causing factor(s) has no direct carcinogenic activity at all ( . the occurrence of utts only in the advanced stages of ben (frequently after renal failure is diagnosed and renal dialysis is performed), however, could be the result of a weak oncogenic agent. while no tumorigenic effect is observed in the earlier phases of ben, when kidney morphology is still preserved, the alteration of the pyelo-and ureterolymphatic drainage system following the kidney destruction leads to stasis and accumulation of the nephrotoxic factor(s) in the urothelium, and a (pro)carcinogenic threshold is reached with subsequent induction/promotion oftumorigenesis. the etiology of ben-linked urinary tumors is still speculative, as is the cause of the background disease. however, no other known human disease, with the possible exception of analgesic nephropathy, is so frequently associated with ureteral and renal pelvis carcinomas. the molecular description of ben-linked tumorigenesis and the influence of a familial or individual susceptibility to utt could offer an important arena of research for molecular and environmentally induced carcinogenesis. aristolochic acid an interesting association has been made between chinese herb nephropathy (chn) and ben ( ) . the incriminated agent in this case is aristolochic acid, a known nephrotoxic and carcinogenic compound synthesized in various portions ofaristolochia plants. the nephropathy was described in several hundred young women in belgium and, more recently, in japan ( ) who followed a slimming diet (accidentally containing plant parts of aristolochia, instead of stephania tetrandra) for several years and a cure for atopic dermatitis, respectively. chn resembles ben in many aspects: dinical-normal blood pressure; aseptic leukocyturia; low grade, low molecular weight proteinuria; early and severe anemia and morphopathological-extensive hypocellular interstitial sderosis; tubular atrophy and global sderosis of table . summary of the main findings that support the role of the low-rank coals (pliocene lignites) and the hydrogeological factor in balkan endemic nephropathy/upper urinary tract tumor (ben/utt) etiology, and disagree with the hypothesis and the additional research needed to confirm the hypothesis pros earlier research ( s) showed the presence of phenolic compounds in drinking well water from endemic regions in romania glogovac was described as a model endemic village composed of two parts with similar ethnical groups, socioeconomic status, and hydric and food preparation habits, but different in the incidence of ben/utt and probably in the hydrogeological conditions and well water chemistry ( ) hyperendemicity of ben is apparently linked to the use of a single or a few well water supplies by an entire village semiquantitative detection in water samples from endemic regions in yugoslavia revealed the presence of pahs and aromatic amines not present in the water samples from the nonendemic regions ( ) characteristic excitation/emission spectra were identified in water samples from endemic regions in yugoslavia and bulgaria ( ) pahs were detected by gc/ms in higher concentrations in water samples from endemic regions than in water samples from nonendemic regions (orem et al., unpublished observations) laboratory leaching experiments showed the highly unaltered character of the pliocene lignites proximal to the endemic regions in yugoslavia and their potential to release pahs and aromatic compounds with functional groups that can be nephrotoxic or carcinogenic (orem et al., unpublished data) the pliocene lignite theory explains peculiar features of the disease, such as the geographic confinement and the seasonal variation in prevalence and death from ben; it also fits epidemiological data, e.g., the fact that ben is encountered only in rural areas that depend entirely or almost entirely on wells for drinking water the pliocene lignite theory has predictive power: a previously unknown ben region was identified based on the proximity to a pliocene coal field ( , ) cons pliocene lignites also occur in regions outside the balkans, although their degree of alteration and their potential to release nephrotoxic/carcinogenic compounds in the ground water may be different from the lignites associated with the ben endemic areas the currently identified pahs and aromatic compounds in the well drinking water from the endemic areas are not solely sufficient to explain the etiology and pathogeny of ben/utt future geochemical analysis of lignites from romania and bulgaria topographically linked to the ben endemic areas may be performed prospects compounds likely to be nephrotoxic/carcinogenic, such as aminophenols, aromatic amines, or other unknown molecules potentially leaching from the pliocene lignites in the well water from the endemic regions, may be identified biomarkers of pah exposure may be assessed, e.g., pah-dna adducts in blood cell dna samples and urinary -hydroxypyrene levels in patients with ben/utt an animal model for ben might be developed by feeding laboratory animals compounds isolated from endemic well water and/or from pliocene lignite leachates abbreviations: pahs, polycyclic aromatc hydrocarbons; gc/ms, gas chromatography/mass spectrometry. glomeruli; and mild to moderate atypia or atypical hyperplasia of the urothelium ( ) . in two cases of chn, utts were described, providing another link to ben. the aristolochic acid hypothesis nevertheless fails to explain one of the defining features of ben, i.e., the geographical confinement. aristolochia plants are widely cultivated and are probably native in many places in europe, asia, and north america, places where no cases of ben or a ben-like disease have been documented.. also, even though many herbal remedies are used locally in the ben areas, it is unlikely that aristolochia leaves, seeds, or roots are ingested on such a widespread scale in the endemic locations ( ) . the pungent repulsive odor of the plant makes it equally unlikely to be accidentally ingested, even on an endemic scale. in addition, the pelviureteric sclerosis found in one of the biopsied cases of chinese herb nephropathy reminds more of analgesic nephropathy than ben. viruses a viral etiology of ben was hypothesized based on the endemic character of the disease, the seasonal variation in the incidence and deaths from ben, and on the discovery of viral particles in the kidneys of some afflicted persons ( ) . the high frequency of urothelial tumors in ben patients may also be explained by infection with slow oncogenic viruses. several viruses have been considered as possible agents: the west nile virus, papilloma viruses (bk and sv ) ( , ) , coronaviruses ( ) , and arenaviruses ( ) . an extensive search for these viruses in ben patients has been unsuccessful so far. for example, the west nile virus (a mosquito-borne flavivirus that caused more than cases of meningoencephalitis in the danube flood lands, including bucharest, romania, in ), is probably not involved in ben because only a few of the investigated ben patients exhibited antiviral antibodies. the same is true for the bk and sv viruses. although high titers of anti-bk and -sv antibodies were found in some of the ben patients, such antibodies are also frequently encountered in the general healthy population. coronaviruses transmitted from pigs in the endemic households were suggested as a possible cause for ben ( ) . however, evidence supporting this hypothesis is lacking. more likely candidates for a viral etiology are rodent-borne viruses such as those responsible for hemorrhagic fever (hf) syndromes. some of these viruses (puumala, hantaan, and dobrava in the balkans) can cause more or less severe hf with renal syndrome in rural or urban areas. the classic hantaan disease in rural china (epidemic hf) and in korea (korean hf) has a seasonal pattern of incidence, being most common in spring or fall, possibly in relation to agricultural practices and rodent density ( ) . infection in humans is acquired through aerosols of rodent urine; the disease is not transmitted from human to human. some of the features of hantaan hf are seen in ben-disease endemicity, incidence in rural settlements, and seasonal variation-but ben is primarily a chronic kidney disease without any acute episode in its evolution. in contrast, the hantaan infection usually evolves with acute renal failure. there is an analogy worth mentioning between ben and the bolivian hemorrhagic fever, an arenaviral hf disease spread by rodents ( ) . as discussed by radovanovic ( ) , both diseases are characterized by similarities in the topography of the disease area (flat plains, in river or stream valleys), clustering only in certain setdements and households, lack of evidence of human-to-human transmission, and the first recognition of both diseases after the beginning of malaria control programs using ddt after world war ii. although the evidence is speculative, radovanovic ( ) postulates that residual ddt sprayed in the balkans in the mid- s led to the reduction in the number of cats, the most important natural barrier against rodents in the endemic (and nonendemic) households. as a result, a decade later, the rodent reservoir expanded and ben emerged and was described for the first time as a disease entity. although improbable based on the previously mentioned findings, a viral etiology for ben cannot be completely rejected until more comprehensive studies are available. in a broader framework, ben and its associated utts would not be unique in having a hydrogeological etiology. for instance, the field of geomedicine also records a possible correlation between digestive cancer mortality rates and the consumption ofwater from coal bearing strata ( ) . from the ben and utt perspective, an intriguing observation is that the top five states in the united states in average annual age-adjusted cancer mortality rates from kidney and renal pelvis cancer ( ) ( ) ( ) ( ) ( ) are alaska, maine, north dakota, south dakota, and minnesota. north and south dakota have extensive lignite deposits and a largely rural population dependent on wells as a water source. alaska, maine, and minnesota have extensive peat deposits (coal precursors) and also considerable rural populations. coal is also considered to be an environmental component in the multicausal etiology of other common diseases besides cancer. an increased rate of heart attacks is encountered in ohio, correlated with the consumption of drinking water from coal-bearing strata ( ) . a significant negative correlation has also been observed between cardiovascular mortality (cvm) rates and the hardness and calcium concentration of potable ground water. apparendy, a harder water and a higher concentration of dissolved calcium salts (from limestone and dolomite) are positively correlated with increased longevity and lower cvm in the northern-midcontinental united states, compared to decreased longevity and higher rates of cardiovascular deaths in the southeastern coastal area where drinking water is softer and ca + concentrations are lower ( . the etiology of cin is variously reported to be secondary to metabolic diseases, drugs (i.e., analgesics or analgesic mixtures), or known environmental factors (e.g., industrial pollution with lead, cadmium, mercury, etc.). about - % of the patients in end stage renal disease (esrd) treatment centers who have been diagnosed with cin have no established etiology. without a known etiology, prevention of disease is an elusive goal. studies that will yield information on the causes of cin could have an obvious immediate prophylactic effect and allow a reduction in the costs of kidney transplantation and dialysis, the current final therapeutical solutions for esrd patients. in this respect, identifying the factor(s) responsible for ben will lead to a deeper understanding of the etiopathogeny of this disease and to a better knowledge of kidney disease mechanisms and carcinogenesis in general. unraveling the etiology of ben will also allow appropriate preventive measures to be taken in the afflicted areas, such as by treatment or filtration of the drinking and cooking water or changing the main water supply. every human disease has a multifactorial causality. usually there is a main factor whose sine qua non action is sufficient for triggering a pathogenic process that individualizes the disease. other factors without direct and feasibly quantifiable effects in disease genesis and evolution are frequently neglected, although in their absence the disease pattern and progression should be different. what is involved and what is not in ben etiology are questions equally difficult to answer, and some of the reasons for this are stated above. similar problems are evident when the long-term and sometimes combined effects of twentieth century environmental pollutants, including pesticides and the largely debated endocrine disruptors, are assessed and straightforward answers are expected. due to the synergistic (or antagonistic) and not infrequently transgenerational action of a multitude of factors (some even unimagined), a cause-effect relationship is not always obvious. causative agents interplay with predisposing or constitutional factors (cofactors or risk factors) toward a complex disease phenotype. defined initially as a lead nephropathy, subsequently considered a viral disease, a genetic or chromosomal disorder, or a form of chronic pyelonephritis, ben is presently thought of by most researchers as an environmental disease. although the environmental etiology is widely accepted, the true factor(s) causing the disease is unclear. multidisciplinary approaches carried on in various countries have excluded some of the factors claimed to cause ben. these studies, however, have also outlined some factors that cannot be disregarded when considering ben etiopathogeny, as they can comfortably explain certain features of the disease (e.g., familial aggregation, geographic confinement, association with uroepithelial tumors, etc.). ben, from these standpoints, may offer a conceptual and mechanistical model for the complex and integrated topics pertinent to the study of environmental diseases. epidemiology of balkan endemic nephropathy on danubian endemic familial nephropathy (balkan nephropathy) some features of balkan endemic nephropathy balkan turmoil delays ben research: disease links to weathered low-rank coals remain speculative epidemiological characteristics of balkan endemic nephropathy in eastern regions of yugoslavia health center control of patients suffering from endemic nephropathy in vratza district chronic nephritis caused by poisoning with lead via the digestive tract (flour) nefrita cronica azotemica endo-epidemica balkan endemic nephropathy: more questions than answers clinical features of balkan endemic nephropathy endemic nephropathy in yugoslavia hematological changes in endemic nephropathy in south-east europe identification of a novel subset of t lymphocytes in patients with balkanic nephropathy balkan endemic nephropathy, the hematopoietic system and the environmental connection cytokine levels in patients with balkan nephropathy some pathomorphological features of balkan endemic nephropathy in croatia eastern europe: missing an opportunity viruses in endemic (balkan) nephropathy evidence of a viral etiology in endemic (balkan) nephropathy cytogenetic studies in balkan endemic nephropathy genetic predisposition to balkan endemic nephropathy environmental renal disease: lead, cadmium and balkan endemic nephropathy selenium deficiency in yugoslavia djujic . selenium deficiency in serbia and possible effects on health possible pathogenic role of low-molecular-weight proteins in balkan nephropathy selenium: mechanistic aspects of anticarcinogenic action understanding gene-environment interactions relationship between weathered coal deposits and the etiology of balkan endemic nephropathy mycotoxic porcine nephropathy: a possible model for balkan endemic nephropathy ochratoxins in food role of ochratoxin in disease causation is ochratoxin a a nephrotoxic in human beings? the toxicology of mycotoxins molecular and epidemiological approaches to the etiology of urinary tract tumors in an area with balkan endemic nephropathy ochratoxin a in human sera in the area with endemic nephropathy in croatia foodstuffs and human blood contamination by the mycotoxin ochratoxin a: correlation with chronic interstitial nephropathy in tunisia ochratoxin a in human blood in relation to nephropathy in tunisia kidney disease beyond the balkans? correlation of balkan endemic nephropathy with fluorescent organic compounds in shallow ground water relation between low-rank coal deposits and balkan endemic nephropathy renal changes in rats and mice caused by total extracts of water and residue obtained from a herd with endemic nephropathy geologia zacamintelor de carbuni - . zacaminte din romania ciba foundation study group no. urinary tract tumors and balkan nephropathy in the south morava river basin tumors of the pielum and ureter in emigrants from endemic nephropathy areas the role of individual susceptibility in cancer burden related to environmental exposure interaction between dose and susceptibility to environmental cancer: a short review polymorphisms in drug-metabolizing enzymes: what is their clinical relevance and why do they exist? genetic predisposition to balkan endemic nephropathy: ability to hydroxylate debrisoquine as a host risk factor characterization of the cytochrome p isozyme that metabolizes ochratoxin a, using metabolic inducers, inhibitors and antibodies chronic interstitial nephritis as a cause of tumors of the upper urinary tract chinese herbs nephropathy: a clue to balkan endemic nephropathy? traditional remedy-induced chinese herbs nephropathy showing rapid deterioration of renal function virus-like particles in the kidneys of three patients with endemic balkan nephropathy serum antibodies to papova viruses (bk and sv ) in subjects from the area with balkan endemic nephropathy further data on the prevalence of serum antibodies to papova viruses (bk and sv ) in subjects from the romanian area with balkan endemic nephropathy epidemiological evidence on balkan nephropathy as a viral disease infections caused by arthropod-and rodent-borne virus the health impact of geochemical alterations associated with coal resource development in midwestern usa heart disease and geologic setting in ohio chemical qualities of water that contribute to human health in a positive way key: cord- - ksfpaf authors: nan title: proceedings of the th european paediatric rheumatology congress: part : virtual. - september date: - - journal: pediatr rheumatol online j doi: . /s - - - sha: doc_id: cord_uid: ksfpaf nan introduction: juvenile idiopathic arthritis (jia) represents the most common pediatric chronic rheumatic disease. children with jia present an increased risk of infections, due to the immune-regulatory effects of disease modifying antirheumatic drugs (dmards); many of these infections are vaccine-preventable. nevertheless, suboptimal vaccinations rates are reported in children with jia. objectives: to evaluate vaccination coverage in a population of children with jia and to describe the prevalence of the adverse events following immunization (aefis) in our cohort. methods: a single-centre retrospective study was conducted by reviewing medical records of all jia patients, diagnosed according to ilar criteria, admitted to the pediatric rheumatology unit of university of naples federico ii from january to december . parents were asked to provide the vaccinations records in form of the vaccination booklet. the occurrence of aefis was explored by telephone interviews. introduction: intrarticular corticosteroid injections (iaci) are widely used in the management of patients with juvenile idiopathic arthritis (jia). general anesthesia can be avoided in case of a small number of joints to inject or in older children. however, pain and anxiety may reduce the patient compliance to iaci, and may compromise the accuracy of the procedure. in order to overcame such problems, the use of appropriate methods of pain and anxiety control is advisable. objectives: to assess the effectiveness and satisfaction of patients undergoing iaci with the use of topical numbing agent or under minimal sedation. methods: patients with jia who underwent an iaci of up to joints were recruited. depending on age and number of joints to treat, a group of patients (group a) were injected with the application minutes prior the procedure of a topical numbing agent (prilocaine+lidocaine) to the skin over the injection site. another group of patients (group b) were treated under minimal sedation (ketorolac/tramadol or morphine + midazolam). the physician was asked to record the degree of motion and pain of the patient during the procedure and the patient (or parents for patients aged less than years) was asked to report the degree of pain and satisfaction on a visual analogue scale (vas) from to . results: twenty-seven patients were enrolled for a total of procedures, and of them in group a and b, respectively. the median age at the procedure was years for group a and years for group b. for group a median pain scores for patients, parents and physicians were , and . , respectively. in patients of group b who underwent the iaci under ketorolac/tramadol the median pain scores for patients, parents and physicians were , . and . , whereas in patients treated with morphine median pain scores were , and , respectively. overall, we found that pain as reported by the patient/parent were higher with increase in the number of sites injected (and, consequently, duration of procedure) and age of patient. amount of motion during procedures was overall negligible. the majority of patients/parents was satisfied for the procedures. only patients treated with midazolam had psychomotor agitation during the iaci. conclusion: iaci in a small number of sites without the use of general anesthesia is well tolerated by patients. the level of pain perceived from patients is irrespective of the power of the painkiller used, but seems to correlate with the duration of the procedures. it is possible that, in the paediatric age, the psychoemotional component seems to be decisive, with a progressive loss of tolerance with the increase in the number of injected joints. for gc-ms analysis of the steroid hormone metabolites age and sexmatched healthy controls were matched to each patient. patients were excluded if they were treated with corticosteroids in the preceding months. results: of the metabolites measured, were significantly lower in jia patients before the etanercept treatment compared to the healthy control group. one day after the injection only metabolites were still significantly lower in the jia patients and all the other metabolites normalized and were similar to the control group. urine metabolite ratios reflecting cyp and β-hsd enzymatic activity indicate that these two enzyme activities were lower in jia patients. the slowest recoveries noted were for metabolites of dheas and oh pregnenolone. conclusion: prior to etanercept treatment almost all urine adrenal metabolites were significantly lower mainly due to the active inflammatory process. immediately after the treatment many metabolites raised to normal values as in the control group. the two adrenal enzymes that were found to be affected in jir are cyp and β-hsd . blocking tn alpha immediately restore adrenal function in jia. introduction: patients with juvenile idiopathic arthritis (jia) receive adalimumab treatment. adalimumab is a monoclonal antibody that blocks tnf-α and is structurally and functionally similar to human igg . nevertheless, there are reports of the development of anti-drug antibodies. the production of these antibodies may be associated with treatment failures (a decrease in the effectiveness of therapy or drug inefficiency that developed over time) and hypersensitivity reactions. to our knowledge, there is currently limited information on the availability of adalimumab antibodies (aaa) in patients with jia. objectives: to evaluate the prevalence rate and the clinical significance of aaa in patients with jia on adalimumab treatment. methods: patients with jia were examined, of whom had the oligoarticular form of the disease, of them with uveitis, and patients had the polyarticular form of the disease, of them with uveitis. among them, there were ( %) girls and ( %) boys. the mean age was . ± . years; the mean disease duration was . ± . years. patients received adalimumab (at least year before the study) with concomitant administration of methotrexate (mtx) or adalimumab only - children who did not receive mtx for at least months prior to the study as a result of either adverse events of mtx administration ( patients) or permanent drug remission ( patients). before starting adalimumab therapy, all participants were treated with mtx. the mean duration of adalimumab treatment for these patients was . ± . years. the serum aaa level of antibodies was determined using the enzyme immunoassay (eia) method. this method determines both free and bound antibodies to adalimumab at reference values less than au/ml. a and was used every weeks for months. the values were presented as mean ± standard deviation. data processing and analysis were carried out using pearson's chi-squared test and spearman's correlation test. results: ( %) of the patients enrolled in the study had aaapositive results. the mean aaa level in positive patients was . ± . au/ml. further disease relapses tended to occur significantly more often in aaa-positive patients than in aaa-negative ones (χ = . , p = . ). thus, of ( . %) aaa-positive children had at least exacerbation of the disease within months, compared with of ( . %) in aaa-negative ones. out of ( . %) aaapositives did not take mtx for at least months compared to out of ( . %) in aaa-negative ones. thus, aaas are found to be significantly more frequent without concomitant administration of mtx in the treatment of jia (χ = . , p = . ). there were no observed adverse events or side effects during adalimumab therapy. no significant correlation was found between the presence of aaa and sex, introduction: advances on molecular medicine, illumination of the cytokine network and the immune pathways shed light on the etiopathogenesis for a better understanding of juvenile idiopathic arthritis (jia). however, the fact that the course of the disease differs individually strongly suggests the effect of external factors. objectives: the current study was undertaken to evaluate sociodemographic and sociocultural features, parent behavior, the gestation and breastfeeding period, nutritional status of early childhood in our patients with jia, and to determine their relationship with disease activity, damage index, remission time, and relapse rate. methods: the study was conducted with a face-to-face questionnaire method with the parents of patients with jia and healthy children. the medical patient records were reviewed. juvenile arthritis disease activity score (jadas) , wallace clinical inactive disease criteria, juvenile arthritis damage index (jadi), and relapse rates were used to assess the general medical condition of each patient. results: the median age of jia patients (n = ) was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , with a female ratio of , %. age at disease onset was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) years. the first remission time was ( - ) months. the patients were evaluated according to disease subtypes and treatment modalities. there was no difference in the duration of breastfeeding according to the distribution of the subtypes (p = , ). when the breastfed and formula-fed patients were compared, there was a marginally significant difference in terms of first remission time (p = , ), whereas there was a significant difference in relapse rate in patients who introduced to cow milk early (< months) (p = , ). the early risk factors and their relationship with the disease are presented in table . both breastfeeding durations and maternal literacy levels showed a significant difference in terms of relapse rates (p = , ; p= , , respectively). there was no significant difference in breastfeeding durations and gestational risks between the patients and the healthy group (p = , ; p= , ), respectively. however, the smoking rate among family members was significantly higher in the patient group (p = , ). conclusion: in patients with juvenile idiopathic arthritis, breastfeeding rate and duration did not differ when compared to healthy controls. however, breastfeeding duration, cow's milk commence age, and maternal literacy appeared to be relevant to the relapse rates. going to preschool both influence the remission time and relapse rate. these findings suggest a role for parental attitude and nutritional status during early childhood in the course of jia. none declared introduction: immunogenicity and low trough concentrations have been associated with adalimumab treatment failure in several studies of paediatric inflammatory diseases, indicating the possible value of therapeutic drug monitoring (tdm). adalimumab efficacy may be improved by changing dose or treatment intervals based on drug concentrations. however, lack of standardization, assay heterogeneity, and paucity of research hinder the implementation of tdm in clinical practice. objectives: to assess the relationship of trough concentrations, immunogenicity and adalimumab response in paediatric patients with jia. methods: monocentric cohort study of patients ≤ years with jia treated with adalimumab due to active arthritis. clinical data and plasma samples were collected during routine follow-up. adalimumab trough concentrations were measured by liquid chromatographytandem mass spectrometry (lc-ms/ms). anti-adalimumab antibodies were measured in samples with trough concentrations < mg/l. disease activity was evaluated using the clinical juvenile arthritis disease activity score with joint count (cjadas ). response to adalimumab was defined as at least % reduction of disease activity within months of therapy followed by clinical inactive disease or minimal disease activity after months. the latter was defined as cjadas ≤ . and ≤ . , for oligoarthritis and polyarthritis, respectively, or an active joint count equal to zero when cjadas was unavailable. results: adalimumab trough samples were available from jia patients. although there was no significant difference in median adalimumab dose, trough concentrations were significantly lower in patients with secondary failure compared to primary failure or an adequate adalimumab response (p-values < . ). in addition, there were samples with trough concentrations < mg/l, in the group with secondary failure and in the group with adequate adalimumab response (table ) . conclusion: adalimumab trough concentrations were significantly lower in jia patients with secondary failure compared to primary failure or an adequate response to adalimumab. anti-adalimumab antibodies were present in out of samples with trough concentrations < mg/l. adalimumab trough concentration measurements may identify jia patients that would benefit from increased doses or shorter treatment intervals. in addition, jia patients with primary failure and adequate adalimumab trough concentrations may respond better to biologic agents with other therapeutic targets. although biologic agents have improved disease outcome of patients with jia, concentration measurements using reliable and cost-effective methods, such as lc-ms/ms, could further improve efficacy of biologic agents and guide treat-to-target strategies. introduction: most studies of physical fitness (pf) in juvenile idiopathic arthritis (jia) have shown decreased levels of maximal oxygen consumption (vo max) compared to healthy peers. in jia, boys have higher pf-levels than girls and younger children have higher levels than adolescents, congruently with data of healthy peers. previously, we have shown that more than half of jia-patients had below normative average of vo max. however, monitoring physical activity (pa) using accelerometry, % of boys and % of girls with jia fulfilled the recommendations of who of ≥ hour of pa per day, which was comparable to normative values ( %/ %). moreover, patients reporting engagement more than hours per week in club-sports exceeded accelerometry values of healthy peers. objectives: to explore the association between pf and specific sport habits in to -year-old jia-patients, related to gender, bmi, disease activity, and pain, and comparing the most fit quartile (q ) of patients (respectively boys and girls) to the least fit quartile (q ). methods: sixty patients with jia performed an indirect ergometertest of vo max (watt max test) and answered the physical activity and sport questionnaire (pasq). objective pa-monitoring for one week was conducted using the gt m accelerometer. cut-offs for moderate-high and high intensity pa were set to > and > counts per minute, respectively. disease activity was assessed with the jadas- , current pain and worst pain last week were measured on visual analogue scales (vas) and in a one-week pain diary using the faces pain scale-revised (fps-r). results: girls with jia (n= ) had lower mean pf than the boys (n= ) ( . ± . / . ± . ml/kg/min), being below normative values, respectively. in both genders the most fit boys and girls (q ; . - / . - ) had average to well-above normative average pf. the least fit boys (q ; . - . ) all had pf-levels well-below normative average. in girls q -levels ( . - . ) were well-below to below normative average. we found significant differences between most fit (q ) and least fit (q ) patients regarding patient´s global wellbeing (p= . ) and pain diary (p= . ). these differences were not significant when separating genders, though differences were more pronounced in girls. the least fit girls (q ) had significantly higher disease activity (jadas- ) than the most fit girls (q )(p= . ). the most fit boys and girls (q ) engaged equally in high intensity sports (soccer: / ; / , handball: / ; / , gymnastics: / ; / , rowing: / ; / ). however, more boys than girls played soccer ( / ; / ), whereas more girls preferred sports of lower intensity (riding: / ; / ). eight of boys in soccer and boys in gymnastics or rowing had below average to well-above normative average of pf (q +q : . - ). three girls in gymnastics, girls in soccer, and girls in handball were in q ( . - ) with levels of average to well-above average pf. the third girl in soccer was in q ( - . ) with levels of well-below to below normative average. none of the riding girls were in q and only was in q ( . - . ) (below to average normative pf). comparing accelerometer-monitored values of pa-intensity in girls with low (q ) and high (q ) pf, pavalues of q were significantly lower than in q . the same tendency was observed in boys, but not to significance. conclusion: our results are in accordance with most other studies of pf in jia, adding to the knowledge of gender-specific differences in pf and type and behavior in sport activities. it emphasizes the need of regular pf-testing and guidance in high intensity pa and sport in order to improve pf and avoid the risks of inactivity and lifestyle diseases in jia. pg/ml), which also showed the highest the frequency of detection of its increase. it was absent in sjia ( . ± . pg/ml). the highest values of il- r ( . ± pg/ml) were in the middle age group. the data obtained suggest the compensatory value of increasing il- r and the simultaneous initiation of inflammatory and anti-inflammatory processes during exacerbation of jia. assessment of the ratios of stimulating and inhibiting cytokines showed in patients with uveitis, the ratio of ifn-γ/il- β ( . ± . ) was higher than with other jia (from . ± . in ojia to . ± . in pjia) and ifn-γ/il- r ( . ± . versus from . ± . in ojia to . ± . in sjia). an increase of il- β/il- r ratio was characteristic only for sjia ( . ± . ). all of these ratios increased with disease activity (r= . - . ) & they did not reflect the unpleasant course of the disease. methods: in this multicenter, case-control study, fecal samples were collected from children with jia, with of these samples collected from untreated children ( of whom were treatment-naïve children). samples from children with jia were collected during treatment with mtx as single treatment and samples from children during treatment with etn. of those children, four were treated with etn as single treatment and nine had a combination of etn and mtx. we compared children on single treatment with mtx with untreated children ( treatment-naïve), and children on treatment with etn (nine in combination with mtx) with untreated children ( treatment-naïve). we also did pairwise comparisons of samples taken before any medication was given (n = ) and samples taken during ongoing treatment with mtx (n = ) or etn (n = , four in combination with mtx). the microbiota was characterized by sequencing amplicons from the v and v regions of the s rrna gene. alpha diversity of the fecal samples was measured using the chao- index and the shannon diversity index. to compare these indices between treated children and untreated children, we used a logistic regression model with age at sampling as a covariate. for pairwise analyses, we used the wilcoxon signed-rank test. to analyze the community composition of the microbiota, principal coordinate analysis (pcoa) plots based on bray-curtis distances were generated for visual comparisons, and analysis of similarity (anosim) was used to test for differences. analyses for relative abundances of taxa were performed at three taxonomic levels (phyla, families, and genera), and logistic regression with age as a covariate was used for calculations of differences between treated and untreated children, while the wilcoxon signedrank test was used for pairwise comparisons. significance was set to p < . and corrections for multiple comparisons were made using the benjamini-hochberg method. results: analyses showed no significant differences in α-diversity between children treated with mtx or etn and untreated children, and pairwise comparisons of samples before and during treatment with mtx or etn also showed no differences. pcoa plots for children treated with mtx or etn, in comparison with untreated children, did not show any clustering. anosim showed no significant differences between treated and untreated children. pcoa plots were also made for the pairwise comparisons of children sampled before and during treatment, and according to that analysis the community compositions of microbiota did not change in any uniform way during treatment with either mtx or etn. furthermor, analyses of relative abundances of specific taxa did not reveal any significant results in any of the comparisons, after adjustment for multiple analyses. conclusion: treatment with mtx or etn did not alter the composition of fecal microbiota in children with jia. introduction: juvenile idiopathic arthritis (jia) is the most common rheumatic disease in childhood and an important cause of shortterm and long-term disability if patients are not treated appropriately. by definition, jia clinically presents with peripheral joint inflammation of unknown origin, persisting for at least six consecutive weeks and starting before the age of years. the predominant subtypes, i.e. oligoarticular (oligo) and polyarticular (poly) jia, have long been assumed autoimmune diseases caused by dysregulation of the adaptive immune system, with a central role for autoreactive t cells belonging to the th and th lineages and autoantigens that may include aggrecan, fibrillin, matrix metalloproteinase (mmp)- and heat shock proteins. nevertheless, the original t cell-centered hypothesis has been challenged since it does not cover nor completely explain the full spectrum of immune-pathological phenomena observed in patients. lien.desomer@uzleuven.b objectives: emerging evidence suggests a potentially important role for neutrophils in jia pathogenesis. here, we investigated extensively the phenotypical features of neutrophils present in the peripheral blood and inflamed joints of jia patients. methods: synovial fluids and parallel blood samples from patients with oligo-or polyjia and blood samples from healthy children were collected. multicolor flow cytometry panels allowed for in-depth phenotypical analysis of neutrophils, focusing on the surface expression of adhesion molecules, activation and maturation markers, chemoattractant-and toll-like receptors. multiplex technology was exploited to quantify pro-and anti-inflammatory cytokines in plasma and synovial fluids. results: the vast majority of synovial fluid neutrophils displayed a strongly activated, hypersegmented phenotype with decreased lselectin (cd l) expression and increased numbers of nuclear lobes, upregulation of adhesion molecules cd b, cd b and cd and downregulation of chemokine receptors cxcr / . an elevated percentage of cxcr -expressing aged neutrophils was detected in synovial fluids from patients. strikingly, significant percentages of synovial fluid neutrophils showed a profound upregulation of atypical neutrophil markers, including cxcr , icam- and hla-dr. conclusion: our data indicate that neutrophils present in inflamed joints of jia patients are strongly activated cells with elevated proinflammatory and antigen presenting potential. this detailed molecular analysis supports the notion that a complex intertwining between these innate immune cells and adaptive immune events drives jia. none declared the main factors, associated with incomplete vaccination againts measels, parotitis, rubella and diphtheria in juvenile idiopathic arthritis patients n. lyubimova , i. objectives: the aim of our study was to evaluate the rate and the main factors of incomplete vaccination against measels,parotitis, rubella (mmr) and diphtheria in jia patients. methods: in the present study were included data jia( boys and girls)aged from to years,who received scheduled vaccination before the age of years and before jia onset against measles,parotitis,diphtheria and rubella.incomplete vaccination means the reduced number of vaccine to age.in all patients the ig g anti-vaccine antibodies levels were detected with elisa.jia categories were:oligoarthritis - ,polyarthritis - ,systemic- and enthesitisrelated arthritis- .data presented with median and %> % results: incomplete vaccination against mmr was in ( %)diphtheria in ( %) of the jia patients. the main differences in the studied groups are in the table.there were no differences in sex,jia categories and treatment, except biologics compare to complete and complete vaccination in all vaccines.no differences in antimeasels(p= . ),antiparotitis (p= . ) and anti-rubella(p= . )vaccination between complete and incomplete vaccination group.number of patients with protective level of anti-vaccine antibodies was similar, except parotitis( % vs . %, p= . ).the anti-diphtheria antibodies igg level was lower in the patients with incomplete vaccination group ( . iu/ml [ %ci: . ; . ] vs . [ %ci: . ; . ], p= . )as well as number of patients with protective level( % vs %, p= . ). in the multiple regression model only jia onset age(p= . )and methotrexate treatment duration(p= . ) were predictors of incomplete vaccination against mmr and methotrexate treatment duration(p= . ) and biologic treatment(p= . ) for diphtheria incomplete vaccination.incomplete mmr vaccination influence on the maintenance of the protective anti-parotitis level(p= . )in regression model.in correlation analysis the number of vaccination influences on anti-diphtheria antibodies level(p= . )and number of patients with protective level of anti-diphtheria antibodies(p= . ). the main predictors in logistic regression for incomplete vaccination for mmr were:onset age< years(or= . conclusion: younger onset of jia age, longer duration of jia and methotrexate treatment, biologics and more than biologics are the main predictors of incomplete vaccination againt mmr and diphtheria. introduction: the prevalence of autoimmune thyroid disorders (aitd) has been reported to be higher in patients with juvenile idiopathic arthritis (jia) in comparison to the general population. nevertheless, there is a lack of studies investigating risk factors for aitd development in children with jia. objectives: to investigate the co-occurrence of jia and autoimmune thyroiditis in southern italy and to identify potential predisposing factors to anti-thyroid antibodies (ata) positivity in a jia population. methods: a single-centre retrospective study was conducted. all jia patients admitted to the pediatric rheumatology unit of the university of naples federico ii, from january to december , tested for ata at least once and with a minimum of -months follow-up, were included. for each patient, demographic, clinical and laboratory data were extracted from clinical charts. differences between patients affected by jia with or without ata were analyzed. results: three hundred thirty jia patients ( females; median age . years, iqr . - . ) were included in study. median age at jia onset was years (iqr: . - . ). twenty-three patients [ % ( % ci . - . )] presented ata positivity. twenty-one of them ( . %) were females. anti-thyroperoxidase was positive in / patients ( . %) while patients presented anti-thyroglobulin positivity ( . %). both antibodies were present in / ( . %). patients showed the typical ultrasound findings of autoimmune thyroiditis, resulting in a prevalence of hashimoto's thyroiditis of . % ( % ci . - . ) in our cohort. three female patients developed subclinical hypothyroidism, whereas one male patient presented subclinical hyperthyroidism. the remaining patients were euthyroid. no statistically significant difference was observed in regard to age of jia onset, follow-up duration and jia subtype between the patients with or without ata. the proportion of females was marginally significantly higher (p= . ) in the group with ata positivity compared to children without thyroid antibodies ( . % vs . %, respectively). . % of patients with ata showed ana positivity compared to . % of patients without ata (p= . ). family history for aitd was significantly higher in children with thyroid antibodies positivity (p= . ). anti-tnf-alpha inhibitors were administered in only children ( %) with thyroid antibodies before their detection compared to . % of patients without thyroid antibodies (p= . ). multivariate regression analysis showed that patients with a family history for aitd were about four times more likely to develop ata (or . , % ci . - . , p= . ) and confirmed that ata positivity is less likely to occur in patients undergone anti-tnf-alpha therapy (or . , % ci . - . , p= . ). conclusion: a high prevalence of ata positivity and hashimoto's thyroiditis in patients with jia was found in our wide southern italian cohort. as expected, a positive family history of aitd was found to be associated with a higher risk to ata development during the follow-up. this finding supports the usefulness of an active screening for aitd in jia children, in particular in patients with relatives affected by thyroid disorders. notably, patients treated with tnf-alpha inhibitors resulted less likely to develop thyroid antibodies. further studies are needed to investigate the effect of anti-tnf-alpha therapy on thyroid autoimmunity in jia. introduction: the knee is considered by far the joint most frequently affected at jia onset. nonetheless, jia onset may present with unusual musculoskeletal involvement, eventually leading to a delay in the diagnosis and treatment. objectives: to identify the type and number of musculoskeletal sites affected at jia onset in consecutive patients seen at the study center in an years period. methods: records of patients with new diagnosis of jia from june to may available information in the medical history and standardized joint assessment at diagnosis, were retrospectively reviewed. systemic jia subtype according to ilar classification criteria were excluded. demographic and clinical features, including the type and number of joints at disease onset and diagnosis, were registered. data were analyzed through descriptive statistics. results: of a total of caucasian patients included in the study ( . % females), patients ( . %) had oligoarthritis, ( . %) rf-negative polyarthritis, ( . %) rf-positive polyarthritis, ( . %) psoriatic arthritis, ( . %) enthesitis-related arthritis (era). antinuclear antibody (ana) were positive in patients ( . %). the median age at onset was . years (iqr . - . ). at diagnosis ( . %) patients had only active joint, ( . %) had - active joints, ( . %) had ≥ . as expected the knee, the tibiotalar and the wrist were the most frequently affected joints ( . %, . %, . %, respectively); cervical spine was involved only in patients with polyarthritis (n= ). notably, of patients with monoarthritis at diagnosis presented with large joints involvement, among which n= isolated elbow and n= isolated wrist, and with small joints involvement (table ) . no sufficient data were available regarding the involvement of tendons and bursae, since the standard joint assessment form did not include them. nonetheless, additional patients, not included in the sample analysis, had isolated tenosynovitis involvement at diagnosis (n= both-sided ulnar extensor tendons; n= isolated tenosynovitis of the flexor digiti proprius; n= tenosynovitis of flexors digiti proprii). conclusion: our study confirms the knee, the tibiotalar and the wrist as the most frequently affected joints at jia diagnosis. on the other hand, musculoskeletal sites, such as small joints of hands and feet, the hip and the shoulder, usually involved in polyarticular jia, can be the site of disease presentation in oligo-and also mono-articular jia. further, jia may present with isolated tendon involvement. our results foster not to delay jia diagnosis in persistent synovitis occurring in infrequent joints and to include musculoskeletal sites, other than joints, in the standard articular evaluation. this could be realized by merging clinical and imaging (i.e. ultrasound) musculoskeletal examinations in the same assessment. none declared introduction: treatment response in jia is currently viewed as a binary outcome: response or non-response. however, jia is a heterogeneous disease and it is likely that different, identifiable subgroups of children and young people (cyp) may demonstrate different patterns of disease following treatment. identifying these response subgroups can assist the tailoring of stratified treatment approaches in jia. objectives: to identify subgroups of cyp defined by different trajectories of juvenile arthritis disease activity score (jadas) components following methotrexate (mtx) initiation for jia. methods: mtx-naïve cyp with jia were selected if enrolled prior to january in the bspar etanercept cohort register or the biologics for children with rheumatic diseases study at point of starting mtx. jadas components (active joint count, physician's global assessment (pga, - cm), parental global evaluation (pge, - cm) and standardised esr ( - ) were calculated based on data collected in the year following mtx initiation. multivariate group-based trajectory models were used to explore mtx response clusters across the different jadas components, which were log p transformed for analysis. optimal models were selected based on a combination of model fit (bic, relative entropy, average posterior probabilities), parsimony and clinical plausibility. clinical and demographic characteristics and achievement of acr pedi / by six months were compared across identified groups. results: of cyp selected, the majority were female ( %) and of white ethnicity ( %), with rf-negative jia the most common disease category ( %). six subgroups of cyp were identified with differing patterns of disease activity following mtx initiation. two groups improved across all jadas components: fast improvers ( %), and slow improvers ( %). persistent pga ( %), and persistent pge ( %) groups maintained one persistent disease feature but otherwise improved. one group relapsed ( %) and a final group had persistent disease overall ( %). there were no differences in active joint counts at mtx initiation between subgroups and all ilar categories were represented across each subgroup. however, cyp in persistent disease and slow improver groups had higher chaq, pga and pge scores at mtx initiation. those with persistent disease were also older at mtx initiation. the majority of cyp fulfilled acr pedi response (> % across every group). acr pedi achievement was low at months for slow improvers ( %) and high in the relapse group ( %). between % and % achieved acr pedi response in groups with persistent disease in one jadas component. we identify different patterns of disease activity within cyp initiating mtx, suggesting a simple responder/non-responder analysis at a set point may be over-simplistic. commonly used response measures did not adequately describe these heterogeneous response patterns. understanding both clinical factors associated with, and biological mechanisms underpinning, these subgroups would aid stratified medicine in jia. introduction: despite modern treatment and improved disease control, pain is the most common complaint in juvenile idiopathic arthritis (jia). knowledge about pain thresholds and pain sensitivity among young adults with jia is sparse. objectives: to study pressure pain thresholds (ppts) in young adults with jia, years after disease onset compared with controls. methods: consecutive newly diagnosed children with jia and a disease onset between - from central norway, were included in this prospective population-based long-term follow-up study. children with onset - were part of the nordic jia cohort , . age-and sex-matched controls were drawn from the national population register of norway. inactive disease and remission were defined according to wallace , . at the follow-up between - , data from a clinical examination and blood tests were included in addition to an investigator-blinded quantification of ppts. a digital algometer was used to manually apply pressure three times with an even rate at the upper and lower limb. ppts from jia and controls, and from subgroups of jia defined by disease status, were compared with multilevel models in stata. results: among the participants with jia, % were female, median age was . (iqr . - . ) years, median disease duration was . (iqr . - . ) years, % had an oligoarticular disease (persistent or extended), and % were in remission off medication. in the control group, % were female and median age was . (iqr . - . ) years. results from the multilevel regression model showed significantly lower ppts among participants with jia compared to controls (table ). in the jia group, participants with inactive disease had lower ppts than both jia in remission off medication and jia with active disease ( table ). the results were consistent for both upper and lower limb. conclusion: in this long-term follow-up study of young adults with jia, we found significantly lower ppts compared to controls. this may indicate that young adults with jia have altered pain sensitivity possibly due to accumulated earlier pain experiences. introduction: juvenile idiopathic arthritis (jia) represents the most common chronic rheumatic disease in childhood. non-steroidal antiinflammatory drugs (nsaids) and intra-articular steroids are the first line treatment for jia. systemic steroids, disease modifying antirheumatic drugs (dmards) and biologic drugs are used in children with severe disease. it is not possible at onset of disease to predict when a child can suspend pharmacological treatment, so children affected from jia have to continue pharmacological treatment for several months or years. anecdotal reports showed that rarely jia could present renal involvement due to uncontrolled inflammation or to long exposure to drugs. objectives: because no cohort studies investigating renal injury in children with jia are available, we designed this kind of study in our population. methods: we retrospectively evaluated patients suffering from jia. jia diagnosis was made according to ilar criteria, treatment was assigned with acr recommendations. for each patient we recorded the type and the duration of pharmacological treatment and the presence of renal injury. renal injury was defined by the presence of hypertension (systolic and/or diastolic blood pressure > th percentile for age, sex and height), proteinuria (persistentconfirmation within months-urinary protein/creatinine ratio> . mg/mg for children < years old and > . introduction: juvenile idiopathic arthritis (jia) is a pediatric rheumatic disease with partially unknown etiology and pathogenesis. neutrophils are the most common immune cell present in synovial fluid from inflamed joints in oligoarticular jia, but the role of neutrophils in the immunopathogenesis of oligoarticular jia has not been investigated. objectives: to characterize neutrophil phenotypes and effector functions in the circulation and in the inflamed joint during active arthritis in children with oligoarticular jia. methods: paired samples of blood and synovial fluid from children with oligoarticular jia were investigated regarding surface markers, phagocytic ability and oxidative burst. healthy blood neutrophils exposed to cell-free jia synovial fluid and healthy oral cavity neutrophils were studied as controls for synovial fluid exposure and transmigration respectively. results: synovial neutrophils had a shifted phenotype, characterized by high surface levels of neutrophil activation markers cd b and cd b, and mannose receptor cd and decreased levels of adhesion molecule cd l compared to circulating neutrophils. in comparison to oral cavity neutrophils, synovial neutrophils had higher levels of cd b and a different overall phenotype, suggesting that the phenotype shift in synovial compared to circulating neutrophils is not dependent on transmigration alone. jia synovial fluid in itself induced activation of healthy blood neutrophils, measured as increased cd b levels. synovial fluid neutrophils had impaired ability to phagocytose opsonized e. coli and to produce oxygen radicals upon neutrophil activation with phorbol-myristateacetate (pma) compared to circulating neutrophils. the impaired effector functions in synovial neutrophils was not dependent on the synovial fluid alone, as addition of cell-free synovial fluid to blood neutrophils did not alter phagocytosis and oxidative burst. conclusion: jia synovial fluid neutrophils are activated, have a "polarized" phenotype and impaired effector functions compared to neutrophils in the circulation. this study will help bridge the current knowledge-gap regarding the role of neutrophils in the immunopathogenesis in oligoarticular jia. methods: a case report is described. data was extracted from the medical chart of the patient and a literature review was undertaken. results: a -year-old girl was transferred to our tertiary center after being admitted for prolonged intermittent fevers, abdominal pain, fatigue and polyarthralgias. on examination, there was symmetrical proximal muscle weakness, a vasculitic lower limb rash, facial erythema with eyelid edema (fig. ) and oral mucositis. initial laboratory exams revealed pancytopenia, high muscle enzymes, increased erythrocyte sedimentation rate with moderately elevated reactive c-protein, and hypocomplementemia. she also had non-nephrotic proteinuria, without hematuria.further investigations showed a positive direct antiglobulin test, antinuclear antibodies, antidouble-stranded dna, anti-mi and anti-ku. serositis (pericardial and pleural effusions, ascitis) and hepatosplenomegaly were present. lower limb mri documented diffuse muscle edema. the diagnosis of an overlap syndrome of jsle and iim was established. while being treated for concomitant bacteremia, the patient became ill-appearing, with persistent fevers, worsened cytopenias, low fibrinogen and high ferritin and triglycerides, and a macrophage activation syndrome (mas) diagnosis was assumed. the patient received antibiotics and intravenous immunoglobulin, followed by methylprednisolone pulses, iv cyclosporine (cyc), hydroxychloroquine and supportive therapy with progressive improvement. due to hypertension (possibly related to cyc) and persistent proteinuria a renal biopsy was performed showing class iv lupus nephritis. after achieving clinical stability, cyc was switched to mycophenolate mofetil as an induction treatment, which is ongoing. conclusion: imm with sle os is uncommon, and has seldom been described in children. in addition to fulfilling sle criteria, our patient had clinical, laboratory and imagiologic evidence of imm. the presence of myositis specific antibodies (especially anti-mi ) further supports the diagnosis of an os rather than an atypical presentation of a lupus myopathy. juvenile dermatomyositis appears to be the imm subtype -it is associated with anti-mi , and mild heliotrope and eyelid edema are compatible. facial rash sparing the nasolabial folds is more suggestive of sle. mas is a rare but life-threatening condition that should be suspected in rheumatologic conditions and might be triggered by infections or disease flares. its identification may be particularly challenging at presentation, especially in sle where cytopenias are common. the reported prevalence in adult sle ranges from . % to . %; disease-specific criteria have been proposed. mas has occasionally been described in iim. in a patient with a predisposing condition, persistent fevers and illappearance must always prompt a mas workup, since early diagnosis and treatment are paramount. due to an early referral to a pediatric rheumatology center, the patient received a prompt diagnosis and treatment, which probably improved her prognosis. results: four of the five patients were female ( %) and all aged between and years. four of them had calcinosis at the time of diagnosis, although they may have had symptoms for to months prior to diagnosis. skin involvement was severe requiring multiple systemic and topical therapeutic agents in four out of the five patients -significantly more affected than the muscles. one patient had amyopathic subtype with normal childhood myositis assessment score (cmas) throughout. none of them had cardiac involvement. all had weakly positive anti-nuclear antibodies (ana); but were negative for myositis antibodies except the patient with most severe skin involvement and calcinosis (patient ), who was positive for anti-tif gamma antibodies. two of the three patients with calcinosis at onset had cyclophosphamide as the second line agent (chosen due to calcinosis) following systemic corticosteroids with complete resolution of the lesions after six cycles at mg/m . one patient responded to infliximab, which failed to work after months, following which cyclophosphamide was tried with good response. the other two patients were given cyclophosphamide after they failed to respond to rituximab, which did work for muscle disease. one patient had recurrent episodes of calcinosis needing surgical curettage despite initial response to cyclophosphamide and later ivig. introduction: systemic juvenile idiopathic arthritis (sjia) is a unique form of childhood arthritis. according to current understanding sjia is primarily driven by innate immune mechanisms at disease onset, but can progress towards chronic destructive arthritis, which can involve t cellular immunity. for yet incompletely understood reasons, sjia can be complicated by macrophage activation syndrome (mas), a severe hyperinflammatory condition characterized by a catastrophic cytokine storm resulting in multiple organ failure and high mortality. objectives: the sjia/mas working party (wp) aims to promote knowledge and international multidisciplinary collaboration among experts in the field of mas and sjia and to foster translational research in order to improve the care and outcome of these patients methods: currently pres members participate to the mas/sjia wp. the wp arranges an annual meeting during the pres congress, open to all members activities. the mas/sjia wp core team frequently report about ongoing activities by email. results: several studies are currently ongoing. a project aimed to establish and validate a risk score for mas in sjia patients using routine laboratory parameters of disease activity and severity has already completed the construction phase. recently, building of a validation cohort comprising data form patients from paediatric rheumatologic centers has been accomplished and is awaiting analysis (claudia bracaglia). a second project focused on mas patients with systemic thrombotic microangiopathy (tma) has just completed the collection of patients with mas and tma from centers in countries and results will soon be published (francesca minoia). furthermore, the mas/sjia wp participated in the data collection phase of a project on the development of new criteria for primary hlh (jan-inge henter and annacarin horne introduction: hemophagocytic lymphohistiocytosis (hlh) is an immunological disorder characterized by clinical signs and symptoms of severe uncontrolled inflammation, due to massive release of inflammatory cytokines. a delay in diagnosis is common, and is one of the factors that determine the poor outcome. hlh is classified into primary (phlh) and secondary (shlh). it is important to differentiate between the two as management differs. objectives: to describe the clinical and laboratory profile of hlh in infancy. methods: the electronic case files of children (age< year) diagnosed with hlh at the aims, kochi, kerala, between january and december , was retrospectively reviewed and described. results: eight infants, with age range . months to months, were clinically diagnosed with hlh. all were immunised and had normal development for age. none had a family history suggestive of hlh. third degree consanguinity was present in parents of patient no. and second degree for patient no. . duration of symptoms before presentation ranged from days to days. duration of follow up with us ranged from days to days, for those who expired. all, eight of them, had fever, anemia, thrombocytopenia, hyperferritinemia, transaminitis, raised ldh and crp. lymphadenopathy was present only in patient no. . before starting specific treatment patient no. had pseudomonas sepsis, patient no. had roseomonas gilardii infection; patient no. and were igm cmv positive but their pcr was negative. both of them had received prior blood transfusion. before making a definitive diagnosis of hlh patients were treated for puo, sepsis ? cause and acute liver failure. there was a delay in diagnosis for all patients except patient no. . all of them were treated with hlh protocol with modification according to clinical status of the patient. later, broad spectrum antibiotics, antifungals and antivirals were used for all. anakinra was tried for patient no. . five patients (phlh) succumbed to sepsis and mods and three (one phlh and two shlh) are continuing follow up. hsct was not done in any of them. other clinical features are shown in table . conclusion: making a timely diagnosis of hlh is difficult. differentiating phlh from shlh is very important as the management differs. genetic testing should be done for all infants with hlh. negative genetic study doesn't rule out phlh. the only curative treatment for phlh is hsct. shlh infants, once their primary condition is treated, can have normal survival. hyperbilirubinemia, splenomegaly, neutropenia, hepatomegaly, tissue hemophagocytes and hypertriglyceridemia were more common in phlh. health, kolkata diagnosed as having mas, admitted between july and april was tabulated and retrospectively analyzed . objectives: to evaluate the clinical features, laboratory findings and outcomes in pediatric mas, assess the response to different pharmacological therapies, and finally to identify possible factors associated with an unfavourable outcome. methods: the data of patients diagnosed with mas over the study period was analyzed for the clinical and laboratory features, treatment details, response to therapy and outcome. results: patients were diagnosed as having mas. primary illness was sjia in ( %), sle in ( %) and kawasaki disease (kd) in ( %). all had fever with varying degrees of multi systemic involvement. hyperferritinemia was universally present. in the absence of anakinra in india, pulse methylprednisolone with cyclosporine was used for treating the majority. patients ( . %) expired. patients on biologics and steroids can present with a silent mas which may be difficult to diagnose. conclusion: mas is a near fatal complication with protean manifestations and multi organ dysfunction. hyperferritinemia is characteristic, higher values being associated with increased mortality. patients resistant to steroids and cyclosporine had a poor prognosis. early recognition with aggresive management forms the backbone of a successful outcome as reflected by improved prognosis over successive years. late presentations with multiorgan dysfunction are associated with the poorest outcomes. methods: case report's description results: a two-year-old boy presented with one month history of fever associated with limping gait, cervical lymphadenopathy and skin rash. laboratory tests showed elevation of inflammatory markers and ferritin. by exclusion criteria, sjia was diagnosed and steroid therapy started. after a soft tissue bacterial infection, fever relapsed and laboratory tests were consistent with mas (day ): hb . g/dl, plt /mm ; fdp ug/l, crp mg/l, ferritin ug/l. high doses intravenous metilprednisolone and oral cyclosporin a (csa) were started. on day he presented a systemic capillary leak syndrome and acute myocarditis. he was admitted into the pediatric intensive care unit (picu) where intravenous immunoglobulin and subcutaneous anakinra (ana) were added. on day , due to an introduction: sjögren's syndrome is a systemic autoimmune disease characterized by dry syndrome and lymphocytic infiltration of the exocrine and extraglandular glands. pulmonary involvement in primary sjögren's syndrome occurs in - % of patients, with very heterogeneous manifestations, and occasionally as an initial mani-festation¹. diffuse interstitial lung involvement is one of the most characteristic pulmonary manifestations and the most frequent subtypes in lung biopsy are interstitial lymphocytic pneumonia and nonspecific interstitial pneumonia². objectives: -year-old girl presented to our hospital because of bilateral interstitial involvement with ground glass areas in lower lobes of both lungs on thorax and abdominal ct scan after for kidney stones follow-up. the patient had grade mmrc dyspnoea and dry cough but denied having symptoms of arthralgia or arthritis, photosensitivity, oral and genital ulcers, raynaud's phenomenon or episodes of dry mucosa. she had no history of autoimmune disease nor family antecedents of any autoimmune disease. a physical examination disclosed no finger clubbing or swollen superficial lymph nodes but indicated crackles on pulmonary auscultation. laboratory work showed elevated acute phase reactants, positive rheumatoid factor, positive antinuclear antibodies ( / ), positive cytoplasmic antineutrophil antibodies ( / ) and igg and iga hypergammaglobulinemia. an examination for autoantibodies were negative for anti-ss-a, anti-ss-b, anti jo- , anticentromere and anti-scl- antibodies. iontophoresis with pilocarpine and -minute walk test was also normal. pulmonary function tests demonstrated a mild restrictive impairment and a reduced percent diffusion capacity for carbon monoxide of %. fibreoptic bronchoscopy showed acute inflammation in bronchial mucosa. flow cytometry of bronchoalveolar lavage and cytology showed lymphocytosis with a % of cd and % of cd lymphocytes in bronchoalveolar lavage fluid. finally, a transbronchial lung biopsy lead to a definitive diagnosis, showing mixed interstitial inflammation and lymphocytic follicular hyperplasia with formation of germinal centers, suggestive of a lymphoid interstitial pneumonia of unreleased autoimmune etiology. throughout time, the patient reported progression of her symptoms with increasing dyspnoea, persistent dry cough, xerostomia and arthralgia. schirmer and rose bengal dye test were negative, and a salivary gland biopsy showed interstitial plasmacytosis and no igg plasma cells expression which suggested sjogren's disease. a high resolution computerized axial tomography was requested, suggesting organizing pneumonia in the context of sjogren's disease. methods: several studies indicate that lung involvement in sjögren is more frequent in advanced stages of the disease and rarely as an initial manifestation. sjögren's syndrome in paediatric age is rare and the subtype of secondary sjogren's is the most common. the course is longer, and the symptoms are more heterogeneous than in adulthood . the diagnosis in children is delayed, because children less frequently report dryness and frequently present with extraglandular clinical features suggestive of other autoimmune diseases. a systematic review on primary sjögren's syndrome in male and paediatric population reported a . % of pulmonary involvement in paediatric patients. pulmonary involvement is associated with an increase in the mortality of patients with sjögren's, therefore, it is essential to periodically monitor patients with respiratory symptoms, making an early diagnosis and treatment of the disease. results: -conclusion: we present a case of a patient with childhood sjögren's disease with atypical onset of disease with lung involvement. introduction: sarcoidosis is a multi-system disorder. little is known about its pathogenesis. in children, the early onset sarcoidosis phenotype including blau syndrome is more often seen. , the diagnosis of sarcoidosis is confirmed by demonstrating a typical non-caseating granuloma on a biopsy specimen. other granulomatous diseases should be excluded, in particular mycobacterial infections, crohn's disease and immunodeficiencies. the clinical presentation may vary depending on the organs involved and the age of the patient. , objectives: we are reporting the case of a boy with a presentation of bone sarcoidosis at a young age. this is a rare phenotype in children. methods: clinical details were retrospectively collated using routine clinical records. confirmation of diagnosis was confirmed with bone biopsy. results: a year old non-identical twin boy of ghanaian descent born in the uk had a slowly growing, painless frontal bone mass which started to develop from months of age. he was developmentally normal, with no history of fever, rashes or joint pains. examination findings revealed frontal bossing while the remainder of the musculoskeletal examination was normal. there was no evidence of rashes, hepatosplenomegaly and ocular examination was normal. the patient was initially referred for neurosurgical review with suspected fibrous dysplasia, after an initial mri scan of the head revealed abnormal marrow signal and expansion of the frontal bone, with no soft tissue swelling. however, the ct scan of the calvarium was not suggestive of fibrous dysplasia. consequently, bone biopsy was performed demonstrating inflammation with granuloma formation. he was referred to infectious diseases and rheumatology. there was no travel history and no tb contact. quantiferon tb was negative. infectious work-up was negative especially for mycobacterial infections. rheumatology work-up identified on skeletal survey another bone location: a well-defined lytic lesion in the right distal fibula that was biopsied. infection cultures and pcr were negative. histopathology identified fibrous tissue and poorly formed granulomas. laboratory investigations revealed a mild microcytic anaemia with iron deficiency and eosinophilia. he had normal serum calcium and vitamin d and his esr was mm/hr. ana, anca and rheumatoid factor were negative, and complement c and c were normal. his serum angiotensin converting enzyme (ace) level was raised at nmol/ ml/min (normal < nmol/ml/min). investigations revealed mild renal impairment with normal urinary tests including normal calcium, protein and tubular proteins. ultrasound of the kidneys was normal. chest x-ray was normal. lung function was performed and was normal. dlco couldn't be performed due to low lung volume. vascular and inflammation genetic panel identified a variant in the nemo gene. functional studies excluded nemo deficiency and patient did not display any of the clinical features. however, a pattern of dysregulated t cells response was identified. he was treated with oral steroids and methotrexate. the oral steroids were successfully weaned off. he has been successfully treated with methotrexate mg s/c to initially stabilise disease with no bone growth, and had no significant side effects. repeat mri years later showed increased burden of disease with other newly affected sites however, including the right femoral diaphysis and signal changes in the left tibial metaphysis. based on the mri and increasing musculoskeletal pain, decision was made to escalate to anti-tnf (adalimumab) with good clinical response. conclusion: bone sarcoidoisis is rare in children but this should be considered in the differential diagnoses when granulomatous inflammation is identified on histopathology. response to steroids and methotrexate is usually good but some patients will need escalation to anti-tnf. the most worrisome non-rheumatic condition causing persistent night pain in children which closely mimics arthritis is malignancy , . it is vital to pick up subtle clues at an early stage especially in absence of hematological manifestations , organomegaly and lymphadenopathy. to reveal early clinical clues in pediatric patients with predominant musculoskeletal (msk) night pains who were initially diagnosed as suffering from some form of chronic arthritis but ultimately turned out to be affected by malignancy. methods i gathered a data of five pediatric patients fulfilling above mentioned criteria who were seen at dev children's hospital between january and march . it included demographics, clinical presentation and laboratory results. all above cases reemphasize the need for an extremely detailed history pertaining to characteristics of pain & pattern recognition in pediatric rheumatology. prolonged fever , persistent msk night pain, persistent limp, upper limb and hip joint involvement which is unlikely for jia at onset are proven to be the earliest subtle clues which should not be missed. other constitutional symptoms, respiratory, cardiovascular, ophthalmologic or osteoarticular involvement were absent. growth was unaffected. auditory tests were normal. systemic antibiotic treatment and local steroids were ineffective. laboratory findings were unremarkable, with only mild elevation of esr ( mm/ st hr). ana and anca were absent in repeat meausrements ( months intervals). cardiovascular disease was excluded. abdominal us was normal. on the basis of relapsing bilateral auricular chondritis and confirmatory histological findings revealing inflamed cartilage from the pinna of the ear with chondrocyte degeneration, perichondrial infiltrates of lymphocytes, plasma and polymorphonuclear cells and replacement of cartilage with fibrous tissue perivascular infiltrates of polymorphonuclear cells and lymphocytes, relapsing polychondritis was diagnosed. one month nsaids trial, pending histology results was ineffective. methotrexate sc and steroids mg/kg/d gradually tapered over a -month period were given with significant improvement of auricular inflammation and normalization of markers of inflammation. auricular chondritis worsened after steroid withdrawal and adalimumab was added to treatment with significant improvement of auricular inflammation in months. in the following months auricular chondritis relapsed during uris with mild elevation of esr ( mm st hr) and crp ( mg/l). after months of treatment, in an effort to prolong the intervals of adalimumab administration, bilateral auricular chondritis relapsed. after months of mtx and months of adalimumab administration inflammation was put in complete remission. the following year no flares or involvement of other systems were observed, under methotrexate and adalimumab treatment. conclusion: in this patient isolated auricular relapsing polychondritis was unresponsive to nsaids. steroids and methotrexate greatly improved inflammation but did not induce complete remission. complete remission was achieved by addition of adalimumab to methotrexate treatment, which also allowed for steroids discontinuation. none declared first ever single center study revealing spectrum of rheumatic diseases in children from an indian state of gujarat d. b. pandya, on behalf of dr mehul mitra, pankaj buch, sonal shah, there is very limited information and awareness about pediatric rheumatic and immunodeficiency diseases amongst primary physicians , , in gujarat and to make this matter even worse, we are not having a single exclusive pediatric rheumatology and immunology centre for a population of around million. to guesstimate a status of children with rheumatic and immunodeficiency diseases in gujarat and spectrum of these diseases at dev children's hospital. methods i gathered a retrospective data of patients who attended dev children's hospital between january and january . out of these, children with confirmed diagnosis of inflammatory rheumatic diseases and suspected primary immunodeficiencies were included. patients with non-inflammatory musculoskeletal(msk) pains and non-rheumatic diseases causing msk pains were excluded. my collected data included referral details, demographics, clinical presentation, laboratory results and diagnosis. majority of the cases were referred by pediatricians, orthopedicians, hemato-oncologist and general physicians. main reasons for referral were joint involvement , undiagnosed fever , multisystem disease and elevated inflammatory markers. many physicians had put a diagnosis like rheumatoid/rheumatic arthritis, autoimmune disease or connective tissue disease. almost % of patients had been evaluated with rf, aso titer, ana and joint imaging irrespective of clinical pattern by their primary physicians before referral. fever , msk involvement, extreme fatigue, constitutional symptoms, skin and mucosal involvement were prominent complaints noted by me. family history of rheumatic, primary immunodeficiency (pid) or consanguinity was found in / of patients. anemia of chronic disease, elevated esr and thrombocytosis were almost universal laboratory findings in our cohort. rheumatic diseases in children are not anymore rare but due to lack of expertise and awareness , these children are not getting diagnosed. many cases were advised unnecessary rheumatological investigations even before referral. results: a -year-old female patient was referred to the rheumatology clinic at our hospital with a previous history of fever of °c ( . ºc), loss of appetite, and acute polyarthritis of wrist, knees, and ankles. at that time, laboratory exams revealed a hemoglobin of . g/dl, c reactive protein . mg/l, and antistreptolysin o titers of ui/ml (normal range < ui/ml. clinical symptoms were relieved only after using nsaids. after months, the patient returned to our hospital with a -month history of weight loss and claudication related to pain and daily morning stiffness ( minutes) on her right ankle. new laboratory findings demonstrated positive antinuclear antibodies : , negative rheumatoid factor, and alpha- -acid glycoprotein of mg/dl (normal range: - mg/dl). clinical signs suggestive of chronic arthritis with exuberant swelling of the ankles were observed on physical examination (figure a). she was screened for tuberculosis (tb) and had a positive ( mm) tuberculin skin test (figure b). chest ct revealed infiltrative soft tissue mass in the posterior mediastinum, with homogeneous contrast enhancement (figure c). magnetic resonance imaging of both ankles was performed and demonstrated bilateral and symmetrical tibiotalar arthritis and prominent tenosynovitis of extensors, flexors, and fibularis tendons (figure d). right ankle synovial biopsy revealed no granulomas and joint fluid culture was negative for mycobacterium tuberculosis, confirming reactive arthritis (poncet's) and tenosynovitis, that may follow mycobacterial infection with no infective agent in the joints. conclusion: to our knowledge, there is no report of poncet's disease associated with inflammatory tenosynovitis, showing the particularity of this case. the patient's symptoms resolved after two months of anti-tb therapy. introduction: cacp is characterized by congenital or early-onset camptodactyly (usually bilateral); non-inflammatory arthropathy (more frequently in the wrists, knees, ankles, elbows, and hips); coxa vara (reduction of the angle between the neck and shaft of the femur); and non-inflammatory pericardial effusion (a late manifestation, less frequently reported). recognizing the radiological aspects of this syndrome and differentiating it from jia is crucial since cacp has no effective treatment and jia is usually treated with nsaids and methotrexate ( , ) . objectives: to report a rare case of cacp syndrome mimicking jia. methods: case report and literature review. results: a -year-old male patient presented with arthropathy characterized by painless progressive swelling and restricted movement of the hands, hips, knees, and ankles since the first year of life. he had a family history of camptodactyly from his paternal grandfather. on physical examination, symmetric camptodactyly of the hands and feet was observed (a). he had no history of rash or weight loss and inflammatory markers were unremarkable. the echocardiogram was normal. the pelvic radiograph showed a widening of the joint space and bilateral coxa vara. magnetic resonance imaging (mri) of the hips (b) and knees (c) was performed and depicted large joint effusions (arrows, b and c) with normal synovial thickness and mild synovial enhancement in all joints, without bone marrow edema-like signal. a synovial biopsy of the knee was performed and revealed mild synovial hyperplasia without inflammatory cells. the patient was diagnosed with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (cacp -omim ), a recently described genetic disorder with no gender predominance identified to date ( ). conclusion: an important differential diagnosis of cacp is juvenile idiopathic arthritis (jia), a painful inflammatory chronic arthritis that can cause not only joint effusions due to synovial inflammation, but arthritis was the most frequent extraglandular manifestation. renal tubular acidosis represented the typical expression of renal involvement ( cases). neuromyelitis optica and aseptic meningoencephalitis ( and cases, respectively) were the most typical neurologic manifestations. two cases of interstitial lung disease and one of pulmonary hypertension were reported. almost all patients had autoantibodies, mostly ana ( / patients) and anti-ssa/ro ( / patients). the schirmer test was performed in less than half of the patients, of whom % tested positive. a positive result of minor salivary biopsy was reported in / cases with available data. juvenile idiopathic arthritis was the most frequently associated disease, followed by systemic lupus erythematosus ( and cases, respectively). no significant differences between patients with or without parotitis were found except that patients with parotitis showed increased levels of crp more frequently than those without it (p= . ). patients with anti-ssa/ro had more frequently a positive schirmer test (p= . ). the presence of rf was significantly associated with dry mouth (p= . ), arthritis (p= . ), and rash (p= . ). a positive minor salivary biopsy was more common in children with dry eyes than in those without this clinical feature (p= . ). arthritis was more frequent in patients with other diseases than in those with primary ss (p= . ). we further investigated ss features according to the age groups (≤ years, - years, ≥ years). parotid involvement was inversely proportional to the age and occurred more frequently in younger patients ( % of those ≤ years; p= . ). interestingly, the rate of anti-ssa/ro positivity increased with age ( % of those ≥ years; p= . ). conclusion: even though parotitis was the most frequently reported feature, a wide range of clinical manifestations in children with ss has been reported so far. a better knowledge of css features will help to pave the way for the development of css specific diagnostic criteria. none declared introduction: pachydermodactyly (pdd) is a rare benign fibromatosis, characterized by progressive painless swelling of soft tissue of proximal interphalangeal (pip) joints without inflammation signs. generally pdd affects pip joints of the fingers, rarely of the thumb. the involvement is typically symmetrical, in few cases unilateral. it usually occurs more frequently in young males. etiology is unknown, but it arises from mechanical stimulation of periarticular skin (i.e repetitive rubbing, interlacing, and cracking of fingers). pdd has to be considered in the differential diagnosis of arthritis (i.e. juvenile idiopathic arthritis, jia) and many syndromes (i.e. progressive pseudorheumatoid dysplasia). prognosis is good with cessation of mechanical stimulation the recurrent paroxysmal appearance of inflammatory lumps (local erythematous tender swellings, which partially respond to antiinflammatory agents), accompanied by elevated inflammatory markers during flares, suggest that fop may be an autoinflammatory disease. the episodic formation of bone, often following a trivial injury, suggests that innate immune-related triggers induce tissue transformation through the bmp pathway. moreover, interleukin- β (il- β), a well-known mediator of the innate immune system, has been linked to ho and mineralization in mesenchymal stem cell cultures derived from human bone marrow. we hypothesized that treating fop patients with anti-il- agents could help ameliorate the progression of this devastating disease. we report our experience treating two fop patients with anakinra and canakinumab. objectives: to decrease the frequency of fop paroxysms, and/or limit the symptoms and extent of residual lesions, by using anti-il- agents. methods: patients' data and blood il- levels were analyzed to characterize the efficacy of anti-il- treatments in ameliorating the natural progression of fop. results: a . year old boy and a year old girl were diagnosed with fop, both clinically and genetically (the typical r h mutation was found). various treatments, including high-dose corticosteroids, pamidronate infusions, celecoxib, monteleukast and sirolimus, did not change the course of the disease. both patients are receiving canakinumab (the male patient was initially treated with anakinra). the male patient has been treated for over years. flare rate was markedly reduced from one new lump every days to approximately one every days ( figure ). the lumps involved in almost all of these flares are the same: at the left scapular base and within the sternocleidomastoid muscle. the female patient has been treated for a year, and has not experienced any ho flares during canakinumab treatment. temporarily withholding canakinumab in both patients, led to serious flares weeks after the last dose. notably, while undetectable levels of il- β (< . pg/ml) were found in the three plasma samples obtained from the male patient during treatment with anakinra or canakinumab, high levels (up to . pg/ml, about -fold higher compared to average levels measured in healthy controls) were found in his plasma samples collected during the flare ( figure ). in contrast, il- and il- plasma levels, measured before, during and after withholding treatment, were comparable or slightly higher than those observed in healthy controls ( figure a , b). conclusion: we report here, for the first time, that anti-il- agents were found efficacious in treating two fop patients. we also found markedly increased il- β levels during flares, which normalized following the treatment. we suggest a role for il- β in the pathogenesis of this disease. although it is too soon to conclude whether fop may be included under the umbrella of auto-inflammatory syndromes, anti-il- agents can be effective in ameliorating the natural progression of fop. introduction: musculoskeletal symptoms are one of the common reasons for applying to rheumatology departments in general practice . although inflammatory causes are generally considered in the foreground, it is known that non-inflammatory causes including genetic diseases may also be responsible. the absence of signs of inflammation (morning stiffness, redness, tenderness) and normal inflammatory markers in laboratory findings may support nonrheumatologic diseases . objectives: to present genetic disorders that can mimic rheumatologic symptoms and to answer when genetic diseases should be considered in the differential diagnosis in patients presenting with rheumatological complaints. methods: we retrospectively evaluated patients who applied to hacettepe university pediatric rheumatology department with musculoskeletal compliants between january and december and had been consulted to genetics departmant. the rate and degree of consanguinity, clinical diagnosis, indication for consultation, accompanying musculoskeletal and other findings had been recorded. the diagnosis of genetic diseases were based on physical examination, radiological evaluations and genetic analysis. results: a total of patients, boys ( . %), with a mean age . ± . years were included in the study. the rate of consanguinity was . %. the most frequent referral to the genetic department was the presence of skeletal anomalies (n: ) such as camptodactyly, clinodactyly, and bone shortness accompanying joint findings. other causes include short stature (n: ), joint deformity (n: ), joint hyperlaxicity (n: ), dysmorphic findings such as atypic facial appearance (n: ), accompanying diseases that may be part of a syndrome (n: ), genetic diagnosis suspicion according to the results of radiological examination (n: ) and joint findings without clinical and laboratory signs of inflammation (n: ). distribution of joint involvement in patients with genetic disease were hands, knees, and hips respectively. in the laboratory evaluation of patients presenting with joint swelling and arthralgia, acute phase reactants (erythrocyte sedimentation rate and c-reactive protein concentrations) were within normal reference values. one third of the patients ( . %) had a final diagnosis of a genetic disease. the diagnoses of these patients were as follows; cacp (camptodactyly, arthropathy, coxa vara deformity and pericarditis) syndrome (n: ), trichorhinophalangeal syndrome (n: ), progressive pseudoromatoid dysplasia (n: ), lig syndrome (n: ), m syndrome (n: ), h syndrome (n: ), spencd (spondyloenchondrodysplasia, n: ), and nonspecific connective tissue disease (n: ). conclusion: genetic syndromes with musculoskeletal findings are often unrecognized and misdiagnosed as rheumatologic diseases leading to unnecessary procedures and treatments. summarizing the genetic diagnosis spectrum that can be detected in these patients will increase the awareness of physicians. results: according to the results of observation, the disease was more common in the age group of - years ( %), to a lesser extent among children in the group of - years ( %), less often in the group of - years ( %). when examining infectious agents, zoonotic infection was detected in % (listeria monozytogenes, yersinia enterocolitica). clinical course of nodular erythema in this group was characterized by an expressed activity of the inflammatory process with multiple elements in the lower and upper extremities, joint syndrome, increased esr to ± . mm per hour, crp ± . mg\l. the disease was preceded by an episode of acute infection with an increase in body temperature, intoxication, in some cases with short-term intestinal syndrome, pharyngitis. the rashes were persistent and recurrent, with a slow regression of laboratory activity. streptococcal etiology of nodular erythema was detected in % of cases. there was an increase in esr to ± . mm per hour, crp ± . mg/l, a significant increase in antistreptolysin on average ± % iu / ml. with an increase in individual cases to iu/ml. in % of cases, erythema nodosum developed after an intestinal infection. among the pathogens were identified sh. disenteria, e. coli, yersinia enterocolitica, enterovirus. the disease was characterized by moderate activity, a good response to etiological therapy and a short course of nsaids . an interesting fact was the development of nodular erythema in % of cases caused by the epstein-barr virus in groups of children from to years and - years. they had clinic picture with normothermia, no symptoms of intoxication, periodically occurring elements of nodular erythema on the shins, no blood changes. therapy aimed at eliminating the virus gave a positive result and did not require specific anti-rheumatic therapy. in % of cases, the etiology of nodular erythema was not defined. the clinical course of nodular erythema in children depends on the infectious agent that was the trigger of the pathological process. the higher activity and duration of the disease is caused by zoonotic infection, which requires more active antiinflammatory therapy with corticosteroids, which may be associated with the activation of autoimmunity. this group of children was taken for further observation as a group at risk of developing systemic connective tissue disease. changes in the etiological structure of nodular erythema and treatment tactics require further study. introduction: sjögren syndrome (ss) is a chronic autoimmune disorder characterized by inflammation of the lacrimal and salivary glands leading to oral and ocular dryness. childhood ss is rare and poorly defined and underdiagnosed owing to the lack of childspecific diagnostic or classification criteria. objectives: the purpose of this study is to describe cases with pediatric ss in order to better clarify the characteristics of the disease in the pediatric age. methods: we retrospectively reviewed medical records of patients (pts) with pediatric ss referring to three italian pediatric rheumatology centers. due to lack of childhood validated ss-specific criteria, physician diagnosis was the only inclusion criteria. results: we collected data on pts ( females). the mean age of disease onset is . yrs (median . , range - ). the mean age of diagnosis is . (median . , range - ). the follow up period varied from . to . yrs (mean . , median . ). the most common manifestations were articular involvement (mainly with arthralgia) ( / pts) and parotid/salivary glands swelling ( / pts). xerostomia and xerophthalmia were found in / pts and in / respectively. vaginal dryness was reported only by one pt. fever and fatigue occurred in / and / pts respectively. we also recorded cases of circulating immune complexes manifestations in pts, purpura (n= ) and glomerulonephritis (n= ). we observed an endocrine involvement in pts ( metabolic syndrome, autoimmune thyroiditis). abdominal pain was found in / pts. all pts were positive for autoantibodies (positivity for ana or anti-ssa or anti-ssb or fr) at presentation. rf test results were available in pts, all positive. positive ana (titer> / ) and anti-ssa were present in / pts and in / respectively. hypergammaglobulinemia (range , - . g/dl) was found in / pts ( na). abnormal schirmer test was observed in the half of cases ( / ). minor salivary gland biopsy was performed in pts resulting in histological evidence of focal lymphocytic sialadenitis in / . sonographic evaluation of salivary glands was abnormal in all of the patients ( / ). with regard to treatment, / pts received corticosteroids and eight were also treated with one or more dmards such a hydroxychloroquine (n= ), methotrexate (n= ), azathioprine (n= ), leflunomide (n= ). biological therapy was used in patients for systemic involvement: received belimumab and then rituximab, while the other patients received rituximab. conclusion: xerostomia and keratoconjunctivitis sicca were not common in our series while recurrent parotid swellings were more frequent than what reported in adults. pediatric recurrent parotitis should increase the suspicion for sjögren syndrome. current diagnostic criteria for ss do not include parotitis and therefore, the incidence of ss may be under-recognized in childhood. the disease is not always benign and patients with severe course may need second line treatment including immunosuppressant and biologics. introduction: improving our understanding of pediatric rheumatological (pr) patient population is crucial for pediatric rheumatologists to know rheumatic disease epidemiology and to raise awareness leading to early detection. we didn't find studies of pr disorders presenting in the first year of life. objectives: the aim of this study is to assess the prevalence of pr disorders with onset in the first year of life. methods: we retrospectively studied patients observed in our pediatric rheumatology unit between january st of and december st of . we defined acute (< weeks), subacute (≥ and < weeks) and chronic (≥ weeks). results: a total of patients were observed in years. diseases' onset occurred in the first decade of life in patients ( %) and in the first year of life in ( , %). among the latest group, chronic inflammation was the most frequent group of diagnosis ( %), followed by recurrent inflammation ( %), acute inflammation ( %), infection ( %), infiltrative/ degenerative disorders ( %) and subacute inflammation ( %). the remaining patients ( %) were diagnosed with other disorders classified as miscellaneous. among chronic inflammation group, patients were diagnosed with juvenile idiopathic arthritis ( systemic); had neonatal lupus and one patient had polyarteritis nodosa. among recurrent inflammation group, patients were later diagnosed with pfapa (periodic fever, aphthous stomatitis, pharyngitis and adenitis), were diagnosed with behçet disease and had an autoinflammatory disorder. acute vasculitis was diagnosed in patients ( kawasaki disease and acute hemorrhagic edema of infancy). among infectious diseases group, there were two cases of congenital syphilis with arthritis and two cases of osteomyelitis secondary to bcg vaccination. conclusion: rheumatological diseases presenting in the first year of life are not exceptional. although many patients didn't have a definitive diagnosis at the beginning of the symptoms, many of them were later diagnosed with rheumatic disorders, mostly chronic inflammation ( %), which requires early diagnosis, specific treatment and long-term follow-up. rheumatic diseases must be considered as differential diagnosis in the first year of life in order to avoid delayed intervention and long term disabilities and sequelae. ( ), on the other side measles-induced mas has rarely been reported ( ). objectives: we present the case of a child known to have sjia in remission, who presented a measles primary infection and a secondary kd complicated by mas. methods: a years old girl, not fully vaccinated and known to have sjia in remission under methotrexate, presented for frequent high grade fever of days duration associated with flat flash red spots on the face and trunk as well as the palms and soles. a koplik's spot was identified. conjunctivitis and coryza were also present. initial viral serology, including measles, returned negative. fever persisted and on day , edema of both hands and feet appeared with bilateral cervical adenopathy, erythematous tonsils, gingivitis, cracked lips and hepatomegaly was noted. all cultures were negative and chest x-ray was normal. inflammatory markers rose up. viral serology was repeated and measles igm came back positive. cardiac ultrasound ruled out coronary aneurism and the ophthalmic exam showed no uveitis. kd criteria were met and g/kg of intravenous immunoglobulins (ivig) were administered. after hours of clinical improvement, fever reappeared and the patient returned to be ill looking although the rash regressed. we noted high ferritine( ng/ml) together with low c , decrease in platelets( x /ml) and elevation of hepatic enzymes, ldh and cpk, without increase in the inflammatory biomarkers. mas was suspected and a bone marrow aspirate showed the presence of mild macrophage hemophagocytosis. antibodies for lupus and auto-immune myositis were all negative. steroids were given, fever disappeared, and spectacular clinical and biological improvements were objected. weeks later, desquamation of all extremities was noted. sars-cov- was not investigated because historically this case presented year earlier than the pandemic. results: we hereby report, for the first time, kd and mas triggered by measles infection in a child with sjia in remission. the exact mechanism involved in kd-induced mas and measles-induced mas has not yet been defined but a defective immune response is suspected ( ). conclusion: significant similarities and overlap between measles, kd, sjia and mas make an early diagnosis very challenging ( )( ). the recent covid pandemic emphasizes how a viral illness can be responsible of kd and sometimes degenerating in mas. we report this clinical case as an example of a systemic inflammatory syndrome (sis) taking place after a viral infection to measles. in the era of covid pandemic and secondary sis in children, an additional challenge is present in regions lacking measles vaccine coverage. none declared the musculoskeletal manifestations of scurvy: a diagnostic challenge for the rheumatologist p was a -year-old boy, with autism spectrum disorder, malnutrition and severe food selectivity, admitted to our unit for refusal to bear weight and bruises in lower limbs. the auxological evaluation showed a strongly dystrophic aspect. coagulation profile and main organ function markers were normal. at nutritional biochemical parameters evaluation, iron and vitamin c deficiencies were detected (vitamin c: μmol/l). oral vitamin c therapy was started, with prompt clinical response. p was a -year-old boy with autism spectrum disorder, admitted to our unit for lameness and difficulty in walking for a month. at clinical examination, a mottled skin at lower limbs was noted. joint examination was normal. auxological parameters and main blood tests were adequate for age. given the presence of food selectivity, he underwent serum vitamin c dosage ( μmol/l); hence he started oral vitamin c therapy, with rapid clinical improvement. p was a years old boy who was referred for coxalgia and fever. at clinical examination, pale skin, gingival hyperemia, and pain in mobilization of the left hip were present. microcytic anemia was detected, but main organ and inflammatory markers were normal. no evidence of infection was present. x-ray of femur and knee showed morpho-structural alteration of the distal metaphysis bilaterally. a low intake of fruit and vegetables was reported; hence, dosage of vitamin c was performed, resulting reduced ( . μmol/l). he started vitamin c oral therapy with clinical response. p was a -year-old girl with behavioral disorder and intellectual disability, admitted for fever and right knee swelling which appeared two days after a right leg burning. c-reactive protein and esr were elevated and ultrasound exam confirmed intra-articular knee effusion. suspecting a septic arthritis, antibiotic therapy was started with laboratory normalization and partial clinical improvement. considering the persistence of knee swelling after nine days of intravenous antibiotic therapy, the presence of gingival hyperemia and history of food selectivity, vitamin c dosage was practiced ( μmol/l). oral vitamin c was administered with complete clinical resolution. conclusion: although scurvy is considered a disease of the past, it still occurs nowadays. food selectivity associated to autism is a major risk factor for vitamin c deficiency in childhood. rheumatologists should take into account the diagnosis of scurvy in the diagnostic approach of musculoskeletal disorders in children, especially when development disorders are present. . %), juvenile dermatomyositis (n= ), sarcoidosis (n= ), granulomatous polyangiitis (gpa) (n= ), sting-associated vasculopathy with onset in infancy (savi) (n= ), and oligoarticular jia (n= ). respiratory symptoms were present in ( . %) patients at the time of primary diagnosis. in other patients, the time period between the diagnosis of the rheumatic disease and the onset of the respiratory symptoms ranged from to years. cough, the most common symptom, was present in ( . %) patients. six patients manifested with cough and sputum. six ( . %) patients had shortness of breath and one patient had hemoptysis. on the physical examination of one patient, rales and clubbing were detected. high resonance computerized tomography (hrct) was performed in all patients. hrct findings were as follows; lymphadenopathy in patients ( . %), ground glass appearance in patients ( . %), consolidation in one patient, pleural effusion in one patient, pulmonary nodule in patients ( . %), fibrosis in one patient, cystic lesions in patients ( . %), septal thickening in patients ( . %), bronchiectasis in one patient, and reverse halo sign in one patient. in echocardiographic examination, only one patient had pulmonary hypertension. three patients underwent open lung biopsy, and diagnosis was made with pathological examination of the lung tissue. of these three patients, two ( . %) had lymphocytic interstitial pneumonia (lip), and one patient had chronic inflammation and focal fibrosis. infectious lung disease was not detected in any patient. ten patients ( . %) had interstitial lung disease associated with rheumatic disease, one patient had pulmonary hemorrhage, one patient had pulmonary involvement of gpa, one patient had pulmonary involvement of sarcoidosis. there was no statistically significant difference between the first and last spirometry and dlco values during the follow-up period. mortality was . % ( / ) in this cohort. active disease was significantly associated with abnormal tc, hdl, and tg levels (p= . *), (p= . *) and (p= . *) respectively. multivariate analysis of the factors affecting abnormal cholesterol level revealed that sle is a significant predictor of abnormal cholesterol level . presence of jsle increase risk of abnormal cholesterol times more than cases without jsle. the overall percent predicted was %. active disease is a significant risk factor for abnormal tg with increased risk of abnormal tg by . among cases with active disease than cases with inactive disease. the overall percent predicted was . %. conclusion: children with rheumatic diseases showed significant lipid profile abnormalities. abnormal tc, hdl and tg are significantly associated with active disease. presence of jsle increase risk of abnormal cholesterol. active disease is a significant risk factor for abnormal tg. therefore, lipid levels should be monitored regularly and managed in patients with paediatric rheumatic diseases to minimize the longterm risk of cvd. methods: non-experimental, cross-sectional and descriptive study. a confidential survey was conducted online, aimed at residents and attendings who deal with musculoskeletal pain. were addressed with the definitions of arthralgia, arthritis, myalgia, allodynia and hyperesthesia (between five to seven options) with only one correct answer. correct definitions: arthralgia (pain localized to the joint or periarticular structures, as a only manifestation); arthritis (criterion one or criterion two: -joint swelling or intra-articular effusion / -limitation of joint mobilization associated with at least one of the following: a) pain b) tenderness c) swelling d) heat); myialgia (pain with muscular origin or referred to muscle, regardless of its etiology); allodynia (pain resulting from usually non-painful stimulus); hyperesthesia (coexistence of allodynia plus hyperalgesia -exaggerated responses to tactile and thermal nociceptive and nonnociceptive stimuli the association of pure red cell aplasia (prca) with thymoma led to the discovery of the autoimmune mechanisms involved in the pathogenesis of this rare disease. till date many adult case reports have revealed a strong link between prca and autoimmune diseases, endocrine disorders, rheumatic diseases, autoinflammation and immune dysregulation. [ ] [ ] [ ] [ ] [ ] objectives to stimulate a search for the genetic and immunological roots for a . years old girl with syndromic face, pure red cell aplasia, type diabetes and polyarthritis. methods this is a story of a . years old girl with pure red cell aplasia, type diabetes and polyarthritis. she was normal till months of age. at the age of months, she was diagnosed with type diabetes. she was evaluated by her paediatrician in view of generalized hypotonia, deformed pinna, low set ears, midfacial hypoplasia, blue sclera, umbilical hernia and retracted eyelids. she had multiple episodes of seizures during next few months. to me, she was presented with one year history of polyarthritis with severe pallor requiring frequent blood transfusions. family history was inconclusive. musculoskeletal examination showed polyarthritis involving right knee, bilateral ankles, fingers and toes. further examination revealed haemolytic facies and hepatosplenomegaly. i was not able to make out any facial dysmorphism mentioned earlier by her paediatrician. results: table conclusion early age of onset, pure red cell aplasia, autoimmune and endocrine manifestations with some doubtful facial dysmorphism inspired me to suspect some known or unknown immune dysregulation syndrome in this child. genetic analysis would be the best possible option in this scenario if financial condition permits. introduction: galactosialidosis (gs) is a rare inherited lysosomal storage disorder (lsd) which is characterized by a defect in the lysosomal glycoprotein catabolism. here we report, for the first time, a case of a child affected by gs who presented with recurrent episodes of extensive joint inflammation in both knees. knowledge on gs related inflammatory joint pathology is lacking, which hampers evaluation of possible mechanisms that could give an explanation for the significant arthritic joint abnormalities as observed in our patient. objectives: the aim of this study is to describe the clinical presentation as well as the laboratory, radiologic and microscopic features of this extremely rare presentation of gs. furthermore, we conduct a literature review on lsd's complicated by arthritis in order to evaluate potential mechanisms that could explain the extensive inflammatory joint swelling observed in our patient. methods: in this study we present a -year-old turkish boy who was diagnosed with gs (late infantile form) at months of age. from the age of years, the boy presented with episodes of inflammatory joint pathology of the knee. informed consent was obtained. alongside the case report, a literature review using medline was conducted. an extensive list of known lsd's was combined with the terms: "arthritis", "joint inflammation", "synovitis" and "synovial inflammation". cases in which joint inflammation was based on a probable cause other than the underlying lsd were excluded. results: in the present case, owing to comprehensive examinations (i.e. laboratory tests, imaging and microscopic examination) multiple possible causes for the recurrent inflammatory joint pathology could be rejected (i.e. no signs of infectious arthritis, reactive arthritis, osteoarthritis, arthritis secondary to a malignancy or crystal induced arthritis). a diagnosis which could explain the clinical picture is the jia subtype: ana negative oligo-articular jia. however, microscopic examination showed numerous foamy macrophages with extensive vacuolization in the synovial tissue of the inflamed joint, which is not associated with jia. given the evidence of storage products within the macrophages of the inflamed synovial tissue and no conclusive diagnosis, gs itself should be considered as the primary cause for the recurrent arthritis. an in-depth literature review using medline for data on inflammatory joint pathology in lsd's showed that lsd subtypes (i.e. fabry disease, farber lipogranulomatosis, gaucher disease type , mucopolysaccharidosis ix, a-mannosidosis, fucosidosis and cystinosis) could present with disease related arthritis. multiple potential arthritic mechanisms secondary to storage product accumulation in lsd's have been described, such as: dysregulation of innate immunity and increased upregulation of numerous pro-inflammatory proteins. conclusion: given the evidence of storage products within macrophages of the inflamed synovial tissue and the absence of other etiological clues, our hypothesis is that gs itself is the primary cause for the inflammatory joint pathology in our patient. although, gs cannot be linked directly to joint inflammation, lysosomal defects have been associated to pro-inflammatory effects that possibly could result in arthritic disease. future identification of other patients with gs is required to support the hypothesis of an arthritic clinical phenotype of gs and to assess underlying pathophysiology. introduction: joint pain (jp) is a relatively common complaint among children and adolescents. a painful joint in children for many years continues to maintain the status of the most common symptom of juvenile arthritis. however this symptom should not always be interpreted as a manifestation of rheumatic diseases. objectives: the aim of current review is to debate of the structure in children with the chief complaint of jp. methods: we retrospectively analysed our series of children which attending outpatient department with complaint about pain lasting longer than two months in one or more joints. the clinical, instrumental and laboratory pictures were collected. special attention was paid to certain aspect of medical complaints, a complete and accurate history and physical examination. different categories as possible etiologies of jp in children were systematize and detailed. results: all children were divided into several groups based on their anatomical and physiological characteristics of osteoarticular system: the first group consisted of children under - years old, the second group - children - years old, the third group - children over years old. research suggests that more preschool children were experience bilateral lower extremity pain by "post-walk genesis" due to natural hypermobility, immaturity of sensory innervation of the joints and imbalance of the leg muscles (e.g. growing pains). the second most common cause of jp was associated with intra -or postinfectious factor (viral, streptococcal and chronic focal of infection). the frequency of juvenile arthritis and other rheumatic diseases in children of this age group did not exceed %. special attention was paid to fever, chills, malaise, nightpain and constitutional symptoms with changes in blood lab tests to exclude osteomyelitis (inc specific cause), malignancies manifestation and other bone tumors (less %). the most common causes of joint pain of school-age children were hypermobility syndrome and enthesopathy (primary, secondary). secondary enthesopathy were result of changes in nutrition, rapid growth and excessive exercise. also enthesopathy were manifestation of endocrine, gastrointestinal or infectious diseases. the proportion of children with the onset of chronic inflammatory arthropathy also did not exceed %. hypermobility child's syndrome was characterized by harmless pain (inc low back pain), linked to physical activity (less morning stiffness). over the past decade, we've seen a gradual increase in the number of children ( % were girls) with knee pain by diagnosed patellofemoral and mediopatellar plica syndromes, patellar tendinitis or idiopathic cause. in most cases children was complicated by syndrome of increased anxiety. the share of true chronic inflammatory arthropathies, including spondylitis, in children of this age group did not exceed %. fibromyalgia were diagnosed less %. introduction: a significant part of patients in rheumatologist's practice is children and teenagers with complaints of pain. the further volume of examination and the choice of treatment course depends on the capability of the rheumatologist to define the inflammatory and non-inflammatory genesis of pain. that makes the problem of differential diagnosis very important. objectives: to conduct a comparative analysis of patients with a principal pain complaint to determine if there are significant differences in the groups with the inflammatory and noninflammatory pain genesis. methods: the retrospective study included children who consulted a rheumatologist in the outpatient clinic in the period - without preliminary selection (n = ). of them there were selected children with principal pain complaint (n = ). according to the diagnosis, the children were divided into groups: those who have inflammatory genesis of pain (a, n = ) and those with noninflammatory genesis of pain (b, n = ). the group a included children with such diagnoses as: reactive, poststreptococcal and juvenile idiopathic arthritides. the group b included children with arthralgia, chronic pain syndrome, orthopedic pathology, fibromyalgia. results: . groups a and b differ in the average age of the first complaints onset (t-criterion for equality of means) with a high degree of statistical significance (group a = , years; group b = , years; p = . ). which means that in group a more often than in group b first complaints appear in the age between to while in group b more often than in group a it happens in the age between to . . there was a statistically significant difference in the means between groups a and b in time between the onset of first complaints and the first visit to a rheumatologist (p = . ) also in favor of this conclusion speaks the fact that in group a the number of visits to a rheumatologist in the same year when the first complaints appear is almost times higher than in group b. % of cases in group a consulted the rheumatologist the same year when the first complaints appeared in comparison to group b where only % of patients did the same. below is the table with distribution of cases by the number of years between the first complaints onset and the first visit to a rheumatologist in both groups: conclusion: in children with arthritides, the first pain complaints appear at an earlier age (an average of . ), and in group b (an average of . ). patients with arthritis more often visit a rheumatologist earlier (within year after the first complaints) than those with non-inflammatory genesis of pain complaints. the most common cause of recurrent musculoskeletal pain is growing pain (gp) in children. differential from rheumatic diseases could be challenging in some cases since there are no diagnostic criteria for gp. objectives: to analyze gp characteristics in a large cohort of patients in comparison with other non-inflammatory and inflammatory diseases causing limb pain, and to simplify the gp's diagnosis process by using machine learning (ml) techniques. methods: this is a multicenter cross-sectional study. introduction: it is a well-known fact that the period of intensive growth in children is associated with the processes of active bone mass accumulation and coincides with them in time. one of the most distinctive indicators of an increase in the disease incidence among children for the recent decade (+ , %) can be found in the skeletal disorders resulting from disrupted calcium metabolism and vitamin d deficit. the latter is widespread in ukraine as it is observed in % of schoolers. objectives: establish the specifics of the structural and functional status of the bone tissue in children during the growth spurt, taking account of the degree of vitamin d sufficiency. methods: the examination covered children aged - who were divided into three groups depending on the presence of the growth spurt (gs) and its intensity: group - children who had become - cm taller for the year in question; group - children who had become taller by cm or more, group - children who had experienced no growth spurt. inclusion criteria were the following: no chronic somatic or endocrine pathologies, no musculoskeletal disorders or mineral homeostasis disruptions; physical exertion corresponding to their age; the children had not been taking any complexes of vitamins and minerals, including vitamin d for months before the examination. conclusion: children aged - showed deficiency of vitamin d reaching % which had no correlation with the presence or intensity of the growth spurt. in children who experienced growth spurt, a reduced bmd proved more frequent and correlated with the spurt intensity, however, it did not depend on sufficiency of vitamin d . therefore, during the growth spurt, disrupted mineralization of the bone tissue was influenced not only by the vitamin d deficit but also by the correlation between the bone tissue mineralization rate and intensity of growth in the children. methods: a self-reported question online survey on qol of patients with sjia and aosd was developed by the non-profit organizations, the autoinflammatory alliance, kaisz/vaisz, enca and sjia foundation in english and translated to dutch. respondents were recruited by convenience sampling through online social media posts. data on flares, triggers, family history, and correlation of symptoms with labs were collected in addition to detailed information on qol during and in-between flares. results: between and , there were responses; were from parents of children with sjia, from adults with sjia, and from adults with aosd. interestingly, adults (whether diagnosed with sjia or aosd) were more likely to report pain, fatigue, joint swelling or arthritis, nausea & vomiting, and diarrhea during flares than children. adults were also more likely to describe flares >one month. % of patients reported being "greatly" or "severely" limited during flares. between flares, % reported being "greatly" or "severely" limited while % were "somewhat" limited. % felt their condition affected their studies, job, and career, including % of children with sjia, % of adults with sjia, and % with aosd. respondents were asked open-ended questions regarding their experience with disease flares and impact on their lives, and specifically how sjia and aosd affected work, career and schooling. responses regarding the disease experience were classified into theme areas: ) experience with disease onset and process of diagnosis; ) health care access, quality, and drug safety concerns; ) physical impact of the disease including pain and chronic fatigue; ) social impact of the disease; ) mental health and emotional impact of the disease; ) impact on work, career, and employment; and ) broad impact on life and lifestyle. responses regarding effect on work, career, and schooling were categorized into theme areas: ) physical impact negatively influencing school/work productivity; ) lost work and wages, including unemployment and needing disability benefits, and parents missing work to care for the child; and ) the socialemotional impact as well as negative effects on mental health. about half of patients regardless of age reported the name sjia did not represent well the disease, specifically that it did not emphasize the systemic symptoms, and that the disease gets confused with other types of arthritis. adult patients with sjia did not like to have juvenile in the name. conclusion: children and adults with sjia and aosd report high levels of qol limitation and effect on school, work, and career, both during and between flares. our qualitative data emphasizes the importance of multidimensional evaluation of disease with ongoing input from the patients, which will provide a foundation for asking more relevant research questions to foster better care and improve qol. results: in total participants were included in the study : juvenile idiopathic arthritis (jia) patients, and their parents. the mean age of the patients was . ± . years. significant differences were found between patients and healthy children in such hrqol survey categories like "autonomy" and "financial resources" (p < . ). although quality of life in children's with juvenile idiopathic arthritis was lower than in healthy children in hrqol survey category "self perception" (p < . ). after analyzing data no significantly differences were found between patients and parents' assessment scores in hrqol survey categories (p > . ). conclusion: juvenile idiopathic arthritis has a moderate negative influence on hrqol survey categories "self perception", "autonomy" and "financial resources" (p < . ) according kidscreen- queationnaire. introduction: juvenile dermatomyositis (jdm) is often first identified by parents and carers as the red facial rash develops. the rash can progress and lead to young people being misdiagnosed with eczema, scarlet fever or psoriasis. however, over time the obvious signs of jdm can become "invisible" as treatment calms the rashes and masks the outward signs of jdm, until a flare occurs, when the rashes can be a marker for disease activity or progression. as part of a larger study, children around the united kingdom were asked to discuss their views on whether they wanted people to be able to see their jdm. objectives: to understand the implications for children and young people from having a disease that has visible and invisible phases and whether they want others to see their jdm, or not. methods: children and young people around the united kingdom who were already consented and enrolled into the uk juvenile dermatomyositis cohort and biomarker study were asked to complete a bespoke questionnaire. there was a mix of open and closed questions, and it was administered in paper format to all children and young people between the ages of and years of age for self-completion, either on the paper forms, or via a secure web-based software system. the questionnaires were administered at the end of . numeric data were described and qualitative data were analysed using standard content analysis. results: questionnaire packs were sent out, with ( %) being returned. of these could not be used due to practical reasons, such as only demographic data being completed, which left a sample of . ( %) of the who responded said other people could not see their jdm, with only ( %) saying it was visible and ( %) saying they did not know if others can see it. did not respond to the question as said their jdm has gone away. they were then asked whether it was a good or bad thing for others to be able to see their jdm or for others not to be able to see it. young people left comments as to why it was a good thing, left comments as to why it was a bad thing and left comments to why they said don't know, table presents the top ranking response for the three multichoice answers. conclusion: this study has highlighted the disparity between young people wanting others to see their jdm so that they gain more understanding and empathy from those around them, but equally, wanting their jdm to be invisible, so that they feel the same as their peers. whilst many paediatric rheumatic conditions are in fact invisible, our data illustrate that jdm often gives children and young people a taste of both visible and invisible phases of disease activity. as one young person said "it's not good, nor badit's good that it's invisible sometimes so i can blend in without the disabled stereotype. however, sometimes it needs to be seen so i can be understood and not challenged". disclosure of interest: none declared multidimensional assessment report (j-fimar) which includes comprehensive patient self-report questionnaire and numerical rating scales to measure pain, fatigue, headache, sleep quality, physical function, psychological state, health-related quality of life, satisfaction with illness course. the j-fimar has been devised according to the outcome measure in rheumatology (omeract) guidelines. discriminant ability of the multidimensional tool was evaluated by testing it in a control group including healthy controls and patients affected by active juvenile idiopathic arthritis (jia). the psychosocial consequences of chronic pain were evaluated by using the children depression index (cdi) and the multidimensional anxiety scale for children (masc). the objective sleep quality was measured by overnight polysomnography. results: table shows characteristics and the most represented somatic symptoms in our cohort of jfs patients at the study enter. polysomnography was performed in patients with sleep disturbance; / ( . %) showed an electroencephalographic pattern of alpha wave intrusion in slow wave sleep (sws). the presence of objective sleep disorders was significantly correlated to cdi score rs - , (p≤ , ) and masc , (p= , ). from november to april j-fimar was completed by jfs patients (f ( . %), median age . years [ . - . ], median disease duration . years [ . - . ]) in visits. all patients filled out the questionnaire in a short time (< minutes) and considered it simple and easy to understand. jfs patients showed significantly higher score for pain, fatigue, poor physical function and measure of psychological distress than healthy controls and jia patients (p< . for each item). conclusion: jfs patients presented significantly higher pain experience, functional disability, and impaired quality of life than patients with active jia. a relevant percentage of jfs patients experience sleep disturbances, which were correlated with mood and anxiety disorders. our multidimensional tool was feasible and able to quantify global jfs severity. this multidimensional tool, by measuring the main domains affected by the disease, could be promising to individualize treatment strategy and to test its efficacy. disclosure of interest: none declared introduction: fatigue is a subjective state of overwhelming, sustained exhaustion and decreased physical and mental capacity, which is not relieved by rest. fatigue is the most common complaint in children and teens with an autoinflammatory disease, besides the disease related flares. the purpose for this study was to show that fatigue is a serious issue for children and young people with autoinflammatory diseases. we hypothesized that age, gender and/ or the type of autoinflammatory disease could have differing effects on the fatigue experience. objectives: we aimed to investigate fatigue in children and young people (cyp) with autoinflammatory disease, including how this affected them on a daily basis. methods: cyp with autoinflammatory diseases were invited to complete an online survey, providing details about their fatigue and how it affected them. the survey was developed by the non-profit organizations autoinflammatory alliance and kaisz/vaisz, in english. respondents were recruited by convenience sampling through online social media posts. data on age, gender and disease were collected in addition to information on their experience of fatigue on school and social interaction. a total of cyp (age range - years) with an autoinflammatory disease responded to the survey ( % female). results: the majority of respondents ( %) reported experiencing both mental and physical fatigue. respondents were asked how much their fatigue affected them, on a scale of to ; overall, the mean fatigue score was . . however, young people aged or over reported a significantly higher impact than those aged - years (mean . , p= . ). different autoinflammatory diseases were surveyed: crmo %, caps %, pfapa % also unclassified said (usaid) with %. in the open-response portion of the survey, % of respondents reported that fatigue was physical, as well as mental, in their experience. most ( %) reported that someone had doubted their fatigue in the past; % had found their teachers had doubted them, % had friends who doubted them, and % reported that they felt their doctors had doubted them. children and young people also felt a number of activities made their fatigue worse (table ) ." conclusion: cyp with autoinflammatory diseases experience physical and mental fatigue. health professionals and teachers should listen to patients reporting fatigue, validate their experience, and help find ways to support them. identifying resources to help the patients with fatigue, and referrals to therapy and mental health resources as needed may help the patients to better cope and manage their chronic disease. further studies will include patient engagement in designing questionnaires about all aspects of life and autoinflammatory disease will help our understanding of these complex conditions and how they affect patients. introduction: scleromyositis is the most common overlap syndrome but is rarely observed in childhood. this disorder involves two different autoimmune diseases: systemic scleroderma (ssc) and polymyositis (pm). objectives: to describe the clinical course of a ssc/pm syndrome in a young girl. methods: case report results: an -year-old female was admitted to the neurological unit of our hospital for creatine phosphokinase (cpk) increase and hypertransaminasemia associated to sporadic episodes of right calf pain. familiarity for muscular dystrophy was reported in the maternal branch. muscle tone and trophism were preserved at initial neurological evaluation. laboratory investigation confirmed increased muscle enzyme levels, including cpk (x ) (ck-mm . %, ck-mb . %), aldolase (x ), cardiac troponin (x ) and myoglobin (x ). suspecting a primary muscle disease, she underwent a total body stir-mri which showed a diffuse edema of gluteus medius bilaterally and a muscle biopsy revealing a marked muscle damage with dystrophic aspects and normality of the tested proteins. a genetic extended panel for congenital myopathies resulted negative. after months, a new clinical examination showed the occurrence of general skin induration, sclerodactyly and tightening of the face skin. appearance of dysphagia was also reported, and muscle enzyme increase persisted. in suspicion of an ssc/pm overlap syndrome, she was referred to our unit. nailfold capillaroscopy showed capillary dilatation and branching, megacapillaries and diffuse microhemorrhages. reduction of esophageal contractions amplitude and hypotensive lower esophageal sphincter pressure were observed at esophageal manometry test. high-resolution ct of lungs and pulmonary function testing were normal. skin biopsy showed sclerodermiform findings. immunological studies revealed a positivity of antinuclear antibody ( : ) and anti-ku. anti-pm-scl resulted negative. an oral corticosteroid therapy (prednisone, . mg/kg/day) was started in association with subcutaneous methotrexate ( mg/m /week) and intravenous immunoglobulins (ivig) ( gr/kg every two weeks). improvement of skin manifestation, joint mobility, as well as normalization of serum cpk levels were observed. over months, prednisone and ivig were slowly discontinued up to the ongoing dosage of . /mg/day and gr/kg every weeks, respectively. mtx is still ongoing at the same dosage. conclusion: the diagnosis of overlap connective tissue disease syndromes may be challenging in pediatric age. different symptoms may be prevalent at different stages throughout the course of the disease. in our patient, a localized myositis preceded the ss onset by about four months. even though the use of high dose of corticosteroids is associated to a higher incidence of renal crisis in patients with css, a combined therapy with high doses of oral steroids, ivig and mtx was safe and effective in skin, muscle and joint symptoms in our patient. results: a total of images were obtained from healthy children included in the study and capillary measurements were made. capillary density was significantly higher in group than in groups and . arterial width was significantly lower in group as compared to group and , and in group as compared to group . apical loop width and capillary distance were significantly lower in group compared to group and and . there was no significant difference between the age groups in terms of capillary length and venous width. there was no difference between the groups in terms of capillary morphology. in total image evaluations, capillary tortuosity was < % in . %, and > % in . %, and capillary crossing were < % in . % and> % in . %. while the enlarged capillary was . % and the avascular area was . %, capillary branching, capillary meandering, microhemorrhage, and giant capillary were not detected in any case. there was a good level of agreement between the researchers, as cases with capillaries were evaluated with a good level of agreement (table ) . conclusion: this is the first study to evaluate capillary morphology in healthy turkish children. this study also adds that some special forms such as enlarged capillary and avascular area, which is always named as pathological in adult age, can be seen in healthy children. these data will be guiding in capillaroscopic studies in various patient groups, particularly in children with collagen vascular diseases. methods: patients with jia were tested for hla-b . they were divided into groups: ) hla-b positive and ) hla-b negative. results: patients ( , %) were hla-b positive and all of them are fulfilled the eular criteria of entesitis-related arthritis (era). the group consists of patients ( . %). there's no statistical difference between both groups in active joint count, ana-positivity and uveitis frequency, the rate of use methotrexate and time before biologics. no difference in axial cervical spine ( . %) vs ( . %) (p= . ) and sacroiliac joints ( . %) vs / ( . %) (p= . ) involvement was observed. hla b (+) patients often received pulse therapy with methylprednisolone due to increased inflammatory activity and severe arthritis ( % vs . %, p= . ). other parameters are listed in table . conclusion: patients with hla-b positivity were characterized by male predominance, more often hip involvement, higher laboratory activity and the need for more frequent use biologics. the rate of axial involvement wasn't different in hla-b positive and negative patients, that needs further study and creating more accurate classification criteria for jsa. disclosure of interest: none declared introduction: although gut is increasingly recognized as origin and/or target of inflammation in adult onset spondyloarthritis (spa), the incidence of gut involvement in juvenile spa (jspa) patients is still largely unknown, mostly due to the lack of reliable non-invasive tests. objectives: we performed a cross-sectional study of fecal calprotectin (fcal), a surrogate marker of gut inflammation, in patients with jspa, other forms of juvenile idiopathic arthritis (jia) and noninflammatory (ni) conditions. methods: fcal was measured by commercially available assay in stool samples of enthesitis related (era), psoriatic (psa) and patients with other jia subtypes (oligo-and poly-articular) who fulfilled ilar criteria, as well as in children with ni causes of musculoskeletal pain (ni-msd), regardless of the gastrointestinal (gi) symptoms (table ). fcal was compared among different groups of patients and correlated with demographic data, clinical characteristics, treatment modalities and disease activity measured by jspada. the values were also dichotomized to < mg/kg, - mg/kg, and > mg/kg, which was regarded as normal, slightly increased and increased, respectively. ileocolonoscopy was performed in one patient. our study has shown that fcal levels are significantly higher in era patients compared to other jia (p= . ) and/or ni-msd (p= . ) patients. moreover, almost a third of patients with era had levels of fcal above the range regarded as normal, which adds to the number of evidences for a gut inflammation in this particular type of jia. besides, the fcal levels were higher in those with axial involvement, which further suppots the association of gut and axial inflammation in children with era. although endoscopy remains a gold standard for the diagnosis of gut inflammation, fcal can help to select children with era who might benefit from this invasive procedure, regardless of the gi symptoms, as shown in one patient with the highest fcal concentration in our study. moreover, fcal levels seems not to be influenced by disease characteristic and/or concomitant therapy intake. therefore, fcal should be a part of diagnostic workup in children with any type of jia, but most importantly in those with era. evaluate potential predictor variables of magnetic resonance imaging (mri) sij remission methods: retrospective review of prospectively collected data. we included patients with era (according to ilar criteria) continuously treated with anti-tnf agents for ≥ months who had at least two mris of the sacroiliac joints performed before starting anti-tnf therapy (baseline) and during the follow up ( > months after anti-tnf treatment). si joints were examined using t -weighted images, t fastsuppressed and short-tau inversión recovery. the sparcc-sis was scored by two pediatric radiologists. sparcc-sis assessed the presence, depth and intensity of bone marrow edema (bme) on consecutive six slices in the iliac and sacrum bones . scoring is composed by: bme ( - ), bm intensity ( - ), bm depth ( - introduction: several paediatric patients manifest conditions commonly misdiagnosed as spider bites, which however, can include other arthropods bites; bacterial, viral, and mycotic infections; vasculitis; dermatological diseases; miscellaneous conditions as drug reactions, chemical injuries. objectives: in italy, spiders which are likely to be associated with severe toxin mediated tissue damage are uncommon, especially in urban zones. however, a minor trauma may be a precipitating factor for pyoderma gangrenosum particularly over the legs, in association with inflammatory bowel disease, haematologic diseases and juvenile idiopathic arthritis (jia). methods: we describe a -years old boy with pyoderma gangrenosum complicated spider bite in association with systemic jia (sjia). the patient was in clinical remission after the start of the sjia, occurred two months before, still treated with tapering doses of steroids and canakinumab, with the normalization of inflammatory parameters (crp, esr, saa, ferritin) and clinical manifestations. only a mild arthritis of the knee persisted and for this reason he was still treated with steroids. furthermore, he developed hyperglycemia, requiring insulin treatment. the first dermatological manifestation which he referred was a red dot of the leg skin. in a few days, the erythema enlarged, involving an area of x cm, with oedema, pain, and blisters, evolving in a necrotic lesion, with purulent exudate, surrounded by a haemorrhagic zone. results: haematological controls revealed neutrophilic leucocytosis, increased crp and procalcitonin. he started treatment with intra venous administration of teicoplanin plus ceftriaxone, with no resolution of the clinical manifestations and the reduction of leukocytosis, crp, procalcitonin. a culture swab was performed and was positive for pseudomonas aeruginosa, confirmed by pcr on the culture. he started ciprofloxacin and surgical curettage of the lesion, with the resolution of the lesion and the normalization of biochemical parameters. conclusion: the aspect of the lesion and its evolution were evocative of a spider bite suggested by anamnestic records, complicated by a pyoderma gangrenosum secondary to pseudomonas aeruginosa. the underlying disease, the immune suppressive treatment, with steroids and biological drugs, the hyperglycaemic pattern of the patient allowed the severe evolution of the spider bite. children in treatment with immune suppressive and/or biologic drugs are at high risk of infections. skin lesions, as arthropods bites, can be a facility for superinfection, with possible haematological and systemic diffusion. the strict application of the ilar requirement for the presence of documented arthritis for the diagnosis of sjia, early in the disease course, may result in unnecessary delays in initiating appropriate treatment. in preliminary printo classification criteria for sjia, this mandatory requirement of documented arthritis has been modified. to measure performance of preliminary printo classification criteria for sjia in our indian cohort. methods i gathered a data of seven sjia patients who attended dev children's hospital between jan and jan . my data included demographics,clinical presentation, laboratory parameters and outcome of these patients. all these patients were diagnosed at an early stage by clinical judgement irrespective of fulfilment of ilar criteria. i applied preliminary printo classification criteria for all. average age of selected children ( girls and boys) was . years. conclusion a preliminary printo classification criteria for sjia has been validated in our cohort. there are many raised inflammatory markers in most of these patients other than wbc count. these markers should be considered to be added in supportive laboratory criteria to be more specific towards the diagnosis. it is important to add pid in exclusion list especially in a case of sjia with mas at onset. trial registration identifying number leningrad's regional children' two patient stopped treatment within months from therapy start: due to primary inefficiency ( ) and allergic reaction ( ). five ( / ) patients were-co-administered with cdmards, other with oral gc, and subjects had been previously exposed to other biologic drugs. whole patients stopped therapy due to secondary inefficiency: patients were switched on toc, other children were switched on eta ( ) introduction: systemic juvenile idiopathic arthritis (sjia) is a rare, complex auto-inflammatory disease with significant morbidity including fever, rash, serositis and articular problems. with the availability of interleukin- (il- ) and il- inhibitor treatment, morbidity has significantly reduced and the outcome for sjia patients has improved. however, differences in access to care and differences in treatment strategies between countries in and outside of europe remain a concern. objectives: the single hub and access point for paediatric rheumatology in europe (share) consortium aimed to develop best practices for paediatric rheumatic diseases in order to decrease differences in care between european countries. here, we present the final results of the literature review and a series of consensus meetings on defining overarching, diagnostic and therapeutic recommendations for diagnosis and treatment of sjia. methods: the share methodology has been previously published, including the use of the eular standardized operating procedure for developing best practice recommendations. as per these guidelines, a methodologist provided supervision during the process and consensus meetings. conclusion: hscore seems to perform slightly better than ms-score for the diagnosis of mas in our cohort. early inhibition of il- is discussed to play an important role in the disease course of sjia . assuming that pretreatment with other dmards leads to a later start of therapy with canakinumab, this analysis evaluates the effectiveness of canakinumab as first-line vs. second-line dmard. to evaluate the effectiveness of canakinumab as first used biological dmard in sjia compared to canakinumab in sjia-patients pretreated with other dmards. methods sjia-patients documented in the german biologic registry for pediatric rheumatology (biker), who were exposed to canakinumab, were identified. for the first-line (fl) group dmard naïve patients were selected, prior treatment with corticosteroids and/or nsaids was allowed. patients receiving any dmard prior to canakinumab entered the second-line (sl) group. both groups were compared in a retrospective intention-to-treat-analysis. effectiveness canakinumab treatment showed good effectiveness in sjia both as first-and second-line dmard. after months the use of canakinumab as first-line dmard is associated with higher response rates compared to second-line use. our data support the hypothesis that early treatment with canakinumab is associated with good therapeutical response and a positive effect on the disease course of sjia. results: a total of children ( girls) with sle were identified. median age of symptom onset and diagnosis was years(range - years) and years respectively. the presenting manifestations were fever( ), oral ulcers( ), alopecia( ), malar rash( ), photosensitivity( ), renal involvement ( ), seizures( ) and gastrointestinal complaints ( ) apart from some unusual manifestations of isolated peripheral arthritis( ), isolated bilateral pleural effusion( ), macrophage activation syndrome( ). laboratory investigations: hemogram revealed anemia in children and thrombocytopenia in . urine examination showed nephrotic range proteinuria in child and subnephrotic proteinuria in . microscopic hematuria was noted in pateints. renal function tests were deranged in cases. ana, anti dsdna positivity and hypocomplimentemia were present in all. renal biopsy was done in patients, had class iv, one class iii and one had class v lupus nephritis. all patients were initiated on hydroxychloroquine and photoprotection. children with renal involvement were given pulse methylprednisolone followed by tapering doses of oral prednisolone and intravenous, monthly cyclophosphamide. azathioprine was used as maintenance therapy in all. subcutaneous weekly methotrexate was used in patients. one child (mas) died during disease course. disease continues to be in remission in rest. conclusion: we found a significant female preponderance in our study group. renal involvement was the commonest presentation. some unusual presentations were also seen. early recognition of sle is critical for timely initiation of appropriate treatment. this is the first report of a cohort of pediatric sle from this part of india. introduction: autoantibodies in ahai may be igg/igm/iga. ahai can be divided into primary or secondary (e.g. sle, lymphoproliferative diseases, infections, medications). it is also classified based on the temperature at which the antibody reacts to erythrocytes, and can be warm (igg or iga) or cold (igm or c ). in warm ahai, the antibodies react at temperatures ≥ ºc, not activating the complement system and not undergoing agglutination in vitro. in cold ahai, antibodies react at temperatures below ºc, activating the complement system with in vitro agglutination.mixed aiha (warm and cold) is rare and occurs in < % of aiha cases and can occur at any age, but is extremely rare in children. the prevalence of the mixed form is less than / , , patients with ahai. objectives: to report a rare case of mixed ahai and idiopathic intracranial hypertension(iih) in a -years old female patient with a previous diagnosis of sle and aps. methods: case report and literature review. results: a -years old female adolescent previously diagnosed with sle/aps since was in remission on hydroxychloroquine( mg);azathioprine( mg);aspirin( mg);vitamind ( . iu);calcium( g), and sunscreen. in april , she had a relapse presenting with fatigue, myositis, headache, hypocomplementemia, and severe autoimmune hemolytic anemia (hb of g/dl) (sledai- k= points). mixed ahai was diagnosed base on a direct/indirect coombs test / +;directantiglobulintesting showing anti-iga(weak),anti-igm( +/ +),anti-igg( +/ +),anti-c c(weak),anti-c d ( +/ +);igg / subclasses with a reaction of : ( +/ +);an eleven cell antibody panel positive revealing a cold and warm antibody, and adsorption technique revealing a cold and warm autoantibody. chest ct showed bibasilar subsegmental atelectasis, head ct/mri was normal and lp showed a high opening pressure of cmh o with a normal cell count. after the procedure, the patient reported improvement in the pain and was diagnosed with iih. the patient was screened for secondary causes for ahai (table ) due to the unusual mixed type pattern and serology was positive for chlamydia trachomatis (igm) and mycoplasma pneumoniae (indeterminate-igm/positive-igg) suggesting a recent infectious trigger causing reactivation of the underlying disease with a probable cross-reactivity. the patient treated with -days of clarithromycin. before the infectious screening came back negative, ahai was treated with a single dose of ivig( g/kg) and then, with -days of methylprednisolone( g/day). azathioprine was replaced by mycophenolate mofetil. due to headache recurrence, acetazolamide( mg/day) was started, and the patient referred no pain. the patient was discharged with a resolution of the symptoms. objectives: to our knowledge, the association of gbs and bbe has been described in adults only. methods: we here describe a child presenting at sle disease-onset with an overlap of peripheral (gbs) and central (bbe) nervous system manifestations, highlighting the possible association between these two entities in children. results: an -year-old healthy girl presented with acute ataxia, ophtalmoparesis and altered level of consciousness, rapidly followed by areflexia, facial paresis, swallowing difficulties, sensory deficits, paresis in all four limbs and respiratory insufficiency. these symptoms were accompanied by pleuro-pericardial serositis, proteinuria and hypertension. immunological investigations revealed the presence of positive ana and ds-dna antibodies. the renal biopsy showed a stage iii lupus nephritis. hence, the clinical, laboratory findings and biopsy report led to the diagnosis of psle. brain and spine mri did not show any abnormalities; diffuse slowing compatible with nonspecific encephalopathy was seen on eeg. nerve conduction studies (ncs) confirmed the clinical suspicion of acute polyradiculoneuropathy with proximal interruption of motor nerve conduction, compatible with guillain-barré-like syndrome. csf analysis (performed twice) remained normal. the patient was treated with glucocorticoids, intravenous immunoglobulins, cyclophosphamide as well as plasmapheresis. the neurological and physical symptoms improved gradually with complete neurological recovery four months after onset. conclusion: overlapping forms of bbe/gbs have never been described in association to sle in children. our patient's presentation and evolution fulfilled the criteria for such an overlap, occurring at psle onset. although sle and bbe/gbs are rare entities, our case suggests that there may be a common underlying immune background. this association should be recognized early for rapid and appropriate treatment initiation. infantile antiphospholipid antibody syndrome: acquired and de novo apl appearance in four infants t. giani , g. ferrara , a. mauro , r. cimaz introduction: antiphospholipid syndrome (aps) is a rare condition in the neonatal age. in most cases it is considered a passively acquired autoimmune disease, due to a transplacental passage of maternal antiphospholipid antibodies (apl). exceedingly unusual is the de novo production of apl in newborns and infants. objectives: to describe four infants who developed an early brain stroke with increased and persistent levels of apl, even after six months of life. methods: we reviewed the clinical charts of four such infants, followed from diagnosis up to two years after the disappearance of apl. conclusion: common characteristics of these four children are the development of brain stroke and the increased and persistent apl levels even after six months of life. this opens the window on a gray zone related to the origin of these antibodies (maternal or neonatal) and on their role in the pathogenesis of the infantile brain stroke. patients had over % of their monitoring completed but only had over %. aspects of monitoring that were more time intensive or were required less regularly were most frequently overlooked. there was a statistically significant increase in the percentage of completed monitoring in those patients for whom the lupus checklist was used compared to patients where a checklist was not used (p= . ). conclusion: there is significant room for improvement in the monitoring of these patients with jsle in the rheumatology clinic. this audit illustrates that more diligent use of the lupus checklist and an overall improvement in sustained use of the checklist will help to improve monitoring of these patients. evidence suggests that checklists are underutilised in medicine and wider implementation of this simple tool could improve patient outcomes. , , interventions such as in person or electronic reminders, or audits with feedback to physicians could improve usage over time. the application of the lupus checklist or a similar document in other paediatric clinics is important for comprehensive monitoring of a condition as complex as jsle and has the potential to prevent ongoing damage and medication toxicity in this high-risk population. juvenile onset and this cluster have may more severe kidney, neuropsychiatric or hematological involvement. objectives: the aim of this study was to assess the clinical and laboratory characteristics, disease activity, and treatment response of patients with juvenile sle (jsle). methods: this is a retrospective study involving patients between july and january . the data of patients diagnosed with jsle and followed up for a minimum of months, were collected. the sledai- k scores at initiation and at the follow-up ( st, rd, th, and th months of treatment) were examined. the sledai- k score was considered to be ≤ , for disease remission status. results: a total of children were included in to the study. the female/male ratio was . / and the median age of the patients at the diagnosis was (iqr: . - . ) years. the median follow-up of patients was (iqr: - ) month. four of the patients were diagnosed with monogenic sle. two siblings were diagnosed with c deficiency and two were diagnosed with familial chilblain lupus. the most common clinical findings were found musculoskeletal complaints ( . %), malar rash ( %), oral ulcers ( . %), and fever ( . %), respectively in over all the group. the frequency of involvement of the system and organs was as follows; mucocutaneous . %, musculoskeletal . %, renal . %, hematological . %, serous membranes . %, neuropsychiatric . %, respectively. all patients had anti-nuclear antibody positivity, while . % had anti-ds dna, . % had anti-sm and . % had antiphospholipid antibody positivity. while all patients received hydroxychloroquine treatment, . % of the patients were received were mycophenolate mofetil, . % were azathioprine, . % cyclophosphamide, . % methotrexate and . % were rituximab. the median sledai- k score was (iqr: - . ) at admission, besides it was found to (iqr: - ), (iqr: - ), (iqr: - ) in the st, th and th months of treatment, respectively. while % of the patients had active disease at admission, . % at months, . % at months and . % at months still had active disease (sledai- k > ). patients with initially high sledai- k scores had significantly lower remission rates in the first month (p= . ). it was observed that patients with high sledai- k scores in admission were more resistant to conventional immunosuppressive treatments and the use of rituximab was more frequent in these patients. at least one major organ (renal, hematological, neurological) were affected in % of patients. the remission rate of these patients at months was found significantly decreased compared to the others (p < . ). renal biopsy was performed in patients ( . %). of them had type lupus nephritis (ln), had type , had type , and had type . it was observed that patients with renal involvement were the group that reached remission latest. conclusion: the presence of high initial sledai- k scores and the major organ involvement have poor predictive value to achieve inactive disease. a two year old girl of consanguineous parents presented to hospital at months of age with fever and erythematous macular rash on her cheeks which spread to her nose, chin, and ears. the rash started a month prior, and progressed over her entire body. a skin swab grew staphylococcus aureus but the rash didn't respond to topical antibiotics. review of systems was unremarkable except for longstanding oral thrush and diaper rash. birth and family history were unremarkable. on exam she had a diffuse, erythematous, morbilliform eruption over her face and body. she had facial swelling, orbital edema and vasculitic oral ulcers. she had leukopenia mainly neutropenia, low hemoglobin, with normal platelets. her liver enzymes and erythrocyte sedimentation rate (esr) were high while c-reactive protein, immunoglobulins, c and c were normal. cultures were negative, however she was positive for adenovirus, mycoplasma and ebv (ebv load was iu/ml ). autoimmune hepatitis work up was negative. the direct coombs test, antinuclear antibodies ( : ), ro, rnp and smd were positive. ch came low as well as c q level of mg/dl (normal range - mg/dl). lymphocyte subsets showed reduced cd and nk cells. bone marrow aspiration showed active marrow. skin biopsy showed chronic non-specific inflammation (immunofluorescence and electron microscopy were not available). echocardiogram showed dilatation of the left coronaries. she was treated with intravenous immunoglobulin (ivig) for kawasaki disease with no improvement. therefore pulse steroid mg/kg followed by mg/kg was initiated. her rash, facial swelling and abnormal blood counts improved dramatically. whole exome sequence showed homozygous variant c. g>t p.g c at the c qa gene. while tapering steroids she flared so subcutaneous methotrexate was started. unfortunately, she continued to have rash, leukopenia and high liver enzymes, so treatment was switched to mycophenolate mofetil and hydroxychloroquine. however she did not improve and started to have recurrent bacterial and viral infections that included cellulitis, gastroenteritis and upper respiratory tract infection. we started her on regular ivig, which helped with infections and allowed for weaning of steroids. however she developed alopecia and lower limb spasticity with delayed walking. mri brain and spine was normal. upon reanalysis of the wes, two other homozygous mutations at kif c and apg were identified and associated with spastic paraplegia, but reported as variants of unknown significant. fresh frozen plasma (ffp) transfusions were started, initially weekly, then every two weeks and subsequently every four weeks. the rash disappeared, leukopenia and esr improved and we were able to discontinue steroids conclusion: early-onset sle with a severe course of disease raises the possibility of a genetic etiology. we are reporting, for the first time, a rare missense mutation g>t in exon of the c qa gene that resulted in an amino acid substitution that is pathogenic. interestingly, she had other mutations associated with neurological manifestation that never reported together before and altered her phenotype. she has responded well to ffp as has been reported in a few case reports results: a total of psle under the age of years were included, % (n = ) were males. the overall mean age at diagnosis was . ± . years and median disease duration was . ( - ) years. huv was diagnosed in . % (n = ) of psle cohort. psle with uv were more likely to be males ( % vs %; p < . ), diagnosed at a younger age ( . vs . years; p < . ), have a family history of sle ( % vs %; p = . ) and have conjunctivitis more frequently ( % vs . %; p < . ) than psle without uv. psle with uv were also less likely to have cns involvement ( . % vs %; p = . ) and hematological manifestations such as leukopenia ( . % vs %; p = . ) and thrombocytopenia ( . % vs %; p = . ). in addition, psle with uv were more likely to be associated with low c complement count ( % vs %; p < . ) and positive cytoplasmic anca ( % vs %; p = . ).however, the psle with uv cohort were less likely to be associated with ana ( % vs %; p = . ), dsdna ( % vs %; p = . ) and perinuclear anti-neutrophil cytoplasmic antibodies ( % vs %; p = . ). conclusion: we report a high occurrence of huv in psle cohort ( . %) associated with unique demographic, clinical features and laboratory features. the debate regarding whether huv is a rare subset or unusual type of sle, or is a separate entity altogether, continues. however, the overlap in clinical, laboratory and genetic mutation supports the notion that huv and sle fall into the same spectrum of autoimmune disease with similar disease pathogenesis. however, further studies are needed to reach clear conclusions regarding the relationship between huv and sle. introduction: the last decade has brought a lot to the approaches to the diagnosis and treatment of juvenile arthritis. in russia, the actualization of the problem of diagnosis and treatment of jia required the development of federal standards, which provide the most detailed algorithms for medical care, both at the stage of inpatient and outpatient care. in the regions of the russian federation, the effective use of these documents required a whole range of additional educated activities, both with students of medical universities, as well as with the medical and nursing community, in addition, a set of work was carried out to create a regional regulatory framework. in the total biological therapy pool, % of patients receive tnf-alpha inhibitors, antibodies to il- receive % of patients, antibodies to il- - , %. it is worth noting that when using biological agents in % of cases, the criterion of an inactive disease was achieved by - months, which was characterized by the absence of acute inflammatory symptoms, normalization of esr and crp. monitoring of patients with jia receiving biological agents required the conduct of a number of educational activities for medical personnel, the creation of an additional methodological base. for further training of young specialists at the regional medical university, a program of an additional educational course in pediatric rheumatology was developed and introduced. a regional patient organization was established and also required a set of information activities by the medical community. conclusion: in the saratov region of the russian federation, about % of patients with jia receive biological therapy, which corresponds to the average indicators according to the literature. in the structure of the biological drugs used, the group of tnf-alpha inhibitors is preserved - %. the introduction of modern methods of treatment using biological agents in jia has significantly increased the effectiveness of treatment, but it required the organization of additional information support for medical personnel. disclosure of interest: none declared introduction: immunogenicity and development of anti-drug antibodies have been associated with treatment failure and adverse events during biologic treatment. anti-drug antibodies (adas) have been reported in % of juvenile idiopathic arthritis patients treated with adalimumab. however, their role in reducing adalimumab efficacy is still debated due to conflicting results. no study has been directed toward identification of neutralizing adas in paediatric rheumatic disorders. objectives: aim of our study was to detect adas, along with their clinical relevance, using a new theranostic peptide-base assay in a cohort of children with inflammatory chronic diseases on adalimumab treatment. methods: six candidate adalimumab derived peptide antigens (hc-cdr , hc cdr , hc cdr , lc cdr , lc cdr , lc cdr ) have been developed and optimized to be tested. their performance has been compared with commercial elisa kit and a spr-based optical assay (biacore®). assays have been performed in sera of a cohort of children receiving adalimumab due to an inflammatory chronic disease. mean age, disease duration, concomitant treatment with methotrexate (mtx), ana positivity, disease activity parameters and scores at the time of ada determination have been recorded. chisquare, and fisher exact test were used to compare data. pearson's and spearman's correlation tests were used to determine correlation coefficients for entered variables. results: eighteen ( f, median age . , range . - , yrs) patients were enrolled: affected by juvenile idiopathic arthritis, of whom complicated by jia -associated chronic uveitis, and patients affected by chronic idiopathic uveitis. peptide assay revealed adas in children, biacore in , commercial elisa in . of note, we found total concordance among the tests just in patients. no significant correlation has been proven among the ada determinations. biacore and elisa determination showed significant concordance (r s : . , p< . ). the presence of hc cdr and lc cdr resulted significantly correlated with disease activity (r s : . , p< . ), and, inversely, with disease remission on treatment (r s = - . , p< . ). no patient experienced severe adverse events and no correlation with adas has been revealed conclusion: in chronic rheumatic disorders, novel reliable methods are urgently required to guide clinical decision and support decisions about switching within or between drugs in refractory children. the different methods, since based on different antigenic probes, detect different antibody populations. the present peptide-based assays might contribute to identify neutralizing adas in patients treated with adalimumab. further validation in larger cohort is required. introduction: non-bacterial multifocal osteomyelitis (nbo) is a rare polygenic autoinflammatory disease, which is difficult to diagnose and treat. because of combination of bone lesions with arthritis and/ or axial skeleton damage in most cases the diagnosis of juvenile idiopathic arthritis (jia) or juvenile ankylosing spondylitis (jas) may be establish as a concurrent diagnosis, so this allows to legal use of biologics (ba) for the treatment. objectives: to analyze the single center experience of clinical and laboratory features of multifocal nbo in patients (pts) who were treated by ba for the last years. methods: the study involved a retrospective cohort of multifocal nbo pts treated by different ba in our clinic from to . all of them underwent standard rheumatological examination. in order to examine all localizations of the bone damage, a scintigraphy and/ or "whole body" mri scan was performed. results: among the whole group of pts with nbo (n= ) we identified pts treated by ba (tnf-inhibitors only). the majority were girls (n= , %). age at disease onset was . years in average (me . range . - . ). for legal reason of ba administration, we classified our patients according to rheumatological features as jia or jas. pts had jia ( girls), pts had jas ( girls). among pts had oligoarthritis ( %), had polyarthritis of low limbs (hip, knee, ankle). axial involvement was represented by active erosive sacroiliitis with deep bone marrow edema on mri scan in pts ( %), active spondylitis of several bodies in thoracic spinein ; erosive arthritis with partial ankyloses of facet joints of neck in pts, multiple syndesmophytes in girl. we found that definite axial lesions in nbo developed in very young children (in y.old at minimum), much earlier than in "idiopathic" jas. hla b was presented in pts ( %), pts had ana in high titer (all of those hla b -negative). the pts had bone lesions in different parts of skeleton: vertebral bodies - pts, clavicle - , sternum, ribs - , extremities bones, metaphysic mostly (tibial, fibular - pts), sacroiliac region - pts. extraskeletal manifestations were observed in pts, one in each condition -uveitis, psoriasis pustulosus, acnae conglobate. in a girl with very severe course of disease, not responded to any therapy nbo was combined with familial mediterranean fever. high level of laboratory activity were detected before biologics in pts ( %): esr acceleration up to mm/h, increase of crp up to mg/l. treatment included nsaids (all), methotrexate ( pts), sulfasalazine ( pts, but it was withdrawn in all pts), bisphosphonates ( pt), prednisolone ( pts). because of high activity of nbo with appearance of new bone lesions and persistent arthritis tnf inhibitors were administrated: etanercept in pts, adalimumab - ( as first line, second line), golimumab - . at the start of ba the average age was . years (range . - . ); mean disease duration was , years (range . - . ). there were cases of withdrawals. due to inefficacy etanercept was switched to adalimumab. disease activity decreasing was reached in the most of the patients ( from ). among them pts developed the whole remission with resolving of active arthritis and bone marrow edema spots. skin lesions (psoriasis pustulosis and acnae conglobate) were significantly improved. there were no adverse events during the tnf therapy. conclusion: our experience of the therapy with tnf inhibitors in patients with high nbo activity has shown that this is a good and safe therapeutic option that is expected to prevent progression and bone destruction. . ae were reported for . % of patients, most within to hours after the first or second injection: flu-like symptoms ( . %), hypocalcaemia ( . %) and hypophosphatemia ( %). underweight patients (body mass index < . kg/m²) accounted for % of hypocalcaemia. the frequency of all the ae not significantly decreased with the reduction of the first dose. only one serious hyponatremia occurred corresponding to a patient with renal failure before treatment. conclusion: our results were similar to those previously published: bisphosphonates are safe for osteoporosis in children. in the literature, sae are very rare in children, being limited to anecdotal osteopetrosis in cases of higher doses and long-term treatment, and delayed bone healing. anecdotal osteonecrosis of the jaw in adults has never been described in children. the use of bisphosphonates beforehand requires dietary measures (vitamin d and calcium supplementation). furthers systematic collection on efficacy and safety parameters for each bisphosphonates drug should confirm these data. introduction: the use of biosimilars in rheumatology has increased significantly over the last years and has resulted in considerable cost savings. objectives: to assess the effectiveness and tolerability of the adalimumab biosimilar abp in patients with jia. methods: a database of patients prescribed adalimumab in our service has been screened to identify patients with jia, who switched from the originator to the biosimilar. only patients who had a clinical review since they had started the biosimilar were included. a paired-samples t-test was conducted to compare the number of active joints at the clinic appointment before and after the initiation of the biosimilar treatment. the frequency and type of side effects, the clinical response and the number of patients who switched back to the originator have been collected. results: sixty-one patients who switched to the biosimilar abp between february and february were included. they were comprised of enthesitis-related arthritis (era), polyarthritis, oligoarthritis, psoriatic and systemic jia patients. their baseline characteristics and outcomes are summarised in table. the mean duration of follow-up after the switch to biosimilar was months (range - ). eleven patients ( %) reported side effects; the most common side effect (n= , . %) was injection site reactions and the remaining consisted of anaphylaxis, druginduced lupus, dizziness and bone pain, respectively. seven patients ( . %) reverted to the adalimumab originator, as a result of side effects, because of ineffectiveness and one patient for both reasons. in addition, patients were changed to a different biologic, one patient due to allergy to both the originator and biosimilar and the other two patients had active disease on the originator and biosimilar adalimumab. two patients stopped the biosimilar and remained off any biologic, in the first case this was due to a side effect and in the second case it was patient's choice. on the whole, . % of patients had remained on abp at their last visit. there was no significant difference in the active joint count before the biosimilar was started (mean . +/- . ) and after the switch (mean . +/- . ), (p= . ). introduction: golimumab (gol) is approved for polyarticular juvenile idiopathic arthritis (pjia) in patients of ≥ years but long-term safety data are limited. objectives: prospective monitoring of long-term safety and effectiveness of gol in routine care using the biker-registry. methods: baseline demographics, clinical characteristics, disease activity and safety parameters were compared to a contemporary : matched control cohort using alternative tnf inhibitors or methotrexate without exposure to a biologic. efficacy outcomes were jadas , joint counts and functional status. safety assessments were based on adverse events (ae) reports. results: in this ongoing study, pts initiating gol were matched to with alternative tnfi and biologic-naïve pts. pts starting gol had a longer disease duration (p< . ) and use of gol was significantly more often second line ( . % vs . %, p< . ) and thus disease activity was lower at baseline. pts in the gol cohort used less corticosteroids, otherwise patients were comparable with pts treated with other tnfi (table ). in gol treated ps a marked clinical response was noted at months and beyond, indicating the effectiveness of gol in the treatment of pjia. a significant decrease of the mean jadas . to . (p= . ) after months of treatment was observed, as well as jia acr / / / response rates of / / / %. jadas remission and minimal disease activity was observed in % and . % after months and in % and % after months of treatment. rates of ae, sae and infectious ae were comparable in the gol cohort ( . / py, . / py and . / py), the alternative tnfi cohort ( . / py, . / py and . / py) and the mtx only cohort ( . / py, . / py and . / py). sae reported in the gol cohort were flares of uveitis and of jia (each ) and fibromyalgia syndrome ( ) . sae reported in the alternative tnf cohort was two serious infections (both influenza), one knee ligament injury, one flare of arthritis and one hyperventilation . no case of pregnancy, malignancy or death was reported. conclusion: golimumab seems an effective in treatment of pjia. tolerability was acceptable and comparable to alternative tnfi or mtx. recruitment to the project is ongoing. disclosure of interest none declared introduction: methotrexate (mtx) is one of the most commonly used disease-modifying anti-rheumatic drug in rheumatology practice. it has some side effects that can impair quality of life. the most common of them is associated with the gastrointestinal tract. objectives: the aim of the study is to evaluate and compare the frequency of methotrexate intolerance in adult and pediatric patients. methods: patients with rheumatologic diseases followed in hacettepe university pediatric rheumatology and rheumatology departments who used oral or parenteral methotrexate for at least months were included in the study. methotrexate intolerance was assessed using 'methotrexate intolerance severity score (miss) questionnaire. the miss questionnaire consisted of parts: abdominal pain, nausea, vomiting, fatigue and behavioral symptoms. the patients scored the severity of each symptom separately; : no symptoms, : mild symptoms, : moderate symptoms, : severe symptoms. a total score of or more was defined as mtx intolerance. visual analogue scale (vas) ranging from cm to cm was performed to each patient concurrently with the miss questionnaire. in the pediatric patient group, miss questionnaire and vas assessment were applied to both patients and families. results: a total of patients, of whom were children, enrolled in the study. the mean age for children and adults were . (± . ) and . (± . ) respectively. the most frequent diagnosis of patients was juvenile idiopathic arthritis ( . %) in children and rheumatoid arthritis in adults ( . %). the mean mtx dose in adults and pediatric group was . (± ) mg vs . (± . ) mg (p: . ). the prevalence of mtx intolerance in children and adults were . % (n: ) and . % (n: ) respectively. the mean miss score in the pediatric group was higher compared with the adults ( . ± . vs . ± . , p< . ). similarly, the mean vas scores were higher in pediatric group ( . ± . vs . ± . (p< . )). there was a strong correlation between miss and vas scores between family and child evaluations (p < . , r = . / p < . , r = . ). abdominal pain, nausea, vomiting and behavioral symptoms were observed more frequently in children compared to adults. results: ( %) out of patients were diagnosed with psoriasis denovo. one patient was treated with ada (a girl with undifferentiated arthritis who had positive hla-b , anf and family history of psoriasis -her grandmother had psoriasis), patients were treated with eta (both female, one patient had undifferentiated arthritis, the other had enthesitis-related arthritis; both patients had positive hla -b and anf negative). patients achieved significant improvement after changing tnfalpha inhibitor ( -ada, -eta), patient (was treated with eta) had significant improvement after discontinuation of biological therapy. conclusion: this single-center observational study demonstrates the possibility of developing psoriasis de-novo in patients with jia receiving tnf-alpha inhibitors. although more extensive research is needed, our data suggest that discontinuing the tnf-alpha inhibitor or switching to another tnfalpha inhibitor in patients with psoriasis de-novo should be considered as a treatment strategy in such cases. objectives: long-term surveillance of patients newly initiating toc treatment for at least years compared to a cohort of patients newly initiating a comparator biologic using the biker-registry. methods: baseline demographics, clinical characteristics and disease activity, efficacy and safety parameters were compared. efficacy outcomes were jadas , joint counts and functional status safety was assessed by adverse events (ae) reports. results: patients with matched controls have been recruited. patients starting on toc were older at treatment start ( . vs. . years (y); p< . ) and had a longer disease duration (p< . ). toc was significantly more often a second line biologic (p< . ). baseline jadas ( +/- vs +/- ), chaq-di ( . +/- . vs . +/- , ), esr +/- mm/h vs. +/- mm/h and active joint counts ( +/- vs. +/- ) were comparable. upon toc a substantial response with a significant reduction in jadas from . to . (p< . ) after months of treatment was observed. there were no significant differences between patients from the toc cohort and their matched controls in the jia acr / / / criteria, jadas , jadas remission and minimal disease activity was reached by comparable numbers (toc % and %; control cohort % and %). the total number of ae was comparable (toc cohort n= ae; ( / py); control cohort n= ; ( / py; rr . ; %ci . - . ). more serious ae (sae) were reported with toc. serious infections were documented at lower frequency with toc. uveitis events were documented at significantly higher frequency with tnf inhibitors most likely due to a selection bias (table ) . sae with toc were depression (n= ) in with suicidal intent, exacerbation of jia (n= ), septic arthritis, gastrointestinal infection, abdominal pain, colitis, paronychia and fracture. sae in the control cohort were depression, osteomyelitis, gastrointestinal infection and disease flare. no significant differences regarding cytopenias and elevated transaminases were observed. no gastrointestinal perforation, no vascular events and no deaths occurred. conclusion: toc was effective and comparable to treatment with alternative biologics. tolerability was acceptable. as toc was given as a second-line biologic in the vast majority of patients comparisons between the cohorts have to be interpreted carefully. observation is ongoing. conclusion: in this retrospective cohort study in pediatric patients on rtx-treatment, we found undetectable low drug levels in adapositive patients, indicative for their neutralizing capacity. consequently, the lack of b-cel depletion leads to reduced treatment efficacy. patients with sle seem more susceptible to develop ada. if ada are detected, continuation of treatment seems non-effective and changing medication is advised. certainly when considering that, in this study, anaphylactic reactions only occurred in ada-positive patients. none declared objectives: the aim of this study was to evaluate retrospectively the long-term efficacy and safety of adalimumab in patients with jiaassociated uveitis. methods: we have retrospectively analysed nineteen jia patiens data with associated uveitis from our centre registry between and , treated with adalimumab after failure of treatment with corticosteroids and metotrexate. demografic data and blood samples were collected at different time points while uveitis activity was evaluated by slit-lamp biomicroscopy. adverse events were recorded. results: registry records provided years follow up of jia patients data with associated uveitis. eleven patients were females ( . %) diagnosed as oligo/extended oligoarticular jia while eight ( . %) were males diagnosed as enthesitis related arthritis (era). before adalimumab was prescribed, all patients were previously treated with metotrexate during . years in avarage dose of mg/ m weekly. the mean uveitis duration, before adalimumab administration was months. ten years long follow up period have showed that there were no new relapsis of uveitis while patients were receiving adalimumab and metotrexate. all of our patients were able to gradually tapper and stop treatment with topical steroids two months after adalimumab commencing. seven patients were able to stop biological treatment after . years of adalimumab usage. uveitis relapsed three monts after the adalimumab discontinuation only in one patient. two patient were lost to follow up during the transitional period. no serious adverse events were recorded. conclusion: during the long term follow up period adalimumab have shown good efficacy and safety profile in jia patients with active inflammatory ocular disease. introduction: post-streptococcal syndrome is a systemic immunemediated complication of beta-haemolytic streptococci infection, mostly seen as post-streptococcal arthritis, rheumatic fever or glomerulonephritis. uveitis is an uncommon manifestation of this syndrome. objectives: case report methods: case report results: a previously healthy -year-old female was admitted at the emergency department with prolonged fever, arthritis and red eye. she had a -month history of febrile episodes every two weeks, with axillary temperature ranging from , to ºc. migratory arthralgia affecting both knees and tibiotarsal joints showed up two months after the fever onset and worsened in the previous week, with refusal to walk. non-painful bilateral red eye for several weeks was mentioned. other symptoms were absent. recent infections were denied and family history was irrelevant. physical examination revealed lower limb muscular atrophy, knees pain and impaired function and bilateral tibiotarsal arthritis with inability to walk. ophthalmological observation showed a bilateral non-granulomatous anterior uveitis. sequential laboratory work up revealed a maximum eritrocitary sedimentation rate of mm/h, maximum c-reactive protein of , mg/dl, microcytic hypochromic anemia, positive antistreptolysin o titer (asot) (initial result of that increased to in weeks and later decreased to ) and negative anti-nuclear antibodies. cardiac involvement was excluded. the diagnosis of rheumatic fever with concomitant poststreptococcal uveitis was assumed and the patient was treated with oral and topical ophthalmic corticosteroids with prompt clinical resolution of fever, acute polyarthritis and uveitis. no relapse occurred in a -year follow-up. conclusion: juvenile idiopathic arthritis (jia) is the most common cause of uveitis in childhood. although our patient clinical course could initially raise the possibility of systemic jia (sjia), the criteria that define this entity weren't all present and clinical and laboratory findings were more supportive of rheumatic fever. besides, uveitis occurs exceptionally in sjia, which turned this diagnosis even less reasonable. in our rheumatology unit, among patients diagnosed with jia in years, had uveitis. however, in the group of patients with sjia only one had ocular involvement, a boy with isolated vitritis. post-streptococcal uveitis (psu) typically presents as bilateral, non-granulomatous anterior uveitis, as described in this case. as streptococcal infection is very common among children and many patients may experience subclinical infection. psu should be considered in all patients with uveitis along with positive asot and negative routine investigations for other causes. although psu has been described in literature, to the best of our knowledge, this is the first reported case of concomitant rheumatic fever and psu. . ada was first tapered to every weeks by % of the responders and then to every weeks by % before discontinuing. fewer respondents used or tapered ifx, toc or aba. around % tapered the interval and % tapered the dose and interval for aba, % for toc and % ifx there were differences in the duration of tapering prior to discontinuation of specific medications. for ada it was months in %, months in % ,and months in %. for ifx it was months in %, months in %, and months in %. for toc it was % after weeks, % after weeks and % after weeks. for aba i.v. it was % after weeks, and % after weeks. if combination therapy was used, % tapered the bdmard first, % csdmard first, and % both simultaneously. conclusion: this is the first survey to describe "real world" medication tapering and discontinuation practices of pediatric rheumatologists and ophthalmologists globally. most physicians start to taper medication after months of remission on medication and discontinue after the to months of tapering. we would like to thank all the participating colleagues, who took time to fill out our surve introduction: jia-associated uveitis (jia-u) occurs in - % of children with juvenile idiopathic arthritis (jia) and typically asymptomatic, and sight-threatening complications occur in % of children, (i.e. cataracts, vision loss). frequent ophthalmic examinations are important for early diagnosis and monitoring of uveitis activity. even after uveitis is controlled, risk of disease exacerbation still exists. therefore, frequent ophthalmic screening and monitoring is important for detection and management of jia-associated uveitis (jia-u). s proteins, cytokines, and chemokines detected in aqueous humor of patients with uveitis are also detected in tears. biomarker discovery using tears is promising since collection is noninvasive, feasible, well-tolerated, and close to the target organ. objectives: we aim to determine if s proteins, cytokines, and chemokines levels differ in tears of children with jia and jia-u and in children with jia-u by uveitis activity. methods: tears were collected using schirmer strips from children ≥ years old with oligo-or polyarticular rf negative jia with (jia-u) and without uveitis (jia-no-u), and in children with jia-u at time of active and inactive eye disease. activity was defined by standardization of uveitis nomenclature (sun) criteria. active uveitis was anterior chamber inflammation grade ≥ . + cells. s a , a , and a were measured by elisa, and il- , il- , ip- , mcp- , rant es, and sicam- by luminex assays. biomarker levels were compared in children with ) jia-no-u (n= ) to active jia-u (n= ), and ) jia-u (n= ) at time of active and inactive uveitis. results: children with jia-no-u and jia-u were matched by jia subtype and arthritis activity. they had primarily oligoarticular jia ( %), active arthritis ( %), and were on systemic medication ( %). at time of active uveitis, % had grade . +, and % had + and mean interval between time of active and inactive disease was months. we found that levels of biomarkers in tears of children with jia-no-u compared to active jia-u were similar. although not statistically significant, levels of s a (mean difference , pg/ml [ % ci - to , ], p= . ) and sicam- ( pg/ml [ % ci - to , ], p= . ) were higher when uveitis was active compared to inactive. conclusion: our results suggest that s a and sicam- are potential biomarkers of uveitis activity in jia-u, but not uveitis diagnosis. thus, neutrophils may play a role in the pathogenesis of anterior uveitis which has been reported in an animal model of acute anterior uveitis. identifying biomarkers using tears provides a noninvasive and objective method of monitoring uveitis. limitations are our heterogeneous cohort that varied by arthritis severity and immunosuppression, and minimally active uveitis. we were underpowered to detect statistically significant differences and continue to collect tears prospectively in children with jia-u with goal of n= . despite low uveitis activity, we were still able to detect differences. further studies in larger and diverse cohorts are necessary to assess the role of s a and sicam- in jia-u. objectives: to report an extremely rare presentation of gpa in a year old with acute digital ischemia. a year old boy, with a background of poorly controlled type diabetes and hypothyroidism, initially presented to hospital unwell with diabetic ketoacidosis. treatment was initiated promptly with good response. furthermore, he was found to have weight loss, productive cough and hearing loss over the past months. he was haemodynamically stable, but very pale and cachectic. he had reduced air entry and crackles on the right. there was hypertonia and clonus in his lower limbs. blood tests showed microcytic hypochromic anaemia (hb g/l), normal white cell count, thrombocytosis and raised inflammatory markers (crp mg/l and esr mm/hr). his chest x-ray showed enlargement of the right hilum with consolidation/ atelectasis extending into the middle and lower lobes. mri scans of head and spine were normal apart from fluid opacification in the paranasal sinuses. he was screened for infections including tuberculosis and started on intravenous antibiotics. on day , he developed painful bluish discolouration of his left hand, particularly his thumb, index and middle fingers. his left radial and brachial pulses weren't palpable. a heparin infusion was started. a doppler scan showed occlusion of radial and ulnar arteries proximal to the wrist with no clear thrombus. he had a ct thoracic aorta with contrast which showed proximal left radial artery occlusion and distal ulnar artery occlusion with no evidence of proximal embolic source or vasculitis. it showed multiple perihilar masses (lymph nodes) in the right lung and peripheral parenchymal masses in both lungs, suggestive of atypical infection or connective tissue disease. blood tests still showed raised inflammatory markers(crp mg/l, esr mm/hr and platelets /l). an autoantibody screen showed positive anca with strongly positive anti pr (> u/ml); other autoantibodies, including ana, ds dna and anti-phospholipid antibodies, were negative. he developed further ischaemia with bluish, painful discoloration of his right foot, especially right great toe, with a weakly palpable dorsalis pedis pulse. doppler scan revealed occlusion/narrowing of the posterior tibial artery cm proximal to the ankle. following vascular team advice, he was started on ilioprost infusion to aid reperfusion of the extremities involved, with good results. based on clinical and lab features of systemic inflammation, evidence of upper airway involvement(bilateral conductive hearing loss and sinusitis on mri scan), parenchymal lesions on ct chest and strong pr positivity, a diagnosis of gpa was made. results: our patient responded well to therapy including multiple pulses of high dose methylprednisolone and cyclophosphamide, with improvement of all organs involved and no further digital ischemia. conclusion: although gpa is very rare in children, it is associated with high morbidity and mortality. many studies show that the spectrum of paediatric gpa is not vastly different from adults, except for higher gender bias towards female, more constitutional and musculoskeletal symptoms and higher risk of subglottic stenosis. although there are a handful of case reports of digital ischaemia in adults with gpa, to our knowledge this is the first case report of acute digital ischaemia in paediatric gpa. early diagnosis and prompt treatment with a multidisciplinary team approach is paramount for good outcome. introduction: adenosine deaminase- deficiency (dada ) is a monogenic vasculitis syndrome whose presentation ranges from recurrent fevers and livedo reticularis to systemic vasculitis, hematologic and immunologic abnormalities, and early-onset stroke. it is characterized by biallelic loss-of-function mutations in the encoding gene of ada protein and low levels of ada enzymatic activity in the peripheral blood. the genotype and phenotype features of dada has a wide spectrum. treatment with anti-tnf inhibitors is effective in controlling vascular inflammation and reducing strokes. objectives: to describe two sisters with different presentations of dada and a deletion mutation on exon of the ada gene. methods: medical data was used to describe the clinical manifestations of two siblings. parental informed consent was obtained. results: patient : a -year-old female had presented with fever, rash, arthralgia, hepatosplenomegaly, and coombs positive autoimmune hemolytic anemia (aiha) at the age of years. she had been followed with a suspected diagnosis of systemic lupus erythematosus (sle) and steroids, azathioprine, mycophenolate mofetil had been used. her ana and complement levels were normal. because of unmet classification criteria of sle, genetic testing had been done, and no mutation found in the ada gene. cranial mr and mr angiography was normal. she was referred to our clinic after . years of the first manifestation. physical examination revealed raynaud phenomenon on both hands and feet, livedo reticularis, arthritis, and splenomegaly. laboratory tests indicated an increase in acute phase reactants, cd , cd , and switched memory b cell lymphopenia, and hypogammaglobulinemia. because of prolonged fevers, a thorax ct was obtained and aneurisms of the renal artery were seen. abdominal ct angiography indicated multiple aneurysms of both renal, intercostal, and hepatic arteries. repeated genetic analysis of the ada gene showed a homozygous deletion mutation on exon . she has been followed on anti-tnf and iv immunoglobulin without severe symptoms for a year. patient : the older sister had been followed with a diagnosis of familial mediterranean fever with e q heterozygous mutation because of recurrent fever, abdominal pain, erysipelas-like erythema, elevated acute phase reactants, and splenomegaly. she did not have any other cutaneous or systemic findings. because of parental consanguinity, the ada gene was analyzed and a homozygous deletion mutation on exon was found. she has been followed without any symptoms after anti-tnf treatment. throat swab was negative. abdomen ultrasound showed bowel wall thickening, testis ultrasound was normal. hsp diagnosis was confirmed. methylprednisolone iv was administered for three days, then oral prednisone was started. purpuric lesions, abdominal pain persisted, so we decided to add mmf ( mg/ m /day) and prednisone was tapered in a month. results: thanks to mmf vasculitis lesions and abdominal symptoms disappeared in few days. mmf was continued for a month, tapered in months. there was no evidence of relapse in a months follow up. conclusion: these cases suggest that mmf may be useful to induce and maintain remission of recurrent hsp with gastrointestinal involvement. multicenter clinical trials with long-term follow up to confirm the efficacy of mmf in the treatment of hsp with gastrointestinal involvement are needed. introduction: henoch-schönlein purpura (hsp), the most common childhood vasculitis. cholecystitis is extremely rare in patients with hsp. this is the first case of a libyan child presenting with hsp complicated by calculus cholecystitis hsp nephritis. objectives: our aim is to present an unusual case of gall bladder involvement in an -year-old libyan female affected by hsp. methods: a case reports study results: : we report an unusual case of gall bladder involvement in an -year-old libyan female with hsp. she was referred to a rheumatology clinic due to hsp with chronic calculus cholecystitis and distended small bowel with fluid-like fecal material with no evidence of intussusception on an abdominal ultrasound. the patient had a one-month history of abdominal pain, purpuric lesion on lower limbs and swelling in both feet. she was admitted times to another hospital before being referred to the rheumatology clinic. an abdominal sonography revealed a distended small bowel with fluid-like fecal material with no evidence of intussusception and chronic calculus cholecystitis; they treated her with urosdoxycholic acid tab at mg per day and ibuprofen syrup. then referred to our rheumatology clinic. after days, she showed a purpuric rash over her lower extremities, mainly over her thighs and buttocks, microscopic hematuria, no arthritis, no fever, no abdominal pain; her blood pressure was normal at \ mmhg, and she had normal laboratory tests (cbc, wbc . , hgb . , platelets esr ml\hour, crp mg\dl was negative, c was mg\dl within normal range - mg\dl, c was . mg\dl within normal range - , anca, ana, as well antidsdna ab yielded negative, antistreptolysin-o (aso) titer was todd , lft included total bilirubin , direct , indirect gpt,got, u, creatinine ) except urine routine showed mild microscopic hematuria rbc hpf , protein was nil ) urine for pro-tein disclosure of interest none declared p correlation of serum neopterin levels with disease activity and moneta division of rheumatology, irccs, ospedale pediatrico bambino gesù, roma; university of genova a multinational study of thrombotic microangiopathy in macrophage syndrome clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis laboratory biomarkers to facilitate differential diagnosis between measles and kawasaki disease in a pediatric emergency room: a retrospective study a rare case of measles-associated hemophagocytic lymphohistiocytosis in an infant. cureus children's interstitial and diffuse lung disease respiratory complications of the rheumatological diseases in childhood on behalf of dr nishant dharsandiya and dr j.p. keshrani paediatric rheumatology & immunology, dev children's hospital arthritis care res (hoboken) disclosure of interest: none declared development and initial validation of the ms score for diagnosis of macrophage activation syndrome in systemic juvenile idiopathic arthritis development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome double-blind, placebo-controlled study of anakinra in pediatric and adult patients with still's disease l. schanberg , p. nigrovic duke children's hospital & health center, durham; boston children's hospital, boston; children's mercy kansas city, kansas city; university of alabama at birmingham, birmingham; nationwide children's hospital, columbus; university of iowa hospitals and clinics children and adolescents with sle: not just little adults severe disease presentation and poor outcomes among pediatric systemic lupus erythematosus patients in south africa the checklist manifesto: how to get things right clinical review: checklists -translating evidence into practice the who surgical safety checklist: a review pehlivanoğlu p gastrointestinal henoch-schönlein purpura treated with mycophenolate mofetil: description of two case reports venous vessel wall thickness in lower extremity is increased in male patients with behcet's disease increased vein wall thickness in behçet disease polyarteritis nodosa: a contemporary overview eular / printo / pres criteria for henoch -schönlein purpura , childhood polyarteritis nodosa , childhood wegener granulomatosis and childhood takayasu arteritis : ankara six patients were treated with canakinumab and patients with anakinra. conclusion: it is known that excessive production of il- β can cause inflammatory bone loss and abnormality. vitamin d deficiency and osteopenia/ osteoporosis may cause additional musculoskeletal problems besides arthritis and joint destruction in caps. we think that ca metabolism and bone mineral density measurements should be a part of routine controls in patients with caps. disclosure of interest none declared ab clinical and genetic features of patients with periodic syndrome associated with mutation of the tumor necrosis factor receptor gene and juvenile arthritis having mutations in tnfrsf a gene m active arthritis in / , it was poly in , oligo in . when assessing the clinical symptoms and laboratory activity of patients with jia, it was revealed that in the onset of the disease, systemic manifestations were observed in / : fever in / , rash in / , hepatosplenomegaly in / , pneumonitis in / , carditis in / and lymphadenopathy in / . high laboratory activity was recorded in / . active arthritis in / , it was polys in , oligo in . in all % of patients, the nucleotide variants of the tnfrsf a gene were identified in the study. / of patients were diagnosed with traps. the most frequent heterozygous variant of tnfrsf a gene with nucleotide substitution of c. g>a was found in / of patients, in / of patients it was found homozygous variant with nucleotide substitution of c. g>a, in / of children it was found heterozygous variant with deletion of c. _ del. all of these variants are pathogenic. / of patients were diagnosed with ja: juvenile arthritis with a systemic onset was in / , paucarticular arthritis was in / , in / it was poly rf-and in / it was psoriatic arthritis. it is worth noting to note that in / a heterozygous version of the tnfrsf a gene was detected with a nucleotide substitution c. g> a, however, considering the absence of clinical manifestations of autoinflammatory disease and active articular syndrome in these patients, children were diagnosed with ja. in addition dvoryakovskaya: none declared, a. mamutova speaker bureau of: novartis, k. isayeva: none declared, r. denisova speaker bureau of: novartis covid- and relapsing kawasaki disease: a case report during the pandemia m. c. maggio ab introduction: the pandemia of covid- remains a global health alarm with high incidence of lethality, especially in older age groups who suffer from underlying medical conditions. however, children are less likely to manifest severe conditions. objectives: covid- was correlated to a higher incidence and a suspected increased risk of kawasaki disease (kd) in children anamnestic records revealed a previous kd, without coronary artery lesions (cal), year before. results: he was treated with antibiotics, intravenous infusion of immunoglobulins (ivig) ( gr/kg), acetylsalicylic acid (asa) ( mg/kg in doses/day) and reached defervescence into days. echocardiography excluded cal. the nasopharyngeal swab for sars-cov- was doubt. the second throat swab done the day after ivig infusion, was negative; however, the third nasopharyngeal swab for sars-cov- , done days after ivig infusion, was positive. chest x-ray showed a significant lung interstitial thickening. il- levels were < . pg/ml (n.v. < . pg/ml). he continued treatment with antibiotics, asa ( mg/kg/day), with the progressive resolution of the clinical symptoms and of the normalization of laboratory findings. conclusion: the peculiar outcome of the patient is the correlation of covid- with kd, recently reported as associated. kd is considered as a multifactorial autoinflammatory disease, induced by a cytokine hypersecretion with a systemic vasculitis. covid- is considered a cytokine storm syndrome, with a severe systemic vasculitis. sars-cov- infection could be the trigger that could lead to hyperinflammation of kd. the ivig infusion could explain the transient negative swab for sars-cov- , with the successive positive relieve lasting days, and the normal levels of il- , detected after ivig infusion. relapsing kd is rare ( . - . %); in our patient this event could be triggered by the documented sars-cov- infection. disclosure of interest none declared disclosure of interest none declared ab spectrum of systemic inflammatory syndrome in children during covid pandemic in india d. b. pandya, on behalf of dr haresh dobariya pediatric rheumatology & immunology, dev children's hospital ab rituximab for treatment of resistant paediatric mctd v. paisal, s. compeyrot-lacassagne paediatric rheumatology the diagnosis and classification of of mixed connective tissue disease mixed connective tissue disease in children -case series the value of rituximab treatment in primary sjögren's syndrome juvenile idiopathic arthritis a multifaceted approach is essential for robust rehabilitation m methods: in a retrospective study children ( % girls) aged median (iqr) , ( , - , ) years with oligoarticular onset jia without extra-articular manifestations (oligo-ja) who did not received dmards were monitored. all children were met ilar criteria. ttriamcinolone acetonide (ta) was administered intra-articular at a dose of - mg with an injection interval of - - months which was depended on the activity of the disease. the maximum allowable number of consecutive isolated intra-articular injections (is-iai) was - . a total of active joints were injected with ta: knees - injections, ankles - injections. all children were divided into two groups: active / inactive arthritis based on the effectiveness of local corticosteroid treatment. the average follow-up was and physicians' assessment of jia disease activity efficacy is-iai of ta was no associated significantly with number of active joint of onset oligo-ja, cjadas , serum level of crp mg/ml, esr mm/h, il pg/ml and tnf-α pg/ml, titer of anf. the mean inflamed synovial fluid of il levels abstracts from conferences and relevant studies were added. rcts were included if (i) patients were aged ≤ years, (ii) patients had a previous defined pediatric rheumatic diagnosis and (iii) rct met predefined outcomes. studies were excluded in case of (i) observational or single arm study or (ii) sample size ≤ patients. study design, location, duration, treatment, population, sample size, age criteria, gender, concomitant treatments and primary outcome was extracted. results: out of screened references, references reporting unique rcts in pird. all rcts reported efficacy while / rcts provided safety outcomes and / rcts provided pk data. ten of reviewed bdmards are approved for pirds by the food and drug administration (fda). of these, seven had ≤ rcts. the most common intervention was tnf inhibitors ( %) treatment with intravenous immunoglobulin (ivig) significantly reduces the risk of caas. however, up to % of cases are ivig resistant with a higher risk of cardiovascular complications. currently several second-line treatments are available for refractory kd. nonetheless, the existing literature is still unable to identify which treatment is the most effective. recent studies suggest that a il- receptor antagonist (anakinra) may be an effective therapy in refractory kd. objectives: we report the case of a year-old boy diagnosed with kd refractory to conventional treatment, who developed giant caas successfully treated with subcutaneous (sc) anakinra. methods: case report. results: a year-old boy was referred to our pediatric rheumatology unit days after the onset of a typical refractory kd. he had been previously treated at a local hospital with two doses of ivig ( g/kg), infused respectively and days after the onset of the fever. afterwards, given the persisting fever, doses of pulse intravenous (iv) methylprednisolone (mpdn mg/kg/day) have been used for days followed by oral prednisone ( mg/kg/day). treatment with acetylsalicylic acid ( mg/kg/day q h) was also started. following a transient defervescence the day after the first iv pulse mpdn, fever relapsed and the echocardiography showed caas of left main coronary artery (lmca), left anterior descending (lad) and right coronary artery (rca) rationale and study design for a phase i/iia trial of anakinra in children with kawasaki disease and early coronary artery abnormalities (the anakid trial) the use of interleukin receptor antagonist (anakinra) in kawasaki disease: a retrospective cases series there are few reports of acute kidney injury (aki) in kd, defined as serum creatinine level elevation to more than . times of baseline level. objectives: to describe the case of kawasaki disease complicated by aki methods: a -year-old female was admitted to our rheumatology unit with persistent fever ( days), widespread polymorphous exanthema, change in lips and in oral mucosa. family history was unremarkable. she had no chronic underlying disease nor history of previous hospitalization. at admission, she appeared stable. body temperature was . °c, o saturation was % in ambient area, blood pressure was / mm hg, heart rate was bpm, respiratory rate was breaths per minute. on examination she presented widespread polymorphous exanthema, changes in lips and in oral mucosa, cervical lymphadenopathy and bilateral conjunctival injection. results: exams revealed: white blood cells /μl, hb . g/dl, platelets . /μl, albumin . g/dl, serum sodium meq/l, serum chloride meq/l. transaminases were in normal range. creatinine was . mg/dl disclosure of interest none declared ab paediatric extra-pulmonary large vessel arteritis, a forme fruste of pediatric behcet's disease? we presented two siblings from a consanguineous marriage with different clinical presentations of dada . further, we emphasize that genetic testing should be repeated in the presence of clinical suspicion. introduction: there are several scoring systems developed in japan that are clinically used to stratify high risk kd patients and thus identify the ones that may benefit from early adjunctive therapy. there are increasing reports from all over the world on poor performance of these scores in other ethnic populations. objectives: the aim of our study was to evaluate the kobayashi, egami, sano and kawamura scores in our population which is homogenous caucasian. methods: hospital database was retrospectively searched for code m . of the international classification of diseases, th revision, clinical modification code: mucocutaneous lymph node syndrome [kawasaki] , over the period from january to december . all patients who were seen in this period for the first time for complete or incomplete kawasaki disease, as defined by the american heart association, were included. we applied ivig resistance prediction scores (kobayashi, sano, egami and kawamura scores) to our cohort. only patients who received g/ kg ivig within the first days of the disease were included in this analysis. the scores of prediction models were calculated for each patient and patients were assigned to high-or low-risk group accordingly. results: during the study period a total of children were diagnosed with kd ( . % males, median age . years). all of them were caucasian except one child who was biracial (caucasian and african american). among them, children were hospitalized in the acute phase of the disease and children were seen in the subacute phase of the disease. children were followed-up for at least one year to evaluate persistent coronary artery aneurysms (caa), which were observed in ( . %) patients. among them, were not treated with ivig and received ivig after days of illness. patients were treated with ivig within first days of illness and were included in the calculation of ivig resistance prediction scores. ( . %) were ivig resistant. sensitivity of kobayashi, sano, egami and kawamura scores were . , . , . and . , respectively. specificity of those scores were . , . , . and . , respectively. we found no difference in demographic or clinical characteristics between ivig resistant and ivig responsive patients. patients with ivig resistance had significantly higher alt (p = . ), neutrophil-to-lymphocyte ratio (p = . ) and lower serum sodium (p = . ). conclusion: by applying the japanese scores to our population, we were able to identify most of the low-risk, but missed many of the high-risk patients. our results are consistent with caucasi n based population studies available to date. introduction: varicella zoster virus (vzv) related arterial ischemic stroke (ais) has been described in literature in pediatric age. however, the long-term course of post-vzv vasculopathy need to be inquired: clear information about prevalence of recurrence and severity of clinical outcome are lacking, even if a favorable evolution was initially described, and therapeutic protocols are not currently standardized. objectives: we aimed to describe the clinical, laboratory and neuroradiologic features of children affected by ais due to post-vzv referred to our institute and to present our experience in their therapeutic management. methods: we selected pediatric patients ( females) with ais and a cns confirmed vzv reactivation and/or with a vzv history in the previous months. other causes of pediatric stroke (systemic disease, cardiac disease, trauma, major thrombophilia) were excluded. clinical, neuroimaging, laboratory and treatment data were reviewed, focusing on pediatric score outcome measure (psom) and executive functions final outcome. results: average age of ais onset, vzv primary infection and interval between infection and ais were: years mo (range: year and mo- years and months), years and months (range months- . years), and months (range days- months), respectively. the ais involved the nucleo-capsular region in cases, the cerebral cortex in cases, the thalamus in cases, and the pons in subjects. seventeen patients had inflammatory focal cerebral arteriopathy (ifca). virological confirmation (vzv-dna or anti-vzv igg in the cerebrospinal fluid) was obtained in patients. three patients were treated with trombectomy and one with rtpa. thirteen patients were treated with antiviral agents associated with steroids in cases, with different administration schedules. only in one case steroid treatment was given without association with antiviral agents. one patient received a short course of steroid and antiviral treatment at the time of the stroke and then a more prolonged course after six months at the time of the virological diagnosis. prophylactic antiaggregants were administered to all patients. mean follow-up was years and months (range mo - years) ; ifca was persistent in cases and transient in subjects. four patients presented a recurrence of post vzv arteriopathy, two of them presenting new stroke events. twelve patients presented a variable motor deficit at last follow up. the mean psom score of the cohort at the last visit was (range - ). executive functions were evaluated at last follow up in twelve patients, showing no deficit in seven patients, a mild deficit in two patients and a severe deficit in the last three. conclusion: albeit a favourable evolution was initially described, our experience suggests that vzv-related ais may result in persistent fca and significant neurological impairment in the majority of cases. therapeutic approach, particularly involving steroid administration, still need to be validated. introduction: iga vasculitis/ henoch schönlein purpura (igav/hsp) is the most common vasculitis of childhood and renal involvement is the most serious long-term complication. a better understanding of the pathophysiology of the progression to kidney disease is required for better treatment to be achieved and current biomarkers of ig a vasculitis with nephritis (igavn) lack the predictive value. objectives: in this study, an untargeted metabolomics approach was performed to reveal the underlying molecular mechanism of disease pathogenesis and to find potential biomarkers of plasma samples from patients with igav and igavn.methods: igav was diagnosed according to the ankara criteria in ( ). forty-five patients, including active igav patients (h), igavn (n), and age-and gender-matched healthy controls (c), were enrolled in the study. plasma samples from subjects were collected on the same day of igav(hsp) diagnosis and before steroid or other immunosuppressive treatment initiated. this study has utilized liquid chromatography-mass spectrometry (lc-ms/ q-tof) to investigate the alterations in plasma metabolomic profiles. three separate pools, health controls, active igav , and igavn were created. peak picking, grouping, and comparison parts were performed (metabolite profiling) via xcms (https://xcmsonline.scripps.edu/) software. results: totally peaks were detected for group h, n and c. among them peaks were found to be statistically significant and reliable (p< . ) and of these peaks were found to be changed (fold change > . ) between the groups c and h. on the other hand, peaks were found to be changed (fold change > . ) between the groups h and n. the number of the peaks on the intersection of the peaks found to be changed between the groups (c and h) and (h and n) was . based on putative identification results, peaks were matched with metabolites. we found an up-regulated level of dhap( : ), prostaglandin d /i , methyltetrahydrofolic acid, porphobilinogen and n-acetyl- -o-acetylneuraminic acid/n-acetyl- -o-acetylneuraminic acid, -aminopentanamide / -aminopentanoic acid, glycocholic acid, saccharopine, n -succinyl-l-ornithine, gamma tocopherol, and galactosylsphingosine /glucosylsphingosine in igav patients. in conclusion, we have identified a number of metabolites that may be associated with the pathogenesis of igav. we also suggest that dhap ( : ), prostaglandin d /i , porphobilinogen, -methyltetrahydrofolic acid and n-acetyl- -oacetylneuraminic acid/n-acetyl- -o-acetylneuraminic acid may serve as biomarkers for predicting kidney disease since they were increased only in the patients who developed renal involvement at follow-up. children were divided into four groups: those with jia who didn't receive mtx yet (group ); those who received mtx less than one gram during whole treatment (group ); those who received mtx from to grams (group ); children, received more than grams of mtx (group ). the autoimmune inflammatory process in jia can cause formation of pathological changes in the liver, even before the start of treatment. it is confirmed by a statistically significant correlation of bfgf level in st group with liver steatosis according to ultrasound examination (r = . ) and the level of c-reactive protein (r = . ). this indicates a close relationship between the intensity of the inflammatory process and collagen synthesis activation, which can further provoke liver fibrosis. alterative processes in the liver associated with autoimmune inflammation, as evidenced by the presence of a positive correlation between the level of alt and bfgf (r = . ). upon reaching mtx dose gram and grams, it is possible that compensatory processes in the liver are triggered, as evidenced by the negative correlation between the content of bfgf and hgf (r = - . ).conclusion: the use of modern markers with routine laboratory and instrumental studies is appropriate for the timely determination of the risks of developing irreversible pathological changes in the liver during jia treatment with mtx. objectives: the aim of our study is to evaluate the efficacy of (iag) injections in hip in children with (jia) and to assess the factors predicting the improvement of this management. methods: this is a retrospective study, between and , including patients with jia diagnosed according to the ilar criteria. the socio-demographic data were collected as well as the parameters of the disease. the activity was evaluated by jadas. the functional impact was assessed by the lequesne score. the treatments taken have been specified as well as the infiltrations received. the improvement after infiltration was assessed by jadas and lequesne score.results: fourteen patients were included, with mean age . + . . the mean age at the onset of symptoms was + . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . subtypes of jia according to the ilar were: enthesitis-related arthritis in cases, seropositive polyarticular jia in cases, seronegative polyarticular jia in cases, oligoarticular jia in cases and juvenile psoriatic arthritis in one case. all the patients had hip arthritis, inaugural in % of the cases. of these, . % had a flexion deformity and lower limb inequality. the average lequesne index was . + . . the treatments taken were methotrexate in . % of the cases, sulfasalazine in . % of the cases, and the combination of the two in . % of the cases. eleven patients underwent hip infiltration, and three of them required more than one. eighty one percent improved thereafter. the number of infiltrations was not statistically associated with the lequesne index (p = . ). improvement after infiltration was negatively associated with the prior existence of an inequality of the lower limbs (p = . ). the existence of a flexion deformity was not associated with good results after infiltration (p = . , r=- , ). ten patients ( %) among those who had an infiltration did not have to resort to surgery. conclusion: iag injection is an adjunct therapy in aji with hip involvement offering a good results and delay surgery in the majority of cases. the presence of lower limb inequality is associated with less improvement of iag. conclusion: synovial rice bodies are rarely described in juvenile idiopathic arthritis, even less at disease onset. their presence has not been associated to a worse disease prognosis or joint outcome but awareness of the existence of this particular form of intraarticular loose bodies may encourage the clinician to use lower gauge needle during arthrocentesis procedure; this can prevent arthroscopy, as occurred in our case . arthroscopy may be necessary in some cases to achieve full drainage of the joint. in our series the duration of arthritis correlated with the size of rice bodies and the number and agressiveness of procedures needed to evacuate them. objectives: we described a case of liver involvement in sle presenting with emphasis on the differential diagnosis with autoimmune hepatitis. methods: case report study results: : an -year-old female patient was referred to the rheumatology clinic with complaints icteric sclera for months anorexia, malaise, pain in the both knees, ankles joints and both wrists accompanied by swelling, and remarkable motion limitations. laboratory revealed t bilirubin . mainly direct . with elevated liver enzymes got , gpt , alkp , high glutamyl transpeptidase u\l her wbc . hgb , ptl , except very high esr ml\hr, crp was positive mg\dl, viral screen (hcv, hbsag, hiv) was normal, serology tests ana was positive with high titer , anti ds-dna ab was positive , anti-sm was negative, anti lkm antibodies negative, anti smooth muscle ab negative soluble liver antigen were negative, antimitohondrial ab( m ,m ,m ). ultrasound abdomen revealed mild enlarged spleen, abnormal diffused increased liver echogenicity with early stage of liver cirrhosis treated her by fresh frozen plasma times, vit k mg once\ day then was referred to rheumatology clinic regarding her serology tests & developed arthritis of her joints suspected psle! she was performed liver biopsy showed lesions necrotic inflammatory portal and lobular severe in eosinophilic polynuclear with cirrhosis evoking a syndrome of overlap associating a primary biliary cirrhosis and an autoimmune hepatitis. laboratory data revealed liver dysfunction and liver biopsy suggesting autoimmune hepatitis, and she underwent treatment for hepatitis (prednisolone with azathioprine), urosdoxycholic acid with fat-soluble vitamins k, d&a, e. however, with the elimination of jaundice and decreased hepatic enzyme levels, the prednisolone dose was tapered within months and stopped before they were referred to rheumatology clinic. on her review of systems, she has malar rash, generalized fatigability. on physical examination, we found malar rash, levidoreticularis of her skin, swelling and limitation of movement in the knees, ankles, wrists joints. there was hepatosplenomegaly. laboratory data revealed liver treatment for hepatitis, ana still high titer : , antids dan positive with titer iu\ml, antisma was negative .wbc . , hgb . , plt , esr ml\hr, her ultrasound abdomen: revealed slightly heterogeneous liver with coarse echotexture without focal lesion with liver span cm.these paraclinical results together with the clinical findings strongly suggested systemic lupus erythematosus (sle) as the definitive diagnosis. indeed, in this case, aih was associated with sle, prednisolone orally for months, after that dose was tapered and continued, rapid clinical improvement in arthritis, malaise, and general condition. azathioprine was continued. in addition, daily hydroxycholoquine sulfate overlapping of sle and aih should be suspected when aih patients present with a malar or other skin rash. the prompt diagnosis and adjustment of further treatment plans can improve disease outcomes and prevent liver disease progression. introduction: juvenile systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by multi-visceral involvement with an unpredictable prognosis. the diagnosis is usually made in young women aged between to years, however, it can affect people at any age and it is classified as a juvenile illness when it starts before the age of . objectives: we are reporting the epidemical, clinical, therapeutical and evolutional characteristics of a series done in the pediatric pole in setif with girls and boy. methods: the average age of onset is years. the average time limits of the diagnosis is months. the clinical features is done with cutaneous, articular manifestations and fever respectively in % % and % of the cases ,followed by kidney damage in % of the cases , the cardiac, pulmonary and ophthalmological participations are reported with low percentage. haematological involvement was detected in % of the patients and the inflammatory syndrome was almost constant. a positive titer of anti-nuclear antibodies and anti-dna is objectified, as well as a reduction in the complement rate. antibodies anti gp and anti cardiolopine are positive in % of cases. kidney damage was diagnosed in % of the cases , and only one case of overlap syndrome with dermatomyosits was reported. concerning the neurological form it was present in only one addolecent girl ,and only one case of familial lupus.results: the diagnosis is based on the classification of the american college of rheumatology (acr) revised on and the new criteria slicc"systemic lupus international collaborating clinics" . the clinical characteristics of our series relies on global data of literature with the predominance of cutaneous and articular involvement. with however some specific characteristics which are individualized by a more advanced age of onset, years on average in our study versus years and years, the rarity of familial forms ( case), a lower percentage of kidney damage ( % versus % and %).the therapeutic management was based on corticosteroid therapy and hydroxychloroquine in the majority of cases, the use of immunosuppressants has been reserved for severe forms. conclusion: lupus is an autoimmune disease with protean clinical manifestations, the prognosis of which is dominated by renal, neurological and thrombotic disorders. cortisonic treatments and immunosuppressants have significantly improved the prognosis for life . trial registration identifying number: lupus is an autoimmune disease with protean clinical manifestations, the prognosis of which is dominated by renal, neurological and thrombotic disorders. cortisonic treatments and immunosuppressants have significantly improved the prognosis for life . onset of inactive and active oligo-ja were not significantly differ. the analysis revealed a correlation between a short phase of beneficial effect after is-iai of ta and risk of activity disease (with an inactive phase of arthritis less than months, the risk activity was or = . , p < . ; with an inactive phase less than months -or = . , p < . ). rtx was administered to patients who had received high-dose cs with - dmards; in all cases combined pulse therapy cs № - was preliminarily used. rtx mg № was applied after mo- y from the debut of the disease. in all cases, its use led to clinical improvement after - mo with normalization of laboratory activity indicators, in cases a decrease in the level of b cells to - . in μl was noted ( with agammaglobulinemia). after months patients had severe infectious complications, of them ended fatally. another patients had a second stroke. the st patient survived, had a kidney allotransplantation, there is no disease activity. the nd patient, in connection with the development of the demyelinating process of cns, attempted to continue therapy using golimumab with ivig. it led to an increase in the infectious syndrome, therefore, we decided to refrain from continuing with itnf as well. the patient died after years from the administration of rtx due to the progression of neurological disorders. cases with auto-inflammatory syndromes were: chronic infantile neurologic cutaneous and articular syndrome received tcz; it was unsuccessful (hyperthermia and rash persisted, eye lesions progressed, there were no increase in height), later switched to anakinra. family mediterranean fever, received adalimumab (ada). the -year-course of ada leaded to the disappearance of articular and abdominal syndrome while maintaining persistent increased levels of esr and crp and periodic fever. the use of tcz in patients with ssd was more successful. the first patient received it subcutaneously for year, cs&dmards ( were used) had already been canceled, lung and kidney lesions were contained, blood pressure normalized, escsg-ai decreased from to , mrss decreased from to . in the second case, the patient received tcz for months i/v, decrease of escsg-ai . to , mrss to were noted, the dose of cs was halved, he also continued treatment with cyclophosphamide. we introduce a -year-old girl patient who has been admitted to our clinic with suspicion of an erythema nodosum. she had painful subcutaneous nodules for weeks, especially on the lower extremities and her face. macroscopically, central necrotizing skin rashes could be seen. she had frank arthritis of both knee and ankle joints. the comprehensive serological diagnosis (including hepatitis serology and anti-streptolysin titer) were normal except for a slight increase in crp , mg/dl and esr mm/h. the patient also complained of abdominal pain and bloody stools. calprotectin was μg/g. a gastro-coloscopy revealed a small mariske and a minimal inflammation of the ileocecal valve, without signs of vasculitis or chronic bowel disease. a skin biopsy revealed leukocytoclastic vasculitis of the small arteries. angiography of the intestinal arteries was rejected by the family. initially we started a treatment with methylprednisolone pulses followed by oral prednisolone. the patient showed a very good response with quick resolution of the skin symptoms and abdominal pain. the medication could be quickly tapered and discontinued at full remission after one month results: pan is classified as a cutaneous pan (cpan) when there are exclusive skin manifestations, besides arthralgia or arthritis. a systemic pan must be diagnosed with the involvement of internal organs. however, cutaneous pan may evolve into systemic pan. in our patient, the skin and joints were primarily affected. if the existing gastrointestinal complaints are part of a systemic pan or chronic bowel disease could not be cleared yet, due to refusal of further investigations. conclusion: cpan must be considered as a suspected diagnosis in patients with necrotizing skin nodules. as transition of the cutaneous into the systemic form cannot be predicted regular monitoring is mandatory. introduction: prevalence of behcet's disease in children is not known, but is probably very low. extra-pulmonary large vessel arteritis in these cases is even rarer as a presenting manifestation. objectives: to report two cases of paediatric extrapulmonary large vessel arteritis with a 'behcet like disease'. methods: we present case reports of two cases who presented to paediatric rheumatology opd to our department. ms. f, a year old girl was referred to us with history of short duration of fever, generalized lymphadenopathy, neutrophilic leucocytosis, thrombocytosis, hyperglobulinemia and high inflammatory markers. on detailed history and examination she was found to have a healed palatal ulcer and her maternal aunt was found to have a history of recurrent oral ulcer, genital ulcer and enthesitis. patient's montoux test was positive but the gene expert for mtb was negative. md-ct showed a circumferential thickening of aorta, subclavian and bilateral renal artery with stenosis at origin of both renal arteries indicating a vasculitis. few necrotic nodes were also noted in lungs. lymph node biopsy suggested a reactive hyperplasia. tissue typing showed presence of hla b , b . she improved clinically with oral prednisolone and mycophenolate mofetil and had no recurrence till her recent follow up visit. second case, master fkn an year old child was referred to us with a background of week history of fever, non migratory arthritis, raised inflammatory markers and a symptomatic severe aortic regurgitation with pandiastolic flow reversal on d echo. his evaluation showed negative montoux, normal igg levels and hla b b on tissue typing. his aortic wall thickness resolved with mg/kg oral prednisolone and mycofenolate mofetil. results: both these cases have features similar to behcet's disease. these cases do not fulfil isg, icbd or icbd criteria for pediatric behcet's disease. however, the aortitis and other clinical features responded well to the treatment in both cases.conclusion: paediatric case with extra-pulmonary large vessel arteritis that do not meet criteria for behcet's disease but have specific clinical or laboratory features do respond well to immunosuppression. therefore, after ruling out other causes of the large vessel vasculitis, a possibility of form fruste of behcet's disease should be under consideration. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. these abstracts have been published as part of pediatric rheumatology, volume , supplement , : proceedings of the th european paediatric rheumatology congress (pres ). the full contents of the supplement are available at https://ped-rheum. biomedcentral.com/articles/supplements/volume- -supplement- . please note that this is part of . key: cord- -ehq qnoo authors: nan title: liver date: - - journal: canine and feline gastroenterology doi: . /b - - - - . - sha: doc_id: cord_uid: ehq qnoo nan the basic elements of the biliary tract are the hepatic canaliculi, bile ductules, intralobular ducts, interlobular ducts, hepatic ducts, cystic duct, gallbladder, common bile duct, and the pancreaticobiliary sphincter (of oddi). there are many variations on this central theme, the most important of which are (a) the pancreaticobiliary sphincter is a common physiologic and anatomical channel at the duodenal papilla in the cat and (b) there are many anatomic variations in the feline gallbladder, from single gallbladder to bilateral gallbladders, body duplication, fundic duplication, complete duplication, septate, and y-shaped gallbladder. hepatocytes account for % to % of the liver cell mass (see table - ) and contribute to a wide range of metabolic activity, including carbohydrate, protein, lipid, nucleic acid, porphyrin, metal, vitamin, glutathione, hormone, and xenobiotic metabolism; coagulation factor synthesis; biliary secretion; and immune surveillance. , hepatocytes have an eosinophilic cytoplasm reflecting numerous mitochondria, and basophilic stippling caused by large amounts of rough endoplasmic reticulum and free ribosomes. hepatocyte nuclei are round with dispersed chromatin and prominent nucleoli. anisokaryosis is common and often reflects various degrees of polyploidy, a normal feature of more than % of hepatocytes. the average life span of the hepatocyte is to months reflecting their ability to regenerate. hepatocytes are organized into plates separated by vascular channels (sinusoids), an arrangement supported by a reticulin (collagen type iii) network. the sinusoids have a discontinuous, fenestrated endothelial cell lining. the endothelial cells have no basement membrane and are separated from the hepatocytes by the space of disse, which drains lymph into the portal lymphatics. hepatocytes are supported by a number of other cell types, which account for % of the liver cell mass. representing % to % of liver cell mass, cholangiocytes are also known as biliary epithelial cells. they secrete water, bicarbonate, and cations into the bile in the physiologic state, but they may also participate in the immune response as antigen-presenting cells in disease states. the biliary tract is a convergent system of canals that begins in the canaliculi, followed by the bile ducts, and ending with the common bile duct. bile secretion depends on the function of membrane transport systems in hepatocytes and cholangiocytes and on the structural and functional integrity of the biliary tract. the hepatocytes, constituting the most abundant liver cell population, generate the so-called primary bile in their canaliculi. biliary canaliculi are blind tubular structures, with a very high surface-to-volume ratio that by means of osmotic gradients favors the formation of bile flow. cholangiocytes, which constitute % to % of the liver cells, modify the canalicular bile by secretory and reabsorptive processes as bile passes through the bile ducts, and they are responsible for approximately % of bile volume. in contrast to hepatocytes, where secretion is constant and poorly controlled, cholangiocyte secretion is broadly regulated. , the immunoglobulin family, without which the liver cannot clear complement system-coated pathogens. in disease states, kupffer cells contribute to the pathology of ethanol and other toxic principles through production of inflammatory mediators, activation of toll-like receptors, and elaboration of tumor necrosis factor (tnf-α). kupffer cell activation is responsible for early ethanol-induced liver injury, common in chronic alcoholics. ethanol activates the toll-like receptor and cd , receptors on the kupffer cell that internalize the endotoxin lipopolysaccharide. internalization activates the transcription of tnf-α and production of superoxide (a prooxidant). tnf-α then enters the stellate cell in the liver, leading to collagen synthesis and fibrosis. fibrosis eventually causes cirrhosis or loss of function of the liver (see the role of the stellate cell, which is discussed in "stellate cells" section that follows). hepatic stellate cells (hscs) (also referred to as vitamin a-storing cells, lipocytes, interstitial cells, fat-storing cells, and ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store % to % of vitamin a in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. [ ] [ ] [ ] [ ] in physiologic conditions, these cells play pivotal roles in the regulation of vitamin a homeostasis. in pathologic conditions, such as hepatic fibrosis or liver cirrhosis, hscs lose vitamin a and synthesize a large amount of ecm components, including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. the morphology of these cells also changes from that of the star-shaped stellate cell to that of the fibroblast or myofibroblast. hscs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. activation of hscs, a key event in liver fibrosis, is caused by diminished adipogenic transcription. wnt signaling inhibits antiadipogenic activation of hscs and liver fibrogenesis; wnt antagonism inhibits hsc activation and liver fibrosis. also known as natural killer (nk) cells or large granular lymphocytes, pit cells represent % of liver cell mass, and serve as part of the immune surveillance mechanism in the hepatic sinusoids. pit also known as browicz-kupffer cells or stellate macrophages, these cells represent % to % of the liver cell mass, and are specialized macrophages localized to the sinusoids as part of the mononuclear phagocyte system. kupffer cells begin their development in the bone marrow with the genesis of promonocytes and monoblasts into monocytes, and then on to peripheral blood monocytes, completing differentiation into kupffer cells within the liver. in health, kupffer cells are involved in the metabolism of erythrocyte hemoglobin. during perfusion of the liver, senescent red blood cells are phagocytized by the kupffer cells, and the hemoglobin molecule is further metabolized into its component parts. globin chains and amino acids are reutilized; the iron-containing portion of heme is removed, transported, and stored; and heme is further oxidized into bilirubin, conjugated with glucuronic acid within hepatocytes, and secreted into the bile. kupffer cells also express a complement receptor of progenitor cells have multilineage potential and similar characteristics to stem cells. the late progenitor cells have differentiated further and produce progeny in only a single lineage. although they divide rapidly, they are capable of only a short-term tissue reconstitution and they do not self-renew. early studies in hepatocyte turnover and liver regeneration showed that the parenchymal cell, the hepatocyte, was the primary and only cell involved in tissue renewal. however, new studies of liver regeneration, hepatocarcinogenesis, liver transplantation, and various cell lines show that a variety of cell types participate in maintaining hepatocyte number and mass. recent studies indicate the presence of both intrahepatic and extrahepatic stem/progenitor cell populations that serve to maintain the normal organ and to regenerate damaged parenchyma in response to a variety of insults. the intrahepatic compartment most likely derives primarily from the biliary tract, particularly the most proximal branches, that is, the canals of hering and smallest ductules. the extrahepatic compartment is at least in part derived from diverse populations of cells from the bone marrow. embryonic stem cells are considered as a part of the extrahepatic compartment. the precise role(s) of each of these individual cells remains to be determined, but it is clear that in the aggregate they confer the vast regenerative capacity of the liver . the liver is involved in many aspects of intermediary metabolism. the liver is at the center of carbohydrate metabolism through its role in maintaining normoglycemia. glucose is the primary energy source for most mammalian cells, and its metabolism is tightly regulated to guarantee that a sufficient supply is available to glucosedependent organs, particularly the brain. glucose can be made available from two sources: absorption of dietary glucose from the intestine, and release of glucose from organs such as the liver and kidney. early in fasting, the majority of endogenous glucose is generated by glycogenolysis where glycogen in the liver is converted to glucose- -phosphate under the regulation of debranching enzyme, hepatic glycogen phosphorylase, and phosphorylase kinase. with more prolonged fasting, endogenous glucose is generated by gluconeogenesis from certain substrates such as amino acids, lactate, and glycerol. both processes generate glucose- -phosphate, which must then be dephosphorylated in order to transport glucose out of the cell. • early fasting: glycogen → glycogenolysis → glucose → normoglycemia • prolonged fasting: amino acids → gluconeogenesis → glucose → normoglycemia the enzyme responsible for the dephosphorylation of glucose- phosphate is glucose- -phosphatase-α. alterations in quantity, location, or activity of glucose- -phosphatase, such as those seen in type glycogen storage diseases, effectively result in a lack of all endogenous glucose production and severe hypoglycemia develops during periods of fasting. the liver is an important site of protein metabolism. amino acids and proteins absorbed from the intestine or produced in the body are delivered to the liver. the liver deaminates amino acids and cells belong to the group of sinusoidal cells, together with kupffer, endothelial, and fat-storing (stellate) cells. pit cells use the fasl fas ligand (fasl) and perforin/granzyme pathway to kill target cells. fasl on effector cells binds the fas that is present on the target cell membrane, which results in oligomerization of fas and activation of caspase . perforin and granzymes, of which granzyme b is the most potent, reside in granules of the cytotoxic lymphocytes and are released by exocytosis. intracellular delivery of granzyme b results in the initiation of the caspase cascade by proteolytic activation of caspase , either directly or through a mitochondrial-dependent pathway. caspases play a central role in the execution of apoptosis. lymphocyte recruitment from the circulation into tissue is dependent on the ability of the lymphocyte to recognize and bind molecules on the endothelial cell surface that promote transendothelial migration. a multistep model of leukocyte adhesion to vascular endothelium has been described and is broadly applicable, although the details of the signals involved differ between tissues. , in a generally accepted model, tethering or rolling receptors expressed on endothelial cells capture free-flowing leukocytes. these receptors may be either selectins or members of the immunoglobulin superfamily. once captured, the leukocyte can receive activating messages presented by endothelial cells in the form of chemokines that activate specific g-protein-coupled receptors on the leukocyte surface. occupancy of these receptors triggers a cascade of intracellular signals that results in the presentation of high-affinity integrin receptors on the leukocyte surface that bind to immunoglobulin family of counterreceptors on the endothelium to promote leukocyte arrest on the vessel wall. in the presence of the appropriate migratory signals the leukocyte will migrate across the endothelium into tissue, where it follows a hierarchy of chemotactic signals toward the focus of inflammation. representing % to % of the liver cell mass, smooth muscle cells are located primarily in the hepatic artery and portal vein and their tributaries, and serve primarily to regulate the hepatic microcirculation. hepatic fibrosis is a common outcome of hepatic injury in the dog. activated fibroblasts that develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: (a) portal or septal myofibroblasts, (b) interface myofibroblasts, and (c) the perisinusoidally located hscs. it is difficult to arrive at a universally applicable definition of a stem cell because some of the defined properties of a stem cell can be exhibited by the stem cells in some tissues or organisms but not in others. in spite of that, a generally acceptable consensus defines a stem cell as an undifferentiated cell that has capacity to self-renew, for production of progeny in at least two lineages, for long-term tissue repopulation after transplantation, and for serial transplantability. in addition, stem cells exist in a mitotically quiescent form and clonally regenerate all of the different cell types that constitute the tissue in which they exist. they can undergo asymmetrical cell division, with production of one differentiated (progenitor) daughter and another daughter that is still a stem cell. the offspring of stem cells are referred to as progenitor cells, also named as transit amplifying cells and therefore cannot be serially transplanted, and are classified as early and late. the early progenitor or stem/ either removed from the circulation by the liver or undergo further transformation by lipoprotein lipase and/or hepatic lipase to form intermediate-density lipoproteins and ldls. ldls, which are relatively depleted of triglyceride and enriched in cholesteryl esters and phospholipid, circulate in the blood and bind to specific ldl receptors that are widely distributed throughout tissues in order to deliver cholesterol. hdls produced by the liver play an important role as donors and acceptors of apo c, apo e, and various lipids from other lipoproteins in the circulation. reverse transport. hdls play an important role in the reverse transport of cholesterol from the periphey to the liver. lecithin cholesterol acyl transferase esterifies hdl cholesterol and cholesteryl esters move to the core of the hdl molecule to allow more free cholesterol to be absorbed into the particle. continued absorption of free cholesterol and subsequent esterification by lecithin cholesterol acyl transferase leads to the formation of the larger, cholesteryl ester-rich hdl s. hdl molecules continuously acquire cholesteryl esters, resulting in the formation of the hdl molecules. on hdl , cholesteryl esters are transferred from tissues to the liver for disposal or reuse, and not to ldl or vldl molecules (as in humans), which transfer cholesterol to peripheral tissues. this function of hdl s may account for the lower incidence of atherosclerotic disorders in dogs compared with humans. , nucleic acids pyrimidine biosynthesis is one of the classic roles of the liver in nucleic acid metabolism. more recently, micrornas have been impugned in the normal development and regeneration of the liver, as well as in hepatic pathology. micrornas are small noncoding rnas that regulate both messenger rna and protein expression of target genes, which results in alterations in messenger rna stability or translation inhibition. micrornas influence at least one-third of all human transcripts and are known regulators of various important cellular growth and differentiation factors. micrornas recently emerged as key regulatory molecules in chronic liver disease. porphyrins porphyrins are intermediates of the heme biosynthetic pathway. porphyrins are found in hemoglobin, myoglobin, cytochromes, catalase, and peroxidase enzyme. the liver and biliary tract serve as an excretory route for the porphyrins. the liver stores iron, which can be toxic in excessive amounts (hemochromatosis). the amount of iron in the body is largely determined by regulation of its absorption in the upper small intestine. iron is stored intracellularly as ferritin in a number of tissues, with the liver having a large storage capacity. when the capacity of the liver is exceeded, iron accumulates as hemosiderin. the liver incorporates copper into specific copper proteins such as cytochrome c oxidase, mitochondrial monoamine oxidase, and ceruloplasmin. mobilization of copper from hepatocytes takes place by at least two mechanisms: ceruloplasmin and bile secretion. cholestatic liver disease is associated with secondary copper retention, which may then induce hepatocyte injury. , vitamins the liver is importantly involved in vitamin metabolism. the liver produces bile for absorption of fat-soluble vitamins (a, d, e, k), and the liver is an important site for vitamin storage. converts them to carbohydrates and lipids. [ ] [ ] [ ] [ ] deamination produces α-keto acids, which can be metabolized for energy or used for synthesis of monosaccharides and fatty acids. the liver synthesizes amino acids from intermediates of carbohydrate and lipid metabolism by amination and transamination. examples of amino acid transaminations include: • alanine + α-ketoglutarate ↔ pyruvate + glutamate • aspartate + α-ketoglutarate ↔ oxaloacetate + glutamate the liver synthesizes many proteins, including albumin and fibrinogen, most α globulins, and some of the β globulins. prothrombin and clotting factors v, vii, viii, ix, and x are produced in the liver, as well as ceruloplasmin, ferritin, and many serum enzymes. lipid metabolism and transport is organized into three basic transport systems: (a) exogenous transport, which is associated with the metabolism of exogenous (dietary) lipids, (b) endogenous transport, which is associated with the metabolism of endogenously produced lipids, and (c) reverse transport, which is associated with the transport of lipids from the periphery (e.g., skeletal muscle, adipose, connective tissue) to the liver. exogenous transport. triglyceride is the major dietary lipid, along with cholesterol, phospholipids, and fat-soluble vitamins. the digestion of dietary lipids begins in the proximal gi tract with the action of lingual and gastric lipases, and is completed in the small intestine with the actions of pancreatic lipase, cholesterol ester hydrolase, and phospholipase a . lipid digestion and absorption is more complicated than carbohydrate and protein digestion and absorption because of lipid solubility characteristics, and involves emulsification of lipids by bile salts, hydrolysis by pancreatic lipase and colipase, solubilization of fatty acids and monoglycerides into mixed micelles, absorption, reesterification, chylomicron formation, and transport into the intestinal lymphatics or portal capillaries. chylomicrons containing short-and long-chain triglycerides, and the newly incorporated b- apoprotein, are preferentially absorbed into the intestinal lymphatics where they are transported into the cisterna chyli, thoracic duct, and systemic circulation where they acquire apolipoproteins c and e from circulating high-density lipoproteins (hdls). apolipoprotein (apo) c-ii activates lipoprotein lipase in the capillary beds of adipose and skeletal muscle, where they are stored as is or hydrolyzed into free fatty acids, β-monoglyceride, and glycerol. the cholesterol-rich remaining particles (now referred to as chylomicron remnants), return apo c-ii molecule to hdl and are recognized by specific hepatic apo e and apo b- receptors that rapidly remove them from the circulation by endocytosis. the cholesterol found in chylomicron remnants can be used in very-low-density lipoprotein (vldl), lipoprotein synthesis, bile acid formation, or cholesteryl storage. endogenous transport. while chylomicrons are the apoprotein responsible for transport of dietary lipids, vldls, intermediatedensity lipoproteins, low-density lipoproteins (ldls), and hdls are instead involved in the metabolism of endogenously produced lipids. triglycerides and cholesterol produced by the liver combine with phospholipids, apo b- , and apo b- to form vldls. when secreted from the liver, vldls acquire the apo c and apo e from hdl. vldl apo c-ii activates lipoprotein lipase located in the capillary beds, where once again triglyceride hydrolysis takes place with the production of free fatty acids and glycerol. the vldl molecules remaining after hydrolysis of vldl triglycerides are intestinal bacteria to produce the secondary bile acids, deoxycholic acid, and lithocholic acid. , , prior to secretion, bile acids are conjugated with taurine and/or glycine to form tauro-and glycoconjugated bile salts (see figure - , c). conjugation lowers the pk a to well below the physiologic range of biliary and intestinal ph, and conjugated bile acids become ionized anions (referred to as bile salts) rather than undissociated bile acids. in the ionized form, they are less likely to be absorbed by the small intestine and so maintain a higher intraluminal concentration appropriate for emulsification, digestion, and absorption of lipids. dogs and cats conjugate primarily with taurine. dogs can convert to glycine conjugation if taurine is deficient, but cats cannot. cats are obligate taurine conjugators, and have an essential dietary taurine requirement. , , bile salts are amphipathic molecules with polar and nonpolar domains imparting two important functions. bile salts have an initial detergent effect on fat particles in food permitting the breakup of fat globules into smaller sizes. this is the initial emulsification phase of bile salts that facilitates intraluminal lipid hydrolytic digestion. bile salts further assist in the absorption of fatty acids, monoglycerides, cholesterol, and other lipids through the formation mixed micelles. these micelles serve to transfer digested lipids across the unstirred layer of the mucosa. following emulsification and micellarization of fat, most of the secreted bile salts are transported along the gi tract to the ileum where they are absorbed into ileal enterocytes and portal blood flow via na + -bile salt cotransporters. , the liver plays an important role in maintaining hemostasis. the liver produces procoagulant, anticoagulant, and fibrinolytic proteins, and also removes normal and abnormal clotting factors from the circulation. hepatocytes synthesize most of the clotting factors including clotting factor i (fibrinogen), ii (prothrombin), v, vii, ix, x, xi, and xiii. the site of biosynthesis of factor viii remains controversial, but it is probable that the liver plays an important role in this factor, too. the liver is also responsible for the activation of the vitamin k-dependent factors ii, vii, ix, and x and protein c. in addition to the production and activation of coagulation factors, the liver is also essential for the clearance of activated coagulation water bilirubin -vitamin a is stored in both stellate cells and hepatocytes. approximately % of total body vitamin a is stored in the liver, representing a -to -year supply. the liver continues to release vitamin a to maintain normal blood concentrations despite reductions in its content. liver and plasma vitamin a concentrations are reduced by malnutrition, liver disease, and intestinal malabsorption, but signs of deficiency do not appear until abnormalities become severe. fat-soluble vitamins a, d, e, and k require normal bile secretion for absorption. vitamin k is particularly essential for synthesis of the prothrombin-complex clotting factors. water-soluble vitamins, with the exception of vitamin b (cobalamin), are readily absorbed from the small intestine. these vitamins are used primarily as coenzymes in metabolic processes. vitamin phosphorylation, occurring primarily in hepatocytes, is required to produce some coenzymes. thiamine is phosphorylated to thiamine pyrophosphate, for example, primarily in the liver and kidney. nicotinic acid is a precursor in pyridine nucleotide synthesis, and an initial step in its conversion is nicotinamide synthesis in the liver. pyridoxine is phosphorylated to its active form in the liver, as is the transformation of pantothenic acid to coenzyme a. folic acid is converted to its active form in the liver. large amounts of all water-soluble vitamins except vitamin c are stored in the liver. glutathione is synthesized in most if not all mammalian cells. the liver is particularly active and has relatively high levels of glutathione. glutathione performs a variety of physiologic and metabolic functions, including thiol transfer reactions that protect cell membranes and proteins; thiol-disulfide reactions involved in protein synthesis, protein degradation, and catalysis; reduction of capacity; detoxification of hydrogen peroxide, organic peroxides, free radicals, and foreign compounds; and metabolism of various endogenous compounds. seen in dogs with chronic hepatitis plus cirrhosis, which may be a result of reduced synthesis rather than increased consumption of coagulation factors. numerous foreign compounds, including drugs, are so hydrophobic that they would remain in the body indefinitely were it not for hepatic biotransformation. cytochrome p (p or cyp) comprises a superfamily of enzymes that catalyze oxidation of a variety of xenobiotic chemicals such as drugs, toxic chemicals, and carcinogens, as well as endobiotic chemicals including steroids, fatty acids, prostaglandins, and vitamins. the cytochrome p enzymes in families one to three mediate % to % of all phase i-dependent metabolism of clinically used drugs and participate in the metabolism of a huge number of xenobiotic chemicals. there are known active p genes and pseudogenes in the human genome. with active genes, dogs are phylogenetically closest to the human. although there are many similarities between dogs and humans, there also are many important differences. [ ] [ ] [ ] [ ] dogs present an products and the production of clotting factor inhibitors, such as antithrombin and α -antitrypsin, as well as fibrinolytic proteins like plasminogen. in liver disease, factor and inhibitor synthesis and clearance of activated factors in both the coagulation factors and fibrinolytic system may be impaired. the extent of coagulation abnormalities depends upon the degree of disturbed liver function. patients with hepatic failure may present with the entire spectrum of factor deficiencies and may even develop disseminated intravascular coagulation. in a study of dogs with histologically confirmed liver disease, one or more coagulation abnormalities were found in % of dogs with liver disease. activated partial thromboplastin time was significantly prolonged in dogs with chronic hepatitis with or without cirrhosis. mean platelet numbers, antithrombin, and factor ix activity were significantly lower in dogs with chronic hepatitis with cirrhosis, compared to dogs with other hepatopathies. d-dimers were not significantly increased in any group. only three dogs, all with different histologic diagnoses, satisfied the criteria for disseminated intravascular coagulation. hemostatic abnormalities were primarily where nh = ammonia; adenosine triphosphate = adenosine triphosphate; adp = adenosine diphosphate; p i = inorganic phosphate; and amp = adenosine monophosphate. mineralocorticoids (aldosterone), glucocorticoids (cortisol, corticosterone), and sex steroids (androgens, estrogens, progesterone) are metabolized by the liver. changes in the concentrations of total and free cortisol and of the binding capacity of corticosteroid-binding globulin have been reported in canine liver disease. as a consequence of hypercortisolemia, dogs with liver disease and hepatoencephalopathy have clinical and biochemical characteristics of pdh, including polyuria, high basal cortisol levels, and α-melanotropin. , chronic hypercortisolism is associated with impaired osmoregulation of the release of vasopressin and inadequate urinary concentration. the multiple physiologic functions of the liver require an immune response that is locally regulated. pathogenic microorganisms must be efficiently eliminated, while the large number of antigens derived interesting challenge in the assessment of p -mediated drug-drug interactions because most of the enzymes have not been completely characterized, diet and aging induce significant changes in gene expression, and dogs are often treated off-label with a number of human drugs with little idea of risk for drug-drug interaction. drug metabolism takes two general forms: phase i metabolism (modification reactions) and phase ii metabolism (conjugation reactions). phase i metabolism typically subjects a drug to oxidation or hydrolysis. it involves the cytochrome p (cyp) enzymes, which facilitate reactions that include n-, o-, and s-dealkylation; aromatic, aliphatic, or n-hydroxylation; n-oxidation; sulfoxidation; deamination; and dehalogenation. phase ii metabolism conjugates the drug to hydrophilic substances, such as glucuronic acid, sulfate, glycine, or glutathione. phase i metabolism usually precedes phase ii metabolism, but this is not always the case. the liver is an important site of drug toxicity and oxidative stress because of its proximity and relationship to the gi tract. seventyfive percent to % of hepatic blood flow comes directly from the gi tract and spleen via the main portal vein. portal blood flow transports nutrients, bacteria and bacterial antigens, drugs, and xenobiotic agents absorbed from the gut to the liver in a more concentrated form. drug-metabolizing enzymes detoxify many xenobiotics but might activate the toxicity of others. hepatic parenchymal and nonparenchymal cells may all contribute to the pathogenesis of hepatic toxicity. the toxicity of drugs can be considered in five contexts: on-target toxicity, hypersensitivity and immunologic reactions, off-target pharmacology, bioactivation to reactive intermediates, and idiosyncratic drug reactions. , ammonia ammonia is an important by-product of amino acid metabolism. organisms that cannot easily and quickly remove ammonia usually have to convert it to some other substance, like urea or uric acid, which are much less toxic. insufficiency of the urea cycle occurs in from the gi tract must be tolerated. the liver favors the induction of tolerance rather than the induction of immunity. although hepatocytes constitute the major cell population of the liver, direct interaction of hepatocytes with leukocytes in the blood is unlikely. sinusoidal endothelial cells, which line the hepatic sinusoids and separate hepatocytes from leukocytes in the sinusoidal lumen, and kupffer cells, the resident macrophage population of the liver, can directly interact with passenger leukocytes. in the liver, clearance of antigen from the blood occurs mainly by sinusoidal endothelial cells through very efficient receptor-mediated endocytosis. liver sinusoidal endothelial cells constitutively express all molecules necessary for antigen presentation (cd , cd , cd , major histocompatibility complex [mhc] classes i and ii, and cd ) and can function as antigen-presenting cells for cd + and cd + t cells. , thus, these cells probably contribute to hepatic immune surveillance by activation of effector t cells. antigen-specific t-cell activation is influenced by the local microenvironment. this microenvironment is characterized by the physiologic presence of bacterial constituents such as endotoxin and by the local release of immunosuppressive mediators such as interleukin- , prostaglandin e , and transforming growth factor-β. regeneration liver regeneration after partial hepatectomy is a very complex and well-orchestrated phenomenon. it appears to be carried out by the participation of all mature liver cell types. , the process is associated with signaling cascades involving growth factors, cytokines, matrix remodeling, and several feedbacks of stimulation and inhibition of growth related signals. , the liver manages to restore any lost mass and adjust its size to that of the organism, while at the same time providing full support for body homeostasis during the entire regenerative process. in situations when hepatocytes or biliary cells are blocked from regeneration, these cell types can function as facultative stem cells for each other. gene expression in the regenerating liver is a multistep process with at least two critical steps: the transition of quiescent hepatocytes into the cell cycle ("priming"), and the progression beyond the restriction point in the g phase of the cell cycle. hepatocytes must first be primed before they can fully respond to growth factors. as many as different genes participate in the early response to hepatectomy, but tumor necrosis factor (tnf), interleukin (il)- , and interleukin- appear to be the major cytokines involved in the priming of hepatocytes. the proliferative effect of tnf on hepatocytes is further influenced by reactive oxygen species, nitric oxide, and glutathione content, and multiple transcription factors (e.g., nuclear factor kappa b, stat , ap- , and c/ebpβ) play major roles in the initiation of early liver regeneration. progression through the cell cycle beyond the initiation phase requires growth factors, primarily hepatocyte growth factor and transforming growth factor-α (tgf-α). the subsequent expression of cell-cycle genes establishes the stage at which replication becomes growth factorindependent and autonomous. at this point, the hepatocyte is irreversibly committed to replicate and the cell cycle replication machinery takes over. the proliferation of hepatocytes advances from periportal to pericentral ares of the lobules, as a wave of mitoses. hepatocytes surrounding the central veins are the last ones to undergo cell replication. proliferation of biliary epithelial cells occurs a little later than hepatocytes. proliferation of endothelial cells starts at to days and ends around to days after partial hepatectomy. the kinetics of proliferation of stellate cells is incompletely understood. the regenerative capacity of the residual hepatocytes may restore liver mass and function after as much as % to % hepatectomy and it takes place over to days in most animal species. a small wave of apoptosis in hepatocytes occurs at the end of regeneration. the liver is the second largest organ in the body and performs an estimated essential biochemical functions. these diverse functions include drug metabolism; removal of exogenous and endogenous toxins (e.g., ammonia, food antigens); synthesis of vital substances such as albumin and blood clotting factors; protein, fat, and carbohydrate metabolism; vitamin storage and activation; glycogen, triglyceride, and mineral (e.g., copper, iron) storage; activation, conversion, secretion, deactivation, and excretion of various hormones; bile salt synthesis; conjugation and excretion of bilirubin in bile; among others. symptoms (defined here as abnormalities noted by the owner), clinical signs (defined here as abnormalities found during the physical examination), and diagnostic results reflect impairments in these functions. hepatitis represented approximately % of the clinical population of the companion animal teaching hospital of utrecht university. box - summarizes the most common liver diseases with their possible etiologies in dogs and cats. a properly taken history is pivotal to defining the most clinically relevant problems that need to be resolved. a structured interview process and understanding the basics of communication are important success factors to retrieve this crucial information. fortunately, the knowledge about communication in the medical profession and the focus on the veterinary curriculum, has increased considerably during the last few years. , some basic principles should be kept in mind to understand the symptoms in dogs and cats with diseases affecting the hepatic parenchyma, portal vasculature, and the biliary system. first, for most of its functions, the liver has a tremendous (approximately %) reserve capacity and a remarkable potential to regenerate. symptoms occur only when progressive disease exhausts hepatic reserves. diseases often remain subclinical for lengthy periods of time; symptoms may be relatively mild and nonspecific because the liver reserve prevents overt abnormalities. symptoms such as lethargy, vomiting, or mild polyuria and polydipsia (pu/pd) may alert the clinician that a liver disorder could be developing. serious symptoms may indicate loss of hepatic reserves. the onset of symptoms may be acute, but they may be the end result of a disease that has been present for many weeks or months. because no specific physical abnormalities occur with most liver diseases, it is important to remember that liver disease may be present when symptoms of illness are unexplained or nonspecific. sensitive and specific laboratory tests may easily detect such liver diseases. second, most liver diseases cause similar signs and symptoms (table - ). one of these is acholic feces, which occurs nearly exclusively in dogs with common bile duct obstruction. owners b documented in humans, may occur in dogs and cats. a more likely to present with chronic rather than acute liver disease. the clinical signs listed in table - are candidates for having a primary hepatopathy. in all such cases, further diagnostic studies should be performed to confirm or exclude liver disease ( figure - ). breed, sex, age, and drugs may predispose dogs and cats to hepatopathies. the presence of numerous risk factors should be a stimulus for an extended diagnostic workup; other diseases should be investigated in the absence of such suspicions. caused by hypersensitivity to sulfonamides, destructive cholangiolitis is the most common drug-induced liver disease. a recent history of therapy with sulfonamides or other potentially hepatotoxic drugs, combined with icterus makes this condition likely and should prompt immediate discontinuation of the medication. breed associations may occur when a disease is (in part) determined by genetic factors. breeders may, by chance, increase the incidence of hepatic diseases by familial selection. because dog breeds may represent more or less closed populations in a country, breed predispositions may vary among countries. therefore this section only mentions generally applicable predispositions; locally, other breed associations may be more pertinent. chronic hepatitis and cirrhosis, both of which are, as a rule, different stages of one disease, occur more frequently in certain breeds. hepatitis may develop at any age, but typically not before years of age. only lobular dissecting hepatitis tends to occur at a young age (i.e., often before year of age). breeds associated with hepatitis are doberman pinschers, bedlington terriers, west highland white terriers, american and english cocker spaniels, labrador retrievers, and many other breeds. recent copper excretion studies have shown that hepatitis is caused by copper retention and not vice versa in doberman pinschers. the cause of copper retention remains unclear; many of the tested candidate genes (including murr , the affected gene in bedlington terriers) were excluded as monogenetic causes for copper-associated subclinical hepatitis in may note the light-gray appearance of stool, which in combination with icterus is virtually diagnostic for extrahepatic cholestasis. different combinations of clinical signs and symptoms may occur with any liver disease. statistically, one disease may be associated with a typical pattern of signs and symptoms in dogs and cats. however, overlapping patterns are so great that it is useless to try to identify the exact disease based on clinical signs and symptoms alone (table . clinical signs and symptoms associated with liver diseases of cats are similar to those in dogs, except for pu/pd, which is not clinically overt in most cats. certain liver diseases cause neurobehavioral signs associated with hepatic encephalopathy, and these signs may wax and wane in their frequency and severity. any medication given may appear to be effective because of the natural fluctuation of signs. therefore signs of hepatic origin may be easily missed. seizures alone are never caused by hepatic encephalopathy; if they do occur, they occur in combination with other signs seen with this syndrome. furthermore, very few, if any, medications to treat liver diseases have been tested in double-blind, placebocontrolled studies, making decisions about the best therapeutic regimens difficult. it is usually not possible to differentiate between hepatic diseases and diseases of other organs based on symptoms and clinical signs. signs associated with hepatic diseases are nonspecific; similar signs may occur in diseases of many other organ systems, most notably the gi, neurologic, renal, and hematologic systems (see table - ). of the gi tract-related symptoms, nausea expressed as vomiting in acute cases or reduced, irregular appetite with occasional vomiting and weight loss over time, is very common in liver and biliary diseases of dogs and cats. for biliary diseases these are always the most prominent symptoms. diarrhea, however, is not a major symptom of liver disease, and in cases in which diarrhea is the leading symptom the liver is only rarely the causative organ (except for rare cases with complete common bile duct obstruction). a rare sign (not included in table - ) is an ulcerative form of dermatosis, so-called superficial necrolytic dermatitis, hepatodermal, or hepatocutaneous syndrome. this syndrome occurs rarely in dogs with liver cirrhosis and nodular hyperplasia and whose pathogenesis is poorly understood. this symptom and the more common symptoms of lethargy, inappetence, vomiting, diarrhea, weight loss, pu/pd, and neurobehavioral symptoms are frequently associated with diseases of other organs. therefore the history often discloses symptoms that may suggest hepatic disease, but may also be caused by other disorders. two main reasons account for the nonspecificity of liver-related symptoms. first, the liver is the central organ for many metabolic and detoxifying pathways; consequently, failing liver function may cause dysfunction of other organs. one example is hepatic encephalopathy; metabolic dysfunctions of the liver cause neurotransmitter dysfunctions of the brain, resulting in neurobehavioral signs. second, toxic factors resulting from diseases of other organ systems (especially from the gi tract) often secondarily affect the liver. examples include hepatic lipidosis in diabetes mellitus, steroidinduced hepatopathy in cushing syndrome, reactive hepatitis in gi diseases, and centrolobular liver necrosis in acute, severe anemia. therefore signs and symptoms of liver disease may be hidden within signs of other organ dysfunction, and vice versa. because clinical and physical examination findings may be compatible with hepatic disease, and because laboratory tests to detect hepatic disease are also abnormal with primary and secondary hepatopathies, it is often necessary to make a histologic diagnosis of the liver disorder to resolve this dilemma. , lack of specific physical examination findings may prevent recognition of a primary liver disease. most dogs with illnesses causing pathetic innervation; therefore dilation (e.g., extrahepatic cholestasis), cholecystitis, or cholelithiasis should be suspected in vomiting dogs and cats. vomiting is also common in upper gi disease. in many gi diseases, translocation of bacteria and endotoxins may cause secondary, nonspecific, reactive hepatitis. this occurs frequently in dogs, but rarely in cats. reactive hepatitis is characterized by intrahepatic canalicular cholestasis, liver cell necrosis, and an exudative inflammatory reaction. clinical signs, symptoms, and diagnostic results associated with primary liver disease and reactive hepatitis are similar; therefore, a further diagnostic workup is important to reveal the primary cause. small bowel-type diarrhea occurs frequently with hepatic diseases (see table - ). two primary mechanisms may account for clinical signs. first, cholestatic diseases (intrahepatic or extrahepatic caused by common bile duct obstruction) disrupt the normal enterohepatic cycle of bile acids; therefore less bile reaches the duodenum. , decreased resorption of dietary fat may cause hyperosmotic intestinal contents and diarrhea. however, studies in rats show that cholestasis must be severe before steatorrhea as a result of disruption of the enterohepatic bile acid cycle occurs. another mechanism for diarrhea in liver disease is increased resistance to portal blood flow, resulting in portal hypertension and congestion of splanchnic organs. intestinal vasculature congestion reduces intestinal water resorption and increases intestinal volume content. this is the predominant mechanism underlying diarrhea in diseases such as chronic hepatitis, lobular dissecting hepatitis, portal vein thrombosis, and portal vein hypoplasia. alternatively, when the primary cause of diarrhea is intestinal disease, the liver may be affected secondarily. in those cases, the hepatic macrophage system should remove the increased absorption of (endo)toxins or bacteria by the affected intestinal wall. increased exposure, however, can lead to secondary, nonspecific, reactive hepatitis. endotoxins also effectively inhibit bile formation and flow, leading to cholestasis. it is therefore common to find increased plasma liver enzyme activities and bile acid levels in cases of reactive hepatitis; clinical icterus may even be apparent. the cause of diarrhea can be determined only by further diagnostic methods, including histologic evaluation of liver biopsy specimens. reactive hepatitis resolves rapidly when the primary disease is treated successfully. in the authors' experience it is very rare to find diarrhea as single or the leading symptom in cases of liver disease. if present, it is usually one of the less prominent symptoms in the spectrum of other more prominent symptoms such as apathy, pu/pd, or vomiting. one may therefore elect to follow liver laboratory values after treatment of the intestinal disease and perform a liver biopsy if liver parameters fail to improve within a few weeks. hepatic encephalopathy is a complex of neurobehavioral signs resulting from portosystemic shunting of blood in combination with a reduction of functional liver mass. it may occur in animals with cpss or in those with apscapsc because of portal hypertension. diseases associated with the latter form are chronic hepatitis, cirrhosis, portal vein hypoplasia, lobular dissecting hepatitis, and portal vein thrombosis. cats, because of their dependence on some essential amino acids (e.g., arginine), may develop hepatic encephalopathy without portosystemic shunting, especially when they have a severe form of hepatic lipidosis. doberman pinschers. the hepatitis in doberman pinschers is sex linked, confined to females, and aggressive. it is responsive to medication with penicillamine, but may terminate in micronodular cirrhosis. this form of cirrhosis, predominantly seen in copper toxicosis, differs from other forms of chronic hepatitis, in which patients typically develop macronodular cirrhosis with large hyperplastic nodules. hepatitis is overrepresented in female doberman pinschers by a factor of ; a study in finland showed that approximately % of doberman pinschers may be affected. inherited copper toxicosis is also a well described entity in bedlington terriers worldwide. both sexes may be affected. clinical signs usually develop after years of age as a result of the gradual accumulation of copper. it is caused by a defect in the murr gene, leading to a severely decreased excretion of copper by hepatocytes. other affected breeds are west highland white terriers (particularly in the united states), skye terriers, dalmatians, anatolian shepherd dogs, and labrador retrievers. [ ] [ ] [ ] [ ] [ ] siamese cats may also be predisposed to copper-associated hepatopathies. although essential for life, copper is usually ingested to excess and must be eliminated by the liver to prevent toxicity. the central role of the liver in copper homeostasis makes it vulnerable if elimination processes fail. furthermore, increased copper levels add to the oxidative stress, which is an important component in chronic inflammatory and cholestatic diseases in dogs. spaniels seem to have the form of chronic hepatitis unrelated to copper toxicosis and develop macronodular cirrhosis when left untreated. no sex predisposition exists, but there seems to be a worldwide overrepresentation of hepatitis in this breed. congenital portosystemic shunts (cpss) are seen in both sexes in various breeds. intrahepatic shunts predominate in large breeds, whereas extrahepatic shunts predominate in small and toy breeds. although cpss are most likely inherited in some fashion in all affected breeds, this has only been proven in irish wolfhounds and cairn terriers. worldwide predispositions occur in irish wolfhounds, australian cattle dogs, labrador retrievers, dachshunds, yorkshire terriers, cairn terriers, maltese terriers, and miniature schnauzers. , in the united states, an increased prevalence of shunts has also been reported to occur in german shepherd dogs, doberman pinschers, and golden retrievers. cpss occur most often in mixed-breed cats; however, persian and himalayan cats are frequently overrepresented. clinical signs are usually seen in young dogs and cats (< year old) with congenital shunts. , pathogenesis of common symptoms of primary liver diseases vomiting is one of the most common symptoms noted in dogs and cats with liver disease. vomiting may be caused by direct stimulation of the vomiting center via the chemoreceptor trigger zone in the fourth ventricle by (endo)toxins that are not cleared by the liver. this typically occurs when toxins from the gi system bypass the liver and access other body systems. vomiting is common in all conditions that share portosystemic shunting and liver dysfunction (e.g., congenital shunts and acquired shunts because of hepatitis, fibrosis, cirrhosis, and portal vein hypoplasia or thrombosis). hepatic diseases that cause an abnormal liver shape may reposition the upper gi tract and induce nausea and vomiting by vagal stimulation. causes include hepatic tumors, especially liver cell (or hepatocellular) carcinomas, and unilateral collapse and contralateral hypertrophy, which may occur with thrombosis of a main branch of the portal vein. the gallbladder and larger bile ducts have a rich sym-form ammonium urate. affected dogs usually have clinical signs related to shunting and liver dysfunction, such as hepatic encephalopathy, pu/pd, or vomiting. in the other category, the enzyme uricase, which forms allantoin, is inactive because of an inborn error affecting only this function. ammonium urate urolithiasis occurs commonly in dalmatians but may occur in other breeds. affected dogs only have signs related to urolithiasis (e.g., pollakiuria, stranguria, dysuria). an owner may note acholic feces, which can provide a direct clue to the underlying diagnosis. steatorrheic feces that do not contain normal bile pigment are seen only when bile flow into the intestinal tract is completely disrupted, usually as a consequence of extrahepatic obstruction of the common bile duct. destructive cholangiolitis is the only intrahepatic process severe enough to seriously disrupt bile flow. the latter disease is caused by a hypersensitivity reaction to sulfonamide-containing drugs. the smaller bile ductules become necrotic and liver lobuli may be disconnected from the biliary tract. affected dogs have a history of recent medication with sulfonamides. acholic feces contain excess fat because resorption is impaired. the lack of normal black-brown fecal pigments occurs because their precursor, bilirubin, does not reach the duodenum. therefore, the feces from affected animals are gray-white and soft. animals with this condition often are icteric. the presence of icterus reduces the likelihood of exocrine pancreatic insufficiency as a diagnosis. abdominal distention may occur in dogs and cats with liver disease for several reasons. first, ascites is a frequent finding associated with liver disease in dogs as a result of portal hypertension, but is less common in cats. abdominal distention may also result from organ enlargement, which in the case of liver disease may include the liver and, in the spleen in the presence of portal hypertension. in contrast to dogs, cats often have hepatomegaly with liver disease. nonspecific symptoms, such as apathy, reduced appetite or anorexia, and weight loss, may occur in dogs and cats with liver disease. retarded growth is common in young animals. these problems reflect the central role of the liver in many metabolic and detoxifying functions. in addition, nausea, inappetence, vomiting, and diarrhea can result in a catabolic state, which, in turn, may aggravate hepatic encephalopathy. signs of early hepatic encephalopathy include depression and other nonspecific problems. anemia, another common finding in liver disease (see below), can cause general malaise. dogs with liver cell carcinoma often are hypoglycemic, which may be the primary problem underlying apathy and weakness. production of insulin-like growth factors by the tumor may be responsible. as with historical findings, physical examination findings rarely provide enough information to pinpoint the liver as definitive cause of the presenting problems. possible findings include icterus, hepatomegaly, splenomegaly, ascites, and pale mucous membranes. petechiae of the skin or mucous membranes occur infrequently. of these possible findings, only icterus and hepatomegaly are more or less specific for liver diseases; other abnormalities on the physical examination occur more frequently with diseases of other organ systems. hepatic encephalopathy is caused by derangement of neurotransmitter systems caused by defective metabolic processes in the liver. inadequate metabolism of ammonia and aromatic amino acids by the liver may reduce the excitatory glutamatergic and monoaminergic neurotransmitter system tones, respectively. in addition, there is an increased tone of the inhibitory γ-aminobutyric acid (gaba) system. these neurotransmitter derangements make anesthesia risky in some animals with liver disease. the liver inactivates many anesthetics and the unforeseen delay of recovery from anesthesia may suggest an underlying liver disease as a cause for nonspecific clinical signs. this occurs especially in dog and cats with portosystemic shunting, either congenital or acquired. in addition to reduced hepatic clearance, anesthetics exert their action via various neurotransmitter systems in the brain, which may already be functioning abnormally as a consequence of hepatic encephalopathy. this is especially true of drugs that act via the gaba-benzodiazepine pathway. that pathway is already overstimulated and may provoke an exaggerated and prolonged anesthetic effect. pu/pd is one of the most frequent signs ( % of cases) in dogs with liver disease, but is less common in cats. pu/pd is most common in diseases associated with congenital or acquired portosystemic shunting and, therefore, with hepatic encephalopathy. in affected dogs, abnormal neurotransmitter disturbances lead to increased secretion of adrenocorticotropic hormone (acth) from the anterior and intermediate pituitary lobes. chronically elevated acth stimulates increased levels of free cortisol. increased levels of free cortisol, in turn, affect the posterior lobe of the pituitary creating an increased threshold for the release of arginine vasopressin. , thus, a higher plasma osmolality is required to stimulate antidiuresis through arginine vasopressin, and before reaching that threshold, affected dogs become thirsty and start drinking. pu/pd is not only present in cases with hepatic encephalopathy, but also frequently in all other liver diseases. this may be caused by certain bile acids, which are often increased in plasma of animals with liver diseases. bile acids may inhibit the activity of β-hydroxyl steroid dehydrogenase. this enzyme protects the aldosterone receptor from occupation by cortisol, by converting cortisol into cortisone, which cannot bind to the receptor. present in plasma in tenfold excess compared with aldosterone, cortisol can occupy and stimulate the aldosterone receptor thereby inducing pseudohyperaldosteronism and pu. reduced hepatic formation of urea is another possible but undocumented mechanism that may play a role in the pathogenesis of pu/ pd. in urea deficiency states, the kidney does not have sufficient urea available to build up an osmotic gradient in the medulla. apart from this mechanism, pd also occurs in liver diseases not associated with hepatic encephalopathy (e.g., extrahepatic cholestasis and liver tumors). the mechanism is unclear; however, nausea with an impulse to drink and compensation of water loss by vomiting and diarrhea may play a role. dysuria may occur as a result of insufficient liver function when nonmetabolized uric acid is excreted by the kidneys and precipitates to form uroliths. such calculi are seen in dogs but rarely in cats. there are two main categories of liver dysfunction that cause ammonium urate urolithiasis. most frequently it is caused by congenital portosystemic shunting, whereby the liver is underdeveloped and fails to metabolize uric acid into allantoin. in urine, uric acid flocculates easily in the presence of high ammonia concentrations to a result of congestive heart disease can, in most cases, be recognized easily by physical examination of the circulatory system. measurement of central venous pressure is diagnostic. the exception is liver congestion caused by a thrombus in the caudal vena cava proximal to the liver, which is assessed by other methods. when the liver is overtly enlarged because of congestion, ascites is usually present. ascitic fluid has the typical slightly hemorrhagic appearance of congestive fluid. dogs with enlarged livers and no signs of congestive disease often have liver cancer, which may be primary, metastatic, or a form of malignant lymphoma. with most tumors, the liver is diffusely enlarged, but primary hepatocellular carcinomas or adenomas may cause enlargement of the affected lobe only. bile duct carcinomas disseminate easily over the biliary system and usually cause pronounced icterus and hepatomegaly. most cats with hepatic disease develop pronounced enlargement of the liver. in cats, liver enlargement occurs with cholangitis, hepatic lipidosis, amyloidosis, hepatic tumors (primarily malignant lymphoma), and congestive disease. when the liver is involved in feline infectious peritonitis, it may not be enlarged. cats with cpss have small livers. splenomegaly and ascites in association with liver disease are nonspecific findings. they occur especially with hepatic diseases causing portal hypertension. both findings are frequent in dogs but rare in cats. there is a positive undulation test with distinct ascites; slight ascites can be found with ultrasonography rather than physical examination. the liver may be enlarged with central causes of venous congestion. canine liver diseases associated with portal hypertension include chronic hepatitis and cirrhosis, portal vein hypoplasia, and lobular dissecting hepatitis. portal hypertension is sometimes seen with cirrhosis because of advanced cholangiohepatitis in cats. in these diseases, hepatic encephalopathy is also common. portal vein thrombosis is a prehepatic cause of portal hypertension that usually causes ascites. although as a rule the liver is small in these cases, unilateral obstruction of a main branch of the portal vein may cause hypertrophy of the rest of the liver, which may be palpable. diseases of the hepatic parenchyma, hepatic vasculature, and biliary tract are relatively common in dogs and cats. because the symptoms and signs accompanying liver disease are quite nonspecific, and the liver may be secondarily involved in diseases of other organs, liver disease can easily go undetected. therefore, after taking a thorough history and performing a physical examination, it is critical to perform additional biochemical tests with the highest possible diagnostic accuracy whenever liver disease is included in the differential diagnosis. if liver disease cannot be ruled out based on these examinations, additional testing is necessary to define the type of liver disease, most notably ultrasonography of the cranial abdominal quadrant and examination of a liver biopsy specimen. the algorithm in figure - summarizes this approach. diagnosis usually depends on histopathologic examination of liver tissue, especially for parenchymal liver diseases, many biliary tract diseases, and tumors of the liver or biliary tract. although biopsy methods are beyond the scope of this chapter, excellent sources exist. one note of caution: blood coagulation testing is vital before collecting a liver biopsy specimen for histopathology. most animals, for example, have one or more abnormal coagulation tests. [ ] [ ] [ ] [ ] [ ] [ ] factors involved may include vitamin k deficiency, reduced ascites and hepato-and splenomegaly may have been noted by the owner as abdominal enlargement. biochemistry analyses are an integral part of the diagnostic process for liver diseases. most of these analyses are not a decisive factor in the diagnosis of liver disease, but serve to rule out liver disease from the differential diagnosis. icterus is the most frequently encountered specific abnormality noted on the physical examination in dogs and cats with liver disease. however, only approximately % of dogs with hepatobiliary diseases and % to % of cats are icteric. icterus results from bilirubin accumulation in the blood and extravascular space as a result of increased production, reduced clearance, impaired conjugation by the liver, and/or impaired bile flow. in most cases, a combination of these factors is involved. cholestasis is predominant; therefore conjugated bilirubin is the fraction present in greatest quantity. hemolysis alone does not result in icterus with normal liver function. when hemolysis is severe, however, it may result in such a degree of portal hypoxia that the centrolobular zones of the liver lobules become necrotic. in those cases, icterus results from the combination of increased production and reduced liver function and cholestasis. if hemolysis is the primary cause of icterus, it must be severe, and the mucous membranes will be extremely pale. primary liver diseases that may cause icterus are commonly accompanied by hemolysis. whereas the erythrocyte lifetime is reduced to to days in dogs with severe primary hemolytic disease; it is to days (normally days) in hepatobiliary disease. increased production of bilirubin and liver dysfunction with cholestasis result in a combined conjugated and unconjugated hyperbilirubinemia in dogs and cats with primary hepatic or hemolytic disease. icterus caused by hemolytic disease is characterized by pale mucous membranes, whereas the mucous membranes in animals with primary liver disease are normal or only slightly pale. the combined evaluation of icterus and the color of the mucous membranes immediately reveal the nature of the underlying process. as previously discussed, most hepatobiliary diseases are accompanied by increased degradation of red blood cells. the mechanisms behind hemolysis in liver disease are not completely clear. hypersplenism and reduced portal blood flow due to portal hypertension may drastically prolong the transit time of erythrocytes through the spleen, with a greater chance that they will be trapped when they are slightly abnormal. increased fragility of red cell membranes may be a result of the high bile acid levels in most liver diseases, whereas a reduced clearance of enteral endotoxins and bacteria by the liver may also induce immune-mediated hemolysis. apart from hemolysis, nonregenerative anemia also may occur as part of the syndrome of anemia of chronic disease as an expression of catabolism and slight deficiencies of iron and b vitamins. although common in liver diseases, anemia, in contrast to icterus, is nonspecific. like icterus, hepatic enlargement is a distinct sign of an abnormal liver. in dogs, most liver diseases do not cause hepatomegaly. exceptions include tumors of the liver, liver congestion, and secondary liver involvement in metabolic diseases. examples of the latter conditions are glycogen accumulation in the liver in cushing disease, fatty liver with diabetes mellitus, and rare cases of amyloidosis of the liver. the more chronic liver diseases of dogs tend to reduce liver size, and acute diseases cause little change in size. liver enlargement as hormones and drugs, leading to an increase in their serum activities in patients without primary hepatic disease. additionally, serum hepatic enzyme activities can be increased as a consequence of secondary hepatopathies. the magnitude of hepatic enzyme activity increases may aid in the assessment of the severity or the extent of hepatic injury but should not be considered to be prognostic. the liver has a large capacity for regeneration, so even in cases of severe hepatic injury, with dramatically raised hepatic enzyme activities, a full recovery is possible. this is especially true when the injury is acute. conversely, in cases of chronic end-stage liver disease, such as cirrhosis, serum hepatic enzyme activities may not be markedly increased, or may even be within the reference interval as a result of the replacement of hepatocytes with fibrous tissue. consequently, serial evaluation of serum hepatic enzyme activities is more useful for assessing prognosis than measurement at a single point in time. consistent decreases of a previously increased activity are considered a favorable sign in acute liver injury, whereas a decreasing hepatic enzyme activity in a patient with chronic liver disease that is clinically deteriorating suggests loss of hepatocytes because of fibrosis. it is important to note that serum hepatic enzyme activities do not provide an assessment of liver function. alt is an enzyme found primarily in the cytosol of hepatocytes. alt is released into the serum when hepatocyte membrane permeability is increased, or if there is hepatocellular necrosis. alt is considered to be the most liver-specific enzyme. alt is also produced by cardiac muscle, skeletal muscle, and the kidneys. apart from the hepatic form, only the muscle isoenzyme is clinically significant. although uncommon, severe muscle injury can result in an increased serum alt activity. hepatic microsomal induction in response to some drugs can also produce small increases in alt activity. some controversy exists regarding the serum half-life for alt in dogs. the mean serum half-life of alt was reported as being minutes in one study and hours in another. the serum half-life of alt is generally believed to be shorter in the cat than in the dog. a mean serum half-life of minutes was reported in an experiment involving three healthy cats. the shorter half-life in cats means that increases in serum alt activity are considered more clinically important in this species. as alt is metabolized in the liver its serum half-life may be longer in patients with liver disease. increased cell membrane permeability in the absence of hepatocyte destruction can cause a rapid increase in serum alt activity. because of this, alt activity is a considered to be a highly sensitive marker of hepatocyte injury. this also means that an increased alt activity does not imply severe or irreversible hepatocellular injury. the highest increases in alt activity are seen during acute hepatic inflammation or necrosis, but because of the capacity for the liver to regenerate these do not indicate irreversible damage. consequently, a single measurement of alt activity does not provide an accurate prognosis. however, the degree of the alt activity increase is believed to have some correlation with the number of hepatocytes that have been injured. cholestasis can also result in an increased serum alt activity because of hepatocellular damage caused by the accumulation of bile acids. certain drugs can lead to increases in serum alt activity. these are usually minor, for example, phenobarbital used at therapeutic doses frequently leads to small increases in serum alt activity, in the absence of hepatic insufficiency. these increases are thought to occur as a result of subclinical hepatic injury rather than induction of hepatic microsomal enzymes. toxic doses production of clotting factors, some degree of disseminated intravascular coagulation (dic), and severe protein deficiency. , it should be noted that taking a fine-needle aspiration biopsy should be considered safe, even if abnormalities in coagulation are present. however, its diagnostic value is limited as the liver architecture is lost. diagnosis of circulatory hepatic diseases depends on information obtained from laboratory results, ultrasonography (most reliable diagnostic test to date), and histopathology. a recent paper found that fasting ammonia concentration was superior to fasting bile acids for diagnosing portosystemic shunting in dogs. ammonia is easily measured in practice with dry chemical methods, some of which provide reliable results. another recent publication showed ultrasonography to be a reliable diagnostic method to noninvasively characterize the underlying hepatic disease in dogs with hyperammonemia. diagnostic evaluation of the hepatobiliary system has several aims: (a) to determine if hepatobiliary disease is present, (b) to assess liver function, (c) to determine if liver disease is primary or secondary, (d) to definitively diagnose hepatobiliary disease, and (e) to monitor response to treatment. despite the apparent clarity of these aims, hepatobiliary disease can present a diagnostic challenge for a number of reasons. first, as clinical signs can be nonspecific, hepatobiliary disease should be a consideration when evaluating any patient with signs of systemic disease. dogs and cats with hepatobiliary disease may not have any clinical signs. furthermore, because of the liver's central role in metabolism and detoxification of endogenous toxins and xenobiotic agents, a number of extrahepatic diseases can secondarily affect the liver. it is important to distinguish these secondary hepatopathies from diseases that originate in the liver (primary hepatopathies). additionally, serum markers of hepatocellular damage, cholestasis, and hepatic function can be abnormal in the absence of hepatobiliary disease. finally, the liver's large reserve capacity means that detectable loss of liver function often occurs late in the course of disease. thus, when assessing a patient with suspected hepatobiliary disease, it is important to consider the clinical presentation, results of laboratory testing, diagnostic imaging findings, and the results of cytologic and/or histopathologic evaluation together. hepatic enzymes can be divided into markers of hepatocellular damage and markers of cholestasis. serum alanine aminotransferase (alt) and aspartate transaminase (ast) activities are the two most commonly measured markers of hepatocellular leakage, while serum alkaline phosphatase (alp) and γ-glutamyltransferase (ggt) activities are the two most commonly measured markers of cholestasis. although increased serum hepatic enzyme activities are considered to be sensitive, they are not specific for primary liver disease because they are produced by extrahepatic tissues. the relative importance of these extrahepatic isoenzymes varies, but their extrahepatic release can lead to increased serum activities. also, the production of some hepatic enzymes can be induced by certain alp (b-alp), and kidney alp (k-alp) are transcribed from the tissue nonspecific alp gene. the other gene encodes intestinal alp (i-alp) and probably glucocorticoid-induced alp (g-alp). in dogs the serum half-lives of placental alp, k-alp, and i-alp are less than minutes. in cats the serum half-life of i-alp is less than minutes. the half-lives of placental alp and k-alp are also assumed to be short in the cat as they have similar structures to i-alp. because of this, only l-alp, b-alp, and, in the dog but not the cat, g-alp, are believed to contribute significantly to serum alp activity. the serum half-life of l-alp is approximately hours in the dog , and hours in the cat. l-alp is bound to the membranes of the hepatocytes by glycosylphosphatidylinositol linkages. cleavage of these links by glycosylphosphatidylinositol-phospholipase allows the enzyme to be released into the bloodstream. as bile acids have detergent-like properties; accumulation of bile acids during cholestasis facilitates this process. cholestasis can also result in the induction of synthesis of l-alp (and g-alp in the dog). consequently, serum alp activity is often severely increased in patients with cholestatic disorders. in the dog, alp is considered to be a sensitive marker for cholestasis with a sensitivity of %. the short half-life of l-alp in cats means that increases in alp during cholestasis are not as high as in the dog. consequently, alp is a less-sensitive marker of cholestasis in the cat than in the dog, with a reported sensitivity of only %. however, the shorter half-life in cats and the absence of g-alp means that any increase in alp activity should be considered clinically important in this species. an increased serum alp activity does not differentiate between intrahepatic or extrahepatic cholestasis. a wide variety of liver diseases can cause intrahepatic cholestasis. this is generally caused by hepatocyte swelling, causing obstruction of the small bile canaliculi. the increase of alp following a hepatic insult is delayed compared to rises in markers of hepatocellular leakage. the reason for this is that it takes time for the enzyme to be synthesized and released into the systemic circulation. alp often remains increased for some time after the resolution of liver injury. b-alp is released into the bloodstream as a result of the activity of osteoblasts. therefore any condition that results in increased bone formation can lead to increased serum alp activity. in animals that are skeletally immature, mild increases in serum alp activity are commonly observed. animals with increased osteoblast activity, such as those with hyperparathyroidism, neoplasia involving bones, and osteomyelitis, may have mild to moderate increases in alp activity. these causes of increased b-alp activity are unlikely to be confused with primary liver disease because the increases are smaller than would be expected with cholestasis, and because bone diseases are often clinically apparent. finally, an increased serum activity of b-alp was reported in a family of asymptomatic huskies. in dogs, but not in cats, g-alp and tissue-nonspecific alp may be induced by corticosteroids. g-alp is believed to be an isoform of i-alp with a prolonged serum half-life that is produced by the liver. posttranslational glycosylation of the g-alp is believed to be responsible the prolonged half-life. induction of g-alp may cause an increase in total serum alp activity after administration of exogenous corticosteroids. synthesis of this isoenzyme can also be induced by the administration of anticonvulsant drugs, such as phenobarbital. similarly, hypercortisolemia frequently causes an increased serum alp activity because of induction of g-alp. however, in dogs with excess serum concentrations of endogenous or exogenous corticosteroids, hepatocyte swelling caused by glycogen accumulation (vacuolar hepatopathy) may lead to intrahepatic cholestasis, another potential contributor to increased serum alp of phenobarbital can cause dramatic increases of serum alt activity and hepatic insufficiency. prednisone and other glucocorticoids can cause an induction of alt (and steroid hepatopathy) and consequently small increases in serum alt activity. serum alt activity can also be increased with any secondary hepatopathy. however, a persistently increased serum alt activity, even with an apparently normal liver function, is an indication for further diagnostic testing. serial evaluation of serum alt activity can be helpful to prognosticate but must be done while considering the patient's clinical signs and other laboratory values. in general, a declining serum alt activity after acute liver injury is considered a good sign. ast is another aminotransferase enzyme that is used as a marker of hepatocellular leakage. ast is found in skeletal muscle, the brain, liver, kidney, cardiac muscle, and to a lesser extent within other tissues. the extrahepatic isoenzymes of ast are relatively more important than they are for alt. muscle disease can cause an increase in serum ast activity. because of this, ast is considered less liver specific than alt. however, by looking at serum ast activity in conjunction with the activities of other hepatic enzymes and muscle enzymes, it is usually possible to differentiate increases caused by muscle damage from increases caused by hepatic damage. again, there is controversy regarding the serum half-life of ast. in dogs one study reported the half-life to be a mean of minutes; another study reported a mean of hours. one study reported the mean half-life to be minutes for cats. unlike alt, a considerable proportion of ast (approximately %) is found within hepatocyte mitochondria rather than the cytosol. the cytosolic fraction of ast is released into the serum from hepatocytes when cell membrane permeability is increased, or in case of hepatocellular necrosis. in contrast, the mitochondrial fraction is only released during hepatocellular necrosis. release of ast from hepatocytes into the serum parallels the release of alt. therefore, like serum alt activity, serum ast activity is considered a sensitive marker for hepatocyte injury. it has been suggested that increased ast activity may be more sensitive than increased alt activity for the detection of hepatocellular injury in cats. corticosteroids and phenobarbital may cause mild increases in serum ast activity. because of the considerable overlap in the information provided by the measurement of serum alt and ast activities, measurement of serum ast activity may be redundant. alp is an enzyme bound to the membranes of the hepatocytes that comprise the bile canaliculi and the sinusoidal membranes. it is considered a sensitive marker for cholestasis, especially in the dog, but is not liver specific. cholestasis, canalicular cell necrosis, and increased hepatic synthesis may lead to the release of this enzyme into the circulation. synthesis of this enzyme can be induced by certain drugs, most notably corticosteroids. the possibility that an increase in serum alp activity could be caused by extrahepatic disease, or could be induced by glucocorticoids in the dog, can make the interpretation of this finding challenging. in the dog a wide variety of tissues exhibit alp activity, including intestinal mucosa, kidney (cortex), bone marrow, pancreas, testicle, brain, lung, kidney (medulla), lymph node, liver, skin, spleen, skeletal muscle, and cardiac muscle. there is disagreement in the literature regarding the relative contributions of alp activity from each of these tissues in cats. [ ] [ ] [ ] there are two genes encoding alp in the dog. different forms of alp arising from the same gene are called isoforms. differences among these isoforms arise because of differing posttranslational processing. liver alp (l-alp), bone globulins are produced in the liver, but not exclusively so. the liver produces α-globulins and β-globulins, whereas lymphoid cells produce immunoglobulins (γ-globulins). hepatic insufficiency rarely leads to a decrease in serum globulin concentration. conversely, inflammatory liver disease may be associated with hyperglobulinemia because the nonimmunoglobulin fraction produced by the liver includes several acute-phase proteins (c-reactive protein, haptoglobin, and serum amyloid a). the hepatic synthesis of these proteins is increased during systemic inflammation [ ] [ ] [ ] [ ] possibly leading to a rise in the total serum globulin concentration. additionally, immunoglobulin production may be increased in infectious, neoplastic, or autoimmune diseases. coagulation factors (except factor viii), anticoagulation factors (antithrombin and protein c), and the fibrinolytic protein plasminogen, are all synthesized by the liver. the liver is also the site of activation of the vitamin k-dependent clotting factors: ii, vii, ix, x, and protein c. furthermore, as bile acids are needed to emulsify fat and aid in the absorption of vitamin k from the intestine, vitamin k malabsorption may develop secondary to cholestasis. consequently, hepatobiliary disease can affect hemostasis in more than one way. in canine and feline liver disease, coagulation parameter abnormalities have been reported in specific clotting factor activities, prothrombin time, aptt, [ ] [ ] [ ] proteins induced in the absence of vitamin k, , fibrin degradation products, fibrinogen, and protein-c activity. these abnormalities of hemostasis are not specific for liver disease but may support its presence. patients with liver disease may develop dic, which can be difficult to distinguish from coagulopathy because of reduced hepatic synthesis of clotting factors alone. although spontaneous bleeding seldom occurs in patients with liver disease, the assessment of the coagulation status of these patients is important, especially when an invasive procedure such as a liver biopsy is being considered. a recent study investigated the diagnostic value of serum protein c as a marker for hepatobiliary disease and portosystemic shunting in dogs. serum protein-c measurement was reported to aid in the differentiation of portal vein hypoplasia without portal hypertension (formerly called microvascular dysplasia) from portosystemic shunt (pss). dogs with portal vein hypoplasia without portal hypertension had a significantly higher serum protein-c concentration than those with portosystemic shunting. urea is produced from ammonia in the liver, released into the systemic circulation, and subsequently excreted by the kidneys. serum urea nitrogen concentration may be close to or below the lower limit of the reference interval in patients with hepatic insufficiency, pss, or urea cycle enzyme deficiencies. however, serum urea nitrogen concentration may also be decreased because of medullary solute washout caused by diuresis, malnutrition, or a protein-restricted diet, and is a normal finding in neonates. in a patient with liver disease, a high fasting serum urea nitrogen concentration relative to the serum creatinine concentration suggests gi hemorrhage. ammonia (nh ) is produced in small intestinal enterocytes from the catabolism of glutamine and in the colon as a consequence of bacterial deamination. ammonia is a highly diffusible gas and passes readily through the bowel wall into the bloodstream. in the blood, at a ph of . , most of the ammonia exists in the form of ammonium ions (nh + ). the ammonium is transported in the blood from the intestines through the hepatic portal circulation to the liver. the extraction of ammonia from the portal circulation is highly efficient. endogenous ammonia is produced from the breakdown of activity. before diagnosing primary liver disease in a dog with an increased serum alp activity, induction of g-alp by endogenous or exogenous steroids should be ruled out. recently a group of scottish terriers were found to have increased serum alp activity with no identifiable underlying cause. it is technically possible to selectively measure the activity of g-alp using techniques such as levamisole inhibition. measurement of g-alp activity was initially investigated as a way to differentiate increases in alp caused by corticosteroids from those caused by cholestasis. unfortunately, measuring g-alp is not clinically useful as g-alp activity may be increased in a variety of conditions, including hepatic disease, diabetes mellitus, hypothyroidism, and pancreatitis. ggt is a glycoprotein enzyme that is bound to the membranes of those hepatocytes that form the bile canaliculi and bile ducts and also periportal hepatocytes. in comparison to alp its distribution includes more distal areas of the biliary tract, but measurement of serum ggt activity is not useful to distinguish between intrahepatic and extrahepatic cholestasis. ggt is also produced by a number of extrahepatic tissues. most of the ggt activity in serum is thought to be a result of the hepatic isoenzyme. colostrum also contains ggt, which is responsible for the mild increases in serum ggt activity that are seen in suckling animals. changes in serum ggt activity generally parallel those in serum alp activity, in that activity is often increased in patients with cholestasis. because ggt is also induced by glucocorticoids, its activity may be increased in patients with hyperadrenocorticism or those exposed to exogenous steroids. in dogs, an increased serum ggt activity is considered to be more specific, but less sensitive than alp activity for the presence of liver disease. in cats, measurement of serum ggt activity is more sensitive but less specific for the detection of liver disease than alp. cats with hepatic lipidosis may be an exception to this as they often have a normal serum ggt activity but an increased serum alp activity. the liver plays a central role in protein metabolism. it is responsible for the synthesis of plasma proteins, deamination of amino acids, conversion of ammonia to urea, amino acid synthesis, and interconversion of amino acids. consequently, in patients with hepatic disease these functions may be compromised. albumin is an important plasma protein that is produced exclusively by the liver. the rate of albumin synthesis must equal the rate of albumin loss to maintain serum albumin concentrations. mild decreases in serum albumin concentration can occur from a variety of conditions. however, the differential diagnoses for severe hypoalbuminemia (< g/dl) are limited to hepatic insufficiency, severe exudative skin disease, protein-losing enteropathy, and proteinlosing nephropathy. it is possible to determine the cause of severe hypoalbuminemia from a combination of clinical findings, measurement of the serum globulin concentration, urinalysis (including protein creatinine ratio), tests of gi protein loss, and tests of liver function. as albumin contributes significantly to colloid oncotic pressure, severe hypoalbuminemia can lead to ascites, pleural effusion, and/or subcutaneous edema. the liver has a large reserve capacity for the synthesis of albumin and albumin has a serum halflife of approximately days in dogs. consequently, hypoalbuminemia is a relatively insensitive marker for hepatic insufficiency and is only likely to be seen in patients with advanced chronic liver disease or portosystemic shunts (psss). for hepatobiliary disease in dogs or cats. in patients with severe hepatic insufficiency or pss serum cholesterol, concentration may be decreased as a consequence of impaired hepatic synthesis. hypocholesterolemia might also occur as a result of inadequate dietary intake, maldigestion, malabsorption, or hypoadrenocorticism. the serum cholesterol concentration of patients with hepatobiliary disease may be within the reference interval. patients with cholestatic disease can become hypercholesterolemic. fasting hypercholesterolemia also may be observed in patients with various endocrinopathies, obesity, protein-losing nephropathy, pancreatitis, or primary hyperlipidemias. serum triglyceride concentration may be increased or normal in patients with liver disease. however, an increased fasting serum triglyceride concentration is not a sensitive or specific marker for hepatobiliary disease in dogs or cats. a mild increase in serum triglyceride concentration may develop in patients with cholestasis. there is some evidence that hypertriglyceridemia is associated with gallbladder mucocele formation. hypertriglyceridemia is associated with increased serum hepatic enzyme activities in miniature schnauzers. increased fasting serum triglyceride concentrations are also observed in patients with endocrinopathies, obesity, pancreatitis, and primary hyperlipidemias. the liver plays a central role in carbohydrate metabolism and is responsible glycogen storage, conversion of galactose and fructose into glucose, gluconeogenesis, and the synthesis of many compounds from carbohydrates. blood glucose measurement is not a sensitive or specific marker for liver disease. the liver has a large reserve capacity for glucose production. consequently, hepatic insufficiency must be severe for hypoglycemia to occur. hypoglycemia occurs in a proportion of patients with congenital pss. hepatic neoplasia can also lead to hypoglycemia. this is thought to be caused by release of insulin-like substances. a variety of extrahepatic conditions can also lead to hypoglycemia. bilirubin is a yellow pigment produced from the breakdown of heme-containing compounds. measurement of serum bilirubin concentration can be used to assess liver function. hyperbilirubinemia can be the result of hepatobiliary or extrahepatic disease. icterus is the yellowish pigmentation caused by the retention of bilirubin in the soft tissues. laboratory assessment is the most sensitive way to detect increased serum bilirubin concentrations. hyperbilirubinemia is classified as prehepatic, hepatic, or posthepatic in origin. bilirubin may be artifactually increased by in vitro hemolysis or by the administration of synthetic hemoglobin polymers. when assessing a hyperbilirubinemic patient it is critical to localize the underlying cause. bilirubin is the major product of the degradation of hemecontaining compounds by cells of the mononuclear phagocyte system. bilirubin is released from the mononuclear phagocyte system and is transported in the plasma. the bilirubin is reversibly bound to albumin as it is water insoluble. the unconjugated bilirubin is absorbed through the hepatocyte cell membranes and is bound to glucuronic acid (conjugation). conjugated bilirubin is water soluble and is actively excreted from the hepatocytes into the bile canaliculi, eventually being excreted into the intestines. once in the intestine some of the bilirubin is converted into urobilinogen by bacteria. some of the urobilinogen is reabsorbed from the intestines, but most of this is immediately reexcreted by the liver. when nitrogenous substances in the body, especially glutamine. in the liver the ammonium is converted to urea by the enzymes of the urea cycle, or is used during the conversion of glutamate to glutamine. urea enters the circulation and is excreted by the kidneys. ammonium that is not removed by the liver enters the systemic circulation. the liver has a large reserve capacity for the conversion of ammonia into urea. because of this, plasma ammonia measurement is a relatively insensitive marker for hepatic insufficiency. however, measurement of blood ammonia concentration is a sensitive test for congenital psss and apsc shunts (also known as acquired psss). this is because when portosystemic shunting occurs, the ammonia absorbed from the intestines bypasses the liver and reaches the systemic circulation directly. the sensitivity of plasma ammonia measurement for the detection of pss is reported to be between % and % in dogs [ ] [ ] [ ] [ ] and % in cats. the measurement of postprandial venous ammonia is more sensitive than the measurement of fasting ammonia (sensitivities of % and %, respectively) for the detection of congenital pss. however, the sensitivity for detecting dogs with hepatocellular disease is only %. generally, hyperammonemia is considered specific for hepatic insufficiency or pss. however, although they are uncommon, urea-cycle enzyme deficiencies may also cause an increased blood ammonia concentration. these enzyme deficiencies can be hereditary as a result of the absence of a particular enzyme or secondary to cobalamin or arginine deficiency. , arginine deficiency is especially relevant in cats with hepatic lipidosis. ammonia is one of the substances that cause hepatic encephalopathy (he). therefore blood ammonia measurement is a useful marker for he. however, other substances can also cause he and the plasma ammonia concentration of a patient with he may be within the reference interval. ammonium ions are labile in plasma, so sample handling is critical when measuring plasma ammonia concentration. samples should be collected, placed immediately on ice, and the plasma separated from the red blood cells as soon as possible. the plasma must be kept cooled and should be analyzed within minutes of collection. these handling requirements have meant that ammonia measurement has been mainly confined to practices with immediate access to a commercial laboratory. measurement of plasma ammonia is available on an in-house dry chemistry analyzer (vettest, idexx laboratories, westbrook, me) although this method was only considered to reliably agree with a reference method for serum ammonia concentrations greater than µm. a recent study found that a point of care blood ammonia analyzer (pocketchem ba, menarini diagnostics, florence, italy) may be suitable for the measurement of blood ammonia concentrations in dogs and cats. ammonia tolerance tests (atts) have been investigated in an attempt to increase the sensitivity of ammonia measurement for detecting hepatic insufficiency and pss. however, the oral administration of ammonium salts can cause vomiting and potentially worsen he signs. ammonium chloride or sulfate can also be given rectally, which is less likely to produce adverse. this method is sensitive for the detection of pss in dogs. the liver plays a central role in lipid metabolism and is responsible for oxidation of fatty acids, synthesis of cholesterol, synthesis of lipoproteins, and synthesis of fatty acids from proteins and carbohydrates. serum cholesterol concentrations may be increased, normal, or decreased in patients with liver disease. increased or decreased fasting serum cholesterol concentrations are not sensitive or specific (fasting or postprandial) suggest hepatic dysfunction, pss, or cholestasis, but they are not specific for any particular liver disease. bile acids are exclusively synthesized in the liver from cholesterol. nearly all of the bile acids that are produced by the hepatocytes are conjugated to an amino acid. in both dogs and cats conjugation is primarily to taurine, but dogs may also convert to a conjugation with glycine. in contrast, even taurine depleted cats conjugate their bile acids almost exclusively to taurine. the conjugated bile acids produced by the liver are called primary bile acids. these are secreted in the bile, and then stored in the gallbladder. cholecystokinin is released from endocrine cells in the small intestine. this hormone stimulates gallbladder contraction and the flow of bile into the duodenum. when the gallbladder contracts the bile acids are released into the intestines. spontaneous gallbladder contraction also occurs during the interdigestive phase. bile acids act as ionic detergents, aiding the emulsification of dietary lipids and their subsequent intestinal absorption in micelles. bile acids are recycled in a process known as enterohepatic circulation ( figure - ). primary bile acids are lipid insoluble and thus are only absorbed from the intestines when they bind to specific high affinity ileal mucosal receptors. this ileal reabsorption is very efficient. the reabsorbed bile acids enter the portal circulation and upon reaching the liver they are efficiently extracted from the plasma and subsequently reexcreted. the total bile acids pool can be recirculated several times in a day. consequently, the rate of hepatic bile acid synthesis and the fasting serum bile acids concentration in dogs and cats with normal hepatic function is low. because of the increased release of stored bile acids during the postprandial period, small increases in total sba concentration occur in animals with normal hepatic function. primary conjugated bile acids can undergo bacterial deconjugation in the intestinal lumen. the resulting unconjugated bile acids are called secondary bile acids. these are readily absorbed from the colon by passive diffusion. first-pass extraction and reexcretion of secondary bile acids is less efficient than that for primary bile acids. consequently, secondary (unconjugated) bile acids are often present in postprandial serum samples. hepatobiliary disease can cause increased sba concentrations by interfering with hepatocellular function, by causing decreased bile flow (cholestasis), or by altering the hepatoportal blood flow. the main clinical use of sba measurement is to assess hepatic function in patients suspected to have hepatic disease, with serum exposed to air the urobilinogen remaining in the intestines is altered and oxidized into the brown pigment sterocobilin. prehepatic hyperbilirubinemia is caused by increased production of bilirubin as a result of hemolysis. the liver has a large reserve capacity for bilirubin excretion so, for hemolysis to cause hyperbilirubinemia, hepatic bilirubin clearance must be decreased. this occurs if the hemolytic anemia results in hepatocyte dysfunction because of hypoxia. if hepatic hypoxia occurs serum hepatic enzymes activities are often increased. prehepatic hyperbilirubinemia is mainly distinguished from other causes of hyperbilirubinemia by the presence of severe anemia. other supportive evidence includes the presence of a regenerative erythroid response, characteristic changes in red blood cell morphology, and possibly the detection of red blood cell bound antibodies. hepatic hyperbilirubinemia is caused by a decreased rate of hepatocyte bilirubin uptake, conjugation, or excretion (as a result of intrahepatic cholestasis). usually, hepatocyte dysfunction and intrahepatic cholestasis occur concurrently. hepatic enzyme activities (both hepatocellular leakage markers and cholestatic markers) are often increased, although they can also be increased with both prehepatic and posthepatic hyperbilirubinemia. because of the hepatic reserve capacity, hepatic disease must be severe in order to cause hyperbilirubinemia. a range of primary and secondary hepatopathies can cause hepatic hyperbilirubinemia. hepatic hyperbilirubinemia can usually be distinguished from prehepatic hyperbilirubinemia by assessment of the patient's hematocrit, and from posthepatic hyperbilirubinemia by abdominal ultrasound. other markers of hepatic insufficiency, when present, provide additional support for the presence of hepatic hyperbilirubinemia. posthepatic hyperbilirubinemia is a result of extrahepatic bile duct obstruction. this is often caused by pancreatic inflammation or, much less commonly, neoplasia. the main diagnostic tool for documenting extrahepatic bile duct obstruction is abdominal ultrasound. typically, extrahepatic bile duct obstruction leads to dramatic increases in serum cholestatic enzyme activities (compared to hepatocellular leakage enzyme activities) and hypercholesterolemia. when the bile duct is completely obstructed, acholic (pale-colored) feces may be noted. rupture of the biliary tract frequently leads to hyperbilirubinemia as bilirubin accumulates in the abdomen. it is possible to measure the concentration of serum conjugated bilirubin. however, this test is not considered to be clinically useful for distinguishing between prehepatic, hepatic, or posthepatic hyperbilirubinemia, and is thus rarely performed. bilirubin can covalently (nonreversibly) bind to albumin. this biliprotein cannot be cleared by the liver and thus persists in the plasma. biliprotein has a serum half-life comparable to albumin. this is of clinical importance because it means hyperbilirubinemia (and icterus) may persist for several weeks after the resolution of its cause. bile acids (or bile salts when they are deionized) are formed from cholesterol in the liver and are the major constituent of bile. serum bile acids (sbas) measurement is a useful test of liver function in dogs and cats. sbas are either measured as a fasting sample (after withholding food for hours) or by collecting paired fasting and -hour postprandial samples. both of these tests are simple to perform and safe. enzymatic measurement of the concentration of total bile acids in serum has become widely available and has replaced other techniques such as radioimmunoassays. , once collected, the samples of serum can be stored at room temperature, making it possible to send them to an outside laboratory for evaluation. lipemia and hemolysis of the blood samples should be avoided as both can interfere with the assay. increased sba concentrations recent study did not support the use of a c-labeled galactose breath test for assessment of liver function in dogs. the metabolism of endogenous substances has been investigated to find possible markers of hepatic cellular metabolism. dogs with hepatic disease (hepatitis and neoplasia) had significantly higher serum l-phenylalanine concentrations than did healthy dogs and those with nonhepatic diseases. further investigations are needed to determine the utility of these tests for assessing liver function in veterinary patients. urine specific gravity can be decreased in patients with hepatic insufficiency or pss. this can be caused by an inability to fully concentrate urine, resulting in pu, or from primary pd. bilirubin is commonly measured semiquantitatively in canine and feline urine using urine dipsticks. bilirubinuria (< + on a dipstick) can be a normal finding in dogs (especially males). bilirubinuria in dogs without hemolytic or hepatobiliary disease can occur as a consequence of the loss of unconjugated bilirubin that is bound to albumin in proteinuric patients and renal filtration of small amounts of conjugated bilirubin that has leaked from the liver. additionally, the renal tubular cells of male dogs have the enzymes needed to produce and conjugate bilirubin. as cats have a higher renal threshold for bilirubin than dogs, bilirubinuria should always be considered abnormal in cats. bilirubinuria in cats and excessive bilirubinuria in dogs implies hemolytic or hepatobiliary disease. because dogs have a relatively low renal threshold for bilirubin, bilirubinuria is often detected before bilirubinemia or jaundice. ammonium biurate crystals are detected in the urine sediment by light microscopy. uric acid is a product of purine catabolism and is converted to allantoic acid by hepatic urate oxidase. in cases with severe hepatic insufficiency or pss, the serum uric acid concentration may be higher than the renal threshold. this combined with hyperammonemia may lead to ammonium biurate precipitation in the urine. urate urolithiasis seems to be more common in patients with pss than those with other types of hepatic dysfunction. between % and % of dogs with pss were found to have urate crystalluria. however, it should be noted that urate crystalluria is not specific for hepatobiliary disease. the erythrocyte series may be affected by hepatobiliary disease, resulting in erythrocyte dysmorphias and anemia. these abnormalities are suggestive of, but are not specific for, hepatobiliary disease. patients with hepatobiliary disease can be anemic as a result of blood loss, in which case signs of a regenerative response are normally present within days of hemorrhage. acute, severe hemorrhage may occur in patients with hepatobiliary disease following invasive procedures such as liver biopsy or as a consequence of hemorrhage from a hepatic neoplasm or hepatic rupture. less-severe anemia may occur as a result of gi bleeding. chronic gi blood loss may eventually lead to iron-deficiency anemia. this is characterized by microcytic hypochromic erythrocytes and a variable regenerative response. additionally, hepatobiliary disease may lead to anemia of chronic disease, which is typically nonregenerative with normocytic normochromic erythrocytes. red blood cell morphologic changes are sometimes observed in dogs with hepatobiliary disease. poikilocytosis, characterized by the presence of acanthocytes and target cells, may be seen in patients with chronic hepatic disease. this is thought to be a result of altered phospholipid metabolism. patients with pss can have microcytic red blood cells. this is more common in dogs than in bilirubin concentrations that are within the reference interval. measurement of postprandial sba concentration does not seem to have an advantage over fasting sba concentrations or vice versa. sensitivity can be increased by collecting paired preprandial and twohour postprandial samples. numerous studies show that sba measurement is a useful test for diagnosing hepatobiliary disease, including pss in dogs and cats. , [ ] [ ] [ ] [ ] [ ] a recent study found the sensitivity of fasting sba measurement for diagnosing pss (using a cutoff value of µmol/l) to be % for dogs and % for cats. the reported specificities were % for dogs and % for cats. however, the sensitivity of sba measurement for detecting hepatic insufficiency is lower than that for detecting pss. measurements of sba concentrations have several limitations. first, this test does not allow differentiation between various types of hepatobiliary disease. also, measurement of serum bile acids in a patient with proven cholestasis is of no clinical benefit. additionally, there is limited utility in measuring sbas concentrations in patients with hyperbilirubinemia, although potentially sba measurement could be useful in distinguishing prehepatic from hepatic or posthepatic causes of hyperbilirubinemia. with prehepatic causes of hyperbilirubinemia, the bile acid concentrations should be within the reference interval. however, in most cases prehepatic hyperbilirubinemia is easily distinguished by the presence of severe anemia. it should also be noted that the magnitude of increases of sba concentration are not correlated with prognosis or disease severity. it is important to note that fasting sba concentrations may be higher than the upper limit of the reference interval or higher than the postprandial value because of spontaneous contraction of the gallbladder in the fasting state, or because of delayed gastric emptying. this could result in an increased fasting sba concentration in the absence of hepatobiliary disease. increased fasting and postprandial serum bile acids concentrations can be the result of increased bacterial deconjugation of primary bile acids into secondary bile acids. false-negative results may occur if enterohepatic circulation of bile acids does not occur from a lack of gallbladder contraction. this could be a problem if a patient is anorectic, does not eat enough food, consumes a diet with insufficient protein or fat, vomits the test meal, or has delayed gastric emptying. ceruletide is an injectable cholecystokinin analogue that has been used to stimulate gallbladder contraction when using sba measurement to diagnose hepatobiliary disease. , this test circumvents many of the factors that influence postprandial sba concentrations. urine bile acids measurement has been described in dogs and cats. the diagnostic performance was similar to that of sba measurement in both species. this test does not offer any advantages over sba measurement. [ ] [ ] [ ] [ ] excretion of exogenous tracers, such as the anionic cholephilic dyes, bromsulphalein, and indocyanine green have been used historically to assess hepatic function in veterinary patients. however, these tests are considered unreliable and have been replaced by the measurement of sba concentrations. the metabolism of exogenous substances can be used to assess liver function. a variety of substances have been investigated as markers for hepatic metabolism in human medicine. assessment of the metabolism of aminopyrine has been investigated in dogs and to a lesser extent in cats. the c-labeled aminopyrine demethylation blood test involves intravenous administration of c-labeled aminopyrine to the subject. the aminopyrine is metabolized by the liver, resulting in the production of co . this is measured in the blood by fractional mass spectroscopy. , further investigation of the utility of this test for assessment of liver function is needed. a in dogs with liver disease than in dogs with extrahepatic disease, and were higher in dogs with cirrhotic liver disease than in dogs with noncirrhotic liver disease. blood ha concentration may prove be a useful marker for hepatic fibrosis in dogs but further studies are necessary to evaluate its clinical utility. histopathologic analysis of liver biopsies or identification of a shunting blood vessel is often required to definitively diagnose hepatic disease. however, the pattern of laboratory test abnormalities, particularly when interpreted in conjunction with the patient's clinical presentation, and the results of diagnostic imaging, can increase or decrease a clinician's index of suspicion for specific liver diseases (table - ) . it is important to note that there is considerable overlap between the patterns for different diseases. to avoid misinterpretation and misdiagnosis when evaluating a patient for liver disease, it is essential to consider the limitations of the laboratory tests discussed above. diagnostic imaging is an important part of the investigation of hepatobiliary disease in dogs and cats. diagnostic imaging may help to determine whether or not hepatobiliary disease is present, identify the cause of a secondary hepatopathy, aid in the diagnosis of specific hepatobiliary diseases, and provide prognostic information. however, with the exception of diagnosis of a pss, imaging seldom yields a definitive diagnosis. radiography and abdominal ultrasound are the most frequently used imaging modalities for assessment of the hepatobiliary system in dogs and cats, but alternative imaging techniques are now being used more frequently. abdominal radiographs allow assessment of hepatic size, shape, opacity, and location in most patients. radiography may also allow identification of extrahepatic abnormalities that affect the liver. however, radiographs provide limited information about the hepatic parenchyma. it is important to note that patients with hepatobiliary disease often have normal abdominal radiographs. radiography allows subjective assessment of liver size. cranial displacement of the gastric axis may be observed on lateral abdominal radiographs when microhepatia is present. however, subtle microhepatia is unlikely to be appreciated radiographically. psss and hepatic cirrhosis are the most common conditions causing microhepatia. mild bilateral renomegaly may also be cats. microcytosis also occasionally occurs in patients with hepatocellular disease. altered iron metabolism is thought to lead to a delay in red blood cell precursors gaining a sufficient amount of hemoglobin to be released into the circulation. this delay leads to the precursors undergoing an extra cell division in the bone marrow, resulting in microcytosis. microangiopathy can occur as a result of hepatic neoplasia or dic, and may lead to the formation of schistocytes. the leukocyte series may be affected by hepatobiliary disease in a variety of ways. the resultant abnormalities are inconsistent and are not specific for hepatobiliary disease. leukocytosis, leukopenia, and sometimes an inflammatory leukogram may be present when infectious and, less commonly, inflammatory or neoplastic processes affect the hepatobiliary system. a leukocytosis was found to be present in % of dogs with chronic hepatitis. the thrombocyte series is occasionally affected by hepatobiliary disease, but changes are both inconsistent and nonspecific. mild to moderate thrombocytopenia may occur in patients with severe liver disease. this may be the result of a decreased production of thrombopoietin by the liver. disseminated intravascular coagulopathy associated with liver disease also may lead to thrombocytopenia. additionally, infectious diseases affecting the liver, such as leptospirosis may result in thrombocytopenia. genetic testing for copper hepatotoxicosis has been developed in bedlington terriers. affected bedlington terriers have an autosomal recessive defect of their commd gene. dogs with a homozygous affected genotype develop copper hepatopathy as a result of impaired biliary excretion of copper. initially, a microsatellite marker that is in linkage disequilibrium with the mutation was discovered and used to identify affected dogs and select dogs homozygous unaffected dogs for breeding. subsequently, a mutation of the commd gene (a deletion of exon ) was identified as the cause of the condition in the majority of bedlington terriers. a genetic test for this disease has become commercially available (vetgen, ann arbor, mi). this test is run alongside the linked marker as a small proportion of bedlington terriers do not have the deletion but are nevertheless affected by the disease. these dogs are likely to have a rare second mutation of their commd gene which the linkage markers may track. hyaluronic acid is a major constituent of the ecm and hyaluronic acid (ha) concentration in blood has been used as a marker for hepatic fibrosis in humans. a recent study investigated the use of ha concentration as a marker for hepatic disease in dogs. this study found that blood ha concentrations were significantly higher the size of the liver can be subjectively assessed by abdominal ultrasonography. the findings of a small liver and cranial displacement of the stomach suggest microhepatia. hepatomegaly is another subjective finding and can be generalized or focal. the finding of rounded liver lobe margins suggests hepatomegaly. hepatic parenchymal changes can be classified as being diffuse, multifocal, or focal. a wide variety of disease processes can cause diffuse changes to the hepatic parenchyma. these changes can be isoechoic, hypoechoic, hyperechoic, or of mixed echogenicity. in some cases the architecture of the liver will not be altered, but in other cases changes will occur. examples of diseases in which the echogenicity of the hepatic parenchyma is diffusely changed but no changes in architecture occur include cholangitis, neoplasia, hepatic lipidosis, other vacuolar hepatopathies, toxic hepatopathy, and early micronodular hyperplasia with various degrees of fibrosis. hyperechogenicity of the liver compared to the falciform fat, poor visualization of the intrahepatic blood vessels, and increased attenuation of the ultrasound beam have been used as criteria for the sonographic diagnosis of feline hepatic lipidosis. diseases where the hepatic architecture is altered are easier to detect sonographically. these include neoplasia, micronodular hyperplasia, and chronic hepatitis with fibrosis. cystic structures, abscesses, hematomas, and granulomas are examples of focal parenchymal liver disease. these lesions are usually easily detected sonographically. sonography seldom allows a definitive diagnosis of hepatic parenchymal disease to be made. in one study the overall accuracy of ultrasound for discrimination among different categories of diffuse liver disease was . % for dogs and . % for cats. hepatic lipidosis in cats could be diagnosed slightly more accurately than other diffuse hepatic diseases. cytologic or histologic evaluation of a hepatic tissue sample is usually needed to make a definitive diagnosis. hepatic neoplasia, whether primary or metastatic, can be diffuse, multifocal, or focal in its distribution. round cell tumors are the most likely tumor type to diffusely infiltrate the liver. these tumors can cause hypoechoic, hyperechoic, or mixed-echoic changes, or appreciated radiographically for patients with pss. urate uroliths can be radiolucent so they might not be visible on plain abdominal radiographs. hepatomegaly can be generalized or focal. generalized hepatomegaly can be caused by a number of conditions including neoplasia, vacuolar hepatopathies, congestion, or amyloidosis. focal hepatomegaly can be caused by neoplasia, abscesses, granulomas, or a liver lobe torsion. radiographic signs associated with hepatomegaly are rounded hepatic borders, caudal displacement of the gastric axis, and extension of the hepatic silhouette beyond the costal arch. radiography does not allow appreciation of mild hepatomegaly. additionally, it can be normal for the hepatic silhouette to extend beyond the costal arch in brachycephalic breeds, chondrodystrophic breeds, neonatal animals, or geriatric animals. the liver is normally appreciated as an area of homogenous softtissue opacity on radiographs. radiolucent areas within the liver indicate accumulation of gas within the hepatic parenchyma, biliary tract, or portal vasculature. gas in the parenchyma of the liver can be associated with an hepatic abscess. gas in or around the gallbladder has been reported in dogs with emphysematous cholecystitis. although uncommon in dogs and cats, if choleliths or choledocholiths contain enough calcium, they may be appreciated as mineral opacities within the hepatic silhouette. mineralization of the gallbladder wall can be associated with a biliary adenocarcinoma in the dog. parenchymal mineralization can be associated with granulomas, abscesses, hematomas, neoplasia, or hepatic necrosis. angiography allows the visualization of the hepatoportal vasculature, including abnormal vessels. this often provides a definitive diagnosis of pss and is indicated in patients that are suspected of having a pss, where the shunt cannot be adequately evaluated by abdominal ultrasound. anatomical characterization of congenital pss is important when planning attenuation, and angiographic procedures often allow this. there are several techniques for mesenteric portography. operative mesenteric portography is commonly performed immediately prior to surgical attenuation of a congenital pss and involves catheterization of a mesenteric vein and injection of a contrast agent. operative contrast portography allows evaluation of the portal vasculature before and after shunt attenuation. this technique has the disadvantage of being relatively invasive. cranial mesenteric portography can be accomplished less invasively by using ultrasound guidance to percutaneously catheterize the splenic vein. however, this can be technically demanding and may not be possible in smaller patients. transvenous retrograde portography has been described and involves the catheterization of the jugular vein. this technique allows selective catheterization of the shunting vessel and measurement of portal pressures (figure - ) . transvenous retrograde portography has been applied during percutaneous transjugular coil embolization of intrahepatic shunts. percutaneous splenoportography involves percutaneous injection of contrast media into the spleen. this technique is simple to perform, but there is a risk of complications such as splenic infarction or hemorrhage. abdominal ultrasonography is the most commonly used imaging modality for evaluating small animal patients with suspected hepatobiliary disease. ultrasonography allows assessment of the hepatic parenchyma and biliary tract. evidence of extrahepatic disease causing a secondary hepatopathy may also be detected. additionally, ultrasound guidance is often used when collecting samples for cytologic and histologic evaluation of the liver. gallbladder mucoceles can also lead to biliary obstruction, which might also be appreciated sonographically. the sensitivity of ultrasound for the detection of a gallbladder wall rupture in dogs with gallbladder mucoceles is reported to be . %. abdominal ultrasound can be used to assess the liver for vascular disease. congenital pss is classified as being intrahepatic or extrahepatic. although angiographic techniques are considered to be the gold standard for the detection and characterization of pss, abdominal ultrasound is being used increasingly for this purpose. sonographic assessment of the portal vasculature is time-consuming and highly operator dependent. because of this there should be a high index of suspicion for pss before performing these studies. secondary findings consistent with pss include mild bilateral renomegaly, urolithiasis (because of urate crystalluria), and microhepatia. ascites and hepatic parenchymal changes are not consistent with congenital pss. extrahepatic shunts typically occur in small-breed dogs and arise from the splenic vein or the right gastric vein while intrahepatic shunts typically occur in larger breeds of dogs and arise from the right or left portal branch. an intrahepatic pss is usually easier to detect sonographically than an extrahepatic pss in dogs. cats typically have single extrahepatic shunts with a wider degree of anatomical variation than in the dog. ultrasonography has been reported to have a sensitivity of % and a specificity of % for detecting pss in dogs. portal hypertension can develop as a result of chronic hepatitis with fibrosis, hepatic arterioportal fistulas, portal vein thrombosis, primary portal vein hypoplasia, extraluminal compression of the portal vein, or after ligation of a congenital pss. the finding of hepatofugal or reduced velocity hepatopetal blood flow using doppler ultrasound is consistent with portal hypertension. however, not all patients with portal hypertension will have these changes. ascites frequently, but not always, develops secondary to portal hypertension and this can be readily detected on abdominal ultrasound examination. acquired portosystemic collaterals (also known as acquired pss) may develop when sustained prehepatic or hepatic portal hypertension is present. sonography may allow detection of portal hypertension and apscapsc although apsc vessels are more difficult to identify than congenital pss. posthepatic portal hypertension does not result in the development of may not affect the echogenicity of the liver at all. neoplasia can also lead to the appearance of nodules within the hepatic parenchyma. malignant liver nodules have a variable appearance and size and can be difficult to distinguish from nonmalignant conditions such as cysts, hematomas, benign hyperplastic nodules, granulomas, or abscesses. the finding of one or more target lesions in the liver or spleen had a positive predictive value of % for detecting malignancy, and thus should not be considered a specific finding. cytologic or histologic evaluation of a tissue sample is needed to differentiate between malignant and benign liver nodules. tumors, such as hepatoma or hepatocellular carcinoma, can also focally infiltrate the liver. contrast-enhanced harmonic ultrasound allows assessment of tissue perfusion patterns. gas-filled microbubbles are administered intravenously to the patient. the microbubbles are relatively echogenic. when they reach the tissue of interest, they produce a more potent harmonic signal than the surrounding tissue. this technique allows enhanced differentiation between tissues with varying perfusion patterns. in one study the sensitivity of contrast enhanced ultrasound for differentiation between benign and malignant liver nodules in dogs was reported to be % and the specificity was reported to be . %. sonography is also a valuable tool for the evaluation of the biliary system. biliary disease can be classified as being obstructive or nonobstructive. the term cholangitis refers to a group of nonobstructive biliary diseases, which are more common in cats than in dogs. typical ultrasound findings in cats include a hypoechoic hepatic parenchyma and prominent portal vasculature. additional findings can include evidence of pancreatic inflammation, thickening of the gallbladder wall, and dilation of the intrahepatic and extrahepatic biliary system. it is important to note these changes are not always present. cytologic or histologic confirmation and bacterial culture are needed to confirm this diagnosis. generalized gallbladder wall thickening can occur as a result of cholecystitis, cholangitis, or hepatitis. however, the gallbladder wall can also appear to be thickened when peritoneal effusion or hypoproteinemia are present. gall bladder wall masses can be identified sonographically as a focal thickening of the gallbladder wall. sonography has also been used to assess gallbladder motility in dogs. it should be noted that gravity dependent gallbladder sludge can be found in dogs without hepatobiliary disease, so this finding should be considered incidental. abdominal ultrasound is the most commonly used imaging modality for the detection of biliary obstruction in dogs and cats. findings consistent with biliary obstruction include common bile duct distention, intra-and extrahepatic bile duct distention, and/or gallbladder dilation. a retrospective study showed that common bile duct dilation greater than mm was % sensitive for the detection of biliary obstruction in cats. sonography can also aid in identifying the cause of biliary obstruction. biliary obstruction can be classified as being luminal or extraluminal. extraluminal causes include nonneoplastic pancreatic disease, abdominal adhesions, and, rarely, pancreatic neoplasia. luminal causes include gallbladder mucocele, biliary neoplasia, inflammation, and cholecystolithiasis. biliary tract obstruction can progress to biliary rupture and bile peritonitis. sonographic signs of biliary rupture include loss of gallbladder wall continuity, free peritoneal fluid, and signs of localized peritonitis. gallbladder mucoceles occur in the dog, but have not been described in cats. mucoceles have a variable sonographic appearance; typical findings include a stellate or finely striated bile pattern with a hypoechoic rim, which is not gravity dependent, and gb lt specificity of %. the disadvantages of ct and mri include their limited availability, cost, and the need for anesthesia. although cytologic evaluation of the liver provides a definitive diagnosis, often histologic examination is also required. there are a variety of techniques to collect cytologic samples of the hepatobiliary system. abdominal effusion, when present, can be collected percutaneously. fine-needle aspirates (fna) of the liver can be collected percutaneously under ultrasound guidance. cholecystocentesis can also be performed percutaneously with ultrasound apsc vessels, but can lead to a distention of hepatic veins and ascites. nuclear scintigraphy involves administering a radioactive tracer substance (radiopharmaceutical) to the patient, which localizes to a specific organ or tissue. the radioactive decay of this substance is detected by a gamma camera and used to form images. scintigraphy has been used to detect pss and to assess gallbladder emptying in small animals. however, specialized equipment and a license for the use of radioisotopes are required. consequently, availability of this imaging modality is currently limited to academic institutions and specialty referral hospitals. technetium- m pertechnetate is the most commonly used radiopharmaceutical for assessing the portal circulation of small animal patients. two techniques have been described: per-rectal portal scintigraphy and transsplenic portal scintigraphy. by analyzing the radiation emitted from regions of interest drawn over the patient's liver and heart, pss can be detected and a shunt fraction can be calculated. this allows for the minimally invasive diagnosis of pss, differentiation of pss from portal vein hypoplasia without portal hypotension (previously known as microvascular dysplasia), and comparison of the degree of shunting before and after shunt attenuation. transsplenic portal scintigraphy is preferred over perrectal portal scintigraphy as it is simpler to perform, uses lower doses of the radiopharmaceutical, and is more sensitive (figure - ). transsplenic portal scintigraphy is % sensitive and specific for the diagnosis of congenital pss, and significantly more likely than per-rectal portal scintigraphy to detect shunt number and termination in dogs. nuclear scintigraphy has been used to quantify liver function and to assess biliary tract patency in dogs. in a retrospective study hepatobiliary scintigraphy was found to be % sensitive and % specific for the detection of extrahepatic biliary obstruction in dogs and cats. computed tomography (ct) (figure - ) and magnetic resonance imaging (mri) have been used to detect hepatic parenchymal neoplasia in humans. compared to abdominal ultrasound these techniques have an improved accuracy for the diagnosis of hepatic neoplasia in humans. however, there is limited data in the veterinary literature evaluating their diagnostic performance. in one study the diagnostic accuracy of ct for detecting hepatic masses was not found to be significantly different from that of abdominal ultrasound in dogs. in another study mri was found to have a sensitivity of % and a specificity of % for the differentiation between benign and malignant liver lesions in dogs. ct angiography is being used increasingly in dogs for the diagnosis of congenital pss and other hepatic vascular diseases. it offers the advantage of being less invasive than operative angiography, allows for improved assessment of the portal vasculature, and allows the creation of a three-dimensional reconstruction. the vasculature detail afforded by ct angiography is particularly useful when planning attenuation of a congenital pss. the diagnostic utility of ct angiography for detecting and characterizing pss in dogs was shown to compare favorably to that of other techniques, including surgical exploration. transsplenic ct portography has been described in dogs without pss. this technique offers more intense enhancement of the splenic and portal veins than ct angiography. mri angiography diagnoses pss in dogs with a sensitivity of % and a the findings above may aid in making a diagnosis of liver disease but are not a substitute for histopathologic analysis, as cytologic specimens do not allow assessment of the hepatic architecture. furthermore, only a tiny proportion of the liver is sampled when cytologic samples are examined. these limitations are reflected by the results of a retrospective study that found the overall agreement between the histopathologic and cytologic diagnosis of liver disease to be . % for dogs and . % for cats. cytologic evaluation of bile can also be useful for the diagnosis of biliary disorders, particularly in cats. world small animal veterinary association (wsava) standards for the clinical and histological diagnosis of canine and feline liver disease suggest that the cytologic evaluation of bile forms part of the minimum diagnostic requirement for cats with extrahepatic cholestasis and for dogs guidance. these techniques are minimally invasive and the risk of complications is relatively low but caution should be exercised in patients with bleeding disorders. liver disease can cause abdominal effusion by several mechanisms. in cases with hepatic insufficiency, severe hypoalbuminemia (< . g/dl) can occur. this can lead to the formation of a pure transudate as the result of a reduced plasma colloid oncotic pressure. increased capillary hydrostatic pressure because of portal hypertension may lead to formation of a pure or modified transudate. hepatic neoplasia can also lead to a formation of a modified transudate. biliary tract rupture can lead to bile peritonitis and abdominal effusion. an exudate with a bilirubin concentration greater than twice that of the plasma is suggestive of bile peritonitis. cytologic evaluation of hepatic fna can aid in making a diagnosis of liver disease. suppurative, mixed inflammatory, lymphocytic and, more rarely, eosinophilic patterns of inflammation can be appreciated cytologically. each pattern of inflammation suggests a group of possible diagnoses. the finding of dark green or black bile casts suggests cholestasis. infectious diseases such as histoplasmosis can be definitively diagnosed based on the cytologic finding of the infectious agent ( figure - ). hepatocellular vacuolation can be classified as being caused by lipid or not. lipid vacuolation of hepatocytes is characterized by colorless cytoplasmic vacuoles. severe lipid vacuolation is suggestive of hepatic lipidosis in cats ( figure - ). however, feline hepatic lipidosis often occurs secondary to another disease process. a group of cats with cytologic findings suggestive of hepatic lipidosis were reported to have underlying infiltrative liver disease. nonlipid vacuolation is characterized by generalized hepatocyte swelling and lacy vacuolation ( figure - ). vacuolar hepatopathy occurs secondary to a wide variety of extrahepatic disease processes in dogs. metastatic tumors and round cell tumors, such as lymphoma ( figure - ) , affecting the liver can often be diagnosed cytologically. additionally, cytologic evaluation can aid in distinguishing liver nodules because of extramedullary hematopoiesis from those caused by neoplasia. however, it is not possible to distinguish hepatic nodular hyperplasia from hepatic adenoma or well-differentiated carcinoma cytologically. some cases of hepatocellular carcinoma can be diagnosed cytologically if criteria for malignancy are present. and cats suspected to have cholangitis. the finding of neutrophils and bacteria on bile cytology supports a diagnosis of feline neutrophilic cholangitis. cytology is essential for the diagnosis of this disease, as cats with neutrophilic cholangitis may not have typical hepatic histopathologic changes and it can be difficult to distinguish these cats from those with lymphocytic cholangitis. bile should also be submitted for aerobic and anaerobic bacteriologic culture. histopathologic evaluation is required to make a definitive diagnosis of most liver diseases. histopathologic evaluation of the liver allows a morphologic and sometimes an etiologic diagnosis to be made (see chapter ) . in addition to routine staining with hematoxylin and eosin, a variety of other staining techniques can be employed to demonstrate hepatic pathology. to optimize the value of histopathologic evaluation of the liver, particular attention should be paid to specimen collection, specimen handling, and communication between the clinician and the pathologist. although liver biopsy is considered to be relatively safe, the patient should be assessed for bleeding disorders before this procedure. this assessment should include a platelet count, coagulation times, and a buccal mucosal bleeding time. liver biopsies can be collected in a number of ways. each method has advantages and disadvantages, and there is controversy in the veterinary literature as to which technique is optimal. laparotomy allows collection of relatively large wedge biopsies, with direct visualization. this technique does not require specialized equipment or training, and excessive bleeding can be readily identified. however, laparotomy requires general anesthesia and is the most invasive biopsy technique. percutaneous needle biopsy techniques have been described. these techniques may be possible under heavy sedation and are the leastinvasive method for collecting liver biopsies. ultrasound guidance is often used, allowing biopsy of focal lesions. it is also possible to biopsy tissue that is deeper within the hepatic parenchyma than is possible with other techniques. however, the specimens that are collected are relatively small and may be inadequate for accurate assessment in some patients. a prospective study showed that there was agreement between the histomorphologic diagnoses made upon examination of needle biopsies and those made on wedge biopsies collected during laparotomy or necropsy for only % of dogs and cats. excessive hemorrhage after biopsy may not be identified immediately. laparoscopy allows collection of biopsies using forceps with laparoscopic guidance. this technique requires general anesthesia, but is less invasive than laparotomy. the biopsies collected are larger than needle biopsies and excessive bleeding can be visualized. however, laparoscopy requires specialized equipment and training. the use of biopsy forceps may result in crushing artifact and the tissue collected may be too superficial to identify lesions that lie deeper within the hepatic parenchyma. regardless of the technique used, a tiny proportion of the organ is sampled and, because liver disease can affect the hepatic parenchyma in a heterogeneous manner, sampling error is possible. to reduce the effect of sampling error, several biopsies from different areas of the liver should be collected and focal lesions should be specifically biopsied. the clinician should provide the pathologist with all the pertinent information from the patient's history, physical examination findings, the results of laboratory testing, and the findings from diagnostic imaging. in turn the histomorphologic diagnosis that the pathologist makes should be interpreted by the clinician along with the other clinical data. when the histopathologic diagnosis does not fit the clinical picture, the pathologist should be consulted and when necessary a second opinion should be requested. variation in the assessment of hepatic pathology between pathologists was highlighted by a study that found agreement between examiners for only % of needle biopsies and % of wedge biopsies examined. hopefully, the adoption of wsava standards for the clinical and histological diagnosis of canine and feline liver diseases since the aforementioned study will reduce this interobserver variation. quantification of hepatic metal concentrations requires submission of tissue for flame atomic absorption spectroscopy. although zinc has a role as an antioxidant, hepatic copper and iron retention can lead to oxidative liver injury. copper is the most frequently course of the evaluation. in patients that are asymptomatic and have abnormal biochemical testing, repeat evaluation is sometimes warranted. a general guideline is to obtain a liver biopsy in asymptomatic patients if there are moderate to severe elevations in serum hepatic enzyme activities that persist for at least months, or if there are mild to moderate elevations in serum hepatic enzyme activities that persist for at least months. if clinical signs of hepatic disease develop, then biopsy should not be unreasonably delayed. if there are concurrent elevations in serum bile acids, biopsy should also not be delayed. other indications for hepatic biopsy are ultrasound imaging abnormalities. if there are focal hepatic masses or diffuse echotextural changes, a biopsy may be warranted, depending on results of laboratory testing. in one study, abdominal ultrasound findings alone were not reliable for obtaining a diagnosis of infiltrative hepatic disease with diffuse changes in echogenicity (either hypoechoic or hyperechoic, uniform or mottled). in another study, sonographic detection of a hepatic mass greater than or equal to cm, ascites, abnormal hepatic lymph node(s), and abnormal spleen were predictive of liver neoplasia based on cytology. conversely, sonographic detection of hepatic nodules less than cm was predictive of vacuolar hepatopathy on cytology. thus several sonographic findings, alone or combined, may be predictive of liver ultrasound-guided fine-needle aspiration cytology results. in light of the fact that ultrasound-guided fine-needle aspiration cytology of the liver has limitations , the results of ultrasound and cytology should be adjuncts to other findings. another indication for hepatic biopsy is the need to assess response to therapy. in cases of chronic hepatitis in dogs, it is often difficult to determine if there is ongoing inflammation and resolution/ progression of fibrosis during long-term therapy. this is particularly true when the patient is receiving glucocorticoid therapy as these medications cause variable increases in serum alp and transaminase activities independent of the underlying disease. repeat or serial hepatic biopsy analysis is often helpful to guide therapeutic decisions in these cases. among the most serious complications of liver biopsies are hemorrhage, infections, and injury to the adjacent viscera. consequently the clinician must take into account the clinical question, the appropriate invasive biopsy method, and methods of managing postbiopsy complications. postbiopsy hemorrhage is often the first concern, although it is unclear as to what the best predictor of hemorrhage is in patients about to undergo hepatic sampling. in one study of human patients in which bleeding was evaluated laparoscopically, there was no correlation between any in vitro coagulation test and "liver bleeding time." other studies in man have used laparoscopy and ultrasonography to assess hepatic bleeding time following needle biopsy, and most have shown similar poor correlation between coagulopathies and hepatic bleeding times. similar studies have not been reported in veterinary medicine. there also have been studies in human and veterinary medicine evaluating risk factors for bleeding complications (as opposed to "hepatic bleeding times"). , bigge et al. correlated coagulation profile findings and bleeding complications after ultrasound-guided biopsies in dogs and cats. there was no apparent correlation between coagulation parameters and major complications following liver biopsy. studies show that clotting times assessing proteins induced by vitamin k antagonism are more sensitive in detecting coagulopathies in patients with hepatic disease. , the measured of these metals and quantification is essential for the diagnosis of hepatic copper retention. these measurements are usually performed on freeze-dried pieces of liver. specimens for metal measurement should not be stored in saline and should be kept in metal-free containers. recently, it was shown that measurements of the concentration of copper and iron, but not zinc, can be ascertained from deparaffinized-archived liver tissue. hepatobiliary diseases can be challenging to diagnose. although diagnostic tests that employ biochemical, molecular biologic, serologic, functional, as well as imaging techniques are capable of establishing the etiology of some chronic or acute liver diseases, in most instances the gold standard for definitive diagnosis and the assessment of stage and severity of liver diseases is the histologic evaluation of a liver sample. recent advances in imaging technology, the use of multiple imaging modalities, and newer biopsy methods have resulted in improvement in the ability to safely procure hepatic tissue for evaluation. there are several means of obtaining hepatic samples including fine-needle aspiration, ultrasound-guided biopsy, laparoscopy, and laparotomy. all techniques have both advantages and disadvantages, which should be carefully considered before choosing the appropriate sampling method. many biochemical tests are available to evaluate the anabolic and/ or catabolic function of the liver and the hepatic circulation. these include measurement of concentrations of bile acids, ammonia, bilirubin, and the ability to excrete organic dyes. other tests of hepatic function include measurement of serum albumin, glucose, urea nitrogen, and clotting factor analysis. hepatic function can be markedly abnormal despite maintenance of the hepatocellular membrane and therefore normal serum activities of hepatic enzymes. examples include psss, terminal cirrhosis, and metastatic hepatic neoplasia. likewise, the liver can continue normal anabolic or catabolic function despite severe hepatocyte leakage of intracellular enzymes because of its marked reserve capacity. this can occur, for example, in certain cases of hepatocellular necrosis, blunt abdominal trauma, or primary hepatic neoplasia. thus, the limitations of serum hepatic enzyme activities must be taken into consideration. hepatocellular leakage enzyme activities include alt and ast. enzyme activities that increase with biliary tract obstruction include serum alp and ggt. no laboratory test identifies a specific problem, helps determine specific therapeutic management, or predicts an outcome. this is because different diseases produce similar alterations in hepatic function or in laboratory tests. once biochemical tests identify the presence of hepatic disease, the diagnosis must be pursued further. in some instances, diagnostic imaging can reveal specific abnormalities (e.g., psss and extrahepatic bile duct obstruction). when results of imaging do not give a specific etiology, the next step is often to pursue a morphologic diagnosis obtained by analysis of a biopsy specimen. often it is a judgment call as to when to pursue hepatic biopsy. in cases with severe clinical signs and/or severe biochemical abnormalities, biopsy is usually warranted early in the fine-needle aspiration has several advantages. little to no sedation is usually required. because the size of the needle is so small, there is little risk of hemorrhage. therefore multiple sites can easily be sampled. the procedure is rapid and can usually be performed on an outpatient basis. there is also less cost to the client. the primary disadvantage of fine-needle aspiration is its questionable accuracy. the sample size often limits the number of available cells to obtain an accurate diagnosis, and hemodilution makes it difficult to assess whether inflammatory cells were present in the liver or peripheral blood. there are several important elements used to interpret pathologic information including lobular architecture, presence and location of inflammation within a lobule, presence and severity of fibrosis, metal accumulation, vascular abnormalities, and lobule heterogeneity. these criteria cannot be accurately determined using a cytologic sample obtained using fna. several studies have compared fine-needle aspiration cytology with biopsy with histopathology. [ ] [ ] [ ] [ ] in one study with a total of cases, there was good correlation in % of cases, partial correlation in % of cases, and no correlation in % of cases. poor correlation was found with a variety of histologic changes, including vacuolar change, lipidosis, cholestasis, inflammation, and neoplasia. in a similar study with cases, complete agreement between fineneedle aspiration and histopathology was seen in only % of cases in dogs: % agreement with inflammation, % agreement with neoplasia (mainly carcinoma), and % agreement with vacuolar hepatopathy. in cats, there was overall agreement in % of cases: % agreement with inflammation, % agreement with neoplasia (lymphoma), and % agreement with vacuolar hepatopathy. although vacuolar hepatopathy was the most sensitive diagnosis, it was also the most common misdiagnosis using cytology. in another study, the best correlation between hepatic cytology and biopsy was seen with lipidosis, lymphoma, and carcinoma, whereas the worst performance was seen with inflammatory and fibrotic disorders. another study found high sensitivity and specificity with fine-needle aspiration in detecting inflammatory hepatic disease in dogs. however, further information was not provided such as the severity of the inflammation or other histopathologic features. additionally, for noninflammatory hepatic disease, cytology was inaccurate in % of cases. proteins-induced-by-vitamin-k-antagonism test is more than twice as sensitive in dogs and more than three times as sensitive in cats in detecting coagulopathies compared with prothrombin time (pt) and aptt. , however, in a pilot study performed by me, hepatic bleeding times assessed via laparoscopy did not correlate with proteins induced by vitamin k antagonism times. thus it appears that indices of coagulation in the peripheral blood are generally unreliable guides of the risk of bleeding after liver biopsy, and hence, are of limited value in determining contraindications to this procedure. this lack of correlation may be explained by the high concentration of clotting factors in the hepatic parenchyma and by mechanical compression of the needle tract by the elastic tissue within the liver. in most cases of significant hemorrhage, technical errors such as damaging a large vessel are the cause rather than persistent oozing from a needle biopsy site. controlled studies in veterinary patients will be necessary to make final conclusions regarding postbiopsy hemorrhage in the patient with a coagulopathy. in one study of normal dogs, biopsies taken from the left lateral hepatic lobe using a biopsy punch, biopsy needle, ligature method, laparoscopic biopsy forceps, and ultrasonically activated scalpel resulted in minimal hemorrhage (< ml). however, this investigation did not assess the risk of hemorrhage in dogs or cats with hepatic disease. these risks will be discussed later under each sampling method. with the exception of fine-needle aspirations, each patient should have a prebiopsy packed cell volume and and hours postbiopsy packed cell volume for close monitoring of potential hemorrhage. fine-needle aspiration involves obtaining a small amount of hepatic tissue for cytologic analysis, and is typically performed in conjunction with, and guided by, ultrasound. ultrasound imaging helps determine if there is a diffuse abnormality (e.g., increased or decreased echogenicity, diffuse mottling) or if there are focal abnormalities (e.g., discrete nodules, cysts, masses, or focal areas of heterogenous mottling). an appropriate site to be sampled is chosen. often multiple sites are chosen to represent different lobes, and in the case of focal lesions, to sample more than one area of abnormal tissue and sample seemingly normal tissue. the sites are also chosen based on accessibility. for example, a solitary nodule in the dorsocranial aspect of the liver in a large deep-chested dog would be impossible to reach with a . -inch needle. a lesion adjacent to the gallbladder or caudal vena cava would involve considerable risk. the clinician would need to decide whether the relative risk of sampling such lesions is the appropriate decision, or whether other methods of sampling would be more appropriate such as laparoscopy or laparotomy. figure - depicts a typical setup for fine-needle aspiration. usually a -gauge, . -inch needle is used. for most patients, the procedure is performed without sedation or local anesthetic. if it is determined that the animal is moving too much during the initial ultrasound examination, a sedative may be necessary (or an anesthetic in extreme cases). the needle is inserted without a syringe using ultrasound guidance. the needle is rapidly agitated in and out (sometimes referred to as mimicking the action of a sewing machine) and simultaneously twisted multiple times for a few seconds to obtain a sample. this method relies on capillary action rather than suction to get tissue into the needle, resulting in less hemodilution. after removing the needle from the liver, a syringe is attached and cells are expelled onto a glass slide for cytologic examination. often three to five separate attempts are made to increase the sample size and diversity. examination is performed prior to biopsy. this allows planning of the procedure based on echo pattern, lesion size, proximity to other organs, proximity to blood vessels, determination of cystic or solid tissue, and optimal approach of the needle path. care must be taken prior to taking samples to ensure that vessels and other organs are not within the path of the needle. for diffuse lesions, the transducer is typically placed caudal and to the left of the xiphoid, and aimed at the left medial or lateral lobes. in patients with a small liver, it may be difficult to adequately visualize the needle without gastric gas interference. placing these animals in a -degree right lateral oblique position can reduce this interference. if the animal is under general anesthesia, an assistant can compress a rebreathing bag to hold the animal in deep inspiration, which serves to move the diaphragm and liver caudally to improve visualization. the area is surgically prepared. the ultrasound transducer is covered with sterile wrap and sterile lubricant is used to enhance skin contact. a small stab incision is made in the skin at the desired needle insertion site. while one hand maneuvers the transducer, the other hand advances the needle into the liver under direct ultrasound visualization. the image should be optimized to maximize the chance of recognizing the needle within the liver. to allow distinction of the needle from other echogenic structures, the needle can be gently moved in and out with minimal movements (attempting to move the liver within the abdominal cavity rather than the needle within the liver). occasionally the needle cannot be seen, and indirect evidence of organ penetration must be used such as movement of the liver or visualization of movement at the liver border. the needle is then directed so the trajectory will avoid other structures when it is fired. the needle is then fired, and immediately removed. for most cases, four to five samples are obtained, and are submitted for aerobic/anaerobic culture, histopathologic evaluation, and metal (copper, zinc, and iron) quantification. in one study, liver tissues with high metal concentrations had significantly lower copper and iron in needle-core versus wedge biopsy specimens. consequently the value of needle-core biopsy specimens for measurement of metal concentrations is questionable. careful examination for post-biopsy hemorrhage is then performed. external digital pressure may be used to help control hemorrhage in smaller patients. usually an abdominal compression wrap is ineffective for controlling hemorrhage. ultrasound-guided biopsy has many of the advantages of fine-needle aspiration, including the need for minimal sedation in some patients, the ability to sample multiple sites, and low to moderate cost to the client. additionally, tissue is obtained for histopathology. one disadvantage of ultrasound-guided biopsy is the risk of bleeding (especially when multiple sites are sampled and largergauge needles are used). in one study, percutaneous transabdominal hepatic needle biopsy samples were obtained with no adverse consequences noted ; however, this study was performed in normal dogs, and still carries high risk. additional disadvantages of ultrasound-guided biopsy include the needing sedation or anesthesia in some patients, difficulty of imaging small livers, difficulty of obtaining liver tissue in patients with fibrosis, and, most importantly, the obtaining of samples that have a questionable representation of the underlying hepatic pathology. the diagnostic accuracy of needle biopsy has been questioned by many clinicians, observing that results of needle biopsy analysis often do not adequately reflect the clinical and laboratory features of the patient. this questionable accuracy is in most part a result of potential for sampling error. this method still results in a relatively small sample size, possible although fine-needle aspiration is easy to perform, involves little risk, and little to no sedation, the information is of little value if it is inaccurate as often as it is accurate. there is institutional bias regarding its accuracy, which may relate to the experience and expertise of the cytologists. given its clear limitations, fine-needle aspiration is best used as an adjunctive diagnostic modality in conjunction with other techniques or clinical findings, and does not replace histopathology. the clinician must be aware of its inherent inaccuracy before undertaking fine-needle aspiration and relying on the cytologic findings. ultrasound-guided hepatic biopsy uses a cutting-type needle as a sampling tool. automated needles are preferred and should be either completely automated or semiautomated. these are spring-loaded needles similar in style to the manual tru-cut needle. completely automated needles thrust the inner obturator (containing the biopsy tray or specimen notch) followed by the outer cutting sheath into the liver in a fraction of a second. these needles can be operated with one hand while the other hand operates an ultrasound transducer to allow precise placement of the biopsy instrument. there is minimal displacement of the liver, a shorter intraparenchymal phase, and a more reliable yield of tissue. this allows a smaller diameter needle to be used and a lighter degree of sedation in some cases. using the rapid cutting action, the hepatic tissue tends to be less fragmented. semiautomated needles require manual placement of the internal obturator into the liver, followed by an automatic thrusting of the outer cutting sheath by a spring-loaded mechanism. these needles have the additional advantage of control over the final needle position, as the tip of the needle can be precisely localized before the outer cutting sheath is deployed. i generally use a -gauge needle for ultrasound-guided hepatic biopsy. figure - depicts a typical setup for ultrasound-guided biopsy. in most dogs, the liver can be biopsied using local anesthesia and minimal sedation. most cats require general anesthesia to safely obtain tissue. it must be emphasized that the degree of sedation must be tailored to each individual patient. a careful ultrasound peritoneal space through a stab incision using a number scalpel blade. after ensuring no obstruction and negative pressure using the infusion and aspiration of saline, the abdomen is insufflated with carbon dioxide gas and maintained at a pressure of approximately mm hg. a scope port (cannula) is then placed cm right lateral to the veress needle. the veress needle is then removed and replaced with an instrument port. hepatic sampling is achieved using a "spoon" or oval cup biopsy forceps. multiple samples are obtained under direct visualization, and samples are submitted for aerobic/anaerobic culture, histopathologic evaluation, and metal (copper, zinc, and iron) quantification. following procurement of all the biopsy specimens, the sites are inspected for hemorrhage. the abdomen is then decompressed, and lidocaine and bupivacaine are infused into the peritoneal cavity through either port. both the instrument and scope ports are removed, and the port site incisions are closed using either a cruciate or simple interrupted pattern in the body wall, subcutaneous tissue, and skin. this technique enables gross evaluation of the entire liver, extrahepatic biliary system, and surrounding structures while obtaining multiple large specimens of liver. the ability to obtain multiple samples decreases the risk for sampling artifact in cases of regional diversity within the liver. additionally, by directly visualizing the hepatic parenchyma, the clinician can correlate the histopathologic findings and clinical data with the gross appearance of the liver to render the most accurate diagnosis. this method also enables the visualization of smaller masses and irregularities that may not be evident with ultrasonographic imaging. these masses can also be individually sampled. laparoscopy also gives the clinician an excellent view of the liver regardless of the hepatic size or conformation of the patient, making it an easy method to sample the liver in patients that are difficult to image with ultrasound. there is generally minimal bleeding during this procedure, even in patients with in vitro coagulopathies. using a "spoon" or oval-cup biopsy forceps typically results in a marked decrease in the amount of hemorrhage when compared with needle biopsies. any hemorrhage can be directly visualized for adequate clot formation. if hemorrhage persists, direct pressure using a blunt probe for minutes can be used. if the site continues to bleed, electrocautery can be applied to the biopsy site or a topical hemostatic agent (gelfoam) can be placed directly on the biopsy site using laparoscopic forceps. disadvantages of laparoscopy include the need for expensive equipment, the need for extensive training, the need for general anesthesia in most cases, and higher cost to the client. laparoscopy gives the clinician the advantages of a laparotomy (large sample size, ability to best direct sampling, and ability to take multiple samples, thus resulting in the highest diagnostic accuracy), though with a relatively minimally invasive procedure. the complication rate (especially hemorrhage) is far less than with ultrasoundguided biopsy in my practice. for these reasons, it is my method of choice for obtaining hepatic biopsy specimens in most cases. wedge biopsy via laparotomy is another potential method for obtaining hepatic biopsies. if a random liver biopsy is needed and a section of liver is protruding, a guillotine suture can be used. a preformed encircling ligature of - monofilament absorbable suture material is placed around the protruding section of liver. fragmentation of fibrous tissue, and may not enable sampling of abnormalities located in other lobes (the left medial or lateral lobes are generally sampled because of their ease of imaging). in one study, percutaneous hepatic sampling using core biopsies resulted in % diagnostic quality samples, however these were not compared with large wedge biopsy to assess the accuracy of this method. in another study, the diagnostic accuracy of the tru-cut-type needle biopsy was compared with the gold standard of surgical wedge biopsy of the liver in patients. the overall discordance between the two methods was % in dogs and % in cats, with a greater than % discrepancy occurring with chronic hepatitis or cirrhosis, cholangitis/ cholangiohepatitis, portosystemic vascular anomalies, microvascular dysplasia, fibrosis, and miscellaneous disorders. these disorders are the most commonly seen among dogs and cats with hepatobiliary disease. the greatest accuracy was with neoplasia ( % concordance). figure - is an example of a mass amenable to an ultrasound-guided needle biopsy. use of a -gauge versus -gauge needle may reduce this discordance as it raises the number of portal triads sampled from an approximate mean of four to seven, though larger needles carry the risk of increased hemorrhage. in summary, ultrasound-guided hepatic biopsy is relatively easy to perform, but involves more risk to the patient (primarily bleeding). like fine-needle aspiration, ultrasound-guided biopsy has questionable accuracy. the accuracy may be increased by using a larger-gauge needle, but this carries a greater risk of postbiopsy hemorrhage. if the patient is suspected of having inflammatory disease, vascular abnormalities, or significant fibrosis, or is at risk for hemorrhage, laparoscopy or laparotomy should be considered. chapter provides a detailed description of laparoscopic liver biopsy. briefly, laparoscopy is performed under general anesthesia with the patient in dorsal recumbency tilted degrees to the left. a veress needle is placed at the level of the umbilicus into the acute hepatitis and necrosis are common morphologic hepatic lesions in dogs and cats presenting with acute liver disease caused by infectious, toxic, metabolic, and ischemic disorders (box - ). however, acute liver disease can also be associated with other pathologic processes such as severe hepatic lipidosis (cats), granulomatous hepatitis (fungal infections), intrahepatic cholestasis (bacterial cholangitis, leptospirosis), and malignant infiltration (lymphoma, malignant histiocytosis). canine adenovirus i, canine and feline herpesvirus in the neonate, clostridium piliforme, and toxoplasma gondii are specific examples of infectious agents that cause acute hepatic necrosis (with variable inflammation), often as part of a multisystemic disorder. although leptospirosis is a well-recognized infectious cause of acute liver disease in dogs, hepatic necrosis is an uncommon histologic feature and hepatic lesions are typically characterized by cholestasis, liver cell dissociation, and nonspecific reactive hepatitis. despite the large number of potential causes of acute hepatitis and necrosis, a specific etiology is often not determined. , in a recent case series of dogs with primary hepatitis (acute and chronic hepatitis) that were presented to a referral clinic, dogs were diagnosed with morphologic features of acute hepatitis. a cause could not be determined in the majority of these cases, although increased hepatic copper was detected in five dogs with acute hepatitis, suggesting that copper accumulation could be a significant contributing factor. despite numerous potential causes of hepatocyte death, two general mechanisms are recognized: apoptosis and necrosis. these two mechanisms have traditionally been considered to be distinct events. however, it now appears that apoptosis and necrosis are alternate outcomes of the same initiating causes and signaling pathways. apoptosis is adenosine triphosphate-dependent (caspasedependent) programmed cell death that causes shrinkage of the cell (apoptotic bodies or acidophil bodies) with orderly resorption of cellular contents, minimal leakage of cellular components, and minimal secondary inflammation. , necrosis occurs when depletion of adenosine triphosphate results in cellular swelling, loss of integrity of the cell membrane and cell lysis, with release of cell contents and secondary inflammation. , diffuse hepatic necrosis is the most consistent histological lesion detected in dogs and cats with acute liver failure. acute liver failure (alf) is a rare clinical syndrome (usually fatal) that occurs when a sudden severe insult to the liver compromises at least % of functional hepatic mass. liver cell death exceeds hepatic regenerative capacity, resulting in clinical signs of liver failure. the clinical and laboratory features of alf are not specific for the inciting cause but reflect disruption of one or more major hepatic functions. once hepatocellular injury has occurred (and assuming the patient survives), the morphologic hepatic response to injury may include parenchymal regeneration, fibrosis, and ductular proliferation. nearly complete hepatic regeneration is possible if hepatocyte injury is limited and the reticulin network remains intact. with severe parenchymal destruction or extensive loss of hepatocytes, periportal ductular proliferation, hepatic fibrosis, postnecrotic scarring, and regenerative hepatic nodules are more likely. dogs with acute hepatitis may also progress to chronic hepatitis. the clinical presentation of dogs and cats with acute hepatitis and necrosis varies with the underlying cause and the extent and severity of the hepatic lesions. the spectrum of hepatic involvement may the ligature is then tightened until it has crushed the hepatic parenchyma. after completing several throws in the knot, the sample is excised to mm distal to the ligature using metzenbaum scissors or a scalpel blade. if a specific area of liver is needed, a sample can be obtained using the transfixation method or a biopsy punch. the transfixation method entails placing a ligature through the liver lobe approximately to mm from its edge. the ligature is tightened to crush through the hepatic parenchyma along one border of the desired biopsy specimen. an additional throw is made at a right angle to the first ligature, and this throw is tightened to crush the parenchyma of the second border of the specimen. the sample is removed to mm distal to the crushed area using a scalpel blade or metzenbaum scissors. if the desired area does not lie near the edge of a liver lobe, a -mm biopsy punch can be used. the biopsy punch should be inserted into the hepatic parenchyma ensuring not to penetrate the opposite surface. if the biopsy site is close to the hilus, extra caution must be used so that no more than half of the thickness of the liver is penetrated. the biopsy sample is removed from the liver using scissors. hemorrhage can be controlled by filling the defect with a topical hemostatic agent (gel foam) and applying digital pressure for to minutes, or by suturing the hepatic capsule with fine, absorbable monofilament suture in a cruciate pattern. akin to laparoscopy, this method has similar advantages and disadvantages as listed previously. although it is more invasive, it allows easier biopsy of other abdominal organs (such as intestine and mesenteric lymph node) and the ability to perform therapeutic maneuvers (such as hepatic mass removal or biliary diversion). etiology hepatocyte death (necrosis and apoptosis) in dogs and cats occurs secondary to a broad variety of insults, including infectious agents, drugs and toxins, hypoxia, immunologic events, and metabolic disorders. hepatic necrosis and acute inflammation often occur together and the relationship between these two processes is complex. acute inflammation may be the primary event, or necrosis of hepatocytes can be followed by a substantial inflammatory response, the "hallmark" of necrotic cell death. the term acute hepatitis traditionally has been used when infectious agents cause hepatocellular necrosis, even though in the early stages, hepatic inflammation can be minimal or absent. controversy exists among veterinary pathologists regarding the preferred terminology (acute hepatitis versus acute hepatic necrosis), when necrosis predominates and is caused by noninfectious insults such as toxins or ischemia. for the purposes of this discussion, lesions of acute hepatitis and acute hepatic necrosis are discussed together, recognizing that the primary contributions of each lesion may be variable, depending on the cause, host response, and passage of time. acute hepatitis, a form of primary hepatitis, should be differentiated from "nonspecific reactive hepatitis," a response of the liver to a variety of extrahepatic disorders that is characterized by focal inflammation without necrosis. nonspecific reactive hepatitis is discussed in a later section of this chapter. synthesis. increased activity of the cholestatic liver enzymes, alkaline phosphatase, and ggt, also commonly occur with acute hepatitis and necrosis, but the magnitude of the increase is much less than for the alt and ast. abnormalities in biochemical tests such as hyperbilirubinemia, increased sbas, hypoglycemia, and hyperammonemia indicate compromised hepatic function. hyperbilirubinemia and bilirubinuria support more significant hepatic injury once prehepatic (hemolytic) causes have been discounted. primary biliary tract disorders including posthepatic mechanisms of hyperbilirubinemia should also be considered in the differential diagnosis. other considerations for hypoglycemia in conjunction with acute liver disease include xylitol toxicity (excess insulin release) and sepsis. hypoalbuminemia usually suggests chronic rather than acute liver disease, because of the long serum half-life of albumin. if azotemia is detected, dehydration, gi blood loss, and concurrent renal damage (e.g., leptospirosis, nonsteroidal antiinflammatory drugs [nsaids]) should be considered. interpretation of azotemia is facilitated by concurrent urinalysis. renal injury is supported by findings of cellular or granular casts, glucosuria, isosthenuria, and proteinuria. the complete blood cell count may reveal an inflammatory response suggesting underlying infectious or inflammatory disorders, and it is also useful for ruling out hemolytic anemia as cause of jaundice. documentation of a coagulopathy is required for the clinical diagnosis of alf. laboratory findings indicative of a coagulopathy include prolonged pt and activated partial thromboplastin time (aptt), decreased fibrinogen, increased fibrin degradation products, and thrombocytopenia. abdominal radiographs are often unremarkable in dogs and cats with acute hepatitis and necrosis. the liver may appear normal or increased in size. on abdominal ultrasound, the liver may appear normal or hypoechoic. thoracic and abdominal imaging may be helpful to evaluate for other causes of acute hepatic disease, and biliary tract disorders. because dogs and cats with acute hepatitis and necrosis present with nonspecific signs of acute liver disease, the clinician should maintain a broad perspective regarding the many potential diseases and processes that can acutely affect the liver. prior to obtaining a liver biopsy, ancillary testing (cytology or biopsy of more accessible lesions, infectious disease titers or molecular tests, diagnostic imaging) should be performed to evaluate for systemic disorders with secondary hepatic effects or multisystemic infections, thus providing a diagnosis of other causes of acute liver disease in a less-invasive manner. when acute hepatitis or hepatic necrosis is suspected (or confirmed by liver biopsy), a thorough history is essential to identify exposure to potential hepatotoxins and infectious agents. the owner should be questioned regarding recent medications, including prescription and over-the-counter drugs, and alternative medicines such as herbal and dietary supplements. the potential for exposure to chemicals or hepatotoxins (amanita mushrooms, blue-green algae, sago palms, aflatoxins, or xylitol) should be assessed (for more details, see "drug and toxin-induced liver injury" section). other pertinent historical questions include current vaccination history (canine adenovirus, leptospirosis), travel history (fungal infections or tick-borne diseases), and exposure to other animals (infectious causes). liver biopsy is required to document the presence of acute hepatitis and necrosis; evaluate for specific causes; and differentiate acute from chronic disease. in patients with mild (or absent) clinical signs and liver enzyme elevations that correspond to recent medication administration, a liver biopsy may be postponed, the medication discontinued, and clinical signs and liver enzymes monitored for include (a) subclinical (biochemical abnormalities only), (b) clinical signs of acute liver disease, or (c) the clinical syndrome of alf. when liver injury is mild (focal necrosis and inflammation), clinical signs may be absent, mild, or related to an underlying cause in another organ system. in this setting, hepatic involvement may not be recognized until biochemical evaluation reveals increased liver enzyme activity or mild hyperbilirubinemia. it has been suggested that many dogs with acute hepatitis are not recognized clinically, because signs are mild and self-limiting, and dogs recover spontaneously regardless of treatment. clinical signs of acute hepatitis include acute onset of lethargy, anorexia, vomiting, diarrhea, pu, and pd, in a previously healthy animal. these are nonspecific findings of acute liver disease, which overlap those of other systemic disorders. the finding of icterus on the physical examination is a more specific indicator of hepatobiliary disease, especially in the absence of anemia. dogs and cats with acute diffuse hepatic necrosis often present with alf. in addition to the signs of acute liver disease described above, animals in alf show signs of he (depression, behavioral changes, dementia, ataxia, pacing, circling, blindness, hypersalivation, seizures, and coma) and clinical evidence of a bleeding tendency (melena, hematemesis, or cutaneous and mucosal hemorrhages), which suggest severe hepatic dysfunction. signs of alf are rapidly progressive (over hours to days) and this clinical syndrome is often fatal, with reported mortality varying from % to %. with acute hepatic disease, the history typically reveals acute onset of signs in a previously healthy animal. however, liver failure that is recently recognized may not necessarily be recent in onset. with occult chronic liver disease, clinical signs may be vague and go unrecognized by the owner until a final phase of hepatic decompensation. the owner should be questioned about any subtle signs of chronic illness that would suggest the underlying liver disease may be chronic rather than acute, and that the current illness may be an exacerbation or decompensation of chronic liver disease. dogs and cats with alf are generally in good nutritional status compared with those with chronic hepatic disease. findings of cachexia, emaciation, ascites, or edema suggest a more protracted illness and are characteristic of chronic rather than acute liver disease. it is important to make a distinction between acute and chronic liver disease as the intensive supportive care indicated in alf might not be warranted in chronic end-stage liver disease. the long-term prognosis is better for acute hepatitis than chronic hepatitis. an initial database consisting of complete blood cell count, serum chemistry, and urinalysis should be obtained in dogs and cats with acute liver disease. liver enzyme elevations are a common finding in dogs and cats with acute hepatitis and necrosis. with mild hepatic injury or focal hepatic necrosis, increased alt activity may be the only finding on an otherwise unremarkable biochemical profile. alt and ast activities are moderately to markedly increased, because of enzyme leakage from damaged hepatocytes. although alt activity increases with many hepatic diseases, the largest magnitude of increase is seen with acute hepatic necrosis and roughly correlates with the number of involved cells. alt activity may be increased as much as times the upper range of normal, with increases in ast activity that parallel but are generally lower ( times the upper limit of normal) than the alt. it should be noted that some recognized hepatotoxins (aflatoxin and microcystin in blue-green algae) are not associated with severe or protracted increases in alt activity because of toxin-suppressed transaminase improvement over a -to -week period. for patients with alf and coagulopathy, the clinician must carefully weigh the benefits of histologic characterization versus the risk of excessive bleeding from the procedure. acute hepatitis is characterized histologically by a mononuclear or mixed inflammatory pattern, accompanied by hepatocellular apoptosis or necrosis. necrosis should be further characterized by the pathologist as to the morphologic pattern of injury (focal, multifocal, confluent, bridging, massive, or piecemeal) because the pattern of necrosis may provide insight into the pathogenesis of the lesion. for example, because centrilobular hepatocytes have an abundance of cytochrome p enzymes, these hepatocytes are preferentially affected in drug-induced hepatotoxicity, when cytochrome p metabolism of the parent drug results in toxic metabolites. quantitative copper analysis and histochemical staining for copper are recommended, as copper accumulation may be an underappreciated cause of acute hepatitis in dogs. infectious causes of acute hepatitis may be diagnosed on liver biopsy, or by additional tests performed on liver tissue (culture, immunohistochemistry, polymerase chain reaction [pcr], virus isolation; table - ). unfortunately, in most cases, routine liver biopsy is unlikely to reveal a specific cause of acute hepatitis. , findings of inflammation and necrosis/apoptosis accompanied by nodular regeneration and fibrosis suggests chronic rather than acute hepatitis. the long-term prognosis is better for acute hepatitis than for chronic hepatitis. if a probable cause of acute hepatitis and hepatic necrosis can be determined, then specific treatment is directed at the primary etiology (e.g., discontinuing potentially hepatotoxic medications, treating for leptospirosis with doxycycline, or chelating hepatic copper with penicillamine). in most cases specific therapy is unavailable and treatment is directed at more general supportive and symptomatic treatment of liver disease. glucocorticoid therapy is not typically indicated in the treatment of acute hepatitis. empirical treatment with antioxidants such as s-adenosylmethionine (same; mg/ kg po q h), milk thistle (siliphos; to mg/kg po q h), or vitamin e ( to iu/kg q h) may be warranted, as oxidative stress is believed to play a role in drug (carprofen, potentiated sulfonamides, diazepam, methimazole, lomustine, others), and toxin (aflatoxin, organic solvents, and heavy metal toxicity) induced hepatic injury. same and milk thistle have additional cytoprotective properties that could be beneficial in necroinflammatory hepatopathies and hepatotoxicity. antioxidants and cytoprotective agents are discussed in more detail in chapters and , respectively. liver biochemistries should be monitored to assess patient response to therapy. repeat liver biopsy performed to weeks after the initial diagnosis has been recommended, to confirm that acute hepatitis has improved or resolved, or to document a progression toward chronic hepatitis. , it has been suggested that most dogs with mild idiopathic acute hepatitis (not in alf) recover after several days, regardless of treatment. for patients with alf, aggressive supportive treatment is required. goals of therapy are to treat the underlying cause when possible, allow adequate time for hepatic regeneration and repair, and prevent or control complications of liver failure, such as hypoglycemia, coagulopathy and anemia, he, gi ulcers, and septicemia. intravenous n-acetylcysteine (nac), a glutathione source/ antioxidant, is the antidote of choice for treatment of acetaminophen toxicity. nac also appears to have additional potential benefits (improved systemic hemodynamics and tissue oxygen delivery), and should be considered for use in any dog or cat with alf. the optimal dose regimen when nac is used for this purpose has not been determined. treatment of complications of liver failure are discussed in "complications of liver disease" section. the prognosis for recovery in dogs with acute hepatitis is good, as most dogs recover uneventfully. however, there is a potential for dogs with acute hepatitis to develop chronic disease. if animals present with signs of advanced liver failure (e.g., he, coagulopathy, hypoglycemia), the prognosis is guarded. if the animal survives, hepatic lesions such as periportal ductular proliferation, hepatic fibrosis, postnecrotic scarring, and regenerative hepatic nodules are likely. if a hepatic drug reaction is suspected, reexposure of the patient to the suspect drug should be avoided. etiology hepatic abscesses from bacterial infection of the liver occur uncommonly in dogs and cats. [ ] [ ] [ ] [ ] abscesses may form as solitary or multiple macroscopic masses or microabscesses. in newborn animals, gram-positive and gram-negative bacteria cause hepatic abscesses, presumably related to postpartum umbilical infections. in adult animals, gram-negative enteric bacteria (especially escherichia coli) and anaerobes (especially clostridia spp.) are most commonly identified; multiagent infections are frequent. , other organisms such as yersinia spp., actinomyces spp., nocardia asteroides can also cause hepatic abscesses as part of a systemic infection. the pathogenesis of hepatic abscesses in dogs and cats is unclear. hepatic abscesses are usually associated with extrahepatic infections or regional hepatic parenchymal damage. small numbers of bacteria, including clostridium spp., can be cultured from liver tissue of healthy dogs. hypoxia of hepatic tissue caused by hepatic neoplasia, liver lobe torsion, or trauma may predispose to abscess formation, because small numbers of existing anaerobes (e.g., clostridium spp.) can proliferate under these conditions. other potential sources of bacteria include hematogenous spread (via the umbilical vein, hepatic artery, or translocation of intestinal bacteria into the portal blood), ascension via bile ducts, penetrating abdominal and caudal thoracic wounds, and direct extension from local suppurative diseases. concurrent diseases or potential predisposing factors in dogs include systemic infections (pneumonia, pyelonephritis, prostatitis, pyometra, endocarditis), gallbladder rupture, pancreatitis, diabetes mellitus, liver lobe torsion, coexisting hepatic disease such as hepatic neoplasia (infected necrosis), longterm phenobarbital administration, long-term corticosteroid administration, and previous surgical biopsy. , concurrent diseases in cats include cholecystitis, pyothorax, and hepatic neoplasia. solitary abscesses are more common in dogs, whereas cats are more likely to be septic and have multiple hepatic abscesses. , no association with feline leukemia virus or feline immunodeficiency virus infection has been made. solitary liver abscesses are more likely to involve the right liver lobe in cats and the left liver lobe in dogs. , clinical examination when adult dogs and cats are diagnosed with hepatic abscesses, they are usually older than years of age. [ ] [ ] [ ] clinical signs are nonspecific and can be attributed to sepsis, inflammation, and hepatic dysfunction. the most common signs are anorexia, lethargy, vomiting, and diarrhea. , clinical signs of hepatic involvement may be overshadowed by signs of the associated disease process (e.g., neoplasia, pyelonephritis, pancreatitis). dogs with hepatic abscess may have a history of failure to respond to antibiotics or improvement that relapsed when antibiotics are discontinued. physical examination findings are often vague and include depression, dehydration, fever, abdominal pain, hepatomegaly, abdominal mass, and abdominal effusion. [ ] [ ] [ ] hypothermia is a more common finding than fever in cats with hepatic abscesses. because the clinical findings are vague and nonspecific, hepatic abscesses often go undetected until an abdominal ultrasound is performed or they rupture and are discovered during laparotomy. rupture of a hepatic abscess leads rapidly to peritonitis, septic shock, and death. clinicopathologic abnormalities are consistent with an inflammatory hepatic disease. potential findings on the complete blood count include neutrophilia with a left shift (or neutropenia and degenerative left shift if rupture occurs), mild anemia, and thrombocytopenia. , increased alt and alp activity are common findings although the alt may be in the normal range. liver enzyme elevations are a less-consistent finding in cats with hepatic abscesses and % in cats. the survival rate appears to be better when solitary abscesses are detected. , granulomatous hepatitis is characterized histologically by focal or multifocal aggregates of activated macrophages with an epithelioid appearance, usually accompanied by lymphocytes and plasma cells. this inflammatory response is distinct from that encountered in canine chronic hepatitis. systemic infectious diseases are an important cause of granulomatous hepatitis and this lesion has been described with fungal infections (histoplasmosis, coccidioidomycosis, many others), bacterial infections (mycobacteria, bartonella, nocardia, actinomyces, rhodococcus), protozoal diseases (cytauxzoonosis, leishmaniasis); parasitic diseases (visceral larval migrans, schistosomiasis, alveolar echinococcus, hepatozoon americana), and disseminated protothecosis (see table - ). , in cats, feline infectious peritonitis (coronavirus) is an important cause of multisystemic granulomatous or pyogranulomatous inflammation. other causes of granulomatous inflammation include a local response to foreign material (crystalline material, sutures, plant material) or a drug reaction. in humans, granulomatous liver lesions have been associated with administration of diltiazem, sulfonamides, quinidine, allopurinol, interferon-α, and phenytoin. , however, drug therapy as a cause of granulomatous hepatitis in dogs and cats has not been specifically reported. granulomatous lesions in the liver has been described in a small number of dogs with lymphangiectasia, lymphosarcoma, and histiocytosis. many cases of granulomatous hepatitis are idiopathic. hepatic lipogranulomas ("fatty cysts"), which are often found in dogs with congenital portosystemic shunt, are aggregates of pigment-laden foamy macrophages and should not be confused with granulomatous hepatitis. clinical findings with granulomatous hepatitis are highly variable, depending on the underlying cause. when granulomatous hepatitis is identified on liver biopsy, special stains for fungal and mycobacterial organisms should be performed. other diagnostics to either identify an organism (cytology, culture, fecal exam, pcr) or detect antibodies against the organism (serology) vary widely with the underlying agent (see table - ). if a cause cannot be found after a thorough diagnostic evaluation, consideration should be given to presumptive treatment for undiscovered infectious agents such as atypical mycobacteria, bartonella spp., or systemic fungal infection. corticosteroids or other immunosuppressant agents should only be used when diagnostic testing and empirical treatment have been unsuccessful, as steroid-induced immunosuppression may exacerbate an underlying infection. eosinophilic hepatitis occurs rarely in dogs and cats. potential causes include visceral larval migrans (toxocara), schistosomiasis, liver fluke infections, sarcocystis canis, and possibly, fungal infections (see table - ). with parasitic causes, eosinophils are often located at or near the site of the parasitic lesion in the liver. dogs and cats with systemic allergic, parasitic (heartworms), or hypereosinophilic syndromes, may also have scattered eosinophils in the liver, a variant of nonspecific reactive hepatitis. hepatic druginduced liver injury should also be considered. phenytoin and minocycline are associated with eosinophilic infiltrates in humans with drug-induced liver injury. , potentiated sulfonamides have been suggested to cause drug-induced eosinophilic hepatitis in dogs, although a more typical pattern is acute hepatocellular necrosis or a cholestatic hepatopathy. when eosinophilic infiltrates are identified, efforts should be directed at diagnosing parasitic causes (fecal, (increased alt and alp activity occurred in less than % of cats). other potential biochemical findings include hyperglobulinemia, mild hyperbilirubinemia, and hypoglycemia (sepsis). laboratory abnormalities may also reflect the associated disease processes (e.g., hyperglycemia with diabetes mellitus, increased pancreatic lipase immunoreactivity with acute pancreatitis). if an abscess ruptures, cytology of the abdominal infusion reveals septic suppurative inflammation. abdominal radiographs may be normal or reveal hepatomegaly, hepatic mass lesion, or decreased abdominal detail or effusion associated with secondary peritonitis. with proliferation of gas-producing organisms, radiolucent areas may be seen in the liver. ultrasonographic examination permits earlier detection of hepatic abscesses. ultrasonographically, a liver abscess appears as a hypoechoic or anechoic structure with irregular, hyperechoic margins. , the ultrasonographic pattern is similar to that seen with hepatic hematomas, cysts, neoplasia, and biliary cystadenoma. gas may be seen within the abscess. if abscess rupture has occurred, concurrent abdominal effusion may be detected. additional ultrasonographic findings may reflect associated disorders such as pancreatitis, cholecystitis, or pyelonephritis. ultrasound-guided fine-needle aspiration of a suspected liver abscess can be safely performed to obtain samples for cytology and culture to confirm the diagnosis. if ultrasonography is not available, the diagnosis of hepatic abscesses is usually established during exploratory laparotomy (or at necropsy). an attempt should be made to isolate and identify the organism(s) associated with abscessation so that appropriate antibiotic therapy can be instituted based on sensitivity testing. aerobic and anaerobic cultures can be performed on abscess contents (by fine-needle aspiration), abdominal exudate, blood or hepatic tissues. treatment of hepatic abscesses consists of surgical resection or drainage of focal lesions, administration of appropriate antibiotics, correction of associated fluid, electrolyte, and acid-base imbalances, and identification and treatment of any underlying disease process. treatment of large unifocal hepatic abscesses has typically involved surgical resection of affected tissue, which may necessitate partial or full lobectomy. , if perforation and peritonitis are present, surgical abdominal drainage and lavage are indicated. ultrasound-guided percutaneous drainage of a solitary abscess may resolve the abscess or allow stabilization until surgical resection can be performed. the successful management of focal hepatic abscesses (up to cm in diameter) by ultrasound-guided percutaneous drainage and alcoholization has been described in five dogs and one cat. broad-spectrum combination antibiotic therapy (directed toward both aerobic and anaerobic bacteria) should be initiated as soon as cultures have been obtained. results of a gram stain on the exudate may provide preliminary information as to type of organism and guide the empirical choice of potentially effective antibiotics. recommendations for broad-spectrum antimicrobial coverage of hepatobiliary infections include either a fluoroquinolone combined with amoxicillin/clavulanate or a fluoroquinolone combined with penicillin and metronidazole, until culture results are available. the dose of metronidazole should be adjusted in animals with hepatic dysfunction ( . mg/kg po q - h). antibiotic therapy should be continued for at least to weeks. response to treatment can be monitored with serial ultrasound examinations and repeated blood work. historically, hepatic abscesses have carried a grave prognosis, with an overall reported mortality rate of approximately % in dogs case series (see chapter ) . breeds of dogs at increased risk for chronic hepatitis include the bedlington terrier, , west highland white terrier, , doberman pinscher, [ ] [ ] [ ] american and english cocker spaniel, [ ] [ ] [ ] [ ] [ ] skye terrier, dalmatian, labrador retriever, [ ] [ ] [ ] and english springer spaniel. unfortunately, with the exception of hereditary copper-associated liver disease in bedlington terriers, information is lacking for most of the breed-related disorders. female dogs appear to be at increased risk in some studies, , while others report that male and female dogs are equally affected. , within particular breeds, sex differences have been noted (female doberman pinschers, labrador retrievers, and english springer spaniels; male cocker spaniels). , , , , dogs diagnosed with chronic hepatitis are generally to years of age, but adult dogs of any age (or breed) can be affected. , , etiology and pathogenesis ideally, canine chronic hepatitis should be classified on an etiologic basis. however, with the exception of copper-associated liver disease in bedlington terriers, the cause, pathogenesis, natural history, optimal treatment, and prognosis of these disorders are unknown (table - ) . idiopathic chronic hepatitis is the most common clinical diagnosis. , , , infectious causes. viral infections are a common cause of chronic hepatitis in humans, but are not currently recognized as an important etiology in dogs. in humans, viruses have the potential heartworm test; see table - ), systemic eosinophilia, and hypersensitivity reactions. if no specific cause can be determined, empirical treatment with fenbendazole should be considered, followed by corticosteroid therapy as described for idiopathic chronic hepatitis. the term nonspecific reactive hepatitis is used to describe the slight to moderate widespread inflammatory infiltrates of the liver that occur secondary to a spectrum of extrahepatic disease processes. lesions of nonspecific reactive hepatitis are associated with febrile and inflammatory disorders, especially those involving the gi tract and pancreas, or they may represent residual evidence of a previous intrahepatic inflammatory disorder. inflammation occurs in portal or parenchymal areas and necrosis is absent. neutrophils predominate with acute extrahepatic disorders, whereas mononuclear inflammation occurs with chronic extrahepatic disorders or residual hepatic inflammation. the liver may be secondarily affected by systemic disorders because of changes in liver blood flow, portal blood delivery of bacteria, drugs, hormones, cytokines, or other substances from the gi tract, or activation of intrahepatic kupffer cells (monocyte-macrophage system) involved in the hepatic immune response. it may be challenging to differentiate nonspecific reactive hepatitis from resolving acute hepatitis or mild chronic hepatitis, without supportive clinical information. clinical signs in dogs and cats with nonspecific reactive hepatitis are usually referable to the extrahepatic disorder. liver enzyme elevations (alt-two times the upper limit of normal; alpthree-to fourfold increases) are common, thus mimicking primary hepatic disease. however, tests that reflect liver function, including serum bile acids, are usually normal. it is important to consider extrahepatic disorders that can secondary affect the liver, prior to focusing on primary hepatic disease. treatment is directed at the underlying extrahepatic disorder. chronic hepatitis, a heterogeneous group of inflammatorynecrotizing diseases of the liver, occurs commonly in dogs, but is rare in cats. cholangitis, which is inflammatory liver disease that targets the biliary tract, rather than hepatocytes, is more common in cats but also occurs in dogs.the term chronic hepatitis, rather than chronic active hepatitis or chronic persistent hepatitis, is recommended. , if the etiology is known, it should be included as an adjective, such as "drug-induced chronic hepatitis," or "copper-associated chronic hepatitis"; otherwise, it is considered "idiopathic chronic hepatitis." chronic hepatitis in dogs is defined based on histopathologic features of hepatocellular necrosis or apoptosis associated with inflammation and evidence of regeneration and fibrosis. lymphoplasmacytic inflammation is characteristic, but a neutrophilic component may be present. the histopathologic features of chronic hepatitis are similar, regardless of the underlying cause. chronic hepatitis has the potential to progress to cirrhosis. , recommendations have been made to include a clinical component to the definition of chronic hepatitis, such as documenting an increase in alt activity along with histologic evidence of hepatic inflammation for a minimum of months. however, many dogs with chronic liver disease are not clinically apparent until the advanced stages, so duration can be difficult to evaluate. the early stages may not be recognized unless biochemistries are monitored for hepatic injury. a familial predisposition to develop chronic hepatitis has been suggested by demographic studies, pathologic surveys, and clinical hepatitis (acute and chronic) accounted for one-third of all dogs with primary hepatitis. hepatic copper accumulation and hepatopathy have been described in cats but appears to be rare. , the severity of hepatic injury correlates with the amount of hepatic copper, but subcellular localization of molecules and the molecular association also plays a role. serum copper levels do not accurately reflect hepatic copper content and quantitative analysis of copper in the liver is required. hepatic copper concentration in normal dogs is between and µg/g dry weight (parts per million). , inflammatory hepatic injury does not consistently occur until copper concentrations exceed µg/g dry weight. , however, there may be breed variations; for example, in doberman pinschers hepatic inflammation is present with copper concentrations of less than µg/g. , transient acquired fanconi syndrome has been described in dogs with excess hepatic copper accumulation. , copper granules were demonstrated on renal biopsy in some but not all dogs. potential mechanisms for hepatic copper accumulation include primary metabolic defects in hepatic copper metabolism, cholestasis causing impaired biliary excretion of copper, and excess copper absorption. , a primary defect in hepatic copper metabolism occurs in bedlington terriers with a genetic mutation in the gene encoding the copper transport protein, commd (formerly murr ), resulting in a defect in biliary copper excretion. , in to cause hepatitis either because of a persistent hepatic infection or as a transient infection that triggers an immune response because of a cross-reaction between the virus and liver antigens. in an attempt to identify infectious causes of canine hepatitis, pcr screening of liver tissue was performed in dogs with various stages of hepatitis to look for canine adenovirus type , hepadnaviridae, hepatitis a virus, hepatitis c virus, hepatitis e virus, helicobacter spp., leptospira spp., and borrelia spp. based on negative results, the authors concluded that canine hepatitis is not typically caused by these infectious agents. however, dogs that are experimentally infected with canine adenovirus type i, but are partially immune, can develop chronic hepatitis that progresses to cirrhosis. the virus could not be detected beyond the first week postinfection, although the disease progressed over a period of months. canine adenovirus antigen has been demonstrated by immunohistochemical techniques in formalin-fixed liver sections from five of dogs with various hepatic inflammatory lesions, suggesting that canine adenovirus (cav- ) may play a role in spontaneous chronic hepatitis. in contrast, pcr and immunohistochemistry failed to detect canine adenovirus in liver tissue of dogs with chronic liver disease. whether cav- is a significant cause of chronic hepatitis under natural conditions is unknown. another proposed viral cause of chronic hepatitis and cirrhosis is the "canine acidophil cell hepatitis virus," reported from great britain in the s. , this transmissible agent, most likely a virus, is distinct from cav- . it was transmitted experimentally by subcutaneous injection of serum or liver extracts from affected dogs, resulting in experimentally induced acute and chronic hepatitis. no further studies have been published to clarify the nature of this infectious agent or the associated hepatitis. canine leptospirosis is typically associated with acute cholestatic hepatic disease and acute renal failure. however, persistent infection can cause chronic hepatitis in the absence of azotemia. , leptospira serovar grippotyphosa was incriminated as a cause of chronic hepatitis in a kennel of american foxhounds, based on serologic evidence and demonstration of spirochetes in the liver. leptospira serogroup australis (serovars australis, bratislava, and muenchen) infection was suspected to cause chronic hepatitis in young beagle dogs in a breeding colony routinely vaccinated against leptospirosis serogroups canicola and icterohaemorrhagica. canine leishmaniasis has been associated with histologic evidence of chronic hepatitis, but clinical features suggestive of hepatic involvement (hepatomegaly, ascites, or icterus) were absent. histologic findings revealed granulomatous hepatitis in most dogs, but some dogs had marked portal infiltration with lymphocytes and plasma cells, and mild portal fibrosis. leishmania amastigotes were routinely identified in macrophages in liver or other affected tissues. bartonella clarridgeiae dna was amplified from a liver biopsy of a doberman pinscher with copper-associated chronic hepatitis, although the significance of this finding is unclear. copper accumulation. copper is an essential trace element in diets and is required for a number of physiologically important enzymes. cells have highly specialized and complex systems for maintaining intracellular copper concentrations. at toxic concentrations, free intracellular copper initiates oxidative damage causing hepatocellular necrosis and inflammation. , normal copper metabolism has been reviewed in detail elsewhere. , copper accumulation in the liver can be associated with significant hepatic injury resulting in acute hepatitis, chronic hepatitis, and cirrhosis ( figure - ) . , , it is one of the few well-documented causes of canine chronic hepatitis. in one study, copper-associated a b liver of dogs but was not identified in any control samples from healthy livers. none of the dogs had decreased serum levels of α antitrypsin. positive α -antitrypsin staining was a more consistent finding in english and american cocker spaniels with chronic liver disease, than in other breeds. the authors concluded that accumulation of α -antitrypsin might play a role, but it could not be determined if it was the cause or a result of chronic liver disease. drugs and toxins. drug or toxin exposure is a potential cause of canine chronic hepatic disease. drugs that have been incriminated include anticonvulsants (phenobarbital, primidone, phenytoin), oxibendazole-diethylcarbamazine, lomustine, and possibly carprofen. [ ] [ ] [ ] [ ] chronic hepatitis and cirrhosis from long-term phenobarbital therapy is most widely recognized. , exposure to aflatoxin from contaminated commercial dog food is usually associated with alf, but low-level long-term exposure in dogs can result in chronic hepatic injury (biliary hyperplasia, fibrosis, nodular regeneration). a breeding colony of german shepherd dogs developed chronic hepatitis and cirrhosis that was suspected (but never confirmed) to be a result of exposure to a porphyrinogenic substance, based on the finding of aggregates of crystalline pigments with orange birefringence with polarized light. early recognition of drug-or toxin-induced chronic hepatic injury requires biochemical monitoring of liver enzymes, as dogs are clinically asymptomatic in the early stages. autoimmune/immune mechanisms. autoimmune hepatitis has not been documented in dogs. however, some dogs with chronic hepatitis appear to respond to corticosteroid therapy and thus may correspond to autoimmune hepatitis in humans. autoimmune hepatitis in humans is a progressive chronic hepatitis of unknown cause that is believed to occur when an environmental agent (viruses, medications) triggers a cascade of t-cell-mediated events directed at liver antigens, in a genetically predisposed individual. women are more commonly affected than men. hyperglobulinemia is a common finding. an infectious cause is difficult to document, as exposure may have occurred many years prior to the overt autoimmune disease. certain drugs may induce or unmask an autoimmune hepatitis, or simply cause hepatocellular injury that mimics autoimmune hepatitis. an autoimmune component to doberman pinscher hepatitis has been speculated, because of the breed's predisposition, high female predominance, and the finding that expression of mhc class ii antigens on hepatocytes of affected dogs correlates with degree of inflammation and decreases after treatment with prednisolone. dogs with chronic hepatitis may have concurrent disorders associated with immune aberrations (immune hemolytic anemia, hypothyroidism, atopy, glomerulonephritis), but whether this is coincidental or indicative of the presence of multiple immune disorders as seen with autoimmune hepatitis in humans is unknown. , , autoimmune hepatitis in humans is diagnosed when other causes of acute or chronic hepatitis have been excluded and serum autoantibodies (antinuclear, antismooth muscle, antibody to liver/ kidney microsomes type , antibody to liver cytosol type ) are detected. a number of studies have evaluated the role of liverassociated antibodies and cell-mediated response in dogs with chronic hepatitis, but none answers the question of whether the immune response is the primary cause of the hepatitis or a secondary phenomenon. twenty-four dogs with chronic hepatitis were evaluated for circulating autoantibodies (against cell nuclei, smooth muscle, liver membrane, and mitochondria) by indirect immunofluorescence. antibodies to cell nuclei and liver membranes were the early stages, copper is sequestered in hepatic lysosomes and hepatic damage is minimal. however, with progressive accumulation of copper, hepatic injury becomes significant. the average copper concentration in bedlington terriers with chronic hepatitis is approximately µg/g dry weight and values up to , µg/g dry weight have been reported. , inherited copper-associated liver disease is also described in the west highland white terrier, skye terrier, doberman pinscher, dalmatian, and labrador retriever, but with the possible exception of dalmatians, the hepatic copper levels are much lower than in bedlington terriers. [ ] [ ] [ ] , , the pathogenesis of copper accumulation and the relationship to chronic liver disease in these breeds is poorly understood. it seems likely that these breeds have a hereditary disorder of copper handling, but it is unlikely to be the same as described for the bedlington terrier. hepatic copper accumulation in the liver may also be a consequence rather than the cause of chronic hepatitis. because copper is normally excreted in the bile, chronic cholestasis and impaired bile flow can result in secondary copper accumulation. , secondary copper accumulation is predominantly periportal and is usually less than µg/g dry weight. , the effect of cholestasis on hepatic copper content was evaluated in three groups of dogs: bedlington terriers with copper toxicity, dogs with extrahepatic biliary obstruction (the prototype example of a cholestatic disorder) and chronic hepatitis in breeds not known to be at risk for copper-associated liver disease. hepatic copper content was evaluated by a semiquantitative method based on copper staining of liver tissue with rubeanic acid, using a scale of (no copper) to . copper staining revealed absent to mild increases (scores of to +) in dogs with biliary obstruction and chronic hepatitis when compared with bedlington terriers (scores of +). it was concluded that copper scores of + or higher were suggestive of a primary copper storage disease. unfortunately, quantitative copper analysis was not evaluated. markers of oxidative injury and altered defense mechanisms were similar in the three groups, consistent with the concept that copper, inflammation, and cholestasis can all contribute to oxidative injury. high dietary copper intake appears to be an unlikely explanation for hepatic copper accumulation and liver disease in dogs. however, the copper content of commercial dog foods ranges from to mg/kg dry matter, which is relatively high compared with recommended minimum daily copper requirements in dogs. there is speculation that the recent increase in pathologically elevated hepatic copper concentrations (specifically evaluated in labrador retrievers), may coincide with a pet food industry recommendation to replace cupric/cuprous oxide in feed formulations because of its low bioavailability. many dogs with copper-associated chronic hepatitis also have increased hepatic iron concentrations. hepatic iron accumulation usually correlates with degree of inflammation. , whether iron, as an oxidant, interacts with copper to contribute to lesions seen in copper-associated hepatitis remains to be determined. α -antitrypsin deficiency. inherited α -antitrypsin deficiency is a well-recognized cause of chronic hepatitis and cirrhosis in humans, and may play a role in the pathogenesis of chronic hepatitis in some dogs. α -antitrypsin is a circulating protease inhibitor that is synthesized and secreted by the liver. α -antitrypsin deficiency in affected humans results in defective formation and impaired hepatic secretion of α -antitrypsin, resulting in hepatic accumulation of α -antitrypsin and hepatic injury. serum levels of α -antitrypsin are typically low. in a study of dogs with chronic liver disease, α antitrypsin was detected by immunohistochemical staining in the early stages of chronic hepatitis, ultrasonography of the liver may be normal or reveal nonspecific changes in echogenicity. when chronic hepatitis has advanced to cirrhosis, potential ultrasonographic findings include microhepatia, irregular hepatic margins, focal lesions representing regenerative nodules, increased parenchymal echogenicity associated with increased fibrous tissue, and ascites. splenomegaly and acquired psss may also be detected. a liver biopsy is essential for the diagnosis of chronic hepatitis. wedge biopsies are preferred over needle biopsies because they provide more tissue and are more likely to represent pathologic process(es) in the liver. when cirrhosis is present, laparotomy or laparoscopy often provide a better appreciation for the gross nodularity of the liver than can be ascertained from blind percutaneous needle biopsy (figure - ). chronic hepatitis is characterized histologically by moderate to severe inflammation (usually combinations of lymphocytes and plasma cells) associated with piecemeal necrosis. piecemeal necrosis, also referred to as interface hepatitis, is necrosis involving the layer of hepatocytes adjacent to the portal tract or "limiting plate." the term bridging necrosis is used when necrosis and inflammation dissect across the hepatic lobule from portal areas to central veins or to adjacent hepatic lobules and suggests a severe form of chronic hepatitis. histopathologic evaluation of the liver should not only consider etiology, but the pathologist should also comment on the activity (amount of inflammation, extent of apoptosis and necrosis) and the stage of disease (extent and pattern of fibrosis; architectural distortion suggestive of cirrhosis). biopsies from dogs with chronic hepatitis should routinely be evaluated for copper accumulation. on hematoxylin and eosin staining, excess copper appears as golden brown refractile granules. histochemical stains, such as rhodanine or rubeanic acid, can be used to semiquantitatively evaluate for copper in the liver (see figure - ) . these stains consistently detect copper when amounts exceed µg/g dry weight. values obtained by quantitative copper analysis have a strong correlation with the number and size of granules seen with histochemical stains within the range of to µg/g of liver tissue. zonal distribution of copper detected, but were also found in dogs with other types of hepatic disease, suggesting a nonspecific secondary response. patterns of circulating autoantibodies found in dogs differed significantly from those found in humans with chronic liver disease. in another study, serum anti-liver-membrane-protein antibody-positive dogs ( : to > : ) had higher alt activity, total bilirubin concentration, and more severe hepatic lesions than did anti-liver-membraneprotein antibody-negative dogs, but it was not determined whether autoantibodies were primary or secondary. cd + lymphocytes are the most common hepatic lymphoid cells in dogs with chronic hepatitis and are associated with hepatic necrosis, - but also account for % of hepatic lymphocytes in normal dogs. historical and physical examination findings in dogs with chronic hepatitis are indicative of chronic hepatic disease, and are similar regardless of the underlying cause. signs are often initially vague and nonspecific, such as anorexia, lethargy, vomiting, diarrhea, weight loss, pu, and pd. , with increased severity of hepatic dysfunction, signs of overt liver failure develop, such as ascites, jaundice, and he. the presence of ascites and he suggest that chronic hepatitis has progressed to cirrhosis, and ascites is a negative prognostic indicator. , melena associated with gastroduodenal ulceration or coagulopathy is also more likely with advanced liver disease. because of the large functional reserve capacity of the liver, the onset of signs may appear very recent, initially suggesting an acute rather than chronic hepatic disorder. clinicopathologic features that support chronicity include poor body condition, ascites, microhepatia, hypoalbuminemia, and histologic evidence of fibrosis. in the early (subclinical) stages, dogs are asymptomatic and only identified by biochemical screening for liver enzyme elevations. increased serum alt activity, reflecting ongoing hepatic injury, is reported in % to % of dogs with chronic hepatitis. , serum alt activity may exceed times the upper normal limit. periods of normal alt activity may reflect cyclic disease activity and the varying severity of necrosis. serum alp activity is also commonly increased, but the magnitude of the increase is generally lower than seen with alt activity. when chronic hepatitis advances to cirrhosis, liver enzyme activity may be normal, indicating decreased viable parenchymal mass. abnormalities in biochemical tests such as hyperbilirubinemia, hypoalbuminemia, decreased blood urea nitrogen (bun), hypoglycemia, and increased sba indicate hepatic dysfunction and a more advanced stage of disease. hyperglobulinemia can be seen in dogs with cirrhosis, but it remains to be determined whether this corresponds with increased autoantibodies as occurs in humans with autoimmune hepatitis, or whether it reflects nonspecific systemic antibody production in response to antigens from the portal blood which bypass the liver through acquired psss. mild nonregenerative anemia may be a reflection of chronic disease. regenerative anemia can occur from blood loss secondary to a coagulopathy or bleeding gi ulcers. copper-associated hemolytic anemia has only been documented in bedlington terriers. abnormal hemostatic parameters (prolonged aptt and pt) are indicative of severe hepatic dysfunction or dic. a prolonged pt and thrombocytopenia may be negative prognostic indicators. , analysis of ascitic fluid reveals a transudate or modified transudate. , abdominal radiographs are unremarkable except when advanced stages of disease are accompanied by microhepatia or ascites. in the kg every hours is most often recommended for treatment of canine chronic hepatitis. complications of corticosteroid therapy include gi bleeding (which may precipitate he), secondary infections, iatrogenic cushing disease, and worsening of ascites. dexamethasone ( . mg/kg po q h) may be preferred in dogs with ascites or edema, because it lacks mineralocorticoid activity, which could exacerbate these signs. prednisone is often used in combination with azathioprine, especially if side effects of prednisone become objectionable. azathioprine is an antimetabolite with antiinflammatory and immunemodulating effects, and is commonly used in combination with prednisone in humans with autoimmune hepatitis. the dose of azathioprine in dogs is to mg/kg/day po every hours for to weeks, then tapered to every hours for maintenance therapy. prednisone ( . to . mg/kg/day) is given on the alternate days. because azathioprine may cause bone marrow suppression and acute hepatotoxicity, the complete blood count and biochemical profile should be monitored. antiinflammatory agents and immunosuppressive drugs are discussed in more detail in chapters and , respectively. because glucocorticoids increase liver enzyme activity (especially serum alp activity), response to therapy is best evaluated by a followup liver biopsy performed to months after starting therapy. if glucocorticoid therapy is eventually discontinued, clinical and biochemical parameters should be periodically monitored to detect a relapse. dogs with hepatic copper concentrations greater than µg/g, should be treated with the copper chelator penicillamine at a dose of to mg/kg po every hours. treatment usually requires months to years to produce significant decreases in hepatic copper. a mean decrease in copper of approximately µg/g was achieved in bedlington terriers treated for months. dogs with secondary copper accumulation appear to respond more rapidly, possibly because hepatic copper content is lower in these breeds. doberman pinschers with subclinical hepatitis treated with penicillamine for months had a mean decrease in copper from µg/g to µg/g. penicillamine has additional effects beyond copper chelation, which may be beneficial in dogs with chronic hepatitis, including inhibition of collagen deposition, stimulation of collagenase activity, immunosuppression, and immunomodulation. common side effects of penicillamine therapy include anorexia, nausea, and vomiting, which can be minimized by giving the medication with a small amount of food. the copper chelator, trientine ( to mg/kg po q h), is also effective for reducing hepatic copper concentrations. it has fewer side effects than penicillamine and is effective in dogs with hemolytic anemia caused by copper release from necrotic hepatocytes. iatrogenic copper deficiency (microcytosis and hepatic dysfunction) has been described in a dog treated with long-term copper chelation therapy (trientine) and a copper-restricted diet. decisions on duration of chelator therapy are based on followup liver biopsies with periodic monitoring of quantitative hepatic copper content. oral zinc salts can be used for maintenance therapy after copper chelation, or as initial therapy in dogs with hepatic copper concentrations between µg/g dry weight and µg/g dry weight. zinc supplementation is typically used in conjunction with dietary copper restriction. zinc decreases intestinal copper absorption by inducing the intestinal copper-binding protein, metallothionein, within intestinal epithelial cells, which preferentially binds dietary copper and prevents its absorption. zinc acetate is given at a dose of mg po bid for to months, then at a maintenance dose of mg po bid. a minimum of months of zinc therapy is required before copper uptake from the intestinal tract is blocked. accumulation should be noted, as copper accumulation starting in the centrilobular area is more likely with a primary metabolic defect in copper metabolism. copper granules can also be detected on cytology of hepatic aspirates or impression smears stained with rhodanine or rubeanic acid. quantitative analysis for copper, by atomic absorption analysis on fresh hepatic tissue, is the definitive method to document increased hepatic copper content. needle core biopsy specimens may not be reliable for metal analysis, as copper and iron values are consistently lower in needle core versus wedge biopsy samples. formalin-fixed tissues should be avoided, because formalin may contain copper or leach copper from the tissue. hepatic copper can be reliably determined retrospectively on deparaffinizedarchived liver biopsy specimens. once chronic hepatitis has been confirmed, a careful consideration of known causes of chronic hepatitis is essential (see table - ) . findings that would support a primary metabolic defect in copper metabolism include a previously recognized breed predisposition, copper accumulation that precedes cholestasis or inflammation, centrilobular (zone ) distribution of copper, histochemical score for copper of + or greater, or quantitative copper measurements that exceed µg/g dry weight. , , special stains of the liver should be requested to evaluate for infectious agents such as leptospirosis; serum antibody titers for leptospirosis may be indicated. a history of chronic drug therapy should be sought, especially long-term anticonvulsant therapy or other drugs listed in table - . recommendations for treatment of chronic hepatitis are empirical at best, because of the lack of controlled therapeutic studies on a well-defined population of dogs with this disorder. if a probable cause or category of injury can be determined, then specific treatment is directed at the primary etiology, for example, discontinuation of phenobarbital, treatment of leptospirosis, or chelation of hepatic copper with penicillamine. in most cases, specific therapy will be unavailable. treatment of chronic hepatitis in dogs has traditionally centered on the use of corticosteroids, presuming that, as in humans with the autoimmune form of hepatitis, immunologic mechanisms (inflammatory cells and mediators, local cytokines), contribute to hepatic inflammation and progression to cirrhosis. corticosteroids have antiinflammatory, immune-modulating, and antifibrotic effects, which may be beneficial in chronic hepatitis. a large retrospective study suggested that corticosteroid therapy at initial immunosuppressive doses ( . mg/kg/day; eventually tapered to . mg/kg/day) improved survival in dogs with chronic hepatitis. however, many concurrent drugs were given and, undoubtedly, a heterogeneous group of disorders were included under the diagnosis of "chronic hepatitis." corticosteroid therapy appears warranted in dogs with histologic features of active inflammation and persistent increases in serum liver enzyme activity, for which known causes of chronic hepatitis (including infectious causes) have been excluded. , glucocorticoid therapy is not indicated for treatment of chronic hepatitis caused by drug therapy, infectious agents, or primary hepatic copper accumulation. the optimal dose and duration of corticosteroid therapy for treatment of canine chronic hepatitis is unknown, including whether immunosuppressive doses are required, or whether lower, anti-inflammatory levels would suffice. even in humans with autoimmune hepatitis, immunosuppressive doses of corticosteroids may not be required. prednisone (or prednisolone) at an initial dose of to mg/kg/day po and then gradually tapered to . to . mg/ is correlated with the amount of hepatic copper. hepatic injury is believed to occur when progressive copper accumulation exceeds the storage capacity of the lysosomes; copper is released to the cytoplasm, damaging mitochondria, initiating lipid membrane peroxidation, and eventually causing cell death. affected dogs can be asymptomatic (in the early stages) or show signs of acute hepatic necrosis, chronic hepatitis, or cirrhosis. in young dogs, copper accumulates in centrilobular (zone ) hepatocytes and is sequestered in hepatic lysosomes. during this first stage, copper concentrations are between and µg/g, dogs are asymptomatic, biochemical testing is within normal limits, and liver biopsy findings are unremarkable. in the second stage, when hepatic copper concentrations are between and µg/g, copper granules are also found in midzonal (zone ) and periportal (zone ) hepatocytes. although dogs are still asymptomatic, focal hepatic inflammation (centrilobular mixed cell foci, with necrotic hepatocytes, lymphoplasmacytic inflammation, and copper-laden macrophages) is seen on biopsy, and increased serum alt activity reflects hepatocellular injury. in the most advanced stage, when hepatic copper concentration exceeds µg/g, morphologic changes reveal chronic hepatitis that may progress to cirrhosis, and clinical and biochemical evidence of liver disease become apparent. clinical signs include anorexia, lethargy vomiting, and weight loss. with progression to cirrhosis, findings of jaundice, ascites, and he may develop. biochemical findings vary with the stage of disease. increased serum alt activity is the most sensitive laboratory indicator, although findings will be normal in young dogs in stage i, because of the lack of hepatic inflammation. other serum biochemical abnormalities typical of chronic hepatic dysfunction eventually develop. in some cases, acute hepatic necrosis and alf occur. hepatocellular necrosis may be associated with release of copper from necrotic hepatocytes, resulting in hemolytic anemia. during episodes of hemolysis, plasma copper levels are increased; other findings include low packed cell volume, hemoglobinemia, and hemoglobinuria. liver biopsy and quantitative analysis of hepatic copper concentrations is required for definitive diagnosis and staging of the disease. serum copper or ceruloplasmin concentrations are not helpful to make a diagnosis. liver biopsies should be performed in all bedlington terriers considered for breeding, in order to identify and remove affected dogs from breeding programs. screening of asymptomatic dogs with a liver biopsy at months and months of age can determine if an affected dog is homozygous or heterozygous (a carrier). affected dogs (both homozygous and heterozygous) typically have increased hepatic copper by months of age. however, copper concentrations in dogs who are carriers (heterozygous) return to normal by year of age, whereas copper concentrations in homozygous dogs continues to increase. selective breeding programs in the netherlands has decreased the prevalence of bedlington terrier copper-associated liver disease from % ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to % ( - ) . dna testing of bedlington terriers is available from vetgen (www.vetgen.com). this assay evaluates a linkage-based dna marker (co , allele ) that is located in the chromosome close to the gene for copper toxicity. the test can identify normal, affected, and carrier dogs with % accuracy. however, the marker can only be relied on for diagnosis of the genetic status of an individual dog when supported by a pedigree study. , significant discrepancies were reported in bedlington terriers, when comparing results of liver biopsy and the dna marker. this may be attributed to different subpopulations of bedlington terriers with variations in the disease-causing mutation of the commd gene or a second mutant copper gene could play a role. liver biopsy for quantitative zinc administration should be separated from meals by at least hour and should theoretically not be prescribed at the same time as a copper chelator. serum zinc concentrations should be monitored to achieve a level of to µg/dl. zinc concentrations greater than µg/dl may be toxic (hemolytic anemia). low-copper diets are most beneficial for managing early (subclinical) copper accumulation in dogs affected with primary metabolic defects in hepatic copper metabolism. feeding a low-copper diet decreases hepatic copper content in labrador retrievers with subclinical copper-associated liver disease. additional treatment with zinc does not appear to increase the copper-lowering effect of dietary management. foods containing large amounts of copper (liver, other organ meats, shellfish, eggs, bean/legumes, chocolate, nuts, cereals, and copper-containing vitamin supplements) should be avoided. because oxidative stress is a significant mechanism for hepatic damage associated with copper accumulation and necroinflammatory hepatic disorders, , antioxidant therapy with vitamin e ( to iu/kg/day), or same ( mg/kg/day) has been advocated. other cytoprotective agents such as silymarin (milk thistle) and ursodeoxycholic acid may also be beneficial. chapters and discuss cytoprotective agents used in the treatment of hepatobiliary disease in detail. when end-stage cirrhosis is diagnosed, treatment is mainly supportive, as cirrhosis itself is essentially irreversible. measures should also be instituted to control the complications of chronic liver failure, such as ascites, he, gastroduodenal ulcers, and coagulopathy, which are discussed in more detail in "complications of liver disease" section. the response to treatment of chronic hepatitis is variable, which is not unexpected as it is likely a heterogeneous group of diseases. some dogs can eventually be taken off medication and remain in remission, but more often, therapy must be continued indefinitely. other dogs fail to respond, especially those that have advanced disease with cirrhosis. , in one study, the estimated median survival time in dogs with idiopathic chronic hepatitis was months (range: to months) and in dogs with copper-associated chronic hepatitis it was months (range: to months). mean survival time in dogs with cirrhosis was week. bedlington terriers develop chronic hepatitis and cirrhosis from copper toxicity, as a consequence of an inherited metabolic defect resulting in impaired biliary copper excretion. , , the disorder is transmitted by autosomal recessive inheritance. the gene responsible for this metabolic disorder is commd , which is different than that described for copper toxicity (wilson disease) in humans, in which the gene involved is atp b. there is no gender predilection. at one time, it was speculated that as many as % of the breed might be affected. hepatic copper concentration in normal bedlington terriers ranges from to µg/g with a mean of ± µg/g dry weight. bedlington terrier copper-associated liver disease is associated with progressive, hepatic copper accumulation (copper levels of up to , µg/g) unless treatment is instituted. the lowest hepatic concentrations of copper are found in the youngest dogs and concentrations increase with age, peaking at around years. copper content usually declines thereafter in affected dogs, but not to normal. this decline may be a result of replacement of copper-containing hepatocytes by fibrous tissue or regenerative nodules that do not contain copper. the severity of hepatic disease recent efforts have focused on identification of affected doberman pinschers prior to advanced hepatic disease. in finland, a survey of randomly selected, clinically healthy doberman pinschers, revealed that . % of dogs had increased alt activity, and . % had hepatitis (parenchymal and portal mononuclear inflammation and positive stains for copper). , the mean age of dogs with subclinical hepatitis was . years, compared with clinically affected dogs ( . years). the asymptomatic period lasted an average of months. the prevalence of subclinical doberman hepatitis was investigated in randomly selected year old doberman pinschers in the netherlands. subclinical hepatitis was identified in dogs ( females and three males); hepatic copper concentration was higher in dogs with hepatitis ( ± µg/g dry weight) than those without liver disease ( ± µg/g). serial liver biopsies over at least a -year period, revealed that hepatitis persisted only in dogs with copper levels greater than µg/g dry weight, and copper levels continued to increase in these dogs ( ± µg/g), supporting a relationship between copper, inflammation and hepatitis. it has also been proposed that hepatic copper is incidental to chronic hepatitis in this breed, based on the findings that five of doberman pinschers with chronic hepatitis had normal copper levels, and histologic changes were similar regardless of copper status. , , an immune-mediated mechanism has been suggested, based on the finding that expression of mhc class ii antigens on hepatocytes of dogs with doberman hepatitis was correlated with degree of inflammation. aberrant mhc class ii molecule expression on nonlymphoid cells could be a result of toxins, drugs, viral infection, or autoimmunity, and hepatocytes with mhc class ii expression might become a target as an antigen-presenting cell for cd + t cells. dogs treated with low-dose prednisolone ( . to . mg/kg/day) for to months had significantly decreased expression of mhc class ii antigens. chronic hepatitis should be suspected in any doberman pinscher (especially females) with clinical or biochemical evidence of hepatic disease. definitive diagnosis requires liver biopsy. other causes of chronic hepatitis should also be considered, since doberman pinschers appear to be at risk for drug-induced hepatitis. early detection of chronic hepatitis provides the best opportunity for treatment. it has been recommended that all doberman pinschers older than year of age be screened for alt activity. persistent increases in alt activity suggest further evaluation including liver biopsy is warranted. the magnitude of increased alt activity is not different between subclinical and clinically affected dogs. hyperbilirubinemia is suggestive of more advanced disease. , effective treatment for doberman pinschers with chronic hepatitis has not been established. however, a preliminary study showed that if diagnosed in the subclinical stage, treatment with penicillamine ( mg total dose po bid for months) lowered hepatic copper content and improved hepatic histopathology. traditionally, antiinflammatory or immunosuppressive drugs such as prednisone with or without azathioprine have been instituted. the efficacy of this treatment remains to be determined but generally, the response is poor if dogs are presented in advanced stages of liver failure. the use of ursodeoxycholic acid ( mg/kg po bid) deserves special consideration in this chronic cholestatic disorder, but has not yet been objectively evaluated. treatment of copperassociated hepatitis in doberman pinschers with advanced disease is usually unsuccessful. most dogs die within weeks to months. the prognosis appears more favorable if the disease is detected in the early stages, but the optimal therapeutic regimen remains to be determined. copper and morphologic examination remain the best option for diagnosis in the individual dog. a database for certification of bedlington terriers is maintained on the web site (www.caninehealthinfo.org) of the canine health information center, which is sponsored by the akc/canine health foundation and the orthopedic foundation for animals. affected bedlington terriers who are asymptomatic (copper > µg/g dry weight but less than µg/g dry weight) should have dietary copper restriction and zinc supplementation. bedlington terriers with copper accumulation (copper > µg/g dry weight) and chronic hepatitis should be treated with a copper chelator such as penicillamine or trientine. early diagnosis and treatment with either zinc or copper chelators will allow most dogs to lead a normal life. treatment of hemolytic anemia may require a blood transfusion. trientine dihydrochloride (but not penicillamine) may be effective in chelating circulating copper during a hemolytic episode. chapter discusses copper-chelating agents in more detail. doberman pinschers are at increased risk for the development of severe chronic hepatitis and cirrhosis. , , , doberman hepatitis accounted for % of all deaths in a dutch population of dobermans. middle-aged ( to years) female dogs are at increased risk, but males also may be affected. although a hereditary mechanism is suspected, the pathogenesis of this disorder is unclear. copper accumulation appears to be associated with hepatic damage, but the pathogenesis is different from the bedlington terrier disorder. immune mechanisms may also play a role. , many doberman pinschers are diagnosed in the advanced stages of hepatic failure. evidence of excessive bleeding (gingival bleeding, epistaxis, and melena) are common. signs of he often predominate in the terminal stages. common physical examination findings include ascites, jaundice, and weight loss. splenomegaly (associated with portal hypertension) is common. laboratory findings included increased alt and alp activity, hyperbilirubinemia, hypoalbuminemia, hyperammonemia, coagulopathy, and thrombocytopenia. typical histologic lesions include portal inflammation (lymphocytes, plasma cells, and macrophages), piecemeal necrosis, bridging necrosis, bile duct proliferation, and portal fibrosis. hepatic copper concentrations are increased in most affected dogs and are typically between and µg/g dry weight, although values as high as µg/g have been reported. , the significance of the increased hepatic copper concentration in this breed remains controversial. copper accumulation was originally attributed to secondary mechanisms, as doberman pinschers with advanced disease (chronic hepatitis and cirrhosis) have biochemical and histologic evidence of cholestasis. however, evaluation of affected dogs in the early (subclinical) stage, reveals that copper accumulation precedes cholestasis, , and decreased biliary excretion of radiolabeled copper has been documented. the hepatic distribution of copper and location of inflammation varies with the stage of disease. in the early stages, the copper (and focal inflammation) is centrilobular. , , as the disorder progresses, copper accumulation and inflammation are more pronounced in periportal regions and areas of bridging necrosis. although copper appears to be related to the hepatic inflammatory reaction, copper levels are typically less than µg/g dry weight, the minimum amount of copper that is believed to cause hepatocellular injury in bedlington terriers and west highland white terriers. , a primary copper retention disorder has been proposed, but the genes associated with bedlington copper toxicity (commd ) and wilson disease in humans (atp b) have been excluded. same, milk thistle), and symptomatic therapies that are not designed to lower hepatic copper. feeding a low-copper diet to labrador retrievers with hepatic copper accumulation (seven of dogs had varying degrees of hepatitis) was effective in decreasing hepatic copper concentrations, but severity of inflammation remained unchanged. additional treatment with zinc did not appear to increase the copper-lowering effect of dietary management. long-term survival appears variable. , dogs who died within months of diagnosis were more likely to have a prolonged pt and thrombocytopenia. dalmatians are reported to have acute hepatic necrosis, chronic hepatitis, and cirrhosis associated with increased hepatic copper concentrations. , cholestasis is not a prominent biochemical or histologic feature until later in the disease, suggesting that hepatic copper accumulation is more likely to be caused by a familial metabolic disorder rather than secondary to altered hepatic biliary copper excretion. most dogs presented initially with acute gi signs (anorexia, vomiting, and diarrhea). biochemical findings revealed markedly increased alt activity with lesser increases in alp activity. hyperbilirubinemia and hypoalbuminemia were seen with advanced disease. glucosuria (in the absence of hyperglycemia) and proteinuria were identified in some dogs. ultrasound findings were usually unremarkable. liver biopsy revealed piecemeal necrosis, bridging fibrosis, and inflammation (predominantly lymphocytes or neutrophils). the mean hepatic copper level was µg/g dry weight (normal < µg/g dry weight) with a range of to µg/g dry weight. in five of nine dogs, copper exceeded µg/g. rapid progression of the disease was characteristic. copper chelation therapy may be beneficial if diagnosed before advanced liver disease occurs. chronic hepatitis and cirrhosis associated with hepatic copper accumulation ( to µg/g dry weight) in genetically related skye terriers has been described. in the early stages, copper accumulation is absent, and biopsy findings indicate hepatocellular degeneration with cholestasis and mild inflammation. chronic lesions are associated with intracanalicular cholestasis, chronic hepatitis, and cirrhosis. skye terrier hepatitis is speculated to be a disorder of disturbed bile secretion with subsequent accumulation of copper. american and english cocker spaniels have an increased incidence of chronic hepatitis and cirrhosis. , the cause is unknown. hepatic copper accumulation does not appear to be a consistent feature. it is unclear whether accumulation of α -antitrypsin in hepatocytes, a well-recognized cause of cirrhosis in humans, is important in the pathogenesis. male cocker spaniels (average age: years) are at increased risk. , despite the chronicity and severity of the underlying hepatic lesions, most affected dogs have a short duration of clinical illness, usually less than weeks. ascites is the most consistent presenting complaint. profound hypoalbuminemia (mean: . g/dl) is a consistent laboratory finding. total serum bilirubin concentration is normal or only mildly increased, supporting that cholestasis is not a key feature of the disorder. ascitic fluid analysis is consistent with a transudate or modified transudate. on liver biopsy, hepatic lesions are consistent with chronic hepatitis and cirrhosis. treatment of cocker spaniels with chronic hepatitis consists of general supportive therapy for the complications of liver failure. corticosteroid therapy prior to progression to cirrhosis may west highland white terrier west highland white terriers are at increased risk to develop chronic hepatitis and cirrhosis. , , males and females are equally affected. the mode of inheritance for the familial copper-associated disorder has not been established. decreased biliary excretion of radiolabeled copper occurs in affected dogs. centrilobular (zone ) copper accumulation occurs during the first year of life, but rarely exceeds µg/g dry weight. in contrast to the bedlington terrier, west highland white terriers do not continuously accumulate copper over their lifetime; in fact, copper content may actually decrease with time. in one report of clinically normal west highland white terriers, most dogs had hepatic copper levels between and µg/g dry weight with normal liver biopsies. in west highland white terriers with chronic hepatitis, histologic lesions include multifocal hepatitis, subacute bridging necrosis, massive necrosis, and cirrhosis. the relationship of hepatic copper to chronic hepatitis in the west highland white terriers is unclear. there appears to be at least two types of chronic hepatitis. some dogs have copper-associated hepatitis with elevated copper content (> µg/g) and multifocal centrilobular hepatitis. copper concentrations do not usually exceed µg/g dry weight. lesions of chronic hepatitis can also be seen in the absence of substantial copper accumulation, and have been described as "idiopathic chronic hepatitis." quantitative copper analysis is necessary to determine if copper accumulation is a significant (> µg/g dry weight) contributing factor. if chronic hepatitis and cirrhosis are associated with increased hepatic copper content (> µg/g dry weight), treatment for hepatic copper accumulation should be instituted. mature west highland white terriers with chronic hepatitis and less than µg/g dry weight of copper may not require chelation therapy, as hepatic copper accumulation is not continuous throughout life. other therapeutic options for treatment of idiopathic chronic hepatitis, such as glucocorticoids, should be considered in these dogs. labrador retrievers are at increased risk for chronic hepatitis. , age at presentation ranges from . to years, with an average age of to years. [ ] [ ] [ ] a female predisposition was noted in two studies, , whereas in another study, males and females were equally affected. most affected dogs have increased hepatic copper, which has been described as centrilobular (zone ) or diffuse. [ ] [ ] [ ] copper concentrations exceeded µg/g dry weight in of dogs (mean copper: µg/g; range: to ; reference interval: to µg/g). most dogs also had elevated iron levels with a mean of µg/g (reference interval: to ). a genetic basis is suspected, based on the finding of increased copper concentrations in asymptomatic related dogs, but the genetic defect remains to be determined. a retrospective survey of hepatic copper content in labrador retrievers during two time periods ( - and - ) , revealed significantly higher copper concentrations in the more recent period both in dogs with chronic hepatitis and in control dogs; no difference in age or gender was noted. it was speculated that increased hepatic copper might reflect increased dietary copper bioavailability, because of pet food industry recommendations to replace cupric/cuprous oxide in feed formulations. treatment with penicillamine ( mg/kg po q h) appears to be effective in decreasing hepatic copper content and inflammation. , however, some dogs appear to respond to immunosuppressive (prednisone, azathioprine), supportive (ursodeoxycholic acid, clinical features of cirrhosis in dogs include ascites (portal hypertension, hypoalbuminemia), he (intrahepatic and extrahepatic portosystemic shunting of blood), and evidence of decreased hepatic function (hypoalbuminemia, increased sba, coagulopathy, hyperbilirubinemia). findings on hepatic ultrasonography (small nodular liver, splenomegaly, and acquired psss) are suggestive for cirrhosis, but liver biopsy is required for confirmation. because cirrhosis is essentially irreversible, treatment is mainly supportive, emphasizing measures that control complications of severe generalized liver failure, such as ascites, encephalopathy, gastric ulcers, coagulopathy, and infection (see "complications of liver disease" section). if clinical signs of liver failure are already present, the prognosis is poor. prevention of fibrosis, an important long-term goal, is best achieved by early specific treatment directed at the probable cause of injury (e.g., discontinuing a suspect drug, penicillamine for copper-associated liver disease, antiinflammatory drugs for idiopathic chronic hepatitis, surgical relief of extrahepatic biliary obstruction). many therapeutic agents used for treatment of liver disease, such as penicillamine, prednisone, azathioprine, milk thistle, ursodeoxycholic acid, and zinc, have potential antifibrotic properties, and are discussed in more detail in other chapters. colchicine, a microtubule assembly inhibitor which increases collagenase activity, has been recommended for treatment of hepatic fibrosis, but its effectiveness in dogs has not been critically evaluated. the recommended dose in dogs is . to . mg/kg/day po. reported side effects include nausea, vomiting, and diarrhea. bone marrow toxicity and myoneuropathy have been reported in humans. lobular dissecting hepatitis is a specific histologic form of cirrhosis seen in neonatal or young adult dogs. , , [ ] [ ] [ ] it is suggested to be a nonspecific response to a variety of hepatic insults. the age at presentation is younger than for dogs with either acute or chronic hepatitis. in affected dogs, the median age was months, with dogs ( %) being months or younger. , females appear to be at increased risk. , lobular dissecting hepatitis may occur in an isolated dog or in groups of dogs from the same litter or kennel. standard poodles may be at increased risk. , clinical features are those of advanced hepatic failure and portal hypertension. the most consistent clinical finding is ascites. liver enzymes are typically increased and hypoalbuminemia and increased sba concentrations are common. , liver biopsy is required for diagnosis and to differentiate it from other types of chronic hepatitis and cirrhosis. the lesion is characterized histologically by lobular hepatitis: inflammatory cells (lymphocytes, plasma cells, macrophages, and neutrophils) are scattered throughout the hepatic lobule rather than concentrated in periportal regions. bands of collagen and reticulin fibers dissect around single or small groups of hepatocytes and disrupt hepatic lobular architecture. copper stains are negative or moderately positive, consistent with secondary copper accumulation. specific treatment has not been reported, but general measures for management of chronic liver failure are appropriate. in a small group of dogs with lobular dissecting hepatitis, the mean survival time was approximately months, which was significantly shorter than for dogs with acute or chronic hepatitis. infection of the liver is an important cause of hepatic disease in dogs and cats. the liver may be the primary target of infection (e.g., infectious canine hepatitis, bacterial cholangitis, hepatic abscess) or be beneficial. the prognosis is poor and most dogs die within a month of diagnosis. a recent report described seven american cocker spaniels with histologic features resembling lobular dissecting hepatitis. males and females were equally affected. in contrast to previous reports of hepatitis in cocker spaniels, most dogs in this study improved with corticosteroid therapy. english springer spaniel a preliminary report has described chronic hepatitis in english springer spaniels from norway and the united kingdom. female dogs were overrepresented. copper does not appear to play a role. the prognosis appears to be poor, with most dogs dying to months after diagnosis. hepatic cirrhosis (end-stage liver disease), is characterized by fibrosis, regenerative nodules that alter liver architecture and intrahepatic (microscopic) psss (see figure - ) . hepatic fibrosis is not synonymous with cirrhosis. cirrhosis is common in dogs but less so in cats. cirrhosis can result from postnecrotic scarring after acute massive necrosis or from chronic hepatic injury caused by a variety of insults such as infection (e.g., leptospirosis, cav- ), hepatotoxins (e.g., copper, phenobarbital, aflatoxin), inflammation (chronic hepatitis), or hypoxia. the common denominator is hepatocyte death, which leads to repair by fibrosis and nodular regeneration. when cirrhosis is fully developed, the histologic features of the original inciting injury often are obscured by the cirrhotic changes. substantial hepatic fibrosis (without "cirrhosis") can be seen with long-standing extrahepatic biliary obstruction, noninflammatory fibrosis, congenital hepatic fibrosis (a disorder of biliary system development), and congenital portal vein hypoplasia. , - a unique form of macronodular cirrhosis, characterized by noninflammatory regenerative hyperplastic nodules and diffuse vacuolar hepatopathy, is seen in dogs with hepatocutaneous syndrome (superficial necrolytic dermatitis). hepatic fibrosis was once considered irreversible, but is now recognized to be a dynamic process, which exists in a balance between synthesis and degradation. a better understanding of the underlying mechanisms may provide potential therapeutic targets. the major fibrogenic cell in the liver is the activated hsc (ito cell, vitamin a-storing cell), which is normally present in the perisinusoidal space. , under the influence of fibrogenic stimuli (inflammation and the immune response, oxidative stress, apoptosis, hypoxia, steatosis), the hsc is activated to a myofibroblast, which produces collagen and other extracellular matrix (ecm) constituents. the cytokine, tgf-β, appears to play a central role in fibrogenesis in humans and dogs. , perisinusoidal fibrosis decreases the permeability of normal sinusoids, impairing metabolic exchange between hepatocytes and sinusoidal blood further compromising hepatic function. excess fibrous tissue also limits the ability of vessels and sinusoids to distend, resulting in increased resistance to hepatic blood flow and portal hypertension. when fibrotic septae become vascularized, these microscopic communications (between portal vein or arterial artery and hepatic vein) lead to portosystemic shunting of blood. reversal of hepatic fibrosis and improvement in liver function can occur, especially if the underlying cause of injury is treated or removed. examples in human medicine include antiviral drugs for hepatitis b and hepatitis c and prednisone for autoimmune hepatitis. although fibrosis is potentially reversible, cirrhosis for all practical purposes is not, because of the accompanying architectural changes and psss. these ocular complications occur in approximately % of naturally infected dogs, and are caused by corneal endothelial damage and antigen-antibody complexes. ich should be suspected in any young, unvaccinated dog with evidence of alf. ich must be differentiated from diseases with similar clinical signs, such as canine distemper, parvoviral enteritis, and hepatotoxicity. abnormalities on the leukogram are common and vary with the clinical stage of infection. during viremia, neutropenia and lymphopenia are often present. neutropenia is also a common a feature of canine parvovirus, a much more prevalent disease of puppies. rebound lymphocytosis and neutrophilia occur in the recovery stages of ich ( days after infection). biochemical findings are characteristic of acute hepatic necrosis and include increased serum alt and alp activity, and abnormal liver function tests. hyperbilirubinemia is a less-consistent finding. hypoglycemia may complicate the terminal stages of the disease. coagulation parameters are consistent with dic. other potential findings include proteinuria secondary to glomerular damage, abdominal fluid consistent with an exudate, and an increase in protein and mononuclear cells in the cerebrospinal fluid. the clinical diagnosis of ich is usually suspected on the basis of age, vaccination history, clinical signs, and laboratory findings, and is confirmed by liver biopsy or necropsy findings. additional diagnostic tests that are used less frequently include serologic testing, virus isolation, and direct immunofluorescence. therapy for alf caused by ich is primarily supportive care and control of complications that frequently occur such as dic, he, and hypoglycemia. the prognosis in dogs with ich depends on the severity of hepatic necrosis and the incidence of serious complications such as dic. hepatic regeneration and recovery is possible unless widespread coagulation necrosis destroys entire lobules. ich can be effectively prevented by vaccination. canine herpesvirus causes an acute, afebrile, rapidly fatal disease in neonatal puppies ( to weeks of age). hepatic necrosis is one manifestation of the widespread multiorgan necrosis and hemorrhage that occurs in this systemic viral infection. clinical signs include acute onset of depression, diarrhea, failure to suckle, crying, and abdominal pain in previously healthy puppies. other findings include petechial hemorrhages and vesicles of the mucous membranes. jaundice is rare. seizures and loss of consciousness may be present in the terminal stages, and most pups die within hours of onset of clinical signs. typical gross pathologic findings include focal areas of necrosis and hemorrhage in the liver, kidneys, lungs, and serosal surfaces of the intestines. microscopically, these areas are characterized by foci of necrosis with occasional intranuclear inclusions. neonates are infected by oronasal exposure to the virus in utero or by secretions from an infected bitch or littermates. neonates are particularly susceptible, possibly because of their low body temperature and immature mechanisms for temperature regulation. the diagnosis of canine herpesvirus is primarily based on the history, physical examination, and pathologic findings. laboratory findings are inconsistent but include neutrophilia or neutropenia, and increased serum alt activity. treatment of affected puppies is generally unsuccessful because of the acute fulminant nature of the disease. maintenance of body it may be one of several organ systems involved in a multisystemic disease process such as feline infectious peritonitis (coronavirus), toxoplasmosis, or histoplasmosis (see table - ). infectious agents can be associated with widespread invasion of organs with a large mononuclear phagocytic component, including the liver, spleen, lymph nodes, and bone marrow, although clinically significant liver disease is uncommon. liver biopsy can be diagnostically useful for identification of these organisms in infected animals. etiology infectious canine hepatitis (ich) caused by cav- has long been recognized as a cause of acute hepatic necrosis in dogs. this virus is genetically and antigenically distinct from cav- , a cause of infectious canine respiratory disease. the incidence of clinical disease caused by cav- is now very low because of effective vaccination procedures. neutralizing antibodies to cav- are also found in mature, unvaccinated dogs, suggesting that natural exposure to the virus is widespread. cav- has a special tropism for vascular endothelial cells and hepatocytes. dogs with sufficient immunity (neutralizing antibodies > : ) do not develop clinical signs of disease. susceptible dogs (titer < : ) develop widespread centrilobular to panlobular hepatic necrosis, which is often fatal. distinctive intranuclear inclusions are present in hepatocytes and the endothelium of other tissues. experimentally, dogs with an intermediate titer (between : and : ) develop chronic hepatitis that can progress to cirrhosis. whether cav- is a significant cause of chronic hepatitis under natural conditions is unknown. cav- antigen was demonstrated in formalinfixed liver sections from five of dogs with various naturally occurring hepatic inflammatory lesions, suggesting that cav- may play a role in spontaneous chronic hepatitis. other attempts to identify cav- in dogs with chronic hepatitis have been negative. , clinical examination ich is seen most commonly in unvaccinated dogs younger than year of age. clinical signs vary with the stage of disease. dogs that are peracutely ill do not have clinical evidence of hepatic disease but simply become depressed and moribund, and die within a few hours. dogs with a more extended clinical course ( to days) have signs associated with acute hepatic necrosis that include vomiting, diarrhea, and abdominal pain. a hemorrhagic diathesis may occur during the viremic phase and is manifested by epistaxis, petechial or ecchymotic hemorrhages of the skin, or excessive bleeding from venipunctures. failure of the liver to clear activated clotting factors and impaired hepatic synthesis of clotting factors probably also contributes to development of dic. signs of central nervous system (cns) dysfunction include depression, disorientation, seizures, and coma and have been attributed to he or nonsuppurative encephalitis. common physical examination findings include fever, enlarged tonsils, pharyngitis, laryngitis, cervical lymphadenopathy, and subcutaneous edema of the head, neck, and trunk. hepatomegaly, abdominal pain, and abdominal effusion can occur. jaundice is rare but can develop in dogs that survive the acute fulminant stage of ich. an uncomplicated clinical course lasts approximately to days before recovery begins. unilateral or, less frequently, bilateral corneal edema and anterior uveitis ("hepatitis blue eye") are complications that may become evident during the recovery period. diagnosis of fip should be supported by cytologic or histologic evidence of pyogranulomatous inflammation. the currently recommended "gold standard" for fip diagnosis is immunohistochemistry performed on effusions or lesions containing infected macrophages. the prognosis for recovery is poor. etiology canine leptospirosis is caused by leptospira interrogans sensu lato, with at least serovars appearing to have clinical significance in dogs. serovars canicola and icterohemorrhagica have been included in vaccines for more than years and the incidence of clinical disease from these serovars has decreased accordingly. an epidemiologic shift in serovars causing clinical disease has since occurred, with increasing reports of disease associated with serovars grippotyphosa, pomona, and bratislava. it has been suggested that serovars icterohemorrhagica and pomona are more likely to be associated with hepatic damage. however, other reports have been unable to correlate serogroups with specific clinical features. , chronic hepatitis has been associated with serovar grippotyphosa and serogroup australis. reports of clinical leptospirosis in cats are rare, although antibodies to several serovars have been demonstrated. pathophysiology acute renal failure is the most common clinical disease syndrome in dogs with leptospirosis. the liver can also be a target organ and alf may occur concurrently in % to % of dogs with acute renal failure or independent from renal involvement. with acute hepatic involvement, the liver is enlarged, friable, and yellowbrown. tissues are often markedly jaundiced. microscopic changes in the liver include intrahepatic cholestasis, liver cell dissociation, and nonspecific reactive hepatitis. , hepatic necrosis is an uncommon histologic feature. the liver may not show striking changes, presumably because hepatic dysfunction can be caused by a toxin that produces mainly subcellular damage. organisms can be identified in tissues with a warthin-starry stain. common clinical signs include anorexia, depression, and vomiting. hepatocellular involvement is suggested by jaundice. other findings may include arthralgia or myalgia, pu, pd, fever, and dehydration. widespread petechial and ecchymotic hemorrhages of the mucous membranes, sclera, and skin are caused by thrombocytopenia and dic. the terminal stages include signs of cardiovascular collapse, shock, coma, and death. a diagnosis of leptospirosis should be considered in dogs with acute cholestatic liver disease, especially when accompanied by acute renal failure. hematologic findings vary with the stage and severity of disease. leukocytosis and left shift are frequent, but in the early stages of leptospiremia, leukopenia is more likely. thrombocytopenia can also be seen. coagulation parameters are normal unless complicated by dic. routine serum chemistry and urinalysis findings reflect involvement of the liver or kidney. serum liver enzyme activity is usually increased with hepatic involvement, and the magnitude of the increase in serum alp activity is usually greater than that of serum alt activity owing to intrahepatic cholestasis. other findings include hyperbilirubinemia, bilirubinuria, and abnormal liver function tests. an increase in bun or creatinine may result from renal failure or prerenal uremia. the urinalysis is often compatible with acute nephritis with findings of proteinuria and increased temperature ( . °c to . °c [ °f to °f]) may be helpful. intraperitoneal infusion of to ml of hyperimmune serum obtained from bitches with previously infected litters may reduce mortality rates. vaccination for herpesvirus infection is not routinely performed because of the low incidence of disease. canine acidophil cell hepatitis, which encompasses a spectrum of hepatic lesions ranging from acute and chronic hepatitis to cirrhosis and liver failure, has been reported in great britain. , it is caused by a transmissible agent, suspected to be a virus that is distinct from cav- , although a specific virus has never been identified. the disease is experimentally transmissible by serum or liver extracts from affected dogs. in the experimentally induced disease, acute hepatitis can progress to chronic hepatitis in the absence of clinical signs. episodic increases in serum alt activity and fever spikes correspond with histologic evidence of acute hepatitis. the liver is enlarged and friable in the acute stages, and becomes progressively smaller and nodular with chronicity. the most notable histologic feature, regardless of the stage of disease, is the acidophil cell. acidophil cells are dying hepatocytes with an angular shape, reduced volume, hyperchromatic nucleus, and strongly acidophilic cytoplasm caused by small acidophilic coalescing granules. end-stage hepatic disease is accompanied by typical findings of cirrhosis. most dogs with spontaneous disease are presented with signs of chronic hepatic failure. the duration of clinical signs can exceed year. based on experimental studies, it is speculated that the early mild stages may go unrecognized until advanced hepatic disease and failure is present. biochemical findings are consistent with hepatic inflammation and necrosis, evidenced as increased serum alt activity. with advanced disease, severe hepatic dysfunction is noted. the diagnosis requires liver biopsy. recommendations for specific therapy of acidophil cell hepatitis await further information on the causative agent. supportive measures should be instituted as needed. feline infectious peritonitis (fip) is a highly fatal coronaviral infection of both domestic and wild cats. the liver is one of many organs (kidneys, spleen, pancreas, mesenteric lymph nodes, cns, uveal tract, omentum, serosal surfaces) that can be affected by widespread immune complex vasculitis and granulomatous or pyogranulomatous inflammation (see table - ). clinical findings in cats with hepatic involvement are nonspecific and include lethargy, depression, anorexia, dehydration, weight loss, and fever. jaundice is a common finding. extrahepatic findings include nodular renomegaly, abdominal mass (lymph node), and ascites or dyspnea (pleural effusion). ophthalmoscopic examination may detect chorioretinitis or anterior uveitis, which must be differentiated from similar ocular changes seen with the other systemic disorders that involve the liver such as lymphosarcoma, toxoplasmosis, and the systemic mycoses. serum hepatic enzyme (alp and alt) activities are usually normal or only mildly increased. mild to moderate increase in serum bilirubin concentration is common. other findings indicating hepatic dysfunction include bilirubinuria and increased sba concentrations. hyperglobulinemia, neutrophilia, and mild to moderate nonregenerative anemia are other laboratory features of fip. abdominal and pleural effusions, when present, are usually pyogranulomatous exudates with greater than g/dl protein. serologic detection of a high coronaviral antibody titer may support a diagnosis of fip, but is not definitive because of its lack of specificity. the serum bile acid concentration can be markedly increased (> µmol/l). extrahepatic bacterial infection-induced hepatic damage should be considered when evidence of cholestatic hepatopathy is found concurrently with extensive bacterial infection in other organ systems (e.g., pyometra, peritonitis) or with extrahepatic disorders likely to be associated with endotoxemia (e.g., parvoviral enteritis). associated clinical findings that would be compatible with endotoxemic crisis include shock, fever or hypothermia, hypoglycemia, neutrophilia or neutropenia with left shift, toxic changes of the neutrophils, and hyperbilirubinemia that is disproportionately increased in comparison to serum alp activity. it is important to recognize that jaundice can occur secondary to extrahepatic infection from a diagnostic standpoint, so that the clinician is not misled into considering that the cause is a primary hepatic or biliary disease. in jaundiced patients with evidence of an inflammatory process, key differential diagnoses include acute pancreatitis, extrahepatic bacterial infections, and primary hepatobiliary disorders such as leptospirosis (dogs only), cholangiohepatitis, cholecystitis, ruptured gallbladder mucocele, and hepatic abscesses. specific therapy for hepatic disease is not usually required, and hepatic damage is reversible with control of sepsis. morbidity is related to the underlying disease process and not overt hepatic failure. etiology hepatotoxicity can be caused by a variety of drugs (prescription or over-the-counter), herbal and dietary supplements, or biologic toxins or chemicals (see box - ). , , the liver is uniquely susceptible to xenobiotic substances because it is directly exposed to them following absorption from the gi tract. the liver is also vulnerable to toxic injury because it plays a central role in the metabolism of many substances. hepatic metabolism renders lipophilic substances more hydrophilic, which promotes excretion via the urine or bile. the process is controlled by phase i and phase ii reactions. phase i reactions are catalyzed by the cytochrome p enzyme systems, which activate or detoxify (oxidize, reduce, or hydrolyze) a drug or toxin. phase i reactions may lead to generation of unstable chemically reactive intermediates, which can be toxic. phase ii reactions conjugate drugs or metabolites and produce products that are nontoxic. during biotransformation, the liver can either reduce or enhance the toxicity of the parent compound. for example, after carbon tetrachloride ingestion, the liver converts the nontoxic parent compound into toxic metabolites, which subsequently cause severe hepatocellular damage. genetic polymorphisms of phases i and ii enzymes have the potential to influence drug metabolism in the individual animal. there has been an increased awareness and recognition that drug-induced liver injury can be a significant cause of liver disease in dogs and cats (see box - ). this information has been gained from isolated case reports, retrospective clinical studies, and experimental studies. unfortunately, for many of these drug reactions, characterization of the clinical and pathologic features are lacking because only small numbers of affected animals have been described, and liver biopsies are not typically obtained when drug withdrawal results in clinical and biochemical improvement. it is possible that drug-induced liver injury is underrecognized in dogs and cats, as in humans, drug-induced hepatic injury accounts for more than % of the cases of alf in the united states, and is the most frequent reason cited for withdrawal of an approved drug from the market. leukocytes, erythrocytes, and granular casts. increased serum creatine kinase activity may indicate leptospiral-induced muscle damage. leptospirosis is most easily diagnosed in the clinical setting by demonstration of a fourfold rise in serum antibody titer (microscopic agglutination test) in paired samples taken at initial presentation and to weeks later. the rise in titer indicates recent or active infection and differentiates a titer from previous exposure or previous vaccination. the optimum treatment regimen for leptospirosis is unknown. traditionally, intravenous ampicillin, mg/kg every hours (with dose reduction in dogs with renal failure), or penicillin g ( , to , units/kg iv q h), has been used for initial treatment of leptospirosis. doxycycline ( mg/kg po q h for to weeks), was recommended as followup therapy to eliminate organisms from renal tubules. however, doxycycline, mg/kg orally or intravenously every hours for weeks, appears to be effective in clearing all phases of leptospiral infection and may be the most effective treatment strategy. management of fluid, electrolyte, and acidbase imbalances is important supportive therapy. the prognosis generally depends on the degree of renal dysfunction and is poor when oliguria develops. c. piliforme (formerly known as bacillus piliformis), a spore-forming gram-negative bacteria, is a rare cause of multifocal hepatic necrosis and necrotizing ileitis in dogs and cats. the infection is mainly opportunistic in stressed or immunocompromised animals with a predisposing disorder (e.g., canine distemper, feline panleukopenia, feline leukemia) or familial hyperlipoproteinemia in kittens. clinical signs include an acute onset of anorexia, lethargy, depression, and abdominal discomfort. jaundice may be observed, especially in cats. these signs rapidly progress to a moribund state; death occurs within to hours. marked increases in alt activity may be detected. histopathology reveals multifocal periportal hepatic necrosis and necrotic ileitis or colitis. bacilli are best seen with special staining techniques (warthin-starry or giemsa), or methylene blue-stained impression smears of fresh tissue. organisms appear as large, slender, intracellular filamentous organisms within hepatocytes surrounding areas of necrosis and in intestinal epithelial cells. routine culture techniques are ineffective for isolation of this organism. the disease is rapidly fatal, and successful therapy has not been reported. extrahepatic bacterial infection associated with sepsis and endotoxemia is an important cause of acute functional cholestatic hepatopathy. however, morbidity is generally related to the underlying infection and not overt hepatic failure. , studies in humans and experimentally in dogs, suggest that endotoxemia and the subsequent release of cytokines induces functional changes that interrupt the transport and excretion of conjugated bilirubin. , microscopically, hepatic lesions are often mild and nonspecific. intrahepatic cholestasis, characterized by bile canalicular plugs and bile pigment accumulation in hepatocytes, is the most consistent finding. a mild periportal lymphocytic infiltrate can be seen, with scattered foci of macrophages or neutrophils, and occasional individual necrotic hepatocytes. total serum bilirubin concentrations as high as mg/dl can be seen and are disproportionately high compared to the mild to moderate increases in serum alp activity. increased serum alt activity is a less consistent finding. hepatic injury caused by drugs, herbal and dietary supplements, biologic toxins, or chemicals can occur via a number of mechanisms, which influence the histologic pattern of disease. , although hepatic necrosis is the most common histologic response, drug-and toxin-induced liver injury can also potentially mimic the full spectrum of acquired hepatic disorders, including acute and chronic hepatitis, granulomatous hepatitis, cholestatic hepatopathy, vacuolar hepatopathy (lipid or glycogen accumulation), hepatic fibrosis and cirrhosis, and venoocclusive disease. hepatic necrosis occurs when covalent binding of a drug or toxin to intracellular proteins disrupts cellular functions, or formation of drug-enzyme adducts stimulates an immunologic response (antibody-or t-cell-mediated cytotoxicity). hepatic inflammation (typically lymphoplasmacytic, but may be granulomatous or eosinophilic), suggests an underlying immunoallergic mechanism. intrahepatic cholestasis occurs when drugs or toxins interfere with hepatic transport proteins at the canalicular membrane, which interrupts bile flow. hepatic lipid accumulation results when direct damage to mitochondria disrupts fatty acid oxidation and energy production, and is the predominant hepatic lesion seen with stanozolol hepatotoxicity in cats, and tetracycline in dogs and cats. hepatic vacuolation caused by glycogen accumulation occurs in dogs treated with corticosteroids, and typically does not cause significant clinical evidence of hepatic dysfunction. damage to the sinusoidal epithelium can result in peliosis hepatic or venoocclusive disease. mechanisms of drug-induced liver injury (and also injury from herbal and dietary supplements) can be characterized as either intrinsic (predictable) or idiosyncratic (unpredictable) reactions. intrinsic hepatotoxic reactions are dose-related and occur shortly after a consistent threshold of toxicity is reached. because intrinsic hepatotoxins predictably damage the liver in an exposed population, they can be experimentally reproduced and studied. hepatic injury is caused by a direct toxic effect of the parent compound (or a reliably generated toxic metabolite) on vital cell targets. acetaminophen is an intrinsic hepatotoxin in dogs and cats and is discussed in more detail later in this section. with intrinsic hepatotoxins, lowering the dose, rather than stopping the drug can be tried. in many cases, most such drugs or chemicals, (e.g., carbon tetrachloride, phosphorus, and chloroform), are no longer used for therapeutic purposes once their intrinsic hepatotoxicity is recognized, but accidental exposures could still occur. in contrast, idiosyncratic hepatotoxic reactions occur at therapeutic doses in only a small number of individuals in the exposed population. these reactions are unpredictable and infrequent; most individuals treated with the drug do not have a reaction, even at high doses. however, toxicity may be more pronounced at higher doses in susceptible individuals. , examples of drugs causing idiosyncratic drug-induced liver injury in dogs and cats that have a dose-related effect are phenobarbital, itraconazole, amiodarone, and lomustine (ccnu). idiosyncratic reactions are characterized by a variable latency period ( to days, but may be longer for some drugs, such as phenobarbital, ccnu, amiodarone) from initial drug ingestion to recognition of hepatic injury. because of the infrequent occurrence (approximately in , ), the potential for hepatotoxicity may not be recognized in preclinical screening of a new drug, and cannot usually be reproduced in an experimental setting. individual differences in susceptibility to idiosyncratic drug-induced liver injury may reflect genetic differences in either (a) alternate metabolic pathways by which a drug is converted to different (potentially hepatotoxic) metabolites, (b) the ability of the individual to detoxify the toxic intermediates, (c) an underlying the center for veterinary medicine of the food and drug administration, washington, dc, maintains a registry (http://www.fda.gov/ animalveterinary/default.htm) for reporting adverse drug reactions in animals. this service has been useful to accumulate data and to alert veterinarians to suspected drug effects, including hepatotoxicity. however, because reporting of adverse drug reactions is voluntary and the information obtained may be incomplete, only subjective trends can be identified. furthermore, because mild hepatic injury may not be associated with clinical signs, these cases will not be detected unless biochemical testing is performed while the animal is receiving the drug. evaluating the incidence of druginduced liver injury in dogs and cats is further clouded by the fact that there are no pathognomonic clinical, laboratory, or biopsy findings to distinguish drug-induced liver injury from other causes of liver disease. specific drugs that have been reported to cause druginduced liver injury are listed in box - . herbal and dietary supplements (herbs or other botanicals, nutraceuticals, vitamins, minerals) have the potential to cause hepatotoxicity, similar to drug-induced injury. [ ] [ ] [ ] in humans, herbal and dietary supplements are reported to account for % of patients with "drug-induced" liver injury. the incidence of hepatotoxicity may be underrecognized, as only a third of humans taking herbal and dietary supplements reported their use of these products to their health care provider. causality is difficult to prove, because herbal and dietary supplements are often dispensed without medical supervision, fda oversight of product quality is minimal, multiple active ingredients contribute to product variability, and product contamination with hepatotoxic substances (toxic herbs or heavy metals) may occur. [ ] [ ] [ ] drug interactions between herbal and dietary supplements and prescription medications may also occur, because medicinal plants can have effects on hepatic p enzyme systems. the rate of dietary supplement use in dogs and cats appears to be lower than that reported for humans. however, because these products are often marketed as "all natural," pet owners may assume they are safe and underreport their use to the veterinarian. reports of herbal and dietary supplement hepatic injury in animals are rare. pennyroyal oil, a volatile oil derived from plants of the labiatae family (pennyroyal, squaw mint, or mosquito plant) was associated with fatal acute hepatic necrosis in a dog after topical application for use as a flea repellent. clinical signs occurred within hour after application and included vomiting, diarrhea, hemorrhage, seizures, and death. other reports include the finding of increased liver enzyme activity in dogs consuming st. john's wort, and increased alt activity and hypoglycemia after accidental ingestion of high doses of α-lipoic acid in two dogs. , the paucity of reported hepatic reactions does not necessarily mean herbal and dietary supplements are safe, and the clinician should maintain a high level of suspicion regarding their potential toxicity. box - lists the herbal and dietary supplements that have been incriminated as causing hepatic injury in humans with potential relevance to dogs and cats. for more detailed information on hepatic injury and herbal and dietary supplements, additional sources should be consulted. [ ] [ ] [ ] hepatic injury may also occur after exposure to a wide variety of industrial chemicals, organic solvents, pesticides, heavy metals, and biologic toxins. most information on chemical hepatotoxins is derived from experimental studies in dogs or extrapolated from information in other species. very few clinical case reports are available in the veterinary literature. isolated reports and case series of clinical liver disease in dogs associated with exposure to biologic toxins, such as aflatoxin, amanita mushrooms, blue-green algae, and cycads (sago palms) have been published. selected hepatotoxins are listed in box - . increased liver enzyme activity is a common finding with hepatotoxicity. increased alt activity (more than three times the upper limit of normal, but can be as high as times the upper limit), suggests hepatocellular injury (often necrosis), and is of more concern than an isolated increase in alp activity (reflecting cholestasis), although mixed patterns commonly occur. progressive increases in alt activity or those accompanied by evidence of hepatic dysfunction (hyperbilirubinemia, increased serum bile acids, coagulopathy, hypoglycemia, hyperammonemia, hypoalbuminemia) are more likely to represent serious hepatic injury. when drug-induced liver injury is suspected, the diagnostic approach is determined by the clinical presentation. if clinical signs are absent or mild, a minimum database consisting of complete history and physical examination, complete blood cell count, serum chemistry, and urinalysis should be performed. if the only abnormality detected is increased liver enzyme activity, and these increases correspond to the recent administration of a drug (especially those listed in box - ), the drug should be discontinued and serum biochemistries should be repeated in to days. in many instances, clinical and biochemical abnormalities resolve after the suspected hepatotoxic drug is discontinued and a liver biopsy is not performed. further evaluation of the liver, including sba concentrations, abdominal radiographs, ultrasonography, and liver biopsy, may be warranted if biochemical abnormalities persist, or if initial clinical and biochemical findings suggest hepatic dysfunction. with suspected drug-or toxin-induced liver disease, a liver biopsy can be helpful to (a) characterize the histologic changes (are they consistent with previously described lesions caused by this particular drug or toxin?), (b) determine the severity (focal or diffuse necrosis?) or reversibility (is cirrhosis present?) of the lesions for prognostic purposes, and (c) rule out known causes of liver disease. histologic changes secondary to drug-and toxin-induced hepatic injury are nonspecific and similar to those seen with other nondrug-related causes of acute and chronic liver disease. the most common pathophysiologic response is necrosis without inflammation. hepatic necrosis may be centrilobular (zone ) or panlobular (figure - ) . centrilobular hepatocytes have an abundance of p enzymes, and are preferentially affected in drug-induced hepatotoxicity when p metabolism of the parent drug results in toxic metabolites. drugs or toxins can also cause a variety of other hepatic lesions, such as cholestasis, lipidosis, or mild inflammation. a chronic response to injury is reflected by findings of biliary immunologic or allergic reaction, or (d) an individual's tolerance or ability to "adapt" to hepatocellular injury (mechanisms unknown) with resolution of injury despite continued medication administration. , oral medications with substantial hepatic metabolism are more likely to be associated with adverse hepatic events in humans, presumably because of hepatic generation of reactive toxic metabolites. because of the unpredictability of an idiosyncratic reaction and the low incidence of occurrence, a cause-and-effect relationship is difficult to establish. if an idiosyncratic reaction occurs, the drug must be discontinued or it could result in death of the patient. an idiosyncratic mechanism is suspected for most of the drugs that cause hepatic injury in dogs and cats. susceptibility to hepatotoxicity in humans is influenced by a number of factors such as age, sex, nutritional status, and concurrent drugs. the most important factor may be the effect of genetic polymorphisms on hepatic drug metabolism. in humans, preexisting liver disease does not appear to enhance susceptibility to druginduced liver injury, but impacts the patient's ability to recover. this may be because drug-metabolizing enzyme systems are remarkably preserved in hepatic disease. similar information on risk factors for hepatotoxicity in dogs and cats has not been determined. however, because many toxic metabolites are normally detoxified by glutathione, some metabolites may become more toxic when hepatic glutathione stores are depleted (e.g., animals with preexisting chronic necroinflammatory and cholestatic liver disease). a breed predisposition has been suggested for doberman pinschers (sulfonamides, amiodarone, diethylcarbamazine/oxibendazole) and labrador retrievers (carprofen), which may be a reflection of a genetic predisposition. , , , , clinical examination the spectrum of drug-and toxin-induced liver injury and, thus, the associated clinical presentation, can vary from subclinical hepatic injury with only increased serum liver enzyme activity, to severe liver damage manifested as alf, or chronic end-stage liver disease. acute rather than chronic liver injury is more likely for most of the drugs and toxins listed in box - . clinical features often include acute onset of lethargy, anorexia, vomiting, diarrhea, pu, pd, or jaundice in a previously healthy animal, which corresponds to hepatotoxin exposure. alf (signs of acute liver disease plus he and coagulopathy) is most likely with drugs or toxins that cause diffuse hepatic necrosis. drug-and toxin-induced injury is an important diagnostic consideration in dogs and cats presenting for acute hepatitis and hepatic necrosis. drugs or toxins also have the potential to cause chronic hepatic disease, if the initial hepatic injury is mild and goes unrecognized, and exposure to the drug or toxin is continued. for example, phenobarbital, ccnu, or chronic aflatoxicosis can cause chronic liver injury in dogs. , , diagnosis there are no pathognomonic clinical, laboratory, or biopsy findings that distinguish drug-or toxin-induced liver injury from other causes of liver disease. the diagnosis of drug-induced liver injury often relies on the clinician maintaining a high level of suspicion, and obtaining an accurate and thorough medication history (including prescription and over-the-counter drugs, and herbal and dietary supplements), in every animal with unexplained increases in liver enzyme activity or clinical liver disease. a definitive diagnosis of hepatic injury caused by biologic toxins or chemicals is rarely possible in a clinical setting, unless the owner specifically observes ingestion of a substance that is a known hepatotoxin. prevented. treatment of drug-induced hepatic disease consists of discontinuing the suspect drug. after a drug is discontinued, clinical (and biochemical) improvement usually occurs within a few weeks, even with chronic drug administration. however, exceptions can occur. for example, in dogs with amiodarone toxicity, liver enzyme elevations can transiently progress despite discontinuation of the drug, and biochemical abnormalities may not resolve for to weeks. with the exception of nac for acetaminophen, and silymarin for amanita mushroom toxicity, no specific antidotes are available, and treatment of drug-and toxin-induced liver injury is primarily supportive and symptomatic. however, nonspecific hepatoprotective therapy with antioxidants (vitamin e), glutathione replacement (nac, same), or milk thistle (silymarin) may be helpful and are discussed in more detail in chapter . use of nac (or same) may be beneficial, as glutathione depletion may predispose to hepatotoxicity (e.g., methimazole) or impair metabolism of toxic metabolites to a nontoxic form. nac has been suggested to be beneficial for treatment of alf associated with toxicities such as diazepam, methimazole, carprofen, and trimethoprim-sulfa. in addition to treating amanita mushroom hepatotoxicity, silymarin may be beneficial in the treatment of carbon tetrachloride and acetaminophen toxicity. , , corticosteroids are not typically indicated for treatment of drug-and toxin-induced hepatotoxicity. it is important to consider a drug-induced cause of liver injury because rapid recognition and prompt discontinuation of an hepatotoxic drug can lead to improvement or complete resolution of hepatic disease, depending on the specific drug and the stage of the lesion. when drug-or toxin-induced hepatic injury causes severe or widespread hepatic necrosis, rapid deterioration and death in to days often occur. with less-severe hepatic injury, complete recovery is possible. acetaminophen acetaminophen is well known as an intrinsic hepatotoxin in dogs and cats. , although acetaminophen is occasionally used as an analgesic in dogs (therapeutic doses up to mg/kg tid), most toxicity occurs because of accidental ingestion of improperly stored medication (dogs) or owner administration without veterinary supervision (dogs and cats). toxic metabolites of acetaminophen cause oxidative injury to erythrocytes and hepatocytes, resulting in methemoglobinemia, anemia, and hepatic necrosis. in therapeutic doses, acetaminophen is detoxified by a combination of hepatic glucuronidation and sulfation and renal excretion. after acetaminophen overdosage, these pathways become saturated and a greater proportion of acetaminophen is metabolized through the p system, leading to production of the toxic metabolite, n-acetylp-benzoquinoneimine (napqi). glutathione detoxifies napqi and thus protects hepatic cellular constituents from its direct toxic effect. however, once glutathione levels are depleted by large amounts of napqi, centrilobular necrosis occurs. toxicity occurs in a dose-dependent manner. there are substantial species differences in both the metabolism of acetaminophen and the toxic manifestations. cats are uniquely sensitive to acetaminophen because of a deficiency of glucuronyl transferase and limited sulfation capabilities. clinical signs in cats may develop after administration of as little as . mg ( tablet). signs of methemoglobinemia usually dominate the clinical picture, such as cyanosis, dyspnea, facial edema, depression, hypothermia, hyperplasia, fibrosis, and cirrhosis. although individual drugs and drug classes may follow the same pattern, there is often not a consistent reaction for any given drug. for example, hepatic injury in dogs secondary to potentiated sulfonamides may cause hepatic necrosis, primary cholestasis, or marked inflammation. for many of the potentially hepatotoxic drugs listed in box - , histologic features have not been fully characterized. it should be emphasized that for most drug-induced disorders, the diagnosis is presumptive and cannot be proved. it can be especially difficult to pinpoint the causative agent when the patient is receiving a combination of drugs. a clinical diagnosis of drug-induced hepatic injury is easier to establish when the hepatotoxicity of the drug has been previously described and the associated clinical and pathologic features have been characterized (see discussion of specific drugs). the diagnosis may be less convincing when the suspected drug has not been previously incriminated as causing liver damage. however, a drug reaction should still be considered, since an idiosyncratic reaction could occur with any drug. the clinician should also maintain a level of suspicion regarding the potential for hepatotoxicity in newly marketed drugs that have not yet been used widely in the population, where idiosyncratic reactions are often first detected. the suspected hepatotoxic drug should be discontinued while a complete diagnostic evaluation is pursued for other causes of liver disease, for which a specific treatment might be available. a diagnosis of drug-induced injury is supported by the following: (a) evidence of liver injury that occurred within the first months of drug therapy (especially if predrug liver enzyme activity was within normal limits); (b) clinical and biochemical improvement when the drug is discontinued; (c) exclusion of other causes of liver disease; (d) reccurrence of hepatic damage after a challenge dose of the same drug (or inadvertent reexposure). it should be emphasized that rechallenge with a suspected hepatotoxic drug is not recommended as a diagnostic consideration, because it is potentially dangerous, especially with a drug that causes acute hepatic necrosis. rechallenge should only be considered if the association of the drug with hepatic injury is highly questionable and there is no alternative drug available for a significant medical condition. with hypersensitivity or immunologic reactions, the hepatic reaction is more rapid and severe with repeated exposure. hepatic injury because of a biologic toxin or chemical is suspected when exposure to a potential hepatotoxin has been documented. a clinical diagnosis of "toxic" hepatic injury is often made when an episode of acute hepatic injury occurs, hepatic biopsy indicates diffuse hepatic degeneration and necrosis, and no other cause for liver disease can be identified. in selected cases, tissues, blood, or food (aflatoxin) samples can be submitted to a toxicology lab to confirm a suspected toxin. toxin-induced injury should also be considered in the absence of known exposure to toxins, because potential hepatotoxins can be present in contaminated dog food or garbage (aflatoxins), pond water (blue-green algae), and many other unobserved sources. when ingestion of a potential hepatotoxin (e.g., toxic mushrooms, sago palms) has occurred within the preceding hours, general procedures for gi decontamination are recommended, including induction of emesis or gastric lavage (within first hours), followed by administration of activated charcoal ( to g/kg). , induction of vomiting is contraindicated if the patient is comatose or debilitated in such a way that the gag reflex is diminished, which could predispose to aspiration pneumonia. whenever possible, the source of toxin exposure should be identified and further exposure or given as alternate-day therapy), with careful monitoring of liver parameters every weeks. hepatotoxicity appears to be at least partly dose related, as dogs receiving higher daily doses of itraconazole ( mg/kg) are more likely to be affected. icterus and evidence of hepatic dysfunction suggests a more serious, potentially fatal hepatopathy, requiring discontinuation of medication and symptomatic and supportive care. it is recommended that liver enzymes be monitored on a monthly basis in all animals receiving ketoconazole or itraconazole. azathioprine, a purine analogue commonly used for treatment of immune-mediated disorders in dogs, is commonly listed as a potential hepatotoxin. however, few clinical details regarding the hepatotoxic reaction are available. in a clinical study of dogs with atopic dermatitis treated with azathioprine ( . mg/kg daily for weeks) as a single agent, an increase in alt or alp activity was noted in the first weeks in ( %) of the dogs. three dogs had clinical signs suggestive of liver disease, which resolved uneventfully when azathioprine was discontinued. in an experimental study of dogs given azathioprine at a dose of to mg/kg po daily for days, all dogs had increased liver enzyme activity (alt >> alp) within to days of initiating therapy. values peaked within the first weeks and then declined, but not to normal, despite continued medication administration. hyperbilirubinemia was absent. liver biopsies in most dogs revealed centrilobular degeneration and necrosis, with intrahepatic cholestasis but no inflammation. these findings raise the possibility that azathioprine may be an intrinsic (dose related) hepatotoxin in dogs, with possible adaptive tolerance to liver injury. it should be noted that doses used in this study exceeded current clinical recommendations of to mg/kg daily or every other day for maintenance therapy. hepatotoxicity is considered a class characteristic of nsaids, despite the fact that there are many different chemical classes of nsaids, and no consistent mechanism of liver injury. with the exception of aspirin, which is an intrinsic (dose-related) hepatotoxin, the mechanism with other nsaids is believed to be idiosyncratic (either immune or as a consequence of toxic metabolites). , toxicity does not appear to be related to prostaglandin inhibition like the renal or gi side effects. preexisting hepatic disease has not been shown to be a risk factor for nsaid-induced liver injury. all nsaids have the potential to cause idiosyncratic hepatotoxicity in dogs, but hepatic reactions appear to be rare. carprofen has specifically been reported as a cause of drug-induced liver injury in dogs. labrador retrievers were overrepresented in the series, but it is not clear whether this is a true breed predisposition. clinical signs (anorexia, lethargy, vomiting, pu/pd) occurred within the first weeks of therapy and icterus was a common finding on physical examination. biochemical evaluation revealed marked increases in liver enzymes (alt activity usually exceeded alp activity) and hyperbilirubinemia. hepatic biopsy findings revealed multifocal to diffuse hepatic necrosis, mild to moderate lymphocytic-plasmacytic inflammation, secondary cholestasis, and variable biliary hyperplasia and bridging fibrosis. concurrent renal toxicity (glucosuria without hyperglycemia, proteinuria, granular casts) also was noted in some dogs. most dogs recovered with discontinuation of carprofen and appropriate supportive care, although some dogs died of alf. general hepatoprotective therapy with same or silybin has been recommended, although the benefits are unproven. nac has been suggested for ancillary treatment when carprofen causes alf. and vomiting. although increases in serum alt activity may be detected, centrilobular hepatic necrosis appears to be uncommon. clinical signs in dogs are more likely when doses exceed mg/kg and can be indicative of methemoglobinemia and/or centrilobular necrosis. laboratory features include methemoglobinemia, anemia, increased serum alt activity, and hyperbilirubinemia. intravenous nac is the treatment of choice for acetaminophen toxicity in dogs and cats. nac increases the synthesis and availability of glutathione, which when conjugated to napqi, decreases toxicity. for maximum effectiveness, nac should be given within hours of acetaminophen exposure; however, there may still be a benefit if given to hours after exposure. nac ( % solution) is diluted : or more with saline and given intravenously through a nonpyrogenic . µm filter at an initial dose of mg/kg over a -to -minute period. a maintenance dose of mg/kg is given iv or orally every hours for seven treatments. same also serves as a glutathione source and has been shown to have protective effects against acetaminophen-induced oxidative stress on the erythrocytes in cats and dogs. , in an experimental study in cats, silymarin ( mg/kg po) was as effective as nac for treatment of acetaminophen toxicity when given up to hours after exposure. vitamin c ( mg/kg iv q h) may be helpful in the treatment of acetaminophen toxicity because of its antioxidant effects. cimetidine ( mg/kg iv q h) is also recommended as adjunctive therapy in the early stages (first hours) because it inhibits hepatic p enzymes and decreases napqi formation. the antiarrhythmic drug amiodarone is associated with a reversible hepatotoxicity in dogs. , doberman pinschers may be at increased risk. toxicity, which appears to be at least partially dose related (doses of mg/day), was identified in % of doberman pinschers treated with amiodarone in one clinical series. clinical signs (anorexia, lethargy, vomiting, diarrhea) and biochemical abnormalities (increased alt and alp activity ± hyperbilirubinemia) developed days to months after initiation of therapy. liver biopsy in one dog revealed multifocal hepatocellular necrosis with mild lipidosis and lymphoplasmacytic inflammation. clinical improvement usually occurs within a few days of stopping the drug, but liver enzyme elevations may not return to normal for months. transient progression of enzyme abnormalities despite discontinuing the drug has also been noted, which may reflect the long half-life of amiodarone causing a delay in systemic elimination. biochemical changes precede clinical signs, so monitoring of liver enzymes at least monthly is recommended. the azole antifungal drugs ketoconazole and itraconazole (and rarely fluconazole) are associated with increased liver enzyme activity and icterus in dogs and cats. hepatotoxicity is more likely with ketoconazole than with itraconazole. cats are more sensitive to the hepatotoxic effects than are dogs, but considerable individual variation occurs. histologic findings are poorly characterized but include bile duct proliferation and infiltration of mononuclear cells. transient mild subclinical elevations of liver enzymes (alt and alp activity) are common, and do not necessarily require a change in therapy. a clinically significant hepatic reaction is suggested by alt activity that exceeds two to three times the upper limit of normal, especially when accompanied by clinical signs of anorexia and vomiting. drug therapy should be stopped for to weeks until appetite and liver enzymes return to normal. a rapid recovery usually occurs, and treatment can be restarted at a lower dose ( % of previous dose decreased appetite, weight loss, pu/pd, vomiting, ascites, and pleural effusion. ascites was due to a combination of hypoalbuminemia and portal hypertension. common biochemical abnormalities included increased liver enzyme activity (alt, ast, alp, ggt) and hypoalbuminemia. other less-consistent findings included hyperbilirubinemia, hypercholesterolemia, and increased serum bile acid concentrations. glucosuria (without hyperglycemia) and renal failure were noted in some dogs, possibly attributable to ccnu renal toxicity. liver biopsy findings were nonspecific (hemosiderinladen kupffer cells, hepatocellular vacuolization, mild to moderate periportal inflammation, and fibrosis) but suggested chronicity. the majority of affected dogs died from progressive chronic liver disease. in a recent study, routine monitoring of alt activity prior to each subsequent dose of ccnu suggested that subclinical elevations of alt activity (greater than five times the upper limit of normal) are common. thirty-two of dogs ( %) had increased alt activity, which developed most commonly after one to three doses of ccnu. increases in alt activity were not associated with cumulative dose. the lower incidence of clinical hepatotoxicity in this study ( of or . %) versus the kristal study ( of or . %), was attributed to prompt cessation of ccnu treatment in dogs with significant increases in alt activity. however, it was noted that chronic administration of ccnu could be associated with chronic irreversible hepatopathy, in the absence of a significant alt elevation. the mechanism of hepatotoxicity is suspected to be a result of generation of toxic intermediate metabolites (e.g., isocyanates, diazonium hydroxide), and depletion of glutathione may play a role. preliminary results of a clinical study using denamarin (same and sylibin; nutramax labs, lancaster, sc) for prevention of ccnu hepatotoxicity, suggested that dogs receiving denamarin had less-severe liver enzyme elevations. methimazole, an antithyroid drug, is associated with hepatic injury in cats with hyperthyroidism. clinical findings include anorexia, vomiting, lethargy, jaundice, markedly increased serum liver enzyme activity, and hyperbilirubinemia that usually occurs within the first month of therapy. histologic lesions have not been fully characterized, although biopsy findings in one cat revealed hepatic degeneration and necrosis. clinical signs resolve within a week of discontinuing therapy but biochemical resolution may take up to days. reduced hepatic glutathione concentrations, which have been documented in other species with hyperthyroidism, may predispose to hepatic injury. treatment with same may be beneficial. phenobarbital is associated with chronic hepatic disease and cirrhosis in dogs. most dogs have been treated with phenobarbital for more than a year before the liver disease is apparent. the mechanism of hepatic injury is not known but higher doses, higher blood levels (> µg/ml), and long duration appear to be important risk factors. clinical signs in dogs reflect chronic liver disease and include sedation, ataxia, anorexia, weight loss, weakness, ascites, jaundice, coagulopathy, and encephalopathy. phenobarbital-induced hepatic injury should be suspected in any dog with a history of chronic phenobarbital therapy and clinical and biochemical evidence of hepatic dysfunction. routine biochemical screening (every to months) of dogs on long-term phenobarbital therapy is recommended for early detection of hepatic injury. however, mild liver enzyme elevations (especially alp) are commonly seen in dogs treated with phenobarbital, who do not have clinical or histologic evidence of significant liver early recognition of hepatotoxicity (including periodic monitoring of liver enzymes during the first months) and discontinuation of drug therapy provides the best opportunity for full recovery. whether dogs with previous carprofen hepatotoxicity can be safely switched to another nsaid without experiencing a hepatic reaction is unknown. oral diazepam has been incriminated as a cause of acute idiosyncratic fatal hepatic necrosis in cats. , intravenous diazepam, and oral oxazepam, clonazepam, and zolazepam also have been implicated. , onset of signs occurs within to days of initiating therapy. clinical signs and biochemical evaluation are consistent with acute hepatic necrosis and liver failure. most cats die within days of initial administration of the drug. if treatment of a cat with oral diazepam is unavoidable, liver enzymes should be checked before and within days after starting therapy. if liver enzymes are increased, the drug should be discontinued and symptomatic therapy should be started. ancillary treatment of alf with nac may be beneficial. glucocorticoid therapy in dogs is commonly associated with increased serum alp activity and development of a reversible vacuolar ("steroid") hepatopathy as a result of hepatic glycogen accumulation. these hepatic effects can be seen with virtually any glucocorticoid preparation (including topical ophthalmic and otic preparations) and are influenced by drug preparation (e.g., repositol versus short-acting), dose, duration of therapy, and individual dog susceptibility. in contrast, cats are quite resistant to the hepatic effects of glucocorticoids and only rarely develop these hepatic changes. increased serum alp activity can occur within days after initiating glucocorticoid therapy in dogs and is often striking (up to times normal). glucocorticoids are associated with the induction of a specific corticosteroid-induced isoenzyme of alp, which may account for % to % of the total alp activity. in contrast, serum alt activity is often normal or only mildly increased. in most dogs, glucocorticoids do not cause significant hepatic dysfunction or clinically relevant hepatic disease and biochemical tests reflecting hepatic function (serum bilirubin, albumin, glucose, blood ammonia concentration, and coagulation tests) are typically normal. serum bile acid concentrations are normal or only mildly increased (< mmol/l). hepatic glycogen accumulation causes hepatomegaly (which can be detected on abdominal radiographs), and diffuse or multifocal increases in hepatic echogenicity (detected on ultrasonography). the hepatic effects of glucocorticoids are reversible after drug withdrawal. the length of time required for complete resolution is unpredictable, varying from weeks to months. lomustine ccnu [ -( -chloroethyl)- -cyclohexyl- -nitrosourea] is an oral nitrosourea alkylating agent that is used for chemotherapy of lymphoma, mast cell tumor, histiocytic sarcoma, and brain tumors in dogs. idiosyncratic dose-related hepatotoxicity was described in of ( . %) dogs given an oral dose of ccnu ( to mg/m ), with a dosing interval of to weeks. the median time to detection of hepatic disease (from the last dose of ccnu) was weeks and ranged from to weeks. delay in onset was noted, with an inverse relationship between the size of dose, and length of time before abnormal serum alt was detected. a cumulative dose effect was suspected. clinical findings of hepatotoxicity included glutathione precursors (nac, same) and vitamin c in treatment. dogs with hepatopathy are less likely to recover ( %) than are dogs with nonhepatic manifestations of sulfonamide hypersensitivity ( %). tetracycline can predispose to hepatic lipid accumulation because it inhibits protein synthesis and interferes with hepatic secretion of triglyceride-rich lipoproteins. however, it does not appear that these hepatic effects are clinically significant in most dogs and cats, although clinically significant idiosyncratic hepatic injury has been reported. increased liver enzyme activity was a common finding in dogs treated with doxycycline (increased alt activity in . %; increased alp activity in . %), but the clinical significance remains to be determined. aflatoxicosis aflatoxins are metabolites, produced primarily from strains of the saprophytic fungus, aspergillus, which cause toxic hepatitis in dogs and many other species. , exposure in dogs may occur through the inadvertent use of aflatoxin-contaminated corn or peanut meal during the commercial production of dog food, or after ingestion of homemade pet foods, moldy garbage, or improperly stored dog food. dogs are relatively susceptible to aflatoxins and the liver is the target organ. clinical cases of aflatoxicosis in cats have not been reported. aflatoxin b is most commonly implicated in hepatotoxicity and toxic effects are seen when levels exceed µg/kg of food. aflatoxin b is readily absorbed from the gi tract and undergoes hepatic metabolism by cytochrome p enzymes, to a toxic intermediate (aflatoxin b , -epoxide) , which binds to essential molecules within the cell, leading to hepatocyte necrosis and decreased protein synthesis. detoxification of aflatoxin b , epoxide occurs by conjugation to glutathione. depending on the amount consumed, dogs may present with acute, subacute, or chronic liver disease. high-dose exposure is associated with acute hepatic failure, jaundice, dic, and death. repeated exposure to low doses can lead to chronic liver disease and cirrhosis. in , an outbreak of aflatoxicosis occurred in at least dogs, as a result of eating a commercially available dog food manufactured with aflatoxin-contaminated corn. severity of clinical signs varied significantly among dogs. some dogs died suddenly without preexisting signs of illness. other dogs were presented with signs of anorexia, lethargy, vomiting, jaundice, diarrhea (including melena and hematochezia), abdominal effusion, he, and evidence of a bleeding disorder. common biochemical features included increased liver enzyme activity (especially alt), hyperbilirubinemia, electrolyte disturbances, hypoalbuminemia, hypocholesterolemia, and prolonged clotting times. reduced plasma antithrombin iii and protein c activities and hypocholesterolemia were suggested to be the most sensitive biomarkers of aflatoxin ingestion in dogs with minimal clinical signs, possibly reflecting an early effect of aflatoxin on biosynthesis of certain proteins and cholesterol. in dogs with acute or subacute aflatoxicosis, the liver is enlarged and pale yellow with histologic features of diffuse hepatic vacuolation (lipid accumulation), scattered individual hepatocyte necrosis, biliary hyperplasia, and modest inflammation. collapse of zone hepatocytes around the central vein, associated with perivenular inflammation, may explain clinical features of portal hypertension. with chronic low-level exposure, findings include a small liver with regenerative nodules, acquired pss, and histologic evidence of marked biliary hyperplasia and periportal fibrosis. disease. potential indicators of clinically significant liver injury include increases in alt and alp activity that exceed five times the upper limit of normal; alt activity that exceeds alp activity; any elevation in ast activity; or enzyme elevations accompanied by evidence of hepatic dysfunction (hyperbilirubinemia, hypoalbuminemia, hypocholesterolemia, increased sba). hepatic cirrhosis associated with chronic phenobarbital therapy is characterized grossly by a small, nodular liver and histologically by bridging portal fibrosis, nodular regeneration, biliary hyperplasia, and mild inflammation (see figure - ) . these lesions are by no means pathognomonic for phenobarbital-induced hepatic damage; however, in the absence of other known causes of hepatic damage, circumstantial evidence would support drug therapy as a likely cause. chronic phenobarbital therapy also is associated with superficial necrolytic dermatitis (hepatocutaneous syndrome) in dogs. liver biopsy changes were typical of those seen with hepatocutaneous syndrome (marked vacuolar change and parenchymal collapse), which are distinct from the characteristic chronic hepatitis and cirrhosis as described above. phenobarbital should be decreased or discontinued if possible in dogs with biochemical and histologic evidence of hepatic disease. in dogs with phenobarbital-associated toxicosis, clinical, biochemical, and histologic improvement can occur if the drug is discontinued or used at a reduced dosage prior to severe, end-stage liver disease. improvement in clinical signs can be noted within days to weeks of decreasing serum phenobarbital levels. primidone also is associated with chronic liver disease in dogs, likely as a consequence of metabolism of primidone to phenobarbital. phenytoin can cause acute or chronic hepatitis in dogs, as well as jaundice and death. the risk of hepatotoxicity is increased with combination therapy of phenobarbital, primidone, and phenytoin. sulfonamides potentiated sulfonamides (trimethoprim-sulfadiazine, trimethoprimsulfamethoxazole, and ormetoprim-sulfadimethoxine) are associated with the acute idiosyncratic drug-induced liver injury in dogs. trimethoprim-sulfadiazine was implicated in over % of hepatic drug reactions in dogs that were reported to the center for veterinary medicine between and . doberman pinschers are suggested to be at risk for development of polyarthropathy from sulfonamide hypersensitivity, but not necessarily the idiosyncratic hepatic reaction. onset of clinical signs occurs within to days (mean: days) from starting the drug. previous exposure to sulfonamides is not required. doses of potentiated sulfonamides are generally higher in dogs who develop the idiosyncratic hepatic reaction, as compared with other systemic manifestations of sulfonamide hypersensitivity (thrombocytopenia, fever, polyarthropathy, other). biochemical findings include increased liver enzyme activity (alt > alp) and hyperbilirubinemia. liver biopsy usually reveals marked hepatic necrosis; however, cholestasis and marked lymphocytic-plasmacytic inflammation also have been described. the pathogenesis of the idiosyncratic hepatic reaction is unclear. dogs in general may be at increased risk for sulfonamide reactions because they lack genes that express the n-acetylation enzymes, which is a major metabolic pathway of detoxification of sulfonamides in humans. however, this does not explain individual risks among dogs. hepatotoxicity may be a result of p oxidation of sulfonamides to reactive metabolites, such as hydroxylamine and a nitroso metabolite, which may be associated with hapten formation, t-cell proliferation, or direct cytotoxicity. impaired detoxification of reactive metabolites via a deficiency in glutathione, cysteine, and ascorbate, may play a role, and theoretically supports the use of hepatocyte uptake of amanitins and has been shown to be protective against experimental a. phalloides liver damage in beagles, when given at a dose of mg/kg iv twice, at and hours after exposure. however, an intravenous form of silymarin is not currently available for clinical use in the united states. experimental studies suggest that penicillin g may also reduce hepatic uptake of amanitins, even several hours after ingestions. iv nac may be beneficial as described for treatment of acetaminophen toxicity. overall mortality rate with amanita mushroom toxicity is high. ingestion of toxin-producing blue-green algae (microcystis aeruginosa) is a rare cause of hepatotoxicity and alf in dogs. algae proliferate in shallow, stagnant water, especially in hot, dry weather. dead or dying algae form a thick blue-green scum on the water's surface, and release the toxic principle, microcystins. toxicity is caused by ingestion of algae-contaminated water. signs occur rapidly (within hour of ingestion) and include vomiting, diarrhea, and lethargy, followed by progressive tachypnea and dyspnea, icterus, and coma. biochemical features reflect hepatocellular injury with increased alt and ast activity (that typically exceed increases in alp activity), and hyperbilirubinemia. however, profound or protracted increases in alt activity may not be detected, because microcystins can interfere with transaminase biosynthesis. hepatic lesions consist of massive hepatic necrosis of the centrilobular to midzonal hepatocytes. treatment is symptomatic and supportive. oxidative injury may play a role, which suggests that glutathione supplementation (nac or same) may be of benefit. the prognosis is guarded. cycads (sago palms) are native to tropical and subtropical regions, and are used as houseplants and in residential landscaping. concentrations of cycasin, the primary toxin in cycads, are highest in the seeds and roots, but present in all parts of the plant. ingestion of as few as one to two seeds can be fatal in dogs. following ingestion, cycasin is metabolized by gi bacteria to its active compound, methylazoxymethanol, which causes gi and hepatic toxicity in dogs. most dogs that ingest cycads develop gi signs, including vomiting, diarrhea, and abdominal pain. neurologic signs (weakness, ataxia, depression, proprioceptive deficits, seizures, coma) are also common, but it is not clear if they are a result of a neurotoxin or he. onset of clinical signs ranges from minutes to days and may last from hours to days. hepatic injury is suggested by findings of progressive depression, icterus, he, and excessive bleeding accompanied by increased liver enzyme activity, hyperbilirubinemia, hypoglycemia, and hypoabuminemia. , centrilobular hepatic necrosis is found on liver biopsy. no specific treatment is available. mortality has been reported to vary between % and %. , xylitol xylitol, a -carbon sugar alcohol used as a sugar substitute, is associated with hypoglycemia and hepatic necrosis in dogs. , xylitol is safe in humans and is commonly used in sugar-free gum and other oral care products, and is available as a granulated powder for baking. xylitol was first introduced into the united states in , and since that time, reports of toxicity to the aspca animal poison control center have increased from two dogs in to dogs in . ingestion of more than . g/kg in dogs is associated with a rapid, severe, increase in blood insulin, which results in signs of hypoglycemia within to minutes of ingestion. when amounts exceed . g/kg, alf may occur within to aflatoxicosis associated with consumption of a commercially manufactured pet food product should be suspected when there is a geographic or temporal cluster of cases in a household, kennel, or region. the history may reveal recent changes in diet or feeding from a new bag. it should be noted that some dogs may consume contaminated food for weeks to months before signs develop. definitive diagnosis of aflatoxicosis is based on chemical detection of increased levels of aflatoxin (> µg/kg) in the food. when aflatoxicosis is suspected, the owner should be advised to retain kg of food in an airtight zippered plastic bag (or four cans of food), for laboratory testing. it is also recommended to save packaging information, including product and date code, to help identify contaminated lots of food. if a sample of food is no longer available, serum or liver samples can be submitted for testing for aflatoxin m (aflatoxin metabolite), although usefulness may be limited because of the rapid metabolism and excretion of aflatoxin. detection of aflatoxin m in urine is only useful if the dog is still consuming the contaminated diet, as levels fall below detectable levels within hours. there is no specific antidote for aflatoxicosis and treatment consists of symptomatic and supportive management of liver failure. nonspecific hepatoprotective therapy with antioxidants (vitamin e), glutathione replacement (nac, same), milk thistle (silymarin), and l-carnitine have been recommended. the prognosis is guarded if clinical signs of aflatoxicosis are present, with a reported mortality rate of % in a series of dogs that consumed aflatoxincontaminated dog food. dogs that survive acute liver injury have the potential to develop chronic liver disease. consequently, monitoring of liver function is recommended in recovering dogs and treatment with thiol donors such as same for months has been empirically recommended. amanita mushrooms amanita phalloides (and other varieties such as amanita verna and amanita bisporigera), are poisonous mushrooms found throughout north america that can cause acute hepatic necrosis in dogs and cats. , toxicity is attributed to extremely toxic cyclopeptide toxins called amanitins. ingestion of two a. phalloides mushrooms can be lethal to an adult dog. clinical signs occur within to hours after ingestion and are characterized initially by gi signs such as vomiting, bloody diarrhea, and abdominal pain. the late phase ( to hours after exposure) is characterized by alf (hemorrhage, marked hypoglycemia, he, and terminal coma) caused by severe massive hepatic necrosis (see figure - ) . toxin-induced renal tubular necrosis may also result in renal failure. biochemical features reflect severe hepatic injury and include increased liver enzyme activity (alt exceeds alp), refractory hypoglycemia, and hyperbilirubinemia. diagnosis is usually made based on positive identification of the suspect mushroom, evidence of its ingestion, and consistent clinical features. mushroom pieces in gastric contents can confirm exposure, but are difficult to identify. accurate mushroom identification requires consultation with an experienced mycologist. the suspect mushrooms should be wrapped in paper towels and stored in a paper (not plastic) bag. definitive confirmation can be established by detecting amanitins in liver or kidney tissue (or serum and urine samples collected during the gi phase) by liquid chromatography-mass spectrometry, through the california animal health and food safety laboratory. gi decontamination procedures are recommended as soon as possible after exposure, as described in the "treatment" section of "drug and toxin-induced liver injury". symptomatic and supportive treatment for alf is indicated, including close monitoring and treatment of marked hypoglycemia. silymarin is believed to reduce lower urinary tract disease ammonium biurate uroliths are formed most of the time in the bladder (and rarely in the renal pelvis) and can cause dysuria, urethral obstruction in males, and seldom uroabdomen as a result of chronic inflammation and subsequent devitalization of the bladder wall. ammonium biurate crystalluria is not pathognomonic for portosystemic shunting, and it may occasionally occur in normal dogs and cats, or be found in certain breeds, such as dalmatians, because of an inborn error of metabolism. prolonged recovery from anesthesia or sedation may occur in seemingly normal dogs and cats that have cpss. the liver plays a crucial role in the detoxification process of toxins and drugs, including anesthetics. patients with cpss or apsc have insufficient functional hepatic mass for detoxification. in addition, dogs with portosystemic shunting have increased endogenous benzodiazepine and gabaergic activities. therefore administration of diazepam or barbiturate to these animals may have prolonged and exaggerated effects (see "pathogenesis" section). recurrent fever together with resultant depression and anorexia can be the only clinical manifestation of cpss in some dogs. portosystemic shunting should be excluded in dogs experiencing recurrent fever. this rare manifestation has only been reported in a few dog. its pathogenesis is not understood. elderly dogs with extrahepatic cpss may present with characteristic signs of hypercortisolism (e.g., pu/pd, polyphagia, thin skin, symmetric alopecia, muscle wasting, pot belly) as primary complaints. these are unusual clinical manifestations of cpss. although small body size is observed in cats with cpss, it infrequently occurs in dogs. many dogs with extrahepatic cpss show no or only nonspecific clinical signs throughout their lives and the shunt is detected as a coincidental finding at the time of necropsy. it is not understood why certain dogs become clinically ill and others do not with the same type of portal vein anomaly. cpss never cause icterus, ascites, or spontaneous hemorrhages. portal vein disorders are much less common in cats than in dogs. most cats are younger than months of age when the first signs appear. cats with cpss are often of smaller stature and have unkempt hair coat. episodic salivation and/or central neurologic signs, for example, compulsive pacing or seizures, as manifestations of he are the most typical presenting complains. , portal hypertension clinical signs can develop at any age and arise from the presence of increased hydrostatic portal venous pressure, portosystemic hours. alf is not necessarily preceded by early signs of hypoglycemia. the mechanism of hepatotoxicity is unknown, but has been speculated to be caused by cellular depletion of adenosine triphosphate resulting in hepatocellular necrosis, or production of reactive oxygen species causing oxidative injury. in addition to signs of hypoglycemia, dogs with alf may have vomiting, icterus, and evidence of excess bleeding. biochemical findings include markedly increased alt and ast activity, mild to moderate increased alp activity, hyperbilirubinemia, hypoglycemia, hyperphosphatemia, prolonged pt and aptt, and thrombocytopenia. if ingestion occurred within the last few hours, induction of emesis is recommended (unless showing signs of hypoglycemia). activated charcoal may be of limited value in adsorbing xylitol, but is still recommended if large amounts have been ingested. treatment recommendations include hospitalization for observation and monitoring, dextrose supplementation for control of hypoglycemia, and symptomatic and supportive care for complications of liver failure. hepatoprotective therapy with nac, same, and silymarin may be beneficial. the prognosis is good for recovery in dogs with uncomplicated hypoglycemia and guarded to poor for dogs in liver failure. however, survival after liver failure does not necessarily correlate with amount of xylitol ingested. vascular hepatic diseases include congenital and acquired disorders of the portal vein. congenital anomalies result from (a) hypoplasia or aplasia of the portal vein, (b) macroscopic communications between the portal vein and a systemic vein, or (c) between the portal vein and an artery. acquired diseases result from conditions that increase the hydrostatic pressure in the portal vein (i.e., portal hypertension). macroscopic venous connections between the portal and systemic venous systems result in portosystemic shunting (i.e., blood flows from the portal to the systemic veins) via a congenital portosystemic shunt (cpss) or acquired portosystemic collaterals (apsc). neurologic signs hepatoencephalopathy (he) is a reversible central neurologic manifestation of hepatic insufficiency. cpss causes chronic he. the following grades of chronic he are recognized : grade -depression, behavior changes; grade -ataxia, compulsive pacing, circling, hypersalivation, head pressing, blindness; grade -stupor and seizures; and grade -coma. chronic he is characterized by periods of severe (grades to ) signs (lasting usually several hours to a few days alternating with longer periods (days to weeks) of no or mild (grade ) symptoms. periods of cortical blindness are accompanied by apparent mydriasis. signs of he may be triggered by ingestion of protein-rich meals. pd means excessive fluid intake (in dogs > ml/kg body weight/ h and in cats > ml/kg body weight/ h). pu may be more difficult to diagnose than pd, as pet owners cannot readily measure urine volume. repeated low specific gravity of morning urine (< . in dogs and < . in cats) is compatible with pu. macroscopic connections exist between major vessel systems or their tributaries. single or multiple portosystemic venous connections allow the portal venous blood to flow directly to the systemic venous system without first flowing through the hepatic sinusoids. this toxin-rich blood will be delivered to all cells of the body through the following route: gut > portal vein > shunt > systemic vein > right heart > lungs > left heart > arteries. a connection through a single, or rarely double, large-bore vein without the presence of portal hypertension is considered to be a cpss. single or multiple connections in the presence of portal hypertension are apsc. whenever a macroscopic venous connection is present between the portal vein (or one of its tributaries) and a systemic vein, the blood will flow from the portal vein to the systemic vein, because the pressure in the portal vein is higher ( to mm hg) than that in the systemic veins ( - mm hg). normal portal venous pressure is approximately to mm hg ( to cm h o). increased pressure in the portal venous system results in portal hypertension. anatomically, portal hypertension can be classified as (a) prehepatic (i.e., portal vein), (b) intrahepatic or (c) posthepatic (i.e., hepatic veins, thoracic caudal vena cava, or heart). posthepatic (postsinusoidal) portal hypertension. all cardiac diseases that result in right-sided congestive heart failure cause posthepatic portal hypertension. these diseases include the (a) congenital or acquired severe insufficiency of the tricuspid valve (e.g., dysplasia, myxomatous degeneration, annulus dilation caused by dilated or arrhythmogenic cardiomyopathy, pulmonary hypertension of various etiologies), (b) pericardial diseases (pericardial tamponade caused by idiopathic or neoplastic effusion or constrictive pericarditis), (c) various congenital anomalies such as cor triatriatum dexter, atrial septum defect, tricuspid or pulmonic stenosis, (d) intracardiac tumors affecting the right heart, and (e) caval syndrome (caused by dirofilaria immitis heart worms). kinking or compression of the thoracic caudal vena cava (by diaphragmatic hernia or a mass) occurs rarely. compression, stenosis or thrombosis of the hepatic veins (the so-called budd-chiari syndrome) does not occur in dogs and cats. , a in posthepatic portal hypertension the portal and caval pressures increase equally. therefore no apsc develop, and the blood ammonia concentration remains within reference range. the high postsinusoidal hydrostatic pressure causes a large amount of protein-rich hepatic lymph to be filtered through the hepatic capsule into the abdominal cavity causing accumulation of a modified transudate. modified transudate has high protein content because the fenestrated endothelial cells of the hepatic sinusoids keep only % to % of the plasma proteins in the capillary lumen. marked generalized hepatomegaly and dilated jugular and hepatic veins as well as caudal vena cava are hallmarks of postsinusoidal portal hypertension. intrahepatic portal hypertension. chronic acquired parenchymal liver diseases lead to hepatocyte necrosis and subsequent collagen deposition. the resultant disorganization of the hepatic architecture and contraction of the connective tissue results in obstruction of the intraparenchymal vessels. the underlying disease processes include viral, bacterial, protozoal, immune-mediated, or copperassociated chronic hepatitis; toxic, drug-induced, or idiosyncratic liver damage; lobular dissecting hepatitis; and chronic bile duct shunting via apsc, and the underlying disease that led to portal hypertension. clinical signs of portosystemic shunting are similar, regardless whether it is congenital or acquired in origin (see "clinical manifestations" section). accumulation of a large amount of pure transudate or modified transudate (clear, straw-colored, or slightly turbid blood-tinged fluid) in the abdominal cavity is commonly detected with physical examination and abdominocentesis. the absence of free abdominal fluid does not exclude portal hypertension. depending on the etiology and the anatomic location of the underlying disease that led to the development of portal hypertension various signs may be seen such as jaundice, periodic vomiting, and anorexia. jaundice and hemorrhagic diathesis are absent in congenital vascular disorders. congenital arterioportal fistula causes transudative ascites in puppies between and months of age. primary hypoplasia of the portal vein (phpv), also described as "noncirrhotic portal hypertension," may result in ascites, vague gi signs, and he, or may be entirely subclinical. , when the phpv is not severe enough to cause portal hypertension, clinical signs may not be obvious. this mild form of phpv is also known as hepatic microvascular dysplasia and may be detected serendipitously if plasma bile acid concentrations are measured for unrelated reasons. portal hypertension is rare and is not typically associated with ascites in cats. portosystemic shunting because of cpss or apsc tend to cause similar signs, for example, periodic salivation and seizures, as manifestations of chronic he. the most common acquired disease causing intrahepatic portal hypertension is biliary cirrhosis caused by chronic bile duct obstruction. jaundice and acholic feces are typical findings in extrahepatic cholestasis. the liver receives its blood supply from the portal vein (approximately %) and the hepatic artery (approximately %). with diversion of portal venous blood flow, a compensatory increase of the hepatic arterial flow occurs. arterial vasodilation is mediated by adenosine from energy-depleted hepatocytes. the portal vein transports blood from the spleen and the gi tract to the liver. the smallest portal venous and hepatic arterial branches terminate in the capillary system of the liver, the so-called hepatic sinusoids. a large amount of plasma is filtered through the fenestrated walls of the sinusoids to the space of disse. from here the filtered plasma is taken by lymphatic vessels to the systemic venous system. the remaining sinusoidal blood is then collected by the hepatic veins, which enter the caudal vena cava. normally no the hepatofugal portal flow prevents the splanchnic venous blood from entering the liver and causes the development of alternative pathways and often times accumulation of pure transudate in the abdomen. interestingly, phpv always accompanies congenital arterioportal fistulas both in dogs and cats. , , when a cpss and arterioportal fistulas are concomitantly present, apsc and ascites do not develop. arterioportal fistula can be extrahepatic in cats. acquired portosystemic collaterals. in healthy mammals, multiple nonfunctional venous connections may exist between the portal and systemic veins. these virtual communications become functional when their lumen becomes sufficiently widened. gradual dilation takes place when sustained increase of the portal pressure takes place without the simultaneous increase of caval pressure. the resultant apsc are multiple, usually thin, tortuous veins with species-specific anatomical locations; however, large-bore veins may also develop. these latter cases should not be misinterpreted as simultaneous cpss and apsc. in the presence of an existing cpss, no apsc would develop even if hepatic cirrhosis develops as there is an already existing portosystemic connection that is able to drain % of the portal blood without allowing the development of portal hypertension. splenorenal collaterals are consistently present in almost every dog with apsc. , these apsc drain the portal venous blood via the splenic vein through acquired connections to the left gonadal vein. the left gonadal vein enters the left renal vein, which later empties into the caudal vena cava. these splenorenal apsc are thought to prevent the spleen from undergoing congestion, which is why splenomegaly is not a feature of canine pre-and intrahepatic portal hypertensive disorders. shunting of the portal blood is not only detrimental for the brain, but for the liver as well. normal hepatic development and function requires sufficient amount of portal venous perfusion of the hepatic sinusoids. increased arterial perfusion is unable to compensate for portal hypoperfusion. regardless of whether the insufficient portal venous perfusion is caused by cpss or by prehepatic portal hypertension, the result is the same: reduced hepatic mass and function. in both cases, histopathologic evaluation of the liver shows stereotypical reaction: small or invisible portal branches, increased number of arterioles and sometimes bile ductules in the portal tracts, hepatocellular atrophy, and periportal sinusoidal dilation. this secondary portal vein hypoplasia is reversible and is histologically indistinguishable from phpv. because primary and secondary portal vein hypoplasia show identical microscopic features, histopathologic evaluation of liver biopsy specimens is unable to diagnose simultaneous phpv and cpss. glycine and gaba are the most important inhibitory neurotransmitters, whereas glutamate and dopamine are the most abundant excitatory neurotransmitters in the brain. in he, a net increase in inhibitory transmission occurs. this results from upregulation of gaba receptors and downregulation of dopamine receptors. activation of gaba receptors causes opening of chloride channels, which leads to hyperpolarization of the postsynaptic membrane. in the presence of gaba, benzodiazepines increase the frequency, whereas barbiturates increase the duration of the chloride channel opening. the cause of increased gabaergic tone in he is thought to be gut derived, but gaba may also be formed in the neurons from glutamate. endogenous benzodiazepines are proven to be obstruction. the resultant increase in portal pressure may cause development of apsc as well as ascites. modified transudate may accumulate in the abdominal cavity when the disease process predominantly obstructs the postsinusoidal hepatic venules. however, when the disease process affects predominantly the intrahepatic portal vein branches (presinusoidal portal hypertension) pure transudate will accumulate in the abdomen. primary hypoplasia of the portal vein is caused by insufficient development of the intrahepatic portal venous branches, the left portal vein branch, or the whole portal venous system. , when the hypoplasia is severe enough to cause intra-or prehepatic portal hypertension, apsc develop. this condition is also known as "noncirrhotic portal hypertension." if hypoplasia is not severe enough to cause portal hypertension, clinical signs will not develop. because no portosystemic shunting is present, the results of rectal ammonia tolerance test (att), portal scintigraphy, and portography are all normal (see "differential diagnosis" section). if either macroscopic or microscopic portosystemic communications is present, hyperammonemia or abnormal att should also be present, but this has never been documented. the only abnormality that could be detected in these dogs is elevation of serum bile acids levels. the most plausible explanation for this is that the hepatic clearance of bile acids by the hypoperfused liver is probably less effective than that of ammonia. the disease can only be diagnosed by histopathologic examination of liver biopsy specimens. this condition also has been described as "hepatic microvascular dysplasia." although the terms noncirrhotic portal hypertension and hepatic microvascular dysplasia have been used to describe this syndrome, both have been replaced by the term phpv. the occurrence of phpv in the cat is very rare and poorly documented. the most common congenital cause of portal hypertension in cats is congenital hepatic fibrosis as a part of polycystic kidney and liver disease complex. this disease may cause clinical signs with or without the presence of macroscopic hepatic or renal cysts. prehepatic portal hypertension. narrowing of the portal vein lumen may be caused by (a) extravascular compression by a tumor, enlarged lymph node, cyst, abscess, or hematoma, (b) idiopathic circumscribed stenosis, or (c) intravascular obstruction by a thrombus or parasites, all of which can lead to portal hypertension. portal vein thrombosis is always secondary either to neoplasia, or to a systemic disorder that causes hypercoagulability such as nephrotic syndrome, immune-mediated hemolytic anemia, hypercortisolism, acute pancreatitis, peritonitis, or sepsis. parasites in the portal vein also may occur, for example, heterobilharzia americana in north america and schistosoma japonicum in east asia. depending on the degree of portal vein occlusion, increased portal pressure with apsc and ascites may develop. as the hydrostatic pressure in the sinusoids does not increase (in contrast to posthepatic portal hypertension), the resultant ascitic fluid will have a low protein content. the narrowed lumen of the portal vein causes reduced portal flow to the liver, resulting in reduction in the size of the liver. portal vein thrombosis itself rarely causes clinical signs and is usually a coincidental finding. when the liver receives little portal venous blood, an insufficient amount of substrate (i.e., ammonia) is available for hepatic urea production. this theoretically results not only in a low plasma urea concentration, but also in a lower renal medullary urea concentration, which impairs renal concentrating ability and causes pu. increased basal plasma concentrations of acth and cortisol as well as increased urinary cortisol-to-creatinine ratios are invariably present in dogs with portosystemic shunting. [ ] [ ] [ ] [ ] cortisol interferes with the action of arginine-vasopressin at the renal tubule, causing a nephrogenic-type diabetes insipidus. hypersecretion of acth (and α-melanocyte stimulating hormone [α-msh]) has been shown to arise predominantly from the intermediate lobe of the pituitary. , the hormone secretion of this lobe is regulated by tonic dopaminergic inhibition. acth-hypersecretion can be explained by the production of "false" neurotransmitters (e.g., octopamine), whose effect is about one-fiftieth that of dopamine on the dopamine receptors. central diabetes insipidus also contributes to pu in dogs with he. impaired release of arginine-vasopressin from the posterior lobe of the pituitary is caused by a reduced magnitude of response and a highly increased threshold to increased plasma osmolality. release of arginine-vasopressin is inhibited by the gaba inhibitory neurotransmitter system, whose activity is increased in he. , pseudohyperaldosteronism cortisol and aldosterone have similar affinities to bind aldosterone receptors. however, cortisol is normally inactivated by β-hydroxysteroid dehydrogenase in tissues where aldosterone action is required. high serum bile acids concentrations inhibit this enzyme, and cortisol can bind to aldosterone receptors resulting in increased mineralocorticoid effect. plasma cortisol concentrations are -fold those of aldosterone, causing constant and inappropriate pseudohyperaldosteronism. the resultant sodium retention causes secondary water retention and subsequent pu by pressure diuresis. hypokalemia caused by hyperaldosteronism also contributes to pu , according to the following mechanism. the presence of aquaporin- channels in the renal collecting ducts' cell membranes is necessary for water reabsorption. intracellular signaling pathways through cyclic adenosine monophosphate regulate the insertion of these channels. hypokalemia decreases the sensitivity of cyclic adenosine monophosphate to arginine-vasopressin, which results in decreased insertion of aquaporin- channels into the cell membrane. this leads to nephrogenic diabetes insipidus and pu. congenital portal venous anomalies in dogs are typically associated with enlarged kidney volume. increased renal gluconeogenesis as a compensation of insufficient hepatic gluconeogenesis may cause the kidneys to enlarge. in addition, increased systemic circulating growth factor concentrations released from the pancreas may play a role in this increased volume. normally, these growth factors act only in the liver, as they do not reach the systemic circulation in high concentrations. behavior changes and abnormalities in the thirst center due to he may contribute to pd; however this is difficult to prove in individual patients. produced in the intestines of dogs with cpss. their source is not quite clear: they could arise from the diet, intestinal flora, or by endogenous modification of inactive gut precursors. in all forms of portosystemic shunting the concentration of aromatic amino acids are increased in the systemic circulation whereas the concentration of branched-chain amino acids is decreased. high aromatic amino acid concentration results from impaired hepatic clearance and increased production caused by muscular breakdown exacerbated by hyperglucagonemia. , the decreased concentration of branched-chain amino acids results from their increased utilization for gluconeogenesis. , this amino acid imbalance may result in the development of "false" neurotransmitters (e.g., octopamine) in the brain, which contribute to the development of he. , these "false" neurotransmitters have a fraction of dopamine's excitatory effect on dopamine receptors. drugs with dopaminergic effect, such as bromocriptine, may improve signs of he. [ ] [ ] [ ] intestinal toxins such as ammonia and bacteria are normally inactivated by hepatocytes and hepatic macrophages (kupffer cells), respectively, so that toxin-and bacterium-free blood can enter the systemic circulation. in patients with portosystemic shunting the majority of the portal venous blood bypasses the liver, allowing the toxin-and bacterium-rich blood to enter the systemic circulation. ammonia is a neurotoxin and can contribute to the clinical signs of he. other toxins that are believed to play a role in he are endogenous benzodiazepines, gaba, tryptophan, glutamine, serotonin, mercaptans, indoles, and skatoles. , , hyperammonemia also leads to impaired glial function through increased intracellular glutamine concentration. glutamine is made in the glial cells through incorporation of ammonia into glutamate by glutamine synthetase. chronically increased glutamine concentrations cause swelling of the glial cells resulting in so-called alzheimer ii type degeneration of astroglias. , glial dysfunction contributes to the development of he. , during hyperammonemia, the reserve capacity of the astrocytic glutamine synthetase is exceeded, and ammonia can enter the neurons. in the neurons ammonia inhibits glutaminase, an enzyme that converts glutamine to glutamate. because glutamate is an excitatory neurotransmitter, its reduced level contributes to the development of he. portosystemic shunting or an atrophic liver alone is insufficient to allow he to develop; he can only develop when they are both simultaneously present. the reserve capacity of the liver ensures that even in advanced chronic parenchymal liver diseases, he does not develop in the absence of portosystemic shunting. alkalosis and hypokalemia can worsen the signs of he. in alkalosis the nh + h + = nh + reaction shifts to the left, causing the more lipophilic ammonia to enter the cells of the cns. during hypokalemia, potassium ions (k + ) move from the cells to the extracellular space in exchange for h + , which later results in extracellular alkalosis and intracellular acidosis. intracellular acidosis causes nh + trapping within the cells. this might explain why the blood concentration of ammonia is not closely related to the severity of the clinical signs of he in individual cases. several mechanisms contribute to the development of pu/pd in portosystemic shunting. high concentrations of sodium and urea in the renal medullary interstitium are essential for the production of concentrated urine. these create a high osmotic gradient between the renal tubular lumen and interstitium, which is necessary for water reabsorption. to result from reduced enzyme activity of the urea cycle. as no clinical signs are present, no treatment is required. the condition resolves spontaneously with age because these pups develop enhanced incorporation of ammonia into glutamine. peritoneal absorption of ammonia-containing urine can cause hyperammonemia, however in such a patient the clinical signs of acute uremia predominate. the presence of urease-producing bacteria (e.g., staphylococci) are necessary to split urinary urea to ammonia. fulminant hepatic failure ingestion of blue algae or certain mushrooms (e.g., a. phalloides) results in peracute insufficiency of hepatic function. these animals develop hepatic coma and die shortly thereafter. hyperammonemia, dic, and icterus are present in most affected patients. arginine is an essential amino acid in the cat. in anorectic cats hyperammonemia may develop along with hepatic lipidosis because of the insufficient amount of hepatic arginine needed for urea synthesis in the urea cycle. thus, in contrast to adult dogs, hyperammonemia in cats can occur without portosystemic shunting. methylmalonic acidemia associated with cobalamin deficiency in dogs, a a cat and a suspected "transient hyperammonemic syndrome" in a german shepherd dog have been reported as extraordinarily rare causes of hyperammonemia. if blood sampling and sample processing are performed inappropriately hyperammonia may be erroneously diagnosed. high bile acids concentration may result not only from portosystemic shunting, but also from any primary or secondary hepatic diseases associated with intra-or extrahepatic cholestasis. therefore, the presence of high serum bile acids concentrations is very sensitive, but not a specific indicator of portal venous disorders. the simultaneous presence of hyperammonemia and a large amount of pure or modified transudate in the abdominal cavity indicates the presence of severe pre-or intrahepatic portal hypertension with apsc. biochemical and cytologic analysis of the peritoneal effusion, ultrasonography and central venous pressure measurement may be useful in identifying the source of ascites. suspected intrahepatic causes should be further evaluated by histopathologic examination of liver tissue. in any animal that is presented with central neurologic signs, portosystemic shunting (along with other metabolic encephalopathies, e.g., hypoglycemia or electrolyte imbalance) should be investigated with appropriate blood tests. no single finding is pathognomonic for diagnosing vascular liver disorders. therefore, a combination of history, physical hyperammonemia results in a higher filtered load and urinary concentration of ammonia. high urinary concentration of uric acid results from decreased hepatic conversion of uric acid to allantoin because of reduced hepatic mass and function. the portal vein carries bacteria and endotoxins from the intestines, which are normally phagocytized by the kupffer cells. shunting of portal blood permits these bacteria and/or endotoxins to enter the systemic circulation causing bacteremia and/or endotoxemia, and subsequent fever. systemic antibiotics may result in temporary resolution of clinical signs, however give no definitive cure. if a cpss occurs within hepatic parenchyma it is called intrahepatic. , intrahepatic cpss arise either from the left or right portal branches. a left divisional intrahepatic cpss results from the failure of postnatal closure of the embryological ductus venosus. , the intrahepatic cpss arising from the right portal branch and all extrahepatic cpss are thought to be developmental anomalies with poorly understood etiology. in dogs the anatomy of the shunting vessel is fairly consistent: intrahepatic cpss may be left, right, or central divisional, whereas extrahepatic shunts drain the portal venous blood via the splenic or the right gastric vein to the caudal vena cava or the azygos vein. the course of the shunting vein in the cat is much more variable. , the inherited nature of the disease has been established in several breeds including the irish wolfhound and the yorkshire and cairn terriers. [ ] [ ] [ ] phpv is a congenital anomaly of unknown etiology. although arterioportal fistulas may develop as a result of neoplasm or trauma (e.g., shot wound or liver biopsy), only the congenital form has been reported in dogs and cats. congenital hepatic fibrosis caused by polycystic kidney disease (pkd) is a genetic disease. , differential diagnosis high fasting venous blood ammonia concentrations together with high fasting serum bile acid concentrations are very specific and sensitive tests for diagnosing portosystemic shunting. urea cycle enzyme deficiency reduced activity of one or more enzymes of the urea cycle may result in elevated blood ammonia concentration. , serum bile acids levels should remain within reference range in patients with urea cycle enzyme deficiencies. normal hepatic scintigraphy and liver histopathology results should confirm the absence of portosystemic shunting. certain metabolites (e.g., citrulline) and enzyme activities (e.g., argininosuccinic acid synthetase) can be measured in urine and liver biopsy specimens, respectively, to establish a definitive diagnosis. the condition is rare in dogs. one suspected feline case has been reported. irish wolfhound puppies healthy irish wolfhounds commonly have moderate hyperammonemia (< µmol/l) at the age of to weeks. this is thought chloride (nh cl) solution is administered at a dose of ml/kg fifteen cm into the descending colon using a soft feeding tube. venous blood ammonia is measured before and and minutes thereafter. in the presence of portosystemic shunting the ammonia concentration will increase at least twofold by the -and/or -minute sampling times. normal att result excludes portosystemic shunting (figure - ) . the degree of increment is a semiquantitative measure of the degree of portosystemic shunting. it should be noted that rectal administration of nh cl solution may cause transient irritation of the colonic mucosa during the first minutes. rectal att is a safe procedure, with signs of he rarely occurring. att should not be performed in patients with very high (> µmol/l) blood ammonia levels. postprandial measurement of ammonia is thought to decrease the possibility of iatrogenic hyperammonemic he; however the increase of blood ammonia concentration after feeding takes longer and its peak time point is poorly predictable. markedly increased preprandial (i.e., -hour fasting) bile acids concentrations are often found with portosystemic shunting as a result of interrupted enterohepatic circulation of the bile acids. , high -hour fasting plasma bile acid concentration is a sensitive, but nonspecific indicator of portosystemic shunting. specificity can be increased by simultaneous measurement of venous ammonia. as plasma bile acids concentrations are invariably high in icteric animals, the presence of apsc can only be justified or excluded by measuring blood ammonia concentration or performing an att. in case of a normal -hour fasting bile acids concentration (< µmol/l), measuring an increased postprandial plasma bile acids concentration (> µmol/l) hours after a meal increases the sensitivity, but not the specificity of the bile acids test in diagnosing portosystemic shunting. meal-induced gallbladder contraction causes an endogenous bile acid load, which will be absorbed in the ileum and will substantially increase the plasma bile acids concentration when portosystemic shunting is present. an abnormal postprandial increase of bile acids occurs also in cholestatic liver diseases. in patients with portosystemic shunting hypoalbuminemia, hypoproteinemia, hypocholesterolemia, and low plasma urea concentration may be found as a result of their reduced hepatic synthesis. hypoglycemia may be seen as a result of reduced hepatic glyconeogenesis and glycogen storage. low creatinine concentration reflects the increased glomerular filtration rate. none of these biochemical changes is specific for portosystemic shunting; furthermore, many of these findings may be present in healthy pups. the cause of mild increase of plasma alt and alkaline phosphatase activities is not fully understood. microcytosis with or without mild nonregenerative anemia and leukocytosis may accompany portosystemic shunting. relative iron deficiency is thought to cause the microcytosis [ ] [ ] [ ] and bacteremia may induce the leukocytosis. although aptt is often moderately prolonged in dogs with cpss, no spontaneous bleeding tendency or hemorrhage occurs. , hemoabdomen is a possible postoperative complication after surgical attenuation of a cpss. acquired parenchymal hepatic diseases examination, laboratory tests, diagnostic imaging results and histopathology of liver biopsy specimens are often required to establish a specific diagnosis. intrahepatic cpss tend to cause clinical signs between months and year of age in large-breed dogs. bernese mountain dogs, irish wolfhounds, hovawarts, and retrievers are predisposed. australian cattle dogs and male dogs more likely have right-sided than leftsided intrahepatic cpss, whereas irish wolfhounds tend to have left-sided intrahepatic cpss. intrahepatic cpss in small-breed dogs are very rare. extrahepatic cpss usually occur in small breeds and can cause clinical signs at any age. the most commonly affected breeds are maltese dogs, miniature schnauzers, dachshund, yorkshire terrier, jack russell terrier, and cairn terrier. no clear sex predisposition is known. the waxing-waning nature of central neurologic signs is suggestive of chronic he. symptoms may improve spontaneously. physical examination often fails to detect abnormalities. occasionally an enlarged left kidney is palpated. the presence of ascites detectable with physical examination should exclude cpss. some authors suggest that copper-colored iris is a typical finding in cats with cpss; however, no reports exist about its positive or negative predictive value. dogs with intra-or prehepatic portal hypertension may have ascites. jaundice is absent in congenital portal hypertensive disorders, but may be present in acquired parenchymal liver diseases. blood ammonia concentration fasting ( -hour) venous hyperammonemia is a very specific and sensitive indicator of portosystemic shunting. as a single test, increased blood ammonia level is usually sufficient to diagnose portosystemic shunting. because ammonia is formed from amino groups of proteins and urea also in the collection tube, the blood sample (in an ethylenediaminetetraacetic acid tube) should be placed directly on ice after sampling and the measurement should be performed within minutes. because contamination of the sample with airborne ammonia may cause false-positive results, samples should be taken using a closed system (needle on a syringe or directly into a vacutainer). hemolysis may artificially increase ammonia concentration because erythrocytes contain two to three times more ammonia than the plasma. measurement of ammonia should be performed in a clean location, where the air is not contaminated with ammonia from congested urine or cigarette smoke. a number of analyzers offer the possibility of ammonia measurement; however some of them are unreliable. measuring arterial ammonia concentration provides no additional value. if venous ammonia concentration is within the reference range or only slightly elevated, rectal att is the cheapest and quickest test to exclude or justify the presence of portosystemic shunting in patients with a suggestive history. most of these "suspicious" animals also have high fasting plasma bile acids levels. a % ammonium anomalies, a high-resolution grayscale ultrasound is often sufficient to establish a definitive diagnosis. although certain secondary changes (such as small liver, large kidneys with hyperechoic medulla, and sediment or stones in the urinary bladder) suggest the presence of cpss, diagnosing a cpss requires the visualization of the anomalous vein from its origin to its termination. the urinary bladder should always be evaluated for the presence of urinary calculi. ultrasonography can be used not only during the initial diagnostic workup, but also during the surgical treatment and postoperative followup of cpss. intraoperative grayscale ultrasonography facilitates localizing the course of an intrahepatic cpss, whereas intraoperative color and spectral doppler examination helps to determine the optimal degree of shunt attenuation. scintigraphy free m tc-pertechnetate administered into the colon is absorbed into the portal vein and appears in the liver first in animals without portosystemic shunting, or in the heart in patients with portosystemic shunting. isotopically labeled albumin macroaggregates may be directly administered into a splenic vein with ultrasound guidance. the macroaggregates are trapped in the first capillary bed, which is normally the liver. the fraction of portal may lead to spontaneous hemorrhages because of the presence of dic. plain radiographs give very limited additional information (such as small liver, possibly enlarged kidneys, and sometimes visible uroliths) to the history and laboratory results, so they don't have to be part of the routine diagnostic workup of vascular liver diseases. pure ammonium biurate uroliths are radiolucent. abdominal ultrasonography is the first choice of diagnostic imaging modality, once the presence of portosystemic shunting has been established by fasting hyperammonemia or abnormal rectal att (see figure - ). the major advantage is that ultrasonography requires no sedation or anesthesia. its drawback is in the operator dependence. by using a systematic examination protocol one can accurately differentiate cpss from apsc, and intrahepatic from extrahepatic cpss, as well as readily diagnose arterioportal fistulas and prehepatic portal hypertensive disorders. , , although colorflow doppler highly facilitates evaluation of portal vascular injection of iodinated contrast agent into the portal vein or into one of its tributaries under fluoroscopy used to be the only form of diagnostic imaging available to diagnose anomalous vessels. angiography allows identification of extra-and intrahepatic cpss as well as apsc. the major drawback of selective portography is its invasiveness and the need for general anesthesia. whereas mesenteric portography requires catheterization of a mesenteric vein during laparotomy, splenic portography can be performed by ultrasoundguided percutaneous injection of contrast material into a parenchymal splenic vein. visualizing portal vein segments with hepatofugal flow or shunt segments with hepatopetal flow (figure - ) is problematic, as no contrast reaches these parts of the vessels. blood that bypasses the liver will be trapped in the pulmonary capillaries. splenic venous injection of isotopes makes exact calculation of the shunting fraction possible (i.e., activity in lungs/activity in liver + lungs); however, the procedure requires anesthesia. scintigraphy is the gold standard diagnostic imaging method to justify or exclude the presence of portosystemic shunting. however, differentiating congenital from acquired portosystemic shunting or intrahepatic from extrahepatic cpss is not possible. information provided by a scintigram is a "yes" or "no" answer to the question: does the patient have portosystemic shunting? this answer can be reached much more easily and without using radiopharmaceuticals by documenting a single high blood ammonia concentration or by rectal att. images (a, b) . arrows indicate the direction of blood flow. a and b, intraoperative color-flow doppler ultrasound images of the portal vein at the point where the congenital splenocaval shunt originates. note that the diameter of the shunt (sh) is larger than that of the portal vein (pvcaudsh). the portal vein cranial to the origin of the shunt (pvcrsh) becomes narrower than the portal vein caudal to the shunt origin (pvcaudsh) because of hypoperfusion. a and c, because blood always flows toward the lowest resistance, % of the portal venous blood flows through the shunt (sh) to the caudal vena cava (cvc). note that the blood from the gastroduodenal vein (gdv) finds lower resistance to flow caudally toward the shunt, than toward the liver. this creates a hepatofugal flow (i.e., flow away from the liver) in the portal vein segment between the entering point of the gdv and the origin of the shunt (pvcrsh). note that the portal vein becomes even narrower cranial to the point where the gdv enters it (pvcrgdv). the diameter of the various portal vein segments varies because of the varying amount of blood that flows through a certain segment. b and d, note that partial occlusion of the shunt increases the resistance in the shunt to such an extent that the blood from the splenic vein (splv) finds lower resistance to flow through the shunting vessel toward the portal vein. this reversed flow in the shunting vessel (*) prevents the portal venous blood from shunting. thus, even though the shunt is only partially closed, it is nonfunctional. also note that the blood in the whole length of the portal vein is forced to flow toward the liver (i.e., hepatopetal flow direction), establishing normal perfusion of the sinusoids. a portion of the splenic venous blood will continue to flow through the attenuated shunt to the cvc, but the splenic blood contains no more toxins than any systemic vein, so no hyperammonemia develops. rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. pine receptor increases the effect of gaba on its gaba areceptor. the cornerstone of therapy in cases of cpss (until definitive surgical therapy) and in all cases of apsc is a high-quality, low-protein diet. , , , commercially available renal prescription diets are ideal and preferable to most hepatic diets, as the protein content is higher in the latter. to decrease colonic transit time and reduce the production of ammonia, oral administration of lactulose is recommended. the dosage of lactulose should be titrated in individual patients to yield soft feces, but not diarrhea. an initial dosage . ml/kg q h is recommended. if diarrhea results the dosage should be reduced. lactulose also stimulates the growth of colonic bacteria that can incorporate ammonia into bacterial protein. additional use of antibiotics (e.g., neomycin, metronidazole) has been recommended by some to diminish ammonia-producing colonic flora. antibiotic administration is usually not necessary to control clinical signs of he; moreover, neomycin is thought to antagonize the action of lactulose and may cause the release of endotoxins. furthermore, chronic antibiotic use may contribute to the development of antibiotic resistance. surgical closure of apsc is generally impossible because of their multiple nature, but also contraindicated, as apsc are usually a compensatory mechanism to resolve high portal venous pressures. surgical narrowing (i.e., banding) of the caudal vena cava to reduce shunting by increasing the caval pressure is currently not recommended. in cases of cpsss, attenuation or complete closure of the shunting vein is the choice of treatment. the goals of shunt occlusion are (a) reducing portal flow via the cpss and (b) simultaneously increasing portal venous perfusion of the liver. the shunt should be attenuated as close as possible to the point where it enters the systemic circulation. attenuating intrahepatic cpss is more risky because of the possibility of major bleeding because of excessive dissection of hepatic parenchyma. there are several techniques described, of which none are perfect. the major problem with shunt reduction is that blood from the portal vein will be redirected to newly perfuse the liver and its vasculature. poorly developed portal branches have insufficient capacity to accept even normal amounts of portal venous blood, and in the case of complete shunt occlusion, splanchnic congestion will develop. the goal of surgery is to sufficiently reduce the amount of shunting blood without causing portal hypertension to develop. although complete shunt occlusion would theoretically be ideal, partial shunt attenuation is often sufficient because (a) partial attenuation often results in functional closure (see figure - ), and moreover (b) complete anatomic occlusion may follow in many cases. partial occlusion of extrahepatic cpsss often results in a better outcome because the chance for development of postligation portal hypertension is much smaller compared to complete occlusion. the liver, which may be less than % of its normal size, grows very quickly following successful surgery. regeneration may take to weeks to complete in uncomplicated cases. the regenerating liver receives progressively more portal blood flow usually resulting in spontaneous complete closure of the cpss. helical ct, especially the multiscale versions, makes excellent images of the abdominal vasculature (including cpss) relatively quickly after intravenous injection of iodinated contrast agents. three-dimensional reconstruction provides impressive anatomic details of shunting vessels. the drawback is the limited availability of the new-generation scanners and the need for patient anesthesia. although magnetic resonance angiography can provide highquality images of the abdominal vessels, it has not become popular because of its limited availability and high costs. patents require general anesthesia and the examination lasts longer than ct angiography. characteristic mri changes have been described in the brains of dogs and cats with cpss, but these findings are more of research than of clinical interest. histopathologic examination of liver biopsy specimens is essential in identifying the underlying disease process in intrahepatic portal hypertensive disorders. in the routine diagnostic workup of canine cpss liver histopathology gives no additional information. currently, the presence of coexistent phpv cannot be diagnosed preoperatively in dogs with cpss. this is because the histologic findings of liver biopsies are identical in the following conditions: cpss, phpv, cpss with phpv, congenital arterioportal fistula, any prehepatic portal hypertensive disorder (e.g., portal vein thrombosis). however, taking liver biopsies for histopathologic examination is recommended in cats before deciding about surgical closure of a cpss. this is because congenital hepatic fibrosis as part of pkd can be present simultaneously, especially in persian and persian crossbreeds. surgical shunt attenuation is not recommended when congenital hepatic fibrosis and a cpss are simultaneously present in a cat. patients with grades to he may present on an emergency basis. the source of ammonia and other protein breakdown products, which cause the clinical signs of he, is the colon. the purposes of the treatment are (a) to reduce the production and amount of ammonia in the colon by removing its content with warm water enema, and (b) to inhibit the absorption of ammonia by administering lactulose syrup into the emptied colon. lactulose may be given as a retention enema with a soft feeding tube . to . ml/kg deep rectally). lactulose is a nonabsorbable disaccharide, which is metabolized by the colonic bacteria into short-chain fatty acids. these fatty acids acidify the intraluminal content causing to form ammonium ion (nh + ) from ammonia (nh ). because of its polarity, nh + will not pass the enterocystic membrane, and is instead trapped in the colonic lumen. it is also essential (c) to correct the acid-base and electrolytic disorders of the patient with iv fluid therapy as alkalosis and hypokalemia can worsen the signs of he. seizures caused by he can be controlled with intravenous propofol. administration of benzodiazepines (e.g., diazepam) and barbiturates should be avoided as they can worsen the clinical signs. binding of a benzodiazepine molecule to its neuronal benzodiaze- acute portal hypertension. variable degrees of portal hypertension necessarily develop subsequent to any cpss attenuation. if portal hypertension is severe, circulatory collapse may develop because of sequestration of blood in the splanchnic veins. slight abdominal enlargement as a result of ascites requires no intervention. this usually resolves spontaneously within week of surgery. severe ascites with signs of shock (e.g., depression, tachycardia, hypotension, prolonged capillary refill time, hemorrhagic diarrhea) necessitates emergency surgery and removal of the ligature or constrictor ring from around the shunt. these signs usually develop within hours postoperatively. unfortunately, the prognosis following emergency surgery is poor. portal vein thrombosis. this rare postoperative complication causes sudden onset of shock usually within several days of shunt attenuation. , exaggerated shunt occlusion, severe portal hypertension, and stasis of portal venous blood are believed to be the cause of the thrombosis. no survivals have been reported. chronic portal hypertension. when the growth of the liver and development of portal branches is insufficient following partial shunt closure, chronic portal hypertension may induce formation of apsc. this may occur as late as to weeks postoperatively. in such cases, evaluation usually reveals partially patent shunt and increased portosystemic shunting as a result of newly formed apsc. the development of apsc has been documented with all surgical methods. apsc can develop as a result of underdeveloped portal branches or exaggerated shunt attenuation. in most dogs, these apsc remain clinically silent because the hepatic mass has substantially increased following shunt attentuation. all patients should be re-evaluated at month after shunt attenuation by measuring fasting blood ammonia concentration and performing an abdominal ultrasonography. if fasting blood ammonia concentration is within the reference range, rectal att should be performed. if fasting hyperammonemia is present or the att is abnormal, ultrasonography should identify whether shunting occurs via the narrowed cpss, apsc, or both. in the latter cases, lifelong conservative therapy with dietary modifications and lactulose is recommended. a normal att result implies complete shunt occlusion and the pet will generally have a very favorable prognosis. a second surgery to reach further shunt attenuation should only be attempted in patients with persistent clinical signs that have high fasting blood ammonia concentration or abnormal rectal att result months after the first surgery. , spontaneous gradual shunt closure would not occur beyond months after surgical ligation. this rare and usually fatal complication of shunt attenuation causes generalized seizures to days postoperatively, almost exclusively in cats and small-breed dogs (often in maltese dogs). , , , its occurrence is unpredictable. the pathogenesis is unknown, but the sudden decrease of endogenous benzodiazepine ligands is thought to play a role (i.e., "benzodiazepine withdrawal syndrome"). because cerebral edema is suspected to be the initial disorder, intravenous mannitol ( . to . g/kg iv, during minutes) may be administered when a patient shows subtle central neurologic signs during the first three postoperative days. once seizures have developed, the prognosis is usually very poor. most patients are euthanized because of uncontrollable seizures or persistent neurologic defects. propofol gradual shunt attenuation is believed to reduce the risk of portal hypertension by allowing the portal venous branches to adapt gradually to the increased flow. , apsc do develop with gradual shunt attenuation techniques (e.g., cellophane banding and ameroid constrictor ring). cystic calculi should be removed during the same surgical procedure, because resolution of ammonium biurate uroliths with dietary management can only be achieved in % of cases. the shunting vessel is identified by midline laparotomy and narrowed or completely occluded by a nonabsorbable ligature. portal hypertension is determined by direct measurement or estimated based on subjective criteria including severe intestinal cyanosis, increased intestinal peristalsis, reduction in arterial blood pressure, and compensatory tachycardia as a result of stasis in the splanchnic circulation. intraoperative doppler ultrasonography will greatly facilitate determination of the optimal degree of shunt attenuation in extrahepatic cpss, helping to prevent severe portal hypertension. , cellophane banding instead of a ligature, a -mm-thin, three-layer-thick cellophane band is placed around the shunting vessel with or without narrowing of the shunt diameter. gradual shunt occlusion takes place as a result of inflammation induced by the cellophane. , ameroid constrictor ring a metal ring filled with a thick layer of casein is placed around the shunting vessel. swelling of the casein occurs as it absorbs fluid. because of the outer metal ring, the casein can only expand centripetally causing gradual occlusion of the shunt over to months. , , the major drawback of this simple technique is that the rate and magnitude of occlusion is uncontrollable and kinking caused by the weight of the device may cause acute fatal portal hypertension. moreover, because of its relatively large size it can only be easily applied on extrahepatic cpss. , laparoscopy laparoscopic shunt narrowing (by clips) is a less-invasive alternative for shunt ligation (see chapter ) . with this minimally invasive intravascular technique metal spirals with thrombogenic fibers are placed in the shunting vessel. the coils are delivered via a catheter, which is inserted through the jugular vein into the shunt under fluoroscopic guidance in anesthetized animals. , the coils cause thrombus formation in the shunt resulting in its partial or complete occlusion. to prevent coil dislodgment from the shunt, an intravascular stent is often placed in the caudal vena cava to cover the point where the shunt enters the caudal vena cava. coiling is an especially attractive method for treating intrahepatic shunts because liver dissection is thereby avoided. in my opinion, the safest and most effective way for attenuation of extrahepatic cpss is doppler ultrasound-guided partial attenuation via surgical ligature. , for intrahepatic cpss, coil embolization appears to be an excellent method. ameroid constrictor and cellophane banding would be ideal in extrahepatic cpss, when the portal vein cranial to the shunt origin is severely hypoplastic and the patient is at risk for severe portal hypertension. spironolactone alone does not resolve ascites, furosemide may be added to the therapy. abdominocentesis for removal of abdominal effusion is not recommended because of loss of protein and exacerbation of starling forces permitting further fluid accumulation. dietary and medical management relieves clinical signs only temporarily in most symptomatic young dogs. without surgical shunt attenuation, gradual deterioration of liver function occurs; consequently, conservative therapy alone offers a guarded prognosis in young dogs. in older animals (> years) with newly reported signs of cpss, lifelong conservative treatment may be recommended because of the significantly higher complication rates of surgical therapy in these patients. the prognosis depends on (a) whether the cpss is intra-or extrahepatic, (b) the coexistence of phpv, (c) the extent of shunt attenuation, (d) experience of the surgeon, and (e) the age of the dog at the time of diagnosis. intrahepatic cpss generally has poorer prognosis. complete resolution of clinical signs can be expected in approximately % to % of dogs with extrahepatic cpss in the hands of an experienced surgeon. this same parameter is approximately % to % for intrahepatic cpss. [ ] [ ] [ ] [ ] with extrahepatic cpss, an excellent prognosis can be expected when blood flow is hepatopetal (i.e., toward the liver) in the portal vein segment, which is cranial to the shunt origin. this can be established preoperatively with doppler ultrasound. in cats, regardless of the shunt type and the surgical method, the success rate is approximately % to % because of the development of postoperative central neurologic signs. , , the prognosis of portal hypertensive disorders depends on the underlying disease. in the majority of acquired diseases the underlying disorder is chronic and so severe that even stopping the disease process will not cause regression of apsc. primary liver neoplasms are infrequent in the dog and cat, with an estimated prevalence in necropsy studies of . % to . % in the dog and . % to . % in the cat. liver metastases are more frequent than primary hepatic tumors in the dog, and tend to originate from the spleen, pancreas, and gi tract. primary hepatobiliary tumors are more common than metastatic disease in the cat. [ ] [ ] [ ] [ ] [ ] the etiology of liver cancer in dogs and cats is incompletely understood. potential causes such as aflatoxins, nitrosamines, food additives, parasites, and radioactive compounds have been reported. [ ] [ ] [ ] liver cancer in the dog has many clinical, pathologic, and histologic homologies with liver cancer in humans. [ ] [ ] [ ] [ ] in human medicine, chronic diseases of the liver, such as hepatitis b or c infection, as well as cirrhosis, are often associated with ( to mg/kg, iv) followed by constant rate infusion [cri]) may be used to control seizures. prophylactic treatment with phenobarbital does not reduce the risk of development of postligation neurologic complications, but may be used in long-term seizure management. preventive use of potassium bromide has not been shown to reduce the possibility of postoperative seizures. in every patient with central neurologic signs in the early postoperative period, hypoglycemia and he should be excluded by measuring venous glucose and ammonia concentrations, respectively. blindness and other types of central neurologic signs may develop in cats shortly after surgery. the pathogenesis of these changes is unknown. hemorrhage from liver biopsy sites or shunt dissection in cases of intrahepatic cpss can lead to hypovolemic shock and death in the early postoperative period. dogs with hepatic insufficiency are prone to develop hemorrhagic complications because of decreased concentrations or abnormal synthesis of coagulation factors. postoperative portal hypertension may also contribute to bleeding tendency from hepatic parenchymal dissection. hemoabdomen should be carefully differentiated from severe portal hypertension and septic peritonitis as they all can cause shock and variable degrees of abdominal distention. coagulation parameters (e.g., pt, aptt) should be routinely monitored after surgery and if they are abnormal or if there is evidence of clinical bleeding, fresh-frozen plasma transfusion should be administered ( to ml/kg, iv). toy-breed dogs are prone to develop hypoglycemia during or shortly after surgery. blood glucose concentrations should be regularly monitored and hypoglycemia should be treated with glucosecontaining infusions. specific treatment should address the underlying parenchymal liver disease based on the histologic results of liver biopsy. he can often be controlled with dietary modification and lactulose. currently, no specific treatment exists for portal venous hypoplasias. renal or hepatic prescription diets and lactulose may alleviate clinical signs of he in case of apsc. diuretic agents may be useful in dogs with ascites. liver lobe resections have been reported in animals with congenital arterioportal fistulas. however, simultaneous presence of phpv in the whole liver prevents postoperative resolution of portal hypertension and the portosystemic shunting via apsc. , therefore, the pet owner should be educated that partial hepatectomy may not result in complete recovery of portosystemic shunting, and that lifelong dietary support and/or lactulose will likely be required. severe abdominal effusions should be treated with diuretic agents. the first choice is spironolactone ( to mg/kg q h), an aldosterone receptor antagonist as (a) chronic hepatic insufficiency is associated with hyperaldosteronism, (b) concurrent hypercortisolemia is associated with cortisol binding to the mineralocorticoid receptor, and (c) potassium-sparing diuretics prevent development of hypokalemia and alkalosis, both of which would worsen signs of he. if show no clinical signs, and liver cancer is suspected only with increases in serum liver enzyme activities. , , , the most common physical examination findings are cranial abdominal mass ( %), abdominal bloating ( %), and jaundice ( %). jaundice is a less common finding in cases of metastatic cancer. other manifestations include neurologic signs as a result of he; paraneoplastic syndromes such as hypoglycemia and myasthenia gravis; and skin alterations consistent with hepatocutaneous syndrome. , no clear breed predisposition is observed with canine liver cancer, although poodles, fox terriers, miniature schnauzers, labrador retrievers, and male dogs are overrepresented in some reports of hepatocellular carcinoma. , , , , labrador retrievers and female dogs are overrepresented in reports of bile duct carcinoma. , [ ] [ ] [ ] whether male and female cats are equally at risk for bile duct carcinomas is unsettled, , but male cats appear to be at greater risk for bile duct adenomas. , neuroendocrine tumors are generally observed in younger animals. , diagnosis definitive diagnosis of liver cancer can be made only by liver biopsy. asymmetrical enlargements of the liver detected on physical examination, abdominal ultrasonography, or survey radiography should not be assumed to be neoplastic in origin. similarly, laboratory data do not distinguish hepatic neoplasia from other liver pathologies. the clinical approach to an animal with suspected liver cancer should include basic information such as a complete blood cell count, serum biochemistry, coagulation tests, urinalysis, thoracic and abdominal radiographs, abdominal ultrasound ( figures - and - ) , and fine-needle or core biopsy of the liver. a tnm tumor classification system has been used for staging of liver cancer where t represents tumor (t , no evidence of tumor; hepatocellular tumors; however, there is no established association between hepatic tumors and viral infections in the dog or cat. moreover, canine hepatic cirrhosis does not appear to predispose to hepatocellular carcinoma (hcc). a possible association between hookworm or whipworm infection and liver cancer has been reported, and cats with chronic cholangitis may have an increased predisposition to biliary carcinoma. , several liver mitogens and tumor suppressor genes such as epidermal growth factor, tgf-α, vascular endothelial growth factor (vegf), p , and tgf-β and its receptors (tgf-β-r) have been associated with liver cancers in humans and these may play a similar role in the dog. [ ] [ ] [ ] [ ] in human medicine a small percentage of hccs or cholangiocarcinomas originate from hepatic progenitor stem cells. dogs diagnosed with hcc or cholangiocarcinoma do demonstrate activation of hepatic progenitor stem cells in response to liver injury, but hepatic progenitor stem cell expression in liver tumors is relatively low. the hypothesis of cancer development as a multistep process applies to liver tumors in the dog and the cat, as well. , precancerous lesions, such as dysplastic nodules, can be identified before the development of overt malignancy in humans. dysplastic nodules are characterized by cell atypia, cellular crowding, trabecular thickness, microacini, and histochemical markers. dysplastic nodules have not been reported in the dog or cat and further studies are needed to understand the chronology of hepatic malignancy in domestic animals. preliminary reports of histochemical markers have been reported in dogs with hyperplastic hepatic lesions and hepatocellular and biliary neoplasms. liver tumors cause damage to the liver by several mechanisms: inflammatory effects, obstruction of the biliary system, obstruction of the vascular compartment or adjacent organs, and spontaneous rupture with hemoabdomen. hepatic tumors are usually resistant to chemotherapy. , in one recent study, p-glycoprotein was more highly expressed in hcc than in cirrhosis, which is consistent with the known resistance of hcc to chemotherapy. p-glycoprotein, which is encoded by the multidrug resistance gene (mdr- ), is normally expressed in tissues with excretory function, including the jejunum, kidney, liver, and adrenal gland. primary liver neoplasms are usually classified according to their cellular origin and macroscopic appearance. with respect to cellular origin, these tumors may be hepatobiliary, hematopoietic, sarcomas, or metastases of other tumors (box - ). in relation to macroscopic appearance, they can be classified as lobular, multiple nodular, or diffuse (). the combination of histopathologic and morphologic classification has consequences for prognosis and treatment strategy in these animals (tables - and - ) , and the clinician must always address these factors to arrive at correct management decisions. in dogs, malignant tumors are more common than benign lesions. in cats, biliary neoplasms are the most common presentation, particularly intrahepatic benign forms. , most animals with liver neoplasia present with nonspecific clinical signs such as anorexia and weight loss. less-frequent clinical signs include vomiting and diarrhea, pd and pu, pale mucosal membranes, and acute weakness because of anemia and hypovolemia coincident with tumor rupture. , up to % of affected animals diffuse or infiltrating, multiple coalescent nodules in all the lobes, or diffuse disappearance of the liver parenchyma; lobular, nodule or large mass in a single liver lobe; nodular, several nodules throughout the liver parenchyma, or several affected liver lobes. t , tumor involving one lobe; t , tumor involving more than one lobe; and t , tumor invading neighboring structures); n represents regional lymph nodes (rlns) (n , no evidence of rln involvement; n , rln involved; n , distant ln involved); and m represents distant metastasis (m , no evidence of metastasis; m , distant metastasis detected). although recommended, this system has not been universally adopted. table - illustrates the most typical hematologic and biochemical findings in dogs and cats affected with liver neoplasia. leukocytosis is a result of inflammation and necrosis of large tumors; anemia tends to be moderate and nonregenerative and is thought to be caused by chronic illness, inflammation, or iron deficiency ; thrombocytosis is attributable to a paraneoplastic syndrome characterized by thrombopoietin production, iron deficiency, or anemia. serum liver enzyme elevation is a frequent, but not universal, finding in animals with liver neoplasia. it should be noted, however, that the degree of serum enzyme elevation does not correlate with the degree of liver involvement or severity of disease. in one survey, animals with primary liver tumors tended to have greater elevations in serum alt and alp activities than animals with metastatic disease, while the latter tended to have greater elevations in serum bilirubin and ast. it also has been suggested that an ast-to-alt ratio of less than is more compatible with carcinoma, while a ratio of greater than is more indicative of a sarcoma or carcinoid. other reported biochemical changes include hypoglycemia, hypo-or hyperalbuminemia, and increased serum bile acids. hypoglycemia as a paraneoplastic syndrome associated with hepatocellular carcinoma is attributed to the secretion of insulin-like growth factor ii. unlike dogs, cats usually present with a high incidence of serum creatinine and bun elevations. , coagulation factor abnormalities are more commonly associated with hemangiosarcoma, although dic may be evident in end-stage liver cancer or in decompensated patients. coagulation studies should always be performed before undertaking invasive diagnostic procedures. serum α-fetoprotein has been evaluated in the dog, and increases are reported in % of animals with hepatocellular carcinoma, and in % of those with biliary carcinomas. the use of this biomarker is limited by the fact that it is increased in cases of hepatic lymphoma and other liver pathologies, and only very dramatic elevations in α-fetoprotein may be taken to indicate hepatocellular carcinoma. [ ] [ ] [ ] abdominal radiographs often reveal a mass effect in the cranial abdomen, although this finding will depend upon the size of the neoplasm and the number and size of metastatic tumors. other reported findings include dorsal displacement of the stomach, hepatomegaly, loss of abdominal detail (because of the presence of free abdominal fluid), and, occasionally, biliary tract calcification. thoracic radiography should be considered as part of the staging procedure for animals with metastatic disease. , changes in the ultrasound density of the liver may take a variety of forms (box - ). most changes are not pathognomonic for a given disease process, and the final diagnosis is established only on the basis of clinical findings, laboratory testing, and results of cytology or histopathology (see . ultrasound is also very useful for evaluating other abdominal structures, and for the staging of cancer. [ ] [ ] [ ] • diffuse or multifocal liver neoplasms tend to present with hepatomegaly, but this depends on the degree of infiltration. liver carcinomas can be diffuse or affect multiple lobes, with variable ultrasound characteristics depending on the presence of necrosis, inflammation, hemorrhage, or cavitation. in these malignant tumors it is common to observe a mixed echogenicity pattern. lymphoma can affect the liver without detectable ultrasound changes, or cause diffuse hypoechogenicity, hyperechogenicity, or mixed echogenicity with or without hypoechoic nodules. consequently, if lymphoma is suspected, even if the liver ultrasound findings appear normal, fine-needle aspiration cytology is recommended. histiocytic neoplasms are more often associated with multiple nodules and hypoechoic masses, although diffuse liver hypoechogenicity has also been described. mast cell infiltration of the liver tends to produce diffuse hyperechogenicity. • nodular patterns • benign nodular hyperplasia is common, particularly in dogs, and accounts for many of the focal liver lesions identified at ultrasound exploration. it has been estimated that % to % of all nodular masses detected in the liver are nodular hyperplasia. • benign liver adenomas or hepatomas can manifest as a focal mass of variable size and of normally hyperechoic characteristics. • the liver is a frequent location of metastatic spread, fundamentally through the portal system that drains most of the abdominal structures. • primary liver neoplasms such as hepatocellular carcinoma can present as focal or multifocal masses, although less often so than in the case of metastases. focal hypoechoic lesions with a hyperechoic center or core (referred to as target or bull's-eye lesions) are usually associated with metastases, although some benign processes, such as nodular hyperplasia, can generate similar patterns. • biliary obstruction: ultrasound has become an important tool for evaluating biliary obstruction in icteric dogs and cats. primary tumors of the liver, biliary tract, duodenum, or pancreas are capable of causing biliary obstruction. liver cytology has obvious limitations in that it cannot distinguish between liver adenomas and regenerative nodules, and even some hepatocellular carcinoma aspirates may be composed entirely of normal-appearing hepatocytes. in many cases it may prove necessary to resort to ultrasound-guided biopsy, laparoscopy, or exploratory laparotomy. however, cytology may prove useful in determining the presence of lymphoma, mastocytoma, and histiocytic sarcoma, as well as contribute to the initial classification of tumor type. concordance rates between cytology and histopathology findings may be good for some disease processes, but the reported concordance rate varies from % to %. , the treatment to be provided and the prognosis of animals with primary liver cancer depend on the cell of origin, degree of malignancy, and clinical presentation. the clinician should quickly determine if surgery, chemotherapy, radiation therapy, or palliative care is the treatment of choice in individual patients. palliative treatment is the option for animals that are not surgical candidates, for example, tumors with poor response to systemic chemotherapy, and for whom pain management and general liver failure treatment are the best recommendations. the success of newer options such as chemoembolization, metronomic therapy, antiangiogenic drugs, and tyrosine kinase inhibitors in the treatment of these patients has not been clearly established. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the macroscopic presentation is clinically very important (see figure - ), as % of the diffuse forms have metastasis at the time of diagnosis, versus % of the isolated (massive or nodular) clinical presentations. it should be noted, however, that some dogs with massive hcc present without metastasis, and deaths in these cases may be unrelated to hcc. , , histopathologic subtype and anaplastic characteristics in general influence the prognosis and predictability of metastasis. , , metastatic spread usually affects the regional lymph nodes, lungs, and peritoneum. , high-field mri scanning has an accuracy of % in differentiating malignant from benign lesions with a sensitivity and specificity of % and %, respectively. mri classified malignant hepatic lesions as hcc in all confirmed cases and correctly predicted the histologic grade of five hcc lesions. these results suggested that mri is a useful modality for abdominal imaging in veterinary patients, and that mri accurately differentiates benign from malignant focal hepatic lesions. liver cytology is useful in the initial evaluation of hepatomegaly and usually permits differentiation between primary tumors, metastatic disease, and focal infection (see . however, cytology does not distinguish between benign focal inflammatory disease and progressive chronic liver disease, and it cannot establish the extent and distribution of disease. likewise, a definitive diagnosis of regenerative nodular hyperplasia cannot be established, and the technique is unable to differentiate a benign inflammatory reaction from cell changes associated with other pathologies. contraindications to ultrasound-guided cytology include the following: • coagulation abnormalities-if one or more coagulation test parameters are altered, it is advisable to administer vitamin k via the subcutaneous route hours before cytology. • cavitary masses-the ultrasound detection of a large cavitary lesion in an elderly dog usually contraindicates cytology, a b tumors are more common in cats. , , , three morphologic forms or presentations have been described: lobular, multifocal, and diffuse. in general, only the lobular form should be considered for surgical removal as long as there is no evidence of metastasis. the prognosis for multifocal and diffuse bile duct carcinomas is very poor, surgery is usually not feasible, and most animals die within months of surgery. no effective chemotherapeutic options have been described for these malignancies in dogs or cats. also known as biliary cystadenomas, biliary adenomas, cholangiocellular adenomas, and cholangiomas, these tumors are common findings in aging cats. males appear to be more frequently affected than females (figure - ). in cats, % of these lesions are isolated or lobular and % are multifocal. biliary duct adenomas usually do not cause clinical signs unless they grow and compress other structures. despite the benign nature of these tumors, surgical removal is usually recommended because malignant transformation is always possible and because expansion into the porta hepatis may cause life-threatening consequences. liver lobectomy is recommended for cats with single bile duct adenoma or multifocal tumors confined to one or two lobes. in cats, surgical resection of biliary adenomas may provide cure or tumor-free survival of several years. , , , , , carcinoid tumors neuroendocrine (carcinoid) tumors are infrequent in the dog and cat. in dogs, carcinoids have an aggressive biologic behavior and are usually not amenable to surgical resection as they tend to present as diffuse lesions (see figure - ). , carcinoid tumors in dogs have also been described in the gallbladder, and these have been managed successfully with cholecystectomy. , carcinoid tumors in cats can be intrahepatic or extrahepatic involving the bile duct and occasionally the gallbladder. the extrahepatic form of carcinoid tumors may cause biliary tract obstruction, icterus, and increases in serum hepatic enzyme activities. biliary tract diversion procedures should be considered for obstructive lesions involving the extrahepatic biliary tract. unlike the circumstance in dogs and humans, female cats are more often affected by these tumors than males (female-to-male ratio of : ). the prognosis of carcinoid liver tumors in dogs and cats is generally poor, and metastatic disease is present in % of the cases at prognostic factors in dogs with massive hcc include need for surgery, liver lobe involvement, serum alt and ast activities, and ratios of alp to ast and alt to ast. liver lobectomy is recommended for cats and dogs with hepatic tumors that have a massive morphologic appearance without metastases. however surgical complications are reported in more than % of cases, with a mortality rate of almost %. the predilection of massive hcc for left-sided liver lobes has been reported. , advanced imaging and intraoperative ultrasonography may provide useful information on the relationship of right-sided and central liver tumors to the caudal vena cava prior to liver lobectomy. , , even though right-sided liver tumors have a poorer prognosis because of intraoperative death, there is no difference in the survival time after successful surgery. the considerable regenerative capacity of the liver can permit successful resection of up to % of hepatic mass if the remaining tissue is functionally normal and critical supportive care is provided. the median survival time for dogs with massive hcc following liver lobectomy is greater than years. without surgery the average life expectancy is days and the prognosis is generally considered poor. tumor recurrence in dogs with massive hcc is rare and reported to be % to % after lobectomy. , the prognosis for dogs with nodular and diffuse hcc is poor. surgical resection is usually not possible because of involvement of multiple liver lobes. no effective systemic chemotherapy or radiation therapy protocols have been described for hcc treatment. hcc is considered chemoresistant in humans although mitoxantrone has been reported to be helpful in some cases. , , the most likely reason for the poor response to systemic chemotherapy is the expression of p-glycoprotein in hepatocytes. treatment options for nodular and diffuse hcc in humans include liver transplantation and minimally invasive procedures for regional control, such as ablation, chemoembolization, immunotherapy, hormonal therapy, and low-dose metronomic chemotherapy. , a recent report recommends therapy with sorafenib, a multikinase inhibitor and antiangiogenic agent. , chemoembolization is a procedure commonly used in the treatment of diffuse hepatocellular carcinoma in humans with median survival times of to years compared with to months with systemic chemotherapy. , in veterinary medicine, chemoembolization has been reported with moderate success in the palliation of four dogs with hcc. , in cats, hepatocellular carcinoma is less frequent, and less data are available. , hepatocellular adenomas these tumors are also known as hepatomas and are more common in cats than in dogs. in the dog it is sometimes very difficult to distinguish adenoma from reactive nodular hyperplasia, and biopsy is needed to clarify the diagnosis. the prognosis for adenomas is usually good, but it is advisable to remove focal mass lesions because they can grow and spontaneously rupture with severe bleeding. bile duct carcinoma (adenocarcinoma and cholangiocarcinoma) bile duct carcinoma is the most common liver malignancy in the cat, and the second most common liver malignancy in the dog (see figure - ) . tumor behavior is very aggressive in both species, and metastases are present at the time of diagnosis in % to % of cases. bile duct carcinomas usually metastasize to the regional lymph nodes, lungs and peritoneum, kidneys, heart, adrenal glands, eye, and bone. , bile duct carcinoma can be intrahepatic or extrahepatic, but rarely occurs within the gallbladder. intrahepatic bile duct tumors are more common in dogs, and extrahepatic bile duct must always be considered as a possibility when a hepatic tumor is diagnosed. benign mesenchymal neoplasms, such as fibroma and hemangioma, have been described but are quite rare. , , , lymphoma in dogs the liver can be involved in variable forms of lymphoma, including multicentric, alimentary, and hepatosplenic forms. a study in cats documented that abdominal lymphoma is currently the most common anatomic location and the liver occasionally is the only organ involved. , many protocols are recommended for treatment of lymphoma in dogs and cats; most include vincristine, cyclophosphamide, and prednisone, with variable combinations of l-asparaginase, methotrexate, and doxorubicin. careful evaluation of liver function is necessary before starting chemotherapy because many drugs undergo hepatic metabolism and altered hepatic clearance may lead to unpredictable and potentially increased toxicity. surgical resection with liver lobectomy is recommended for cats with primary hepatic myelolipoma and the prognosis is excellent with prolonged survival time and no reports of local recurrence. in dogs with advanced disease, mast cell tumors can metastasize to the liver. primary visceral mast cell tumors are more common in cats than dogs. the spleen is usually the primary site with metastasis to the liver and bone marrow, and the survival time with splenectomy alone can be a year or more. the overall prognosis for disseminated mast cell tumor in the dog is grave. the median survival time reported in one study was days despite therapy with various chemotherapy agents. canine mastocytoma involving the liver can be controlled with cyclophosphamide, vinblastine, and prednisone. recently, tyrosine kinase inhibitors have shown some promise and ccnu has been shown to be active against feline mast cell tumors. , hepatic nodular hyperplasia is a common benign lesion observed in the liver of older dogs that can occasionally be observed in some cats. it is characterized by a discrete accumulation of hyperplastic hepatocytes arising as either macroscopic or microscopic hepatic nodules. it reportedly occurs in % of dogs older than years and % of dogs over years. [ ] [ ] [ ] the wsava standards for clinical and histologic diagnosis of canine and feline liver diseases include hepatic nodular hyperplasia in its classification system of hepatocellular neoplasia so that it may be differentiated from true neoplasia. the etiology of hepatic nodular hyperplasia is unknown. it has been suggested to be a preneoplastic lesion, but this has not yet been reported in the dog. because of hepatocyte microscopic changes, it is suggested that nutritional and metabolic disorders play a role in the pathogenesis of this lesion. hepatic nodular hyperplasia is characterized microscopically by well-differentiated hyperplastic hepatocytes with increased mitotic activity. [ ] [ ] [ ] hyperplastic nodules may be accompanied by concurrent focal intrahepatic cholestasis, mechanical compression on a higado b the time of diagnosis. a better prognosis is observed with extrahepatic carcinoids with a life expectancy of more than a year. , liver sarcomas primary liver sarcomas are rare in the dog and cat. hemangiosarcoma is the most frequent primary hepatic sarcoma in cats and leiomyosarcoma the most common in dogs. , there also have been reports of hepatic fibrosarcoma, rhabdomyosarcoma, osteosarcoma, liposarcoma, and histiocytic sarcomas in both animal species. , , , these are usually very aggressive tumors, metastasizing in % to % of cases to the spleen and lungs, or spreading diffusely within the liver. chemotherapy has not been studied in the treatment of primary hepatic sarcomas, although, similar to other solid sarcomas, response rates are likely to be poor. histiocytic sarcomas respond partially to ccnu, with a mean duration of remission of days and a survival of days. continuous low-dose oral chemotherapy may be an effective alternative to conventional high-dose chemotherapy for adjuvant therapy of dogs with hemangiosarcoma. mass resection may offer some palliation in the circumstance of tumor hemorrhage despite irrefutable evidence of metastasis. a cat with a primary extraskeletal hepatic osteosarcoma was treated with surgery and carboplatin and was alive months after diagnosis with no clinical evidence of disease. on the other hand, metastases feline hepatic lipidosis (hl) is a metabolic syndrome found in obese, middle-aged cats that undergo a period of acute anorexia and catabolism. morbidly obese cats are at increased risk and more than % of cats with hl suffer from an underlying disorder that contributes to the initial anorectic event. [ ] [ ] [ ] [ ] [ ] although the underlying pathogenesis of hepatic lipid accumulation in cats has not yet been completely elucidated, several unique biochemical and nutritional features place this obligate carnivore at risk for fat mobilization and fatty infiltration of the liver during periods of anorexia or starvation (box - ). , [ ] [ ] [ ] [ ] [ ] [ ] there is a general consensus that reduced caloric intake and protein-calorie malnutrition are important predisposing factors. the result is a rapid mobilization of peripheral fat culminating in fatty accumulation in the liver. intracellular processing of fats is an important function of the hepatocyte. during fasting or starvation, fatty acid metabolism becomes deranged in an obligate carnivore as a result of obesity, catabolism, chronic overnutrition, impaired fatty acid oxidation or vldl secretion, and enhanced hepatic fatty acid synthesis ( figure - ). , - hl is a disorder of middle-aged to older cats; domestic short-haired cats are more commonly affected. cats with hl often present with a history of acute stress and/or near-complete anorexia of several days duration. [ ] [ ] [ ] icterus is a variable feature of hl. when serum bilirubin concentrations exceed . mg/dl, clinical icterus can be observed on the pinnae, mucous membranes, sclera, and hard palate in the cat. in general, most cats with hl are obese at the time of presentation, with many cats being % to % over ideal body weight prior to an episode of hl. other physical features of hl include hepatomegaly, dehydration, vomiting, and weakness. if he develops as a consequence of hl, neurologic abnormalities such as ptyalism, stupor, coma, ataxia, and seizures may be observed. [ ] [ ] [ ] a minimum database, including complete blood cell count, serum chemistry, and urinalysis, almost always reveals severe liver enzyme elevation and other abnormalities such as nonregenerative anemia, stress leukogram, poikilocytosis, and bilirubin crystalluria. the pattern of liver enzyme elevation is typically cholestatic in nature and characterized by marked increases in serum alp activity, followed by smaller increases in serum alt and serum ast activities. serum ggt activity is often normal in affected cats. increased serum bile acids and bilirubin are often observed in cats with hl, and electrolyte abnormalities, such as hypophosphatemia and surrounding hepatic parenchyma, as well as alterations in the microvascular circulation. vacuolar changes are seen frequently, suggesting a reactive or metabolic condition such us hyperadrenocorticism, lipidosis, or hypothyroidism. nodular hyperplasia affects older dogs with a mean age of years without gender or breed predisposition. hepatic nodular hyperplasia does not appear to cause clinical signs or illness. laboratory findings may include mild to marked increases in serum alkaline phosphatase activity and, less commonly, increases in serum alt activity. liver function tests are usually normal with hepatic nodular hyperplasia. hepatic nodular hyperplasia is usually discovered as an incidental finding during a diagnostic workup for other medical problems. nodular hyperplasia is clinically important because it may easily be confused with primary or metastatic hepatic neoplasia during abdominal ultrasound or at surgery. even microscopically, it may be impossible to differentiate hepatic nodular hyperplasia from hepatocellular adenomas, and a large sample (wedge rather than needle biopsy) may be required to confirm hyperplasia from welldifferentiated hccs. [ ] [ ] [ ] routine abdominal radiographs are generally unremarkable and ultrasonographic features are inconsistent because of the varied hepatocellular morphologic characteristics and size of the nodules. multiple nodules varying in size, distributed randomly among the liver lobes, being superficial or deep within the parenchyma are found in most cases. hyperplasic nodules of hepatocytes need to be differentiated from regenerative nodules. hyperplasic nodules develop in livers of normal mass, whereas regenerative nodules arise as a result of compensatory hyperplasia of surviving hepatocytes in a background of hepatic injury, atrophy, and fibrosis. no treatment is usually required. rupture of large nodules may require emergency mass removal and blood transfusion (rare). hepatic nodular hyperplasia has no significance in the morbidity of affected patients. metabolic disorders of the liver are commonly encountered in companion animal practice. this section focuses on the metabolic liver disease induced by concurrent endocrinopathies (hyperthyroidism, hypothyroidism, diabetes mellitus, and hyperadrenocorticism), lipid disturbances (lipoproteinemias, feline hepatic lipidosis, and hyperlipidemias), and metabolic infiltration (amyloidosis). hepatic lipidosis and hyperthyroid hepatopathy are the primary metabolic hepatopathies in cats. in dogs, steroid (or glycogen vacuolar) hepatopathy is the most frequent metabolic liver disorder; diabetic hepatopathy and hyperlipidemic hepatopathies (lipoproteinemias, hypothyroidism) occur less commonly. • essentiality of dietary arginine • low levels of hepatic ornithine • high dietary protein requirements • lack of hepatic enzyme adaptation to low protein • insufficiency of hepatic glutamate reductase • insufficiency of intestinal ornithine transcarbamylase • diversion to orotic acid metabolism • differences in lipoprotein metabolism (hdls) , to kcal/kg body weight in most cats. , unless he is present, dietary protein should not be restricted (ideal is % to % protein on a dry matter basis) and even then protein restriction is controversial as protein is needed to support hepatic regeneration. feeding multiple small frequent meals may help to maintain euglycemia and lessen the metabolic impact on the liver. the protein content of the diet should be considered when he is present (see chapter ). dairy and vegetable-based proteins are higher sources of branchedchain amino acids than meat-derived proteins and may lessen the signs of he. diets high in fiber generally should be avoided because they decrease the nutrient density of the diet. cats with hl occasionally may experience a refeeding syndrome, a condition that results in metabolic and electrolyte disturbances. with the reintroduction of food, insulin secretion promotes intracellular uptake of phosphorus, potassium, and magnesium. hypophosphatemia can result in muscle weakness and hemolytic anemia. gradual reintroduction of food and correction of electrolytes diminishes the risk of refeeding syndrome. glucose intolerance and hyperglycemia are common in cats with hl and can be addressed by decreasing the carbohydrate content of the diet. canned low-carbohydrate, high-protein formulations without added fiber are ideal for the treatment of feline hl as they provide amino acids, limited carbohydrates, and water, and are easily administered through a feeding tube. small amounts of food should be administered via the feeding tube after residual gastric fluid contents have been removed. trickle feeding can be performed by placing liquefied food into an empty fluid bag and allowing gravity to force flow into the feeding tube. alternatively, a large-bore hypokalemia, may be frequently observed. in particular, the presence of hypophosphatemia should alert the clinician to the possibility of refeeding syndrome. presumptive diagnosis of feline hl can be made on the basis of clinical history, physical examination, clinicopathologic features, ultrasound examination, and liver aspirates. , [ ] [ ] [ ] ultrasound examination of the liver often reveals hepatic parenchyma that is hyperechoic to that of falciform fat, but a thorough ultrasound evaluation of the gallbladder, pancreas, intestines, kidneys, bladder, and other abdominal structures is essential to rule out other primary disorders, such as acute pancreatic necrosis, which may be the basis of the anorectic event precipitating an episode of hl. definitive diagnosis is best achieved through liver biopsy ; however, anesthesia and biopsy may not be possible in acutely ill patients because of the presence of coagulopathies from vitamin k deficiency. a liver aspirate that reveals more than % fatty infiltration of the hepatocytes may be used for presumptive diagnosis of hl. if there is no response to treatment after to days, liver biopsy may be necessary to rule out other underlying hepatobiliary conditions such as cholangitis. a catabolic state develops quickly in the anorexic cat and prompt measures should be taken to place an enteral feeding tube (table - ) . nasoesophageal, esophageal, and gastrostomy tubes can be used for this purpose. the caloric needs should be approximately increases in serum ast and alt activities have been reported in approximately % of hyperthyroid cats. , liver enzyme elevation has been attributed to increased liver metabolic activity compared to blood flow. long-term untreated hyperthyroidism in human beings can ultimately lead to cirrhosis. [ ] [ ] [ ] middle-aged to older cats are typically affected, and there is no breed or sex predilection. because hyperthyroidism is characterized by hypermetabolism, polyphagia, weight loss, pd, and pu are prominent features of the disease. , hyperactivity, tachycardia, pupillary dilation, and behavioral changes are also characteristic of the disease and are associated with activation of the sympathetic nervous system. long-standing hyperthyroidism leads to hypertrophic cardiomyopathy, high-output heart failure, and cachexia. long nails, dermatologic conditions, panting, elevated body temperature, and poor grooming or overgrooming are additional clinical signs of feline hyperthyroidism. clinicopathologic features of hyperthyroidism include erythrocytosis and stress leukogram (neutrophilia, lymphocytosis) caused by increased circulating catecholamine concentrations. increased catabolism of muscle tissue in hyperthyroid cats may result in increased bun, but not serum creatinine. most cats will have decreased urine specific gravity, particularly if they are exhibiting pu as a clinical sign. increased metabolic rate results in liver hypermetabolism, therefore serum activities of liver enzymes (alt, ast) are increased in more than % of hyperthyroid cats. , diagnosis diagnosis of feline hyperthyroidism is achieved by measurement of serum total thyroxine (tt ) concentration. serum thyroxine concentrations are elevated in more than % of hyperthyroid cats, making this a very sensitive test of thyroxine-induced hypermetabolism. , false-positive test results are rare to nonexistent, suggesting that hyperthyroxinemia is a specific test for feline hyperthyroidism. in a clinically hyperthyroid cat, thyroid hormones still fluctuate on a daily (and hourly) basis with hormone concentration intermittently decreasing into the normal range. to avoid this type of diagnostic error the clinician should repeat blood sampling to weeks after the first test. nonthyroidal disease can have a significant effect on circulating thyroid hormone concentrations. [ ] [ ] [ ] in the case of persisting nonthyroidal illness (e.g., renal disease), the syringe attached to a syringe pump may be useful in delivering the food through the feeding tube. crystalloid fluids supplemented with fortified b vitamins, including thiamine, riboflavin, niacinamide, d-panthenol, pyridoxine, and cyanocobalamin, should be used. nutritional supplements to enhance antioxidant function, such as vitamin e and glutathione precursors (e.g., same) may also be beneficial. amino acid supplements that support hepatic regeneration and metabolism include carnitine and taurine. , [ ] [ ] [ ] carnitine functions in the transport of fatty acids into hepatic mitochondria for energy production. taurine is an essential nutrient for cats and is involved in cns, cardiac, and biliary functions. signs of taurine deficiency may be similar to those associated with he. antiemetic therapy is necessary to control vomiting and facilitate feeding of an appropriate type and quantity of diet (see chapters and ) . injectable antiemetics, such as maropitant (cerenia, pfizer animal health, kalamazoo, mi), a selective nk- receptor antagonist, at a dosage of mg/kg sc or iv on a daily basis, is preferred. oral maropitant at the same dosage or oral ondansetron, a -ht receptor antagonist at a dosage of . to . mg/kg q - h may be used in cats with larger-bore feeding tubes. persistent vomiting should be investigated to identify feeding tube occlusion or other undiagnosed disease. the prognosis depends upon duration of illness, and the time frame of resolution of hepatic enzyme elevation, hyperbilirubinemia, and other biochemical changes. cats that survive an episode of hl have a greater than % reduction in liver enzyme and bilirubin concentrations within days of therapy, whereas cats that die usually do so within days of hospitalization. long-term prognosis for recovery is good with the majority of cats having resolution of hl as long as the underlying disease process (e.g., pancreatitis) is identified and treated. hyperthyroidism in cats is caused by adenomatous hyperplasia of the thyroid gland resulting in increased circulating concentrations of thyroxine and triiodothyronine. , hyperthyroxinemia increases hepatic metabolism without proportionate increases in hepatic blood flow with the overall consequence of reduced oxygen delivery to hepatocytes. characteristic lesions of superficial necrolytic dermatitis (hard, cracked foot pads and elbows). painful feet caused by footpad lesions are common. clinicopathologic features include mild nonregenerative anemia, microcytosis (with advanced liver dysfunction), increased serum liver enzyme (alp and alt) activities, hypoproteinemia, hypoalbuminemia, and fasting hyperglycemia. serum bile acids are usually increased. serum glucagon is inconsistently elevated, but plasma amino acid concentrations are often less than % of normal. , diagnosis abdominal ultrasonography may reveal small, normal or increased liver size; however, there usually is a characteristic "swiss cheese" appearance of the hepatic parenchyma as a result of hepatic degeneration, nodularity, and collapse. , pancreatic imaging and biopsy are indicated if a glucagonoma is suspected. symptomatic palliative therapies may be beneficial and include high-protein diets with egg white (approximately to egg whites/ day for a -kg dog), zinc ( mg/kg q h po) niacinamide ( to mg/dog q h po), ursodeoxycholic acid ( to mg/kg/day po), vitamin e ( iu/kg daily po), same ( mg/kg/day po hours before feeding), and fatty acid supplementation. some patients will respond to % parenteral amino acid solutions (aminosyn, abbott laboratories, chicago) given at a dose of ml over to hours intravenously through a large-bore central venous catheter. if no response is observed following the initial amino acid infusion, therapy should be repeated every to days for a total of four treatments. prognosis is poor for most cases; however, remissions of longer than years have been reported with intensive amino acid and hepatic support therapy. steroid hepatopathy develops following exogenous corticosteroid therapy, or from endogenous hyperadrenocorticism of pituitary or adrenal origin. the dog liver is uniquely susceptible to both glucocorticoid-and sex steroid-induced liver enzyme elevation, glycogen accumulation, and vacuolar degeneration. , pathophysiology in healthy dogs, glucocorticoid administration results in significant liver enzyme (alp and alt) elevation in to days. increased alp and ggt activities develop in parallel as the enzymes undergo induction and release from sinusoidal and canalicular membranes. within days of glucocorticoid administration, the glucocorticoidinduced alp isoenzyme increases significantly. glycogen accumulates within the hepatocyte resulting in a vacuolar degeneration typical of the syndrome. , , steroid hepatopathy occurs primarily in the dog. there is only one reported case of steroid hepatopathy in the cat. a history of corticosteroid administration or signs consistent with endogenous steroid overproduction (cushing syndrome) are usually evident, for example, pd, pu, panting, potbellied appearance, bilaterally symmetric alopecia on the trunk, and polyphagia. in dogs affected with measurement of unbound thyroxine (t ) or free t may be preferable to repeated tt measurements. free t concentrations are a very sensitive test for the diagnosis of hyperthyroidism with % of hyperthyroid cats exhibiting elevated serum free t concentrations. the specificity of free t is not as good as its sensitivity; as many as % of euthyroid cats with concurrent illness will have high free t concentrations for reasons that remain unclear. as a result, free t should not be used as a screening test, and free t values should be interpreted in light of the tt concentrations. the combination of a high free t with a low tt is indicative of nonthyroidal illness; however a high free t with a high-normal tt is suggestive of hyperthyroidisim. methimazole (tapazole) is the antithyroid drug most often recommended ( . to mg q h). it is available as a transdermal gel or as an oral tablet. methimazole is often used to prepare the patient for surgical thyroidectomy or radioiodine therapy. antithyroid drugs have several side effects. anorexia and vomiting are common side effects of methimazole, whereas rare side effects include self-induced excoriation of the face, thrombocytopenia, bleeding diathesis, agranulocytosis, development of serum antinuclear antibodies, and cholangitis. bleeding, jaundice, and agranulocytosis necessitate immediate withdrawal of the drug. hepatic injury related to antithyroid therapy such as methimazole is well documented in humans and reported in the cat. , mild histologic changes are common, but cases of fulminant hepatic failure with central lobular necrosis have been described. prognosis is excellent with definitive therapy of the hyperthyroidism (surgery or radioactive iodine). hepatic reactions to methimazole will necessitate discontinuation of therapy. the etiology is unknown, but hypoaminoacidemia may play a role in the development of diabetic hepatopathy. , fatty acid, niacin, and zinc deficiencies also may be involved in the pathogenesis. increased serum glucagon, originally thought to be the cause of diabetic hepatopathy, is found in only one-third of the reported cases. a much stronger association between the skin lesions of superficial necrolytic dermatitis and glucagonoma, hyperglucagonemia, and poorly regulated diabetes mellitus have been observed in both humans and dogs. , pathophysiology hepatopathy is thought to occur secondary to the metabolic abnormalities associated with diabetes mellitus, glucagonoma, or nutritional deficiencies. [ ] [ ] [ ] [ ] hepatic features include vacuolar hepatocyte degeneration, hepatic parenchymal collapse, and hepatic nodularity. the disorder is seen most frequently in middle-aged male dogs, and has been reported in one cat. [ ] [ ] [ ] [ ] acute presentations may include clinical signs such as vomiting, diarrhea, lethargy, weight loss, pd, pu, icterus, and lameness because of dermatopathy of the footpads. in some cases clinical signs are mild or nonexistent. physical examination may reveal poor body condition, lethargy, and lipoproteinemias etiology genetic abnormalities in lipid metabolism lead to diffuse vacuolar hepatopathy and biliary mucoceles. increased circulating cholesterol and triglyceride cause a vacuolar hepatopathy associated with excess lipid accumulation and/or hepatocyte glycogen synthesis and storage. chronic hypercholesterolemia increases biliary cholesterol content and predisposes to cystic hyperplasia, dysmotility of gallbladder smooth muscle, and biliary mucocele. familial hypercholesterolemia and other hyperlipidemias are found in certain breeds of dogs including the miniature schnauzer, shetland sheepdog, briard, west highland white terrier, scottish terrier, cairn terrier, and beagle. mixed-breed dogs may also be affected. clinical signs are usually associated with necrotizing cholecystitis and may include icterus and cranial abdominal pain. more often, dogs are asymptomatic and biliary mucoceles are identified serendipitously during ultrasound evaluation for some other medical problem (such as pancreatitis). clinical pathology findings usually include hypercholesterolemia or hypertriglyceridemia, and elevated liver enzyme activities, particularly alp. necrotizing cholecystitis may be accompanied by leukocytosis, neutrophilia, and hyperbilirubinemia. diagnosis may be made by characteristic ultrasound findings of nongravitational gallbladder sludge, increased gallbladder wall thickening, "kiwi"-shaped mucosal image, and bi-or trilaminar appearance of the gallbladder wall. the hepatic parenchyma may have a pattern of multifocal hyperechogenicity and hypoechoic nodules. the best treatment for biliary mucoceles is surgical removal of the mucocele and/or cholecystectomy and may become an emergency procedure if the clinical signs of necrotizing cholecystitis are severe. medical therapy following surgical removal is usually necessary and includes a fat-restricted diet and lifelong treatment with ursodeoxycholic acid ( mg/kg po q h). prognosis is good for patients undergoing successful removal of the mucocele as long as lifelong medical therapy is continued. in dogs and cats, amyloid deposition is usually secondary to sustained systemic inflammatory response, for example, chronic infection, chronic inflammation, immune disorders, and malignancy. amyloidosis is a familial disorder in the chinese shar-pei dog, and in abyssinian, oriental, and siamese cats. [ ] [ ] [ ] [ ] hepatic amyloidosis has also been reported secondary to vitamin a toxicity in cats. pathophysiology deposition of amyloid fibrils within and between hepatic sinusoids results in progressive organ dysfunction. light deposits are found in the space of disse and heavier deposits are often found in the atypical hyperadrenocorticism caused by sex steroid overproduction, dermatologic changes (alopecia, poor hair coat) and reproductive manifestations (perianal adenoma in a castrated male or female dog) are often the only signs suggestive of sex steroid imbalance. atypical hyperadrenocorticism with sex steroid excess may present with no clinical signs except increased serum liver enzyme activities. diagnosis of steroid hepatopathy should be based on a history of exogenous steroid administration or endocrine function testing with or without liver biopsy. classically, liver enzyme elevations consist of moderate to marked increases in alp and ggt, and mild to moderate increases in alt and ast. bile acids may also be increased. the low-dose dexamethasone suppression (ldds) test is considered the screening test of choice for endogenous canine hyperadrenocorticism. , the ldds test has a high sensitivity at % to %. only % to % of dogs with pdh will exhibit suppressed cortisol concentrations at hours. in addition, % of dogs with pdh will exhibit suppression at or hours followed by "escape" of suppression at hours. this pattern is considered diagnostic for pdh, making further testing unnecessary. the major disadvantage of the ldds test is the lack of specificity in dogs with nonadrenal illness. the corticotropin (acth) stimulation test is used to diagnose a variety of adrenopathic conditions, including endogenous or iatrogenic hyperadrenocorticism, as well as spontaneous hypoadrenocorticism. , , as a screening test for the diagnosis of naturally occurring hyperadrenocorticism, the acth response test has a diagnostic sensitivity of approximately % to % and a higher specificity than the ldds test. , in a study by kaplan and peterson, only % of dogs with nonadrenal disease exhibited exaggerated response to acth stimulation. i prefer the acth response test over the ldds test as the acth response test is more accurate for the diagnosis of iatrogenic hyperadrenocorticism (if the history is incomplete) and sex steroid imbalance in addition to pdh or adrenal-dependent hyperadrenocorticism. the urine cortisol-to-creatinine ratio (uccr) is highly sensitive in separating normal dogs from those with hyperadrenocorticism; however, the test is not highly specific for hyperadrenocorticism because dogs with moderate to severe nonadrenal illness also exhibit elevated ratios. [ ] [ ] [ ] an elevated uccr should always be confirmed with an ldds test. in the uccr test, urine is collected for days for a baseline uccr. the animal then is given three doses of dexamethasone ( . mg/kg, po q - h) and the final uccr is collected hours after the first dose of dexamethasone. failure of the uccr to suppress into the normal range is diagnostic for hyperadrenocorticism. treatment for exogenous hyperadrenocorticism consists of discontinuation of exogenous steroids by slowly weaning the patient to prevent the development of addisonian crisis. treatment for endogenous hyperadrenocorticism can be achieved with chemotherapy (o,p′-ddd, or trilostane) or surgery (hypophysectomy or adrenalectomy). treatment of sex steroid imbalance can be achieved with mitotane or trilostane. prognosis for steroid hepatopathy is good to excellent if diagnosed early and if corticosteroid injury can be abated by discontinuation of steroid therapy or treatment of the underlying disorder. threefold increases in the frequency of hypothyroidism. the pathogenesis of stone formation in hypothyroidism is believed to involve hypercholesterolemia, gallbladder dysmotility, and bilirubin retention. the most common clinical symptoms of hypothyroidism are lethargy, weight gain, depression, hypothermia, and bradycardia. gi signs such as reflux esophagitis, gastric atony, constipation, diarrhea, and hepatopathy with mucocele formation are rare clinical signs of hypothyroidism in dogs. symmetric truncal or tail-head alopecia are a classic findings in hypothyroid animals. hyperkeratosis, hyperpigmentation, secondary pyodermas, and demodicosis are also observed. clinicopathologic findings such as normocytic normochromic anemia, hypertriglyceridemia, and hypercholesterolemia are seen in the majority of hypothyroid animals because of altered lipid metabolism and binding proteins (increased hdls), decreased fecal excretion of cholesterol, and decreased conversion of lipids to bile acids. total serum t concentration and endogenous thyroid-stimulating hormone (tsh) may be used to confirm the diagnosis of hypothyroidism. this combination of tests has been shown to have the highest specificity, sensitivity, and lowest overall cost. if the tt is in the low normal or below normal range and the tsh is high, the animal is suffering from primary hypothyroidism. , if the tt and tsh are both low, free t by dialysis should be determined to distinguish euthyroid sick syndrome (normal free t ) from true secondary hypothyroidism (low canine thyroid stimulating hormone [ctsh] resulting from pituitary tsh deficiency). treatment synthetic thyroid hormone supplementation is the treatment of choice for hypothyroidism. levothyroxine sodium therapy is started at a dosage of . mg/kg given orally twice daily. thyroid function should be monitored every to weeks for the first to months of treatment and then once or twice yearly thereafter. in stable well-controlled animals, the total treatment may be given once daily with excellent clinical results, as long as adequate peak hormone concentrations are achieved. with thyroid hormone replacement therapy in hypothyroid dogs, the prognosis is excellent. inflammatory disease involving the intrahepatic bile ducts is commonly encountered in veterinary practice. cholangitis is recognized more commonly in cats than in dogs, but both species can be affected. the wsava liver standardization group suggests that cholangitis be considered in the following four groups: neutrophilic cholangitis (nc), lymphocytic cholangitis (lc), chronic cholangitis associated with liver fluke infestation, and destructive cholangitis. sinusoidal lumen. amyloid fibrils are readily detected on routine hematoxylin and eosin or diff-quik staining. amyloidosis is confirmed on examination of congo red-stained aspirates or biopsies under polarized light where the extracellular material shows characteristic green birefringence. concurrent amyloid deposition in the kidneys, liver, spleen, and adrenal glands can occur, but clinical manifestations of liver failure are most common. chronic progressive liver failure with clinical signs of anorexia, weight loss, and lethargy, is the typical clinical course in many cases. some animals may instead present with acute collapse following hepatic rupture and intraabdominal hemorrhage. pallor of mucous membranes, hypothermia, and hepatomegaly are the most frequently recognized physical examination findings. typical laboratory findings include regenerative anemia, leukocytosis, thrombocytopenia, marked elevations in serum alt and ast, and marked prolongations in aptt and pt times. radiography is useful in detecting free peritoneal fluid, hepatomegaly, and irregular hepatic borders. ultrasonography reveals a diffuse, heterogeneous echogenicity with highly echogenic ("sparkling") areas and hypoechoic foci. definitive diagnosis requires tissue biopsy and congo red staining. there are no specific treatments for this disorder. colchicine has been recommended because it may block formation of amyloid in the early stages of the disease, but it is of unproven benefit and has been associated with significant side effects. dimethyl sulfoxide has been recommended because it may promote resorption of amyloid. as there are no specific therapies for this disease, treatment is instead largely symptomatic and supportive. with progressive amyloidosis lesions, the prognosis for long-term survival is poor. a familial hyperlipoproteinemia has been reported in cats that is characterized by fasting hyperchylomicronemia, elevated circulating concentrations of vldls, and hypertriglyceridemia. , serum cholesterol is only minimally elevated. the underlying biochemical lesion is a reduction in the activity of lipoprotein lipase, and the disorder is transmitted as an autosomal recessive gene. xanthomas accumulate in the soft tissues, including the liver, but clinical signs are more often related to involvement of the peripheral nerves. dietary fat restriction improves clinical signs in some affected animals. etiology decreased circulating thyroid hormone concentration affect hepatic metabolism and cholesterol turnover in the liver. liver function tests are mildly disturbed in almost % of patients with hypothyroidism despite normal histologic findings. pathophysiology decreased hepatic metabolism in hypothyroidism is reflected by reduced oxygen consumption. , , patients with a common bile duct stone and gallbladder stone have, respectively, sevenfold and infection of bile has been commonly identified in cats with ehbdo, , clinical examination. previous literature has highlighted differences in clinical presentation between cats with different forms of cholangitis. however, we have recognized few differences between the various forms , and suggest that any statistically significant differences cited previously hold little clinical relevance given the large degree of overlap within data ranges. nc can occur in cats of any age, breed, or sex. clinical signs are nonspecific and include anorexia, lethargy, vomiting, and weight loss. the duration of these clinical signs ranges from a few days to a few months and may be shorter in cats with anc than in those with cnc, but this is not a consistent finding. , physical examination findings commonly include dehydration and icterus. fever is present in % to . % of cases. , some reports suggest that fever is more commonly associated with anc than cnc, while others recognize no difference. , hepatomegaly is seen in fewer than half of the cases. abdominal pain is noted occasionally. , , diagnosis. definitive diagnosis is made by examination of liver biopsy specimens, with ancillary diagnostics providing supportive information. hematologic findings are variable and may include poikilocytosis, neutrophilia, and left shift, although these abnormalities are present in fewer than one-third of cases. , , , biochemical analysis commonly reveals increased activity of alt, ast, alp, and ggt ranging in severity from mild to severe. however, increased liver enzyme activity may be absent in some cases. serum total bilirubin is increased in most cases. serum cholesterol may become increased in cases with ehbdo. imaging findings are nonspecific for cholangitis, but may provide useful information regarding concurrent disease. abdominal radiographs are rarely helpful. ultrasonographic appearance of the liver in cats with nc can vary greatly, with the most common abnormality being a diffuse change in echogenicity ranging from hypo-to hyperechoic. dilation of intra-and/ or extrahepatic bile ducts, gallbladder distention, increased gallbladder sediment, and thickening of the gallbladder or bile duct walls may be seen. gallbladder distention and bile duct dilation may indicate ehbdo, but these changes may occur in cats with cholangitis lacking obstruction. ultrasonography will also provide information regarding the presence of concurrent disease, such as pancreatitis and inflammatory bowel disease. wedge liver biopsy during laparotomy is the optimal method for obtaining a definitive diagnosis. other biopsy techniques that may be considered include laparoscopic and ultrasound-guided tru-cut needle approaches. tru-cut needle biopsy diagnoses correlate with wedge biopsies in fewer than % of cases. diagnostic accuracy of laparoscopic liver biopsies compared with wedge biopsies have not been evaluated. laparotomy and laparoscopy provide the additional benefit of evaluation and sampling of extrahepatic structures. laparotomy should be performed in any cat suspected of having ehbdo. while the optimal sampling strategy is unknown, biopsies should be obtained from multiple liver lobes, as we have recognized wide ranges of severity between different lobes in the same cat. in patients that are not stable enough for liver biopsy, such as those with hypotension, coagulopathy or he, fine-needle aspiration with cytology offers a less-invasive diagnostic approach as it can usually be performed quickly with light sedation. however, liver cytology correlates with biopsy results in only % to % of cases. , cytology is sensitive for identifying the presence of hl, however this is the most common misdiagnosis when using cytology. cytology is cholangitis is a common hepatobiliary disorder of cats, second only to hl. although varying terminology has created some confusion regarding this syndrome, it is clear that feline cholangitis includes a spectrum of disease processes, including forms displaying neutrophilic inflammation and those lacking neutrophilic inflammation. histologically, nc is characterized by the presence of neutrophils in the lumen and/or epithelium of the bile ducts. the disease is recognized to occur in acute and chronic forms. in acute neutrophilic cholangitis (anc) edema and neutrophilic inflammation are seen in the portal areas, with occasional extension of inflammation to the hepatic parenchyma. in chronic neutrophilic cholangitis (cnc) there is a mixed inflammatory infiltrate consisting of neutrophils, lymphocytes, and plasma cells. varying degrees of bile duct hyperplasia and fibrosis will be present depending on the chronicity of disease. etiology. although the true etiology remains unknown, nc is largely suspected to be caused by ascending bacterial infection from the intestine. - rates of bacterial isolation using traditional methods have varied greatly, from less than % to more than % in affected cats. , recently, fluorescence in-situ hybridization (fish) with a s rdna probe that recognizes bacteria in general has been used to identify and localize bacteria in cats with cholangitis. combining traditional culture and fish, bacteria were isolated in three of three ( %) cats with anc and eight of ( %) with cnc. the localization of the bacteria identified using fish supports translocation of enteric bacteria as the cause of infection. although it appears that bacteria play an important role in the etiology of nc in many cases, it is important to note that they are not identified in all affected cats. some authors theorize that nc, and cnc in particular, may have an immune-mediated etiology with persistent inflammation following an initial bacterial infection or other unknown initiating factor. , pathophysiology. nc in cats is commonly associated with inflammatory bowel disease and pancreatitis. , , the pathophysiology underlying the relationship of these diseases is unknown, but rational theories revolve around the unique anatomy of the feline biliary and pancreatic duct systems. in the cat, the common bile duct and pancreatic duct merge prior to entering the duodenum at the major duodenal papilla. cholangitis may develop secondary to reflux of ascending bacteria from the duodenum during vomiting. pancreatitis may result from bacterial reflux into the pancreatic duct, or from pancreatic duct obstruction secondary to cholangitis. in most reported cases, the inflammatory bowel disease associated with cholangitis is moderate or severe, whereas the pancreatitis tends to be mild chronic interstitial disease. nc is also commonly associated with extrahepatic bile duct obstruction (ehbdo). ehbdo has been identified in % of cats with anc and % of cats with cnc. cholangitis and/or pancreatitis are the most common cause of ehbdo in the cat. in one study, % of cats with ehbdo had cholangitis, representing % of cats that did not have a neoplastic cause. it is unknown whether cholangitis is the cause or the result of ehbdo. histologic changes consistent with cnc have been seen in the livers of cats with ehbdo secondary to pancreatic carcinoma, cholelithiasis and surgical occlusion of the common bile duct. in contrast, cholangitis has been implicated as the sole cause of ehbdo resulting from proliferation of mucosa within the common bile duct. bacterial choleretic properties that make it a rational choice for treating cholangitis. because of the possibility of immune-mediated mechanisms in the perpetuation of nc, particularly with cnc, corticosteroids may be appropriate in some cases. initial treatment should always involve antibiotics in cats with nc. failure to improve within weeks of antibiotic therapy, or clinical deterioration prior to that time, warrants initiation of corticosteroid therapy. prednisolone at to mg/ kg twice daily is given initially and gradually tapered to the lowest effective dose. antibiotics should be continued concurrently with corticosteroids for a minimum of weeks. the duration of corticosteroid therapy varies between individual patients. many cases can be gradually tapered off of corticosteroids over to months, while others require lifelong therapy. surgical intervention is required in cats with ehbdo; however, the optimal surgical procedure is unknown. biliary diversion (cholecystocholedochostomy, choledochoduodenostomy, or cholecystojejunostomy) and choledochal stenting are the most common procedures. surgery in cats with ehbdo is associated with significant perioperative morbidity. in many cases, profound hypotension develops intraoperatively after to minutes as a result of decreased vascular responsiveness and decreased myocardial contractility and is often refractory to interventions such as fluid or vasopressor therapy. , , whichever surgical procedure is chosen, it is clear that anesthesia time should be minimized and long-term medical management will be necessary. biliary diversion is associated with short-term mortality rates of % to % , and is associated with long-term complications. , in a small case series describing choledochal stenting in cats with pancreatitis and cholangitis, five of seven experienced long-term survival (≥ months), but reobstruction occurred in two of seven and chronic vomiting and recurrent cholangitis were reported. prognosis. the prognosis for cats with nc is typically good. , , survival to discharge was reported in % of all cats with cholangitis in one study. median survival time of . months has been reported in cats with nc, with no difference between anc and cnc. prognostic factors have not been identified. given the high rate of perioperative morbidity and mortality, it seems likely that cats with ehbdo have a worse prognosis than those without ehbdo. thirty percent to % of cats with ehbdo secondary to inflammatory disease die within a week of surgery. , however, in those that survive to discharge, long-term survival has been reported. the wsava liver standardization group describes lc as a common, slowly progressive, chronic disease of cats characterized histologically by infiltration of small lymphocytes (and occasionally plasma cells or eosinophils) restricted to the portal areas associated with varying degrees of fibrosis and bile duct hyperplasia. they remark that inflammation centered on the bile ducts may be present, but is not a hallmark of the disease. it is also stated that welldifferentiated lymphoma may be difficult to differentiate from lc. based on the existing literature regarding lc in cats, the description from the wsava group includes several clinically and histopathologically different subsets that may or may not revolve around a common pathogenesis. recognition of these different subsets within the umbrella of lc may have therapeutic and prognostic ramifications. several investigators describe a group of cats with lc where inflammation is confined to portal regions and there is a lack of targeting of bile ductules or biliary epithelium. , , this has been insensitive for identifying cholangitis in cats, diagnosing fewer than % of cases. cytologic examination of bile may prove more sensitive for the diagnosis of nc in cats. in five of seven cats with cnc evaluated at this institution, bile cytology revealed neutrophilic inflammation, presence of bacteria, or both. techniques have been described for safely obtaining bile via ultrasound-guided percutaneous cholecystocentesis in lightly sedated cats. samples for aerobic and anaerobic bacterial cultures should be obtained in any cat suspected of having cholangitis. gallbladder bile is preferred to liver tissue as the culture source. in a group of cats suspected of having hepatobiliary disease, bile cultures isolated pathogens in % compared with only % of liver cultures. in the same study, dogs and cats had both liver and bile cultured and none had a positive liver culture in the absence of a positive bile culture. in a group of cats with cholangitis, bile cultures were more likely to isolate pathogens ( % vs. %) and less likely to yield contaminants ( % vs. %) than liver cultures. in a small study comparing bile versus liver cultures in cats with various hepatobiliary diseases, bile culture was positive in five (four had cnc) while liver culture was positive in only two. in the two cats with positive liver cultures, the same organism was isolated from bile. it is important to recognize that many cats with nc (and other hepatobiliary diseases that mimic it clinically) are not stable enough to tolerate diagnostic testing. in such patients, the risk of aggressive diagnostics may outweigh the benefits of obtaining a definitive diagnosis. in these cases, the diagnosis may be suspected based on clinical response to supportive care, including broad-spectrum antibiotic therapy. treatment. optimal treatment protocols for cats with nc are unknown and the recommendations herein are based solely on anecdotal clinical experience. antibiotics are the mainstay of treatment. drug selection is ideally based on results of bacterial culture and susceptibility testing. in cases where cultures are not performed, or while results are pending, broad-spectrum coverage should be provided. the most commonly isolated pathogens are aerobic and anaerobic bacteria of enteric origin, including e. coli, enterococcus spp., and clostridium spp., among others. , , effective empiric antibiotic combinations would include a penicillin, a fluoroquinolone, and metronidazole. the optimal duration of antibiotic therapy is unknown, but we recommend a -to -week course for initial treatment. supportive care and treatment of specific sequelae of liver disease should be included as indicated. nutritional support is required in many cats and is best accomplished by use of enteral feeding tubes. we recommend placement of esophageal feeding tubes in cats with cholangitis if they are anorexic and stable enough for general anesthesia. in unstable patients, nasoesophageal feeding tubes offer a less-invasive method of providing short-term support. several medications and nutritional supplements (including ursodeoxycholic acid [udca], same, milk thistle, vitamin e, vitamin c, carnitine, taurine, and phosphatidylcholine) have been suggested for treating cats with cholangitis. while most of these compounds have theoretical benefits, a clinical benefit has not been proven. to optimize client compliance and avoid adverse drug reactions, i prefer to minimize the number of medications given to feline patients. because most cats with anc respond well to antibiotic therapy, i rarely include other medications in our treatment protocol. however, in cats with anc that do not quickly respond to antibiotics and in many cats with cnc, i like to use udca. among its theoretical benefits, udca has immunomodulatory and distinction between lc and well-differentiated (small cell) lymphoma can be a challenge even for experienced pathologists. preliminary data using immunohistochemistry and pcr for t-cell receptor clonality has not proven useful in differentiating between the two conditions. surprisingly, cats with both lc and lymphoma had monoclonal t-cell receptors, oligoclonal t-cell receptors, and polyclonal t-cell receptors. using light microscopy, the following features were unique to lc and not present in cats with lymphoma: ductopenia, bile duct targeting by lymphocytes, and the presence of lipogranulomas within portal regions (representing a residual marker of cell death). until more studies are done evaluating molecular techniques, these features may prove useful in differentiating the two conditions. interestingly, bile duct hyperplasia and fibrosis were present in cats with lc and those with lymphoma. this may suggest that an inflammatory state precedes the development of lymphoma, which has been reported anecdotally. treatment. the therapeutic approach to cats with lc should be similar to that described for cats with nc in regards to supportive care and symptomatic treatment of the sequelae of liver disease. because bacteria have been isolated from some cats with lc, i recommend treatment with broad-spectrum antibiotics while awaiting results of bacterial cultures. in contrast to nc, long-term treatment of culture negative cats with antibiotics is not warranted. immunomodulation and immune suppression are the major components of treatment based on a presumed immune-mediated etiology. cats that are culture negative, or that have failed to respond to antibiotics within a few days, should be treated with prednisolone at to mg/kg twice daily. responders should be tapered gradually over to months to the lowest effective dose. other drugs that are useful for immunomodulation include metronidazole and udca. cats that fail to respond completely to corticosteroids and/ or other immunomodulators, or who relapse while being treated, may require additional immunosuppressive drugs. although these drugs have not been well evaluated in cats with lc, chlorambucil and methotrexate are suggested by some authors. cats with small cell lymphoma often respond to combination therapy with prednisolone and chlorambucil, but they may require a multidrug weekly sequential chemotherapy protocol. prognosis. cats with lc have a variable prognosis, , likely a result of being diagnosed at different stages of a chronic disease process. survival of greater than years has been reported, and many cats that die appear to succumb to disease unrelated to the liver. other cases have been reported that fail to respond to treatment and die more acutely, though this is uncommon in my experience. this is a disease that likely requires lifelong management and monitoring with relapse of illness possible as medication doses taper. trematode parasites of the families dicrocoeliidae and opisthorchiidae may inhabit the gallbladder and bile ducts of cats and rarely dogs. there are multiple species with worldwide distribution. the most commonly identified species include the dicrocoelid platynosomum concinnum and the opisthorchid amphimerus pseudofelineus. [ ] [ ] [ ] p. concinnum is mainly found in tropical and subtropical areas, including the southeastern united states. a. pseudofelineus has a wider area of distribution throughout north and south america. the life cycle is similar for both dicrocoelids and opisthorchids. parasite eggs are ingested by a land snail (subulina octona or eulota [bradybaena] similaris), develop into cercariae and enter a second referred to as lymphocytic portal hepatitis. , the connection between this histopathologic finding and clinical disease in cats is unknown, as it may represent a common change associated with aging. it was identified in % of cats older than years of age and % of cats older than years of age from a necropsy population that did not have primary liver disease. it is also possible that this lesion represents a response to inflammation at a distant site, as it is similar to the lesion of nonspecific reactive hepatitis associated with chronic extrahepatic disease. although clinical signs have been described in cats with lymphocytic portal hepatitis, the common occurrence of concurrent disease in these cats makes it difficult to know if the clinical signs are attributable to the lesions in the liver. in another subset of cats, lc is marked by inflammation targeting bile ductules and infiltrating biliary epithelium, leading to progressive ductopenia. , , these cases seem more likely to have clinical disease attributable to their liver pathology, although side-by-side comparisons of cases with and without bile duct targeting have not been performed. cats with this form of lc in the united states have a similar clinical picture to cats with nc. in the united kingdom, this lesion has been associated with ascites, icterus, and hyperglobulinemia in young cats and termed progressive lymphocytic cholangitis. , etiology. although the etiology of lc is unknown, theories suggest that it is an immune-mediated or infectious phenomenon. genetic factors may also play a role, as persian cats are overrepresented in the united kindgom. , immunohistochemistry in affected cats has provided evidence for an immune-mediated pathogenesis, although the inciting antigen is unknown. , bacteria have been identified in the liver or bile of fewer than % of cats with lc. [ ] [ ] [ ] , although helicobacter pylori has been isolated from the liver and bile of cats with cholangitis, the evidence for this organism playing an important role in feline cholangitis is not compelling at this time. , pathophysiology. as with nc, concurrent inflammatory bowel disease and pancreatitis appear to be common in cats with lc, , although some authors report it to be uncommon. the theory that reflux of duodenal bacteria into the biliary and pancreatic ducts incites inflammation may hold true for cats with lc, although a common immune mechanism must be considered. clinical examination. the clinical picture of cats with lc varies widely and has significant overlap with other forms of hepatobiliary disease in cats, including nc. , although some studies describe a predominance of older cats, others describe more younger cats. , nonspecific clinical signs, including anorexia, lethargy, vomiting, and weight loss, may be chronic and intermittent. , , physical examination findings may include icterus, hepatomegaly, or ascites, but none are consistent findings. signs of he (dullness, ptyalism, seizure) may develop in severely affected cats. diagnosis. definitive diagnosis is made by liver biopsy. as discussed for nc, ancillary diagnostics will provide information to support hepatobiliary disease, but are not specific for lc. hematology results may be unremarkable, even though marked lymphocytosis has been described in some cases. activity of serum liver enzymes is increased in many, but not all cases and varies in severity. hyperglobulinemia has been described. , , abdominal radiographic and ultrasonographic findings are nonspecific, but may aid in the recognition of concurrent disease. amoxicillin-clavulanate, milbemycin oxime, and amitraz prior to the onset of clinical signs. , the proposed toxic etiology is based on these case histories and histopathologic similarity to humans with idiosyncratic drug toxicity. other toxic insults and viral infection, such as canine distemper, can also result in destructive cholangitis. affected dogs typically present for signs referable to cholestasis, including anorexia, icterus, vomiting, and acholic feces. , activities of serum liver enzymes and total bilirubin are moderately to markedly increased. abdominal ultrasound may be unremarkable, showing only mild dilation of intrahepatic bile ducts. definitive diagnosis is made by liver biopsy. optimal treatment options are unknown, but should undoubtedly involve discontinuation of any medications that preceded illness. corticosteroids for immune suppressive and antiinflammatory effects and udca for immunomodulatory and choleretic effects would be rational treatments options, but their efficacy has not been documented. the prognosis appears to be poor, as six of the eight reported cases were euthanized within weeks and the remaining two had only shortterm followup (< months). , nc, as described in the cat, has been rarely reported in dogs. [ ] [ ] [ ] [ ] [ ] bacteria have been isolated from the majority of cases reported, including e. coli, klebsiella spp., proteus mirabilis, streptococcus spp., and clostridium spp. [ ] [ ] [ ] [ ] although the bacterial species would support ascending infection from the intestine, the literature is too sparse to make conclusions about the pathophysiology of this disease in dogs. bacterial infection could also spread hematogenously or via translocation from the portal circulation. the clinical presentation and diagnostic findings are similar to those reported for cats with nc. affected dogs present with lethargy, anorexia, vomiting, and icterus usually of acute onset. fever is reported in approximately half of the cases. [ ] [ ] [ ] [ ] [ ] neutrophilia with or without a left shift is common. activity of serum liver enzymes is typically increased and most dogs have mild to moderate elevation of serum total bilirubin. ultrasonography is nonspecific, with the liver varying from normal to hyperechoic with some heterogeneity. , thickening and hyperechogenicity of the gallbladder wall is common. definitive diagnosis is made by liver biopsy with changes similar to those reported in cats with nc. most dogs have some degree of mixed inflammatory infiltrate, similar to cnc. [ ] [ ] [ ] [ ] [ ] this infiltrate extends into the hepatic parenchyma in the majority of cases. aerobic and anaerobic culture of bile or hepatic tissue should be performed, though bile has been the source of bacterial isolation in most cases. treatment involves antibiotic therapy guided by culture and susceptibility results. duration of treatment should be prolonged, as antibiotic courses of to weeks or greater have been required to completely eliminate bile infections. clinical improvement precedes bacterial eradication. the prognosis appears to be good in most cases, although dogs with concurrent disease may have a worse prognosis. liver cysts arising from the intrahepatic bile ducts are rarely encountered in veterinary practice. although cysts may be acquired secondary to trauma, neoplasia, inflammation, or biliary obstruction, , the vast majority of cases described in the literature are congenital in origin. congenital cystic liver diseases result in dilation of various segments of the intrahepatic bile ducts, and they are associated with varying degrees of hepatic fibrosis and cysts in other organs (most commonly the kidneys). little is known about inheritance patterns of cystic disease in dogs and cats. the various morphologic patterns of cystic disease likely represent abnormalities of bile duct intermediate host. , , the dicrocoelids tend to use an arthropod, while opisthorchids utilize fish. typically cats acquire infection by ingesting the second intermediate host. in the case of p. concinnum, the sporocysts leaving the snail intermediate host may be eaten by a paratenic terrestrial isopod host (pill, sow, or dung bugs). , cats are infected by ingesting this form in a variety of lizard or amphibian intermediate hosts. cercariae migrate from the cat intestine to the gallbladder and bile ducts, where they develop into adults. eight weeks or more after infection eggs are passed in the feces to complete the life cycle. clinical signs are proportional to parasite burden. cats with light infections are often asymptomatic. , clinically ill cats may present with nonspecific signs such as anorexia, lethargy, vomiting, or diarrhea. severely affected cats present with signs of ehbdo such as icterus and acholic feces. [ ] [ ] [ ] [ ] preliminary diagnostics are nonspecific for fluke infestation. eosinophilia proportional to the parasite burden may be present. serum liver enzyme activity may be mildly to moderately increased, although it is normal in many cases. [ ] [ ] [ ] abdominal ultrasound often reveals evidence of ehbdo. [ ] [ ] [ ] definitive diagnosis by identification of flukes or fluke eggs in the feces is difficult as small numbers of eggs are shed daily, the eggs have varying morphology at different stages of development, and the eggs are quite small. fecal concentration-sedimentation using the formalin-ether technique is the most reliable method of identifying eggs in stool. , eggs may also be identified in cytologic preparations of bile. , eggs or adults may be seen in liver biopsy specimens, but they are inconsistently identified. , , , histopathologic changes seen in the liver are characterized by dilation of larger intrahepatic bile ducts associated with papillary projections and marked periductal and portal fibrosis. mild to moderate inflammation may be present within the ducts (neutrophils and macrophages) and in the portal areas (neutrophils, lymphocytes, plasma cells). eosinophils may be present in limited numbers. rarely, chronic cholangitis associated with liver flukes can result in the development of cholangiocarcinoma. , optimal treatment protocols have not been established, but praziquantel at to mg/kg daily for days appears to be the most effective. [ ] [ ] [ ] [ ] [ ] doses as high as mg/kg daily have been used successfully, but this dose has also been fatal in cats. sporadic resumption of egg shedding following praziquantel has been reported, suggesting that it does not completely eliminate infection. , for this reason, continued treatment at -week intervals has been recommended. symptomatic and supportive therapy should be tailored to the individual patient. cats with ehbdo require surgical decompression. glucocorticoids and udca may have some benefit in controlling inflammation and providing choleresis. although infected cats may remain asymptomatic, patients with ehbdo appear to have a grave prognosis. long-term survival has only been reported in rare cases. , cholangitis is rarely reported in dogs. reports of cholangitis in dogs include two distinct entities: destructive cholangitis and nc. destructive cholangitis is characterized histopathologically by a loss of bile ducts (ductopenia) within the smaller portal areas, associated with cholestasis, portal inflammation consisting primarily of macrophages, neutrophils, and occasionally eosinophils, and progressive portal fibrosis. , , this is a rarely reported lesion of unknown etiology. it has been postulated that the lesion represents an idiosyncratic drug toxicity. however, of the eight cases reported in the literature only three had a prior drug history: two having received potentiated sulfonamides and the other having received adult polycystic disease is most commonly recognized as polycystic kidney disease in persian cats. , it has also been reported in cats of other breeds and in dogs. , this is similar to autosomal dominant polycystic kidney disease in humans. inheritance in persian cats is autosomal dominant. liver cysts are thought to represent a late defect in the development of peripheral intrahepatic bile ducts. the liver may contain multiple cysts ranging from less than mm to greater than cm in diameter. these cysts typically contain clear, colorless fluid. discrete fibrotic areas containing small, irregularly formed bile ducts, referred to as von meyenburg complexes, may be present. in the kidneys, multiple cysts may form in any segment of the kidney but may involve only a small percentage of the nephron population. this is in contrast to the diffuse cysts seen with congenital dilation of the large and segmental bile ducts and juvenile polycystic disease. , hepatic cysts are present in % to % of cats with polycystic kidney disease, while hepatic fibrosis is recognized in up to %. , the hepatic cysts are usually incidental findings and the animals are not clinically ill unless they develop renal failure secondary to cysts in the kidneys, which happens in adulthood. biliary atresia is an extremely rare congenital disorder, having been reported in only one dog and one cat. , in both cases, the common bile duct was not patent because of atresia. in the dog, the occluded segment of bile duct was histologically comprised of fibrous tissue with minimal inflammation. the etiology is unknown, but this lesion likely represents an embryologic nonfusion of the cranial (hepatic) and caudal (cystic) anlages of the bile ducts during development. other possible explanations include ischemic, toxic, traumatic, or infectious insults occurring pre-or postnatally. affected animals have presented at to months of age with clinical signs of depression, anorexia, vomiting, or lameness associated with rickets because of inadequate vitamin d absorption. , affected animals show icterus, hepatomegaly and acholic feces. serum biochemistry abnormalities are consistent with ehbdo. definitive diagnosis is made at exploratory laparotomy. surgical biliary diversion is a viable treatment option depending on the location of atresia, but it was unsuccessful in the one case reported. a guarded prognosis should be given as for any animal undergoing a biliary diversion procedure (see discussion under "neutrophilic cholangitis"). cholestasis is impaired bile flow resulting in the accumulation of bile components in the blood. intrahepatic cholestasis occurs secondary to a variety of primary or secondary hepatobiliary diseases. , , increased activity of serum liver enzymes, particularly alp and ggt, is common with intrahepatic cholestasis but is not specific for the condition. clinically patients may appear jaundiced, but the predominant clinical sign will be related to the underlying disease process. cholestasis is marked by the presence of bile plugs in canaliculi, phagocytosed bile in kupffer cells, and bile granules within hepatocytes. these changes are easily recognized in cytologic and frozen preparations, but are less apparent in paraffin-embedded specimens, particularly in cats. when cholestasis is identified, ehbdo should be ruled out. this should be easily accomplished by abdominal ultrasonography as animals with intrahepatic cholestasis lack the dilation of intra-and extrahepatic bile ducts that is typical of ehbdo. however, exploratory laparotomy should be considered in highly suspicious cases for confirmation. development at different stages of their formation. the wsava liver standardization group suggests that cystic disorders be classified into one of the following groups: congenital dilation of the large and segmental bile ducts; juvenile polycystic disease/congenital hepatic fibrosis; and adult polycystic disease. congenital dilation of the large intrahepatic bile ducts (i.e., the hepatic ducts and segmental ducts) has been described in dogs. [ ] [ ] [ ] the lesion is similar to that of caroli disease in humans and it is thought to represent an early defect in the formation of the intrahepatic bile ducts. , the disease is marked by extreme, diffuse, grossly evident dilation of the extrahepatic portion of the large intrahepatic bile ducts containing pale-yellow viscous fluid. the gallbladder and common bile duct are normal, as these have a separate embryologic origin from the intrahepatic bile ducts. the liver is normal to mildly increased in size, with diffuse cysts of varying sizes throughout. histologically there are areas of marked bridging portal fibrosis containing multiple dilated bile ducts. the lobular architecture of the liver is normal. although concurrent ascending cholangitis commonly occurs in humans, this is rarely reported in dogs. [ ] [ ] [ ] in addition to the hepatic lesions, affected dogs have fusiform, radially arranged renal cysts with moderate to marked fibrosis throughout the renal cortex and medulla. [ ] [ ] [ ] affected dogs are presented early in life, ranging from weeks to . years. [ ] [ ] [ ] clinical signs include vomiting, weight loss/failure to thrive, decreased appetite, lethargy, ascites, and rarely icterus and neurologic signs. the clinical signs are typically chronic in nature. gi signs, neurologic signs, and ascites are likely a result of portal hypertension caused by pressure of the cysts on the portal vein. hepatomegaly may be noted on physical examination. activity of serum liver enzymes is typically normal to mildly increased, although marked increases in activity of alt and alp have been reported. renal azotemia is present in some patients. ultrasonographically, cystic dilations of the intrahepatic ducts (most with associated calcification) are easily recognized, even though the renal cysts are not always apparent. definitive diagnosis is made on the basis of the gross and histologic findings described above. rational treatment options and prognosis are unknown, as only one dog in the literature has been treated. this dog was doing well on a low-protein diet at months of followup. most of the affected dogs have been fairly stable, and supportive care may be warranted despite the appearance of severe cystic disease. juvenile polycystic disease/congenital hepatic fibrosis has been described in litters of cairn terriers, west highland white terriers, and cats. [ ] [ ] [ ] this form is analogous to autosomal recessive polycystic kidney disease in humans, and the inheritance appears to be autosomal recessive in the few families of veterinary patients that have been described. , the liver cysts are thought to represent an intermediate defect in the development of the intrahepatic bile ducts. , the liver involvement is primarily microscopic including fibrotic portal areas containing abnormally structured, dilated small bile ducts. the result is a grossly enlarged and firm liver. renal cysts are present and are identical to those described for dogs with dilation of the large and segmental bile ducts. affected animals are usually presented at less than weeks of age for abdominal distention because of renomegaly and hepatomegaly. , however, one -yearold cat with similar lesions has been reported. most affected animals are ill or have died at the time of presentation. , liver enzyme activity has been increased in the few animals in which it was evaluated. abdominal ultrasound may identify cysts and definitive diagnosis is made based on histologic findings as described. treatment has not been attempted, and the prognosis appears to be grave. hepatic progenitor cells. they may be identified at intrahepatic or extrahepatic locations. they appear to have a more aggressive course than cholangiocellular carcinomas, with the majority being present in multiple lobes and greater than % having metastasized at the time of diagnosis. , , the major pathogenetic mechanisms of canine and feline extrahepatic biliary tract disease are obstruction, inflammation, and exudation. the major causes of extrahepatic biliary tract obstruction (ehbo) are pancreatitis, gallbladder mucoceles (in dogs), cholelithiasis, parasitic infections (in cats), and tumors. ehbo is more common in dogs than cats. biliary tract inflammation (e.g., cholecystitis, cholangitis) is primarily caused by bacterial infection, but can be nonseptic or parasitic, especially in cats. gallstones may be associated with biliary tract disease (e.g., infection, obstruction), but the majority of them appear to be clinically silent. stones in the gallbladder are termed choleliths while stones in the biliary tract are termed choledocholiths. biliary tract exudation or leakage can be caused by traumatic (primarily of the bile ducts) or spontaneous rupture (primarily of the gallbladder). the latter is caused by necrotizing cholecystitis, which can be caused by sepsis (e.g., infectious cholecystitis), pressure necrosis (e.g., mucocele), or infarction. biliary tract tumors are rare and of uncertain cause. the common bile duct passes through the lesser omentum and the pancreatic parenchyma before entering the mesenteric wall of the duodenum. in the dog, it empties near the opening of the minor pancreatic duct at the major duodenal papilla, whereas in the cat it joins with the major pancreatic duct before emptying into the duodenum. inflammation and edema with pancreatitis may be sufficient to cause compression and obstruction of the bile duct. this is probably the most common cause of canine ehbo. there is, however, no consistent relationship between clinical severity of the pancreatitis and likelihood of ehbo, possibly because pancreatitis can affect different regions of the pancreas. pancreatitis is a rare cause of ehbo in the cat. chapter discusses the breeds at increased risk for pancreatitis and causes. gallbladder mucoceles occur primarily in dogs. in such cases, the gallbladder is filled with inspissated, semisolid mucus that may extend into the bile ducts causing obstruction. mucoceles can exceed the storage capacity of the gallbladder thereby causing pressure necrosis on the wall of the gallbladder. they can also spontaneously rupture (often at the fundus) , causing bile peritonitis. the cause of gallbladder mucocele is unknown, but might include dysfunction/hyperplasia of mucus-secreting cells in the gallbladder mucosa. mucoceles may become secondarily infected. gallstones are often clinically silent and observed incidentally only at the time of abdominal imaging. they can be associated with cholecytitis, but they rarely cause ehbo because they must be small enough to enter the cystic bile duct but large enough to lodge there. intrahepatic cholestasis is associated with extrahepatic bacterial infection in dogs. this syndrome is well characterized in humans and may occur in other species such as the cat. it represents an important differential diagnosis for hyperbilirubinemia in animals without primary liver disease, as over -fold increases in total bilirubin have been reported. the physiology behind this mechanism is incompletely understood, but it is thought to result from reduction of bile salt-dependent and -independent bile flow caused by bacterial toxins and/or inflammatory mediators. tumors of biliary origin in dogs and cats include cholangiocellular adenoma, cholangiocellular carcinoma, and carcinoid. they are uncommon, representing less than % of all canine and feline neoplasms. [ ] [ ] [ ] [ ] the tumors of epithelial origin, cholangiocellular adenoma and carcinoma, are the most common, comprising % of all hepatic neoplasms in dogs [ ] [ ] [ ] and % to % of all hepatic neoplasms in cats. [ ] [ ] [ ] tumors showing characteristics of both hepatocellular and cholangiocellular carcinoma have been reported rarely. , in dogs, % to % of biliary epithelial tumors are malignant, , while in cats % to % are malignant. [ ] [ ] [ ] cholangiocellular adenomas commonly contain cystic components, especially in cats, and have been referred to as biliary or hepatobiliary cystadenomas in this species. , cholangiocellular tumors arise predominantly from intrahepatic bile ducts in both species. extrahepatic location is more common in cats than in dogs and is always associated with malignancy. [ ] [ ] [ ] [ ] in both species, cholangiocellular carcinomas are more likely to present as multiple or diffuse tumors than are adenomas. , , carcinomas are highly metastatic ( % to % rate) with local lymph nodes, peritoneum, and lung being the most common sites of metastasis. , , the etiology of biliary neoplasia is unknown. affected dogs and cats are typically middle-aged to older, although animals with malignancy may present at a younger age than those with benign disease. , clinical signs are usually vague (such as anorexia and lethargy) and somewhat chronic. malignant tumors are more likely to cause clinical signs, as many benign tumors are incidental findings not associated with illness. , [ ] [ ] [ ] hepatomegaly or the presence of a cranial abdominal mass may be identified on physical examination. increased activity of serum liver enzymes is more commonly associated with malignancy, often being absent with benign tumors. , , even though abdominal radiographs will often identify the presence of hepatomegaly or an hepatic mass, ultrasonography is the preferred imaging method for identification of biliary neoplasms. this modality allows for determination of the cystic nature of the tumors and for the evaluation of metastatic potential. fine-needle aspiration and cytology are of limited utility for diagnosis of biliary epithelial tumors. carcinoma was correctly identified via liver mass aspiration in only % of cases in one study. however, cytology is recommended as it likely exhibits high specificity, especially for metastatic lesions. surgical excision and biopsy is the optimal diagnostic and therapeutic technique for tumors confined to one or two liver lobes. surgical excision appears to be curative in cats with benign tumors, , but malignant tumors carry a poor prognosis in both dogs and cats with many cases not surviving to discharge and survival greater than months not reported in any case. , adjunctive chemotherapeutic protocols have not been reported. carcinoids, also referred to as neuroendocrine tumors, are far less prevalent in dogs and cats than the tumors of biliary epithelial origin. , , these tumors are thought to develop from neuroendocrine cells in the epithelium of bile ducts or gallbladder or from anorexia and vomiting as more prominent symptoms. fever is uncommon, icterus inconsistent, and leukocytosis is often insignificant, even with marked bacterial infections of the biliary tract. gallstones are generally asymptomatic. they can be associated with cholecystitis or ehbo. bilious abdomen can be a clinically mild condition, or it can be associated with life-threatening signs. septic bilious abdomen causes extremely severe peritonitis with systemic inflammatory response syndrome (e.g., anorexia, vomiting, abdominal pain, poor perfusion, fever, and death). these patients may be in the initial hyperdynamic state (e.g., red mucus membranes, bounding pulse, fever, or hypothermia) or, if initially undiagnosed, can be in the late hypodynamic state (e.g., pale mucus membranes, weak pulse, and hypothermia). intraperitoneal bile seems to make septic peritonitis more severe. in contrast, some animals with sterile bilious abdomen (e.g., as a consequence of automobile trauma) are essentially normal except for ascites and icterus. plain radiography is occasionally diagnostic of biliary tract disease. some gallstones are radiopaque (figure - ). finding air in the gallbladder or in the wall of the gallbladder (i.e., emphysematous cholecystitis; figure - ) is diagnostic of infection with a gas-producing bacterium. "porcelain" gallbladder (i.e., a radiopaque gallbladder because of intramural mineralization of the gallbladder) is associated with carcinoma. ultrasound is generally accepted as the most important and sensitive method for diagnosing extrahepatic biliary tract diseases. many patients that are not suspicious for biliary tract disease are fortuitously diagnosed when ultrasound or radiographs are requested for various reasons. in difficult cases (e.g., partial ehbo versus complete ehbo), nuclear scintigraphy techniques can be used ; however, this seems to be rarely required. diagnosis of ehbo generally relies upon the use of ultrasound. dilation of the bile ducts (normal canine bile ducts are ≤ mm; normal feline bile ducts are ≤ to . mm) is primarily caused by ehbo, and is generally seen by days postobstruction. if one is unsure whether the ducts are dilated, repeating the examination in days should be helpful. enlargement of the gallbladder is not diagnostic of ehbo because anorexia and starvation of any cause may do the same thing. however, failing to find a dilated gallbladder parasites occasionally cause obstruction. platynosomum fastosum (i.e., p. concinnum) is a fluke that inhabits the gallbladder and/or bile ducts of cats that are infected by eating lizards or toads. natural infections are found primarily in florida, hawaii, and the caribbean. it may be asymptomatic or may cause obstruction or fibrosis. they are rare causes of cholecystitis. tumors may cause obstruction, and may be one of the more common causes in cats. , cholecystitis is most commonly caused by bacterial infection, ostensibly from bacterial migration up the bile duct. various grampositive, gram-negative, aerobic, and anaerobic bacteria (e.g., clostridium, staphylococcus spp., enterococcus spp., streptococcus spp., klebsiella spp., e. coli, helicobacter spp.) have been reported. [ ] [ ] [ ] [ ] infections caused by gas-producing bacteria can produce emphysematous cholecystitis. because of a shared biliary and pancreatic ductal system in the cat, hepatobiliary disease is a well-established risk factor for pancreatitis in the cat. aseptic inflammation of the gallbladder (necrotizing cholecystitis) has been reported, and infarction is one such cause. , exudation leakage of bile into the abdomen can be a result of mechanical forces (e.g., automobile trauma) that cause a shearing effect resulting in transaction of the common bile duct or one of the other bile ducts. mechanical rupture has also been reported following gunshot trauma. necrosis of the gallbladder raises the risk for rupture of the gallbladder and occasionally the bile ducts. canine and feline gallstones are typically composed of cholesterol, bilirubin, or may be mixed (as opposed to human gallstones which are usually caused by cholesterol). feline gallstones are typically calcium carbonate or mixed stones. biliary tract diseases can cause severe clinical signs (e.g., anorexia, depression, vomiting, icterus, abdominal pain) or they may be relatively asymptomatic. most symptomatic patients have serum biochemical abnormalities (e.g., increased serum alt, alp, and serum bilirubin). hypercholesterolemia is common in patients with ehbo. hyperbilirubinemia by itself does little besides cause icterus; therefore, patients with extremely high serum bilirubin concentrations can be relatively asymptomatic. renal failure has been attributed to excessively high serum bilirubin concentrations, but it is unclear that bilirubin is the cause or that this is common. shetland sheepdogs appear to have an increased risk for biliary tract disease, and cocker spaniels might also (see chapter ) . besides icterus, clinical signs in patients with ehbo are primarily a result of the cause of the obstruction, not the obstruction itself. canine pancreatitis in particular may cause severe clinical signs (e.g., anorexia, vomiting, abdominal pain-see "complications of liver disease" section). however, not all patients with pancreatitisinduced ehbo have severe pancreatitis. causes of ehbo that are insidious (e.g., mucoceles, tumors, and stones) are often unsuspected until the patient becomes icteric. biliary tract inflammation such as septic cholecystitis is welldocumented in dogs and cats. shetland sheepdogs appear to be at a greater risk of inflammatory biliary tract disease than most other breeds. clinical signs vary, but most animals are clinically ill with cholecystitis is often diagnosed by ultrasound-guided percutaneous aspiration of gallbladder bile for cytology and culture. this is a relatively sensitive and specific procedure for diagnosing biliary tract infection. , the finding of a dilated bile duct coincident with a normal-size gallbladder suggests cholecystitis, prior ehbo, or a rare congenital problem such as caroli disease. it is important to note that patients with bacterial cholecystitis may have no ultrasonographic or gross abnormalities. ultrasound may be suggestive, but is relatively insensitive for cholecystitis. , consequently, it is probably best to routinely aspirate bile for cytology and culture in patients with hepatobiliary disease. gallstones are relatively easy to diagnose, some can be found by radiographs, but almost all can be found by ultrasonography ( figure - ). with ehbo, the underlying cause is always the primary concern. ehbo in and of itself is not the primary consideration when deciding upon therapy. pancreatitis, for example, is primarily a medical disease (see chapter ) . surgery is rarely appropriate in the management of pancreatitis even when it is causing ehbo. if deemed necessary, ehbo caused by pancreatitis can be relieved by percutaneous aspiration or placement of a biliary tract stent. if absolutely necessary, a cholecystoduodenostomy may be performed, but this surgery should be avoided if possible. these procedures are seldom necessary because almost all patients with ehbo caused by pancreatitis will experience resolution of the obstruction with medical therapy. gallstones should be removed only if they are causing obstruction or cholecystitis. it is usually better to perform a cholecystectomy , as opposed to a cholecystotomy; the former has a lower morbidity and mortality rate in people and presumably in dogs and cats as well. biliary tract tumors can seldom be cured surgically. if a patient has ehbo caused by something that cannot be treated medically (e.g., tumor, pancreatic stricture, traumatically torn bile duct), then a biliary bypass procedure can be performed. cholecystoduodenostomy can relieve the obstruction, although the surgery requires special surgical skills. this surgery can predispose the patient to recurrent, ascending cholecystitis or other complications , and should only be performed in patients that in a patient with clearly dilated bile ducts suggests biliary tract inflammation or prior ehbo. any dog with ehbo should be suspected of having acute pancreatitis until proven otherwise. chapter details the diagnosis of pancreatitis. if acute pancreatitis is eliminated in a patient with ehbo, then one should look for gallstones and tumors, first by imaging and then by exploratory surgery or laparoscopy if imaging fails to provide a diagnosis. mucoceles are readily diagnosed by ultrasound, although there is some debate about what constitutes a mucocele. "classic" mucoceles are described as producing a "kiwi fruit" appearance without gravitydependent bile movement. "sludge" in the gallbladder is a common finding (and it has gravity-dependent movement), but is not clinically significant. the gallbladder of clinically normal dogs may appear abnormal on ultrasound examination (figure - ) , while patients with significant biliary tract disease may appear essentially normal. whether or not patients with ultrasonographic abnormalities of the gallbladder will become symptomatic cannot be reliably predicted. the gallbladder is filled with hyperechoic material (arrows) that was not gravity dependent. the dog was asymptomatic for biliary tract disease and was still in good health without therapy for the gallbladder months after this image was taken. . the stones were found fortuitously during an abdominal ultrasound; there was no evidence that they were causing any clinical signs. gb that has a risk of dehiscence. in general, one should either remove the gallbladder, aspirate gallbladder bile, gently express the gallbladder, or leave it undisturbed. if the leakage is associated with septic peritonitis, then that is a true emergency and requires immediate, aggressive medical and surgical therapy. gallstones may be monitored if the patient is asymptomatic. tumors can rarely be resected; they are generally inoperable when found. rare examples exist of patients with biliary tumors being cured surgically. biliary flukes can be treated with praziquantel ( to mg/kg sq for days). chapter discusses pancreatitis in greater detail. biliary mucoceles that have not ruptured often have a good prognosis; however, one report found a % mortality rate in patients without a rupture versus % mortality in patients with bile peritonitis. the report authors recommended a more preemptive approach (i.e., versus waiting until the patent is symptomatic). another group found no difference in mortality between rupture and nonruptured mucoceles (i.e., %). gallstones generally are innocuous, and even when they are causing signs have a good prognosis as long as rupture has not occurred. bacterial cholecystitis has a good prognosis as long as the gallbladder is intact and not at risk for rupture. septic bilious peritonitis has a very guarded to poor prognosis, depending upon the severity of the peritonitis. gallstones are usually asymptomatic, but can occasionally cause a problem. they are usually easily removed and resolved. biliary tumors are usually a poor prognostic finding , because of late diagnosis. flukes can be treated, but without early recognition, extensive tissue injury may or may not resolve after therapy. the liver serves many important functions including metabolism (carbohydrate, protein, lipid, nucleic acid, xenobiotics, porphyrins, vitamins, minerals, glutathione, endogenous hormones), coagulation factor synthesis, biliary secretion, and immune surveillance (see chapter ). it is not surprising, therefore, that animals with liver disease experience a broad range of complications reflecting perturbations in one or more of these functions. the hepatic portal venous system accounts for up to % of the total hepatic circulation and serves to transport nutrients from the gi tract to hepatic sinusoidal capillaries (and hepatocytes) before coalescing once again into central veins, hepatic veins, and caudal vena cava. in health, the multiple branching of the main portal vein, venules, and capillaries reduces the overall resistance to blood flow (circuits in parallel reduce resistance), and therefore the pressure required to perfuse these capillaries is maintained at a low level of less than to mm hg. , disease processes that obstruct flow through the intrahepatic branches of the portal vein or sinusoids elevate this pressure and result in significant portal hypertension. hepatocyte swelling impedes portal flow in acute pathophysiologic states, and fibrosis further impedes this flow in absolutely require it. generally speaking, ehbo caused by pancreatitis that is not resolving as quickly as desired is not necessarily a good indication for this procedure; patience and medical therapy generally resolve the problem. in fact, pancreatitis can be a complication of surgery in this region. many animals with hepatic disease are concurrently treated with antioxidants and other hepatoprotectants. although most of these drugs will not hurt patients with biliary tract disease (in fact, they may be beneficial if the disease is extending into the hepatic parenchyma, such as cholangitis/cholangiohepatitis), care must be taken before administering udca. udca is a choleretic agent that stimulates bile flow. this could be disadvantageous in a patient with complete ehbo. mucoceles usually need to be removed surgically. , , medical management may be attempted (e.g., fat-restricted diet plus udca), but there is a substantially increased mortality rate for patients that experience gallbladder rupture (figure - ) ; consequently, surgery is probably the safest course. most patients are not at risk of immediate rupture. as long as ultrasonography does not suggest impending rupture, one should make sure the patient is an optimal anesthetic risk. bacterial cholecystitis, uncomplicated by ehbo or stone, should generally first be treated with antibiotics, usually for to weeks. however, some patients with bacterial cholecystitis cannot be cured with antibiotic therapy, ostensibly because the infection has localized in the gallbladder mucosa. these patients consistently relapse after discontinuation of antibiotics, and therefore a cure may be achieved only with antibiotics plus cholecystectomy. when performing this surgery, great care should be taken to avoid causing a stricture or obstruction of the common bile duct. postcholecystectomy bile duct obstruction may prove fatal. it is also very important to be avoid traumatizing the pancreas to avoid severe pancreatitis. biliary tract leakage is treated based upon the underlying cause. if the gallbladder has ruptured, cholecystectomy is usually most appropriate. if the bile duct has ruptured, it is very difficult to successfully anastomose the two ends. in that instance, one generally must perform a cholecystoduodenostomy and ligate both ends of the torn bile duct. it is important to avoid biopsying the gallbladder as clinical signs associated with he in dogs and cats include depression, behavioral changes, circling/head-pressing, ataxia, apparent blindness, abnormal swallowing or salivation, stupor, seizures, and coma (table - ) . salivation is much more common in cats than dogs with he. brain dysfunction in he was historically considered to be a neurotransmitter dysfunction but current evidence suggests lowgrade cerebral edema caused by astrocyte swelling is the predominant pathologic change. there is consensus that ammonia is the key toxin in he but blood ammonia concentrations do not always correlate with severity of clinical signs. this is because a large number of other factors interact with the effects of ammonia to precipitate he. astrocytes play a central role because they express glutamine synthetase and detoxify the ammonia that reaches the cns. intraastrocyte accumulation of osmotically active glutamine in he results in astrocyte swelling and thus low-grade cerebral edema. this is largely reversible if the precipitating factors are treated, but edema can become severe and result in irreversible cns changes in severe and acute he. precipitating factors include inflammatory cytokines, benzodiazepine-type sedatives, and disturbances in amino acid metabolism and dopaminergic neurotransmission. [ ] [ ] [ ] the source of ammonia is primarily absorption from the gut, although other sources also exist as a result of interorgan metabolism. gut-derived ammonia was traditionally assumed to be a by-product of intestinal bacterial metabolism in the colon. this remains an important source in some conditions such as melena. however, recent studies in other species suggest that small intestinal enterocyte metabolism of glutamine as their main energy source is the most important source of postprandial ammonia absorption in the portal vein. , this is also likely the case in most dogs on normal diets as it is very unusual for undigested protein to reach the colon, although the source of gut-derived ammonia has never been investigated in dogs. in normal dogs, ammonia is transported to the liver via the main portal vein, and further metabolized chronic pathophysiologic states. sustained portal hypertension is associated with many, but not all, of the complications of liver disease. portal hypertension is an important, potentially life-threatening complication of liver disease in the dog. it is rare or poorly documented in the cat. portal hypertension develops most often in dogs with chronic liver disease and cirrhosis. it is occasionally recognized as a congenital lesion in young animals with arteriovenous fistulas, or as a hypoplastic disorder of the intrahepatic portal vein branches resulting in a condition referred to as noncirrhotic portal hypertension. , prehepatic portal hypertension can develop secondary to portal vein thrombosis or congenital hypoplasia of the extrahepatic portal vein, but these are less common. sustained portal hypertension may progress to splanchnic congestion, gi ulceration, ascites, and encephalopathy. portal venous hypertension produces vascular stasis and venous congestion and increases the risk of gi ulceration, particularly in conjunction with other risk factors such as anorexia and steroidal and nonsteroidal antiinflammatory drug usage. portal hypertensionrelated ulceration in the dog is typically duodenal although bleeding esophageal varices, similar to those reported in humans, are occasionally observed. glucocorticoids should be used only with great caution in dogs with portal hypertension. reduced systemic blood pressure is another consequence of portal hypertension and splanchnic venous congestion. changes in systemic blood pressure activate the renin-angiotensin-aldosterone system (as described in chapter ) and renal sodium retention, and thus increase total circulating fluid volume. the increase in circulating fluid volume (the "overfill" hypothesis) is believed to be the triggering event for the development of ascites in animals with portal hypertension. this is why aldosterone antagonists are the initial treatment of choice in ascites caused by portal hypertension (for more details see chapter ). ascites is a negative prognostic indicator in dogs with chronic hepatitis, although individual animals with chronic hepatitis and ascites can be managed and maintained for many months. with sustained portal hypertension, multiple acquired psss develop and serve as a conduit for portal blood flow directly into the systemic circulation. shunts serve to dissipate some of the increased portal pressure thus reducing the risk of adverse complications such as venous congestion, gi hemorrhage, and ulceration. they do, however, raise the risk of yet another complication of liver disease-hepatoencephalopathy. he is a syndrome of potentially reversible brain dysfunction resulting from impaired liver function. it results from either severe hepatocyte dysfunction or more commonly the presence of portosystemic collateral circulation, either congenital or acquired, where a variable combination of shunting of portal blood and hepatocyte dysfunction contributes to the clinical signs. it can be acute or chronic in presentation. acute he is most often a result of acute fulminating liver failure (see "consequences of hepatocyte and biliary tract injury" section) and carries a poor prognosis. more chronic he is usually a result of congenital or acquired psss. platelet counts, d-dimers, fibrinogen, or protein c) in ( %) of dogs affected with liver disease. coagulation abnormalities are also common in cats with liver disease and one study found abnormalities in ( %) of cats with liver disease. multiple mechanisms of coagulopathy are possible in liver disease patients. in alf cases such as xylitol toxicity in dogs, hl in cats, and cirrhosis in dogs, loss of normal hepatocyte function results in severe coagulation factor deficiency. vitamin k deficiency has also been implicated in coagulopathy particularly in cats in which cholestasis impedes bile salt secretion, emulsification, and micellarization of fat and fat-soluble vitamins and fat-soluble vitamin absorption. concurrent inflammatory bowel disease and pancreatitis exacerbate this condition in many cats with cholangitis. finally, platelet abnormalities (cytopenia and cytopathy) may contribute to coagulopathy in dogs with liver disease. , the liver has the unique ability to regulate its growth and mass. hepatocyte loss caused by viral, bacterial, or chemical injury, or partial hepatectomy triggers hepatocyte replication. , liver injury not only stimulates hepatocyte turnover but may also stimulate biliary proliferation and activation and proliferation of hscs. these changes usually occur together in an orchestrated wound-healing response. in the case of hepatocyte loss, normally quiescent hepatocytes replicate to restore the liver functional capacity and mass. these are the main cells that regenerate liver mass. however, in severe injury or where hepatocyte turnover is inhibited by senescence, a progenitor cell reserve may also replicate and regenerate liver mass. although hepatocytes are capable of replication, they have very slow turnover in a normal liver and there are negative consequences of long-term increased stimulation and turnover in chronic liver disease. it has been shown that cycling hepatocytes suffer irreversible erosion of telomeres, which leads to senescence. functional capacity is a relative rather than absolute parameter. the set point for growth regulation is the ratio between liver mass and body mass rather than liver mass per se. the optimization of the ratio indicates that the liver reaches a state in which it performs the amount of metabolic work needed to meet the functional requirements of the body. gene expression in the regenerating liver is a multistep process with at least two critical steps: the transition of quiescent hepatocytes into the cell cycle ("priming"), and the progression beyond the restriction point in the g phase of the cell cycle. hepatocytes must first be primed before they can fully respond to growth factors. as many as different genes participate in the early response to hepatectomy, but tnf and il- appear to be the major cytokines involved in the priming of hepatocytes. the proliferative effect of tnf on hepatocytes is further influenced by reactive oxygen species, nitric oxide, and glutathione content, and at least four transcription factors (nuclear factor kappa b, stat , ap- , and c/ebpβ) play major roles in the initiation of early liver regeneration. , progression through the cell cycle beyond the initiation phase requires growth factors, primarily hepatocyte growth factor and tgf-α. the subsequent expression of cell-cycle genes, particularly cyclin d , establishes the stage at which replication becomes growth factorindependent and autonomous. at this point, the hepatocyte is irreversibly committed to replicate and the cell-cycle replication machinery takes over. the regenerative capacity of the residual hepatocytes may restore liver mass and function after as much as % to % hepatectomy. , progenitor cells are only activated in severe liver injury. they appear to reside in a "niche" that is a particular regulatory environment. a recent immunohistochemical to urea by hepatocytes in the krebs-henseleit cycle. with portosystemic shunting or severe hepatocyte dysfunction, ammonia accumulates in the brain (and other tissues) where it is taken up by astrocytes and results in edema as described previously. dogs with he also show disturbances in cns aromatic amino acid metabolism. , the aromatic amino acids (tyrosine, tryptophan, and phenylalanine) accumulate in, the cns in portasystemic shunting. in the brain, β-phenylalanine and tyrosine are metabolized to phenylethanolamine and octopamine, both of which can act as false neurotransmitters. however, dietary supplementation with branched-chain amino acids (e.g., leucine, isoleucine, valine) does not convincingly improve he in either dogs or humans. , however, some dietary protein sources appear to be better than others in dogs with he. dogs on soya protein diets show a lower plasma ammonia concentration than those fed meat protein. dogs with he have also traditionally been fed a protein-restricted diet. however, protein restriction is no longer advocated in humans with he and it may be the digestibility and type of protein rather than a reduced amount that are most important in dogs. more studies are needed to investigate this. several other metabolic alterations exacerbate clinical signs associated with he, including acid-base disorders, electrolyte abnormalities, particularly hypokalemia, hypoglycemia, hypoxemia, and arginine deficiency (cats). an important trigger in humans and rodents is inflammation: recent studies confirm that inflammatory cytokines are synergistic with ammonia in precipitating he and that controlling inflammation in other organs is an important part of managing the patient with he. , there is anecdotal evidence that this is also true in dogs. the liver has significant structural and functional reserve capacity to support ongoing metabolic needs during mild to moderate forms of liver injury. moreover, the liver has the ability to regenerate liver volume and cell mass during the recovery phase of most forms of liver injury. signs of liver failure develop earlier with acute forms of liver injury than with chronic, progressive liver disease. zones , , and hepatocytes of the hepatic acinus have differing functions. zone (periportal) hepatocytes, for example, have a high capacity to cycle ammonia through the urea cycle thereby reducing the toxicity of ammonia. zone hepatocytes (nearer the hepatic vein) have a lesser capacity for ammonia and instead convert it to glutamine. , in health, zonation permits flexibility in hepatic function such that in metabolic acidosis, for example, the liver can rapidly divert ammonia toward glutamine production, which is necessary for h+ ion excretion in the kidney. in severe acute liver injury, this becomes an important "tradeoff" because acute selective destruction of periportal (zone ) hepatocytes more readily results in signs of encephalopathy because of the reduced ability of zone hepatocytes to detoxify ammonia. in chronic liver disease, if hepatocytes undergo piecemeal necrosis at different rates in different zones, the remaining hepatocytes can assume some of those functions, so that clinical signs of deficiency are not seen until later in chronic disease processes. coagulopathy is a complication of both acute and chronic liver disease in dogs and cats. a recent study reported one or more coagulation abnormalities (prolongation of coagulation times, changes in the cause of chronic hepatitis is usually unknown. there is increasing evidence that fibrosis and even some forms of cirrhosis in humans and rodent models are reversible if the underlying cause is removed. , the challenges are removing the cause and also defining the point at which cirrhosis moves from a reversible to irreversible state. increased fibrous septal thickness, smaller nodule size, and reduced cellularity together with increased collagen cross-bridging have all been associated with an irreversible cirrhotic state in rodents and humans. it is unknown whether liver fibrosis or cirrhosis in dogs is reversible clinically. cases of chronic hepatitis in dogs very rarely have sequential liver biopsies over a long period of time to assess progression of disease and noninvasive markers of fibrosis remain to be validated. serum hyaluronic acid is increased in dogs with cirrhosis as is tgf-β but the usefulness of these markers in following progression in clinical cases has not been assessed. identifying a reliable noninvasive marker of fibrosis for sequential studies in humans and dogs remains a challenge. study suggests that canine and human liver progenitor cells are functionally very similar. further characterization of the molecular events regulating hepatocyte replication and liver regeneration should improve outcome in animals affected with severe liver disease. the normal ecm of the liver provides cells with positional information and a mechanical scaffold for adhesion and migration. the ecm consists of collagens, glycoproteins, proteoglycans, glycosaminoglycans and molecules that are bound specifically by the ecm, such as certain growth factors, cytokines, matrix metalloproteinases, and processing enzymes such as tissue transglutaminase and procollagen propeptidases. a normal liver contains a very small amount of fibrous tissue as a percentage of its total mass. , acute or chronic liver injury causes a dynamic wound-healing response with both production and removal of fibrosis. hscs are the major source of the collagens that comprise fibrosis and cirrhosis, as well as of the tissue inhibitors of metalloproteinases (timps) that inhibit collagen degradation. it is the balance between collagen production by hscs and its degradation by matrix metalloproteinases that determines the severity and reversibility of the fibrotic response. following acute or chronic liver damage, hscs are stimulated to multiply and to undergo a complete phenotypic transformation from quiescent vitamin a-storing cells to contractile myofibroblasts which synthesize large amounts of ecm. , important stimuli for hsc transformation and multiplication in liver injury include oxidative stress; chemokines including platelet-derived growth factor; vegf and tgf-β; adipokines; and parts of the innate immune system including toll-like receptor ligands. the role of adipokines in stimulating fibrosis is increasingly being recognized in humans, where nonalcoholic fatty liver disease can lead to fibrosis and cirrhosis. they are produced by hscs themselves, as well as fat cells, and increased leptin and reduced adiponectin drive fibrosis. their importance in dogs is unknown and it is also unknown whether vacuolar or fatty liver diseases progress to fibrosis in dogs, but these are important questions to answer in the future given the widespread occurrence of obesity in dogs. the contractile function of activated hscs contributes significantly to the development of portal hypertension, and increases in angiogenic chemokines such as vegf and platelet-derived growth factor not only contribute to fibrogenesis by hscs, but also to the development of portal hypertension, so that the two pathologic processes are inextricably linked. activated hscs have greatly increased production of timps, particularly timp and timp , which prevent the action of matrix metalloproteinases in the ecm. the degree of fibrosis and reversibility then depend on the balance between perpetuation of hsc proliferation and secretion and resolution of hsc by either apoptosis or senescence of hscs or, indeed, their reversion to an inactive state. many factors contribute to hsc apoptosis, senescence, or reversion, including reduction in timps and nuclear factor kappa b and increased fas and p . however, in spite of all this understanding of the molecular mechanisms of fibrosis, a truly effective treatment for hepatic fibrosis in either humans or dogs has yet to be found. it is important to remember that a normal fibrotic response (scar) is important in walling off pathogens and tissue injury and inhibiting this response without removing the inciting cause (e.g., a viral cause) could lead to spread of the pathology. future treatment strategies for fibrosis should therefore incorporate treatment of the underlying cause of disease. this is clearly a problem in dogs where organizational principles of the liver bile formation and enterohepatic circulation functional and morphological relationships between the feline main pancreatic and bile duct sphincters the extrahepatic biliary tract in common domestic and laboratory animals hormonal regulation of bile secretion physiology of cholangiocytes hepatic stellate cell (vitamin a-storing cell) and its relative -past, present, and future hepatic stellate cells wnt antagonism inhibits hepatic stellate cell activation and hepatic fibrosis morphological characterisation of portal myofibroblasts and 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ultrasonography of portosystemic shunting in dogs doppler studies before, during and after surgery ultrasonographic assessment of hemodynamic changes in the portal vein during surgical attenuation of congenital extrahepatic portosystemic shunts in dogs use of transcolonic mtechnetium-pertechnetate as a screening test for portosystemic shunts in dogs quantification of portosystemic shunting in dogs by ultrasound-guided injection of m tc-macroaggregates into a splenic vein portal vein anomalies in the dog: their angiographic diagnosis use of intraoperative mesenteric portovenography in congenital portosystemic shunt surgery helical computed tomographic angiography of canine portosystemic shunts three-dimensional multiscale helical computed tomography techniques for canine extra-hepatic portosystemic shunt assessment use of magnetic resonance angiography for diagnosis of portosystemic shunts in dogs brain magnetic resonance imaging characteristics in dogs and cats with congenital 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shunts rectal ammonia tolerance test in the evaluation of portal circulation in dogs with liver disease postprandial venous ammonia concentration in the diagnosis of hepatobiliary disease in dogs long-term biochemical and physiologic effects of surgically placed portocaval shunts in dogs outcome of ameroid constrictor occlusion of single congenital extrahepatic portosystemic shunts in cats: cases evaluation of ameroid ring constrictors for the management of single extrahepatic portosystemic shunts in cats: cases clinicopathologic features of primary and metastatic neoplastic disease of the liver in dogs a retrospective study of feline neoplasms a survey of feline neoplasms hepatobiliary neoplasia in dogs and cats tumors of the liver and gallbladder small animal clinical oncology primary neoplasms in dog liver induced by diethylnitrosamine translation of new cancer treatments from pet dogs to humans the establishment and characterization of the first canine hepatocellular carcinoma cell line, which resembles human oncogenic expression patterns immunohistochemical characterization of canine hyperplastic hepatic lesions and hepatocellular and biliary neoplasms with monoclonal antibody hepatocyte paraffin and a monoclonal antibody to cytokeratin canine hepatic neuroendocrine carcinoma: an immunohistochemical and electron microscopic study canine biliary carcinoma: epidemiological comparisons with man comparative analysis of hepatocellular carcinoma in men and dogs inactivation of tgfbeta signaling in hepatocytes results in an increased proliferative response after partial hepatectomy mdm and p polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis b virus infection epidermal growth factor-induced hepatocellular carcinoma: gene expression profiles in precursor lesions, early stage and solitary tumours identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma withrow 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occlusion after placement of an ameroid constrictor in dogs evaluation of ameroid ring constrictors for treatment for single extrahepatic portosystemic shunts in dogs: cases evaluation of a portocaval venograft and ameroid ring for the occlusion of intrahepatic portocaval shunts in dogs surgical management of left divisional intrahepatic portosystemic shunts: outcome after partial ligation of, or ameroid ring constrictor placement on, the left hepatic vein in twenty-eight dogs laparoscopic portosystemic shunt attenuation in two dogs transvenous coil embolization of portosystemic shunt in dogs transjugular coil embolisation of an intrahepatic shunt in a cat complications associated with the diagnostic, medical, and surgical management of portosystemic shunts portal vein thrombosis as a complication of portosystemic shunt ligation in two dogs neurologic dysfunction in dogs following attenuation of congenital extrahepatic portosystemic shunts neurologic dysfunction in three dogs and one cat following attenuation of intrahepatic portosystemic shunts use of propofol to manage seizure activity after surgical treatment of portosystemic shunts portal hypertension part ii: clinical assessment and treatment diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats magnetic resonance imaging of focal splenic and hepatic lesions in the dog accuracy of ultrasound guided aspiration of the liver and cytologic findings in dogs and cats: cases a multistep approach in the cytologic evaluation of liver biopsy samples of dogs with hepatic diseases metronomic therapy with cyclophosphamide and piroxicam effectively delays tumor recurrence in dogs with incompletely resected soft tissue sarcomas sorafenib (bay - , nexavar), a dual-action inhibitor that targets raf/mek/erk pathway in tumor cells and tyrosine kinases vegfr/pdgfr in tumor vasculature sorafenib in advanced hepatocellular carcinoma: we have won a battle not the war phase i dose escalating study 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evaluation of biliary cystadenomas in cats canine hepatic carcinoids hepatic neoplasms tnm classification of tumours in domestic animals diagnostic efficacy of serum alkaline phosphatase and gamma-glutamyltransferase in dogs with histologically confirmed hepatobiliary disease: cases ( - ) paraneoplastic hypoglycemia due to an insulinlike growth factor type-ii secreting hepatocellular carcinoma in a dog detection of serum alpha-fetoprotein in dogs with hepatic tumors alpha-fetoprotein in serum and tumor tissues in dogs with hepatocellular carcinoma serum alpha-fetoprotein values in dogs with various hepatic diseases two-dimensional, grayscale ultrasonography for assessment of hepatic and splenic neoplasia in the dog and cat correlations between ultrasonographic findings and specific hepatic disease in cats: cases ( - ) acute pancreatitis in cats with hepatic lipidosis ammonia intoxication in the near adult cat as a result of a dietary deficiency of arginine nutrition of the domestic 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and decreases ketosis during fasting and induced hepatic lipidosis dietary taurine content changes liver lipids in cats safety, pharmacokinetics and use of the novel nk- receptor antagonist maropitant (cerenia) for the prevention of emesis and motion sickness in cats textbook of veterinary internal medicine: diseases of the dog and cat feline hyperthyroidism: pretreatment clinical and laboratory evaluation of cases liver changes in patients with hyperthyroidism serum enzyme disturbances in thyrotoxicosis and myxoedema liver tests in hyperthyroidism: effect of antithyroid therapy serum thyroid hormone concentrations fluctuate in cats with hyperthyroidism effect of nonthyroidal illness on serum thyroxine concentrations in cats surgical treatment of hepatobiliary cystadenomas in cats: five cases ( - ) biliary cystadenoma of cats a carcinoid tumor in the gallbladder of a dog hepatobiliary neuroendocrine carcinoma in cats: a clinicopathologic, immunohistochemical, and ultrastructural study of cases neuroendocrine carcinoma of the gallbladder in a dog ccnu for the treatment of dogs with histiocytic sarcoma primary extraskeletal hepatic osteosarcoma in a cat feline epitheliotropic intestinal malignant lymphoma: cases feline lymphoma in the post-feline leukemia virus era feline systemic mastocytosis: a review and results of splenectomy in seven cases clinicopathological features and outcome for dogs with mast cell tumors and bone marrow involvement hepatic neoplasia in the dog and cat lomustine for treatment of mast cell tumors in cats: cases ( - ) nodular hyperplasia in the liver of the dog: an association with changes in the ito cell population nodular hyperplasia of the liver in the beagle dog hepatic nodular hyperplasia wsava standards for clinical and histological diagnosis of canine and feline liver disease correlation of ultrasonographic and pathomorphologic findings in canine hepatic diseases toxic, metabolic, infectious, and neoplastic liver diseases metabolic and hormonal alterations in cats with hepatic lipidosis a retrospective study of cats with severe hepatic lipidosis: - feline hepatic lipidosis hepatic amyloidosis in two chinese shar pei dogs inheritance of renal amyloidosis in chinese shar-pei dogs a canine febrile disorder associated with elevated interleukin- familial renal amyloidosis in chinese shar-pei dogs amyloidosis associated with chronic hypervitaminosis a in cats lipid and lipoprotein analysis of cats with lipoprotein lipase deficiency the pathology of an inherited hyperlipoproteinemia of cats dietary diacylglycerol oil has no effect on hypertriglyceridemia in lipoprotein lipase-deficient cats relationship between resting metabolism and hepatic metabolism: effect of hypothyroidism and hours fasting hypothyroidism in dogs: cases epidemiological, clinical, haematological and biochemical characteristics of canine hypothyroidism measurement of serum total thyroxine, triiodothyronine, free thyroxine, and thyrotropin concentrations for diagnosis of hypothyroidism in dogs evaluation of serum free thyroxine and thyrotropin concentrations in the diagnosis of canine hypothyroidism serum total thyroxine, total triiodothyronine, free thyroxine, and thyrotropin concentrations in dogs with nonthyroidal illness treatment and therapeutic monitoring of canine hypothyroidism reexamination of dosage regimens for l-thyroxine (t ) in the dog: bioavailability and persistence of tsh suppression morphological classification of biliary disorders of the canine and feline liver histopathologic evaluation of feline inflammatory liver disease the cholangitis/cholangiohepatitis complex in the cat wsava classification and role of bacteria in feline inflammatory hepatobiliary disease culture-independent detection of bacteria in feline inflammatory liver disease feline cholangiohepatitis complex effect of illness not associated with the thyroid gland on serum total and free thyroxine concentrations in cats influence of systemic 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(abstract) plasma cortisol response to exogenous acth in dogs with hyperadrenocorticism caused by adrenocortical neoplasia evaluation of the hypothalamic pituitary-adrenal axis in clinically stressed dogs urinary cortisol in the assessment of pituitary-adrenal function: utility of -hour and spot determinations urine cortisol:creatinine ratio as a screening test for hyperadrenocorticism in the dog evaluation of a urine cortisol:creatinine ratio as a screening test for hyperadrenocorticism in dogs spontaneous hepatic rupture in six cats with systemic amyloidosis severe cholestatic liver disease secondary to liver fluke (platynosomum concinnum) infection in three cats destructive cholangiolitis in seven dogs suspected druginduced destructive cholangitis in a young dog primary cholangiohepatitis in a dog cholangiohepatitis in a dog gallbladder aspirate from a dog bacterial cholangitis/ cholangiohepatitis with or without concurrent cholecystitis in four dogs concurrent bacterial 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infarction: cases surgical treatment of cholelithiasis in cats: a study of nine cases gallbladder disease in shetland sheepdogs: cases porcelain gallbladder associated with primary biliary adenocarcinoma in a dog correlation between hepatobiliary scintigraphy and surgery or postmortem examination findings in dogs and cats with extrahepatic biliary obstruction, partial obstruction, or patency or the biliary system: cases prevalence of gallbladder sludge in dogs as assessed by ultrasonography percutaneous ultrasound-guided cholecystocentesis in healthy cats cross-species immunohistochemical investigation of the activation of the liver progenitor cell niche in different types of liver disease is liver fibrosis reversible now there are many (stages) where before there was one: in search of a pathophysiological classification of cirrhosis evolving challenges in hepatic fibrosis chronic hepatitis in dogs: a review of current understanding of the aetiology, progression, and treatment reversibility of liver fibrosis blood hyaluronic acid as a marker for canine cirrhosis transforming growth factor beta- signalling in canine hepatic diseases: new models for human fibrotic liver pathologies therapeutic targets in liver fibrosis structural-functional organization of hepatic glutamine and ammonium metabolism coagulation disorders in dogs with hepatic disease coagulation abnormalities in cats with naturally occurring liver disease acute hepatic failure and coagulopathy associated with xylitol ingestion in eight dogs proteins invoked by vitamin k absence and clotting times in clinically ill cats correlation between coagulation profile findings and bleeding complications after ultrasoundguided biopsies: cases ( - ) liver regeneration new concepts in liver regeneration defining therapeutic targets for liver fibrosis: exploiting the biology of inflammation and repair key: cord- - f ofdsc authors: alsaied, tarek; aboulhosn, jamil a.; cotts, timothy b.; daniels, curt j.; etheridge, susan p.; feltes, timothy f.; gurvitz, michelle z.; lewin, mark b.; oster, matthew e.; saidi, arwa title: coronavirus disease (covid‐ ) pandemic implications in pediatric and adult congenital heart disease date: - - journal: j am heart assoc doi: . /jaha. . sha: doc_id: cord_uid: f ofdsc the corona virus disease ‐ (covid‐ ) is a recently described infectious disease caused by the severe acute respiratory syndrome corona virus with significant cardiovascular implications. given the increased risk for severe covid‐ observed in adults with underlying cardiac involvement, there is concern that patients with pediatric and congenital heart disease (chd) may likewise be at increased risk for severe infection. the cardiac manifestations of covid‐ include myocarditis, arrhythmia and myocardial infarction. importantly, the pandemic has stretched health care systems and many care team members are at risk for contracting and possibly transmitting the disease which may further impact the care of patients with cardiovascular disease. in this review, we describe the effects of covid‐ in the pediatric and young adult population and review the cardiovascular involvement in covid‐ focusing on implications for patients with congenital heart disease in particular. corona viruses are important pathogens that can infect humans and animals. at the end of , a novel corona virus was isolated in wuhan, a city in china, as a cause of a cluster of severe cases of pneumonia and acute respiratory distress syndrome . the virus rapidly spread around china and the world. on march th , covid- was declared as a pandemic by the who and the virus has infected hundreds of thousands of patients and thousands of health care providers , . while children in general seem to have milder or even asymptomatic forms of the illness, there is little to no data in children with chronic medical conditions, specifically heart and lung disease , . the knowledge and experience in caring for patients with covid- is rapidly evolving and thus it is crucial for all health care providers to be aware of the potential impact of covid- on patients with congenital heart disease (chd). covid- is particularly important for cardiac team members for the following reasons: -covid- has higher morbidity and mortality in adult patients with acquired cardiac disease. -covid- may affect and impact the cardiovascular system. -covid- may cause symptoms that mimic symptoms of cardiovascular disease like cyanosis and shortness of breath commonly seen in forms of worsening cardiac disease. -cardiac care team members are at risk for acquiring covid- and may play a role in spreading the disease between patients and in the society at large. -patients with chd are known to have higher risk for complications with viral illnesses , . -covid- may stretch our health care system and our resources which may impact the care of patients with cardiovascular disease. based on the covid- pandemic trends it is expected that many of the cardiovascular care team members will be exposed to patients with covid- in the coming weeks and months. in anticipation of the spread of covid- many hospitals and cardiology practices have this article is protected by copyright. all rights reserved changed their care models and policies to accommodate the care of patients impacted by covid- and prevent the spread of the disease. the objective of this review is to explore the current knowledge about covid- potential effects on the cardiovascular system in the pediatric cardiology and adult chd population. in addition, we will review the prevention practices and the impact on the health care team wellness. due to the emergent and evolving nature of covid- we reviewed the published literature in medline through pubmed and critically assessed early reports using a search for covid- and sars-cov- in medrxiv and biorxiv from january st until march rd . we had personal communications with multiple organizations around the world caring for children and adults with chd and we reviewed social media reports from leaders in the field of congenital cardiology. in addition, the websites of the health organizations including who and cdc were reviewed to provide up to date numbers and infection control recommendations. a congenital cardiology email list serve was initiated on the th march to facilitate discussion and share experiences and ideas on topics such as: strategies to manage outpatient clinic, inpatient service, procedure prioritization, screening, telemedicine and dealing with staff illness and school closures and we share some thoughts and information from physicians on the frontline of care of patients with covid- . sars-cov belongs to the coronaviridae family. as a group this family has a single strand rna genome . the viral rna closely resembles the rna of two bat corona viruses ( % identical) . the hypothesis is that the bat is likely the primary source of the virus although the this article is protected by copyright. all rights reserved mechanism of transfer from bat to human is still unclear (i.e. whether it was through direct transmission or whether there was an intermediate host) , . the virus is also closely related ( % identical) to the sars corona virus which was responsible for an outbreak in china in [ ] [ ] and % identical to the middle east respiratory syndrome corona virus that caused an outbreak in the arabian peninsula in [ ] [ ] [ ] . importantly the virus enters the cells using the angiotensin converting enzyme (ace- ) , . sars-cov spike protein binds to the ace- and the virus enters the cells through endocytosis , . ace- are expressed on the surface of epithelial cells within the alveoli and small intestine which explain the respiratory and gastrointestinal symptoms associated with the disease , . ace is a membrane bound protein that is also expressed in vascular endothelium, renal tissue and cardiovascular tissue . ace converts angiotensin i to angiotensin while ace- deactivates angiotensin . angiotensin stimulates lung injury and ace- serves a role in lung protection as it deactivates angiotensin . viral binding to ace- deactivates the protective role of ace- and that may contribute to severe lung injury seen with covid- , . more than . million cases of covid- have been confirmed worldwide with more than , deaths attributed to covid- according to the who website as of / / , . of these cases , cases were reported from china where the disease peaked between mid-january and early february , , . the initial patients had worked or visited a seafood "wet" market in wuhan that sold live animals and was closed subsequently for infection control , . human to human spread subsequently became the main source of transmission , . the disease then spread around the world initially through patients travelling from china followed by local transmission . as of april rd , there were , confirmed cases in the united states and over , deaths due to covid- according to the centers for disease this article is protected by copyright. all rights reserved control and prevention (cdc) website . transmission is thought to occur mainly via respiratory droplets produced during talking, coughing or sneezing similar to other corona viruses and similar to the influenza virus . transmission from asymptomatic individuals or patients in the incubation period has been documented , . the virus can also stay viable in aerosols up to . hours and can stay for hours to days on surfaces, especially plastic and stainless steel . in adult patients the incubation period is up to two weeks with a median of - days [ ] [ ] [ ] . more than % of infected individuals will remain asymptomatic or develop mild pneumonia . about % of patients will develop severe pneumonia and another % of patients will develop critical disease with respiratory and multi-organ failure and shock . fever is the most common symptom reported in most adult patients followed by fatigue and dry cough , . other symptoms include myalgia, anorexia, dyspnea, loss of sense of taste and smell (anosmia) and less commonly rhinorrhea, headache and sore throat [ ] [ ] [ ] . gastrointestinal symptoms including diarrhea and nausea have also been reported in up to % of patients , . the median time to hospital admission is days from the start of symptoms . according to the who, recovery happens in two weeks in mild cases and in up to weeks in severe cases , . the median time from symptoms to death in severe cases is estimated to be between - days . estimating case fatality rate is challenging and is evolving depending on the testing policies in each country and the age and risk profile of the infected patients. the rates reported range from . % up to . % , - . this article is protected by copyright. all rights reserved while the severity and mortality due to covid- is higher in older patients, the virus can also infect children and young adults , . overall only % of the reported cases from china were in individuals younger than years and less than % were younger than years , . fortunately, the disease in children is reported to be milder than adults although severe cases with multi-organ involvement have been reported , . a nationwide case series from china included pediatric patients with covid- . of those, had positive testing while the rest had highly suspicious clinical picture . severe disease with hypoxia was reported in % of the cases while a total of patients had critical disease with acute respiratory distress syndrome or multi-organ failure and one death was reported in a year-old . compared to older children, infants had a high risk for severe disease . another study looked at all the cases tested and treated at the wuhan children's hospital which is the only hospital to test and treat children in wuhan for covid- . of patients tested, ( . %) were confirmed to have sars-cov infection at a median age of . years. of these % were asymptomatic and only % were febrile at some point during the illness. during the course of the admission only of patients required intensive care unit and mechanical ventilation and all had preexisting conditions (intussusception, leukemia on maintenance chemotherapy and hydronephrosis). one month old patient died but the patient also had intussusception with multi-organ failure . the data on cardiovascular involvement with covid- is evolving . covid- may result in cardiac injury through multiple potential mechanisms, including: . cardiomyocyte injury due to an acute and severe inflammatory response as seen during a cytokine storm this article is protected by copyright. all rights reserved a recent meta-analysis evaluated the prevalence and effect of preexisting cardiovascular disease in patients with covid- . the study evaluated patients and reported the prevalence of hypertension, cardiac and cerebrovascular disease, and diabetes to be %, % and % respectively . studies from china and italy consistently show that preexisting cardiovascular disease increase the risk of mortality in patients with covid- , . the mechanism for increased risk of mortality is likely multifactorial including older age in patients with cardiac morbidities and altered immunologic response due to age and diabetes , . a population with potentially increased risk is patients with heart transplantation due to immunosuppression and two case reports describe covid- in heart transplant patients [ ] [ ] [ ] . one patient had mild disease while the other had severe disease. both patients were successfully treated with steroids, intravenous immunoglobulins and antibiotics and both survived without rejection , . multiple cardiovascular sequelae are reported in patients with covid- which may lead to or exacerbate cardiac disease . myocardial injury evidenced by elevated troponin is common in covid- especially in patients with severe disease , . arrhythmia is also commonly seen in covid- patients and while nonspecific, palpitations are one of the presenting symptoms in % of the patients , , . arrhythmia was noted in % of a group of hospitalized adult patients in china and was more common in the icu setting . there remains uncertainty about the specifics of the type of arrhythmia and there are no data in children. arrhythmia could be related to stress, inflammation and metabolic abnormalities but the development of new malignant arrhythmias should raise the suspicion for an underlying myocarditis , . myocarditis is a significant contributor to mortality in up to one third of cases with severe covid- and was the primary cause of death in % of the patients . autopsy found evidence this article is protected by copyright. all rights reserved of fulminant myocarditis with inflammatory mononuclear infiltrates in the myocardial tissue in some cases although the prevalence and risk factors for myocarditis are yet to be described . heart failure was reported in up to % of patients with covid- with a high prevalence of mortality . whether this is due to unmasking of pre-existing left ventricular dysfunction or new onset of cardiomyopathy due to myocarditis remains unclear . finally some severe cases may progress to cardiogenic shock and extracorporeal membranous oxygenation support may be considered in these cases although the prognosis is poor with % mortality in one report , . population chd affects up to % of the general population and remains the leading cause of infant mortality due to congenital malformations this article is protected by copyright. all rights reserved summarized in table . in addition to the cardiac morbidity, many patients with chd also have other organ involvement including chronic lung disease, cirrhosis and renal disease which may also increase the risk of covid- . we want to emphasize that there is very limited evidence to make firm recommendations and that these are based on the experience with previous viral diseases , . of note cardiovascular morbidities like diabetes and hypertension may also increase the risk of covid- in pediatric patients similar to that seen in adult patients. additionally, diabetes, hypertension, and acquired cardiovascular disease may coexist in adults with chd, potentially further increasing their risks. it is also important to note that many symptoms of covid- may mimic symptoms of worsening cardiac conditions in the chd population. symptoms such as shortness of breath, palpitations and fever are commonly seen in endocarditis which is a major concern in patients with chd . the same symptoms can also be seen in heart failure decompensation due to a viral illnesses and these considerations should be kept in mind during the covid- pandemic. aspirin is commonly used in the pediatric and adult population with chd for its antiplatelet effects at a low dose of - mg/kg/day . there is an association between the use of aspirin and the development of reye syndrome, a rare form of hepatic encephalopathy, in young children taking high dose aspirin for kawasaki disease who have concurrent infection with influenza or varicella , . there are no reports of reye syndrome in patients on low dose aspirin typically used with chd or with covid- . due to that we currently do not stop aspirin in children or adults with chd and covid- . this article is protected by copyright. all rights reserved there is no convincing evidence that nsaids are harmful in covid- although some clinicians suggested a possible negative impact if used early in the course of the disease for fever . the who and the european medicines agency currently approve the use of ibuprofen and nsaids in covid- , . the british congenital cardiac association suggested avoiding nsaids to treat fever in covid- . the ace- is used by the virus for cell entry . at this point there is insufficient data to suggest that ace-i/arb could affect the course of covid- , . ace-i/arbs suppress angiotensin ii or blocks its effect on its receptor. this is a positive effect to blunt the pulmonary inflammatory response and may result in upregulation of ace- which can be a positive effect for the same reason but at the same time carries the potential risk of increasing sars-cov- entrance to the alveolar cells . in the chinese data the rate of hypertension was significantly higher in patients who died post covid- . whether this is because of the older age and multiple comorbidities in the patients with hypertension or if it has any relationship to the ace-i/arbs use is unclear , . it is our practice currently not to stop ace-i/arb in patients who have covid- as stopping these medications could have harmful effects. the acc, american heart association and the heart failure society of america issued a statement recommending not adding or removing these medications for patients who are taking these medications if diagnosed with covid- and to follow an individualized approach . this article is protected by copyright. all rights reserved coagulation cascade abnormalities and disseminated intravascular coagulation (dic) are reported in patients hospitalized with severe covid- disease , , . we suggest continuing anticoagulation in mild cases with monitoring from the prescribing physician. adjusting anticoagulation can be done as needed in cases of severe covid- . there is no data to suggest any interaction of covid- with these medications and we suggest continuing these medications and adjusting as needed by the prescribing physician. the interaction between covid- treatments and the cardiovascular system are previously reviewed in jacc . several medications are under active investigation to treat covid- . antiviral medications being trialed include ritonavir/lopinavir which can cause qt prolongation . ribavirin, another antiviral being investigated, and ritonavir/lopinavir can also interact with anticoagulants , . remdesivir, an investigational antiviral medication used to treat ebola virus, is being trialed and although cardiovascular toxicities have not been reported, among patients one developed hypotension and cardiac arrest after the loading dose , . chloroquine and hydroxychloroquine, antimalarial agents, have been shown in vitro to have inhibitory activity on sars-cov due to increasing cellular endosomal ph and thus preventing the viral endocytosis into the cells , [ ] [ ] [ ] . this medication has been trialed in vitro and has been shown to be effective. however, there is limited human data to support effectiveness in humans , . one small study treated patients who were asymptomatic or had mild covid- with hydroxychloroquine in combination with azithromycin and showed a significant decrease in viral load in treated patients . the results should be taken with this article is protected by copyright. all rights reserved caution as the medication was previously tried in cases of influenza, hiv and zika virus and showed effectiveness in vitro without any positive effect in humans . of note these agents have qt prolonging effects which should be taken into consideration especially when combined with hypokalemia, hypomagnesemia, myocarditis or other qt prolonging medications. this includes azithromycin, another commonly used antibiotic in respiratory infections with qt prolonging effects that is being trialed in covid- infections , . this would be of particular concern in patients in an intensive care unit setting where a variety of qt affecting medications and physiologic conditions might put patients at risk of myocardial hypersensitivity and arrhythmia risk. a list of these medications can be found at https://www.crediblemeds.org/. screening all patients entering a health care facility for covid- symptoms including fever and respiratory symptoms, travel and exposure to covid- cases is essential for early identification of potential covid- patients and to allow care team members to wear appropriate personal protective equipment (ppe) (figure ) . screening may be through the phone before the patient arrives and should employ the use of interpretation services when needed. non-urgent cardiac visits should be rescheduled for a later date, particularly if the patient has viral symptoms without concern for possible worsening cardiovascular disease. urgent cardiac visits with symptomatic patients (or if asymptomatic but with a high-risk exposure) should include the use of a facemask by the patient and ideally a separate waiting rooms . any caregiver entering the room for a confirmed or suspected covid- case should wear a gown, gloves, eye protection and a respirator (e.g. n ). when the supply of respirators is limited a facemask is an acceptable alternative for routine interaction but a respirator this article is protected by copyright. all rights reserved (preferably a powered or controlled air purifying respirator) should be worn for any aerosol generating procedures . these procedures include intubation, cardiopulmonary resuscitation, and manual ventilation before intubation , . transesophageal echocardiography (tee) has been included on this list; but the data is currently unavailable as to whether the risk of aerosolization during tee is similar in both the setting of the paralyzed and intubated patient as opposed to the sedated patient without cuffed intubation who might be expected to gag or cough during the procedure. in light of this data gap, a conservative approach should be employed. during this pandemic, patient to provider, provider to patient and provider to provider transmission represent major challenges. it is important to know that . % of the cases reported from china were of healthcare team members suggesting that health care providers are at a significantly increased risk of contracting covid- , . this emphasizes the need for selfprotection by all providers. this is especially a challenge in cases where ppe are limited and in emergent interventions such as cardiopulmonary resuscitation; providers should prioritize following ppe guidelines and should limit exposure. similarly, elective and nonessential visits and procedures should be postponed. in the chd settings, these include non-urgent cardiac surgeries, cardiac catheterization, tee and electrophysiological procedures. office visits can be postponed or scheduled through phone calls or telemedicine. telemedicine technologies limit the patient and provider exposure while still allowing for patient care . teams must go through the exercise of considering unintended consequence to delayed access to care for the patient and put into place safeguards in their strategy. the duration of the corona virus pandemic is uncertain and therefore requires frequent reassessment of care plans. specific definitions of urgent versus non-urgent should be created so that schedulers and care providers this article is protected by copyright. all rights reserved are in alignment. the definition of non-urgent should include the concept that "delay of a certain duration unlikely to result in patient harm". other strategies may include limiting the number of team members with exposure to suspected cases by allowing phone and chart review consults in patients admitted to the hospital. most congenital cardiac centers hold weekly multidisciplinary conferences to discuss the cases and all team members including cardiologist, surgeons and anesthesiologists attend these conferences. due to the requirement of social distancing to limit viral transmission management, and teaching conferences should be limited and virtual meetings should replace in person meetings (figure ) . in addition, many hospitals are implementing staff screening strategies for symptoms and taking temperature for all employees and is assigning those staff non-essential for patient care to work remotely during these times. as age is a risk factor for severe disease, decisions must be made as to whether to restrict older physicians and care team members to work in settings with lower potential for exposure. in addition to the physical risks this is a stressful time for the care team members and it is important to keep provider wellness in mind. resources can be found on the american college of cardiology website and other professional societal websites . during this time of "physical" distancing it is important to maintain "social connectivity" by supporting each other through our professional societies, our teams and our personal support networks. this has become increasingly via available video communication enterprises , . new information is emerging every day on covid- . it is important to continue to follow the guidelines from the who, the cdc, your local health department, hospital and practice. it is also crucial to make this information easily available and accessible in real time and utilize data visualization tools to allow smooth transformation of the data to the public. in addition, social distancing has limited trainee education. online education is now accessible and this article is protected by copyright. all rights reserved resources from the acc virtual national meeting will be available free of charge. efforts to make other recorded material available for all providers are ongoing. finally technology allows virtual meetings with video communications and conferences to allow for fellows teaching and continue to make multidisciplinary discussions accessible to all care team members. while data are emerging on covid- effect on the cardiovascular system, there are several areas where further research is needed. identifying high risk patients among children and adults with chd is a priority. furthermore, studies to understand the mechanisms of cardiovascular involvement in covid- will facilitate the efforts to discover treatment options. in addition, several trials are ongoing searching for identification of the significance of the infection in patients with chd, treatments and post exposure prophylaxis. an example is a collaboration effort between the adult congenital heart association and the international society for adults with congenital heart disease in order to better understand the impact and burden of covid- on adults with chd in the united states and globally. during these trying times of covid- pandemic, cardiovascular team members will play a central role in controlling the disease. patients with chd are likely to become increasingly affected with covid- and may have more grave outcomes than other populations during the pandemic due to their perturbed physiologic state and co-morbidities. infection control processes are crucial. continuing research and education about the disease may provide us with insights to alleviate morbidity and mortality in the near future. includes simple native lesions (isolated small asd or vsd, mild isolated pulmonary stenosis) or repaired lesions (ligated or occluded pda), repaired asd or vsd without residual shunting or chamber enlargement. repaired or unrepaired lesions including avsd, aortic or mitral valve disease and coarctation of the aorta, anomalous pulmonary venous return, alcapa, aaoca, ebstein, tof, ps (moderate or greater), vsd with moderate or greater shunt. cyanotic congenital heart defect (unrepaired or palliated, all forms), double-outlet ventricle, fontan procedure, iaa, single chd: congenital heart disease. this article is protected by copyright. all rights reserved theoretical concern for reye syndrome although the only reports were at high doses used in kawasaki disease in the setting of influenza or varicella. no reports with covid- . suggest continuing aspirin. theoretical concern due to potential upregulation of ace- . no data to support an interaction with the disease. suggest continuing the medication. covid- may cause dic and coagulation cascade abnormalities in severe cases. suggest continuing the medication in mild cases and adjust as needed in severe cases. some clinician suggested worsening course of the disease if given early. there is no data to show any association of nsaids with the course of the disease. the who does not recommend avoiding nsaids. there is no information to suggest any harmful effect in covid- patients thus it is reasonable to continue these medications. this article is protected by copyright. all rights reserved clinical features of patients infected with novel coronavirus in wuhan, china world health organization declares global emergency: a review of the novel coronavirus (covid- ) who director-general's opening remarks at the media briefing on covid- - covid- (coronavirus) american academy of pediatrics bronchiolitis guidelines c. updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection frequency and consequences of influenza vaccination in adults with congenital heart disease a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic 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cardiovascular medicine this article is protected by copyright. all rights reserved sources of funding: none. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord- -gmcugd h authors: song, jian-xin; zhu, lin; zhu, chuan-long; hu, jin-hua; sun, zi-jian; xu, xiang; xin, min-you; zhang, qiong-fang; zhang, da-zhi; shang, jia; huang, jia-quan; xu, dong title: main complications of aechb and severe hepatitis b (liver failure) date: - - journal: acute exacerbation of chronic hepatitis b doi: . / - - - - _ sha: doc_id: cord_uid: gmcugd h this chapter describes the clinical features, and diagnosis of complications in aechb including secondary bacterial infections, coagulation disorder, water electrolyte disorder, hepatorenal syndrome, hepatic encephalopathy, hepatopulmonary syndrome and endotoxemia: . patients with severe hepatitis have impaired immunity and are therefore vulnerable to all kinds of infections. after infection, these patients may experience shock, dic and multiple organ failure, all of which seriously affect their prognosis and are major causes of death. concurrent infections consist primarily of infections of the lungs, intestines, biliary tract, and urinary tract, as well as spontaneous bacterial peritonitis and sepsis. . severe hepatitis may reduce the synthesis of coagulation factors and enhance their dysfunction and increase anticoagulants and platelet abnormalities, leading to coagulopathy. infection, hepatorenal syndrome and complications can further aggravate coagulopathy, resulting in dic and seriously affecting patient prognosis. . hepatorenal syndrome, which is characterized by renal failure, hemodynamic changes in arterial circulation and abnormalities in the endogenous vascular system, is a common clinical complication of end-stage liver disease, and one of the important indicators for the prognosis of patients with severe hepatitis. . water electrolyte disorder (water retention, hyponatremia, hypokalemia, hyperkalaemia) and acid-base imbalance are common in patients with severe hepatitis. these internal environment disorders can lead to exacerbation and complication of the illness. . hepatic encephalopathy is a neurological and psychiatric anomaly syndrome based on metabolic disorder, and an important prognostic indicator for patients with severe hepatitis. . the hepatopulmonary syndrome is an important vascular complication in lungs due to systemic hypoxemia in patients with cirrhosis and portal hypertension. the majority of patients with hps are asymptomatic. long-term oxygen therapy remains the most frequently recommended therapy for symptoms in patients with severe hypoxemia. . endotoxemia, an important complication of severe hepatitis, is not only a second hit to the liver, but also leads to other complications including sirs and mods. (nacseld) for survival analysis of prospectively enrolled cirrhosis patients hospitalized with infections, bajaj found that there is . % of nosocomial infection in all patients [ ] . domestic data showed significant difference of infection incidence in different times. it is related to raising diagnosis and treatment level. according to the data from shanghai public health clinical center, in patients who were treated as severe hepatitis from january to december , there were patients obtained nosocomial infection with a rate of . % [ ] . the incidences of infection complicated with acute liver failure, acute on chronic liver failure or chronic liver failure are similar. patients with old age or long hospitalization are more easily to get infection. there was rare report about infection in children patients with severe hepatitis. in , godbole et al. from uk reported that % in children patients with severe hepatitis were complicated with infection, and mostly are sepsis, respiratory tract infection and urinary infection. most of the infections occurred within weeks of admission, while patients with infection had longer hospitalization [ ] . reduced innate immunity the innate immune system is the first line of defense against premier environmental challenges and injury. in liver, it is a complex system that includes nk cells, nkt cells, kcs, neutrophils, eosinophils and complement components. the innate immune response acts much more rapidly compared with adaptive immunity. monocytes and macrophages: the liver plays an important role in defense and immune function. the main cellular components of the innate immune system within the liver are the kupffer cells. kupffer cells represent - % of the tissue macrophages in the human body [ ] . in normal condition, kupffer cells in liver help to clear the macromolecular substance such as pathogen, endotoxin, heteroantigen and immune complex to defense infection. in severe hepatitis, due to massive hepatocytes necrosis, the number and function of monocytes/macrophages are impaired, thus the activity of fibronectin, which is opsonin of macrophages, is decreased. therefore, bacterium, endotoxin and other poison from gut directly access into circulation. subrat kumar acharya from india reported that the plasma fibronectin (fn) level in severe hepatitis patients was significantly lower than that in healthy controls ( . μg/ml ± . μg/ml, vs. . μg/ ml ± . μg/ml). the fn level was remarkably correlated with incidence of infection and mortality [ ] . complement: liver is the organ where complements are mainly produced, such as c , c , c , c . the complements help to expand phagocytosis of phagocytes by chemotaxis, opsonization or adhesion, as well as help antibody to kill or solute some gram negative bacilli [ ] . report from wyke showed that the defect of complement closely correlated with impaired opsonization [ ] . defect of c or c can result to weakened movement of polymorphonuclear leucocyte. in severe hepatitis, the ability of liver to produce complement has been weakened due to massive injury of parenchymal hepatic cells, which leads to decreased activity of complement to % of normal condition. meanwhile, serum opsonization to e. coli or saccharomycetes are also diminished. besides, high ammonia level in severe hepatitis also restrains complement activity to impact germicidal effect. the most direct and also the most important result for decreased complement production and reduced opsonization is the susceptibility to infection. it was also reported that complement and the alternative pathway play an crucial role in lps/d-galn-induced fulminant hepatic failure [ ] . neutrophils: a majority of patients with severe liver disease have altered function of neutrophil granulocytes [ ] . the most common manifestation include abnormal ultrastructure or function of neutrophil granulocytes, as cytoplasm degeneration, organelle reduction, mitochondrial swelling, pyknotic nuclei, etc. decrease filtration and phagocytosis of reticuloendothelial system, as well as impaired chemotaxis of blood cells, making immunity weakened, lead to invasion of bacterium. therefore, patients with severe hepatitis are vulnerable to be infected with bacterium or fungi due to decreased phagocytosis and germicidal effect of neutrophil granulocytes, and impaired adhesion of macrophages and white cells [ ] . data from liu h demonstrated pretreatment neutrophil-lymphocyte ratio was associated with the prognosis of patients with hbv-aclf, and elevated nlr predicted poor outcome within weeks [ ] . natural killer and natural killer t cells: natural killer (nk) and natural killer t cells (nkt) are important components of the innate immune response. natural killer cells have potent cytolytic activities that are exerted through the death receptor and perforin/granzyme pathways. activated nkt cells have both perforindependent and fas-ligand dependent cytotoxic function that are triggered upon tcr recognition of an antigen [ ] . nk cells and nkt cells play an important role in many experimental models of liver injury, such as viral hepatitis, alcoholic liver disease, and autoimmune liver disease [ ] . however, their role in aclf has not yet been clearly elucidated, it was reported the median percentage of nk cells in the lymphocytes of patients with acute and fulminant liver failure were significantly lower compared to healthy controls. meanwhile, patients with acute and fulminant liver failure had significantly high and comparable nkt cells compared to control group [ ] . the important pathophysiological role of innate immune dysfunction in patients with acute-on-chronic liver failure (aclf) has been investigated in recent years. however, dysregulation of adaptive immunity remains poorly elucidated [ ] . patients with severe hepatitis has varying degrees of impaired cellular immunity, manifested as decreased cd + cell number and declined cd +/cd + ratio, which is pathogenesis of opportunistic infection. it was reported that there exists a reduction in cd (+) t lymphocytes in hbv-aclf patients. these cd (+) t cells predominantly are cd (+) tconv, and the development of suppressive cd (+) tregs greatly surpass tconv, which constitutes important characteristics of adaptive immune dysfunction of hbv-aclf [ ] . a report from china showed total amount of lymphocytes, cd (+) t cells, cd (+) t cells and nk cells in circulation were lower in the hbv-aclf patients compared to the chb patients [ ] . hbv-specific cd (+)t-cell responses are considered to be of great importance in viral control and immune-mediated liver damage [ ] . however, cd (+) t cell has seldom been studied in aclf. ye [ ] reported decreased activated cd (+) t cells may be related to poor outcomes in patients with sh. the frequency of circulating th cells increased with disease progression from chb to aclf patients compared to healthy control. th cells were also found largely accumulated in the livers of chb patients. the increases in circulating and intrahepatic th cells were positively correlated with plasma viral load, serum alanine aminotransferase levels and histological activity index. in addition, the serum concentration of th -associated cytokines was also augmented in both chb and aclf patients [ ] . in process of diagnosis and treatment for severe hepatitis, repeatedly abdominal paracentesis, retention catheterization, venous cannula, hemofiltration, or trachea cannula are usually necessary. unthoroughly sterilization or nonstandard sterile operation will lead to pathogen invading to develop an infection. in addition, artificial liver treatment is also an important cause of infection. it was reported that the incidence of fungal infection in severe hepatitis correlated with the number of artificial liver treatment. application of broad spectrum antibiotics is also a major cause of infection in severe hepatitis. antibiotics inhibit or kill sensitive normal bacterium as well as pathogens, especially normal bacterium colonized in natural orifice, leading to flora disproportionality. this time, nonpathogen could cause infection, or mass produced pathogen become dominant colony to develop infection. it was proved that dosage, exposure time, or varieties of antibiotics used in patients was closely correlated with severity of dysbacteriosis and incidence of sbp [ ] . nosocomial origin of infection, longterm of norfloxacin prophylaxis, history of recent infection by multiresistant bacteria and recent use of β-lactams were independent inducements associated with the development of multiresistant infections. bacterial translocation is defined as the migration of viable microorganisms or bacterial products (i.e., bacterial lps, peptidoglycan, and lipopeptides) from theintestinal lumen to the mesenteric lymph nodes and other extraintestinal sites [ ] . there are multiple mechanisms which are involved in defective gut functions and altered microbiota in patients with cirrhosis or liver failure. these include small intestine bacterial overgrowth (sibo), increased intestinal permeability, and impaired antimicrobial defense. additionally, decreased bile acids, due to decreased synthesis and defective enterohepatic circulation, contribute to altered gut microbiota [ ] . small intestinal bacterial overgrowth (sibo) was defined as ≥ total colonyforming units per milliliter of proximal jejunal aspirations, which presents in % of cirrhotic patients and is associated with systemic endotoxemia [ ] . in the condition of hepatic failure, due to impaired immunity, bacterium overgrowth and translocation happened, which increased incidence of infection [ ] . in vivo study from wang showed phagocytosis of monocytes were prominently and immediately diminished after resection of liver tissue up to %, accompanied by decreased oxygen ingestion [ ] . mass propagation of e. coli in the lower part of the small intestine and bacterium translocation were all happened h after resection. in rats with ascites in severe liver disease induced by carbon tetrachloride, the bacteria easily transferred from the intestine to extra-intestine, including local lymph nodes and blood circulation. these evidences proved that liver failure prompted the proliferation of intestinal bacterial overgrowth and translocation, which may be the main reason for the endogenous infections in patients with liver failure. another reason for bacterial translocation is that patients with cirrhosis or liver failure display increased intestinal permeability. in patients with severe hepatitis, gastrointestinal mucosa membrane has impaired ability for regeneration and repair, leading to dysfunction of intestinal barrier and declined anti-infection ability. patients with severe hepatitis have weakened regeneration and repair capacity of gastrointestinal mucosa, as well as decreased gastrointestinal barrier function and resistance to infection. the intestinal mucosa often manifested as congestion, edema or erosion. especially when intestine get infection, bacteria can invade to abdominal cavity directly or through slight mucosal defect [ ] . besides, ascites often present in severe hepatitis patients, which provide a good medium for bacteria to multiply. patients with severe hepatitis often have ascites combined with portal hypertension. due to lobular structural damage, abnormal construct of liver sinusoids or blood vessels, bacteria directly access into the systemic circulation without filtration and phagocytosis by liver, leading to formation of bacteremia. meanwhile, bacteria in blood circulation may access into the abdominal cavity and cause abdominal infections. hence, patients with severe hepatitis are not only prone to get endogenous infection but also prone to have intestinal endotoxemia. patients with severe hepatitis have decreased bile secretion, changes of bile composition, so that infection are prone to occur in epithelium of bile duct and gallbladder. in patients with severe hepatitis, intestinal edema and cellulitis are obvious, some may develop acute serositis. for patients with portal hypertensive gastrointestinal disease, regeneration and repair capacity of gastrointestinal mucosa are decreased, accordingly, the natural immune barrier function is reduced. once esophageal and gastric venous bleeding occur, gastrointestinal ischemia aggravate, which result in decreased resistance to infection. invasive procedures, complications, prophylactic use of broad-spectrum antibiotics, underlying diseases, long hospital stay, and old age are risk factors for infection in patients with severe hepatitis. elderly patients with other underlying diseases, decreased immune function, more severe primary disease, have a high risk of infection and may predispose to severe infections. in addition, albumin level is closely related to ascites production and sbp occurrence. irrational uses of immunosuppressive agents such as corticosteroids suppress immune function, lead to flora, and promote the formation of drug-resistant strains and pathogenic bacteria. mortality of severe hepatitis is closely related to infection, which directly affects the prognosis of severe hepatitis. to reduce the incidence of infection in patients with severe hepatitis, we should aim at a variety of risk factors, improve patients' immunity, rectify hypoproteinemia, treat the primary disease, prevent complications, strengthen disinfection and isolation, operate with strict aseptic technique and strict indications for invasive procedures, and use antibiotics rationally. once patients with severe hepatitis are infected, the consequences are often serious, as infection easily diffuse, which is difficult to control. after infection, the bacteria produce more toxins, which aggravate liver disease and cause a series of adverse effects, finally lead to severe complications (hepatic encephalopathy, hepatorenal syndrome, and etc.), which is the major cause for death in patients with severe hepatitis. bacterial endotoxin plays a major role in development and prognosis of severe hepatitis when infection happened [ ] . endotoxemia may directly or indirectly worsen liver injury. intrahepatic cholestasis positively correlated with endotoxin levels. endotoxin can trigger hepatorenal syndrome. for kidney, endotoxin is a potent renal vasoconstrictor substance, which can make a strong contraction of the renal artery and renal blood flow reduction. in addition, endotoxin can cause kidney capillary thrombosis and acute renal tubular necrosis, or even renal cortex necrosis. endotoxin can activate coagulation factor vii and factor vi, making the intrinsic coagulation system startup, as well as directly damage hepatocytes which release tissue thromboplastin to start the extrinsic coagulation system. meanwhile, endotoxin can also damage the vascular endothelial cells to cause bleeding, especially upper gastrointestinal bleeding. in the event of gastrointestinal bleeding, infections are more prone to happen or primary infection aggravates. in recent years, due to extensive use of broad-spectrum antibiotics, the mortality of severe hepatitis has been significantly reduced. however, infection is still an important cause of death. nosocomial infection control directly affects the prognosis of patients with severe hepatitis, which is an important part for the treatment of severe hepatitis. once the etiology and other evidence of infection appear, patients should be treated with antibiotics. effective prevention and treatment for nosocomial infections are positive to promote recovery and reduce mortality. bacterial translocation (bt) is the key mechanism in the pathogenesis of sbp, which is because of the concurrent failure of defensive mechanisms in liver failure [ ] . investigators have demonstrated pronounced impairment of gastrointestinal tract motility in cirrhosis [ ] . the disturbance of gut flora microecology was associated with changes in the (ultra)structure of the gastrointestinal tract [ ] . meanwhile, reduced cellular and humoral immunity make it easier for the free flow of microorganisms and endotoxins to the mesenteric lymph nodes [ ] . besides, it was reported that hypoalbuminemia was related to sbp. a prospective study from runyon reported that the incidence of sbp in patients with ascites protein less than . g/l was %, while the number in patients with ascites protein more than . g/l was only %. after years of follow-up, sbp incidence in patients with ascites protein higher than . g/l were negligible [ ] . the level of ascites protein correlates with opsonin activity, which means ascites protein < . g/l was an independent risk factor for sbp. the clinical manifestations of sbp are diverse. most patients have subtle and insidious onset, which usually manifested as abdominal pain and fever. acute onset may burst chills, fever and abdominal pain. generally the body temperature of patient is around °c, sometimes as high as °c. fever type usually presents as remittent fever. abdominal pain is always around the navel or lower abdomen, paroxysmal or persistent. nausea and vomiting are obvious, sometimes with diarrhea. ascites can occur in patients without ascites, or increased significantly. patients may also have significant bloating, abdominal muscle tension, tenderness, rebound tenderness, diminished or disappeared bowel sounds, and so on, or even shock in severe cases. according to clinical manifestations, sbp can be divided into five types: . common type: acute onset, protruding abdominal pain, followed by fever. or irregular fever take place followed by abdominal pain, abdominal tenderness and rebound tenderness, mild to moderate abdominal tension and growing ascites. increased wbc count and nuclear left shift. routine examination of ascites showed acute inflammatory changes. . shock: septic shock break out in a few hours to one day after abdominal pain or fever. the clinical manifestations include low temperature, lip cyanosis, abdominal tenderness, and hardly relieved shock. wbc count increases, blood culture is positive. . encephalopathy: fever and abdominal pain are often obscure, the early emergence of trance and other neuropsychiatric symptoms rapidly develop into a coma. careful examination of the abdomen in patients with light coma may still find in patients with pain expression. jaundice is deep, liver damage is serious. . refractory ascites: ascites progressively increase, which is difficult to subside with invalid diuretic therapy. abdominal distension is obvious, often without pain. carefully check the abdomen may still find slight peritoneal irritation. . asymptomatic: symptoms are mild, exhibited as slight bloating, occasional fever. mild tenderness can be found by deep palpation. in addition, a considerable part of the patients showed non-specific symptoms and signs, such as deepened coma, deepening jaundice, oliguria, azotemia or dramatically increased ascites. diagnosis of sbp should be considered if following conditions exist: ( ) fever, which can't be explained by other reasons or other parts of infection; ( ) abdominal pain, abdominal tenderness or rebound tenderness, but not serious; ( ) sudden increased ascites or poor diuretic effect manifested as refractory ascites; ( ) sudden onset of septic shock; ( ) rapid deterioration of general condition, or deteriorated liver and kidney function in a short term, deepening jaundice, or hepatic encephalopathy. the diagnoses of sbp mainly rely on ascites puncture. easl clinical practice guidelines recommend that a diagnostic paracentesis should be performed in all patients with new onset grade or ascites, and in all patients hospitalized for worsening of ascites or any complication of cirrhosis (level a ) [ ] . for patients with severe hepatitis, diagnostic paracentesis indications are: . in liver cirrhosis patients with ascites that paracentesis should be performed after admission to determine whether sbp exist. . if the following conditions happened during hospitalization, diagnostic paracentesis should be performed: ( ) abdominal sighs suggest abdominal infections, such as abdominal pain, rebound tenderness and gastrointestinal symptoms (such as vomiting, diarrhea, intestinal paralysis); ( ) systemic infection signs such as fever, leukocytosis, or septic shock; ( ) no clear incentive for hepatic encephalopathy, or rapidly emerged renal dysfunction. . in patients with ascites and gastrointestinal bleeding, paracentesis should be performed before prophylactic antibiotics. once ascites was acquired, polymorphonuclear cells (polymorphonuclears, pmn) count and ascites culture should be performed. diagnosis of sbp is made according to international guidelines [ , ] in patients with liver cirrhosis if the ascites polymorphonuclear (pmn) cell count exceeds cells/μl and other forms of peritonitis have been excluded. an ascites fluid polymorphonuclear (pmn) leukocyte count ≥ /mm should be considered as sbp, if pmn > /mm can be confirmed as sbp. if there is bloody ascites (erythrocytes more than , /mm ), pmn count are as / of red blood cells. the leukocyte esterase reagent strips (lers) test is based on the esterase activity of the leucocytes. since , studies have examined the validity of using leukocyte esterase reagent strips (lers) in sbp diagnosis. lers appeared to have low sensitivity for sbp. on the other hand, a high negative predictive value (> % in the majority of the studies)supported the use of lers as a preliminary screening tool for sbp diagnosis [ ] . there is bacteria colonization in ascites but no inflammation. the diagnosis is based on positive ascites culture, ascites pmn countless than /mm , without evidence of systemic or local infection. bacterascites have two outcomes: a shortterm or transient bacterial ascites (mostly asymptomatic), or the development of sbp (mostly with symptoms). once the diagnosis was established, paracentesis examination should be conducted again after - days. take appropriate action under the circumstances. if the second sample has a pmnl count > /mm , treat as for sbp. if the pmnl count is< /mm and a second set of cultures is positive, treat as for sbp. if the pmnl count is < /mm and the second set of cultures is negative, no further action is recommended [ ] . if ascites culture is positive but ascites pmn < /mm and there were signs of abdominal infection, it usually progress to sbp within a few days. these patients should be given appropriate antibiotic therapy. brolin first proposed the concept in . the mechanism: in early stage of severe hepatitis, ascites has not been exist yet, however because of necrosis of hepatocytes, dysfunction of kupffer cell, impairment of liver immune barrier, intestinal bacteria easily invasive into the systemic circulation through liver, causing spontaneous bacteremia, and then sbp. diagnostic criteria: ( ) primary disease was severe hepatitis, no ascites was detected by strict examination and ultrasound. ( ) clinical manifestation include fever, varying degrees of abdominal pain, diarrhea, diffuse abdominal tenderness and rebound tenderness, increased peripheral blood leukocyte, positive rivalta's test, ascites fluid leukocyte count> /mm , or pmn > /mm . ( ) exclude abdominal organ perforation or primary foci. it was used to describe the clinical situation when the ascitic pmnl count is > /mm but cultures fail to grow any bacteria. however, the term is now considered obsolete. in severe hepatitis with secondary infection, pneumonia is most common. patients with hepatic encephalopathy are susceptible to pulmonary infections as pneumonia since bed-ridden, impaired cough reflex and inadequate ventilation, especially comatose patients with intubation and tracheotomy. inpatients underwent thoracentesis, paracentesis and other invasive procedures, non-specific damage to immune barrier provide conditions for the bacterial invasion. reported in patients with invasive procedures, lung infection risk increased significantly, which demonstrated blood infection is an important way for pulmonary infection. furthermore, there was a significant increase of pulmonary infection in patients with intestinal infections or abdominal infection. pathogen: in recent years, pneumonia was classified as "community acquired pneumonia" (community acquired pneumonia, cap) "and" nosocomial pneumonia (hospital acquired pneumonia, hap). cap is the pneumonia acquired outside hospital [ ] . although cap can be caused by a wide variety of micro-organisms, the pneumococcus, mycoplasma pneumoniae and chlamydophila pneumoniae, staphylococcus aureus and certain gram-negative rods are more usual pathogens encountered [ ] . hap is the pneumonia that develops h or more after hospital admission and that was not incubating at hospital admission. hap infected with gram-negative bacilli accounts for more than %, of which pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, other aeruginosa are common bacteria. the primacy is pseudomonas aeruginosa, while gram-positive cocci accounted for about %, mainly are staphylococcus aureus, coagulase-negative staphylococci, viridans and streptococcus pneumoniae. anaerobic is rare. clinical manifestations and diagnosis: clinical manifestations are characterized as fever, cough, expectoration, dyspnea, and cyanosis. diagnostic criteria: . chest percussion of dullness, auscultation of rales, along with one of the following conditions: ( ) purulent sputum or change of phlegm; ( ) positive pathogens in sputum culture. . the chest x-ray and/or ct examination revealed new or progressive exudative lesions, and the emergence of one conditions above. urinary tract infection is divided into upper urinary tract infection and lower urinary tract infection. upper urinary tract infections are mainly pyelonephritis, which can be manifested as acute pyelonephritis and chronic pyelonephritis. lower urinary tract infections include urethritis and cystitis. the most common pathogen of urinary tract infection is escherichia coli, followed by enterococcus faecalis, klebsiella pneumoniae and streptococcus agalactiae. urinary tract infections often occur in patients with indwelling catheter, therefore, aseptic urethral catheterization and management and timely replacement of catheterization are effective to prevent such infections [ ] . . acute pyelonephritis: ( ) acute onset; ( ) chills, chills; ( ) fever; ( ) malaise, headache, fatigue; ( ) loss of appetite, nausea, vomiting; ( ) urinary frequency, urgency, dysuria; ( ) low back pain, kidney area discomfort; the ( ) tenderness of upper ureter point; ( ) tenderness of rib waist point; ( ) percussion pain in kidney area or bladder. . chronic pyelonephritis: ( ) acute onset of acute pyelonephritis with the same, but usually much lighter, even without fever, malaise, headache and other systemic manifestations, urinary frequency, urgency, dysuria and other symptoms are not obvious; ( ) edema; ( ) hypertension. . urocystitis and urethritis: frequent urination, urgency, dysuria, urinary bladder pain. urethral secretions. laboratory tests and diagnosis: diagnosis elements include: ( ) tenderness in rib waist point, percussion pain in kidney area. ( ) urine leukocytosis, pyuria. ( ) urinary sediment smear find bacteria. ( ) positive in urine culture. ( ) urinary colony counts> /ml; in patients with urinary frequency and other symptoms, colony counts> /ml are meaningful; counts - /ml also helpful in diagnosis; ( ) one hour urine wbc count> , . ( ) blood test showed leukocytosis, neutrophil nucleus left. ( ) increased esr. biliary tract infection is a common complication in severe hepatitis, which is often in company with cholelithiasis to reinforce each other. hepatitis b virus can directly violate bile duct cells and cause cholecystitis. on this basis, cholelithiasis and secondary bacterial infections are easy to happen and become a major focus, which can cause other parts of infection in severe hepatitis patients. furthermore, severe hepatitis patients often have reduced gastric acid secretion, so that e. coli in duodenum easily reproduce and cause ascending infection. biliary tract infection is usually a mixed infection of aerobic and anaerobic infections. enteric gram-negative bacteria include escherichia coli, klebsiella, enterobacter, proteus and enterococcus. anaerobic bacteroides include clostridium and fusobacterium, in which bacteroides is common, about - %, particularly bacteroides fragilis. common symptoms of biliary tract infection include chills, fever, nausea, vomiting, right hypochondrium pain and tenderness in gallbladder area. clinical performance of concurrent biliary tract infection in severe hepatitis is not always very clear, often unconfirmed by pathogen test. symptoms were epigastric or right upper quadrant pain, radiating to the right shoulder, hours after a heavy meal or a high fat meal. the patient may have severe colic, often accompanied by nausea and vomiting. patients with chronic biliary tract infection have indigestion symptoms as heartburn, belching, acid reflux and bloating, or sometimes fever and right upper quadrant pain. severe hepatitis patients have weakened intestinal resistance, especially for reduced immunoglobulin a secretions, which creates good invasion opportunities for bacterium. in addition, as mentioned above, intestinal flora are prone to happen in severe hepatitis patients, which makes intestinal infection very common [ ] . few patients exhibit cellulitis like colitis, which can further result in peritonitis and septicemia, and finally lead to death. pathogens in intestinal infections could be shigella spp., salmonella, campylobacter jejuni, clostridium difficile, and salmonella typhi etc. clinical manifestations are nausea, vomiting, abdominal pain, diarrhea, watery stool or bloody mucopurulent stool. some patients may have fever and tenesmus. depending on the pathogenesis and clinical manifestations, intestinal infections are classified as enterotoxigenic bacterial enteritis and invasive bacterial enteritis. pathogenesis of enterotoxigenic bacterial enteritis is that bacteria adhere but not invade intestinal mucosa. intestinal toxins are secreted in the process of bacteria growth and reproduction, which stimulate small intestinal epithelial cells secreting large amount of water and electrolytes. overuptake of water and electrolyte and retention in intestine makes watery stool, which is called "secretory diarrhea." secretory diarrhea is exhibited as frequent, large amount stool with no pus, usually without pain or tenesmus. it is often accompanied by vomiting, which is prone to bring out dehydration, electrolyte imbalance and acidosis, but less severe systemic toxic symptoms. stool examinations show less erythrocytes or leukocytes. invasive bacterial enteritis refers to pathogenic bacteria adhere and invade the intestinal mucosa and submucosa, causing significant inflammation. different pathogens violate different parts of intestine, small intestine, or colon, and sometimes cause inflammation both of small intestine and colon. the basic clinical manifestations include obvious systemic sepsis, high fever, and even septic shock in severe patients. stool can be mucus bloody or purulent, less amount and more frequency. abdominal pain are often severe, paroxysmal colic. if the lesion invades the rectum and distal colon in particular, there may be tenesmus. sigmoidoscopy examination showed diffuse inflammation and ulceration. if only the small intestine or upper part of colon are invaded, the stool is more moisture, and without tenesmus. stool examination show large amount of leukocytes. although diagnosis of intestinal infection is not difficult, it should be careful to distinguish the site of infection, make sure of pathogens, and pay particular attention to water, electrolyte imbalance and acid-base imbalance. therefore, in addition to routine examinations and stool culture, keeping abreast of the general condition is also important to avoid disturbance of the internal environment which aggravate liver damages. in severe hepatitis, the more severe hepatic damage and immune dysfunction, the higher of incidence of sepsis. bacterium most commonly enters through intestine into the portal vein and then into the systemic circulation, followed by skin, respiratory tract, urinary tract or other intrusion. pathogens are often opportunistic bacteria, in which gram-negative bacteria are more than gram-positive bacteria, especially escherichia coli. clinical manifestations of nosocomial infection concurrent with severe sepsis are not specific, easily masked by the primary disease and complications. in some cases primary focus is not obvious, therefore the diagnosis mainly rely on blood culture. clinical manifestations include: ( ) unexplained sudden chills, fever, shock, increased peripheral blood leukocytes or neutrophils; ( ) deepening jaundice, an increase in ascites, or appearance of hepatic coma, hepatorenal syndrome in a short term. when complications appear in severe hepatitis patients, sepsis should be alert. the mortality of sepsis in severe hepatitis is high. nosocomial infection not only aggravates liver damages, but also induces hepatic coma, hepatorenal syndrome, upper gastrointestinal bleeding and other serious complications, leading to multiple organ failure. nancy rolando reported mortality of septicemia in patients with liver failure was as high as %, in which % with septic shock [ ] . fungal infections can be classified into endogenous and exogenous infection. according to the source of the fungus, the former belongs to opportunistic pathogens, while the latter is in the environment, being infected through various routes of exposure. fungal infections in severe hepatitis are invasive fungal infection in most occasions, and the majority are nosocomial infection and endogenous pathogenic fungi infection [ ] . candida infection is most common, followed by aspergillus, again neoformans and histoplasma monocytogenes. candida albicans is widely present in normal human digestive tract. other fungi such as cryptococcus neoformans, aspergillus are widely found in nature, which can colonize in body surface, or non-enclosed cavity. they also exist in hospital work environments, increasing the chance of nosocomial infection in hospitalized patients. clinically, severe hepatitis with fungal infections are mostly superinfection. the rate of fungal infection in severe hepatitis is increasing in recent years. nancy rolando reported fungal infection was present in of acute severe hepatitis patients ( candida, aspergillus) and in seven cases was considered the major cause of death [ ] . domestic data reported that in a group of patients with liver failure, fungous infection was found in cases in which the rate of nosocomial infections was . %. in separated fungous strains, strains ( . %) were candida albicans, strains ( . %) were aspergillus fumigatus and ( . %) strains were non-candida albicans. the main sites of fungus infection were lungs ( cases) and oral cavity ( cases) [ ] . for severe hepatitis complicated by fungal infection, the mechanism is complex. there are many influencing factors such as immune dysfunction and reduced defensing ability, besides, application and abuse of broad-spectrum antimicrobial drugs are also related [ ] . because of broad-spectrum antibiotics destroy the imbalance between bacteria and fungi in digestive system, which inhibit the growth some gram-negative bacteria that had an anti-fungal effect and some bacteria that are able to synthesize vitamin b family. lack of vitamin b can lead to inhibition of oxidation coenzyme, enabling weakened immunity, which is conducive to fungal growth. research and experience have demonstrated that repeatedly use of glucocorticoid is also an important factor to induce fungal infection in patients with severe hepatitis [ ] . and therefore, the use of corticosteroids also need special care of. currently the use of glucocorticoids in patients with severe hepatitis is still controversial. the majority do not advocate glucocorticoids, or consider a short-term use in the early stages of the disease and be removed as soon as possible. otherwise it will cause a large increase of possibility of fungal infection. fungal infections are among the leading causes of death in patients with severe hepatitis. according to an analysis from rolando, among cases of severe hepatitis, seven cases of deaths directly related to fungal infections [ ] . a recent domestic research analyzed outcomes of patients with severe hepatitis, it was found that the mortality of patients with fungal infection was significantly higher than those without fungal infections ( . % compared to . %) [ ] . according to statistics, fungal infections often occur eight days after admission ( - days) or . days after broad-spectrum antibiotics usage ( - days). symptoms of fungal infection are often covered by severe liver injury related symptoms, while clinical systemic manifestation such as fever is also difficult to identify with the bacterial infection. the site of infection often occurs in mouth, respiratory, digestive or urinary tract. severe immunocompromised persons may appear disseminated infection. oropharyngeal is the site that fungal infections mostly occur, and candida albicans is the most common pathogen, followed by non-candida albicans and aspergillus infection. bedridden patients with severe hepatitis can not properly maintain oral hygiene, which results in change of oral local environmental ph and blood flow. candida albicans easily retains in the mouth, and multiplies, causing oral flora and opportunistic infections. usually general symptoms are mild. patients often have abnormal taste or loss of taste, followed by xerostomia, mucosal burning and other symptoms. candida stomatitis may have pseudomembrane formation which is not easy to peel, accompanied by angular cheilitis, or sometimes manifests as mucosal congestion, erosion or clumps shrink of tongue papillae, thickening of coating on the tongue. there are clear lines between oral pseudomembranous damages. if remove the pseudomembrane it will leave a bright red base, sometimes thick film is like a layer of cheese. take the film directly under microscopic examination shows hyphae and spores. oral fungal infection is often a prelude of deep fungal infection, which should be on the alert. simple oral candida albicans infection is not always accompanied with fever. oral fungal infection is easy to be found, therefore, if oral fungal infection exists with fever and increased leukocytes, it should be paid attention to merger of deep fungal infections such as the lungs or other organs [ ] . aspergillus is ubiquitous in nature which can be spread through air flow. the most common pathogen that causes invasive aspergillosis is aspergillus fumigatus, while aspergillus flavus, aspergillus niger and aspergillus soil are less common. inhalated aspergillus spores can reproduce in healthy or immunity weakened people. respiratory tract is an infective route of invasive aspergillosis, accounted for %. once tissue infection exists, blood vessels violation and bloodstream invasion are common. invasive aspergillosis infection has three characteristics: tissue necrosis, hemorrhage, dissemination [ ] . the mortality of invasive pulmonary aspergillosis accompanied by severe hepatitis is up to % [ ] . it is lack of specific clinical manifestations. in most patients fever is the first arisen symptom, mostly with middle or low heat, sometimes with sudden high fever. hemoptysis occurs with fever simultaneously or - days later, often accompanied with purulent tan sputum or bloody sputum, sometimes with a pinhead size of gray-green particles in it. shortness of breath and chest tightness often exist in late stage of infection, as well as dyspnea, cyanosis, hypoxemia, and expectoration of a lot of bright red bloody sputum or blood clots. pulmonary signs appear later, manifests as pulmonary wet or dry rales, occasional pleural friction sound. in some cases there are no pulmonary signs. x-ray examination of invasive pulmonary aspergillosis showed patchy infiltrates and (or) nodular lesions. typical nodules were like cotton, which can occur in unilateral or bilateral lobes. the lesion progresses rapidly to cause expanded infiltrates, and segmental or lobar consolidation. ct examination showed mass shadow, nodules, or exudative lesions. there are two typical imaging performance: ( ) a characteristic finding on chest ct is the halo sign: a solid nodule surrounded by a halo of ground-glass attenuation [ ] . ( ) the formation of voids, which appear in late infection. signs are hollow cavity lesions, the balloon "crescent" sign was seen around necrosis tissue [ , ] . in order to prevent hepatic encephalopathy, patients can be given high doses of laxatives such as lactulose, which can reduce the intestinal ph value, creating an environment for growth of fungi, thereby increasing the chances of fungal infection of the intestine. in particular, some antibiotics combination increases the incidence of intestinal candidiasis. patients usually have watery or jerry-like diarrhea, with foam or blood. diarrhea is accompanied by bloating, sometimes with vomiting and fever, however, abdominal pain is not obvious. in patients with severe hepatitis, due to decreased intestinal mucosal defense capability, candida may invade the muscle and cause intestinal bleeding, or even perforation. in part of patients, it may even progress to fungal peritonitis, which is similar to clinical manifestations of bacterial peritonitis. in patients with oral candidiasis, if there is dysphagia or pain, especially retrosternal burning, it should be considered that esophagus is invaded. incoordination motor of the upper and lower esophageal can be found by esophageal barium enema. gastroscopy helps to confirm the diagnosis. urinary fungal infection usually involves bladder and kidney, among which candida infection is the most common. however, all pathogenic fungi (such as cryptococcus neoformans, aspergillus species, mucor species, histoplasma, blastomycete, coccidioides) can spread to the urinary system as a systemic or part of disseminated fungal infection, which is more related to usage of broad-spectrum antibiotics and indwelling catheters. clinical manifestations include fever and urinary tract irritation, while some patients were asymptomatic with candidiasis urine. candida and bacterial infections often occur simultaneously. candida infections of the kidney, mostly secondary, are caused by the spread of blood candida. renal cortex and medulla abscesses can occur, which affect renal function in severe cases. patients may have low back pain, abdominal pain, fever and chills, accompanied by urgency, urinary frequency, proteinuria and hematuria. urine tests may find hyphae and fungal spores, culture for candida is positive. candida albicans is common, but now there is a growing trend of candida glabrata [ ] . the main pathogen of fungal sepsis is saccharomyces. most patients have high fever, often above °c. the thermal type varies, of which intermittent fever or remittent fever is more common. wbc count and neutrophil are usually increased. disease in patients with fungal sepsis followed by severe hepatitis is deteriorating rapidly, even progressing to shock. fungal septicemia may invade all the tissues and organs. involved organs have corresponding performance, such as fungal pneumonia, oral fungal infections, intestinal and urinary tract infections. disseminated fungal infection often occurs in severe immunocompromised patients who have long-term use of antimicrobial agents. candida, cryptococcus, aspergillus can disseminate along with blood to all of the organs, such as kidneys, lungs, heart, and liver. the condition is dangerous, which often lead to death in a short term. in addition to common bacterial and fungal infections, severe hepatitis can also be complicated by other pathogens, such as viruses, tuberculosis, protozoa and others. cmv (cytomegalovirus, cmv), herpes simplex virus (herpes simplex virus, hsv), or varicella -zoster virus (varicella-herpes and zoster virus, vzv) infections are three of common herpes viruses infections in severe hepatitis. their common characteristics rely on that once the host is infected, the virus can persist for long periods in the host. when the host immunity is weakened, the virus can re-proliferative, which leads to disease resurgence [ ] . hsv infections manifest as perioral or external genital herpes, oral and esophageal mucosa inflammation and ulcers, also viremia which leads to pneumonia and encephalitis. common clinical symptoms of herpes simplex are mild, only a few people show fatigue, fever and other symptoms. local manifestations are single or multiple blisters on skin or mucous membrane, with tingling. due to reduced immunity, skin rashes in patients with severe hepatitis perform as varicella-like rash, vaccination herpes, herpes keratoconjunctivitis and disseminated herpes simplex. severe herpes usually manifests as herpes simplex virus encephalitis with high mortality. there are fever, headache, mental disorders, coma and other clinical symptoms, often without skin herpes lesions. the sites of infection are commonly in the frontal and temporal lobes. elevated serum antibodies help confirm the diagnosis. cytomegalovirus infections are common in cirrhosis of the liver [ ] . interstitial pneumonia is the most common clinical manifestation in severe hepatitis concurrent with cmv infection. chest ct examinations mainly perform as diffuse interstitial or alveolar infiltrations, very few case show as nodular shadows, occasionally as pleural effusion [ ] . pulmonary consolidation reminds complicated bacterial or fungal infections. pathology manifestations show alveolar interstitial edema, with varying degrees of fibrosis, lymphocyte infiltration and epithelial cell proliferation. blood examination shows leukopenia. because viral pneumonia shares a certain similarity in clinical manifestations, diagnosis mainly depends on pathologic examination. pathogenic examinations for cmv often use methods below: ( ) detect of cmv inclusion body cells and viral particles: eosinophilic nuclear inclusions giant cells are found in respiratory secretions and bronchoscopy lung biopsy specimens. respiratory secretions, saliva, urine, cervical secretions, liver or lung biopsy specimens were inoculated to human embryonic fibroblast cell culture medium, where cytomegalovirus was separated. ( ) immunological methods: cmv antigen from secretions was detected by fluorescent antibody assay, or elisa, which is conducive to the early diagnosis. serum antibodies can also be detected by complement combined experiment, in which acute and convalescent serum antibody titer more than times are positive. ( ) molecular biology methods: pcr technology and nucleic acid hybridization, which helps to make distinction between a variety of different subtypes of the virus. most of vzv infections in patients with severe hepatitis are due to latent virus reactivated. in patients who have had chickenpox, there is a small amount of virus lurking in the spinal cord dorsal root ganglia or cranial nerve sensory ganglia. when severe hepatitis happened, latent virus is reactivated in ganglia due to decreased immunity. the activated viruses spread along with sensory nerve axons to downstream disposal areas, proliferate and cause shingles. in early time, there is paresthesia, itching, and pain in local skin. and then rashes and herpes break out, chaining into a strip, which distribute in denomination or trunk, unilateral, with duration of about three weeks or several months. part of patients with severe hepatitis show severe disseminated herpes zoster. varicella-like rashes appear in a few days, often accompanied by fever, which may be complicated by lung, brain damage with a high mortality rate. latent tuberculosis can burst to tuberculosis and extrapulmonary tuberculosis when cellular immune function gets weakened. under normal host immune function, lymphocytes, macrophages and common langerhans cell may promote granuloma formation and make infection localized. when host immunity is dysfunctional, tissue reaction is very small or even disappeared, leading to mycobacterium growing rather than formation of granulomas, nor any effective defense against infection. severe hepatitis patients with m. tuberculosis infection may develop acute miliary tuberculosis that manifest as fever, cough, expectoration, bloody sputum, chest pain, and shortness of breath in addition to deteriorated liver function. moreover, tuberculosis easily spread in patients with severe hepatitis, with poor anti-tuberculosis efficacy. common extrapulmonary tuberculosis can be lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, renal tuberculosis, epididymal tuberculosis, genitourinary tuberculosis, nervous system tuberculosis, tuberculous meningitis [ ] . in patients with severe hepatitis concurrent with tuberculous mycobacterial infections, tuberculin reaction is negative in about half of patients, especially in those applied with glucocorticoid. diagnosis relies on sputum acid-fast staining or pcr, but the diagnostic yield is not high. interferon gamma release assays (igras), including quantiferon ® -tb gold in-tube, and the t-spot tb, have been extensively used for the auxiliary diagnosis of tuberculosis infection in adults. igras detect circulating t-cells responsive to specific mycobacterium tuberculosis antigens, which are absent in bcg and other non-tuberculosis mycobacteria, and exhibited similar sensitivity and higher specificity than tst in adults [ , ] . however, these igra tests are also affected by host immune status [ ] . in addition, the decision regarding whether to treat ltbi should be dependent not only on igras results but also on clinical histories. ntm generally causes local wound infections. however, in severe hepatitis patients with impaired immune function, non-tuberculous mycobacteria can invade the lungs, causing tuberculosis-like diseases, but rarely causes hematogenous dissemination. histological examination of the lesion is mainly characterized by epithelioid cell granulomas and foam cell-like balls of tissue proliferation, detection of non-tuberculous mycobacteria [ ] . protozoa and worms, such as toxoplasma gondii, giardia lamblia, cryptosporidium and stercoralis, can also infect patients with severe hepatitis, especially those with immunosuppression drugs or combined with cancer. main lesions of toxoplasmosis manifest as lymphadenopathy, hepatosplenomegaly, encephalitis and pneumonia [ ] . clinical manifestations of giardia lamblia infection are chronic diarrhea and malabsorption, also fever and cholecystitis [ ] . pathological changes are deformation of small intestine villi and lymphoid hyperplasia. parasites present in small intestinal surface and gallbladder, and the detection of parasites from the stool and duodenal drainage fluid that is eligible confirmed. strongyloides stercoralis is a weak virulent worm, there is few clinical stercoralis infection [ ] . but this worm infection in patients with severe hepatitis is a serious threat, even causing death. clinical manifestations include long-term nausea, vomiting, diarrhea, bloating, intestinal paralysis, dehydration, electrolyte imbalance, edema, weight loss and difficulty breathing in cases with extensive lung lesions. all patients have hypoproteinemia and anemia. patients with increased eosinophils have good prognosis, whereas eosinophils reduction often is a dangerous signal. varieties of complications, such as system or local infections, coagulopathy, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, acid-base imbalance and water-electrolyte imbalance, are main causes of hbv-associated mortality during deterioration of liver function of aechb. reducing and effective treatments of these complications are still nodules in therapy of severe phase of aechb. infection is one of usual complications of aechb, which shows - % morbidity in civil china. the most common localities are respiratory system, especially lungs, and abdomen, which shows the incidence of spontaneous bacterial peritonitis (sbp) as much as %. others include urinary tract and bloodstream. gram-negative bacteria are the predominant causes including aerobic gram-negative bacilli, escherichia coli, klebsiella, enterococcus and anaerobic bacteroides fragilis, etc. however in recent years, gram-positive bacterial infections are found increasing in patients with aechb, including pneumococcus and other streptococcus, but staphylococcus aureus infection is relatively infrequent. fungal infections are usually secondary to bacterial infection. candida, aspergillus, sporotrichosis, histoplasmosis and coccidioidomycosis infection, especially candida albicans, are most frequent in occurrence. but, infections of cryptococcus and mucormycosis are relatively rare. beyond of bacterial and fungal infection, other infectious pathogens in aechb are virus, mycobacterium tuberculosis, mycoplasma, chlamydia, parasites and protozoa. bacterial infection is the most frequent complication in aechb. infections are more likely occurred in abdomen, including abdominal cavity, biliary tract, gastrointestinal tract, or other parts like respiratory tract, and urinary tract. keeping oral and perineal asepsis, unobstructed respiratory tract and urine tract, anti-bedsore care for patients with limited mobility, rational use of antibiotics, avoiding long-term use of broad-spectrum antibiotics, strict controlling usage of glucocorticoid, aseptic practices in invasive operation, parenteral nutrition and protecting the intestinal mucosa are all necessities to prevention of bacterial infection in aechb. empirical use of antimicrobial agents could be determined by the localities of infection without antibiotic susceptibility testing results. gram-negative infections are more frequent in peritoneal or biliary tract, in which cephalosporin or quinolone could be the first choice. penicillin and vancomycin could be considered in pulmonary infection. azithromycin and quinolone could be adopted in urinary tract infection. metronidazole or tinidazole could be used in anaerobic infection. broad-spectrum antibiotics can be used in serious infection, such as ceftriaxone, cefoperazone, cefepime, and carbapenems, but the secondary infections need to be highly concerned. initial antibiotics should be adjusted according to antibiotic susceptibility testing results as soon as possible. non-specific immune enhancer agents, such as thymosin, are well used in treatment of infection during aechb, but it currently still lack of evidence-based support. the incidence of fungal infection ranks secondly in aechb associated infection. respiratory tract, gastrointestinal tracts and genitourinary system are the major sites. keeping wards dry and ventilated can help to reduce environmental fungal growth. oral and perineal cleaning and regularly dental check are necessary. if oral fungal spots are found, alkaline mouthwash and nystatin with glycerol can be used in treatment. for patients with limited mobility, anti-bedsore care is terribly necessary. rational use of antibiotics, especially avoiding long-term usage of broadspectrum antibiotics, can prevent secondary fungal infection. glucocorticoid should be used strictly according to indication. during invasive procedures, aseptic operations are obligated. regular check of sputum, lungs, urine and feces will facilitate early diagnosis. empiric antifungal treatment is generally not recommended, but to patients with aechb with aids, especially with count of peripheral blood cd + t cell-less than /μl or oropharyngeal candidiasis found, the sulfamethoxazole (smz-tmp) should be chosen to prevent pneumocystis pneumonia. fluconazole should be considered when moderate amount of candida albicans has been indicated by sputum culture. empirical treatment. if candida albicans infection is highly susceptible, the initial treatment choice is fluconazole ( - mg/days). but if aspergillus infection is preferred, amphotericin b liposome, itraconazole, caspofungin or voriconazole could be considered as the initial treatment, in which process liver and kidney function should be intensively monitored. to patients with cryptococcal encephalopathy combining severe liver damage, fluconazole or flucytosine combined with intrathecal injection of amphotericin b treatment could be implemented. evidence based treatment. initial antifungal strategy should be adjusted according to antibiotic susceptibility testing results. for certain invasive pulmonary aspergillosis, combination treatment of several different types of antifungal agents should be considered under monitoring of liver and kidney function. however, for pulmonary mucormycosis infection, the combination of amphotericin b and flucytosine is the only effective strategy. amphotericin b is a type of polyene antifungals, mainly for aspergillus, candida, cryptococcus, histoplasma infection, but is invalid in aspergillus terreus or ringworm fungus infection. intravenous or intrathecal injection of amphotericin b should be administrated because of non-digestive absorbance. the recommended initial dose of intravenous administration is - mg/days, and gradually increases to . - mg/kg.days. the infusion track needs to be dark and process needs not less than h. the initial dose of intrathecal injection is . mg/days and gradually increases to . - mg/days. amphotericin b has an extra toxicity to liver and kidney, and also causing hypokalemia. intensive monitoring of liver and kidney function and serum potassium levels is necessary during treatment, and be sure largely avoiding combination with other agents with liver and kidney toxicity. itraconazole is one of triazole antifungal agents, mainly for aspergillus, candida, cryptococcus and histoplasma infection, but it is invalid in fungi fusarium or zygomycetes infection. the intravenous dose for the initial days is mg/days, administrated by twice, and then followed by mg/days for weeks. the oral doses of mg bid could be subsequent. the total curative course could be determined by the improvement of symptoms and largely absorption of radiographic lesions of infection. the monitoring of liver function is necessary and recommended, especially in long duration treatment. furthermore, combination treatment with other hepatotoxic drugs should be avoided. fluorocytosine is one of bacteriostatics, mainly for cryptococcosis and candida infection, frequently based on the combination with amphotericin b. the daily dose for adults is . g with intravenous dripping speed of - ml/min. a half dose should be conducted in renal insufficiency. the inhibited administration includes severe liver or kidney dysfunction and allergy to fluorocytosine, and also cautious treatment for pregnant or breastfeeding women. no combination treatment of fluorocytosine with cytarabine or bone marrow suppression agents is recommended. fluconazole is a triazole antifungal, mainly for candida albicans or cryptococcus infection, but not as good in candida glabrata infection, totally invalid in aspergillus or candida krusei infection. the recommended daily dose for adult is - mg, and the initial dose should be doubled. voriconazole belongs to triazole antifungals, mainly for candida, cryptococcus, aspergillus, fusarium and histoplasma capsulatum infection, especially used for invasive aspergillosis and invasive fluconazole-resistant candida infection, but is invalid for mucor or rhizopus infection. the recommended intravenous initial dose in adult is mg/kg, q h, with infusion rate of mg/kg within - h. the maintenance dose from the second day is mg/kg, q h. to patients without tolerance to normal dose, it could be reduced to mg/kg, q h. the reduction of daily dose could not be necessary in mild to moderate liver dysfunction. but intravenous administration should be avoided in patients with severe renal dysfunction. the side effects of voriconazole include transient visual disturbances, mental disorders, thrombocytopenia and so on. caspofungin belongs to echinocandin antifungals with antibiotic spectrum of pathogenic aspergillus, candida and pneumocystis, without in cryptococcus neoformans, fusarium and mucor. it is mainly used for invasive aspergillosis. the initial dose for adults is mgqd, and with subsequent mg qd. the infusion time is no less than h. no fixation curative course is suggested. caspofungin should be avoided for patients with severe liver function, because of highly hepatic distribution and metabolic pathways during treatment. liver is the largest solid organ in the body, and it plays a central role in the clotting process, except for general function such as metabolism, detoxification and choleresis [ ] . a majority of the coagulation factors are synthesized almost exclusively in the liver. in the pathogenesis of severe hepatitis, massive hepatic necrosis leading to reduced production and dysfunction of coagulation factor. in addition, the increased levels of anticoagulation and platelet abnormalities also contribute to occurrence of coagulopathy, which were further exacerbated by severe complications such as spontaneous bacterial peritonitis (sbp) and hepatorenal syndrome (hrs). coagulation abnormalities, even disseminated intravascular coagulation (dic) often occur in those patients with severe hepatitis. therefore, the changes in coagulation factors were usually used to evaluate prognosis of severe hepatitis [ ] . the normal procedure of coagulation includes two independent coagulation process, namely intrinsic and extrinsic pathway, which initiated by coagulation factor xii, and factor viia/tissue factor, respectively. the two pathways reach a confluence at the points of factor xa and va. wherein factor xa activates prothrombin to thrombin in the presence of ca + and factor va bound to membrane surfaces, and then thrombin converts fibrinogen to fibrin. thus, blood becomes clotted and the "y" shaped pathway was established [ ] . simultaneously, there are some anticoagulation systems existing in our body, which prevents the formation of thrombus, then keeps normal circulating of blood flow. it also participates in maintaining of normal permeability of the blood vessel [ ] . among anticoagulation systems, the most important one is the fibrinolytic system, whose basic process can divided into two stages, i.e. plasminogen activation and fibrin degradation [ ] . plasminogen activators include tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator (upa), and the principal function of these two plasminogen activators is to convert plasminogen to plasmin, then plasmin cleaves fibrin into soluble small peptides, namely fibrin degradation products [ ] . moreover, the process of fibrinolysis was affected by fibrinolysis inhibitors, such as plasminogen activator inhibitor (pai) and alpha antiplasmin (α -ap). those fibrinolysis inhibitors can either inhibit plasminogen activation, or reduce the function of plasmin [ ] . thus, there is a dynamic balance between coagulation and anti-coagulation systems under physiological conditions ( fig. . ) [ ] . many factors involved in coagulation process, i.e. clotting factors, thrombin inhibitor, fibrinolytic system and so on, are synthesized almost exclusively in the liver. thus, liver plays a pivotal role in remaining the balance between hemorrhage and coagulation under physiological conditions (table. . ) [ ] . it is the important basis for pathogenesis of coagulopathy that massive hepatocyte necrosis occurs in severe hepatitis patients, resulting in reduced production and dysfunction of those blood clotting factors [ ] . coagulation abnormalities, such as decreased production and dysfunction of coagulation factors, are commonly found in severe hepatitis, there are at least types of factors participated in the coagulation process, including classical coagulation factors, namely factor i/fibrinogen, factor ii/prothrombin, factor iii/tissue factor, factor iv/calcium ions, as well as factor v, vii, viii, ix, x, xi, xii, and factor xiii. some bradykinin factors, such as rk (prekallikrein) and hmwk (high molecular weight kininogen) are also associated with coagulation. among above factors, the others belong to proteins excepting factor iv. plasma coagulation factors are synthesized exclusively in the liver, excepting factor iii/tissue factor, factor iv, factor vi/activated factor v, factor viii and factor viiia [ ] . massive hepatic necrosis leads to the decreased production of clotting factor in patients with severe chronic hepatitis. moreover, the coagulation factors are sensitive indicators for clinical evaluation of severe hepatitis. it is common that the serum levels of factor v, vii, ix, x, xi and prothrombin decreased in the patients with severe hepatitis. on the contrary, clotting factor viii is synthesized, and then excreted increasingly by the mononuclear phagocytic cells with stimulation of various inflammatory cytokines in the patients with severe hepatitis [ ] . anorexia and antibiotics overuse lead to obstacles in assimilation, absorption, and application of vitamin k in these patients. vitamin k dependent coagulation factors includes factor ii, vii, ix, and x. the function of γ-hydroxylation was weaken by the deficiency of vitamin k and damage of hydroxylase, then the abnormal clotting factors without γ-hydroxy glutamate were synthesized. finally, these abnormal clotting factors lead to dysfunction of the vitamin k dependent coagulation factors [ ] . coagulation factor vii eliminated significantly in the initial stage of liver injury was usually used as an early diagnosis index [ ] . in addition, coagulation factors ii and x, then factor ix decreased markedly, along with exacerbation of liver injury. the deficiency of vitamin k induced by obstructive jaundice, malabsorption syndrome, can be rescued after intravenous injection of vitamin k, which is different from coagulopathy caused by liver injury. the plasma level of fibrinogen is within normal range in patients with compensatory cirrhosis. however, patients with severe hepatitis or liver failure have a significant reduction in the level of fibrinogen, and then develop dysfibrinogenemia [ ] . fibrinogen, as the main hydrolysis substrate of thrombin, is the crucial factor in the coagulation process. thus, decreased levels or abnormal structure of fibrinogen leads to coagulation abnormalities in patients with severe hepatitis [ ] . there are two clotting factors associated with thrombin except for fibrinogen, i.e. factor v and factor xiii. factor xiii induce a crosslink of sfm (soluble fibrin monomer), resulting in the formation of insoluble fibrin [ ] . it reports that the levels of circulating coagulation factor xiii were decreased in % of patients with liver disease, resulting in fibrin decline or abnormality. these patients with liver disease will receive a bad prognosis if the plasma level of fibrin was at level below % of normal concentration [ ] . it is uncommon that the plasma level of plasma factor v decreased markedly in patients with severe hepatitis, except for those complicated with dic or hyperfibrinolysis. the low level of factor v cannot induce the formation of the enzyme-substrate complex, which delay the activation of prothrombin [ ] . contact factors include classical coagulation factor xii and xi, as well as some bradykinin factors, i.e. pk and hmwk. these factors contact with vascular intima damage or abnormal surface in blood vessel walls, then active the intrinsic coagulation pathway [ ] . moreover, the contact factors can connect with bradykinin, fibrinolysis and complement system. in addition, coagulation factor xiii also activates fibrinolysis system as the initiating factor. factor xii deficiency does not cause excess bleeding, but induce thrombus due to its decreasing activation of fibrinolytic system [ ] . there is a dynamic balance between pro-and anti-coagulation systems in physiological conditions, which keep normal blood circulation in the body. the anticoagulation system includes physiological anti-coagulation factors (e.g. antiprothrombin-iii, protein c, and so on) and fibrinolysis factors (e.g. plasminogen, α- plasmin inhibitor, and so on) [ ] . at-iii, with a half-life period of . days, synthesized mainly in hepatocytes and partly in endotheliocyte, is responsible for about % of anticoagulation in plasma. it is the main reason for low plasma level of at-iii that decreased production and increased consumption of at-iii caused by hepatocytes necrosis in patients with severe hepatitis. the activity of at-iii obviously decreased in severe hepatitis [ ] . therefore, heparin, thrombin, activated coagulation factors (e.g. factor x, ix, xi, xii) cannot be inhibited, due to rare at-iii combining with them [ ] . there is a negative correlation between at-iii: a (the activity of at-iii) and pt (prothrombin time), which indicate that the level of at-iii: a decreased obviously along with exacerbation of liver injury [ ] . the plasma levels of pro-and anti-coagulant factors are low in patients with liver injury. when the necrotic tissue and cytolysis are released in the blood, the balance of pro-and anti-coagulant is destroyed. finally the depletion of at-iii by massive activated coagulation factors will lead to dic [ ] . massive hepatocytes necrosis, vitamin k deficiency, and protein c without γ-hydroxyglutamic acid result in blocking activation of protein c. thus, va, viiia and pai cannot be degraded, and the plasma level of them will rise up, due to reduction of activated protein c (apc) in the patients with severe hepatitis [ ] . plasminogen synthesis will decreased by about % in the liver when sever hepatitis occurs. as the activators of plasminogen, tpa and upa are synthesized by vascular endothelial cells, and their production will increase after vascular endothelial cells initiated with virus, immunocomplex or endotoxin in the patients with severe hepatitis b [ ] . with exacerbation of liver injury, pai synthesis decreased significantly in the liver. the main physiological activity of pai is to inhibit tpa induced plasminogen activation. therefore, the activity of tpa will increase with reduction of pai synthesis, resulting in promoting the conversion of plasminogen into plasmin [ ] . plasmin, as a kind of powerful proteolytic enzyme, can hydrolyse fibrinogen into fdps, degrade coagulation factors, and inhibit platelet aggregation, resulting in the aggravation of bleeding [ ] . hyperfibrinolysis can be caused by congenital or acquired reason, and it commonly leads to a rapid depletion of coagulation and anticoagulation factors, especially in those patients with dic [ ] . synthesis and secretion of tpa and upa markedly increase, while pai, namely tpa or upa inhibitor, has been a decrease in the plasma of severe hepatitis patients, resulting in hyperfibrinolysis [ ] . at the same time, mononuclear phagocyte system cannot degrade plasminogen activator, also leading to hyperfibrinolysis. it is not necessarily accompanied by hemorrhagic tendency, although there are many risk factors that can cause hyperfibrinolysis, even hyperfibrinolysis occurred in severe hepatitis [ ] . in addition, fdps inhibit fibrin monomer polymerize function, and also block platelet aggregation, then further worse the deficiency of hemostasis and coagulation, finally leading to the aggravation of bleeding tendency [ ] . studies have shown that low-level endogenous small molecule heparin in patients with chronic hepatitis may be associated with many factors, such as increased mastocyte, decreased production of heparinase in the liver and reduced activity of ph (platelet factor ) [ ] . when the disease progress to cirrhosis or chronic severe hepatitis, pt was significantly prolonged, while endogenous heparin wasn't increased markedly, indicating that low-level endogenous heparin has little effect on pt elongation and hemorrhagic tendency [ ] . however, if the patient is undergoing the following condition together, the endogenous small molecule heparin will increase significantly. ( ) esophageal variceal bleeding with serious infections (such as abdominal infection, biliary tract infection and pulmonary infection) or portal hypertension. ( ) combining with the significant increased white blood cell count. ( ) percentage of neutrophils > %. the level of endogenous small molecule heparin in plasmin will decrease after those infections were cured. taken together, these data indicate that increased level of endogenous small molecule heparin in blood circulation was closely related to severe hemorrhagic tendency when combining with infection [ , ] . platelet significantly promotes blood coagulation through connecting with many coagulation factors (such as fibrinogen, factor v, factor xi, factor xiii and so on). α-granule includes fibrinogen, factor xiii, and some platelet factor such as platelet factor (pf ) and platelet factor (pf ), which promote coagulation activation process of factor xii and factor xi can be accelerated by activated platelets [ ] . it is estimated that pf provided by platelets could accelerate activating of thrombin by twenty thousand times. xa and factor v could not be inhibited by at-iii and heparin if they were linked with pf . when platelets aggregated and formed platelet plugs, the process of coagulation was initiated at this site, and then platelets reveal a large number of phospholipid surfaces, which were helpful for activating of factor x and fibrinogen [ ] . various platelet factors were released from α-granule after platelet aggregating, and then hemostyptic fibre was produced increasingly. those hemostyptic fibre finally produced blood clot formation after capturing the other hemocytes. thus, platelet plugs would progress independently of platelet disintegrating gradually [ ] . two aspects of platelet abnormalities consist of depletion and dysfunction. in patients with chronic hepatitis and cirrhosis, the occurrence of decreased platelet level is usual through hypersplenism accompanied with other hemocyte decreasing [ ] . the ratio of which reaches - %. its incidence rate in severe hepatitis and explosive hepatic failure also reaches approximately by %. when severe hepatitis occur, myelosuppression, decreased megacaryocyte replication leading short-life platelet, lacking of hemopoietic material such as vitamin b, folate and so on, all of these can initiate thrombocytopenia [ ] . other reasons leading to thrombocytopenia contain low expression and metabolic disturbance of thrombopoietin (tpo), as well as platelet antibody production. researches show that the serum level of tpo related positively to platelet expression [ ] . we next studied platelet associated immunoglobulin (paig) and its sorts such as paigg, paiga, paigm etc., with chronic hepatitis. in line with previous reports, we found that serum levels of paig and paigg negatively correlated with blood platelet count, corroborating the crucial role of the paig-mediated autoimmunization in controlling thrombocytopenia in viral hepatitis [ ] . various factors impact platelet function forwardly or passively. increased expression of oxide and prostacyclin, two kinds of platelet inhibitor derived by endothelium, may inhibit platelet activation in vivo [ ] . on the other hand, increased serum level of von willebrand factor (vwf) can promote platelet adhering and aggregation in patients with cirrhosis. when severe hepatitis occur, . % and . % of patients have decreased levels of platelet adhesion rate and platelet aggregation rate (par), respectively. in addition, reduced effectiveness of pf and abnormal clot contraction occur in patients with severe hepatitis by an incidence of . % and . %, severally [ ] . patients with terminal liver disease significantly exert hemorrhagic tendency, especially in the digestive tract [ ] . the latest report indicated that basic laboratory examinations for coagulation function testing in common use at present, such as pt, aptt, international normalized ratio (inr) etc., have little correlation with occurrence of gastrointestinal bleeding in these patients, thereby revealing the importance to search and pay close attention to those complicating disease upregulating bleeding risk, such as bacterial infection, renal failure, hemodynamic change after portal hypertension, dysfunction of endotheliocyte as well as macrophagocyte and so on [ ] . it is common to see renal failure occurrence in advanced hepatopathy. when it happened, acquired platelet dysfunction, abnormal activation of platelet and vascular wall, anemia and so on, all of them significantly promote hemorrhage [ ] . as another severe complication, bacterial infection is also very important and common [ ] . when tumor necrosis factor (tnf) were injected into healthy individuals, we found that endotoxin have an important role to exert its function directly in clotting cascade reaction. early researches indicated that the body can express endogenous heparin-like substance through stimulation by endotoxin in patients with cirrhosis [ ] . furthermore, some studies revealed the relevance of endotoxin with prothrombin fragment, indicating that infection promoted occurrence of dic-like status in laboratory examination in cirrhosis [ ] . coagulation system became weaker in patients with chronic hepatitis. it can hardly mediate factors due to the relative deficiency of pro-and anticoagulation factors. when the balance was destroyed, it may tend to hemorrhage or thrombosis depending on related risk factors which were in the ascendant. to assess abnormality of blood coagulation in patients with liver disease, we should be in consideration of hypercoagulability, one state may easily be overlooked. otherwise, it will be unfair. current literatures indicated that, unlike previous concept that the body of patients initiated anticoagulation state automatically when infected by the hepatitis virus. various clinical evidences revealed that hepatitis virus infecting cannot inhibit thrombus forming. furthermore, it may increase dangerous to thrombus forming especially in the portal system, for existence of individuals having hereditary mutation to promote thrombus forming [ ] . slower bloodstream, abnormal fibrinolysis initiated by blood stasis, and decreased activity of anticoagulant accelerate formation of venous thrombosis. moreover, the changes of platelet phospholipids membrane activity were also helpful to the formation of thrombosis in patients with chronic liver disease [ ] . it is reported that the incidence of periphery deep venous thrombosis and pulmonary embolism was . and . % in patients with cirrhosis, respectively [ ] . the rate of portal vein thrombosis is about % in patients with compensated cirrhosis, but it reaches - % in the candidates waiting for liver transplantation [ ] . for mutations (such as leiden mutation of factor v, g a mutation of prothrombin, c mutation of methylenetetrahydrofolate reductase) or the existence of antiphospholipid antibody syndrome, the hypercoagulable state will be represented in patients with liver disease [ ] . the hypercoagulable state represents diverse modes in the body of patients with chronic hepatitis. among them, thrombosis is more traditional and common. the mechanism of dic complicated with severe hepatitis may contain several aspects as follows. . with attacks from endotoxin, virus and immune complex etc., endotheliocyte was injured, and then it activated plasmakinin system and complement system, leading to the aggravation and participation of dic progress. damaged endotheliocyte can simultaneously activate factor vii, intrinsic coagulation pathway and platelets, and participate in micro-thrombosis with platelets adhesion and aggregation beneath endothelium [ , ] . . in patients with severe hepatitis, massive necrotic hepatocytes activated extrinsic coagulation system with the releasing of various tissue thromboplastin-like substances [ ] . . hepatocellular necrosis or dysfunction decreased expression of anti-coagulation factors such as at-iii, pc, protein s and so on, and then it enhanced activation of thrombin and plasmin [ , ] . . impaired function of the mononuclear phagocyte system. mononuclear phagocytes can express activated tissue factor with the releasing of tnf, il- and platelet activating factor (paf) on its surface after stimulation by endotoxin, inflammatory cytokines and complement activation. tnf and il- can decrease the activation of protein c through its function to increase the expression of plasminogen activator and plasminogen activator inhibitor- and to inhibit the production of thrombomodulin (tm) [ ] . activated clotting factor and other factors with promoting coagulation can lead to the occurrence and aggravation of dic, since it cannot be promptly removed [ ] . . the onset and enhancement of fibrinolysis. massive clotting factors and platelets were depleted in the extensive formation in vivo microthrombus. thrombin promoted the conversion of fibrinogen into fibrous protein [ ] . simultaneously, it activated fragments which dropped in the formation of activated factor xiii, factor xa and factor xiia. all of them can activate plasmin, and it enhanced fibrinolysis with tpa, one factor released by damaged blood vessel endothelium [ ] . fibrinogen and fibrous protein were degraded after plasminogen activation to generate the corresponding fdps, one factor inhibit blood coagulation and also block platelet aggregation [ ] . taken together, the above process exacerbates bleeding initiated by coagulation factors depletion and platelet deficiency. coagulopathy, characterized by prolongation of blood clotting time, tendency of hemorrhage, or even dic, were commonly found in severe hepatitis patients. it may cause uncontrolled external or internal hemorrhage. as common clinical symptoms, the external hemorrhages include gingivale or nasal mucosal bleeding, skin petechiae, the punctures or injection-site ecchymosis, and so on. the internal hemorrhages include esophagogastric varices, intracranial, subcutaneous, and muscle interval bleeding. except for esophagogastric varices hemorrhage, the other internal hemorrhages rarely occur in those patients. in addition, massive hemorrhage from esophagogastric varices is a serious medical emergency, can potentially cause death and cardiac arrest if proper medical treatment is not received quickly [ ] . when dic occur, a wide range of thrombogenesis in microvasculature can cause circulatory collapse, characterized by low blood pressure and shock. microcirculatory dysfunction occur after microthrombosis producing, resulting in hypofunction of multiple organs (e.g. kidney, liver, lung and pancreas), which perform from dysfunction to failure, with illness progressing [ ] . crushed by the fibrous protein in the vessels, red blood cell destruction can lead to intravascular hemolysis. at the early stage of disease, it shows hypercoagulability. the blood in the needle is easy to coagulate, when getting blood sampling from the vein. after that, it comes with the stage of consumed hypocoagulation. the consuming of a great number of blood coagulation factors leads to significant tendency of hemorrhage [ ] . it is difficult to stop bleeding in many parts of the body, including visceral organs, operative sites, injection sites, puncture sites, and mini-invasiva sites [ ] . when the third stage, namely secondary fibrinolytic stage comes, a great number of the blood coagulation factors have already been used up, resulting in severe bleeding under the condition of low coagulation. shock, acidosis and mods make the patient's condition continue deteriorating, and are also the main reasons for death [ ] . the latest study indicates that thrombosis is also a noticeable state in cirrhosis and end-stage liver disease. portal thrombosis and peripheral vein thrombosis (e.g. deep vein thrombosis and lung embolism) are commonly seen. the deep vein thrombosis is more danger than lung embolism. anyway, the incidence rate of thrombosis in cirrhosis and end-stage liver disease is still very low. the clinical symptoms depend on the embolizing position after the thrombosis occurs [ ] . presently blood clotting factors test is the most maturely and frequently used test in the term of blood coagulation function in the world. the indicators including prothrombin time (pt), international normalized ratio (inr) and prothrombin time activity (pta) have always been chosen in the patients with severe hepatitis. prothrombin time (pt) reflects whether there are anticoagulant substances in the extrinsic blood coagulation system and blood circulation or not. the elongation of prothrombin time (pt) presents the declined activity of several blood clotting factors including factorii (fii), factorv (fv), factor vii (fvii), factorx (fx) or the existence of anticoagulant substances. in severe hepatitis patients, the incidence rate of prothrombin time (pt) elongated can reaches to %, thus it is regarded as a sensitive and frequently used indicator in the term of liver function [ ] . another index international normalized ratio (inr), a reckoned ratio calculated from prothrombin time (pt) and international sensitivity index (isi), making prothrombin time (pt) between different laboratories and different reagents comparable, is an international general indicator. the guides about acute-on-chronic liver failure and acute liver failure take the index, inr ≥ . , as one of the most significant diagnostic criteria in the american association for the study of liver diseases (aasld) and the asian pacific association for the study of liver (apasl). international normalized ratio (inr) can also be used as a monitor on blood coagulation function [ ] . hepaplastin test (hpt), reflecting not only blood coagulation mechanism in hepatitis patients but also the function of hepatocytes to synthetize vitamin k dependent clotting factors including factorii (fii), factor vii (fvii), factorix (fix), factorx (fx) synthetically, is a test about liver reserve function; but this indicator can't reflect the change of factorv (fv). when severe hepatitis occur, the function of liver to synthetize above-mentioned clotting factors declines and the time of hepaplastin test (hpt) elongates. with illness progressing, hpt continues elongating. survivors undergoing effective therapies can have gradual recovery in the time of hepaplastin test (hpt). so this test is regarded as the specific test of liver diseases or the optimal indicator reflecting liver reserve function [ ] . the severity of liver damage is positively correlated with the decline degree of prothrombin time activity (pta): the more severe damage occurs in hepatocytes, the more significantly prothrombin time activity (pta) will decline. therefore prothrombin time activity (pta) < % and total bilirubin (tbil)> μmol/l have always been used as the main laboratory indicators to diagnose severe hepatitis domestically. pta < % is also regarded as diagnostic criterion of blood coagulation dysfunction in the guideline of acute-on-chronic liver failure in the asian pacific association for the study of liver (apasl) [ ] . in severe hepatitis, total bilirubin (tbil), total cholesterol, prothrombin time activity (pta) and complications (e.g. rectory hyponatremia, hepatic encephalopathy, hepatorenal syndromes and so on) are all independent risk factors to evaluate prognosis. the lack of any factors including factori (fi), factorii (fii), factorv (fv), factorvii (fvii), factor x (fx), can lead to the decline of prothrombin time activity (pta). moreover the half-life time of those factors are extremely short, factorii (fii) - h, factorv (fv) - h, factorv (fv) - h, factorv (fv) - h, respectively. it means that when hepatocytes suffer from severe serious damage and necrosis in severe hepatitis, prothrombin time activity (pta) will have dramatic decline just in a few days. as a result, prothrombin time activity (pta), characterized by significant advantages in evaluating patients condition and judging prognosis over other laboratory indicators, has been widely used. elongation of aptt prompts the lack of any clotting factor belonging to intrinsic coagulation system or the existence of anticoagulant substances. the incidence of the elongation of aptt reaches to - % in severe hepatitis patients [ ] . fxii:c reflects liver synthetic function. fvii, characterized by the shortest halftime - h, is the first one to be affected when facing liver synthetic dysfunction. on the contrary, fv:c, characterized by a relatively long half-time, is one of the latest factors to be affected and is correlated with the degree of liver damage. it prompts severe liver failure, bad prognosis and even easy death when plasma levels of fv:c under %. some literatures report that fv:c and pta can be used as significant prognostic factors in liver failure and significant screening indicators in liver transplantation. however, the indicators-fxii:c and fv:c-are not listed as routine examination items. they are just selected on the condition of illness demand [ , ] . the main test about anti-clotting factors is the determination of antithrombin iii activity (at-iii:a). in the state of pathosis, the decline of antithrombin iii activity (at-iii:a) is not parallel with the decline of antithrombin iii (at-iii) content, namely the depletion of antithrombin iii activity (at-iii:a) more apparent. owe to this, it has more clinical value to determine antithrombin iii activity (at-iii:a) rather than antithrombin iii (at-iii) content. moreover, anti-clotting factors tests include the determination of protein c activity (pc:a) as well [ ] . serum levels of fdps are very low in normal people. significantly elevating of fdps indicates the existence of hyperfibrinolysis and reflects the occurring of dic indirectly. there are many assay methods including immunization fi test (namely latex particle agglutination test, normal titer< : ), fdps flocculation test, radial immunodiffusion staphylococcal clumping test, indirect hemagglutination inhibition test, enzyme-linked immunosorbent assay (elisa) and so on. if the serum levels of fdps elevate, it indicates acute dic may occur [ ] . plasma protamine paracoagulation test ( p test) and ethanol gel test (egt) reflect the soluble fibrin complexes in plasma. soluble fibrin complexes, combination of fdps and fibrin monomer, can't be solidified by thrombase. but protamine is able to make the complexes isolate and then fibrin monomers separate out again. the paracoagulation test means self-polymerization between fibrin monomers and fdps, then forming macroscopic flocks. ethanol gel test (egt) has the same principle with plasma protamine paracoagulation test ( p test), whereas the former has a lower positive rate. the two methods may have false negative results and false positive results. in contrast, egt has a relatively lower sensitivity, while p test has a relatively lower specificity. for example, relative small molecular mass of the shreds of fdps may lead to negative result using p test. so it is more valuable to compare the two indicators simultaneously [ , ] . euglobulin, a protein (including fibrinogen, plasminogen and other activins except for fibrinolysis inhibitor) separating out from plasma in acid circumstances, can be used to determine whether levels of plasminogen activators increase or not [ ] . when hyperfibrinolysis occurs, plasma levels of plasminogen decline, plasma levels of plasmin increase, and euglobulin suffer from accelerated dissolution by a great number of plasmin. the normal value of euglobulin lysis time (elt) is above h. that is to say dissolution within h means the occurrence of hyperfibrinolysis. domestic population data report the positive rate of elt test reaches - . %, when dic occur [ ] . furthermore there are other tests about fibrinolysis including tissue-type plasminogen activator test (t-pa), plasminogen activator inhibitor test (pai), plasminogen antigen test (plg:ag), plasmin activity test (pl:a), α -plasmin inhibitor test (α -pi) and so on [ , ] . blood platelet count reflects the absolute number of platelet in peripheral blood circulation. according to the reports at home and abroad, platelet count have a significant decline in patients with chronic severe hepatitis. moreover, studies on domestic population find that platelet count ranges from × /l to × /l in peripheral blood of severe hepatitis patients. some studies have already compared the platelet count among early stage, typical stage and late stage in severe hepatitis, and they have turned out to be × /l, × /l and × /l respectively. as a result, it indicates that platelet count is positively correlated with the severity of hepatitis [ ] . except for platelet count, other routine indicators including mean platelet volume (mpv), plateletcrit (pct) and platelet distribution width (pdw) also have significant reference value. when severe hepatitis occurs, the above-mentioned three indicators will be dramatically lower, and they have the tendency to continue declining with illness progressing. what's more, platelet quality tests include platelet aggregation rate, platelet factor validity tests and blood clot retraction test (table . ) [ ] . diagnosis of blood hypercoagulability in the early stage of dic relies on several molecular marks including plasma thrombinogen segment + (f + ), thrombinantithrombin complex (tat) and d-dimer, due to no significant changes in general laboratory tests. this stage is characterized by the elevated levels of those three molecular marks, and levels will increase more significantly with the occurrence of typical dic symptoms. dynamic monitoring of above-mentioned indicators is helpful for early diagnosis of dic [ ] . the stages are mainly characterized by the decline of blood clotting factors (including factorv (fv), factorvii (fvii), factorxii (fxii), factorix (fix), fac-torx (fx)), platelet count and plasminogen, and increasing of fibrinolytic the elevating levels of fibrin(−ogen) degradation products (fdps) and d-dimer; the shortening of euglobulin lysis time (elt) and the positive reaction of p test [ , ] . with the occurrence of dic, there will be a wide range of blood coagulation and highly-activated fibrinolysis in the patient's body [ ] . what's more, abnormal increased soluble fibrin monomer and fdp fragments will exist in plasma [ ] . the level of soluble fibrin monomer complex (sfmc), a complex combined fibrin monomer with fdp, is determined by p test. the p test shows positive with the occurrence of secondary fibrinolysis, whereas it shows negative with the occurrence of primary fibrinolysis. this means p test is negative when there is no blood coagulation. domestic population data report that the positive rate of p tests reach - %. however, the test can't be used as the ideal diagnostic indicator for dic, owing to many affected factors. false positive reactions are mainly found in the following conditions: gastrointestinal bleeding, massive hemoptysis, malignant carcinoma or blood sampling reserved improperly, whereas false negative reactions are usually found at the late period of the stage of secondary fibrinolysis [ , ] . as mentioned above, plasma thrombinogen segment + test (f + ), thrombinantithrombin complex test (tat) directly reflects production of intracorporeal thrombase, which increased in the early stage of dic. plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the d-dimer antigen, which reflects production of intracorporeal plasmin. d-dimer will have an apparent increase with the occurrence of secondary fibrinolysis, but d-dimer test shows negative when primary hyperfibrinolysis occurs. monitoring the above molecular markers dynamically is helpful to estimate therapeutic efficacy and guide treatment [ ] . as mentioned above, clinical manifestations of blood coagulopathy in patients with severe hepatitis are lack of specificity. the most common manifestation is bleeding, not only little hemorrhage from superficial sites, but also massive hemorrhage from internal sites, such as esophagogastric varices [ ] . the diagnosis of severe hepatitis complicated with blood coagulopathy is mainly based on the results of laboratory tests and clinical manifestations [ ] . according to current guidelines, basic diagnose conditions of dic contain the following points [ ] . . severe or multiple bleeding tendency. . microcirculation collapse or shock which is difficult to explain using protopathy. . a wide range of embolism in skin and mucosa, focal ischemic necrosis and ulcer, or unexplained dysfunction of kidney, lung, brain. . anticoagulant therapy is effective. if severe hepatitis b patients have one of the above-mentioned points except for ( ) and exhibit blood coagulating easily or prothrombin time (pt) shortening over s simultaneously, it can be considered as the early stage of dic in which the tendency of bleeding is not obvious. in addition, if severe hepatitis b patients have two of the above-mentioned four points, dic can be considered as the preliminary clinical diagnosis. furthermore, it can be definitely diagnosed when combined with the aforementioned items of laboratory tests ( table . ). firstly, we should use anti-viral therapy as a mainly method to treat primary disease of severe hepatitis b [ ] . then, we must eliminate the incentive, maintain the balance of water and electrolyte, and correct hypoxia and acidosis. it is very important to focus on massive upper gastrointestinal hemorrhage and disseminated intravascular coagulation when treating coagulation disorders [ ] . gastrointestinal bleeding, including esophageal varices bleeding and non-bleeding esophageal varices, were correlated with coagulation dysfunction and portal hypertension. prevention is still focused on improving the coagulation function, including adequate vitamin k supplements, coagulation factors, fibrinogen, fresh plasma or platelets supplements. it is particularly critical to control diet in patients with severe hepatitis. in which, light and easily digestible diet was recommended, but rough, hard and too greasy food was prohibited. for these patients, appropriate antacids can be used to protect the gastric mucosa [ ] . . general treatment: bed rest is necessary, meanwhile vital signs were closely monitored. the patients can eat liquid diet when bleeding mildly or having no active bleeding. however, abrosia is required when the patients have a heavy bleeding. . fluid resuscitation: firstly, intravenous access should be established rapidly in patients with gastrointestinal bleeding, then, the patients were given intravenous infusion of normal saline, lactated ringer's solution, plasma, whole blood or plasma substitute [ ] . . vaso-active agents: vaso-active agents such as dopamine and alamin may be given to maintain normal blood pressure, if blood circulation is still not stable after fluid resuscitation [ ] . . hemostatic: if mucosal bleeding was caused by portal hypertension, oral administration of norepinephrine and ice normal saline can promote mucosal vascular contraction and hemostasis. in addition, oral administration of yunnan baiyao may be effective [ ] . . acid suppression therapy: the proton pump inhibitors, such as omeprazole, pantoprazole and esomeprazole, are commonly used to inhibit gastric acid secretion [ ] . . reduction of portal pressure: patients with severe hepatitis complicated by gastrointestinal bleeding often accompanied with portal hypertension, so it may be considered to give them drugs to decrease portal pressure. especially in patients with portal hypertension gastropathy, reduction of portal pressure is more important than acid suppression therapy [ ] . . compression hemostasis via using three-chamber double-balloon catheter: after the above treatment, if there is still active bleeding in patients with bleeding esophageal varices, it can be considered to use three-chamber double-balloon catheter [ ] . . endoscopic hemostasis: if the above treatments have no effect in upper gastrointestinal bleeding, endoscopic hemostasis, including endoscopic hemostatic agents spray, endoscopic ligation or endoscopic injection sclerotherapy may be used [ ] . . others: surgery or interventional therapy may be considered, if internal medicine therapy is ineffective [ ] . firstly, we should treat original disease, then improve coagulative function in those patients. in addition, prevention and control of infection, correction of electrolyte disturbance, avoiding the hemorrhage and reduction of allergies and transfusion reactions should be considered [ ] . the key is early diagnosis and early treatment. . anticoagulant drugs: low molecular weight heparin is the most commonly used drug in earlier stage of dic. it is recommended to periodic test blood routine and coagulation, and dynamically observe coagulation status during medicine therapy. others such as dextran, anti-platelet aggregation ticlopidine, salvia injection, urokinase may be effective, but it should be support by more evidence-based medicine [ ] . . plasma and blood products: during the process of dic formation, the patients should be transfused with fresh plasma, each - ml/kg. when they developed the stage of secondary fibrinolysis, prothrombin complex containing coagulation factors, cryoprecipitate and platelets can be supplemented due to a large consumption of coagulation factors [ ] . . others: such as hemodialysis, anti-fibrinolytic -aminocaproic acid and tranexamic acid should be supported by more evidence-based medicine. hepatorenal syndrome (hrs) is a common and serious complication occurring in patients with decompensated cirrhosis and liver failure, who have overt circulatory dysfunction. the -year incidence of hrs in patients with ascites is about % [ ] . hrs may predict a poor prognosis in spite of it being functional reversible [ , ] . the clinical characteristics of hrs were first described in [ ] . in , hrs was defined by a group of international investigators as a progressive renal dysfunction that occurred in severe liver diseases, with features of prerenal failure (low urine sodium concentration and hyperosmolar urine) but without any improvement following volume expansion [ ] . the international ascites club (iac) developed hrs definition in , as a syndrome that occurs in patients with cirrhosis, portal hypertension and advanced liver failure, characterized by impaired renal function with marked abnormalities in the arterial circulation and activity of endogenous vasoactive systems [ ] . hrs is a potentially reversible syndrome that occurs mainly in patients with liver cirrhosis, ascites and all kinds of liver failure [ ] . it's clinical features include impaired renal function, marked changes in cardiovascular function and over activity of the renin-angiotensin systems and the sympathetic nervous. progressive hrs with severe renal vasoconstriction is able to cause a decrease of gfr [ ] . clinically, hrs can be divided into two types ( and ). type- , so-called acute hrs is a rapid progressive form of renal failure defined by doubling of the initial serum creatinine concentrations to a level higher than mmol/l ( . mg/dl) or a % reduction of the first h creatinine clearance to < ml/min within weeks [ ] . it appears mainly in patients with acute liver failure, but often develops after a precipitating event, such as bacterial infections (especially spontaneous bacterial peritonitis, sbp). the prognosis of type is poor with the median survival about month [ ] [ ] [ ] . type- , so-called chronic hrs is a steady or slowly progressive form of renal failure defined by serum creatinine from to mmol/l or from . to . mg/ dl [ ] . type- hrs is mostly related to refractory ascites. survival of patients with type- hrs is generally around - months, which is better than that of patients with type- hrs but shorter than that of non-azotaemic cirrhotic patients with ascites. patients with type- hrs tend to develop type- hrs while infections or other trigger events occurred [ , [ ] [ ] [ ] ]. portal hypertension is the essential factor of haemodynamic changes, which resulted from the development of cirrhosis associated with distortion, compression and even obliteration of the hepatic sinus and vessels [ ] . in patients with portal hypertension, bacterial translocation is increased and intrahepatic hypercontractile stellate cells activated [ , ] . this overall increased resistance to portal hypertensional causes increased local production of various vasodilators such as nitric oxide, leading to splanchnic vasodilation [ , ] . there are several other factors contributing to the splanchnic vasodilation, including hyporesponsiveness of the splanchnic vessels and mesenteric vascular hyperplasia [ ] . in addition, portal hypertension per se can cause renal vasoconstriction by activating sympathetic nervous. for example, when tips is used to reduce portal hypertension and improve renal blood flow, a corresponding reduction in sympathetic nervous activity has been observed [ , ] . as a result of splanchnic vasodilation, blood is accumulated in the splanchnic vascular bed just like a splanchnic steal syndrome [ ] . the combined effect leads to reduction in the effective arterial blood volume (relative hypovolemia) causing a relative inadequacy in the systemic circulation, which triggers a hyperdynamic circulation in these patients [ , ] . vasodilatation induces activation of neurohumoral systems including the reninangiotensin-aldosterone system (rass); sympathetic nervous system (sns); and non-osmotic release of antidiuretic hormone (adh) [ ] . relative hypovolemia initially causes sodium and water retention, increases intravascular volume, and simultaneously increases cardiac output. as cirrhosis progresses, vasodilatation aggravates, which activated vasoconstrictive systems, causing renal vasoconstriction and decreased renal blood flow [ , ] . local release of potent vasodilators such as nitric oxide (no) leads to splanchnic visceral vasodilation, as well as enables the splanchnic circulation against a variety of vasopressor agents, including norepinephrine, vasopressin, angiotensin ii and endothelin [ ] . the resistance of the splanchnic circulation to these vasopressor agents makes the control of arterial pressure during cirrhosis dependent on the extra-splanchnic effects produced by the endogenous vasoconstrictor systems. the role of vasoconstrictors in maintaining haemodynamic stability becomes pivotal as arterial vasodilatation increases during cirrhosis, which makes clear why cirrhotic patients with hrs are prone to develop renal, hepatic and cerebral vasoconstriction [ ] . the reduction of effective arterial blood volume leads to the compensatory activation of various vasoconstrictor systems. normally, the kidneys increase the production of renal vasodilators including prostaglandins and kallikrein to maintain blood flow. however, renal vasodilator production is generally reduced in patients with cirrhosis, thus contributing to renal vasoconstriction. this type of renal hypoperfusion further increases the production of various intrarenal vasoconstrictors such as angiotensin ii and endothelin, causing further decline of renal haemodynamics and renal function, occasionally accompanied by glomerular ischaemia and mesangial constriction [ ] . when blood pressure fluctuates, renal auto-regulation regulatory mechanisms initiate to make sure that the kidneys receive a relatively constant blood supply. when the critical threshold is below mmhg, renal blood flow is proportional to renal perfusion pressure which, in turn, is dependent on mean arterial pressure. in cirrhosis, with the development of liver disease in patients of cirrhosis, the renal auto-regulation curve gradually shifts to the right -which means as liver disease advances, the renal blood flow gradually decreases for each given renal perfusion pressure [ ] . furthermore, lumbar sympathetic blockade increases renal blood flow in patients with hrs, suggesting that the renal sympathetic activity is involved in this outgoing hepatorenal arm. insufficient cardiac output is considered one of the leading causes for renal hypoperfusion in patients with hrs in recent years. despite control of infection, the cardiac output of cirrhotic patients with sbp who developed progressive renal failure was lower than that in similar sbp patients without renal failure. similarly, when patients with non-azotaemic cirrhotic patients who developed hrs are compared with similar patients who did not, it is observed that low cardiac output and high plasma renin activity (pra) were independent predictors of hrs. in addition, in patients developing hrs, the evolvement of circulatory dysfunction leading to arterial hypotension and renal failure occurs in the setting of a continued decline in cardiac output and increase in pra. therefore, effective hypovolaemia occurs when cardiac output decreases, resulting in renal hypoperfusion and hrs [ ] . to summarize, the principal mechanisms leading to renal vasoconstriction include systemic circulation changes, accompanying portal hypertension which are characterized by decreased peripheral vascular resistance with subsequent vasodilatation, hyperkinetic circulation and the activation of compensatory mechanisms, i.e., the sns, raas, and adh. with the progression of cirrhosis, the combined effective of all the above factors will result in the gradual deterioration in renal function. any event that leads to a sudden deterioration in hemodynamics can cause a rapid renal dysfunction, precipitating type hrs [ ] . the diagnostic criteria for hrs have been first defined by iac in [ ] . the main findings include reduced glomerular filtration (creatinine clearance) less than ml/min or serum creatinine increased more than μmol/l after excluding the other causes of renal dysfunction. however, estimation of renal function by using creatinine clearance is not reliable, because these patients have lower levels of serum creatinine and higher renal tubular creatinine secretion compared with filtered creatinine. furthermore, it is often incomplete for the collection of a h urine. iac developed the new definition and diagnostic criteria for hrs in , which ( ) excludes creatinine clearance because of its inaccuracy of renal function estimation and the complicity to perform; ( ) includes renal failure at the time of combined bacterial infection (but absence of septic shock), indicating that hrs can be diagnosed before antibiotic treatment; ( ) determines by using albumin for plasma volume expansion better than saline. ( ) excludes minor diagnostic criteria (urinary indices) because of its poor sensitivity and specificity for the diagnosis [ , , ] . the diagnostic criteria of hrs for patients with liver cirrhosis are as follows: . cirrhosis with ascites; . serum creatinine > mmol/l ( . mg/dl); . no improvement in serum creatinine (decrease to a level of ≤ mmol/l or . mg/dl) after at least days of diuretic withdrawal and volume expansion with albumin. the recommended dose of albumin is g/kg body weight/day up to a maximum of g/day; . absence of shock; . no current or recent treatment with nephrotoxic drugs; . absence of parenchymal kidney disease as indicated by proteinuria > mg/ day, microhematuria (> red blood cells/high power field) and/or abnormal renal ultrasonography [ , ] . there are some other causes of renal failure in patients with cirrhosis that were not included, such as membranoproliferative glomerulonephritis and/or iga nephropathy in patients with chronic liver diseases. these chronic forms of kidney disease can cause acute rises in serum creatinine. determining whether it is a potential kidney disease or hrs that causes a sudden increase in serum creatinine in patients with cirrhosis and chronic kidney disease could be difficult [ ] . naturally, liver transplantation is the only rational solution in cases of advanced liver disease while it is also the treatment of choice for both type- and type- hrs. as calcineurin inhibitors (ciclosporin and tacrolimus) may induced gfr impairment, it is recommended to delay their administration until a partial recovery of renal function is recorded, usually - h after transplantation [ ] . clinically, the haemodynamic associated with hrs as well as neurohormonal abnormalities fade away within one month of transplantation, and the patients recover their ability to excrete sodium and free water. compared with patients without hrs, patients with hrs tend to have more complications, take more days in intensive care units and have higher in-hospital mortality rates after liver transplantation. nevertheless, their -year survival rate is acceptable ( % vs. - % in liver transplant patients without hrs [ ] . the primary limitation of liver transplantation is that most patients with type- hrs die before transplantation due to the shortage of donor liver and their extremely short survival time. reference to the model of end-stage liver disease (meld, including scr, tbil, inr) for organ prioritisation has partially addressed this problem, since patients with hrs have a high priority on the waiting list. in addition, the use of vasoconstrictors and albumin in the treatment of type- hrs can improve survival rate of these patients, and increase the likelihood of their transplantation [ ] . in patients with advanced liver disease and the renal insufficiency, simultaneous liver and kidney transplant (slkt) is taking into consideration. however the reversibility of renal function in some patients when they receive slkt should be taken into account. therefore, to ensure allocation of transplants only to those truly in need, the transplant community proposed an evaluation algorithm in , whose purpose is to determine the presence of kidney disease with structural damage (preferably on biopsy) before giving slkt. in the case of chronic kidney disease, a decreased creatinine clearance at ml/minute or less is considered an indication of slkt. slkt should not be performed for the patients with simple hrs, but for the patients with hrs who become dialysis dependent and without any recovery after to weeks of dialysis [ , ] . vasoconstrictors combined with albumin are the first line of therapy for type- hrs patients. it was recognized long time ago that the effective plasma volume was reduced when patients of advanced liver diseases complicated with hrs, and this led to many attempts to improve the patients' renal function by expanding their plasma volume, including a large dose of albumin or saline perfusion. with the advent of safer compounds including terlipressin, a vasopressin analogue with longer activity, and the α -agonist midodrine combined with octreotide the analogue, vasoconstrictors is widely used in the patients with hrs. these vasoconstrictors are able to ameliorate vasodilatation while increasing effective arterial blood volume, improving renal vasoconstriction and improving renal flow. in order to further increase effective blood volume, vasoconstrictors have been used in conjunction with intravenous albumin. the clinical results from uncontrolled studies including patients with hrs (type- , ) showed that a total of % were observed complete response (mostly defined as a decrease in scr to . mg/dl). interestingly, once the treatment is stopped, hrs relapses only in a few "responders" [ , ] . there were several randomized controlled trials (rcts) published, suggesting that terlipressin was associated with an increase in gfr compared with albumin alone or with a placebo. the rate of hrs reversal in the terlipressin group was higher than that in the control group ( % vs. %). as survival rate was not improved in the two largest rcts, liver transplantation is still the preferred treatment for hrs, but terlipressin seems to serve as a "bridging" treatment. two recent small, open-label rcts suggested that the incidence of hrs reversal and the rate of side effects showed no significant difference between the two groups of norepinephrine and terlipressin [ , , ] . the initial dose of terlipressin recommended in many studies ranged from . to mg per - h [ , ] . if the creatinine level did not decrease by % on the third day, the dose could be increased to mg every h or mg/days by continuous intravenous infusion, respectively. in some studies, the daily dose of albumin was generally - g by a load of g/kg body weight. some mentioned central venous pressure to establish albumin doses and to prevent body fluid from overloading. this treatment was maintained until hrs is reversed, but did not exceed weeks [ ] . about % of patients relapsed after the treatment withdrawal. however, these patients should be given repeated treatment with terlipressin, which is often effective [ ] . several studies have evaluated the role of transjugular intrahepatic portosystemic stent-shunt (tips) in hrs. these studies show that tips help decreasing in scr in most patients, even in a minority of organic renal failure, but it is slower compared to those obtained using terlipressin combined with albumin [ ] . recrudescence of hrs is rare provided the shunt remains patent, while hepatic encephalopathy often comes. it is worth noting that the vast majority of patients included in these studies suffered from alcoholic cirrhosis, many of whom have active alcoholism, and therefore the improvement observed may be caused by the improvement in an acute-on-chronic process. in addition, since all these studies excluded patients with a child-pugh score ≥ , resulting in a lack of data, the efficacy of tips should be further explored in rcts [ ] . extracorporeal albumin dialysis molecular adsorbent recirculation system (mars) is designed for making clearance of water-soluble cytokines (i.e., il- ) and albumin-bound toxins (i.e., bile acids) which is implicated in the pathogenesis of hrs. two studies showed that mars was ineffective in improving survival rate and emic haemodynamics in type- hrs [ , ] . another clinical observation including patients with type- hrs reported a rate of complete renal response of % [ ] . extracorporeal albumin dialysis (ecad) reduces serum creatinine levels, but it is not clear whether this effect is due to a real improvement of renal function or merely a filtration process. several studies demonstrated that patients' systemic haemodynamics improved after ecad, manifested as an increase in systemic vascular resistances and arterial pressure, as well as a decrease in cardiac output, pra and levels of norepinephrine. however, there were too few studies on the effect of ecad on survival in type- hrs patients to draw any definitive conclusions [ , ] . in addition, ecad is very expensive and therefore not suitable for wide and rapid clinical application [ ] . the treatment of type- hrs should take into account the survival rate as well as controlling the ascites. both hrs- and hrs- are indications for the tips treatment. the therapeutic effect of tips is excellent for its better controlled of complications of portal hypertension compared with other treatments. tips have been reported not only to improve renal function in patients with type- hrs but also to treat refractory ascites in patients with type- hrs [ ] [ ] [ ] . the contraindications to the creation of tips are shown in the followings [ ] . • contraindications to placement of a tips: - there were only a few studies evaluated the role of tips in type- hrs and the number of cases was quite low. in most patients, tips could decrease scr, even in some with organic renal failure [ ] . hrs recurrence is rare as long as the shunt remains patent, but hepatic encephalopathy often occurs [ ] . nine patients were followed-up for month after the treatment of tips in a study, eight cases were found with decreased scr decreased and notably controlled ascites. four patients died, two of them died within month, the other two died at months and months respectively. the remaining five patients survived for a long time. although tips can be used in improving refractory ascites which often contributes to type- hrs, data on the effect of tips on survival are still insufficient. therefore, the efficacy of tips should be further explored in randomized controlled trials (rcts) [ ] . the information about combining albumin and vasoconstrictive agents treated in type- hrs is limited. only a few patients with type- hrs have been specifically treated with terlipressin and albumin. in one clinical study, patients with hrs- were assigned to receive this treatment and of them achieved improvement of renal function. however after the treatment withdrawal, hrs patients showed relapsed during the follow-up. the most common side effects during terlipressin therapy are cardiovascular and ischemic and reported as an incidence of nearly %. the high recurrence rate of hrs after terlipressin and albumin treatment discontinuation suggests that they are less effective in treating type- hrs compared to type- hrs [ ] . prevention of hrs is important because it develops at a constant frequency in cases of spontaneous peritonitis (sbp) and advanced liver disease [ , ] . it becomes possible to prevent hrs if sbp is diagnosed and treated promptly [ ] . according to current data, using albumin in combination with antibiotics for the treatment of patients with sbp seems to be warranted but only for those with jaundice or renal dysfunction. the prophylactic use of antibiotics in cirrhosis with gastrointestinal bleeding also seems to be necessary, because the use of antibiotics contributes to reducing incidence of infection and rebleeding whereas improving survival rate. furthermore, the incidence of hrs in sbp patients decreases by albumin administration, and prevention of hrs can also be related to increased survival. the recommended dose of albumin is . g/kg body weight on the first day then g/kg body weight on the third day, a maximum of g and g, respectively. albumin administration is strongly recommended in sbp patients with serum bilirubin levels higher than . mmol/l ( mg/dl) or serum creatinine more than . mmol/l ( mg/dl). a placebo-controlled rct that enrolled the patients with low (< . g/l) ascites protein who also had advanced liver diseases or "renal dysfunction"(defined as scr ≥ . mg/dl or blood urea nitrogen≥ mg/dl, or serum sodium level ≤ meq/l) suggested that oral norfloxacin contributed to a reduced hrs incidence within year ( % vs. %) and an improvement in survival at the end of months [ ] . norfloxacin may ameliorate or prevent vasodilatation by reducing bacterial translocation and overt infections, as well as suppressing plasma renin activity, thereby prevent these patients from developing hrs. the concept that the severity of the clinical course of patients with cirrhosis complicated with serious bacterial infection is related to the degree of an impairment of circulatory function, which has led to new and effective approaches in the prevention and treatment of these complications. in patients with severe hepatitis, multiple causes may lead to disorders of internal environment, mostly manifesting fluid and electrolyte imbalance as well as acidbase imbalance, usually resulting in deterioration, greater complexity and even death. accurately recognizing the occurrence of severe hepatitis with complications such as fluid and electrolyte imbalance and/or acid-base imbalance, and therefore giving appropriate treatment to maintain balance of internal environment, is of great importance for improving prognosis of the patients [ ] . water is the major component of human body. electrolytes are substances that dissociate in solution to form charged particles, orions. body fluid comprise mainly of water and electrolytes and electrolytes comprise mainly of na + , k + , ca + , mg + , cl − , hco − , hpo − and so − . the primary function of electrolyte include: ( ) to maintain osmotic pressure and acid-base balance of body fluids; ( ) maintain nerve, muscle, cardiac cells resting potential, involved in the formation of action potentials; ( ) involved in metabolism and physiological activities [ ] . body fluid include intracellular fluid and extracellular fluid, the latter can be divided into plasma and interstitial fluid. intracellular and extracellular fluid differ in ion components. na + is major cation in extracellular fluid and its main anions are cl − and hco − . k + is major cation in intracellular fluid and its major anion is hpo − . the total number of ions in body fluids is called osmolality, its unit is mosm/l. if the osmolality on both sides of a semipermeable membrane is not equal, water moves toward the side with the higher osmolality. this phenomenon is called osmosis [ ] . osmosis of water can be opposed by applying a pressure across the semipermeable membrane in the direction opposite to that of the osmosis. the amount of pressure required to oppose the osmosis is defined to be osmotic pressure. in spite of various solute concentrations are different in extracellular and intracellular fluid, the osmotic pressure remain equal. normal plasma osmotic pressure is - mosm/l. steady osmotic pressure is the basic guarantee to maintain the fluid balance across the cell membrane. multiple mechanisms in nervous and hormonal system are involved in the regulation of body fluid and electrolyte balance [ ] . ( ) there exists sensation of thirst in central nervous system, which plays an important role in regulating body water. ( ) there is a powerful feedback system for regulating plasma osmolarity and sodium concentration that operates by altering renal excretion of water independently of the rate of solute excretion. a primary effector of this feedback is called antidiuretic hormone (adh) which plays an important role in regulation of renal concentration and dilution to maintain the body fluid homeostasis. ( ) reninangiotensin-aldosterone system (raas) is an important regulator of sodium reabsorption and potassium secretion by the renal tubules. human body fluid environment must be suitable ph value for maintaining normal metabolism and physiological function, under normal conditions, human body take in acidic and basic food and drinking water, produce acids and bases during metabolism and eliminate acidic or basic substances by the kidneys and lungs. in the plasma, the normal ph value ranges from . to . with an average value at . . the regulation of the acid-base balance is accomplished by the buffer system of the body fluid, the respiration of the lungs and the excretion of the kidney [ ] . ( ) blood buffer system is composed by a weak acid and its corresponding buffer base, includes bicarbonate buffer system, phosphate buffer system, plasma protein buffer system, hemoglobin and oxygen synthetic hemoglobin buffer system. ( ) the role of the lung in acid-base balance is to adjust the concentration of plasma carbonic acid by changing the amount of co , so that the ratio of hco − and h co in the plasma is close to normal, so that the ph is relatively constant. ( ) the major role of the kidneys in maintaining acid-base balance is to conserve circulating stores of bicarbonate and to excrete h + . the kidneys maintain ph by increasing urinary excretion of h′ and conserving plasma hco − when the blood is too acidic, or increasing urinary excretion of hco − , and decreasing urinary excretion of h + when the blood is too alkaline. patients with severe hepatitis are prone to develop water retention, with the main manifestation of seroperitoneum (ascites) as well as body weight gain. with the acatharsia of water becoming more serious, oliguria and edema of lower extremities occur. sbp (spontaneous bacterial peritonitis) can also occur, which is manifested with symptoms such as fever and abdominalgia. several factors contribute to ascites include an increase in capillary pressure due to portal hypertension, obstruction of venous and lymph flow through the liver, decrease in colloidal osmotic pressure due to impaired synthesis of albumin by the liver, salt and water retention by the kidney [ ] . some theories have been used to explain the increased salt and water retention by the kidney. because of vasodilation or an actual loss of fluid into the peritoneal cavity, the effective blood volume maybe reduced, which may in turn decrease the renal blood flow leading to a lower glomerular filtration rate (gfr) and an activated rennin-angiotensin-aldosterone system (raas). the diagnosis of water retention depends on typical clinical symptoms such as ascites, pleural effusion, and edema of lower extremities, when the body begins to excess water, blood pressure is increased, which leads to many complications such as congestive heart failure and pulmonary edema. hyponatremia is a common complication in severe hepatitis patients, and always incorporate with the retention of water and sodium, but the total sodium can be decreased, normal or even increased, that is dilutional hyponatremia. in laboratory test, serum sodium is below . mmol/l. the mechanism of hyponatremia probably depends on the following factors: ( ) the decreased function of adh inactivation in liver brings adh increase, enhancing the reabsorption of water in renal tubule, which causes the formation of water retention. this is the main cause of dilutional hyponatremia. ( ) water retention brings about volume extension, causing the aldosterone secretion decrease, which leads to the sodium egestion increase in urine. ( ) some severe hepatitis patients frequently vomit, and can not eat, bringing about a major loss of body fluid and electrolyte. ( ) severe hepatitis patients, serum albumin reduces, combining with the factors such as poor appetite, anorexia, fasting or limit sodium, bring about a state of low permeability in the cell, which causes the extracellular na + moving into the cell. ( ) iatrogenic factors, exhaust potassium diuretic such as hydrochlorothiazide and furosemide and spironolactone have a strong role in the excretion of sodium, so a large number diuretic is liable to hyponatremia in ascites patients, and in the treatment of cerebral edema, a large infusion of mannitol may cause hyponatremia either [ ] . hyponatremia due to the osmotic pressure of extracellular fluid decreased, water moves to the cells, causing cell edema, especially brain edema. so the symptoms of nerve system are the main manifestation in hyponatremia patients [ ] . generally dilutional hyponatremia in severe hepatitis patients develops slowly and progressively, and the symptoms are often covered by primary disease symptoms, like weak, feeble, nausea, vomiting, lethargy, significant body weight increase, pale and moist skin and sometimes saliva, tears increase. improper treatment will bring about a sharp serum sodium decrease in the short term, such as serum sodium rapidly decreasing to below mmol/l, acute hyponatremia syndrome comes, the manifestation include convulsions, coma, hypotension, pulse narrowing, tachycardia, oliguria even respiratory arrest and death. if cerebral hernia happens, corresponding nerve location signs will follow. hypokalemia refers to the condition in which the concentration of potassium (k + ) in serum is less than . mmol/l. it could occur during the whole period in severe hepatitis patients and is more common in early metaphase of disease. hypokalemia can be the result from one or more following medical conditions: ( ) insufficient intake of potassium due to the poor appetite or anorexia in the patients with severe hepatitis. ( ) frequent vomiting leads to excessive loss of stomach acid, which causes alkalosis and extracellular potassium is transferred into cells. ( ) the reduce of effective circulating blood volume can cause to high aldosterone levels and excessive urinary losses of potassium. ( ) the decreased function of aldosterone inactivation in liver brings aldosterone increase, enhancing urinary losses of potassium. ( ) some medications such as diuretics can also cause urinary losses of potassium. the clinical syndromes of hypokalemia are related to the degree of the shortage of intra/extracellular potassium and disorders of other electrolytes and acid-base, but more depends on how soon it occurs. in the early time it shows muscle weakness, first in limbs, then develops to the torso and respiratory muscle. deficiency of potassium also can lead to weak peristalsis, poor appetite, sick and constipation in mild hypokalemia but abdominal distention and paralytic ileus in severe situation. in cardiac syndromes, it mainly presents atrioventricular block and arrhythmia which including premature ventricular contraction or atrial premature beats, sinus bradycardia, paroxysmal auricular tachycardia or junctional tachycardia, even ventricular fibrillation. in hypokalemia state an increasing shift of potassium from extracellular fluids into cells and an obligate loss of potassium from kidney can cause metabolic alkalosis and abnormal acidic urine. long-term hypokalemia also can lead to hypokalemic nephropathy with proteinuria and cylindruria syndrome. the changes of ecg [ ] : in the early stage, flattened t wave and an obvious u wave, st-segment depression can be found, qu interval is widen. in severe situation, a wide pr interval, low voltage, wide qrs interval and ventricular arrhythmia can occur. hyperkalemia refers to the condition in which the concentration of potassium (k + ) in serum is higher than . mmol/l. it is more common in the middle and late period of severe hepatitis. the mechanism of hyperkalemia: ( ) the most usual way lead to hyperkalemia is oliguria or uroschesis which are caused by renal dysfunction among the patients have severe hepatitis with hepatorenal syndrome. ( ) metabolic acidosis and na + -k + -atp enzyme inactive lead to a shift of potassium out of cells also contribute to develop hyperkalemia. ( ) long-term and high dose potassium-sparing diuretics applied during the treatment lead to hyperkalemia is not rare, and easy to get sudden death in patients. hyperkalemia mainly influences myocardium and skeleton muscle. the most dangerous situation is fatal arrhythmia. when the concentration of k + is higher than . - . mmol/l, there are peaked t waves. when it is over - mmol/l, pr interval is widen and p wave is flattened even vanish. when it is up to - mmol/l, t waves and qrs complex can evolve to sinusoidal shape and cardiac arrest. hyperkalemia is a common critical and severe symptom in clinic. when it happens, all of the potassium-sparing diuretics and potassium uptake should be stopped. at meantime, the treatment against the toxicities to myocardium and skeleton muscle should be taken to accelerate a shift into cells and potassium excreting. there also can happen hypocalcemia, which shows neuromuscular excitability, cardiac electrical instability and instable emotion. the concentration of calcium in serum under mmol/l is significant in diagnosis. hypomagnesemia can be found too. it mainly presents similar symptoms as hypocalcemia such as weakness, muscle cramps, increased irritability, tetany and chvostek positive. hypomagnesemia can cause cardiac arrhythmia, when it occurs, the concentration of magnesium is less than . mmol/l, and it should be urgently treated. severe hepatitis patients prone to acid-base imbalance [ ] , mainly to alkalemia. the main types of acid-base imbalance include respiratory alkalosis, metabolic alkalosis, respiratory alkalosis plus metabolic alkalosis, secondly include respiratory alkalosis plus metabolic acidosis, triple acid-base disorders (tabd), metabolic alkalosis plus metabolic acidosis [ ] . history and clinical manifestations provides important clues for the judgment of acid-base imbalance. the result of blood gas monitoring is the decisive basis for judging the type of acid-base imbalance. serum electrolyte examination is an important reference. anion gap (ag) has important diagnostic value in determining the type of acidbase imbalance [ ] . ag = na + -(cl − + hco − ) is a simple formula for the value between the number of cations and anions in serum. its normal value was ± mmol/l. ag can not only help diagnose "potential" metabolic acidosis and to distinguish different types of metabolic acidosis, can also help determine special types of mixed acidosis, and has its unique role in the judgment of tabd. sometimes the indicators of blood gas analysis are normal, the calculation of ag value become the only evidence of diagnosis of metabolic acidosis. in addition, ag value can be used as reference for correction of acid-base imbalance. in severe hepatitis, the change of ag values can be used as an indicator to estimate the complications and prognosis. clinical observations indicate: ag value significantly increased often suggestive of severe infection, kidney dysfunction or severe bleeding, and the prognosis is poor. respiratory alkalosis refers to arterial paco decrease and ph > . as well as compensatory decrease of blood hco − . respiratory alkalosis occurred in early stage of severe hepatitis. in severe hepatitis, respiratory alkalosis related to hyperventilation: accumulated ammonia and other vasoactive peptides excited respiratory center, ascites and pleural fluid increase respiratory rate, hypoxemia excited respiratory center. compensatory mechanisms: co reduction, breathing shallow and slow, so that co retention, h co compensatory rise; when last longer, reduce renal row h + , hco − excretion increased, hco − /h co equilibrium at a low level. most patients have the performance of shortness of breath and heart rate increase. can have vertigo, hand, foot and mouth numbness, muscle tremor, hand and foot convulsions. convulsions associated with low calcium. dysfunction of nervous system is related to the damage of brain function and cerebral blood flow decrease. respiratory alkalosis diagnosis relies on the following: ( ) ph is normal when fully compensated, increased underdecompensation. ( ) paco lower (typically < mmhg or . kpa). ( ) hco − compensatory decline. ( ) ag value may have a slight increase. ( ) blood cl − may increase. metabolic alkalosis refers to the type of acid-base imbalance characterized by an increase hco − in extracellular liquid. the inappropriate application of basic drugs, potassium-sparing diuretics, dehydrating agents, hormones can often induce or aggravate metabolic alkalosis. severe gastrointestinal symptoms, anorexia, vomiting or diarrhea are also the reasons for the occurrence of metabolic alkalosis. compensatory mechanisms: when alkaline substances increased in body, buffer system instantly transfer strong base into weak base, increase hco − consumption, h co increased. inhibit respiratory center, decrease pulmonary ventilation, co retention, hco − compensatory increase. renal carbonic anhydrase activity decreased and h + formation and excretion decreased, nahco reabsorption is also reduced, so hco − /h co compensatory restore to : , ph value is normal. patients with mild metabolic alkalosis usually have no obvious symptoms. many disorders can occur in severe metabolic alkalosis. ( ) functional changes in the central nervous system, the patient may have irritability, confusion, delirium, consciousness disorders. ( ) slow and shallow breathing, hypoxemia. brain tissue is particularly sensitive to hypoxia, thus neurological symptoms first appeared. ( ) hypocalcemia and neuromuscular stress increased, the performance of tendon hyperreflexia, face and muscle twitching, and limbs twitching. ( ) hypokalemia can cause neuromuscular symptoms and arrhythmias. according to ph value, paco , hco − , level of k + and cl − , effective circulating blood volume and performance of primary disease, diagnosis of metabolic alkalosis is no difficult to make. metabolic alkalosis should be divided into two categories based on the urinary level of cl − . ( ) chloride positive metabolic alkalosis: supplement sodium chloride can correct the alkalosis. it indicates that the body has cl − deficiency, urinary cl − < mmol/l. ( ) chlorine negative metabolic alkalosis: alkalosis can not be corrected by supplement sodium chloride, urinary cl − > mmol/l. respiratory alkalosis plus metabolic alkalosis tend to occur on the early stage of severe hepatitis. in most cases, there is no obvious complication, more often metabolic alkalosis happens on the basis of respiratory alkalosis, or the other way around. due to respiratory and metabolic factors are inclined to alkaline change, name as reduce paco and elevated plasma hco − , there is no mutual compensation between them, so it is easy to present as severe decompensation and poor prognosis. main point of diagnosis: ( ) ph value of blood increase significantly. ( ) paco decrease. ( ) hco − increases, the value should be greater than . ×( -paco ) + . . ( ) hypokalemia and hypochloremia are common phenomenon. respiratory alkalosis plus metabolic acidosis is relatively rare. metabolic acidosis can be divided into types: ( ) value of ag is normal (high chlorine acidosis), commonly seen at long-term diarrhea, combined with renal tubular acidosis and a large amount of physiological saline input in patients or water intoxication. ( ) high ag value type (normal blood chlorine acidosis), regularly present in combination of hepatorenal syndrome, lactic acidosis and patients with ketoacidosis. ( ) a hybrid type (high ag merged high blood chlorine), mainly in patients with severe diarrhea following lactic acid or ketoacidosis. when respiratory alkalosis plus metabolic acidosis happens, paco and plasma concentration of hco − are higher than scope of compensation to each other. its characteristics as follows: ( ) the range of blood ph change is not large, normal, slightly higher or slightly lower. ( ) paco reduce to less than . × hco − + or hco − < -( -paco ) × . - . . ( ) ag values can be normal or elevated, the latter is more common. if the elevated blood cl − value is equal to the hco − decrease, ag value normal metabolic acidosis type can be diagnosed; the cases that the rising value of ag is equal to the decline of hco − values can be diagnosis as ag increased metabolic acidosis type. on the third occasion, the increase value of ag is equal to the sum of hco − and cl − drop, the diagnosis is mixed metabolic acidosis. for this type of offset mixed acid-base balance disorders, treatment should be moderate, the measure of the metabolic factors correcting should be precede to respiratory factors, avoid paco quickly returning to normal in the process of treatment, which would lead to blood ph drop rapidly and acidosis more worse. to patients with severe hepatitis, the harm of alkalosis is greater than acidosis, thus the blood ph should be kept slight acidic in a normal state. generally, the target of alkali supplement can be arterial blood ph value recovered to . . respiratory alkalosis tabd refers to respiratory alkalosis, metabolic acidosis and metabolic alkalosis three primary imbalances coexist in the same patient, which is one sort of serious acid-base imbalance, mostly develops in the late stages of severe hepatitis, fatality rate is high. respiratory alkalosis tabd characteristics as follows: ( ) blood ph value depends on the relative severity of these three primary imbalances, which can be normal, or slightly high generally. ( ) reduce paco , its value is less than . xhco − + . ( ) hco − can raise, normal or lower. ( ) value of ag rise significant, and extent of ag raise is greater than the hco − lower. ( ) cl − often lower than normal. the occurrence of metabolic alkalosis plus metabolic acidosis in patients with severe hepatitis is not uncommon. usually it is accompanied with existing lactic acidosis or ketoacidosis, and the patient may manifest frequent vomiting. since the causes for raising and lowering hco − coexist, they tend to cancel one another. the ph and hco − concentration can be normal, increased or decreased, depending on the relative severity of the two kinds of imbalances. severe hepatitis can also be accompanied by metabolic acidosis, metabolic acidosis plus respiratory acidosis, tabd of respiratory acidosis, and etc. pure metabolic acidosis refers to arterial blood ph < . and compensatory decline of paco due to primary decrease of hco − . typical manifestation is known as kussmaul breathing, characterized by deeper and faster breathing, as well as obvious contraction of respiratory muscle, and also ketone-smelled exhaled breath. the patients often flush, companied by increased heart rate and decreased blood pressure. there may be reduced or disappeared tendon reflexes, confusion or stupor. due to respiratory and metabolic factors both towards to acidic changes, there is no respiratory compensation for decrease in hco − , nor renal compensation for increase in paco , hence presenting severe decompensated status. the resulting distinct decrease in ph and vicious circle are the characteristics for metabolic acidosis plus respiratory acidosis. the characteristics for tabd of respiratory acidosis include significantly increased paco , elevated hco − , ag > mmol/l, and significantly decreased cl − . the incidence of the above types of acid-base imbalance is very low in patients with severe hepatitis. once it happens, it should be actively treated with corresponding methods, so that the blood ph quickly restores to the safety range. during therapeutic process against various pure acid-base imbalance, interactions among various treatments need to be taken into consideration, in order to avoiding the possibility that the treatment for one type of acid-base imbalance causes or aggravates another type. in summary, water-electrolyte imbalance and acid-base imbalance have a relatively high morbidity in patients at various stages of severe hepatitis. this often results in deterioration and complication of the disease, evermore, the death of the patient. therefore, the functions of heart, lung, kidney, blood circulation as well as changes of body weight in the patient need to be intently monitored. regular detection of k + , na + , cl − , carbon dioxide combining power (co cp), blood urea nitrogen, creatinine, ph, data about arterial blood gas analysis, and also detailed records of patient's input and output are demanded. during the process of diagnosis and treatment, careful analysis of the history, clinical manifestations and laboratory examination are necessary to achieve correct diagnosis, early prevention and prompt treatment. in normal condition, proper amount fluids can make a lubrication action on organs in peritoneal cavity. but in those patients who have severe hepatitis, especially with cirrhotic portal hypertension, too many fluids over ml can lead to ascites. the ascites can be categorized into uncomplicated ascites and refractory ascites. there is no infection in uncomplicated ascites and won't lead to hepatorenal syndrome, but refractory ascites is in the contrast. the refractory ascites includes diureticresistant and diuretic-intractable ascites. the diuretic-resistant ascites shows no response to diuretic and diuretic-intractable ascites limits the application of diuretic due to the complications induced by diuretic. mg antisterone per day as an initial dose can be given to those patients with moderate ascites, if it goes to no satisfied effect, mg can be added after every days till the maximum dose to mg/d. if the patients show a hyperkalemia or aldosterone antagonist-resistant, nicorol can be combined and with an increasing dose from to mg/d gradually. the patients without edema losing weight should be less than . kg and those with edema should be less than kg per day to avoid electrolyte disturbance or hepatorenal syndrome during the whole treatment period. diuretics should be withdrawn on the patients with severe hepatic encephalopathy, severe hyponatremia, progressive renal failure or severe muscle spasm. the patients should only take the minimum dose of diuretics to maintain the state after the syndrome controlled, or withdraw when it is necessary. due to the poor outcome and living quality, the median survival time of refractory patient is half year. so liver transplantation can be considered in the patients with refractory ascites induced by cirrhosis, which required more cautious before make a diagnosis. generally, if the patients meet the following conditions when they are receiving mg/days antisterone and mg/days nicorol treatment over week and sodium uptake limited in mmol/days, refractory ascites can be diagnosed. ( ) losing weight less than . kg/days over days ( ) sodium uptake is more than elimination ( ) grade - ascites is arisen again after -week long treatment ( ) hepatic encephalopathy, hepatorenal syndrome and severe electrolyte disorder induced by diuretics are shown up. refractory ascites patients without complications can be treated with abdominocentesis, but it will possibly induce circulation failure, and increases the risk of hepatic coma and hemorrhage. so low rate infusion of albumin with abdominocentesis is combined to avoid circulation failure, meantime diuretics also need to give after abdominocentesis. aldosterone antagonist combined with nicorol is a suitable strategy: the dosage of antisterone is increased from to mg/days and nicorol from to mg/days gradually. the goal of this strategy is to maintain the situation without ascites under the minimum dosage. but when severe hepatic encephalopathy and electrolyte disorder show up, which means serum sodium concentration is less than mmol/l, serum potassium concentration is less than . mmol/l, nicorol should be withdrawn, if serum potassium concentration is more than . mmol/l, antisterone should be withdrawn. to those patients who need abdominocentesis repeatedly, transjugular intrahepatic portosystemic shunt (tips) can be considered, but the risk of hepatic encephalopathy will be higher and the outcome is poor. so the patients with severe liver and renal failure, cardiorespiratory function failure or active infection should be in cautious. the mean survival time of refractory ascites patients complicated with hepatorenal syndrome is months, preventive antibiotics combined with albumin is an option for these patients. to those patients who have already showed hepatorenal syndrome, terlipressin combined with albumin could be useful. meantime, abdominocentesis, tips or artificial liver supporting treatment can improve patients' living quality in short term, but long-term outcome won't be good, so liver transplantation should be execute as soon as possible. in general, medicine can improve the symptoms in short term but with poor outcome, liver transplantation is more meaningful. patients with hypovolemic hyponatremia can have a supplement with sodium and decrease the dosage of diuretics, patients with hypervolemic hyponatremia can restrict fluids uptake (less than ml/d) and combined with vasopressin v receptor blocker or antidiuretic hormone receptor blocker. currently, vaptans, tolvaptans, conivaptan and satavaptan have already applied in clinical practice. there was research showed that vaptans could improve - % patients' symptoms significantly after patients took it for week to month, and main side reaction was thirsty. the patients complicated with hepatic encephalopathy should be used vaptans with cautious due to its high risk in dehydration and hypernatremia. meantime, vaptans is metabolized by cyp a, so rifampin, barbital and phenytoin can decrease its effect, and ketoconazole, clarithromycin can increase its plasma concentration. tolvaptans can give some relief but increase the risk of hemorrhage. satavaptan will decrease patients' survival rate. so the proper treat period and side reactions of these drugs in long-term using need to make clear. potassium uptake should be withdrawn immediately after hyperkalemia occurs, emergency treatment for detoxicating potassium should be taken to protect cardiac. the treatment depends on the plasma concentration of potassium. the treatment for patients with fulminant hepatitis b with cirrhosis complicated with hyperkalemia is to restrict the uptake of potassium, improve the microcirculation, correct renal filtration decrease induced by hepatorenal syndrome and increase the elimination of potassium. there are also some patients have abnormal distribution of potassium due to hypoxia, acidosis, catabolism, and deficiency of energy supplies, which leads to intracellular potassium is transferred into extracellular. the treatment for these patients is to correct hypoxia and acidosis, high glucose, insulin and atp are administered to boost glycogen synthesis to transfer potassium from extracellular to intracellular. peritoneal dialysis and plasmapheresis can be given to the patients with intractable hyperkalemia. hypokalemia can present during the whole period of fulminant hepatitis b, it will occur more often on the early and middle stage. long-term inappetency and abdominal distension lead to insufficient potassium uptake. nausea, vomiting and diarrhea lead to increase of potassium losing. renal filtration rate decreasing and aldosterone increasing lead to potassium elimination increase. complicated with alkalosis and anabolism increase also can cause hypokalemia. the treatment for hypokalemia should focus on comprehensive therapy, correcting alkalosis, increasing potassium supply, improving microcirculation. disturbance of acid-base balance occurs quit often in hepatitis b patients, especially with alkalosis. during the early stage, it can present in pure respiratory alkalosis, also can complicated with metabolic alkalosis. during the middle and late stage, both of above symptoms and metabolic acidosis occurs concurrently. treating idiopathy and correcting hyperventilation is a treatment for respiratory alkalosis. arginine hydrochloride injection is used to treat metabolic alkalosis to avoid secondary metabolic alkalosis. the general regulation is prefer acid to base, till ph value of arterial blood back to . . in prevention of disturbance of acid-base balance, the effective strategy is to correct hypokalemia and hypochloremia, control vomiting. meantime, controlling infection, endotoxemia and upper gastrointestinal hemorrhage are necessary. hepatic encephalopathy (he) due to metabolic disturbance is a complex neuropsychiatric syndrome caused by severe liver dysfunction or disorder and is one of the common complications and causes of death in severe liver diseases. patients with he mainly present with neuronal or mental abnormalities and disturbance of consciousness, even coma and death. the clinical manifestations and the severity of the disease vary because of its complex pathogenesis. hepatic encephalopathy is the result of acute and chronic hepatic failure caused by cirrhosis or various kinds of portosystemic shunt (pss) created. a diagnosis of he can be made after excluding encephalon diseases. the syndrome is caused by metabolic disorders and is potentially reversible. he clinical features differ due to the wide degree and range of neuropsychiatric symptoms that vary from subtle abnormalities detected only by intelligence tests or electrophysiological methods geared for detecting personality changes to abnormal behavior, intellectual impairment, and even different degrees of consciousness disorders. he was previously known as hepatic coma, but that is only one of the worst severe signs of he and does not represent all types of he. in , the world congress of gastroenterology (wcog) suggested that based on the cause he can be divided into three types (a, b, and c) [ , ] . type a: type a is acute liver failure-related he and the symptoms occur within weeks. in subacute liver failure-related he, the symptoms of he occur within - weeks with or without predisposing factors. type b: patients with type b he have obvious pss and normal liver histology without associated intrinsic liver disease. these clinical manifestations are similar to those in patients with he and cirrhosis. the pss may be spontaneous or caused by surgical or interventional procedures [ ] . common causes of pss include congenital vascular malformation, intrahepatic or extrahepatic portal vein obstruction (including trauma, carcinoid, and bone marrow hyperplastic disease caused by a high coagulation state due to portal vein branch embolization and thrombosis) and generation of portal hypertension by oppression of lymphoma, metastatic tumors, and bile duct carcinoma. type c: type c he is related to chronic liver diseases, with cirrhosis being the most common type, and is generally accompanied by portal hypertension and pss. type c he is mainly caused by liver function failure, rather than by pss. according to the clinical manifestations, duration and characteristics, type c can be divided into three types: episodic he, persistent he, and minimal he [ ] . episodic he, related to chronic hepatic disease, is defined as a disturbance of consciousness and cognitive change in a short time and can be alleviated by spontaneous remission or drug treatment in the short term, which cannot be explained by a relevant preexisting mental disorder. episodic he can be divided into three types according to the presence of known risk factors: ( ) incentive type: there is a clear history of predisposing factors; ( ) spontaneous type: there is no history of predisposing factors. ( ) recurrent type: he attacks more than two times within a year. persistent he related to chronic hepatic disease is defined as an occurrence of continuous neural mental abnormality, including cognitive decline, disturbance of consciousness, coma and even death. persistent he can be further divided into three types according to the severity of the disturbance in the patient's self-control and self-discipline: , mildest type, namely west haven level ; , severe type: namely west haven level - ; and , therapeutic resistance type: medication can alleviate he quickly, but withdrawal can aggravate he rapidly. patients with minimal he, with normal clinical manifestations and routine biochemical tests, have mild cognitive and psychomotor deficits detected by neuropsychology and neural physiology tests, and these patients usually have a history of chronic hepatic disease [ ] . the prevalence of minimal he in patients with cirrhosis is - %. patients with minimal he with reduced physical and mental ability have gained more and more attention recently because they have a high risk of accidents when engaged in occupations involving mechanical, or driving work. the pathogenesis of he has not been fully elucidated so far, and many theories have been put forward. it is generally believed that he is caused by acute and chronic liver failure and/or pss. when toxic substances absorbed by the intestines cannot be detoxified and cleared by (or through) the liver, they directly enter into the systemic circulation and pass through the blood-brain barrier to reach the brain tissue and cause central nervous system dysfunction. a variety of the risk factors mentioned above can result in he. hyperammonemia is still recognized as one of the most important factors, especially in he related to chronic liver disease, liver cirrhosis and/or pss. according to the ammonia intoxication theory several factors including false neurotransmitters, such as γ-aminobutyric acid/benzodiazepine (gaba/bz) receptor complex, an imbalance in the ratio of branched chain amino acids to aromatic amino acids, brain cell edema, astrocyte dysfunction, mercaptan, short chain fatty acid toxicity and manganese deposition are all involved in the occurrence of he [ ] . ammonia intoxication caused by an ammonia metabolism disorder is the most important factor in the pathogenesis of he [ ] . ammonia comes mainly from the gut and the generation and absorption of ammonia increase in a serious liver disease when excess ammonia cannot be cleared sufficiently by ornithine cycle due to serious damage to liver parenchyma. when pss occurs, intestinal ammonia directly enters the systemic circulation without liver detoxification, resulting in increased blood ammonia. high levels of blood ammonia can enter the brain through the blood-brain barrier and generate central nervous system toxicity by interfering with cerebral energy metabolism, neurotransmitter and nerve cell membrane ion transport; increasing cerebral edema; and changing gene expression (such as stellate cell glutamate carrier, stellate cell structural protein, glial fibrillary acidic protein, peripheral benzodiazepine receptor and aquaporin- ) and inducing the mitochondrial permeability transition (mpt). the main way of removing ammonia from the brain is through urea cycle. during glutamine synthesis, glutamic acid is formed from ammonia and α-ketoglutaric acid and the glutamic acid combines with ammonia to generate glutamine. this process requires atp and consumes a large amount of α-ketoglutaric acid, which interferes with the brain energy metabolism and causes an energy supply shortage in brain cells. glutamate is an important excitatory neurotransmitter in the brain, and lack of glutamate increases inhibition in the brain. glutamine synthetase is present in astrocytes, where glutamic acid is detoxified to glutamine. glutamine is a strong intracellular osmotic agent, and increases in glutamine can lead to brain cell swelling. reports have identified a strong correlation between the content of glutamine in cerebrospinal fluid (csf) and the degree of he [ ] . during he, excess ammonia under the effect of glutamine synthetase, not only reduces the formation of active glutamate but also consumes a lot of energy, leading to the accumulation of glutamine, which increases intracellular osmotic pressure and causes brain cell swelling. swollen astrocytes with impaired function further affect ammonia metabolism, reduce the ability of neurons to efficiently uptake or release extracellular ions and neurotransmitters, and stimulate glial cell synthesis of neurosteroids by upregulating their expression of the peripheral-type bz receptor (translocator protein, kda). neurosteroid is an endogenous bz that can enhance gaba nerve tension and cause symptoms in patients with he [ ] (fig. . shown that the metabolic rate of cerebral ammonia in he patients is increased. increased levels of blood ammonia enter the brain through the blood-brain barrier. brain dysfunction also occurs even if blood ammonia levels appear normal; this partially explains the occurrence of he in the case of normal blood ammonia and invalidates he treatment by simply reducing blood ammonia. in addition, increasing evidence suggests a synergistic effect between blood ammonia and its metabolic disorders with systemic inflammation, nerve steroids, oxidative stress, nitrification stress, manganese poisoning, and gaba/bz [ ] . the main inhibitory neurotransmitter in the mammalian brain is gaba. plasma gaba is derived from the conversion of glutamic acid by glutamate decarboxylase in intestinal bacterial. notably, gaba has dual role. on one hand, during liver function failure and pss, the removal of gaba in liver is significantly decreased; on the other hand, gaba can directly enter the systemic circulation bypassing the liver, resulting in increased concentration of gaba in blood. the concentration of gaba in csf and brain tissue increases as more gaba crosses the abnormal blood-brain barrier. in addition, endogenous bz was found in the blood and csf, and the gaba receptor on the membrane surface of the brain's postsynaptic neurons increased significantly in some patients with he and in animal models. this receptor not only combines with gaba but also binds to barbiturates (barb) and bz on different parts of the receptor surface; thus, it has been named the gaba/bz complex receptor or the super receptor complex. when liver function is severely impaired, the binding affinity of this complex receptor to its three ligands is also increased. binding of gaba, barb, or bz with the complex receptor can promote entry of chloride ions from neuronal membrane ion channels into the cytoplasm of postsynaptic neurons, causing membrane hyperpolarization and nerve conduction inhibition. he symptoms were relieved in about % of patients treated with a gaba receptor antagonist or bz receptor antagonist, and gaba/bz and ammonia were reported to act synergistically in he. recently, some studies focused on peripheral type bz receptors, which are different from central gaba [ , ] . some questions, including the source of endogenous bz, and the correlation between the increased degree of gaba or bz and the disease, remain to be answered. therefore, therapy targeted at reducing the blood ammonia concentration in patients with he and significantly reducing the increased gaba nerve tension seems reasonable [ ] , but may not be completely effective. treatment effects of reducing ammonia vary, because of the different levels of ammonia in he patients that can be produced by the interaction between various known or unknown factors and the different effects of bz receptor antagonists. this theory is related to the metabolism of aromatic amino acids (aaa), the precursors of true neurotransmitters, including norepinephrine and dopamine. due to the reduction in the liver's detoxification function or formation of pss, the amines (phenylethylamine and tyramine) produced in the intestine cannot be cleared completely, resulting in elevated concentrations of these amines in the systemic circulation and increased levels in the brain through the blood-brain barrier. under the effect of β-hydroxylase, phenethanolamine and β-hydroxytyramine (β-dopamine) are generated from phenylethylamine and tyramine, respectively and are similar to norepinephrine and dopamine in chemical structure. these amines can be taken up, stored and released by adrenergic neurons in the brainstem reticular structure. phenethanolamine and β-hydroxytyramine are called false neurotransmitters because of their low physiological effects on the postsynaptic membrane, which is about / of norepinephrine. when these false neurotransmitters accumulate in the nerve synapse, they can outcompete or replace normal neurotransmitters, resulting in a disorder of nerve conduction. it was reported that plasma aaa (such as phenylalanine, tyrosine, and tryptophan) increased and branched-chain amino acids (bcaa, such as valine, leucine, isoleucine) decreased in patients with decompensated liver cirrhosis, leading to an imbalance of amino acid metabolism. aaa are decomposed and metabolized in the liver, and liver failure decreases aaa decomposition resulting in an elevated concentration of aaa in the plasma. insulin can promote bcaas entering muscle, which is then broken down and metabolized in the skeletal muscle instead of the liver. insulin inactivation is decreased in patients with liver failure, promoting a large number of bcaas entering the muscle tissue and decreasing the concentration of bcaas in plasma. finally, the bcaa/aaa ratio is reduced from a normal - . : to : or lower. the above process reduces the bcaa concentration, but increases the aaa concentration, leading to an increase in synthesis of false neurotransmitters and reduction of the normal neurotransmitter [ ] [ ] [ ] . the epidemiological data suggests that manganese poisoning and he extrapyramidal have common clinical symptoms. the liver is an important organ for manganese excretion. the concentration of blood manganese can be increased when liver function is affected, during pss, or when excretion of bile is reduced. manganese content in plasma was sharply increased in more than % of patients with acute hepatitis and liver cirrhosis and the density of globus pallidus increased in the brain basal ganglia of he patients (partially - times higher by mri). based on histological results, the above changes were caused by manganese deposition, which disappears after liver transplantation. it has been suggested that manganese deposition may cause dopamine dysfunction. deposition of manganese not only cause direct brain injury, it can influence the function of -hydroxytryptamine ( -ht), norepinephrine and gaba neurotransmitters; impair astrocyte function; and have a synergistic effect with ammonia. however, there is no reliable correlation between the concentration of serum manganese and he severity, which may be due to the chronic deposition of manganese [ ] . the characteristic change in mri imaging as the deposition of manganese remains to be verified. the effectiveness of manganese removal to improve the symptoms and neurological signs of patients with he needs further validation. the synergistic toxic effects between toxins (ammonia and mercaptan) and short chain fatty acids [ ] , the -ht hypothesis, the effect of helicobacter pylori urease, opioids, endotoxin, tumor necrosis factor, melatonin, and hepatitis b virus termed additional theories of he syndrome. this theory also suggests the same hypothesis mentioned in the above theories. due to the extensive amount of liver cell damage caused by acute liver failure in type a he, the residual liver cells cannot effectively remove toxins leading to central nervous system dysfunction. type a he, known as non-ammonia encephalopathy, is endogenous he without clear causative agents. simple type b he is rare in mainland china; the liver can clear limited metabolic toxins in patients with chronic liver failure or pss, but once these toxins exceed the compensatory capacity of the liver, type c he occurs. the occurrence of type c he is largely related to the following risk factors, which are the most important factors in the prevention and treatment of he. patients with chronic liver failure or pss are less tolerant to the protein found in food, especially animal protein. a large amount of ammonia and aaa are produced by the decomposition of intestinal bacteria, which can induce he. oral ammonium salts, urea, and methionine can induce he by increasing the absorption of nitrogenous substances and elevating blood ammonia. intestinal production of ammonia can be increased by hemorrhage in the intestine ( ml of blood contains - g protein). at the same time, because of the lack of isoleucine in the blood, after digestion and absorption of a hemorrhage, extra blood leucine and valine increase bcaa decomposition by enhancing the activity of bcaa dehydrogenase, thereby exacerbating the imbalance in the bcaa/aaa ratio. loss of blood volume, cerebral ischemia and hypoxia also increase the sensitivity of the central nervous system to ammonia and other toxic substances [ ] . infections such as spontaneous peritonitis, pneumonia, and urinary tract infection can increase tissue decomposition and production of ammonia. secondary sepsis or sirs induce he through tnf-α, il- , il- and other inflammatory factors, exacerbates oxidative stress, and increases the blood-brain barrier permeability of ammonia and other toxic molecules to liver and brain [ ] . studies have shown that sirs is directly related to the deterioration of he in patients with liver cirrhosis, and its extent and mortality increase with the deterioration of sirs [ ] . similarly, sirs is a common factor in triggering chronic liver failure characterized by he and renal failure. in a study of patients with liver cirrhosis, artificially-induced hyperammonemia by oral administration of glutamine may have worsened the results of psycho-mental testing in cases of sepsis patients; while brain toxicity was not obvious after the inflammation was relieved, the observation of decreased cytokine levels indicated that infection and induced inflammatory mediators enhanced brain toxicity of hyperammonemia. accordingly, some researchers suggested that sirs could be an independent pathogenesis of he rather than a risk factor [ ] . hyponatremia can affect the intracellular osmotic pressure and lead to brain edema, which induces he. hypokalemia is often associated with metabolic alkalosis [ ] . mass use of diuretics or extraction of ascites can also cause alkalosis. ammonia is easily absorbed by the intestinal tract or through the blood-brain barrier inducing he [ ] . a variety of reasons can cause pre-renal azotemia such as hypovolemia, anorexia, diarrhea, limiting the amount of liquid, mass use of diuretics, or extraction of ascites. hepatorenal syndrome or other causes can result in renal azotemia. pre-renal azotemia and renal azotemia caused by hepatorenal syndrome or other causes can increase the concentration of ammonia in the blood. several other predisposing factors can contribute to he such as constipation, hypoglycemia, the use of sedatives and proton pump inhibitors, and epilepsy. after the occurrence of constipation and intestinal obstruction, the patient's intestinal mucosa is exposed to ammonia longer thus increasing the absorption of ammonia. hypoglycemia can reduce brain deamination. the binding of sedatives, hypnotics and the brain gaba/bz receptor produce an inhibitory effect on the brain. it was reported that proton pump inhibitors increase the risk of he in patients with cirrhosis in a population study [ ] . another study also suggested that epilepsy was associated with an increased risk of he in patients with cirrhosis [ ] . patients with type a he often have no obvious anatomical abnormalities in their brains, but - % of patients have brain edema, which may be a secondary change of the disease. hypertrophy and hyperplasia of the original plasma astrocytes in gray matter and subcortical tissue can be found in patients with type c he. patients with longer course of the disease will exhibit brain atrophy (especially in patients with alcoholic cirrhosis) of different degrees, thinning of the cerebral cortex, loss of neurons and nerve fibers, and deep cortical sheet necrosis, even the cerebellum and the base may also be involved. the majority of patients with cirrhosis may have different degrees of he at some stage in the course of the disease. the incidence of he in patients with liver cirrhosis is at least - % in mainland china while the incidence of post-tips (transjugular intrahepatic portosystemic shunt) he is - %. if patients with chronic liver disease have he, the outcome is poor; the one year survival rate is lower than % and the year survival rate is less than % [ , ] . the incidence of mild he is . % in mainland china in patients with liver cirrhosis, . % in patients with child-pugh a, . % in patients with child-pugh b, and . % in patients with child-pugh c. the incidence of mild he is not significantly associated with cirrhosis; however, with the increased degree of decompensated liver cirrhosis, the incidence of mild he increase. several studies have found that the incidence of depression and anxiety in patients also increased, with the increase of liver function damage, the incidence also increased, and the outcome is poor [ , ] . the clinical manifestations of he vary, because of the difference in the nature of underlying disease, the degree of liver cell damage, the speed of injury and incentives. they are not specific to he compared with other metabolic encephalopathies. early pathological changes of he are mild he. the neuropsychological and intelligence tests detect mild form of he, which exhibit no clear clinical symptoms and often develop symptomatic he. the main clinical manifestations seen in acute liver failure induced by type a he are rapid-onset jaundice, bleeding, decrease in prothrombin, and eventually, change in mental status that can start as mild confusion but progress to coma and even death. type c he is characterized by chronic recurrent episodes of changes in personality and behavior [ ] , stupor and coma, which is often accompanied by increased muscle tone, hyperreflexia, hepatic flap, ankle clonus or positive babinski sign and nervous system abnormalities. most patients in the early stages relapse, but then their symptoms become persistent. he often has a variety of risk factors such as consuming a high-protein diet or discontinuing treatment of he. patients with type c he not only have the clinical manifestations of encephalopathy, they also have chronic liver injury, cirrhosis and other clinical manifestations [ ] . observation of encephalopathy dynamic changes is beneficial for early diagnosis, treatment and analysis of treatment efficacy. he can be graded and quantified according to the degree of disturbance of consciousness, nervous system performance and eeg changes. according to the edition of the "consensus on the diagnosis and treatment of hepatic encephalopathy" in china, he is divided into - periods, but each period can be overlapping or distinct but each period can be overlapping (table . ). at present, scholars have stressed that the occurrence of he is a continuous progression of the disease and should be viewed as a continuum of a wide range of neuropsychiatric abnormalities, rather than isolated clinical stages. according to the traditional west haven criteria diagnosing grade he is based on clinical signs and physician assessments, resulting in diagnostic criteria confusion [ ] . (fig. . ) . covert he is diagnosed by a variety of neuropsychological and intelligence tests; the evaluation of overt he widely uses the modified west haven semi-quantitative grading table for the analysis of patients with neuropsychiatric state (table . ), the glasgow coma scale for the analysis of the degree of consciousness of patients, and the simple he severity rating scale for the disease in addition to abnormal liver function (such as increased bilirubin, enzyme bile separation, and decreased prothrombin activity) commonly used auxiliary examinations for he diagnosis include: determination of ammonia, amino acid analysis of plasma and csf, psychological intelligence test, neurophysiological test, electroencephalogram and neuroimaging. the normal level of fasting venous ammonia is - μg/l (serum) or - μg/l (whole blood) and arterial ammonia concentration may be . - times that of venous ammonia. generally, the determination of arterial ammonia is common in clinical practice than intravenous determination; however, if venous blood has been transported on ice box and detected in a timely manner after proper collection, the result is expected to be as effective as arterial detection. ammonia levels are increased in type b and c he, but are normal in type a he. thus, he cannot be ruled out based on having a normal ammonia level. the increased level of ammonia was reported to be associated with the degree of type a he, but significant overlaps in different clinical stages of patients were also found [ , ] . therefore, ammonia detection is not routinely recommended in the diagnosis of he. notably, we need to rule out falsely elevated levels of ammonia caused by lab error, renal failure, complete parenteral nutrition, gastrointestinal bleeding, the use of steroid hormones and other extrahepatic factors. the fischer ratio (bcaa/aaa) is used as a marker of he, the plasma bcaa levels decrease while aaa levels increase; resulting bcaa/aaa: < (normal > ). it was reported that the concentration of glutamate in csf in he patients is increased compared to healthy controls. the concentrations of phenylalanine and tyramine in csf were also significantly increased, and the level of phenylalanine was closely related to the degree of he [ ] . recently, it was reported that h-nuclear magnetic resonance spectroscopy could select biomarkers for these diseases, such as in patients with he [ ] , but this is not commonly used clinically because the the characteristic manifestations of cognitive dysfunction in patients with covert he are lack of attention, working memory problems, and deficits in executive function. therefore, various intelligence tests are used to assess the subtle changes in a patient's cognitive or precise movement, which is important for the diagnosis of covert he, but not for overt he. [ ] . at present, computer-aided psychological tests such as information and communication technology (ict), cognitive drug research test (cdr), and critical flicker fusion test (cff) are not influenced by the factors mentioned above and easily operated, which can be used as an alternative choice for pen and paper tests. ict with sensitivity % and specificity % was one of the most commonly used tests to diagnose minimal he. cff was originally used to detect the critical flicker frequency of alert patients, reflecting brain conduction dysfunction. based on a spanish study of cases, including patients with cirrhosis and healthy controls, cff was a sensitive method to diagnose covert he with sensitive, simple and reliable advantages [ , ] . because the diagnosis of minimal he has just started, the related diagnostic value still needs to be further evaluated. an abnormal eeg is often observed before biochemical abnormalities or mental abnormalities [ ] . the main abnormalities by eeg are slowed rhythm, sporadic or universal θ wave ( - times/s) and the occasional α wave ( - times/s). with the deepening of consciousness, symmetrical δ-wave and three-phase waves appear on both sides simultaneously. this change usually occurs on both sides of the forehead and the top, gradually moving backwards. although these eeg changes are not specific to he and can appear in uremic encephalopathy and other metabolic encephalopathy, the severity of changes have a good correlation with clinical stages of he. computer analysis of eeg frequency distribution, such as artificial neural network-expert system (aness) and short epoch dominant activity cluster analysis (sedaca), is more objective and valuable in diagnosing minimal he than conventional eeg [ ] . there are many kinds of evoked potential tests, including visual evoked potential (vep), brainstem auditory evoked potential (baep), somatosensory evoked potential (ssep) and endogenous event evoked potential (event-related potentials, erps) p , of which the p is the most sensitive test for the diagnosis of he. compared with intelligence tests, neurophysiologic tests, independent of age and education background, are more objective. however, they are only used in clinical studies and are limited by equipment, and professional operation. based on cerebral ct and mri, brain edema can be found in patients with type a he while brain atrophy in the frontal cortex, and the t -weighted signal enhancement in the globus pallidus can also be found, which may be associated with manganese deposition. detected by h-magnetic resonance spectroscopy (h-mrs), the metabolic changes of he patients in the brain include increased levels of glutamate and glutamine, and decreased levels of inositol, taurine and choline [ ] . using fluid attenuation inversion recovery (flair) and diffusion weighted imaging (dwi) techniques, diffuse t -weighted signal enhancement is found in the hemisphere white matter and corticospinal tracts, which may be associated with cerebral ischemia. however, the sensitivity and specificity of the above-mentioned imaging abnormalities remain unknown, and the correlation with he staging is not clear. therefore, the main significance of cranial nerve imaging is to exclude cerebrovascular accident, intracranial tumors and other diseases, rather than diagnose he. there is no gold standard diagnostic criteria of he, and diagnosis is mainly based on the exclusion of other diseases. but one should consider the following five factors [ ]: several forms of hepatic diseases may lead to different kinds of he. type a he is caused by acute hepatic failure, but without chronic hepatic disease. type b is caused by pss, but without any history of hepatic diseases. type c is caused by serious hepatic diseases and/or widespread pss, such as cirrhosis, liver cancer, post-tips and so on. psychiatric symptoms can be found such as change of mood and personality, dementia, behavior disorder and disorientation. drowsiness alternating with excitability, hypermyotonia, asterixis, ankle clonus, insanity and coma are physical signs could be present in progressed patients. some patients may lack related physical signs and psychiatric symptoms, but have deficits in ability of learning, understanding, concentration, and quick verbal response. upper gastrointestinal hemorrhage, ascites tapping, excessive diuresis, high protein diet, medicine (such as sedatives) and infection could lead to he. previous he symptoms could be helpful for the diagnosis. type a usually does not have any risk factors. metabolic encephalopathy includes ketoacidosis, hypoglycemia, uremia, pulmonary encephalopathy, serious electrolyte disturbances and toxic encephalopathy. nervous system diseases include intracranial hemorrhage, infection or tumors, mental diseases and excessive use of sedatives [ ] . but one should also watch out for the coexistence of he in these situations. an overt he should be considered if ( ), ( ), ( ), and ( ) coexist; and covert he is based on ( ), ( ), ( ), and ( ) [ ] . then, based on the degree of neuropsychiatric symptoms, determine the stage of he, or for he classification refer to the west have semi-quantitative classification table or ishen scores. the flow chart of diagnosis is shown in fig. . . he is a complex metabolic disorder caused by many factors and comprehensive measures should be taken to cure it from different aspects. according to the clinical type, inducements and the severity of the disease, different plans of treatment should be designed for he. at present, the treatment of overt he generally includes the following aspects: ( ) supportive treatment; ( ) identification of possible concurrent encephalopathy and removal of other precipitants; ( ) cause of treatment; and ( ) empirical treatment (fig. . ). the point of nutritional therapy is to promote anabolism, inhibit catabolism, and maintain a positive nitrogen balance, rather than simply limiting protein intake. to reduce the source of ammonia, it has been suggested that patients with he should limit protein intake. in critically ill patients, it has been suggested that they should stop all protein intake and, after the disease improves, gradually increase protein intake to the maximum clinical tolerance. these recommendations are now being questioned because most cirrhotic patients are malnourished and all long-term protein-restricted diets increase the severity of malnutrition. in addition, a negative nitrogen balance increases mobilization of skeletal muscle, resulting in a reduction in ammonia metabolism that may increase blood ammonia levels. recent studies have shown that normal ingestion of protein . g/(kg • d) can also improve the health-related quality of life (hrqol), especially in mhe [ ] ; and have no adverse effects on the recovery of blood ammonia and he fig. . the flow chart of diagnosis of he compared with the restricted protein intake. according to the guidelines of the european society of enteral nutrition in , the intake of protein should be guided by the following principles: patients with acute phase he on the first day should be put on a prohibited protein diet and given glucose to ensure energy supply and those who cannot eat food may be fed through a nasogastric tube without short-term fasting; patients with chronic he do not need to fast and their intake of protein should be - . g/(kg • d); oral or intravenous use of bcaa and essential amino acid preparations can be administered to adjust the balance of aaa/bcaa, promote the balance of nitrogen, and also reduce the risk of he recurrence [ ] ; probiotics and prebiotics can enhance the body's tolerance to protein; plant protein is superior to animal protein because it contains methionine, has less aaa, and more bcaa, but it also contains cellulose, which is conducive to maintain the normal flora in the colon and acidize the intestinal tract, shortening the transit time of the colon and reducing absorption of ammonia. the above points need further verification. additional supportive treatments include: maintaining adequate hydration, electrolytes and acid-base balance; ensuring an energy supply of - kal/(kg • d), which should be composed of - % sugar, - % protein, and - % fat; administering appropriate vitamins and trace elements; treating for hypokalemia, hyperkalemia, hyponatremia, hypocalcemia, hypomagnesemia and metabolic alkalosis as needed; strengthening the basis of treatment with the appropriate infusion of fresh plasma or albumin, increased plasma colloid osmotic pressure; treating for hypoxemia and cerebral edema; and preventing and treating any bleeding and bacterial infection. type c he has a variety of precipitents. actively finding and eliminating triggers can effectively prevent the development of he, such as esophageal variceal bleeding that can develop into he. active hemostasis, anemia correction, and removal of intestinal blood are also conducive to controlling he. in addition, active control of infection, correction of water and electrolyte imbalance, elimination of constipation and improving renal function are essential to control he. anesthetics, painkillers, sedatives, sleeping pills, and other drugs should be strictly controlled. patients with mania or convulsions can reduce the use of diazepam and scopolamine, and the frequency of administration can also be reduced for promethazine, chlorpheniramine, and other antihistamines. toxic substances causing he mainly come from the intestine. thus, in order to prevent and control he, it is very important to clean the intestinal tract to reduce the generation and absorption of ammonia and other toxic substances. saline or weak acidic solution enemas (such as a dilute acetic acid solution), or oral or nasal feeding of % magnesium sulfate ( - ml) can be used to clear intestinal hemorrhage, intestinal impaction, and other toxic substances. an enema composed of non-absorbable lactulose ( - ml) plus water ( ml) is also useful, especially when applied for type b he. a recent clinical trial suggested that polyethylene glycol was more effective than the current standard first-line therapy in treating these patients [ ] . another two studies showed that polyethylene glycol was more effective than the standard lactulose therapy in treating patients with acute he by cirrhosis [ , ] . other available drugs include pear liquors, mannitol, rhubarb, and so on, but excessive use of these substances may lead to dehydration and aggravate he. non-absorbable disaccharides include lactulose and lactitol. lactulose, a kind of synthetic ketone disaccharide, cannot be broken down in the stomach and small intestine due to a lack of enzymes that can break down galactose in the digestive tract. after entering the colon, lactulose can be broken down into acetic acid and lactic acid with the help of gut bacteria, leading to a reduction in the colonic ph and inhibition of the absorption of ammonia in the intestine. these non-absorbent disaccharides are decomposed into organic particles in the intestinal tract, which can increase the osmotic pressure of the intestine, and their acidic products stimulate the intestinal wall and can slightly promote intestinal excretion. these non-absorbent disaccharides, acting as prebiotics in the intestine, can inhibit the growth of bacteria, which can produce ammonia and urea, finally reducing the production of ammonia and reversing low-grade cerebral edema when combined with rifaximin [ ] . however, probiotics can benefit patients in the long-term [ ] . oral or nasal feeding ( - ml, or times daily) was recommended to adjust the daily defecation appropriately, about - times daily. main adverse reactions include abdominal discomfort, abdominal distension, abdominal pain, loss of appetite, nausea, vomiting, and diarrhea. lactulose can even be used in patients with diabetes or lactose intolerance when the purity of non-absorbable disaccharide was high (≥ %), but is not used in patients with intestinal obstruction. numerous randomized controlled studies showed that lactulose or lactitol can significantly alleviate overt he and improve the patient's cognitive function and quality of life [ , ] . lactulose is still the first-line therapy of anti-he, although its effect on improving the survival rate of patients is uncertain. antimicrobial agents can be used as a substitute for non-absorbable disaccharides in treating acute and chronic he. in the past, oral aminoglycoside antibiotics, such as neomycin, which are rarely orally ingested, were used to inhibit the overgrowth of bacteria in the intestine. however, recent randomized placebo-controlled studies have shown that neomycin may not benefit patients with he compared with placebo-treated patients and that long-term use of neomycin may lead to increased ear and renal toxicity risk and impair the function of small intestinal mucosa [ ] . metronidazole can inhibit anaerobic bacteria in the intestine and alleviate he, but long-term use may lead to disruption in the intestinal flora, gastrointestinal discomfort, or neurotoxicity. rifaximin, a derivative of rifamycin with a broad-spectrum, has a potent inhibitory effect on intestinal bacterial growth, is a minimally-absorbed oral antibiotic and only plays a role in the gastrointestinal part. administration of rifaximin ( mg, twice a day) can significantly prevent the occurrence of he compared with placebo-treated patients [ ] [ ] [ ] ; rifaximin was equivalent to or better than lactulose and neomycin in treating patients with chronic he [ ] . a study indicated that rifaximin-α in combination with lactulose was a cost-effective therapy for patients who had experienced at least two prior overt he episodes [ ] , and this therapy could also improve the driving ability of patients with covert he without toxicity to the auditory nerve and renal function [ , ] . thus, rifaximin has been recommended by the us food and drug administration (fda) for the prevention of recurrent he. the efficacy of rifaximin and relation between longterm use of rifaximin and intestinal flora in the treatment of he needs to be further investigated. however, a recent study in mice showed that rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression in mhe [ ] . the study may provide a new opportunity to study intestinal flora in the treatment of he. microecologics with bifidobacterium and lactobacillus can regulate intestinal flora structure to inhibit the growth of bacteria that produce ammonia and urease. in combination with prebiotics, microecologics can reduce the production and absorption of intestinal ammonia and other toxic substances [ , ] . in a recent openlabel study, patients with cirrhosis were randomized to three groups and treated with lactulose ( - ml daily), probiotic capsules, or with both drugs. after a month of treatment, patients with he showed better results in the neuropsychological test, p auditory evoked potentials, and blood ammonia. however, there was no difference in the therapeutic effect among the three groups [ , ] . clinicians commonly use sodium glutamate, potassium glutamate, arginine hydrochloride and potassium magnesium aspartate, but the exact efficacy is highly controversial at present and effective drug reduced ammonia is described below. (a) l-ornithine-l-aspartate lola, a dipeptide, can lower blood ammonia by promoting ammonia consumption and the synthesis of urea, glutamic acid and glutamine in brain, liver and kidney [ , ] . ornithine, a substrate of the ornithine urea cycle, can increase activity of carbamyl phosphate synthetase and ornithine carbamyl transferase, and promote urea synthesis. n-methyl-d-aspartate (nmda) is a substrate of glutamine synthesis, and the conversion of glutamic acid to glutamine in the body can remove blood ammonia [ ] . nmda is also involved in nucleic acid synthesis in liver cells and indirectly improves the metabolism of the krebs cycle process in liver cells to facilitate the repair of liver cells. clinical studies show that, compared with a placebo group, g/days lola intravenously could noticeably reduce fasting blood ammonia (fnh ), postprandial blood ammonia, and improve the mental status of patients with he [ ] . patients with oral lola also had improved he examination results for the digital connection test, the flapping tremor, and eeg results [ ] . in addition, glycerol phenylbutyrate (gpb) can safely reduce the incidence of he as well as ammonia in patients with cirrhosis and he. the results showed that gpb had therapeutic potential in this population [ ] . zinc is an important cofactor in urea cycle enzyme catalysis. a study in he patients showed that serum zinc concentration is reduced, and showed a negative correlation with the blood ammonia concentration; the serum ammonia level is much lower after zinc supplementation in patients, and he can be improved in some patients. a new study suggests that antioxidant and zinc supplementation can improve mhe in patients with liver cirrhosis [ ] . oral zinc preparation can also reduce absorption of divalent cations such as manganese in the intestine; however, it has not been determined if zinc has a positive therapeutic effect on he. (c) sodium benzoate sodium benzoate can lower the blood ammonia concentration by activating the urea cycle for ammonia detoxification and promoting urinary ammonia. randomized controlled studies showed that sodium benzoate had the same efficacy as lactulose in treating patients with he. the recommended sodium benzoate dose is g twice a day; nevertheless, few patients can tolerate this dose because of its high gastrointestinal side effects [ ] . a recent study showed that tranilast could protect patients from thioacetamide-induced acute liver injury and alleviate he [ ] . endogenous bz analogues combine with the inhibitory neurotransmitter gaba receptor to depress the action on the cns, and is one of the occurring hallmarks of he pathogenesis. a large-scale clinical study on he cases showed that the improvement rate in brain function in treatment and control groups were % and %, respectively [ ] . the study showed that treatment of he with receptor antagonists such as fluorine marcie is feasible. a meta-analysis which included casecontrol studies of patients show that fluorine marcie can noticeably improve he, but didn't show any long-term benefits or improve patient survival rate. so fluorine marcie should only be considered for he patients who had used bz. although the reduction of dopamine neurotransmitter activity is also one of the pathogenesis, the application of bromocriptine, levodopa, has been unable to bring more benefits besides partly improving symptoms of patients. oral or intravenous infusion with a bcaa-based amino acid mixture can theoretically correct an imbalance in amino acid metabolism and control false neurotransmitter formation in the brain [ ] . a meta-analysis which included five studies showed that intravenous bcaa did not reduce the mortality rate of he. three studies with bcaa did not reduce the mortality rate of he; however, two larger studies (randomized controlled study about patients with liver cirrhosis in cases and cases, respectively) show that the application of bcaa not only reduced the occurrence of he and liver failure, but also improved the nutritional status, liver function and survival rate in patients. another study showed that bcaa could stimulate liver cell regeneration thus reducing the occurrence of liver failure. supplementation with a bcaa-rich amino acid mixture showed improved restoration of the patients' positive nitrogen balance, and increased the patient's susceptibility to protein food, improving cerebral perfusion [ ] . considerable progress has been made to understand treatment of he, studies of basic and clinical research are underway using newly discovered treatment strategies, such as toll-like receptor antagonists (with the ability to reduce systemic inflammation and oxidative stress) as well as non-steroidal anti-inflammatory drugs (ibuprofen) [ , ] , nmda antagonists, anticholinesterase. however, research using gene therapy should not be ignored [ ] . after tips, lola can significantly reduce the increase of venous ammonia concentration in patients with he [ ] . and the positive dietary intervention can significantly reduce the incidence of he [ ] . for patients with refractory he, embolization of pss is a safe and effective treatment strategy [ ] . improving liver function antiviral treatment with nucleos(t)ide analogues can reduce or eliminate liver inflammation and necrosis, promote the regeneration of liver cells, and help restore the functions of hepatic metabolism and detoxification in chronic liver failure caused by hepatitis b virus. artificial liver support systems can be divided into three types including nonbiological type, biological type and mixed type. the non-biological liver support system is the most widely used type, and consists of hemodialysis, hemofiltration, plasma exchange, blood perfusion, plasma adsorption, and the molecular adsorption recirculation system (mars) [ ] . the artificial liver support system can replace the partial function of the liver, remove the poison accumulated in the body, create conditions that allow for the regeneration of liver cells and provide enough time to wait for liver transplantation for patients with he. an artificial liver support system can be used to treat acute and chronic he, but patients with overt stage he should be especially careful with plasma exchange. liver transplantation remains the only promising therapy for patients with an acute liver failure or endstage liver disease. liver transplantation is an effective means for all kinds of persistent and severe he; however, in patients with he there is a significant increase in mortality among patients awaiting liver transplantation [ ] . recently, it has been reported that cognitive function was not fully recovered after liver transplantation in some patients with severe he [ ] . the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. theoretically, for patients with serious pss, interventional therapy, surgery or permanently/temporarily and partially/totally blocking the pss can improve the patient's symptoms. the use of this therapy should be carefully weighed because it can increase the risk of gastrointestinal bleeding in case of portal hypertension. covert he has gained an increasing amount of attention in recent years. patients with covert he do not exhibit obvious signs and symptoms; however, their quality of life is reduced because of reduced operational ability or sleep disorders. without treatment, covert he will progress to overt he over time. the population with high risk should be examined and treated early, especially those engaged in potentially dangerous occupations. the following solutions can be referred to: (a) adjusting dietary structure (vegetable protein is the main intake); (b) oral administration of lactulose ( - ml, - times daily); (c) oral administration of rifaximin ( mg, twice a day); (d) oral administration of lola ( g, times a day); (e) oral administration of baaa; and (f) oral administration of probiotic preparations [ , , ]. although medical technology has made great progress and the research into he is also increasing in recent years, the pathogenesis of he is still unclear. due to a lack of specific methods, combination treatment is still the main therapy for he. it is generally believed that the onset of he may be a result of the synergistic effects of many factors. therefore, it is difficult to implement and draw convincing conclusions from randomized controlled trials with a single intervention targeting a specific pathogenesis and risk factor. ongoing issues remain, such as standardizing the research design of he treatment and evaluating the efficacy of he treatment more scientifically and objectively. some clinical studies may bring new hope for he treatment by new ongoing strategies of targeted systemic inflammation, oxidative stress, and neurosteroids. in addition, the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. it is difficult to popularize the cognitive dysfunction detection methods of latent he. so the key and difficult point is to develop new method of assessments for clinicians in the future. jia shang hepatopulmonary syndrome (hps) is a syndrome of shortness of breath and hypoxemia induced by vasodilation in the lungs of patients with a variety of acute and chronic liver disease. essentially primary liver disease, pulmonary vasodilation and arterial oxygen lack of co-triad constituted. due to abnormal increase of vasodilators、ventilation/ blood flow disproportion and pulmonary hypertension caused by liver disease, the hypoxemia (pao < . kpa) ( mmhg) is included in hps. when fluckiger reported a -year-old syphilis female patient as early as in , he described cirrhosis, cyanosis and clubbing at the same time, while he was not aware of the intrinsic relationship between these clinical manifestations [ ] . in , snell reported decreased arterial oxygen saturation (sao , less than %) with abnormal hemoglobin in patients with liver parenchymal lesions and biliary obstruction, and years later, he proposed that such a phenomenon was associated with decreased affinity of o with hemoglobin. in , rydell and hoffbauer reported the detailed clinical diagnostic and treatment process of a -year-old male with "juvenile cirrhosis", and found multiple arterial-venous anastomoses in the lungs during autopsy, which he thought contributed to clinical cyanosis mainly. this provided a histological basis for the patient, and people conducted a large amount of studies thereafter. in , berthelot et al. injected opaque glue into the pulmonary vascular beds at the time of biopsy after the patient's death for the first time, and he found abnormal small arterial dilation in the lungs of the patient with cirrhosis, which he termed lung spider nevus. the term hepatopulmonary syndrome (hps) was first proposed by kennedy and knudson in [ ] . after nearly years of studies in a large number, people gradually developed a clear understanding of the mechanisms underlying its pathogenesis. in , eriksson used the term functional hepatopulmonary syndrome for the first time. in , the famous liver disease expert sherlock formally used this diagnostic term in his monograph hepatobiliary diseases, which has been recognized by many scholars [ ] . hps can occur in patients of any age groups, and various literature reports show conflicting incidences of hps in patients with cirrhotic portal hypertension, with the average incidence of various chronic liver diseases being about - %. the incidence of cirrhosis in patients is high, and - % of patients can additionally develop mild arterial hypoxia and - % develop arterial hypoxia. in the study by binay on indian cirrhotic populations arising from hepatitis b mainly, the incidence of this disease is relatively low ( . %) [ ] . the differences in incidence were mainly attributable to the different diagnostic criteria adopted. schenk et al. studied the incidence of hps by performing transthoracic contrast echocardiography (ttce), pulmonary function tests and blood gas analysis on patients with cirrhosis patients. the results showed that the incidence of hps patients in whom alveolar-arterial partial pressure of oxygen (aapo ) was used as an indicator of hypoxemia was significantly higher than those in whom arterial partial pressure of oxygen (pao ) was used [ ] . when arterial partial pressure of oxygen (pao ) was reduced to reflect hypoxemia, hps incidence was % when < mmhg and % when < mmhg, respectively. while when increase in alveolar-arterial partial pressure of oxygen (aapo ) was used to reflect hypoxemia, the incidence of hps was high, with % when > mmhg and % when > mmhg, respectively. hps is most common in cirrhosis due to various causes. pulmonary vascular abnormalities and arterial hypoxemia can occur in a variety of acute and chronic liver diseases, and this is true mainly when it comes to cirrhotic patients due to chronic liver diseases, especially cryptogenic liver cirrhosis, alcoholic cirrhosis, hepatitisinduced cirrhosis and primary biliary cirrhosis. besides, hps can also occur in chronic hepatitis, acute severe hepatitis, cholestasis, ɑ-anti-trypsin deficiency [ ] , tyrosinemia, wilson disease, and non-cirrhotic portal hypertension (such as idiopathic portal hypertension and schistosomiasis cirrhosis, etc.). arterial hypoxemia can also occur in extrahepatic portal vein occlusion. the observation of these patients suggests that portal hypertension may be the main factor for the pathogenesis of hps. hps can also occur in non-cirrhotic portal hypertension, and even cirrhosis-and portal hypertension-free chronic viral hepatitis. in , binay et al. found that patients with progressive liver failure with hyperdynamic circulation are most likely to suffer from hps, while they did not find the correlation with the severity of liver cirrhosis. hps is, in essence, hypoxemia due to anomaly in pulmonary vascular dilatation and arterial oxygenation when liver disease occurs. arterial hypoxemia occurs as the result of insufficient oxygenation by blood cells in the blood when blood flows through the lungs, or a proportion of blood fail to flow through the alveoli [ ] . since primary heart and lung diseases have been excluded when hps occurs, the abnormal pathways that red cells may pass through include: ( ) passing through the pleural and hilar bronchial vessels while not reaching the alveoli; ( ) blood flows directly into the pulmonary veins due to the high pressure portal system in the mediastinum, thereby bypassing the pulmonary circulation; ( ) flowing directly into the pulmonary veins through the expanded alveolar capillaries or the pulmonary-venous fistula. alveolar telangiectasia may be more important to the formation of hypoxemia, and existing study data show that the development of hps is at least associated with the systemic hyperdynamic state, portal hypertension, hepatic encephalopathy, hepatorenal syndrome and pulmonary hypertension [ ] . therefore, it is believed that the main causes of hps are systemic metabolism and hemodynamic disorders, and that it is involved in the formation of systemic metabolism and hemodynamic disorders, which is of important pathophysiological significance. . the basic pathological change of hps is pulmonary vascular dilatation, which is manifested as: (a) dilation of anterior capillaries in a large number. (b) formation and opening of the pulmonary basilar arterial -venous communicating branches. (c) formation of pleural "spider mole", which is mainly manifested as dilation of anterior capillaries. in autopsies, it was found that the basic pathological changes in patients with liver cirrhosis and other chronic liver diseases were extensive pulmonary vascular dilatation and arteriovenous communicating branches. some people found the pathological changes through vascular shaping, with pleural vasodilation at the basal aspect of the lungs or the formation of subpleural spider nevus. domestic professor gu changhai summarized these pathologic changes in as arterial dilation within the pulmonary acinus in a pattern of inhomogeneous distribution, thin-walled blood vessels, - μm in diameter, in the lower lobes of the whole lungs, extensive dilation of pulmonary vascular beds adjacent to the alveolar gas at the anterior capillary level, and significantly expanded pulmonary artery branches and pulmonary capillaries up to μm in diameter. electron microscopy showed thickened pulmonary capillaries, pulmonary arterial walls and the basal layers of small veins. . factors that affect the dilation of blood vessels: the mechanisms underlying pulmonary vascular dilatation have not yet fully elucidated, and the possible influencing factors include: (a) increased activity of vascular dilators various acute and chronic liver diseases, liver cell failure and metabolic disorders, particularly reduced inactivation of vasoactive substances which can enter directly into the systemic circulation through abnormal anastomotic collateral vessels, result in disorder of the systemic hemodynamics and increased contents of vasodilators in the blood circulation. just as visceral congestion in patients with portal hypertension, they can act on the intrapulmonary vessels, causing pulmonary vascular dilatation and pulmonary congestion. substances that cause vasodilation include glucagon, prostaglandin, vasoactive intestinal peptide, nitric oxide, angiotensin, bradykinin and endotoxin, etc. (b) reduced vasoconstrictors or decreased sensitivity of intrapulmonary vascular beds to the endogenous vasoconstrictors, such as norepinephrine, endothelin, atrial natriuretic peptide, vasopressin, serotonin and tyrosine, etc. the contents of the substances are not absolutely reduced because maybe their sensitivity is reduced. when chronic liver disease occurs, the anterior communicating branches of the originally closed non-functional capillaries may be opened, and a disorder occurs in the hypoxic pulmonary vascular systolic dysfunction which should have been normal, and it is only % of the normal state [ ] . (c) neurological factors cirrhotic patients show sympathetic nerve hyperactivity, but after the formation of portal hypertension, their sympathetic nerve function may be damaged, which play an important role. animals with portal hypertension often show abnormal pressure responses and reduced sensitivity of blood vessels to norepinephrine, resulting in increased cardiac output, and dilated pulmonary vascular volumes. besides, hemodynamics within the lungs is also a manifestation of the body's hyperdynamics. (d) decreased reactivity of intrapulmonary blood vessels to hypoxia recent inert gas dispersion tests show that cirrhotic patients with over two spider nevus are manifested as not only liver damage, but also decreased systemic intrapulmonary vascular resistance, decreased reactivity of blood vessels to hypoxia and dilated pulmonary vessels. however, it was also found using pulmonary angiography that in spite of the dilated vessels at the ending of arteries, the responses of vessels to oxygen were almost normal, which did not support this view. (e) intrahepatic angiogenesis or dysplasia may also be one of the factors for the formation of hps. to date, the mechanisms underlying pulmonary vascular dilatation caused by hps is still unclear. however, long-term administration of intrapulmonary vasoactive substances can cause significantly increased intracellular cyclic adenosine monophosphate (camp) and/or cyclic guanosine monophosphate (cgmp), resulting in hypoxic pulmonary vasomotor dysfunction and pulmonary artery dilatation, which may be an important cause of this disease and also pulmonary manifestations of systemic hyperdynamic circulation. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, some of the blood around the capillaries in contact with the alveoli can still undergo exchanges with gases, while the central blood, due to the increased diffusion distance from the alveoli, leads to insufficient gas exchange, resulting in insufficient arterial oxygenation and thus a series of hypoxemic manifestations. to date, the pathogenesis underlying the pathogenesis of hps has not yet been elucidated. in view of the above pathophysiological changes and current studies, it is believed that the disease may be caused by insufficient ventilation, diffusion disorder, ventilation/blood flow imbalance and decreased oxygenated hemoglobin affinity, or the above factors in combination. under normal circumstances, insufficient ventilation due to a variety of reasons causes insufficient oxygen inhaled into the alveoli and reduced blood oxygen exchanges, which can result in hypoxemia [ ] , such as chronic bronchitis, foreign bodies in trachea, atelectasis and respiratory muscular paralysis, etc. and the presence of insufficient ventilation in patients with chronic liver disease and cirrhosis or not is still controversial. in , fujiwara studied the lung function in patients with decompensated liver cirrhosis and reported that vital capacity (vc), functional residual capacity (frc) and respiratory reserve volume (evr) in the patients were significantly reduced, that r/t was mildly increased, and that there was no changes in s forced expiratory volume (fev ). therefore, it was believed that mechanical compression and insufficient ventilation due to pulmonary interstitial edema in patients with liver cirrhosis was the main reason for impaired lung function. subsequently, edison et al. studied the pulmonary function of patients with decompensated liver cirrhosis, and found that their vc, maximum ventilation volume (mvv), frc, total lung volume, and r/t were significantly reduced, and they believed that patients with cirrhosis had obvious obstructive and limited insufficient ventilation, which were mainly caused by compression of lung tissue due to increased abdominal pressure, elevated diaphragm and increased chest volume and pressure when patients had additional ascites, and atelectasis [ ] . however, decreased fev resulted from compression of small trachea due to pulmonary interstitial edema and vasodilation, and early closure of expiration. theoretically, all of the above factors can lead to insufficient ventilation, one of the factors resulting in this disease. this was also substantiated by significantly increased arterial partial pressure of oxygen and decreased co partial pressure in cirrhotic patients with pleural effusion after pleural effusion extraction and recovery from atelectasis. however, there are also some people who do not think that hypoxemia results from insufficient ventilation, but because cirrhotic patients are not complicated by high concentrations of co when their arterial partial pressure of oxygen is decreased [ ] . this is likely because when patients have hypoxemia, compensation of hyperventilation causes arterial blood co partial pressure not to increase, and results in decreased paco or even respiratory alkalosis. besides, in some patients without decompensated liver cirrhosis, arterial hypoxemia can also occur. even it has been found that the lung function tests in patients with decompensated liver cirrhosis are normal. therefore, the majority of scholars currently believe that insufficient ventilation is not the main cause of hypoxemia in cirrhotic patients. for patients with hps, the inert gas exclusion technique should be performed to prove that there is a disorder in the diffusion of oxygen, which is determined by the basic pathological changes of hps -pulmonary vasodilatation. pulmonary angiography can show small spider-like to obviously cavernous diffuse vasodilation within the lungs. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, the diffusion distance of the blood flow in central blood vessels and the alveoli is increased, preventing the gases in the alveoli from entering the pulmonary capillaries, thereby affecting the gas exchanges. studies have shown that hypoxemia often occurs in patients with cirrhosis or aggravates during exercises, and it is believed that diffusion disorder or limitation of oxygen occurs in patients. in fact, factors that affect o diffusion do occur in patients with cirrhosis, but they are still not sufficient to explain the apparent hypoxemia. although vascular dilatation occurs at arterial endings in patients with hps, their arterial partial pressure of oxygen can be reduced while inhaling air and increased when they are given oxygen inhalation, which further proves that although diffusion disorder does exist and it plays a role in the formation of this disease, the role is not important. to engage in gas exchanges is the most important biological function of lung tissues, and this gas exchange must be completed when there is an appropriate ventilation/blood flow ratio. under the normal circumstance (normal adult resting state), the most appropriate ventilation/blood flow ratio physiologically is . . changes in the ratio due to any cause can affect the gas exchange, and the imbalanced ventilation/blood flow ratio in hps patients with hypoxemia is mainly because of pulmonary vascular dilatation and arteriovenous shunt [ ] . . intravascular vascular dilatation: pulmonary vascular dilatation has been confirmed pathologically and by angiography. dilated blood vessels in the lungs leads to gas diffusion disorder. besides, since the oxygen molecules in the air can not be diffused to the central dilated blood for the gas exchange, causing decreased ventilation/blood flow ratio and pulmonary arterial partial pressure of oxygen. this decreased ventilation/blood flow, together with increased amount of reactive cardiac output, shortens the duration of blood that flows through the capillary network and insufficient oxygenation [ ] . excessive ventilation can in part enhance patients' pao . if the alveolar oxygen partial pressure is increased at this time, some oxygen molecules can reach the central areas of dilated blood vessels, increasing the arterial partial pressure of oxygen. therefore, it is called the diffusion -perfusion disorder or pulmonary arteriovenous functional shunt rather than the true lung shunt. . arterial -venous shunt: intrapulmonary vascular fistula and pleural spider nevus can occur in cirrhosis of the liver and allow the pulmonary arterial blood to circumvent gas exchange and directly flow into the pulmonary vein so that patients may develop hypoxemia. this hypoxemia can not be corrected by oxygen inhalation and represents the true pulmonary shunt, which has been confirmed by pulmonary histopathology, angiography, transthoracic echocardiography and other examinations. it is now believed that pulmonary vascular casting is still the most direct evidence for determining the arterialvenous shunt. this intrapulmonary arterial -venous shunt is the main cause of abnormal ventilation/blood flow ratio and insufficient gas exchange. although pleural spider nevus can also cause arterial -venous shunt, it generally does not suffice to cause significant hypoxemia due to the small amount of shunt. in addition, studies in recent years also show that portal-pulmonary vein shunt in a small amount occurs in some patients with cirrhosis, in whom blood flow circumvents the alveolar gas exchange and enters directly the systemic circulation. this can also cause ventilation/blood flow abnormalities, causing insufficient gas exchange . airway closure: in , ruff et al. proved that cirrhotic patients had significantly increased closed volume (cv) and total amount of closed gas (cc) and increased gases trapped in the lower fields of the lungs, resulting in an extremely low ratio of ventilation/blood flow, and they believed these were due to reduced airway ventilation [ , ] . in , furukawa et al. measured the lung function of patients with liver cirrhosis and did not find abnormalities; however, most patients had flow -volume abnormalities and significantly increased cv, suggesting the closed the airway in advance and decreased ratio of ventilation/blood flow, which might important causes of hypoxemia. . decreased affinity of oxygen with hemoglobin: some reports showed that patients with cirrhosis (mostly alcoholic cirrhosis) patients had mild systemic vascular or pulmonary vascular dilatation, normal pao , mild hypocapnia, mild right shift of the oxygenated hemoglobin dissociation curve, normal amount of carbon monoxide diffusion [ ] , and mild imbalance of the ventilation/pao blood flow, indicating that the right shift of the oxygen dissociation curve due to decreased affinity of oxygen with hemoglobin in the patients. this was possibly caused by the increased concentration of , -diphosphate glyceride in red blood cells, which, however, is not an important factor in the occurrence of hypoxemia [ ] . in summary, hypoxemia can result from many factors, while none of the factors can completely explain the pathogenesis underlying the disease. since the basic pathological changes in patients with hps are intrapulmonary vascular dilatation and opening of arterial -venous communicating branches, together with recent findings, it is suggested that the diffusion disorder of the alveoli and pulmonary capillaries and ventilation/blood flow imbalance may coexist, and are the main cause of hypoxemia in this disease. other factors may aggravate hypoxia and are secondary factors. therefore, it is believed that the disease occurs as result of the above factors. the pathological features of hps are dilation of capillaries in the anterior aspect of the lungs and telangiectasia. autopsies show arterial-venous short circuit within the lungs, vasodilation and thickened pulmonary muscles [ ] . at the same time, arterial hypoxemia is common in liver diseases, often attributable to a variety of factors (such as ascites, hepatic pleural effusion, and copd in patients with alcoholism); it shows unique pathophysiological characteristics under specific circumstances of hps. its prominent features are dilation of micro-arteries in the anterior aspect of pulmonary capillaries and true capillaries (the normal diameter of these vessels is to μm, which can reach - μm when patients rest), with the number of dilated vessels increased macroscopically. some patients show arterial-venous communicating between the pleura and lungs, vascular anastomosis in the liver and the lungs, and thickened walls of small veins and capillaries. pulmonary vascular dilatation is promoted, and mixed venous blood quickly or directly enter the pulmonary veins through the anastomosis in the lungs, leading to oxygenation defects. increased nitric oxide (no) is a key cause for pulmonary vasodilation, and whether other mediators, such as heme oxygenase-derived carbon monoxide, are causes of pulmonary vasodilatation are not yet confirmed. abnormal arterial oxygenation seriously affects the survival of patients, and is an important indicator that determines the timing and risk of liver transplantation as well as an important basis for the grading of severity of hps. causes of deaths associated with hps are often multifactorial and are associated with basic liver diseases, and there are few cases of respiratory failure due to severe hypoxemia. hps is a triad composed by intrapulmonary vascular dilatation and insufficient arterial oxygenation due to primary liver disease, and it is mainly clinically manifested as primary liver disease and pulmonary lesions. hps can occur in various liver diseases, mostly in chronic liver disease, especially cirrhosis caused by various causes, such as cryptogenic cirrhosis, alcoholic cirrhosis, liver cirrhosis, viral cirrhosis, postnecrotic cirrhosis and biliary liver cirrhosis, etc. the most common clinical manifestations include liver palms [ ] , spider nevus, jaundice, ascites, hepatosplenomegaly, gastrointestinal bleeding and abnormal liver function, etc., while they are not significantly correlated with hps. some patients with clinically stable liver disease may also develop the clinical manifestation of progressive pulmonary insufficiency. since hps patients have no primary cardiopulmonary diseases, most ( - %) patients gradually develop respiratory manifestations on the basis of various liver diseases, such as cyanosis, dyspnea, clubbing, orthodeoxidation and platypnea, etc. among them, progressive dyspnea is the most common lung symptoms of hps. binay et al. believed that cyanosis was the only reliable clinical sign, and that platypnea and orthostatic hypoxia are the most characteristic manifestations. pulmonary examinations generally show no obvious positive signs [ ] . a small number of patients (about - %) can present complaining dyspnea on exertion in the absence of clinical manifestations of a variety of liver diseases, to which attention should be paid clinically so as to prevent misdiagnosis. the domestic researchers gao zhi et al. reported that patients presented to hospitals with cyanosis, palpitation after exercises and shortness of breath; meanwhile, they found that the patients had clinical manifestations of liver cirrhosis [ ] (such as liver palms, spider nevus, hepatosplenomegaly and ascites), which were conducive to the diagnosis of this disease. if liver disease patients have other lung diseases (such as chronic bronchitis, emphysema and pneumonia, and pleural effusion, etc.), then significant respiratory symptoms may occur. therefore, differential diagnosis should be made. data show that the duration of initial dyspnea to the conformed diagnosis in patients with hps average - years; that is to say, about % of patients already have dyspnea at the time confirmed diagnosis. . orthodeoxidation: pao is decreased by > % when patients switch from the supine position to the standing position. . platypnea: when patients switch from the supine position to the standing position, they have palpation, chest tightness and shortness of breath, and when patients resume the supine position, the above symptoms are improved [ ] . krowka reported that about - % of patients with hps had the above two manifestations because vascular dilatation in the patients was mainly distributed in the middle and low lung fields. when patients switch from the supine position to the standing position, the blood flow in the middle and lower lobes of the lungs is increased under the action of gravity, aggravating hypoxemia [ ] . although the two manifestations are not unique to hps, they suggest the significant abnormality in the patients' pulmonary vascular system. if patients with a variety of liver diseases present with the above two manifestations, further examinations are needed for confirmation. patients may present with liver palms, hepatosplenomegaly, spider nevus and ascites; patients show palpitation, chest tightness, shortness of breath when switching from the supine position to the standing position due to hypoxemia. schenk et al. defined the values of pao for the diagnosis of hps, thinking that pao < mmhg suggested a high possibility of hps, and that for pao < mmhg, the diagnosis of hps could be made [ ] . pulmonary function tests mainly showed significantly decreased vc, frc, mvv and fev , but sometimes the total lung volume and fev were normal. chest x-ray, ct scan and transthoracic contrast echocardiography (ttce) hps patients are mostly normal on chest radiography or show diffuse small millet shadows predominantly in both lower lobes of the lungs, nodular shadows in both lower lung interstitium, dilated pulmonary arterial trunks, and thickened pulmonary markings, whereas these manifestations have no specific values to the diagnosis of this disease. ct scan shows certain diagnostic values in that it demonstrates distal vasodilation and even pleural blood vessels, and can suggest the presence of hps. arteriovenous communicating occurs in hps patients due to pulmonary vascular dilation, indicating that subclinical pulmonary vascular dilatation and abnormal gas exchanges occur in cirrhotic patients with normal pao [ ] . contrast echocardiography: when hps is suspected, transthoracic echocardiography can be used as a preliminary screening to determine whether the intrapulmonary vascular dilation occurs or not. the microbubble contrast material in the right atrium, after intravenous injection of dioxane isotonic saline, will develop images in the left atrium through the dilated vascular beds after - cardiac cycles, while the microbubbles cannot pass through the normal capillaries (normal capillaries are < - μm in diameter). approximately % of patients with cirrhosis had positive changes on contrast echocardiography, while only a small proportion of patients are in line with the diagnosis of hps due to the influence of dilated blood vessels. if contrast echocardiography is positive for liver cirrhosis or portal hypertension patients with hypoxemia and cardiopulmonary diseases in them can be ruled out, then the diagnosis of hps is established. this means is used to confirm the diagnosis of intrapulmonary vasodilatation. the pulmonary vascular abnormalities in hps patients are as follows: ( ) diffuse spider nevus images. patients of this type have severe hypoxemia and erectile hypoxia and respond well to inhalation of % oxygen; ( ) cavernous or spotted arterial dilatation mainly seen in the basal aspect of the lungs. patients during this period respond poorly to % oxygen; and ( ) intermittent local arterial malformation or communicating branches, isolated earthworm-like or bulk images. in addition to severe hypoxemia and erect hypoxia, patients of this this type respond extremely poorly to the inhalation of % oxygen. when hypoxemia is caused by hps and cardiopulmonary diseases concurrently, cm tc-maa scan can determine hypoxemia resulting from hps more likely. radiolabeled albumin cm tc, which is administered through intravenous injection, is about pm in diameter. when pulmonary vascular shunt occurs, a proportion of the polymerized albumin passes through the lungs and enters the systemic circulation, the intake of albumin by other organs can be simultaneously determined by scintigraphy [ ] . therefore, the amount of shunt can be calculated. a study showed that cm tc-maa scan was positive for hps patients with pa < mm hg, while the scan was negative for chronic obstructive pulmonary disease (copd) patients with the same degree of hypoxemia, a result indicative of the good specificity of this means. compared with contrast echocardiography, cm tc-maa scan, in spite of its low sensitivity, can be used for the diagnosis of hps in patients with copd. pathological examinations are the most reliable means for the diagnosis of hps, whose basic pathological change is pulmonary vasodilation manifested as diffuse anterior capillary dilation or discontinuous formation of arteriovenous branches [ ] . in addition, pulmonary perfusion scan and right cardiac catheterization are also valuable for the diagnosis of hps to a certain extent. there is no unified standard for hps diagnosis to date. diagnosis should be based on clinical manifestations plus imaging evidence of pulmonary angiography. (c) the domestic scholars gao zhi et al. thought in that the diagnosis of this disease should be based on the following manifestations in patients, hepatosplenomegaly, ascites, liver palms, spider nevus, dyspnea on exertion, hypoxia while breathing in the supine and orthostatic positions, increased mesenchyma in the basal aspects of the lungs and vascular markings on chest radiography, patchy or nodular shadows, dilated basilar pulmonary vessels and increased pulmonary vascular branches on ct, severe hypoxemia or not on blood gas analysis, increase in alveolar -arterial oxygen gradient by ≥ kpa ( mmhg), and % diffusion disorder on pulmonary function tests [ ] . in addition, shunt-related examinations should be performed, such as cm tc-maa scanning, contrast-enhanced two-dimensional echocardiography and pulmonary angiography, etc., while the last one does not show the same sensitivity as that of the former two because the small blood vessels in the lungs may not develop on angiography. most hps patients have a slow onset of the disease, are difficult to treat, and have a poor long-term prognosis, with a mortality of more than % after years. therefore, early diagnosis of the disease and its differential diagnosis are vital to improving the prognosis of patients. first of all, the previous liver and lung diseases in the patients should be ruled out, such as chronic obstructive pulmonary emphysema, pulmonary infection, interstitial pneumonia and silicosis, etc. at the same time, cirrhosis with pulmonary hypertension, infections secondary to pleural effusion, interstitial pulmonary edema, atelectasis and hyperventilation syndrome, etc. need to be ruled out. hps should be differentiated mainly from the following diseases: . liver cirrhosis following pulmonary heart disease: this is mainly because pulmonary diseases result in cardiac insufficiency and thus increased pulmonary venous pressure. repeated or long-term existence of liver congestion can lead to central venous hypertrophy and lobular central connective tissue hyperplasia, and further progression of the lesion will lead to the formation of liver cirrhosis following pulmonary heart disease. patients with pulmonary cirrhosis often have a long history of chronic lung disease and signs of cardiac insufficiency, such as edema of lower extremities, palpitation, shortness of breath and other symptoms. this patient has no history of chronic lung disease or edema of lower extremities, making him inconsistent with liver cirrhosis following pulmonary heart disease. . left heart insufficiency: both hps and left heart insufficiency can cause severe dyspnea and hypoxemia. a history of liver disease or evidence of chronic liver damage and decreased po can be found in patients with hps, especially such characteristics as orthostatic hypoxemia and intrapulmonary vascular dilatation. patients with left heart insufficiency have a history of heart disease, orthopnoea, pink frothy sputum and moist rales in the lungs, etc. this patient is inconsistent with such manifestations. . primary pulmonary hypertension: after inhalation of pure oxygen, hypoxemia in most of patients with hps will be significantly alleviated. the effects of oxygen are poor in patients with hypoxemia [ ] , and hps is manifested as orthostatic hypoxemia. the characteristics of hemodynamics of patients with hps are hyperdynamics and normal or decreased pulmonary artery pressure and pulmonary vascular resistance, while those in hypoxemic patients are increased. . others: ductus arteriosus, eisenmenger syndrome and pulmonary embolism, etc., need to be differentiated from this disease, and comprehensive judgments should be provided based on other clinical data of the medical history. hps patients can also have the above-mentioned diseases, and careful and meticulous examinations are needed for differentiation. because hps is developed on the basis of original liver disease, the frequency of its occurrence and its severity are mostly associated with the liver cell function of patients, while there are also hps patients in whom chronic liver disease is relatively stable and liver functions are normal. besides, pleural effusion, ascites and infections secondary to pulmonary edema after liver function decompensation can aggravate patients' respiratory function injury. therefore, under the current circumstances in which there are no effective measures for hps, active and effective treatment of primary liver diseases is the basis for the treatment of hps. therapy of primary diseases, including correction of hypoproteinemia, elimination of pleural effusion, improvement of liver function and treatment of complications, etc., can improve tissue oxygenation and improve arterial oxygen saturation. on this basis, the following treatment can be given. oxygen therapy also helps the differential diagnosis of pulmonary shunt: if pao is resumed after oxygen inhalation, then the diagnosis of intrapulmonary vascular dilatation (ipvd) can be made; for patients with partial improvement, pulmonary anatomical shunt and functional shunt may coexist; for patients in whom the oxygen therapy proves inefficacious, pulmonary arteriovenous fistula is a possible diagnosis. it is now believed that once the diagnosis is established, treatment should be given as soon as possible. in the early stage of correcting hypoxemia in patients with mild conditions, even in patients in whom the critical value of hypoxemia (pao , - kpa ( - . mmhg) is reached and who have ascites, the hemoglobin saturation may still be less than % when patients are in activities or even sleep. that is to say, nasal catheter oxygen inhalation at - l/min is needed so as to improve hypoxemia [ ] . with the development of the disease, oxygen flow needs to be gradually increased, and intratrachea oxygen supply can be offered when necessary. during the late stage, patients can receive pressurized oxygen through a ventilator or a hyperbaric oxygen chamber. for patients whose conditions are severe, the efficacy of oxygen therapy alone is not obvious. . vasoactive drugs vasoactive drugs for the treatment of patients with hps are most studied; however, since its pathogenesis has not been clarified to date and primary liver disease is difficult to reverse, it is hard to define the clinical efficacy of these drugs. the commonly used drugs include: used aerosolized ephedrine hydrochloride for the treatment of patients with hps, and the preliminary efficacy was significant. the mechanisms were that ephedrine could excite the pulmonary vascular α receptor, resulting in contracted bronchial mucosa and pulmonary capillaries and alleviated bronchial edema, so that the dilated blood vessels within the lungs were contracted and intrapulmonary shunt was reduced. meanwhile, the bronchial β receptors were excited and the bronchi were dilated so as to improve the ventilation/blood flow ratio and relieve hypoxia. further studies are merited. (f) others: there have been reports on sympathomimetic drugs (isoproterenol) and β-blockers (propranolol), etc. that improve the symptoms of hps. theoretically, vascular endothelin, estrogen suppressor (tamoxifen) and so on can relieve the spider nevus and pulmonary vascular dilatation in patients with liver cirrhosis and improve their respiratory symptoms, while further studies are needed. no is most studied currently, and there are reports indicating that no synthesis inhibitors can increase pulmonary vascular resistance. alexander et al. used no for the treatment of severe hypoxemia in patients after liver transplantation, and obtained good results. durand et al. also reported that an hps patient was cured by inhaling no, while its mechanisms and clinical efficacy needed to be further confirmed. . pulmonary embolism it is generally considered that pulmonary vascular dilatation can vanish after liver transplantation in hps patients who are normal on pulmonary angiography or who have cavernous vessels on imaging [ ] ; for patients who show diffuse pulmonary vascular dilation features on pulmonary angiography, embolization is usually not adopted since patients' lesions are extensive and the efficacy is poor; for patients with isolated and severe pulmonary vascular dilation or arterial-venous communicating branches, local pulmonary embolism therapy can yield a satisfactory effect. . liver transplantation it is currently considered that liver transplantation is still a possible fundamental measure for the treatment for hps. in the past, it was believed that serious hypoxemia was an absolute contraindication against liver transplantation, while recent studies show that liver transplantation is preferred for patients who have good alveolar gas diffusion function, who can respond well to pure oxygen inhalation and who can undergone oxygenation safely during anesthesia. recent reports further prove that hypoxemia can be cured after liver transplantation [ ] . through literature review and case reports, krowka et al. believed that progressive hypoxemia in hps could be used as an indication of liver transplantation. temporary hypoxemia following liver transplantation can be adjusted by using no and taking the head-down supine position and the alternate lateral decubitus position. and for hps patients who fail to respond to the inhalation of pure oxygen, who have direct pulmonary arterial communicating branches on pulmonary angiography and who have severe clinical hypoxia, liver transplantation cannot improve their hypoxic status, has limited efficacy, or even increases intraoperative and postoperative risks. therefore, liver transplantation should not be performed on them. tips was an effective method for the treatment of hps, and its effects of improving symptoms, enhancing oxygenation and reducing intrapulmonary shunt could last up to months. riegler et al. performed tips on an hps patient with diffuse intravascular dilatation who was not suitable for vascular embolization, and the results showed significantly increased pao and significantly improved hypoxemia. however, coley et al. also reported that a patient failed to respond to tips, and therefore, its exact effects remain to be studied. . other treatment options one patient with hps was once treated with garlic, and months later, his oxygenation was significantly improved and his symptoms were relieved. there are also patients who receive plasma replacement therapy, which has limited effects on the oxygenation of patients with hps [ ] . to sum up, no effective treatment options are currently available for hps. since the basic cause of hps is liver cell failure, the usual cause of patients' deaths is not lung failure, mostly complications such as gastrointestinal bleeding, renal failure, hepatic encephalopathy and sepsis. therefore, we consider that the therapy of primary liver disease is particularly important. oxygen inhalation alone can be given in the early stage of hypoxemia, or conservative treatment can be provided if additional drugs are effective. liver transplantation is the best solution whenever possible. it is generally accepted that liver transplantation is the most promising regimen with confirmed efficacy. if oxygen inhalation is less satisfactory and patients are diagnosed with local intrapulmonary vascular dilatation or arterial -venous fistula by such means as pulmonary angiography, pulmonary embolism should be carried out as soon as possible. for patients with additional obvious portal hypertension, tips treatment can also be given. the interval from chronic liver disease and cirrhosis in patients to the confirmed hps due to such respiratory symptoms as anoxic dyspnea is usually several years or even more than years [average interval, ( . ± . ) years], and a small number of patients can develop such a disease acutely in the short term. besides, signs of chronic liver disease can be traced in patients complaining breathing difficulties. once hps is established, obvious hypoxemia has occurred already. it confers a poor prognosis, and most patients die within - years often due to other complications of liver disease [ , ] . if patients' oxygenation is satisfactory and they have undergone liver transplantation, or with the improvement in liver function, their hypoxemia can be resolved or improved of its own volition with good prognosis. if patients' oxygenation deteriorates severely and they have a very poor prognosis, most of them will die in the short term. hps often progresses slowly. although it is not a direct cause of death in patients with cirrhosis, it can significantly aggravate the disease. therefore, cirrhotic patients, especially those with positive liver palms and spider nevus as well as patients with portal hypertension, should be careful of the possibility of hps. timely detection and symptomatic treatment (such as low flow oxygen inhalation) can improve the prognosis of patients. active and effective treatment of primary liver disease forms the basis for the prevention of this disease. education of common sense should be given to patients with liver diseases so as to avoid factors inducing hps in their life. for patients with liver disease, mild hps should be found as soon as possible and appropriate treatment should be given. jia-quan huang and dong xu lipopolysaccharide (lps) is a constituent of bacteria cell wall which plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as nitrous oxide, cytokines, superoxide anions, and lipid mediators. despite the recent advances in antibiotic treatment and hemodynamic monitoring, septic shock still remains a serious disorder that is associated with a high mortality rate, to more comprehensive definition of the mechanisms that underlie innate immunity against bacterial pathogens, lps has been extensively studied [ ] . the pathophysiological consequences of bacterial sepsis are contributed by the dysregulation of these same mechanisms. before we can hope to design effective anti-sepsis therapies, greater insight into the nature of host interactions with lps is extremely essential. the gram-negative bacterial envelope is composed of two bacterial membranes, outer and inner membrane. the outer membrane consists of the following substances, like lipopolysaccharide (lps), several kinds of outer membrane proteins, lipid a and metal ions. for most gram-negative bacteria, lps is a major component in the outer monolayer of the outer membrane which works like a tight shield. the shield is composed by unique molecules, such as polysaccharide, or long chain of sugar, and lipid a. during the process to evoke the signaling events of lps, lipid a plays a pivotal role. the entire lipid component of lps molecule, however, is required for optimal activity [ ] . the basic principles of lps bioactivity are nowadays well understood. endotoxins do not elicit their toxic effects -as we might suspect and as it is known for many proteinous exotoxins which can kill host cells or inhibit cellular functions. rather, lps requires the active response of host cells. according to present knowledge we get, lps interacts with various host cell types through lipid a, those cells include mononuclear cells, thrombocytes, endothelial and smooth muscle cells, and polymorphonuclear granulocytes, among which macrophages/monocytes are of particular importance. through the lps-induced activation, macrophages produce many substances, like bioactive lipids, reactive oxygen species, and in particular, cytokines such as tumor necrosis factor a (tnf), interleukin- , il- , il- , and il- . it appears that when low levels of mediators are produced, beneficial effects (e.g., induction of resistance to infection, adjuvant activity) are elicited and when high levels of mediators reach the circulation that detrimental effects (e.g., high fever, hypotension, irreversible shock) are induced. however, when the host organism is in a hyperreactive state lps, low mediator concentrations may also become harmful. the hyperreactivity to endotoxin may be caused by exo-toxins, chronic infection, and by growing tumors, and interferon-γ. to function properly, organism requires an immune system that must detect pathogens, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. the immune system can be classified into humoral immunity versus cell-mediated immunity or the innate immune system versus the adaptive immune system. when microbes invade organism, the innate response is usually triggered by pattern recognition receptors, which recognize components that are conserved among broad groups of microorganisms, or when injured, damaged, or stressed cells send out alarm signals, many of which (but not all) are recognized by the same receptors as those that recognize pathogens. the innate immune system defenses are nonspecific, meaning that the system responds to pathogens in a generic way. this system does not confer long-lasting immunity against a pathogen. in most organisms, the innate immune system is the dominant system of host defense [ ] . they activate innate immune responses by identifying some conserved non-self molecules, so as to protect the host from infection,. bacterial lipopolysaccharide (lps), an endotoxin which is found on the bacterial cell membrane, is considered to be the prototypical pamp. lps is specifically recognized by toll-like receptor (tlr) , a recognition receptor of the innate immune system. the interaction of the lipid a moiety of lps with macrophages appears to be especially important because subsequent cellular activation results in the release of systemically active pro-inflammatory molecules, which in turn mediate systemic toxicity. lps has extreme potential in macrophages activated at concentrations of lps as low as pg/ml [ ] . host-defense peptides (hdps) could be a possible alternative solution since they possess the antimicrobial, antiseptic, and immunomodulatory properties [ ] . endotoxins lipopolysaccharide is released not only from dead gram-negative bacterial, but also from the growing ones. endotoxins are very stable molecules, which are resisted to extreme temperatures and ph values in comparison to proteins. endotoxins are shed largely during cell death as well as growth and division. they are highly heat-stable and are not destroyed under regular sterilizing conditions. endotoxin can be inactivated through exposed at temperature of ° c for more than min or ° c for more than h. acids or alkalis of at least . m strength can also be used to destroy endotoxin in laboratory scale [ ] . gut microbiota is composed of strict anaerobes, facultative anaerobes and aerobes. recent reports suggest the existence of over , bacterial species in the human gut microbiota. an important characteristic of gut microbes is their heterogeneity [ ] . the composition and the frequency of the microbiome changes with the different segments of the elementary tract. the composition is influenced by the environment, consumed diet and host factors. endotoxin to surrounding tissues and organs or blood shift that shift pathway include: ( ) via the portal vein, liver into the systemic circulation; ( ) through the intestinal tract into the lymphatic system lymphatic; ( ) through the intestinal wall into the peritoneal cavity and then absorbed into the bloodstream. under physiological conditions, although a small amount of toxins continued to parenteral shifted via the portal vein into the liver, but it does not cause endotoxemia; mild in gram-negative bacilli infections, although bacteria continue to release to the tissue or blood endotoxin, but it does not give rise to a strong inflammatory response, the above are dependent on the presence of an effective mechanism within the body to remove toxins and detoxification. the liver is the main organ of clearance of endotoxin, and the spleen, also removes toxins. molecules in lps removed include cationic antimicrobial peptides (cationic antimicrobial peptides, cap), acyloxy acyl hydrolase (acyloxyacyl hydrolase, aoah) lipoprotein binding protein and anti-lps antibodies are important endotoxin clearance methods [ , ] . liver blood endotoxin clearance, primarily through kupffer cells, hepatocytes internal toxin endocytosis achieved, but the specific metabolic process is not entirely clear. mediated by kupffer cells engulf toxins may be scavenger receptor, it may be a molecular weight of , and , protein; mediate phagocytosis liver toxin structure may be the lectin-like receptor (lectin-like receptor). endotoxin receptor hepatocyte sinusoidal plasma membrane on the surface: after one to one binding, is taken up within the liver cells endocytosis way to microtubule-dependent vesicular transport through the liver cells, transported to the liver cells bile duct surface to exocytosis into the bile duct, and then discharged into the biliary system through the intestine [ ] . splenic macrophages containing approximately % of the body to settle within the organization, and macrophages are important endotoxin removal cells. when endotoxin intravenously into the body, except gathering in the liver, a lot of endotoxin can be quickly gathered and taken up into macrophages in the spleen, the spleen and therefore equally important endotoxin removal organs. in addition to its clear role in the performance of its direct effect, but more importantly, spleen macrophages is the precursor cells of kupffer cells in the liver, having a very big impact on removing toxins within the liver [ ] . mechanisms for removing toxins in the intestine are related to the intestinal villus tip epithelial cells. under normal circumstances, injected into the intestinal, endotoxin in intestine does not enter intestinal epithelial cells, but after intravenous injection of endotoxin, endotoxin may enter intestinal epithelial cells inside. therefore, endotoxin receptor may identify ways by endotoxin and (or) simple diffusion way into the intestinal epithelial cells. endotoxin way into the intestinal epithelial cells intravenously may have two: ( ) displaced from the lamina propria macrophages to intestinal epithelial cells basolateral; and enter intestinal epithelial cells from the side; ( ) including lower toxin, intestinal lamina propria macrophages, intestinal epithelial cells release large amounts of no and oxygen free, resulting in intestinal lamina propria microvascular injury, increased permeability, extravasation of endotoxin and ultimately displaced into the outer intestinal epithelial cells. villus tip epithelial cells within a stronger uptake of toxins, thus endotoxin can be started from the crypt, moving along the intestinal villi, and finally to the top of the inner hair cells of the intestinal epithelium. uptake of endotoxin villus tip epithelial cell loss, while the endotoxin into the intestine, which constitutes one of the effective clearance mechanisms of endotoxin [ , ] . plasma lipoproteins in endotoxin detoxification mechanisms play an important role, in which the lipopolysaccharide binding protein (lipopolysaccharide binding protein, lbp) and high-density lipoprotein (high density lipoprotein, hdl) play a major role. endotoxin into the bloodstream within minutes there were half white blood circulation due to binding to the edge of the pool or the pool is cleared, the remaining residual endotoxin and rapidly bound to plasma lipoprotein is inactivated. in plasma, lipoproteins and endotoxin when hdl plays a major role in its binding of endotoxin to reach more than % of the total, hdl, and thus research endotoxin important [ ] . cationic antimicrobial peptides are an ancient ingredients in the natural evolution of the immune system, including bactericidal/permeability-increasing protein (bactericidal/permeability-increasing protein, bpi), cathelicidin, lactoferrin, defensins and other substances, with not only the activity against gram-negative bacteria, but also the ability to combine internal toxins. cationic antimicrobial peptides are mainly in regular contact with the pathogen site mammalian skin, digestive tract, respiratory tract, and inherently express or express induced by pathogens and their products in the blood, secretions and neutrophil granules. cationic antimicrobial peptides have two types of three-dimensional structure; one is a α-helix, having such a molecular structure include cathelicidin and lactoferrin; the other is β-fold, including mammals α and β-defensins, etc. [ ] aoah aoah is a glycoprotein produced by white blood cells with weight of . - , , the large subunit of , and small subunits of . million to , , the large and small subunits connected by covalent disulfide bond. aoah as a lipase with hydrolysis for toxin, can selectively hydrolyze the secondary acyl chain on lipid a acyl groups acyloxy. when hydrolyzing endotoxins, both the large and small subunits of aoah play an important role, and both are indispensable. in addition to directly destroying toxin, the deacylated lps after the hydrolysis of endotoxin by aoah is also involved in aoah's detoxification mechanism on endotoxin; the material can accumulate and inhibit endotoxin-induced inflammatory response in the cell. however, due to a limited number of secretion by local infiltration of leukocytes, the internal detoxification of toxins is also limited [ ] . when the body respond with endotoxin, one trigger inflammation, on the other hand can be cleared to produce or activate, specific polysaccharide inactivate toxins, including antimicrobial-specific polysaccharide antibody and anti-core polysaccharide antibody. after two antibodies binding with endotoxin, and then with the fc receptors on the cell membrane, inner source of the toxin-mediated, so that the endotoxin inactivated intracellularly. anti-endotoxin antibodies can interfere with toxin within lbp binding, thus preventing lbp endotoxin transport [ ] . in the body's defense system, the shift from the inhibition of intestinal endotoxin ingredients include intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, the normal intestinal flora and liver hepatocytes and kupffer cells, in which intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, hepatocytes and kupffer cells play a direct inhibitory effect, while the normal flora plays an indirect inhibition. the intestine is huge "endotoxin library", a special anatomical location determines the intestinal mucosa must be an effective defense barrier. immunological barrier intestinal barrier formed by the epithelial cells of mechanical barrier and secretory iga (siga) and the like components [ ] . intestinal epithelial cells and tight junction formation mucosal mechanical barrier, is a significant barrier in the intestine endotoxin translocation defense to maintain its integrity is it to play an important role in defense guarantee. in severe trauma, burns, infection, considerable loss of body fluids, hypovolemia, cause the body ischemia and hypoxia. in order to maintain blood pressure, to ensure that the blood supply to the heart brain and other vital organs, compensatory splanchnic vascular contraction, including gastrointestinal ischemia and hypoxia longer time than other organs, even after shock patients after resuscitation to restore normal hemodynamics, stomach intestinal still in a state of shock occult. therefore, when the intestinal microvascular perfusion recovery, intestinal ischemic/reperfusion injury, epithelial cells produce large amounts of reactive oxygen species and other media, resulting in intestinal epithelial cell apoptosis, destruction of tight junctions between cells, thus rapidly increasing intestinal permeability mechanical barrier function weakens, thus contributing to the intestine of the endotoxin absorbed through the intestinal wall to parenteral tissue displacement [ ] . intestinal mucosal intestinal immunology barrier is a defense of the invasion of pathogens and endotoxin important line of defense, siga plays an important role in intestinal mucosal immunity. siga is an important component of the protection of the intestinal mucosa, both to prevent bacteria in the intestine mucosal surface colonization, but also in endotoxin. studies have found that e. coli o infection suffered intestinal mucosa, the anti-endotoxin core polysaccharide-specific siga secretion, in convalescent patients has been particularly evident, suggesting siga endotoxin to prevent the transfer of the body has a protective effect. in addition, studies suggest that nitric oxide (nitrogen monoxide, no) preventing endotoxin translocation in intestinal mucosal barrier oxide formed locally. under physiological conditions, nitric oxide synthase (inducible nitric synthase, inos) expression only in the respiratory epithelium, the pregnant uterus and ileal mucosa and a few other parts. induced by endotoxin including but under normal colonic epithelial cells also express inos and catalytic synthesis of no, are formed in the local oxidation barrier to prevent bacterial translocation colon, thus effectively preventing bacterial translocation, also indirectly prevents endotoxin shift [ ] . under physiological conditions, intestinal flora forms a relatively balanced microecosystem. flora distribution in the intestine has certain rules: deep close to the intestinal mucosal surface, parasitic anaerobic bifidobacteria and lactobacilli, these anaerobic bacteria are sugar coated, relatively stable, known as membrane flora; middle class bacteria, streptococcus digest, veillonella and excellent bacilli; the surface of e. coli and enterococci, can swim in the intestine, known as cavity flora. the antagonism between the layers flora, mutual cooperation, to maintain a dynamic equilibrium, in which the film anaerobic flora is a very important body's natural defense barrier that can prevent opportunistic pathogens such as e. coli colonization in the mucosa, but also can inhibit the overgrowth of opportunistic pathogens. intestinal flora micro-ecosystem is a very sensitive system, in severe stress or long-term systemic administration of large doses of broad-spectrum antibiotics, etc., the film significantly reduced the number of anaerobic bacteria, e. coli and other bacteria thrive conditions and continuous release of endotoxin to the intestine, since the film flora defense decreased, these opportunistic pathogens to colonize the intestinal mucosa, resulting in intestinal mucosal barrier damage, followed by the occurrence of intestinal bacteria, endotoxin translocation [ ] . under normal circumstances, the liver is one of the major barriers preventing endotoxin translocation, via the portal vein into the liver hepatocytes and a small amount of the toxin can be kupffer cell depletion. in conditions such as stress, not only liver cell dysfunction, so the ability to reduce endotoxin detoxification and collaterals between the portal vein and the vena cava, causing an overflow of endotoxin from the liver into the systemic circulation. endotoxin absorbed into the bloodstream, which in turn may increase the intestinal epithelial cells of the intestinal microvascular endothelial cell damage and, in a vicious cycle [ ] . when the body's defense system to produce responses in endotoxin, the innate immune system plays a major role. pathogens can be identified conserved receptors called pattern recognition molecules (pathogen-associated molecular patterns, pamps), including endotoxins of gram-negative bacilli, peptidoglycan grampositive cocci, lta and other cell wall composition and gram-negative bacteria, gram-positive bacteria such as dna. although a variety of pattern recognition molecules of different chemical structures, but they have similar characteristics: ( ) characteristic structure in which different types of pathogens in a relatively constant conserved; produced by a pathogen, the host body without these molecules; survival or disease-causing pathogen is generally the essential, such as mutations, death or loss of a pathogen will pathogenicity. natural immune system to recognize the receptor molecule called pattern recognition receptors (pattern-recognition receptors, prrs), including cd , toll-like receptor family (toll-like receptor, tlrs) and scavenger receptors. but in recognition of toxins, some differences exist between the different kinds of cells [ ] . macrophages in addition to expressing cd , tlrs and scavenger receptors and other associated endotoxin receptors on the cell surface, but in the cytoplasm also express the protein molecules nod recognizing toxins. cd , tlrs are key receptors that mediate endotoxin within macrophage activation; and scavenger receptor has relationship with macrophage clearing and inactivating toxins [ ] . kupffer cell is the main cell that clears the endotoxin in the liver. under physiological conditions, although there is still a small amount of bacteria and endotoxin via the portal vein into the liver, but kupffer cells will clear. kupffer cells are the most resident macrophages in the liver and are the largest number of resident cells in tissues. there is a theoretical speculation that if kupffer cells and on is very sensitive to endotoxin as other macrophages, the cell will be in constant activation, but in fact when kupffer cell engulfs, removes endotoxin, its itself is not activated by endotoxin, which suggests that in the treatment of endotoxin, kupffer cells have different mechanisms with other macrophages: kupffer cells treats endotoxin mainly depending on its phagocytosis. in the absence of serum, the phagocytic effects of kupffer cell on endotoxin can play a normal; and with the appropriate increase in endotoxin concentration, the phagocytic activity of kupffer cell was enhanced. the effect has something to do with phosphorylation events of two protein tyrosine residue individually weighting . million and . million. cd is the main receptor that mediates endotoxin activating kupffer cell, scavenger receptor is kupffer cells' important defense of receptor mediated kupffer cell to remove and inactivate endotoxin. there are four stages of the activation of kupffer cells, of which cd is the characteristic marker of cellular activation and function change. ( ) the stationary phase; the performance of less number of kupffer cells, small shape, a number in the hepatic sinusoids, cd staining negative; ( ) reaction period: for the local kupffer cells stimulate hyperplasia and systemic mononuclear macrophage intrahepatic accumulation; ( ) pre excitation period: kupffer cell phenotype occurred transformation period, expressed cd cell membrane receptor, kupffer cell functional changes; ( ) the activation period: the performance of nuclear transcription factor nf kb activation, cellular secretion cytokines [ ] . the cd (cd membrane-bound, mcd ) and tlr endotoxin were activated by the neutrophil surface to activate the neutrophils by binding with the receptor. in addition to the expression of the high affinity endotoxin receptor cd , the surface of the neutrophils also expressed the low affinity endotoxin receptor l-and the activated cells were activated by the receptor. in addition, the integrin is considered to be a low affinity endotoxin receptor for the surface of the neutrophils [ ] . it is generally believed that the expression of mcd was not on the surface of endothelial cells and serum soluble cd (soluble cd (scd ) is mediated endothelial cell recognition of lps molecules, and tlr is involved in lps induced endothelial cell activation. lbp was transported to scd by endotoxin, and tlr was activated by lps/scd and activated endothelial cells in the endothelial cell membrane. scd in addition to mediated endothelial cell activation and also mediated by endothelial removal of endotoxin and lps/scd /lbp form trimers and binding to endothelial cells, following the lps/scd endogenise, thus the removal of endotoxin [ ] . the epithelial cells of the intestinal mucosa were consistently associated with the bacterial and its products, and the bacteria and its products could stimulate other types of cells and induce inflammatory response, but did not induce intestinal epithelial cell defense, this feature for colonic epithelial cells is particularly important, because if can react to the normal intestinal flora in intestinal epithelial cells, it will cause adverse effects on the body. but this does not mean that intestinal epithelial cells are immune cells, when suffered pathogens and their products invasion, intestinal epithelial cells produce normal response. description: intestinal epithelial cells with normal differentiation of natural bacteria and pathogenic ability, and the recognition system of subcellular localization. there are different endotoxin recognition mechanisms in the intestinal epithelium and the myeloid cells [ ] . uncontrolled inflammatory responses (uncontrolled inflammatory response) has a relationship with infection, bacteremia, septicemia, sepsis, systemic inflammatory response comprehensive sign (systemic inflammatory response syndrome (sirs), compensatory anti-inflammatory response syndrome (compensatory antiinflammatory response syndrome, cars) and other related terms used for a long time, but also has an essential difference. out of control including inflammatory reaction syndrome (msas anti-inflammatory response syndrome, mars), a dynamic process of sirs and cars and the mixed antagonistic response syndrome, at present clinical many diseases occurrence and development are closely related. uncontrolled inflammation is a common pathological phenomenon in clinic, which is the important mechanism of the development of the complication after trauma. lps is one of the main factors that induce the uncontrolled inflammatory reaction in the most common. lps receptor on the monocyte/macrophage surface is the he initial factor for the body to recognize and start inflammatory reaction, also is one of the key links for the induction of uncontrolled inflammatory response. the concept of uncontrolled inflammatory response refers to inflammatory disorders then resulting in multiple organ dysfunction syndrome (multiple organ dysfunction syndrome. mods), emphasizes the importance of the of balance inflammatory/anti-inflammatory mechanism in the body, changes the limitations that in the past we only attached importance of the pathogenic effects of inflammatory factors. it is believed that the response of the body to the inflammatory factor is the dominant factor in the development of the whole body inflammatory reaction and mods. this concept is more focused than the previous focus on the dynamic changes of the whole process of inflammation. this can be divided into two types of mods: one is the early stage after the injury, that is, the speed hair style. the main blame is a strong inflammatory reaction induced by proinflammatory factors, and the other is a late phase of the disease, which is "late style", mainly due to the immune paralysis or worse immune disorders caused by cars or mars. the inflammatory is actually a kind of medium disease mainly caused by the chain reaction of cytokine. endotoxin is thought to be one of the most important predisposing factors in the chain reaction and can be referred for chain reaction "trigger" (trigger). endotoxin induced inflammation mechanism is mainly mediated by pamps that can induce cytokines such as il- , tnf alpha and other active molecules synthesis, the formation of the cytokine network, has a very important role in the occurrence and development of infection. the excessive activation of cytokines can cause septic shock, and is the leading cause of death in patients with bacterial infections. accordingly, prrs plays an important role in innate immunity and inflammation, and it can distinguish the pathogens from self organization through prrs organism, which is characteristic of immune response [ ] . inflammatory response syndrome systemic (sirs) is a systemic inflammatory response caused by any pathogenic factor to the body. the concept is first proposed by coris in . august american college of chest physicians and critical care medicine to present the diagnostic standard of sirs, think to have the following each of the two or more than two, sirs can be established: ( ) temperature > deg c or < deg c; ii heart rate beats/min; ( ), the breathing frequency > times per minute or arterial blood carbon dioxide into pressure (paco ) < . kpa mmhg; ( ) peripheral white blood cell count > × /l or × /l < or immature myeloid cells > %. what should be paid attention to is that sirs is a common athophysiological state of body with severe inflammatory reactions, and should be differentiated from some abnormal factors such as leukemia or cause increase or reduction of white cells after chemotherapy. although the naming of sirs has been generally concerned, but some scholars have raised objection to the concept, for example sirs has following problems: the sensitivity and specificity of the diagnostic criteria is poor, has the same meaning with the "critical"; can not understand the pathophysiology of the original disease; is difficult to guide clinical trials and practice. the production of sirs can be divided into two cases, the sirs caused by the infection and the non infectious sirs. from the point of view of the clinical development process, sirs can be followed by injury immediately aroused, then known as the "single phase velocity hairstyle; also to start local, and later developed into a systemic sirs, namely after the initial shock is brief period of stability, later gradually intensified when sirs is called" dual phase delayed onset. either of the factors or the clinical development process, the systemic inflammation of the control of the uncontrolled, and ultimately can lead to mods [ ] . the intestine is the biggest bacterial and endotoxin warehouse in the body. in severe trauma, systemic infection, intestinal ischemia and liver disease, there may be the occurrence of endotoxin. the main source is due to intestinal gram-negative bacteria in the excessive growth and reproduction, or due to increased intestinal permeability lps entry into the portal vein increased. if hepatic kupffer cell phagocytic function is low, the amount of endotoxin over the liver ability to remove endotoxin can "flood" (spill over) into the body of the loop and the endotoxemia formated. because of the endotoxin from the gut, so it is called intestinal endotoxemia (intestinal endotoxemia, ietm) [ ] . hepatitis b patients are often accompanied with ietm. its formation mechanism is: the production and absorption of intestinal endotoxin increased. there is a large number of gram negative bacteria in the body's normal intestinal, so endotoxin in intestinal contents is very high, but the intestinal mucosal epithelial cells have stronger resistance to toxins so that endotoxin is not easy to run through the intestinal mucosa into the blood, even a small amount of endotoxin breaking through the intestinal mucosal barrier into the portal vein, will be swallowed up by the kupffer cells in the liver. severe hepatitis b when the intestinal flora disturbance, endotoxin increased, increased intestinal hyperemia, edema and the permeability of the intestinal mucosa; endotoxin itself can damage the mitochondria and lysosome of intestinal epithelial cells, leading to epithelial cell autolysis; endotoxin can cause intestinal microvascular contraction of the intestinal mucosa, reduce blood, intestinal ischemia, hypoxia, cause the intestinal mucosal barrier function decreased, increased the absorption of endotoxin; severe hepatitis, due to intrahepatic bile acid and bilirubin deposition in kupffer cell phagocytosis was inhibited, resulting in the removal of endotoxin in the endotoxin decreased; through the door body circulation circuit into the systemic circulation, resulting in blood within liver cells to escape kupffer toxin the phagocytosis and clearance, which aggravate endotoxemia; the endotoxin can also pass through the celiac lymphatic system into systemic circulation by thoracic duct. in addition, severe hepatitis patients with sepsis, spontaneous bacterial peritonitis, etc., in the release of endotoxin, so the formation of the endotoxin is exogenous [ ] . in viral hepatitis and other basic diseases complicated with ietm and liver function failure are closely related and endotoxin can directly cause arterial vasoconstriction, the organ ischemia; endotoxin can activating endogenous clotting system, coupled with kupffer cell dysfunction, decrease delimination of blood coagulation or fiber soluble substances, easily lead to dic, so as to damage to multiple organs. endotoxin activated phospholipase a mediated membrane phospholipid degradation and lipid peroxidation, which is an important part of liver cell damage. nolan has pointed out that the effect of kupffer cell dysfunction induced by intestinal endotoxemia on liver and body, far more than the direct action the endotoxin, and production and release of inflammatory mediators and factors from kupffer cells activated by endotoxin are closely related. the occurrence of ietm affect hepatic energy metabolism, resulting in liver cell damage and necrosis, also caused hepatic microcirculatory disturbance, performing liver hemorrhagic necrosis. on the basis of severe hepatitis, it can accelerate liver function failure [ ] . hepatic cellular jaundice the acute and chronic liver function is often accompanied by intrahepatic cholestasis jaundice, and ietm plays an important role in the occurrence of intrahepatic cholestasis.. endotoxin involves in liver cell damage mainly through activation of phospholipase a , and inhibits the activity of na + − k + − atpase on liver canalicular membrane to make the bile excretion disorder, and then to cause intrahepatic cholestasis in the liver cells. endotoxin can start the peroxidation of liver parenchymal cells mitochondrial membrane lipid so that an increase in the content of oxygen free radical in blood, resulting in the disorder of energy generation, atp was reduced, so that the active uptake, metabolism and secretion of bile acid by liver parenchymal are short of energy, resulting in cholestasis [ ] . the liver disease with ietm often causes the coagulation dysfunction, the serious person appears the different degree bleeding, in particular the severe liver disease patient may concurrent dic, endangers the life. violi found that, when liver dysfunction in patients with ietm, the expression of tissue factors on the surface of macrophages and endothelial cell factor induced by endotoxin increased, promoting the synthesis of tumor necrosis factor (tumor necrosis, factor, tnf) to increase, and thrombin generation increasing, activation of coagulation system in about % of patients, followed by hyperfibrinolysis, suggesting that ietm in liver dysfunction can be used as the warning signal as the activation of coagulation and fibrinolysis system and activate, and with the increased hepatic lesions, plasminogen activation decreases with endotoxin levels increased, thus plasminogen may decline with endotoxin induced by chronic consumption of dic on the microstructure of ii related factors, new blood can not correct. in fact, before variceal rupture bleeding, patients with liver function severely damaged already have the gastrointestinal mucosa extensive ischemia and erosion, which is the potential causes of bleeding, ietm in the process. and gastric h + at this time can occur abnormal reverse diffusion and stimulate mast cells to release histamine, may lead to mucosal blood vessel dilation and permeability enhanced. as a result, hemorrhage, edema; histamine and directly stimulate the secretion of gastric acid, so that an increase in the number of h + and reverse diffusion, lesions persisted, form a vicious circle [ ] . it is important for the formation of ascites in ascites due to the obstruction of the hepatic vascular outflow tract obstruction. the initial vascular response to endotoxin was the rapid obstruction of the hepatic venous outflow tract and increased the portal pressure which may be related to endotoxin induced swelling of kupffer cells, liver cells with microvilli swelling, platelet aggregation and fibrin deposition effect, while others think that is endotoxin of blood vessels of the liver has a direct effect. ietm continuous damage to the liver cells, resulting in albumin synthesis and of hormones such as aldosterone de live function obstacles, thus affecting the renal function, and led to the emergence of the refractory ascites plays an important role in the process [ ] . patients with severe liver disease always are complicated with the functional renal failure, hepatorenal syndrome (hrs). the patients with severe liver disease can be associated with the pre-renal azotemia and acute renal tubular necrosis, and there is a certain relationship with ietm. the clinical studies showed that the levels of no --no and endotoxin in serum of patients with liver cirrhosis were significantly higher. at the same time, plasma renin activity, aldosterone and vasopressin levels increased and urinary sodium excretion decreased. the mechanism about ietm induced by hrs is not clear, may be related to the following factors: leukotrienes (leukotrienes, lts) lts can lead to renal vasoconstriction, increased renal vascular resistance, reduced renal blood flow and renal blood redistribution, decreased glomerular filtration rate induced by hrs, in ietm lts generation and release increased obviously. in addition, liver dysfunction, liver's uptake, inactivation and excretory function of lts decline, causing blood concentration increased; the thromboxane a (thromboxane a txa )/i (prostaglandin i , prostaglandin pgi ) can contract renal arterioles, decrease glomerular filtration rate, while pgi and pge (prostaglandin e , pg e ) is caused by on the role of txa . in patients with severe hepatitis with ietm, elevated systemic levels of pgi , reducing the renal vascular resistance, leading to renal vascular resistance txa , reduce the renal blood flow and glomerular filtration rate, promote the formation of hrs; third, nitric oxide, nitrogen monoxide) nono can through vasodilatation of the systemic, resulting in effective circulating blood volume reduction and evoked hrs; endothelial endothelin (et) et caused renal cortical blood priming hrs; and platelet activating factor (growth factor (paf) endotoxin and platelet activating factor (paf) can lead to a decrease in cirrhotic rats cardiac ejection fraction, reducing blood flow to the kidney, and paf antagonist can improve hemodynamics changes [ ] . the mechanism of endotoxin induced hepatic encephalopathy is not clear. we have known that lps can increase the permeability of blood brain barrier, promote intestinal toxic substances through the blood brain barrier (bbb), damage mitochondrial oxidative metabolism in brain cells, reduce oxygen utilization in patients with liver cirrhosis and disrupt energy metabolism of brain cells, induce brain edema. the clinical symptoms of chronic severe hepatitis with endotoxemia included in addition to fatigue, anorexia, tiresome of the oil, nausea, vomiting, yellow skin and sclera and, performance of endotoxemia. endotoxin can cause the release of histamine, -hydroxytryptamine ( -ht), prostaglandin, bradykinin, resulting in micro circulation expansion, venous blood volume reduction, decreased blood pressure, inadequate tissue perfusion, hypoxia and acidosis, and main symptoms and signs are: fever, elevated white blood cell count, bleeding tendency, heart failure, renal dysfunction, hepatic injury, nervous system symptoms and shock [ ] . improve liver function this is the basic treatment of ietm. liver function improved can strengthen mononuclear phagocyte system function to help the endotoxin removal. it can also decrease portal vein pressure to relieve intestinal congestion, edema, hypoxia, improve the intestinal microenvironment, reduce production and lymph reflux, and lower door shunt. these all contribute to the prevention and treatment of ietm. clean the gut saline is available as enema if severe liver disease, which helps reduce intestinal endotoxin generation and absorption. decompensated cirrhosis is often accompanied by small intestinal bacterial overgrowth and intestinal flora disturbance. thus, the promotion of intestinal flora back to normal state help prevent and treat intestinal endotoxemia. a variety of bifidobacterium, lactobacillus can be selected. a synthetic disaccharide, it is not digested and absorbed in the small intestine, but can be broken down into lactic acid, acetic acid and other small molecules by the bacteria into the colon. such acidification of the intestine reduces the generation and absorption of endotoxin, and promotes the growth of intestinal bacteria, stimulates bowel movements so as to increases tool frequency and so on. in addition, the lactulose may have internal direct inactivation of toxins, prevents activation of macrophages to release cytokines. oral absorption of antibiotics can effectively suppress the generation of intestinal endotoxemia. patients with liver cirrhosis taking oral polymyxin e or neomycin, the level of plasma lps and no -/no -horizontal declines in synchronization. polymyxin b has an internal direct antitoxin effect [ ] . it play a role by reducing intestinal absorption of toxins, inactivating toxins and inhibiting those lps-induced media by monocyte macrophages. the new anti-endotoxin therapy including interrupt endotoxin synthesis, binding or neutralizing its activity, preventing its interaction with the host effector cells, or interfering with toxin-mediated signal transduction pathways. therapeutic formulations include endotoxin analogs, antibodies, subunit vaccines, polymyxin combination column, recombinant human protein, small molecule inhibitors of endotoxin synthesis and intracellular signal transduction. bacteriophage producing a piece of short nucleotide sequence which plays the role of an antisense rna, blocking the synthesis of bacterial lps synthase. current clinical studies carried out an experiments in cloning of human anti-endotoxin lipid a light and heavy chain variable region. it laid the foundation for the next screening and expression that recombination between dna of antibodies and phage's succeeded. the clone is one kind of anti-polymyxin b (polymyxin b) monoclonal antibody of the igm class. it can play the role of anti-endotoxin shock by imitating the surface antigen structure of lipid a so as to substitute receptor antagonist of lipid a and lps blocking the causative link that the endotoxin induces inflammatory mediators [ ] . isolating antibodies having a high affinity of various g-bacteria to prepare a chimeric monoclonal igg antibody sdz - which have a therapeutic effect on the human endotoxemia. anti-endotoxin core glycolipid monoclonal antibody (antimonoclonal antibody r ) can prevent and treat the metabolic disorders in peritoneal infection with mods; it plays a significant role in conditioning in high catabolism, and can significantly improve metabolic disorders under the condition of abdominal infection associated with mods. bactericidal/permeability-increasing protein (bpi) bpi is a human endogenous protein, found primarily in neutrophils primary particles. its molecular amino-terminal and carboxy-terminal appear v-shaped structure planar symmetry. many amino acids to form a hydrophobic capsule hold lps's lipid a. it was reported that bpi has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin [ ] . reconstructing hdl (high density lipoprotein, hdl) hdl can be used as an endogenous lps scavenging system, binding of bacterial endotoxin with high affinity to form a stable hdl-lps. lps-hdl complexation may contribute to a reduction in endotoxic activities in vivo by preventing lps (lipid a) from generating important transmembrane signals after binding to cells [ ] . e is the first generation lipid a analogue, which is derived from the lipid a structure from the endotoxin of rhodobacter capsulatus. it can block lps in cell culture without any endotoxin-like activity. e can protect mice from lethal doses of lps, and viable e. coli infections in combination with antibiotics. in human healthy volunteers who are exposed to intravenous lps. e also blocks the endotoxin response [ ] . an anti-cd antibody cd has a very important role in monocyte-macrophage cell signaling. since epitopes of lps on the cell membrane at the binding site is the same material with soluble cd , so we can develop a monoclonal antibody interfering with lps binding to cd and blocking to pass activation signals from immune effector cell [ ] . tyrosine kinase and mitogen-activated protein kinase are involved in lps cellular signal transduction. build anti-endotoxin (lps) single-chain antibody gene and attempt to make it express in e, coli. the scfv gene was successfully constructed and gst scfv fusion protein highly expressed in e. coli was obtained. glucocorticoids, including the synthetic glucocorticoid dexamethasone, are recognized for their anti-inflammatory properties and have the ability to inhibit the production of proinflammatory cytokines such as tnf-α. in intestinal ischemia-reperfusion methylprednisolone pretreatment can prevent endotoxemia. combined lps and dexamethasone treatment at h significantly changed tnf-α [ ] . this points that glucocorticosteroids added before or during stimulation of macrophages can prevent tnf release, after which the administration would be invalid. in fact, it is very difficult to use corticosteroids before tnf release. current clinical anti -tnf antibodies and tnf antagonist use exists, and before many scholars have obtained a more satisfactory results of blocking or neutralizing excessive tnf with anti-tnf. although the clinical symptoms improved, yet the survival rate is not higher than expected. the effect of anti--tnf clinical application needs further evaluation. the first proposed concept is sequential organ failure, based on which multiple organ failure (multiple system organ failure, msof or mof) is put forward, and in diagnostic criteria is developed, but it reflected the end-stage, denied reversibility, ignore the dynamic development from organ dysfunction to failure. therefore, the us accp/sccm proposed to replace the concept with mods. mods emphasized an early phase of organ dysfunction before overt failure occurred. it is defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention." [ ] organ dysfunction may be relative, or can be absolute; with extension of time, mods can increase or reverse. thus, the term. mods was coined to indicate the wide range of severity and the dynamic nature of this disorder, which contributes to early diagnosis and treatment of patients and is more in line with clinical practice. there are two relatively distinct (although not mutually exclusive) pathways by which mods can develop: in primary mods, there is a direct insult to the organ that becomes dysfunctional. examples of such direct insults include gastric aspiration in the lungs or rhabdomyolysis in the kidney. this direct insult causes: an inflammatory response that is localized, at least in the beginning, to the affected organ. secondary mods is a consequence of trauma or infection in one part of the system that results in the systemic inflammatory response and dysfunction of organs elsewhere [ ] . secondary mods means not directly caused by damage, but to experience the "second strike", the first blow can make the immune system in a preactivated state under which inflammation lost control and significant sirs appears, then the following second strike can quickly cause multiple distant organ dysfunction and easily form sepsis with the basis of sirs/cars. this type of mods often develops as the following model: the original cause → stress → immune wpw → sirs/cars → infection → sepsis → mods → mof. the process of severe hepatitis, can cause intestinal damage and massive release of endotoxins into the blood, leading to intestinal endotoxemia (ietm). endotoxin is a powerful trigger for complement activation, and then these complement can activate "cascade effect" (cascade) to release oxygen free radicals, prostaglandins, endorphins, paf, cytokines and other inflammatory mediators to cause cytotoxicity, the error of microcirculation and tissue metabolism, eventually leading to the occurrence of mods. in this case, we emphasize mods originated in continuous, uncontrolled inflammation and factors causing systemic inflammatory response can be induced mods, including bacteria, fungi, parasites, viruses, toxins and other infectious agents. it is a debate of long-century problems that whether endotoxin has effects on hearts. the late s a large number of experiments prove endotoxins has a role of heart. it enables reduction of coronary blood flow, decrease of total coronary vascular resistance, and have meanings in heart failure. studies have shown that endotoxin can damage myocardial mitochondria, muscle paddle net, muscle membrane, contractile proteins etc., leading to cell membrane system damage, energy metabolism. past research in cardiac dysfunction under endotoxin shock did not attract enough attention, for they think that the heart is the final failure among all organs so that clinical treatment value is little. now people's awareness has completely changed that heart dysfunction can occur early in sepsis or septic shock. therefore, early identification and prevention of the occurrence and development of cardiac dysfunction may have some clinical value for treatment of septic shock and other serious complications [ ] . clinical gram-negative bacteria sepsis often complicated by adult respiratory distress syndrome, or server development of mods. in this pathological process, the high biological activity of endotoxin plays an important role. endotoxins often involving the lungs firstly, and the pathogenesis of non-injury may be associated with the direct damage on endothelial cell by endotoxin through complement pathway and induction of cytokines [ ] . the liver is the major site of endotoxin on clearing and detoxifying and the place where clinical gram-negative bacteria sepsis often can be complicated, and it is also the primary organ suffering attacks by toxins. it is generally believed that toxins in the liver circulating mainly from the gut and liver dysfunction is closely related to the formation of endotoxemia. histological examination revealed the inner toxins damage the liver cells, showing sinusoidal congestion, dieldrin expansion chamber, kupffer cell swelling, endoplasmic reticulum、mitochondria swelling, crest destroy and lysosomal activation etc. [ ] . the liver is the body's largest metabolic organ, and many cases of acute liver failure often involve other organ complications, which has become an important factor in determining the prognosis. endotoxins may cause the reduction of liver nutritional blood flow, mitochondrial oxygen metabolism, interfering with sugar metabolic pathways in liver leading to metabolic disorders. various damaging factors (such as gastrointestinal disorders, ischemia, immunocompromised, dysbiosis) promote absorb of intestinal bacteria and toxins and displacement via the portal vein, the lymphatic system into the systemic circulation. on the one hand these infectious agents can directly damage liver cells, or mediate hepatic injury whether by kupffer cell; on the other hand it can induce systemic inflammatory response by the monocyte-macrophage cells to release the media, both leading to organ perfusion disorder, affecting protein synthesis and energy metabolism, eventually resulting in severe sepsis, mods and even death. the effect of endotoxin on kidneys is not clear yet. in the early stage, it can affect the kidneys, decreased its blood flow renal via vasoconstriction. when endotoxemia is complicated by renal failure, the mechanism of glomerular filtration rate decreasing is unclear. the pathophysiology of aki in sepsis is complex and multi-factorial and includes intrarenal hemodynamic changes, endothelial dysfunction, infiltration of inflammatory cells in the renal parenchyma, intraglomerular thrombosis, and obstruction of tubules with necrotic cells and debris [ ] . the relationship between endotoxin and dic is quite complicated. dic is considered to be an important incentive of mods, especially patients with severe sepsis with dic having a highly possibility of developing mods, what's more, the prognosis is very poor, and the mechanism is multifaceted. endotoxin can start the endogenous coagulation system directly or via activating factor xii (hageman factor) by damaged endothelial cells, also can act on the monocyte-macrophage cells, stimulate the release of tissue factor to trigger the extrinsic coagulation pathway [ ] . in clinical practice it has been noted that serious infections is very possible to be complicated by gastrointestinal failure. the intestine is the biggest reservoir of bacteria in the body and leakage of bacteria or microbial products, notably lps, from the lumen of the gut into the systemic compartment, leads to initiation or amplification of a deleterious inflammatory response and mods [ ] . after endotoxins challenging the gastrointestinal mucosa, it initially shows mucosal telangiectasia, interstitial edema and hemorrhage. microcirculation leading to damage of lysosomal and release of proteases in the cell, cell degeneration and necrosis. in addition, mucosal cellular energy metabolism decrease, h + reverse diffusion, prostaglandins and bradykinin further aggravate mucosal damage. at the same time destroy of the gastric mucosal barrier also make a lot of bacteria and endotoxins pass through the gastrointestinal mucosa, migrate to the blood circulation, the lymphatic system and the abdominal cavity etc., leading to systemic multi-system organ damage. clinical characteristics of mods . organ failure usually do not result directly from the primary injury. there is a certain time interval from the primary injury to organ failure. . not all of infection have bacteriological evidence, and more than % of patients and autopsy found no infected lesions. thus, to identify and treat the infection may not be able to improve the patient's survival. . mods may have perfectly healthy organ involved, and it is ferocious and rapidly progresses. once happened, it is difficult to depress in the event almost, so often with a high mortality rate. . in pathology, mods lacks features, the affected organ only showing acute inflammation, such as inflammatory cell infiltration and so on, and these changes are very inconsistent with severe clinical manifestations, and once restored, patients do not have any clinical sequelae. . mods is closely with shock and infection. shock, infection, injury (including trauma and surgery, etc.) are the three main causes of mods. . generally the later period of shock will typesetting idc and mods, and the order of occurrence of mods usually is the lungs, liver, kidney, gastrointestinal tract, finally the heart. the characteristic clinical manifestations . instability of circulation due to a variety of inflammatory mediators have effects on the cardiovascular system, the circulation is most likely involved. almost all cases, at least in the course of the early and middle will be in highpower type of cycle of "high ranked low resistance". cardiac output up to l/ min or more and low peripheral resistance cause shock and need vasopressors to maintain blood pressure. . high metabolic systemic infection and mods are usually accompanied by severe malnutrition. its metabolic mode has three salient features: ( ) persistent high metabolism, metabolic rate up to . times more than normal; ( ) abnormalities of energy pathway. in starvation, the body obtain energy mainly through the decomposition of. however, with systemic infection, the body will get energy by breaking down proteins while the use of sugar is limited and fat utilization may increase early, fall later; ( ) poor response to exogenous nutrient, supplement of exogenous nutrition can not effectively prevent itself consumption, which suggests that a high metabolism itself has a "mandatory" also known as "autophagy metabolism." high metabolic may have serious consequences. first, protein malnutrition result from it will cause serious damage to the structure and function of the enzyme system of organs; secondly, imbalance of branched-chain amino acids and aromatic amino acid which makes the latter formate into a pseudo-neurotransmitter, then further lead to dysfunction of nerve. . hypoxia in tissue cells at present many scholars believe that the high metabolic and circulatory disorders often cause oxygen supply and oxygen demand does not match, so that the tissues of bodies are in a hypoxic state, mainly clinically manifesting "oxygen supply dependency" and "lactic acidosis. ". currently mods still lacks an unified diagnostic criteria, and any one of the diagnostic criteria of mods is difficult to reflect the entire contents of organ dysfunction, so in clinical practice we can select one according to our own specific situation. . the main contents from national critical care medicine conference standard in are: ( ) respiratory failure: r > /min; pao < . kpa; pco > . kpa; pao /fio ≤ . ( mmhg); p (aa) do (fio . ) > . kpa ( mmhg); x-ray of chest shows alveolar consolidation ≥ / lung (which have more than three or three); ( ) renal failure: except prerenal factors, little or no urine, serum creatinine, increased blood urea and nitrogen levels, exceeding more than twice the normal value; ( ) heart failure: systolic blood pressure < mmhg ( . kpa), sustained more than h; ci < . l/(min · m ); ventricular tachycardia; ventricular fibrillation; degree atrioventricular block; resuscitation after cardiac arrest (with which three or more); ( ) liver failure: total bilirubin> μmol/l; liver enzymes increased more than times compared with the normal; prothrombin time > s; with or without hepatic encephalopathy; ( ) dic: platelets × /l; prothrombin time and partial thromboplastin time prolong . times, and fibrin degradation products increase; systemic hemorrhage; ( ) brain failure: glasgow score below means coma, and less than points means brain death. . the sooner the primary diseases or the primary risk factors are eliminated or controlled, the greater the possibility of organ recovery is. . to effectively rescue and debride as soon as possible, prevent infection, prevent ischemia-reperfusion injury, use a variety of supportive care; . to reduce stress response, mitigate and shorten high metabolism and the magnitude and duration of glucocorticoid receptor; . to pay attention to the patient's breathing and circulation, as soon as possible to correct hypovolemia and hypoxia; . to prevent infection is an important measure of preventing mods; . if possible, improve the nutritional status of patients. . early treatment of any starting organ failure. mods is a problem in the medical field with an acute onset, rapid progression, and high mortality rate. so far for mods, there is no specific treatment, but through clinical monitoring, early detection of possible organ dysfunction, early intervention, and taking effective measures can slow down or block the course, 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interlinked biodiversity and climate crisis. here, we report on another risk: the accelerated infectious disease risk associated with the number and geographic spread of human infectious disease outbreaks. using the most complete, reliable, and up-to-date database on human infectious disease outbreaks (gideon), we show that the number of disease outbreaks, the number of diseases involved in these outbreaks, and the number of countries affected have increased during the entire anthropocene. furthermore, the spatial distribution of these outbreaks is becoming more globalized in the sense that the overall modularity of the disease networks across the globe has decreased, meaning disease outbreaks have become increasingly pandemic in their nature. this decrease in modularity is correlated with the increase in air traffic. we finally show that those countries and regions which are most central within these disease networks tend to be countries with higher gdps. therefore, one cost of increased global mobility and greater economic growth is the increased risk of disease outbreaks and their faster and wider spread. we briefly discuss three different scenarios which decision-makers might follow in light of our results. the anthropocene has also been nicknamed the 'great acceleration' because various socioeconomic and earth-system related indicators experienced a continuous and often exponential growth after the second world war (steffen et al., a; mcneill and engelke, ; steffen et al., ) . while this relentless growth of the human enterprise improved human well-being around the world in many aspects (waage et al., ; barrett, ; permanyer and scholl, ; zheng and qian, ) , negative impacts have likewise increased (ipcc, ; ipbes, ) . in tandem, warnings about a climate emergency (lenton et al., ; ripple et al., ) , a sixth mass extinction (pereira et al., ; barnosky et al., ; ceballos et al., ) , increasing ocean acidification and dead zones (hoegh-guldberg et al., ; diaz and rosenberg, ; branch et al., ) , and even widespread ecosystem collapse (jackson, ; barnosky et al., ; unep, ) and transgression of safe planetary boundaries (steffen et al., b) have grown increasingly urgent. to avoid or at least dampen the anticipated or already realized changes, scientists and many others have called out for radical changes to how human economies operate and relate to human societies and their environments (unmüßig, ; simms, ; walther, ; ripple et al., ) . we here want to draw attention to another important and noteworthy feature of the anthropocene which greatly affects public health, human well-being, and economic performance. these findings are especially pertinent as the world reels from the health, social and economic impact of the current sars-cov- pandemic (el zowalaty and järhult, ; ghebreyesus and swaminathan, ; lorusso et al., ) . the increasing connectivity of human populations due to international trade and travel (guimerà et al., ; colizza et al., ; brockmann and helbing, ; gabrielli et al., ) , the rapid growth of the transport of wild and domesticated animals worldwide (rosen and smith, ; schneider, ; rohr et al., ; levitt, ) , and other factors such as the increasing encroachment of human populations on hitherto isolated wild animal populations through loss and fragmentation of wild habitats (patz et al., ; despommier et al., ; pongsiri et al., ; myers et al., ) have led to a great acceleration of infectious disease risks, e.g., the increase in emerging infectious diseases and drug-resistant microbes since (jones et al., ) and the increase in the number of disease outbreaks since (smith et al., ) . to expand the previous analysis (smith et al., ) to the beginning of the anthropocene, we investigated whether the number of disease outbreaks has increased since the second world war. in addition, we examined whether the global pattern of infectious disease outbreaks changed possibly due the increasing connectivity of human populations. in other words, have the disease outbreaks become more globalized in the sense that these outbreaks are increasingly shared by countries worldwide? to investigate these questions, we used a the most complete, reliable, and up-to-date global dataset (gideon informatics, ) which had already been used in the previous analysis (smith et al., ) . this dataset can be used to enumerated the recorded annual number of disease outbreaks. to investigate the changing global patterns of disease outbreaks, we used this dataset to calculate two measures which have been recently introduced into ecological and parasitological studies. these two measures, namely modularity and centrality, quantify the connectivity of bipartite networks. modularity is defined as the extent to which nodes (specifically, sites and species for presenceabsence matrices) in a compartment are more likely to be connected to each other than to other nodes of the network (thébault, ) . the calculation of a modularity measure is useful for global phenomena because it allows the overall level of compartmentalization (or fragmentation) into compartments (or clusters, modules, subgroups, or subsets) of an entire dataset to be quantified. high modularity in a global network means that subgroups of countries and disease outbreaks interact more strongly among themselves (that is, within a compartment) than with the other subgroups (that is, among compartments) (bordes et al., ) . centrality is defined as the degree of the connectedness of a node (e.g., a keystone species in ecological studies; jordán, ; gonzález et al., ) . in the context of our study, centrality is the degree of the connectedness of a country and those countries connected to it. we estimated the countries which are the potential centres of disease outbreaks by investigating the eigenvector centrality of a given country in a network of countries which share disease outbreaks among each other. eigenvector centrality is a generalization of degree centrality, which is the number of connections a country has to other countries in terms of sharing disease outbreaks. eigenvector centrality considers countries to be highly central if the connected countries to them through shared outbreaks are connected to many other well-connected countries (bonacich and lloyd, ; wells et al., ) . modularity and centrality analyses have been used to investigate various ecological, parasitological and epidemiological questions (e.g., tylianakis et al., ; jordán, ; gonzález et al., ; anderson and sukhdeo, ; bascompte and jordano, ; poisot et al., ; bordes et al., ; genrich et al., ) . using a widely used world dataset on infectious disease outbreaks, we here present results which demonstrate that the accelerated number of disease outbreaks and their increased global spread are two further threatening aspects of the accelerated infectious disease risk associated with the globalization process which characterizes the anthropocene. to collate the total number of infectious disease outbreaks over the years - , we extracted the relevant data from the medical database called gideon (gideon informatics, ) which contains information on the presence and occurrence of epidemics of human infectious diseases in each country as well as the number of surveys conducted in each country. the gideon data are curated as records of confirmed outbreaks, are continually updated using various sources such as who and promed, and are accessible via subscription. this dataset is generally considered to be the most complete, reliable, and up-to-date in the world and has been regularly used in previous macro-scale studies of infectious disease, epidemics, and pathogen diversity (e.g., smith et al., ; dunn et al., ; morand et al., ; morand et al., ; poisot et al., ; smith et al., ; morand, ; morand and walther, ) . each row in the gideon dataset specifies the disease 'species', the year and the country of the outbreak. the 'annual total outbreak number' is simply the annual total number of outbreaks regardless of the disease designation and including all countries. the 'annual total disease number' uses the same data for each year as the 'annual total outbreak number' but then counts only the different infectious diseases which had at least one outbreak in that respective year. the 'annual total country number' uses the same data for each year as the 'annual total outbreak number' but then counts only the different countries which had at least one outbreak in that respective year. our entire - dataset contains outbreaks of human infectious diseases in nations. in our case, we built yearly bipartite networks of presence-absence matrices which link countries with all the recorded epidemic outbreaks. we then transformed these bipartite networks where separate nodes from countries were connected with nodes of epidemic outbreaks into unipartite networks using the tnet package (opsahl, ) the calculation of modularity of bipartite networks of shared epidemic outbreaks among countries allowed us to identify modules of countries that share common epidemic outbreaks in each respective year (see introduction) (blondel et al., ; bordes et al., ) . we calculated our modularity measure of unipartite network for each year using igraph (lehoucq et al., ) . high modularity calculated in this context means that an epidemic remains relatively constrained within a few countries while low modularity means that an epidemic has spread across relatively more countries. the calculation of the eigenvector centrality of the unipartite network of each respective year allowed us to determine the number of connections a country has to other countries in terms of epidemic outbreak sharing. eigenvector centrality is a measure of the degree of the connectedness of a country and those countries connected to it. high centrality calculated in this context means that a country is connected to many countries which are also well-connected (bonacich and lloyd, ; wells et al., ) . a visual examination of the time trend of the annual modularity measure suggested a discontinuous trend over time. to detect such a discontinuity (or breakpoint) in the trend over time, we used the r package segmented (muggeo, ; muggeo, ) . this package allows the identification of one or more discontinuities using the bootstrap method; in other words, the decomposition of a relationship into one of more piece-wise linear relationships and the identification of breaking point(s). the standard errors of the breakpoint estimates were computed with the procedure of clegg et al. ( ) which is implemented in the r package segmented. finally, we used a smooth regression to visualize the patterns of changes over time (harrell, ) (e.g., for air transport, modularity, etc.). increase in global measures of mobility and gdp. the increase in measures of mobility during the anthropocene has been staggering, with growth percentages of up to % (table ) . many more impressive local and regional examples exist, see, e.g., (wilson, ; wilson, ) . for further analyses below, we only used the world bank data on flight passengers and air freight; therefore, we detail them here. since the s, the total global number of flight passengers ( increase in the number of outbreaks. since the s, the annual total outbreak number ( fig. d ), the annual total disease number (fig. e) , and the annual total country number ( fig. f ) have increased exponentially. however, for the annual total disease number, this increase has slowed since the s. while there is some annual up-and-down variation, the overall trends are well demonstrated by the smooth line regressions. furthermore, around , the trends for the three variables began a decrease or stagnation in the actual data, but not in the smooth line regressions. we also plotted all the annual centrality values for each nation within six regions. despite a large amount of variation over the investigated time period, the three north american countries had the highest mean of centrality values, followed by the three pacific countries and then the european, south american, asian and african countries (fig. ). these differences in centrality between the six regions are statistically significant (kruskal-wallis χ = . , df = , p = . e- ). our results further support the hypothesis that the anthropocene is associated with to a great acceleration of infectious disease risks. first, we showed that the number of disease outbreaks, the number of diseases involved in these outbreaks, and the number of countries affected have increased during the entire anthropocene (thus expanding on the results of previous studies which were more limited in time or space, e.g., morand et al., ; morand et al., ; smith et al., ; morand, ) . furthermore, these increases have mostly been exponential, although with some recent slowdowns (see discussion below). second, we demonstrated that the spatial distribution of these outbreaks has become more globalized in the sense that the overall modularity of the disease networks across the globe has decreased since around . in other words, clusters of disease outbreaks began to increasingly become connected with other clusters so that the fragmented nature of outbreak clusters diminished over time. before , a disease outbreak usually remained confined to one or a few closely connected countries; thereafter, disease outbreaks have become increasingly pandemic in their nature. we thus revealed a long-term, worldwide change in the biogeographic structure of human infectious diseases associated with outbreaks. we further found that this decrease in modularity is correlated with the increase in air traffic. the increase in global mobility and especially in air traffic (table ) allows an outbreak to rapidly spread across several national and continental borders within a short period of time (see also results from modelling and real-world data below). third, we demonstrated which countries and regions are most central within these disease networks. countries which are more centrally located within these disease networks tend to be also the more developed and emerging countries with significantly higher gdps. therefore, one cost of increased global mobility (which is currently tightly linked to economic growth and globalization, see discussion below) is the increased risk of disease outbreaks and their faster and wider spread (although we note that the risk per capita may be decreasing, smith et al., ) . before we discuss the implications of our results, we address possible limitations and biases. while gideon is generally acknowledged to be the most complete, reliable, and up-to-date global dataset on infectious disease outbreaks, we nevertheless should consider that ( ) there may have been some underreporting in the early part of the anthropocene, and ( ) recent outbreaks may not have been entered into gideon yet. ( ) there may have been some underreporting of infectious disease outbreaks during the early parts of the anthropocene in developing countries. however, the imposition in the s of the so- while we cannot exclude the possibility of some underreporting of disease outbreaks during the early part of the anthropocene, it is rather unlikely that the large increases of several hundreds of percent which we documented in figures d-f are entirely due to underreporting. since the overall trends are so consistent and so large over a relatively long period of time, we argue that these trends are real even if the actual numbers may be off by a few percentage points. ( ) the recent slowdowns shown in figures d-f could be real or due to the most recent outbreaks not having been entered into gideon yet. if they are real, they are not really influencing the overall decade-long trends documented here. however, if they are due to underreporting, then the documented trends would be even stronger. although it is generally acknowledged that correlation does not prove causation, the correlation between air travel and modularity specifically (fig. ) , and the relationship between increased mobility and the faster and wider spread of disease outbreaks (table , figures and ) in general make sense given theoretical models and real-world evidence (see discussion below). however, we acknowledge that other factors may be responsible, especially variables which may covary with mobility measures. further causal analyses are therefore required, but these are beyond the scope of this study. the starting point of a disease outbreak is due to a variety of local conditions or factors (morand and lajaunie, ; morand and figuié, ) . however, after emergence, the local, regional, or global spread of a disease is of course dependent on many other factors of which host mobility is usually one of the most important ones. this is of course especially true for directly transmitted human pathogens (walther and ewald, , and studies cited below), although mobility of humans as well as vectors are also important for the global spread of vector-borne diseases (tatem et al., ; brown et al., ; eritja et al., ; golnar et al., ; oliveira et al., ) . theoretical models predict that increased mobility leads to a faster and more wide-ranging spread of a disease outbreak, and, vice versa, decreased mobility slows and contains the spread of an outbreak. modelling the spread of the sars-cov- pandemic, hufnagel et al. ( ) demonstrated that two control strategies, shutting down airport connections and isolating cities, reduced the spread of the virus. drastic travel limitations also delayed a pandemic by a few weeks in a model of the global spread of influenza (colizza et al., ) . similarly, increasing levels of ( ) isolation of infectious hosts, household quarantine and related behavioral changes which reduce transmission rates and ( ) air traffic reduction increasingly slowed the global spread of influenza, although the latter control strategy required the almost complete halt of global air traffic (cooper et al., ; ferguson et al., ; flahault et al., ; hollingsworth et al., ; epstein et al., ; bajardi et al., ) . epstein et al. ( ) emphasized that a combination of both control strategies would be even more effective, a result mirrored by mao ( ) for a model at the city scale. crucially, hollingsworth et al. ( ) also showed a significant decrease in the number of countries affected if travel reductions are combined with other control strategies to reduce transmission rates. this result was confirmed by cooper et al. ( ) and flahault et al. ( ) who found that fewer cities (distributed around the world) were affected by major outbreaks if sufficiently early and significant travel and transmission reductions were implemented. in a simulated smallpox attack, even gradual and mild behavioral changes had a dramatic impact in slowing the epidemic (del valle et al., ) . another model suggested that public health policies that encourage self-quarantine by infected people can lower disease prevalence (chen et al., ) . real-world examples also demonstrate the link between increased mobility and faster disease outbreaks. real data on influenza in the usa showed that a reduction in air travel resulted in a delayed and prolonged influenza season (brownstein et al., a; brownstein et al., b) . similarly, the presence of airports and railway stations significantly advanced the arrival of influenza during the pandemic in china (cai et al., ) . global connectivity due to air traffic allows an outbreak to rapidly spread across several national and continental borders within a short period of time. for example, the ebola virus outbreak was brought into the continental usa onboard a commercial flight from liberia because the host was asymptomatic during the flight (cdc, ) . sevilla ( ) reviewed and modelled how air travel can aid the global spread of ebola, h n influenza, sars-cov- , and pneumonic plague. a systematic review of the effectiveness of travel reductions concluded that internal travel restrictions as well as international border restrictions both delay the spread of influenza epidemics (mateus et al., ) . therefore, given the staggering global increase in global mobility during the anthropocene documented in table , the increase of the number of disease outbreaks, of diseases involved in these outbreaks, and of countries affected, as well as the decrease of modularity of these disease outbreaks make sense because the mobility of humans, other living beings, and goods (which can act as carriers or vectors) all facilitate the spread of disease and species (e.g., smith and guégan, ; findlater and bogoch, ; sardain et al., ) . given the lack of any antiviral or vaccine treatment, the current sars-cov- pandemic has forced governments to drastically curtail people's mobility and introduce continent-wide social distancing and lockdowns in order to at least slow its spread by lowering transmission rates. thus, the governments' responses to this acute global health emergency actually mirror many of the recommendations which were given by the various modelling studies cited above. it should also be noted that the restriction of people's mobility (and its most extreme form, quarantine) and social distancing were already used before the advent of the germ theory of disease and are even used to some extent by other species (hart, ; tognotti, ; bashford, ) . given the link between mobility and disease outbreaks documented by our study, the key question which decision-makers and society at large should ask are which of the following three scenarios (which we outline in very broad terms only) should we aim for in the coming decades. ( ) once the current sars-cov- pandemic is over, we continue on our path of ever increasing mobility without regard to the costs in terms of the accelerated infectious disease risk. ( ) we attempt to slow down or even reverse mobility rates of infected hosts and vectors. ( ) we attempt to slow down or even reverse mobility rates of humans and other carriers and vectors (in other words, decrease many or all of the mobility measures in table ). we briefly discuss the implications of each scenario. however, this discussion is by no means exhaustive, but meant to stimulate further discussion and study which are urgently needed to come to terms with the accelerated infectious disease risk of the anthropocene. ( ) most likely, at least in the short-term, economic and political decision-makers will return to 'business-as-usual' which means increasing mobility rates even further. after all, various projections predict more immense increases of mobility within the next few decades. for example, international tourist arrivals worldwide are expected to increase by . % a year between and to reach . billion by (unwto, ) from the . billion recorded in (table ) in addition to the environmental and social costs and risks of this scenario (e.g., increasing landuse change, greenhouse gas emissions, resource use and waste production, etc.), including the risk of widespread ecosystem collapse (see introduction), we can now add the cost of an increasing infectious disease risk. while our study only focused on human infectious diseases, this cost related to increased mobility is also increasing for animal and plant disease outbreaks as well as alien species introductions (e.g., anderson et al., ; fisher et al., ; bélanger and pilling, ; sardain et al., ) . while outbreaks of animal and plant diseases may be amenable to a cost- benefit analysis (tildesley et al., ) , the current sars-cov- pandemic has clearly shown that simple cost-benefit analyses cannot be applied when the lives of millions of people are at stake (note that another emerging infectious disease, the global hiv pandemic, has claimed million lives so far). given that another pandemic becomes more likely with increasing rates of emergence and increasing global mobility, a 'business-as-usual' scenario is automatically associated with further epidemics and pandemics, possibly killing further millions of humans and devastating local and regional economies or even the global economy. if a 'business-as-usual' scenario is followed with regards to global mobility, countries and the world community should at least invest in better detection and surveillance methods to catch and contain the next pandemic as early as possible, and in better preparedness of public health facilities in case the next pandemic nevertheless gets out of hand (jain et al., ; bedford et al., ; di marco et al., ) . however, this scenario nevertheless will likely be associated with an increased number of epidemic outbreaks (some of which may become devastating pandemics), given the results of our study. ( ) consequently, the most realistic and agreeable scenario may be to slow down or even reverse the mobility rates of infected hosts and vectors. again, the current sars-cov- pandemic has demonstrated that identifying infected hosts and reducing secondary infection rates caused by these infected hosts appear to be the most successful strategies to achieve elimination of the outbreak (e.g., . the required measures, such as mass home quarantine, restrictions on travel, expanded testing and contact tracing, and additional surveillance measures, are thus mostly focused on ( ) identifying and isolating infected hosts (which means to drastically restrict their mobility) and ( ) drastic restrictions of mobility for uninfected hosts. while the latter is possible in crisis situations, it cannot be a long-term solution to the quandary of the increased infectious disease risk of the anthropocene unless we want to decrease our total global mobility (see scenario below). therefore, much improved identification and isolation infected hosts may be the way forward. already, such measures have been adopted during the current sars-cov- pandemic, e.g., body temperature checks for every air travel passenger even though they appear to be ineffective (cohen and bonifield, ) . however, if efficient and reliable health checks which can identify various diseases and which can be administered relatively timeand cost-efficiently to large numbers of passengers could be implemented, we may be able to significantly restrict the mobility of infected hosts. while such a proposal may sound like "pie in the sky" at the moment, rapid advances in diagnostic techniques, such as translational proteomics, may soon allow us to identify infected hosts using simple breathalyzer, saliva, or urine tests (athlin et al., ; nakhleh et al., ; tao et al., ; zainabadi et al., ) . furthermore, hygienic measures, such as the enforcement of handwashing and the wearing of facemasks in public transport hubs, complete and regular disinfection of important traffic hubs and vehicles (including the air and all surfaces), and much better vector control should become mandatory global standards of public health (e.g., grout and speakman, ; nicolaides et al., , reviewed in huizer et al., , especially in the most central of traffic hubs, such as the world's most connected airports (guimerà et al., ; bajardi et al., ) . such measures would certainly help to decrease the mobility of infected hosts and thus the transmission and global spread of diseases. ( ) the most sustainable scenario is, however, to decrease or even reverse global mobility rates of humans and other carriers and vectors, especially if it is part and parcel of a much larger movement towards global sustainability by reducing humanity's environmental footprint and replacing unsustainable economic growth with sustainable economic degrowth (schneider et al., ; daly and farley, ; alexander, ; czech, ; galaz, ; cosme et al., ; weiss and cattaneo, ; chiengkul, ; sandberg et al., ; schmid, ) . such a general, comprehensive and global slowdown of mobility of both uninfected and infected people and vectors would be opposed for many reasons and by many interest groups, mainly based on economic arguments based around the need for continuous economic growth which has so far almost always been positively linked with increased mobility (e.g., arvin et al., ; hakim and merkert, ; unwto, ; saidi et al., ; nasreen et al., ) . it is to some extent possible to decouple mobility from economic growth (loo and banister, ; lane, ) , but even if such a decoupling was achieved, it would not sufficiently reduce mobility to significantly decrease infectious disease risks. as the modelling results cited above and the experience with the current sars-cov- pandemic clearly show, only a huge reduction in mobility and contact rates is sufficient to achieve a slowdown or halt of a highly contagious disease outbreak which is already under way. yet, a decrease in global levels of mobility should also decrease the overall number of disease outbreaks, according to our results (which is different to just slowing and containing the spread of an outbreak, see discussion above). therefore, the many environmental benefits of economic degrowth and deglobalization would be augmented by a global health benefit, the almost certain decrease of infectious disease outbreaks. since economic degrowth and deglobalization have been advocated by many sustainability experts to deal with the currently converging environmental crises (climate change, ocean acidification, biodiversity, etc., see references above), our results further strengthen the argument for such a 'not-business-as-usual' scenario. moreover, the economic degrowth scenario would also ameliorate many of the local conditions or factors associated with the emergence of outbreaks (such as increased livestock production and contact rates with wildlife, climate change, loss and fragmentation of natural habitats caused by urbanization and agricultural intensification, etc.) thus further decreasing the likelihood of disease outbreaks. in addition, we have a growing understanding that the presence of abundant biodiversity and healthy ecosystems has an overall positive effect on human well-being and health (chivian and bernstein, ; wood et al., ; sandifer et al., ; walther et al., ; morand and lajaunie, ; mcmahon et al., ) which should count as an additional health benefit of the economic degrowth scenario. naturally, decreasing mobility is a moral and political choice which can be informed by science, but not answered by science. however, given all the current negative impacts of high mobility (greenhouse gas emissions, land-use and land-cover change and the resulting habitat loss and fragmentation due to transportation infrastructure and energy production, transport of alien species, etc.), maybe it is time to ask whether it is morally justified, for example, to move the equivalent of all the inhabitants of a small town across a continent so that a football team can be supported by its fans during an away game? is it necessary to fly halfway around the world for a weekend shopping trip? is it really most cost-efficient for supply chains to cover the entire globe when all the externalities are included? is long-term sustainability achievable with ever higher rates of mobility? the demand for ever-increasing mobility is putting many stresses on the earth system and therefore also on many aspects of human well-being and health. in this study, we documented another one: the public health risks of an increasing number of disease outbreaks and their increasingly global spread. even without the devastating current impacts of the sars-cov- pandemic, the additional disease outbreak burden associated with our highly mobile and migratory human societies is a definite cost which must be considered in its moral and ethical implications as we consider the future trajectory of the anthropocene (ehrlich and ehrlich, ; steffen et al., ; schill et al., ) . the medicine that might kill the patient: structural adjustment and its impacts on health care in bangladesh planned economic contraction: the emerging case for degrowth emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers host centrality in food web networks determines parasite diversity transportation intensity, urbanization, economic growth, and co emissions in the g- countries comparison of the immuview and the binaxnow antigen tests in detection of streptococcus pneumoniae and legionella pneumophila in urine human mobility networks, travel restrictions, and the global spread of h n pandemic elimination: what new zealand's coronavirus response can teach the world. the guardian new zealand's elimination strategy for the covid- pandemic and what is required to make it work approaching a state shift in earth's biosphere food security and sociopolitical stability mutualistic networks quarantine: local and global histories a new twenty-first century science for effective epidemic response the state of the world's biodiversity for food and agriculture. fao commission on genetic resources for food and agriculture assessments fast unfolding of community hierarchies in large network eigenvector-like measures of centrality for asymmetric relations forecasting potential emergence of zoonotic diseases in south-east asia: network analysis identifies key rodent hosts habitat fragmentation alters the properties of a host-parasite network: rodents and their helminths in south-east asia impacts of ocean acidification on marine seafood the hidden geometry of complex, network-driven contagion phenomena assessing the risks of west nile virus-infected mosquitoes from transatlantic aircraft: implications for disease emergence in the united kingdom. vector-borne zoonotic dis air travel and the spread of influenza: authors' reply empirical evidence for the effect of airline travel on inter-regional influenza spread in the united states roles of different transport modes in the spatial spread of the influenza a(h n ) pandemic in mainland china cdc and texas health department confirm first ebola case diagnosed in the u accelerated modern human-induced species losses: entering the sixth mass extinction public avoidance and epidemics: insights from an economic model the degrowth movement: alternative economic practices and relevance to developing countries sustaining life: how human health depends on biodiversity estimating average annual per cent change in trend analysis no us coronavirus cases were caught by airport temperature checks. here's what has worked modeling the worldwide spread of pandemic influenza: baseline case and containment intervention the role of the airline transportation network in the prediction and predictability of global epidemics delaying the international spread of pandemic influenza assessing the degrowth discourse: a review and analysis of academic degrowth policy proposals supply shock: economic growth at the crossroads and the steady state solution ecological economics: principles and applications effects of behavioral changes in a smallpox attack model the role of ecotones in emerging infectious diseases sustainable development must account for pandemic risk spreading dead zones and consequences for marine ecosystems global drivers of human pathogen richness and prevalence can a collapse of global civilization be avoided? 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air transport, freight (million ton-km) air transport maritime highways of global trade maritime logistics: a complete guide to effective shipping and port management an efficient and costeffective method for purification of small sized dnas and rnas from human urine development and poverty reduction: a global comparative perspective a pneumonia outbreak associated with a new coronavirus of probable bat origin this work was part of thefuturehealthsea project funded by the french anr (anr- -ce - - ). s.m. is supported by the thailand international cooperation agency (tica) "animal innovative health". we sincerely thank dieter stockmann from the institute of shipping economics and logistics (isl) and claire thackeray from container trades statistics (cts) for providing data about container movements, jean tournadre for providing data about ship numbers, and ting-wu chuang for providing references. traffic vehicles for transportation < to > million motor vehicles (> %) (steffen et al., a) - international tourism < to > million arrivals (> %) (steffen et al., a) table are approximations whenever numbers are given with a ~, < or > sign because these numbers were taken from graphs in steffen et al. ( a) and isl ( ). abbreviations: dwt = deadweight tonnage is a measure of how much weight a ship can carry; mt = metric ton; teu = twenty-foot equivalent unit, a standard size for . m long containers. key: cord- -f xc uu authors: milinovich, gabriel j; avril, simon m r; clements, archie c a; brownstein, john s; tong, shilu; hu, wenbiao title: using internet search queries for infectious disease surveillance: screening diseases for suitability date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: f xc uu background: internet-based surveillance systems provide a novel approach to monitoring infectious diseases. surveillance systems built on internet data are economically, logistically and epidemiologically appealing and have shown significant promise. the potential for these systems has increased with increased internet availability and shifts in health-related information seeking behaviour. this approach to monitoring infectious diseases has, however, only been applied to single or small groups of select diseases. this study aims to systematically investigate the potential for developing surveillance and early warning systems using internet search data, for a wide range of infectious diseases. methods: official notifications for infectious diseases in australia were downloaded and correlated with frequencies for internet search terms for the period – using spearman’s rank correlations. time series cross correlations were performed to assess the potential for search terms to be used in construction of early warning systems. results: notifications for infectious diseases ( . %) were found to be significantly correlated with a selected search term. the use of internet metrics as a means of surveillance has not previously been described for ( . %) of these diseases. the majority of diseases identified were vaccine-preventable, vector-borne or sexually transmissible; cross correlations, however, indicated that vector-borne and vaccine preventable diseases are best suited for development of early warning systems. conclusions: the findings of this study suggest that internet-based surveillance systems have broader applicability to monitoring infectious diseases than has previously been recognised. furthermore, internet-based surveillance systems have a potential role in forecasting emerging infectious disease events, especially for vaccine-preventable and vector-borne diseases. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. prudent detection is a cornerstone in the control and prevention of infectious diseases. traditional infectious disease surveillance systems are typically characterised by a bottom-up process of data collection and information flow; these systems require a patient to recognise illness and seek treatment and a physician or laboratory to diagnose the infection and notify the relevant authority [ , ] . for emerging infectious disease events, this process is reported to take, on average, days from onset to detection and a further - hours for the world health organization to be notified [ ] . the development and implementation of more efficient systems for gathering intelligence on infectious diseases has the potential to reduce the impact of disease events. internet-based surveillance systems are one such system [ ] . internet-based surveillance systems produce estimates of disease incidence through analysis of various digital data-sources. targeted sources include internet-search metrics, online news stories, social network data and blog/ microblog data [ ] . currently, the most promising approach appears to be those based upon monitoring of internet search behaviour. this approach works on the premise that people will actively seek information on diseases they develop and that estimates of disease activity with the community may be developed by monitoring the frequency of related internet searches. through targeting people earlier in the disease process, internet-based systems are able to access a larger fraction of the community and produce more timely information. furthermore, internet-based surveillance systems are intuitive and adaptable, cheap to run and maintain (once established), do not require a formal public health network and have the capacity to be automated and operate in near-real time. despite these advantages, internet-based surveillance systems have a number of significant shortcomings and must not be considered an alternative to traditional surveillance approaches [ ] . firstly, as these systems crowd-source data, resolution will be contingent on the size of the population serviced and may be further limited by national communications infrastructure availability and distribution [ ] . secondly, as internetbased surveillance systems are limited to people who use the internet to source health information, there is the potential that estimates produced by these systems may not accurately reflect the entire community [ ] . finally, as internet-based surveillance systems essentially rely upon self-reporting, bias may be introduced through differences in internet usage between sectors of the community (the elderly, for example, may not use the internet as a source of health information, despite being a high-risk group for many infectious diseases) and/or through media driven interest in emerging disease events [ ] . infectious diseases surveillance systems have been developed using internet search metrics to estimate incidence of influenza (google flu trends) [ ] and dengue (google dengue trends) [ ] . currently, operational systems that utilise this approach are limited, however, studies of the potential for internet-based surveillance have been conducted for a range of other infectious diseases, including: acute respiratory illness [ ] , aids [ ] , chickenpox [ , ] , cryptosporidiosis [ ] , dysentery [ ] , gastroenteritis [ ] , hepatitis [ ] , listeriosis [ ] , lyme disease [ ] , methicillin-resistant staphylococcus aureus [ ] , norovirus [ ] , respiratory syncytial virus [ ] , rotavirus [ ] , scarlet fever (streptococcus pyogenes) [ , ] , salmonella [ ] , tuberculosis [ , ] and west nile virus [ ] . previous studies have focused on single diseases, or a small number of diseases, and the justification of the focus on a particular disease has been specific to each study. the published results have largely been promising; however, to date there has been no systematic, generalizable analysis to identifying diseases that are suited to monitoring through the analysis of internet-search metrics. the underpinning goal of this study was to provide direction for future approaches to developing digital surveillance systems; such as the development of predictive models and/or integrative surveillance models that draw upon multiple traditional and digital data source to create estimates of disease within the community. this study, however, did not aim to develop actionable surveillance systems, produce predictive models of infectious disease based on internet-based data or to identify the best search terms for use in these models. rather, this study aimed to determine which diseases have most promise for monitoring by surveillance systems built on internet search metrics; this was achieved by assessing the level of correlation between a wide range of infectious diseases and internet search term metrics. finally, this study aims to identify diseases for which internet-based data could be used to create early warning systems. surveillance data on notifiable infectious diseases were collected from the national notifiable disease surveillance system (nndss) which is maintained by the australia government department of health (doh) [ ] . monthly notifications (case numbers) aggregated at state/territory and national level, were downloaded for the period of january to september . a full list of notifiable diseases in australia and case definitions can be accessed through the doh webpage [ ] . sixty-four diseases are monitored and these are categorised in the nndss as belonging to one of eight groups: bloodborne diseases; gastrointestinal diseases; other bacterial diseases; quarantinable diseases; sexually transmissible infections; vector-borne diseases; vaccine preventable diseases; and zoonoses. for the purpose of consistency, we have reported diseases according to these groupings. whilst notifiable, data were not downloaded for human immunodeficiency virus infection/acquired immunodeficiency syndrome, creutzfeldt-jakob disease or variant creutzfeldt-jakob disease because surveillance for these diseases is not performed by doh or for severe acute respiratory syndrome, because reporting to the doh is informal; as such, these diseases are not listed on the nndss. in the construction of google flu trends model, the authors identified search terms by performing correlations between influenza-like illness data from the us cdc and the top million google search queries performed in the us over the corresponding period [ ] . such data is not available to the public and an alternative approach to identification of search terms was required; two approaches were used. firstly terms related to diseases, the aetiological agents and colloquialisms (such as "hep" for hepatitis or "flu" for influenza) were manually identified. secondly, google correlate (www.google.com/trends/correlate) was queried using monthly surveillance data (described above). google correlate provides a list of up to search terms that correlate most highly with the query data. to account for potential language shifts that may have affected search behaviour [ ] , this was performed three times using surveillance data covering the periods - , - and - . up to search terms were downloaded from google correlate for each notifiable disease ( search terms per period analysed) and manually sorted; any term related to the queried notifiable disease was included, regardless of the nature of the potential association suitable terms were combined with the manually identified search terms to create a list of search terms (see additional file ). no attempt was made to filter search terms based upon biological plausibility; any term that may be perceived to have any association with the disease of interest was included. search frequencies for terms of interest were collected through google trends (www.google.com/trends/). all data extractions were performed on the nd of october, . google trends was queried using each of the identified terms at a national and state/territory level using the entire time range available ( -present). google trends presents search frequency as a normalised data series with values ranging from to (with representing the point with the highest search frequency and other points scaled accordingly); functionality for exporting search frequency data as a .csv file is provided. for the purpose of privacy, data are aggregated at a daily, weekly or monthly level (or are restricted if there is insufficient search volume). the level of aggregation applied is determined by the period analysed and the search frequency; the level of aggregation is not able to be specified by the user. as the notifiable disease surveillance data used was in monthly format, monthly indices of query search frequencies were required. monthly indices are displayed graphically by google trends when querying periods greater than months; rather than downloading. csv files, a script was developed to scrape data from the google trends webpage, allowing the problems associated with the level of data aggregation to be overcome. analyses were performed at both national and state levels for the period - . as state-level search frequency data were not always available, particularly for less common diseases (due to low search frequency at this level of disaggregation), correlations between state-level notification data and national search frequency data were also performed. owing to the large number of correlations performed in this study, bonferroni adjustments [ ] were applied to significance levels by the equation -( -α) /n ; all p-values reported in this document correspond to onetailed tests. spearman's rank correlation coefficients were used to rank performance. time-series cross correlations were performed to assess linear associations between disease notifications and google trend search indices. cross correlations were calculated using lag values for google trends data ranging from − to . this range allowed for assessment of biologically plausible associations that were relevant to the development of early warning systems. cross correlations were performed on national data using ibm spss version (spss inc; chicago, il, usa). seasonal differencing was applied (value ) to all analyses to remove cyclic trends. whilst all available data ( - ) were downloaded, analyses for this study were focused on the most recent five years ( - ) as preliminary data analyses indicated that google trends data were not available prior to for numerous search terms ( figure ; panels , , , , and ). additionally, shifts in language are known to affect surveillance systems built upon textual data [ ] . the shortened period ( - ) was selected to minimise the effects of language shifts. however, this period still provides the requisite pairs of observations for performing cross correlations [ ] . in this section we discuss analyses of time series data. briefly, the time series analysed were monthly case numbers for the infectious diseases monitored by the australian government's national notifiable disease surveillance system (nndss) and google trends monthly search metrics for related internet search terms. in total, search terms were analysed in this study; this ranged from a single term for some diseases, up to search terms for influenza and search terms for pneumococcal disease. the majority of terms could be categorised as diseases or aetiological agents ("brucellosis" or "brucella"), colloquialisms ("flu", "hep" or "tb"), symptoms ("cough", "white discharge" or "cervical mucus") or medication or general health/treatment related queries ("whooping cough treatment", "symptoms of dengue" or "flu and pregnancy"). a few terms that may have environmental ("flash floods" for leptospirosis) or behavioural ("african tours" for malaria) meanings were also included. a full list of the search terms analysed is presented in the supplementary material. evaluation of the bivariate associations between surveillance and corresponding search frequency data was performed using the spearman's rank correlation. spearman's rank correlations for the top ranked notifiable diseases and terms are presented in figure and raw data for the corresponding diseases and search terms are presented in figure . results of spearman's correlations indicated diseases to be significantly correlated (p < . ; bonferroni corrected: p < . e − ) with at least one search term; p-values for of these were < . (bonferroni corrected: p < . e − ). marked differences were observed in correlations between the various disease groups. correlations for vaccine-preventable diseases were generally highest with six of fourteen exhibiting strong (rho = . - . ) or very strong (rho = . - . ) correlations, followed by sexually transmitted infections ( / ), the vector-borne diseases ( / ), blood-borne diseases ( / ), other diseases ( / ), zoonoses ( / ), gastrointestinal infections ( / ) and, finally, quarantinable diseases ( / ). state level correlations are also reported in figure . consistency between state correlations were variable with some diseases exhibiting reasonable consistency (pertussis; rank ), whilst others were inconsistent (hepatitis c; rank ). results of cross correlations are demonstrated in figure . cross correlation results should be interpreted as product-moment correlations between the two time series; they allow dependence between two time series to be identified over a series of temporal offsets, referred to as lags. lag values indicate the degree and direction of associations. a lag value of − indicates that correlations were performed using time series data for which the first series (google trends' data) has been shifted backwards one unit (a month). conversely, a lag value of indicates that the primary series had been shifted forward one unit. significant positive correlations for lag vales of ≥ or above are of most interest in the context of this study as they indicate a positive relationship between the two time series with google trends data leading the notifications (a pre-requisite for google trends data to be a suitable early warning tool). it should also be noted that seasonal differencing was applied to cross correlations to remove cyclic seasonal trends. disease notifications positively correlated at a lag of one month (lag ) with search term frequency for of the diseases that exhibited significant spearman's rank correlations. overall, of the notifiable diseases exhibited significant, positive correlations at lag of one month. significant positive associations were observed for four of the nine vector-borne diseases (barmah forest virus infection, dengue virus infection, murray valley encephalitis virus infection and ross river virus infection), six of the vaccine preventable diseases (haemophilus influenzae type b, influenza, pertussis, pneumococcal disease and varicella zoster (chickenpox and shingles)), two of the six blood-borne diseases (hepatitis b (unspecified) and c (unspecified)), two of gastrointestinal diseases (campylobacteriosis and cryptosporidiosis) and one zoonosis (leptospirosis). positive significant correlations were not observed at a lag of one month for any of the quarantinable diseases (n = ), sexually transmissible infections (n = ) or other bacterial infections (n = ). it should be noted that positive significant correlations were observed at lags of over one month (but not at lag ) for two of the top ranked diseases (gonococcal infection and meningococcal disease) and diseases overall (see additional file ). additionally, the terms "haemolytic uraemic syndrome" and "leprosy" exhibited significant negative correlations with the respective disease notifications at a lag of one month. the development and application of internet-based infectious disease surveillance systems has the potential to enhance infectious disease control and prevention. whilst this is widely recognised [ , , , , , , , ] the investigation and application of internet-based surveillance has not been systematically applied across infectious diseases; the lack of systemic knowledge regarding the potential breadth of internet-based surveillance appears to have restricted the development of systems to a small number of diseases. to our knowledge, assessments of the use of internet-based surveillance have only been performed for five of the diseases that were demonstrated to have a significant association with internet search terms (influenza [ ] , dengue [ , ] , chickenpox [ , ] , hepatitis b [ ] and cryptosporidiosis [ ] the authors of the final study were, however, not able to detect signals from internet search queries). our study suggests that internetbased surveillance systems have potential application to a wider range of diseases than is currently recognised. however, correlations alone should not be viewed as definitive evidence that such systems are viable; some discretion must be applied, particularly as the analyses performed were univariate. correlations between internet metrics and both gonococcal infection and chlamydia (figure , boxes and ) were high; this appears to be due to a general upward trend in both and internet metrics appears to have little value in detecting perturbations in cases beyond this. this is supported by the cross correlation results (which are seasonally differenced); despite being ranked nd and th by spearman rho (figure ), no positive correlations were observed for these disease/search term cross correlations, even at lag ( figure ) . further research needs to be performed; however, this study suggests surveillance systems build on internet search data to have significant promise for a number of diseases beyond those previously described, most notably pneumococcal disease, ross river virus infection, pertussis, barmah forest virus and invasive meningococcal disease. the application of internet-based data to monitoring systems of interest has been termed "nowcasting"; this approach does not predict the occurrence of future events, but rather seeks to produce more timely information on the systems of interest [ ] . for infectious disease surveillance, this is typically achieved through the ability of internet-based surveillance systems to collect data at an earlier time point than is possible for traditional systems or by circumventing bureaucratic structures inherent to traditional systems that impede information flow [ ] . search terms that exhibit a high level of correlation with disease notifications are of value as they may be used to provide faster intelligence on emerging disease events. results of cross correlations (figure ) , however, indicated that forecasting of infectious disease events may also be possible using internet-based data. of the diseases that exhibited significant spearman's correlations, also had significant positive cross correlations at a lag of one month. overall, cross correlations indicated that forecasting of notification rates using internet-based metrics would be most realistic for the vaccine-preventable and vector-borne diseases. despite search terms offering strong or very strong correlations for two of the sexually transmissible diseases, neither exhibited significant correlations at a lag of one month. whilst internet metrics may provide valuable information regarding disease status, it is important to view these within context. the term "dengue mosquito" (figure , panel ) leads notifications by up to one month. the data imply dependence of dengue notifications on searches for the term "dengue mosquito". the mechanism of this dependence is more likely that environmental conditions that increase the abundance of mosquitos in dengue risk areas correlate with both an increase in dengue notifications and increased search interest for "dengue mosquito", allowing the search term to be used as an indicator for notifications. in this context the internet metrics also provide information that is of potential significance with respect to control of dengue fever; there is increased interest regarding mosquitos in the community and this may be driven by an increase in mosquito numbers. conversely the incidence of disease in the community may also affect search habits. the search term "chikungunya" lags notifications for chikungunya virus infection (figure , panel ). searches for "chikungunya" are probably driven by media exposure. media bias has previously been reported to adversely affect internet-based surveillance systems [ , [ ] [ ] [ ] [ ] [ ] and an increase in cases of a disease in the community will likely result in the publication of stories about the disease in the media; in turn, media exposure will drive internet searches on the topic. these processes, however, are not necessarily mutually exclusive. searches for a disease may lead notifications, however, increased notifications and reporting of an emerging disease event in the media may also drive internet searches. the complexity of this relationship may make interpretation of google trends' data more difficult. for pertussis (figure , (see figure on previous page.) figure cross correlation results for the diseases with the highest spearman's rho values . cross correlations for two search terms are displayed for each disease. coloured bars correspond to the search term with the highest spearman's rho value for each disease (red bars indicate values that exceed the % confidence interval, whereas blue bars do not). unfilled bars indicate cross correlation results for alternative search terms with highest cross correlation values at a lag value of . confidence intervals ( %) are indicated by the grey lines. panel ), the term "whooping" exhibits a significant positive correlation with disease notifications from lag − through to lag . it appears that both mechanisms occur for the same term, demonstrating a potential difficulty in interpreting these data. it is imperative that any terms used in the development of forecasting models are heavily screened to address the complexities of the driving forces behind health-information seeking and routinely re-evaluated to account for any shifts in search behaviour which may occur [ ] . there were a number of obvious limitations to this study. the temporal resolution of the data used was monthly. internet-based surveillance systems built upon monthly data are unlikely to provide better intelligence than existing traditional surveillance systems; these commonly rely upon weekly or daily reporting. this was a function of the availability of the notification data. secondly, the analyses were performed for a specific setting: australia. the nuances of language will create differences in the applicability, not just for different countries, but also within a country and between different settings (such as during an influenza pandemic) [ ] . australia was selected as the study area because internet penetration in australia is very high (> %) [ ] and use is largely restricted to a single search engine; google maintains a market share of over % in australia [ ] . these features reduce biases associated with unequal patterns of use and/or access. additionally, owing to its extensive size, australia exhibits a range of climates and varying environmental conditions, making it susceptible to a wide range of infectious diseases, including endemic and nonendemic vector-borne diseases. additionally, australia has a strong public health network and comprehensive infectious disease surveillance systems which compile high quality data on a range of diseases. combined, these features of internet usage and availability, infectious disease surveillance systems and diseases susceptibility patterns make australia an ideal system in which to study the potential application of internet-based surveillance systems. it is hoped that this work will stimulate further research into internet-based infectious disease surveillance systems beyond australia. even within our own study, however, we observed variation in correlations between internet search metrics and disease notifications for the various states ( figure ). it is imperative to develop models specific to the region of interest and to assess the performance of any internet-based system against traditional surveillance data specific to the region being monitored. thirdly, this study analysed the performance of only single search terms in estimating infectious disease notifications. whilst google has not revealed the terms utilised, or the weightings applied, google flu trends is reported to incorporate around search terms [ ] . despite using only a single search term for each analysis, notifications for diseases were identified as having a strong or very strong correlation with the selected search terms. compounding this is the fact that bonferroni adjustments were applied in assessing significance. bonferroni adjustments have previously been criticised for being overly conservative and for increasing the occurrence of type ii errors (false negatives) [ ] . as such, whilst this study provides a base for future research, it would be remiss to limit future investigations to just these diseases. this study identified numerous infectious diseases of public health significance that had not previously been investigated to have potential for monitoring using internetbased surveillance systems however, this study did not seek to produce robust, accurate, internet-based surveillance systems or early warning systems that are able to produce actionable and timely data for public health units. the aim of this study was to identify the diseases for which this is possible and to focus future research efforts into these. to achieve this aim, this study used univariate analyses to determine the usefulness of internet search metrics for monitoring a wide range of infectious diseases. whilst this simplistic approach was useful for screening diseases, it will not suffice in monitoring or forecasting incidence. future studies should focus on developing composite indexes incorporate multiple search terms, or data sources (such as weather data). models built in such a manner are more resilient to media-driven behaviour, fear-based searching and evolutions in language [ ] . internet-based surveillance systems have the potential to be applied to more than just enumerating disease cases within the community or predicting the onset, peak and magnitude of outbreaks. internet-based systems also have value as tools for planning emergency department staffing and surge capacity [ , ] or for healthcare utilisation [ ] . future research needs to also investigate to application of internet-based data; the greatest challenge in this field may not actually be creating models for forecasting or monitoring disease within the community, but rather applying and articulating the significance in a manner that is beneficial. internet-based surveillance systems have broader applicability for the monitoring of infectious diseases than is currently recognised. furthermore, internet-based surveillance systems have a potential role in forecasting of emerging infectious disease events. additional file : complete tables of results for google correlate searches, google trends data, spearman correlations and cross correlations. trends and directions of global public health surveillance modeling the effects of epidemics on routinely 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the salary for gjm was provided through the australian national health the authors declare that they have no competing interests.authors' contributions gjm and wh developed the original idea for this study. development of the script for data collection was performed by smra. data analysis was performed by gjm with the assistance of wh, jsb, st and acac. the manuscript was primarily written by gjm with editorial advice from wh, smra, jsb, st and acac. all authors read and approved the final manuscript. key: cord- -qqrhvlm authors: shoghri, ahmad el; liebig, jessica; jurdak, raja; gardner, lauren; science, salil s. kanhere school of computer; engineering,; wales, university of new south; sydney,; australia,; data ,; scientific, commonwealth; organization, industrial research; brisbane,; science, school of computer; technology, queensland university of; civil, department of; engineering, systems; university, johns hopkins; baltimore,; usa,; innovation, research center for integrated transport; sydney, unsw title: identifying highly influential travellers for spreading disease on a public transport system date: - - journal: nan doi: nan sha: doc_id: cord_uid: qqrhvlm the recent outbreak of a novel coronavirus and its rapid spread underlines the importance of understanding human mobility. enclosed spaces, such as public transport vehicles (e.g. buses and trains), offer a suitable environment for infections to spread widely and quickly. investigating the movement patterns and the physical encounters of individuals on public transit systems is thus critical to understand the drivers of infectious disease outbreaks. for instance previous work has explored the impact of recurring patterns inherent in human mobility on disease spread, but has not considered other dimensions such as the distance travelled or the number of encounters. here, we consider multiple mobility dimensions simultaneously to uncover critical information for the design of effective intervention strategies. we use one month of citywide smart card travel data collected in sydney, australia to classify bus passengers along three dimensions, namely the degree of exploration, the distance travelled and the number of encounters. additionally, we simulate disease spread on the transport network and trace the infection paths. we investigate in detail the transmissions between the classified groups while varying the infection probability and the suspension time of pathogens. our results show that characterizing individuals along multiple dimensions simultaneously uncovers a complex infection interplay between the different groups of passengers, that would remain hidden when considering only a single dimension. we also identify groups that are more influential than others given specific disease characteristics, which can guide containment and vaccination efforts. human mobility continues to play a vital role in spreading infectious diseases within a population [ ] [ ] . ongoing population growth and the high reliance of individuals on public transport services in highly populated cities provide a suitable platform for contagious diseases, such as measles, the recently emerged coronavirus and influenza, to spread widely and rapidly [ ] [ ] [ ] . for example, individuals travelling on a bus are in close enough proximity to infect each other and can carry the infection to distant locations across the public transport network [ ] [ ] . additionally, some pathogens may remain in the environment (e.g. a bus) for a prolonged period and can infect susceptible individuals after the infectious person has left the area [ ] [ ] . furthermore, transport services shorten distances and times and strongly connect different suburbs, potentially exposing communities to a high infection risk [ ] . the risk of disease spread due to human movement is evident from the current novel coronavirus outbreak in china and internationally, with chinese authorities shutting down public transportation within the affected area [ ] . the recent uptake of smart travel cards and the availability of this data have created an unprecedented proxy to elicit different travelling behaviours and to study their effects on disease spread [ ] [ ] . the analysis of such data is critical to understand the spreading dynamics of a disease and consequently to develop effective containment strategies [ ] . previous studies investigated several spreading dynamics of infectious diseases, however, to the best of our knowledge none of the studies has incorporated different aspects and dimensions of mobility behaviour simultaneously. in this paper we study three aspects of mobility behaviour, i.e. the degree of exploration, the distance travelled and the number of encounters of passengers using the sydney bus network in the context of infectious disease spread. by considering the three dimensions simultaneously, we identify previously unknown mobility behaviours. the high spatiotemporal resolution of the dataset allows us to construct a time resolved physical human contact network to simulate disease spread. specifically, we trace the infection flows between groups of passengers who display different mobility behaviours to investigate the change in the spreading dynamics. in addition, we investigate how changes in the infection probability and the time pathogens remain suspended in the environment affect the spreading of the disease. this study identifies the most influential passenger groups in a disease spread scenario for different disease characteristics and types. our simulation results identify four dominant transmission paths between the mobility groups that should become a focus of containment efforts. in addition, we find that highly connected passengers who regularly visit the same places have the highest spreading power when pathogens do not remain in the environment. however, with an increase in the suspension time of pathogens, highly connected passengers who visit new locations become the most efficient spreaders. an increase in the infection probability on the other hand, amplifies the spreading power of all mobility groups, especially for passengers who regularly visit the same places and travel short distances, until reaching a saturation point at a probability of . . the remainder of the paper is organised as follows: we begin by discussing relevant previous work in section ii. in section iii we present our framework for modelling infectious diseases on human contact networks. we explain the approach for classifying the individuals based on their movement behaviour and introduce the dataset that we use for our case study. in section iv we run extensive trace driven simulations to investigate the underlying interactions and disease transmission dynamics between the different groups of passengers. furthermore, we study the effect of changing the infection probability and the time pathogens remain in the environment on the transmission dynamics. finally, we identify the most influential mobility behaviours for various disease characteristics, which can guide intervention strategies. section v concludes the paper. studies of epidemiology have long recognized that human mobility plays a key role in fostering severe disease epidemics that may result in high rates of morbidity and mortality [ ] . furthermore, these studies have acknowledged the importance of identifying the most influential individuals, as it can aid to predict outbreaks before their occurrence [ ] [ ] . health related datasets and detailed patient mobility profiles present informative data that may be used to reflect the status of a disease and its progression [ ] [ ] . however, accessing such information is challenging due to privacy concerns and other related issues [ ] [ ] . in the absence of health related data, previous work has studied alternative data sources. an important body of research has explored the use of call detail records (cdrs) and data from the global positioning system (gps) to build epidemiological models and to study the spatial transmission of various diseases in a population at both city and country levels [ ] [ ] . in [ ] cdr and gps datasets were exploited to extract two types of mobility behaviours. the authors used the recurrent mobility and the total mobility characteristics to group individuals into returners and explorers. returners are individuals who can be characterized by their most visited locations as these dominate their movement behaviour, whereas explorers are individuals who often visit new places and cannot be characterized by their most frequently visited locations. the statistical measure used to compute the total mobility of an individual is the total radius of gyration r g , defined as [ ] : where l is the set of all visited locations by the individual, r i is the coordinates of the visited location i, n i is the individuals visitation frequency of location i, r cm is the centre of mass of all visited locations and n is the total number of visited locations. the authors also defined the k-radius of gyration, denoted r g (k) , which is similar to the overall mobility formula, with the difference that the set of locations l is reduced to the k most visited locations. the value of r g (k) represents the recurrent mobility of the individual. the correlation between the recurrent and the total mobility values distinguishes between the two mobility patterns, namely returners and explorers. if the recurrent mobility of an individual dominates the total mobility, that is r g (k) > r g / , the individual is classified as a returner. otherwise, the individual is an explorer. the authors of [ ] found that explorers have more impact on disease spread than returners. their experiments consisted of , individuals chosen randomly from a pool of , individuals. to study the impact of each mobility behaviour on the spreading, different proportions of returners and explorers were used. the extent of disease spread is computed through the global invasion diffusion threshold r * . this experimental setup presents three main limitations. first, changing the proportion of the mobility groups alters the topology and the characteristics of the network being studied. second, the contact links connect geographical areas rather then actual human physical encounters. third, their study of spreading power was performed on a static network in which if a link existed at any point in time that link is considered present during the entire period of study. these limitations make the experiments theoretical as they study a snapshot of a possibly unrealistic contact network. several other limitations emerge when cdr and gps datasets are used in the context of disease spread [ ] [ ] . most importantly, these datasets lack accurate localization of the individuals due to the distant positioning of cellular towers and poor satellite signals [ ] . hence, these datasets do not guarantee the existence of real physical encounters between the individuals [ ] . in addition, individuals who are tracked via gps may be driving a car and hence are not in physical contact with other individuals [ ] . recent studies of epidemiology showed an increasing interest in dynamic networks that guarantee the existence of real physical human contact when studying disease spread [ ] . a well suited source of data to study the spreading dynamics of diseases in dense cities are public transit records [ ] . several studies have confirmed the presence of a risk factor between the use of bus transportation services and the spreading of many airborne diseases such as tuberculosis, measles and influenza [ ] [ ] . the authors of [ ] stated that bus routes "are veins connecting even the most diverse of populations" and showed that individuals who reported regular use of buses are more likely to be infected by tuberculosis. in fact, the congregated and enclosed setting of buses presents a suitable environment for any contagious respiratory disease to spread widely. the infectious pathogens can easily be transmitted onward among passengers through coughing and sneezing [ ] . in addition, natural and artificial air flow can move suspended pathogens through space. this makes all individuals in an enclosed space like a bus susceptible. in our previous work [ ] we confirmed the existence of explorers and returners in the public bus transit dataset of sydney, australia. furthermore, through extensive simulations, we showed that explorers are generally more influential in spreading a disease through the network in comparison to returners. also, long distance travellers were found to be more influential than short distance travellers. however, when only long distance travellers are considered returners showed a greater propensity in spreading the disease over explorers. the work proved the presence of a deeper and more complex interplay between various mobility aspects when it comes to spreading a disease on a public transport system. in our previous work we did not consider the connectivity aspect of the individuals, which holds critical information in contact based spreading scenarios. further, our simulations only considered direct encounters between passengers and assumed an infection probability of . while disease transmission is possible through direct encounters (i.e. the infected and the susceptible individual are present in the same place at the same time), pathogens can remain in the environment for an extended period of time [ ] [ ] . therefore, an infectious person can infect susceptible individuals without a direct encounter. contact networks where only direct encounters are considered are commonly called spst (same place same time) networks. networks that in addition to direct contacts capture indirect encounters caused by suspended pathogen are called spdt (same place different time) networks. previous studies have shown that considering the suspension time of pathogens changes the underlying topology of the contact network and alters the spreading dynamics of contagious diseases significantly [ ] [ ] . this paper addresses the limitations of our previous work through the addition of the connectivity dimension and by considering different suspension times of particles and infection probabilities. we identify groups of passengers that have a high potential to spread a disease through a public bus network. although there are several studies that recognized the importance of human mobility data to identify the most influential individuals in a network, none of the studies tried to use a comprehensive mobility dataset to extract patterns along different movement aspects simultaneously and study the detailed interaction between the different patterns. in particular, we consider the passengers' total mobility, recurrent mobility and connectivity. the impact of each group on the spreading will be evaluated as all the infectious activities occurring in the background of the simulations are traced. to understand the disease spread dynamics on a public transport network we construct spst and spdt contact networks from the smart card data and run a susceptible-infected-recovered (s-i-r) disease spread model on top. at the beginning of the simulation all bus passengers, except a given number of randomly chosen seed nodes, are susceptible. the seed nodes are infectious and able to transmit the disease to susceptible individuals. when a susceptible individual encounters an infectious individual or, in the case of spdt networks, comes in contact with pathogens that remain in the environment, the susceptible individual moves to the infectious state with a given probability. the individual remains infectious for a given period of time before recovering from the disease. once in the recovered state the individual is no longer susceptible and remains in the recovered state until the end of the simulation. figure exemplifies the s-i-r disease spread simulation on the bus network. we demonstrate how the spreading dynamics are affected by changing two key parameters, namely the probability of infection, denoted β and the suspension time of pathogens, denoted d t . the case d t = corresponds to an spst disease spread scenario and hence a susceptible passenger will be infected only if both individuals meet on the same bus at the same time. when d t > the infectious particles remain on the bus for an additional time d t , allowing the infectious passenger to infect susceptible individuals after disembarking. while simulating the empirical movements of individuals, we track all encounters and infection transmissions. at every encounter the identification number of the two passengers in contact are recorded. similarly, when an infection is transmitted the identification numbers of the infectious and the susceptible individuals are recorded. to understand how different mobility behaviours influence the transmission paths of the simulated disease we classify the bus passengers into different mobility groups. we modified the opportunistic network environment (one) simulator [ ] to carry out our trace driven simulations and spread a disease on a large scale real-world transport network. before running the disease simulations on the constructed networks, we cluster the bus passengers into different groups, based on their mobility behaviour. to do so, we simultaneously consider the degree of exploration, the distance travelled and the number of encounters during the period of study. first, we plot passengers' mobility profiles in three-dimensional space, with the x-axis corresponding to the passenger's total radius of gyration, the y-axis corresponding to the k-radius of gyration (i.e. the recurrent mobility) and the z-axis corresponding to the number of encounters. next, we cluster the individuals into two groups along each dimension. the degree of exploration is divided into returners and explorers, the distance travelled into short distances and long distances and the number of encounters into low connected and highly connected individuals. classifying our passengers along the three dimensions results in = different types of movement behaviours. in order to identify each of the groups, we normalize the values of the three dimensions between [ , ] and use the approaches detailed in the following subsections. ) returners and explorers: to split the population based on the degree of exploration, we project all the points onto the xy-plane and use the bisector method to differentiate between returners and explorers. when plotting the passengers' total mobility and recurrent mobility values on the cartesian plane, points along the x-axis correspond to explorers as their recurrent mobility does not dominate their total mobility and points along the y = x line correspond to returners whose total mobility can be well represented by their recurrent mobility as r g (k) ≈ r g . our clustering approach results in . % explorers and . % returners. ) short distances and long distances: to cluster the passengers based on their travelled distance, we project the points onto the x-axis and apply a standard k-means clustering algorithm [ ] with k= . this results in two groups, namely, passengers who travel short distances and have a relatively low radius of gyration ( %) and passengers who travel long distances and have a relatively high radius of gyration ( %). ) low connected and highly connected individuals: in order to cluster the passengers based on their degree centrality (i.e. the number of encounters) we use a similar approach as in the previous section. we project the points onto the z-axis and apply the standard k-means clustering algorithm [ ] with k= , which splits the population in low connected passengers ( . %) and highly connected passengers ( . %). specifically, we differentiate between passengers who encounter a high number of other passengers and those who experience fewer encounters with other passengers during the month of study. the public transport dataset consists of , , trips made by million bus users. the dataset is recorded in the greater sydney area of new south wales, australia during the month of april in . each trip record records the following information: the passenger's smart card identification, the bus number in use and the time and location the passenger entered and exited the bus. sydney for a short time or travellers who lost or damaged their card. as infrequent travellers cannot be classified accurately due to the lack of sufficient data records, we remove these passengers from our analysis. in order to explore how a threshold on the number of trips affects the total number of passengers included in our analysis, we plot in fig. the population size against varying threshold values between and the maximum number of trips observed in the data. the population size drops rapidly with the increase of the threshold especially at low values. this is due to the high number of passengers who use the bus only occasionally (see fig. ). for our analysis, we set the threshold to trips per month. that is, individuals who travelled less than times with the bus during april are excluded from the analysis. the threshold of trips is chosen so that the passengers have travelled at least on half of the days of the month. the final dataset has , , records belonging to , bus passengers. in fig. we compare some key topological aspects of the original and the resulting networks. figure .a shows the distribution of connected component sizes for both networks. we notice that the two networks have similar structures, with a giant component and several smaller components. there are fewer components of size one in the resulting network, which can be attributed to the fact that the original network contained many infrequent travellers who were isolated from the rest of the network. this observation also applies to the isolated components consisting of less than eleven individuals. figure .b shows the degree distribution of the original and the resulting networks. the degree of a passenger is the total number of direct encounters experienced during the month of april. we notice that the degree distributions of the original and the resulting networks increase linearly until reaching maximum values of , and , respectively. then both distributions drop exponentially. the drop in the frequencies of the resulting network is due to the removal of passengers who travelled less than trips. the difference between the two distributions is especially clear at low degree values as passengers with few number of trips are less likely to have higher number of contacts. in this section, we present the identified mobility patterns and discuss the results of our disease spread simulations. the different groups resulting from our classification and clustering tasks are visualized in fig. . all subfigures display the same plot from a different angle. each point in fig. corresponds to one individual in the dataset and its coordinates represent the values of the three-dimensional movement behaviour of the corresponding passenger. in the coming figures and tables we refer to the groups using the notation {degree of exploration} {connectivity} {distance travelled}. the pie chart in fig. summarizes the percentage of each of the eight classified groups of passengers in the network. we notice that highly connected returners who travel short distances constitute the major portion ( . %) of the population. this group of individuals are regular commuters who tend to use public transportation to commute between home and work during peak hours and rarely explore or visit other places during the month. . % of public transport users are classified as low connected returners who travel short distances. we believe that these individuals regularly travel to specific locations that are less crowded or during off-peak hours, for example people who go to shopping malls in the afternoon. on the other hand, explorers are individuals who in addition to their regular commute visit other places, for example going to malls to shop or going to touristic attractions for leisure. the following subsections present and discuss our simulation results. for every experiment, we randomly choose individuals who are infectious at the beginning of the simulation and can transmit the disease to susceptible individuals. individuals remain infectious for five days (which is the average infectious period for influenza [ ] ) before recovering. every experiment is simulated times and the results averaged. in our first experiment we set the infection probability β to and the pathogen suspension time d t to , corresponding to an spst disease spread scenario. this experiment serves as a baseline for comparison to other parameter settings that are explored in further simulations. in table i , we present the total number of encounters and the total number of infections that were transmitted and received by every mobility group. in addition, we compute the average number of encounters, the average number of transmissions and the average number of infections received per individual for each group. dividing the total number of infections caused by a given group by its population size results in the average number of infections that one individual from that group causes during the simulation period. this average value of infections is not constrained to a specific target group but to all groups in the network. similarly, dividing the total number of infections received by a given group by its population size, results in the average number of infections that one individual of that group receives during the simulation period. interestingly, the averages of received infections per individual is nearly the same across all the groups with a value just divide individuals into explorers and returners, but to distinguish them further along other dimensions such as the distance travelled and the connectivity as their spreading abilities differ. in order to visualise the disease transmission dynamics between the groups we use a chord diagram (see fig. ). the diagram shows cumulative disease flows between the different groups. the eight different groups are represented by circle segments, with each group being associated to a unique colour. for example, the red segment corresponds to highly connected explorers who travel long distances (see label "a" in fig. ) . the links indicate the volume of disease transmissions between any two groups and are assigned the same colour as the source group. the thickness of each link is proportional to the average number of people that one individual from the source group infects in the target group. for example, the link labeled "b" shows the volume of disease flow transmitted from the group of highly connected explorers who travel long distances (red segment) to the group of highly connected returners who travel short distances (blue segment). links that start and end at the same segment represent disease transmissions between individuals of the same mobility group. for scaling purposes, we multiply all average number of infections caused per individual by and show the resulting values in the chord diagram. the diagram in fig. clearly identifies four dominant infection paths. these occur amongst highly connected returners who travel long distances (cyan segment) with individuals causing on average . infections within their own mobility group and highly connected returners who travel short distances (blue segment) with individuals causing on average . infections within their own mobility group. highly connected explorers who travel long distances (red segment) infect on average . highly connected returners who travel short distances (blue segment) during the simulation period. highly connected explorers who travel short distances (orange segment) infect on average . highly connected returners who travel short distances (blue segment). furthermore, low connected returners who travel short distances form a group that is prone to receive infections, but less likely to infect individuals from other groups (see the incoming non-pink links that occupy the majority ( %) of the pink segment in fig. and table i ). on the other hand, highly connected explorers who travel long distances have caused the greatest number of infections per individual on average. however, this group is less likely to get infected in comparison to other groups (see red segment in fig. ) . we observe that the disease transmissions from highly connected explorers who travel long distances dominate this group's activity as the red outgoing links going to all other groups constitute the majority of the segment with more than %. that is, even a low number of infected individuals of this group would be sufficient to infect other groups and spread the disease through the entire network. highly connected explorers who travel long distances infect . individuals on average during the simulation period. highly connected returners who travel short distances receive a high number of infections and mostly infect individuals within their own group (see blue segment in fig. ). this behaviour is expected, as this group consists of regular commuters who display consisted movement behaviour. highly connected returners who travel long distances (see cyan segment in fig. ) display a similar behaviour of mostly infecting individuals within their own group. highly connected explorers who travel the mobility groups are represented by differently coloured circle segments (e.g. the red segment, labelled "a", corresponds highly connected explorers travelling long distances). links represent disease flow between mobility groups and are coloured by the source group. the link with label "b" shows the average number of infections that highly connected explorers who travel long distances transmit to highly connected returners who travel short distances. "c" refers to the start of the link coloured by the receiving group. "d" refers to the end of the link coloured by the transmitting group. "e" refers to the relative infection transmissions, receptions and overall total for each segment. long distances spread infections to all other groups (see red segment in fig. ) , although the average number of encounters is lower than groups who infect specific target groups. our results highlight important interactions between the eight identified groups and shed light on disease spread dynamics that should be given more attention while monitoring a disease and applying prevention measures. to understand how different disease types and characteristics change the spreading dynamics between the eight groups, we perform two additional experiments. in this experiment, we run the simulations with different suspension times of pathogens, i.e. d t = , , and minutes, while keeping β = . for each value of d t we construct a matrix that shows the difference in the average number of infections caused and received by each mobility group in comparison to experiment (β = , d t = ). positive values refer to a gain in disease transmissions, whereas negative values indicate a loss. the rows of the matrix correspond to the groups that cause the infections and the columns correspond to the groups that receive the infections. figures .a and .b show the matrices for a suspension time of minutes and minutes, respectively. the matrix in fig. .a shows that the average number of infections caused per individual increased or remained the same for the four groups of explorers (top four rows of the matrix). the spreading potential of the four groups of returners (bottom four rows of the matrix) generally decreased. as the suspension time is increased to minutes, we observe further increases in the average number of infections caused by explorers and further decreases for returners (see fig. .b). we highlight that the increase in d t weakens the spread of infections within (self-loops) the two groups of highly connected returners. the loss in the infection power of returners coincides with an increase in infections caused by explorers, especially for highly connected explorers who travel long distances. since almost every individual of the population is infected at the end of the simulation period, we conclude that an increase in the time that pathogens remain in the environment favours the infection power of explorers. that is, explorers are even more influential in an spdt disease spread scenario. we only show the results for d t = and as no change in the behaviour was seen for d t = and , the values for explorers keep increasing and those of returners decrease. in the third experiment, we vary the infection probability β, while setting the pathogen suspension time d t = . the considered probabilities are . , . , . , . , . , . and . to understand the effect of the infection probability on the disease spread we construct matrices that show the differences in the average number of infections caused per individual between each two consecutive values of β. the average number of infections caused per individual increases rapidly for all groups with an increase of β from . to . . this result is visualised in fig. with all matrix elements being positive. when β is increased from . to . we see only a slight increase in the spreading power of all groups. further increases of β to . and do not result in significant changes in spreading powers. figure shows that all individuals who travel short distances experienced the most increase in the number of received infections, whether they are low or highly connected. this pattern can be seen through the dark coloured columns of the short distances groups. the observation is due to these groups constituting the highest percentages in the network allowing them to have the highest total number of encounters (see fig. and table i ). in addition, the increase in the probability of infection strengthens the self-loops of the groups (infections within the same group), especially those of the short distance returners. we conclude that increasing the infection probability favours the spreading power all mobility groups. the increase of spreading power is relative to the interaction between each pair of mobility groups. for increasing infection probabilities each element in the matrix increases until ultimately reaching the values presented in section iv-b in which the probability is set to . this is the first study to identify mobility patterns along three dimensions simultaneously, namely the degree of exploration, the distance travelled and the number of encounters. we found previously unknown mobility patterns that were thoroughly investigated to understand the spreading dynamics of contagious diseases on a city wide public transport system. we ran extensive disease spread simulations with varying values for the infection probability and the suspension time of pathogens. our results show that characterizing individuals along multiple dimensions simultaneously uncovers a complex infection interplay between the different groups of travellers. furthermore, the infection probability and the suspension time of pathogens play different roles in the spread. highly connected passengers who regularly return to the same places play the most important role in the spreading when pathogens do not remain in the environment. however, with an increase in the suspension time of pathogens, highly connected passengers who visit new locations are the most influential. unlike the suspension time, increasing the infection probability does not affect particular mobility groups, but increases the infection power of all groups especially for returners who travel short distances. our simulation experiments are abstractions of the real-world and flexible to adapt to different contexts. we presented a framework that can be applied to model any disease that is spread through a physical contact network. our findings are especially beneficial to advise health authorities on the design of more efficient intervention and containment strategies depending on the characteristics of the emerging diseases. we plan to open-source the modified simulator in order to be used broadly for similar types of datasets and scenarios. impact of indirect contacts in emerging infectious disease on social networks disease and mobility: a neglected factor in epidemiology investigating physical encounters of individuals in urban metro systems with large-scale smart card data the roles of transportation and transportation hubs in the propagation of influenza and coronaviruses: a systematic review wuhan shuts public transport over outbreak airborne biological hazards and urban transport infrastructure: current challenges and future directions bernard mans, and frank de hoog. indirect interactions influence contact network structure and diffusion dynamics connecting mobility to infectious diseases: the promise and limits of mobile phone data big data for infectious disease surveillance and modeling a comparison of spatial-based targeted disease containment strategies using mobile phone data commentary: containing the ebola outbreak-the potential and challenge of mobile network data beyond doctors: future health prediction from multimedia and multimodal observations quantifying the impact of human mobility on malaria integrating rapid risk mapping and mobile phone call record data for strategic malaria elimination planning impact of human mobility on the emergence of dengue epidemics in pakistan using mobile phone data to predict the spatial spread of cholera quantifying seasonal population fluxes driving rubella transmission dynamics using mobile phone data the impact of human mobility on hiv transmission in kenya unveiling spatial epidemiology of hiv with mobile phone data returners and explorers dichotomy in human mobility using gps technology to quantify human mobility, dynamic contacts and infectious disease dynamics in a resource-poor urban environment on the use of human mobility proxies for modeling epidemics efficient detection of contagious outbreaks in massive metropolitan encounter networks giving tb wheels: public transportation as a risk factor for tuberculosis transmission how mobility patterns drive disease spread: a case study using public transit passenger card travel data the one simulator for dtn protocol evaluation scikit-learn: machine learning in python key: cord- - dfbhsl authors: sweet, michael j.; bateman, kelly s. title: reprint of ‘diseases in marine invertebrates associated with mariculture and commercial fisheries’ date: - - journal: j sea res doi: . /j.seares. . . sha: doc_id: cord_uid: dfbhsl diseases in marine invertebrates are increasing in both frequency and intensity around the globe. diseases in individuals which offer some commercial value are often well documented and subsequently well studied in comparison to those wild groups offering little commercial gain. this is particularly the case with those associated with mariculture or the commercial fisheries. specifically, these include many holothuroidea, and numerous crustacea and mollusca species. pathogens/parasites consisting of both prokaryotes and eukaryotes from all groups have been associated with diseases from such organisms, including bacteria, viruses, fungi and protozoa. viral pathogens in particular, appear to be an increasingly important group and research into this group will likely highlight a larger number of diseases and pathogens being described in the near future. interestingly, although there are countless examples of the spread of disease usually associated with transportation of specific infected hosts for development of aquaculture practices, this process appears to be continuing with no real sign of effective management and mitigation strategies being implicated. notably, even in well developed countries such as the uk and the us, even though live animal trade may be well managed, the transport of frozen food appears to be less well so and as evidence suggests, even these to have the potential to transmit pathogens when used as a food source for example. globally, documented cases of disease outbreaks are increasing in both frequency and intensity in many marine taxa (burge et al., ; harvell et al., ) . these recent increases in disease outbreaks may be caused by either the introduction of new pathogens or changes within the environment (burge et al., ; harvell et al., ) . although there are many recorded disease outbreaks in invertebrates, here, we are only focusing on those diseases associated with mariculture and other commercially important marine species. we will also focus on the potential of spread of certain well documented pathogens, through specific pathways such as the transportation of commercially important species from country to country, a result which inadvertently often includes the transfer of specific pathogens capable of infecting native populations of similar species. the three most commonly cultured phyla or subphyla include that of echinodermata, crustacea and mollusca. the world organisation for animal health (office international des epizooties (oie)) currently lists notifiable diseases for only the two latter groups, crustaceans and molluscs, as these are arguably the most important invertebrates on a commercial basis. however, holothuroidea (i.e. sea cucumbers) are increasing in popularity throughout many areas of the globe and likely warrant being added to oies' list of notifiable diseases in the not too distant future. the ability to assess the effects that disease may have on wild fisheries in terms of production losses is far more challenging than in farmed stocks. therefore, as a result, there is limited information available on the pathogens and disease of echinoderms, crustaceans and molluscs, and this has led to a deficit in knowledge on causes of mortalities within the wild populations. furthermore, recent work has also highlighted a difference in pathogens present in juvenile and adult populations of wild organisms (bateman et al., ) . such data is important in understanding the potential for disease to cause 'silent mortalities' (i.e. unobserved) in commercially exploited stocks. this data, also highlights that the ability to accurately assess the effect disease may have in terms of production losses in the wild is far more challenging than in a farmed environment (stentiford et al., ) . in this respect an emerging disease within wild fisheries may be more difficult to identify and classify when compared to the farmed stocks. in this review, we are not listing all known diseases for the three main commercially important phyla/ sub-phyla and/or class (echinoderms, crustaceans and molluscs), but instead focus on those which likely pose a major threat and/or are infecting large populations of both wild and farmed organisms around the world. we have therefore opted to split the review into sections outlined by major pathogenic and/or parasite causal agents. the impacts of disease on numerous echinoderm species have been relatively well documented over previous years and have in some cases provided compelling examples of major shifts in ecosystem state and cascading community effects following disease-induced die-offs in wild systems (uthicke et al., ) . in addition to the fact that such outbreaks in the wild can illicit dramatic changes to whole ecosystems, the increase in mariculture for certain species particular those from the class holothuroidea (sea cucumbers) has further exacerbated the need to understand diseases in this phylum. recent improvements in artificial breeding techniques of sea cucumbers around the world have occurred rapidly. in areas of asia, in particular the northern coast of china, rearing currently results in over - billion seeds being produced with~ , tonnes of sea cucumber (live weight) being harvested every year. such rapid expansion and intensification of sea cucumber farming have subsequently led to the occurrence of various diseases, causing serious economic losses and becoming one of the limiting factors in the sustainable development of this industry. although, little research has been conducted on these diseases so far, it is clear that in the next few years the causes and consequences of outbreaks in this group of organisms need to be understood for commercial practices to continue unabated. . . . viral diseases in holothuroidea . . . . acute peristome edema disease (aped). agent: to date, although there have been no definitive results, the most likely candidates for pathogenesis appear to be viruses. hosts affected: cultured sea cucumbers in particular apostichopus japonicus (wang et al., ) . clinical signs and pathology: diseased individuals first show edema in their peristomes, the tentacles cannot retract completely and adhesion capacities are weakened. this latter ailment results in diseased individuals dropping to the bottom of the ponds. grazing rate and activity decrease and upwards of % eviscerate (i.e. eject their internal organs). about - days later, small white lesions usually appear on the surface of the skin and gradually expand, and increased mucus secretion is often noticed over large areas of the body wall. about - days after occurrence of the first symptoms the sea cucumbers die, with mortality often in excess of % (wang et al., a) . virus-like particles (vlps) have been found associated within the epithelium of the intestine of diseased individuals (wang et al., ) . transmission electron microscopic examinations showed that the virions are spherical, - nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. detailed studies on the morphogenesis of these viruses found many characteristics previously described for other coronaviruses (wang et al., ) . virus particles were found to be congregated, and always formed a virus vesicle with an encircling membrane (wang et al., ) . the most obvious cellular pathologic feature appears to be large granular areas of cytoplasm, relatively devoid of organelles. tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also routinely found in the cytopathic cells. furthermore, in support of a viral pathogen being the causal agent for this disease, no rickettsia, chlamydia, bacteria, or other parasitic organisms have been seen to be associated with any of the diseased tissues sampled (wang et al., ) . furthermore, these vlps are only present in diseased individuals and absent in healthy animals. however, no infection trials have been conducted to date. therefore, further evidence is required to prove that these vlps are the aetiological agent. interestingly, the same vlps have also been observed in diseased larvae ( days old). this latter finding suggests that the disease may be transmitted vertically from parents, which may have severe implications for transportation of brood stock from pond to pond. epidemiology: first reported in cultured sea cucumbers along the shangdong and liaoning province coasts in china in , it has now caused a significant number of deaths in cultured sea cucumbers throughout china. diagnosis and/or treatment: based on disease signs only at the current time. . . . . stomach atrophy syndrome (sas). agent: an unknown virus approximately - nm in diameter (deng et al. ). hosts affected: a. japonicus. clinical signs and pathology: the stomach of diseased larvae can be observed to shrink gradually exhibiting thick, rough and distorted walls. virus like particles have been observed only in diseased individuals, these are predominantly spherical or hexagonal with a welldefined envelope, and exhibit dense core structures (yin-geng et al., ) . sizes range from to nm in diameter. the vlps have also been found in the gonad, body wall, alimentary canals and the respiratory trees of corresponding parents suggesting akin to aped (above), a possible route for vertical transmission of this pathogen. further examination of larvae by electron microscopic observations showed that the virus affects mainly the connective tissue cells and epithelial cells (yin-geng et al., ) . epidemiology: in , sas was first observed in a. japonicus throughout the liaoning area (yin-geng et al., ) . sas affects mainly larvae, approximately - days after hatching (deng et al. ). diagnosis and/or treatment: based on disease signs only at the current time. . . . bacteria diseases in holothuroidea . . . . skin ulceration disease (sud) aka bacterial ulceration syndrome (bus) aka acute peristome tumescence (apt). agent: numerous bacterial pathogens have been proposed (deng et al., ; lu et al., ; ma et al., ; wang et al., a; wang et al., ; zhang et al., ) . commonly members of the genus vibrio are isolated from diseased lesions, including vibrio cyclitrophicus, vibrio splendidus, vibrio harveyi, vibrio tasmaniensis, vibrio tapetis, and vibrio alginolyticus (deng et al., ; ma et al., ; wang et al., a; wang et al., ; zhang et al., zhang et al., , . however others such as a photobacterium sp. (deng et al. ; , pseudomonas nigrifacien (deng et al., ; wang et al., a) , aeromonas salmonda and aeromonas media (wang et al., ) , a pseudoalteromonas sp. and pseudoalteromonas tetraodonis (liu et al., ) and a marinomonoas dokdonensi (deng et al., ; ma et al., ) have also been implicated to play some role. the latter pathological agent, has been described under one of the alternative names associated with disease, apt (deng et al., ; ma et al., ) . inoculation experiments have been fulfilled for a few of these candidate pathogens including the pseudoalteromonas sp., p. tetraodonis (liu et al., ) and v. splendidus (lu et al., ) , suggesting that at the very least these three play a significant role in the disease aetiology. furthermore, phage therapy has been used to treat this disease targeting v. alginolyticus again suggesting a role for this bacterium. in addition, like many marine diseases, researchers are only recently searching for viruses associated with these diseases. indeed, two distinct virus like particles have now been observed associated with the disease lesions (wang et al., a; wang et al., ) . these include, a spherical virus with a diameter of - nm and a spherical virus with a nucleocapsid and diameter of - nm (wang et al., a; wang et al., ) . the latter of the two has been named, skin ulceration and peristome tumescence syndrome virus (suptsv) (liu et al., ) . crude experimental inoculation trials, which included the addition of filtered tissue extracts (i.e. supposedly only containing viruses) to otherwise healthy individuals elicited similar disease signs, suggesting that the proposed virus does indeed play an important role in disease onset. interestingly, the disease signs produced from the bacterial pathogens and viral pathogen, although resulting in the same overall pathology had certain differences. for example, inoculation with the bacterial pathogens included ulcer spots on the dorsal skin and abdominal parapodia, followed by an increase in the number and merging of ulcer spots and a decrease or loss of tentacle activity. in comparison symptoms caused by inoculation of the viral pathogen included an initial decrease in tentacle activity or loss of activity all together, peristome tumescence and the decay of dorsal papillate podia and peristome, along with abdominal ulceration. these differences suggest that the virus first affects the function of cells in tentacles and peristome, whereas bacteria initially cause ulceration of the skin. finally, its worthy of note here that unidentified platyhelminths are often observed in diseased tissue and they are thought to cause heavy damage to the skin. the size of the worms is variable, normally ranging from to mm. histological observations demonstrate that there is an abundance of worms in the tissues of the lesion area. they occupy a large space within the tissues and cause topical necrosis and scattering of the musculature (wang et al., a; wang et al., ) . however, exactly how these worms fit into the overall pathology of this disease remains unknown. hosts affected: numerous sea cucumber species including; juvenile holothuria scabra, a. japonicus and isostichopus fuscus (mercier et al., ; yin-geng et al., ) . clinical signs and pathology: due to its wide spread distribution, numerous names have been proposed for what some say is likely the same disease. however there are slight differences in the way the disease often manifests itself, suggesting that there may actually be multiple diseases currently under this one banner. for example in australia and madagascar, white lesions are observed to appear near the cloacal orifice and extends over the whole body, whilst in china, the white lesions have been noted to occur on any part of the body, begin canking and then extend over the whole body surface (mercier et al., ; yin-geng et al., ) . this disease usually results in chronic mortalities, with cumulative rates reported of - %. generally, infected individuals die within - days of the first clinical signs being observed. skin ulceration begins with the appearance of small white patches (ca. mm in diameter), which enlarge and eventually expose the underlying muscle and spicules. yet, the mesothelium, the muscles and the internal organs remain unaffected. podia inside lesions are often totally destroyed. juveniles have been reported to show less motivation once they had contracted the disease and become translucent (mercier et al., ; yin-geng et al., ) . scanning electron micrographs show two distinct 'zones' associated with this disease. the first, where the epidermis and cuticle are totally destroyed and where disorganized connective tissue is exposed has been refered to as the 'affective zone' and appears to be colonised by numerous different types of microorganisms. the second 'zone' is the borderline area of infestation, a few tens of micrometres wide where the surface is again colonised by a mix of microorganisms and where patches of degrading epidermis are mixed with degrading, exposed connective tissue. in the 'affected zone', collagen fibres run in all directions, breaking off from each other and ossicles, some of which appear highly degraded, are exposed to the external medium. epidemiology: skin ulceration disease was first described by (becker et al., ) affecting juvenile h. scabra reared in the hatchery of toliara, madagascar. since then, the same disease has been found in numerous species of cultured sea cucumbers in australia, new caledonia, ecuador and china. diagnosis and/or treatment: based on disease signs only at the current time. however preventive measures have been suggested for this disease and include many general husbandry practices such as: ( ) good hatchery management operations; ( ) disinfection of tanks, plates and tools before use; ( ) removal of excess food, faeces and other organic matter; and ( ) provision of high quality water. furthermore, the disease can be treated with antibiotics ( - ppm) such as ofloxacin, terramycin, acheomycin, levofloxacin hydrochloride, cefobid, doxycycline, novobiocin and sulphanilamides. this latter treatment suggestion may favour the roles of the bacteria as primary pathogens, over that of the viruses. . . . . stomach ulceration symptom (sus). agent: reportedly associated with pathogenic bacteria. hosts affected: the disease is known to target the larvae, with the auricularia being notoriously susceptible to the infection. the mortality of affected larvae has been shown to rise up to % in certain instances. clinical signs and pathology: stomach walls of the diseased juveniles appear thick, rough, and visibly atrophic . the ulceration of the stomach usually results in reduced growth and a lower metamorphosis rate. the disease often leads to mortality during the metamorphosis from the auricularia to the doliolaria stage. epidemiology: stomach ulceration symptom (sus) has been shown to have strong linkages with increases in temperature, occurring more commonly during the warmer summer months. the disease is also often triggered by feeding with unsuitable feeds and when the animals are kept in high stocking densities zhang and liu, ). diagnosis and/or treatment: based on disease signs only at the current time. interestingly, although no specific pathogens have been identified, numerous treatments have been suggested, including using appropriate feeds from the onset (i.e. marine yeast) or treating with antibiotics such as penicillin or streptomycin, in the range of - ppm (zhang and liu, ) . similar to sud, described above, finding a successful treatment for such diseases can aid in the identification of pathological agents. in this instance as antibiotics succeed in stopping the disease (again, similar to sud), the most likely causal agents appear to be bacterial. . . . . viscera rejection syndrome (vrs). agent: to date only, two bacterial isolates have been implicated in this disease: arthrobacter protophormiae, and staphylococcus equorum compared to the in sud (see above). however, as antibiotics rarely have a significant effect on treatment of this disease, these bacteria may actually turn out to be secondary causal agents or alternatively opportunistic pathogens. again similar to sud a virus has also been implicated in this disease, although little information is available with regard to morphology, structure etc. however, one study has shown that exposure to media containing this virus results in mortality of upwards of - % of individuals infected (deng et al., ). hosts affected: various sea cucumber species. clinical signs and pathology: first identified in , viscera rejection syndrome (vrs) usually starts with only a few infected individuals at the bottom of ponds, but the disease often spreads quickly. after - days, the viscera are ejected out of the body and the sea cucumbers succumb shortly afterwards. more than % of hatcheries have been known to exhibit this disease, with more than % mortality in some areas. previously vrs was characterised in the same disease group as sud (see above), however deng et al. ( ) argued that due to substantial differences in pathology, these diseases should be classed as separate diseases/syndromes. there are four main reasons which led deng and colleagues to suggest a separation of these diseases. primarily, sea cucumbers suffering from sud rarely exhibit signs of viscera ejection but do exhibit peristome edema expanding to the whole body. a second reason for this proposed separation of diseases is that after dissection of infected individuals the majority of those with sud (over %) had satiation intestines in coelom. in comparison, those with vrs (again upwards of % of infected individuals) had no intestines within their coeloms. furthermore, both diseases show differences in their mortality rates and have different methods proposed for prevention. sud for example has been shown to be treatable with antibiotics (slowing the spread), with mortality usually around %. in contrast vrs shows high infectivity, rapid spread, little response to antibiotic treatment and usually - % mortality. finally, although no definitive causal agents have been proposed for either disease state, the general consensus over the causal agents seems to differ between diseases. epidemiology: there is currently, very limited information on the spread of this disease. diagnosis and/or treatment: based on disease signs only at the current time. . . . . rotting edges symptom (res). agent: only one dominant bacterium (vibrio lentus), has been highlighted as the likely causal agent in this disease (zhang et al., ) . hosts affected: again, this disease predominantly occurs during the auricularia stages of development of many different sea cucumber species, with mortality being recorded as high as % in certain cases (zhang et al., ) . clinical signs and pathology: this symptom was first detected in commercial sea cucumber hatcheries in the shandong province in china. compared with healthy larvae, diseased individuals exhibit a darkening of the body edges, giving this disease its name. these individuals then undergo autolysis and the body completely disintegrates within days of initial signs. if metamorphosis is achieved, the pentactulae are weak and the survival rate is significantly lower than healthy individuals. infected larvae show a dark purple discolouration when stained with haematoxylin and eosin. cells appear inflamed and often infiltrate the spheroplasts. the epithelial tissues appear ulcerated with epithelial cell necrosis. no obvious pathological changes have yet to be observed in the internal organs, apart from shedding of the gastric mucosal cells from the epithelial cells. early ulceration also often occurs on the skin. epidemiology: to date, there is limited information on the spread of this disease. diagnosis and/or treatment: based on disease signs only at the current time. treatment with antibiotics including deomycin appears to cure this disease and again suggests that bacteria are the likely causal agents (zhang et al., ) . . . . . off-plate syndrome (ops). agent: three dominant rod shaped gram-negative bacteria have been isolated from specimens collected from different hatcheries, all of which are thought to come from the genus vibrio (zhang et al., ). hosts affected: shown to occur in settled juveniles (normally on pvc plates) of many different species. ops causes significant mortality of juveniles metamorphosing from the doliolaria to the pentactula stage, with rates often reaching % (zhang et al., ) . clinical signs and pathology: the affected juveniles shrink and gradually lose the ability to remain attached onto the available substrate, hence the name of this disease. subsequently, the epidermis of infected individuals usually disappears, however in more extreme cases the whole body can dissolve with the autolysing process. in such cases, the spicules can be found on the bottom of the infected tanks as they drop from the dissolved carcasses. epidemiology: limited information on the spread of this disease. diagnosis and/or treatment: based on disease signs at the current time. agent: many different protozoa have been described to be associated with holothuroids yet these are often disregarded as commensals (snyder, ) . although there are no specific described diseases associated with protozoa, infections do often manifest themselves. infected individuals often appear weak and sluggish but rarely show any conspicuous lesions, hence why names have most likely not been assigned. that said, one species worthy of note, diplodina gonadipertha, infests the gonads of cucumaria frondosa and could partially destroy the gonads completely (djakonov, ) . a result which would obviously have knock on effects in brood stocks. furthermore, diseases caused by platyhelminthiasis have been shown to infect both aestivated juveniles (larger than cm) and adults of many different sea cucumber species. however, similar to protozoa (above), no specific disease states have been named. numerous umagillids for example have been shown to ingest intestinal host tissue (similar to that observed with protozoan parasites). hosts affected: many sea cucumber species. clinical signs and pathology: such infestations have been recorded as being very high, with instances of upwards of flatworms for example being observed within a single individual. infestation by turbellarian egg capsules in particular have been recorded in over % of inspected individuals, with numbers being in excess of , in some instances. the majority of infestations usually manifest themselves in the form of small internal wounds with no lethality, however in rare instances high mortality has been noted (over %) within one month for example. epidemiology: limited information as no specific names have been described linked to these parasites. diagnosis and/or treatment: based on disease signs only at the current time. agent: although, no fungal pathogens have been definitively described for sea cucumbers, two have been described based on morphology alone. one is large with branched hyphae and macroconidia that contain more than spores, whilst the second species is thin, with straight hyphae and small sporangium (yin-geng et al., ) . hosts affected: many sea cucumber species. clinical signs and pathology: although fungal diseases have not been reported to cause widespread death, they will result in an unhealthy appearance and poor quality of the final product. the papillae of the sea cucumbers become white during the early stage of fungal infections. with large areas of the body wall often appearing bluish white as the skin is eroded by the fungi. unlike bacterial infections, there is no obvious mucus around the lesions. in some cases, the whole body surface becomes discoloured and transparent; the body wall becomes thinner and the affected individuals develop edema. histopathological observations have shown that fungal hyphae and spores can be detected in the muscular tissues, indicating that these fungi can invade the body wall and grow deep into the body tissues (yin-geng et al., ) . connective fibre tissue has also been reported to turn necrotic and disintegrate during heavy infections. epidemiology: fungal diseases frequently occur in sea cucumbers during summer months. both juveniles and adults have been shown to be infected, yet larvae appear to be unaffected to date. diagnosis and/or treatment: based on disease signs only at the current time. agent: the main causal agent in these incidences is the predatory copepod, from the genus microsetella zhang and liu, ) . hosts affected: many sea cucumber species. clinical signs and pathology: in the presence of high numbers of copepods, the abundance of juveniles decrease acutely within or days. usually, these predatory copepods will attack juveniles smaller than cm, and often cause high mortalities. juveniles often have lesions on their body and become weak. eventually, the juveniles die off, the body walls dissolve and the spicules disseminate on the bottom of the rearing tanks. epidemiology: summer often marks the peak of copepod reproduction as the larvae of sea cucumbers develop into juvenile stages. normally, the rearing conditions for juveniles are favourable for the growth and reproduction of this parasite. at temperatures between and °c, one adult copepod can produce individuals in days. generally, the mature female can produce new oocysts within a few minutes following the release of an initial batch. the copepods compete for food and space, as well as bite the young sea cucumber juveniles. diagnosis and/or treatment: based on disease signs only at the current time. however, the use of chlorophos has been proposed as the best option to control the problem. a dosage from to ppm has been shown to be effective; at this concentration all copepods can be killed in h without harming the sea cucumber juveniles zhang and liu, ) . several diseases pose significant threats to many different crustaceans, in both the wild and within post-capture holding facilities (shields, b; stentiford and neil, ) . annual production of farmed and captured crustaceans exceeds m metric tonnes with a first sale value of $ bn, with the sector being dominated by farmed tropical marine shrimp. strikingly, current estimates predict that up to % of total shrimp production is lost annually (n$ bn) and this loss is mainly accredited to disease. the world organisation for animal health (office international des epizooties (oie)) currently lists notifiable crustacean diseases, of which are viral in origin, bacterial and protistan. three of the viral diseases in particular are also listed within european legislation, ec council directive / /ec. although viral pathogens appear to exert the most significant constraints on the growth and survival of crustacea under culture conditions, protozoan pathogens appear to elicit the greatest detriment effects in wild populations, and affect the marketability of products harvested from these hosts (stentiford et al., ) . . . . . white spot syndrome virus (wssv). agent: white spot disease (wsd) is caused by the white spot syndrome virus (wssv) and is probably the most extensively studied crustacean virus to date. this is primarily due to the devastating effect it has had on the shrimp farming industry, with cumulative losses exceeding $ bn since (stentiford et al., ) . wssv was originally classified as an unassigned member of the baculoviridae family. however, it was quickly reclassified to be from a new genus whispovirus in the family nimaviridae and was named white spot syndrome virus by the ictv (mayo, ) . to date, wssv is currently the only member of this new genus. hosts affected: the virus appears to be non-specific, with all decapod and non-decapod crustaceans being listed as susceptible. to date, more than species of arthropods have been reported as hosts or carriers of wssv, either from culture facilities, the wild or experimental infection (sánchez-paz, ) . from the susceptible species, stentiford et al. ( ) identified which were eligible to be described as susceptible using the european food standard agency (efsa) principles. however, although all these species are susceptible, they have recently been found to vary in their levels of susceptibility (bateman et al., a) . research is also concentrating on potential vectors of this disease, which may be facilitating the spread. numerous aquatic organisms such as rotifers (yan et al., ) , bivalves, polychaete worms (vijayan et al., ) , non-decapod crustaceans such as artemia sp. and copepods have all been reported as potential vectors (stentiford et al., ) . although, the viruses have been shown to accumulate in these vectors in large viable numbers, there has been no evidence of replication within these hosts meaning they are currently classed as only being 'mechanical' vectors (chang et al., ; lo et al., ; yan et al., ) . clinical signs and pathology: the name of the disease refers to the clinical signs that have been reported in some (but not all) species of shrimp. white spots, associated with a calcium deposit, appear on the inner surface of the cuticle. wssv infects all tissues of mesodermal and ectodermal origin, such as gills, lymphoid organ, mid-gut but especially cuticular epithelium and sub-cuticular connective tissues. virions are ovoid or ellipsoid to rod-shaped and consist of an electron dense nucleocapsid, with a tight fitting capsid layer, surrounded by a loose-fitting trilaminar envelope. virions measure - nm in diameter and - nm in length and in some cases a tail like projection can be seen extending from one end. wssv virions possess a very distinctive capsid layer giving the dna core a cross-hatched or striated appearance (wu et al., ) . penaeus japonicus in china and taipei, and was shown to spread rapidly throughout asia and then to america (shengli et al., ; stentiford et al., ). due to this rapid spread, and the isolation and identification by numerous independent laboratories, the virus was referred to by a variety of different names; however it was agreed that these infections are actually all caused by the same agent and the name white spot syndrome virus was adopted (joseph et al. ) . wsd is listed as an oie notifiable disease and as a non-exotic disease within europe, ec council directive / /ec. although not officially reported (i.e. in scientific publications), outbreaks of wsd have been observed in shrimp farms in southern europe, and these reports provided the basis that wsd should be classified as a non-exotic disease to europe. however, the prevalence and spread of this virus throughout europe remain unclear; with the last known/reported outbreak of the disease being in on a shrimp farm in italy (stentiford and lightner, ) . interestingly and also alarmingly, a recent study investigated the viability of wssv in supermarket commodity shrimp which were imported into the uk (bateman et al., b) . unsurprisingly, the shrimp in the supermarkets harboured viable viral particles which where transmissible to live penaeid shrimp. furthermore, wssv was also successfully transmitted to homarus gammarus, which were fed on these infected supermarket-derived shrimp, suggesting a possible pathway for wssv to infect other species such as lobsters and highlighting a pathway for this disease to infect european crustacean stocks in the future (bateman et al., a) . finally, anecdotal evidence also suggests that some anglers are using commodity product as bait. as commodity product is not covered or controlled by existing legislation, we suggest that this practice needs to be investigated and regulated, as there is the potential for spread of pathogens such as wssv in this way. diagnosis and/or treatment: clear pathological signs and pcr of the virus. . . . . taura syndrome. agent: the causal agent, taura syndrome virus (tsv), is currently classified by the ictv as an unassigned species in the family dicistroviridae, from the order picornavirales. to date, at least four genetic variants have been identified and it is likely that more will be assigned in the near future. similarly to wssv, tsv is listed as an oie notifiable disease and as an exotic disease within europe, ec council directive / /ec. hosts affected: the principal host for tsv is litopenaeus vannamei, although the susceptible host range of tsv is thought to include most farmed marine shrimp species. however, susceptibility has been shown to vary between species and virulence also varies for the different strains of the virus. the virus mainly affects small shrimp at the nursery or during the grow-out phase in shrimp production, withiñ - days after stocking, although larger shrimp have been shown to become affected in some instances (lightner et al., ) . to date, other crustaceans including freshwater shrimp and crabs appear to be resistant to the disease but may also be potential carriers. certain species of birds and the water-boatmen, trichocorixa reticulata have been proposed as possible mechanical vectors (brock, ) which are capable or transporting the disease from pond to pond, therefore making control of this disease very difficult. the focal origin of the tsv panzootic and absence of evidence of infection prior to the first outbreak suggest that, as for wssv, penaeid shrimp are not likely to be the natural host and this remains unidentified to date. clinical signs and pathology: there are three main stages of the infection, acute, transition and chronic. mortalities in the acute phase can be as high as %, but surviving shrimp remain infected and have been shown to carry the virus for the rest of their life. therefore, these survivors act as a potential source of virus transmission between stocks. tsv causes necrosis of cuticular epithelium, haemopoietic tissues and the antennal gland, infected cells are rounded with pyknotic nuclei. the lymphoid organ itself is not infected but lymphoid organ spheroids (los) develop during infection. virions are non-enveloped, icosahedral in shape and measure nm. epidemiology: first documented in l. vannamei in ecuador in (lightner and redman, ) . the disease is named after the taura river where the disease was first identified, however it was initially thought to be caused by a toxin (hasson et al., ) . the disease spread rapidly throughout shrimp farming regions in central and south america and in , it was detected in taiwan and has now spread throughout much of asia (tu et al., ) . the rapid spread of this virus, similar to that of wssv, has been attributed to the international trade in live shrimp (walker and winton, ) . diagnosis and/or treatment: clear pathological signs and pcr of the virus . . . . yellow head disease (yhd). agent: yellow head disease (yhd) is caused by the yellow head virus (yhv). to date, there are six known genotypes in the yellow head complex, however only yhv (genotype ) has been shown to be the causal agent of yhd. interestingly, gillassociated virus (gav) is designated as genotype and along with four of the other known genotypes (genotypes - ) are commonly associated with healthy penaeus monodon in east africa, asia and australia and are rarely or never associated with disease outbreaks (walker and winton, ) . furthermore, despite this variance in pathogenicity, all the genotypes in the yellow head complex are currently classified by the ictv as a single species (gill-associated virus), from the genus okavirus, family roniviridae, order nidovirales (oie, ) . this is mainly because there is strong evidence of genetic recombination between the different genotypes (wijegoonawardane et al., ) . again similar to wssv and tsv, yhd is listed as an oie notifiable disease and as an exotic disease within europe, ec council directive / / ec. hosts affected: yhd mainly affects black tiger shrimps (p. monodon), however many other penaeid and palemonid shrimp species have also been shown to be susceptible to experimental infection with yhv or gav. p. monodon appears to be the natural host, yellow-head complex viruses are detected rarely in other penaeid shrimp species (walker and winton, ) . clinical signs and pathology: yhd is characterised by high rapid mortality and was named after the gross appearance of yellowing of the cephalothorax and general bleaching of body colour in some affected shrimp. yhd causes necrosis of ectodermal and mesodermal tissues such as cuticular epithelium, haemocytes, haemopoietic tissue, gills and connective tissues. yhv also infects the lymphoid organ, stromal matrix cells that comprise the tubules become infected leading to loss of tubular structure, and tubules appear degenerate. virions measure nm and appear rod-shaped with prominent projections at surface. epidemiology: yhd first emerged in p. monodon in central thailand in and has since been reported in most major shrimp farming countries in asia, including india, indonesia, malaysia, the philippines, sri lanka, vietnam and taiwan (boonyaratpalin et al., ) . however, there is also a recent unconfirmed report that yhd is also present in farmed penaeus vannamei and penaeus stylirostris in mexico, showing again that transportation of stock likely spreads these pathogens around the globe. although, it must be noted that there are no official confirmed reports of this virus causing disease outbreaks in shrimp farms in the americas at the time of writing (lightner, ) . diagnosis and/or treatment: clear pathological signs and pcr of the virus. hypodermal and haematopoietic necrosis virus (ihhnv) was first described by lightner et al. ( ) from a disease outbreak in cultured p. stylirostris in the americas. ihhnv has been renamed as p. stylirostris brevidensovirus (pstdnv), from the genus brevidensovirus and in the family parvoviridae, although this listing has not been completely accepted by the ictv (kaufmann et al., ; shike et al., ) . the original name of this virus was given to describe the principal lesions observed and is still often referred to as ihhnv by many researchers as this is how it is commonly known. this viral infection remains an oie notifiable disease since the original discovery of pstdnv in . hosts affected: multiple penaeid species appear to be susceptible to the virus and have been shown to carry the virus, however, the severity of infection and mortality rate often differs between species, with p. stylirostris for example appearing most susceptible (bell and lightner, ) . clinical signs and pathology: in addition to necrosis, the virus was also found to cause stunted growth and deformities called by another disease name, runt deformity syndrome (rds) in p. vannamei and p. monodon (bell and lightner, ; primavera and quinitio, ) . typical signs of pstdnv in these species were shown to develop after indirect exposure via contaminated water or direct feeding of infected p. stylirostris carcasses. pstdnv infects the haematopoietic tissue, nervous tissue, gills, gonad, connective tissues and antennal gland. hypertrophied nuclei containing eosinophillic inclusion bodies can usually be seen distributed throughout the tissues. virions are nonenveloped, icosahedral in shape and measure - nm. epidemiology: the disease has caused substantial economic losses in shrimp farming in infected areas (wyban, ) . three distinct genotypes of pstdnv have now been identified lightner, , ; tang et al., ) , type from the americas and east asia, type from south-east asia and type from east africa, india, australia and the western indo-pacific region. types and are infectious to penaeids, whilst type appears to not be infectious to p. vannamei and/or p. monodon lightner, , ; tang et al., ) . non-infectious inserts of the partial pstdnv genome into the shrimp genome have been discovered in p. monodon which were sampled in east africa and australia (krabsetsve et al., ; tang and lightner, ) . diagnosis and/or treatment: clear pathological signs together with a modified pcr method which was developed to detect the infectious type (tang et al., ) . false positives were found to occur under standard pcr conditions. . . . . infectious myonecrosis virus (imnv). agent: infectious myonecrosis virus (imnv) is the most recently emerging of the major viral diseases of marine shrimp. imnv although still un-described is thought to be most closely related to members of the family totiviridae, and belonging to the genus giardiavirus. interestingly, the only other known members of this family infect yeasts and protozoa. hosts affected: litopenaeus vannamei late pl, juveniles and subadults, experimentally infected litopenaeus stylirostris and p. monodon. clinical signs and pathology: shrimp with the acute form of the disease display various degrees of skeletal muscle necrosis, which is visible as an opaque, whitish discolouration of the abdomen. imnv causes significant disease and mortalities in juvenile and sub-adult pond reared stocks of l.vannamei and has been responsible for millions of dollars in losses in the affected regions. surviving shrimp progress to a chronic phase with persistent low-level mortalities. virions are nonenveloped, icosahedral in shape and measure~ nm. epidemiology: it first appeared in farmed white pacific shrimp, l. vannamei at pernambuco in brazil in and has subsequently spread throughout coastal regions of north-east brazil and more recently to indonesia, thailand and the hainan province in china (walker and winton, ) . the original source of infection is unknown but the trans-continental spread has almost certainly been due to the voluminous trade in l. vannamei. although, to date several other farmed marine shrimp species have been reported to be susceptible to infection by imnv, only l. vannamei has shown pathological signs. the increasingly common practice in parts of asia of co-cultivation of l. vannamei and the black tiger shrimp (p.monodon) is likely to present opportunities for adaptation and further spread of this disease. diagnosis and/or treatment: clear pathological signs and pcr of the virus. . . . . panulirus argus virus (pav ). agent: panulirus argus virus (pav- ) was the first description of a viral infection in lobsters (shields and behringer, ) . although, given a name, the exact classification of this virus still remains to be resolved. hosts affected: the infection was initially observed in juvenile p. argus. clinical signs and pathology: juveniles appear lethargic, displaying milky haemolymph, which does not clot and infected individuals sometimes exhibit a discoloured, heavily fouled carapace. pav has been shown to be transmitted to juvenile lobsters via inoculation, ingestion of diseased tissue and through close contact with infected individuals. the virus has also been shown to be transmitted via the water over small distances although contact and waterborne transmission were shown to be the least efficient methods (butler et al., ) . this ease of transmission is worrying and highlights the likely instances of more cases being reported in the near future. interestingly, juveniles with light infections and adult lobsters do not appear to show any obvious external signs of infection. it is only upon histological investigation whereby researchers can observe nuclear hypertrophy with cowdry-type a viral inclusions in infected haemocytes. in heavily infected individuals, virtually all the hosts hyalinocytes and semi-granulocytes are destroyed, the granulocytes however appear to not be affected. furthermore, fixed phagocytes, blood vessels and surrounding connective tissues appear necrotic and often destroyed in these heavily infected individuals. li et al. ( a) and li et al. ( b) showed that the infection initially infects the fixed phagocytes and the connective tissues within the hepatopancreas. in heavily infected lobsters virally infected cells have also found in the spongy connective tissues surrounding most organs (li et al., a) . histological evidence has also suggested that the prevalence of the virus is highest (~ %) among crevice-dwelling juveniles measuring - mm carapace length. this has been shown to decline to % once they reach - mm cl and is virtually undetectable in adults (b %). virions are icosahedral in shape and measure approximately nm. envelopes possess an electron lucent inner layer and an electron dense outer layer on which there are possible projections when located extracellularly. epidemiology: pav was first shown to be present throughout the florida keys, and infections have since been found in belize, mexico and the us virgin islands, so it is thought to be widespread throughout the caribbean (butler et al., ) . prevalence of pav is usually highest among the smallest juveniles and has been shown to decline with increasing lobster size (behringer et al., ) . early benthic juveniles appear to be highly susceptible; with field experiments indicating that prevalence in wild populations exceeded % in focal outbreaks (butler et al., ) . diagnosis and/or treatment: clear pathological signs and pcr of the virus. . . . . sighs disease (sd). agent: the condition has been found to be caused by a roni-like virus infection of the connective tissues and was named eriocheir sinensis ronivirus (esrnv). hosts affected: chinese mitten crabs (eriocheir sinensis) are an economically important species in china and have been extensively cultured in recent years. as demand increased over the years, the culture practices were developed and in conjunction with this step up in production, outbreaks of disease suddenly increased. a viral infection specifically associated with e. sinensis was one of these diseases and named 'sighs disease' (zhang and bonami, ) . clinical signs and pathology: the disease is named after the sounds which can be heard at night when affected crabs release a slow extrusion of bubbles reminiscent of a sigh. the disease is also known as black gill syndrome (bgs), due to the discolouration seen within the gills (dark grey or partly black colouration, usually at the tips of the gill filaments). signs of infection can be noted in the, connective tissues of gills, hepatopancreas (hp), heart and gut. infected cells display pale to deep basophilic staining in affected areas showing tissue degeneration, abnormal nuclei and dense bodies that could be karyorrhexic and pyknotic nuclei, or inclusion bodies. virions are rod shaped, enveloped and measure - × - nm. epidemiology: to date, very little information is available on this disease and we will likely find that it is more widely spread and infects other species than currently identified. diagnosis and/or treatment: clear pathological signs. . . . . early mortality syndrome/acute hepatopancreatic necrosis disease (ems/ahpnd). agent: ems/ahpnd is caused by a bacterial agent, which has been shown to be transmitted orally. the causative agent is reported to be a pathogenic vibrio belonging to the harveyi clade, presumably vibrio parahaemolyticus. the vibrio is also thought to be infected by a virus known as a phage, which causes the release of a potent toxin (tran et al., ) , a result which causes the particular signs of the disease. a similar phenomenon occurs in the human disease cholera, where a phage makes the vibrio cholerae bacterium capable of producing a toxin that causes cholera's life-threatening diarrhoea. to date, it remains unclear as to whether or not all incidences of ems/ahpnd are caused by one or more strains of v. parahaemolyticus. the vibrio has been shown to colonize the shrimp's gastrointestinal tract and produces a toxin that causes tissue destruction and dysfunction of the hepatopancreas. hosts affected: ems/ahpnd outbreaks typically affect the postlarvae of both black tiger shrimp (litopenaeus monodon) and white pacific shrimp (l. vannamei), appearing within - days after initial stocking. clinical signs and pathology: diseased shrimp become lethargic and anorexic, are slow growing and display a 'corkscrew' swimming behaviour. they show a significant atrophy of the hepatopancreas (hp) which often becomes yellowish or white within the hp connective tissue capsule. the hp does not squash easily between thumb and finger and black spots or streaks are sometimes visible. other signs of the disease include a soft, generally darker shell, mottling of the carapace and moribund shrimp sink to the bottom of the ponds. mortalities have been shown to reach as much as % in severely affected ponds. epidemiology: the condition was first reported in china in ; then subsequently spread to vietnam, malaysia and thailand (fao, ) . furthermore, and akin to many of the viral diseases reviewed above, ems/ ahpnd has also recently been reported in mexico (nunan et al., ) . it now causes annual losses of more than us$ billion. diagnosis and/or treatment: the current practice after an outbreak has occurred is to disinfect the ponds to try to remove any potential pathogens or their carriers, however a recent study by de schryver et al. ( ) suggests that this practice may do more harm than good. upon restocking, the increase in nutrient availability, decreased microbial community and consequent lack of competition may favour fast growing bacteria, such is the case with the genus vibrio. the authors go on to suggest that microbial management practices may be the best solution to control this disease (de schryver et al., ) . the development of diagnostic tests for rapid detection of the ems/ahpns pathogen is ongoing, which will enable improved management of hatcheries and ponds, and help lead to a long-term solution for this disease. this will also enable a better evaluation of risks associated with importation of frozen shrimp or other products from countries affected by ems/ahpnd. in fact, some countries have already implemented policies that restrict the importation of frozen shrimp or other products from ems/ahpnd-affected countries. however, research currently indicates that ems/ahpnd does not survive cooking or freezing, and repeated attempts to transmit the disease using frozen tissue have so far been unsuccessful (lightner et al., ) . therefore, frozen shrimp are likely to pose a low risk for contamination of wild shrimp or the environment because ems/ahpnd-infected shrimp are typically very small and do not enter international commerce, so although this disease is damaging and worthy of note in this review, the potential for spread to other countries should be relatively limited. the condition is thought to be caused by a rickettsia-like bacteria. hosts affected: it has been shown to infect multiple spiny lobsters (panulirus spp. especially panulirus ornatus, panulirus homarus and panulirus stimpsoni) from tropical regions but the condition has only been reported in vietnam to date. the condition affects younger lobsters (juveniles approximately months old) and appears to be a similar disease to that reported in other crustacean species (nunan et al., ) such as carcinus maenas (eddy et al., ) and l. monodon (nunan et al., ) . clinical signs and pathology: the bacterium causes the haemolymph of infected lobsters to appear milky white and lose the ability to form clots. onset of the disease is relatively rapid, with affected lobsters becoming lethargic and showing signs of the milky haemolymph within - days. furthermore, the lobsters die soon after clinical signs become apparent. epidemiology: the disease emerged in vietnam in (shields, a) and has caused massive economic losses, with farmers suffering high losses (up to %) and failed crops causing between us$ and k in losses. diagnosis and/or treatment: diagnostic tests have been developed and the disease is under consideration for listing as a notifiable disease by the oie. infection with a gram-negative, pleomorphic intracytoplasmic rod shaped rickettsial like organism which is a member of the α-subclass of proteobacteria, yet remains unclassified (frelier et al., ; lightner and redman, ; loy et al., ) . genetic analysis of the pathogenic agent from outbreaks in north and south america suggests that the isolates are either identical or very closely related subspecies (loy et al., ) . hosts affected: most penaeid species can be infected with nhp, infections in l. vannamei appear most severe where the intracellular bacterium can cause acute epizootics and mass mortality (n %). juvenile, broodstock and sub-adult life stages are the most severely affected (oie, ) . clinical signs and pathology: the disease has four distinct phases; initial, acute, transition and chronic. in the initial and chronic stages of the disease, molecular and antibody diagnostic techniques are necessary to confirm infection as there are no pathognomonic lesions. in the acute and transition phase, disease pathognomonic lesions are typically present in histological sections of the hepatopancreas (vincent et al. ). epidemiology: the bacterium has been detected in the western hemisphere in both wild and cultured penaeid shrimp and is commonly found in cultured penaeid shrimp in belize, brazil, colombia, costa rica, ecuador, el salvador, guatemala, honduras, mexico, nicaragua, panama, peru, united states of america, and venezuela, highlighting the current and global extent of the issue. natural transmission of nhp has been shown to occur via the practice of cannibalism, however cohabitation and dissemination of nhp via the water column is also thought to play a part (frelier et al., ) . similar to many of the diseases highlighted in this review, outbreaks of the disease are often preceded by prolonged periods of high water temperature (approximately °c) and salinity (up to parts per thousand [ppt]) (oie, ). diagnosis and/or treatment: the use of antibiotics has been suggested as the best treatment for nhp which is currently available, particularly if the disease is detected in the initial phase (oie, ). . . . . gaffkaemia. agent: gaffkaemia or red tailed disease is a fatal systemic bacterial disease. first reported in (stewart, ) the pathogen has been identified as the gram positive coccus aerococcus viridians var. homari. homarus americanus. similar to many diseases in this group, it has been particularly well studied due to the disease impacting on stocks of such a commercially viable species. although, predominantly associated with h. americanus, the pathogen has been recorded in low prevalence in other crustacean species in the wild such as libinia emarginata, carcinus maenas, cancer borealis, cancer irroratus and penaeus aztecus (alderman, ) . furthermore, experimentally, the disease has been transferable to panulirus interruptus, pandalus platyceros, cancer irroratus, cancer magister, geryon quinquedens, chionoecetes opilio and callinectes sapidus (alderman, ) . the diverse number of species this pathogen has been associated with clearly shows the dramatic influence epidemics of this disease can have on the crustacean populations and the whole ecosystem in general. enzootics in both wild populations and captive animals are relatively common off the american/canadian coast line and reports have occurred in cultured lobsters in california, however prevalence was reportedly low (stewart, ) . despite the high number of species which have been shown to be susceptible to this disease, the pathogen had not been reported to be present in european waters until relatively recently. in , the press reported the capture of an american lobster off the coast of devon, southern uk (stebbing et al., a) . the lobster caught, had blue bands around its claws, indicative of being caught and tossed overboard by a passing ship. this lobster tested positive for gaffkaemia and this spread real concern of the possibility that european lobsters h. gammarus, could have been exposed to this devastating disease. since this first report, and a further positive outbreak in a european lobster holding facility in south wales, a base line survey was conducted to test the presence or absence of the disease in wild populations of h. gammarus around the east coast of england and wales (stebbing et al., b) . results showed that although gaffkaemia was detected in wild populations, its prevalence was low (positive in b % of those tested). pulsed-field gel electrophoresis conducted on the strains of a. viridians var. homari collected from the uk wild lobsters, those in the welsh holding facility and examples from the usa and canada showed that the isolates were identical, supporting the theory that this pathogen has been introduced into the uk, likely with the release of american lobsters, h. americanus. although the increase in h. americanus in uk waters is of great concern and has the potential to cause a pandemic of gaffkaemia, there are reports that there was a previous outbreak in england in the s, although this is difficult to confirm (stebbing et al., b) . to date, however no other european crustaceans have been tested for the presence of this pathogen and future studies should focus on assessing the extent of the problem in european waters. clinical signs and pathology: infections occur when the bacteria gain entry through breaks in the exoskeleton. eventually, large numbers of cocci, often arranged in chains or tetrads, are visible in the haemolymph (steenbergen et al., ) . the disease gets its common name, 'red tail', from the dark orange discoloration of the ventral abdomen of affected lobsters. this is the haemolymph seen through the thin ventral arthrodial membranes. the red discoloration comes from astaxanthin, acarotenoid pigment, which is exported to the blood during times of stress. epidemiology: the bacterium has been detected in the western hemisphere in both wild and cultured penaeid shrimp and is commonly found in cultured penaeid shrimp in belize, brazil, colombia, costa rica, ecuador, el salvador, guatemala, honduras, mexico, nicaragua, panama, peru, united states of america, and venezuela, highlighting the current and global extent of the issue. similar to many marine diseases, it has been proposed that a. viridians var. homari is temperature-dependent with strains lying dormant when lobsters are held at temperatures as low as °c whilst warming to only °c can cause the signs of classical disease (steenbergen et al., ) . generally diseased lobsters are found in waters ranging from to °c, interestingly above temperatures of °c, virulence of a. viridians var. homari has been shown to reduce significantly (steenbergen et al., ) . diagnosis and/or treatment: bacteriology and pcr. . . . . epizootic shell disease (esd). agent: despite its devastating potential, the pathogen has only just been identified as the bacterium aquimarina homaria (quinn et al., ) . however, there is the strong possibility that this bacterium is not the only causal agent, with other co-infecting pathogens and conditions (environmental or physiological) confounding a simple cause-effect relationship . clinical signs and pathology: many infected individuals display deep shell lesions, sometimes covering most of their carapace (castro et al., ) a result which renders them unmarketable. the cuticle is eroded by bacteria growing horizontally into the endocuticle. epidemiology: east coast of north america, in particular long island sound. in , an epizootic outbreak caused such a decimation of the population of american lobsters that the fishery was recommended to be closed. although the disease has subsided, to a degree, esd prevalence remains relatively high but varies temporally and geographically between and % (castro and somers, ) . similarly to gaffkaemia, esd has also recently been reported in h. gammarus populations off the coast of norway and within public aquaria. although little research has been conducted on these european diseased lobsters, it is hypothesised that the pathogen (again similar to that for gaffkaemia) has been transported over with their american cousins (jørstad et al., ) and there is a very high potential that a similar epidemic (to that recorded in the east atlantic) could occur in european waters. diagnosis and/or treatment: pathology and pcr. . . . . spiroplasma. agent: spiroplasma is a relatively new pathogen occurring predominantly in farmed crabs (nunan et al., (nunan et al., , . spiroplasmas belong to a unique genus of the family spiroplasmataceae and members of this genus are some of the smallest prokaryotes in the world. although only recently identified as a cause for concern in the marine environment, spiroplasmas are not new to science and were first identified in associated with terrestrial plants and animals (williamson and whitcomb, ) . in , the first study showed the isolation of a novel spiroplasma in the marine environment and was associated with the now widely distributed chinese mitten crab, e. sinensis. since the initial discovery in e. sinensis, other spiroplasmacaused diseases have been identified in freshwater crayfish, procambarus clarkii, (wang et al., b) and in shrimp such as the white pacific shrimp, p. vannamei (nunan et al., ) and rimicaris exoculata (zbinden and cambon-bonavita, ) , highlighting the likelihood of more cases being confirmed in the coming years. however, despite the pathogen being described in , it took a further seven years before the pathogen was actually named, spiroplasma eriocheiris (wang et al., ) . hosts affected: numerous species of crabs, shrimps and crayfish. clinical signs and pathology: little is known about the specific signs caused by these pathogens. one named disease caused by spiroplasmas is known as tremors disease (td) (wang et al., ) . although the proposed pathogen (s. eriocheiris) was only identified in , td was initially recorded in , with mortalities in populations ranging from between and % (wei, ) . epidemiology: now, pathogens from this novel genus are being found throughout the world, for example cultured shrimps (p. vannamei) in colombian farms have just been described as showing similar disease signs which have been accredited to a further species of spiroplasma, s. penaei (nunan et al., ) . these studies indicate that spiroplasmas have a wide distribution in the aquatic environment and may be serious threats to crustaceans, especially those in cultured ponds. diagnosis and/or treatment: pathology and pcr. . . . . paramikrocytos canceri. agent: initially this infection was thought to be caused by a haplosporidian, however phylogenetic analysis robustly grouped the parasite as a sister ( % similar) to mikrocytos mackini, causative agent of denman island disease in oysters. the pathogen has now been described as belonging to a new taxon, paramikrocytos canceri n.gen. et n. sp. on the basis of its phylogenetic affinity to the genus mikrocytos, its difference in host range, and its ability to form plasmodia (hartikainen et al., ) . hosts affected: the pathogen has only been observed in juvenile life stages of the edible crab, cancer pagurus, and to date has not been observed within adult populations. juvenile crabs have shown high prevalence ( %) for the pathogen with peaks (at % prevalence) occurring in march (bateman et al., ) . the fact that the parasite is only observed in juvenile life stages may indicate that the parasite is causing mortality within these infected crabs, hence removing them from the population. if this is the case, the disease could likely have a devastating impact upon the fishery and further work is urgently needed to investigate the effect this parasite may have upon edible crab populations around the uk. clinical signs and pathology: there does not appear to be any visible signs of infection externally, however upon dissection the parasite can be observed infecting the antennal gland (bladder) of the edible crab causing the organ to become massively hypertrophied, distinctly yellow coloured and gelatinous in appearance. histology of infected individuals has revealed massive proliferation of both the major lateral lobes of the antennal gland and the bladder, in the regions next to the hepatopancreatic lobes. uninucleate and multicellular parasitic plasmodial stages have also been found to be distributed throughout the epithelial cells of the infected glandular tubules. electron microscopy confirmed that infection of these epithelial cells occurred with unicellular parasite stages developing to multicellular plasmodia. epidemiology: first discovered in juvenile edible crabs (c. pagurus) sampled originally from the shoreline in weymouth, uk (bateman et al., ) . a similar parasite has also recently been reported in edible crabs from south wales (thrupp et al., ) . mikrocytid specific primers have now been utilised to screen invertebrate tissues and environmental dna (edna) samples from marine, freshwater and soil environments from the uk, continental europe, south africa, panama and borneo. to date, p. canceri has not been detected in any of the environmental samples, however it was detected in a range of shoreline invertebrates, demonstrating that these newly recognised parasites are common, diverse and widespread. therefore, they should be considered when assessing risks of aquaculture activities, invasive species spread and movements of ballast water and sediments with associated invertebrates (hartikainen et al., ) . diagnosis and/or treatment: pathology and pcr. hosts affected: shown to infect more than crustacean host species (pagenkopp lohan et al., ; small, ) . specifically, devastating impacts have been recorded for numerous crab populations worldwide (small, ) , as infections are more often than not fatal. there have been various disease names associated with this parasite, which is due to the various characteristics of the disease in each species. these include: bitter crab disease (in chionoecetes spp.) (meyers et al., ) , pink crab disease (in cancer pagurus) (stentiford et al., ) , milky disease (in portunus trituberculatus, scylla serrata) (eddy et al., ; li et al., b) , yellow water disease (in s. serrata) and milky shrimp disease (in exopalaemon carinicauda) . this condition causes massive losses to wild crustacean populations and associated fisheries (small, ) and has recently been associated with losses in cultured crabs and shrimp in china, with losses of up to % having been reported in these farms. the now increasingly common practice of rearing several different species in the same pond (known as polyculture) is likely a significant contributing factor towards transmission of the parasite between species and the occurrence of hematodinium infections within e. carinicauda has obvious implications for the culture of other economically important shrimp species (small, ) . following the emergence of hematodinium in aquaculture in china li et al., b) , small ( ) proposed that hematodinium meets the criteria for listing as an emerging aquatic animal disease and suggested that the listing of this disease should be considered by the oie. clinical signs and pathology: grossly affected hosts are lethargic and often present a hyperpigmented or 'cooked' appearance of the host carapace and appendages. advanced infections can usually be characterised by the appearance of white or cream coloured haemolymph due to the high numbers of parasites being present. the parasite causes a biochemical alteration of the crab meat causing them to become bitter tasting and hence unmarketable which causes significant commercial losses. epidemiology: three genotypes have been identified infecting hosts from different geographical locations: the english channel, east coast of the united states and gulf of mexico, and eastern china. identification of hematodinium species appears to be largely dependent upon geographical location, rather than host species. however it is important to note that this is not exclusive, as both hematodinium species can be found infecting multiple species from same location in the english channel. diagnosis and/or treatment: pathology and pcr. similar to crustacea, there are many diseases which pose significant threats to commercially important mollusk species both farmed and in the wild. aquacultured shellfish include various oyster, mussel and clam species. these bivalves are filter and/or deposit feeders, which rely on ambient primary production rather than inputs of fish or other feed. as such, shellfish aquaculture is generally perceived as benign or even beneficial in some cases. depending on the species and local conditions, bivalve molluscs are either grown on beaches, on longlines, or suspended from rafts and harvested by hand or by dredging. overfishing and poaching have reduced wild populations of many commercially important species such as abalones to the brink of extinction and increased farming activity is proposed as a measure to reduce wild collections and ultimately conserve wild stocks. however, as with other farming practices, increased densities of mollusca in these farms often result in disease outbreaks and the pathogens effectively cultured in these packed conditions can easily affect wild stocks as well. . . . viral diseases in mollusca . . . . pacific oyster mortality syndrome (poms). agent: the cause of the mortalities appears to be from a microvariant of type osterid herpes virus (oshv- μvar) (segarra et al., ) . oshv- is a member of the family herpesviridae known to infect a variety of different oysters, clams and scallop species (farley et al., ) . recently, it was proposed to include oshv- in the newly created family malacoherpesviridae in the order herpesvirales (davison et al., ) . the ostreid herpes virus variant, oshv- μvar, is defined on the basis of partial sequence data exhibiting a systematic deletion of base pairs in orf of the genome in comparison with oshv- (genbank # ay ) (renault et al., ) . hosts affected: most commonly the pacific oyster, crassostrea gigas, however the disease has been recorded to infect numerous other species as well. interestingly, a few species are however starting to show levels of resistance to this virus with the most recent being the blacklip pearl oyster (tan et al., ) and the pacific oyster (pernet et al., ) . clinical signs and pathology: interestingly, prior to death there are no visibly identifiable gross pathological lesions. however upon histological analysis, a high viral load can be seen causing multifocal to coalescing ulceration with attenuation of the epithelium and pyknotic nuclei throughout the connective tissue (renault and novoa, ) . epidemiology: horizontal transmission has been shown to play a role in spreading the disease from infected adults to larvae and subsequently the spat, however vertical transmission may also play a role in transmission, yet this remains to be demonstrated. as is becoming a common theme throughout this review, prevalence of this disease has been associated with increases in average sea surface temperatures and overcrowding (sauvage et al., ) . to date, this condition has been recorded throughout much of europe and especially in france and around ireland. mortality associated with oshv- is primarily observed among oysters less than one year of age, although all ages of oysters have been recorded as contracting the disease at some point. a harmonised approach has also now been implemented between eu member states with regard to managing and mitigating the spread of this disease, particularly focusing at trying to keep the virus from uk waters (murray et al., ) , which at the time of writing show only a few isolated cases of infection (murray et al., ) . given the economic importance of oyster aquaculture in many european countries, the eu commission regulation n. / was enacted in march to identify the presence of oshv- μvar associated with mortality in oysters in order to reduce the spread of the virus to uninfected regions. according to the regulation, disease control measures must be implemented which include the establishment of containment areas and the restriction of movement from these areas if oshv- μvar and accompanying mortality is identified. in addition to affecting the pacific oyster in europe, reports of poms have also been made in two estuaries in new south wales, australia (paul-pont et al., ) highlighting another potential problem area which needs to be monitored and managed to mitigate further spread. diagnosis and/or treatment: pathology and pcr. however, very recently challenge experiments, involving the inoculation of oysters with oshv- μvar at both high and low levels showed that infected oysters exhibited an increase in glycolysis and voltage-dependent anion channel (vdac) accumulation, which reflects the 'warburg effect', first reported in cancer cells and more recently in shrimps infected with other types of viruses (corporeau et al., ; su et al., ) . these latest results should allow the identification of potential biomarkers of disease resistance which will aid in the development of antiviral measures. . . . . abalone viral ganglioneuritis (avg). agent: abalone herpesvirus (abhv) has been tentatively assigned as the second member of the malacoherpesviridae, following comparisons between abhv and oshv- that showed similarities of to % over some common coding regions (savin et al., ) . avg is currently another oie notifiable disease. hosts affected: susceptible species are listed as the greenlip abalone (haliotis laevigata), the blacklip abalone (haliotis rubra), the diversicolor abalone (haliotis diversicola) and hybrids of greenlip and blacklips (oie, ) . clinical signs and pathology: affected abalone exhibit a distinct curling of the foot when infected. most die within - days of showing this sign. ganglioneuritis has been observed in sections of neural tissue by light microscopy and confirmation of the presence of abhv can be obtained by quantitative polymerase chain reaction (qpcr) and/or insitu hybridisation (corbeil et al., ) . in on-farm epizootics, cumulative mortality has been recorded in all age classes and has been known to reach in excess of %. in experimental trials, % mortality has been shown to occur within days post-exposure. the major histopathological lesion identified in abalone affected with avg is ganglioneuritis (i.e. inflammation which is confined to the neural tissue). the cerebral, pleuropedal and buccal ganglia can also be affected as well as the cerebral commissure and associated peripheral nerves (hooper et al., ) . epidemiology: to date, the condition has been reported in australia and taiwan and causes mortalities of up to % in all size classes of animals. due to the high prevalence of this disease occurring in multiple species and the increasing number of aquaculture farms being developed around the world for this group, the chance of spread of this disease is significantly high and we are therefore likely to see an increase in reports of this disease. diagnosis and/or treatment: due to the typical curling of the foot (see above), false positives of this infection are therefore rare based on pathology alone. however, as there are currently no effective anti-viral treatments, preventive measures should always be taken. these consist of implementing high levels of on-farm and live-holding facility biosecurity and regional movement restrictions. following an on-farm outbreak, the destruction of infected stock, disinfection of water and equipment, and fallowing procedures appear to be effective at preventing reinfection. . . . . vibriosis. agent: among several pathogens thought to be involved in these mortalities, the gamma-proteobacterium, vibrio aestuarianus, is frequently isolated from adult moribund oysters (labreuche et al., ) . furthermore, isolated cultures of v. aestuarianus have been shown to induce similar disease pathologies in inoculation trials under controlled laboratory conditions. alarmingly, prevalence of this pathogen in certain populations appears to be increasing. in for example, v. aestuarianus was detected in % of moribund adults tested, whereas only a few individuals tested positive in and (wendling et al., ) . interestingly, all oysters which tested positive in had isolates which matched those from french oysters (wendling et al., ) . furthermore the virulence of specific isolates also appears to be increasing, with this particular french isolate being particularly pathogenic (wendling et al., ) . similarly, isolates of v. aestuarianus from infected oysters in ireland have shown to induce very high mortalities of otherwise healthy c. gigas within only a few days of inoculation. however, despite this apparent increase in virulence, there is no evidence to suggest that new strains are responsible for the current outbreaks and future research should focus on assessing the environmental conditions that could potentially favour this apparent increase in virulence. hosts affected: primarily the pacific oyster (c. gigas) in europe, however some clam species have also been described with this disease. clinical signs and pathology: v. aestuarianus causes necrosis of the adductor muscle and epithelial atrophy within the digestive gland. organisms which contract this disease often exhibit setting and a decrease in larval motility. furthermore adults suffer sudden mortalities. in oysters, progressive growth of the bacterium can be seen along the mantle and into the visceral mass. velar deformation occurs without tissue invasion and immobility and progressive visceral atrophy often occur. in clams, entry of the pathogen appears to occur via the oesophagus. epidemiology: occurs in europe and the west coast of north america. diagnosis and/or treatment: pathology and pcr. once disease signs become obvious it is often too late for any treatment. however various antibiotics such as chloramphenicol, polymyxin b, erythromycin and neomycin can be used in an attempt to control the infection. (friedman et al. ; haaker et al. ) . the rickettsial bacterium infects the gastrointestinal epithelia of wild and farmed abalones, particularly haliotis spp. there is limited information available on strain variations of this bacterium, however it is thought that x. californiensis may be infected with a phage highlighting a similar mode of pathogenesis as that which occurs in early mortality syndrome/acute hepatopancreatic necrosis disease in crustaceans (friedman and crosson, unpublished) . hosts affected: infects members of the genus haliotis. susceptibility is known to vary between species, but can cause upwards of % mortality in some (e.g. the white abalone, haliotis sorenseni). all post-larval life stages have been shown to be susceptible to this condition but disease is typically observed in animals less than year old and often in farms rather than the wild (oie, ) . clinical signs and pathology: gross signs of disease include pedal atrophy, mottled digestive gland and weakness and lethargy. infection is characterised by intracytoplasmic bacterial inclusions within the oesophagus, intestine and epithelia of the digestive gland. epidemiology: the disease occurs along the south-west coast of north america, infected abalones have been reported to have been transported to other areas and as such the geographical range is thought to be extensive in areas where haliotis rufescens are cultured. interestingly, infections have been shown to persist for long periods without any evidence of disease when the host is maintained in cool conditions, and the disease only manifests itself at elevated water temperatures (those above °c). diagnosis and/or treatment: the bacterium can be detected in tissue squashes stained with propidium iodide, microscopic examination of stained tissue sections, pcr or in situ hybridization. 'candidatus xenohaliotis californiensis' can be differentiated from other closely related alpha-proteobacteria by its unique s rdna sequence. the bacterium is not cultivable on synthetic media or in fish cell lines (e.g. chse- ) and may be controlled by tetracyclines (oxytetracycline) but not by chloramphenicol, clarithromycin or sarafloxicin (friedman et al., ) . recently a new methodology has been designed to simplify detection of this pathogen in abalone (cruz-flores et al., ) . vacuoles can be visualized by staining with nucleic acid fluorochrome and their identity confirmed utilising the combined techniques of laser capture microdissection and pcr. . . . . marteiliosis (aber disease/qx disease). agent: marteiliosis is caused by the paramyxean protist pathogen marteilia refringens. currently three types, o, m and c have been defined based on the basis of the presence or absence of a restriction profile in the its- region of the genome. interestingly, both o and m appear to be generally specific to either oysters or mussels, with type m more often detected in mussels and type o in flat oysters. however, co-infection has also been shown to exist. when such co-infection occurs, it does however appear to be restricted to those areas where the prevalence of the disease is significantly higher. type c, in contrast is predominantly associated with mass mortalities of cockles in delta del ebro (carrasco et al., ) and galicia in spain. although classed under the same disease name, this latter type has been subsequently named marteilia cochillia. although very little is known about this more recently described type, mixed infections of all three have been shown to occur in rare cases. the life cycles of both marteilia species are not completely known, however it appears to involve at least one intermediate host. multiple taxa of zooplankton in the same areas as the oysters, mussels and cockles are likely to play important roles at different stages of the life cycle, along with at least two copepods from the genus paracartia, p. latisetosa and p. grani (audemard et al., ) . in the latter instances, marteilia have been shown to be transmitted from infected oysters and infected mussels to healthy copepods. mature sporangia of m. refringens have recently been observed in spring and autumn in mussels suggestive that the parasite has two cycles per year and that the mussels release the parasites from spring to autumn. in conjunction, m. refringens has also been detected in p. grani copepodid stages between june and november strengthening the hypothesis of the transmission of the parasites from copepods to mussels. in addition, in-situ end labelling showed that the marteilia were present within the digestive tracts and gonads of the rd copepodid stages . as stated above, very little is really known about these pathogens, but as they appear to use multiple hosts in different parts of their lifecycle, management and mitigation of this disease will be difficult to say the least. hosts affected: shown to infect the flat oyster, ostrea edulis, and two mussel species; mytilus edulis and mytilus galloprovincialis (murray et al., ) . it is listed as an oie notifiable disease. qx disease is the term used to describe this pathology for the sydney rock oyster, saccostrea glomerata. clinical signs and pathology: basically these pathogens have various effects on their hosts via two major target systems, digestive and reproductive. for an in-depth review on this disease we refer you to berthe et al. ( ) . severe infections often cause loss of condition as a consequence of reduced energy acquisition. the parasite may also interfere directly with the host feeding and absorption simply by its physical presence. death is usually attributed to direct blockage of the digestive gland by the parasite and consequent host starvation. mature spores of marteilia spp. are released into the environment via the lumen of the digestive tubules and intestine. this release is associated with destruction of the host digestive gland epithelia with cases observed whereby the secondary digestive tubules have been totally destroyed by the release of the spores. the parasite evolves from early stages, usually observed in the epithelia of the upper digestive tract, such as the palps and stomach. in some rare cases they have been observed to develop in the gills. sporulation takes place within the epithelium of the digestive gland tubules. sporangia are released in the lumen of the digestive tract and this is when the destruction of the digestive gland epithelia usually occurs. epidemiology: this parasite has been identified in various countries around europe. diagnosis and/or treatment: pathology and pcr. the effect of marteilia spp. on their hosts is very variable making diagnosis difficult. . . . protistan molluscan diseases . . . . dermo disease. agent: the genus perkinsus includes protozoan parasites of marine molluscs in many different locations around the world and infections are often associated with high mortality rates. two species, perkinsus marinus and perkinsus olseni, are given particular attention due to their impact on aquaculture practices (cho and park, ; ford, ) , and each parasite is listed as a separate oie notifiable disease. however, with regard to p. marinus (originally described as dermocystidium marinum, from which the abbreviation 'dermo' refers), differences in virulence have now been shown to occur between isolates separated geographically (thompson et al., ) . this suggests that there may be numerous strains with differences in genetic composition, geographic distribution and virulence. similarly, variability in the pathogenicity of p. olseni raises questions on the existence of different strains as well. alternatively, or additionally there may also be differences in host responses under different environmental conditions, which further study should be able to highlight. hosts affected: p. marinus has been shown to infect the eastern oyster (crassostrea virginica), pacific oyster (c. gigas), suminoe oyster (crassostrea ariakensis), mangrove oyster (crassostrea rhizophorae), cortez oyster (crassostrea corteziensis), softshell clam (mya arenaria) and baltic macoma (macoma balthica) (oie, ). p. olseni, in particular has a wide geographical presence, occurring from the pacific islands through australia, southeast asia, to europe and to uruguay (cho and park, ) . in addition, p. olseni has been shown to infect an equally large number of hosts (clams, oysters, cockles, and abalones). clinical signs and pathology: affected oysters are typically thin, pale and 'wasted' in appearance, as tissues are overrun by the explosively proliferating parasites. uninucleate trophozoites of to μm develop into multinucleate 'rosette' forms ( μm) that rupture to release more uninucleate trophozoites. this process occurs most rapidly and causes the greatest mortality during the warmer months of the year. infections usually remain in the gut epithelium but have often been seen to become systemic, resulting in significant destruction of epithelia and connective tissues. this destruction is thought to be primarily caused by the digestion of host tissues by certain enzymes the parasite secretes. epidemiology: wide geographic presence occurring from the pacific islands through australia, southeast asia, to europe and to uruguay (cho and park, ) . in addition to severely affecting wild populations, this disease has been shown to occur in farms. however outbreaks can be reduced by culturing oysters in waters with salinity below to ppt (cook et al., ) . in the wild, season salinities, around ppt, appear favorable for supporting full dermo disease outbreaks, so in theory areas most at risk from these disease outbreaks can be identified by local salinity patterns (cook et al., ) . perkinsus proliferates at temperatures higher than °c and in warm, southern waters 'dermo' has been recorded as decimating up to % of oyster beds each year (cook et al., ) . the parasites are transmitted directly between individual oysters, primarily when the oysters are in advanced stages of infection, die and then disintegrate. new infections are acquired as oysters feed, the parasite infecting its hosts through the gut epithelia. interestingly, it has been reported that most serious outbreaks occur during drought years, so as climate change is predicted to increase periods of draught in many areas of the globe, increases in this disease may also likely occur. areas such as in the gulf of mexico are also likely to see increases in this disease, as dermo has been linked to the el nino-southern oscillation (enso) cycle (cook et al., ) , with serious outbreaks regularly following the arrival of warm, dry, la nina climatic conditions. interestingly, it has been proposed that p. olseni, has recently been introduced into florida and infections in giant clams has been documented. it had only previously been associated with clams in vietnam and it has been hypothesised that the parasite has been transported to florida by the aquarium trade. furthermore, a recent study has highlighted that certain perkinsus species are sympatric and have been shown to occur within close proximity to each other (fernández-boo et al., ) . the same study also highlighted that perkinsus chesapeaki (a parasite previously only detected in north america) was present in france (fernández-boo et al., ) . this latter study highlights the real possibility that the rest of europe may begin to show outbreaks of dermo in a variety of species of clams, oysters, cockles, and abalones. diagnosis and/or treatment: currently based on the pathology and epidemiology of diseased individuals. however, the effect of dermo disease in aquaculture can be minimized by culturing oysters in waters where the salinity is below to ppt (cook et al., ) . however, prospects for successful culture in waters of higher salinity are improving with the development of fast-growing, relatively disease-resistant, domesticated oyster strains. . . . . bonamiosis in oysters. agent: the genus bonamia includes smallsize ( - μm) uninucleate protozoan parasites also called microcells (carnegie and cochennec-laureau, ) . hosts affected: two of these microcells are thought to be of particular concern, including bonamia ostreae, which affects populations of the european flat oyster o. edulis and bonamia exitiosa which infects ostrea chilensis from new zealand and ostrea angasi from australia. both species cause diseases which are listed as notifiable by the oie. other known species of the genus are bonamia roughleyi infecting saccostrea commercialis from australia and bonamia perspora described in ostreola equestris from north carolina, usa. however, at the current time both these two species are thought to not be of high enough risk warranting notifiable status by the oie. clinical signs and pathology: although, we seem to be getting a better understanding of the diversity of bonamia species (see above), their mode of pathogenesis remains largely unknown. we are, however, beginning to understand how certain species may play specific roles. for example, b. ostreae appears to mainly infect the haemocytes of infected oysters and transmission appears to be direct from infected to naïve oysters. the parasites enter through epithelia from palleal organs including the gills. once they have passed the epithelium barrier, they are internalized within haemocytes which in turn contribute to the spread of the parasite in all the other organs. multiplication of b. ostreae occurs inside the haemocytes and is associated with a decrease of esterases and production of reactive oxygen species. furthermore, the modulation of apopotosis and decrease of phagocytosis has also been proposed as being important mechanisms related to resistance to these parasites (engelsma et al., ) . b. ostreae, affects the granular blood cells (haemocytes) of the host. lesions occur in the connective tissue of the gills, mantle and digestive gland. the parasite multiplies by binary fission. ten or more parasites have been observed within an infected haemocyte. more rarely, during the terminal stages of the disease, a larger plasmodial form can occur, this contains between and nuclei. epidemiology: to date, oysters such as o. chilensis in chile, o. puelchana in argentina, c. ariakensis in north carolina, and o. stentina in tunisia have shown no presence of any bonamia species (hill et al., ) . however, in , b. exitiosa was detected in spain and in the adriatic sea in italy (nardsi et al., ) in samples of o. edulis which led credence to the theory that bonamia have a global distribution already and questions were asked with regard to this parasite impact on european flat oyster populations (narcisi et al., ) . importantly, as both parasites; b. exitiosa and b. ostreae have been shown to occur in the same locations and sometimes even in the same individuals, specific diagnostic tools are urgently needed to understand the spread of these parasites and improve our understanding of specific infections (murray et al., ) . diagnosis and/or treatment: diagnosis of infected oysters is made by histological or cytological examination and molecular techniques. there is no known treatment for bonamiosis and therefore the only effective measure is to prevent the introduction of the disease. principally, methods of oyster health surveillance will allow for the limitation of the spread. furthermore, the use of predictive models of disease progression in infected areas could help to improve stock management and minimize the impact of these particular parasites (engelsma et al., ) . moreover, with respect to aquaculture practices, the development of resistant animals has been suggested and may help to revive specific production. in this context, studies are currently being undertaken to understand how b. ostreae interacts with o. edulis. . . . . denman island disease. agent: mikrocytos mackini is the final pathogen we will deal with here in this review and again causes another oie notifiable disease, known as denman island disease. hosts affected: the disease causes mortalities in several economically important oyster species (c. gigas, c. virginica, o. edulis, o. lurida). clinical signs and pathology: infected oysters display yellow/green pustules within the mantle tissues, labial palps and adductor muscle. histologically, the microcell parasite appears morphologically similar to the bonamia sp. (mentioned above) and can be identified within the cytoplasm of vesicular connective tissues and the muscle fibres. areas of haemocyte infiltration within the mantle, labial palps and adductor muscle have also shown the presence of the parasite. the parasites are often associated with intense inflammatory reaction around the site of infection. epidemiology: reports of this disease have occurred along the west coast of north america and canada (oie, ) . the disease usually affects oysters when water temperatures are cooler; below °c (hervio et al. ) . all life stages of the oyster appear to be susceptible, with mortalities reaching up to %. although the life cycle outside the host is currently unknown, the disease has been transmitted experimentally under laboratory conditions by cohabitation or by intamuscular inoculation of purified parasites. furthermore, a novel mikrocytos infection has been recently recorded in the uk (hartikainen et al., ) . in fact the causative agent was initially suspected to be that of m. mackini due to the similarities in gross pathology to that of denman island disease. pacific oysters (crassostrea gigas) were shown to suffer mortality events with upwards of % of animals reportedly being lost. infected individuals were seen to be gaping and were thin and watery, with green pustules observed on the adductor muscle and on the surface of the mantle tissue. furthermore, histology revealed the presence of microcell parasites throughout the cytoplasm of vesicular connective tissues and within the fibres of the adductor muscle. however, diagnostic tests for m. mackini came back negative and sequencing revealed that it was only % similar to m. mackini. hartikainen et al. ( ) was the first to describe this recent outbreak and highlighted that it is the first time that a mikrocytos infection has been described infecting pacific oysters in europe. this recent discovery therefore represents a new potential threat to the molluscan industries throughout europe. diagnosis and/or treatment: due to the risk of spread, a diagnostic test has been designed in the attempts to screen oysters and prevent any further spread (oie, ). a combined approach of both pcr and fluorescent in situ hybridization (fish) would give the greatest chance of detecting this pathogen in hosts. for primers and protocols see carnegie et al. ( ) and meyer et al. ( ) . interestingly, this latter study showed that only % of the positive oysters diagnosed with these techniques were found infected via the normal histological procedure, highlighting the importance of well validated diagnosis technqiues. finally, it is worthy of note that a very closely related parasite, initially identified in c. gigas has also been found causing a similar pathology to denman island disease in o. lurida in canada. this has now been named as a new species from the same genus, mikrocytos boweri sp. nov. (hartikainen et al., ) . this review highlights the importance and strikingly large numbers of diseases prevalent in three main commercially important marine invertebrate phyla/sub-phyla and/or class. we highlight a dramatic skew in the knowledge available between different phyla, particularly associated with the level of importance these groups are given based on their importance in aquaculture and as a sustainable food source. specifically, species in the sub phylum crustacea and the phylum mollusca are extensively studied in contrast to holothuroidea, although there remains a large amount of unknowns with regard to specific diseases and parasites/pathogens even in these two groups. interestingly, especially with regard to mariculture, researchers appear to be well advanced with regard to viral work for both crustacea and mollusca. even with regard to bacterial induced diseases, the discovery of how phages interact with the proposed pathogens, highlights the importance viruses play not just in disease but also with regard to healthy systems. finally, and more importantly, this review highlights the very real risk of the continued spread of specific pathogens around the globe. this is likely to occur either via the introduction of the hosts (during set up of different aquaculture practices for example) or alternatively via the transmission of the pathogens themselves (by transport in the ballast waters of ships or through the aquarium trade). despite the well documented cases of disease spread, it is surprising that we appear to not be able to learn from these past mistakes and are continuing the transport diseased individuals or vectors around the globe. specifically a call for more control appears to be needed, implemented in the movement of live individuals and also frozen food to attempt to seriously minimize further spread of novel pathogens and those already described. as far as prevention and treatment of outbreaks are concerned, this review highlights that there is very little information available to draw on. therefore, we can only suggest that the best strategy to minimize stocks contracting disease at the current time should be robust management. for example, broodstocks used for reproduction should be healthy without any signs of pathogens which will avoid vertical infections occurring. stocking density should be adapted to the environmental conditions, culture method and experienceovercrowded conditions would lower resistance to diseases. individuals should be fed with high quality diets that are supplemented with necessary elements including vitamins and minerals. water quality should be maintained by keeping an optimum stocking density, avoiding over feeding and increasing water exchange rates whilst monitoring the water quality on a daily basis. all equipment and tools should be disinfected before use or before transfer from one tank to another and excess food, faeces and other organic wastes should be siphoned and removed as 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bacteriophages on vibrio alginolyticus infection in the sea cucumber key: cord- -a prku k authors: mao, liang; yang, yan title: coupling infectious diseases, human preventive behavior, and networks – a conceptual framework for epidemic modeling date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: a prku k human-disease interactions involve the transmission of infectious diseases among individuals and the practice of preventive behavior by individuals. both infectious diseases and preventive behavior diffuse simultaneously through human networks and interact with one another, but few existing models have coupled them together. this article proposes a conceptual framework to fill this knowledge gap and illustrates the model establishment. the conceptual model consists of two networks and two diffusion processes. the two networks include: an infection network that transmits diseases and a communication network that channels inter-personal influence regarding preventive behavior. both networks are composed of same individuals but different types of interactions. this article further introduces modeling approaches to formulize such a framework, including the individual-based modeling approach, network theory, disease transmission models and behavioral models. an illustrative model was implemented to simulate a coupled-diffusion process during an influenza epidemic. the simulation outcomes suggest that the transmission probability of a disease and the structure of infection network have profound effects on the dynamics of coupled-diffusion. the results imply that current models may underestimate disease transmissibility parameters, because human preventive behavior has not been considered. this issue calls for a new interdisciplinary study that incorporates theories from epidemiology, social science, behavioral science, and health psychology. despite outstanding advance in medical sciences, infectious diseases remain a major cause of death in the world, claiming millions of lives every year (who, ) . particularly in the past decade, emerging infectious diseases have obtained remarkable attention due to worldwide pandemics of severe acute respiratory syndrome (sars), bird flu and new h n flu. although vaccination is a principal strategy to protect individuals from infection, new vaccines often need a long time to develop, test, and manufacture (stohr & esveld, ) . before sufficient vaccines are available, the best protection for individuals is to adopt preventive behavior, such as wearing facemasks, washing hands frequently, taking pharmaceutical drugs, and avoiding contact with sick people, etc. (centers for disease control and prevention, ) . it has been widely recognized that both infectious diseases and human behaviors can diffuse through human networks (keeling & eames, ; valente, ) . infectious diseases often spread through direct or indirect human contacts, which form infection networks. for example, influenza spreads through droplet/physical contacts among individuals, and malaria transmits via mosquitoes between human hosts. human behavior also propagates through inter-personal influence that fashions communication networks. this is commonly known as the 'social learning' or 'social contagion' effect in behavioral science, i.e., people can learn by observing behaviors of others and the outcomes of those behaviors (hill, rand, nowak, christakis, & bergstrom, ; rosenstock, strecher, & becker, ). in the current literature, models of disease transmission and behavioral diffusion have been developed separately for decades, both based on human networks (deffuant, huet, & amblard, ; keeling & eames, ; valente, ; watts & strogatz, ) . few efforts, however, have been devoted to integrating infectious diseases and human behaviors together. in reality, when a disease breaks out in a population, it is natural that individuals may voluntarily adopt some preventive behavior to respond, which in turn limits the spread of disease. failing to consider these two interactive processes, current epidemic models may under-represent human-disease interactions and bias policy making in public health. this article aims to propose a conceptual framework that integrates infectious diseases, human preventive behavior, and networks together. the focus of this article is on issues that arise in establishing a conceptual framework, including basic principles, assumptions, and approaches for model formulization. the following section (section ) describes the conceptual framework and basic assumptions, which abstract essential aspects of a disease epidemic. the third section discusses approaches to formulize the model framework into a design. the fourth illustrates a computing model upon various human network structures and compares the simulation results. the last section concludes the article with implications. the conceptual model consists of two networks and two diffusion processes (fig. ) . the two networks include an infection network that transmits disease agents (dark dash lines), and a communication network that channels inter-personal influence regarding preventive behavior (gray dash lines). both networks are composed of same individuals but different types of interactions. these two networks could be non-overlapping, partially or completely overlapping with one another. the two diffusion processes refer to the diffusion of infectious diseases (dark arrows) and that of preventive behavior (gray arrows) through the respective network. as illustrated in fig. , if individual # is initially infected, the disease can be transmitted to individual # and # , and then to individual # , following the routes of infection network. meanwhile, individual # may perceive the risk of being infected from individual # , and then voluntarily adopt preventive behavior for protection, known as the effects of 'perceived risks' (becker, ) . further, the preventive behavior of individual # may be perceived as a 'social standard' by individual # and motivate him/her toward adoption, i.e., the 'social contagion'. in such a manner, the preventive behavior diffuses on the communication network through inter-personal influence. during an epidemic, these two diffusion processes take place simultaneously and interact in opposite directions. the diffusion of diseases motivates individuals to adopt preventive behavior, which, in turn, limits the diffusion of diseases. this two-network two diffusion framework is dubbed as a 'coupled diffusion' in the subsequent discussion. the conceptual framework entails five assumptions. first, individuals differ in their characteristics and behaviors, such as their infection status, adoption status, and individualized interactions. second, both infection and communication networks are formed by interactions among individuals. third, the development of infectious diseases follows disease natural history, such as the incubation, latent, and infectious periods. fourth, individuals voluntarily adopt preventive behavior, dependent on their own personality, experiences, and inter-personal influence from family members, colleagues, as well as friends (glanz, rimer, & lewis, ) . fifth and lastly, the infection status of surrounding people or their behavior may motivate individuals to adopt preventive behavior, which then reduces the likelihood of infection. of the five assumptions, the first two provide networks as a basis for modeling. the third and fourth assumptions are relevant to the two diffusion processes, respectively. the last assumption represents the interactions between the two processes. corresponding to the five assumptions, this article introduces a number of approaches to represent individuals, networks, infectious diseases, and preventive behavior, as four model components, and depicts the relationships between the four. the first model assumption requires a representation of discrete individuals, their unique characteristics and behaviors. this requirement can be well addressed by an individual-based modeling approach. in the last decade, this modeling approach has gained momentum in the research community of both epidemiology and behavioral science (judson, ; koopman & lynch, ) . specifically, the individual-based approach views a population as discrete individuals, i.e., every individual is a basic modeling unit and has a number of characteristics and behaviors. the characteristics indicate states of individuals, e.g., the infection status, adoption status, and the number of contacts, while the behaviors change these states, e.g., receiving infection and adopting preventive behavior. by simulating at an individual level, this approach allows to understand how the population characteristics, such as the total number of infections and adopters, emerge from collective behaviors of individuals (grimm & railsback, ) . from an implementation perspective, the characteristics and behaviors of individuals can be easily accommodated by object-oriented languages, a mainstream paradigm of programming technologies. various tools are also available to facilitate the design and implementation of individual-based approach, such as the netlog and repast (robertson, ) . with regard to the second assumption, both the infection and communication networks can be abstracted as a finite number of nodes and links. nodes represent individuals and links represent interactions among individuals. the network structure is compatible with the aforementioned individual-based approach, in that the individual nodes directly correspond to the basic modeling units, while links can be treated as a characteristic of individuals. interactions between individuals (through links) can be represented as behaviors of individuals. to be realistic in modeling, both networks can be generated to fit observed characteristics and structures of real-world networks. important characteristics of networks include: the number of links attached to a node (the node degree), the minimum number of links between any pair of nodes (the path length), the ratio between the existing number of links and the maximum possible number of links among certain nodes (the level of clustering), and so on (scott, ) . particularly for human networks of social contacts, empirical studies showed that the average node degree often varies from to , dependent on occupation, race, geography, etc (edmunds, kafatos, wallinga, & mossong, ; fu, ) . the average path length was estimated to be around , popularly known as the 'six degrees of separation' (milgram, ) . the level of clustering has typical values in the range of . e . (girvan & newman, ) . besides these characteristics, studies on human networks have also disclosed two generic structures: "small-world" and "scale-free" structures. the "small-world" structure is named after the 'small-world' phenomena, arguing that people are all connected by short chains of acquaintances (travers & milgram, ) . theoretically, the small-world structure is a transition state between regular networks and random networks (watts & strogatz, ) . the regular networks represent one extreme that all nodes are linked to their nearest neighbors, resulting in highly clustered networks. the random networks are the other extreme that all nodes are randomly linked with each other regardless of their closeness, resulting in short path lengths. a typical small-world structure has characteristics from both extremes, i.e., most nodes are directly linked to others nearby (highly clustered), but can be indirectly connected to any distant node through a few links (short path lengths). the "scale-free" structure has also been commonly observed in social, biological, disease, and computer networks, etc. (cohen, erez, ben-avraham, & havlin, ; jeong, tombor, albert, oltvai, & barabási, ; liljeros, edling, amaral, stanley, & aaberg, ) . it depicts a network with highly heterogeneous degrees of nodes, whose distribution follows a power-law decay function, p < k > wk Àg (k denotes the node degree and empirically < g < ). in other words, a few individuals have a significantly large number of links, while the rest of individuals only have a few (albert, jeong, & barabasi, ) . all of these observed characteristics and structures can be used to calibrate the modeled networks, which then serve as a reliable basis to simulate the coupled-diffusion process. in epidemiology, the development of infectious diseases has been characterized by a series of infection statuses, events, and periods, often referred to as the natural history of diseases (gordis, ) . the progress of an infectious disease often starts with a susceptible individual. after having contact with an infectious individual, this susceptible individual may receive disease agents and develop infection based on a transmission probability. the receipt of infection triggers a latent period, during which the disease agents develop internally in the body and are not emitted. the end of the latent period initiates an infectious period, in which this individual is able to infect other susceptible contacts and may manifest disease symptoms. after the infectious period, this individual either recovers or dies from the disease. among these disease characteristics, the transmission probability is critical for bridging infectious diseases to the other model components: individuals, networks, and preventive behavior. this probability controls the chance that the disease agents can be transmitted between individuals through network links. meanwhile, the reduction of transmission probability reflects the efficacy of preventive behavior. the individual-based modeling approach enables the representation of disease progress for each individual. the infection statuses, periods, and transmission probability per contact can be associated with individuals as their characteristics, while infection events (e.g., receipt of infection and emission of agents) can be modeled as behaviors of individuals. each individual has one of four infection statuses at a time point, either susceptible, latent, infectious, or recovered (kermack & mckendrick, ) . the infection status changes when infection events are triggered by behaviors of this individual or surrounding individuals. the simulation of disease transmission often starts with an introduction of a few infectious individuals (infectious seeds) into a susceptible population. then, the first generation of infections can be identified by searching susceptible contacts of these seeds. stochastic methods, such as the monte carlo method, could be used to determine who will be infected or not. subsequently, the first generation of infections may further infect their contacts, and over time leads to a cascade diffusion of disease over the network. to parameterize the simulation, the transmission probability of a disease, the lengths of latent period and infectious period can be derived from the established literature or from observational disease records. like other human behaviors, the adoption of preventive behavior depends on the individual's own characteristics (e.g., knowledge, experience, and personal traits) and inter-personal influence from surrounding individuals (e.g., family supports and role model effects) (glanz et al., ) . because individuals vary in their willingness to adopt, human behaviors often diffuse from a few early adopters to the early majority, and then over time throughout the social networks (rogers, ) . a number of individual-based models have been developed by sociologists and geographers to represent such behavioral diffusion processes, e.g., the mean-information-field (mif) model (hägerstrand, ) , the threshold model (granovetter, ) , the relative agreement model (deffuant et al., ) , etc. the mif model populates individuals on a regular network (or a grid), and assumes that a behavior diffuses through the 'word-of-mouth' communication between an adopter and his/ her neighbors. the mif is a moving window that defines the size of neighborhood and the likelihood of human communications to every adopter. the simulation centers the mif on every adopter and uses the monte carlo method to identify a new generation of adopters (hägerstrand, ) . the threshold model assumes that individuals observe their surroundings and adopt a behavior based on a threshold effect (granovetter, ; valente, ) . the threshold is the proportion of adopters in an individual's social contacts necessary to convince this individual to adopt. the behavioral diffusion begins with a small number of adopters, and spreads from the low-threshold population to the high-threshold population. the recently proposed relative agreement model assumes that every individual holds an initial attitude, which is a value range specified by a mean value, maximum and minimum. based on the value ranges, individuals' attitudes are categorized as positive, neutral, and negative. individuals communicate through a social network, and influence their attitudes (value ranges) reciprocally according to mathematical rules of relative agreement. if individuals can hold positive attitudes for a certain time period, they will decide to adopt a behavior (deffuant et al., ) . due to the individual-based nature of all these models, they can be easily incorporated under the proposed conceptual framework. to further discuss the individual-based design of behavioral models, this research chose the threshold model for illustrations. in terms of complexity, the threshold model lies midway between the mif model and the relative agreement model, and its parameters can be feasibly estimated through social surveys. the mif model has been criticized for its simplicity in that it assumes an immediate adoption after a communication and oversimplifies the decision process of individuals (shannon, bashshur, & metzner, ) . by contrast, the relative agreement model is too sophisticated: many parameters are difficult to estimate, for example, the ranges of individual attitudes. the threshold model can be formulized as follows so as to become an integral part of the coupled-diffusion framework. first, individuals are assumed to spontaneously evaluate the proportion of adopters among their contacts, and perceive the pressure of adoption. once the perceived pressure reaches a threshold (hereinafter called the threshold of adoption pressure), an individual will decide to adopt preventive behavior. second, in order to relate the preventive behavior to infectious diseases, individuals also evaluate the proportion of infected individuals (with disease symptoms) among their contacts, and perceive the risks of infection. once the perceived risk reaches another threshold (hereinafter called the threshold of infection risk), an individual will also adopt preventive behavior. these two threshold effects can be further formulized as three characteristics and two behaviors of individuals. the three characteristics include an adoption status (adopter or non-adopter) and two individualized thresholds toward adoption. the two behaviors represent the individual's evaluation of adoption pressure and infection risk from surrounding contacts, which in turn determines their adoption status. the individualized thresholds toward adoption reflect personal characteristics of individuals, while the behaviors of evaluation represent the inter-personal influence between individuals. to build a working model, the individualized thresholds toward adoption can be best estimated by health behavior surveys as illustrated below. based on the discussion above, the conceptual framework ( fig. ) can be transformed into a formative design with four model components and their relationships (fig. ) . individuals are building blocks of the proposed model, and their interactions compose networks as a core of the model. through the infection network, individuals may receive infection from others and have their infection status changed, propelling the diffusion of diseases. meanwhile, individuals may perceive risks and pressure from the communication network, and gradually adopt preventive behavior, resulting in the behavioral diffusion. the adoption of preventive behavior reduces the disease transmission probability, thus controlling and preventing the disease transmission. in this manner, the diffusion of diseases and preventive behavior in a population are coupled together. to illustrate the proposed coupled-diffusion model, an influenza epidemic was simulated in a hypothetic population of individuals (n ¼ ), each with characteristics and behaviors as described in fig. . influenza was chosen because it is common and readily transmissible between individuals. the simulation simply assumes that the population is closed, i.e., no births, deaths, or migrations. with regard to the network component, the average number of links per individuals was set to , reasonably assuming that an individual on average has contact with family members and colleagues. for the purpose of sensitivity analysis, the illustrative model allowed the disease and communication networks to take either a small-world (sw) structure or a scale-free (sf) structure. the generation of sw structures started with a regular network where all individuals were linked to their nearest neighbors. then, each individual's existing links were rewired with a probability to randomly selected individuals (watts & strogatz, ) . the rewiring probability p ranged from to , and governed the clustering level and average path lengths of resultant networks (fig. a) . the sf structures were created by a preferential attachment algorithm, which linked each new individual preferentially to those who already have a large number of contacts (pastor-satorras & vespignani, ) . this algorithm produces a power-law degree distribution, p < k > wk Àg (k is the node degree), with various exponents g (fig. b) . based on fig. a and b, the rewiring probabilities p were set to . , . , and . to typically represent the regular, small-world, and random networks, respectively (fig. cee) . the exponent g were set to , , and to represent three scale-free networks with high, medium, and low levels of node heterogeneity (fig. feh) . a sensitivity analysis was performed to examine every possible pair of <p, p>, <g, g>, <p, g> and <g, p> as a network combination ( p-values  g-values  ¼ combinations in total), where the first parameter indicates the structure of infection network and the second specifies the structure of communication network. to simulate the diffusion of influenza, the latent period and infectious period were specified as day and days, respectively, based on published estimates (heymann, ) . the transmission probability per contact was varied from . to . (with a . increment) to test its effects on the coupled-diffusion processes. % of infected individuals was assumed to manifest symptoms, following the assumption made by ferguson et al. ( ) . only these symptomatic individuals could be perceived by their surrounding individuals as infection risks. recovered individuals were deemed to be immune to further infection during the rest of the epidemic. with respect to the diffusion of preventive behavior, the use of flu antiviral drugs (e.g., tami flu and relenza) was taken as a typical example because its efficacy is more conclusive than other preventive behavior, such as hand washing and facemask wearing. for symptomatic individuals, the probability of taking antiviral drugs was set to % (mcisaac, levine, & goel, ; stoller, forster, & portugal, ) , and the consequent probability of infecting others was set to be reduced by % (longini, halloran, nizam, & yang, ) . susceptible individuals may also take antiviral drugs due to the perceived infection risk or adoption pressure. if they use antiviral drugs, the probability of being infected was set to be reduced by % (hayden, ) . the key to simulate the diffusion of preventive behavior was to estimate thresholds of infection risk and that of adoption pressure for individuals. a health behavior survey was conducted online for one month (march eapril , ) to recruit participants. voluntary participants were invited to answer two questions: ) "suppose you have close contacts, including household members, colleagues, and close friends, after how many of them get influenza would you consider using flu drugs?", and ) "suppose you have close contacts, including household members, colleagues, and close friends, after how many of them start to use flu drugs would you consider using flu drugs, too?". the first question was designed to estimate the threshold of infection risks, while the second was for the threshold of adoption pressure. the survey ended up with respondents out of participants (a % response rate), and their answers were summarized into two threshold-frequency distributions (fig. ) . the monte carlo method was then performed to assign threshold values to the modeled individuals based on the two distributions. this survey was approved by the irb in university at buffalo. to initialize the simulation, all individuals were set to be non-adopters and susceptible to influenza. one individual was randomly chosen to be infectious on the first day. the model took a daily time step and simulated the two diffusion processes simultaneously over days. the simulation results were presented as disease attack rates (total percent of symptomatic individuals in the population), and adoption rates (total percent of adopters in the population). another two characteristics were derived to indicate the speed of coupled-diffusion: the epidemic slope and the adoption slope. the former is defined as the total number of symptomatic individuals divided by the duration of an epidemic (in day). similarly, the latter is defined as the total number of adopters divided by the duration of behavioral diffusion (in day). they are called slopes because graphically they approximate the slopes of cumulative diffusion curves. a higher slope implies a faster diffusion because of more infections/adoptions (the numerator) in a shorter time period (the denominator). all simulation results were averaged by model realizations to average out the randomness. simulation results were presented in two parts. first, the coupled-diffusion process under various transmission probabilities was analyzed, and compared to an influenza-only process that is fig. . (a) standardized network properties (average path length and clustering coefficient) as a function of rewiring probability p from to , given n ¼ ; (b) the power-law degree distributions given g ¼ , and , given n ¼ ; (cee) an illustration of generated sw networks for three p values, given n ¼ for figure clarity; (feh) an illustration of sf networks for three g values, given n ¼ . widely seen in the literature. the influenza-only process was simulated with the same parameters in the coupled-diffusion process except that individual preventive behavior was not considered. for the ease of comparison, a typical "small-world" network (p ¼ . ), was chosen for both infection and communication networks, assuming the two are overlapping. the second part examined the dynamics of coupled-diffusion under various structures of infection and communication networks, i.e., the pairs of network parameters <p, p>, <g, g>, <p, g> and <g, p> while fixing the influenza transmission probability to . (resultant basic reproductive number r ¼ e . ). fig. a indicates that the diffusion of influenza with and without the preventive behavior differs significantly, particularly for medium transmission probabilities ( . e . ). for the influenzaonly process (the black curve with triangles), the disease attack rate rises dramatically as the transmission probability exceeds . , and reaches a plateau of % when the probability increases to . . the coupled-diffusion process (the black curve with squares) produces lower attack rates, which slowly incline to the maximum of %. this is because individuals gradually adopt preventive behavior, thereby inhibiting disease transmission from infectious individuals to the susceptible. meanwhile, the adoption rate (the gray curve with squares) also increases with the transmission probability, and can achieve a % of the population as the maximum. this is not surprising because the more individuals get infected, the greater risks and pressure other individuals may perceive, motivating them to adopt preventive behavior. individuals who have not adopted eventually may have extremely high-threshold of adoption (see fig. ), and thus resist adopting preventive behavior. fig. b displays an example of the coupled-diffusion process (transmission probability ¼ . ), ending up with nearly symptomatic cases and approximately adopters of flu antiviral drugs. despite differences in magnitude, the two diffusion curves exhibit a similar trend that follows the -phase s-shaped curve of innovation diffusion (rogers, ) . the 'innovation' phase occurs from the beginning to day , followed by the 'early acceptance' phase (day e ), 'early majority' (day e ), 'late majority' (day e ) and 'laggards' (after day ). this simulated similarity in temporal trend is consistent with many empirical studies regarding flu infection and flu drug usage. for example, das et al. ( ) and magruder ( ) had compared the temporal variation of both influenza incidence and over-the-counter flu drug sales in the new york city and the washington dc metropolitan area, respectively. both studies reported a high correlation between over-the-counter drug sales and cases of diagnosed influenza, and thus suggested that over-the-counter drug sales could be a possible early detector of disease outbreaks. the consistency with the observed facts, to some extent, reflects the validity of the proposed model. in addition to the transmission probability, the coupled-diffusion process is also sensitive to various combinations of network structures, i.e., pairs of network parameters <p, p>, <g, g>, <p, g> and <g, p> (fig. ) . the z axis represents either the epidemic or adoption slope, and a greater value indicates a faster diffusion process. in general, both epidemic and adoption slopes change dramatically with the structure of infection network, while they are less sensitive to the variation of communication networks. given the small-world infection network ( fig. aeb and e-f), the epidemic and adoption slopes increase quickly as the rewiring probability p rises from . to . . when p ¼ . (a regular network), almost all individuals are linked to their nearest neighbors, and influenza transmission between two distant individuals needs to go through a large number of intermediate individuals. the slow spread of influenza induces a low perception of infection risks among individuals, thereby decelerating the dissemination of preventive fig. . the sensitivity of coupled-diffusion processes to various network structures, including sw infection Àsw communication as <p infection , p communication >, sf infection Àsf communication as <g infection ,g communication > sw infection Àsf communication as <p infection , g communication > and sf infection Àsw communication as <g infection , p communication >. each combination is displayed in one row from top to bottom. the sw and sf denote the network structure, while the subscripts indicate the network function. parameter p is the rewiring probability of a sw network, taking values ( . , . , . ), while parameter g is the exponent of a sf network, taking values ( , , ). the z axis denotes epidemic slopes (the left column) and adoption slopes (the right column) as a result of a network structure. a greater z value indicates a faster diffusion process. behavior. as p increases to . (a random network), a large number of shortcuts exist in the network, and the transmission of influenza is greatly speeded by shortcuts. as a result, the diffusion of preventive behavior is also accelerated, because individuals may perceive more risks of infection and take actions quickly. likewise, given a scale-free infection network ( fig. ced and g-h) , both influenza and preventive behavior diffuse much faster in a highly heterogeneous network (g ¼ ) than in a relatively homogeneous network (g ¼ ) . this is because a highly heterogeneous network has a few super-spreaders who have numerous direct contacts. super-spreaders act as hubs directly distributing the influenza virus to a large number of susceptible individuals, thus speeding the disease diffusion. as individuals perceived more risks of infection in their surroundings, they will adopt preventive behavior faster. human networks, infectious diseases, and human preventive behavior are intrinsically inter-related, but little attention has been paid to simulating the three together. this article proposes a conceptual framework to fill this knowledge gap and offer a more comprehensive representation of the disease system. this twonetwork two diffusion framework is composed of four components, including individuals, networks, infectious diseases, and preventive behavior of individuals. the individual-based modeling approach can be employed to represent discrete individuals, while network structures support the formulization of individual interactions, including infection and communication. disease transmission models and behavioral models can be embedded into the network structures, and simulate disease infection and adoptive behavior, respectively. the collective changes in individuals' infection and adoption status represent the coupled-diffusion process at the population level. compared to the widely used influenza-only models, the proposed model produces a lower percent of infection, because preventive behavior protects certain individuals from being infected. sensitivity analysis identifies that the structure of infection network is a dominant factor in the coupled-diffusion, while the variation of communication network produces fewer effects. this research implies that current predictions about disease impacts might be under-estimating the transmissibility of the disease, e.g., the transmission probability per contact. modelers fit to observed data in which populations are presumably performing preventive behavior, while the models they create do not account for the preventive behavior. when they match their modeled infection levels to those in these populations, the disease transmissibility needs to be lower than its true value so as to compensate for the effects of preventive behavior. this issue has been mentioned in a number of recent research, such as ferguson et al. ( ) , but the literature contains few in-depth studies. this article moves the issue towards its solution, and stresses the importance of understanding human preventive behavior before policy making. the study raises an additional research question concerning social-distancing interventions for disease control, such as the household quarantine and workplace/school closure. admittedly, these interventions decompose the infection network for disease transmission, but they may also break down the communication network and limit the propagation of preventive behavior. the costs and benefits of these interventions remain unclear and a comprehensive evaluation is needed. the proposed framework also suggests several directions for future research. first, although the illustrative model is based on a hypothetical population, the representation principles outlined in this article can be applied to a real population. more realistic models can be established based on the census data, workplace data, and health survey data. second, the proposed framework focuses on inter-personal influence on human behavior, but has not included the effects of mass media, another channel of behavioral diffusion. the reason is that the effects of mass media remain inconclusive and difficult to quantify, while the effects of interpersonal influence have been extensively studied before. third, the proposed framework has not considered the 'risk compensation' effect, i.e., individuals will behave less cautiously in situations where they feel safer or more protected (cassell, halperin, shelton, & stanton, ) . in the context of infectious diseases, the risk compensation can be interpreted as individuals being less cautious of the disease if they have taken antiviral drugs, which may facilitate the disease transmission. this health psychological effect could also be incorporated to refine the framework. to summarize, this article proposes a synergy between epidemiology, social sciences, and human 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stages of pandemic response date: - - journal: nan doi: nan sha: doc_id: cord_uid: h au h some of the key questions of interest during the covid- pandemic (and all outbreaks) include: where did the disease start, how is it spreading, who is at risk, and how to control the spread. there are a large number of complex factors driving the spread of pandemics, and, as a result, multiple modeling techniques play an increasingly important role in shaping public policy and decision making. as different countries and regions go through phases of the pandemic, the questions and data availability also changes. especially of interest is aligning model development and data collection to support response efforts at each stage of the pandemic. the covid- pandemic has been unprecedented in terms of real-time collection and dissemination of a number of diverse datasets, ranging from disease outcomes, to mobility, behaviors, and socio-economic factors. the data sets have been critical from the perspective of disease modeling and analytics to support policymakers in real-time. in this overview article, we survey the data landscape around covid- , with a focus on how such datasets have aided modeling and response through different stages so far in the pandemic. we also discuss some of the current challenges and the needs that will arise as we plan our way out of the pandemic. as the sars-cov- pandemic has demonstrated, the spread of a highly infectious disease is a complex dynamical process. a large number of factors are at play as infectious diseases spread, including variable individual susceptibility to the pathogen (e.g., by age and health conditions), variable individual behaviors (e.g., compliance with social distancing and the use of masks), differing response strategies implemented by governments (e.g., school and workplace closure policies and criteria for testing), and potential availability of pharmaceutical interventions. governments have been forced to respond to the rapidly changing dynamics of the pandemic, and are becoming increasingly reliant on different modeling and analytical techniques to understand, forecast, plan and respond; this includes statistical methods and decision support methods using multi-agent models, such as: (i) forecasting epidemic outcomes (e.g., case counts, mortality and hospital demands), using a diverse set of data-driven methods e.g., arima type time series forecasting, bayesian techniques and deep learning, e.g., [ ] [ ] [ ] [ ] [ ] , (ii) disease surveillance, e.g., [ , ] , and (iii) counter-factual analysis of epidemics using multi-agent models, e.g., [ ] [ ] [ ] [ ] [ ] [ ] ; indeed, the results of [ , ] were very influential in the early decisions for lockdowns in a number of countries. the specific questions of interest change with the stage of the pandemic. in the pre-pandemic stage, the focus was on understanding how the outbreak started, epidemic parameters, and the risk of importation to different regions. once outbreaks started-the acceleration stage, the focus is on determining the growth rates, the differences in spatio-temporal characteristics, and testing bias. in the mitigation stage, the questions are focused on non-prophylactic interventions, such as school and work place closures and other social-distancing strategies, determining the demand for healthcare resources, and testing and tracing. in the suppression stage, the focus shifts to using prophylactic interventions, combined with better tracing. these phases are not linear, and overlap with each other. for instance, the acceleration and mitigation stages of the pandemic might overlap spatially, temporally as well as within certain social groups. different kinds of models are appropriate at different stages, and for addressing different kinds of questions. for instance, statistical and machine learning models are very useful in forecasting and short term projections. however, they are not very effective for longer-term projections, understanding the effects of different kinds of interventions, and counter-factual analysis. mechanistic models are very useful for such questions. simple compartmental type models, and their extensions, namely, structured metapopulation models are useful for several population level questions. however, once the outbreak has spread, and complex individual and community level behaviors are at play, multi-agent models are most effective, since they allow for a more systematic representation of complex social interactions, individual and collective behavioral adaptation and public policies. as with any mathematical modeling effort, data plays a big role in the utility of such models. till recently, data on infectious diseases was very hard to obtain due to various issues, such as privacy and sensitivity of the data (since it is information about individual health), and logistics of collecting such data. the data landscape during the sars-cov- pandemic has been very different: a large number of datasets are becoming available, ranging from disease outcomes (e.g., time series of the number of confirmed cases, deaths, and hospitalizations), some characteristics of their locations and demographics, healthcare infrastructure capacity (e.g., number of icu beds, number of healthcare personnel, and ventilators), and various kinds of behaviors (e.g., level of social distancing, usage of ppes); see [ ] [ ] [ ] for comprehensive surveys on available datasets. however, using these datasets for developing good models, and addressing important public health questions remains challenging. the goal of this article is to use the widely accepted stages of a pandemic as a guiding framework to highlight a few important problems that require attention in each of these stages. we will aim to provide a succinct model-agnostic formulation while identifying the key datasets needed, how they can be used, and the challenges arising in that process. we will also use sars-cov- as a case study unfolding in real-time, and highlight some interesting peer-reviewed and preprint literature that pertains to each of these problems. an important point to note is the necessity of randomly sampled data, e.g. data needed to assess the number of active cases and various demographics of individuals that were affected. census provides an excellent rationale. it is the only way one can develop rigorous estimates of various epidemiologically relevant quantities. there have been numerous surveys on the different types of datasets available for sars-cov- , e.g., [ ] [ ] [ ] [ ] , as well as different kinds of modeling approaches. however, they do not describe how these models become relevant through the phases of pandemic response. an earlier similar attempt to summarize such responsedriven modeling efforts can be found in [ ] , based on the -h n experience, this paper builds on their work and discusses these phases in the present context and the sars-cov- pandemic. although the paper touches upon different aspects of model-based decision making, we refer the readers to a companion article in the same special issue [ ] for a focused review of models used for projection and forecasting. multiple organizations including cdc and who have their frameworks for preparing and planning response to a pandemic. for instance, the pandemic intervals framework from cdc describes the stages in the context of an influenza pandemic; these are illustrated in figure . these six stages span investigation, recognition and initiation in the early phase, followed by most of the disease spread occurring during the acceleration and deceleration stages. they also provide indicators for identifying when the pandemic has progressed from one stage to the next [ ] . as envisioned, risk evaluation (i.e., using tools like influenza risk assessment tool (irat) and pandemic severity assessment framework (psaf)) and early case identification characterize the first three stages, while non-pharmaceutical interventions (npis) and available figure : cdc pandemic intervals framework and who phases for influenza pandemic therapeutics become central to the acceleration stage. the deceleration is facilitated by mass vaccination programs, exhaustion of susceptible population, or unsuitability of environmental conditions (such as weather). a similar framework is laid out in who's pandemic continuum and phases of pandemic alert . while such frameworks aid in streamlining the response efforts of these organizations, they also enable effective messaging. to the best of our knowledge, there has not been a similar characterization of mathematical modeling efforts that go hand in hand with supporting the response. for summarizing the key models, we consider four of the stages of pandemic response mentioned in section : pre-pandemic, acceleration, mitigation and suppression. here we provide the key problems in each stage, the datasets needed, the main tools and techniques used, and pertinent challenges. we structure our discussion based on our experience with modeling the spread of covid- in the us, done in collaboration with local and federal agencies. • acceleration (section ): this stage is relevant once the epidemic takes root within a country. there is usually a big lag in surveillance and response efforts, and the key questions are to model spread patterns at different spatio-temporal scales, and to derive short-term forecasts and projections. a broad class of datasets is used for developing models, including mobility, populations, land-use, and activities. these are combined with various kinds of time series data and covariates such as weather for forecasting. • mitigation (section ): in this stage, different interventions, which are mostly non-pharmaceutical in the case of a novel pathogen, are implemented by government agencies, once the outbreak has taken hold within the population. this stage involves understanding the impact of interventions on case counts and health infrastructure demands, taking individual behaviors into account. the additional datasets needed in this stage include those on behavioral changes and hospital capacities. • suppression (section ): this stage involves designing methods to control the outbreak by contact tracing & isolation and vaccination. data on contact tracing, associated biases, vaccine production schedules, and compliance & hesitancy are needed in this stage. figure gives an overview of this framework and summarizes the data needs in these stages. these stages also align well with the focus of the various modeling working groups organized by cdc which include epidemic parameter estimation, international spread risk, sub-national spread forecasting, impact of interventions, healthcare systems, and university modeling. in reality, one should note that these stages may overlap, and may vary based on geographical factors and response efforts. moreover, specific problems can be approached prospectively in earlier stages, or retrospectively during later stages. this framework is thus meant to be more conceptual than interpreted along a linear timeline. results from such stages are very useful for policymakers to guide real-time response. consider a novel pathogen emerging in human populations that is detected through early cases involving unusual symptoms or unknown etiology. such outbreaks are characterized by some kind of spillover event, mostly through zoonotic means, like in the case of covid- or past influenza pandemics (e.g., swine flu and avian flu). a similar scenario can occur when an incidence of a well-documented disease with no known vaccine or therapeutics emerges in some part of the world, causing severe outcomes or fatalities (e.g., ebola and zika.) regardless of the development status of the country where the pathogen emerged, such outbreaks now contains the risk of causing a worldwide pandemic due to the global connectivity induced by human travel. two questions become relevant at this stage: what are the epidemiological attributes of this disease, and what are the risks of importation to a different country? while the first question involves biological and clinical investigations, the latter is more related with societal and environmental factors. one of the crucial tasks during early disease investigation is to ascertain the transmission and severity of the disease. these are important dimensions along which the pandemic potential is characterized because together they determine the overall disease burden, as demonstrated within the pandemic severity assessment framework [ ] . in addition to risk assessment for right-sizing response, they are integral to developing meaningful disease models. formulation let Θ = {θ t , θ s } represent the transmission and severity parameters of interest. they can be further subdivided into sojourn time parameters θ δ · and transition probability parameters θ p · . here Θ corresponds to a continuous time markov chain (ctmc) on the disease states. the problem formulation can be represented as follows: given Π(Θ), the prior distribution on the disease parameters and a dataset d, estimate the posterior distribution p(Θ|d) over all possible values of Θ. in a model-specific form, this can be expressed as p(Θ|d, m) where m is a statistical, compartmental or agent-based disease model. in order to estimate the disease parameters sufficiently, line lists for individual confirmed cases is ideal. such datasets contain, for each record, the date of confirmation, possible date of onset, severity (hospitalization/icu) status, and date of recovery/discharge/death. furthermore, age-and demographic/comorbidity information allow development of models that are age-and risk group stratified. one such crowdsourced line list was compiled during the early stages of covid- [ ] and later released by cdc for us cases [ ] . data from detailed clinical investigations from other countries such as china, south korea, and singapore was also used to parameterize these models [ ] . in the absence of such datasets, past parameter estimates of similar diseases (e.g., sars, mers) were used for early analyses. modeling approaches for a model agnostic approach, the delays and probabilities are obtained by various techniques, including bayesian and ordinary least squares fitting to various delay distributions. for a particular disease model, these are estimated through model calibration techniques such as mcmc and particle filtering approaches. a summary of community estimates of various disease parameters is provided at https://github.com/midas-network/covid- . further such estimates allow the design of pandemic planning scenarios varying in levels of impact, as seen in the cdc scenarios page . see [ ] [ ] [ ] for methods and results related to estimating covid- disease parameters from real data. current models use a large set of disease parameters for modeling covid- dynamics; they can be broadly classified as transmission parameters and hospital resource parameters. for instance in our work, we currently use parameters (with explanations) shown in table . challenges often these parameters are model specific, and hence one needs to be careful when reusing parameter estimates from literature. they are related but not identifiable with respect to population level measures such as basic reproductive number r (or effective reproductive number r eff ) and doubling time which allow tracking the rate of epidemic growth. also the estimation is hindered by inherent biases in case ascertainment rate, reporting delays and other gaps in the surveillance system. aligning different data streams (e.g., outpatient surveillance, hospitalization rates, mortality records) is in itself challenging. when a disease outbreak occurs in some part of the world, it is imperative for most countries to estimate their risk of importation through spatial proximity or international travel. such measures are incredibly valuable in setting a timeline for preparation efforts, and initiating health checks at the borders. over centuries, pandemics have spread faster and faster across the globe, making it all the more important to characterize this risk as early as possible. formulation let c be the set of countries, and g = {c, e} an international network, where edges (often weighted and directed) in e represent some notion of connectivity. the importation risk problem can be formulated as below: given c o ∈ c the country of origin with an initial case at time , and c i the country of interest, using g, estimate the expected time taken t i for the first cases to arrive in country c i . in its probabilistic form, the same can be expressed as estimating the probability p i (t) of seeing the first case in country c i by time t. data needs assuming we have initial case reports from the origin country, the first data needed is a network that connects the countries of the world to represent human travel. the most common source of such information is the airline network datasets, from sources such as iata, oag, and openflights; [ ] provides a systematic review of how airline passenger data has been used for infectious disease modeling. these datasets could either capture static measures such as number of seats available or flight schedules, or a dynamic count of passengers per month along each itinerary. since the latter has intrinsic delays in collection and reporting, for an ongoing pandemic they may not be representative. during such times, data on ongoing travel restrictions [ ] become important to incorporate. multi-modal traffic will also be important to incorporate for countries that share land borders or have heavy maritime traffic. for diseases such as zika, where establishment risk is more relevant, data on vector abundance or prevailing weather conditions are appropriate. modeling approaches simple structural measures on networks (such as degree, pagerank) could provide static indicators of vulnerability of countries. by transforming the weighted, directed edges into probabilities, one can use simple contagion models (e.g., independent cascades) to simulate disease spread and empirically estimate expected time of arrival. global metapopulation models (gleam) that combine seir type dynamics with an airline network have also been used in the past for estimating importation risk. brockmann and helbing [ ] used a similar framework to quantify effective distance on the network which seemed to be well correlated with time of arrival for multiple pandemics in the past; this has been extended to covid- [ , ] . in [ ] , the authors employ air travel volume obtained through iata from ten major cities across china to rank various countries along with the idvi to convey their vulnerability. [ ] consider the task of forecasting international and domestic spread of covid- and employ official airline group (oag) data for determining air traffic to various countries, and [ ] fit a generalized linear model for observed number of cases in various countries as a function of air traffic volume obtained from oag data to determine countries with potential risk of under-detection. also, [ ] provide africa-specific case-study of vulnerability and preparedness using data from civil aviation administration of china. challenges note that arrival of an infected traveler will precede a local transmission event in a country. hence the former is more appropriate to quantify in early stages. also, the formulation is agnostic to whether it is the first infected arrival or first detected case. however, in real world, the former is difficult to observe, while the latter is influenced by security measures at ports of entry (land, sea, air) and the ease of identification for the pathogen. for instance, in the case of covid- , the long incubation period and the high likelihood of asymptomaticity could have resulted in many infected travelers being missed by health checks at poes. we also noticed potential administrative delays in reporting by multiple countries fearing travel restrictions. as the epidemic takes root within a country, it may enter the acceleration phase. depending on the testing infrastructure and agility of surveillance system, response efforts might lag or lead the rapid growth in case rate. under such a scenario, two crucial questions emerge that pertain to how the disease may spread spatially/socially and how the case rate may grow over time. within the country, there is need to model the spatial spread of the disease at different scales: state, county, and community levels. similar to the importation risk, such models may provide an estimate of when cases may emerge in different parts of the country. when coupled with vulnerability indicators (socioeconomic, demographic, co-morbidities) they provide a framework for assessing the heterogeneous impact the disease may have across the country. detailed agent-based models for urban centers may help identify hotspots and potential case clusters that may emerge (e.g., correctional facilities, nursing homes, food processing plants, etc. in the case of covid- ). formulation given a population representation p at appropriate scale and a disease model m per entity (individual or sub-region), model the disease spread under different assumptions of underlying connectivity c and disease parameters Θ. the result will be a spatio-temporal spread model that results in z s,t , the time series of disease states over time for region s. data needs some of the common datasets needed by most modeling approaches include: ( ) social and spatial representation, which includes census, and population data, which are available from census departments (see, e.g., [ ] ), and landscan [ ] , ( ) connectivity between regions (commuter, airline, road/rail/river), e.g., [ , ] , ( ) data on locations, including points of interest, e.g., openstreetmap [ ] , and ( ) activity data, e.g., the american time use survey [ ] . these datasets help capture where people reside and how they move around, and come in contact with each other. while some of these are static, more dynamic measures, such as from gps traces, become relevant as individuals change their behavior during a pandemic. modeling approaches different kinds of structured metapopulation models [ , [ ] [ ] [ ] [ ] , and agent based models [ ] [ ] [ ] [ ] [ ] have been used in the past to model the sub-national spread; we refer to [ , , ] for surveys on different modeling approaches. these models incorporate typical mixing patterns, which result from detailed activities and co-location (in the case of agent based models), and different modes of travel and commuting (in the case of metapopulation models). challenges while metapopulation models can be built relatively rapidly, agent based models are much harder-the datasets need to be assembled at a large scale, with detailed construction pipelines, see, e.g., [ ] [ ] [ ] [ ] [ ] . since detailed individual activities drive the dynamics in agent based models, schools and workplaces have to be modeled, in order to make predictions meaningful. such models will get reused at different stages of the outbreak, so they need to be generic enough to incorporate dynamically evolving disease information. finally, a common challenge across modeling paradigms is the ability to calibrate to the dynamically evolving spatio-temporal data from the outbreak-this is especially challenging in the presence of reporting biases and data insufficiency issues. given the early growth of cases within the country (or sub-region), there is need for quantifying the rate of increase in comparable terms across the duration of the outbreak (accounting for the exponential nature of such processes). these estimates also serve as references, when evaluating the impact of various interventions. as an extension, such methods and more sophisticated time series methods can be used to produce short-term forecasts for disease evolution. formulation given the disease time series data within the country z s,t until data horizon t , provide scale-independent growth rate measures g s (t ), and forecastsẐ s,u for u ∈ [t, t + ∆t ], where ∆t is the forecast horizon. data needs models at this stage require datasets such as ( ) time series data on different kinds of disease outcomes, including case counts, mortality, hospitalizations, along with attributes, such as age, gender and location, e.g., [ ] [ ] [ ] [ ] [ ] , ( ) any associated data for reporting bias (total tests, test positivity rate) [ ] , which need to be incorporated into the models, as these biases can have a significant impact on the dynamics, and ( ) exogenous regressors (mobility, weather), which have been shown to have a significant impact on other diseases, such as influenza, e.g., [ ] . modeling approaches even before building statistical or mechanistic time series forecasting methods, one can derive insights through analytical measures of the time series data. for instance, the effective reproductive number, estimated from the time series [ ] can serve as a scale-independent metric to compare the outbreaks across space and time. additionally multiple statistical methods ranging from autoregressive models to deep learning techniques can be applied to the time series data, with additional exogenous variables as input. while such methods perform reasonably for short-term targets, mechanistic approaches as described earlier can provide better long-term projections. various ensembling techniques have also been developed in the recent past to combine such multi-model forecasts to provide a single robust forecast with better uncertainty quantification. one such effort that combines more than methods for covid- can be found at the covid forecasting hub . we also point to the companion paper for more details on projection and forecasting models. challenges data on epidemic outcomes usually has a lot of uncertainties and errors, including missing data, collection bias, and backfill. for forecasting tasks, these time series data need to be near real-time, else one needs to do both nowcasting, as well as forecasting. other exogenous regressors can provide valuable lead time, due to inherent delays in disease dynamics from exposure to case identification. such frameworks need to be generalized to accommodate qualitative inputs on future policies (shutdowns, mask mandates, etc.), as well as behaviors, as we discuss in the next section. once the outbreak has taken hold within the population, local, state and national governments attempt to mitigate and control its spread by considering different kinds of interventions. unfortunately, as the covid- pandemic has shown, there is a significant delay in the time taken by governments to respond. as a result, this has caused a large number of cases, a fraction of which lead to hospitalizations. two key questions in this stage are: ( ) how to evaluate different kinds of interventions, and choose the most effective ones, and ( ) how to estimate the healthcare infrastructure demand, and how to mitigate it. the effectiveness of an intervention (e.g., social distancing) depends on how individuals respond to them, and the level of compliance. the health resource demand depends on the specific interventions which are implemented. as a result, both these questions are connected, and require models which incorporate appropriate behavioral responses. in the initial stages, only non-prophylactic interventions are available, such as: social distancing, school and workplace closures, and use of ppes, since no vaccinations and anti-virals are available. as mentioned above, such analyses are almost entirely model based, and the specific model depends on the nature of the intervention and the population being studied. formulation given a model, denoted abstractly as m, the general goals are ( ) to evaluate the impact of an intervention (e.g., school and workplace closure, and other social distancing strategies) on different epidemic outcomes (e.g., average outbreak size, peak size, and time to peak), and ( ) find the most effective intervention from a suite of interventions, with given resource constraints. the specific formulation depends crucially on the model and type of intervention. even for a single intervention, evaluating its impact is quite challenging, since there are a number of sources of uncertainty, and a number of parameters associated with the intervention (e.g., when to start school closure, how long, and how to restart). therefore, finding uncertainty bounds is a key part of the problem. data needs while all the data needs from the previous stages for developing a model are still there, representation of different kinds of behaviors is a crucial component of the models in this stage; this includes: use of ppes, compliance to social distancing measures, and level of mobility. statistics on such behaviors are available at a fairly detailed level (e.g., counties and daily) from multiple sources, such as ( ) the covid- impact analysis platform from the university of maryland [ ] , which gives metrics related to social distancing activities, including level of staying home, outside county trips, outside state trips, ( ) changes in mobility associated with different kinds of activities from google [ ] , and other sources, ( ) survey data on different kinds of behaviors, such as usage of masks [ ] . modeling approaches as mentioned above, such analyses are almost entirely model based, including structured metapopulation models [ , [ ] [ ] [ ] [ ] , and agent based models [ ] [ ] [ ] [ ] [ ] . different kinds of behaviors relevant to such interventions, including compliance with using ppes and compliance to social distancing guidelines, need to be incorporated into these models. since there is a great deal of heterogeneity in such behaviors, it is conceptually easiest to incorporate them into agent based models, since individual agents are represented. however, calibration, simulation and analysis of such models pose significant computational challenges. on the other hand, the simulation of metapopulation models is much easier, but such behaviors cannot be directly represented-instead, modelers have to estimate the effect of different behaviors on the disease model parameters, which can pose modeling challenges. challenges there are a number of challenges in using data on behaviors, which depends on the specific datasets. much of the data available for covid- is estimated through indirect sources, e.g., through cell phone and online activities, and crowd-sourced platforms. this can provide large spatio-temporal datasets, but have unknown biases and uncertainties. on the other hand, survey data is often more reliable, and provides several covariates, but is typically very sparse. handling such uncertainties, rigorous sensitivity analysis, and incorporating the uncertainties into the analysis of the simulation outputs are important steps for modelers. the covid- pandemic has led to a significant increase in hospitalizations. hospitals are typically optimized to run near capacity, so there have been fears that the hospital capacities would not be adequate, especially in several countries in asia, but also in some regions in the us. nosocomial transmission could further increase this burden. formulation the overall problem is to estimate the demand for hospital resources within a populationthis includes the number of hospitalizations, and more refined types of resources, such as icus, ccus, medical personnel and equipment, such as ventilators. an important issue is whether the capacity of hospitals within the region would be overrun by the demand, when this is expected to happen, and how to design strategies to meet the demand-this could be through augmenting the capacities at existing hospitals, or building new facilities. timing is of essence, and projections of when the demands exceed capacity are important for governments to plan. the demands for hospitalization and other health resources can be estimated from the epidemic models mentioned earlier, by incorporating suitable health states, e.g., [ , ] ; in addition to the inputs needed for setting up the models for case counts, datasets are needed for hospitalization rates and durations of hospital stay, icu care, and ventilation. the other important inputs for this component are hospital capacity, and the referral regions (which represent where patients travel for hospitalization). different public and commercial datasets provide such information, e.g., [ , ] . modeling approaches demand for health resources is typically incorporated into both metapopulation and agent based models, by having a fraction of the infectious individuals transition into a hospitalization state. an important issue to consider is what happens if there is a shortage of hospital capacity. studying this requires modeling the hospital infrastructure, i.e., different kinds of hospitals within the region, and which hospital a patient goes to. there is typically limited data on this, and data on hospital referral regions, or voronoi tesselation can be used. understanding the regimes in which hospital demand exceeds capacity is an important question to study. nosocomial transmission is typically much harder to study, since it requires more detailed modeling of processes within hospitals. challenges there is a lot of uncertainty and variability in all the datasets involved in this process, making its modeling difficult. for instance, forecasts of the number of cases and hospitalizations have huge uncertainty bounds for medium or long term horizon, which is the kind of input necessary for understanding hospital demands, and whether there would be any deficits. the suppression stage involves methods to control the outbreak, including reducing the incidence rate and potentially leading to the eradication of the disease in the end. eradication in case of covid- appears unlikely as of now, what is more likely is that this will become part of seasonal human coronaviruses that will mutate continuously much like the influenza virus. contact tracing problem refers to the ability to trace the neighbors of an infected individual. ideally, if one is successful, each neighbor of an infected neighbor would be identified and isolated from the larger population to reduce the growth of a pandemic. in some cases, each such neighbor could be tested to see if the individual has contracted the disease. contact tracing is the workhorse in epidemiology and has been immensely successful in controlling slow moving diseases. when combined with vaccination and other pharmaceutical interventions, it provides the best way to control and suppress an epidemic. formulation the basic contact tracing problem is stated as follows: given a social contact network g(v, e) and subset of nodes s ⊂ v that are infected and a subset s ⊂ s of nodes identified as infected, find all neighbors of s. here a neighbor means an individual who is likely to have a substantial contact with the infected person. one then tests them (if tests are available), and following that, isolates these neighbors, or vaccinates them or administers anti-viral. the measures of effectiveness for the problem include: (i) maximizing the size of s , (ii) maximizing the size of set n (s ) ⊆ n (s), i.e. the potential number of neighbors of set s , (iii) doing this within a short period of time so that these neighbors either do not become infectious, or they minimize the number of days that they are infectious, while they are still interacting in the community in a normal manner, (iv) the eventual goal is to try and reduce the incidence rate in the community-thus if all the neighbors of s cannot be identified, one aims to identify those individuals who when isolated/treated lead to a large impact; (v) and finally verifying that these individuals indeed came in contact with the infected individuals and thus can be asked to isolate or be treated. data needs data needed for the contact tracing problem includes: (i) a line list of individuals who are currently known to be infected (this is needed in case of human based contact tracing). in the real world, when carrying out human contact tracers based deployment, one interviews all the individuals who are known to be infectious and reaches out to their contacts. modeling approaches human contact tracing is routinely done in epidemiology. most states in the us have hired such contact tracers. they obtain the daily incidence report from the state health departments and then proceed to contact the individuals who are confirmed to be infected. earlier, human contact tracers used to go from house to house and identify the potential neighbors through a well defined interview process. although very effective it is very time consuming and labor intensive. phones were used extensively in the last - years as they allow the contact tracers to reach individuals. they are helpful but have the downside that it might be hard to reach all individuals. during covid- outbreak, for the first time, societies and governments have considered and deployed digital contact tracing tools [ ] [ ] [ ] [ ] [ ] . these can be quite effective but also have certain weaknesses, including, privacy, accuracy, and limited market penetration of the digital apps. challenges these include: (i) inability to identify everyone who is infectious (the set s) -this is virtually impossible for covid- like disease unless the incidence rate has come down drastically and for the reason that many individuals are infected but asymptomatic; (ii) identifying all contacts of s (or s ) -this is hard since individuals cannot recall everyone they met, certain folks that they were in close proximity might have been in stores or social events and thus not known to individuals in the set s. furthermore, even if a person is able to identify the contacts, it is often hard to reach all the individuals due to resource constraints (each human tracer can only contact a small number of individuals. the overall goal of the vaccine allocation problem is to allocate vaccine efficiently and in a timely manner to reduce the overall burden of the pandemic. formulation the basic version of the problem can be cast in a very simple manner (for networked models): given a graph g(v, e) and a budget b on the number of vaccines available, find a set s of size b to vaccinate so as to optimize certain measure of effectiveness. the measure of effectiveness can be (i) minimizing the total number of individuals infected (or maximizing the total number of uninfected individuals); (ii) minimizing the total number of deaths (or maximizing the total number of deaths averted); (iii) optimizing the above quantities but keeping in mind certain equity and fairness criteria (across socio-demographic groups, e.g. age, race, income); (iv) taking into account vaccine hesitancy of individuals; (v) taking into account the fact that all vaccines are not available at the start of the pandemic, and when they become available, one gets limited number of doses each month; (vi) deciding how to share the stockpile between countries, state, and other organizations; (vii) taking into account efficacy of the vaccine. data needs as in other problems, vaccine allocation problems need as input a good representation of the system; network based, meta-population based and compartmental mass action models can be used. one other key input is the vaccine budget, i.e., the production schedule and timeline, which serves as the constraint for the allocation problem. additional data on prevailing vaccine sentiment and past compliance to seasonal/neonatal vaccinations are useful to estimate coverage. modeling approaches the problem has been studied actively in the literature; network science community has focused on optimal allocation schemes, while public health community has focused on using meta-population models and assessing certain fixed allocation schemes based on socio-economic and demographic considerations. game theoretic approaches that try and understand strategic behavior of individuals and organization has also been studied. challenges the problem is computationally challenging and thus most of the time simulation based optimization techniques are used. challenge to the optimization approach comes from the fact that the optimal allocation scheme might be hard to compute or hard to implement. other challenges include fairness criteria (e.g. the optimal set might be a specific group) and also multiple objectives that one needs to balance. while the above sections provide an overview of salient modeling questions that arise during the key stages of a pandemic, mathematical and computational model development is equally if not more important as we approach the post-pandemic (or more appropriately inter-pandemic) phase. often referred to as peace time efforts, this phase allows modelers to retrospectively assess individual and collective models on how they performed during the pandemic. in order to encourage continued development and identifying data gaps, synthetic forecasting challenge exercises [ ] may be conducted where multiple modeling groups are invited to forecast synthetic scenarios with varying levels of data availability. another set of models that are quite relevant for policymakers during the winding down stages, are those that help assess overall health burden and economic costs of the pandemic. epideep: exploiting embeddings for epidemic forecasting an arima model to forecast the spread and the final size of covid- epidemic in italy (first version on ssrn march) real-time epidemic forecasting: challenges and opportunities accuracy of real-time multi-model ensemble forecasts for seasonal influenza in the u.s realtime forecasting of infectious disease dynamics with a stochastic semi-mechanistic model healthmap the use of social media in public health surveillance. western pacific surveillance and response journal : wpsar the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak basic prediction methodology for covid- : estimation and sensitivity considerations. medrxiv covid- outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand. imperial college technical report modelling disease outbreaks in realistic urban social networks computational epidemiology forecasting covid- 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interval for coronavirus disease (covid- ) based on symptom onset data the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application estimating clinical severity of covid- from the transmission dynamics in wuhan, china the use and reporting of airline passenger data for infectious disease modelling: a systematic review flight cancellations related to -ncov (covid- ) the hidden geometry of complex, network-driven contagion phenomena potential for global spread of a novel coronavirus from china forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study. the lancet using predicted imports of -ncov cases to determine locations that may not be identifying all imported cases. medrxiv preparedness and vulnerability of african countries against introductions of -ncov. medrxiv creating synthetic baseline populations openstreetmap american time use survey multiscale mobility networks and the spatial spreading of infectious diseases optimizing spatial allocation of seasonal influenza vaccine under temporal constraints assessing the international spreading risk associated with the west african ebola outbreak spread of zika virus in the americas structure of social contact networks and their impact on epidemics generation and analysis of large synthetic social contact networks modelling disease outbreaks in realistic urban social networks containing pandemic influenza at the source report : impact of non-pharmaceutical interventions (npis) to reduce covid mortality and healthcare demand the structure and function of complex networks a public data lake for analysis of covid- data midas network. midas novel coronavirus repository covid- ) data in the united states covid- impact analysis platform covid- surveillance dashboard the covid tracking project absolute humidity and the seasonal onset of influenza in the continental united states epiestim: a package to estimate time varying reproduction numbers from epidemic curves. r package version google covid- community mobility reports mask-wearing survey data impact of social distancing measures on coronavirus disease healthcare demand, central texas, usa current hospital capacity estimates -snapshot total hospital bed occupancy quantifying the effects of contact tracing, testing, and containment covid- epidemic in switzerland: on the importance of testing, contact tracing and isolation quantifying sars-cov- transmission suggests epidemic control with digital contact tracing isolation and contact tracing can tip the scale to containment of covid- in populations with social distancing. available at ssrn privacy sensitive protocols and mechanisms for mobile contact tracing the rapidd ebola forecasting challenge: synthesis and lessons learnt acknowledgments. the authors would like to thank members of the biocomplexity covid- response team and network systems science and advanced computing (nssac) division for their thoughtful comments and suggestions related to epidemic modeling and response support. we thank members of the biocomplexity institute and initiative, university of virginia for useful discussion and suggestions. this key: cord- -yxqbe dj authors: ren, yunzhao r.; golding, amit; sorbello, alfred; ji, ping; chen, jianmeng; bhawana, saluja; witzmann, kimberly; arya, vikram; reynolds, kellie s.; choi, su‐young; nikolov, nikolay; sahajwalla, chandrahas title: a comprehensive updated review on sars‐cov‐ and covid‐ date: - - journal: j clin pharmacol doi: . /jcph. sha: doc_id: cord_uid: yxqbe dj this literature review aims to provide a comprehensive current summary of the pathogenesis, clinical features, disease course, host immune responses, and current investigational antiviral and immunomodulatory pharmacotherapies, in order to facilitate the development of future therapies and measures for prevention and control. this article is protected by copyright. all rights reserved the disease name -covid- ‖ and the associated virus name -sars-cov- ‖ were coined by the world health organization (who) and the coronavirus study group of the international committee on virus taxonomy, respectively, on february , . currently no specific drug has been approved by the fda for treating covid- , and the current management of patients is mainly supportive. fda has issued only emergency use authorizations (eua) to permit the emergency use of chloroquine phosphate, hydroxychloroquine sulfate, and remdesivir. therapeutic development for covid- includes repurposing existing medications and developing investigational candidates. the first reported confirmed covid- case was presented as atypical pneumonia on december , in wuhan, china. the patient was among a cluster of cases reported to who on january , . as of may , , sars-cov- has infected over . million people worldwide . covid- had resulted in a global death toll of more than , . an epidemiology study noted that of , confirmed covid- cases in china through february , . % and . % of patients were in severe and critical condition, respectively . the reported median incubation period of covid- is days , and the median period from symptom onset to hospital admission ranges from to days , . median time from onset of the first symptom to dyspnea is - days , ; to pneumonia, days ; to icu admission, days ; and to acute respiratory distress syndrome (ards), days . the median duration of hospitalization is days to days ; median length of icu stay, days ; median time from admission to invasive mechanical ventilation, . days ; and median time from admission to death was . days . the currently estimated reproductive number (r ) of sars-cov- , the average number of people to which one infected individual will pass the virus, ranges from . to . [ ] [ ] [ ] , whereas the reported r for sars-cov is approximately . sars-cov- is an airborne virus which can be transmitted by aerosol . a hospital survey detected the maximum transmission distance of sars-cov- aerosol might be meters from the covid- patients . in the contaminated area of the hospital, the viral nucleic acid positive rate was % for computer mice, % for floor swabs, % for trash cans, % for sickbed handrails, and % for doorknobs. in a virus viability test, authors used a nebulizer to generate artificial aerosols with small particle size (< μm) containing sars-cov- . the results showed that sars-cov- remained viable in the artificial aerosols for at least hours. the virus is most stable on plastic and stainless steel surfaces, on which viable virus had been detected for up to hours. no viable virus was detected after hours on copper and after hours on cardboard. the who, therefore, advises the public not to touch their eyes, nose, or mouth with their hands to limit self-contamination . in addition, the centers for disease control and prevention (cdc) recommends wearing cloth face coverings in public settings where other social distancing measures ( feet) are difficult to maintain . a case study reported that of passengers on a coach bus contracted the virus from an infected patient who did not wear a protective face mask. however, the same patient bought a mask and wore it before transferring to a mini-bus, and none of the passengers on the mini-bus contracted the virus . live sars-cov- was isolated from nasal/pharyngeal swabs and sputum, but not from stool, in patients with covid- . the live viral copies peaked during the early stage of symptom onset (≤ days); and could not be detected after day in samples from mild cases of infection. this discovery suggests that the viral transmission occurs primarily through the airborne route rather than the fecal-oral route during early stages of the disease. indeed, there have been sporadic case reports on the human-to-human transmission from asymptomatic or pre-symptomatic subjects [ ] [ ] [ ] . one study estimated that the transmissibility of the asymptomatic cases is comparable to that of symptomatic cases . an epidemiology report from china indicated that . % ( ) of , tested/suspected/diagnosed cases were asymptomatic . identification and isolation of asymptomatic subjects has helped reduce the pandemic in an italian village . these reports suggest that a panpopulation screening for viral exposure is an effective way to critically contain the spread of the disease. two meteorology models consistently found that higher relative humidity favored sars-cov- transmission , . the two models differed regarding the trend of temperature effect on viral transmission, probably due to the different ranges of temperature studied in china (winter) and brazil (autumn). an epidemiology study from china found that although the proportion of male patients ( . %) was comparable to that of females ( . %), male patients ( . %) comprised almost two-thirds of the total deaths . data from the cdc website as of may , showed that among approximately , covid- cases in the u.s., % were in children (< years old); % were in the elderly (≥ years old), and most ( %) were in patients between and years old . of hospitalized patients in u.s. states from march - , ( %) were non-hispanic white; ( . %) were non-hispanic black; ( . %) were hispanic; ( . %) were asian, two ( . %) were american indian/alaskan native, and ( . %) were of other ethnic origins or unknown . culture of bronchoalveolar lavage fluid collected from early wuhan cases identified the etiology of the virus to date, the virus phylogenetically closest to sars-cov- by genetic homogeneity is a coronavirus isolated from the horseshoe bat (bat cov ratg ) with an overall genome sequence identity of . %, which is higher than that of sars-cov (< %) . angiotensinconverting enzyme (ace ) was identified as a shared receptor required for cell entry both for sars-cov and sars-cov- , with higher binding affinity for sars-cov- . sequence comparison of spike (s) protein, the viral ligand of ace , identified three short insertions located at the n-terminus region that are conserved in sars-cov- and bat cov ratg , but not in sars-cov . examination of the receptor-binding domain (rbd) of s protein surprisingly identified that a malayan pangolin coronavirus had a higher degree of similarity ( . %) than bat cov ratg ( . %), indicating that recombination may have occurred during the evolution of sars-cov- . variation analysis based on sequences of sars-cov- up to february revealed very high homology (> . %) among different strains . another group estimated that the evolution rate of sars-cov- is approximately . × - per base per year , which indicates that sars-cov- transmission in humans is a recent event. the sars-cov- genome sequence can be found at https://www.ncbi.nlm.nih.gov/genbank/sars-cov- -seqs/. the direct diagnosis of covid- requires detection of sars-cov- -specific rna from patients' samples. reverse transcription-polymerase chain reaction (rt-pcr) is the most widely used technique for diagnosis. a commercial rt-pcr test kit usually uses to pairs of primers detecting the different regions of sars-cov- genomic rna to increase the test specificity. the sensitivity of this method is not optimal. one paper noted that the sensitivity of rt-pcr ( %), even after % of patients had multiple tests, was lower than that of a ct scan ( %) . a test report of , wuhan cases with typical covid- symptoms and history of close patient contact demonstrated that the positive rate was about % for nasal and pharyngeal swabs, % for sputum samples, and % to % for bronchoalveolar lavage fluid . another study screened subjects in wuhan and found that the positive rate from nasopharyngeal swabs was . -fold higher than that of oropharyngeal swabs . interestingly, pharyngeal swab viral nucleic acid screening results of , patients between january and february from a hospital fever clinic in hunan province (a neighboring province of hubei) demonstrated that the positive rate of sars-cov- ( . %) was lower than that of influenza a ( . %) and influenza b ( . %) . it is unclear whether the lockdown status of hubei province or the sensitivity of the detection methods between different viruses contributed to the result. the disease course also affects viral nucleic acid detection results. one study closely followed throat swab samples or deep nasal cavity swab samples from hospitalized covid- patients and found that the positive rate was the highest ( %) within week since the symptom onset . however, the positive rate reduced to about one-third at week . similar results were obtained from another study, in which the positive rate of throat swabs from patients was > % when tested within - days since symptom onset, but decreased to < % on day , and < % after day . other than the traditional rt-pcr, other viral rna detecting methods such as loop-mediated isothermal amplification (lamp) were expeditiously developed and approved by the fda . the apparent advantage of lamp is the much shorter waiting time for the results (< minutes) compared to the traditional rt-pcr ( hours). crispr, the powerful gene editing technique, premiered in this pandemic and was also approved by fda, though the commercial kit requires an isothermal amplification step . reports on the relationship between viral load in respiratory tracts and disease severity showed conflicting results. one study (n= ) reported that the high viral load from a patient's respiratory tracts is moderately associated with a high murray score for acute lung injury and low pao /fio . the same study also reported that the high viral load is associated with high plasma angiotensin ii concentration. however, two other studies (n= and n= ) did not find significant differences in viral load between mild and severe cases , . one study demonstrated that the speed of viral clearance differs significantly in mild and severe cases . the average time of viral nucleic acid turning positive to negative was about days in mild cases and days in severe cases. in non-survivors, persistent viral rna was detected until death . however, another study with intensive testing was able to detect viral nucleic acid in throat/deep nasal cavity swab samples from of hospitalized patients with mild-to-moderate confirmed covid- weeks after symptom onset . sars-cov- was detected in the whole blood and serum , . more studies are needed to investigate the correlation between viremia with blood viral load and disease severity. patients on admission, and discovered that the cutoff of . µg/ml had a sensitivity of % and a specificity of % in predicting in-hospital mortality . higher levels of hypersensitive troponin i in patients with severe covid- (table ) indicates an association of sars-cov- infection and cardiomyopathy , . a review summarized that cardiovascular complications associated with covid- include myocardial injury, myocarditis, acute myocardial infarction, heart failure, dysrhythmias, and thromboembolic events two covid- -associated kawasaki disease cases were reported in a -month-old girl and a -year-old boy, respectively il- , il- , tnf-α and ifn-γ were found between the two groups. whether the age differences between the two groups also contributed to these observed differences is unclear. two rsv-infected children ( . %) and one sars-cov- -infected child ( . %) developed severe pneumonia. all children survived. various case reports, case series, retrospective and case-controlled studies of pregnant women with covid- presented clinical and laboratory data on maternal and neonatal manifestations and outcomes [ ] [ ] [ ] [ ] [ ] . reviews and analyses of published reports provided additional insights [ ] [ ] [ ] [ ] . most of the pregnant women in these studies and reports were in their third trimester, and many of their babies were delivered by caesarean section. in general, they experienced signs and symptoms of covid- much like those in non-pregnant women. no maternal deaths were reported. fetal distress, premature births, premature rupture of membranes, respiratory difficulties, and low birth weight were observed among some babies born to mothers with covid- , , . no definitive cases of vertical transmission of the sars coronavirus (sars-cov- ) from mother to fetus have been identified, although two highly suspect cases have been reported. wang and colleagues described a male infant delivered by emergency cesarean section to a -year-old woman with covid- confirmed by pharyngeal swab . the infant had a viral nucleic acid detected from pharyngeal swab approximately hours after birth. tests of the cord blood, placenta, and the mother's breast milk were negative for sars-cov- . both mother and infant recovered. alzamora and colleagues reported on a -year-old diabetic woman with covid- -induced respiratory failure whose neonate was positive for sars-cov- nucleic acid from nasopharyngeal swab hours after a cesarean section delivery . serologies for sars-cov- were negative for the mother and the baby at the time of delivery. the mother converted to seropositive status on postpartum day ; whereas the neonate, who had not been breastfed, remained seronegative at that time. in relation to neonatal survival, zhu and colleagues reported one neonatal death in which a male baby born to a mother with confirmed covid- developed refractory shock, gastric bleeding, multi-organ failure, and disseminated intravascular coagulation approximately days after birth three publications reported findings with inconsistent results from systematic reviews or meta-analyses. a systematic review by vardavas and nikitara found an association between smoking and covid- illness progression . emami and colleagues in a meta-analysis reported a high prevalence of smoking ( . %) in hospitalized patients with covid- . however, a meta-analysis by lippi and henry found no association between smoking and covid- , although they acknowledged the findings reported by liu above . the inconsistent results on smoking may be attributed to lack of data on smoking quantity and duration, small population size and/or few smokers in certain studies, and the presence of other concurrent comorbid conditions. future research should consider including documentation of nicotine exposures through vaping and ecigarettes. the scientific community recently has debated a possible therapeutic role for nicotine in treating covid- . some epidemiologic data has shown lower numbers of smokers among patients with covid- , indicating that nicotine may mediate the viral transmission by lowering ace levels , . a randomized clinical study is being planned in france to more formally assess if nicotine could reduce the risk of contracting the disease . an analysis of deceased patients with covid- revealed that deaths were due to complications directly related to the viral infection . among them, ards was most prevalent ( %), followed by myocardial injury ( %), liver injury ( %), and renal insufficiency ( %). multiple organ dysfunction syndrome occurred in % of cases. autopsy and biopsy of covid- cases have been sporadically reported. a report of complete autopsy in consecutive covid- -positive deaths ( males and females with median age of years) in germany found that half of the cases had coronary heart disease and a quarter of the cases had respiratory diseases (asthma/copd) . the cause of death was found within the lungs or the pulmonary vascular system in all cases. deep venous thrombosis was identified in of patients ( %) in whom venous thromboembolism was not suspected before death. pulmonary embolism was the direct cause of death in patients. the histopathology examination of lungs found diffuse alveolar damage in cases. the lesions included hyaline membranes, activated pneumocytes, microvascular thromboemboli, capillary congestion, and protein-enriched interstitial edema, which were consistent with ards diagnosis. however, another immunohistology investigation of lung tissues from covid- patients who died with respiratory failure found that the pattern of covid- pneumonitis was predominantly a pauci-inflammatory septal capillary injury with significant septal capillary mural and luminal fibrin deposition and permeation of the inter-alveolar septa by neutrophils without hallmarks of classic ards three clinical studies recorded the baseline cytokine plasma concentrations in , , and patients with covid- on admission or from initial tests. the median time from symptom onset to admission in these three papers was - days , days , and - days , respectively. however, the cytokine examination date from a fourth study may be even later , since the median time for blood sample collection in this paper was days since subjects were transferred to a designated hospital. one study reported that ifn-γ, tnf-α, il- β, and il- plasma concentrations in covid- patients were significantly higher compared to results from four healthy in addition to the consistent trend of lower lymphocyte counts observed in severe cases of covid- (table ) from different studies, total t cells, cd + t cells, and cd + t cells also were significantly lower in severe/critical covid- cases than in non-severe cases , , . subgroups of cd + t cells did not show significant proportion changes of cd ra + naïve t cells and cd ro + memory t cells between severe cases and moderate cases . however, the proportion of cd ra + regulatory t cells in severe cases ( . %) was only half the value of that in moderate cases ( . %). one study noted a slight improvement of mean t cell counts (including cd + or cd + subpopulations) in comparison to baseline values after - days of in-hospital treatment . the count improvement appears baselineproportional, regardless of disease severity. several published studies have observed features of cellular exhaustion in t cells analogous to that described for nk cells . in two related studies, the authors showed that healthy individuals could be distinguished from mild and severe covid- t cells; specifically, the severe group had much lower levels of non-exhausted (pd- -ctla- -tigit -) cd + t cells , . they also found that cd + t cells in covid- patients exhibit many aspects of exhaustion and reduced function, such as once in the cell, the virus is not killed or neutralized; instead, it may continue to replicate, and/or stimulate or kill the target cells, causing more inflammation and damage. of note is that ade is not necessarily associated with past humoral response to a related pathogen, such as dengue virus , and can occur during the primary humoral response when the neutralizing antibody is at a suboptimal level. this was documented in some severe cases following sars-cov infection , . in a study reporting patients with sars, the lung radiographic worsening of some severe cases was correlated with the time of igg seroconversion . the pattern is consistent with that of covid- , in which the disease in some severe cases suddenly worsened around one to two weeks , , when seroconversion of anti-sars-cov- occurred (around to days after the onset of the first symptom) . in addition, the recovered sars patients had higher and sustainable or steadily increasing levels of both anti-n antibody and anti-s neutralizing antibody since the seroconversion . however, the titer of anti-n antibody in the sras non-survivors was low, and the titer of anti-s antibody decreased rapidly approximately days after the peak, an observation much like that in some patients with severe cases of covid- . in vitro studies demonstrated that human anti-s serum enhanced sars-cov infection in human monocyte-derived macrophages (mdm) . the infection mechanism is very different from that of the ace -mediated, endosomal/lysosomaldependent pathway, and can be blocked by anti-fcγr ii antibody on april , the national institutes of health (nih) issued general treatment guidelines for covid- , with the recommendations based on scientific evidence and expert opinion. we do not intend to repeat the previously well-documented work in our review, but prefer to focus on two important topics: antiviral and immunomodulatory pharmacotherapies. because results from clinical trials currently are being generated at such a blazing pace, this review was up to date at the time it was written. siddiqi hk et al. many viral cellular adherence/endocytosis blocking reagents were proposed. the cellular infection mechanism of sars-cov- is believed to be the same as sars-cov, which is an ace -mediated, endosomal-dependent pathway. chloroquine and hydroxychloroquine were first identified through in vitro drug screening to reduce viral titers from the supernatant of infected cell cultures. the mechanism probably is through interference with viral entry/endocytosis by increasing the ph of the endosome . two series of open-label, non-randomized studies in france reported only one death in patients with relatively mild disease treated with hydroxychloroquine sulfate ( mg tid for days) and azithromycin ( mg on day followed by mg qd) , . the authors justified the use of azithromycin because it had been shown to be effective against zika and ebola viruses in vitro. of note is that azithromycin also prolongs the qt interval. based on limited scientific information, it is reasonable to believe that hcq may be an effective treatment. fda issued an eua on march to permit the emergency use of chloroquine phosphate and hydroxychloroquine sulfate supplied from the strategic national stockpile to treat adults and adolescents who weigh kg or more and are hospitalized with covid- for whom a clinical trial is not available, or participation is not feasible . it should be noted that the fda's typical process for eua is to review its circumstances and appropriateness periodically. the review would include regular assessments, based on additional information from the sponsor, regarding progress on the unapproved product's -or unapproved use of an approved product's-approval, licensure, or clearance. an observational study in patients from new york city did not find a significant difference in the rate of intubation or mortality between patients who received hydroxychloroquine and those did not o intravenous immunoglobulin (ivig) has been used to treat sars patients [ ] [ ] [ ] the covid- pandemic is still ongoing, and a long time may pass before we can fully grasp the complete picture of the pathogen's characteristics; including its vulnerabilities, which can be used to inform development of effective and efficient treatments. development of antiviral therapeutics, led by dna/rna polymerase and protease inhibitors, has been streamlined since their invention in combatting hiv. given worldwide extensive efforts, we are hopeful that anti-sars-cov- 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disease, medical frailty, and renal failure date: - - journal: braddom's physical medicine and rehabilitation doi: . /b - - - - . - sha: doc_id: cord_uid: tcobane cardiopulmonary rehabilitation includes essential interventions that help patients maximize functional potential due to progressive deconditioning or acute decompensation following an acute medical event. the population of patients who benefit from both cardiac and pulmonary rehabilitation is increasing as the population ages and heart disease remains a leading cause of global morbidity and mortality. the principles of exercise physiology when applied to this population can reverse deconditioning, build cardiopulmonary reserve, and ultimately reduce morbidity and mortality in these populations. the physically disabled also benefit from exercise conditioning. the model of cardiac rehabilitation can also be applied to improve functional status of stroke patients, and this is an emerging area of interest supported by the shared pathophysiology and risk factors of cardiovascular conditions. the benefits of supervised exercise can also be extended to patients with clinically recognized frailty and post-transplant decline in function. frailty is a complex diagnosis with multiple tools and approaches used to describe this syndrome. medical rehabilitation medicine needs to encompass the needs of patients with cardiopulmonary disorders as well as patients with debility and renal dysfunction. there are ever greater numbers of patients who are now presenting with these issues, especially as the population ages. cardiac disease is still the number one cause of mortality and disability in the united states, with chronic obstructive pulmonary disease (copd) currently being the third leading cause of mortality. even if it is not primary rehabilitation for patients with these diseases, many patients with other disabilities will have cardiac, pulmonary, and renal disabilities, and a common theme among all of these conditions is a degree of frailty. this issue of frailty underlies a great number of the common features of disability in these underlying conditions and will be discussed in the section on frailty, later. key areas that will be addressed include sarcopenia, acute and chronic deconditioning, and the role of exercise as medicine to improve patients with all of these conditions. important contributors to the prevalence of all of these conditions are the aging of the population and the effects of combinations of these conditions on the ability to rehabilitate patients. the overall approaches to exercise treatment in these patients will be discussed as well as how to approach these conditions in the acute, rehabilitation, and outpatient settings. it must be remembered that there are two types of cardiopulmonary patients, those with primary cardiac and pulmonary disease who need cardiac/pulmonary rehabilitation and those patients with other disabilities who have a cardiac or pulmonary secondary disability. patients with respiratory failure and patients who have need for ventilatory support are also in this group but are beyond the scope of this chapter. dual disability patients are more prevalent than ever in rehabilitation because more patients are currently older and have multiple comorbidities. many patients with stroke, vascular disease, or other conditions can be included in active cardiac and pulmonary rehabilitation programs or benefit from the application of cardiopulmonary rehabilitation principles to their rehabilitation. remember also that cardiopulmonary rehabilitation is one of the most underused yet most effective treatments for patients with cardiopulmonary disease. because we work with frail older adults and other compromised populations, it is important for rehabilitation specialists to know how to provide cardiopulmonary rehabilitation in patients with either primary or secondary cardiopulmonary disability. to use cardiac and pulmonary rehabilitation principles for patients with cardiopulmonary disease, whether it is a primary or secondary disability, it is necessary to review some of the basic principles of cardiac and pulmonary physiology and learn how to apply these principles to improve the exercise capacity of these patients. it is also essential to have an understanding of normal exercise physiology to appreciate the issues of patients with abnormal cardiopulmonary physiology. a complete cardiopulmonary history and physical examination are essential parts of the evaluation of patients with cardiopulmonary disease who participate in rehabilitation. key parts of the history include both verbal and nonverbal cues and will allow the establishment of goals and improve patient compliance with the treatment program. the history should include emotional state, concurrent illnesses, other disabilities, functional history, occupational history, social history, personal habits, family dynamics, and the effect of disability and cardiopulmonary illness on the patient in the community. both rest and activity symptoms are reviewed with particular emphasis on the following. dyspnea. shortness of breath is usually the prime complaint for patients with cardiopulmonary disease. the history of dyspnea helps to differentiate the role of cardiac or pulmonary disease in the patient's symptoms. cardiac dyspnea can be from ischemic heart disease, congestive heart failure, valvular heart disease, and arrhythmias. pulmonary dyspnea can come from pulmonary vascular disease, restrictive lung disease, and obstructive lung disease. in some patients both cardiac and pulmonary issues may be present, and in all cases physical conditioning needs to be assessed. because psychological factors are also important, patients also should be screened for anxiety and depression. finally, an assessment for hypoxemia should be done. see table . for a list of common causes of dyspnea. chest pain. chest pain and tightness is not only a mark of coronary insufficiency but can also be seen with valvular heart disease or arrhythmia. assessing the duration, quality, provocation, location of the pain, and any ameliorating factors can help to assess functional limitations and help to design the appropriate therapy program. in addition, lung conditions can cause chest pressure in both obstructive and restrictive lung disease and is very common in pulmonary vascular disease. palpitations. symptoms of palpitations can be indicative of serious arrhythmias. syncope. cardiac syncope is usually abrupt with no warning or only a brief warning (with the patient feeling as if he or she were about to pass out) and can be caused by aortic stenosis, idiopathic hypertrophic subaortic stenosis, primary pulmonary hypertension, hypercarbia, hypoxemia, ventricular arrhythmias, reentrant arrhythmias, high-degree atrioventricular block, or sick sinus syndrome. pulmonary syncope is often gradual in onset and can be caused by hypercarbia, hypoxemia, or pulmonary vascular disease. orthostatic syncope can be caused by autonomic dysfunction, neurologic disease, vagal stimuli, or psychological stimuli. edema. peripheral edema may be an indication of heart failure and may indicate the onset of right ventricular failure in pulmonary vascular disease. fatigue. fatigue is likely to be the most common complaint in cardiopulmonary disease and may be worsened by the presence of depression, physical exhaustion, medication side effects, and deconditioning. cough. cough can be from both cardiac and pulmonary diseases. "cardiac" cough is often nocturnal and postural, with little to no sputum production and relieved by assuming an upright position. cough is common in both restrictive and interstitial lung disease, with or without sputum production. a description of the complete and detailed physical examination of the patient with cardiopulmonary disease is beyond the scope of this chapter (see chapter ) . still, some important elements of the examination include a general survey of the patient for exophthalmos (possible thyrotoxicosis), xanthelasma (hypercholesterolemia), acrocyanosis (chronic hypoxemia), clubbing (chronic hypoxemia), ankylosis (aortic valve disease and conduction defects), down syndrome (cardiac abnormalities), myasthenia gravis, or neuromuscular disease (cardiomyopathy, conduction disease, and ventilatory failure). a good cardiopulmonary examination and history can help to prevent complications in a cardiopulmonary rehabilitation site of pathology pathophysiology program and should be done as a part of the physiatrist's initial history and physical examination. a few key cardiopulmonary examination findings are highlighted here. cardiac auscultation can indicate an atrial septal defect, a midsystolic click may indicate mitral valve prolapse, and a murmur may indicate valvular heart disease. pulmonary hypertension typically produces a heightened second heart sound, a noncompliant ventricle can be detected via an atrial gallop at the cardiac apex, and a left ventricular gallop may reveal heart failure. the pulse contour, split heart sounds, and the quality of the murmur can help to differentiate aortic sclerosis from aortic stenosis. in younger patients, pulmonary stenosis or valvular heart disease needs to be differentiated from idiopathic hypertrophic subaortic stenosis. diastolic murmurs may be mitral stenosis or pulmonary hypertension with pulmonary valve regurgitation, and continuous murmurs may be from ventricular septal or atrial septal defects. new findings or changes in findings are important as they may indicate the need for further evaluation or alterations in the program of cardiopulmonary rehabilitation. lung physical examination may have decreased breath sounds and/or barrel chest in obstructive disease, whereas interstitial lung disease may have diffuse or basilar crackles. inspiratory stridor may indicate upper airway obstruction, whereas expiratory wheezing and rhonchi can be seen with obstruction or secretions. it is also important to assess symmetry of breathing, accessory muscle use, and possible compromise to diaphragmatic function. to supervise cardiac rehabilitation, it is essential to be familiar with the normal distribution of the major arteries of the heart, the anatomy of the heart valves, and the distribution of ischemia or infarction from the coronary arteries. the heart has paired atria and ventricles, with deoxygenated venous blood entering the right atrium, traversing the right ventricle through the tricuspid valve, and entering the pulmonary artery through the pulmonic valve. oxygenated blood enters the left atrium, goes to the left ventricle through the mitral valve, and is ejected into the aorta through the aortic valve. the cardiac valves ensure unidirectional unobstructed flow of blood, with atrial contraction adding up to % to % to the total cardiac output (co). atrial contribution to blood flow is greater with increased heart rate and in conditions with decreased ventricular compliance. atrial fibrillation can cause a loss of this atrial "kick" and may contribute to cardiac dysfunction. the cardiac conduction system allows coordinated contraction of the atria and ventricles at a controlled rate. the normal heartbeat is initiated at the sinoatrial node and then travels through three atrial internodal pathways to the atrioventricular node, where conduction is delayed to cause sequential atrial and ventricular contraction. below the atrioventricular node, the signal passes into the bundle of his and divides into left and right bundles. all cardiac fibers then end in terminal branches, which excite the myocytes and cause contraction. the conduction system can be injured by myocardial infarction (mi), aging, and other conditions and can cause heart block or sick sinus syndrome. accessory pathways that bypass the atrioventricular node can be seen in wolff-parkinson-white syndrome. there are three main distributions of coronary circulation. normally the left main coronary artery divides into the left anterior descending and the circumflex arteries, whereas the right coronary artery continues on as a single vessel. right dominant circulation is seen in % of individuals, whereas left dominant circulation with the posterior descending artery arising from the left circumflex is seen in % to % of individuals. the remaining % of individuals have balanced circulation with the posterior descending artery coming from the left circumflex and right coronary arteries. cardiac myocytes extract nearly % of oxygen from the blood at all levels of activity (compared with % for brain and % for the rest of the body). cardiac myocytes prefer carbohydrates as an energy source ( %), with fatty acids making up most of the remaining %. with high oxygen extraction and coronary blood flow only during diastole, the heart is at high risk of ischemic injury, especially in the endocardium. coronary vasodilation with exercise is normally done via nitric oxide-mediated pathways and increases blood flow with exertion. the goal of most medical and surgical therapies for ischemia is to restore or preserve myocardial perfusion through vasodilation, bypass, or endovascular procedures. exercise can increase cardiac collateral circulation and improve arteriolar vasodilation and has long been known to be a primary therapy for cardiac ischemia. , , , another important issue to manage with patients with cardiac disease is fluid volume. appropriate venous return can maintain appropriate cardiac "preload," whereas fluid overload can lead to too much venous return and exacerbate heart failure. in cases of mechanical cardiac constriction, surgery can restore co, and in dilated heart failure, medical treatment aims to decrease the size of the ventricles to increase co. in refractory or end-stage disease, left ventricular assist devices (lvads) and cardiac transplantation are options. important pulmonary anatomy includes the upper and lower airways (the oropharynx, larynx, trachea, main stem bronchi, and smaller bronchi), the lung parenchyma, the chest wall, and musculature (diaphragm, accessory muscles of breathing, rib cage, and pleura). pulmonary limitations can come from abnormalities in any of these structures. the lungs also have a dual circulation with pulmonary arteries and veins, which deliver deoxygenated blood to the lungs and deliver oxygenated blood to the left atrium and intrinsic pulmonary artery circulation delivering oxygenated blood to the respiratory tree. stridor can result from upper airway obstruction from vocal cord paralysis or tumor, whereas asthma, bronchitis, or reactive airway disease may cause dyspnea from lower airway obstruction. emphysema is a result of parenchymal lung disease with a loss of alveoli leading to decreased intrinsic recoil of the lung and subsequent hyperinflation and dyspnea. in interstitial lung disease and pulmonary fibrosis, there is interstitial scarring with increased recoil and decreased ability to diffuse oxygen through the lung tissues. in some patients, both restrictive and obstructive diseases can be present with one predominant over another (cystic fibrosis and sarcoidosis). in these cases, it is important to evaluate the lung parenchyma with imaging or physiologic testing (pulmonary function tests) to assess which condition may predominate. normal breathing is regulated in the medulla oblongata by the respiratory center. respiratory signals are carried by the phrenic and other somatic nerves to the diaphragm and secondary inspiratory muscles (intercostals, sternocleidomastoids, and pectorals) and cause rhythmic breathing by generating negative pressure in the chest wall. normal exhalation is passive, resulting from the elastic recoil of the chest wall and the lung parenchyma. copd and emphysema can create the need for active exhalation, markedly increasing the work of breathing. interstitial lung disease with scarring decreases compliance of lung tissue so severely that lung volumes decrease and hypoventilation can result. any disease affecting the brain, spine, phrenic nerves, respiratory muscles, or changing the mechanical properties of the chest wall or diaphragm can affect normal respiration. , pulmonary vascular disease can result in either primary or secondary pulmonary hypertension. primary pulmonary hypertension can be idiopathic or can result from vasculitis, thromboembolic disease, or intrinsic parenchymal disease. secondary pulmonary hypertension is from vascular congestion, often a result of left heart failure. secondary pulmonary hypertension can lead to intrinsic vascular compromise if the condition is chronic. chronic hypoxemia may also create pulmonary hypertension in individuals with obesity, obstructive sleep apnea, or high-altitude exposure through a mechanism of pulmonary vascular constriction. chronic hypoxemia can lead to vascular intimal hypertrophy with resultant fixed pulmonary vascular resistance and pulmonary hypertension. aerobic capacity (vo max ) is the measure of the work capacity of an individual and is expressed as the oxygen consumed by the individual (liters of oxygen per minute or milliliters of oxygen per kilogram per minute). oxygen consumption (vo ) increases linearly with workload, up to the vo max , where it reaches a plateau. maximal exercise capacity assessment can assist in rating disability and planning exercise and recovery programs. heart rate is a useful guide for exercise as a result of having a linear relationship to vo . maximum heart rate is best determined by testing and decreases with age. it can be estimated either by the karvonen equation or by the equation heart rate = − age. physical conditioning can alter the slope of the relationship of heart rate and vo with improved conditioning lowering the slope (less heart rate increase for a given vo ). a limitation to using heart rate can be the alteration of heart rate response in the setting of medications that alter vagal and sympathetic tone. stroke volume (sv) is the volume of blood ejected with a left ventricular contraction. maximal sv can be increased with exercise and is sensitive to postural changes (least increases in supine), with the greatest increase during early exercise. normally sv increases in a curvilinear manner, achieving maximum at approximately % of vo max . sv declines with advancing age, with decreased cardiac compliance, after mi, and in heart failure. co is the product of the heart rate and sv. it has a linear relationship with work and is the primary determinant of vo max . at maximum exercise, left ventricular ejection fraction (lvef), and thus co, is greater in the upright position compared with the supine position. , myocardial oxygen consumption (mvo ) is the vo of the heart muscle increasing in proportion to workload. when the mvo exceeds the maximum coronary artery oxygen delivery, an individual will have myocardial ischemia and angina. the rate pressure product (rpp) = [heart rate × systolic blood pressure (sbp)]/ and has a direct relationship to the mvo . another consideration is that arm exercise, isometric exercise, and exertion in the cold, extreme heat, after eating, and after smoking all have a higher mvo for a given mvo . supine exercises have a higher mvo at low intensity and a lower mvo at high intensity compared with erect exercises. basic static lung volumes and dynamic responses to exercise are helpful in the assessment of exercise capacity in individuals with lung disease. although complete pulmonary function evaluation is beyond the scope of this chapter, some important values include: total lung capacity (tlc): volume of air in the lungs at full inspiration vital capacity (vc): volume of air between full inspiration and full expiration the best evaluation of the capacity to exercise in cardiac and pulmonary conditions is with a cardiopulmonary exercise test (cpet). the cpet yields diagnostic, prognostic, and exercise prescription guidance in patients with cardiopulmonary disease. the interpretation of pulmonary exercise testing in a number of conditions is shown in table . . physical exercise that increases the cardiopulmonary capacity (vo max ) allows for aerobic training. all aerobic training prescriptions must include four components: intensity, duration, frequency, and specificity. intensity. how hard an exercise is. it can be prescribed by a target heart rate, metabolic (met) level, or intensity (wattage). usual intensity target for cardiac primary prevention is a heart rate of % to % of the predicted maximum heart rate/peak heart rate from the exercise tolerance test (ett). for secondary prevention in patients with known cardiopulmonary disease, exercise should be at a safe level at % or more of the maximum heart rate to achieve a training effect. duration. how long a given bout of exercise is. usual cardiopulmonary conditioning requires -to -minute sessions and should have a -to -minute warm-up and cool-down period. the lower the intensity of an exercise, the longer the duration will need to be to achieve a similar training effect. frequency. how often exercise is performed over a fixed time period (usually a week). moderate-intensity training programs should be done at least three times per week, and low-intensity programs should be done five times per week. specificity. the activity to be done in exercise. training benefits are specifically related to the activities performed. thus elliptical exercise is not as beneficial for walking as treadmill training. specificity in prescription should be altered to adapt to the needs of each patient. for a patient with spinal cord injury, upper arm ergometry would be more functional, and for a patient with severe leg arthritis, cycle ergometry would be better than a treadmill. the law of specificity of conditioning should be remembered when designing a cardiopulmonary conditioning program. the benefits of aerobic training include the following: aerobic capacity: maximum capacity increases with training. resting vo is stable as is the vo at a given workload. the changes are specific to the trained muscles. cardiac output: maximum co increases, whereas resting co is stable. resting sv increases with a corresponding decrease in resting heart rate. heart rate: heart rate is lower at rest and at any given workload, whereas maximum heart rate is unchanged. the lower heart rate at rest and submaximal exercise causes a lower mvo with normal activity. stroke volume: sv increases at rest and at all levels of exercise. co is thus maintained at a lower heart rate and causes a lower rpp for a given level of exertion. myocardial oxygen capacity: after training, maximum mvo does not usually change but is less at a given workload. this reduces episodes of angina and increases safety for moderate activity. mvo can also increase after pharmacologic treatments or revascularization procedures. peripheral resistance: exercise training decreases peripheral vascular resistance (pvr) by reducing "afterload" through lowering arterial and arteriolar tone. the reduction in pvr results in a lower rpp and a lower mvo at a given workload and at rest. minute ventilation: with improved conditioning, individuals will require a lower vo and thus a lower minute ventilation for a given activity. for patients with pulmonary and cardiac disease, this can lead to a large reduction in dyspnea. tidal volume: exercise can lead to a higher tv on exertion, with a subsequent decrease in respiratory rate and decreased dyspnea. respiratory rate: as tv is improved, respiratory rate will be lower for a given minute ventilation, decreasing dyspnea. the application of basic physiologic principles to the design of cardiopulmonary rehabilitation programs can improve function, decrease symptoms, and improve outcomes for patients with cardiopulmonary disease. the prime effect of cardiac conditioning is in reduction of cardiac risk and improved cardiac conditioning. reduction of cardiac risk has been well established since , when pooled data from randomized studies of exercise in patients following acute mi demonstrated a % to % reduction in all-cause mortality, fatal mi, and cardiac mortality in a -year follow-up study. these benefits of cardiac rehabilitation apply across populations, including older adults, women, and patients after bypass. similar benefits have also been shown for pulmonary rehabilitation in copd with decreased hospitalizations, improved function, and improved quality of life, , , , and new studies are showing that interstitial disease and pulmonary vascular disease can also benefit from exercise. , , , , pulmonary rehabilitation patients with pulmonary disease demonstrate three main impairments: ( ) obstructive lung disease, ( ) restrictive lung disease, and ( ) pulmonary vascular disease. often more than one type of limitation may be present in a given patient and will increase the complexity of their condition. understanding the underlying physiology can assist in the design of a specific exercise program for an individual patient. for primary pulmonary disease, it is essential to know if the patient has primarily an obstructive or restrictive condition. obstructive lung disease is marked by an inability to exhale resulting from either upper airway or large airway disease (sleep apnea, tracheomalacia, vocal cord disease, asthma, and bronchitis) or as a result of lower airway disease from either secretions or lung parenchymal disease (emphysema and bronchiectasis). obstruction can also be exacerbated by a component of acute obstruction (asthma) combined with a chronic condition (copd). the hallmark of severe copd is carbon dioxide retention and active exhalation. medical treatments are limited for copd, with steroids and bronchodilators offering incomplete relief. lung reduction surgery is appropriate only in selected individuals, and transplant is only for end-stage disease. for all levels of obstructive disease, pulmonary rehabilitation is appropriate, and in the "gold" recommendations for treatment of copd, pulmonary rehabilitation is recommended for all patients with moderate to severe disease. , in restrictive lung disease, the primary limitations are low tvs from an inability to expand the chest wall (extrinsic restriction) or from very noncompliant lung tissue (intrinsic restriction). in extrinsic restrictive disease (neuromuscular disease, paralysis, and kyphoscoliosis), the parenchyma of the lung is normal and gas exchange is preserved, meaning that treatment is usually respiratory muscle training and mechanical ventilatory support as needed. with intrinsic restrictive lung diseases (pulmonary fibrosis, sarcoidosis, etc.), there may be a profound associated hypoxemia from severely decreased diffusion capacity of scarred lung tissue. patients with parenchymal restrictive disease classically have severe hypoxemia and may need highflow supplemental oxygen. patients with end-stage intrinsic restrictive disease can have ventilatory failure with hypercarbia and hypoxemia and may be refractory to ventilatory support, and lung transplantation is then often the only remaining treatment option. table . shows some of the lung pathologies and effects on inspiratory reserve and rv (obstructive diseases) and the effects of various conditions on lung compliance (restrictive diseases). finally, patients with pulmonary vascular disease have a similar presentation in many ways to patients with heart failure, and in the end stages of the disease, right ventricular heart failure is a major part of the condition and leads to excess mortality and morbidity. rehabilitation is focused on a program that resembles exercise for patients with heart failure, with the addition of close monitoring of oxygen saturation and the use of appropriate levels of supplemental oxygen to prevent hypoxemia. for patients with either intrinsic restrictive or obstructive disease, pulmonary rehabilitation is an important treatment to consider and should be offered for patients whether or not they have their pulmonary condition as a primary or a secondary disability. a brief overview of pulmonary rehabilitation programs for primary pulmonary disease is shown in table . . an understanding of abnormal cardiac physiology in disease is necessary for appropriate cardiac rehabilitation. in general, cardiac limitation is caused by either decreased co, or ischemic disease, or a combination of these. ischemia causes the myocardium to have lower contractility and lower compliance reducing sv. valvular heart disease lowers maximum co through stenotic valves (e.g., aortic or mitral stenosis) or valvular regurgitation (e.g., aortic or mitral insufficiency). finally, heart failure is a state of low co, often as a result of low sv, and is associated with a reduction of vo max , increased resting heart rate, and often a greater mvo for a given vo . arrhythmias decrease co by lowering sv and increasing heart rates. for atrial arrhythmias, the mechanism can be by a loss of atrial ventricular filling (atrial "kick") during atrial fibrillation or supraventricular tachycardias, or from high heart rates without atrial coordination as in ventricular tachycardias and ventricular bigeminy. surgical treatments for heart disease either restore coronary circulation (e.g., bypass and intravascular procedures) or restore normal anatomy (e.g., valve replacement). surgical treatment for heart failure can include lvads or transplantation. , medical treatment for heart disease either aims to improve coronary circulation for ischemia or works to improve blood flow and restore co for heart failure by lowering afterload, reducing fluid overload, and increasing inotropy. although medical treatment of ventricular arrhythmias has been limited, implantable defibrillators and pacemakers have been very successful. severe end-stage heart disease of all types may require cardiac transplantation or an lvad. in all of these conditions and treatments, cardiac rehabilitation has an important role to play. some basic concepts to remember include that patients before transplantation are similar to patients with heart failure, whereas patients after transplantation have several physiologic changes that are unique, including high resting heart rate, limited increase in sv, and peak heart rate with exercise. the basic principles of cardiac rehabilitation are discussed as follows. cardiac rehabilitation is either primary prevention, which includes risk factor modification and education before a cardiac event, or secondary prevention, which is cardiac rehabilitation after the onset of cardiac disease including both exercise and risk factor modification. primary prevention is usually performed in primary care settings rather than a rehabilitation setting. the focus is on the reduction of cardiac risk factors with a combination of education and exercise for patients in the community. primary prevention can have a profound effect on the rate of cardiac disease with a decrease in obesity, blood pressure, and lipid profiles. , , ideally, behavior modification should begin in childhood with the establishment of healthy behavior and then maintained throughout life. because populations who are disabled are generally sedentary and may have other risk factors, primary prevention should be an important component of the care of the disabled and should include management of hypertension and lipids along with encouraging exercise and consideration of antiplatelet agents. these are all cost-effective approaches and can decrease mortality and morbidity on a population-based scale, in addition to the individual benefits. , , after an episode of cardiac disease, it is essential to have secondary risk factor modification, which includes all of the features of primary prevention programs discussed earlier. in addition, disease-specific education and formal exercise is a part of the secondary prevention program. in both cardiac and pulmonary disease, smoking cessation is crucial as part of both primary and secondary prevention programs. , , rehabilitation programs for patients with pulmonary disease are similar to cardiac rehabilitation programs. after severe acute exacerbations, some patients can benefit from a short acute inpatient rehabilitation, but the majority of pulmonary rehabilitation is done in an outpatient setting. for patients who are in an intensive care setting, early mobilization programs are now being used to limit debility in these vulnerable patients. , , outpatient pulmonary rehabilitation programs also have primary prevention for pulmonary disease with smoking prevention and cessation, occupational safety, and prevention of exposure to environmental and infectious agents. secondary pulmonary prevention involves medication adherence and education, smoking cessation, supplemental oxygen use and education, and environmental modification for known environmental triggers. , for patients with ventilatory failure that cannot be supported with noninvasive ventilation, lung transplant may become necessary. rehabilitation before transplantation is focused on both the underlying condition and transplant-specific education, whereas rehabilitation after transplantation includes education and restoration of muscle strength, which is impaired from the medical regimen for patients after transplantation. the standard model for cardiac rehabilitation after mi was first described by wenger et al. in . because revascularization is currently common and infarcts are smaller than in the past, there loss of inspiratory reserve have been modifications to the classical program with a reduction to three phases, eliminating the classical stage recovery phase. a modern acute phase mobilization program is illustrated in table . . the exception to bypassing the recovery phase for cardiac rehabilitation comes for surgical patients with sternotomy who may require recovery from their surgery before starting the training phase of rehabilitation. in summary, phase rehabilitation is the acute phase in hospital immediately following a cardiac event and ends at discharge. phase is an outpatient training phase, with secondary prevention, intense education, and aerobic conditioning. phase is the most difficult, the maintenance phase in which patients seek to achieve continued aerobic exercise and maintenance of lifestyle modifications. risk factor modification is performed at all phases. this model is similar for patients with pulmonary disease. for patients with cardiopulmonary disease who are not hospitalized, the goal is essentially phase and phase for all patients at the time of diagnosis. a more detailed description of each of the phases follows. the basics of the phase program are illustrated in table . . education about cardiopulmonary risk factor modification is introduced at the time of acute hospitalization. for patients with cardiac disease, all acute mobilizations should be done with cardiac monitoring with appropriate supervision by trained therapists or nurses. a post-mi heart rate increase with activity should be kept to within beats/min of baseline and sbp kept within mm hg of baseline. a decrease of mm hg or more is indicative of further medical issues, and exercise should be halted. the target intensity at the end of the phase i program exercise is to a level of four mets, covering most of the daily activities patients may perform at home after discharge. for patients with pulmonary disease, similar phase goals exist and there is new emphasis on early mobilization in the intensive care unit (icu) to prevent frailty and deconditioning. patients are aggressively mobilized, some while still on the ventilator. innovations, including extracorporeal membrane oxygenation, are also now allowing more aggressive mobilization of patients because sedation is less, and patients may maintain better nutritional status. these patients with pulmonary disease should be enrolled in outpatient pulmonary programs to maintain their early gains and complete a full program of education and exercise. to distinguish between patients who have a rapid recovery after their cardiopulmonary event (pure phase ) and those patients who require either acute or subacute rehabilitation treatment before discharge home, the designation of phase b rehabilitation has been established. with advanced age or substantial comorbidities or other disabilities that make mobilization more difficult, many rehabilitation specialists will care for these patients in phase b. the guidelines for exercise are the same as they are for patients in phase but with a longer recovery period extending their hospitalized care to an acute or subacute rehabilitation setting before discharge. classically, phase cardiopulmonary rehabilitation starts after a symptom limited full-level ett for patients with cardiac disease or a cpet for patients with complex pulmonary disease. this allows for setting target heart rates and target exercise intensity from the exercise. a target heart rate of % of the maximum heart rate on an ett or a cpet is generally regarded as safe for patients at low risk. exercise intensity targets are lower for patients at higher risk or those with underlying conditions. in patients with life-threatening arrhythmias or chest pain, target heart rates are chosen that are below notable end points. because hypoxemia can add risk and limits participation with exercise, it is important to provide supplemental oxygen as needed (up to a rate of l/ min as needed) to maintain saturation greater than % for safe exercise. a target heart rate of % to % of maximum is safe and effective in a regular exercise program for patients at higher risk and, with target rates as low as %, still provides a training benefit. monitoring also needs to be customized to accommodate the underlying risk profile. classically, a cardiopulmonary training program is three sessions per week for to weeks. cardiac rehabilitation is covered by most insurance plans, but the major limitation is a lack of referral and/or a lack of facilities in many areas. creative and innovative care delivery programs have been developed to increase access and include home programs (patients at low risk), telemedicine programs, and community-based programs in nonmedical facilities. because training continues after the -to -week period, it is important for patient self-efficacy that they learn to perform self-monitoring following the guidelines presented in standard references. , patients need to learn to begin exercise with a stretching session, then a warm-up session, a period of training exercise at designated intensity, followed by a cool-down period. the principles of specificity of training need to be remembered because training benefits generally are seen in the specific muscles exercised. although the maintenance phase of cardiopulmonary rehabilitation is the most important part of the program, it often receives the least attention. the benefits of a phase program can be lost in as little time as a few weeks if a patient ceases to exercise. because of this, patient education of the importance of making exercise a part of their new health habits has to be emphasized and the patient needs to integrate exercise as a part of a healthy lifestyle. to maintain capacity, patients should perform moderate exercise at the target intensity learned in their rehabilitation program for at least minutes three times a week. with low-level exercise, the frequency has to be increased to five times a week for maintenance of gains. although telemetry monitoring is usually not used with patients with cardiac disease, patients with pulmonary disease can benefit from the use of home pulse oximetry and should be taught to adjust their supplemental oxygen as needed with exercise to maintain adequate oxygenation. cardiac rehabilitation for angina aims to lower heart rate at rest and with given levels of activity to decrease angina by improving fitness. exercise benefits for patients with angina include improved peripheral efficiency and improved coronary artery collateralization. cardiac rehabilitation after coronary artery bypass grafting (cabg) emphasizes secondary prevention aims to improve conditioning and fitness. for patients with low ejection fractions and heart failure, closer telemetry monitoring should be done. if a patient had a sternotomy, arm exercises will have to be limited until sternal healing occurs, usually at approximately weeks after surgery. patients who have had percutaneous interventions usually pursue the program immediately and it is similar to the program after cabg. because most patients after cardiac transplantation have severe heart failure and debility before transplantation, involvement in a heart failure pretransplant program can help to limit deconditioning and help to treat depression and anxiety. heart transplantation usually improves cardiac function; therefore a posttransplant program can focus on conditioning, education, and secondary prevention. an added feature is that many patients after transplantation may have vascular and neurologic complications, which may mean a phase b program is needed before starting the phase outpatient program. this is often done in either acute or subacute rehabilitation settings. remembering the alterations of cardiac physiology in the patients after transplantation is important. transplanted hearts are denervated and have no direct sympathetic or vagal central regulation. in many patients, the loss of vagal inhibition creates a resting tachycardia of to beats/min. by contrast, because there is a loss sympathetic innervation, the chronotropic response to exercise is in response to circulating catecholamines, leading to a delayed and blunted heart rate response to exercise. posttransplant, peak heart rates are usually % to % lower than in matched controls. other cardiovascular effects that are seen include resting hypertension from the renal effects of calcineurin inhibitors (e.g., cyclosporine and tacrolimus) and prednisone, along with diastolic dysfunction in some patients. combined, these effects usually reduce maximum work output and maximum oxygen by approximately one-third compared with age-matched individuals. of interest, despite no denervation of the heart, similar decreases in exercise capacity are also seen in patients after lung transplantation. with exercise, patients after transplantation have a lower work capacity, reduced co, lower peak heart rate, and lower oxygen uptake and a higher resting heart rate and sbp than normal individuals. in addition, resting and exertional diastolic blood pressures are usually higher for patients after heart transplantation. , the net effect of these alterations in exercise response is higher than normal perceived exertion, minute ventilation, and ventilatory equivalent for oxygen at submaximal exercise levels. the focus of a cardiopulmonary rehabilitation program after transplantation is on conditioning and education. target intensity for aerobic exercise is usually approximately % to % of peak effort for to minutes three to five times weekly. intensity can be regulated with rating of perceived exertion target at to on the borg scale, approximately to on the modified borg scale, with the goal being to consistently increase the level of activity. education focuses on learning the complicated medical regimen and vocational and psychological needs. for patients after cardiac transplantation, a program of rehabilitation can help to assist them to improve work output and exercise tolerance, with some patients able to participate in competitive athletics. , , cardiomyopathy fortunately, cardiac rehabilitation for heart failure is currently covered by insurance plans, since medicare regulations started to cover rehabilitation for heart failure in march of ( c.f.r. § . (b) ( )(vii)). an important consideration for heart failure rehabilitation is the increased risk of complications such as sudden death, depression, and chronic cardiac disability. closer monitoring of telemetry and vital signs is also needed because some patients with heart failure have inconsistent responses to exercise with increased fatigue, possible exertional hypotension, and syncope. most patients also exhibit low endurance and chronic fatigue as a result of their low-exercise capacity. however, a positive effect can be realized in their fatigue and function with even a small improvement in vo . these changes in capacity can lead to a marked improvement in quality of life and may help patients with heart failure to continue to live independently. because of the increased risk for complications in patients with heart failure, a graded ett is helpful before starting a cardiac rehabilitation program. long warm-up and cool-down periods with gentle exercise at a limited workload helps to compensate for an impaired ability to generate a dynamic exercise response, and dynamic exercise is preferred to isometric exercise because isometric exercise can lead to an increase in diastolic pressure and cardiac afterload. heart rate targets are usually set beats/min less than any notable end point found with cardiopulmonary exercise testing. cardiac rehabilitation begins with cardiac monitoring especially when severe left ventricular dysfunction is present. once the patient has demonstrated stability with an exercise program and has learned how to self-monitor, the patient should be taught a self-monitored program. education of patients with heart failure also includes doing a daily body weight (to observe for fluid accumulation) and monitoring their blood pressure and heart rate responses to exercise. patients who are on pharmacologic inotropic support or left ventricular mechanical support for end-stage heart failure can also exercise in a cardiac rehabilitation program with similar precautions to other patients with congestive heart failure. rehabilitation after an lvad usually follows a classical postsurgical course and may include phase and phase b rehabilitation followed by phase and phase programs. patients with an lvad seen in acute and subacute units require staff training, close cooperation with the lvad team, and familiarity with the devices that are used locally. because an lvad often restores a reasonable co, exercise tolerance is often only limited by the peak flow of the device. in addition to normal secondary prevention education, lvad-specific family and patient education are also essential parts of post-lvad rehabilitation. cardiac rehabilitation for valvular heart disease resembles the program for cardiac heart failure. postsurgical considerations are the same as for cabg, with the added consideration of anticoagulation for patients with mechanical valves. because anticoagulation increases the risk of hemarthrosis and bruising, patients need to avoid impact exercises and need education regarding injury avoidance. the overall training program is similar to that discussed for the patient post-cabg. an essential consideration for patients with cardiac arrhythmias is the need for telemetry monitoring with increases in intensity of exercise and new exercises. patients at high risk can benefit from an automatic implantable cardiac defibrillator (aicd), which may offer protection form ventricular arrhythmias. cardiac rehabilitation for patients with aicd needs to be done at intensities that avoid the heart rates at which the device is set to respond to ventricular tachyarrhythmias. an exercise stress test can help to set appropriate target heart rates for an exercise program. in addition to secondary prevention and education, aicd-specific education and emotional support are important to include in the rehabilitation program. an emerging area of scientific exploration is using the cardiac rehabilitation model for stroke recovery and prevention. , this is a logical extension of the effectiveness of the cardiac rehabilitation model in reducing known coronary artery disease risk factors in regular participants. because the risk factors for coronary artery disease are the same as the risk factors for stroke, it is logical to apply the same group exercise-based behavior modification model to a similar at-risk population. participants must be screened carefully for balance impairments and muscle strength asymmetry that could increase the risk of falling in a group exercise setting. asymmetric muscle fatigue must be scrupulously monitored and can contribute to degradation of gait or exercise form as exercise progresses. subtle cognitive deficits may require close observation until it is determined that the stroke patient care set machine intensity levels accurately and independently. meticulous blood pressure control must be maintained and may require more frequent measurements than regularly collected in cardiac patients. any questions regarding safe blood pressure range should be directed to the referring neurologist. this is analogous to target heart-rate range directed by referring cardiologist when indicated. a new and exciting area of emerging research is the conditioning of cardiac patients with high-intensity interval training (hiit). , following appropriate screening and evaluation, patients are enrolled in a traditional cardiac rehabilitation program. once they are familiar with the execution of exercise intervals, an accurate setup of exercise machines trials of hiit can be introduced. patients are instructed to exercise at high intensity (> % of vo max ) for short or ultra-short intervals followed by low-intensity or rest intervals. it is recommended to start hiit training on seated machines to reduce likelihood of falls on standing machines due to loss of balance, distraction during interval initiation or termination, and dizziness from blood pressure fluctuations. meticulous blood pressure and telemetry monitoring should take place until the practitioner is confident they have established an individualized baseline response for each patient. additional considerations are the potential for exercise-induced hypoglycemia-an expected and physiologically beneficial outcome. symptomatic hypoglycemia can be quickly treated with any source of oral glucose. it is important to reassure cardiac patients that hypoglycemic symptoms are not the onset of cardiac ischemia to reduce negative associations with exercise. hiit has been shown to provide significant conditioning benefits in the cardiac rehabilitation population with medically supervised selection of candidates. demand for cardiac rehabilitation services is unable to keep up with the current and projected future supply throughout the world leading to innovative approaches to delivery included home-based and "hybrid" programs. , , numerous innovative models are emerging in both cardiac and pulmonary rehabilitation. all models are initiated with an in-person evaluation and followed by various technology driven follow-up contacts. the telerehabilitation approach allows one-on-one live supervised sessions or weekly coaching via video link. a less technology-driven approach includes weekly phone calls and reviews of self-kept activity logs that may also include additional communication via text message. hybrid programs begin as a traditional facility-based, telemetry-monitored exercise program followed by rapid transition to home-based continuation. most hybrid programs do not extend telemetry monitoring into the home setting, although with technologic advances this has been demonstrated in at least two foreign studies. , in addition, current technology allows physiologic data to be collected and relayed to the provider via wearable technology and app-or web-based interfaces. , , regular follow-up appointments, whether in person or via video link or phone call, are recommended to ensure continuity of care. it has been demonstrated that homebased programs are not inferior to facility-based programs, but the medical literature is still developing regarding conclusions of superiority. home-based cardiac rehabilitation is an emerging and exciting trend. there is a wide variety of solutions being explored throughout the international cardiac rehabilitation community. exercise has been repeatedly demonstrated to be safe for patients in the cardiac rehabilitation population-extrapolation from this well-accepted practice is leading to new delivery methods to an everexpanding population. , , , rehabilitation for patients with copd is the standard for pulmonary rehabilitation. goals of a pulmonary rehabilitation program include improving disease management and exercise capacity. because pulmonary rehabilitation does not improve lung function, the goal of the rehabilitation program is to improve peripheral efficiency and decrease dyspnea. energy conservation education (how to do a given activity at a lower level of exertion), anxiety reduction, and improved endurance all contribute to improved function and decreased dyspnea. longer-duration exercise of moderate intensity is often used rather than high-intensity exercise. investigations have started to evaluate a possible role for hiit for patients with copd, but this has not yet been proven to be more effective than the standard training program. because isometric exercises increase intrathoracic pressures, they should be avoided in patients with copd. appropriate supplemental oxygen should be given to maintain saturation greater than %, with education to lower supplemental oxygen after exercise back to baseline levels to prevent resting hypercarbia. patients with copd generally have relatively modest oxygen needs and can often maintain their oxygen saturation levels with to l of oxygen via a nasal cannula. bilevel ventilation may have a role for patients with sleep apnea or ventilatory failure, and education for these patients should include the proper use of this modality. for patients being considered for lung volume reduction surgery, pulmonary rehabilitation is considered essential both to qualify for the surgery and after surgery to ensure adequate outcomes. airway clearance and chest physical therapy has a role in the pulmonary rehabilitation of patients with substantial secretions. a combination of external percussion devices, vibration devices, and inhaled saline in combination with cough training and huffing may help to mobilize secretions. it is also important to include family training and education about inhaled medications, supplemental oxygen use, and management of equipment. the basics of a program of pulmonary rehabilitation for interstitial lung disease are the same as for copd. an essential issue for patients with interstitial lung disease is often profound hypoxemia that requires high-flow oxygen with exercise to maintain adequate saturation for activity. it is essential in this group of patients to avoid chronic hypoxemia to prevent secondary pulmonary hypertension because the coexistence of interstitial lung disease and pulmonary hypertension can lead to a markedly decreased life expectancy. exercise intensity is often limited in patients with interstitial lung disease by oxygenation rather than dyspnea, and airway secretions are usually not an important issue. for some individuals with severe end-stage disease, there may be ventilatory failure with hypercarbia, but in those patients, rehabilitation may no longer be possible. because interstitial lung disease is often progressive, transplant evaluation and education or end-of-life planning may be needed to permit as many patient goals as possible to be achieved. patients with pulmonary hypertension have similar limitations as patients with heart failure and share many similar precautions. effective medical treatment for pulmonary hypertension has made a once-fatal condition into a chronic disease for many patients. patients with pulmonary hypertension currently have a much longer life expectancy, and improved functional status is essential for maintaining an active life. major concerns for pulmonary rehabilitation are preventing debility and improving dyspnea. because hypoxemia can worsen pulmonary hypertension, it is important to maintain oxygen saturation with exercise, and cardiac monitoring may be needed for patients with a history of arrhythmias and right ventricular failure. education for this group of patients should include a review of their vasodilating medications and supplemental oxygen use. intravenous and continuous subcutaneous vasodilator infusion is appropriate for a pulmonary rehabilitation program, but, similar to patients with heart failure, there may need to be long warm-up and cool-down periods. for patients with severe pulmonary vascular disease, the program should start with moderate-to low-level exercise. definitive research of the efficacy and safety of pulmonary rehabilitation for patients with pulmonary hypertension is still ongoing. for alert patients on either invasive or noninvasive ventilation for ventilatory failure, a program of pulmonary rehabilitation can help to increase mobility and prevent complications. exercise programs for patients on nocturnal or intermittent ventilatory support aim to improve efficiency and decrease fatigue while off the ventilator. the details of ventilatory support for patients requiring noninvasive ventilation is beyond the scope of this chapter. as the population ages and more patients survive a disabling condition, there is an increase in patients with both physical disability and cardiopulmonary disease. an issue for cardiac rehabilitation for patients with dual disability is the impaired mobility that can impair both evaluation and participation in a rehabilitation program. individuals who are disabled tend to have lower activity levels, which puts them at increased risk of cardiac and pulmonary disease and may present obstacles for a standard rehabilitation program for a person who is newly disabled and who has preexisting cardiopulmonary limitations. for new-onset cardiac or pulmonary disease, cardiopulmonary rehabilitation is just as important and needs to be considered. cardiopulmonary primary and secondary prevention also overlaps with the education needed for stroke and peripheral vascular disease and is especially important for patients with physical disabilities because they are often more sedentary with a higher prevalence of obesity and deconditioning. finally, because mobility in individuals who are disabled requires greater energy expenditure, compromised work capacity from cardiopulmonary disease may impose an even greater degree of disability on an individual who is disabled than an individual who is able bodied. cardiopulmonary exercise prescription for individuals who are disabled has to be adapted for the individual disabilities that the patient has. individuals who have a lower extremity impairment resulting from neurologic or orthopedic conditions can perform upper extremity ergometry or use modified lower extremity exercise equipment, whereas an adapted bicycle ergometer or airdyne may be helpful for a patient with hemiplegia. the higher mvo requirements for upper extremity exercise compared with lower limb exercise should be considered to adapt the cardiac rehabilitation program for patients who are disabled. patients who are disabled also need to focus on task-specific activities while increasing their aerobic conditioning and endurance, with a goal of lowering the mvo for any given task. because of the expertise in dealing with physical disabilities and understanding the mechanics of motion, physiatrists are particularly well positioned to lead cardiopulmonary rehabilitation programs for the disabled. it is especially important when many traditional cardiac rehabilitation program teams are hesitant to work with patients who are physically disabled because of their lack of experience with physical disability. , cardiopulmonary rehabilitation is an area where physiatry is uniquely positioned to help manage the patient who is multidisabled, and the multidisciplinary approach is well suited to address the education and team management needed for successful cardiopulmonary rehabilitation. a goal for cardiopulmonary rehabilitation is to increase the access to cardiopulmonary rehabilitation to a greater number of patients, including populations who are underserved in rural and urban areas, women and minority groups, and patients with dual disabilities as they become a larger proportion of the patients seen with cardiopulmonary disease. movement is an essential part of human life and is important for the preservation of function throughout the entire life cycle. since the beginning of human history, a high degree of physical activity has been required to maintain a livable environment and to secure adequate nutrition to ensure survival. it is only following industrialization, a relatively recent event from an evolutionary point of view, that the diseases and conditions associated with inactivity and immobility began to manifest themselves throughout human societies. obesity and the resultant conditions of diabetes, hyperlipidemia, and decreased cardiopulmonary reserve have increased steadily throughout the past century. much attention is focused on the "westernization" of diet, although less attention is focused on the "westernization of physical activity levels." as physicians concerned with function, physiatrists intuitively understand the dangers of activity reduction in all settings from all causes, both medical and environmental. in fact, often physiatrists are the only physicians who have familiarity with the maintenance of function via physical activity using therapists, nurses, and family members. the knowledge of how to modify physical and social environments to maximize functional movement and overall function for their patients allows physiatrists to improve and maintain function in their patients. the physiatric focus on activities of daily living (adls) is an effort to return functional movements to an individual who is disabled, allowing them to maintain their baseline degree of physical activity required for autonomy and independent movement. the link between physical activity and cardiovascular disease has been well described since the s, , when the relationship between workplace activity levels were directly related to higher rates of cardiovascular events. it is no surprise that less physically active daytime behavior (e.g., mail sorters vs. mail deliverers) affected the development of cardiovascular disease. a more interesting finding is the strength of this association; primarily seated workers developed almost twice the rate of cardiovascular disease. more research investigating and elucidating the cellular biology of inactivity needs to be done until this area of physiology is as well understood as exercise physiology. currently, the global workforce is becoming more sedentary in numerous sectors as desk-based work responsibilities dominate the work day and after-hours couch-based recreational activities, including home theaters, media centers, and ubiquitous social media, create prolonged voluntary immobilization after work. in the united states the amount of daytime sedentary hours is high, as revealed by data from the national health and nutrition examination survey (nhanes) database, which showed that . % of waking hours in the population studied was spent during sedentary activities, with late adolescents and older individuals being the most sedentary. rising "sedentarism" is not only an american phenomenon. in australia a large population-based study found that sedentary behavior (television viewing time) was positively associated with abnormal glucose metabolism and metabolic syndrome. , more concerning is that these associations were preserved even when controlling for what would be considered active individuals who participated in sustained and moderate-intensity recreational activities. prolonged physical inactivity appears to be a unique risk factor for maladaptive energy metabolism. in the future it is possible that number of hours spent sitting will be recognized as a risk factor for the development of cardiovascular disease. in the context of this discussion, these observational data support the hypothesis that the physiology of inactivity is a risk factor for poor health outcomes in numerous settings. this supports the idea that there should be a paradigm shift in how all physicians view physical inactivity in their patients, especially in the hospital setting where the physiatrist is in a key position to increase patient mobility. in addition to increased cardiovascular risk, there are numerous associations between inactivity and poor health outcomes, including the metabolic syndrome, deep vein thrombosis, obesity, and serum insulin levels. although there have not been studies to elucidate the molecular biology linking prolonged sitting to metabolic syndrome, epidemiologic data are compelling. the metabolic syndrome is the presence of three out of five of the following findings: central obesity, elevated blood pressure, low serum high-density lipoprotein cholesterol, high triglycerides, and elevated fasting glucose. patients with this constellation of findings are at increased risk for the development of cardiovascular disease and diabetes. in recent years, the recognition of the metabolic syndrome and its prevention has produced a rich literature specific to this syndrome. prolonged inactivity (sitting) more than doubles the risk for development of metabolic syndrome. development of the metabolic syndrome has been shown to increase with each additional hour of sedentary television viewing, as opposed to having television in the background during other household activities. the correlation with prolonged sitting and development of deep vein thrombosis has been well described. this can occur even in active individuals who are immobile for prolonged periods of time. case reports from varied settings have reported seated individuals who developed deep vein thrombosis since the s. these have included observations from air raid shelters, sitting in theater, sitting on extended airplane flights, and even prolonged sitting during video game playing. , presumed causes include rheologic changes and hemoconcentration. , obesity the relationship between physical activity and body mass index is supported by the medical and epidemiologic literature. in varied populations, pediatric, adult, or older adult individuals who have a lower level of baseline physical activity generally have a higher rate of obesity. sedentary behavior is a reversible cause of obesity in all populations, , , and improvement of physical activity levels should begin with school-aged children. , this is especially true because there is evidence that an obese child has a significantly greater likelihood of continuing life as an obese adult. , even high levels of physical activity in athletic adults may not be able to compensate for the deleterious effects of sedentary behavior. insulin resistance is a component of the metabolic syndrome, and thus it is reasonable to assume that an association would exist between amount of insulin present and sedentary behavior. reduced leisure time physical activity levels are associated with higher levels of insulin at baseline. numerous studies have demonstrated the relationship between prolonged sitting, obesity, and the development of type diabetes, an insulin resistance state. more interestingly, however, is the suggestion that insulin levels are elevated in individuals who have prolonged sitting, even in the presence of regular exercise. the frailty syndrome is a recognized syndrome of decreased ability to adapt to stressors accompanied by reduced physiologic reserves and reduced energy metabolism. the frailty syndrome has been reported to vary widely depending on the population studied, ranging from % to %. the frailty syndrome is clearly associated with advanced age and becomes more prevalent in older groups studied; however, it is considered to be a separate syndrome and not a variant of normal aging. if it were considered the end result of normal aging, it would be reasonable to expect all individuals to acquire the frailty syndrome if they lived long enough, which they do not. the potential for confusion exists when the descriptor "frail" is confused with the frailty syndrome. a universally accepted definition or set of variables to diagnose the frailty syndrome has not yet been agreed upon; however, the conversation in the medical literature is ongoing and evolving. substantial opportunities exist for physiatric contribution to this discussion because the frailty syndrome is defined in most tools as having a strong functional component. a true consensus on the definition of the frailty syndrome has yet to be agreed upon internationally; however, the following findings are discussed in the medical literature as possible variables in evaluating individuals thought to be at risk for the frailty syndrome: there are numerous frailty screening tools being used in a variety of settings. two of the most commonly cited tools in the medical literature include the fried frailty phenotype and the "rockwood indices" from the canadian study of health. these measurement tools can be of use to physiatrists depending on the practice setting in which they are used. fried and colleagues at johns hopkins university developed a screening tool that identifies frailty based on a positive score in three out of five possible domains: weight loss, exhaustion, low physical activity, slow walking speed, and reduced grip strength. a positive response was assigned a score of or for each category. a positive value of was assigned when participants responded "yes" to the following question, "in the last year, have you lost more than pounds unintentionally?" because there is considerable room for self-reported bias, follow-up measurements to confirm weight loss are recommended to confirm the initial findings. two questions extracted from the center for epidemiologic studies short depression scale (ces-d ) were used as indicators of exhaustion: "i felt that everything i did was an effort" and "i could not get going." scoring was based on the strength of participants' agreement with these statements. a positive score was assigned a value of . kilocalories per week expended were calculated with a self-reported description of voluntary activities adapted from the minnesota leisure time activity questionnaire. this included questions about walking, chores, outdoor gardening, and numerous types of exercise. interpretation of the answers should be clarified to ensure that negative answers are not based on different settings (urban vs. suburban) or culture. a positive value of was assigned for participants with the lowest % of activity. measurement was the time in seconds required to walk feet. stratification by gender and height took place. the slowest % of the population studied was defined as receiving a positive score and a value of . results were stratified based on gender and body mass index with values recorded in kilograms with a standard dynamometer. participants who scored in the lowest % after adjustment for gender and body mass index were assigned a positive value of . after scores are totaled, individuals who score are considered "robust." the presence of one to two of the criteria listed earlier are considered to be "prefrail," and three or more positive criteria are considered "frail." this phenotypic description has been correlated with notable clinical outcomes that are recognized as important in the geriatric population including falls, hospitalizations, and mortality. this clinical applicability and relevance to important measurable outcomes has made the fried frailty measure a popular tool in clinical research. there are no laboratory tests or psychosocial components taken into account when determining a score with this scale. using these criteria, fried identified individuals who met the criteria for the frailty syndrome in % of the participants included in the cardiovascular health study, % of the participants older than years of age, and % of the women's health and aging studies. the "rockwood index," also known as the canadian study of health and aging (csha) frailty index, and the clinical frailty scale were both derived from the csha dataset, a prospective cohort of more than , participants. both of these scales have been widely used in medical studies. the -item csha frailty index is driven by clinical judgment. this is a detailed tool in which clinical deficits are scored based on a -item index. the items include self-reported functional activities, mood, and motor symptoms, as well as signs and symptoms derived from medical history and physical examination. each deficit is assigned a value between and . to give a total score, which is then divided by . some examples of variables collected are listed in table . , with division into categories provided as a conceptual framework. this index determines clinical deficits and allows scoring based on evaluating participants for accumulation of impairments. the clinical frailty index was developed as an attempt to integrate clinician judgment into a formal and universally applicable model to evaluate frailty. the authors recognized that simply evaluating frailty based on a limited number of phenotypic variables may correlate with mortality but does not give considerably more information across all populations. although it is of benefit to identify participants at risk for increased overall mortality, it does not guide the physician to develop interventions that can address specific and more importantly correctable impairments. the frailty index gives structure to an intensive review of deficit accumulation that can serve as a starting point for interventions. these same authors recognized the utility for a shorter more clinically oriented scale that could be used by clinicians across numerous specialties. in , they developed and validated the csha clinical frailty scale. this is a descriptive scale with seven categories from "very fit" to "severely frail." it has been demonstrated to be an effective measure of frailty and offers predictive information about probability of survival and likelihood of institutionalization. this is a judgment-based scale that would be more applicable in the physiatric setting, whereas the more timeintensive frailty index would give more specific information to a primary care provider or geriatrician. both tools are validated instruments of benefit in the research setting (table . ). in addition to the functional, psychological, and musculoskeletal changes, there are also altered organ system and homeostatic responses in the frailty syndrome. these include a reduced capacity to maintain homeostasis and an increased vulnerability to stressors caused by lower energy metabolism, sarcopenia, altered hormonal activity, and decreased immune function. changes in the endocrine axis, specifically the growth hormone/insulin-like growth factor (gh/igf- ) axis, affect numerous metabolic systems. hepatic igf- production is controlled by pituitary growth hormone secretion and is essential for normal metabolic processes in adults. because growth hormone secretion declines with normal aging, igf- levels also decline; this is often referred to as the "somatopause," which accounts for the normal age-related decline in endocrine function. decreasing circulating levels of endocrine hormones contribute in part to osteoporosis, alteration in muscle/fat ratio, and cognitive decline in addition to sarcopenia. there is an association between the frailty syndrome and abnormally low igf- levels. routine testing of igf- levels is not recommended because it is neither diagnostic nor cost effective. however, appreciating the metabolic setting in which the frailty syndrome is more likely to occur provides a framework for understanding this complex syndrome. in the women's health and aging study, participants who had disabilities in mobility and disabilities in adls were more likely to have an increase in the proinflammatory cytokine interleukin- (il- ). the combination of decreased igf- and increased il- could contribute to a theoretical shift from the normal slowly decreasing anabolic state of aging to a rapidly increasing catabolic state seen in the frailty syndrome. as the population ages, the incidence of the frailty syndrome will also increase. this complex syndrome will continue to be elucidated as advances in the molecular biology of normal aging progress. for the physiatrist, early recognition of the frailty syndrome may allow multidisciplinary intervention with the intended goal of preserving function as long as possible for these patients. traditional hospital practices (bed rest, sedation, and immobilization). there has been a long-standing culture of bed rest in hospital culture. the concept of "convalescence" is that an ill person's strength returns gradually and is enhanced through greater than normal rest. this word is believed to have entered regular use in the late th century at a time when patients who were ill had few medical options other than rest. the deeply held belief that hospitals are places to "rest" influences sedation practices and encourages the overuse of bed rest despite there being a clear understanding of the dangers of immobility. this is especially true in the traditional critical care setting. long-standing provider beliefs include the idea that undersedation during mechanical ventilation is painful, traumatic, and panic-inducing despite ample data to the contrary. , in a recently published study that explored nurse sedation practices in the icu, % of nurses surveyed believed that sedation is necessary for patient comfort and % of the nurses surveyed would want sedation if they were ventilated themselves. although some degree of sedation is usually required, % of the nurses surveyed believed that patients who are "spontaneously moving hands and feet" are undersedated. although overuse of sedation is associated with an increase in posttraumatic stress disorder (ptsd) and prolonged mechanical ventilation with the accompanying complications of total immobility, it is still a widely held belief in many institutions that "rest is best." these practices lead to accelerated and iatrogenic deconditioning resulting from immobilization. iatrogenic immobilization and deconditioning. the dangers of immobilization have been understood for a long time (table . ). the often-cited % to % loss of muscle strength for each week of bed rest was derived from studies that involved young healthy test individuals without underlying disease or musculoskeletal conditions. it is likely that the rate of deconditioning is even faster in older adult patients with multiple comorbidities, because ambulatory function and ability to perform basic adls have been shown to decline in one-third of hospitalized patients older than the age of years. some of the complications of immobility include orthostatic intolerance, skeletal muscle changes, joint contractures, pulmonary atelectasis, urinary stasis, glucose intolerance, and pressure ulcers. traditionally, physiatrists have served as advocates for increased patient activity in the hospital setting because they evaluate and identify patients who can benefit from physical and occupational therapy. rationale. the importance of early mobilization is well accepted as a "best clinical practice" in every hospital setting, not just the icu. as the complications of immobilization became more widely understood, the importance of early mobilization throughout hospital organizations becomes a logical institutional goal, ideally approached through the quality improvement methodology. less universally agreed is how to design and implement a multidisciplinary early mobilization program and how to effect the accompanying culture change that is required for success. the physiatrist, working closely with colleagues in nursing, critical care, physical therapy, and occupational therapy is ideally suited to take a major role in the effort to bring mobilization to all patients who are hospitalized. the icu is an ideal setting in which to initiate such a program because of the increased staffing to patient ratios, extended length of stay, and awareness within the critical care community as to the important role of physical medicine and rehabilitation in these programs. culture of immobility. to successfully mobilize patients who are hospitalized, one must understand the many reasons that patients are ordered to bed rest and immobilization. only then can the root causes of reduced patient activity be addressed. the concept of therapeutic bed rest can be a difficult idea to challenge. therapeutic bed rest has been recommended for almost every medical problem at some point in medical history, including a variety of cardiac and pulmonary conditions in both the pediatric and adult populations. , , in the intensive care setting, longheld beliefs that the experience of mechanical ventilation was traumatic and frightening for patients gave rise to a culture of complete sedation and resultant immobility. the idea that patients who were unconscious would recover faster, "fight the vent" less, and be spared psychological suffering was essentially unchallenged. similar to the experience of patients with cardiac disease before unit-based cardiac rehabilitation efforts began in the s, survivors of critical illness were profoundly weak with substantial disabilities resulting from their prolonged immobilization. this was often interpreted as proof of how tenuous their conditions were at the time of presentation, rather than the side effects of immobilization and oversedation. culture of mobility. the first step to increase patient activity is gaining the trust and "buy-in" of colleagues in medicine and nursing. the importance of having evidence-based discussions with colleagues cannot be overstated. the common ground of all healthcare providers is commitment to patient care and improved functional outcomes. there is currently a developing medical literature on the benefits of early mobilization. , , journal clubs, consultative rounds, and a strong inpatient presence contribute to an understanding of the physiatric approach and will provide an appropriate venue for discussions about early mobilization. physiatric involvement. as a physician who understands the important role of physical, occupational, and speech therapy, the physiatrist is ideally suited to emphasize the vital role that these services play in the hospital setting. because of hospital-bundled payments, physical therapy expenditures have traditionally been seen as cost centers and not profit centers by hospital administrators. to address this perception, the physiatrist should be well aware of the importance and robust discussion taking place in the medical literature about the cost-effectiveness as well as clinical use of early mobilization programs in a variety of medical settings. , , , familiarity with the current literature demonstrating the multiple savings to an institution is key in acquiring the resources needed to implement an early mobilization program. having a physiatrist as part of the implementation team is optimal to represent the interests and contributions of the entire spectrum of physical medicine and rehabilitation providers. ambulatory devices. hospitalized patients who use assistive devices normally are generally unsafe to ambulate without their usual devices while in the hospital. because of staffing ratios and constant surveillance, ambulation in the icu would not take place unassisted; however, in a noncritical unit a mobilization program would need accommodation for patients who can ambulate independently as well as with assistance. a recently developed fall prevention tool kit includes bedside signs identifying risks and required assistive devices if they are determined to be necessary for safe ambulation. in one study, % of falls were related to attempts by patients to reach the bathroom. it is logical that removing assistive devices from ambulatory patients entering the hospital will result in increased falls when patients attempt to ambulate independently. training. coordinated interdisciplinary training before implementation is essential for any mobilization program. although early mobilization is often focused on physiatric oversight and physical therapy services, without appropriate coordination by all disciplines involved, successful early mobilization cannot take place. multidisciplinary simulation training with case-based scenarios should take place in the unit where mobilization is planned. all members of the healthcare team should have clearly defined roles before simulation training to experience the difference between standard multidisciplinary care and interdisciplinary coordination of care, which is at the heart of early mobilization. unit-based simulation training, ideally with an actor as the patient, is optimal when possible. cases provided should address medical emergencies and the challenges of physical coordination of care. allocating time following simulation training for team members to discuss their experiences further facilitates the team-building experience. patients hospitalized in critical care units almost universally have hemodynamic instability. the systemic inflammatory response syndrome causes peripheral vasodilation, cardiac dysfunction, capillary leak, and circulatory shunting leading to hypovolemia. a constantly changing cardiovascular environment makes daily evaluation essential. as a result of vasodilation, patients who are critically ill may be unable to tolerate bed elevation, let alone seated positioning. observing hemodynamic response to simple turning is a bedside test of hemodynamic tone that is easily carried out by a single provider. patients who cannot tolerate trunk elevation can be treated in a supine position with range of motion and progressive resistance. it is important for treating therapists to continually observe blood pressure response to intervention. it is strongly recommended that treating therapists consult with nursing providers to discuss any changes since last treatment resulting from rapid changes in physiologic state. with constant surveillance and gradual progression, mobilization of the patient who is critically ill is unlikely to produce any unexpected events. hypoxemic failure causes numerous pathophysiologic effects culminating in requirement for ventilator support. these include hypoxemic failure caused by ventilation/perfusion (v/q) mismatch, shunting, and altered oxygen exchange properties. hypercapnic failure causes decreased minute ventilation relative to physiologic demand, especially in the setting of critical illness complicated by increased dead space ventilation. coordination of therapy in conjunction with ventilator management should take place between physical medicine and rehabilitation and respiratory therapy. in some cases, therapists may be given parameters by the primary team as to titration of oxygen during treatment sessions. oxygen titration should always take place in coordination with respiratory therapy or nursing. similar to hemodynamic monitoring, ventilator status and oxygen saturation can vary quickly in response to activity and should be monitored at all times. during active ambulation, a respiratory therapist is essential to monitor oxygenation and the position of an endotracheal tube. icu-acquired weakness is common following hospitalization in the intensive care setting. approximately % of patients with prolonged mechanical ventilation, sepsis, or organ failure have some degree of neuromuscular dysfunction. the presence of icu-acquired weakness can cause abnormalities at any point in the gait cycle. endurance is reduced in all patients following prolonged bed rest with or without paralytics. ambulation trials should begin with standing at bedside and progress only out of the patient room once the entire team is assembled. this will ensure patient safety and continuous monitoring in the face of global weakness. early and aggressive physical therapy intervention has been shown to improve recovery of muscle strength in patients in the icu. , psychology of the patient in the intensive care unit survivors of critical illness often have symptoms of ptsd. in a recently published study, % of icu survivors following admission for acute lung injury reported ptsd symptoms during the -year period following critical care admission. symptoms of ptsd can be persistent, given that % of patients who reported symptoms confirmed their persistence at months. of these, % had taken psychiatric medications and % had seen a psychiatrist since hospital discharge. clearly, the complications following an episode of critical illness are multifactorial. although it is too early to definitively state that early mobilization programs reduce symptoms of ptsd, it is likely that the benefits of such a program include reduced psychiatric complications. delirium in the intensive care setting frequently complicates patients' hospital courses with resulting negative health outcomes. it is important that all providers in the intensive care setting recognize delirium and understand how it can impact on early mobilization programs. by definition, delirium is a change in cognitive function hallmarked by a fluctuating course over a short period of time (hours to days). in the critical care setting, delirium is not just a descriptive term; it is a measurable medical syndrome associated with poor outcomes. the confusion assessment method (cam) and cam-s (short form) are measurement tools that have been validated for use in this setting. it is important for therapists to understand that the interventions the provider has been shown reduce delirium and improve functional outcomes. recent critical care practice guidelines state, "we recommend performing early mobilization of adult icu patients whenever feasible to reduce the incidence and duration of delirium." for the rehabilitation provider, even though the fluctuating nature of delirium has the potential to interfere with physical and occupational therapy, it is essential that therapy intervention be provided whenever possible. this often necessitates frequent reevaluations throughout the day until an appropriate window of intervention can be found. the importance of care transitions is receiving greater attention in both the medical literature and the administrative realm of hospital management. an appreciation for the impact of poorly structured systems to transition care from one medical setting to another has become an area of intense focus across the healthcare spectrum. transitional care programs have been studied using different combinations of licensed providers including social workers, pharmacists, nurses, and physicians with most of these programs showing some degree of benefit. , , the use and feasibility of transitional care programs has been studied in varied environments where the transition begins including icu, hospital, nursing home, emergency department, and rural care centers. , , , , the effect of early mobilization programs on care transition has not been studied, but the current interest in ensuring continuity of communication emphasizes the importance in developing and studying systems to ensure transition of physical and occupational therapy. without attention to appropriate transition of care of rehabilitation services, the benefits gained during early mobilization are rapidly lost following transfer. this is an area of emerging interest that justifies research efforts on the part of all disciplines involved in early mobilization programs. although the importance of early mobilization in the critical care setting has received substantial attention, the principles of early mobilization are applicable throughout the healthcare continuum. evaluating patients at risk for immobility or reduced mobility followed by appropriate referral to trained providers can increase patient activity levels throughout a medical system. the type of provider selected is determined by the degree of assistance required for a patient to safely increase their activity level. in the hospital setting, "bed rest" orders should be replaced by systemized evaluations regarding safe patient activity levels. allowing patients who are hospitalized to use bedside commodes and assistive devices where appropriate is an economical way to increase patient activity without skilled intervention. providing assistive devices to appropriate and carefully selected patients who are hospitalized can increase mobility and reduce incidence of falls in the context of a structured fall-prevention program. healthy people . the us department of health and human services has initiated five "healthy people" initiatives since . these are comprehensive public health programs designed to provide structure and guidance to achieve more than objectives to improve the health of all americans. each health objective has a reliable data source, baseline measurement, and target for specific improvements to be achieved by the year (table . ). the healthy people website is a rich resource that includes leading health indicators, tips and tools for implementation of programs, and a consortium of organizations and agencies committed to achieving the healthy people goals. community mobilization programs can play a role in achieving targets for some of the most important leading health indicators listed as follows. wellness centers. chronic diseases and their cost are responsible for approximately % of us healthcare costs. the prevention and public health fund was established by the patient protection and affordable care act of to administer the community transformation grant program. more than grantees have been funded to support americans in adopting healthier lifestyles including healthy eating, active living, and tobacco-free living. funding opportunities exist within this new structure to explore evidence-based community wellness initiatives. although the causes of preventable chronic diseases in the united states are clear (cigarette use, lack of physical activity, calorie-dense/nutrient-poor dietary patterns), the solutions are as varied as the communities throughout the country. there are many unique and innovative programs that intervene at different stages of the disease process. preventative programs are some of the most appealing as long as the cost-benefit ratio strongly supports ongoing investment. some programs may involve disease-specific secondary prevention, such as a traditional cardiac rehabilitation program. others may be more broadly based and focus on increasing fitness in the context of a community. although wellness programs with general physical activity goals may be common in senior centers, some of the most innovative approaches to the overarching problem of obesity in the united states are taking place in community centers with a focus on modifying heath behavior in the context of community-based family education and counseling. the growing right onto wellness (grow) program in nashville, tennessee, represents an innovative program with a population of parent-child pairs who received educational interventions from to with a goal of preventing childhood obesity from developing in children. wellness centers can be freestanding but with community resources (community centers, houses of worship, schools, and employers) because locations for population-specific wellness programs are an efficient way to improve health outcomes. regardless of the population being targeted, all wellness programs can be developed based on a standard approach, listed as follows: step : organize advocates and advisors wellness programs are multidisciplinary in nature. identifying a group of committed and interested leaders and creating a working group is a logical first step. having the input and involvement of advocate community members at an early stage can improve awareness and uptake in later stages. step : determine the target population the most common wellness programs are either community or workplace based in scope. a work-based program will by definition be multidisciplinary. a physiatrist would be ideally suited to spearhead this type of intervention within a medical center based on their understanding of function, occupational health concerns, and expertise in multidisciplinary work. surveying the target population by questionnaire will yield the most accurate and direct information regarding what types of conditions a population is at risk for. this in turn will guide the development of appropriate interventions. in the community setting, an open forum or informal sampling of health concerns and interests can serve the same purpose. a needs assessment survey is also an opportunity to determine the preferences and interests of the target community. for example, there are numerous ways to increase physical activity levels. early identification of prevailing interests will guide the working group to direct its efforts where they will yield the greatest participation. information from health risk appraisals will direct the priorities for each organization or community. some examples include: exercise/physical fitness, tobacco reduction, stress management, back care, nutrition, weight control, and mental health. there is no way to predict what topics will be of importance to a population without sampling opinion. in a study that queried medical students regarding priorities for a health promotion program, % were interested in financial planning initiatives and only % were interested in alcohol and drug abuse programming. targeted polling will inform where resources should be directed for maximum impact. human behavior is driven by reinforcement. creating sustainable incentives (e.g., t-shirts vs. weekend getaways) will contribute to success over time. newsletters, parties, recognition meals, snacks, and gift cards are all popular workplace incentives that have the added value of increasing visibility of a new program for nonparticipants. workplace competition can be a healthy way to promote a program, especially if a substantial incentive is being offered as reward to the winning team. geographic competitions, departmental teams, and intradisciplinary teams can all contribute to employee "buy-in" and team building. step : implementation before implementation, additional community members, division heads, or other leaders should be included as part of the implementation team. it is just as challenging to develop programs with too many members as it is to implement a program launch with too few members on the launch team. leaving some choices to be decided by the entire implementation team will improve participation and sense of involvement for all team members, leading to better enthusiasm and community participation following deployment. spearheading or participating in implementation of an employee wellness program can be a gratifying and worthwhile way to improve function on a population level rather than at the individual level. highlighting the importance of the unique physiatric approach in the healthcare setting, as well as educating communities as to the important role physiatrists can play in keeping their community members healthy and active, are additional benefits to involvement in these types of programs. scientific advances have expanded our understanding of what constitutes renal failure. once thought to be solely attributable to volume overload, urea buildup, and hypocalcemia, it is currently known that uremia is a complex syndrome caused by the accumulation of organic waste products, some of which have yet to be identified. hemodialysis cannot fully duplicate the complex actions of the nephron, which filters blood, reabsorbs water and solutes, secretes toxic substances, and excretes essential hormones. chronic kidney damage leading to kidney failure has numerous causes; however, the most common causes are uncontrolled hypertension, poorly controlled diabetes, and glomerulonephritis. uncontrolled hypertension leads to nephrosclerosis, or localized damage to the glomeruli. there are two proposed mechanisms of hypertensive nephrosclerosis, the first glomerular ischemia and the second hypertension-induced damage and resultant hyperfiltration. diabetic glomerulopathy has excessive extracellular matrix as the most important pathologic feature. glomerulonephritis can be primary or secondary, each of which has many causes, a full discussion of which is beyond the scope of this chapter. infections, immune diseases, and vasculitides can all cause primary glomerulonephritis with resulting scarring of the nephrons. secondary causes include diabetes, hypertension, and lupus, among others. alternatively, chronic kidney disease can be classified based on the anatomic part of the nephron that is affected. glomerular, interstitial, tubulointerstitial, vascular, and obstructive are all anatomic classification examples that are listed in table . . fluid overload with hypertension can occur when the glomerular filtration rate falls below ml/min. the ability to maintain normal potassium levels is preserved until the glomerular filtration rate reaches approximately % of normal. although patients with chronic kidney failure can secrete potassium until an advanced stage of renal failure, the rate of excretion is reduced, causing prolonged elevations in potassium following ingestion. caution must be used when a patient is noted to be receiving spironolactone. spironolactone is an aldosterone agonist that can result in dangerous hyperkalemia if not monitored closely. sodium metabolism is maintained until renal failure becomes advanced. as renal failure progresses, the ability to conserve sodium is compromised, resulting in hyponatremia. uremia literally means "urine in the blood." it is a general term that began to be used before the understanding that end-stage kidney disease was more complex than just the inability to filter urea from the blood. as nephrons die, the remaining units increase their capacity in a process known as compensatory hyperfiltration. increased glomerular permeability is another adaptation to the reduced number of functioning nephrons. metabolic acidosis is caused by the reduced ability of the failing kidney to excrete acid. this is exacerbated by decreased ability to resorb bicarbonate. bicarbonate serves as the main ph buffer in the body and is derived from bone stores. hypocalcemia. hypocalcemia is loss of calcitriol and leads to decreased calcium absorption and stimulation of parathyroid hormone release. hyperphosphatemia. hyperphosphatemia is caused by impaired excretion. in the face of abnormal bone metabolism, soft tissues became the phosphate reservoir, causing increased vascular calcification. this leads to increasing pulse pressure in advanced disease. anemia of chronic disease resulting from decreased erythropoietin and iron deficiency contributes to fatigue and weakness. supplementation with erythropoietin is recommended and overseen by the primary nephrologist. studies have shown that a glomerular filtration rate of less than ml/min is associated with reduced well-being and overall function. , patients who are dependent on dialysis have multiple possible sources of debility depending on the type of dialysis they receive. peritoneal dialysis using the peritoneum as a membranous filter typically takes place in the patient's home, causing less functional impact than hemodialysis, a facility-based intervention. both methods of dialysis have potential for infection resulting from indwelling catheters, but peritoneal dialysis is more likely to be complicated by subacute bacterial peritonitis as opposed to bacteremia in hemodialysis. sbp is more likely to result in hospitalization, whereas hemodynamically stable bacteremia will be treated on an outpatient basis with antibiotics and with less likelihood of iatrogenic complications during hospitalization. patients who are hemodialysis dependent often report substantial fatigue following dialysis sessions; this impacts on their ability to participate not only with adls but also with rehabilitation efforts. the term "residual syndrome" has been applied to the syndrome of partially treated uremia, because dialysis cannot fully replace all renal functions. electrolyte imbalances and the resultant acid-base abnormalities are responsible in part for the uremic symptoms that are seen in patients who are dialysis dependent. uremic sarcopenia has been described in chronic kidney disease. , the presence of uremia causes changes in skeletal muscle fibers including mitochondrial depletion and atrophy of both slow-and fast-twitch muscle fibers. , , this in turn contributes to the overall sense of fatigue and weakness reported by these patients. the role of exercise in maintaining health-related quality of life and exercise capacity has been described, and patients should be enrolled in an exercise program whenever possible to preserve skeletal muscle function as soon as possible following the diagnosis of renal insufficiency. , patients with chronic renal insufficiency are often candidates for rehabilitation in both the hospital as well as outpatient settings. chronic deconditioning resulting from uremic sarcopenia, a predisposition to chronic pain and gait abnormalities, represents areas for substantial rehabilitative intervention. , , patients on dialysis should be instructed in a regular stretching program because of the potential for hip and knee contractures from prolonged sitting. patients on hemodialysis are at risk for a substantial degree of sedentary behavior as a result of extended immobility during hemodialysis sessions and postdialysis fatigue following dialysis sessions. three hours of dialysis followed by hours of resting while watching television constitutes up to hours of additional sedentary time a week. early referral to a rehabilitation professional for institution of a home exercise program before dialysis is warranted for all patients with uremia. it has been shown to be beneficial for patients on hemodialysis to exercise during dialysis sessions, although this is rarely standard practice. similar to cardiac transplantation, patients following renal transplantation are substantially disabled as a result of a long-standing chronic medical condition: uremia instead of congestive heart failure. although a patient on hemodialysis is typically more functional than a patient with end-stage heart failure, the principles of preprocedure rehabilitation or "prehab," posttransplant evaluation, and coordination of rehabilitative care are the same. following transplantation, reduced mobility, prescribed corticosteroids, and a sedentary lifestyle during recovery can greatly accelerate debility even in the face of a normally functioning kidney. unlike cardiac transplantation, there is no expected increase in co to boost energy metabolism. it is essential that all patients be evaluated following renal transplantation for potential rehabilitation intervention whether it be for aggressive mobilization and extended monitoring or for discharge to acute or subacute facilities. patients following renal transplantation have complex rehabilitative needs similar to patients who are critically ill in the medical intensive care setting. both populations have multisystem organ damage, distributive shock, hemodynamic instability, and severe deconditioning and are at risk for functional decline. unlike the medical icu where a comprehensive early mobilization program may be implemented previously, the patient following renal transplantation may be more reliant on physiatric consultation and coordination of care to receive required services. applying the early mobilization model to this population would necessitate evaluation for participation with physical medicine and rehabilitation on postoperative day . patients requiring ventilator support can receive physical and occupational therapy if sedation is interrupted long enough to participate with the treating therapist. physical therapy is often automatically ordered for all appropriate patients postoperatively and coordination with the primary team should take place in the morning with an agreed upon time for treatment should an interruption in sedation be needed. daily physiatric consultation and review of treatment session notes can guide expectations for the following treatment session. it is crucial to ensure uninterrupted therapy at least three times a week because posttransplant physical reserve is reduced and potential for accelerated deconditioning in the presence of an immunosuppressive regimen that includes corticosteroids is substantial. outpatient rehabilitation programs following renal transplantation should focus on controlled conditioning and education regarding the importance of exercise in maintaining exercise capacity and lifelong function. the same target heart ranges can be used for patients following both cardiac and renal transplantation: % to % of peak effort for to minutes three to five times weekly. rate of perceived exertion as described in the borg scale is a validated method to regulate intensity. it is important to provide consistent encouragement because patients following transplantation can be extremely debilitated and depressed. advising transplant survivors that functional improvement may not be seen for weeks or more following initiation of conditioning will help to maintain participation. a comprehensive rehabilitative program will offer numerous benefits following renal transplant. physical therapy can be focused on achieving a baseline improvement to facilitate participation in activities that the patient following transplantation may have forgone in the face of declining function. perhaps more than any other solid organ transplant, successful candidates for renal transplantation can hope to achieve maximum function with optimal rehabilitation. the coronavirus disease (covid- ) pandemic caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) has been the most significant public health crisis in years, and it has affected all aspects of healthcare, including every area of rehabilitation medicine. at the time of publication, covid- is still increasing worldwide, and with the high number of survivors who have suffered a severe acute respiratory distress syndrome (ards) with prolonged intubations or severe pneumonia, the need for rehabilitation interventions has never been higher. a even though recent evidence has suggested that most patients hospitalized with covid pneumonia can be successfully treated without mechanical ventilation, there are severe pulmonary implications for many patients. all hospitalized patients should be discharged with clear recommended guidelines for safe and progressive conditioning at home as standard of care is now to provide medical oversight and guidance for patients at home whenever possible. a, a this has led to a rapid expansion of telehealth services across the medical continuum that includes all disciplines within physical medicine and rehabilitation even as the medical complexity of patients admitted to acute rehabilitation has increased. a the common admitting diagnoses to acute rehabilitation units during the pandemic has been deconditioning following acute hypoxic respiratory failure secondary to covid pneumonia. a key principle guiding care of covid- patients is the maintenance of adequate oxygenation at all times. for hospitalized patients the principles of early mobilization should be utilized whenever possible to prevent rapid deconditioning and further need for already limited rehabilitation services. a oxygen saturation should be maintained above % with liberal use of supplemental oxygen. continuous oximetry monitoring may be indicated for patients whose oxygenation level declines with trial mobilization on room air. covid- patients have also been found to be at higher risk for venous thromboembolic events (vtes), and baseline laboratory studies for all patients admitted to a rehabilitation unit should include a panel screening for prothrombotic states that may indicate need for prophylaxis. patients are at elevated risk for vtes for an extended period following intensive care hospitalization for covid- pneumonia, so a high index of suspicion should be maintained throughout the course of rehabilitation. a as the incidence of covid- decreases and acute rehabilitation units reopen, there will be ongoing opportunities to generate more objective data and high-quality literature on characteristics, complications, and outcomes of the covid- population in the rehabilitation setting. harmonizing the metabolic syndrome: a joint interim statement of the international diabetes federation task force on epidemiology and prevention american heart association; world heart federation; international atherosclerosis society; and international association for the study of obesity american association of cardiovascular and pulmonary rehabilitation: guidelines for cardiac rehabilitation and secondary 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preventive cardiovascular nurses association, and the society of thoracic surgeons the benefits and risks of exercise training in patients with chronic coronary artery disease effect of chronic uraemia on skeletal muscle metabolism in man understanding the prevalence of inpatient falls associated with toileting in adult acute care settings cardiac rehabilitation and artificial heart devices cardiovascular complications of outpatient cardiac rehabilitation programs safety of medically supervised exercise in a cardiac rehabilitation center interventions for preventing obesity in children cardiac conditioning after myocardial infarction. an early intervention program high-intensity interval training for patients with cardiovascular disease-is it safe? a systematic review predicting obesity in young adulthood from childhood and parental obesity secondary prevention of coronary heart disease in the elderly (with emphasis on patients > or = years of age): an american heart association scientific statement from the council on clinical cardiology subcommittee on exercise, cardiac rehabilitation, and prevention effect of individualized exercise during maintenance haemodialysis on exercise capacity and healthrelated quality of life in patients with uraemia the full reference list for this chapter is available online. key: cord- -d at y authors: ghasemiyeh, parisa; mohammadi-samani, soliman title: covid- outbreak: challenges in pharmacotherapy based on pharmacokinetic and pharmacodynamic aspects of drug therapy in patients with moderate to severe infection date: - - journal: heart lung doi: . /j.hrtlng. . . sha: doc_id: cord_uid: d at y the new coronavirus (covid- ) was first detected in wuhan city of china in december . most patients infected with covid- had clinical presentations of dry cough, fever, dyspnea, chest pain, fatigue and malaise, pneumonia, and bilateral infiltration in chest ct. soon covid- was spread around the world and became a pandemic. now many patients around the world are suffering from this disease. patients with predisposing diseases are highly prone to covid- and manifesting severe infection especially with organ function damage such as acute respiratory distress syndrome, acute kidney injury, septic shock, ventilator-associated pneumonia, and death. till now many drugs have been considered in the treatment of covid- pneumonia, but pharmacotherapy in elderly patients and patients with pre-existing comorbidities is highly challenging. in this review, different potential drugs which have been considered in covid- treatment have been discussed in detail. also, challenges in the pharmacotherapy of covid- pneumonia in patients with the underlying disease have been considered based on pharmacokinetic and pharmacodynamic aspects of these drugs. wuhan city of china. the most common clinical signs and symptoms of these patients were dry coughs, fever, dyspnea, and bilateral infiltration in chest ct. all these patients were associated with wuhan's huanan seafood wholesale market which sells fish and other live animals such as bats, poultry, snakes, etc. the causative agent, new coronavirus, was first detected through a swab sample which was drawn from the throat of these patients . this new coronavirus was subsequently named severe acute respiratory syndrome coronavirus (sars-cov- ). soon this disease, which called coronavirus disease (covid- ) by world health organization (who), promptly spreads around the world , and to date over . million cases have been diagnosed with covid- and this disease became a pandemic. on the late january covid- chinese outbreak, were introduced as a public health emergency of international concern . so although previously coronaviruses were considered as a potential cause of the common cold now we know that they are more than just the common cold! most of the covid- infected patients have an average age of s, it is slightly more predominant in the male sex, approximately % of infected patients involved with severe disease were required to intensive care unit services and % of them were required to mechanical ventilation . a published report from italian patients revealed that covid- was predominant in men ( . % in male and . % in female), most of the patients (about %) were over years old, approximately % of all confirmed patients had mild disease, % had severe disease, % were in a critical situation, and rest of the patients showed few symptoms, unspecified symptoms or were completely asymptomatic . according to recently published researches, the most common clinical presentations in covid- patients were fever in % to % of patients, dry cough in to %, and fatigue or myalgia in to % of them. other signs and symptoms which have been reported include sore throat, headache, confusion, rhinorrhea, sneezing, ageusia, anosmia, chest pain, hypoxemia, pneumonia, hemoptysis, acute cardiac injury, neurologic complications , , and gastrointestinal presentations such as nausea, vomiting, diarrhea and abdominal pain , - . patients with underlying diseases are highly prone to present with severe infection especially with organ function damage such as acute respiratory distress syndrome (ards), acute kidney injury (aki), septic shock, and ventilator-associated pneumonia (vap) , . severe covid- could cause death due to huge alveolar damage and highly progressive respiratory failure . covid- particles could spread through the respiratory mucosa and fecal-oral route . the nucleic acid of the virus was detected in stool, saliva, and respiratory specimens . this virus could be transmitted between humans during the epidemic and then pandemic of covid- . human-to-human transmission could highly accelerate the spread of this virus around the world. this type of transmission among humans is restricted to close contact and through sneezing or coughing of the infected patients who are capable to spread the respiratory droplets. then these respiratory droplets could settle in oral mucosa and lung of the people who inhaled the contaminated air near (about feet) to the infected patients , . although some researches have been focused on the airborne transmission of this virus but this route of transmission has not been approved yet and further studies are required. researches revealed that covid- could also be transmitted through asymptomatic carriers with an incubation period of to days . in order to prevent spreading of this new virus: hands should be washed frequently, the face should not be touched with unwashed hands, regular surface disinfecting is required, social distancing from people with respiratory symptoms is essential, sneezing or coughing should be done into the elbow or soft tissue if available . based on the published reports, in most of the patients with covid- , the absolute value of lymphocytes was reduced, which indicated that this novel coronavirus (covid- ) acts more on lymphocytes especially t lymphocytes, just similar to sars coronavirus . it seems that covid- could induce a cytokine storm and activate immune responses which could be appeared as changes in the number of white blood cells and immune cells especially lymphocytes, the clinical outcome of such events would be respiratory distress syndrome, septic shock and finally end-organ damage . covid- could also affect the liver which could be presented as hypoproteinemia, elevated aminotransferases, and prolonged prothrombin time. hepatotoxicity could be attributed to the higher expression of angiotensin converting enzyme ii (ace ) in cholangiocytes, ace could act as an entry receptor for covid- . so it seems that this new virus can directly damage the intrahepatic bile ducts . pathological findings of a liver biopsy from a patient with covid- showed moderate micro-vesicular steatosis and also a mild portal and lobular activity which could be a result of direct sars-cov- liver damage or antiviral drug-induced hepatotoxicity . almost all covid- patients had abnormal lung ct when diagnosed. according to the recently published article, an average of . ± . segments were involved in patients and the number of involved lung segments was significantly higher in symptomatic patients group in comparison to asymptomatic ones. ct findings revealed that affected covid- patients could present as bilateral lung involvement, peripheral distribution, or diffuse distribution. the most common presentation in chest ct was ground-glass opacity pattern, consolidation, and ill-defined margins , . laboratory confirmation of steatosis could be performed by real-time reverse-transcription polymerase chain reaction (rrt-pcr) , . according to who approved laboratory testing for covid- diagnosis is based on nucleic acid amplification test (naat) such as rrt-pcr which could detect the sequence of the rna of covid- . governments need to appreciate people to obey social distancing and isolation. in some situations, quarantine of major cities is also suggestive. global health governance should apply the least restrictive measures for people according to the international health regulations (ihr) , . scientists around the world are looking for drugs that could be beneficial in covid- treatment. many drugs have been studied that are listed in table with the usual dosage ranges in adults and pediatrics. the latest guidelines for the prevention, diagnosis, and treatment of novel coronavirus-induced pneumonia, have been suggested antiviral agents containing: interferon alpha (ifn-α), lopinavir/ritonavir, chloroquine phosphate, ribavirin, and arbidol as potential options in covid- treatment . drugs that have been considered in covid- management have been classified as investigational drugs, drugs under clinical trials, and drugs that have received u.s. food and drug administration (fda) as shown in table . . although many previous studies emphasized the potential therapeutic effects of these drugs in covid- management, unfortunately some recent publications reported that the efficacy of chloroquine/hydroxychloroquine in covid- management is not consistent. also, their safety is still remaining a major concern for physicians and pharmacists, since chloroquine/hydroxychloroquine could cause qt prolongation and arrhythmia. since the possible risks of these drugs could overweigh their potential benefits and efficacy, united states food and drug administration (fda) no longer recommended these two drugs as potential options for covid- management . results of a systematic review on the efficacy of hydroxychloroquine or chloroquine on the prevention or treatment of covid- revealed that the available evidences on their benefits and risks are weak and controversial . results of another systematic review and meta-analysis on randomized clinical trials on administration of hydroxychloroquine in covid- management revealed that hydroxychloroquine administration (case group) was significantly associated with higher incidence of total adverse effects in comparison to placebo or no treatment (control group) in overall population of patients with covid- . so, the recruitment of chloroquine/hydroxychloroquine in covid- management is still controversial and further larger multi-center randomized clinical trials are required to evaluate their efficacy, safety, risk-benefit ratio, dose and duration of individualized pharmacotherapy. also, close patient monitoring, especially cardiac, ocular, and neurotoxicity assessments, are required and strongly recommended during drug administration . recently published studies revealed that chloroquine could highly reduce covid- replication . chloroquine is a weak base that could be entrapped in organelles that are membrane-enclosed and have low-ph, so interfering with their acidification process. therefore chloroquine could inhibit ph-dependent viral fusion and replication. also, it might inhibit viral assembly in endoplasmic reticulum-golgi intermediate like structures . another possible antiviral mechanism of chloroquine is its immunomodulatory effect through cell signaling pathways and regulating the action of proinflammatory cytokines that can enhance its antiviral effect synergistically , , . chloroquine/hydroxychloroquine could prevent from covid- -induced ards by attenuating the pro-inflammatory cytokines and receptors . hydroxychloroquine can enhance intracellular ph and avoid lysosomal activity in antigen presenting cells containing b cells, also they can avoid antigen processing and mhc-ii presentation to t cells. so, t cell activation could be reduced by the action of hydroxychloroquine. it can suppress the cytokine release syndrome (crs), which is a result of immune system over-activation, caused by covid- . according to this mechanism, hydroxychloroquine could alleviate symptoms of mild to severe covid- pneumonia . chloroquine has different adverse reactions such as cardiovascular adverse reactions . also the results of a systematic review on dermatologic adverse effects of hydroxychloroquine emphasized that the most common dermatologic reactions due to hydroxychloroquine administration were rash, sjs, toxic epidermal necrolysis (ten), pruritus, hyperpigmentation, and hair loss. these dermatologic reactions were mostly occurred after cumulative dosages of hydroxychloroquine . in overall, since hydroxychloroquine has lower tissue accumulation potential in comparison with chloroquine, it has fewer adverse drug reactions and would be better choice . contraindications in chloroquine use contains hypersensitivity to chloroquine ( -aminoquinolone compounds) and the presence of retinal or visual field changes . chloroquine and hydroxychloroquine have a narrow therapeutic index and poisoning could be occurred with cardiovascular features so it should be used with caution in patients with predisposing cardiovascular disease . long-term exposure to these drugs could induce cardiomyopathy . chloroquine in patients consuming heparin, prone the patients to risk of bleeding. also, chloroquine in patients with digitalization (using digoxin) could cause cardiac block . there is no dosage adjustment available for chloroquine or hydroxychloroquine in patients with hepatic failure but it should be used with caution . there is no dosage adjustments available for chloroquine in patients with renal failure from the manufacture's labeling but according to uptodate some clinicians use the following guideline : a) patients with gfr ≥ ml/min: no dosage adjustment is required. b) patients with gfr < ml/min: dosage should be reduced to %. c) patients with peritoneal-or hemodialysis: dosage should be reduced to %. required. there is no dosage adjustments available for hydroxychloroquine in renal failure but it should be used with caution . although some studies showed a low risk of congenital abnormalities in patients receiving chloroquine during pregnancy because of the lack of a pattern in these congenital defects, the possible association is unlikely and it seems that the benefits of its use are higher than risks . hydroxychloroquine use during pregnancy could not be accompanied by risks for fetuses, especially in low doses. but patient monitoring during pregnancy is required . in general, since chloroquine may induce severe side effects during fetal development, so hydroxychloroquine would be a better option in pregnant women with covid- infection because of its safety profile during pregnancy . according to the american academy of pediatrics, chloroquine is compatible with breastfeeding. although it could be excreted into the milk, this amount was not considered harmful for nursing infants . according to the american academy of pediatrics, hydroxychloroquine is compatible with breastfeeding. small amounts of hydroxychloroquine could be excreted to the milk, but because of the slow elimination rate and the possibility of drug accumulation and toxicity, breastfeeding during hydroxychloroquine therapy should be done with caution . umifenovir is a broad-spectrum antiviral agent which is effective against enveloped and nonenveloped rna or dna viruses especially against influenza virus type a and b, respiratory syncytial virus, sars-cov, adenovirus, hepatitis c virus (hcv), etc. umifenovir was first developed in russia and now its usage is more common in russia and china and is less common in western countries. its possible antiviral mechanism is the inhibition of viral fusion with targeted membrane and preventing from the viral entrance to targeted cells . umifenovir has a dual pharmacologic action: first is its beneficial effect on respiratory viruses such as the covid- virus and the second is its immune-stimulating function which can activate serum interferon and phagocytes. since , umifenovir was patented for its beneficial effect in the treatment of severe acute respiratory distress (sars) coronavirus-induced atypical pneumonia . results revealed that umifenovir can induce direct viricidal effect so it would be a promising direct-acting antiviral (daa) agent. umifenovir could affect critical stages of viral life cycles such as cell attachment, cell internalization, viral replication, assembly, and budding so it also would be a promising host targeting agent (hta). its dual pharmacologic function is related to its potential interaction with both cell membranes and with cellular and viral lipids and proteins . the most important adverse reactions associated with umifenovir are diarrhea, nausea, vomiting, dizziness, confusion, and elevated liver enzymes (serum aminotransferases) . umifenovir is an indole derivative with poor water solubility which could affect its bioavailability and pharmacokinetics. after oral administration of umifenovir, it could rapidly distribute to organs and tissues, maximum plasma concentration (c max ) was achieved after to . hours. in the russian population, it had elimination half-life (t ½) of to hours, but t ½ was shorter in the chinese population. after multiple-dose administration of umifenovir, little drug accumulation could be predictable. the main site of drug metabolization is the liver. umifenovir could undergo several metabolism pathways such as oxidation at the s site, ndemethylation, glucuronidation, and conjugation at -hydroxy moiety. the potential antiviral effects of umifenovir metabolites are unknown until now . since the major site of umifenovir metabolization is in the liver, so it should be used with caution in patients with predisposing liver diseases. animal data revealed that umifenovir therapy couldn't induce embryo-toxic effects during pregnancy. according to these results umifenovir would be a promising safe and well-tolerated antiviral agent in pregnancy with a wide therapeutic index in administration for a few days up to one month . ribavirin is a nucleoside antihepaciviral agent (anti-hcv) which has been suggested for covid- treatment. ribavirin is a direct-acting antiviral (daa) agent . ribavirin is a nucleoside analog that has antiviral action against a variety of rna and dna viruses. the potential antiviral activity of ribavirin is inhibition of inosine monophosphate dehydrogenase (impdh) cellular protein and therefore intracellular gtp would be diminished which inhibits rna replication of viral genomes, so viral growth might be stopped. another possible antiviral activity of ribavirin is its immunomodulatory effects by suppression of il- . ribavirin also could inhibit rna polymerase activity and therefore inhibition of rna fragments' initiation and elongation, so viral protein synthesis could be inhibited. the ribavirin is contraindicated in patients with hypersensitivity to ribavirin, pregnant women and their partner, patients with severe renal failure, patients with severe hepatic failure, and patients with major hemoglobinopathies such as sickle cell anemia and major thalassemia . ribavirin distribution could significantly prolonged in erythrocytes for about to days, which is responsible for ribavirin-induced anemia . ribavirin has hepatic metabolism. its oral bioavailability (f) is about %. ribavirin elimination half-life (t ½) in the normal population is hours but in patients with pre-existing chronic hepatitis c infection, half-life could be increased to hours. so because of its prolonged half-life and potential overdose toxicity, ribavirin is contraindicated in patients with hepatic failure (child-pugh class b and c). time to peak level (t max ) after oral administration is between to hours. ribavirin excretion could take place through both urine and feces routes. because of its renal elimination, dose adjustment in patients with underlying kidney disease is highly essential. according to the previous pharmacokinetic/pharmacodynamic study, bayesian therapeutic drug monitoring would be a suitable approach to control ribavirin-induced anemia [ ] . one of the most important side effects of ribavirin is hemolytic anemia which could worsen cardiac disease in patients with underlying cardiac diseases and it could induce fatal or non-fatal myocardial infarction in them. so ribavirin should be avoided in patients with a history of unstable or severe cardiac diseases. ribavirin is contraindicated in patients with hepatic decompensation (child-pugh class b and c). ribavirin dosage adjustment in patients with renal failure highly depends on different formulations which are available. these data are shown in table . in children, if serum creatinine level rises over mg/dl during administration, ribavirin should be discontinued promptly . ribavirin has teratogenic and mutagenic effects. so it is a high-risk drug in pregnancy according to animal data. also because of its half-life of hours in multiple-dose drug therapy and the possibility of drug accumulation in tissue compartments for up to months, ribavirin administration is contraindicated in pregnant women and also in men who are pregnant women's partner. it was suggested that pregnancy should be avoided during ribavirin therapy and at least months after completion of therapy in women or men . ribavirin because of the prolonged plasma elimination half-life and molecular weight of da, potentially would have toxicity in nursing infants but there are no human data available . ribavirin may precipitate hematologic adverse effects of organ transplantation regimen such as immunosuppressive agents (mycophenolate mofetil, azathioprine, mtor inhibitors), trimethoprim/sulfamethoxazole, and valganciclovir. these hematologic adverse reactions would also worsen hematologic reactions related to covid- . so close patient monitoring is essential. . also, lopinavir would be a promising drug of choice in children with covid- . lopinavir/ritonavir are protease inhibitor, anti-retroviral agents. the potential antiviral mechanism of lopinavir/ritonavir is inhibition of viral protease, which is a critical enzyme in viral maturation and infectivity. low dose ritonavir in combination with lopinavir act as a pharmacokinetic enhancer by inhibition of lopinavir inactivation metabolism . the since lopinavir undergoes hepatic metabolism through cyp a enzyme, potential drug-drug interactions could occur with all drugs that are strong inhibitors or inducers of cyp a enzyme and p-glycoprotein inhibitors. also, ritonavir has hepatic metabolism via cyp a and cyp d . ritonavir has serious and life-threatening drug interactions with sedative-hypnotic agents, antiarrhythmic drugs, and ergot alkaloid agents because of the effect of ritonavir on their hepatic metabolism through cyp a and cyp d . concurrent use of these agents with ritonavir is absolutely contraindicated and should be avoided . lopinavir/ritonavir is contraindicated in patients with a history of hypersensitivity reactions such as toxic epidermal necrolysis (ten), stevens-johnson syndrome, angioedema, etc. to lopinavir/ritonavir or its components . absorption and oral bioavailability are highly affected by fasting or fed state, its absorption, bioavailability, and peak level (c max ) could be significantly increased with food. but food can delay t max from hours in fasting state to hours in the fed state. ritonavir also has small renal elimination so dosage adjustment is not required in patients with underlying kidney disease . lopinavir/ritonavir have major interactions with drugs used in cardiovascular diseases such as anti-coagulating agents (anti-factor xa inhibitors), none dihydropyridine calcium channel blockers, digoxin, antiarrhythmic agents such as amiodarone, etc. so close patient monitoring is required in covid- patients with pre-existing cardiovascular disease who are planned to treat with lopinavir/ritonavir and sometimes alternative drugs might be considered . in patients with mild to severe hepatic failure, there is no dosage adjustments available but lopinavir has primary liver metabolism and its auc will be increase by about %, so it should be used with caution . there is no dosage adjustments based on renal function according to the manufacture's labeling . results revealed that embryo-fetal risk of lopinavir/ritonavir is low, so it is compatible with pregnancy and should not be stopped during pregnancy . lopinavir and ritonavir with a molecular weight of and da respectively and their lipid solubility nature, are good candidates for excretion to milk during lactation period but their high plasma protein binding could limit this excretion. a comprehensive data is not available yet. it has been recommended that breastfeeding during lopinavir/ritonavir therapy is better to be avoided especially in developed countries . immunosuppressive agents are critical drugs in patients undergone solid organ transplantation. lopinavir/ritonavir cannot be administered in combination with immunosuppressive agents because of the occurrence of strong drug interactions. lopinavir/ritonavir can enhance the plasma level of immunosuppressive agents such as calcineurin inhibitors and mtor inhibitors. so if co-administration is essential, immunosuppressive agents dose reduction and therapeutic drug monitoring, to maintain optimum immunosuppressive plasma level, is highly recommended. it has been suggested that during covid- treatment, calcineurin inhibitors (ex. cyclosporine and tacrolimus) and mtor inhibitors (ex. sirolimus and everolimus) could be discontinued and replaced with lopinavir/ritonavir but it seems that the benefit of this drug discontinuation could not overlay the risk of allograft transplant rejection . tocilizumab is an interleukin- (il- ) inhibitor which is a disease modifying anti-rheumatic agent . a small retrospective observational study on covid- pneumonia patients receiving tocilizumab revealed that this drug would have potential benefits in these patients such as since an active immune response against respiratory viruses such as covid- is highly dependent on cytotoxic t cells' action, so in patients with total t cell count of fewer than cells/µl, aggressive intervention is essential. one of the possible approaches in enhancing t cell count in these patients is the administration of tocilizomab, because there is a reverse relationship between t cell count and the number of cytokines such as il- . ifn-α is a broad-spectrum antiviral agent that is commonly used in hepatitis management. after corticosteroids are a double-edged sword, they can inhibit our immune response and so the clearance of covid- could be delayed, but on the other hand, they can suppress our inflammatory response which is highly responsive to the lung damage and ards during viral convalescent plasma or immunoglobulins would be a promising therapy in covid- patients. previous results revealed that convalescent plasma therapy during viral infection outbreaks, it has been recommended that patients with covid- who are suffering from refractory hypoxemia should be managed with extracorporeal membrane oxygenation (ecmo in some countries such as switzerland, tocilizumab has been considered in patients with multiorgan failure and inotropic support . ace has a critical role in cardiac and immune systems. ace is related to heart function and it could be a potential cause of hypertension and diabetes mellitus development . since ace is a functional receptor for covid- , in patients with underlying cardiovascular diseases, clinical symptoms of covid- are more severe and fatal than the general population because, in cardiovascular diseases, ace secretion might be enhanced. administration of reninangiotensin-aldosterone system inhibitors, such as angiotensin converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs) could enhance ace level. thiazolidinediones and ibuprofen might also enhance the ace level pneumonia but arbs, through blockade of angiotensin receptors, may have beneficial effects in these patients. most of the hepatic metabolites of drugs considered in covid- treatment such as chloroquine, hydroxychloroquine, and lopinavir/ritonavir would be found in urine due to renal elimination. so in patients with chronic kidney disease (ckd), the accumulation of drug metabolites would be expected if administered in routinely recommended doses for the normal population. therefore, individualized dose adjustment based on kidney function is required for each drug as mentioned above . a small study on covid- patients with end-stage renal disease (esrd) who undergone hemodialysis, revealed that the number of total t cells (cytotoxic and helper t cells), natural killer (nk) cells, and inflammatory cytokines were significantly lower than these levels in non-hemodialysis patients with covid- . this study revealed that esrd patients with hemodialysis who infected with covid- had a good prognosis and they had mild symptoms of pneumonia, it might be related to the fewer number of inflammatory cytokines and reduced immune function which can avoid crs but further studies are required to confirm this hypothesis . according to the recent studies, liver abnormalities (such as elevated ast and alt serum levels) have been occurred after and during infection with covid- . these abnormalities would be related to viral infection pathogenesis and direct liver injury or it may be drug-induced . almost all of the potential drugs in covid- treatment containing chloroquine, hydroxychloroquine, ribavirin, and lopinavir/ritonavir have hepatic metabolism. so injury to the liver because of pre-existing liver disease or acute hepatic failure would impair drug metabolism and therefore drug accumulation and enhancement in plasma level, which can lead to drug toxicity. in these patients, frequent liver function monitoring is essential to achieve an optimal serum drug level . also, dosage adjustment for each drug should be done individually according to the patients' liver function as mentioned above. the impact of chronic liver diseases such as chronic viral hepatitis, alcoholic and non-alcoholic liver diseases on occurrence of liver injury related to covid- infection, still is not clear. it seems that in patients with underlying liver disease, with the immunocompromised condition, who infected with covid- , more intensive and individualized pharmacotherapy is required. further studies would be also helpful to explain the exact role of pre-existing liver diseases in covid- prognosis . the new coronavirus (covid- ) was first detected in wuhan city of china in december . soon this coronavirus disease (covid- ) spreads around the world and became a pandemic. now many patients around the world are suffering from this disease. patients with underlying diseases are highly prone to severe covid- pneumonia. till now many drugs have been considered in the treatment of covid- pneumonia, but pharmacotherapy in patients with pre-existing comorbidities is highly challenging. in this review, different potential drugs which have been considered in covid- treatment have been discussed in detail. also, challenges in the pharmacotherapy of covid- pneumonia in patients with underlying disease especially heart diseases have been considered based on pharmacokinetic and pharmacodynamic aspects of drugs. patients with covid- who have cardiac diseases such as coronary heart disease and those who are aceis consumers should be highly considered in treatment options. also, patients with liver and kidney disease and those with organ transplants should be considered to avoid the occurrence of drug overdose toxicities and potential drug-drug interactions. funding: this work was not funded. coronavirus infections-more than just the common cold the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status covid- -new insights on a rapidly changing epidemic the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? characteristics of and 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results of an open-label non-randomized clinical trial chloroquine or hydroxychloroquine for management of coronavirus disease : friend or foe? hydroxychloroquine or chloroquine for treatment or prophylaxis of covid- : a living systematic review safety of hydroxychloroquine in covid- and other diseases: a systematic review and meta-analysis of randomized trials hydroxychloroquine and chloroquine: a potential and controversial treatment for covid- insights from nanomedicine into chloroquine efficacy against covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine-induced stevens-johnson syndrome in covid- : a rare case report characterizing the adverse dermatologic effects of hydroxychloroquine: a systematic review american pharmaceutical association drugs in pregnancy and lactation: a reference guide to fetal and neonatal 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feasibility of convalescent plasma therapy in severe covid- patients: a pilot study high-dose intravenous vitamin c treatment for covid- a review of the novel coronavirus (covid- ) based on current evidence preparing for the most critically ill patients with covid- : the potential role of extracorporeal membrane oxygenation are patients with hypertension and diabetes mellitus at increased risk for covid- infection? coronavirus disease (covid- ) and cardiovascular disease clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study. the lancet liver and kidney injuries in covid- and their effects on drug therapy; a letter to editor liver injury in covid- : management and challenges. the lancet gastroenterology & hepatology ethical approval: not required. p.g. and s.m. were contributed equally in data gathering, writing-original draft, reviewing, and revising the final version of this manuscript. key: cord- -y pzsoqa authors: adalja, amesh a. title: biothreat agents and emerging infectious disease in the emergency department date: - - journal: emerg med clin north am doi: . /j.emc. . . sha: doc_id: cord_uid: y pzsoqa the challenges faced by the emergency physician with recognizing and treating category a biothreat agents and emerging infectious disease are summarized and reviewed. emergency physicians in every location in the world, in developed and developing countries alike, will undoubtedly be confronted with the possibility of an emerging infectious disease in their career. a subset of these physicians may be faced with a patient who has potentially been exposed to biological weapons. of the myriad infectious disease emergencies an emergency physician contends with, these possibilities are the gravest and most impactful. in such scenarios, the emergency department (ed) clinician can be the key in recognizing or containing an outbreak. the challenge inherent with emerging infectious diseases presenting in the ed is that such cases can be camouflaged, lurking amongst innumerable infectious disease clinical syndromes, from common colds to viral rashes. this article provides guidance to emergency physicians as to how to approach this challenging problem as well as familiarizing readers with specific microbial threats of high consequence. a key method for detecting the presence of an emerging infectious disease syndrome or a biological weapons exposure in an ed patient is to develop a general approach that seeks out key historical and physical examination clues. this approach is not different from what is included in a full history and physical examination but requires meticulous attention to certain aspects of the history. travel history becomes a key focus of the history because many infectious diseases, especially of the emerging variety, have delimited borders in which they are prevalent. the travel history must be coupled, however, with situational awareness as to what infections are known to be present in specific parts of the world. such a task is daunting for most physicians and, therefore, it is important that they know where such resources can be found. both the centers for disease control and prevention (cdc) (www.cdc.gov/travel) and promed (program for monitoring emerging diseases) (www.promedmail.org) are such resources that are easy to access and continually updated. using these resources, a busy provider can quickly assess which specific infection risks any given country might confer. an important component of the travel history is understanding the dates of travel and how they relate to the incubation period of specific infections. travel must be contextualized and integrated with incubation period, because domestic infections acquired before or after travel might be mistaken for a travel-related infection. additionally, eds in a given geographic locale (eg, metropolitan area, county, or state) should develop a mechanism to have insight into changes in ed volume, chief complaint mix, and unusual diagnoses at other eds in the region. much of this can be accomplished through leveraging emergency health care coalitions and local or state health departments to develop tools to enhance insight into the vicissitudes of a given region's ed-relevant infectious disease problems through syndromic surveillance programs. an important component of an individual's risk for particular infections is related to exposures. attention must be paid to animal exposures (domestic and wild), eating habits, occupation, and hobbies. additionally, it is essential to determine if a person has had any sick contacts or has attended a mass gathering, because an ed physician might be seeing one of the first formal presentations of a wider outbreak. of the specific biological agents, the category a agents (anthrax, plague, tularemia, and botulism), and certain viral hemorrhagic fevers (vhfs) (eg, ebola, marburg, machupo, and lassa fever) are of the highest priority. table provides salient points regarding the treatment of the category a biothreat agents. in all cases of uncertainty, prompt consultation with an infectious disease physician is recommended. anthrax is caused by the gram-positive bacillus, bacillus anthracis. it is a ubiquitous spore-forming gram-positive bacterium that is found naturally in the soil worldwide. it is a disease of herbivores. humans can contract of forms of the infection: cutaneous, inhalational, injectional, and gastrointestinal. , of these forms, cutaneous is by far the most common and represents a majority of cases. an intentional release of anthrax is expected to result in primarily inhalational cases. anthrax is not contagious from person to person and no special precautions are required. adalja cutaneous anthrax is characterized by a painless black ulceration ( fig. ) that occurs on the site of exposure. infection is more common in those exposed to animal products contaminated with spores, such as meat, drum skins, or wool. after an incubation period of approximately days, the lesion characteristically begins as a papule and progresses to a black eschar. diagnosis is often clinical, although culture, biopsy, polymerase chain reaction (pcr), and serology confirm the diagnosis. mortality is low if the disease is recognized and treated with appropriate antimicrobials. treatment regimens include oral ciprofloxacin or doxycycline (although penicillin may be used if susceptibility is known) for days. if exposure was through a biological attack, treatment duration is extended to days to cover incubating spores that may have been inhaled. injectional anthrax has been exclusively linked to use of contaminated illicit drugs whereas gastrointestinal anthrax is due to ingestion of contaminated food. , inhalational anthrax is the deadliest form of anthrax and occurs on inhalation of as little as spore. anthrax was historically known as wool sorter's disease because of its linkage with the occupation of wool sorting, in which spores on sheep's wool became aerosolized. the disease is characterized not by pneumonia but by mediastinal widening (fig. ) that can progress rapidly to shock. toxin-laden pleural effusions may be present. the disease begins after a week-long incubation period and is typically biphasic with flulike symptoms (with the notable exception of rhinorrhea) occurring before a terminal phase. when anthrax of any form progresses, the grave complication of hemorrhagic meningitis can occur. the treatment of systemic anthrax syndromes (inhalational, gastrointestinal, and injectional) involves first ruling out the presence or absence of meningitis via a lumbar puncture. if meningitis is confirmed or cannot be ruled out, the treatment regimen should include central nervous system penetrating drugs, of which should be a protein synthesis inhibitor (eg, linezolid) and of which should be bactericidal (eg, meropenem). the third drug could be ciprofloxacin. if meningitis has been ruled out, ciprofloxacin and clindamycin or linezolid could be used for treatment (with deescalation of ciprofloxacin to penicillin once drug susceptibility is known). treatment is for weeks to weeks. adjunctive antibody therapies, available from the cdc, such as anthrax immune globulin, raxibacumab, and obiltoxaximab, also should be given. additionally, if present, pleural effusions, pericardial effusions, and ascites should be drained, a factor that has likely improved survival rates from inhalational anthrax in the modern era. postexposure prophylaxis, for those exposed to anthrax spores, includes both an abbreviated -dose regimen of the vaccine coupled with days of oral ciprofloxacin or doxycycline (antibody therapies can be used in this manner when no other prophylaxis method can be used). in a mass event the post-exposure prophylaxis regimen for adults can be shortened to days after the first vaccine dose or weeks after the last vaccine dose, which ever comes later. plague is caused by the gram-negative bacillus yesinia pestis and is endemic in many parts of the world, including the western united states. this zoonotic infection is naturally spread from rodents, such as prairie dogs, to humans via the bite of a flea. there are forms of plague: bubonic (the most common), pneumonic, and septicemic. if used as a bioweapon, plague is expected to present in its pneumonic form. bubonic plague is characterized by marked painful lymphadenopathy (fig. ) that develops after a -day to -day incubation period whereas pneumonic plague (fig. ) may be indistinguishable from ordinary community-acquired pneumonia but has a mortality rate that can reach %. pneumonic plague has a -day to -day incubation period. pneumonic plague is transmissible from person to person through respiratory droplets and requires patients be placed in droplet isolation. , diagnosis of pneumonic plague in an intentional attack requires a high index of suspicion and can be made through pcr, serology, and/or culture. , the treatment of plague is with aminoglycoside antibiotics, such as gentamicin or streptomycin, for days to days, whereas postexposure prophylaxis of those exposed to an aerosol in a bioweapon attack consists of oral ciprofloxacin or doxycycline. , tularemia tularemia is caused by the gram-negative bacillus francisella tularenesis and is naturally a zoonotic infection that is common in many parts of the united states. naturally, tularemia may occur through tick, fly, and mosquito bites or through contact with reservoir animals (eg, rabbits). contaminated uncooked food or water can also be a vehicle for spread. the most common presentation of tularemia is the ulceroglandular cutaneous form whereas a biologic attack likely results in pneumonic tularemia. tularemia is not contagious between humans. tularemia is notable for its low infectious dose in which inhalation of just a small number of bacilli can result in disease. because of this, it is important to notify laboratory personnel about the possibility of tularemia, so they are able to don appropriate personal protective equipment when working with clinical specimens. pneumonic tularemia occurs after a -day to -day incubation period and is essentially indistinguishable from community-acquired pneumonia. even physicians who live in endemic areas often miss the diagnosis of tularemia in its various form, highlighting the need for tularemia to be in the differential diagnosis of compatible syndromes in endemic areas. temperature-pulse disassociation may be present and can serve as a clue to diagnosis. the treatment of tularemia is aminoglycoside antibiotics, such as gentamicin or streptomycin, for days to days. postexposure prophylaxis, to those exposed to an aerosol in a biological weapons attack, is with oral doxycycline or ciprofloxacin. , botulism botulism is caused by the acetylcholine release-blocking neurotoxin released by clostridium botulinum, a ubiquitous spore-forming gram-positive rod. there are several forms of naturally occurring botulism: infant, wound, and gastrointestinal. these forms result from exposure to spores of the bacteria, which then germinate and elaborate toxin. in a biological attack, inhalational botulism is expected, and it manifests similar to gastrointestinal botulism. botulism is not contagious. , after hours to hours postexposure to spores, clinical botulism occurs. it is characterized by a symmetric, flaccid, descending paralysis without sensory symptoms. patients are afebrile and not tachycardic. cranial neuropathies are common. paralysis progresses to involve respiratory muscles and can be prolonged, requiring long durations of mechanical ventilation. diagnosis is largely clinical. confirmatory mouse bioassay testing is used to determine which of the several botulinum toxinotypes is responsible. , heptavalent antitoxin, which neutralizes toxinotypes a to g, is obtainable from the cdc and can neutralize toxin. antibiotics and babybig (california department of public health, a bivalent botulinum antitoxin used in infant botulism) are not indicated. , there was controversy over the existence of an eighth toxinotype (h) but it has been shown to be a hybrid toxin and is neutralized by a-type antitoxin. more recently, a toxinotype x has been described and is unable to be neutralized by any available antitoxin. smallpox is the only human infectious disease that has been eradicated from the planet. as such, there is little current clinical experience with this disease. smallpox is a significantly contagious disease that is spread via airborne, respiratory droplet, or direct contact route. fomites are also known to spread the virus. , the clinical presentation of smallpox begins with flulike symptoms after a -day incubation period which is followed by the characteristic rash. the rash begins in a papular form and then progresses to umbilicated lesions and finally to pustules that crust and scab. a person with smallpox is contagious only from the appearance of the rash until the rash is scabbed, a key factor that led to its control. , the rash of smallpox (fig. ) must be distinguished from similar rashes that can be seen with varicella. several points of distinction are important. the rash of smallpox is centrifugal with more lesions on the face and extremities while the varicella rash is centripetal. the lesions of the smallpox rash are all at identical stages with identical appearances whereas the rash of varicella may have lesions of different stages. the case fatality rate of smallpox was historically %. , a diagnosis of smallpox would be a national security emergency of the highest order because even case represents either a laboratory accident or a biological attack. because smallpox vaccination is no longer routine, there is a sizable amount of the world population that lacks immunity. any suspicion of smallpox should prompt infectious disease consultation, airborne isolation procedures, and notification of local, state, and national public health authorities. the cdc has a telephone consultation service in place to discuss potential cases with experts. a diagnosis of smallpox initially is based on clinical suspicion while confirmatory testing by pcr, viral culture, or electron microscopy is performed under appropriate biosafety conditions. , there is no food and drug administration-approved treatment of smallpox currently, although several experimental antiviral compounds are in late stages of clinical development and might be accessible. the smallpox vaccine is effective as postexposure prophylaxis, even during the incubation period, and should be given to all patient contacts, who also will be placed under public health surveillance. the vaccine is contraindicated in the immunosuppressed and pregnant and those with eczema. experimental attenuated vaccines may be more suitable for these individuals. [ ] [ ] [ ] additionally, the smallpox vaccine carries a risk of myocarditis. vhfs are caused by a diverse group of viruses, each with its own unique microbiological, epidemiologic, and clinical features. of this group, which ranges from yellow fever to ebola, certain are more important as potential biological weapons than others. in the biological weapons context, it is the filoviruses (ebola and marburg) as well as the arenaviruses (lassa fever, machupo, and others) that merit concern. despite their differences, this group of viruses is characterized by a clinical presentation that often includes general malaise, fever, rash, prostration, pharyngitis, nausea, vomiting, and diarrhea. disease can rapidly progress to shock and multiple organ dysfunction syndrome with disseminated intravascular coagulation and hemorrhagic manifestations. diagnosis can be made using molecular tests, but a high index of suspicion is needed to differentiate these infections from ordinary septic shock. in the ed patient, travel to endemic areas, exotic animal exposure, or laboratory work with vhfs might be the only clue to the etiology. in a biological attack, a cluster of patients with similar symptoms may present to several eds in a given region. any suspicion of a vhf should prompt immediate consultation with an infectious disease physician and state and local health authorities. although these viruses are spread via blood and body fluid exposure and do not spread between humans via the airborne route, the experience of the united states during the west africa ebola outbreak has influenced infection control recommendations. the nosocomial infections at a hospital in texas have led to recommendations for strict airborne and body fluid isolation for patients suspected of having a vhf, with transfer to definitive care at specialized units for confirmed cases. treatment is generally supportive and has proved life-saving in the case of ebola. the recent experience with ebola highlighted the fact that simple supportive care with fluids and electrolytes brought fatality rates down from % to less than %. there are several experimental treatments and vaccines (which can be used for postexposure prophylaxis) that are available for filovirus infections and arenavirus infections that would likely be used in any domestic vhf cases caused by these groups of viruses. for ebola exposures, the experimental vaccine would be indicated for postexposure prophylaxis whereas a combination of experimental antiviral agents (eg, favipiravir) and antibody-based therapies, such as zmapp, might be indicated after consultation with cdc. lassa fever can be treated with intravenous ribavirin, which is available via cdc. the possibility of vhf infection should be considered in those with severe illness and travel to areas in which these infections are endemic, such as parts of africa (eg, democratic republic of congo, uganda, and nigeria) or south america (eg, brazil and argentina). consultation of the cdc travel web site (www.cdc.gov/travel) is advised to determine specific vhf travel risks. coronaviruses (covs) are major causes of the common cold and rarely cause severe disease in immunocompetent hosts. there are, however, covs that have the capacity to cause severe disease: severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov. although both sars and mers present to the ed as ordinary upper or lower respiratory tract infections, they have distinct geographic and epidemiologic features that should alert an ed physician to the possibility of their presence. sars, which emerged in china in , was a worldwide infectious disease emergency that led to more than cases worldwide, with approximately % of cases fatal-including in the united states. the virus was zoonotic in origin and linked to human consumption of palm civet cats. the spread of the virus was abetted by the presence of superspreading events in which certain individuals infected a disproportionate number of others. the epidemic extinguished once infection control measures were instituted in health care settings and the consumption of palm civet cats ceased. mers is also a zoonotic respiratory cov that emerged in the arabian peninsula in and has been linked to contact with both bats and camels. all cases have an epidemiologic link to the arabian peninsula, including a multiple-ed superspreading event that occurred in south korea. mortality rates are approximately %. in the united states, imported mild cases have been diagnosed in travelers returning from the middle east. mers should be suspected in individuals with upper or lower respiratory infection after travel to the middle east in the prior weeks, and confirmatory molecular testing can be done in conjunction with state and local health authorities. many respiratory viral panels have the capacity to identify the presence of a cov and may be helpful in the work-up. infectious disease consultation and institution of droplet or airborne precautions are advised. there are no antivirals or vaccines available for any cov. influenza is often considered one of the highest pandemic threats. prior influenza pandemics have killed millions and have caused severe societal disruption. each modern pandemic ( , , , and ) has been linked to the emergence of a novel influenza a variant of zoonotic (avian, swine, or a combination) origin. zoonotic influenza viruses, in their first forays into humans, can cause a range of illness, ranging from ordinary influenza to fulminant disease, including pneumonia and acute respiratory distress syndrome. poor to limited nonsustained human-tohuman transmission characterizes these viruses in the prepandemic stage, with most cases linked directly or indirectly to poultry exposure. it is when sustained human-to-human transmission occurs that a pandemic is eminent. because of this threat, monitoring and surveillance efforts exist for avian influenza infections in humans and poultry. currently, of the myriad zoonotic influenza infections, the h n strain of influenza a has been deemed the highest threat amongst these viruses currently, although others (such as h n ) are also important to track. the ed physician should suspect avian influenza in travelers from china and other areas in which avian influenza is known to circulate, who present within approximately week after travel with upper or lower respiratory tract infection. additionally, domestic agricultural workers or those with agricultural contact with flulike symptoms (eg, children at fairs) also may harbor zoonotic influenza infections. diagnosis is similar to ordinary influenza, but a rapid or standard molecular test may or may not be able to detect influenza. confirmatory testing is via health authorities. treatment involves supportive care coupled to antiviral therapy with either oral oseltamivir or, if disease severity is high, intravenous peramivir. infectious disease consultation and institution of droplet precautions is advised. mosquito-borne arboviruses have increasingly taken on importance in the field of emerging infectious disease with the explosion of cases of west nile, chikungunya, and zika in the western hemisphere. additionally, local transmission of dengue fever has occurred in florida, texas, hawaii, and new york. , chikungunya, dengue fever, and zika are all spread by the aedes species of mosquitoes, which have habitats both within and outside the united states and cause clinically indistinguishable syndromes. these syndromes all involve fevers, rash, myalgias, and arthralgias. conjunctivitis has been noted with zika. prolonged debilitating arthralgias can occur with chikungunya whereas severe illness, including shock and hemorrhagic manifestations, can occur with dengue fever (especially with repeat infection with disparate strains). , zika has been linked to guillain-barré syndrome and requires special counseling regarding sexual transmission and pregnancy given its ability to cause a devastating congenital syndrome. , travel history and residence in an endemic area (eg, key west, hawaii, and texas) are important elements of the history. diagnostic testing is commercially available for each infection. consultation with the cdc travel web site is advised to assess specific risks for individual patients' travel history. no vaccines or antiviral therapies are available for these infections. emergency physicians play a crucial and unique role in the defense against emerging infectious disease and are most likely to encounter the first cases of any new infectious disease syndrome. the challenge that the emergency physician faces is that these cases do not announce themselves and are hidden among the sea of chief complaints that any ed sees on a given day. with many clinical scenarios, a busy physician may not be inclined to pursue a specific diagnosis if it "doesn't change treatment," may lengthen ed length of stay, and will not produce a result while a patient is in the ed, creating follow-up logistical problems. failing to diagnose an epidemiologically important emerging infectious disease or biological attack, however, can have tremendous cascading effects involving all sectors of society (health care, government, and economy) that can be minimized or averted with a proactive approach. in the current era, there are several molecular multianalyte tests available, some of which are clinical laboratory improvement amendments (clia) waived and available at point of care for specific infectious disease syndromes, such as gastrointestinal infections, meningitis, and respiratory infections, that can be used to increase the rate of microbial specific diagnoses in ed settings. biodefense cartridges, which probe for select bioagents, are also available. these tests, in the hands of an astute physician, can improve the nation's resiliency to infectious disease emergencies, and negative results in the right clinical context may prompt further investigation for a specific etiology. by having a working knowledge of emerging infectious disease and biothreat landscape, an emergency physician becomes a key component of the infectious disease emergency system. injectional anthrax in heroin users clinical management of potential bioterrorismrelated conditions mandell, douglas, and bennett's principles and practice of infectious diseases centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults systematic review: a century of inhalational anthrax cases from to mandell, douglas, and bennett's principles and practice of infectious diseases s principles and practice of infectious diseases clinical recognition and management of tularemia in missouri: a retrospective records review of cases mandell, douglas, and bennett's principles and practice of infectious diseases a novel botulinum neurotoxin, previously reported as serotype h, has a hybrid-like structure with regions of similarity to the structures of serotypes a and f and is neutralized with serotype a antitoxin identification and characterization of a novel botulinum neurotoxin orthopoxviruses: vaccinia (smallpox vaccine), variola (smallpox), monkeypox, and cowpox acam : the new smallpox vaccine for united states strategic national stockpile safety and immunogenicity of imvamune smallpox vaccine using different strategies for a post event scenario safety and immunogenicity of lc m , an attenuated smallpox vaccine in vaccinia-naive adults acam prescribing information mandell, douglas, and bennett's principles and practice of infectious diseases systems for rapidly detecting and treating persons with ebola virus disease -united states evidence-based guidelines for supportive care of patients with ebola virus disease experimental therapies for ebola virus disease: what have we learned? including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) summary of influenza risk assessment tool (irat) results outbreak of influenza a (h n ) variant virus infections among persons attending agricultural fairs housing infected swine -michigan and ohio lessons learned during dengue outbreaks in the united states health commissioner reports dengue virus case japanese encephalitis, west nile encephalitis, st. louis encephalitis, tick-borne encephalitis, kyasanur forest disease, alkhurma hemorrhagic fever, zika) mandell, douglas, and bennett's principles and practice of infectious diseases guillain-barre syndrome associated with zika virus infection in columbia zika virus and birth defects-reviewing the evidence for causality key: cord- -qw atrx authors: bhattacharyya, rajat; iyer, prasad; phua, ghee chee; lee, jan hau title: the interplay between coagulation and inflammation pathways in covid- -associated respiratory failure: a narrative review date: - - journal: pulm ther doi: . /s - - - sha: doc_id: cord_uid: qw atrx the novel coronavirus disease (covid- ) pandemic has caused an unprecedented worldwide socio-economic and health impact. there is increasing evidence that a combination of inflammation and hypercoagulable state are the main mechanisms of respiratory failure in these patients. this narrative review aims to summarize currently available evidence on the complex interplay of immune dysregulation, hypercoagulability, and thrombosis in the pathogenesis of respiratory failure in covid- disease. in addition, we will describe the experience of anticoagulation and anti-inflammatory strategies that have been tested. profound suppression of the adaptive and hyperactivity of innate immune systems with macrophage activation appears to be a prominent feature in this infection. immune dysregulation together with endotheliitis and severe hypercoagulability results in thromboinflammation and microvascular thrombosis in the pulmonary vasculature leading to severe respiratory distress. currently, some guidelines recommend the use of prophylactic low molecular weight heparin in all hospitalized patients, with intermediate dose prophylaxis in those needing intensive care, and the use of therapeutic anticoagulation in patients with proven or suspected thrombosis. strong recommendations cannot be made until this approach is validated by trial results. to target the inflammatory cascade, low-dose dexamethasone appears to be helpful in moderate to severe cases and trials with anti-interleukin agents (e.g., tocilizumab, anakinra, siltuximab) and non-steroidal anti-inflammatory drugs are showing early promising results. potential newer agents (e.g., janus kinase inhibitor such as ruxolitinib, baricitinib, fedratinib) are likely to be investigated in clinical trials. unfortunately, current trials are mostly examining these agents in isolation and there may be a significant delay before evidence-based practice can be implemented. it is plausible that a combination of anti-viral drugs together with anti-inflammatory and anti-coagulation medicines will be the most successful strategy in managing severely affected patients with covid- . suppression of the adaptive and hyperactivity of innate immune systems with macrophage activation appears to be a prominent feature in this infection. immune dysregulation together with endotheliitis and severe hypercoagulability results in thromboinflammation and microvascular thrombosis in the pulmonary vasculature leading to severe respiratory distress. currently, some guidelines recommend the use of prophylactic low molecular weight heparin in all hospitalized patients, with intermediate dose prophylaxis in those needing intensive care, and the use of therapeutic anticoagulation in patients with proven or suspected thrombosis. strong recommendations cannot be made until this approach is validated by trial results. to target the inflammatory cascade, low-dose dexamethasone appears to be helpful in moderate to severe cases and trials with anti-interleukin agents (e.g., tocilizumab, anakinra, siltuximab) and non-steroidal anti-inflammatory drugs are showing early promising results. potential newer agents (e.g., janus kinase inhibitor such as ruxolitinib, baricitinib, fedratinib) are likely to be investigated in clinical trials. unfortunately, current trials are mostly examining these agents in isolation and there may be a significant delay before evidence-based practice can be implemented. it is plausible that a combination of anti-viral drugs together with anti-inflammatory and anti-coagulation medicines will be the most successful strategy in a novel coronavirus infection caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), now called coronavirus disease- , has led to a global pandemic in . it has affected , , people worldwide with , deaths directly attributed to the virus at the time of writing this paper (june , ) , and the rate of new infections continues to increase at a very fast pace [ ] . while most of those infected have mild cases of the disease, up to % can have a severe clinical picture (i.e., dyspnea, tachypnea, hypoxemia and/or lung infiltrates on radiological imaging), and approximately % of the infected patients will require admission to the intensive care unit (icu) for respiratory failure, septic shock, and/ or multiple organ failure [ ] . the severe respiratory failure caused by covid- infection results in acute hypoxemia, which is associated with severe ventilation/ perfusion (v/q) mismatch and overt intrapulmonary shunting [ , ] . however, the exact mechanism of respiratory failure remains unclear and there is evidence that pulmonary microvascular thrombosis due to thrombo-inflammation may be a major factor contributing to respiratory decompensation in these patients [ ] . this narrative review aims to summarize the current available evidence on the interplay between hypercoagulability, thrombo-inflammation, and pulmonary microvascular thrombosis in covid- infection resulting in respiratory failure and how this information can be used to design clinical trials to optimize patient outcomes. we begin with a concise review of the unique properties of sars-cov- that impacts the coagulation and inflammation pathways before proceeding with an in-depth discussion on the current available medical literature. we will also discuss anticoagulation and other pharmacological strategies described for these patients that pertain to the role of the coagulation pathway in the prevention and management of covid- -induced respiratory failure. for the purpose of this review, we performed a literature search on pubmed using the following keywords: covid- , coronavirus, sars-cov- , thrombosis, and respiratory failure. this review is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. the virulence of sars-cov- sars-cov- is a novel betacoronavirus [large ribonucleic acid (rna) virus] that is similar to the earlier sars-cov virus that caused the severe acute respiratory distress (sars) outbreak in [ ] . multiple spike glycoproteins (s) protrude from the viral surface and give it a halo-like appearance (hence corona). the spike s protein enables the virus to engage with its target cell receptor, angiotensin-converting enzyme (ace ) [ ] . the host's transmembrane protease serine (tmprss ) primes the spike s protein of the virus to facilitate its internalization into the cell [ ] (fig. ) . after binding to the ace receptor, the virus enters the cell through endocytosis, releases ribonucleic acid (rna) into the cytosol, replicates by using the cell machinery, and finally, by the process of exocytosis, is excreted from the cell [ ] . ace is mostly expressed in the airway and type ii pneumocytes explaining the tropism of the virus to the respiratory system. not surprisingly, one of the major features of covid- infection is involvement of the respiratory system. importantly, ace receptors are also expressed by vascular endothelial cells [ ] . while most patients will have symptoms of respiratory tract infection and start to improve within a week, sudden clinical deterioration - days after initial symptom onset and development of severe respiratory failure often combined with multiorgan failure is commonly recognized [ ] . the group at highest risk of deterioration includes the elderly population, people with pre-existing chronic health conditions (e.g., cancer, chronic kidney disease, chronic obstructive pulmonary disease, immunocompromised state, obesity, cardiovascular comorbidities, sickle cell disease and type diabetes mellitus) [ ] . in addition, patients from certain ethnicity groups (e.g., afro-americans, south asians, and minority ethnic groups) and blood groups (e.g., blood group a with p . gene cluster) have been fig. indirect and direct injury to the lungs by the severe acute respiratory syndrome coronavirus involving the interplay between coagulation and inflammation pathways. ace angiotensin-converting enzyme , crp c-reactive protein, esr erythrocyte sedimentation rate, ldh lactate dehydrogenase, nets neutrophil extracellular traps, sars-cov- severe acute respiratory syndrome coronavirus , tmprss transmembrane protease serine shown to be at higher risk of worse outcomes [ ] [ ] [ ] (fig. ) . current literature suggests that a unique pattern of immune dysfunction is responsible for this disease progression in high-risk patients. suppression of lymphocytes by the virus leads to a complicated immune mechanism with activated macrophages which releases cytokines (fig. ). this creates a cytokine storm [mostly interleukin il- b and il- ], promoting expression of adhesion molecules for endothelial activation, inflammatory cell infiltration, and vascular inflammation. endothelial cells release pro-inflammatory cytokines that contribute to propagation of microcirculatory lesions. the dysfunctional endothelium then becomes proadhesive and pro-coagulant [ ] . presence of viral elements within endothelial cells causes endotheliitis and accumulation of inflammatory cells with increasing cell death, leading to impairment of microcirculatory function in different vascular beds and their consequent clinical effects [ ] . a study that examined the serum of covid- patients found elevated markers of neutrophil extracellular traps (nets) [ ] . nets are microbicidal proteins, extracellular webs of chromatin, and oxidant enzymes that are released by neutrophils to contain infections. nets have potential to cause inflammation and microvascular thrombosis in the lungs, causing respiratory failure. highly specific markers of nets such as myeloperoxidase (mpo)-deoxyribonucleic acid (dna), and citrullinated histone h (cit-h ) were found to be elevated in this study. these collective findings suggest a unique ability of the virus to cause intense inflammatory reaction that makes the virus lethal. severe hypercoagulability with respiratory failure is a recognized feature of covid- patients admitted in intensive care units (icu) [ ] . a combination of profound inflammation fig. currently known clinical and laboratory biomarkers of severity to predict disease progression, combining with timely antiviral, anti-inflammatory, and anticoagulation intervention to optimize outcome. chd chronic heart disease, cld chronic lung disease, ckd chronic kidney disease, doacs direct oral anticoagulants, fdps fibrinogen degradation products, htn hypertension, ifn interferon, jak janus kinase, ldh lactate dehydrogenase, lmwh low molecular weight heparin, nsaids nonsteroidal anti-inflammatory drugs, pt prothrombin time, tnf tumor necrosis factor, vw ag von willebrand antigen and microvascular thrombosis appears to be responsible for the clinical picture that leads to progressive multi-organ failure in a small percentage of patients, ultimately causing fatalities. initial response to the viral infection is by the activation of the innate immune system with neutrophils, macrophages, and cytokines. this results in elevation of acute-phase reactants like c reactive protein (crp), erythrocyte sedimentation rate (esr), and ferritin [ , ] . this can be considered as the first cytokine wave or hypercytokinemia, which is essential in controlling viral infection. dysregulated type interferon response has been demonstrated in mice models of sars-cov infection [ ] . it is postulated that similar suppressed type interferon response is present in covid- infection, which when combined with suppression of the adaptive immune system [t,b and natural killer (nk) lymphocytes] creates an immunodeficient state that results in poor and delayed viral clearance [ ] . initial hypercytokinemia, suppressed type interferon response, and the inability of the adaptive immune system to clear the virus sets the stage for hyper activation of the innate immune system and cytokine storm in some patients. cytokine dysregulation is a recognized feature of this infection. studies in hospitalized patients have shown that plasma interleukin beta(il b), il- receptor antagonist (il ra), il , il , il , il , granulocyte colony-stimulating factor (gcsf), granulocyte monocyte colony-stimulating factor (gmcsf), interferon gamma (ifnc), interferon gamma-induced protein (ip ), monocyte chemoattractant protein (mcp ), macrophage inflammatory protein alpha (mip a), macrophage inflammatory protein beta (mip b), platelet-derived growth factor (pdgf), tumor necrosis factor alpha (tnfa), and vascular endothelial growth factor (vegf) concentrations on admission were higher in patients with covid- infection compared to healthy adults [ ] . in the same study, plasma concentrations of il , il , il , gcsf, ip , mcp , mip a, and tnfa were found to be higher in patients needing icu admission compared to those who did not. disease severity has been found to positively correlate with serum levels of tnfa, il , and il , and there was also a strong negative correlation of levels of tnfa and il with the number of cd ?, cd ? t lymphocytes [ ] . in a study by diao et al., higher levels of the exhausted marker pd- were seen in covid- patients, suggesting not only that lymphocyte numbers are lower, but existing t cells are functionally exhausted. in a larger study of patients, of whom were classified as having severe disease, dysregulation of the immune system was seen with a high leucocyte count and high neutrophil-to-lymphocyte ratio [ ] . regulatory t cells that help control inflammation were also found to be low in this study, especially in patients with severe disease. these mechanisms partly explain the unabated progression of the inflammatory process, the ongoing cytokine storm, and the progressive tissue damage. the similarity of severe covid- disease to thrombotic microangiopathic anemia with elevated lactate dehydrogenase (ldh), d-dimer and bilirubin, decreased platelets, anemia, and renal and cardiac injury is suggestive of excessive complement activation. hence, complement inhibition as a therapeutic option has also been suggested [ ] . another multi-center study conducted in europe investigated immune dysregulation in patients ( with severe respiratory failure). clinical features suggestive of macrophage activation syndrome (mas) associated with low cd ? t-cells, cd ? b-cells, and nk cells were found in all the patients. sustained elevation of tnfa and il- was also found, representing a hyperactive monocyte macrophage system [ ] . in another study, lymphocyte subset analysis in covid- patients showed a statistically significant lower number of cd ? helper t cells, cd ? cytotoxic t cells, nk cells, and b cells when compared to healthy controls [ ] . in this study, patients with severe disease ( % cases) had significantly lower cd ? and cd ? t cells and b cells, although no significant differences were seen in the cd /cd ratio or nk cell numbers when compared to those with mild symptoms. these studies suggest that in this disease, a suppression of the adaptive immune system along with hyper activation of the innate immune system causes a disease phenomenon that is similar to mas/hemophagocytic lymphohistiocytosis (hlh). comparing the laboratory features, hyperferritinemia and high lactate dehydrogenase (ldh) levels are common in both covid- patients and patients with primary hlh. on the other hand, low fibrinogen and cytopenias due to bone marrow hemophagocytosis is not commonly found in covid- patients [ ] . it is likely that the pathophysiology of covid- overlaps with low-grade hlh, and further studies addressing this aspect will be important to evaluate the role of immunosuppression and hlh type therapy in these patients. the acronym covid- -associated coagulopathy (cac) is being used to describe the coagulation changes in infected patients [ ] . abnormal coagulation parameters including increased d-dimer, fibrin degradation products, and prolonged pro-thrombin time have been found to be associated with poor prognosis [ ] . in a study of patients with covid- disease, out of ( . %) non-survivors had disseminated intravascular coagulation (dic) as per the criteria set by the international society of thrombosis and haemostasis (isth). this was in contrast to only one ( . %) survivor fulfilling the criteria for dic during the period of hospitalization, indicating that non-survivors have a higher incidence of dic. described incidence of thrombocytopenia has been variable, reported as % in an initial case series of patients [ ] and . % in another series of patients [ ] . a meta-analysis analyzing pooled data from nine studies of patients including with severe disease has demonstrated that thrombocytopenia is prognostic of the severity of the disease. although there was notable heterogeneity between studies, severe thrombocytopenia was common in non-survivors [ ] and increased mortality has been associated with lower platelet counts [ ] . activated partial thromboplastin time (aptt)based clot waveform analysis (cwa) is an easily available form of global hemostatic assay and remarkably high cwa were noted despite prophylactic anticoagulation in patients admitted to the icu although there was no thrombosis or death in a study from singapore [ ] . a study on critically ill patients analyzed whole-blood thromboelastography (teg) which showed decreased r (reaction time) and k (kinetics time) values but increased k angle and ma (maximum amplitude) values, which is consistent with hypercoagulability. factor viii, von willebrand factor, fibrinogen, d-dimer, platelets, and protein c were increased in most patients, and antithrombin marginally decreased, while prothrombin time (pt) and aptt were mostly normal [ ] . the authors concluded that the above findings do not support features of acute dic but are consistent with severe hypercoagulability secondary to inflammation. another similar study in patients admitted to the icu with covid- showed fibrinolysis shutdown as evidenced by markedly raised d-dimer and complete absence of clot lysis in teg at min; this was predictive of high rate of venous thrombosis) and need for hemodialysis [ ] . this may suggest that functional coagulation assessment like teg may be more useful in assessing hypercoagulability rather than conventional coagulation tests in patients with covid- . a high rate of venous thrombosis has been observed even in anti-coagulated patients with this disease. in a french study of patients on mechanical ventilation, prophylactic anticoagulation was used in eight ( %) cases while therapeutic dose anticoagulation with low molecular weight heparin (lmwh) was started on admission in ( %) patients [ ] . all of these patients were screened by doppler ultrasound of the lower limbs. despite anticoagulation, high rates of deep vein thrombosis were noted in all patients in the prophylactic dose group and % in the therapeutic dose group that was associated with high rates of pulmonary embolism [eight ( %) in the whole cohort]. a larger study involving patients admitted in three hospitals in the netherlands and treated with prophylactic enoxaparin reported an incidence of % and % of deep vein or arterial thrombosis and pulmonary embolism, respectively [ ] . a follow-up of the same cohort of patients was published, which confirmed the high incidence of pulmonary embolism ( / , %) in those patients diagnosed with deep vein thrombosis; and pulmonary embolism was associated with higher risk of all-cause mortality [ ] . a high rate ( %) of lupus anticoagulant activity was identified in a study of patients when screened by a sensitive aptt test and direct russel viper venom test (drvvt). this may be a contributing factor in the development of thrombosis in some patients [ ] . peripheral arterial disease in the form of arteriosclerosis obliterans of lower extremity in combination with deep vein thrombosis has also been reported in severe infection, which confirms the presence of a severe hypercoagulable status in these patients [ ] . the pathophysiological mechanism of respiratory failure in covid- infection is not clear at present but there is an increasing body of evidence suggesting microthrombosis of the pulmonary vasculature is a key player (fig. ) . in covid- pneumonitis, histological changes showed pauci-inflammatory septal capillary injury with significant septal capillary fibrin deposition and neutrophil infiltration of alveolar septa [ ] . viral cytopathic and classical acute respiratory distress syndrome (ards) changes such as diffuse alveolar damage with hyaline membranes or hyperplasia of type ii pneumocyte were not prominent. deposits of terminal complement components c b- , c d, and mannose binding lectin-associated serine protease (masp) in the microvasculature was noted, suggesting activation of the alternative and lectin-based complement pathways. another study with lung autopsy findings from ten patients showed predominantly proliferative diffuse alveolar damage, epithelial viral cytopathic effects of small airway epithelium, but minimal lymphocytic infiltration [ ] . the lung injury observed in this study was secondary to formation of fibrinous thrombi in small pulmonary arteriole, endothelial tumefaction, and megakaryocyte aggregation in pulmonary capillaries, suggesting activation of coagulation cascade with microthrombus formation. the term ''microvascular covid- lung vessels obstructive thrombo-inflammatory syndrome (microclots)'' has been proposed to describe the thrombo-inflammatory lung pathology [ ] . the working hypothesis is that in individuals at risk of severe disease, an inflammatory reaction and pulmonary microvascular thrombosis leading to alveolar damage is initiated. because of the systemic endotheliitis, this thrombo-inflammatory syndrome may progress to involve a microvascular bed of other major organs contributing to multi-organ failure. it is increasingly recognized that in this infection, pulmonary thrombus develops due to a local inflammatory reaction. platelets interacting with the vascular wall, leukocytes, factor xiia, von willebrand factor and complement leads to an intense thrombo-inflammation, which is a direct consequence of vascular damage associated with viral infection [ ] . because of the unusually high incidence of thrombotic complications despite prophylactic anticoagulation, some centers recommend a higher dose for prophylactic anticoagulation (e.g., enoxaparin mg twice a day rather than the conventional dosing of mg once a day) [ ] . although there appears to be a consensus about treating all hospitalized patients with some form of anticoagulation, dosing strategies are not yet clear. the isth interim guidelines and some other expert opinions have proposed a day) . however, this approach of either standard or intermediate dose prophylaxis has been questioned and systemic anticoagulation with unfractionated heparin infusion is routinely used for critically ill patients in some centers [ ] . this approach is recommended because cac is considered to be an overwhelmingly thrombotic dic with pulmonary embolism being difficult to diagnose in patients who require mechanical ventilation. also, bleeding is noted to be rare even in sick patients, and hence the risk of therapeutic anticoagulation is not as great as other patients with sepsis-induced coagulopathy. hyperfibrinogenemia with fibrin deposition in the air spaces and lung parenchyma due to a hypofibrinolytic state is well recognized in these patients [ ] . fibrin deposition can occur even before appearance of symptoms of infection as identified in lung biopsy specimens of two patients with lung cancer [ ] . there is limited experience in the use of fibrinolytic therapy with tissue plasminogen activator (tpa). it has been used in off-label fashion in three patients with a noted improvement in oxygenation [increase in partial pressure of oxygen (pao )/ fraction of inspired oxygen (fio ) (p/f) ratio] [ ] . nebulized tpa may be effective with less risk of bleeding and this is currently being examined in a clinical trial (nct ). a recent review has recommended standard prophylactic dose of anticoagulation in hospitalized but well patients, intermediate dose prophylaxis to those admitted in icu and ards, and therapeutic anticoagulation to those with presumed or confirmed deep vein thrombosis [ ] . currently, there are trials registered with clinicaltrials.gov that are investigating anticoagulation in these patients [ ] (table ) . these are mainly adult trials with only one pediatric study. one registered study aims to look at antiplatelet agents to prevent cardiac complications. outcomes from these trials (table ) will be important for our understanding of the optimum anticoagulation strategy in this infection to prevent thrombosisassociated complications. glucocorticoids have been investigated in the treatment of covid- disease. unfortunately, the first randomized controlled trial of methylprednisolone against standard of care in beijing, china (nct ) had to be stopped because of lack of patients, with the pandemic ending in china. at the time of writing this review, reports but not the full peer-reviewed manuscript for the randomised evaluation of covid- therapy (recovery) conducted in the united kingdom provided preliminary data that low-dose dexamethasone reduced deaths by one-third in ventilated patients and by onefifth in other patients receiving oxygen, but made no difference to those with milder disease [ ] . there are several other ongoing trials of systemic steroids (nct , nct , nct , nct ) and some others looking at efficacy of inhaled steroids (nct , nct , nct ). similar to patients with sars and middle eastern respiratory syndrome (mers), the cytokine responses seen in patients with covid- have features of a cytokine storm. one study involving adult patients found significant correlation of increased baseline il levels with disease severity [ ] and this is supported by similar findings in another study [ ] . hence, some investigators have proposed using il as a biomarker for assessing disease severity and blocking il as a therapeutic strategy has been suggested [ ] . tocilizumab, a monoclonal antibody against the il receptor, has been used safely and effectively in patients with rheumatoid arthritis [ ] . a pilot trial of adding tocilizumab therapy in patients with severe covid- disease in combination with standard therapy of lopinavir, methylprednisolone, and oxygen therapy showed remarkable short-term clinical responses in out of patients within weeks of tocilizumab therapy [ ] . no adverse drug reactions attributable to tocilizumab were noted. following the success of this preliminary trial, a larger multicenter clinical trial is currently being undertaken (chictr ). another trial in belgium has been designed to observe the efficacy of the il blockers tocilizumab and siltuximab individually and in combination with the il blocker anakinra (nct ). indeed, il , il blockade, ifn-c inhibition, and tnfa inhibition are also amongst consideration for randomized control trials in patients with severe disease. high-dose intravenous immunoglobulin (ivig) has been used as an immunomodulator in patients with severe disease with good outcomes [ ] . there are several trials currently exploring the utility of ivig (nct , nct , nct , nct , and nct ). the role of non-steroidal anti-inflammatory drugs (nsaids) as therapy is extremely controversial. ibuprofen is a commonly used nsaid, which is used for fever control, usually with acetaminophen. however, ibuprofen increases the synthesis of ace enzyme and may potentially increase the risk of severity and fatality of covid infection [ ] . however, currently there is no definite evidence for or against the use of ibuprofen. the european medicine agency (ema) guidelines published on march , suggest using acetaminophen for fever but also had advised not to stop ibuprofen if the patient is on it for other reasons [ ] . currently, there are ongoing trials to assess the efficacy of inhaled ibuprofen (nct ) and also oral lipid formulation of ibuprofen (liberate trial, lipid ibuprofen versus standard of care for acute hypoxemic respiratory failure due to covid , nct ). the efficacy and safety of other nsaids are currently being evaluated, the details of which are beyond the scope of this article. combining anti-inflammatory agents with anti-viral agents is another attractive option. artificial intelligence (ai)-powered algorithm predictions have identified baricitinib, ruxolitinib, and fedratinib as potential candidates [ ] . these drugs belong to the janus kinase (jak) inhibitor class of medications that have been approved and used safely in conditions like myelofibrosis and rheumatoid arthritis. these drugs are powerful anti-inflammatory agents that target the jak-stat pathways and are likely to be effective against the deleterious consequences of elevated cytokines. of particular interest is baricitinib, which is predicted to interfere with the ability of the virus to enter and infect lung cells by receptor-mediated endocytosis [ ] . due to the favorable pharmacological and safety profile of baricitinib, it can be considered in combination with direct-acting anti-viral agents like lopinavir, remdesivir, and ritonavir to decrease the viral replication, infectivity, as well as to calm the cytokine responses. statins, another group of drugs that have anti-inflammatory and antithrombotic properties, have also been studied in covid- patients; a retrospective study showed a favorable recovery profile and lower all-cause mortality with the use of statins in hospitalized patients with covid- [ ] . convalescent plasma has historically been used to improve outcomes in a variety of viral epidemics [ ] . in theory, recovered covid- patients' plasma containing anti covid- neutralizing antibodies should provide passive immunity to patients with ongoing disease. however, a recent systematic review failed to demonstrate any benefit of this approach in patients hospitalized with covid- , in addition to uncertainty of the safety profile of convalescent plasma therapy [ ] . it has been a steep learning curve for the medical fraternity in learning how to manage critically ill patients with covid- . the existing knowledge of abnormalities in inflammatory and coagulation pathways offers opportunities for identifying biomarkers for severe disease, which combined with clinical risk factors will help in prompt identification of patients at risk of severe disease who require early and targeted intervention. better understanding of the pathophysiology suggests suppression of the adaptive immune system on one hand and a hyperactive innate immune system on the other as the two principle severity-driving mechanisms in covid- infection. this leads to a mas-like picture associated with a hypercoagulable state that causes both micro and macrovascular thrombosis in the pulmonary as well the systemic circulation. this understanding will help us develop treatment protocols targeting various inflammatory pathways as well as to optimize anticoagulation therapies. current anticoagulation practice suggests that all hospitalized patients should receive prophylactic anticoagulation, those with severe disease needing intensive care with respiratory compromise should have intermediate dose prophylaxis and therapeutic anticoagulation reserved for those with proven or suspected thrombosis. results of prospective trials will only answer if this strategy is optimum in preventing thrombotic complications and improving pulmonary and overall outcome. a multi-pronged approach combining anti-inflammatory drugs and anticoagulation with specific antiviral medications is likely the approach that will work in critically ill patients (fig. ) . many trials are currently trying to address efficacy of different antiviral, anti-inflammatory, and anticoagulation agents in isolation. unfortunately, these trials may take a significant amount of time for completion and analysis before the evidence-based recommendations can be produced. because of the enormous number of new patients over a short period, we have a unique opportunity to develop an international collaborative network and conduct large trials with many combination arms to answer the all-important question of which combination of antiviral, anti-inflammatory drugs with anticoagulation therapies are going to be the most effective in treating patients with severe disease. early intervention strategy is likely to be helpful in preventing disease progression and thereby reducing the morbidity and mortality from this pandemic. funding. no funding or sponsorship was received for this study or publication of this article. authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. disclosures. rajat bhattacharyya, prasad iyer, ghee chee phua, and lee jan hau have nothing to disclose. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study open access. this article is licensed under a creative commons attribution-noncommercial . international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images 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for covid- infection? ema advice on the use of nsaids for covid- covid- : combining antiviral and anti-inflammatory treatments baricitinib as potential treatment for -ncov acute respiratory disease in-hospital use of statins is associated with a reduced risk of mortality among individuals with covid- convalescent plasma: new evidence for an old therapeutic tool? convalescent plasma or hyperimmune immunoglobulin for people with covid- : a living systematic review key: cord- - fu cp authors: ligon, b. lee title: outbreak of marburg hemorrhagic fever in angola: a review of the history of the disease and its biological aspects date: - - journal: seminars in pediatric infectious diseases doi: . /j.spid. . . sha: doc_id: cord_uid: fu cp transmission of a dangerous infectious disease threatens not merely a local population but the world at large as the result of immigration and increased and faster travel. any outbreak elicits considerable concern and demands that various precautionary methods be instituted and that the disease be contained as quickly as possible. recently, an old disease, one that may have been present for centuries and was identified decades ago, reared its ugly head, killing more than people before it was contained. fortunately, the disease, marburg hemorrhagic fever, was limited to a small geographic area, but the devastation of lives was much greater than that of many epidemics and was a warning of the numerous factors, including fear, lack of understanding, and deception, that can exacerbate the spread of disease and hinder implementation of restraints. this article reviews the history of the disease caused by marburg virus and its biological components. a t a time when the transmission of a dangerous infectious disease threatens not merely a local population but the world at large as the result of immigration and, particularly, increased and faster travel, any outbreak elicits considerable concern and demands that various precautionary methods be instituted and that the disease be contained as quickly as possible. we recently experienced such a situation with the outbreak of a new disease, severe acute respiratory syndrome (sars), which caused the death of numerous people and raised awareness of the speed with which a disease can reach pandemic proportions. most recently, an old disease, one that may have been present for centuries and was identified decades ago, reared its ugly head, killing more than people before it was contained. fortunately, the disease, marburg hemorrhagic fever (mhf), was limited to a small geographic area, but the devastation of lives was much greater than that of many epidemics and was a warning of the numerous factors, including fear, lack of understanding, and deception, that can exacerbate the spread of disease and hinder implementation of restraints. this article reviews the history of the disease caused by marburg virus and its biological components. discovery and early outbreaks mhf was described first in , when outbreaks in germany and the former yugoslavia were linked to african green monkeys (cercopithecus aethiops) imported from a primate export facility in entebbe, uganda. a total of cases, including six from secondary transmission, ultimately were reported; seven deaths occurred in primary cases. in england, an infectious agent, unlike any previously seen, was recovered from the blood and organs of these persons. researchers called the agent marburg virus. [ ] [ ] [ ] [ ] the first recognized outbreak of marburg virus in africa occurred in february (fig ) , eliciting considerable press coverage (fig ) . the index case was a young australian who had been touring rhodesia. he was admitted to the johannesburg hospital and died on the seventh day from hemorrhage resulting from a combination of disseminated intravascular coagulation and hepatic failure. tests performed by the centers for disease control and prevention (cdc) confirmed that he had contracted marburg virus ( fig ) . two secondary cases, the first patient's traveling companion and a nurse, also were reported. both of these patients survived after being given vigorous supportive treatment and prophylactic heparin. in , two other cases occurred, one of which was in western kenya not far from the ugandan source of the monkeys implicated in the outbreak. the patient's attending physician in nairobi became the second case. yet another infection was diagnosed in when a young man who had traveled extensively in kenya became ill and died. in , a related disease, ebola hemorrhagic fever (ehf), reemerged in kikwit, democratic republic of the congo. the disease was contained by the implementation of house-tohouse searches, interviews of healthcare personnel, retrospective contact tracing, and direct follow-up of suspect cases, as well as other measures (fig ) . the onset of the earliest documented case occurred on january , , and the last patient died on july , . during that period, a total of cases of ehf were reported, including cases among healthcare workers, with an percent case fatality rate. two individuals were reported to be the source of infection in more than cases, highlighting the potential for fast and widespread transmission of diseases. three years later, an outbreak of mhf occurred in in northeastern democratic republic of the congo; it was the largest outbreak to have occurred at that time. , the epicenter of the outbreak was the village of durba in the haut-uélé district, oriental province. because of the remoteness of the area and the fact that a civil war that was occurring, access to the area and evaluation of the disease were delayed until may , when an international team of investigators was able to perform studies. these investigators identified cases ( laboratory-confirmed and suspected and later identified retrospectively). later follow-up surveillance identified more than cases through december . on march , , the bbc news service reported that at least people had died in the previous months in angola from an outbreak of a disease reported to have symptoms similar to those of ebola and thought to be marburg disease. the news service also reported that vice health minister jose van dunem had said that cases of the illness had been reported in a hospital in the city of uife, that people had died, and that two people had left the hospital without having been properly discharged. on march , , the world health organization (who) confirmed that marburg virus was the causative agent of the outbreak of viral hemorrhagic fever in uige province in northern angola. on march , , the cdc reported that their special pathogens branch had identified the presence of marburg virus in of specimens from patients who had died during the outbreak in angola. the cdc branch is a who collaborating center on viral hemorrhagic fevers. testing done in that laboratory had confirmed the presence of the virus using a combination of reverse transcriptase polymerase chain reaction (pcr), antigen-detection enzyme-linked immunosorbent assays (elisas), and virus isolation. the cdc immediately posted an outbreak notice on their travelers' health website. on that same day (march ), the bbc news service reported that the virus had reached the capital, luanda, where six victims, all of whom had visited uige recently, had been diagnosed. the news service also reported that the european union had announced that it would donate $ , us. between october , , and march , , a total of cases were identified, of which were fatal. all of the cases had originated in uige province. approximately percent of the reported cases had occurred in children aged years or younger. , by april , people were reported dead, and working conditions were extremely difficult for healthcare providers, who were facing a huge challenge. swathed in head-to-toe protective medical gear that requires half an hour to put on and minutes to remove, physicians were contending with extreme heat as well as a feeling of helplessness, knowing that there is no cure for the disease. exacerbated by fear and ignorance among the people, who feared going to the hospital because they considered it the source of the disease, the death toll continued to climb. news reports told of a crisis situation in which some angolans were taking out their anxiety on healthcare providers, requiring mobile surveillance teams in uige to suspend operations when vehicles were attacked and damaged by residents. on april , the news media reported that the organization staff in uige was notified of several workers' deaths but that teams were unable to investigate the causes of death or collect the bodies for burial. one report said that residents who erroneously thought the workers were exposing them to the virus had killed them. a spokesperson for who was reported to have explained that "the dramatic symptoms of marburg hemorrhagic fever and its frequent fatality are resulting in a high level of fear, which is further aggravated by a lack of public understanding about the disease. moreover, because the disease has no cure, hospitalization is not associated with a favorable outcome, and confidence in the medical care system has been eroded." , days later, news reports were providing details of the results of fear and ignorance that were fueling the outbreak, including that terrified residents had stoned who worker's vehicles and that they were hiding patients at home, contributing to the spread of the disease. fatomata diallo, a who country repre- sentative was quoted as explaining that the fear is seen "especially in this kind of epidemic where you have to have special clothes, like an astronaut, and come into the family to take a sick person or suspected case (fig ) . when you come to take away a body, a dead body, with all this kind of clothing, sometimes it is not easy for the community to accept it." on friday, april , who issued through the united nations an appeal for $ . million us to support the emergency response to the outbreak. who already had established an international network of laboratories, including two portable field laboratories in angola, to help in the investigation of marburg and other viral hemorrhagic fevers on april , , a report was issued stating that the outbreak of the virus that had claimed lives in angola had been confined to the province of uige, as no new cases had been detected outside the northern region. deputy health minister jose van dunem was quoted as saying that they had "circumscribed the epidemic to the province of uige" and that four provinces and the capital luanda had not reported any new cases. of the persons who had died (of a total of cases), were from the northern province of uige. he also was quoted as saying that "the disease is on a decline" and that "the epidemic is under control because we know how it is transmitted, how to break the chain of transmission." update: as of june, the ministry of health in angola had reported a total of cases and deaths in uige province. twenty-one contacts were being followed in uige municipality, and were being followed in other municipalities. alerts to potential cases existing in difficult-to-reach locations outside uige municipality had been received and plans were underway for a team to travel by helicopter to investigate these cases. http:// www.who.int/csr/don/ _ _ /en/index.html the disease presents as an acute febrile illness. it can progress within to days to severe hemorrhagic manifestations. the incubation period of to days is followed by a sudden onset marked by fever, chills, headache, and myalgia. approximately the fifth day after the onset of the symptoms, a maculopapular rash may manifest, after which the individual may experience nausea, vomiting, chest pain, sore throat, abdominal pain, and/or diarrhea. patients often develop severe hemorrhagic manifestations between days and , and fatal cases usually have some form of bleeding, often from multiple sites. symptoms become increasingly severe and can include jaundice, inflammation of the pancreas, severe weight loss, delirium, shock, liver failure, massive hemorrhaging, and multiorgan dysfunction. the fatality rates have ranged from to percent, with higher rates occurring in outbreaks lacking effective case management. , in the case of the outbreak in angola, who has reported a greater than percent case fatality rate, rendering this outbreak the deadliest on record for this rare disease: the outbreak in the democratic republic of congo from to had a case fatality rate of percent. the marburg virus, the natural reservoir for which remains unknown, causes mhf. thought to be of zoonotic origin, it is from the same family (filoviridae) as the virus that causes ehf. both viruses are among the most virulent pathogens known to infect humans, and, although both diseases have been rare occurrences, they have the capacity to cause dramatic outbreaks with high fatality rates. except for the ebola virus subtype reston, all members of the filoviridae are thought to be african viruses. the viruses have similar morphologies: long filamentous particles often have bizarre configurations and may be as long a , nm. they are nm in width and contain a single negative-sense genome that codes for seven polypeptides (fig ) . peters and coworkers have published a review of the molecular organization and replication strategy of this virus group and comparison with other closely related families. the viruses are largely destroyed by heat and acidity, but they may persist for weeks at room temperature. the surface glycoprotein self-associates to form the virion surface spikes and has high sugar content that may contribute to its low capacity to elicit neutralizing antibodies. because the origin of every index case of human filovirus disease is unknown, outbreaks thus far have been based on person-to-person transmission, which is fueled by the high and persistent viremia that makes the syndrome and by the presence of virus in other body fluids. in the original marburg outbreak, sources were tissues of monkeys used to prepare cell cultures for manufacture of poliovirus vaccine; secondary infection was limited primarily to medical workers who failed to take appropriate precautions to prevent direct contact of skin with body fluids. although available evidence suggests a nonprimate reservoir for the virus, intensive search has failed to elucidate what it might be. epidemiologic studies have yielded no evidence for an important role of airborne particles in either ebola or marburg disease, al- marburg virus replicates well in virtually all cell types, including endothelial cells, macrophages, and parenchyma cells of multiple organs, in both humans and animal models. in vivo and in vitro viral replication is associated with cellular necrosis. significant findings at the light-microscopic level include liver necrosis with councilman bodies, interstitial pneumonitis, cerebral glial nodules, and small infarcts. antigen and virions are found in abundance in fibroblasts, interstitium, and the appendages of the subcutaneous tissues in fatal cases. they may escape through small breaks in the skin or possibly through sweat glands, in which case they may be correlated with the established epidemiologic risk of close contact with patients and the touching of the deceased. inflammatory cells are not prominent. primary transmission of the virus from the natural reservoir appears to occur only in sub-saharan africa. secondary personto-person transmission occurs in both community and nosocomial settings. bausch and associates conducted two antibody surveys to assess risk factors for mhf in the democratic republic of the congo, where confirmed transmission had occurred. although the number of antibody-positive survey participants was small, these investigators were able to systematically identify and quantify several risk factors. the local mines were found to be a probable site of primary infection with the virus, most likely through exposure to the primary zoonotic reservoir; this conclusion was based on the preponderance of antibody in male miners without obvious evidence of person-to-person transmis-sion, as well as other factors. close contact with case patients with the disease or corpses was identified as a risk factor for secondary transmission of marburg virus. another possible risk factor was having received an injection in the past year, but the actual association was not clear. unprotected exposure to dead bodies is a significant cause of further spread. hence, in the outbreak in angola, mobile teams, dressed in their protective gear, prepared, transported, and buried the bodies of individuals who were fatal victims of the disease (fig ) . cultural practices of burying the dead, despite warnings otherwise, have contributed to the spread of the disease. aerosol transmission has not been described, but it cannot be eliminated as a possible means of transmission. physicians should consider the diagnosis of mhf among febrile patients who, within days before onset of fever, have traveled in an area where marburg virus is endemic; have had direct contact with blood, other body fluids, secretions, or excretions of a person or animal suspected of having mhf; or have worked in a laboratory or animal facility that handles hemorrhagic fever viruses. diagnosis is established by isolation of virus because antibodies to the agent usually are not present before death or defervescence. an elisa method correlates well with viral presence in blood, and pcr has proved to be successful. as with ehf, mhf must be differentiated from other viral hemorrhagic diseases in africa, a well as from many bacterial, rickettsial, and protozoal diseases that may have similar presentations early in the clinical course. yellow fever and rift valley fever can be eliminated by the absence of jaundice. for patients outside africa, a travel history is a primary diagnostic tool. no vaccine or curative treatment is available for mhf. supportive treatment, including balancing the patient's fluids negative stained transmission electron micrograph depicts numerous filamentous marburg virions, which had been cultured on vero cell cultures and purified on sucrose, rate-zonal gradients. note the virus' morphologic appearance with its characteristic "shepherd's crook" shape. magnification ϫ , . (photograph provided by drs. erskine palmer and russell regnery; public domain, available through the cdc.) and electrolytes, maintaining the oxygen status and blood pressure, replacing lost blood and clotting factors, and treating them for any complicating infections, should be provided. treatment also has included transfusion of fresh-frozen plasma and other preparations to replace the blood proteins important in clotting. a controversial treatment is the use of heparin to prevent the consumption of clotting factors because some researchers argue that the consumption is part of the disease process. because of the means of transmission, hospital practices should include implementing contact and droplet precautions, in addition to wearing eye protection or a face shield. , numerous public health measures were initiated during the outbreak in angola. who and international partners in the global outbreak alert and response network (goarn) worked with the ministry of health in angola to conduct an investigation and public health response to the outbreak. outbreak-control efforts were directed at providing technical support for case management, strengthening infection control in hospitals, improving surveillance and contact tracing, and educating residents about the disease and its mode or transmission. as part of a public health response, in addition to posting information for travelers, the cdc sent personnel to join the who-coordinated goarn response team. the cdc representatives assisted with epidemiologic investigation, infection control, and laboratory diagnosis. the cdc also provided laboratory and other scientific and logistical support. in most western countries, mhf is a reportable or notifiable disease. cases or outbreaks also should be reported to the who. the virus is a hazard category virus and should be handled in a containment level laboratory. strict isolation and barrier nursing are recommended, and the handling of biological specimens for diagnosis and patient management should be performed according to regulations governing risk assessment and control. case contacts or individuals with exposure in laboratories should be placed under health surveillance for days after their last exposure to infection. if they should become feverish, they should undergo risk assessment and perhaps be admitted to strict isolation pending the results of diagnostic tests. in conjunction with the who, the cdc has developed guidelines entitled "infection control for viral hemorrhagic fevers in the african health care setting." english and french versions of the manual can be obtained online at http://www. cdc.gov/ncidod/dvrd/spb/mnpages/vhfmanual.htm. an infectious disease transmitted by cercopithecus aethiops a previously unknown infectious disease contracted from monkeys: marburg virus disease fatal human disease from vervet monkeys filoviral hemorrhagic fevers: marburg and ebola fevers questions and answers about marburg hemorrhagic fever outbreak of marburg virus disease in johannesburg the reemergence of ebola hemorrhagic fever, democratic republic of the congo world health organization. marburg fever, democratic republic of the congo world health organization. viral haemorrhagic fever/marburg, democratic republic of the congo democratic republic of the congo: between civil war and the marburg virus. international committee of technical and scientific coordination of the durba epidemic risk factors for marburg hemorrhagic fever democratic republic of the congo. emerg infect dis angolans die from ebola-like bug ebola-like virus reaches luanda world health organization. marburg haemorrhagic fever in angolaupdate fears grow over angola marburg outbreak marburg virus death toll hits . cnn.com fear, ignorance fuel marburg outbreak in angola. reuters marburg outbreak contained. agence france-presse world health organization. marburg haemorrhagic fever in angolaupdate communicable disease surveillance & response (csr) filoviridae: marburg and ebola viruses in part , section , harrison's online images from the outbreak of marburg haemorrhagic fever in uige province key: cord- -d ei l authors: geddes, duncan title: the history of respiratory disease management date: - - journal: medicine (abingdon) doi: . /j.mpmed. . . sha: doc_id: cord_uid: d ei l lung diseases have shifted from infections – tuberculosis, pneumonia – to diseases of dirty air – chronic obstructive pulmonary disease, asthma and lung cancer. new diseases have emerged from industrial pollution and hiv, while better imaging has revealed others previously unrecognized. scientific advances in microbiology, imaging and clinical measurement have improved diagnosis and allowed better targeted treatment. advances in treatment have been dramatic, the most important being drugs (antibiotics, cortisone, β( )-adrenoreceptor agonists), ventilatory support (from iron lung to nasal positive-pressure ventilation), inhaled therapy (metered dose inhalers, nebulizers) and lung surgery (resections, video-assisted thoracoscopic surgery, transplantation). delivery of care has shifted from sanatoria for the rich but nothing at all for the poor, to hospitals and universal coverage. generalists have turned into super-specialists and doctors have been joined by growing numbers of professions allied to medicine (pams). management of lung disease has vastly improved but the impact of disease remains. the lungs are the most exposed of the internal organs and throughout the past years have occupied centre stage for disability and death. the diseases have changed: some fade, some emerge and others come and go, but all through this period our knowledge has grown. we understand these breathing bags better than ever before, we can see into them in ways unimagined in the th century and we have a growing armamentarium to fight their disorders. some key events are listed as a historical timeline of respiratory medicine (table ) . change is sometimes real and sometimes where the spotlight falls. lung diseases surge and sometimes seem to go. pneumonia finished off sir francis bacon in as he stuffed a chicken full of snow, rene descartes in as he taught freezing philosophy lessons to queen christina of sweden and leo tolstoy in in a lonely railway station. tuberculosis (tb) gave john keats a far from easeful death in but provided violetta in with the best last act of all in la traviata. and then these two captains of the men of death drifted away. antibiotics cured them and left the theatre empty for the diseases of dirty air. foul smoke inhalation lifted lung cancer from zero to hero, promoted chronic obstructive pulmonary disease (copd) from the wings to centre stage, and gave small cameo parts to interstitial lung diseases. the air became opaque with coal dust underground, while up above asbestos and other factory chemicals were inhaled, lingered in the lungs and slowly scarred them. new chapters were written into books of lung pathology, new x-ray patterns described. asthma annoyed william of orange and charles dickens but was never a th-century celebrity. however, years later, with the help of bill clinton and david beckham, it became a star; allergy replaced psychology as the cause, and big pharma smelt and dealt in gold. sleep apnoea moved on from dickens' fat boy snoring in the background to crest the wave of obesity and became the fastest growing branch of lung disease. and then, just as the audience was beginning to forget them, came the triumphal re-entry of the infections. tb, declining long before the discovery of streptomycin e but only in the countries of the rich e returned. of course, it had never really left at all, surging on among the malnourished and overcrowded in poorer countries. drug-resistant bacteria emerged and tb deaths rose again. pneumonia, too, had never gone away. osler in the s named it the 'old man's friend' and may have been right for donald bradman in or ronald reagan in . but pneumonia was no friend to the increasing numbers of immunocompromised individuals, their defences weakened by malnutrition (george orwell in 'how the poor die'), cancer treatments or, from , hiv (freddie mercury in ). other viruses hit the headlines. spanish flu killed million people in , and since then smaller flu epidemics, severe acute respiratory syndrome and middle eastern respiratory syndrome have shown that, in spite of vaccination, viruses are still very effective killers. in , eyes, hands, nose and ears e minimally aided by a stethoscope e were all the doctor had. he could see pallor, cyanosis or a flapping tremor but did not understand them. he could feel chest movements and tap out different tones with over the past years: c infections have declined but returned while asthma, chronic obstructive pulmonary disease and lung cancer have surged c scientific advances, especially in imaging and microbiology, have improved diagnosis c new targeted treatments with antibiotics, corticosteroids, ventilatory support and lung surgery have revolutionized management c delivery of care has shifted from inefficient remedies for the rich to specialized treatment for all duncan geddes md frcp cbe is an honorary consultant at the royal brompton hospital, london and professor of respiratory medicine at imperial college, london, uk. his main clinical and research interests are cystic fibrosis, lung cancer and emphysema. he was president of the british thoracic society and chairman of asthma uk. competing interests: none declared. elegant percussive fingers but had no idea of what was going on inside. he could smell anaerobes (new-mown hay with an arri ere gout of stale faeces) and he could wield a stethoscope as an instrument of erudition rather than knowledge. his drug treatments were herbs e poppy, willow bark, stramonium, wormwood e and his surgical skills were few. tb was treated with rest, varying diets and lifestyle changes. love and sex in particular had to be avoided e difficult as in sanatoria there was little else to do. acute infections and fevers were cupped, bled or scarified, partly in order to remove bad humours and partly to structure time while waiting for nature to kill or cure. did the doctor do more harm than good? no one knew. then came the age of scientific discovery. three key advances were pasteur's germ theory of disease, roentgen's discovery of the th century saw a cascade of scientific discoveries, but much more important was development and application of the earlier discoveries. microbiologists gave us precise diagnosis and the tools to develop vaccines and antibiotics. radiologists showed what was really going on, culminating in computed tomography scanning ( ), with its ever-greater resolution rivalling histopathology (figure ). public health improved with housing laws, school meals, free milk, mass mini x-ray vans and programmes of vaccination. lung function testing developed more slowly, taking years to move from hutchison's lung volumes to tiffeneau's flow rates. this was followed by a major academic push in the s to understand gas exchange, with attempts to make carbon monoxide transfer and body plethysmography clinically useful. more important, however, were blood gas measurements. today spirometry is encouraged in primary care, with occasional benefits over peak flow meters, but simple oximeters have become an essential tool of lung medicine. endoscopy took off slowly from the first laryngoscopy in to rigid bronchoscopy ( ), toughed out with either no anaesthesia at all or, for wimps, cocaine spray. all changed in with japanese fibreoptics; by , diagnostic bronchoscopy was being carried out by physicians, not surgeons. science and luck precede new treatments by e years. the three main types e drugs, devices and surgery e hit the healthcare market in completely different ways. new drugs get the most attention as they are launched with a fanfare, they can be prescribed as soon as they are approved, and all doctors can use them. patients report instant improvement and the makers make money. device development is slower but at least as important. specialists start to use them and slowly the word goes round. slower still and much more invisible to patients and primary care are advances in surgery. three out of thousands of new chemical drugs revolutionized lung treatment. antibiotics came first and deserve first prize. synthetic sulphonamides in the s were toxic and unsuitable for systemic use. moulds had been used to treat infections for centuries before fleming investigated penicillin in (poland, france, italy, belgium and costa rica can all claim to have got there first); florey and pfizer were essential in taking it on to manufacture in . then came streptomycin ( ), tetracycline ( ), choramphenicol ( ), erythromycin ( ), isoniazid ( ) and rifampicin ( ) . the transition from watching an infection to treating it was dramatic, and it must have been a wonderful era to be a doctor. close behind came the miracle of cortisone. in , patients with rheumatoid arthritis picked up their beds and walked, and the wonder drug began to cure all known diseases; recreational use led to cortisone parties in new york. by , the benefits and adverse effects had been quickly established clinical observation e controlled trials take longer. asthma attacks responded to treatment, and with inhaled corticosteroids came long-term control. third prize goes to the b-adrenoreceptor agonists, first isoprenaline, then salbutamol and finally the long-acting badrenoreceptor agonists. selective b -adrenoreceptor agonists are among the world's best-selling drugs for asthma, copd, sporting performance and beef production. again, there are thousands of these; some assist ventilation, some get drugs in or mucus out, but two types dominate the field: ventilators and inhalers. the simplest positive-pressure support devices, bag and mask, work, but endotracheal tube and intensive care are better. drinker's iron lungs ( ) countered respiratory paralysis from polio, other negativepressure devices e turtles, ponchos and cuirasses e being less effective. in the s, non-invasive positive-pressure ventilation took off, and devices have been improving ever since (figure ) , keeping hundreds of thousands of people alive who would, years ago, have died. linked to this are oxygen delivery devices; cylinders, liquid oxygen reservoirs and cumbersome concentrators have been replaced by neat portable versions weighing less than kg. old inhalers were inefficient: steam with pleasant-smelling herbs is soothing and still enjoyed today but delivers little to the lungs. nebulizers started in the s with a squeeze-bulb version, but the particle size needed for penetration into the lungs was not understood until s. the first efficient metereddose inhaler, based on perfume spray technology, was launched in and has lasted well. dry powder inhaler research started in s; the aerohaler Ò ( ) did not work, the rotahaler Ò ( s) and spinhaler Ò ( s) worked to some degree, but the s turbohaler and diskhaler were good and there are now over different versions. big pharma did all the research as it was rather too hum-drum for academia. it takes many small steps to climb a mountain. key were endotracheal intubation ( ), anaesthetic gases (chloroform , halothane ), antisepsis ( s), blood groups and safe transfusion ( ), antibiotics ( s) and, most important of all, brave pioneers. each new operation builds on the experience of others; artificial pneumothorax ( s) moved on to thoracoplasty ( s) and plombage ( s). the first survival from pneumonectomy was in ; the next patients died. eventually, surgery for tb turned into lung cancer resection in the s. drainage of pleural fluid was routine from (axel munte describes draining an empyema in a pet ape during a home visit in the story of san michele in ). thoracoscopy started in and advanced in the s with fibreoptics. it has since developed into video-assisted thoracoscopic surgery for investigation of pleural disease, lung biopsy, treatment of pneumothorax and simple resections. lung transplantation opened new and wonderful prospects for endstage lung disease with the first successful operations in the s and has now become routine, albeit limited by scarcity of donor organs. thoracic surgery led on to heart surgery, and by the s there was a danger that lung surgery would be left behind. however, in the s, lung surgery developed separately and has flourished. organizational change can be as important as technical. delivery of care lung medicine was a major part of the general doctor's workload in the th century until the s sanatorium movement e well the rack e and this specialization led on to chest clinics. these were alongside but separate from hospitals and endured professional snobbery. general physicians looked down on chest physicians ('chest e yes, physician e never') before the systems were integrated in the s. today, the lung specialist is often the best general physician. tb nurses morphed, after a brief spell in the wilderness, into respiratory nurses with their own training programmes. some work in primary care and some are specialist nurses in hospitals and have been joined by respiratory therapists, specialist physiotherapists, lung function measurers, psychologists and the rest. the room can now be very full as a single patient faces hoards of multi-disciplinary professionals sharing out the management. in spite of all this change lung disease, a major killer years ago, remains a major killer today. the sanitary condition of the labouring population the rack. vallencourt books london: everyman's library the story of san michele, new edition. london: john murray in: shooting an elephant; and other essays key: cord- -yy abob authors: chavez, summer; long, brit; koyfman, alex; liang, stephen y. title: coronavirus disease (covid- ): a primer for emergency physicians date: - - journal: am j emerg med doi: . /j.ajem. . . sha: doc_id: cord_uid: yy abob introduction: rapid worldwide spread of coronavirus disease (covid- ) has resulted in a global pandemic. objective: this review article provides emergency physicians with an overview of the most current understanding of covid- and recommendations on the evaluation and management of patients with suspected covid- . discussion: severe acute respiratory syndrome coronavirus (sars-cov- ), the virus responsible for causing covid- , is primarily transmitted from person-to-person through close contact (approximately ft) by respiratory droplets. symptoms of covid- are similar to other viral upper respiratory illnesses. three major trajectories include mild disease with upper respiratory symptoms, non-severe pneumonia, and severe pneumonia complicated by acute respiratory distress syndrome (ards). emergency physicians should focus on identifying patients at risk, isolating suspected patients, and informing hospital infection prevention and public health authorities. patients with suspected covid- should be asked to wear a facemask. respiratory etiquette, hand washing, and personal protective equipment are recommended for all healthcare personnel caring for suspected cases. disposition depends on patient symptoms, hemodynamic status, and patient ability to self-quarantine. conclusion: this narrative review provides clinicians with an updated approach to the evaluation and management of patients presenting to the emergency department with suspected covid- . on january , , the world health organization (who) designated an outbreak of a novel coronavirus not seen before in humans to be a "public health emergency of international concern" (pheic); this was followed by the declaration of a pandemic on march , [ , ] . severe acute respiratory syndrome coronavirus (sars-cov- ), previously referred to as -ncov, is the virus responsible for causing coronavirus disease (covid- ) [ ] [ ] [ ] [ ] [ ] . the pandemic traces its early beginnings to the report of a cluster of unexplained pneumonia cases in late december originating from a seafood and live animal market in wuhan, hubei province, china [ ] [ ] [ ] [ ] . from the outset, the causative agent was thought to be viral, with most patients reporting fever or dyspnea [ , ] . with unprecedented numbers of individuals under travel restrictions or quarantine, worldwide spread, and no known cure or vaccine yet available, covid- has proven a formidable adversary [ , [ ] [ ] [ ] . the ebola virus disease (evd) outbreak of in west africa provided valuable lessons with regards to emergency preparedness, personal protective equipment use, and triage processes and underscored the important role that emergency physicians play on the frontlines of emerging infectious diseases [ ] [ ] [ ] . we describe the virology, epidemiology, clinical presentation, radiographic and laboratory findings, current testing protocols, and management of patients presenting with covid- to the emergency department (ed). in this review article, we provide emergency physicians with best practices based on the rapidly evolving body of literature surrounding covid- . sars-cov- is a member of the coronavirus family, named for the crown-like appearance of spikes on the virus surface [ , ] . other members of the coronavirus family include middle east respiratory syndrome coronavirus (mers-cov) and sars-cov- , as well as coronaviruses responsible for the common cold (figs. and ) [ , , , ] . like mers-cov and sars-cov- , sars-cov- is a betacoronavirus and is likely associated with an animal reservoir (e.g., bats) [ , , ] . while an exact animal source has not been confirmed for covid- , many of the early cases in china were linked to a live animal and seafood market [ , , , ] . american journal of emergency medicine xxx (xxxx) xxx the majority of initial covid- cases were associated with travel to hubei province, china; however, a growing number of cases due to person-to-person transmission have been reported both in and outside of china (fig. ) [ , , , ] . up to % of covid- cases were reported to originate from hubei province in december ; as of march , the greatest number of new cases are now being reported in italy, spain, germany, and the united states (u.s.) (figs. and ) [ ] [ ] [ ] . based on what is known about other coronaviruses, experts believe covid- primarily spreads from person-to-person through close contact (approximately ft) by respiratory droplets [ , , , , ] . transmission of the virus through contaminated surfaces or fomites with subsequent contact with the eyes, nose, or mouth may also occur [ , , ] . patients are felt to be at highest risk of spreading the illness when they are most symptomatic [ , ] . limited data support viral shedding in asymptomatic patients while increased levels of viral shedding may be more pronounced in those critically ill [ ] [ ] [ ] . current epidemiologic patterns of covid- in china indicate that it is highly contagious with sustained spread; the extent of person-to-person transmission within the u.s. was initially limited but has progressed now to community transmission in many parts of the country [ ] . the current r or basic reproduction number is estimated to be n . ; for every case of covid- identified in the population, n additional cases are possible in the absence of adequate isolation. [ , [ ] [ ] [ ] in an early epidemiologic analysis of covid- cases in wuhan, china, the median patient age was years, and % were male [ ] . in the largest study to date of covid- , comprising over , patient records (up to february , ) from china, . % of patients were - years of age [ ] . while . % of these cases were reported to be mild, the overall case fatality rate was . % [ ] . few pediatric cases of covid- have been reported, with patients aged - years representing just . % of all cases [ , ] . approximately . % of laboratory confirmed cases of covid- occurred in healthcare personnel, and . % of these cases were either severe or critical [ ] . to be classified as severe, the following characteristics were required: pao /fio b , oxygen saturation ≤ %, presence of n % lung infiltrates within - h, respirations ≥ breaths/min, or dyspnea [ ] . critical patients, defined as those with septic shock, multiple organ dysfunction/failure, and/or respiratory failure, accounted for approximately % of the study population with a case fatality rate of . % [ , ] . the highest case fatality rate was observed in those older than years ( . %) [ ] . patients without comorbidities had a case fatality rate of just . %, in contrast to those with comorbid conditions such as cardiovascular disease ( . %) [ ] . caution should be exercised in interpreting early findings, as underreporting and variable testing practices have been a concern with covid- [ , ] . case fatality rates in hubei province have been reported to be % ( % confidence interval [ci] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , while those outside mainland china range from . - . % [ ] . mortality rates have been calculated to be as high as - % [ , ] . when compared to other recent epidemics such as sars ( . ) or evd ( . ), the average case fatality rate ( . %) for covid- is much lower (table ) [ , ] . in comparison, the h n influenza pandemic and influenza season were responsible for approximately times more cases [ , ] . based on what is known about similar coronaviruses, the longest potential incubation period for covid- is thought to be days from initial exposure [ , ] . the mean incubation period is . days ( % ci . - . ) but can range from to days [ , , ] . co-infections occur in - % of patients and may be higher in critical patients [ , ] . risk factors for severe covid- disease include advanced age, chronic medical conditions, immunocompromise, and cancer [ ] . data regarding pregnancy and covid- are limited [ ] . pregnant women and fetuses may be more vulnerable to covid- infection compared to the general population [ ] . there are case reports of pregnant women diagnosed with covid- complicated by adverse outcomes including preterm birth [ ] . historically, infants born to mothers with other coronaviruses such as mers-cov or sars-cov- have been small for gestational age or preterm [ ] . newborn infants are also an at-risk population [ ] . occupational exposure to pathogens is an inherent risk of working in healthcare settings [ ] . during the sars outbreak, % of healthcare professionals (hcps) in toronto, canada participating in endotracheal intubation of infected patients became infected themselves [ ] . in another study, % of sars patients in toronto had ties to the hospital setting, and % of cases were hcps [ ] . during the h n influenza pandemic, hcps were significantly more likely to develop infection (odds ratio [or] . , % ci . - . ) with a pooled prevalence of . % [ ] . as covid- has disproportionately affected hcps, emergency physicians must be vigilant about potential exposure risks and adhere to appropriate infection prevention precautions [ , ] . in italy, anywhere from to % of hcps have been infected with sars-cov- [ , ] . the symptoms of covid- are similar to other viral upper respiratory illnesses and include fever, cough, fatigue, and dyspnea [ , , ] . the differential diagnosis for covid- should be tailored to the patient and their presenting symptoms and comorbidities. influenza, respiratory syncytial virus (rsv), other viral illnesses, and bacterial pneumonia should be considered, as well as other pulmonary diseases (ie, pulmonary embolism). completing a thorough yet focused history and physical examination and obtaining collateral history from family members are vital. aside from pulmonary symptoms, patients with covid- may initially present with more vague complaints including diarrhea, lethargy, myalgias, and nausea [ , ] . patients may also experience headache, confusion, vomiting, pleurisy, sore throat, sneezing, rhinorrhea, and nasal congestion [ , ] . a case series of patients (median age . years) with covid- from wuhan, china found the most commonly reported symptoms were cough ( %), fever ( %), or dyspnea ( %) [ ] . in the same case series, patients also reported myalgias/fatigue ( %), productive cough ( %), headache ( %), hemoptysis ( %), and diarrhea ( %) [ ] . in a nationwide study of covid- cases from across china, the most common presenting symptoms included cough ( %), fever ( %), fatigue ( %), sputum production ( %), and shortness of breath ( %) [ ] . fever was not a predominant symptom at the time of initial presentation. in patients with more severe disease, dyspnea may be present in % of patients and progress to acute lung injury in % of patients [ ] . one study of patients with confirmed covid- suggests . % of patients have gastrointestinal (gi) symptoms [ ] . these symptoms may include anorexia ( . %), diarrhea ( . %), vomiting ( . %), and abdominal pain ( . %). seven of the patients had only gi symptoms with no respiratory symptoms [ ] . atypical presentations of infection in general may be more likely in the elderly and immunocompromised, who may not mount a febrile response [ , ] . to increase sensitivity and identify potential covid- patients sooner, the u.s. centers for disease control and prevention (cdc) recommends using a temperature cutoff of . f o [ ] . patients older than years of age and those with comorbidities may also present with more severe disease compared to other populations [ ] . three major trajectories for covid- have been described: mild disease with upper respiratory symptoms, non-severe pneumonia, and severe pneumonia complicated by acute respiratory distress syndrome (ards) necessitating aggressive resuscitative measures [ ] . anywhere from to % of patients may develop ards [ , ] . other complications of covid- include secondary bacterial infection, acute kidney injury, septic shock, ventilator-associated pneumonia, and cardiac injury [ , ] . an emergency medicine approach to covid- should focus on identifying and isolating patients at risk for infection, informing hospital infection prevention and local public health authorities, and engaging infectious disease and other specialists early in care. the world health organization (who) has established case and contact definitions for covid- to standardize global surveillance ( table ). most patients with confirmed covid- have had a subjective or confirmed fever and/or symptoms of acute respiratory illness (e.g., cough, difficulty breathing) [ ] . in concert with clinician judgment regarding patient presentations compatible with covid- , cdc guidelines prioritize patients from defined populations for further evaluation and testing as persons under investigation (pui) ( table ). these criteria are not exhaustive, and patients with an unestablished etiology or equivocal history of exposure may be considered for further testing on an individual basis [ ] . confirmed local covid- cases in the setting of known community transmission should reduce the threshold for further covid- evaluation in the ed. collaboration with local and state public health departments is strongly recommended [ , ] . a pui should be asked to wear a facemask to reduce risk of transmission to others in the immediate vicinity. fig. details cdc recommendations for identifying and assessing suspected covid- . emergency medical services (ems) directors and public health authorities working in conjunction with the cdc will need to modify emergency preparedness strategies to address covid- [ ] . emergency medical dispatchers should consider whether callers describing risk factors and symptoms concerning for covid- should be a contact is a person who experienced any one of the following exposures during the days before and the days after the onset of symptoms of a probable or confirmed case: • face-to-face contact with a probable or confirmed case within m and for n min; • direct physical contact with a probable or confirmed case; • direct care for a patient with probable or confirmed covid- disease without using proper personal protective equipment; or • other situations as indicated by local risk assessments. note: for confirmed asymptomatic cases, the period of contact is measured as the days before through the days after the date on which the sample was taken which led to confirmation. identified as a potential pui [ , ] . if so, ems personnel arriving onscene as well as hcps at the receiving hospital should be notified immediately to ensure proper personal protective equipment (ppe) use and confirm that appropriate isolation facilities are available [ , ] . once contact is made with the patient, initial triage and assessment should be done at least ft or meters away and minimized until the pui dons a facemask [ ] . in addition to limiting the number of ems personnel in the patient compartment, those providing any direct patient care should follow standard, droplet (surgical mask), and contact precautions (gown and gloves) while wearing eye protection (face shield or goggles) [ ] . airborne precautions (n respirator) should be employed if the patient is critically ill and/or if an aerosol-generating procedure is anticipated during transport. ideally, transport vehicles with isolated compartments or high efficiency particulate air (hepa) filtration should be used, and the patient should be transferred directly to a treatment room on arrival at the receiving healthcare facility [ ] . after the patient has been transported and ems documentation is being completed, patient compartment doors should be left open to allow proper ventilation [ ] . when cleaning the vehicle, disposable gown, gloves, surgical mask, and face shield should be worn [ ] . routine cleaning should be followed by application of a hospital-grade disinfectant, preferably one approved by the u.s. environmental protection agency (epa) for use against emerging viral pathogens including sars-cov- [ ] . patients presenting with symptoms concerning for covid- to the ed should be separated from other patients by at least ft or m and asked to wear a facemask [ , ] . ideally, stable covid- puis would be identified at time of check-in or triage and then placed in a private room with the door closed [ , ] . critically ill patients and those requiring aerosol-generating procedures should be placed in an airborne infection isolation room (aiir), also known as a negative pressure isolation room, with hepa filtration of the recirculated air [ , ] . once a pui is identified, the appropriate health department or agency and institutional personnel should be notified in an expeditious manner [ , ] . movement into and out of the patient's treatment room should be limited to only essential hcps involved in patient care [ ] . while in the room, the pui may remove their facemask [ ] . however, it is reasonable to ask the patient to wear a facemask during interactions with hcps (e.g., performing a physical examination) in the room as tolerated to contain respiratory droplets generated from coughing. hcps should either use alcohol-based hand sanitizer or wash their hands with soap and water before and after contact with a covid- pui [ , ] . they should be trained in the appropriate use of ppe per hospital guidelines, including techniques to safely doff equipment protecting mucous membranes [ ] . when caring for a stable pui, hcps should adhere to droplet (surgical mask), contact (gown and gloves), and standard precautions with the addition of eye protection (face shield or goggles) [ , , , , ] . if a pui is critically ill or an aerosol-generating procedure (e.g., endotracheal intubation, suctioning of the airway, sputum induction) is necessary, hcps should escalate to airborne precautions with the use of a fitted n respirator in place of a surgical mask [ , , ] . reusable respirators such as powered air purifying respirators (paprs) may also be used, but should be disinfected and maintained appropriately [ ] . patients with a history of covid- exposure presenting with non-infectious symptoms may be evaluated and treated in adherence to standard precautions alone [ ] . if portable studies (e.g., plain radiography) cannot be completed within the patient's room or the patient requires transport elsewhere within the ed or hospital by wheelchair or stretcher, hcps should don appropriate ppe [ , ] . healthcare professionals at the destination or receiving location should be made aware of the patient's arrival and likewise don appropriate ppe [ , ] . patients leaving their treatment room should wear a facemask, be dressed in a clean hospital gown (when possible), perform hand hygiene, and be educated in proper respiratory hygiene [ ] . personnel cleaning empty pui rooms should follow droplet, contact, and standard precautions with eye protection as infectious particles may be present [ ] . it is unclear how long sars-cov- remains in the air, but drawing parallels from other airborne disease such as tuberculosis can be helpful, particularly if an aerosol-generating procedure has been performed [ ] . frequently used surfaces should be cleaned at least twice daily with implementation of standard institutional cleaning procedures [ ] . intubation is a high-risk procedure due to the aerosolization of respiratory droplets [ , ] . rescue intubations should be avoided whenever possible as complete adherence to ppe may be inadequate in a timesensitive critical scenario [ ] . society of critical care medicine (sccm) surviving sepsis covid- guidelines recommend performing endotracheal intubation under airborne precautions, including use of a fitted n respirator and placement of the patient in an aiir [ ] . based on prior cases of hcps infected with sars-cov- while using n respirators, some experts recommend using a papr [ ] . the most experienced provider should intubate [ , ] . to reduce inadvertent contamination by touching one's face or hair, a full face shield and head cover is recommended if a papr is not used [ , ] . wrist exposure can be minimized by using longer-sleeved gloves or vertically taping gloves to the gown [ ] . applying tape circumferentially makes removing ppe more difficult and does not have added benefit [ ] . shoe covers should be avoided, as they can lead to accidental selfcontamination. instead, impermeable shoes that can be appropriately decontaminated should be worn [ ] . if available, coveralls with or without a hood may be used, but processes and training in safe doffing should be established beforehand as hcps may be less experienced in using these ppe ensembles [ ] . in order to inform decisions related to infection control. . other symptomatic individuals such as, older adults and individuals with chronic medical conditions and/or an immunocompromised state that may put them at higher risk for poor outcomes (e.g., diabetes, heart disease, receiving immunosuppressive medications, chronic lung disease, chronic kidney disease). . any persons including healthcare personnel a , who within days of symptom onset had close contact b with a suspect or laboratory-confirmed c covid- patient, or who have a history of travel from affected geographic areas d within days of their symptom onset. notes: a for healthcare personnel, testing may be considered if there has been exposure to a person with suspected covid- without laboratory confirmation. because of their often extensive and close contact with vulnerable patients in healthcare settings, even mild signs and symptoms (e.g., sore throat) of covid- should be evaluated among potentially exposed healthcare personnel. hcps may consider double gloving and positioning waste and other transport receptacles close by to limit droplet and/or contact transmission when securing contaminated equipment for disposal or reprocessing [ ] . preoxygenation should be optimized with nonaerosol generating strategies including head of bed elevation, jaw thrust, and use of positive end expiratory pressure valves. fiberoptic laryngoscopy should be avoided unless absolutely necessary as atomization of anesthetic will cause the virus to become aerosolized [ ] . preoxygenation for at least min with % oxygen before performing rapid sequence intubation (rsi) may be used with nasal cannula, though this may increase the risk of contamination [ , ] . to reduce this risk, a surgical mask can be placed on the patient over the device. non-invasive positive pressure ventilation (nippv) may increase risk of aerosolization and is not recommended for preoxygenation [ , ] . a high efficiency hydrophobic filter should be used between the facemask and the rest of the respiratory circuit [ ] . video laryngoscopy is preferred to direct laryngoscopy to increase distance between the intubator and the patient [ , ] . a closed system should be utilized for suctioning. once the intubation is complete, the emergency physician should immediately place the laryngoscope in their outer glove along with all other equipment used for intubation in a double ziplocked plastic bag [ ] . the presence of a high-efficiency particulate air (hepa) filter should be verified in the expiratory limb of the mechanical ventilator prior to patient use. the cdc has developed a real time reverse transcription polymerase chain reaction (rt-pcr) assay for detecting sars-cov- in upper and lower respiratory specimens obtained from covid- puis [ ] . [ ] a nasopharyngeal swab specimen should be collected for testing [ ] . for lower respiratory tract specimens, sputum can be obtained from patients with productive cough, otherwise bronchoalveolar lavage or tracheal aspirate can be substituted [ ] . serum samples are not necessary [ , ] . there are few data available regarding sensitivity and specificity for the test, but false negatives may be seen in asymptomatic individuals or those early in the course of their disease who may not have high viral burden [ ] . patients who test negative for covid- using a sample taken while they were symptomatic likely do not have the disease [ ] . however, the sensitivity of rt-pcr has been reported to range from % to % [ ] . a single negative rt-pcr should not be used to exclude the diagnosis, especially if the patient is in the early stages of the disease with no severe symptoms. a patient with negative rt-pcr with continued suspicion of covid- should be isolated and rechecked several days later. molecular testing (e.g., respiratory virus panel) for alternative diagnoses such as influenza should be considered for all puis [ ] . however, co-infection with other viruses may occur. anemia, lymphopenia, hypoxemia, abnormal kidney and liver function, elevated creatine kinase and d-dimer, thrombocytopenia, and increased lactate dehydrogenase can be present [ , , ] . lymphocytopenia can occur in up to % of patients [ ] . interestingly, one study found procalcitonin was elevated in just % of patients, while other inflammatory markers like serum ferritin and c-reactive protein were elevated [ ] . troponin and brain natriuretic peptide may be elevated in those with cardiac involvement and should be obtained in patients with suspected myocardial infarction or heart failure [ ] . advanced imaging such as computed tomography (ct) is not required for diagnosis and may create additional infection prevention challenges in the ed. however, if obtained, ct may demonstrate several findings. lung findings may be present on imaging before patients develop clinical manifestations. in a case series of patients from wuhan, china admitted with covid- , % had chest ct findings consistent with pneumonia [ ] . patients may also have radiographic groundglass opacities [ ] . another study of covid- patients found % of patients had bilateral pneumonia, % had unilateral pneumonia, and % had mottling and ground-glass opacities on chest x-ray and ct imaging [ ] . ultrasound can be utilized as well, as it is repeatable and reliable, has no radiation, and is inexpensive. ultrasound findings depend on the stage and severity of the disease, and it cannot detect lesions deeper in the lung. patients with covid- typically demonstrate an irregular/thickened pleural line, scattered/confluent b lines, consolidations of various sizes, and both non-translobar and translobar consolidations on lung ultrasound [ ] . pleural effusions are typically small and localized if they are present, and abnormalities are typically found in multiple lung zones. currently, no specific treatments exist nor are recommended for patients with covid- [ , , ] . several vaccines are under study, including dna-based, vector-based, and protein based vaccines [ ] . supportive care is the mainstay of treatment, preferably with acetaminophen [ , ] . if pneumonia is present on imaging or the patient is critically ill, antibiotics are recommended. patients presenting with respiratory insufficiency in the setting of potential covid- infection should be given supplementary oxygen to maintain an oxygenation saturation ≥ % but no higher than % [ , ] . up to % of patients require oxygen therapy [ ] . for those with acute hypoxemic respiratory failure who require intubation, endotracheal intubation should be performed. for those with hypoxemic respiratory failure who do not require intubation but who do not improve with conventional oxygen therapies, high flow nasal cannula (hfnc) is recommended over noninvasive positive pressure ventilation (nippv) [ ] . if hfnc is not available and there is no urgent need for intubation, a trial of nippv ventilation is recommended with frequent reassessment, though nippv increases the risk of aerosolization [ ] . nippv may result in patient improvement. the sccm does not make a clear recommendation for helmet nippv compared to mask nippv [ ] . while most recommend avoiding nippv due to the risk of aerosolization, it can be utilized safely if high risk patients are cohorted and clinicians use appropriate ppe [ ] . patients who decline despite use of hfnc or nippv should be intubated [ ] . if intubation is indicated, airborne precautions should be used with the patient ventilated using tidal volumes of - ml/kg of predicted body weight and plateau pressures b cm h o [ ] . if available, patients with severe ards may benefit from prone ventilation n h per day [ , ] . over-resuscitation with intravenous fluids should be avoided, which can potentially worsen oxygenation [ ] . even when covid- is suspected as the cause of the patient's symptoms, the who recommends administering empiric antibiotics and a neuraminidase inhibitor within h of identifying sepsis [ ] . early recognition of septic shock is critical, with management of sepsis focusing on intravenous fluid resuscitation and antibiotics [ ] . a conservative resuscitation strategy with buffered/balanced crystalloids is recommended for those in shock, and hypotonic crystalloids should be avoided [ , ] . vasopressors, preferentially norepinephrine, are indicated for persistent shock with a goal map of - mmhg [ , ] . for those with continued shock despite norepinephrine, vasopressin should be added, rather than increasing norepinephrine dose [ ] . if cardiac dysfunction is present and there is persistent hypoperfusion, dobutamine is recommended [ ] . systemic steroids (hydrocortisone mg per day) should be considered in those with vasopressor-refractory shock or for those with another indication for steroids such as chronic obstructive pulmonary disease exacerbation [ , , ] . without delaying antibiotic administration, bacterial blood cultures should be obtained [ ] . clinical trials of investigational drugs and antivirals are underway, although none are currently approved by the u.s. food and drug administration (fda) ( table ) [ , ] . remdesivir has demonstrated activity against mers-cov and sars-cov in vitro and animal models [ , ] . an in vitro study found that remdesivir and chloroquine inhibit viral infection, but further study is required [ , ] . results from a single study of over covid- patients found chloroquine was superior to control in reducing pneumonia exacerbation, improving imaging findings and virus-negative conversion, and shortening the course of the disease [ ] . a study evaluating lopinavir-ritonavir found no improvement in patient survival or differences in detectable viral rna [ ] . hydroxychloroquine and azithromycin are also under study [ ] . in a single prospective, observational study of patients with covid- , those receiving hydroxychloroquine demonstrated higher rates of viral load reduction/disappearance, though no patient centered outcomes were assessed [ ] . other medications under study include tocilizumab and favipiravir [ , ] . there are no clear data supporting harm or benefit with angiotensin converting enzyme inhibitors or ace receptor blockers (arbs) [ , ] . unless authorized through a clinically approved trial or monitored emergency use of unregistered interventions framework (meuri), unlicensed treatments should not be administered [ ] . continuous renal replacement therapy (crrt), extracorporeal membrane oxygenation (ecmo), and immunoglobulin have been utilized for management, but have not been definitively shown to be beneficial [ ] . patients with severe symptoms, hypoxemia requiring oxygen supplementation, or high risk for clinical deterioration (i.e. pneumonia on radiograph, severe comorbidities) may require admission for further management and monitoring. patients with mild symptoms and no significant comorbidities without concern for deterioration of clinical condition may be candidates for discharge, self-quarantine for two weeks, and home monitoring [ ] . these patients must have the ability to be safely isolated at home to prevent transmission to others and be carefully monitored [ ] . social distancing is a vital component of reducing the spread of the virus, comprised of limiting events, mass gatherings, and even small group meeting [ ] . individuals should remain ft or meters apart from other individuals. health departments should be involved early in the care of these patients and can assist with decisions regarding disposition, further surveillance, and testing, especially until confirmatory test results are available [ , ] . emergency physicians should counsel these patients to return for worrisome symptoms including new or worsening pulmonary complaints and fever [ , ] . development of a clinical pathway among emergency physicians, infectious disease specialists, and health departments is critical to safely evaluate covid- puis in the community. covid- is a novel coronavirus that has affected an unprecedented number of people to date. patients typically present with a combination of fever or cough and have a history of exposure to either a close contact with covid- or travel to an affected geographic area. while most patients will have mild disease, some may develop severe complications including ards and multi-organ failure, with some succumbing to the disease. special consideration should be given to those at the extremes of age, the immunocompromised, or pregnant women. no curative treatment is currently approved. emergency physicians should obtain a detailed travel history from all patients and suspect covid- in patients presenting with symptoms of an acute upper respiratory illness and fever. early recognition and isolation of a patient with covid- in the ed may help decrease exposure to other patients and healthcare personnel. future research is necessary to expand our collective knowledge of covid- and optimize patient outcomes. none. author contributions sc, sl, ak, and bl conceived the idea for this manuscript and contributed substantially to the writing and editing of the review. none. statement on the second meeting of the international health regulations ( ) emergency committee regarding the outbreak of novel coronavirus world health organization. who director-general's opening remarks at the media briefing on covid- - world health organization. who director-general's remarks at the media briefing on -ncov on q&a on coronaviruses new images of novel coronavirus sars-cov- now available|nih: national institute of allergy and infectious diseases ovel coronavirus ( -ncov) frequently asked questions and answers naming the coronavirus disease (covid- ) and the virus that causes it a timeline of the coronavirus. the new york times chp closely monitors cluster of pneumonia cases on mainland coronavirus travel restrictions, across the globe. the new york times million people in china face travel restrictions over coronavirus outbreak novel coronavirus ( -ncov) situation summary novel coronavirus ( -ncov) prevention & treatment. centers for disease control and prevention the impact of a case of ebola virus disease on emergency department visits in metropolitan dallas-fort worth ebola virus disease: preparedness and infection control lessons learned from two biocontainment units inform: emergency department evaluation and management for patients under investigation (puis) for ebola virus disease (evd)|emergency services|clinicians|ebola (ebola virus disease early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia covid- ) covid- ). date novel coronavirus ( -ncov) transmission. centers for disease control and prevention general's remarks at the media briefing on covid- outbreak on february world health organization novel coronavirus ( -ncov) cases in the u.s. centers for disease control and prevention transcript for cdc media telebriefing sars-cov- viral load in upper eespiratory specimens of infected patients presumed asymptomatic carrier transmission of covid- practical recommendations for critical care and anesthesiology teams caring for novel coronavirus ( -ncov) patients. can j anesth can anesth covid- -new insights on a rapidly changing epidemic pattern of early human-to-human transmission of wuhan how does the new coronavirus compare with the flu? livescience the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) -china more outbreak details emerge as covid- cases top , facts vs. fears: five things to help weigh your coronavirus risk report : estimating the potential total number of novel coronavirus cases in wuhan city report : severity of -novel coronavirus (ncov) clinical features of patients infected with novel 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physicians. two emergency physicians in critical condition er doctors at rush oak park hospital test positive for coronavirus. nbc chic italy has a world-class health system. the coronavirus has pushed it to the breaking point doctors: covid- pushing italian icus toward collapse novel coronavirus ( -ncov) flowchart for healthcare professionals a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical characteristics of coronavirus disease in china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of covid- patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected covid- ) interim infection prevention and control recommendations novel coronavirus ( -ncov) healthcare 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different types of clinical specimens correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases. radiology troponin and bnp use in covid- . latest in cardiology. http% a% f% fwww.acc.org% flatest-in-cardiology% farticles% f % f % f % f % f % ftroponin-and-bnp-use-in-covid findings of lung ultrasonography of novel corona virus pneumonia during the - epidemic. intensive care med world health organization. draft landscape of covid- candidate vaccines episode -covid- update. an interview with andrea duca, md. ebmedicine the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus compounds with therapeutic potential against novel respiratory coronavirus remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro drug treatment options for the -new coronavirus ( -ncov) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies a trial of lopinavir-ritonavir in adults hospitalized with severe covid- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial tocilizumab vs crrt in management of cytokine release syndrome (crs) in covid- (tacos) favipiravir combined with tocilizumab in the treatment of corona virus disease angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target are patients with hypertension and diabetes mellitus at increased risk for covid- infection? home care for patients with suspected novel coronavirus (ncov) infection presenting with mild symptoms and management of contacts covid- ): interim us guidance for risk assessment and public health management of persons with potential coronavirus disease (covid- ) exposures: geographic risk and contacts of laboratory-confirmed cases this manuscript did not utilize any grants, and it has not been presented in abstract form. this clinical review has not been published, it is not under consideration for publication elsewhere, its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in english or in any other language, including electronically without the written consent of the copyright-holder. this review does not reflect the views or opinions of the u.s. government, department of defense, u.s. army, u.s. air force, brooke army medical center, or saushec em residency program. key: cord- -cyul w authors: mcguirk, sheila m. title: disease management of dairy calves and heifers date: - - journal: veterinary clinics of north america: food animal practice doi: . /j.cvfa. . . sha: doc_id: cord_uid: cyul w this article focuses on the most important diseases of dairy calves and heifers and presents clinical approaches that can improve detection, diagnosis, and treatment of herd-based problems. a systematic herd investigation strategy is pivotal to define the problems, understand important risk factors, develop a plan, and make recommendations for disease management accurately. a review of records, colostrum and feeding routines, housing and bedding management, routine procedures, vaccination, and treatment protocols begins the investigation and determines which diagnostic procedures and testing strategies are most useful. disease management is most effective when the problem source is well defined and the exposure can be limited, calf immunity can be enhanced, or a combination of both. screening examinations performed regularly or done at strategic time points improves detection of disease, can be used to monitor treatment outcomes, and can avoid disease outbreaks. analysis are used to define problems, sources of infection, opportunities for improving resistance, disease detection, and prevention. the investigation of herd-based calf diarrhea begins with an accurate understanding of the age of onset, morbidity, and mortality data. for an endemic herd problem, it is optimal to review months of retrospective data. the minimum database includes the total number of calves born; the number of heifer calves alive at or hours (depending on when they leave the calving pen); the number affected; primary age-group affected; treatment history; and the mortality rate. for calf enteritis outbreaks, it is useful to see at least months of similar data. calf records may not be kept or may provide minimal information but a review of the adult cow records can provide enough information to calculate calf mortality rate. prospective record keeping may be necessary and forms that are simple and useful (table ) can be provided to the dairy before the investigation. a verbal clinical history is necessary and important but the scope, which is frequently dominated by the most recent cases, requires some validation from a minimum of months of records. other records of potential importance to the investigation are laboratory results from calf fecal specimens, blood cultures, tissue specimens, or postmortem examinations. most calf diarrhea problems are caused by a combination of factors, not all of which are infectious. the purpose of the herd investigation is to elucidate the potential enteric pathogens and to focus on the environment, calf immune status, nutrition, and management to define other contributing factors. colostral immunity is an essential part of enteric disease management and is discussed elsewhere in this issue. most calf diarrhea herd problems are caused by mixed infections [ ] and the agents may change over time, depending on season of the year and population dynamics within the environmental site of exposure. by analyzing the fecal shedding patterns of calves in the affected age groups, potential pathogens can be identified. knowledge of the agents can better define sources or sites of exposure, can result in the development of more effective treatment plans, and can result in more specific preventive recommendations. to determine the potential enteric pathogens to which calves have been exposed, fecal specimens are obtained from untreated calves within the affected age group. a good clinical history and calf health records provide the initial evidence for the age group of calves from which fecal specimens are obtained. the population at-risk can be confirmed by identifying the age of calves being treated for diarrhea on the day of the farm investigation. unless, the age-of-onset of diarrhea is in calves less than days of age, diagnostic tests for enterotoxigenic escherichia coli are not performed. for calf diarrhea problems with age-of-onset between and days, the age group most commonly affected in most calf diarrhea investigations, samples are submitted for rotavirus, coronavirus, salmonella spp, and cryptosporidium parvum. diarrhea problems in calves older that days or in weaned heifers may include diagnostic tests for attaching and effacing e coli, salmonella spp, eimeria spp, and giardia spp. fecal samples are obtained from a minimum of six calves by inserting a gloved finger into the rectum carefully to extract feces that are present or by gently massaging the rectal lining. most calves defecate with the stimulation and the feces can be collected into a -oz specimen cup. one should remove gloves before sampling the next calf, clean the outside surface, and seal the cap of the specimen cup. four cotton swabs can be used to obtain a rectal smear from calves that do not produce manure. the calf identification, age or birth date, and fecal consistency score are recorded as shown in table . sample handling should follow the directions of the diagnostic laboratory receiving the samples, but within minutes of collection it is best to place feces for salmonella culture into transport or selective salmonella media like tetrathionate, selenite, or both. we bring media to the farm and inoculate each with where fecal score is ¼ normal consistency; ¼ semiformed or pasty; ¼ loose but enough consistency to remain on bedding; ¼ watery feces that sift through bedding material; b ¼ blood is present. a -to -g (pea-size) portion of fresh feces. c parvum and viral samples can be prepared according to the laboratory's specifications. for acid-fast stained smears, a dry cotton swab is dipped into the fecal specimen and then used to make a thin fecal smear on each of two glass slides that are appropriately labeled and submitted for c parvum. the remainder of the fecal sample is submitted for rotavirus and coronavirus testing. from expected level of exposure to potential fecal pathogens in the environment [ ] [ ] [ ] , it is anticipated that up to % of the calves sampled may be shedding rotavirus, coronavirus, or c parvum. fecal shedding of virus in orally vaccinated calves is uncommon [ ] , so when two or more of six calves sampled are positive for the enteric viruses or c parvum or if any calf is salmonella spp positive, the exposure is considered abnormally high. enteric pathogens revealed in fecal shedding profiles can be validated as the cause of herd diarrhea problems when intestinal microscopic lesions described in postmortem specimens are consistent with the pathogens present in the feces. as shown in table , fecal shedding results show evidence of increased exposure to c parvum and salmonella newport in a herd with calf diarrhea problems in -day-old calves. previous postmortem examinations from the herd had demonstrated some intestinal villus blunting and clumping, consistent with c parvum infection, but the fibrino-purulent and necrotizing enteritis from three recently examined dead calves had not been linked to a salmonella spp isolate. with abnormal fecal shedding patterns present, locating the source of infection is important and provides incentive to find solutions that bypass, dilute, or distance calves from that site. finding the sources of infection for a dairy herd with calf diarrhea must take into account the health status of the dam, routes of infection of the potential pathogens, the traffic pattern of the calves, incubation period of the potential pathogens, and the behavior of calves. sick and bacteremic calving cows are more likely to have septicemic calves than calves with enteritis. salmonella dublin carrier cows, whether or not they have clinical manifestations, are a high risk for colostrum transmission of the disease to their calves, in which clinical disease is most common between weeks and months [ ] but can also occur at an earlier age. most enteric pathogen transmission between the dam and calf, however, occurs by fecal-oral spread by colostrum or the environment. even healthy cows have a large increase in fecal coliform bacterial counts during the periparturient period [ ] , putting the calves that commingle with cows in the calving environment at much higher risk for enteric infection. fecal-oral transmission of enteric pathogens to calves can occur by contaminated bedding; commingled animals; pets; pests; colostrums; feeds; feeding utensils; esophageal feeders; or the hands, boots, or clothing of calf caregivers. salivary secretions from sick calves that reach the mouth of susceptible calves can transmit salmonella spp and other enteric pathogens, making the disposal of refused milk, water, and feed away from the calf environment an essential aspect of disease management. esophageal feeders, balling guns, clothing, and hands can facilitate salivary-oral transmission in a dairy herd experiencing calf diarrhea problems and must be cleaned and disinfected between successive calf uses. as the newborn calf moves from the birthing pen to its final preweaning home, every place of short-term occupancy is regarded as a potential source of infection. from the maternity pen to the warming or drying area, to the transport vehicle or temporary hutch, all of the environments should be evaluated for enteric pathogens. considering the percentage of time that calves most at risk for enteric infections spend lying [ ] , the most likely environmental source of an enteric infection is the bedding. qualitative assessments of bedding cleanliness are subjective, unreliable, difficult to communicate, and easy to dismiss. objective data that can be reported and compared with appropriate benchmarks provide motivation for change. the concept of quantifying bedding bacteria as a risk assessment tool for mastitis [ , ] is well established, whereas the interest in identifying specific bacteria like multidrug-resistant salmonella in bedding material is more recent [ ] but is useful for locating an environmental site of infection. an evaluation of different calf bedding materials [ ] also demonstrates the use of this approach to assessing disease risks in the environment where calves spend most of their time. for herd problems of enteric disease in calves, bedding materials from each environment that has housed calves are submitted both for bacterial counts (university of minnesota laboratory for udder health, minnesota veterinary diagnostic laboratory, st. paul, mn) and salmonella spp culture. samples are taken with gloved hands from the perimeter of the pen in each of the four quadrants and from the center of the pen, specifically avoiding sampling fecal material. samples for bacterial count are collected in a sealable storage bag and stored in the refrigerator overnight before shipment to the laboratory. bedding samples for salmonella culture are placed in room temperature buffered peptone water pre-enrichment media, which is sealed tightly and shipped overnight to the laboratory in biohazard bag containers. bedding sample results from a dairy with diarrhea that starts in -day old calves is shown in table . on that dairy, calves leave the maternity pen, move to a currently unoccupied maternity pen hutch, from which location they are taken by truck to a second farm, where they are placed in a clean hutch. because of a -to -day incubation period for the fecal pathogens identified in the calves, bedding from a -day occupied hutch is also sampled. from the data in table , the bedding from the unoccupied hutch in the maternity pen and the truck are the most likely sources of infection for the calves on that dairy. for the enteric pathogens of most concern to calves, the incubation periods range from hours to days. when a herd diarrhea problem affects calves within the first days of life, the source of infection is usually encountered before the calf reaches its final preweaning pen. alternatively, the source of infection for diarrhea that begins after days of age is usually found in the calf housing area. the optimal bedding material for calves depends on age of the calf, temperature, cost, bedding, season, and management. when granite fines, sand, rice hulls, long wheat straw, and wood shavings were compared [ ] , performance indices were similar under the moderate temperatures of the study period but calves on sand and granite fines had more scours. adding clean, dry bedding to maintain a minimum of in between the calf and the base of the pen and the removal of all feed refusals from the calf housing area are two very effective ways to dilute and distance calves from potential enteric pathogens. continuous occupancy of calf raising facilities is a major risk factor that increases both the number and survival time of enteric pathogens in the environment. a goal of having % more calf pens than calves at maximum occupancy [ ] provides time for cleaning, sanitizing, and resting pens between successive occupants. strategic filling of calf raising facilities to empty large areas of the barn, rather than a single row at a time, can reduce endemic enteric disease of calves. disinfection protocols are useful if prior cleaning of facilities and pens has removed all organic debris; if the disinfectant is effective for the agents encountered in that facility; and if contact application, time, and surface are as specified. safe, broad-spectrum disinfectants that can be used in housing facilities, have penetration into soil or porous surfaces, can be cross-protective for boots, and can be applied in novel ways lead to greater compliance and improved calf disease management [ ] [ ] [ ] . in addition to bedding contamination, other sources of enteric disease pathogens for calves are feeds, feeding equipment, pathogens on the skin, and the pen itself. colostrum bacterial contamination is discussed elsewhere in this issue. milk replacer and pasteurized milk have a low risk for bacterial contamination, especially fecal coliform bacteria, when there is proper mixing, storage, delivery, and feeding with clean equipment. unpasteurized whole milk can present a high risk for enteric infection when it is nonsaleable milk that, if not fed immediately, has not chilled. to determine the level of risk coming from the liquid feed, a bulk tank milk culture can be performed. of most interest in reviewing the culture results is the total bacterial count and the lactose-positive (fecal) coliform count. table shows goals and the ranges in milk and milk replacer bacterial numbers from bucket . culture swabs are used to assess cleanliness of feeding equipment, esophageal feeders, nipples, feeding bottles, and buckets for potential enteric pathogens. only lactosepositive coliform growth is reported as evidence of inadequate sanitizing procedures. self-grooming, a normal behavior of calves, can introduce enteric pathogens from the skin of calves. although this is not considered a major risk factor for transmission of enteric disease, commingled calves, calves with contact across open pen dividers, or calves housed in barns that are power-washed while still occupied may be at risk. aerosolized bacterial spread of enteric pathogens, although possible [ ] , is rarely the primary source of enteric disease in calves. a review of current vaccination, routine health management, and treatment protocols is an important part of enteric disease management in calves. colostrum management, as discussed elsewhere in this issue, is the most effective way to transfer immunity to the specific enteric pathogens, enterotoxigenic e coli, coronavirus, rotavirus, and clostridium perfringens types c and d from vaccinated cows to newborn calves. similarly, vaccinated cows may transfer the benefit of gram-negative core antigen vaccine and siderophore receptor porin s newport vaccine immunity to calves. because most calf diarrhea problems occur within the first weeks of life, immune colostrum may be the only way effectively to protect young calves. the vaccines labeled for administration to the young calf to aid in preventing diarrheal diseases are limited and, with one exception (entervene-d, fort dodge, fort dodge, iowa), are administered at birth. although many extralabel protocols attempt to improve the immunity of colostrum-deprived or susceptible calves against diarrheal diseases, there is little scientific basis for safety, efficacy, or disease protection. where the veterinarian investigating calf diarrhea problems can be influential is in eliminating practices that have potential to do harm or that make calves more susceptible to disease. avoid gram-negative bacterial vaccines not labeled for young calves. do not routine medications, feed additives, and well calf treatments should be reviewed closely in calf herds with enteric disease problems. individually, feed additives like immunoglobulins, mannan oligosaccharides, coccidostats, antibiotics, direct-fed microbials, immune modulators, charcoal, amino acids, and other ingredients may benefit calves, but unrestrained combinations, concentrations, and packaged remedies may change intestinal flora, transport time, digestibility, absorption, and intestinal health of calves. simplicity and consistency is a good starting point for most calf health programs. the treatment protocol for calf diarrhea is based on early and effective detection followed by appropriate intervention. calves with diarrhea (fecal score or , with or without blood as described in table ) should be identified and currently on a treatment protocol. as part of a calf diarrhea investigation, determine the disease detection rate by dividing the number of calves currently being treated for diarrhea by the number of calves with fecal scores (loose but enough consistency to remain on bedding) and (watery feces that sift through bedding material). the goal of an % or greater detection rate can be achieved by twice weekly fecal scoring of all calves weeks of age or less. in table , the detection of one of four calves with diarrhea is below expectations. diarrhea treatment protocols for farm use must be straightforward and trainable. it should penalize neither the calf nor the person administering treatments. for compliance, it must be effective, frequently monitored, and updated. the most important component of the treatment protocol is rehydration, and intravenous and oral fluid and electrolyte therapy of calves have been reviewed [ , ] . feeding calves through the course of diarrhea maintains caloric intake and adds fluid volume and electrolytes to supplemental fluid administration. continued feeding may facilitate the induction of digestive enzymes but may not be beneficial if force-feeding is required [ ] . therapeutic antibiotics are recommended for calves with diarrhea that have signs of systemic illness [ , ] . for the herd calf diarrhea protocol, criteria for treatment is clearly established as any calf with a fecal score or as described in table . if the examination of the calf reveals blood in the feces, a temperature greater than or equal to f, or the calf is dull, depressed, or off feed a -day course of antibiotics is started. if the diarrheic calf has no signs of systemic illness, fluid therapy is the basis of the treatment protocol. to encourage voluntary suckling, dividing the normal feeding volume into three or four smaller volume feedings may be better tolerated by sick calves. milk or milk replacer is not given to diarrheic calves with a distended abdomen or to one that is down and cannot be assisted to maintain sternal recumbency. either a veterinarian is called or the recumbent calf is given intravenous fluid therapy. in addition to feeding milk, calves with diarrhea need (diarrhea score ) or qt (diarrhea score ) of warm electrolyte solution each day. the electrolyte solution can be fed immediately after (but not mixed in) the milk replacer or it can be fed at a time different than the milk replacer feeding. the approach i prefer is feeding four times per day: qt milk followed by qt oral electrolyte solution at the regular morning feeding, at noon, again at the regular afternoon feeding, and last thing in evening. alternatively, the four-time-a-day feeding schedule can provide two feedings for milk and two feedings for oral electrolyte solution for calves with a fecal score of . oral electrolyte solution (always mixed in water, not milk replacer) is fed until the diarrhea score returns to or . as the fecal consistency improves, the -qt electrolyte solution feeding can be placed between the two milk feedings. fresh warm water should be available to all calves but especially to diarrheic calves. water is either fed at pleasure or within to minutes of a milk feeding so that calves drink before they leave the buckets to lie down. the selection of a therapeutic antibiotic is based on the fecal culture results or its gram-negative bacterial spectrum [ , ] . once started, an antibiotic protocol is not changed before the -day treatment is completed. the antibiotic recommendation may look like one of the three extralabel protocols shown next. dose: mg/kg ¼ tablet ( -mg size) for a -lb calf twice daily. give two pills on the first dose. route: oral, crushed and added to milk, crushed and dissolved in water-karo syrup combination, or bolus administered by balling gun used slowly and gently. frequency: . calves ! weeks: two times daily for days . calves - weeks: three times per day . naxcel/excenel (ceftiofur) dose: mg/kg ¼ . ml for a -lb calf. this dose is . times the dose for respiratory disease and is specific for salmonella. route: in the muscle frequency: two times daily for days . nuflor (florfenicol) dose: mg/kg ¼ ml per lb. unlike the protocol for respiratory disease, calves with diarrhea receive a daily dose for days. route: subcutaneously frequency: one dose daily for days treatment is successful if the calf is aggressively eating and has a bright attitude, even if the feces stay somewhat loose (score or ). it may take to days for return to normal intestinal function and fecal consistency. pneumonia is responsible for . % and . % of preweaned and weaned heifer deaths, respectively, at an estimated cost of almost $ per calf year [ , ] . despite the importance of the disease, early detection is hampered by use of diagnostic criteria that are poor predictors of pneumonia in the preweaned calf age group. delayed diagnosis results in prolonged use of antibiotics, a high recurrence rate, the development of refractory sequelae, such as pulmonary abscessation, ear infections, and endemic herd problems. dairy calf and heifer pneumonia has serious economic consequences because subclinical, clinical, and chronic pneumonia of calves has a negative impact on growth, reproductive performance, milk production, and longevity [ ] [ ] [ ] . pneumonia is typically viewed as a postweaning problem of dairy heifers but preweaned calves are frequently affected [ , ] and are the source of pneumonia outbreaks in group pens. early detection of pneumonia is a significant problem in dairy calves, however, because typical signs of illness, such as depressed appetite, dull attitude, or an infrequent cough, are not exhibited. in investigating a dairy calf or heifer pneumonia problem, the review of records to determine morbidity and mortality data, seasonal patterns, health, management, housing, number of calves at maximum occupancy, nutrition, vaccinations, procedures, case definition, and treatment protocols is important. the site for disposal of liquid and solid feed refusals and pen management between successive calf occupants is also important. calf housing, with the number of calf pens or hutches, barn and pen construction, layout and dimensions, type, amount and condition of the bedding, calf traffic patterns, and stocking density have an impact on respiratory disease that is described elsewhere in this issue. weaning parameters, age of weaning, and routine health management procedures are additional data of importance. tests for colostral immunity, infectious disease testing, and laboratory or postmortem data are assembled and reviewed. the true age of onset of respiratory disease and prevalence is determined on the day of the herd investigation using a respiratory disease screening tool [ , ] (http://www.vetmed.wisc.edu/dms/fapm/fapmtools/calves. htm). individual calves in pens are examined and assigned a clinical score of (normal), (variation of or slightly abnormal), (abnormal), and (severely abnormal) for temperature, nasal discharge, cough and eye discharge, and ear position. calves with a total respiratory score over are considered to have respiratory disease based on bronchoalveolar fluid cytology and culture validation (mcguirk, unpublished data, ) . all of the calves are scored if there are less than calves. for larger groups, a representative sample up to calves are screened by the scoring system to determine the earliest age of onset and barn, pens, or location of most of the affected calves. in group pens, respiratory disease evaluations are similar but based on the percentage of calves in the pen with abnormal ocular or nasal discharge, abnormal ears, or coughing as shown in table . with completion of the scoring examinations, a detection rate is calculated by dividing the number of calves currently on treatment for respiratory disease by the total number of calves with a total respiratory score greater than . as with enteric disease, the goal is detection of at least % of the calves that require treatment, but this goal is rarely met until the farm is trained to use the respiratory screening procedure. with digital thermometers that have a -second reading time, an individual examination can be completed in less than minutes per calf. calves are easiest to examine between milk feedings when they are resting. the nasal discharge, eye, and ear scores can be assigned without entering the calf pen. spontaneous coughing can also be noted from outside the pen, giving the calf points in that category and obviating the need to use tracheal compression for cough induction. proactive use of the respiratory scoring system improves early detection, provides more reliable information on case rate, monitors for treatment efficacy, and determines which calves can move into the postweaning group pen. having identified the age of onset of respiratory disease through scoring, the youngest age group of affected calves is used for further diagnostic testing. if the goal of the investigation is simply to improve early detection and initiate a more effective treatment protocol, nasal swabs are obtained from six untreated calves with respiratory disease. from each calf, two deep nasal swabs are taken using flexible culturettes that contain a transport system for aerobic and anaerobic bacteria (bbl culture swab plus, benton dickenson, sparks, maryland). one of the swabs is submitted for bacterial culture and the second is submitted for mycoplasma bovis culture. from the nasal pasteurella multocida, mannheimia haemolytica, and histophilus somnus isolates, the antibiotics to which all isolates are susceptible are used to predict the susceptibility of bacterial isolates from the lung [ ] . if more than one of six calves cultures m bovis from the nose [ ] , b-lactam antibiotics are not recommended for the routine treatment protocol. from the nasal swab antibiotic susceptibility patterns shown in table , ceftiofur, florfenicol, trimethoprim-sulfonamide combination, and tulathromycin are considered suitable for respiratory disease treatment if fewer than two of six calves cultures m bovis from the nasal swab. prioritization of the antibiotic protocols is based on the farm, age of calves being treated, compliance, acceptance, and cost of the drugs. bronchoalveolar fluid collection from preweaned calves in a herd with respiratory disease is a relatively efficient way to confirm and characterize the type and severity of respiratory inflammation and provide fluid for bacterial culture. the bronchoalveolar fluid is collected from sedated calves using a sterilized, flexible  -in french catheter with a -ml balloon cuff (mila international, medical instrumentation for animals, florence, kentucky). five to minutes after administration of . mg/kg xylazine intramuscularly, the sedated calf is restrained and the nostrils are cleaned with a dry  -in gauze sponge. the head and neck of the calf are extended to facilitate passage of the sterile bronchoalveolar catheter by a person wearing surgical gloves. before catheter introduction into the nostril, sterile saline is dripped into the catheter to lubricate the guidewire stylette. the bronchoalveolar catheter is introduced into the ventral meatus of the nose through which it is advanced until it encounters resistance in the caudal pharynx. at that point, the restrainer pushes the poll of the calf's head ventrally while simultaneously elevating the ventral mandible and the catheter is advanced down the trachea during the inspiratory phase of the respiratory cycle. repeated coughing is induced with proper catheter placement and it is rapidly advanced until resistance is met as it wedges in a cranial lung lobe bronchus. a failure to induce spontaneous coughing subsequent to passage beyond the pharynx usually implies passage into the esophagus. in the wedged position, the catheter is held firmly in place while the guidewire stylette is removed. the balloon cuff is then inflated with ml of air and ml of sterile saline is infused using -ml syringes with a stopcock and catheter-tipped adapter attached. immediately after the -ml infusion, negative pressure is applied to aspirate fluid, a process that usually yields to ml of clear to mildly turbid, foamy fluid. the returned fluid sample is placed into a sterile -oz specimen cup. a second -ml infusion is introduced and aspirated as described and the pooled fluid is sealed in the specimen cup and preserved in a cooler until it can be processed. the fresh bronchoalveolar fluid sample is processed within hours of collection or refrigerated until it can be analyzed. a -ml aliquot of the pooled sample is used for bacterial and mycoplasma cultures. the remaining fluid is submitted for cytologic interpretation, which is based on routine staining of cytospin and direct smear preparations. bronchoalveolar fluid that yields homogenous (o cfu/ml) bacterial or positive m bovis culture is considered abnormal. a disproportionate lowering of macrophages (! %) or elevation of neutrophils (o %) provides evidence of an inflammatory response with or without a positive culture (mcguirk, unpublished data, ) . the ability to troubleshoot respiratory problems in calves is hampered if the problem is not respiratory disease; the methods for and detection of the problem are neither sensitive nor specific; or the treatments use inappropriate drugs, routes of administration, dose administered, duration of therapy, or storage. the perceived problem of high morbidity or poor cure rates may be a problem of poor disease definition, inaccurate diagnosis, overwhelming exposure, unusual susceptibility, ineffective treatments, or a combination of these factors. respiratory disease management of calves and heifers is not complete without a thorough review of the vaccination protocols for the herd. with preweaned respiratory disease problems, the emphasis is placed on vaccination of the adult cows and an effective colostrum feeding program. as more and more is learned about the effectiveness of vaccinating calves for respiratory pathogens in the face of maternal immunity, vaccination of preweaned calves may become more common [ ] . where colostral immunity is consistently good, most dairy heifers have the first modified live virus vaccines at or months of age. in the absence of adequate colostral immunity, earlier vaccination schedules have been instituted and, at least for viral respiratory pathogens, are relatively safe. respiratory disease investigations present three opportunities to reduce endemic problems in calves and heifers. regular implementation of a screening examination can find calves at an early age when treatment is extremely effective. scoring calves after a -or -day treatment protocol can determine which calves are cured and which require additional treatment. calves scored before moving into a group pen can result in fewer uncured pneumonia cases causing a respiratory disease outbreak in the weaning pen. nasal swab results can guide the implementation of effective treatment protocols and bronchoalveolar fluid can more specifically characterize respiratory inflammatory changes. finally, characterizing and resolving calf housing risk factors for respiratory disease can reduce the exposure to aerosolized bacteria and lower the prevalence of respiratory disease. descriptive epidemiology of morbidity and mortality in minnesota dairy heifer calves partitioning the mortality risk associated with inadequate passive transfer of colostral immunoglobulins in dairy calves part i: reference of dairy health and management in the united states mortality in swedish dairy calves and replacement heifers abortion and calf mortality in danish cattle herds morbidity from nonrespiratory diseases and mortality in dairy heifers during the first three months of life dairy replacement programs: cost and analysis what affects the costs of raising replacement dairy heifers: a multiple component analysis custom dairy heifer grower industry characteristics and contract terms pathology of calves with diarrhoea in southern britain potential risk factors for cryptosporidium infection in dairy calves prevalence of bovine group a rotavirus shedding among dairy calves in ohio association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves rotavirus shedding in feces of gnotobiotic calves orally inoculated with a commercial rotavirus-coronavirus vaccine evaluation of an indirect serum elisa and a bacteriological faecal culture test for diagnosis of salmonella serotype dublin in cattle using latent class models fecal shedding of coliform bacteria during the peripartum period in dairy cows growth performance and health of dairy calves bedded with different types of materials growth of environmental mastitis pathogens in various bedding materials bacterial counts associated with recycled newspaper bedding long-term persistence of multi-drug resistant salmonella enterica serovar newport in two dairy herds neonatal diarrhea in calves: investigation of herd management practices efficacy of two peroxygen-based disinfectants for inactivation of cryptosporidium parvum oocysts broad-spectrum microbicidal activity, toxicologic assessment, and materials compatibility of a new generation of accelerated hydrogen peroxide-based environmental surface disinfectant efficacy of directed misting application of a peroxygen disinfectant for environmental decontamination of a veterinary hospital transmission of salmonellae among calves penned individually oral electrolyte therapy intravenous fluid therapy of calves effects of additional milk replacer feeding on calf health, growth and selected blood metabolites in calves antimicrobial use in the treatment of calf diarrhea a clinical trial evaluating prophylactic and therapeutic antibiotic use on health and performance of preweaned calves efficacy of ceftiofur for treatment of experimental salmonellosis in neonatal calves veterinary and nonveterinary costs of disease in california dairies participating in the national animal health monitoring system from to calf and disease factors affecting growth in female holstein calves in florida, usa the effect of early calfhood health status on survivorship and age at first calving the relationship of calfhood morbidity with survival after calving in new york holstein herds epidemiologic and pathologic characteristics of respiratory tract disease in dairy heifers during the first three months of life calf respiratory disease and pen microenvironments in naturally ventilated calf barns in winter troubleshooting dairy calf pneumonia problems comparison of pasteurella spp. simultaneously isolated from nasal and transtracheal swabs from cattle with clinical signs of bovine respiratory disease the nasal mycoplasmal flora of healthy calves and cows evaluation of protection against virulent bovine viral diarrhea virus type in calves that had maternal antibodies and were vaccinated with a modified-live vaccine key: cord- - lakgpxp authors: yoon, sung‐won title: sovereign dignity, nationalism and the health of a nation: a study of china's response in combat of epidemics date: - - journal: stud ethn natl doi: . /j. - . . .x sha: doc_id: cord_uid: lakgpxp this paper seeks to understand the role of nationalism in china's policy towards the combat of emerging infectious diseases. by locating nationalism as a factor which facilitates or impedes global governance and international collaboration, this paper explores how nationalism influences china's political decision‐making. given her historical experience, china has in its national psyche an impulse never to become ‘the sick man of the east’ again. today, china's willingness to co‐operate with international bodies emanates out of reputational concerns rather than technical‐medical considerations. this was clearly manifested in her handling of two epidemics in recent years: the severe acute respiratory syndrome (sars) and hiv/aids episodes. this paper concludes that china's nationalism plays an inhibiting role in china's attempts to further incorporate herself into the architecture of global health governance in the long run. as well as a regulatory approach to a new disease outbreak. sars demonstrated that simple divisions between national and global health policy do not work in practice. during the outbreak, governments had to realise that a disease in any one part of the world is a threat to the rest of the world. however, ironically governments were in general reluctant to acknowledge the existence of the outbreak. most notably, the chinese government's response to sars was critical to the functioning of global governance in infectious disease control. it is a classic textbook example and a fundamental test case of how nationalism can impede or facilitate global governance and international collaboration. china had initially covered up the outbreak, but later reversed its stance to fully co-operate with the world health organization (who). scholars viewed china's initial response to sars within the context of china's poor public health infrastructure, ineffective and fragmented bureaucratic system, and political transition in leadership at the time of the outbreak. at the same time, china's remarkable u-turn towards international collaboration was demonstrated in the framework of the tremendous power of international organisation (i.e. who) in facilitating china's submission and the changing nature of international politics where sovereignty has been curtailed (eckholm : ) . some scholars have even gone further to argue that china's sars episode demonstrated the governance transition from westphalian to post-westphalian strategies (fidler ; ). yet, despite some scholars' claims based on china's initial reluctance and subsequent acquiescence to international forces, there is no evidence that china's sovereignty has been curtailed or that china has been integrated into global health governance. while the existing literature has focused on china's stunning reversal during the sars outbreak, less attention has been paid to the extent to which this turnaround has continued during the aftermath of sars. indeed, in light of the chinese leadership's attitude and commitment towards other infectious diseases such as hiv/aids and the recent cases of avian influenza, china's newfound openness did not seem to be genuine. this leads to the following questions: what were the factors that inhibited and then facilitated the collaboration during the outbreak? what eventually brought china back to 'business as usual' after the outbreak? what ultimately motivates the government's agenda and actions towards infectious diseases? this paper argues that nationalism in the form of national pride and security consciousness in china are enduring driving forces that have studies in ethnicity and nationalism: vol. , no. , shaped chinese policy towards emerging infectious diseases. by locating nationalism as a factor which facilitates or impedes international collaboration, this paper explores how the chinese leadership has exhibited ways in which nationalism affected much of their political decision-making in their quest to restore national pride and to secure china's developmental goals and national interests. it is argued that more often than not, nationalism hindered the formulation and implementation of health policy at both the provincial and national levels. maintaining a positive image of china in the international arena and securing the interests of the regime were key driving forces that affected policy-making. more specifically, this usually revolves around the ruling elites' competency in the handling of a national crisis and in its foreign policy. given the historical experiences of china, it has in its national psyche an impulse never to become the 'sick man of asia' again. the reaction of the chinese authorities at both the central and provincial levels towards unknown health threats is often to deny and cover up the disease's existence. this could be in part to avoid the stigma of being a 'sick' nation as well as to buy time in order to search for an indigenous solution to the problem. this sort of mentality belies a negotiated basis of existence behind the modern chinese nation -as if to say that there are no problems that modern china cannot handle or solve. yet it is also the same desire to appear 'healthy', 'confident' and a 'responsible' member of the international community that often swings chinese response to disclosure and collaboration. however, the latter only happens when the disease in question and china's handling of it is put under international scrutiny and often criticism. in short, china's compulsion to co-operate with international bodies emanates out of reputational concerns rather than medical considerations. nationalism can then be rapidly conjured up as a force which legitimises the draconian measures taken in the name of the nation to defend its sovereignty. one of the most immediate effects is that public health threats are often securitised and political-security solutions are sought rather than technical-medical ones. this means that any information pertaining to the outbreak is treated as classified, and is revealed on a 'need to know' basis, and collaboration is discouraged. it also means that the development of treatments is often seen as an opportunity to showcase the work of indigenous scientists who are 'able' to come up with a cure. an interrelated point pertains to the prospect of china's integration into global health governance. unless and until the chinese leadership examines the nationalistic element embedded in their approach towards growing disease sung-won yoon: sovereign dignity, nationalism and the health of a nation epidemics and globalising health challenges, china's ascendance to great power status will actually be harmed rather than helped. history is replete with examples of nationalist wars, where historical enemies and entire cities were wiped out, territories annexed and glory won, often at an exorbitant cost. history is equally manifested with examples where massive genocide has taken place in the name of the nation to realise some deranged nationalist blueprint, such as hitler's efforts to achieve a pure and superior aryan race or pol pot's infamous year zero project to restart civilisation in pursuit of a communist utopia. yet, such apocalyptic devastation is not always wrought by genocides or wars, but often through inaction, ineptness or impotence by national leaders who fail to defend the nation against aggression, man-made disasters, and pandemics. just as the great athenian statesman pericles learnt from the athenian plague in the fifth century bc and the roman emperor marcus aurelius from the outbreak of smallpox, epidemics could besiege large segments of any given population with quick and lethal consequences on a scale far greater than imperialistic wars or disastrous famines. the primitive state of medical science, limited understanding of hygiene, poor sanitary conditions, and widespread poverty often enable plagues and epidemics to thrive and spread with ease. the plague of justinian (sixth century ad), the black death (fourteenth and fifteenth centuries ad), and the bubonic plague ( - ) stand in testimony throughout the ages to remind the world of the imagery of the four horsemen of the apocalypse described in revelation : : 'and i looked, and behold, a pale horse; and his name that sat on him was death, and hell had followed him'. it was not until the advent of modern advances in science and technology that the verse was more associated with nuclear weapons than with epidemics. since the peloponnesian war in bc, when a plague that originated in ethiopia spread to the persian empire and to athens, the world has seen nation-states combat these epidemics at a localised and, at most, a national level (hays : ; porter ; watts ) . health policy on disease epidemics was a matter of sovereign discretion and exclusively dependent on the concerned nation's capabilities. when the black death (bubonic plague) in the fourteenth century spread through international travel and trading routes afflicted europe and north america, quarantine measures aimed exclusively at preventing disease threats from entering a nation from outside its borders were taken in major european nations. as in defence or foreign policy, health policy is most certainly an exclusive jurisdiction of the national government or ruling regime, and on numerous occasions, great rulers who have built their empires on military conquests have seen their legitimacy to rule crumble very quickly with the unbridled spread of pandemics in their nations. religion, as opposed to science, became the salvation from diseases and epidemics until the eighteenth century. nonetheless, the combat of plagues and epidemics has always invariably been seen and recognised as a national 'problem' for leaders, even though pathogens, bacteria and epidemics in general have no respect for political borders or sovereign rights over territory or people. however, as the rapid growth of populations and intensified human interactions in the sixteenth century and the process of industrialisation from the eighteenth century offered many communicable diseases opportunities to spread more widely, nations began to contemplate types of global response. one dimension of this response was a nascent form of international conferences. these early conferences were widely supported by the economic and political elites, as they believed that the spread of epidemic diseases would hamper the expansion of trade and the development of commerce. therefore, in retrospect, most nations' responses to epidemics were not born out of historically nationalistic gestures underpinned by a statist discourse to protect and defend against westphalian notions of sovereign dignity. instead, the nations' responses were traditionally based on more rationalistic and interest-based considerations. elites regard the protection of the nation's health to be of paramount importance, sometimes not so much to defend the citizen's right to life or liberty, but out of a less altruistic desire to ensure that the state's interests are maintained (e.g. keeping the economy vibrant, harvesting crops, maintaining troop levels, and rendering services and goods). as norman howard-jones observed, the very first international health conferences in the nineteenth century were not motivated by a wish for the general enhancement of the world's health, but by the desire to protect certain favoured (european) nations from contamination by their less favoured (eastern european) counterparts (howard-jones : - ) . therefore, earlier forms of collaborative action illuminated these politicians' concerns about the impact of outbreaks on their nationals and, in particular, the rationale that underpinned the sense of nationalism behind the ruling elite's orientation towards healthy policy. one of the fundamental pillars of understanding in the westphalian notion of sovereignty in the modern fraternity of nation-states is the very fact that sung-won yoon: sovereign dignity, nationalism and the health of a nation all nation-states are sovereign entities, with their own jurisdiction over territories and peoples. extrapolating from this, all nations are therefore born 'equal' in this fraternity of nations, as enshrined in article of the charter of the united nations that membership in the un is 'based on the principle of the sovereign equality'. the stark reality is that the members in this community of nations can hardly be equal. we hear of 'great powers' all the time, as much as we hear of the influence of the 'bi-polar' or the 'uni-polar' world. the logic that drives and motivates nations in this fraternity today is the quest to become 'great', and this desire for nationalism underpins most nationalist thinking and discourse within any given state. while there is no definite statistical evidence for this, it would not be an exaggeration to say that most nations wished that they could at least play in the finals of the world cup. for that matter, americans beam with pride as the us is consistently regarded as the only superpower left in the world, just as a significant number of russians look back nostalgically with pride to a glorious past. the french are convinced that they are the most superior civilisation and culture left in the world, and point to the vibrancy and the romance of their capital, paris, and the french dominance in luxury and designer goods industry. it is also quite clear that the chinese are extremely proud of their economic rise and perceive the hosting of the olympic games as a sign that it has made it in the world. in short, nations thrive on pride and sovereign dignity. most nations regard themselves as 'strong', founded on science, rationality, and progress. this vocabulary is found not only in discourse pertaining to health but also politics and economics. a healthy nation is strong, resilient, and able to withstand any political, economic, or health crisis. in , the french medical community thought that the disease creating havoc in the eastern mediterranean would never affect france, expecting that it be confined to weaker and less civilised populations experiencing a poor and unhealthy climate. the cholera killed , people in four days in paris (delaporte ) , thus confounding most of the elites. today when political leaders speak of a healthy nation, they more often use 'health' as an analogy to draw comparisons, alluding to their aspirations for the country to be strong in all dimensions. the 'health' of a nation is therefore founded on both symbolic and substantive terms, and they are often intertwined in reality. in symbolic terms, a 'sick' nation is one that is weak. tsar nicholas i described the crumbling ottoman empire as the 'sick man of europe', just as russia has been tagged with the same label in the last two decades after the demise of the soviet union. in asia, one saw the japanese labelling the decaying qing china the 'sick man of east asia'. qing china was not only weak economically, militarily, and politically, but was also plagued by internal stifle, unrest, and opium addiction. the most potent symbol of this 'sick man of east asia' was the imagery of chinese men hooked on opium, a drug introduced by the british to reduce its trade deficit as its demand for chinese tea, silk, and porcelain increased. this imagery has underpinned various versions of chinese nationalism ever since. william a. callahan ( ) eloquently discusses the role of shame in chinese nationalism. as callahan argues, nationalism in china very often commemorates its weaknesses rather than celebrating the glories of chinese civilisation ( : ) . the official narrative of modern china is generally a tragic tale of its fall from being the 'centre of the universe' beginning with the opium wars, to the incursion of western powers into imperial china, to the grand finale of the invasion of china by the japanese. this is intricately linked with the rise of the communist party of china (cpc) and the founding of the people's republic of china (prc), and provides the very basis of the legitimacy with which the cpc stakes its political right to reign. it is this deeply seeded shame that motivates the modern chinese state never to fall behind again -whether in economic or political terms -as modern china seeks to erase the 'shame and humiliation' today. this quest for greatness is a significant impetus for china's economic rise. in essence, 'achievement' is used as a remedy to get rid of the shame that china has written into much of its official discourse over the past developmental trajectory. one need not look far for evidence. china's national anthem written in (translated below), one year after the japanese invasion of china, poignantly documents the sense of shame behind the 'sick' nation: arise, ye who refuse to be slaves! with our very flesh and blood, let us build our new great wall! the peoples of china are in the most critical time, everybody must roar his defiance. arise! arise! arise! millions of hearts with one mind, brave the enemy's gunfire, march on! brave the enemy's gunfire, march on! march on! march on! sung-won yoon: sovereign dignity, nationalism and the health of a nation numerous scholars have documented how the obsession with 'humiliation' and the goal of turning 'grief' to 'strength' have significantly influenced china's foreign and security policy towards the united states and japan (dittmer and kim ; gries ; shambaugh : - ) . one of the most important dimensions is china's obsession with sovereignty or perceived infringements of her sovereignty or pride. this has made china hypersensitive to any sort of international criticism, it as an infringement of her sovereignty or meddling in her internal affairs. obviously one could argue that this is a political strategy of the cpc to deflect any sort of political challenge, but given the rise of nationalism within china over the last decade, one cannot help but realise that these sentiments might be more widespread than conceived. while china maintains a discourse of equality and camaraderie of friendship with other nations of the world, its actions and its policies often belie china's real intention of ascendance and greatness. as such, china is not only concerned with building capabilities associated with a great power, it is also extremely concerned with portraying an image that it is one. most interestingly, china's obsession with sovereign dignity and pride and its place in chinese nationalism is clearly manifested in its handling of two health-related issues: china's approach towards the handling of the aids epidemic and its response to the sars epidemic in - . the impulse for china's reticence on sars perhaps the most pertinent question discussed in this paper is why the chinese leadership tried to cover up the extent of sars and later took decisive action. answering this entails an understanding of a number of factors, but it is argued that nationalism and sovereign dignity played a part in china's response to sars. despite all the changes that the country has undergone, the traditional so-called 'face-saving' approach seemed to be as intact as ever and even deeply engrained in the mentality of the chinese leadership. therefore, when there is an event that could damage national pride, it is highly embarrassing for them to admit. the best way to resolve the troublesome event is to actively avoid or deny. thus the mode of dealing with national calamity is to identify a problem before the outside world discovers it, take measures to address it, and only afterwards report the improved situation. these mechanics have been generally at the heart of the chinese leadership's crisis management, particularly with regard to emerging infectious diseases. this was evident in china's response to sars. the crucial feature of the sars outbreak in china from early january through early february was that the provincial authorities kept the lid on the situation. the cpc has tended to suppress negative news such as diseases and disasters out of fear that such information would disrupt national stability. cover-ups are usually started by local officials who want to avoid embarrassment in front of their superiors due to their incapability to control the situation themselves. therefore, there is no incentive for local officials to pass the information up the chain of command. when early cases occurred in guangdong province, the local authority was at least aware of the situation. anxious to avoid criticism for their mishandling of earlier outbreaks and in an effort to maintain guangdong as a location of commercial dynamism and economic growth, the provincial leaders concealed the gravity of the disease. later, provincial party secretary zhang dejiang, the highest-ranking official in guangdong, stated: 'if we made a contrary decision, it would have been impossible to achieve a gdp growth rate of . per cent' (cctv interview, june ) . this denotes the fact that the guangdong leadership feared the impact of the disease's outbreak on national development. the potential 'loss of face' also contributed to provincial leaders' concealment, as the disastrous public health problem would eventually contribute to the negative effects on the cpc's legitimacy. sars was considered something that needed to be defeated before it became embarrassing. therefore, the suppression of information at the provincial level was not only motivated by development goals for the nation but was also prompted by the desire to save face and maintain reputation. once the virus spread internationally, the nature of the problem and the possibilities for resolution broadened significantly. when the chinese government failed to address the growing epidemic, the health problem in china became a political issue and an embarrassment for the central government. however, the central government's long-overdue response to growing infectious diseases was not reversed abruptly. beijing's initial reaction to the epidemic was silence and an unwillingness to co-operate. until early april , an international team of experts was not permitted to investigate hospitals in beijing. china's health minister zhang wenkang declared at a press conference that there were twelve sars cases in beijing but claimed that the disease had not spread to other parts of china (abraham : ) . the discourse that sars was under control was also backed by hong tao, the esteemed chinese microbiologist, who asserted that the cause of the disease was chlamydia and that the outbreak was dying down. due to sung-won yoon: sovereign dignity, nationalism and the health of a nation systematic problems in the chinese scientific community -a lack of coordination; stifling political influence; hesitation to challenge authorities; and isolation from the rest of the world -the chlamydia hypothesis was firmly established in china (enserink : ) . when the world-leading scientific labs confirmed that the causative agent was a coronavirus that had never before been seen in humans, china's persistent assertion had to be withdrawn. it was another national embarrassment that the emerging power was short of a scientific solution. there was a major turning point on april . faced with widespread international criticism of china's unresponsiveness, president hu jintao and premier wen jiabao finally declared a war against sars, calling for accurate and timely information about the disease to be provided and shared amongst units and international partners. this announcement was widely reported by chinese newspapers and television. shortly after the declaration, health minister zhang wenkang and beijing mayor meng xuenong were dismissed, ostensibly for their inadequate response to sars. premier wen went on to attend the asean-china leaders meeting in bangkok on april and stated that 'the chinese government is here in a spirit of candour, responsibility, trust and cooperation' (macan-markar ) . in hindsight, as the country's reputation suffered abroad, the leadership needed to re-establish itself as a responsible member of the international community in the eyes of its international counterparts. having exposed the emerging power's incapability of handling a disease crisis, the only way of restoring national pride was to show the world that the great china had the capacity to deal with the national threat efficiently and effectively in a short period of time. the chinese leadership made a lot of effort to inform the general public of the dangers of sars and to mobilise society in the name of the 'national' spirit. depicting the combat against sars as a 'baptism of fire' for the entire nation and urging the public to unite around the communist leadership to defeat the national crisis, the chinese government set up the 'sars control and prevention headquarters of the state council' headed by vice premier wu yi. the government created a fund for new building projects and the provision of more healthcare services (balasegaram and schnur, : ) . amazingly, the government was able to build a dedicated hospital for the treatment of sars within a week. apart from government initiatives, the propaganda department made an effort to 'nationalise' a regional outbreak into a national crisis and, in doing so, mobilised an entire nation into the battle against sars virus. the people's daily ( may ) used traditional maoist revolutionary rhetoric, such as calling for the people to 'build out a new great wall -on the great spirit of the fight against sars' (ren zhongping, ) . the propaganda department worked towards alerting the people about the disease and instilling in every individual a sense of patriotism and national duty to rally around the cpc. in guangxi province, minority groups sang songs about sars; in inner mongolia, murals were painted to depict the sars experience; and in beijing, banners spurred comrades on, harkening back to mao's campaigns during the cultural revolution (balasegaram and schnur : - ) . having decided to be transparent, the media sprung into full action with reports of 'whitecoated warriors' and 'angels in white coats', describing heroic stories of doctors and nurses working for love of their nation and its people. community leadership also re-introduced the traditional neighbourhood committee by revitalising the grassroots party structure. this committee, mainly consisting of elderly residents, barred outsiders and checked for sars symptoms in their neighbourhood. these committees created groups of ten households and appointed one volunteer. the volunteers were in turn grouped in tens and reported to a higher authority. this structure continued upwards until it meshed seamlessly with the communist party system that ruled the country (south china morning post ) . yet, what is important to note here is that this nationalism extends beyond making sars an immediate enemy of the nation. the image of a china being 'sick', the inability of chinese scientists and people to eradicate the virus independently, the ineptness of the chinese health system to effectively contain the outbreak were all smudges on the chinese image that the leadership wanted erased. by encouraging the national spirit and mobilising the energy of the entire population, the government was able to show the international community that china in fact had the capacity to deal with the national crisis, thereby restoring the national pride and dignity that was commensurate with china's international reputation and image as a rising great power. the success of this reversal did ameliorate the damage done to china's image by its previous bungling. china's handling of the sars crisis, unfortunately, does not represent its approach to epidemics in general. scrutinising the prc's approach to the hiv/aids epidemic in the country, china's reticent approach to international co-operation is clear. compared to the recent sars episode, the aids epidemic has been around for more than two decades. although its sung-won yoon: sovereign dignity, nationalism and the health of a nation effects are less 'visible' than the sars outbreak, in reality, the aids epidemic is probably more devastating and has had a much higher death toll than sars. aids, however, did not receive much attention from the central authorities until very recently. however, the actual number of aids carriers is believed to be much greater. the inaccuracy of the estimate is due to the fact that there is massive underreporting of the disease, especially the rural areas. there are many reasons for underreporting, but a shortage of adequate resources and a lack of openness in confronting the epidemic at many levels of government (provincial and local levels) are some of the major factors that contribute to china's slow response. exact figures are difficult to gauge because the government at the local level is very reticent to report on actual cases (human rights watch ). when aids was first reported in beijing in , the initial cases were treated with disdain and the disease was labelled as 'foreign'. the government warned that young women having sexual relations with foreigners could be in danger, and that 'foreigners' would facilitate aids becoming an epidemic in the prc. the government also tightened immigration controls and required all foreign students entering china to present a certificate from their country of origin testifying that they were not infected with aids. during the s, although aids began to spread from yunnan province to other parts of country, the chinese government officially denied that it had an hiv/aids problem. this was exposed as a lie by a few whistleblowers, at least one of whom was imprisoned for revealing 'state secrets' (watts : ) . therefore, the real extent of hiv/aids cases remains unknown as the government's long-standing position towards this epidemic has always been that aids is a 'foreign' disease requiring surveillance and border control, as well as action against social undesirables such as prostitutes, drug users, and blood brokers. the chinese government's response to aids was initially persistent silence and a refusal to acknowledge that the issue was serious. a major factor behind the government's recent change in its attitude towards the aids epidemic seemed to be the outbreak of sars in china in studies in ethnicity and nationalism: vol. , no. , , which exposed the dangers of not reacting to emerging infectious diseases. yet, it took almost two decades and a lot of pressure, internationally and domestically, before the chinese government decided to institute measures to 'contain' the aids epidemic. despite some candidness surrounding the discussion of aids in china, the scale of the problem is still being played down, mainly due to the government's tight control on media and infected aids patients not coming forward for fear of discrimination. thus the true extent of the problem remains unclear. yet, the chinese government was actually forced to address the aids epidemic for reasons very similar to those that led to the disclosure of the sars epidemic. it was sparked by the revelation of the very controversial case of an entire aids village in henan province, where thousands of poor farmers were infected by hiv while selling blood to the health authorities. initially the villagers were isolated and ostracised, and local authorities acted in the name of the national interest and the public good to cover up this outrageous negligence on the part of the health authorities. due to the mounting weight of evidence that china is in the grip of a major epidemic, the chinese government came under tremendous international criticism for their inaction over aids. the government seems to have increased efforts to fight aids by reversing some policies. despite enhanced intervention measures and infrastructure, stigmatisation, fear, and hidden infection constitute a vicious circle that fuels the aids epidemic in china. the prevalent societal attitude in china towards aids was and still is very prejudiced. aids is regarded as a 'foreign' disease and is associated with promiscuity, perversion, and homosexuality. hiv carriers are shunned by society as social outcasts and are seen as 'deserving' the disease because of their 'morally' decadent lifestyles. according to a survey conducted in china, seventy-five per cent of respondents said they would sung-won yoon: sovereign dignity, nationalism and the health of a nation avoid hiv/aids carriers and forty-five per cent responded that the disease was a consequence of moral degeneration (the un theme group on hiv/ aids in china ). the dominant narrative in china of these unknown diseases is characterised by a lack of understanding and education, and is 'nationalistic' in the sense that biological threats and diseases are still perceived by the majority of people to be 'foreign', even though germs do not respect political boundaries. it is very unlikely that health policy towards epidemics will be reconfigured overnight without a corresponding change in societal attitudes and government perceptions. there are other factors at work that have prevented the chinese state from addressing the aids issue openly and candidly. chinese nationalism has often compelled the chinese state to securitise any issue it perceives to be damaging to its national interests. the literature on aids has long highlighted that it is a significant security threat. significantly, other than the direct impact of aids on the constituent population, aids has grave consequences for the 'affected' nation. aids heightens the prospect of wars internally and externally and hollows out military and state capacities, weakening both to the point of failure as this is a disease that targets the most economically active and demographically most reproductive segment of any nation's populace (singer ) . moreover, aids will also have a tremendous and significant impact on the demographics of the population, as the disease has demonstrably killed off the most productive and strongest segment of the population first, rather than the infirm and weak segments. aids will dramatically increase health costs per capita within a relatively short period of time. it could possibly affect prospects for inward investment and long-term economic development. the chinese state realises that the aids epidemic might well have more disastrous and far-reaching consequences than previously thought. yet, at the same time, the chinese government realises that the inability to handle and contain any epidemics would have repercussions for their legitimacy to govern internally and china's reputation externally. given china's historical experiences, china has an obsession with sovereign dignity which has been rigidly built into her strategic and political culture. china therefore has a propensity to construe issues critical to china's interests as a zero-sum game, as the chinese outlook is very much influenced by neorealist thinking. this has rendered china hypersensitive to any threats and often to frame its responses to any challenges to its well-being by securitising these challenges. as such, china's initial reaction to the infectious diseases is most distinctly characterised by securitising the disease, as opposed to taking a biomedical and technical approach. china's incomplete turnabout on aids is in large part due to her reticence and inability to adjust her approach to perceive aids as something beyond a security issue. this is also significantly highlighted in the sars episode. right from the beginning of the sars outbreak, information about the disease was deemed to be a state secret. divulging data regarding infectious disease outbreaks could make one a defendant in a treason case (saich : ) . therefore, there was no reason to go public regarding a curious incident or with rumours of a new disease. despite recent changes, china is still characterised by its obsession with the notion of security and much information remains confidential, including information about infectious diseases. the scope of classified information is wide and can be flexibly applied to anything considered related to national security (human rights watch/asia and human rights in china : - ) . while a regulation had amended a regulation that classified high-level infectious diseases as highly secret with the secrecy extending from the first occurrence of the disease until the day it was announced, infectious diseases still remained national security matters. when a report on earlier cases of sars was produced in january in guangdong, the report was labelled neibu or 'top secret', which meant that information about the situation must be kept among only the highest national officials (human rights watch/asia and human rights in china : ). the classification of sars as a secret may have been motivated not only by the post-cold war legacy (i.e. their obsession with security), but also the leadership's desire to hide from other nations vulnerable issues which could be regarded as national threats. in order to deal with the national secret, the top officials needed to close the lid tight. an array of actions and policies demonstrated this. for example, the ministry of health explicitly ordered the heads of beijing hospitals to report sars only through channels upward on a confidential basis but not to any media. according to one ministry of health official, they had been told by higher levels that the outbreak was a closed matter: 'this came from quite high up in our ministry. . . . we did not have that information and once we were told that the outbreak was officially closed, we could not secure cooperation' (greenfeld : ) . hiv/aids has been securitised in much the same way as sars. aids activists in china have been either detained or arrested by the chinese officials for 'harming the state security' or 'revealing state secrets' sung-won yoon: sovereign dignity, nationalism and the health of a nation (benjamin kang lim ) . with regard to releasing official figures on aids patients, the government withheld information from the public while they checked political matters and prepared for immediate economic repercussions. national interests and security maintained precedence over transparency. this sort of 'secrecy' with regard to national security issues is typical of post-communist regimes and of most countries. it also characterises what might be a first response for any country that is sensitive to the judgement of international opinion. interviews with the chinese scientists involved in the aftermath of sars revealed that many of them felt that it was a great shame that the virus was not first identified by chinese scientists, since the virus outbreak occurred primarily in china and the majority of the victims were chinese (ensernik : - ) . in fact, many of the scientists were frustrated by the way information was 'partitioned' and the epidemic 'securitised'. yet, it is not only that securitisation of this epidemic that would pose a problem. a more important problem is how the prc elites could possibly believe that there could be a response other than a bio-medical solution to a health threat. this situation may be slowly changing as the chinese government becomes more amenable to outside co-operation as long as their regime and defined national interests are not compromised. there seems to be an inverse correlation between china's propensity to cooperate internationally and her desire to protect her core national interest and national aspiration i.e. reunification with taiwan. this aspiration is intricately linked to the regime's legitimacy and political survival and if threatened would lead the chinese government to become more hardline and nationalistic, and less willing to discuss any form of cross-border cooperation. the sars episode saw the regime's legitimacy challenged by the fact that the authorities appeared to be quite inept at handling the crisis, but more importantly the sars episode was also politicised as a reunification and a security issue. chinese nationalism complicated the handling of this medical crisis across the taiwan straits. preventing taiwanese independence has been a foremost chinese national priority. in that respect, china has consistently exerted tremendous political and diplomatic pressure to prevent taiwan from gaining membership in any international organisation. taiwan has made seven efforts to join the who. china insists that taiwan is an 'inalienable' part of china and should therefore not be recognised as an individual entity. when sars cases mounted in taiwan, the taiwanese government asked the who for assistance. in response to this, zhang wenkang, china's minister of health, stated: 'we hope that the leaders of taiwan authority no longer spread rumours with ulterior motives, or even use the disease as an excuse and in the name of human rights to try to enter the who, which is only opened to sovereign nations' (mirsky ) . this symbolised china's long-standing position towards taiwan, which denies taiwan's place in the international system. but it also reflected how chinese leaders regarded sovereignty issues over other impending issues such as the immediate crisis of the epidemic. for china's leadership, the health crisis was no longer a health issue but a political one. there is no guessing how the ruling elites would choose when deciding between political survival and enhancing international co-operation. the process of globalisation significantly impacts the socio-political context of health. emerging infectious diseases, in particular, have triggered the political aspect of public health because the threat of the trans-border spread of disease challenges the traditional state-centric approach to infectious disease policy. the pathogenic threats highlight the inability of states to act alone to prevent the spread of infectious diseases amid globalisation. as a consequence, emerging infectious diseases have forced a reconceptualisation of public health governance both nationally and globally, leading to an increase in the process of global collaboration. the concept of global health governance has therefore emerged in this context, characterised by the relative decline in the salience of states alone and the increased involvement of new ways of norm setting and compliance processes. the question that this article considers is whether nations and national dignity have been virtually impacted and eventually weakened by the new health governance mechanism which is able to set and control the rules of health at a global level. it is argued that global health governance may influence the nation's response to the threats posed by emerging infectious diseases such as sars or aids as a mode of building political compromises but does not considerably alter the nation's behaviour, at least for china. it is argued that in case of china, sovereign dignity and nationalism outweighed the global values in the response to infectious diseases. the chinese government's initial silence on sars and aids, and its lack of co-operation despite its awareness of the extent of the epidemic, demonstrated the nature of national pride inherent in china's response to national crisis. in pursuit of national prosperity through foreign investment and international trade, any discourse or narrrative on potential disasters such the sars or aids epidemics would naturally be suppressed at the first instance. in addition, the chinese leadership's obsession with issues of national security further allowed the sars virus to spread across the world. no one could disclose information about the epidemic unless they had security clearance, because sharing information pertaining to or even acknowledging the existence of any infectious disease is regarded as a crime by revealing 'state secrets'. if anything, the sars and aids episodes have shown that the existence of epidemics cannot be contained by censorship, regulation or legislation alone. however, once the information about the sars outbreak was divulged, the growing epidemic was beyond the national public health capacity, and to make matters worse, other nations began to criticise china's unresponsiveness, the chinese government had to institute 'damage control' measures by denying that it had any role in intentionally concealing the disease's existence. this would undoubtedly further damage china's national pride and dignity by announcing to the world that china was not able to deal with domestic public health problems. by the same token, the chinese government therefore had to show the international community that as an emerging power, it could still address the public health crisis, this time through mobilising the nation to participate in international efforts to stem the epidemic. with the help of international health expertise, china was able to successfully curb the sars epidemic in a very short period and with remarkable efficiency. its successful efforts somewhat ameliorated the embarrassment it caused itself by the bungled handling of the disease outbreak, and to a certain extent restored some of its lost national pride and international reputation. in the aftermath of the incident, china's successful story of controlling sars was a sign that china was incorporated into the global health governance where international health problems are resolved for the greater global public good. it seems, however, that china's leadership was only prepared for limited and selective openness. in light of china's response to recent cases of avian influenza, the need to preserve an infallible national image still takes precedence over public health concerns in the minds of china's leaders (cyranoski a: - ; cyranoski b cyranoski : . indeed, the incidents surrounding reporting of avian influenza in china clearly demonstrate how the chinese government has hindered progress towards halting the epidemic, denied the presence of an outbreak, prevented the exchange of information on the flu virus, and allegedly promoted widespread misuse of antiviral vaccination in chickens. scientists were often faced by less than cooperative local and central officials, whose primary concern was how health problems would negatively impact on national development goals. therefore, china's long tradition of avoiding sensitive questions and denying negative developments has yet to change. rather, it appears that national pride in a great china invariably contributes to the national response to emerging infectious diseases. in an era of rapid globalisation, it remains uncertain to what extent the chinese leadership's concern with nationalism and sovereign pride would modify itself to fit into the global governance if a more serious threat posed by a new transnational but more lethal disease than sars emerges. clearly, china's national pride and security would do little to further the achievement of china's incorporation into global health governance, not to mention the health of the population of an emerging world power. twenty-first century plague: the story of sars china: from denial to mass mobilization', in world health organization, western pacific region, sars: how a global epidemic was stopped china rejects internet claims of human cases national insecurities: humiliation, salvation, and chinese nationalism flu in wild birds sparks fears of mutating virus china's chicken farmers under first for antiviral abuse' disease and civilization: the cholera in paris, china's quest for national identity nationalism and the health of a nation eckholm, erik china's missed chance sars, governance and the globalization of disease from international sanitary conventions to global health security: the new international health regulations china syndrome: the true story of the st century's first great epidemic china's new nationalism: pride, politics and diplomacy the burdens of disease: epidemics and human response in western history the scientific background of the international sanitary conferences locked doors: the human rights of people living with hiv/aids in china sars strengthens china-asean ties joint united nations programme on hiv/aids, and world health organization containing sars: the scandal over taiwan', international herald tribune health, civilisation and the state: a history of public health from ancient to modern times building our new geat wall -on the great spirit of resisting and attacking sars', people's daily, may is sars china's chernobyl or much ado about nothing? beautiful imperialist: china perceives america neighbourhood watchers join sars battle hiv/aids: china's titanic peril aids in china: new legislation, old doubts epidemics and history: disease, power, and imperialism summary of probable sars cases with onset of illness from sung-won yoon is a phd candidate at the who collaborating centre on global change and health, london school of hygiene and tropical medicine (lshtm). prior to coming to lshtm, she completed her graduate studies at seoul national university and ewha womans university in korea. sung-won has worked as a policy researcher for various korean universities, think-tanks and the parliament of the republic of korea for several years, principally researching on social and health care issues. she was awarded a fully funded research fellowship by the korean government to undertake research work at london school of economics and political science before she embarked on her work at lshtm. key: cord- -uoek pba authors: peset, josé l. title: plagues and diseases in history date: - - journal: international encyclopedia of the social & behavioral sciences doi: . /b - - - - . - sha: doc_id: cord_uid: uoek pba in spite of the development of the medical science, during the twentieth century, individuals have observed the spread of new or reemerging diseases, from plague, cholera, and flu; measles, cancer, and malaria; to acquired immune deficiency syndrome, west nile fever, resistant tuberculosis, virus of ebola, creutzfeldt-jakob disease, and others. as individual illness is rooted in society and the environment, human life is tied up with the history of main endemic and epidemic diseases. human health is very sensitive and adaptable to changes, so the history of disease and hygiene is the core of the new ecological history. the 'sweating sickness' (sudor anglicus) . on yet other occasions, these changes are merely due to cultural appreciations, as it has happened with certain sexual practices such as masturbation or homosexuality, which used to be framed as stigmatized diseases. on the other hand, disease is not always considered only harmful: thus it may be considered a distinction of the gods, as was the case of epilepsy in ancient times, or among some historical aboriginal tribes. nevertheless, the hippocratic text on the sacred disease established the natural condition of this illness, similar to other medical affections. the same disease was associated with the devil by the christians, and historically, it had always been a supposed distinction of great personages from caesar to napoleon. disease can also be considered a way toward perfection or transformations, leading to the creation of art or to the salvation of the soul, as melancholy or sorrow were for centuries. disease may also have political repercussions: it is claimed that george the third's madness gave rise to problems for the british crown, while roosevelt's fragile health was considered to have placed him in a weak position in the yalta negotiations. socially, disease has sometimes unjustly been considered the stigma of groups (as acquired immune deficiency syndrome (aids) for homosexuals, and poor or marginal groups, or alcoholism for black or aboriginal peoples). medicine and politics have endeavored to preempt social changes by means of the contested eugenic theories and practices of social engineering, inspired by francis galton at the end of nineteenth century, which began with recommendations or laws to avoid marriages entailing risks (real or imaginary), and went on to such processes as sterilizations. heredity is today considered an important predisposition for disease, but mostly not in a deterministic way. hereditary conditions are thus intertwined with cultural, social, religious, and moral ones. besides, in contrast with the stark determination of former times, there is today an attempt to draw new optimism from the possibilities of genetic modifications. yet, in this latter respect, there is fear of a future where such a formidable force could rest in the hands of the rich and powerful. an important renewal in the study of infectious diseases was driven by bacteriological discoveries, and the theories about infection and immunity. in this respect, hans zinsser in rats, lice, and history combined them with human history, proposing biographical interpretations of epidemiological history. the study of the transmission of pathogens through animals and human beings (as vectors and hosts), living in a physical, biological, social, and cultural environment, was crucial in bringing about a new history of disease and also much later in fueling the most recent ecological history. in the meantime, the essential contributions of historical demography and social history licensed the consideration of the history of diseases as 'biographies,' shared by human cultures, living beings, and natural environment. but with the development of accurate technologies of diagnosis, the real identification of old plagues is more and more demanding. paleopathology enables us to discover the high incidence of diseases in the early inhabitants of the earth, especially where such diseases left their mark on bones. it is thus possible to detect remnants of tumors, infections, necrosis, osteoporosis, and malformations, as also traumatisms, dental alterations, rickets, rheumatism, and other diseases of the bones. today, laboratory analysis allows the possibility of detecting remnants of other diseases, germs, or lesions, in animal or human remains, including of course the famous mummies. from the first settlements in the fertile crescent, changes have taken place in the relationship of man and animals with their environment and it is possible to establish an extensive catalog of diseases that have largely subsisted down to this very day. particularly significant are the forms of settlement, the crops and hunting, and the contact of man with cattle, pets, and parasites, as well as the relations between peoples, through mixing and trade, wars, and migrations. malaria originated in tropical africa, accompanying the dawn of humanity, from plasmodia infecting animals and hominids. caused by different plasmodia (plasmodium vivax and plasmodium falciparum among others, with different geographical distribution), its relation with human populations is shaped by migrations and deforestation, hunting and farming, climate and soils, crops, animals and foods, and a complicated immunity resulting from ancient genetic mutations and new infections. thus, with the early human emigrations, it expanded through eurasia, and in the third millennia bc, malaria had already set in the early civilizations in marshy areas where the water, the climate, and the crops provided the conditions for the anopheles mosquitoes to infect in crowded populations. evidence for the existence of intermittent fevers is already to be found in ancient cultures, finding suitable conditions along the yellow, indus, ganges, euphrates, or nile rivers. different interpretations considered the disease to originate from climatic or environmental factors, from particles or small animals, because of spirits or gods, or from unbalanced or disharmonic alterations. the connection between fevers, splenomegaly, and marshy areas appears in the hippocratic texts, such as on airs, waters and places. deforestation, agriculture, temperature, and a greater population density, as well as military and commercial movements, favor the spread of the disease. in italy, in republican times, the disease becomes acute; and it was recommended that the marshy lands be either abandoned or sold and that people should dwell on high groundthe latter being traditional advice and here defense reasons also counted. also, the cleaning and draining of swamps was considered, this being the origin of systems of engineering sanitation. the spread of malaria got worse and worse in rome owing to negligence and flooding, climate and agricultural changes, wars, travels, and migrations, and it reached its zenith at the time of the fall of the roman empire, a cataclysm to which it may have contributed. but when the old empire declined, another frightening ghost traveled through the mediterranean sea. plagues were considered in ancient cultures as diseases originating in god's punishment, with a violent pattern, and quick and terrible diffusion, affecting and killing a large number of persons. destructive epidemic catastrophes were narrated frequently in mesopotamian, biblical, egyptian, indian, or chinese sources, and in - bc, thucydides describes the plague of athens in the war against sparta, but it is doubtful that this case refers to the bubonic plague. this epidemic death is considered the beginning of the decline of athenian hegemony, the crisis of the democracy, and culture of the pericles era. fear, war, siege of the city, and its maritime port were accompanied by the death of the great ruler pericles. later on, the first reliable description of this disease is that given by rufus of ephesus in the ad first to the second century, in the epoch of trajan; and in the sixth century, the plague of justinian initiates the first great epidemic cycles of bubonic plague. soldiers and merchants, animals and merchandises, and slaves and prisoners all contribute to linking up the extremes of the known world. a consequence of this contact was the antonine plague in the second century. this plague -and othersis attributed to smallpox, a disease that had already existed as far back as years ago, as certain mummies show. in the middle ages, leprosy spread widely, allegedly as a result of the increasing east-west relations trough trade, travels, and wars. this biblical and present day disease is accompanied by social repulse and malignant connotations since dirtiness and overcrowding provided the conditions for the spread of the otherwise not extremely contagious mycobacterium leprae. in this respect, the way to santiago de compostela where pilgrims slept in groups and under unhealthy and dirty conditions provided a fertile ground for this disease. another markedly religious character is to be found in the 'sacred fire,' 'st anthony's fire,' or ergotism; a disease that developed in people eating rye infected by ergot, it was soon prevented, but reappeared during the course of severe famines. these diseases are linked to many others that owed their spread to squalor and poverty, for example, parasites and infections, scabies, mycoses and lice, anthrax and ophthalmia. natural catastrophes, wars, poverty, and famines were escorted by mental diseases, tuberculosis and pneumonia, traumatisms and poisonings, diarrheal diseases and fevers, as well as smallpox and measles. in the mediterranean basin, malaria continued to be endemic with the population tending to resort to residing on the hills and mountains, far from marshy lands. the extension of malaria involved south and southeast of asia, central china and japan, and also north europe. the plague set in the growing cities with the burden of rats and fleas, making this disease an explicative example par excellence of epidemic history. a second epidemic cycle began with the black death in . commerce and the cities collapsed, giving rise to the origin of the modern european kingdoms. at this time, venice established control over persons and ships, in order to avoid the spread of plague, arriving from distant lands. dark ages are ending, and new worlds are opening in culture and geography. european expansion meant a sudden traffic of diseases mostly toward the new world. the discovery of america, with its scattered population devoted to agriculture, where there were no dirty cities, and where the inhabitants were devoid of immunity to the diseases of old europe, produced an enormous demographic crisis. certain diseases, such as smallpox (and measles) ravaged the indigenous population; this very contagious and harsh illness was introduced in the caribbean and mexico, and destroyed american indigenous populations. syphilis came supposedly from america (although other human treponematoses were present in other continents), brought back by the spaniards and it caused havoc since renaissance in europe and soon all around the world. troop movements spread an unknown disease called 'tabardillo,' or 'typhus exanthematicus,' bringing about serious suffering for armies and poor people. sea voyages led to the development of scurvy, due to the lack of fresh food. other diseases also voyaged overseas, such as yellow fever; although it originated from africa, due to the commerce of slaves and merchandise, special temperature conditions and mosquitoes as vectors were required for the contagion. in the seventeenth century, it took root in brazil and also spread to the caribbean and throughout america, reaching new york and boston and likewise colombia, ecuador, and peru. it affected warm america and europe, through the iberian peninsula. immunity and cutoffs in commerce stalled the disease, but it remains endemic in tropical areas of america and africa. also, malaria arrived to america with the european conquest in sixteenth century, beginning in the caribbean and central america and soon spreading to south and north america, becoming endemic in hot, wet, and low lands. malaria was combated since the seventeenth century by the use of cinchona bark (peruvian bark), found in peru, as an indigenous medical practice. later on, the quininethe alkaloid obtained from the bark in french laboratoriesgave rise to rich industry and trade. trees were cultivated in british and dutch colonies, mainly in java. after being taken in the second world war by the japanese army, synthetic products such as atebrine and chloroquine were obtained. getting worse with agriculture, mining and livestock changes, settlements, and slavery trade, malaria becametogether with yellow fevera scourge along the warm and hot reaches. throughout the american conflicts between european empires, and later during the american independence wars, both diseases played an important role, infecting and killing nonimmune soldiers. plague slowed demographic growth in europe since the wars between modern and powerful nations, the crowded and dirty cities, and the developing trades impeded protection against the disease. the great writer daniel defoe remembered the terrible contagion in london in in journal of the plague year. after the great european plague of marseille in , the relative peace of the eighteenth century allowed western europe to set up sea and land defenses, with austria becoming a solid bulwark in the face of the ottoman empire, thus sealing off all possible spread of the plague. toward the middle of the nineteenth century, the third wave of the disease broke out and, with the exception of europe, it spread to all countries including asia, africa, and paradises like america and australia, leaving remnants in many places. the eradication of plague in europe was a result of the advent of public hygiene, immunity to disease, and the disappearance of rats and of the old and dirty wooden buildings. the great london fire in 'purified' the city, since the hygienic rebuilding was carried out in stone, eliminating animals and dirt. the disappearance of plague from europe was followed by the outbreak of cholera, which had been endemic in india for centuries. this disease was described by western travelers in the sixteenth century and its spread to europe and america was a consequence of pilgrimages, trade, and a lack of cleanliness in water. a great pandemic broke out in from india and spread through eastern africa and southern asia, arriving to china and the philippines, and in a second wave, it spread through persia reaching russia and poland in . then this second wave swept across europe and reached america by . throughout years, terrible waves spread from the east. however, the timely and necessary cleansing of urban water supplies helped to progressively stall its advance. nevertheless, as late as , the free hanseatic city of hamburg suffered an outbreak of cholera epidemic, while the neighboring altona, governed by the prussian reich, was able to avoid the disease, thanks to the successful filtering of its water. local and national governments advanced in hygiene and they attempted to bring about healthier cities by means of appropriate public health measures. the nineteenth century sees the beginning of the demographic revolution in europe, followed by developing countries, in america for example, with a sharp decline in mortality, especially maternal-infant mortality. old inoculation and the new vaccination proposed in by edward jenner fought smallpox successfully. studies about human immunity since the last decades of nineteenth century began a new medical approach to the prevention and understanding of illness, being successful in the contention of many diseases, which have a long record of burdens and deaths. mother care also advanced both throughout pregnancy and at childbirth and during lactation. the rise of great and industrial cities with low-grade outskirts and the emigration of peasants to the city favored the spread of maladies associated with poverty, hard work, squalor, lack of appropriate food, and exposure of people devoid of defenses. some of the diseases were strictly occupational as in the case of miners and textile workers who were exposed to injury, and to industrial poisons that affected the proletariat. sir percivall pott described in the eighteenth century the cancer produced in professional cleaners (chimney sweep) by chemical toxics contained in soot. many other diseases, such as cancers, will be attributed to chemical and physical aggressions, including radioactivity. other diseases were closely related to the harsh and dirty conditions of life such as spread of typhoid fever and pulmonary diseases. tuberculosis spread over the turn between the eighteenth and nineteenth centuries, and while it was represented as the disease in fashion, affecting notable and distinguished people, it struck the proletariat much more severely. the same can be claimed regarding the enormous spread of syphilis, and drug abuse, beginning with alcohol and continuing with cocaine and morphine. the discovery of the microbiological origin of infectious diseases, and of effective therapies against them, and the development of public health changed the pathological landscape in developed countries. the long way between ignaz semmelweis and alexander fleming arrived to the contention of infections in health care. the twentieth century marked the descent of the high mortality rates in countries that reached high standards of sanitary development, investing in health care and public health. such advances have been due to public health services, hospitals, antibiotics, surgery, and vaccination, and they have been reinforced by the developments in immunology and microsurgery, pharmacology, and biotechnology and with the promising future of genetic engineering. the international health solidarity promoted by the world health organization (who), and other governmental and nongovernmental organizations (ngos), including church missionaries, were accompanied by better governance of nations, and internationalization of information and resources. the twentieth century established a serious change in geopathology of diseases. smallpox is the first disease to be considered totally eradicated with only some samples of the virus being kept at a few laboratories for study purposes. a cuban doctor (carlos finlay) found the method by which yellow fever is transmitted through mosquitoes (aedes aegypti). after the cuban war and during the opening of the panama channel, yellow fever and malaria were studied and faced by us army and american sanitarians (walter reed, william gorgas), and later by the rockefeller foundation. impeding mosquito reproduction and avoiding bites and spreading chemical products were useful. fortunately, an effective vaccine against yellow fever was later discovered. during nineteenth century, malaria expanded all around the world, arriving to the central extensions of america and eurasia. emigrations and settlements, wars, famines, climate change, and revolutions in travels, such as railroads and steamships, contributed. during the napoleonic wars and the american civil war, malaria was seriously extended, as it was also during wars and revolutions in twentieth century. in the interwar years, the rockefeller foundation and the league of nations sponsored international campaigns against the disease. many governmental campaigns also fought against malaria; several national programs were effective, from taiwan and china to the united states, brazil, and argentina, passing across the mediterranean basin, from italy to egypt. opinions and campaigns oscillated between quinine treatment, fighting against mosquitoes (by dichlorodiphenyltrichloroethane (ddt)), and improvements in life, education, and land sanitation, clearing up the marshy areas. malaria has been eradicated from europe thanks to drainage, improvements in crop cultivation, mechanical barriers, quinine and modern drugs such as atebrine and chloroquine, and insecticides. colonial settlements entered tropical areas backed by quinine, nets in beds, hygiene, and sanitation, but transmissible diseases were continuously a serious burden for colonial armies, in america, africa, or asia. some chemical products such as ddt were a successful support for soldiers, travelers, merchants, or settlers. nevertheless, in , the world health assembly, meeting in mexico, warned against the resistance of the mosquitoes to insecticides. mosquitoes are still today carrying both diseases, and also dengue. chagas disease produced by trypanosoma cruzi is endemic in america, and sleeping sickness caused by trypanosoma brucei (rhodesiense and gambiense) occurs in africa. today, an effort is being made to involve governments in the fight against malaria, a disease that represents a danger to nearly half of the world's population. in africa, the situation is very serious due to the changes in agriculture and irrigation as well as in work and migrations, the political and economic problems, the severe droughts, famines and wars, the bad sanitary conditions following in the wake of housing expansion and deforestation, and harsh social and economic exploitation. together with the who and the united nations international children's emergency fund (unicef), regional institutions such as the pan american health organization and the us government are financing programs of eradication. the who, through the st world health assembly of , set up the program 'roll back malaria' and, with aid from unicef and the world bank, is endeavoring to bring about economic and sanitary improvements in developing countries. in twentyfirst century, the global fund to fight aids, tuberculosis and malaria is attaining important amount of resources and success. nets treated with insecticides are very useful for protection against mosquitoes. today, hope is also placed on vaccination and in the sterilization, or genetic modification, of the mosquitoes. yet, malaria continues to be endemic in warm zones of america, asia, and with severe cruelty, in africa. the rapid increase of world population in huge metropolitan areas was accompanied by new settlements and emigrations, wars, conflicts and revolutions, and marginalization of aboriginal cultures and disinherited peoples. throughout the past two centuries, economical, social, and political expansion of the west led to studies on tropical medicine, creating hospitals and laboratories and institutions on public health and sanitary departments. cholera continues to be feared in asia, between india and the far east, and also in warm zones in america and africa. wars and catastrophes, travels and migrations, as well as famines and unhealthy conditions allowed the expansion of the cholera germ, the vibrio cholerae isolated by robert koch in , as was the case of the recent tragedy of haiti. at the end of nineteenth century, the third outbreak of bubonic plague allowed its bacteriological and epidemiological description. the germ of bubonic plague yersinia pestis, discovered by yersin and kitasato, is still to be found throughout the world. the germ is carried by rats, rattus rattus: rat fleas are the arthropod vectors transmitting 'epizootic' plague to humans from rodent hosts, and related species, but transmission among humans is also possible. almost forgotten in the developed world, with several and very effective antibiotic therapies, there is, nevertheless, always the possibility that the devil may once more send his rats to the old, rich cities. but other viruses have taken its place in the twentieth century. thus the very old and common influenza caused several cruel and heavy outbreaks; among many others, influenza pandemics began in and reappeared in , , and : these were known, respectively, as the 'spanish flu,' which perhaps originated in the united states, the 'asian flu,' the 'hong kong flu,' and the 'avian flu,' mostly coming from asia. vaccines are very useful in its prevention, as they are also in the control of many infectious childhood diseases, such as measles, rubella, mumps, chicken pox, whooping cough, diphtheria, and so on. old diseases such as poliomyelitis exploded in the united states and europe, affecting nonimmune children, and this disease has only been controlled in developed countries by means of vaccination. during the past century, individuals have observed astonished the spread of new or reemerging diseases, from plague, cholera, influenza, measles, and malaria, to aids, west nile fever, avian flu, severe acute respiratory syndrome, resistant tuberculosis, hemorrhagic fever (ebola virus disease), transmissible spongiform encephalopathy (creutzfeldt-jakob disease), and others. unfortunately today, many frequent or rare diseases do not have adequate treatment, and many germs are developing resistance to antibiotics, a serious threat for its affectivity. aids has meant a development in the study of viral infections, and this disease has associations with the exploitation of poor people, unsafe sex, and drug trading, but it also affected an elite sector, which has sprung rapid research in the field. the world commotion surrounding this disease has served both to reveal human altruism on the one hand, and, on the other, contempt toward those infected by the disease, since aids became more and more the lot of the poor, mostly in large areas of africa, or india, and of downcast or marginal groups such as the chronic patients, drug addicts, prostitutes, and homosexuals. nevertheless, heterosexual transmission through unsafe sex and transmission from mother to child are today serious dangers. safe sex and antiretroviral treatment are the best contention, while a vaccine will be obtained perhaps in the coming future. mental affections were considered till modern times, as devilish, criminal, or vicious behaviors, more an ethical or social than a medical problem. in the eighteenth century, the natural explanation of mental disease was established, according to alexander crichton, or philippe pinel, and in the nineteenth and twentieth centuries, its psychogenic process, following sigmund freud. from pinel to freud, the possibility of treatment and remedy of mental disease was established, leading the soul of the patient with convictions and health measures to the cure. the discovery in recent times of some effective pharmacological drugs acting on human mind, and conduct, has allowed better treatment. reclusion was considered a doubtful possibility, restricted to some severe problems. the old lunatic asylums, founded since middle ages, were being abolished or completely renewed. nevertheless, mental illnesses wreak serious havoc at present times all around the world, affecting all ages, genders, and conditions, without any distinction. sometimes, the misunderstanding regarding mental diseases still produces cruel treatments, harsh restrictions, or punishment. during the past decades, economic and social improvements led to quick globalization and urbanization, with longer human life and changes in disease patterns. political, sanitary, and economic development managed to stamp out infectious morbidity, and forms of suffering or living illnesses are changing. unicef and who, in collaboration with governments, foundations, and ngos, promote successful campaigns of vaccination, especially focusing on children. the burden of infectious, parasitic, and transmissible diseases changed to chronic diseases and sufferings related with aging and lifestyles, likewise, nutritional disorders, cancer, or heart and brain vascular diseases. on the other hand, the increase in life expectation favors the development of alzheimer and other chronic and degenerative diseases, neurological and muscular diseases, mental affections such as schizophrenia, mania and depression, diabetes, vascular diseases, and of course, the terrible presence of cancer. accidents and traumatisms, due to traffic and sports, factories and radiation, or even home accidents, are something that the twenty-first century is inheriting. blindness, deafness, dumbness, and other frequent physical and mental disabilities, with multiple origins in traumatisms or accidents, genetic, metabolic and degenerative diseases, infectious diseases such as poliomyelitis, or cerebral and vascular affections, make daily life difficult and, at the same time, make economical, institutional, and social support necessary. incapacitating and chronic diseases lead to severe dependency and so the need for protection of disabled individuals is leading to the founding of patients' associations looking for help and justice, creating new rights and demands. ghettos and migrations, hard work, poverty and unemployment, and some of the old drugs such as alcohol, heroine, and tobacco or new synthetic ones are causing havoc even to the rich world. but in developing countries, the old morbidity due to transmissible diseases continues to exist, maintaining a very low life expectation. certainly, the increase of migrations and of urban population are requiring more water, food, and energy, producing climate warming, deforestation and agriculture changes, and dangerous issues such as waste, toxics, and pollutants. potable water, health services (medical care or medical drugs), sanitation and hygiene are urgently requested. environmental degradation and the confrontations between national, social, and ethnic groupings are a serious danger for healthy life. china, japan, and south korea and other expanding countries have lived through these challenges in different ways from diverse british colonies such as australia or india. different traditions and cultures are extremely important to understand the relations between peoples and diseases. death is most rampant amid the least protected and poorest people, especially women, children, and the elderly, with harsh sufferings such as wars, famine, and exploitation. under such circumstances, diseases caused by deprivation and infection continue to be the most rampant, causing high death rates among the population. old and new diseases, such as malaria and aids, are ruining great stretches of africa. leprosy and cholera, tuberculosis and tetanus, and many child diseases, such as mumps, measles, tetanus, or meningitis are in poor countries and populations the salt of the earth. private and public funds, and international solidarity, are always necessary for relieving these harsh sufferings. the united nations millennium development goals are also facing them. according to the who report global health risks ( ), the better or worse conditions for mortality and for the burden of disease are seriously conditioned by several circumstances, related to lifestyles: blood pressure, blood glucose, physical activity, alcohol and tobacco, weight, safe sex, safe water, sanitation, and hygiene. obviously, these circumstances and their consequences are very different, depending on the social level of individuals and the public health governance of nations and peoples. if developing countries are freeing themselves from transmissible diseases, now they are fighting against noncommunicable diseases, related to social level, health organization, and hygienic customs. some american, asian, and african countries are still supporting a terrible burden of disease, which is also shared by low-income population in developed countries. the rio political declaration in the world conference on social determinants of health (who-rio de janeiro, brazil, october ) is a new call looking for equity, justice, and universality of health. it has recommended adopting better governance for health and development; promoting information, justice, and participation in policy making and during the implementation process; including civil society like indigenous people; and reducing health inequities in the health sector. this declaration promotes research on the relationships between social determinants and differences (economic, ethnic, and gender inequalities) and health equity. we are all convinced about the relation between poverty, social discrimination, low education and low sanitation, and diseases and death. history of western; science, history of; welfare state, history of plagues in world history the great pox: the french disease in renaissance europe les hommes et la peste en france et dans les pays européens et méditerranéens, vols la malaria tra passato e presente the columbian exchange: biological and cultural consequences of médecins, climat et épidémies à la fin du xviiie siècle the cambridge world history of human disease public health in asia and the pacific. historical and comparative perspectives plague and the end of antiquity: the pandemic of - mosquito empires: ecology and war in the greater caribbean plagues and peoples inescapable ecologies: a history of environment, disease, and knowledge humanity's burden: a global history of malaria rats, lice and history. printed and pub. for the atlantic monthly press by little, brown, and company key: cord- -fwh g authors: leggio, loredana; paternò, greta; vivarelli, silvia; l’episcopo, francesca; tirolo, cataldo; raciti, gabriele; pappalardo, fabrizio; giachino, carmela; caniglia, salvatore; serapide, maria francesca; marchetti, bianca; iraci, nunzio title: extracellular vesicles as nanotherapeutics for parkinson’s disease date: - - journal: biomolecules doi: . /biom sha: doc_id: cord_uid: fwh g extracellular vesicles (evs) are naturally occurring membranous structures secreted by normal and diseased cells, and carrying a wide range of bioactive molecules. in the central nervous system (cns), evs are important in both homeostasis and pathology. through receptor–ligand interactions, direct fusion, or endocytosis, evs interact with their target cells. accumulating evidence indicates that evs play crucial roles in the pathogenesis of many neurodegenerative disorders (nds), including parkinson′s disease (pd). pd is the second most common nd, characterized by the progressive loss of dopaminergic (daergic) neurons within the substantia nigra pars compacta (snpc). in pd, evs are secreted by both neurons and glial cells, with either beneficial or detrimental effects, via a complex program of cell-to-cell communication. the functions of evs in pd range from their etiopathogenetic relevance to their use as diagnostic tools and innovative carriers of therapeutics. because they can cross the blood–brain barrier, evs can be engineered to deliver bioactive molecules (e.g., small interfering rnas, catalase) within the cns. this review summarizes the latest findings regarding the role played by evs in pd etiology, diagnosis, prognosis, and therapy, with a particular focus on their use as novel pd nanotherapeutics. parkinson's disease (pd) is the second most common chronic neurodegenerative disorder (nd) after alzheimer's [ ] . pd affects about - % of the population over the age of years [ ] . globally, pd incidence is increasing because the life expectancy is longer than in the past, which results in a larger elderly population [ , ] . in fact, according to the world health organization, the total of individuals over years will double between and [ ] . in the near future, pd will affect not only people in the western industrialized countries, but also those living in the developing countries. for this reason, the number of people with pd is estimated to exceed the million count by [ , , ] . the causes and mechanisms involved in pd onset and progression are ill-defined, and currently there is no cure available to stop or reverse pd progression [ ] . the clinical symptoms of pd were characterized for the first time in by the british doctor james parkinson, who described this condition as a "shaking palsy" [ ] . fifty-five years later, in , as outlined, other types of vesicles have been characterized, such as microvesicles, apoptotic bodies, ectosomes and others [ ] . these vesicles differ from the exosomes in their biogenesis, being released directly from the plasma membrane with a shedding mechanism. these shedding vesicles have a dimension between and nm that partially overlaps with the exosome size. for this reason, to date, it is not possible to separate pure subtypes of evs [ ] . this issue may be solved by the identification of specific ev surface markers. initially, tetraspanins such as cd and cd were recognized as specific markers for exosome biogenesis. further studies found these proteins expressed by additional categories of vesicles, making the identification of specific subtypes not yet feasible [ , ] . therefore, the literature in the last years reflects all the issues mentioned above. the latest guidance paper, released in from the international society for extracellular vesicles, defined new important advices for scientists working with evs [ ] . in particular, a new nomenclature has been proposed, based merely on the size of the evs, thus dividing them into small vesicles (s-evs < nm) and medium and large vesicles (m/l-evs > nm) ( figure ). biomolecules , , of as outlined, other types of vesicles have been characterized, such as microvesicles, apoptotic bodies, ectosomes and others [ ] . these vesicles differ from the exosomes in their biogenesis, being released directly from the plasma membrane with a shedding mechanism. these shedding vesicles have a dimension between and nm that partially overlaps with the exosome size. for this reason, to date, it is not possible to separate pure subtypes of evs [ ] . this issue may be solved by the identification of specific ev surface markers. initially, tetraspanins such as cd and cd were recognized as specific markers for exosome biogenesis. further studies found these proteins expressed by additional categories of vesicles, making the identification of specific subtypes not yet feasible [ , ] . therefore, the literature in the last years reflects all the issues mentioned above. the latest guidance paper, released in from the international society for extracellular vesicles, defined new important advices for scientists working with evs [ ] . in particular, a new nomenclature has been proposed, based merely on the size of the evs, thus dividing them into small vesicles (s-evs < nm) and medium and large vesicles (m/l-evs > nm) ( figure ). another important aspect deeply investigated in the ev field concerns the analysis of their content. the cargoes delivered via evs reflect the current "status" of the donor cell and can change in response to specific modifications in the microenvironment. in particular, evs contain nucleic acids, both dna (chromosomal or mitochondrial) [ , ] and rnas (mrnas, small ncrnas, such as micrornas etc.), potentially able to regulate the gene expression of the target cells [ , ] . moreover, these vesicles transport proteins, metabolites, and lipids, whose identity changes in response to several stimuli [ ] . however, the role played by evs in both physio-and pathological conditions is still debated, since they can be either beneficial or detrimental, depending on the specific context in which they are investigated [ ] . in nds, evs were initially characterized as vehicles for misfolded or dysfunctional mutant proteins, such as amyloid-beta oligomers in alzheimer's disease (ad) [ ] , sod in amyotrophic lateral sclerosis (als) [ ] , or α-syn in pd [ ] . in line with the dual role described for glial cells, this view was gradually challenged in the last decade by the demonstration that evs can play relevant neuroprotective functions in several degenerative conditions (including pd), as we and others recently reviewed [ , ] . in this review, the latest findings regarding the roles played by evs in the development of pd, and their possible use as novel pd biomarkers are provided. in particular, we will focus on the another important aspect deeply investigated in the ev field concerns the analysis of their content. the cargoes delivered via evs reflect the current "status" of the donor cell and can change in response to specific modifications in the microenvironment. in particular, evs contain nucleic acids, both dna (chromosomal or mitochondrial) [ , ] and rnas (mrnas, small ncrnas, such as micrornas etc.), potentially able to regulate the gene expression of the target cells [ , ] . moreover, these vesicles transport proteins, metabolites, and lipids, whose identity changes in response to several stimuli [ ] . however, the role played by evs in both physio-and pathological conditions is still debated, since they can be either beneficial or detrimental, depending on the specific context in which they are investigated [ ] . in nds, evs were initially characterized as vehicles for misfolded or dysfunctional mutant proteins, such as amyloid-beta oligomers in alzheimer's disease (ad) [ ] , sod in amyotrophic lateral sclerosis (als) [ ] , or α-syn in pd [ ] . in line with the dual role described for glial cells, this view was gradually challenged in the last decade by the demonstration that evs can play relevant neuroprotective functions in several degenerative conditions (including pd), as we and others recently reviewed [ , ] . in this review, the latest findings regarding the roles played by evs in the development of pd, and their possible use as novel pd biomarkers are provided. in particular, we will focus on the therapeutic potential of both natural and engineered evs, and the possibility to use them as advanced drug delivery systems in pd. currently, the causes responsible for the progressive degeneration of midbrain daergic neurons are poorly understood. aging, inflammation, genetics, and environmental toxicity are prominent etiological factors in pd development. in particular, aging represents the most critical vulnerability factor for pd, whereby the age-dependent dysregulation of critical cellular functions within the midbrain microenvironment (e.g., inflammation, oxidative and nitrosative stress, proteasome/lysosome dysfunctions) significantly contributes to the progressive neuronal deterioration observed in pd [ ] [ ] [ ] . notably, emerging studies on aging mouse models of pd, based on the exposure to the environmental neurotoxins -methyl- -phenyl- , , , -tetrahydropyridine (mptp) or -hydroxydopamine ( -ohda), clearly indicate that aged glial cells lose their neuroprotective, pro-neurogenic, and regenerative functions, thereby contributing to the inflammatory and degenerative processes during pd onset and progression [ , , [ ] [ ] [ ] [ ] [ ] [ ] . in fact, both reactive astrocytes and microglial cells play key roles during pd nigrostriatal degeneration/self-repair. these glial cells are located within the striatal subventricular zone (svz), a major neurogenic niche of the adult brain, and the peri-aqueductal ventral midbrain (vmb) region, enriched in daergic neuroprecursor cells [ , , , , [ ] [ ] [ ] [ ] . significantly, in the context of snpc, reactive astrocytes and microglia represent the key "cellular-hub" able to handle crucial cellular functions-including oxidative and endoplasmic reticulum stress, mitochondrial, lysosomal, proteasomal, and autophagic activities-all converging to α-syn aggregation and spreading [ , , , , [ ] [ ] [ ] [ ] . astrocytes are pivotal cells in maintaining the homeostasis of the microenvironment in the central nervous system (cns). they provide physical and metabolic support to neurons by helping them to accomplish their complex functions. moreover, astrocyte functions are selectively linked to the brain region, the type of brain injury, as well as to the age and the sex of the individual [ ] [ ] [ ] [ ] . importantly, they mediate neuroprotective effects via the release of growth and neurotropic factors, antioxidants and anti-inflammatory molecules [ , , , , , , ] . for instance, astrocytes from the ventral midbrain are pivotal for the development of daergic neurons, thanks to the release of an array of pro-survival and neuroprotective molecules [ ] [ ] [ ] [ ] [ ] , such as the glial-derived neurotrophic factor (gdnf) [ ] and the basic fibroblast growth factor (bfgf) [ ] , whose levels are altered in pd patients [ , ] . remarkably, both astrocyte-derived wingless-type mouse mammary tumor virus (mmtv) integration site (wnt ) and wnt / β-catenin signaling network contribute to astrocyte-neuron interactions in pd. in this context, the wnt pathway represents a vital cascade, in turn able to promote daergic neurogenesis, daergic neuron survival and immunomodulation via a bidirectional glia-neuron cross-talk [ , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] . following brain injury, astrocytes undergo gene expression changes resulting in an activated phenotype and a "reactive astrogliosis" [ ] . almost two decades ago, the concept of a dual beneficial/harmful role of reactive astrocytes and microglia in nds, including pd [ ] , paved the way to further characterization of the phenotype-dependent ability of glial cells to deliver either neuroprotective or detrimental molecules for neuronal health and survival [ , , , , ] . as for microglial cells [ ] , liddelow and colleagues in described two different kind of reactive astrocytes, the a harmful phenotype and the a protective one [ ] . accordingly, a astrocytes are involved in many nds, particularly pd, and display detrimental and neurotoxic functions induced in turn by activated microglia [ ] . microglia-the resident macrophage-like immune cells of the cns-monitor the cerebral microenvironment through a constant interaction with astrocytes, neurons, and blood vessels, contributing to maintain the homeostatic balance in the brain [ , , [ ] [ ] [ ] . with age and under neurotoxic stimuli, the microglial cells-similarly to the polarization described for astrocytes-shift to the harmful m phenotype and release pro-inflammatory cytokines such as tumor necrosis factor α (tnf-α), interleukin β (il- β) and il- [ , , ] . the simultaneous production of reactive oxygen species (ros) results in a higher oxidative stress and inflamed microenvironment for surrounding neurons [ , , , , , , ] . notably, microglia shows the highest density in the snpc, suggesting a likely harmful milieu that may predispose daergic neurons to neuroinflammation-dependent degeneration [ , , , , , , , ] . on the contrary, the m polarized microglia are associated with the release of anti-inflammatory cytokines (e.g., il- and il- ), neurotrophic factors (e.g., bdnf and igf- ) and extracellular matrix proteins (e.g., fibronectin) [ , , ] . as a corollary, both activated and aged glial cells secrete evs capable of exerting both pro-and anti-inflammatory mechanisms to balance immune reactivity in the brain [ , ] . hence, glial-derived evs powerfully regulate the inflammatory microenvironment thanks to a panel of immunomodulatory cargoes, which may further direct the glial status to an a /m or a /m phenotype, with crucial consequences for neuronal survival and health, as recently reviewed in [ ] . beside aging, a substantial proportion of pd cases is driven by genetics. indeed, about - % of patients have a familial history of pd, which indicates the possibility of inheriting such risk factors. on the contrary, the majority of pd cases are considered idiopathic, and likely result from a complex array of interactions between dysfunctional genes and a growing list of environmental risk factors [ , , , , , ] . in both the familial and idiopathic forms of pd, common molecular pathway alterations have been found. as mentioned before, dysfunctions in the lysosomal and/or ubiquitin-proteasome system, increased levels of oxidative stress, impairments in the mitochondrial respiratory mechanisms and synaptic vesicle-recycling pathway, represent only a few examples of the altered functions existing in both idiopathic and familial pd. as a matter of fact, a complex panel of gene-environment interactions heavily contribute to pd onset and/or progression [ , , , ] . for example, living in rural areas and farming may increase the chances to be exposed to pesticides. some examples are: rotenone, which leads to mitochondrial complex i dysfunction, and paraquat, which induce ros formation. both environmental exposures represent risk factors for pd [ ] [ ] [ ] . as anticipated, genetics plays an important role in the etiology of pd [ ] . many genes are linked to pd pathogenesis, including an increasing number of novel genetic mutations found associated with specific geographic areas [ ] . the first and most characterized pd-linked gene is the snca gene (park ), encoding for α-syn [ ] [ ] [ ] . toxic aggregates of this protein are found in pd brains, heavily involved in the insurgence of neuroinflammation and, ultimately, in the clinical symptoms of pd. as expected, several mutations for this gene have been found in pd patients, some of them in familial early onset pd (eopd), including the missense mutation a t [ , ] . in particular, the a t mutation is the most prevalent one, showing faster aggregation kinetics than wild type α-syn [ ] . park or parkin is another gene whose mutations are found in approximately % of familial cases with eopd and~ % of sporadic eopd [ ] [ ] [ ] . park encodes for the parkin rbr e ubiquitin protein ligase which is involved in the proteasomal degradation pathway [ , ] . it works closely with the product of the park gene-pten-induced putative kinase (pink )-which depolarizes mitochondria to induce mitophagy. mutations in both pink and parkin result in mitochondrial dysfunctions linked to eopd [ ] [ ] [ ] [ ] [ ] . together with park , park , and park , other genetic mutations have been detected in pd patients within the so-called "park" genes. dj- , encoded by the park gene, is a ubiquitous protein principally expressed by neural and glial cells, whose function is not well understood. it is mainly involved in the protection against oxidative stress, as mutations in this gene might cause mitochondrial dysfunctions [ ] [ ] [ ] [ ] [ ] . the leucine-rich repeat kinase (lrrk ), encoded by park gene, when mutated, acquires a gain of function of its catalytic activity, due to cis-phosphorylation (or autophosphorylation) and trans-phosphorylation. the point mutation g s is one of the most common lrrk mutations found in the genetic forms of pd. however, not all people carrying this mutation have the same risk to develop pd, and different strategies are currently evaluated to find a method to better predict the risk of pd when expressing the g s-lrrk mutant [ ] . glucocerebrosidase (gcase) is a lysosomal glycoside enzyme encoded by the gba gene and, although it does not belong to the "park" genes, it represents a common genetic risk factor for pd. gcase catalyzes the hydrolytic cleavage of the β-glycosidic bond of glucosylceramide (glccer), producing free ceramide and glucose [ ] . thus, gcase plays a central role in the degradation of complex lipids and the overall turnover of cellular membranes. deficiencies in this enzyme lead to accumulation of glccer and to the development of the lysosomal storage disease, known as gaucher s disease [ ] . it has been speculated that alterations in gcase functions lead to a decreased lysosomal/proteolytic activity resulting in a structural change of α-syn, from soluble to aggregated [ ] . in the contest of pd, gba mutations are associated with the appearance of cognitive impairments and motor disabilities [ ] [ ] [ ] . other genes have been discovered to play a role in the pathogenesis of pd, including uchl (park ) [ ] and atp a (park ) [ ] , as reviewed elsewhere [ ] . more recently, other factors have been correlated with pd, including the specific gastrointestinal microbiota composition. gut microbiota has been reported to have a pivotal role in the development of several pathologies, including cancer, inflammation and nds [ , ] . in particular, the gastrointestinal microbial population may play a crucial role in influencing the brain via a two-way interaction with the neural, neuroendocrine and immune systems, in a network called "gut-brain" axis [ ] . several studies highlighted significant differences existing between the intestinal microbial population in pd patients vs. healthy controls. these alterations mainly involve the diversity and the abundance of some species, such as lactobacilli, fecalibacteria and others, which can be over-or under-represented in pd-affected patients [ ] [ ] [ ] [ ] . additionally, the commonly stomach-infecting pathogen helicobacter pylori, causative agent of peptic ulcers, has been recently associated with pd. it has been demonstrated that these bacteria may affect the absorption of levodopa (l-dopa), thus causing motor fluctuations in pd patients [ ] [ ] [ ] . in general, imbalances in the intestinal microflora-condition called dysbiosis-and, in particular, a small intestinal bacterial overgrowth may be correlated with gastrointestinal symptoms and motor function impairment in pd-affected subjects [ , ] . moreover, the alteration of the intestinal permeability and the impairment of the natural intestinal barrier could facilitate the action of toxins deriving from inner or outer intestinal environment, resulting in an enhanced inflammatory response within the enteric nervous system (ens) [ ] . this finally may trigger an exacerbated immune response both at the intestinal and at the cns levels [ ] . importantly, intestinal dysfunction (i.e., constipation) is one of the most common non-motor symptoms of pd, even years before cns degeneration, thus representing an early indicator of pd onset [ ] . in , shannon and colleagues discovered that α-syn aggregates were present in the colon tissue before the onset of pd [ ] . this observation led to the theory that pd could originate within the ens, and hence spread to the vague nerve, reaching the cns [ ] . additional in vivo studies gave further strength to the theory of an intestinal origin for pd [ , , ] . even an abnormal pro-inflammatory activity, caused by the inflammatory bowel disease (ibd), may lead to hyperinflammation, dysfunctional immune response and, finally, favor the onset of pd [ ] [ ] [ ] [ ] . for all these reasons, different strategies to manipulate the gut microbiota composition, including the use of probiotics, are currently under study as valuable adjuvants in the treatment of pd (see below). this approach may contribute to decrease the local gastrointestinal inflammation and subsequent ros-related impairment, as demonstrated by several clinical observations [ ] . in , a study from théry and colleagues demonstrated for the first time the presence of evs in biological fluids, such as blood and urine [ ] . since then, several protocols have been developed to isolate and purify biofluid-derived evs-which stably protect their cargoes from degradation-for the discovery of novel vesicle-associated biomarkers [ ] . virtually all biofluids are considered important sources of vesicles, where also cns-derived evs can be detected. so far, in pd studies, evs have been isolated from cerebrospinal fluid (csf), plasma, serum, saliva, and urine. in addition, evs have been isolated, in vitro, from the culture supernatant deriving from a variety of cell types, including brain and peripheral cells [ ] . as such, evs and ev-derived cargoes may be relevant as diagnostic and/or prognostic tools in pd, as herein summarized. many of the above-mentioned defective/mutated pd proteins have been seen associated with circulating evs. the possibility to noninvasively evaluate the presence of such molecules captured the interest of researchers who actively investigate ev-associated pd biomarkers [ ] . the first report indicating that α-syn is secreted inside evs dates back to [ ] . the presence of α-syn in the vesicular fraction may have two effects in the context of pd: (i) to prevent the accumulation of aggregates inside the cell [ ] ; but also (ii) to spread the pathology to other districts [ , ] . ev-associated α-syn has been detected in saliva, plasma, csf, and serum from pd patients. although sometimes its levels were found controversial in different reports, it is possible to envisage the use of ev-α-syn as a valid pd biomarker in the near future [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to α-syn, lrrk has been discovered inside evs, especially the mutated form g s which displays phosphorylation at the ser(p)- residue. the functions associated with this protein are still not well defined, although it seems to be involved in the regulation of biogenesis of vesicular and membranous cellular structures [ , ] . the levels of ser(p)- -lrrk has been found higher in evs from urine samples of pd patients compared to healthy controls, and reflected the severity of the disease [ ] . also, the presence of dj- inside evs has been analyzed in urine and plasma samples from pd patients. ev-dj- levels are higher in pd subjects compared to controls, even if further studies need to confirm its validity as a novel pd biomarker [ , , ] . another interesting finding about ev-based biomarkers can be ascribed to the presence of cellular prion protein (prpc) in evs derived from pd patient plasma samples [ ] . prpc is a glycosylphosphatidylinositol-anchored membrane protein mainly located in the cns and involved in the transmission of α-syn to neurons [ ] . recently, in , leng and colleagues demonstrated that the levels of evs-prpc were higher in pd patients compared to controls and correlated with the progression of the cognitive decline [ ] . besides dysfunctional proteins, specific small non-coding rnas (i.e., mirnas) have been associated with the development and/or the progression of pd. of note, the profiling of ev-mirnas in both serum and csf bear the potential to become a reliable diagnostic tool for pd [ , ] . finally, very recent reports showed that gut microbiota-derived evs may have specific effects in several pathologies, including nds. more specifically, bacteria-derived evs in blood have been found dramatically altered in an ad mouse model, supporting their use for the metagenomic analysis of the gut microbiota in ad [ ] . similar findings were obtained using mucosal-luminal interface samples from pediatric ibd patients, suggesting that the alteration of intestinal microbe-derived evs may be associated with an aberrant host-microbiota interaction also in other inflammatory conditions, including pd [ ] . it is evident that a better understanding of the different risk factors in pd-and their relative contribution to the onset and progression of the disease-is urgently needed. the precise identification of the molecular triggers responsible for the daergic neurodegeneration will help to predict, and possibly to prevent, pd. this is a key point, as pd is usually diagnosed when already % of the neurons in the snpc are lost, making their recovery a major challenge for regenerative medicine. in this scenario, the identification of novel biomarkers associated with evs may help the early diagnosis of pd. importantly, compared with biomarkers identified in conventional specimens, ev-associated biomarkers may provide the highest amount of sensitivity and specificity, which can be attributed to their excellent stability in biofluids. at present, daergic drugs designed to replace the action of da in the depleted striatum represent the pharmacological treatment of pd [ ] [ ] [ ] [ ] . currently, various options are available to achieve this goal, either indirectly-through drugs acting at a presynaptic level, via da metabolism, inhibiting the breakdown of endogenous da-or directly, through daergic agonists acting at a post-synaptic daergic receptor level [ ] [ ] [ ] [ ] [ ] [ ] . unfortunately, there are no effective neuroprotective therapies for pd, but different clinical trials designed to develop potential disease-modifying strategies are currently on their way [ , ] . the complex topic of pharmacological therapies and pd management is out of the scope of this work and recent reviews have expanded on the pharmacological advances in pd therapeutics, including medication regimens tailored to the individual patient, based on the severity and temporal nature of their symptoms, as well as the side effects that they experience and how to manage them [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . numerous other medications have a role as adjunctive treatment, including surgical treatments, such as deep brain stimulation (dbs). these approaches are being assessed both to provide relief to patients with advanced pd, as well as to ameliorate their quality of life [ ] . below, the advances in cell-based therapies will be discussed, with major emphasis on ev-related approaches, either by targeting key pd pathogenic mechanisms or/and by slowing or preventing its progression. cell therapy-consisting of the transplantation of relevant cell types-was proposed following the establishment of the -ohda-animal disease model in the s [ ] . -ohda is a synthetic neurotoxic compound widely used to reproduce the nigrostriatal lesion characteristic of pd. since this molecule cannot pass the blood-brain barrier (bbb), it is intracranially injected and, through the da active transporter (dat), it induces neuronal death within the ventral midbrain with consequent da reduction and insurgence of motor dysfunctions [ ] . the selective and irreversible lesion of the nigrostriatal pathway produced by -ohda may be used as a useful parameter to evaluate the restoration of the daergic tone following cell transplantation [ ] . the first clinical trials evaluating cell therapy in pd were conducted in the s, by using da-producing adrenergic medullary cells. the results were not satisfactory, as the cells poorly grafted in the caudate nucleus and patients experienced postoperative psychiatric disturbances [ ] . more interesting results were instead achieved by transplanting human fetal ventral mesencephalic tissue into the striatum of pd patients. although some patients displayed a long-term efficacy, most of them developed graft-induced dyskinesia (gid) that, together with the ethical and safety issues connected to the use of fetal tissue, made this approach not feasible for many years [ ] . nevertheless, in , this particular branch of cell therapy was reconsidered by the transeuro consortium, which developed a currently ongoing open-label study (nct ), in which patients with mild pd will be transplanted with human fetal daergic cells [ ] . however, in the context of cell therapy, the use of stem cells represents the most promising approach for transplantation in pd, as they may be differentiated into specialized phenotypes, such as daergic neurons. moreover, these cells, thanks to their neurotrophic/immunomodulatory properties, are also able to reduce inflammation and inhibit apoptosis of damaged tissues. embryonic stem cells (escs) were the first type evaluated for transplantation in [ ] . again, the adverse immune reactions and ethical issues limited their use [ ] , although an ongoing phase i/iia clinical trial is evaluating the safety and the efficacy of intracranial transplantation of human esc-derived neural precursor cells in pd patients (nct ), following their positive outcome in primate pd models [ ] . induced pluripotent stem cells (ipscs) represent a valid alternative for cell therapy in pd. these cells are collected from skin (fibroblasts) or red blood cells and they are reprogrammed to differentiate into daergic neurons or replicative astrocytes [ , ] . this can be achieved by transfection with lentiviral vectors carrying specific transcription factors [ ] , or by exposure to specific growth conditions [ , ] . although these cells are recovered from the same patients, thus overcoming the immune rejection problems, teratoma formations have been observed after transplantation, leaving open questions about their safety [ , ] . on the contrary, other reports demonstrated that ipsc-derived daergic progenitor cells transplanted in primate pd models survived and worked as daergic neurons without any tumor formation in the brain, therefore demonstrating their clinical relevance for transplantation in pd patients [ , ] . another strategy to achieve the rescue of damaged daergic neurons was evaluated in pre-clinical models by transplanting adult syngeneic neural progenitor stem cells (nscs) in the snpc of aged mptp-treated mice [ ] . one third of these cells acquired an astrocytic phenotype and, synergistically with endogenous astrocytes, they activated the wnt/β-catenin signaling in snpc-daergic neurons, finally inducing neurorescue and immunomodulation [ ] . remarkably, a robust migration of nscs and nsc-derived astrocytes to the wnt-sensitive midbrain daergic niche was accompanied by a time-dependent daergic neurorescue [ ] . importantly, in nsc-grafted mice, the nsc-derived astrocytes and the endogenous astrocytes expressed wnt , mediating daergic neurorescue and microglia down-regulation [ ] . following a similar line of research, serapide and coworkers addressed the ability of ventral midbrain astrocytes, used as a graft source for unilateral transplantation above the sn of middle-aged mptp mice, to ameliorate the aged and mptp-injured microenvironment, thus mitigating nigrostriatal toxicity [ ] . here, grafting vmb astrocytes rejuvenated the sn microenvironment via a downmodulation of microglial pro-inflammatory status. these data suggest a chief role for vmb astrocytes and astrocyte-derived molecules in favoring neurorepair in pd [ ] . a first phase i clinical study, which evaluated the safety and the efficacy of human parthenogenetic derived nscs (isc-hpnsc) as therapy for pd, started in in australia. these cells were intracerebrally implanted into the striatum and snpc of individuals affected by moderate to severe pd (nct ). this intrinsic ability of nscs to induce neurorepair and immunomodulation may be ascribed, at least in part, to nsc-derived evs that could be used as substitute in a cell-free approach (see below) [ , ] . in line with these findings, another ongoing clinical trial is evaluating the safety and the efficacy of human fetal nscs injected intranasally in pd patients (nct ). mesenchymal stem cells (mscs) are another cell type that potentially might be used for pd cell therapy. these cells have a great plasticity and easily integrate into the host tissue as demonstrated in different contexts, including ad [ ] , als [ ] , multiple sclerosis (ms) [ ] , autoimmune diseases [ ] , diabetes [ ] , and pd [ ] . several studies demonstrated their potential for trans-differentiation in daergic neuronal phenotypes [ ] , and two ongoing clinical trials are evaluating the efficacy and the safety of undifferentiated or differentiated umbilical cord-derived mscs in pd patients. these cells are being administered intravenously (nct ) or intrathecally following their differentiation in nscs (nct ). as already mentioned for nscs, the benefits of mscs seem to be mediated also via their secretome, including the ev component, as demonstrated by several studies [ ] [ ] [ ] . despite mscs transplantation ameliorated pd symptoms, the trophic effects were often only transient [ , ] . moreover, in some cases, the systemic injection of these cells showed severe side effects, such as pulmonary thrombosis [ ] [ ] [ ] [ ] , whereas the alternative intracranial transplantation is a very invasive procedure [ ] . overall, the issues and risks potentially connected with the implantation of "living materials" within the cns stimulated the scientists to evaluate alternative strategies for brain repair. indeed, different cell-free strategies are under study to ameliorate the clinical picture in pd patients. for instance, lentivirus-based gene therapies have been evaluated in phase i-ii pd clinical trials with prosavin [ ] , a lentiviral vector carrying three enzymes for da biosynthesis [ ] . the safety of this approach was demonstrated together with motor improvements in mid-to late-stage pd patients. however, da replacement needs to be better addressed to maximize the benefits and, again, intracranial injection is a highly invasive procedure [ ] . following the first pioneer studies, the subsequent functional characterization of the evs remained elusive for many years, with scarce interest in the field. in , an important publication from raposo and colleagues, reported for the first time the presence of surface antigens on evs released by b lymphocytes. importantly, the evs were able to induce a specific t-cell response [ ] . on the same line, zitvogel et al. in demonstrated that dendritic cells (dcs) release antigen-presenting vesicles, carrying major histocompatibility complex proteins and t-cell co-stimulating cytokines and chemokines [ ] . the ev field has grown exponentially after these seminal findings, and several clinical trials are currently evaluating the efficacy of the ev-based treatments for different pathologies [ ] , including the recent severe acute respiratory syndrome coronavirus (sars-cov- ) infection (nct ). in the context of cns, one of the main challenges for candidate pd drugs is to cross the bbb and to target the brain. this is potentially achievable by using lipid-based vehicles, such as evs, which display an innate low immunogenicity and an intrinsic ability to cross biological barriers [ ] . moreover, an increasing body of evidence supports the neuroprotective/neuroregenerative potential of evs, with specific therapeutically relevant outcomes depending on the donor cells and the microenvironmental milieu [ , ] . during the last decade, several strategies based on natural or modified evs have been proposed, also for treating pd [ ] (figure and table ). biomolecules , , of from raposo and colleagues, reported for the first time the presence of surface antigens on evs released by b lymphocytes. importantly, the evs were able to induce a specific t-cell response [ ] . on the same line, zitvogel et al. in demonstrated that dendritic cells (dcs) release antigenpresenting vesicles, carrying major histocompatibility complex proteins and t-cell co-stimulating cytokines and chemokines [ ] . the ev field has grown exponentially after these seminal findings, and several clinical trials are currently evaluating the efficacy of the ev-based treatments for different pathologies [ ] , including the recent severe acute respiratory syndrome coronavirus (sars-cov- ) infection (nct ). in the context of cns, one of the main challenges for candidate pd drugs is to cross the bbb and to target the brain. this is potentially achievable by using lipid-based vehicles, such as evs, which display an innate low immunogenicity and an intrinsic ability to cross biological barriers [ ] . moreover, an increasing body of evidence supports the neuroprotective/neuroregenerative potential of evs, with specific therapeutically relevant outcomes depending on the donor cells and the microenvironmental milieu [ , ] . during the last decade, several strategies based on natural or modified evs have been proposed, also for treating pd [ ] (figure and table ). naturally occurring and engineered evs as cell-free treatment for pd. evs may be manipulated to deliver: (i) anti-oxidant agents (e.g., curcumin, catalase or apod) which protect neurons from oxidative stress; (ii) growth factors (e.g., gdnf) to stimulate proliferation of daergic neurons; (iii) da to ameliorate behavioral parameters; (iv) sirnas silencing the expression of snca gene to decrease α-syn levels. different routes of administration (systemic injection, intranasal injection and intraperitoneal injection) may be used to treat pd mouse models. naturally occurring and engineered evs as cell-free treatment for pd. evs may be manipulated to deliver: (i) anti-oxidant agents (e.g., curcumin, catalase or apod) which protect neurons from oxidative stress; (ii) growth factors (e.g., gdnf) to stimulate proliferation of daergic neurons; (iii) da to ameliorate behavioral parameters; (iv) sirnas silencing the expression of snca gene to decrease α-syn levels. different routes of administration (systemic injection, intranasal injection and intraperitoneal injection) may be used to treat pd mouse models. several studies showed the promising effects of cell therapy for pd treatment. in line with these encouraging results, evs derived from clinically relevant cells have been explored to reproduce the beneficial effects mediated by the corresponding donor cells, thereby reducing the possible side effects linked with the administration of whole cells. for example, a particular kind of mscs-recovered from the dental pulp of human exfoliated deciduous teeth (sheds)-has been used by jarmalavičiūtė and colleagues in [ ] . these particular cells, arising from the embryo neural crest, have unique neurogenic properties, as they are able to differentiate into daergic neuron-like cells and schwann cells [ ] . based on these properties, the authors selected sheds as ev donor cells. the rencell vm immortalized human neural progenitor cell line-opportunely differentiated to assume a daergic neuronal phenotype-was chosen as model of target cell, for evaluating the potential neuroprotective effects of shed-derived evs. to reproduce the parkinsonian/oxidative environment, the neurons were treated with the neurotoxin -ohda [ ] . the authors compared two strategies to culture sheds: (i) classic growth on two-dimensional flasks in serum free medium; and (ii) three-dimension growth on laminin-coated micro-carriers into a bioreactor, in a suspension-like environment. they also analyzed vesicles obtained with two different protocols: , × g centrifugation (l-evs) and , × g centrifugation (s-evs). interestingly, the authors demonstrated that s-evs only, specifically derived from sheds cultured on d micro-carriers, were able to suppress apoptosis of -ohda treated neurons. in fact, this anti-apoptotic effect was not observed with s-evs deriving from sheds cultured with standard methods, demonstrating that the culture conditions may affect the nature and amount of cargoes contained inside the evs [ , ] . recently, in , the same group published a follow-up work where they evaluated the effect of shed-evs in vivo, in a pd rat model [ ] . the animals were subjected to unilateral cranial injection of -ohda in the medial forebrain bundle and then intranasally treated with evs. by using the apomorphine test, the authors evaluated the effects of shed-evs on the dysfunctional consequences deriving from -ohda mediated striatal lesions. apomorphine injection is known to induce involuntary contralateral rotations in -ohda lesioned animals, while controls do not rotate at all. importantly, the treatment with shed-evs in -ohda treated rats halved the number of contralateral rotations in the apomorphine test, together with a significant improvement in gait parameters. moreover, a notable increase in the density of tyrosine hydroxylase (th, the rate-limiting enzyme converting l-tyrosine to l-dopa) positive cells was observed in both the striatum and the snpc of -ohda rats, as a result of the ev treatment [ ] . the authors suggested that the neuroprotective effects of shed-evs might primarily due to their protein content: for instance anti-oxidant molecules-such as the superoxide dismutase (sod ) [ ] , the thioredoxin (txn), the peroxiredoxin- (prdx ) and hsp -but also other proteins belonging to the family of annexins. thanks to the above-listed features, evs have been proposed by the authors as potent tools for a safe and non-invasive therapy to delay the progression of the pathology and to improve the motor impairments in pd patients [ ] . it is important to highlight the potential combinatorial effects mediated by the diversity of ev-cargoes, which represents one of the most relevant advantage for ev-based therapeutics, as reported in several contexts [ ] . remarkably, besides shed-evs, glial-derived evs may play key roles in both neurodegeneration and neuroprotection in pd experimental models, as recently reviewed by our group [ ] . for example, the studies of leggio and collaborators [ ] revealed that astrocytes from the ventral midbrain, which induce nigrostriatal rescue in mptp-treated mice [ ] , were able to secrete vesicles in the size range of s-evs and positive for classical ev markers (e.g., cd , cd , tsg ) [ ] . interestingly, in basal conditions, ventral midbrain astrocytes release more evs than striatal astrocytes. this diversity in the secretion of evs seems to suggest the existence of specific brain region-linked differences which may have possible functional implications in the intercellular communication within the nigrostriatal area [ ] . moreover, the authors identified a specific subset of mirnas and proteins enriched in the ventral midbrain astrocyte-derived evs, with a potential impact on daergic neuroprotection and nsc differentiation. overall, these data support the importance to further characterize the potential of astrocyte-derived evs as advanced therapeutics tools for promoting neuroprotection and neurorepair in pd (leggio et al., manuscript in preparation). as mentioned above, da cannot be administered directly to pd patients given its inability to cross the bbb. other prodrugs, including l-dopa, have been developed to address this issue. moreover, novel approaches based on lipophilic coating have been further investigated. in , pahuja and colleagues prepared nanoparticles (nps) of poly(lactic-coglycolicacid) (plga) incorporating da. this material has been chosen given the high biocompatibility. in fact, its degradation products (i.e., lactic and glycolic acids) are extremely biodegradable since they are eliminated as carbon dioxide and water through the krebs cycle. da, once incorporated within the nps, was more stable in the blood and with an improved capability of entering the brain. importantly, da-nps, when injected in the -ohda pd rat model, reversed neurobehavioral and neurochemical impairments of injured animals without any increase in ros production. no additional neurodegeneration in the striatum and in the snpc was observed, thus suggesting da-nps as a viable approach for a sustained delivery of da in the brain [ ] . a similar technology has been characterized by qu and colleagues in [ ] . they prepared fresh serum-derived evs, which were loaded with da and injected intravenously into a -ohda mouse model. to evaluate the brain-targeting potential, evs were labeled with the lipophilic fluorescent compound , -dioctadecyl- , , , -tetramethylindodicarbocyanine, -chlorobenzenesulfonate salt (did). as expected, h post-injection, evs were detected in both the striatum and snpc. the therapeutic efficacy was instead evaluated three weeks following da-ev administration. the results showed a significant amelioration of behavioral parameters compared to -ohda control mice. importantly, an enhanced accumulation of da was observed specifically in the striatum. moreover, the number of th-positive cells in the lesioned striatum raised, demonstrating that this approach was efficient and safe [ ] . one year later, tang and colleagues synthetized borneol and lactoferrin-modified nanoparticles (lf-bnps) used as da carrier. the authors intranasally administered the da-lf-bnps to -ohda pd rats, showing that these nps restored the da levels in the striatum of pd animals. as a consequence, a decrease in the number of contralateral rotations via the apomorphine test was also observed in treated rats, demonstrating the efficacy of nanoparticle-mediated da delivery [ ] . considering the pivotal role played by oxidative stress in the progression of pd, many studies are currently evaluating feasible strategies to inhibit the exacerbated ros production to restore their balance in the brain. for this purpose, recent in vitro and in vivo studies have been carried out to find novel approaches to scavenge ros. curcumin is a natural polyphenol widely used as anti-oxidant, antiseptic, antibacterial and cytostatic agent in several diseases [ ] . curcumin can also protect neurons from oxidative stress, by inhibiting protein aggregation and promoting neurogenesis in vivo [ ] . however, its poor solubility significantly reduces the overall bioavailability. taking advantage from its hydrophobicity and the preferential interaction with lipid membranes, the group of zhang in and developed an ev-based model to encapsulate curcumin [ , ] . first, they assessed the increased solubility and stability of curcumin-evs in vitro and in vivo [ ] . then, to evaluate the anti-inflammatory properties of curcumin encapsulated within evs, they delivered intranasally curcumin-evs in mice challenged with lps, used as a model of neuroinflammation. the authors observed a reduction of microgliosis following the treatment, thereby supporting the possibility to use curcumin-evs to treat nds [ ] . notably, the promising results obtained by using curcumin-loaded vesicles have been translated into clinics with an ongoing phase i clinical trial in cancer patients (nct ). in the study, curcumin-evs are orally administered, as diet supplement, to patients diagnosed with colon cancer [ ] . another molecule with important anti-oxidant activity is the enzyme catalase, which hydrolyzes the hydrogen peroxide to form hydrogen and oxygen [ ] . for this purpose, haney and colleagues in developed a cell-based delivery system in order to induce the catalase expression in brain cells [ ] . they used the macrophage cell line raw . stably transfected with a plasmid encoding a fluorescent catalase. these cells were intravenously injected into the -ohda pd mice. once in the brain, the cells were able to induce profound anti-inflammatory and neuroprotective effects, as confirmed by the improved motor functions [ ] . to evaluate whether the neuroprotective effects were mediated by macrophage-derived evs, the authors isolated and treated the pd animals with the vesicles produced by macrophages overexpressing the fluorescent catalase. evs were shown to carry the catalase at dna, rna, and protein levels. this important finding supports the notion that cells can exchange both nucleic acids and proteins by using their evs as carriers. the authors demonstrated that contiguous neurons can efficiently transfer evs, resulting in de novo protein synthesis in target cells, in turn responsible to counteract the inflammation [ ] . two years later, in , the same group used again the raw . macrophages to obtain evs loaded with catalase by using saponin-based permeabilization. these vesicles were intranasally injected into -ohda-treated mice and the effects were evaluated days later. first, they found reduced microgliosis and astrocytosis in mice treated with catalase-loaded evs compared to free catalase treated mice. in addition, the authors observed a significant increase of daergic neurons in ev-treated mice. moreover, in the apomorphine test, pd mice intranasally injected with catalase-loaded evs rotated less than control mice. these results demonstrated that the evs improved the bioavailability, as well as the targeting capability of catalase in the inflamed brain, with protective effects towards daergic neurons [ ] . in , kojima et al., developed an interesting cell-based system equipped with an "ev production booster" [ ] . they used the hek t cell line as donor cells to produce evs loaded with the catalase mrna, to specifically target the brain. as expected, these vesicles attenuated both the neuroinflammation and the neuronal death induced by the -ohda treatment [ ] . more recently, in , pascua-maestro and colleagues focused on another relevant player in the context of neuroinflammation, evaluating how evs are involved in the transport of lipocalin apolipoprotein-d (apod) from astrocytes to neurons [ ] . the role of apod in context of cns has been widely documented. this molecule binds lipids (e.g., cholesterol, arachidonic acid and steroids) and it is highly expressed by glial cells in response to oxidative stress conditions, including pd [ ] . apod is a small glycoprotein with a strong anti-oxidant activity, since it prevents lipid peroxidation, and therefore protects membranes from the oxidative damage [ ] . it was observed that apod expression increases in order to respond to the enhanced demand of lipids during brain development [ ] . in fact, apod expression is five to ten-fold higher in adult brains compared to neonatal ones, following expression changes which occurs during brain maturation and aging [ , ] . apod is located inside the endo-lysosomal compartment and, not surprisingly, it is found associated with evs [ ] . in their work, pascua-maestro et al. demonstrated that apod is conveyed from astrocytes towards neurons carried within the secreted evs. the authors treated neurons with the herbicide paraquat, which is a well-known ros generator (especially the superoxide free radical). hence, they found that astrocyte-derived evs were able to restore the viability in damaged neurons, via the direct transfer of apod [ ] . strikingly, apod was detected in blood-circulating evs from people affected by inflammatory-related conditions, such as pd patients, further suggesting a potential use of evs as biomarkers [ ] . as discussed above, the α-syn aggregates contribute both to motor and non-motor dysfunctions in pd patients. for this reason, research is trying to mitigate the formation and the accumulation of α-syn in the brain. among the methodologies investigated, one of the most explored is the use of micrornas (mirnas) or small interfering rnas (sirnas), which operate at post-transcriptional level by silencing the expression of the snca mrna [ , [ ] [ ] [ ] . the first report describing the ev-mediated delivery of sirnas to specific target sites have been proposed by alvarez-erviti and colleagues in [ ] . to avoid the development of adverse immune reactions in the host, they used immature dendritic cells to produce evs. notably, the authors developed a molecular system to increase the ev targeting to the brain. for this purpose, they fused the rabies viral glycoprotein peptide (rvg)-which specifically binds the acetylcholine receptor within the cns-to the n-term of the exosomal protein lamp . this construct was used to transfect the dendritic cells before ev isolation. once obtained the rvg/lamp -modified vesicles, the authors used the electroporation to introduce sirnas within evs [ ] . the same protocol was used by the same group in , to deliver α-syn sirnas to the brain of transgenic mice expressing the human phospho-mimic s d α-syn. when systematically injected in normal mice, evs loaded with α-syn sirnas were able to reach the midbrain, the striatum, and other cortical brain areas, where the sirnas produced a significant decrease of α-syn at both mrna and protein levels. importantly, when injected in transgenic mice, these vesicles induced a complete depletion of α-syn mrna, but they were less effective at the protein level. the human α-syn s d protein is more prone to aggregate, and probably the presence of α-syn inclusions requires a prolonged down-regulation of the expression to obtain an efficient depletion. moreover, as described by the authors, this sirna-based treatment demonstrated a short-term efficacy [ ] . five years later, in , the same group designed a novel short hairpin rna (shrna) minicircle (mcs) specifically delivered in the brain through rvg modified evs [ ] . mcs are small double-stranded dna vectors that can be easily inserted inside the evs. in fact, mcs contain only the transgene cassette, favoring a higher expression of the transgene and thus allowing a prolonged down-regulation of the target gene [ , ] . the authors used again the transgenic mouse models expressing the human phospho-mimic s d α-syn to evaluate the efficacy of shrna-mcs-loaded evs injected intravenously. this time they obtained a prolonged down-regulation of s d α-syn mrna and a significantly lower level of α-syn protein, compared to controls. notably, these brain-targeting evs were able to down-regulate the α-syn expression in several areas of the cns, where they prevented protein aggregation and consequently the death of neurons [ ] . moreover, the authors tried to evaluate the effect of this approach on pd mice subjected to intrastriatal injections of murine α-syn pre-formed fibrils. even in this case, the α-syn expression was efficiently down-regulated, at both mrna and protein level compared to controls, with a substantial protection against daergic terminal loss in the striatum and a consequent improvement of the clinical parameters [ ] . taken together, these data supported the great potential of this long-term therapy based on ev systemic administration. however, as discussed by the authors, this approach is effective at the early stages of the pathology, and therefore the efficacy during pd progression remains to be further evaluated [ ] . more recently, in , zhao et al. developed a nanoparticle (np)-based system able to inhibit α-syn aggregation and also to attenuate microgliosis [ ] . in fact, as mentioned earlier, upon sensing oxidative stress, microglia become activated, which may further exacerbate neuroinflammation. in addition, microglia play a crucial role in α-syn clearance and degradation. np preparation included two anti-oxidant molecules, the ferulic acid diacid (faa, a strong anti-inflammatory molecule), and the tannic acid (ta, a protein aggregation inhibitor). following in vitro studies, the authors observed that these nps were able to inhibit fibrillation and aggregation of acetylated α-syn (ac-α-syn, one of the most common post-translational modification of α-syn, with a potentially relevant pathological role). moreover, the authors applied these nps on bv microglial cells previously treated with monomers of ac-α-syn or mutated a t α-syn. the results showed that in the presence of nps, the levels of intracellular oligomer formation significantly decreased for both ac-α-syn and a t α-syn triggers. to evaluate microgliosis attenuation, bv cells were stimulated with a t α-syn, which increased the production of pro-inflammatory molecules, such as ros, tnf-α and il- . np treatment lowered the secretion of tnf-α and il- , as well as ros production, thus confirming the attenuation of α-syn induced microgliosis in the presence of faa-ta nps [ ] . considering that few approaches have been designed to target both α-syn aggregation and microglial activation, this work represents a valid anti-oxidant-based nanotherapeutic alternative to treat nds, such as pd. as illustrated above, gdnf is one of the "beneficial" growth factors produced by astroglial cells that stimulates proliferation and provides support and protection to daergic neurons. following the activation of astrocytes and microglia during neurodegeneration, gdnf levels drop. in vitro and in vivo studies aiming to restore gdnf levels demonstrated the great potential of this approach. consequently, gdnf administration to pd patients led to promising results [ , ] . moreover, the beneficial effects of gdnf on neuronal survival has been recently evaluated in -ohda rodent models transplanted with esc-derived daergic neurons [ , ] . however, the invasiveness of the procedure (i.e., the intracranial infusion) makes this approach not ideal for its usage in clinical routine [ ] . for this reason, other strategies have been proposed to reduce the invasiveness and guarantee the safety. in , an alternative route for gdnf administration has been developed by biju and colleagues. they transfected ex vivo bone marrow stem cell-derived macrophages transduced with a lentiviral vector overexpressing gdnf. these cells were then transplanted in mice before the mptp-mediated daergic neuron degeneration. the transplanted cells, which were able to cross the bbb, were recruited within sn area lesioned by mptp and efficiently differentiated in microglial cells. the results showed a remarkable neuroprotection of daergic neurons together with an increase of da production, which, in turn, resulted in motor function improvements [ ] . based on these results, the group of batrakova and colleagues-the same team investigating the role of catalase in macrophage-derived evs [ ] -in , developed an improved system based on autologous macrophages transfected ex vivo to produce gdnf [ ] . moreover, to avoid the triggering of an m pro-inflammatory status, macrophages were cultured in the presence of il- . the treatment with this cytokine induces the m phenotype, as already discussed for the m /m microglia polarization. the deriving m gdnf-producing macrophages were intravenously injected in the -ohda pd mouse model, where they were able to cross exclusively the bbb of inflamed brains, inducing both neuroprotective and anti-inflammatory effects with consequent motor function improvements. as in [ ] , to evaluate the possible role played by macrophage-derived evs, the authors analyzed the content of vesicles, finding a significant enrichment in gdnf carried by the vesicles. therefore, they suggested that evs might be directly involved in the release of gdnf within the proximity of damaged neuronal membranes, thereby favoring gdnf binding to its receptor expressed by the target neuronal cells. as a consequence, gdnf pathway was activated in recipient neurons with subsequent enhancement of the downstream protective effects [ ] . another strategy to increase gdnf levels in the inflamed brain has been developed in by aly et al. [ ] . they induced brain cell transfection in vivo by delivering plasmid dna (pdna) encoding gdnf, either alone or mixed with a gelatine solution to form nps, in a pd rat model. both the naked-pdna and the pdna-nps were then intranasally injected in rats one week before the -ohda toxin treatment, and the effects on pd rat brains were analyzed three weeks later. they found that the nps, but also-to a lesser extent-the naked-pdna, reached the brain with a consequent significant increase of the gdnf expression. the high concentration of gdnf within the sn protected daergic neurons and dendritic fibers against the damage induced by -ohda. in addition, the authors observed significant improvements in motor functions [ ] . therefore, these promising findings corroborate the important role played by gdnf to preserve daergic neurons from cytotoxic triggers. moreover, this approach seems to be as effective as invasive intracranial infusion, with the advantage to be more robust and easily applicable. combining the safety and targeting properties of evs with the reproducibility of the synthetic nps could lead to the development of an improved drug delivery system not only for pd, but also for other conditions. figure illustrates the ev-based therapeutics for pd and the related outcomes are listed in table . biomolecules , , of safety and targeting properties of evs with the reproducibility of the synthetic nps could lead to the development of an improved drug delivery system not only for pd, but also for other conditions. figure illustrates the ev-based therapeutics for pd and the related outcomes are listed in table . following their administration, evs cross the bbb to reach the brain, where they target neurons and glial cells. among the effects in ev recipient cells: (i) production of anti-oxidant molecules, (ii) reduction of neuroinflammation, (iii) decrease in α-syn levels, (iv) increase in da bioavailability. pd is a progressive and debilitating nd, which mainly affects the elderly people, but it is additionally diagnosed in younger subjects. despite its long history, the causes of pd remain elusive and no cure is currently available. moreover, no clear-cut diagnostic biomarkers for pd exist yet, and current methods to track pd progression are missing. indeed, pd neurodegenerative processes typically begin decades before the appearance of debilitating clinical symptoms. therefore, the diagnosis is achievable only when most of the relevant neurons have already died. the identification of novel pd biomarkers is key to develop early robust pd diagnostic methodologies, as well as to design neuroprotective therapies for at-risk populations. in this context, evs are good candidates as promising biomarkers, as well as functional nanodrugs carriers. first, evs are stably released within the blood and other biofluids, where their cargoes-protected from the action of nucleases and proteases present in the external environmentcan be easily isolated. secondly, thanks to their cargoes, circulating evs may mirror pathological changes, thus representing potential prognostic tools. this application of evs is currently tested in clinics, where evs are explored as "sensors" to follow the outcome of a selected therapeutic treatment [ ] . finally, evs are currently explored as effective bbb-permeable carriers to deliver in a targeted fashion nanodrugs within pd-damaged brains. following their administration, evs cross the bbb to reach the brain, where they target neurons and glial cells. among the effects in ev recipient cells: (i) production of anti-oxidant molecules, (ii) reduction of neuroinflammation, (iii) decrease in α-syn levels, (iv) increase in da bioavailability. pd is a progressive and debilitating nd, which mainly affects the elderly people, but it is additionally diagnosed in younger subjects. despite its long history, the causes of pd remain elusive and no cure is currently available. moreover, no clear-cut diagnostic biomarkers for pd exist yet, and current methods to track pd progression are missing. indeed, pd neurodegenerative processes typically begin decades before the appearance of debilitating clinical symptoms. therefore, the diagnosis is achievable only when most of the relevant neurons have already died. the identification of novel pd biomarkers is key to develop early robust pd diagnostic methodologies, as well as to design neuroprotective therapies for at-risk populations. in this context, evs are good candidates as promising biomarkers, as well as functional nanodrug carriers. first, evs are stably released within the blood and other biofluids, where their cargoes-protected from the action of nucleases and proteases present in the external environment-can be easily isolated. secondly, thanks to their cargoes, circulating evs may mirror pathological changes, thus representing potential prognostic tools. this application of evs is currently tested in clinics, where evs are explored as "sensors" to follow the outcome of a selected therapeutic treatment [ ] . finally, evs are currently explored as effective bbb-permeable carriers to deliver in a targeted fashion nanodrugs within pd-damaged brains. in this scenario, naturally occurring evs (without any in vitro manipulation) have been demonstrated to bear an intrinsic neuroprotective potential. of note, evs derived from relevant cell types, such as astrocytes, were shown to mediate the same (or even improved) therapeutic efficacy, compared with the administration of the corresponding donor cells. indeed, evs have a low immunogenicity and they can cross the bbb, thus emerging as novel cell-free nanotherapeutics for brain repair. the beneficial properties displayed by evs may be conjugated with the possibility to properly engineer them. these vesicles may be opportunely modified to deliver anti-oxidant/inflammatory proteins, as well as da or molecules able to block α-syn synthesis or aggregation. moreover, several routes of administration (e.g., intranasal, intravenous, implantable devices) may improve the delivery and the desired effects. in pre-clinical models of pd, these drug-loaded vesicles can induce anti-inflammatory effects and motor function improvements, ameliorating the overall course of the disease. the research in the field is moving at a fast pace towards the development of a new generation of vesicles, combining the beneficial properties of evs with the versatility of artificial nps, such as liposomes and polymersomes. the availability of several biodegradable materials and the possibility to artificially manipulate their chemical structure (e.g., to improve the drug release efficacy or the targeting efficiency) makes nps very promising tools for pd treatment. a hybrid natural-artificial vesicle, clearly inspired by naturally occurring vesicles, but enriched with polymers and lipids, may enhance the overall responsiveness of vesicles to environmental changes (e.g., ph, temperature, 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mimicking extracellular vesicle-mimetic ghost nanovesicles for delivering anti-inflammatory drugs to mitigate gram-negative bacterial outer membrane vesicle-induced systemic inflammatory response syndrome this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - u v nlr authors: mansueto, gelsomina; niola, massimo; napoli, claudio title: can covid disease induces a specific cardiovascular damage or it exacerbates pre-existing cardiovascular diseases? date: - - journal: pathol res pract doi: . /j.prp. . sha: doc_id: cord_uid: u v nlr a novel coronavirus sars-cov- causes acute respiratory distress syndrome (ards) with cardiovascular and multiple organ failure till death. the main mechanisms of virus internalization and interaction with the host are down-regulation or upregulation of the ace receptor, the surface glycoprotein competition mechanism for the binding of porphyrin to iron in heme formation as well as interference with the immune system. the interference on renin–angiotensin–aldosterone system (raas) activation, heme formation, and the immune response is responsible for infection diffusion, endothelial dysfunction, vasoconstriction, oxidative damage and releasing of inflammatory mediators. the main pathological findings are bilateral interstitial pneumonia with diffuse alveolar damage (dad). because ace receptor is also present in the endothelium of other districts as well as in different cell types, and as porphyrins are transporters in the blood and other biological liquids of iron forming heme, which is important in the assembly of the hemoglobin, myoglobin and the cytochromes, multiorgan damage occurs both primitive and secondary to lung damage. more relevantly, myocarditis, acute myocardial infarction, thromboembolism, and disseminated intravasal coagulation (dic) are described as complications in patients with poor outcome. here, we investigated the role of sarscov- on the cardiovascular system and in patients with cardiovascular comorbidities, and possible drug interference on the heart. coagulation (dic) are described as complications in patients with poor outcome. here, we investigated the role of sarscov- on the cardiovascular system and in patients with cardiovascular comorbidities, and possible drug interference on the heart. between the december and the beginning of february , a novel coronavirus sarscov- spreads from china all over the world. sars-cov- induced a disease called covid- by the world health organization (who) which declared the pandemic status (https://www.who.int/emergencies/diseases/novel-coronavirus- /events-as-they-happen). the sars-cov- is an rna virus (family coronaviridiae), similar to sars-cov (severe acute coronavirus) ( , ) . the main symptoms of covid- disease are fever, cough, and shortness of breath. less common symptoms are muscle pain, anorexia, malaise, sore throat, nasal congestion, dyspnoea, and headache with an onset between and days. the clinical severity ranges widely from asymptomatic infection to fatal disease. the involvement of the upper respiratory district and lung is typical and primary. radiological features include interstitial thickening, lung opacities, lower lobe predominance, and bronchiectasis; multifocal peripheral subpleural ground-glass opacification or consolidation has been commonly observed ct feature up to pleural and pericardial effusion, with symptoms ranging from flu syndrome to acute respiratory distress syndrome (ards), caused by diffuse alveolar damage (dad), with cardiovascular and multiple organ failure till death ( , , ) . a multicenter study evaluated pulmonary and extrapulmonary manifestations such as hepatic and renal dysfunction, lymphopenia, thrombocytopenia, and elevated inflammatory biomarkers on a large series of hospitalized patients. the study shows that the main risk factors of clinical behavior j o u r n a l p r e -p r o o f are diabetes hypertension and coronary heart disease ( ) . the mortality rate of covid- is estimated at . %, according to the national official statistics in china ( ) . preliminary reports in the united states indicate that the highest mortality is found in people aged and over ( % to %), followed by people aged - ( % to %), people aged to ( % to %) and people aged to (< %), with no few deaths among people aged or under. however, contrary to previous reports from china, % of deaths occurred among adults aged to , and % of those hospitalized were between and years of age ( , ) . according to pubmed (https://www.ncbi.nlm.nih.gov/pubmed/), in less than two months, over papers have been published on sars-cov- including its virology, epidemiology, pathogenesis, diagnosis, and treatment. from the literature, it emerges that many deceased patients are old, with oncological, immunological, or dysfunctional systemic diseases linked to the cardiovascular system; it also emerges that multi-organ complications take place in the lung infection, including cardiovascular complications especially that often constitute the real cause of death. this scenario is a survival challenge for patients and a challenge for all those who work to find the right therapy and prevent the unfortunate event of death. here, we examine pathophysiological effects of sars-cov- , on the cardiovascular system as well as drug mechanisms with side effects on cardiovascular complications. it is important to focus that coronaviruses mainly infect epithelial cells of the upper respiratory tract and pulmonary pneumocytes but also other types of epithelial cells such as endothelial cells of arteries and veins, smooth muscle cells, intestinal epithelium cells and immune cells ( , ) ( , ). the rna genome is released into the cytoplasm of the cells through the fusion of the capsid with the cell plasma membrane. after interaction with its proposed receptors angiotensinconverting enzyme (ace ), genomic rna accompanied by envelope glycoproteins and nucleocapsid proteins forms virion-containing vesicles, which then fuse with the plasma membrane j o u r n a l p r e -p r o o f to release the virus ( ). physiologically, ace counters renin-angiotensin-aldosterone system (raas) activation by the degradation of angiotensin ii to angiotensin which attenuates its effects on vasoconstriction, sodium retention, and fibrosis. ace also cleaves angiotensin i to angiotensin and participates in the hydrolysis of other peptides and may be up-regulated in certain clinical states ( , , ) the interaction between sars viruses and ace which is localized above all on the endothelium of the pulmonary capillaries, causes alterations of the circulation with the variation of the pressure levels ( fig. ) . on the other hand, sars-cov- also generates interference in the native immune response (toll-like receptors (tlrs), rig-i-like receptors (rlrs) and nod-like receptors (nlrs), which trigger the expression of interferon (ifn) and activation of anti-viral effectors such as natural killer cells, t cd + cells, and macrophages); generates interference in the recruitment of inflammatory cells, in the regulation of host dna replication with transcription anomalies and with triggering of the apoptotic pathways, and in dysregulation of the renin-angiotensin system ( ). ( fig. ) . mainly, the sars-cov- is responsible for bilateral interstitial pneumonia with dad and primary pulmonary hypertension from direct alveolar and endothelial damage. the morphological changes are represented in the first phase by an interstitial inflammatory infiltrate, diffuse vascular congestion, edema, and subsequently from proteinaceous edema with or without hyaline membranes lining alveolar walls, inflammatory mononuclear infiltrate with multinucleated giant cells, exfoliation to also intra-alveolar granulocyte inflammation and position of fibrinoid material which are the true signs of direct damage. the foci of reactive alveolar epithelial hyperplasia, fibroblastic proliferation with fibrosis, and of alveolar rupture are present as a consequence ( ) . these changes in the parenchyma induce primary pulmonary hypertension due to an increase in lung resistance and overload of the right and left circulation. the clinical manifestation is j o u r n a l p r e -p r o o f characterized by systemic hypertension and ventricular hypertrophy, as it happens in all lung diseases from direct damage with increased of resistance. cardiovascular mortality is known to be higher in all influenza pandemics than in all other causes. acute respiratory viral infections, such as coronaviruses are well known to trigger factors for cardiovascular disease (cvd) ( ) . during covid- pandemic disease, the literature produced in a few months report that the increase in morbidity and mortality is found in particular in the elderly and in those who present comorbidities. the most prevalent comorbidity were hypertension, diabetes, and cardiovascular diseases ( , ) . compared to lung, data concerning cardiovascular involvement are less described ( ) . this makes it difficult to draw a line between complications of comorbidity and possible direct cardiovascular damage by covid- . there are autopsy data relating to the infection produced by sars cov in and by mers-cov in that can help better understand the involvement of the cardiovascular system. however, these data are affected by the difficulty of autopsy diagnosis made in a particular background. for example, an autopsy study reported post-mortem examinations in patients who died of sars in which patients had pulmonary thromboembolism and patients with deep vein thrombosis. one patient had a subendocardial infarction with occlusive coronary artery disease. one patient had valve vegetations along with heart attack, kidney, spleen, and brain ( ) . the reported presence of increased d-dimer and alteration of coagulation parameters highlights the need to understand whether positive cov- patients have a greater risk of hypercoagulability than negative patients with comorbidity and the need to understand the correct use of the antiplatelet therapy. some clinical trials have compared the clinical characteristics, coagulation parameters, and mortality between the covid and non-covid groups both with underlying chronic diseases including hypertension, diabetes, heart disease, and lung disease, and antiplatelet therapy and d-dimer evaluation. in the covid group, significantly j o u r n a l p r e -p r o o f lower mortality was found in users of heparin with markedly high d-dimer compared to non-users, while no difference in mortality among heparin users compared to non-users was found in the non-covid group. no significant improvement in clinical symptoms. however, further studies on the role of anticoagulants are still needed ( ) . some authors have paid attention to surface structural proteins and non-structural proteins of cov- not only in the mechanism of integration into the host cell but also indicated inhibition of the normal metabolic pathway of heme with competition mechanism for the binding of porphyrin to iron. heme is the degradation product of hemoglobin through the link with porphyrin ( ). this mechanism is not yet clear, but it explains the finding of the virus also in biological liquids such as urine, saliva, feces, and blood. the reported data requires clinical and experimental demonstration. ace is the putative receptor of sars viruses which is present mainly in the epithelial cells of the upper respiratory district, in pulmonary endothelium and pulmonary alveolar pneumocytes, but also present in other cell types in different organs as heart and kidneys ( ). it is possible to hypothesize direct cardiac damage mediated by the interaction of the virus with ace or much more reasonably mediated by the imbalance caused by the alteration of the functioning of the raas. based on these considerations, it is difficult to distinguish between death from direct cardiac or systemic vascular damage induced by sars-cov- in patients without comorbidity and death in patients with previous cardiovascular disease. because the pandemic infection broke out in a very short time, there are no large autopsy studies able to shed light on the real damage to the cardiovascular system in subjects who died from covid- disease ( , ) . can sars-cov- induce new heart conditions or can it only exacerbate pre-existing cardiovascular diseases? we believe that attention must be paid to three fundamental entities that can cause sudden death in patients infected by sars-cov- : myocarditis, coronary syndrome, thromboembolism. only a few cases with myocarditis and pericarditis involvement were reported, without consistent histological evidence ( , ) . it is clear that in a condition of myocarditis during covid- disease, with inflammatory infiltrate and myocardial necrosis according to dallas criteria ( ), ventricular dysfunction, arrhythmias, and sudden cardiac death can occur as occurs in enterovirus or adenovirus myocarditis due to an exuberant inflammatory response in subjects without pre-existing diseases. only one case of cardiac tamponade in a -year-old man sars-cov- infected without cardiovascular risk is reported in the literature as a complication of myocarditis and pericarditis ( ) . the causes of hydropericardium and cardiac tamponade also include infectious and inflammatory causes ( %) and mechanical complications of myocardial infarction ( %) ( ) . our autoptic experience leads us to some considerations. myocarditis is a cause of sudden death in children and in young adults with undiagnosed or underestimated viral infections ( ) . patients who died for complications of covid- are old and have often comorbidity due to cardiovascular diseases. we can, therefore, hypothesize that myocarditis with the presence of inflammatory infiltrates in the myocardial interstitium and with the structural damage validated by the laboratory markers of damage and cardiac necrosis, can be a secondary complication of the immune response and not of the direct action of the virus on cardiomyocytes. to date is not evidence of rna coronaviruses in the heart. considering the reported presence of ace in different cell types and also in the heart ( ), we can only hypothesize a hematogenous diffusion of the pathogen and a similar interaction as happens in the lung; the hypothesis remains that the sars-cov- action on the heart in old people is mediated by systemic imbalance caused by the alteration of the functioning of the raas on comorbidity background. the hypothesis that influenza viruses can trigger myocardial infarction has long been known ( , ) . large and more recent studies have reported that previous myocardial infarction, diabetes, j o u r n a l p r e -p r o o f dyslipidaemias, hypertension, and other cardiovascular risk factors can predispose to an acute ischemic event in respiratory virus infections such as recently reported during the pandemic covid- disease ( , , ) . a cohort study compared patients with and without heart damage and analyzed the association between heart damage and mortality. the results of the study showed that out of hospitalized patients, . % had heart damage, and, compared to patients without heart damage, these patients were older with comorbidity, leukocytosis, high sensitivity troponin i increase regardless of abnormalities in electrocardiography and echocardiography. the study demonstrated that cardiac injury and mortality in patients with covid- were significantly associated ( ). heart damage patients had higher mortality than those without heart damage. another study evaluated the association of underlying cardiovascular disease (cvd) and myocardial injury with fatal outcomes in patients with covid- . the study showed that of patients with sars-cov- , . % of patients had a myocardial injury, resulting in cardiac dysfunction and arrhythmias and an unfavorable prognosis while the prognosis of cvd patients without myocardial damage was relatively favorable. a percentage of . had underlying cvd including hypertension, coronary artery disease, and cardiomyopathy, and . % showed myocardial damage as indicated by elevated tnt levels. the study showed that myocardial damage and mortality was markedly higher in patients with high tnt levels than in patients with normal tnt levels and that the patients with underlying cvds had the highest mortality ( . %) and the shortest survival period ( ) . this data favor the role of the pre-existence of disease as a risk factor and of high mortality in adults and old people. since covid- binds to ace which is expressed in different cell types and the kidney and heart, a mechanism of direct damage to cardiomyocytes is conceivable but the systemic inflammatory responses, destabilized coronary plaque and aggravated hypoxia constitute now the only obvious modes of heart damage (fig. ) positive probably leading to activated t-helper- (th ) cell responses ( , ) , and increased of thelper- (th ) cytokines (eg, il and il ) that suppress inflammation ( ) . the inflammatory state can aggravate acute coronary syndrome with myocardial infarction until sudden death. it is crucial to emphasize that in this context, only biopsy and autopsy studies could provide information to understand the disease. acute myocardial infarction has been indicated as a complication, as well as laboratory parameters reported as the increase in cardiac troponin. the clinical data do not explain the type of heart damage. the increase in cardiac troponin is a marker of myocardial damage without distinguishing between necrosis and apoptosis. the identification of histological features could certainly be useful. we believe that it is fundamental to be able to distinguish in the heart between necrosis from hypoxic, ischemic damage, myocarditis and direct virus damage, or other cardiomyopathies responsible for heart failure and sudden death. there is currently no evidence of the presence of cov- in human myocardiocytes with the pcr method. some autopsies were performed on old subjects (> years old) and the morphological data highlighted the presence of cardiomyocyte hypertrophy, degeneration, and necrosis of some cardiomyocytes, mild hyperemia and edema of the interstitial cells and infiltration of a small amount of lymphocytes cd +, macrophages neutrophils but no evidence of sars-cov- in tissue ( ) . these aspects do not allow to make a correct differential diagnosis because they lack a conspicuous consistency of the data also in light of the same histological diagnostic criteria. it is known that patients with cardiovascular disease have a higher risk of a thrombo-embolic event as it is known that all viral infections have a potential role in disseminated intravascular coagulation j o u r n a l p r e -p r o o f (dic) the endothelial damage, the blood flow turbulence, and hypercoagulability are the basis of the mechanism. some clinical studies have highlighted that sars-cov- positive and symptomatic patients have more thrombus-embolic and dic risk. there is evidence for abnormal coagulation parameters with significantly higher levels of d-dimer and fibrin degradation, longer prothrombin times, and activated partial thromboplastin time in hospitalized patients with severe covid- disease and non-survivors; dic diagnosis was performed according to clinical and laboratory criteria ( , , ) . the greatest incidence of an adverse event is however detected in patients with underlying diseases including cardiovascular disease. vascular inflammation and hemodynamic instability can contribute to the hypercoagulable state and endothelial dysfunction resulting in thromboembolism or dic in patients with underlying cardiovascular diseases. also, in this case the literature is deficient in the morphological data that could be taken from an autopsy case study. some clinical studies have shown that one-third of patients with covid- disease, aged to years, have comorbidities such as cvd, hypertension and atherosclerotic cardiovascular disease and have shown that a predisposition to acute cardiac complications related to comorbidities already in this age group ( ) although clinical studies are limited by the short time and the absence of an autoptic finding, some meta-analyses have identified a movement towards intensive care and increase the mortality risk with the increase in average age and patients whit cardiovascular comorbidities such as diabetes, hypertension and coronary disease ( ) . a high proportion of severe to critical cases and high fatality rates were observed in the elderly patients (aged ± years), with hypertension ( . %), diabetes ( . %), and cardiovascular disease ( . %) ( ) many patients may be immunocompromised due to the underlying malignancy or anticancer therapy or primitive immunodeficiencies and are at higher risk of developing infections. as with other infections, the risk of covid- disease is increased in these patients. clinical studies in cancer patients report a higher risk of infection than in a non-cancerous population (median age years) ( ) . however, some data suggest that there is no greater multiorgan compromise from sars-cov- in immunocompromised individuals as a consequence of chemotherapy ( ) cardiovascular effects of current therapy against covid- to date, there are no specific effective therapies for covid- , but pharmacologic agents are under active investigation. old antiviral drugs of which the pharmacokinetics and side effects are already known to have been re-evaluated in the treatment ( - ) . the real dilemma is the role of the basic drugs used in cardiological comorbidities and their possible interference in worsening the prognosis. lopinavir/ritonavir (kaletra) inhibits the replication of the rna virus and has evidence of a synergistic in vitro effect with ribavirin ( , , ) ; has been used for years as components of treatment for hiv ( , ) it may interfere with the dosage of anticoagulants such as ribavirin ( ) . moreover, it may also interact with hmg-coa reductase inhibitors (statins) due to the risk of rhabdomyolysis ( ). favipiravir (avigan) is an anti-influenza virus that selectively and potently inhibits the rnadependent rna polymerase (rdrp) of rna viruses ( ) . the preliminary results indicated that favipiravir had more potent antiviral action and not significant adverse reactions ( ) . remdesivir is an antiviral agent that binds to the active site on rdrp has broad-spectrum activities against viruses including ebolav, mers-cov, and sars-cov with controversial results and in the absence of reported cardiovascular toxicity. it was the first drug used in sars-cov . clinical trials have shown no cytotoxicity or adverse reactions related ( ) ( ) ( ) ( ) . ribavirin that binds to the active site on rdrp has been used for years as components of treatment for hepatitis c ( ), has broad-spectrum activities against viruses including mers-cov, sars-cov- ( , ); it can influence the dosage of anticoagulants and can also influence the activity of p y inhibitors through the inhibition of cyp a , which results in a reduction of the serum concentrations of the active metabolites of and increased serum concentrations ( ) . hydroxychloroquine (hcq) was originally developed and used for the treatment of malaria and it is increasingly used in the management of a variety of autoimmune disorders, with well-established roles in dermatology and rheumatology and emerging roles in oncology ( , ) . hcq is a modulator of the immune response through several mechanisms. hcq inhibits the toll-like receptors and in dendritic cells, inhibiting the production of interferon-alpha, counteracts the effect of extracellular oxidants, and stops the signaling of t cell receptor-dependent calcium within t cells and hence antigen processing. it also induces apoptosis and interferes with lysosomal function, influencing the processing of antigenic peptides necessary to trigger autoimmune responses ( , ) ; and has to interfere with heme which is essential for the parasite ( ). retinopathy and some cardiac effects such as bundle-branch block, ventricular tachycardia, and cardiomyopathy often with hypertrophy, restrictive physiology, and congestive heart failure are known ( ) . in the cytoplasm, a consequence of lysosomal dysfunction, are described ( , ) . cardiotoxicity with potential conduction or structural abnormalities on electrocardiogram (ecg) has been reported. an association between cumulative hcq dose above the median and structural ecg abnormalities (left ventricular hypertrophy or atrial enlargement) was reported; however, it is not shown a statistically significant association with ecg structural abnormalities. the cumulative am decreases the odds of ecg conduction abnormalities (prolonged corrected qt interval, short pr interval, left bundle branch block (lbbb), right bundle branch block (rbbb) and atrioventricular block (avb), premature atrial complex, tachycardia, bradycardia, atrial fibrillation, ectopic atrial rhythm, premature ventricular complex and ventricular bigeminy ( , ) . since the dose of hqc used in covid- therapy is lower, and its use is not chronic, we can rule out a cardiotoxic effect. recent studies have shown that azithromycin (azm), and the closely related drug roxithromycin, both act as drugs that can target and selectively remove senescent cells, with an efficiency of nearly % ( ) . used in cystic fibrosis, is has an anti-fibrotic effect by targeting myofibroblast cells and functionally acts as an anti-inflammatory drug and reduces mediators, such as il- beta and il- ( , ) . it also inhibits the replication of other viruses, such as ebola and hiv- ( , , ) . azithromycin has been linked to an increased risk of ventricular arrhythmia, prolonged qt interval such as rifampicin. clinical trials indicate that the risk of ventricular arrhythmia with the use of azithromycin is likely to be due to the patient's health rather than due to the drug itself. the qt interval prolongation is not observed in the association between chronic azm therapy ( ) ( ) ( ) ( ) ( ) . ace is essential in the mechanism by which anti-raas inhibitor drugs work to reduce the risk of cardiovascular disease due to hypertension and diabetes. concerning cardiovascular risk in positive sars-cov- subjects taking antihypertensive and hypoglycaemic therapy, the literature produced so far is confusing. contrary, some authors indicate overexpression of ace in sars-cov infection ( , ) , which we could explain as a rebound effect ( ) . ace is expressed in different cell types and also in the kidney and heart, but mainly in the lung as well as ace (angiotensinconverting enzyme) ( , ). considering the pathogenesis and the effects produced by sars-cov- in the lung, it is more reasonable to believe that there is a down-regulation. in both cases, there is no scientific evidence that patients using anti-raas or ace inhibitors have a higher risk of covid- infection or that they have a higher cardiovascular risk. furthermore, an up-regulation would favor greater treatment efficacy and protection rather than risk. some authors suggest the protective role of ace inhibitors against sars-cov- infection as opposed to others. however, we still have doubts about how to truly interpret the role of ace inhibitors which, unlike anti-raas ( ) ( ) ( ) , act on the angiotensin-converting enzyme. instead, sartan antagonizes the angiotensin ii receptor, which acts by blocking the activation of the angiotensin at receptors. the scenario is very confusing regarding anti-ras and ace inhibitors, but even more so regarding ( , ) . the use of statins in ebola virus infection is described as a protective factor ( , ) . besides, for anti-inflammatory effects, statins have been proposed as adjunctive therapy in influenza although extensive clinical studies in patients with ards have shown no clinical benefit ( , ) . this data suggest the protective effect of statins in sars-cov infections although few ( ) ( ) ( ) ( ) ) . the main mechanisms of virus internalization and interaction with the host are regulated by ace receptor and porphyrin binding. because ace receptor is also present in the endothelium as well as in different cell types, and as porphyrins are transporters in the blood and other biological liquids of iron forming heme, which is important in the assembly of the hemoglobin, myoglobin and the cytochromes, multiorgan damage occurs both primitive and secondary to lung damage. the absence of important predisposing factors in covid- pneumonia seems to confirm the role of serious infections as a precipitating factor for acute thromboembolism and heart failure through the inflammatory state and dysregulation of the immune response. the presence of cardiovascular comorbidities seems to aggravate the prognosis until death by myocarditis, coronary syndromes, and diffuse thromboembolism or dic. no large autopsy studies able to shed light on the real damage to the cardiovascular system in subjects who died from covid- and our autoptic experience suggests that the autopsy exam is very important ( , [ ] [ ] [ ] . there is no substantial data to say that anti-raas, ace inhibitors, statins increase the risk of cardiovascular damage in covid patients. the role of anticoagulants can be useful especially in people with cardiovascular disease. hqc therapy appears to be effective and the risks for cardiovascular complications are related to the long-term cumulative dose. this work was supported by prin f zb from the italian ministry of university and research (miur) (pi prof napoli). the funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. we thank dr. giuditta benincasa, phd student, for help with ithenticate software. none declared. sars-cov- infects host cells through ace receptors. the virus can act on the cardiovascular system increases the risk of damage and sudden death in people with comorbidities. the figure shows the main hypothesized mechanisms of interaction between the virus and the cardiovascular system with the causes of damage most reported in the literature. damage mediated by the release of inflammation mediators, by direct action on the vascular endothelium that acts on the determinism of hypercoagulability and hypoxic damage by alterations of perfusion (acute coronary syndrome, ima, thromboembolism, dic), and by the immune response responsible in the first instance of myocarditis with myocardial damage. the damage mediated by the proposed downregulation or up-regulation of the ace receptor with a consequent imbalance of the normal circulatory and inflammatory homeostasis. the surface glycoprotein competition mechanism for the binding of porphyrin to iron in hema formation. in red the inhibition pathways, and green the main activation pathways both of the virus infection modality, of the pathogenetic mechanisms of the damage, and when taking the drugs used in people with hypertension, diabetes, and vascular disease. a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: 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after alemtuzumab treatment for multiple sclerosis epigenetic hallmarks of fetal early atherosclerotic lesions in humans key: cord- - jxvngvz authors: kunii, osamu title: the okinawa infectious diseases initiative date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: jxvngvz at the kyushu–okinawa group of eight summit in , japan announced the okinawa infectious diseases initiative (idi) and pledged to spend us$ billion over a five year period to combat infectious and parasitic diseases in developing countries. the idi has exceeded expectations, spending more than us$ billion over four years. the idi is a unique initiative with its own philosophical basis and specifically tailored interventions and measures that helped to initiate worldwide political and financial commitments in the fight against infectious diseases. notably, it promoted partnerships among stakeholders and emphasized comprehensive and inter-sectoral approaches (i.e. coordination and collaboration between health and other sectors). it helped to create a new vision of what is possible in the global effort against communicable diseases and has been instrumental in shaping the changing environments of development assistance, poverty reduction and other trends to reduce the impact of infectious and parasitic diseases. the okinawa infectious diseases initiative osamu kunii institute of tropical medicine, nagasaki university, sakamoto - - , nagasaki - , japan at the kyushu-okinawa group of eight summit in , japan announced the okinawa infectious diseases initiative (idi) and pledged to spend us$ billion over a five year period to combat infectious and parasitic diseases in developing countries. the idi has exceeded expectations, spending more than us$ billion over four years. the idi is a unique initiative with its own philosophical basis and specifically tailored interventions and measures that helped to initiate worldwide political and financial commitments in the fight against infectious diseases. notably, it promoted partnerships among stakeholders and emphasized comprehensive and intersectoral approaches (i.e. coordination and collaboration between health and other sectors). it helped to create a new vision of what is possible in the global effort against communicable diseases and has been instrumental in shaping the changing environments of development assistance, poverty reduction and other trends to reduce the impact of infectious and parasitic diseases. since the group of seven (g ) summit in denver, usa, world leaders have made major commitments and agreed to collaborate to combat infectious and parasitic diseases [ ] . at the kyushu-okinawa group of eight (g ) summit in july , infectious diseases were on the main agenda for the first time, and major progress was made in securing stronger commitments from all g nations [ ] . the commitments included an agreement to establish new partnerships to help maximize the impact of health and medical interventions, and recognition of the need to expedite the mobilization of additional resources. one of the outcomes of this new-found political appreciation of the urgent need to tackle infectious diseases was the establishment in january of the global fund to fight aids, tuberculosis and malaria (gfatm: http://www. theglobalfund.org/) [ ] . japan remained the top overseas aid donor throughout the s and is still the second biggest after the usa [ ] . even before the denver summit, the japanese government was committed to fighting infectious diseases. in , together with the usa, japan launched the common agenda for cooperation in global perspective [ ] to address global problems such as increasing environmental damage, overpopulation and damage from both natural and manmade disasters. this was followed, in , by the global issues initiatives on population and aids [ ] . japan underscored its undertaking to boost parasite control efforts by launching the global parasite control initiative, also known as the hashimoto initiative (hi), at the g summit in birmingham, uk [ ] . the subsequent okinawa infectious diseases initiative (idi) was designed using the successes and challenges experienced during hi activities, expanding targets and strategies towards broader infectious disease control and prevention, and deploying a broad spectrum of interventions. in this article, i review the idi, focusing on its philosophy, components, activities and achievements. the idi incorporated the following basic philosophy, which greatly influenced the manner in which the programme was designed and implemented. infectious and parasitic diseases as a central issue in economic and social development infectious and parasitic diseases not only threaten the lives of individuals in developing countries but also are an impediment to the social and economic development of those nations [ ] [ ] [ ] , particularly affecting the poor [ ] . the risk of infection in developing countries is increased by several factors, including high population growth rates, poverty, gender disparities, fragile medical systems, inadequate preventive care and treatment services, lack of safe water supply, and malnutrition [ ] [ ] [ ] . these factors are compounded because poor health exacerbates poverty [ ] . fighting infectious and parasitic diseases should be a central part of all development programmes and a crucial element in efforts to reduce poverty [ ] . global partnership and community-based action infectious and parasitic diseases should be viewed as a global issue that requires collective approaches based on international, multisector partnerships [ ] . effective measures to tackle these diseases also require action at the community level based on the concept of primary health care [ ] . as such, it is also important to incorporate measures against infectious and parasitic diseases in all community-level development programmes. the experiences of japan in public health activities and its future role following world war ii, japan developed a public health centre system, trained public health workers extensively, promoted measures for maternal and child health care and enhanced health care services in schools [ , ] . these steps contributed to the rapid reduction in infant mortality rates [ ] . japan also mounted major initiatives to eliminate infectious and parasitic diseases nationwide; for example, by linking public health activities with measures to control tuberculosis (tb), japan substantially reduced the number of tb-related deaths [ ] . okinawa, which comprises the southernmost islands of japan, has a history of successful eradication of malaria, filariasis and other parasitic diseases through active public health measures, including community participation and mobilization [ ] . drawing upon these experiences, japan has provided experts, technical assistance and capacity development programmes to developing countries through the idi by modification and application of the methods and technology that have proved so successful in japan. strengthening the health sector in developing countries the most important philosophy of japanese development assistance is to support the self-help efforts of developing countries. japan supports the development and implementation of health sector plans and/or infectiousdisease-specific intervention programmes designated by the countries themselves. through infrastructure and institution building, in addition to provision of technical assistance, japan supports and facilitates the development of health systems and sector reforms, thereby reinforcing health development planning and programmes, building capacity and helping to ensure sustainability of infectious disease control. the idi has given high priority to supporting human resources development, which is the most crucial component in making health systems function, and to creating quality services and high performance in infectious disease control. japan has provided several training programmes and courses in response to local needs in both developing countries and in japan aimed at individuals from all levels (e.g. from policy makers to field site personnel). to tackle a formidable global issue, japan has consolidated partnerships with various stakeholders, including the united nations (un: http://www.un.org) and other multilateral organizations, donor agencies, nongovernmental organizations (ngos) and civil organizations such as private nonprofit organizations and community groups. japan has also promoted partnerships with developing nations that have already shown progress and in which high-quality human resources in infectious disease control already exist. these partnerships have been developed into a cooperative network in developing countries that facilitates sharing of knowledge, skills and expertise among the cooperating countries. for decades, japan has promoted research activities in developing countries that are designed to create new, appropriate technology and quality clinical and laboratory work [ , ] . therefore, the promotion of research activities became one of the key components of the idi. however, because of japanese official development assistance budget constraints (i.e. reduced aid to developing countries) and the increasing trend of targeting worldwide assistance towards poverty reduction [ ] , there has been a priority shift away from scientific research and towards an operational focus, with the goal of providing rapid and direct support to those most in need. consequently, support for research has declined. promotion of public health at the community level japan has paid special attention to the improvement of basic sanitation, clean water, basic education and primary health care within communities, concentrating on interventions that lead to the reduction of infectious diseases. this infrastructure support, ostensibly assistance to boost primary and community health care, has emphasized the need for comprehensive, integrated approaches and has included the construction of health centres, the distribution of medical and laboratory equipment and supplies, the training of community personnel and the facilitation of community participation. major achievements of the idi the formation of the idi stimulated an increase in worldwide political and financial commitment for the fight against infectious diseases [ ] . according to the g performance assessments conducted by the g information between and the start of , japan spent us$ . billion on the idi. idi spending is classified under two categories: direct assistance (us$ . billion) and indirect assistance (us$ . billion). direct assistance involves projects and interventions that directly affect disease prevention. these include treatment and control (i.e. the provision of medicine), diagnostics and vaccines, technical assistance for disease control and training a workforce for disease control. indirect assistance involves projects and interventions that indirectly influence infectious disease control. these include clean water and sanitation, provision of basic education and renovation of health facilities. with regard to the geographical allocation of the idi inputs (table ) , japan has focused mainly on asia. however, more attention is now being paid to africa because it represents the biggest obstacle to the achievement of the millennium development goals (mdgs: http://www. un.org/millenniumgoals/), which are a series of global development targets set by the un millennium assembly. in the direct assistance section, % of spending was allocated to hiv/aids-related projects, whereas < % was allocated to the control of malaria and other parasitic diseases. this reflects the political commitment worldwide and especially the efforts that have been prevailing in recent years to combat hiv/aids [ , ] . japan had a japan has put great emphasis on the promotion of public health and the improvement of underlying social and economic conditions that enable infectious diseases to flourish and spread. these include illiteracy, lack of potable water and sanitation, and inadequate access to basic health services. major importance has been assigned to water supply projects, which facilitate and promote infectious and parasitic disease control and improve the socioeconomic status of communities through reduction of the time and labour required for drawing water [ ] . japanese leadership in this respect has placed japan as the top global donor in the sector, having contributed to more than one-third of water supply and sanitation projects in the developing world to provide > million people with access to safe drinking water and basic sanitation in the past five years [ ] (http://www.mofa.go.jp/mofaj/ gaiko/oda/index.html). the idi also provided extensive support to basic education, not only through the building of schools and the training of teachers but also through equipping schools with basic sanitation and a clean water supply, and developing school-based programmes (e.g. deworming, hygiene education and hiv/aids awareness and education). for discussion of japanese initiatives in school health, see ref. [ ] . the creation of new and multifaceted partnerships has been integrated throughout all idi activities. first, japan has provided small grants to local, international and japanese ngos in > countries. second, japan has cemented and extended its partnerships with other donors, in particular with the usa. japan signed the japan-usaid partnership for global health document in june and sent joint project-formulation missions to nigeria, nepal, honduras and other countries to initiate collaborative efforts to combat infectious diseases. third, japan has strengthened partnerships with the world health organization (who: http://www.who.int), united nations children's fund (unicef: http://www.unicef.org) and other un agencies in various programmes such as the roll back examples japan has provided diagnostic kits, equipment, voluntary counselling, testing centres, training of laboratory technicians and clinicians, and other types of support to control hiv/aids in many hiv-epidemic countries. for vaccine-preventable diseases, the idi has supplied vaccines with a cold chain and provided technical assistance for surveillance and laboratory and clinic management. in support of the global malaria programme and rbm, japan has donated anti-malarial drugs, diagnostic equipment and long-lasting insecticide-treated bed nets, and offered technical assistance and training for laboratory and clinical management, especially in sub-saharan africa and the greater mekong subregion to combat lymphatic filariasis in the pacific region, japan has contributed to pacelf by offering the know-how learned from japanese experience in eliminating this disease in the s. the idi has also provided diethylcarbamazine and immunochromatographic test cards and deployed the japan overseas cooperation volunteers for this programme [ ] . japan has also been one of the largest donors in controlling chagas disease in central america through the provision of equipment and supplies, in addition to technical assistance for programme management and individual interventions, including surveillance, materials for information, education and communication, and insecticide spraying [ ] . in partnership with the who and the carter center (http://www. cartercenter.org), japan has contributed to the guinea worm eradication program by establishing community-based surveillance systems and offering health education in infected villages. the idi also provided indirect assistance to improve water supply systems using new water-filtering equipment. in response to the tremendous impact of severe acute respiratory syndrome (sars) and avian flu on neighbouring asian nations and the urgent need for the containment and control of these disorders, japan provided quick and intensive assistance to china, vietnam and other affected nations. japan provided preventive, diagnostic and curative supplies, in addition to equipment and expertise [ ] . as the largest donor in the western pacific region of the who, the idi has supported the polio eradication programme. this region obtained polio-free status in october [ ] . japan has made the second biggest contribution, after the usa, towards the eradication of guinea worm and continues to support this programme [ ] . the number of people infected with guinea worm worldwide declined by %, from . million cases in to in . good results have also been achieved in chagas disease control in central america. the vector rhodnius prolixus has been eliminated from villages in nine health areas, and the house infestation rate of another vector, triatoma dimidata, has decreased by % following the first large-scale vector control project in guatemala. this project was supported by japan and involved two cycles of residual spraying in > houses in (http://www.paho.org/english/ ad/dpc/cd/dch-jica-pjt.doc). the idi direct assistance programme has provided several projects and interventions, varying from provision of supplies and equipment to technical assistance and training. these were planned and implemented with the recipient governments and development partners according to the needs of the countries. some examples of the idi projects are listed in box . it is difficult to measure the impact of the idi in terms of mortality or morbidity reduction with regard to each infectious disease because the inputs of the idi vary according to the needs of each country. in addition, the contribution by idi is just a part of all the efforts and interventions made by the recipient countries, donors and other development partners. however, there are some examples of the idi having made a major contribution and having shown considerable impact (box ). since the un adopted the mdgs, global development efforts have been increasingly focused and result orientated [ ] . substantial funding has gone to the control of hiv/aids and several other infectious diseases through the gfatm, the us president's emergency plan for aids relief (pepfar: http://www.pepfar.org) and other initiatives. however, relatively little funding is provided to control several parasitic diseases that are now increasingly being labelled as the neglected diseases [ , ] . although some of the recently formed foundations and agencies such as the carter center focus on the eradication or elimination of individual parasitic diseases [ ] , most of the donors and aid agencies focus their attention on hiv/aids and other high-profile killer diseases [ ] . under these circumstances, japan needs to pay special attention to the neglected diseases. apart from the mdgs, there are other approaches that focus on poverty reduction and that promote coordinated efforts and actions involving the recipient country, donor agencies and other stakeholders; these include poverty reduction strategy papers and sector wide approaches [ , ] . these are characterized by a set of operating principles, including broadening policy dialogue, developing a private sector policy (e.g. health and education, and a common realistic expenditure programme), common monitoring arrangements and additional coordinated procedures for funding and procurement [ , ] . this trend has been gradually preventing a single donor from providing specific projects in developing countries [ ] . rather, it is recommended to enable donors to support overall health planning and financing in the recipient countries [ ] . however, many developing countries still have several specific needs that range from the central government to individual communities and from financial assistance to technical matters. donor agencies should carefully observe the real needs of recipient countries and be aware of the short-and long-term effects of external assistance. infectious and parasitic diseases are more prevalent among the poor; therefore, unless special attention and assistance are given for the prevention, treatment and care of the poor, health inequities will broaden and expand, even in a country undergoing rapid development. japan has upheld the concept of 'human security', which is a human-centred approach that protects and improves the welfare of people most in need and empowers them to cope with adversity [ ] . professionals who work on infectious or parasitic disease control in or for developing countries also need to broaden their views to encompass the more comprehensive and integrated approach that is needed if poverty reduction and sustainable development of communities, nations and the world are to be successfully achieved. true to its long-term commitment, japan announced a new health and development initiative (hdi) in . this will build on the foundations and successes of the idi and will commit a further us$ billion over a five-year period [ ] . the hdi will provide substantial resources to help the global community achieve the mdgs and to help many developing countries improve the overall wellbeing of their populations and, thereby, encourage them to work towards attaining a higher quality of life. a world wide web resource for plasmodium vivax will see the launch of www.vivaxmalaria.com, a site for the plasmodium vivax research community. the website will be divided into five sections. (i) disease: data, graphics and links about the history of p. vivax malaria, its incidence, life cycle, morphology of infected cells, parasite strains and therapeutics. (ii) genomics: describing the p. vivax genome project and related genomics initiatives, and comparative genomics of plasmodium species. (iii) issues: highlighting outstanding questions about p. vivax biology, pathology and epidemiology. (iv) resources: protocols, reagents and resources for p. vivax research. (v) meetings: details of previous and upcoming conferences of interest to p. vivax researchers. a supplementary section will categorize links on the site and other links of interest to the community. for information and to offer suggestions, please contact the webmaster at: webmaster@vivaxmalaria.com collaboration in the fight against infectious diseases g countries accused of ignoring debt relief global plagues and the global fund: challenges in the fight against hiv, tb and malaria japan's official development assistance: recent issues and future directions the united states and japan pursue a common agenda japan/united states collaboration in international hiv prevention and care global parasite control initiative of japan (hashimoto initiative) infectious diseases, non-zero-sum thinking, and the developing world social and economic impact of infectious diseases -united states infectious diseases -new and ancient threats to world health the disease profile of poverty: morbidity and mortality in northern uganda in the context of war, population displacement and hiv/aids factors and determinants of disease emergence emerging zoonoses and pathogens of public health significance -an overview infection control: old problems and new challenges global health inequalities: an international comparison ) health, development and the millennium development goals health is a global issue impact of infections on primary caregreater than expected monitoring motherhood: sociocultural and historical aspects of maternal and child health in japan population strategy trends and regional variations in infant mortality rates in japan epidemiological review of tuberculosis okinawan health sector human resources experiences since the mid- s and their applications to international health; final report. ministry of education ghana/jica/who training course in polio diagnostic procedures and vaccine potency testing international cooperation with islamabad children's hospital. pakistan: the jica project halving global poverty okinawa infectious disease initiative (idi) -summary of mid-term evaluation report the global hiv/aids pandemic, structural inequalities, and the politics of international health the global fund to fight aids, tuberculosis and malaria (gfatm). health policy plan water quality and health in the new millennium: the role of the world health organization guidelines for drinking-water quality international cooperation for the improvement of environmental sanitation by japan reconsidering the underestimated burden caused by neglected tropical diseases donor funding priorities for communicable disease control in the developing world. health policy plan neglected' diseases but unrecognised successes -challenges and opportunities for infectious disease control priorities in global assistance for health, aids, and population new approaches to development cooperation: what can we learn from experience with implementing sector wide approaches? working paper , centre for aid and public expenditure poverty reduction strategy papers pro-poor health policies in poverty reduction strategies. health policy plan the sector-wide approach: a blessing for public health? the sector-wide approach in health: what is it? where is it leading? better health in developing countries: are sector-wide approaches the way of the future? rethinking human security health and development initiative (hdi) -japan's contribution in achieving the health-related mdgs pacific collaboration to eliminate lymphatic filariasis control of chagas disease experience and review of sars control in vietnam and china certification of poliomyelitis eradication. who western pacific region dracunculiasis (guinea worm disease) eradication key: cord- -fippk p authors: palmeiro, brian s.; roberts, helen title: clinical approach to dermatologic disease in exotic animals date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: fippk p skin disease is an extremely common presenting complaint to the exotic animal practitioner. a systematic diagnostic approach is necessary in these cases to achieve a diagnosis and formulate an effective treatment plan. in all exotic species, husbandry plays a central role in the pathogenesis of cutaneous disease, so a thorough evaluation of the husbandry is critical for successful management. the clinical approach to skin disease in exotic animal patients is reviewed with specific focus on structure and function of the skin, diagnostic testing, and differential diagnoses for commonly encountered cutaneous diseases. skin disease is an extremely common presenting complaint to the exotic animal practitioner. a systematic diagnostic approach is necessary in these cases to achieve the appropriate diagnosis and formulate an effective treatment plan. in all exotic species, husbandry plays a central role in the pathogenesis of cutaneous disease, so a thorough evaluation of the husbandry is critical for successful management. there are vast differences in the structure and function of the skin in exotic species; an understanding of these unique properties is important when treating skin disease in exotic pets. this article focuses on the clinical approach to skin disease in exotic pets including structure and function of the skin, appropriate diagnostic testing, and differential diagnoses for commonly encountered cutaneous diseases. study of dermatologic lesions in reptiles found that from % to % (dependent on institution and reptile group) of the cases had underlying husbandry-related deficiencies. reptile skin is modified into scales and composed of a three-layered epidermis and a dermis that typically is aglandular. [ ] [ ] [ ] the three layers of the epidermis are ( ) stratum corneum (six to eight cell layers, heavily keratinized); ( ) stratum intermedium; and ( ) stratum germinativum (deepest). [ ] [ ] [ ] two types of keratins compose the stratum corneum. the softer more flexible a-keratins are elastic and pliable and form the suture/hinges and spaces between scales. the b-keratins (unique to birds and reptiles) compose the hard horny scale. the skin is protected by scales produced by the stratum germinativum; scales are separated by scale pockets. the keratinized layers of chelonians are modified into scutes. the scales or scutes of chelonians and some lizards (plated and girdled lizards, skinks, and crocodilians) are underlain by dermal bony plates referred to as osteoderms or osteoscutes. [ ] [ ] [ ] [ ] in tortoises, the stratum corneum produces the shell, which consists of the carapace (dorsal) and plastron (ventral); the keratinized scutes cover osteoderms that fuse with the vertebrae and sternebrae. [ ] [ ] [ ] chromatophores (pigment cells) are found in the dermis and melanocytes are present within the stratum germinativum. reptiles shed their skin at regular intervals in a process called ecdysis. the skin of lizards and chelonians shed in several smaller pieces, whereas snakes typically shed their entire skin as one piece. chelonians and crocodilians shed their epidermis continuously, whereas lizards and snakes shed their epidermis periodically. a detailed clinical history is important in all cases of reptile skin disease; important husbandry-related questions include those pertaining to diet, substrate and housing, lighting, heating, humidity, and temperature. common findings during clinical examination of reptiles with dermatologic disease include abrasions, erosions, ulcers, wounds, swellings, pustules, blisters/vesicles/ bullae, crusts, dysecdysis, petechial and ecchymoses, discoloration, macroparasites, and edema. in some cases, cutaneous changes can be secondary to systemic disease; petechia and ecchymoses are commonly seen with septicemia and ventral edema may be seen with renal or liver disease. in one study, % of all reptiles with confirmed or suspected cases of sepsis had petechiae, with the highest association seen in chelonians ( %). commonly used dermatologic diagnostic tests in reptiles include the following - : most useful for swellings and growths . clinicopathologic evaluation including complete blood count (cbc) and biochemistry analysis . radiographs are useful when assessing damaged osteoderms and for the presence of bony changes associated with secondary nutritional hyperparathyroidism or other internal disease see table for a review of common differential diagnoses for dermatologic diseases in reptiles, including bacterial dermatitis, shell rot, bacterial ulcerative dermatitis, snake mite, and secondary nutritional hyperparathyroidism (figs. [ ] [ ] [ ] [ ] [ ] . the thin, relatively unprotected skin of amphibians combined with the significant diversity of amphibian habitats and their biphasic life cycles render them particularly susceptible to a wide range of infectious and noninfectious cutaneous diseases. amphibians belong to three distinct orders: anura (frogs); caudata (salamanders); and gymnophiona (caecilians). the skin of amphibians is clinically the most important organ system of amphibians and varies depending on the life stage (premetamorphosis or postmetamorphosis); habitat (generally divided into aquatic or terrestrial); and the species. the skin functions in osmoregulation, gas respiration, and water absorption. , amphibian epidermis is typically thin; keratinized; and consists of the stratum corneum, stratum granulosum, stratum spinosum, and the stratum basale. [ ] [ ] [ ] [ ] [ ] modifications of amphibian skin include the presence of dermal scales (caecilians); folds and grooves for increased surface area (salamanders); partial ossification of the cranial skin and adherence to the skull (bufonids); a specialized highly vascularized ventral dermal organ for water absorption ("drinking patch" in anurans); and the presence of dermal bones (some anurans). , the stratum corneum is typically shed in one piece at regular intervals and consumed (dermatophagy) unless the animal is ill. , , the skin of anurans is loosely adhered to the body and can become edematous in disease states. two key features separate adult caecilians and anurans from their larval form: the epidermis is keratinized in adults and the dermis contains a variety of dermal glands. , , mucus, produced by mucous glands and epithelial cells, aids in respiration, prevents evaporative water loss, contains antibacterial and antifungal properties, can be defensive noxious or contain toxic chemicals, may act as pheromones, and can aid in reproduction. , [ ] [ ] [ ] [ ] dermatologic examination and diagnostic testing a thorough history and dermatologic examination are important when evaluating any case of amphibian skin disease. husbandry-related factors often underlie the development of many skin diseases in amphibians. important questions to consider include recent introductions into the collection; diet; and tank setup including filtration, aeration, water quality, and temperature. during examination, it is important to always handle amphibians with rinsed gloves to avoid damaging their skin and prevent cutaneous absorption of potentially toxic glandular secretions. [ ] [ ] [ ] many amphibian skin diseases can have a similar appearance with cutaneous hyperemia and discoloration, dermal papules and nodules, ulceration, hemorrhages, edema, and excess mucus being the most common findings. clinical approach to dermatologic disease commonly used dermatologic diagnostic tests in amphibians include the following: . skin scraping , , using a coverslip, blunt scalpel blade, or edge of a glass slide, gently scrape over the surface of the skin samples taken from lesions may be more diagnostic place the sample on a slide if needed, wet the slide with physiologic saline for a wet mount preparation examine immediately using lowest power objective first shed skin can also be examined as a wet mount preparation samples can also be dried and stained for later examination . impression or swab smears, fine-needle aspirates , , typically these samples are air dried and stained less traumatic than skin scrapings . bacterial culture , , , gentle irrigation of the lesion with sterile physiologic saline or getting a deep sample can reduce contamination of normal surface microflora and environmental bacteria dermal glandular secretions and normal microflora may inhibit bacterial growth because of antibacterial properties swabs can be moistened with sterile saline or transport media to minimize skin damage and maximize recovery of bacteria optimal temperature for sample growth is c/ f most isolates are gram-negative bacteria but gram-positive and mycobacterial infections also occur . fungal cultures , tissue sections can be placed directly onto fungal culture media sabouraud dextrose agar media is a good choice for most fungal isolates culture at room temperature . polymerase chain reaction (pcr) of skin swabs [ ] [ ] [ ] [ ] consult laboratory for availability; verification of positive results; type of pcr (conventional, taqman, real-time, and so forth); use of negative and positive controls; sample collection and swab type; and shipping details avoid cross- see table for a review of common differential diagnoses for dermatologic diseases in amphibians. cutaneous disease is an extremely common presenting complaint to the fish veterinarian. many owners notice abnormalities in the integumentary system as the first sign of disease in their pet fish. in addition, the skin is an extremely common target for many infectious diseases of ornamental fish. the skin of fish provides a protective barrier against infection, osmotic pressure, and injury. disruptions of the skin can result in osmotic disturbance, disruption of internal homeostasis, morbidity, and mortality. the skin can be divided histologically into the cuticle, epidermis, dermis, and subcutis. the cuticle (outermost layer) is approximately mm thick and contains mucus, sloughed cells, and cellular debris. it has antimicrobial properties mediated by antibodies (igm), free fatty acids, and lysozymes. , this layer is commonly referred to as the "slime coat" by aquarium hobbyists because of its high concentration of mucus. this layer is usually lost during routine processing for histopathology. together with the cuticle, the epidermis produces a waterproof barrier. the epidermis is a nonkeratinizing (most species) stratified squamous epithelium that contains to cell layers. , it contains many mucus-producing goblet cells and, in some species, club cells that secrete an "alarm substance" when the skin is damaged. unlike mammals, epidermal cells are not keratinized and are capable of mitotic division in all layers; however, division most commonly occurs in cells adjacent to the basement membrane where the epidermis junctions with the dermis. , the upper dermis contains collagen and reticulin and forms a supportive network; the deeper dermis contains more compact collagen and provides the main structural strength to the skin. , scales are flexible bony plates that develop in scale pockets in the dermis; they are not shed regularly. , as scales emerge they are covered by a layer of epidermis, and often overlap one another, providing structural support and protection. two main types (ctenoid and cycloid scales) are described that differ in surface sculpture. , ultrastructurally, scales contain collagen fibers interspersed with an organic matrix in which hydroxyapatite crystals are deposited. , some fish are scaleless and histologically have a thicker epidermis. chromatophores (pigment cells) are present in the dermis and include melanophores; xanthophores (yellow); erythrophores (orange-red); leucophores (white); and iridophores (reflective/iridescent/silver). the pigments consist mainly of carotenoids. the subcutis contains connective tissue and fat and is highly vascular; bacterial disease can spread rapidly along this layer. , dermatologic examination and diagnostic testing the diagnostic approach to a fish with dermatologic disease should include a complete history, direct observation of the fish in its aquarium or pond, dermatologic examination, complete water quality, skin scrapings, and a gill biopsy. as with other species, historical evaluation is extremely important. because infectious disease is very common in pet fish, questions pertaining to quarantine protocol, most recent fish introduction, and number of fish affected are extremely important. husbandry-related questions (water changes, filtration, tank or pond setup, water quality testing, and so forth) are extremely important because many diseases in fish are related to poor husbandry and water quality. the owner should be questioned regarding prior treatments because many fish hobbyists attempt numerous overthe-counter remedies before consulting with a veterinarian. direct observation is best performed in the home aquarium or pond. isolation is often an early indication of disease in schooling fish. other signs that can be seen during direct observation include piping (gasping for air at the surface) and flashing (a sign of pruritus in which the fish rubs against objects in the aquarium or pond). the skin and fins can also be evaluated for abnormalities. during the dermatologic examination, the skin, fins, and scales should be evaluated thoroughly. some fish require sedation for this procedure. latex gloves should be worn to protect the cuticle. abnormalities that are commonly seen on the dermatologic examination include skin discolorations; erythema; frayed and irregular fins; erosions and ulcerations; petechial and ecchymoses; edema and raised scales; macroparasites (anchor worm, fish lice); papules and nodules; excess mucus production; scale loss; and white-to-gray irregular patches. commonly used dermatologic diagnostic tests in fish include the following: . water quality evaluation a. poor water quality is the most common cause of morbidity and mortality in pet fish b. poor water quality is the most common underlying cause of immunosuppression and opportunistic infections in pet fish c. parameters that should be monitored include temperature, ph, ammonia, salinity, nitrite, nitrate, dissolved oxygen, and alkalinity . skin scrapings and gill biopsy a. skin scrapings i. if there are lesions on the skin, a coverslip should be dragged across lesional skin in a head-to-tail direction, collecting mucus on the coverslip. the coverslip is then placed onto a slide with a drop of tank water. some fish require sedation for this procedure. sedation may reduce the number of ectoparasites found on skin scrapings. ii. when there are no obvious lesions on the skin, sites commonly sampled include just caudal to the pectoral fin, operculum, and the ventrum. samples should be taken from two to three different sites; when possible, several fish should be sampled. b. gill biopsy i. gill is epithelial tissue and many ectoparasites affect the gills and skin. occasionally, ectoparasites are found only on the gills. ii. typically requires sedation iii. the operculum is lifted and a small snip of distal gill lamellae is taken (usually with iris scissors) and placed onto a slide with a drop of tank or pond water to examine. c. skin scrapings and gill biopsies are examined under the microscope; superior results are obtained with the condenser down. most parasites can be seen on  or  magnification. however, with some smaller parasites, such as ichythobodo, and bacteria, such as flavobacterium columnare,  magnification is required. . bacterial culture and sensitivity a. tissue biopsy for culture sampling is preferred over superficial swabbing of ulcerative lesions . histopathology . clinical pathology (complete blood count, biochemistry panel) . viral testing a. koi herpes virus serology and pcr . necropsy see table for a review of common differential diagnoses for dermatologic diseases in fish, including gyrodactylus and ulcerative bacterial dermatitis (figs. and ) . cutaneous disease is extremely common in pet birds; assessing the skin can be difficult given the variation in species presenting to the avian practitioner. avian clinical approach to dermatologic disease avian skin is composed of an epidermis and dermis; the skin is thicker in nonfeathered areas. the layers of the epidermis include the stratum germinativum and the stratum corneum. the stratum germinativum (bottom most layer) produces cells that mature to form the keratinized stratum corneum and can be divided into three distinct layers: ( ) the stratum basale, ( ) the stratum intermedium, and ( ) the stratum transitivum. the cells show signs of keratinization in the stratum transitivum. feathers are formed from feather follicles in the dermis. the dermis is thicker than the epidermis and contains structurally supportive collagen, blood vessels, fat, nerves and neuroreceptors, feather follicles, and associated smooth muscle. , avian skin is aglandular with the exception of the uropygial (or preen) glands; the pericloacal glands (secrete mucus); and the sebaceous glands of the ear canal. , the uropygial gland is a holocrine gland found at the base of the tail that secretes a liposebaceous material important in protecting and waterproofing feathers; it is spread through the feathers in a process called preening that is also necessary for interlocking of feather barbules. , feathers are arranged into tracts known as pterylae that are separated by featherless areas of skin called apteria. [ ] [ ] [ ] table illustrates the common feather types and their properties. the calamus is the part of the feather that attaches to the follicle. [ ] [ ] [ ] the main shaft of the feather is called the rachis; where the rachis meets the calamus is a pulp cap referred to as the superior umbilicus. [ ] [ ] [ ] there may be a smaller feather attached to the superior umbilicus that is referred to as the after feather. [ ] [ ] [ ] projections from the rachis are referred to as barbs, which bear projections called barbules. [ ] [ ] [ ] most barbules contain hooks called barbicels that hold the barbs and barbules together. [ ] [ ] [ ] molting occurs when the growth of a new feather in the follicle forces out the older feather; all feathers of adult birds are replaced regularly during molting. most species of pet birds molt once to twice yearly. , dermatologic examination and diagnostic testing a thorough history and dermatologic examination are important when evaluating any case of avian skin disease. dermatologic examination in birds should include evaluation of feathers, skin, beak and cere, ears, legs and claws, preen gland, and cloaca. common findings during clinical examination of birds with skin disease include feather abnormalities (broken or absent feathers, dystrophic and discolored feathers); scaling; crusting; ulceration; redness; and nodules and masses. commonly used dermatologic diagnostic tests in pet birds include the following: . feather pulp cytology , feather pulp cytology is collected from a freshly plucked feather and used to assess for the presence of folliculitis. the calamus can be removed from the feather and contents smeared onto a microscope slide. possible findings include bacteria, inflammatory cells, viral inclusion bodies, and dermatophytes. . gross and microscopic examination of feathers , evaluate for overall condition, ectoparasites, fret marks and stress bars, evidence of self-trauma . feather preparation with potassium hydroxide to improve mite identification, the calamus of the feather can be placed into a % potassium hydroxide solution, gently heated, and then centrifuged, followed by microscopic examination of the sediment . acetate tape impressions used to detect ectoparasites, yeast, and bacterial infections. feather dander and keratinaceous debris is very abundant on these samples and in some cases can be difficult to differentiate from bacteria and yeast. for moist, exudative, or crusted lesions, direct slide impressions are often used for drier lesions, direct impressions can be attempted but acetate tape impressions may be preferred. alternatively, a moistened swab can be used to collect a sample and contents rolled onto a slide. . skin scrapings . culture and sensitivity (bacterial, fungal) calamus and feather plucking, sterile tissue biopsy, or superficial swabs . biopsy avian skin is much thinner than dogs and cats. in some cases, it is easier to biopsy the skin with a scalpel compared with a punch biopsy. if a punch biopsy is to be performed, a technique has been described where acetate tape is placed over the biopsy site to maintain the structure of the skin. . clinical pathology evaluation including complete blood count and biochemistry panel and heavy metal testing , systemic diseases can cause cutaneous changes including feather picking testing for lead and zinc levels may be needed in some cases . crop washes can identify trichomonas or candida, which can present in birds that feather pluck over the crop area . fecal examination certain intestinal parasites may result in feather plucking , . intradermal allergy testing codeine phosphate at : , wt/vol preferred over histamine as a positive control in birds further research is needed to evaluate appropriate protocols for intradermal allergy testing in pet birds and establish correct allergen dilutions and thresholds . viral testing including pcr for polyoma virus and psittacine beak and feather disease , common differential diagnoses for cutaneous diseases see table for a review of common differential diagnoses for dermatologic diseases in pet birds, including feather picking (fig. ) . clinical approach to dermatologic disease the skin is a common site of disease in small mammals and a very common presenting complaint to the exotic animal practitioner. parasites, bacterial infections, and husbandry-and environmental-related conditions are most commonly seen. the basic structure and function of exotic small mammal skin is very similar to that of the dog and cat. relevant differences are discussed. the skin is divided into a fourlayered avascular epidermis (stratum corneum, stratum granulosum, stratum spinosum, stratum basale) and the underlying, structurally supportive, collagenous, and vascular dermis. the subcutis is below the dermis and consists of connective tissue and fat. in rodents, brown fat is located between the scapulae, in the ventral neck, and in the axillary and inguinal regions; it is more prominent in smaller rodents, rabbits, and ferrets and less so in guinea pigs and chinchillas. fig. . gyropus ovalis from a guinea pig. fig. . cystic ovarian disease resulting in symmetric noninflammatory flank alopecia. hairs can be divided into primary (guard) hairs; secondary (undercoat) hairs; and tactile hairs. the number of hairs per follicle varies with the species, breed, age, and other external factors; chinchillas have as many as hairs per follicle, producing the characteristic dense soft coat. the keratinized hair consists of the innermost medulla, pigmented cortex, and outermost cuticle. primary hairs are associated with sebaceous glands, apocrine sweat glands, and an arrector pili muscle. rodents and ferrets have no epitrichial (apocrine) sweat glands. secondary hairs are typically only accompanied by sebaceous glands. the rat and mouse tail is very sparsely haired. in interfollicular regions, there is surface parakeratosis and no stratum granulosum, whereas follicular ostia contain the typical orthokeratosis and stratum granulosum; these changes give the tail its characteristic scaly appearance. the footpads are areas of specialized thickened epidermis with underlying shockabsorbing fat deposits. atrichial (eccrine) sweat glands are located only in the footpad. rabbits lack foot pads, but instead have coarse fur on their distal limbs. sebaceous scent glands are a common feature in many small mammal species and are important in scent marking and communication. hamsters have large darkly pigmented glands on their flanks, more prominent in males. gerbils have large oval-shaped yellowish hairless scent glands on the ventrum. guinea pigs have a large gland over the rump that can secrete an oily substance, especially in boars. rabbits have sebaceous scent glands on the chin (mental gland) that is used for territorial marking, anal glands, and androgen-dependent inguinal scent glands. ferrets have active sebaceous glands throughout their skin that results in their typical musky odor and greasy coat; they also have two prominent perianal scent glands. as with other exotic species, a thorough questioning and evaluation of the husbandry is critical for successful diagnosis and treatment of small mammal dermatoses. important questions include those pertaining to the environment and husbandry (type of housing, indoor or outdoor, substrate or bedding, diet, and so forth), and more targeted questions pertaining to skin disease. in small mammals, it is important to know whether the condition is pruritic and whether any other animals are affected. a thorough dermatologic examination is necessary in all patients with skin disease. common lesions include hairloss, erythema, scaling, crusting, excoriations, erosions, and ulcers. restraint to obtain quality diagnostic samples from small mammal skin can be challenging in some cases, so anesthesia or sedation may be needed. commonly used dermatologic diagnostic tests in small mammals include the following: for moist, exudative, or crusted lesions, direct slide impressions are often used for drier lesions, direct impressions can be attempted but acetate tape impressions may be preferred. alternatively, a moistened swab can be used to collect a sample and contents rolled onto a slide. . skin scrapings a. very useful for detection of ectoparasites b. given the thin skin of many exotic patients, some practitioners prefer to use scraping spatulas to perform skin scrapings . bacterial culture and sensitivity . fungal culture . wood lamp a. limited usefulness in small mammals given that trichophyton mentagrophytes is the most common dermatophyte isolate in clinical cases . trichogram a. useful to evaluate hair structure b. evaluation for broken or fractured hair ends that would help determine whether hair loss is traumatic. evaluate for ectoparasites. c. evaluation for evidence of dermatophytosis (fungal hyphae/ectothrix spores) . acetate tape impression a. useful for collection of surface-dwelling mites, such as cheyletiella and myobia . skin biopsies for histopathology . clinical pathology testing including complete blood count and biochemistry panel . testing for adrenal disease in ferrets see table for a review of common differential diagnoses for dermatologic diseases in rabbits, including cheyletiella parasitivorax (fig. ) . see table for a review of common differential diagnoses for dermatologic diseases in guinea pigs, including gyropus ovalis and noninflammatory flank alopecia (figs. and ). see table for a review of common differential diagnoses for dermatologic diseases in gerbils and hamsters, including demodicosis, (demodex aurati and demodex criceti, [fig. ] ). see table for a review of common differential diagnoses for dermatologic diseases in mice and rats, including myobia musculi and polyplax spinulosa (figs. and ) . see table for a review of common differential diagnoses for dermatologic diseases fig. . alopecia in a ferret with hyperadrenocorticism. in ferrets, including alopecia (fig. ). see table for a review of common differential diagnoses for dermatologic diseases in chinchillas. skin disease is an extremely common presenting complaint to the exotic animal practitioner. these cases may be challenging because dermatologic diseases are often multifactorial and many have underlying husbandry or environmental deficiencies that must be identified. a thorough diagnostic evaluation is critical for successful management of exotic animal cutaneous disease. reptiles with dermatological lesions: a retrospective study of cases at two university veterinary teaching hospitals dermatology in reptiles skin diseases of exotic pets. blackwell: ames (ia) scales and sheds: the ins and outs of reptile skin disease bsava manual of small animal dermatology. gloucester (england): bsava medicine and surgery of amphibians amphibian medicine and captive husbandry cytologic diagnosis of disease in amphibians frog skin epithelium: electrolyte transport and chytridiomycosis amphibian emergency medicine amphibian medicine and captive husbandry amphibian medicine and captive husbandry amphibian medicine and captive husbandry host-defense peptides in skin secretions of african clawed frogs (xenopodinae, pipidae) co-habiting amphibian species harbor unique skin bacterial communities in wild populations skin glands, poison and mimicry in dendrobatid and leptodactylid amphibians oribatid mites and skin alkaloids in poison frogs clinical approach to dermatologic disease bsava manual of exotic pets. gloucester (united kingdom): british small animal veterinary association common procedures in reptiles and amphibians amphibian medicine and captive husbandry an overview of amphibian skin disease bacterial and parasitic diseases of amphibians clinical microbiology of amphibians for the exotic practice amphibian medicine and captive husbandry amphibian medicine and captive husbandry amphibian virology proceedings from workshop on infectious diseases in amphibian survival assurance colonies and reintroduction programs diagnostic testing differential host susceptibility to batrachochytrium dendrobatidis, an emerging amphibian pathogen skin to fins: diving into pet fish dermatology bsava manual of ornamental fish bsava manual of small animal dermatology. gloucester (england): bsava skin diseases of exotic pets anatomy, clinical presentation and diagnostic approach to the feather picking pet bird a modified biopsy technique to improve histopathological evaluation of avian skin intradermal skin testing in hispaniolan parrots (amazonia ventralis) skin diseases of exotic pets bsava manual of small animal dermatology. gloucester (england): bsava amphibian medicine and captive husbandry guinea pigs, chinchillas, degus and duprasi disease problems of guinea pigs disease problems of small rodents bsava manual of exotic pets. gloucester (united kingdom): british small animal veterinary association cytologic diagnosis of diseases of rabbits, guinea pigs, and rodents ferret oncology: disease, diagnostics, and therapeutics dermatologic diseases emergency medicine of the ferret endocrine diseases bsava manual of exotic pets. gloucester (united kingdom): british small animal veterinary association ferrets, rabbits, and rodents: clinical medicine and surgery key: cord- -ytj cit authors: hoyo, javier del; aguas, mariam title: implementing telemedicine in inflammatory bowel disease: is covid- the definitive trigger? date: - - journal: gastroenterol hepatol doi: . /j.gastrohep. . . sha: doc_id: cord_uid: ytj cit nan the pandemic, we already lived times of overwhelmed consultations with financial constraints, and the promise of telemedicine for improving access to better health services at lower costs drew attention to its use. paradoxically, the exponential increase in the number of articles over years led to an asymptotic evolution that rarely reaches the implementation of telemedicine in daily practice and policy [ ] . the use of information and communication technologies (icts) for health practice faced several challenges explaining why many telemedicine projects fail to scale-up, despite the technical advances made since the term "telemedicine" was coined about years ago. then, will this pandemic trigger a deep implementation of telemedicine never seen earlier? during the first steps of modern telemedicine, the limitations were mainly technical or procedural, with high costs associated to the communication tools that only allowed their use in restricted settings such as spatial or military applications. over time, the digitalization in telecommunications and the progressively wider access to the internet offered an opportunity to reorganize healthcare services. the increase of data transmission and storage capacity, as well as the evolution of mhealth with the development of wireless communications, provided us a broad range of easy-to-use devices adaptable to many aspects of our practice remotely. furthermore, the incorporation of artificial intelligence and big data to analyze massive volumes of information could potentially improve healthcare systems to facilitate tailored medicine. it has been a long way to go, and still the development of more powerful and cheaper communication tools turns technical challenges into legal, ethical, economical and professional issues. unlike the use of icts in other fields (streaming entertainment services, grocery delivery, e-banking, etc.), telemedicine interventions deal with the need to integrate patient-generated data into electronic health records, while privacy is essential in the processing of these sensitive data. moreover, the efficacy of telemedicine on health outcomes is inconsistent across different programs used in inflammatory bowel disease (ibd), and their value is difficult to establish when only few economic data are available. thus, decision-makers have difficulties to support the implementation and investment on telemedicine due to a lack of solid evidence. in addition, these decisions become even more complicated in areas were reimbursement is an important factor in the setup of clinical activity. at this point, are we ready to transform covid- crisis into a revolution? as other disasters, the pandemic leads to a surge in demand for healthcare services, which directly and indirectly could collapse health systems. to solve this problem, telemedicine offers two main advantages. on the one hand, the classical benefit of providing healthcare at a distance may serve to start-up efficient triage services without exposure to sars-cov [ ] . on the other hand, components like tele-education and telemonitoring that provide action plans can promote patients´ empowerment and self-management. the combination of these two benefits could alleviate our previously overwhelmed healthcare capabilities not only during the pandemic, but also in our daily practice. in this sense, our research group developed a web-based platform called teccu ("telemonitorización de la enfermedad de crohn y colitis ulcerosa" or telemonitoring of crohn's disease and ulcerative colitis). in a previous pilot trial, teccu showed to be a safe strategy to improve health outcomes of complex ibd patients [ ] , with a high probability of being more cost-effective in the short term compared to standard care and telephone care [ ] . in view of these results, a new project in collaboration with other hospitals and investigators from the spanish working group on crohn´s disease and ulcerative colitis (geteccu) and the confederation of associations for patients with crohn's disease and ulcerative colitis of spain (accu) is currently underway (image ). in any case, to reorganize ibd health practice definitely we should waive the previous brakes in the adoption of telemedicine, but we must also know how to drive the new situation (image ). first, it is necessary to standardize remote medical practice. in the us, the interstate medical licensure compact was created to increase efficiency in multistate licensing of physicians [ ] , but such a proposal is lacking in europe. second, those organizations that previously investigated the value of telemedical innovations should lead this revolution [ ] , with the collaboration between centers and regions to develop the european health strategies. third, institutions lacking telemedicine programs can outsource these services, but the provision of remote health safely also requires a uniform legal framework regarding medical liability. finally, in order to maintain adherence to follow-up it is essential to adapt telemedicine programs according to patients´ requirements. maybe the pandemic has reduced reluctance amongst physicians to use telemedicine, but funders, policy-makers, providers and patients need to align their interests to implement remote healthcare successfully. as an example, on march , it was signed into law an emergency bill of more than $ billion passed by the us congress to face covid- . periods act of [ ] , in order to temporarily lift certain restrictions on medicare telemedicine coverage in the efforts to contain the virus. page of j o u r n a l p r e -p r o o f therefore, telemedicine offers many opportunities to overcome healthcare challenges posed in the management of ibd during the covid- outbreak, as long as we know how to use these resources properly. in spite of the use of telephone and e-mail in many centers, the development of mature telemedicine programs integrated with electronic health records requires further collaborative efforts between different investigators. telemedicine intends to reorganize (not to replace) healthcare systems, and decisionmakers still need more evidence on the efficacy and cost-effectiveness of its use in ibd prior to perform important changes. even if we assume that the pandemic could reduce reluctance to use telemedicine, specific european regulation is required to protect remote medical practice and to lift some existing legal barriers. why do entrepreneurial mhealth ventures in the developing world fail to scale? british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic a web-based telemanagement system for improving disease activity and quality of life in patients with complex inflammatory bowel disease: pilot randomized controlled trial telemonitoring of crohn's disease and ulcerative colitis (teccu): cost-effectiveness analysis virtually perfect? telemedicine for covid- , coronavirus preparedness and response supplemental appropriations act key: cord- -p ysi authors: davis-wurzler, gina m. title: update on current vaccination strategies in puppies and kittens date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: p ysi vaccines remain one of the practitioner’s greatest tools in preventing disease and maintaining individual and population health. this article is an update to “current vaccination strategies in puppies and kittens” published in veterinary clinics of north america, small animal practitioner, in may . there are now comprehensive guidelines readily available for small animal practitioners regarding canine and feline pediatric (and adult) vaccination recommendations. perhaps more importantly, there is an increased dialogue regarding all aspects of preventive medicine, of which vaccination is only a small, yet significant portion; and an increased drive to provide scientific evidence for developing vaccination recommendations. recommendations, and adverse events; as such, a comprehensive discussion is not possible here. as veterinarians we should look forward to the ongoing growth of this area of interest within clinical practice and within the research community, to eventually provide practitioners with answers currently sought by pet owners and veterinarians alike. it is far better to prevent than experience disease. this tenet should be the philosophy and goal of every veterinarian and every pet owner. for decades the veterinary profession has diligently educated pet owners about the benefits of preventing infectious disease, so well that there has been a significant decline in many of these diseases, in large part attributable to the development and use of effective vaccines. veterinary practice staff members have done remarkable jobs sending reminder cards to ensure that canine and feline patients are current on their vaccinations. in fact, vaccines have become such a priority that many pet owners are inclined to forfeit other, indicated medical care in lieu of vaccines lest their beloved pets fall behind on their vaccine schedule. veterinarians should commend themselves on a job well done, and commend pet owners for such conscientious stewardship of their pets. now, however, the veterinary community must reflect on what has been accomplished, and make decisions for current and future patient care based on scientific, rational merit. with the advent of knowledge on demand (ie, the internet), pet owners have access to information regarding all issues of animal care. however, such information may not be accurate. it is our duty to educate pet owners; in fact, it should be seen as an opportunity. who better to disseminate knowledge about veterinary medicine to the general public than veterinarians? no other group of individuals is as equipped with knowledge, skills, and insight as the veterinary community. to adequately discuss and understand how to make appropriate choices regarding pediatric vaccinations, a brief review and discussion of terms relative to basic immunology are warranted. passive transfer of immunity occurs when maternal antibody is transferred by the dam or queen to the fetus via the placenta, which occurs minimally in dogs and cats. it also occurs during initial suckling through the ingestion of colostrum, which has more significant effects in these species. this maternal immunity does provide initial protection against many pathogens, but of course depends on the health and immune status of the mother and the health of the fetus and neonate. although this may result in temporary protection for the neonate, in the long term it may be deleterious to that individual's health by essentially keeping the animal naïve to different antigens (eg, maternal antibody interference with vaccination of the neonate). maternal or passive immunization is effective in protecting neonates for the first several weeks of life, but begins to decline and lose the ability to protect against diseases rapidly as the maternal antibodies are degraded through natural catabolic processes. between the ages of and weeks, depending on multiple factors (including species, amount of maternal antibody produced, transferred, and absorbed, and the individual health status of the neonate), most puppies and kittens have maternal antibody levels below protective levels. however, if present at high enough levels, maternal antibodies can interfere with the neonate's ability to respond to vaccination, as the circulating maternal antibody within the puppy or kitten may effectively respond to and neutralize the vaccine antigen, or render it ineffective by preventing recognition of the antigen by the immune system. this is one reason why multiple, sequential vaccines are recommended in kittens and puppies until davis-wurzler they are at least to weeks of age. , of importance is that maternal antibodies can interfere with immunization, although the level of maternal antibody present may not be protective against pathogens. a functioning immune system is composed of multiple parts. innate immunity is the oldest (evolutionarily), least specific, and most immediate (in terms of response to potential invaders/pathogens) form of immunity. macrophages, neutrophils, dendritic cells, and natural killer (nk) cells combined with numerous products produced by these cells comprise the innate immune system. examples of some of the chemical components produced and released by these cells in response to microbial invasion include lysozyme, complement, various cytokines such as tumor necrosis factor a and interleukins, and various vasoactive molecules such as histamine. active immunization is the process of the individual responding to an antigenic stimulus appropriately, by either natural infection or vaccination. active immunization is processed through the acquired immune system. the main types of acquired immunity are cell-mediated immunity and antibody, or humoral, immunity. cell-mediated immunity is predominantly directed against pathogens that typically are obligate, intracellular organisms. examples include viruses, some obligate intracellular bacteria, some fungi, and protozoa. t lymphocytes are the predominant effector cells, and depend on foreign protein (antigen) being presented to them before they can take effect against the pathogens; thus, multiple cell types are involved in forming cellmediated immunity. antibody or humoral immunity is predominantly directed against pathogens that can survive outside the host, or at least survive extracellularly. examples include most bacteria, fungi, protozoa, and helminths. multiple cells act in concert to confer humoral immunity as well, but the primary effector cell is the b lymphocyte. having stated this, in actuality humoral immunity is extremely important in protection against viral infections, and is intricately and definitively dependent on competent cellmediated immunity. kittens and puppies will have varying degrees of ability to respond to antigens, whether resulting from natural or vaccine exposure, based on antigen load, route of exposure, antigenic virulence, genetics of the individual animal, and levels of persistent maternal immunity. in naïve animals whose maternal immunity has declined sufficiently so as not to interfere with an immune response; the first vaccine should stimulate a primary immune response (priming of the immune system). this initial exposure and recognition process and the ability to produce antibody to respond to the antigen typically takes to days; however, the maximum response takes up to weeks. this primary response must not be confused with the animal having been immunized. a subsequent dose of vaccine (exposure) will lead to immunologic memory. subsequent exposures to the same antigen elicit a stronger response: a greater amount of antibody is produced and the subsequent response is more rapid. this process is known as the secondary or anamnestic immune response, which results in immunity. although multiple cell lines are involved in this response, subsets of t and b lymphocytes known as memory cells preserve the host's ability to recognize and respond to antigens to which the animal had previously been exposed. to design, recommend, and actuate an effective plan for each patient, a practitioner must have familiarity with multiple variables. those variables include duration of protection conferred on the neonate by the mother; the typical length of time maternal antibody may persist and pose interference with the young animal's ability to respond fully to a vaccine; and the length of time needed for an appropriate response. in vaccination strategies in puppies and kittens addition, knowledge of the various diseases that pose risks to pediatric patients and knowledge of available safe, efficacious vaccines is critical. in essence, each patient must be assessed as an individual within the population to provide optimal wellness over the lifetime of each individual, as well as the population. this rationale has led to the concepts of core and noncore vaccines, terms commonly used when discussing vaccination within the veterinary field. criteria for assigning vaccines into these categories, and a third category, "generally not recommended," are based on: ( ) morbidity and mortality associated with the specific disease (does the organism cause serious illness or does it cause a mild, transient disease that may pose only minimal risk to the individual or population?); ( ) the prevalence and/or incidence rate of the disease (although a specific disease may not commonly be seen, the organism is ubiquitous in the environment and therefore poses risk to the individual or population); ( ) the risk of the individual for exposure to the disease (indoor-only animal vs free-roaming individual, regional variations of occurrence); ( ) the efficacy of the vaccine (does the vaccine prevent infection or simply ameliorate some signs or length of disease?); ( ) the risks associated with administering the vaccine (are the risks associated with that vaccine greater than the risk of the disease?); ( ) the potential for zoonotic disease; ( ) the route of infection or transmissibility. , , - when these criteria are assessed, general guidelines may be generated for the individual practitioner and the veterinary community at large. again, guidelines are not to be thought of as absolutes, nor are they to be used to establish standard of care. simply stated, they are tools for each of us to use to promote optimal wellness for our patients when considering all factors affecting the individual's health (environmental, organismal [both pathogen and host], owner concerns, and current vaccine technologies). , , - multiple vaccines are available for canine and feline patients, although most fall within basic categories. assignment of vaccine products (which are considered biological agents, not drugs, and are therefore assessed and approved under the united states department of agriculture [usda] animal and plant health inspection service rather than the food and drug administration) into these categories is based on how the product is created. simply stated, modified live virus (mlv) vaccines are vaccines created by altering (attenuating) the pathogen in some way so that it is no longer able to cause serious or clinical disease in the targeted species. killed vaccines are vaccines produced by inactivating the pathogen completely, rendering it incapable of reproducing and thereby unable to cause disease. the third category of vaccines consists of recombinant vaccines, of which there are multiple types, and this category itself has subcategories. these vaccines use genetic technologies to either introduce genetic material directly into the host (no vector, eg, purified subunit vaccines or type i recombinant, is used), alter the genetic material to change its virulence (gene deletion, type ii recombinant), or incorporate genetic material from the desired pathogen into an attenuated vector organism (eg, feline rrabies [r recombinant], type iii recombinant). , within the near future, multiple new technologies are likely to provide even more choices, potentially providing patients with better protection against disease with minimal vaccine-associated risks. a more recent discussion for categorizing vaccines has evolved, and assigns vaccines to of groups: infectious or noninfectious. simply stated, infectious vaccines include those biologics that have the ability to enter host cells and undergo replication within the host (ml, rcanary poxvectored vaccines). noninfectious vaccines do not have the ability to undergo replication within the host. for a comparison between vaccine types, the reader is referred to table . vaccines are available in single-dose and multiple-dose (tank) vials. the use of singledose vial vaccines is highly recommended in these species. conversely, the use of multiple-dose vials is discouraged because of the increased risk of contamination and the inability to assure consistent levels of antigen and adjuvant in individual doses from a single vial. , multivalent vaccines are not recommended in cats other than the core feline vaccine designed to protect against feline panleukopenia, feline herpesvirus i, and feline calicivirus. owing to increased inflammation at the site of multivalent vaccines, all other vaccines should be given as a separate vaccine, at the indicated site (see later discussion on feline core and noncore vaccines). , allowing vaccines to acclimatize to room temperature before administration, particularly in cats, is recommended, as the administration of cold vaccines was found to have an increased association for tumorigenesis in cats. use of sterile, single-use syringes is also recommended, as vaccines may become inactivated and/or ineffective with exposure to various products used to clean and sterilize syringes. mixing of more than vaccine within a syringe should not be performed because of the potential for inactivation of vaccine material, in addition to increasing the amount of antigen deposited within a single site. moreover, administration of reconstituted vaccines (mlv r) should be done within hour of reconstitution or otherwise discarded, owing to the potential inactivation of product and loss of efficacy. the practitioner is advised to always follow the manufacturer's directions for dose and route of administration. using a topical product parenterally or splitting doses should never be done. a full dose is required to stimulate the immune system; there is no medical basis for giving a smaller dose to a toy breed dog, and this practice could lead to vaccine failure in that animal. if done with a rabies vaccine the practitioner is not following federal requirements, which carries potential legal implications. , the interval between various vaccines, whether using the same product serially in the initial series or whether using different products in an adult animal, should never less than to weeks. interference between the first product administered and a second vaccine product may lead to failure to optimally respond to the second vaccine. the exact mechanism of this interference is unknown, but may be associated with interferon produced by cells processing an mlv agent, or by transient immunosuppression by an mlv agent. multiple vaccines administered at the same time do not appear to elicit this interference and is therefore an acceptable practice. , the reader is referred to tables and for comparison between pediatric canine and feline core, noncore, and generally not recommended vaccines. the diseases that fall within this category carry high rates of morbidity and/or mortality, are of public health concern, or are readily transmissible or may be ubiquitous in the environment. in addition, safe, efficacious vaccines are available and either provide sterile immunity (prevent infection) or confer a high degree of protection (do not prevent infection, but may confer protection such that the animal will not develop clinical signs of disease). , essentially, the vaccines that fall within this category are recommended for each individual within the population regardless of the animal's lifestyle or locale. canine distemper virus (cdv), an enveloped morbillivirus, has been well controlled because of the widespread vaccination programs over the last several decades. however, the disease still persists and, in addition to high virulence, it is readily vaccination strategies in puppies and kittens transmissible. infection with the virus causes respiratory, gastrointestinal, and neurologic signs, and is often fatal. the distemper vaccine is commonly administered as part of a multivalent product. the general recommendation is to use a modified live or recombinant, multivalent product (cdv, canine adenovirus type ii [cav-ii], canine parvovirus [cpv]) beginning at to weeks, and to give serial vaccines every to weeks until the puppy has reached to weeks of age. , , many studies support the improved ability of recombinant vaccines to overcome maternal antibody interference in comparison with modified live virus vaccines. , most puppies will receive or distemper vaccinations, depending on the age at which they are first presented to the veterinarian. however, it is the interval between or the timing of the vaccinations, rather than the number, that is important. serial vaccinations help to increase the likelihood of a complete response of the patient and thereby decrease the risk of vaccine failure that may occur when only vaccine is administered. in addition, by eliciting a secondary immune response, they may help to increase the level of circulating antibody and decrease the lag time between exposure to an antigen and achievement of maximal antibody level. potential causes for vaccine failure include: a modified live vaccine that was improperly stored and therefore has lost its efficacy; the vaccine was improperly administered (wrong route or accidental loss of vaccine onto the skin of the patient); the patient's immune system did not respond (the immune system may have been responding to another antigenic challenge or the vaccine may have been given too soon after a previous vaccine); or maternal interference. in theory, if a puppy were kept sequestered from exposure to this virus, modified live distemper vaccine administered after weeks of age would confer protection for at least year. , however, in reality most pet owners are not inclined to isolate their puppies for the first months of life, nor should they. early socialization is an important part of families bonding with their puppies. exposure to various people, other dogs, and new places helps decrease behavioral problems in the young adult and mature dog. as long as the last distemper vaccine is administered after weeks of age, the puppy should be able to mount a strong active response and fully overcome any residual maternal antibody. the current recommendation is to have the puppy return year later (when approximately months old) for another distemper vaccine. after the first annual vaccination, triennial immunization is recommended, regardless of vaccine type used. , , canine adenovirus there are types of adenovirus that cause disease in canine patients. canine adenovirus type i (cav-i), a nonenveloped virus in the family adenoviridae, causes the potentially fatal disease infectious canine hepatitis. clinical signs include fever, depression, vomiting and diarrhea, and potential petechiation and ecchymotic hemorrhage secondary to hepatic dysfunction. in addition, uveitis and renal disease are associated with infection with this virus. cav-ii causes respiratory tract disease. cav-i is associated with severe, potentially fatal disease, and protection against this disease is recommended. transmission is via the oronasal route and exposure to infected secretions. cav-ii infection typically results in mild self-limiting disease and is therefore considered to be a noncore disease; however, the modified live vaccine designed for prevention of cav-i has been associated with adverse effects such as uveitis and corneal edema (an arthus reaction, similar to effects caused by natural infection). , the current recommendation is to use the cav-ii modified live virus product, as it stimulates the immune system to protect against both cav-i and cav-ii, without the associated adverse reaction caused by the type i vaccine. , , the modified live adeno-type ii virus is typically included in a multivalent injection (as mentioned earlier) and is therefore usually administered at intervals of to weeks, beginning between and weeks of age and ending between and weeks old. a vaccination year later is recommended before instituting triennial vaccinations. cpv is a nonenveloped type parvovirus. the predominant form currently causing infection in the united states is type b, but other subtypes exist and cause disease elsewhere. because the virus is nonenveloped, it may exist (outside of a host) under certain environmental conditions, and is somewhat resistant to many disinfectants. transmission is via the fecal-oral route, and clinical signs include lethargy, anorexia, pyrexia, vomiting, and diarrhea (typically hemorrhagic). young animals appear to be at highest risk for developing severe, life-threatening disease. the current recommendation for vaccination is to use a multivalent mlv vaccine beginning at to weeks and to repeat the vaccine at intervals as already stated (every - weeks, until the puppy is - weeks old). in the past there was concern that certain breeds may have been at increased risk for contracting and developing severe parvoviral disease (doberman pinschers, rottweilers), but it is generally agreed that these breeds will mount an appropriate response to a quality product if the last vaccine is given between and weeks of age. , , there is, however, a small population of dogs that is genetically unable to respond to vaccination against cpv , regardless of the number of vaccinations (nonresponders). one benefit of having a well-vaccinated population is that even those nonresponders are at decreased risk of exposure and subsequent infection by parvovirus, based on strong herd immunity. studies using mlv cpv b strains showed a higher antibody response to cpv and cpv b, and were better able to overcome maternal antibody interference than the cpv -strain vaccines used , ; however, all cpv vaccines currently available should produce strong immunity in immunocompetent dogs. immunization year after completing the initial puppy series is recommended, with subsequent triennial vaccinations. , , there is also emerging evidence that weimaraner puppies are at increased risk of developing a severe form of hypertrophic osteodystrophy (hod) in association with vaccination with mlv distemper, adenoviral, and parvoviral products. the exact vaccination strategies in puppies and kittens mechanism is unknown, but the current recommendation is to use killed products in this breed for their pediatric vaccinations, and consider starting vaccinations when they are slightly older. rabies virus, an enveloped virus in the rhabdoviridae family, is capable of infecting all mammals. because it is an enveloped virus, it is not stable in the environment and is readily inactivated by most common disinfectants. the virus is transmitted through infected saliva, most commonly from a bite by an infected animal. clinical signs range from anxiety or other vague behavioral changes to pica, dysphagia, photophobia, and paralysis. because of the zoonotic potential and implications regarding public health, canine vaccination programs are strongly regulated and enforced. the current recommendation is to vaccinate puppies using a killed-virus vaccine at a minimum of or weeks of age. state regulations vary as to the minimum age for canine rabies vaccination: in california the legal minimum age of canine vaccination against rabies is weeks. a second rabies vaccine (killed product) is administered year later and then annually or triennially thereafter, depending on local regulations. , it is the practitioner's professional responsibility for knowledge of and adherence to regional laws regarding rabies vaccination frequency. vaccines in the noncore category may have limited efficacy, or the organism causing disease is not readily transmissible or may have limited geographic distribution or prevalence. in addition, the diseases these vaccines are designed to prevent may be so mild or self-limiting that the risks associated with administering the vaccines may be greater than the actual disease. lastly, some vaccines may interfere with common screening methods for disease detection, and are therefore not recommended unless absolutely warranted for a specific individual. it is the burden of the practitioner, along with the pet owner, to make decisions regarding which, if any, of the noncore vaccines should be administered to a puppy. , [ ] [ ] [ ] leptospirosis a bacterial pathogen that causes acute hepatic and renal disease, leptospirosis is typically transmitted through urine of infected animals (reservoir hosts include dogs, rats, wildlife, and livestock), and in contaminated water. there are at least different species (leptospira interrogans and leptospira kirschneri) that can infect dogs, with multiple serovars (variants of the same species) of l interrogans causing disease in dogs. although these organisms have the potential to cause serious disease, dogs are not likely to be at risk in a mostly urban, controlled environment (housed in a fenced yard with no exposure to wildlife or livestock). however, a dog that frequents rural environments or has exposure to waterways or livestock is definitely at risk of infection and should therefore be protected against the disease. again, the initial puppy appointments should involve a through history and include the owner's plans for the dog's future use. if an owner brings a labrador retriever puppy to the veterinarian for "whatever vaccines he needs," it is up to the practitioner to ask "will he be a hunting dog, will he be used in field trials, will he be exposed to wildlife and waterways?" the border collie who lives on a working sheep ranch surely should be vaccinated appropriately against leptospirosis. conversely, a long-haired miniature dachshund who will spend her days on her owner's lap in an urban setting will be at minimal risk of exposure and, therefore, vaccination is most likely not warranted. in essence, regional distribution, seasonality (increased prevalence during and immediately following the rainy season), and lifestyle of the puppy will be factored into the decision as to whether the puppy should be vaccinated. if the decision is made to vaccinate against leptospirosis, the general recommendation is to wait until the puppy is at least weeks old, at which time a killed or purified subunit vaccine is administered. infection is serovar specific, and no cross-protection is seen between different serovars; therefore, vaccination with as many serovars known to cause disease in a given region is recommended. an initial series of vaccinations should be administered to weeks apart and repeated at least annually thereafter, as long as the risk of exposure to the agent exists. the recommendation to wait until the puppy is at least weeks old before administering the leptospirosis vaccine is based on the increased potential for adverse events associated with killed vaccines, and to increase the likelihood of a complete immune response. , bordetella bordetella bronchiseptica is a bacterial agent that causes infectious tracheobronchitis. infection with this agent may occur in concert with other agents infecting the respiratory tract (canine parainfluenza virus [cpiv], cav-ii). transmission occurs via direct contact or through aerosolized microdroplets from infected dogs, and is most likely to occur under crowded conditions such as boarding and grooming facilities and dog-show venues. the current recommendation is to vaccinate puppies at risk a minimum of week before potential exposure with a combination vaccine containing both an avirulent live bacterin for b bronchiseptica and a modified live cpiv. the vaccine can be administered to puppies as young as to weeks of age, but is generally not indicated unless the puppy is in a kennel environment. many organized puppy socialization and obedience classes commonly require proof of vaccination against bordetella at the time of enrollment or before beginning the course. the general consensus is that intranasal vaccines are superior to parenteral vaccines, as they stimulate rapid local immunity (which is not affected by persistent maternally derived antibody). , intranasal vaccines should never be given subcutaneously, owing to the potential for severe (in some cases fatal) reactions (fig. ) . if the puppy will be intermittently exposed throughout the year (traveling to shows, boarding or grooming facilities) the vaccine should be repeated every months to annually. as already stated, cpiv may occur in concert with other respiratory tract agents. the vaccine recommendations are as stated for b bronchiseptica if indicated. there are vaccination strategies in puppies and kittens multiple products available, but the product currently recommended is the combination of intranasal vaccine containing a modified live parainfluenza virus with an attenuated b bronchiseptica bacterin. intranasal vaccines can be used in puppies aged to weeks for individuals at high risk of exposure (depending on vaccine manufacturer label restrictions). for optimal protection, the vaccine should be administered every months to annually if indicated. alternatively many multivalent, parental products containing modified live cdv, cav-ii, cpv, and parainfluenza are available and appropriate for use. , borreliosis borrelia burgdorferi is a vector-borne, spirochete bacterium responsible for lyme disease (borreliosis). transmission occurs when an infected tick (various species within the ixodes genera, also referred to as hard ticks) bites and remains attached to a host, in this case a puppy. direct, horizontal transmission is not likely to occur, so the risk to humans and other pets is thought to be minimal. if a puppy has a significant burden with infected ticks, it of course increases the exposure to others in the household but, as ticks typically do not reattach once they have taken a complete meal, the risk is considered to be fairly small unless appropriate tick control is not instituted. vaccination to protect against lyme disease is controversial, as the duration of immunity and degree of protection provided by vaccination is unknown, and vaccination with some vaccines interferes with standard screening diagnostics. therefore, vaccination against lyme disease is warranted only if a puppy will be expected to be at high risk for tick exposure, and only if it lives in a borrelia-endemic area. there are killed and recombinant (ospa subunit) vaccines available for use against b burgdorferi, and if vaccination is deemed warranted, the current recommendation is to use one of the subunit vaccines before exposure to ticks. the vaccine can be given as early as weeks and should be repeated to weeks later. the best prophylaxis is likely achieved by using appropriate tick prevention, such as fipronil with methoprene spray or spot-on products (eg, frontline top spot; merial ltd, iselin, nj), amitraz collars (eg, preventic collar; virbac animal health, fort worth, tx), or an imidacloprid/permethrin topical product (eg, canine advantix; bayer animal health, shawnee mission, ks). , these products should be chosen and recommended carefully by the veterinarian based on household situations, owner concerns, and the age of the puppy. this virus, also a morbillivirus, can stimulate an immune response that is crossprotective against cdv. the indication for using this vaccine is for puppies that may have maternal antibody to distemper virus sufficient to cause interference with distemper vaccination but inadequate to protect against infection. if indicated (see later discussion on special circumstances), a single vaccination with a modified live vaccine should be given intramuscularly as early as weeks of age. subsequent immunizations with mlv cdv vaccines should be given serially as recommended (see cdv section). , , canine measles vaccines should never be administered to female puppies older than weeks, as they may develop an acquired immune response to the virus, which could be problematic if a female puppy vaccinated against measles at weeks of age later became pregnant. if she developed antibodies to the measles virus and maintained immunologic memory, she would confer measles antibody to her puppies via passive transfer, thus rendering measles vaccination in those puppies ineffective. a more appropriate alternative to administering a measles vaccine to a young puppy thought to be at risk for infection but too young to receive an mlv cdv vaccine would davis-wurzler be to use a recombinant cdv vaccine, thereby decreasing the likelihood of maternal antibody interference. , canine influenza virus canine influenza virus has been seen in various countries, most notably in enzootic outbreaks. this virus is typically seen in puppies and dogs in shelter, boarding, and day-care facilities, and often occurs as a coinfection in canine infectious respiratory disease (cird) with bacterial pathogens. there is a commercially available inactivated vaccine available for use in puppies as young as weeks. vaccination with this product should be used only in puppies with a high risk of exposure, such as to shelters and areas known to be dealing with current/recent outbreaks (typically not in client-owned puppies). in addition, some countries now require an initial vaccination series ( doses, given - weeks apart) before importation. , rattlesnake vaccine a vaccine designed to protect against envenomation by crotalus atrox, the western diamondback rattlesnake, was released onto the market several years ago. the original provisional licensure was granted to provide possible protection against this single species of snake, and was granted for use only in california. the company was later granted extended licensure for multiple states, and has extended its claim for potential protection against multiple species of members of the crotalidae (pit vipers). to date, no challenge studies have been performed in the canine species to validate efficacy claims. all claims are based on antibody titer to the venom component included in the vaccine, to murid challenge studies, and to field reports of protection of naturally occurring envenomation. no controlled, independent studies exist concerning the impact of prior vaccination on therapeutics after envenomation. the manufacturer does not claim that vaccination with this product will completely protect against effects of envenomation; rather, they claim it may slow the onset of clinical signs and decrease the severity of signs. immediate veterinary care is still the gold standard for any snake bite. because of the great potential for variability in envenomation (site of bite on animal, size and age of snake, amount of venom injected into animal, and species of snake), field observations and anecdotal reports of protection are difficult to substantiate. challenge studies conducted under controlled conditions will likely be necessary to validate the efficacy of this product. at present, owing to the preceding statements, this vaccine is not recommended for general use. aversion training and keeping dogs out of areas known to favor rattlesnake habitation, and immediate veterinary evaluation and care are still the standard recommendations for preventing and treating disease associated with rattlesnake envenomation. if an owner is extremely concerned about the potential for exposure and envenomation by a western diamondback rattlesnake, the decision to vaccinate should be made after a discussion between the veterinarian and owner, with full disclosure of vaccine efficacy and a risk/benefit analysis, understanding the potential for adverse events from vaccination. this vaccine has been shown to be safe for use in puppies as young as months. an enveloped virus belonging to the family coronaviridae, this virus is transmitted via the fecal-oral route. vaccination against this disease is generally not recommended because the vaccines provide questionable protection, and the actual prevalence vaccination strategies in puppies and kittens and severity of the disease are unknown. those most likely to be infected and develop clinical disease are neonates younger than weeks. clinical signs may include diarrhea, possibly hemorrhagic, but typically self-limiting. the general recommendation is to vaccinate puppies against cpv (as recommended in the section on cpv), as this practice appears to confer protection against coronavirus in addition to preventing infection with cpv . , canine adenovirus type i as stated in the canine core vaccine section, cav-i causes serious disease in dogs; however use of the cav-i is associated with a high incidence of adverse events. vaccination with cav-ii induces an immune response that is protective against both cav-i and cav-ii without the adverse effects. the recommendation is to use cav-ii as part of the canine core vaccination program; cav-i should not be used. feline panleukopenia, a nonenveloped parvovirus closely related to canine parvovirus, causes serious, often fatal disease in kittens. transmission typically occurs from direct contact with infected animals, although in utero infection and fomite transmission also occurs. clinical signs typically include pyrexia, anorexia, lethargy, vomiting, and diarrhea. kittens may be immunosuppressed subsequent to pancytopenia associated with this viral infection. kittens infected in utero may exhibit cerebellar disease. prevention is achieved by using modified live virus vaccines beginning between and weeks of age. the standard recommendation is to use a parenteral product (as opposed to intranasal products, which have higher incidences of postvaccinal viral shedding and potential for clinical disease induced by the more virulent viruses in these vaccines). , , as is the case for canine distemper, adenovirus, and parvovirus, the core feline diseases, with the exception of rabies, are typically administered in a multivalent product in series. there are numerous vaccine products containing feline panleukopenia virus, herpesvirus i, and calicivirus (see later discussion). the current recommendation is to choose an mlv or killed product from a reputable manufacturer. vaccines are administered subcutaneously in the distal aspect of the right thoracic limb (elbow or distally) and given every to weeks until the kitten is at least to weeks old. repeat administration is recommended year later before instituting a triennial schedule. , feline herpesvirus i feline herpesvirus i (fhv-i), also known as feline viral rhinotracheitis virus, is an enveloped virus causing respiratory tract disease in cats. clinical signs include sneezing, nasal congestion and discharge, conjunctivitis, and ocular discharge. in addition, kittens may exhibit pyrexia, anorexia, and lethargy along with oral/lingual ulcerations and associated hypersalivation. in some cases ulcerative, crusting dermatitis occurs, which may mimic other dermatologic disease. the virus typically causes upper respiratory disease but the lower respiratory tract may become involved, especially in neonates or debilitated animals. infection with this virus is lifelong, although many cats will "recover" and not show clinical signs. however, cats infected with fhv-i may have recurrent outbreaks, especially under times of stress or if their immunity is otherwise compromised. cats may persistently shed the virus and act as a source of infection in shelters, catteries, and multiple-cat households. therefore, prevention before exposure is key to controlling this disease. , vaccination with a modified live virus (or killed product) beginning as early as to weeks is recommended, this being commonly administered as part of a multivalent product, given subcutaneously, in the right thoracic limb. the current recommendation is for kittens to receive a second vaccination weeks later. the last vaccine in the series should be given when kittens are to weeks of age. a vaccine should be given year later before beginning the triennial schedule. feline calicivirus causes respiratory tract disease in kittens and cats. because it is a nonenveloped virus, it is more resistant to disinfectants and may therefore persist in the environment. signs are similar to those associated with fhv-i, but lameness and stomatitis are also commonly seen. transmission of both fhv-i and calicivirus is through direct contact, exposure to contaminated secretions, aerosolization, and fomites. , another, highly virulent, strain of feline calicivirus was identified several years ago and carries a higher incidence of mortality. transmission is through either direct contact or via fomites. prior vaccination against feline calicivirus does not appear to be protective against this strain, and adult cats appear to be more severely affected than kittens. , the current recommendation is as for panleukopenia and fhv-i: administering a modified live virus inactivated-virus parenteral vaccine beginning at or weeks with a subsequent vaccine weeks later (the last vaccination should be when the kitten is at least to weeks old). a booster vaccine should be administered year later, and then every years. as stated earlier, rabies virus affects all mammals and in the united states, with most documented cases of rabies in pet animals occurring in cats. because of the significant risk to pets, wildlife, and humans, vaccination against rabies virus is highly recommended for all kittens and cats, even those kept inside. , local requirements vary, but the general recommendation is that all kittens should be vaccinated beginning at weeks of age with either the recombinant rabies vaccine (preferable) or a killed rabies virus vaccine. , , the recombinant product uses gene-splicing technology: reverse transcriptase is applied to rabies viral rna to create complementary dna. the segment of rabies virus dna that codes (a codon) for the immunogenic protein associated with the virus (glycoprotein g) is then spliced from the rabies dna and inserted into a canarypox virus. the canarypox virus, which is attenuated, is nonpathogenic to mammalian cells and therefore carries no potential to cause disease in this species. because the vaccine is essentially a modified live product, the canarypox virus can enter cells, delivering the codon for rabies virus glycoprotein g to its targeted site. once inside the cell the canarypox virus is unable to replicate, but the rabies glycoprotein g codon is preserved, leading the host cell to express the glycoprotein on its surface; this stimulates both cell-mediated and humoral immune responses. besides the benefit of stimulating both types of immunity, because this product is adjuvant-free there may be a decreased risk of local inflammation associated with vaccination, thereby potentially decreasing the risk of subsequent vaccine reactions and tumorigenesis. the current recommendation is to use either the rrabies virus vaccine (preferred when possible) or a killed-virus vaccine in a case where increased duration of immunity is required (not pertinent to kittens because all pediatric/initial rabies vaccinations provide only months of protection, regardless of label claims). , rabies vaccines should be administered subcutaneously in the right pelvic limb, as distally as is reasonably possible: the level of the stifle is acceptable and areas distal to the tarsus are difficult to inject, and therefore not really feasible or appropriate. administering vaccines (or any injections for that matter) in the tail should be avoided. giving injections in the tail is difficult because of the scant amounts of loose skin and subcutaneous tissue, which is vaccination strategies in puppies and kittens likely to cause more discomfort in patients during vaccination. more importantly, if a tumor does arise proximally on the tail, the potential for complete resection and cure are decreased because of the potential for tumor infiltration into the vertebral column. at present there is only one recombinant rabies vaccine approved for use in cats (pure-vax feline rabies vaccine'; merial ltd, duluth, ga). the current usda approval/label states that this product should be administered annually. there are multiple killed-virus rabies vaccines approved for use in cats, with initial vaccination occurring at weeks of age with a subsequent vaccination year later. because regulations vary depending on state or region, the veterinary practitioner must be familiar with local laws regarding rabies vaccination in this species. feline leukemia virus (felv) is a retrovirus primarily affecting cats of any age, but kittens and juvenile cats appear to be most susceptible to infection. clinical signs are numerous and nonspecific, and include pyrexia, failure to thrive, chronic or recurrent respiratory tract, and gastrointestinal disease. infection in kittens occurs via vertical transmission from the queen to the fetus, but may also spread horizontally from queen to kitten during lactation and grooming. transmission also occurs through direct and usually prolonged contact with other infected cats from behaviors such as grooming and sharing food, and water bowls, and litter boxes. viral screening using an enzymelinked immunosorbent assay (elisa) test designed to detect antigenemia should be performed on all kittens, even if their owners plan to house them strictly indoors. because the elisa test detects antigen, maternal antibody and vaccination do not interfere with test results. therefore kittens of any age may be tested, and the current recommendation is to test every kitten (and adult cats with an unknown viral status) prior to felv vaccination. if a kitten is antigen negative, the current recommendation is to administer either a killed or a recombinant vaccine on the first or second kitten visit. a second vaccine should be administered weeks later followed by vaccination year after the last felv kitten vaccine. , the recommended site for administration of any felv vaccine is the left pelvic limb, as distally as is reasonably possible. at present there is only one recombinant felv vaccine available (purevax recombinant leukemia vaccine; merial ltd, duluth, ga). although felv is considered a noncore vaccine in adult cats because kittens are most vulnerable to infection and may be exposed if outdoors, and immunity increases with age, it is rational to vaccinate all kittens against this disease with a repeat vaccination year later. if the cat is subsequently housed strictly indoors and does not live with an infected (felv) cat, additional vaccinations are not indicated. chlamydophila felis, formerly known as chlamydia psittaci, is a bacterium that causes upper respiratory tract disease in kittens and cats. the most common sign is conjunctivitis, but sneezing and nasal discharge may also be present. transmission is typically through direct contact with infected cats. kittens are most commonly affected, but usually recover fully with appropriate antibiotic therapy: either topical oxytetracycline (terramycin ophthalmic ointment) or systemic tetracycline (panmycin aquadrops) or doxycycline (vibramycin). vaccination against this agent typically does not prevent infection but may prevent clinical signs of disease. because the vaccine does not fully prevent infection and carries an association with adverse events that may be greater than the actual disease, routine vaccination of household pets with this product is davis-wurzler generally not recommended. however, it may be of use in some environments where the risk of infection is high, such as shelters or catteries with recent outbreaks. , if vaccination is deemed appropriate by the practitioner, an attenuated parenteral vaccine can be given to kittens beginning at weeks, with a second dose given to weeks later. bordetella this bacterial agent causes respiratory tract disease in cats, and cats affected by stress, poor nutrition, or overcrowding seem more susceptible. many kittens infected show mild, self-limiting disease with signs including pyrexia, sneezing, and nasal and ocular discharge, although bronchopneumonia has been documented. there is a topical, modified live bacterin vaccine designed for use in this species, but it is generally not recommended for routine use. if the practitioner feels protection against b bronchiseptica is warranted based on the kitten's risk of exposure, such as attendance at cat shows or visiting a boarding facility, or is in a shelter with potential contact with dogs (with a recent b bronchiseptica outbreak), administration of the vaccine designed for use in cats may be considered. a single dose of the modified live intranasal vaccine can be given to kittens as young as weeks of age. the product designed for use in canines should not be used in cats. there are multiple vaccines in addition to those described and recommended here; however, many of these diseases pose a minimal risk to most of the feline population or the vaccines are minimally efficacious at preventing infection or disease, and therefore are generally not recommended. additional reasons not to use some of these products are vaccine interference with screening tests and adverse events associated with some vaccines. a retrovirus, feline immunodeficiency virus (fiv) primarily affects cats by compromising their immune system, leaving them vulnerable to opportunistic infections. in addition to immunosuppression, with most of the effect targeted against the cellmediated (t-cell) immune response, infection with fiv also carries an increased risk for development of certain types of neoplasia, b-cell lymphoma being the most common. transmission occurs most commonly from breeding and fighting. the virus is not spread through casual contact between housemates not engaging in the behaviors stated, nor is it spread through casual encounters between nonbreeding, nonfighting cats outside. naturally occurring infection of kittens from queens is rare; however, kittens can become fiv-antibody positive via passive transfer from ingestion of colostrum of fiv-positive queens or queens previously vaccinated against fiv. , fiv-antibody levels acquired from maternal transfer in kittens who are actually fivvirus negative decline over the first several months of life. the standard screening test for fiv is an elisa test designed to detect fiv antibody. the elisa was designed to detect antibody rather than antigen, because infected cats produce high levels of circulating antibody in contrast to low levels of circulating virus. because kittens may have circulating fiv antibody although actually may be fiv-antigen negative, it is generally not recommended to test kittens younger than months. if a kitten is tested and a positive result is obtained, the test result should be repeated with a different methodology (western blot or polymerase chain reaction [pcr] ) and should be repeated once the kitten is more than months old. if a kitten is truly not infected, the maternal antibody will wane by months of age, leading to seroconversion. if, however, a kitten or cat remains seropositive, the recommendation is made to keep the cat indoors only from that point, both to prevent infection of other cats and to decrease exposure to potential environmental pathogens. fiv-infected cats can live for years and, unless otherwise indicated by concurrent disease, euthanasia is generally not indicated for most owned pets. there is a killed fiv vaccine available, but the efficacy of this product is still unknown. there are known subtypes of fiv virus, and the vaccine has been formulated to protect against subtypes a and d; however, the predominant subtype infecting cats in north america and europe appears to be subtype b. it is unknown whether cross-protection exists between the different subtypes. because the vaccine elicits a strong antibody response, vaccinated kittens and cats will become seropositive on both elisa and western blot tests, as both tests detect antibody. a pcr test is available but is currently only performed at certain laboratories, and results and reliability vary with testing centers. because of the increased technological needs and increased costs of this test, it is not considered the standard screening test. if done under specific conditions it can detect virus, and therefore may be of benefit in differentiating between cats with viremia (truly infected cats) and kittens or cats with circulating antibody, attributable either to maternal transfer or vaccination. because of the nature of transmission of the virus and interference with the standard screening methods for infection, the vaccination against fiv is not currently recommended. keeping cats indoors if possible, neutering all cats going outside, and preventing exposure to stray or feral cats that may be more likely to engage in fighting behaviors remain the gold standards for preventing this disease. , feline infectious peritonitis the disease feline infectious peritonitis (fip) is caused by a member of the coronaviridae. feline enteric coronavirus (fecv) and fip virus are phenotypes of the same virus. fecv transmission occurs through the fecal-oral route where it typically infects intestinal epithelium, but the organism can be transmitted via fomites and persists for long periods of time in the environment. most cats infected with fecv either do not show clinical signs of disease or may have transient diarrhea, and some will persistently shed the virus in their feces. fecv can, however, undergo random mutations within a host, creating fip virus, although in most cats the virus does not mutate into this form and most cats will not develop fip. the fip virus enters and replicates within macrophages where it can then be disseminated throughout the body. clinical signs are numerous, but commonly include weight loss, failure to thrive, diarrhea, pyrexia, and chronic respiratory tract disease. two main types of the disease exist, the dry (noneffusive) and the wet (effusive) forms. both are ultimately fatal diseases. although there is a vaccine available, its efficacy and indication for use is believed to be minimal, if at all. the current recommendation is not to use this vaccine, based on efficacy concerns and the minimal risk of infection in most kittens and cats. infection with fecv and mutation with subsequent development of disease occurs most commonly in multiple-cat households ( ), catteries, and shelters. the standard screening test for fip is a serologic, indirect immunofluorescent antibody (ifa) test designed to detect antibody. this test may be of some value, but results need to be interpreted with caution, and concomitantly with signalment, clinical signs, and other laboratory data. prior vaccination against fip will yield positive ifa results, further posing potential complications in routing screening of this disease. in general, kittens are most vulnerable to this disease, with greater than % of cats with fip being younger than years. prevention is directed toward decreasing stress in kittens and cats in multiple-cat households, preventing exposure of naïve kittens and cats in environments known to have high endemic levels of feline enteric corona virus, and at depopulating catteries known to have high prevalence rates of fecv and fip. , because of the complexity of this disease and the limited space and objectives of this discussion, readers are encouraged to review infectious diseases of the dog and cat, rd edition, by greene, and textbook of veterinary internal medicine, th edition, by ettinger and feldman, for a more comprehensive review of this disease. vaccines are potent biological agents designed to prevent disease. any foreign product administered to an animal has the potential to be associated with an unexpected response by that animal. while vaccines must meet usda requirements for safety, efficacy, potency, and purity, there still exists the potential for adverse events with products that have met these standards. veterinarians should always report adverse events associated with vaccination to the vaccine manufacturer. some adverse events are more likely to occur with certain agents, whereas others appear to have an increased rate of occurrence in certain breeds. still others may be idiosyncratic and are not predictable. the following is offered as a brief overview of some types of adverse events associated with vaccination, with suggestions as to how a practitioner might best respond to and prevent such events from recurring. the reactions seen most commonly are local inflammation at the site of the injection or general malaise, pyrexia, and anorexia for to days after vaccination. most of these reactions are self-limiting and require nothing more than monitoring by the animal owner. it is appropriate for the practitioner to note any reaction along with a description of signs documented in the medical record, and offer supportive care if indicated. in some instances administration of an mlv vaccine will cause transient mild clinical disease. supportive care and isolation from unvaccinated animals is recommended, as the vaccinated animal showing clinical disease will shed the vaccinal organism and is potentially infectious to other animals. contact information for vaccine manufacturers, support agencies, and disease-reporting organizations is included in table . feline injection-site sarcomas (fiss), formerly known as feline vaccine-associated sarcomas or fibrosarcomas, develop secondarily to local inflammation at injection sites. originally it was thought that there was an increased risk for development of these tumors associated with specific adjuvants and vaccines; however, it is now accepted that all vaccines and repositol agents such as long-acting penicillin and corticosteroid injections, in addition to other injections, can be associated with the formation of fiss. measures to prevent these tumors are aimed at decreasing the local inflammatory response by avoiding the use of adjuvants in this species and administering only those vaccines indicated for the individual animal. , multiple vaccines should not be administered in one site, as this may increase the amount of inflammation in that site. following the recommended sites for injection is strongly recommended (see individual vaccine sections for specific sites) , and avoiding adjuvanted products when there is a reasonable alternative (mlv or recombinant) product available is ideal, as a recent study confirmed an increased association of tumor formation with adjuvanted vaccines compared with recombinant vaccines, although no vaccine was risk free. , there are specific guidelines as to how a practitioner should proceed if a cat develops a swelling at the site of a vaccine or injection. the practitioner is advised to monitor the patient closely, documenting -dimensional measurements and temporal association if a mass or swelling develops at the site of a vaccine. the - - rule developed by the table feline vaccine-associated sarcoma task force should be closely applied. "three" refers to persistence of the mass for months or greater; " " refers to a size of cm or greater; and " " applies if the mass increases in size after month. if any of these criteria are met, the mass should be biopsied with wedge technique or needle biopsy allowing for complete resection of the biopsy margins in the future, and subsequent referral to an oncologist or surgical oncologist if fibrosarcoma is confirmed. fine-needle aspiration is not recommended for evaluation of potential injection-site sarcomas. , most vaccine manufacturers have programs established to help defray the medical and surgical costs associated with these tumors, and the practitioner is advised to always notify the vaccine manufacturer any time an adverse event is seen. type i hypersensitivity, also known as immediate hypersensitivity and, in some cases, anaphylaxis, is mediated by immunoglobulin e antibody. the host's immune system may react to anything contained within the vaccine product, including cellular products used for culture, adjuvant, preservative, and the antigen itself, and reaction typically occurs within to hours after the administration of a vaccine. in the dog, signs range from urticaria, angioedema, and pruritus ( fig. ) to respiratory distress and fulminant vascular collapse (anaphylaxis). in the cat, acute onset of vomiting and diarrhea with associated hypovolemia and respiratory and vascular shock may be seen. if an animal develops any of these signs within the first several hours after vaccination, it should be presented to the veterinarian immediately for emergency medical care and support. it is not the goal of this review to offer therapies for shock, so the reader is referred to emergency veterinary literature for recommended therapies. the point here is to advise the practitioner to proceed with caution when using vaccines that may have a higher incidence of these reactions, or in breeds that may be at increased risk for immediate hypersensitivity. the increased association between killed bacterin vaccines and type i reactions is well documented, and there are reports that toy breeds may be at increased risk for type i reactions associated with these vaccines. if an animal does have a type i reaction to a vaccine, the signs shown by the patient, interval between vaccine and onset of signs, and therapeutics administered should be well documented in the medical record, as well as plans for future vaccination of the patient in question. once an animal has this type of reaction to a vaccine, ideally the product should not be used again in that patient. all subsequent vaccines should be administered after a complete physical examination, and the vaccine should be given early in the day to allow monitoring of the patient in the hospital for several hours. however, if this is not possible the patient should remain in the veterinary hospital for monitoring for at least minutes followed by subsequent monitoring by the owner at home for several hours. pretreatment with diphenhydramine (benadryl) is an option, given parenterally (subcutaneous or intramuscular routes) at the dose of . mg/kg to minutes before vaccination if hypersensitivity is a concern. however, administration of corticosteroids concurrently with vaccination to prevent a hypersensitivity reaction is neither appropriate nor recommended because of potential immunosuppression and vaccine interference. the patient's medical record should be identified, outside and inside, to prevent future accidental readministration of the product. advising the owner that the patient should never receive that product again is important. type ii hypersensitivity reactions (autoimmune reactions) are suspected to occur in dogs secondarily to vaccine administration. although this theory is yet unproved, there are reports of dogs developing immune-mediated thrombocytopenia and immunemediated hemolytic anemia temporally associated with recent vaccination. if a dog develops either of these conditions within to months after vaccine administration, the practitioner is advised to strongly consider the risk/benefit ratio of subsequent use of that product in the patient. , type iii hypersensitivity type iii hypersensitivity reactions are immune complex reactions. examples include the anterior uveitis associated with use of the cav-i vaccine and the complement-mediated rabies vaccine induced vasculitis-dermatitis seen in dogs. other examples include glomerulonephritis and polyarthritis. antihistamine administered at the time of vaccine will do nothing to prevent the reaction, nor is it recommended to administer corticosteroids concurrently with vaccination. once an animal has had this type of reaction, subsequent use of the product should be avoided in that patient. , type iv hypersensitivity type iv hypersensitivity reactions are cell-mediated responses occurring locally or systemically. examples include sterile granulomas at the sites of vaccine administration or polyradiculoneuritis. many sterile granulomas resolve without any intervention, but for more severe reactions the practitioner is referred to various medicine texts for recommendations. , the foregoing discussion applies mainly to puppies and kittens owned by individuals. puppies and kittens housed in shelters face unique challenges, as do orphaned animals. these animals may not have received colostrum, and it is more likely that their mothers were not adequately vaccinated. the implications are that these animals are less likely to have received maternal antibodies, leaving them more vulnerable in the earliest stages of life. in addition, they frequently are malnourished, have an increased parasite burden, and are placed in crowded environments possibly with high numbers of endemic pathogens. the american animal hospital association canine vaccination task force and american association of feline practitioners have developed recommendations specifically designed for puppies and kittens in these environments. in general, neonates who may not have vaccination strategies in puppies and kittens received colostrum or who are housed under the aforesaid conditions may be vaccinated at an earlier age, and ideally should be vaccinated before or at the time of entry into the shelter. use of recombinant products may be of benefit in these animals, as well as additional vaccines (noncore vaccines). husbandry is extremely important in these animals: providing proper nutrition, anthelmintics, and clean, dry housing is paramount. in general, these animals are special subsets of the general population facing challenges most young animals do not experience. fiscal considerations and overall population health applies in these cases much more so than to individual, client-owned pets. vaccines are perhaps one of the practitioner's greatest tools in preventing disease and maintaining individual and population health. vaccination is to be used with forethought based on the risk of disease to the population and the individual, balanced with assessment of the risks associated with individual vaccines. it is the practitioner's role to educate pet owners regarding actual risks associated with both undervaccination and overvaccination. the goal is to reach the highest level of overall animal health with the minimum number of adverse events, based on scientific and epidemiologic merit. immunity in the fetus and newborn aafp feline vaccination advisory panel report american animal hospital association canine vaccination task force the defense of the body cells and their response to antigen avma council on biologic and therapeutic agents' report on cat and dog vaccines vaccines & vaccinations: guidelines vs. reality vaccines and vaccinations: the strategic issues infectious diseases of the dog and cat vaccines and their production multicenter case-control study of risk factors associated with development of vaccine-associated sarcomas in cats the use of vaccines canine viral diseases canine vaccination vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection using recombinant attenuated poxvirus vaccines quedgeley (gloucester): british small animal veterinary association infectious canine hepatitis and canine acidophil cell hepatitis infectious diseases of the dog and cat seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges duration of serologic response to five viral antigens in dogs national association of state public health veterinarians. compendium of animal rabies prevention and control prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) leptospirosis: a re-emerging zoonotic disease st louis (mo): elsevier saunders infectious diseases of the dog and cat canine borreliosis saunders infectious diseases of the dog and cat protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins efficacy of the canine influenza virus h n vaccine to decrease severity of clinical disease after cochallenge with canine influenza virus and streptococcus equi subsp rattlesnake vaccine to prevent envenomation toxicity in dogs. presented at the dr ross o. mosier th annual western veterinary conference use of serologic tests to predict resistance to feline herpesvirus , feline calicivirus, and feline parvovirus infection in cats other feline viral diseases infectious diseases of the dog and cat update on feline calicivirus: new trends an outbreak of virulent systemic feline calicivirus disease rabies surveillance in the united states during epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats feline leukemia virus american association of feline practitioners' feline retrovirus management guidelines infectious diseases of the dog and cat feline immunodeficiency virus infection feline immunodeficiency virus infection infectious diseases of the dog and cat feline infectious peritonitis and feline coronavirus infection vaccine-associated feline sarcoma task force. vaccine-associated feline sarcomas vaccine-associated feline sarcoma task force. the current understanding and management of vaccine-associated sarcomas in cats resistance to tumors comparative vaccine-specific and other injectable-specific risks of injection-site sarcomas in cats vaccine-associated adverse events immune complexes and type iii hypersensitivity key: cord- - q eg z authors: keller, mikaela; blench, michael; tolentino, herman; freifeld, clark c.; mandl, kenneth d.; mawudeku, abla; eysenbach, gunther; brownstein, john s. title: use of unstructured event-based reports for global infectious disease surveillance date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: q eg z free or low-cost sources of unstructured information, such as internet news and online discussion sites, provide detailed local and near real-time data on disease outbreaks, even in countries that lack traditional public health surveillance. to improve public health surveillance and, ultimately, interventions, we examined primary systems that process event-based outbreak information: global public health intelligence network, healthmap, and epispider. despite similarities among them, these systems are highly complementary because they monitor different data types, rely on varying levels of automation and human analysis, and distribute distinct information. future development should focus on linking these systems more closely to public health practitioners in the field and establishing collaborative networks for alert verification and dissemination. such development would further establish event-based monitoring as an invaluable public health resource that provides critical context and an alternative to traditional indicator-based outbreak reporting. i nternational travel and movement of goods increasingly facilitates the spread of pathogens across and among nations, enabling pathogens to invade new territories and adapt to new environments and hosts ( ) ( ) ( ) . offi cials now need to consider worldwide disease outbreaks when determining what potential threats might affect the health and welfare of their nations ( ) . in industrialized countries, unprecedented efforts have built on indicator-based public health surveillance, and monitoring of clinically relevant data sources now provides early indication of outbreaks ( ) . in many countries where public health infrastructure is rudimentary, deteriorating, or nonexistent, efforts to improve the ability to conduct electronic disease surveillance include more robust data collection methods and enhanced analysis capability ( , ) . however, in these parts of the world, basing timely and sensitive reporting of public health threats on conventional surveillance sources remains challenging. lack of resources and trained public health professionals poses a substantial roadblock ( ) ( ) ( ) . furthermore, reporting emerging infectious diseases has certain constraints, including fear of repercussions on trade and tourism, delays in clearance through multiple levels of government, tendency to err on the conservative side, and inadequately functioning or nonexistent surveillance infrastructure ( ) . even with the recent enactment of international health regulations in , no guarantee yet exists that broad compliance will be feasible, given the challenges associated with reporting mechanisms and multilateral coordination ( ) . in many countries, free or low-cost sources of unstructured information, including internet news and online discussion sites (figure) , could provide detailed local and near real-time data on potential and confi rmed disease outbreaks and other public health events ( , , ( ) ( ) ( ) ( ) ( ) ( ) . these eventbased informal data sources provide insight into new and ongoing public health challenges in areas that have limited or no public health reporting infrastructure but have the highest risk for emerging diseases ( ) . in fact, event-based informal surveillance now represents a critical source of epidemic intelligence-almost all major outbreaks investigated by the world health organization (who) are fi rst identifi ed through these informal sources ( , ) . with a goal of improving public health surveillance and, ultimately, intervention efforts, we (the architects, developers, and methodologists for the information systems described herein) reviewed of the primary active systems that process unstructured (free-text), event-based information on disease outbreaks: the global public health intelligence network (gphin), the healthmap system, and the epispider project (semantic processing and integration of distributed electronic resources for epidemics [and disasters]; www.epispider.net). our report is the result of a joint symposium from the american medical informatics association annual conference in . despite key differences, all systems face similar technologic challenges, including ) topic detection and data acquisition from a high-volume stream of event reports (not all related to disease outbreaks); ) data characterization, categorization, or information extraction; ) information formatting and integration with other sources; and ) information dissemination to clients or, more broadly, to the public. each system tackles these challenges in unique ways, highlighting the diversity of possible approaches and public health objectives. our goal was to draw lessons from these early experiences to advance overall progress in this recently established fi eld of event-based public health surveillance. after summarizing these systems, we compared them within the context of this new surveillance framework and outlined goals for future development and research. background gphin took early advantage of advancements in communication technologies to provide coordinated, near real-time, multisource, and multilingual information for monitoring emerging public health events ( , ) . in , a prototype gphin system was developed in a partnership between the government of canada and who. the objective was to determine the feasibility and effectiveness of using news media sources to continuously gather information about possible disease outbreaks worldwide and to rapidly alert international bodies of such events. the sources included websites, news wires, and local and national newspapers retrieved through news aggregators in english and french. after the outbreak of severe acute respiratory syndrome (sars), a new, robust, multilingual gphin system was developed and was launched november , , at the united nations. the gphin software application retrieves relevant articles every minutes ( hours/day, days/week) from news-feed aggregators (al bawaba [www.albawaba.com] and factiva [www. factiva.com]) according to established search queries that are updated regularly. the matching articles are automatically categorized into > gphin taxonomy categories, which cover the following topics: animal, human, or plant diseases; biologics; natural disasters; chemical incidents; radiologic incidents; and unsafe products. articles with a high relevancy score are automatically published on the gphin database. the gphin database is also augmented with articles obtained manually from openaccess web sites. each day, gphin handles ≈ , articles. this number drastically increases when events with serious public health implications, such as the fi nding of melamine in various foods worldwide, are reported. although the gphin computerized processes are essential for the management of information about health threats worldwide, the linguistic, interpretive, and analytical expertise of the gphin analysts makes the system successful. articles with relevancy below the "publish" threshold are presented to a gphin analyst, who reviews the article and decides whether to publish it, issue an alert, or dismiss it. additionally, the gphin analyst team conducts more in-depth tasks, including linking events in different regions, identifying trends, and assessing the health risks to populations around the world. english articles are machine-translated into arabic, chinese (simplifi ed and traditional), farsi, french, rus- sian, portuguese, and spanish. non-english articles are machine-translated into english. gphin has adopted a best-of-breed approach in selecting engines for machine translation. the lexicons associated with the engines are constantly being improved to enhance the quality of the output. as such, the machine-translated outputs are edited by the appropriate gphin analysts. the goal is not to obtain a perfect translation but to ensure comprehensibility of the essence of the article. users can view the latest list of published articles or query the database by using both boolean and translingual metadata search capabilities. in addition, notifi cations about events that might have serious public health consequences are immediately sent by email to users in the form of an alert. as an initial assessment of data collected during july through august , who retrospectively verifi ed outbreaks, of which % were initially picked up and disseminated by gphin ( ) . outbreaks were reported in countries, demonstrating gphin's capacity to monitor events occurring worldwide, despite the limitation of predominantly english (with some french) media sources. one of gphin's earliest achievements occurred in december , when the system was the fi rst to provide preliminary information to the public health community about a new strain of infl uenza in northern people's republic of china ( ) . during the sars outbreak, declared by who in march , the gphin prototype demonstrated its potential as an early-warning system by detecting and informing the appropriate authorities (e.g., who, public health agency of canada) of an unusual respiratory illness outbreak occurring in guangdong province, china, as early as november , . gphin was further able to continuously monitor and provide information about the number of suspected and probable sars cases reported worldwide on a near real-time basis. gphin's information was ≈ - days ahead of the offi cial who report of confi rmed and probable cases worldwide. in addition to outbreak reporting, gphin has also provided information that enabled public health offi cials to track global effects of the outbreak such as worldwide prevention and control measures, concerns of the general public, and economic or political effects. gphin is used daily by organizations such as who, the us centers for disease control and prevention (cdc), and the un food and agricultural organization. operating since september , healthmap ( , ) is an internet-based system designed to collect and display information about new outbreaks according to geographic location, time, and infectious agent ( ) ( ) ( ) . healthmap thus provides a structure to information fl ow that would otherwise be overwhelming to the user or obscure important elements of a disease outbreak. healthmap.org receives , - , visits/day from around the world. it is cited as a resource on sites of agencies such as the united nations, national institute of allergy and infectious diseases, us food and drug administration, and us department of agriculture. it has also been featured in mainstream media publications, such as wired news and scientifi c american, indicating the broad utility of such a system that extends beyond public health practice ( , ) . on the basis of usage tracking of healthmap's internet site, we can infer that its most avid users tend to come from government-related domains, including who, cdc, european centre for disease prevention and control, and other national, state, and local bodies worldwide. although the question of whether this information has been used to initiate action will be part of an in-depth evaluation, we know from informal communications that organizations (ranging from local health departments to such national organizations as the us department of health and human services and the us department of defense) are leveraging the healthmap data stream for day-to-day surveillance activities. for instance, cdc's biophusion program incorporates information from multiple data sources, including media reports, surveillance data, and informal reports of disease events and disseminates it to public health leaders to enhance cdc's awareness of domestic and global health events ( ) . the system integrates outbreak data from multiple electronic sources, including online news wires (e.g., google news), really simple syndication (rss) feeds, expertcurated accounts (e.g., promed-mail, a global electronic mailing list that receives and summarizes reports on disease outbreaks) ( ) , multinational surveillance reports (e.g., eurosurveillance), and validated offi cial alerts (e.g., from who). through this multistream approach, healthmap casts a unifi ed and comprehensive view of global infectious disease outbreaks in space and time. fully automated, the system acquires data every hour and uses text mining to characterize the data to determine the disease category and location of the outbreak. alerts, defi ned as information on a previously unidentifi ed outbreak, are geocoded to the country scale with province-, state-, or city-level resolution for select countries. surveillance is conducted in several languages, including english, spanish, russian, chinese, and french. the system is currently being ported to other languages, such as portuguese and arabic. after being collected, the data are aggregated by source, disease, and geographic location and then overlaid on an interactive map for user-friendly access to the original report. healthmap also addresses the computational challenges of integrating multiple sources of unstructured information by generating meta-alerts, color coded on the basis of the data source's reliability and report volume. although information relating to infectious disease outbreaks is collected, not all information has relevance to every user. the system designers are especially concerned with limiting information overload and providing focused news of immediate interest. thus, after a fi rst categorization step into locations and diseases, a second round of category tags is applied to the articles to improve fi ltering. the primary tags include ) breaking news (e.g., a newly discovered outbreak); ) warning (initial concerns of disease emergence, e.g., in a natural disaster area; ) follow-up (reference to a past outbreak); ) background/context (information on disease context, e.g., preparedness planning); and ) not disease-related (information not relating to any disease [ - are fi ltered from display]). duplicate reports are also removed by calculating a similarity score based on text and category matching. finally, in addition to providing mapped content, each alert is linked to a related information window with details on reports of similar content as well as recent reports concerning either the same disease or location and links for further research (e.g., who, cdc, and pubmed). healthmap processes an average of . most alerts come from news media ( . %), followed by promed ( . %) and multinational agencies ( . %). the epispider project was designed in january to serve as a visualization supplement to the promed-mail reports. through use of publicly available software, epispider was able to display topic intensity of promed-mail reports on a map. additonally, epispi-der automatically converted the topic and location information of the reports into rss feeds. usage tracking showed, initially, that the rss feeds were more popular than the maps. transforming reports to a semantic online format (w c semantic web) makes it possible to combine emerging infectious disease content with similarly transformed information from other internet sites such as the global disaster alert coordinating system (gdacs) website (www.gdacs.org). the broad effects of disasters often increase illness and death from communicable diseases, particularly where resources for healthcare infrastructure have been lacking ( , ) . by merging these online media sources (promed-mail and gdacs), epispider demonstrates how distributed, event-based, unstructured media sources can be integrated to complement situational awareness for disease surveillance. epispider connects to news sites and uses natural language processing to transform free-text content into structured information that can be stored in a relational database. for promed reports, the following fi elds are extracted: date of publication; list of locations (country, province, or city) mentioned in the report; and topic. epispider parses location names from these reports and georeferences them using the georeferencing services of yahoo maps (http:// maps.yahoo.com), google maps (http://maps.google.com), and geonames (www.geonames.org). each news report that has location information can be linked to relevant demographic-and health-specifi c information (e.g., population, per capita gross domestic product, public health expenditure, and physicians/ , population). epispider extracts this information from the central intelligence agency (cia) factbook (www.cia.gov/library/publications/the-world-factbook/index.html) and the united nations development human development report (http://hdr.undp.org/en) internet sites. this feature provides different contexts for viewing emerging infectious disease information. by using askmedline ( ) , epispider also provides context-sensitive links to recent and relevant scientifi c literature for each promed-mail report topic. after epispider extracts the previously described information, it automatically transforms it to other formats, e.g., rss, keyhole markup language(kml; http://earth.google.com/ kml), and javascript object notation (json, a human-readable format for representing simple data structures; www. json.org). publishing content using those formats enables the semantic linking of promed-mail content to country information and facilitates epispider's redistribution of structured data to services that can consume them. continuing along this transformation chain, the simile exhibit api (http://simile.mit.edu) that consumes json-formatted data fi les enables faceted browsing of information by using scatter plots, google maps, and timelines. recently, epispider began outsourcing some of its preprocessing and natural language processing tasks to external service providers such as opencalais (www.opencalais.com) and the unifi ed medical language system (umls) web service for concept annotation. this action has enabled the screening of noncurated news sources as well. built on open-source software components, epispi-der has been operational since january . in response to feedback from users, additional custom data feeds have been incorporated, both topic oriented (by disease) and format specifi c (kml, rss, georss), as has semantic annotation using umls concept codes. for example, the epispider kml module was developed to enable the us directorate for national intelligence to distribute avian infl uenza event-based reports in google earth kml format to consumers worldwide and also to enable an integrated view of promed and world animal health information database reports. epispider is used by persons in north america, europe, australia, and asia, and it receives - visits/hour, originating from - sites and representing - countries worldwide. epispider has recorded daily visits from the us department of agriculture, us department of homeland security, us directorate for national intelligence, us cdc, uk health protection agency, and several universities and health research organizations. in the latter half of , daily access to graphs and exhibits surpassed access to data feeds. epispider's semantically linked data were also used for validating syndromic surveillance information in openrods (http://openrods. sourceforge.net) and populating disease detection portals, like www.intelink.gov and the research triangle institute (research triangle park, nc, usa). despite their similarities, the described event-based public health surveillance systems are highly complementary; they monitor different data types, rely on varying levels of automation and human analysis, and distribute distinct information. gphin, being the longest in use, is probably the most mature in terms of information extraction. in contrast, healthmap and epispider, being comparatively recent programs, focus on providing extra structure and automation to the information extracted. their differences and similarities, summarized in the table, can be analyzed according to multiple characteristics: what data sources do they consider? how do they extract information from those sources? and in what format is the information redistributed and how? for completeness, the broadest range of sources is critical. gphin's data comes from factiva and al bawaba, which are subscription-only news aggregators. their strategy is to rely on companies that sell the service of collecting event information from every pertinent news stream. in contrast, healthmap's strategy is to rely on open-access news aggregators (e.g., googlenews and moreover) and curated sources (e.g., promed and eurosurveillance). epispider, until recently, has concentrated on curated sources only (e.g., promed, gdacs, and cia factbook). this distinction between free and paid sources raises the question of whether the systems have access to the same event information. after the data sources have been chosen, the next step is to extract useful information among the incoming reports. first, at the level of the report stream, the system must fi lter out reports that are not disease related and categorize the remaining (disease-related) reports into predefi ned sets. then, at a second level of triage, the information within each retrieved alert (e.g., an event's location or reported disease) is assessed. gphin does this data characterization through automatic processing and human analysis, whereas healthmap and epispider rely mainly on automated techniques (although a person per- forms a daily scan of all healthmap alerts and a sample of epispider alerts). after a report in the data stream is determined to be relevant, it is processed for dissemination. gphin automatically translates the reports into different languages and grants its clients access to the database through a custom search engine. gphin also decides which reports should be raised to the status of alerts and sent to its clients by email. healthmap provides a geographic and temporal panorama of ongoing epidemics through an open-access user interface. it automatically fi lters out the reports that do not correspond to breaking alerts. the remaining alerts are prepared for display (time codes and geocodes as well as disease category and data source) to allow faceted browsing and are linked to other information sources (e.g., the wikipedia defi nition of the disease). these data are also provided as daily email digests to users interested in specifi c diseases and locations. although gphin and health-map provide their own user interface, epispider explores conventional formats for reports, adding time-coding, geocoding, and country metadata for automatic integration with other information sources and versatile browsing by using existing open-source software. these reports are displayed under the name of web exhibits and include, for example, a mapping and a timeline view of the reports and a scatter plot of the alerts with respect to the originating country's human development index and gross domestic product per capita. a division arises between the healthmap and epispi-der strategies and the gphin strategy regarding the level of access granted to users. this division is due in part to the access policies of the data sources used by the systems, as discussed previously. a discrepancy also exists in the amount of human expertise, and thus in the cost, required by the systems. these differences also raise the question of whether information from one system is more reliable than that of the others. undertaking an evaluation of the systems in parallel is a critical next step. also, all systems are inherently prone to noise because most of the data sources they use or plan to use (figure) for surveillance are not verifi ed by public health professionals, so even if the system is supervised by a human analyst, it might still generate false alerts. false alerts need to be mitigated because they might have substantial undue economic and social consequences. eventbased disease surveillance may also benefi t from algorithms linked by ontology (formal representation of a set of concepts within a domain and the relationships between those concepts) detecting precursors of disease events. measurement and handling of input data's reliability is a critical research direction. future development should focus on linking these systems more closely to public health practitioners in the fi eld and establishing collaborative networks for alert verifi cation and dissemination. such development 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and the healthmap project world wide wellness: online database keeps tabs on emerging health threats technology and public health: healthmap tracks global diseases get your daily plague forecast public health information fusion for situation awareness the threat of communicable diseases following natural disasters: a public health response infectious diseases of severe weather-related and fl ood-related natural disasters askmedline: a free-text, natural language query tool for medline/pubmed use of trade names is for identifi cation only and does not imply endorsement by the public health service or by the key: cord- -pbxd tt authors: bamias, giorgos; boletis, john title: balkan nephropathy: evolution of our knowledge date: - - journal: am j kidney dis doi: . /j.ajkd. . . sha: doc_id: cord_uid: pbxd tt balkan endemic nephropathy (ben), originally described in the late s as a chronic tubulointerstitial kidney disease, is identified by its unique epidemiological features. the most remarkable characteristic of ben is the focal topographical nature that characterizes its occurrence at the global, national, and even household level. ben affects only certain endemic rural foci along tributaries of the danube river in the balkan countries of bosnia, bulgaria, croatia, romania, and serbia. the spatial distribution has remained astonishingly unchanged with time because the disease affects the same endemic clusters as years ago. the natural course of the disease is characterized by universal development of end-stage renal disease and the frequent development of upper urinary tract tumors, posing a substantial disease burden to the afflicted areas. the greatest challenge in the study of ben has been the elucidation of its cause. the unique features of the disease, in particular its endemic nature and the long incubation period required for the disease to develop, have led to the proposal that ben represents a unique environmental disease. the quest for the responsible environmental factor has been long and diverse, and although no definitive answer has been provided to date, converging lines of evidence support the theory that long-term consumption of food contaminated with aristolochic acid underlies the pathogenesis of ben. the present review describes the evolution of our knowledge of ben in relation to the development of the main theories for its pathogenesis. i n , the first description of a novel form of interstitial kidney disease appeared in the non-english literature. the story had started a decade earlier, when local physicians noted a high prevalence of kidney disease in certain settlements in northwest bulgaria in the district of vratza. this prompted a thorough investigation by tanchev, w h o studied patients with renal disease at the regional hospital from to . he was the first to observe the remarkable clustering of the patients in villages, families, and even households. after presenting the condition at local meetings in and proposing the term "endemic vratza nephritis" in , tanchev et a l published the first detailed clinical description of the new entity in . a year later, a "family outbreak" of renal disease was noticed in the neighboring country of (what was at the time) yugoslavia; a mother and daughters had died of renal failure, and the father and son also had severe renal damage. it was soon recognized that a disease with almost identical clinical and epidemiological phenotype to the vratza nephritis existed in confined rural areas of yugoslavia. this series of original publications was completed in , as it became evident that a similar nephropathy was also prevalent in discrete regions of romania. as a consequence of these early reports, scientific conferences were organized to address the facts for the new disease. the first was held by the world health organization in , and the second, years later, by the ciba foundation. , as a result of these meetings, a novel nosological entity was recognized that was designated balkan endemic nephropathy (ben). the term epitomized the predominant features of the new disease; first, it existed in the balkans exclusively, and, second, it occurred in endemic clusters. in the following years, the clinical and epidemiological characteristics of ben were clarified. - it represents a discrete form of tubulointerstitial nephropathy with insidious presentation and slow progression. nonspecific symptoms and anemia typically develop before significant renal dysfunction. , the latter ensues eventually and manifests as reduced tubular transport, low-molecular-weight proteinuria, and an increase in serum urea nitrogen level, but no high blood pressure or edema, findings usually encountered in every form of advanced nephropathy. , histologically, interstitial fibrosis and tubular atrophy are prominent features, as opposed to the absence of significant inflammatory changes (fig ) . [ ] [ ] [ ] in the final stages, the kidneys are reduced in size, sometimes weighing as little as g and measuring just to cm. , the outcome is universally fa-tal unless renal dialysis therapy is introduced. it therefore is evident that from the clinical stand point, the new entity only added one more subcategory to the long list of interstitial renal diseases. ben would have never achieved such scientific attention and such nominated adjectives as "mysterious" and "enigmatic" if not for its unique epidemiological characteristics. from the very beginning, it was recognized that the most remarkable feature of ben is its focal nature. , [ ] [ ] [ ] [ ] [ ] at the global level, the disease has been described in balkan countries only: the aforementioned bulgaria and romania, as well as bosnia, serbia, and croatia, countries formed after the division of the former yugoslavia. on a national scale, ben cases are not distributed evenly throughout each affected country. they are strictly confined in settlements in the former yugoslavia, in bulgaria, and in southwest romania, the total area not exceeding miles in length or , km in surface (fig ) . this geographic distribution is so stable that years after the original description, no new endemic areas have been reported and no endemic areas became free from ben. all affected regions consist of villages or small towns built on the alluvial planes of tributaries of the danube river. in accord with that observation, the disease affects only rural farming populations, but never inhabitants of big cities. the focal nature of ben is so remarkably firm that afflicted villages are in close proximity to unaffected ones, only to km apart. finally, the focal nature is even preserved at the local level. that means that within an affected village, one can find diseased households that exist next to disease-free ones. in a single household, only individuals "living under the same roof and eating the same food" may be affected. however, ben does not show a preference for specific ethnic or religious groups. this was shown when croatian aboriginals were compared with ukrainian immigrants in the area of slavonski brod in regard to several parameters of renal function. it was clearly shown that the sole factor determining the presence or absence of pathological values was residency in an endemic versus a nonendemic area. apart from its focal nature, ben is also characterized by a long incubation period. for an individual to have the disease, he or she must live in the endemic area for at least to years. this probably explains why the disease has never been diagnosed in children. accordingly, a native who leaves the area before reaching the age of years is spared from developing ben. conversely, immigrants into an afflicted focus become susceptible after living there for to years. after being exposed to the risk of developing ben, it takes an equally long time before clinical manifestation occurs. therefore, the typical peak of the disease takes place between the third and fifth decades. this may explain why ben was not recognized before world war ii; the average life expectancy at the time ( to years) may simply have been too short to allow the disease to reach the clinical stage. finally, even in the first reports of ben, it was recognized that such patients were at increased risk of developing upper urinary tract tumors. [ ] [ ] [ ] [ ] this association was already known in the s, but became more pronounced in later years when the life expectancy of patients with ben increased significantly as a result of the broad use of dialysis therapy. the estimated incidence of tumors of urothelial origin has been times greater in ben endemic regions between and compared with nonendemic areas; similarly, bladder cancer was times more frequent. these epidemiological studies provided a strong link between ben and upper urinary tract tumors. it was not long after the original description of ben that the search for its cause started. for a disease with such an intriguing epidemiological profile, it was inevitable that there was room for wide speculation. as far as the inhabitants of the endemic villages were concerned, the reason was simple. the sudden misfortune that was laid upon their lives after the end of world war ii could not have any other explanation but the interference of supernatural forces, divine or otherwise. it therefore was not unexpected that they tried to calm these forces with rituals and ceremonies and protect themselves by wearing amulets. however, for the health scientists, the unique characteristics of ben offered different opportunities. this was a disease with a distinct, homogeneous phenotype, affecting a small part of the world, confined to certain recognized areas, and with all the afflicted people easily identifiable and therefore amenable to thorough investigation and research. it therefore is not unexpected that one of the first reports of ben also announced a definitive answer to its pathogenesis, because serbian investigators considered it to be the result of lead poisoning of the flour in endemic regions. ironically, years later, the precise cause of ben has not been definitely established. meanwhile, perhaps no other human disease has produced so many hypotheses in an effort to elucidate its causal factors. the quest for the cause of ben proved not to be an easy task because of many factors. first, the epidemiological data from the countries where the disease was prevalent were not of perfect quality. second, collaboration between investigators from these different countries did not take place. as a stefanovic and cosyns. ) consequence, results from studies in one country were never confirmed in the others. third, no matter how fascinating its nature may be, ben is a rare disorder affecting only some , s of people in confined balkan villages, with a practical consequence being a lack of funding resources for its study. finally, political and social events that took place in the troubled region of the balkan peninsula had their own negative effect on the efforts to find a solution to the mystery of ben. one has to keep in mind that the political tensions that followed the end of world war ii created a situation in the balkan area that made a rare nephropathy much more a local disturbance than a national priority. no matter what the obstacles were, research about the causative factor(s) of ben started almost immediately after its recognition as a discrete nosological entity. for a disease with such a narrow, defined, and well-preserved geographic distribution, it was a logical assumption that the causative factor should be an environmental one endemic to the afflicted regions. nonetheless, along with this ecological influence, other factors had to be taken into consideration. for example, an obvious question was how could such an environmental trigger selectively affect certain villages and households and leave the ones just next to them undisturbed? could these differences be explained by the existence of genetic variability between diseased and healthy families? or could it be an infectious agent prevalent to the inflicted foci? another pivotal question related to the long incubation period of the disease, which pointed to a long exposure to a low-dose harming agent that existed in the environment for many years. if that is the case, why then did ben appear only during the second half of the century in the endemic areas and not before? such a temporal association requires a major local environmental change before the first appearance of the disease, and such an alteration is not easy to identify in the history of the region. in addition, any etiologic hypothesis should take into consideration not only the nephropathy itself, but also the increased incidence of upper urothelial neoplasms in the patients. finally, if somebody were to solve the mystery of ben, he or she should use information from both animal models of interstitial nephropathy and the recognized effects of the candidate environmental factors in human health and disease. given all these varied questions, it is not surprising that throughout the years, the effort to solve the riddle of ben involved not only nephrologists, but also scientists from such diverse fields as occupational medicine, animal models of human disease, environmental sciences and epidemiology, oncology, genetics, and geology. one of the first comprehensive hypotheses regarding the pathogenesis of ben appeared in the literature in the early s. akhmeteli and then krogh proposed that ben was the result of contamination of the food chain in endemic ar-eas by ochratoxin a (ota), a toxic product of molds that belong to the aspergillus or penicillium fungal genera. this "mycotoxin" theory was built around the remarkable similarity between ben and porcine nephropathy. the latter has a similar confined geographic distribution because it mainly occurs in northern europe and shares many pathological characteristics with ben. porcine nephropathy is caused by ota, raising the possibility for a similar connection between ben and mycotoxins. therefore, an effort to provide evidence for such an association was started. from a general point of view, the establishment of a cause-and-effect link between an environmental factor and a disease, the so-called exposure analysis, consists of a stepwise approach. , briefly, the agent has to be present in an endemic area and in quantities large enough to induce health damage. in addition, there should be evidence regarding how the offending agent moves from the environmental source to the human organism. moreover, topographical specificity should exist, meaning that the agent must be more prevalent in endemic compared with nonendemic regions. finally, if the same agent occurs in other areas of the world, a similar disease would be expected to be found there as well. if not, an explanation should be offered. the application of these principles in the case of the ochratoxin/ben association has produced ambivalent results. contamination of food with ota is very frequent, and widespread exposure is common in several places in the world, including western nations. , some, but not all, studies have detected greater levels of ota in the foodstuff consumed in endemic versus nonendemic areas, as well as in affected versus nonaffected households. , - other studies reported that patients with ben have increased serum and urine concentrations of ota, indicating greater consumption of the mycotoxin. it therefore could be the case that (although the contamination of the food chain is ubiquitous) patients with ben are exposed to greater quantities of ota as a result of some still unidentified practice in food processing and consumption. nonetheless, it was shown in the same studies that the variation in ota values within the affected population was very high and often overlapped greatly with values from nonendemic areas of the world. moreover, there was no constant association between consumption of ota and its levels in serum or urine. a strong argument against the mycotoxin theory is that ota had never been linked to any type of nephropathy in humans. however, this argument may be subject to challenge after the proposal in the s that other forms of interstitial nephropathy may be associated with ota: the first is endemic in tunisia, , and the second is the karyomegalic interstitial nephropathy. both types of renal disease share clinicopathologic similarities with ben and support the hypothesis that ota toxicity may underlie the pathogenesis of the latter, as well. the evidence for the role of ota as the causative factor for ben was critically reviewed in a recent international symposium held in zagreb. a series of arguments against such a role were presented; the strongest were the uneven geographic distribution of food contamination with ota and ben and the lack of definitive proof for ota-dna adduct formation. a different pathogenetic hypothesis was proposed by kazantzis in during the conference on ben organized by the ciba foundation. he claimed that contamination of the baking flour in endemic areas by seeds of the birthwort aristolochia clematitis was causing the disease (fig ) . apparently, a toxic constituent was contained in the seeds of the plant and induced the renal damage. the initial exploration of this theory is attributed to ivić, who analyzed available data and performed field and laboratory studies to provide a well-documented hypothesis on the pathogenesis of ben. again, animal studies set the background. ivić was aware of publications of such croatian scientists as dumic and martincic reporting that horses consuming hay that contained seeds of a clematitis experienced renal disease with proteinuria. more strikingly, when kidneys from these horses were examined histologically, they showed tubulointerstitial damage with minimal inflammation, a picture that was analogous to ben. , to prove his hypothesis, ivić performed field studies and observed that seeds from a clematitis were inter-spersed among wheat grains during the harvest. the local villagers did not make an effort to remove the contaminants during flour preparation. because bread is the major constituent of the local diet, contamination of the wheat with even a few seeds of a clematitis could result in low-dose long-term intoxication with the offending agent. continuing his sound scientific approach, ivić went on and fed rabbits with flour prepared from a clematitis seeds. the rabbits developed nephropathy, which, at the histological level, resembled the findings of ben. ivić even proved the carcinogenetic potential of the plant because rats developed sarcomas at the site of injection of aqueous extracts of a clematitis. it is surprising that ivić's thorough approach and well-documented results failed to attract more interest from the scientific community until many years later. however, this was to change in , when many cases of chinese herb nephropathy erupted. several hundred young belgian women developed end-stage renal disease after receiving slimming pills at a single medical clinic in brussels. the regimen contained chinese herbs, and it eventually was proved that it was contaminated with aristolochic acid, a main toxic product of aristolochia species. its presence in the slimming regimen was the result of accidental substitution of the prescribed herb stephania tetrandra ('han fang-ji') by aristolochia fangchi ('guang fang-ji'). therefore, the name of the belgian disorder changed to chinese herb nephropathy, or aristolochic acid nephropathy. it was soon realized that this disease had many similarities to ben, especially in the morphological characteristics, which were almost identical in the conditions. this similarity suggested that the same factor, ie, aristolochic acid, could be responsible for both conditions. this association was supported further by the development of renal failure in japanese patients receiving an aristolochic acidcontaining remedy for atopic dermatitis. interestingly, upper urothelial neoplasms also developed in some patients with chinese herb nephropathy. , these intriguing reports renewed scientific interest and stimulated new research that resulted in strong evidence supporting a dominant role for aristolochic acid in the pathogenesis of ben. after its metabolic activation, aristolochic acid reacts with dna to generate covalent aristolactam-dna adducts. some forms of these adducts are stable and can be detected in the affected tissues. therefore, if aristolochic acid were to blame for ben, renal tissue from these patients should contain such adducts. arlt et al were the first to prove that this hypothesis is correct by detecting aristolochic acid-specific adducts in all urinary tract tissues from patients with ben that they examined. conversely, ota-specific adducts were detected with much lower density and in only some of the examined tissues. recently, work by grollman et al , expanded these results by showing that aristolochic acidspecific adducts were present in the renal cortex of patients with ben from an endemic region in croatia, but not in patients with other forms of chronic renal disease or patients with upper urinary tract transitional cell cancer living in a nonendemic area of croatia. in addition, when a p mutational spectra analysis was performed in urothelial cancer specimens from patients living in endemic foci, a "signature" mutation (a:t¡t:a) was detected. interestingly, this molecular mark is also induced by aristolochic acid in animal models of carcinogenesis and was observed in one patient with aristolochic acid nephropathy and ureteral tumor. therefore, aristolochic acid appears to be responsible not only for the development of renal injury, but also for the high incidence of urothelial cancer in patients with ben. a recent publication by lemy et al provides convincing evidence for the carcinogenic potential of aristolochic acid. this was a case series in belgian patients undergoing renal transplantation for end-stage renal failure caused by slimming pill-related aristolochic acid nephropathy. in the pathological examination of the resected specimens, upper-tract urothelial carcinoma was diagnosed in % of patients. more interestingly, on long-term follow-up, there was a % incidence of bladder carcinoma. it was concluded from this study that the pathogenic properties of aristolochic acid exist for a long time after cessation of exposure. to date, the aristolochic acid hypothesis perhaps offers the best-characterized model for the pathogenesis of ben. , the pivotal work of ivić combined with modern state-of-theart biochemical and molecular techniques have promulgated a pathogenetic concept that explains most of the epidemiological and clinical parameters of ben. however, a critical question still is unanswered; why do only % to % of the residents in an endemic area develop the disease? this cannot be attributed easily to preferential exposure of such a small minority of the population to aristolochic acid, but it could result from genetic polymorphisms. nevertheless, if the aristolochic acid hypothesis is true, the most important clinical conclusion is that ben is preventable with simple measures. one can safely predict that contamination of the flour with a clematitis seeds has already been eliminated as a result of the changing ways of life and work in the endemic areas. the widespread use of herbicides and use of new harvesting techniques has led to decreased growth of a clematitis in the harvesting fields. nowadays, fewer and fewer families bake their own bread, rendering the making of contaminated flour highly improbable. if this hypothesis proves to be true, we can be optimistic that a decrease in the incidence of ben should be expected in the years ahead. no review of the evolution of our knowledge of ben is complete if it does not address the so-called "lignite hypothesis." this theory was developed in the s by scientists of the us geological survey. , it originated from the primary observation that there was a spatial relationship between the endemic villages with ben and the locations of pliocene lignite deposits in the balkans. all endemic areas are in close vicinity to low rank coals. the lignite theory claims that toxic organic substances from these coals leak into groundwater and are transported to the wells that exist in the alluvial valleys, below the lignite deposits. this was shown to be true because studies reported greater concentrations of these organic compounds in the water from wells in the endemic areas compared with those in nonendemic ones. the final link in the exposure chain is that people in the villages use water from their wells for drinking and cooking purposes. , , in line with this, settlements where most inhabitants use the same limited number of wells show hyperendemicity for ben. the presence of small concentrations of organic toxic products in the water of the wells is compatible with a slow low-level, but relentless, poisoning of the villagers, a concept that fits very well with the natural history of ben. the increased incidence of urothelial cancers also can be explained by this theory because these toxic organics are well-known carcinogenic factors. maybe the most impressive application of the lignite hypothesis is that it may be used as a predictive model. an area in serbia that was not known to be endemic was proved to be such after it was recognized to lay close to a low rank coal field. research on the role of the pliocene lignites in the pathogenesis of ben required a lot of field studies and thus it was abruptly interrupted when the war broke out in yugoslavia in . although the environmental factor(s)-centered theories dominated the efforts to explain the etiopathogenesis of ben, alternative explanations were sought, as well. the infection theory claims that ben is caused by a coronavirus. uzelac-keserović et al developed epithelial cell monolayer cultures from kidney biopsy specimens obtained from patients with ben and control patients. they were able to detect a novel virus, named the ebn virus, that was present in ben-derived epithelial cell cultures, but from none of the control cultures. it was proposed that the microorganism was a novel coronavirus based on its cross-reactivity with human coronaviruses oc and e, as well as a pig coronavirus known as transmissible gastroenteritis virus (tgev). when seroreactivity to antigens from the new coronavirus was tested, it was shown to be very high in patients with ben on dialysis therapy ( % by neutralization activity and % by immunofluorescent assay, respectively) and controls from endemic areas ( % and %), but not in controls from nonendemic regions ( . % and . %). nonetheless, the coronavirus theory was put in dispute when vero cells infected with ebn virus were tested further with various methods, including electron microscopy studies. no similarities between ebn virus and coronaviruses were observed in that study. the strict clustering of ben cases in selected families has raised the possibility that genetic factors may determine its clinical and epidemiological profile. this theory was established by detailed family investigations in bulgarian patients with ben. after studying , patients from ben-affected families, toncheva et al were led to the conclusion that all patients with ben belong to certain families. even residents of nonendemic foci who were found to have ben were identified to be members of ben families and to have moved from their birthplaces. in addition, ben shows some epidemiological characteristics typical of genetic disorders. for example, the proportion of sick offspring increases according to the number of parents affected. accordingly, the risk of developing the disease is much greater in first-degree than second-degree relatives and decreases substantially in remote relatives. to further elaborate the genetic component of ben, cytogenetic studies were performed by the same group of scientists. they pro-posed that a specific ben-associated locus exists in q combined with instability of the long arm of chromosome . , interestingly, this alteration in q may also determine the genetic susceptibility for the development of the disease in relatives of patients with ben. additional data generated from the same group link the increased prevalence of urothelial neoplasms that occurs in patients with ben to genetically determined aberrations in oncogenesis. patients with ben were tested for chromosomal aberrations induced by x-rays or folic acid deprivation. there was increased frequency of the aforementioned abnormality in q , but also in other areas that all contain oncogenes, namely c-src (csk, q ), raf- (raf , p ), and myb (myb, q ). it has been estimated that , people are at risk of ben, whereas , have the disease. it is not clear what the current trend for ben incidence is because results from studies performed in different endemic areas produced conflicting information. some epidemiological reports reported an increase between and , a plateau between and , and a final decrease in disease prevalence in some endemic areas. similarly, a decreasing incidence with time was found in another endemic area during a surveillance period between and . nevertheless, for certain endemic regions, ben continues to pose a major health problem, and it seems that the incidence of new cases remains stable over time. probably, these differences are related to differences in the study design or true epidemiological differentiation between endemic areas. fifty years after its original description, ben has shown impressive stability in regard to its epidemiological profile and clinical phenotype. although this stability initially generated optimism for the identification of the causative agent(s), this proved to be a much more difficult task. the most widely accepted theories have failed to date to answer the main critical question: what is the basis for the focal-topographical nature of ben? ben is definitely not a genetic disorder in that it does not follow a pattern of mendelian inheritance. conversely, ben clusters in familial foci, indicating exposure to a common environmental offense. nevertheless, it is difficult to appreciate which environmental agent could be so selective as to affect only a small minority of closely residing individuals. in regard to that question, one needs to consider the possibility of ben-like diseases existing in other parts of the world. supporters of the mycotoxins or the lignite hypothesis have already proposed that ben is part of and may be the most obvious example of a panendemic nephropathy that takes place in locations characterized by the contamination of the food or water supplies by ota or coal-derived organic toxic substances, respectively. , as the case is for most human diseases, it is likely that ben is a multifactorial disorder. an environmental factor probably is superimposed on a certain genetic background to create the phenotype of the disease. a triggering agent must exist in the environment in sufficient quantity and with an available exposure route that allows it to be introduced to the human host. there should be a defined population focus isolated enough to render exposure to the agent to be universal in that population and constant. there should be stability of the population at risk, so that individuals are exposed for a long period and an average lifespan that should far exceed years of age, allowing for the long incubation time of the disease. finally, there should be a health care system with the ability for proper establishment of the diagnosis. it therefore is possible that the combination of all these prerequisites in the post-world war ii balkans resulted in the identification of ben. from this perspective, it can be said that ben represents a unique geophysical experiment in which all the components must meld for the disease to develop. the optimistic view from that perspective is that only one of these factors needs to be eliminated for the disease to disappear. we speculate that certain changes in the lifestyle of the people, agricultural practices, or dietary habits may remove one or more cofactor(s) for the development of ben. such a scenario eventually would eliminate the burden of this unfortunate condition from the balkans. studies on the nephritides in the district of vratza porodicna oboljenia bubrega u selu sopic izvazvana hronicnom intoksikacijom olovom world health organization: memorandum: the endemic nephropathy of south-eastern europe ciba foundation study group no. : the balkan nephropathy. boston, ma, ciba foundation clinical features of balkan endemic nephropathy djukanović l: erythropoietin and anemia in the progression of balkan endemic nephropathy and other renal diseases creatinine clearance and kidney size in balkan endemic nephropathy patients some pathomorphological features of balkan endemic nephropathy in croatia chernozemsky in, stoyanov is, petkova-bocharova tk, et al: geographic correlation between the occurrence of endemic nephropathy and urinary tract tumours in vratza district maaroufi k, achour a, betbeder am, et al: foodstuffs and human blood contamination by the mycotoxin ochratoxin a: correlation with chronic interstitial nephropathy in tunisia is ochratoxin a a nephrotoxic in human beings? kazantzis g: comment in the general discussion: possible nephrotoxic agents ivić m, lovriae b: carcinogenic action of aristolochia rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including chinese herbs urothelial carcinoma associated with the use of a chinese herbs analyses of dna adducts formed by ochratoxin a and aristolochic acid in patients with chinese herbs nephropathy aristolochic acid and the etiology of endemic (balkan) nephropathy late onset of bladder urothelial carcinoma after kidney transplantation for end-stage aristolochic acid nephropathy: a case series with -year follow-up petropoulos ea: evaluation of the hypothesis that balkan endemic nephropathy is caused by drinking water exposure to contaminants leaching from pliocene coal deposits isolation of a coronavirus from kidney biopsies of endemic balkan nephropathy patients stefanović v: balkan endemic nephropathy: a need for novel aetiological approaches epidemiology of endstage renal disease and current status of hemodialysis in yugoslavia health effects of toxic organic substances from coal: toward "panendemic" nephropathy acknowledgements support: none. financial disclosure: none. key: cord- -pz lsaw authors: rodrigues, helena sofia title: optimal control and numerical optimization applied to epidemiological models date: - - journal: nan doi: nan sha: doc_id: cord_uid: pz lsaw the relationship between epidemiology, mathematical modeling and computational tools allows to build and test theories on the development and battling of a disease. this phd thesis is motivated by the study of epidemiological models applied to infectious diseases in an optimal control perspective, giving particular relevance to dengue. dengue is a subtropical and tropical disease transmitted by mosquitoes, that affects about million people per year and is considered by the world health organization a major concern for public health. the mathematical models developed and tested in this work, are based on ordinary differential equations that describe the dynamics underlying the disease, including the interaction between humans and mosquitoes. an analytical study is made related to equilibrium points, their stability and basic reproduction number. the spreading of dengue can be attenuated through measures to control the transmission vector, such as the use of specific insecticides and educational campaigns. since the development of a potential vaccine has been a recent global bet, models based on the simulation of a hypothetical vaccination process in a population are proposed. based on optimal control theory, we have analyzed the optimal strategies for using these controls, and respective impact on the reduction/eradication of the disease during an outbreak in the population, considering a bioeconomic approach. the formulated problems are numerically solved using direct and indirect methods. the first discretize the problem turning it into a nonlinear optimization problem. indirect methods use the pontryagin maximum principle as a necessary condition to find the optimal curve for the respective control. in these two strategies several numerical software packages are used. a relação entre a epidemiologia, a modelação matemática e as ferramentas computacionais permite construir e testar teorias sobre o desenvolvimento e combate de uma doença. esta tese tem como motivação o estudo de modelos epidemiológicos aplicados a doenças infeciosas numa perspetiva de controloÓtimo, dando particular relevância ao dengue. sendo uma doença tropical e subtropical transmitida por mosquitos, afecta cerca de milhões de pessoas por ano, eé considerada pela organização mundial de saúde como uma grande preocupação para a saúde pública. os modelos matemáticos desenvolvidos e testados neste trabalho, baseiamse em equações diferenciais ordinárias que descrevem a dinâmica subjacentè a doença nomeadamente a interação entre humanos e mosquitos.É feito um estudo analítico dos mesmos relativamente aos pontos de equilíbrio, sua estabilidade e número básico de reprodução. a propagação do dengue pode ser atenuada através de medidas de controlo do vetor transmissor, tais como o uso de inseticidas específicos e campanhas educacionais. como o desenvolvimento de uma potencial vacina tem sido uma aposta mundial recente, são propostos modelos baseados na simulação de um hipotético processo de vacinação numa população. tendo por base a teoria de controloÓtimo, são analisadas as estratégiasótimas para o uso destes controlos e respetivas repercussões na redução/erradicação da doença aquando de um surto na população, considerando uma abordagem bioeconómica. os problemas formulados são resolvidos numericamente usando métodos diretos e indiretos. os primeiros discretizam o problema reformulando-o num problema de optimização não linear. os métodos indiretos usam o princípio do máximo de pontryagin como condição necessária para encontrar a curvá otima para o respetivo controlo. nestas duas estratégias utilizam-se vários pacotes de software numérico. ao longo deste trabalho, houve sempre um compromisso entre o realismo dos modelos epidemiológicos e a sua tratabilidade em termos matemáticos. introduction "mathematical biology is a fast-growing, well-recognized, albeit not clearly defined, subject and is, to my mind, the most exciting modern application of mathematics." -j. d. murray, mathematical biology, epidemiology has become an important issue for modern society. the relationship between mathematics and epidemiology has been increasing. for the mathematician, epidemiology provides new and exciting branches, while for the epidemiologist, mathematical modeling offers an important research tool in the study of the evolution of diseases. in , a smallpox model was proposed by daniel bernoulli and is considered by many authors the first epidemiological mathematical model. theoretical papers by kermack and mckendrinck, between and about infectious disease models, have had a great influence in the development of mathematical epidemiology models [ ] . most of the basic theory had been developed during that time, but the theoretical progress has been steady since then [ ] . mathematical models are being increasingly used to elucidate the transmission of several diseases. these models, usually based on compartment models, may be rather simple, but studying them is crucial in gaining important knowledge of the underlying aspects of the infectious diseases spread out [ ] , and to evaluate the potential impact of control programs in reducing morbidity and mortality. after the second world war, the strategy of public health has been focusing on the control and elimination of the organisms that cause the diseases. the appearance of new antibiotics and vaccines brought a positive perspective of the diseases eradication. however, factors such as resistance to the medicine by the microorganisms, demographic evolution, accelerated urbanization, increased travelling and climate change, led to new diseases and the resurgence of old ones. in , the human immunodeficiency virus (hiv) appears and since then, become as important sexually transmitted disease throughout the world [ ] . futhermore, malaria, tuberculosis, dengue and yellow fever have re-emerged and, as a result of climate changes, has been spreading into new regions [ ] . recent years have seen an increasing trend in the representation of mathematical models in publications in the epidemiological literature, from specialist journals of medicine, biology and mathematics to the highest impact generalist journals [ ] , showing the importance of interdisciplinary. their role in comparing, planning, implementing and evaluating various control programs is of major importance for public health decision makers. this interest has been reinforced by the recent examples of sars -severe acute respiratory syndrome -epidemic in and influenza pandemic in . although chronic diseases, such as cancer and heart diseases have been receiving more attention in developed countries, infectious diseases are still important and cause suffering and mortality in developing countries. these, remain a serious medical burden all around the world with million deaths per year estimated to be directly related to infectious diseases [ ] . the successful containment of the emerging diseases is not just linked to medical infrastructure but also on the capacity to recognize its transmission characteristics and apply optimal medical and logistic policies. public health often asks information such as [ ] : how many people will be infected, how many require hospitalization, what is the maximum number of people ill at a given time and how long will the epidemic last. as a result, it is necessary an ever-increasing capacity for a rapid response. education, vaccination campaigns, preventive drugs administration and surveillance programs, are all examples of prevention methods that authorities must consider for disease prevention. whenever the disease declares itself, the emergency interventions such as disinfectants, insecticide application, mechanical controls and quarantine measures must be considered. intervention strategies can be modelled with the goal of understanding how they will influence the disease's battle. as financial resources are limited, there is a pressing need to optimize investments for disease prevention and fight. traditionally, the study of disease dynamics has been focused on identifying the mechanisms responsible for epidemics but has taken little into account economic constraints in analyzing control strategies. on the other hand, economic models have given insight into optimal control under constraints imposed by limited resources, but they are frequently ignored by the spatial and temporal dynamics of the disease. therefore, progress requires a combination of epidemiological and economic factors for modelling what until here tended to remain separate. more recently, bioeconomic approaches to disease management have been advocated, since infectious diseases can be modelled thinking that the limited resources involved require trade-offs. finding the optimal strategy depends on the balance of economic and epidemiological parameters that reflect the nature of the host-pathogen system and the efficiency of the control method. the main goal of this thesis is to formulate epidemiological models, giving a special importance to dengue disease. moreover, it is our aim to frame the disease management question into an optimal control problem requiring the maximization/minimization of some objective function that depends on the infected individuals (biological issues) and control costs (economic issues), given some initial conditions. this way, will allow us to propose practical control measures to the authorities to assess and forecast the disease burden, such as an attack rate, morbidity, hospitalization and mortality. the thesis is composed by two parts. the first part, comprising chapters to , gives a mathematical background to support the original results presented in the second part, that is composed by chapters to . in chapter , the definition of optimal control problem, its possible versions and the adapted first order necessary conditions based on the pontryagin maximum principle are introduced. simple examples are chosen to exemplify the mathematical concepts. with the increasing of variables and complexity, optimal control problems can no longer be solved analytically and numerical methods are required. for this purpose, in chapter , direct and indirect methods are presented for their resolution. direct methods consist in the discretization of the optimal xii control problem, reducing it to a nonlinear constrained optimization problem. indirect methods are based on the pontryagin maximum principle, which in turn reduces the problem to a boundary value problem. for each approach, the software packages used in this thesis are described. in chapter , the basic building blocks of most epidemiological models are reviewed: sir (composed by susceptible-infected-recovered) and sis models (susceptible-infected-susceptible). for these, it is possible to develop some analytical results which can be useful in the understanding of simple epidemics. taking this as the basis, we discuss the dynamics of other compartmental models, bringing more complex realities, such as those with exposed or carrier classes. the second part of the thesis contains the original results and is focused on dengue fever disease. dengue is a vector borne disease, caused by a mosquito from the aedes family. it is mostly found in tropical and sub-tropical climates, mostly in urban areas. it can provoke a severe flu-like illness, and sometimes, in severe cases can be lethal. according to the world health organization about % of the world's population is now at risk [ ] . the main reasons for the choice of this particular disease are: • the importance of this disease around the world, as well as the challenges of its transmission features, prevention and control measures; • two portuguese-speaking countries (brazil and cape verde) have already experience with dengue, and in the last one, a first outbreak occurred during the development of this thesis, which allowed the development of a groundbreaking work; • the mosquito aedes aegypti, the main vector that transmits dengue, is already in portugal, on madeira island [ ] , which without carrying the disease, is considered a potential threat to public health and has been followed by the health authorities. in chapter information about the mosquito, disease symptoms, and measures to fight dengue are reported. an old problem related to dengue is revisited and solved by different approaches. finally, a numerical study is performed to compare different discretization schemes in order to analyze the best strategies for future implementations. in chapter , a seir+asei model is studied. the basic reproduction number and the equilibrium points are computed as well as their relationship with the local stability of the disease free equilibrium. this model implements a control measure: adulticide. a study to find the best strategies available to apply the insecticide is made. continuous and piecewise constant strategies are used involving the system of ordinary differential equations only or resorting to the optimal control theory. chapter is concerned with a sir+asi model that incorporates three controls: adulticide, larvicide and mechanical control. a detailed discussion on the effects of each control, individually or together, on the development of the disease is given. an analysis of the importance of each control in the decreasing of the basic reproduction number is given. these results are strengthened when the optimal strategy for the model is calculated. bioeconomic approaches, using distinct weights for the respective control costs and treatments for infected individuals have also been provided. in chapter simulations for a hypothetical vaccine for dengue are carried out. the features of the vaccine are unknown because the clinical trials are ongoing. using models with a new compartment for vaccinated individuals, perfect and imperfect vaccines are studied aiming the analysis of the repercussions of the vaccination process in the morbidity and/or eradication of the disease. then, an optimal control approach is studied, considering the vaccination not as a new compartment, but as a measure control in fighting the disease. finally, the main conclusions are reported and future directions of research are pointed out. xiv part i state of the art chapter the optimal control definition and its possible formulations are introduced, followed by some examples related to epidemiological models. the pontryagin maximum principle is presented with the aim of finding the best control policy. optimal control (oc) is the process of determining control and state trajectories for a dynamic system over a period of time in order to minimize a performance index [ ] . historically, oc is an extension of the calculus of variations. in the seventeenth century, the first formal results of calculus of variations can be found. johann bernoulli challenged other famous contemporary mathematicians -such as newton, leibniz, jacob bernoulli, l'hôpital and von tschirnhaus -with the brachistochrone problem: "if a small object moves under the influence of gravity, which part between two fixed points enables it to make the trip in the shortest time?" other specific problems were solved and a general mathematical theory was developed by euler and lagrange. the most fruitful applications of the calculus of variations have been to theoretical physics, particularly in connection with hamilton's principle or the principle of least action. early applications to economics appeared in the late s and early s by ross, evans, hottelling and ramsey, with further applications published occasionally thereafter [ ] . the generalization of the calculus of variations to optimal control theory was strongly motivated by military applications and has developed rapidly since . the decisive breakthrough was achieved by the russian mathematician lev s. pontryagin ( pontryagin ( - and his co-workers (v. g. boltyanskii, r. v. gamkrelidz and e. f. misshchenko) with the formulation and demonstration of the pontryagin maximum principle [ ] . this principle has provided research with suitable conditions for optimization problems with differential equations as constraints. the russian team generalized variational problems by separating control and state variables and admitting control constraints. in such problems, oc gives equivalents results, as one would have expected. however, the two approaches differ and the oc approach sometimes affords insight into a problem that might be less readily apparent through the calculus of variations. oc is also applied to problems where the calculus of variations is not convenient, such as those involving constraints on the derivatives of functions [ ] . the theory of oc brought new approaches to mathematics with dynamic programming. introduced by r. e. bellman, dynamic programming makes use of the principle of optimality and it is suitable for solving discrete problems, allowing for a significant reduction in the computation of the optimal controls (see [ ] ). it is also possible to obtain a continuous approach to the principle of optimality that leads to the solution of a partial differential equation called the hamilton-jacobi-bellman equation. this result allowed to bring new connections between the oc problem and the lyapunov stability theory. before the arrival of the computer, only fairly simple the oc problems could be solved. the arrival of the computer age enabled the application of oc theory and some methods to many complex problems. selected examples are as follows: • physical systems, such as stable performance of motors and machinery, robotics, optimal guidance of rockets [ , ] ; • aerospace, including driven problems, orbits transfers, development of satellite launchers and recoverable problems of atmospheric reentry [ , ] ; • economics and management, such as optimal exploitation of natural resources, energy policies, optimal investment of production strategies [ , ] ; • biology and medicine, as regulation of physiological functions, plants growth, infectious diseases, oncology, radiotherapy [ , , , ] . today, the oc theory is extensive and with several approaches. one can adjust controls in a system to achieve a goal, where the underlying system can include: ordinary differential equations, partial differential equations, discrete equations, stochastic differential equations, integro-difference equations, combination of discrete and continuous systems. in this work the goal is the oc theory of ordinary differential equations with time fixed. a typical oc problem requires a performance index or cost functional (j[x(t), u(t)]), a set of state variables (x(t) ∈ x), a set of control variables (u(t) ∈ u ) in a time t, with t ≤ t ≤ t f . the main goal consists in finding a piecewise continuous control u(t) and the associated state variable x(t) to maximize a given objective functional. the development of this chapter will be closely structured from lenhart and workman work [ ] . x(t f ) could be free, which means that the value of x(t f ) is unrestricted, or could be fixed, i.e, x(t f ) = x f . for our purposes, f and g will always be continuously differentiable functions in all three arguments. we assume that the control set u is a lebesgue measurable function. thus, as the control(s) will always be piecewise continuous, the associated states will always be piecewise differentiable. we have been focused on finding the maximum of a function. we can switch back and forth between maximization and minimization by simply negating the cost functional: an oc problem can be presented in different ways, but equivalent, depending on the purpose or the software to be used. there are three well known equivalent formulations to describe the oc problem, which are lagrange (already presented in previous section), mayer and bolza forms [ , ] . definition (bolza formulation). the bolza formulation of the oc problem can be defined as where φ is a continuously differentiable function. definition (mayer formulation). the mayer formulation of the oc problem can be defined as proof. ( ) ⇒ ( ) to get the proof, we formulate the bolza problem as one of lagrange, using an extended state vector. let (x(·), u(·)) an admissible pair for the problem ( . ) and let z(t) = (x a (t), . so, (z(·), u(·)) is an admissible pair for the lagrange problem thus, the value of the functionals in both formulations matches. ( ) ⇒ ( ) conversely, each admissible pair (z(·), u(·)) for the problem ( . ) corresponds the pair (x(·), u(·)), where x(·) is composed by the last component of z, admissible for the problem ( . ) and matching the respective values of the functionals. ( ) ⇒ ( ) for this statement we also need to use an extended state vector. let be thus we have (z(·), u(·)) an admissible pair for the following mayer problem: this way, the values of the functional for both formulations are the same. ( ) ⇒ ( ) conversely, to all admissible pair (z(·), u(·)) for the mayer problem ( . ) corresponds to an admissible pair (x(·), u(·)) for the bolza problem ( . ) , where x(·) consists in the last component of z(·). for the proof of the previous theorem it was not necessary to show that the lagrange problem is equivalent to the mayer formulation. however, in the second part of the thesis, and due to computational issues, some of the oc problems (usually presented in the lagrange form) will be converted into the equivalent mayer one. hence, using a standard procedure is possible to rewrite the cost functional (cf. [ ] ), augmenting the state vector with an extra component. so, the lagrange formulation ( . ) can be rewritten as max the necessary first order conditions to find the optimal control were developed by pontryagin and his co-workers. this result is considered as one of the most important results of mathematics in the th century. pontryagin introduced the idea of adjoint functions to append the differential equation to the objective functional. adjoint functions have a similar purpose as lagrange multipliers in multivariate calculus, which append constraints to the function of several variables to be maximized or minimized. definition (hamiltonian). let the previous oc problem considered in ( . ). the function h(t, x(t), u(t), λ(t)) = f (t, x(t), u(t)) + λ(t)g(t, x(t), u(t)) if u * (t) and x * (t) are optimal for problem ( . ), then there exists a piecewise differentiable adjoint variable λ(t) such that for all controls u at each time t, where h is the hamiltonian previously defined and proof. the proof of this theorem is quite technical and we opted to omit it. the original pontryagin's text [ ] or clarke's book [ ] are good references to find the proof. remark . the last condition, λ(t f ) = , called transversality condition, is only used when the oc problem does not have terminal value in the state variable, i.e., x(t f ) is free. this principle converted the problem of finding a control which maximizes the objective functional subject to the state ode and initial condition into the problem of optimizing the hamiltonian pointwise. as consequence, with this adjoint equation and hamiltonian, we have at u * for each t, namely, the hamiltonian has a critical point; usually this condition is called optimality condition. thus to find the necessary conditions, we do not need to calculate the integral in the objective functional, but only use the hamiltonian. here is presented a simple example to illustrate this principle. example (from [ ] ). consider the oc problem: the calculus of this oc problem can be done by steps. step -form the hamiltonian for the problem. the hamiltonian can be written as: step -write the adjoint differential equation, the optimality condition and transversality boundary condition (if necessary). try to eliminate u * by using the optimality equation h u = , i.e., solve for u * in terms of x * and λ. using the hamiltonian to find the differential equation of the adjoint λ, we obtained the optimality condition is given by in this way we obtain an expression for the oc: as the problem has just an initial condition for the state variable, it is necessary to calculate the transversality condition: λ( ) = . step -solve the set of two differential equations for x * and λ with the boundary conditions, replacing u * in the differential equations by the expression for the optimal control from the previous step. by the adjoint equation λ = − − λ and the transversality condition λ( ) = we have hence, the optimality condition leads to u * = −λ ⇔ u * = − e −t and the associated state is if the hamiltonian is linear in the control variable u, it can be difficult to calculate u * from the optimality equation, since ∂h ∂u would not contain u. specific ways of solving these kind of problems can be found in [ ] . until here we have showed necessary conditions to solve basic optimal control problems. now, it is important to study some conditions that can guarantee the existence of a finite objective functional value at the optimal control and state variables, based on [ , , , ] . the following is an example of a sufficient condition result. suppose that f (t, x, u) and g(t, x, u) are both continuously differentiable functions in their three arguments and concave in x and u. suppose u * is a control with associated state x * , and λ a piecewise differentiable function, such that u * , x * and λ together satisfy on t ≤ t ≤ t f : then for all controls u, we have proof. the proof of this theorem is available on [ ] . this result is not strong enough to guarantee that j(u * ) is finite. such results usually require some conditions on f and/or g. next theorem is an example of an existence result from [ ] . theorem . . . let the set of controls for problem ( . ) be lebesgue integrable functions on t ≤ t ≤ t f in r. suppose that f (t, x, u) is convex in u, and there exist constants c , c , c > , c and β > such that then there exists an optimal control u * maximizing j(u), with j(u * ) finite. proof. the proof of this theorem is available on [ ] . for a minimization problem, g would have a concave property and the inequality on f would be reversed. note that the necessary conditions developed to this point deal with piecewise continuous optimal controls, while this existence theorem guarantees an optimal control which is only lebesgue integrable. this disconnection can be overcome by extending the necessary conditions to lebesgue integrable functions [ , ] , but we did not expose this idea in the thesis. see the existence of oc results in [ ] . in some cases it is necessary, not only minimize (or maximize) terms over the entire time interval, but also minimize (or maximize) a function value at one particular point in time, specifically, the end of the time interval. there are some situations where the objective function must take into account the value of the state at the terminal time, e.g., the number of infected individuals at the final time in an epidemic model [ ] . definition (oc problem with payoff term). an oc problem with payoff term is in the form where φ(x(t f )) is a goal with respect to the final position or population level x(t f ). the term φ(x(t f )) is called payoff or salvage. using the pmp, adapted necessary conditions can be derived for this problem. proposition (necessary conditions). if u * (t) and x * (t) are optimal for problem ( . ), then there exists a piecewise differentiable adjoint variable λ(t) such that for all controls u at each time t, where h is the hamiltonian previously defined and proof. the proof of this result can be found in [ ] . a new example is given to illustrate this proposition. example (from [ ] ). let x(t) represent the number of tumor cells at time t, with exponential growth factor α, and u(t) the drug concentration. the aim is to minimize the number of tumor cells at the end of the treatment period and the accumulated harmful effects of the drug on the body. this problem is formulated as let us consider the hamiltonian the optimality condition is given by the adjoint condition is given by using the transversality condition λ(t f ) = (note that φ(s) = s, so φ (s) = ), we obtain the optimally state trajectory is (usingẋ = αx − u and x( ) = x ): many problems require bounds on the control to achieve a realistic solution. for example, the amount of drugs in the organism must be non-negative and it is necessary to impose a limit. for the last example, despite being simplistic, makes more sense to constraint the control as ≤ u ≤ . definition (oc with bounded control). an oc with bounded control can be written in the form where a, b are fixed real constants and a < b. to solve problems with bounds on the control, it is necessary to develop alternative necessary conditions. proposition (necessary conditions). if u * (t) and x * (t) are optimal for problem ( . ), then there exists a piecewise differentiable adjoint variable λ(t) such that for all controls u at each time t, where h is the hamiltonian previously defined and λ(t f ) = (transversality condition). by an adaptation of the pmp, the oc must satisfy (optimality condition): i.e., the maximization is over all admissible controls, andũ is obtained by the expression ∂h ∂u = . in particular, the optimal control u * maximizes h pointwise with respect to a ≤ u ≤ b. proof. the proof of this result can be found in [ ] . if we have a minimization problem, then u * is instead chosen to minimize h pointwise. this has the effect of reversing < and > in the first and third lines of optimality condition. remark . in some software packages there are no specific characterization for the bounds of the control. in those cases, and when the implementation allows, we can write in a compact way the optimal controlũ obtained without truncation, bounded by a and b: u * (t) = min{a, max{b,ũ}}. so far, we have only examined problems with one control and with one dependent state variable. often, it is necessary to consider more variables. definition (oc with several variables and several controls). an oc with n state variables, m control variables and a payoff function φ can be written in the form max u ,...,um x i (t ) = x i , i = , , . . . , n ( . ) where the functions f , g i are continuously differentiable in all variables. from now on, to simplify the notation, let x(t) = [x (t), . . . , x n (t)], u(t) = [u (t), . . . , u m (t)], x = [x , . . . , x n ], and g(t, x, u) = [g (t, x, u), . . . , g n (t, x, u)]. so, the previous problem can be rewritten in a compact way as x(t ) = x , i = , , . . . , n ( . ) using the same approach of the previous subsections, it is possible to derive generalized necessary conditions. proposition (necessary conditions). let u * be a vector of optimal control functions and x * be the vector of corresponding optimal state variables. with n states, we will need n adjoints, one for each state. there is a piecewise differentiable vector-valued function λ(t) = [λ (t), . . . , λ n (t)] , where each λ i is the adjoint variable corresponding to x i , and the hamiltonian is it is possible to find the variables satisfying identical optimality, adjoint and transversality conditions in each vector component. namely, u * maximizes h(t, x * , u, λ) with respect to u at each t, and u * , x * and λ satisfy x(t f )) for j = , . . . , n (transversality conditions) ∂h ∂u k = at u * k for k = , . . . , m (optimality conditions) by φ xj , it is meant the partial derivative in the x j component. note, if φ ≡ , then λ j (t f ) = for all j, as usual. similarly to the previous section, if bounds are placed on a control variable, a k ≤ u k ≤ b k (for k = , . . . , m), then the optimality condition is changed from ∂h ∂u k = to below, an optimal control problem related to rubella is presented. rubella, commonly known as german measles, is a most common in child age, caused by the rubella virus. children recover more quickly than adults, and can be very serious in pregnancy. the virus is contracted through the respiratory tract and has an incubation period of to weeks. the primary symptom of rubella virus infection is the appearance of a rash on the face which spreads to the trunk and limbs and usually fades after three days. other symptoms include low grade fever, swollen glands, joint pains, headache and conjunctivitis. it is presented now an optimal control problem to study the dynamics of rubella in china over three years, using a vaccination process (u) as a measure to control the disease (more details can be found in [ ] ). let x represent the susceptible population, x the proportion of population that is in the incubation period, x the proportion of population that is infected with rubella and x the rule that remains the population constant. the optimal control problem can be defined as: it is very difficult to solve analytically this problem. for most of the epidemiologic problems it is necessary to employ numerical methods. some of them will be described in the next chapter. in this chapter, some numerical approaches to solve a system of ordinary differential equations, such as shooting methods and multi-steps methods, are introduced. then, two distinct philosophies to solve oc problems are presented: indirect methods centered in the pmp and the direct ones focus on problem discretization solved by nonlinear optimization codes. a set of software packages used all over the thesis is summarily exposed. in the last decades the computational world has been developed in an amazing way. not only in hardware issues such as efficiency, memory capacity, speed, but also in terms of the software robustness. groundbreaking achievements in the field of numerical solution techniques for differential and integral equations have enabled the simulation of highly complex real world scenarios. this way, oc also won with these improvements and numerical methods and algorithms have evolved significantly. the next section concerns on the resolution of differential equations systems. a dynamic system is mathematically characterized by a set of ordinary differential equations (ode). specifically, the dynamics are described for t ≤ t ≤ t f , by a system of n odeṡ . . , y n (t), t) f (y (t), . . . , y n (t), t) . . . the problems of solving an ode are classified into initial value problems (ivp) and boundary value problems (bvp), depending on how the conditions at the endpoints of the domain are specified. all the conditions of an initial-value problem are specified at the initial point. on the other hand, the problem becomes a boundary-value problem if the conditions are needed for both initial and final points. there exist many numerical methods to solve initial value problems -such as euler, runge-kutta or adaptive methods -and boundary value problems, such as shooting methods. one can visualize the shooting method as the simplest technique for solving bvp. supposing it is desired to determine the initial angle of a cannon so that, when a cannonball is fired, it strikes a desired target. an initial guess is made for the angle of the cannon, and the cannon is fired. if the cannon does not hit the target, the angle is adjusted based on the amount of the miss and another cannon is fired. the process is repeated until the target is hit [ ] . suppose we want to find y(t ) = y such that y(t f ) = b. the shooting method can be summarized as follows [ ] : step . guess initial conditions x = y(t ); step . propagate the differential system equations from t to t f , i.e., shoot; step . evaluate the error in the boundary conditions c(x) = y(t f ) − b; step . use a nonlinear program to adjust the variables x to satisfy the constraints c(x) = , i.e., repeat steps - . despite its simplicity, from a practical standpoint, the shooting method is used only when the problem has a small number of variables. this method has a major disadvantage: a small change in the initial condition can produce a very large change in the final conditions. in order to overcome the numerical difficulties of the simple method, the multiple shooting method is presented. in a multiple shooting method, the time-interval [t , t f ] is divided into m − subintervals. then is applied over each subinterval [t i , t i+ ] with the initial values of the differential equations in the interior intervals being unknown that need to be determined. in order to enforce continuity, the following conditions are enforced at the interface of each subinterval: a scheme of the multiple shooting method is shown in figure with the multiple shooting approach the problem size is increased: additional variables and constraints are introduced for each shooting segment. in particular, the number of nonlinear variables and constraints for a multiple shooting application is n = n y (m − ), where n y is the number of dynamic variables y and m − is the number of segments [ ] . both shooting and multiple shooting methods require a good guess for initial conditions and the propagation of the shoots for problem of high dimension is not feasible. for this reason, other methods can be implemented, using initial value problems. the numerical solution of the ivp is fundamental to most optimal control methods. the problem can stand as follows: compute the value of y(t f ) for some value of t < t f that satisfies ( . ) with the known initial value y(t ) = y . numerical methods for solving the ode ivp are relatively mature in comparison to other fields in optimal control. it will be considered two methods: single-step and multiple-step methods. in both, the solution of the differential system at each step t k is sequentially obtained using current and/or previous information about the solution. in both cases, it is assumed that the time t = nh moves ahead in uniform steps of length h [ , ] . the most common single-step method is euler method. in this discretization scheme, if a differential equation is written likeẋ = f (x(t), t), is possible to make a convenient approximation of this: this approximation x n+ of x(t) at the point t n+ has an error of order h . clearly, there is a trade-off between accuracy and complexity of calculation which depends heavily on the chosen value for h. in general, as h is decreased the calculation takes longer but is more accurate. for many higher order systems it is very difficult to make euler approximation effective. for this reason more accurate and elaborate techniques were developed. one of these methods is the runge-kutta method. a runge-kutta method is a multiple-step method, where the solution at time t k+ is obtained from a defined set of previous values t j−k , . . . , t k and j is the number of steps. if a differential equation is written likeẋ = f (x(t), t), it is possible to make a convenient approximation of this, using the second order runge-kutta method or the fourth order runge-kutta method x n+ x n + h (k + k + k + k ) where this approximation x n+ of x(t) at the point t n+ has an error depending on h and h , for the runge-kutta methods of second and fourth order, respectively. numerical methods for solving oc problems date back to the s with bellman investigation. from that time to present, the complexity of methods and corresponding complexity and variety of applications has substantially increased [ ] . there are two major classes of numerical methods for solving oc problems: indirect methods and direct methods. the first ones, indirectly solve the problem by converting the optimal control problem to a boundary-value problem, using the pmp. on the other hand, in a direct method, the optimal solution is found by transcribing an infinite-dimensional optimization problem to a finite-dimensional optimization problem. in an indirect method, the pmp is used to determine the first-order optimality conditions of the original oc problem. the indirect approach leads to a multiple-point boundary-value problem that is solved to determine candidate optimal trajectories called extremals. for an indirect method it is necessary to explicitly get the adjoint equations, the control equations and all the transversality conditions, if there exist. notice that there is no correlation between the method used to solve the problem and its formulation: one may consider applying a multiple shooting method solution technique to either an indirect or a direct formulation. in the following subsection a numerical approach using the indirect method is presented. this method is described in a recent book by lenhart and workman [ ] and it is known as forwardbackward sweep method. the process begins with an initial guess on the control variable. then, the state equations are simultaneously solved forward in time and the adjoint equations are solved backward in time. the control is updated by inserting the new values of states and adjoints into its characterization, and the process is repeated until convergence occurs. considering x = (x , . . . , x n + ) and λ = (λ , . . . , λ n + ) the vector approximations for the state and the adjoint. the main idea of the algorithm is described as follows: step . make an initial guess for u over the interval ( u ≡ is almost always sufficient); step . using the initial condition x = x(t ) = a and the values for u, solve x forward in time according to its differential equation in the optimality system; step . using the transversality condition λ n + = λ(t f ) = and the values for u and x, solve λ backward in time according to its differential equation in the optimality system; step . update u by entering the new x and λ values into the characterization of the optimal control; step . verify convergence: if the variables are sufficiently close to the corresponding in the previous iteration, then output the current values as solutions, else return to step . for steps and , lenhart and workman used for the state and adjoint systems the runge-kutta fourth order procedure to make the discretization process. on the other hand, wang [ ] , applied the same philosophy but solving the differential equations with the solver ode for matlab. this solver is based on an explicit runge-kutta ( , ) formula, the dormand-prince pair. that means the numerical solver ode combines a fourth and a fifth order methods, both of which are similar to the classical fourth order runge-kutta method discussed above. these vary the step size, choosing it at each step an attempt to achieve the desired accuracy. therefore, the solver ode is suitable for a wide variety of initial value problems in practical applications. in general, ode is the best method to apply as a first attempt for most problems [ ] . let consider the open problem defined in chapter (example ) about rubella disease. with x(t) = (x (t), x (t), x (t), x (t)) and λ(t) = (λ (t), λ (t), λ (t), λ (t)), the hamiltonian of this problem can be written as using the pmp the optimal control problem can be studied with the state variableṡ with initial conditions x ( ) = . , x ( ) = . , x ( ) = . and x ( ) = and the adjoint variables: the optimal control is here it is only presented the main part of the code using the backward-forward sweep method with fourth order runge-kutta. the completed one can be found in the website [ ] . .. beta*(x (i)+h *m )*(x (i)+h *m )-( . *(u(i) + u(i+ )))*(x (i)+h *m ); m = b*p*(x (i)+h *m )+beta*(x (i)+h *m )*(x (i)+h *m )-(e+b)*(x (i)+h *m ); m = e*(x (i)+h *m )-(g+b)*(x (i)+h *m ); m = b-b*(x (i)+h *m ); m = b-b*(p*(x (i)+h *m )+q*(x (i)+h *m ))-b*(x (i)+h *m )-... beta*(x (i)+h *m )*(x (i)+h *m )-( . *(u(i) + u(i+ )))*(x (i)+h *m ); m = b*p*(x (i)+h *m )+beta*(x (i)+h *m )*(x (i)+h *m )-(e+b)*(x (i)+h *m ); m = e*(x (i)+h *m )-(g+b)*(x (i)+h *m ); m = b-b*(x (i)+h *m ); m = b-b*(p*(x (i)+h *m )+q*(x (i)+h *m ))-b*(x (i)+h *m )-... beta*(x (i)+h *m )*(x (i)+h *m )-u(i+ )*(x (i)+h *m ); m = b*p*(x (i)+h *m )+beta*(x (i)+h *m )*(x (i)+h *m )-(e+b)*(x (i)+h *m ); m = e*(x (i)+h *m )-(g+b)*(x (i)+h *m ); m = b-b*(x (i)+h *m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); x (i+ ) = x (i) + (h/ )*(m + *m + *m + m ); end for i = :m j = m + -i; n = lambda (j)*(b+u(j)+beta*x (j))-lambda (j)*beta*x (j); n = lambda (j)*b*p+lambda (j)*(e+b-p*b)-lambda (j)*e; n = -a+lambda (j)*(b*q+beta*x (j))-lambda (j)*beta*x (j)+lambda (j)*(g+b); n = b*lambda (j); n = (lambda (j) -h *n )*(b+u(j)+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- ))); n = (lambda (j) -h *n )*b*p+(lambda (j) -h *n )*(e+b-p*b)-(lambda (j) -h *n )*e; n = -a+(lambda (j) -h *n )*(b*q+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- )))+(lambda (j) -h *n )*(g+b); n = b*(lambda (j) -h *n ); n = (lambda (j) -h *n )*(b+u(j)+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- ))); n = (lambda (j) -h *n )*b*p+(lambda (j) -h *n )*(e+b-p*b)-(lambda (j) -h *n )*e; n = -a+(lambda (j) -h *n )*(b*q+beta*( . *(x (j)+x (j- ))))-... (lambda (j) -h *n )*beta*( . *(x (j)+x (j- )))+(lambda (j) -h *n )*(g+b); n = b*(lambda (j) -h *n ); n = (lambda (j) -h *n )*(b+u(j)+beta*x (j- ))-(lambda (j) -h *n )*beta*x (j- ); n = (lambda (j) -h *n )*b*p+(lambda (j) -h *n )*(e+b-p*b)-(lambda (j) -h *n )*e; n = -a+(lambda (j) -h *n )*(b*q+beta*x (j- ))-... (lambda (j) -h *n )*beta*x (j- )+(lambda (j) -h *n )*(g+b); n = b*(lambda (j) -h *n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); lambda (j- ) = lambda (j) -h/ *(n + *n + *n + n ); end u = min( . ,max( ,lambda .*x / )); the optimal curves for the states variables and optimal control are shown in figure there are several difficulties to overcome when an optimal control problem is solved by indirect methods. firstly, is necessary to calculate the hamiltonian, adjoint equations, optimality condition and transversality conditions. besides, the approach is not flexible, since each time a new problem is formulated, a new derivation is required. in contrast, a direct method does not require explicit derivation neither the necessary conditions. due to these practical difficulties with the indirect formulation, the main focus will be centered on the direct methods. this approach has been gaining popularity in numerical optimal control over the past three decades [ ] . a new family of numerical methods for dynamic optimization has emerged, referred to as direct methods. this development has been driven by the industrial need to solve large-scale optimization problems and it has also been supported by the rapidly increasing computational power. a direct method constructs a sequence of points x , x , . . . , x * such that the objective function in minimized and typical f (x ) > f (x ) > · · · > f (x * ). here the state and/or control are approximated using an appropriate function approximation (e.g., polynomial approximation or piecewise constant parameterization). simultaneously, the cost functional is approximated as a cost function. then, the coefficients of the function approximations are treated as optimization variables and the problem is reformulated to a standard nonlinear optimization problem (nlp) in the form: where c i , i ∈ e e c j , j ∈ i are the set of equality and inequality constraints, respectively. in fact, the nlp is easier to solve than the boundary-value problem, mainly due to the sparsity of the nlp and the many well-known software programs that can handle with this feature. as a result, the range of problems that can be solved via direct methods is significantly larger than the range of problems that can be solved via indirect methods. direct methods have become so popular these days that many people have written sophisticated software programs that employ these methods. here we present two types of codes/packages: specific solvers for oc problems and standard nlp solvers used after a discretization process. the oc-ode [ ] , optimal control of ordinary-differential equations, by matthias gerdts, is a collection of fortran routines for optimal control problems subject to ordinary differential equations. it uses an automatic direct discretization method for the transformation of the oc problem into a finite-dimensional nlp. oc-ode includes procedures for numerical adjoint estimation and sensitivity analysis. considering the same problem (example ), here is the main part of the code in oc-ode. the completed one can be found in the website [ ] . the achieved solution is similar to the indirect approach, therefore we will not present it. the dotcvp [ ] , dynamic optimization toolbox with vector control parametrization is a dynamic optimization toolbox for matlab. the toolbox provides environment for a fortran compiler to create the '.dll' files of the ode, jacobian, and sensitivities. however, a fortran compiler has to be installed in a matlab environment. the toolbox uses the control vector parametrization approach for the calculation of the optimal control profiles, giving a piecewise solution for the control. the oc problem has to be defined in mayer form. for solving the nlp, the user can choose several deterministic solvers -ipopt, fmincon, fsqp -or stochastic solvers -de, sres. the modified sundials tool [ ] is used for solving the ivp and for the gradients and jacobian automatic generation. forward integration of the ode system is ensured by cvodes, a part of sundials, which is able to perform the simultaneous or staggered sensitivity analysis too. the ivp problem can be solved with the newton or functional iteration module and with the adams or bdf linear multistep method. note that the sensitivity equations are analytically provided and the error control strategy for the sensitivity variables could be enabled. dotcvp has a user friendly graphical interface (gui). considering the same problem (example ), here is a part of the code used in dotcvp. the completed one can be found in the website [ ] . the solution, despite being piecewise continuous, follows the curve obtained by the previous programs. functional for non-stiff problems data.odes.linearsolver with the gui interface this method was the preferred to be tested, due to its simple way to implement the code. in neos [ ] platform there is a large set of software packages. neos is considered as the state of the art in optimization. one recent solver is muscod-ii [ ] (multiple shooting code for optimal control) for the solution of mixed integer nonlinear ode or dae constrained optimal control problems in an extended ampl format. ampl [ ] is a modelling language for mathematical programming and was created by fourer, gay and kernighan. the modelling languages organize and automate the tasks of modelling, which can handle a large volume of data and, moreover, can be used in machines and independent solvers, allowing the user to concentrate on the model instead of the methodology to reach solution. however, the ampl modelling language itself does not allow the formulation of differential equations. hence, the taco toolkit has been designed to implement a small set of extensions for easy and convenient modeling of optimal control problems in ampl, without the need for explicit encoding of discretization schemes. both the taco toolkit and the neos interface to muscod-ii are still under development. probably for this reason, the example could not be solved by this software which crashed after some runs. anyway, we opted to also put the code, for the same example that is being used, to show the differences of modelling language used in each program. include optimalcontrol.mod; var t ; var x , >= <= ; var x , >= <= ; var x , >= <= ; var x , >= <= ; var u >= , <= . suffix type "u "; the three nonlinear optimization software packages presented here, were used through the neos platform with codes formulated in ampl. the ipopt [ ] , interior point optimizer, is a software package for large-scale nonlinear optimization. it is written in fortran and c. ipopt implements a primal-dual interior point method and uses a line search strategy based on filter method. ipopt can be used from various modeling environments. ipopt is designed to exploit st and nd derivative information if provided, usually via automatic differentiation routines in modeling environments such as ampl. if no hessians are provided, ipopt will approximate them using a quasi-newton methods, specifically a bfgs update. continuing with example the ampl code is shown for ipopt. the euler discretization was the selected. indeed, this code can also be implemented in other nonlinear software packages available in neos platform, reason why the code for the next two software packages will not be shown. the full version can be found on the website [ ] . , short for "nonlinear interior point trust region optimization", was created primarily by richard waltz, jorge nocedal, todd plantenga and richard byrd. it was introduced in as a derivative of academic research at northwestern, and has undergone continual improvement since then. knitro is also a software for solving large scale mathematical optimization problems based mainly on the two interior point (ip) methods and one active set algorithm. knitro is specialized for nonlinear optimization, but also solves linear programming problems, quadratic programming problems, and systems of nonlinear equations. the unknowns in these problems must be continuous variables in continuous functions; however, functions can be convex or nonconvex. the code also provides a multistart option for promoting the computation of the global minimum. this software was tested through the neos platform. snopt [ ] , by philip gill, walter murray and michael saunders, is a software package for solving large-scale optimization problems (linear and nonlinear programs). it is specially effective for nonlinear problems whose functions and gradients are expensive to evaluate. the functions should be smooth but do not need to be convex. snopt is implemented in fortran and distributed as source code. it uses the sqp (sequential quadratic programming) philosophy, with an augmented lagrangian approach combining a trust region approach adapted to handle the bound constraints. snopt is also available in neos platform. choosing a method for solving an oc problem depends largely on the type of problem to be solved and the amount of time that can be invested in coding. an indirect shooting method has the advantage of being simple to understand and produces highly accurate solutions when it converges [ ] . the accuracy and robustness of a direct method is highly dependent upon the method used. nevertheless, it is easier to formulate highly complex problems in a direct way and can be used standard nlp solvers as an extra advantage. this last feature has the benefit of converging with poor initial guesses and being extremely computationally efficient since most of the solvers exploit the sparsity of the derivatives in the constraints and objective function. in the next chapter the basic concepts from epidemiology are provided, in order to formulate/implement more complex oc problems in the health area. in this chapter, the simplest epidemiologic models composed by mutually exclusive compartments are introduced. based on the models sis (susceptible-infected-susceptible) and sir (susceptible-infected-recovered), other models are presented introducing new issues related to maternal immunity or the latent period, fitting the features to distinct diseases. illustrative examples are presented, with diseases that can be described by each model. the basic reproduction number is calculated and presented as a threshold value for the eradication or the persistence of the disease in a population. in the th century, occurred one of most famous epidemic events: the black death. it killed approximately one third of the european population. from - , twenty to forty percent of the world's population suffered from the spanish flu, the most severe pandemic in history. in , the united nations promoted an ambitious agreement between the countries forecasting that in the year infectious diseases would be eradicated. this conjecture failed, mainly due to the assumption that the microorganisms were biologically stationary and consequently they were not modified and became resistant to the medicines. besides, the improvements in the transportation allowing for a faster movement of individuals and the population growing especially in developing countries, led to the appearance of new diseases and the resurgence of old ones in distinct places. nowadays, aids is the most scrutinized. in there were an estimated . million sufferers worldwide, and . million deaths with over three quarters of these occurring in sub-saharan africa [ ] . epidemiology -the study of patterns of diseases including those which are non-communicable of infections in population -has become more relevant and indispensable in the development of new models and explanations for the outbreaks, namely due to their propagation and causes. in epidemiology, an infection is said to be endemic in a population when it is maintained in the population without the need for external inputs. an epidemic occurs when new cases of a certain disease appears, in a given human population during a given period, and then essentially disappears. there are several types of diseases, depending on their type of transmission mechanism, of which stand out: • bacteria, which do not confer immunity against the reinfection and frequently produce harmful toxins to the host; in case of infection, the antibiotics are usually efficient (examples: tuberculosis, meningitis, gonorrhea, syphilis, tetanus); • viral agents, that confer immunity against reinfection; here the antibiotics do not produce effects and usually it is hoped that the immune system of the host responds to an infection by the virus or it will be necessary to take antiviral drugs that retard the multiplication of the virus (examples influenza, chicken pox, measles, rubella, mumps, hiv / aids, smallpox); • vectors, that are usually mosquitoes or ticks and are infected by humans and then transmit the disease to other humans (examples: malaria, yellow fever, dengue, chikungunya). the transmission can happen in a direct or indirect way. the direct transmission of a disease can happen by physical proximity (such as sneezing, coughing, kissing, sexual contact) or even by a specific parasite that penetrates the host through ingestion or the skin. the indirect transmission involves the vectors that are intermediaries or carriers of the infection. in most of the cases, the direct and indirect transmission of the disease happens between the member that coexists in the host population; this is called the horizontal transmission. when the direct transmission occurs from one ascendent to a descendent not yet born (egg or embryo) it is said that vertical transmission happens [ ] . when formulating a model for a particular disease, we should make a trade-off between simple models -that omit several details and generally are used for specific situations in a short time, but have the disadvantage of possibly being naive and unrealistic -and more complex models, with more details and more realistic, but generally more difficult to solve or could contain parameters which their estimates cannot be obtained. choosing the most appropriated model depends on the precision or generality required, the available data, and the time frame in which the results are needed. by definition, all models are "wrong", in the sense that even the most complex will make some simplifying assumptions. it is, therefore, difficult to definitively express which model is right, though naturally we are interested in developing models that capture the essential features of a system. the art of epidemiological modelling is to make suitable choices in the model formulation making it as simple as possible and yet suitable for the question being considered [ ] . mathematical models are a simplified representation of how an infection spreads across a population over time, and generally come in two forms: stochastic and deterministic models. the first ones, employ randomness, with variables being described by probability distributions. deterministic models split the population into subclasses, and an ode with respect to time is formulated for each. the state variable are determined using parameters and initial conditions. the main focus in this chapter will be the deterministic models, neglecting the others. most epidemic models are based on dividing the population into a small number of compartments. each containing individuals that are identical in terms of their status with respect to the disease in question. here are some of the main compartments that a model can contain. • passive immune (m ): is composed by newborns that are temporary passively immune due to antibodies transferred by their mothers; • susceptible (s): is the class of individuals who are susceptible to infection; this can include the passively immune once they lose their immunity or, more commonly, any newborn infant whose mother has never been infected and therefore has not passed on any immunity; • exposed or latent (e): compartment referred to the individuals that despite infected, do not exhibit obvious signs of infection and the abundance of the pathogen may be too low to allow further transmission; • infected (i): in this class, the level of parasite is sufficiently large within the host and there is potential in transmitting the infection to other susceptible individuals; • recovered or resistant (r): includes all individuals who have been infected and have recovered. the choice of which compartments to include in a model depends on the characteristics of the particular disease being modelled and the purpose of the model. the exposed compartment is sometimes neglected, when the latent period is considered very short. besides, the compartment of the recovered individuals cannot always be considered since there are diseases where the host has never became resistent. acronyms for epidemiology models are often based on the flow patterns between the compartments such as mseir, mseirs, seir, seirs, sir, sirs, sei, seis, si, sis. there are three commonly used threshold values in epidemiology: r , σ and r. the most common and probably the most important is the basic reproduction number [ , , ] . definition (basic reproduction number). the basic reproduction number, denoted by r , is defined as the average number of secondary infections that occurs when one infective is introduced into a completely susceptible population. this threshold, r , is a famous result due to kermack and mckendrick [ ] and is referred to as the "threshold phenomenon", giving a borderline between a persistence or a disease death. r it is also called the basic reproduction ratio or basic reproductive rate. definition (contact number). the contact number, σ is the average number of adequate contacts of a typical infective during the infectious period. an adequate contact is one that is sufficient for transmission, if the individual contacted by the susceptible is an infective. it is implicitly assumed that the infected outsider is in the host population for the entire infectious period and mixes with the host population in exactly the same way that a population native would mix. definition (replacement number). the replacement number, r, is the average number of secondary infections produced by a typical infective during the entire period of infectiousness. note that the replacement number r changes as a function of time t as the disease evolves after the initial invasion. these three quantities r , σ and r are all equal at the beginning of the spreading of an infectious disease when the entire population (except the infective invader) is susceptible. r is only defined at the time of invasion, whereas σ and r are defined at all times. the replacement number r is the actual number of secondary cases from a typical infective, so that after the infection has invaded a population and everyone is no longer susceptible, r is always less than the basic reproduction number r . also after the invasion, the susceptible fraction is less than one, and as such not all adequate contacts result in a new case. thus the replacement number r is always less than the contact number σ after the invasion [ ] . combining these results leads to note that r = σ for most models, and σ > r after the invasion for all models. for the models throughout this study the basic reproduction number, r , will be applied. when r < the disease cannot invade the population and the infection will die out over a period of time. the amount of time this will take generally depends on how small r is. when invasion is possible and infection can spread through the population. generally, the larger the value of r the more severe, and possibly widespread, the epidemic will be [ ] . in table . are some example diseases with their estimated basic reproduction number. due to differences in demographic rates, rural-urban gradients, and contact structure, different human populations may be associated with different values of r for the same disease [ ] . in the next section some of the epidemiologic models will be presented. numerous infectious diseases confer no long-lasting immunity. the sis models are suitable for some bacterial agent diseases like meningitis, sexually transmitted diseases such as gonorrhea and for protozoan agent diseases where malaria and the sleeping sickness are good examples. for these diseases, an individual can be infected multiple times throughout their lives, with no apparent immunity. here, recovery from infection is followed by an instant return to the susceptible compartment. throughout this chapter we will consider that population is constant, neglecting the tourism and immigration factors. also it is considered that the population is homogeneously mixed, which means that every individual interacts with another at the same level and therefore all individuals have the same risk of contracting the disease. the number of individuals in each compartment must be integer, but if the population size n is sufficiently large, it is possible to treat s and i as continuous variables. calculating the proportion of these compartments, varying from to , it is considered that the total population is constant over time, i.e., = s + i. the compartment changes are expressed by a system of differential equations. the sis model can be mathematically represented as follows. definition (sis model). the sis model can be formulated as subject to initial conditions s( ) > and i( ) ≥ . it is considered β the transmission rate (per capita) and γ the recovery rate, so the mean infectious period is /γ. the vital dynamics (births and deaths) were not considered, but a similar model can be constructed with these effects [ ] . despite this lack of susceptible births, the disease can still persist because the recovery of infected individuals replenishes the susceptible class and will guarantee the long-term persistence of the disease. remark . the si model is a particular case of the sis model when the recovery rate (γ) is null. a new infected individual is expected to infect another at a transmission rate β, during γ that is the infectious period, so the expected basic reproduction number is trachoma is an infectious disease causing a characteristic roughening of the inner surface of the eyelids. also called granular conjunctivitis or egyptian ophthalmia, it is the leading cause of infectious blindness in the world. adapting a model from [ ] , and using β = . as transmission rate and the recovery rate γ = . , we have the basic reproduction number r approximately . and the following representation of the state variables (figure . ). the sir model was initially studied in depth by kermack and mckendrick and categorizes hosts within a population as susceptible, infected and recovered [ ] . it captures the dynamics of acute infections that confers lifelong immunity once recovered. diseases where individuals acquire permanent immunity, and for which this model may be applied, include measles, smallpox, chickenpox, mumps, typhoid fever and diphtheria. once again we will consider the total population size constant, i.e., = s + i + r. two cases will be studied, distinguished by the inclusion or exclusion of demographic factors. having compartmentalized the population, we now need a set of equations that specify how the sizes of compartments change over time. definition (sir model without demography). the sir model, excluding births and deaths, can be defined as in addition, the transmission rate, per capita, is β and the recovery rate is γ. since population is constant and as r does not appear in the first two differential equations, most of the times the last equation is omitted, indeed, r(t) = − s(t) − i(t). by assuming equations from ( . ) is possible to notice that ds dt then a newly introduced infected individual can be expected to infect other people at the rate β during the expected infectious period /γ. thus, this first infective individual can be expected to infect consider an epidemic of influenza in a british boarding school in early [ ] . three boys were reported to the school infirmary with the typical symptoms of influenza. over the next few days, a very large fraction of the boys in the school had contact with the infection. within two weeks, the infection had become extinguished. the best fit parameters yield an estimated active infectious period of /γ = . days and a mean transmission rate β = . per day. therefore, the estimated r is . . figure . represents the dynamics of the three state variables. it was presented a sir model given the assumption that the time scale of disease spread was sufficiently fast that births and deaths can be neglected. in the next subsection we explore the long-term persistence and endemic dynamics of an infectious disease. the simplest and most common way of introducing demography into the sir model is to assume there is a natural host lifespan, /µ years. then, the rate at which individuals, at any epidemiological compartment, suffer natural mortality is given by µ. it is important to emphasize that this factor is independent of the disease and is not intended to reflect the pathogenicity of the infectious agent. historically, it has been assumed that µ also represents the population's crude birth rate, thus ensuring that total population size does not change through time, or in other words, ds dt + di dt + dr dt = . putting all these assumptions together, we have a new definition. definition (sir model with demography). the sir model, including births and deaths, can be defined as the epidemiological scheme is in figure . . it is important to introduce the r expression for this model. the parameter β represents the trans-mission rate per infective and the negative terms in the equation tell us that each individual spends an average γ+µ time units in this class. therefore, if we assume the entire population is susceptible, then the average number of new infectious per infectious individual is determined by the inclusion of demographic dynamics may allow a disease to die out or persist in a population in the long term. for this reason it is important to explore what happens when the system is at equilibrium. definition (equilibrium points). a model defined sir has an equilibrium point, if a triple e * = (s * , i * , r * ) satisfies the following system: if the equilibrium point has the infectious component equal to zero (i * = ), this means that the pathogen suffered extinction and e * is called disease free equilibrium (dfe). if i * > the disease persist in the population and e * is called endemic equilibrium (ee). with some calculations and algebraic manipulations, it is possible to obtain two equilibria for the system ( . ): dfe: when r < , each infected individual produces, on average, less than one new infected individual, and therefore, predictable that the infection will be cleared from the population. if r > , the pathogen is able to invade the susceptible population [ , ] . it is possible to prove that for the endemic equilibrium to be stable, r must be greater than one, otherwise the disease free equilibrium is stable. more detailed information about local and global stability of the equilibrium point can be found in [ , , , ] . this threshold behavior is very useful, once we can determine which control measures, and at what magnitude, would be most effective in reducing r below one, providing important guidance for public health initiatives. the sir model below, figure . , is plotted using similar parameters and initial conditions, except the transmission rate β (adapted from [ ] ). it is shown that in case (a), using β = , the basic reproduction number is greater than one. with demographic effects could have damped oscillations with a decreasing amplitude, in the ee direction. in case (b), with β = , we obtain r < and the system tends to go to a dfe. the next three sections present a brief description of other possible refinements of the basic models sis and sir. in the seir case, the transmission process occurs with an initial inoculation with a very small number of pathogens. then, during a period of time the pathogen reproduces rapidly within the host, relatively unchallenged by the immune system. during this stage, pathogen abundance is too low for active transmission to other susceptible host, and yet the pathogen is present. the time in this stage is very difficult to quantify, since there is no symptomatic features of the disease. it is called the latent or exposed period. assuming the average duration of the latent period is given by ν , the seir model can be described as follow. definition (seir model). the seir model is formulated as the parameter β and γ were defined in the previous section. the epidemiological scheme for seir model is presented in figure this threshold is the product of the contact rate β per unit time, the average infectious period adjusts to the population growth of γ+µ , and the fraction ν ν+µ of exposed people surviving the latent class e. finding steady states of the system, we obtain the following equilibrium points: although the sir and seir models behave similarly at equilibrium, when the parameters are suitably adapted, the seir model has a slower growth rate after pathogen invasion. this is due to the fact that individuals need to stay some time in the exposed class before their contribution in the disease transmission chain [ ] . an infected or vaccinated mother transfers some antibodies across the placenta to her fetus, so that the newborn infant has temporary passive immunity to an infection. since the infant can not produce new antibodies, when these passive antibodies are gone at a rate δ, the baby passes from the immune state m to the susceptible state s. some childhood diseases have this feature. the birth rate µs into the susceptible class of size s corresponds to newborns whose mothers are susceptible, and the other newborns µ( − s) enter passively immune class of size m , since their mothers were infected or had some type of immunity [ ] . the transfer diagram for the mseir model is shown in the mseir is also composed by a system of differential equations. definition (mseir model). the seir model can be described as thus, the basic reproduction number is equal to previous model seir, once the m compartment does not affect the transmission chain of the disease: the equations ( . ) always have a dfe given by there are several models with different epidemiological states, such as mseirs, seirs, sei, si, sirs, depending on the specific features and the level of detail that is wished to introduce in the model. these models are similar to the previous ones presented. other epidemiological models can be studied using more compartments such as quarantine-isolation (q), treatment (t ), carrier (c) or vaccination (v ). besides, most of the populations can be subdivided into different groups (sex, age, health weaknesses, ...), depending upon characteristics that may influence the risk of catching and/or transmitting an infection. more details and examples can be found in [ , , , ] . other diseases can be caught and transmitted by numerous hosts or even is required a second different population to complete the transmission cycle, such as vector borne-diseases, using double models. this last case will be explored in the second part of the thesis. in cases that include multiple compartments of infected individuals, in which vital and epidemiological parameters depend on factors as stage of the disease, spatial position, age, behavior, multigroups, the next generation method is the more generalized approach to calculate r . the definition of r has more than one possible interpretation, depending on the field (ecology, demography or epidemiology) and there exist distinct methods and estimations to calculate this threshold. the next generation method, introduced by diekmann et al. [ ] , defines r as the spectral radius of the next generator operator. the formation of the operator involves the determination of two compartments, infected and non-infected from the model. recent examples of this method are given in [ , , , ] . let us assume that there are n compartments of which m are infected. we defined the vector x = (x , . . . , x n ) t , where x i ≥ denotes the number of proportion of individuals in the ith compartment. for clarity we sort the compartments so that the first m compartments correspond to infected individuals. the distinction between infected and uninfected compartments must be determined from the epidemiological interpretation, and not by the mathematical expressions. it is necessary to define the set the disease transmission model consists of nonnegative initial conditions together with the following system of equations:ẋ note that f i should include only infections that are newly arising, but does not include terms which describe the transfer of infectious individuals from one infected compartment to another. let us consider the following assumptions: (a ) if f(x) is set to zero, then all eigenvalues of df (x ) have negative real parts and df (x ) is the derivative ∂fi ∂xj evaluated at the dfe x . assuming that f i and v i meet the assumptions above, we can form the next generation matrix f v − from matrices of partial derivatives of f i and v i . specially, where i, j = , . . . , m and x is the dfe. the entries of f v − give the rate at which infected individuals in x j produce new infections in x i , times the average length of time an individual spends on a single visit to compartment j. definition (basic reproduction number using the next generator operator). the basic reproduction number is given by where ρ denotes the spectral radius (dominant eigenvalue) of the matrix f v − . the following theorem states that r is a threshold parameter for the stability of the dfe. proof. the proof of this theorem can be found in [ ] . consider a simple model seit for tuberculosis with a treated compartment (adapted from [ ] ). tuberculosis, is a common, and in many cases lethal, infectious disease caused by various strains of mycobacteria. tuberculosis typically attacks the lungs but can also affect other parts of the body. most infections are asymptomatic and latent, but about one in ten latent infections eventually progresses to active disease which, if left untreated, kills more than % of those so infected. in this model a constant population is considered where n = s + e + i + t . exposed individuals progress to the infectious compartment at a rate ν. the treatment rates are r for exposed individuals and r for infectious individuals. however only a fraction q of the treatments of infectious individuals are successful. unsuccessfully treated infectious individuals re-enter the exposed compartment ( − q). the dynamics are illustrated in figure . and the differential equations are the following: e and i, which gives m = , and we will construct the matrices f and v only related to these compartments. let this way, epidemiological models can be understood as a framework to explain the mechanisms of the disease procedures and test ideas to implement control measures. r is a key concept, used as a threshold parameter to predict where an infection will spread and how these controls can be effective. in the second part of the thesis, the epidemiological models will be used for studying dengue disease. applying oc theory, the repercussions of several controls in the development of the disease will be analyzed. during the last decades, the global prevalence of dengue progressed dramatically. in this chapter, dengue details are given, such as disease symptoms, transmission and epidemiological trends. an old oc model for dengue is revisited, as a first approach to the disease. due to the software robustness improvements and the higher computational capacity, a better solution for this problem is proposed. in order to study different discretization schemes for an oc problem, taking into account time performance and resources used, some numerical simulations are made using euler and runge-kutta methods. the origins of the word dengue are not clear. some researchers think that it is derived from the swahili phrase "ka-dinga pepo", meaning "cramp-like seizure caused by an evil spirit". the first recognized dengue epidemics occurred almost simultaneously in asia, africa, and north america in the s, shortly after the identification and naming of the disease in [ ] . dengue transcends international borders and can be found in tropical and subtropical regions around the world, predominantly in urban and semi-urban areas. dengue is a disease which is now endemic in more than one hundred countries of africa, america, asia and the western pacific. in figure . it is possible to see the areas that in have more surveillance. nevertheless, some studies have indicated that countries with a mild climate, such as in the mediterranean, are at risk due to future climate conditions that may be favorable to this kind of disease [ ] . in europe there are no registered cases, but the main vector of the disease is already in the old continent and has been followed on madeira island [ , ] . this risk may be aggravated further due to climate changes and to the globalization, as a consequence of the huge volume of international tourism and trade [ ] . travelers play an essential role in the global epidemiology: they act as viremic travelers, carrying the disease into areas where mosquitos can transmit the infection. dengue is a vector-borne disease transmitted from an infected human to a female aedes mosquito by a bite. then, the mosquito, that needs regular meals of blood to feed their eggs, bites a potential healthy human and transmits the disease making it a cycle. there are two forms of dengue: dengue fever (df) and dengue hemorrhagic fever (dhf). the first one is characterized by a sudden high fever without respiratory symptoms, accompanied by intense headaches, painful joints and muscles and lasts between three to seven days. humans may only transmit the virus during the febrile stage [ ] . dhf initially exhibits a similar, if more severe pathology as df, but deviates from the classic pattern at the end of the febrile stage [ ] . the hemorrhagic form has an additionally bleeding from the nose, mouth and gums or skin bruising, nausea, vomiting and fainting due to low blood pressure by fluid leakage. it usually lasts between two to three days and can lead to death [ ] . nowadays, dengue is the mosquito-borne infection that has become a major international public health concern. according to the world health organization (who), to million dengue fever infections occur yearly, including dengue hemorrhagic fever cases and deaths, mostly among children [ ] . there are four distinct, but closely related, viruses that cause dengue. the four serotypes, named den- to den- , belong to the flavivirus family, but they are antigenically distinct. recovery from infection by one virus provides lifelong immunity against that virus but confers only partial and transient protection against subsequent infection by the other three viruses. there is good evidence that a sequential infection increases the risk of developing dhf [ ] . unfortunately, there is no specific effective treatment for dengue. activities, such as triage and management, are critical in determining the clinical outcome of dengue. a rapid and efficient front-line response not only reduces the number of unnecessary hospital admissions but also saves lives. although up until now there is no effective and safe vaccine for dengue, a number of candidates are undergoing various phases of clinical trials [ ] . with four closely related viruses that can cause the disease, there is a need for a vaccine that would immunize against all four types to be effective. the main difficulty in the vaccine production is that there is a limited understanding of how the disease typically behaves and how the virus interacts with the immune system. another challenge is that some studies show that some secondary dengue infection can leave to dhf, and theoretically a vaccine could be a potential cause of severe disease if a solid immunity is not established against the four serotypes. research to develop a vaccine is ongoing and the incentives to study the mechanism of protective immunity are gaining more support, now that the number of outbreaks around the world is increasing [ ] . the spread of dengue is attributed to the geographic expansion of the mosquitoes responsible for the disease: aedes aegypti and aedes albopictus [ ] . due to its higher interaction with humans and its urban behavior, the first mosquito is considered the major responsible for dengue transmission and, our attention will be focused on it. aedes aegypti, in figure . , is an insect species closely associated with humans and their dwellings, thriving in crowded cities and biting primarily during the day. humans not only provide blood meals for mosquitoes, but also nutrients needed for them to reproduce through water-holding containers, in and around their homes. in urban areas, aedes mosquitoes breed on water collections in artificial containers such as cans, plastic cups, used tires, broken bottles and flower pots. with increasing urbanization, crowded cities, poor sanitation and lack of hygiene, environmental conditions foster the spread of the disease that, even in the absence of fatal forms, breed significant economic and social costs (absenteeism, immobilization, debilitation and medication) [ ] . dengue is spread only by adult females that require a blood meal for the development of their eggs, whereas male mosquitoes feed on fruit nectar and other sources of sugar. in this process the female acquire the virus while feeding on the blood of an infected person. after virus incubation from eight to twelve days (extrinsic period), an infected mosquito is capable, during probing and blood feeding, of transmitting the virus for the rest of its life to susceptible humans, and the intrinsic period for humans varies from to days. the life cycle of a mosquito has four distinct stages: egg, larva, pupa and adult, as it is possible to see in figure . . in the case of aedes aegypti, the first three stages take place in or near water whilst air is the medium for the adult stage [ ] . female mosquitoes lay their eggs, but usually do not lay them all at once: it releases them in different places, increasing the probability of new births [ ] . the eggs of aedes aegypti can resist to droughts and low temperatures for up to one year. although the hatching of mature eggs may occur spontaneously at any time, this is greatly stimulated by flooding. larvae hatch when water inundates the eggs as a result of rains or an addition of water by people. in the following days, the larvae will feed on microorganisms and particulate organic matter. when the larva has acquired enough energy and size, metamorphosis is done, changing the larva into pupa. pupae do not feed: they just change in form until the adult body is formed. the newly formed adult emerges from the water after breaking the pupal skin. the entire life cycle, from the aquatic phase (eggs, larvae, pupae) to the adult phase, lasts from to days at room temperature, depending on the level of feeding [ ] . the adult stage of the mosquito is considered to last an average of eleven days in an urban environment, reaching up to days in laboratory environment. studies suggest that most female mosquitoes may spend their lifetime in or around the houses where they emerge as adults. this means that people, rather than mosquitoes, rapidly move the virus within and between communities. aedes aegypti is one of the most efficient vectors for arboviruses because it is highly anthropophilic, frequently bites several times before complete oogenesis [ ] . the extent of dengue transmission is determined by a wide variety of factors: the level of herd immunity in the human population to circulating virus serotype(s); virulence characteristics of the viral strain; survival, feeding behavior, and abundance of aedes aegypti; climate; and human density, distribution, and movement [ ] . it is very difficult to control or eliminate aedes aegypti mosquitoes because they are highly resilient, quickly adapting to changes in the environment and they have the ability to rapidly bounce back to initial numbers after disturbances resulting from natural phenomena (e.g., droughts) or human interventions (e.g., control measures). we can safely expect that transmission thresholds will vary depending on a range of factors. primary prevention of dengue resides mainly in mosquito control. there are two primary methods: larval control and adult mosquito control, depending on the intended target [ ] . larvicide treatment is done through long-lasting chemical in order to kill larvae and preferably have who clearance for use in drinking water [ ] . the application of adulticides can have a powerful impact on the abundance of adult mosquito vector. however, the efficacy is often constrained by the difficulty in achieving sufficiently high coverage of resting surfaces [ ] . this is the most common measure. however the long term use of adulticide has several risks: the resistance of the mosquito to the product reducing its efficacy, the killing of other species that live in the same habitat and has also been linked to numerous adverse health effects including the worsening of asthma and respiratory problems. larvicide treatment is an effective way to control the vector larvae, together with mechanical control, which is related to educational campaigns. the mechanical control must be done by both public health officials and residents in affected area. the participation of the entire population is essential in removing still water from domestic recipients and eliminating possible breeding sites [ ] . the most recent approach for fighting the disease is biological control. it is a natural process of population regulation through natural enemies. there are techniques that combine some parasites that kill partially the larval population; however there are some operational resistance, because there is lack of expertise in producing these type of parasites and there are some cultural objections in introducing something in the water for human consumption [ ] . another way of insect control is to change the reproduction process, releasing sterile insects. this technique, named as sterile insect technique, consists in releasing sterile insects in natural environment, so that the result of mating produces the non-viability of the eggs, and thus can lead to drastic reduction of the specie. this way of control, shows two types of inconvenience: it is expensive to produce and release insects, and can be confronted with social objections, because an uninformed population could not correctly understand the addition of insects as a good solution [ , , ] . mathematical modeling became an interesting tool for understanding epidemiological diseases and for proposing effective strategies in fighting them. a set of mathematical models have been developed in literature to gain insights into the transmission dynamics of dengue in a community. while zeng and velasco-hérnandez [ ] investigate the competitive exclusion principle in a two-strain dengue model, chowell et al. [ ] estimates the basic reproduction number for dengue using spatial epidemic data. in [ ] the author studies the spread of dengue thought statistical analysis, while in tewa et al. [ ] global asymptotic stability of the equilibrium of a single-strain dengue model is established. the control of the mosquito by the introduction of a sterile insect technique is analyzed in thomé et al. [ ] . more recently, a study in disease persistence was made [ ] in brazil and otero et al. [ ] studied dengue outbreaks. all these studies were made with the aim of providing a better understanding of the nature and dynamics of dengue infection transmission. in the next section a temporal mathematical model that explores the dynamics between hosts (humans) and vectors (mosquitoes) is analyzed. the aim of this section is to present a mathematical model to study the dynamic of the dengue epidemics, in order to minimize the investments in disease's control, since financial resources are always scarce. quantitative methods are applied to the optimization of investments in the control of the epidemiologic disease, in order to obtain a maximum of benefits from a fixed amount of financial resources. the used model depends on the dynamic of the mosquito growing, but also on the efforts of public management to motivate the population to break the reproduction cycle of the mosquitoes by avoiding the accumulation of still water in open-air recipients and spraying potential zones of reproduction. the dengue epidemic model described in this paper is based on the one proposed in [ ] . it has four state variables and two control variables as follows. state variables: to describe the model it is also necessary to introduce some parameters: parameters: α r : average reproduction rate of mosquitoes α m : mortality rate of mosquitoes β : probability of contact between non-carrier mosquitoes and infected individuals η : rate of treatment of infected individuals µ : amplitude of seasonal oscillation in the reproduction rate of mosquitoes ρ : probability of individuals becoming infected θ : fear factor, reflecting the increase in the population willingness to take actions to combat the mosquitoes as a consequence of the high prevalence of the disease in the specific social environment τ : forgetting rate for goodwill of the target population ϕ : phase angle to adjust the peak season for mosquitoes ω : angular frequency of the mosquitoes proliferation cycle, corresponding to a weeks period p : population in the risk area (usually normalized to yield p = ) γ d : the instantaneous costs due to the existence of infected individuals γ s : the costs of each operation of spraying insecticides γ e : the cost associated to the instructive campaigns the model consists in minimizing subject to the following four nonlinear time-varying state equations [ ] : . . , . equation ( . a) represents the variation of the mosquitoes density per unit time to the natural cycle of reproduction and mortality (α r and α m ), due to seasonal effects µ sin(ωt + ϕ) and to human interference −x (t) and u (t). equation ( . b) expresses the variation of the mosquitoes density carrying the virus x (t) denotes the rate of the infected mosquitoes and β [x (t) − x (t)] x (t) represents the increase rate of the infected mosquitoes due to the possible contact between the uninfected mosquitoes x (t) − x (t) and infected individuals denoted by x (t). the dynamics of the infectious transmission is presented in equation ( . c). the term −ηx (t) is related to the rate of cure and ρx (t) [p − x (t)] describes the rate at which new cases spring up. the factor [p − x (t)] is the number of individuals in the area, that are not infected. equation ( . d) is a model for the level of popular motivation (or goodwill) to combat the reproductive cycle of mosquitoes. over time, the level of people motivated will have changed. as a consequence, it is necessary to invest in educational campaigns designed to increase consciousness of the population under risk. the expression −τ x (t) represents the decay of the people's motivation over time, due to forgetfulness. the term θx (t) describes the natural sensibilities of the public due to increase in the prevalence of the disease. the goal is to minimize the cost functional ( . ). this functional includes social costs related to the existence of ill individuals -like absenteeism, hospital admission, treatments -, γ d x (t), the recourses needed for the spraying of insecticide operations, γ s u (t), and for educational campaigns, γ e u (t). the model for the social cost is based on the concept of goodwill explored by nerlove and arrow [ ] . due to computational issues, the optimal control problem ( . )-( . d), that was written in the lagrange form, was converted into an equivalent mayer problem. hence, using a standard procedure (cf., section . ) to rewrite the cost functional, the state vector was augmented by an extra component x , leading to the following equivalent terminal cost problem: with given t f , subject to the control system ( . a)-( . d) and ( . ). two different implementations were considered. in a first approach, the oc problem is solved by a specific optimal control package, oc-ode, already described in section . . the oc problem considers ( . a)-( . ) and the code is available in [ ] . a second approach uses the nonlinear solver ipopt [ ] , also described in section . . in order to use this software, it was necessary to discretize the problem. the euler discretization scheme was chosen (see section . for more details). the discretization step length was h = / , because it is a good compromise between precision and efficiency. thus, the optimal control problem was discretized into the following nonlinear programming problem: the error tolerance value was − using the ipopt solver. the discretized problem, after a presolve done by the software, has variables, of which are nonlinear; and constraints, of which are nonlinear (see the ampl code for this problem in [ ] ). [ , ] that was used by the authors of the paper [ ] . it is important to salient that, at the time of the initial paper [ ] , the authors had not the same computational resources that exist nowadays. the results with oc-ode and ipopt are better since the cost to fight the dengue disease and the number of infected individuals are smaller. [ ] . packages were tested, and they could not reach a solution -some crashed at the middle or some bad scaling issues were observed. until some years ago, due to computational limitations, most of the models were run using codes made by the authors themselves as [ ] . nowadays, one can choose between several proper software packages "out of the box", that already take into account specific features of stiff problems, scaling problems, etc. with this work it is possible to realize that "old" problems can again be taken into account and be better analyzed with new technology and approaches, with the goal of finding global optimal solutions, instead of local ones. for this purpose, and at an initial research stage, it is important to understand if different kinds of discretization for an oc problem have influence on the problem resolution. this section aims to study the costs of different discretization processes, in terms of time performance, number of variables and iterations used. for the purpose of this analysis two discretization schemes, euler and second order runge-kutta's scheme [ ] (cf. section . ), are considered to solve the problem described in the previous section. this discretization process transforms the dengue epidemics problem into a standard nonlinear optimization problem, with an objective function and a set of nonlinear constraints. this nlp problem was codified, for both discretization schemes, in the ampl modeling language [ ] and can be checked in [ ] . two nonlinear solvers with distinct features were selected to solve the nlp problem: the knitro (ip method) and the snopt (sqp method). the neos server [ ] platform was used as interface with both solvers. the ipopt (ip method), used in the previous section, was the first choice for our research. however, at the time of this investigation, the neos platform was moved to another center of research and some software packages were unavailable for long periods of time. so, we had to choose another interior point robust software. table . reports the results for both solvers, for each discretization method using three different discretization steps (h = . , . , . ), rising twelve numerical experiences. the columns # var. and # const. mean the number of variables and constraints, respectively. the next columns refer to the performance measures -number of iterations and total cpu time in seconds (time for solving the problem, for evaluate the objective and the constraints functions and for input/output). as the computational experiments were made in the neos server platform, the selected machine to run the program remains unknown as well as its technical specifications. the optimal cost reached was ≈ × − for all tests. comparing the general behavior of the solvers one can conclude that the ip based method (knitro) presents much better performance than the sqp method (snopt) in terms of the measures used. regarding the knitro results, one realizes that the euler's discretization scheme has better times for h = . and h = . and similar time for h = . , when compared to runge-kutta's method. another obvious finding, for both solvers, is that the cpu time increases as far as the problem dimension increases (number of variables and constraints). with respect to the number of iterations, snopt presents more iterations as the problem dimension increases. however this conclusion cannot be taken for knitro -in fact, there does not exist a relation between the problem dimension and the number of iterations. the best version tested was knitro using runge-kutta with h = . (best cpu time and fewer iterations), and the second one was knitro with euler's method using h = . . an important evidence of this numerical experience is that it is not worth the reduction of the discretization step size because no significative advantages are obtained. at this moment, as a result of major demographic changes, rapid urbanization on a massive scale, global travel and environmental change, the world faces enormous future challenges from emerging infectious diseases. dengue illustrates well these challenges [ ] . in this work we investigated an optimal control model for dengue epidemics proposed in [ ] , that includes the mosquitoes dynamics, the effect of educational campaigns. the cost functional reflects a compromise between financial spending in insecticides and educational campaigns and the population health. for comparison reasons, the same choice of data/parameters in [ ] was considered. the results obtained from oc-ode and ipopt are similar, improving the ones previously reported in [ ] (cf. section . ). indeed, the obtained control policy in this work presents an important progress with respect to the previous best policy: the percentage of infected mosquitoes vanishes just after four weeks, while mosquitoes are completely eradicated after weeks (figures . and . ); the number of infected individuals begin to decrease after four weeks while with the previous policy this only happens after weeks (figure . ) . despite the fact that our results are better, they are accomplished with a much smaller cost with insecticides and educational campaigns (figure . ) . the general improvement, which explains why the results are so successful, rely on an effective control policy of insecticides. the proposed strategy for insecticide application seems to explain the discrepancies between the results here obtained and the best policy of [ ] . our results show that applying insecticides in the first four weeks yields a substantial reduction in the cost of fighting dengue, in terms of the functional proposed in [ ] . the main conclusion is that health authorities should pay attention to the epidemic from the very beginning: effective control decisions in the first four weeks have a decisive role in the dengue battle, and population and governments will both profit from it. we successfully solved an oc problem by direct methods using nonlinear optimization software based on ip and sqp approaches. the problem was discretized through euler and runge-kutta schemes. the implementation efforts of higher order discretization methods bring no advantages. the reduction of the discretization step and consequently the increase of the number of variables and constraints do not improve the performance with respect to the cpu time and to the number of iterations. we can point out the robustness of both solvers in spite of the dimension problem increase. the conclusions drawn in section . were helpful for decision-making future processes of discretization all over the work. as future work we intend to analyze how different parameters/weights associated to the variables in the objective function can influence the spread of the disease. this chapter was based on work available in the peer reviewed journal [ ] and the peer reviewed conference proceedings [ ] . a model for the dengue disease transmission is presented. it consists of eight mutually-exclusive compartments representing the human and vector dynamics. it also includes a control parameter, adulticide spray, as a measure to fight the disease. the model presents three possible equilibria: two disease free equilibria (dfe) and an endemic equilibrium (ee). it has been proved that a dfe is locally asymptotically stable, whenever a certain epidemiological threshold, known as the basic reproduction number, is less than one. in this work we try to understand which is the best way to apply the control in order to effectively reduce the number of infected humans and mosquitoes. a case study, using outbreak data in in cape verde, is reported. in chapter the dengue epidemic was studied, mostly centered in people, specially in the goodwill of the individuals and spraying campaigns. however, the virus transmission scheme was overlooked: it was only considered two compartments for people and two compartments for adult mosquitoes. here, the aim is to deepen the relationship between human and mosquitoes, creating a better framework to explain the development and transmission of the disease. the mathematical model is based on [ , ] , that describes the chikungunya disease transmitted by aedes albopictus. the notation used in our mathematical model includes four epidemiological states for humans: it is assumed that the total human population (n h ) is constant, so, n h = s h + e h + i h + r h . there are also four other state variables related to the female mosquitoes (the male mosquitoes are not considered in this study because they do not bite humans and consequently they do not influence the dynamics of the disease): similarly, it is assumed that the total adult mosquito population is constant, which means n m = s m + e m + i m . in this way, we put our model more complex and reliable to the reality of dengue epidemics. for this study we introduced a control variable: the control variable, c(t), varies from to . however, the model does not fit completely the reality. epidemiologist and policy makers need to be aware of both strengths and weakness of the epidemiological modeling approach. an epidemiological model is always a simplification of reality. so, some assumptions were made to built this model: • the total human population (n h ) is constant; • there is no immigration of infected individuals into the human population; • the population is homogeneous, which means that every individual of a compartment is homogenously mixed with the other individuals; • the coefficient of transmission of the disease is fixed and does not vary seasonally; • both human and mosquitoes are assumed to be born susceptible, i.e., there is no natural protection; • for the mosquito there is no resistant phase, due to its short lifetime. to completely describe the model it is necessary to use parameters, which are: total population b : average daily biting (per day) β mh : transmission probability from i m (per bite) β hm : transmission probability from i h (per bite) /µ h : average lifespan of humans (in days) /η h : mean viremic period (in days) /µ m : average lifespan of adult mosquitoes (in days) ϕ : number of eggs at each deposit per capita (per day) µ a : natural mortality of larvae (per day) η a : maturation rate from larvae to adult (per day) /η m : extrinsic incubation period (in days) /ν h : intrinsic incubation period (in days) m : female mosquitoes per human k : number of larvae per human k : maximal capacity of larvae for notation simplicity, the independent variable t will be omitted when writing the dependent variables, example given, will be written s h instead of s h (t). the dengue epidemic can be modelled by the following nonlinear time-varying state equations: and vector population with the initial conditions for the previous set of differential equations is now analyzed the equilibrium points of the system and is determined the threshold phenomena. let the set proof. system ( . )-( . ) can be rewritten in the following way: as m (x) has all off-diagonal entries nonnegative, m (x) is a metzler matrix. using the fact that f ≥ , the system ( . ) is positively invariant in r + [ ] , which means that any trajectory of the system starting from an initial state in the positive orthant r + remains forever in r + . theorem . . . let Ω be defined as above. consider also the system ( . )-( . ) admits at most two disease free equilibrium points: • if m ≤ , there is a disease free equilibrium (dfe), called trivial equilibrium; • if m > , there is a biologically realistic disease free equilibrium (brdfe), proof. the equilibrium points are reached when the following equations hold: using the mathematica software to solve the system ( . ), we obtained four solutions. the first one is known as the trivial equilibrium, since the mosquitoes do not exist, so there is no disease: in the second one, mosquitoes and humans interact, but there is only one outbreak of the disease, i.e., over time the disease goes away without killing all the mosquitoes. we have called this equilibrium point a biologically realistic disease free equilibrium (brdfe), since it is a more reasonable situation to find in nature than the previous one: which is equivalent to e * = n h , , , kn hm η a µ b , kn hm µ b µm , , . this is biologically interesting only if m is greater than . the third solution corresponds to a situation where humans and mosquitoes live together but the disease persists in both populations, which means that it is not a dfe. this equilibrium will be explained after (see theorem . . ). thus the disease is not anymore an epidemic episode, but transforms into a endemic one. with some algebraic manipulations we obtained the following point: with the mathematica software we obtained a fourth solution. but some of the components are negative, which means that they do not belong to the Ω set. remark . the condition m > is equivalent, by algebraic manipulation, to the condition which corresponds to the basic offspring number for mosquitoes. thus, if m < , then the mosquito population will collapse and the only equilibrium for the whole system is the trivial equilibrium. if m ≥ , then the mosquito population is sustainable. the amount of mosquitoes is also related to an epidemic threshold: the basic reproduction number of the disease, r . following [ ] , we prove: . the equilibrium point brdfe is locally asymptotically stable if r < and unstable if r > . proof. to derive the basic reproduction number, we use the next generator approach. the basic reproduction number is calculated in a disease free equilibrium. in this case we consider the most realistic one, brdfe. following [ , ] , let consider the vector x t = (e h , i h , e m , i m ) which corresponds to the components related to the progression of the disease. the subsystem used is: this subsystem can be partitioned, represents the components related to new cases of disease (in this situation in the exposed compartments) and v(x) represents the other components. thus the subsystem ( . ) can be rewritten as let us consider the jacobian matrices associated with f and v: according to [ ] the basic reproduction number is r = ρ(j f (x ) j v − (x ) ), where x is a disease free equilibrium (brdfe) and ρ(a) defines the spectral radius of a matrix a. using mathematica and we obtain the value for the threshold parameter, with m > . in the model we have two different populations (human and vectors), so the expected basic reproduction number reflects the infection human-vector and also vector-human, that is, r = r hm × r mh . the term bβ hm sm n h represents the product between the transmission probability of the disease from humans to vectors and the number of susceptible mosquitoes per human; η h +µ h is related to the human's viremic period; and ηm c+ηm+µm represents the proportion of mosquitoes that survive to the incubation period. analogously, the term bβ mh s h n h is related to the transmission probability of the disease between mosquitos and human, in a susceptible population; when r < , each infected individual produces, on average, less than one new infected individual, and therefore it is predictable that the infection will be cleared from the population. if r > , the disease is able to invade the susceptible population [ , ] . theorem . . . if m > and r > , then the system ( . )-( . ) also admits an endemic equilibrium (ee): . proof. see proof of theorem . . . from a biological point of view, it is desirable that humans and mosquitoes coexist without the disease reaching a level of endemicity. good estimates of dengue transmission intensity are therefore necessary to compare and interpret dengue interventions conducted in different places and times and to evaluate options for dengue control. the basic reproduction number has played a central role in the epidemiological theory for dengue and other infectious diseases because it provides an index of transmission intensity and establishes a threshold criteria. we claim that proper use of control c, can result in the basic reproduction number remaining below unity and, therefore, making brdfe stable. in order to make effective use of achievable insecticide control, and simultaneously to explain more easily to the competent authorities its effectiveness, we assume that c is constant. the goal is to find c such that r < . for this purpose we have studied the reality of cape verde. an unprecedented outbreak was detected in the cape verde archipelago in september . this is the first report of dengue virus activity in that country. as the population had never had contact with the virus, the herd immunity was very low. dengue type spread throughout the islands of the archipelago, reaching four of the nine islands. the worst outbreak occurred on the santiago island, where most people live. the number of cases increased sharply since the beginning of november, reaching cases per day. the cape verde ministry of health reported more than cases of dengue fever within the archipelago between october and december , which is about % of the total population of the country. from cases of dengue hemorrhagic fever reported, six people died [ , ] . it represented a challenge to the performance of the national health care system. government officials launched a plan to eradicate the mosquito including a national holiday during which citizens were being asked to clear out standing water and other potential breeding areas used by the mosquitoes. the intense and spontaneous movement of solidarity from the civil society was another noteworthy dimension, not only cleaning but also voluntary donating blood to strengthen the stock of the central hospital dr. agostinho neto, in praia. we used the data for human population related to cape verde [ ] . due to low surveillance and the fact of being the first ever of dengue outbreak in the country, it was not possible to collect detailed data from the mosquito. however, the authorities speak explicitly about mosquitoes coming from brazil [ ] . also the information that comes from the ministry of health in the capital of cape verde, praia, confirms that the insects responsible for dengue came most probably from brazil, transported by means of air transport that perform frequent connections between cape verde and brazil, as reported by the radio of cape verde. with respect to aedes aegypti, we have thus considered data from brazil [ , ] . the simulations were carried out using the following values: n h = , b = , β mh = . , β hm = . , µ h = /( × ), η h = / , µ m = / , ϕ = , µ a = / , η a = . , η m = / , ν h = / , , m = , k = . the initial conditions for the problem were: considering nonexistence of control, i.e. c = , the basic reproduction number for this outbreak in cape verde is approximately r = . , which is in agreement to other studies of dengue in other countries [ ] . the control c affects the basic reproduction number, and our aim is to find a control that puts r less than one. this value was obtained by mathematica, calculating the inequality with the parameters values above. the computational investigations were carried out using c = . , which means that the insecticide is continuously applied during a twelve week period. the software used was scilab [ ] . it is an open source, cross-platform numerical computational package and a high-level, numerically oriented programming language. for our problem we used the routine ode to solve the set of differential equations. by default, ode uses the lsoda solver of package odepack. it automatically selects between the nonstiff predictor-corrector adams method and the stiff backward differentiation formula (bdf) method. it uses the nonstiff method initially and dynamically monitors data in order to decide which method to use. the graphics were also obtained using this software, with the command plot (see code in [ ] ). figures . and . show the curves related to human population, with and without control, respectively. the number of infected people, even with small control, is much less than without any insecticide campaign. figures . and . show the difference of the mosquito population with control and without control. when the control is applied, the number of infected mosquitoes is close to zero. note that the intention is not to completely eradicate the mosquitoes but instead the number of infected mosquitoes. it has been algebraically proved that if a constant minimum level of a control is applied (c = . ), it is possible to maintain the basic reproduction number below unity, guaranteeing the brdfe. this value is corroborated in another numerical study [ ] . the values of infected humans obtained by the model are higher when compared to what really happened in cape verde. despite the measures taken by the government and health authorities were not accounted, they have had a considerable impact in the progress of the disease. until here, it was considered a constant control. using a theoretical approach [ ] , we intend to find the best function c(t), using oc theory. instead of finding a constant control it will be possible to study other types of control, such as piecewise constant or even continuous but not constant. additionally we could consider another strategy, a more practical one: due to logistics and health reasons, it may be more convenient to apply insecticide periodically and at some specific hours at night. in this section we investigate the best way to apply the control in order to effectively reduce the number of infected humans and mosquitoes, using pulse control. in literature it has been proven that a dfe is locally asymptotically stable, whenever a certain epidemiological threshold, known as the basic reproduction number , is less than one. in the previous section, it was proven that if a constant minimum level of insecticide is applied (c = . ), it is possible to maintain the basic reproduction number below unity, guaranteeing the dfe. in this section, other kinds of piecewise controls that maintain the basic reproduction number less than one and could give a better solution to implement by health authorities are investigated. to solve the system ( . )-( . ), in a first step, several strategies of control application were used: three different frequencies (weekly, bi-weekly and monthly) for control application, a constant control (c = . ) and no control (c = ). the first three different frequencies mean that during one day (per week, bi-week and month), the whole ( %) capacity of insecticide (c = ) is used during all day. besides, also was used a constant control strategy (c = . ) that consists in the application of . % capacity of insecticide hours per day all the time ( days). in this work, the amount of insecticide is an adimensional value and must be considered in relative terms. the numerical tests were carried out using scilab [ ] , with the same ode system and parameters of the previous section (code available in [ ] ). figures . and . show the results of these strategies regarding infected mosquitoes and individuals. without control, the number of infected mosquitoes and individuals increase expressively. it is also possible to see that the weekly pulse control had the closest results to the continuous control. therefore, realizing the influence of the insecticide control, further tests were carried out to find the optimum periodicity of administration which, from gathered results, must rest between six and seven days. the second phase of numerical tests, figures . and . , considered four situations: days, days, days and continuously c = . . to guarantee the dfe, the curves must remain below the one corresponding to c = . . the amount of insecticide, and when to apply it, are important factors for outbreak control. table . reports the total amount of insecticide used in each version during the days. the numerical tests conclude that the best strategy for the infected reduction is every / days application. the value spent in insecticide is similar to the continuous strategy, but is much easier to implement in this work, several piecewise strategies were studied to find the best way of applying insecticide, but always having in mind a periodic application of the product. but if the best strategy is not a periodic one? in the next section the optimal control solution for this problem will be studied. the aim of this section is the study of optimal strategies for applying insecticide, taking into account different perspectives: thinking only on the insecticide cost, focusing on the cost of infected humans or even combining both perspectives. to take this approach, and after several numerical experiences, we considered that it was better to normalize the ode system ( . )-( . ). the reason for this transformation is due to the bad scaling of the variables: some vary from to , and others from to , which can influence the software performance. consider the following transformations: the ode system ( . )-( . ) is transformed into: with the initial conditions let us consider the objective functional j considering the costs of infected humans and costs with insecticide: where γ d and γ s are positive constants representing the costs weights of infected individuals and spraying campaigns, respectively. using oc theory, let λ i (t), with i = , . . . , , be the co-state variables. the hamiltonian for the present oc problem is given by by the pontryagin maximum principle [ ] , the optimal control c * should be the one that minimizes, at each instant t, the hamiltonian given by ( . ), that is, h (x * (t), λ * (t), c * (t)) = min c∈[ , ] h (x * (t), λ * (t), c). in this way, the optimal control is given by it is also necessary to consider the adjoint system λ i (t) = − ∂h ∂x i , i.e., as the oc problem just has initial conditions it is necessary to find the transversality conditions, that correspond to a terminal condition in the co-state equation, replacing the optimal control c * in the state system ( . ) and in the adjoint system ( . ) is possible to solve the differential system taking into account the initial and transversality conditions. in order to solve this oc problem three approaches were tested. the first one is a direct method, dotcvp toolbox [ ] for matlab. it uses the differential system ( . ), the initial conditions ( . ) and it is necessary to transform the problem into a mayer form (see section . ). to solve the discretized oc problem, the ipopt software is chosen as an option inside the dotcvp. the functional is divided into time intervals, in this case ten intervals, with an initial value problem tolerance of − and a nlp tolerance of − . the optimal control function given by this toolbox is piecewise constant. another direct method is oc-ode [ ] . it uses the differential system ( . ), the initial conditions ( . ) and it includes procedures for numerical adjoint estimation and sensitivity analysis and the feasibility tolerance considered was − . the last method used, an indirect one, it is coded in matlab environment. it involves the backwardforward method (see section . ) and the resolution of the ode systems are made by the ode routine. the state differential system ( . ) is solved forward with initial conditions ( . ), while the adjoint system ( . ) is solved backwards using the terminal conditions ( . ). the absolute and relative tolerances were fixed in − . the three codes are available at [ ] . all the parameters are assumed equal to the previous section. for this first simulation, the values for the weights of the costs are γ d = . and γ s = . . figure . shows that, despite having distinct philosophies of resolution, the curves obtained by the three solvers are similar, which reinforces the confidence in the result. the optimal functional is . , . and . , for dotcvp, oc-ode and backward-forward, respectively. the last solver achieved better value for the functional. this is expected, once its resolution contains more information about the oc problem because the adjoint system is supplied. the study considers three situations: a, b, and c. situation a, that was previously presented, regards both perspectives in the functional (human infected and insecticide application). situation b concerns only to infected humans whereas case c only considers insecticide campaigns. table . resumes the three situations. functional values for weights case a both perspectives since the three solvers presented similar solutions, only one of them was chosen to solve these cases, that was dotcvp. figures . , . and . show the results for optimal control c, infected humans and total costs, respectively. in the medical perspective (case b), when only the costs related with ill people (absenteeism, drugs, ...) are considered, the number of infected is the lowest; however a huge quantity of insecticide is used, because it is considered cheaper. on the other hand, when people just think on the economical perspective (case c), the treatment for people is neglected. optimal control is low, but the number of infected humans is high. the total cost is higher when both perspectives are considered. epidemiological modelling has largely focused on identifying the mechanisms responsible for epidemics but has taken little account on economic constraints in analyzing control strategies. economic models have given insight into optimal control under constraints imposed by limited resources, but they frequently ignore the spatial and temporal dynamics of the disease. nowadays the combination of epidemiological and economic factors is essential. the bioeconomic approach requires an equilibrium between economic and epidemiological parameters in order to give an efficient disease control and reflecting the nature of the epidemic. for this, the study goes on implementing both perspectives, but taking into account distinct weights of the parameters associated to the variables i h and c. table . resumes these approaches. values for weights table . : different weights for the functional in case d, it is studied a situation where the lack of insecticide in a country could be a reality and as a consequence its market value is high. this could happen due to an unprecedent outbreak where the authorities were not prepared or even due to financial reasons by the fact that the government does not have financial viability for this kind of measure. in case e, once again the human perspective gains strength and, as human life and quality of life is an expensive good, it was considered that it is more expensive to treat humans then apply insecticide. the analysis from the figures from . to . is consistent with what we expected in reality. in case d, as insecticide is expensive, the function for optimal control is lower than the other perspective. as consequence, the number of infected people is higher. in case e, where the human factor is preponderant, the number of infected humans is low but expenses with insecticide are higher. curiously the total cost, in case d and e, are of the same order of magnitude, with a slightly higher cost for case e. the total cost is reported in table . . when both perspectives are considered, the total cost is higher than the single perspective. for a last analysis, a mathematical perspective was carried out: what values should the γ d and γ s have, in order to minimize the functional? let us call this perspective as case f. in this perspective, we not only want to minimize the control c, but also the parameters γ d and γ s , enforcing the equality constraint γ d + γ s = . show the comparison of this case with the first one. as expected, the total cost and the number of infected humans are the lowest ones. giving freedom to the parameters it is possible to see that the optimal control function is not periodic (as studied in the previous section), but gives a practical solution in applying insecticide. in this chapter a model based on two populations, humans and mosquitoes, with insecticide control is presented. it has shown that as time goes by, depending on several parameters, the outbreak could disappear (leading to a dfe) or could transform the disease into an endemic one (leading to an ee). this assuming that the parameters are fixed, the only variable that can influence this threshold is the control variable c, it has shown that with a steady insecticide campaign it is possible to reduce the number of infected humans and mosquitoes, and can prevent an outbreak that could transform an epidemiological episode to an endemic disease. for a steady campaign has proven that c = . it is enough to maintain the r below unit. however, this type of control is difficult to implement. a pulse insecticide campaign was studied to circumvent this difficulty. it has proven that applying insecticide every / days, is a better strategy to implement by health authorities and has the same efficacy level and financial costs. finally, the oc theory was used to find the best optimal control function for the insecticide. the optimal function varies, giving a different answer depending on the main goal to reach, thinking in economical or human centered perspective. as future work it is important to study different kinds of controls. the accelerated increase in mosquito resistance to several chemical insecticides and the damage caused by these to the environment, has resulted in the search for new control alternatives. among the alternatives available, the use of bacillus thuringiensis israelensis (bti) has been adopted by several countries [ ] . laboratory testing shows that bti has a high larvicide property and its mechanism of action is based on the production of an endotoxin protein that, when ingested by the larvae, causes death. to ensure the minimization of the outbreaks, educational programmes that are customized for different levels of health care and that reflect local capacity should be supported and implemented widely. people should be instructed to minimize the number of potential places for the mosquito breeding. educational campaigns can be included as an extra control parameter in the model. this chapter was based on work available in the peer reviewed journal [ ] and the peer reviewed conference proceedings [ , ] . a new model with six mutually-exclusive compartments related to dengue disease is presented. in this model there are three vector control tools: insecticides (larvicide and adulticide) and mechanical control. the human data for the model is again related to cape verde. due to the rapid development of the outbreak on the islands, only a few control measures were taken, but not quantified. in this chapter, some of these measures are simulated and their consequences are analyzed. in chapter , a model with eight compartments and a single control was analyzed. however, after discussion with some researchers in this area, many of them suggested removing the compartment exposed for three main reasons: first, it is difficult to collect data for this compartment, since the disease at this stage does not show symptoms; second, the curve obtained is similar to the infected compartment with only an advance of time, not bringing novelty to the model, but possible difficulties to the numeric resolution; and finally, as the main goal is to study the effects of several controls centered in the infected humans, this compartment plays a secondary role. thus, it was decided to remove the exposed compartments, in humans and mosquitoes, adjusting the other parameters to this new model and including three controls. taking into account the model presented in [ , ] and the considerations of [ ] - [ ] , a new model more adapted to the dengue reality is proposed. the notation used in the mathematical model includes three epidemiological states for humans: it is assumed that the total human population (n h ) is constant and n h = s h (t) + i h (t) + r h (t) at any time t. the population is homogeneous, which means that every individual of a compartment is homogeneously mixed with the other individuals. immigration and emigration are not considered. there are three other state variables, related to the female mosquitoes: due to the short lifespan of mosquitoes, there is no resistant phase. it is assumed homogeneity between host and vector populations, which means that each vector has an equal probability to bite any host. humans and mosquitoes are assumed to be born susceptible. to analyze the effect of campaigns in the disease fight, three controls are considered: proportion of mechanical control, < α ≤ . larval control targets the immature mosquitoes living in water before they become biting adults. a soil bacterium, bacillus thuringiensis israelensis (bti), is applied from the ground or by air to larval habitats. this bacterium is used because when properly applied, it has virtually no effect on non-target organisms. the control of adult mosquitoes is necessary when mosquito populations cannot be treated in their larval stage. it is the most effective way to eliminate adult female mosquitoes that are infected with human pathogens. depending on the size of the area to be treated, either trucks for ground adulticide treatments or aircraft for aerial adulticide treatments can be used. the purpose of mechanical control is to reduce the number of larval habitat areas available to mosquitoes. the mosquitoes are most easily controlled by treating, cleaning and/or emptying containers that hold water, since the eggs of the specie are laid in water-holding containers. the aim is to simulate different realities in order to find the best policy to decrease the number of infected humans. a temporal mathematical model is introduced, with mutually-exclusive compartments, to study the outbreak occurred on cape verde islands in and improving the model described in [ ] . the model uses the following parameters: average daily biting (per day) β mh : transmission probability from i m (per bite) β hm : transmission probability from i h (per bite) /µ h : average lifespan of humans (in days) /η h : mean viremic period (in days) /µ m : average lifespan of adult mosquitoes (in days) ϕ : number of eggs at each deposit per capita (per day) /µ a : natural mortality of larvae (per day) η a : maturation rate from larvae to adult (per day) m : female mosquitoes per human k : number of larvae per human the dengue epidemic is modelled by the following nonlinear time-varying state equations: with the initial conditions due to biological reasons, only nonnegative solutions of the differential system are acceptable. more precisely, it is necessary to study the solution properties of the system ( . )-( . ) in the closed set it can be verified that Ω is a positively invariant set with respect to ( . )-( . ). the proof of this statement is similar as in [ ] . the system ( . )-( . ) has at most three biologically meaningful equilibrium points (cf. theorem . . ). is said to be an equilibrium point for system ( . )-( . ) if it satisfies the following relations: an equilibrium point e is biologically meaningful if and only if e ∈ Ω. the biologically meaningful equilibrium points are said to be disease free or endemic depending on i h and i m : if there is no disease for both populations of humans and mosquitoes (i h = i m = ), then the equilibrium point is said to be a disease free equilibrium (dfe); otherwise, if i h = or i m = (in other words, if i h > or i m > ), then the equilibrium point is called endemic. theorem . . . system ( . )-( . ) admits at most three biologically meaningful equilibrium points; at most two dfe points and at most one endemic equilibrium point. more precisely, let , . if m ≤ , then there is only one biologically meaningful equilibrium point, e * , which is a dfe point. if m > with ξ ≥ χ, then there are two biologically meaningful equilibrium points, e * and e * , both dfe points. if m > with ξ < χ, then there are three biologically meaningful equilibrium points, e * , e * , and e * , where e * and e * are dfes and e * endemic. proof. system ( . ) has four solutions easily obtained with a computer algebra system like maple: e * , e * , e * and e * . the equilibrium point e * is always a dfe because it always belongs to Ω with i h = i m = . in contrast, e * is never biologically realistic because it has always some negative coordinates. the other two equilibrium points, e * and e * , are biologically realistic only for certain values of the parameters. the equilibrium e * is biologically realistic if and only if m ≥ , in which case it is a dfe. for the condition m ≤ , the third equilibrium e * is not biologically realistic. if m > , then three situations can occur with respect to e * : if ξ = χ, then e * degenerates into e * , which means that e * is the dfe e * ; if ξ > χ, then e * is not biologically realistic; otherwise, one has e * ∈ Ω with i h = and i m = , which means that e * is an endemic equilibrium point. by algebraic manipulation, m > is equivalent to condition is related to the basic offspring number for mosquitos. thus, if m ≤ , then the mosquito population will collapse and the only equilibrium for the whole system is the trivial dfe e * . if m > , then the mosquito population is sustainable. from a biological standpoint, the equilibrium e * is more plausible, because the mosquito is in its habitat, but without the disease. an important measure of transmissibility of the disease is now introduced: the basic reproduction number. it provides an invasion criterion for the initial spread of the virus in a susceptible population. for this case the following result holds. theorem . . . the basic reproduction number r associated to the differential system ( . )-( . ) is proof. in agreement with [ ] , just the epidemiological compartments that have new infections, i h and i m , are considered. the two differential equations related to these two compartments can be rewritten where f is the rate of production of new infections and v is the transition rates between states: the jacobian derivatives are the quantity j f (x) j − v(x) gives the total production of new infections over the course of an infection. the largest eigenvalue gives the fastest growth of the infected population, which means that r is the spectral radius of the matrix j f (x) j − v(x) in a dfe point. maple was used to obtain the basic reproduction number r in ( . ) is obtained, replacing s h df e and s m df e in ( . ) by those of the dfe e * . the model has two different populations (host and vector) and the expected basic reproduction number should reflect the infection transmitted from host to vector and vice-versa. accordingly, r can be seen as r = (r hm × r mh ) if r < , then, on average, an infected individual produces less than one new infected individual over the course of its infectious period, and the disease cannot grow. conversely, if r > , then each individual infects more than one person, and the disease can invade the population. proof. the only solution of ( . ) with i h > or i m > , the only endemic equilibrium, is e * . that occurs, in agreement with theorem . . , in the case m > and χ > ξ. the condition χ > ξ is equivalent, by theorem . . , to r > . using the methods in [ , ] , it is possible to prove that if r ≤ , then the dfe is globally asymptotically stable in Ω, and thus the vector-borne disease always dies out; if r > , then the unique endemic equilibrium is globally asymptotically stable in Ω, so that the disease, if initially present, will persist at the unique endemic equilibrium level. assuming that parameters are fixed, the threshold r is influenceable by the control values. figure . gives this relationship. it is possible to realize that the control c m is the one that most influences the basic reproduction number to stay below unit. besides, the control in the aquatic phase alone is not enough to maintain r below unit: an application close to % is required. the simulations were carried out using the following numerical values: n h = , b = . , β mh = . , β hm = . , µ h = /( × ), η h = / , µ m = / , ϕ = , µ a = / , η a = . , m = , k = . the initial conditions for the problem were: s h = n h − , i h = , r h = , a m = kn h , s m = mn h , i m = . with these values, one has m > . as in the previous chapter the values related to humans describe the reality of cape verde [ ] and the information about mosquitoes is based on brazil [ , ] . all computational calculus consider one year for time interval. although the final time was t f = days, the figures show graphics in suitable windows, in order to provide a better analysis. all the simulations and graphics were done in matlab. to solve the differential equation system, the ode routine was used. this function implements a runge-kutta method with a variable time step for efficient computation (see [ ] for more details about the code). the number of infected humans from the model ( . )-( . ) is higher when compared with what really happened in cape verde. as far as it was possible to investigate in the local news, the government of cape verde has done their best to banish the mosquito, with media campaigns appealing to people to remove or cover all recipients that could serve to breed the mosquito, and to use insecticide in critical areas. however, it was not possible to quantify those efforts in precise terms. next, follows a set of simulations using different controls. in each figure, only one control is used, continuously, which means that the others are not applied. the aim of this simulation is to see the importance of the control and what repercussions has on the model. figures . and . concern the adulticide control, figures . and . the larvicide control, and figures . and . the mechanical control. using a small quantity of each control, the number of infected people falls dramatically. in some cases, in spite of all graphs displaying five simulations, some curves are so close to zero that it is difficult to distinguish them. figures . and . show that excellent results for the human population are obtained by covering only % of the country with insecticide for adult mosquitoes. the simulations were done considering that the aedes aegypti does not become resistant to the insecticide and that it is financially possible to apply insecticide during all time. figures . - . are related to the applied controls in the aquatic phase of the mosquito. in these graphics the controls were studied separately, but one is closely related to the other. the application of these controls are not sufficient to decrease the infected human to zero, but the removal in the next section, using oc strategy we will find the best solution for the controls. epidemiological models may give some basic guidelines for public health practitioners, comparing the effectiveness of different potential management strategies. in reality, a range of constraints and trade-offs may substantially influence the choice of practical strategy, and therefore their inclusion in any modelling analysis may be important. frequently, epidemiological models need to be coupled to economic considerations, such that control strategies can be judged through holistic cost-benefit analysis. control of livestock disease is a scenario when cost-benefit analysis can play a vital role in choosing between cheap, weak controls that lead to a prolonged epidemic, or expensive but more effective controls that lead to a shorter outbreak. normalizing the previous ode system ( . )-( . ), we obtain: with the initial conditions the cost functional considered was where γ d , γ s , γ l and γ e are weights related to costs of the disease, adulticide, larvicide and mechanical control, respectively. in a first approach to this problem, it is assumed that all weights are the same, which means γ d = γ s = γ l = γ e = . (case a). the oc problem was solved using two different packages: dotcvp [ ] and muscod-ii [ ] . the mathematical formulation of the sir+asi problem, for the both packages, is available on [ ] . the simulation behavior is similar, and we decided to show only the dotcvp results. the optimal functions for the controls are given in figure . . the adulticide was the control that more influenced the decreasing of that ratio and, as consequence, the decreasing of the number of infected people and mosquitoes, matching with the results obtained for the basic reproduction number in section . . therefore, the adulticide was the most used. we believe that the other controls do not assume an important role in the epidemic episode, because all the events happened at a short period of time, which means that adulticide has more impact. however the mosquito control in the aquatic phase can not be neglected. in situations of longer epidemic episodes or even in an endemic situation, the larval control represents an important tool. figure . presents the number of infected humans. comparing the optimal control case with a situation with no control, the number of infected people decreased considerably. besides, in the situation where oc is used, the peak of infected people is minor, which facilitates the work in health centers, because they can provide a better medical monitoring. a second analysis was made, taking into account different weights on the functional. table . resumes the weights chosen for perspectives: not only economic issues (cost of insecticides and educational campaigns), but also human issues are considered. in case a, all costs were equal. in case b is given more impact on the infected people, considering that the treatment and absenteeism to work is very prejudicial to figure . : comparison of infected individuals under an optimal control situation and no controls the country, when compared with the cost of insecticides and educational campaigns. in case c, the costs with killing mosquitoes and educational campaigns have more impact in the economy. higher total costs were obtained when human life had more weight than controls measures as can be checked in table table . : different weights for the functional and respective values figure . shows the number of infected human in each bioeconomic perspective. we can realize that case a and case c are similar. it can be explained by the low weight given to the cost of treatment (cases a and c) when compared with the heavy weight given in case b. figure . presents the behavior of the controls for the a, b and c cases. again, as adulticide is the one that has more influence on the model, this is the control that most varies when the weights are changed. a third analysis was made to the model: it changed the functional in order to study the effects of each control when separately considered. therefore, the new functional also considers bioeconomic perspectives, but only includes two variables: the costs with infected humans (with γ d = . ) and the costs with only one control (with γ i = . , i ∈ {s, l, e}). thus, in figure . are presented the proportion of adulticide (a) and infected humans (b), when the functional is dengue disease breeds, even in the absence of fatal forms, significant economic and social costs: absenteeism, debilitation and medication. to observe and to act at the epidemics onset could save lives and resources to governments. moreover, the under-reporting of dengue cases is probably the most important barrier in obtaining an accurate assessment. a compartmental epidemiological model for dengue disease composed by a set of differential equations was presented. simulations based on clean-up campaigns to remove the vector breeding sites, and also simulations on the application of insecticides (larvicide and adulticide), were made. it was shown that even with a low, although continuous, index of control over time, the results are surprisingly positive. the adulticide was the most effective control, since with a low percentage of insecticide, the basic reproduction number is kept below unit and the number infected humans was smaller. however, to bet only in adulticide is a risky decision. in some countries, such as mexico and brazil, the prolonged use of adulticides has been increasing the mosquito tolerance capacity to the product or even they become completely resistant. in countries where dengue is a permanent threat, governments must act with differentiated tools. it will be interesting to analyze these controls in an endemic region and with several outbreaks. we believe that the results will be quite different. aedes aegypti eradication is not considered to be feasible and, from the environmental point of view, not desirable. the aim is to reduce the mosquito density and, simultaneously, amount the level of immunity on the human population. the increase of population herd immunity can be reached by two ways: increasing the resistant people to the disease implying an increasing of infected individuals or with a vaccination campaign. no commercially available clinical cure or vaccine is currently available for dengue, but efforts are underway to develop one [ , ] . the potential of prevention of dengue by immunization seems to be technically feasible and progress is being made in the development of vaccines that may protect against all four dengue viruses. in the next chapter, a model of this disease with a vaccine simulation as a new strategy to fight the disease will be analyzed. this chapter was based on work accepted in the peer reviewed journal [ ] and the peer reviewed conference proceedings [ ] . as the development of a dengue vaccine is ongoing, it is simulated an hypothetical vaccine as an extra protection to the population. in a first phase, the vaccination process is studied as a new compartment in the model, and some different types of vaccines are simulated: pediatric and random mass vaccines, with distinct levels of efficacy and durability. in a second step, the vaccination is seen as a control variable in the epidemiological process. in both cases, epidemic and endemic scenarios are included in order to analyze distinct outbreak realities. in , the swiss mathematician daniel bernoulli published a study on the impact of immunization with cowpox upon the expectation of life of the immunized population [ ] . the process of protecting individuals from infection by immunization has become a routine, with historical success in reducing both mortality and morbidity. the impact of vaccination may be regarded not only as an individual protective measure, but also as a collective one. while direct individual protection is the major focus of a mass vaccination program, the effects on population also contribute indirectly to other individual protection through herd immunity, providing protection for unprotected individuals [ ] (see scheme in figure . ). this means that when we have a large neighborhood of vaccinated people, a susceptible individual has a lower probability in coming into contact with the infection, being more difficult for diseases to spread, which decreases the relief of health facilities and can break the chain of infection. vector control remains the only available strategy against dengue. despite integrated vector control with community participation, along with active disease surveillance and insecticides, there are only a [ ] . besides, the levels of resistance of aedes aegypti to insecticides has increased, which implies shorter intervals between treatments, and only few insecticide products are available in the market due to the high costs for development and registration and low returns [ ] . dengue vaccines have been under development since the s, but due to the limited appreciation of global disease burden and the potential markets for dengue vaccines, industry interest languished throughout the th century. however, in recent years, the development of dengue vaccines has accelerated dramatically with the increase in dengue infections, as well as the prevalence of all four circulating serotypes. faster development of a vaccine became a serious concern [ ] . economic analysis are now conducted periodically to guide public support for vaccine development in both industrialized and developing countries, including a previous cost-effectiveness study of dengue [ , , ] . the authors compared the cost of the disease burden with the possibility of making a vaccination campaign; they suggest that there is a potential economic benefit associated with promising dengue interventions, such as dengue vaccines and vector control innovations, when compared to the cost associated to the disease treatments. constructing a successful vaccine for dengue has been challenging: the knowledge of disease pathogenesis is insufficient and in addition the vaccine must protect simultaneously against all serotypes in order to not increase the level of dhf [ ] . nevertheless, several promising approaches are being investigated in both academic and industrial laboratories. vaccine candidates include live attenuated vaccines obtained via cell passages or by recombinant dna technology (such as those being developed by the us national institutes of allergy and infectious diseases, inviragen, walter reed army institute of research/glaxosmithkline, and sanofi pasteur) and subunit vaccines (such as those developed by merck/hawaii biotech) [ , ] . recent studies indicate that, by the progress in clinical development of sanofi pasteur's live attenuated tetravalent chimeric vaccine, a vaccine could be licensed as early as [ ] . the team is carrying out an efficacy study on a vaccine covering four serotypes on children aged four to eleven years old in muang district, thailand. at this time, the features of dengue vaccine are mostly unknown. so, in this chapter we opt to present a set of simulations with different types of vaccines and we have explored also the vaccination process under two different perspectives. the first one uses a new compartment in the model and several kinds of vaccination are considered. a second perspective is studied using the vaccination process as a disease control in the mathematical formulation. in this case the theory of oc is applied. both methods assume a continuous strategy vaccination. in this section, a new compartment v is added to the previous sir model related to the human population. this new compartment represents the new group of human population that is vaccinated, in order to distinguish the resistance obtained through vaccination and the one achieved by disease recovery. two forms of random vaccination are possible: the most common for human disease is pediatric vaccination to reduce the prevalence of an endemic disease; the alternative is random vaccination of the entire population in an outbreak situation. in both types, the vaccination can be considered perfect conferring % protection for all life or else imperfect. this last case can be due to the difficulty of producing an effective vaccine, the heterogeneity of the population or even the life span of the vaccine. for many potentially human infections, such as measles, mumps, rubella, whooping cough, polio, there has been much focus on vaccinating newborns or very young infants. dengue can be a serious candidate for this type of vaccination. in the sv ir model, a continuous vaccination strategy is considered, where a proportion of the newborn p (where ≤ p ≤ ), was by default vaccinated. this model also assumes that the permanent immunity acquired through vaccination is the same as the natural immunity obtained from infected individuals eliminating the disease naturally. the population remains constant, i.e., n h = s h + v h + i h + r h . the new model for human population is represented in figure we are assuming that it is a perfect vaccine, which means that it confers life-long protection. as a first step, it is necessary to determine the basic reproduction number without vaccination (p = ). theorem . . . the basic reproduction number, r , associated to the differential system ( . ) without vaccination is given by proof. the proof of this theorem is similar to the one in the previous chapter (see proof of theorem . . ). just consider in this chapter, we make all the simulations in two scenarios: an epidemic and an endemic situation (programming codes available in [ ] ). for these, the following parameter values of the differential system and initial conditions were used (tables . and . ). there were two main differences between the epidemic episode and an endemic situation. firsty, in the endemic situation there was a slight decrease in the average daily biting b and transmission probabilities β mh and β hm , that could be explained by the fact that the mosquito could have more difficulties to find a naive individual. the second difference is concerned with the strong increase of the initial human population that is resistant to the disease. this may be explained by the fact that the disease, in an endemic situation, already creates an immune resistance to the infection, i.e., the population already has herd immunity. with these values we obtain approximately r = . and r = . for epidemic and endemic scenarios, respectively. during an outbreak, the disease transmission assumes different behaviors, according to the distinct scenarios, as can been seen in figure . . in one year, the peak in an epidemic situation could reach more than cases. in contrast, instead in the endemic situation the curve of infected individuals has a more smooth behavior and reaches a peak less than cases. figure . relates to the mosquito population. in the endemic scenario, because a substantial part of the human population is resistant to the disease, the infected mosquitoes bite a considerable percentage of resistant host, and as consequence, the disease is not transmitted. suppose that at time t = a proportion p of newborns are vaccinated with a perfect vaccine that causes no side effects. since this proportion, p, is now immune, r is reduced, creating a new basic reproduction number. definition (basic reproduction number with pediatric vaccination). the basic reproduction number with pediatric vaccination, r p , associated to the differential system ( . ) is given by where r is defined in ( . ). observe that r p ≤ r . equality is only achieved when p = , i.e., when there is no vaccination. the constraint r p < implicitly defines a critical vaccination portion p > p c that must be achieved for eradication, where since vaccination entails costs, to choose the smallest coverage that achieves eradication is the best option. this way, the entire population does not need to be vaccinated in order to eradicate the disease. this phenomenon is called herd immunity. vaccinating at the critical level, p c , does not instantly lead to disease eradication. the immunity level within the population requires time to build up and at the critical level it may take a few generations before the required herd immunity is achieved. thus, from a public health perspective, p c acts as a lower bound on what should be achieved, with higher levels of vaccination leading to a more rapid elimination of the disease. figure . shows the simulations related to the proportion of newborns vaccinated (p = , . , . , . , ) in both scenarios. notice that at time t = no person was vaccinated. in the epidemic situation, as the outbreak reached a peak at the beginning of the year, the proportion of newborns vaccinated at that time is minimum and cannot influence the curve of infected individuals, giving the optical illusion of a single curve. on the other hand, in the endemic case, as the outbreak occurs later, the vaccination campaign starts to produce effects, decreasing the total number of sick humans. this last graphic illustrates that a vaccination campaign centered in newborns is a bet for the future of a country, but does not produce instantly results to fight the disease. to produce immediate results, it is necessary to use random mass vaccination, which means that it is necessary to vaccine a significant part of the population. a mass vaccination program may be initiated whenever there is an increase of the risk of an epidemic. in such situations, there is a competition between the exponential increase of the epidemic and the logistical constraints upon mass vaccination. for most human diseases it is possible, and more efficient, to not vaccinate those individuals who have recovered from the disease because they are already protected. another situation could be the introduction of a new vaccine in a population that lives an endemic situation. let us consider the control technique of constant vaccination of susceptibles. in this scheme a fraction ≤ ψ ≤ of the entire susceptible population, not just newborns, is being continuously vaccinated. it is assumed that the permanent immunity acquired by vaccination is the same as natural immunity obtained from infected individuals in recovery. the epidemiological scheme is presented in figure the mathematical formulation for human population (the differential equations related to the mosquito remain equal to the previous subsection) is given by: for this model, we define a new basic reproduction number. definition (basic reproduction number with random mass vaccination [ ] ). the basic reproduction number with random mass vaccination, r ψ , associated to the differential system ( . ) is where r is defined in ( . ) . comparing this model with the constant vaccination of newborns model, it is apparent that instead of constantly vaccinating a portion of newborns, a part of the entire susceptible population is now being continuously vaccinated. since the natural birth rate µ h is usually small, the fraction pµ h of newborns being continuously vaccinated will be small, whereas in this model, will be also a larger group of susceptibles can be continuously vaccinated, ψs h . due to this, we expect that this model should require a smaller proportion of ψ to achieve eradication. notice that r ψ ≤ r . equality is only achieved in the limit ψ = , that is, when there is no vaccination. the constraint r ψ < implicitly defines a critical vaccination portion ψ > ψ c that must be achieved for eradication, where ψ c = (r − ) µ h . observe that in spite of the calculations done in the period of days, the figures only show suitable windows, in order to provide a better analysis. in both scenarios, even with a small coverage of population, vaccination dramatically decreases the number of infected. the epidemic scenario has changed from less than cases (with no vaccination, figure . ) to less than cases vaccinating only % of population. in the endemic scenario, the decrease is more accentuated. until here, we have considered a perfect vaccine, which means that every vaccinated individual remains resistant to the disease. however, a majority of the available vaccines for the human population does not produce % success in the disease battle. usually, the vaccines are imperfect, which means that a minor percentage of cases, in spite of vaccination, are infected. most of the theory on the disease evolution is based on the assumption that the host population is homogeneous. individual hosts, however, may differ and they may constitute very different habitats. in particular, some habitats may provide more resources or be more vulnerable to virus exploitation [ ] . the use of models with imperfect vaccines can describe better this type of human heterogeneity. another explanation for the use of imperfect vaccines is that until now we had considered models that assumed that as soon as individuals begin the vaccination process, they become immediately immune to the disease. however, the time it takes for individuals to obtain immunity by completing a vaccination process cannot be ignored, because meanwhile an individual can be infected. in this section a continuous vaccination strategy is considered, where a fraction ψ of the susceptible class was vaccinated. the vaccination may reduce but not completely eliminate susceptibility to infection. for this reason we consider a factor σ as the infection rate of vaccinated members. when σ = the vaccine is perfectly effective and when σ = the vaccine has no effect at all. the value − σ can be understood as the efficacy level of the vaccine. the new model for the human population is represented in figure . . figure . : epidemiological sv ir model for human population with an imperfect vaccine therefore, the differential system is as follows: ( . ) for this system of differential equations, we have a new basic reproduction number. [ ] ). the basic reproduction number with an imperfect vaccine, r σ , associated to the differential system ( . ) is where r is defined in ( . ) . notice that r ψ ≤ r σ and when the vaccine is perfect (σ = ), r σ degenerates into r ψ . in other words, a high efficacy vaccine leads to a lower vaccination coverage to eradicate the disease. however, it is noted in [ ] that it is much more difficult to increase the efficacy level of the vaccine when compared to controlling the vaccination rate ψ. figure . a, in the epidemic scenario with a perfect vaccine, the number of human infected has reached to a maximum peak of cases per day, in the worst scenario (ψ = . ). using an imperfect vaccine, with a level of efficacy of % (figure . a), with the same values for ψ, the maximum peak increases until cases. we conclude that production of a vaccine with a high level of efficacy has a preponderant role in the reduction of the disease spread. figures . c and . d reinforce the previous sentence. assuming that % of the population is vaccinated, the numbers of infected cases decreases sharply with the increasing of the effectiveness level of the vaccine. according to [ ] , an acceptable level of efficacy is at least % against all four serotypes, and to years for the length of protection. these are commonly considered across countries, as the minimum acceptable levels. in the next subsection we study another type of imperfect vaccine: a vaccine that confers a limited life-long protection. until the s, this was an universal assumption of mathematical models of vaccination: there is no waning of vaccine-induced immunity. this assumption was routinely made because, for most of the major vaccines against childhood infectious diseases, it is approximately correct [ ] . suppose that the immunity, obtained by the vaccination process, is temporary. assume that immunity has the waning rate θ. then the model for humans is given by definition (basic reproduction number with an imperfect vaccine). the basic reproduction number with an imperfect vaccine, r θ , associated to the differential system ( . ) is where r ψ is defined in ( . ). according to [ ] , the basic reproduction numbers are the same, r θ and r ψ , because the disease will still spread at the same rate with or without temporary immunity. however, we should expect that the convergence rate will be different between the random mass vaccination and random mass vaccination with waning immunity, since the disease will be eradicated faster in the constant treatment model without waning immunity compared to the other with waning immunity. depending on the vaccine that will be available on the market, it will be possible to choose or even combine features. in the next section we will define the vaccination process as a control system. in this section we consider a sir model for humans and an asi model for mosquitoes. the parameters remain the same as in the previous chapter. the vaccination is seen as a control variable to reduce or even eradicate the disease. let u be the control variable related to the proportion of susceptible humans that are vaccinated. a random mass vaccination with waning immunity is selected. in this way, a parameter θ associated to the control u represents the waning immunity process. figure . shows the epidemiological scheme for the human population. figure . : epidemiological sir model for the human population using the vaccine as a control the model is described by an initial value problem with a system of six differential equations: the main aim is to study the optimal vaccination strategy, considering both the costs of treatment of infected individuals and the costs of vaccination. the objective is to where γ d and γ v are positive constants representing the weights of the costs of treatment of infected and vaccination, respectively. using oc theory is possible to solve the problem. let us consider the following set of admissible control functions: ∆ = {u(·) ∈ (l ∞ ( , t f ))| ≤ u(t) ≤ , ∀t ∈ [ , t f ]}. table ( . ), admits a unique optimal solution (s * h (·), i * h (·), r * h (·), a * m (·), s * m (·), i * m (·)) associated with an optimal control u * (·) on [ , t f ], with a fixed final time t f . moreover, there exists adjoint functions, λ * i (·), i = ... such that with the transversality conditionsλ i (t f ) = , i= , . . . . furthermore, u * = min , max , proof. the existence of optimal solutions (s * h (·), i * h (·), r * h (·), a * m (·), s * m (·), i * m (·)) associated to the optimal control u * (·) comes from the convexity of the integrand of the cost function ( . ) with respect to the control u and the lipschitz property of the state system with respect to state variables (s h , i h , r h , a m , s m , i m ) (for more details see [ ] ). according to the pontryagin maximum principle [ ] , if u * (·) ∈ ∆ is optimal for the problem considered, then there exists a nontrivial absolutely continuous mapping λ : [ , t f ] → r, λ(t) = (λ (t), λ (t), λ (t), λ (t), λ (t), λ (t)), called the adjoint vector, such that ( . ) and holds almost everywhere on [ , t f ]. moreover, the transversality conditions λ i (t f ) = , i = , . . . , hold. system ( . ) is derived from ( . ), and the optimal control ( . ) comes from the minimality condition ( . ). the simulations were carried out using the values of the previous section. the system was normalized, using the same strategy as in chapter . it was considered that the waning immunity was at a rate of method epidemic scenario endemic scenario direct (dotcvp) . . indirect (backward-forward) . . table . : optimal values of the cost functional ( . ) θ = . . the oc problem was solved using two methods: direct [ , ] and indirect [ ] . the direct method uses the cost functional ( . ) and the state system ( . ) and was solved by dotcvp [ ] . the indirect method used is an iterative method with a runge-kutta scheme, solved through ode of matlab. figure . shows the optimal control obtained by both methods. notice that dotcvp only gives the optimal control as a constant piecewise function. table . shows the costs obtained by the two methods in both scenarios. the indirect method gives a lower cost. this method uses more mathematical theory about the problem, such as the adjoint system ( . ) and optimal control expression ( . ). therefore it makes sense that the indirect method produces a better solution. using the optimal solution as reference, some tests were performed, regarding infected individuals and costs, when no control (u ≡ ) or upper control (u ≡ ) is applied. table . shows the results for dotcvp in the three situations. in both scenarios, using the optimal strategy of vaccination produces better costs with the disease, when compared to not doing anything. once there is no control, the number of infected humans is higher and produces a more expensive cost functional. figure . shows the number of infected humans when different controls are considered. it is possible to see that using the upper control, which means that everyone is vaccinated, implies that just a few individuals were infected, allowing eradication of the disease. although the optimal control, in the sense of objective ( . ), allows the occurrence of an outbreak, the number of infected individuals is much lower when compared with a situation where no one is vaccinated. we conclude that a vaccination campaign in the susceptible population, and assuming a considerable efficacy level of the vaccine, can quickly decrease the number of infected people. the worldwide expansion of the dengue fever is a growing health problem. dengue vaccine is an urgent challenge that needs to be overcome. this may be commercially available within a few years, when the researchers find a formula that protect against all four dengue viruses. a vaccination program is seen as an important measure used in infectious disease control and immunization and eradication programs. in the first part of the chapter, different types of vaccine, as well as their features and some of coverage thresholds, were introduced. the main idea was to study some types of vaccines in order to cover most of the future vaccine features. the main goal of a vaccination program is to reduce the prevalence of an infectious disease and ultimately to eradicate it. it was shown that eradication success depends on the type of vaccine as well as on the vaccination coverage. the imperfect vaccines may not completely prevent infection but could reduce the probability of being infected, thereby reducing the disease burden. in this study all the simulations were done using epidemic and endemic scenarios to illustrate distinct realities. a second analysis was made, using an oc approach. the vaccine behaved as a new disease control variable and, when available, can be a promising strategy to fight the disease. dengue is an infectious tropical disease difficult to prevent and manage. researchers agree that the development of a vaccine for dengue is a question of high priority. in the present study we have shown how a vaccine results in saving lives and at the same time in a reduction of the budget related with the disease. as future work we intend to study the interaction of a dengue vaccine with other kinds of control already investigated in the literature, such as insecticide and educational campaigns [ , ] . this chapter was based on work accepted in the peer reviewed proceedings [ ] . mathematical models can be a powerful tool to understand epidemiological phenomena. these models can be used to compare, plan, implement and evaluate several programs related to detection, prevention and control of infectious diseases. indeed, one of the most important issues in epidemiology is to improve control strategies with the final goal to reduce or even eradicate the disease. in this thesis were constructed and analyzed some models for the spreading of diseases, particularly for dengue fever. the links between health and sustainable development are illustrated for this disease. any attempt in predicting and preventing the disasters caused by the disease will imply a global strategy that takes into account environmental conditions, levels of poverty and illiteracy and, eventually, degree of coverage by vaccination programs [ ] . although vector control strategies were already available before the second world war, dengue pandemic was underestimated. it became a global public health problem in the past years and a major concern for who. the main contributions of this thesis can be classified into three main categories, namely, model formulation, mathematical and computational analysis, and contributions to public health/intervention design. deterministic models for assessing the combined impact of several control measures in dengue disease were considered. in chapter an old oc problem for dengue was revisited. it was shown that new and robust tools bring refreshing solutions to the problem. some analysis with discretization schemes were carried out in order to understand the best way to implement the direct methods in future approaches. for this problem, is was better to use robust solvers than to implement higher order discretization methods, due to the increasing of problem's dimension. in chapter , a seir+asei model was studied. threshold criteria was established ensuring the disease eradication and hence convergence to the so-called disease free solution. using real data from cape verde, the application of insecticide in the country during the outbreak was simulated and its repercussions were analyzed. the study of this outbreak was performed in several phases: firstly, using a previously calculated constant control for the whole period, with the aim of having a basic reproduction number below unity; then, several periodic strategies were studied in order to find the best logistics approaches to implement that keep r less than one; finally, an oc approach to compare with the previous suboptimal approaches was used. for this final phase, several perspectives of the problem were analyzed, including bioeconomic, medical and economic approaches. depending of the main target to achieve, the results for the control and infected individuals vary. in chapter , a sir+asi model for dengue was presented. the lost of two differential equations, was compensated by the introduction of two more controls: in addition to adulticide, larvicide and mechanical control were introduced. similarly to chapter , a threshold criteria for the eradication of the disease was established. the influence of the controls in this threshold was analyzed. then, varying the controls, separately and simultaneously, an analysis of the importance/consequence of each control in the development of the disease was made. finally, an oc approach using different weights for the variables in the functional was studied, in order to establish the best optimal curve for each control and the respective effects in the development/erradication of the disease. in chapter , some simulations with different types of vaccines were made. as a dengue vaccine is not yet available, distinct hypothetical models to introduce the vaccine were studied. in section . , was considered a new compartment v h producing a new model svir+asi. this research comprised svir models with perfect vaccines -constant vaccination of newborns and constant vaccination of susceptibles -and imperfect vaccines -using a level of efficacy below % and also with waning immunity. in section . , the vaccination process was studied as a control of the epidemic model. for this, as in the previous chapter, an oc approach was presented. all the simulations were done in epidemic and endemic scenarios, in order to understand what type of repercussions could bring each kind of vaccines. in this work, there was a concern in producing a mathematical analysis for all new models presented. epidemiological concepts, such as the basic reproduction number and equilibrium points, were calculated. the equilibrium points were classified and their local stability analyzed. the oc theory was used in order to provide the best strategies for each model, involving direct and indirect approaches. throughout the thesis, a set of software packages were used, showing the importance of the these tools in the development of some mathematical fields. to solve ode systems, codes in matlab and scilab were implemented. to calculate the equilibrium points and the basic reproduction number, mathematica and maple were used. for the oc approach, several packages were also selected: from programmed codes in ampl and run in neos server (ipopt, snopt, knitro, muscod-ii) to fortran codes in the linux environment (oc-ode) or even programs coded in matlab (dotcvp and indirect methods). the software choice was varying during the research process, due to availability, robustness and solving speed. the main codes developed in this thesis are available at: https://sites.google.com/site/hsofiarodrigues/home/ phd-codes- [ ] . using direct and indirect methods, the solutions obtained were similar, reinforcing the confidence in the results. the study provides some important epidemiological insights into the impact of vector control measures. dengue burden decreases with the increasing of vector control measures (adulticide, larvicide and mechanical control). furthermore, the adulticide should be the first/main measure to apply when an outbreak occurs, whereas the other two measures should be considered as a long time prevention. the last control measure to be studied was the vaccination. it was shown that the vaccine, when available, could bring advantages not only in the reduction of infected individuals, but also decreasing the disease costs. a phd thesis is always an unfinished process, but some day it is necessary to stop. therefore, some topics were not explored and can be understood as future directions of the work. a first suggestion is to use heterogeneity for both populations, dividing each one in more compartments [ ] . human population is not immunologically homogeneous, presenting groups with distinct level of risk, related with age, sex or the presence of a disease or immunosuppressive drugs which would be the case with transplant patients or cancer suffers. for example, the transmission probability in young children is higher and generally with more severe symptoms when compared to adult transmission. moreover, the mosquito population does not have the same behavior during the whole year, depending specially on weather conditions. temperature and humidity are key variables on vector population dynamics. it will be interesting to add some seasonality factors into the last models [ , ] . another open question is the introduction of immigration and tourism issues. along the work it was considered a constant population, but the addition of new individuals could induce new outbreaks. other aspect is related to the disease development in the presence of several serotypes. while in cape verde only one serotype was found, the interaction of several serotypes in asia is already a reality. this will induce changes in the model, not only increasing of the number of variables but also causing a more expressive number of dhf cases [ ] . currently, portuguese researchers are developing a new repellent for mosquito that could be considered as a new control for the disease. this product does not kill the mosquito, but prevents the bite by deviating it from the target and consequently decreasing the chain of the disease transmission. with the viability of the vaccine, it will be possible to fit a better model according to the vaccine used. one of the possibilities is using pulse vaccination, where children at a certain age cohorts are periodically immunized [ ] . the theoretical challenge of pulse vaccination is the a priori determination of the pulse interval for specific values of r , the proportion of vaccinated p and the population birth rate µ. most of the analysis and models presented in this thesis can be adapted for other vector-borne diseases -such as malaria, yellow fever, west nile virus, chikungunya, japanese encephalitis -by just fitting some variables/parameters and some initial assumptions intrinsic to the disease [ , , ] . when attempting to model epidemics and control for public health applications, there is the compelling urge to make models as sophisticated as possible, including many details about the host and the vector. although this strategy may be useful when such details are known or exist suitable data, it may lead to a false sense of accuracy when reliable information is not available. another approach is to keep the model simple and, instead of using the conventional differential calculus, to apply fractional calculus to fit to the disease reality [ ] or to use the general theory of time scales [ ] . 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ure authors: lim, tow keang; siow, wen ting title: pneumonia in the tropics date: - - journal: respirology doi: . /resp. sha: doc_id: cord_uid: ure pneumonia in the tropics poses a heavy disease burden. the complex interplay of climate change, human migration influences and socio‐economic factors lead to changing patterns of respiratory infections in tropical climate but also increasingly in temperate countries. tropical and poorer countries, especially south east asia, also bear the brunt of the global tuberculosis (tb) pandemic, accounting for almost one‐third of the burden. but, as human migration patterns evolve, we expect to see more tb cases in higher income as well as temperate countries, and rise in infections like scrub typhus from ecotourism activities. fuelled by the ease of air travel, novel zoonotic infections originating from the tropics have led to global respiratory pandemics. as such, clinicians worldwide should be aware of these new conditions as well as classical tropical bacterial pneumonias such as melioidosis. rarer entities such as co‐infections of leptospirosis and chikungunya or dengue will need careful consideration as well. in this review, we highlight aetiologies of pneumonia seen more commonly in the tropics compared with temperate regions, their disease burden, variable clinical presentations as well as impact on healthcare delivery. pneumonia continues to be an important cause of death and accounts for % of all deaths in children under the age of . the disease burden, especially in children, is heaviest in south asia and sub-saharan africa. these regions have tropical climates, which are characterized by a hot climate present all year round, whilst large volumes of rainfall each year result in the spread of zoonotic diseases. besides environmental influences, socio-economic factors also impact heavily on the epidemiology of tropical diseases. the tropics, which is the geographical region of the earth centred on the equator and limited by the tropic of cancer on the north and the tropic of capricorn on the south, is also home to some of the poorest nations. together with the movement of people triggered by globalization, mass migrations and climate change, these dynamics have a profound effect upon the patterns of respiratory tract infections in the tropics. global temperatures have risen through the past decades due to greenhouse gas emissions, and we have also seen a rise in a number of extreme meteorological events such as tropical super typhoon haiyan in . warmer temperatures and altered rainfall patterns are anticipated to promote outbreaks of infectious diseases due to more hospitable environments for pathogens and lack of ready access to health care. prevalence and mortality from pneumonia in children are the highest during rainy months in tropical and subtropical regions of asia and africa, which again highlights the pattern of pneumonia against climate. global warming and climate change also result in an increase in average as well as nadir temperatures, even in temperate regions. , hence, areas that were previously free from tropical diseases may now encounter such entities. this is shown by an emergence of tropical diseases in temperate regions, such as an outbreak of leptospirosis in in springfield, illinois in the united states, and an outbreak of human pneumonic plague in colorado in . natural disasters have been linked to disease outbreaks including pneumonia, due to an increased risk of water-, air-and vector-borne diseases. in the post-disaster period, phase is the impact phase ( - days) where victims are rescued and immediate treatment is provided for disaster-related injuries. phase which is the postimpact phase ( days to weeks post-disaster) is the period when the first surge of infectious diseases may surface, and phase the recovery phase (after weeks) when symptoms of diseases with longer incubation periods may declare. examples of such diseases are influenza and leptospirosis. a tertiary hospital reported an increase in patient admissions for pneumonia and tuberculosis (tb) in the aftermath of typhoon haiyan especially in the impact and post-impact phases, and the entity 'tsunami lung' has been described in victims who have pneumonia following a near-drowning episode after surviving a tsunami event. victims described by inoue et al. were rescued immediately after the tsunami and were in respiratory distress. the term tsunami lung describes both pneumonia from various organisms (such as stenotrophomonas maltophilia, burkholderia cepacia and pseudomonas aeruginosa), as well as severe systemic after-effects such as disseminated aspergillosis. there are no clear differences in viral aetiologies of pneumonia when comparing tropical with temperate climates. [ ] [ ] [ ] [ ] [ ] different authors have found varying impacts of precipitation on viral pneumonias. [ ] [ ] [ ] respiratory syncytial virus respiratory syncytial virus (rsv) is a common causative pathogen in respiratory tract infections and was the most commonly isolated virus in children with respiratory tract infections in a malaysian study, accounting for . % of patients. in adults, most manifestations are in the upper respiratory tract, although about % will develop pneumonia. the virus is detected using polymerase chain reaction methods, immunofluorescent or immunoassay antigen detection, cultures or serology analysis. treatment is mainly supportive. aerosolized ribavirin can be used as a specific antiviral therapy for rsv, but this is less well studied in adults compared with infants. influenza a influenza, which remains a global health burden, displays seasonality. a recent study of viral infectious patterns according to time of the year in a singaporean medical intensive care unit by siow et al. showed peaking of influenza cases around the start and middle of the year. in this study, the most common viral isolate was the influenza a h n / virus, followed by human rhinovirus. the seasonal distribution of influenza a in this study echoes the results of tang et al. who found the incidence of influenza a in singapore to peak during january and june to july period, as well as chew et al. who noted two peaks during the november to january and june to july period. in singapore, months with heaviest rainfall are clustered around year-end (october to december), and drier months are clustered around mid-year, in june and july. pharmacological treatments include antivirals such as oseltamivir and zanamivir to help shorten the duration of illness if administered within h of onset of illness. the seasonality of influenza therefore cannot be fully explained by rainfall alone. multiple contributory reasons have been hypothesized, including host behaviour such as spending more time indoors during adverse weather conditions, and altered host defences, but uncertainties remain. we must consider the complex relationship of climate and human behaviour when determining patterns, not just for influenza, but for other respiratory viruses as well. the h n strain of avian influenza was the causative agent of an influenza pandemic in asia in . prevalent in poultry and wild birds, animal-to-human transmission occurs to cause a spectrum of pneumonia/ pneumonitis, culminating in acute respiratory distress syndrome (ards). as of april , the world health organization (who) recorded a total of confirmed human cases of h n with deaths with a % mortality rate. as recently as october , who was notified of the influenza a (h n ) virus outbreak in china. since then, it has been noted to have an increased number of cases in december and january. , the major risk factor of infection was live poultry exposure. as live poultry markets are commonplace in china, and with chinese new year festivities the consumption of poultry in the populace will increase, there will be a higher risk of continued exposure leading to sporadic infections or small clusters of human cases. hantavirus is associated with the hantavirus pulmonary syndrome (hps). its manifestation is as a rapidly progressing non-cardiogenic pulmonary oedema and can mimic that of a severe pneumonia clinically and radiologically. hps was first discovered in in the southwestern united states, and since then has been described in latin america as well. risk factors are exposure to rural activities and rodents, and treatment is largely supportive. other viruses associated with pneumonias are the corona viruses, such as the middle eastern respiratory syndrome corona virus (mers-cov) and severe acute respiratory syndrome corona virus (sars-cov). these entities are beyond the scope of discussion of this paper, but suffice to say their impact on global health has been daunting given confirmed deaths out of confirmed mers-cov infections and more than cases of sars-cov, of which a large proportion were concentrated within asia (in particular, china, hong kong and taiwan). there are differences between causative organisms encountered in the tropics compared with temperate climates. due to both environmental and socioeconomic factors, diseases such as melioidosis, leptospirosis and tb are more widespread in the tropics-these will be discussed later. observation of the aetiologies of community-acquired pneumonia (cap) in an asian outpatient setting showed that the most common isolates were chlamydophila pneumoniae, followed by mycoplasma pneumoniae and streptococcus pneumoniae. that same systematic review by peto et al. identified s. pneumoniae as the most common pathogen in hospitalized patients with cap. although there was great variation in terms of proportion between countries in this study, frequency was similar to the trends observed in european studies quoted in the review and also comparable to findings by siow et al. interestingly, the most common gram-negative bacillus isolated in the studies was klebsiella, with higher proportions being reported in india and southeast asia. the authors found that asian patients with cap requiring hospitalization yielded a larger proportion of gramnegative bacilli ( . % vs . %) and staphylococcus aureus ( . % vs . %) isolates compared with referenced european studies. however, there was no comparison made between the asian countries, as certain countries such as korea and japan experience a temperate climate compared with the tropical climates of thailand and malaysia. siow et al. found the top two causative pathogens to be s. pneumoniae and klebsiella pneumoniae in a recent study looking at bacterial isolates from severe cap patients in a singaporean medical intensive care unit. streptococcus pneumoniae was the main grampositive bacterium isolate, and s. aureus was the next common gram-positive organism. otherwise, gramnegative organisms such as k. pneumoniae, escherichia coli and p. aeruginosa represented the majority of cases detected. similarly, lin et al. described k. pneumoniae not only as a prevailing cause of cap with bacteraemia in a taiwanese tertiary hospital, but also showed that it was associated with a more fulminant clinical course and worse prognosis when compared with patients with s. pneumoniae cap with bacteraemia. in a series of severe cap cases in singapore, patients who had gram-negative organisms isolated tended to have a worse outcome including a higher mortality, especially for patients with pseudomonas and burkholderia pseudomallei infections. this has changed the way local clinicians initiate their empiric treatment for patients admitted for severe cap, with antibiotics deliberately chosen to cover gram-negative organisms, melioidosis as well as gram-positive pathogens. a systematic review by goyet et al. looking at resistance patterns of cap pathogens in cambodia and neighbouring countries showed that up to % of s. pneumoniae and . % of k. pneumoniae were resistant to amoxicillin-clavulanic acid. streptococcus pneumoniae also displayed a high resistance to trimethoprim/sulfamethoxazole (average of . %) and wide range of resistance patterns to cephalosporins: between . % and . % to ceftriaxone, and up to . % to cefuroxime. there was also a mean high-level resistance rate to penicillin g of . %. burkholderia pseudomallei did not show resistance to first-line treatments ceftazidime, carbapenems and trimethoprim/sulfamethoxazole. as a result of this study, the authors have advocated the preservation of fluoroquinolones as they are not warranted as first-line therapy, and they are also used to treat tb, which importantly is endemic in this region. this highlights the importance of continued surveillance of regional resistance patterns and revision of therapeutic guidelines. melioidosis was first described by krishnaswami and whitmore in , when they noticed a 'glanders-like' disease afflicting opiate addicts in rangoon. today, melioidosis still poses a threat to public health due to mortality rates up to % if early treatment is not instituted. in an endemic country like thailand, it can account for up to % of the pathogens identified in adult patients with pneumonia. currie and kaestli estimated global mortality from melioidosis in to be , comparable with deaths from measles, and higher than those from dengue and leptospiral disease. the causative pathogen, b. pseudomallei, is a gramnegative rod that is found in soil and fresh water. it occurs mainly in northern australia, southeast asia, china and south asia with increased incidences during rainy seasons. this is in contrast to temperate countries where melioidosis is extremely rare and almost always encountered in migrants or travellers. presentation of melioidosis can be either acute or subacute. acute illnesses usually present with pneumonia which can be associated with ards and shock. subacute presentations may take a more insidious course, mimicking tb. there is also a propensity for the pathogen to spread haematogenously, and patients may present with extrapulmonary manifestations such as solid organ and skin abscesses, and even septic arthritis and encephalomyelitis. diagnosis of melioidosis is confirmed on positive cultures. with potential mortality rates approaching %, a clinician's suspicion must be high when faced with a patient with severe cap coupled with an appropriate travel history. in the local context, because of being in an endemic region, intensive care units including those in singapore have adopted empirical antibiotic treatment to include specific coverage for melioidosis (ceftazidime and meropenem would be appropriate) for patients admitted with severe cap. , indeed, in a prospective study over years in darwin, mortality rates have improved over time ( % described by the authors) with better recognition of the disease as well as early treatment with appropriate antibiotics. leptospirosis is a zoonotic disease prevalent in the tropics, with a much higher incidence than in temperate regions. within the asia pacific region, high-incidence countries include thailand, bangladesh and cambodia. there are also certain regions such as korea and china where leptospirosis incidences, although low, are increasing. leptospira are aerobic spirochetes. both feral and domesticated animals can host the diseasecommonly, dogs, cattle, rodents, swine, but interestingly, and rarely in cats. human infection typically occurs after contact with contaminated urine, animal tissue, water or soil. in the tropics, it especially affects low-income regions with poor sanitation, low education and poor housing, where outbreaks are common and morbidity is high. even in higher income regions, heavy rainfall leading to flooding increase the risk of both humans and livestock exposure to contaminated water. for example, there was an outbreak in anuradhapura, sri lanka. anuradhapura is a region with abundant paddy fields for rice farming and was not previously known as an endemic area, so the diagnosis and outbreak of leptospirosis which followed flooding was initially challenged by local clinician. recreational events such as caving, canoeing and freshwater swimming could expose humans to contaminated sources. an example highlighting the impact of these activities would be the leptospirosis outbreak in springfield, illinois, involving triathletes who were exposed to lake water. clinical features are variable. it can take a subclinical, self-limited course, or can progress to severe and potentially life-threatening illness complicated by jaundice, renal failure and ards, with reported mortality rates up to %. typical presenting complaints include fever, myalgia, headaches and conjunctival insufflation. cough, nausea, vomiting and diarrhoea are common. dall'antonia et al. described cough and haemoptysis in patients with serologically proven leptospirosis. severe forms of the disease with multiorgan dysfunction and ards-like syndromes may be fatal. chest roentgenogram findings are non-specific; they commonly show non-segmental patchy or even nodular infiltrates with poorly defined margins usually in the lower lobes, which can be unilateral or bilateral. interestingly, leptospirosis and chikungunya coinfection can potentially lead to a delayed diagnosis and subsequent deleterious outcomes. nhan et al. described a fatal case of leptospirosis and chikungunya co-infection in a french-polynesian outbreak during the rainy season, where diagnosis was delayed due to overlapping symptoms. co-infections with dengue have also been described by pan et al., where three cases of co-infection were detected during a dengue outbreak. again, diagnosis was challenging because of nonspecific symptoms such as fever, chills and myalgia. adding on to the diagnostic challenge, sathiyakumar et al. described a case of haemorrhagic pneumonitis secondary to leptospirosis, which showcases the spectrum of clinical presentation. the diagnosis of leptospirosis is both clinical and microbiological, but the gold standard is the microscopic agglutination test (mat). cumberland et al. found the mat to have a sensitivity of between % and % (depending on when samples were taken in the disease's time course), and % specificity. leptospira can be grown in vitro from blood, cerebrospinal fluid and urine from infected hosts. however, the laboratory needs to be notified if leptospira needs to be isolated as it requires specialized culture media, and time to positive cultures can take between week and months. should the clinician strongly suspect leptospirosis clinically, empiric antibiotics such as doxycycline or ceftriaxone should be started. the strain of rickettsial illness encountered in the tropics is scrub typhus-a mite-borne disease caused by orientia tsutsugamushi, a gram-negative coccobacillus. it is predominantly found in the asia pacific rim, with larval mites ('chiggers', from the genus leptotrombidium) that live on vegetation and rodents. wu et al. have described a rise in the incidence of scrub typhus in mainland china between and with a . times increase. on top of seasonal peaks, the authors postulated that the increase of popularity in ecotourism have exposed more people to vector habitats. clinical manifestations typically include myalgia, high fevers, headaches, as well as a rash and eschar at the chigger bite. scrub typhus is usually self-limiting over - weeks but is sometimes associated with severe illness and multiorgan failure leading to death, although this is rare. [ ] [ ] [ ] pneumonia can occur in the late phase of the disease , , as well as in an ardslike picture. pulmonary involvement is well described. chest roentgenograms may be abnormal in - % of patients, and may show bilateral diffuse reticulonodular opacities, septal lines and hilar lymphadenopathy. consolidation is not common, and would tend to appear in the lower zones. pleural effusions can be found in up to . % of patients. , the diagnosis is confirmed on serological testing or eschar biopsy, but there should be a suspicion of scrub typhus infection if there is an appropriate exposure history. patients who have been started on appropriate antibiotics (such as doxycycline, chloramphenicol and azithromycin) usually have defervescence of the fever within h. countries in the tropics bear the brunt of tb, and south east asia holds approximately one-third of the global burden of tb. peto et al. found more than % of cases of cap in asia were attributable to mycobacterium tuberculosis. however, with the rise in tourism and immigration, tb is now seen in higher income countries, with a substantial proportion of cases diagnosed in immigrants in the united states and england. , people at risk include those with poor nutrition, immunocompromised and those living in poorly ventilated and overcrowded environments. the american thoracic society and infectious diseases society of america recommends repeat examinations of expectorated sputum for acid fast bacilli (afb) augmented by a nucleic acid amplification test such as the xpert mtb/rif assay (cepheid, sunnyvale, ca, usa) in the rapid diagnosis of pulmonary tb. in smear-negative cases, they suggest the testing of induced sputum instead of proceeding to bronchoscopy and lavage which seems to be a very popular option. sputum induction is more cost effective than bronchoscopic examination and should be the test of choice if smear-negative pulmonary tb is the most likely diagnosis. we must also consider that in certain settings, it is common to lack access to high-quality chest roentgenograms and people who can reliably interpret them, and there may not be access to the xpert mtb/rif assay. who has an alternative algorithm to reference in settings where chest roentgenograms and/or xpert mtb/rif assays are not available, and it is largely based upon careful history taking, clinical examination and sputum smear analysis. treatment regimens using first-line drugs include rifampicin, isoniazid, ethambutol and pyrazinamide. directly observed therapy (dot) has been utilized in some countries to ensure compliance as this is the crux of treatment success, but a cochrane review of trials in found no significant differences in cure rates, treatment completion when comparing dot and selfadministered therapy. the authors have stated that given the costs and personnel involved in dot, policymakers may wish to have alternative strategies to help improve adherence to treatment. multidrug-resistant tb and extensively drug-resistant strains are beyond the scope of this review and will not be examined. helminthic and protozoal parasitic diseases are common in the tropics. pulmonary disease typically presents as an eosinophilic lung disease, with or without peripheral blood eosinophilia. lung infiltrates may be fleeting on radiology-this was famously described by loffler in . lymphatic filariasis lymphatic filariasis can manifest as a syndrome known as tropical pulmonary eosinophilia (tpe). the disease is seen mainly in south asia, southeast asia and the south pacific islands. three species of filarial nematodes cause tpe: wuchereria bancrofti, brugia malayi and brugia timori. , mosquitoes transmit the disease and humans are the definitive hosts. microfilariae trapped in the lungs lead to an immune hyper-responsiveness, leading to symptoms of cough, fever, night sweats, wheezing and weight loss. pulmonary radiology can appear miliary or nodular, mimicking tb. however, imaging can be normal in up to % of patients. spirometry tends to demonstrate a restrictive pattern with airways obstruction, and airway obstruction is usually reversed by bronchodilators. treatment with diethylcarbamazine is associated with rapid improvement in signs and symptoms, as well as a gradual trend towards normal in spirometric values, although permanent impairment in lung function can occur. the causative pathogen, paragonimus westermani, is endemic in much of asia and south america. a foodborne trematode, infection is cause by ingestion of raw of improperly cooked freshwater crabs. patients may be asymptomatic although can also experience a chronic cough, chest pain and haemoptysis which can be recurrent. radiological findings include pleural effusions, pneumothorax, ring shadows and consolidation on chest roentgenograms. , computer tomography scanning can reveal cysts within the consolidated lung. paragonimiasis is treated with triclabendazole or praziquantel. strongyloides infection is common in the tropics, sub tropics and warmer temperate climates. nematode larva spread haematogenously as well as via the lymphatics to the heart and lungs. patients can present with loeffler's syndrome and peripheral eosinophilia during larval migration through the lungs. respiratory signs and symptoms include cough, bronchospasm and in some cases haemoptysis. chest roentography can be normal. during larval migration, miliary nodules or ill-defined patchy consolidation may be present. in an overwhelming infection especially in the immunocompromised host, a marked bilateral alveolar pattern similar to that of pulmonary oedema can be seen, and clinically the patient would be in ards. , , diagnosis of strongyloides can be strengthened with examination of several stool samples on several days. larvae may also be demonstrated on duodenal aspirates, sputum and bronchoalveolar lavage fluid. ivermectin and albendazole can be used for effective treatment. , additionally, clinicians need to be aware of gut translocation of enteric organisms especially in immunocompromised hosts, leading to further complications of sepsis. malaria is caused by the intraerythrocytic protozoa plasmodium. it is transmitted to humans by the bite of the female anopheles mosquito, and falciparum malariae is the most severe of all malarial infections. the symptoms leading to a suspicion of malarial infection are fever which can be cyclical, breathing difficulties and anaemia. once the disease is suspected, light microscopy is the standard tool used to detect parasites on blood smears. rapid diagnostic tests utilize antigen detection technology as an alternative when reliable light microscopy is unavailable, and the who is recommending its use as a field alternative when rapid diagnosis is paramount. there is a wide range of pulmonary manifestations, from a non-productive cough to ards, occurring in up to % of adults with severe falciparum malaria infection although any strain of plasmodium can lead to ards. the development of ards portends an extremely grave prognosis , -gachot et al. described a % mortality rate in patients with malaria and acute lung injury despite admission to an intensive care unit. chest roentgenogram findings are non-specific and can range from confluent nodules to basal and/or bilateral pulmonary infiltrates, mimicking pulmonary oedema, although this is usually non-cardiogenic. resistance to antimalarial drugs especially chloroquine and sulfadoxine-pyrimethamine has become widespread. who now recommends artemisinin-based combination therapy (act) as the firstline treatment in uncomplicated falciparum malaria. for uncomplicated, non-falciparum malaria in regions with low choroquine resistance, chloroquine can be used. we have summarized the common pathogens causing pneumonia in tropical regions in table . other differentials of pneumonia in the tropics would be tpe, pulmonary plague, histoplasmosis, cryptococcosis, thoracic actinomycosis, nocardiosis and pulmonary anthrax. however, these are beyond the scope of this review, and we would urge clinicians to practice careful history taking including a travel and exposure history, and conscientious examination to lead them towards the correct diagnosis. the burdens of pneumonia in tropical and subtropical regions remain high, especially when coupled with global warming and climate change. with the advances in air travel, immigration patterns and international tourism would mean tropical diseases including pneumonias would be encountered in the temperate countries as well. it is important for clinicians to recognize these relations and conditions so that correct treatment can be instituted early, as some of the tropical diseases such as leptospirosis, melioidosis and malaria with ards herald a poor prognosis if treatment is delayed. clinicians will need to be cognizant of co-infections with overlapping symptoms such as chikungunya or dengue co-infection with leptospirosis, as late diagnosis would potentially lead to deadly consequences. t.k.l. is professor of medicine and senior consultant in the department of medicine, national university hospital singapore. his research interests include effective implementation of clinical evidence and improving clinical reasoning expertise by deliberate practice and structured reflection. w.t.s. is an associate consultant in the department of respiratory and critical care medicine, national university hospital singapore. her clinical interests include critical care medicine and biomedical technology. world health organization. pneumonia factsheet climate change and respiratory infections characterizing hospital admissions to a tertiary care hospital after typhoon haiyan human health: impacts, adaptation, and co-benefits childhood pneumonia: a neglected, climate-sensitive disease? diurnal temperature range and daily mortality in shanghai leptospirosis working group. outbreak of leptospirosis among triathlon participants and community 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qualitative study date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: ax i d f background: infectious diseases are common problems in mass gatherings, especially when there is a lack of health system preparedness. since iran is one of the most important countries on the walking path of arbaeen and has a vital role in providing health services to pilgrims, the experiences of health challenges by participants is of key importance. the aim of this study is to explore stakeholders’ experiences on the health system's preparedness and challenges, and to provide suggestions for preventing infectious diseases during the arbaeen mass gathering. methods: a qualitative research method was used with a conventional content analysis approach. the number of participants was , including executive managers and health policymakers who entered the study among participants. semi-structured interviews were used to generate the data. interviews were analyzed by means of content analysis after face-to-face interviews. results: data analysis resulted in the extraction of four main themes and sub-themes. health infrastructure defects in iraq has three sub-themes (health abandonment in iraq, the weaknesses in health culture and problems related to the health system); poor control of the causative factors of infectious diseases has three sub-themes (the underlying factors of the prevalence of contagious diseases, health system response to communicable diseases and ignoring the risks of the arbaeen ceremony); the low perception of risk in pilgrims has three sub-themes (lack of awareness in pilgrims, fatalism in pilgrims and unhygienic belief in pilgrims); and the ineffectiveness of health education has two sub-themes (training shortage in the targeted group and educational content problems) that shows participant’s experiences of the health system's challenges for coping with infectious diseases during the arbaeen ceremony. conclusion: pilgrim-based training, planning and controlling other challenges may change these threats to opportunities and improve the health of participants of the mass gathering of arbaeen in the region. according to the definition of the world health organization (who), any structured or spontaneous event leading to a certain number of people gathering in a particular site, for a specific aim in a determined period, putting pressure on the response resources and social programs, is called a mass gathering. mass gatherings are divided into different types based on their purpose. the expansion of interconnectivity between societies and increases in the number of national and international events in communities has led to an increase in the number of mass gatherings, which, despite the benefits like cultural exchange, have health challenges such as infectious disease transmission and, should therefore be considered by health planners . one of the health challenges of mass gatherings is the prevalence of infectious diseases and the outbreak of diseases, which, along with, complicated health needs of participants increases the health burden on the host country. the public health system can be under severe pressure, even with advanced equipment and the proper resources for prevention and control of infectious diseases - . various factors, such as the type and location of gathering, the number of participants and the lack of access to health facilities, can affect the incidence of infectious diseases in mass gatherings. planners must therefore pay attention to these factors in preparation , [ ] [ ] [ ] . since a mass gathering is a collection of many people together in one particular site, the possibility of infectious disease transmission due to the high population density always exists. studies on mass gatherings such as hajj, ashura day in karbala, and kumbh mela and sabarimala in india show the prevalence of infectious diseases in these ceremonies [ ] [ ] [ ] [ ] [ ] [ ] . the dates of religious ceremonies like hajj and arbaeen are decided using the lunar calendar (the islamic or hijri calendar) which is not only shorter than the gregorian calendar, meaning that these events occur days earlier each year and can synchronize with different seasons and season-associated diseases. accordingly, planners and policy makers of public health are faced with changing goals, requiring health system preparedness , . one of the world's largest religious gatherings is the arbaeen ceremony, which happens on the th day after the anniversary of imam hussein's martyrdom, the third shiite imam. in the ashura event, pilgrims walk to karbala, south of baghdad. based on the statistics of , the number of iranian pilgrims taking part in the arbaeen ceremony was , , . iran is a neighboring country of iraq and shares a common land border with it. pilgrims from other neighboring countries of iran, such as afghanistan and pakistan, cross iran to reach iraq and the arbaeen ceremony. on the basis of the mutual memorandum of cooperation between the two countries of iran and iraq, iran is committed to providing health services to other pilgrims in addition to iranian pilgrims . therefore, it is necessary to have a plan for preparedness in dealing with infectious diseases during arbaeen ceremony. if a mass gathering is not carefully managed, it can lead to the distribution of infectious diseases. in mass gatherings, infectious diseases are threats to global health security and even the political security of countries. therefore, planning, communication and public health supervision are important in these religious ceremonies , . mass gatherings are different from structured disasters so that in case of occurrence, many people will be affected . since the arbaeen ceremony is held with the presence of many pilgrims from many different countries and, like the hajj pilgrimage, is based on the lunar calendar, and it is held in iraq, there is the possibility of the occurrence and transmission of infectious diseases. it is therefore essential to be prepared to control and prevent these diseases. dealing with infectious diseases in arbaeen is considered a challenge for policy makers . according to arbaeen's social and cultural context, it seems essential to take a deeper look in this field. on the other hand, there is relatively little knowledge about the arbaeen ceremony; therefore, a qualitative method for clarifying the concept and challenges of health system preparedness in arbaeen ceremony is necessary. the aim of this study was to explore challenges of health system preparedness for communicable diseases in arbaeen ceremony. we collected data from june to march . since the study attempted to explore preparedness challenges of health systems, a qualitative research method with the approach of conventional content analysis was used . the health system's challenge in arbaeen is multidimensional, owing to the cultural difference between iran and iraq, the challenges faced by the health system during the arbaeen pilgrimage should be investigated in both countries. indeed, the cultural practices of the participants, especially those surrounding health, differs during the arbaeen ceremony. therefore, a qualitative research method, with the aim of describing phenomena, providing new knowledge, insight and a practical health guide, is the method used in this study , . the study was conducted using in-depth interviews, based on stakeholder's experiences in iraq-iran land terminals (mehran, shalamche, and chazaba) and also in health care posts in iraq. the interviews were conducted with health care providers and policy makers, as well as pilgrims in the arbaeen ceremony. participants were chosen among executive managers and policymakers of the ministry of health and medical education, medical training and treatment and other related organizations, including the red crescent organization, mobilizing the medical society, the hajj and pilgrimage organization, medical universities in the border cities and the social security organization. in total, participants, consisting of executive managers and health policymakers, were selected in this study through purposeful sampling with the aim of exploring challenges of health system preparedness for communicable diseases in the arbaeen ceremony. the existence of practical experience in planning or participate in arbaeen ceremony and the ability to communicate and willingness to participate were inclusion criteria in the research. we recruited participants either by phone call or by approaching them in person. "maximum variety sampling" was used to explore the experiences of the participants in those selected so that they were chosen from the ministry of health and medical education, medical training and treatment and other related organizations, including the red crescent organization, mobilizing the medical society, the hajj and pilgrimage organization, medical universities in the border cities and the social security organization, with different experiences of work, education and gender. inclusion criteria included the existence of practical experience in planning or participate in arbaeen ceremony, the ability to communicate and willingness to participate in the research (table ). the study was done through face-to-face interviews followed by telephone interviews for concept saturation. the data was collected using audio recorders with permission from the participants. ak conducted the interviews, ak and dkz transcribed the data. ak and dkz and zgh coded the data. dkz and zgh performed rigor. initially the first two interviews were conducted in a non-structured format, with the following interviews being semi-structured. open questions used to generate the data were developed by experienced and/or knowledgeable policy makers and health care providers. the individual's experiences and beliefs were used without considering their specialty . interviews were continued until data and concept saturation were reached . the interview duration was between and minutes, based on the tolerance, amount of information and desire of the participants. interviews were performed individually and based on participants' willingness in terms of time and site. firstly, interview questions began with the following general questions based on the participants' level and the main questions of the research: "how was your organization preparedness plan to deal with infectious diseases in arbaeen ceremony?"; "please express your experiences of related challenges in infectious diseases in arbaeen ceremony"; "what problems were in your preparedness plan?"; "what problems did you face in the vaccination program of the health team and pilgrims in arbaeen ceremony?"; and "what is your offer to pilgrims for a safe pilgrimage?" following this, exploratory questions were gradually used to clarify the concept and deepen the interview process: e.g. "please explain further what you mean?" and "why?" at first, the interviews were transcribed verbatim and then typed up using microsoft word office. the data were analyzed through a conventional content analysis method . first, the main researcher converted the interviews to written texts. the digital files were listened to several times and the texts were read repeatedly. next, the meaning units were determined based on the aim and the question of the research. meaning units were a collection of words and sentences that were related to each other in terms of content and were grouped together. meaning units reached the level of abstraction and conceptualization and were coded considering the research question. the key points and subjects were extracted as open codes. these codes have been put under the broader headings based on the present similarities and differences; in other words, the data were reduced in order to describe the phenomenon and gain a better understanding, and this abstraction process continued until concept extraction . data first emerged as meaning units, then condensed meaning units, codes, sub-themes and finally themes. the study was approved by ethics committee of shahid beheshti university of medical sciences on / / , no. ir.sbmu. retech.rec. . . the interviews were conducted and recorded with participants' consent. written or verbal consent was taken from participants to participate in the study. verbal consent was only taken for telephone interviews; this was due to the distance between interviewer and interviewee. anonymity, confidentiality and the right of resignation were informed to participants and considered during the study. the interview time was set according to participants' willingness. the researchers used the trustworthiness criteria recommended by guba and lincoln to establish rigor . all authors were engaged in the environment and field of research. in addition, the principle investigator always had suitable involvement with the participants for in-depth interviews. credibility was established by the prolonged engagement of researchers with participants. researcher triangulation was also used to verify the accuracy of the coding process. the research team also retained raw data, codes, and themes for control of the reliability. at the same time, sampling was carried out with maximum diversity in order to provide triangulation by means of credibility and confirmability. a detailed description of the method was used to establish transferability. engaging participants in the research increases the interaction between researchers and participants and then the credibility . the research supervisor monitored the data collection and data analysis process. it is necessary to mention that the research team participated in the arbaeen ceremony as pilgrim and conducted field notes. also, memberand peer-checking were used to ensure credibility. therefore, many interviews with related topics were sent to some external expert reviewers and participants (policymakers of the ministry of health and medical education, medical training and treatment and the red crescent organization) to be checked, and they were requested to assess the degree of relevance between the findings and raw data. moreover, transferability was established by sampling with maximum variation from various centers, including the ministry of health and medical education and other peer organizations including the red cross organization, the medical community mobilization, the hajj and pilgrimage organization, the country's medical universities in the border cities and the social security organization with different experiences of work, education and gender. a completed srqr checklist is available in supplementary file . themes and sub-themes the mean age of participants was and the mean time of work experience was years (table ) . overall, original codes were extracted and after integration using conventional concept analysis, four original themes consisting of sub-themes were identified. the theme of "health infrastructure defection in iraq" had three sub-themes: "health abandonment in iraq", "the weakness of health culture" and "problems related to health system". the theme of "poor control of factors effective in infectious diseases" had three sub-themes: the "underlying factors in prevalence of contagious diseases", "health system response to communicable diseases" and "ignoring the risks of the arbaeen ceremony". the theme of "low perception of risk in pilgrims" had three sub-themes: "lack of awareness in pilgrims", "fatalism in pilgrims" and "unhygienic belief in pilgrims". the theme of "ineffectiveness of health education" had two sub-themes: "training shortage in the targeted group" and "educational content problems" (table ) . according to the findings of this study, the main theme is pilgrim-based education. it seems that the biggest issue with the prevention of communicable diseases in the arbaeen ceremony is pilgrim-based education. educating pilgrims can directly help people to create or reform health infrastructures in iraq. education can also help to identify health risks and respond to them by identifying effective factors in combating infectious diseases. the training of health instructors and guidelines from people who are trusted and accepted by pilgrims, such as missionaries and religious leaders, can have a positive impact on their beliefs. the main point of training is to determine the targeted group both at the level of pilgrims and health directors and consider the training needs of each group in preparing training guidelines. health abandonment in iraq. most of the participants believed that the iraqi health system has been abandoned because foodstuff distribution is not under the supervision of a special organization and does not have a special trustee in the execution and monitoring of health rules or, if there is one, he is inconspicuous. the existence of a trustee or supervisor in the health system can prevent the delivery and distribution of unsafe food and reduce the prevalence of infectious diseases. unhealthy foodstuff preparation, production and distribution, and a lack of food evaluation and supervision system can lead to gastroenteric disease. based on the participants' experiences, the lack of a health system trustee means system weakness. the following quotation is an example of the above: "sometimes donations are prepared in an unhealthy manner and so lead to acute digestive problems… the health system is weak in iraq because health rules are not enforced and there is no supervision for these centers…" (executive manager, male, - years, - years job experience). the weakness of health culture. according to the participants' point of views, observation of unhealthy behaviors, such as neglect of the individual and public health standards, and the existence of cultural differences between iran and iraq, are considered by pilgrims to bring about an unsafe culture. policy makers and executives should be familiar with the kind of health culture in iraq so that they can develop a program to prevent contagious diseases. in iranian culture, the non-use of spoons and forks is considered as lack of health belief and neglect of health, whereas in iraq, eating food with the hands is part of the food culture. iran's health system can help train the iraqi people in sanitary practices alongside iranian pilgrims. the following quotation is an example of the above: "some food providers don't meet health … the culture of using spoons and forks for food serving is different in iran and iraq… one of the cultural works which we can do in iraq is health education…" (executive manager, male, - years, - years job experience). problems related to health system. most participants acknowledged that despite annual health care improvements in iraq, there are also some shortages in this field due to the lack of a long history of a health service system. health system weakness, incomplete health service implementation and insufficient supervision of environment health are indicative of the weakness of the health infrastructure; this has made it impossible to provide environmental health, sanitation and waste disposal. the following quotation is an example of the above: "health background in iraq was poor… there were no waste bins and waste sanitation there… failure to implement health services has led to failure in meeting health conditions … there was no health the probability of an outbreak of endemic and non-native diseases the ineffectiveness of the health system in screening the impossibility of requirement flu vaccination the inability of the system to provide services in an epidemic the underlying factors in prevalence of contagious diseases. most participants have identified underlying factors of infectious diseases as one of the challenges affecting the preparedness of the health system in dealing with infectious diseases. various factors, such as population density and diversity, not paying attention to the principles of personal and general health, for example through a lack of health facilities, weather conditions during the trip and changes in nutrition can cause the spread of infectious diseases. identifying these factors helps pilgrims and planners to prevent infectious diseases. the following quotation is an example of the above: "there were few toilets or no healthy facilities. somehow, pilgrims would have to sleep in the desert or in a limited space with a lot of people… congestion of pilgrims from different countries increases the risk of spreading infectious diseases…" (executive manager, male, - years, over year job experience). health system response to communicable diseases. on the basis of the view of participants, mass population movements from different countries and their gathering with different population diversity can transfer and spread endemic diseases and also emerging diseases such as plague and anthrax. there is also the possibility of bioterrorism events occurring during the arbaeen ceremony. the health system needs facilities such as equipped laboratories to diagnose and treat infectious diseases in a timely manner. the inaccessibility or lack of experimental equipment for disease identification leads to the failure of timely diagnosis of diseases, a lack of disease control and finally the incidence of epidemics. syndromic surveillance system can be used to diagnose infectious diseases. there is a need to correctly pass the treatment period of infectious diseases in order to prevent epidemics, although drug shortages can affect treatment completion and is one of the causes of epidemics. vaccination also helps to prevent infectious diseases, but the requirement of pilgrims to vaccinate is always up to the host country and since vaccination is not one of iraq's priorities, the ministry of health and medical education can only advise pilgrims to vaccinate. the following quotation is an example of the above: "there is the probability of spread of local and new-appeared diseases and even bioterrorism due to the gathering of pilgrims from different countries… some diseases can't be diagnosed due to lack of facilities but syndromic surveillance system can be used. …the large number of pilgrims and lack of medication have led to not having complete course of antibiotic treatment… the need for vaccination is one of the requirements of the host country". (policy makers, male, - years, - years job experience). the arbaeen pilgrimage has special features that distinguish it from other mass gatherings. participation of different peoples with a diverse range of socio-demographic statuses, cultures and nationalities makes this distinction. the arbaeen pilgrimage has some specific hazards like other trips that are sometimes neglected. the population of arbaeen pilgrims and its time of year are changeable. financial management of travel expenses at the ceremony is carried out by volunteers. considering these features is essential for the readiness program. in the opinion of the majority of participants, one of the challenges of the health system is the lack of attention to the risks of arbaeen ceremony and the lack of planning based on these features. the following quotation is an example of the above: "arbaeen is a new phenomenon that children and adults, men and women with different cultures and ethnicities take part in the ceremony … even with the knowledge of the dangers of the route, people attend arbaeen ceremony… arbaeen is a spontaneous and popular event and doesn't cost much … the population is moving and the time of the ceremony changes every year …" (executive manager, male, - years, - years job experience). lack of awareness in pilgrims. one of the challenges in the view of the participants, especially executives, is the pilgrims' low awareness of health hazards, such as not using personal hygiene products and non-compliance with health standards. unhealthy and dangerous practices, such as unsanitary food consumption, are not understood by pilgrims, and this low awareness and inadequate knowledge of the risks are the causes of infectious diseases in the pilgrims. respect for personal and public health, such as hand washing, providing food from health food centers and avoiding overeating, is effective in preventing digestive diseases. the following quotation is an example of the above: "some pilgrims do not wash their hands or do not use personal hygiene products …or they do not get foods from healthy centers … overeating and then digestive problems are one of the pilgrims' problems … the other problem is not being familiar with arabic language …" (executive manager, female, - years, less than years job experience). participants believed that one of the health challenges is belief in destiny and fatalism; so that most pilgrims who participate in the arbaeen walking ceremony, based on their belief in destiny, have a relatively low understanding of the dangers and diseases, and begin their trip merely confident in allah and without any plan for dealing with infectious diseases, such as vaccination, and continue the way using food and drinks that are often unhealthy. the following quotation is an example of the above: "it's enough to decide to travel and you do not need to have a special plan … if you think openly, you won't get sick, and nothing bad will happen…" (policy makers, male, - years, - year job experience). based on the participants' experiences, low perception of danger in pilgrims is sometimes seen as insanitary beliefs in preventing medication consumption and disregarding hygiene recommendations. believing in the use of medication during illness, preventing self-curing and trying to abide by hygiene recommendations prevents infectious diseases. the following quotation is an example of the above: "sometimes we see pilgrims using traditional treatments instead of antibiotics consumption… they don't pay attention to hygiene recommendations such as masking and not using suspicious food…" (executive manager, male, - years, - year job experience). training shortage in the targeted group. based on stakeholders' views, one of the present challenges is the quantitative and qualitative shortage in the stakeholder's training level. this means that providing a personal and public health plan should cover all stakeholders, including executives, policymakers and volunteers in the arbaeen ceremony, and be considered with respect to each participant. furthermore, the timing of training is also important and should be before the days of arbaeen ceremony in order to have a greater effect on the individuals' knowledge. indeed, training courses should be held separately for each of the groups, pilgrims, executive managers, and policymakers, and at a proper time before arbaeen. the following quotation is an example of the above: "training should not exactly be in the days of arbaeen. personal and public health training must be held several months before arbaeen… the training is not only for pilgrims, but also anyone involved in arbaeen ceremony. everyone should be trained from pilgrims to policymakers…" (policy maker, male, - years, - year job experience). another problem was the provision of educational content. most of the participants believed that training should be fitted with pilgrims' needs and respond to the problems of pilgrims. pilgrims should be divided into different groups based on the level of education, their problems and illnesses, and individual and general education should be planned accordingly. participants also believed that training in the area of personal, general and nutritional health should be provided more comprehensively. the following quotation is an example of the above: "we should divide pilgrims to diverse groups and send targeted training messages to each group, not the same training for everyone… the benefactors should be trained…pilgrims should have more training…" (policy maker, male, - years, - year job experience). the aim of this study was to explore the challenges of health system preparedness for infectious diseases in the arbaeen ceremony as the first qualitative study in iran. the most important findings of the study are the ineffectiveness of health training, the low perception of risk in pilgrims, poor control of the causative factors of infectious diseases, and deficient and defective health infrastructure in iraq. based on the views of the majority of participants, pilgrim-based training is the most effective factor in health system readiness in dealing with infectious diseases in the arbaeen ceremony. ineffectiveness of health training is one of the challenges of health system preparedness. one of the plans which should be considered to ensure arbaeen ceremony preparedness is health training. educational planning must be done before holding the arbaeen ceremony, with consideration of the training content and targeted groups. indeed, according to the needs of the targeted group, training must be given to pilgrims, executives and volunteer treatment teams, and the training content for pilgrims should be different to that of executives and policy makers. past conducted studies of religious gatherings such as the hajj in saudi arabia and ashura day in iraq, as well as other mass gatherings, such as the tamworth country music festival, australia, indicate the reality that a crowd of people from diverse nations and cultures is a source of infectious disease; disease transmission is one of the most important challenges of public health in these kinds of events, so using training strategies in relation to hand washing, masking and vaccination is an important factor in preventing the diseases and health improvement , , , - . since religious ceremonies are rooted in people's beliefs and have great popularity among people, people-centered education therefore has great potential to reduce the gap between knowledge and practice in pilgrims, empowering individuals and enhancing their ability to deal with health threats. this goal is achieved by identifying accurate and targeted needs, developing relevant content and through educational planning . in this study, like other studies on mass gatherings, a personal health training plan, such as the importance of hand washing, using healthy food and masking, should be included in the pilgrims' training plan, and public health training such as vaccination, environmental health, monitoring donations, cooking and distribution, controlling bioterrorism and establishing mobile toilets should be considered in executives and policymakers' preparedness plan. regarding the aim of holding the arbaeen ceremony, which is a popular religious-ideological ceremony, training can be performed in mosques before the ceremony by clergy. a low understanding of the risks of infectious diseases in pilgrims is one of the other challenges mentioned by the majority of participants, especially policymakers. a lack of risk understanding refers to the inability to identify and respond to dangerous situations. top documents such as sendai framework have identified that understanding risks is the first priority to decrease the incidence of disasters, and that it needs people-based and vast preventive approaches. besides this, the hyogo framework and sustainable development goals emphasize the role of training in increasing risk understanding and decreasing the vulnerability of individuals to hazards - . in this study, one of the health issues was fatalism and a low understanding of risk. a study concerning the beliefs and methods of infection control in hajj pilgrims residing in australia also showed that the majority of participants had low understanding of the occurrence of respiratory infections and the need for an influenza vaccine in hajj, and refused the vaccine, using trust in allah as an excuse and belief in destiny when dealing with the risk of disease . another problem is also the prevalence of self-treatment among pilgrims. in a study aimed at assessing the knowledge, attitude and the performance of australian pilgrims on using antibiotics in hajj showed that they did not have proper understanding of using these drugs and used medications arbitrarily, meaning that more training on proper use is needed . a lack of understanding of health instructions and disregard for public and personal health in any situation can endanger human health. it seems that the fatalism of pilgrims with regards to diseases increases their vulnerability if the understanding of danger is reduced. islamic instructions state that the person is obliged to preserve his health and life in any location and position, even in holy lands, and to avoid risks . given that arbaeen is a religious gathering, religious leaders have an important position in ceremony implementation and can affect the pilgrims' beliefs and understanding of risks during pilgrimage. the influence of religious leaders on the people's beliefs can provide the opportunity to promote health. we should consider that religious scholars must be trained first and then transfer health instructions and methods of infectious disease control to the people in cultural and religious gatherings such as mosques. another problem in this field is the poor control of the effective factors on infectious diseases. one of the most common causes of infectious disease is the epidemiology triangle, which has three components-pathogen, host and environment. the interaction of these agents causes infectious diseases, and considering these factors can help control infectious diseases . considering the three factors in arbaeen is very important. the 'host' factor includes different pilgrims with diverse cultures and nationalities. the 'environment' factor includes holding the ceremony in iraq, which, due to the many years of internal and external conflicts, has had little attention paid to its health infrastructure, and also the overall environmental factors effective in the occurrence of infectious diseases. different studies , , have shown that factors such as crowd size, equipment, climate, the event duration and location, the type of ceremony, and features and behavior of participants affect the occurrence of diseases in mass gatherings, and planners must consider it during preparations. to prevent the occurrence of contagious diseases and their consequences, comprehensive planning, rapid diagnosis and effective management is required , , . one of the factors that affects the occurrence of disease is the time of year that the arbaeen ceremony is held. arbaeen ceremony is held based on the lunar calendar and so its needs and challenges differ and are dependent on the season that the ceremony is held, so that if the ceremony is held in cold seasons, respiratory diseases are more common and if is held in hot seasons, the majority of infectious diseases are of the digestive system. additionally, population movement among different countries leads to the transfer of local diseases, so health considerations and cooperation between states are needed.. policymakers should consider three components-pathogen, host and environment-before the beginning of arbaeen mass gathering. it is also necessary that the health system is aware of all possible scenarios and the methods for dealing with them. the preparedness plan at the local level includes assessing the risk, resources capacity, equipment, surveillance system and an expert team for providing services to pilgrims. health infrastructure defects in iraq was another challenge to the health system from the participants' perspective. the term 'health infrastructures' refers to health facilities and their related factors. the infrastructure includes staff instructions, processes and the development of systematic approaches related to personnel resources and medical support plans . various studies indicate that readiness for structured mass gatherings depends on investing in health infrastructures and the size of gatherings, and strengthening infrastructures and post-event coordination of mass gatherings must be continued. the inappropriate location of gatherings, the weakness of facilities and the lack of infrastructure increase the vulnerability of communities , . the remoteness of health facilities and a lack of needed road infrastructure can make medical services and emergency assistance ineffective. limitation of infrastructure and medical care system, increase the incidence of injuries , . arbaeen is held in a country that has long been involved with interior and exterior wars; therefore, it seems that due to economic difficulties, it does not have the capacity to support the necessary health infrastructure required by pilgrims. although according to the participants, the health system in iraq seems somewhat weak, given that arbaeen ceremony is of particular popularity among shia muslims and is held annually, therefore, some of the activities serving pilgrims and its management during the arbaeen ceremony is voluntarily conducted. in recent years, numerous health facilities have been constructed on religious places, as well as along the path of pilgrimage using pilgrims' donations. management of pilgrims' donations can help to build and maintain health infrastructure in iraq. with this policy, the iranian pilgrims will benefit from the arbaeen ceremony, and the level of health in the region will be improved. this study is the first qualitative study on the experiences of arbaeen, so it provides rich information in this regard; however, since the results have been collected from semi-structured interviews, it is considered subjective. it is recommended that in future studies, by creating a quantitative instrument for examining the challenges and measuring, the subjective concepts can be objectively transformed and analyzed. the present study can be used as a basis for this purpose. the ineffectiveness of health training, low perception of risk in pilgrims, poor control of the effective factors on infectious diseases and deficient health infrastructure in iraq are important challenges of the health system in dealing with contagious diseases in the arbaeen ceremony from the stakeholder's perspective. therefore, pilgrim-based educational planning, along with the control of other challenges, represents an opportunity to improve the health of pilgrims taking part in the arbaeen ceremony. the full data for this study are not provided because the transcripts of the interviews contain identifiable and sensitive information. researchers can apply to access limited deidentified transcripts of interviews from the first author, arezou karampourian (a.karampourian@sbmu.ac.ir) under no strict conditions. please note that transcripts are only available in persian. the study was supported by the shahid beheshti university of medical sciences, tehran, iran. . . the paper is interesting and adds to the medical literature of arbaeen ceremony which has been poorly addressed from a public health research perspective. we hope this paper will serve as a key reference on health aspect of arbaeen ceremony in near future. a few minor issues should be addressed before the manuscript is published. the reference list needs to be revised, we note some misplacement error. for example, in the third paragraph of the introduction (p ), "based on the statistics of , the number of iranian pilgrims taking part in the arbaeen ceremony was , , ", the reference given for this is number . ineffectiveness of health education. emerging of all four themes has been verified by the participants' comments which made results acceptable. the method of analysis is appropriately explained which can facilitate the reproducibility. the authors conclude that the main factor in preventing communicable disease is pilgrim-based training long before the ceremony. this conclusion is adequately supported by the results. is the study design appropriate and is the work technically sound? yes are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. i have read this submission. i believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. the benefits of publishing with f research: your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and more the peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f .com establishment of public health security in saudi arabia for the hajj in response to pandemic influenza a h n knowledge, attitude and practice (kap) survey concerning antimicrobial use among australian hajj pilgrims fatalism at the arbaeen ceremony methodology of applied research in medical sciences letter to editor: mortality trends of pilgrims in hajj: an implication for establishment of surveillance system. health in emergencies and disasters qurterly should cities hosting mass gatherings invest in public health surveillance and planning? reflections from a decade of mass gatherings in pubmed abstract | publisher full text | free full text human stampedes during religious festivals: a comparative review of mass gathering emergencies in india pattern of morbidity and mortality in karbala hospitals during ashura mass this study is part of a phd thesis. the authors thank shahid beheshti university of medical sciences for approving the study, as well as all participants in this study who participated in the study, despite being busy. click here to access the data. competing interests: we have read this submission. we believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. it is better to discuss the results respectively as the results mentioned. so, the reader can insure . it is better to discuss the results respectively as the results mentioned. so, the reader can insure that all results have considered in the discussion part. it is better to explain what pilgrim-based education is and how it controls cd in discussion part and conclusion as well. are all the source data underlying the results available to ensure full reproducibility?no source data required no competing interests were disclosed. this is an interesting and well-written piece of work. infectious disease prevention strategies in mass gatherings, especially religious or cultural gathering, need to start with such the qualitative study. having a predetermined program, implementation, evaluation, and upgrade of the strategies in preventing and responding to communicable diseases requires the proper recognition of the contextual factors and challenges, experiences and perception of healthcare providers and decision-makers of the key agencies involved. the authors carried out a qualitative study with conventional content analysis approach about the arbaeen ceremony, a religious mass gathering in iran and iraq and found four main themes: -health infrastructure defects in iraq, poor control of the causative factors of infectious diseases, -the low perception of risk in pilgrims, and ineffectiveness of health education. emerging of all four themes has been verified by the participants' key: cord- -x js vqe authors: callan, robert j; garry, franklyn b title: biosecurity and bovine respiratory disease date: - - journal: vet clin north am food anim pract doi: . /s - ( ) -x sha: doc_id: cord_uid: x js vqe although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. several animal husbandry and production management practices can be used to minimize pathogen shedding, exposure, and transmission within a given population, however. various combinations of these control measures can be applied to individual farms to help decrease the morbidity and mortality attributed to respiratory disease. and treatment, it seems that respiratory disease remains one of the foremost cattle health concerns. the challenge that the authors were presented with in writing this article was to consider the role that biosecurity could play in reducing the occurrence or effect of respiratory disease. it seems that little research has specifically evaluated the effects of biosecurity management practices on the occurrence of the problem in livestock operations. indeed, recognizing the multifactorial etiology of infectious respiratory disease and the ubiquitous presence of the pathogens involved leads to the conclusion that attempts to decrease disease prevalence must incorporate multiple management steps, of which biosecurity practices are only a single component. although biosecurity practices have equal potential to decrease respiratory disease losses in all food animal species, the authors focus this article primarily on bovine respiratory disease complex. this article addresses major areas of respiratory pathogen control and provides some suggestions for practical intervention. bovine respiratory disease is not a single entity, nor is it attributable to a single cause [ ] . one useful scheme for characterizing respiratory tract diseases in a practical manner distinguishes three different categories of problems [ ] . these include the bovine respiratory disease complex (brdc), epitomized by shipping fever pneumonia and enzootic calf pneumonia; acute interstitial pneumonias; and metastatic pneumonia. this scheme excludes many problems that involve only the upper respiratory tract, although these problems may predispose to lower tract infections. the interstitial pneumonias are most commonly attributed to toxicoses, and metastatic pneumonias are secondary complications of disease in other organ systems that spread hematogenously to the lung. although these disease problems are frequently fatal for affected cattle, they occur sporadically and are generally not considered to be contagious. the authors focus their attention for this discussion on brdc. this problem has an infectious origin, and it is by far the most frequently occurring form of cattle respiratory disease. cattle of all ages and in a variety of circumstances can be affected by brdc, but the disease most commonly manifests in young dairy calves (enzootic calf pneumonia) and in beef calves recently arrived at feedlots (shipping fever pneumonia). research over the past several decades has provided an increasingly clear picture of how brdc occurs and why it is so common. unfortunately, this knowledge has not led to a commensurate decrease in the morbidity and mortality associated with this problem, primarily because animals are commonly managed in ways that predispose to disease development. bovine respiratory disease complex refers to bacterial bronchopneumonia that may or may not be complicated by previous or concurrent viral or mycoplasma infection [ ] . numerous bacterial species can be isolated from the lungs of affected animals. in feedlot cattle and adult cattle, mannheimia (pasteurella) haemolytica is considered the most important pathogen, with lesser roles attributed to pasteurella multocida and hemophilus somnus. in younger calves, these same pathogens play a role, but mycoplasma spp. are also considered to be important. arcanobacterium pyogenes, fusobacterium spp., and bacteroides spp. are frequently isolated from animals with chronic, abscessing lung lesions but do not play a major role in acute bronchopneumonia. less common bacterial isolates, including streptococcus spp., staphylococcus spp., pseudomonas aeruginosa, and chlamydia spp., are also occasionally identified in young calves. all of the bacterial pathogens considered important in brdc can be isolated from the upper respiratory tract of healthy cattle and calves. these pathogens are considered ubiquitous in cattle populations, not because they can be found in each animal, but because they are readily identified in the nasopharynx of some animals in most populations. in the absence of other predisposing causes of disease, it seems that the simple presence of these bacterial agents is not of major significance. the disease complex is best characterized as being multifactorial, only occurring when a combination of factors involving the animal, environment, and infectious agents are present. viral pathogens are implicated in the development of brdc, although the final pulmonary pathology is primarily caused by bacterial pathogens [ ] . the principal viruses involved in brdc include bovine herpesvirus (infectious bovine rhinotracheitis), bovine parainfluenza virus type , bovine respiratory syncytial virus, and bovine viral diarrhea virus. lesser roles are attributed to bovine coronavirus, adenovirus, rhinovirus, reovirus, and enterovirus. these viral pathogens primarily infect the upper respiratory tract, resulting in rhinitis, tracheitis, and bronchitis. their ability to cause direct pulmonary disease is generally limited except for bovine respiratory syncytial virus, which can also cause severe lung damage as the primary agent. all of these viral pathogens predispose the lung to bacterial infection and bronchopneumonia. the primary role of these agents in brdc is to promote bacterial challenge to the lungs by compromising respiratory tract defense mechanisms. the predisposing causes of brdc act synergistically and are most commonly identified in combination rather than as single causative problems. the list of predisposing animal factors is long and includes animal age, decreased immune responsiveness due to animal stress, lack of previous viral exposure or vaccination, inadequate passive immunoglobulin transfer in young calves, nutritional deficiencies, and dehydration. environmental risk factors include high air humidity or dust content, rapidly changing environmental temperatures, extreme heat or cold, and high concentrations of noxious gases such as ammonia. several risk factors may increase pathogen density or pathogen exposure, although these risk factors probably act by other means as well. for example, commingling cattle from multiple sources may increase exposure to antigenically heterogeneous viral pathogens, while also increasing animal stress. poor ventilation and high humidity can increase pathogen density and survival time but also can increase noxious gas concentrations and adversely affect pulmonary function. animal crowding increases airborne pathogen exposure but also induces animal stress and reduces immune responsiveness. when evaluating the rate of brdc occurrence in cattle populations, it is clear that efforts to prevent this disease have not been effective on an industry-wide basis, although some individual producers have successfully used prevention strategies. the two biggest areas of brdc effect are in the form of enzootic calf pneumonia of dairy calves and shipping fever pneumonia of feedlot cattle. given our current understanding of this disease problem, it is clear that the animal management systems employed for these groups of animals (i.e., dairy calf-rearing systems and feedlot cattle-receiving systems) have failed to rigorously apply knowledge of disease pathogenesis and prevention into their processes. this situation may be changing currently, as the beef production industry increasingly uses quality-assurance principles in production systems and develops marketing procedures and animal-purchasing practices that reward improvements in animal health [ , , ] . similarly, the dairy industry has begun to recognize the economic benefit of improved calf health and increasingly uses specialized calf-rearing systems [ , ] . because the purpose of this article is to examine the role of biosecurity management in respiratory disease prevention, the authors do not attempt to provide a complete review of brdc preventive practices. many of the important means of preventing brdc do not employ biosecurity but are targeted toward enhancing animal immune preparedness and enhancing animal response to infectious challenge. effective respiratory disease preventive practices are those targeted at reducing identified risk factors for disease development [ , , , ] . these practices include management to improve animal nutrition with special emphasis on micronutrient nutrition, practices that reduce animal stress, reduced commingling of animals, improved animal transportation and feedlot receiving practices, improved preconditioning and vaccination programs that emphasize vaccination before shipment and during times of low calf stress, and improved ventilation with reduced crowding. it is important to consider the factors that drive the development and implementation of disease prevention and biosecurity programs. the most apparent of these factors is the effect on animal production and growth; however, all interventions have their cost, and these costs must always be considered relative to the potential economic returns. unfortunately, information regarding the financial impact of herd biosecurity programs is limited, and estimates based on clinical experience must often be applied. other issues, including herd pathogen status and its effect on livestock marketing, food product quality assurance, drug residues, injection site lesions, antimicrobial resistance, and animal welfare also contribute to the forces that drive the development of biosecurity programs. ultimately, a biosecurity program must be integrated into the overall herd management. it must be developed using a team approach that addresses the concerns of the producer, the economic effect on the production unit, the influence on product quality, and public health concerns. the veterinarian is best suited to effectively develop and implement such programs. the multifactorial nature of brdc and the ubiquitous presence of respiratory pathogens are important concepts when considering the role that biosecurity can play in decreasing the prevalence of disease. for infectious diseases in which point source pathogen exposure, high susceptibility, and high virulence are prominent features of disease transmission (e.g., anthrax, footand-mouth disease, rabies, and so forth), limiting animal contact with the pathogen is a key feature of disease prevention and may even provide the means of disease eradication. alternatively, when the causative pathogens are endemic in a population and individual susceptibility is dependent on numerous interrelated factors, the management of animal resistance and risk factors may be proportionally more important for disease prevention than biosecurity practices. it appears that brdc prevention requires a combination of management to enhance animal resistance plus management to reduce exposure to the pathogens. the important point is not to de-emphasize the value of reducing pathogen introduction, exposure, and transmission (i.e., biosecurity) but to also stress the importance of other management features that promote animal resistance. it is particularly important that preventive management practices be coordinated and used in combination, because no single management procedure will be successful without the complement of other practices. it is likely that our inability to reduce the prevalence of respiratory disease in cattle is, in part, attributable to our failure to integrate multiple aspects of respiratory disease prevention practices, including biosecurity. the fundamental concept of biosecurity is to decrease pathogen transmission between animals. transmission of respiratory pathogens occurs by close nose-to-nose contact, environmental or fomite exposure, and airborne exposure. increased contact between shedding and susceptible individuals increases pathogen spread. environmental exposure through common areas and equipment that involve oral or nasal contact such as feed bunks, water troughs, and salt blocks may be an even greater risk, however. total environmental pathogen load is extremely important in considering respiratory pathogen transmission. environmental contamination from animals in contact is the primary source of most respiratory pathogens. individual animal shedding is quite variable and depends on the etiologic agent, the time course of the disease, the clinical severity, and the immune response of the host. in general, clinically ill animals shed greater numbers of pathogens than normal or asymptomatic animals; however, it must be recognized that individuals periodically shed both viral and bacterial respiratory pathogens without evidence of disease. well-vaccinated animals may also periodically shed pathogens and should not necessarily be considered completely safe from disease transmission. the persistence of the pathogen in the environment also contributes to pathogen exposure. environmental pathogen survival times depend on many factors, including organic material, moisture, direct sunlight, and exposure to disinfectants. environmental survival times for most viral respiratory pathogens are probably on the order of minutes to several hours [ , ] . survival times for bacterial pathogens may be longer depending on the environmental conditions and the organism. airborne transmission is dependent on numerous factors, including ambient temperature, relative humidity, airborne particle (dust) density, ventilation, prevailing wind, and structural or geographic obstructions [ ] . airborne transmission of typical viral respiratory pathogens can occur over distances as far as meters and possibly further [ , ] . airborne transmission of other viruses such as footand-mouth disease virus or pseudorabies virus has been shown to occur over many miles, however [ , , , , ] . adding to the complexity of pathogen transmission, it seems that the efficiency of transmission is different between different strains of a given pathogen [ ] . understanding how management practices can reduce either pathogen shedding or exposure is the key to creating effective biosecurity programs. the term biosecurity is used for those management and hygiene practices that reduce introduction, exposure, and transmission of infectious agents. although biosecurity may not provide the single most important component of respiratory disease prevention, reducing pathogen exposure is a valuable part of any infectious disease management system. little information is available to specifically evaluate the effect of individual biosecurity practices in prevention of brdc, but there are some important respiratory disease prevention practices that limit pathogen exposure and good reason to more closely evaluate the role that biosecurity could play in the future. the authors emphasize five areas of biosecurity management that should be more rigorously applied for the reduction of respiratory disease prevalence in cattle, including ( ) strategic vaccination, ( ) calf biosecurity, ( ) housing ventilation, ( ) commingling and animal contact, and ( ) bovine viral diarrhea virus control. many improvements in vaccine technology have occurred over the past few decades, and practitioners have an array of improved bovine respiratory pathogen vaccines at their disposal [ ] . unfortunately, the current respiratory pathogen vaccines have not all been scrutinized for efficacy to the most desirable degree, and many do not protect against respiratory disease nearly as effectively as some veterinarians and producers would like to believe. although vaccines directed at specific conserved proteins, such as toxoid vaccines, may completely prevent a particular disease, vaccines against complex disease agents that have multiple antigenic strains are unlikely to be capable of such levels of protection. respiratory vaccines are better viewed as disease modifiers than absolute preventive agents. vaccines are usually used as a means to decrease the likelihood or severity of disease occurrence in the individual animal receiving the vaccination. indeed, vaccine efficacy may be evaluated in many ways, but the more rigorous evaluations involve the ability of a vaccinated animal to withstand a challenge of disease or pathogen exposure [ ] . practitioners tend to view vaccination as one of the management factors that enhance animal resistance to infection and thus augment the value of biosecurity management by working to reduce susceptibility to infectious disease rather than decrease exposure and transmission. for respiratory disease prevention, however, effective vaccination can also serve as part of a biosecurity management system. in addition to preventing disease, a vaccine's efficacy might also be considered for its ability to limit pathogen shedding when infection does occur. vaccineinduced immunity often results in decreased magnitude and duration of pathogen shedding [ , , ] . because exposure is directly related to pathogen concentration in the environment, it follows that vaccine-induced reductions in shedding should decrease transmission within a susceptible population. proper vaccine use and a well-managed vaccination program can be viewed as part of a complete biosecurity program. at a minimum, a good vaccination program should include the following: • proper storage and administration of the vaccine as indicated by the manufacturer's labeled recommendations. • vaccination of all susceptible animals, including both resident and incoming animals. • application of the vaccine to systemically healthy, well-nourished, minimally stressed, and immunocompetent cattle. • strategic timing of vaccination so that it precedes contact with new animals long enough to allow an appropriate immune response. • revaccination as recommended for the particular vaccine product. biosecurity management of calves is extremely important for development of healthy animals. many of the biosecurity recommendations for newborn calves focus on decreasing the transmission of enteric pathogens; however, these same principles can be important for minimizing respiratory disease problems. several details of calf biosecurity management deserve emphasis. environmental and housing factors significantly affect calf health and viability. differences in calf management for cow-calf herds versus dairies are related to the relative risk of respiratory disease between these two production groups. beef calves are generally raised in open-range situations that effectively dilute the exposure to respiratory pathogens. although beef calves are continually exposed to pathogens shed from adult cattle and other calves, the magnitude of pathogen exposure before weaning is generally low, resulting in relatively little respiratory disease. in contrast to many enteric pathogens, the environmental survival time of the respiratory pathogens is limited [ ] , and accumulation of pathogens in the environment is not considered a primary concern. dairy calf housing has a significant effect on the incidence of respiratory disease in neonatal calves. although the common viral respiratory pathogens can be transmitted over distances up to meters [ , ] , properly spaced calf hutches seem to effectively limit aerosol transmission of respiratory pathogens. the short survival of these pathogens in the environment limits the transmission between successive occupants of an individual hutch. disinfection procedures that are used for enteric diseases should be more than sufficient to decrease respiratory pathogen transmission (see article by barrington et al. in this issue). in contrast, there is a high risk of respiratory disease transmission in group-raised neonatal calves. factors including the number of animals, relative animal density, housing facilities, and ventilation conditions significantly contribute to transmission in grouped calves and are discussed in subsequent sections of this article. numerous management practices can decrease exposure and transmission of respiratory pathogens to calves in dairy operations. feeding pasteurized milk or milk replacer is a useful biosecurity practice for minimizing the spread of enteric agents such as salmonella spp. or mycobacterium avium subsp. paratuberculosis. these practices are also effective at limiting ingestion of potential respiratory pathogens. mycoplasma spp. bacteria are commonly implicated in newborn calf disease, including enzootic calf pneumonia [ , , , ] . although mycoplasma spp. may spread by the airborne route, it is also a common mastitis pathogen and can be shed from clinically or subclinically infected cows [ , , ] . nasopharyngeal colonization occurs after oral ingestion of contaminated milk, potentially resulting in clinical respiratory disease in calves [ ] . mycoplasma spp. and other pathogens can also spread hematogenously after ingestion by a susceptible calf [ , ] . similarly, other potential respiratory pathogens such as streptococcus spp., staphylococcus spp., salmonella spp., and escherichia coli can be recovered from milk and spread hematogenously to the lungs after oral ingestion. bovine viral diarrhea virus is shed in the milk of persistently infected cattle. ingestion of bovine viral diarrhea virus-contaminated milk can result in respiratory and systemic infections, possible immune suppression, and respiratory disease. proper cleaning and disinfection of calf feeding equipment, including nursing bottles, buckets, and mixing utensils, should be performed. equipment should be cleaned with a detergent and disinfected between uses. a common and economical disinfectant is standard household bleach used at a : dilution. bottles and equipment that are potentially shared between multiple animals should be soaked for to minutes in this solution. although bleach will not completely kill all potential pathogens, it is effective at significantly decreasing viable numbers and thus contributing to decreased exposure and transmission between feedings. prompt removal of dairy calves from the maternity pen environment, where they are exposed to numerous adult cow pathogens, can also decrease transmission of potential respiratory pathogens. newborn calves should not have direct contact with older calves and adults. calf hutch spacing should be evaluated, with a minimum of feet of separation between calves. worker hygiene can minimize contamination of calf feed and the calf environment. appropriate vaccination of dams before colostral production can increase passive transfer of effective antibodies, reducing the risk of exposure and potential shedding after infection. it has been demonstrated that good colostral transfer to beef calves was associated with decreased occurrence of disease episodes and improved calf performance all the way through the growing and finishing period in feedlot animals [ ] . it is unlikely that the passive transfer of immunoglobulins per se is specifically responsible for beneficial effects on the long-term health of animals, but profound effects may result from management that improves newborn health and disease resistance. this in turn provides for improved nutrition, growth, physiologic well-being, and decreased total pathogen load. numerous calfhood husbandry procedures should be considered as standard biosecurity protocols for all infectious diseases, including respiratory disease. sick animals should be identified and separated from healthy animals. a specific calf-isolation area should be established, with consideration to animal comfort and ease of cleaning and disinfection. where practical, individual equipment should be used for each separate calf. specific care and treatment personnel should be identified, and animals with suspected infectious diseases should be treated after handling healthy animals. additional personnel hygiene protocols include dedicated coveralls to be used in the sick pens, the use of rubber overboots, and disinfectant footbaths. personnel should be encouraged to wash their hands before and after entering the sick pens and between caring for animals with dissimilar disease conditions. in many cases, equipment and facilities need to be made available to help establish such procedures. similar biosecurity management practices can be used in cow-calf herds. although feeding pasteurized milk or milk replacer is obviously not a practical management practice, milk-borne exposure to pathogens can be minimized by proper attention to the adult cows. adult cattle must be appropriately vaccinated to provide optimal colostral immunity to the calves and to decrease adult cow infections and shedding. adult cow nutrition should be optimized to improve colostrum quality. adult cow nutrition can also have a dramatic effect on calving ease and decrease the incidence of dystocia. special attention should be placed on high-risk calves, including calves delivered with manual assistance, cesarean section, born in inclement weather, weak or premature calves, and multiple births. such calves often do not nurse colostrum in a timely fashion or have impaired absorption of immunoglobulin. cows should be evaluated for evidence of clinical mastitis and treated or culled as appropriate. decreased morbidity can be observed by minimizing the time that beef cow-calf pairs spend in a designated calving area, where pathogen loads tend to increase throughout the calving season. bovine viral diarrhea virus surveillance and eradication in cows and calves should also be used (see discussion in a following section). as can be seen from the preceding discussion, many of the management practices that contribute to biosecurity of respiratory disease are standard quality-assurance practices that are recommended for basic calf health. good ventilation is a critical aspect of animal management and can profoundly affect respiratory health. several discussions of ventilation and its effect on animal health are present in the literature [ ] [ ] [ ] [ ] , , , , , , , , , , ] . proper ventilation serves eight primary functions: . it decreases the airborne pathogen concentration . it eliminates noxious gases (ammonia, hydrogen sulfide, carbon dioxide, carbon monoxide, and methane) . it decreases airborne dust contamination . it decreases airborne endotoxin levels . it maintains optimum ambient temperature . it maintains optimum environmental humidity levels . it eliminates drafts . it eliminates areas of stagnant air with respect to biosecurity, one of the most important aspects of proper ventilation is the reduction in the concentration of airborne pathogens. all of the important viral and bacterial respiratory pathogens can spread aerogenously and can attain high concentrations in poorly ventilated housing areas. airborne pathogen concentration is a function of many factors, including animal type, housing system, stocking rate, bedding, humidity, dust particle density and size, and finally, elimination through ventilation. improved ventilation is one important means whereby airborne pathogen concentration can be readily decreased within the given constraints of an operation; however, pathogen removal is not a linear function, and practical and theoretical limits are often observed [ ] . studies of building ventilation for humans demonstrate potential reductions in airborne exposure of pathogens and disease incidence, although improved ventilation beyond that which provides comfort may not be practical or provide significant additional benefit [ ] . as the airborne pathogen load rises, ventilation provides progressively less protection against respiratory infections. it is important to realize that stocking rate has a more dramatic effect on airborne pathogen density than ventilation [ , ] . for example, a two-fold increase in stocking rate requires nearly a -fold increase in ventilation to maintain the same airborne pathogen density [ ] . ventilation cannot overcome grossly inadequate housing, management, or hygiene within a production unit. along with stocking density, there are other practical concerns that contribute to airborne pathogen density and transmission. one of these is related to animal handling and excitement. it is extremely important to handle grouped animals in a calm environment with minimal animal activity and stress. increased animal activity not only increases dust exposure (which contains airborne pathogens) but also increases ventilatory rate, ventilatory effort, and tidal volume, which in turn increases the amount of aerosolized pathogen shed by infected animals and the amount of pathogen inhaled by susceptible animals. the increased dust exposure will also adversely affect mucociliary clearance and respiratory defense mechanisms. part of the effect of ventilation is to minimize airborne contaminants that can impair respiratory function and defense mechanisms [ , , , ] . significant airborne contaminants include ammonia, hydrogen sulfide, carbon dioxide, carbon monoxide, methane, dust particles, and endotoxin. ammonia and hydrogen sulfide are toxic gases and can contribute to respiratory damage, decreased mucociliary clearance, decreased alveolar macrophage activity, and overall compromise to respiratory defense mechanisms. carbon dioxide, carbon monoxide, and methane contribute primarily as asphyxiative gases and generally do not contribute to significant impairment of the respiratory tract. dust particles also contribute to the impairment of respiratory defense mechanisms. dust particles can arise from both organic and inorganic sources. in general, particles greater than lm are filtered out by the nasal passages; most particles from to lm are removed by the mucociliary clearance of the trachea and bronchi, and particles less than lm can penetrate to the alveolar spaces [ , ] . organic and inorganic dust particles can impair mucociliary clearance and overload alveolar macrophage phagocytic clearance [ ] . organic dust particles are generally of more concern in confinement and intensive housing situations. in animal housing environments, most of the organic dust arises from fecal material, skin, and hair. organic dust is significant in that it often contains high endotoxin and pathogen levels [ , ] . inhaled endotoxin can contribute to pulmonary compromise by initiating inflammatory reactions within the alveoli and alveolar vascular endothelium. appropriate ventilation is also important in maintaining acceptable humidity and ambient temperature levels within confinement or semi-open housing. observed thermoneutral ranges (the range of air temperature that sustains optimal performance) for a variety of domestic livestock are available (table ) [ ] . in general, livestock can perform adequately within a fairly wide thermoneutral range. higher temperatures, especially when combined with high humidity, tend to be more problematic than low temperatures [ ] . depending on the given climate and temperature ranges of a geographic region, housing ventilation will need to be designed to provide either heating or cooling or both. cold temperatures and perhaps temperature fluctuations can decrease mucociliary clearance and predispose animals to respiratory disease [ ] . often, wide temperature fluctuations are more detrimental to animal health because they do not allow suitable adaptation over time. there is minimal information on how ambient temperature directly relates to airborne pathogen biosecurity. increased ambient temperature results in increased respiration and may increase pathogen shedding from infected animals. the direct effects of ambient temperature on pathogen survival are relatively unknown. some studies suggest that the concentration of airborne particles is increased at low temperatures, and airborne bacterial concentrations were higher in winter than in summer [ ] . there is slightly more information concerning the effects of relative humidity on pathogen survival and thus, airborne biosecurity [ , ] . in general, viruses with a hydrophobic lipid outer shell (i.e., enveloped viruses) survive better in lower humidity, and lipid-free viruses (i.e., foot-and-mouth disease virus) are more stable in moist air [ , ] . the four primary viral respiratory pathogens in cattle (bovine herpsevirus , bovine parainfluenza virus type , bovine respiratory syncytial virus, and bovine viral diarrhea virus) are all enveloped viruses and would be considered more stable in dry air, although the authors are unaware of specific studies documenting this conclusion. gram-negative bacteria have outer phospholipid membranes and are also expected to be more stable in dry air [ ] . mycoplasma are reported to be sensitive to relative humidity between % and % [ ] . extrapolation of these limited data suggests that typical airborne pathogens associated with respiratory disease in domestic animals survive better in cool, dry air such as is observed in the late fall, winter, and early spring months. although this correlates with clinical observations concerning the relative seasonal incidence of respiratory disease, a direct association has not been established. in beef cattle, seasonal increases in respiratory disease also correlate with seasonal management practices associated with movement of cattle to feedlots and increased animal density. it is likely that climate and management factors act together to dramatically increase pathogen exposure and transmission in feedlots. alternatively, the high humidity that can be observed with dairy confinement housing in cold weather probably contributes to increased respiratory disease because of the higher pathogen density associated with increased aerosolized particle concentrations. ventilation systems should be constructed to provide even airflow throughout the structure without areas of air stagnation or drafts. pockets of air stagnation have higher levels of airborne contaminants and contribute to the exposure and transmission of respiratory pathogens. air stagnation can often be remedied by appropriate use of inexpensive fans. correcting draft conditions can be more problematic and often requires complete evaluation of the housing structure for air leaks and evaluation of the ventilation system, especially air intake vents. guidelines for housing of livestock have been reported, including recommendations for ventilation (table ) [ , , , , , , ] . appropriate ventilation should flow from younger to older animals to minimize spread of pathogens to the more susceptible animals. the total air volume should be completely changed times per hour in winter, and it should be changed up to times per hour in summer [ , ]. the ventilation system should confinement housing • minimum of four air changes per hour (winter) • total exhaust capacity for up to air changes per hour (summer) • continuous (not intermittent) ventilation • single-speed fans, not variable-speed fans, should be used • fans must be able to sustain / -inch static pressure • one must allow for two to four different ventilation rates using multiple fans • enough inlet slot area should be provided to allow minimal inlet velocity of fpm (winter) and fpm (summer) • thermostats should be used to control ventilation fans • thermostats should be located at eye level near the center of the barn • the ventilation rate should be altered by stepping up the number of fans used for each level • wall fans should be mounted near the ceiling but collect air using ducts from within cm ( in) of the floor • the fresh air intake should be located near the ceiling but at least feet from any exhaust fan • adjustable eave slot inlets should be used to distribute incoming air uniformly provide constant rather than intermittent airflow. in the winter, the goal of ventilation is to minimize airborne pathogen density, remove excess moisture from animal respiration, and maintain adequate ambient temperature ( - °c, - °f). although higher ventilation rates improve air quality, they are inefficient because they require excessive heating costs. supplemental heating may be necessary as the outside temperature falls or stocking density decreases. at optimal stocking densities, livestock generally produce enough animal heat to maintain adequate ambient temperature in confined housing when outside temperatures remain above ÿ °c [ ] . winter ventilation is a compromise between the removal of airborne contaminants and the maintenance of ambient temperature. the primary goal of summer ventilation is to minimize ambient temperature and relative humidity. this requires high ventilation flow rates, which also enhance air quality. the goal is to maintain an ambient housing temperature to no more than °c above the outside temperature [ , ] . relative humidity levels should be maintained between % and %, and ammonia levels should not exceed ppm [ , , , , ] . maximum recommended stocking densities should not be exceeded (see table ) [ ] . separate age groups of cattle should be maintained in separate barns or be separated by barrier walls. calf hutches for individual dairy calves provide an ideal means of managing relative calf isolation and limiting airborne transmission if they are properly positioned and spaced. recommendations for calf hutches include one calf per hutch with a minimum separation of feet between hutches. hutches should be placed at least feet from oldercattle enclosures and feet from livestock building exhaust fans. many cattle management systems provide numerous opportunities for exchange of respiratory pathogens from animal to animal. assembling groups of beef calves for a feedlot often involves mixing calves from different origins, congregation of animals at sale barns or other holding pens, and movement in congested cattle transports. these activities are well known to increase the rate of respiratory disease occurrence by stressing the animals and providing circumstances that decrease disease resistance. these same animal contact and crowding circumstances can dramatically increase exposure to pathogens, often including pathogens to which the animal has not developed prior immunity. in a recent national survey, more than % of dairy producers housed sick animals in a manner that allowed direct nose-to-nose contact with healthy herdmates [ ] . many dairy producers expand their herds by purchasing animals from other sources, but less than % of them provide any quarantine time for the incoming animals. for producers who introduced % or more of their total animal inventory during an expansion, . % reported an increase in occurrence of respiratory disease during the year [ ] . during the early phases of respiratory disease, the shedding rates of pathogens via respiratory secretions increases dramatically. commingling, crowding, and the animal stresses that are involved in animal movement can precipitate respiratory problems. these same factors can increase spread of pathogens to other animals with close contact. although quarantine may not be effective against diseases with chronic carrier states such as johne's disease, it can substantially decrease the risk of spreading respiratory pathogens. furthermore, the duration of respiratory pathogen shedding has also been well characterized. in general, nasal shedding of viral respiratory pathogens is significantly reduced by days after infection but may persist longer in individual animals, which suggests that quarantine for approximately to days should significantly reduce the exposure and transmission of these pathogens within an operation. practical suggestions for limiting pathogen spread by contact include quarantine of incoming livestock, maintenance of hospital areas that do not allow contact with healthy animals, prevention of animal contact between different age groups of cattle, minimizing the time animals spend in market channels, and limiting the introduction of new animals to assembled herds or pens of cattle. the concepts of pathogen transmission within grouped housing can be effectively applied to weaned dairy calves. calves receive relatively low pathogen exposure while in calf hutches. on weaning and grouping in calf pens, the risk of exposure increases dramatically. it is important to appreciate that the risk of exposure rises with the number of calves housed together. for example, if one estimates that % of calves born in a herd with bovine viral diarrhea virus are persistently infected, then the probability of bovine viral diarrhea virus exposure in a group of calves is approximately . ( - . ). if the stocking rate increases to calves, the probability nearly doubles to . ( - . ). limiting the number of calves per pen to less than seven is associated with decreased respiratory disease mortality [ ] (also see article by smith in this issue). it must be emphasized that one animal can expose an entire pen of animals by simple close contact, airborne transmission, or environmental transmission at common housing areas such as feed bunks and water troughs. by dividing animals into smaller groups, the number of animals exposed is lowered significantly. using the same example of bovine viral diarrhea virus exposure, if one splits the calves into three separate pens, the probability of having all calves exposed to bovine viral diarrhea virus falls from . to the comparatively negligible level of . [( - . ) ]. simple segregation of animals is not sufficient unless physical barriers for fence line contact, separation of food and water troughs, segregation of likely fomites, and blocking airborne spread are used. these same principles can be applied to any group-housing situation. such management and housing decisions must be made based on a balance between the risk and cost of disease versus the availability and cost of facilities and labor. it was noted previously that the common bovine respiratory pathogens are considered to be ubiquitous in cattle populations in the united states and most other countries. although this does not suggest that every animal harbors each pathogen, these agents can be found routinely in the nasopharynx of healthy and diseased animals within most herds. in contrast, some european countries have successfully eradicated some viral respiratory pathogens such as bovine herpesvirus and bovine viral diarrhea virus (bvdv) from cattle populations. under the currently prevailing practices within the united states and many other countries, the authors do not suggest testing or identification of most respiratory pathogens as a viable means to identify carriers or to exclude the animals from introduction into a herd. the exception to this is bvdv. although bvdv is not considered a primary pneumopathogen, it is considered to have an important role in respiratory disease of cattle [ , ] . the immunosuppressive effects of the virus and the close association of bvdv infection and respiratory disease occurrence in some epidemiologic studies suggest that the virus plays a role by promoting secondary bacterial lung infection. although bvdv vaccines have been improved over the past several years, vaccination alone rarely eliminates bvdv from an infected herd. an effective bvdv biosecurity program must include the identification and removal of persistently infected animals, bvdv screening of incoming animals and their calves, and a comprehensive vaccination program [ , , , ] . persistently infected cattle do not mount an effective immune response against the virus and are capable of shedding large amounts of the virus into the environment through multiple routes. persistently infected animals have been implicated as the primary means by which bvdv infection is maintained in assembled dairy herds, and they are also considered a significant threat for transmission in cow-calf and feedlot operations [ ] [ ] [ ] [ ] , , ] . with the development of new tests over the past several years, our ability to accurately and expediently identify persistently infected animals has dramatically improved [ , ] . the serum immunoperoxidase monolayer assay (ipma) and antigen capture elisa tests and the immunohistochemistry test of skin biopsy material have appropriate sensitivity and specificity for detecting persistently infected cattle. the authors do not know of significant published research that evaluates the effect of test-and-cull strategies for bvdv on the occurrence of brdc; however, elimination of persistently infected animals from herds can have significant positive effects in decreasing other bvdv manifestations such as reproductive failure. implementing test-and-cull procedures for persistently infected animals may prove to be a powerful means of decreasing brdc prevalence. in general, all cattle introduced into a herd should be tested for bvdv before purchase or entry. acute bvdv infections of pregnant cattle can result in animals that are bvdv negative at the time of testing while the fetus is persistently infected. it is critical that all calves from newly introduced pregnant animals also be tested immediately after birth. to establish a bvdv-negative herd, it is generally more effective and economical to test calves as they are born rather than screen adult populations. a negative result for a calf indicates that not only the calf but also all of the calf's maternal ancestors are not persistently infected. a single positive test on a calf does not differentiate between acute and persistent infection. a confirmatory test may be performed in weeks, or the animal may be assumed to be persistently infected and euthanized or sold for slaughter. the dams of all persistently infected calves should be traced and tested as well to determine their status. in most cases, these animals will test negative, indicating fetal exposure due to acute infection during gestation. bulls should also be tested because they can contribute to animal exposure within a herd. although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. several animal husbandry and production management practices can be used to minimize pathogen shedding, exposure, and transmission within a given population, however. various combinations of these control measures can be applied to individual farms to help decrease the morbidity and mortality attributed to respiratory disease. dairy calf pneumonia: the disease and its impact the bronchopneumonias (respiratory disease complex of cattle, sheep, and goats) appraising the adequacy of environment for confined animals influence of improved ventilation on health of confined cattle calculation of ventilation needs for confined cattle a new inactivated bvdv genotype i and ii vaccine: an immunisation and challenge study with bvdv genotype i control of bovine viral diarrhea virus infection without vaccines control of bovine viral diarrhea infection by use of vaccination diagnosis of bovine viral diarrhea virus infections further evidence of long distance airborne transmission of aujeszky's disease (pseudorabies) virus evidence of long 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respiratory syncytial virus vaccines in experimentally infected calves persistent bovine viral diarrhoea virus infection in us beef herds key: cord- -yq dtf n authors: samaranayake, lakshman p.; peiris, malik title: severe acute respiratory syndrome and dentistry a retrospective view date: - - journal: the journal of the american dental association doi: . /jada.archive. . sha: doc_id: cord_uid: yq dtf n abstract background severe acute respiratory syndrome, or sars, which has created panic in asia and in some parts of north america, is the first epidemic of the new century. although it has been well-contained, sporadic cases continue to emerge. objectives the authors trace the emergence of the sars outbreak from southern china and its spread worldwide, discuss the viral etiology of the infection and its clinical features, and review the infection control guidelines issued during the outbreak by the health authorities in hong kong, the centers for disease control and prevention, the world health organization and the american dental association. they also review the prospects for a new outbreak and preventive measures. overview the disease, which is caused by a novel coronavirus termed the “sars coronavirus,” or sars-cov, essentially spreads through droplet infection and affects people of any age. it has a mortality rate ranging from to percent. a major hallmark of this disease has been the rate at which it has affected health care workers through nosocomial transmission; in some countries, up to one-fourth to one-third of those infected were in this category. however, no dental health care worker has been affected by sars in a nosocomial or dental setting. conclusions and clinical implications researchers believe that a combination of factors, including the universal infection control measures that the dental community has implemented and/or the low degree of viral shedding in the prodromal phase of sars, may have obviated the spread of the disease in dental settings. the dental community should reflect on this outbreak to reinforce the currently applied infection control measures. m icrobial threats continue to emerge, reemerge and persist. some organisms are newly recognized pathogens that have existed for centuries (for example, helicobacter pylori, which causes gastric ulcers). others are old organisms that have learned new tricks (for example, multidrug-resistant tubercle bacilli). a third category consists of totally new organisms. this last group of alarming new infectious agents that are virulent and deadly have emerged in rapid succession during the last few years. some of these, such as the ebola virus infection, are still smoldering in some remote corners of the world, while others, such as the h n (and h n ) influenza a bird flu virus and the west nile virus infections, are emerging in different parts of the world. severe acute respiratory syndrome, or sars, is the latest addition to this deadly assortment of new diseases. in the face of these infectious threats, in particular the pandemic of hiv infection, the dental community has reacted swiftly by adopting standard precautions. dentists follow a uniform infection control protocol to treat all patients, irrespective of their medical histories. however, in the face of a new infection that is considered highly contagious, it is prudent to review infection control procedures. the objective of this article, therefore, is to describe the epidemiology, clinical features, etiology and prevention of sars, as well as to evaluate the current infection control protocols used in dentistry in view of the facts related to the spread of this infection. we also explore the prospects for recrudescence of the disease, its treatment modalities and the promise of a sars vaccine. we do not know with certainty how, where and when the disease now known as sars manifested in humans, although theories abound. in february , the world health organization, or who, coined the term "severe acute respiratory syndrome" for the flulike condition that developed into pneumonia. nonetheless, researchers and clinicians generally believe that the first few cases may have originated in china. these sporadic cases were described sometime in the fall of in the guangdong province in southern china. for decades, the guangdong province had a large concentration of people, pigs and fowl living in close proximity because of mixed farming traditions that date back for centuries. this region also has the dubious distinction of being the deadly source of the asian flu, caused by the h n virus, which killed about million people worldwide in and . in , the avian flu (caused by the h n virus), which killed six people, also originated in the guangdong province. the recent outbreak. the sars outbreak has been identified in more than countries in five continents, affecting more than , people, predominantly in asia (especially china), with mini-outbreaks in north america and a few cases in europe. the disease has led to more than deaths worldwide. clusters of cases are particularly common among close associates of patients and the health care workers who treated them and their household contacts. because of the alarming global spread of the disease, who issued a global alert in march and instituted worldwide surveillance. patient characteristics. most patients identified up to now were previously healthy adults aged through years. a few cases of sars have been reported among children (≤ years of age), in whom the clinical course now is thought to be less aggressive. we provide a summary of the major clinical characteristics of patients with sars, although the information should be considered preliminary because of the broad and nonspecific case definition. clinical features. the incubation period for sars is widely considered to be two to seven days, but occasionally may last up to days. symptomatically, the illness appears to have two phases: an early, prodromal febrile phase and a secondary lower-respiratory phase. in pathological terms, however, it is a triphasic disease with a primary viral replicative phase, a secondary immune hyperactive phase and a pulmonary destructive phase. the disease generally begins with a prodrome of typically high fever (> c) that may be accompanied by chills and rigors. headache, malaise and myalgia also are common. at the onset of the illness, some patients have mild respiratory symptoms. in a few cases, the febrile prodrome may be accompanied by diarrhea, although rash and neurologic or gastrointestinal findings are absent. after three to seven days, the secondary lower-respiratory phase begins with a dry, nonproductive cough or dyspnea that may be accompanied by, or progress to, hypoxemia. in up to one-fifth of the cases, the respiratory illness is severe enough to require intubation and mechanical ventilation. the fatality rate among patients with illness that meets the current who definition for probable and suspected cases of sars ranges from to percent, depending on the age group and possibly other, yet unconfirmed, factors. furthermore, the mortality rate is higher among those with underlying illnesses and among the very elderly. typically, chest radiographs appear normal during the febrile prodrome and, in some patients, throughout the course of the illness. however, in the majority of patients, the respiratory phase is characterized by early focal infiltrates that progress to more generalized, patchy, interstitial infiltrates, sometimes leading to consolidation in the very late stages. in general, in the early phase of the disease, patients may have either a normal or decreased white blood cell count, with a reduction in the absolute lymphocyte count. at the peak of the jada, vol. , september c l i n i c a l p r a c t i c e symptomatically, the illness appears to have two phases: an early, prodromal febrile phase and a secondary lowerrespiratory phase. c l i n i c a l p r a c t i c e nomenon not common among other human coronaviruses. medical workers in hong kong, toronto and germany noted this effect when they inoculated lung tissue of patients into cultured monkey kidney cells. [ ] [ ] [ ] this phenomenon leads to a classic cytopathic effect in which the confluent cell layers in laboratory cell cultures are broken down, causing patchy denudation and detachment of cells. [ ] [ ] [ ] immunofluorescence testing of the infected cells indicated that they were indeed infected with a new form of the coronavirus, which has been termed "sars coronavirus," or sars-cov. furthermore, antibodies to the sars-cov were found almost exclusively in patients with sars during their convalescence, but not in human serum samples from healthy patients or in samples banked before the outbreak, suggesting that the infection is new to humans. until now, human coronaviruses have caused the relatively innocuous common cold. however, coronaviruses that infect other mammals and birds are more virulent. these include avian infectious bronchitis (a major problem in the poultry industry), transmissible gastroenteritis of pigs and feline infectious peritonitis. although there was initial speculation that close contact between poultry and humans in the chinese province of guangdong (where sars is thought to have originated) may have resulted in the virus' crossing the species barrier from poultry to humans, evidence now indicates that the himalayan palm civet cats that are consumed as a delicacy and sold widely in animal markets in china are the source of the infection. however, the sars-cov is not a recombinant of known coronaviruses. analyses of the genetic signatures of the viral strains from different geographic regions indicate that immunological pressure might modulate the evolution of the virus in human population cohorts. , other candidate organisms such as paramyxovirus and chlamydia have been implicated in the disease process, but the consensus is that they play a very small role, if any, in the pathogenesis of sars. general properties of coronaviridae. coronaviridae are a family of rna viruses that have been associated etiologically with respiratory ill-lower respiratory phase, up to one-half of patients exhibit leukopenia and thrombocytopenia or platelet counts at the low end of the normal range ( , to , per microliter). renal function appears to remain normal in the vast majority of patients. the box shows the second interim case definition for sars, provided by the centers for disease control and prevention, or cdc. it is based on clinical, epidemiologic and laboratory criteria. however, in areas such as hong kong, where there has been significant disease activity, the cdc criteria have been amended to include patients who do not respond to appropriate antibiotic therapy for atypical pneumonia caused by conventional agents (such as mycoplasma pneumoniae and chlamydia pneumoniae) and/or are in direct contact with another patient with sars. treatment and prevention. a number of treatment regimens have been explored for sars. these include a variety of antibiotics to presumptively treat known bacterial agents of atypical pneumonia, as well as antiviral agents such as oseltamivir and ribavirin. steroids also have been administered in combination with these antimicrobial agents. however, the most beneficial regimen remains to be determined. until reliable diagnostic tests, an effective vaccine and antiviral drugs are available, control of the epidemic depends on early identification of suspected and probable cases, quarantine of patients (and their close contacts) and effective infection control measures, particularly after patients are admitted to a health care facility. etiology. researchers have confirmed that a new strain of virus belonging to the family coronaviridae is the prime agent of this disease. [ ] [ ] [ ] [ ] although other viruses belonging to paramyxoviruses such as metapneumovirus have been implicated, these appear to play only a secondary role, if any, in the disease process. the coronaviruses-so named for the crown of spikes they carry on their surface -attracted the interest of researchers when they noted that the virus rapidly infected cells in culture, a phe- is the prime agent of severe acute respiratory syndrome. copyright © american dental association. all rights reserved. one or more of the following exposures in the days before the onset of symptoms: dtravel to a foreign or domestic location with documented or suspected recent transmission of sars-cov; dclose contact † with a person with mild-to-moderate or severe respiratory illness and history of travel in the days before onset of symptoms to a foreign or domestic location with documented or suspected recent transmission of sars-cov likely exposure to sars-cov one or more of the following exposures in the days before onset of symptoms: dclose contact with a person with confirmed sars-cov disease; dclose contact with a person with mild-to-moderate or severe respiratory illness for whom a chain of transmission can be linked to a confirmed case of sars-cov disease in the days before onset of symptoms laboratory criteria tests to detect sars-cov are being refined and their performance characteristics assessed; therefore, criteria for laboratory diagnosis of sars-cov are changing. the following are general criteria for laboratory confirmation of sars-cov: ddetection of serum antibody to sars-cov by a test validated by the centers for disease control and prevention, or cdc (for example, enzyme immunoassay); or disolation in cell culture of sars-cov from a clinical specimen; or ddetection of sars-cov rna by a reverse transcriptase polymerase chain reaction test validated by cdc and with subsequent confirmation in a reference laboratory (for example, cdc) dprobable case: meets the clinical criteria for severe respiratory illness of unknown etiology and epidemiologic criteria for exposure; laboratory criteria confirmed or undetermined dsuspect case: meets the clinical criteria for moderate respiratory illness of unknown etiology and epidemiologic criteria for exposure; laboratory criteria confirmed or undetermined a case may be excluded as a suspect or probable sars case if: dan alternative diagnosis can fully explain the illness; dthe case has a convalescent-phase serum sample (that is, obtained > days after symptom onset), which is negative for antibodies to sars-cov; dthe case was reported on the basis of contact with an index case that was subsequently excluded as a case of sars, provided other possible epidemiologic exposure criteria are not present * adapted from the centers for disease control and prevention. † close contact is defined as having cared for or lived with a person who has sars, or having a high likelihood of being in direct contact with respiratory secretions and/or body fluids of a person with sars (during encounters with the patient or through contact with materials contaminated by the patient), either during the period in which the individual was clinically ill or within days of resolution of symptoms. examples of close contact include kissing or embracing, sharing eating or drinking utensils, close conversation (less than feet apart), physical examination, and any other direct physical contact between people. close contact does not include activities such as walking by an individual or sitting across a waiting room or office for a brief time. ness in humans and with other diseases in domestic animals. interestingly, they also are associated to some extent with human diarrheal diseases. structurally, they are to nanometers in diameter, positive-stranded and about kilobases in length. their genome is the largest of all rna viruses, and highfrequency recombination between related coronaviruses leads to the generation of much genetic diversity. the virus has three major proteins. the nucleocapsid protein is enclosed within the viral envelope with the rna in a helical nucleocapsid. the other two proteins are the membrane glycoproteins and the spike glycoprotein. the spike glycoprotein is responsible for the characteristic fringe of crownlike projections. antibodies that elicit spike glycoprotein are thought to confer protection against infection. because the human strains are relatively difficult to culture compared with animal strains, the extent of strain variation in human coronaviruses is unclear. there are three serologically and genetically distinct groups of coronaviruses associated with animal and human disease. in general, they are species-specific, although there are a number of examples of viruses crossing species barriers and establishing themselves in new hosts. , once the host is infected, the virus may produce localized disease that often is restricted to the respiratory epithelium or the gastrointestinal tract, or they may produce disseminated infection causing systemic disease. coronavirus was confirmed as the etiologic agent in sars via serologic techniques demonstrating a rise in antibody titer, its growth in tissue culture, a determination of reverse transcriptase-polymerase chain reaction, or rt-pcr, specific for this virus using molecular genetic techniques, and animal studies. animal studies have helped to satisfy koch's postulates, which are necessary to prove disease causation. these postulates stipulate that to be the causal agent, a pathogen must meet four conditions: it must be found in all cases of the disease, it must be isolated from the host and grown in pure culture, it must reproduce the original disease when introduced into a susceptible host, and it must be found in the experimental host so infected. however, further studies that include control groups are required to determine the role of other agents, if any, in causality or as cofactors for severe disease. virus infectivity and survival. the rapid spread of sars worldwide within a few months points to the contagious nature of the disease. the infectivity during the incubation period is still unclear, and it appears that the risk of transmission during the prodrome is low. in contrast, in coronaviruses that cause the common cold, the viral shedding period usually precedes the onset of clinical symptoms by one to two days, although the peak viral excretion occurs during the symptomatic phase. , , the infectivity of sars during convalescence appears to be low and remains to be determined. some data on the survival and infectivity of the sars coronavirus indicate that, unlike other coronaviruses, it is a rather robust organism that is stable in feces (and urine) at room temperature for at least one to two days. it is more stable (up to four days) in stool from patients with diarrhea (which has a higher ph than does normal stool). however, the virus loses infectivity five minutes after being exposed to commonly used disinfectants and fixatives, including percent formaldehyde, percent hypochlorite, percent ethanol and percent phenol. heat at c kills the sars coronavirus at around , units per minutes (considered to be a quick reduction). spread of the disease. the available epidemiologic data suggest strongly that the main routes of virus spread are droplets, direct contact and fomite (indirect contact) transmission, although airborne transmission has not been ruled out completely. researchers believe that the cause of the large outbreaks among health care workers was the transmission of droplets through aerosol-generating medical procedures, such as the use of nebulizers. , no firm data exist regarding the infectivity of contaminated saliva (as opposed to sputum from the respiratory tract) through the droplet route. in some patients, the infection manifests itself as a mild form of diarrhea, and coronavirus particles have been recovered from fecal matter. hence, it is possible that fecal contamination could lead to the spread of the disease, although more data are needed to confirm this route of transmission. it is interesting that some animal coronaviruses are spread through the fecal-oral route. laboratory diagnosis. the mainstay of the sars diagnosis is its characteristic clinical features mentioned above. however, a number of laboratory tests-including serologic tests, cell culture and molecular diagnostics-can be used to confirm the clinically suspicious or probable cases. , these tests include the following. enzyme-linked immunosorbent assay, or elisa, test. from about days after the onset of clinical signs, elisa tests can be used to detect immunoglobulin, or ig, m and iga antibodies in the serum samples of patients with sars. early antibodies are detected in some patients within two weeks. immunofluorescence assay. sars virusinfected vero cells can be used to detect igm antibodies in serum samples of patients after about day of the onset of the disease. this test is reliable, yet demanding, because the live virus must be grown in cell culture; in addition, subsequent immunofluorescence needs to be demonstrated. cell culture. laboratory workers can detect virus in specimens (for example, respiratory secretions, blood) from patients with sars by infecting cultured vero-e or fetal rhesus kidney , or frhk- , cells. molecular tests. laboratory workers can use pcr assays that detect genetic material of coronavirus in patient specimens (such as respiratory secretions, blood or stool samples). primers that are required for this test now are available widely through various web sites (for example, the cdc, the university of hong kong and the governmental viral unit of hong kong). interpretation of test results. clinicians must exercise caution when interpreting laboratory test results, because the key to diagnosis is clinical evaluation and possible exposure to an infected person. a positive laboratory test result indicates that the patient is, or has been, infected with the sars-cov, while a negative test result does not necessarily rule out sars. , seroconversion of paired serum samples with convalescent serum samples obtained more than days after onset of symptoms is a reliable, sensitive and specific diagnostic method. however, the current diagnostic option of choice for early and rapid diagnosis is rt-pcr detection of virus in respiratory or fecal specimens. this test has low sensitivity, and a negative test result does not exclude the diagnosis. many laboratories are addressing the problem of sensitivity and specificity of the sars diagnostic tests, and it will take some time before a highly sensitive, specific, quick and simple diag-nostic test is available. it is possible that, as is the case with hiv infection, saliva could be used as a diagnostic fluid in this context. many people have been alarmed by the spread of sars in clinical facilities, where a disproportionately large number of health care workers (sometimes up to one-third) have been infected. however, it is reassuring that, to date, there have been no documented cases of sars transmitted in a dental setting. this may be the result of a combination of factors. first, transmission has not been documented during the incubation period before the appearance of febrile symptoms. it is unlikely that patients with sars would visit a dentist for elective treatment while they are in the acute phase of the disease, because of the high fever and other, rather debilitating, attendant symptoms. seto and colleagues conducted a case-control study in which they showed that proper use of standard precautions is adequate to prevent the nosocomial spread of sars in the absence of aerosol-producing procedures. however, as health care providers, dental personnel should be wary of the disease and should know how it is spread, how to identify patients with sars and what modifications need to be made to the practice to prevent transmission of the disease. although sars is well-controlled now, it may emerge insidiously, as has been the case with many other coronavirus infections. we review below the infection control measures that dentists and dental staff members now follow, in light of new epidemiologic data about sars, particularly its spread through aerosols and droplets. our recommendations are based on the recent ada guidelines, the cdc's recent report of recommended infection control practices for dentistry and our own experience in hong kong related to the last outbreak. identification of patients with sars. as health care providers, dentists should be able to identify a suspected case of sars. the cdc's current interim diagnostic criteria for sars are shown in the box. they are subject to change as more is learned about the disease, and should be reviewed periodically by visiting the ada or cdc web sites. to date, there have been no documented cases of severe respiratory syndrome transmitted in a dental setting. as stated above, we doubt that patients with sars who are in the acute febrile phase of the disease will visit a dentist. in the unlikely event that this does occur, the dentist should not treat the patient in the dental office, but should refer him or her to a health care facility as soon as possible for diagnosis and care. dentists also have a duty to report the case to state or local health departments. patient evaluation. as always, dentists should take a thorough medical history from each patient and update it at each recall appointment. the questionnaire used for this purpose may have to be modified to incorporate targeted screening questions regarding sars. although these questions may appear superfluous during the current abeyance of the outbreak, they are important as a guide if there is another outbreak of sars or an outbreak of a similar new disease. these questions may include the following: ddo you have fever? dhave you experienced a recent onset of a respiratory problem, such as a cough or difficulty breathing? dhave you, within the last days (that is, the incubation period for sars), traveled internationally or visited an area where documented or suspected community transmission of sars is occurring? dhave you come into contact with a patient with sars in the past days? in the event that the patient recently has returned from a geographic region with documented or suspected community transmission of sars, the clinician can defer elective treatment until the incubation period is over. dentists can provide emergency treatment, provided they use routine barrier precautions and avoid spatter or aerosol-generating procedures. this emergency treatment should be limited to the control of pain and infection. dentists should not treat patients in the dental office who are suspected of having sars. if a patient replies "yes" to the first two screening questions, the dentist should wear a surgical mask, discuss his or her potential concerns with the patient, call an area medical facility (such as a hospital) and inform the staff that he or she is referring a patient suspected of having sars so that arrangements can be made for transportation and care of the patient. patients with sars need ground emergency medical services. these screening questions should be asked routinely of all patients, because questioning only a select group of patients, for whatever reason, may undermine the early detection of infection and might be construed as a discriminatory practice. clinicians should delay treating convalescing patients for at least one month after they are released from the hospital. convalescing patients are instructed to remain at home for seven days after discharge from the hospital, and during this period they are requested to stay indoors and keep contact with others to a minimum. preprocedural rinsing. a preprocedural antimicrobial mouthrinse (with . to . percent chlorhexidine gluconate) is believed to reduce the number of microbes that are released into the operatory environment. this has been shown in a number of studies in which a longlasting mouthrinse (for example, chlorhexidine gluconate with povidone iodine and essential oils) has reduced the disseminated microbial load during procedures such as ultrasonic scaling. , however, no concrete data show that a preprocedural mouthrinse reduces infection among dental health care workers or patients. a preprocedural rinse would be most useful in situations in which a rubber dam cannot be used, such as when a prophylaxis cup or an ultrasonic scaler is used, and in the absence of assisted, high-volume suction. hand hygiene. microflora on the skin can be divided into two categories: the transient flora colonizing the superficial layers of the skin and mainly acquired through environmental routes, and the residential flora thriving on the deeper layers of the skin and hair follicles. the exogenous, superficial flora are harmful and pathogenic, but are removed easily with clinical hand-washing procedures. by contrast, the endogenous residential flora are almost impossible to remove completely, but are less likely to be associated with infections. the single most important method of preventing transmission of any infectious agent, including the sars coronavirus, is hand washing and appropriate hand care. studies have found that even in critical care units, hand-washing compliance is relatively low, sometimes approaching percent. by contrast, a dramatic reduction in the prevalence of health careassociated infections has been shown when regimented hand hygiene measures were introduced. thus, appropriate hand hygiene is the mainstay of a good dental infection control program. furthermore, recent data indicate that the sars virus, compared with other coronaviruses, is a relatively robust organism and may survive on nonporous surfaces for up to hours. this reinforces the need for good hand hygiene, as well as the importance of thorough surface disinfection. hand hygiene for routine dentistry. for routine dentistry, which entails examinations and nonsurgical procedures, plain soap and water are adequate. recently, the cdc recommended that if the health care worker's hands are not visibly soiled, an alcohol-based hand rub could be used for routine decontamination, because this is as effective as hand washing and also saves time. also, clinicians should decontaminate their hands both before and after removing gloves, because humidity and moisture cause bacteria to multiply rapidly under the glove surface. hand rubs that are based on alcohol alone should not be used owing to their rapid evaporation and lack of residual effect. consequently, hand rubs must be laced with agents such as chlorhexidine, octenidine or triclosan to achieve the needed effect. after using an alcohol-based hand rub, the clinician must dry his or her hands thoroughly before putting on gloves, because any residual alcohol may increase the risk of glove perforation. personal protective equipment. personal protective equipment, or ppe, is designed to protect the skin and mucous membranes of the eyes, nose and mouth from exposure to potentially infectious material. recent experience with the sars coronavirus has shown that vast numbers of health care workers acquired the infection in hospital settings, either as a result of inadequate barrier protection methods or the improper use of these methods. this barrier protection equipment consists of protective eyewear, masks, gloves, face shields and protective overwear. we should note that general work clothes such as uniforms do not protect against a hazard and should not be considered ppe. we describe below the relevant aspects of ppe that pertain to protection against airborne hazards. masks. face masks were first worn by surgeons to minimize postsurgical infection in patients due to microbes that were exhaled or shed by the surgical team. however, the realization that face masks protect the health care worker as well as the patient has led to the routine use of this protective measure in many clinical settings including dentistry. transmission of airborne infection depends on factors such as the virulence of the organism, as well as the number of organisms, transmitted. in the case of coronavirus-induced pneumonia leading to sars, airborne droplet transmission of infection is considered to be the main route of spread. various types of masks and face shields are available. surgical masks usually provide adequate protection in dental care settings, where highly transmissible infectious diseases are not typically encountered. particulate respirators. however, surgical masks are not designed to provide adequate protection against exposure to airborne infectious agents such as tubercle bacilli or droplet nuclei smaller than micrometers. for such purposes, particulate respirators (for example, n- masks) must be used. during the sars outbreak in hong kong, the vast majority of dental practitioners in that country used n masks for routine dentistry. however, these masks are uncomfortable to wear for extended periods because of the difficulty in breathing through a thick impervious fabric, and are not recommended for routine dental office settings. rubber dam isolation. rubber dams help minimize the production of saliva-and bloodcontaminated aerosol or spatter. samaranayake and colleagues reported an up-to- -percent reduction in airborne particles around a -foot diameter of the operational field when a rubber dam was used. a split-dam technique may be used in situations in which gingival areas are involved, such as class v restorations and crownand-bridge preparations. aerosol-generating procedures should be avoided as much as possible if rubber dam isolation is not feasible. some of these procedures include ultrasonic scaling, root-surface débride- ment, and high-or low-speed drilling with water spray. there is no doubt that coronavirus research has gained an unprecedented and urgent momentum owing to the lethality of sars and its nearly worldwide spread within a few months. consequently, laboratories throughout the world are working in unison to provide answers to many unresolved questions, as well as to develop a new preventive vaccine. in dentistry, in particular, a number of questions remain to be resolved, including the following: although the global threat of sars has peaked for the most part, it is helpful to review the response of the community to this novel disease. it is fortunate that sars was not sufficiently infective to cause a repeat of the influenza pandemic that killed millions. even so, we might be able to attribute the relatively low death rate in large part to the worldwide surveillance networks and patient isolation efforts that were introduced rapidly in most countries. in retrospect, an overreaction seems to have been a better option than allowing the disease to run out of control, as was the case with the aids pandemic. culmination of the outbreak. the who lifted all its travel advisories as of june , , and since then, only three new cases of sars have been reported. two of these-one in singapore and the other in taiwan-were acci-dental, laboratory-acquired infections in research technicians working with the organism, while the third patient-from guangdong province in southern china-is thought to have acquired the infection through contact with contaminated rodents. because the initial symptoms of sars mimic those of many variants of atypical pneumonia, a high degree of suspicion by the medical establishment, intense surveillance and immediate quarantine of all close contacts of patients should ward off another, large-scale winter outbreak. if sars does return, its epidemiology may be different from that of the current strain. for instance, the genome of the new sars-cov may differ, and the virus may be more or less infective than the original strain that emerged in . furthermore, we do not know how long the acquired immunity to sars persists. also, will those exposed to the virus be carriers of the disease in the face of a new infection? how many will be silent healthy carriers of the virus? will an emergent strain or strains behave similarly to the older counterpart? we do not have the answers to these questions. mutation of the sars-cov. the reason for the pandemic spread of hiv is its ability to mutate rapidly from one generation to another so it can escape the immune surveillance mechanisms of the host, as well as the prescribed antiviral medications. the sars virus, on the other hand, seems to be remarkably invariant; the genome sequence of isolates from patients in singapore, toronto, china and hong kong has not revealed any changes of major consequence. this does not mean that the sars virus is incapable of mutation; rather, because the virus has encountered little resistance from new human hosts, there is less selective pressure for new mutants to emerge and persist. drugs and vaccines for sars. many researchers are working on potential drugs and vaccines to treat patients with sars. however, their approach has been scattered for the most part, as they screen the multitude of available drugs and compounds for their ability to destroy the sars-cov. thus far, a few have had success. one group reported that the compound glycrrhizin, which is derived from licorice roots, can rid cultured monkey kidney cells of the sars virus. other researchers, using in silico research, have proposed that the newly described proteinase of the sars virus (which converts viral proteins into the active form required for viral replication) could be inhibited by drugs. animal models are essential for experimentation and drug discovery; thus far, the only validated model has been cynomolgus macaque (macaca fascicularis). a smaller and less expensive animal model for sars research has yet to be defined. although vaccines exist for animal coronavirus infections, it may take a few years before a vaccine for sars is developed. it is comforting to note that the existing technology and know-how for animal coronavirus vaccines could be translated directly toward the manufacture of a sars vaccine. furthermore, implicit evidence shows that the vaccine approach to preventing sars is feasible, because patients' conditions appear to improve when they are given hyperimmune serum from recovered patients with sars. sars vaccines could be based on a killed sars virus or on an attenuated virus that is sufficiently potent to replicate itself in humans and initiate a successful antibody response, but not potent enough to cause disease. another option would be to re-engineer a harmless candidate virus to contain genetic sequences of the sars virus. this approach has been used to produce a prototypic vaccine against a coronavirus that causes bronchitis in chickens. should there be a renewed threat of sars, a vaccine would be a mostwelcomed weapon by health care workers. sars is the first readily transmissible infectious disease that the global community has confronted in the new millennium. this, surely, will not be the last contagion that we will encounter. the fact that no dental health care worker or dental patient has thus far contracted sars in a dental setting is a testament to good infection control measures that have been implemented in the vast majority of dental offices. however, the dental community cannot let down its guard, and must be constantly aware of impending infectious threats in various guises, as well as recrudescence of disease, that may challenge the current infection control regimen. i institute of medicine; . . samaranayake lp, peiris js, scully c. ebola virus infection: an overview the ebola virus and the challenges to health research in africa west nile virus: statistics, surveillance, and control. available at infection control for the dental team cumulative number of reported probable cases of severe acute respiratory syndrome (sars) there is nothing permanent except change: the emergence of new virus diseases centers for disease control and prevention. isolation of avian influenza a (h n ) viruses from humans: hong kong epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children sars: what do we know about the disease updated interim u.s. case definition for severe acute respiratory syndrome (sars) (appendix b ) sars bulletin from hong kong coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome sars-associated coronavirus koch's postulates fulfilled for sars virus coronaviruses of man isolation and characterization of viruses related to the sars coronavirus from animals in southern china identification of severe acute respiratory syndrome in canada comparative full length sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection centers for disease control and prevention. interim domestic infection control precautions for aerosol-generating procedures on c l i n i c a l p r a c t i c e patients with severe acute respiratory syndrome (sars) effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) world health organization. who hospital discharge and followup policy for patients who have been diagnosed with severe acute respiratory syndrome (sars) reduction of viable bacteria in dental aerosols by preprocedural rinsing with an antiseptic mouthrinse reduction of bacteria-containing spray produced during ultrasonic scaling essential microbiology for dentistry how good are hand-washing practices? world health organization. summary on major findings in relation to coronavirus by members of the who multicenter collaborative network on sars aetiology and diagnosis. available at healthcare infection control practices advisory committee, hicpac/shea/apic/idsa hand hygiene task force. guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force: society for healthcare epidemiology of america/association for professionals in infection control/infectious diseases society of america hospital epidemiology and infection control the integrity of latex gloves in clinical practice the surgical mask unmasked: a review medical progress: how contagious are common respiratory tract infections? evidence of airborne transmission of the severe acute respiratory syndrome virus the efficacy of rubber dam isolation in reducing atmospheric bacterial contamination sars: breaking the chains of transmission chemical modification of glycyrrhzic acid as a route to new bioactive compounds for medicine coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs a recombinant fowl adenovirus expressing the s gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus dr. peiris is a professor of virology, faculty of medicine, the university of hong kong, hong kong, and the chief clinical virologist, queen mary hospital, hong kong. key: cord- -s hc fxs authors: ostaszewski, marek; niarakis, anna; mazein, alexander; kuperstein, inna; phair, robert; orta-resendiz, aurelio; singh, vidisha; aghamiri, sara sadat; acencio, marcio luis; glaab, enrico; ruepp, andreas; fobo, gisela; montrone, corinna; brauner, barbara; frischman, goar; monraz gómez, luis cristóbal; somers, julia; hoch, matti; gupta, shailendra kumar; scheel, julia; borlinghaus, hanna; czauderna, tobias; schreiber, falk; montagud, arnau; de leon, miguel ponce; funahashi, akira; hiki, yusuke; hiroi, noriko; yamada, takahiro g.; dräger, andreas; renz, alina; naveez, muhammad; bocskei, zsolt; messina, francesco; börnigen, daniela; fergusson, liam; conti, marta; rameil, marius; nakonecnij, vanessa; vanhoefer, jakob; schmiester, leonard; wang, muying; ackerman, emily e.; shoemaker, jason; zucker, jeremy; oxford, kristie; teuton, jeremy; kocakaya, ebru; summak, gökçe yağmur; hanspers, kristina; kutmon, martina; coort, susan; eijssen, lars; ehrhart, friederike; rex, d. a. b.; slenter, denise; martens, marvin; haw, robin; jassal, bijay; matthews, lisa; orlic-milacic, marija; senff ribeiro, andrea; rothfels, karen; shamovsky, veronica; stephan, ralf; sevilla, cristoffer; varusai, thawfeek; ravel, jean-marie; fraser, rupsha; ortseifen, vera; marchesi, silvia; gawron, piotr; smula, ewa; heirendt, laurent; satagopam, venkata; wu, guanming; riutta, anders; golebiewski, martin; owen, stuart; goble, carole; hu, xiaoming; overall, rupert w.; maier, dieter; bauch, angela; gyori, benjamin m.; bachman, john a.; vega, carlos; grouès, valentin; vazquez, miguel; porras, pablo; licata, luana; iannuccelli, marta; sacco, francesca; nesterova, anastasia; yuryev, anton; de waard, anita; turei, denes; luna, augustin; babur, ozgun; soliman, sylvain; valdeolivas, alberto; esteban-medina, marina; peña-chilet, maria; helikar, tomáš; puniya, bhanwar lal; modos, dezso; treveil, agatha; olbei, marton; de meulder, bertrand; dugourd, aurélien; naldi, aurelien; noel, vincent; calzone, laurence; sander, chris; demir, emek; korcsmaros, tamas; freeman, tom c.; augé, franck; beckmann, jacques s.; hasenauer, jan; wolkenhauer, olaf; wilighagen, egon l.; pico, alexander r.; evelo, chris t.; gillespie, marc e.; stein, lincoln d.; hermjakob, henning; d’eustachio, peter; saez-rodriguez, julio; dopazo, joaquin; valencia, alfonso; kitano, hiroaki; barillot, emmanuel; auffray, charles; balling, rudi; schneider, reinhard title: covid- disease map, a computational knowledge repository of sars-cov- virus-host interaction mechanisms date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: s hc fxs we hereby describe a large-scale community effort to build an open-access, interoperable, and computable repository of covid- molecular mechanisms - the covid- disease map. we discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. we highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. we describe the contents of the map and their relevance to the molecular pathophysiology of covid- and the analytical and computational modelling approaches that can be applied to the contents of the covid- disease map for mechanistic data interpretation and predictions. we conclude by demonstrating concrete applications of our work through several use cases. the coronavirus disease (covid- ) pandemic due to severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] has already resulted in the infection of over million people worldwide, of whom one million have died . the molecular pathophysiology that links sars-cov- infection to the clinical manifestations and course of covid- is complex and spans multiple biological pathways, cell types and organs [ , ] . to gain the insights into this complex network, the biomedical research community needs to approach it from a systems perspective, collecting the mechanistic knowledge scattered across the scientific literature and bioinformatic databases, and integrating it using formal systems biology standards. with this goal in mind, we initiated a collaborative effort involving over biocurators, domain experts, modelers and data analysts from institutions in countries to develop the covid- disease map, an open-access collection of curated computational diagrams and models of molecular mechanisms implicated in the disease [ ] . to this end, we aligned the biocuration efforts of the disease maps community [ , ] , reactome [ ] , and wikipathways [ ] and developed common guidelines utilising standardised encoding and annotation schemes, based on community-developed systems biology standards [ ] [ ] [ ] , and persistent identifier repositories [ ] . moreover, we integrated relevant knowledge from public repositories [ ] [ ] [ ] [ ] and text mining resources, providing a means to update and refine contents of the map. the fruit of these efforts was a series of pathway diagrams describing key events in the covid- infectious cycle and host response. we ensured that this comprehensive diagrammatic description of disease mechanisms is machine-readable and computable. this allows us to develop novel bioinformatics workflows, creating executable networks for analysis and prediction. in this way, the map is both human and machine-readable, lowering the communication barrier between biocurators, domain experts, and computational biologists significantly. computational modelling, data analysis, and their informed interpretation using the contents of the map have the potential to identify molecular signatures of disease predisposition and development, and to suggest drug repositioning for improving current treatments. covid- disease map is a collection of diagrams containing interactions between elements, supported by publications and preprints. the summary of diagrams available in the covid- disease map can be found online in supplementary material . the map is a constantly evolving resource, refined and updated by ongoing efforts of biocuration, sharing and analysis. here, we report its current status. in section we explain the set up of our community effort to construct the interoperable content of the resource, involving biocurators, domain experts and data analysts. in section we demonstrate that the scope of the biological maps in the resource reflects the state-ofthe-art about the molecular biology of covid- . next, we outline analytical workflows that can be used on the contents of the map, including initial, preliminary outcomes of two such workflows, discussed in detail as use cases in section . we conclude in section with an outlook to further development of the covid- map and the utility of the entire resource in future efforts towards building and applying disease-relevant computational repositories. the covid- disease map project involves three main groups: (i) biocurators, (ii) domain experts, and (iii) analysts and modellers: i. biocurators develop a collection of systems biology diagrams focused on the molecular mechanisms of sars-cov- . ii. domain experts refine the contents of the diagrams, supported by interactive visualisation and annotations. iii. analysts and modellers develop computational workflows to generate hypotheses and predictions about the mechanisms encoded in the diagrams. all three groups have an important role in the process of building the map, by providing content, refining it, and defining the downstream computational use of the map. figure illustrates the ecosystem of the covid- disease map community, highlighting the roles of different participants, available format conversions, interoperable tools, and downstream uses. the information about the community members and their contributions are disseminated via the fairdomhub [ ] , so that content distributed across different collections can be uniformly referenced. the biocurators of the covid- disease map diagrams follow the guidelines developed by the community, and specific workflows of wikipathways [ ] and reactome [ ] . the biocurators build literature-based systems biology diagrams, representing the molecular processes implicated in the covid- pathophysiology, their complex regulation and the phenotypic outcomes. these diagrams are main building blocks of the map, and are composed of biochemical reactions and interactions (further called altogether interactions) taking place between different types of molecular entities in various cellular compartments. as there are multiple teams working on related topics, biocurators can provide an expert review across pathways and across platforms. this is possible, as all platforms offer intuitive visualisation, interpretation, and analysis of pathway knowledge to support basic and clinical research, genome analysis, modelling, systems biology, and education. table lists information about the created content. for more details see supplementary material . communicating to refine, interpret and apply covid- disease map diagrams. these diagrams are created and maintained by biocurators, following pathway database workflows or standalone diagram editors, and reviewed by domain experts. the content is shared via pathway databases or a gitlab repository; all can be enriched by integrated resources of text mining and interaction databases. the covid- disease map diagrams, available in layout-aware systems biology formats and integrated with external repositories, are available in several formats allowing a range of computational analyses, including network analysis and boolean, kinetic or multiscale simulations. both interactions and interacting entities are annotated following a uniform, persistent identification scheme, using either miriam or identifiers.org [ ] , and the guidelines for annotations of computational models [ ] . viral protein interactions are explicitly annotated with their taxonomy identifiers to highlight findings from strains other than sars-cov- . moreover, tools like modelpolisher [ ] , sbmlsqueezer [ ] or memote help to automatically complement the annotations in the sbml format and validate the model (see also supplementary material ). the knowledge on covid- mechanisms is rapidly evolving, as demonstrated by the rapid growth of the covid- open research dataset (cord- ) dataset, a source scientific manuscript text and metadata on covid- and related coronavirus research [ ] . cord- currently contains over , articles and preprints, over four times more than when it was introduced . in such a quickly evolving environment, biocuration efforts need to be supported by other repositories of structured knowledge about molecular mechanisms relevant for covid- , like molecular interaction databases, or text mining resources. contents of such repositories may suggest improvements in the existing covid- disease map diagrams, or establish a starting point for developing new pathways (see section "biocuration of database and text mining content"). interaction and pathway databases contain structured and annotated information on protein interactions or causal relationships. while interaction databases focus on pairs of molecules, offering broad coverage of literature-reported findings. pathway databases provide detailed description of biochemical processes and their regulations of related interactions, supported by diagrams. both types of resources can be a valuable input for covid- disease map biocurators, given the comparability of identifiers used for molecular annotations, and the reference to publications used for defining an interaction or building a pathway. table text-mining approaches can help to sieve through such rapidly expanding literature with natural language processing (nlp) algorithms based on semantic modelling, ontologies, and linguistic analysis to automatically extract and annotate relevant sentences, biomolecules, and their interactions. this scope was recently extended to pathway figure mining: decoding pathway figures into their computable representations [ ] . altogether, these automated workflows lead to the construction of knowledge graphs: semantic networks incorporating ontology concepts, unique biomolecule references, and their interactions extracted from abstracts or full-text documents [ ] . the covid- disease map project integrates open-access text mining resources, indra [ ] , biokb , ailani covid- , and pathwaystudio . all platforms offer keyword-based search allowing interactive exploration. additionally, the map benefits from an extensive protein-protein interaction network (ppi) generated with a custom text-mining pipeline using opennlp and gnormplus [ ] . this pipeline was applied to the cord- dataset and the collection of medline abstracts associated with the genes in the sars-cov- ppi network [ ] using the entrez gene reference-into-function (generif). for detailed descriptions of the resources, see supplementary material . molecular interactions from databases and knowledge graphs from text mining resources discussed above (from now on called altogether 'knowledge graphs') have a broad coverage at the cost of depth of mechanistic representation. this content can be used by the biocurators in the process of building and updating the systems biology focused diagrams. biocurators can use this content in three main ways: by visual exploration, by programmatic comparison, and by direct incorporation of the content. first, the biocurators can visually explore the contents of the knowledge graphs using available search interfaces to locate new knowledge and encode it in the diagrams. moreover, solutions like covidminer project , pathwaystudio and ailani offer a visual representation of a group of interactions for a better understanding of their biological context, allowing search by interactions, rather than just isolated keywords. finally, indra and ailani offer assistant bots that respond to natural language queries and return meaningful answers extracted from knowledge graphs. second, programmatic access and reproducible exploration of the knowledge graphs is possible via data endpoints: sparql for biokb and application programming interfaces for indra, ailani, and pathway studio. users can programmatically submit keyword queries and retrieve functions, interactions, pathways, or drugs associated with submitted gene lists. this way, otherwise time-consuming tasks like an assessment of completeness of a given diagram, or search for new literature evidence, can be automated to a large extent. finally, biocurators can directly incorporate the content of knowledge graphs into sbml format using biokc [ ] . additionally, the contents of the elsevier covid- pathway collection can be translated to sbgnml preserving the layout of the diagrams. the sbgnml content can then be converted into other diagram formats used by biocurators (see section . below). the biocuration of the covid- disease map is distributed across multiple teams, using varying tools and associated systems biology representations. this requires a common approach to annotations of evidence, biochemical reactions, molecular entities and their interactions. moreover, the interoperability of layout-aware formats is needed for comparison and integration of the diagrams in the map. the covid- disease map diagrams are encoded in one of three layout-aware formats for standardised representation of molecular interactions: sbml [ ] [ ] [ ] , sbgnml [ ] , and gpml [ ] . these xml-based formats focus to a varying degree on user-friendly graphical representation, standardised visualisation, and support of computational workflows. for the detailed description of the formats, see supplementary material . each of these three languages has a different focus: sbml emphasizes standardised representation of the data model underlying molecular interactions, sbgnml provides standardised graphical representation of molecular processes, while gpml allows for a partially standardised representation of uncertain biological knowledge. nevertheless, all three formats are centered around molecular interactions, provide a constrained vocabulary to encode element and interaction types, encode layout of their diagrams and support stable identifiers for diagram components. these shared properties, supported by a common ontology [ ] , allow cross-format mapping and enable translation of key properties between the formats. therefore, when developing the contents of the map, biocurators use the tools they are familiar with, facilitating this distributed task. the covid- disease map community ecosystem of tools and resources (see figure ) ensures interoperability between the three layout-aware formats for molecular mechanisms: sbml, sbgnml, and gpml. essential elements of this setup are tools capable of providing cross-format translation functionality [ , ] and supporting harmonised visualisation processing. another essential translation interface is a representation of reactome pathways in wikipathways gpml [ ] and sbml. the sbml export of reactome content has been optimised in the context of this project and facilitates integration with the other covid- disease map software components. the contents of the covid- disease map diagrams can be directly transformed into inputs of computational pipelines and data repositories. besides the direct use of sbml format in kinetic simulations, celldesigner sbml files can be transformed into sbml qual [ ] using casq [ ] , enabling boolean modelling-based simulations (see also supplementary material ). in parallel, casq converts the diagrams to the sif format , supporting pathway modelling workflows using simplified interaction networks. notably, the gitlab repository features an automated translation of stable versions of diagrams into sbml qual. finally, translation of the diagrams into xgmml format (the extensible graph markup and modelling language) using cytoscape [ ] or ginsim [ ] allows for network analysis and interoperability with molecular interaction repositories [ ] . thanks to the community effort discussed above supported by a rich bioinformatics framework, we constructed the covid- disease map, focussing on the mechanisms known from other coronaviruses [ ] and suggested by early experimental investigations [pmid: ]. then, we applied the analytical and modelling workflows to the contributed diagrams and associated interaction databases to propose initial map-based insights into covid- molecular mechanisms. the covid- disease map is an evolving repository of pathways affected by sars-cov- . figure . it is currently centred on molecular processes involved in sars-cov- entry, http://www.ebi.ac.uk/sbo/main/ http://www.cbmc.it/fastcent/doc/sifformat.htm replication, and host-pathogen interactions. as mechanisms of host susceptibility, immune response, cell and organ specificity emerge, these will be incorporated into the next versions of the map. the covid- map represents the mechanisms in a "host cell". this follows literature reports on cell specificity of sars-cov- [ , [ ] [ ] [ ] [ ] [ ] . some pathways included in the covid- map may be shared among different cell types, as for example the ifn- pathway found in cells such as dendritic, epithelial, and alveolar macrophages [ ] [ ] [ ] [ ] [ ] . while at this stage, we do not address cell specificity explicitly in our diagrams, extensive annotations may allow identification of pathways relevant to the cell type of interest. the sars-cov- infection process and covid- progression follow a sequence of steps ( figure ), starting from viral attachment and entry, which involve various dynamic processes on different time scales that are not captured in static representations of pathways. correlation of symptoms and potential drugs suggested to date helps downstream data exploration and drug target interpretation in the context of therapeutic interventions. human host ilc , ilc- , ilc natural killer renin-angiotensinaldosterone system (raas) granulocytes nasal mucosa disease map golgi er cd + cd + ace tmprss integrative stress response dendritic cells transmission of sars-cov- primarily occurs through contact with respiratory drops, airborne transmission, and through contact with contaminated surfaces [ ] [ ] [ ] . upon contact with the respiratory epithelium, the virus infects cells mostly by binding the spike surface glycoprotein (s) to angiotensin-converting enzyme (ace ) with the help of serine protease tmprss [ ] [ ] [ ] [ ] . importantly, recent results suggest viral entry using other receptors of lungs and the immune system [ , ] . once attached, sars-cov- can enter cells either by direct fusion of the virion and cell membranes in the presence of proteases • dendritic cells. • nk cells. • monocytes and macrophages. • t cells, th and th response. • b cells, antibody production. asymptomatic/pre -symptomatic. vaccine? pre-exposure prophylaxis? antivirals? sirs, shock. shortness of breath. anosmia, ageusia, cough, fever, diarrhea. multiple organ dysfunction ards, complications. host response • cellular stress. • apoptosis. systemic and ventilation support oxygen therapy host raas ards; acute respiratory distress syndrome. raas; renin-angiotensin-aldosterone system. sirs; systemic inflammatory response syndrome. pathophysiology virus-host cell interactions and host response disease map critical asymptomatic (lung, heart, kidney) (nasal and respiratory epithelium, alveoli, vascular endothelial) tmprss and furin or by endocytosis in their absence. regardless of the entry mechanism, the s protein has to be activated to initiate the plasma or endosome membrane fusion process. while in the cell membrane, s protein is activated by tmprss and furin, in the endosome s protein is activated by cathepsin b (ctsb) and cathepsin l (ctsl) [ , ] . activated s promotes the cell-or endosome-membrane fusion [ ] with the virion membrane, and then the nucleocapsid is injected into the cytoplasm. these mechanisms are represented in the corresponding diagrams of the map . within the host cell, sars-cov- hijacks the rough endoplasmic reticulum (rer)-linked host translational machinery. it then synthesises viral proteins replicase polyprotein a (pp a) and replicase polyprotein ab (pp ab) directly from the virus (+)genomic rna (grna) [ , ] . through a complex cascade of proteolytic cleavages, pp a and pp ab give rise to non-structural proteins (nsps) [ ] [ ] [ ] . most of these nsps collectively form the replication transcription complex (rtc) that is anchored to the membrane of the double-membrane vesicle [ , ] endoplasmic reticulum stress and unfolded protein response as discussed above, the virus hijacks the er to replicate. production of large amounts of viral proteins exceeds the protein folding capacity of the er, creating an overload of unfolded proteins. as a result, the unfolded protein response (upr) pathways are triggered to assure the er homeostasis, using three main signalling routes of upr via perk, ire , and atf [ ]. their role is to mitigate the misfolded protein load and reduce oxidative stress. the resulting protein degradation is coordinated with a decrease in protein synthesis via eif alpha phosphorylation and induction of protein folding genes via the transcription factor xbp [ ] . when the er is unable to restore its function, it can trigger cell apoptosis [ , ] . the results are er stress and activation of the upr. the expression of some human coronavirus (hcov) proteins during infection, in particular the s glycoprotein, may induce activation of the er stress in the host cells [ ] . based on sars-cov results, this may lead to activation of the perk [ ], ire and in an indirect manner, of the atf pathways [ ] . processes of degrading malfunctioning proteins and damaged organelles, including the ubiquitin-proteasome system (ups) and autophagy [ ] are essential to maintain energy homeostasis and prevent cellular stress [ , ] . autophagy is also involved in cell defence, including direct destruction of the viruses via virophagy, presentation of viral antigens, and inhibition of excessive inflammatory reactions [ , ] . sars-cov- directly affects the process of ups-based protein degradation, as indicated by the host-virus interactome dataset published recently [ ] . this mechanism may be a defence against viral protein degradation [ ] . the map describes in detail the nature of this interaction, namely the impact of orf virus protein on the cul ubiquitin ligase complex and its potential substrates. interactions between sars-cov- and host autophagy pathways are inferred based on results from other covs. a finding that covs use double-membrane vesicles and lc -i for replication [ ] may suggest that the virus induces autophagy, possibly in atg -dependent manner [ ] , although some evidence points to the contrary [ ] . also, the cov nsp restricts autophagosome expansion, compromising the degradation of viral components [ ] . recently revealed mutations in nsp [ ] indicate its importance, although the exact effect of the mutations remains unknown. based on the connection between autophagy and the endocytic pathway of the virus replication cycle [ ] , autophagy modulation was suggested as a potential therapy strategy, either pharmacologically [ , [ ] [ ] [ ] , or via fasting [ ] . apoptosis, a synonym for programmed cell death, is triggered by virus-host interaction upon infection, as the early death of the virus-infected cells may prevent viral replication. many viruses block or delay cell death by expressing anti-apoptotic proteins to maximize the production of viral progeny [ ] . in turn, apoptosis induction at the end of the viral replication cycle might assist in viral dissemination while reducing an inflammatory response. for instance, sars-cov- [ ] and mers [ ] are able to invoke apoptosis in lymphocytes, compromising the immune system. apoptosis follows two major pathways [ ] , called extrinsic and intrinsic. extrinsic signals are transmitted by death ligands and their receptors (e.g., fasl and tnf-alpha). activated death receptors recruit adaptors like fadd and tradd, and initiator procaspases like caspase- , leading to cell death with the help of effector caspases- and [ , ] . in turn, the intrinsic pathway involves mitochondria-related members of the bcl- protein family. cellular stress causes bcl- proteins-mediated release of cytochrome c from the mitochondria into the cytoplasm. cytochrome c then forms a complex with apaf and recruits initiator procaspase- to form the apoptosome, leading to the proteolytic activation of caspase- . activated caspase- can now initiate the caspase cascade by activating effector caspases and [ ] . the intrinsic pathway is modulated by sars-cov molecules [ , ] . as intrinsic apoptosis involves mitochondria, its activity may also be exacerbated by sars-cov- disruptions of the electron transport chain, mitochondrial translation, and transmembrane transport [ ] . the resulting mitochondrial dysfunction may lead to increased release of reactive oxygen species and pro-apoptotic factors. another vital crosstalk is that of the intrinsic pathway with the pi k-akt pro-survival pathway. activated akt can phosphorylate and inactivate various pro-apoptotic proteins, including bad and caspase- [ ] . sars-cov uses pi k-akt signalling cascade to enhance infection [ ] . moreover, sars-cov could affect apoptosis in a cell-type-specific manner [ , ] . sars-cov structural proteins s, e, m, n, and accessory proteins a, b, , a, a, and b have been shown to act as crucial effectors of apoptosis in vitro. structural proteins seem to affect mainly the intrinsic apoptotic pathway, with p mapk and pi k/akt pathways regulating cell death. accessory proteins can induce apoptosis via different cascades and in a cellspecific manner [ ] . sars-cov e and a protein were shown to activate the intrinsic pathway by blocking anti-apoptotic bcl-xl localized to the er [ ] . sars-cov m protein and the ion channel activity of e and a were shown to interfere with pro-survival signalling cascades [ , ] . the viral replication and the consequent immune and inflammatory responses cause damage to the epithelium and pulmonary capillary vascular endothelium and activate the main intracellular defence mechanisms, as well as the humoral and cellular immune responses. resulting cellular stress and tissue damage [ , ] impair respiratory capacity and lead to acute respiratory distress syndrome (ards) [ , , ] . hyperinflammation is a known complication, causing widespread damage, organ failure, and death, followed by a not yet completely understood rapid increase of cytokine levels (cytokine storm) [ ] [ ] [ ] , and acute ards [ ] . other reported complications, such as coagulation disturbances and thrombosis are associated with severe cases, but the specific mechanisms are still unknown [ , [ ] [ ] [ ] , although some reports suggest that covid- coagulopathy has a distinct profile [ ] . the sars-cov- infection disrupts the coagulation cascade and is frequently associated with hyperinflammation, renin-angiotensin system (ras) imbalance and intravascular coagulopathy [ , [ ] [ ] [ ] . hyperinflammation leads in turn to detrimental hypercoagulability and immunothrombosis, leading to microvascular thrombosis with further organ damage [ ] . importantly, ras is influenced by risk factors of developing severe forms of covid- [ ] [ ] [ ] . ace , used by sars-cov- for host cell entry, is a regulator of ras and is widely expressed in the affected organs [ ] . the main function of ace is the conversion of angii to angiotensin - (ang - ), and these two angiotensins trigger the counter-regulatory arms of ras [ ] . the signalling via angii and its receptor agtr , elevated in the infected [ , ] , induces the coagulation cascade leading to microvascular thrombosis [ ] , while ang - and its receptor mas attenuate these effects [ ] . the innate immune system detects specific pathogen-associated molecular patterns (pamps), through pattern recognition receptors (prrs). detection of sars-cov- is mediated through receptors that recognise double-stranded and single-stranded rna in the endosome during endocytosis of the virus particle, or in the cytoplasm during the viral replication. these receptors mediate the activation of transcription factors such as ap , nfkappab, irf , and irf , responsible for the transcription of antiviral proteins, in particular, interferon-alpha and beta [ , ] . sars-cov- reduces the production of type i interferons to evade the immune response [ ] . the detailed mechanism is not clear yet; however, sars-cov m protein inhibits the irf activation [ ] and suppresses nfkappab and cox transcription. at the same time, sars-cov n protein activates nfkappab [ ] , so the overall impact is unclear. these pathways are also negatively regulated by sars-cov nsp papain-like protease domain (plpro) [ ] . the map contains the initial recognition process of the viral particle by the innate immune system and the viral mechanisms to evade the immune response. it provides the connection between virus entry (detecting the viral endosomal patterns), its replication cycle (detection cytoplasmic viral patterns), and the effector pathways of pro-inflammatory cytokines, especially of the interferon type i class. the latter seems to play a crucial but complex role in covid- pathology: both negative [ , ] and positive effects [ , ] of interferons on virus replication have been reported. interferon type i signalling interferons (ifns) are central players in the antiviral immune response of the host cell [ ] , specifically affected by sars-cov- [ ] [ ] [ ] [ ] . type i ifns are induced upon viral recognition of pamps by various host prrs [ ] as discussed earlier. the ifn-i pathway diagram represents the activation of tlr and ifnar and the subsequent recruiting of adaptor proteins and the downstream signalling cascades regulating key transcription factors including irf / , nf-kappab, ap- , and isre [ , ] . further, the map shows irf mediated induction of ifn-i, affected by the sars-cov- proteins. sars-cov nsp and orf interfere with irf signalling [ , ] and sars-cov m, n, nsp and nsp act as interferon antagonists [ , , , , ] . moreover, coronaviruses orf a, orf and nsp proteins can repress interferon expression and stimulate the degradation of ifnar and stat during the unfolded protein response (upr) [ , ] . another mechanism of viral rna recognition is rig-like receptor signalling [ ] , leading to sting activation [ ] , and via the recruitment of traf , tbk and ikkepsilon to phosphorylation of irf [ ] . this in turn induces the transcription of ifns alpha, beta and lambda [ ] . sars-cov viral papain-like-proteases, contained within the nsp and nsp proteins, inhibit sting and the downstream ifn secretion [ ] . in line with this hypothesis, sars-cov- infection results in a unique inflammatory response defined by low levels of ifn-i and high expression of cytokines [ , ] . the ifnlambda diagram describes the ifnl receptor signaling cascade [ ] , including jak-stat signaling and the induction of interferon stimulated genes, which encode antiviral proteins [ ] . the interactions of sars-cov- proteins with the ifnl pathway are based on the literature [ ] or sars-cov homology [ ] . metabolic pathways govern the immune microenvironment by modulating the availability of nutrients and critical metabolites [ ] . infectious entities reprogram host metabolism to create favourable conditions for their reproduction [ ] . sars-cov- proteins interact with a variety of immunometabolic pathways, several of which are described below. heme catabolism is a well-known anti-inflammatory system in the context of infectious and autoimmune diseases [ , ] . the main effector of this pathway, heme oxygenase- (hmox ) was found to interact with sars-cov- orf a, although the nature of this interaction remains ambiguous [ , ] . hmox cleaves heme into carbon monoxide, biliverdin (then reduced to bilirubin), and ferrous iron [pmid: ]. biliverdin, bilirubin, and carbon monoxide possess cytoprotective properties, and have shown promise as immunomodulatory therapeutics [ , ] . importantly, activation of hmox also inhibits the nlrp inflammasome [ ] [ ] [ ] , which is a pro-inflammatory and prothrombotic multiprotein system [ ] highly active in covid- [ ] [ ] [ ] . it mediates production of the pro-inflammatory cytokines il- b and il- via caspase- [ ] . the sars-cov orf a, e, and orf a incite the nlrp inflammasome [ ] [ ] [ ] [ ] . still, the potential of the hmox pathway to fight covid- inflammation remains to be tested [ , , ] despite promising results in other models of inflammation [ , , [ ] [ ] [ ] . the tryptophan-kynurenine pathway is closely related to heme metabolism. the ratelimiting step of this pathway is catalysed by the indoleamine , dioxygenase enzymes (ido and ido ) in dendritic cells, macrophages, and epithelial cells in response to inflammatory cytokines like ifn-gamma, ifn- , tgf-beta, tnf-alpha, and il- [ ] [ ] [ ] . crosstalk with the hmox pathway also increases the expression of ido and hmox in a feed-forward manner. metabolomics analyses from severe covid- patients revealed enrichment of kynurenines and depletion of tryptophan, indicating robust activation of ido enzymes [ , ] . depletion of tryptophan [ , , ] and kynurenines and their derivatives affect the proliferation and immune response of a range of t cells [ , [ ] [ ] [ ] [ ] [ ] . however, despite high levels of kynurenines in covid- , cd + t-cells and th cells are enriched in lung tissue, and t-regulatory cells are diminished [ ] . this raises the question of whether and how the immune response elicited in covid- evades suppression by the kynurenine pathway. the sars-cov- protein nsp interacts with three human proteins: gla, sirt , and impdh [ ] . the galactose metabolism pathway, including the gla enzyme [ ] , is interconnected with amino sugar and nucleotide sugar metabolism. sirt is a naddependent desuccinylase and demalonylase regulating serine catabolism, oxidative metabolism and apoptosis initiation [ ] [ ] [ ] . moreover, nicotinamide metabolism regulated by sirt occurs downstream of the tryptophan metabolism, linking it to the pathways discussed above. finally, impdh is the rate-limiting enzyme in the de novo synthesis of gtp, allowing regulation of purine metabolism and downstream potential antiviral targets [ , ] . the pyrimidine synthesis pathway, tightly linked to purine metabolism, affects viral dna and rna synthesis. pyrimidine deprivation is a host targeted antiviral defence mechanism, which blocks viral replication in infected cells and can be regulated pharmacologically [ ] [ ] [ ] . it appears that components of the dna damage response connect the inhibition of pyrimidine biosynthesis to the interferon signalling pathway, probably via sting-induced tbk activation that amplifies interferon response to viral infection, discussed above. inhibition of de novo pyrimidine synthesis may have beneficial effects on the recovery from covid- [ ] ; however, this may happen only in a small group of patients. covid- pathways featured in the previous section cover mechanisms reported so far. still, certain aspects of the disease were not represented in detail because of their complexity, namely cell-type-specific immune response, and susceptibility features. their mechanistic description is of great importance, as suggested by clinical reports on the involvement of these pathways in the molecular pathophysiology of the disease. the mechanisms outlined below will be the next targets in our curation roadmap. cell type-specific immune response covid- causes serious disbalance in multiple populations of immune cells. some studies report that covid- patients have a significant decrease of peripheral cd + and cd + cytotoxic t lymphocytes (ctls), b cells, nk cells, as well as higher levels of a broad range of cytokines and chemokines [ , [ ] [ ] [ ] [ ] . the disease causes functional exhaustion of cd + ctls and nk cells, induced by sars-cov- s protein and by excessive pro-inflammatory cytokine response [ , ] . moreover, the ratio of naïve-to-memory helper t-cells, as well as the decrease of t regulatory cells, correlate with covid- severity [ ] . conversely, high levels of th and cytotoxic cd + t-cells have been found in the lung tissue [ ] . pulmonary recruitment of lymphocytes into the airways may explain the lymphopenia and the increased neutrophil-lymphocyte ratio in peripheral blood found in covid- patients [ , , ] . in this regard, an abnormal increase of the th :treg cell ratio may promote the release of pro-inflammatory cytokines and chemokines, increasing disease severity [ ] . sars-cov- infection is associated with increased morbidity and mortality in individuals with underlying chronic diseases or a compromised immune system [ ] [ ] [ ] [ ] . groups of increased risk are men, pregnant and postpartum women, and individuals with high occupational viral exposure [ ] [ ] [ ] . other susceptibility factors include the abo blood groups [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and respiratory conditions [ ] [ ] [ ] [ ] [ ] [ ] . importantly, age is one of the key aspects contributing to the severity of the disease. the elderly are at high risk of developing severe or critical disease [ , ] . age-related elevated levels of pro-inflammatory cytokines (inflammation) [ ] [ ] [ ] [ ] , immunosenescence and cellular stress of ageing cells [ , , , , ] may contribute to the risk. in contrast, children are generally less likely to develop severe disease [ , ] , with the exception of infants [ , [ ] [ ] [ ] . however, some previously healthy children and adolescents can develop a multisystem inflammatory syndrome following sars-cov- infection [ ] [ ] [ ] [ ] [ ] . several genetic factors have been proposed and identified to influence susceptibility and severity, including the ace gene, hla locus, errors influencing type i ifn production, tlr pathways, myeloid compartments, as well as cytokine polymorphisms [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we aim to connect the susceptibility features to specific molecular mechanisms and better understand the contributing factors. this can lead to a series of testable hypotheses, including the role of vitamin d counteracting pro-inflammatory cytokine secretion [ ] [ ] [ ] in an age-dependent manner [ , ] , and modifying the severity of the disease. another example of a testable hypothesis may be that the immune phenotype associated with asthma inhibits pro-inflammatory cytokine production and modifies gene expression in the airway epithelium, protecting against severe covid- [ , , ] . in order to understand complex and often indirect dependencies between different pathways and molecules, we need to combine computational and data-driven analyses. standardised representation and programmatic access to the contents of the covid- disease map enable the development of reproducible analytical and modelling workflows. here, we discuss the range of possible approaches and demonstrate preliminary results, focusing on interoperability, reproducibility, and applicability of the methods and tools. our goal is to work on the computational challenges as a community, involving the biocurators and domain experts in the analysis of the covid- disease map and rely on their feedback to evaluate the outcomes. in this way, we aim to identify approaches to tackle the complexity and the size of the map, proposing a state-of-the-art framework for robust analysis, reliable models, and useful predictions. visualisation of omics data can help contextualise the map with experimental data creating data-specific blueprints. these blueprints could be used to highlight parts of the map that are active in one condition versus another (treatment versus control, patient versus healthy, normal versus infected cell, etc.). combining information contained in multiple omics platforms can make patient stratification more powerful, by reducing the number of samples needed or by augmenting the precision of the patient groups [ , ] . approaches that integrate multiple data types without the accompanying mechanistic diagrams [ ] [ ] [ ] produce patient groupings that are difficult to interpret. in turn, classical pathway analyses often produce long lists mixing generic and cell-specific pathways, making it challenging to pinpoint relevant information. using disease maps to interpret omics-based clusters addresses the issues related to contextualised visual data analytics. footprints are signatures of a molecular regulator determined by the expression levels of its targets [ ] . for example, a footprint can contain targets of a transcription factor (tf) or peptides phosphorylated by a kinase. combining multiple omics readouts and multiple measurements can increase the robustness of such signatures. nevertheless, an essential component is the mechanistic description of the targets of a given regulator, allowing computation of its footprint. with available sars-cov- related omics and interaction datasets [ ] , it is possible to infer which tfs and signalling pathways are affected upon infection [ ] . combining the covid- disease map regulatory interactions with curated collections of tf-target interactions like dorothea [ ] will provide a contextualised evaluation of the effect of sars-cov- infection at the tf level. the virus-host interactome is a network of virus-human protein-protein interactions (ppis) that can help understanding the mechanisms of disease [ , [ ] [ ] [ ] . it can be expanded by merging virus-host ppi data with human ppi and protein data [ ] to discover clusters of interactions indicating human mechanisms and pathways affected by the virus [ ] . these clusters first of all can be interpreted at the mechanistic level by visual exploration of covid- disease map diagrams. in addition, these clusters can potentially reveal additional pathways to add to the covid- disease map (e.g., e protein interactions or tgfbeta diagrams) or suggest new interactions to introduce into the existing diagrams. computational modelling is a powerful approach that enables in silico experiments, produces testable hypotheses, helps elucidate regulation and, finally, can suggest via predictions novel therapeutic targets and candidates for drug repurposing. mechanistic models of pathways allow bridging variations at the scale of molecular activity to variations at the level of cell behaviour. this can be achieved by coupling the molecular interactions of a given pathway with its endpoint and by contextualising the molecular activity using omics datasets. hipathia is such a method, processing transcriptomic or genomic data to estimate the functional profiles of a pathway conditioned by the data studied and linkable to phenotypes such as disease symptoms or other endpoints of interest [ , ] . moreover, such mechanistic modelling can be used to predict the effect of interventions as, for example, the effect of targeted drugs [ ] . hipathia integrates directly with the diagrams of the covid- map using the sif format provided by casq (see section . ), as well as with the associated interaction databases (see section . ). the drawback of approaches like hipathia is their computational complexity, limiting the size of the diagrams they can process. an approach to large-scale mechanistic pathway modelling is to transform them into causal networks. carnival [ ] combines the causal representation of networks [ ] with transcriptomics, phosphoproteomics, or metabolomics data [ ] to contextualise cellular networks and extract mechanistic hypotheses. the algorithm identifies a set of coherent causal links connecting upstream drivers such as stimulations or mutations to downstream changes in transcription factor activities. analysis of the dynamics of molecular networks is necessary to understand their dynamics and deepen our understanding of crucial regulators behind disease-related pathophysiology. discrete modelling framework provides this possibility. covid- disease map diagrams, translated to sbml qual (see section . ), can be directly imported by tools like cell collective [ ] or ginsim [ ] for analysis. preserving annotations and layout information ensures transparency and reusability of the models. importantly, cell collective is an online user-friendly modelling platform that provides features for real-time in silico simulations and analysis of complex signalling networks. the platform allows users without computational background to simulate or analyse models to generate and prioritise new hypotheses. references and layout are used for model visualisation, supporting the interpretation of the results. the mathematics and code behind each model, however, remain accessible to all users. in turn, ginsim is a tool providing a wide range of analysis methods, including efficient identification of the states of convergence of a given model (attractors). model reduction functionality can also be employed to facilitate the analysis of large-scale models. viral infection and immune response are complex processes that span many different scales, from molecular interactions to multicellular behaviour. the modelling and simulation of such complex scenarios require a dedicated multiscale computational architecture, where multiple models run in parallel and communicate among them to capture cellular behaviour and intercellular communications. multiscale agent-based models simulate processes taking place at different time scales, e.g., diffusion, cell mechanics, cell cycle, or signal transduction [ ] , proposed also for covid- [ ] . physiboss [ ] allows such simulation of intracellular processes by combining the computational framework of physicell [ ] with maboss [ ] tool for stochastic simulation of logical models to study of transient effects and perturbations [ ] . implementation of detailed covid- signalling models in the pysiboss framework may help to better understand complex dynamics of multi-scale processes as interactions and crosstalk between immune system components and the host cell in covid- . in this case study, we combine computational approaches discussed above and present results derived from omics data analysis on the covid- disease maps diagrams. we measured the effect of covid- at the transcription factor (tf) activity level by applying viper [ ] combined with dorothea regulons [ ] on rna-seq datasets of the sars-cov- infected cell line [ ] . then, we mapped the tfs normalised enrichment score (nes) on the interferon type i signalling pathway diagram of the covid- disease map using the sif files generated by casq (see section . ). as highlighted in figure , our manually curated pathway included some of the most active tfs after sars-cov- infection, such as stat , stat , irf and nfkb . these genes are well known to be involved in cytokine signalling and first antiviral response [ , ] . interestingly, they are located downstream of various viral proteins (e, s, nsp , orf a and orf a) and members of the mapk pathway (mapk , mapk and map k ). sars-cov- infection is known to promote mapk activation, which mediates the cellular response to pathogenic infection and promotes the production of proinflammatory cytokines [ ] . altogether, we identified signaling events that may capture the mechanistic response of the human cells to the viral infection. in this use case, the hipathia [ ] algorithm was used to calculate the level of activity of the subpathways from the covid- apoptosis diagram, with the aim to evaluate whether covid- disease map diagrams can be used for pathway modelling approach. to this end, a public rna-seq dataset from human sars-cov- infected lung cells (geo gse ) was used. first, the rna-seq gene expression data was normalized with the trimmed mean of m values (tmm) normalization [ ] , then rescaled to range [ ; ] for the calculation of the signal and normalised using quantile normalisation [ ] . the normalised gene expression values were used to calculate the level of activation of the subpathways, then a case/control contrast with a wilcoxon test was used to assess differences in signaling activity between the two conditions. the activation levels have been calculated using transcriptional data from gse and hipathia mechanistic pathway analysis algorithm. each node represents a gene (ellipse), a metabolite (circle) or a function (square). the pathway is composed of circuits from a receptor gene/metabolite to an effector gene/function, with interactions simplified to inhibitions or activations (see section . , sif format). significantly deregulated circuits are highlighted by color arrows (red: activated in infected cells). the color of the node corresponds to the level of differential expression of each node in sars-cov- infected cells vs normal lung cells. blue: down-regulated elements, red: up-regulated elements, white: elements with not statistically significant differential expression. hipathia calculates the overall circuit activation, and can indicate deregulated interaction even if interacting elements are not individually differentially expressed. results of the apoptosis pathway analysis can be seen in figure and supplementary material . importantly, hipathia calculates the overall activation of circuits (series of causally connected elements), and can indicate deregulated interactions resulting from a cumulative effect, even if interacting elements are not individually differentially expressed. when discussing differential activation, we refer to the circuits, while individual elements are mentioned as differentially expressed. the analysis shows an overactivation of several circuits, specifically the one ending in the effector protein bax. this overactivation seems to be led by the overexpression of the bad protein, inhibiting bcl -mcl -bcl l complex, which in turn inhibits bax. indeed, sars-cov- infection can invoke caspase -induced apoptosis [ ] , where bax together with the ripoptosome/caspase- complex, may act as a pro-inflammatory checkpoint [ ] . this result is supported by studies in sars-cov, showing bax overexpression following infection [ , ] . overall, our findings recapitulate reported outcomes. with evolving contents of the covid- disease map and new omics data becoming available, new mechanism-based hypotheses can be formulated. in the covid disease map community we strive to produce interoperable content and seamless downstream analyses, translating the graphic representations of the molecular mechanisms to executable models. we are also aware of parallel efforts towards modelling of covid- mechanisms, which we plan to include as a part of our ecosystem. these efforts are not yet directly interoperable with the covid disease map content as they use either different notation schemes or require parameters not covered by our biocuration guidelines at the same time, they provide a complementary source of information and the opportunity to create an even broader toolset to tackle the pandemic. the modified edinburgh pathway notation (mepn) scheme [ ] allows for the detailed visual encoding of molecular processes using the yed platform but diagrams are constructed in such a way as to also function as petri nets. these can then be used directly for activity simulations using the biolayout network analysis tool [ ] . the current mepn covid- model details the replication cycle of sars-cov- , integrated with a range of host defence systems, e.g. type interferon signalling, tlr receptors, oas systems, etc. simulations of altered gene expression, interactions with drug targets or changes to interaction kinetics can be represented by introducing relevant transitions or nodes directly in the diagram. currently, models constructed in mepn can be saved as sbgn.ml files, however is a loss of information and the features associated computationally are not compatible with other covid- disease map diagrams (not modelled as petri nets). the covid- disease map can support dynamic kinetic modelling to quantify the behaviour of different pathways and evaluate the dynamic effects of perturbations. however, it is necessary to assign a kinetic equation or a rate law to every reaction in the diagram to be analysed. this process is challenging because any given reaction depends on its cellular and physiological context, which makes it difficult to parameterise. software support of tools like sbmlsqueezer [ ] and reaction kinetics databases like sabio-rk [ ] are indispensable in this effort. nevertheless, the most critical factor is the availability of experimentally validated parameters that can be reliably applied in sars-cov- modelling scenarios. the covid- disease map is both a knowledgebase and a computational repository. on the one hand, it is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. on the other hand, it is a computational resource of curated content for graph-based analyses and disease modelling. it offers a shared mental map for understanding the dynamic nature of the disease at the molecular level and also its dynamic propagation at a systemic level. thus, it provides a platform for a precise formulation of models, accurate data interpretation, monitoring of therapy, and potential for drug repositioning. the covid- disease map spans three platforms and assembles diagrams describing molecular mechanisms of covid- . these diagrams are grounded in the relevant published sars-cov- research, completed where necessary by mechanisms discovered in related beta-coronaviruses. this unprecedented effort of community-driven biocuration resulted in over forty diagrams with molecular resolution constructed since march . it demonstrates that expertise in biocuration, clear guidelines and text mining solutions can accelerate the passage from the published findings to a meaningful mechanistic representation of knowledge. the covid disease map can provide the tipping point to shortcut research data generation and knowledge accumulation, creating a formalized and standardized streamline of well defined tasks. this approach to an emerging pandemic leveraged the capacity and expertise of an entire swath of the bioinformatics community, bringing them together to improve the way we build and share knowledge. by aligning our efforts, we strive to provide covid- specific pathway models, synchronize content with similar resources and encourage discussion and feedback at every stage of the curation process. with new results published every day, and with the active engagement of the research community, we envision the covid- disease map as an evolving and continuously updated knowledge base whose utility spans the entire research and development spectrum from basic science to pharmaceutical development and personalized medicine. moreover, our approach includes a large-scale effort to create interoperable tools and seamless downstream analysis pipelines to boost the applicability of established methodologies to the covid- disease map content. this includes harmonisation of formats, support of standards, and transparency in all steps to ensure wide use and content reusability. preliminary results of such efforts are presented in the case studies. the covid- disease map community is open and expanding as more people with complementary expertise join forces. in the longer run, the map's content will help to find robust signatures related to sars-cov- predisposition or response to various treatments, along with the prioritization of new potential drug targets or drug candidates. we aim to provide the tools to deepen our understanding of the mechanisms driving the infection and help boost drug development supported with testable suggestions. we aim at building armor for new treatments to prevent new waves of covid- or similar pandemics. a pneumonia outbreak associated with a new coronavirus of probable bat origin covid- and the kidney: from epidemiology to clinical practice receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected 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and flexible graphical system for rendering biological pathways a graphical and computational modeling platform for biological pathways sabio-rk: an updated resource for manually curated biochemical reaction kinetics key: cord- -yu qw l authors: burgner, david; harnden, anthony title: kawasaki disease: what is the epidemiology telling us about the etiology? date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: yu qw l kawasaki disease (kd) is an important and common inflammatory vasculitis of early childhood with a striking predilection for the coronary arteries. it is the predominant cause of paediatric acquired heart disease in developed countries. despite years of research, the aetiology of kd remains unknown and consequently there is no diagnostic test and treatment is non-specific and sub-optimal. the consensus is that kd is due to one or more widely distributed infectious agent(s), which evoke an abnormal immunological response in genetically susceptible individuals. the epidemiology of kd has been extensively investigated in many populations and provides much of the supporting evidence for the consensus regarding etiology. these epidemiological data are reviewed here, in the context of the etiopathogenesis. it is suggested that these data provide additional clues regarding the cause of kd and may account for some of the continuing controversies in the field. kawasaki disease; vasculitis; genetics; epidemiology; inflammation summary kawasaki disease (kd) is an important and common inflammatory vasculitis of early childhood with a striking predilection for the coronary arteries. it is the predominant cause of paediatric acquired heart disease in developed countries. despite years of research, the aetiology of kd remains unknown and consequently there is no diagnostic test and treatment is non-specific and sub-optimal. the consensus is that kd is due to one or more widely distributed infectious agent(s), which evoke an abnormal immunological response in genetically susceptible individuals. the epidemiology of kd has been extensively investigated in many populations and provides much of the supporting evidence for the consensus regarding etiology. these epidemiological data are reviewed here, in the context of the etiopathogenesis. it is suggested that these data provide additional clues regarding the cause of kd and may account for some of the continuing controversies in the field. # international society for infectious diseases. published by elsevier ltd. all rights reserved. no predefined order and some may even be absent on presentation. in many children the clinical presentation is striking and kd patients are often misdiagnosed with severe sepsis. however, kd can have similar clinical features to other common childhood illnesses, such as measles, rubella and scarlet fever. these children may present a diagnostic challenge and the lack of a specific diagnostic test may delay treatment and thus worsen prognosis. moreover, to confuse the clinical picture further, there is increasing concern that the diagnostic criteria are too narrow. 'atypical' or 'incomplete' kd is a description used for children presenting with the characteristic fever but fewer than four classical signs. a better descriptive term is 'incomplete kd' because these children do not demonstrate atypical signs, just fewer of them. cervical lymphadenopathy is the least commonly observed of the diagnostic criteria, occurring in about three quarters of usually older children, whilst prolonged fever and peripheral desquamation have been reported as the commonest diagnostic features. incomplete kd may be poorly recognised and occurs more often in infants. children presenting with incomplete kd are at higher risk of developing coronary artery lesions both because of their young age and their potential for not receiving timely immunoglobulin treatment. interestingly, as many as % of the children reported in the original report of the syndrome would not fulfil the current case definition. the clinical diagnostic criteria need refinement to increase their positive predictive value and recent guidelines have been developed in an attempt to increase the sensitivity of the clinical diagnosis. it is unclear how these will perform in clinical practice. although these guidelines are intended only as a clinical tool, they are likely to have an impact on the reported epidemiology of kd. kawasaki disease is clearly not the benign childhood exanthem initially proposed , and has significant long-term implications. kawasaki disease is the most common cause of paediatric acquired heart disease in the world. coronary artery lesions (predominantly aneurysms) occur in up to % of untreated and - % of treated children; , , the poor outcome despite adequate treatment reflects an incomplete understanding of the etiopathogenesis. acute mortality in kd is increased significantly, with deaths predominantly occurring from myocardial infarction following occlusion of giant coronary aneurysms. , overall, myocardial infarction occurs in % of those with coronary artery lesions. lifelong medical therapy, coronary artery grafting and even heart transplantation may be required. crucially, in children without coronary artery lesions who die of other causes, the coronary arteries are almost invariably markedly abnormal, with striking pro-atherosclerotic changes. , abnormal in vivo function in non-coronary arteries suggests that cardiovascular damage post-kd is both pervasive and persistent, even in the absence of acute coronary artery lesions. thus, there is intense speculation that kd is pro-atherosclerotic, but definitive long-term data are lacking. regressed coronary artery lesions result in abnormal coronary artery function and histological changes that are pro-atherosclerotic. , the similarities between kd and adult cardiovascular pathology suggest that kd may be a useful paradigm for investigating the etiopathogenesis of atherosclerosis. does kawasaki disease represent an extreme phenotype of a more pervasive phenomenon? it is possible that kd is not a distinct entity, but the more clinically obvious end of a spectrum of pathogenic processes. kd may therefore reflect an extreme clinical phenotype where childhood infections predispose to subsequent endothelial damage and cardiovascular pathology. thus in genetically susceptible children, acute infections such as those causing fever and rash, may result in unrecognised damage to the cardiovascular system that later manifests itself as adult cardiovascular disease. adult atherosclerotic disease (like kd) has not been reliably associated with a single infectious etiology, but correlates with overall infectious burden. furthermore, acute childhood infections are accompanied by pro-atherosclerotic phenomena and subsequent thickening of the arterial intima. understanding the etiopathogenesis of kd may therefore identify common gene-environment interactions that are involved in adult cardiovascular disease. why is it important to understand the etiology and pathogenesis of kawasaki disease? the timely diagnosis of kd is essential in maximising the prevention of overt coronary damage; treatment beyond ten days of onset is associated with a worse outcome and an increased incidence of coronary abnormalities. , the lack of a specific diagnostic test and the limited clinical utility of the current clinical diagnostic criteria mean that the diagnosis is often delayed, even in populations where the condition is well recognised. the currently accepted best treatment (intravenous immunoglobulin and aspirin (table ) fails to prevent coronary artery abnormalities (identifiable by imaging) in up to % of cases. specific diagnostic test(s) and rational interventions could be readily developed if the etiopathogenesis of kd was fully understood. moreover, preventative treatments such as vaccines would be justified in populations with the highest kd incidence, where kd affects - % of all children, such as korea and japan. the consensus view is that kd results from a widely distributed infectious agent (or possibly agents) that causes the clinical syndrome in genetically susceptible children. much of the supporting data for this viewpoint is provided by epidemiological studies in a variety of populations. kd is described in all ethnic groups, but the incidence varies dramatically (see below). the homogeneity of the clinical phenotype and epidemiology suggest that kd arises from common disease processes, although the antigenic trigger(s) and/or the genetic determinants may differ between populations. kawasaki disease shows a striking age distribution reminiscent of other childhood infections. over % of cases occur between the ages of six months and four years, although the condition occurs rarely both earlier and later in life. the low incidence of kd in both the first six months suggests that most infants are protected by passively acquired maternal antibody against the causative agent(s). a transient immunological immaturity may also account for the low incidence in the first few months postnatally. the low incidence of kd beyond mid-childhood suggest a ubiquitous antigen(s) that most children encounter uneventfully in early childhood and to which they mount an appropriate and protective immune response. kawasaki disease is more common in boys (male:female ratio . : ) a feature observed in many infectious diseases , and also in coronary atherosclerosis, where sex differences in immune responses are suggested to mediate susceptibility. seasonal variation in kd incidence is well recognised, but the predominant season varies in different countries. in the uk, australia and the usa , kd is most common in winter and spring. in china, spring and summer predominate and in korea kd incidence is highest in summer months. in japan, which reports the highest kd incidence, table treatment of kawasaki disease (for detailed discussion of the treatment of kawasaki disease see references , , ). at a dose of g/kg given as a single infusion over - hours (unless cardiac status necessitates infusing the dose more slowly or in divided doses). failure to respond to this initial ivig dose is usually treated with a second dose (usually g/kg as a single infusion). failure to respond to the second ivig dose is often treated with intravenous methylprednisolone under expert supervision the dose of aspirin is controversial and its utility has never been proven in a randomised controlled trial. it remains, however, part of the standard management of kd. generally 'high dose' aspirin ( - mg/kg/day in divided doses) is given acutely until the fever defervesces, when 'low dose' aspirin ( - mg/kg/day) is given until an echocardiogram at six weeks after the kd diagnosis is normal. if the six week echocardiogram is abnormal, aspirin is usually continued under cardiological supervision anti-cytokine therapies and other interventions are generally unproven but have occasionally been used. for a review see newburger and fullton the seasonal variation is less marked. one possible explanation for these divergent data is that season is a marker for weather conditions that have a more direct role in determining the incidence. in the us, kd incidence clearly correlates negatively with average ambient temperature and positively with average rainfall in the preceding month. studies are underway investigating similar parameters in the uk. it is unknown whether the meteorological conditions themselves predispose to kd, or, more plausibly, if they alter the epidemiology of etiological agents. the lack of consistent seasonal associations in different countries raises the possibility that various etiological agents may be involved in the etiology of kd. geographical clustering of kd cases and epidemics have been reported from a number of countries, - although they have been much less frequently reported in the past decade, possibly suggesting a changing epidemiology. in japan, which has provided the most comprehensive epidemiological data, epidemics of kd have been described with a clear epicentre and documented geographical spread across the whole nation within six months. these epidemiological data clearly indicate an infectious etiology for kd. the clinical features of the disease are also characteristic of a severe acute childhood infection. it seems likely that the causative agents are widely distributed and are also highly immunogenic, at least in most children, as more than one episode of kd is rare. recurrent kd is reported in - % of children, although it appears less common in caucasians. it may reflect a specific immunological deficiency in these children or exposure to more than one causative agent. various environmental causes of kd have been repeatedly suggested (table ) , but none has been consistently replicated. however, the possibility of environmental factors influencing etiology, possibly by modulating infection risk, remains a possibility. the search for a single unifying microbiological cause has been unrelenting but, to date, fruitless. standard microbiological techniques, molecular methods and serological investigations have so far failed to identify an etiological agent. molecular techniques fail to detect circulating conserved microbial sequences in kd, indicating that the antigenic stimulus may arise from a distant site (e.g. colonising pathogens in the nasopharynx) and/or may represent host-derived factor(s) that induce or promote the pro-inflammatory cascade. the list of discarded and/or unproven etiological agents in kd is long (table ) . a recent report of an association between the presence of genetic material from a novel coronavirus and kawasaki disease in a handful of cases remains unproven and may reflect an epiphenomenon; the putative etiological agent is a relatively common viral pathogen in young children and it is unclear how long the dna persists. the lack of a unifying etiological agent despite a significant research effort suggests that kd can follow exposure to more than one infectious agent, or that a novel infectious agent is involved. alternatively the clinical phenotype may reflect a stereotyped response in a genetically-susceptible host to one of a variety of infectious agents. much of the continuing debate in the literature concerns whether kd is caused by a superantigen or a conventional antigen. kd shares many clinical features with superantigen-mediated diseases (for example, rash, conjunctivitis and skin peeling) and kd has occasionally been reported concurrently in children with toxic-shock syndrome, which is caused by superantigens. however, unequivocal epidemiological and laboratory support for a role for superantigens in kd is lacking. in one small study, maternal antibodies against toxic shock syndrome toxin- appeared protective against early-onset kd. however, the carriage rates of superantigen-producing bacteria by children with kd are not consistently increased, , although these data may reflect the involvement of as yet unidentified d. burgner, a. harnden superantigens, with more than one superantigen capable of causing kd. superantigens bind to the vb region of the t-cell receptor and clonal expansion of vb -expressing t-cells has been reported in some studies of kd, but again the finding is inconsistent. other studies have reported oligoclonal igaproducing plasma cells infiltrating bronchial and intestinal tissues in fatal kd, which suggests the involvement of a conventional antigen. much of the controversy and inconsistency surrounding the nature of the infectious trigger in kd might reflect multiple etiological agents resulting in the same clinical phenotype. it is possible, for example, that a viral upper respiratory tract infection may alter local immunity and allow elaboration of superantigens by colonising bacteria in the nasopharynx. certainly the epidemiology of kd, with rapid changes in incidence, seasonal variation and the relationship between incidence and weather conditions is more redolent of acute viral infections , than bacterial colonisation, which alters more slowly. in meningococcal disease, influenza infection acts in an analogous way, and meningococcal epidemics often follow influenza outbreaks. , this hypothesis could be addressed through large detailed prospective epidemiological studies. another possibility is that either pathogen or host factors modulate the behaviour of an antigen, so that it behaves both as a conventional antigen and as a superantigen. heat shock proteins are increased in acute inflammatory conditions, including kd and cross-reactivity with certain heat shock proteins is thought to be responsible for the inflammation of the bcg scar in kd. heat shock proteins have been reported to alter the behaviour of superantigens, so that the immune system recognises them as conventional antigens and also can greatly up-regulate pro-inflammatory responses to conventional antigens. the possibility of endogenous stimuli that profoundly suppress or enhance antigenic effects has not previously been considered in kd, but might account for much of the controversy surrounding the roles of conventional or superantigens. whatever the etiological trigger(s) for kd, there is clear evidence that host genetic determinants play a major role in both susceptibility and probably outcome in kd. genetic studies of kd are therefore likely to be highly informative about etiology and pathogenesis. although kd is reported in all ethnic groups, the variation in incidence of kd between (and to a lesser extent within) countries is striking. the annual incidence varies from approximately three (per , children < years of age) in south america, to four in australia, eight in the uk, - in the us, - in china and hong kong in taiwan, in korea and > in japan the reported incidence is probably underestimated in many countries as atypical cases are not included. australian data (burgner et al., unpublished) suggest an incidence % higher than that recorded through active surveillance. in a number of countries the incidence of kd appears to be increasing. , , whilst this may be partly attributable to increased awareness, the increasing incidence is reported in countries where the disease has been widely recognised for several years and where a standard case definition is employed and may therefore reflect changing epidemiology. the incidence of kd is therefore greatest in north-east asians, especially in koreans and japanese. it is estimated that - % of all hospitalised korean children have kd (park yw, personal communication) and that kd affects one in japanese children. this indicates genetic factors may be central in determining susceptibility, especially as the incidence rate remains high in those migrating to lower incidence countries. for example, the incidence of kd in japanese americans in hawaii ( / , < years) is identical to the highest rates reported from japanese living in japan. the incidence rate in siblings of affected children is - fold higher than the population incidence in japan. the ratio of sibling to population incidence is termed the 'heritability' or 'l s '. sibling rates outside japan are unknown, but reports from caucasians support this trend and sibling rates in korea (burgner, unpublished observations) also support this high heritability. this figure is slightly less than the heritability for insulin-dependent diabetes (l s ) and times higher than that of asthma, suggesting a striking genetic predisposition to develop the disease in a minority of children across different ethnic groups. in addition, the incidence of previous kd in parents of japanese children with kd is significantly increased and these families are much more likely to have other affected children and children with recurrent disease. taken together, these epidemiological data provide convincing evidence for a major role for host genetics in kd susceptibility. whilst there are concerns that cardiovascular damage may be pervasive in kd, overt coronary artery lesions only develop in a minority of children. in acute kd all arms of the innate and adaptive immune response are activated, but lymphocytes, macrophages and neutrophils are central. the extent and kinetics of host inflammation strongly correlates with the risk of coronary damage. the duration of fever prior to treatment, - the maximal erythrocyte sedimentation rate, the extent of pro-inflammatory cytokine production , and the degree of neutrophil activation have all been shown to be risk factors for coronary damage. the extent of the host inflammatory response is partly genetically determined. genetic factors are therefore likely to be important in determining outcome in kd and genetic studies may identify key pathogenic mediators and ultimately guide the development of new interventions. kawasaki disease is likely to be a genetically 'complex disease', with contributions from a number of genetic loci to susceptibility and outcome. associations between genetic variants at candidate loci and kd susceptibility and outcome may be extremely informative about the role of specific mediators in etiopathogenesis, allowing investigation of hypotheses suggested by the clinical data, but untestable by conventional clinical or laboratory studies. the consensus view supports the concept of a genetically-susceptible host in kd , and there is growing realisation that immunogenetic studies may reveal much about the disease and improve diagnosis, treatment and prognosis. immunogenetic data suggest a number of plausible associations. many studies focus on putative downstream outcome determinants and suggest a role for mediators of innate inflammation, endothelial activation and cardiovascular homeostasis. studies of susceptibility determinants are more limited. there are associations with class i regions of the human leucocyte antigen (hla) in japanese, with different alleles associated in caucasians. however, these hla studies are largely historical and further work using modern hla typing techniques are warranted. unfortunately, genetic studies of kd have often been undermined by methodological problems that dog many such studies of human complex disease. thus some of the reported associations are likely to be false positive results. in particular, the studies often lack statistical power, employ multiple uncorrected statistical comparisons and many do not replicate findings in an independent population. , , [ ] [ ] [ ] case-control methodology in a multi-ethnic disease may yield spurious disease associations (type i errors) due to population admixture, unless this is actively identified. inadequate marker density in candidate loci, where the functional variants are unknown, may increase type ii errors. kawasaki disease is a fascinating and important paediatric illness, which presents a significant diagnostic challenge. it is the most common cause of heart disease acquired in childhood and an important paradigm for understanding the determinants of adult cardiovascular pathology. the epidemiology is well characterised and clearly supports the view that the disease results from an inappropriate immunological response to one or more infectious triggers in genetically-susceptible individuals. the search for the microbial etiology has been disappointing and unsuccessful and all that remains of over three decades of such studies is a 'long list of discarded pathogens'. understanding the genetic determinants of susceptibility to kd and those involved in mediating coronary artery damage may be a more profitable approach. the methodological issues that have undermined genetic analyses can be largely overcome by international collaborative studies that employ standardised phenotypic definitions and large sample sizes derived from different ethnic groups. the use of familybased genetic association analyses circumvents the problems of population stratification and the use of trans-racial mapping (i.e. investigating genetic determinants in different ethnic groups) may prove important to defining the critical genetic determinants, particularly in regions of high linkage disequilibrium. newer molecular techniques, particularly gene expression profiling and proteomics may identify novel molecular 'fingerprints' that differentiate kd from other febrile and inflammatory illnesses. the mystery of kd may ultimately be solved by looking within the host. kawasaki disease: a brief history acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children (japanese) kawasaki syndrome kawasaki disease characterized by erythema and induration at the bacillus calmette-guerin and purified protein derivative inoculation sites kawasaki disease kawasaki disease: an evidence based approach to diagnosis, treatment, and proposals for future research effects of gamma-globulin on the cardiac sequelae of kawasaki disease incomplete (atypical) kawasaki disease fate of coronary aneurysms in kawasaki disease: serial coronary angiography and long-term followup study cardiovascular involvement in kawasaki disease: evaluation and natural history mortality among patients with a history of kawasaki disease: the third look. the kawasaki disease follow-up group clinical spectrum of kawasaki disease in infants younger than months of age long-term consequences of kawasaki disease. a -to -year follow-up study of patients with special reference to the heart and coronary arterial lesions pathological study of postcoronary arteritis in adolescents and young adults: with reference to the relationship between sequelae of kawasaki disease and atherosclerosis endothelial dysfunction late after kawasaki disease long term consequences of regressed coronary aneurysms after kawasaki disease: vascular wall morphology and function noninvasive assessment of the early progression of atherosclerosis in adolescents with kawasaki disease and coronary artery lesions kawasaki disease-from a mystery to a paradigm impact of infectious burden on extent and long-term prognosis of atherosclerosis acute infections in children are accompanied by oxidative modification of ldl and decrease of hdl cholesterol, and are followed by thickening of carotid intima-media a single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute kawasaki syndrome the treatment of kawasaki syndrome with intravenous gamma globulin kawasaki disease in australia, - immunoglobulin failure and retreatment in kawasaki disease kawasaki disease in children classical kawasaki disease in a neonate adult onset kawasaki disease diagnosed by the echocardiographic demonstration of coronary aneurysms kawasaki disease: a maturational defect in immune responsiveness the male predominance in the incidence of infectious diseases in children: a postulated explanation for disparities in the literature genetic susceptibility to infectious diseases host response to cytomegalovirus infection as a determinant of susceptibility to coronary artery disease: sex-based differences in inflammation and type of immune response rising incidence of kawasaki disease in england: analysis of hospital admission data kawasaki syndrome in the united states to hospitalizations for kawasaki disease among children in the united states epidemiologic picture of kawasaki disease in beijing from through epidemiologic study of kawasaki disease in korea, - : comparison with previous studies during - results of nationwide epidemiological incidence surveys of kawasaki disease in japan relationship of climate, ethnicity and socioeconomic status to kawasaki disease in san diego county possible ''outbreak'' of kawasaki disease in victoria temporal and geographical clustering of kawasaki disease in japan nationwide epidemic of kawasaki disease in japan during winter of - epidemiologic study of kawasaki disease in korea an epidemic of kawasaki syndrome in hawaii cardiac sequelae of kawasaki disease among recurrent cases recurrent kawasaki disease search for highly conserved viral and bacterial nucleic acid sequences corresponding to an etiologic agent of kawasaki disease association between a novel human coronavirus and kawasaki disease superantigens: microbial agents that corrupt immunity superantigens, conventional antigens and the etiology of kawasaki syndrome kawasaki disease and toxic shock syndrome --at last the etiology is clear? maternal antibody against toxic shock syndrome toxin- may protect infants younger than months of age from developing kawasaki syndrome prevalence of superantigen-secreting bacteria in patients with kawasaki disease chips with everything: dna microarrays in infectious diseases evidence for a superantigen mediated process in kawasaki disease characterization of the t-cell receptor v-beta repertoire in kawasaki disease detection of antigen in bronchial epithelium and macrophages in acute kawasaki disease by use of synthetic antibody seasonal trends of viral respiratory tract infections in the tropics epidemiology of respiratory viral infection among paediatric inpatients over a six-year period in north-east england the nasopharyngeal bacterial flora in infancy: effects of age, gender, season, viral upper respiratory tract infection and sleeping position outbreak of meningococcal disease after an influenza b epidemic at a hellenic air force recruit training center influenza a and meningococcal disease increased expression of human -kd heat shock protein gene in kawasaki disease determined by quantitative reverse transcription-polymerase chain reaction t cells recognize an immunodominant epitope of heat shock protein in kawasaki disease a toxic shock syndrome toxin- peptide that shows homology to amino acids - of mycobacterial heat shock protein is presented as conventional antigen human heat shock protein induces maturation of dendritic cells versus a th -promoting phenotype summary and abstracts of the seventh international kawasaki disease symposium surveillance of kawasaki disease in taiwan and review of the literature physical and psychosocial health in children who have had kawasaki disease kawasaki disease in families kawasaki disease in siblings the value of isolated populations in genetic studies of allergic diseases kawasaki disease in parents and children kawasaki syndrome kawasaki disease: review of risk factors for coronary aneurysms coronary risks after high-dose gamma-globulin in children with kawasaki disease clinical relevance of the risk factors for coronary artery inflammation in kawasaki disease coronary artery involvement in kawasaki syndrome in manhattan cytokines predict coronary aneurysm formation in kawasaki disease patients raised serum interleukin levels in kawasaki disease urinary neopterin as a predictive marker of coronary artery abnormalities in kawasaki syndrome genetic influence on cytokine production and fatal meningococcal disease kawasaki disease: the mystery continues increased frequency of alleles associated with elevated tumor necrosis factor-alpha levels in children with kawasaki disease high incidence of angiotensin i converting enzyme genotype ii in kawasaki disease patients with coronary aneurysm methylenetetrahydrofolate reductase polymorphism in kawasaki disease hla antigens in kawasaki disease hla antigens in mucocutaneous lymph node syndrome in new england problems of reporting genetic associations with complex outcomes the hla class ii region and susceptibility to kawasaki disease polymorphism of transmembrane region of mica gene and kawasaki disease polymorphism of slc a (formerly nramp ) gene confers susceptibility to kawasaki disease mapping disease genes: family-based association studies control of confounding of genetic associations in stratified populations the tdt and other family-based tests for linkage disequilibrium and association identification of susceptibility loci for insulindependent diabetes mellitus by trans-racial gene mapping the diagnosis and management of kawasaki disease adenovirus infection in patients with kawasaki disease could a herpesvirus be the cause of kawasaki disease? mycoplasma infection and kawasaki disease probable role of streptococcus pyogenes in kawasaki disease serologic evidence that streptococcal superantigens are not involved in the pathogenesis of kawasaki disease variant streptococcus sanguis as an etiological agent of kawasaki disease toxic shock syndrome toxin-secreting staphylococcus aureus in kawasaki syndrome the absence of evidence of staphylococcal toxin involvement in the pathogenesis of kawasaki disease variant strain of propionibacterium acnes: a clue to the etiology of kawasaki disease pathogenicity of propionibacterium acnes isolated from kawasaki disease patients-cytopathogenic protein (cpp) isolated from p. acnes culture filtrates and measurement of the antibody against cpp positive ehrlichia canis serology in kawasaki disease ehrlichia chaffeensis and rochalimaea antibodies in kawasaki disease rickettsia-like bodies and splenitis in kawasaki disease failure to confirm the rickettsial etiology of mcls (kawasaki disease) epstein-barr virus genome-positive tubulointerstitial nephritis associated with kawasaki disease-like coronary aneurysms kawasaki disease, epstein-barr virus and coronary artery aneurysms kawasaki disease and epstein-barr virus the etiology of kawasaki disease: retrovirus? polymerase activity in lymphocyte culture supernatants from patients with kawasaki disease virus-like particles with reverse transcriptase activity associated with kawasaki disease a measles virus isolate from a child with kawasaki disease: sequence comparison with contemporaneous isolates from 'classical' cases isolation of measles virus from child with kawasaki disease demonstration of chlamydia pneumoniae in cardiovascular tissues from children with kawasaki disease is there an association between kawasaki disease and chlamydia pneumoniae failure to demonstrate chlamydia pneumoniae in cardiovascular tissue from children with kawasaki disease is kawasaki disease a variant of q fever? kawasaki disease in european adult associated with serological response to coxiella burneti role of house dust mites in kawasaki disease urine mercury levels in kawasaki disease rug shampoo and kawasaki disease association of rug shampooing and kawasaki disease clinical and epidemiologic characteristics of patients referred for evaluation of possible kawasaki disease. united states multicenter kawasaki disease study group the authors' kawasaki key: cord- -f j bb authors: sabater gonzález, mikel; calvo carrasco, daniel title: emergencies and critical care of commonly kept fowl date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: f j bb fowl are birds belonging to one of the biological orders, the game fowl or land fowl (galliformes) and the waterfowl (anseriformes). studies of anatomic and molecular similarities suggest these two groups are close evolutionary relatives. multiple fowl species have a long history of domestication. fowl are considered food-producing animals in most countries and clinicians should follow legislation regarding reportable diseases and antibiotic use, even if they are pets. this article reviews aspects of emergency care for most commonly kept fowl, including triage, patient assessment, diagnostic procedures, supportive care, short-term hospitalization, and common emergency presentations. refill time (crt). normally, when the finger is removed from the vein, refilling cannot be witnessed visually. if it can be witnessed visually the bird is considered approximately % dehydrated, and if second can be counted, then the bird is about % dehydrated or in shock. in chickens, the comb should be firm and red. a crt can be assessed on the comb. it should refill within seconds. mucous membrane color can be assessed by everting the vent or the eyelid. respiratory monitoring includes auscultation of the upper and lower respiratory tracts, assessing breathing frequency and quality, as well as detection of signs of dyspnea (eg, orthopneic gait or tail bobbing). the levels of brightness, alertness, and response should be evaluated as part of a first neurologic examination. birds showing depression or severe weakness should be placed immediately in a prewarmed incubator with % to % humidified oxygen and complete physical examination or diagnostic procedures may be delayed until the bird is stable enough to tolerate them. secondary survey includes obtaining a complete medical history, a full physical examination, assessment of the response to initial therapy, and more diagnostic procedures, which may provide a comprehensive diagnostic and therapeutic plan as well as orientate the owner about the potential economic costs and prognosis. a complete anamnesis should include, but is not restricted to, species; breed; age; gender; presenting complaint; source of the bird; diet; number of birds in the household; open or closed flock; acquisition date; date of the last addition to the flock; number and species of animals affected; potential exposure to toxins; length of illness; changes in behavior; history of previous diseases, treatments, and outcomes; reproductive history; and clinical signs, including their duration and progression. physical examination in fowl is similar to that of other avian species. careful observation of the bird before handling is mandatory in order to determine the length and depth of the physical examination and further diagnostics that the patient is likely to tolerate. all equipment and supplies are readied before removing the bird from the holding container or the intensive care unit. if the patient is debilitated, examination can be performed in a stepwise fashion with small breaks given to the bird between handling, examination, diagnostics, and treatments. in general, fowl species may be handled without chemical restraint. precautions should be taken in order to avoid physical injuries to the bird or the handler (bites, scratches [eg, from tarsal spurs], or blows from the wings [larger species]). fowl should be grasped across the back with or without a towel to avoid wing flapping. then, the legs should be firmly grasped placing finger between them to prevent pressure damage. the bird should then be restrained close to the handler's body or against a hard, nonmovable surface. fractious birds may benefit from having their heads covered with a cloth. smaller species of waterfowl may be held singlehandedly by restraining the animal with the wings folded or with fingers of one hand under each wing supporting the proximal humerus and the other hand supporting the bird's abdomen. larger species, such as geese and swans, are typically restrained keeping the wings folded and facing backward under the arm of the handler. large, calm species may also be straddled on the floor (fig. ) . the position of the bird during physical examination, diagnostic procedures, and therapeutic procedures may affect its cardiorespiratory function. dorsal recumbency in conscious chickens decreases tidal volume by % to % and increases breathing frequency by % to %. birds showing signs of respiratory distress should be held upright. respiratory compromise may be worsened in fowl by the inertial resistance of the large pectoral muscle mass to respiratory excursions of the keel. enlarged viscera, excessive intracoelomic fat, or fluid within the coelom may compress the air sacs, reducing their effective volume and potentially leading to hypercapnia, respiratory acidemia, and death. cloacal or body core temperature can be measured. cloacal temperature depends on body temperature and cloacal activity over time. normal range for body temperature in waterfowl is c to c. to read body core temperature, the probe of a thermistor thermometer should be inserted along the esophagus until it passes the thoracic inlet. normal range for core body temperature in chickens is . c to . c. the body condition should be assessed and an accurate weight obtained on a gram or appropriately sized scale in order to correctly calculate potential drug dosages or to compare with previous or future weights (fig. ) . the patient's needs must be prioritized. despite preferring that samples for hematologic and biochemical analysis be obtained before treatment for the best diagnostic ability, fowl in shock must be stabilized before extensive diagnostic sampling. a conservative minimum database includes determination of packed cell volume, total solids, and estimated white blood cell count. there is intraspecies variation in blood volume ( ae ml/kg for common pheasants and ae ml/kg for redhead and canvasback ducks). in healthy patients, the amount of blood that can be removed without deleterious effects is % of body weight in ducks, % in chickens, and % in pheasants. in compromised patients, this should be reduced to . % of body weight. reference values for multiple avian species can be found in the literature. intravenous or intraosseous fluid administration is essential when treating critical patients. catheters can be placed under general anesthesia if necessary. sites for intravenous catheterization include the medial metatarsal vein, the ulnar vein, and the jugular vein (fig. ) . intraosseous catheters can be placed in the distal ulna or proximal tibiotarsus. pneumatic bones, such as the femur or humerus, should be avoided. most birds benefit from intravenous or intraosseous administration of warmed crystalloids at ml/ g body weight. because fluid resuscitation in critically ill birds is difficult, administration of bolus of crystalloids with oxyglobin to hypovolemic birds may be beneficial. different types of colloids may be used as an alternative to oxyglobin. capnography, direct and indirect blood pressure, electrocardiography, and blood gas analysis are additional monitoring techniques that may be useful in assessing unstable patients. deciding the instrumentation to use depends on practicality and procedure length. capnography measures end-tidal carbon dioxide concentrations in expired air and is a useful indicator of arterial carbon dioxide concentrations. the use of capnographs with sidestream sensors is recommended for small avian patients. pulse oximetry has not yet been validated for birds. the characteristics of oxygenated and deoxygenated avian and human hemoglobin are different, resulting in underestimation of hemoglobin saturation. an ultrasonic doppler flow detector is most commonly used for cardiac monitoring but can also be used for indirect blood pressure measurement. indirect blood pressure measurement techniques used in fowl include doppler, photoplethysmographic/photoacoustic probes with a sphygmomanometer, and oscillometric monitors. systolic blood pressure determination via ultrasonic doppler flow detection correlates well with direct blood pressure measurements in ducks (a platyrhynchos). diastolic, and therefore mean, blood pressure cannot be obtained with this method. direct arterial pressure measurement is ideal but not commonly used because of the need for specific technical skill, the invasive nature of the procedure, and the cost of equipment. for medium to large birds (> g), the deep radial artery is the preferred site for arterial catheter placement, whereas for smaller birds (< g) the superficial ulnar artery is preferred. for waterbirds or long-legged birds, the cranial tibial or dorsal metatarsal arteries are acceptable sites for catheterization. catheterization of the external carotid artery usually requires a cut-down for proper visualization. a study performed on anesthetized galliformes comparing glomerular filtration rate and blood pressure found that galliformes were able to maintain their glomerular filtration rate when mean arterial pressure (map) ranged between and mm hg. when map decreased to less than mm hg, chickens were unable to sustain glomerular filtration and urine output ceased. unlike chickens that have normal systolic, mean, and diastolic arterial blood pressures of ae , ae , and ae mm hg, respectively, values for normotension are higher in other galliformes (eg, turkeys). , if the definition of hypotension in humans (reduction of % from the baseline of conscious maps) is extrapolated to birds, the level of blood pressure at which birds are considered hypotensive would have a tendency to be higher than that recorded in mammals, with the exception of some galliformes and anseriformes species. hypovolemia is treated with intraosseous or intravenous bolus administration of crystalloids ( - ml/kg) or colloids ( ml/kg) until systolic pressures are restored. , reference blood pressure values have been determined for different species of fowl. , electrocardiography can be used to monitor cardiac rate and rhythm. electrocardiographic parameters can be highly variable between fowl species, as shown by electrocardiographic studies published for several species including the chicken, turkey, quail, duck, swans, muscovy ducks, guinea fowl, and rock and chukar partridges. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] arterial blood gasometry is the gold standard for assessing the acid-base status, ventilation, and tissue perfusion. it provides essential physiologic information for patients with critical illness or respiratory disease and is vital in the correction of any metabolic respiratory disorders. detailed information about blood gases in birds has been published. sedation or anesthesia may minimize stress to fractious or painful patients. it also may aid in minimizing risk of capture myopathy in canada geese or turkeys. , midazolam is increasingly used in birds to produce sedation, hypnosis, anxiolysis, anterograde amnesia, centrally mediated muscle relaxation, and anticonvulsion activity. the pharmacokinetics of midazolam hydrochloride following intravenous administration at mg/kg were determined in broiler chickens, turkeys, ring-necked pheasants, and bobwhite quail. several articles regarding fowl anesthesia and analgesia have been published. , , inhalation anesthesia with isoflurane or sevoflurane is the most common in-hospital method for anesthetizing fowl. oxygen flow rates of to l/min allow rapid changes in anesthetic concentration if vaporizer setting is altered. induction is typically via a face mask. the apnea and bradycardia that occur when an induction mask is placed over the beak and face are consequences of a stress response caused by stimulation of trigeminal nerve receptors. [ ] [ ] [ ] preoxygenation with % oxygen for several minutes reduced this response in dabbling but not diving ducks. the isoflurane vaporizer is set to % to % for induction. intubation with a noncuffed endotracheal tube is recommended for anesthetic procedures lasting more than minutes. waterfowl females may require an endotracheal tube . to full size larger than males of the same species. if intubation is not feasible because of the nature of the procedure to be performed or anatomic structures preventing intubation (eg, presence of crista ventralis), ventilation can be achieved via air sac perfusion. airway patency should be regularly checked during waterfowl anesthesia because the thickening of mucus in the trachea or glottis may completely obstruct the endotracheal tube, leading to death of the patient. anticholinergic drugs reduce pharyngeal and tracheal secretions but also increase their viscosity, and so are only recommended for treatment of bradycardia. in chickens and ducks, isoflurane has a minimum anesthetic concentration (mac) of . % and . %, respectively. , isoflurane produces dose-dependent cardiopulmonary depression in birds and in pekin ducks induces tachycardia and hypotension. in geese, an average paco of mm hg was necessary for spontaneous respiration to occur, and no respiration occurred with a paco less than or equal to mm hg. intermittent positive pressure ventilation may be used in anesthetized birds, even if some spontaneous breathing is present, to ensure adequate oxygenation of the blood. ventilation is assisted manually using the reservoir bag on the breathing system or a mechanical ventilator. a spontaneously breathing bird is given greater than or equal to assisted beats per minute. if an anesthetized bird is apneic, the assisted ventilation rate is greater than or equal to to beats per minute depending on size (large birds require fewer breaths than small birds). analysis of blood gases showed that effective gas exchange is achieved using mechanical ventilation. in chickens, sevoflurane mac is . %. at mac, heart rate did not change significantly and cardiac arrhythmias were not observed at less than or equal to times mac. in another study in chickens, hypotension was observed during both spontaneous and controlled ventilation. however, this effect was only dose dependent during critical care of commonly kept fowl controlled ventilation. tachycardia occurred during spontaneous ventilation, whereas heart rate remained unchanged during controlled ventilation. analgesia species variability occurs because of differences in pain sensitivity, the conscious response to pain, and the physiologic response to analgesic therapy. dosages and effects of opioids and nonsteroidal antiinflammatory drugs in fowl have been reviewed. , , hospitalization many birds benefit from symptomatic treatment such as oxygen supplementation, nebulization, fluid therapy, broad-spectrum antibiotics, antifungals, and/or nutritional support and observation for to hours in a warmed incubator before diagnostic tests are performed. separate equipment and housing should be used for birds with suspected contagious diseases and all equipment and cages should be thoroughly disinfected after use to minimize the risk of disease transmission. the optimum temperatures for ill birds are . c to . c. administration of oral or subcutaneous fluids is reserved for stable fowl that are less than % dehydrated. oral fluid administration requires a patient that can maintain an upright body position and has a functional gastrointestinal tract to avoid regurgitation and aspiration of fluids. subcutaneous fluids may be administered in the inguinal web, interscapular area, axillary region, lateral flank, or midback. replacement fluid therapy is critical before nutritional support is instituted. diets for stable hospitalized fowl should ideally be selected according to the natural diet of the species. commercially available feeding formulas, such as a formulated critical care diet lafeber's critical care diet (lafeber company, cornell, il) or hill's a/d diet (a/d canine/feline; hill's pet nutrition, topeka, ks) can be used on a short-term basis. the use of emeraid exotic carnivore diet improves postsurgical recovery and survival of long-tailed ducks (fig. ) . the most common causes of blood loss in birds include traumatic injury and hemorrhagic lesions of internal organs. the ld (lethal dose, %) for acute blood loss in mallard ducks was % of total blood volume. after chronic blood loss, the ld of mallard ducks was reached when % of blood volume was removed, compared with an ld in pheasants and chickens of % to % loss of total blood volume. recommendations for fluid resuscitation after severe blood loss in birds have included the administration of crystalloids, colloids, and whole blood. although no statistical difference in mortality was appreciated among the fluid resuscitation groups (crystalloids, hetastarch, or a hemoglobin-based oxygen-carrying solution [hbocs]) in the acute blood loss study, a trend of decreased mortality was observed in the hbocs group. an early regenerative response was apparent following acute blood loss. trauma traumatic injuries occur fairly frequently in fowl kept outdoors, either as a result of predator attack, gunshot, electrocution, or as a result of inappropriate housing. a thorough physical examination is essential to determine the extent of trauma and the best approach for treatment. prioritize therapy (oxygen therapy, fluid therapy, and analgesia), control active hemorrhage, cleanse wounds, and stabilize fractures initially until patient stabilization allows a more specific treatment. any animal that has a bite wound should be provided with antibiotics after a sample has been taken for microbiological culture and sensitivity. clinical signs of head trauma may include, but are not restricted to: anisocoria, head tilt, depression, or other neurologic signs, skull fractures, retinal detachment, or hemorrhage from the nares, oral cavity, ears, and/or anterior chamber of the eye. mentation, pupil symmetry and size, and pupillary light reflex should be constantly monitored. changes in pupil size to dilated and loss of pupillary light reflex along with mentation progression to stupors or coma indicate neurologic deterioration. soft tissue injuries in the head and neck are commonly seen, and may require surgical repair. posttraumatic exposed epibranchial bones, part of the hyoid apparatus, can be surgically excised without significant postsurgical impairment, allowing easier surgical repair of wounds in the crown. fractures of the skull bones (eg, mandible, quadrate, jugal arches, palatine, pterygoid, and maxilla) can also occur. if the animal is able to groom and feed, healing by second intention can create a false joint allowing normal function. surgical repair of the beak and the use of prostheses have been reported. , anseriformes are prone to foreign body injuries. ingestion of fishing hooks and lines is common in swans (fig. ) . lesions can be observed in the rhamphotheca, tongue, skin of the neck, and gastrointestinal tract. management varies depending on the severity of the injuries. endoscopy can be attempted, but sometimes surgery is required. management of a neck injury caused by a nail shot from a pneumatic nail gun in a young muscovy duck (cairina moschata) has been reported. ocular injuries can also be seen after head trauma. if the eye is not visual and is severely damaged, enucleation may be considered. injuries over the coelomic cavity should always be assessed to make sure no penetrating injuries are present. in such cases the prognosis is poor. skin and muscle injuries can be surgically repaired when indicated, or managed medically to allow healing by second intention. fracture repair follows the same principles as in other avian species. in laying hens, pathologic fractures caused by hypocalcemia and metabolic bone disease might occur, and calcium status should be assessed and deficiencies corrected before surgical repair. vitamin c deficiency can also cause secondary fractures. emergency care for bone fractures should align the fracture as fully as possible so further damage of the surrounding tissues and pain are minimized, and weight bearing can occur as soon as possible, avoiding excess stress and load on the unaffected limb. luxations should be reduced as soon as possible to provide the best chance for joint mobility. bandaging includes soft bandage material and splints applied for temporary support or permanent fixation of fractures. bandaging techniques commonly used in other avian species (tape splint, football-type bandage, plastic spica bandage, modified robert jones bandage, schroeder-thomas splint, ehmer-type bandage, and figure-of-eight bandage) can be also used in fowl (figs. and ) . damage to the cervical air sac can cause emphysema because of the leakage of air into the subcutaneous space. this condition is often self-limiting. a cauterized skin defect can also be created over the swelling to allow the air to escape. the cauterized hole takes longer to heal than the air sac lesion, preventing recurrence. older hens quickly run for shelter when the weather conditions are not desirable. however, juvenile animals may stand on the wet ground, becoming hypothermic rapidly, especially those with thin skulls and crest, such as polands and those with wooly feathering, such as silkies. nevertheless, adequate shelter must be always provided. hypothermic patients may be warmed externally and via infusion of warmed fluids. poultry experience heat distress when high temperatures accompanied by high humidity increase beyond their comfort zone. when the environmental temperatures are between c and c, birds use nonevaporative cooling in ways: ( ) increasing the surface area by relaxing the wings and hanging them loosely at their sides, and ( ) increasing the peripheral blood flow. as the environmental temperature approaches the body temperature of the bird ( c), the rate of respiration increases and the bird open-mouth breathes in order to increase evaporative cooling or water evaporation. if panting fails to prevent body temperature from increasing, birds become listless, comatose, and finally die of respiratory, circulatory, or electrolyte imbalances. respiratory disease is a common presentation in avian practice. clinical signs are often unspecific, and hardly ever pathognomonic. respiratory signs are not only seen with primary respiratory disease but also with any organomegaly or distended coelom as a result of the pressure to the air sacs, or secondary to other disorders, such as cardiovascular disease. sinusitis is a common presentation in chickens and waterfowl, and often presents because of swelling of the periocular sinuses. different agents could cause sinusitis, such as mycobacterium spp, pasteurella spp, escherichia coli, pseudomonas spp, and some viral agents, such as avian influenza and newcastle disease (both reportable diseases in the united kingdom, european union [eu], and united states (us); fig. ). however, newcastle disease, avian influenza, and infectious laryngotracheitis are all rare in backyard poultry, and the most common causative agent of sinusitis in fowl in the us is mycoplasma. [ ] [ ] [ ] different agents are often isolated from the nasal cavity, aggravating the clinical presentation. in such cases, the authors recommend performing an initial sinus flush with sterile saline, in order to obtain samples for culture and polymerase chain reaction (pcr) identification. sinus flush should be performed under general anesthesia with an uncuffed endotracheal tube placed, to avoid fluid going into the respiratory tract. once samples have been obtained, f , enrofloxacin (not to be used in the usa and only to be used on label in egg laying chickens and turkeys in europe), amikacin or gentamicin flush could be performed, and repeat it as necessary. if purulent material is present in the sinus, the author recommends surgical access to remove as much purulent material as possible, because antibiosis alone is unlikely to resolve it. mycoplasmosis is the most common respiratory condition seen in backyard poultry. , poultry is mainly affected by species of mycoplasma: mycoplasma gallisepticum, mycoplasma synoviae, mycoplasma meleagridis, and mycoplasma iowae. m gallisepticum is often the pathogen causing respiratory signs, although m synoviae can also cause respiratory signs (sneezing, foamy nasal and ocular discharge, conjunctivitis, sinusitis, and/or purulent aural discharge). mycoplasma spp can be latent within the flock and often causes disease when there is immunosuppression, stressful factors, and concomitant infections. tylosin is the recommended treatment because it is licensed (at least in the united kingdom/eu and united states). antibiotic therapy does not eliminate mycoplasma, but it can resolve clinical signs; equally important is to assess and treat any other stressful factors (ammonia and dust sabater gonzá lez & calvo carrasco levels, densities, overall hygiene, food and water quality). however, if symptoms persist despite treatment, euthanasia should be considered for the well-being of the flock. anseriformes are an important reservoir for avian influenza, often being asymptomatic carriers even from some of the high-pathogenic strains. avian influenza or fowl plague is a rare disease in wild waterfowl, with few records in the wild. despite being uncommon, in particular in mixed flocks or flocks exposed to wild waterfowl, avian influenza should be considered and investigated in cases with compatible clinical signs, such as mucopurulent or caseous sinusitis. important management factors to control this disease, such as hygiene and density levels, should be assessed in captive waterfowl showing clinical signs. newcastle disease or avian paramyxovirus can also present with signs of upper respiratory disease, such as conjunctivitis or tracheitis, and it can also cause central nervous system and gastrointestinal signs. , zoonosis can also occur, although this only causes mild conjunctivitis in humans. it is a relevant disease given the high economic losses it can produce in the commercial poultry industry, because there is no effective treatment. vaccinations are available to reduce the likelihood of outbreaks. vaccination against newcastle disease is not currently allowed in the uk, but seems to be standard practice in the us. infectious laryngotracheitis (ilt) is caused by a herpesvirus, as well as marek disease. ilt can affects chickens (mainly meat breeds) as well as pheasants, and is similar in presentation to other respiratory diseases. it is characterized by the formation of a diphtheritic membrane in the trachea that can cause obstruction; animals can present gasping. vaccination can be attempted in an outbreak to reduce morbidity and mortality. early vaccination prevents clinical manifestation, but not latent infection. modified live vaccines are available in the uk, eu, and us. aspergillosis is a common condition affecting waterfowl, although it can also affect gallinaceous birds, such as chickens. as in other avian species, aspergillus fumigatus is the main isolated pathogen, although others species of the genus aspergillus can also cause disease. , in chickens, despite most healthy birds coping with a moderate exposure to the aspergillus conidia, infection may occur in immunocompromised birds or when exposed to an overwhelming quantity of spores. common sources of aspergillus are contaminated food and moldy substrates. clinical signs might include dyspnea, but it can present as lethargy, anorexia, and significant weight loss. diagnosis and treatment present similar challenges to those faced in other avian species. treatment is based in antifungal therapy, often an azole drug, together with supportive care. infectious bronchitis is caused by a highly infectious coronavirus and is characterized by having main presentations depending on the age of the infected animals; in young chicks, respiratory disease is the predominant manifestation, whereas salpingitis and the subsequent decrease in egg production is most commonly seen in older laying hens. soft, irregular, or rough-shelled eggs are often seen. in certain animals the lesions caused might impair normal laying for the rest of the animal's life, or even cause secondary problems, such as egg coelomitis. c psittaci is a well-known pathogen among avian practitioners worldwide, not only relevant for its high prevalence but also for its zoonotic potential. more than a species have been shown to be infected, including galliformes. because of its very wide infection range, many different species can act as a reservoir, such as pigeons and waterfowl. a recent study in pigeons showed a prevalence of % in adult animals, which was twice as high in juvenile birds. outbreaks in fowl occur only occasionally. in poultry, infection is often systemic, and occasionally fatal. clinical signs, incubation periods, morbidity, and mortality vary widely depending on the virulence of the strain infecting the flock. common clinical signs observed with chlamydiosis are sinusitis, rhinitis, diarrhea, and weakness. postmortem findings in affected birds include splenomegaly, hepatomegaly, airsacculitis, pericarditis, and peritonitis. , in turkeys, the disease pattern differs from other species and tends to present as an explosive outbreak. clinical signs can be aggravated by concurrent infections, such as salmonella or pasteurella. ideally, a combination of serology and pcr identification is used to diagnosis chlamydia. however, after adequate therapy, there is no currently available test to ascertain whether affected birds are no longer carriers; therefore, treatment should be carefully considered, because of its zoonotic risk, especially in collections open to the public. chlortetracycline ( ppm; ie, . g/kg food daily for days) has been recommended in turkeys, although doxycycline ( mg/kg po twice a day or ppm in food daily for days; or - mg/kg im weekly on occasions) is also used in outbreaks to reduce mortality in turkeys as well as in other species. , avian tuberculosis can present as a respiratory emergency when lesions are localized in the pharynx or trachea. certain parasites can also cause respiratory disease in fowl, such as syngamus trachea (commonly known as gapeworm), duck leeches (theromyzon tessulatum), streptocariasis, (streptocara spp), and air sac mites (cytodites nudus). [ ] [ ] [ ] if upper airways are affected, animals present gasping for air or open-mouth breathing, coughing, or retching. diagnosis is based on identification of the parasites (adult forms, ova, or larvae). riemerella anatipestifer can cause a peracute infection in ducklings, which might present with upper respiratory clinical signs, such as dyspnea, or nasal or ocular discharge. this condition evolves quickly and can cause sudden death. samples should be obtained for culture and sensitivity, to allow adequate antibiotic therapy. neurologic disease is common in fowl. clinicians must be vigilant and aware of the reportable diseases that can present with neurologic signs, such as newcastle disease, avian influenza, and chlamydiosis. marek disease is common in unvaccinated chickens, and heavy metal poisoning should always be considered in waterfowl. other possible causes are trauma, nutritional deficiencies, central nervous system ischemia, vascular insult, and other intoxications (fig. ) . marek disease is caused by gallid herpesvirus , and has recently been described as the most common disease diagnosed in backyard poultry. the disease is characterized by the presence of t-cell lymphoma as well as mononuclear infiltration of nerves, organs, reproductive tract, internal viscera, iris, muscle, and skin. the mononuclear infiltration of peripheral nerves, in particular the sciatic nerve, causes paralysis. however, there is no treatment of affected birds and euthanasia should be considered in unvaccinated suspicious cases. early vaccination (within the first days of hatching) does not stop infection (the virus is considered ubiquitous worldwide) but achieves more than % protection under commercial conditions. lead poisoning is thought to be one of the most significant causes of neurologic disease in waterfowl. a recent report estimated between , and , (approximately . %- . % of the wintering population) wildfowl deaths each winter are caused by lead poisoning. that number represents a quarter of all recorded deaths regarding migratory swans. not only waterfowl are affected by this, because other terrestrial game birds and fowl may ingest lead pellets that they mistake for grit or food; lead pellets may be buried in mud, in areas where fishing or hunting has previously taken place. animals can experience chronic intoxication when ingesting small numbers of lead pellets intermittently, but can also present acutely and in the form of an outbreak when reduced water levels or other circumstances expose lead that was previously unavailable. in the united kingdom, the sale and use by fishermen of lead leger weights and split shot weighing less than g has been banned since . since then, the incidence of lead poisoning has reduced significantly. however, the environmental contamination will still have an effect for many years. characteristic clinical signs of lead toxicity include weight loss, weakness, and green faces; weakness of the neck muscles causes a typical posture with the head resting on the bird's dorsum. whole-body radiographs might reveal the presence of metallic objects in recent cases; however, the grinding action and ph of the ventriculus dissolves the lead pellets within a few days. other common findings in chronic cases on radiographs are dilatation and impaction of the proventriculus. a blood sample should always be tested for lead levels to achieve a definitive diagnosis (normal, < . ppm; diagnostic, . - . ppm; severe, > . ppm). moderate anemia ( %- % hematocrit) can be observed. delta amino levulinic acid dehydrase activity has been suggested as a more sensitive diagnostic indicator for lead intoxication. early treatment of lead toxicosis should include stopping any further lead absorption; lead particles within the gastrointestinal tract can be removed by lavage under general anesthesia with warm fluid via a gastric tube. some investigators recommend repeating gastric lavage within to hours if lead particles are still present in postlavage radiographs, because fragments of lead can be trapped in crevices in the koilin. those particles precipitate when muscle activity has restarted. chelation therapy should be started in all affected animals. sodium calcium edetate ( - mg/kg intramuscularly every hours for days, with a -day break at day ) is the recommended treatment of lead and zinc toxicosis. penicillamine can be used as an alternative if sodium calcium edetate (nacaedta) is not available, or at the same time in severe cases. zinc toxicosis is less common in animals housed outdoors, and is similar in diagnosis and treatment to lead intoxication. botulism occurs when animals are kept in water with anaerobic conditions, particularly in warm, dry periods. clostridium botulinum overgrows and produces toxin type c, causing flaccid paralysis. other clinical signs are similar to other heavy metal poisoning, such as weakness. a good anamnesis and water analysis allows a presumptive diagnosis. other intoxications are common in fowl, such as coccidiostats in waterfowl (found in chicken-formulated commercial diets) or pesticides (dimetridazole and organophosphorus pesticides). , diarrhea diarrhea can have many different causes; after physical examination, clinicians should perform a direct observation, flotation, and diff-quik examination of a fresh fecal sample. samples should also be taken for viral identification. duck plague or duck viral enteritis is caused by a herpesvirus, and can cause significant losses in waterfowl collections. presentation can be peracute, including sudden death without previous obvious signs. other described clinical signs are cloacal lethargy, diarrhea, hemorrhage, prolapse of the penis, photophobia, ataxia, and tremors. , outbreaks are often seasonal (may to june in the united kingdom). it can cause morbidities between % and % in unvaccinated collections. in affected animals, the prognosis is very poor with no effective treatment. annual vaccination is recommended in endemic areas. avian or fowl cholera, caused by pasteurella multocida, is the most common pasteurellosis in poultry. chickens, duck, geese, and turkeys can be affected. outbreaks in turkeys can cause up to % mortality. clinical signs include oral and nasal discharge, dyspnea, diarrhea, and sudden death. this condition seems to be less frequent in the united kingdom than in north america, where annual outbreaks can cause significant mortalities. impaction of the crop, proventriculus, or gizzard has occasionally been reported in poultry and waterfowl. affected birds commonly present showing lethargy, emaciation, and esophageal or crop distension. despite the crop/esophagus, proventriculus, and/or gizzard being full of a solid mass of interwoven fibrous material, the intestines of birds with this condition are frequently empty. poultry have been known to ingest poorly digestible items (eg, grass, newspaper, sawdust shavings/ wood chips, and feathers) out of curiosity or as a response to stress, causing crop impaction. crop impaction is most frequently seen in spring, when chickens ingest long stems of grass that get impacted in the crop. captive waterfowl, especially geese, suddenly exposed to new environments may ingest nondigestible items like newspaper or plant products, like grasses. ingestion of grains that have low moisture content with concurrent exposure to water can lead to grain swelling and result in impaction of the esophagus. gizzard impaction can cause high mortality during the first weeks of life in turkey flocks. although rehydration of the impaction, gentle massage or flushing (only for crop or esophageal impactions), and liquid paraffin may help to resolve the impaction in early cases, surgical intervention might be necessary (fig. ) . these conditions occur sporadically in domestic fowl. intussusception occurs most frequently in the intestine, but it has also been reported in the proventriculus. in young birds volvulus of the small intestine may be caused by twisting around the yolk sac. intussusception and volvulus have been reported in chickens secondary to enteritis or abnormal peristalsis caused by nematode or coccidial infection. intestinal torsion has also been associated with pedunculated neoplastic stalks. clinical signs are anorexia and progressive weight loss, which may lead to death within a few days. diagnosis may be achieved by ultrasonography, radiography, or endoscopy. if an early diagnosis is made, resection of the affected intestine can be performed. coelomitis is an occasional cause of morbidity and mortality in waterfowl and a common condition in chickens, particularly seen in laying or ex-battery hens. infection of the coelom can become established following infection of the respiratory system, penetrating injuries, neoplasia, heavy parasitism, or reproductive diseases. in chickens, e coli is often responsible of the oviduct infections. salmonella pullorum or infectious bronchitis can also cause lesions in the reproductive tract. diagnosis may be achieved by aspiration of coelomic fluid (ultrasonographically guided if possible). powerful antibiotic therapy is recommended (fig. ) . egg coelomitis may occur because of an ectopic ovulation, when the follicle or yolk misses the infundibulum, or when the follicle in the oviduct moves back in a retroperistaltic manner. this condition can be caused by an underlying disorder or can occur fig. . ultrasonography in a chicken with distended coelom. critical care of commonly kept fowl after a stressful event while the egg was forming within the oviduct. in both situations the yolk reaches the coelomic cavity causing a coelomitis. secondary bacterial colonization can occur. often this occurs because of pathologic changes in the oviduct, with either infectious or neoplastic causes, or because of oviductal damage in battery hens. a recent study performed in backyard poultry in the united states revealed that the most common condition diagnosed was marek disease. in that study, the most common finding observed in gross postmortem was the presence of tumors affecting internal organs or carcinomatosis, which can affect the ovaries. equally, non-viralinduced reproductive neoplasia, despite having significantly different findings in the different institutions involved in the study, is also considered common. salpingitis was one of the most common presentations in of the institutions, with . % of the presented cases. initial treatment can include coelomoentesis when dyspnea is observed; this technique, although not free of risk, also helps in achieving a diagnosis by analyzing the fluid drained. fluid therapy, antibiosis, analgesia, and assisted feeding are required at initial stabilization. salpingohysterectomy is likely to be required for long-term treatment because this condition is likely to reoccur. stress, age, obesity, and poor nutrition can contribute to the presentation of this condition, and good layers seem to have a higher predisposition. this condition can be seen in animals with egg binding. often animals had experienced trauma of exposed tissue from the other animals in the flock. medical management is often unsuccessful and salpingohysterectomy is the preferred treatment option according to the investigators. alternatively, a gonadotrophin-releasing hormone agonist implant (deslorelin acetate) can be used, once the prolapsed tissue has been repositioned and infection and inflammation controlled. repeated applications are required longterm, and in certain animals the duration of the implant seems to decrease after repeated applications. this condition may result from inflammation of the oviduct, partial paralysis of the muscles of the oviduct, or production of an egg so large that it is physically impossible for it to be laid. young pullets laying an unusually large egg are most prone to the problem. as in other avian species, this condition is often linked to calcium imbalance, caused by a combination of dietary deficiencies, stress, and other husbandry-related problems. treatment includes fluid therapy, calcium, and oxytocin administration. if initial medical management is unsuccessful, ovocentesis (either directly into the egg shell or via the coelomic wall) should be the next step. the egg should not be manually broken or pulled, because iatrogenic damage to the oviduct may occur. if the shell of the egg is not eliminated within hours, salpingohysterectomy is indicated because the remnants of the shell might adhere to the oviduct, inevitably causing further complications in future oviposition. phallus prolapse is occasionally seen in anseriformes associated with mechanical damage, infection (ie, cryptosporidum spp., mycoplasma spp, neisseria spp.), hypersexsuality or immunosuppression. frostbite and bacterial infection may occur as a sequela of phallus prolapse. treatment may include analgesics, local and/or systemic antibiotherapy, and decongestive and lubrifying local therapies which allow reposition of prolapsed healthy tissues. severe cases may require amputation of the phallus. euthanasia might be required in cases with a poor prognosis and when certain infectious diseases have been confirmed. euthanasia should always be performed in a humane manner. the authors' preferred method is intravenous administration of barbiturates, but other methods can be used. fowl are stoic patients that commonly mask signs of illness in the early stages of disease and are not commonly presented as emergencies until the acute or chronic condition is severe. an understanding of intraspecific and interspecific anatomic and physiologic variations is crucial to the successful management of critically ill fowl. stabilization of the patient should be prioritized over diagnostic procedures. clinicians treating fowl should be aware of infectious and noninfectious conditions causing emergencies in fowl. a classification of the living birds of the world base on dna-ddna hybridization studies a phylogenetic supertree of the fowls (galloansera, aves) bsava manual of canine and feline emergency and critical care zoonoses, public health, and the backyard poultry flock free-living waterfowl and shorebirds emergency care of raptors backyard poultry medicine and surgery. a guide for veterinary practitioners the maximum capacities of the lungs and air sacs of gallus domesticus evaluation of three heat sources for their ability to maintain core body temperature in the anesthetized avian patient updates in anesthesia and monitoring avian clinical biochemistry emergency and critical care of pet birds evaluation of pulse oximetry as a monitoring method in avian anesthesia arterial blood pressure monitoring in anesthetized animals determination of indirect blood pressure in the companion bird arterial catheterization, interpretation and treatment of arterial blood pressures and blood gases in birds principles of shock and fluid therapy in special species intraosseous cannulation and drug administration for induction of anesthesia in chickens physiological studies on the electrocardiogram of the chicken i: bipolar leads physiological studies on the electrocardiogram of the chicken iii: on the normal values of the electrocardiogram of laying hens the electrocardiogram of birds (chicken, duck, pigeon) electrocardiographic observation on spontaneously occurring arrhythmias in chickens comparative electrocardiographical studies on the wave form of qrs complex in vertebrates the electrocardiogram of the chicken cardiac muscle mass distribution in the domestic turkey and relationship to electrocardiogram the electrocardiogram of the turkey scalar electrocardiographic measurements in unrestrained young japanese quail hyperpotassemia and electrocardiographic changes in the duck during prolonged diving normal electrocardiogram patterns and values in muscovy ducks (cairina moschata) analysis of electrocardiographic parameters in helmeted 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the use of emeraid exotic carnivore diet improves postsurgical recovery and survival of long-tailed ducks comparison of fluid types for resuscitation after acute blood loss in mallard ducks (anas platyrhynchos) bsava manual of raptors, pigeons and waterfowl. cheltenham (united kingdom): british small animal veterinary association distraction osteogenesis correction of mandibular ramis fracture malunion in a juvenile mute swan (cygnus olor) bsava manual of farm pets. gloucester (united kingdom): british small animal veterinary association what is your diagnosis? clinical avian medicine volume ii bandaging, endoscopy, and surgery in the emergency avian patient bsava manual of farm pets. gloucester (united kingdom): british small animal veterinary association developmental, metabolic, and other noninfectious disorders postmortem survey of disease conditions in backyard poultry prevalence and differentiation of diseases in maryland backyard flocks avian medicine: principles and application. lake worth (fl): wingers publishing bsava manual of farm pets. gloucester (united kingdom): british small animal veterinary association bsava manual of raptors, pigeons, and waterfowl. cheltenham (united kingdom): british small animal veterinary association aspergillus infections in birds: a review outbreak of psittacosis in a group of women exposed to chlamydia psittaci-infected chickens avian chlamydophilosis (chlamydiosis/psittacosis/ornithosis) chlamydia psittaci in ducks: a hidden health risk for poultry workers epidemiological investigations on the possible risk of distribution of zoonotic bacteria through apparently healthy homing pigeons experimental ornithosis in turkeys respiratory and pericardial lesions in turkeys infected with avian or mammalian strains of chlamydia psittaci chlamydiosis (psittacosis, ornithosis) use of ovotransferrin as an antimicrobial in turkeys naturally infected with chlamydia psittaci, avian metapneumovirus and ornithobacterium rhinotracheale leech parasitism of waterfowl in north america laryngeal streptocariasis causing death from asphyxiation in ducks cytodites nudus infestation of chickens dose response study of enrofloxacin against riemerella anatipestifer septicaemia in muscovy and pekin ducklings marek's disease protective synergism among marek's disease vaccine viruses report of the nature conservancy council working group poisoning of birds and other wildlife from ammunition-derived lead in the uk bsava manual of raptors, pigeons and waterfowl. cheltenham (united kingdom): british small animal veterinary association progress on lead-free shot in the uk pathological study of lead poisoning in whooper swans (cygnus cygnus) in japan lead poisoning in trumpeter swans toxicities in waterfowl treatment of lead poisoning in swans emergency care and managing toxicoses in the exotic animal patient triage of botulism in wild birds toxicity of dimetridazole in waterfowl identification of differentially expressed proteins related to organophosphorus-induced delayed neuropathy in the brains of hens duck, geese, swans, and screamers: infectious diseases the comprehensive diagnosis and prevention of duck plague in northwest shandong province of china fowl cholera in turkeys persistence of pasteurella multocida in wetlands following avian cholera outbreaks backyard poultry medicine and surgery. a veterinary guide for veterinary practitioners bsava manual of raptors, pigeons, and waterfowl. cheltenham (united kingdom): british small animal veterinary association key: cord- -lmf h oc authors: light, r. bruce title: plagues in the icu: a brief history of community-acquired epidemic and endemic transmissible infections leading to intensive care admission date: - - journal: critical care clinics doi: . /j.ccc. . . sha: doc_id: cord_uid: lmf h oc the ability to diagnose and treat infectious diseases and handle infectious disease outbreaks continues to improve. for the most part, the major plagues of antiquity remain historical footnotes, yet, despite many advances, there is clear evidence that major pandemic illness is always just one outbreak away. in addition to the hiv pandemic, the smaller epidemic outbreaks of legionnaire's disease, hantavirus pulmonary syndrome, and severe acute respiratory syndrome, among many others, points out the potential risk associated with a lack of preplanning and preparedness. although pandemic influenza is at the top of the list when discussing possible future major infectious disease outbreaks, the truth is that the identity of the next major pandemic pathogen cannot be predicted with any accuracy. we can only hope that general preparedness and the lessons learned from previous outbreaks suffice. intensive care, the epitome of the application of modern technology to medicine, arguably began as a response to the increasing numbers of older children and young adults developing paralytic polio during the early s. severe paralytic polio itself turns out to have been among the products of increasing modernity in the western world. clinical recognition of the syndromes associated with the polio virus date back about years. the fact that the disease was caused by a transmissible viral particle was demonstrated by . until early in the twentieth century the virus was readily transmitted throughout the population almost continuously by personal contact and by the fecal-oral route via water. the result was that most people's first exposure to the virus occurred in infancy. at this age most infections resulted in a clinically unapparent infection, in part due to partial protection by maternal antibody, after which lifelong immunity was established. only a few suffered a paralytic episode with the infection, at the time termed ''infantile paralysis.'' as hygienic standards of the advanced economy nations rose throughout the first half of the century, early childhood exposure to the virus declined. an increasing fraction of the population had their first exposure in late childhood or during young adulthood. in these age groups the likelihood that the infection will cause a paralytic syndrome is greatly increased, so both the incidence of clinically recognized infection and that of paralysis rose. by the early s clinically recognized cases had reached - annually for every , people in the united states, making it a major public health concern and a source of a great deal of fear in the general populace. more than % of polio virus infections are asymptomatic. however, at the more severe end of the clinical spectrum are the paralytic syndromes which range from paralysis of one or more limbs (''spinal polio'') to syndromes with respiratory muscle or bulbar paralysis (''respiratory polio'' or ''bulbospinal polio'') with loss of respiratory or upper airway muscle function or both. these more severe outcomes rise in incidence from about . % in infants to more than % in older children and adults. in the early part of the century development of polio with bulbar involvement was associated with a death rate of greater than %, generally from respiratory failure. use of a mechanical respirator to try to avert death was first attempted at the children's hospital in boston in , using an ''iron lung.'' the machine was basically a sealed box with a hole at one end for the patient's head to protrude, attached to two vacuum cleaners. the motors were then cycled to alternately create vacuum inside the box, expanding the patient's chest and causing the patient to inhale through the mouth (outside the box), then allowing air back into the box to permit exhalation. the design was further improved in , and the machines came into increasingly broad use throughout north america and europe during the severe outbreaks of the s. adoption of this therapy resulted in a significant reduction in mortality during these years. iron lungs, however, were cumbersome, difficult to use when trying to provide nursing care, and expensive. a more cost-effective and user-friendly approach to providing respiratory support was clearly needed. this finally came by bringing the positive pressure ventilation (ppv) concept out of the operating room. ppv was first used for respiratory support for polio victims at blegdam hospital in copenhagen, denmark, an innovation attributable to danish anesthetist bjorn ibsen. during this large outbreak in , some medical students were put to work hand-ventilating dozens of patients through tracheostomies until the worst of the paralytic phase of the illness had passed, often several weeks. the concept quickly spread elsewhere and was widely adopted, yielding substantial reductions in mortality. for reasons of efficiency and convenience, patients needing respiratory support were often grouped in a single location where the necessary expertise and equipment were available. the introduction of ppv into a defined area of the hospital used to support respiratory failure was the genesis of the modern icu and represents a signal event in the development of the field of critical care medicine. the introduction of effective vaccines, the salk (inactivated) vaccine in and later the sabin (live attenuated oral) vaccine in , immediately and dramatically reduced the incidence of polio to less than one per , population by the early s and the incidence continued to fall thereafter. the last case of wild strain polio in north america was reported in , and since that time the only cases of paralytic polio have been rare instances of disease due to variants of the live oral vaccine strain. polio does, however, still contribute to illnesses that may require intensive care in the form of the ''post-polio syndrome.'' this occurs in patients who survived paralytic polio decades ago and who, over the years, develop a gradual decline in function in the originally affected nerves and muscles which can years later once again threaten them with disability and, in some cases, respiratory failure requiring intensive care. in july , american legion members attending a convention at a hotel in philadelphia suddenly began falling ill with an acute febrile illness with pneumonia, often associated with extrapulmonary symptoms such as myalgia or diarrhea. many developed acute respiratory failure requiring mechanical ventilatory support in icu. over were affected and died, an alarming mortality rate, especially since physicians caring for the patients had no idea what was causing the illness. conventional microbiologic investigations yielded no convincing pathogens despite intensive investigation for the usual bacteria and viruses and other potential pathogens. epidemiologic and various biologic investigations were quickly implemented by local health authorities and by the centers for disease control and prevention (cdc). these showed that the disease was likely airborne and that it occurred more frequently in older individuals who had underlying lung disease, smoked, or were relatively immunocompromised. analysis of the likelihood of death revealed that those who received tetracyclines or macrolide antibiotics were more likely to survive than those who received only betalactams. however, no causal agent was uncovered, though many potential causes were excluded-most known bacteria and viruses, many biologic toxins and many environmental agents such as toxic chemicals and metals. in , joseph mcdade and charles shepard of the cdc reported the isolation of a novel fastidious gram-negative bacillus from the available clinical specimens. they named it legionella pneumophila. this discovery was quickly followed by an explosion of knowledge about the organism and its ecology, antimicrobial susceptibility, and of other bacteria within the genus. over time, demonstration that it was a widely occurring colonizer of brackish water, particularly in air conditioners, cooling towers, and water heaters and pipes, led to the implication of these reservoirs in several hundred outbreaks of the disease worldwide, especially in hospitals and other public health institutions, and hotels. thus began widespread development of regulations and guidelines for limiting the degree of colonization of these water sources by legionella, resulting in a reduction in the size, number, and scope of subsequent outbreaks. since the initial description of the disease, legionella has, of course, been shown to be one of the major causes of community-acquired pneumonia (cap), particularly in the more severe subset requiring icu care; this fact underlies the major lesson from plagues in the icu the outbreak-the nearly universal recommendation for inclusion of antimicrobial therapy for legionell spp in any treatment regimen for severe cap without another obvious cause. although we now know that no amount of continuing effort can completely eliminate this organism from our environment, and that we will continue to see endemic cases, we also know that clusters of cases should trigger an investigation into finding the point source of the outbreak, a situation that continues to occur several times a year somewhere in the world. in addition, the philadelphia outbreak that defined ''legionnaire's disease'' was the first in the modern icu era to demonstrate that major unknown infectious disease syndromes of severe consequence still exist, presaging the new infectious disease syndromes to be discovered in the years that followed. in the late s, emergency rooms and icus throughout north america began to see an increasing number of young menstruating women presenting with a previously little-known syndrome characterized by sudden onset of a high fever, often associated with vomiting and diarrhea, quickly followed by severe hypotension. early in the course most patients developed a diffuse macular rash, often with mucous membrane inflammation, with subsequent desquamation during convalescence. patients frequently required massive fluid resuscitation because of systemic capillary leak, as well as vasopressor support, mechanical ventilation for adult respiratory distress syndrome, and even renal replacement therapy for acute renal failure, complicating the shock episode. one of the early clusters of observed cases was reported in , and the term ''toxic shock syndrome'' was coined based on the isolation of an exotoxin-producing staphylococcus aureus from mucosal surfaces or the site of a minor infection in the absence of bacteremia. as the case numbers rapidly increased case definitions for the syndrome were formulated and epidemiologic studies mounted. by , over cases had been formally reported to the cdc. the case fatality rate was reported to be as high as % in the more severe cases included in the earliest reports, falling to about %- % as recognition of the syndrome improved. by this time there were clear epidemiologic links between the syndrome and menstrual use of high-absorbency tampons which were often left in place longer than less absorbent products. colonization of the tampon with staphylococcus aureus was also implicated, consistent with the postulated toxin-mediated disease mechanism. within months of these revelations the main manufacturer of the implicated tampons withdrew them from the market and women began changing tampons with greater frequency or stopped using them at the urging of public health authorities. the incidence of the syndrome immediately began to fall and within a few years, with the changing of use patterns of tampons and changes in their manufacture, toxic shock syndrome disappeared, for the most part, but not entirely, from the icu. even at the height of tss incidence in the united states, about % of the cases reported were nonmenstrual and % were in males. subsequent development of the knowledge that the clinical syndrome was due to strains of staphylococcus aureus that secrete a particular toxin (toxic shock syndrome toxin , or tsst- ), which is both absorbable from mucosal surfaces and capable of producing a profound shock syndrome even in the absence of significant invasive infection, soon led to the more widespread recognition of the nonmenstrual toxic shock syndrome. this syndrome, which was almost certainly extant before but little-recognized, was perhaps the main lesson from the outbreak: even trivial staphylococcal skin or wound infections light or mucosal surface colonization in the presence of a foreign body such as a nasal pack for nosebleed can lead to a severe shock syndrome if the organism is present and produces this toxin or one of several related ones. the recognition of the staphylococcal toxic shock syndrome also led to increasing understanding of the role of ''superantigens'' as a mechanism of disease-bacterial toxins capable of activating a large fraction (up to %) of the total t-lymphocyte population. such superantigens have since been implicated in a number of other disease syndromes, among them the streptococcal toxic shock syndrome (see below). over the past two decades, the incidence of menstrual and nonmenstrual staphylococcal tss has been about one per , population in most areas. busy icus will, therefore, continue to see occasional cases. however, there is some recent evidence that case numbers may be on the rise again in at least some areas, possibly because of a resurgence in the prevalence of toxin-producing strains in the community. in , physicians working in infectious diseases and critical care medicine thought they knew all about pneumocystosis. the organism, then thought to be a protozoon, had been first described in by carlos chagas in brazil, and since then had been clearly implicated as a cause of interstitial pneumonia in debilitated and malnourished children (in the aftermath of world war ii) and, later, a cause of severe opportunistic pneumonia in immunocompromised patients, usually those being treated with highdose corticosteroids for connective tissue diseases or lymphoreticular neoplasms. in these patients it caused an impressively aggressive bilateral pneumonia leading to acute respiratory failure. this pneumonia was notoriously difficult to definitively diagnose, requiring bronchoscopy or open lung biopsy to demonstrate the small numbers of characteristic pneumocystis organisms on special silver stains of clinical specimens. the mainstay of treatment at that time was pentamidine, generally given intramuscularly, giving way to trimethoprim/sulfamethoxazole after the publication in of a randomized clinical trial showing that it was at least as effective and generally better tolerated. in the early s, a new form of the infection began to be seen with regularity. young men began to present to hospital with a rather more indolent diffuse bilateral pneumonia that nevertheless went on to cause respiratory failure and which, when investigated, proved to be due to pneumocystis. the course of the disease was quite different from what physicians had been used to up to then. it began more gradually, progressed at a slower pace and was associated with a much lesser systemic inflammatory response. microscopy of respiratory specimens revealed exponentially more organisms than previously seen, such that many patients could be diagnosed from sputum specimens rather than bronchoscopy, and biopsy was virtually never needed. nobody had any idea why this was happening, although it quickly became apparent that the underlying cause of the infection was a new form of severe deficiency of cell-mediated immunity. epidemiologic investigations were soon underway. patterns began to emerge. many of the young men were haitian or had been to haiti. many were homosexual, bisexual, or had worked in the sex trade; others had abused intravenous drugs. the many fewer women with the disease had similar exposures. theories proliferated. was it immunologic exhaustion from exposure to too many microbial stresses? toxins? drugs used in the sex trade? multiple and synergistic viral infections? through the early s, the case load grew. icus throughout north america and, later, in europe and elsewhere, saw increasing numbers of young people, mainly men, plagues in the icu with severe respiratory failure due to pneumocystis pneumonia. by they were being called patients with acquired immunodeficiency syndrome (aids) on the basis of demonstration of low numbers of cd lymphocytes in the blood, but the cause remained unclear. then, in , montagnier and barre-sinoussi at the pasteur institute in france isolated a viral pathogen that they named lymphadenopathy-associated virus (lav). at the national institutes of health in the united states, gallo demonstrated that the virus (which he referred to as human t-cell leukemia virus iii, based on an unproven relationship to other viruses he had previously discovered) definitively caused aids. the virus, now called human immunodeficiency virus (hiv)- was isolated and described and the first diagnostic kits devised, resolving the mystery of causation. montagnier and barre-sinoussi would eventually win the nobel prize in medicine in for their contribution. during the s, patients continued to present with severe pneumonia requiring respiratory support and intensive antimicrobial therapy, often with less than satisfactory results. as knowledge progressed, hiv-associated pneumocystis infection in the icu changed its face several times over the years. at the beginning of the epidemic, most patients presenting for care with hiv/aids and pneumocystosis were severely ill with diffuse pneumonia and hypoxemic respiratory failure and many died, %- % in most centers, prompting widespread debate about whether such patients should even be admitted to icu for mechanical ventilatory support. however, as experience with the disease developed it became clear that an early and aggressive approach could improve prognosis. it was found that in the aids population even minor respiratory symptoms with few or no abnormalities on chest radiograph could be due to pneumocystis infection in the earlier stages, and that even modest degrees of arterial oxygen desaturation signaled impending respiratory failure. earlier bronchoscopy for diagnosis followed by prompt antimicrobial therapy, with pentamidine predominantly in the early s and later primarily with trimethoprim/sulfamethoxazole, led to overall mortality rates falling to the %- % range by the middle of the decade. the advent of systemic corticosteroids therapy for early respiratory failure in aidsassociated pneumocystosis was then shown to further reduce the numbers of patients progressing to advanced respiratory failure, leading to reductions in the numbers of cases needing icu admission and further reducing overall mortality rates to the %- % range. but for patients requiring icu care mortality rates were as high as before the use of steroids and often higher, likely related to the fact that most patients developing respiratory failure had already failed to improve or had progressed despite intensive antimicrobial and corticosteroid therapy. along with these developments in management of the disease, progress was being made on hiv itself. following identification of the virus in , there soon followed increasingly reliable diagnostic tests for the infection, leading to earlier identification of cases and monitoring of cd lymphocyte counts. by the early s, studies supporting widespread use of chemoprophylaxis against pneumocystis in all patients with cd counts < /mm were available and became standard public health agency recommendations. pneumocystosis, which in the s and s was one of the principal causes of hypoxemic respiratory failure in many icus in north america and western europe, began to decline rapidly in incidence, becoming relatively uncommon even before the widespread adoption of highly active antiretroviral therapy, which has, since the mid- s, caused the disease to all but disappear from our icus. although many lessons can be drawn from the battle against aids-related pneumoncystis pneumonia during the s and s, for icu and infectious diseases practitioners one of the main ones comes from the sad fact that once patients had developed full-blown hypoxemic respiratory failure even the best intensive care could only deliver % survival rates. the really large gains in survival came not from better icu technology but from pre-empting the disease on multiple fronts, including earlier diagnosis of hiv infection, early diagnosis and antimicrobial treatment of pneumonia, steroid treatment of early respiratory failure, antimicrobial prophylactic regimens and, later, effective antiretroviral therapy. until , the only members of the bunyaviridae family of viruses known to cause disease in north america were members of the genus bunyavirus, all causing mosquito-borne viral encephalitis, mainly in children (california encephalitis). other members of the family were known to cause serious febrile illnesses, encephalitides and hemorrhagic-fevers in africa and asia (rift valley fever, crimean-congo hemorrhagic fever, hemorrhagic fever with renal syndrome). however, in the spring of wetter-than-usual conditions in the american southwest led to increased availability of food for deer mice, leading to a population explosion and increasing movement of rodents into human-occupied spaces, increasing the chance that humans might be exposed to the rodents and their excreta. in rapid succession, several previously healthy young people, mainly navajos, presented to health care institutions in the four corners area of the southwestern united states, all with fulminant illnesses leading to shock and acute respiratory failure requiring icu care. by early june that year, cases had been identified and had died. in most cases the illness had started with fever and widespread myalgia, soon followed by cough, then by cardiovascular collapse due to a severe systemic capillary leak syndrome and by acute respiratory failure due to low-pressure pulmonary edema. in some cases the time from onset to icu or death was as little as hours, in others a few days. remarkably, although no pathogen was initially identified from blood or tissues, in less than a month after the first report of a possible outbreak, serologic testing had demonstrated antibody cross-reactivity with a range of known pathogens of the hantavirus group, suggesting that the disease was due to a previously unknown member of this group. shortly thereafter exposure to deer mice and their excreta was implicated as the likely source of the infection. the mortality rate for the early cases of hantavirus pulmonary syndrome (hps) was extremely high- % in the initially reported group of patients-mostly due to intractable shock and unsupportable hypoxemic respiratory failure due to acute respiratory distress syndrome (ards). however, this improved with clinical experience as it became evident that administration of large amounts of intravenous fluids in the face of profound capillary leak only resulted in much worse generalized and pulmonary edema, with little improvement in the shock state and only worsening of the respiratory failure. management changed to an approach limiting the amount of fluid administered early in the course together with earlier institution of inotropic support, resulting in a much improved survival rate of about %, generally with minimal or no long-term sequelae in survivors. in subsequent years development of increasingly specific serologic and virologic testing has demonstrated that this disease had been present but unrecognized throughout north and south america long before this outbreak, and that there are several related viruses, each associated with a particular rodent, causing endemic disease and the occasional outbreak. by the mid- s, over cases were reported in states, mainly in the southwest, and cases have since been reported plagues in the icu in small numbers in most other states, canada, mexico, and south america, where several outbreaks have occurred. whereas occasional cases continue to be seen in icus in all these areas, no further major outbreaks have yet occurred in the united states or canada, though clearly remaining a threat under the right conditions; the only currently available preventive measure is avoiding rodent contact. steven simpson, md, one of the intensivists at the health sciences center in albuquerque, new mexico, who was closely involved in the initial four corners outbreak, points out that the event highlights several trends in subsequent disease outbreaks in north america. one is the extreme rapidity with which novel pathogens and potential pharmacotherapeutic agents can now be identified. whereas the pathogen in the legionnaire's outbreak took almost a year to identify, researchers identified the hps pathogen and its source in just months. computerized access to data and data analysis along with virtually instantaneous electronic transmission of information plays a central role in this development. the initial hps outbreak has several icu-related lessons to teach us. while the aforementioned treatment strategies effective in a systemic capillary leak syndrome have been absorbed by the critical care community, it appears that one lesson taken to heart by the local icu teams failed to disseminate to the broader icu community. the initial outbreak was accompanied by a marked element of fear and concern among health care workers, including those in the icu, and a significant level of panic in the local community; a combination of this fear, the requirement for rigorous quarantine precautions, and a marked increase in transfers to the icu of any severely ill patients with symptoms remotely compatible with hps resulted in some compromise of icu operations, due to being completely overwhelmed. this might potentially have been avoided by an awareness that for an effective epidemic response, it is essential to include both hospital and icu operations in each locale. the outbreak also reinforces the principle that nearly all old and most new epidemic infectious diseases have their origin in close contact between humans and other species of animal, both wild and domestic, and new kinds and quantities of such contact are likely to cause new, or newly recognized, disease syndromes. streptococcus pyogenes was one of the first bacteria ever to be conclusively linked to human disease (puerperal infection associated with childbirth). however, over the past years the nature of the diseases stemming from it has changed dramatically on several occasions. at the turn of the last century, it was well known as a cause of streptococcal pharyngitis, erysipelas, and wound infections. it also caused severe septicemic illnesses that frequently led to death. osler knew streptococcus pyogenes as a principal cause of thoracic empyema following pneumonia or severe cases of scarlet fever, and also as a major cause of primary bacteremia with sepsis. these more severe manifestations of streptococcal infection became increasingly uncommon as the twentieth century progressed, particularly after the arrival of antibiotics mid-century. notably, osler did not mention streptococcus as a cause of necrotizing fasciitis or being associated with soft tissue necrosis in wound infections. this syndrome was first described by meleney in ; at that time, it was characteristically a slowly evolving gangrenous infection, usually of surgical wounds, which often responded well to debridement and was associated with a mortality rate of only %. for over a generation after the advent of the modern antibiotic era, streptococcus pyogenes was seldom a problem that led to critical illness-soft tissue infections and light the occasional bacteremia were generally very amenable to treatment; extensive surgery or drainage was seldom required, and cases requiring icu support for shock or respiratory failure were rare. beginning in the mid- s, medical practitioners in centers across north america and europe began seeing previously unknown forms of severe streptococcal disease, soon labeled streptococcal necrotizing fasciitis and streptococcal toxic shock syndrome. streptococcal toxic shock syndrome (strep tss) is any infection with streptococcus pyogenes that is associated with a rapidly progressing systemic toxic response characterized by early onset of high fever and myalgia, often with prominent gastrointestinal symptoms, and by rapid progression to hypotension and multiple organ system failure. the illness usually requires icu support for massive fluid resuscitation, vasopressor and inotropic support and mechanical ventilation. although some cases have primary bacteremia, many others have a localized focus of infection, most often in soft tissues, that only becomes clinically apparent after the onset of shock. streptococcal necrotizing fasciitis is often associated with strep tss and, as mentioned, is often only correctly diagnosed after the onset of shock. the most characteristic story is presentation to a physician or an emergency room with abrupt onset of severe pain, often in an extremity with minimal or no evidence of cutaneous injury. at this stage severe systemic toxicity is usually not present and, since examination of the painful site is also at this stage quite unremarkable, patients are frequently sent home with analgesics and reassurance. over the next - hours pain at the site of infection continues to increase, soft tissue swelling and redness appear above the deeper tissues that are undergoing ongoing necrotizing infection, eventually resulting in full-thickness necrosis evidenced by ecchymosis, cutaneous necrosis, and bullae formation. early or later in this course strep tss frequently occurs. when these cases first began to appear, clinicians' approach to both the sepsis and the tissue necrosis was essentially the same as that used for apparently similar syndromes caused by other bacteria. a broad spectrum antimicrobial was started, fluid resuscitation begun and imaging studies ordered to better define the source of infection causing pain or localized swelling. imaging frequently demonstrated only soft tissue swelling consistent with cellulitis, so surgery was often deferred until superficial signs of tissue necrosis became obvious, and then when surgery was done it was often performed using the conventional approach of trying to conserve as much tissue as possible. the result was that treatment was often too little and too late, with mortality rates exceeding % in many reported series. with the realization that treatment, to be successful, must be swift and aggressive, approaches to therapy changed. emergency physicians were increasingly alerted to the fact that severe pain at any body site, even with relatively minimal localized physical findings and particularly if accompanied by signs of systemic inflammation, could represent necrotizing fasciitis. surgeons began to be consulted much earlier, and any localized pain with swelling more often led to diagnostic surgical exploration rather than imaging and waiting. antimicrobial strategies changed. addition of clindamycin to the usual penicillin or other beta-lactam therapy was advocated and widely adopted, based on results from animal models of the syndrome and on pharmacologic and physiologic considerations, including its ability to inhibit bacterial protein (ie, toxin) synthesis, penetrate necrotic tissues, and inhibit inflammatory cytokine synthesis. toxin neutralization using pooled intravenous gamma globulin was also advocated with the support of primarily historical case-control studies. in most centers, implementation of these approaches has led to dramatic reductions in mortality rates to about %- % although, in the absence of any adequate controlled trials, it remains unclear what the relative contribution of each of these measures has been to the improved outcome. unlike several of the other ''plagues'' discussed above, this is one that is still very much with us. the streptococcus pyogenes strains most strongly associated with severe invasive disease (m-protein types and ) have increasingly been supplanting those associated with less severe disease resulting in an endemic sporadic case-rate for severe disease of one to cases/ , population yearly, with intermittent larger-scale community outbreaks, both of which will continue to require vigilance and an aggressive therapeutic stance from the critical care community. the first case of this apparently novel severe viral respiratory infection occurred in guangdong province in southern china in november . the victim, a farmer, died of an undiagnosed ''atypical pneumonia.'' over the ensuing weeks several more cases of severe respiratory syndromes began to appear in the region, also undiagnosed. by the end of november there had been enough such cases to generate considerable alarm among the medical community in china, generating internet communications between institutions which were picked up by international monitoring agencies. this led to a request from the world health organization (who) for information about the outbreak, but no information was forthcoming from chinese authorities. the first official report about the outbreak was made to public health authorities in guangdong in early january , with a later report to the who in february that, in retrospect, did not fully make clear either the nature or the scale of the problem. transmission of the disease within china continued to occur, leading to rapidly increasing numbers of cases in south china, then throughout the country and to the capitol beijing (where one of the largest outbreaks occurred). exposure of chinese travelers and visitors to the country was inevitable, given the scale of the outbreak. one exposed individual was a physician from mainland china who, incubating the disease during his travel, stayed at the metropole hotel in hong kong in early march. later investigations showed that he transmitted the virus to at least other guests at the hotel, who then carried it by international air travel to taiwan, singapore, vietnam, and canada. one of these contact cases was an american businessman headed for singapore. becoming ill while in transit, he stopped in hanoi where he was admitted to hospital with a severe pneumonia, to which he eventually succumbed. soon after, a number of health care workers who had been in contact with him also became acutely ill. fortunately for the course of the outbreak, one of the consultants on the case was an italian physician working with the who in vietnam, dr. carlo urbani. he immediately recognized that this was a previously unknown severe atypical pneumonia that was relatively easily transmissible and reported it to the who; this led to immediate mobilization of investigative efforts and worldwide alerts about the threat. unfortunately, in the course of caring for the victims of the disease in hanoi, dr. urbani himself contracted the infection and died of it later. as information from china became more available, it became clear that by this time there had already been hundreds of cases and numerous deaths. the majority of the initial wave of cases were noted to have occurred primarily in farmers and food handlers, particularly those working in food markets where live wild animals were kept and sold for food. the second large wave of those affected were health care workers exposed in hospital to patients with the disease. the illness was characterized by fever and myalgia with gastrointestinal symptoms in the initial phase, occurring an average of days after exposure (range - days). many cases got no worse than this, but others went on to develop dyspnea associated with radiographic evidence of a diffuse, patchy pneumonitis which, in some, progressed to ards. an average of light % required mechanical ventilatory support, and when the data were all in from later phases of the outbreak, mortality rates averaged about % overall, worse in the aged and debilitated, lower in the young and healthy. the largest outbreak outside asia occurred in toronto, canada. the index case, a visitor to china, returned to canada and died of pneumonia at home, undiagnosed, in early march . shortly thereafter, one of his sons was admitted to hospital with a severe respiratory illness and died a few days later. by this time, four other family members had become ill and had been admitted to hospital; the first cases of affected health care workers appeared soon after among those who had cared for the dying son of the index case. within days, other instances of transmission from undiagnosed contacts of the initial cases in hospitals, doctors' offices, emergency rooms, and at social events were leading to admission of cases to several hospitals throughout toronto. the response of the public health authorities, beginning soon after the who global alert and coincident with the recognition of the first local cases, was quick and vigorous, including closure of the main affected hospital, intensive follow-up of probable contacts, quarantine of suspected cases based on a fairly inclusive case definition and strict institution of barrier contact protection for health care workers. by mid-april the number of new cases was rapidly declining, although there was one cluster of late cases related to exposure of a large number of health care workers during the resuscitation and difficult intubation of a critically ill patient. a later cluster of cases also occurred in a rehabilitation hospital, where it appeared that unrecognized contacts from the first phase of the outbreak had been transferred and transmitted the disease to other patients and staff. the worldwide outbreak was essentially over by july . there were a total of reported cases from countries, with deaths. intensive epidemiologic and laboratory study of the disease by investigators and laboratories worldwide led to unprecedented rapid growth in knowledge about the causative agent. the virus, more or less simultaneously characterized at a number of laboratories around the world, proved to be a previously unknown coronavirus (severe acute respiratory syndrome [sars]-cov) with capacity to infect and spread from a variety of wild animals to humans. epidemiologic, serologic, and virologic evidence was developed linking human cases to exposure to infected wild animals, including masked palm civets, raccoon dogs, ferrets and ferret badgers, all being sold for human consumption in markets in china. control of their transport and sale and exposures to humans by chinese health authorities was probably one of the major factors in bringing the first outbreak under control, the partial failure of which later led to a second, much smaller outbreak late in . although the initial speculation was that one or more of these wild animals were the reservoir in nature for the infection, it now appears more likely that the viral reservoir is actually bats, with crosstransmission of the virus between bats, food animals, and humans in crowded markets leading to development of strains with the capacity to transmit between humans. public health authorities worldwide learned much from sars about the importance of effective international communication in developing a rapid and effective response to outbreaks of novel viruses, and more about how to go about containing such infections within communities and hospitals. several intensivists involved in the outbreak credit e-mail communications from other international outbreak sites for effective advice on critical elements of disease protection (eg, powered air purifying respirators and full contact rather than droplet precautions) and therapy. for the critical care community, perhaps one main lesson was the importance of ''super-spreading incidents'' in propagating the disease in hospitals. many of these occurred in critically ill patients undergoing resuscitation with difficult or traumatic intubation, generating aerosols in closed spaces which contained many superfluous and inadequately protected health care workers. handling these situations safely depends crucially on identifying the potential risk and undertaking the resuscitation and intubation using the most experienced operators available, adequately protected with basic barrier precautions (eye protection, gloves and surgical face-masks), using sedation or paralysis as necessary to minimize trauma and aerosol generation, and with only essential and adequately protected staff in the room. this likely applies to many other situations with potential for disease transmission to health care workers. unfortunately, this epidemic again points out the primary lesson that was not absorbed from the earlier hps outbreak, namely, the need for detailed preplanning and preparation for a major infectious disease epidemic that is inclusive of hospital and icu operations in each locale. according to participants, the sars outbreak demonstrated many of the same early icu operational problems that plagued the hps outbreak albeit on a larger scale. in the icu era, there has yet to occur a true influenza pandemic with a high attack rate in all age groups and associated high hospitalization and mortality rates, as was seen in the great pandemic. in that worldwide disaster, it is estimated that % of all people became ill with the virus and an estimated - million died. minor recent pandemics in and had less than one twentieth of the impact of the influenza, not greatly different from the yearly interpandemic influenza the world has been experiencing in the years since. interpandemic influenza epidemics since have been caused primarily by h n and h n influenza viruses, to which most of the population has developed some degree of immunity from prior infection or vaccination. the result is what public health authorities have become used to seeing: each year a slightly different influenza a appears in asia with minor antigenic changes in the ha or na surface proteins (termed drift), making it infectious once again for humans whose immune systems have yet to be exposed to the new variant, and a new epidemic is launched. when the ''flu'' arrives in an area, cases begin to appear suddenly and there is rapid spread in the population, usually with %- % becoming infected over a -week period with a peak in case numbers at week two or three. about half of those infected will seek medical attention, many more than once, and one to about per thousand infected will be admitted to hospital with a respiratory syndrome such as pneumonia, chronic obstructive pulmonary disease exacerbation, asthmatic attack, or cardiac failure, the rate depending on age and underlying comorbidities. overall, about . % of those infected die, with mortality rates among those with major comorbidities up to %. these latter cases constitute most of the increase in the icu case load which most units experience every winter. the load is sometimes taxing but usually not overwhelming. a true pandemic is unlikely to play out this way. how different it would be depends on a number of factors: the antigenic difference in the new influenza virus compared with the old (ie, the antigenic ''shift'' to a different one of ha or na protein subtypes due to introduction of a variant from another influenza-susceptible species), how transmissible the new virus is, how virulent it is, how susceptible it is to antiviral drugs, and whether the world is prepared for it with drug availability and vaccines. the prototype severe pandemic was the spanish influenza of , an h n virus. the most recent circulating influenza virus just before that time was an h n that had arrived in . current evidence suggests that an avian influenza virus underwent a period of evolutionary adaptation, possibly in another susceptible species such as swine, fitting it for transmission to humans, which it then did. , this h n virus had not been previously experienced by any segment of the population except the very old, so nearly everyone, particularly non-elderly adults and children, was without immunity and was at risk of severe infection. attack rates, as noted earlier, were extremely high everywhere as were rates of primary influenza pneumonia, complicating bacterial pneumonia, and death. in the united states, death rates were more than -fold higher than in any influenza pandemic since. an outbreak of influenza on this scale, if unchecked by effective antiviral therapy or vaccines, would render icu care such as mechanical ventilatory support for respiratory failure irrelevant. even today, with maximal respiratory support, most patients with diffuse primary viral pneumonia complicated by respiratory failure cannot be saved, and the numbers presenting for such care in a short space of time, if comparable to the pandemic, would overwhelm our current icu capacity within days. currently the main apparent threat of a new pandemic comes in the form of the h n influenza virus. this virus is now present nearly worldwide in migratory and, intermittently, domestic bird populations. from time to time, transmission of the virus from birds to humans occurs, generally from close contact situations. who data indicate that there have been laboratory-confirmed cases of such transmission from to mid- . the mortality rate has exceeded %, although it is likely that many less severe cases do not come to medical attention and are therefore not counted as confirmed case survivors. to date no instances of transmission to humans by humans or other mammals has been documented. however, the threat remains that if this virus were to become capable of human-to-human transmission by adaptation in another susceptible mammalian host such as swine, a pandemic on the order of the event could occur. with no true pandemic for over years, including all of the icu era, health authorities worldwide are deeply engaged in trying to learn the lesson of this new ''plague'' before it actually occurs. it is clear that we will need excellent international communication, rapidly enactable containment and quarantine plans and, if possible, effective antivirals and vaccines to deal with the h n virus. if it evolves as feared and becomes easily transmissible while retaining its current virulence; modern life-sustaining technology alone will be no shield at all. the last years have seen remarkable advances in the ability to diagnose and treat infectious diseases and handle infectious disease outbreaks. for the most part, the major plagues of antiquity remain historical footnotes. however, despite these advances, there is clear evidence that major pandemic illness is always just one outbreak away. in addition to the hiv pandemic, the smaller epidemic outbreaks of legionnaire's disease, hantavirus pulmonary syndrome, and sars, among many others, points out the potential risk associated with a lack of preplanning and preparedness. although pandemic influenza is at the top of the list when discussing possible future major infectious disease outbreaks, the truth is that the identity of the next major pandemic pathogen cannot be predicted with any accuracy. we can only hope that general preparedness and the lessons learned from previous outbreaks suffice. a history of poliomyelitis polio: an american story an apparatus for the prolonged administration of artificial respiration the physiologic challenges of the copenhagen poliomyelitis epidemic and a renaissance in clinical respiratory physiology post-poliomyelitis syndrome legionnaire's disease: description of an epidemic of pneumonia legionnaire's disease: isolation of a bacterium and demonstration of its role in other respiratory diseases toxic-shock syndrome associated with phagegroup- staphylococci toxic-shock syndrome -united states toxic-shock syndrome in menstruating women: association with tampon use and staphylococcus aureus and clinical features in cases epidemiologic notes and reports, toxic-shock syndrome, united states reemergence of staphylococcal toxicshock syndrome in intensity of immunosuppression and the incidence of pneumocystis carinii pneumonia comparison of pentamadine isothionate and trimethoprim/sulfamethoxazole in the treatment of pneumocystis carinii pneumonia pneumocystis pneumonia -los angeles a controlled trial of early adjunctive treatment with corticosteroids for pcp in aids aids-related pneumonia in the era of adjunctive steroids outbreak of acute illness -southwestern united states hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease cardiopulmonary manifestations of hantavirus pulmonary syndrome hantavirus pulmonary syndrome -united states: updated recommendations for risk reduction the principles and practice of medicine hemolytic streptococcus gangrene hemolytic streptococcal gangrene: the importance of early diagnosis and operation clinical and bacteriological observations of a toxic-shock-like syndrome due to streptococcus pyogenes streptococcal necrotizing fasciitis: comparison between histological and clinical features antibiotic effects on bacterial viability, toxin production and host response intravenous immunoglobulin therapy for streptococcal toxic-shock syndrome -a comparative observational study. the canadian streptococcal study group epidemiologic analysis of group a streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis who -epidemic and pandemic alert and response (epr) acute respiratory syndrome in hong kong special administrative region of china/vietnam public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses critically ill patients with severe acute respiratory syndrome influenza: the mother of all pandemics molecular basis for the generation in pigs of influenza a viruses with pandemic potential who-epidemic and pandemic alert and response (epr) key: cord- - uvawth authors: barth, rolf f.; buja, l. maximillian; parwani, anil v. title: the spectrum of pathological findings in coronavirus disease (covid- ) and the pathogenesis of sars-cov- date: - - journal: diagn pathol doi: . /s - - - sha: doc_id: cord_uid: uvawth nan the coronavirus disease (covid- ) is now a worldwide pandemic and is the most significant global health crisis of our time. covid- disease continues to present us with major healthcare challenges with over million cases around the world and still less than optimal treatment options [ ] . the virus initially was identified in wuhan, hubei province, china in . covid- disease is caused by a novel coronavirus, which has been named "severe acute respiratory syndrome corona virus- (sars-cov- )" [ ] . covid- is highly transmissible among humans and primarily, but not only, affects the lower respiratory tract and lungs. the virus attaches to the angiotensin converting enzyme- (ace- ) receptor in order to help enter the target cell by attaching its spike-like surface projections ("corona") to the receptor. this receptor is expressed on various cell types such as myocardial cells, type ii pneumocytes, enterocytes and vascular endothelium. most of the symptomatic patients have mild flu-like features but a significant subset develop a bronchopneumonia, which clinically is the acute respiratory distress syndrome (ards), and leads to significant morbidity and mortality [ ] . the fatality rates of covid- are highest amongst older patients with concomitant comorbidities and/or patients who are immunosuppressed [ ] . our current understanding of the pathology and the pathogenesis of covid- disease and sars-cov- transmission is at an early stage and much still remains to be learned [ , ] . additionally, there are also published reports on the pathogenesis of other coronaviruses such as sars-cov and mers-cov [ ] [ ] [ ] . in early april a letter to the editor appeared online in chest entitled "a call to action: the need for autopsies to determine the full extent of organ involvement associated with covid- " [ ] . this was prompted by the stunning lack of publications relating to autopsy findings in decedents who had succumbed to covid- infections despite the fact that several hundred thousand individuals already had died by that time [ ] . since then, at the time of this writing in late june and to the best of our knowledge, there have been at least reports describing autopsy findings in approximately decedents who have succumbed to covid- [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the meantime, as of june , , the number of confirmed cases has climbed to over . million and over , deaths in the united states and million cases and more than , deaths worldwide. therefore, the total number of autopsies performed is miniscule compared to the number of deaths, but nevertheless they are both very revealing and important in order to better understand the multi-organ involvement associated with covid- infection and for the development of better treatment strategies [ , ] . the three largest and most recent of these are reports from the mount sinai hospital in new york city of decedents [ ] , the l. sacco hospital in milan, italy of , limited to the lungs [ ] and the university hospital in basel switzerland of decedents [ ] . these three reports taken together have provided a more complete picture of the various organs that can be involved in individuals who succumb to covid- . what initially was thought to be a disease almost exclusively involving the lungs now is being recognized as one that involves multiple organ systems including the heart, kidneys, bone marrow, lymph nodes and brain. the underlying pathology of covid- infection appears to be involvement of vascular endothelium of multiple organs [ ] . it is noteworthy that in the chinese medical literature there was, as of march , only one autopsy report of a decedent who had succumbed to covid- and this was published in chinese in the journal of forensic medicine [ ] . unfortunately, no diagnoses were given nor was microscopic examination carried out making this report of very limited usefulness. this in part is due to the fact that very few autopsies are carried out in china and most of these seem to be forensic autopsies. several other reports from china have provided some detailed histopathologic information based on "biopsies" of the lungs and kidneys of a few decedents taken following their death [ , , ] . even with this limitation, very important information was obtained, which described the major hallmark of covid- infection, namely diffuse alveolar damage (dad) of the lungs and a glomerulopathy involving the kidneys. although autopsies have shown a steep decline in almost all countries of the world, they still remain a very powerful tool [ ] to develop a full understanding of a new and previously unknown diseases such as sars-cov . it is beyond the scope of this editorial to go into a detailed discussion of the specific pathologies associated with covid- infection, but, based on the autopsy reports to date, a much clearer picture is emerging and will be briefly summarized here. readers interested in more detailed information are referred to the report cited in the references in this editorial. for a more comprehensive overview of sars-cov and covid- disease readers are referred to a soon to be published review by mohanty et al., [ ] . acute severe covid- respiratory disease develops as a severe acute respiratory distress syndrome (ards) that autopsy studies have shown to be related to an underlying severe form of dad in the acute, exudative phase. covid- -induced dad is characterized by damage to alveolar capillary endothelium and type ii pneumocytes leading to alveolar septal edema and the formation of hyaline membranes, accumulation of numerous megakaryocytes, platelets, and neutrophils in alveolar capillaries and precipitation of fibrin inside and outside the alveolar capillaries with a relatively mild accumulation of lymphocytes and macrophages within alveoli. the former provides evidence for a pulmonary thrombotic microangiopathy that often results in fibrinplatelet thrombi in alveolar capillaries and small pulmonary arteries. similar changes have been identified in dad of other etiologies, including influenza and sars-cov [ ] . however, the changes in full blown covid- dad are more extensive and severe, and collectively constitute a distinct and characteristic type of covid- dad [ ] . the pulmonary thrombotic microangiopathy progresses to a diffuse hypercoagulable state in some patients that can lead to deep vein thrombosis and large pulmonary thromboemboli. a clinical marker for patients at risk for this coagulopathy is elevated plasma d-dimer at the time of presentation. a postulated underlying mechanism for severe covid- associated pneumonia is a state of virally-induced hyper-inflammation that has been variously designated as macrophage activation syndrome (mas), cytokine storm and secondary hemophagocytic lymphohistocytosis (shlh) [ , ] . this hyperinflammatory response most likely involves activation of the innate and acquired immune systems [ , ] hence, the initial pulmonary pathology is a florid dad with an immuno-thrombotic microangiopathy [ , , ] . patients who succumb after a more prolonged clinical course are likely to show late stage dad and/or organizing pneumonia. consensus guidelines are now available for prevention, antithrombotic therapy, and follow-up for thrombotic and thromboembolic disease in covid- patients [ ] and the damping of the hyperimmune inflammatory response by the administration of dexamethasone [ ] . the chinese clinical studies early on identified serum troponin as a marker for an adverse outcome and this led to the suggestion of a myocarditis [ , ] . however, cardiac involvement has proven to be more subtle with most hearts showing multifocal individual cardiomyocyte injury without overt interstitial inflammation and only a few cases of classical myocarditis have been described [ ] . the pathological basis for severe acute renal failure in some patients requires further evaluation. some decedents had prominent thrombi in glomerular capillaries [ ] although in many cases, glomerular involvement has been limited. it is likely that the acute renal failure is a secondary form of acute tubular necrosis [ ] . the spleen has been found to have a dimunition of white pulp with loss of peripheral cuff lymphocytes. this is consistent with a viral attack on immunocytes and the lymphopenia that may be seen at the time of presentation. hemophagocytic lymphohistiocytosis (hlh) has been postulated to have an important role in the progression of severe covid- disease [ , ] . however, so far morphological evidence of prominent erythrophagocytosis has been found in only a few cases [ ] . of special interest is involvement of the brain in individuals infected by covid- . there certainly was a hint of this in the very common presenting symptoms of anosmia and ageusia suggesting the neurotropism of the virus. magnetic resonance imaging of the brain of one patient with these two symptoms revealed abnormalities in the right gyrus rectus and the olfactory bulbs [ ] . even more striking has been numerous reports of individuals with covid- infection who have had strokes, and in some instances these were the presenting symptom. bryce et al. have reported that of brains that were examined showed a range of abnormal pathologic findings, the most striking of which was the presence of microthrombi and acute infarcts in the brains that were examined. in some brains this was accompanied by acute parenchymal microthemorrhages in areas of necrotic infarcts, which may be due to the high expression of ace in vascular endothelium, not only in the brain [ ] but also in the lungs. in contrast to these findings, solomon et al. [ ] have reported that neuropathologic examination of the brains of decedents failed to reveal any changes that could specifically be related to covid- infection. however, if covid- is anything like sars-cov [ ] , then the possibility of neuronal infection also must be considered. more detailed neuropathologic examination of a larger number of decedents with a broader representation in age, gender, and duration of clinical course should provide a more definitive answer to the question of brain involvement. finally, the involvement of other organ sites such as the testes, gastro-intestinal tract, liver, endocrine system and musculo-skeletal system are yet to be determined. we still are at an early stage of understanding the effects of covid- on the immune system but clearly this is of great importance considering the occurrence of a hyperactive immune response to the virus [ , ] . the autopsy reports that already have been published provide a solid base for a better understanding of the consequences of covid- infection but much more remains to be learned about this complex disease in order to develop better treatment strategies. this includes a better understanding of the epidemiology, pathology, molecular profile and immunology of the virus and the need for better diagnostic tests including serological assays to monitor the host response to the virus, recovery and the persistence of disease in infected individuals. all of these would help in the design of better therapeutics and vaccines [ , ] . in closing, this editorial announces the inauguration of a new thematic series on the pathological spectrum and pathogenesis of sars-cov- . as the editor-in-chief of diagnostic pathology, i, anil parwani, am very excited about launching this important series as it will serve to attract key articles in covid- pathophysiology, diagnostics, molecular pathology, immunology with an emphasis on the cliniopathological correlation of this disease. the series will also solicit articles on the most up to date understanding of the histopathological features of a sars-cov- infection including an understanding of the ultrastructure of the virus. we hope that our readers will find the collection of articles in this series both interesting and useful in disseminating important information relating to the pathology of covid- . the trinity of covid- : immunity, inflammation and intervention epidemiology and transmission of covid- in cases and of their close contacts in shenzhen, china: a retrospective cohort study covid- pathophysiology: a review pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak a review on sars-cov- virology, pathophysiology, animal models, and anti-viral interventions pathology and pathogenesis of severe acute respiratory syndrome pulmonary pathology of early-phase novel coronavirus (covid- ) pneumonia in two patients with lung cancer immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates a call to action: the need for autopsies to determine the full extent of organ involvement associated with covid- no autopsies on covid- deaths: a missed opportunity and the lockdown of science pathophysiology of sars-cov- : targeting of endothelial cells renders a complex disease with thrombotic microangiopathy and aberrant immune response. the mount sinai covid- autopsy experience the emerging spectrum of cardiopulmonary pathology of the coronavirus disease (covid- ): report of autopsies from houston, texas, and review of autopsy findings from other united states cities pulmonary post-mortem findings in a series of covid- cases from northern italy: a two-centre descriptive study time to consider histologic pattern of lung injury to treat critically ill patients with covid- infection pulmonary and cardiac pathology in african american patients with covid- : an autopsy series from new orleans pulmonary arterial thrombosis in covid- with fatal outcome: results from a prospective, single-center complement associated microvascular injury and thrombosis in the pathogenesis of severe covid- infection: a report of five cases post-mortem examination of covid patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings of lungs and other organs suggesting vascular dysfunction multiorgan and renal tropism of sars-cov- postmortem examination of patients with covid- autopsy findings and venous thromboembolism in patients with covid- the autopsy report of the demise of patient of novel corona virus full spectrum of covid- severity still being depicted pathological findings of covid- associated with acute respiratory distress syndrome the importance of the autopsy in medicine: perspectives of pathology colleagues severe acute perpiratory syndrome coronavirus- (sars-cov- ) and coronavirus disease (covid- )-anatomic pathology perspective on current knowledge immune mechanisms of pulmonary intravascular coagulopathy in covid- pneumonia the role of cytokines including interleukin- in covid- induced pneumonia and macrophage activation syndrome-like disease covid- and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review covid- : low dose steroid cuts death in ventilated patients by one third, trial finds magnetic resonance imaging alteration of the brain in a patient with coronavirus disease (covid- ) and anosmia severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace neuropathological features of covid- covid- vaccine development pipeline gears up covid- pandemic: an overview of epidemiology, pathogenesis, diagnostics and potential vaccines and therapeutics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements i would like to acknowledge the expertise and knowledge of our guest authors (rolf f. barth and l. maximillian buja for this editorial. the author(s) read and approved the final manuscript. all authors contributed in preparing and review of the entire manuscript. the authors declare that they have no competing interests. key: cord- -onghrm y authors: nevarez, javier title: chapter crocodilians date: - - journal: manual of exotic pet practice doi: . /b - - . - sha: doc_id: cord_uid: onghrm y publisher summary this chapter presents a general overview of the anatomy, physiology, and treatment methodology for crocodilians. most crocodilians grow to be larger than other reptile species and, therefore, have significant space requirements. like most animals requiring an aquatic environment, crocodilians need water that is clean and free of disease. crocodilians have a true hard palate in the roof of the mouth that ends caudally in a soft palate. this soft palate has a ventral flap which is referred to as the velum palati. the respiratory system of crocodilians consists of well-developed lungs benefiting from a very effective inspiration aided by the intercostal muscles and the septum post hepaticum. crocodilians have a four-chambered heart as opposed to the three-chambered heart found in other reptiles and amphibians. the temperature and humidity requirements for crocodilians in captivity vary with the species. an understanding of crocodilian biology and natural history is needed to try and duplicate their natural environment. an important consideration is the allowance of circadian variations in light cycle and temperatures to mimic their natural environment. this is not the case in many commercial operations, where they are maintained at a fairly constant temperature and humidity to achieve faster growth. c h a p t e r crocodilians javier nevarez modern-day crocodilians date back to the mesozoic era over million years ago, surviving to the present with relatively few evolutionary changes. their biology, physiology, and anatomy are unlike those of any other reptiles. however, some species have suffered the effects of a changing environment and human encroachment; this situation has led to the formation of international programs dedicated to the preservation of threatened or near-extinct species. of the species of crocodilians, can be found on the red list of endangered species. taxonomic identifi cation varies among all species, and there is debate over the proper classifi cation of species and subspecies (tables - and - ; box - ). in addition to having wild crocodilian populations, countries such as australia, india, mexico, papua new guinea, south africa, and the united states maintain intensive production operations for various species. in north america, the american alligator (alligator mississippiensis) is the best-known crocodilian. second to it is the american crocodile (crocodylus acutus), a vulnerable species found in small numbers in florida. the american alligator was considered a threatened species during the s, but a captive rearing program in louisiana has been successful at maintaining the estimated population at over million animals. the fi rst type of captive rearing operations consisted of alligator farming. in farming operations, breeding pairs of alligators were kept in enclosed areas where they could mate and nest (figure - ). the eggs were collected from the nests and incubated. the farming operations led to ranching operations in which eggs are harvested from the wild and incubated in private facilities ( figure - ). the alligators hatched on an "alligator ranch" are then raised for their hide and meat. to help maintain the wild populations, % of the hatched alligators are eventually returned to the wild. louisiana is the primary producer of american alligators in the world. captive rearing operations can also be found in florida, texas, georgia, and other southern states within the united states where alligators naturally inhabit. in louisiana the majority of the operations are ranches, whereas many farms still exist in florida. other crocodilian species, such as the nile crocodile (crocodylus niloticus), are primarily raised under farming operations in other countries. in those states where alligator productions are present, there are opportunities for veterinarians to get involved with the industry. some veterinarians, regardless of practice concentration, have alligator farmers or ranchers as part of their clientele and meet new challenges in their daily practice. a population management approach is necessary when working with these alligator production facilities. it is also advisable for veterinarians and farmers alike to be aware of the rules and regulations imposed by the local department of wildlife and fisheries. in addition to being found in their natural environment, crocodilians are also found in zoos. zoologic parks house different species of crocodilians for educational purposes, reproductive efforts, or both. as is the case with many other threatened species, zoologic institutions play a critical role in the captive reproduction of threatened species of crocodilians. here the animals can be observed by specialists in a more controlled environment that mimics their natural habitat, although design and maintenance of the enclosures can be diffi cult. disease prevention and control are other important considerations when it comes to oversight of threatened species. veterinarians may also encounter crocodilians as privately kept "pets." in the past, the spectacled caiman (caiman crocodilus) and even the american alligator (a. mississippiensis) have been sold to the general public. there are many reasons why crocodilian species should not be maintained as pets. in addition to the physical dangers and risks associated with keeping crocodilians, there is the misfortune of inadequate husbandry, adversely affecting the reptile's health. most crocodilians grow to be larger than other reptile species and therefore have signifi cant space requirements. like most animals requiring an aquatic environment, crocodilians need water that is clean and free of disease. another husbandry issue is an incorrect diet, which can lead to metabolic abnormalities and overall poor health. many private owners are unable or unwilling to provide these conditions for their crocodilians. in addition to these issues, most states will require special permits to own these animals. most people lack the proper ownership permits, and veterinarians could be liable if they treat these illegal patients. the biggest challenge that veterinarians face when treating illegally owned animals is that they are often the last chance for humane treatment for these animals. it is the duty of most of the material presented in this chapter specifi cally relates to the american alligator (a. mississippiensis). although most of the alligator information will apply to other crocodilian species, there are variations among groups that will be mentioned. the natural environment and geographic distribution of a species will often determine disease exposure. once in captivity, all species are susceptible to the same diseases within that environment. in addition, there are some inherent differences among the three families of crocodilians-alligatoridae, crocodylidae, and gavialidae-that have emerged from studying them in captivity. one of the most common questions a veterinarian may encounter regarding reptiles is "what is the difference between alligators and crocodiles?" the fi rst difference is that they belong to two different families: the family alligatoridae (see table - ) includes the alligators and caimans, and the family crocodylidae (see table - ) includes all crocodiles. there is a third family, the gavialidae (see box - ), which contains the gharial, or gavial. geographic location may help in the identifi cation of some species. alligators are thought to tolerate colder temperatures and live at higher latitudes, whereas crocodiles and caimans are less cold resistant and live in warmer areas. however, there are some anatomic features that will be most useful in differentiating alligators from crocodiles. the alligators and caimans have a broad, u-shaped snout, whereas crocodiles have a more narrow, v-shaped snout. this difference can be observed by looking at the dorsal aspect of the head (figure - ) . a more obvious distinction can be made when looking at their mouth from the side (figure - ) . alligators and caimans have notches in the maxilla that fi t the mandibular teeth. therefore, they have no mandibular teeth visible if observed from the side with their mouth closed. on the other hand, crocodiles have the fourth mandibular tooth exposed when looking at them from the side with their mouth closed. integumentary sensing organs, also known as dome pressure receptors (dprs), are clear to gray pits present on the skin of crocodilians. their function is not completely understood, but they may play a role as mechanoreceptors in prey detection or even as chemoreceptors aiding in detection of salinity levels. , alligators and caimans have dprs only on the lateral aspect of the mandible ( figure - ), whereas crocodiles and gharials have dprs all over the body, most noticeably on the ventral scales ( figure - ) . the presence of dprs can be used to differentiate the two main groups of crocodilian skins in the leather market. an additional feature that could be used for differentiation of alligators and crocodiles is the salt glands, which are absent from the tongue of alligators and caimans but well developed in crocodiles and gharials. an interesting anatomic feature of crocodilians is the palatal valve, also known as the gular valve. there is some discrepancy as to the name of this structure and its two components, but i will attempt to describe them based on the anatomic location. crocodilians have a true hard palate in the roof of the mouth that ends caudally in a soft palate. this soft palate has a ventral fl ap that is referred to as the velum palati. the velum palati is the dorsal component of the palatal valve, with its second and ventral component being the gular fold. this structure projects in a craniodorsal direction from the base of the tongue and has a cartilaginous base to it that is part of the larynx. together, the velum palati and the gular fold form what is known as the palatal, or gular, valve ( figure - ) . the function of this valve is to seal the pharyngeal cavity while under water to prevent aspiration. crocodilians also have control of the nares and are able to open and close them as needed to prevent aspiration of water. the respiratory system of crocodilians consists of welldeveloped lungs benefi ting from a very effective inspiration aided by the intercostal muscles and the septum post hepaticum. the septum post hepaticum is a diaphragm-like muscle that creates a partial separation of the thoracic and abdominal viscera. a number of membranous connections separate the lungs and the liver, and an intricate mesentery system encompasses the gastrointestinal tract and viscera. all of these tissue structures may be necessary for allowing the changes in pressures that occur during diving. the cardiovascular system of crocodilians also has special characteristics. crocodilians have a four-chambered heart as opposed to the three-chambered heart found in other reptiles and amphibians. the circulation of blood through the crocodilian heart is like that in the heart of mammals, but the crocodilians possess a viable foramen of panizza. this opening is located at the base of the heart between the left and right aortic arches and allows for venous admixture, which is essential during periods of diving to conserve oxygen. during diving, there is pulmonary hypertension. this in turn creates increased pressure in the pulmonary artery and the right ventricle, which forces deoxygenated blood through the foramen of panizza into the left side of the heart and the aorta to be distributed through the body. this mechanism allows for conservation of oxygen and supplies oxygenated blood to those organs that require it the most, allowing some crocodilian species to stay submerged for up to hours. a second anastomosis may be present in other crocodilian species as a vessel connecting the two aortic arches. submandibular (figure - ) and paracloacal glands and a gall bladder are present in crocodilians. the hard dorsal scales are known as osteoderms, bony plates lined by skin. crocodilians have a smaller gastric compartment distal to the stomach that is a gizzard-like structure in which rocks and other materials may be found. it does not appear as evolved as the ventriculus in birds, however. crocodilian intestines have a thick wall and can have well-developed diffuse aggregates of lymphoid tissues like peyer's patches. there is no urinary bladder, but the colon can hold large amounts of urine and water. sexing can be performed by palpation of the cloaca; males have a phallus that can be palpated and extracted from the cloaca. females have a well-developed clitoris that, depending on size, can be confused with a phallus. internally paired gonads are found near the ventral surface of the kidneys. physiologic data, including body temperature, heart rate, and respiration, vary with species, age, and environmental factors (e.g., environmental temperature, season of the year). environmental considerations are variable depending on where a crocodilian species lives or is farmed. the aim of captive rearing operations is to produce a large number of animals in the most effi cient way possible. in a zoologic or other educational institution, the goal is to exhibit the animals in an environmentally accurate artifi cial environment. the underlying policy of any aquatic enclosure should be clean water, appropriate diet, and enough space to accommodate the growth of the animals. as with most exotic animals, the challenge is to mimic a captive animal's natural environment. there are no specifi c references for the enclosure size of crocodilian species in captivity. the size of the enclosure will largely depend on the species of reptile and the purpose of their captivity. a general understanding of biology and natural behavior of the captive species is essential to designing appropriate enclosures. although a zoologic institution housing the species might provide more specifi c advice, there are some general enclosure guidelines for the commercial production of american alligators: square foot per alligator up to inches in length (snout to tip of tail), square feet per alligator for those between and inches in length, and an additional square foot of space for every inches in body length beyond inches. these are the recommendations for the maximum stocking rate for alligators in commercial operations. the recommendations for a zoo or educational facility are to make the exhibit as large as possible, taking into consideration the species being housed. a consideration for larger species is territoriality requiring an expanded enclosure. male crocodilians may become more aggressive during the reproductive season, and keeping them separated should be a consideration if space is a concern. exhibits can be outdoor, indoor, or a combination of both. outdoor exhibits can closely mimic the natural environment but also present more challenges for maintaining water and environmental quality as well as for controlling diseases. geographic location will also play a role in the creation of outdoor exhibits, as not all species of crocodilians can tolerate cold weather. finally, some species can dig considerably, and measures must be taken to prevent an escape. the temperature and humidity requirements for crocodilians in captivity vary with the species. once again, an understanding of crocodilian biology and natural history is needed to try and duplicate their natural environment. an important consideration is the allowance of circadian variations in light cycle and temperatures to mimic their natural environment. this is not the case in many commercial operations, where they are maintained at a fairly constant temperature and humidity to achieve faster growth. from a health standpoint, this may allow for cross exposure of reptiles in commercial operations to infectious organisms that typically affect mammals. as the commercial reptiles are maintained at higher temperatures, new diseases commonly associated with mammals may adapt to living inside a reptile host and lead to clinical disease. in an enclosure, the temperature can be maintained via heating elements contained within the concrete slab, in line water heaters, or both. the water temperature must also be maintained during the refi lling of the pen or enclosure to avoid signifi cant temperature variations. light requirements for reptiles are still a controversial subject. in general, a source of ultraviolet b (uvb) light for herbivorous and omnivorous reptiles is recommended. ultraviolet light is essential for the synthesis of vitamin d , specifi cally its active form , dihydroxyvitamin d, which is essential for the metabolism of calcium and phosphorus. a lack of vitamin d can lead to inappropriate calcium absorption, which in turn creates a metabolic imbalance resulting in metabolic bone disease. metabolic bone disease, specifi cally secondary nutritional hyperparathyroidism, is recognized in many reptile species housed with an inappropriate source of uvb light, fed a diet defi cient in calcium, or both. carnivorous reptiles may also benefi t from uvb light but are thought to obtain enough vitamin d and calcium from their prey. the uvb light requirement of crocodilians is unknown, but as true carnivores they may thrive with minimal exposure to uvb light. it is a common practice on alligator ranches to raise animals in darkness with no source of uvb light or a normal light cycle. most animals will grow well under these conditions, and some have reached adulthood without signs of metabolic diseases. however, i have also observed evidence of metabolic bone disease in a subset of captive american alligators being fed a commercial diet with no exposure to uvb light. in these cases veterinarians must also consider the possibility that the commercial diet may be defi cient in calcium. anecdotal stories from alligator ranches claim that weak, anorectic animals appear to improve after being exposed to sunlight over a period of time. further research is needed to determine the uvb light requirements of crocodilians and the potential benefi ts of exposure to uvb light. natural unfi ltered sunlight is the best source of uvb light, but various artifi cial sources are available (e.g., fl uorescent uvb light bulbs, mercury vapor light bulbs). the two main substrates in crocodilian exhibits are water and soil/sand. the species, age, and feeding habits must be taken into account; avoid substrates that may be ingested by accident and may lead to impactions. it is also important to prevent the public from throwing coins and trash into exhibits, as this may represent a source of toxicity and a cause of impactions. in commercial operations, a smooth covering is applied to the concrete to preserve the quality of the hide. either an epoxy coating or plastic liners are routinely used as substrate. crocodilians are true carnivores, as evidenced by their short gut and oral cavity. as such, they require a high protein diet, low in fi ber. their feeding habits in the wild will vary with age and food availability. early on, their diet will consist of small invertebrates, amphibians, and reptiles. as they grow, they will eat larger prey of the type described earlier and will incorporate fi sh and birds into their dietary regime. with time, and depending on the species, size, and food availability, crocodilians will start eating mammals. there have been few studies investigating the nutritional requirements and feeding protocols of alligators. [ ] [ ] [ ] various commercial feeds are available for alligators maintained in captivity. the commercial rations consist of dry pelleted diets that try to provide full nutritional requirements. these diets can be found with a %, %, or % protein content, less than % fat, and approximately % fi ber content. refi ned commercial alligator diets are widely used in production operations but may prove too expensive and/or inappropriate for the long term. a variety of whole prey feeds, such as chicken, nutria, and fi sh, are also recommended. if using nutria, be sure that it has not been killed using lead shot; if the lead is ingested, toxicosis can occur. when feeding frozen fi sh to crocodilians, provide a vitamin b supplement or another meat source to prevent thiamine defi ciency. to prevent dietary associated problems, purchase meat from a reputable source. all animals should be quarantined before their introduction to a production or zoologic facility. a detailed history should be obtained from the source facility, including information regarding diseases to which the animals in question may have been exposed. one should always purchase animals from a reputable individual or institution. little information will be known about wild-caught animals. a minimum quarantine period of to days is recommended in a building that is separate from the main facility. during the quarantine period, the animals can be examined for any sign of illness, and diagnostic tests (complete blood count [cbc], plasma or serum chemistry, west nile virus antibodies, etc.) can be performed to assess their overall health status. if the alligators originated from an area where wnv is endemic, it is advisable to test for previous exposure to this virus. unfortunately, a true quarantine process does not often occur. quarantine is limited by the availability of appropriate facilities, as well as by the time and production budget. quarantine is also a challenge in commercial operations where there may be a large population of animals. nonetheless, zoologic institutions and production facilities should try to establish an adequate quarantine program for their crocodilians. routine physical exams of crocodilians may be incorporated into a zoologic institution's preventive health program. as animals get larger in size, performing physical exams becomes more hazardous. chemical immobilization can be used, if needed, to perform necessary examinations, particularly on large crocodilian species. in commercial operations, the skins of a subset of the production animals will be examined at intervals before the anticipated time of slaughter. the time of hide examination presents an opportunity for veterinarians to examine the animals in more detail. other than hide examinations, routine physical exams are not commonly performed in crocodilians from commercial operations. biosecurity is an essential part of disease prevention in any animal facility. the creation of a sanitation station at the entrance of each building is recommended to prevent introduction of disease organisms. these stations should contain a foot bath and a hand-washing station to decrease the opportunity of disease transfer between exhibits or buildings. a brush should be provided at each foot bath to thoroughly clean boots and shoes. the solution for the bath can be made of bleach or other commercial disinfectants, preferably with virucidal activity, and should be changed daily. organic material will contaminate a foot bath, rendering it ineffective. the foot bath should be used before and after entering the building. a hand-washing station should consist of a water source, a sink, hand soap, and disposable hand towels. a waterless hand sanitizer product or exam gloves will suffi ce in lieu of a handwashing station. in addition to the sanitation station, separate tools for working in each building are required. separate working tools also prevent the transfer of diseases via nets, brooms, rakes, and so on. the buildings themselves must also be maintained free of pests and thoroughly cleaned whenever possible. in commercial operations, it is common practice to empty and disinfect the buildings after the slaughter period, before the introduction of new animals. water quality is one of the main issues of concern when keeping crocodilians in captivity, and many health problems can be prevented if attention is paid to acceptable water quality. manual restraint of crocodilians is essential for physical examination, administration of medications, administration of anesthetics, and relocations. the size and species of crocodilian will determine the best and safest restraint methods to be used. an experienced crocodilian handler must be available to help restrain the animal. although alligators and caimans are usually thought of as being less aggressive than crocodiles, this may not always be true. all sizes and species should be handled with the safety of the people as well as the animal in mind. crocodilians less than m in length (snout to tip of tail) may be handled by one or two individuals. those between and m in length should be handled by at least two or three indi-viduals. those longer than m in length will require at least four to fi ve individuals. various tools (e.g., pole snares, nets, squeeze cages, traps) also can be used to restrain crocodilians. the head, tail, and limbs must be immobilized and controlled. once the animal is under control, the mouth is secured with strong tape or a rope. albino and leucistic animals can have increased skin sensitivity compared to the normal pigmented individual; therefore, additional care should be employed to avoid irritation of the skin. restraint is stressful for the animal, and contact must be limited to the time it takes for the procedures being performed. (see anesthesia for more discussion on chemical restraint.) signs of illness in captive crocodilians are usually nonspecifi c. anorexia, lethargy, a change in behavior, or death may be the fi rst indication that something is wrong in a collection of animals or commercial operation. adequate observations of the animals made by the personnel in the facility should be taken seriously. a visit to the facility is best undertaken during feeding time to avoid additional stress to the animals. at this time, the feeding and water quality policies can be evaluated. a thorough history should include information about the number of animals, source, age, most recent introduction, quarantine practices, feed, frequency of feeding, water quality parameters, clinical signs, time since fi rst signs were observed, recent changes in management techniques, and any treatments such as salt, bleach, or antibiotics. within a commercial operation, a subset of animals should be collected for disease diagnostics and necropsy. in addition to the obtaining of routine samples, tissues should be frozen for possible bacterial, fungal, or viral cultures. within a zoologic institution, sacrifi cing live animals may not be possible, but veterinarians should obtain diagnostic samples from those with and without clinical signs. necropsies should be performed in all dead animals. live animals should undergo physical examination. once the animal is properly restrained, a physical examination can be performed. for safety purposes, veterinarians must be aware of the location of the head and tail at all times when performing the examination. a protocol should be followed for the examination process in crocodilians as in any other species. the oral examination can be performed if a speculum (e.g., pvc pipe, piece of wood) is inserted in the mouth before securing it with tape or a rope (figures - and - ). examine the eyes for evidence of discharge and assess their function ( figure - ). examine the skin for any evidence of trauma or dermatitis, and then palpate the extremities, joints, musculature of neck, pelvic region, and tail. joint swelling is often noted with infectious disease such as mycoplasmosis or trauma. poor body condition may be refl ected in atrophy of the muscles. in commercial operations, it is important to examine the skin on the animal's ventral aspect because this is the area where many disease manifestations will be noted. it is also common to fi nd tooth marks, scratches, and lacerations on the ventral epithelial surface. finally, examine the vent and cloaca for abnormalities. if working in a commercial operation, a veterinarian must examine multiple animals to determine if an observation is associated with disease. if working in a zoologic institution and any fi ndings are suspected to be infectious in origin, other animals in the exhibit should be examined. as with any species, an examiner must know what a normal presentation is in order to recognize clinical signs of disease. diagnostic tests used to determine crocodilian health status are no different than those used with other animal groups. cbcs, chemistry panels, and bacterial cultures can all be performed in crocodilians. however, there are limitations when it comes to the interpretation of test results because of the lack of reference ranges available for the multiple crocodilian species. in addition, other diagnostic tests, such as those based on polymerase chain reaction (pcr) technology, enzyme-linked immunosorbent assay (elisa), and other antibody or antigen serologic tests, are usually not validated for crocodilians. a clinician must inquire about the specifi cs of the tests being performed to make an accurate interpretation of the results. published literature and the experience of other colleagues are invaluable for interpreting diagnostic test results of crocodilian species. also there may be generally accepted diagnostic tests for diseases that have been recognized and studied in crocodilians, such as mycoplasmosis and wnv. there are various sites for venipuncture in crocodilians. the ventral coccygeal vein can be accessed from either the ventral or the lateral aspect of the tail ( figure - ). this vessel lies ventral to the vertebral processes and on midline with the vertebrae. a second alternative is the supravertebral sinus, located on midline at the junction of the head and the neck ( figure - ). the examiner must be careful not to go too deep at this site, or physical damage may occur. a third site is an unnamed vessel located on both the left and right sides of the neck. this vessel can be approached from the dorsal aspect of the neck and is surrounded by muscles, decreasing the risk of coming in contact with nervous tissue (figure - ) . a -ml syringe and a -gauge needle are recommended for collecting blood from crocodilians. lithium heparin and edta tubes are used for plasma chemistry analysis and cbcs, respectively. if collecting serum, the tubes may have to sit for at least to minutes before centrifugation to allow proper separation of the serum from the blood cells. as with other species, cbcs and chemistry panels are a fundamental part of diagnostic testing of crocodilians. these tests can help assess the overall health status of the animals. a cbc can show evidence of acute or chronic infl ammation that may prompt further investigation or lend direction to a defi nitive diagnosis. chemistry panels can give insight into the hydration status and the health of the liver and kidneys. also of importance are electrolyte values, which can show abnormalities related to poor diet and husbandry. a measurement of packed cell volume and total solids and/or total proteins is also an essential health indicator for crocodilian species. fine needle aspirates, impression smears, and fl uid analysis are diagnostic tools that can be useful in determining a defi nitive diagnosis. urinalysis is not a practical test in crocodilians due to the absence of a urinary bladder and the fact that urine will be highly contaminated in a voided sample. the hemocytometer with the unopette eosinophil determination for manual methods stain (becton dickinson and company, franklin lakes, nj - ) is often used to obtain a cbc of crocodilian species and other reptile and avian species. a blood smear stained with diff-quick (quik-dip stain, mercedes medical physician and laboratory products, commerce ct., sarasota, fl ) is also needed to complete the total estimated white blood cell count and obtain the cell differential count. interpretation of cbcs from crocodilians can be challenging for veterinarians unfamiliar with the morphology of their white blood cells, which can vary from that of other reptile species. this variation found in crocodilian species is typical and more pronounced among reptiles. to become familiarized with the different cell types and their appearance, veterinarians should spend time scanning blood smears. this will often help veterinarians differentiate heterophils from eosinophils before the initiation of the differential count. alternatively the samples can be submitted to a commercial laboratory that runs cbcs on blood collected from exotic animal species. most imaging modalities (radiographs, computed tomography [ct] scan, magnetic resonance imaging [mri], ultrasound, etc.) can be used on crocodilian species as long as there is a general understanding of their anatomy. knowledge of normal anatomy is crucial for the interpretation of radiographs, ct scans, and mri images. knowledge of the anatomy will also help locate organs during ultrasound examination. radiographs can be used to locate foreign bodies as well as diagnose fractures. contrast studies can also be performed if the facilities and personnel are allowed to hospitalize the animal for an extended period of time. barium sulfate (liquid e-z-paque, e-z-em inc., westbury, ny ) and iohexol (omnipaque, amersham health inc., princeton, nj ) can be administered at to ml/kg po. iohexol can be diluted with water at a : to : ratio depending on the concentration. depending on the size of the animal and the site of interest, some imaging procedures may be performed with manual restraint or, if needed, chemical restraint. neuromuscular blocking agents may be benefi cial as chemical restraint agents if used appropriately and if the animal is monitored closely. stress has been defi ned as "a physiological answer to a perceived threat that includes, but is not restricted to, increased adrenal secretion." stress can also be any event that challenges homeostasis. the response of the body to that event is complex and involves more than an adrenal response. the autonomic nervous system, the hypothalamic adrenal axis, neuropeptides, neurotransmitters, and neuroimmunologic mediators all have a role in the response of the immune system to stress. measuring stress and immunosuppression is a challenge in veterinary medicine. there are no specifi c tests available to provide a clinical measure of stress. veterinarians concentrate on identifying a combination of physiologic changes that give them an idea of what is involved in a stress response. the stress response in crocodilians has been examined in relation to restraint, long-term corticosterone implants, cold shock, and stocking densities. , - lance et al. provides an overview of the physiology and endocrinology of stress in crocodilians. catecholamines, glucocorticoids, glucose, and lactate have been implicated in the stress response of crocodilians. changes in the white blood cells have also been implicated with immunosuppression and the stress response. [ ] [ ] [ ] there is enough evidence to suggest that stress plays an important role in the physiology of crocodilians and may indeed predispose them to illness. overcrowding, handling, excessive noise, diet changes, temperature irregularities, and so forth, should be considered as predisposing or confounding factors of disease. all of these factors must be considered in the history of a clinical case and when determining treatment. an example of how stress can play a role in disease susceptibility was observed in a case at a commercial alligator facility. this facility had animals with a history of chronic dermatitis. the alligators with the most severe lesions were consistently located on one end of the building, whereas those at the other end were unaffected or only mildly affected. the location of the severely affected animals was consistent in all buildings. one building had no affected animals. based on a thorough history and observation of the operation, a possible explanation was determined for the occurrence of the dermatitis problem. all of the buildings with affected animals had pvc pipes on the inside walls that delivered water to each individual pen. a strong water stream fell from the pipes down into the water and the strength of the stream decreased from one end of the building (infl ow) to the other end (outfl ow). the strength of the water stream was considerable near the infl ow. the building with no affected animals did not have any source of falling water into the pens. water quality, temperature, and feed were the same in affected and nonaffected buildings. the most affected animals happened to be in the pens at the infl ow side of the building with the number of affected animals decreasing toward the outfl ow side of the building. this constant fl ow of water was creating a constant movement of the water surface and consequently stimulating the alligators via the dprs. once this watering system was changed, the cases of dermatitis decreased and no new cases were reported. although other factors, such as water quality, may have contributed to the dermatitis, the change in the watering system decreased the progression and occurrence of the disease. this case demonstrates the importance of addressing the environment, as well as the animals, when working in commercial operations. bacterial diseases in captive crocodilians occur primarily as opportunistic infections. poor water quality, trauma, and stress are some of the factors that contribute to bacterial infections in crocodilians. in their natural environment, crocodilians appear to have a stronger ability, compared with other species, to withstand bacterial infections. reports of antibacterial properties in serum and tissues of crocodilians , offer a possible explanation. the possibility of increased antibacterial properties in the serum and tissues of alligators should not mislead people into thinking that crocodilians are resistant to bacterial infections. it is true that they appear to tolerate trauma and other lesions that would be fatal in many species, but crocodilians are still capable of succumbing to an array of microorganisms, including bacteria. in fact, a number of bacterial infections have been reported in crocodilians, and septicemias are thought to be a frequent fi nding. septicemias can be diagnosed in postmortem examinations and are often associated with a wide number of bacteria, many of them normal gut fl ora. there is an abundance of information about bacteria recovered from the american alligator (a. mississippiensis) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table - ) and from african dwarf crocodiles (osteolaemus tetraspis). salmonella spp. are normal inhabitants of the gut in most reptile species and crocodilians. [ ] [ ] [ ] the importance of salmonella sp. arises more from its zoonotic potential rather than its ability to cause disease in crocodilians, but it has been reported to cause deaths in nile crocodile (c. niloticus) hatchlings. in most commercial operations the meat is sold as a by-product of the hide production. various species of salmonella and other bacteria have been isolated from the meat of crocodiles in commercial operations in an attempt to address the concern of zoonoses. , , [ ] [ ] [ ] [ ] [ ] zoologic institutions that have smaller crocodilians as part of a "petting" station should exercise caution, as fecal shedding of salmonella sp. occurs. although the zoonotic potential exists, there are no well-documented cases of human infections originating from crocodilians. chlamydiosis has also been reported in crocodilian species. there is a report of an isolate, closely associated to chlamydophila psittaci, obtained from the livers of nile crocodiles (c. niloticus) in zimbabwe. this infection is thought to have an acute course characterized by a hepatitis in which hatchling mortalities are observed. a chronic form of reptilian chlamydiosis characterized by conjunctivitis has also been reported and may be more common. there have been reports of concurrent chlamydia sp. isolates in cases of mycoplasmosis and adeno viral infections. dermatophilosis or "brown spot disease" is thought to be caused by dermatophilus sp., with most of the cultures resembling dermatophilus congolensis, and affects both crocodiles and alligators. , , [ ] [ ] [ ] the characteristic brown to red lesions are located usually at the junction of the ventral abdominal scales, which may become ulcerated over time. this has occurred in both alligator and crocodile operations. this disease does not respond well to antimicrobial therapy; therefore, intensive hygiene practices are required to prevent and control outbreaks. mycoplasma alligatoris is a recognized respiratory pathogen of crocodilians. it has been documented in a. mississippiensis and in the broad-nosed caiman (caiman latirostris). , , other crocodilian species closely related to alligators also may be susceptible to this intracellular organism. clinical signs are nonspecifi c and include lethargy, weakness, anorexia, white ocular discharge, paresis, and edema (facial, periocular, cervical, limbs). virus identifi cation and diagnosis in reptiles has lagged compared to virus identifi cation and diagnosis in other species. this delay has occurred primarily because of diffi culties in developing diagnostic tests and lack of knowledge of viruses that affect reptiles and crocodilians. many conditions that go undiagnosed may be caused by viruses. these may be new viruses or just viral diseases unknown to occur in reptilian or crocodilian species. there are only a few recognized viruses that have been documented in crocodilians. jacobson et al. described an adenovirus-like infection in captive nile crocodiles (c. niloticus) characterized by nonspecifi c clinical signs, lethargy, and anorexia. conjunctivitis and blepharitis were also observed in one of two crocodiles. , histopathologic examination revealed intranuclear inclusions, primarily in the liver but also in the intestines, pancreas, and lung. both horizontal and vertical transmission have been postulated for this adenovirus-like infection. diagnosis is obtained through postmortem examination, and no treatment regimes have been established. coronavirus, infl uenza c virus, and paramyxovirus have been identifi ed by transmission electron microscope in the feces of crocodilians. herpesvirus-like particles were identifi ed by electron microscopy in the skin of a salt water crocodile (c. porosus). there is a second fi nding of herpesvirus identifi ed from the cloaca of an american alligator (a. mississippiensis) via pcr. the clinical signifi cance of these fi ndings is unknown. seroconversion to paramyxovirus and eastern equine encephalitis virus has also been reported in crocodilians. parapoxvirus or pox-like viruses have been identifi ed in fi ve different crocodilian species: spectacled caiman (caiman crocodilus fuscus), , brazilian caiman (caiman crocodilus acre), nile crocodile (crocodylus niloticus), , saltwater crocodile (crocodylus porosus), and freshwater crocodile (crocodylus johnstoni). pox lesions in caimans will be to mm in diameter, gray to white in color, and coalescing to macular. they may appear on the head, palpebra, maxilla, mandible, limbs, palate, tongue, and gingiva. [ ] [ ] [ ] palpebral and generalized edema also may be present. resolution of clinical signs has been observed with and without changes in husbandry practices. , in crocodiles the lesions are described as to mm in diameter, yellow to brown, wart-like, sometimes fi rm, and unraised to raised nodules with occasional shallow ulcers. in crocodiles pox lesions appear on the head, palpebra, nostrils, sides of the mouth, oral cavity, limbs, ventral neck and coelom, and at the base of the tail. , resolution of lesions was reported to occur as early as to weeks from the onset of clinical signs. histopathologic fi ndings include epithelial hyperplasia, acanthosis, hyperkeratosis, and necrosis, with intracytoplasmic borrel and bollinger's bodies being visible in some cases. [ ] [ ] [ ] [ ] [ ] secondary bacterial and fungal infections may occur concurrently with the viral infection. at this time there are no specifi c treatment recommendations. the use of an autogenous vaccine to treat poxvirus in nile crocodiles (c. niloticus) has had some success. mosquito control and good hygiene are essential in preventing and controlling poxvirus outbreaks. west nile virus (wnv) has been reported to affect various crocodilian species, including the american alligator (a. mis-sissippiensis), the nile crocodile (c. niloticus), and the morelet's crocodile (c. moreletii). crocodilians likely become infected, as birds and mammals do, via a mosquito bite. there is also the possibility of infection after ingestion of an animal with a high viral load of wnv, as reported by klenk et al. this last scenario is more likely to occur when crocodilians are housed outdoors and not in enclosed buildings, as in most ranching operations. it has been demonstrated that alligators can serve as amplifi ers of wnv. although there is a lot to be learned about wnv in croco dilians, it is believed that once infected, they can develop high viremias and shed the virus in the feces. fecal shedding leads to horizontal transmission of the virus. there is clinical evidence for horizontal transmission to occur in commercial operations. fecal shedding and high viremias also raise the concern of zoonosis, especially in commercial operations where animals are being slaughtered and people come in contact with blood and tissues. a strict building quarantine and hygiene strategies should be implemented to prevent the spread of wnv to other animals in the facility as well as to the personnel. in the state of louisiana there are a number of recommendations provided to help alligator producers cope with episodes of wnv and prevent spread of the disease (box - ). . buildings with affected animals should be maintained under isolation from other buildings in the farm. . feeding and cleaning of affected buildings should be performed last, after all other nonaffected buildings. . a foot bath ( part water, part bleach) should be placed at the entrance of the building to disinfect the shoes and boots of farm employees before and after entering the building. this foot bath should be changed on a daily basis. in addition, a set of shoes or boots may be kept only to be used inside affected buildings. . hands should be washed before and after entering the building. . the water should be changed more frequently in affected buildings, at least once a day. this may create additional stress on the animals but should decrease the amount of feces and therefore viral organisms possibly present in the water. any special treatment of the discharge water still remains to be determined. . affected animals should not be transported to other alligator farms for processing of hides or meat. . additional care should be exercised during the slaughter process to avoid direct contact with the blood and organs of animals known as exposed to, or affected with, west nile virus. . dead animals should be disposed of by burning the carcasses. . aggressive mosquito control is required to help in the prevention of the disease. affected animals in captive operations have ranged in age from month to over months. in younger animals infection is usually acute and severe, with as much as % mortality. the pattern of deaths is peracute, usually seen as a sudden onset of mortalities followed by a peak and subsequent decline in the number of deaths. however, sporadic mortalities may also be noted, especially in older animals. clinical signs of wnv in alligators include swimming in circles, head tilt, muscle tremors, weakness, lethargy, and anorexia. bloating and diffi culties swimming have also been observed but occur less commonly. gross fi ndings from wnv-infected alligators are nonspecifi c. light microscopy of tissues from infected animals may reveal diffuse severe heterophilic, histiocytic, and necrotizing enterocolitis; heterophilic meningoencephalitis; necrotizing and heterophilic hepatitis; heterophilic and histiocytic splenitis; generalized heterophilic and histiocytic lymphoid folliculitis; and necrotizing and heterophilic pancreatitis. veterinarians may also observe a mild multifocal heterophilic and lymphohistiocytic interstitial nephritis, gastritis, and mild pulmonary congestion and edema. strong immunopositivity results have been observed in the brain, liver, spleen, pancreas, kidney, and gastrointestinal tract after immunohistochemistry testing for wnv. proliferative enteritis has been diagnosed in a group of alligators positive for wnv. the colon lesions tested positive for wnv via reverse transcriptase polymerase chain reaction (rt-pcr), culture, and immunohistochemistry , ( figure - ). these affected alligators were bloated and unable to submerge themselves; these symptoms may have been due to a blockage caused by the fi brinous membrane in the colon. there is no known treatment for wnv-infected crocodilians. mosquito control, strict quarantine, and biosecurity are essential for the prevention of wnv. once wnv is present within an operation, it is critical to maintain infected and exposed animals in strict isolation. the ill and exposed animals must not be moved to other areas or buildings where wnv has not been observed. failure to maintain strict isolation may result in the spread of the virus throughout the facility. in addition, there should be strict biosecurity measures, especially in the affected areas or buildings. tools such as nets, feeding utensils, boots, and so forth, must be left in the affected area and not introduced to other, "clean" areas or buildings. the surviving animals will continue to thrive after the wnv infection has run its course, but it is unknown for how long thereafter they can shed the virus. antibody titers of : and : were observed in two alligators months after exposure to wnv. these antibody titer results were obtained via a plaque reduction neutralization test. defi nitive diagnosis of wnv infection should be based on history, clinical signs, and the results of diagnostic tests. postmortem diagnosis can be performed via rt-pcr and/or viral culture of brain, spinal cord, or liver, with immunohistochemistry providing additional supportive evidence of wnv infection. immunohistochemistry may prove useful as an antemortem test when applied to biopsies of the colon mucosa. fungal infections also occur with some frequency in captive crocodilians. most fungi are opportunistic invaders of the integument, respiratory system, and gastrointestinal tract. poor water quality, trauma, stress, and extreme temperatures can contribute to the occurrence of fungal disease. it is thought that most fungal infections in crocodilians are of enteric origin and occur in other tissues secondary to an immunocompromised state. it is important to remember that fungi are considered ubiquitous organisms in nature. therefore, it is not uncommon to isolate fungi from tissues (e.g., skin, intestines) that are in contact with water and soil. the presence of fungi will vary with geographic location and management techniques that affect the different environments for their growth. there are a number of fungi that have been isolated from crocodilians , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (table - ). some of these fungal organisms were associated with disease, whereas others were incidental fi ndings. diagnosis of fungal disease requires the identifi cation of a fungal organism via culture or special stains of infected tissues. positive identifi cation of genus and species may be diffi cult. treatment is expensive and may be affordable only when administering to a small number of animals. it also requires a long treatment period of at least or months. for these reasons, many fungal infections go untreated, and recommendations are made for prevention of disease. a number of parasites are known to affect crocodilians. protozoa, nematodes, trematodes, and pentastomes have all been reported in crocodilians. cestode larvae have been found in some species, but no adult tapeworms are reported from crocodilians. external parasites, such as leeches, fl ies, mosquitoes, ticks, and mites, can also affect crocodilians. the presence of scales has created a misconception about the possibilities for external parasitism in crocodilians. however, there are a number of areas in a crocodilian's body that have soft skin and allow an external parasite to attach and/or obtain a meal. the recent cases of wnv in the american alligator (a. mississippiensis) are a reminder that arthropods can play an important role in disease transmission. internal parasites are not a common problem in ranching operations where animals are maintained in concrete buildings with no organic substrate. parasites are of major concern in wild crocodilians or those kept captive outdoors on organic substrates. external parasites are more ubiquitous, affecting both captive and wild populations. to determine the best prevention and control methods to use, veterinarians must assess the environment as well as the life cycle of the parasite. with the increased knowledge of crocodilian dietary requirements, nutritional diseases are diagnosed less frequently than they were in the past. better understanding of the biology, natural history, and physiology of the different crocodilian species has allowed implementation of better feeding schemes and diets. over time, there has been a desire to develop commercial diets that will allow for high feed to weight gain ratios. these commercial diets are available for feeding american alligators (a. mississippiensis), and although the diets are not perfect, if fed appropriately, captive-hatched american alligators (a. mississippiensis) will grow up to inches in length within a -month period. for zoologic institutions the goal is not rapid growth but rather normal development and a foundation for excellent health. zoologic institutions and crocodile farms commonly provide crocodilians with fresh and/or frozen prey (e.g., poultry, swine, beef, fi sh, nutria, native prey, etc.) with bones providing a source of calcium. to prevent lead toxicity in crocodilians, veterinarians must use a reputable fresh or frozen prey source that has not been killed with lead projectiles (e.g., shotgun pellets, bullets). despite better feeding schemes and diets, veterinarians will still see crocodilians with diseases associated with nutritional defi ciencies. a common nutritional disease of reptiles is metabolic bone disease (mbd). clinical signs of mbd include weakness, lethargy, kyphosis, scoliosis, osteodystrophy, pathologic fractures, paresis, and tooth decalcifi cation. various possible causes of mbd have been proposed, but it is usually associated with a calcium-phosphorus imbalance that leads to increased bone resorption. calcium and vitamin d are the two main nutritional compounds defi cient in reptiles with mbd. vitamin d defi ciencies are associated with a lack of exposure to uvb spectrum light, which comes naturally from the sun. light bulbs and light tubes are commercially available to help provide adequate amounts of uvb light for reptiles. mbd is primarily observed in herbivorous or omnivorous reptiles. it appears that mbd is not commonly observed in commercial crocodilian operations, even in those were animals are raised in the dark with no source of uvb light. the carnivorous nature of crocodilians may allow them to obtain vitamin d from the diet without a true requirement for uvb light. however, if an appropriate calcium source is not offered, these animals will develop mbd. adult alligators without mbd have been observed growing in enclosed buildings with no light source while being fed a commercial diet supplemented with fresh prey meat with bones. however, anecdotal comments from various ranchers indicate that the animals appear to thrive better if exposed to sunlight. this is an area where more research is needed to determine the uvb and calcium requirements of crocodilians and their effects on growth and health. articular and visceral gout also have been reported in crocodilians. some predisposing factors for gout include high protein diets, dehydration, and stress. clinical signs associated with chronic gout in crocodilians are primarily nonspecifi c, but limb paresis or paralysis and joint enlargement can occur. ariel et al. reported a case of visceral and articular gout with concurrent hypovitaminosis a in crocodile hatchlings. defi ciencies of vitamins a, b, c, or e also can lead to a variety of musculoskeletal disorders. these are thought to be less common in crocodilians fed commercial diets in addition to fresh meat products. respiratory disease is one of the most common presentations of captive crocodilians in commercial operations. it is common to fi nd lesions in the lungs associated with respiratory disease when performing postmortem examinations (figure - ) . although most of the lung pathology is incidental, some animals will have a history of clinical signs associated with respiratory disease before death. clinical signs of respiratory disease in crocodilians are nonspecifi c anorexia, lethargy, and weakness but may also include dyspnea, tachypnea, nasal secretion, excessive basking, respiratory stridor, and abnormal swimming (either in circles or on one side of the body). in some cases the animals may appear neurologic as they become weak and ataxic. a clear distinction must be established between respiratory disease and neurologic disease. respiratory disease may be secondary to neurologic disease as a consequence of weakness, predisposing the animal to aspiration pneumonia. most respiratory infections are either bacterial or fungal in origin. upper respiratory diseases, including rhinitis and pharyngitis, also have been reported in crocodilians. neurologic diseases are not common in crocodilians. however, the causes implicated by clinical neurologic presenting symptoms deserve special attention. the most common neurologic clinical sign is abnormal swimming behavior (e.g., swimming in circles, swimming on one side of the body, etc.). once outside the water, these animals may show signs of lethargy, ataxia, head tilt, and muscle tremors. anorexia may be the earliest sign observed by the facility personnel. the most recent explanation of signs of neurologic disease in crocodilian species is wnv. this virus should be in the differential diagnosis list for any crocodilian that has neurologic signs and is housed in an area where wnv is endemic. hypoglycemia also has been reported to cause neurologic signs in alligators. neurologic signs and deaths of alligators (a. mississippiensis) were identifi ed from a commercial operation where a building was not cleaned according to the maintenance schedule. the water in the building had not been changed in a -hour period, and there was evidence of food debris from previous feedings. the owner found a few hundred dead alligators, and of those that remained alive, some had neurologic signs (e.g., slow refl exes, abnormal swimming patterns, ataxia, and lethargy). necropsy and histopathologic evaluation leaned toward toxicity as the cause of death. both dead and live animals tested negative for wnv. all other buildings were cleaned on schedule, and there was no evidence of disease in those buildings. mortalities decreased after the building housing the sick animals was cleaned. based on the history, clinical observations, and histopathologic fi ndings, veterinarians believe these animals developed neurologic signs as a result of either increased ammonia level in the water, oxygen depletion in the air, or both. musculoskeletal disease can be caused by changes in the incubation temperature or humidity; trauma from fi ghting, transport, or restraint; and infectious diseases ( figure - ) . deformities of the head, limbs, and tails are common malformations infl uenced by incubation rather than true genetics ( figure - ). fractures and limb amputations can occur as a result of trauma and fi ghting, with many healing without major complications. healed traumatic injuries are observed in captive and wild crocodilians. if the trauma is severe enough, nerve damage, muscle damage, paresis, and/or paralysis may result. the most common infectious disease known to have effects on the musculoskeletal system is mycoplasmosis. both m. alligatoris and m. crocodyli can cause polyarthritis in affected animals. ingestion of foreign bodies, gastric ulcers, enteritis, and trauma to the oral cavity are gastrointestinal diseases associated with crocodilian species. anorexia is a common sequelae to the gastrointestinal diseases listed above. ingestion of foreign bodies occurs in both wild and captive crocodilians. construction, malfunction of water pumps and fi lters, forgotten objects in the enclosure, and tossed objects by the public are some of the sources of foreign bodies for captive animals. sharp objects such as nails may cause severe gastrointestinal problems, while other less sharp or pointed ingested material may pass without major diffi culties. infectious enteritis is usually diagnosed post mortem based on gross and histopathologic evaluation. crocodilians appear to have an aggressive response to insult of the gastrointestinal tract. grossly one may observe accumulation of fi brinous or fi brous material and/or necrosis of the mucosa (figure - ) . in some instances, this can even lead to obstruction due to accumulation of fi brinous/fi brous material. fecal impactions and torsions are probably rare but can also occur in crocodilians, while gastric ulcerations may be associated with stress and diet. the intestinal tract of crocodilians appears to contain a signifi cant amount of gut associated lymphoid tissue like peyer's patches. this lymphoid tissue may allow for an aggressive infl ammatory response to infectious agents in the intestinal tract. integumentary disease is arguably the most signifi cant disease process affecting captive crocodilians. water quality, temperature, and stressors contribute to the occurrence of integumentary diseases. secondary bacterial and fungal dermatitis in crocodilians can have a devastating economic impact on com- mercial operations. lacerations, abscesses, and draining tracts can serve as a nidus for microorganisms. an additional predisposing factor of integumentary disease in captive operations is the accumulation of fat in the upper water column. this concentration of fat creates a slime layer on the walls and water surface, which is then transferred to the skin of the animals where it creates an ideal environment for fungal and bacterial growth. this is more of a problem when meat is the sole source of food or is provided in addition to a commercial diet. a surfactant disinfectant can be used to break down the fat accumulation in the water and on the walls of the enclosure. in most instances both bacteria and fungi will be present in the skin lesions associated with fat accumulation in the captive environment. it is important to recognize the fat accumulation problem early to institute appropriate cleaning measures and possible therapy. mixed fl ora is often found in the dermal lesions, making identifi cation of a single causal agent diffi cult. antimicrobial therapy is indicated if bacterial involvement is suspected. although the use of systemic antifungal medications is cost prohibitive in commercial operations, it can be used if only a small number of animals are affected. improved hygiene, better water quality, stable temperatures, and decreased stress are essential for managing these dermatitis cases. in most instances those animals that are already showing clinical signs may not recover. the key to preventing and decreasing the spread of disease is improving and implementing appropriate husbandry methods. various alligator (a. mississippiensis) ranches in louisiana have reported hatching animals with normal pigmentation that becomes "white" after a few weeks of life (figure - ) . physical examination of these animals does not reveal any abnormalities other than an apparent depigmentation or hypopigmentation of the skin. the white discoloration is not unlike that described as a result of fungal or bacterial dermatitis. upon palpation, the skin appears thinner and has a fl aky nature similar to that observed in leucistic alligators. affected animals do not appear to grow at different rates than normally pigmented animals. the processing of the hides is not affected by the pigment defi cit. a nutritional defi ciency or genetics are suspected in this presentation, but there are anecdotal reports of improvement of the condition after vitamin supplementation with a multivitamin formulation. lymphohistiocytic proliferative syndrome of alligators ("pix" disease) lymphohistiocytic proliferative syndrome of alligators (lpsa), also known as pix disease, has been recently studied in captivereared alligators (a. mississippiensis) from florida and louisiana. lpsa affects the quality of the hides in commercial operations, leading to decreased profi ts for alligator producers. gross lesions of lpsa are multifocal, -to -mm, gray to red foci on the ventral mandibular and abdominal scales ( have been found in the lungs, intestines, kidneys, ovaries, testicles, thyroid, gall bladder, mesentery, eyes, and nervous tissue. ongoing research has revealed a strong relationship between exposure to/infection by west nile virus and the occurrence of pix lesions. toxicities are not common in captive crocodilians. however, there have been cases of lead toxicity in alligators being fed nutria that had been killed with lead shotgun pellets. clinical signs include weakness, lethargy, anorexia, and death. alligator ranchers and farmers are now more diligent in inquiring about the source and kill method of nutria before feeding their animals. wild alligators, on the other hand, can be exposed to a number of toxic substances, mostly pesticides and heavy metals. many of these substances are thought to be endocrine dis rupters that affect the development and function of the reproductive organs and result in developmental defects of young crocodilians. [ ] [ ] [ ] [ ] [ ] there is an abundance of information to support the impact of environmental contamination on crocodilians as well as other species. this reiterates the critical state of the natural habitat of many species around the world. crocodilians have served as sentinels for anthropogenic changes that ultimately may come back to affect human health. runting is a phenomenon observed in captive crocodilian operations. it describes a general state of unthriftiness, lack of growth, and weakness. there is a visible size difference in same-age animals between the "runts" and the otherwise normally developing animals. it is not unusual to fi nd some buildings in a commercial operation that contain the runt animals for that year. it is also in this group of animals that disease problems may be observed with higher frequency. stress factors, including dominance by other animals, environment, and even incubation temperature, contribute to the presence of runts. fluid therapy of crocodilians is accomplished as in other reptile species. veterinarians must determine hydration status, percentage of fl uid defi cit, and maintenance requirements to develop a plan for fl uid therapy. a maintenance rate of to ml/kg can be administered intracoelomically (ice), orally (po), or intravenously (iv). in some instances, food prey items can be injected with fl uid and then fed to the animal. if the animal is anorexic, manual restraint will be required for fl uid administration. it is important to remember that restraint itself will be an additional stressor that may hinder the recovery of an animal. therefore, to limit the amount of stress in the animal, treatment protocols including fl uid therapy must be planned appropriately and administered quickly. antimicrobial therapy presents a challenge in crocodilians because of their extra label use. in commercial alligator facilities, antimicrobial treatment is complicated by the lack of drug withdrawal times for crocodilian species. currently the u.s. , enrofl oxacin was found to be effective when administered at mg/kg iv every hours. the longlasting oxytetracycline was found to be effective at mg/kg both iv and intramuscularly (im) every days. both of these treatment regimes may be practical in a zoologic institution or a commercial operation with a small number of animals. in a large commercial operation where a higher number of animals may need treatment, it may be time, cost, and labor prohibitive to administer medications intramuscularly or intravenously to individual animals. spectrum of coverage, method and frequency of administration, and cost should be considered when selecting an antimicrobial agent(s). consideration should be given to those drugs that can be mixed with, or injected into, the feed. one technique of antibiotic administration is making a sausage fi lled with the animal's feed and placing the drug in the sausage. manual medication by mouth or injection can be performed in smaller animals. whenever possible, culture and sensitivity test should be obtained to identify the organism in question and determine the effi cacy of the antimicrobial selected. unless a bacterial infection is suspected, use of antibiotics is strongly discouraged. most antimicrobial agents and their metabolites will be transported in the wastewater, potentially creating an avenue for future antimicrobial resistance. nutritional support of crocodilians should be approached as in other carnivorous reptiles. the main diffi culties result from the handling and restraint required to force-feed an ill crocodilian. nutritional support of an ill patient is more likely to occur at a zoologic institution than in a commercial operation. placing an esophagostomy tube should be considered as a less stressful alternative method of feeding the critical crocodilian. nutritional support through an esophagostomy tube will require restraint while feeding, but the stress level is reduced because the mouth does not have to be opened. any highprotein feed (e.g., commercial feed, fresh prey) that can be blended may be used as long as it can easily pass through the tube. in larger and aggressive animals, force-feeding is a serious challenge and must be performed carefully. various factors should be considered before performing a surgery procedure on a crocodilian, with the initial consideration being the ultimate purpose of the animal. is the animal part of a zoologic collection, is it from a commercial operation, or will it be released back to the wild? the next concern is the expected prognosis and time of recovery. ultimately the animal should be able to thrive in its future environment whether it be an exhibit or in the wild. cost of the procedure also is a major presurgical consideration. whereas cost may not be a major decision factor in some zoologic institutions, in other programs such as conservation programs, the money needs to be carefully allocated to obtain the highest return on the investment. the available facilities, especially for postoperative management, are also an important consideration. if the procedure requires placement of sutures, the animal should be maintained in a clean environment with shallow water for a few days to prevent infection and infi ltration of water into the surgical site. depending on the species, it may be possible to spray the animal with water throughout the day instead of keeping it in water. the fi nal consideration is the ability of the personnel to carry out the postoperative care, especially if force-feeding is required. it would be benefi cial to obtain a cbc and chemistry panel to assess the overall health status of the animal before surgery is performed. diagnostic test results will help determine if the animal is fi t for surgery and can be compared to postsurgical blood work to monitor recovery and healing, although in many instances diagnostic evaluation is an unrealistic expectation. to anesthetize crocodilian species, a basic understanding of their physiology and anatomy is needed to ensure safe delivery, induction, and maintenance of the anesthetic agent. there are various anesthetic agents, both injectable and inhalant, that can be used to anesthetize and sedate crocodilians. procedure type, species, and size are factors that will infl uence the choice of anesthetic agent(s). there are no established anesthetic protocols for crocodilians, and information is often derived from fi eld research as well as clinical experience of practitioners. injectable anesthetic agents can be delivered in the tail muscles via a pole syringe or hand syringe or administered intravenously after immobilization or restraint. neuromuscular blocking agents, such as gallamine and succinylcholine, are widely used in crocodilians. when transporting large, aggressive animals, veterinarians might use these agents to prevent injury to both the handlers and the animal. however, these drugs do not have analgesic properties and should not be used alone for any surgical procedures. when using neuromuscular blocking agents, veterinarians should ensure the animal is supported with a backboard to minimize injury to the spine. succinylcholine is a depolarizing neuromuscular blocker with primary action at the nerve end plate, and fl accid or rigid paralysis may occur depending on the dose. , there is no reversal agent for succinylcholine, so it must be used with caution and the animals monitored closely. effects can be observed within minutes of administration, and recovery takes up to . hours. recovery will depend on the excretion of the drug in the urine and metabolism of the crocodilian. gallamine is a nondepolarizing neuromuscular agent that competitively blocks acetylcholine at the motor end plate, resulting in fl accid paralysis. crocodilians immobilized with gallamine will open their mouths following relaxation of the muscles; this is known as the flaxidil reaction. increased respiratory rate and heart rate may be observed with gallamine immobilization. , an overdose of gallamine may lead to bradycardia, mydriasis, increased gastrointestinal motility, and paralysis of respiratory muscles. increased salivation and emesis can be prevented with presurgical use of atropine. an advantage of gallamine over succinylcholine is that it can be reversed with neostigmine methylsulfate. fatalities have been reported in american alligators (a. mississippiensis), chinese alligators (a. sinensis), and false gharials (tomistoma schlegelii) that were administered gallamine. diazepam can also be administered to minutes before succinylcholine or gallamine for added muscle relaxation. administration and recovery of animals with neuromuscular agents should be done far from the water to avoid drowning. noise and light stimuli should be minimized when animals have been sedated with gallamine or any other neuromuscular agent or anesthetic. table - shows doses for various neuromuscular agents. following administration of a neuromuscular agent, it may be possible to intubate a crocodilian for general anesthesia with an inhalant anesthetic such as isofl urane. remember to always provide analgesics if performing a painful procedure. injectable anesthestics have the added benefi t of providing analgesia and decreasing the amount of inhalant anesthetics used during surgery. dissociatives, opioids, benzodiazepines, and barbiturates have been administered to reptiles and croco-dilians with various rates of success. alpha- adrenergics are gaining popularity in reptile medicine and show promising results for their continued use as anesthetics in reptiles. a combination of medetomidine and ketamine works well in american alligators (a. mississippiensis) and has the added benefi t of being able to reverse the medetomidine with atipamezole. in a study involving juvenile alligators, the young animals had a higher requirement of medetomidine ( . ± . μg/kg im), atipamezole ( . ± . μg/kg im), and ketamine ( . ± . mg/kg im), compared to adult alligators' requirement of medetomidine ( . ± . μg/kg im), atipamezole ( ± μg/kg im), and ketamine ( . ± . mg/ kg im). these fi ndings are similar to those observed for other anesthetic agents used in reptiles. metabolic differences between age classes usually require higher doses for juvenile animals. an additional anesthetic agent that has gained popularity in reptile medicine is propofol (abbott laboratories, abbott park rd., abbott park, il - ). propofol is a lipid soluble drug with fast action and short duration of effects, which makes it an ideal anesthetic agent that allows for intubation of the animal to be followed with inhalant anesthesia. propofol can be administered at to mg/kg iv , and as a constant rate infusion. the iv route of administration of propofol presents a disadvantage in cases where manual restraint is not possible or is undesired. when choosing an anesthetic or immobilizing agent, veterinarians should take into account all the factors previously mentioned while keeping the safety of the animal and personnel in mind. fortunately, surgeries are not routinely performed in crocodilian species. the most common surgical procedure performed in zoologic institutions is probably the removal of foreign bodies through gastroscopy. this may be performed under immobilization or general anesthesia. enterotomies are rare but may be required if a foreign body lodges itself beyond the stomach, is too large to pass on its own, or both. the next most common procedure is the repair of fractures and lacerations caused by trauma. crocodilian fracture repair can present a real challenge to the veterinarian because of an animal's large size, demeanor, and need to live in an aquatic environment. therefore, it is preferred to perform fracture repairs with internal fi xation methods, such as intramedullary pins, cerclage wire, and bone plates. before attempting a surgical procedure, any veterinarian must realistically determine the prognosis for the animal. huchzermeyer fw: crocodiles: biology, husbandry and diseases an ancient sensory organ in crocodilians crocodilian anesthesia reptile medicine and surgery stocking rates of ranched american alligators (alligator mississippiensis) essential fatty acid nutrition of the american alligator (alligator mississippiensis) protein nutrition in the alligator alligator feed evaluations elevated lead levels in farmed american alligators (alligator mississippiensis) consuming nutria (myocastor coypus) meat contaminated by lead bullets unpublished description of a new site for venipuncture in the american alligator exotic animal formulary biotic and abiotic factors in crocodilian stress: the challenge of a modern environment stress-mechanisms of immunosuppression plasma catecholamines and plasma corticosterone following restraint stress in juvenile alligators effects of long-term corticosterone implants on growth and immune function in juvenile alligators (alligator mississippiensis) hormonal and metabolic response of juvenile alligators to cold shock growth rate and plasma corticosterone levels in juvenile alligators maintained at different stocking densities physiology and endocrinology of stress in crocodilians antibacterial properties of serum from the american alligator (alligator mississippiensis) naturally occurring antibacterial activities of avian and crocodile tissues isolation of aeromonas hydrophila from the american alligator (alligator mississippiensis) aeromonas-induced deaths among fi sh and reptiles in an eutrophic inland lake diseases of the eye and ocular adnexae in reptiles immobilization, blood sampling, necropsy techniques and diseases of crocodilians: a review initial antibiotic therapy for alligator bites: characterization of the oral fl ora of alligator mississippiensis gram-negative septicemia in american alligators (alligator mississippiensis) isolation of edwardsiella tarda from a sea lion and two alligators treatment of multiple cases of pasteurella multocida and staphylococcal pneumonia in alligator mississippiensis on a herd basis pathology of experimental mycoplasmosis in american alligators adenovirus-like infection in two nile crocodiles morbidity and mortality associated with a new mycoplasma species from captive american alligators (alligator mississippiensis) clinical values associated with opportunistic bacterial diseases in farm-raised alligators brown spot disease in the louisiana alligator industry: what we know about the disease and possible control protocols brown spot disease of commercially-raised alligators: a preliminary report colibacillosis in captive wild animals mycoplasma alligatoris sp nov, from american alligators aerobic intestinal fl ora of wild-caught african dwarf crocodiles (osteolaemus tetraspis) salmonella isolation from reptilian faeces: a discussion of appropriate cultural techniques recovery rates, serotypes, and antimicrobial susceptibility patterns of salmonellae isolated from cloacal swabs of wild nile crocodiles (crocodylus niloticus) in zimbabwe salmonella isolated from crocodiles and other reptiles during the period - in south africa hepatitis in farmed hatchling nile crocodiles (crocodylus niloticus) due to chlamydial infection public health risks of the fl esh of farmed crocodiles prevalence and serovar distribution of salmonella in fresh and frozen meat from captive nile crocodiles (crocodylus niloticus) microbial fl ora of frozen tail meat from captive nile crocodiles (crocodylus niloticus) salmonella in captive crocodiles (crocodylus johnstoni and c porosus) microbiological evaluation of dressing procedures for crocodile carcasses in queensland isolation of dermatophilus sp from skin lesions in farmed saltwater crocodiles (crocodylus porosus) pathology of skin diseases in crocodiles brown spot disease in the american alligator (alligator mississippiensis) experimental inoculation of broad-nosed caimans (caiman latirostris) and siamese crocodiles (crocodylus siamensis) with mycoplasma alligatoris in vitro susceptibility pattern of mycoplasma alligatoris from symptomatic american alligators (alligator mississippiensis) mycoplasma-associated polyarthritis in farmed crocodiles (crocodylus niloticus) in zimbabwe vaccination of farmed crocodiles (crocodylus niloticus) against mycoplasma crocodyli infection vaccination to control an outbreak of mycoplasma crocodyli infection a color atlas of diseases of the crocodile unpublished data from clinical and research cases, la herpes virus-like particles in the skin of a saltwater crocodile (crocodylus porosus) poxlike skin lesions in captive caimans pox virus infection in captive juvenile caimans (caiman crocodilus fuscus) in south africa poxvirus dermatitis outbreak in farmed brazilian caimans (caiman crocodilus yacare) poxvirus in farmed nile crocodiles poxvirus infection in nile crocodiles (crocodylus niloticus) poxvirus infection in two crocodile west nile virus in farmed alligators west nile virus infection in crocodiles alligators as west nile virus amplifi ers west nile virus in alligator (alligator mississippiensis) ranches from louisiana unpublished data on fungal isolates from the skin of american alligators (alligator mississippiensis) disease-husbandry associations in farmed crocodiles in queensland and the northern territory fatal mycotic pulmonary disease of captive american alligators cephalosporiosis in three caimans mycotic pneumonia caused by fusarium moniliforme in an alligator phycomycoses resulting in death of crocodiles in a common pool systemic mycotic disease of captive crocodile hatchling (crocodylus porosus) caused by paecilomyces lilacinus concurrent gout and suspected hypovitaminosis a in crocodile hatchlings hypoglycemic shock in captive alligators lymphohistiocytic proliferative syndrome of captive-reared american alligators (alligator mississippiensis) from louisiana unpublished data on lymphohistiocytic proliferative syndrome of alligators (lpsa) interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the american alligator abnormal bone composition in female juvenile american alligators from a pesticide-polluted lake sex reversal effects on caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol a affi nity of the alligator estrogen receptor for serum pesticide contaminants achieving environmentally relevant organochlorine pesticide concentrations in eggs through maternal exposure in alligator mississippiensis incubation temperature affects body size and energy reserves of hatchling american alligators (alligator mississippiensis) pharmacokinetics of enrofl oxacin after single-dose oral and intravenous administration in the american alligator (alligator mississippiensis) pharmacokinetic disposition of a long-acting oxytetracycline formulation after single-dose intravenous and intramuscular administrations in the american alligator (alligator mississippiensis) physical and chemical restraint of crocodilians gallamine reversal in cuban crocodiles (crocodylus rhombifer) using neostigmine alone versus neostigmine with hyaluronidase crocodilian anesthesia diazepam and succinylcholine chloride for restraint of the american alligator evaluation of medetomidine-ketamine anesthesia with atipamezole reversal in american alligators (alligator mississippiensis) key: cord- -utbuj iz authors: dear, jonathan d. title: bacterial pneumonia in dogs and cats date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: utbuj iz bacterial pneumonia is a common clinical diagnosis in dogs but seems to occur less commonly in cats. underlying causes include viral infection, aspiration injury, and foreign body inhalation. identification of the organisms involved in disease, appropriate use of antibiotics and adjunct therapy, and control of risk factors for pneumonia improve management. bacterial pneumonia remains one of the most common clinical diagnoses in dogs with either acute or chronic respiratory disease. new research suggests a complex relationship between viral respiratory diseases and development of bacterial pneumonia in dogs. over the past decade, much has been discovered about the convoluted interplay between host and environmental factors that leads to this complex of diseases. in cats, bacterial pneumonia is less commonly identified than inflammatory feline bronchial disease. aspiration pneumonia results from the inadvertent inhalation of gastric acid and/or ingesta and remains a common cause of bacterial pneumonia, accounting for roughly % of clinical diagnoses in a study of human patients admitted to the intensive care unit. although inhalation of gastroesophageal material is a common theme, different factors lead to the development of this phenomenon. risk factors that have been identified for the development of aspiration pneumonia include esophageal disease, refractory vomiting, seizures, prolonged anesthesia, and laryngeal dysfunction ( table ) . in a healthy animal, physiologic and anatomic features reduce the chance of aspiration. during a normal swallow, fluid and food are propelled caudally in the oropharynx and through the upper esophageal sphincter by contraction of the oral cavity and tongue. at the same time, the epiglottis retracts to cover the laryngeal aditus and protect the trachea from particulate inhalation. adduction of the arytenoid cartilages then contributes to further occlusion of the upper airways. any process impeding these primary defenses or inhibiting the normal swallowing reflexes increases the likelihood of aspiration. aspiration injury results from inhalation of either sterile, acidic gastric contents (resulting from vomiting or gastric regurgitation) or of septic material from gastric or oral secretions. irritation induced by acid inhalation promotes a local environment in which bacterial colonization can develop and lead to bacterial pneumonia. the severity of disease varies depending on the quantity and nature of the material aspirated as well as the length of time between the event and its diagnosis. conscious patients with intact airway reflexes tend to cough and prevent massive aspiration injury. animals under anesthesia or with reduced airway reflexes because of neurologic disorders are less likely to cough in response to the aspiration event and are, therefore, more likely to develop diffuse pulmonary infiltrates and acute lung injury. in many instances aspiration injuries occur under general anesthesia and the presence of a cuffed endotracheal tube does not prevent inadvertent aspiration. infectious, or community-acquired, pneumonias in dogs commonly begin with viral colonization and infection of the upper respiratory tract (canine respiratory coronavirus, herpesvirus, pneumovirus, and parainfluenza virus, among others). often, such diseases are acute and self-limiting, but in a subset of dogs inflammation associated with these organisms immobilizes the host's immune defenses and predisposes infection with other (often bacterial) respiratory pathogens. many bacteria have been implicated in canine infectious respiratory disease (cird), although special focus has been directed toward streptococcus (specifically streptococcus equi subsp zooepidemicus and s canis), mycoplasma cynos, and bordetella bronchiseptica. cird is especially prevalent in dogs naive to the pathogens and exposed in overcrowded, stressful environments such as animal shelters, boarding kennels, and treatment facilities. the pathophysiology associated with this disease and infectious lower respiratory tract disease in cats is discussed later in this article (boxes and ). inhaled foreign bodies carry mixed bacterial and fungal organisms into the lung and are associated with focal pneumonias that are often initially responsive to antimicrobial medications but relapse shortly after discontinuation of therapy. , foreign bodies reported in the veterinary literature include grass awns, plant materials, or plastic materials. organisms associated with grass awn inhalation include pasteurella, streptococcus, nocardia, actinomyces, and anaerobic bacteria. , most often, foreign material remains at the carina or enters caudodorsal principal bronchi (accessory, right and left caudal lobar bronchi). features associated with pulmonary foreign bodies include: young, sporting breeds environmental exposure to grass awns focal, recurrent radiographic alveolar pattern history of other cutaneous or visceral foreign bodies spontaneous pneumothorax or pyothorax box cird complex: changing the nature of kennel cough cird complex (formerly known as kennel cough) is a syndrome in which multiple pathogens, both viral and bacterial, coinfect either naive, immunocompromised dogs or previously vaccinated dogs. this complex is multifactorial and it is likely that both host and environmental factors play a role in the development of illness. organisms associated with this disease are ubiquitous, especially in overcrowded housing facilities such as animal shelters and training facilities. it is likely that stress induced by the new environment and exposure to novel pathogens both play a role in development of disease. in most cases, respiratory signs are present for days to weeks and most animals show mild to moderate clinical signs. viral infections typically cause either a bronchopneumonia or bronchointerstitial pneumonia because of their propensity to infect and damage type i pneumocytes. as the condition progresses, desquamation of the respiratory epithelium and aggregation of inflammatory cells further reduce the lungs' natural defenses, increasing the potential for secondary bacterial colonization and infection. previous studies have implicated viral organisms such as canine adenovirus or canine parainfluenza as major participants in cird, although recent studies have proposed novel respiratory pathogens such as canine respiratory coronavirus, , , , canine influenza virus, and canine herpesvirus as additional important pathogens associated with cird. b bronchiseptica, streptococcus canis, s equi subsp zooepidemicus, and m cynos , have been implicated as secondary bacterial infections associated with cird. s equi subsp zooepidemicus infections, in particular, have been associated with a rapidly progressive and often fatal hemorrhagic pneumonia. , some strains identified in outbreaks of this pathogen have been identified as resistant to tetracycline antibiotics, which are often the drug of choice prescribed for other bacterial pathogens associated with this complex. normal thoracic radiographs do not rule out the possibility of an airway foreign body and even computed tomography (ct) can fail to identify an affected bronchus. chronic pulmonary foreign bodies are associated with marked inflammation that can lead to massive airway remodeling and bronchiectasis that, when seen on radiographs, should raise the degree of suspicion for foreign body. ventilator-associated pneumonia (vap) is a common cause of hospital-acquired pneumonia in people, although there are few veterinary reports in the literature. colonization of the oropharynx by pathogenic and multidrug-resistant bacteria occurs and the endotracheal tube acts as a conduit to transmit pathogens into the airways, which leads to tracheobronchitis and potentially pneumonia. in addition, any animal with a compromised respiratory tract or serious systemic disease is particularly prone to development of infectious airway disease while hospitalized. the use of mechanical ventilation in human patients raises the risk of nosocomial infection by -fold to -fold. no published studies assess the risk in ventilated veterinary patients, although a study investigating differences in bacterial sensitivity between ventilated and nonventilated patients suggested that patients requiring mechanical ventilation were more likely to be infected with bacteria resistant to the antimicrobials most commonly used in veterinary practice. this finding parallels the increase in incidence of multidrug-resistant vap in human medicine. both the innate and adaptive immune systems protect against the development of infectious airway disease, and a breakdown in either increases the likelihood of opportunistic infection ( table ). congenital immunodeficiencies have been recognized that make an animal particularly sensitive to infectious disease. young animals are especially prone to the development of bacterial pneumonia because of their naive immune systems, and when coupled with alterations to the innate immune system, such as primary ciliary dyskinesia (pcd) or complement deficiency, the risk of life-threatening infection increases greatly (see veterinary clinics of north america ; ( ): - for a comprehensive review of respiratory defenses in health and disease). any cause of systemic immunocompromise increases the risk for bacterial pneumonia, and any additional alterations to the body's natural defense mechanisms increase the risk. medications such as chemotherapy, immunosuppressive therapy, or antitussive therapy significantly increase the likelihood of bacterial pneumonia. organisms that have been reported as lower respiratory pathogens of cats include pasteurella spp, escherichia coli, staphylococcus spp, streptococcus spp, pseudomonas spp, b bronchiseptica, and mycoplasma spp, and specific attention has been paid to mycoplasma spp because of a possible association with the induction and exacerbation of asthma in adult and pediatric human patients. however, the association between lower respiratory infection and chronic inflammatory lower airway disease in cats is unclear and is a topic of ongoing interest. mycoplasma species are considered normal flora in the upper respiratory tract and their role is controversial in lower respiratory tract infection. because they are rarely identified cytologically, and specific culture or polymerase chain reaction is needed to document the presence of these organisms, the role of mycoplasma in cats (as well as in dogs) remains difficult to define. underlying respiratory viruses or systemic viruses such as feline leukemia virus and feline immunodeficiency virus have the potential to enhance the severity of respiratory illness. clinical signs of bacterial pneumonia vary depending on its cause, severity, and chronicity of disease. they can be acute or peracute in onset or can display an insidious onset, resulting in chronic illness. early in disease, mild signs such as an intermittent, soft cough might be the only evidence of disease. as infection spreads, clinical signs worsen and often include a refractory, productive cough, exercise intolerance, anorexia, and severe lethargy. owners can note a change in the respiratory pattern, with increased panting or rapid breathing and, in cases of severe infection, cyanosis and orthopnea can be observed. in general, these systemic signs are more obviously displayed in dogs than in cats. cats with pneumonia can display similar clinical signs, although the cough can be misinterpreted as a wretch or vomit by owners. clinical signs and radiographic findings can also be considered consistent with inflammatory airway disease. as disease worsens, cats can become tachypneic with short, shallow breaths and nasal flaring. owners rarely notice exercise intolerance associated with bacterial pneumonia. as with the history and clinical signs of bacterial pneumonia, physical examination findings vary with the state and severity of disease. dogs or cats with mild disease can have no abnormalities detected on physical examination. an early clue to the diagnosis might be a change in the respiratory pattern, with an increase in rate and effort. the clinician needs to pay close attention to thoracic auscultation because adventitious lung sounds (crackles and wheezes) can be subtle, focal, or intermittent. in many cases, only harsh or increased lung sounds are detected rather than crackles. the examination should also include a thorough auscultation of the trachea and upper airway for evidence of upper airway signs (eg, nasal congestion or discharge) that can result from lower airway infection, either as an extension of epithelial infection or from nasopharyngeal regurgitation of lower airway secretions. animals with bacterial pneumonia generally present with mixed inspiratory and expiratory signs, similar to those seen with other diseases of the pulmonary parenchyma. fever is detected in % to % of cases, so it is not a reliable indicator of disease. , [ ] [ ] [ ] diagnosis bacterial pneumonia implies sepsis of the lower airway and lungs, so the diagnosis is confirmed by showing septic suppurative inflammation on airway cytology obtained through bronchoalveolar lavage (bal) or tracheal wash, along with a positive microbiology culture. in some cases, this is completed easily and yields results consistent with clinical suspicion. however, financial limitations or patient concerns can inhibit the ability to collect samples needed to document specifically a bacterial infection, and in those cases a clinical diagnosis of bacterial pneumonia might be presumed based on available information. a clinical diagnosis of bacterial pneumonia should be reached after obtaining compelling evidence to suggest a bacterial cause for the animal's clinical signs (after excluding other causes), with appropriate resolution of signs following suitable antimicrobial therapy. acute bacterial pneumonia is a common diagnosis in the small animal clinic and can often be easily identified; however, early and chronic pneumonias are more challenging to recognize because clinical signs can be subtle. the complete blood count is a useful diagnostic test in animals with respiratory signs. bacterial pneumonias are typically associated with an inflammatory leukogram, characterized primarily by a neutrophilia, with or without a left shift and variable evidence of toxic changes, , although the absence of inflammatory change does not exclude the possibility of pneumonia. , furthermore, the leukogram and differential can provide clues to suggest that bacterial pneumonia is less likely. for example, eosinophilia in an animal with respiratory signs would suggest eosinophilic bronchopneumopathy or parasitic lung diseases as an underlying cause rather than a bacterial cause. the erythrogram and platelet evaluation are generally not helpful in determining a bacterial cause of respiratory disease. a biochemistry panel, urinalysis, and fecal flotation do not always contribute to the diagnosis of bacterial pneumonia but can provide clues to the presence of metabolic or endocrine diseases that could make the development of bacterial pneumonia more likely. thoracic radiographs are crucial diagnostic tests in the evaluation of lower airway and pulmonary parenchymal disease. radiographic evidence of bacterial pneumonia can appear as a focal, multifocal, or diffuse alveolar pattern, although early in the disease process infiltrates might be primarily interstitial (figs. and ) . ventral lung lobes are most commonly affected in aspiration pneumonia, and a caudodorsal pattern is expected with inhaled foreign bodies or hematogenous bacterial spread. a lobar sign can be seen in cases of aspiration pneumonia in which the right middle lung lobe is most often affected ( table ) . three-view thoracic radiographs (left lateral, right lateral, and either dorsoventral or ventrodorsal views) should be obtained when screening for pneumonia because differential aeration associated with positional atelectasis can either mask or highlight pulmonary changes. for example, a radiograph taken in left lateral recumbency is preferred when aspiration is suspected because it increases aeration of the right middle lung lobe, the most commonly affected lobe. diffuse radiographic involvement is expected to suggest more severe disease, although radiographic changes lag behind clinical disease. consequently, bacterial pneumonia cannot be ruled out in patients with acute onset of clinical signs and unremarkable radiographs. advanced imaging is rarely necessary in the diagnosis of uncomplicated bacterial pneumonia, although it can be helpful in more complicated cases. thoracic ultrasound can be used to characterize peripheral areas of consolidation and to obtain fine-needle aspirates for cytology. cytology is often helpful in distinguishing inflammation from neoplastic infiltration. in addition, sonographic evaluation is particularly fig. . dorsoventral (a) and right lateral (b) thoracic radiographs from a dog with an alveolar pattern in the cranioventral lung lobes, suggesting aspiration. in this case, the left cranial lobes were most affected which are most easily examined on the right lateral view. in many cases the right middle lung lobe is most affected, necessitating a left lateral orthogonal view. useful in the detection of superficial foxtail foreign bodies when they remain in the periphery of the lobe (fig. ) . ct provides greater detail and resolution of lesions within the pulmonary parenchyma and gives the clinician better spatial information regarding the severity and extent of pulmonary involvement (fig. ) . in some cases, ct can be useful to identify migration tracts associated with inhaled foreign bodies. however, in most cases, general anesthesia is required for ct acquisition and prolonged recumbency can dear lead to atelectasis, which is difficult to differentiate radiographically from infiltrates. repeating the ct in a different position after providing several maximal inspirations can alleviate atelectasis. nuclear scintigraphy can be useful for the evaluation of ciliary dyskinesia, although secondary causes of mucociliary stasis (ie, infection with mycoplasma or bordetella, as well as exposure to smoke) must be excluded before assuming the diagnosis of pcd. because of the time necessary for image acquisition, magnetic resonance imaging is not commonly used for the diagnosis of most respiratory diseases. examination of the trachea and bronchial tree should be performed systematically. the endoscopist should note the color and character of the mucosa and any airway sections, making sure to evaluate all branches of the lower airways for evidence of foreign bodies, bronchiectasis, or collapse (diffuse or focal changes). airway mucosa in a normal animal should be pale pink with visible mucosal and pulmonary vessels. airway bifurcations should appear as narrow, crisp mucosal margins. animals with pneumonia can have hyperemia of the epithelium, prominent mucosal vessels, and evidence of airway inflammation, appearing as rounded, thickened airway bifurcations and airway nodules. airway secretions are usually opaque, viscous, and discolored (brown, yellow-green, or red tinged). when available, bal is preferred for collection of a lower airway sample rather than tracheal wash because the trachea and carina are not sterile, even in healthy dogs. in addition, the sensitivity for detecting cytologic features of sepsis is greater with bal than tracheal wash. however, when only a tracheal wash specimen can be obtained, because of the lack of equipment for bal or because of patient instability, collection of a lower airway sample is desirable to identify infecting bacteria and to determine appropriate antibiotic therapy through susceptibility testing. fig. . ct image of a dog with severe, diffuse pneumonia resulting from a chronic foxtail foreign body (see fig. ). the foreign body was not visible on thoracic radiographs, but is clearly evident in the left principal bronchus on this image. bal cell counts in animals with bacterial pneumonia are markedly higher than in patients with chronic bronchitis or other respiratory disease. septic, suppurative inflammation is a reliable indicator of bacterial pneumonia in dogs and is likely to indicate bacterial pneumonia in cats. in those cases that lack evidence of airway sepsis (intracellular bacteria), bal cytology generally reveals suppurative or mixed inflammation. in animals with suspected or confirmed foreign bodies, a bal sample should always be obtained from the affected airway and submitted individually for cytologic analysis. airway bacteria are more likely to be found in the cytologic sample from the site of the foreign body than from an alternate site. furthermore, cytology of bal samples obtained from multiple lobes can reveal different findings, even in cases of sterile inflammatory diseases like feline bronchial disease, thus reliance on a single-segment bal cytology could lessen the chance of yielding diagnostic results. diagnosis of bacterial pneumonia relies on identification of septic inflammation in conjunction with a positive bacterial culture. aerobic and mycoplasma culture and sensitivity are typically requested, and, in cases with markedly purulent secretions or a history of known aspiration or foreign bodies, anaerobic cultures should also be requested. samples should be refrigerated in sterile containers until submitted. if multiple alveolar segments are sampled during bal, these are usually are pooled for culture submission. cultures should always be performed when possible in order to guide appropriate antimicrobial therapy. with the liberal use of antibiotics, increasing populations of resistant microbes are being identified, particularly in patients with hospitalacquired pneumonia. , however, airway samples cannot be collected in all animals and, in those instances, judicious use of antibiotics must be followed. common bacteria cultured from lung washes of cats or dogs with bacterial pneumonia include enteric organisms (escherichia coli, klebsiella spp), pasteurella spp, coagulase-positive staphylococcus spp, beta-hemolytic streptococcus spp, mycoplasma spp, and b bronchiseptica ( table ) . , , pulmonary function testing arterial blood gas analysis is a useful test to measure the lung's ability to oxygenate. for patients with significant respiratory compromise, arterial blood samples ideally should be collected and analyzed to determine the severity of pulmonary disease. furthermore, trends in arterial oxygen partial pressures can be used to track progression or resolution of disease. in many cases, blood gas analysis is not available or patient factors preclude the acquisition of samples. pulse oximetry is a quick, noninvasive evaluation of oxygen delivery to body tissues that measures percentage of hemoglobin saturation with oxygen. it provides only a crude assessment of oxygenation and is subject to variability; however, trends in hemoglobin saturation can provide additional clinical support to progression or resolution of disease. treatment treatment of bacterial pneumonia varies considerably with the severity of disease, and appropriate antibiotic therapy is essential. the international society for companion animal disease is currently constructing guidelines for antibiotic therapy for respiratory infections. pending those guidelines, antibiotic recommendations from previous literature should be considered ( table ) . for stable animals with mild disease, outpatient therapy consisting of administration of a single, oral antibiotic is often all that is necessary. antimicrobial choices should ideally be based on culture and sensitivity results from airway lavage samples, although sometimes empiric therapy is more practical. regardless, in cases of severe pneumonia, initial empiric therapy should be instituted while awaiting culture results. antibiotics are typically administered for to weeks, and at least to weeks beyond the resolution of clinical and/or radiographs signs of disease. animals with more advanced disease require more intensive care, including hospitalization with intravenous fluids to maintain hydration. adequate hydration is essential to facilitate clearance of respiratory exudates. nebulization to create particles that enter the lower airways (< mm) can also enhance clearance of secretions. nebulizer types include ultrasonic devices, compressed air nebulizers, and mesh nebulizers. nebulization with sterile saline can be achieved by directing the hosing from the pneumonia in dogs and cats nebulizer into a cage or animal carrier covered in plastic. depending on how viscous secretions are, therapy can be provided for to minutes to times daily. in many cases, nebulization coupled with coupage helps the animal expectorate airway secretions. coupage is performed by cupping the hands and gently and rhythmically pounding on the lateral thoracic walls in dorsal to ventral and caudal to cranial directions. coupage should not be performed in animals with regurgitation because any increase in intrathoracic pressure could exacerbate regurgitation and subsequent reaspiration. supplemental oxygen is necessary for animals with moderate to marked hypoxemia (documented by a pao less than mm hg or oxygen saturation via pulse oximetry less than % on room air) in conjunction with increased respiratory effort. oxygen supplementation at % to % is provided until respiratory difficulty lessens and the animal can be weaned to room air. animals with refractory pneumonia that fail to improve on supplemental oxygen can succumb to ventilatory fatigue and need to be referred to an intensive care facility for mechanical ventilation. administration of an oral mucolytic agent such as n-acetylcysteine can be useful for animals with moderate to severe bronchiectasis that are prone to recurrent pneumonia. decreasing the viscosity of airway secretions might improve expectoration of fluid and debris that accumulates in dependent airways, although no published information is available on use of mucolytics in animals. n-acetylcysteine is typically not used via nebulization because of the risk of bronchoconstriction and epithelial toxicity. under no circumstances are cough suppressants (such as butorphanol or hydrocodone) appropriate for use in the management of bacterial pneumonia, particularly when it is complicated by bronchiectasis. by decreasing the cough reflex, these drugs perpetuate retention of mucus, debris, and other material in the airways and therefore hinder clearance of infection. also, furosemide should not be used because drying of secretions traps material in the lower airway and perpetuates infection. in cases in which aspiration pneumonia is suspected, strategies should be used to reduce the chance of reaspirating through appropriate treatment of the underlying condition. with disorders of esophageal motility, upright feedings of either slurry or meatballs can enhance esophageal transit. furthermore, diets low in fat can increase gastric emptying. in patients with refractory vomiting, antiemetic and prokinetic agents can be used to reduce the episodes of vomiting. drugs like maropitant (cerenia; mg/kg subcutaneously once daily) or ondansetron (zofran; . - mg/kg intravenously or subcutaneously once to twice daily) act peripherally and centrally to decrease the urge to vomit and are safe to use in both cats and dogs. the role of antacids in management of aspiration pneumonia remains controversial. by neutralizing the ph of gastric secretions, animals with refractory vomiting or regurgitation are less likely to succumb to chemical injury related to aspiration. however, in cases treated with acid suppression, the aspirant is likely to contain a greater concentration of bacteria that can colonize the lower airways and lead to bacterial pneumonia. no controlled studies have assessed the severity of aspiration pneumonia or the relative risk of using antacid therapy in dogs or cats. because radiographic findings lag behind clinical disease, recheck radiographs are not helpful early into the disease process, although they are useful to document resolution of disease and should be obtained within a week of discontinuation of antimicrobial therapy. in cases of refractory pneumonia, recheck radiographs midway through therapy can be used to assess resolution or progression of disease and help to guide further therapy. in animals suspected of having contagious or multidrug-resistant pathogens, appropriate contact precautions should be used. isolation gowns, examination dear gloves, and good hand washing technique along with appropriate quarantine facilities are essential to preventing transmission of disease to other patients or members of the health care team. prognosis for animals with bacterial pneumonia varies depending on the severity of disease, the animal's immunocompetence, and the virulence of the infectious agent. in general, between % and % of patients diagnosed with pneumonia are discharged from the hospital. , no long-term studies assess the overall prognosis of patients with multidrug-resistant bacteria or recurrent pneumonia. the outcome associated with these cases presumably will be worse. an -year-old female spayed chihuahua mix presented for a wet cough. cough had been present for months and there had been minimal response to antibiotics combined with a cough suppressant and no response to heart failure medication (furosemide, enalapril, and pimobendan). temperature ( . c [ f]), pulse ( beats per minute [bpm]), and respiratory rate ( breaths per minute) were normal. no murmur was auscultated and lung sounds were normal. chronic cough in a small-breed dog is often associated with airway collapse or chronic bronchitis; however, infectious and neoplastic disease must remain on the differential list. congestive heart failure is unlikely given the lack of a heart murmur and the lack of response to diuretic therapy. a white blood cell count was normal ( cells/ml) with neutrophils. thoracic radiographs revealed scattered bronchial markings in the caudal thorax (fig. ) . fluoroscopic examination did not reveal evidence of tracheal or airway collapse. laryngoscopy indicated lack of abduction of the arytenoid cartilages consistent with bilateral laryngeal paralysis. secretions were evident throughout the upper and lower airways. diffuse airway hyperemia and irregularities of the mucosa were apparent. bal cytology was remarkable for septic suppurative inflammation, and bacterial cultures were positive for pasteurella, mycoplasma spp, and anaerobic bacteria, consistent with an aspiration cause. a -year-old male castrated domestic medium hair cat was presented for evaluation of acute respiratory distress. lethargy and anorexia had been noted days before the onset of respiratory signs. temperature ( . c [ . f]) and pulse ( bpm) were normal. tachypnea was noted (respiratory rate, breaths per minute) with increased respiratory effort on inspiration and expiration. diffuse expiratory wheezes were auscultated. acute onset of respiratory difficulty in a cat is most commonly related to inflammatory airway disease. the physical examination is consistent with this diagnosis, although it is uncommon for affected cats to show lethargy and anorexia. infectious and neoplastic diseases were also on the differential diagnosis list, along with aspiration and foreign body pneumonia. thoracic radiographs revealed a focal opacity in the left caudal lung lobe and a diffuse bronchial pattern (fig. ) . complete blood count revealed a normal white blood cell count ( /ml) with a left shift ( /ml neutrophils, /ml bands). bronchoscopy with lavage was performed. a moderate amount of airway hyperemia and edema was noted along with purulent material obstructing several airways. bal cytology showed increased cellularity ( , normal cells/ml) with neutrophilic inflammation ( %, normal %- %). neutrophils contained dark blue granular debris, suspicious for sepsis. aerobic and anaerobic cultures were negative but a pure culture of mycoplasma was isolated on special medium. a diagnosis of mycoplasma bronchopneumonia was made. community-acquired aspiration pneumonia in intensive care units. epidemiological and prognosis data potential risks, prognostic indicators, and diagnostic and treatment modalities affecting survival in dogs with presumptive aspiration pneumonia: cases mycoplasmas and novel viral pathogens in canine infectious respiratory disease community-acquired infectious pneumonia in puppies: cases capnocytophaga cynodegmi in a rottweiler dog with severe bronchitis and foreign-body pneumonia the role of bronchoscopy in foreign body removal in dogs and cats: cases radiographic, computed tomographic, and ultrasonographic findings with migrating intrathoracic grass awns in dogs and cats airway microbial culture and susceptibility patterns in dogs and cats with respiratory disease of varying severity ventilator-associated tracheobronchitis and pneumonia: thinking outside the box bordetella bronchiseptica infection in cats. 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korean dogs clinical factors associated with death before discharge and overall survival time in dogs with generalized megaesophagus age-related thoracic radiographic changes in golden and labrador retriever muscular dystrophy primary ciliary dyskinesia in newfoundland dogs immunoglobulin deficiency in cavalier king charles spaniels with pneumocystis pneumonia x-linked severe combined immunodeficiency in the dog comparison of clinical signs, diagnostic findings, organisms isolated, and clinical outcome in dogs with bacterial pneumonia: cases ( - ) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus textbook of respiratory disease in dogs and cats. st louis (mi): saunders the association of streptococcus equi subsp zooepidemicus with canine infectious respiratory disease the seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in new zealand tropism and pathological findings associated with canine respiratory coronavirus (crcov) nosocomial outbreak of serious canine infectious tracheobronchitis (kennel cough) caused by canine herpesvirus infection recent advances in canine infectious disease. international veterinary information service mycoplasmas associated with canine infectious respiratory disease streptococcus zooepidemicus: an emerging canine pathogen lower respiratory tract infections in cats: cases ( - ) mycoplasma pneumoniae in children with acute and refractory asthma key: cord- -bgygebgx authors: lundgren, a.-l. title: feline non-suppurative meningoencephalomyelitis. a clinical and pathological study date: - - journal: journal of comparative pathology doi: . / - ( ) -m sha: doc_id: cord_uid: bgygebgx abstract a spontaneous neurological disease in cats characterized by behavioural and motor disturbances was investigated by clinical, morphological and immunological methods. neuropathological examination showed a marked inflammatory reaction in the cerebral leptomeninges and the grey matter of the brain. in the white matter, the reaction was moderate. the changes consisted of perivascular cuffing by mononuclear cells and neuronal damage. the brain stem (thalamus, mesencephalon, caudal colliculus) was most severely affected. the spinal cord and its leptomeninges were involved to a lesser degree. the histopathological picture as well as the laboratory findings suggests a viral cause of the disease. the morphology of the disease and serological as well as immunohistochemical results indicate that this disorder is different from previously known feline viral encephalitides. feline non-suppurative meningoencephalomyelitis comprises a group of diseases which apparently are related. the syndrome seems to be geographically widespread since cases have been reported from australia (borland and mcdonald, ) , the united states (vandevelde and braund, ) and switzerland (hoff and vandevelde, ) . suspected cases have also been recorded in morocco (martin and hintermann, ) and sri lanka (mcgaughey, ) . a similar clinical condition affects lions, tigers and other large cats (flir, ; melchior, ; gutter, wells and baskin, ; truyen, stockhofe-zurwieden, kaaden and pohlenz, ). in , kronevi, nordstrgm, moreno and nilsson reported the occurrence in sweden of a feline neurological disorder with the histological features of a non-suppurative meningoencephalomyelitis. thirty cats were affected, seven of which were examined post .mortem. these showed, throughout the brain and spinal cord, mononuclear perivascular cuffing, gliosis and meningitis. the lesions were most pronounced in the brain stem. the authors thought the disease was a specific entity, but did not express an opinion as to its aetiology. serological screening for viral agents was not performed by kronevi et al. ( ) and efforts to isolate a virus yielded negative results. since the report by kronevi et al. ( ) , the disease has become recognized in certain parts of sweden and is referred to as "staggering disease". in , strijm reviewed the clinical findings in cases of staggering disease recorded from till . the author performed serological screening for feline leukaemia virus (felv), feline immunodeficiency virus (fiv) , feline coronavirus (fcov), toxoplasma gondii and borrelia burgdorfeeri in some of the diseased cats. the results were negative with the exception of one cat, which was positive for antibodies to fiv. there have been doubts as to whether staggering disease is a specific entity. it has been argued that the syndrome may include several aetiologically unrelated conditions affecting the central nervous system of cats, e.g. toxoplasmosis (hirth and nielsen, ) and the cerebral form of feline infectious peritonitis (slauson and finn, ; kornegay, ) . in order to establish whether staggering disease is a specific entity, it was decided to perform a systematic and thorough neuropathological examination of a larger number of cats than were included in the original report by kronevi et al. ( ) . furthermore, it was decided to discuss the possible aetiology of the disease based on modern knowledge of feline infectious agents. the present investigation details the pathological and clinical features of staggering disease and includes a search for aetiological agents with the use of immunohistochemistry and serology. from january till march a total of cats was referred to the department of pathology, faculty of veterinary medicine, uppsala, for post mortem examination. sixty-three of the cats were diagnosed as having staggering disease. from this group, cases that were clinically well documented were chosen for the present investigation. clinical examination and treatment of most of the cats took place at the university clinics, faculty of veterinary medicine, uppsala. the author performed clinical examination of six cats as well as interviews with their owners. fifteen cats were examined by several small animal veterinarians at the university clinics. four cats were clinically examined at private veterinary clinics and afterwards referred to the department of pathology for necropsy. one cat died of the disease. the others were killed by pentobarbital overdose. all cats with staggering disease were investigated post mortem by the author. tissue specimens were fixed in buffered per cent formalin for light microscopy. the brain and spinal cord were removed from all cats for examination. coronal sections of the brain included the frontal, parietal, temporal and occipital lobes, basal ganglia, hippocampus, thalamus, mesencephalon, caudal colliculus, cerebellum and obex. from the spinal cord, sections were taken from cervical, thoracic and lumbar segments. the number of cases in which the different parts of the brain and spinal cord were examined are shown in table . in cases, portions of the proximal sciatic nerve were taken for histological examination. portions of the eyes and sartorius muscles were taken from eight cats. the cranial mesenteric ganglion was examined in four cases. samples were removed from the liver in , the kidney, heart and spleen in , the lung, mandibular gland and small intestine (jejunum) in , the pancreas in and the retropharyngeal lymph nodes in five cats. paraffin wax sections were cut l.trn thick and stained with haematoxylin and eosin (he). sections of the basal ganglia, hippocampus, thalamus, mesencephalon, caudal colliculus, obex and the spinal cord were stained with luxol fast blue, nissl, giemsa, phosphotungstic acid haematoxylin, gomori's reticulin and lendrum's stain for inclusion bodies in selected cases. to estimate the inflammatory changes, the presence of perivascmar cuffs and inflammatory nodules consisting of lymphoid cells and macrophages (as described by leestma, ) was recorded in each examined section of the central nervous system (table ) . perivascular cuffs were counted in at least low power fields (lpf) with a diameter of mm and rated + = to j/lpf, + + = to g/lpf, + + + = > g/lpf. the thickness of the perivascular cuffs was rated from to where cone cell thick, = to cells, = to cells, = more than cells. the size of the inflammatory nodules was rated from to where =a few cells, =moderate cluster of cells, = extensive cellular infiltration. the presence of inflammation in the leptomeninges was graded from to with regard to the degree of cellular infiltration where = slight, = moderate, =severe. in five cats, material from the parietal lobe, basal ganglia, thalamus, mesencephalon, caudal colliculus, cerebellum and pons were fixed in buffered per cent formalin for h and then examined for the presence of pseudorabies virus antigen, canine distemper virus (cdv) antigen and toxoplasma gondii antigen, by the peroxidase-antiperoxidase (pap) immunohistochemical method (sternberger, ) with polyclonal primary rabbit antibodies kindly su rijksuniversiteit, ghent, belgium (pseudorabies), lied by p. de groot c. rmell swedish national bacteriological laboratory (cdv) institute ( toxoplasma gondii). and a. uggla, swedish national veterinary blood chemistry and csf analysis complete blood analysis was performed in cats. the tests included haemoglobin concentration, total white blood cell count (wbc) and differential cell count, packed cell volume (pcv) and alanine aminotransferase and urea concentrations. in three other cats, only haemoglobin concentration and wbc were analysed. cerebrospinal fluid (csf) of seven cats was obtained by cisternal puncture and examined for colour, turbidity, protein content, total red (rbc) and white blood cell (wbc) counts and differential cell count. serology serum from cats was tested for the presence of felv antigen and antibodies to fiv using an enzyme-linked immunosorbent assay (elisa) kit (cite-combo-test, idexx corp., portland, me, u.s.a.). s erum was tested for antibodies to fcov in nine cats with an elisa kit supplied by svanova biotech, uppsala, sweden. in seven cats, serum was tested for antibodies to borrelia burgdorferi with an indirect immunofluorescent antibody (ifa) test (burgess, ) with a human borrelia burgdorferi isolate as antigen. in five cats, serum was tested for antibodies to tick-borne encephalitis virus (tbev) by the haemagglutination inhibition (hi) method with l-day-old chicken erythrocytes starting with serum diluted in (kunz, hofmann and dippe, ) . some of the cats with staggering disease came from urban, but mostly ( per cent) from rural surroundings of uppland and malardalen, sweden. cases of the disease occurred throughout the year, but most often from december to may. the cats were aged to years, the mean age being . years. fifteen cats were male ( per cent), twelve of them neutered. twenty-one cats ( per cent) were short or long haired domestic cats, two were siamese, one abyssinian and one a norwegian forest cat. twenty cats ( per cent) were allowed to roam freely outdoors. one cat came from a home where two other cats previously had been affected by staggering disease. four cats came from households where there were other cats unaffected by the disease. one cat had experienced several upper respiratory tract infections during a period of months before showing signs of staggering disease. three to months before showing the first signs of staggering disease, eight cats ( per cent) were infested with ticks. the vaccination status of most cats was unknown; six had been vaccinated against feline panleukopenia. the most striking clinical manifestations (table ) were a stiff staggering gait, inability to jump up and down normally and into-ordination of the hindlegs followed by weakness and paresis. some cats lost their ability to retract their claws. in addition to these motor disturbances, many cats showed mental changes. cats previously shy became social and affectionate, mewing more than usual, while cats that were customarily cheerful and affectionate became introverted and shy. aggressive behaviour was rare. depression, loss of appetite and dehydration were seen in many cases. less frequent signs included pruritus, hypersensitivity to sound and light, increased salivation, impaired vision, staring gaze, hyperaesthesia, constipa- tion, tremor, circling and seizures. the rectal temperature was measured in cats. nine of these ( . per cent) were febrile ( > * "c). in the final stages of the disease the hindlegs were paralysed. all cats remained conscious to the end. most cats were treated with antimicrobial drugs such as tetracycline, ampicillin and chloramphenicol. intravenous fluids and b-vitamins were administered in many cases. some cats were given corticosteroids. despite treatment, the condition of the cats deteriorated and most cats had to be killed after to weeks illness. two cats were severely ill from the onset of the disease and had to be put down in less than a week. a few cats recovered incompletely. the only cat who died naturally of the disease did so after weeks. a moderate leukopenia ( . to . x " cells per ) was found in five of cats examined. in two of the cats with leukopenia, the differential cell count was normal. the other three cats with leukopenia showed mild neutropenia and mild lymphocytosis; the neutrophil-lymphocyte ratio being : , : and : , respectively (normal range of the neutrophil count: to per cent; normal range of the lymphocyte count: to per cent). one cat in the final stage of the disease exhibited a wbc count of . x ' cells per and a neutrophil-lymphocyte ratio of : . haemoglobin concentration (normal value to g per ) and pcv (normal value to per cent) were normal except for slightly raised values in dehydrated animals. alanine aminotransferase (normal value < . pkat per ) was moderately raised in four cats (n= ). urea concentration (n= ) was normal ( . to -o mmol per ) in cats and slightly raised in one cat ( -o mm per ). cerebrospinal fluid (table ) was transparent and colourless in all cases, showed a moderate elevation in protein content (normal value < mg per dl) in six cats and an increased wbc count (normal value < cells per pl) in four cats. all sera examined for antibodies against fiv and presence of felv antigen were negative. six of the cats tested for antibodies against fcov were negative (titre < in ). in two cats the fcov antibody titre was in and in , respectively. all sera examined for antibodies against borrelia burgdorferi and tbev were negative. pathology central nervous system. one cat that had been ill for months exhibited a slight thickening and opacity of the cerebral leptomeninges. in the other cases, the brain and spinal cord did not show any abnormal changes on naked eye inspection. histopathological examination revealed throughout the central nervous system a non-suppurative inflammation characterized by perivascular mononuclear cuffing, presence of inflammatory nodules and neuronal degeneration in all cats. perivascular cuffs occurred within virchow-robin spaces and rbc= red blood cells, * after correction for blood contamination (fenner, around small vessels (fig. ) . the cell population of the cuffs consisted of lymphoid cells, histiocytes, occasional plasma cells and macrophages laden with lipid-like material. the cuffs were one to six cells thick and sometimes seemed to compress the lumen of the vessels. the endothelial cells were swollen, but vascular thrombosis and vasculitis were not seen. neurones adjacent to cuffed vessels sometimes appeared shrunken and uniformly acidophilic. perivascular cuffing was less prevalent and less severe in the white matter than in the grey matter. inflammatory nodules consisting of aggregates of lymphoid ceils and macrophages, as described by leestma ( ) , were frequently seen in the grey matter. neuronal degeneration and neuronophagia (fig. ) occurred in all parts of the cns except for the cerebellum. inclusion bodies were not identified. some large neurones, such as the hypoglossal nerve cells, contained small, cytoplasmic, eosinophilic granules. these were not further characterized. meningitis was present over all parts of the brain, but was most prevalent over the cerebral cortex and cerebellum (fig. ) . spinal cord meningitis was slight and sometimes absent. the meningeal inflammatory cells were of the same type as in the cuffs. in the cat that had been ill for months there was a moderate fibrosis of the cerebral leptomeninges. on the basis of classification and scoring of lesions, a localization pattern emerged (table ; fig. ). the most severe inflammatory changes were seen in the grey matter of the brain stem (thalamus, mesencephalon, caudal colliculus), basal ganglia and hippocampus. moderate inflammation was observed in the cerebral cortex. parenchymal lesions in the cerebellum were slight or absent, whereas cerebellar meningitis was severe. in the medulla oblongata, lesions were not as extensive as in the more proximal sections of the brain stem. at all levels of the spinal cord, inflammatory changes were moderate and mostly confined to grey matter. the lesions occurred in the ventral and dorsal horns with no apparent predilection for either place. degeneration of myelin was sometimes observed in the spinal ventrolateral tracts. the spinal nerve roots did not show any abnormalities. immunohistochemical examination by the pap method for the antigens of pseudorabies virus, cdv and toxoplasma gondii yielded negative results. positive control sections were stained and examined in all cases. other tissues. five cats were in poor physical condition. the urinary bladder of two cats was severely distended with urine. marked constipation of the rectum was evident in four cats. one cat had multiple small cysts in the cortex of the kidneys. other gross lesions were not observed. the liver of three cats showed varying degrees of fatty change. the cortex of the kidney of cats exhibited small interstitial collections of lymphocytes, histiocytes and plasma cells. the retropharyngeal lymph nodes showed follicular hyperplasia and abundant accumulation of lymphocytes in cortex and paracortex in five cats. in eight cats, the lymph follicles of the spleen were reduced in number. the germinal centres, although large in size, were markedly lacking cells and in some cases showed degenerative changes of the central area (karyorrhexis, karyolysis and formation of an amorphous eosinophilic material). depletion of lymphoid cells was evident in the marginal zones of the follicles and in the periarteriolar lymphatic sheath (t cell area). small perivascular lymphocytic accumulations were seen in the cranial mesenteric ganglion of two cats (n = ). the eyes and skeletal muscle showed no pathological changes. there were no abnormalities in the sciatic nerve of any of the cats examined. neuropathological examination of the cats of the present study showed a marked inflammatory reaction in the cerebral leptomeninges as well as in the grey matter of the brain and spinal cord. in the white matter, inflammatory changes were moderate. the reaction was characterized by perivascular mononuclear cuffing, neuronal damage and presence of inflammatory nodules consisting of lymphoid cells and macrophages. these changes are typical of viral infection (leestma, ) . the findings of degeneration and depletion of lymphoid cells in the follicles of the spleen also seem to point to an infectious agent. the laboratory findings, including leukopenia and elevations in protein content and wbc count of the csf, are not specific for viral infection, but at least suggest that the disease in the cats of this study is infectious in its nature. the abundance of neurological signs in cats with staggering disease is a reflection of a widespread failure of the nervous system, typically seen in disseminated inflammations. the most notable viral agents causing progressive multifocal involvement of the cns in cats are feline infectious peritonitis virus (fipv), feline leukaemia virus (felv) and feline immunodeficiency virus (fiv). any of these viruses as well as some other agents known to cause cns disorders in cats could be the cause of staggering disease. the central nervous system form of feline infectious peritonitis (fip) is manifested clinically in personality changes, posterior paresis, nystagmus and seizures (pedersen, ; kornegay, ) . lesions in the brain and spinal cord consist of pyogranulomatous meningitis, encephalitis and ependymitis (slauson and finn, ; legendre and whitenack, ; kornegay, ) . inflammation of choroid plexuses and the mesencephalic aqueduct results in blockage of normal csf flow and secondary hydrocephalus (krum, johnson and wilson, ; bar-lough and summers, ) . in the present investigations, cns lesions showed a different pattern of localization compared with fip. there was no predilection for choroid plexuses and ependyma. vasculitis, a common finding in fip, was not present. although cases of pure neurological fip exist, most cats also have involvement of the eyes and/or other organs (kornegay, ) . in the present investigation, all major lesions were confined to the cns. the results of serological investigation for coronavirus antibodies do not support the view that fipv or other coronaviruses are plausible aetiological agents in feline non-suppurative meningoencephalomyelitis. the finding of a coronavirus antibody titre of in in one cat is not significant since titres of this size are also seen in many cats that have had previous infections with feline enteric coronavirus (fecv), a virus closely related to fipv (pedersen, a) . feline leukaemia virus (felv) has been reported as a cause of encephalitis in cats (sottiaux and pialat, ) . however, felv is more commonly associated with epidural lymphosarcoma, which is usually manifested as acute posterior paresis or paralysis (pedersen, b) . neither the serological results nor the clinical and histopathological findings in the cats with staggering disease indicate a felv infection. feline immunodeficiency virus (fiv) has emerged as an important cause of neurological disease in cats (dow, poss and hoover, ; sparger, ) , often in association with clinical syndromes typical of an immunodeficient state (chronic stomatitis, enteritis, dermatitis, etc). neurological signs include psychotic behaviour, dementia, seizures and ataxia. brain lesions are confined to the thalamus and midbrain, sparing the cerebral cortex and cerebellum (dow et al., ) . non-suppurative encephalitis with perivascular mononuclear cuffing and glial nodules is commonly observed. antibodies against fiv were not found in the cats of the present study. seroconversion in fiv infection may be delayed for months or even years (sparger, ) . also, cats in the terminal stages of fiv infection will sometimes show undetectable concentrations of antibody. however, the absence of clinical signs of immunodeficiency in cats with staggering disease argues against fiv as a causative agent. the nervous system in cats is susceptible to feline panleukopenia virus infection during the prenatal and early postnatal period, resulting in cerebellar hypoplasia (greene and scott, ) . both clinical and histopathological data (csiza, de lahunta, scott and gillespie, ) are different from the cats of this report. in addition, six of the cats with staggering disease had been vaccinated against feline panleukopenia. sweden is a rabies-free country and staggering disease has neither the clinical signs nor histological features typical of rabies. aujeszky's disease (pseudorabies), which is fatal in carnivores (dow and mcferran, ) , is excluded because of differences in the clinical picture, absence of intranuclear acidophilic inclusion bodies in neurones and astroglia and negative immunohistochemical staining for virus antigen. canine distemper virus (cdv) has been reported as a cause of encephalomyelitis in tigers (blythe, schmitz, roelke and skinner, ) . domestic cats are susceptible to experimental infection with cdv, but natural disease has not been reported in cats (appel, sheffy, percy and gaskin, ) . immunohistochemical staining for cdv antigen yielded negative results in cats with staggering disease. encephalitis has been induced experimentally in cats with the viruses of newcastle disease (luttrell and bang, ) ) b orna disease (ihlenburg, ) , near eastern equine encephalomyelitis (daubney and mahlau, ) and human poliomyelitis (salvioli, gotti and sternini, ) . it is not known whether these viruses naturally infect domestic cats, but there are no reported cases in the literature. keane, parent and little ( ) inoculated one cat intracerebrally and six cats intravenously with powassan virus of the flavivirus serogroup. none of the cats developed neurological signs although histological lesions of a nonsuppurative encephalomyelitis were observed in two cats. tick borne encephalitis virus (tbev) of the flavivirus group is transmitted by the tick ixodes ricinus in sweden. most of the cats of the present study were allowed to roam freely outdoors in regions harbouring ixodes ricinus and there is a possibility that this tick could be the vector of the aetiological agent causing staggering disease. however, cats with staggering disease have tested negative for antibodies to tbev, which indicates that this virus and probably other related viruses of the flavivirus serogroup can be excluded as the primary cause of the disease. toxoplasma gondii, the most important non-viral infectious agent causing cns disease in cats, is ruled out because of negative immunohistochemical staining for . gondii antigen, absence of tissue cysts and absence of extraneural lesions. borrelia burgdorferi, a cause of arthritis and possibly meningitis in dogs (greene, ) has not been reported as a cause of neurological disease in cats. all cats tested for antibodies to borrelia burgdorfeeri in the present study were negative. in conclusion, the neuropathological features as well as the serological and immunohistochemical results in the cats with staggering disease are not compatible with any of the known spontaneous feline viral diseases affecting the cns. neither are they compatible with toxoplasmosis. the feline meningoencephalomyelitis of the present investigation is doubtless identical with that described by kronevi ef al. ( ) and shows many similarities to the cases of unknown aetiology reported in switzerland by hoff and vandevelde ( ) . six cases of polioencephalomyelitis in cats were reported in the united states (vandevelde and braund, ) . clinical signs included ataxia, tremors and seizures. although the pathological lesions in the cns were qualitatively similar to the lesions in the cats of the present study, the localization pattern was different. in the american cats, the most severe lesions were observed in the spinal cord and consisted of a marked degeneration and loss of neurones as well as white matter degeneration. in a german safari park, an outbreak of fatal encephalomyelitis in lions and tigers occurred in the early s (flir, ; melchior, ) . the clinical as well as the histopathological picture in these large cats closely resembled that of our cats. many lions became more affectionate and infantile and assumed a strange staring gaze. this increased affection, a phenomenon often observed in staggering disease, is not reported as a conspicuous feature of any of the known feline infectious cns disorders; neither is it mentioned in the other reports of feline meningoencephalomyelitis of unknown aetiology cited above. the histological features in the lions and tigers were those of non-suppurative polioencephalomyelitis, with the most severe lesions in the brainstem. efforts to isolate virus from the cns failed. in recent years, a further outbreak of the disease was observed in the same park (truyen et al., ) . attempts to isolate a virus from the cns again failed, but feline herpes virus type (fhv- ) was isolated from the tonsil of one lion. this was interpreted as an infection superimposed on the encephalomyelitis as a result of reduced resistance and was not regarded as the primary cause of the disease. fhv- is well known as the cause of feline rhinotracheitis. what is against fhv-i as the aetiological agent in staggering disease is the fact that fhv-i is regarded mainly as a breeding cattery problem, the virus being transmitted through intimate contact between cats (pedersen, c) . in contrast, staggering disease is almost unheard of-in breeding catteries and does not appear to be as contagious as feline rhinotracheitis. kittens are more severely affected by fhv- than adult cats, while staggering disease has not been observed in cats younger than months. the role of fhv- in feline cns disorders has not yet been clarified, however, and the possibility that fhv- or related herpesviruses could be involved in the cause of staggering disease remains open. in conclusion, some previously recorded cases of idiopathic, feline, non-suppurative meningoencephalomyelitis presented clinical and pathological similarities to our cases suggesting a common or related aetiology. viral infection is the most likely cause of staggering disease. the possibility of an arthropod-borne mode of transmission has been suggested. given the consideration that most cats were freely roaming outdoors, rodents could be alternative sources of infection since they are the hosts of some neurotropic viruses, e.g. theiler's virus and lymphocytic choriomeningitis virus. another possible mode of transmission may be through bites and scratches exchanged between cats in fights over territory. inoculation by bite wounds has been shown to be the major mode of transmission of feline immunodeficiency virus (fiv) and the fact that male cats are more often affected by staggering disease than females gives some support to the theory that staggering disease could be transmitted by bites in a similar way. further serological and virological investigations in order to identify the cause of the presently described feline non-suppurative meningoencephalomyelitis are required. this work is now in progress. canine distemper virus in domesticated cats and pigs encephalitis due to feline infectious peritonitis virus in a twelve-week-old kitten chronic encephalomyelitis caused by canine distemper virus in a bengal tiger feline encephalomyelitis experimental inoculation of dogs with borrelia burgdorferi. zentralblatt f$r bakteriologie spontaneous feline ataxia near-eastern equine encephalomyelitis aujeszky's disease in the dog and cat feline immunodeficiency virus: a neurotropic lentivirus the neurologic evaluation of patients encephalomyelitis bei grosskatzen. deutsche tier&tliche wochenschrifi feline panleukopenia. in infectious diseases of the dog and cat lyme borreliosis. in infectious diseases of the dog and cat neurologic disease in three cats at the audubon park zoo pathology of feline toxoplasmosis non-suppurative encephalomyelitis in cats suggestive of a viral origin experimentelle prufung der empfanglichkeit der katze fur das virus der bornaschen krankheit california serogroup and powassan virus infection of cats feline infectious peritonitis: the central nervous system form feline ataxia due to nonsuppurative meningoencephalomyelitis of unknown aetiology hydrocephalus associated with the noneffusive form of feline infectious peritonitis viral infections of the nervous system feline infectious peritonitis with spinal cord involvement in two cats newcastle disease encephalomyelitis in cats infectious myelitis of cats: preliminary communication sur i'existence, au maroc, d'une maladie contagieuse du chat, non encore d&rite: la myilite infectieuse meningo-enzephalitis beim lowen und tiger feline infectious peritonitis: something old, something new feline infectious peritonitis virus infection feline leukemia virus infection feline herpesvirus type-l infection effetti polimorfi nel gatto da inoculazione di materiale poliomielitico. rivista dell'lstituto sieroterapico italiano meningoencephalitis and panophthalmitis in feline infectious peritonitis meningo-encephalite associte a une infection felv chez un chat. pratique medicale et chirurgicale de i'animal de compagnie feline immunodeficiency virus immunocytochemistry, nd edit vingelsjuka hos katt a case report: encephalitis in lions. pathological and virological findings polioencephalomyelitis in cats the author is grateful to the staffs of the departments of virology, pathology and bacteriology of the swedish national veterinary institute for carrying out the serological and immunohistochemical investigations. thanks are due to dr per bierke for assistance with evaluation of the lymphatic tissues and to drs egenvall, stavenborn and tistedt of the department of medicine and surgery, faculty of veterinary medicine, for performing clinical examination of cats with staggering disease. this work was supported by grants from the albert hjarre foundation, agria insurance company and the swedish fund for research without animal experiments. key: cord- -tfktgy authors: creech, c buddy title: it’s true even in a pandemic: children are not merely little adults date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: tfktgy nan m a n u s c r i p t though separated from family and friends due to physical distancing, my wife's grandfather celebrated his th birthday in april . born in , in the middle of the first world war, he is now experiencing his nd global pandemic and the attendant reality of human mortality. in his toddler years, he belonged to a high-risk population for pandemic influenzachildren less than years old, adults > , and uniquely, young adults - years of age were disproportionately affected. now, a century later, he finds himself again in a high-risk populationadults > years of age and living in a long-term care facility. strangely, and to his delight, his great-great grandchildren (of whom there are many) do not seem to share this increased risk for disease. his endurance and repose during the intervening years point to a principle that each new global threat to human health brings with it something old and something new. pandemic influenza and coronavirus share transmission characteristics that allow for rapid and dense spread through communities. they share a predilection for severe disease, particularly in high risk hosts, while they also benefit from asymptomatic hosts and minimally symptomatic individuals who are capable of transmission prior to obvious signs of disease. an unusual and new feature of sars-cov- is the apparent reduced infectivity and relatively low frequency of severe disease in children, an unusual characteristic of a highly contagious respiratory virus. understanding these similarities and differences will be critically important as we move forward through this pandemic. in this issue of cid, mehta et al (cid paper) provide a systematic review of pediatric covid- , evaluating the available literature to date to glean characteristics of disease and transmission. though limited by the availability of case reports and case series, many from epicenters in china and italy, the authors were able to identify a number of studies that were of sufficient quality and granularity to warrant inclusion in the review. consistent with local and national experiences, children appear to be less affected by covid- than adults. the authors report that children represent only % or less of diagnosed covid cases and the data available at the time of review suggest that children are less likely to develop either severe pneumonia or the laboratory alterations commonly associated with severe disease, such as lymphopenia and elevated inflammatory markers. a c c e p t e d m a n u s c r i p t the authors also report that intrauterine transmission appears to be extremely uncommon and that newborns born to infected mothers are likely to experience either asymptomatic disease or mild disease. taken together, it would appear that children experience a very different response to sars-cov- infection than adults and raises the hypothesis that dysregulated host responses may be the primary driver of disease severity. in fact, targeting the host response to infection has become the focus of multiple clinical trials, including baricitinib, tocilizumab, ravulizumab, eculizumab, and a host of other drugs aimed at blunting an exuberant immune response. using a similar approach, zimmermann et al recently evaluated available cases of disease in children and neonates [ ] . in this review, approximately % were asymptomatic and coinfections (namely mycoplasma and influenza) have been identified in many children, complicating how providers attribute symptoms to sars-cov- . pregnancy outcomes were similarly encouraging, though fetal distress was reported in up to % of pregnancies and preterm delivery complicated approximately % or reported cases. the authors raise a number of hypotheses that may explain the differences in phenotypes from adults, including differences in immune response, fewer co-morbidities, microbial interactions in the nasal mucosa, differences in ace receptor density in the nasal epithelium [ ] , and others. the report, and emerging data from the uk and us, particularly new york city, are particularly intriguing given previous hypotheses regarding the potential role of coronaviruses in the pathogenesis of kawasaki disease [ ] . in addition, larger case series of pediatric cases are becoming available [ ] , giving even greater clarity on epidemiology, transmission characteristics, and disease manifestations. teachers, administrators, and support staff in local schools and childcare facilities belong to high risk groups as a result of age or comorbidities. we have a duty to protect these individuals as much as possible. moreover, we must recognize that some proportion of children that are infected will experience either moderate disease, severe disease, or immunologically mediated complications, such as mis-c. defining the true burden of disease across pediatrics will be essential as we consider best practices for the start of the - academic year. my wife's grandfather would likely summarize the issue by saying there is 'nothing new under the sun.' unfortunately, the vast majority of us have not been around the sun as many times as he has. we are still learning what we know, what we don't know, and what we think we know that just isn't so. as it relates to children, early reports are encouraging, but the emergence of immunologically mediated complications must give us pause. in this pandemic, at least thus far, children are not merely mirrors of the adult experience but bring their own challenges in diagnosis and treatment. a c c e p t e d m a n u s c r i p t children, pregnancy and neonates nasal gene expression of angiotensin-converting enzyme in children and adults an outbreak of severe kd-like disease at the italian epicentre association between a novel human coronavirus and kawasaki disease epidemiology of covid- among children in china key: cord- - mw kpmr authors: mcvey, scott; shi, jishu title: vaccines in veterinary medicine: a brief review of history and technology date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: mw kpmr the use of vaccines in veterinary medicine has progressed from an experimental adventure to a routine and relatively safe practice. the common and aggressive use of efficacious vaccines has been responsible for the control and eradication of several diseases. despite progress in research technologies, diagnostic capabilities, and manufacturing methods, there remain many infectious diseases for which no effective vaccines exist. global availability, field compliance, effectiveness, and safety are also significant concerns. this review addresses the history, current practices, and potential future improvements of vaccine use in veterinary medicine. become a routine component of medicine. problems with consistent potency, available supply, purity, and safety were common. nevertheless, both the effectiveness and imperfections of vaccination lead to the eventual global eradication of smallpox, and was the inspiration for development of the products and programs for immunization against several diseases in humans and animals. louis pasteur first used the term vaccine in for immunogens directed at other diseases besides smallpox. pasteur directed many investigations that demonstrated the feasibility of attenuating or inactivating microbes. studies with fowl cholera and anthrax led to the concepts of chemical inactivation as a means to reduce the virulence of microorganisms. , studies with erysipelas and rabies explored serial passage in animals (lapinization or passage through rabbits) or other animal derived tissues as an alternative strategy to reduce or eliminate virulence. thus, the virulence of infectious microbes could be completely or partially reduced. these studies have led to the eventual successful control of anthrax and rabies in particular. the work of salmon and smith ( ) clearly demonstrated that some microbes could be completely inactivated (killed). these developments eventually led to successful immunization programs against typhoid fever, tuberculosis, rinderpest, and foot and mouth disease (fmd). attenuation and inactivation principles were extended to microbial toxins by the work of gaston ramon at the pasteur institute. a tetanus toxoid was developed in through heat and formalin inactivation of the toxin to form an ''anatoxin.'' also, enhanced efficacy was provided by absorbing the toxoid to an aluminum hydroxide, providing an adjuvant effect. these process and formulation improvements were developed and refined in the early twentieth century, first through production of equine sera with antidiphtheria and antitetanus toxin-neutralizing antibody for prophylactic use. in the modern era of vaccine use, these same basic technologies are still the mainstays of vaccine production. however, new generations of recombinant, nucleic acid and subunit vaccines have become available. it is remarkable that the principles of developmental research, registration, and manufacture still follow the techniques of the grand heritage. during the early years of the modern era of vaccine production, infected tissues were often used as a source of microbial antigens through grinding, inactivation (typically with formaldehyde solutions), and subsequent filtration or clarification. more often than not these vaccines were produced in regional research institutions. industrialization of the processes began in the s and s when large-scale, controlled processes were used to produce fmd antigens in germany by waldmann and colleagues. the development of first primary and subsequently clean cell lines occurred in the s and s. development of high-volume roller bottle methods and later large-scale bioreactors has made possible the production of millions of doses of vaccines. further, production has been maintained in secure, closed systems, enhancing the security for the environment as well as the technical staff. in like manner, improvements in inactivation technologies (cyclized binary ethyleneimines), purification and concentration of antigens, storage of bulk antigens, improved aluminum gels, and oil suspension adjuvants in the formulation of polyvalent antigens have been critical achievements in the steady advancements in vaccinology. as these technical advances were employed in the industry, independent and collaborative efforts by numerous governmental authorities created regulatory frameworks that have established regulations and guidelines for registration of new biologicals as well as consistent manufacture of pure, safe, and potent vaccines. under these regulations all released lots of vaccines are tested to ensure consistent formulation characteristics and potency (immunologic strength), safety, and purity (sterility and freedom from contamination with extraneous biologic agents). development of good manufacturing practices guidelines and master seed and master cell stock concepts has further ensured consistent manufacture of vaccines that will provide consistent immunogenicity and efficacy. a veterinary clinician therefore may use with confidence any approved vaccine as recommended by the manufacturer to achieve the anticipated clinical outcome of protection. as the vaccine manufacturing processes improved with regard to consistency of biologic activity, robustness, and efficiency, routine clinical use of vaccines became more practical and economical. there is no doubt that widespread use of efficacious vaccines has been associated with the global eradication of smallpox in humans and the regional control of fmd and rabies. the routine use of processed immunoglobulins (usually in the form of processed horse serum) preceded the use of vaccines. although passive protection by the immunoglobulins is still employed (particularly for rabies and tetanus post exposure prophylaxis), the advantages of active immunity (immunologic memory and reduced risk of infection) have significantly reduced the use of passive immunity. in the mid- s, veterinarians were commonly using rabies vaccines of brain tissue origin in dogs. the principal biologic products used in practice at that time were rabies vaccines, ''viabilized'' canine distemper/hepatitis virus vaccine and antisera, hog cholera and erysipelas vaccines and antisera, leptospirosis bacterins, and clostridial toxoids (fig. ) . as the development and manufacturing capacity increased with time, vaccination of companion animals expanded to include rabies for cats, feline herpesvirus, parvovirus in cats and dogs, and feline calicivirus. table describes the types of vaccines currently available to companion animal practitioners in most regions of the world - (http://www.aphis.usda.gov/animal_health/vet_biologics/ vb_licensed_products.shtml) these vaccines include very traditional inactivated antigen formulations, multiple attenuated agents, and new technologies such as poxvectored vaccines, defined subunit vaccines, and nucleic acid vaccines (see table ). the term vaccine is now used to describe many therapeutic or prophylactic formulations and products that stimulate active immunity in the vaccinated animal. this discussion focuses on vaccinations associated with infectious diseases. routine clinical use of these vaccines usually includes immunization of puppies and kittens at approximately -week intervals after maternal-derived antibody decreases to noninterfering titers. these immunization series are usually administered between the fourth and th weeks of life. , puppies and kittens associated with unusual risk may be vaccinated at younger ages or at more frequent intervals. rabies vaccination is usually first given at months of age. it is a common and efficacious practice to provide booster doses at year of age for most vaccines. these immunization practices will provide a solid duration of immunity of at least to years and longer in some cases. general recommendations (world small animal veterinary association) are to vaccinate every third year after the initial immunization series, and these recommendations are consistent with product label guidelines. these initial immunization guidelines are derived from the initial registration immunogenicity and efficacy studies for any individual vaccine product. the efficacy studies define the minimum immunologic strength for the vaccine (the potency that must be present when the vaccine lot goes out of date). these same types of studies also define the minimum age of animals that can be successfully immunized as well as the specifics of the initial and booster immunization regimens (part , code of federal regulations). it has vaccines in veterinary medicine become very clear that many vaccines provide effective and long-term immunity for an extended period of time. over the past decades, cumulative evidence for extended duration of immunity has been provided to support the -year booster intervals for most vaccines in dogs and cats. however, as described in table , the relative efficacy of some vaccines is less than ideal. ''ideal'' immunity would be not only protection from clinical disease (morbidity and mortality) but also blocking the infection/replication/spread or progression of infectious agents. some vaccines do achieve this degree of protection. some, however, may only reduce morbidity and/or mortality without generating a sterilizing immunity. based on clinical and microbiological outcomes of an efficacy study challenge of immunity, various degrees of protection may be achieved and therefore claimed. the united states department of agriculture (usda) has recognized these differences through a hierarchy of efficacy claims that may be allowed for a vaccine based on the outcomes of efficacy studies (box ). the degree of efficacy and claim structures are usually derived from direct investigations of efficacy and challenge of immunity studies in their respective host animal species. vaccinated and nonvaccinated animals are challenged with fully virulent organisms, and the degree of protection (efficacy) is determined under controlled settings. these classic studies are adequate to establish the efficacy of the vaccine but are not always sufficient to estimate the field effectiveness of a vaccine, or, in other words, the ability of a vaccine to control disease in the field. effective control of infectious disease should result in reduced incidence and prevalence. , this would be true of not only clinical disease but also of infection and spread of the infectious agent. it is very clear that use of efficacious products has reduced incidence of rabies, particularly in dogs. immunization of dogs has reduced the incidence of canine rabies to essentially nil in the united states and western europe. the rabies immunization programs in these countries have been so effective that most manufacturers of rabies vaccine for dogs and cats have switched to master seeds from canine street strains of virus to other types of terrestrial rabies (bat strains, for instance) to protect from the most significant current threats in these regions. vaccination has also greatly reduced the incidence of canine distemper, canine parvovirus, infectious canine hepatitis, feline panleukopenia, and feline herpes virus infections as well as other diseases. when these diseases do occur, there are usually issues with vaccine dose compliance, vaccination of sick or immunocompromised animals, exposure to wildlife, or problems associated with vaccine handling and/or administration. in situations where vaccines do not provide prevention of infection, concurrent infections may exist and vaccine failures are therefore more common. there are often issues with type-specific protections. for instance, it is not clear that available vaccines can protect cats against all types of calicivirus infections. continual vigilance is required to ensure continued protection of animals in the face of potential newly evolving and emerging pathogens (eg, rabies and other lyssaviruses, canine distemper and parvoviruses, and feline calicivirus). the vaccines used in veterinary medicine generally fall into of categories: inactivated vaccines (in which antigens are typically combined with adjuvants); attenuated, live vaccines; and recombinant technology vaccines, which may include subunit antigens or genetically engineered organisms. in practice, combination and multivalent vaccines may employ all approaches. all of these technologies have been used successfully, and each approach has inherent advantages and disadvantages. the protective mechanisms associated with vaccines are also becoming clearer. historically, the most common correlate of immunity to derive from vaccination has been measurements of antibody responses. , antibodies have several functions including facilitating opsonization, complement-mediated cellular lysis, neutralization-blocking adherence or replication, and facilitating cytotoxic cells. however, mature, well-differentiated immune responses are the consequence of cumulative, regulated interactions between phagocytic cells, antigen-presenting cells, and both b and t lymphocytes. therefore, a well-differentiated antibody response with isotype switching, affinity maturation to high avidity, and memory requires some effective initial stimulation involving dendritic cells and expansion of regulatory t lymphocytes a claim that it is intended to prevent disease may be made only for products shown to be highly effective in preventing clinical disease in vaccinated and challenged animals. the entire % interval estimate of efficacy must be at least %. if so, a label statement such as ''for the prevention of disease due to [specific microorganism]'' may be used. - . . aid in disease prevention. a claim that it is intended to aid in disease prevention may be made for products shown to prevent disease in vaccinated and challenged animals by a clinically significant amount which may be less than that required to support a claim of disease prevention (section . . ). if so, a label statement such as ''as an aid in the prevention of disease due to [specific microorganism]'' may be used. - . . aid in disease control. a claim that it is intended to aid in disease control may be made for products which have been shown to alleviate disease severity, reduce disease duration, or delay disease onset. if so, a label statement such as ''as an aid in the control of disease due to [specific microorganism]'' or a similar one stating the product's particular action may be used. (likely cd ) and b lymphocytes. this stimulation phase is followed by a phase of differentiation into effector/memory t cells, b cells, and plasma cells. with respect to the nature of pathogenesis of many infectious agents, the adaptive immune response to the vaccine often blocks or interferes with a specific segment of the infection process. for instance, antibody-mediated neutralization of rabies virus in extracellular spaces inhibits transmission to neurons and subsequent axonal progression of the virus to the central nervous system. in this case the presence of preformed, neutralizing antibody is critical for protection. a summary of protective characteristics of the immune responses to vaccines (as potential correlates of protection and disease prevention) is provided in table . although antibody responses are good correlates of protection, they do not always reflect all available protective mechanisms provided by a well-differentiated immune response. in some cases, other correlates are available. it is clear that the presence of neutralizing, vaccine-derived antibody will reduce mucosal virus replication, virus shedding, and viremia in kittens vaccinated with modified live feline herpes vaccines. [ ] [ ] [ ] however, regulated cd and cd cellular responses are required to control tissue damage and reactivation of disease. in this case, antibody may be a protective correlate of infection while cellular immunity is a protective correlate of disease. the ability of modified live vaccines to generate a very rapid onset of cytokines and interferons (and rapid antigen focusing in dendritic cells in lymphoid tissues) is associated with a rapid onset of protection, even though antibody responses may not be detectable in serum for up to weeks. [ ] [ ] [ ] therefore, the early response of multiple cytokines and concurrent activation of the innate immune system may serve as early correlates of protection. there are also documented cases in which functional immunity outlasts detectable circulating antibody; this is true with many herpesvirus infections. however, the presence of detectable neutralizing serum antibody is correlated with protection against recrudescent disease. in situations where vaccinated animals may be exposed to heterotypic viruses or bacteria, the presence of immune cd t cells specific to conserved antigens may be very important for protection. it is possible that the effective mechanisms for development of protection associated with a vaccine may be specific to the nature of the disease and infectious process. recent studies have provided important information regarding this phenomenon. a common hypothesis is that vaccine-induced immunity should reflect convalescent immunity following natural infection. for example, it is known that recovery from primary poxvirus infections requires robust cytokine responses, natural killer cells, and antibodies as well as t helper (cd ) and cytotoxic t (cd ) lymphocyte effector functions. however, recovery from a secondary infection requires only t-and b-lymphocyte interaction and an anamnestic antibody response. again, neutralizing antibody will reduce infection, viremia, and spread of a virus (and may do so to the extent of blocking infection) while t-cell-mediated responses will allow survival and recovery. it seems clear that balanced antibody and cellular responses are necessary for complete protection from infection and disease as well as spread to other animals. it should be mentioned that not all antigen-binding antibodies are protective. in some cases, such as with influenza virus, canine distemper virus, and herpesvirus vaccines, nonneutralizing antibody may be produced that does not contribute to the blocking of infection or enhancing clearance of the infectious agent. , for this reason, correlates or surrogates of protection should be linked to protective mechanisms; this can be done through retrospective analysis of data from efficacy and immunogenicity studies or through associational studies in immune populations (such as with primary vaccinates in an efficacy study). veterinary vaccinology has realized significant successes that have affected human and animal well-being, and the ability to coexist. the virtual elimination of canine rabies in north america and western europe has indirectly led to human-animal bonding at a very intimate level that was not feasible when canine rabies was relatively common. however, there remain many diseases for which no efficacious or effective vaccine exists. many parasitic diseases as well as diseases of a chronic, intracellular nature are not covered by any available vaccine. in some cases safety profiles or efficacy characteristics of existing vaccines are not acceptable. fortunately, there are promising technologies that may close the technical gaps for prevention of these challenging diseases. the processes of absorption of antigens such as chemically inactivated toxoids or viruses to aluminum gels, or the creation of water-in-oil emulsions of antigen particles have been the principal methods used for veterinary vaccine formulations. in some cases compounds such as crude or purified saponins (quil a), squalenes, or pluronic block copolymers have been added to enhance immune stimulation. , although these practices have been successful, newer technologies such as cpg dna, defense peptides, imidazoquinolones, and polyphosphazenes may enhance both safety and vaccines in veterinary medicine efficacy. , further, additional cholesterol and phospholipids may be combined with antigens and saponins to create immunostimulating complexes (iscoms) particles. similar adjuvant particles can be generated with no antigen (iscomatrix) that can be admixed directly with antigen suspensions. these advanced formulations may be used to provide very efficient adjuvants to in turn allow development of microvolume formulations as well as transdermal applications. also, as better understanding of immune genotypes and phenotypes in animal populations emerges, individualized formulations of vaccines may be developed and produced that may enhance safety and efficacy. proteomic technologies may very well provide methods to identify antigen subsets from among complex organisms and infectious agents such as bacteria and protozoa. these organisms contain large, complex genomes. antigen expression is often dependent on growth conditions, and the medium may be very complex. these conditions are difficult to reproduce and regulate in vitro. the combination of transcriptional and proteomic analysis may provide a means to identify key antigens associated with tissue or cellular persistence and potential virulence. such analyses could provide means to simplify vaccine formulations to include only protective antigens and reduce the presence of nonprotective, potentially interfering bacterial proteins. not only would this potentially improve efficacy, but it could also improve safety profiles by reducing the antigenic mass in a vaccine dose. the continued use of alternative expression systems has many potential advantages. transgenic expression of protein antigens and plant-based systems may provide access to oral vaccines as well as enhanced stability of antigens. expression of antigens in avirulent viruses, bacteria, and yeast and insect cells may provide both manufacturing and user safety by eliminating the need to use a virulent or partially virulent microbe to provide immunity. further development of nucleic acid vaccines may provide even greater formulation simplicity and biosecurity. viral particles such as capsids from avirulent viruses may serve as building blocks to deliver nucleic acids, protein subunit antigens, and microadjuvants directly to secondary lymphoid tissue. not only would these biologically engineered vaccines provide targeted immunity and eliminate the need to work with dangerous microbes, they very likely would reduce the time required for the onset of immunity, with excellent safety characteristics. one of the most pressing problems associated with manufacturing vaccines is the requirement to rapidly modify antigen formulations as new diseases emerge or as older pathogens mutate and reemerge. transcriptomics and proteomics combined with established recombinant or synthetic approaches could potentially provide antigens that could be rapidly formulated with approved new-generation adjuvants to produce novel and efficacious vaccines. these technologies are commonplace in experimental laboratories. using combinations of proteomics, reverse genetics, recombinant or molecular syntheses, and stable, consistent adjuvant platforms will allow development of ''first line of defense'' vaccines for a rapidly emerging disease in a short time. such a use-inspired approach to vaccines would allow the use of assembly-line techniques to manufacture vaccines. as new antigens are required they could be selected, evaluated, and produced in a short period of time, and inserted directly into an established production system. this process would greatly reduce the time required for exploratory research and early development. classic development cycles may require to years and sometimes may require even longer times for unusual or new types of pathogenic microbes. a reduction of the development time by % to % may be achievable using newer research and development technologies. it is clear that new methods to assess efficacy and definitive, direct correlates of immunity also need to be identified. it is also clear that use of the many new technical achievements and discoveries will require advances in the regulatory framework to ensure more efficient but adequate evaluation of new biologicals. vaccine development faces many technical, political, and ethical challenges. the history of vaccine research and development as well as the continued use of immunization as the principal method to prevent infectious disease predict that the innovative experimental procedures of today will lead to common clinical applications tomorrow. china and the origins of immunology a brief history of vaccines and vaccination an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england. particularly gloucestershire, and known by the name of the cow pox. london: samson low; de l'atttenuation du virus du cholera des poules compte rendu sommaire des experiences faites a pouilly-le fort, pres melun, sur la vaccination charbonneuse. comptes rendus de l'academie des method pour prevenir la rage apres morsure. comptes rendus de l'academie des on a new method of producing immunity from contagious diseases sur la toxine et l'anatoxine diphtheriques. pouvir floculant et proprietes immunoantes die aktive immunisierung des rindes gegen maul und klauenseuche vaccines and vaccination: the principles and the polemics duration of immunity for canine and feline vaccines: a review guidelines for the vaccination of dogs and cats. compiled by the vaccination guidelines group (vgg) of the world small animal veterinary association (wsava) review of companion animal viral diseases and immunoprophylaxis current vaccination strategies in puppies and kittens emerging aspects of rabies infection: with a special emphasis on children use of molecular epidemiology in veterinary practice rabies re-examined vaccines: correlates of vaccine-induced immunity: an official publication of the infectious diseases society of america vaccination and antigenic drift in influenza onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus feline panleukopenia virus, feline herpesvirus- , and feline calicivirus antibody responses in seronegative specific pathogenfree cats after a single administration of two different modified live fvrcp vaccines antibody and cell-mediated immune responses to feline herpesvirus following inactivated vaccine and challenge long-term immunity in cats vaccinated with an inactivated trivalent vaccine correlates of protection: novel generations of influenza vaccines correlates of protective immunity in poxvirus infection: where does antibody stand? identification of two proteins associated with virulence of streptococcus suis type strategies to link innate and adaptive immunity when designing vaccine adjuvants innate immunity and new adjuvants vaccine immunopotentiators of the future personalized vaccines: the emerging field of vaccinomics proteomic technology in the design of new effective antibacterial vaccines plant-based oral vaccines: results of human trials innovative vaccine production technologies: the evolution and value of vaccine production technologies trends affecting the future of vaccine development and delivery: the role of demographics, regulatory science, the anti-vaccine movement, and vaccinomics key: cord- - quf td authors: jung, sung-mok; kinoshita, ryo; thompson, robin n.; linton, natalie m.; yang, yichi; akhmetzhanov, andrei r.; nishiura, hiroshi title: epidemiological identification of a novel pathogen in real time: analysis of the atypical pneumonia outbreak in wuhan, china, – date: - - journal: j clin med doi: . /jcm sha: doc_id: cord_uid: quf td virological tests have now shown conclusively that a novel coronavirus is causing the – atypical pneumonia outbreak in wuhan, china. we demonstrate that non-virological descriptive characteristics could have determined that the outbreak is caused by a novel pathogen in advance of virological testing. characteristics of the ongoing outbreak were collected in real time from two medical social media sites. these were compared against characteristics of eleven pathogens that have previously caused cases of atypical pneumonia. the probability that the current outbreak is due to “disease x” (i.e., previously unknown etiology) as opposed to one of the known pathogens was inferred, and this estimate was updated as the outbreak continued. the probability (expressed as a percentage) that disease x is driving the outbreak was assessed as over % on december , one week before virus identification. after some specific pathogens were ruled out by laboratory tests on january , the inferred probability of disease x was over %. we showed quantitatively that the emerging outbreak of atypical pneumonia cases is consistent with causation by a novel pathogen. the proposed approach, which uses only routinely observed non-virological data, can aid ongoing risk assessments in advance of virological test results becoming available. a cluster of cases of atypical pneumonia with unknown etiology in wuhan, china attracted global attention towards the end of [ , ] . an impressive series of rapid virological examinations ruled out common pneumonia-causing viruses such as influenza viruses, adenoviruses, and the coronaviruses associated with middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) [ ] [ ] [ ] [ ] . early in the outbreak, the causative agent was suspected to be a coronavirus of non-human origin [ , ] . the coronavirus was subsequently found to be a relative of sars and named the severe acute respiratory syndrome coronavirus (sars-cov- ) [ , ] . while examination of the viral genome was critical for identifying the pathogen, information made publicly available in real time describing clinical characteristics and other outbreak-related factors also allowed experts to consider the etiology and thereby differential diagnoses. for instance, most cases shared a history of visiting or working at a seafood market in wuhan [ ] , where exposure to the novel coronavirus is suspected to have occurred with no evidence of direct human-to-human transmission [ ] , although human-to-human transmission was found later to be common. observed characteristics of the outbreak led us to believe that the cluster of cases was due to "disease x" (i.e., an infectious disease of previously unknown viral etiology). however, rigorous quantitative assessment based on these characteristics of the chance that the manifestations of atypical pneumonia were in fact disease x has not previously been undertaken. the present study addresses this, demonstrating that non-virological information can lead to an objective classification of disease x, using a simple statistical model that exploits the well-known bayes' theorem. as the outbreak unfolded, we calculated in real-time the probability that the pathogen responsible for the atypical pneumonia cases was novel (disease x), as opposed to the outbreak instead being generated by a previously known pathogen that can cause atypical pneumonia. our analysis began on december , when the wuhan municipal health commission announced that there had been a surprisingly large number of atypical pneumonia cases. at that time, we assumed the causative agent could have been one of eight known viral or three known bacterial pathogens, along with the chance that it was instead disease x. we tracked two active medical social media sites (promed [ ] and flutracker [ ] ) that collected reports of the non-virological characteristics of the outbreak as it progressed. these characteristics were basic observations from the outbreak and do not necessarily represent the features that were causing symptoms. given these characteristics, we then calculated the probability that the ongoing outbreak was due to a known pathogen or unknown disease x. on the first day of calculation (i.e., december )-the day that we became aware of the outbreak-the only explanatory factor we included was diagnosis of atypical pneumonia, which was common to all pathogens considered in our dataset. our analysis represents simple logical deductions from the limited data that were available during the outbreak in a quantitative manner and was updated to reflect new information about the outbreak as it became available in real time. table shows the information compiled about the current outbreak, and the dates on which each of these characteristics were discovered. each characteristic listed was assigned a value of zero or one, denoting whether or not the outbreak characteristic was likely in general (rather than for individual cases) for the emerging outbreak, and the equivalent values for outbreaks of previously observed pathogens were also noted. we note that some information believed at the time was later found to be untrue; for example, it was believed that human-to-human transmission was infrequent. consequently, inclusion of a large number of characteristics is important for our analysis. once pathogens were ruled out as the causative agent of the current outbreak, they were removed from our analysis: for example, highly pathogenic avian influenza (hpai; h n ) was confirmed not to be the causative agent by laboratory testing on january . hence, we omitted this pathogen from our analysis from that date onwards. we performed two versions of our analysis to demonstrate how our results might change with the inclusion of different outbreak characteristics. in the first, all characteristics in table were included in the analysis. in the second, information about the exposure location (i.e., exposure at a wet market) was excluded from the analysis. zeros represent characteristics that are unlikely for outbreaks of that pathogen, and ones represent characteristics that occur. dates and characteristics for the ongoing outbreak were obtained from two online information systems [ , ] , and information for other pathogens was summarized from the pathogen-specific pages on the who and cdc websites. to assess the probability that the emerging outbreak was caused by a known pathogen, we first calculated the distance between the set of characteristics of the ongoing outbreak and those of previously known pathogens. the distance between the characteristics of the ongoing outbreak and cases due to pathogen j is denoted by d j. we assumed that the probability that the outbreak is due to a variant of pathogen j decreased exponentially with distance d j . then, by bayes' theorem, pr(pathogen j | observed characteristics) = pr(observed characteristics pathogen j)q j i pr(observed characteristics | pathogen i)q i ( ) in which the sum in the denominator is over all possible pathogens i (i.e., each of the columns of table , including the column describing the current outbreak). the constants q i represented a priori probabilities that the outbreak is due to pathogen i [ , ] . we set uninformative priors for all pathogens considered, so that q i was simply the reciprocal of the number of pathogens being considered (including disease x) on each date in our analysis. we initially estimated the distance between observed characteristics of the outbreak and each known candidate pathogen using the hamming distance (i.e., the sum of squares differences between the entries in the columns of table corresponding to the disease x and the candidate pathogen). then, we assumed that the probability that the outbreak is driven by pathogen j was governed by a negative exponential function, where d j is the calculated hamming distance, although in principle any decreasing relationship, and any metric describing the distance between two vectors, could have been used. we also repeated our analysis using an alternative measure of the distance between observed characteristics of the outbreak and each known candidate pathogen, namely the euclidean distance (i.e., the square root of the hamming distance). in each case, we assumed that the importance of each characteristic had an identical weight in our analysis, so that a simple quantitative assessment could be obtained in a probabilistic manner without the need for subjective judgement. combining equations ( ) and ( ), and assuming uninformative priors for q i , gives, the probability that the outbreak was driven by disease x corresponds to the distance d x = , and represents a risk score taking values between the reciprocal of the number of candidate pathogens including disease x itself and one: if there are n known pathogens that can potentially cause atypical pneumonia, the probability of observing disease x without any information would be identical to the probability of observing any other listed pathogen (i.e., /( + n)). as pathogens were ruled out by laboratory testing, that uninformative probability increased (i.e., / until january , / from january and / from january in the current outbreak). in addition, if the probability of observing disease x according to equation ( ) takes a value close to the probability of observing other candidate pathogens, the overall probability that the outbreak is due to a novel pathogen should be interpreted as being low. a result of significant practical importance, however, is when the probability of observing disease x is close to one or much larger than the probability corresponding to each previously observed candidate pathogen. in that case, all candidate pathogens are not similar to the causative agent of the ongoing outbreak, and so the outbreak is likely to be due to a novel pathogen. we converted the probability of disease x into the equivalent percentage value (so that, for example, a result of . in equation ( ) is assumed to mean an % probability) and refer to the percentage value as the "probability of disease x" hereafter. we show temporal changes in estimates of the probability that the ongoing outbreak is driven by each candidate pathogen in figure . because the only information on december was that cases had symptoms of atypical pneumonia, the distances between the ongoing outbreak and the eleven known pathogens were all zero; thus, all eleven candidate pathogens initially showed an identical probability of . % (i.e., / , when the possibility of disease x is accounted for). if no further information had become available during the outbreak, other than the gradual ruling out of candidate pathogens through laboratory tests, then the inferred uninformative probability for each candidate pathogen would have been given by the dotted gray lines in figure . we converted the probability of disease x into the equivalent percentage value (so that, for example, a result of . in equation ( ) is assumed to mean an % probability) and refer to the percentage value as the "probability of disease x" hereafter. we show temporal changes in estimates of the probability that the ongoing outbreak is driven by each candidate pathogen in figure . because the only information on december was that cases had symptoms of atypical pneumonia, the distances between the ongoing outbreak and the eleven known pathogens were all zero; thus, all eleven candidate pathogens initially showed an identical probability of . % (i.e., / , when the possibility of disease x is accounted for). if no further information had become available during the outbreak, other than the gradual ruling out of candidate pathogens through laboratory tests, then the inferred uninformative probability for each candidate pathogen would have been given by the dotted gray lines in figure . real-time estimation of the probability that the ongoing pneumonia outbreak is driven by each candidate pathogen, given available information on different days. the probability that the outbreak is due to an unknown pathogen (disease x) increases as more information becomes available, for two reasons: (i) the current outbreak can be seen to exhibit characteristics that are not similar to those observed in previous outbreaks, and; (ii) previously observed pathogens are ruled out by laboratory test results. arrows indicate new information available on each date. results are shown for different metrics describing the distance between characteristics of the ongoing outbreak and each candidate pathogen, and either including or excluding initial exposure information for the current outbreak (i.e., worked at/visited a wet market), specifically: (a) hamming distance (the sum of squares difference between the entries in the columns of table corresponding to the ongoing outbreak and the candidate pathogen considered) with wet market exposure; (b) euclidean distance (the square root of the hamming distance) with wet market exposure; (c) hamming distance without wet market exposure; (d) euclidean distance without wet market exposure. dashed grey lines show the probability for every pathogen (including disease x) if the only information included is the ruling out of different pathogens through laboratory tests (i.e., a probability of /( + number of candidate pathogens remaining on that day)). note that the probability corresponding to different pathogens can be identical, for example, severe acute respiratory syndrome (sars) and mycoplasma pneumoniae were assessing as being equally likely as the causative pathogen from december to january, and legionellosis and chlamydia pneumoniae had equal probability from december to january (details in supplementary material s ). real-time estimation of the probability that the ongoing pneumonia outbreak is driven by each candidate pathogen, given available information on different days. the probability that the outbreak is due to an unknown pathogen (disease x) increases as more information becomes available, for two reasons: (i) the current outbreak can be seen to exhibit characteristics that are not similar to those observed in previous outbreaks, and; (ii) previously observed pathogens are ruled out by laboratory test results. arrows indicate new information available on each date. results are shown for different metrics describing the distance between characteristics of the ongoing outbreak and each candidate pathogen, and either including or excluding initial exposure information for the current outbreak (i.e., worked at/visited a wet market), specifically: (a) hamming distance (the sum of squares difference between the entries in the columns of table corresponding to the ongoing outbreak and the candidate pathogen considered) with wet market exposure; (b) euclidean distance (the square root of the hamming distance) with wet market exposure; (c) hamming distance without wet market exposure; (d) euclidean distance without wet market exposure. dashed grey lines show the probability for every pathogen (including disease x) if the only information included is the ruling out of different pathogens through laboratory tests (i.e., a probability of /( + number of candidate pathogens remaining on that day)). note that the probability corresponding to different pathogens can be identical, for example, severe acute respiratory syndrome (sars) and mycoplasma pneumoniae were assessing as being equally likely as the causative pathogen from december to january, and legionellosis and chlamydia pneumoniae had equal probability from december to january (details in supplementary materials table s ). however, additional characteristics of the ongoing outbreak were observed on december . these characteristics allowed the ongoing outbreak to be distinguished from outbreaks due to previous pathogens, and consequently the inferred probability that the outbreak was driven by a novel pathogen increased substantially to . % and . % for hamming and euclidean distance metrics, respectively ( figure a,b) . if instead the exposure characteristic (i.e., exposure at a wet market) was excluded from the analyses, the probability of observing disease x given observed characteristics was still as high as . % and . % for the hamming and euclidean distance metrics ( figure c,d) . adenoviruses, hpai (h n and h n ) and other influenza viruses were ruled out on january , leading to an estimated probability that the outbreak was due to disease x of . % and . % for the hamming and euclidean distance metrics when all factors were considered. excluding the characteristic corresponding to wet market exposure, the probability that the outbreak was due to disease x was assessed to be . % and . % for the hamming and euclidean distance metrics, respectively. sars and mers coronaviruses were ruled out as possible causative agents on january , leading to a very high estimate for the probability that the outbreak was caused by a novel pathogen once all information was collected. on january , the probability the outbreak was due to disease x was estimated to be . % and . % according to the model considering all the characteristics (again, for the hamming and euclidean distances, respectively), while the model excluding the characteristic of exposure at the wet market suggested probabilities of . % and . %. in this analysis, we showed how the outbreak of pneumonia cases in wuhan was assessed in early january as being caused by a novel pathogen. this was demonstrated using a series of clinical, occupational, and behavioral observations extracted from fragmented reports describing the cases as these reports became available in real time [ , ] . although virological investigation is the gold standard for pathogen identification, and the virus has now been confirmed to be a novel coronavirus that is a relative of sars, laboratory-based outcomes can only be obtained after successfully sequencing the novel virus, which can sometimes be a lengthy process. at the time of writing, it still remains for the microbiological causal link to be established, for instance by ensuring that koch's postulates are met (as seen, e.g., in a study of zika virus [ ] ). in the ongoing outbreak, the provisional identification of a novel coronavirus was performed on january and announced formally on january [ ] . we have shown that non-virological information can indicate that the cause of the outbreak is likely to be a novel pathogen ("disease x"), and that this conclusion was obtained before virological test results were announced. disease x was inferred to be very likely on all dates from december onwards-the date on which descriptions of outbreak characteristics began to emerge. when sufficient clinical details of cases (e.g., complete blood cell counts) are available, the number of causative pathogens considered can be limited to a reasonable number. in this instance, atypical pneumonia combined with reduced white blood cell counts and the lack of response to antibiotics indicated that the pathogen was consistent with viral rather than bacterial infection. with such information, non-virological data can be used for convenient quantification of the probability that the outbreak was due to a novel pathogen, while awaiting the results of virological tests. we believe that the proposed approach can improve risk assessment practices across the world. it is important to consider two issues about the compilation of table . first, a critical underlying assumption is that table represents general outbreak characteristics of the ongoing outbreak and previously known outbreaks. the representation does not reflect observations from all confirmed cases nor epidemiological findings from a case control study (e.g., statistically significant risk factors). rather, zeros and ones in the table were defined in a phenomenological manner, and values may change as the ongoing outbreak continues. depending on the opinions of different experts (e.g., [ ] ), the defined nominal values could have been different to those shown in table ; in this study, we are simply demonstrating how such an approach might work in practice. second, as we have shown, quantitative estimates depend on the precise characteristics that are used. we showed results including and not including information on wet market exposure. in table , infections due to previously observed pathogens other than hpai were assumed not to be associated with exposure to wet markets. since this assumption was not derived from empirical observations, it could be debated. in the past, descriptive outbreak information has been used to generate outbreak case definitions, and causative agents have been pinpointed without using statistical methods in combination with epidemiological observations. in the present study, we have shown that such assessments can be made quantitatively using a simple statistical model, allowing for comparisons between the possible causative agents among different candidates. when outbreak characteristics are shared and updated in real-time (table ) , these data can contribute to efforts to narrow down the possible range of causative agents. in the case of the outbreak in wuhan, our calculation of the probability that each pathogen is the causative agent indicates that virological exclusion of influenza viruses, adenoviruses and known virulent coronaviruses associated with sars and mers on and january can be regarded as an "unsurprising" finding. as important limitations, the precision and credibility of the input data and the method for calculating the distance between the candidate pathogens and the observed outbreak, must be refined in future. first, our proposed approach used very limited data in table for logical quantification of the probability that each pathogen was the causative agent. however, with more clinical data, the binary characteristics could be replaced by continuous frequencies (e.g., the proportion of cases experiencing coughing and/or breathing difficulties). second, with sufficient data it would also be possible to estimate the probability that each pathogen is the causal agent (equation ( )) not by arbitrarily measuring the distance but by using classification models involving regression or more sophisticated machine learning approaches. third, the erroneous input of incorrect information may be a challenge in real time analyses. the veracity of the sources of information for future analyses could have an impact on the resulting probability calculations. fourth, the estimated probability that an outbreak is driven by a novel pathogen might be slightly over-or underestimated due to limited information about the mode of transmission and small numbers of observed cases. of note, the respiratory syncytial virus (rsv) was not completely ruled out as a candidate pathogen in our real-time analysis. however, rsv was an unlikely candidate since the majority of cases in the ongoing outbreak are adults [ ] while most rsv infections are observed in infants and young children. finally, we had to restrict ourselves to assuming the a priori probability that the ongoing outbreak driven by each candidate pathogens (q i ) is identical for each pathogen. however, since no alternative information was available, we believe such uninformative priors to be the optimal choice. despite the future improvements to our statistical modelling framework that are required, including the need to test our approach using data from outbreaks of previously known pathogens, this short study demonstrated clearly that the ongoing outbreak is consistent with causation by a novel pathogen, "disease x". we reached this conclusion after only a few days of the outbreak had passed. attention has now rightly turned towards identifying the pandemic potential of this outbreak [ ] [ ] [ ] , as well as planning control interventions within china and elsewhere [ , ] . however, at the start of the next outbreak of an unknown pathogen, virological testing and quantitative analyses of clinical data are two complementary methods that can be used. thus, analyses of the type conducted in this study can greatly support efforts to characterize causal agents in future outbreaks, with the benefit that analyses like this one can be carried out extremely quickly. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : estimated values of the probability of disease x, given available information at different timepoints using hamming distance and including wet market exposure, table s : estimated values of the probability of disease x, given available information at different timepoints using euclidean distance and including wet market exposure, table s : estimated values of the probability of disease x, given available information at different timepoints using hamming distance and excluding wet market exposure, table s : estimated values of the probability of disease x, given available information at different timepoints using euclidean distance and excluding wet market exposure. wuhan municipal health commission's briefing on the current pneumonia epidemic who statement regarding cluster of pneumonia cases in wuhan wuhan municipal health commission. wuhan municipal health and health committee's report on unexplained viral pneumonia novel human virus? pneumonia cases linked to seafood market in china stir concern mystery virus found in wuhan resembles bat viruses but not sars pneumonia cases possibly associated with a novel coronavirus in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus probabilistic differential diagnosis of middle east respiratory syndrome (mers) using the time from immigration to illness onset among imported cases who zika causality working group. zika virus infection as a cause of congenital brain abnormalities and guillain-barre syndrome: systematic review initial cluster of novel coronavirus ( -ncov) infections in wuhan, china is consistent with substantial human-to-human transmission clinical features of patients infected with novel coronavirus in wuhan, china. lancet pandemic potential of -ncov nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study real-time estimation of the risk of death from novel coronavirus (covid- ) infection: inference using exported cases novel coronavirus outbreak in wuhan, china, : intense surveillance is vital for preventing sustained transmission in new locations estimation of the transmission risk of the -ncov and its implication for public health interventions the authors declare no conflicts of interest. key: cord- - g n e authors: steele, james harlan title: veterinary public health: past success, new opportunities date: - - journal: preventive veterinary medicine doi: . /j.prevetmed. . . sha: doc_id: cord_uid: g n e abstract animal diseases are known to be the origin of many human diseases, and there are many examples from ancient civilizations of plagues that arose from animals, domesticated and wild. records of attempts to control zoonoses are almost as old. the early focus on food-borne illness evolved into veterinary medicine's support of public health efforts. key historical events, disease outbreaks, and individuals responsible for their control are reviewed and serve as a foundation for understanding the current and future efforts in veterinary public health. animal medicine and veterinary public health have been intertwined since humans first began ministrations to their families and animals. in the united states, the veterinary medical profession has effectively eliminated those major problems of animal health that had serious public health ramifications. these lessons and experiences can serve as a model for other countries. our past must also be a reminder that the battle for human and animal health is ongoing. new agents emerge to threaten human and animal populations. with knowledge of the past, coupled with new technologies and techniques, we must be vigilant and carry on. farmer keeping animals in his midst. the people who domesticated the animals were thus the first to be victims. those early humans then developed resistance to some zoonotic diseases that had emerged (diamond, ) . the relation of animal diseases to human disease was observed in the ancient civilizations of babylon, the nile valley, and china and noted by leviticus in the old testament, and later by hippocrates in greece, and virgil and galen in rome. millions of people across europe during the middle ages suffered from plague carried by rat fleas. the invasion of europe by rinderpest in the th century disrupted commerce and government so much that the papal authority created a medical commission to advise the vatican on what measures should be taken to control the animal plague/rinderpest . the movement of animal diseases into the americas is believed to have been in the support of the settlements founded by columbus in santo domingo in . these livestock were the foundation animals for spanish colonies in the americas. in the next century, de soto, the spanish explorer of florida and the southeast, brought cattle, horses, and swine, as well as dogs that thrived. farther north, the virginia colonists brought animals to roanoke island, but none survived, neither humans nor animals. later the jamestown colonists imported domestic animals that survived and became valuable foundation stock, but no zoonotic diseases are recorded in any of these earliest settlements. not until was rabies the first zoonosis recorded in the us colonies, and later as an epizootic in both the colonies and the federation of states in the late th century (smithcors, a,b) . in , the newly founded medical repository editors were the first to inquire about emerging diseases in the united states and territories. they asked for information on human diseases, diseases among domestic animals, accounts of insects, the condition of the vegetation, and even the state of the atmosphere. they hoped to put the facts together as an annual report on the status of health in the united states. surgeon general luther terry ( ) of the us public health service (usphs) in his address at the american veterinary medical association (avma) centennial called this report the first reference to veterinary medicine in support of public health. a few years after this report, benjamin rush called for the establishment of veterinary medical education at the university of pennsylvania. the united states sanitary commission, organized during the civil war by public-spirited women, was concerned largely with sanitary conditions, including food hygiene. they were the first to call attention to the putrid meat and later embalmed beef sent to the army. the commission was to be a forerunner of public health in the years following the civil war (furman, ) . by the s, there was interest in developing a national health service. yellow fever epidemics were frightening as they spread up the mississippi river from new orleans. the possibility that yellow fever involved animals brought professor john gamgee, a famous veterinarian, from england to investigate the epidemic. he recognized the seasonal occurrencethat cold weather stopped the epidemic -and even suggested river traffic be limited to the colder months. however, he failed to associate the effect of cold weather with the decline of the numbers of mosquito populations, the vector of yellow fever (furman, ) . the us board of health came into being largely because of the yellow fever epidemic and the morbidity and mortality that it caused. by the time of the board's inception in , malaria was widespread in the south, and tuberculosis was a recognized disease. typhoid fever and enteric diseases were also common. in addition, animal diseases were present, especially the spread of glanders and anthrax following the civil war (furman, ) . in the president of the us board of health, dr. j.l. cabell, asked james law, professor of veterinary medicine at cornell university, to advise the board on how they should supervise the diseases and movements of domestic animals. law's report ( ) was the first comprehensive recognition of the effects of zoonotic diseases upon public health published in the united states (steele, ) . the organization of public health in the post civil war period has been reviewed by miles ( ) , former historian of the national institutes of health (nih). his report discusses the struggle between public health and agricultural interests in the decade leading up to the inauguration of the bureau of animal industry in . the interest of the bureau was to protect animal health, and later to provide a meat inspection service for public health, international trade, and subsequently interstate commerce. the relation of animal diseases to the public health and their prevention by frank s. billings ( ) was the first book to review the problems and the state of bacteriology as well as parasitology in the s. although the book is limited to trichinosis, hog-cholera, tuberculosis, anthrax, texas fever, rabies, and glanders, his knowledge of these diseases is remarkable for the time. billings gained this knowledge through education in berlin, where he learned about the history of animal diseases in the greco-roman period and the latin origin of ''veterinarians,'' which he says first appeared in the th century writings of vegetii. he also traveled extensively in europe, where he observed veterinary activities. billings makes a strong plea for the development of veterinary public health to control the animal diseases that affect man. he stated that this could be accomplished only by having trained veterinarians who were scientifically educated. he was one of the veterinarians who was active in the early years of the american public health association (apha), during which discussions of trichinosis, tuberculosis and other animal diseases took place at the early annual meetings. a true visionary of veterinary public health, billings pointed out that milk from diseased cows is dangerous. he appealed to the government to set up laboratories to use the new science of bacteriology to find the cause of illness of milk origin. food hygiene came into being only with the new science of bacteriology (billings, ) . the frightful toll of milk borne disease is reviewed by stenn ( ) . in his report, he cites the shocking figure of deaths per births in new york city in . spoiled milk accounted for the deaths of thousands of children in the early s, and in many other cities. the records of , cited by stenn, list many milk borne outbreaks of typhoid fever and diphtheria. he goes on to state that % of the milk cans sampled contained tubercle bacilli, and in cities, % of the milk had tubercle bacilli. in a milk borne typhoid epidemic occurred in washington, dc, that caused president theodore roosevelt to order the usphs to investigate the local supply. surgeon general walter wyman ordered his staff to examine not only the washington milk problem but to examine the national milk problem. the report milk and its relation to public health by milton rosenau, issued by the usphs, brought reform to the dairy industry and support for the bureau of animal industry program to control bovine tuberculosis (myers and steele, ) . pasteur took milk safety even further, changing science and veterinary medicine by creating a new concept of the origin of disease. no longer would the myth of spontaneous origin of disease guide society, although there were as many objections to scientific advances then as now. the centennial celebration of the rabies vaccine revealed in pasteur a man of many accomplishments. he was a chemist who discovered the cause of fermentation and applied it to the beer and wine industries, a process that led to milk pasteurization. he was an artist who was known to the impressionists of the th century as the man who prepared better paint colors. he was also a genius who gave public health the science and vaccines to combat th century diseases and prepare for the th century's emerging problems (koprowski and plotkin, ) . although the concept of pasteurization of beer and wine brought fame to pasteur, the application to milk was less known, and it was accepted no more readily than the concept of evolution. it was asserted that all kinds of illness and changes in well being would ensue from pasteurization. the eradication of bovine tuberculosis and brucellosis (bang's disease) insured a safe milk supply and protected the health of farmers, dairymen, veterinarians and the handlers of milk and milk products. the case for pasteurized milk and milk products is conclusive. in the late th century, a new array of milk borne zoonoses is of concern to public health and veterinarians. some date back to the th century, such as salmonella. the salmonella were identified in by one of the most distinguished public servants of the veterinary profession, dr. daniel salmon. as the first chief of the bureau of animal industry (bai) from to , he assembled and trained a great staff. this included theobald smith, v.a. moore and e.c. schroeder, who solved the epidemiology of texas fever caused by babesia bigemina, which is carried by the tick boophilus annulatus. salmon was the leading proponent of veterinary public health in the s. he asked for, and received from congress, authority for a federal meat inspection service in to meet the demands of foreign commerce. however, his national program was circumvented by local interests citing states' rights; therefore, the meat inspection act of was ineffective nationally. salmon sought support from the apha and the american medical association for these early veterinary efforts to protect public health. unfortunately, these agencies did not support him (schwabe, a) . the federal meat inspection service act of came about only after sinclair ( ) exposed the filthy conditions of the chicago stockyards. salmon was blamed for the local hygiene failure over which he had no authority and was removed from office. however, he is remembered today by the usda's salmon award for leadership. in , the bai initiated tuberculin testing of dairy cattle in the district of columbia, a demonstration that revealed an infection rate of almost %. this was the beginning of a successful tuberculosis control campaign that led to its eradication under john r. mohler, bai director from to . the late jay arthur myers memorialized the near eradication of bovine tuberculosis in his book, entitled ''man's greatest victory over tuberculosis'' (myers, ) . at the start of the th century, pathologists were greatly interested in comparative medicine. they were led by karl f. meyer, a swiss veterinarian who was to become one of the leaders and outstanding scientists of the th century. he was among the early public health scientists to delve into virology as professor of pathology at the university of pennsylvania (penn), and in he may have been among the first to recover a virus causing equine encephalitis. as director of the pennsylvania livestock sanitary board laboratory, he published on glanders, anthrax, anaplasmosis, sporotrichosis, paratuberculosis, septicemia, and many other diseases of animals. in he left the university of pennsylvania to accept a position at the university of california's newly established tropical medical center. the following year, he accepted an appointment to the george williams hooper foundation for medical research at the university of california medical center. he remained there the rest of his life and become a legend. his lectures introduced medical students to the biologically active world, including the zoonoses, plant life, the atmosphere and all that is called the environment today. at the hooper foundation, meyer researched a wide spectrum of animal diseases of public health importance. after being active in the investigation of human influenza in - , he went to the field to define the epidemiology of malaria, dysentery, and even dental diseases. his study of the bacterial causes of abortion in animals resulted in bringing together brucella abortus, brucella melitensis, and brucella suis in a new genus honoring david bruce. another important event was his report on clostridium botulinum in nature. botulism became a national concern in the s when california canned fruit and vegetables were found to contain botulinum toxin. the industry asked meyer to resolve the problems and underwrote a laboratory to maintain surveillance. thereafter, meyer was active in food safety, but he was also concerned with humane animal care in which he maintained an interest all his life. in , meyer and his long time lab associate bernice eddie began their series of psittacosis reports in birds. these reports eventually led to control years later with tetracycline-impregnated seed. the same antibiotic is now used to prevent ornithosis in domestic fowl (meyer, ) . one of meyer's most memorable lectures was in when he called attention to the importance of the animal kingdom as a reservoir of diseases that endanger the health and welfare of people throughout the world (meyer, ) . in , he first reviewed the state of the animal reservoir of diseases, by then referred to as zoonotic diseases, before the world health organization (who) general assembly. he repeated the same theme before the who expert committees for the zoonoses, plague, food hygiene and for the pan american health organization (paho) until his th year. meyer's work on plague was reported in the special supplement of the journal of infectious diseases to commemorate his th birthday. this was underwritten by max stern, president of hartz mountain, which supported the psittacosis control investigations at the hooper foundation (steele, ) . meyer died in san francisco on may , , less than a month before his th birthday. larry altman ( ) , the medical editor of the new york times, wrote a lengthy obituary from which the following excerpt is taken. it also appears on the fore page of the journal of infectious diseases (supplement), may : ''dr. karl fredrich meyer was regarded as the most versatile microbe hunter since louis pasteur and a giant in public health [. . .] . public health leaders yesterday called his contributions to medicine 'monumental.' his scientific work had such broad implications that it touched on virtually all fields of medicine.'' the obituary was placed in the congressional record that same month. in , albert sabin ( ) wrote a biographical memoir of meyer for the national academy of science, of which meyer was a member from l to . sabin explains that as a youth in basel, switzerland, pictures of the black death so fascinated meyer that he became an outdoor scientist instead of following in the aristocratic business world in which he grew up. he told friends that in choosing to become a veterinarian he could ''be a universal man and study all diseases in all species.'' the s were memorable for public health growth and scientific advances. the viral etiology of influenza was uncovered by richard shope and thomas francis at the rockefeller institute. the use of egg embryos was a new method of growing viruses that would lead to the chick-embryo rabies vaccine and other viral vaccines. the development of the strain brucella vaccine and the stern anthrax vaccine in south africa were important to the control of brucellosis and anthrax worldwide. earlier investigation of toxoids by gaston ramon, a french military veterinarian, led to the discovery of tetanus toxoid for both horses and humans. discovery of the sulfa drugs and penicillin gave the clinician medication he had not dreamt of, a prelude to great advances in medicine. to be a veterinary student in the late s was both exciting and slightly dangerous. the brucellosis epidemic among veterinarians -both students and clinicians -raised epidemiological questions as to how brucellosis was spread. in michigan state college experienced an epidemic among veterinary students and others in the bacteriology building (holland, ) . up to then the disease was thought to be caused by direct exposure or ingestion of milk borne brucella, and airborne brucella was not given much consideration. the episode at michigan state college would change that oversight. as a student in the brucellosis testing laboratory, i heard discussion of the means of spread being water borne and back siphonage. professor i.f. huddleson, whose research laboratory was the focus of this investigation, disagreed with the state investigators, who were public health scientists and engineers focusing on the water borne theory. these investigators suggested contaminated glassware was not being autoclaved properly, and in turn, viable brucella was getting into the water system (newitt et al., ) . the discussion of the epidemic, which affected most of the people in the building, along with other public health interests of dean ward giltner, professor h.j. stafseth, professor i.f. huddleson and dr. w.t.s. thorp of the michigan state university college of veterinary medicine, led me to think about a career in public health. dr. stafseth encouraged many students to consider public health as a career (stalheim and steele, ) . when he learned of my interest, he and dean giltner worked out a program to make me eligible for a usphs fellowship. i was excused from senior clinics to pursue the fellowship. my assignment was an internship at the michigan health department. there i observed and learned from health department veterinarians, pathologists and bacteriologists how to remove and examine an animal brain for rabies and to inoculate mice to further confirm the diagnosis. vaccinia were grown on the belly of a calf that had been shaved, scrubbed and disinfected. after harvesting the scabs, the vaccinia would be tested for contaminants. it was a lengthy procedure. the same high standards were maintained for the pertussis/whooping cough vaccine, equine antiserum for tetanus and rabbit pneumococcal antiserum. it was a learning period that would serve me well. dean giltner and c.c. young, the director of the michigan public health laboratories, put together my fellowship application to the usphs and harvard school of public health. approval came the week before graduation, and my bride-to-be aina oberg and i were elated. we were married the evening after graduation, with many of the faculty and classmates in attendance two days later i took the michigan examination to practice veterinary medicine. the summer of was spent as an intern at the petoskey animal hospital. there i learned about swimmer's itch-a common affliction of man and pets caused by an avian schistosome. i was exposed to the parasite while swimming in inland lakes. at harvard it became my thesis subject. later it was the first subject i reported on at the avma convention in chicago ( ) with dean giltner in the audience. at harvard, there was talk of war. president conant addressed the incoming class with the admonition there would be important world changes during their student years. the school of public health's dean cecil drinker, the faculty, and the students were stimulating. i was the only veterinarian, which attracted some attention, and the medical school librarian was delighted to know there was a veterinarian around. she showed me a remote section of the library that contained many old books on veterinary medicine-harvard had a veterinary faculty from to . we students were delighted with our newly found classmates, and many of us would remain lifetime friends. to me, the academic work was not demanding except for statistics, which took much time. after all, in those days, we used hand-cranked machines for tabulations. my wife, aina, first worked at the harvard co-op and then with the british american ambulance volunteers. she enjoyed the students and compliments and being invited to fundraisers for the volunteers. then tragedy struck. a sudden collapse with fever hospitalized her. the diagnosis was advanced tracheal-bronchial tuberculosis that would confine her to sanitariums and hospitals for the next years, from january to april . after innumerable surgeries, the newly discovered streptomycin saved her life after months of treatment. eventually, we established a home and family with two sons, jay and david, in atlanta, georgia, for years. there aina died from the complications of arrested pulmonary tuberculosis in . the new year ( ) brought unforeseen problems, mainly medical bills, even though student health expenses were covered to a lesser extent. i sought work at the angel memorial animal hospital, where i knew some of the staff. when dean drinker heard of my after-school work plans, he called me into his office and told me to concern myself with school and taking care of my wife. a check signed by dean drinker awaited me in his secretary's office, a practice that continued until graduation. the drinker society still honors his contributions at the harvard school of public health, which we support. as graduation neared, many of us knew we were going into uniformed service job opportunities. i was deferred by the lansing michigan draft board, but i volunteered for the army veterinary corps and the navy special services, an epidemiology unit. both declined my services, and in the meantime, i found no positions of interest. i wanted to do epidemiology of the animal diseases affecting human health, but all the positions i was interested in required a medical degree. finally i brought my dilemma to dean drinker's attention. shortly thereafter, he asked me to his office to talk over my future. i was upset that it seemed i must have a medical degree to be an epidemiologist. should i get an md? dr. drinker and his wife, also a physician, heard me out. their reply was to list my attributes: good student, industrious, good appearance, good speaker, and creative ambitions. that said, they followed with memorable advice: ''jim, fly under one flag.'' before leaving harvard, i met kf meyer who was lecturing at the school of public health. some days later, i learned he had asked about me because he anticipated some research contracts with the us army epidemiology board and would need staff. i was elated when he offered me a position, and aina and i left boston with high expectations. some days later we arrived in chicago, only to find out a week later that meyer had not received the contract and had no funds to support the research position. i was depressed: no job and a sick wife. a few days later i visited the usphs chicago regional offices to seek their help in finding work. i appeared unannounced and asked a secretary to see the director. while waiting, a medical officer appeared and asked if he could help. i explained that i was a usphs fellow they had supported in getting an mph, and my objective was to find a position where i could investigate the epidemiology of animal diseases that affect the public health. dr. henry holle, the medical officer, listened and replied he never heard about such a situation. so he took me to see the medical director, mark ziegler, a tall, soft-spoken, southern gentleman. the availability of a young mph graduate led them to call washington. a week later after a review of my qualifications and evidence of my education, i was offered an internship as a civilian sanitarian in the ohio department of health. there i would spend the next year, july -october . the challenges were milk sanitation problems, food borne diseases, diarrhea, typhoid, rabies and the ohio river flood, a great learning experience. in september , the us army offered me a commission as a veterinary officer which i planned to tentatively accept. within days, medical director frank meriwether told me since the usphs had given me a fellowship, they should have a first call to commission me. i received a commission as a sanitarian on november , . afterward, i spent a short tour of duty in the midwest region with senior sanitarian william h. haskell, an authority on pasteurization methods and practice. he was one of the civil service veterinarians brought in by the usph milk specialists early in . in , other newly recruited veterinarians raymond helvig, ray fagan, and ted price were also commissioned as sanitarians. they were the only veterinarians in the usphs except for a veterinarian who was an animal control officer in world war i in and two parasitologists willard wright and maurice hall at the nih in the s. from chicago, i was ordered to report to washington, dc, for orientation. there i learned of my assignment to puerto rico and the virgin islands where i was to be responsible for coordinating milk and food sanitation and evaluating any zoonotic diseases in areas that had been isolated by the war. brucellosis and bovine tuberculosis were widespread. the diagnosis of venezuelan equine encephalitis and bat rabies in trinidad caused some concern in the islands but did not spread beyond trinidad. rabies was indigenous in the dominican republic and cuba in the s. in march , the pan american sanitary bureau asked the usphs san juan, puerto rico, office to do an assessment of the post-war veterinary public health problems in the dominican republic and haiti, neither of which had a functional veterinary service. i was directed to make a report on their problems. in the dominican republic, there were no reported diseases, but bovine tuberculosis, brucellosis and mastitis were known. no veterinary laboratory support existed, and the abattoirs kept no records. rabies had been reported in dogs, and possibly in horses, and some years later there was an epizootic of equine encephalitis. president trujillo kept some racing horses near ciudad trujillo (santo domingo), and i was asked to examine them. these old horses were brought to the dominican republic before the war in - . all were broken down and hardly fit to run. regardless, the dominican republic officials thought i could repair their ailments. when i told them i could not, they complained that i was not cooperative to the u.s. embassy, who then told me to be cooperative. later i visited the trainer, who told me, ''we will do what we can.'' thereafter, i was anxious to leave and went to haiti within a few days. port-au-prince was a rundown but hospitable capital. the country had been ravaged by tropical fevers for decades; malaria and filariasis were widespread. animal diseases were mainly fever and parasites, but an epizootic of anthrax in the early s was still present in : the disease was sporadic in the countryside. to my amazement, the dead animals were salvaged regardless of what they died from. there were no veterinarians in the government or in practice. still, the abattoir in port-au-prince was an elegant open iron structure. the cattle were immobilized by pithing, in which a small blade severs the spinal cord after which they are bled and eviscerated. the procedure was done rapidly, usually late at night, and the meat was distributed early the next day. however, a serious shortage of animal products existed, and few shops had any meat for sale. all in all, my stay at port-au-prince and the rural areas was a distressing experience. some weeks later i was in washington for further assignment as the war wound down. while there, i visited the pan american sanitary bureau to discuss my report with surgeon general hugh s. cumming who served the pan american sanitary bureau for a decade, after retiring from the usphs. at our meeting, i emphasized the need for a veterinary public health program to help in updating the animal health, preventing zoonotic diseases, and enhancing food safety. dr. cumming suggested i discuss the need for a veterinary public health program with his medical staff, where the proposal was enthusiastically accepted. the veterinary public health program was initiated with dr. aurelio malaga alba, a peruvian military veterinarian, as a consultant. dr. fred soper, the post-war director of the reorganized paho appointed dr. ben blood to organize a veterinary public health program in june . he carried on until and was followed by the outstanding public health veterinarian dr. pedro acha. the temporary duty in dc left my future uncertain. i was to be assigned to kansas city to prepare for the problems that might evolve with the invasion of japan. i took leave to spend some weeks with my hospitalized wife whose health was failing. the end of the war in europe and the pacific shortly thereafter changed my reassignment. i returned to dc to meet with assistant surgeon general joe mountin, whom i met earlier in puerto rico. dr. joe dean, his deputy, had arranged the interview. after a few inquiries about my wife's health, dr. mountin came to the point: ''what are you veterinarians going to do for the public health now that the war is over?'' the follow-up to that interview is in the appendix of ''the th anniversary of the veterinary medical corps officers of the u.s. public health service.'' after the approval of a veterinary public health section in the states relation division in december , i spent some months at the national institutes of health. i also worked to establish liaisons with the usda, bai, federal agencies, congressional interests, state relations, the avma and apha. in september after surgeon general parran's approval of the veterinary medical officer cadre, dr. mountin felt my washington activities were successful. he told me i was to be assigned to the newly created communicable diseases center, formerly the malaria control in war areas. there the veterinary public health program was established as a division, but it was a challenge to integrate. the new director of the centers for disease control (cdc) was dr. r.a. vonderlehr, previously chief of the puerto rico regional office, who i served under. he gave excellent support as did his deputy, dr. justin andrews, who succeeded dr. vonderlehr a year later. rabies was a national problem after the war. there was a great movement of people as war industries and encampments closed, and as a result, pets were lost or abandoned. the incidence of human rabies was the highest ever recorded, and unfortunately, human vaccine therapy was not always effective. canine rabies vaccine protection was short, with the vaccines being given every months. therefore, rabies became the lead program of the veterinary public health division. to head the activity, dr. ernest tierkel, a university of pennsylvania graduate who had completed his mph at columbia school of public health in , was recruited. he, dr. robert kissling and martha eidson along with a staff of animal handlers became the nucleus of the national rabies program at the rockefeller rabies investigation center in montgomery alabama (steele and tierkel, ) . the center was transferred to the cdc for $ . . they successfully demonstrated the effectiveness of a new chicken embryo rabies vaccine in the laboratory (tierkel et al., ) and in epidemic situations in memphis, tennessee (tierkel et al., ) . dr. mountin had learned from the public health authorities of indiana, michigan and others that brucellosis in man was of concern. they went so far as to say that as the sanitariums lost tuberculosis patients, brucellosis patients would take their place. the indiana health department was to be a brucellosis project site under dr. sam damen, the director of laboratories. the goal was to determine what action the health agencies should take. the federal bovine brucellosis control program was active in all states, so it became apparent that if the health authorities gave their support, the federal state brucella control program could eliminate the animal source of the human disease. late we brought the problem to the attention of dr. herman bunderson, chicago's dynamic health officer who remembered the struggle to eradicate bovine tuberculosis in the chicago milk shed, which included dairy herds in six midwestern states. in , he had required all milk coming into chicago to be from tb-free herds regardless of whether the milk was to be pasteurized. he recognized the brucella problem and shortly thereafter instituted the same standards for the elimination of bovine brucellosis in the s. the brucella eradication program was supported by the usphs milk code, which required that all grade a milk be from disease-free herds (us public health service, ) . the chicago brucella control program was soon adopted by big city health authorities, which gave impetus to the joint state federal brucella programs. as a result of these efforts, human brucellosis declined rapidly in the midwest from a high of thousands of human infections to hundreds in less than a decade. thereafter most of the human cases were of occupational origin, in travelers or in people using raw milk in rural areas. in the s there was a scare of brucellosis at dugway proving grounds, a military research center in western utah. dr. herbert stoenner investigated the alleged contaminated area and found the problem to be a rodent disease caused by brucella neotoma. this organism does not cause disease in man or domestic animals, but will cause antibody formation in cattle (stoenner and lackman, ) . after world war ii, there was great interest in the application of atomic energy for civilian use. professor s.f. gould ( ) at wayne state medical school initiated studies on the use of irradiation to destroy trichinella. he persuaded the american medical association to host a trichinosis symposium in in which the cdc participated. the evidence was conclusive that gamma radiation was effective at low doses (gould et al., ) . this was the beginning of my interest in promoting food irradiation, but it was not until that irradiation for commercial use was approved by federal agencies. the zimmerman human tissue survey - revealed the lowest rate of trichinosis ever (zimmerman et al., ) . modern pig raising, the prohibition of garbage feeding of swine, and consumer education are all contributing factors in the decline of the disease in pigs and humans (steele, ) . trichinosis has continued to decline in the states except in wild animals especially bears. other veterinary public health studies of parasitic diseases involved creeping eruption, also known as cutaneous larva migrans. this condition is due to the common dog hookworm larva ancylostoma caninum entering the skin and causing intense itching. this disease was common in the southeast states among persons exposed to damp, sandy soil; children playing in sandboxes; bathers at the beach, and utility men (cypess, b) . toxocariasis or visceral larva migrans is another parasite due to the dog, and sometimes the cat, roundworm larva migrating in the body of a foreign host, human beings (cypess, a) . dr. peter schantz confirmed these findings as a world health problem. toxoplasmosis was recognized as a human infection, and the domestic cat is recognized as a common source of human infection. infection is more likely to be caused by consumption of raw or undercooked meat. irradiation is effective in destroying this oocyst in meat (gould et al., ) . in the early s, a large equine encephalitis epizootic in central california required the assignment of all cdc veterinary officers. later another equine encephalitis epizootic occurred in new jersey in . since then there have been only occasional epizootics of the equine encephalitides. although the principal reservoir is birds, there is also survival of the virus in mosquito eggs that over winter. the cdc-fort collins laboratory has been at the forefront of these investigations. the most recent mosquito born disease is the introduction of west nile virus into north america in . wild birds and common city birds are the reservoirs, and the culex mosquito is involved in the transmission. horses may show clinical signs. control of the vector mosquito breaks the transmission cycle. plague, primarily a disease of rodents, is sporadic in the united states. the appearance of plague in domestic and feral cats and squirrels has brought the ancient scourge to households in the western states (poland and barnes, ) . however, dogs were never identified as carriers of the disease to man. an unusual epidemic of anthrax caused alarm in animal and human public health circles in the s. the anthrax was introduced by contaminated bone meal used in animal feed to improve lactation in sows. a radio announcer in cincinnati raised the question if cows' milk could be a vehicle for anthrax to be carried to humans. a search of literature found that milk was never a vehicle or cause of human or animal anthrax disease because the high fever of the disease stops lactation (steele and helvig, ) . salmonellosis was a recognized public health problem early in the s as well as during world war ii and afterwards among the civilian populations (galton et al., ) . after the war, investigators demonstrated it was widely disseminated. dr. phil edwards led the way at the university of kentucky and later at the cdc. mildred galton, chief of the veterinary public health laboratory contributed with her unusual ability to find evidence that others had overlooked. she demonstrated salmonella in many animals. her studies of transported pigs revealed how stress caused latently infected pigs to become shedders. the same reaction was found in other species. her work on raw eggs and meats led to the pasteurization of egg slurry used in baked or cooked products. she was among the first to find salmonella in raw milk years ago, and her work on the frequent presence of salmonella in poultry led to the federal poultry inspection program in the late s (steele and galton, ) . thirty years before weil described leptospirosis in humans in , animal leptospirosis was identified as its own problem. a record of an canine epidemic in stuttgart, germany, exists, but the etiologic agents were not determined. years after the canine epidemic, it was discovered that microorganisms morphologically identical caused the disease in both dogs and humans. leptospirosis proved confusing to all health professionals partly because ''isolated serovars were given names denoting the clinical signs observed in the patients from whom they were isolated'' (torten, ) . therefore, it was thought that serovar grippotyphosa would cause signs similar to catarrhal fever, and serovar icterohemorrhagia would cause hemorrhagic jaundice. it was not recognized that both serotypes are capable of causing both signs (torten, ) . in the us, there were numerous outbreaks among animal handlers, veterinarians and swimmers as well as people whose occupation exposed them to contaminated waste water in the - period. leptospirosis is now recognized as a problem associated with disasters such as flooding and earthquakes. there is wide agreement that vaccination of cattle and dogs has reduced environmental contamination (stoenner et al., ) . galton ( ) edited the ''leptospiral serotype distribution list '' through , and sulzer ( ) carried it up to . they were truly dedicated in keeping these records. listeriosis was first recorded in in sheep, and the first reported human case was in denmark in (bomer et al., ) . prevention of listeriosis is still not possible with the knowledge available, as there are no immunizing agents of proven worth. killed bacterins have been disappointing, and living attenuated vaccines have not been evaluated properly nor have they shown promise in limited experiments. good physical hygiene is essential to prevention (bomer et al., ) . groups at high risk of infection are pregnant women, neonates, diabetics, alcohol dependents, persons with neoplastic disease, or those being treated with corticosteroids or antimetabolites. among animals, ewes are at the highest risk late in the first pregnancy. sheep in late pregnancy should not be fed ensilage of doubtful quality nor be exposed to severe cold or inclement weather and crowding (bomer et al., ) . improved measures for preventing and controlling human listeriosis depend on increasing awareness of its diverse clinical manifestations and an increasing index of suspicion. because l. monocytogenes, the causative agent of listeriosis, is sensitive to most antibiotics, their early administration, once the diagnosis has been established, significantly decreases mortality. cortisone and its derivatives may, however, cause asymptomatic listeria infections to become overt (bomer et al., ) . after the end of the war in europe, the breakdown of food hygiene there allowed salvaged food to spread zoonotic diseases. at the same time there were numerous cases of listeriosis reported in france that caused abortion, stillbirths and reproductive tract disease (seeliger, ) . the disease remains prevalent in western europe to the extent that all midwives and obstetricians alert their patients to report symptoms. since there has been a steady decline of reported cases. food borne listeriosis elsewhere was virtually forgotten until when an outbreak occurred in the maritime provinces of canada and was associated with consumption of contaminated coleslaw (schleck et al., ) . then years later, a major outbreak in massachusetts between june and august of was epidemiologically linked to consumption of a particular brand of pasteurized whole and % milk (fleming et al., ) . although questions have been raised about the adequacy of the epidemiologic study (ryser and marth, ) , no other food has emerged as the vehicle that transmitted l. monocytogenes in this outbreak. in mexican-style cheese made in a factory near los angeles was definitively linked to a large outbreak listeriosis (linnan et al., ) . this was followed in by the linking of consumption of vacherin mont d'or, a variety of cheese, to an outbreak of listeriosis in the canton of vaud in switzerland (bille et al., ) . in recent years, food borne outbreaks continue to be reported in north america and europe. during the s, many more human cases and deaths were reported in the united states. the vehicles reported as contaminated were cold cuts, canned meats and frankfurter sausages. worldwide, listeriosis is a problem mostly in the temperate zones. another emerging zoonotic food borne disease is escherichia coli o :h , the enterohemorrhagic strains as well as those characterized by cytotoxins. these e. coli and others of human origin are major causes of the human enteric disease. however, they are less causative in food producing animals that may be infected but show few or no clinical signs. pasteurization of milk is effective in the control of e. coli spread. irradiation has proven effective for pasteurization of food of animal origin for the protection of the public health. recently improved inspection and hygiene have reduced reported human diseases, even though toxic e. coli is wide spread among cattle. the same can be said for newly identified emerging food borne zoonotic diseases. cryptosporidia parvum is a coccidian protozoa found worldwide. giardia are found in numerous animals, and during the late th century, the flagellate protozoan was identified worldwide as a water borne disease of humans and animals. old problems new to the states are taenia saginata and t. solium, largely found in immigrant workers. the tapeworm cysts found in meat, beef and pork are easily destroyed by irradiation, a technology that slowly is being accepted in the southern countries where tapeworm disease is recognized as both an economic as well as a public health problem. the acceptance of veterinary public health internationally by the paho has been previously discussed. the inauguration of veterinary public health as a national program in the usphs in stimulated interest worldwide, especially in the newly created international agencies. the united nations health office organizing committee chaired by surgeon general tom parran met in new york in june to further public health worldwide. the public health service officers and personnel were asked by the surgeon general's chief of staff, g.l. dunnahoo, to suggest topics. veterinary public health was new, but a few weeks before the organizing group was to meet, i was directed to make a veterinary public health presentation and answer questions at the surgeon general's staff meeting. after the meeting, i asked dr. dunnahoo if he would be interested in a recommendation for a veterinary public health program for the who organizing committee. he urged me to give him a memo recommending a veterinary public health activity. that may , , i wrote a memo paraphrased as follows: ''regarding our conversation and your encouragement, i propose that in the organization of the united nation's health office there be a veterinary public health (vph) program. the vph program would be concerned with animal diseases transmissible to man. the vph would carry on liaisons with veterinary activities in the agriculture agencies and collect information on animal health.'' some months later i asked how the vph recommendation was received. dr. dunnahoo said there were no objections or discussion: the vph item was accepted and placed in the records. years later i learned an american veterinarian, martin m. kaplan, was recruited by an english physician with whom kaplan worked with in the united nations relief and rehabilitation administration (unrra) in greece. in kaplan came to the newly established who in geneva, switzerland. he developed a vph program in the communicable disease division that is a model for a public health program in the developing world. during the next years, he organized the expert committee meetings and technical reports. the first was in l (who, ) to review tuberculosis, which was a major disease problem in humans and animals at the end of world war ii. an american tuberculosis authority, dr. franklin top, a us army consultant, had reported that % of the human cases in occupied germany were caused by mycobacteria tuberculosis bovis. the problem was referred to the who expert zoonoses committee by the who expert tuberculosis committee. there was no consensus on what recommendation to make. the danish veterinarian dr. plum spoke for the classical tuberculin test and identification. the french urged the use of bacille calmette-guérin (bcg) vaccinations. the success of bovine tuberculosis eradication in the united states was recommended as the ideal method. eventually the committee recommended test and removal, with the caveat for developing countries to try other methods, including the bcg vaccination, which had no success in field trials. a number of other diseases were reviewed with the recommendation for control. there was a consensus on the following: q fever, anthrax, psittacosis, and hydatidosis. another issue was to settle on a definition of veterinary public health. a current definition of public health is summarized as diseases that are naturally transmitted between animals and man. the following year, , who called together a panel of rabies experts, including e.s. tierkel of the cdc (who, ). tierkel and others who followed from the cdc, namely george baer, keith sikes, jerry winkler and currently charles rupprecht, contributed to rabies control and prevention. the first of the who expert committees on the zoonoses was followed by zoonoses study groups in , which meyer chaired in stockholm (who, ) . he was most effective in leading the committee, and in his closing remarks he passed the leadership to james steele. at the next meeting of the who zoonoses expert committee in geneva in , i was the chairman (who, ) . the next meeting in was chaired by calvin schwabe (who, ) , professor of epidemiology at the university of california school of veterinary medicine and the school of human medicine. schwabe ( b) summarizes the who veterinary public health in his monumental third edition of veterinary medicine and human health: ''the final objective of veterinary medicine does not lie in the animal species that the veterinarian commonly treats. it lies very definitely in man, and above all in humanity.' ' we in veterinary public health recognize the contributions of acha and szyfres ( ) for their invaluable book, zoonoses and communicable diseases common to man and animals in spanish and english. it has been the foundation of veterinary public health epidemiology and surveillance in the spanish speaking countries of the americas. at this time, dr. george beran is to be recognized as one of the consultants to paho and who, and for his work in the philippines. he has carried on in admirable style for more than years in teaching, research, health promotion and consulting, and as author and editor. he has updated the chemical rubber company (crc) handbook of zoonoses series (beran, ) and the paho zoonoses reports, and hopefully will continue to do so. he is a historian of veterinary public health. in closing we pay tribute to the american veterinarians who demonstrated and promoted veterinary public health in the united states. most of these early pioneers years ago were recruited by the cdc and assigned to states that had zoonotic disease problems, mainly rabies. among the early cdc recruits assigned to a state was ernest wine. he was sent to pennsylvania, where he remained for years, rising to the position of state epidemiologist. oscar sussman went to arizona, and later the new jersey health department recruited him, where he built an outstanding program. martin baum served colorado for many years after leaving the cdc. john mason served in new mexico. art wolff did excellent service in michigan before returning to washington, where he became a leader at the usphs in environmental health as a radiation authority and assistant surgeon general. herbert stoenner went to utah and raymond fagan to indiana as described earlier. monroe holmes followed stoenner to utah, and john scrugs went to indiana when fagan went to the harvard school of public health. john winn, francis abimanti, don mason, and lauri luoto were among the early investigators of q fever in california. stoenner, in addition to his investigation of brucellosis and leptospirosis, was also a leader in q fever studies. don mason, john richardson, and paul arnstein worked on the control of psittacosis in k.f. meyer's laboratory at ucsf. dick parke, joe held and robert huffaker kept the cdc office responsive to many inquires and provided service to the states. james glosser closed his career at the cdc in . his work coordinating the venezuelan equine encephalitis epizootic and epidemic with the u.s. department of agriculture veterinary services earned him the united states department of agriculture's outstanding service award. the veterinarians service to public health in the th century resulted in better health in all humans and animals. what are the st century challenges? animal medicine and veterinary public health have been intertwined since humans first began ministrations to their families and animals. dr. william foege, former director of the communicable disease center and professor at emory school of public health and now consultant to the bill gates foundation center, expressed this more forcefully in saying that we cannot have good public health unless we have good animal health. we can invert that and say we cannot have good animal health unless we have good public health. in the united states, the veterinary medical profession has carried on effectively in eliminating those major problems of animal health that had serious public health ramifications, namely bovine tuberculosis and brucellosis. in recent years the advances in rabies immunization have eliminated the disease from our pets, and humans have benefitted. the new human cases that occur are mainly the result of bat exposure. looking beyond that, we can see there is a sizable list of parasitic diseases, namely trichinosis and tapeworms, that have been brought under control in the united states. however, tapeworms are now being introduced by the recruitment of workers from mexico, central america and south america. these problems affect society in the united states, but it is apparent that we have an obligation to share our knowledge with our neighbors of the americas as well as africa and asia. all of these countries face the same problems the united states, solved in the past century. now as we move into the st century, the technology for controlling these diseases is available. these proven effective procedures in the united states can be used worldwide. some challenges exist, however, for methods that control bovine tuberculosis. there is a continuous demand for vaccines to prevent tuberculosis in animals, but there is little evidence there is any value in routine vaccination. these procedures are quite costly, and the best examples are in europe in the past years. after world war ii, tuberculosis was a major problem in central europe especially in germany, eastern europe, what is now russia, and western europe. there has been an uncalled-for degree of confidence in the tuberculosis vaccine, bcg, but with constant pressure from the world health organization, the world animal health organization (oie), the food and agricultural organization and united states agencies and consultants, the use of vaccines has been put aside. the old test and removal strategies have proven to be the most successful. to introduce that method into mexico, central america, south america and asia is difficult at this time because they are hopeful that a good vaccine will be developed. unfortunately we have lived with that hope for years. the major problem that remains is to compensate farmers for diseased animals that are removed. the neighboring countries of mexico, central america and south america have the opportunity to further their own disease control by employing the proven techniques used in the united states, canada and europe. the control of brucellosis in the developing world is a much bigger problem than tuberculosis. in veterinary epidemiologist george baer described the human disease in mexico and said that most rural people who had reached the age of had evidence of past infection with brucellosis. the same can be said for the countries of latin america where goats have a high rate of b. melitensis infections. to control b. melitensis is a difficult task and is a matter of the governments facing up to the issue. a new vaccine developed in the united states, the rb rough strain, had been researched for years or more before the united states department of agriculture veterinarians were able to find a solution to producing an effective vaccine for cattle. the vaccines have not proven valuable for goats and sheep. the control of widespread brucellosis in north africa and the middle east across asia has been given little attention. the who, through their consultancies and expert committees on rabies, has spread the knowledge of dog vaccination throughout the world. we can say with some degree of pride that the technology developed by veterinarians at the communicable disease center and carried to other parts of the world by authorities such as the late ernie tierkel and others who worked with him and george baer have made a great contribution to the world scene. we do see the light at the end of the tunnel for worldwide control of canine rabies. other efficient rabies vaccines have been developed in south america and europe. looking at the parasitic infections of the world, there is certainly a great deal of interest in control of trichinosis, which has been fostered through scientific congresses every few years. the world wide results are favorable today with a drastic reduction in north america and europe. unfortunately new problems have arisen in connection with the disease in wild animals, especially those found in the arctic zones of the world. taenia saginata and t. solium are receiving more attention as we face worldwide problems with the measurements of disease. in the americas the problem has been carried from one country to another by human carriers and then spread to animals. new foci have been established in north america, where there have been meetings to plan for initiating a worldwide control program. in my own way of thinking, the control of t. saginata is a measurement of good hygiene and good waste control in any country where it is present. dr. peter schantz has advocated world control of tapeworm and hydatid disease with the goal of eradication. many other new problems arising in zoonotic and parasitic diseases are constantly coming to our attention. the continuous migration of workers seeking better opportunities in industrialized countries also carries the risk of infections being brought with them. the surgeon general has spoken for the globalization of public health. the veterinary public health program of the cdc has been active in globalization of veterinary public health, namely in the control of rabies, parasitic diseases and food borne diseases. many of the veterinary officers have served on who expert zoonosis committees have carried out detailed missions for who. the cdc program has been supportive of paho veterinary activities with assignments of veterinary officers to mexico, panama, peru, argentina, and most recently david ashford and hugh mainzer to brazil for foot and mouth disease control and other problems. the number of emerging diseases increased in the latter part of the th century. infectious disease scientists have found that acquired immune deficiency syndrome (aids) is a disease that makes people more susceptible to zoonotic diseases, including bovine tuberculosis and related mycobacterial infections, toxoplasmosis, cryptosporidiosis, food borne salmonella and enteric infections including campylobacter, listeria and yersinia. it is possible that other zoonotic diseases that are dormant or infrequent may emerge in individuals with aids, human immunodeficiency virus infection, or other immune-compromised conditions. related latent or nonpathogenic viral diseases have been described in tropical cats of africa including lions as well as domesticated cats. in australia and malaysia new diseases which also affect humans have been reported in horses and swine. these diseases are caused by the morbilliviruses, a measles-like virus that causes canine distemper and rinderpest. another virus that killed the wild felids in the cairo zoo has not been identified. could this be another form of distemper? some of the emerging viral diseases that have a rodent or unknown animal host have caused fatal, devastating diseases in humans in africa and south america, namely lassa fever and south american hemorrhagic diseases in argentina and bolivia. in africa, ebola virus hemorrhagic fever and marburg hemorrhagic fever virus infection, linked to monkey disease, caused disease in medical personnel, handlers and people who had only casual exposure. an incident that surprised us many years ago was the deaths of workers in middle east abattoirs caused by crimean hemorrhagic fever carried by ectoparasites. one example of developing, emerging, or relatively unknown diseases is severe acute respiratory syndrome (sars), a disease that erupted a few years ago in china and was carried to many parts of the world. recently information has suggested that bats are a natural reservoir of a sars like coronavirus. even though sars may have been an occasional emerging disease that disappeared as rapidly as it appeared, there may have been other infections from bats that have been around the world for millenia. naturally, an infection that has been given much attention now is where we stand with the influenza virus. are wild birds the true reservoir? apparently, birds are the reservoir based on the information we have gathered showing that wild birds transmitted the virus to avian domestic flocks. all this new information is challenging. the emerging diseases of the world are reasonably covered in the table of the last chapter of merck veterinary manual's ninth edition zoonosis section, . in addition to infectious diseases, we have a new class of diseases that are caused by prionsproteinaceous infectious parties that transfer diseases without any dna or rna. transmissible diseases are not the same as infectious diseases which are characterized by replication of dna or rna. this is certainly a bewildering situation especially when we read that saliva may be a means of transfer. immediately veterinarians think of rabies which is transmitted by saliva. is it possible the prion of the diseased brain can be secreted through nerve fibers that innervate the salivary gland? the prions are of great and continuing concern as a cause of concern as new types of diseases. our associates in chemistry, physics, and physiology may offer clues to other neurological diseases. one last subject i want to mention is humaneness. it is important that we abide by sensible, humane policies, but humaneness can be carried to such an extreme that it destroys values that we hold so high for protecting our pets, farm animals, and the wild animals around us. periodically we all read about overpopulations in different areas. society calls for conservative measures for population control that applies to all pets, wild animals and domestic animals. in a broader sense, it has applications to the human race. we are aware of the collapse of earlier civilizations that have overpopulated their given area or were destroyed by natural events such as starvation. so i say all veterinarians, especially those in public health, have a responsibility in developing humane regulations for animal population control and public guidance. in the united states, - % of veterinarians treat our animal associates or pets for various diseases. it is important that veterinarians have a broad, basic knowledge of public health issues and are alert for new public health issues that can be resolved with tender loving care, new antibiotics, and new procedures. the , or more veterinarians in the avma in the united states are key to the control of zoonotic diseases by public health agencies. the health of our animal population is tied to the emotional and mental well being of those humans who are close to animals in their lives. animals are vital companions to those homebound, and animal health becomes a family concern. an area i have stressed is the need for basic veterinary science. we see in current publications that most research is based on support from nih. at the avma meeting in july , the speaker us senator hatch of utah spoke highly of the public health activities of veterinary medicine. he went on to say that there may be a nih veterinary institute in the future. it behooves us all that the agricultural interest in public health be recognized as an important issue to the american public. i think highly of the importance of animal health in providing good public health. public health should not be guided by economic interest but by the welfare of all society. i go back to my earlier statement that animal health and public health are of great importance to all, and we must have good animal health to have good public health. good public health provides a means for good animal health. as we look to the future, we have to have open minds and think in terms that anything can occur in biology. i would like to quote my dean from michigan state, ward giltner, who said the only thing about biology we can accept that remains a firm truth is there always is new information that provides exceptions. looking at it broadly, all infectious things in nature, and prions which may cause disease are always looking for a new host. i like to say they are seeking social security, as most of the world is. carry on in the st century. i wish i could continue to be a part of it, but it seems time has a way of saying, ''you have been here. you have enjoyed it.'' i especially enjoy the recognition of years of public health service, i am elated. to the audience, especially to the teachers of public health science, thank you. carry on. dr. steele does not have a financial or personal relationship with other people or organisations that could inappropriately influence or bias the paper entitled ''veterinary public health: past success, new opportunities.'' zoonoses and communicable diseases common to man and animals. pan american health organization viral scientist dies-public health giant handbook of zoonoses section a: bacterial, rickettsial, chlamydial and mycotic. section b: viral, second ed epidemic food-borne listeriosis in western switzerland. ii. epidemio ogy the relation of animal diseases to the public health and their prevention listeriosis visceral larva migrans cutaneous larva migrans guns, germs, and steel: the fates of human societies pasteurized milk as a vehicle of infection in an outbreak of listeriosis a profile of the us public health service - . pub. no. nih- - . us department of 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evidence for transmission by food food safety veterinary medicine and human health listeriosis the jungle. double, page & company the veterinarian in america - the socioeconomic responsibilities of veterinary medicine biographical notes on the occasion of karl f. meyer's th birthday trichinosis zoonoses - : an update of james law's report on diseases of animals epidemiology of food borne salmonellosis present status of anthrax rabies: problems and control. a nationwide program nurture turned to poison a new species of brucella isolated from the desert wood rat neotoma lepida (thomas) the epizootiology of bovine leptospirosis in washington leptospiral serotype distribution lists ( to ). veterinary public health laboratory. us department of health, education, and welfare public health service a century's progress in public health effective control of an outbreak of rabies in memphis and shelby county a brief survey and progress report of controlled comparative experiments in canine rabies immunization leptospirosis milk ordinance and code. public health bulletin no. . department of the treasury joint who/fao expert group on rabies. who technical report series no. . who joint who/fao expert group on zoonoses: bovine tuberculosis, q fever, anthrax who, . joint who/fao expert committee on zoonoses, report on the nd session. who technical report series no. . who, geneva. who, . joint who/fao expert committee on zoonoses trichiniasis in the u.s. population, - . prevalence and epidemiologic factors the symposium at which this paper was presented was sponsored by bayer animal health. the author also wishes to acknowledge and thank dr. cynthia hoobler for assistance with the preparation of this paper. key: cord- -s oxr es authors: najar nobar, niloufar; goodarzi, azadeh title: patients with specific skin disorders who are affected by covid‐ : what do experiences say about management strategies? : a systematic review date: - - journal: dermatol ther doi: . /dth. sha: doc_id: cord_uid: s oxr es background and aim: in patients with dermatologic disorders who are affected by new corona virus, we know little about course (underlying disease and new onset‐infection) and the most proper management strategies include both issues that are what this systematic review targets. method: databases of pubmed, scopus, google scholar, medscape and cebd coronavirus dermatology resource of nottingham university searched completely and initial articles selected to further review and finally nine articles (including patients) entered to this study. result: from patients with chronic underlying dermatologic disease treated with systemic therapies, only one patient required icu admission, the others have been treated for mild‐ moderate symptoms with conventional therapies. the biologic or immunosuppressive/ immunomodulator agents has been ceased during the course of disease. the course of covid‐ its management was as similar as normal populations. their underlying dermatologic disease was exacerbating from mild to moderate. their treatment has been continued as before, after the symptoms improved. conclusion: exacerbation of patients underlying dermatologic disease is mild‐ moderate. discontinuing the treatment in the acute period of covid and the restart after recovery may prevent severe recurrence in these patients. this article is protected by copyright. all rights reserved. manifestations. there are growing reports of dermatologic manifestation among children like morbiliform or urticarial rash and conjunctivitis ( , ) or non-specific exanthematous, urticarial rashes and facial ulceration in affected infants may born from infected mothers ( , ) . hydroxychloroquine-induced rashes solely or in combination with other therapies, are responsible for the main cutaneous drug reactions especially generalized pustular eruptions ( ) ( ) ( ) . there are some secondary covid- related dermatoses like incidence of an acute new dermatologic entity for a limited time period that could be stress-related (e.g. herpes simplex, herpes zoster, alopecia areata ad etc…) or physical-environmental-related (e.g. contact dermatitis, acute urticaria…) or presence of a new dermatoses which have tendency to become chronic, like telogen-effluvium, various types of dermatitis, neurocutaneous or psychocutaneous disorders. in the pandemic, some preexisting chronic dermatoses may become poorly-controlled or aggravated by the stress, irregular visits, treatment interruptions, delayed therapies, physical and environmental situations like wearing masks and latex gloves, frequent washing and disinfectants, excessive sweating and etc ( ) . dermatologic visits and cosmetic or elective surgical procedure have been affected largely by a decreasing manner during pandemic ( ) . adherence to personal and social hygiene strategies, social isolation and distance are the rules of pandemic for better controlling any situations and the role of teledermatology is really important in this area regarding educational and therapeutic issues ( ) . in dermatology, one of the most important concerns in the pandemic setting is the manner of disease management especially in the case of patients under treatment with immunosuppressive immunomodulators. non-infected non-at risk patients do not need to any change in their therapeutic protocols but at-risk patients or who suspected to being infected (like having suggestive symptoms) need altering drug regimen as dose reduction, increase dose interval or transient stop of drug usage for at least weeks. it is not any doubt that, every patient with an active corona virus infection should discontinue systemic biologic or non-biologic immunosuppressive for at least month or completely being symptom-free. a more severe covid- course is usually predictable in the setting of dermatologic diseases which are treating by systemic immunomodulators. presence of any comorbidities related to underlying dermatologic disorder (like older age, metabolic syndrome and vital organ dysfunctions especially respiratory and cardiovascular involvements that may be seen in psoriatic or atopic patients or who with hidradenitis suppurativa, immunobullous or collagen vascular disorders), is associated with poorer outcomes in the case of being infected by covid- . some studies suggested that more sever skin conditions are at risk for higher rate of pneumonia and symptomatic respiratory involvement. in future vaccination of these groups of patients is one of priority issues. it seems that in patients with any severe and serious dermatologic disorders, under treatment with systemic agents, if there is not any suspicion about concurrent infection or any high risk exposures, not only it is not recommend to cessation therapy but only emphasize that these drugs could prevent disease flare-up and control cytokine storm that both in a negative direction, affect the covid- course ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . one of the most important perspectives in the field of dermatology is to approach patients with any documented skin or mucosal disorders especially who are using immunomodulators and now are affected by covid- . for consensus and more exact expert recommendations in this group of patients, we need data gathering about people who were in this situation and know more about what experience say about dermatologic disease course, covid- course and manner of managing both conditions in the best possible way? so in this systematic review we focused on specific patient groups with a dermatologic disorder (usually under therapy) that concomitantly have been infected by the new corona virus and summed up their data in all aspects of underlying and infectious disease course and management. this study is implemented according to the prisma (preferred reporting items for systematic reviews and meta-analyses) statement. inclusion criteria comprised all studies about patients with any documented skin or mucosal disorders especially who were using immunomoduators and now were affected by covid- . the exclusion criteria consisted of all publications not meeting the above contents and non-english literature. we searched databases of pubmed, scopus, google scholar, medscape and cebd coronavirus dermatology resource of nottingham university (https://www.nottingham.ac.uk/). our key words were ( "covid- " ) or ( "severe acute respiratory syndrome coronavirus " ) and ( "skin" ) or ( "skin diseases" ) or ( "skin manifestations" ) or ( "dermatology" ) or ("skin and connective tissue diseases" ) or ( "eczematous" ) or ( "eczema" ) or ( "eczematous dermatitis" ) or ( "eczematous eruption" ) or ( "eczematous skin" ) or ( "dermatitis, atopic" ) or ( "papulosquamous" ) or ( "psoriasis" ) or ( "hidradenitis, suppurativa" ) or ("alopecia" ) or ( "pemphigus" ) or ( "pemphigoid" ) or ( "immunobullous" ) or ( "skin cancer" ) or ( "dermatologic agents") or (" immunomodulator") or ( " systemic drugs" ) or ( "biologics" ) or (" immunomodulator") or ( "immunospress" ) or ( "immunospressive" ). in the search of cebd coronavirus dermatology resource of nottingham university we focused on the link of skin manifestations of coronavirus and coronavirus and specific patient groups. we finally complete our search up to may/ / . in the first screening articles assigned to the inclusion and exclusion groups and then the titles and abstracts of articles were review by two expert dermatologists and acceptable articles ( ) searched to find their full text and finally articles (including patients) selected to data entry for this systematic review ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . after researching, we have found nine related articles and the information of these articles are prepared as a table. of the patients reviewed in these articles, ten had psoriasis/ psoriatic arthritis and two had immunobullous disease included pemphigus and mucous membrane pemphigoid. their underlying disease was treated with the following drugs:  from patients with psoriasis/psoriatic arthritis, eight patients were treated with biologic agents (guselkumab: three patients, secukinumab: two patients, apremilast: one patient, adalimumab: one patient, ustekinumab: one patient); two patients were on other immunosuppressive/immunomodulators agents include cyclosporine and methotrexate.  one patient with mucous membrane pemphigoid was treated with prednisolone, rituximab, mycophenolate mofetil and high dose ivig  one patient with pemphigus was treated with mycophenolate mofetil this article is protected by copyright. all rights reserved. at the time of the pandemic, many patients were diagnosed with various dermatoses treated with biological agents or immunosuppressive/ immunomodulators ( ) . although, there is still no strong evidence of a higher risk of infection with covid- in these patients, and clinicians emphasizes that the symptoms and management of disease in these patients are similar to those in the general population ( ) . in addition, for many dermatoses, any mental or physical stress, such as infection or anxiety, may exacerbate the disease ( - ) . this study has evaluated nine case reports of patients with chronic dermatologic diseases treated with systemic agents. of the patients reported, the two patients did not show any symptoms despite close contact with people with confirmed cases of covid- ; at the time of quarantine, these patients showed no signs of the disease and their treatment continued as before. of the remaining ten patients, only one required hospitalization in the intensive care unit. five patients were treated with conventional therapies such as hydroxychloroquine, oseltamivir, lopinavir/ritonavir, antibiotics. other patients were treated with supportive/symptomatic therapies. at the time of the covid- , the biologic agents were discontinued except for one case (which was treated with guselkumab) but the patients did not report any severe exacerbation of their underlying dermatologic disease despite treatment discontinuation. there was only one case of severe relapse of psoriasis after covid- , in which the patient was previously treated with cyclosporine and methotrexate. investigation of these articles showed that the severity of covid- in patients with underlying dermatologic disease treated with biological agents was not more than in the general population. the management of these patients was in the form of discontinuation of the biological agents or immunosuppressive/ immunomodulators and the use of routine and conventional treatments. after the symptoms have improved, treatment of the underlying dermatologic disease can be recommenced as before. our knowledge about managing patients with dermatologic disorders (especially chronic disorders under treatment with systemic immunomodulators) who are affected by covid- in the pandemic is really scarce so for better controlling and managing both underlying dermatoses and concurrent corona virus infections, we need to sum up current relevant data that we did in this systematic review so far. we found that the severity of the disease or the need for hospitalization in these patients was not greater than in normal population. the treatment of the disease in confirmed cases with covid- were similar to that of normal population, with the difference that it is better to stop the patient's previous medications until the symptoms improve. in patients who have already been treated with biological drugs, recurrence of underlying dermatologic disease was mild. after the symptoms of the disease have improved, the patient's treatment could be continued as before. covid- : 'co' stands for corona, 'vi' for virus, 'd' for disease, and ' ' for . cebd: centre of evidence based dermatology classification of the cutaneous manifestations of covid- : a rapid prospective nationwide consensus study in spain with cases cutaneous manifestations in covid- : a new contribution cutaneous lesions in a patient with covid- : are they related? cutaneous manifestations in covid- : a first perspective cutaneous manifestations in covid- : a first perspective " by recalcati s clinical and histopathological study of skin dermatoses in patients affected by covid- infection in the northern part of italy cutaneous manifestations of covid- : report of three cases and a review of literature dermatologic findings in two patients with covid- cutaneous manifestation of covid- in images: a case report alert for non-respiratory symptoms of coronavirus disease (covid- ) patients in epidemic period: a case report of familial cluster with three asymptomatic covid- patients occupational skin disease among health care workers during the coronavirus (covid- ) epidemic a distinctive skin rash associated with coronavirus disease a case of covid- pneumonia in a young male with full body rash as a presenting symptom morbilliform exanthem associated with covid- rash as a clinical manifestation of covid photographs of a patient sars-cov- infection presenting as a febrile rash urticarial eruption in covid- infection urticarial exanthem as early diagnostic clue for covid- infection a case of covid- presenting in clinical picture resembling chilblains disease. first report from the middle east chilblain-like lesions on feet and hands during the covid- characterization of acute acro-ischemic lesions in non-hospitalized patients: a case series of patients during the covid- outbreak chilblain-like lesions during covid- epidemic: a preliminary study on patients complement associated microvascular injury and thrombosis in the pathogenesis of severe covid- infection: a report of five cases acral cutaneous lesions in the time of covid- acute acro-ischemia in the child at the time of covid- coagulopathy and antiphospholipid antibodies in patients with covid- covid- can present with a rash and be mistaken for dengue": petechial rash in a patient with covid- infection petechial skin rash associated with severe acute respiratory syndrome coronavirus infection covid- can present with a rash and be mistaken for dengue varicella-like exanthem as a specific covid- -associated skin manifestation: multicenter case series of patients varicella-like exanthem associated with covid- in an -year-old girl: a diagnostic clue? a dermatologic manifestation of covid- : transient livedo reticularis cutaneous manifestations in the current pandemic of coronavirus infection disease (covid ) a child confirmed covid- with only symptoms of conjunctivitis and eyelid dermatitis characterizing the adverse dermatologic effects of hydroxychloroquine: a systematic review a case of exacerbation of psoriasis after oseltamivir and hydroxychloroquine in a patient with covid- : will cases of psoriasis increase after covid- pandemic? generalized pustular figurate erythema: a newly delineated severe cutaneous drug reaction linked with hydroxychloroquine an assessment of united states dermatology practices during the covid- teledermatology: a useful tool to fight covid- should patients stop their biologic treatment during the covid- pandemic advice regarding covid- and use of immunomodulators, in patients with severe dermatological diseases covid- and the use of immunomodulatory and biologic agents for severe cutaneous disease: an australia/new zealand consensus statement managing cutaneous immune-mediated diseases during the covid- pandemic dermatology patients' knowledge and concerns regarding their immunomodulatory medication during the covid- pandemic the prompt use of rituximab could decrease adverse effects in patient with pemphigus vulgaris: a preliminary evaluation biologic therapy for psoriasis during the covid- outbreak: the choice is to weigh risks and benefits no evidence of increased risk for covid- infection in patients treated with dupilumab for atopic dermatitis in a high-epidemic area systemic isotretinoin therapy in the era of covid evolution of covid- infection in psoriatic patients treated with biological drugs occurrence of sars-cov- during mycophenolate mofetil treatment for pemphigus mucous membrane pemphigoid and covid- treated with highdose intravenous immunoglobulins: a case-report covid- pulmonary infection in erythrodermic psoriatic patient with oligodendroglioma: safety and compatibility of apremilast with critical intensive care management a challenging case of psoriasis flare-up after covid- infection covid- in an elderly patient treated with secukinumab improvement of sars-cov symptoms following guselkumab injection in a psoriatic patient sars-cov- infection in a psoriatic patient treated with il- inhibitor systemic immunobiological, immunosuppressant and oncologic agents for the treatment of dermatologic diseases during the sars-cov- (covid ) pandemic emergency: a quick review for a quick consultation evaluating the role of stress in skin disease. stress and skin disorders association between stress and skin disease the authors would like to thank rasoul akram hospital clinical research development center (rcrdc) for its technical and editorial assists. the authors contribute equally to all stages of this study. the team has reviewed the manuscript and the data, and all contributors were in full agreement. the authors declare there is no conflict of interest in this study. this study had no funding. key: cord- -cz m iqk authors: cheng, tsung o. title: the current state of cardiology in china date: - - journal: int j cardiol doi: . /j.ijcard. . . sha: doc_id: cord_uid: cz m iqk cardiology in china has shown significant changes in the last decade or so. interventional cardiology, in particular, has shown remarkable advances, especially in the management of coronary artery disease, which, unfortunately, has shown a disconcerting increase in incidence in a country traditionally known for very low incidence of coronary artery disease. important contributing factors include increasing affluence, westernization of dietary habit and lifestyle, and rampant cigarette smoking. at present, the chinese population has an annual coronary mortality of one sixth of that reported in the west, an incidence of acute myocardial infarction of one tenth to one eighth, and a mortality of acute myocardial infarction of one eighth. the prevalence of coronary artery disease among the general chinese population ( – %) is roughly one quarter of that among the caucasians in the west, but this will get worse for sure. china still has a lot of catching up to do to reach full modernization. there is a price that every developing country must pay for modernization. however, let the price the chinese pay not exceed the benefits derived from modernization. can we achieve a utopian stage in the st century in which the modern chinese retain their ancestral low rates of coronary artery disease while adapting the positive aspects of a modern western lifestyle? i used to return to china to lecture, demonstrate, and exchange scientific and medical information every - years since . i have previously reported my observations on several occasions [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . due to increasing demand on my time by other countries around the world in the past decade, i have not been back to china as frequently in recent years. the last time i visited china before my latest visit in october was in . so much changes have taken place that i felt it important to report a few of the more significant ones in modern china. incidentally, severe acute respiratory syndrome (sars), the recent epidemic that spread through populous china then spread around the world and posed as a potential threat to china's stability, is said to have originated in the southern china province of guangdong in the fall of . despite the accusations that the chinese authorities have been underreporting or covering up cases of sars, i saw no evidence of such an occurrence during my visit in october , which included hong kong, guangzhou, beijing, and shanghai. it only came to surface early in when, according to a good colleague of mine in guangzhou who apologized for not promptly responding to my correspondences, as he always did over the many years of our acquaintance, his hospital ''had been kept busy with quite a few cases of atypical pneumonia since the spring festival,'' another term for the chinese new year, which fell on february , . most cases of atypical pneumonia reported during that period were indeed cases of sars. as a matter of fact, the chinese term for sars is still atypical pneumonia . personally, i do not believe for a moment that china was underreporting or covering up cases of sars. china was simply caught offguard by the sudden appearance of this mysterious disease, which, on the surface, resembled common colds or ordinary flu. because it occurred during the usual flu season, i could empathize with china's initial ''what's the big deal?'' attitude. to me, the most important outcome of this whole incident of sars is that china's press and news media have now become completely transparent-a sign of chinese ''glasnost''. of course, sars is all history now (fig. ). as i pointed out in my earlier publications [ ] [ ] [ ] , coronary artery disease, which used to be extremely rare in old china [ ] , has increased considerably in prevalence in china in the past several decades. coronary artery disease in china has climbed from the fifth most common form of heart disease in - , to the second most common in - and - , and to the most common in - (table ) , where it remains until this date. this changing pattern of heart disease in china was also reflected in the changing etiologies of congestive heart failure in modern china. whereas rheumatic heart disease used to be the most common cause of congestive heart failure in the s, coronary artery disease took over in the s and (table ). myocardial infarction, which was extremely rare in old china, has been the fifth leading cause of death in modern china [ ] . i still recall that when i was a medical student, i saw only one case of acute myocardial infarction during my entire years of schooling. it was such a rarity that the patient became the subject of medical grand rounds, cardiology grand rounds, ecg conference, radiology conference, physiology seminar, pharmacology round table, journal club, and research conference. i felt so sorry for that poor patient who had to be present in each of these didactic conferences. at present, the chinese population has an annual coronary mortality of one sixth of that reported in the west, an incidence of acute myocardial infarction of one tenth to one eighth, and a mortality of acute myocardial infarction of one eighth [ ] . the prevalence of coronary artery disease among the general chinese population ( - %) is roughly one quarter of that among the caucasians in the west [ ] . but this will change for the worse in the years to come. part of the reason for this changing pattern is, of course, relative due to more accurate diagnosis, less people dying from malnutrition and infectious diseases, and more people living to an older age. but most of the increases are really due to the following contributing factors. modern chinese love the atherogenic fast food and devour them at an ever-faster rate. the huge success of the world's two of the best known fast-food giants, mcdonald's ( fig. ) and kentucky fried chicken, also known as kfc (fig. ) , is the result of a change of chinese lifestyles, which are becoming more geared to speed, convenience, and choice. although most of the chinese do not like the taste of either hamburgers (chinese prefer pork to beef) or cheeseburgers (chinese compare cheese to vomitus), they possess enough curiosity to try them. furthermore, consumers in china are buying at mcdonald's and kfc because they are fashionable in the west, not because they represent [ ] . more than one third of adults in china and over half of adults in china's urban areas consume over % of their energy from fat [ ] . as a consequence of the recent change in the dietary habits in china, the normal plasma cholesterol values in modern china have shown a steady increase. in , the upper limit of normal was . mmol/l or mg/dl, and the mean value was . mmol/l or mg/dl [ ] . these ''normal'' values, which were very similar to those reported recently from another study of retirees in beijing [ ] and the chinese monica project involving million chinese from provinces [ ] , were considerably higher than the normal values in china published in ( mg/ dl) [ ] , ( mg/dl) [ ] , and ( mg/dl) [ ] . in a country known traditionally for its low plasma cholesterol values, low incidence of coronary artery disease, and lean body build, this upward adjustment of the so-called ''normal'' values in modern china represents an alarming trend that deserves special attention. this assumes an even greater significance in view of the recent report that blood cholesterol concentration is directly related to mortality from coronary artery disease even in those with-what was, by western standards-a ''low'' cholesterol concentration [ ] . in addition to the recent fast-food onslaught, there is also a new craze in china for milk. china is starting to consume more milk, a basic change in the national diet that reflects an upward shift in the expanding chinese middle class. in old china, milk was considered a luxury, costing more than water or tea. with the improvement of living standards, near doubling of milk production during the last years [ ] , and the fitness craze sweeping across the country, chinese are drinking more milk in record numbers- % increase from to (q.y. ge, personal communication, april , ) . china's annual milk production has finally caught up with that of its grain alcohol [ ] . so, instead of ''gan bei'' (''bottoms up'') with the traditional mao-tai at most of the state banquets, chinese hosts are now holding up glasses filled with milk. unfortunately, while the older generations of chinese have benefited from a diet high in vegetables and fruits and low in saturated fats, a move to too much milk undoubtedly would accelerate the development of atherosclerosis in modern china. such changes in china's dietary intake will have a great influence on the coronary artery disease burden, as was supported by the recently reported singapore experience in which the higher consumption of dietary saturated fat is associated with a higher serum cholesterol and higher coronary artery disease mortality in singaporean chinese compared with the chinese in hong kong and mainland china [ ] . china used to be known for her slender people. i never saw a fat person in china until recent years (fig. ). now china is fighting obesity, especially childhood obesity, which is as high as % [ ] as the rest of the world [ ] . excessive caloric intake from fast food [ ] ; insufficient exercise from increasing availability of, and increased reliance on, automobiles [ ] ; and the popularization of television result in weight gain. the effect of television viewing on pediatric obesity has recently been documented: each hourly increment of tv viewing is associated with a - % increase in the prevalence of obesity in urban china [ ] . the prevalence of obesity continues its upward trend in china as the rest of the world. by the end of , the obesity rate of male students in beijing reached %, doubling that of and approaching that of developed countries [ ] . traditionally, a fat child in china meant a healthy child, one who was likely to survive the rigors of undernourishment and infections. this misconception still prevails today in many parts of china, which, unfortunately, is a participant in an international epidemic of childhood obesity [ , ] . fat children grow up to be fat adults. overweight in china [body mass index (bmi) z ] has shown a progressive increase in both men and women over the past two decades (table ) [ ] . it is estimated that overweight is present in - % of northern chinese and % of the total chinese population [ ] . obesity (bmi z ) is present in % of the population in northern china and % in southern china; the total number of obese people in china is now estimated to be - million [ ] . the chinese have a lower baseline bmi to begin with (baseline value = [ ] ; mean = . - . [ ] ), and it takes less increment to reach an obese level, so that a bmi of - . is considered as overweight and z as obese [ ] . it takes smaller increments to increase the risk of hypertension, coronary artery disease, and type ii diabetes in the chinese population [ , , ] . china is in the midst of an obesity epidemic [ ] . obesity in china has reached such a serious stage that the chinese government has started a national campaign against it [ ] . furthermore, the chinese medical association just convened its first national symposium on the prevention and treatment of obesity on march , in suzhou [ ] . the recent report that obesity significantly raises cancer risk [ ] will undoubtedly add impetus to china's war against obesity because of the chinese's inordinate fear of cancer. china is the greatest producer and consumer of cigarettes in the world [ ] . one of every three cigarettes manufactured in the world is consumed in china (fig. ). even more alarming is the prevalence of teenage smoking in china (fig. ) ; three of every five chinese smokers begin smoking at the age of - years [ ] . although cigarette smoking is far less common among the chinese women than men [ ] , women smoke almost as much as men in the autonomous region of inner mongolia. cigarette smoking becomes the only pastime for both men and women who are housebound by the inclement weather in the winter in the inner mongolia grasslands (fig. ) . as the chinese saying goes, ''women uphold half of the sky''; therefore, women smoke as much as the men in inner mongolia [ ] . countries such as china, with its million smokers, are being targeted by the tobacco industry as stricter control on tobacco began to bite in the united states. the young smokers in china not only like to smoke foreign brands of cigarettes but also name brands. next to coca cola and mickey mouse, marlboro is the third most well-known american name in china. advertisements in the form of billboards are conspicuously shown in every major city in china (fig. ) . despite health hazard warnings on the cigarette packs as required by the ministry of public health and uncontroversial evidence that cigarette smoking is a major cause of death in china [ ] , a direct cause of coronary artery disease in china [ ] , and an indirect cause of coronary artery disease by causing hypertension [ ] and diabetes [ ] , many chinese continue to smoke, including medical professions. on each china visit, i always inquired why so many chinese continue smoking despite the health hazards. they always pointed out to me that the late chairman mao zedong smoked (fig. ) and the late chairman deng xiaoping also smoked (fig. ). mao and deng were china's two greatest leaders in the th century and are still revered throughout china. so i believe that example-setting is very important. furthermore, despite the recent unanimous approval of the global antitobacco treaty by all countries in the world health organization [ ] , of which china is a member, china is still not enforcing the ban, claiming that ''individuals' rights should be respected.'' since the introduction of selective cine coronary arteriography into china in [ ] , the field of interventional cardiology has been growing by leaps and bounds and is now an established discipline in china (table ). selective coronary arteriography is now routinely performed in every major medical center throughout china with a very respectable complication rate. as was recently reported from the fu wai hospital and cardiovascular institute, chinese academy of medical sciences, beijing [ ] , where i performed china's first selective cine coronary arteriogram in [ ] , in an analysis of cases from october to december , death, myocardial infarction, stroke, serious arrhythmias, peripheral vascular complications, and allergic reactions to the contrast media were . %, . %, . %, . %, . %, and . %, respectively, as compared to . %, . %, . %, . %, . %, and . % in an analysis of , cases between and from the registry of the society for cardiac angiography and interventions [ ] . percutaneous transluminal coronary angioplasty (ptca) was first performed in xian, china, in . percutaneous coronary intervention (pci) is growing rapidly in china ( fig. ) at an annual rate of increment of around % [ , ] . in , , procedures were performed with a success rate of % [ , ] . this, of course, is rather a small number as compared with the united states where, in , , procedures were performed [ ] . similar to the trend in the western world, the chinese cardiologists have also become more aggressive in the management of acute coronary syndromes. according to a recent report of a study, which is a part of the international multicenter registry, selective coronary arteriography was carried out in % of these patients, in half of whom (or %) ptca was performed; thrombolytic therapy was carried out in only % of the patients and coronary artery bypass grafting (cabg) in % [ ] coronary stenting has also rapidly expanded in number; according to the third national coronary intervention registry, published at the end of , over a -year period from to , stents were implanted in % ( , of , ) of pcis with a success rate of % [ ] . the number of pcis has surpassed that of percutaneous balloon mitral valvuloplasty, which used to be the most frequently performed interventional cardiologic procedure in china [ - ] . this reversal in trend reflects the change in the etiologies of heart diseases in modern china over the past two decades, namely, an increase in coronary artery disease and a decline in rheumatic heart disease ( table ) . chinese cardiologists learn fast. hypertrophic cardiomyopathy was an unfamiliar diagnosis in china until i introduced selective coronary arteriography in [ ] ; of patients catheterized because of exertional chest pain were found to have normal coronary arteriograms but hypertrophic obstructive cardiomyopathy with a significant systolic gradient across the left ventricular outflow tract and a typical angiographic appearance. this disease entity is encountered more and more often now in china. as a matter of fact, the interventional cardiologists in china have also become quite aggressive in treating these patients with the percutaneous technique of transluminal septal myocardial ablation with alcohol, with one center alone having performed such a procedure in patients with excellent short-and long-term results [ ] . according to the chinese national registry, over such procedures have been performed among hospitals in china by the end of . the chinese cardiologists have also been actively engaged in laboratory investigations to determine whether it is the volume of the alcohol injected, or the rate of alcohol injection into the first septal perforator of the left anterior descending coronary artery during septal ablation that is important. it was concluded that the size of the iatrogenically induced myocardial infarct in piglets is directly related to the volume of intracoronary alcohol injection, rather than the rate of alcohol injection [ ] . frequent symposia on interventional cardiology are held regularly at both national and local levels to keep the cardiologists abreast of the latest developments in interventional cardiology. these meetings are usually supported by foreign pharmaceutical companies or/and device manufacturers, which also take care of the travel expenses of chinese cardiologists going overseas to attend international cardiologic meetings. unlike the united states where such practices are now forbidden, many chinese cardiologists who otherwise would not be able to afford such international trips on their meager salaries are able to attend these cardiologic meetings in both europe and north america. upon returning to china from such meetings, they promptly report the highlights of such meetings in national or local medical journals in china. such practices serve to educate the rest of the cardiologists in china who otherwise would not have an opportunity to keep abreast of the recent advances in cardiology due either to lack of despite frequent gifts from their grateful patients, which may range from a chicken from a farmer to a mercedes benz automobile from a wellto-do businessman. financial means to go overseas, or to unfamiliarity with the english language. china's first cabg operation was performed in october at beijing's fu wai hospital where china's first selective coronary arteriogram was performed in june [ ] . it was a single-vessel saphenous vein graft to the left anterior descending coronary artery in a -year-old male patient who survived for years (zhu xiaodong, md, fu wai hospital, personal communication, august , table ) . one of the patients in harbin, china, has survived for more than years now following the heart transplantation. as is the case with the rest of the world, the principal limiting factor in heart transplantation is scarcity of donors. the latter factor was further complicated in china by the traditional buddhist teaching that a dead person must be buried with all the organs intact to ensure the spirit's happiness. there exist rumors that hearts and other organs from executed prisoners in china have been harvested as donor organs without the explicit consent of their family members. but i am unable to confirm such rumors. however, such practices provide a useful donor source in taiwan where the condemned prisoners voluntarily initiated the process of organ donation [ ] . pulmonary embolism used to be thought of as rare in china [ ] . but this is no longer the case. in a recent analysis of documents of chinese language case studies involving misdiagnoses of pulmonary embolism published from january to june and identified by searching the chinese biomedical literature database [ ] , patients with misdiagnosed pulmonary embolism were identified. the five commonest misdiagnoses were coronary artery disease in %, pneumonia in %, primary pulmonary hypertension in %, cardiomyopathy in %, and pleurisy in % [ ] . china is launching a campaign to increase awareness on this disease, as evidenced by several recent publications in major medical journals in china [ - ] . there are several explanations for underdiagnosing or misdiagnosing pulmonary embolism in china: ( ) traditional teaching by such authorities as snapper (fig. ) , who was highly regarded in old china as one of the most astute diagnosticians, was that pulmonary embolism was rare in china. he did not even mention it in his book ''chinese lessons to western medicine'' [ ] . ( ) even as recent as s, pulmonary embolism was thought to be rare in china [ ] the rarity of pulmonary embolism was even more remarkable when one considered the widespread use of birth control pills among the chinese women of child-bearing age [ ] . perhaps the use of acupuncture anesthesia [ , - ] , which permitted early ambulation after major surgery, accounted for the low incidence of postoperative pulmonary embolism in china. because acupuncture anesthesia is seldom employed in modern china, this protective factor is no longer operative. ( ) more accurate in vivo diagnosis of pulmonary embolism is now possible because of improved diagnostic techniques. the publishing industry in china has shown an extraordinary growth in recent years. in , china published , books; years later, the number was , -a little more than twice that of the united states [ ] . similarly, exponential growth took place for magazines (from to ) and newspapers (from to ) over the same period [ ] . by comparison, the united states published , magazines in and newspapers in [ ] . this publishing explosion was mirrored in medical publishing as well. not only the quantity but also the quality of medical books showed a great deal of improvement within a relatively short period of time. some textbooks in cardiology that were originally published as a single volume (fig. a ) had to be published in a twovolume set because of increased pages required to cover all the latest advances (fig. b) . other books, such as the one on congestive heart failure, which originally appeared as monographs (fig. a) , later were expanded to become textbooks (fig. b) . the chinese medical doctors are thirsty for new knowledge; because attendance at national and international medical meetings was restricted by financial constraints, they have to rely mostly on books and journals for their postgraduate medical education. china published some medical journals in [ ] (table ) [ ] . the top five journals represented nearly one third of the citations. this list is somewhat reminiscent of the omnipotent science citation index impact factors of these journals. according to the chinese scientific and technological journal citation reports , which was published in , impact factor and total citation frequency for the chinese journal of cardiology ranked th and st, respectively, among chinese scientific and technological journals (y.m. jiang, chinese journal of cardiology, personal communication, june , ) . a word about international access to the chinese medical literature through medline: users doing author searches may have difficulties with common chinese surnames, especially if only one given name initial is used [ ] . the difficulty may be further compounded by the fact that chinese authors customarily spell their last names first and first names last [ - ] . china, in both basic research and clinical investigation in cardiology, has made significant improvements and advances over the years in the following several broad areas. ( ), and other countries. the growth in the number of participants from china reflects an increased interest in biomedical collaboration between us and chinese scientists. over a -year period from to , the number of abstracts submitted from china increased from to , and the number accepted for presentation showed a parallel increase from three to eight (table ) . a similar trend was noticed for the american college of cardiology annual scientific meetings. such increases in contributions of our chinese colleagues to these national heart meetings are quite impressive. china is a huge country with its . billion population; one of every five persons on earth lives in china. china's unique environment, therefore, constitutes a tremendous opportunity for intervention trials. ethnic, demographic, and cultural diversities make it easier to conduct major epidemiologic studies in china. because the chinese population is more compliant than the western population, high participation rates are certain. because the chinese population is more stable than the western population, follow-up is easier and more complete. finally, the studies are much cheaper to carry out, ''practically nothing compared to what a similar trial will cost in the united states,'' as noted by fraumeni of nih according to mervis [ ] . besides participating in many international trials, china also conducts several national trials on her own [e.g., tissue plasminogen activator/urokinase comparisons in china (tucc)] [ ] . some of these trials carry rather cute acronyms [e.g., cast (chinese active stroke trial), cat (chinese angiotensin-converting enzyme inhibitor in acute myocardial infarction trial), cathay (chinese atherosclerosis study in the aged and young), and stone (shanghai trial of nifedipine in the elderly)], similar to the fascination with the creation of attractive trial acronyms by the western investigators [ - ] . the use of statistical methods in chinese medical journals has been improving [ ] . according to wang and zhang [ ] , the percentages of original articles reporting clinical trials, prospective studies, or basic science research increased from % in to % in ( p < . ), of articles using statistical methods increased from % in to % in ( p < . ), and of those articles using appropriate statistical methods increased from % to % ( p < . ). in both and , the most commonly used statistical methods in the chinese medical journals were t tests and contingency tables [ ] . according to wang and zhang [ ] , the most common errors the chinese authors committed were presentations of p values without specifying the test used, use of multiple t tests instead of analysis of variance, and use of unpaired t tests when paired tests were required. medical genetic research in china started in the late s [ ] . molecular genetic research started in china in the s [ ] . as the human genome project was initiated in the s, china also launched a national human genome project as part of its contribution to international efforts to sequence the human genome [ ] . china's contribution in the human genome project could be significant. according to one official from the national science foundation of china, ''the chinese account for over one fifth of the world's population, and the country has ethnic groups. as a result, no databank of human genetic information can be said to be complete without a detailed study of the chinese human genome'' [ ] . the project will have important practical implications, as the chinese, with their powerful sequencing capacity and capability in the genome project, are expected to contribute vital information that may lead to the early diagnosis and treatment of more than hereditary diseases [ ] . in , china's scientific leaders overcame skepticism from some members of the human genome project to become the only developing country to take a role in sequencing the human genome by contributing % of the published sequence-an achievement that is of huge sym- table american heart association scientific sessions abstracts submitted and accepted from china, china, - china, china, china, china, china, china, china, china, china, china, china, submitted bolic importance [ ] . chinese researchers are now setting up programs in everything from stem cell research, through large-scale efforts to determine protein structures, to population studies to hunt for human disease genes [ ] . there is even talk of trying to clone the endangered giant panda [ ] . recently, china presented her new edition of a genetic map illustrating the no. human chromosome, as part of the human genome project [ ] . the new completed mapconsidered . % accurate-would help improve medical diagnosis and also aid the discovery of new drugs and new treatments [ ] . in the field of cardiology, china has made much progress in research work for gene therapy of hypertension [ ] , myocardial infarction [ ] , and congestive heart failure [ ] . on the long list of organs that should be sequenced, the beijing genomics institute has already started investigations into proteomics and drug discovery, including a project to isolate the active compounds in the herbs used in traditional chinese medicine [ ] . there are many chinese herbs that are widely used in this country, especially for treatment of coronary artery disease and congestive heart failure. unfortunately, they tend to interact unfavorably with commonly prescribed western cardiac drugs, including warfarin [ - ] , digoxin [ - ] , and hypoglycemic agents [ ] . therefore, the isolation of the active compounds in these herbs will facilitate the understanding of the pharmacodynamics of these herbs and thus avoid their inadvertent adverse interactions with the western medications. cardiology in china has shown significant changes in the last decade or so. interventional cardiology, in particular, has shown remarkable advances, especially in managing coronary artery disease, which, unfortunately, has shown a disconcerting increase in incidence in a country traditionally known for its very low incidence of coronary artery disease. important contributing factors include increasing affluence, westernization of dietary habit and lifestyle, and rampant cigarette smoking. changes in china acupuncture anesthesia a view of modern chinese medicine. observations by a shanghai-born physician during his recent visit after years of absence medicine in modern china china medicine as we saw it. bethesda: the fogarty international center white lecture-cardiology in people's republic of china acupuncture anesthesia for open heart surgery cardiology in people's republic of china in cardiology in china revisited barefoot doctors cardiovascular disease in china coronary heart disease in china price of modernization of china chinese lessons to western medicine suicide rates in china - principles and practice of clinical medicine in asia. treating the asian patient. philadelphia: lippincott, williams and wilkins china swallows fast food at an ever more rapid rate risk of having heart disease soars in china experts differ on healthiness of 'fast' salad a new stage of the nutrition transition in china extensive association analysis between polymorphisms of pon gene cluster with coronary heart disease in chinese han population a long-term followup study of serum lipids and coronary heart disease in the elderly epidemiologic study of blood lipids in the chinese population the international textbook of cardiology the textbook of modern cardiology serum cholesterol concentration and coronary heart disease in population with low cholesterol concentrations new craze seizes china's consumers: a glass of milk the emergence of coronary heart disease in populations of chinese descent obesity: a warning to chinese children the obesity epidemic is a worldwide phenomenon fast food and obesity in china effect of television viewing on pediatric obesity jama patient page. obesity treatment of pediatric and adolescent obesity advances in research on hypertension and obesity. program of national congress on prevention and treatment of obesity obesity, coronary heart disease risk factors and diabetes in chinese: an approach to the criteria of obesity in the chinese population chinese medical association subsection of cardiovascular disease epidemiological study on obesity and its comorbidities in urban chinese older than years of age in overweight is an independent risk factor for cardiovascular disease in chinese populations an obesity epidemic in modern china what is china doing in policy-making to push back the negative aspects of the nutrition transition? national conference on prevention and treatment of obesity to be held in suzhou overweight, obesity, and mortality from cancer in a prospectively studied cohort of us adults smoking and diabetes in chinese men teenage smoking in china smoking and health do photographs lie? smoking in china mortality attributable to cigarette smoking in china a review on studies of smoking and coronary heart disease in china and hong kong meta-analysis of risk factors on hypertension in china wha approves historic tobacco accord complications in selective coronary arteriography: analysis of cases coronary arteriography - : a report of the registry of the society for cardiac angiography and interventions: i. results and complications furthering the healthy development of percutaneous coronary interventions in china section of interventional cardiology, chinese society of cardiology, editorial office of chinese journal of cardiology: a data analysis of the third national coronary intervention registry treatment of coronary artery disease in the year clinical characteristics of acute ischemic syndrome in china for the multicenter study group. percutaneous balloon mitral valvuloplasty using inoue technique: a multicenter study of patients in china percutaneous balloon mitral valvuloplasty by the inoue balloon technique: the procedure of choice for treatment of mitral stenosis late results of percutaneous balloon mitral valvuloplasty: the chinese experience percutaneous transluminal septal myocardial ablation for hypertrophic obstructive cardiomyopathy. the chinese experience in patients from a single center experimental study of relationship between intracoronary alcohol injection and the size of resultant myocardial infarct the gift of life from prisoners sentenced to death: preliminary report an investigation on the misdiagnosis of pulmonary embolism in china nomenclature and definition of pulmonary thromboembolism guidelines to the diagnosis and treatment of pulmonary thromboembolism the role of the media in a market economy chinese medical journals; getting in touch. chinese editors and medical journals have much to offer a preliminary study of reference citations of foreign journals in articles published in the chinese journal of cardiology international access to the chinese medical literature through medline the chinese last name what's in a name-the chinese name? word order: oriental or occidental? chinese name use of surnames to identify individuals of chinese ancestry chinese last name comes first china's unique environment favors large intervention trials a randomized trial confirming the efficacy of reduced dose recombinant tissue plasminogen activator in a chinese myocardial infarction population and demonstrating superiority to usual dose urokinase: the tucc trial acronyms of clinical trials in cardiology- acronymophilia: the exponential growth of the use of acronyms should be resisted acronymania, acronymophilia and acronymophobia acronyms of clinical trials in cardiology- acronyms of cardiologic trials- research design and statistical methods in chinese medical journals the achievements of medical genetic research in china during the past years china launches genome project a great leap forward genetic map the textbook of modern cardiology relationship between apolipoprotein e gene polymorphism and myocardial infarction or its action on clinical courses a textbook on congestive heart failure from standing start to sequencing superpower warfarin danshen interaction warning about a medicinal herb herbal remedies interaction of herbal medicine with coumadin ginseng -warfarin interaction comment: drug -herb interaction herbal interactions with cardiac drugs john's wort interaction with digoxin interaction of herbal drugs with digoxin panax (ginseng) is not a panacea key: cord- - tbrl a authors: hawkes, c. h.; del tredici, k.; braak, h. title: parkinson's disease: a dual‐hit hypothesis date: - - journal: neuropathol appl neurobiol doi: . /j. - . . .x sha: doc_id: cord_uid: tbrl a accumulating evidence suggests that sporadic parkinson's disease has a long prodromal period during which several non‐motor features develop, in particular, impairment of olfaction, vagal dysfunction and sleep disorder. early sites of lewy pathology are the olfactory bulb and enteric plexus of the stomach. we propose that a neurotropic pathogen, probably viral, enters the brain via two routes: (i) nasal, with anterograde progression into the temporal lobe; and (ii) gastric, secondary to swallowing of nasal secretions in saliva. these secretions might contain a neurotropic pathogen that, after penetration of the epithelial lining, could enter axons of the meissner's plexus and, via transsynaptic transmission, reach the preganglionic parasympathetic motor neurones of the vagus nerve. this would allow retrograde transport into the medulla and, from here, into the pons and midbrain until the substantia nigra is reached and typical aspects of disease commence. evidence for this theory from the perspective of olfactory and autonomic dysfunction is reviewed, and the possible routes of pathogenic invasion are considered. it is concluded that the most parsimonious explanation for the initial events of sporadic parkinson's disease is pathogenic access to the brain through the stomach and nose – hence the term ‘dual‐hit’. sporadic parkinson's disease (pd) is the most frequent degenerative disorder of the human nervous system after alzheimer's disease. it is not known to occur spontaneously in other vertebrates and does not directly affect other organs apart from the nervous system. motor dysfunction (hypokinesia, postural imbalance, cogwheel rigidity, resting tremor) indicates the presence of disease, but also can appear in the guise of 'parkinsonism' in other disorders that are associated with a significant reduction of dopamine in the central nervous system (cns). parkinsonism may develop as a sequel to intoxication, trauma, vascular disease and infections. there are genetically based familial forms and degenerative or sporadic forms, the last of which include the tauopathy progressive supranuclear palsy (psp) and synucleinopathies such as multiple system atrophy (msa) and lewy body disease. lewy body disease has been further subdivided into pure autonomic failure, sporadic pd and dementia with lewy bodies (dlb) [ ] [ ] [ ] [ ] [ ] . this review will refer mainly to the sporadic form of pd. the pathological process that underlies sporadic pd is linked to the development of a-synuclein-containing inclusion bodies in the form of lewy bodies (lbs) in perikarya and lewy neurites (lns) in neuronal processes [ ] [ ] [ ] . of the diverse neuronal types within the human nervous system, only a few develop pathological inclusions, and this selective involvement is reflected in the regional pattern of the pathology. vulnerable cells are distributed throughout the peripheral, enteric and central portions of the nervous system (ens/cns) [ ] [ ] [ ] [ ] . all of the susceptible cells are projection neurones that generate a long and thin axon, which is unmyelinated, or poorly myelinated [ ] . despite their greater prevalence with advancing age, pd-associated inclusion bodies do not occur consistently in all aged non-symptomatic cases and, as such, they are pathological rather than protective or neutral age changes. the presence of these pathognomonic inclusions is a prerequisite for the post mortem diagnosis of sporadic pd [ , ] . these disease-related inclusions occur in symptomatic pd patients as well as in individuals who did not manifest any of the characteristic motor symptoms in life. thus, the pathological process has a pre(motor)-symptomatic and a symptomatic phase [ ] [ ] [ ] . the term 'presymptomatic phase' implies that even when only a few lns/lbs are detectable in non-symptomatic cases, such 'incidental' inclusion bodies represent incipient pd or the harbinger of the symptomatic disease phase [ , ] . it has been postulated that sporadic pd might be a primary disorder of olfaction, given that smell loss is an early event in the course of this disorder [ ] . this theory was derived from sources based on psychology, physiology, anatomy and pathology. additional studies not only have corroborated the initial involvement of anterior olfactory structures, but also have pointed to an early involvement of the enteric nerve cell plexuses as well as of the dorsal motor nucleus of the vagus and the intermediate reticular zone in the lower brainstem [ ] [ ] [ ] [ ] [ ] . to be plausible, any theory attempting to explain sporadic pd must incorporate these extra-nigral sites, which consistently become affected in the course of the disorder [ ] . furthermore, any speculation regarding the cause and beginnings of pd must take into account the involvement of olfactory and autonomic systems that generally develop prior to the onset of the classical somatomotor symptoms [ ] [ ] [ ] . this article will review the evidence for such involvement and summarize several hypotheses developed on the basis of these findings. the first case-control study that demonstrated smell abnormality in pd was conducted by ansari and johnson [ ] in clinically diagnosed pd patients. a subsequent larger study used detection threshold tests to amyl acetate in subjects and controls [ ] . thresholds were reduced, but no correlation was found with age, gender or treatment with levodopa. unlike the first study, there was no association with disease duration. the next sizeable investigations using the university of pennsylvania smell identification test (upsit) showed that age-matched olfactory dysfunction did not relate to odour type, was independent of disease duration, and did not correspond with motor function, tremor or cognition [ , ] . the authors also demonstrated that the deficit was of the same magnitude in both nostrils, and not influenced by anti-parkinsonian medication. a comparable survey was undertaken using upsit in cognitively normal, depression-free pd patients, and age-matched controls [ ] . upsit scores for pd patients were dramatically lower than those for controls. there was no correlation between disease duration and upsit score (r = . ). impairment of smell sense has also been documented in pd patients using sniffin sticks [ , ] . although the psychophysical evidence provides overwhelming support for olfactory involvement in pd, it does not completely eliminate the possibility of confounding factors from cognitive dysfunction, nor can it be determined whether the smell defect is initially peripheral or central [ ] [ ] [ ] [ ] . a further measure of smell sense is the olfactory eventrelated response (oerp) pioneered by kobal and plattig [ ] , which has the advantage of minimizing the potential effect of cognitive dysfunction. an initial examination compared oerp recording of pd patients with that of controls of similar age and gender [ ] . in patients ( %), responses were either absent or unsatisfactory for technical reasons. analysis of the with a measurable trace showed that, for hydrogen sulphide (h s), a highly significant latency difference existed between diagnostic groups. similar results were obtained in patients with clinically labelled pd tested by oerp to vanillin and h s [ ] . prolonged latencies were seen in these individuals whether they were taking anti-parkinson medication or not. the above-cited evidence from psychophysical and neurophysiological sources gives virtually unassailable support to the presence of olfactory dysfunction in established pd, a feature that occurs more frequently [ ] than tremor ( % vs. %). if the previous observations about early olfactory involvement are correct, this should be reflected by tests in individuals at risk for future disease or by prospective studies of those with prior olfactory impairment [ ] . montgomery and colleagues [ , ] implemented a test battery comprising motor function, olfaction (upsit) and mood for pd first-degree relatives. there were significant differences in sons and daughters, particularly where the affected parent was the father.this work has been criticized [ ] because there may have been self-selection in allegedly unaffected relatives -who may have had undisclosed motor complaints resulting in the unusually high -year positive prediction rate ( out of subjects). ponsen et al. [ ] evaluated prospectively asymptomatic firstdegree relatives of non-familial pd patients by olfactory tests and dopamine transporter scan (datscan). forty were hyposmic at baseline and, when reviewed years later, four had abnormal datscans and showed clinical signs of pd. in the remaining hyposmics, who displayed no sign of pd, the rate of decline of dopamine transporter binding was higher than in normosmic relatives. others [ ] tested patients with unexplained and isolated smell impairment to determine whether any might be in the premotor phase of pd. apart from detailed olfactory testing, subjects were evaluated by datscan and transcranial sonography of the substantia nigra. eleven displayed increased (that is, abnormal) echogenicity on transcranial sonography characteristic of pd. ten subjects volunteered for datscan and, of them, five had abnormal scans and an additional two were borderline, suggesting that they might be in a presymptomatic phase of pd. two of the five scanpositive patients have now developed clinically confirmed pd (hummel, pers. comm., ) . the first long-term, community-based prospective study has been published, in connection with these relations [ ] . the authors used the cross-cultural brief smell identification test (bsit) [ ] in healthy japanese-american men aged - years who participated in the honolulu-asia ageing study. after -year follow-up, men developed pd at an average latency of . years from baseline assessment. adjustment for multiple confounders gave relative odds for pd in the lowest tertile of bsit score of . ( % ci . - . ; p = . ), thus indicating a moderate predictive power of olfactory testing. in the same cohort, those who later died underwent brain autopsy to detect the presence of brainstem lbs [ ] . of autopsied men without clinical pd or dementia, were found to have incidental lbs. those who scored in the lowest tertile of the bsit were significantly more likely to have lbs at autopsy. potentially conflicting findings were obtained in a study of male twin pairs discordant for pd [ ] , of whom agreed to undergo olfactory tests. at baseline, the authors confirmed impaired upsit scores in the affected twins, but not in their brothers who were rated normal. after a mean of . years, brothers were still alive and, of them, were retested using the odour bsit, whereby had developed pd. neither had impaired upsit at baseline, but the average decline in upsit percentile score in both was greater than the remaining , who had not developed the disease. it is suggested that smell testing may not be a reliable predictor. the dropout rate was unusually high and smell assessment was by different methods, that is, initially by upsit ( -item) then by bsit ( -item). on the first occasion, the test was unsupervised; moreover, a . -year interval may have been too short. despite such evidence, theories regarding olfactory dysfunction as a characteristic symptom of pd have had difficulty gaining acceptance, because - % with a clinical diagnosis of pd have normal smell identification. in some pd patients with normal upsit scores, the oerp was absent or delayed [ ] , which implies that the olfactory deficit may be more frequent than indicated by upsit. pathology-based studies indicate that the diagnostic error rate for pd is % or higher, and that olfactory identification is sometimes abnormal in other diseases showing parkinsonism [ ] . mistaken diagnosis of pd occurs most often in msa, psp, vascular parkinsonism and, occasionally in individuals with essential tremor. all these disorders may be characterized by either normal or slight impairment of olfaction. it is not yet known whether persons with normal upsit score are the very patients who have received an incorrect diagnosis, but prospective studies would resolve this issue. an understanding of the applied anatomy of the vagus is necessary in view of its early and severe pathological involvement. three major components of the vagus can be distinguished in the medulla oblongata: (i) the ambiguus nucleus, a motor nucleus that innervates muscles of the palate, pharynx, larynx and heart; (ii) the dorsal motor nucleus, which harbours the preganglionic visceromotor neurones that control the postganglionic parasympathetic nerve cells in internal organs of the chest and abdomen; and (iii) the complex of nuclei accompanying the solitary tract. the parvocellular nuclei of the latter complex receive taste information and an abundance of visceral sensations. the dorsal motor nucleus is damaged severely and early on in pd, the solitary tract nuclei exhibit little change until later stages, and the nucleus ambiguus remains intact [ ] (figure , right). the gastrointestinal tract is supplied by two major nerves: the vagus, which is for excitatory parasympathetic control, and sympathetic nerves, which are inhibitory. there is abundant evidence of pathological involvement in both systems, as detailed below, and clinically features of this pathology become increasingly obvious [ ] [ ] [ ] [ ] . dysphagia is a common feature sometimes demonstrable in asymptomatic individuals [ ] . in one survey of autopsy-confirmed cases of pd, swallowing difficulties appeared after an average period of years from onset of initial extrapyramidal symptoms [ ] . a variety of mechanisms account for swallowing difficulty, with evidence of dysfunction at either oropharyngeal or oesophageal level. although dysphagia may relate to mechanical problems (e.g. zenker's diverticulum) or dry mouth, the majority can be explained on the basis of vagal dysfunction. reduction of appetite and weight loss are common features that typically affect more advanced cases, and this may also relate to impairment of smell and taste [ ] , or to poor diet, increased energy expenditure or depression [ ] . although many patients drool saliva, the consensus view is that the volume of saliva produced is either unchanged or decreased, although it may be increased by levodopa preparations [ ] . gastric dysfunction is likewise a recognized feature of established pd [ , ] . these investigators reported frequent bloating or nausea suggesting delayed gastric emptying ,and this phenomenon has indeed been confirmed by measurement of gastric emptying time in patients whether they are receiving levodopa or not [ ] . while little is known about small intestinal motility in pd, there are several studies relating to colonic disorder. [ ] . (a-b, right hand side) to show the topographical distribution pattern of a-synuclein pathology in the human medulla in stages and of sporadic parkinson's disease, adapted and reproduced in part with permission from del tredici et al. [ ] . amb, ambiguus nucleus; dmo, dorsal motor nucleus of the vagus (marked in green); gig, gigantocellular reticular nucleus, iop, inferior olivary nucleus, principal subnucleus; irz, intermediate reticular zone; rob, nucleus raphes obscurus; sol, solitary tract; xii, motor nucleus of the hypoglossal nerve. if ens involvement and disease of both the parasympathetic and sympathetic systems is an early event in pd, there should be relevant symptoms in the premotor phase. there is presently only one investigation in support of this, namely the prospective study of bowel habit in elderly patients who took part in the honolulu heart program [ ] . this comprised men without extrapyramidal disease at enrolment, followed up for years, of whom developed pd. the adjusted risk of pd in those with less than one bowel movement per day, compared with those with one or more per day, was increased almost threefold (or . ; % ci . , . ), implying that constipation is a harbinger of pd [ ] . prevalence estimates of constipation in pd have been inconsistent because of variable definitions of constipation, but a conservative estimate would be at least % [ ] . colonic transit time is raised from control values of around h to - h [ , ] , probably worsening as the disease advances. in established pd, several investigators have found reduced cardiac uptake of the noradrenaline analogue, metaiodobenzylguanidine (mibg) [ ] [ ] [ ] [ ] [ ] . this procedure evaluates postganglionic noradrenergic cardiac sympathetic function. heart rate variability may be abnormal in pd and is thought to reflect involvement of both sympathetic and parasympathetic pre-and postganglionic neurones [ ] . incidental lbs have been found in the cervical sympathetic ganglia and cardiac plexus [ ] . in another study, it was clearly shown that tyrosine hydroxylase-immunoreactive nerve fibres in the heart had almost entirely disappeared in patients with pd (and dlb), whereas they were well preserved in all those with psp and pure alzheimer's disease [ ] . only one patient with pd displayed abnormalities in the sympathetic ganglia, which indicates that cardiac sympathetic denervation precedes neuronal loss there. autonomic failure presaging a parkinsonian syndrome has been shown in two patients [ ] , but, to date, there have been no long-term prospective epidemiological studies of cardiac function. one investigation showed a significant correlation between mibg uptake and olfactory disorder in patients with pd (but not msa), thereby confirming the close association of these two modalities [ ] . a variety of sleep disorders are recognized in established pd [ ] , of which the most studied are excessive daytime sleepiness and rapid eye movement sleep behaviour disorder [ ] . the pathophysiology is not understood fully but most likely reflects cellular changes found in the parkinson neuropathological stages and , involving the reticular formation, coeruleus/subcoeruleus complex, pedunculopontine nucleus and hypothalamus [ ] . in the prospective honolulu-asia ageing study [ ] , of men aged - years developed pd over a -to -year period, and those reporting excessive daytime sleepiness were approximately three times more likely to develop pd (or . ; % ci . , . ). according to one source [ ] , / subjects with unexplained rem sleep behaviour disorder (rbd) subsequently developed parkinsonism, complementing the finding of decreased striatal transporter uptake in rbd by others [ ] . in other studies of olfaction and sleep [ ] [ ] [ ] , many rbd patients had significantly impaired smell function, once more implying that olfaction and rbd are early features. a difficulty with most of these studies, however, is the lack of post mortem confirmation. the investigation by iranzo and co-workers [ ] is of particular interest in this regard. apart from corroborating the suspected status of rbd as a precursor of pd, they document the time interval between onset of rbd and clinically manifest sporadic pd. in seven patients, the latent period was on average years (range - years). given that rbd is a sign of pd stage pathology, this estimate provides a lower time limit for the presymptomatic phase and suggests that the earliest evidence of pd pathology (stage ) would be - years before the onset of typical clinical manifestations. braak and colleagues [ ] performed detailed pathoanatomical analyses of cases of pd by a-synuclein immunostaining. a similar approach was taken in subjects who had no pd-associated somatomotor symptoms in life but displayed lns and/or lbs (incidental cases). a third group consisted of age-and gender-matched cases without lns or lbs and no history of neurological or psychiatric illness. the results of the cross-sectional study led the authors to propose that the pd-related pathological process in the cns progresses, apparently without remission, through presymptomatic and symptomatic disease phases [ ] . a subsequent study identified lns and lbs in enteric nerve cell plexuses in both presymptomatic and clinically diagnosed pd cases [ ] (figure ).this led to the dual-hit hypothesis suggestion that the pathological process begins at two sites simultaneously, that is, in the olfactory bulb/anterior olfactory nucleus and within enteric nerve cell plexuses. following damage of these predilection sites, the preganglionic parasympathetic projection neurones of the dorsal motor nucleus of the vagus (figure ) and, shortly thereafter, the post-and preganglionic sympathetic projection neurones in the coeliac ganglion and intermediolateral nucleus of the spinal cord may become drawn into the disease process [ ] . next to show the lesions are superordinate supraspinal centres such as the coeruleus/subcoeruleus complex, magnocellular portions of the reticular formation, and posterior raphe nuclei ( figures b and , right) . additional cns regions might then follow successively: central subnucleus of the amygdala [ ] [ ] [ ] [ ] , pars compacta of the substantia nigra, and magnocellular nuclei of the basal forebrain [ ] . in other words, all of the vulnerable sites do not become involved at the same time but, rather, in a predictable topographic sequence. within the brain, the disease process displays a stereotypical caudal-rostral advance from the lower brainstem through basal portions of the mid-and forebrain, finally reaching the cerebral cortex ( figure a) . a distinctive lesional pattern, including neuronal loss, emerges. with one exception [ ] , subsequent studies have confirmed, for the most part, the results of this pathoanatomical study [ ] [ ] [ ] [ ] , and interrater reliability of the pathological samples was high [ ] . pd-associated a-synuclein-containing inclusion bodies as yet have not been found in the olfactory epithelium of autopsied pd patients [ ] . nasal biopsy specimens from seven patients with symptomatic pd [ ] were compared with four anosmic controls using antibodies against olfactory marker protein (omp), neurotubulin, protein gene product . (pgp . ) and mrna for omp. irregular areas of olfactory epithelium were positive for pgp . and neurotubulin but mostly negative for omp, although mrna for omp was found in the olfactory cleft and respiratory mucosa. in this small series, there was no clear difference between pd and anosmic controls; however, sections were not examined for presence of lns/lbs, and it could be argued that those with anosmia may have been in the presymptomatic phase of pd. daniel and hawkes [ ] examined olfactory bulbs and tracts in eight controls as well as eight patients with a clinical and pathological diagnosis of pd taken from the united kingdom parkinson's disease brain bank. all pd cases contained lbs, which were most numerous in the anterior olfactory nucleus but also were found in mitral cells, the first projection neurones to receive input from the bipolar neurones in the olfactory epithelium. it was subsequently shown that loss of neurones in the anterior olfactory nucleus correlated with disease duration [ ] . one report [ ] suggested that expression of tyrosine hydroxylase in the olfactory bulb is increased -fold, and that the consequent excess of dopamine might explain the hyposmia that develops in pd. braak and colleagues [ ] confirmed the presence of pd-related lesions in mitral cells and tufted neurones of the olfactory bulb and in projection neurones of the anterior olfactory nucleus, which is dispersed throughout the olfactory tract. a tightly woven network of lns rapidly develops within the anterior olfactory nucleus. from there, the pathology tends to spread slowly into more remote olfactory sites (olfactory tubercle, piriform and periamygdalear cortex, entorhinal cortex of the ambient gyrus) [ ] without advancing into non-olfactory cortical areas [ , , ] . it is known that ens lesions occur in symptomatic cases of pd [ ] [ ] [ ] [ ] [ ] . as in the brain, only a few of the many neuronal types within the ens [ ] [ ] [ ] are prone to develop pd-associated lesions (figure a,b) . once again, the vulnerable cells apparently are projection neurones with a long and unmyelinated axon [ ] . the inhibitory nitrergic vasoactive intestinal polypeptide neurones have been shown to develop lns and lbs [ , ] . axons containing a-synuclein from the submucous plexus have been seen to protrude through the muscle layer of the mucosa, extending widely and ramifying within the mucosal lamina propria [ ] (figures c,d and ) . axons of affected ens cells contain thread-like or spindle-shaped lns of varying size. in the mucosal lamina propria, the unmyelinated axons are only micrometers away from the body's innermost environment, their only protection being a single layer of epithelial cells (figures d and ) . such involvement of the enteric nerve cell plexuses may represent a particularly early event -if not the earliest vagal-associated event -because lns were observed both in clinically diagnosed cases and in non-symptomatic individuals with pd-related brain lesions limited to the lower brainstem [ ] . in summary, the studies indicate that early involvement of the ens with widespread and thinly distributed lesions throughout the walls of the upper gastrointestinal tract is accompanied by mild cns lesions. it remains to be seen whether ens lesions also occur in the absence of lesions in the brain. the very first brainstem lesions appear in the dorsal motor nucleus of the vagus [ ] . the preganglionic parasympathetic projection neurones there generate long and thin unmyelinated axons [ , ] . pathological a-synuclein aggregates are detectable in both proximal intramedullary and peripheral portions of these axons. other nuclei in the dorsal vagal area, including the nucleus gelatinosus, area postrema, and the nuclei surrounding the solitary tract, are minimally affected or uninvolved. the catecholaminergic melanoneurones in this area and those in the intermediate reticular zone [ ] remain intact, at least initially [ ] . these neurones do not project to the periphery but to higher levels of the brain [ ] . the multipolar motorneurones of the ambiguus nucleus have thickly myelinated axons and remain free of lns/lbs. pd-associated pathological changes in sympathetic pathways (intermediolateral column of the spinal cord, coeliac ganglion and other peripheral sympathetic ganglia) have been studied and, to some extent, characterized already [ , ] . recently, such lesions also have been shown in presymptomatic patients dying of non-neurological causes, thereby confirming the early involvement of the autonomic system [ , , ] . all cases examined also displayed pd-related involvement of the lower brainstem, which, in turn suggests that the spinal cord abnormalities within the sympathetic nervous system may occur after the parasympathetic preganglionic projection neurones of the vagus have become affected. it is unlikely that the lesions in the olfactory system and in other predilection sites within the ens and cns develop independently of each other. a more plausible explanation is that a common pathologic insult, a hitherto unknown neurotropic factor or pathogenic substance, induces the disease and triggers the sequential involvement of vulnerable regions. anatomical analysis reveals a continuous chain of long-axoned and sparsely myelinated projection neurones that interconnect not only the olfactory epithelium but also the ens with the brain (figure ) and, within the brain, all of the vulnerable regions [ ] . after uptake, such a neurotropic pathogen might utilize these pathways and progress within the nervous system by way of axonal transport and trans-synaptic transmission [ ] [ ] [ ] [ ] neuroactive substances, including neurotropic viruses, unconventional pathogens with prion-like properties, or slow neurotoxins, usually are taken up at synapses, where they are frequently controlled by receptor-mediated endocytosis. the substances are transported to the cell body via the axon [ ] [ ] [ ] [ ] . neurotropic viruses that pass from the surroundings into axons of susceptible nerve cells can be prevented from doing so by the existence of a myelin sheath, which functions as a physical barrier against virus penetration [ ] . thus, the absence of a myelin sheath around axons of the first neurones in the potential chain of vulnerable projection neurones may facilitate entrance and damage by viruses or other pathogens [ , , [ ] [ ] [ ] . in addition, most of the neuronal types located within the cns are protected against uptake of substances from the extracellular milieu beyond the cns by the blood-brain barrier. only axons of nerve cells that project from the olfactory epithelium into the cns and those that project from the cns into the periphery, such as the preganglionic parasympathetic and sympathetic fibres, lack such a protective barrier. accordingly, an intravenous injection of horseradish peroxidase (hrp) results in retrograde labelling of the dorsal motor vagal nucleus [ ] . despite enormous gaps in the present state of knowledge, the main line of reasoning that favours a 'dual-hit' pd-related process (with olfactory and enteric means of access) is that both are in close and constant contact to the (potentially hostile) outer environment. from the enteric plexuses, the prospective pathogen may gain access retrogradely to the cns via parasympathetic pathways (vagus; figure ) and, thereafter, through post-and preganglionic sympathetic fibres. once in the cns, the disease process could ascend from spinal cord and lower brainstem through vulnerable portions of the basal mid-and forebrain until it reaches the cerebral cortex ( figure a ). from the olfactory epithelium, on the other hand, the pathogen would follow an anterograde route, to reach medial amygdala, olfactory tubercle, as well as piriform and periamygdalear cortex. the possibility that pd might be of viral aetiology was formally addressed by elizan and casals [ ] . the inability to transfer pd to primates and the lack of viral antibodies in the then newly described guam pd-dementia individuals, who were usually anosmic [ ] , argued against a viral aetiology for pd, although the theory was not entirely discounted. influenza virus has been associated with pd, as discussed in detail by takahashi and yamada [ ] . despite many negative studies that searched for direct evidence of influenza a in pd [ ] [ ] [ ] [ ] [ ] , nearly all pointed out that influenza a behaves as a persistent virus possibly capable of initiating autoimmunity. on the basis of human and experimental models, these authors proposed that the virus shows a predilection for the substantia nigra, cerebellum and hippocampus, and may be responsible for the formation of lbs. parkinsonism is seen on rare occasion during or after infection with herpes simplex encephalitis [ ] , and a link between chronic herpes simplex type encephalitis, pd and tic doloureux has been suggested [ ] . there is no evidence of antibodies to herpes simplex virus (hsv) type and when compared with measles or cytomegalovirus antibodies in the serum and spinal fluid of pd subjects [ , ] . similar findings were documented by elizan and casals [ ] . one unconfirmed report suggests an association between pd and coronavirus infection on the basis of enzyme-linked immunosorbent assay in cerebrospinal fluid in patients [ ] . there is recent evidence that diseases of intermediate type hypersensitivity (asthma, allergic rhinitis, seasonal rhinitis) may be associated with pd [ ] . retrospectively, the authors reviewed medical records in cases of pd and healthy controls and found a significant, approxi-dual-hit hypothesis mately twofold increase of prior intermediate-type sensitivity disorder in general, but particularly for allergic rhinitis (or . ; % ci . , . ; p < . ). there was a trend towards protection against pd in those who had used anti-inflammatory drugs. it is suggested that patients with pd might initiate an inflammatory response that could be directed towards the cns. there is some evidence of impaired smell sense in those with allergic rhinitis [ ] , raising the possibility that allergic rhinitis might facilitate entry of a pathogen from the nose into the olfactory bulb and tract. altered protein handling may be a factor in the pd-associated pathogenic process. many viruses, e.g. epstein-barr virus, encode proteins that exploit the ubiquitin-proteasome system to regulate virus latency and allow the persistence of infected cells in immunocompetent hosts [ , ] . there is longstanding evidence that neurotropic virus can enter the brain via the nasal route. it was shown that hsv type placed intranasally in -week-old mice was detectable in the trigeminal root entry zone and olfactory bulbs days later [ ] . in some mice, virus which had entered the olfactory bulb, spread via axons as far as the temporal lobe, hippocampus and cingulate cortex. another study in the rat showed that hrp applied intranasally can be transported to the bulb, anterior olfactory nucleus, as well as to cholinergic neurones of the diagonal band, serotonergic raphe neurones, and noradrenergic cells of the locus coeruleus [ ] . it is not widely appreciated that direct connections exist between primary olfactory areas and the substantia nigra. for example, application of hrp into the olfactory tubercle results in anterograde labelling of the ventral tegmental area, the pars reticulata of the substantia nigra, and anterior olfactory nucleus. as anticipated, there was retrograde labelling in the olfactory bulb and anterior olfactory nucleus [ ] . blessing and colleagues [ ] studied connections of the vagus using hsv type in the rat (figure , left) . live hsv- was injected into the cervical vagus, and its distribution was examined using polyclonal antiserum. on day , virus was detected in glial cells of the area postrema, in nuclei of the solitary tract, the vagal dorsal motor nucleus, spinal trigeminal nucleus, and the great raphe nucleus, whereas the hypoglossal nucleus was spared, as in pd. on day , there was rostral spread to involve the locus coeruleus, parvocellular reticular nucleus and periaqueductal grey, but not the substantia nigra. hsv- -positive neurones were seen in an oblique area corresponding to the human intermediate reticular zone [ ] . this route of infection is shown in figure (left) and should be compared with the similar distribution of pd-associated brainstem pathology (figure , right). there are clear differences between this animal model and the pathology in humans. for instance, in contrast to the situation in pd, the trigeminal nerve in the rodents is involved and the substantia nigra is spared. nevertheless, the rostral migration of hsv- has remarkable similarity to the suggested progression of the a-synuclein pathology in pd ( figure ) and reaffirms speculation that a neurotropic agent may utilize the vagus as a means of entering the medulla [ , ] . the experiments of blessing and co-workers [ ] are of additional interest in that they show that the first site of attack is in glial cells, which raises the possibility that residual virus in these cells may be responsible for the slow progression of pd. transvagal spread in animal models has been reported for other neurotropic enteroviruses, reovirus, pseudorabies virus and haemagglutinating encephalomyelitis virus [ ] . the same group was unable to introduce cns infection in mice with influenza a virus through non-autonomic routes, such as the anterior chamber of the eye, the brachial plexus, knee joint, sciatic nerve and hindlimb footpad. conversely, pseudorabies virus spreads though both somatic and autonomic nerves. a strain of hong kong influenza virus inoculated intranasally in mice reached the cns through afferent fibres of the olfactory, vagal, trigeminal and sympathetic nerves following replication in the respiratory mucosa [ ] . intranasally inoculated avian influenza virus in mice gave rise to lesions in lung and brainstem [ ] . the main areas involved were the nuclei of the solitary tract and the trigeminal ganglia. although haematogenous cns spread is well documented in many viral strains, the findings of these experiments indicate that influenza viruses have preferential routes of access to the cns along the vagus and olfactory nerves following replication in the lungs, but the pattern of brainstem involvement is variable; that is, the trigeminal nerve is sometimes involved, at other times spared. these observations have some parallels with the pattern of proposed cns invasion in pd. although no virus mirrors exactly the pathological profile seen in pd, it is possible that a particular viral strain might display a predilection for the olfactory and vagal routes of entry. we propose that an unknown neurotropic pathogen initiating the pathological process underlying sporadic pd adopts a two-pronged attack on the nervous system: anterogradely, via olfactory pathways; and retrogradely, via enteric plexuses and preganglionic vagal fibres. if the pathogen is viral, then brain entry via the nasal route or uptake through the gastrointestinal tract has ample precedent, as described above. a nasal infection would have direct access to the olfactory nerve; infected saliva or mucous could be swallowed and reach the upper digestive tract to infect axons of meissner's plexus and, after transneuronal passage, ascend retrogradely in preganglionic parasympathetic fibres of the vagus nerve to the lower brainstem. direct access to the medulla via the viscerosensory fibres of the vagus in the pharynx or via the trigeminal nerve, while anatomically appealing, is not compatible with virtual sparing of the solitary tract and trigeminal nuclei during the entire course of the disorder. our observations relate to the commonest, sporadic variety of pd. as mentioned earlier, not all neuropathologists are in accord with the frankfurt pd classification [ , ] , the main objection 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striatum of the golden hamster. ii. neuronal connections of the olfactory tubercle transneuronal transport of herpes simplex virus from the cervical vagus to brain neurons with axonal inputs to central vagal sensory nuclei in the rat the invasion routes of neurovirulent a/hong kong/ / , h n ; influenza virus into the central nervous system after respiratory infection in mice avian influenza virus intranasally inoculated infects the central nervous system of mice through the general visceral afferent nerve critical appraisal of the braak staging of brain. pathology related to sporadic parkinson's disease the authors wish to thank ms inge szász-jacobi for expert assistance with the graphics. this work was made possible in part by funding from the deutsche forschungsgemeinschaft (dfg) and hilde-ulrichs foundation (florstadt-staden, germany). key: cord- - ovqhypt authors: iqbal, umar h.; zeng, emma; pasinetti, giulio m. title: the use of antimicrobial and antiviral drugs in alzheimer’s disease date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: ovqhypt the aggregation and accumulation of amyloid-β plaques and tau proteins in the brain have been central characteristics in the pathophysiology of alzheimer’s disease (ad), making them the focus of most of the research exploring potential therapeutics for this neurodegenerative disease. with success in interventions aimed at depleting amyloid-β peptides being limited at best, a greater understanding of the physiological role of amyloid-β peptides is needed. the development of amyloid-β plaques has been determined to occur – years prior to ad symptom manifestation, hence earlier interventions might be necessary to address presymptomatic ad. furthermore, recent studies have suggested that amyloid-β peptides may play a role in innate immunity as an antimicrobial peptide. these findings, coupled with the evidence of pathogens such as viruses and bacteria in ad brains, suggests that the buildup of amyloid-β plaques could be a response to the presence of viruses and bacteria. this has led to the foundation of the antimicrobial hypothesis for ad. the present review will highlight the current understanding of amyloid-β, and the role of bacteria and viruses in ad, and will also explore the therapeutic potential of antimicrobial and antiviral drugs in alzheimer’s disease. alzheimer's disease (ad) is a progressive neurological disorder that accounts for the greatest number of dementia cases. as of , . million people were living with ad, with its prevalence predicted to increase to . million by [ ] . the vast majority of cases are concentrated in ages over , impacting % of people in this age group. in addition, the economic toll of ad on the united states economy is significant, estimated to be roughly usd billion in [ ] . as the number of cases is only expected to rise over the coming decades, research in this field is critical in order to understand the pathology of this disease, as well as potential therapeutics. the understanding and characterization of ad can be traced back over years to alois alzheimer, from whom the disease takes its name. after completing an autopsy of a patient with progressive dementia, alzheimer noticed a severe amount of cortical degeneration and an accumulation of protein deposits, specifically extraneuronal plaques and intraneural tangles [ ] . by , the buildup of extraneuronal amyloid-β (aβ) plaques became the hallmark trait of the pathogenesis of alzheimer's disease [ ] , initiating the development of the amyloid cascade hypothesis [ ] . in parallel to aβ plaques formation, the accumulation of other naturally unfolded proteins is central to ad and other cerebral proteopathies [ ] . the intracellular aggregation of tau proteins in the form of neurofibrillary tangles (nfts) is also an essential trait in the pathogenesis of alzheimer's disease [ , ] . a recent study alzheimer's disease [ , ] . a recent study found that neuroinflammation could play a role in the aggregation of tau, as dna extracted from various bacterial species promoted tau misfolding [ ] . whereas aβ plaques are more critical to ad pathogenesis, the tau protein appears to be more responsible for subsequent cognitive impairment and dementia symptoms associated with ad [ ] . indeed, tau hyperphosphorylation and nft levels are closely correlated with cognition, and exhibit potential as therapeutic targets for ad treatment [ ] . furthermore, tau protein production has been shown to have a positive correlation with the production of aβ plaques [ ] , with the formation and lack of clearance of aβ plaques also being proposed to induce tau protein formation into nfts [ ] . coupling this with the bi-directional relationship between aβ plaques and neuroinflammation [ ] would cement aβ's key role in driving ad pathology. aβ formation begins with the breakdown of the amyloid precursor proteins (app) embedded in the membranes of cells, such as neurons, as a type transmembrane glycoprotein [ ] . aβ peptides are produced through a two-step cleavage process, in which app is metabolized into smaller fragments. in the first step, app is cleaved by β-secretase into a membrane-bound ctfβ fragment (containing amino acids) and an extracellular fragment sappβ. ctfβ is then further cleaved by γsecretase to create the final aβ peptide [ ] [ ] [ ] , as illustrated in figure . the length of aβ peptides is not fixed, and can consist of anywhere between and amino acids, depending on where the cleavage was done by β-secretase and γ-secretase [ , ] . the most abundant length is aβ - , representing approximately - % of aβ peptides, whereas the least soluble of the aβ peptides, aβ - , represents roughly - % [ , ] . as a greater number of aβ peptides form, they begin to aggregate into oligomers, which then form fibrils, and eventually the insoluble plaques characteristic of ad [ ] . of the different isoforms of aβ peptides, aβ - and aβ - are the most common in plaques. regarding the comparative role of aβ - and aβ - peptides in the pathogenesis of ad, aβ - peptides have been found in higher concentrations in ad. furthermore, aβ - peptides have been found to be more prone to forming insoluble amyloid fibrils than aβ - [ ] . this is further supported by a study using transgenic mice that expressed either aβ - (bri-aβ ) or aβ - (bri-aβ a). the authors found that the mice that selectively expressed aβ - did not develop ad pathology at any age. however, the same did not hold true for bri-aβ a mice, which had developed aβ deposits [ ] . in addition, another study found aβ - peptides to promote aβ plaques formation, and aβ - to decrease aβ deposition [ ] . these findings would indicate the key role aβ - peptides play in the pathogenesis of ad. even with the tremendous effort that has been put into developing potential therapies for ad over the past few decades, there has been little success in reaching an effective therapy, with no new drug being approved in over a decade. while cholinesterase inhibitors and memantine are fda-approved drugs for ad, and do address some of its symptoms, they lack the ability to attenuate disease progression. over the past years, a majority of the therapies have been based on the amyloid cascade hypothesis, and hence have focused on depleting aβ peptides. theses therapies often aim to inhibit γ-secretase or β-secretase activity in order to limit aβ peptide production. therapies that use such methods have, however, seen an increase in the rate of infection during clinical studies, with one study seeing % of participants develop meningoencephalitis [ , ] . tarenflurbil, for instance, had been administered clinically, after it was shown to modulate γ-secretase and increase production of the less toxic aβ - peptide, rather than aβ - peptide [ , ] . however, in addition to not showing any significant benefit in individuals with mild ad, participants in the treatment group experienced an increase in upper respiratory infections and dizziness compared to the placebo group [ ] . additionally, in the time of covid- infection, respiratory-related side effects, such as the ones related to tarenflurbil, are of growing concern. the γ-secretase inhibitor semagacestat has not only been associated with increased levels of infection, but also with a failure to provide any cognitive improvement in patients with probable ad [ ] . furthermore, when patients with mild to moderate ad were administered elnd , a compound that inhibits aβ fibrils and plaque formation, it was observed that higher doses of this treatment led to serious infection. this led to lower dosage recommendations for future trials [ , ] . lastly, the b-site abpp cleaving enzyme (bace )-inhibitor e has also been associated with oralabial herpes relapse [ , ] . a rise in infection occurring in tandem with the reduction of aβ peptide production could indicate these peptides' potential role in immune function. through these past clinical trials, it is evident that therapies largely based on the amyloid cascade hypothesis, which in turn aim to eradicate aβ peptides in the brain, have historically been ineffective. these failures could imply that current approaches either intervene at a stage that is too late, or possess a therapeutic target that is not as relevant to disease progression [ ] . this would make sense in the context of ad especially, as aβ deposition occurs - years before the occurrence of clinical symptoms [ ] . therefore, these treatments that target aβ peptides specifically may already be too late. to create a successful therapy, it may be necessary to consider intervention in the presymptomatic stage of the ad instead. to do so, it would be crucial to identify biomarkers for early identification of ad. in march of , a meeting was convened in which international, interdisciplinary experts identified a list of biomarkers that could be used for identifying ad early on [ ] . csf levels of aβ - and aβ , and the ratio of aβ - /aβ - , were determined to be among the candidates [ , ] . in addition, plasma levels of the same biomarkers were determined to decrease in ad patients when compared to healthy subjects [ ] . other biomarkers to consider would include plasma levels of tau protein and neurofilament light [ ] . plasma levels of the latter have been shown to be able to detect neurodegeneration in presymptomatic ad [ ] . other highly sensitive methods that have shown promise in the early identification of ad include protein misfolding cyclic amplification (pmca) and real-time quaking-induced conversion (rt-quic), to determine aβ oligomers and tau protein levels in csf [ , ] . taking a more preventative approach to ad treatment, and understanding and addressing factors that contribute to the progression of ad, may be favorable for identifying therapeutic targets earlier in the disease. based on evidence of the association between bacterial/viral infection and ad progression, the antimicrobial hypothesis suggests that aβ peptides may be produced as a protective mechanism by the innate immune system, and act as an antimicrobial peptide (amp) against foreign agents. if aβ peptides do in fact play a beneficial role in immunity, then the aim of treatment should not be to eradicate the compound entirely. rather, it should be to target the root cause of its over production, and reduce its deleterious effects and general neuroinflammation in ad. neuroinflammation is inflammation within the brain or spinal cord due to infection, toxins or injury [ ] . in the brain specifically, resident glial cells, such as microglia and astrocytes, along with endothelial cells and mast cells, all aid in defending the brain against foreign pathogens [ ] . microglia, the main immune effector cells of the central nervous system (cns), are constantly surveying their environment for potential threats to the brain [ , ] . when an invading agent is detected, microglia change into an activated state, characterized by an enlarged soma and the production of inflammatory cytokines and chemokines [ ] . astrocytes also play a critical role in mediating neuroinflammation as they are responsible for many neuroprotective functions, such as maintaining blood brain barrier (bbb) integrity and buffering neurotransmitters [ ] . upon injury, astrocytes likewise undergo morphological changes, and exhibit increased reactivity and secretion of cytokines and chemokines [ ] . while acute inflammatory responses are common to healthy individuals, chronic inflammation is damaging to the natural balance of pro-and anti-inflammatory signaling in the brain, and can lead to the development and progression of neurodegenerative diseases like ad [ ] . over the years, there has been increasing evidence of neuroinflammation's role in ad. in addition to aβ plaques and nfts, markers of sustained inflammation and microglial activation have repeatedly been found in ad brain samples [ ] . the cytokines interleukin and interleukin are especially elevated [ ] . one source of the neuroinflammation in ad patients could be the response to invading microorganisms and viruses. in fact, researchers have found evidence pointing to the presence of pathogens, such as viruses, bacteria and fungi, in ad brains [ ] [ ] [ ] [ ] [ ] [ ] . this notion draws parallels with the measles virus, which can lead to the development of the neurological disease known as subacute sclerosing panencephalitis [ ] . these findings include the identification of viral [ ] and bacterial dna in post-mortem brain samples, and the detection of pathogens [ ] and/or their respective antibodies [ ] in the serum or cerebrospinal fluid of patients. furthermore, detection of lipopolysaccharide is commonly used by researchers to measure the presence of gram-negative bacteria, like p. gingivalis specifically, as it is found in their cell walls and can stimulate an inflammatory response in the immune cells [ ] . herpes simplex virus- (hsv- ) [ , [ ] [ ] [ ] [ ] was the first pathogen found to be present in ad brain samples [ ] , and it thereafter became the most widely-researched pathogen regarding the linkage between viral infection and ad. since then, other viruses have been identified in leading to the progression of ad, including human cytomegalovirus [ ] and epstein barr virus [ ] . a recent study found that, in addition to hsv- , herpesvirus types hhv- [ , ] and hhv- were highly present in ad patients [ ] . in this study, by readhead et al., hhv- and hhv- were also observed to be involved in regulatory processes critical to characteristic features of the disease [ ] . bacterial infection has likewise been associated with the progression of ad. the presence of bacteria in the brain has been determined in previous studies, suggesting the presence of a brain microbiome [ ] [ ] [ ] . even though bacterial presence has been seen in the brains of healthy individuals, tissue samples from ad brains have greater levels of bacterial species [ ] , indicating a greater level of infiltration. chlamydia pneumoniae is the most widely-studied bacteria regarding association to ad [ , ] . a clinical investigation, made up of a healthy control group and an ad group, detected c. pneumoniae in % of the ad patients, whereas the control group were all negative [ ] . escherichia coli, likewise identified in ad brains [ ] , has been found to be capable of synthesizing extracellular amyloid [ ] . stains such as borrelia burgdorferi [ ] , spirochetes [ ] , and porphyromonas gingivalis, a pathogen commonly linked to chronic periodontitis, have also been identified in ad brain samples [ ] . interestingly, fungal infection from species primarily associated with periodontal disease has recently been suggested to be involved in ad progression. researchers in a study detected multiple fungal species in ad brain samples, including saccharomyces cerevisiae, malassezia globosa, malassezia restricta, penicillium and phoma [ ] . pisa et al. have since followed up on this initial discovery by analyzing the presence of these species between brain regions [ ] . with any of these studies that have been conducted, however, it is important to recognize the technical limitations that arise when studying microorganisms and neurodegenerative disease. many of these studies are limited to the use of post-mortem brain samples, and thus present the risk of contamination due to death or the passage of microbes from other areas of the body, such as the gut to the brain, due to the lack of a functioning bbb to prevent this leakage. depending on the organism, there are several ways that pathogens can infiltrate the cns and potentially further the progression of ad. the first is through a compromised bbb. whereas a healthy and functional bbb normally provides a selective barrier to the passage of cells and molecules into the brain, a compromised bbb can allow direct entry into the cerebral spinal fluid via the bloodstream [ ] . this places aging populations and those with weakened immune responses especially at risk, as some viruses, such as herpesvirus, can remain latent after initial infection and then reactivate in aging individuals long after, to introduce delayed adverse complications [ ] . even with a healthy bbb, however, bacteria and viruses are still able to be introduced into the brain through various mechanisms. hiv, for example, is carried from the immune system to the brain by infected leukocytes that are able to cross the bbb. p. gingivalis and other oral spirochetes have also been suggested to be capable of invading the cns via the oral cavity, through the trigeminal nerves and ganglia [ ] . additionally, pathogens such as bacteria and viruses can bypass the bbb altogether by entering through the olfactory system, as the nasal cavity connects the peripheral environment to brain regions such as the olfactory bulb [ ] , the entorhinal cortex and the hippocampus, which traditionally receive smell sensory signals. c. pneumoniae, a respiratory pathogen, has specifically been suggested to enter the brain through the olfactory system, with its presence detected in the entorhinal cortex and hippocampal formation of ad patients [ ] . a recent study model demonstrated that exposure to c. pneumoniae via the olfactory system was sufficient to induce aβ plaque and nft formation in the olfactory cortex and hippocampus of immunocompromised individuals [ ] . this is further evidenced by little et al., who found that intranasal inoculation of c. pneumoniae was sufficient to induce ad-like traits in mice [ ] . once in the brain, there are several ways in which these pathogens contribute to the aβ production that is characteristic to ad. one mechanism is through the alteration of gene expression. the study previously mentioned by readhead et al. found that hhv- and hhv- interact with known regulatory genes responsible for amyloid processing, such as the amyloid beta a precursor protein-binding family (apbb ), clusterin (clu), and gamma-secretase subunit presenilin- (psen ) [ ] . similarly, infection of c. pneumoniae within human neuronal cell cultures possibly alters calcium-related gene expression such that they express patterns similar to those reported in ad brain samples [ ] . another way viruses can influence aβ production is through protein misfolding. specifically, viruses such as hhv, cytomegalovirus and epstein barr virus have been shown to contain prion-like domains that may trigger the misfolding of proteins like aβ [ ] . through these varying mechanisms, many microorganisms and viruses have been found to initiate aβ plaque formation. in-vivo studies have noted a correlation between viral and bacterial infections and the accumulation of aβ peptides. mice infected with hsv- [ ] , pseudorabies virus [ ] , c. pneumoniae [ ] and p. gingivalis [ , ] were found to have a significantly increased level of aβ - in the brain. in addition, aβ expression was found to be upregulated in rats exposed to bacterial pathogens. hsv- has also been found to infect the hippocampus region at a greater rate; the same area found to have greater amounts of aβ plaques in ad [ ] . in vitro studies observed cells co-cultured with either hsv- , hsv- , p. gingivalis or b. burgdoferi to have increased intracellular concentrations of aβ [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, hsv- has been associated with the inhibition of the non-amyloidogenic pathway of app metabolism, and the increased expression of β-secretase. this is evidenced by a study indicating the direct and frequent interaction between hsv- and aβpp [ ] . aβ plaques have also been identified in the brains of hiv- -infected individuals. autopsies performed on hiv positive individuals found roughly half of them to contain aβ plaques [ ] . cell culture studies observed an increase in aβ production and secretion following exposure to mrna and proteins from the hiv nef gene [ ] . it must be noted, however, that the association between infection and aβ plaque formation is not consistent across all populations. for example, in the study previously mentioned by sundar et al., younger and healthier individuals exposed to c. pneumoniae did not exhibit the same ab peptide and nft formation as older and immunocompromised subjects [ ] . moreover, it has been observed that genetic discrepancies, especially in the apoe gene, influence one's susceptibility to hsv- infection and subsequent ad development. specifically, the apoeε allele places individuals at a greater risk of developing hsv- -associated ad, with a combination of apoeε and hsv- comprising % of all ad cases [ , ] . this finding has been recapitulated in animal studies, where mice with the apoeε allele display a greater viral load than mice with other allele types after hsv- infection [ ] . it is clear that a relation exists between bacterial and viral infections and aβ production rate, as described in the previous section. aβ peptides have long been thought to lack any physiological function; however, this notion has been challenged in recent years. clinical studies have observed the depletion of aβ peptides, through anti-aβ therapies, to increase the rate of infections in some participants. furthermore, aβ plaques have been found to contain microbial and viral dna, such as hsv- . one study identified hsv- virus dna in roughly % of aβ plaques [ ] . in addition, ad brains have been associated with -to -fold increases in bacterial read compared to control brains [ ] . in the presence of bacterial lipopolysaccharides, microglial cells have also been shown to upregulate aβ production [ ] . from these and similar findings, it has been suggested that the pathogenesis of ad could be triggered by viral and/or microbial infections. these observations led to the recent development of the antimicrobial protection hypothesis for ad, which explores the notion of aβ peptides having a role in innate immunity as an amp that aids in the entrapment and degradation of invading bacteria and viruses. the innate immune system utilizes amps to target invading microorganisms, such as bacteria, viruses, fungi, and in some instances cancerous cells. mammalian amps exist in three main families: defensins, histatins and cathelicidins [ ] . similar to how aβ peptides are generated through the two-step cleavage of app, amps are also formed from the breakdown of larger precursor proteins. examples of amyloid amps that have a role in immunity are present in the human body. amyloidogenic major basic protein- (mbp- ) is implemented in eosinophils against pathogens [ ] . like aβ peptides, mbp- also forms aggregates, specifically at the surface of the bacteria to limit its spread. further support of aβ peptides being amp stems from their similarity to amp ll- , the only cathelicidin identified thus far in humans. both compounds exhibit tendencies to form cytotoxic soluble oligomers and insoluble fibrils, characteristic features of tinctorial amyloid [ ] . additionally, deficiency in the latter can result in kostmann syndrome, an immunodeficiency disorder that, if left untreated, can result in death due to infection within the first year of life [ ] . high levels of ll- are likewise dangerous, as they has been associated with the development of plaques in atherosclerosis and other non-infectious diseases [ ] . protein analyses comparing known amps and aβ peptides demonstrate structural similarities between these peptides, as well sequential similarities pointing to a shared homology between aβ peptides and a specific family of bacteriocins [ ] . this is particularly notable as bacteriocins are traditionally synthesized by bacteria as part of an antimicrobial response to contact with closely-related strains [ ] . if verified as an amp, aβ would not be the only amp suggested to be involved in ad. often expressed in epithelial cells, β-defensin- is significantly elevated in astrocytes of the hippocampus, the choroid plexus, and the granulovacuolar degeneration structures of ad brain samples [ ] . in vitro studies suggest the ability of aβ peptides to be an amp, and inhibit growth of a number of bacteria and viruses. in respect to the latter, aβ has been shown to have antiviral activity against both hsv- and the influenza virus a by inhibiting the infectivity of hsv- [ ] , influenza virus a [ ] , h n [ ] and h n [ ] . researchers found that in mouse and human neural cell cultures, aβ peptide deposition was accelerated in response to hsv- and hhv infection, with the oligomers binding to the viruses as part of a protective entrapment mechanism [ ] . aβ peptides have also been shown to have antimicrobial properties against both gram-positive and gram-negative bacteria, including enterococcus faecalis, escherichia coli, listeria monocytogenes, salmonella typhimurium, staphylococcus aureus, staphylococcus epidermidis, streptococcus agalactiae and streptococcus pneumoniae [ ] . in a study comparing aβ peptides and ll- , soscia et al. found that, against eight different bacteria and viruses, aβ peptides demonstrated an antimicrobial activity equivalent to, and sometimes even greater than, the known amp ll- . the same study also found that, when comparing the brain homogenates of aβ-enriched areas from both ad and non-ad brains, the ad brain samples had elevated antimicrobial and antiviral activity. these discrepancies were eliminated once the ad brain tissues were immunodepleted using anti-aβ antibodies. it is important to consider the sequence length of the aβ peptide when examining its antimicrobial capabilities, as aβ - was shown to be capable of binding to the surface of bacteria and aggregating into clusters, whereas other peptide lengths were not [ ] . animal studies have further evidenced the important potential role that aβ peptides have in protecting against infections. in a study, researchers tested aβ's functionality as an amp in mice and nematode models [ ] . kumar et al. found that transgenic xfad mice, which constitutively express human aβ peptides, survived significantly longer than wild-type mice after the injection of salmonella typhimurium into their brains. the xfad mice were observed to have accelerated aβ deposition that closely co-localized with the bacteria, reducing their cerebral viral load compared to wild-type mice. these findings were recapitulated again in worm models, as nematodes expressing aβ were found to have increased survival following fungal infection of c. albicans, compared to nematodes that did not produce aβ peptides. additionally, it has been observed that impaired mice that lacked the ability to generate aβ peptides have been shown to have increased postpartum mortality, which was only reversed by maintaining a sterile environment [ ] . furthermore, app knockout mice were also observed to have increased rates of mortality. altogether, these studies support the notion that aβ peptides are an amp, and an integral part of the brain's innate immune response against invading pathogens. the mechanism by which aβ peptides have been suggested to exert their antimicrobial and antiviral effect has been based on entrapment and lytic activity, as illustrated in figure . being a self-complimentary peptide containing two distinct hydrophobic and hydrophilic surfaces, aβ peptides can self-assemble into oligomers. as oligomerization continues, a fibril network is created which targets, captures and agglutinates microbes, limiting their proliferation and impact on their environment. aβ peptide affinity towards microbes has been suggested to be due to its positive charge and the microbe's negatively charged membrane [ , , ] . furthermore, the ability of aβ peptides to agglutinate microbes stems from its heparin-binding activity, which is able to target carbohydrates present on the surface of microbes. once entrapped, it is suggested that aβ peptides induce cell membrane disruption by forming cation channels. these channels cause ion dyshomeostasis and subsequent cell death. this mechanism is similar to the activity observed in amps such as ll- , which assert protection through microbial agglutination and entrapment. the oligomerization activity observed with aβ peptides is a common trait of amps, which mediates their ability to entrap and lyse pathogens while maintaining their resistance to protease activity. the entrapment of microbes and viruses can also enhance their uptake by neutrophils and macrophages. in respect to hsv- , aβ peptides have been proposed to interfere with its ability to fuse with the plasma membrane of cells, hindering its infective ability [ ] . int. j. mol. sci. , , x for peer review of in aligning with the antimicrobial hypothesis of ad, the use of antimicrobial and antiviral therapeutics could prove to be effective in targeting the root cause of ad. it should be noted, however, that the chronic over-production of aβ peptides, which form numerous insoluble plaques, would also need to be addressed. as such, the primary aim of these drugs would be to target bacteria and viruses, but a secondary aim would be to reduce the already-present aβ burden that the brain is under. acyclovir is an antiviral drug that is used for hsv- infections, which has been found to be well tolerated and safe [ ] . by inhibiting the virus's replication, this antiviral agent is able to reduce the viral load exhibited by hsv- . administration of acyclovir in hsv- -infected cells has been found to cause a significant reduction of hsv- proteins, along with a reduction of roughly % in aβ accumulation, compared to untreated cells. the study associated this with reduced levels of βsecretase and a component of γ-secretase which metabolizes app into aβ [ ] . acyclovir administration has also shown to prevent hsv- -related neuronal death [ ] . in relation to the cognitive impairment observed in ad, a study by hui et al. investigated the effects of acyclovir on aβ oligomer-induced spatial cognitive impairments. the study found that the co-administration of in aligning with the antimicrobial hypothesis of ad, the use of antimicrobial and antiviral therapeutics could prove to be effective in targeting the root cause of ad. it should be noted, however, that the chronic over-production of aβ peptides, which form numerous insoluble plaques, would also need to be addressed. as such, the primary aim of these drugs would be to target bacteria and viruses, but a secondary aim would be to reduce the already-present aβ burden that the brain is under. acyclovir is an antiviral drug that is used for hsv- infections, which has been found to be well tolerated and safe [ ] . by inhibiting the virus's replication, this antiviral agent is able to reduce the viral load exhibited by hsv- . administration of acyclovir in hsv- -infected cells has been found to cause a significant reduction of hsv- proteins, along with a reduction of roughly % in aβ accumulation, compared to untreated cells. the study associated this with reduced levels of β-secretase and a component of γ-secretase which metabolizes app into aβ [ ] . acyclovir administration has also shown to prevent hsv- -related neuronal death [ ] . in relation to the cognitive impairment observed in ad, a study by hui et al. investigated the effects of acyclovir on aβ oligomer-induced spatial cognitive impairments. the study found that the co-administration of acyclovir with dexamethasone attenuated impairments in spatial cognition. furthermore, this combination reduced the levels of neuroinflammation markers such as tnf-α and il- , along with microglia activation. interestingly, the study found these effects to only occur when acyclovir and dexamethasone were administered together [ ] . penciclovir is another antiviral drug that targets hsv- dna replication by blocking chain elongation. cell cultures infected with hsv- displayed a reduction of virus and aβ accumulation when penciclovir was administered. this was paralleled with a reduction in β-secretase and a component of γ-secretase [ ] . foscarnet has been tested for its ability to reduce hsv- levels in vitro. a study found it was able to reduce aβ accumulation, although only at higher doses. it was also unable to significantly reduce virus levels. furthermore, foscarnet was not as effective as acyclovir or penciclovir, and hence currently is not seen as the optimal antiviral drug available for ad [ ] . valacyclovir, an antiviral medication used in hsv- and hsv- infections, has been determined to positively impact cognition by improving visual object learning, verbal memory and working memory in patients with schizophrenia [ ] . due to its effects on working memory, its effectiveness against hsv- and hsv- , and its generally safe consumption, valacyclovir has been suggested as a potential therapeutic for ad. a clinical study is currently underway in which patients that both have mild ad and tested positive for hsv- or hsv- will receive valacyclovir. the aim of the study is to determine the impact of this treatment on cognition and the accumulation of amyloid and tau [ ] . numerous studies have determined the antiviral agent bay - to be effective in combating hsv- [ ] [ ] [ ] [ ] [ ] . by targeting the helicase-primase complex, bay - can inhibit viral dna replication, and has been found to be more potent than acyclovir. the severity and frequency of recurring hsv was also found to be reduced by use of this drug [ ] . furthermore, it was able to decrease levels of aβ and reduce p-tau production in vero cells infected with hsv- [ ] . our lab has investigated the use of bioflavonoids, including ginkgetin, isoginkgetin and ginkgolic acid, derived from the leaves of ginkgo biloba. the antiviral capabilities of these compounds has been well established in previous studies [ ] [ ] [ ] [ ] [ ] . hayashi et al. determined ginkgetin to successfully inhibit the viral replication of hsv- , hsv- and the human cytomegalovirus, while also suppressing viral protein synthesis [ ] . additionally, a study by miki et al. found ginkgetin to have anti-influenza virus activity [ ] . ginkgetin has been studied for use in ad by zeng et al., who administered the drug to app/ps transgenic mice. they observed a significant reduction in aβ plaques and an improvement in inflammation [ ] . borenstein et al. have demonstrated the ability of ginkgolic acid to limit virus infectivity by inhibiting its fusion. the study found ginkgolic acid to be successful in inhibiting hsv- , human cytomegalovirus and zika virus. furthermore, it was effective in inhibiting viral protein synthesis and genome replication, in hsv- and human cytomegalovirus, respectively [ ] . ginkgolic acid has also demonstrated antimicrobial properties, specifically against e. coli and staphlylococcus aureus [ ] . isoginkgetin has been shown to provide neuroprotection against the cytotoxic effects of excessive aβ accumulation [ , ] , while also having anti-microbial and anti-fungal activity [ ] . our lab's preliminary work in testing these three compounds in ad determined their effectiveness in reducing aβ load in vitro, further supporting their therapeutic potential in ad. doxycycline is a tetracycline antibiotic that has been studied for its therapeutic efficacy in ad models. contrary to other tetracyclines, doxycycline has been determined to be safe and is able to penetrate the bbb [ ] , allowing it to exert its effect directly in the cns. in vivo models, in which doxycycline was administered to mice, observed its accumulation in amyloid deposits, including aβ plaques [ ] . with respect to the production and formation of aβ oligomers, it was observed that although doxycycline administration in transgenic mice did not cause a shift in aβ monomers, there was a significant reduction in aβ -mer levels when compared to control [ ] . the same study also observed a significant memory recovery in animals that received treatment; however, there was no reduction in aβ plaque size [ ] . the paper suggested this was possibly due to the short two-month period of the study, as a previous three-month study found plaque size to be significantly reduced [ ] . in respect to neuroinflammation, a reduction in microglia activation has also been associated with doxycycline administration [ ] . a drosophila model, which administered doxycycline to aβ - -expressing flies, observed that the treated group's locomotor deficits developed slower than the control group. the same study also observed doxycycline administration to be associated with reduced aβ fibrilization, suggesting the production of smaller amyloid structures [ ] . another study associated doxycycline with the destabilization of aβ fibrils [ ] . clinical trials, however, were not as successful. one study, which administered doxycycline and rifampicin, observed improvements in cognitive function, as assessed by the standardized alzheimer's disease assessment scale-cognitive subscale (sadascog) score [ ] . however, a second study did not find any improvements in the cognition or function of patients with mild to moderate ad with doxycycline/rifampicin administration [ ] . further investigations would be needed to understand why the benefits seen in murine models do not translate into clinical trials. propranolol hydrochloride, an antihypertensive drug shown to have antimicrobial properties [ ] , has also been found to impact aβ production. cortico-hippocampal neuronal cultures treated with this drug manifested reduced levels of aβ production. furthermore, the one-month treatment of tg mice resulted in roughly a % reduction of aβ - and aβ - levels in the brain. when administered over a period of months, aβ peptide levels were still reduced in the brain; however, no improvement in spatial memory function was observed [ ] . rifampicin is an antibiotic derived from nocardia mediterranei, which has been investigated for use in neurodegenerative diseases such as parkinson's and ad [ , ] . rifampicin has been found to provide neuroprotection through its anti-oxidant and anti-inflammatory properties [ , ] . furthermore, in vitro studies found that its administration improved neuronal survival and reduced microglial activation [ ] . studies by tomiyama et al. found rifampicin to protect neurons from cytotoxicity by scavenging free radicals [ , ] . in relation to the antimicrobial hypothesis, rifampicin has been previously studied for use in bacterial cerebral infections [ ] . as rifampicin is able to cross the bbb [ ] , it can exert its antimicrobial effect directly in the brain. in the presence of rifampicin, a reduction of aβ fibril formation [ ] has been observed in addition to augmented aβ clearance [ ] . a study by umeda et al., in which rifampicin was administered to apposk mice, found the treatment to reduce aβ accumulation, provide synaptic protection, and reduce microglial activation [ ] . clinical studies exploring the impact of rifampicin on cognitive function have also been investigated, as mentioned in previous sections. even with its many benefits, the oral intake of rifampicin has also been associated with liver injury in humans. to circumvent this limitation, administering rifampicin intranasally or subcutaneously has been suggested [ ] . these routes of rifampicin administration have been shown to be more effective in improving memory than oral administration [ ] . the use of gingipain inhibitors in ad is another approach that has been taken to alleviate the negative impact of the disease. gingipains are virulence factors that are produced by p. gingivalis [ ] . they are made up of a group of cysteine proteinases, specifically arginin-gingipain a, arginine-gingipain b, and lysign-gingipain [ , ] . given the key role gingipains play in host colonization [ ] and the inactivation of host defenses [ ] [ ] [ ] , they are essential for the survival and pathogenicity of p. gingivalis. regarding aβ - peptide production, p. gingivalis infection was found to increase aβ - levels. furthermore, incubating p. gingivalis with aβ - peptides led to a significant increase in p. gingivalis death. these two findings further support the antimicrobial hypothesis for aβ peptides [ ] . gingipain inhibitors, such as cor , cor and cor , have been found to be effective in inducing p. gingivalis death and reducing the bacterial load in the brain, more so than other antibiotics, such as moxifloxacin [ , ] . in addition, cor was found to provide some level of neuroprotection as well [ ] . the administration of gingipain inhibitors has also been associated with a decrease in host aβ - response to p. gingivalis infection [ ] . even with the benefits associated with the antimicrobial and antiviral drugs listed above, insights into their mechanism of action and their impact on aβ peptide levels are needed. a greater understanding as to whether their administration indirectly reduces the presence of aβ peptides by reducing the viral/bacterial load on the brain, or if they act directly in reducing aβ peptides level, is needed. if it is the latter, and the antimicrobial hypothesis for aβ peptides holds true, their efficacy might not be as positive as hoped. in addition, it is important that the chosen antimicrobial or antiviral drug does not have any adverse effects that could take away from its benefits. for example, cefepime is an antibiotic that has shown to be able to cross the blood-brain barrier and cause neurotoxic symptoms [ ] . the findings of the numerous studies highlighted in this review present a clear indication of the role bacteria and viruses can have in ad development. even with this conclusion, it is clear that a specific bacteria or virus alone is not responsible for ad development, as no specific bacteria or virus has been identified to be universally present in all ad brains. rather, a number of viruses and bacteria could exacerbate the progression of neurodegenerative diseases, either independently or along with other pathogens. by exploring the presence of multiple viruses/bacteria in ad brains, future investigations can give insights into which microorganisms are most present, and whether all ad brains have both a detected and increased level of selected bacteria/virus. the use of antiviral and antimicrobial drugs early on, while the patient is still in the presymptomatic phase of ad, could have potential effectiveness in targeting the root cause of ad pathogenesis and alleviating the viral/microbial load on the brain. further investigations into their use in ad would give greater insight regarding their efficacy and limitations. funding: this study was supported by the altschul foundation and in part by grant number p at - from the nccih and ods. we acknowledge that the contents of this review do not represent the views of the nccih, the ods, the nih, or the united states government. 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action as virulence factors: from cleaving substrates with the precision of a surgeon's knife to a meat chopper-like brutal degradation of proteins role of bacterial proteinases in matrix destruction and modulation of host responses cleavage of the human c a receptor by proteinases derived from porphyromonas gingivalis: cleavage of leukocyte c a receptor treatment of porphyromonas gulae infection and downstream pathology in the aged dog by lysine-gingipain inhibitor cor cefepime-induced neurotoxicity: a systematic review key: cord- -j iawzp authors: fitzpatrick, meagan c.; bauch, chris t.; townsend, jeffrey p.; galvani, alison p. title: modelling microbial infection to address global health challenges date: - - journal: nat microbiol doi: . /s - - - sha: doc_id: cord_uid: j iawzp the continued growth of the world’s population and increased interconnectivity heighten the risk that infectious diseases pose for human health worldwide. epidemiological modelling is a tool that can be used to mitigate this risk by predicting disease spread or quantifying the impact of different intervention strategies on disease transmission dynamics. we illustrate how four decades of methodological advances and improved data quality have facilitated the contribution of modelling to address global health challenges, exemplified by models for the hiv crisis, emerging pathogens and pandemic preparedness. throughout, we discuss the importance of designing a model that is appropriate to the research question and the available data. we highlight pitfalls that can arise in model development, validation and interpretation. close collaboration between empiricists and modellers continues to improve the accuracy of predictions and the optimization of models for public health decision-making. m icrobial pathogens are responsible for more than million years of life lost annually across the globe, a higher burden than either cancer or cardiovascular disease . diseases that have long plagued humanity, such as malaria and tuberculosis, continue to impose a staggering toll. recent decades have also witnessed the emergence of new virulent pathogens, including human immunodeficiency virus (hiv), ebola virus, severe acute respiratory syndrome (sars) coronavirus, west nile virus and zika virus. the persistent global threat posed by microbial pathogens arises from the nonlinear mechanisms of disease transmission. that is, as the prevalence of a disease is reduced, the density of immune individuals drops, the density of susceptible individuals rises and disease is more likely to rebound. the resultant temporal trajectories are difficult to predict without considering this nonlinear interplay. for instance, many microbial diseases exhibit periodic spikes in the number of cases that are unexplainable by pathogen natural history or environmental phenomena. by explicitly defining the nonlinear processes underlying infectious disease spread, transmission models illuminate these otherwise opaque systems. forty years ago, nature published a series of papers that launched the modern era of infectious disease modelling , . since that time, these methodologies have multiplied . transmission models now employ a variety of approaches, ranging from agent-based simulations that represent each individual to compartmental frameworks that group individuals by epidemiological status, such as infectiousness and immunity , . accompanying the methodological innovations, however, are challenges regarding selection of appropriate model structures from among the wealth of possibilities . at this anniversary of the publication of these landmark papers , , we reflect on contributions that transmission modelling has made to infectious disease science and control. through a series of case studies, we illustrate the overarching principles and challenges related to model design. with expanding computational capacity and new types of data, myriad opportunities have opened for transmission modelling to bolster evidence-based policy (box ) , . in all pursuits, modelling is most informative when conducted collaboratively with microbiologists, immunologists and epidemiologists. we offer this perspective as an entry point for non-modelling scientists to understand the power and flexibility of modelling, and as a foundation for the transdisciplinary conversations that bolster the field. even within the same disease system, the ideal model design depends on the specifics of the questions asked. here, we highlight a series of models focused on one of the defining infectious agents of our era: hiv. the virus has challenged science, medicine and public health at every scale, from its deft immune evasion to its death toll of more than million over the last four decades . we describe how clinical needs, research questions and data availability have shaped the design of hiv models across these scales. unless otherwise indicated, the term 'hiv' is inclusive of both hiv- and hiv- . within-host models. at a within-host scale (table ) , models can be used to simulate cellular interactions, immunological responses and treatment pharmacokinetics . in such simulations, viral dynamics are often modelled using a compartmental structure, with the growth of one population, such as circulating virions, dependent on the size of another population, such as infected cells. for example, a seminal within-host model fit to viral load data by perelson et al. revealed high turnover rates of hiv- , counter to what was then the prevailing assumption that hiv- remained dormant during the asymptomatic 'latency' phase. the corollary to these high rates of viral turnover was that drug resistance would likely evolve rapidly under monotherapy. further analyses of this model indicated that a combination of at least three drugs was necessary to maintain drug sensitivity . once combination therapy did become available, extension of the perelson et al. model demonstrated that the two-phase decline in viral load observed following treatment initiation was attributable to a reservoir of long-lived infected cells . with this insight also came the realization that prolonged treatment would be necessary to suppress viral load. the incorporation of meagan c. fitzpatrick , , chris t. bauch , jeffrey p. townsend , , and alison p. galvani , , * the continued growth of the world's population and increased interconnectivity heighten the risk that infectious diseases pose for human health worldwide. epidemiological modelling is a tool that can be used to mitigate this risk by predicting disease spread or quantifying the impact of different intervention strategies on disease transmission dynamics. we illustrate how four decades of methodological advances and improved data quality have facilitated the contribution of modelling to address global health challenges, exemplified by models for the hiv crisis, emerging pathogens and pandemic preparedness. throughout, we discuss the importance of designing a model that is appropriate to the research question and the available data. we highlight pitfalls that can arise in model development, validation and interpretation. close collaboration between empiricists and modellers continues to improve the accuracy of predictions and the optimization of models for public health decision-making. stochasticity into this within-host framework allowed model fitting to 'viral blips'-transient peaks in viral load, even under antiretroviral treatment . analysis of this data-driven stochastic model demonstrated that homeostatic proliferation maintained the infected cell reservoir and produced these viral blips, a finding that was later confirmed experimentally , . the implication for clinical care was that intensified antiretroviral treatment would be unable to eliminate the latent reservoir of infected cells as had been hypothesized, sparing patients from potentially fruitless trials with such regimens. individual-based models. whereas the unit of interest for withinhost modelling is an infected cell, the analogous unit for individualbased models is an infected person (table ) , , . individual-based models are often used to explore the interplay between disease transmission and individual-level risk factors, such as comorbidities, sexual behaviours and age. such models are capable of incorporating data with individual-level granularity, including those regarding contact patterns, patient treatment cascades and clinical outcomes. individual-based models are uniquely suited for representing overlap in individual-level risk factors and translating the implications of this overlap for public health policy. for example, an individual-based model was recently used to demonstrate that the majority of hiv transmission among people who inject drugs in new york city is attributable to undiagnosed infections . these modelling results underscore the urgency for the city to invest in more comprehensive screening and improved diagnostic practices. population models. most commonly, models are created at the population scale, capturing the spread of a pathogen through a large group (table ) . at this scale, compartmental models shift in focus from the pathogen to the host. unlike individual-based models, compartmental models will aggregate individuals with a similar epidemiological status. for instance, the archetypical 's-i-r' model separates the entire population of interest into one of three categories: s, susceptible to infection; i, infected and infectious; or r, recovered and protected . in practice, most models will have additional compartments or stratification beyond this simple structure. age stratification is essential when either the disease risk or the intervention is age-specific. as an example, an age-stratified multipathogen model demonstrated that schistosomiasis prevention targeted to zimbabwean schoolchildren could cost-effectively reduce hiv acquisition later in life . this framework was extended to additional countries with a range of age-specific disease prevalence and co-infection rates to assess the potential value of treating schistosomiasis in adults. although adult treatment is not usually considered efficient, the model showed that it could be cost-effective in settings with high hiv prevalence . these models strengthened the investment case for treatment of schistosomiasis, an otherwise neglected tropical disease. network models are also deployed to represent dynamics on the population scale (table ) . these models impose a structure on contacts between hosts, unlike compartmental models which assume that contacts are random among hosts within a compartment. in a network model, nodes represent individuals and the connections between nodes represent contacts through which infection may spread . sources for network parameterization may include surveys, partner notification services or phylogenetic tracing , . as with individual-based models, network models tend to require significant amounts of data to fully parameterize, but various computational and statistical methods have been developed to analyse the impact of uncertain parameter values on model predictions . network models are applied to discern the influence of contact structure on disease transmission and on the effectiveness of targeted intervention strategies. for instance, network models predicted that hiv would spread more quickly through sexual partnerships that are concurrent versus serially monogamous, even if the total numbers of sexual acts and partners remain constant . the study prompted a more rigorous engagement of epidemiologists with sociological data to tailor interventions for specific settings . other network models have focused on the more rapid transmission within clusters of high-risk individuals and slower transmission to lower-risk clusters, a dynamic which explains discrepancies between observed incidence patterns and the expected pattern based on an assumption of homogeneous risks . these studies both illustrate the importance of accounting for network-driven dynamics when individuals are highly aggregated with regards to their risk factors, and when appropriate data for parameterization are available. metapopulation models. metapopulation models represent disease transmission at dual scales, considering not just the interactions of individuals, but also the relationships between groups of individuals, which are typically defined geographically (table ) . transmission intensity is often higher within groups than across groups, especially when the groups are spatially segregated . one metapopulation model of hiv in mainland china considered there are the three principal objectives of modelling, all of which can inform public health policy. predicting disease spread. models can be used to estimate the infectiousness of a pathogen within a given population. a fundamental concept is that of r , the basic reproduction number, which quantifies the number of infections that would result from a single index case in a susceptible population. r governs the temporal trajectory of an outbreak and the scale of interventions required for its containment. models may be used to infer r as well as forecast changes in r that could drive transitions in epidemic dynamics, such as the shift from sporadic outbreaks to sustained chains of transmission. example: assessing real-time zika risk in texas . selecting among alternative control strategies. simultaneous field trials of multiple infectious disease control options are often infeasible. models can simulate a wide range of control strategies and thus optimize public health policies according to translational objectives and real-world constraints. modelling can also extrapolate from the individual clinical outcomes of interventions or novel therapeutics to the population-level impacts. extrapolating to the population level is essential to evaluate the indirect benefits of interventions, including a reduction in transmission, or unanticipated repercussions, such as evolution of resistance. example: comparing antibiotic 'cycling' versus 'mixing' to minimize the evolution of antimicrobial resistance . hypothesis testing. it is often logistically or ethically infeasible to empirically test scientific hypotheses in the field or experimentally. modelling can identify parsimonious explanations of observed phenomena, including complex outcomes that can arise from the nonlinear processes common in microbiological systems. even simple models can be useful to help us understand dynamics that are common to many microbiological systems through identification of basic mechanisms that apply across a range of infections. by examining a new infectious agent through the lens of previously characterized systems, models provide insight into the ways that a particular microbial infection might follow or break from typical patterns. example: investigating whether individual heterogeneity within social networks significantly impacts disease spread . transmission within and between provinces, driven by the mobility of migrant labourers . the study suggested that hiv prevention resources could be most effectively targeted to provinces with the greatest initial incidence, as rising incidence in other provinces is driven more by migration from the high-burden provinces than by local transmission. given that the chinese provinces with employment opportunities for migrants are also those with the heaviest burden of hiv, migrant workers who acquire hiv often do so in the province where they work. however, government policy requires migrants to return to their home province for treatment. the movement of these workers perpetuates the disease cycle, as new migrants move to fill the vacated jobs and themselves become exposed to elevated hiv risk. these results therefore call for reconsideration of provincial treatment restrictions. multinational models. global policies, such as the treatment goals set by the joint united nations programme on hiv/aids (unaids), have been modelled on a global scale (table ) by considering the effectiveness of the policies for each nation. for example, a compartmental model was used to evaluate the potential impact of a partially efficacious hiv vaccine on the epidemiological trajectories in countries that together constitute over % of the global burden . the model was tailored to each country by fitting to country-specific incidence trends as well as diagnosis, treatment and viral suppression data. this model revealed that, even with efficacy as low as %, a hiv vaccine would avert millions of new infections worldwide, irrespective of whether ambitious treatment goals are met. these results identify the synergies between vaccination and treatment-as-prevention, and provide evidence to support continued investment in vaccine development , . from the cellular level to the population level, hiv modelling has led to improvements in drug formulations, clinical care and resource allocation. as scientific advances continue to bring pharmaceutical innovations, modelling will remain a useful tool for illuminating transmission dynamics and optimizing public health policy. hiv was not controlled before it became a pandemic, but our response to future outbreaks has the potential to be more timely . when diseases emerge in new settings, such as ebola in west africa and sars in china, modelling can be rapidly deployed to inform and support response efforts (fig. ) . unfortunately, the urgency of public health decisions during such outbreaks tends to be accompanied by a sparsity of data with which to parameterize, calibrate and validate models. as detailed below, uncertainty analysis-a method of analysing how uncertainty in input parameters translates to uncertainty in model outcome variables-becomes all the more vital in these situations. media attention regarding model predictions is often heightened during outbreaks, ironically at a time when modelling results are apt to be less robust than for well-characterized endemic diseases. we discuss the importance of careful communication regarding model recommendations and associated uncertainty to inform the public without fuelling excessive alarm. despite these challenges, and especially if these challenges can be navigated, the timely assessment of a wide range of intervention scenarios made possible by modelling would be particularly valuable during infectious disease emergencies. ebola virus outbreaks. the ebola virus outbreak struck a populous region near the border of guinea and sierra leone, sparking a crisis in a resource-constrained area that had no prior experience with the virus. as the caseload mounted and disseminated geographically, it became apparent that the west african outbreak would be unprecedented in its devastation. models were developed to estimate the potential size of the epidemic in the absence of intervention, demonstrating the urgent need for expanded action by the international community [ ] [ ] [ ] , and to calculate the scale of the required investment . initial control efforts included a militarily enforced quarantine of a liberian neighbourhood in which ebola was spreading. modelling analysis in collaboration with the liberian ministry of health demonstrated that the quarantine was ineffective and possibly even counterproductive . connecting the microbiological and population scales, another modelling study integrated within-host viral load data over the course of ebola infection and between-host transmission parameterized by contact-tracing data. the resulting dynamics highlighted the imperative to hospitalize most cases in isolation facilities within four days of symptom onset . these modelling predictions were borne out of empirical observations. early in the outbreak, when the incidence was precipitously growing, the average time to hospitalization in liberia was above six days . as contact tracing improved, the concomitant acceleration in hospitalization was found to be instrumental in turning the tide on the outbreak . in another approach, phylogenetic analysis and transmission modelling were combined to estimate underreporting rates and social clustering of transmission . this study informed public health authorities regarding the optimal scope and targeting of their efforts, which were central to stemming the epidemic. although data can be scarce for emerging pathogens, modellers can exploit similarities with better-characterized disease systems to investigate the potential efficiency of different interventions (box ). as vaccine candidates became available against ebola, ring vaccination was proposed based on the success of the strategy in eliminating smallpox , another microorganism whose transmission required close contact between individuals and for which peak infectiousness occurs after the appearance of symptoms. compartmental models had suggested parameter combinations for which ring vaccination would be superior to mass vaccination , and methodological advances subsequently allowed for explicit incorporation of contact network data . modelling based on social and healthcare contact networks specific to west africa supported implementation of ring vaccination , and the approach was adopted for the clinical trial of the vaccine . in , two independent outbreaks of ebola erupted in the democratic republic of the congo. during the initial outbreak in Équateur province, modellers combined case reports with time series from previous outbreaks to generate projections of final epidemic size that could inform preparedness planning and allocation of resources . ring vaccination was again deployed, this time within two weeks of detecting the outbreak. a spatial model quantified the impact of vaccine on both the ultimate burden and geographic spread of ebola, highlighting how even one week of additional delay would have substantially reduced the ability of vaccination to contain this outbreak . the second outbreak was reported in august in the north kivu province. armed conflict in this region has interfered with the ability of healthcare workers to conduct the necessary contact tracing, vaccination and treatment. as conditions make routine data collection difficult and even dangerous, modelling has the potential to provide crucial insights into the otherwise unobservable characteristics of this outbreak. in contrast to the unexpected emergence of ebola in a new setting, the influenza virus has repeatedly demonstrated its ability to cause pandemics. a pandemic is an event in which a pathogen creates epidemics across the entire globe. the pandemic killed an estimated million people worldwide , exceeding the combined military and civilian casualties of world war . while the % case-fatality rate of the strain was approximately times higher than is typical for influenza , pathogenic strains with case-fatality rates exceeding % periodically emerge . modelling has illustrated how repeated zoonotic introductions impose selection for elevated human-to-human transmissibility, which thereby exacerbates the threat of a devastating influenza pandemic . such threats underscore the importance of surveillance systems and preparedness plans, which can be informed by modelling (box ). transmission models are able to optimize surveillance systems, accelerate outbreak detection and improve forecasting [ ] [ ] [ ] [ ] . for example, a spatial model integrating a variety of surveillance data streams and embedded in a user-friendly platform is currently implemented by the texas department of state health services to generate real-time influenza forecasts (http://flu.tacc.utexas.edu/). modelling has also motivated the development of dynamic preparedness plans, which adapt in response to the unfolding events of a pandemic, as models identified that adaptive efforts would be more likely to contain an influenza pandemic than static policies chosen a priori . other pandemic influenza analyses used agestructured compartmental models to study the trade-off between targeting influenza vaccination to groups that transmit many infections but experience relatively low health burdens (for example, schoolchildren) versus groups that transmit fewer infections but experience greater health burdens (for example, the elderly) . such examples illustrate the insights that modelling has provided to the decision makers charged with maintaining readiness against simultaneously rare but catastrophic situations. modelling has also examined the impact of human behaviour, including vaccination decisions and social interactions, on the course of an epidemic. public health interventions are not always sufficient to ensure disease control, as behavioural factors can thwart progress [ ] [ ] [ ] [ ] . for example, reports in of potential neurological side effects from the whole-cell pertussis vaccine led to a steep decline in vaccine uptake throughout the uk, followed by a slow recovery (fig. a) . vaccine uptake ebbed and flowed over the next two decades, with higher rates of vaccination in the wake of large pertussis outbreaks (fig. b) , . compartmental models analysing the interplay between vaccine uptake and disease dynamics confirmed the hypothesis that increases in vaccination were a response to the pertussis infection risk , and showed that incorporating this interplay can improve epidemiological forecasts. network models extending these coupled disease-behaviour analyses types of projection that can be generated include outbreak trajectories, disease burdens and economic impact. d, probabilistic uncertainty analyses convey not only model projections of policy outcomes, but also quantification of confidence in the projections. e, as policies are adopted and the microbiological system is influenced accordingly, the model can be iteratively updated to reflect the shifting status quo, thereby progressively optimizing policies within an evolving system. have illustrated how the perceived risk of vaccination can have greater influence on vaccine uptake than disease incidence . more recently, vaccine refusal has led to the resurgence of measles in the usa , . researchers are turning to social media to gather information about attitudes toward vaccines and infectious diseases, and to glean clues about vaccinating behaviour , , . for instance, signals that vaccine refusal is compromising elimination can be detected months or years in advance of disease resurgence by applying mathematical analysis of tipping points to social media data that have been classified on the basis of sentiment using machine learning algorithms . these and other data science techniques might help public health authorities identify the specific communities that are at increased risk of future outbreaks. on shorter timescales, the near-instantaneous availability of social media data facilitates its integration into models developed for outbreak response , . other behavioural factors that have been incorporated into transmission models include attendance at social gatherings, sexual behaviour and commuting patterns-elements which are also often affected by perceived infection risk , , . antimicrobial resistance. a substantial portion of the increase in human lifespan over the last century is attributable to antibiotics , but the emergence of pathogen strains that are resistant to antimicrobials threatens to reverse these gains. the extensive use and misuse of antibiotics has led to the evolution of multidrug-resistant, extensively drug-resistant and even pan-drug-resistant pathogens across the globe. precariously, this evolution outpaces the development of new antibiotics. mathematical modelling is being used to identify strategies to forestall the emergence and re-emergence of antimicrobial resistance , . models are particularly valuable for comparing alternative strategies, such as administration of different antibiotics within the same hospital ward, temporal cycling of antibiotics and combination therapy [ ] [ ] [ ] [ ] . high-performance computing now permits the rapid exploration of multidimensional parameter space. models can thereby narrow an array of possible interventions down to a subset likely to have the highest impact or optimize between trade-offs, such as effectiveness and cost (box ). by contrast, expense, feasibility and ethical considerations may impose more limitations on in vivo investigations (box ). not only can models identify the optimal strategy for a given parameter set, but they can generate the probability that this intervention remains optimal across variation in the parameters. for example, an optimization routine combined with simulation of hospital-based interventions identified combination therapy as most likely to reduce antibiotic resistance . as a complementary approach, modelling can incorporate economic considerations into these evaluations. a stochastic compartmental model showed that infection control specialists dedicated to promoting hand hygiene in hospitals are cost-effective for limiting the spread of antibiotic resistance . although most models of antibiotic resistance have focused on transmission in healthcare settings, the importance of antibiotic resistance in natural, agricultural and urban settings has been increasingly recognized [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, a metapopulation model of antimicrobial-resistant clostridium difficile simulated its transmission within and between hospitals, long-term care facilities and the community. this model demonstrated that mitigating risk in the community has the potential to substantially avert hospital-onset cases by decreasing the number of patients with colonization at admission and thereby the transmission within hospitals . this study illustrates how models can consider the entire ecosystem of infection to elucidate dynamics that might not be captured through focus on a single setting. during the initial phase of an outbreak, the predictive power of models is often constrained by data scarcity. this challenge is exacerbated for outbreaks of novel emerging diseases given that our understanding of the disease will rely on the unfolding epidemic (fig. ) . not only can the absence of data constrain model design, but sparse data requires extensive sensitivity analyses to evaluate the robustness of conclusions. univariate sensitivity analyses, in which individual parameters are varied incrementally above and below a point estimate, can identify which parameters most influence model output (box ). such comparisons reveal both salient gaps in knowledge and targets for preventing and mitigating the outbreak (box ) . as an outbreak progresses, each day has the potential to provide more information about the new disease, including its duration of latency, the symptomatic period, infectiousness, transmission modalities, underreporting and the case-fatality rate. however, collecting detailed data to inform each of these parameters can strain resources when they are thinly spread during an emergency response. sensitivity analysis can support clinicians and epidemiologists in prioritizing data collection efforts . parameterization challenges are compounded for complicated disease systems, such as vector-borne diseases. for example, models of zika virus infection span both species and scales, as the disease trajectory is influenced by factors ranging from mosquito seasonality and mosquito abundance down to viral and immunological dynamics within human and mosquito hosts , . adding to this complexity, the ecological parameters vary seasonally and geographically-heterogeneities that may be amplified by socioeconomic factors modulating human exposure to infected mosquitoes . in the absence of the high-resolution data that would be ideal to tailor a mosquitodriven disease system to a given setting, uncertainty analysis can unify parameterization from disparate data sources. in contrast to univariate sensitivity analyses, uncertainty analysis simultaneously samples from empirical-or expert-informed distributions for many or all input parameters. collaboration between modellers and disease experts is thus instrumental to ensuring the biological plausibility of these parameter distributions , . the uncertainty analysis produces both a central point estimate and a range for each outcome, a combination which can inform stakeholders about the best-case and worst-case scenarios as well as the likelihood that an intervention will be successful [ ] [ ] [ ] . in constructing models and communicating results, there are common pitfalls which can compromise the rigor and impact of the research. a pervasive pitfall is the incorporation of excessive model complexity, particularly through inclusion of more parameters than can be reliably parameterized from data. intuition might suggest that a complex representation of a microbiological system would more closely represent reality. however, the predictive power of a model can be degraded if incorporating additional parameters only marginally improves the fit to data. this tendency results in complicated transmission models that overfit data in much the same way that complicated statistical regressions can overfit data, replicating not only the relevant trends but also the noise in a particular data set. these overfit models thus become less useful for prediction and generalization , . to guide appropriate model complexity and parameterization, modellers have used the mathematical theory of information to develop criteria which quantify the balance between realism and simplicity. such criteria penalize additional parameters but reward substantial improvements in fit, thereby identifying the simplest model that can adequately fit the data , , . these methods can be applied to select among models or alternatively to calculate weighted average predictions across models. in a similar vein, modelling consortiums serve to address uncertainty surrounding model design [ ] [ ] [ ] . in a consortium, several modelling groups develop their models independently, each applying their particular expertise and perspective. for example, consortia of malaria modellers were convened to predict the effectiveness of interventions, including a vaccine candidate and mass drug administration . congruence of output among models engenders confidence that model results are robust. another pitfall concerns the quality of data used to inform the model. incompleteness of data has been an issue since , when daniel bernoulli published a compartmental model of smallpox and acknowledged that more extended analyses would have been possible if the data had been age-stratified . even today, using data to develop models without knowledge of how the data were collected or the limitations of the data can be risky. data collected for an alternative purpose can contain gaps or biases that are acceptable for the original research question, yet lead to incorrect conclusions when incorporated for another purpose in a specific model. in ideal circumstances, modellers would be involved in the design of the original study, ensuring both seamless integration of the results into the model and awareness on the part of the modeller with regard to data limitations. failing that, it is very helpful for modellers to collaborate with scientists familiar with the details of empirical studies on which their results might depend. this lack of familiarity with the biases or incompleteness of data sources may be particularly dangerous in the era of digital data. 'big data hubris' can blind researchers to the limitations of the dataset, such as being a large but unrepresentative sample of the general population, or the alteration of search engine algorithms partway through the data collection process . some of these limitations can be addressed by using digital data as a complement to traditional data sources. in this way, the weakness of one data source (for example, low sample size of traditional surveys or bias in large digital data) can be compensated by the strengths of another data source (for example, balanced representation in small survey versus large scale of digital data). a final pitfall that often arises in the midst of an ongoing outbreak concerns the interpretation of epidemic projections. initial models may assume an absence of intervention as a way to assess the potential consequences of inaction. such projections may contribute to the mobilization of government resources towards control, as was the case during the west african ebola outbreak , , . in this respect, the projections are intended to make themselves obsolete . in retrospect and without knowledge of the initial purpose of the model, it may appear that the initial predictions were excessively pessimistic . additionally, people living in outbreak zones often change their behaviour to reduce infection risks, thereby mitigating disease spread through, for example, reducing social interactions or increasing vaccine uptake (fig. ) , , . thus, risk assessment constitutes a 'moving target' . for example, input parameters estimated from contact tracing early in an outbreak could require adjustments to reflect these behaviour changes and accurately predict subsequent dynamics . the need for proficient communication skills is heightened during an outbreak. this concern is particularly relevant when presenting sensitivity and uncertainty analyses. although predictions at the extreme of sensitivity analyses also tend to be less probable than mid-range projections, there can be a temptation to focus on the most sensational model scenarios. ensuing public pressure on the basis of misunderstood findings can cause unwarranted alarm and trigger counterproductive political decisions. in both publications and media interactions, underscoring the improbability of extreme scenarios explored during sensitivity analysis, as well as how improved interventions turn a predictive model into a counterfactual one, may pre-empt this pitfall . the role for modelling in supporting epidemiologists, public health officials and microbiologists has progressively expanded since the foundational publications forty years ago, in concert with the growing abundance and granularity of data as well as the refinement of quantitative approaches. models have now been developed for virtually every human infectious disease, as well as in many that affect animals and plants, and have been applied across the globe. interdisciplinary collaboration among empiricists, 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reduce antimicrobial resistance in hospitals the authors gratefully acknowledge funding from the notsew orm sands foundation (grants to m.c.f., j.p.t. and a.p.g.), the national institutes of health (grant nos. k ai and u gm to m.c.f. and a.p.g., respectively) and the natural sciences and engineering research council of canada (grant no. rgpin- - to c.t.b.). the authors also thank c. wells and a. pandey, both members of the yale center for infectious disease modeling and analysis, for their helpful discussions regarding the hiv and ebola modelling literature. m.c.f. and a.p.g. drafted the initial manuscript. m.c.f., c.t.b., j.p.t. and a.p.g. all critically revised the content. the authors declare no competing interests. correspondence should be addressed to a.p.g.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -difauneh authors: rahmani, amir masoud; mirmahaleh, seyedeh yasaman hosseini title: coronavirus disease (covid- ) prevention and treatment methods and effective parameters: a systematic literature review date: - - journal: sustain cities soc doi: . /j.scs. . sha: doc_id: cord_uid: difauneh background and objective: the coronavirus disease (covid- ) outbreak was first identified in wuhan in december , which was declared a pandemic virus by the world health organization on march in . covid- is an infectious disease and almost leads to acute respiratory distress syndrome. therefore, the virus epidemic is a big problem for humanity healthy and can lead die in special people with background diseases such as chronic obstructive pulmonary diseases, chronic heart failure, diabetes mellitus, and kidney failure. different medical, social, and engineering methods have been proposed to face the disease include treatment, detection, prevention, and prediction approaches. methods: we propose a taxonomy tree to investigate the disease confronting methods and their negative and positive effects. our work consists of a case study and systematic literature review (slr) to evaluate the proposed methods against the virus outbreak and disease epidemic. results: our experimental results and observations demonstrate the impact of the proposed medical, prevention, detection, prediction, and social methods for facing the spread of covid- from december to july . conclusion: our case study can help people have more information about the disease and its impact on humanity healthy and illustrate effective self-caring methods and therapies. severe acute respiratory syndrome coronavirus (sars-cov- ) was identified as the infectious disease and first generation of covid- that was inspired sars. the virus was first identified in wuhan in december with common clinical symptoms including fever, dry cough, and tiredness, which are different for people in age categories (such as adults and pediatrics). other disease's clinical symptoms were included fatigue, muscle or body aches, headache, the new loss of taste or smell, sore throat, congestion or runny nose, and nausea or vomiting diarrhea. china researchers confirmed the mutated second-generation with mild symptoms or without clinical symptoms. the world health algorithms, machine learning-based applications (accurate image processing and voice detection), medical and behavioral sciences methods against the disease epidemic [ ] . therefore, an overall review can help evaluate the issue from different angles and give more information to people about self-caring and facing covid- . this paper proposes a taxonomy tree for evaluating different proposed ideas against the virus and their positive and negative impacts on people's lives in various aspects. our slr reviews the issue based on the proposed taxonomy tree and investigates the disease epidemic's useful parameters and its problems considering the tree levels. our research evaluates the treatment, prevention, detection, and prediction methods for facing the spread of covid- extracted from the content from authoritative scientific sources such as ieeexplore, science direct, elsevier, and google scholar. our motivations for providing slr include:  epidemic covid- and its negative impact on people's quality of life in all the world  expanding the filed of the proposed ideas against the virus outbreak  lack of an overall review in provided case studies, slrs, and smss in various aspects  the positive impact of the review in various angles on people's level of awareness for self-caring and facing the disease an overall review has to investigate many proposed approaches to confront covid- disease and the virus outbreak, whereas the previous slr-based case studies focused on the particular fields of facing methods such as different observations of pcr's tests. it can give researchers a comprehensive view about finding various study's areas at a glance that can help them achieve the aim of their favorites for presenting a new facing the disease method in the desired branch. an overall review can also help governments for general assignment the confronting covid- epidemic approaches to decide about managing the issue for year-ahead. moreover, we propose a taxonomy tree covering the roots of many proposed methods for facing the virus outbreak and their side effects. after that, we use a method to categorize the case studies based on the proposed taxonomy tree to analyze the approaches according to the classifications. it is facilitated the research about the issue and presenting a new method or improving a method. we classify the related studies to the covid- outbreak into three baseline categories, including treatment, prevention, and effective parameters in spreading the virus. in contrast, other slrs paid attention to details of a confronting method without considering a root for the issue. hence the previous slr can only inform the researchers about a particular branch of studies, whereas our slr reviews the issue from different aspects. the research challenges during or post-pandemic, which also can see in previous studies, including:  finding the related case studies to the purpose field for achieving more information  finding reliable information between the mass of published manuscripts and registered datasets  providing a new idea with more benefits after analyzing and introducing the previous approaches' advantages  managing the hidden damages after recovery and applying restrictions rules, and therapies' injury of post-pandemic we report the information based on the presented experimental results and observations in the published papers selected due to their highly cited and novelty and accepted in reputable journals. our work focuses on the virus confronting methods and potential problems after applying them to a humane society, which our main contributions as the following:  providing a new-of-the-art taxonomy tree for evaluating the issue based on three baseline fields of treatment, and prevention facing methods, and the effective parameters in the covid- outbreak  providing a systematic literature review based on the proposed taxonomy tree  covering detection, prediction, and management approaches against the disease according to three heads of treatment, prevention, and effective parameters  supporting future scopes according to economic damages, treatment's injuries, and people's physical and mental damages after recovery and regarding social distancing's rules in post-pandemic the remainder of this paper is organized as follows. the second section reviews previous studies on different proposed approaches for facing covid- . the third section describes our method for researching and collecting information. section fourth focuses on investigating the proposed ideas based on the taxonomy tree. section fifth presents a discussion and comparison of the case studies, and the sixth section concludes the paper. in this section, we examined the related survey reviews to the proposed methods for facing the covid- outbreak that is focused on specific fields, such as investigating the impact of artificial intelligence-based confronting approaches. the virus is a big problem and dangerous for people with background diseases due to increasing the probability of getting an acute disease, leading to death. bansal [ ] provided a literature search about the impact of underlying cardiovascular (cv) disease on getting infected covid- and the patient's worsening. this work utilized pubmed and google search engines for collecting information and dataset to analyze the issue. the observations and experimental results demonstrated deteriorating the patient's condition with a cv to get covid- compared to the patients without background diseases. diabetes mellitus and hypertension are likewise the background diseases that are increased the probability of getting acute covid- and death for patients with them. parveen et al. [ ] studied the relationship between the probability of getting acute the disease for patients with diabetes and hypertension and people without background diseases using two databases of pubmed and cochrane. the experimental results and observations demonstrated a direct dependency between severity, icu care requirement, death, and patients with hypertension infected to covid- . in contrast, the acute conditions were not observed for patients with diabetes. the case study achieved the result that was led to suggest employing fast detection methods and icu care for patients with diabetes and hypertension according to their negative impact on cip's health status. more awareness about the coronavirus disease family and its disease complications can help people for self-caring and healthcare rules. ortiz-prado et al. [ ] conducted a comprehensive literature review of clinical, molecular, and epidemiological characterizations of the sars-cov virus and covid- to present more information on the virus ability in creating a risk for people's life quality in all the world. the case study reported the measurement of the concentration and the number of cells in blood cells before and after getting the disease, such as plasma, erythrocytes, leukocytes, and thrombocytes. it is useful for researchers to propose a novel treatment method and develop an antibody with minimal serology time. the rapid and accurate detection of the disease has an impressive effect on reducing the probability of acute respiratory syndrome and death caused by penetrating the virus into the lower respiratory system. investigating the efficiency of detection tools can help propose a suitable diagnostic tool for fast and accurate detecting of the disease [ ] . udugama et al. [ ] provided a review article to evaluate all of the proposed detection tools and inform the researchers, health, and medical staff for utilizing them according to their ability to diagnose covid- . the laboratory tests significantly affect detecting the disease and screening the patients with common initial symptoms. the observations illustrated that serological tests were urgently needed to accurately diagnostic covid- and help fast screening the disease infected people [ ] . bastos et al. [ ] evaluated the impact of various serological tests on detecting the virus using databases of medline, biorxiv, and medrxiv, which consists of enzyme-linked immunosorbent assay (elisa), lateral flow immunoassay (lfia), and clinical laboratory improvement amendments (clia) tests. the matching antibody was measured to diagnose the disease that elisa used to detect human immunodeficiency virus (hiv) before covid- to achieve a result from the tests. the results of lfias proved the lower sensitivity of commercial kits than non-commercial tests and higher sensitivity at least three weeks after symptom onset than the first week. various clinical symptoms were identified to detect the disease, which is different in adults and pediatrics. katal et al. [ ] investigated imaging's characteristics for diagnosing the infection in pediatrics caused by getting covid- . this work reviewed the issue and analyzed the previous studies using online monitoring the databases of medline, scopus, google scholar, and embase, which consists of the results of pediatrics ct. chest ct observations illustrated that ground-glass opacities (ggo) and consolidations were almost seen as ct's anomalies. the case study proved a balancing requirement between the risk of radiation and the need for chest ct according to the higher sensitivity of pediatrics than adults in facing x-ray. ming-yen et al. [ ] studied ct and chest radiologic's roles in detecting covid- 's infections based on retrospective observations in shenzhen and hong kong cities. imaging patterns showed that ggos were almost available in the peripheries, and also were not seen pleural effusions and lymphadenopathy in all cases. the case study demonstrated higher sensitivity of ct in diagnosing the disease's infections than chest radiographs. proving and guaranteeing health safety increased people's mental health in various social positions when community health is threatened by spreading a dangerous virus similar to covid- [ ] . the issue consists of evaluating the ensuring of healthcare professionals' occupational safety, biosafety of laboratories and facilities, social safety, food safety, and mainly mental/psychological health and domestic safety. haghani et al. [ ] proposed analyzing the literature on covid- about investigating providing community health against the disease epidemic from different aspects such as cyber safety, economic safety, and supply-chain safety. various symptoms were identified in patients with covid- , which are not persistent for all infected people such as fever, dry cough, and tiredness. also, acute respiratory distress syndrome (ards) was detected as the disease's injury, and brain abnormalities were identified as the damage of covid- for people's health, which refers to any atypical feature in the brain functioning, structure, or biochemical levels. egbert et al. [ ] reviewed typology and topography of the disease's injury in adults with covid- utilized from databases (pubmed, google scholar, and sciencedirect) for collecting information about the issue. according to neurologic symptoms, this work employed the neuroimaging methods, including investigating brain white matter's discharges (wm) using computed tomography (ct) and magnetic resonance imaging (mri) besides the collected information from the databases. the observations demonstrated brain abnormalities injury for a third of acute/subacute covid- patients, which it was proved the importance of the neuroimaging for the disease infected people. rodriguez-morales aj et al. [ ] investigated various symptoms and injuries of the disease, including fever, dry cough, dyspnea, ards, and polymorbid in cip. also, they evaluated the detection tools' role in the probability of people's recovery or death. case fatality rate (cfr) illustrated the disease's dangers for peoples' health and alerted countries for equipping therapeutic and health facilities. table demonstrates a summary of the related survey reviews on the various ideas against the covid- outbreak based on the proposed methods and publication months in . the previous slrs and survey studies focused on a particular issue with more details, explaining and analyzing it, and helping researchers for awareness of its advantages and weaknesses. nevertheless, the case studies restricted to a particular field at a specific time interval, while the covid- outbreak is a big persistent problem for people's health in all the world and also are not yet proposed the definitive approaches against covid- . providing new techniques is challenging for the introduced benefits and disadvantages in the special issue such as lfia serology tests' highlight role in diagnosing the disease, whereas an oral saliva test was proposed to detect covid- with more advantages than lfia. an overall review can evaluate the disease and the virus outbreak in different aspects and examine each category's pros and cons in general. also, its investigation is easier than the reviewed slr with a particular field. also, presenting new approaches does not create a vacuum in the introduced advantages and weaknesses by an overall review. according to the previously provided survey reviews, we present a systematic literature review for informing more information to people and researchers about various the disease confronting approaches from different angles that our reasons for slr are including:  lack of investigating the case studies against covid- in different aspects  classifying the issue and propose a taxonomy tree  providing slr based on the taxonomy tree j o u r n a l p r e -p r o o f we describe the stages of our research and information collection methods to provide slr for investigating the proposed ideas against the covid- outbreak. this section demonstrates our review method for extracting content from the digital libraries and classifying the proposed approaches for facing the disease epidemic in slr [ ] [ ] [ ] [ ] . our work utilizes particular keywords to find the intended ideas and case studies that define [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] :  ("covid- ") and (("treatment") or ("prevention")) our slr reviews the issue based on planning the analytical questions (aq) and evaluating the proposed ideas to achieve appropriate responses to the questions that are followed: the virus was first identified in december , and it became a persistent problem for humanity's health and people's quality of life in all the world. therefore, the researchers focused on the issue after diagnosing the disease, and the first published idea was proposed in december . our research domain reviews the published papers in december to july using the digital libraries, in which our slr refers to the published case studies from december to july in . we utilized the paper's concepts about the disease's background, and the virus transmission methods in the introduction section, published in december . according to our research method's stages and benchmarks, the published papers are selected from january to july . figure shows the stages of our research and collecting information, which demonstrates the investigating digital databases of ieeexplore, springer, and elsevier to access valid scientific documents. our slr covers three main categories of treatment, prevention, and effective parameters in the spread of covid- . its research domain is vast, including approximately published research papers from december to july . we try to select the case studies in this research domain to analyze the issue from different angles. finally, we extract the content from thirty-nine research papers published in reputable journals, as shown in figure . we provide a slr based on the research studies fig. the stages of our research method we select forty-two research papers for referring the proposed methods, experimental results, and observations in the provided slr after the researching process. our research method screens studies from published papers based on the thematic analysis approach, including investigating their title, abstract, and conclusion concepts. figure illustrates the number of referred articles depending on ieee and elsevier's digital databases, from january to july . according to the field of the reviewed case studies, we utilize the information of published papers by comparing between elsevier, ieee, and springer, as shown in figure . in this section, we present different proposed methods to face the virus outbreak and its problems. the researchers investigated the issue from the various aspects and proposed different approaches for solving the disease epidemic's challenges. since the research spectrum is pervasive in this field and the studies' domains have to classify to present a regular and comprehensive slr. we propose a taxonomy tree for covering the issue from different angles, shown in figure . the root of the tree demonstrates the main challenge that its solutions are investigated in different aspects. we classify the proposed approaches against covid- into three categories of treatment, prevention, and the effective parameters of the virus outbreak, illustrated in level one of the tree. the treatment and prevention methods consist of detection and experimental, and medical and non-medical approaches, respectively, as shown in level two of the taxonomy. the detection methods include laboratory tests, voice detection, and ct, and also the proposed non-medical approaches are categorized into the prediction and facing solutions. we demonstrate the virus facing methods that consist of personal and social recommendations in level four of the tree. we distribute the social recommendations into quarantine and social distance methods, and also, their positive and negative impacts on people's life are mapped on the leaves of the tree. our review maps the dependent approaches to distributed classifications in level three of the taxonomy tree on the leaves, such as accurate image processing, respiratory sound, and rrt-pcr test. after introducing the proposed taxonomy tree, we review the case studies against the spread of covid- based on the presented categories, as shown in figure . we analyze the issue based on three main categories of effective parameters in the spread of covid- , treatment, and prevention methods for confronting the disease, including detection and therapy approaches. this tree covers the heads of all studies in the domain of the issue to investigate the effective parameters in the virus outbreak (individual, ethnic, cultural, and social habits and environmental factors), prevention and detection's role in facing the disease, and various therapies' impact on improving patients' with covid- considering the treatment's injuries. we also evaluate the damages and their impact on people's mental and physical health after recovery and applying restriction rules, including quarantine, social distance, and self-caring that the taxonomy tree helps cover them. dong et al. [ ] evaluated the impact of inf-α, lopinavir/ritonavir, ribavirin, chloroquine, and arbidol drugs in recovering covid- infected people (cip) and estimated duration of treatment due to their dosage prescriptions. the experimental results demonstrated that the required duration of treatment is ten days in exchange for prescribing inf-α, lopinavir/ritonavir, ribavirin, chloroquine, and arbidol considering the antivirals included in the guidelines for covid- therapy. sallard et al. [ ] investigated the impact of type interferons (int- ) on the disease's detection and treatment according to the first cytokines produced during a viral infection. the immune system generates interferon families (α, β, ε, ω, and κ) as the proteins that are alerted to people's immune system. the infs-based drugs have antiviral effects besides activating the immune system. the studies analyzed the impact of interferon families in the disease treatment that the role of ifn-β in the treatment of multiple sclerosis is still debated. touret et al. [ ] investigated the negative and positive impacts of chloroquine prescription on therapy of the disease infected people. the previous studies demonstrated the treatment effect of hydroxychloroquine and chloroquine as the antivirus drug for the therapy of malaria and autoimmune diseases such as lupus. nowadays, the drug is widely used to treat covid- , whereas the harmony between the therapeutic and toxic doses is essential. chloroquine and hydroxychloroquine have to use based on the subject to strict rules because the margin between the therapeutic and toxic dose is narrow and can lead to cardiovascular disorders. voisin et al. [ ] examined the impact of prescribing hydroxychloroquine (hcq) and azithromycin (az) on patients with covid- . the case study analyzed the efficiency of prescribing hcq+az on covid- infected people with fifty-five median age and background diseases, including hypertension and diabetes, according to the drug's negative impact of increasing qt congenital disorder that can lead the increasing risk of an irregular heartbeat. one of hcq and az's side effects is a growing qt that can be dangerous for people with latent congenital qt. since this work proposed cardiac monitoring regularly performed, and hospital settings allow them to do it safely, patients are treated with hcq+az. the result demonstrated the positive impact of prescribing hcq + az drugs for patients with covid- whiteout occurring heart attacks. li et al. [ ] discussed the current potential therapeutic approaches and the clinical value of blood transfusion-related technologies against covid- . as shown in this work, the laboratory test illustrated reducing the number of lymphocytes in the blood of the patients with covid- , whereas the number of peripheral blood leukocytes remains stable and nature. the study explained that the disease's clinical and external symptoms occurred between three to seven days caused by respiratory system pneumonia and internal infections. also, the disease can negatively affect people's digestive system and leads to its infection, according to the study's achieved result. visibility of multiple ground-glass shadows (ggo) in the chest city determined the tissue infection of lungs caused by penetrating the virus into the respiratory system. the symptoms of severe acute respiratory syndrome coronavirus almost occurred after a one-week infection. xu et al. [ ] prescribed tocilizumab as a treatment method for patients with covid- and analyzed the drug's impact on the probability of infected people's recovery. the case study proved reducing the number of lymphocytes in peripheral blood (approximately %) and increasing c-reactive protein before treatment with tocilizumab. as shown in the study's laboratory tests, prescribing tocilizumab reduced crp in . % of patients. the number of lymphocytes returned to normal in . % of patients on the fifth day after treatment without any noticeable adverse reactions caused by therapy with tocilizumab. the observations and results demonstrated a patient's recovery with acute respiratory syndrome coronavirus , on average, . d after giving tocilizumab. panwar et al. [ ] proposed a neural network-based detection method that is employed ncovnet for detecting the infection tissues of lungs in chest ct. the proposed idea can help to fast and accurate disease detection by analyzing visual indicators found in the chest radiography imaging of covid- patients. the virus is identified in covid- infected people in under seconds by employing the proposed method that fast detection has an impressive effect in treatment and reducing the probability of death induced by getting the disease. fan et al. [ ] proposed an artificial intelligence-based method for detecting lower respiratory system infections caused by penetrating the virus into the lungs in chest ct. the study employed patent scale application and infection segmentation deep network (inf-net) for classifying and multi-class labeling of different types of lung infections to diagnose the disease accurately. this method improved the identification of infected regions by providing inf-net, which was utilized implicit reverse attention and explicit edge-attention. the proposed neural network could detect the objects with low-intensity contrast between infections and normal tissues. inf-net quantified the infected regions, monitoring the longitudinal disease changes, and mass screening processing. ozturk et al. [ ] presented the application of advanced artificial intelligence-based, a novel method to accurate and automatic detecting covid- in patients using chest ct. this work utilized darknet for raw chest x-ray image classifying in the face of binary and multi-class with high accuracy achieved to classification accuracy of approximately . % and . % for binary classes multi-class cases. the study can help fast the disease diagnosis to enhance the probability of covid- infected people's recovery using initial screening and cloud computing to screen patients immediately. this work's highlight feature is automatic image processing and diagnosing the disease without expert radiologists. it is ready to be tested j o u r n a l p r e -p r o o f with a larger database according to a lack of expert radiologists in deprived areas involved in covid- . tan et al. [ ] utilized ultrasound for detecting between features of covid- and community-acquired pneumonia (cap) using a review method the statistical population, including the disease patients and cap patients. the experimental results and observations demonstrated the differences between modified lung ultrasound (mlus) score and highresolution computed tomography (hrct). the correlation between mlus and hrct had a significant effect in diagnosing covid- and cap, and also mlus scoring system was used for evaluating the severity and treatment effect of covid . the values of mlus and hrct were increased in case of facing covid- and cap that the results demonstrated increasing their values for the disease compared to acquired pneumonia. kulkarni et al. [ ] focused on the efficiency of point-of-care lung ultrasound (pocus) inaccuracy diagnosing, clinical utility, and physical practicality in the intensive care unit (icu) for the disease infected people with acute symptoms. the case study proposed pocus instead of chest ct to check the patient's condition and symptoms with respiratory syndrome coronavirus because the intubated transfer is critically high risk. also, patient moving increased the probability of getting the disease for other healthcare staff, patients, and visitors, whereas patients with covid- have to move from icu to radiology for ct scan of lungs. other benefits of utilizing pocus consist of test cheap, not use ionizing radiation, and the results are available instantly compared to ct, as shown in this case study. a. kavsak et al. [ ] investigated the experimental results and laboratory tests for providing a detection method based on blood plasma concentrations. first, the case study evaluated the result of the complete blood count (cbc) test, which consists of investigating the number of leukocytosis, thrombocytopenia, and lymphocytopenia cells. cbc tests can help diagnose infectious diseases such as respiratory tract infections caused by penetrating the respiratory system's virus. the disease is diagnosed where the three cbc findings are inadequate, whereas lymphocytopenia below the reference interval or absolute count is unclear. in the acute phase of the disease, the condensation of protein is highly in covid- infected people. also, inflammatory biomarkers are widely seen in the c-reactive protein (crp) test, demonstrating sensitive inflammation and tissue damage biomarkers. the laboratory tests illustrated reducing post-convalescent plasma transfusion and crp levels after seven days of the infected person's recovery. soraya et al. [ ] evaluated various laboratory tests and their impacts on diagnosing the infected covid- that are included the number of leukocyte, neutrophil, thrombocyte, and lymphocyte in addition to crp and procalcitonin (pct) and d-dimer levels. this case study utilized the random-effect model (rem) instead of the fixed-effect model (fem) adjacent to heterogeneity. the observations demonstrated a significant reducing leukocyte, neutrophil, platelet, lymphocyte, and thrombocyte counts for covid- pneumonia compared to other types of pneumonia. the laboratory tests also showed that leukocyte, neutrophil, d-dimer, and crp increased for acute covid- than non-acute the disease. caccialanza et al. [ ] studied people's diet situation and its impact on the probability of getting covid- that the case study proposed a pragmatic protocol for early nutritional supplementation of the non-acute disease situation in infected people. getting the disease reduced j o u r n a l p r e -p r o o f the desire to consume food for infected people and led to malnutrition that was decreased the body's immune system against covid- . this work provided nutrition, including high-calorie dense diets in various consistencies with highly digestible foods, oral supplementation of proteins, and prescript intravenous infusion of the multivitamin, multimineral trace elements. the observations demonstrated the positive impact of recommending suitable nutrition in facing the disease. sher et al. [ ] examined covid- infected people's clinical symptoms admitted to the intensive care unit (icu). the study demonstrated acute respiratory distress syndrome (ards) for hospitalized patients in icu who have been proposed psychiatry manage hyperactive delirium, such as complicated by environmental factors. the distress syndrome was managed by providing psychiatry with the prescription of a combination of high-dose melatonin, suvorexant, guanfacine, intravenous haloperidol, and intravenous valproic acid. the case study could help patients with covid- reduce and manage the delirium caused by hospitalized in icu and the acute disease status in a long time using a systematic approach including prophylaxis, screening, diagnosis, and treatment. we classify the treatment methods-based reviews and define the studies' characteristics, including the main context, advantage, weakness, and new finding, as shown in table . the main context of the upper-reviewed treatment studies mostly focused on detection methods to accurate and fast diagnosis patients with covid- due to a significant effect of detecting in reducing the probability of patient's death. table demonstrates the evaluation parameters in introducing the ability of the presented treatment-based studies. the parameters consist of cost, accuracy, treatment ability, detection ability, direct covid- treatment, and time overhead. the treatment methods most focused on increasing accuracy, treatment ability, and detection ability. wu et al. [ ] proposed an antibody against covid- and studied its impact on patients with covid- and negative covid- . the study utilized four point-of-care (poc) lateral flow immunoassays (lfia) such as alltest -ncov igg/igm rapid test. the results determined suitable time and time dependency between diagnosis and seroconversion using the four poc rapid tests. the seroconversion was different for getting covid- with pneumonia compared to patients without pneumonia symptoms. during antibody development and detection in the blood, it was shorter for the disease infected people with lung infections than without pneumonia. the proposed method of developing antibodies demonstrated its efficiency on covid- infected people with pneumonia, whereas other methods have to test for patients without pneumonia to reduce seroconversion. considering the observations, the response time was three weeks for developing antibodies and became detectable in the blood, and also serological testing could be a detection method for diagnosing patients with covid- . korth et al. [ ] investigated antibody development on three categorize of healthcare workers, including high-risk-group, medium-risk-group, low-risk groups that the classification was based on their contact percent with covid- infected people. the result demonstrated increasing during the time of seroprevalence for intermediate-risk-group compared to the high-risk-group, whereas the study is not a definitive result for deciding about requirement time to develop antibodies in various groups of healthcare workers. the environmental, nutrition, and local hygiene standard factors can affect the probability of getting the disease or developing antibodies. the case study proved that the antibody is not still the definitive prevention method against covid- , according to the impact of different parameters in determining the required time for antibody development and becoming detectable in the blood. jacofsky et al. [ ] studied the impact of background information about the covid- antibody on appropriately and responsibly returning to work and community activity. the case study investigated three types of antibodies founded in plasma and extracellular fluid, including secreted antibodies into the blood and mucosa, facilitate phagocytosis, and antibody activation of the complement system. the antibodies' function is different and effectively strengthens the various parts of the body's immune system. the time of seroconversion has an impressive effect in determining the body's immune system's resistance for different persons that this case is not still definitive. nevertheless, the case study illustrated the positive impact of information about the antibodies in deciding social distancing and personal protective equipment. also, this work can help to risk-based classifying workforce members, such as people with background diseases. maqbool et al. [ ] proposed an evaluation method to analyze the health resources for implementing social distance and public health. the case study classified public health and social distance implementations barriers based on the decision-making trial and evaluation laboratory (dematel) method. the health resources have a significant effect on implementing and managing social and health measures against covid- , such as medical equipment, personnel, and financial dealings. people's awareness of the disease can help to social distance and self-caring that lack of commitment from public or public resilience would increase transmission rates. this work analyzed the impact of the barriers factors (such as lack of safety commitment from the public, poor safety culture, and lack of door to door services during the quarantine period) in implementing prevention measures in the form of statistical calculations. abel et al. [ ] found a relationship between social distance ( meters) and individual and social health care. the case study's results demonstrated strict health protocols by reducing the determined social distance. also, countries' cultural and economic infrastructures significantly affected social distancing's rules, quarantine, and self-caring. the tolerating and accepting the rules were harder for countries' people with poor conditions, including weak economies, weak infrastructures, etc. hence the probability of getting covid- was increased in the countries with inappropriate cultural and economic infrastructures. huynh [ ] examined the role of cultural and demographic factors regarding social distancing rules for facing the virus outbreak using drawing the data from the reported information from google covid- community mobility. the result demonstrated that some cultural habits were recognized as the barriers of implementing social distance such as people gathering in public locations of retail and recreation, grocery and pharmacy, parks, transit stations, workplaces. the case study added the control variable as the wealth status and gross domestic product (gdp) per capita and suggested some effective communications to face the disease epidemic by emphasizing uncertainties. this work utilized two databases of the google trackback and the extended hofstede to collect dataset about the gathering of people in the cities' public location to alert people regarding social distancing. feng et al. [ ] investigated the effective environmental factors in the spread of covid- to provide social distance according to these factors. the case study determined the safe distance margin between any two defined longer -foot people due to different environmental wind velocities and ambient relative humidity (rh). this work employed a validated computational fluid-particle dynamics (cfpd) model for providing the guidelines of social distancing by simulating the transient transport, condensation/evaporation, and deposition of the virus-laden droplets emitted by coughs. according to the dependency between unstable environmental factors in the covid- outbreak, the safe distance margin is not still definitive for accurate determining social distancing that self-caring methods (such as people wear masks and other face coverings) have to consider for preventing and facing the disease epidemic besides regarding social distance. jia et al. [ ] traced population migration between china's cities to investigate the impact of traveling to wuhan from the time the virus identification using a mobile network. the case study demonstrated the negative effect of migration between cities at risk of the virus outbreak on increasing the probability of getting the disease and death caused by penetrating the virus's respiratory system and pneumonia. this work can help to provide prevention methods (quarantine and social distance) against the virus outbreak according to the critical and normal situations of the cities that are determined due to received information from the mobile network. the study proposed an artificial intelligence-based social distancing method using the online monitoring situation of the cities. oosterhoff et al. [ ] investigated the direct relationship between social distancing and effective parameters such as age range using multiple linear regression. the case study proved unique associations between adolescents' motivations, such as engaging in a perceived amount of social distancing, anxiety symptoms, depressive symptoms, burdensomeness, and belongingness, and regarding social distancing. the result demonstrated that accepting social distance and quarantine rules is challenging for adolescents according to their anxiety, depressive, burdensomeness, and belongingness symptoms. this work proposed an approach that includes understanding adolescents' motivations to engage in social distancing to solve the challenges caused by defined constraints of social distance and quarantine. tuli et al. [ ] proposed an improved mathematical model to analyze and predict the cities' situation to alert people and the government about the disease epidemic. the case study utilized machine learning-based methods to predict the potential threat of covid- in countries worldwide and achieved a better fit to develop a prediction framework using generalized inverse weibull distribution. the case study's observations and results are deployed on the cloud computing platform for accurate and real-time predicting the disease epidemic's growth situation. this work also presented an excellent study platform to create opportunities and setup grounds for further practical applications. the study achieved statistically better predictions than the gaussian model using the robust weibull model based on iterative weighting. meinzen-dick et al. [ ] studied the dependency between jobs, sessions, physical proximity requirements, and rural meetings habits such as forming groups for irrigating in nepal and india. the case study proposed employing information communication technologies (ict) and educating villagers to utilize ict instead of physical proximity in quarantine and social distance rules in the spread of covid- . nevertheless, the observations demonstrated inequality in ict accesses, which it is not restricted to poor and rich categorizations and consists of different between employing it in women and men. this inequality was dependent on economic and ethnic cultures that were challenging for regarding social distance's restrictions. schneider et al. [ ] focused on different methods and online resources available for distance learning as an approach regarding quarantine and social distancing' rules to prevent getting infected with the disease. distance learning positively impacted therapeutic aids for reducing going patients to the hospital and the probability of the virus transmission investigated on dermatology trainees with sharing online learning resources. young et al. [ ] evaluated the impact of various technologies on reducing physical communications and social distancing such as telemedicine, social media, mobile applications (apps), chatbots, and biosensors/wearables. the case study considered the particular rules and different social distancing restrictions for risky patients such as hiv in facing covid- . this work prioritized providing remote services based on the disease (such as patients with hiv and patients with different cancers) and habitats (city, metropolitan, and village). the risky patients prioritize getting distance medical advice compared to normal patients (such as patients with common cold) due to their higher sensitivity against covid- . garg et al. [ ] provided a state-of-the-art the internet of things (iot)-based approach for tracing the contact to follow the moving infection cases, including vehicles, moving things, animals, and patients with covid- or vectors without symptoms, which can lead to transfer the virus to others and epidemic the disease. this work utilized radio frequency identification (rfid)'s highlight role in determining risky locations and managing the spread of covid- by alerting people via cellphone-based applications and social media. it was not restricted to identify automated digital contact tracing using mobile service-provider application and their law employing. the case study presented a new iot and blockchainbased commotional method for monitoring moving risky cases and reported information from the different stations for managing the virus outbreak by considering personal privacy. hanumanthu et al. [ ] presented deep learning and machine learning-based approaches to diagnose and predict the situation of the covid- outbreak to inform the governments and people for deciding about management and control of the virus outbreak and self-caring. the study compared the efficiency of other approaches based on statistic, mathematical, and machine learning algorithms to forecast and predict the disease epidemic. this work demonstrated that more information about people's background diseases (chronic obstructive pulmonary diseases, chronic heart failure, diabetes mellitus, and kidney failure) could help present the forecast and predict highly accurate models. a. mujawar et al. [ ] examined biosensor tools' efficiency in low-level detection and prediction of covid- to manage the disease epidemic and decide about confronting methods. employing the nanotechnology-based detection and prediction tools is affordable, rapid, selective, and sensitive for managing health care against the virus at now and a nextgeneration non-invasive disease diagnostics methodology. according to the case study result, utilizing personalized healthcare management-related analytical tools provided access to better health for everyone. also, this study has been useful the employing applications based on the internet of medical things (iomt), artificial intelligence (ai), and iot for bioinformatics analysis, rapid detection, and prediction. david et al. [ ] examined the impact of social distance and quarantine approaches against the disease epidemic on patients with obsessive-compulsive disorder (ocd). the result included investigating symptoms of patients with ocd before and after quarantine using the yale-brown obsessive-compulsive scale (y-bocs) test that observations demonstrated increasing the severity of symptoms after quarantine. the case study proposed various methods to reduce the severity of symptoms for patients with ocd during quarantine, such as online consultations and digital psychiatric management. this work explained different effective parameters, including lack of psychiatric visits during the quarantine, the inadequacy of the living physical space, or financial concerns caused by the covid- pandemic in deteriorating severity for patients with ocd. tan et al. [ ] proposed a method to investigate the positive impact of the target management and psychotherapy on people's mental and physical health in the workplace in the spread time of covid- . psychological parameters reduced people's immune system resistance against the disease that are included event scale-revised, depression, anxiety, stress scale- (dass- ), and insomnia severity index (isi). the case study demonstrated no remarkable difference in the psychiatric symptoms between workers/technicians and executives/managers. this work presented the negative parameters in the severity of psychiatric symptoms, including marital status, physical symptoms, poor physical health, and viewing return to work in the workforce. reducing psychiatric symptoms was caused by employing confidence instilled by psychoneuroimmunology (pni) prevention measures before the resumption of work, as shown in this work's observations. our reviews classify three categories of treatment, prevention, and effective parameters in the spread of covid- in level one of the taxonomy tree analyzed the treatment methods in subsection . . we categorize the prevention methods and define the studies' characteristics, including the main context, advantage, weakness, and new finding, as shown in table . the main context of the examined prevention studies mostly focused on social distancing methods due to lack of providing definition approaches for therapy and antibody against covid- and its significant effect in reducing the probability of getting the disease and transmission. table demonstrates the evaluation parameters in introducing the ability of the presented studies in the prevention category. the parameters consist of definitive prevention method, detection ability, prediction ability, time overhead, and negative impact. the reviewed prevention methods most investigated the effective parameters regarding quarantine and social distance rules and their positive and negative effects. contreras et al. [ ] investigated the different effective parameters in the spread of covid- , such as geographical, behavioral, or economic factors, different sub-groups among a population. this work proposed a multi-group seira-based general method to illustrate heterogeneous populations' impact on the virus outbreak and test it numerically. the case study opened the horizon of vision about the effective parameters in the covid- outbreak that can help people better understand the disease epidemic's evolution and better public-health policies to control it. the observations and numerical results demonstrated a significant effect of individuals interact that is affected worldwide health emergency. the information about the ways of the virus transmission can help the governments provide the control and management approaches to face the disease epidemic and people's self-caring. chakraborty et al. [ ] determined the various types of restricted commutations and migrations affected by the spread of covid- , such as limiting large gatherings and maintaining complete or partial lockdown, etc. one possible way of the virus transmission is transferring via different types of wildlife such as civet, bat, wolf pup, pangolin, etc. that its purchase, sale, and trade are temporarily prohibited. the case study also illustrated the positive impact of the covid- outbreak on improving natural environmental health caused by implementing the rules of social distancing, quarantine, and restricting trade and changing wild animals' environment. yang et al. [ ] proposed a susceptible-exposed-infectious-removed (seir) and artificial intelligence-based method to investigate the migration's impact on the spread of covid- using collecting a dataset of population migration data before and after january . the case study demonstrated that removing the quarantine restrictions led to the second peak of hubei's disease. seir-prediction model has estimated the probability of starting the next peak of the disease outbreak, in which governments can be utilized for applying restriction rules and quarantine before being in alert mode. aguilar et al. [ ] investigated the transmission's role of asymptomatic carriers and patients without acute clinical symptoms in the spread of covid- . they proposed minimizing people's in-town or out-of-town migrations approaches, including quarantine, social distance, and lockdowns to guidelines in individual and social health behaviors. the study presented a mathematical model for achieving a numerical reporting about the migration's impact of asymptomatic carriers and patients without acute clinical symptoms in the virus outbreak that was reduced the risk of their transmissions. huff et al. [ ] presented a people's screening method based on providing periodical tests with priority placed on groups with high-frequency exposure to positive patients (such as health care workers) according to the migration's negative impact of asymptomatic carriers and patients without acute clinical symptoms. the results of periodical tests and observations demonstrated that asymptomatic cases' pcr tests were almost positive and had a highlighted role in the virus outbreak compared to patients with clinical symptoms. varga et al. [ ] studied the probability of covid- outbreak in different workplaces. also, they estimated the percent of the virus outbreak by asymptomatic carriers or patients with various symptoms that were identified as common clinical symptoms, including fever, dry cough, and tiredness. the asymptomatic carriers and patients with different clinical symptoms contributed to the spread of the virus, which observations demonstrated their highlight role in health centers. screening healthcare workers reduced effective health workforces and health services to patients, which remains a challenge. according to the high risk of asymptomatic carriers between healthcare workers, screening in health centers reduced the virus outbreak's probability and was required. guan et al. [ ] focused on the role of cultural factors and psychological perspective in the covid- outbreak. they analyzed them to understand better and manage the proposed methods to face the disease epidemic. the case study included the national culture influencing collective actions and norms, integrating insights from cultural psychology, and orientating internalized culture such as thinking style, regulatory focus, and values. the study aims to analyze the role of national culture in applying to other cultural settings. some personal and social-cultural behaviors have an impressive effect on the spread of covid- . the harmful individual and social behaviors consist of prioritizing personal interests over public benefits and defying collective coping actions, such as escaping from quarantine, hoarding essential hygiene products, or concealing health information. velraj et al. [ ] studied various environmental parameters in the spread of covid- and their role in the disease epidemic. the observations demonstrated increasing the probability of getting the virus via airborne droplet nuclei. the study proved air condition's highlight role in the virus outbreak that cold and dry weather had an impressive effect in the spread of covid- , according to investigate the relationship between the weather condition and increasing the rate of patients in india. according to the weather's significant effect on transferring the virus, this work presented heating, ventilation, and air conditioning (hvac) systems approach against the covid- outbreak with recommending the suitable relative humidity in the range of %- %. rahman et al. [ ] focused on providing a solution for facing and managing economic damages caused by quarantine against the virus outbreak. its highlight role in reducing the disease epidemic's probability compared to provide definitive antibody or treatment method. this work proposed a data-driven dynamic clustering framework for investigating damages and the quarantine's problems to manage the issue using analyzing uncooperative civilians' role in improving the rate of getting infected covid- . the case study identified lockdowns and reduce its coverage using dynamic clustering, which its impact was investigated in malaysia. the observation and experimental results demonstrated improving approximately % for lockdown coverage that was estimated based on employing the machine learning algorithm our reviews classify three categories of treatment, prevention, and effective parameters in the spread of covid- in level one of the taxonomy tree that are examined the treatment and prevention methods in subsection . and . , respectively. we classify the effective parameters in the spread of covid- to two categories of environmental and cultural factors and define the studies' characteristics, including the main context, advantage, weakness, and new finding, as shown in table . table illustrates the evaluation parameters in introducing the presented studies' ability in the disease epidemic category's effective parameters. the parameters consist of cultural factors, environmental factors, individual factors, prevention ability, and management ability. the reviewed effective parameters in the spread of the disease considered all the effective parameters that are led to the virus outbreak or reducing the epidemic probability. we compare the existing literature's based on treatment, prevention, and detection abilities in table , which is demonstrated the common abilities in the reviewed studies. in the previous sections, we explained the research method and reviewed the various case studies about covid- outbreak problems. this section discusses the issue and compares the categorized methods according to the proposed taxonomy tree using responding to the analytical questions presented in section . some of our responses are including:  aq : is the covid- outbreak a problem in all the world, and why? the disease epidemic is a problem worldwide due to the virus's specific feature and the lack of definitive treatments or antibodies to face the covid- outbreak. its lifespan and transmission ways lead to an epidemic worldwide and remain the challenge of confronting the virus outbreak. figure the classified prevention methods consist of facing, predicting, and deploying antibodies that figure shows the maximum attention of approximately % for recommending social distancing compared to antibody and prediction approaches. we estimate the efficiency percent of confronting methods due to the number of published papers in . their subcategories cover the evaluated parameters for determining a suitable method from three main classifications. figure demonstrates that researchers' attention is approximately %, %, and % for treatment, prevention, and effective parameters in the disease epidemic categories. our observations illustrate the importance of treatment methods compared to other approaches against covid- . however, social distancing has a significant role in confronting the virus transmission. nevertheless, quarantine and social distancing approaches depend on cultural, ethnic, and individual habits, and also their implementation in society can lead to psychological injuries. regardless of the issues, patients with covid- need a definitive therapy method to recover their health. providing a definitive antibody assures people about health safety against the virus in the future. so far, the researchers have not proposed a definitive method for patients' therapy or providing a definitive antibody to health safety guarantee against the disease in the future. the virus's specific characteristics lead to its sustainability in the environment worldwide and daily, increasing the number of patients with covid- , as shown in figure . as shown in figure and figure , covid- is a persistent and severe problem that can lead to people's deaths and economic crises worldwide. the virus's next-generation confirmed by who, and the researchers identified with new clinical symptoms, including the patients without symptoms or with different mild symptoms. growing up, death and new cases' daily statistics illustrate the dangerous and negative impact of the disease on humanity's mental and physical health and a big problem for governments worldwide. covid- was identified as an intelligence-based virus with mutability, which leads to a challenge for providing a definitive therapy or antibody. according to the reviewed case studies, the disease's damages and treatment injuries remain for year-ahead post-epidemic. these include economic damage, therapy's injury, physical health damages after recovery, and people's mental health problems (such as depression and traumatophobia ). therefore, this problem is an open issue and future scope to continue the research path to achieve a definitive confronting approach against covid- and improve the virus's effects after clearing the world. according to the observations, analyzing the reviewed studies, and covid- 's daily statistics, we mention the reasons for the issue as a future scope:  identifying and confirming the next generation of the virus without initial clinical symptoms or with different mild symptoms  observing the next peaks of the virus outbreak in different countries in all the world  providing a definitive treatment or antibody  challenging providing a definitive treatment or antibody caused by identifying the next generation of the virus with different characteristics  economic damages induced by the downturn, medical, health, and cleaning's costs in post-pandemic we collect a dataset of the related statistics to covid- include the number of patients, dead people, and recovered cases using online monitoring the digital stations that are reported daily information about the disease in all the world. we shared the dataset on github [ ] (https://github.com/yasamanhosseini/slr-covid- ). nowadays, covid- is a big problem for humanity's health and economy according to the negative impact of the virus on people's quality of life, leading to acute respiratory diseases, death, and financial crises worldwide. the virus's special characteristics lead to its sustainability and transmission between people in a long time and increase the probability of getting the disease for now and ahead years. the researchers proposed many different methods to face covid- . in contrast, they have not until achieved a definitive therapy approach or antibody against the disease since the aim of case studies is almost presenting the disease confronting method for reducing the negative impact of the consequences caused by the covid- epidemic and providing a survey review to manage the crisis induced by the outbreak whereas an overall review is invisible in the previous studies. we proposed the taxonomy tree for classifying the case studies to investigate the issue in various aspects. our work consists of a systematic literature review that was examined the studies based on the taxonomy tree for covering the issue from different angles. our observations demonstrated that researchers paid more attention to treatment and social distancing recommendation categories according to the number of published papers in these fields in . our slr can help people and government control and implement health advice and self-caring by informing them about the issue. background disease (cardiovascular) march parveen et al. [ ] background disease (diabetes and hypertension) june ortiz-prado et al. [ ] history of covid- may udugama et al. [ ] detection tools march bastos et al. [ ] detection with laboratory tests june katal et al. [ ] detection of the infections (ct) july ming-yen et al. [ ] detection of the infections (ct and radiologic) february haghani et al. [ ] health safety may egbert et al. [ ] covid- 's injuries july rodriguez-morales aj et al. 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covid- disease using dematel method the covid- pandemic calls for spatial distancing and social closeness: not for social distancing does culture matter social distancing under the covid- pandemic influence of wind and relative humidity on the social distancing effectiveness to prevent covid- airborne transmission: a numerical study population flow drives spatiotemporal distribution of covid- in china adolescents' motivations to engage in social distancing during the covid- pandemic: associations with mental and social health predicting the growth and trend of covid- pandemic using machine learning and cloud computing collective action and "social distancing" in covid- responses. agriculture and human values distance learning in the era of covid- clinical care, research, and telehealth services in the era of social distancing to mitigate covid- anonymity preserving iotbased covid- and other infectious disease contact tracing model role of intelligent computing in covid- prognosis: a 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case for healthcare worker screening to prevent hospital transmission. the lancet understanding the impact of the covid- pandemic on career development: insights from cultural psychology the contribution of dry indoor built environment on the spread of coronavirus: data from various indian states. sustainable cities and society data-driven dynamic clustering framework for mitigating the adverse economic impact of covid- lockdown practices. sustainable cities and society could fighting airborne transmission be the next line of defence against covid- spread?. city and environment interactions j o u r n a l p r e -p r o o f wu et al. [ ] ✗ √ ✗ √ ✗ korth et al. [ ] ✗ √ ✗ ✗ ✗ jacofsky et al. [ ] ✗ √ ✗ √ ✗ maqbool et al. [ ] √ ✗ ✗ ✗ √ abel et al. [ ] √ ✗ ✗ ✗ √huynh. [ ] √ ✗ ✗ ✗ √ feng et al. [ ] √ ✗ ✗ ✗ √ jia et al. [ ] √ ✗ √ ✗ √ oosterhoff et al. [ ] √ ✗ ✗ ✗ √ j o u r n a l p r e -p r o o f [ ] √ ✗ ✗ ✗ huff et al. [ ] √ ✗ ✗ ✗ varga et al. [ ] √ ✗ ✗ ✗ guan et al. [ ] √ ✗ ✗ ✗ velraj et al. [ ] √ ✗ ✗ ✗ rahman et al. [ ] √ ✗ ✗ ✗ key: cord- - fn ei f authors: hanania, nicola a.; king, monroe j.; braman, sidney s.; saltoun, carol; wise, robert a.; enright, paul; falsey, ann r.; mathur, sameer k.; ramsdell, joe w.; rogers, linda; stempel, david a.; lima, john j.; fish, james e.; wilson, sandra r.; boyd, cynthia; patel, kushang v.; irvin, charles g.; yawn, barbara p.; halm, ethan a.; wasserman, stephen i.; sands, mark f.; ershler, william b.; ledford, dennis k. title: asthma in the elderly: current understanding and future research needs—a report of a national institute on aging (nia) workshop date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: fn ei f asthma in the elderly is underdiagnosed and undertreated, and there is a paucity of knowledge on the subject. the national institute on aging convened this workshop to identify what is known and what gaps in knowledge remain and suggest research directions needed to improve the understanding and care of asthma in the elderly. asthma presenting at an advanced age often has similar clinical and physiologic consequences as seen with younger patients, but comorbid illnesses and the psychosocial effects of aging might affect the diagnosis, clinical presentation, and care of asthma in this population. at least phenotypes exist among elderly patients with asthma; those with longstanding asthma have more severe airflow limitation and less complete reversibility than those with late-onset asthma. many challenges exist in the recognition and treatment of asthma in the elderly. furthermore, the pathophysiologic mechanisms of asthma in the elderly are likely to be different from those seen in young asthmatic patients, and these differences might influence the clinical course and outcomes of asthma in this population. states is currently about % but is projected to grow from about million in to more than million by , accounting for % of the population. the age group with the largest growth will be those older than years, which is estimated to be more than million by . , in , the us prevalence of asthma for those years or older was %, with , , reporting an asthma attack in the previous months. older asthmatic patients are more likely to be underdiagnosed, undertreated, , and hospitalized than younger asthmatic patients. they also have the highest death rate ( . per million persons) of any other age group. older women are hospitalized more than twice as often as older men. asthma in older adults is superimposed on a background of aging-related changes in respiratory and immune physiology and often on multiple diseases and conditions common in older age. recognizing the paucity of research, the many challenges that exist in the recognition and treatment of asthma in older adults, and the opportunity to bridge geriatrics and the clinical specialties that focus on asthma, the national institute on aging (nia) sponsored a workshop on asthma in the elderly in herndon, virginia, on september and , . the workshop was planned by a committee of physician-scientists from us academic institutions or from the division of geriatrics and clinical gerontology in the nia. the planning committee selected speakers and participants for their expertise in asthma, pulmonology, allergy/immunology, primary care, emergency medicine, geriatrics, and/or gerontologic science (see the list of participants in appendix ). the immediate goals of this workshop were to summarize the current understanding of the mechanisms of asthma in older persons and to identify knowledge gaps and research opportunities leading to improved medical care and health outcomes for older persons with asthma. these research opportunities are discussed in the body of this report and summarized in table i . [ ] [ ] [ ] in addition, the nia, in collaboration with the national heart, lung, and blood institute and the national institute of allergy and infectious diseases, recently issued a set of program announcements inviting research proposals on asthma in older adults (http:// grants.nih.gov/grants/guide/pa-files/pa- - .html, http://grants. nih.gov/grants/guide/pa-files/pa- - .html, and http://grants. nih.gov/grants/guide/pa-files/pa- - .html). it is a central principle of gerontology that aging itself is not a disease. yet there are physiological changes within organs, tissues, and cells that result in diminished functional reserve and thereby increased susceptibility to stressors, disease, or both. a second principle is that these aging changes are highly variable and account for the great constitutional heterogeneity among older persons from very ''fit'' to very ''frail.'' in fact, the concept of frailty, both its causes and consequences, has become a focus of concentrated gerontologic investigation. at the root of age-associated physiological changes are a number of genetic, epigenetic, and environmental factors. molecular damage accumulates over time, and the capacity for dna repair decreases. cellular senescence, which is believed to be the consequence of accumulated dna and protein damage and reduced proliferative capacity, is becoming increasingly understood at the molecular level. however, just how this correlates with the phenotypic changes of advanced age remains incompletely understood. there has been much written about cellular senescence and the events that lead to cell death. [ ] [ ] [ ] after a finite number of divisions, normal somatic cells invariably enter a state of irreversibly arrested growth, a process termed replicative senescence. in fact, it has been proposed that escape from the regulators of senescence is the antecedent of malignant transformation. however, the role of replicative senescence as an explanation of organismal aging remains the subject of vigorous debate. the controversy relates, in part, to the fact that certain organisms (eg, drosophila species and caenorhabditis elegans) undergo an aging process, yet all of their adult cells are postreplicative. what is clear is that the loss of the proliferative capacity of human cells in culture is intrinsic to the cells and not dependent on environmental factors or even culture conditions. unless transformation occurs, cells age with each successive division. the number of divisions turns out to be more important than the actual amount of time passed. thus cells held in a quiescent state for months, when allowed back into a proliferative environment, will continue approximately the same number of divisions as those that were allowed to proliferate without a quiescent period. the question remains whether this in vitro phenomenon is relevant to animal aging. one suggestive observation is that fibroblasts cultured from samples of old skin undergo fewer cycles of replication than those from young skin. furthermore, when various species are compared, replicative potential is directly and significantly related to lifespan. an unusual b-galactosidase with activity peaks at ph has proved to be a useful biomarker of in vitro senescence because it is expressed by senescent but not presenescent or quiescent fibroblasts. this particular b-galactosidase isoform was found to have the predicted pattern of expression in skin from young and old donors, with measurably increased levels in dermal fibroblasts and epidermal keratinocytes with advancing age. the nature of the expression of this in vivo biomarker of aging in other tissues will be important to discern. for clinical investigators, frailty has proved hard to define primarily because of the seemingly insurmountable heterogeneity inherent in geriatric populations on the basis of these variable rates of organ system decrease and the presence or absence of or more diseases. yet, regardless of the pathway taken to frailty, the clinical picture has common features, including a reduction in lean body mass (sarcopenia), loss of bone mass (osteopenia), cognitive impairment, functional decline, and anemia. on the basis of data derived from large cohorts of elderly patients, fried et al have offered an operational definition of frailty incorporating an assessment of specific characteristics to ascribe a frailty index. on this -point scale, a score of or more has been shown to be independently predictive of a range of adverse clinical outcomes, including acute illness, falls, hospitalization, nursing home placement, and early mortality. [ ] [ ] [ ] furthermore, simple performance measures, such as the assessment of walking speed, are predictive of important outcomes, including survival. with the phenotype better defined, attention has shifted to pathophysiology. although frailty can occur in the absence of a diagnosable illness, the fact that some become frail and others do not suggests an inherent or acquired variability in homeostatic pathways. recent evidence from observational studies has raised suspicion that dysregulated inflammatory processes are involved in, if not central to, the variable patterns of aging. increased serum levels of certain proinflammatory cytokines, most notably il- , are increasingly present with advancing age and to a greater extent with frailty. , furthermore, the appearance of this and other inflammatory markers has been associated with a number of adverse clinical outcomes, including decreased strength and mobility, falls, dementia, and mortality. life expectancy, lifespan, and maximum survival from the perspective of those who study aging, there is an important distinction made between median (life expectancy) and maximum lifespan. over the past several decades, with the advent of modern sanitation, refrigeration, and other public health measures, including vaccination and antibiotics, there has been a dramatic increase in median survival. early deaths have been diminished, and more patients are reaching old age. in the united states today, life expectancy now approaches years. median survival is what concerns public health officials and health care providers, but for those studying the biology of aging, it is maximum survival that is the focus of greatest attention. it is worthwhile to note that it has been estimated that if atherosclerosis and cancer were eliminated from the population as a cause of death, about years would be added to the average lifespan, yet there would be no change in maximum lifespan. although several theories have been proposed, none suffice to account for the complexities of aging. lifespan is finite and varies generally from species to species and much less so within species. mice live, on average, ½ years, monkeys years, and human subjects about years. among species, larger animals generally live longer than smaller animals, but within species, smaller animals are likely to live longer. it is clear that aging is not entirely explained by dna sequence. for example, mice and bats have only . % difference in their primary dna sequence, but bats live for years, times longer than mice. a commonly held notion is that regulation of gene expression accounts for a longevity difference between species. the aging lung large, longitudinal, and more complete studies to determine the effects of aging on the function of the respiratory system improved knowledge about lung structure-function relationships in older age using techniques of imaging and measures of lung function not requiring effort (eg, high-resolution computed tomographic scanning and forced oscillation) improved assessment of lung processes underlying airflow limitation attributable to aging versus copd or asthma, especially in asthmatic patients who smoke studies to examine the effects of aging in ethnic groups and the role of gender epidemiology, effect, diagnosis, and management determine the true prevalence and cost of asthma in the older population develop a uniform definition of asthma to be applied to health care records that will distinguish asthma from copd and mixed asthma/copd evaluate evidence-based treatment algorithms for older asthmatic patients, such as those developed by the national heart, lung, and blood institute and global initiative for asthma guidelines assess the effect of asthma treatment, including direct medical costs of care, indirect costs of care, and value of treatment in improving quality of life , assess the effect of comorbid conditions, especially copd and congestive heart failure, on asthma characterize phenotypes of elderly asthma with regard to responses to therapy and long-term outcomes based on age of onset, duration of disease, and environmental triggers develop algorithms for electronic medical record systems that are asthma-specific evaluate effects of current asthma medications in older patients compared with younger patients identify pharmacogenetic determinants of response to asthma medications in older adults identify simpler and safer drug delivery systems and schedules for older adults develop simple methods to differentiate copd from asthma exacerbations in older adults understand how environmental or aging-related factors affect epigenetic changes in asthma in older adults identify differences between older and younger asthmatic patients or between lsa and loa with regard to inflammation, remodeling, intracellular mechanisms, responses to environmental pollutants, and allergy sensitization and their effects on the metabolism and action of asthma drugs identify naturally occurring age-related changes in airway cellular patterns develop animal models of age-related airway inflammation understand the significance of allergy sensitization associated with asthma in older adults (eg, through larger prospective studies) identify the utility of allergy tests, either skin tests or serum specific ige measurement, in reflecting allergy sensitization in older adults identify the role of the microbiome in patients with loa understand the role of non-ige mechanisms in older adults' inflammatory responses to inhalant allergens or pollutants (eg, t h lymphocytes producing il- or protease receptor responses to molds and dust mites) determine the roles of adaptive versus innate immune mechanisms on asthma development, progression, and response to treatment in older adults determine whether there are environmental pollutants peculiar to institutional settings identify viruses and other microbiological agents responsible for, and the mechanisms by which they cause, asthma exacerbations in older adults, which might lead to the development of vaccine-or antiviral drug-based interventions determine effects of asthma medications, viral or bacterial load, or allergy status on susceptibility to exacerbations in older patients define rates of infection and specific pathogens in older asthmatic patients distinguish roles of innate immunity in eosinophilic versus neutrophilic asthma it is now clearly established that certain specific genes can alter lifespan, at least in lower animals, but whether these same genes regulate ''aging'' is still in question. for example, transgenic drosophila species expressing increased copies of the free radical scavenging enzymes superoxide dismutase and catalase live on average a third longer than the appropriate controls. in even lower species (eg, yeast and nematodes) the identification of specific genes that influence lifespan , has led to the optimistic impression that analogous genes in higher organisms will lead greater insights into the aging process. yet the identification and functional analysis of analogous genes in human subjects remains elusive. the oldest human being alive today is approximately years old. what is intriguing is that the record has remained stable and unchanged by the public health initiatives mentioned above. in fact, there has been some recent data presented that the maximum survival is actually decreasing in the united states. , what is interesting is that, unlike the public health initiatives in human subjects in which median but not maximum survival has been enhanced, experimental interventions in lower species have resulted in prolongation of maximum survival. as mentioned above, transgenic drosophila species producing extra copies of superoxide dismutase and catalase survived about % longer than controls, and similarly, the maximum survival in c bl/ mice fed a calorically restricted diet enhanced by % or more. , the true mechanisms of aging might well be uncovered with a better understanding of how these interventions affect longer survival. future research in aging should attempt to improve our understanding of the basic biology of aging and interventions that retard the aging process. there is a need for the development and application of a standardized definition of frailty for future clinical investigation. investigations directed at the role of comorbidities in accelerating the aging process are important. furthermore, future research should focus on the development of cellular and animal models of typical, delayed, and accelerated aging and of large collaborative networks in which populations and resources can be shared to study aging and frailty. leveraging on well-characterized existing cohorts, when possible, is recommended. the lungs, like other organs, age and exhibit continued loss of function as a person grows old. lung function is traditionally assessed by means of a number of standardized methods. the most common measurement used is spirometry with the determination of fev and forced vital capacity (fvc). fev and fvc both show continuous decreases of between and ml with each year of life after about age years. the cause of this decrease is usually attributed to the loss of the driving forces for airflow as a result of reduced respiratory muscle performance, loss of static elastic recoil, or both. , the decrease in fev in asthmatic patients is largely a function of the decrease in fvc because of the increase in residual volume. stiffening of the chest wall and reduced respiratory muscle performance result in a decrease in total lung capacity and an increase in residual volume because of ever-increasing closing volume. accordingly, these aging processes lead to airflow limitation that might be hard to distinguish from an active disease process. not all older persons are able to perform spirometry, especially those with decreased cognition, coordination, and frailty. in addition, spirometry is effort dependent, and the very old can tire quickly. techniques of imaging and measures of lung function not requiring effort (ie, forced oscillation) should be used in future studies to extend our knowledge about lung structure-function relationships at the very end of life. bronchodilator responses are known to be less marked in the elderly, perhaps as a consequence of the aging effects attributed to the emphysema-like state of the senile lung ; however, this would not explain the slow temporal response to bronchodilators. other studies do not find such age-related bronchodilator differences. furthermore, although methacholine responsiveness has been reported to increase with aging, the exact mechanism for this is not apparent. increased incidence and prevalence of many lung diseases occur with age. alterations in immune function increase the risk of many of these diseases. studies of systemic immunity suggest that sustained antigenic stress over a lifetime leads to a decrease in naive t-cell numbers, an accumulation of memory t cells, and a decrease in t-cell repertoire and b-cell functions but a lesser decrease in innate immunity. , little is known about what happens to the immune/inflammatory pathways in older asthmatic patients. the immune system changes seen with aging will be discussed in more detail in the section on pathophysiology. in the united states the national health interview survey asks questions regarding lifetime history of asthma, current asthma prevalence, and asthma attacks in the last months. for all age groups, asthma prevalence has been steadily increasing since . for the years and older age group, asthma is consistently more prevalent in female than male subjects. the national center for health statistics tracks data on physician encounters for asthma. the national ambulatory medical care survey reported that those years or older have the second-highest rate of outpatient office visits after those aged to years. those years and older did not have significantly different emergency department visits than the other adult groups. the years and older age group accounts for a greater proportion of hospitalizations ( %) than the size of its population ( %) would indicate. not surprisingly, the elderly population is a high user of medical resources for the treatment of asthma. hospitalizations and emergency department visits are more common for these patients than for other adult cohorts. some of the increased costs are related to comorbid disease. for example, the presence of comorbid chronic obstructive pulmonary disease (copd) increases the risk of an asthma-related hospitalization in medicare patients . -fold, respiratory medical costs almost -fold, and total medical costs -fold. elderly female subjects appear at greater risk than elderly male subjects. [ ] [ ] [ ] [ ] asthma mortality increased steadily from until it peaked in . the highest mortality rates for asthma occur in the years and older group. in fact, the increase in asthma mortality between and was primarily driven by the years and older group. in addition, the decrease in asthma mortality between and was most evident in this age group. elderly women with asthma tend to have higher mortality rates than elderly men with asthma. one reason for the increasing prevalence of asthma in the elderly might be the improved longevity of the population. also, increased office visits for asthma in the elderly might be responsible for fewer attacks. increasing hospital admissions might account for decreased mortality. by continuing to gather surveillance data on asthma, reasons for these trends could become clearer. in addition, surveillance data help to focus intervention efforts in areas of greatest need. in the cardiovascular health study, a large community-based cohort of subjects older than years, questions were asked that were relevant to asthma and provided more insight into the prevalence and effect of asthma in this population. , , definite asthma was defined as a positive response to the questions indicating that the patient had current asthma and that a physician confirmed the diagnosis. probable asthma was defined as a history of wheezing in the past year associated with chest tightness or breathlessness. excluding smokers and those with a diagnosis of congestive heart failure, % of subjects had definite asthma, and % had probable asthma. among those who smoked, % had definite asthma, and % had probable asthma. among nonsmokers, subjects were identified who had definite or probable asthma; % were women, and % were older than years. the age of asthma onset was spread approximately evenly among decades. twenty-seven percent had late onset of disease after age years, and % had onset of disease before age years. as expected, respiratory symptoms in the older asthmatic subjects were more prevalent, with a -to -fold increase in cough, phlegm, wheezing, and dyspnea. dyspnea on exertion was . -fold more likely to be present in asthmatic patients than in those without the diagnosis. lung function was reduced in those with a diagnosis of asthma. mean fev was % of predicted value in those with definite asthma and % of predicted value in those with probable asthma compared with % in those who did not have asthma. forty-one percent of those with a diagnosis of asthma had airflow obstruction below the fifth percentile for the age group, and peak flow lability was increased. elderly asthmatic patients reported the most common trigger was a viral infection in % compared with animal allergies in %. two thirds reported seasonal worsening. asthma had a significant effect on quality of life, with % of patients with definite or probable asthma reporting a fair or poor health status compared with % of elderly patients without asthma. sixty percent of patients with definite asthma reported seasonal allergic rhinitis compared with only % in the nonasthma group. despite the high prevalence and morbidity of asthma in this population, inadequate treatment was common. only % of those with definite asthma had a rescue albuterol inhaler, and only % had inhaled corticosteroid use. , , , the pathophysiology of asthma in the older adult is poorly understood and understudied. many questions about this issue remain: is asthma the same disease in older adults as it is in children and younger adults? is late-onset asthma (loa; asthma that starts in middle age or older) different from longstanding asthma (lsa; asthma of early onset that has persisted into older adulthood)? if loa and lsa are the same disease, then the diagnosis and treatment should be similar. however, if loa and lsa are different phenotypes or at least have a different cause and pathophysiology, then the diagnosis and treatment might differ (table ii) . the traditional view of disease susceptibility has been expanded to include epigenetics to account for the influence of environmental factors and aging on the genomic blueprint. epigenetics is defined as heritable changes in gene expression that occur without alterations in dna sequence. it is the process by which genotype interacts with environment to produce a phenotype and explains differences between cells, tissues, and organs despite identical genetic information. genes function in a milieu determined by the developmental and environmental history of the cell, which constitutes the epigenotype. , epigenetic changes or marks can play a major role in human disease. , the most common examples of epigenetic marks are dna methylation of cpg islands by dna methyltransferases and chromatin modification of histone proteins, particularly acetylation by histone acetyltransferases and histone deacetylases. , the function of epigenetic changes is to regulate gene expression. epigenetic changes are known to contribute to cancer and autoimmune disease and are thought to contribute to common diseases, including cardiovascular disease, diabetes, and the loss of response to stress caused by aging. asthma is a markedly heterogeneous disease, and recent evidence suggests that environmentally induced epigenetic changes contribute to asthma phenotypes and that airway inflammation in patients with asthma and copd might involve epigenetic regulation. , , methylation patterns and chromatin structure change with age and are thought to contribute to the increase in the incidence of common diseases that begin in middle age. , the incidence of asthma in the elderly resembles the incidence of common diseases. moreover, characteristics and asthma drug response in the elderly asthmatic patient differ from those seen in childhood asthma. compared with younger cohorts, elderly asthmatic patients have a higher prevalence, higher rates of bronchial hyperreactivity, more severe asthma, and a lower prevalence of atopy. the symptoms of elderly asthmatic patients are more difficult to control with drug therapy, and these patients have steroid resistance and might respond better to leukotriene receptor antagonists compared with inhaled corticosteroids. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the contribution of epigenetics to differences observed between elderly asthmatic patients and younger cohorts is unknown. unlike genetic variants that contribute to disease, epigenetic changes can be reversed and therefore represent potential drug targets. older asthmatic patients are less responsive to albuterol treatments given in the emergency department and are more frequently admitted for hospitalization. thus it appears that the responsiveness to treatment is diminished and the severity of asthma exacerbations is greater. the exact reason for these disparities is not known. immune cell function decreases with aging, a property often termed immunosenescence. one often-confusing aspect of immunosenescence is the observation that aging might be associated with opposing immunologic effects. for example, t-cell secretion of il- , il- , or ifn-g has been shown to be both decreased with aging and also increased with aging. it is likely that both phenomena are correct but are dependent on the context of the immune function. thus the effect of aging on t-cell function in the context of allergen stimulation might be different than the effects of aging on t-cell function in the context of viral infection. given that asthma is an inflammatory disorder of the airway, it is of interest to determine whether asthmatic airway inflammation of the elderly might differ from that of younger asthmatic patients and thus represent a distinct phenotype of asthma. these changes might have implications for susceptibility to exacerbations because of viruses or other pathogens, as well as response to treatment. the aging process has been shown to exhibit changes in airway inflammation. an examination of the cellular composition of bronchoalveolar lavage fluid from -to -year-old subjects without a history of allergies, pulmonary disease, or gastroesophageal reflux showed increased airway neutrophilia, as well as increased numbers of cd t cells. , the t cells also appeared to be more activated in the elderly, with increased expression of hla-dr and cd . the increase in airway neutrophils with aging has also been observed in asthmatic patients. because there is a phenotype of severe asthma characterized by a predominantly neutrophilic airway inflammation, the question arises as to whether the increased presence of neutrophils contributes to greater asthma severity in the elderly. some of the prominent inflammatory cells recruited into the airway in asthmatic patients are eosinophils, neutrophils, and t cells, which are capable of secreting numerous inflammatory mediators, including leukotrienes and cytokines. it is not known whether immunosenescence affects the production of these mediators in elderly asthmatic patients, either at baseline or during an exacerbation of symptoms. furthermore, it is not known whether age-related changes in their production would have any implication for the clinical presentation or management of asthma in the elderly. peripheral blood eosinophils were isolated from younger ( - years old) and older ( - years old) subjects for in vitro functional assays. the eosinophil effector functions of degranulation and superoxide production were diminished in the older compared with the younger asthmatic patients. in another study examining the expression of neutrophil mediators in younger and older asthmatic patients, there was decreased baseline expression of leukotriene b in the sputum of older asthmatic patients despite greater numbers of neutrophils. whether these findings have implications during an asthma exacerbation has yet to be determined. nevertheless, the results demonstrate agerelated changes in the function of an inflammatory cell considered pathognomonic for allergic asthma and raise the question of whether additional effects of immunosenescence are relevant to airway inflammation in asthmatic patients. there have been several studies of animal models to address age-related changes in the airway inflammation induced by allergen challenge of sensitized aged animals (see experimental approaches section below). these studies yielded conflicting results, and there is concern that the animal models do not accurately represent the chronic features of human asthma with seasonal allergen exposure and intermittent exacerbations. the aged animals were both sensitized and challenged at old age, which is in contrast to the typical elderly human asthmatic patient who might be exposed to allergens for several decades. typically, nasal and ocular symptoms on exposure to allergens diminish with age. allergen-triggered asthma symptoms also diminish with age. the epidemiology and natural history of asthma (tenor) study examined the natural history of asthma in older (> years old) compared with younger patients and found that older asthmatic patients had lower total ige levels, fewer positive skin prick test responses, and less concomitant allergic rhinitis or atopic dermatitis. several studies have demonstrated age-related decreases in total ige and allergen-specific ige levels, [ ] [ ] [ ] [ ] [ ] [ ] suggesting that this might be the explanation for the decrease in allergy symptoms. there is also evidence for an age-related decrease in skin prick test responses to allergens. however, the relationship between total ige levels and allergic disease persists in the elderly, such that subjects with greater ige levels remain more likely to have allergic rhinitis or asthma. , given the changes in allergic inflammation with aging, one might conclude that asthma in the elderly should be milder. however, there are several other common triggers for exacerbations of asthma, including irritants (eg, cold air) and respiratory tract infections. estimates suggest that up to % of asthma exacerbations in adults are caused by viral upper respiratory tract infections. the role of environmental exposures and allergy in older asthmatic patients is largely unknown. in the general population, evidence regarding the effect of indoor pollution on asthma is summarized in ''clearing the air: asthma and indoor air exposure'' by the institute of medicine for the environmental protection agency published in . evidence was reported for asthma development related to house dust mites and for asthma development associated with environmental tobacco smoke in preschool children. the report also showed evidence for causation of asthma exacerbations for house dust mite, environmental tobacco smoke (in preschool children), cat, and cockroach; an association with exacerbations was found for dogs, fungi, formaldehyde, and rhinovirus. in addition, evidence associating exacerbation of asthma-related symptoms with self-reported damp air was reported in a review of damp indoor spaces and health. , there are only a few studies that have evaluated the role of atopy in elderly patients with asthma. one large national study of allergy skin tests that included older adults and several small studies of allergy skin tests in older adults with asthma , - j allergy clin immunol volume , number were reviewed. allergy skin test results were positive in % to % of all older adults. the prevalence of positive skin test results or specific ige levels to at least allergen in older adults with asthma ranged from % to %. those whose asthma had an unknown age of onset ranged from % to %, those with onset before age years ranged from % to %, and those with onset greater than age years ranged from % to % (table iii) . , , although there were no studies of allergen room exposure or bronchial challenge in older adults, neither prick-puncture skin test results nor specific ige levels predicted the nasal challenge response to dust mites. safety concerns for allergen challenges in older adults are unresolved. technical limitations of allergens, environmental measurements, and age-specific norms and cutoff levels for laboratory and physiologic tests are needed. a few epidemiologic studies suggest an association between outdoor environmental exposures and emergency department or hospital admissions in older adults. , in summary, studies of the general population suggest a causation or association between indoor air pollutants and allergy exposure and asthma. there are several small studies suggesting higher levels of positive allergy test results in older adults with asthma than in the general population of older adults. when age of onset is considered, asthma with an early onset (< years of age) has a much higher association with positive allergy test results than late-onset asthma. viral respiratory tract infections are common precipitants of asthma exacerbations during childhood. in approximately % of children with acute asthma exacerbations, a respiratory tract virus can be detected, with rhinovirus being the most frequent pathogen identified. although it is likely that viruses also lead to exacerbations of asthma in older adults, comprehensive studies regarding the rates and specific pathogens are lacking. several issues have made defining the role of viruses in adult asthmatic patients problematic and include difficulty distinguishing copd from asthma, lack of sensitive diagnostic tests, and issues with asymptomatic infection. a number of investigators have explored the incidence of viral infection in adult asthmatic patients. , , older studies using viral culture and serology for diagnosis demonstrated infection rates of % to %. in contrast, more recent studies, which include rt-pcr, have shown significantly higher infection rates of % to %. , similar to results found in children, rhinoviruses are the most frequently detected pathogen. few older persons were included in these studies, in which the mean ages of subjects were to years. the incidence of acute respiratory tract infections decreases steadily with advancing age, and rates of viral infection in older adults are influenced by place of residence. among community-dwelling older adults, rates of acute respiratory tract infections are roughly % to % per year, whereas rates in senior day care centers and long-term care facilities are substantially higher at % to %. in addition, the epidemiology of respiratory tract infections can be quite complex in these semiclosed populations, with multiple pathogens circulating simultaneously. influenza a, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) are the most commonly identified viruses among older persons hospitalized with acute cardiopulmonary conditions. , most patients who require hospitalization during a viral respiratory tract infection have underlying heart and lung conditions. although studies to date have largely focused on the role of viruses in copd exacerbations, it is reasonable to extrapolate infection rates and specific pathogens from these studies to older adults with asthma. johnston, in ontario, canada, found a seasonal peak in emergency department visits for all acute respiratory tract infections, as well as exacerbations of both copd and asthma, for persons younger and older than years. all the common respiratory tract viruses have been associated with copd exacerbations, and depending on the methodology and season of study, the specific rates of influenza, rsv, parainfluenza viruses, coronaviruses, hmpv, and rhinoviruses vary. , wheezing appears to be a common symptom in older adults infected with any of the respiratory tract viruses, particularly rsv and hmpv, and % of adults hospitalized with rsv will have a discharge diagnosis of asthma. , because most adult infections represent reinfection, the viral load in respiratory secretions tends to be low, making detection with conventional techniques difficult. viral culture and rapid antigen testing, which can be used successfully in children, have poor sensitivity in older adults. the use of molecular diagnostics has vastly improved the ability to detect a number of viruses, such as rsv, parainfluenza, and rhinoviruses, and allows the detection of hitherto uncultivable agents, such as hmpv and coronaviruses. viral infections appear to aggravate reactive airway disease through a number of different mechanisms. it has been postulated that viral infection disrupts the negative feedback loop of acetylcholine on the m receptor, leading to increased levels of acetylcholine and increased constriction of bronchiolar smooth muscle. infection of the respiratory epithelium also induces chemokines, cytokines, and immune and growth factors, which result in a proinflammatory state. , immunosenescence might affect the ability of older adults to clear viruses efficiently, and thus greater and more prolonged inflammation can result. in summary, respiratory viral tract infections are common among older adults and are likely precipitants of acute asthma exacerbations. furthermore, viral respiratory tract infections might likely precipitate the onset of loa, although this needs to be further examined. comprehensive studies regarding the rates and specific pathogens are lacking in older adults. distinguishing copd from asthma, lack of sensitive diagnostic tests, and issues with asymptomatic infections make it difficult to define the role of infections in older adults. classic symptoms of asthma in the elderly are mostly similar to those seen in younger asthmatic patients. , data on the clinical features of asthma in the elderly have been derived from both longitudinal community surveys and case studies. , , , , [ ] [ ] [ ] most patients complain of episodic wheezing, shortness of breath, and chest tightness. these symptoms are often worse at night and with exertion and, like those in younger asthmatic patients, are often precipitated by an upper respiratory tract infection. in fact, the majority of elderly patients who have asthma after age years have their first asthmatic symptom preceded immediately by or concomitant with an upper respiratory tract infection. asthma can often be triggered by environmental exposures, such as aeroallergens, irritants (cigarette smoke, household aerosols, and paints), strong odors (perfumes), and inhalation of metabisulfites (found in beer, wine, and food preservatives). asthmatic symptoms can also be triggered by medications, such as aspirin, nonsteroidal anti-inflammatory agents, angiotensin-converting enzyme inhibitors, or b-blockers, which are commonly used by this patient population. this emphasizes the need for the physician to perform a comprehensive review of medications taken by the older asthmatic patient. studies have consistently shown that elderly patients and their physicians frequently overlook symptoms caused by asthma. , , several factors contribute to the underdiagnosis and misdiagnosis of asthma. one reason, as shown in large community studies, is that most patients first have asthma in childhood or adolescence, and many physicians have had the misconception that asthma is a childhood disease. another important reason is that the symptoms of asthma are more commonly associated with other diseases seen in this age group. the symptoms of asthma in the elderly are therefore nonspecific and might be caused by conditions that mimic asthma. the differential diagnosis of asthma in the elderly is greater than seen in younger asthmatic patients and includes congestive heart failure, emphysema and chronic bronchitis (copd), chronic aspiration, gastroesophageal reflux disease (gerd), and tracheobronchial tumors. comorbid illnesses and the psychosocial effects of aging might also profoundly affect the diagnosis, clinical presentation, and care of asthma in the elderly. one particular diagnosis that is often difficult to detect and frequently overlooked by the patient and physician until the condition is advanced is upper airway obstruction, including the extrathoracic and intrathoracic central airways. common causes of upper airway obstruction include malignancy, infection, inflammatory disorders, trauma, and extrinsic compression related to enlargement of adjacent structures (eg, an enlarged thyroid gland). it appears that malignancy and benign strictures related to airway instrumentation (eg, endotracheal intubation and tracheostomy) are becoming increasingly more prevalent in the older age group. distinguishing chronic asthma from copd can be very challenging, and in some patients asthma cannot be distinguished from copd with widely available diagnostic tests. the management of these patients might have similarities to that of asthma. the distinction between loa and copd can be difficult to define precisely. the lung health study showed that methacholine-induced airways reactivity is present in many patients with mild-to-moderate copd (ie, % of men and % of women). approximately % of patients with tobacco-related copd demonstrate bronchodilator reversibility at least once on repeated testing sessions. the distinction between copd and asthma can be confounded by either the coexistence of the common disease entities, the progression of common pathobiologic mechanisms induced by different environmental agents, or different disease mechanisms leading to an overlapping clinical syndrome. it has been known for more than a century that early-morning wheezing is a prominent symptom of congestive heart failure. it has been called cardiac asthma because it can mimic the clinical picture of typical asthma. the usual symptoms of gastroesophageal reflux in the elderly, such as vomiting and heartburn, might be absent. in a study of elderly patients with esophageal reflux proved by means of intraesophageal ph monitoring, chronic cough, hoarseness, and wheezing were present in % of patients. in addition to causing asthma-like symptoms, there is also evidence that gerd might be a cause of worsening asthma. shortness of breath is a common symptom in the elderly and is most commonly caused by heart or lung diseases. it is usually experienced during exertion. shortness of breath at rest is not typical of heart disease or lung diseases, such as copd or interstitial lung disease, except in advanced stages. when present, it should prompt an investigation for asthma because sudden bronchospasm can cause respiratory distress at rest or exercise. paroxysmal nocturnal dyspnea, which is typical of congestive heart failure, is found in a smaller number of elderly patients with asthma. many elderly patients limit their activity to avoid experiencing dyspnea, and others assume that their dyspnea results from their aging process and thus avoid seeking medical attention early in their disease process. however, aging per se does not cause dyspnea, and a cause needs to be always pursued in assessing an elderly patient who complains of breathlessness. there are several other reasons why the diagnosis of asthma in the elderly might be delayed or not made at all. elderly patients have been shown to have a reduced perception of bronchoconstriction, and this might delay medical intervention. many elderly patients are fearful of having an illness and dying and are reluctant to admit they are having symptoms. underreporting of symptoms in the elderly might have many causes, including depression, cognitive impairment, social isolation, denial, and confusing symptoms with those of other comorbid illnesses. cough is a very prominent symptom and might occasionally be the only presenting symptom. wheezing, on the other hand, might not be as prominent, and its presence is not very specific and does not correlate with severity of obstruction. physical examination in elderly patients with asthma is usually nonspecific and might misguide the diagnosis: a negative examination result does not rule out asthma, and wheezing can be found in a number of conditions, such as copd, recurrent aspiration, and ''cardiac asthma'' (congestive heart failure). two distinct clinical presentations have been described for asthma in the elderly. these are based on the onset and duration of the disease state. , , patients with loa start having asthma symptoms for the first time when they are years of age or older (some studies have suggested middle age or older). some studies of elderly asthmatic patients have shown that, as a group, as many as % will have their first attack after the age of years. patients belonging to this group tend to have fewer atopic manifestations, higher baseline fev , and a more pronounced bronchodilator response than those with lsa. patients with lsa start having asthma symptoms early in life. patients belonging to this group tend to have a higher incidence of atopic diseases, more severe and irreversible or partially reversible airway obstruction, and more hyperinflation. the duration of the disease in this group is an important determinant of severity and of the development of irreversible airflow obstruction. longitudinal studies of asthmatic populations, whether new onset or long standing, have shown that remission from asthma is uncommon in older groups, occurring in less than % of patients. this contrasts with asthma in children and adolescents, in whom remission of asthma symptoms is common, especially in the second decade of life, and might be seen in as many as % to % of patients. objective measures to confirm the diagnosis of asthma are uncommonly performed in primary care settings. inhalers are prescribed for patients who are evaluated for asthma-like symptoms, and during a follow-up visit, the patient is asked whether the controller inhaler reduced the frequency of asthma symptoms or whether the albuterol inhaler quickly relieved the symptoms. such an empiric approach might work most of the time for young patients with mild asthma but is more likely to result in an incorrect diagnosis, poorly efficacious treatment, or unnecessary medication side effects in older patients. the onset of wheezing, shortness of breath, and cough in an elderly patient is likely to cause concern. although the adage ''all that wheezes is not asthma'' is true at any age, it is especially true in the elderly. diagnosis based on objective measures is essential. moreover, lung function testing, even in the presence of minimal symptoms, is especially important in this age group because there is thought to be an age-related reduction in the perception of exertional dyspnea in the elderly. an older patient with chronic, untreated, severe airway obstruction caused by asthma might reduce activity to avoid dyspnea and stoically deny impairment of activity. this might reflect either neurocognitive function or changes in lifestyle that favor sedentary activities. there exist some barriers to lung function testing in the elderly. spirometry might be difficult to perform in some situations because of physical or cognitive impairments. however, % to % of elderly persons are able to perform goodquality spirometry when tested by skilled technologists. [ ] [ ] [ ] [ ] [ ] the global initiative for obstructive lung disease guidelines for diagnosing the airway obstruction of copd by using a fixed fev /fvc ratio of less than . caused a high misclassification rate in older persons. however, almost all computerized spirometers automatically calculate the appropriate lower limit of the normal range for fev /fvc ratio and for fev by using race-specific national health and nutrition examination survey iii reference equations. in addition, it is hard to define the lower limits of predicted normal values in this age group. although complete reversibility of airflow obstruction is frequently seen with young asthmatic patients, most elderly asthmatic patients show incomplete reversibility despite continuous intense therapy, and many show fixed airflow obstruction as if they have copd. however, objective measures of lung function, such as spirometric and peak flow measurements, are generally underused in elderly patients, and this also contributes to the delay or absence of diagnosis. , lung function testing is especially important in this age group because of the age-related reduction in the perception of dyspnea seen in the elderly. spirometry is easily performed to determine that fev and fev /fvc ratio are demonstrated with the timed vital capacity maneuver. the flow-volume loop, which also measures inspiratory flow, is especially useful when the cause of respiratory tract symptoms is not known and an upper airway obstruction is in the differential diagnosis. although it might be difficult to perform spirometry in the elderly in some situations because of physical and poor cognitive impairment, studies have demonstrated that between % and % of elderly patients are able to perform the test properly. [ ] [ ] [ ] [ ] [ ] on the other hand, it might be more difficult to define the lower limits of predicted normal values in this age group. traditionally, an fev /fvc ratio of less than % increases the probability of asthma in an elderly patient with asthma symptoms, but this ratio normally decreases with age because of a decrease in elastic recoil, and a ratio lower that % might be a normal finding. a brisk response to a short-acting bronchodilator might demonstrate the second cardinal feature of asthma: reversible airflow obstruction (ie, ''a responder''). when airflow obstruction is found in an elderly patient, attempts should be made to demonstrate reversibility after the inhalation of a short-acting b-adrenergic agent, such as albuterol. evidence of reversibility (postbronchodilator fev or fvc increases of > % and ml) increases the probability of a diagnosis of asthma. elderly asthmatic patients, however, might have an impaired b-agonist bronchodilator response because the number of b-adrenergic receptors on smooth airway muscles is decreased with aging. although the bronchodilator response to inhaled b-agonists decreases with age, this is not the case with anticholinergic agents. airway obstruction might be absent at the time of testing, and further testing might be needed to facilitate the diagnosis. bronchoprovocation testing with a methacholine challenge can be useful, and it is a safe and effective method to uncover asthma in older adults. , a negative test result will rule out asthma; a positive test result must be interpreted and include an assessment of pretest probability. in addition, some studies have shown that bronchial responsiveness is heightened in older adults, and therefore aging might be an independent factor that influences airway responsiveness. there is a relationship between the degree of bronchial hyperresponsiveness and prechallenge pulmonary function; a low fev predicts heightened responsiveness. other factors that might contribute to heightened airway responsiveness in the older population are atopy and current or previous smoking history. peak expiratory flow variability might be helpful in the diagnosis and follow-up of younger patients with asthma, but poor coordination and muscle weakness in some elderly patients might lead to an inaccurate reading. , a prospective study did not demonstrate any advantage of peak flow monitoring over symptom monitoring as an asthma management strategy for older adults with moderate-to-severe asthma when used in a comprehensive asthma management program. other tests, such as measuring the carbon monoxide diffusing capacity of the lung, have been advocated to distinguish between asthma and copd because the diffusing capacity of the lung is reduced by parenchymal destruction found with emphysema. however, studies have shown that differences in lung function tests, although statistically significant, cannot be used clinically to separate the groups of subjects because of a large overlap. there is growing evidence that the airway function of young and middle-aged asthmatic patients decreases at a greater rate than that of healthy subjects. [ ] [ ] [ ] the rate of decrease increases with increasing age and in those who smoke cigarettes. , in patients with loa, there is evidence that lung function is reduced even before a diagnosis is made and decreases rapidly shortly after diagnosis. , thereafter, it remains fairly stable. although the effect on older asthmatic patients with lsa is variable, in a random survey of elderly asthmatic patients older than years, only in patients had normal pulmonary function (fev > % of predicted value), whereas a similar number showed moderate-to-severe airflow obstruction (fev < % of predicted value) after an inhaled short-acting bronchodilator. because structural changes of emphysema are minimal in elderly asthmatic patients, except if they are previous smokers, airway remodeling is thought to be the main cause of fixed airflow obstruction. nitric oxide (no) is a gas generated by the action of no synthase from the substrates molecular oxygen and arginine. it was originally identified as a biologically important signaling molecule with the properties of an activity previously described as endothelial-derived relaxing factor. this molecule is important in regulating vascular integrity and blood flow and is thought to be a regulator of vascular smooth muscle relaxation. more recently, it has been found that no can be generated by a variety of inflammatory cells, including polymorphonuclear leukocytes, mononuclear cells, and, importantly, eosinophils. this finding led to the identification of no as a molecule present in exhaled breath. studies of no exhalation have found that it is increased in infection and inflammation of the airway. although high levels of no are found in nasally expired air, studies in pulmonary inflammation have avoided this by redirecting airflow through the oral airway. it has been found that exhaled no reflects airways inflammation and particularly eosinophilic inflammation. exhaled no levels are increased during the allergy season in atopic subjects. inhaled glucocorticoids promptly suppress exhaled no and do so in conjunction with suppression of eosinophilic inflammatory infiltrates. studies have demonstrated that monitoring exhaled no might permit better regulation of asthmatic symptoms, exacerbations, and total steroid use than treatments based on guidelines or symptoms. furthermore, increases in exhaled no levels might predict asthma exacerbations. it is of interest that no levels in expired air decrease after bronchoconstrictive stimulation of asthmatic airways. little is known of the effects of age on no levels in the expired air. it appears that no production and vascular responses to no might be diminished in the elderly, but that effect might be overcome by exercise to increase fitness. an unanswered question in airways biology is whether no is causative of airways dysfunction, a marker for this dysfunction, or an ineffective homeostatic response to airways constriction. [ ] [ ] [ ] [ ] [ ] challenges in defining asthma in the elderly there is agreement that asthma is both a common and underrecognized health problem for the elderly that leads to impairments of lung function and quality of health and life. the first question that needs to be addressed is why we need to make such a diagnosis rather than just treat the symptoms. there are reasons that physicians must strive to assign a diagnosis to a patient with a symptom complex. the patient is given relief by letting him or her know what is wrong by giving the illness a name, which implies a cause, establishes a prognosis, and initiates a treatment plan. moreover, advancement of the understanding of epidemiology, natural history, pathobiology, and treatment require a definable disease entity. whether the threshold for diagnostic criteria is set at a high level of sensitivity, a high level of specificity, or a high level of accuracy depends entirely on the costs and benefits of an incorrect diagnosis versus a missed diagnosis. for example, enumeration of a disease might require a high level of accuracy, whereas diagnosis of an uncommon and difficult-to-treat disease (eg, metastatic cancer) ought to be highly specific. the diagnosis of a common and easily treatable disease (eg, vitamin deficiency) ought to be highly sensitive, even if there is a risk of overdiagnosis. asthma tends to be one of those disorders that is relatively easy (although not inexpensive) to treat and has morbid consequences if left untreated, suggesting that the diagnostic criteria ought to be highly sensitive. although medical students are taught the rigorous discipline of data collection, differential diagnosis, and test confirmation, most physicians do not practice this way. in practice, physicians typically rely on a constellation of signs and symptoms along with demographic characteristics and recent experiences to establish diagnoses through the process of pattern recognition. there are no shortages of official definitions of asthma, and modifications seem to be added every year. most of these definitions involve the definition of a clinical syndrome (episodic cough, wheezing, and dyspnea), an underlying pathophysiology (airway hyperresponsiveness, variable, and reversible airflow obstruction), an underlying biological process (chronic eosinophilic or neutrophilic inflammation of the airways), and an associated morbid anatomy (basement membrane thickening, smooth muscle hypertrophy, and mucus cell metaplasia). given this, why is it so challenging to diagnose asthma in the elderly? first, the syndrome of asthma is often confused with other common diseases in the elderly, such as copd, congestive heart failure, paroxysmal arrhythmias, pulmonary emboli, recurrent aspiration, and gerd. second, asthma can often coexist with these other conditions, and it can be impossible to determine which of the conditions is responsible for the patient's ill health. this diagnostic confusion can be amplified by the different manifestations of asthma in the elderly. elderly asthmatic patients can be insensitive to exertional dyspnea because of a sedentary lifestyle. they tend to be less atopic and have an incomplete response to bronchodilators. the elderly without asthma tend to show some signs suggestive of asthma: slower emptying of the lung during forced expiration, decreased lung elastic recoil, and a higher prevalence of nonspecific airways reactivity. the hope that formal testing of airways reactivity would prove useful in diagnosing asthma has led to disappointment. in young adults a history of asthma, wheeze, or treatment for asthma plus a positive methacholine challenge test result is highly specific for asthma ( %) but misses about half of the asthmatic population. in epidemiologic studies that have examined various criteria for diagnosing asthma, it turns out that the solution is relatively j allergy clin immunol volume , number simple. patients who answer yes to the question ''have you ever had asthma?'' have nearly % specificity and % to % sensitivity when compared with those receiving an independent expert's diagnosis. , the problem of diagnosing asthma in the elderly is more complicated because of the overlap with copd. asthma is typically considered a disease of onset in youth driven by atopy and eosinophilic inflammation causing reversible airflow limitation. copd, in contrast, is considered to be a disease of onset in middle age driven by cigarette smoking and neutrophilic inflammation and leading to irreversible airflow limitation. as evidence presented in this workshop has shown, asthma in the elderly displays many of the features of copd. the disease can have its symptomatic onset late in life, often is only partially reversible, and is associated with neutrophilic inflammation. moreover, the current cohort of elderly patients has a high prevalence of past smoking, reflecting the health habits in the united states in the s and s. the failure to deal with the population of elderly patients who have overlapping signs of asthma and copd is not just a matter of classification of disease. it has significant health consequences in that such patients are systematically eliminated from clinical trials and are not covered by treatment guidelines. little is known about how best to treat the elderly patient with asthma who smokes or the elderly patient with copd who has reversible airflow limitation. this confusion is manifest by diagnostic coding in older medicaid patients. of those who were hospitalized with an initial diagnosis of copd, % had an asthma diagnosis within years. of those who had an initial hospital diagnosis of asthma, % had a diagnosis of copd within years. price et al attempted to develop a discriminant function using clinical and demographic information that would separate patients with copd from those with asthma by using strict physiologic criteria. although several discriminating characteristics were found, the best diagnostic criteria were only % sensitive and only % specific. we need to ask whether it really is important to make the distinction between asthma and copd in the elderly in terms of prognosis or treatment. one study by hansen et al suggests that regardless of whether a person is given a diagnosis of asthma or copd, the prognosis is mostly determined by the impairment in fev . there are a number of ways to measure the effect of asthma in both young and elderly patients. assessments of symptoms, functional limitations, quality-of-life measures, and risk of adverse events are several that have been suggested by current asthma guidelines. in addition, measuring a patient's satisfaction with his or her asthma symptom control and overall asthma care has been advocated. the use of objective measures of asthma control and satisfaction can be especially important in the elderly because the perception of symptoms might be impaired with advancing age. in addition, many elderly patients unconsciously accommodate to their symptoms or assume that the symptoms are a function of the aging process itself. because the number of unscheduled ambulatory visits, emergency department visits, and hospitalizations are high in elderly asthmatic patients, , and quality-of-life scores are low in elderly patients with persistent asthma when compared with those with mild asthma or no asthma at all, careful assessment of asthma control is essential in this age group. despite severe symptoms and physiologic impairment, most elderly patients with asthma can lead active productive lives if their asthma is appropriately managed. in fact, when elderly patients with severe or difficult-to-treat asthma have been identified by a physician's assessment, they appear to do better than younger patients. in the tenor study, despite lower lung function, older asthmatic patients (mean age, years) had lower rates of unscheduled office visits, emergency department visits, and corticosteroid bursts. patients reported in the tenor study received more aggressive care than younger adults, including higher use of inhaled and oral corticosteroids, and this undoubtedly had an effect on outcomes. the tools to measure asthma outcomes include questionnaires and other self-report tools, such as diaries and standardized medical history forms. standardized questionnaires that assess asthma impairment include the asthma control test, , the asthma control questionnaire, , the asthma therapy assessment questionnaire, , and others. [ ] [ ] [ ] [ ] there are many tools available to clinicians to assess the quality of life of asthmatic patients. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] unfortunately, these psychometric instruments that claim to measure the same outcomes might produce disparate results, and none have been targeted for the elderly. in general, results that measure several domains are more accurate when a composite score is derived rather than when subscores of specific domains are compared. medical, administrative, and pharmacy records have also been used, especially to study larger asthmatic populations; these have proved useful for the assessment of a patient's change over time and to measure group differences. clinical trials of asthma therapy and educational, selfmanagement, and health services interventions have used psychometric instruments to assess elderly patients with asthma. in most of these studies, however, the majority of subjects are younger. there are no studies that have specifically determined the reliability and validity of these instruments in elderly persons. this is true of patient-satisfaction measures that have been used to assess asthma care. [ ] [ ] [ ] this is much needed because using lung function testing to measure outcomes has potential limitations in this age group. there are difficulties in defining normal predicted values at a very advanced age, and many patients with physical or cognitive impairment cannot reliably perform these tests. it is hopeful that newer biomarkers of lung inflammation have a particular role to play in the assessment of asthma control in the elderly. tools assessing physical function: self-reported and objective a major goal of geriatric and gerontologic research is to reduce the decrease in cognitive and physical function and prevent disability among older adults. accordingly, many functional status measures have been developed and used to understand the disabling process, as well as to evaluate interventions to prevent functional decline. it is useful to identify instruments that measure functional limitations and disability to investigate the functional consequences of asthma in older adults and to understand the pathway from asthma to disability. functional limitations are restrictions in performing basic physical and mental actions at the whole-person level (eg, walking or climbing stairs), whereas disability refers to limitations or difficulty in performing socially defined roles or tasks of everyday living in a given environmental context (eg, grocery shopping or bathing). , both self-reported and objective measures can be used to measure these different stages of disablement. self-reported measures can provide an indication of how well a patient is functioning in daily life and provide an assessment of care needs. these measures incorporate self-perception of function and can assess adaptations made to compensate for decrements in function. for disability assessment, self-reported difficulty or inability to perform basic activities of daily living (adl) is commonly used. for example, a composite score of adl items has been used as an outcome to evaluate the efficacy of a program to prevent functional decline in frail older adults. other composite scores assessing difficulty in ambulation, stair climbing, transferring, upper extremity function, and basic and instrumental adls have been developed. these comprehensive instruments of function and disability are amenable to computer adaptive testing. several objective measures of physical performance are used in studies of older adults and in disease-specific patient populations. tests of physical performance eliminate subjective attitudinal differences in the patient's reporting of physical function limitations. they have the advantage of providing an objective measure for comparisons across populations. these tests are sensitive to change over time and can detect decrements in function that might not be observed with self-reported instruments. many studies in older adults have used physical performance tests as predictors of adverse health events, as well as outcomes. for example, the short physical performance battery, which consists of timed balance, walking, and chair-rise tasks, is a powerful predictor of disability, nursing home admission, and mortality. , the short physical performance battery was also used as a screening instrument to identify functionally limited older adults and as an outcome in a randomized controlled trial of exercise. increasingly, objective measures of physical function are used to summarize the effect of total disease burden, including subclinical conditions and impairments, and to identify physiologic reserve that might help some older adults cope with disease burden. clinically meaningful differences have been established for commonly used performance measures. the goals of asthma therapy in elderly patients are not different from those for younger asthmatic patients. they are to treat acute symptoms, prevent chronic symptoms, decrease emergency department visits and hospitalizations, preserve normal activity level, and optimize pulmonary function with a minimal adverse effect from medications. , , optimal management should also focus on improving health status (quality of life) in these patients, which is often complicated by depressive symptoms and side effects from the drugs commonly used for asthma. unlike many younger adults who might require no medication or just asneeded b-agonist therapy for occasional symptoms, most older asthmatic patients need continuous treatment programs to control their disease. at a time when memory loss is common and financial resources are often limited, many older patients require complicated and frequent dosing with multiple expensive drugs. unfortunately, this has led to a significant rate of noncompliance among the elderly population in general. sex, socioeconomic factors, educational level, marital status, and severity of disease do not seem to be good predictors of compliance in elderly asthmatic patients. in summary, there are many challenges in the treatment of asthma in the elderly, which include a greater propensity to experience adverse events from medication use, as well as potential drug interactions with medications used for the treatment of comorbidities, , and thus it is particularly important to treat any disease in the elderly, including asthma, with a minimum of therapy while attaining maximum efficacy. a thorough understanding is required regarding which medications will be most effective in the treatment of asthma in the elderly to achieve this balance. because many current therapeutic options and those in development for asthma focus on specific inflammatory cells and mediators, any age-related changes in the airway inflammatory milieu will likely affect their therapeutic efficacy. therefore a rigorous characterization of age-related changes in airway inflammation will facilitate the management of asthma in the elderly. the therapeutic approach to asthma in elderly patients does not differ from what is recommended for young patients. statements on the standard of care for treating asthma have been published by the national institutes of health and are widely used as guidelines. , treatment protocols use step-care pharmacologic therapy based on the intensity of asthma symptoms and the clinical response to these interventions. as symptoms and lung function worsen, step-up or add-on therapy is given. as symptoms improve, therapy can be stepped down. in this age group special attention should also be given to the potential adverse effects of commonly used medications. corticosteroids are capable of reducing airway inflammation, thereby improving lung function, decreasing bronchial hyperreactivity, reducing symptoms, and improving overall quality of life. oral corticosteroids should be avoided if possible because they place the patient at risk for bone fracture and increased likelihood of cataracts, muscle weakness, back pain, bruising, and oral candidiasis. many studies have shown that inhaled corticosteroids are safe and effective treatment for persistent asthma, but none have specifically targeted the elderly population. long-term use of inhaled corticosteroids has been associated with a good safety profile, but higher doses of inhaled steroids (eg, > mg/d) are capable of causing hypothalamicpituitary-adrenal axis suppression. local adverse effects, such as hoarseness, dysphonia, cough, and oral candidiasis, do occur but can usually be avoided by the use of a spacer or holding chamber with the metered-dose inhaler and by rinsing the mouth after each use. despite the pivotal role of inhaled corticosteroids in asthma, many elderly patients are undertreated with this group of medications. , leukotriene-modifying agents (ltms) are also asthma controllers. these agents have been shown to be effective in preventing allergen-induced asthma, exercise-induced asthma, and aspirin-induced bronchospasm. studies on their use in the elderly are limited. when compared with ltms, low-dose inhaled corticosteroids have favored the latter. the ltms might also reduce asthma exacerbation rates and the need for steroid bursts. the ltms are generally very safe. , b-adrenergic agents are important medications in the acute and chronic management of asthma. elderly patients with asthma j allergy clin immunol volume , number might be less responsive to certain bronchodilators compared with younger patients. , inhaled short-acting b -adrenergic agonists are the treatment of choice for the acute exacerbation of asthma symptoms. despite the minimal systemic absorption seen with these agents, slight tachycardia might be observed. this is presumably because of vasodilatation, which results from the stimulation of b -receptors in vascular smooth muscle. tremor can also occur and is especially troublesome in the geriatric patient. tremor is thought to be caused by stimulation of b -receptors in skeletal muscle. in general, they have been proven to be safe and effective in all age groups. however, b-agonists can cause ( ) a dose-dependent decrease in serum potassium levels and ( ) a dose-dependent increase in the qt interval on electrocardiography. because sudden death from ventricular arrhythmia can be caused by both of these mechanisms, as well as being a complication of ischemic heart disease, the use of b-agonists in the elderly should be closely monitored. short-acting b -agonists should be used for rescue of symptoms, whereas long-acting agents should be used as maintenance medications only as an add-on to inhaled corticosteroids and never as stand-alone therapy. anticholinergics, such as inhaled ipratropium, a short-acting bronchodilator, and tiotropium, a bronchodilator with -hour action, have an excellent safety profile in the elderly. they should be considered when additional bronchodilator therapy is necessary; however, their role in long-term maintenance of asthma in the elderly has not been established. theophylline is an effective bronchodilator and has some antiinflammatory properties. however, its use has been greatly reduced over the past decade because of safety concerns, especially in the elderly. the narrow therapeutic range of theophylline, the frequency of concomitant illnesses that alter theophylline kinetics, and many drug interactions that affect the clearance of theophylline make it essential to closely monitor blood theophylline levels in older asthmatic patients. theophylline toxicity can cause seizures and cardiac arrhythmias, such as atrial fibrillation, supraventricular tachycardia, ventricular ectopy, and ventricular tachycardia. the most common cause for theophylline toxicity is a self-administered increase in medication. controlling triggers. measures should be taken to avoid triggers that can cause worsening of symptoms. as with asthma at any age, education concerning avoidance of aggravating factors that can lead to severe bronchospasm is very useful. although aeroallergens are less important in provoking symptoms in the elderly than in young patients, a program implementing environmental control measures, such as avoiding or minimizing aeroallergen exposure, should be instituted in patients with documented sensitivity to specific allergens. however, such programs might not be successful in all cases, especially because lifestyle changes in the elderly population might be difficult. the most important provocative factors include viral respiratory tract infections and irritants, such as cigarette smoke, paints, varnish, and household aerosols. pharmacologic agents that are often prescribed for concomitant illnesses (ischemic heart disease and hypertension), such as b-adrenoreceptor antagonists (b-blockers), can also provoke bronchospasm. this includes both noncardioselective agents (propranolol, pindolol, and timolol) and, to a lesser extent, cardioselective agents (metoprolol and acebutolol). topical b-blockers are also widely used in the elderly to reduce intraocular pressure in wide-angle glaucoma. with such treatment, sufficient systemic absorption might cause fatal status asthmaticus. the severity of b-blocker-induced bronchoconstriction correlates with the severity of underlying airflow obstruction and the degree of bronchial reactivity and might be reduced by the use of a cardioselective topical b-blocking agent, such as betaxolol. aspirin and nonsteroidal anti-inflammatory agents might precipitate acute bronchospasm in certain asthmatic patients, and angiotensin-converting enzyme inhibitors might cause dry cough in some, worsening the symptoms of asthma. gerd should also be considered a cause of worsening asthma symptoms. asthma education. the complexity of the prescription regimen (number and frequency of medications), coupled with the memory loss and cognitive dysfunction that might be present in this group of patients, contribute partially to poor compliance with therapy. , patient education is an effective tool and should be an integral part in the management of asthma. active participation by a patient and family members in monitoring lung function, avoidance of provocative agents, and decisions regarding medications provide asthma management skills that give that patient the confidence to control his or her own disease. mastering the technique of an inhaled medication delivery device is a challenging problem in elderly patients, and the great majority of elderly patients are unable to properly use the metered-dose inhaler, even after proper instruction. [ ] [ ] [ ] [ ] use of dry powder devices, although simpler, requires the generation of an adequate inspiratory flow that might be suboptimal in frail patients and those with severe airway obstruction. in such situations the use of spacer devices or nebulizers might be beneficial. patients should recognize the rationale behind using the different medications, the correct way to use them, and their side effects, and polypharmacy should also be avoided. asthma in the elderly can be effectively managed, and despite severe symptoms and physiologic impairment, most patients can lead active and productive lives. a demographic study of low-income elderly persons in chicago found that ( %) without a previous diagnosis of asthma or emphysema had symptoms compatible with those of obstructive lung disease. of patients with a previous diagnosis, only % were compliant with medications, and this was largely due to the cost of medications. in addition, health care use was high in this population. telephone intervention offers a simple option in the management of elderly patients with asthma. it has been shown that asthma care by means of telephone triage of adult asthmatic patients can lead to a higher percentage of asthmatic patients being reviewed at less cost per patient and without loss of asthma control when compared with usual routine care in the outpatient clinic. however, it has not been determined whether such an intervention could improve asthma care specifically in persons aged years or older. the following study was designed to evaluate this question. fifty-two elderly asthmatic patients who used their rescue inhalers more than twice a week and had at least emergency department or urgent care visit in the previous year were randomized to an intervention or control group. all patients received telephone calls over a -month period. the intervention group received an asthma-specific questionnaire, and the control group received a general health questionnaire. medication use and health care use were evaluated at the beginning and end of a -month period. the study was completed by control and intervention subjects. baseline data were similar in both groups. after months, % (n ) of the intervention group was taking an inhaled corticosteroid compared with % (n ) of the control group. the intervention group had fewer emergency department visits when compared with the control group. sixtyfour percent (n ) of the intervention group had an asthma action plan compared with % (n ) of the control group. this study provides evidence that using a simple telephone questionnaire can successfully improve asthma care in the elderly. by empowering the elderly with the appropriate knowledge regarding their asthma, an appropriate discussion about their asthma care can be initiated with their primary care physicians. pulmonary rehabilitation. although pulmonary rehabilitation is recommended as the standard of care for patients with copd, there are only a few studies that evaluate the benefit of rehabilitation for asthmatic patients, and none of these consider elderly asthmatic patients. one study looked at the effects of a -week outpatient rehabilitation program for asthmatic patients after years. [ ] [ ] [ ] they found that of subjects continued to exercise regularly all years; there was a decreased number of emergency department visits and a decrease in asthma symptoms. further studies are needed to assess empowerment strategies for elderly patients with asthma, as well as the potential benefits of pulmonary rehabilitation on morbidity and mortality. asthma pathogenesis is complex and incompletely understood. research into the pathophysiologic mechanisms is made more difficult by multiple factors, including the heterogeneity of the disease itself, variable presentations in different stages of life, and the lack of highly relevant animal models. [ ] [ ] [ ] [ ] [ ] in the last decade, increasing interest in asthma in the elderly has triggered more intensive investigation in both human and animal systems by using ever more sophisticated immunologic methodologies. early investigation with rats revealed a lack of total and allergenspecific ige in response to ovalbumin. this was born out by several later in vivo studies. [ ] [ ] [ ] igg subset analysis (igg vs igg ) provided further support for this phenomenon. igg , correlating in the mouse to a t h response (vs igg [t h ]) was shown to follow a similar pattern. , recent studies [ ] [ ] [ ] of cytokine profiles in aged rodents compared with young control animals enhanced the paradigm that age resulted in less robust t h cytokines, particularly il- , il- , and il- , in favor of t h gene and protein expression. , this pattern was not fully supported in a recent chronic murine asthma model wherein il- was greater in aged sensitized mice, making the picture more complex. ifn-g, a key t h cytokine, has been consistently overexpressed in aged versus young rodents. [ ] [ ] [ ] [ ] eosinophilia, which is considered a key component of (allergic) asthma, was more pronounced in younger versus older animals (bronchoalveolar lavage fluid, lung tissue, or both) after most, [ ] [ ] [ ] although not all, sensitization paradigms. molecular genetics and t-cell subset analysis has allowed further insight into possible mechanisms underlying the waning t h response observed in most models. [ ] [ ] [ ] specifically, elderly mice appear to have more memory t cells, less activated cd t h cells, and less activated monocytes. , resident goblet cells also appear to express upregulation of mucin and mucin gene expression. a key to the impaired t h response was recently found in the gata pathway. elderly mice do not phosphorylate components of the extracellular signal-regulated protein kinase/mitogen-activated protein kinase pathway, resulting in lack of downstream signaling with gata , with subsequent impairment of promoter regions for key t h cytokines, including il- . this could be an overarching explanation for many findings in the elderly asthmatic patient, including less ige (il- and il- are needed for opening switch regions for ige production); il- and il- are highly associated with airway hyperreactivity, and il- is associated with eosinophil activation, survival, and, to a lesser extent, trafficking. finally, airway hyperreactivity has been universally found to be greater in young versus aged animals. [ ] [ ] [ ] [ ] the mechanisms might be complex, including both an altered key cytokine milieu and alterations in muscle function at the muscarinic receptor level. [ ] [ ] [ ] clinical and translational research research into the pathogenesis of asthma in recent years has led to the discovery of a number of novel, potentially important targets for the development of new treatment options. much of this research has focused on t h lymphocyte-driven processes underlying allergic asthma and its characteristic eosinophilic airway inflammation. abundant information supporting this research has been derived from bronchial biopsy and bronchoalveolar lavage studies largely carried out in a young adult population. it is recognized, however, that the role of allergy and allergic triggers in asthma diminishes with age. , in addition, loa is often less reversible, more severe, and frequently occurs in response to a viral respiratory tract infection. a distinct asthma phenotype characterized by normal airway eosinophil numbers has been described. moreover, normal airway eosinophilia might also be associated with abnormal sputum neutrophilia. , recent studies have shown that neutrophilic asthma might be associated with activation of innate immune pathways in contrast to the adaptive immune response associated with t h -mediated allergic asthma. thus alternative immune pathways involving natural killer t or t h lymphocyte subtypes have been hypothesized as being potentially important in the pathogenesis of asthma, particularly in adult-onset asthma. , just as the discovery of t h -related pathways has led to important leads in drug discovery for allergic asthma, further clinical research into these alternative pathways should be carried out with the goal of identifying new and exciting targets for future drug discovery. this research should focus not only on the discovery of new molecular targets but also on the identification of noninvasive biomarkers that will help predict the success of any new therapy in an individual patient. asthma is an important disease in the older adult, affecting % of the population older than years, which is understudied and frequently underdiagnosed. there are data to suggest that asthma in older adults is phenotypically different from that in young patients, with a potential effect on the diagnosis, assessment, and management in this population. this workshop brought together many disciplines to further our current understanding, resolve gaps in knowledge, and explore future areas of research and education. table i lists specific areas in need of research and study. the coming acceleration of global population ageing the census bureau on prospects for us population growth in the twenty-first century. population and development review national surveillance for asthma-united states asthma in the elderly: current knowledge and future directions underdiagnosis and undertreatment of asthma in the elderly. cardiovascular health study research group increasing u.s. asthma mortality rates: who is really dying? quality of care for older adults with chronic obstructive pulmonary disease and asthma 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disease murine models of asthma modeling allergic asthma in mice: pitfalls and opportunities promise and pitfalls in animal-based asthma research: building a better mousetrap van der straeten m. the effect of age on ige production in rats decreased expression of th type cytokine mrna contributes to the lack of allergic bronchial inflammation in aged rats induction and maintenance of airway responsiveness to allergen challenge are determined at the age of initial sensitization impaired gata -dependent chromatin remodeling and th cell differentiation leading to attenuated allergic airway inflammation in aging mice effect of ageing on pulmonary inflammation, airway hyperresponsiveness and t and b cell responses in antigen-sensitized and -challenged mice failure of aged rats to accumulate eosinophils in allergic inflammation of the airway age differences in cholinergic airway responsiveness in relation with muscarinic receptor subtypes effect of ageing on nicotine-induced contraction of guinea-pig bronchial preparation effects of age on muscarinic agonist-induced contraction and ip accumulation in airway smooth muscle total serum ige is associated with asthma independently of specific ige levels. the spanish group of the european study of asthma analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin- innate immune activation in neutrophilic asthma and bronchiectasis persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease il- is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines fla co-chair: nicola a. hanania, md, ms section of pulmonary and critical care medicine asthma clinical research center baylor college of medicine houston, tex members sidney s. braman, md warren alpert medical school, brown university division of pulmonary, critical care, and sleep medicine rhode island hospital providence, ri carol saltoun we thank the nia for recognizing the need for this workshop, especially susan nayfield, md, who convened the workshop and provided tremendous support in moving this research field forward; evan hadley, md, the director of the division of geriatrics and clinical gerontology; and basil eldadah, md, who continued the work of dr nayfield and contributed valuable advice and encouragement to the authors in completing these proceedings. key: cord- - ra uda authors: snowden, frank m. title: emerging and reemerging diseases: a historical perspective date: - - journal: immunol rev doi: . /j. - x. . .x sha: doc_id: cord_uid: ra uda summary: between mid‐century and , there was a consensus that the battle against infectious diseases had been won, and the surgeon general announced that it was time to close the book. experience with human immunodeficiency virus/acquired immunodeficiency syndrome, the return of cholera to the americas in , the plague outbreak in india in , and the emergence of ebola in zaire in created awareness of a new vulnerability to epidemics due to population growth, unplanned urbanization, antimicrobial resistance, poverty, societal change, and rapid mass movement of people. the increasing virulence of dengue fever with dengue hemorrhagic fever and dengue shock syndrome disproved the theory of the evolution toward commensalism, and the discovery of the microbial origins of peptic ulcer demonstrated the reach of infectious diseases. the institute of medicine coined the term ‘emerging and reemerging diseases’ to explain that the world had entered an era in which the vulnerability to epidemics in the united states and globally was greater than ever. the united states and the world health organization took devised rapid response systems to monitor and contain disease outbreaks and to develop new weapons against microbes. these mechanisms were tested by severe acute respiratory syndrome in , and a series of practical and conceptual blind spots in preparedness were revealed. in the long contest between humans and microbes, the years from mid-century until marked a distinctive era. in those euphoric decades, there was a consensus that the decisive battle had been joined and that the moment was at hand to announce the final victory. almost as if introducing the new period, the us secretary of state george marshall declared in that the world now had the means to eradicate infectious diseases from the earth. marshall's view was by no means exceptional. for some, in the early postwar years, the triumphant vision applied primarily to a single disease. the heady goal arose first of all within the field of malariology, where the rockefeller foundation scientists fred soper and paul russell thought that they had discovered in ddt (dichlorodiphenyltrichloroethane) a weapon of such unparalleled power that it would enable the world to eliminate the ancient scourge forever. with premature confidence in , russell published man's mastery of malaria ( ), frank m. snowden in which he envisaged a global spraying campaign that would free mankind from malaria -cheaply, rapidly, and without great difficulty. rallying to russell's optimism, the world health organization (who) adopted a global campaign of malaria eradication with ddt as its weapon of choice. the director of the campaign, emilio pampana, elaborated a one-size-fits-all program of eradication through four textbook steps -'preparation, attack, consolidation, and maintenance' ( ). russell's followers alberto missiroli, the director of the postwar campaign in italy, and george macdonald, the founder of quantitative epidemiology, reasoned that so signal a victory over mosquitoes could be readily expanded to include the elimination of all other vector-borne tropical diseases, ushering in what missiroli called a contagion-free eden, where medicine would make man not only healthy but also happy ( ) ( ) ( ) . if malariologists, who dominated the international public health community, launched the idea of the final conquest of infectious diseases, it rapidly developed into the prevailing orthodoxy. e. harold hinman, chief malariologist to the tennessee valley authority and member of the who expert committee on malaria, extrapolated from the conquest of malaria to the conquest of all contagion in his influential work world eradication of infectious diseases ( ) . aidan cockburn, a distinguished epidemiologist at johns hopkins and advisor to the who, gave expression to this new creed in his revealingly titled work the evolution and eradication of infectious diseases ( ) . as cockburn noted, '''eradication'' of infectious disease as a concept in public health has been advanced only within the past two decades, yet it is replacing ''control'' as an objective' ( ) . although not a single disease had yet been destroyed by his time of writing in , cockburn believed that the objective of eradication was 'entirely practical,' not just for individual illnesses but for the whole category of communicable diseases. indeed, he argued, 'it seems reasonable to anticipate that within some measurable time, such as years, all the major infections will have disappeared' ( ) . by that time, he explained, 'the major infections of today should have disappeared, and only remaining should be their memories in textbooks, and some specimens in museums. . . . with science progressing so rapidly, such an end-point is almost inevitable, the main matter of interest at the moment is how and when the necessary actions should be taken' ( ) . cockburn's timetable of total eradication by was, in fact, too slow for some. just a decade later, in , the australian virologist and nobel laureate frank macfarlane burnet went so far as to proclaim, together with his colleague david white, that 'at least in the affluent west,' the grand objective had already been reached. 'one of the immemorial hazards of human existence has gone,' he reported, because there is a 'virtual absence of serious infectious disease today' ( ) . the who also saw the entire planet as ready to enter the new era by the end of the century. meeting at alma ata in , the world health assembly adopted the goal of 'health for all, ' ( ) . what could possibly have led to such overweening confidence in the power of science, technology, and civilization to vanquish communicable disease? one factor was historical. in the industrialized west, rates of mortality and morbidity from infectious diseases began to plummet in the second half of the th century, in large part as a result of 'social uplift' -dramatic improvements in wages, housing, diet, and education. at the same time, developed nations erected the solid fortifications of sanitation and public health: sewers, drains, sand filtration, and chlorination of water as defenses against cholera and typhoid; sanitary cordons, quarantine, and isolation against bubonic plague; vaccination against smallpox; and the first effective 'magic bullet' -quinine -against malaria. meanwhile, improvements in the handling of food, pasteurization, retort canning, and the sanitation of seafood beds, yielded major advances against bovine tuberculosis (tb), botulism, and a variety of food-borne maladies. already by the early th century, therefore, many of the most feared epidemic diseases of the past were in headlong retreat for reasons that were initially more empirical and spontaneous than the result of the application of science. science, however, soon added new and powerful weapons. the foundational work of louis pasteur and robert koch had established the biomedical model of disease that promoted unprecedented understanding and yielded a cascade of scientific discoveries and new sub-specialties (microbiology, immunology, parasitology, and tropical medicine). the dawn of the antibiotic era with penicillin and streptomycin provided means to treat syphilis, staph infections, and tb. the development of a series of vaccines dramatically lowered the incidence of smallpox, pertussis, diphtheria, tetanus, rubella, measles, mumps, and polio. ddt seemed to furnish a means to abolish malaria and other insect-borne pathogens. by the s, therefore, scientific discoveries had provided effective weapons against many of the most prevalent infectious diseases. extrapolating from such dramatic developments, many concluded that it was reasonable to expect that communicable diseases could be eliminated one at a time until the vanishing point was reached. indeed, the worldwide campaign against smallpox provided just such an example when the who announced in that the disease had become the first ever to be eradicated by intentional human action. those who asserted the doctrine of the conquest of infection viewed the microbial world as largely static or only very slowly evolving. for that reason, there was little concern that the victory over existing infections would be challenged by the appearance of new diseases for which humanity was unprepared and immunologically naive. falling victim to historical amnesia, they ignored the fact that the last years even in the west had been punctuated by the appearance of a series of catastrophic new diseases: bubonic plague in , syphilis in the s, cholera in , spanish influenza in - . macfarlane burnet in this regard was typical. burnet was a founding figure in evolutionary medicine who acknowledged, in theory, the possibility of the emergence of new diseases as a result of mutation. but, in practice, he believed that such appearances are infrequent and that they occur only at such distant intervals as to occasion little concern. 'there may,' he wrote, 'be some wholly unexpected emergence of a new and dangerous infectious disease, but nothing of the sort has marked the last fifty years' ( ) . the notion of microbial fixity, that the diseases that we have are the ones that we will face, even underpinned the international health regulations adopted in (ihr ) , which specified that the three great epidemic killers of the th century were the only diseases requiring notification: plague, yellow fever, and cholera. the regulations gave no thought to what action would be required if an unknown but deadly and transmissible new microbe should appear ( , ) . if belief in the stability of the microbial world was one of the major articles of faith underpinning the eradicationists' vision, a second misplaced evolutionary idea also played a crucial role. this was the doctrine that nature was fundamentally benign. over time, eradicationists believed, the pressure of natural selection would drive all communicable diseases toward a decline in virulence. the principle was that excessively lethal infectious diseases would prevent their own transmission by prematurely destroying their hosts. the long-term tendency, the proponents of victory asserted, is toward commensalism and equilibrium. new epidemic diseases are virulent almost by accident as a temporary maladaptation, and they therefore evolve toward mildness, ultimately becoming readily treatable diseases of childhood. examples were the evolution of smallpox from variola major to variola minor; the transformation of syphilis from the fulminant 'great pox' of the th century into the slow-acting disease of today; and the transformation of classic cholera into the far milder el tor biotype. similarly, the doctrine held a priori that, in the family of four diseases of human malaria, the most virulent, i.e. falciparum malaria, was an evolutionary newcomer relative to the less lethal vivax, ovale, and malariae malaria, which were believed to be older and to have evolved toward commensalism. against this background, the standard textbook of internal medicine in the eradicationist era, the th edn of harrison's principles of internal medicine of , claimed that a feature of infectious diseases is that they 'as a class are more easily prevented and more easily cured than any other major group of diseases' ( , ) . the most fully elaborated and most cited theory of the new era was the 'epidemiologic transition' or 'health transition' theory represented by abdel omran, professor of epidemiology at johns hopkins, in and refined by him in and . omran's theory of the transition was an account of the encounter of human societies with disease in the modern period. according to omran and his followers in such journals as the health transition review, humanity has passed through three eras of modernity in health and disease. although omran is ambiguous about the precise chronology of the first era, the 'age of pestilence and famine,' it is clear that it lasted until the th century in the west and was marked by malthusian positive checks on demography: epidemics, famines, and wars. there followed the 'age of receding pandemics' that extended from the mid- th century until the early th in the developed west and until later in non-western countries. during this period there was a declining mortality from infectious diseases in general and from tb in particular. finally, after world war i in the west and after world war ii in the rest of the globe, humanity entered the 'age of degenerative and man-made diseases.' whereas in the earlier stages of disease evolution, social and economic conditions played the dominant role in determining health and the risk of infection, in the final phase medical technology and science played a major part. in this period, mortality and morbidity from infectious diseases have been progressively replaced by the rise of degenerative diseases such as cardiovascular disease, cancer, diabetes, and metabolic disorders, by man-made diseases such as occupational and environmental illnesses, and by accidents ( ) ( ) ( ) . adopting the perspective of 'health transition' theory, us surgeon general julius b. richmond announced in that infectious diseases were simply the 'predecessors' of the degenerative diseases that succeed and replace them. the course of nature, in his view, was simple, unidirectional, and benign ( ) . if memory of the power of public health and science provided a major impetus to overconfidence, forgetfulness also played a vital role. us surgeon general william steward reported in that the time had come to 'close the book on infectious diseases.' this view was profoundly eurocentric. even as medical experts in europe and north america snowden Á emerging and reemerging diseases r the authors journal compilation r blackwell munksgaard immunological reviews / declared final victory, infectious diseases remained the leading cause of death worldwide, and nowhere more disastrously than in the poorest and most vulnerable countries of africa, asia, and latin america. while the tb sanatoria were closing their doors in the developed north, the disease continued its ravages in the south. indeed, the disease continued to ravage the marginalized underclasses of the north itself: the homeless, prisoners, intravenous drug users, immigrants, and racial minorities. as paul farmer has argued, tb was emphatically not disappearing; it was just that the bodies it affected were either distant or hidden from sight ( , ) . indeed, in the best estimates suggest that there are more people ill with tb today than at any time in human history and that nearly two million will die of it during the course of the year ( , ) . ultimately, by the early s, the eradicationist position became untenable. rather than witnessing the rapid fulfillment of the prediction that science and technology would eliminate all infectious diseases from the globe, the industrial west discovered that it remained painfully vulnerable and to a degree that had seemed unimaginable. the decisive event, of course, was the arrival and upsurge of human immunodeficiency virus (hiv)/acquired immunodeficiency syndrome (aids). aids was first recognized as a new disease entity in , and its etiologic pathogen was identified in . by the end of the decade, it was clear that hiv/aids embodied everything that the eradicationists had considered unthinkable. aids was a new infectious disease for which there was no cure, it reached the industrial world as well as developing countries, and it unleashed in its train a series of exotic additional opportunistic infections. furthermore, it had the potential to become the worst pandemic in history as measured not only by mortality and suffering but also by its profound social, economic, and security consequences. from the front lines of the battle against aids, a series of voices sounded the alarm in the s about the severity of the new threat. most famous of all was the case of the us surgeon general c. everett koop, who became the chief federal spokesman on the disease. in he produced the brochure understanding aids and took the pioneering step of having it mailed to all million households in the nation ( ) . working in greater obscurity in sub-saharan africa, peter piot, who later directed unaids, warned in that aids in africa was not a 'gay plague' but an epidemic of the general population. he warned that it was transmitted by heterosexual as well as homosexual intercourse and that in fact it affected women more readily than men. the warnings of the s, however, were confined to the issues of aids: they did not directly confront the larger issue of eradicationism or announce a new era in medicine and public health. that task fell first to the national academy of science's institute of medicine (iom) and its landmark publications on emerging diseases that began in with emerging infections: microbial threats to health in the united states ( ) . once raised by the iom, the cry of alarm was taken up widely and almost immediately: by the centers for disease control and prevention (cdc), which devised its own response to the crisis in and founded a new journal emerging infectious diseases devoted to the issue; by the national science and technology council (nstc) in ; and by of the world's leading medical journals that agreed to take the unprecedented step by which each devoted a theme issue to emerging diseases in january , which they proclaimed 'emergent diseases month' ( ) ( ) ( ) . in , in addition, president bill clinton ( ) issued a fact sheet entitled 'addressing the threat of emerging infectious diseases' in which he declared them 'one of the most significant health and security challenges facing the global community.' there were also highly visible hearings on emerging infections in the us congress ( ) . in opening those hearings before the senate committee on labor and human resources, senator nancy kassebaum, the committee chairperson, noted, new strategies for the future begin with increasing the awareness that we must re-arm the nation and the world to vanquish enemies that we thought we had already conquered. these battles, as we have learned from the year experience with aids, will not be easy, inexpensive, nor quickly resolved. ( ) finally, to attract attention at the international level, the who, which had designated april of each year world health day, declared that the theme for was 'emerging infectious diseases -global alert, global response' with the lesson that in a global village, no nation is immune ( ) . in addition to the voices of scientists, elected officials, and the public health community, the popular press gave extensive coverage to the new and unexpected danger, especially when the lesson was driven home by three events of the s that captured attention worldwide. the first was the onset of a large-scale epidemic of asiatic cholera in south and central america, beginning in peru in and rapidly spreading across the continent until cases and deaths were reported in countries ( ) . since the americas had been free of the disease for a century, the arrival of the unwelcome visitor reminded the world of the fragility of painfully won advances in public health. because cholera is transmitted by the contamination of food and water by fecal matter, it is a 'misery thermometer' -an infallible indicator of societal neglect and substandard living conditions ( ) . its outbreak in the west late in the th century, therefore, caused shock and a sudden awareness of unexpected danger. indeed, the press informed its readers of the 'dickensian slums of latin america,' where the residents of lima and other cities drew their drinking water directly from the 'sewage-choked river rimac' and similarly polluted sources ( , ) . who director-general hiroshi nakajima proclaimed the south american epidemic an 'emergency situation. ' the second news-catching event in the matter of epidemic diseases was the outbreak of plague in the indian states of gujarat and maharashtra in september and october . the final toll for the epidemic was limited - cases and deaths were reported ( ) . nevertheless, the news that plague had broken out in both bubonic and pneumonic forms unleashed an almost biblical exodus of hundreds of thousands of people from the industrial city of surat. it cost india an estimated $ . billion in lost trade and tourism, and it sent waves of panic around the world. the disproportionate fear, as the new york times explained, was due to the fact that the very word plague was explosively charged. it evoked cultural memories of the black death that killed a quarter of the population of europe in the th century. india's plague, the paper continued, 'is a vivid reminder that old disease, once thought to have been conquered, can strike unexpectedly anytime, anywhere' ( ) . the third major epidemic shock of the s was an outbreak of the frightening disease of ebola hemorrhagic fever at the city of kikwit, zaire (now democratic republic of the congo), in . cholera claimed international attention because of the numbers of those it afflicted, even though it had a low case fatality rate if treated early. plague demanded attention because of its all too familiar potential. ebola, by contrast, did not inspire terror by giving rise to a major epidemic: it infected only people between january and july . nor did it create fear because of historical memories of disaster since it was a new disease first recognized in . nevertheless, ebola set off a tidal wave of fear -a 'modern nightmare' in the words of le monde -across the globe. the reasons were that it dramatically revealed the lack of preparedness of both industrial and developing nations to deal with a public health emergency. it ignited primordial western fears of the jungle and of untamed nature, and it fed on racial anxieties about 'darkest' africa. as a result, a prominent aspect of the kikwit outbreak was its capacity to generate what the journal of infectious diseases termed 'extraordinary' and 'unprecedented' press coverage that amounted at times to the commercial 'exploitation' of human misery and a 'national obsession' ( ) . descending onto the banks of the kwilu river, the world's tabloids stressed in vivid hyperbole that ebola was a zoonotic disease that had sprung directly from the jungles of africa as a result of the encounter between native charcoal burners and monkeys and now threatened the west. in the revealing headline of the daily telegraph of sydney, 'out of the jungle a monster comes' ( ) . even the most legitimate investigators, however, were disturbed to discover that ebola had eluded public health attention for weeks between the death of the index case on january and the notification of the international community on april , despite the fact that the disease had left clusters of severely ill and dying patients in its train. with such a porous surveillance network in place, ebola aroused the fear that it might spread unnoticed km from kikwit to kinshasa, and then throughout the world by means of the zairian capital's intercontinental airport. there the virus could be loaded on board as 'a ticking, airborne time bomb' ( ) . most of all, however, the kikwit outbreak commanded attention because ebola is almost invariably fatal and because its course in the human body is excruciating, dehumanizing, and dramatic. commenting on the scenes that he had observed in zaire, the author richard preston explained on television at the height of the outbreak that the mortality rate among sufferers was % and that there was no known remedy or prophylactic. he continued: the victims suffer what amounts to a full-blown biological meltdown. . . . when you die of ebola, there's this enormous production of blood, and that can often be accompanied by thrashing or epileptic seizures. and at the end you go into catastrophic shock and then you die with blood pouring out of any or all of the orifices of the body. and in africa where this outbreak is going on now, medical facilities are not all that great. i've had reliable reports that doctors . . . were literally struggling up to their elbows in blood -in blood and black vomit and in bloody diarrhea that looks like tomato soup, and they know they're going to die. ( ) in combination with the announcement by scientists that the world was highly susceptible to new pandemics of just such infections, these events on three continents generated hordes, and of nature exacting its revenge for human presumption. as forrest sawyer reported on abc news, 'once the western world thought it was safe from these invisible killers. not anymore. we are now biologically connected in a web or a net.' in addition, there was an outpouring of films devoted to the possibility of pandemic disaster such as wolfgang petersen's thriller outbreak and of widely read books on the same theme, including richard preston's best-seller, the hot zone; laurie garrett, the coming plague: newly emerging diseases in a world out of balance; and william close, ebola. in the words of david satcher, director of the cdc, the result was the 'cnn effect' -the perception by the public that it was at immediate risk even at times when the actual danger was small ( ) . in this climate of anxiety, the term 'emerging and reemerging diseases' was coined for the iom by joshua lederberg, winner of the nobel prize for medicine, to mark a new era. lederberg defined these disease entities as follows: 'emerging infectious diseases are diseases of infectious origin whose incidence in humans has increased within the past two decades or threatens to increase in the near future' ( ) . emerging diseases were those that, like aids and ebola, were previously unknown to have afflicted humans; reemerging diseases, such as cholera and plague, were familiar scourges whose incidence was rising, or whose geographical range was expanding. lederberg's purpose in devising a new category of diseases was to give notice that the age of euphoria was over. instead of receding to a vanishing point, he declared, communicable diseases 'remain the major cause of death worldwide and will not be conquered during our lifetimes . . . we can also be confident that new diseases will emerge, although it is impossible to predict their individual emergence in time and place' ( ) . indeed, the contest between humans and microbes was a darwinian contest with the advantage tilted toward the microbes. the stark message of the iom was that, far from being secure from danger, the united states and the west were at greater risk from contagious and epidemic diseases than at any time in history. an important reason for this new vulnerability was the legacy of eradicationism itself. the belief that the time had come to close the books on infectious diseases had produced a pervasive climate that critics labeled variously as 'complacency,' 'optimism,' 'overconfidence,' and 'arrogance.' the conviction that victory was imminent had led the industrial world to premature and unilateral disarmament. assured by a consensus of the leading medical authorities for years that the danger was past, federal and state governments in the united states dismantled their public health programs dealing with communicable diseases and slashed their spending. at the same time, investment by private industry on the development of new vaccines and classes of antibiotics dried up, the training of health care workers failed to keep abreast of new knowledge, vaccine development and manufacture were concentrated in fewer laboratories, and the discipline of infectious diseases struggled to attract its aliquot share of research funds and of the best minds. at the nadir in , the united states spent only $ million for infectious disease surveillance as public health officials prioritized other concerns -chronic diseases, substance abuse, tobacco use, geriatrics, and environmental issues. for these reasons, the assessment of american preparedness to face the challenges of the new era was disheartening. in the words of the cdc in : the public health infrastructure of this country is poorly prepared for the emerging disease problems of a rapidly changing world. current systems that monitor infectious diseases domestically and internationally are inadequate to confront the present and future challenges of emerging infections. many foodborne and waterborne disease outbreaks go unrecognized or are detected late; the magnitude of the problem of antimicrobial drug resistance is unknown; and global surveillance is fragmentary. ( ) more bluntly, michael osterholm, the minnesota state epidemiologist, informed congress in that, 'i am here to bring you the sobering and unfortunate news that our ability to detect and monitor infectious disease threats to health in this country is in serious jeopardy. . . . for twelve of the states or territories, there is no one who is responsible for food or water-borne disease surveillance. you could sink the titanic in their back yard and they would not know they had water' ( ) . a striking example of the effects of complacency on infectious disease is the case of tb in new york city. tb had once been the leading cause of death in the city, but improvements in hygiene and education, followed by the discovery of streptomycin, led to the conviction by the middle of the th century that the disease was on the verge of being entirely conquered. as a result, funding was diverted, and demonstrably effective tb programs were dismantled although the social determinants of the disease worsened dramaticallyimmigration, crowding, homelessness, and rates of incarceration. meanwhile, hiv/aids continued to provide large numbers of patients with compromised immunity. as a result, the risk of infection increased, while access to health care became increasingly difficult, and the city experienced a remarkable and entirely preventable resurgence of the 'white plague,' primarily among african american and hispanic residents. between and , the numbers of cases tripled, while drug resistance developed as a significant additional problem. new york city led the way in a national resurgence of tb as cases increased by % between and . overweening confidence led directly and rapidly to a local epidemic and a partial reversal nationally of decades of tireless campaigning ( ) . if the experience of the united states with tb suggests how fragile advances in health remained even in the industrial world, the situation in developing countries was still more disquieting. there, progress toward the germ-free eden during the eradicationist era was nil. in david satcher's uncompromising observation, 'persons living in tropical climates are still as vulnerable to infectious disease as their early ancestors were' ( ) . the critique of years of hubris went deeper than just a protest against a decline in vigilance. in addition, the theorists of emerging diseases argued that, unnoticed by the eradicationists, society since world war ii had changed in ways that actively promoted the emergence and reemergence of epidemic diseases. one of the leading features most commonly cited was the impact of globalization in the form of the rapid mass movement of goods and populations. as william mcneill noted in plagues and peoples ( ) , the migration of people throughout history has been one of the most dynamic factors affecting the balance between microbes and man. humans are permanently engaged in a kind of war in which the social and ecological conditions that they create exert powerful evolutionary pressure on micro-parasites. by mixing gene pools and by providing access for microbes to populations of non-immunes living in conditions in which the microbes thrive, globalization gave microorganisms a powerful advantage. in the closing decades of the th century and the early years of the st, the speed and scale of this phenomenon amounted to a quantum leap, as . billion passengers boarded airplanes in ( , ) . in the words of the popular press, the daily movement of people around the globe by airplane means that a disease breaking out today in kikwit can arrive in new york, mumbai, and mexico city tomorrow. the numbers of voluntary travelers, moreover, are massively supplemented by millions of involuntary refugees and displaced persons in flight from warfare, famine, and religious, ethnic, or political persecution. for lederberg and the iom, these rapid mass movements have tilted the advantage in favor of microbes, 'defining us as a very different species from what we were years ago. we are enabled by a different set of technologies. but despite many potential defenses -vaccines, antibiotics, diagnostic tools -we are intrinsically more vulnerable than before, at least in terms of pandemic and communicable diseases' ( ) . after globalization, the second factor most frequently underlined was demographic growth, especially because this growth occurred in circumstances that were the delight of microorganisms and of the insects that often transmit them. in the postwar era, population has soared above all in the poorest and most vulnerable regions of the world, with the global urban population growing at four times the rate of the rural. its hallmark has been wholesale, chaotic, and unplanned urbanization, led by the resource-poor nations of sub-saharan africa, which is the most rapidly urbanizing region on the planet ( ) . the results have been escalating poverty, widening social inequality, the birth of 'megacities' exceeding million inhabitants, and the spawning of teeming peri-urban slums without sanitary, educational, or other infrastructures. such places were ready-made for ancient diseases to expand, as cholera demonstrated in the shantytowns and barrios of cities like lima, mexico city, and rio de janeiro, where millions lived without sewers, drains, secure supplies of drinking water, or appropriate waste management. already in the th century, cholera had flourished in the conditions created in european cities by rapid and unplanned urbanization. in the final decades of the th century and the start of the st, a much larger process on a global scale reproduced in the cities of africa, asia, and latin america the anomalous sanitary conditions propitious for cholera ( ) . another clear indication of socio-economic conditions in these new urban ecosystems is the appearance of trench fever (bartonella quintana) among the inhabitants of homeless shelters in north american cities. trench fever first emerged in the filth and crowding of soldiers in the trenches of the western front in the first world war, when millions of combatants were infected by the lice that covered their bodies. bartonella quintana, however, had never been documented apart from the vermin and the grime of wartime. the reemergence of the disease in urban america is therefore a clear measure of the insalubrious conditions of marginalized populations among the urban poor ( , ) . here too in urban poverty were the social determinants that made possible the global pandemic of dengue fever that began in and has continued unabated until today, when . billion people are at risk every year and - million people are infected. dengue is the ideal type of an emerging disease. an arborovirus transmitted primarily by the highly urban, daybiting, and domestic aedes aegypti mosquito, dengue thrives in crowded tropical and semi-tropical slums whenever there is standing and unregulated water. it breeds abundantly in gutters, uncovered cisterns, unmounted tires, stagnant puddles, and plastic containers, and it takes full advantage of societal neglect and the absence or cessation of vector control programs. particularly important for the theorists of 'emerging diseases' was the manner in which dengue demonstrated the hollowness of the reassuring dogma that infectious diseases evolve inexorably toward commensalism and reduced virulence. the dengue virus is a complex of four closely related serotypes (den- , den- , den- , and den- ) that have been known to infect humans since the th century. until , however, dengue infections in any geographical area were caused by a single virus that gave rise to a painful illness marked by fever, rash, headache behind the eyes, vomiting, diarrhea, prostration, and joint pains so severe that the infection earned its nickname 'break-bone fever.' but 'classical' dengue was a self-limiting disease that was followed by lifelong immunity. the movement and mobility of populations, however, have allowed all four serotypes to spread indiscriminately around the globe, so that for the first time individuals who have already experienced infection with one dengue virus can subsequently be infected with one or more of the others, as there is no crossover immunity from one serotype to another. through mechanisms that are still imperfectly understood, the disease is much more severe in patients suffering re-infections with different serotypes. instead of becoming milder, therefore, dengue has become a growing threat, giving rise to far more frequent outbursts and to sudden, devastating epidemics in which large numbers of patients suffer the severe and often lethal complications of dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) that were once unknown. in the americas, the first modern epidemic of dengue fever broke out in in cuba, producing cases, of whom suffered dhf and dss ( ) . moreover, since the dengue vectors a. aegypti and aedes albopictus are present in the united states, scientists at the national institute of allergy and infectious diseases (niaid), such as its director anthony fauci, have noted that dengue fever has broken out in both hawaii and puerto rico, and that they see no inherent reason it could not include the continental united states in its ongoing global expansion ( ) . dengue therefore demonstrates the following important evolutionary lessons: (i) infectious diseases that do not depend on the mobility of their host for transmission (because they are vector-borne, waterborne, or foodborne) are not under selective pressure to become less virulent; (ii) overpopulated and unplanned urban or peri-urban slums provide ideal habitats for microbes and their arthropod vectors; and (iii) modern transportation and the movements of tourists, migrants, refugees, and pilgrims facilitate the process by which microbes and vectors gain access to these ecological niches. paradoxically, the very successes of modern medical science also prepared the way for the emergence of new infections. by prolonging life, medicine gives rise to ever larger numbers of elderly people with compromised immune systems. as part of this process, significant numbers of immunocompromised populations have appeared at earlier ages as well-diabetics, cancer and transplant patients undergoing chemotherapy, and aids patients whose lives have been radically extended by antiretroviral treatment. furthermore, such people are frequently concentrated in settings where the transmission of microbes from body to body is amplified, such as hospitals, facilities for the elderly, and prisons. the proliferation of invasive procedures has also increased the opportunities for such diseases. modern nosocomial infections emerged in these conditions, and have become a major problem of public health as well as an ever growing economic burden. of these infections, the so-called 'superbug' staphylococcus aureus -the leading cause of nosocomial pneumonia, of surgical site infections, and of nosocomial bloodstream infections -is the most important and widespread. a recent study notes that in the united states by : each year approximately two million hospitalizations result in nosocomial infections. in a study of critically ill patients in a large teaching hospital, illness attributable to nosocomial bacteria increased intensive care unit stay by days, hospital stay by days, and the death rate by %. an earlier study found that postoperative wound infections increased hospital stay an average of . days. ( ) a further threatening byproduct of the advance of medical science is the development of ever increasing antimicrobial resistance. already in his nobel prize acceptance speech, alexander fleming, who discovered penicillin, the first antibiotic, issued a prophetic warning. penicillin, he advised, needed to be administered with care, because the bacteria susceptible to it were likely to develop resistance. the selective pressure of so powerful a medicine would make it inevitable. echoing fleming's warning, the emerging diseases theorists argue that antibiotics are a 'non-renewable resource' whose duration of benefit is biologically limited. by the late th century, this prediction was reaching fulfillment. on the one hand, the discovery of new classes of antimicrobials had slowed to a trickle, especially in a market in which profit margins are compressed by competition, by regulations requiring large and expensive clinical trials before approval, and by the low tolerance for risk on the part of regulatory agencies charged with the safety of the public. on the other hand, while antiinfective development stagnates, many microorganisms have evolved extensive resistance. as a result, in one telling metaphor, physicians are rapidly emptying their quiver, and the world stands poised to enter the postantibiotic era ( ) . some of the most troubling examples of the emergence of resistant microbial strains are the emergence of plasmodia that are resistant to all synthetic antimalarials, of s. aureus that is resistant both to penicillin and to methycillin (mrsa), and of strains of mycobacterium tuberculosis that are resistant not only to first-line medications (mdr-tb) but to second-line medications as well (xdr-tb) ( ) . antimicrobial resistance has become a global crisis, and many anticipate the early appearance of strains of hiv, tb, staph a, and malaria that are not susceptible to any available therapy. in part the problem of antimicrobial resistance is a simple result of darwinian evolution. as a rand corporation study ( ) notes, there are tens of thousands of viruses and species of bacteria that are capable of infecting human beings, and many of them replicate and evolve billions of times in the course of a single human generation. evolutionary pressures, in this context, work to the long-term disadvantage of human beings. but unwise human actions have dramatically hastened the process. farmers spray crops with pesticides and fruit trees with antibiotics, and they add subtherapeutic doses of antibiotics such as virginiamycin and avoparcin wholesale to animal feed to prevent disease, promote growth, and increase the productivity of chickens, pigs, and feedlot cattle. indeed, half the world output of antimicrobials by tonnage is used in agriculture ( ) . at the same time, the popular confidence that microorganisms will succumb to a chemical barrage has led to a profusion of antimicrobials in domestic settings where they serve no purpose ( ) . physicians, pressured to give priority in clinical settings to the immediate risk of individual patients over the long-term interest of the species and to meet patients' expectations, have succumbed to profligate prescribing fashions, administering antibiotics even for non-bacterial conditions for which they are unnecessary or entirely useless. the classic case in this regard is the pediatric treatment of otitis media (or middle ear infection), for which the overwhelming majority of practitioners in the s prescribed antibiotics, even though two-thirds of the children derived no benefit from the medication. widespread possibilities of self-medication in countries with few regulations or through opportunities created by the internet amplify the difficulties. in the case of diseases such as malaria and tb that require a long and complicated therapeutic regimen, there is also the issue of patients who interrupt their treatment after the alleviation of their symptoms instead of persevering until their condition is cured. here the problem is not the overuse but the underuse of antibiotics. sometimes described as simple non-compliance by patients, the issue in fact raises complex questions of education, poverty, and lack of access to health care. here the who strategies of dots (directly observed treatment short course) and dots-plus are helpful but cannot solve the underlying problems. a further issue raised by the new era was the overly rigid conceptualization of disease by the eradicationists, who drew too sharp a distinction between chronic and contagious diseases. infectious diseases, it became clear during the s, are a more expansive category than scientists previously realized because many diseases long considered noninfectious in fact have infectious origins. in demonstrating these causal connections, the decisive work was that of the australian nobel laureates barry j. marshall and robin warren with regard to peptic ulcers in the s. peptic ulcers are a significant cause of suffering, cost, and even death, as one american in develops one during the course of a life time, over one million people are hospitalized by them every year, and die. marshall noted in his acceptance speech for the nobel prize in , however, that the chronic etiology of peptic ulcer in the s was universally accepted as scientific truth. in his words, 'i realized that the medical understanding of ulcer disease was akin to a religion. no amount of logical reasoning could budge what people knew in their hearts to be true. ulcers were caused by stress, bad diet, smoking, alcohol and susceptible genes. a bacterial cause was preposterous.' what marshall and warren were able to demonstrate, therefore, was a medical watershed. they proved, in part by means of an auto-experiment, that the bacterium helicobacter pylori was the infectious cause of the disease and that antibiotics rather than diet, lifestyle change, and surgery were the appropriate therapy ( ) . this insight led to the realization that many other non-acute diseases, such as certain forms of cancer, chronic liver disease, and neurological disorders, are due to infections. human papillomavirus, for instance, is thought to give rise to cervical cancer, hepatitis b and c viruses to chronic liver disease, campylobacter jejuni to guillain-barré syndrome, and certain strains of escherichia coli to renal disease ( , ) . there are indications as well that infections serve as an important trigger to atherosclerosis and arthritis, and there is a growing recognition that epidemics and the fear that accompanies them leave psychological sequelae in their wake, including posttraumatic stress ( , ) . this understanding of these processes is what some have termed finally, and most emphatically, the concept of emerging and reemerging diseases was intended to raise the most important threat of all -that the spectrum of diseases that humans confront is broadening with unprecedented and unpredictable rapidity. the number of previously unknown conditions that have emerged to afflict humanity since exceeds , with a new disease discovered on average more than once a year. the list includes such frightening names as hiv, hantavirus, lassa fever, marburg fever, legionnaires' disease, hepatitis c, lyme disease, rift valley fever, ebola hemorrhagic fever, nipah virus, west nile virus, sars (severe acute respiratory syndrome), bovine spongiform encephalopathy, avian flu h n , chikungunya virus, and group a streptococcus -the so-called 'flesh-eating bacterium.' skeptics argue that simply to list the diseases that have emerged since s gives the misleading impression that diseases are emerging at an accelerating rate. this impression, they suggest, is largely an artifact of heightened surveillance and improved diagnostic techniques rather than a new development. the who has countered that not only have diseases emerged at record rapidity as one would expect from the transformed social and economic conditions of the postwar world, but also that they gave rise between the years and to a record worldwide epidemic events ( ) . the most recent and comprehensive examination of the question ( ), published in february in nature, involved the study of emerging infectious disease (eid) 'events' between and , controlling for reporting effort through more efficient diagnostic methods and more thorough surveillance. the conclusion was that, 'the incidence of eid events has increased since , reaching a maximum in the s. . . . controlling for reporting effort, the number of eid events still shows a highly significant relationship with time. this provides the first analytical support for previous suggestions that the threat of eids to global health is increasing' ( ) . there are no rational grounds, the public health community concluded, to fail to expect that as diseases emerge in the future, some of them will be as virulent and as transmissible as hiv or the spanish influenza of / . discussion has therefore shifted dramatically from the question of whether new diseases will emerge and old ones resurge to the issue of how the international community can best prepare to face them. in the stark words of the us department of defense, 'historians in the next millennium may find that the twentieth century's greatest fallacy was the belief that infectious diseases were nearing elimination. the resultant complacency has actually increased the threat' ( ) . a major aspect of the official response to the challenge of emerging and reemerging diseases is that microbes now are regarded as threats to the security of states and to the stability of the international order. for the first time, therefore, not only public health authorities but also intelligence agencies and conservative think tanks have classified infectious diseases as a 'non-traditional threat' to national and global security. they assumed therefore the task of envisaging the future and the challenge that communicable diseases would play. here a turning point was the central intelligence agency (cia)'s national intelligence estimate (nie) for ( ) , which was devoted to the danger posed by disease and presented defense against epidemic diseases as a major security goal for the united states. as a document, nie - d ( ) was divided into four major sections: alternative scenarios, impact, implications, and discussion. in the first section, the cia attempted to outline three possible scenarios for the course of infectious diseases over the next years: (i) the optimistic contemplation of steady progress in combating communicable disease; to (ii) the forecast of a stalemate with no decisive gains either by microbes or by humans in their long war of attrition; and (iii) the consideration of the most pessimistic prospect of deterioration in the position of humans, especially if the world population continues, as seems probable, to expand and if megacities continue to spring up with their attendant problems of crowding, sanitation, and unprotected drinking water. unfortunately, the cia regarded the optimistic first case as extremely unlikely. the probable course of events, in its view, is that americans will die from infectious diseases every year or considerably more if a pandemic of influenza or of a still unknown disease occurs, if there is a dramatic decline in the effectiveness of antiretroviral treatments for hiv/aids. only toward the end of the years did the report foresee possible advances due to enhanced public health initiatives, the development of new drugs and vaccines, and economic development ( ) . against this background, the succeeding sections on 'impact' and 'implications' outlined a series of likely economic, social, and political results that would occur in the new age of increasing disease burdens. in the most afflicted regions of the world, such as sub-saharan africa, the report anticipated 'economic decay, social fragmentation, and political destabilization.' the international consequences of these developments would be growing struggles to control increasingly scarce resources, accompanied by crime, displacement, and the degradation of familial ties. disease, therefore, would heighten international tensions while it weakened forces, such as international peacekeepers, who might otherwise have played a larger role in controlling regional tensions. us or european military forces deployed abroad in support of humanitarian or other operations would be at high risk. because the economic and social consequences of increasing burdens of communicable diseases in the developing world are certain to impede economic development, the nie also predicted that democracy would be imperiled, that civil conflicts and emergencies would multiply, and that the tensions between north and south would deepen. three years later, motivated by the cia's report, an influential national security think tank, the rand corporation, turned to the intersection of disease and security when it attempted to provide 'a more comprehensive analysis than has been done to date, encompassing both disease and security' ( ) . in so doing, it envisaged even more somber probabilities than the cia in the new global environment. the rand corporation intelligence report the global threat of new and reemerging infectious diseases: reconciling u.s. national security and public health policy ( ) had two leading themes. the first was that in the postwar era there was a sharp decline in the importance of direct military threats to security. the second was that there is a corresponding rise in the impact of 'non-traditional challenges,' of which diseases are the major but inadequately recognized component. it has always been accepted, the report stressed, that diseases kill and undermine the quality of individual lives. in addition, it was essential to recognize that the transition to the era of emerging and reemerging diseases marked the opening of a period in which infectious diseases would profoundly affect the ability of states to function and to preserve social order. the most striking portion of the global threat of new and reemerging infectious diseases ( ) was its imagining of a probable scenario in which south africa could become the first modern state to fail specifically because of infectious diseases in general and the hiv/aids pandemic in particular. as the report explained, 'the contemporary hiv/aids crisis in south africa represents an acute example of how infectious diseases can undermine national resilience and regional stability.' in absolute numbers, south africa has the highest number of hiv-positive inhabitants in africa - . million people in , or % of the country's adult population. already, such extreme prevalence of the disease has pervasive impacts, affecting all aspects of south african security. but south africa is just emerging from the first phase of the aids pandemic and is therefore far from experiencing the full effects of the crisis, which even in the absence of resistance to antiretroviral therapy, is expected to produce patients with hiv and with full-blown aids by . in these circumstances, over a quarter of the economically active population will have the disease, causing severe skill shortages, creating poverty, destroying economic development, undermining participation in political life, and giving rise to more than two million orphans who will be impoverished, uneducated, and easily drawn into crime and prostitution. the effects will also be deeply felt in the military, the police, and the legal system, which will be severely deprived of manpower and unable to function just as social tensions deepened. 'the net effect,' it concluded, 'will be entirely negative for south africa's civil stability, possibly reducing the country to widespread social anarchy within the next five to twenty years.' this disturbing outcome, moreover, could be hastened by the public health policies of president thabo mbeki, who espoused the theories of the aids denier peter duesberg and rejected the link between the hiv virus and the disease. the point the rand corporation stressed most about south africa, however, was that it was simply a dramatic illustrative example. what was occurring there as a result of hiv/aids could happen without warning elsewhere. 'a crisis of similar proportions,' it explained, 'could therefore break out in any country at any time.' indeed, in the context of a growing danger of bioterrorist attack, such an outbreak could be launched intentionally. it was precisely this point -the growing vulnerability of all in the age of globalization -that led the world community, the european union, and individual nations to rearm in preparation for the inevitable threats to come. in the new climate of preparedness, the united states took a prominent role, beginning almost immediately in the aftermath of the iom report. in the cdc -the chief monitoring agency -drafted a strategic plan that it then updated in , while niaid -the principal basic research center -established a research agenda. both agencies' plans were endorsed by the white house, where the nstc under the chairmanship of vice president al gore issued a 'fact sheet: addressing the threat of emerging infectious diseases,' which in turn was backed by a presidential decision directive of june , . the result, as gore explained, was the first national policy by the united states to confront the international problem of infectious diseases ( ) . the essential starting point of the plan envisaged by the cdc, niaid, and the white house was the iom's description of the darwinian struggle under way between humans and microbes. in the iom's analysis of that struggle, microbes possess formidable advantages. they outnumber human beings a billionfold, they enjoy enormous mutability, and they replicate, in lederberg's estimate, a billion times more quickly than man, with generations measured in minutes rather decades. in terms of natural evolutionary adaptation, therefore, microbes are genetically favored to win the contest. in lederberg's observation, 'pitted against microbial genes, we have mainly our wits' ( ) . taking this iom analysis as its starting point, the american response to the new challenge is best seen as the attempt to organize and deploy human wit, backed by newly found financial resources, to counter the microbial genetic challenge ( ) . the white house 'fact sheet' declared in clear alarm that, 'the national and international system of infectious disease surveillance, prevention, and response is inadequate to protect the health of u.s. citizens.' to remedy the situation, the white house established six policy goals, as follows: . strengthen the domestic infectious disease surveillance and response system, both at the federal, state, and local levels and at ports of entry into the united states, in cooperation with the private sector and with public health and medical communities. . establish a global infectious disease surveillance and response system, based on regional hubs and linked by modern communications. . strengthen research activities to improve diagnostics, treatment, and prevention, and to improve the understanding of the biology of infectious disease agents. . ensure the availability of the drugs, vaccines, and diagnostic tests needed to combat infectious diseases and infectious disease emergencies through public and private sector cooperation. . expand missions and establish the authority of relevant us government agencies to contribute to a worldwide infectious disease surveillance, prevention, and response network. . promote public awareness of eids through cooperation with non-governmental organizations and the private sector ( ) . in pursuit of goals , , and , nih funding was doubled between and . niaid established a research agenda to develop new weapons to combat epidemic diseases, giving rise to an explosion in knowledge while publications on infectious diseases burgeoned. indeed, the agency director, anthony s. fauci, claimed in that hiv/aids in particular has become the most extensively studied disease in human history. niaid's priority is the development of safe and effective vaccines and medications to combat hiv/aids, malaria, tb, and influenza. to that end, it has evaluated over hiv vaccine candidates, funded clinical trials, and developed antiretroviral medications. in the field of malariology, it has completed the genomic sequencing of plasmodium falciparum and of the feared malaria vector anopheles gambiae with the expectation that this genetic knowledge is the first step toward the capacity to design anti-malarial drugs, vaccines, and pesticides. the work of the federal agency, moreover, has been complemented by the work of private organizations such as the bill and melinda gates foundation, and university laboratories ( ) . at the same time that niaid stressed basic research, the cdc developed a defensive strategy against emerging pathogens in compliance with goal of the president's directive. the cdc articulated its plan in two seminal works published in and . there it articulated its objectives in four principal areas: surveillance; applied research; prevention and control; and the enhancement of the infrastructure and trained personnel needed for diagnostic laboratories at the federal, state, local, and international levels. in addition, the atlanta-based agency strengthened its links with the international public health community and with other surveillance agencies such as the fda and the department of defense. it enhanced its capacity to respond to outbreaks, and it launched the journal emerging infectious diseases as a forum to pool information on communicable diseases. it sponsored a series of major international conferences on the topic of emerging and reemerging diseases, beginning in with the participation of representatives from all states and countries. the cdc initiatives were widely regarded as a model for the establishment of surveillance and response capabilities in other countries as well ( , ) . at the global level, the un and its agency who also took major steps to strengthen international preparedness for the ongoing siege by microbial pathogens. a first step was the creation in of the disease-specific organization unaids with the function of raising awareness, mobilizing resources, and monitoring the pandemic. funding levels in the fight against the disease increased from $ million in to nearly $ billion a decade later ( ). a further step was that like the united states, the united nations announced that it regarded infectious diseases as threats to international security. in acknowledgement of this new development, the security council took the unprecedented step in june of devoting a special session to the hiv/aids crisis. the session adopted a 'declaration of commitment on hiv/aids: global crisis -global action.' the declaration declared the global epidemic a 'global emergency and one of the most formidable challenges to human life and dignity' ( ) . five years later, in june , the general assembly reaffirmed its commitment to the campaign, and adopted the ' political declaration on hiv/aids,' whose chief goal was the establishment of national campaigns to improve access to care and treatment ( ). a third step was the establishment of a new set of international sanitary regulations -ihr ( ) -to replace the outdated ihr ( ). whereas the old framework was disease-specific and required notification only in the event of plague, yellow fever, and cholera, the new rules required notification for any 'public health emergency of international concern,' thereby including unknown pathogens and emerging infections. the regulations specified the nature of the 'events' that should trigger international concern. they also committed all of the who member states to improve their capacity for surveillance and response and to designate 'national ihr focal points' as the units responsible for providing notification while requiring, in exchange, that the who provide assistance to member states in fulfilling their obligations ( , ) . in addition, recognizing that microbes do not acknowledge political frontiers, ihr ( ) called for effective responses wherever necessary to contain an outbreak on the basis of realtime epidemiological evidence instead of concentrating on taking defensive measures at international borders. finally, the who organized a rapid response capacity with the necessary supporting infrastructure. this was the global outbreak alert and response network (goarn), which was established in with the goal of ensuring that even most resource-poor countries would have access to the experts and resources needed to respond to an epidemic emergency. to that end, goarn pooled the resources of countries and organized experts in the field. in addition, it stockpiles vaccines and drugs, and supervises their distribution during epidemic events. between its founding and , goarn responded to outbreaks and attempted to learn from experience by establishing protocols to standardize such matters as field logistics, security, communication, and the deployment of field teams ( ). in addition to goarn, the who set up surveillance systems specifically designed to deal with pandemic influenza, which the un agency determined as its most feared security threat. these disease-specific networks are (i) the global influenza surveillance network, which provides recommendations twice a year on the appropriate vaccine for the subsequent influenza season by collecting samples from patients in countries and forwarding them to who collaborating laboratories for analysis, and (ii) flunet, which compiles the surveillance data thus collected to establish a global real-time early-alert system for the disease ( , ) . in practice, the first test of the effectiveness of the new structures was the sars pandemic of / -the first major emerging disease threat of the st century. after first appearing in the chinese province of guangdong in november , it erupted as an international health threat in march , when the who received notification and declared a global travel alert. between march and the declaration on july that the disease had been contained, sars affected people, caused deaths, brought international travel to a halt in entire regions, and cost $ billion in gross expenditure and business losses to asian countries alone. as retrospective studies have demonstrated, sars presented many of the features that most severely expose the vulnerabilities of the global system: sars is a respiratory disease capable of spreading from person to person without a vector; it has an asymptomatic incubation period of more than a week; it generates symptoms that closely resemble those of other diseases; it takes a heavy toll on caregivers and hospital staff; it readily spreads unobserved aboard aircraft; and it has a case fatality rate of %. at the time this new disease appeared, moreover, its causative pathogen (sars-associated coronavirus) was unknown, and there was neither a diagnostic test nor a specific treatment. for all of these reasons, it dramatically confirmed the iom's prediction that all countries were more vulnerable than ever to eids. sars demonstrated no predilection for any region of the globe and was no respecter of prosperity, education, technology, or access to health care. indeed, after its outbreak in china, sars spread by airplane primarily to affluent cities such as singapore, hong kong, and toronto, where it struck relatively prosperous travelers and their contacts, hospital workers, patients, and hospital visitors, rather than targeting the poor and the marginalized. more than half of the recognized cases occurred in well-equipped and technologically advanced hospital settings such as the prince of wales hospital in hong kong, the scarborough hospital in toronto, and the tan tock seng hospital in singapore ( , , ) . in terms of response to the crisis, the sars outbreak demonstrated and vindicated the reforms taken on both the national and international levels. after the debacle of chinese obfuscation at the start of the epidemic, national governments cooperated fully with ihr ( ). the world's most equipped laboratories and foremost epidemiologists, working in realtime collaboration via the internet, succeeded, with unprecedented speed, in identifying sars-cov in just weeks. at the same time the newly created goarn, together with such national partners as the canadian public health intelligence network, the cdc, and the who global influenza network, took rapid action to issue global alerts, monitor the progress of the disease, and supervise containment strategies before the disease could establish itself endemically. ironically, given the high-tech quality of the diagnostic and monitoring effort, the containment policies were based on traditional methods dating from the public health strategies against bubonic plague by the th century and the foundation of epidemiology as a discipline in the th. these measures were case tracking, isolation, quarantine, the cancellation of mass gatherings, the surveillance of travelers, recommendations to increase personal hygiene, and barrier protection by means of masks, gowns, gloves, and eye protection ( ) . although sars affected countries and every continent, the containment operation coordinated by goarn successfully limited the outbreak overwhelmingly to hospital settings with only sporadic community involvement, so that by july the who could announce that the pandemic was over. although sars tested the newly established global defenses against emerging diseases and the protective ramparts withstood the challenge, doubts relentlessly surfaced. the chinese policy of concealment between november and march had placed international health in jeopardy and revealed that even a single weak link in the response network could undermine the ihr ( ) system. indeed, resourcepoor countries that were compliant with the new framework of obligations nonetheless found it difficult or impossible to maintain the surveillance effort for the full -month duration of the emergency. still more tellingly, it was also clear that a major factor in the containment of sars was simple good fortune. the world was lucky that sars is spread by droplets and therefore requires extended contact for transmission, unlike classic airborne diseases such as influenza and smallpox. it was, relatively, much easier to contain, because except in the infrequent and still poorly understood case of so-called 'super shedders,' it is not readily communicable from person to person. as poorly transmissible as it was, however, sars exposed the absence of 'surge capacity' in the hospitals and health care systems of the prosperous and well-resourced countries it affected. the events of thereby raised the specter of what might have happened had sars been pandemic influenza, and if it had traveled to resource-poor nations at the outset instead of mercifully visiting cities with well-equipped and well-staffed modern hospitals and public health care systems. furthermore, sars arrived in peacetime rather than in the midst of the devastation and the dislocations of war. in that respect, too, it did not repeat the challenge of the spanish lady of - . the physician paul caulford, who fought the sars epidemic in the front lines at scarborough hospital in toronto, raised these matters. in december , after the passing of the emergency, he reflected: sars must change us, the way we treat our planet, and how we deliver health care, forever. will we be ready when it returns? sars brought one of the finest publiclyfunded health systems in the world to its knees in a matter of weeks. it has unnerved me to contemplate what the disease might do to a community without our resources and technologies. without substantive changes to the way we manage the delivery of health care, both locally and on a worldwide scale, we risk the otherwise preventable annihilation of millions of people, either by this virus, or the next. ( ) at the end of the victory over sars, the nagging question therefore remains: even after the impressive efforts at rearmament since , how prepared is the international community for upcoming emerging diseases? have we been forever changed? the reforms introduced since the iom report in have been profound and important. indeed, the manner in which the international community responded to sars was innovative and, in the circumstances, highly successful. there is, however, a disconcerting sense of a systematic blindness in the responses -at all levels -to the crisis described by the iom, the cia, the rand corporation, the who, and the white house. what has been done has been necessary but probably far from sufficient. some of the issues raised by those who sounded the alarm have been forcefully addressed, but others have been largely ignored. the responses to date have fit into two chief categories, both of which are essential and both of which were evident during the sars pandemic. the first is reactive: the ability to respond rapidly and effectively to the outbreak of new epidemic threats. through a series of initiatives, the years since have witnessed the establishment of organized networks for gathering public health intelligence, of an international legal framework to structure emergency interventions, and of well equipped response teams of experts to contain and monitor outbreaks. if one were to compare outbreaks of infectious diseases to forest fires, the world has provided itself with surveillance satellites, advanced communications infrastructures, and a well-equipped fire department. one could question details of the response to sars, such as implementation lapses that risked the spread of the disease from the hospital environment into the community, but overall the world's 'dress rehearsal' demonstrated far-sighted planning and coordination beyond anything ever attempted before on an international scale. the second category of initiatives is proactive and scientific: the attempt to discover new weapons to attack microbial threats. after half a century of dwindling resources for the fight against infectious diseases, the scientific and public health communities have successfully aroused worldwide awareness of the threat to health and security. they have, at least initially, attracted new levels of funding for basic research from both public and private sources, and they have set research agendas. the result has been an explosion of knowledge, grants, and publications with priority given to genomic approaches to microbes and vectors, to the development of vaccines, and to the search for new medications and diagnostic tools. naturally there are grounds for criticism of various aspects of these initiatives. there is, for example, general agreement that overall levels of funding remain inadequate to the extent of the crisis and that after initial enthusiasm, governments have not continued to increase their support. there are also reasonable grounds for disagreement as to the relative distribution of research efforts, with discussion, for example, about the balance struck between research against hiv/aids and that against such other major diseases as malaria, tb, and pandemic influenza. some have also questioned whether developing vaccines is the right paradigm on all fronts. for example, should priority be given to those diseases for which the human immune response gives grounds for optimism thaton the basis of historical experience -a safe and effective vaccine can be developed (e.g. influenza and dengue)? or should other strategies be followed with respect to diseases for which the human immune response makes the development of a vaccine a far more arduous and unpredictable endeavor (e.g. cholera and malaria)? nevertheless, although there is no basis for false confidence, global research efforts have been galvanized, and major advances have been made in the field of infectious diseases in comparison with the early decades after world war ii. there is also a consensus that the effort to find vaccines and medicines is vital and that it must be enhanced in order to replenish the quivers of clinicians and public health officials. what is more troubling in principle is that there are also systematic blind spots -areas of danger raised by those who first sounded the tocsin regarding emerging diseases that have not been addressed at all or only marginally and sporadically. broadly speaking, the global community has chosen to address those issues for which scientific and technological responses are appropriate, while giving little sustained priority to what might be termed the social, economic, and environmental determinants of infectious disease. here there is a considerable irony. the founding figures of the modern concept of emerging and reemerging diseases such as joshua lederberg and robert shope stressed that epidemics do not strike societies randomly or in accord with the caprices of angry gods. diseases instead reflect the relationships that human beings establish with one another and with the natural and built environments. they then spread by taking advantages of the fault lines created by demography, poverty, environmental degradation, warfare, mass transportation, and societal neglect. the very beginning of the iom's discussion of the new dangers was the recognition that our new vulnerability is not accidental but is the logical result of the type of society that we have become. in defining this vulnerability in a keynote speech in , for instance, lederberg stated: to our disadvantage, we have crowding; we have social, political, economic, and hygienic stratification. we have crowded together a hotbed of opportunity for infectious agents to spread over a significant part of the population. this condensation, stratification, and mobility is unique, defining us as a very different species from what we were years ago. ( ) if our problem results from 'condensation, stratification, and mobility,' there is a disturbing silence in the government response. ironically, the various agencies -niaid, the cia, the department of defense -tasked by the presidential directive with augmenting american preparedness in the fight against infectious diseases neither mention socioeconomic factors nor elaborate a long-term strategy to address them. the call to action aroused the will to find new means to attack microbes and their vectors, and to contain disease outbreaks in human populations, but not to ameliorate the underlying conditions that have made modern societies vulnerable in the first place. three crucial examples illustrate the problem. the first is condensation or the press of overpopulation. clearly unrestrained demographic growth as the world population approaches seven billion strains all resources, degrades the environment, gives rise to the megacities and peri-urban slums where dengue, tb, and cholera thrive, drives populations to intrude into forests where they are exposed to new zoonotic infections, and overwhelms educational, housing, and hygienic infrastructures. here, the medical and public health communities agree, is a driving factor in the new human vulnerability to emerging diseases. the remedies, moreover, are already known, involving voluntary universal access for women to family planning education and technologies. one of the few forums even to raise the issue was the 'first international conference on women and infectious diseases' held in atlanta, february - , , where it was noted that, 'women's health, in and of itself, rarely has been at the forefront of international development programs or national health planning and policies' ( ) . in the field of infectious diseases, this lacuna is especially glaring because women are, as the conference stressed, more susceptible to infections than men, both for biological reasons and due to their caregiving roles and their relative burden of unemployment and poverty. women, moreover, suffer more serious complications from infectious diseases, above all during pregnancy. a second illustration is stratification, the burden of poverty and inequality. nearly all of the leading studies on emerging diseases regard poverty and its sequelae of poor diet, substandard housing, lack of education, and inadequate access to health care as one of the chief determinants of epidemic disease. poverty prevents people from taking measures to protect their own health, it undermines the immune system, it complicates access to safe water supplies, it leads to overcrowding in unhygienic housing, and it creates patterns of labor mobility and migration that compromise health. health care workers and clinicians recognize the link between inadequate resources and disease, with the result that many of the leading epidemic infections are widely termed 'diseases of poverty' ( ) . the issue therefore surfaces in who campaigns to combat the three most important contemporary epidemics: hiv/aids, malaria, and tb. as the report addressing poverty in tb control stated: poverty is the greatest impediment to human and socioeconomic development. the united nations and its specialized agencies are focusing on poverty reduction as a leading priority. in the health sector, poverty represents a principal barrier to health and health care and, consequently, the world health organization has committed to integrate the promotion of pro-poor policies throughout its work. ( ) the reduction of extreme poverty and hunger also form part of the un 'millennium development goals' to be achieved by . except for exhortation and moral suasion, however, it is not clear that the who has developed specific plans to tackle the problem of poverty as a primary determinant of public health, and the promotion of greater equality is entirely ignored. more strikingly, neither issue forms part of the strategic public health thinking of the united states. american analyses recognize poverty as a factor creating an environment favorable for infectious diseases, but they avoid both poverty and inequality as matters of practical health policy. here is the antithesis of the strategic recommendation of the south african pediatrician nulda beyers, who commented: the western cape is in some ways a model of tb epidemiology . . .. tb is almost non-existent in the white population, but in the black and coloured populations, where unemployment is running at %, and malnutrition and crowded slum housing are the norm, tb deaths can reach per . if i had to put my money on only one option -science or social upliftthere is no doubt that social uplift would have the bigger impact. ( ) poverty, moreover, reinforces both condensation and mobility. poverty creates a vicious downward spiral by interacting with population pressure because impoverished women are unable to practice effective family planning. the population explosion of the st century is based in the poorest regions of the planet. given a free and informed choice, privileged families in the industrial world limit their fertility. at the same time, however, poverty also augments vulnerability to infectious disease by setting in motion great streams of mobile people -the poor who become migrants, refugees, and displaced persons, and who then crowd into slums, mining compounds, refugee camps, and homeless shelters. these are people who are at disproportionately high risk of falling ill and of transporting their microbial burden with them. finally, there is the question of access to care. here the position of the leading figures in the campaign to recognize the importance of emerging and reemerging diseases is strangely contradictory. the iom examined the managed care revolution in the united states and the implications of for-profit medicine for the preparedness of the nation to face infectious diseases ( ) . by , managed care already enrolled million americans and therefore dominated health care delivery. the performance of the managed care revolution, however, did not inspire the iom. on the contrary, it produced a list of the major problems that, in its view, managed care created for public health. this list was lengthy and devastating. according to the iom, managed care creates severe public health difficulties because it does the following: (i) it places such strict controls on reimbursements that it becomes an impediment to effective collaboration with the public health community; (ii) it lowers costs by fostering management of infectious diseases by nonspecialists; (iii) it promotes the shift from inpatient to outpatient treatment, where there are neither the specialists nor the infrastructure to diagnose or contain infectious diseases; (iv) it proliferates bureaucratic complexities that complicate prompt response to disease outbreaks; (v) it reduces the commitment to training and research; and (vi) it encourages excessive antibiotic use ( ) . by leaving tens of million of people in the united states without insurance coverage and therefore without effective access to care, for-profit medicine effectively removes them from the disease surveillance network. to the extent that uninsured people avoid care entirely or seek it only at a late stage of their illness, the prompt information on which effective public health depends is undermined. in addition, excluding people from coverage drives them further into poverty and creates an underclass of the marginalized. finally, managed care relentlessly cuts costs by squeezing out of the system the surge capacity on which populations depend in the event of a disease outbreak. nevertheless, despite these observations, the iom reached perfectly anodyne conclusions. it did not conclude that only a system that guaranteed universal access is compatible with defense against infectious disease threats. instead, it lamely urged a deeper partnership between the managed care industry and public health officials. for these reasons, one can only conclude that we are not, in fact, forever changed. on the contrary, on both the national and international levels the response to the challenge of emerging disease threats remains partial with major gaps that are potentially costly in terms of human life and suffering. the united states and the world health community have established a sophisticated and necessary rapid response system. they have also proclaimed -and partially funded -a new commitment to basic research aimed at finding new antimicrobial weapons. they have not, however, systematically addressed the underlying causes for the new vulnerability. man's mastery of malaria textbook of malaria eradication 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diseases preventing emerging infectious diseases: a strategy for the st century. atlanta: us department of health and human services declaration of commitment on hiv/aids global public health security who. international health regulations flunet as a tool for global monitoring of influenza on the web global surveillance, national surveillance, and sars bell dmworld health organization working group on international and community transmission of sars. public health interventions and sars spread sars: aftermath of an outbreak infectious disease as an evolutionary paradigm steps for preventing infectious diseases in women targets now set by g countries to reduce ''diseases of poverty addressing poverty in tb control: options for national tb control programmes tuberculosis experts back social reform managed care systems and emerging infections: challenges and opportunities for strengthening surveillance, research and prevention key: cord- -l w nkdo authors: dobbs, r. john; dobbs, sylvia m.; weller, clive; charlett, andré; bjarnason, ingvar t.; curry, alan; ellis, david s.; ibrahim, mohammad a. a.; mccrossan, maria v.; o'donohue, john; owen, robert j.; oxlade, norman l.; price, ashley b.; sanderson, jeremy d.; sudhanva, malur; williams, john title: helicobacter hypothesis for idiopathic parkinsonism: before and beyond date: - - journal: helicobacter doi: . /j. - . . .x sha: doc_id: cord_uid: l w nkdo we challenge the concept of idiopathic parkinsonism (ip) as inevitably progressive neurodegeneration, proposing a natural history of sequential microbial insults with predisposing host response. proof‐of‐principle that infection can contribute to ip was provided by case studies and a placebo‐controlled efficacy study of helicobacter eradication. “malignant” ip appears converted to “benign”, but marked deterioration accompanies failure. similar benefit on brady/hypokinesia from eradicating “low‐density” infection favors autoimmunity. although a minority of uk probands are urea breath test positive for helicobacter, the predicted probability of having the parkinsonian label depends on the serum h. pylori antibody profile, with clinically relevant gradients between this “discriminant index” and disease burden and progression. in ip, h. pylori antibodies discriminate for persistently abnormal bowel function, and specific abnormal duodenal enterocyte mitochondrial morphology is described in relation to h. pylori infection. slow intestinal transit manifests as constipation from the prodrome. diarrhea may flag secondary small‐intestinal bacterial overgrowth. this, coupled with genetically determined intense inflammatory response, might explain evolution from brady/hypokinetic to rigidity‐predominant parkinsonism. in , james parkinson described the shaking palsy, a rigid brady/hypokinetic syndrome with a characteristic tremor and stooped posture [ ] . consequent therapeutic milestones are few and far between: noting the wider antiparkinsonian effect of tinctures of deadly nightshade, used to control excessive salivation ( ) [ ] ; describing dopamine deficiency in basal ganglia ( ) , thereby instigating dopamine-substitution therapy [ ] ; and discovering the antiparkinsonian effect of an antiviral agent, amantadine ( ) [ ] . the way to the helicobacter etiologic hypothesis was paved, before the discovery of h. pyloriassociated gastritis ( ) [ ] , by observation of an excess of previously documented peptic ulcer in parkinson's disease ( ) [ ] , and speculation that an infectious agent was involved in both ( ) [ ] . explicit suggestion of a causal link followed [ ] . presence of intracytoplasmic inclusions, lewy bodies, in a characteristic distribution in brain was, until recently [ ] , considered the gold standard for designation of "parkinson's disease". however, whittling away the population with this syndrome to a core sample, according to clinical predictors of post-mortem brain histopathology, is not a tenable start point for unravelling causality. were the syndrome a manifestation of systemic disease, unifying pathology would be expected outside the brain. moreover, aged brains are frequently affected by more than one "neurodegenerative" pathology: parkinsonian and alzheimer often coexist [ ] . polymorphisms in a single gene are associated with parkinsonism alone or with other distinctive neurologic phenotypes, and with more than one type of pathologic hallmark, even in the same neurone [ ] . the same environmental insult might also result in neuronal deposition of different aberrant proteins, each having a spectrum of associated phenotypes [ ] . we use calne's broad clinical definition of idiopathic parkinsonism (ip) to describe a syndrome of unknown etiology [ ] . he regards the manifestations as the net effect of "doses" of genetic predisposition and environmental risk factors [ ] , their balance varying betweenand within-proband. however, there is no convincing evidence in ip of excessive exposure to environmental chemicals [ ] , and studies of genes controlling their metabolism have been unrewarding [ ] . our strategy and approach led to an infection hypothesis, implicating the gut (see defining and assembling the jigsaw pieces and table ), the initial therapeutic target being small-intestinal bacterial overgrowth (sibo), secondary to slow transit. indeed, constipation features in james parkinson's essay [ ] . in ip, frequency of defecation begins to deviate from that of controls three decades before the median age of neurologic diagnosis, two before diagnosis table generation of an "infection hypothesis" for idiopathic parkinsonism (ip), implicating the gut statistical model findings [ ] observational comparison nocturnal axial rotation in elderly ip probands, their spouses and control couples. rotation in spouses less than in controls, greater than in probands. a / / [ ] [ ] [ ] comparison measured facets of parkinsonism in elderly probands, their spouses, control couples. spouses significantly different from controls (toward parkinsonism) in measures of brady/hypokinesia, postural abnormality, rigidity, and frequency of seborrheic dermatitis. / [ , , ] relationship serum immunoglobulin concentrations to presence/ absence of (i) no overall difference in igm, a, or g with ip. b (ii) in controls, bradykinesia associated with higher iga and lower m, postural abnormality with higher iga, as if exaggerated ageing. (i) diagnosed ip, (ii) a parkinsonian feature in subjects without diagnosed parkinsonism, (iii) specified medication in ip probands. (iii) iga higher in probands with constipation warranting laxatives or taking antimuscarinic c . higher iga in ii and iii explained by a subclass: a systemic rather than mucosal response. / [ , ] (i) comparison between ip probands and controls of current and estimated past frequency of defecation. (i) frequency less in probands prodromally (from fourth decade of life). (ii) association serum iga with a discriminant index for presence/ absence parkinsonism based on current bowel habit. (ii) iga increased with index irrespective of subject group. (iii) differential effect of smoking on "difficulty passing a motion": laxative-like effect greater in controls. (iii) relative effect of tobacco smoking on bowel habit. [ ] comparison serum cortisol in ip probands and controls, taking into account: (i) current or past smoking, (ii) anti-parkinsonian medication and laxatives in ip. cortisol higher in ip. in controls, the lower the cortisol, the shorter the stride and the more the deterioration over years: relationship inverted in ip. (i) smoking tended to be associated with lower cortisol, irrespective of group. (ii) levodopa and dopaminergic agonists did not affect cortisol; selegiline and antimuscarinics d reduced it, constipation warranting laxatives tended to increase it. [ ] comparison serum il- and tnf-α in ip probands and controls. il- increased with age, effect of ip equivalent to more than years of ageing. of the first quartile of probands [ ] : a finding upheld prospectively by the association of infrequent bowel movements and subsequent diagnosis of parkinsonism [ ] . in ip, there is loss of, and damage to, colonic myenteric dopaminergic neurones. these, enteric plexus ganglia and physiologically related sympathetic neurones can contain lewy bodies, as does the dorsal vagal nucleus [ ] [ ] [ ] . pfeiffer, thinking on similar but noninfective lines, homes in on constipation as a marker of the genesis of ip [ ] . as a collateral hypothesis (table ) , we described the epidemiologic fit of helicobacter infection to ip (including familial clusters, evidence for early acquisition, long prodrome and association with water source) and proposed an autoimmune basis [ ] . by , we had proof of principle that infection contributes to ip, through case studies of anti-helicobacter therapy in gastritis, with and without associated helicobacter [ ] , and a hypothesis-testing study [ ] . in essence, "malignant" ip appeared converted to "benign" following successful eradication, but marked deterioration accompanied failure. in cases of late ip, eradicating helicobacter produced u-turns in both parkinsonism and cachexia [ ] . in probands receiving no or only stable long-t / antiparkinsonian medication, the randomized efficacy study contrasted effect, on the time course of ip facets, of week's successful anti-h. pylori therapy against placebo, and against failure [ ] . improvement in the primary outcome, mean stride length at free-walking speed, followed successful blinded active therapy (de-blinding being scheduled for year post-randomization). benefit on brady/hypokinesia did not fall off during the year after de-blinding, and was echoed in those given open-active anti-h. pylori therapy subsequent to placebo [ ] . improvement was irrespective of whether patients were yet receiving background antiparkinsonian medication. figure illustrates that gait can improve dramatically following h. pylori eradication therapy where biopsies are molecular microbiology positive but culture negative (and there is no evidence of sibo or other infection) [ ] . "low-density" infection may be sufficient to perpetuate autoimmunity. persistence even at this level of detection appeared detrimental [ ] . however, we are seeing disease modification, not cure. time trends in objective measurements of facets of parkinsonism dissociate following h. pylori eradication, with insidious increase in rigidity, in mirror image to the pattern for hypokinesia [ ] . mean torque required to extend the relaxed forearm [ ] increased following successful blinded-active therapy compared with placebo, again irrespective of antiparkinsonian medication status. this was echoed in those given open-active. size of effect was clinically relevant compared with baseline measurements. stepping back, peptic ulceration and, presumably, the zenith of gastric inflammatory response to helicobacter precede the diagnosis of ip, often by decades [ ] . in the uk, % of ip probands are serum h. pylori immunoblot positive, % urea breath test positive [ ] . immunoblot positivity is directly related to the blood lymphocyte count, lymphopenia, a feature of ip [ , ] . irrespective of evidence for current helicobacter infection, the serum immunoblot antibody profile predicts not just the presence and severity of ip, but also the progression over years [ ] . in contrast to peptic ulceration, the clinical manifestations of slow gastrointestinal transit become more apparent post-diagnosis [ ] . the h. pylori immunoblot is predictive of abnormal bowel function within ip, irrespective of current infection [ ] . helicobacter -associated autoimmunity might have wrought irreparable damage to the enteric nervous system. sixty percent of our probands, without current helicobacter infection, are lactulose-hydrogen breath test positive for sibo. obvious benefit accrues from helicobacter eradication, but loss of hypersecretion in response to antral gastritis may weaken the gastric acid barrier, making conditions for sibo more opportune. staging accommodates variability between probands in manifestations of ip, and within subject change in predominant manifestations. in stage (fig. ) , we propose that helicobacter infection evokes autoimmunity against mitochondria, and that this mechanism underlies brady/ hypokinesia predominant ip. most helicobacter infections are transmitted where there is close contact, as between parent or sibling and infant [ ] . this accords with the younger the child when a parent develops ip, the greater the risk of ip to that child [ ] , and with probands and their siblings sharing facets of the syndrome and increased prevalence of h. pylori seropositivity [ ] . in younger people, the prevalence of urea breath test positivity is falling (associated with better hygiene/increased exposure to antimicrobials), as is incidence and age-specific mortality of ip [ ] . however, transient h. pylori infection or persistence in low density may be sufficient to trigger/perpetuate autoimmunity, thereby setting the scene for the age-specific increase in ip in older people [ ] . we propose that, in stage , acquisition of sibo causes further mitochondrial dysfunction, resulting in a rigiditypredominant picture. chronic infection over a large mucosal interface ratchets up dose-related mitochondrial damage, its potency being magnified by polymorphisms for intense innate inflammatory response. any adaptive immunodeficiency might predispose to sibo. our early case studies show improvement in rigidity on eradicating sibo (lactulose-hydrogen breath test criterion), but reinfection/recrudescence is probable. the stages may table generation of " helicobacter hypothesis" for idiopathic parkinsonism (ip) year statistical model findings [ ] observational comparison of facets of parkinsonism, and h. pylori anti-urease elisa seropositivity, in ip probands and their siblings with controls. siblings significantly different from controls (toward parkinsonism) in measures of brady/hypokinesia, rigidity, abnormal posture, and frequency seborrhea/seborrheic dermatitis. odds ratio of for seropositivity in probands and siblings cf. controls. a [ ] explanation of facets of syndrome by h. pylori urease antibody in subjects with and without diagnosed parkinsonism. seropositivity unrelated to presence/absence facets in those who have not passed diagnostic threshold, but decreased with abnormal posture in ip. b [ ] relationship of increase in serum il- and tnf-α with age, and in il- and cortisol with parkinsonism, to h. pylori urease antibody. these immune/inflammatory responses not explained by antibodies measured in routine elisa. [ ] explanation of increase in serum cortisol with ip, over that in controls, by presence/absence of antibodies against vaca, caga, and urease-b. effect of antibodies independent of disease status: anti-vaca seropositivity associated with elevated cortisol, ip with additional elevation, neither anti-urease nor anti-caga adding to variance explained. [ ] (i) contrast of relationship of h. pylori urease antibody to age in subjects with and without diagnosed parkinsonism. (i) birth-cohort effect in elisa value (ev), seen in controls, obliterated in ip. probands twice as likely to be seropositive before . years. (ii) relationship of titer to severity ip. (ii) ev lower with greater global disease severity. b [ , ] contrast of relationship of serum immunoglobulin classes to h. pylori urease antibody in subjects with and without diagnosed parkinsonism. in controls, downward shift in igm with anti-urease positivity (equivalent to years ageing). in ip, igm higher than in controls in seropositive, c lower in seronegative. no seropositivity effect on iga and igg, either group. [ ] discrimination for seborrheic dermatitis by h. pylori serum immunoblot antibody profile in subjects without diagnosed parkinsonism. discriminant index for presence characteristic rash contained anti-caga (directly associated) and anti-vaca (inversely). d,e [ ] contrast of relationship between being underweight and inflammatory products in subjects with and without diagnosed parkinsonism. association of low body mass index with serum il- concentration specific to parkinsonism, unlike that with anti-vaca and anti-caga. [ ] explanation of failure of helicobacter eradication in ip by blood lymphocyte subset counts. failed eradication associated with lower b-cell count. / [ , ] comparison blood counts in ip probands and their spouses with routine general practitioner requests total lymphocyte counts in spouses and untreated probands similar, lower than in controls. higher counts in probands and spouses with vaca antibody. count in ip not explained by serum b or folate. probands' cd +, cd + and cd + counts lower than in spouses, but cd + + higher. f [ ] discrimination for parkinsonism, and explanation of its severity and progression, by h. pylori serum immunoblot antibody profile. predicted probability of being labeled parkinsonian greatest with anti-caga seropositivity and anti-vaca and -urease-b negativity. e clinically-relevant association between index and measures of disease facets and their progression in ip, despite potentially confounding effect of anti-parkinsonian medication. [ ] discrimination for abnormal bowel function (constipation and/or diarrhea) in ip probands and their spouses by h. pylori serum immunoblot profile. bowel function abnormal in % probands and in % spouses. fourfold increase in odds for abnormal function with urease-b band, sixfold decrement with outer-membrane protein band, irrespective of urea breath test status, nature of abnormality, or subject group. irritable bowel syndrome ( %) had same band associates. a in contrast elisa seropositivity not increased in (older) spouses (unpublished data further to [ ] ). b h. pylori antigens, other than urease, are known to stimulate cytokine production [ ] . they, like serum immunoglobulin classes [ ] , may be associated with facets of parkinsonism in prodrome/early disease. helicobacter infection is more likely a forerunner of postural abnormality and global severity in ip, than protective. c although cd + count is low [ ] , there may be increase in naïve b-cells, or b cells, producing polyspecific igm. alternatively, local sequestration of igm may fail in ip. there is systemic and basal ganglia (nigro-striatal) immune activation in ip [ , ] . whilst local brain inflammation does not usually signal out [ ] , systemic inflammation can communicate with the brain's immune system [ , ] . communication involving immune cells, antibody, or inflammatory products can occur where the blood brain barrier is naturally deficient, or when permeability is increased by circulating cytokines and cortisol. peripheral afferent nerve stimulation, including vagal, can activate microglia (brain's resident macrophages) [ ] . helicobacter infection could stimulate the vagus, sibo perhaps more so. peripheral infection would increase traffic and signaling into brain. the substantia nigra is regarded as particularly vulnerable to everyday insults, such as products of dopamine oxidation [ ] . its homeostatic mechanisms function at full stretch. nigral glial activation would increase stress, and recruit immune cells locally. mitochondrial dysfunction is described in substantia nigra in ip (complex i) and multiple-system atrophy (complex iv) [ ] , and in platelets in ip [ , ] . indeed "cybrid" cells containing mitochondrial (platelet) and nuclear dna, from donors with and without ip, respectively, had reduced complex i activity [ ] . our evidence for systemic, extraneuronal mitochondrial involvement in ip is of filamentous arrays encapsulated in double membranes in duodenal enterocytes (fig. , upper panels) [ ] . they are found among normal mitochondria in ip probands with current or recent h. pylori infection. our electron microscopists had not previously observed similar bodies, although two examples were found subsequently in archived duodenal biopsies from patients with human immunodeficiency virus (hiv) infection. hiv can be associated with parkinsonian features [ ] . there is an isolated report of similar mitochondrial inclusions in cerebral neurones in creutzfeldt-jakob-like disease [ ] . prion tubulovesicular structures are larger; cluster, free within cytoplasm; and not found at prion infectivity sites outside central nervous system. routine anti-mitochondrial antibody serology has been negative in our ip patients (unpublished observation). the cytoplasmic fluorescence assay may detect only mitochondrial surface binding. autoimmunity in ip may be directed against mitochondrial protein, neoantigen, dna, rna or chromatin. matrilineal inheritance, typical of mitochondrial disorders, is rare in parkinsonism [ ] , but absence of mitochondrial dna repair enzymes gives susceptibility to mutagens [ ] . the modern mitochondrial proteome is widely accepted as originating, in part, from endosymbiont bacteria [ ] . helicobacter dna might be incorporated into host mitochondria. despite evidence for occurrence of bacteriophage in h. pylori , transduction has not been demonstrated, but there is evidence for a conjugation-like mechanism and natural transformation between bacteria [ ] . while helicobacter infection may be the trigger, proinflammatory cytokines, such as tumour necrosis factor (tnf)-α , can intensify the mitochondrial damage [ ] . long thin mitochondria, sometimes with complex branching, become the predominant feature of probands' duodenal enterocytes subsequent to h. pylori infection and in the presence of sibo (fig. , lower panels) . this apparent hypertrophy, associated with rough endoplasmic reticulum, may compensate for hypofunction. a genetically determined, intense systemic, innate inflammatory response would accelerate the natural history of ip (fig. ) . however, an intense interleukin- β response could cause helicobacter to self-destruct, its "protective" urease continuing to produce ammonia in the face of powerful inhibition of gastric acid secretion [ ] . this could explain "low-density" infection but no gastric atrophy [ , ] in ip, and increase susceptibility to sibo. certain polymorphisms increase the risk of noncardiac cancer, seropositivity for antibodies against caga geneproduct escalates it further [ ] . variability in time course of ip may be determined analogously [ ] . exploratory studies in ip suggest greater prevalence of similar polymorphisms, and associations with early onset [ ] [ ] [ ] [ ] [ ] . here too, anti-caga seropositivity is a bad prognostic sign [ ] . mild acquired, adaptive immunodeficiency, flagged in later ip by lymphopenia [ , ] , may attenuate inflammatory response. this may be double-edged, guarding against "parkinsonian effects" of chronic inflammation, but leaving probands open to infection. "quieter" disease is usual in old age. tobacco smoking protects against parkinsonism, as it does for ulcerative colitis [ ] . this may be immunomodulatory; a pharmacologic effect on injurious (but "physiologic") serum cortisol elevation [ ] ; or just a laxative action [ ] . higher prevalence of h. pyloriseropositivity in smokers [ ] , irrespective of ip [ ] , might keep sibo at bay in ip. shifting ip/overlap diseases to a more appropriate nosologic grouping seems important to unravelling them. a systemic mitochondrial disorder may give a spectrum from "functional bowel disease" alone to its co-manifestation with parkinsonism (fig. ) . intensity and nature of inflammatory response is primarily a between-subject determinant [ , ] , acquired or inherited vulnerability of the basal ganglia is a susceptibility factor. the above scheme, with possibilities for pragmatic testing, grew out of an exploratory strategy, approaching from different clinical clues, addressing different questions, and assessing the balance of probabilities (tables and ) . the aim is a scientifically plausible etiologic fit. oversights in interpretation of a given statistical model within the series does not jeopardize the whole. particular hierarchical layers in the modelling are addressed below: there are biologic gradients between measures of ip and markers of inflammation. higher serum cortisol or tnf-α is detrimental in ip compared with controls [ , ] . serum cortisol and interleukin (il)- are elevated overall in ip, tnf-α is not. association of a low body mass index with increased circulating il- is specific to parkinsonism, by contrast with healthy controls [ ] . cachexia in ip is usually intractable and associated with rapid deterioration. where helicobacter infection was found, gastritis was not severe, but successful treatment produced a turnabout [ ] . where body mass index is normal and there is no evidence of helicobacter, ravenous appetite and night sweats are common, and may largely be attributable to sibo. the classical spousal approach to environmental causality was used. figure illustrates the short but highly significant "distance down the pathway" of spouses cohabiting with ip probands for half a century. marked, multifarious, relevant differences (physiologic/psychomotor/dermatologic) between spouses and control couples are difficult to explain by selective mating or learned/reactive behavior [ ] [ ] [ ] [ ] . these and lymphopenia, in a large group of probands and spouses [ , ] , suggest adult transmission. moreover, half of the latter probands and a third of their spouses had chronic bowel abnormality (criteria [ ] ) [ ] . seven percent of probands and % of spouses had diarrhea (unformed stool during at least three-quarters of past year, plus three or more bowel movements/day for half). neuronal damage in probands may evade a diarrheal response to a shared insult. biologic gradients between measures of ip and serum h. pylori immunobot antibody profile strengthen the case for causality [ ] . in controls without diagnosed parkinsonism, there were no such relationships, suggesting undefined bacterial pathogenicity or host susceptibility factors. indeed, the double-membraned bodies containing filamentous arrays in duodenal enterocytes might flag the course to parkinsonism. association of the h. pylori immunoblot profile with abnormal bowel function within ip [ ] , and with seborrheic dermatitis (frequent accompaniment of ip [ ] ) in subjects without parkinsonism [ ] , further implicates helicobacter. increasing prevalence of h. pylori anti-urease seropositivity with age is typical of the general population of socioeconomically advanced countries. this "birth cohort effect" is said to reflect greater early life acquisition of persistent infection in less hygienic times. in ip, there was an increased prevalence of anti-urease seropositivity before old age, allowing no significant age effect to be captured in anti-urease titer, despite demonstration of the birth cohort effect in contemporaneous controls [ ] . lack of birth cohort effect is also documented for peptic ulcer and gastric carcinoma, where causal links with h. pylori are generally accepted. in ip, there is also a differential trend in total serum igm concentration in relation to anti-urease titer [ , ] . h. pylori culture supernatants, whole bacteria, protein products, and h. pylori-specific regulatory t-lymphocytes inhibit human t-cell proliferation [ , ] . since lymphopenia in ip is more marked with h. pylori immunoblot negativity, it is unlikely to be due to direct inhibition, but might relate to helicobacter-triggered autoimmunity. unlike its effect on platelets in idiopathic thrombocytopenic purpura [ ] , h. pylori eradication has little impact on ip lymphopenia. mitochondrial damage in ip, and also oxidative stress, excitatory amino acid production and proteosomal damage [ ] , could be secondary to, or augmented by, infection. substantia nigra microglia are activated in ip [ ] , and secrete tnf-α [ ] . dopaminergic neurones express tnf-α receptors [ ] and up-regulate nuclear factor κb in the apoptotic pathway [ ] . basal ganglia and cerebrospinal fluid (csf) il- β and il- concentrations are raised [ , ] . cytotoxic t-lymphocytes are seen in relation to "degenerating" nigral dopaminergic neurones showing igg binding [ ] . these neurones appeared labelled for destruction, their proportion being less in severe/longer-standing disease. activated microglia persist, presumably continuing to mediate damage. in ip, there are activation markers on circulating tlymphocytes and csf monocytes [ , , [ ] [ ] [ ] [ ] blood and csf γδ + t-cells are increased [ ] . they are biased toward bacterial antigens (e.g. heat-shock proteins), associated with autoimmune conditions, and prominent in gut-associated lymphoid tissue. csf contains antibodies against bacterial heat-shock proteins [ ] . both csf and blood contain antibodies against sympathetic and dopaminergic neurones [ ] . csf from ip probands recognizes rat nigral dopaminergic neurones [ ] . injection of purified igg from ip serum into rat nigra selectively destroys dopaminergic neurones [ ] . purified igg from probands' csf or serum is toxic to them in culture [ , ] . toxicity depends on complement and microglial fcγ antibody receptors [ , ] . serum from ip probands also inhibits high-affinity neuronal dopamine uptake, but not gamma-amino butyric acid uptake [ ] . an underlying viral infection? the pandemic of encephalitis lethargica ( ) sparked the viral hypothesis. parkinsonism occurs in uncomplicated hiv infection, and when precipitated by opportunistic infections (e.g. toxoplasmosis) in acquired immunodeficiency syndrome [ , , ] . hiv sensitizes to anti-dopaminergic medication. parkinsonian features may be associated with hiv dementia. motor dysfunction compatible with basal ganglia damage is found in early hiv, and basal ganglia dopaminergic cell loss is seen without clinical parkinsonism. in simian immunodeficiency virus-infected monkeys, nigrostriatal dopamine is halved within months [ ] . seborrheic dermatitis is associated with both ip [ , ] and hiv [ ] . although the epidemiology of hiv is distinct, a relatively benign retrovirus could be pathogenic in ip. (lewy bodies are not reported in hiv [ ] ). as well as lymphopenia, damage to the enteric nervous system in ip might be viral in origin. jejunal autonomic denervation is described with hiv infection [ ] . enteroviruses infect via the gastrointestinal . means ( % confidence interval) are shown. p and c were matched for age and gender. p < . for predicted probability in ps cf. c + cs combined [ ] . tract and are associated with neurologic syndromes. a combined virologic approach is needed: current standardof-care diagnostic assays to address the clinical features of ip (table ) , pathogen discovery for uncharacterized viruses. antimicrobial medication could produce benefit independent of eliminating target organism(s). in rats, minocycline reduces inflammation, blood-brain-barrier permeability, and dopaminergic neurone damage, produced by intranigral injection of bacterial lipopolysaccharide [ ] . table outlines our strategy and approach to etiology and pathogenesis. investigational medicine yields clinical clues to elucidating processes and mechanisms, and pinpointing the systemic nature of ip has been ignored as a template for intervention: the dogma of a "cold" neurodegeneration demands that constitutional illness be attributed to other causes. a one-step pragmatic rather than an exploratory strategy is the mode. (e.g. an elegant pathologic study table strategy and approach to etiology/pathogenesis of an idiopathic parkinsonism step back consider whole entity reject dividing syndrome by clinical minutiae and nature of cellular protein aggregates define clinical syndrome and be aware of overlap diseases assemble all raw clinical clues, without selectivity avoid focusing on tip of an iceberg in a disease with a long prodrome early disease may hold clues masked in later stages. indeed, solution may be untenable without acknowledging preclinical state. question adequacy of measurement methodology reject subjective global scores. embrace valid, sensitive/specific, reliable measures (objective where possible) of disease facets, which can identify clinically relevant changes with time or intervention. a adopt exploratory statistical methodology for hypothesis generation; defer any pragmatic testing seek to explain clues by associations. measure potential biologic effect in small well-defined subject groups, taking into account candidate confounders and effect modifiers. carefully select control groups, avoiding convenient family (including spouses) and close contacts. collect observational data to generate statistical models, each focusing on different clue(s) or addressing a different question identify associations and effects. adjustments for multiple comparisons are inappropriate: false positives are not anathema at this stage, but failing to notice (or falsely rejecting) leads is. retain "odd-ball" results, pending future insights avoid peer pressures to conform, and demands for repetition. seek coherent explanation of associations with each clinical clue look for biologically plausible explanations and connections identify pieces which appear not to fit present conundrum to diverse experts to gain fresh insights. conduct pragmatic studies when testable cause/effect hypothesis generated obtain case study evidence for appropriateness of interventions licensed for treating suspected etiologic agent (i.e. explore a "new indication"). proceed to per-protocol analysis of randomized efficacy study of effect on facets of syndrome. where novel intervention is needed, seek pharmaceutical collaboration apply stages of pharmaceutical testing to candidate compounds with a view to licensing define chain of events in natural history elucidate process mechanistically define hierarchical ordering of interventions in established cases tailor optimal treatment for an individual by screening tests plan for prophylaxis in early clinical syndrome and preclinical state identify core event(s) and perpetuating circumstance(s) perform effectiveness studies on available interventions. use environmentally/genetically heterogeneous subject groups, with large sample size and outcome criteria appropriate to an effectiveness study. use intention-to-treat analysis to give generalizable results on benefit. continue to explore differential effects of intervention in per-protocol analyses to challenge robustness of hypothesis and suggest refinements. document adverse events and their predictors. control introduction into clinical practice initiate surveillance program. b be alert to unmasking further etiologic insights and unanticipated adverse events in long term. a they allow economy in sample size; give clarity in defining differential time trends between disease facets; and allow pre-and post-presentational states to be considered as a continuum, in a disease widely accepted as having a long prodrome. global scores, and even relevant subscores, are relatively insensitive to intervention [ ] : adding in the immutable can mask important changes. b required for "evidence beyond reasonable doubt" of clinical importance, as with discovery of h. pylori. sees ip as evolving from the gastrointestinal tract [ ] , but readers seize upon just one mechanism, migration along neural pathways.) assessment of ip by "global subjective scores" is ubiquitous. this is analogous to studying diabetes mellitus without blood glucose or cardiovascular risk factors. financial constraints on the risks associated with innovation mean that the same fields are reploughed. traditional funding has favored the laboratory-to-clinic approach of arbitrary selection of putative pathogenic mechanisms for detailed examination. it has been directed to patching up damage (e.g. by seeking to modify components of cellular mechanisms, and replace/restore dopaminergic neurones by surgical implants/intracerebral infusion of neurotrophins), rather than getting to grips with what might be driving the process. such dissociation is unwise: ongoing inflammation may jeopardize patching-up. the work was funded initially by the medical research council, and subsequently by the psychiatry research trust which received grants from the hayward foundation, the cyril corden trust and cecil pilkington charitable trust, and donations from abbott laboratories and astrazeneca. barclay's corporate social responsibility ambassador, nicholas smith, coordinated a fundraising program. malcolm plant coordinated the network of support from patients and carers. dr ron hutton helped with the bibliography. the review is dedicated to the memory of anthony dawson paul. an essay on the shaking palsy sur la paralysie agitante et la sclérose en plaque géneralisée distribution of noradrenaline and dopamine ( -hydroxytyramine) in human brain and its relation to diseases of the extrapyramidal system amantadine in the treatment of parkinson's disease unidentified curved bacilli on gastric epithelium in active chronic gastritis the association of gastro-duodenal ulceration with parkinson's disease dopamine disorder in duodenal ulceration gastric helicobacter pylori infection as a cause of idiopathic parkinson's disease and non-arteric anterior optic ischaemic neuropathy staging of brain pathology related to sporadic parkinson's disease parkinson's disease mutations in the lrrk gene cause autosomal-dominant parkinsonism with pleomorphic pathology parkinson's disease is not one disease criteria for diagnosing parkinson's disease parkinson's disease and exposure to infectious agents and pesticides and the occurrence of brain injuries: role of neuroinflammation candidate genes and parkinson's disease. where to next? stasis in the gut: the source of xenobiotic in idiopathic parkinsonism frequency of bowel movements and the future risk of parkinson's disease dopaminergic defect of enteric nervous system in parkinson's disease patients with chronic constipation parkinson's disease and megacolon: hyaline inclusions (lewy bodies) in enteric ganglion cells gastrointestinal dysfunction in parkinson's disease link between helicobacter pylori infection and idiopathic parkinsonism role of chronic infection and inflammation in the gastrointestinal tract in the aetiology and pathogenesis of idiopathic parkinsonism. part : eradication of helicobacter in the cachexia of idiopathic parkinsonism role of chronic infection and inflammation in the gastrointestinal tract in the aetiology and pathogenesis of idiopathic parkinsonism. part : response of facets of clinical idiopathic parkinsonism to helicobacter pylori eradication. a randomised, double-blind, placebo-controlled efficacy study helicobacter in idiopathic parkinsonism: a template for intervention in the role of inflammation in neuropsychiatric disease differential effect of helicobacter eradication on facets of idiopathic parkinsonism: explanation for predominantly hypokinetic or rigid syndromes lymphopenia in idiopathic parkinsonism and spouses of probands as a clue to an infectious environmental insult role of chronic infection and inflammation in the gastrointestinal tract in the aetiology and pathogenesis of idiopathic parkinsonism. part : predicted probability and gradients of severity of idiopathic parkinsonism based on h. pylori antibody profile duodenal enterocyte mitochondrial involvement and abnormal bowel function in idiopathic parkinsonism intrafamilial clustering of helicobacter pylori infection evidence for environmental causation of parkinson's disease siblings share helicobacter pylori seropositivity and facets of syndrome the epidemiology of parkinson's disease. bailliere's role of inflammation in gastrointestinal tract in aetiology and pathogenesis of idiopathic parkinsonism persistent pathogens in the parenchyma of the brain perceptions, emotions and immunity: an integrated homeostatic network molecular mechanisms for neurodegeneration. synergism between reactive oxygen species, calcium, and excitotoxic amino acids anatomic and disease specificity of nadh coq reductase (complex i) deficiency in parkinson's disease peripheral markers in parkinson's disease. an overview origin and functional consequences of the complex i defect in parkinson's disease parkinsonism in hiv dementia mitochondrial inclusions in neurons of creutzfeldt-jakob-like disease maternal inheritance in parkinson's disease normal oxidative damage to mitochondrial and nuclear dna is extensive on the origin of mitochondria: a genomics perspective helicobacter pylori -a conundrum of genetic diversity immunocytochemical analysis of tumour necrosis factor and its receptors in parkinson's disease the importance of interleukin β in helicobacter pylori associated disease increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms a direct, or surrogate, not necessarily unique, role for helicobacter infection in pathogenesis of idiopathic parkinsonism: analogy and contrast with gastric cancer genetic analysis of immunomodulating factors in sporadic parkinson's disease polymorphisms in the interleukin- alpha and beta genes and the risk for parkinson's disease association of interleukin- β polymorphisms with idiopathic parkinson's disease influence of interleukin- β gene polymorphisms on age-at-onset of sporadic parkinson's disease tumour necrosis factor gene polymorphisms in patients with sporadic parkinson's disease mortality in relation to smoking: years' observations on male british doctors cortisol is higher in parkinsonism and associated with gait deficit does the protective effect of tobacco smoking in idiopathic parkinsonism reside in its laxative action epidemiology of helicobacter pylori infection amongst randomly selected subjects from northern ireland parkinsonism: differential age-trend in helicobacter pylori antibody association of circulating tnf-α and il- with ageing and parkinsonism reduced axial rotation in the spouses of sufferers from idiopathic parkinsonism quantification of the cardinal signs of parkinsonism and of associated disability in spouses of sufferers objective measurement of activation of rigidity: diagnostic, pathogenetic and therapeutic implications in parkinsonism could seborrhoeic dermatitis be implicated in the pathogenesis of parkinsonism? associations of immunoglobulins, igg, iga and igm, with treatment in those with diagnosed parkinsonism, and with parkinsonian-like postural abnormality in those without association of serum immunoglobulin subclasses iga and iga with facets of parkinsonism could antibodies to helicobacter pylori urease explain the facets of parkinsonism quantified in subjects who have and have not passed the diagnostic threshold? does helicobacter pylori account for increase in serum interleukin- and tumour necrosis factor-α with age and interleukin- and cortisol with parkinsonism? systemic cortisol response to helicobacter pylori vacuolating toxin in parkinsonism and controls downward shift in serum igm with helicobacter pylori seropositivity evidence for autoimmunity triggered by helicobacter pylori in idiopathic parkinsonism discriminant index for seborrhoeic dermatitis based on antibody profile against helicobacter pylori the functional gastrointestinal disorders. diagnosis, pathology, and treatment -a multinational consensus helicobacter pylori-specific cd + cd high regulatory t-cells suppress memory t-cell responses to helicobacter pylori in infected individuals inhibition of t-cell proliferation by helicobacter pylori gamma-glutamyl transpeptidase helicobacter pylori and extragastric diseases -other helicobacters reactive microglia are positive for hla-dr in the substantia nigra of parkinson's and alzheimer's disease brains nuclear translocation of nf-kappab is increased in dopaminergic neurons of patients with parkinson's disease interleukin- -beta, interleukin- , epidermal growth-factor-alpha and transforming growth-factor-alpha are elevated in the brain from parkinsonian patients interleukin (il)- -beta, il- , il- , il- and transforming growth-factor-alpha levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and parkinson's disease a possible role for humoral immunity in the pathogenesis of parkinson's disease a correlation study between serum adenosine-deaminase activities and peripheral lymphocyte subsets in parkinson's disease parkinson's disease and immunological abnormalities: increase of hla-dr expression on monocytes in cerebrospinal fluid and of cd ro + t cells in peripheral blood lymphocyte populations in parkinson's disease and in rat models of parkinsonism increase in peripheral cd bright+ cd dull+ t cells in parkinson disease γδ + t cells are increased in patients with parkinson's disease humoral response to hsp and hsp in cerebrospinal fluid in parkinson's disease role of the pallidum: pathophysiological biochemical and therapeutic aspects antibodies in the csf of a parkinson patient recognizes neurons in rat mesencephalic regions experimental destruction of substantia nigra initiated by parkinson's disease immunoglobulins investigations on auto-antibodies in alzheimer's and parkinson's diseases using defined neuronal cultures parkinson serum carries complement-dependent toxicity for rat mesencephalic dopaminergic neurons in culture role of fcγ receptors in nigral cell injury induced by parkinson disease immunoglobulin injection into mouse substantia nigra slowed reaction time in asymptomatic hiv-positive patients hiv dementia: the role of the basal ganglia and dopaminergic systems autonomic denervation in jejunal mucosa of homosexual men infected with hiv presence of human mycoplasma dna in gastric tissue samples from korean chronic gastritis patients immunologic diseases, anti-inflammatory drugs and parkinson's disease: a case control study occupational risk factors in parkinson's disease minocycline reduces the lipopolysaccharide-induced inflammatory reaction, peroxynitrite-mediated nitration of proteins, disruption of the blood-brain barrier, and damage in the nigral dopaminergic system objective evidence for tolerance, against a back-ground of improvement during maintenance therapy with controlled release levodopa/ carbidopa key: cord- - socw hp authors: ortega, miguel Ángel; guzmán merino, alberto; fraile-martínez, oscar; recio-ruiz, judith; pekarek, leonel; g. guijarro, luis; garcía-honduvilla, natalio; Álvarez-mon, melchor; buján, julia; garcía-gallego, sandra title: dendrimers and dendritic materials: from laboratory to medical practice in infectious diseases date: - - journal: pharmaceutics doi: . /pharmaceutics sha: doc_id: cord_uid: socw hp infectious diseases are one of the main global public health risks, predominantly caused by viruses, bacteria, fungi, and parasites. the control of infections is founded on three main pillars: prevention, treatment, and diagnosis. however, the appearance of microbial resistance has challenged traditional strategies and demands new approaches. dendrimers are a type of polymeric nanoparticles whose nanometric size, multivalency, biocompatibility, and structural perfection offer boundless possibilities in multiple biomedical applications. this review provides the reader a general overview about the uses of dendrimers and dendritic materials in the treatment, prevention, and diagnosis of highly prevalent infectious diseases, and their advantages compared to traditional approaches. examples of dendrimers as antimicrobial agents per se, as nanocarriers of antimicrobial drugs, as well as their uses in gene transfection, in vaccines or as contrast agents in imaging assays are presented. despite the need to address some challenges in order to be used in the clinic, dendritic materials appear as an innovative tool with a brilliant future ahead in the clinical management of infectious diseases and many other health issues. infectious diseases are produced by pathogenic microorganisms, mainly bacteria, viruses, parasites, and fungi. since the dawn of civilization, these diseases have persisted as sources of human morbidity and mortality, representing the second cause of death worldwide and the main reason of b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [ , ] . pharmaceutics , , x for peer review of routes are prevalent ( figure ): the divergent strategy and the convergent strategy [ ] . in the divergent growth approach, first developed by tomalia et al. [ ] , the dendrimer synthesis proceeds inside-out from the core. the core reacts with abn monomeric units through the a functional group, while the b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [ , ] . a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [ ] [ ] [ ] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [ ] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [ ] , carbosilane [ ] , poly(phosphorhydrazone) (pph) [ ] , and polyester dendrimers [ ] (figure ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pharmaceutics , , x for peer review of a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [ ] [ ] [ ] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [ ] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [ ] , carbosilane [ ] , poly(phosphorhydrazone) (pph) [ ] , and polyester dendrimers [ ] (figure ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh or oh), figure a . pamam dendrimers exhibit appealing properties for biomedical studies [ ] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [ ] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [ ] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [ ] . however, they share the presence of multiple nh peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [ ] , figure d . they are often decorated with polar groups in order to achieve water solubility. they are pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh or oh), figure a . pamam dendrimers exhibit appealing properties for biomedical studies [ ] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [ ] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [ ] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [ ] . however, they share the presence of multiple nh peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [ ] , pharmaceutics , , of figure d . they are often decorated with polar groups in order to achieve water solubility. they are classified as "inorganic dendrimers" and exhibit relevant differences compared to traditional "organic dendrimers" such as pamam. furthermore, they have a great variability by altering the core and the amount and length of the branches. pph dendrimers, which can be quantitatively prepared up to generation [ ] , present phosphorus atoms throughout the entire dendritic scaffold and have been widely studied for biomedical applications [ ] , figure e . like carbosilane dendrimers, pph are also "inorganic dendrimers" with a huge variability in cores, branches, and peripheral groups, and require the attachment of polar groups in the periphery to become water-soluble. polyester dendrimers attract widespread attention in the biomedical field due to their biocompatibility and biodegradability. in particular, dendrimers based on , -bis(hydroxymethyl) propanoic acid (bis-mpa, figure f ) are commercially available. since the first reports in the early s, bis-mpa dendrimers have undergone an extraordinary increase in their structural complexity and control, which capitalized on a constant evolution of the synthetic strategies, from the traditional divergent and convergent routes to accelerated approaches based on chemoselective reactions [ , ] . since their first reports, dendrimers have been tested in a large number of in vitro and in vivo studies for multiple biomedical applications. the most explored use of dendrimers is their ability to carry drugs to the desired site of action, being an important resource in precision medicine [ ] . dendrimers protect the encapsulated or bound drug and allow the delivery to the desired site of action. as they can be customized, dendrimers improve the drug pharmacokinetics and solubility, control the drug release, enable more comfortable administration routes, and reach target sites with difficult accessibility such as the ocular system [ ] . another application that has raised great interest is the use of dendrimers in gene therapy. several dendrimers have been explored as non-viral vectors for dna and rna, enabling gene transfection to specific cells. this has been especially useful in in vitro cancer studies, where rna transfected to tumor cells can alter their mechanisms, making them more susceptible to treatment or hindering their uncontrolled division [ ] . on the other hand, dendrimers can act as immunomodulators, by either reducing or enhancing the immune response [ ] . the first approach can be very useful towards autoimmune diseases and allergies [ ] , while the second has been employed, for example, in cancer immunotherapy [ ] . the attachment of multiple antigen copies to the dendritic scaffold produces an increase in the immune response related to the multivalency and the decrease in the conformational freedom of the antigen. in infectious diseases, dendrimers can support the development of vaccines by acting as antigens carrier, providing stability, safety, and a sustained release. in addition, they can serve as adjuvants or can promote the uptake of the antigen by the antigen-presenting cells, thus enhancing its recognition and improving the effectiveness of the vaccine [ ] . another outstanding application is the use of dendritic materials in diagnosis [ ] , such as iron oxide magnetic nanoparticles decorated with dendrimers, which can be monitored through magnetic resonance, or the oxygen sensors, very useful in pathologies such as diabetes [ ] . dendrimers also enable a combined therapeutic and diagnostic action in a single platform, the so-called "theranosis" [ ] . the diagnostic capacity is provided by a specific molecule (e.g., a radionuclide) which, bound to the surface or encapsulated inside the nanoparticle, serves to detect its position in vivo by means of diagnostic imaging such as single photon emission computed tomography (spect). in order to fully benefit from the use of dendrimers as nanocarriers, it is essential to understand the mechanisms of interaction between the dendrimer and the different cargo ( figure ) [ ] [ ] [ ] : • encapsulation. the drug is physically trapped within the dendritic scaffold due to the spheroidal or ellipsoidal hollow cavities found between the different branches. these cavities are frequently hydrophobic, so they exhibit affinity towards drugs with poor water-solubility, and can also lead to h-bonding due to the presence of oxygen and nitrogen atoms. the main drawback of this approach is the tendency of the drug to rapidly leak in biological fluids, compared to a covalent conjugation approach [ ] . • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [ ] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [ ] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [ ] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. pharmaceutics , , x for peer review of • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [ ] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [ ] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [ ] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [ ] [ ] [ ] [ ] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section . the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia ( ) [ ] , more than , articles/patents have been published on dendritic materials, with an approximate increase of publications and patents per year since . key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [ ] . these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [ ] [ ] [ ] [ ] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section . the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia ( ) [ ] , more than , articles/patents have been published on dendritic materials, with an approximate increase of publications and patents per year since . key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [ ] . from the low rate of issued patents turning into commercial products, it becomes apparent that dendritic materials must face several challenges for the bench-to-bedside translation in the biomedical field. mignani et al. ( ) summarized the requirements to become a clinical candidate [ ] . to secure a successful development, the authors highlight the importance of complying with the good laboratory practice (glp) requirements to ensure the quality, reproducibility and reliability of in vitro and in vivo data. furthermore, the good manufacturing practice (gmp) is desirable, but emerges as one of the main challenges in dendrimer translation. indeed, dendrimer defects have been related to the failure of relevant preclinical trials [ ] . an accurate dendrimer synthesis and a thorough purification process are deemed necessary to ensure monodispersity and batch-to-batch reproducibility. this is a highly demanding challenge especially for high-generation dendrimers, multipurpose platforms, and large-scale production. many different strategies are currently explored to overcome these challenges in dendrimer translation, including engineering through critical nanoscale design parameters (cndps) [ ] ; accelerated synthetic approaches [ ] ; the improvement of analytical techniques, such as mass spectrometry; the accurate and simplified design of multipurpose platforms [ ] ; or the dendronization of materials to expand their uses [ ] . viruses are simple acellular organisms, which have coevolved with living beings to replicate and reproduce inside their cells, after the binding to specific receptors [ ] . nearly species of viruses have been reported; many of them can cause human diseases [ ] . some viral infections, such as respiratory ones, represent an important economic burden and a serious public health concern [ ] . antiviral drugs are the most common clinical tool to address these pathologies. to date, different drugs have been approved for the treatment of viral infections [ ] . however, some drugs have serious side effects, including nausea, insomnia, vomiting, allergic reactions, behavior disorders, cardiovascular complications, and dependency [ ] . opening new therapeutic windows, decreasing the side effects while maintaining their efficacy, is a key action. dendrimers contribute to the fight against viral infections [ ] , acting as microbicides per se or as drug nanocarriers, with relevant properties such as low systemic absorption, biocompatibility, water solubility, or simple formulation [ ] . the main uses of dendrimers in viral infections are herein addressed, the human immunodeficiency virus (hiv) being one of their most important targets. stis are highly prevalent worldwide, despite the efficient preventive tools (e.g., preservatives) [ ] . one of the most illustrative examples is the human immunodeficiency virus (hiv). hiv is responsible for the deterioration of immune cells, especially the target cd + t cells [ ] , thus aiding the entry of opportunistic pathogens that cause the acquired immunodeficiency syndrome (aids) [ ] . hiv transmission mainly occurs through body fluids exchange, mostly by sexual contact, but blood, breastfeeding or vertical transmission have also been described. according to the who, . million people were infected by hiv in [ ] . fortunately, around % decrease in infections was registered from to , but the treatment, diagnosis, and prevention remain as a global challenge, especially for developing countries with lack of resources. to date, antiretroviral therapy has shown an excellent outcome in the clinical management of aids. however, these drugs produce important side effects, including hiv resistance to the treatment [ ] . dendrimers represent an interesting alternative to minimize these side-effects and prevent the transmission of hiv and other viral or bacterial stis, figure [ ] . a promising approach relies on the use of dendrimers bearing anionic, sugar, or peptide moieties to prevent the entry of the virus in the target cell. these dendrimers block either the host cell or the viral receptors ( figure , top), such as the glycoproteins gp and gp located at the hiv envelope, two key proteins for the interaction and fusion of hiv with cd t cells. the most representative example is the anionic pll dendrimer spl [ ] . spl is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct ). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov- [ ] , enabling a fast-track development of tools to fight covid- . polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [ ] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [ ] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [ ] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [ ] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th response compared to th [ ] . pharmaceutics , , x for peer review of fusion of hiv with cd t cells. the most representative example is the anionic pll dendrimer spl [ ] . spl is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct ). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov- [ ] , enabling a fast-track development of tools to fight covid- . polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [ ] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [ ] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [ ] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [ ] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th response compared to th [ ] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a (ev ). ev belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [ ] . currently, neither vaccines nor therapies have been approved to prevent or treat ev infection, representing an important global problem but specially in the asian southeast [ ] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev in some clinical isolates in the low nm-high pm range [ ] . as ev is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev and hiv [ ] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov- [ ] , figure a ; ebola virus [ ] , figure b ; zika and dengue viruses [ ] , figure c ; hsv [ ] ; cytomegalovirus [ ] ; some flavivirus, such as the responsible of the japanese encephalitis [ ] ; and different human or aviary flu viruses [ ] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a (ev ). ev belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [ ] . currently, neither vaccines nor therapies have been approved to prevent or treat ev infection, representing an important global problem but specially in the asian southeast [ ] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev in some clinical isolates in the low nm-high pm range [ ] . as ev is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev and hiv [ ] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov- [ ] , figure a ; ebola virus [ ] , figure b ; zika and dengue viruses [ ] , figure c ; hsv [ ] ; cytomegalovirus [ ] ; some flavivirus, such as the responsible of the japanese encephalitis [ ] ; and different human or aviary flu viruses [ ] . bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [ ] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [ ] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of , multiresistant bacterial infections were estimated, being responsible for up to , deaths in [ ] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [ ] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [ ] or cystic fibrosis [ ] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [ ] . lectines are proteins which show a high specificity for sugars and bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [ ] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [ ] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of , multiresistant bacterial infections were estimated, being responsible for up to , deaths in [ ] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [ ] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [ ] or cystic fibrosis [ ] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [ ] . lectines are proteins which show a high specificity for sugars and their derivatives, being able to successfully recognize and agglutinate cells with glycosylated proteins or lipids. lecb binds to fucose, a mucin located in the epithelial mucosa, playing a key role in p. aeruginosa biofilm formation, in conjunction with leca, specific to galactose, although this binding is weaker and less important [ ] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd (ic = . µm) depicted in figure a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic ~ µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [ , ] . pharmaceutics , , x for peer review of less important [ ] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd (ic = . µm) depicted in figure a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic ~ µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [ , ] . one of the most representative examples of resistant bacteria is gram-negative bacteria. these microorganisms present a peptidoglycan wall located between the inner and the outer membranes, which is responsible for the higher resistance of gram-negative bacteria to immune system, and in case of lysis, promotes the release of proinflammatory substances known as lipopolysaccharides (lps), exacerbating the infection [ ] . this structure also confers gram-negative bacteria resistance to some external agents through multiple mechanisms, which are heterogeneous between species [ ] . examples of common mechanisms of bacterial resistance are the expelling of toxic residues that eliminate antibacterial agents; a decrease in bacterial permeability, through the alteration of the membrane channels; or the production of antibiotic inactivating enzymes [ ] . cationic dendrimers may be a solution to fight against resistant bacteria, as they can bind efficiently to the negatively-charged walls, destabilize it by displacing sodium and calcium ions and increase the membrane permeability [ , ] , figure bottom. however, despite their promising biocide activity, cationic dendrimers exhibit high toxicity towards mammal cells and require structural modifications to reduce their cytotoxicity, without affecting the efficacy [ ] . cationic antimicrobial peptides (amp) arranged as multiple antigen peptide (map) dendritic structures can also exert a potent antibacterial activity, decreasing the minimum inhibitory concentration (mic) and minimum bactericide concentration (mbc) of the peptide alone, while dramatically increasing the peptide stability to proteolysis [ ] . for example, a dendritic map structure based on the peptide qkkirvrlsa effectively inhibited diverse gram-negative bacteria (mic = - g/ml for e. coli atcc , p. aeruginosa atcc , and a clinical isolate of k. pneumoniae) [ ] . the higher local concentration of amp allows a multivalent binding and enhances the destabilizing effect of the bacterial membrane. dendrimers may also be used in the rapid diagnosis to discern between gram-negative or gram-positive bacteria, through a ph-dependent bacteria-selective aggregation occurring within min of adding the dendrimer to a bacterial suspension [ ] ; and as carriers of different drugs such as vancomycin or agents like led , both specific to gram-negative microorganisms [ , ] . chorioamnionitis is an infection in the amniotic liquid, which may cause neurological problems in the fetus due to the production of proinflammatory cytokines, escherichia coli being one of the most important etiological agents [ ] . this disease often occurs due to the ascent of microorganisms from vagina to uterus, although other pathways have also been reported like transplacental infection, retrograde seeding from the peritoneal cavity through the fallopian tubes or accidental invasive procedures [ ] . the use of antibiotics such as penicillins, cephalosporins, macrolides, and corticosteroids reduce the risk of developing chorioamnionitis [ ] . in this sense, dendrimers can increase their efficacy. for example, a study conducted by wang et al. ( ) in a guinean pig model demonstrated that hydroxyl-and amino-functional pamam dendrimers successfully encapsulate ampicillin [ ] . both dendrimers significantly decreased the uterus cytokines, compared to the usual therapy, but the amino-dendrimer exhibited a higher toxicity. dendrimers have been studied against other bacterial infections like chlamydia trachomatis, increasing the efficacy of vaccines by conjugating a peptide mimic of a chlamydial glycolipid antigen to a g -pamam-oh dendrimer [ ] ; some opportunistic agents, such as s. aureus, using different generation gn-pamam-nh dendrimers [ ] ; or genital infections, through a sustained and localized delivery of amoxicillin in the cervicovaginal region by pamam-peg dendritic hydrogels [ ] . overall, these studies show the potential role of dendrimers in bacterial infections, although a long road is still to cover, especially to decrease their cytotoxicity and increase their specificity. fungi are eukaryotic organisms responsible of a wide range of human infections. the prevalence of these diseases has increased in some countries, particularly in hospital areas and immunocompromised patients, candida, cryptococcus, pneumocystis, and aspergillus being the most representative families [ ] . data collected by the national nosocomial infections surveillance system (nnis) from january to april showed that up to % of nosocomial infections in eeuu were due to fungi, mainly candida species [ ] . however, current trends indicate the prevalence of aspergillus in different european states [ ] . the treatment of fungal infections is performed through antifungal (antimycotic) drugs, which produce some alterations in their cellular structures, thus inhibiting their development, viability and survival in a direct or indirect way. the most representative antifungal drugs include [ ] : polyenes (e.g., amphotericin b); azoles (e.g., imidazole, triazole); allylamines; lipopeptides; and miscellaneous agents, as griseofulvin, which inhibits microtubules and mitotic fuse, affecting cell division. unfortunately, the resistance developed by some fungi may hinder the clinical management of these infections [ ] . in addition, the great similarity between mammal and fungal cells can lead to cytotoxicity problems, being necessary to find molecules which selectively target fungal cells in a particular tissue [ ] . in this context, the use of nanoparticles like dendrimers may be an effective method to carry all these substances, maintaining their benefits and reducing their side effects. candida albicans, which is responsible of more than half of total fungal infections around the world [ ] , is often treated with ketoconazole, a dual-action drug capable of inhibiting both ergosterol synthesis and the transformation of spores to micellar infectious forms [ ] . however, ketoconazole is poorly water-soluble and can greatly benefit from the use of nanocarriers, which increase its bioavailability in the bloodstream. gn-pamam-nh dendrimers improve the administration of ketoconazole (up to -fold increase of antifungal activity using g dendrimer, compared to the drug alone), being even more efficient when used as hydrogel formulation [ ] ; as well as clotrimazole (up to -fold increase with g dendrimer) due to its hydrophobic and electrostatic interactions [ ] . similarly, these dendrimers have significantly improved the antifungal activity of amphotericin b, overcoming the low water solubility and nephrotoxicity issues [ ] . on the other hand, peptide dendrons have shown efficient anti-candida activity per se [ ] . the representative example shown in figure b , which displays four tryptophan residues in the periphery and a dodecyl chain in the focal point, produced % growth inhibition at µg/ml, as well as affected the biofilm viability and the hyphal and cell wall morphology. on the other hand, dendrimer-assisted gene therapy can prevent fungal infections. cationic pamam dendrimers, bearing -nh , -nme , and -nme + peripheral groups, were used with a ribozyme extracted from an intronic region of c. albicans, an rna molecule capable to cut other rna chain or even itself [ ] . these dendrimers inhibited the catalytic activity of candida ribozymes, with a generation-dependent activity (g > g > g ). however, the nature of the peripheral group did not produce a significantly different inhibition. consequently, the construction of the dendrimer depended on the size of the rna to inhibit and the charge ratio between dendrimer and rna [ ] . the use of rna:dendrimer complexes may have multiple applications, such as inhibiting protein synthesis, splicing or even rna delivery in an era where non coding rna are beginning to be used, thus showing the potential of dendrimers in targeted therapy [ ] . unfortunately, the diagnosis of fungal diseases is currently a challenge. the current golden standard for the detection of these infections relies on poorly sensitive and invasive methods such as cell culture and histopathological study of the infected tissue [ ] . new diagnostic tools are demanded, such as polymerase chain reaction (pcr), immunoassays, or tests capable of detecting specific fungal antigens such as beta-glycans [ ] . in this context, the use of nanostructures can play a key role in the development of new techniques that are more sensitive and effective in the early diagnosis of fungal infections. at present, very few studies report the use of dendrimers in the diagnosis of fungal diseases. takano et al. ( ) performed cdna microarray analysis using highly sensitive dendrimer-based technology in the detection of the rice pathogen magnaporthe grisea and the stage of infection in this pathogen [ ] . another potential approach is the dendrichips ® technology (dendris), relying on a phosphorous dendrimers coating which dramatically increases their sensitivity. this tool is capable of discerning up to respiratory bacterial pathogens from a single sample [ ] , and could be refined and targeted to other types of infectious agents such as fungi. parasites are organisms characterized by the need of other living organism or "host" in order to survive. parasites comprise an important variety of species of diverse complexity, from the simplest organisms such as protozoans to the more complex ones, such as plants [ ] . helminths and protozoans entail the main threat of human parasitosis; the clinical expression and its severity depend on the condition of the immune system of the host, as part of a tight interrelation [ , ] . prevention could be the most efficient mechanism of controlling parasitic infections but, despite the considerable efforts, there are no effective vaccines against any of the main parasites. accordingly, antiparasitic drugs are the pillar in protozoan control, when the simple prevention measures fail. however, the drug resistance of protozoans is becoming an alarming public health problem [ ] . dendrimers could be an effective tool in the early diagnosis or prevention of parasitosis, as well as a new treatment for some of these infections. protozoans comprise a diverse group of eukaryotic unicellular microorganisms that belong to protista kingdom. most common human infections caused by protozoans are related to plasmodium spp. and toxoplasma gondii, as well as trypanosoma and leishmania spp. protozoan parasites are responsible for a considerable mortality and morbidity all over the world, that affect more than million people [ ] . malaria is par excellence the main parasitic infection and it is caused by intracellular plasmodium parasites transmitted by mosquitoes of genus anopheles. approximately, % of world population lives in areas where malaria is transmitted, causing - million infections and . million deaths per year. in specific regions, such as sub-saharan africa, children below years-old conform % of the total deaths from malaria [ ] . an important epidemiological study of the prevalence of malaria in salomon islands revealed the high rate of asymptomatic patients, highlighting the need for a diagnostic tool with high sensibility and specificity to detect plasmodium [ ] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [ ] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp ) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [ ] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure . the role of dendrimers against helminthic schistosoma parasites has also been tested. the disease, known as schistosomiasis [ ] , begins when the larvae form penetrates in the organism through the skin and settle in mesenteric and pelvic veins of the host, turning into the adult form. here, the female parasites lay eggs, which could be eliminated through feces or urine or lead to complications like granulomas or intestinal, hepatosplenic and urogenital damage. this highlights the need for an early diagnosis. wright et al. ( ) confirmed the promising activity of magnetic particles coated with g -pamam-nh to concentrate the schistosoma circulating anodic antigen (caa), resulting in a -fold improvement in caa limits of detection for lateral flow assays [ ] . preventive measures like vaccines are key to stop the impact of schistosoma parasites in specific regions where they cause endemic infections. for example, infections by s. haematobium, s. japonicum, or s. mansoni affect over million people worldwide [ ] . lysine-decorated pamam dendrimers showed excellent behavior as vaccine vector, enhancing the immunoreactivity and efficacy of dna vaccine against s. japonicum infection [ ] . along the same lines, anderson et al. ( ) developed a dendrimer-based vaccine platform which encapsulate antigen-expressing replicon mrnas and generate a protective response towards others parasites such as toxoplasma gondii, and relevant viruses like ebola and h n influenza, with a single dose [ ] . the vaccine nanoparticle comprised an ionizable g -pamam dendrimer, a lipid-anchored peg and rna. these studies show the role of dendrimers in the development of new generation vaccines against different infections. specificity to detect plasmodium [ ] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [ ] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp ) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [ ] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure . leishmaniasis is a parasitic disease produced by leishmania protozoans that infects and multiplies in macrophage-rich organs and tissues of the reticuloendothelial system, mainly the liver and spleen. estimations indicate an increase of . to million cases per year [ ] . for decades, leishmaniasis has been treated with pentostam and glucantime, leading to drug resistance and serious side effects like pancreatitis. alternative broad-spectrum drugs with less toxicity, such as amphotericin b, have been used but they present disadvantages such as the high cost, low solubility, the side effects, and its less efficacy as antiparasitic agent. mannose-decorated g -ppi dendrimers improved the activity of amphotericin b for the treatment of leishmaniasis, reducing the toxicity by increasing the targeting in macrophage-rich organs [ ] . other nanocarrier, a dendritic-linear-dendritic hybrid based on peg and citric acid, figure c , also improved the solubility of amphotericin b ( times) and reduced the in vitro/in vivo toxicity. it resulted as potent as free amphotericin and glucantime in reducing the parasite burden and number [ ] . toxoplasmosis is a zoonosis caused by the ingestion of the parasite toxoplasma gondii. this disease has a chronic and silent course in the majority of population without immune system disorders, mainly causing symptoms such as low fever and muscular pain. this infection is usually treated with sulfadiazine [ ] , which has two disadvantages: it requires a high dose of the drug, which can produce severe side effects like high fever or allergic reactions; and it cannot reach target tissues where the parasite is typically localized. cationic g pamam dendrimers and anionic g . dendrimers efficiently solubilize sulfadiazine (up to molecules per dendrimer) and improve the penetration into the parasite, thus greatly reducing the required sulfadiazine dose and localizing the drug in muscle and brain, where t. gondii is usually present [ ] . the main dendrimer-drug interactions found were electrostatic, for cationic dendrimers, and hydrogen bonding, for anionic counterparts. furthermore, the anionic dendrimer showed intrinsic antiparasitic effect. other successful examples of antiparasitic dendrimers include pegylated pll dendrimers coated with chondroitin sulfate a, as targeted unimolecular micelles for the delivery of the antimalarial drug chloroquine phosphate [ ] ; and pamam dendrimers decorated with ethynil estradiol against trypanosoma cruzi, the parasite responsible for chagas disease, where the g dendrimer is times more effective than benznidazole at h and h (ic = . µm) [ ] . amoebae are eukaryotic protozoa extensively distributed in nature and human habitats, often acting as a host and reservoir of other microorganisms like giant viruses and some class of bacteria [ , ] . amoebae infecting humans are classified as parasitic, such as entamoeba organisms, or opportunistic free-living amoebae, such as acanthamoeba spp., balamuthia mandrillaris, sappinia diploidea, and naegleria fowleri (known as the brain-eating amoebae) [ ] . free-living amoebae do not require human infection for their life cycle, which presents two stages: trophozoite (active form) and cyst (inactive form). acanthamoeba and other free-living amoebae are responsible of serious diseases such as keratitis, encephalitis, and infections in immunocompromised patients in the central nervous system, skin, and lungs. fortunately, amoebae infections are relatively rare, although the high mortality associated with meningoencephalitis are of concern, mainly due to the late diagnosis and the lack of effective antimicrobial treatments [ ] . low generation cationic carbosilane dendrimers bearing ammonium or biguanide moieties have shown strong amoebicidal activity against trophozoites and cysts of acanthamoeba spp., figure d (ic = . mg/l; minimum cysticidal concentration mcc = mg/l) [ , ] . furthermore, they exhibit a synergistic effect with traditional drugs such as chlorhexidine, decreasing the required drug concentration - times using g dendrimers [ ] . the dendrimers target the amoeba membrane and produce inhibition and cell death. in this context, dendrimers could be a promising therapeutic alternative to properly manage amoebae infections. prions (prp) are infectious pathogens that cause neurodegenerative transmissible diseases such as spongiform encephalopathies, after a long period of incubation from to years [ , ] . prp sc are glycoproteins with an abnormal folding that are originated from a conformational change of normal prion proteins (prp c ), acquiring pathological properties [ ] . prion diseases can occur for two reasons: the infectious prion agent can transmit the pathological folding to the prp c ; or the prnp gen mutates, leading to a genetic variation of the prion disease [ ] . infectious prion diseases have gained importance since the epidemic bovine spongiform encephalopathy in united kingdom in that was transmitted to humans as a variation of the creutzfeldt-jakob prion disease [ , ] . the spongiform encephalopathies are named after the shape the brain acquires at final stages of the disease, caused by an increase of vacuoles and the holes that appear in the tissue. it is a rare but severe pathology, which leads to neuronal loss and, eventually, dementia. nowadays, there are some pharmacological interventions that delay the progression of prion diseases; however, there is no effective treatment to stop or avoid it in a significant way [ ] . in this field, nanomaterials such as dendrimers can exert a relevant activity, preventing the conversion of prp c to prp sc [ ] . due to the high affinity of amine groups to prions, phosphorus dendrimers decorated with tertiary amines in their surface are promising agents in the therapy against prion diseases, figure e [ ]. these dendrimers showed an inhibitory effect in the generation of prions (ic = (g ), . (g ) and (g ) µg/ml) and they had anti-infectious action in vitro and in vivo for some spongiform encephalopathies, some of them causing creutzfeldt-jakob disease [ ] . other dendritic families with even higher anti-prion activity include g -pamam-nh and g -ppi-nh (both ic = ng/ml) [ , ] . for example, ottaviani et al. ( ) showed that ppi glycodendrimers, comprising maltose or maltotriose units, prevented the aggregation of prp and aβ( - ) proteins due to the interference of dendrimers with the latency phase (nucleation) of the prion protein [ ] . dendrimers can also be useful for the diagnosis of prion infections. usually, elisa is used to determine whether the causing agent of the disease is a prion, but this method has important limitations as it cannot distinguish between prion chains [ ] . the nature of the prion agent will determine the etiology and course of the disease, changing the pathology parameters like the incubation period or the type of lesion [ ] . importantly, different generation pamam and ppi dendrimers were capable of distinguishing between the different types of prions [ ] . this study demonstrated that the susceptibility of a prion towards a particular dendrimer could be used to diagnose and predict the course of a disease. on the other hand, korri-youssoufi et al. developed an electrochemical biosensor comprising multiwalled carbon nanotubes modified with dendrimers and aptamers to detect prion proteins with a high sensibility (min. . pm) [ ] . in conclusion, dendrimers constitute a promising research line for fighting against prion diseases as they could slow down their progression and allow a reliable diagnosis of the responsible etiological agent. dendritic nanosystems could have a principal role as excipients in the pharmaceutical industry, providing that they are compatible, safe and effective nanocarriers in several administration routes [ ] . for example, ocular administration is still a challenge due to the unique physiology of the eye and the presence of numerous barriers. in this context, recent studies showed that dendrimers improved the time that a drug remains in the cornea after topical administration and they supply directed and sustained neuroprotection for retina [ ] . moreover, dendrimers could act as dna vectors, providing safety and reducing cytotoxicity compared to the viral vectors and intraocular injections used at present [ ] . dendritic polymers can easily improve the properties of different materials, such as cotton, to generate antimicrobial activity. for example, the addition of amine-functional dendrimers confers antibacterial activity against gram-positive bacteria (e. coli, p. aeruginosa) and gram-negative bacteria (s. aureus) and fungi (c. albicans) [ , ] , even after several washing cycles. sepsis is a life-threatening response to an infection, which happens when the immune system overreacts and starts to damage the patient's own tissues and organs. peptide-decorated pamam dendrimers inhibited the acetylation of transforming growth factor β-induced protein, thus improving the mortality and organ damage in the septic mouse model [ ] . additionally, gadolinium-containing g dendrimers can be used as mri diagnostic and prognostic biomarker of sepsis-induced acute renal failure [ ] . these studies open up a field in the treatment and diagnosis of sepsis with a not very high cost and that could be protective against infectious pathogens in certain circumstances like during hospital stays, surgeries or risky exposures. the design of metallodendrimers is a promising field to explore, which can open new avenues in the fight against infectious diseases [ ] . dendrimers are excellent platforms to control the attachment of a great variety of metal complexes with antimicrobial activity, such as silver(i), copper(ii), and zinc(ii), among others. the resultant metallodendrimers often exhibit a synergistic activity and improve the therapeutic response of the dendrimer or the metal complex alone. for example, demonstrated the impact of a single metal ion in the prevention of hiv infection [ ] . a carboxylate-decorated g ppi dendrimer, which inhibited hiv- infection of hec- a cells around %, reached % inhibition by attaching a single cu(ii) ion through its ethylenediamine core. the remarkable control on the dendritic structure enables an optimized antimicrobial activity, difficult to accomplish with other nanomaterials. the reader is referred to a recent review on this topic [ ] . dendrimers are an innovative tool in the treatment, prevention and diagnosis of serious infectious pathologies, as summarized in table . they emerge as a unique opportunity to overcome problems of traditional approaches, such as microbial resistance. nevertheless, the transition from the laboratory to the clinical practice still requires facing several challenges, such as the big scale production, the batch-to-batch consistency, the purity for clinical tests, or the regulatory obstacles. it should be kept in mind that most investigations have been conducted in experimental models in vitro or in animal models, so it is not possible to extrapolate these results to humans yet. however, the outstanding results obtained by vivagel ® in the prevention of viral and bacterial infections predict a brilliant future of dendritic materials in the fight against infectious diseases. 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/s - - -z sha: doc_id: cord_uid: c ut nan abstract/case report text oral lichen planus (olp) is a t-cell mediated chronic inflammatory tissue reaction in which presentation can range from asymptomatic plaques to painful, erosive, bullous, or ulcerative lesions. here, we present a year-old female with a novel ctla- variant, multiple autoimmune conditions, and unusual tongue lesions. our patient was healthy until years of age when she developed hashimoto's thyroiditis. at , she developed psoriasis. at , she was diagnosed with alopecia totalis and epstein-barr virus (ebv) with resultant and persistent anemia, thrombocytopenia, lymphopenia and neutropenia. she had chronic abdominal pain and diarrhea since age . esophagogastroduodenoscopy revealed lymphocytic esophagitis and active duodenal inflammation with increased intraepithelial lymphocytes. colonoscopy revealed mildly active chronic colitis with eosinophils. whole exome sequencing revealed a heterozygous c. dela (p.q rfs* ) pathogenic mutation in exon of ctla- . family history is remarkable: father (splenomegaly and psoriasis) and brother (autoimmune hemolytic anemia) have ctla haploinsufficiency with the same mutation. abatacept was initiated with re-growth of hair, improvement in cytopenias, improvement in psoriasis, and some reduction of gastrointestinal symptoms. since her abdominal pain persisted repeat endoscopies after six months of abatacept revealed persistent active lymphocytic esophagitis with some improvement in inflammatory injury in her duodenum and colon. physical exam revealed glossitis with a gel-like coating and ulceration on her tongue, xerosis along her face and scalp without other abnormalities ( figure) . she denied recent dental procedures, appliances, or tongue biting. her wbc ranged from - x ^ cells/l and hemoglobin . - . g/dl. absolute lymphocyte count ranged from . - . x ^ cells/l. immunologic evaluation revealed low iga and pan-low lymphocyte subsets (table) . ebv pcr ranged from - , copies/ml. tongue scraping revealed candida dubliniensis and she responded to days of fluconazole. two months later, she developed painful white patches along her tongue and subsequent kilogram weight loss recalcitrant to viscous lidocaine, antacids, and days of fluconazole. incisional tongue biopsy revealed ulceration with underlying granulation tissue with lymphocyte and plasma cell infiltration consistent with olp ( figure) . periodic acid-schiff diastase stain and grocott stain were negative. aerobic culture was normal. no fungus was isolated within days. epstein-barr encoding region in situ hybridization was negative. two weeks of topical dexamethasone lead to temporary improvement. her tongue lesions waxed and waned over the following months. due to persistent psoriasis, methotrexate was initiated without worsening in her tongue lesion. to our knowledge, this is the first case of olp reported in a patient with ctla- haploinsufficiency. ctla- haploinsufficiency may present with variable clinical phenotypes including increased risk of ebv viremia and malignancies. therefore, after ebv and malignancy are ruled out, olp may be a prudent diagnosis to consider in a ctla insufficient patient with unusual oral lesions. mutations of bcl b appear to be associated with lymphoproliferation and autoimmunity as well as susceptibility to herpes virus infections. additional research focusing on characterization of dna binding sites of bcl b as well as the downstream expression of associated target genes is needed. these data combined with longitudinal analysis of additional patients with confirmed bcl b mutations, will help clarify determinants of bcl b pathogenesis and highlight potential therapeutic strategies. consanguineous marriages in tribal cultures, such as that in the united arab emirates significantly increase the prevalence of autosomal recessive disorders. premarital genetic screening and counseling, thus, are expected to reduce the frequency of these diseases. in this pilot study, diagnostic exome sequencing was used in the premarital screening program to identify recessive pathologic variants preventable by premarital counseling. a total of pathologic or likely pathologic variants were identified in studied emiratis ( couples), averaging . variants per person. four percent of the persons had negative diagnostic exome sequencing; the remaining had one to eight variants per person. of the distinct variants, ( %) were novel. twenty ( %) couples had pathologic or likely pathologic variants of inborn errors of immunity (iei). two couples ( %) had iei pathologic or likely pathologic heterozygous variants in both partners imposing risk for autosomal recessive disease in the offspring. other eighteen couples ( %) had pathologic/ likely pathologic heterozygous variants present in only one person of the couple. total of sixteen ( . %) iei variant identified and eight ( %) were novel. fourteen known phenotypic iei diseases were recognized (table. ). these preliminary results support a need for nationwide premarital genetic screening, and primary immunodeficiency registry to identify common and novel pathogenic variants with high heritability rate. these results will aid adopting a preand post-connectional reproductive carrier counseling to reduce autosomal recessive diseases. also, it will assist the diagnosis of these complex diseases in our community. table pathologic or likely pathologic variants of primary immunodeficiency (pid). abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency disorder caused by defects in the phagocytic nadph oxidase complex, leading to increased susceptibility to infection and inflammatory or autoimmune disease. up to % of patients have gastrointestinal (gi) involvement and meet diagnostic criteria for inflammatory bowel disease (cgd-ibd). objectives: we analyzed cgd patients from the united states immunodeficiency network (usidnet) registry to determine whether ibd may change the presentation, treatment, and outcomes of cgd patients, as compared to those without ibd. methods: a retrospective evaluation of cgd cases from the usidnet registry was completed. cgd-ibd was defined as the presence of any major physician-reported inflammatory, non-infectious gi tract disease manifestation, including crohn disease, ulcerative colitis, ibd endoscopy findings, gi fistulas, gi strictures, gi obstruction, and proctitis. demographic information, genotypes, symptoms and conditions, infections, antimicrobial therapies, immunomodulator use, and allogeneic hematopoietic stem cell transplantation (hsct) data were analyzed. results: patients with a diagnosis of cgd were identified. met criteria for ibd; were categorized in the non-ibd group. crohn disease and colitis were the most common gi disease manifestations in the cgd-ibd group (n= ), followed by gi fistulas (n= ). cgd-ibd patients had an increased average frequency of infections ( . events/patient) compared to the cgdnon-ibd group ( . events/patient). in both groups, lower respiratory tract infections were the most common infection type and aspergillus was the most common organism. enteric organism infections were more common in ibd patients. temporal data regarding the timing of infections were not available. immunomodulators, including biologics and interferon-gamma, were used at a significantly higher rate in ibd patients compared to non-abstract/case report text down syndrome (ds) is characterized by the occurrence of three copies of human chromosome (hsa ). these patients often develop chronic mucocutaneous candidiasis (cmc) and autoimmune thyroiditis, mimicking patients with heterozygous gain-of-function (gof) stat mutations, which enhance cellular responses to the three types of interferon (ifn). hsa contains a cluster of four interferon receptor (ifn-r) genes: ifnar , ifnar , ifngr and il rb. a gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with ds. we report high levels of ifn-αr , ifn-αr and ifn-γr expression on the surface of monocytes and ebv-transformed-b (ebv-b) cells from ds patients. levels of ifn-ɣr , encoded by a gene on chromosome , were similar in the immune cells of ds patients and healthy controls. total and phosphorylated stat (stat and pstat ) levels were constitutively high in unstimulated and ifn-α-and ifn-γ-stimulated monocytes from ds patients, although less so than those in patients with gof stat mutations. following stimulation with ifn-α or -ɣ, but not with il- or il- , pstat and ifn-ɣ activation factor (gaf) dna binding activities were significantly higher in the ebv-b cells of ds patients than in controls, this response resembling the dysregulated responses observed in patients with stat gof mutations. plasma type i ifns concentrations were high in about % of the ds patients tested. a genome-wide transcriptomic analysis involving principle component analysis and a comparison of interferon modules was performed on circulating monocytes. it showed that ifn-stimulated genes (isgs) were expressed more strongly in ds than in controls. ds monocytes have intermediate levels of ifn-α-and ifn-γ-induced isgs relative to monocytes from healthy controls and from patients with gof stat mutations. by contrast to patients with gof stat mutations, circulating th counts were normal and the proportion of terminally differentiated cd + t cells was high in ds patients. the constitutive upregulation of type i and type ii ifn-r, at least in monocytes of ds patients, may therefore contribute to the autoimmune diseases observed in these individuals. scid screens (positive screen defined as trec values less than units/ul per statewide criteria). results forty nine neonates were identified with low trec values. ( %) of these infants had repeat trec screening, ( %) of which were found to have a positive second trec screen. lymphocyte subsets were evaluated in of these infants and of which were noted to have lymphopenia (defined as absolute lymphocyte count less than ). infants were noted to have low cd levels (defined as < cells/ul) and were noted to have low cd levels (defined as less than cells/ul). of note, % of the infants with cd and cd lymphopenia had normal repeat trec levels. of the infants were noted to have low b cell levels (defined as < cells/ul). infants had quantitative immunoglobulin levels and of these two were noted to have igg levels less than . of note one infant was diagnosed with partial digeorge syndrome via microarray. in our study population, no infants were diagnosed with scid. discussion our study shows that testing for trec levels on newborn screen may be beneficial in identifying not only scid, but also other immunologic conditions. infants in our study had evidence for both cell mediated and humoral immunodeficiency which necessitated further workup and follow up from allergy and immunology specialists. it may be beneficial to develop further programs to track infants identified with abnormal trec levels on newborn screens to determine if they develop signs of immunodeficiency syndromes later in life. abstract/case report text the patient was transferred to our adult clinical immunology transition clinic for low igg and iga, elevated igm and b cell lymphopenia, treated with subcutaneous gamma globulin. his medical history was relevant for recurrent respiratory infections since two years-old, failure to thrive and developmental delay. he also developed chronic auto-immune hemolytic anemia (aiha) at ten years old, accompanied by prominent lymphoid hyperplasia. our initial evaluation at the age of twenty years old showed massive polyadenopathy and splenomegaly. work-up confirmed flair-up of aiha. at that point, the diagnosis of apds was raised. he also had mild intellectual impairment and dysmorphic features such as a mild degree of ocular depression, deep-set eyes, vaguely triangular face, small chin, but had normal stature. a customized panel for usual genes involved in classic hyperigm syndromes, apds, noonan and kabuki syndromes came back negative. at years old, an urgent coloscopy was performed because of acute abdominal pain and showed diffuse ileal lymphoid hyperplasia. biopsies confirmed reactional lymphoid hyperplasia without infection nor malignancy. a second genes ngs panel associated with pid identified a heterozygote mutation in tap ; expression of hla class was normal on flow cytometry. the patient was then started on sirolimus for an "apds-like syndrome" despite the lack of genetic confirmation. six months after introduction of mtor inhibitor, his abdominal pain had completely disappeared. tep scan showed complete resolution of axillar, retroperitoneal and inguinal lymph nodes and significant regression of splenomegaly. a third large non-biased + genes ngs panel revealed a pb de novo deletion that included the splice site of pik r exon typically involved in apds : c. _ + del p.(asp glufs* ) , which was missed by the first two panels. indeed, oligonucleotide-selective sequencing technology used for the previous panels was associated to mapping errors of short reads and difficult detection of large deletions. interestingly, the patient also presented some but not all dysmorphic features of short syndrome which is related to pik r haploinsufficiency. in this new era of genetic testing, this case is a reminder that we need to be aware of the pitfalls of genetic tests and that clinical judgment is still our best diagnostic tool. abstract/case report text intro: patients with chronic granulomatous disease (cgd) are theorized to have a lower risk of malignancy related to their lack of free radical formation. there are relatively few reports of malignancy described in patients with cgd. we report three cases of malignancies in the large cohort cgd population at the national institutes of health followed between - to add to the seven cases described in the literature. case : a -year-old man with x-linked chronic granulomatous disease with a history of severe inflammatory bowel disease, who presented with progressive left-sided chest pain in , decreased appetite and weight loss. transthoracic lung biopsy showed atypical cells and a pet/ct showed abnormally dense mesentery and widespread hypermetabolic abnormalities. a mesenteric biopsy showed metastatic pancreatic adenocarcinoma. palliative care was initiated. patient expired four months after diagnosis. case : a -year-old man with x-linked chronic granulomatous disease and severe inflammatory bowel disease requiring total proctocolectomy and who had been remotely treated with infliximab, presented in with right upper quadrant pain. abdominal ultrasound and mri of the liver showed multiple liver lesions. biopsy of these lesions revealed hepatocellular carcinoma. patient underwent two courses of radiolabeled itrium spherules. however, his disease progressed and he expired approximately five months after diagnosis. c a s e : a n -y e a r-o l d m a n w i t h x -l i n k e d c h r o n i c granulomatous disease and inflammatory bowel disease who presented in with fevers, abdominal pain and pancytopenia. during the course of his hospitalization, he developed sepsis which led to his demise. on autopsy, an incidental finding of papillary thyroid carcinoma was made. discussion: these three patients all had poorly controlled inflammatory bowel disease. additionally, patients with cgd are typically exposed to higher doses of radiation, leading one to expect higher rates of radiation induced malignancies. however, there are still relatively few case reports of cancer in the cgd population. tissue biopsy is necessary for diagnosis. due to end organ damage secondary to the underlying disease in the first two cases, treatment options were limited. managing infections during chemotherapy can be complex due to drug interactions with chemotherapeutic agents. abstract/case report text myeloperoxidase (mpo) deficiency is the most common inherited defect of phagocytes that impairs microbial killing since the toxicity of the respiratory burst is dampened without myeloperoxidase release from the azurophilic granules. a significant portion of these patients remain asymptomatic, however there is a clinically variable phenotype that can present if they do become symptomatic. fungal infections with candida strains appear to be the most frequently reported. we present an adulthood case of recurrent invasive candidal disease due autosomal recessive myeloperoxidase deficiency from a pathogenic missense variant in the mpo gene (c. c>t (p.arg trp)). a -year-old caucasian male was in his normal state of health without any major illnesses until years of age when he was diagnosed with candida osteomyelitis of the heel, followed by cryptococcal meningitis the following year which ultimately required a ventriculoperitoneal shunt. in , he had a prolonged hospitalization after presenting with lethargy, headache and vomiting that culminated in seizure activity and prompted an emergency room visit. imaging at the time showed ventriculomegaly, and fluid from the shunt revealed yeast, but no bacteria. he was started on broad spectrum antifungal therapy and admitted for further management. cerebral spinal fluid and blood cultures confirmed invasive candida albicans meningitis. during this hospitalization, he also developed sepsis secondary to serratia marcescens. because of the pathogens that were being isolated, our service was consulted. of note, our patient does not have diabetes mellitus. a neutrophil oxidative burst assay showed an absent respiratory burst compared to control. a primary immunodeficiency panel to identify genetic variants was also sent to invitae. variants in cyba, cybb, ncf , and ncf were not identified, making chronic granulomatous disease less likely. peroxidase staining was negative on neutrophils and normal on eosinophils, suggesting a diagnosis of mpo deficiency. this led to mpo gene sequencing for deletion and duplication analysis. a homozygous pathogenic variant consistent with a molecular diagnosis of a mpo related condition was identified. immunoblotting of patient-derived immune cells demonstrated an absence of mature enzyme. although not typically indicated, given the severity of his presentation, our patient remains on fluconazole for long term prophylaxis. his younger brother also had a history of invasive disease with candidaosteomyelitis and meningitis. a neutrophils oxidative burst assay showed similar results in his brother and similar results with peroxidase staining, also suggesting a diagnosis of mpo deficiency. confirmatory genetic testing has not been performed yet. their father, who reported severe skin infections with candida, had peroxidase stains performed on neutrophils and eosinophils which were both normal. we have presented a patient without a significant history of diabetes mellitus who developed invasive disease from candida and serratia and was ultimately diagnosed with myeloperoxidase deficiency. abstract/case report text introduction: juvenile xanthogranuloma (jxg) is an often benign, histiocytic proliferative disorder of the mononuclear phagocytic system. patients typically present with localized cutaneous lesions. systemic disease, especially central nervous system involvement, rarely occurs but has significant morbidity and mortality risk. no standard evaluation nor therapy regimen exists for systemic jxg and little is known about the genomic alterations underlying its pathology. case report: a full-term male infant presented at months of age with post-prandial abdominal pain, fevers, altered mental status and weight loss. abdominal ultrasound and ct identified renal masses. a chest ct was obtained showing a paraspinal mass with possible neural foramina extension. mri brain and total spine was consistent with diffuse leptomeningeal disease involving the left frontal convexity, brainstem, cerebellum, and multiple cranial nerves. abnormal enhancement was also present along the entire surface of the spinal cord extending into the cauda equina with additional enlargement of the cervical/upper thoracic cord with intramedullary enhancing masses and a right paraspinal mass. renal biopsy yielded a pathologic diagnosis of disseminated jxg. integrative clinical sequencing of the mass identified a somatic driving alk rearrangement (kif b-alk in-frame fusion). tumor and matched germline dna sequencing did not detect any alterations in the ras/mapk pathway. bone marrow biopsy was negative for disease with cerebrospinal fluid analysis showing numerous monocytes and macrophages consistent with jxg. the patient was started on therapy consisting of systemic dexamethasone, intrathecal methotrexate/hydrocortisone and systemic intravenous cytarabine. his first cycle was complicated by pseudomonas aeruginosa bacteremia and gangrenous cellulitis of the perianal region, treated with systemic/topical antibiotics and topical gm-csf. following completion of the initial cycle of therapy, the patient was noted to have declining neurologic status, including seizure-like activity. repeat mr imaging revealed worsening cns disease with new subdural fluid collection and progression of leptomeningeal enhancement and intramedullary cervical lesion. in light of disease progression, the decision was made to continue dexamethasone treatment, but add adjunct intrathecal cytarabine, and transition to targeted alk inhibition via daily oral ceritinib, given its predicted cns penetrance followed by ceritinib in combination with systemic intravenous clofarabine. significant clinical and radiographic improvement was noted with the new targeted treatment regimen. ceritinib therapy was tolerated well overall after a % dosing reduction made for initial grade gastrointestinal toxicity and grade hypertriglyceridemia (non-life threatening but level > mg/dl). following continued treatment with daily ceritinib and completion of cycles of clofarabine therapy, our patient experienced complete disease remission. he continues to do well on daily ceritinib monotherapy with plan to complete an additional year of therapy. conclusion: our report highlights the potential benefit of real-time integrative clinical sequencing in the management of systemic histiocytic lesions, specifically non-langerhans cell conditions. it has the potential to identify novel somatic genetic alterations, other than the typical lchassociated braf mutations of the mapk pathway, that may be therapeutically targetable. treatment with nd generation alk-inhibition in our pediatric disseminated jxg patient was a novel, biologicallyrationale management approach with minimal toxicity and potentially contributed to his complete remission. abstract/case report text background: c glomerulonephropathy (c gn) is a progressive kidney disease with the predominant pathological feature of c deposits around the glomerular capillaries. c gn patients suffer from dysregulated activation of the alternative pathway as the result of autoantibodies or congenital genetic defects that stabilize cleavage of c . despite therapy involving immunosuppression and complement-pathway inhibition, the prognosis for c gn is poor. we report a patient with autoantibody-mediated, refractory c gn who demonstrated no improvement on rituximab but achieved sustained remission on bortezomib. follow up studies after one year demonstrated clearance of the culprit autoantibody, normalization of c levels, and improved pathologic appearance of the kidneys. this case supports the idea that c gn is frequently driven by pathogenic autoantibodies that may not clear with rituximab alone. plasma cell directed therapy has the potential to clear these autoantibodies and halt the progression of disease. case presentation: we report the case of a hispanic male with chronic renal dysfunction initially diagnosed with membranoproliferative glomerulonephritis on renal biopsy at years of age. despite cellcept and prednisone, over the next years, he had worsening proteinuria and an increase in protein-to-creatinine ratio. renal biopsy suggested c gn, and lab studies revealed a factor-h autoantibody and c level below the assay limit of detection. after initiating eculizumab, the proteinuria temporarily improved; however, the proteinuria eventually worsened, and he was referred to immunology. we hypothesized that the factor h-binding autoantibody was the cause of dysregulated c cleavage and disease progression, and blocking the terminal complement pathway with eculizumab would not halt upstream c -mediated kidney injury. at the age of , rituximab and plasmapheresis were administered to clear the factor-h autoantibody. three months after rituximab administration, the factor-h autoantibody level decreased to the normal range, but he continued to have significant proteinuria with low serum albumin and undetectable c level. we concluded that the relevant autoantibody was not solely produced by differentiating memory b cells, so we decided to target the plasma cell compartment. bortezomib was started at the age of , and eculizumab was continued given his initial response to treatment. after adding bortezomib, factor h autoantibody levels dropped below prior levels and serum c level normalized. renal biopsy at the age of showed evidence of imp r o v i n g c d e p o s i t i o n a n d l e s s p r o m i n e n t g l o m e r u l a r hypercellularity, with stable mesangial hypercellularity, interstitial fibrosis, tubular atrophy, and sclerotic glomeruli. although his proteinuria did not worsen, it remained persistent, suggesting that earlier introduction of bortezomib could have prevented disease advancement. there has been no further progression of kidney failure. conclusions: the majority of c gn patients harbor autoantibodies to components of the alternative pathway of complement. this case provides evidence that at least some of these autoantibodies are indeed the cause of complement dysregulation, and thus are prime targets for therapy. b cell targeting therapies may be inadequate to decrease autoantibody levels for some patients. early initiation of bortezomib, or other plasmacell directed therapy, may effectively induce complement normalization and disease remission in these cases. inhaled corticosteroid and a long-acting bronchodilator. he has no family history of immunodeficiencies or congenital disorders. computed tomography(ct) chest showed bronchiectasis. his complement studies and isohemagglutinin titers were also normal. his serum immunoglobulin(ig) and lymphocytes on presentation are shown in table . he had a poor response to polysaccharide pneumococcal vaccination. he was diagnosed with combined igg /igg subclass/iga deficiency and was started on immunoglobulin replacement therapy and prophylactic rotating antibiotic therapy. thereafter, his clinical course markedly improved with a reduction in the frequency of rti's as well as the number of bronchiectasis exacerbations. there was high suspicion for an underlying genetic disorder based on his constellation of neurodevelopment disorders and immunodeficiency. cytogenetic evaluation with array comparative genomic hybridization(cgh) analysis showed duplication of xq and xq consistent with mds. discussion: mds is caused by duplications involving the mecp gene locus of the x chromosome at xq . it has a % penetration rate in males whereas females act as carriers and are usually unaffected. rarely, cases of de novo mutations causing mds have been reported. chromosome microarray analysis is currently the best initial clinical test when mecp duplication syndrome is suspected. management needs a multidisciplinary approach involving geneticists, neurologists, ophthalmologists, physical medicine and rehabilitation specialists, psychologists, gastroenterologists, and allergy and immunology specialists. prophylactic treatment with ivig and antibiotics has been the standard of care for immunodeficiency in these patients. prognosis is guarded and most male patients die in the mid to late 's because of severe rti's secondary to immunodeficiency. conclusions: this case confirms the association of mds with combined iga and igg subclass deficiencies. clinicians should consider pursuing genetic evaluation for mds in patients with neurodevelopmental disorders and immunodeficiency because the diagnosis of the syndrome can change the overall approach to management and expectations in prognosis. abstract/case report text introduction: c nephritic factor is an autoantibody that binds to the alternative pathway c convertase (c bbb). this results in unchecked overactivation of the alternative complement pathway, which can lead to renal disease, partial lipodystrophy, retina disease, and frequent infections. in this case, we present a patient with partial lipodystrophy and low c , subsequently found to have c nephritic factor. case description: a year old female presented with a month history of low c levels. she was diagnosed months ago with poststreptococcal glomerulonephritis (psgn) after presenting with hematuria and elevated aso titers. she had c levels drawn - months after time of diagnosis and c level was low at (normal range - ), which was consistent with psgn. it was rechecked months after time of diagnosis and was still low. she was referred to rheumatology at this time and was found to have a positive ana titer : . tests for lupus and anti-phospholipid syndrome were negative. c normalized to the low-normal range at months after time of diagnosis to . her pediatrician checked to make sure it remained normal around months after initial diagnosis and c was low again at . c was normal at . she was referred to immunology for further evaluation. during this time she was asymptomatic with no fevers, infections, hematuria, rashes, joint pain, or joint swelling. she has no history of hospitalizations other than the first for psgn. mother denied family history of autoimmune disorders. physical exam: physical exam was notable for abnormal subcutaneous facial fat with normal fat distribution in the rest of her body. the rest of the exam was unremarkable with normal cardiac, pulmonary, abdominal, and skin exam. testing: c level was rechecked and low at . c nephritic factor was elevated at . (normal range . - . ). alternate pathway complement (ah ) was confirmed twice and was undetectable, < (normal level greater or equal to ). total hemolytic complement (ch ) was low at (normal level - ). other complement levels were checked and c q, c , c , c , c , c , c , and c complement were within normal range. discussion: the overactivation of the alternative complement pathway by c nephritic factor can result in various clinical manifestations, such as c glomerulopathy and acquired partial lipodystrophy in predominantly the face and the upper torso. the exact mechanism of how c nephritic factor is related to facial and upper body lipodystrophy is not known. one proposed mechanism is that adipocytes in the face and upper body produce more factor d, which is a complement protein utilized by c nephritic factor. overactivation of the alternative complement pathway on the adipocyte then leads to formation of the membrane attack complex, resulting in adipocyte lysis. eye disease, such as retinitis pigmentosa and macular degeneration can develop. c nephritic factor can also lead to more frequent infections and renal disease. patients need to be closely monitored. if patients develop c glomerulopathy, they may need to be considered for immunomodulatory therapy, such as steroids and other immunosuppressants. project manager/ucsf benioff children's hospital senior clinical research associate/ucsf benioff children's hospital associate professor/department of clinical pharmacy, ucsf staff research assistant iv/ucsf benioff children's hospital senior supervisor/ucsf benioff children's hospital laboratory specialist/ucsf benioff children's hospital research specialist/ucsf benioff children's hospital assistant professor/ucsf benioff children's hospital clinical professor/ucsf benioff children's hospital assistant professor/ucsd rady children's hospital staff pediatrician/tuba city indian health service staff pediatrician/phoenix children's hospital associate professor/seattle children's hospital chief, genetic immunotherapy section/niaid, nih professor/university of minnesota professor/ucsf benioff children's hospital abstract/case report text background: artemis-deficient scid (art-scid) represents % of all scid, but occurs in / births in navajo and apache native americans. artemis protein, encoded by dclre c, is essential for repairing dna double-stranded breaks, including those generated during v(d)j recombination of antigen receptor genes as t and b cells develop. artemisdeficiency causes not only t-b-nk+ scid, but also increased sensitivity to alkylating drugs and radiation. art-scid is the most difficult scid to treat with allogeneic hematopoietic cell transplantation (hct) due to high rates of rejection and gvhd, incomplete immune reconstitution, and toxicity following intensive conditioning regimens. as an alternative, we developed a self-inactivating lentiviral vector containing the human artemis promoter and dclre c cdna (aproart). we are evaluating its toxicity and efficacy in a phase i/ii gene transfer trial in art-scid patients. methods: newly diagnosed infants with art-scid and older patients with insufficient immunity despite prior allogeneic hct were eligible if organ function was acceptable. infants needed to have no matched sibling donor and be at least months old at conditioning. cd + cells were isolated from bone marrow or cytokine-mobilized peripheral blood, cultured with cytokines, transduced x with aproart, and cryopreserved. patients received daily doses of busulfan, targeted for a cumulative exposure (cauc) of mg*hr/l, with infusion of thawed cells on the following day. results: we treated newly diagnosed infants (art - & - ) with median age . m (range . - . ) and previously-treated patients (art - ) ( . y, . y and . y), with a median follow-up of . m (range . - . ). the mean (sd) bu cauc was . ± . mg*hr/l. patients received a median of . x aproart-transduced cd + cells/kg (range . - . ). the average vector copy number (vcn) and transduction efficiency in the marrow grafts exceeded those in the pbsc grafts: . ± . copies/cell vs . ± . (p= . ) and ± % vs ± . % (p= . ), respectively. there were no serious busulfan side effects. all patients had transduced peripheral blood leukocytes by w and of developed gene marking in t, b, nk and myeloid cells by w (fig. ) . gene-corrected cd , cd , cd / ra/ccr , cd and cd cells appeared in of patients (fig. ) , with art having t, nk and myeloid marking without b cells at m post infusion. normalization of lymphocyte proliferation to pha occurred in the evaluable (> w) infants (fig. ) , all now outpatients off isolation. two infants and previously treated child developed autoimmune hemolytic anemia (aiha), with requiring immunosuppressive therapy. infections included rhinovirus at presentation in art that resolved with t cell reconstitution. after discharge art acquired and recovered from norovirus and art acquired and recovered from cmv and rotavirus. analyses of insertion sites and t cell receptor diversity are pending. conclusion: infusion of aproart-transduced autologous cd cells into art-scid patients pretreated with very low exposure busulfan resulted in multilineage engraftment of transduced cells with evidence for t and b cell immune development. aiha, the only complication to date, occurred early and appears to resolve following restoration of t cell immunity. these encouraging results suggest potential effectiveness of ex vivo gene therapy for art-scid. ( ) submission id# mailan nguyen, md , susan canny, md, phd , andrea ramirez, md , ivan chinn, md abstract/case report text background: systemic lupus erythematosus is a heterogeneous disorder of the immune system. systematic genetic evaluation of patients with childhood-onset sle (csle) has begun to identify phenotypic clusters of csle patients with classic sle-causing genetic variants, as well as revealed unexpected genetic mimics of lupus. we report patients diagnosed with csle with similar typical and atypical lupus features, who were subsequently found to carry pathogenic nras variants that are the cause of ras-associated autoimmune leukoproliferative disorder (rald). cases: all patients ( females, male) presented at < years of age (average age . months, range - months) with antinuclear antibodies, anti-double-stranded dna antibodies, autoimm u n e h e m o l y t i c a n e m i a , s e v e r e t h r o m b o c y t o p e n i a , antiphospholipid antibodies, hypocomplementemia and nephritis. additionally, the patients all displayed fevers, organomegaly, lymphadenopathy and hypergammaglobulinemia. two out of patients had a malar rash, leukopenia, lymphopenia, anti-smith antibodies, serositis or arthritis. no patient had oral or nasal ulcers or photosensitivity. despite the fevers, lymphoproliferation and systemic autoimmunity, the patients did not display overwhelming immune dysregulation (peak ferritin - ng/ml). interestingly, the patients were found to have monocytosis ( - %), as has previously been reported in rald. double negative t cells were within normal range in the patients in which this was tested. all patients required aggressive immune modulation for control of their disease manifestations. two of the developed severe infections, specifically pneumococcal sepsis, during therapy. current follow-up covers an average of . years (range . to years). the patients responded to corticosteroids and were given sequential trials of various steroid-sparing therapies. in general, they appeared to benefit from both b cell depletion and t cell-directed modalities (cyclosporine, rapamycin), which are not first line therapy in csle. unfortunately, patient developed a fatal pulmonary infection while on treatment; her underlying disease was felt to be quiescent. due to the early-onset of disease, each patient was selected for genetic evaluation ( by exome sequencing, by gene panel). this lead to the discovery of pathogenic nras variants (c. g>a, p.g d) in all patients, assumed to be somatic, although this was confirmed in only case. conclusion: ras-associated autoimmune leukoproliferative disorder can present indistinguishable from csle with positive autoantibodies, immune cytopenias, arthritis, nephritis and hypocomplementemia. clinicians should consider evaluating for rald in csle patients who present at an early age ( < years) with predominant features of lymphoproliferation and hematologic abnormalities, particularly monocytosis. t cell-directed therapy with cyclosporine or rapamycin should be considered for rald. ( ) human ctla loss-offunction causes dysregulation of foxp + regulatory t (treg) cells, hyperactivation of effector t cells, and lymphocytic infiltration of target organs. patients also exhibit progressive loss of circulating b cells, associated with an increase of predominantly autoreactive cd (lo) b cells and accumulation of b cells in non-lymphoid organs. inherited human ctla loss-of-function demonstrates a critical quantitative role for ctla in governing t and b lymphocyte homeostasis. ( ) this case highlights the importance of next generation sequencing (ngs) in diagnosing and managing complex presentations with multi-system involvement. case presentation: patient was diagnosed with diffuse large b cell lymphoma at age and treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop) in / . he underwent autologous stem cell transplant with preparative carmustine, etoposide, cytarabine, and melphalan (beam) in / . he subsequently developed recurrent giant condyloma acuminata following transplantation requiring surgical resections, refractory immune thrombocytopenic purpura (itp) requiring aggressive systemic steroids and high dose ivig at least yearly, experiencing hypogammaglobulinemia, recurrent sinopulmonary infections, disseminated herpes zoster, and kaposi sarcoma. in / , he underwent ct/pet, revealing extensive hypermetabolic lymphadenopathy and splenomegaly. bone marrow biopsies were negative for lymphoma, although showed a slightly hypocellular marrow ( - % cellularity), % blasts, and eosinophilia without peripheral eosinophilia. repeated evaluations for hiv, syphilis, histoplasma, cmv, hhv , hhv , bartonella, coxciella, brucella, htlv, toxoplasma were negative. htlv and antibodies had been negative prior to transplantation. tonsillectomy / due to progressive enlargement showed reactive follicular hyperplasia with focal acute tonsillitis without granulomas or viral inclusions. repeated lymphocyte enumeration and proliferation studies were normal. a repeat pet scan / revealed persistent diffuse lymphadenopathy involving the neck, chest, abdomen, and pelvis. left lung biopsy revealed non-caseating granulomas without lymphoma. stains for ebv were negative. repeat pet scan / indicated disease progression prompting a left axillary excisional lymph node biopsy, revealing ebv lymphadenitis with large, reactive follicles with interspersed inflammation and loosely formed granulomas and cd positive b cells within the follicles. ebv blood pcr was negative. afb and fungal stains were negative on all biopsies. in / , his igg was ( - ), iga ( - ), and igm ( - ) with only out of protective serotypes to pneumococcus post-vaccination at . or greater. in / , igg was ( - ). custom ngs panel showed a heterozygous missense variant in ctla c. c>g (p.s r) located in the transmembrane domain. this variant of uncertain significance is suspicious and strongly suggests the diagnosis of ctla -related autoimmune lymphoproliferative syndrome . patient was referred to the national institute of health, where he received a bone marrow transplant. conclusion: primary immunodeficiency diseases comprise a group of highly heterogeneous immune system diseases and around forms of pid have been described. ngs has recently become an increasingly used approach for gene identification and molecular diagnosis of human diseases guiding treatment to patients who may otherwise have poor outcomes. ( ) ( ) submission id# erik newman, md , cullen dutmer, md allergy and immunology fellow/university of colorado and children's hospital colorado assistant professor of pediatrics/section of allergy & immunology, children's hospital colorado, university of colorado school of medicine, aurora, co, usa abstract/case report text introduction: inherited defects of the complement system are rare disorders that can result in unique susceptibility to infections with select bacteria. patients with a deficiency of a complement protein early in the complement pathway (affecting c qrs, c , c , factor h, or factor i) have increased susceptibility to infection with encapsulated bacteria, most notably streptococcus pneumoniae and neisseria species. in contrast, patients with a deficiency of a complement protein at the terminal end of the complement pathway (affecting c , c , c , c alpha/beta/gamma, or c ) almost universally present with severe, recurrent, or disseminated neisseria species infections. most genes encoding complement proteins are found on autosomes, in which specific complement deficiencies result from biallelic mutations. although particular complement deficiencies occur at higher frequencies in certain populations, the prevalence of specific complement deficiencies is unknown in many parts of the world, especially in underdeveloped regions, including sub-saharan africa. herein, we describe a young congolese boy with an atypical presentation of c alpha deficiency. case description: a -month-old congolese boy with consanguineous parents (first cousins) presented with recurrent infections. prior to an evaluation of his immune system, he was hospitalized five times. his infections included episodes of acute otitis media, bacterial pneumonia, and viral pneumonitis. a bronchoscopy revealed diffusely edematous airways and growth of candida albicans, moraxella catarrhalis, and streptococcus pneumoniae in bronchoalveolar lavage cultures. concurrently, his respiratory pcr panel was positive for adenovirus. his initial immune evaluation included assessments of his serum immunoglobulin levels, vaccine titers (tetanus, diphtheria, haemophilus influenzae, and streptococcus pneumoniae), neutrophil oxidative burst, lymphocyte subsets, and ch , in which only his ch was abnormal ( u/ml). his ch remained low on repeat assessment ( u/ml), at which time an ah was pursued and also returned with a low result ( % of normal). a complement system genetic panel identified a homozygous intronic variant in c a (c. - g>a). functional confirmation of the variant revealed that the patient had a significantly decreased c level ( mcg/ml) and absent c function. discussion: we present a case of a young congolese boy with c alpha deficiency and a clinical presentation atypical for defects in terminal complement proteins. our patient presented primarily with recurrent respiratory infections, including streptococcus pneumoniae pneumonia, but without a preceding history of meningococcal disease. while it is well established that patients presenting with terminal complement pathway defects have an increased susceptibility to meningococcal disease, it is less clear if they have increased susceptibility to pneumococcal infections. occurring between exons and , the homozygous intronic variant in c a identified in our patient is predicted to result in abnormal splicing. while the allele frequency of this mutation is relatively high in the african population ( . ), functional confirmation of the variant demonstrated a decreased c level and absent c function that support the pathogenesis of the mutation. the discrepancy between the allele frequency and reported disease cases could be explained in part by varying clinical manifestations seen in c deficiency or underrecognized disease in sub-saharan africa. abstract/case report text rationale: scid is a syndrome characterized by profound t, b, and (in some cases) nk cell defects that is universally fatal unless immune reconstitution is achieved. a total of scid infants have been given allogeneic bone marrow transplantation at duke university medical center without pre-transplantation chemotherapy or post-transplantation graft-versus-host disease (gvhd); % received t cell-depleted haploidentical parental marrow and ( %) are known to be deceased. post-transplantation follow-up ranged from months to years. the aim of this cross-sectional study is to characterize the clinical status of a large cohort of survivors treated at a single medical center. methods: clinical status was assessed by detailed questionnaires delivered by mail or electronically. adult (≥ years old) and pediatric questionnaires were based on patients' age. patients were also contacted by telephone and evaluated at clinic visits. molecular type of scid, demographics, type, date and age at transplant were obtained from a clinical database. results: fifty questionnaires were completed to date from survivors ranging in age from to years. twenty-nine/ were adults ≥ years at the time of the questionnaire. genetic defects were known for all patients-xlinked scid was the cause in about half ( figure ). twenty-three of patients were on immunoglobulin replacement. thirty of reported having received immunizations, and about half of those received live vaccines. fifteen of reported they were taking no regular medications; reported taking prophylactic antibiotics. >we found substantial scholastic achievement, with / adult patients reporting college attendance. two had post graduate education including doctorate level degrees. occupations included physician, nurse, factory worker, musician, teacher, and engineer. one patient had children. twenty-seven / adult patients shared their height and weight and % ( / ) had a healthy bmi (bmi . - . ), while % ( / ) were overweight, and % ( / ) were underweight (< . ) . in pediatric patients, the average age and sex-adjusted bmi was at the th percentile and only had a bmi that was < th percentile. thirty-four/ patients reported seeing an immunologist regularly. in the adult group, % reported no longer seeing an immunologist. the health conditions reported were similar to those common in the general population, and included rashes, warts and mouth ulcers. most reported these were transient, self-resolving issues. thirteen of ( %) reported having adhd, higher than nih reported rates which estimate adhd in . % of adults and % of children). ten of ( %) reported having anxiety, similar to the nih reported prevalence of . % in the general population. / (~ %) reported having no active concerns about their health. conclusions: overall, our findings are consistent with those in the last update done by railey et al, j. peds. : [ ] [ ] [ ] [ ] [ ] [ ] [ ] in this population. patients are doing well with most problems similar to those common in the general population. most have a healthy bmi. adhd had a higher prevalence than in the general population. more than / of scid patients are not seeing an immunologist regularly, and a majority do not have any active concerns. . genetic causes of scid in the patients whose questionnaire data are presented. x-linked scid was the most common, followed by ada and il- r deficient scid clinical fellow/national institute of allergy and infectious diseases (niaid/nih) research nurse/nih-nhgri professor of pediatrics and allergy and immunology/ann & robert h. lurie children's hospital of chicago chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text rationale: myopathy has been occasionally documented in patients with primary immunodeficiency (pid). however, data on frequency and patient characteristics associated with myopathy are lacking. we performed a descriptive analysis of patients with primary immunodeficiency (pid) in the usidnet having myopathy as a feature of their primary disease. methods: the usidnet registry was queried for the spectrum of myopathic disorders in pid patients that had been entered into the registry as of november , . results: a total of pid patients with myopathy were identified, of which ( . %) were female. median age at onset of symptoms related to pid was years (range . - years, iqr . - years). median age of diagnosis of pid was . years (range . - years, iqr - . years). age at onset of myopathic disorders was not known. twenty-eight ( . %) patients had a diagnosis of common variable immunodeficiency (cvid), ( . %) had agammaglobulinemia, patients each ( . %) were diagnosed with severe combined immunodeficiency, hypogammaglobulinemia, or 'hlh and pigmentary disorders', while patients ( . %)were reported in each category of combined immunodeficiency (cid), autoimmune lymphoproliferative syndrome (alps), and autoinflammatory disease. thirty-five patients ( . %) had a causative gene variant identified attributable to pid. the most common variant identified was btk ( patients) followed by aire, lyst, cybb ( patients each) and pi kcd ( patients). eighteen individual patients had other variants identified ( figure ). patients had cellulitis or skin/ subcutaneous tissue infection, patients had a 'skin or subcutaneous tissue abscess', had pyoderma gangrenosum, and patient with eczema herpeticum. within this cohort of patients, the most common myopathy listed was myositis ( ) followed by 'muscle weakness' ( ), dermatomyositis ( ) , myalgia/s ( ) , myalgia/myositis ( ), myopathy ( ), polymyositis ( ) , steroid-induced myopathy ( ) . no patient had an infectious myositis or muscle abscess listed. eighteen patients ( . %) had a myopathic disorder at the time of diagnosis of their pid. thirtyone patients ( . %) received prednisone, ( . %) received hydrocortisone and ( . %) received dexamethasone. two patients had a diagnosis of adrenal insufficiency. nine patients ( . %) underwent hematopoietic stem cell transplantation. nine patients ( . %) died; median age of death years (range . - . years, iqr . - . years). ). one patient with chronic granulomatous disease had myopathy due to duchenne muscular dystrophy, which was listed as a cause of death. no other myopathic disorders were listed as a cause of death for the other patients. conclusion: myopathy and inflammatory myopathic disorders occur at relatively high frequency in pid, and may be present even at the onset of clinical symptoms. the underlying etiology can be speculated to be multifactorial. further subgroup analysis is warranted to elucidate possible variant-specific or treatment-associated characteristics of myopathy in pid. laboratory studies at years revealed normal igg/iga/igm but markedly elevated serum ige ( , ku/l), anemia (hb . g/dl), thrombocytopenia ( x /l) and lymphopenia ( cells/l), with low t and b cell counts, very low proportion of naïve t cells, skewed repertoire of cd + t cells, undetectable trec levels, and impaired t cell proliferation to mitogens and antigens. there was an elevated percentage of circulating plasmablasts ( . %) and of dysreactive cd low cd low b cells ( . %). at the age of , hsct with reduced intensity conditioning was performed from her phenotypically hla-matched father, with improvement of t and b cell count and function. whole exome sequencing (wes) identified a homozygous missense variant in the mannosidase alpha class b member (man b ) gene (p.asp asn), that segregates with disease in the pedigree. the man b asp residue is evolutionary conserved. the p.asp asn allele has a minor allele frequency of . in gnomad, with no homozygotes. the cadd score for this variant is . , significantly higher than the mutation significance cutoff score ( . ). man b is involved in the lysosomal degradation of glycoproteins and demannosylation of free n-glycans. in particular, man b cleaves man glcnac to generate man glcnac . serum n-glycan profiling revealed elevated man /man and man / man in the patient. n-linked and free glycan profiling by mass spectrometry (ms) showed accumulation of man glcnac , man glcnac and man glcnac glycans in patient fibroblasts as compared to control cells, consistent with defective lysosomal glycoprotein degradation. lentiviral transduction of wild-type man b into patient fibroblasts led to normalization of the n-linked glycan profile, with reduction of man glcnac from . to . times control levels, and of man glcnac from . to . control levels, indicating rescue of the impaired deglycosylation ( figure a ). western-blotting demonstrated defective n-glycosylation of lamp and icam proteins in patient fibroblasts ( figure b) , which were corrected upon lentiviral transduction of wild-type man b ( figure c ). overall, our results indicate that loss of man b enzymatic activity leads to dysregulation of deglycosylation and abnormal mannosylation of glycans. in conclusion, we have demonstrated that man b deficiency accounts for a novel autosomal recessive cdg with prominent features of immune deficiency and immune dysregulation. abstract/case report text heme oxygenase- (hmox ) is a rate-limiting enzyme that catalyzes the degradation of heme to carbon monoxide, ferrous iron, and biliverdin, which becomes bilirubin. these byproducts are implicated in inflammation, cell homeostasis, and antioxidant defense( ). hmox -deficiency is an extremely rare autosomal recessive disorder with a complex presentation of a wide spectrum of symptoms, including hemolytic anemia and hyperinflammation, requiring genetic testing for confirmed diagnosis ( ) . we report the fifth known case of hmox -deficiency ( ) ( ) ( ) , a boy who presented at years of age with aspects of the characteristic phenotype, but also had early onset asplenia, interstitial lung disease, and previously undocumented immune deficiency. patient's presentation was notable for hyperinflammatory exacerbations triggered by viral and bacterial infections as well as vaccinations. episodic flares occurred every few months lasting weeks to months with fevers of - f, hypoxia, leukocytosis above , /mm , hemolytic anemia with negative coombs, thrombocytosis exceeding million/ mm , transaminitis, hemoglobinuria, hyperferritinemia to , ng/ml, and elevated ldh to , iu/l. immune evaluation revealed normal immunoglobulin levels and adequate vaccine titers to both protein and carbohydrate antigens. although class switched populations were normal, b-cell phenotyping showed absent immature and transitional b-cells, low mature memory, and reduced cd + memory b-cells at % (normal > %). mitogen stimulation with phytohemagglutinin and anti-cd were decreased ( . % of control and . % of control, respectively). t-cell phenotyping demonstrated cd population heavily skewed to immaturity with % of cells with naïve phenotype cd ra+cd +ccr +. there were few effector-memory t cells and the cd population was skewed towards immaturity with > % of the cells naïve. liver biopsy was performed secondary to hepatomegaly yielding mild to moderate sinusoidal fibrosis. bone marrow biopsy revealed a normocellular marrow with % blasts, increased megakaryocytes, and extensive hemophagocytosis. natural killer cell function was very low, while soluble il- ra level was normal. further workup for hemoglobinopathies, metabolic defects, congenital disorders of glycosylation, lysosomal storage disorders, wilson's disease, autoimmune hepatitis, inherited and autoimmune hypercoagulability disorders, connective tissue disorders, myositis, and myopathies were all unremarkable. imaging demonstrated asplenia and howell-jolly bodies were present. hemophagocytic lymphohistiocytosis (hlh) genetic testing showed no variants. he was suspected to have systemic juvenile idiopathic arthritis (sojia) with episodes of macrophage activation syndrome. the frequency of his autoinflammatory flares increased such that he was corticosteroid dependent by age , having failed methotrexate, azathioprine, and anakinra. he was started on tocilizumab with laboratory improvements, but his lung disease progressed and became oxygen dependent. lung biopsy confirmed nonspecific interstitial pneumonitis (nsip) with cholesterol granulomas also seen in sojia. ultimately, chronic lung disease led to his death at age . whole exome sequencing yielded a paternal frame shift hmox and maternal splice donor hmox resulting in absence of protein. bone marrow transplantation (bmt) in hmox deficient mice have rectified phagocytotic defects and thereby their autoinflammatory phenotype, but no human reports for bmt treatment of hmox -deficiency has been described. here we describe a phenotype expansion for hmox deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares. abstract/case report text introduction: mutations in the gene encoding signal transducer and activator of transcription (stat ) cause autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) characterized by recurrent skin and sinopulmonary infections, atopic dermatitis, and elevated serum immunoglobulin e (ige) levels. treatment is largely aimed at controlling symptoms and preventing infections with no standard of care. there is a paucity of literature describing the utilization of biologic therapies in the ad-hies patient population. we present patients from one family with ad-hies successfully treated with monoclonal antibody therapies targeted at il- , il- and il- . case descriptions: patient : -year-old female with stat lof c. c>t (p.arg trp) with a history of atopic dermatitis and asthma requiring - steroid courses per year with frequent school absences. she developed a severe pruritic rash covering her upper body months ago that failed to respond to antihistamines and topical antibiotics prescribed by her primary care provider. given her poorly controlled asthma and our concern for a follicular type morphologic variant of atopic dermatitis, dupilumab was initiated. her scoring atopic dermatitis (scorad) prior to initiation of biologic therapy was . and improved to . following doses ( weeks) of dupilumab with clear dramatic improvement in her skin and quality of life ( figure ). she also reports decreased asthma severity with no steroid courses, reduced albuterol usage, and significant decline in school absences since initiation of dupilumab. patient : -year-old female with stat lof c. c>t (p.arg trp) who is the sister of patient . she has a history of severe asthma requiring frequent emergency department visits, hospitalizations, and - steroid courses per year despite therapy with high-dose fluticasone-salmeterol. spirometry prior to april demonstrated an obstructive pattern with an fev ranging from - %. she was initiated on mepolizumab in april . subsequent spirometry demonstrates an fev average of % with a range of - %. she had one hospitalization in early but otherwise no hospitalizations for asthma since initiation of biologic therapy. patient : -year-old female with stat lof c. c>t (p.arg trp) who is the paternal first cousin of patients & . she has a history of severe atopic dermatitis with associated pruritus and picking behaviors, poorly controlled despite daily triamcinolone application. she previously failed ultraviolet therapy and crisaborole. she also has a history of severe asthma requiring - steroid courses per year despite high-dose fluticasone-salmeterol. dupilumab was started in may . scorad prior to initiation monoclonal antibody therapy was . and declined to . following weeks ( doses) of dupilumab therapy with marked improvement in skin appearance and pruritus ( figure ). discussion: we present three cases of ad-hies caused by stat loss-of-function mutations treated successfully with monoclonal antibody therapies targeted at il- or il- and il- . to the best of our knowledge, there is no published data describing the use of these biologic agents in the treatment of ad-hies. future studies are needed to clarify the role of these cytokines in the pathogenesis of ad-hies and to elucidate clinical indications for biologic therapy in this patient population. informed consent was obtained from all individual participants included in the study. abstract/case report text ctla- is a potent inhibitor of t cell proliferation that competes with costimulatory receptor cd for its ligands cd and cd expressed on antigen presenting cells. heterozygous loss-offunction mutations in ctla- have been identified in patients with lymphocytic infiltration of multiple nonlymphoid organs (lo et al). the patient is a -year-old jordanian male born at term to nonconsanguineous parents, hospitalized at mo for lll pneumonia, and at mo and at mo he was evaluated in the ed and diagnosed with non rsv-bronchiolitis with lll infiltrate thought to be secondary to atelectasis. at yo he developed lll pneumonia and respiratory failure requiring picu admission. he was treated with ceftriaxone. after discharge, he had weeks of intermittent fever, progressive fatigue, productive cough, and ftt. he received courses of cefdinir, clindamycin, and tmp-smx without improvement. chest ct revealed left lung consolidation, lll bronchiectasis, lul tree in bud opacities, and hilar lymphadenopathy. bronchoscopy with bal revealed no bacterial growth and no acid-fast bacilli. srrna ngs was positive for h. influenzae. lung biopsy demonstrated acute and chronic bronchiolitis with bronchiolitis obliterans and intraluminal polyps, with lymphocytic infiltration involving the bronchi and bronchioles. the lung parenchyma showed airspace filling with foamy macrophages and chronic interstitial inflammation. acid fast and fungal stains were negative. he was treated with systemic steroids for bronchiolitis obliterans with noted improvement. the severity of lung disease at such an early age prompted an immune evaluation. sweat test, anca, anti-pr and hiv were negative. total immunoglobulins were normal for age, and titers to s. pneumoniae, diphtheria and tetanus were protective. lymphocyte enumeration revealed elevated t and nk cell numbers for age. lymphocyte proliferation to pha, pwm, candida and tetanus were normal. dihydrorhodamine assay was normal. b cell phenotyping was normal. there was normal expression of cd , hla-dr and cd on activated t cells; of note the patient was on systemic steroids when tested. invitae gene pidd panel revealed a variant in ctla : c. g>a (p.gly glu) that has been shown to be pathogenic in one patient (schawb et al). flow cytometry showed normal frequency of t follicular helper cells and t regulatory cells compared with controls, however ctla- expression by t regulatory cells was lower than control. due to the severe and progressive nature of the patient's lung disease, therapy with x weekly azithromycin and abatacept mg sq weekly was initiated. we report a case of ctla- haploinsufficiency presenting with recurrent pneumonia and bronchiolitis obliterans in a -year-old child. based on patient registry data, our patient appears to be the youngest child diagnosed with ctla- haploinsufficiency reported in the literature to date (schawb et al). notably, our patient l a c k s o t h e r f e a t u r e s c o m m o n l y d e s c r i b e d i n c t l a - haploinsufficiency, including autoimmune cytopenias, gastrointestinal disease, lymphoproliferation, and hypogammaglobulinemia. this case illustrates the importance of consideration of this diagnosis in young children with severe lung disease without other evidence of immune dysregulation. our hope is that prompt recognition and early treatment administration will prevent disease progression and further decrease in pulmonary function. splenectomy, he had an episode of pneumococcal meningitis at age , and sepsis of unknown origin at age . over the past several years he has developed chronic tinea corporis, onychomycosis, and otitis externa infections despite numerous antimicrobial regimens. at age , the patient developed urinary retention, walking, and balance difficulties. he was found to have diffuse white matter changes on mri, elevated wbc, and positive oligoclonal bands. initially, he was diagnosed as progressive ms treated with steroids with partial improvement. csf microbiology studies including afb stains, bacterial, fungal, mycobacterial cultures, cryptococcal antigen, vdrl, t. pallidum particle agglutination (tppa), as well as, pcr for cmv, ebv, vzv, enterovirus, hsv - , jc virus and t. pallidum were all negative. peripheral blood studies included mycobacterial blood culture, pcr for cmv, ebv, hhv- , in addition to serology for cryptococcal antigen, and coccidioides species, all of which were negative. additional neurological complications include granulomatous uveitis and oscillopsia, which he developed around age . immune evaluation performed at age revealed low igg and igm, and the patient was started on grams of monthly ivig. cbc with differential was notable for normal monocyte count and thrombocytopenia, mild neutropenia (table ). immunophenotyping revealed absent b cells and nk cells, while the cd t cells were elevated. cd t cells were normal (table ) . at age , whole-exome sequencing identified a heterozygous missense mutation in gata c. c>t, p.(arg trp). after the diagnosis of gata haploinsufficiency, he was found to have myelodysplastic syndrome with multilineage dysplasia (mds-mld) on bone marrow biopsy. he is currently awaiting bone marrow transplant discussion: we present a -year-old male with cytopenias, splenomegaly, leukoencephalomyelopathy, granulomatous uveitis, and recurrent fungal infections found to have a pathogenic heterozygous missense mutation in gata . leukoencephalomyelopathy in gata haploinsufficiency has been associated with jc virus and ebv infection. our patient did not have any evidence of a chronic csf infection. to our knowledge, myelopathies have not been reported with gata c. c>t, p.(arg trp). this case highlights the variable nature of presentation in gata haploinsufficiency, and the need for clinical awareness of this entity in order to facilitate early diagnosis and appropriate therapy. immunoglobulins* white blood cells . x * /l (n: - ) magnetic resonance imaging (mri) of the brain and spine showed numerous enhancing parenchymal nodules (figure one). brain or spinal biopsy was requested, but not recommended by our neurosurgery service. lumbar puncture evaluation was performed. cerebral spinal fluid showed no bacterial or fungal elements. both quantiferon gold for tuberculosis and three consecutive sputum cultures for acid fast bacilli were negative. he continued his sirolimus and the intravenous immunoglobulin replacement was increased to two grams/kg. the patient was cleared from respiratory isolation and discharged after two weeks in our facility with mild improvement in his neurologic status. within five days he was sent to a nationally renowned hospital. at this facility, extensive evaluation for his neurologic deficits were performed including culture and pcr for bacteria, virus, mycobacteria from csf bone marrow, lymph node, blood, and induced sputum. these were noncontributory. he was given high dose corticosteroids for two days. one of our facility's sputum cultures was reported with acid fast bacilli. but sputum mycobacterium tuberculosis pcr was negative. repeat mri scans of the brain and spine showed improvement (figure ), so no brain biopsy was performed. he was sent back to our facility with the recommendation to start a targeted pi kinase inhibitor on compassionate grounds as he was not eligible for the clinical trial because his weight was less than kg. but pretreatment abdominal ct revealed multiple low-density lesions scattered throughout the liver (figure ) not previously seen on prior noncontrast ct four months prior. discussion: although this gain in function mutation of the pi kδ signaling pathway disorder has been well characterized, this is a rare report of a patient with pasli immunodeficiency with central nervous system and later liver lesions pet imaging showed subcarinal, mediastinal, retroperitoneal lymphadenopathy, splenic enlargement to cm and bilateral lung nodules ( figure ). excisional biopsies of left axillary and left lower lobe of lung were performed and showed low-grade b-cell lymphoma (mucosal) and underlying lymphoproliferative disease. invitae alps and cvid panels ( genes) revealed a heterozygous variant of unknown significance in exon of fas (c. a>g(p.asp gly) unlike most fas mutations causing alps, this mutation is in the extracellular region rather than the death domain . the c. a>g variant has been reported in a single patient with alps phenotype, affecting fas protein function by inhibiting binding to fas ligand (fas-l), reducing fas-l induced apoptosis . this fas c. a>g mutation was found in alps affected brother and was absent in the unaffected father. the patient's mother passed away prior to testing. since diagnosis, the patient's malt lymphoma has been treated with rituximab weekly for the first month and then monthly. he continues to receive monthly ivig for hypogammaglobulinemia. after two years, ct demonstrates a significant decrease in pulmonary nodules and splenomegaly ( figure ). the patient's forced vital capacity (fvc) improved from . l ( % predicted) to . l ( % predicted). conclusion: we report the first case of malt lymphoma seen in a patient with alps. it is unknown whether the unique fas c. a>g mutation in the non-death domain contributes to malt lymphoma progression. we propose malt lymphoma is a malignant transformation of chronic inflammation that has the potential to occur in patients with alps. in the future, improved knowledge of mechanistic pathways of inflammation in lymphoma development and progression is important in the optimal management of alps. abstract/case report text background wiskott-aldrich syndrome (was) is a rare x-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and increased risk of hematological malignancies. gene therapy (gt) using autologous cd + cells is an emerging alternative treatment with possible advantages over standard allogeneic hematopoietic stem cell transplant. we report the outcomes of a phase i/ii clinical trial in which was patients underwent gt using a self-inactivating lentiviral (sin-lv) vector expressing the human was cdna under the control of a . kb fragment of the human was promoter. subjects and methods: five patients with severe was (clinical score - ) were enrolled (table ) . cd + cells were transduced ex-vivo and reinfused after conditioning with busulfan and fludarabine. two subjects (p , p ) had autoimmunity pre-gt, manifested as skin vasculitis and autoimmune cytopenias. results: all subjects were alive at median follow-up of . (range . - . ) years. multi-lineage vector gene marking was sustained over time. all had clinical improvement of eczema, infections and bleeding diathesis. was protein (wasp) expression was increased over baseline but remained below normal levels. proliferation of t cells in response to anti-cd improved post-gt. humoral immune deficiency improved, with normalization of igm, and independence from ig replacement and vaccine responses in those tested. platelet levels increased to > x cells/ul in only the two subjects with a vcn ≥ in transduced stem cells. podosome formation in monocyte-derived dendritic cells was near absent pre-gt and improved in all subjects post-gt, but only reached healthy control levels in the subjects with highest vcn. in contrast to other trials using this sin-lv, two patients (p and p ) had flares of autoimmunity post-gt, offering the opportunity to study the poorly understood mechanistic features of immune dysregulation in this disease. selfreactive vh - -expressing b cells and cd lo b cells remained elevated in most patients. however, despite wasp expression in foxp + tregs, those with autoimmunity had poor numerical recovery of t cells and tregs at the time of clinical symptoms ( fig a) . in addition, il- producing regulatory b cells (bregs) were highly deficient pre-gt, recovered in subjects who did not experience autoimmunity, but failed to recover in p and p ( fig b) . moreover, transitional b cells, which are enriched in bregs and are potent inducers of treg populations, also recovered poorly in those two subjects ( fig c) . there have been neither severe gt-related adverse events nor abnormal clonal expansion in transgene-marked cells to date. conclusion in summary, our data confirm and extend the safety and efficacy of gt in correcting disease manifestations associated with was, with the longest overall follow-up reported so far in studies using sin-lv. in addition, our findings suggest that higher vcn is needed in order to correct myeloid compartments such as platelets and monocytes. finally, we report the novel finding of the restoration of bregs and suggest that recovery of this compartment, along with tregs, is protective against development of autoimmunity post-gt. overall, these data suggest a mechanism for breakdown of immune tolerance in was with important therapeutic implications and prognostic value. this is an -year-old hispanic female who initially presented with failure to thrive, recurrent fevers and intermittent cough with episodes of perioral cyanosis. symptoms started at age months and were attributed to recurrent viral and bacterial infections. at months old, she was hospitalized with fever and hypoxemia (o saturations %). cxr showed prominent interstitial lung markings and she was diagnosed with pneumonia. ct scan confirmed cxr findings and ruled out anatomical anomaly. she was lost to follow up for years, and re-presented with worsening respiratory status. a repeat ct scan demonstrated worsening interstitial thickening. immune workup, including quantitative immunoglobulins, ch , lymphocyte subsets and vaccine response titers (pneumococcal and tetanus), was unremarkable, except for elevated igg levels. genetic testing for surfactant dysfunction mutations was negative. thoracoscopic lung biopsy revealed interstitial fibrosis, pas-positive granular alveolar proteinosis, type ii cell hyperplasia, and lymphoid follicles. at age , she was admitted for a pericardial effusion. rheumatology was consulted for evaluation frequent fevers and persistently elevated inflammatory markers, with concern that the pericarditis was autoinflammatory. she had an elevated ana (> : homogeneous pattern), il- ( . pg/ml), and igg ( mg/dl) at that time. she had an atypical anca pattern with positive myeloperoxidase antibodies. anti dsdna, smith and scl were negative. she was treated with steroids and hydroxychloroquine with some improvement in her oxygen requirement. one year later she had an additional episode of pericarditis, treated with colchicine. a few months later, she was admitted with newonset gross hematuria and elevated serum creatinine (to mg/ dl). kidney biopsy showed anca vasculitis with glomerulonephritis ( % crescents, no scarring or fibrosis). she provisionally received a diagnosis of microscopic polyangiitis, with lung and kidney involvement. she did not have peripheral vasculopathy. she was started on cyclophosphamide, rituximab, and iv steroid pulses. cyclophosphamide was discontinued due to recurrent episodes of posterior reversible encephalopathy syndrome (pres) after infusion. her igg level decreased as she developed nephrotic range proteinuria. a primary immunodeficiency genetic panel was sent to evaluate for monogenic immune dysregulation syndromes and revealed a tmem gene mutation (c. g>a) which has previously been reported in other subjects with savi (stingassociated vasculopathy of infancy syndrome). sting is a cytosolic dna sensor that leads to type i interferon production upon stimulation. this gain-of-function mutation was confirmed by measuring interferon signature gene expression at the nih (fig ) , and her diagnosis was revised accordingly. the patient was started on a jak-inhibitor (tofacitinib) to block interferon signaling. unfortunately, the patient is now deceased, due to overwhelming infection and multi-organ system failure. conclusion: genetic testing can be crucial in aiding the diagnosis of complex patients with immune dysregulation and can provide an opportunity for targeted therapy, which should be employed as soon as able to stop disease progression. abstract/case report text background: activated phosphoinositide -kinase δ syndrome (apds- ) was first described in as a monogenetic immune dysregulation syndrome with a variable phenotype. increased sinopulmonary and herpesvirus infections are well described, but fungal infections such as candidiasis have been rare. to date, disseminated histoplasmosis has not been described. history: a yo caucasian male who was previously diagnosed with common variable immunodeficiency (cvid) in late childhood due to recurrent sinopulmonary infections presented with recurrent fever, pancytopenia, severe splenomegaly, and lymphadenopathy. urine histoplasmosis antigen and beta-d-glucan were elevated. a bone marrow biopsy demonstrated granulomatous inflammation. transbronchial biopsy of a subcarinal lymph node was consistent with granulomatous disease. this led to a diagnosis of disseminated histoplasmosis. he was treated with amphotericin b and then months of itraconazole, with improvement of his symptoms. he was admitted to the hospital about years later when he presented with fatigue, fever, chills, dark urine, and scleral icterus. he was found to have an acute worsening of chronic anemia with a hemoglobin of . g/dl. due to elevated ldh, presence of schistocytes on peripheral smear, and undetectable haptoglobin, he was diagnosed with autoimmune hemolytic anemia, despite a negative direct coombs. a bone marrow biopsy specimen was hypercellular with marked erythroid predominance, with normal flow cytometry and no blasts identified. infectious workup was negative. ct chest during the workup revealed new right hilar and mediastinal lymphadenopathy, in addition to calcified right hilar and subcarinal lymph nodes, bronchiectasis, and stable hepatosplenomegaly. transbronchial biopsy of lymph nodes showed benign lymph nodes with calcified necrotizing granulomata and presence of non-viable fungal species, presumably "old" histoplasmosis. family history: family history was significant for mom dying at years-old from undefined cns infection. immune labs: · panlymphocytopenia: absolute lymphocyte count of /ul, cd + t cells /ul, cd + /ul, cd + /ul, cd + /ul, cd +cd + /ul. cd +/cd + ratio . · decreased class-switched memory b cells and plasmablasts · elevated t central memory cells and activated (hla-dr+) cd + and cd + t cells · hemoglobin . g/dl, platelets , /ul, anc ranging from /ul to /ul · iga mg/dl, igm mg/dl; reportedly had low igg prior to initiating ivig in childhood · ebv pcr and cmv pcr negative genetics: · pik cd (c. g>a), consistent with diagnosis of autosomal dominant apds- . discussion: gain-of-function variants leading to increased pi kδ activity have been shown to cause both b and t cell dysfunction, leading to impaired immunologic responses to bacterial and viral infections. recurrent sinopulmonary infections and herpesvirus infections are commonly seen and while mucocutaneous candidiasis has been reported in cohorts of patients with pik cd, other fungal infections are not common. severe disseminated histoplasmosis infections have been described in primary immunodeficiencies characterized by signaling defects in the il- /ifn-γ pathway, stat deficiency, cd l deficiency, gata deficiency and in stat gain-of-function mutations. to our knowledge, disseminated histoplasmosis has not been previously reported in patients with pik cd immunodeficiency. abstract/case report text the reported case represents the first case of nbas disease detected by newborn screening program for primary immunodeficiency, based on krec assay. the patient came to our attention due to the complete absence of krecs and normal trecs on dbs (dried blood spot) while hospitalized for low weight at birth ( , g), intolerance for enteral feeding, hepatosplenomegaly, slightly elevated liver transaminase, head and face eczematous dermatitis. during the st month, he also presented klebsiella pneumoniae urinary tract infection and methicillin-resistant staphylococcus aureus sepsis. peculiar phenotypic features including triangular face, proptosis, flat philtrum, mild retrognatia, hirsutism, loose and slightly wrinkled skin, and apparent reduction of subcutaneous fat were noticed at birth. complete blood count showed lymphocytopenia, marked hypereosinophilia. serum immunoglobulin g (igg) were markedly decreased, iga and igm were undetectable. extended immune-phenotyping showed complete absence of cd + cells, low count of cd + lymphocytes, and reduced natural killer (nk) levels. at month of age a colonoscopy was carried out for persistent diarrhea and reduced tolerance to enteral feeding. the histological examination of mucosal intestinal biopsies showed signs compatible with autoimmune enteropathy. for this reason immunosuppressive therapy with rapamycin was started without consistent clinical amelioration. many cvc-sepsis occurred in the last months, associated with persistent gastrointestinal symptoms and severe growth restriction. despite the absence of experience data in literature for nbas syndrome, we retain that hsct represents the only resolutive therapy for him. abstract/case report text case: a -year-old female with asthma and allergies presented to immunology clinic with a history of chronic fatigue and sinusitis. fatigue occurred daily every - weeks and was described as not feeling rested even after hours of sleep. chronic sinusitis required - prolonged antibiotic courses per year. nasal cultures grew methicillin-sensitive and -resistant staphylococcus aureus and haemophilus influenzae type b (hib). three separate sinus surgeries over the prior few years reduced her sinus symptoms. other infectious history was significant for recurrent urinary tract infections with e. coli and klebsiella, recurrent otitis media as a child, and a diagnosis of transient hypogammaglobulinemia of infancy that resolved at years of age. review of systems revealed axillary lymphadenopathy for - days twice per year not related to infection. she had longstanding eczema that responded to topical tacrolimus, multiple environmental allergies, and recently diagnosed asthma that improved with inhaled budesonide/formoterol. as a teenager she received allergy immunotherapy for a few years but stopped due to frequent adverse reactions. family history revealed that father died from cancer. physical exam was unremarkable. laboratory evaluation demonstrated normal igg mg/dl, igm mg/dl, iga mg/dl, elevated ige mg/dl, normal t and nk cell enumeration, mildly low total b cells ( cells/mcl, . %), and normal b cell subsets (cd +igm+cd - %, cd +igm+cd + %, cd +igm-cd + %). tetanus antibody titer was protective, but hib antibody titer was undetectable at < . mcg/ml with marginal response after vaccination ( . mcg/ml). pneumococcal serotype specific igg levels (mayo) were mostly undetectable with of serotypes protective at baseline and only protective post vaccination with pneumovax . cd / cd blastogenesis was poor. due to poor antibody response and continued sinus infections she was started on igg replacement. her fatigue and sinus symptoms improved moderately but she continued to require antibiotics and sinus ct scans continued to demonstrate significant disease. a focused exome sequencing panel was pursued and a novel heterozygous card variant was found (c. g>t, p.r l). discussion: the card /bcl /malt (cbm) complex is a critical signaling adapter that facilitates several downstream immune responses predominately through nf-kb. mutations in several different domains of card result in a clinical entity collectively referred to as card -associated atopy with dominant interference of nf-kb signaling (cadins). cadins is associated with a broad range of clinical manifestations but most have marked atopy with infections, poor t cell proliferation, and varying levels of poor antibody response. both our variant (p.r l) and a previously reported pathogenic variant in the same amino acid (p.r g) involve a change from a charged arginine to a non-polar amino acid in the critical bcl / card binding interface. iκbα degradation was not present in b cells from our patient ( figure ) confirming the functional defect in nf-kb signaling. thus, we present a novel variant that fits cadins both clinically and genetically. clinicians should be aware of cadins when patients present with recurrent infections in the setting of significant allergic disease. i b degradation assay. stimulation: μl of whole blood was stimulated in a ml facs tube with ng/ml phorbol -myristate -acetate (pma; sigma, cat# p ) at °c for , , or min, at which point ml of pre-warmed x lyse/fix buffer (bd, cat# ) was added. cells were fixed for min at °c, centrifuged and washed twice with facs buffer (pbs supplemented with % fbs and mm edta). staining and permeabilization: fc receptors were blocked for min at rt (human trustain fcx; biolegend), followed by a min stain on ice with anti-cd af (clone rpa-t ; biolegend), and anti-cd bv (clone hib ; biolegend). cells were washed with facs buffer and permeabilized for min on ice with ml phosflow perm buffer ii (bd biosciences, cat# ) that had been precooled to - °c. after permeabilization, two ml facs buffer was added and the samples were centrifuged. after three additional washes, the cells were stained with anti-iкbα pe (clone /ikba/mad- ; bd biosciences) for min at rt. samples were washed three times and data were collected on a cytek dxp flow cytometer. data were analyzed with flowjo software. abstract/case report text introduction: wiskott-aldrich syndrome (was) is a rare, but well-defined x-linked disorder. loss-of-function mutations in the was gene result in classic was and x-linked thrombocytopenia (xlt), while gainof-function mutations lead to x-linked neutropenia (xln). classic was phenotypic features include recurrent infections, microthrombocytopenia and eczema along with increased susceptibility to autoimmune disorders and malignancy. most males with classic was are diagnosed in early childhood and early death can result from its various clinical manifestations. case: we present a -year-old male who was referred to immunology for hypogammaglobulinemia. as an infant he had moderate eczema, and at the age of two was diagnosed with immune thrombocytopenia (itp) with baseline platelets of - x ^ /l. infectious history was notable for one episode of pneumosepsis and recurrent otitis media, influenza, and herpes labialis infections. around the age of , he was diagnosed with common variable immunodeficiency (cvid) based on the finding of low immunoglobulins. he developed diffuse large b cell lymphoma at age , and was treated with cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (chop-r). at age he developed abdominal pain with bloody stools. investigations confirmed an endoscopic and pathologic diagnosis of ulcerative colitis. due to the severity of his disease, he has required maintenance therapy with vedolizumab. he was again noted to have hypogammaglobulinemia at which point he was referred to immunology at our centre. there was no significant family history of immunodeficiency, malignancy or autoimmunity. blood work was notable for normal white blood cell and lymphocyte counts, platelets of x ^ g/l, low igg at . g/l ( . - . g/l) with normal iga, igm and ige. lymphocyte subsets including t, b and nk cells were within the normal range. genetic testing was performed and he was found to have a known pathogenic mutation in the was gene (c. g>a, p.asp asn) which has been previously reported in association with was and xlt. he has since been placed on immunoglobulin replacement and has been referred for consideration of hematopoietic stem cell transplantation. discussion: was is a rare syndrome that can have a similar phenotype to other immunodeficiency disorders including cvid, omenn syndrome and ipex (immune dysregulation, polyendocrinopathy, x-linked). individuals with cvid present with hypogammaglobulinemia and recurrent infections, and these individuals also have an increased susceptibility to autoimmune disorders, gastrointestinal disease and malignancies, especially lymphoma. although eczema is a common disorder, its presence in addition to features of early onset thrombocytopenia, immunodeficiency, autoimmunity and/or malignancy in male patients should heighten the suspicion for was. it is important to make the diagnosis of was as hematopoietic cell transplantation and gene therapy are potentially curative treatment options. abstract/case report text secondary immune deficiencies (sid) are caused by varied mechanisms and are common in patients with hematological malignancies such as chronic lymphocytic leukemia (cll) and multiple myeloma (mm). in this setting, both the disease and its treatment (such as b cell ablation therapy) contribute to the development of secondary antibody deficiency. infections remain a major cause of morbidity and mortality in cll and mm patients. this underscores the need for early recognition and stratification of risks in order to guide appropriate treatment, including immunoglobulin replacement therapy (igrt). new guidelines for the use of human normal immune globulins in sid patients were implemented by the european medicines agency (ema) in . despite these new guidelines, significant variations remain across european countries in the assessment and approaches aiming to achieve reduction in infection burden, including different strategies for initiation, dosing and discontinuation of igrt. the same is true for north america where igrt is widely used off-label to prevent infections in patients with sid due to hematological disease or other reasons. in order to address this variability, a task force comprising both immunologists and hemato-oncologists drafted statements aiming to test for consensus. statements were related to six major areas: definition of infections, measuring igg levels, initiating igrt, igrt dosing, scig usage and discontinuing igrt. this was followed by an international delphi consensus exercise in three rounds which aimed to develop recommendations on how to diagnose, treat and follow-up patients with antibody deficiency associated with hematological malignancies. the first delphi round consisted in testing the statements with a panel of sid specialists and subsequently their comments were used by the task force to refine the statements. in the second delphi round, the refined statements were presented via phone interviews to the same panel to assess their level of agreement with each statement (ranging from "i totally disagree" to "i totally agree"). consensus was considered to be reached per statement if % of the experts agreed with each statement overall. the cut-off for overall agreement was "i somewhat agree". if the expert chose level or less the reasons underpinning his/her choice were discussed. consensus was achieved for all statements on level ("i somewhat agree"). only statements did not achieve consensus on level "i mostly agree". in delphi round , panelists who had not "mostly agreed" with these five statements were given the opportunity to reconsider their assessment based on the feedback from other panelists, which was shared with them. the panelists then chose to maintain or refine their assessment. analysis of the full results on the six key areas identified by the task force will be presented at the conference to offer recommendations and help guide the management of sid in patients with hematological malignancies. abstract/case report text introduction: mast cells (mcs) are hematopoietic-derived immune cells, whose precursors migrate within tissues reaching maturation and differentiation. masitinib, a selective tyrosine kinase inhibitor, is efficient in controlling the survival, differentiation, and degranulation of mcs. aim: to optimize mast cell-differentiation from human bone marrow (bm) hematopoietic stem cells, and to find best cell culture conditions for proliferation, differentiation, and maintenance of mcs, which is important when studying particularly mcs' response to cytotoxic compounds. material-methods: to produce mcs in vitro, the first method (m ) we used was a modified semi-solid culture method ( ). briefly; human bm mononuclear cells (mncs) were obtained with ficoll gradient from bm sample of a patient with idiopathic thrombocytopenic purpura. colonyforming unit (cfu)-mast was developed from mncs in methylcellulose medium supplemented with scf ( ng/ml) + il- ( ng/ml), and il- ( ng/ml; only first week). - weeks later mast cell colonies were transferred into suspension cultures, in which mcs matured and multiplied up to - weeks and were used in experiments till th week of culture. on the other hand, in our second method (m ); mncs were separated by ficoll, seeded in well-plates with imdm containing fbs %, pen/ strep, and a little amount of methylcellulose, and incubated at o c, %co . cultures were then supplemented with imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) on day ; and imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) + il- ( ng/ml) on day . beginning on day till the end, imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) were added to cultures. for both methods, morphological assessment of colonies/cells were evaluated under an inverted microscope (figure and ). verification of mcs was performed by immunoflorescence staining for anti-tryptase andchymase antibodies, and by toluidine blue staining. macrophages were verified by anti-cd- immunoflorescence staining. mcs were exposed to masitinib or dmso for the evaluation of dose-related effects of masitinib, and cytotoxicity was evaluated by mtt assay. results: in m , culture conditions were easier to handle compared to m . in m , high amounts of mcs in immature and pre-mature forms were appeared as early as - days, and peak levels of proliferation rate was around - weeks of culture, which was about weeks earlier than m . culture could be maintained till weeks in both methods. although mcs are non-adherent cells, in liquid method adherent bm cells such as fibroblasts, endothelial cells and mesenchymal stem cells have adhered to the plate and grown up, providing an attachment site for mcs and serving as a natural bm nest, mimicking in-vivo environment, for mcs to grow and proliferate ( figure ). attachment of mcs has provided medium exchange available without changing culture dishes. when mcs were exposed to masitinib ( . , , and μm/μl), approximate survival rates were %, %, %, respectively. discussion: in our liquid medium method, the adherent bm cells not only provided a natural nest supporting mc development and differentiation, they also served as an attachment site for mcs. as the cells slightly adhered, when trypsinized shortly, they easily detached and used for experiments. and we also report for the first time that adding a little amount of methylcellulose to the liquid medium provides ease of aggregation of cfus, and easy development of mcs. we suggest that our liquid culture may be superior to semi-solid method, that it is faster and easier to handle. in studies subjects crossed over to subcutaneous (sc) igiv-c %, and in the third study crossover was to immune globulin sc (human), % caprylate/chromatography purified (igsc %). a total of pi patients from these studies were included in the poppk analysis and serum igg concentrations were included in the final pk analysis. the pk of igg following iv and sc administration was adequately described by a two-compartment model with first-order elimination from the central compartment. administration of igiv was modeled as an infusion directly into the central compartment. absorption of exogenous igg from the depot site of sc infusions into the central compartment was modeled as a first-order process with an absorption rate constant (ka). the full model was constructed by incorporation (forward selection process) of covariates of interest into the model. after completion of the covariate model development, the final model showed that igg pk was not influenced by (a) the igsc formulation used in the different studies ( % vs. %), (b) gender, and (c) age (pediatric vs. adult). body weight was identified as a significant covariate having an effect on clearance and volume of distribution. based on the final pk results, serum clearance of igg for the reference population was estimated to be . l/day. the volume of distribution of the central and peripheral compartments accounted for . l and . l, respectively. the intercompartmental clearance was . l/day, and the absorption constant from the depot (ka) was . day- . the absolute bioavailability of igg after sc administration was calculated as . %. the developed method was used to evaluate alternative dosing intervals following sc administration. the equivalent of a weekly igsc maintenance dose administered , , , , or times per w e e k , o r b i w e e k l y p r o d u c e d o v e r l a p p i n g s t e a d y -s t a t e concentration-time profiles and similar area under the concentration versus time curve (auc), maximum concentration (cmax), and minimum concentration (cmin) values. the results of the evaluation and simulations for igg exposure following a switch from igiv-c % dosing (every -or -weeks) to sc dosing further suggest that a range of dose-adjustment factors (daf), from : to : . would be sufficient to provide clinically effective trough igg concentrations throughout the course of treatment at various treatment frequencies. current us product labeling for igsc % specifies a daf of : . for transitioning immune globulin dosing from iv to sc, and specifies igsc % dosing frequencies of weekly or more frequently ( - times per week). in this poppk analysis all sc dosing regimens evaluated theoretically would provide viable alternative administration options for maintaining adequate immunoprotection in pi patients with dosing flexibility over a range of regimens. however, in % or more, no causative gene can be found going down to undefined inflammatory syndromes (uis). anti-il drugs (ail d) revolutionized some il- mediated diseases, such as traps, caps, hyper-igd/mkd and fmf. nevertheless, treatment response among disorders are not the same as well as no specific study was designed for uis. papa et al, , recently suggested the use of anakinra for the treatment of uis, especially those refractory/intolerant to colchicine with severe or very symptomatic phenotype. this paper aims to retrospectively report for the first time the experience with canakinumab in monogenic and multifactorial disorders in a single, private center in brazil. patient and methods: patients's records that received canakinumab from january to december at clinica croce, ima-brazil, were revised. demographic and clinical data were extracted and descriptively described. all statistical analysis are presented as: average (minimal; maximum; standard deviation). results: a total of patients with autoinflammatory diseases were enrolled and % (n= ) are female. of them, % (n= ) patients had a monogenic disease: % (n= ) caps, % (n= fmf), % (n= ) mkd, % (n= ) homozygous nlcr and % (n= ) pami syndrome. multifactorial disorders were % (n= ) patients : % (n= ) recurrent idiopatic pericarditis, % (n= ) schnitizler syndrome and % (n= ) uis. the average age of the first symptoms was , years ( ; ; , ) and the average age of diagnosis was , years ( ; ; , ) while the aveage of diagnosis delay was , years ( ; ; , ). all patients had used, prior to anti-il , corticosteroids with % prevalence of cushing syndrome and % (n= ) tried at least one steroid sparing agent without clinical success due to: intolerance or non-effective disease control or side effects. in the fmf group (n= ) % tried colchicine prior to canakinumab and this drug was not effective to % because of amyloidosis status and in % colchicine-induced hepatitis was observed. canakinumab was effective for disease control in % (n= ) considering: control of clinical manifestations, amyloidosis reversion and normalization of acute reactants markers. the only side effect observed during the follow up were acute flu-like symptoms and psicomotor agitation ( , % , n = ). the average time of follow up is of , months ( ; ; , ). canakinumab could be discontinued in just one patient with uis. conclusions: this is the first report of canakinumab use for autoinflammatory disorders in brazil. canakinumab is an effective and safe drug for monogenic and multifactorial disorders control. no serious adverse effect could be observed in the years maximum follow up of this drug. neither, no specific infectious disease more prevalent in south america, such as yellow fever, dengue, zika or chikungunya was observed. abstract/case report text a -year-old caucasian male with autosomal recessive hyper igm syndrome type (higm ) due to aicda mutation, diagnosed at age , presented with a newly developed mediastinal mass. he receives routine ivig, pulmonary function tests (pft's) and chest x-rays. at age , patient was noted to have cervical and inguinal lymphadenopathy. ct scan indicated left mediastinal, hilar and pleural lymphadenopathy with soft tissue infiltration around the descending thoracic aorta and esophagus. biopsy indicated no evidence of a lymphoma or infection. years after initial workup, routine pft's showed a declining diffusion capacity by %. patient complained of intermittent chest pain but displayed no clinical symptoms of cough, dyspnea, dysphagia or reflux. ct scan which revealed an extensive illdefined soft tissue mass extending from the thoracic outlet to the level of the esophageal hiatus that encased vascular structures resulting in narrowing and occlusion of left upper lobe pulmonary artery and left lower lobe pulmonary arteries respectively. imaging demonstrated homogenous ventilation to bilateral lungs and decreased perfusion in the left lung compared to the right lung. infectious workup was negative for atypical infections. biopsy revealed miced cellular infiltrate with no predominenant cell type or evidence of malignancy, consistent with previous lymph node biopsy years prior. cd and cd stains revealed aggregates and scattered b-cells and t-cells, respectively. removal of mass was proposed but due to the ambiguous borders and location, surgical excision was not possible. patient was given doses of rituximab ( g), doses mg/kg pulse steroids hours apart, and daily sirolimus (level was adjusted based on sirolimus level). follow-up ct scan indicated significant interval improvement with - % reduction of the soft tissue mass. blood flow in the left lower lobe pulmonary artery has still not returned. this may be due to collaterals and may be a separate problem from compression due to the mass. cytotoxic t-lymphocyte antigen (ctla- ) is an inhibitory immune regulator critical for governing t and b cell homeostasis. heterozygous ctla mutations can cause a syndrome of immune dysregulation with a variable clinical phenotype including hypogammaglobulinemia , autoimmune cytopenia and endocrinopathies, lymphoproliferation, predisposition to malignancy, tissue specific lymphocytic infiltration of brain, lung and gi tract as well as colitis. methods: we retrospectively reviewed medical records of all patients with ctla haploinsufficiency evaluated at the nih between - . a pathologic variant in ctla was confirmed in all patients. we analyzed frequency of campylobacter species detected in the stool samples by pcr based biofilm rapid array as well as reflex bacterial stool and blood cultures when available. results: forty-six patients aged - years were evaluated at the nih between and . six of patients ( %) had at least one episode of campylobacter species associated acute or worsening diarrhea, with one patient also having campylobacter bacteremia. all patients with positive campylobacter species in stool samples had clinical histories and/or endoscopic biopsy findings consistent with enteropathy or colitis predating the incidence of campylobacter infection. two of the six patients ( %) had recurrent or chronic campylobacter infection, while four of the six patients ( %) had multiple gastrointestinal pathogens detected by stool pathogen screening at various times. conclusions: campylobacter species infection of the gastrointestinal tract seem to occur at an increased incidence in our ctla haploinsufficient cohort. to the best of our knowledge, this is the initial report for the association between ctla haploinsufficiency and campylobacter species infection of the gastrointestinal tract. although ctla- is a critical immune checkpoint involved in mucosal immune homeostasis and gut microbiota-immune system cross talk, the underlying mechanism predisposing to campylobacter infection in ctla- deficient patients remains to be explored. our study suggests screening of stool for campylobacter species in patients with ctla haploinsufficiency associated enteropathy. ( ) submission id# abstract/case report text ikaros transcription factor and ikaros family members are critical for development of lymphocytes and other blood cell lineages. full length ikaros (isoform ) contains six c h zinc fingers (zf), four nterminal dna binding zf and two c-terminal dimerization zf. somatic ikaros mutations and deletions have been associated with increased predisposition to b-acute lymphoblastic leukemia (all) as well with poor disease prognosis. recently, germline ikaros mutations affecting the n-terminal dna binding domain and acting in a haploinsufficiency or dominant negative manner were reported to be associated with common variable immunodeficiency (cvid) and combined immunodeficiency (cid), respectively. herein we describe a novel set of germline heterozygous ikaros allelic variants affecting the c-terminal dimerization domains in four unrelated families. clinical manifestations include hematopoietic cytopenias presenting as evans syndrome, and hematologic malignancies including t-cell all and burkitt lymphoma; other manifestations observed were b-cell lymphopenia and hypogammaglobinemia, but recurrent or severe infections were not prevalent or characteristic. we demonstrate that mutants affecting dimerization abolish ikaros homodimerization as well as heterodimerization with ikaros family members aiolos and helios. these variants also affect dna binding at dimerization sites and pericentromeric targeting. opposed to previous allelic variants reported, dimerization changes alter post-translational sumoylation and gene transcription regulation. our data show that mutations affecting ikaros dimerization are mainly associated with cytopenias and/or malignancies, have a different mechanism of action than previously reported variants, present with incomplete clinical penetrance, and contribute to the growing spectrum of genotype-phenotype ikaros associated diseases. introduction: there has been much discussion regarding the return of secondary findings in genetic sequencing research. opinions differ on whether researchers should return secondary findings to participants at all and if so, what the best method is to do so. we have opted to systematically identify and return pertinent secondary findings to participants in our cohort of patients with immune-mediated diseases that undergo exome sequencing. additionally, exome sequencing may determine multiple or other genetic diagnoses in addition to the primary diagnosis, which we call "incidental findings." here, we discuss the secondary and incidental findings discovered in our cohort thus far. methods: individuals in our protocol underwent consent for exome sequencing, including a discussion of the possibility of secondary findings. exome sequencing data was analyzed, and variant pathogenicity was scored using the acmg criteria (richards et al); variants determined to be likely pathogenic, pathogenic, or otherwise clinically important were confirmed via clia-certified sanger sequencing. confirmed variants were returned to participants. we then queried internal databases for cases involving secondary and incidental findings. results: as of november , exome sequencing, interpretation and reporting had been completed for participants. we detected a total of secondary findings in ( . %) participants, including variants in apob, brca ( ), brca ( ), dsp, fbn , kcnh , ldlr, mybpc ( ), ryr , pkp ( ), and vhl. additionally, we detected possible dual/multiple genetic diagnoses in ( . %) participants, some of which explained an unusual clinical presentation or symptom. these included individuals with variants in multiple immune-related genes, including one individual with variants in gata and tnfrsf a, and those with variants in genes related to multiple organ systems, including an individual with variants in ifngr and sco . discussion: exome sequencing in this cohort detects not only important secondary findings, but also discovers a significant portion of individuals with multiple genetic diagnoses. notably, exome sequencing may provide further context or explanation for unusual phenotypic presentation and help determine specific symptom etiology even when a primary genetic etiology is already known. additionally, these secondary and incidental finds may be important to consider when delineating risks and symptoms of novel or recently-discovered conditions. abstract/case report text background: immune dysregulation and lymphoproliferative disorders including alps like disease, hlh ebv driven lymphoproliferative disease leading to rare lymphomas require a multidisciplinary approach utilizing expertise in immunology and hematology/oncology to care for these patients as we learn the molecular etiology of their underlying disorders. at texas children's hospital, the immunology lymphoproliferative evaluation and diagnostic (ilead) clinic was created to provide a comprehensive clinical and research approach to caring for patients with these rare disorders. in an effort to streamline care and access, we recently on-boarded an advanced practice provider (app) . methods: a chart review was conducted months before and after onboarding the app for ilead patient visits. we reviewed the following patient care and access parameters to determine increase in efficient and effective patient care as well as improved access to the clinic. these parameters included: referral process, time of referral placement to appointment, number of patient visits, wait time in clinic, lab interpretation and reporting time for disseminating results to families, and collaboration process with other specialties. results: within months of the app starting our average wait from placing the referral to first appointment fell by an average of %. in addition, we created an algorithm to prioritize patients with immediate need to be seen. by streamlining the referral process and patient priority, we developed a "pre-clinic" conference process by which all patients are reviewed and preliminary plans are made prior to the patient's arrival. this has translated into our ability to increase the number of patients seen in clinic from to and decreased the wait time in clinic by approximately minutes. since the app started, no patient has been in clinic for more than minutes. this has also led to an increase in rvu generation. in terms of efficiency in patient care, all labs are now ordered while in the room with the patient by the app and physician providers. in turn, all labs are resulted directly to the app who reviews labs, collaborates with physicians for care and reports to families in a timely fashion within - weeks of labs being resulted compared to greater than month previously. to improve collaborator communication and post visit plans, a post-visit clinic summary was created. this has been effective in reducing the time to other specialty referrals, follow up visits and effective care for ongoing clinical needs. conclusions: the addition of an app in our ilead multidisciplinary clinic which provides specialized care for patients with immune dysregulation and lymphoproliferative disorders effectively increases work productivity of providers and enhances patient care by increasing access to care, decreasing wait time in clinic and time of reporting of results and future plans. the app with knowledge and expertise in immunology and immune dysregulation is a cost effective way to enhance provider and patient support. with the overwhelmingly positive results, future plans include expanding our multidisciplinary clinic to other services that care for patients with suspected immune deficiency. abstract/case report text introduction: pediatric lymphoproliferative disorders represent a clinically and genetically heterogeneous group of conditions. misdiagnosis and delayed diagnosis can contribute to substantial morbidity and mortality. identification of molecular etiologies and underlying disease mechanisms may facilitate timely interventions and guide targeted or curative therapies. methods: the study was performed through retrospective chart reviews in accordance with all local ethics and irb committees. the study was designed to investigate a cohort of pediatric patients who met criteria for non-malignant lymphoproliferative disorders from texas children's hospital and collaborating centers for underlying genetic etiologies. results: a total of affected individuals from families met criteria. distribution between male and females was nearly equivalent: males (n = ) and females (n = ). approximately half of the cohort was hispanic (n = ). overall kaplan meier survival was % (n = ). whole exome sequencing was performed in all subjects and available family members. likely disease-causing genetic defects were identified in of families ( %). within these families, ( %) carried variants in genes in international union of immunological societies established primary immunodeficiency diseases. potential novel genetic causes of immune deficiency or immune dysregulation were also discovered. mechanistically, all of the implicated genes had roles in modulating lymphocyte activity; initial activation, cytoskeletal organization, or apoptosis of lymphocytes; or regulation of inflammation. all subjects less than one year of age had an identified gene in one of the three mechanistic categories with the dominant mechanistic genetic category being defective control of lymphocyte signaling ( %). in addition, % of patients between and years of age were found to have a potential genetic diagnosis underlying the lpd, with a more equal distribution of mechanistic categories compared to patients greater than years of age where only % have a genetic cause. other important disease manifestations identified were ebv-associated disease in subjects ( %) and subjects ( %) met hlh- criteria. conclusion: primary immunodeficiency diseases and other genetic abnormalities of the immune system underlie a significant percentage of pediatric lymphoproliferative disorder cases. greater than % of patients less than years of age have a genetic etiology underlying the lymphoproliferative disorder. many of these gene defects can be treated with targeted therapies or hematopoietic stem cell transplantation. genetic testing therefore plays an essential role in the diagnosis and management of children with these conditions. abstract/case report text a -year-old gentleman with a history of immune dysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome with known pathogenic variant in foxp presented to our emergency department with two witnessed episodes of tonic-clonic seizures earlier that day. he has had a longstanding history of recurrent infections and autoimmune conditions since birth, and was being treated with monthly ivig infusions and sirolimus while awaiting bone marrow transplantation. his symptoms on admission included foaming at the mouth, generalized shaking, bladder incontinence, and tongue biting that lasted about five minutes. family reported recent sores inside his mouth and lips, but denied any recent fevers, neck pain, headaches, chest pain, abdominal pain, nausea, vomiting, and sick contacts. he lives on a farm with livestock and reportedly had recent tick exposure. his last ivig infusion was two weeks prior to admission, at which time he also received inactivated flu vaccine. in the ed, a third seizure was witnessed by multiple medical providers. he subsequently received lorazepam and lacosamide with interval improvement. he underwent diagnostic lumbar puncture, as well as extensive evaluation for infections. he was started on empiric antibacterial and antiviral meningitis coverage. analysis of the csf showed a lymphocytic pleocytosis; bacterial cultures and hsv / pcr were negative, as was a -pathogen meningitis/ encephalitis panel performed by pcr. eeg was negative for seizure-like activity, and brain mri showed mild atrophy without sclerosis in the left hippocampus. subsequently, anti-infectious therapy was stopped, and patient was discharged with outpatient followup scheduled for suspected non-infectious aseptic meningitis that was potentially triggered by flu vaccination versus ivig. on day four post-discharge, however, pcr for ehrlichia chaffeensis in the serum returned positive, and he was started on oral doxycycline. ehrlichiosis is a rare tick-borne illness that may cause various nonspecific symptoms including fever, headaches, myalgias, and generalized malaise. most prevalent in the mid-atlantic regions of the united states, tick-borne ehrlichia spreads through the mononuclear phagocytic system and can infiltrate many organs including the kidney, liver, lungs, and heart. csf penetration can cause sometimes fatal meningoencephalitis. aseptic meningitis due to ehrlichiosis has been described in recent literature. cases in hiv patients and transplant patients on chronic immunosuppressive therapy have been severe, resulting in organ dysfunction in many instances and death in a few. however, this case marks the first documented ehrlichia infection in a patient with primary immunodeficiency. this patient's presentation of aseptic meningitis and clear exposure history fits the clinical picture. his relatively benign course could be due to preserved t effector function not seen in persons with hiv or transplant patients with significant immunosuppression. patients with ipex usually present with autoimmunity and allergies, but are also prone to significant infections. it is important to perform a comprehensive workup, including testing for atypical infections, in patients with immune dysregulation syndromes who present with symptoms of unclear etiology. special attention should be paid to patients who live in areas with known endemic exposure risks. empiric antibiotic therapy may need to be considered early to prevent delays in treatment. abstract/case report text introduction autosomal recessive hypomorphic mutations in pgm have been described to result most commonly in either hyper-ige or severe combined immunodeficiency (scid) clinical phenotypes in humans, with one report of an individual with combined immunodeficiency without atopy. herein, we describe a series of individuals newly diagnosed with pgm deficiency functionally confirmed using lectin-based flow cytometric analysis of peripheral blood mononuclear cells, that broadens the associated clinical phenotypes to confirm cid without atopy and childhood evans syndrome. in addition, we present new disease-causing pgm variants, and functionally confirm the pathogenicity of a fourth (p.i t). classical hies phenotype cases . and . identify sisters of spanish descent with a classical hyper-ige phenotype. the younger sibling demonstrated severe atopic dermatitis, mild-moderate asthma, multiple food allergies, one episode of itp, and adhd. the older sibling demonstrated atopic dermatitis, skin infections, and c. albicans otomastoiditis. the siblings were found to have the damaging compound heterozygous variants p.t i and p.q x in pgm . case is a year-old guatemalan boy with prominent atopy including asthma, allergic rhinitis, food allergy, elevated ige, atopic dermatitis, as well as oral hsv who was found to be homozygous for the damaging pgm variant p.i t. cid phenotype with a paucity of atopy case is a -year-old turkish girl who is the daughter of a consanguineous union. she presented with infantile nephrotic syndrome at months of age, and subsequently developed leukopenia, neutropenia, and low igg. complications include bronchiectasis, sinusitis, pseudomonas urinary tract infection, and inflammatory skin lesions without atopy. she was found to be homozygous for the damaging pgm variant p.r h evans syndrome case is a -year-old girl from guatemala. she developed multilineage autoimmune cytopenias including immune thrombocytopenic purpura (itp), autoimmune hemolytic anemia (aiha) and autoimmune neutropenia (ain) at the age of years, refractory to multiple treatments and finally responding to mycophenylate mofetil. she has a history of mild eczema but is without other atopy and suffered from multiple invasive bacterial infections. an additional patient, case , was diagnosed with coombs positive aiha and itp at age years refractory to multiple treatments and finally responsive to cyclosporine. cytopenias recurred year later, resulting in hypoxic brain injury. he died of infectious complications at the age of years. both patients were found to be homozygous for the damaging pgm variant p.i t. discussion this is the first report of pgm deficient individuals presenting with evans syndrome as a primary presentation without additional pathology. while disease-associated mutations appear to cluster around the key conserved domains of the protein, no clear genotype-phenotype correlation is readily observed. in addition to autoimmune cytopenias, pgm deficient individuals have also been reported with splenomegaly, lymphoma, and ebv viremia. thus, in particular for children with lymphoproliferative disease, pgm deficiency should also be considered in the differential diagnosis. abstract/case report text introduction: meningitis is a life-threatening manifestation of cryptococcus neoformans (c. neoformans). it occurs in increased frequency in those with impaired cell-mediated immunity, especially those with hiv/aids. infection with c. neoformans has been seen in previously healthy individuals diagnosed with idiopathic cd lymphopenia (icl). icl is defined by an absolute cd + count of less than cells/m on multiple occasions, usually to months apart, without other apparent cause such as hiv infection, immunodeficiency, or immunosuppressive medications. case description: our patient is a previously healthy -yearold female with cryptococcus meningitis and fungemia. her course was complicated by elevated intracranial pressure requiring extraventricular drain. she was treated with amphotericin and flucytosine for month. notably, the patient was also found to have moraxella catarrhalis (m. catarrhalis) bacteremia without identifiable source. she denied history of environmental risk factors, was not up to date on cancer screening, and recently returned from a trip to italy. initial evaluation revealed lymphopenia ( cells/ul), low cd + ( cells/ul), cd + cells ( cells/ul), and cd / + ( cells/ul), but normal cd + ( cells/ul) and cd + cells ( cells/ul). hiv, ana, leukemia/ lymphoma flow cytometry panel was negative. she also had a normal lymphocyte proliferative responses to pha ( . %), normal cd ra:ro, and protective tetanus titers ( . iu/ml), but only / protective pneumococcal serotypes. initial immunoglobulins demonstrated slightly low igg ( mg/dl). laboratory studies months after presentation demonstrated improved lymphopenia ( cells/ul) continued low cd + cells ( cells/ul), but normalized igg levels ( mg/dl). followup labs also demonstrated decreased cd + b cells ( cells/ ul) and insufficient response to polysaccharide vaccine ( / pneumococcal serotypes). three months after discharge, she is continued on daily fluconazole without recurrence of infections although she still has diplopia and headache. discussion: in a review of cryptococcosis in patients with icl, of them had cryptococcal infection in both the cns and blood. of these patients, was cured, improved, relapsed and then improved, and died. three of these patients were treated with amphotericin and flucytosine. five of these patients had underlying disease and had notable infections with vzv, tb, or hpv, however other infections such as m. catarrhalis were not mentioned. m. catarrhalis bacteremia has been described in children with underlying immune dysfunction and respiratory infection as well as secondary to pneumonia with m. catarrhalis. in cases of m. catarrhalis bacteremia in adults, most had underlying malignancy and/or neutropenia, predisposing respiratory factors, or source for infection. conclusion: this report of c. neoformans meningitis and m. catarrhalis bacteremia in the setting of icl is unusual in that to our knowledge, m. catarrhalis bacteremia has not been reported in icl. cases like this also raise the question as to whether some laboratory abnormalities are secondary to infection, treatment, or underlying disease. it is important to report these cases with icl in order to group disease phenotypes, as continued monitoring and data collection of these cases may lead to discovery of new disease processes. abstract/case report text cytokines play critical roles in regulating the development, survival, differentiation and effector function of immune cells. cytokines exert their function by binding specific receptors on the surface of immune cells and typically activating intracellular jak/stat signaling pathways, resulting in induction of specific transcription factors and regulated expression of target genes. in order to differentiate into an appropriate effector fate, lymphocytes need to integrate multiple signals often provided concomitantly by numerous cytokines that activate shared transcription factors. how these signals are balanced and regulated to yield the optimal class of immune response remains to be completely determined. inborn errors of immunity, or primary immunodeficiencies (pids), result from germline mutations in defined genes, leading to loss-of expression, loss-of function, or gain-of function of the encoded protein. pids are characterised by defects in immune cell development, or their differentiation into effector cells during immune responses, thereby rendering patients not only highly susceptible to infectious diseases, but also autoimmunity, autoinflammation, allergy and cancer. pids are thus an unprecedented model to link defined monogenic defects to immune dysregulation in clinical settings. indeed, pids have unequivocally revealed non-redundant roles of single genes, molecules, signaling pathways and lymphocyte subsets in host defense and immune regulation, and formed the basis of better therapies for immunopathologies. our indepth analysis of inborn errors of immunity of cytokine signalling pathways have identified fundamental requirements for generating long-lived humoral immune responses in humans. here, i will present data relating to our recent studies of how inactivating mutations in il r, il r, znf , stat , stat , and stat , disrupt or dysregulate the generation and function of human memory b cells and tfh cells, thereby precipitating humoral immunity, as well as allergic disease and autoimmunity. abstract/case report text introduction: flow cytometry is a powerful diagnostic tool for detecting hematologic malignancies in a variety of patient specimens including body fluids and lymph node aspirates. cytopathologists are frequently confronted with lymphocyterich effusions, and the definite decision of whether the lymphocytosis is of a purely reactive nature or a presentation of an indolent lymphoma may be an extremely difficult based on microscopy alone. moreover, small proportions of malignant cells that may be missed out by routine morphology can be detected by flow cytometry. objective: the purpose of this study was to evaluate the usefulness of multiparametric flow cytometry immunophenotyping (fci) to confirm the presence of leukemia or lymphoma cells in body fluids and fna specimens. methods: body fluids and fna specimens simultaneously obtained for fci, cytologic analysis and real time pcr from patients were submitted to our flow cytometry laboratory from january to september . the samples studied were body fluids ( pleural fluids and ascitic fluids) and fna samples ( enlarged lymph nodes and lung mass).four color fci method was performed and the following fluorescent monoclonal antibodies were used: cd , cd , cd , cd , cd , cd , cd b, fmc , kappa and lambda light chains, cd , cd , cd , cd c, cd , cd a, cd , cd , cd , cd , cd , tdt, cd , cd , cd , cd , cd , cd , bcl , cd . fci analysis was performed on a beckman coulter cytomics fc flow cytometer using software cxp to analyze data. the cases were diagnosed as leukemia or lymphoma as per tuberculosis ( case). ascitic fluid (n= ) samples showed positivity for angioimmunoblastic t-cell lymphoma ( cases) and dlbcl ( case). fna of lymph nodes (n= ) were positive fort-lymphoblastic lymphoma ( cases), angioimmunoblastic tcell lymphoma ( cases), dlbcl ( cases), hodgkin lymphoma ( case), nodular lymphocyte predominant hodgkin lymphoma ( case), peripheral t-cell lymphoma (nos) ( case), splenic b-cell marginal zone lymphoma( case), tuberculosis ( cases). one fna of lung mass were tumor of neural cell origin. both immunophenotype and cytomorphology positive for malignancy were in / ( . %) cases.cytomorphology was negative/ suspicious in / ( . %) cases, of which both cytomorphology a n d i m m u n o p h e n o t y p e n e g a t i v e w e r e ( % ) a n d cytomorphology negative but immunophenotype positive cases were ( . %). mtb dna was detected in pleural fluid in case and fna sample in cases. conclusion: multiparametric flow cytometry by using comprehensive panel of monoclonal antibodies is a useful diagnostic test to evaluate body fluids or fna as it can demonstrate small malignant populations that may be missed out by routine cytomorphology. clinical laboratory geneticist/department of genetics, university of groningen, groningen, the netherlands abstract/case report text the phenotypes of primary antibody deficient (pad) patients range from milder (e.g. specific antibody deficiency) to severe (e.g. x-linked agammaglobulinemia) deficiency of the immune system. pad patients form a clinically, immunologically as well as genetically heterogeneous group. often, the genetic background has not been elucidated; it probably is not monogenetic in a large subgroup of patients. pad patients suffer most frequently from recurrent bacterial infections of the respiratory or gastrointestinal tract due to immune deficiency, but may also have varying degrees of autoimmune and lymphoproliferative comorbidities due to immune dysregulation. unfortunately, a standardized description of pad phenotypes is lacking rendering robust definitions of pad-subtype diagnoses, including cvid, difficult. this impairs the formation of homogeneous cohorts that can form the starting point for future clinical and genetic research. the pad subgroup of the human phenotype ontology (hpo) immune mediated disorders consortium supported by ern rita and esid is addressing the gaps in standardized phenotypic description of pads. using the hpo dataset, literature mining, and esid, iuis and omim classifications, we aimed to reevaluate and complete the pad-related hpo terms to allow efficient data exchange and matching of phenotypically similar pad patients. as a principle, it was decided to avoid the ongoing variance in pad-subtype definitions and to build the pad-related hpo tree based as much as possible on unambiguously interpretable items. 'hypogammaglobulinemia' was deleted as hpo term, and replaced by separate hpo terms such as 'decreased total igg in blood', subdivided in 'transient' vs. 'chronic', and '(near) absent' vs. 'partially decreased' (the same for igg , igg , igg , igg , iga and igm). 'decreased specific antibody level in blood' was specified further into 'decreased natural antibody level to blood group antigens in blood', subdivided in '(near) complete' vs. 'partial' absence (the same for protein, polysaccharide and protein-conjugated polysaccharide vaccination). relevant hpo terms related to infection and to specific organ manifestations like bronchiectasis, autoimmunity and lymphoproliferation were re-evaluated and completed, and will be linked to pad diseases in the hpo online system by the pad subgroup experts. once finalized, existing pad cohorts will be classified according to the new hpo pad-related terms, and studied by clustering technologies (example of two patients shown in figure ; white = absent, color = present). acceptance and widespread use of this pad-related hpo tree for standardized phenotyping will be essential to empower future multicenter clinical research and related genetic discoveries as well as support clinicians in diagnosing pad through the linkage of hpo terms to pad disease entities. senior investigator oral immunity & infection section/nih/nidcr abstract/case report text leukocyte adhesion deficiency type (lad ) is an autosomal recessive disorder characterized by the inability of granulocytes to emigrate from the bloodstream to sites of inflammation. lad is caused by mutations in the itgb gene ( q . ), encoding the beta- -integrin, cd , which is essential for firm adhesion of leukocytes to the endothelium. in lad survival is compromised, morbidity from inflammatory lesions is high, and treatment is poor. the moderate form of lad is often managed with antibiotics for prophylaxis and during acute infections. after infancy severe gingivitis and chronic periodontitis are universal. periodontal findings affect primary and permanent teeth, causing intense oral mucosal (gingival) inflammation and destruction of tooth supporting bone, which are hallmarks of the disease periodontitis. blocking the il- /il cytokines, which are up regulated in lad gingiva, can reduce bacterial load and resolve inflammatory gingivitis. ustekinumab binds to the shared p subunit of human il- and il- , cytokines that modulate lymphocyte function, including t helper (th) cells and th subsets, thereby blocking them. objective: explore the effect of ustekinumab on lad inflammatory disease. method: prospective study using ustekinumab for oral inflammation. patients receive five doses over year, -or mg depending of weight. results: (two patients have enrolled, p is > year post treatment, p is still on study) patient characteristics · patient : age at diagnosis, yrs.(itgb mutation c. delt (null)); cd (%pmn control): . %; cd a(%pmn); . %. at the initiation of the protocol ( yrs old) wcc: . k/ul; crp: . · patient : age at diagnosis, yrs.(itgb mutation c. a>g,p.g s c. c>t,p.a v) cd (%pmn control): %; cd a(%pmn): . %. at the initiation of the protocol ( yrs old) wcc: . k/ul; crp: . response: patient : oral ulcers before treatment: episodes every two months. during ustekinumab therapy: · oral ulcers: episode in a year · reduction in bleeding on probing: . % · gingival index reduction: % patient : oral ulcers before treatment : monthly. during ustekinumab therapy: · oral ulcers: none in first months · reduction in bleeding on probing : . % · gingival index reduction: . % safety: no significant adverse events were documented during the therapy p had a previous skin lesion that flared leading to iv antibiotics. p had a previous sebaceous cyst drain spontaneously. discussion: two patients showed improvement in chronic periodontitis and a substantial decrease in oral ulcers while on ustekinumab. no clear safety signals were seen. durability of these findings is still unknown. ustekinumab in lad deficiency appears to be safe and potentially effective. post doctoral fellow/servicio de inmunología, inst. multidisciplinario de investigación en patologías pediátricas (imipp), hospital de niños ricardo gutiérrez. chief resident/servicio de inmunología-hospital de niños "dr. r.gutierrez" immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños "dr. r.gutierrez" immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo" immunologist/servicio de inmunología-hospital de niños "dr. r.gutierrez" abstract/case report text introduction: primary immunodeficiencies with dysregulation associate defects in the immune homeostasis leading to inappropriate immune response (lack or excess) that causes autoimmunity, allergy and/or inflammation. impairment of different subsets of t and b compartments may be associated with these pids. aim: ) describe t and b memory compartment of pid patients (pts) with dysregulation: cd deficiency, stat gof, stat b deficiency, ctla variant, pi kcd variant and cvid-like (with no molecular defect) and compare them with a group of healthy donors (hd). ) associate ctfh profile with b cell compartment impairment. results: ) pts showed a significant decrease of naïve cd + t cells (cd ra+cd +) ( , % vs , %) (p < . ) with expanded central memory t cells (cd ra-cd +) ( . % vs . %) (p < . ); cd + t cells had higher levels of activation markers (cd +hla-dr+) ( , %vs , %) (p < , ). pts showed a significant increase of circulating follicular t cells (ctfh) (cd ra-cxcr +) compared with hd (mean , % vs , %) (p < , ) with pd- overexpression (p < . ). stat gof, ctla , pi kcd and cvid-like pts showed a skew towards ctfh (cxcr +). regulatory t cells (cd +cd ++ foxp +) were absent in cd and stat b deficiency and decreased in the other pts. within cd + cells, although effector memory (cd ra-cd -) (p < . ), temra (cd ra+ cd -) (p < . ) and hla-dr+ cd + (p < . ) subsets showed a significant increase compared with hd, the behaviour was variable between different mutations. regarding b cell compartment, pts with stat gof, pi kcd and cvid-like showed a severe impairment of switched-memory b cells (sw-mbl) (cd +igd-igm-); the stat b deficient patient had increased frequencies of this subset, while ctla pts had a variable b defect. ) lower sw-mbl values were significantly associated with lower values of ctfh cells (p < . ) (r= . ). cd low b cells were exclusively high in cvid-like pts, and transitional b cells were increase in pi kcd and almost all cvid-like pts. discussion: in summary, patients with dysregulatory syndromes associate a defect of t and b homeostasis (survival, activation and differentiation). specific mutations can differentially affect the quantity and/or the quality of ctfh. there is a strict association between the differentiations of tfh with th profile with the generation of sw-mbl. these alterations may play a role in the pathophysiology of primary immunodeficiencies with b lymphocyte functional impairment. immune monitoring of lymphocyte subsets of patient with dysregulation may approach to the diagnosis of specific monogenic mutations. objective: the purpose of this study is to increase awareness and improve diagnosis of primary immune deficiency (pid) in the heterogenous group of patients with autoimmune cytopenia (aic) by identifying clinical characteristics and laboratory biomarkers that distinguish those with underlying pid, disease activity and guide mechanism-based targeted therapy. methods: patients with aic (autoimmune hemolytic anemia (aiha), immune thrombocytopenia (itp), or autoimmune neutropenia (ain)) were referred to our immune dysregulation team and prospectively enrolled during - . detailed immune phenotyping (igg, iga, igm, lymphocyte subsets, vaccine titers, lymphocyte proliferation to mitogens/ antigens), serum lipopolysaccharide (selps) and autoantibodies were measured and/or collected by chart review and genetic testing for pid was pursued. results: from to , patients were enrolled; two subjects were removed due to parental request or lack of aic diagnosis. of the remaining patients, ( %) were classified as "aic-pid" based on genetic testing and/or immune phenotyping; ( %) were classified as aic-only, and ( %) were asymptomatic family controls. the patients were predominantly children (ages - years, average age . years); % ( / ) were male. among patients who have had genetic testing to date (n= )( %), pathogenic genetic mutations were confirmed in / ( %) of patients. mutations include fas/fasl (n= , including family members without aic), ctla (n= ), q (n= ), and one patient each with nfkb , was, pole- , pi k, casp , card , and cgd; the remainder of aic-pid patients were classified as combined immune deficiency or common variable immune deficiency based on immune phenotyping. lymphocyte subsets (cd +t, cd +t, cd +b, cd + nk) and immune globulins (igg, iga, igm) tended to be lower in aic-pid patients vs aic-only (p < . ). evans syndrome was more common in aic-pid patients ( / , %) compared to aic-only ( / , %). lps was elevated in the serum of aic patients compared to healthy controls (mean vs pg/ml, p < . ). excluding partial digeorge syndrome patients (average lps pg/ml), selps levels were significantly higher in genetically-defined untreated pid patients (average pg/ml) vs. other pid (average pg/ml)(p= . ) or patients with aic alone (average pg/ml)(p= . ). studies are ongoing on specific subsets that are linked to immune dysregulation (switched memory b cells, t-regulatory cells, double negative t cells, t follicular helper cells) and the use of soluble il- as a biomarker of disease activity. conclusions: a high fraction of aic patient were identified with underlying pid in our study. basic immune evaluation with immunoglobulin levels and lymphocyte subsets expedited diagnosis of pid. genetic evaluation distinguished a group of patients with aic-pid and highly elevated lps level, reflecting high bacterial load, which may distinguish them from the rest the aic cohort. the source of bacterial lps can be multifactorial and is yet to be determined. our studies continue focusing on biomarkers that can be applied to the heterogenous group of patients with aic. this will allow early detection and timely initiation of targeted therapies. investigator/dermatology branch, niams, nih head, dermatology consult service/dermatology branch, niams, nih chief, fungal pathogenesis section/laboratory of clinical immunology and microbiology, nih abstract/case report text introduction/background: autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a monogenic autoimmune disease resulting from biallelic mutations in the aire gene. although typically characterized by the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency, we recently reported that the clinical spectrum of the syndrome is far broader and that incorporation of an adjunct triad of apeced rash, intestinal dysfunction, and enamel hypoplasia in the classic triad could lead to earlier diagnosis (ferre et al., jci insight, ). among the adjunct triad manifestations, apeced rash occurs in % of american apeced patients by age , most often developing in the first year of life. objectives: to report and describe the clinical features of apeced rash as the first manifestation in a -month old patient with apeced. methods: following enrollment in a niaid irb-approved protocol ( -i- ) the patient was evaluated with history and physical examination, aire sequencing, measurement of interferon-autoantibodies, and skin biopsy with immunohistochemical analyses. results: a -month-old girl with a family history of genetically confirmed apeced in her -year old sister developed discrete circular, maculopapular erythematous lesions on her torso that spread to the face, arms, and legs while sparing the palms and soles. the rash was partially blanching, non-painful and non-pruritic and was preceded by low-grade fever ( □c) without other accompanying symptoms. she had not received medications or vaccinations prior to the rash onset. the lesions increased in size with associated central clearing and resolved . months after onset. the rash recurred with similar appearance times over months with each recurrence lasting between days and . months. as with the first rash episode, recurrences were often preceded by fever ( - □c) without accompanying symptoms or inciting factors. neither topical nor oral antihistamines improved the rash. aire sequencing identified the same compound heterozygous mutations (c. _ del and c. c>t) that the sister has. high titers of interferon-□ autoantibodies were measured in serum. skin biopsy revealed superficial perivascular chronic inflammation and intraepidermal lymphocytes composed predominantly of mixed cd and cd t lymphocytes with few perivascular b lymphocytes. no eosinophils or vasculitis was observed. myeloperoxidase immunostaining revealed extensive karyorrhexis. laboratory studies revealed normal white count and esr, negative anti-ige receptor antibody, and positive anti-ige antibody. at months, she developed oral candidiasis as second manifestation of apeced, thus reaching a diagnostic dyad when applying our proposed expanded diagnostic criteria. she has not developed hypoparathyroidism or adrenal insufficiency; thus, she has not yet reached a classic diagnostic dyad. systematic screening for these endocrinopathies will be needed to avoid lifethreatening complications of acute endocrine failure. conclusions: we report the clinical and histologic features of apeced rash manifesting as the first disease component of apeced in a month old girl. apeced should be considered in the differential diagnosis of recurrent erythematous maculopapular urticaria-like eruptions characterized by mixed lymphocytic and neutrophilic infiltration unresponsive to antihistamines. our case illustrates the clinical utility of incorporating the expanded diagnostic criteria of apeced rash, enamel hypoplasia and intestinal dysfunction into the classic diagnostic triad, which can lead to earlier apeced diagnosis. who presented with prolonged severe neutropenia despite g-csf and seven hospitalizations for febrile neutropenia in the span of ten months. prior to his neutropenia, patient was on monthly ivig, with igg trough - in the past year. he was evaluated for bmt in but declined. patient was first found to be neutropenic in aug when he was admitted with pseudomonas thigh abscess, hsv stomatitis and rhinovirus infection. he was treated with broad-spectrum antibiotics with improvement in neutropenia. the following month, he was hospitalized again with neutropenic fever, left axilla pseudomonas abscess and rhinovirus infection. he underwent bone marrow biopsy revealing left shifted myeloids with decreased maturing forms and t cell predominant lymphoid aggregates, suggestive of autoimmune neutropenia vs. hyper igm syndrome associated with neutropenia. anti-neutrophils antibodies were negative. he was then admitted the following month ( / ) with febrile neutropenia with cxr concerning for viral pneumonitis vs. atypical pneumonia. he was started on g-csf therapy with significant initial response in anc. however, this response was short-lived as he was again admitted in / with febrile neutropenia and upper respiratory rhinovirus infection. he was continued on daily g-csf. due to persistently normal anc for approximately three weeks, he was weaned off g-csf in / . in / and / , he had two more hospitalizations for febrile neutropenia. g-csf was restarted with dose uptitrated to mcg/kg during his hospitalization in april. he was found to be thrombocytopenic with splenomegaly on abdominal ultrasound. anti-platelet antibodies and repeat anti neutrophil antibodies were not detected. he was discharged with close follow up with immunology and hematology. due to his age, he was transitioned to penn allergy/immunology in / . there was close communication between chop allergy/immunology, chop hematology and penn allergy/immunology during this transition period. patient was admitted to hup in / with febrile neutropenia (despite higher dose of g-csf), rhinovirus infection, pseudomonas sinusitis and ct chest findings suggestive of possible fungal pneumonia. due to persistent neutropenia refractory to g-csf treatment, hematology was consulted and repeat bone marrow biopsy showed hypercellular bone marrow with markedly left shifted granulocytic hyperplasia, compatible with g-csf therapy. flow cytometry showed no evidence of plasma cell neoplasm. dose of ivig was adjusted and increased based on his weight. per hematology, he also received an additional high dose ivig g/kg x days for presumed immune mediated neutropenia with immediate increase in anc. despite anc of for days,anadditional g/kgofivigimprovedhisancto> within hours of his first dose. thrombocytopenia also improved to normal range. since then, patient has been on monthly - mg/kg ivig with no recurrence in neutropenia. this patient's prolonged persistent neutropenia with immediate response to high dose ivig is suggestive of autoimmune neutropenia, which should be taken into consideration in hyper igm patients with persistent neutropenia. abstract/case report text background: children with digeorge anomaly (dga) represent a heterogenous group, often classified as either partial dga (pdga) or complete dga (cdga) based upon the degree of thymic hypoplasia. this paucity of t-cell parameters and function has serious implications for infection risk, autoimmunity, and malignancy. however, there are limited studies stratifying children with dga by these subgroups, especially in regard to immune function and subsequent infection risk. study design: single-center, retrospective cohort analysis evaluating the relationship between pdga and cdga to infection risk with particular focus on infection-related hospitalization, pathogenic organism identification, and antimicrobial resistance profiles. the source population includes all pediatric patients < years of age diagnosed with either pdga or cdga while receiving care at duke university from january , to june , . the final analysis sample included patients. methods: to evaluate the differences in immune function between dga subgroups, we will report the proportion of low ( < th percentile for age) t cell immune biomarkers for both subgroups and compare populations using a chi-squared test. to compare per year incidence of infectionrelated hospitalization for dga subgroups, a poisson model with number of hospitalizations per patient as the outcome, an offset equal to the time at risk for hospitalization, and either pdga or cdga diagnosis as the exposure will be used. models will be bivariate. we will report an incidence rate ratio (irr) and % confidence interval ( % ci). to evaluate the impact of cellular and humoral immune function on infection-related hospitalization, we will use poisson models where the outcome is the number of hospitalizations per patient, an offset equal to the time at risk for hospitalization, and low immune biomarker as the exposure. all models will be bivariate. we will report an irr and % ci. infection type and resistance profiles will be completely descriptive. results: as expected, children with cdga had a significantly higher probability of a low ( < th percentile for age) values for total t cells (cd +), helper t cells (cd +cd +), cytotoxic t cells (cd +cd +), and naïve helper t-cells (cd +cd ra+cd l+) as well as a significantly lower probability of low pan memory t-cells (cd +cd ro+) compared to children with pdga. no differences were detected in the percentage of low natural killer (nk) cells (cd +cd +) or b cells (cd +) between subgroups. cdga patients had a significantly higher incidence of hospitalization per year ( . ( . , . )) compared to pdga patients ( . ( . , . )). the irr is . ( . , . ). across both subgroups, the incidence of hospitalization was higher in dga patients who had low helper and naïve t-cells. there is ongoing analysis into hospital-related infection and resistance profiles. notable frequencies include bacteremia ( > %), invasive viral disease ( > %), and opportunistic infections ( > %). conclusions: children who had cdga were % more likely to have an infection requiring hospitalization than children who had pdga, emphasizing the need for thymus transplant for cdga. further analysis of infection type and patient outcomes is critical to enhancing management of this unique patient population. abstract/case report text antibody cross-reactivity among flavivirus has been documented. in recent times zika virus has been emerging in pockets of the mosquito-infested regions, while southwestern saudi arabia is known for arthropod-borne viral diseases and we do not know the incidence or even presence of zika virus in this region. it is restricted to predict the igm and igg antibody detection ranges owing to limited data and colossal cross-reactivity among the zika and other flaviviruses. we tested sera from pregnant women irrespective of their clinical presentation for zika and dengue igm, igg respectively. the zika positive samples were further confirmed by plaque reduction neutralization tests (prnt). from our results, . % ( ) cases were positive for zika igm against . % ( ) positivity to igg. when these samples were assessed for dengue igm and igg, we observed . % ( ) seropositivity for igm and igg respectively. there was no single sample positive for both igm and igg of zika or dengue. however, we observed one sample positive for both zika and dengue igm. upon mapping the overlapping serotiters, there was no significant correlation observed between the dengue igm and igg. whereas zika igg positive sample showed high serotiter for dengue igg indicating the contribution of cross-reactivity for observed zika positivity. screening for the incidence of zika, therefore, becomes particularly hard in a population that has the presence of pre-exposure of dengue and this cross-reactivity makes it hard to determine the zika incubation and antibody prevalence confounded with other flaviviruses. abstract/case report text introduction humoral pid diagnostic protocol includes the analysis of the immune response to different protein and polysaccharide antigens (ags) ( ) . although the analysis of the immune response against the polysaccharides vaccine from pneumococca has been the standard method, the use of s. typhim vi vaccine has appeared as a good alternative ( ) . in this report we show the results obtained with the use of s.typhim vi in adults patients attending the pid outpatient clinic. material and methods patients with humoral-suspected pids were challenged with typhoid polysaccharide vaccine (typhim vi®; sanofi-pasteur). serum was obtained on basal and after weeks of vaccination. specific igg levels against s.typhim were measured using "vacczyme tm human anti-salmonella typhi vi igg enzyme immunoassay kit" (binding-site). results a total of adult patients attended the pid clinics during nov -nov . from those, patients were fully evaluated using a humoral-suspected pid algorithm that includes the s.typhi vaccination. in total male and female patients completed the protocol and were analyzed. twenty patients were considered as responders (ratio pre/ post > x) whereas patients were non-responders. discussion the main advantage of assessing polysaccharide immune response using s.tyhpim is the usual lack of specific igg at the moment of the initial evaluation. in this serie, just one patient has a high basal level (# -vaccinated in the past). thirteen patients had basal levels below the detection limit of the test ( , u/ml) and patients between , and , u/ml, that has been described as a cut otf level for nonimmunised individuals (personal experience, , ). regarding the polysaccharide immune response as a tool to distinguish pid vs non-pid patients, the results showed a good correlation between those non-responders with more clinical relevant pid diagnostics. seven non-responders patients were subsequently diagnosed with a primary (* on table i ) and/or secondary id (** on table i ). despite this, there were patients that we could have classified as strong responders (ratio > x, absolute specific igg postvaccination level > u/ml) and patients considered as weak responders (ratio post/pre > x, absolute specific igg postvaccination level < u/ml). strong responders were considered non-pid after including other clinical investigations and laboratory tests (cell subpopulation study, pcp response) whereas weak responders group consisted in some "minor" forms of pid, like isolated igm or ig subclasses deficits. more patients are needed to confirm this functional classification of pid patients regarding their s.typhi immune response. abstract/case report text activated pi kδ syndrome (apds) is a primary immunodeficiency characterized by recurrent respiratory infections, as well as increased risk of chronic viremia with herpes family viruses, benign lymphadenopathy and b cell lymphoma. it is caused by heterogeneous germline gain-of-function mutations which ultimately lead to the hyperactivation of the phosphoinositide- -kinase δ (pik δ). pik δ exists as a heterodimer composed of a catalytic and a regulatory subunit. it interacts with b cell receptors, t cell receptors, costimulatory and cytokine receptors, and is a key player in a signaling pathway involved in cell growth, proliferation and survival. apds is caused by mutations in the pik cd gene, affecting its protein product p δ (catalytic subunit). apds is caused by mutations in the pik r affecting p a (regulatory subunit). short syndrome is a rare multisystem disorder characterized by short stature, hypertextensible joints, ocular depression, reiger anomaly and tooth eruption delay. the primary causes of short syndrome are heterozygous loss-of-function mutations in the pik r gene. the combination of apds and short syndrome is very rare, with only few cases described in the literature. in this report we present a teenager with a pathogenic variant in the pik r gene, and phenotypic characteristics of both apds and short syndrome. our patient is a -year-old female with a history of growth delay and short stature, delay tooth eruption, recurrent sinopulmonary infections and hypogammaglobulinemia. evaluation performed at a prior institution for recurrent infections revealed low igg levels. she did not initiate therapy at that time and was lost to follow up for several years. at the time of our initial evaluation she reported continued recurrent episodes of upper respiratory infections and sinus infections requiring antibiotic treatment that often did not clear the infections. her physical exam was relevant for short stature ( %ile, z=- . ), low weight for age ( < %ile, z=- . ) and hyperextensibility. her facial features were significant for prominent forehead and triangular face. given concern for immune deficiency, a complete immune evaluation was obtained. her workup revealed low igg levels, with igm and iga within normal limits. she did not have protective titers to s. pneumoniae, h. influenza or diphtheria and tetanus. after administration of vaccine boosters, she was able to generate a response to all vaccines except for tetanus. she had remarkably low absolute b cells ( cells/ul) and percentage ( %), and low cd :cd ratio ( . ). she was started on amoxicillin prophylaxis and monthly ivig replacement therapy. invitae immunodeficiency panel genetic testing was sent and revealed a pathogenic loss of function variant in an intronic splice site in the gene pik r (c. + g>c). after initiating treatment with ivig, her sinus infections significantly improved and she has not had any further episodes. igg levels have remained within normal limits with monthly ivig therapy. this pathogenic variant had been previously associated with apds ; however, it had not been associated with short syndrome. the mechanisms that link both conditions is yet to be identified. this case report emphasizes the importance of screening for comorbidities associated with short syndrome in apds patients, and vice versa. finding the genetic diagnosis for patients with suspicion of primary immunodeficiency (pid) is becoming increasingly important in the management of primary immunodeficiency and estimating the risk for family members. we constantly increase the diagnostic yield for pids by improving the sequencing technology, updating the panels with new genes discovered related to pid, and finding diagnoses from difficult to sequence regions and regions with high homology. here we report our experiences with nearly patients suspected with pid. moreover, we provide a case example, how we increase the diagnostic yield by developing unique techniques for specific genes which cannot be reliably analyzed by ngs alone. diagnostic yield including all immunology related panels was . % ( / ). the majority of the tested individuals were males ( / , . %) and the most common age of testing was between to years ( / , . %). the highest diagnostic yield . % ( / ) is in children from ages to years, whereas in patients over years of age the diagnosis was found for only . % ( / ) of the patients. in two patient cases, our cnv detection algorithm indicated a homozygous deletion in the index patient samples potentially covering the whole ncf gene. additional bioinformatic analysis targeting specifically two coding positions that differ between the ncf gene and the two pseudogenes showed that all reads in those positions originated from the pseudogenes. homozygous deletion in the ncf gene was further confirmed by sanger sequencing two regions in ncf with primers that specifically bind to either ncf or the pseudogenes. while clean ncf sequences from both regions were obtained for a control sample, no ncf -specific amplification product was obtained for the index patient samples. pseudogenes were amplified and sequenced successfully in both index patient samples and positive control samples. loss-of-function of ncf is a well-established mechanism leading to cgd and by overcoming the difficulties regarding ncf deletion detection by ngs, we can improve diagnostic rate in individuals affected with cgd. gata deficiency can lead to a broad spectrum of clinical and hematological phenotypes; in some cases, nk cell deficiency is the primary manifestation, resulting in a greatly increased susceptibility to viral infections and malignancy. gata -deficient patients, particularly those who suffer from severe viral infections, have reduced frequencies of peripheral blood nk cells and loss of function in the existing nk cells. specific loss of the less mature (cd ^bright) nk cell subset is a hallmark of the immune phenotype in gata deficiency, suggesting that generation or survival of nk cell precursors is impaired. given the remarkable spectrum of clinical phenotypes in gata -deficient patients and the poorly understood biology underlying their nk cell defect, we sought to characterize circulating nk cells on a single-cell level. we performed single-cell (sc)rnaseq of lineage-depleted innate lymphocytes from a patient with gata deficiency. as expected from flow cytometric phenotyping of peripheral blood cells from this and other patients, scrnaseq revealed decreased representation of canonical cd ^bright cells. within the cd ^dim population, we identified two nk cell populations that were seemingly unique to the gata deficient patient relative to a healthy donor. pathway analysis defined the first of these populations (population ) by the expression of genes associated with cellular response to stress, extracellular stimulus and inflammation, as well as programmed cell death and regulation of proliferation and apoptosis. the second population (population ) was defined by genes associated with nk cell chemotaxis, cytokine responses and interferon signaling. to extend our findings, we performed scrnaseq of additional healthy donors and analyzed an additional gata -deficient individual who was clinically asymptomatic (yang et al. ). of note, we detected population in seemingly healthy cmvnegative individuals, suggesting it was not uniquely a result of gata deficiency but associated with an inflammatory response and not related to adaptive nk cells generated in response to cmv infection. population , on the other hand, only appeared in our symptomatic gata -deficient patient. we additionally performed bulk gene expression analyses from an unrelated gata -deficient patient that confirmed the altered expression of genes associated with both novel cell populations. current efforts are focused on better defining the functional response of nk cells in these patients and confirming the identification of our novel populations by mass cytometry (cytof). together, our data define the heterogeneity and complexity of nk cells in gata deficient and healthy individuals. perforations have been reported with tocilizumab, a monoclonal antibody of the interleukin (il- ) receptor, suggesting that il- signaling plays a role in intestinal wall integrity. as il- signals through stat , we sought to investigate the potential association between lof stat and intestinal perforations, as well as the incidence and outcome in our patient cohort. methods: we performed a retrospective chart review of patients with lof stat (n= ) followed at our institution, looking for those with non-malignancy associated spontaneous gastrointestinal perforations. the demographic information, stat mutation, comorbidities at the time of perforation, clinical presentation, management, and clinical outcomes were compiled results: ten lof stat patients were identified as having documented intestinal perforations, an approximate rate of %. one perforation was the initial presentation of diffuse large b cell lymphoma (dlbcl) of the duodenum and liver, and was excluded from the rest of the analysis. the other nine perforations occurred between to years old (mean: ), and % were female. stat mutations were localized to the dna binding domain (n= ) and the sh domain (n= ). two of the perforations occurred while inpatient for lung infection. another occurred while recovering from pneumonia at home. two perforations were associated with the initial diagnosis of diverticulitis (at age and ). one perforation occurred in the terminal ileum, one in the cecum, one in the transverse colon, and six in the sigmoid. five patients underwent primary closure of the bowel. four patients required a temporary ostomy, with subsequent successful ostomy reversal. only one patient has since died of pulmonary hemorrhage, the other patients are alive with a mean of years post perforation follow-up, with no recurrence of perforation. one patient with prior sigmoid resection required ileal resection post perforation due to as massive intestinal bleed. conclusion: spontaneous gastrointestinal perforations occurred in our lof stat cohort at a rate of approximately %. one case was associated with malignant infiltration of the gastrointestinal tract and two cases were associated with diverticulitis both known risk factors for perforation. although the pathogenesis of the perforations in lof stat remains unclear, the connective tissue phenotype likely contributes as well as the association with diminished il- signaling, as has been demonstrated with the perforations and tocilizumab. abstract/case report text background granulomatous and lymphocytic interstitial lung disease (glild) is a life-threatening complication that occurs in patients with common variable immunodeficiency (cvid) and monogenic cvid-like disorders, but the optimal treatment is unknown. objective to determine if the use rituximab and azathioprine (rtx-aza) or rituximab and mycophenolate mofetil (rtx-mmf) would improve the radiographic abnormalities as determined by high-resolution computed tomography (hrct) of the chest and/or pulmonary function tests (pfts) in patients with cvid and glild. methods this is a retrospective study of patients seen from july to december with cvid and glild who completed immunosuppressive therapy (rtx ( mg/m ) for weeks, repeated at -month intervals for or total courses, and aza ( . - . mg/kg/day) or mmf ( mg- mg-bid) for months). complete pfts and hrct scans were performed prior to therapy, at the conclusion of therapy, and periodically thereafter. hrct scans were blinded, randomized, and scored independently (in pairs) by two radiologists. all patients underwent whole exome sequencing (wes). number (percentage) and median (interquartile range) were reported for categorical and continuous variables, respectively. differences between pre-and post-treatment and between relapse and post-relapse hrct scores and pft parameters were analyzed with wilcoxon signed ranks test. kaplan-meier survival curves were also done. unadjusted one-sided p-values < . were considered statistically significant. results the glild cohort (n= ) had a : female predominance, and age at glild diagnosis was ( - ) years (table ) . autoimmunity was present in the majority of patients, with thrombocytopenia ( ( %)) the most common manifestation. enteropathy ( ( %)), inflammatory bowel disease ( ( %)), and nodular regenerative hyperplasia of the liver ( ( %)) were also present. splenomegaly ( ( %)) was present in the majority, but polyarthritis ( ( %)) was notably absent. twenty ( %) patients had been previously treated with systemic steroids. hrct scores substantially improved between pre-and posttreatment for rtx-mmf (p= . ) and rtx-aza (p < . , figure ). fev (p= . ), fvc (p= . ), and tlc (p= . ) also improved, but dlco (p= . ) was unchanged (figures and ). excluding two ( %) patients who died . and years after therapy of respiratory failure ( ( %)) and septicemia ( ( %)) respectively, / ( %) patients relapsed . ( . - . ) years following therapy with an estimated % relapse rate after years ( figure ). as of december , of patients that relapsed showed improvement in hrct scores (p= . ), and the remaining patients are still undergoing retreatment ( figure ). four ( %) pneumonias occurred during immunosuppressive therapy, all with severe restrictive lung disease . eight ( %) patients had a damaging mutation in a gene known to predispose (tnfrsf b, n= ( %)) or cause a cvid-like primary immunodeficiency (ctla : ( %); kmtd : ( %); birc : ( %)). immunosuppressive treatment improved the hrct scores regardless of the absence (p < . ) or presence of a damaging mutation (p= . ) ( figure ). conclusion combination chemotherapy appeared to be effective in improving the radiographic abnormalities and pulmonary function of patients with cvid and glild. a majority of patients had sustained remissions, regardless of the presence or absence of a monogenic disorder. iqr=interquartile range, cvid=common variable immunodeficiency, glild=granulomatous and lymphocytic interstitial lung disease, vats=video-assisted thoracoscopic surgery, tbx=transbronchial biopsy, ms=mediastinoscopy excluding b cell malignancy, pft=pulmonary function test, h/o=history of, dz=disease, nrh=nodular regenerative hyperplasia, ibd=inflammatory bowel disease table . baseline patient characteristics since its first description the number of cases has increased progressively [ ] . although described as a predominant antibody deficiency [ ] , various complex phenotypes have been associated with mutations in this gene [ ] . case description. female patient with no remarkable history until years old, when she suffers from a persistent fever associated with purulent abscesses in venipuncture areas and hyperleukocytosis with neutrophilia, so she was treated for about months in hospitals in the city of barranquilla before she was referred to our institution. the patient's clinical picture consisted of persistent fever unresponsive to broad-spectrum antibiotic treatments, skin abscesses, left subphrenic abscess and toes osteomyelitis. the microbiological studies documented a bacteremia by acinetobacter baumannii and isolation in bone marrow of candida parapsilosis. during her care stay in barranquilla, she was approached as a chronic granulomatous disease versus job's syndrome, she received two doses of immunoglobulin with partial control of symptoms. due to the recurrence o f fever, abs ces ses and hyperleukocytosis, they decided to refer to our institution for further studies. upon admission to our institution, the patient presented nutritional compromise, with spontaneous resolution of fever but persistence of high acute phase reactants, with significant improvement of leukocytosis. all the cutaneous lesions she presented were debrided at the site of remission. immunoglobulin levels, lymphocyte populations and dyhidrorhodamine test were normal. she remained with no weight gain, constipation, abdominal distension and hepatic involvement with elevated liver enzymes and prolonged coagulation times. new bone compromise was documented. inflammatory bowel disease, neoplastic or chronic infectious disease involvement was ruled out. during the stay in our institution no microbiological isolation was documented. skin, colon and bone tissue biopsies were performed and extra-institutionally performed liver biopsy were examined, showing as a single common finding leukocytoclastic vasculitis in all tissues. given the heterogeneous nature of the condition, the diagnostic possibility of an immune dysregulation disorder was considered and a therapeutic trial with nsaids and prednisolone at mg/kg/day was started, as well as genetic studies by exome sequencing. the exome results documented a novel mutation in the pik cd gene [c. t> a (p.phe tyr)] as probably pathogenic. the patient has presented a clinical improvement and a significant decrease in inflammation markers. at the moment, we are waiting for the performance of functional tests to define the definitive therapy for this patient. conclusions. this case description highlights the diagnostic difficulties that face in developing countries, where the nonavailability of functional testing has implications on the diagnosis opportunity and establishment of optimal therapeutic for patients with complex diseases such as primary immune regulatory disorders abstract/case report text background: autoinflammatory syndromes, a wide family of diseases, defined as attacks of inflammation that are unprovoked (or triggered by a minor event) and are primarily related to dysregulation of the innate immune system. periodic/recurrent fever syndromes were the former name of these diseases. however, only in two conditions: cyclic neutropenia (cn) and periodic fever, aphthous stomatitis, pharyngitis, and adenitis (pfapa) are febrile episodes truly periodic. for pfapa, although diagnostic criteria differ and there is no consensus research definition, patients are usually not difficult to recognize based on clinical course and presentation . high index of suspicion and understanding the parental experiences and descriptions of febrile episodes is imperative in facilitating early recognition and timely diagnosis. aims: to standardize and summarized the clinical presentation of pfapa, based on parental descriptions and providers observation of febrile episodes. methods: utilizing a query for the icd- diagnosis code m . ( + ) we identified a cohort of children diagnosed and managed for periodic fever, excluding those with monogenetic mutation (e.g. blau, majeed) and those with chronic illness. we reviewed the charts for documented parental report and provider observation of febrile episodes. standardized signs and symptoms were recorded for each patient [ table ]. results: a cohort of children, boys ( %) and girls ( %) with documented, cyclic episodes of fever > , was identified. the average age at diagnosis was . +/- . years. classic symptoms were reported or observed in % of patients ( ). more than half ( %, patients) had documentations of other symptoms, usually reported by parents to occur sporadically during some fever episodes. decreased oral intake and general "ill appearance" was reported by parents in % of patients. when reporting the time intervals, parents usually reported similar length for each episode, typically between - days, and regular interludes, typically between - weeks. the findings are summarized in table . discussion: the findings presented here are in concordance with previously published data describing pfapa as a syndrome affecting young, generally healthy children with identical episodes of fever lasting for a few days, recur with regularity. our data support the approach that parental observation is fundamental in identifying the unique pattern of illnesses. engaging the parents with directed interview is crucial to establish this clinical diagnosis in a timely fashion, prevent misdiagnoses of future febrile episodes as presumptive infections, and avert unnecessary antibiotics courses. this cohort adds the observation that up to % of patients who display this identifiable pattern of illnesses, do not present with aphthous stomatitis, pharyngitis, or adenitis. these classic symptoms although common, are not the rule. the cyclic chronicity of febrile episodes associated with general ill appearance (but not lethargy), and decreased po, in an otherwise healthy child is the clinical gold-standard of this condition. abstract/case report text heterozygous mutations in nfkb are frequently identified among immunodeficient patients with highly variable clinical symptoms. in a world-wide collaborative effort, we characterized the clinical and cellular phenotype and the management of of these patients harboring distinct nfkb variants. nfkb encodes the transcription factor precursor p which is processed to p (canonical pathway). known pathogenic variants cause p haploinsufficiency (due to protein decay) or p -skipping (with expression of p -like forms). most variants however are single amino acid changes with yet unknown effects. all sequence changes were assessed in silico for their probability of pathogenicity including variants which were additionally tested in vitro. these analyses include the sub-cellular protein localization (microscopy), protein expression, stability and processing (western blotting), transcription factor activity (reporter assay) and dna-binding ( figure ); the associated unadjusted odds ratio (or) was not significant, suggesting igrt had restored cases to a similar baseline infection risk as the controls. in a multivariate conditional logistic regression model adjusting for the significantly higher occurrence of risk factors in cases compared with controls in the preindex period, cases were associated with a % lower adjusted odds of major/severe infections in the post-index period versus controls (or= . ; p= . ). conclusions: patients who required treatment with igrt (privigen®/ hizentra®) had previously experienced more major/severe bacterial infections than those not needing treatment with igrt. however, igrt was associated with a reduction in the risk-adjusted odds of major/severe bacterial infections compared with non-igrt treated sid patients with haematological malignancies. abstract/case report text introduction: real-world data are lacking as far as identifying patients with secondary immunodeficiency (sid)/hypogammaglobulinemia who may benefit most from interventions to protect them from potentially fatal infections. this study aimed to identify risk factors for major/severe infections in patients with sid with underlying haematological malignancies. methods: a retrospective database analysis was conducted using the iqvia real-world data adjudicated claims -us database (study period: january -september ). inclusion criteria were adults newly diagnosed with sid (first diagnosis termed the index date), with ≥ months continuous health plan enrolment pre-index (baseline period) and a minimum of months' data post-index (mean: days), with chronic lymphocytic leukaemia, multiple myeloma and/or non-hodgkin's lymphoma and without claims for any ig therapy in the -month baseline period. patient characteristics in the -month baseline period were assessed. over the post-index period, antibiotic/antiviral use and frequency of infections were assessed. the frequency of major/severe infections was determined using diagnosis codes for bacterial, viral, fungal, parasitic, other or unspecified causal pathogen infections. major/severe infections were defined as those requiring inpatient hospitalisation with an infection diagnosis code and/or use of intravenous (iv) antibiotics or iv antivirals in an outpatient setting. a multivariate cox proportional hazards (ph) model evaluated baseline patient characteristics associated with risk of major/severe infections post-index. results: a total of , patients met the inclusion criteria. the mean age of patients was years and . % were male. in the -month baseline period: . % of patients received cancer treatments and . % of patients received antibiotics ( . % iv antibiotics). a total of . % of patients experienced any infection, . % experienced ≥ infections and . % experienced major/ severe infections. the mean number of infections over the baseline months was . for any infection (at the unique diagnosis code level) and . for major/severe infections (unique hospitalisations with any infection diagnosis code and/or unique days with an outpatient iv antibiotic or iv antiviral). in the post-sid diagnosis period, . % of patients had major/severe infections; of the major/ severe infections, . % were identified as bacterial, . % were viral, . % were fungal, while . % did not have a causal pathogen specified. a total of . % of patients experienced one severe/ major infection and . % experienced ≥ severe/major infections ( figure ). the mean annualised number of major/severe infections post-index was . . receiver operating characteristic (roc) curve analysis to optimise sensitivity versus false positives in identifying those at risk of major/severe infections post-index identified a cutoff point of three bacterial infections in the baseline pre-index period as a potential optimal trigger to consider treatment to avoid major/severe infections post-index ( figure ). the multivariate cox ph analysis suggested that hospitalisations, infections (≥ ), or antibiotic use in the -months pre-index (prior to sid diagnosis) were predictive of major/severe infections post-index (post-sid diagnosis) (all p < . ). conclusion: infections are common in patients with haematological malignancies and sid. key baseline predictors for major/severe infections in patients with an sid diagnosis were a history of infections, hospitalisations or antibiotic use. unfortunately, ms/ms detection is limited by the extremely low (e.g., pmol/l) protein concentrations in blood cells. peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) is a robust method for quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). in a study of patients, immuno-srm reliably identified wiskott-aldrich syndrome (was) and x-linked agammaglobulinemia (xla) patients using direct quantification of proteins responsible for disease (front. immunol., ). we further expanded our approach for x-linked chronic granulomatous disease (x-cgd), ada and dock deficiency. marker proteins representing platelets, nk cells, and t-cells have also been analyzed to provide additional information about disease processes. these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed signature peptide quantification and its potential for pidd newborn screening and clinical diagnosis from dbs. methods: candidate peptides were selected based on ms/ms sensitivity and uniqueness in the proteome. anti-peptide monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, xla, was, xl-cgd, dock and ada deficiency patients was collected after consent on filter paper, dried, and stored at - °c. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed with a waters xevo tq-xs. results: a multiplexed immuno-srm panel has been generated for screening eight signature peptides representing five pidd-specific and three cell-type specific proteins from dbs. limits of detection and quantification were femtomoles of peptide, the assay showed a broad linear range, and intra-assay and inter-assay coefficients of variation were < %. in samples from xla, was, xl-cgd, dock and ada deficiency patients, signature peptides are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. also included in the multiplex panel are cell specific markers for platelets (cd ), t-cells (cd ɛ), and nk cells (cd ). diagnostic cutoffs for each peptide concentration have been established. in was patients, cd levels were significantly reduced consistent with characteristic thrombocytopenia. immuno-srm also has the ability demonstrate the effects of pidd treatment. a was patient analyzed before and after bone marrow transplant showed normalized was protein and cd after treatment. two ada deficiency patients showed normal levels of ada enzyme after rbc transfusion. finally, a high-throughput (ht) immuno-srm method screens pidd-specific peptides in a . -minute runtime meeting high volume nbs workflow requirements. this ht method returned identical results to the standard immuno-srm pidd panel. conclusions: the data herein demonstrate the feasibility of using immuno-srm as a broad clinical diagnostic for identifying and studying pidd patients from easily collected and shipped dbs. significantly, ht immuno-srm workflows represent a promising potential option for nbs of pidds and other congenital disorders. chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text we have previously used the artificial thymic organoid (ato) system, based on the d aggregation and culture of a delta-like canonical notch ligand -expressing stromal cell line (ms -dll ) with cd + cells, to study t cell differentiation from cd + cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag - , ak , il rg) or that affect thymus development (digeorge syndrome). we now report results of in vitro t cell differentiation of cd + cells obtained from patients with either haploinsufficiency or dominant negative (dn) mutations of the ikzf gene. ikzf is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. heterozygous germline mutations in ikzf give rise to distinct clinical phenotypes, depending on the nature of the mutation. in particular patients with ikzf haploinsufficiency present with common variable immunodeficiency (cvid) associated with b cell immune deficiency, b-all susceptibility, and autoimmune manifestations. no clinical t cell defects are evident among these patients, except for elevated naive and central memory cd +cd + t cells. in contrast, patients carrying dn ikzf mutations present with combined immunodeficiency (cid) characterized by the presence of an increased proportion of naïve t cells, associated with defective generation of memory t cells, impaired t cell activation, signaling and proliferation, reduced t-helper (th) polarization, and susceptibility to pneumocystis pneumonia. different mouse models of ikzf mutations have been developed, however their phenotype does not fully match what reported in patients, and in some models indicates a more severe defect in t cell development. to address these controversies and to gain novel insights into the effects of distinct ikzf mutations on human t cell development, we used the ato system to analyze progression of t cell development from cd + cells obtained from one patient with ikzf haploinsufficiency and one patient with dn ikzf mutation. both patients showed a similar early block in t-cell differentiation a t p re -t c el l s ta g e. ho w ev e r, th e p a ti e nt wi t h i kz f haploinsufficiency showed a more pronounced leakiness, with a residual production of cd +tcrab+ cells, which could account for the milder t-cell phenotype presented in this type of patients. interestingly, the dn patient presented an increased accumulation of cd -cd b-cd aa+ cells. these results show an unexpected role for ikzf in humans in early stages of t-cell differentiation and indicate ikzf as a necessary factor for the induction of cd b expression in t cells. abstract/case report text background: pediatric acute liver failure without an identifiable cause (indeterminate palf/ipalf) is associated with increased rates of liver transplant and mortality. aplastic anemia (aa) may develop weeks after the diagnosis. the immunologic mechanisms that contribute to disease pathogenesis have not been clearly elucidated. we report detailed immunophenotyping of a patient with ipalf/aa. case: a previously-healthy -year-old male was admitted for acute hepatitis presenting with jaundice and hepatosplenomegaly. evaluation for infectious, toxic, metabolic, autoimmune, and rheumatologic disorders was negative. he was pan-lymphopenic (cd +alc cells/μl) with an inverted cd :cd ratio of . on admission. liver biopsy showed severe portal, interface, and lobular inflammation characterized by activated sinusoidal macrophages and perforinexpressing cd +t-cells. compared to a healthy control, the percentage and number of peripheral blood cd +t-cells expressing perforin ( %v. %, v. cells/μl) and granzyme-a/b ( %v. %, v. cells/μl) was also increased, while percentages of perforin+ ( %) and granzyme-a/b+( %) nk cells were normal. bone marrow (bm) showed % cellularity with rare hemophagocytosis. serum cytokine analysis demonstrated il- pg/ml, il- -binding-protein pg/ml, cxcl pg/ml, and sil- rα u/ml, consistent with smoldering hemophagocytic lymphohistiocytosis (hlh), but he did not meet hlh diagnostic criteria. genetic sequencing did not identify pathogenic variants in genes associated with primary immunodeficiencies. he was diagnosed with ipalf and treated with three doses of anakinra and two weeks of ruxolitinib, followed by prednisone - mg/kg/day and intravenous immunoglobulin g/kg/month. immunophenotyping performed after two months of therapy showed persistent inversion of the cd :cd ratio with small expansions of cd +cd -cd -cells and tcr··+t-cells. in the cd +t-cell subset, there was a substantial paucity of naïve cells, with effector memory t cells (tem) being more abundant than central memory t cells (tcm). in the cd +t-cell subset, the majority of cd ro+cells were tem with no detectable tcm, and % of all cd +t-cells were cd +temra. in both cd + and cd +t-cell subsets, activated (hla-dr+) and senescent (cd +) subpopulations were increased, and the majority of cells expressed the exhaustion marker pd- . the hepatic inflammatory infiltrate similarly reflected repetitive antigenic stimulation, with expansion of cd +cd +t-cells. quantitative immunoglobulins and total memory b-cells and plasmablasts (cd +cd +) were normal for age. however, there were no circulating iga-memory b-cells and a reduced number of iggswitched memory b-cells (table ) . given the severity of his phenotype and bm hypocellularity ( %), allogeneic hct was performed using a matched-related-donor ( / ) with conditioning of flu+cy+alemtuzumab. at d+ , he shows improved liver function but persistent pancytopenia, with transfusion-dependence for platelets. discussion: to our knowledge, this is the first description of detailed immunophenotyping in blood from a patient with ipalf/ aa. other studies have identified distinguishing hepatic infiltrates and cytokine/chemokine profiles that suggest excessive activation of cytotoxic t-lymphocytes and macrophages contribute to disease pathogenesis (alonso et al, ). our preliminary data supports this hypothesis and expands the spectrum of immune dysregulation in the t and b cell compartments, proposing a primary immune etiology. immune dysregulation may be concordant with hyperinflammation and cytokine storm, the latter offering potential therapeutic targets. early diagnosis and treatment of immune dysregulation may prevent development of aa. background: x-linked agammaglobulinemia (xla) is one of the first inborn errors of immunity identified, with thousands of patients described to date. infections originally dominated the clinical phenotype, but early diagnosis and immunoglobulin replacement allowed for long term survival as well as recognition of late-onset complications. nodular regenerative hyperplasia (nrh) of the liver is a silent cause of non-cirrhotic portal hypertension. nrh underlying pathophysiology remains blurry and the disease has no specific treatment. nrh has been increasingly reported in primary immunodeficiency but data in xla are very limited. objectives: to assess and characterize nrh in patients with xla. methods: we retrospectively reviewed the medical records of all xla patients referred to the nih between and . hepatology evaluation and liver biopsies were performed when clinically indicated. patients were stratified into nrh+ or nrhgroups, according to their nrh biopsy status (patients with no liver biopsies were classified as unknown). laboratory values are presented as medians. fisher's exact test and mann-whitney test were used to compare categorical and continuous variables, respectively. results: twenty-one xla patient records were reviewed, with a median age at start of follow-up (f/u) of y and a median duration of f/u of years. eight patients underwent at least one liver biopsy of whom ( % of nih xla cohort) were nrh+. the median age at nrh diagnosis was y ( - ). among patients who had liver biopsies, alanine aminotransferase (alt) levels were mildly elevated in all, while alkaline phosphatase (alp) levels were only increased in nrh+ patients (p= . ). both nrh+ and nrhgroups had similar aspartate aminotransferase (ast) levels at baseline but higher values were observed at the end of f/u in the nrh+ group ( vs. u/l, p= . ). persistently low platelet count ( < k/μl for more than months), mildly to highly elevated hepatic venous pressure gradient (hvpg) and either hepatomegaly and/ or splenomegaly were present in all nrh+ patients. in opposition, neither persistently low platelet counts, nor hepato-or splenomegaly were present in the two nrh-patients evaluated. hvpg was normal in the only nrh-patient tested. all-cause mortality was higher among nrh+ patients ( / , %) than in the rest of the cohort ( / , % among nrh-and unknown patients, p= . ). conclusions: based on our retrospective analysis, nrh appears as an underreported, frequent and severe late-onset complication in xla, which is highly associated with increased mortality. persistent thrombocytopenia, elevated alp, elevated hvpg, hepato-and/or splenomegaly were common in liver biopsyproven xla/nrh+ patients and distinguish them from xla/ nrh-patients. based on nrh prevalence, severity, lack of specific treatment and poor outcome in xla, immune-reconstitution (rather than igg replacement and infectious prophylaxis) should be considered early in this population in order to prevent fatal long term complications. abstract/case report text introduction: barth syndrome (bths) is an x-linked recessive disorder caused by a mutation in the tafazzin (taz) gene resulting in an inborn error of cardiolipin phospholipid metabolism (an important mitochondrial inner membrane lipid). it is commonly characterized by intermittent neutropenia and cardiac and skeletal myopathies. we present a case of bths with associated lymphopenia and hypogammaglobulinemia, which has not been previously described in the literature. case report: a two-month old male, born full term with normal newborn screening, was first admitted for rsv bronchiolitis. at this time, patient underwent an echocardiogram given his older brother with hydrops had died hours after birth and on autopsy was found to have dilated cardiomyopathy (dcm). patient was similarly noted to have dcm and thus had whole exome sequencing done that showed a hemizygous mutation in the taz gene (c. g>a). this novel variant resulted in early termination of the protein (p.trp ter) with concern for loss of function. in regard to patient's first year of life, he had frequent uri symptoms, episodes of acute otitis media requiring tympanostomy tubes, but no documented pneumonias or other serious bacterial infections. patient also had gross developmental delay, particularly motor, and feeding difficulties with persistent failure to thrive requiring g tube placement. his absolute neutrophil count ranged from - cells/mm in the first year. at age months, patient was found to be in acute decompensated heart failure with concern for myocarditis (ck , u/l, troponin i . ng/ml) as well as acute hypoxic respiratory failure with respiratory cultures growing pseudomonas. he was incidentally found to have an igg level of mg/dl (normal for age - ) and treated empirically with ivig. when seen by immunology, further workup showed persistent b cell lymphopenia (absolute cd of - /mm ). he also had a low initial nk cell count ( - /mm , later normal) with normal cd and cd t cell counts. tetanus and hib titers could not be assessed as he had recently received ivig. his igg trended up to mg/dl a few days after initial ivig and then subsequently dropped to mg/ dl, with a level of mg/dl two weeks following initial dose. workup for gastrointestinal or renal losses of immunoglobulin were negative. he also shortly after developed enterobacter bacteremia. his igg levels at this time continue to remain around mg/dl. he subsequently required a heart transplant at age months for his dcm. after transplant, he continued to improve from a cardiac standpoint, but his lymphopenia persisted and each time he was weaned off ivig, his hypogammaglobulinemia persisted at - mg/dl thus requiring additional ivig replacement over the course of the next months. the remainder of immunoglobulins were normal initially, but the igm slowly dropped over time to - . mg/dl. patient was started on weekly subcutaneous immunoglobulin replacement at months, doing well clinically at age -month follow up. conclusion: here we present a patient with bths, with a novel variant, who had b-cell lymphopenia as part of his presentation with persistent hypogammaglobulinemia requiring ivig replacement. year fellow/ucla associate professor/division of allergy, immunology, and rheumatology, university of california los angeles chief of pediatric allergy and immunology/harbor-ucla chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih biologist/laboratory of clinical immunology and microbiology, division of intramural research, national institute of allergy and infectious diseases, national institutes of health project scientist/ucla abstract/case report text a -year-old female presented for combined immunodeficiency. at years of age she was diagnosed with rag hypomorphism and started on ivig. as a child, she was hospitalized for pneumonias and cryptococcal meningitis. she suffered sinusitis, hepatitis, tooth abscess, cmv and herpes stomatitis. later, she experienced recurrent cutaneous abscesses, utis, vaginal yeast infections, and hidradenitis. she twice hospitalized recently for pneumonias and diagnosed with mycobacterium abscessus on bronchoscopy. she suffers onychomycosis, osteomyelitis and oral and esophageal candidiasis with odynophagia. on exam, she had white plaques on tongue and buccal mucosa. she had hyperpigmented plaques on forehead and cheeks and thickened nails. immune evaluation was significant for lymphopenia with alc and thrombocytopenia with platelets k. b cells were nearly absent ( absolute count) and nk cells were low at absolute count. ige was absent, igm mg/dl, iga mg/dl and igg mg/dl (on replacement). her total cd + count was , cd + t cells were low at %, but cd + cells normal at %. the cd + t cells were mostly memory phenotype, which probably reflects lymphopenia-induced proliferation of a small number of clones. her cd + t cells also had an elevated amount of memory cells for age, but still had presence of naive cd + t cells. as expected with perpetual lymphopeniainduced proliferation, there was evidence of terminal memory (temra) in the cd + lineage. proliferative responses of t cells were modest. cd + t cells did respond to pokeweed, but less to pha and cona. there were no antigen specific responses. trecs were normal. esr was mildly elevated at . of note, her liver enzymes were elevated with alkaline phosphatase and ast , presumably secondary to prolonged fluconazole use. w es r e v e a l e d a k n o w n p a t h o g e n i c v ar i a n t i n s tat (nm_ . : c. c>g (p.n k)) as well as a heterozygous variant in rag p.m t. the stat mutation is de novo and was previously published as a gain of function mutation. however, when we performed validation studies to evaluate cd + cells with stimulation to ifna, the patient had decreased pstat as compared with control. va . analysis was performed to evaluate rag defect and showed % of t cells with va . expression confirming that the rag defect is not clinically significant. she developed severe thrombocytopenia refractory to platelet transfusions and ivig. she was started on ruxolitinib which improved platelet counts. however, she presented with shortness of breath, persistent tachycardia and was found to have cmv carditis and hepatitis significant for echocardiogram with ef %. cmv pcr is improving with last check iu/ml after month of therapy with ganciclovir. we now are looking for evidence of socs to explain the decreased stat phosphorylation. genetic testing is critical when evaluating a patient with immunodeficiency. our patient demonstrates that genetic mutations cannot be taken at face value and should be evaluated and validated fully to optimize patient care. fellow/university at buffalo / oishei children's hospital abstract/case report text opportunistic infections (oi) are commonly seen in patients undergoing hematopoietic cell transplantation (hct). different strategies for antimicrobial prophylaxis are often employed in the transplant setting to reduce the likelihood of encountering infection. the predisposing risks for infections include the expected neutropenia and lymphopenia following conditioning, prolonged defects in cell-mediated and humoral immunity during the engraftment period, and iatrogenic immunosuppression by medications for graft versus host disease (gvhd). we report the case of a -year-old male with acute lymphoblastic leukemia, which relapsed to chronic myelogenous leukemic blast crisis, and failed a subsequent allogeneic hct with central nervous system relapse. he was subjected to a second allogeneic hct. his immediate post-second transplant course was complicated with skin and gut gvhd, and infection and/or reactivation of coronavirus, respiratory adenovirus, epstein-barr virus, and human herpesvirus . while the herpesviral infections were controlled with antivirals and rituximab, adenovirus c infection proceeded to involve the gastrointestinal tract, and proved persistent over several months despite use of cidofovir. the patient's gvhd and transplant-associated thrombotic microangiopathy necessitated use of further immunosuppressants, including the complement protein c -binding eculizumab (an inhibitor of formation of the terminal c b- complex), ruxolitinib (a janus kinase [jak] / inhibitor) and low-dose interleukin- . h i s c l i n i c a l c o u r s e w a s f u r t h e r c o m p l i c a t e d b y stenotrophomonas maltophilia gut colonization and subsequent bacteremia, as well as multiple gram-positive bacteremia courses. at around day + , there was a life-threatening pericarditis with pericardial effusion and respiratory distress, associated with pneumocystis and stenotrophomonas being isolated from bronchoalveolar lavage. this occurred despite the patient being on pentamidine prophylaxis. the patient eventually recovered on trimethoprim/sulfamethoxazole therapy. we discuss the various risk factors potentially contributing to each oi in this illustrative case. in particular, complement and jak inhibitor therapy are fairly new drugs approved for other indications, whose off-label use in transplant patients is increasing. both have recently been associated with certain oi in the literature, as they are in this patient. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and other autoimmune manifestations. typically, the biomarker profile of patients with alps includes elevated tcr αβ+ dnt cells, serum igg, serum b , serum il and soluble fas ligand (sfasl). hdl cholesterol can also be significantly low. alps is caused by lymphocyte accumulation due to defects in the fas-mediated apoptosis signaling pathway. these defects cause resistance to physiological apoptosis in lymphocyte populations that results in chronic lymphoproliferation. the molecular defect underlying most alps etiologies is attributed to heterozygous germline or somatic (limited to dnt cell subpopulation) pathogenic single nucleotide variants (snv) in fas. we describe copy number variants (cnvs) at the fas locus underlying alps in unrelated families. methods: through the centralized sequencing initiative at at the national institute of allergy and infectious diseases (niaid), patients undergo genomic workup to identify molecular defects contributing to clinical phenotypes of immune system disorders. all patients receive exome sequencing and a subset of patients also receive array-cgh analysis. patients and results: we performed exome sequencing on patients with a clinical diagnosis of alps. for patients with no molecular defect through exome, we performed cnv analysis. in this cohort, we identified three patients with a copy number variant involving the fas locus. all patients presented with splenomegaly and lymphadenopathy in childhood with ages of onset ranging from months to years old. all patients experienced anemia, autoimmune neutropenia, and thrombocytopenia. they had biomarker evidence showing elevated serum b levels, sfasl levels, and elevated αβ+dnt cell populations. they were found to have very low hdl cholesterol in early childhood ranging from - mg/dl ( - mg/dl). all patients had negative family histories for lymphoproliferative disorders and immunodeficiency. these patients had clinical presentations and biomarker profiles similar to alps patients with germline and somatic fas variants. patient : we detected a~ . mb copy number loss encompassing all of fas. parental studies were not performed. patient : we detected a~ . mb copy number loss encompassing all of fas. parental studies showed this to be maternally inherited. in addition, prior karyotype testing of the bone marrow showed the same deletion. patient : we detected a~ . mb copy number loss encompassing exons - of fas. parental studies were not performed. these results are consistent with the pathogenic nature of copy number variant losses involving fas. the mechanism of disease in these patients is consistent with haploinsufficiency. in family , the mother harboring the fas deletion is unaffected. this is consistent with prior observation of reduced penetrance within a family in alps. conclusion: these three cases harbored causative deletions in fas in the presence of biomarkers indicative of alps and negative results for germline and somatic genetic variant testing. these patients demonstrate that copy number variant analysis should be pursued if there is robust clinical and biomarker evidence of alps as it can lead to a molecular diagnosis and appropriate treatment when exome or next generation panel based fas sequencing is inconclusive. abstract/case report text rationale: the thymus is essential for the development of tcells. patients with thymoma have decreased aire expression and have an abnormal thymic microenvironment where the negative selection of t-cells is compromised, resulting in a broad spectrum of autoimmune-mediated diseases. besides myasthenia gravis, which is found in to % of patients with thymoma, other autoimmune diseases have been reported including erythroblastopenia, systemic lupus erythematosus, inflammatory myopathies, thyroid disorders and good's syndrome. recent studies have described additional autoimmune conditions such as pneumonitis in thymoma patients. we identified a patient who developed chronic cough post-thymectomy and was found to have lymphocytic pneumonitis with associated autoantibodies against lung antigen kcnrg and lung immunopathology consistent with apeced pneumonitis, which implies a common pathogenic mechanism between these conditions. methods: we describe a patient with thymoma who developed autoimmune pneumonitis associated with kcnrg autoantibodies and a characteristic pattern of immunopathology recently described in patients with monogenic disorder caused by primary aire deficiency (apeced) and secondary aire deficiencies (thymoma, rag deficiency). results: patient is a -year-old male with no significant past medical history who was in good state of health until age when he was diagnosed with and received treatment for guttate psoriasis (resolved with uv therapy) and alopecia areata. at age , he developed severe abdominal pain and weight loss. he had an abdominal ct performed that showed chronic pancreatitis and thymoma. one month later, the patient underwent thymectomy and subsequently, underwent ercp and pancreas biopsy, revealing atrophic pancreatitis with negative staining for lgg and lgg . at that time, he was started on pancreatic enzymes with improvement of abdominal symptoms. following thymectomy, he developed persistent dry cough and recurrent symptoms of sinusitis which did not respond to several courses of oral antibiotics to treat his positive culture for pseudomonas. he had a negative work up for vocal cord dysfunction and cystic fibrosis, and negative autoantibodies against ifn-gamma, il- a, and gm-csf. for work up of chronic cough, the patient underwent ct imaging of the chest which revealed diffuse peri-bronchial thickening, mucus plugging, and tree-in-bud nodularity through most of his lungs. he underwent bronchoscopy with bal which revealed n o r m a l b r o n c h i a l m u c o s a a n d a i r w a y n e u t r o p h i l i a . endobronchial biopsies showed basement membrane thickening and dramatic lymphocyte infiltration in intraepithelial and submucosal areas. his bal cultures revealed mycobacterium intracellulare/chimaera. patient was also tested for autoantibodies against lung-specific bactericidal/permeabilityincreasing fold-containing b (bpifb ) and the potassium channel regulator kcnrg that have been associated with the development of pneumonitis in patient with apeced, thymoma and rag deficiency, and was found to have kcnrg-targeted autoantibodies. conclusions: thymoma is a disease associated with secondary aire deficiency. this case illustrates common clinical, radiographic, histological, and autoantibody features in thymomaassociated and apeced-associated pneumonitis, indicating that disorders with primary and secondary aire deficiencies may have common pathogenetic mechanisms. bpifb and kcnrg should be included in the autoantibody profile testing of patients with thymoma and lung disease. immune suppression and antimycobacterial antibiotic treatment are planned. abstract/case report text introduction/background: activated phosphoinositide -kinase δ (pi kδ) syndrome (apds) is a primary immunodeficiency caused by a gain-of-function mutation in the pik cd gene that encodes the p δ catalytic subunit of pi kδ. it is characterized by recurrent respiratory tract infections, lymphoproliferation, nodular mucosal lymphoid hyperplasia, enteropathy, ebv and/or cmv infection, reduced t cell function and high levels of igm. there is not evidence of this disease in peruvian patients. methods: a case series of two pediatric patients with apds. results: the first patient is a girl of non-consanguineous parents. family history shows four maternal uncles died at pediatric ages with unknown diagnosis. at the age of , she presented lymphadenopathy and fever being treated as cat scratch disease without improvement of symptoms. months later, she was hospitalized due to anemia, mild hepatosplenomegaly, ascites and chronic diarrhea and diagnosed with gastrointestinal tuberculosis (tb). a hepatic biopsy only showed reactive hepatitis. however, the patient did not improve her symptoms despite anti tb treatment. years later, she was hospitalized for lymphadenopathy, pancytopenia, chronic diarrhea, ascites and severe hepatosplenomegaly. cmv igg was positive and lymph node biopsy revealed paracortical and follicular lymphoid hyperplasia due to ebv infection without neoplastic proliferation. low cd + t and cd + b cells and high igg levels were found (table ) . at this time, it was suggested the diagnosis of apds which was confirmed by next generation sequencing (ngs) identifying a heterozygous mutation in the pik cd gene (c. g>a, p.glu lys). she was treated with sirolimus and ivig for years. the symptoms persisted despite treatment and died at the age of . the second patient is a -year-old girl also of non-consanguineous parents. family history includes eczema (father) and colorectal cancer (mother). she has had recurrent respiratory infections, chronic diarrhea and poor weight gain since months old receiving symptomatic treatment only. at the age of , she was hospitalized for persistent pneumonia ( p s e u d o m o n a s p o s i t i v e ) , l y m p h a d e n o p a t h y a n d m i l d hepatosplenomegaly. a ct scan showed bilateral bronchiectasis and the sweat chloride test was negative. based on this, a diagnosis of cystic fibrosis was made and treatment was started. however, a genetic study only showed heterozygous mutations in the cftr gene (g d and g x). year later, she presented a neck-located skin abscess. at the age of , she was hospitalized for complicated pneumonia, diarrhea, lymphadenopathy, ascites and severe hepatosplenomegaly. multiple polyps in the duodenum and colon with lymphoid hyperplasia were detected, ebv igm and igg were positive and a lymph node biopsy showed paracortical hyperplasia without neoplastic proliferation. cd + t cells and igm levels were increased (table ) . a diagnosis of apds was suspected and ivig was started. ngs showed the same mutation as the first patient (c. g>a, p.glu lys). conclusion: apds should be considered in patients with recurrent respiratory tract infections, lymphoproliferation, enteropathy and abnormal immunologic function without another explanation. ngs is a useful tool to identify these cases in low-income countries. acknowledgments: we thank drs. raif geha and janet chou, division of immunology, boston children's hospital, harvard medical school for the genetic diagnosis. background: granulomatous-lymphocytic interstitial lung disease (glild) is an increasingly recognized pulmonary complication associated with common variable immunodeficiency (cvid) but the natural history and long term prognosis remains poorly defined. imaging findings with computed tomography (ct) are heterogeneous and visual features do not consistently predict a patient's progression to fibrotic lung disease. computer-aided lung informatics for pathology evaluation and rating (caliper) provides an objective analysis of lung parenchymal texture and quantifies the extent of normal lung, along with abnormal features such as honeycombing, reticular/consolidative and groundglass opacity. this may be useful in cvid patients to monitor changes in character or extent of disease and may facilitate early intervention before the disease becomes more aggressive or advanced. case description: our patient is a -year-old non-smoking female with cvid who has been followed for her cvid and associated interstitial lung disease. for more than twenty years, she has had varying abnormalities found on chest ct and these appear consistent with glild. specifically, she has had variable regions of mixed consolidation, ill-defined nodularity and septal thickening. the changing morphology and distribution made assessment of overall severity and extent of fibrosis versus parenchymal infiltration inconsistent. for clinical decision support we used caliper to analyze the current ct ( ) and compared caliper results for previous ct data. caliper provided a comprehensive analysis of the extent and characteristics of parenchymal features, and objectively determined normal and abnormal regions, some of which were not visually apparent. the caliper color overlay was able to highlight subtle regions of ground-glass opacity in areas that visually were regarded as uninvolved lung and quantify the extent of the reticular densities/ consolidation over time. caliper does not differentiate reticulation from consolidation, does not detect nodularity or septal thickening, and ct imaging cannot distinguish inflammation from fibrosis. however, caliper has the power to quantitatively assess overall disease extent and demonstrate subtle abnormalities that would otherwise have been dismissed as normal, given relative sparing compared to other regions. caliper may also provide evidence for disease progression or therapeutic response that is not otherwise radiographically apparent. conclusion: caliper assessment may be a useful tool as an adjunct for a patient with glild to help quantify the extent and character of lung parenchymal involvement. this information may serve as an important guide for clinicians in the assessment of successful management and early intervention to prevent irreversible fibrosis. patients had a trial of fingolimod without any beneficial changes in immune status. both patients receive pneumocystis jirovecii pneumonia prophylaxis with sulfamethoxazole-trimethoprim. conclusions: these results indicate that s pl deficiency due to sgpl mutations is a syndromic primary immunodeficiency leading to profound lymphopenia and hypogammaglobulinemia. our data emphasize the importance of sphingolipid metabolism for an efficient immune response and the need for more studies to delineate the exact mechanisms on how this happens in humans. after d at °c there was a median - % (range - % to + %; p= . ) change in activity. after d - % (range - % to + ; p= . ), d - % (range - % to + %; p < . ), d - % (- % to + %; p < . ) and after d - % (- % to + % p < . ). a °c stability of d was determined from the median percentage reduction; total allowable error adjusted stability data indicated a °c stability of d. samples stored at - °c following repeat freeze thawing saw a freeze/thaw cycle dependent decrease in ch activity. after freeze/thaw cycle there was a median - % (range - % to + %; p= . ) change, cycles - % (range - % to + %; p= . ), cycles - % (range - % to + %; p < . ), cycles - % (range - % to - %; p < . ) and after cycles - % (range - % to - %; p < . ). allowable error adjusted stability data indicated a maximum of freeze/thaw cycles. conclusion: sample storage and handling can have a significant impact on functional complement assessments. room temperature storage should be avoided unless samples will be analysed on the day of collection, °c storage is tolerable providing that assessment is within d; freezing samples at - °c with limited freeze/thaw analysis would be optimal. however, further investigations into longer-term storage at - °c and - °c would be beneficial. conclusions: gi disease is common in cvid affecting % of patients in our cohort. gi+ cvid patients have a higher frequency of autoimmune manifestations than those without gi complaints. the odds of itp, hypothyroidism, and evans syndrome all showed significantly increased odds in the gi+ group. the results of our study may have implications for both gastroenterologist and immunologist. recurrent infections especially those of the sinopulmonary tract are often the trigger for cvid evaluation. autoimmune and gi symptoms however may be the initial presentations of cvid and overlooked until other more recognizable manifestations evolve. the combination of gi issues and autoimmunity especially thrombocytopenia, evans syndrome, and hypothyroidism should include cvid in the gi differential. for the immunologist, a cbc is standard in the work-up of cvid and may reveal autoimmune cytopenia. evaluation for autoimmune disease and in particular hypothyroidism is not. given our findings an initial immune work up specifically for thyroid disease may be indicated. is the transcriptional factor for many cytokines such as il- , responsible for t cell and neutrophil defense again fungal infection. stat mutation leads to defect of neutrophil proliferation and chemotaxis to inflammatory site as well as production of antimicrobial peptides by respiratory epithelial cells. the poor tissue repair in the cavitary lesions and bacterial superinfection in patient's lung created a culture dish for fungal growth and dissemination. traveling to the endemic area and patient's noncompliance to antifungal prophylactic treatment further increased the risk of histoplasmosis infection. pediatric immunologist/john hunter children's hospital abstract/case report text we present the case of a month old boy, the first child to his nonconsanguineous parents of european descent. he first presented at months of age with a cellulitis of his right fourth finger culture positive for staphylococcus aureus which responded to a prolonged course of flucloxacillin. at months he presented with norovirus positive gastroenteritis leading to a brief admission and slow resolution. the first of two severe episodes of oral stomatitis and respiratory distress occurred at months of age. hsv was isolated from the oral lesions and blood culture during that admission was positive for kingella kingae. no cardiac or bone involvement was identified. a more severe episode of oral stomatitis occurred two months later (age months) swab positive for an enterovirus (not typed). due to airway compromise and rapid deterioration he was admitted to the pediatric intensive care unit. again, kingella kingae was cultured from blood cultures with no obvious focal systemic source. the only notable clinical finding was rapid deterioration and, in retrospect, the absence of any significant recorded fever ( < oc). crp elevation was observed (max. mg/l) and neutropenia was found with each of the more severe infectious presentations but recovered in the interval. baseline immunological investigations were normal (lymphocyte subsets, naïve t cell populations, lymphocyte proliferation, serum immunoglobulins and vaccine responses). serial measurement of circulating neutrophils did not identify a cyclical pattern and they were morphologically normal. a panel of genes relevant to primary immunodeficiency (invitae©) revealed a homozygous mutation in irak ((c. c>t (p.gln *)) which leads to a premature stop codon. this is a known pathogenic mutation leading to disease and is most prevalent in the european population (allele frequency (gnomad) = . ). prophylaxis with sulfamethoxazole / trimethoprim and amoxicillin was commenced along with monthly ivig. he has been well since diagnosis with no further severe infectious presentations. functional testing is underway to assess in vitro host viral defence in our patient and potential novel mechanisms relevant to this rare innate immunodeficiency. case studies will be presented on the five cases of fmp that were diagnosed and treated in . potential exposures were identified in four out of five cases: gardening exposure in one case and vaping exposure in three cases. all five were male, age range - . four were gp deficient, and one was p -phox deficient. historically, the vast majority of cases of fmp could be traced to a significant gardening exposure such as lawn mowing or spreading mulch. this was the first year that we saw patients with no identifiable gardening exposure in the setting of significant vaping exposure. with vaping at epidemic levels, especially among teenagers and young adults, it is important to consider that a vaping history is potentially a risk factor for fmp and counseling regarding the potential risks of vaping should be included in infection risk modification for all patients with cgd. abstract/case report text background: granulocyte-macrophage colony-stimulating factor (gm-csf) plays a critical role in macrophage and dendritic cell maturation and host defense against fungus. autoantibodies to gm-csf are associated with susceptibility to cryptococcus and nocardia infections as well as pulmonary alveolar proteinosis (pap) in otherwise healthy individuals. we report a case of a -year-old previously healthy female who presented with cryptococcal meningitis and was found to have autoantibodies against gm-csf. case presentation: weeks prior to admission, our previously healthy -yearold taiwanese female developed a headache associated with tinnitus and visual changes. the headache worsened over the next few weeks and she developed photophobia, phonophobia, and severe nausea/vomiting. at presentation, her exam was notable for papilledema, bilateral cn vi palsy and right foot & left hand paresthesia. mri brain showed ring-enhancing lesions in the anterior frontal lobe, caudate head, and the inferior globus pallidus. she underwent a diagnostic and therapeutic lp. opening pressure was elevated at and csf studies were notable for low glucose, elevated protein, pleiocytosis ( % lymphocytes) and positive cryptococcal antigen. csf culture grew cryptococcus gattii. ct chest revealed a right upper lobe and a left lower lobe nodule. workup: cbc with diff was unremarkable. hiv was negative. lymphocyte subsets were unremarkable with only mildly decreased nk cells, normal immunoglobulin panel including ige, protective titers to tetanus, diphtheria, and ppsv . targeted genetic sequencing did not identify any known mutations in primary immunodeficiency. notably, anti-gmcsf autoantibodies were detected by elisa and were able to neutralize gm-csf phosphorylation of stat detected by flow cytometry. autoantibodies to ifn-γ were not detected. management: patient was initiated on a -week course of liposomal amphotericin b and flucytosine. her csf cultures were cleared of cryptococcus after days of treatment, but her hospital course was complicated by persistently symptomatic intracranial hypertension, worsening pleiocytosis, and elevated cytokine levels in the csf, all of which were consistent with post-infectious inflammatory syndrome (piirs). she received therapeutic lps - x/week until subsequent ventriculoperitoneal shunt placement. concurrently, methylprednisolone was administered for days with a gradual prednisone taper. these interventions led to improvements in her symptoms, including diplopia, and reduction in opening pressures and inflammatory markers in the csf. lifelong fluconazole prophylaxis was recommended. from a pulmonary standpoint, she remained asymptomatic without signs of pap and has had normal pulmonary function tests (normal dlco) and stable chest imaging. conclusion: in otherwise healthy hiv-negative patients presenting with extrapulmonary cryptococcus or nocardia infections, autoantibodies to gm-csf should be suspected and testing for functional autoantibodies to gm-csf (and ifn-γ) should be sent, as genetic testing will not pick up this disease entity. genetic testing should be considered to rule out gata deficiency and x-linked cd l deficiency. idiopathic cd lymphopenia can be ruled out with lymphocyte enumeration. immediate treatment of cryptococcosis is not necessarily different from patients without gm-csf autoantibodies. long-term prophylaxis (fluconazole if presenting with cryptococcus; trimethoprim-sulfamethoxazole if with nocardia) is likely warranted in addition to monitoring for the development of pap. recognizing piirs in patients with cryptococcal meningitis and management with corticosteroids are critical steps. abstract/case report text background: non-infectious complications cause most morbidity and mortality in common variable immunodeficiency (cvid). cvid with complications (cvidc) is defined by elevated t helper (th ) responses attributed to increased circulating microbial products resulting from mucosal iga deficiency. however, complications do not uniformly occur in those with iga deficiency. objective: we tested whether cvidc occurs preferentially in those with hyper-responsiveness to microbial stimuli, manifested by elevated nf-κb-driven cytokines and resultant th responses in cvid patients with increased circulating microbial products. methods: we applied unbiased high-throughput seromics and mass cytometry, cellular and molecular biology approaches, and clinical record review in a subject cvid cohort. results: cvidc was defined by increased nf-κb-driven cytokines that promote th immunity in blood in association with elevated soluble cd , a marker of circulating microbial products, and elevated tnf production by peripheral blood mononuclear cells stimulated with lipopolysaccharide. this cytokine upsurge was associated with mutation of full-length nfkb p gene product ( delt) but not mutations that also involved the nfkb p product involved in transactivation. cytokine elevation corresponded with increased cd +cd -monocytes expressing higher cd and hla-dr and more central and effector memory cd + t cells, t cell chemoattractants, and t cellpredominant tissue pathology. those with granulomatous or neutrophilpredominant, rather than t cell, pathology had the highest tnf. tnf antagonism improved neutrophilic gastritis in cvid with nfkb delt after t cell targeted therapy failed. conclusion: nf-κb dysfunction underlies th immunopathology and tnf-associated innate inflammation in cvidc. both forms of nf-κb immune dysregulation may divergently shape cvid immunopathology. staff clinician/laboratory of clinical immunology and microbiology, immunopathogenesis section, national institute of allergy and immunology, national institutes of health, abstract/case report text introduction: patients with autoantibodies to ifn-γ develop severe and progressive infections with intracellular pathogens, despite aggressive antimicrobial treatment. we describe the use of daratumumab (anti-cd , targeting plasma cells) in a patient with autoantibodies to ifn-γ and progressive disseminated mycobacterium avium infection. she had progressive disease despite treatment with multi-drug antimycobacterials rituximab, and bortezomib. methods: clinical symptoms, total cd /cd , anti-ifn-γ autoantibody titers, and specific imaging were obtained before and after treatment with daratumumab. anti-ifn-γ autoantibody titers were determined by serial -fold dilutions of plasma and measuring anti-ifn-γ autoantibody levels by a particle-based technique as previously described. results: a -year-old filipino woman had progressive disseminated m. avium with extensive bone and soft tissue involvement (calvarium, ribs, bilateral arm soft tissue, paraspinal muscles, bilateral glutei, left inferior pubic ramus, bilateral iliac bones, sacrum, and bilateral humeri) and a tracheo-esophageal fistula. she received bedaquiline, azithromycin, ethambutol, tedizolid, moxifloxacin, clofazimine and meropenem as well as rituximab g once monthly for months. despite these she had progression of clinical and radiographic disease. bortezomib . mg/m twice weekly for weeks was added, but discontinued for ast and alt elevations. rituximab was continued to maintain cd numbers undetectable but clinical and radiographic disease progressed. while on rituximab, total igg level and anti-ifn-γ autoantibody levels decreased from mg/dl to mg/dl and to , respectively. while on bortezomib, total igg levels remained stable ( mg/dl to mg/dl) and anti-ifn-γ autoantibody levels fell slightly ( to ). after starting daratumumab, there was clinical and radiographic improvement, with reduced pain and disappearance of multiple soft tissue lesions. igg levels decreased from mg/dl to mg/dl and anti-ifn-γ autoantibody levels decreased from to . adverse effects of daratumumab were urticaria, pruritus and shortness of breath after the first infusion and aseptic meningitis after the th infusion. conclusions: daratumumab resulted in clinical and radiographic improvement of disseminated m. avium in a patient with rituximab and bortezimib-refractory autoantibodies to ifn-γ. daratumumab is another potentially effective therapeutic agent for anti-ifn-γ autoantibodies. abstract/case report text next-generation sequencing (ngs) is now routinely used as a clinical diagnostic tool. however, regions of high sequence homology continue to be a major challenge for short-read technologies. regions within ikbkg, ncf , sbds, c a, c b, coro a, fcgr a, fcgr b, pms , slfn , slfn , stat b, unc b , and ups are not available by standard ngs. we discuss strategies for analysis of these special regions. we have developed a strategy for supplementing our disease targeted panels which are performed using capture chemistry and a standard reference file. the supplemental method uses gene specific long range amplicon and a special gene specific reference file for alignment. the genes of interest are separated from their homologous counterparts using specific long range amplification primers. multiple amplicons may be pooled together and prepared for sequencing on an illumina miseq instrument using truseq nano dna library prep. bioinformatic analysis proceeds with a custom reference file in which non-specific regions of homology have been removed. this allows reads to be uniquely mapped despite significant homology; a requirement for variant calling. we prepared specific amplicon for several homologous gene targets including the ikbkg gene and the ikbkg pseudogene (ikbkgp ). both amplicons were sequenced in separate reactions and were compared with the standard capture method. variants which are not called in the standard-capture method due to poor mapping scores (non-uniquely mapped reads) are called in the amplicon method. in the capture method, the variants are visualized in the bam as a mixture of gene and pseudogene, while gene and pseudogene variants are clearly separated and identified in the amplicon method. due to high variability in alignment, many homologous regions do not provide reliable copy number variant (cnv) results and must be removed from cnv analysis. however in some situations, we are able to creatively leverage cnv analysis to identify alleles that mis-align to the pseudogene. the pathogenic ncf gt deletion in exon appears to resemble a copy number deletion event when present as reads from one allele mis-align to the ncf b and ncf c pseudogenes. complement genes, c a and c b, share alignment due to their high homology with each other. cnv analysis in normal samples represents four alleles rather than two alleles. cnv events may have a weak signal with no indication of which gene is affected. variant frequencies from the capture and supplemental pcr analysis can be used in tandem with cnv analysis to detect events and may indicate which gene is affected. we plan to include these strategies in our new inborn errors of immunodeficiency gene panel (ieigp) which will enable us to provide a more comprehensive analysis than is currently available. the im diagnosed were inflammatory bowel disease-like (n = , with perianal fistula in / ), mouth ulcers (n = ), discoid lupus (n = ), autoimmune dermatitis (n = ) and eczema (n = ), chronic lung disease (n = ) and granulomas (pulmonary n = ; ocular n = ; bladder n = ; oropharynx n = ). three patients presented more than one site of inflammatory disease. all patients were treated with systemic or topical immunosuppressive or immunomodulatory therapy, most of them corticosteroids. five patients underwent hematopoietic stem cell transplantation (hsct), median age at hsct was years ( - ), and two died month after hsct. conclusions: although infections are more frequent and have a major impact on patient morbidity and mortality, im are increasingly prevalent in patients with cgd. awareness regarding this possible comorbidity is of major importance, since earlier diagnosis and adequate treatment may be crucial for patients survival and quality of life. there is a gap in clinical knowledge regarding associations between specific pid and different rheumatological diseases. in this study, we are reporting the incidence of various rheumatological conditions reported in a large pid population using the usidnet (united states immunodeficiency network) registry. methods: we used the retrospective usidnet registry to conduct the analysis. we included all primary immunodeficiency patients with physician diagnosed rheumatological diseases. results: the total number of pid patients in our query was . ( . %) patients had a diagnosis of rheumatological disease. this cohort included ( . %) female and ( . %) male patients. rheumatologic complications were highest in the interferonopathies ( . %), complement deficiencies ( . %) and autoimmune lymphoproliferative syndrome (alps) ( . %). additionally, disease patterns were noted to be different in each pid. dermatomyositis was found to be the most common rheumatologic condition in patients with x-linked agammaglobulinemia (xla) with a rate of . %, which was remarkably higher than the reported prevalence in the united states ( . %). alps patients had a higher ( . %) numbers of sjogren syndrome diagnoses as compared to the general population ( . - . %). systemic lupus erythematosus was increased in patients with mucocutaneous candidiasis ( . %) as compared to the general population ( . %) and other pids. rheumatoid arthritis (ra) was reported in patients with specific antibody deficiency ( . %), common variable immunodeficiency (cvid) ( . %) and alps ( . %). wiskott-aldrich syndrome patients had the highest numbers of cases diagnosed with vasculitis ( . %). . % of patients with severe combined immunodeficiency (scid) had reported rheumatologic disease. juvenile rheumatoid arthritis (jia) and systemic sclerosis were reported in . % of patients with digeorge syndrome. conclusions: this study reports that higher numbers of rheumatologic diseases are diagnosed in pids compared to the general population. the incidence of different rheumatological disease was variable based on the pid diagnosis. early diagnosis of these diseases is crucial, given the high risk of irreversible complications. limitations of our study include possible selection bias as majority of cases were enrolled from tertiary care centers. abstract/case report text background disorders of immune dysregulation are associated with autoimmune features. this feature could potentially have an impact on the outcome post hematopoietic stem cell transplantation (hsct). hsct, although curative, can be challenging with the underlying immune dysregulation resulting in significant morbidity and mortality. we present the journey through hsct for these children and the factors affecting the outcome. we analysed the data on children up to the age of years diagnosed to have a disorder of immune dysregulation through gene mutation analysis and who underwent hsct at our centre from to . results . xiap mutation a -year-old boy underwent a haploidentical hsct from his father using fludarabine, treosulfan, and gray radiotherapy with post-transplant cyclophosphamide. after initial complete chimerism and cytomegalovirus reactivation responsive to valganciclovir, he developed progressive diarrhoea almost months post-hsct. a rectal biopsy confirmed cmv reactivation and features of inflammation. he has since been treated for the same and is on follow up for inflammatory bowel disease. his chimerism had dropped to % and has remained stable. the second child is a -year-old girl who underwent tcr alpha/beta depleted haplo sct and is months post-hsct, with no features of gvhd or infections, and is doing well with complete chimerism. . il r deficiency three boys aged eight months, one year, and two years of age, diagnosed to have il r deficiency underwent hsct. all three children needed nasogastric tube feeding, parenteral nutrition, and vigilant monitoring for electrolyte disturbances. in the first two children, we had performed tcr alpha/beta depleted pbsc transplants from their haplo matched fathers. the -year-old engrafted by d+ and is doing well two years post hsct with complete chimerism, no gvhd, and infections. his autoimmunity, including recurrent skin scarring, has resolved entirely. the -month-old, however, had primary graft failure and succumbed to his illness. the -year-old boy underwent matched unrelated donor hsct and engrafted by d+ with completed chimerism documented on three occasions. he, however, had secondary graft failure around d+ , and he succumbed to the illness. . lrba deficiency an -month-old girl with lrba deficiency had presented at four months of age with excessive sweating, hepatosplenomegaly, and recurrent chest infections. she was started on monthly intravenous immunoglobulin replacement and abatacept. she received myeloablative conditioning with thiotepa, treosulfan and fludarabine and underwent a matched sibling donor hsct. she engrafted by d+ and has been well ten months post hsct with complete chimerism, no gvhd, and infections. conclusion disorders of immune dysregulation are a heterogeneous group with a varied spectrum of immune dysfunction. myeloablative conditioning is essential, and there is a high risk of cytokine release syndrome and the need for supportive care. the autoimmune features need to be followed for progression in organs other than the hematopoietic system and may require interventions. as long-term data evolves, more precise definitions for patient and donor selection will enable improving outcomes. we performed a retrospective observational analysis of case records of children up to years of age, diagnosed to have variants of scid, and underwent hsct at our centre from to . results . zap deficiency a -month-old girl presented with oral thrush and submandibular cellulitis from one week of life with failure to thrive. she underwent a tcr alpha/beta depleted haploidentical hsct. conditioning included treosulfan/thiotepa/fludarabine/anti-thymocyte globulin. she engrafted by d+ ; now three years post-hsct with complete donor chimerism without gvhd or infections. . orai- mutation a -month-old girl presented with failure to thrive, generalized hypotonia, oral thrush, and recurrent respiratory infections. she underwent haplo-sct with post-transplant cyclophosphamide with pbsc from her haplo-matched father. conditioning included fludarabine/treosulfan. she had cytokine release syndrome grade , which responded to tocilizumab. she had hypertension throughout the peri-engraftment period and had an episode of pres with seizures. her symptoms abated with neutrophil engraftment by d+ . the post-transplant period was complicated by grade skin gvhd and cytomegalovirus reactivation. she has remained disease-free with complete chimerism three years post-hsct. her hypotonia is steadily improving with physiotherapy. . cernunnos-xlf deficiency a -year-old male presented with recurrent infections from years of age, aplastic anemia diagnosed at years of age, subsequent transformation to acute myeloid leukemia at years of age. he had developed multiple fusarium abscesses during the neutropenic period post-chemotherapy for aml. he was referred for a matched sibling sister hsct when in remission. conditioning included fludarabine/treosulfan. he engrafted by d+ with complete chimerism. he developed progressively worsening skin, gut, and liver toxicity secondary to chemotherapy and succumbed to the illness two months post-hsct. . ikzf mutation an -month-old girl presented with failure to thrive, massive splenomegaly, persistent pneumonia, anemia, and thrombocytopenia. she underwent a matched sibling donor pbsc transplant after myeloablative conditioning with thiotepa/treosulfan/fludarabine. she engrafted by d+ , following which all her symptoms abated. she had secondary graft failure two months post-hsct and succumbed to her illness. . mhc class ii deficiency (bare lymphocyte syndrome) three children, aged months, two years, and four years underwent matched sibling donor hsct. myeloablative conditioning with thiotepa/treosulfan/fludarabine resulted in engraftment. the first child died of invasive intestinal aspergillosis days post-hsct. the other two children are well months post-hsct with complete chimerism without gvhd or infections. the two-year-old girl received one cycle of pre-transplant immunosuppression with fludarabine/dexamethasone to prevent graft rejection pre-hsct as she was referred for a second transplant. conclusion children with scid have traditionally been transplanted using reduced intensity (ric) conditioning with immunomodulation. scid variants require myeloablative conditioning with a vigilant follow up for the detection of graft rejection. radiation sensitive scid associated with dna breakage repair defects require ric and close monitoring for gvhd. advances in hsct, including supportive care and haplo-sct, have provided a ray of hope for these hitherto rare conditions. j clin immunol abstract/case report text immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) (omim # ) is a monogenic autoimmune disorder that occurs due to loss of function variation in foxp causing dysfunctional t regulatory cells. although immunosuppression is a mainstay of treatment for autoimmunity, ipex treatment is frequently limited by insufficient response to therapy or side effects of immune suppression. we present a year old male with ipex whose prior immunosuppressive treatment was complicated by inefficacy and medication side effects, requiring a new approach to treat his colitis and erosive dermatitis. he initially presented with infantile diabetes and subsequently developed dermatitis, squamous cell carcinomas, alopecia totalis, and colitis. his clinical diagnosis of ipex was confirmed by foxp sequencing, demonstrating known pathogenic variant c. g>a (p.ala thr). this variant has been described in ipex affected individuals in multiple publications (ref ). his variant affects at the frkhead domain of foxp and has been associated with others with severe psoriasiform dermatisis and alopecia universalis (ref ). his prior immunosuppressive therapies included at different times combinations of corticosteroids, tacrolimus, sirolimus, azathioprine, infliximab, adalimumab, rituximab, dupilumab, and oral mesalamine. the relative efficacy of these agents based on experiences in a cohort of ipex patients was reviewed in (ref ), with the exception of duplimab, which was not listed in that review. for our patient, management of his widespread autoimmunity has been limited by toxicity or lack of efficacy of medications. notably, his dermatitis had no improvement with duplimab, consistent his low total ige and lack of allergic manifestations. at age , after initiation of treatment with sirolimus, he had spontaneous colonic perforation requiring descending colectomy. after stabilization of his colonic perforation, his multi-disciplinary team of allergy-immunology, gastroenterology, and dermatology initiated tofacitinib. tofacitinib is small molecule inhibitor of janus kinase (jak) signaling pathways that mediate cytokine driven autoimmune activation. it is fda approved to treat rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. the decision to use this jak inhibitor was due to its fda approved use for ulcerative colitis, to target our patient's colitis and his other autoimmune manifestations, specifically his dermatitis. its off label for primary immune dyregulatory disorders including candle, stat -gain of function and stat -gain of function disorders has been published (ref ), but thus far its use to treat autoimmunity due to ipex has not been published. he experienced leukopenia while on mg of tofacitinib, which resolved after lowering his dose. currently, he has had improvement in his colitis and dermatitis, and partial improvement in alopecia. he has been on tofacitinib mg daily for months, with only prednisone mg daily as additional immune suppression. as the number and types of selective immune modulators increases, there is continued need to share the experiences of treating physicians of which therapies have been successfully able to decrease disease manifestations with tolerable side effect profiles. we present a year old male with ipex syndrome with severe dermatitis and colitis complicated by colonic perforation despite standard immunosuppressive therapy, who is safely and effectively being treated with tofacitinib. is not frequently associated with autoimmunity, likely due to impaired il- and il- pathways. however, in our relatively large cohort of lof stat patients, we have noted an increased incidence of systemic lupus erythematosus (sle) diagnoses and sle-like symptoms. herein, we characterized the clinical and laboratory features of the patients in our cohort with sle and sle-like disease, with the aim to better understand the pathogenesis by evaluating ifn stimulated genes and neutrophil net formation. methods a retrospective chart review was performed of patients with lof stat to identify those with sle and sle-like presentations, and included clinical features, laboratories including inflammatory markers, auto-antibodies, and complement levels. rt-pcr was performed for interferon stimulated genes (isgs) from neutrophils and pbmcs of lof stat patients with and without sle, and healthy controls. neutrophil net formation was assessed for lof stat patients with and without sle, and healthy controls. results out of a cohort of patients, five patients (ages - ) were identified who carried the diagnosis of sle, and with slelike disease (ages - ). for those with sle, age of presentation was - years, of were female. clinical features included nephritis ( ), alopecia ( ), autoimmune cytopenias ( ), arthritis ( ), discoid rash ( ), and raynaud ( ). all had positive auto-antibodies, and of had low c and/or c . for those with sle-like disease, age of presentation was - , and of were female. clinical features included alopecia ( ), autoimmune cytopenias ( ), raynaud( ), and nephritis ( ). all had positive autoantibodies, and of had low complements. lof stat patients with and without clinical features of sle had increased expression of isgs from both pbmcs and neutrophils. increased spontaneous net formation was observed for lof stat patients both with and without sle symptoms. discussion although autoimmunity is not a common finding in lof stat , we have identified sle or sle-like disease in about % of our cohort, with a high incidence of kidney disease, including one patient who required kidney transplant. the interferon signature and net formation were unexpectedly high in both the patients with and without the sle features. ongoing studies include whole exome sequencing for possible second mutations or modifiers, the role of ige in the kidney disease, and further autoantibody detection. the increased ifn signature raises the question about jak-stat modulation for therapy. chief, genetic immunotherapy section/niaid, nih abstract/case report text chronic granulomatous disease (cgd), a rare immunodeficiency with decreased reactive oxygen species (ros) production, increased susceptibility to infection, and increased mortality is caused by mutations in any one of distinct phagocyte oxidase (phox) components of the nadph oxidase, nox . in the past, identification of the specific protein defect was primarily determined by immunoblotting using specific antibodies to the phox proteins. recently, however, we have shown using fluorescenceactivated cell sorting (facs) analysis of neutrophils in whole blood permeabilized and stained with specific anti-p phox antibody that p phox protein expression was absent in p phox cgd patients and significantly reduced in p phox cgd carriers [kuhns et al. . blood adv. ( ): - ]. these findings demonstrated that determination of phox protein expression by facs analysis provide an alternative to immunoblotting and can aid in the identification of p phox cgd patients and carriers. we now have extended these studies to patients and carriers with p phox cgd. facs analysis of p phox expression in permeabilized neutrophils demonstrated that p phox expression was absent in four patients with different mutations in ncf [two patients homozygous for c. e (+ ) g>a, one patient homozygous for c. _ del aag, p.glu del; and one patient compound heterozygous for the mutations, e (+ ) g>a and c. _ del aagaaggac]. moreover, the expression of p phox in nine p phox cgd carriers was significantly reduced > % compared to expression in neutrophils from healthy volunteers. another cytosolic phox protein, p phox, has been shown to associate with p phox in a : molar ratio [tsunawaki et al. . biochem biophys res comm. ( ): - ]. the expression of p phox was reduced in both carriers and patients with mutations in ncf . despite reduced expression of p phox and p phox, neutrophils isolated from carriers of p phox cgd exhibited normal dihydrorhodamine (dhr) oxidation after stimulation with phorbol ester and fell within the normal range for ros production (measured by luminol-enhanced chemiluminescence) after stimulation with either fmlf, opsonized zymosan, or phorbol ester with one notable exception. included in this cohort of p phox carriers was a p phox cgd patient (homozygous for a gt deletion at the start of exon in ncf ) who also carried a heterozygous damaging mutation in ncf [c. a>t; p. asn ile]. normal ros production in the presence of reduced p phox and p phox expression suggest that these proteins are not rate-limiting components for maximum nox activity in neutrophils. finally, determination of the expression of specific phox components by facs analysis of permeabilized neutrophils from whole blood provides a rapid and alternative approach to immunoblotting to determine the specific protein defect in cgd, and, importantly, one that could be easily established in most clinical labs. funded by nci contract no. n d . the original clinical observation that defined patients with hyper-ige syndrome (hies) was the presentation of cold abscesses ("job's syndrome"), which indicated a deficient inflammatory response. mutations in the stat gene have now been identified in most classic autosomal dominant hies patients, but we do not fully understand how these mutations cause the clinical presentation. since the discovery of stat mutations, research on hies focused largely on the adaptive arm of the immune system and suggested that the innate immune defects could be secondary. for example, the discovery that there is a th cell and il- cytokine deficiency in hies provided a possible explanation to the neutrophil chemotaxis defects in hies, as il- is one of the chemokines critical for neutrophil recruitment in vivo. the goal of this study was to investigate myeloid cells from hies patients. first we used c a, fmlp, il- , cxcl , and cxcl to study neutrophil chemotaxis in vitro. responses to c a, fmlp, and il- were equally robust in hies compared to healthy controls, demonstrating that neutrophils from patients are capable of efficient directed migration in vitro. neutrophils from all hies patients responded to cxcl and cxcl significantly below that of the healthy controls. cxcl and cxcl are cxcr -specific chemokines. these results indicated a neutrophil intrinsic cxcr -specific defect. we also found that patient-derived cells express comparable levels of cxcr on the cell surface, suggesting a cxcr chemokine receptor signaling defect. after identifying a neutrophil defect in hies, we wanted to get a broader view of myeloid cells in hies in addition to identifying the cxcr specific defect. stat is a transcriptional regulator, therefore we performed transcriptional profiling of hies and healthy control-derived neutrophils and monocytes. as it was shown before, the expression of stat was not different between patients and controls, since hies is usually caused by the decrease in stat activity not by decrease in expression. we found, however, an increase in stat and stat expression as well as significant changes in the expression of genes regulated by interferons. increased expressions of stat / in both neutrophils and monocytes likely provide and explanation for the increase in interferon regulated genes. multiple genes were identified as potential regulators of cxcr signaling. the balance between the stat and stat signaling has long known to be a regulator of immune cell activation, especially in t cells, but less studied in myeloid cells. stat and stat / signaling pathways crossregulate each other in healthy cells. we propose that in hies the decreased stat signaling leads to not only changes in expression of effector (e.g. inflammatory) genes, but also decreases expression of genes in the regulatory (negative) feed-back loop, which are required for decreasing stat / activity. therefore, the immune cell defects caused by decreased stat activity are compounded by the increase in stat / activity. increase in stat / signaling can cause pathologies in the absence of stat defects, as well as further decrease stat signaling, thus contributing to hies. interfering with stat / signaling in hies may represent a therapeutic opportunity. abstract/case report text rationale: t-cell receptor excision circles (trecs) testing on newborn screening (nbs) has been vital for identifying patients with severe combined immunodeficiency (scid). we aimed to determine whether one or more abnormal trecs result on a nbs might predict higher mortality rates despite the absence of an identifiable underlying etiology. methods: newborns with a positive trecs nbs result without the diagnosis of scid or q . deletion syndrome born from october to december were included (n= ). newborns were divided into three groups: group infants had a subsequent normal repeat screen (n= ); group infants did not undergo repeat screening as the majority expired before a repeat screen could be conducted (n= ); group infants had a normal initial screen but subsequent abnormal screen (n= ). cases were matched : to controls on gestational age, birth weight, nicu status, race, birth quarter, and birth year. nbs records were linked to birth and death certificate records. demographic characteristics were compared and mortality rates were calculated between the groups. results: the mortality rate of group was . %, group was . % and group was . %. when compared with matched controls, there was no difference in the mortality rate of group when compared to the control group. there was a significant difference in the mortality rate between cases and controls in both group (p < . , % ci . , . ) and group (p < . , % ci . , . ). the apgar scores in group infants were comparable to their matched controls. infants in group (p = . ) and group (p = . ) had significantly lower apgar scores than the controls. the majority of the infants in all three groups were less than weeks gestation, however, group had a higher percentage of infants born very premature (less than weeks). there was no significant difference in maternal age, maternal education, prenatal care status, cigarette use, or maternal steroid use between the cases and controls in all three groups. conclusions: infants with an initial abnormal screen who had a subsequent normal repeat screen did not have an increased rate of mortality compared to their matched controls (group ). however, group infants (with unresolved repeat screen) and group infants (with a first abnormal value on a repeat screen) did have increased mortality rates when compared to their controls. overall, an abnormal trecs level on nbs without a confirmed negative repeat screen, was associated with higher mortality in our study population. further studies will be needed to determine if the trecs assay can serve as a predictor for mortality in newborns with an abnormal screen. abstract/case report text introduction: primary immunodeficiency refers to a heterogeneous group of diseases characterized by altered function or composition of the immune system, and are grouped into adaptive or innate system defect. immunoglobulin g subclass immunodeficiencies (iggscs) are classified as a b-cell-related adaptive system disorder and are therefore associated with recurrent sinopulmonary infections with encapsulated bacteria, presenting with pneumonia, recurrent bronchitis, rhinosinusitis, and herpes zoster. its primary mechanisms are still unclear, although the cause for this deficiency might be related to gene deletions, transcription errors, or be an effect of allotype. igg immunodeficiency reaffirms its association with the patient's clinical condition and is often associated with igg deficiency. objective: to evaluate the prevalence of igg immunodeficiency in ferraroni's clinic, classify it by gender, age, igg dosage and other subclasses, correlate it with igg immunodeficiency and the clinical presentations presented by the patients under analysis. method records of patients with igg immunodeficiency whose clinical pictures were followed throughout years were evaluated, patients aged from to years. all tests were done at the same laboratory and all patients have consented to be part of this study, which has been approved by the ethics committee. results twenty-four patients with igg deficiency, , % (n= ) were women and , % (n= ) were male, with average of and years, respectively. the average of igg was . mg/dl, and that of igg was mg/dl. of the patients evaluated, . % had upper airway infections (sinusitis, rhinitis, otitis and tonsillitis), % herpes simplex, . % asthma. less prevalent cases were reported as . % of patients had bronchiectasis, . % candidiasis and . % herpes zoster. . % presented the association of igg and igg deficiency. discussion: the role of specific igg deficiency in the infectious setting is still unknown, but it usually occurs in association with other isotypic deficiencies and sinopulmonary infections. furthermore, the igg subclass is relevant on the study of environmental antigens -suggesting its involvement with allergic disordersand has been described in association with other diseases, such as chronic mucocutaneous candidiasis, ataxia-telangiectasia and allergic colitis. igg deficiency is related to increased susceptibility to bacterial infections. studies show a correlation between igg and igg immunodeficiency that generally imply clinical features characterized by recurrent infections by encapsulated bacteria. the data obtained through the analysis of patients' charts corroborated this information, since it was evident that most of the patients had really similar clinical conditions. conclusion: igg deficiency has a direct correlation with higher prevalence of upper airway infections, such as rhinitis, sinusitis and pneumonia, and with an increased incidence of allergic disorders, here presented by our cohort. additionally, research suggests that hies may cause impaired cd + t cell function. we hypothesized that a low percentage of both th and th cells would be predictive of hies and would differentiate hies from atopic disorders. to evaluate this hypothesis, we examined the percentage of th , th , and ifng+cd + t cells, laboratory parameters, and genetic diagnoses from a large cohort of patients to determine which parameters distinguish patients with stat loss-of-function variants. methods: we conducted a retrospective, multi-institutional chart review of over patients who received a th assay at the medical college of wisconsin clinical immunology research laboratory. the th assay is performed by activating pbmcs with pma/ionomycin/brefeldin a and staining for cd , cd , ifng and il- a. the following parameters were included in the chart review: the percentage of th , th , and cd +ifng+ cells, immunoglobulin levels, atopy scores, infectious history, and genetic diagnoses. results: using logistic regression, we demonstrated that the percentage of th , cd +ifng+, and th cells were positively correlated with age, and percentage of cd +ifng+ cells was higher in females than males. we found that the percentage of th and th cells were decreased in both atopic disease and hies, with hies having the lowest values. interestingly, one subject with a stat gain-of-function (gof) variant had an elevated percentage of th and th . in addition, we determined that ige levels were inversely correlated with the percentage of th , cd +ifng+, and th cells, while iga and igm were positively correlated with the percentage of th cells. several different monogenic defects characterized by increased fungal infections exhibited a low percentage of th including tatton-brown-rahman syndrome and cornelia de lang syndrome. conclusions: we confirmed that the percentage of th cells is low in both hies and atopy in a large cohort of subjects, and that the percentage of th cells may be helpful in distinguishing hies from atopic disease. however, since the percentage of th , cd +ifng+, and th cells correlate with age, caution should be used when testing young children. the inverse correlation between ige levels with th and th responses suggests that similar pathway(s) may drive both hies and atopy. additionally, the decreased th responses in stat lof and increased th responses in stat gof hies raise questions about the role of stat in regulating ifng levels. we also identified patients with different genetic disorders with fungal infections in which the th percentages were low, suggesting that the th test may be useful in evaluating individuals with unusual fungal infections. abstract/case report text background:primary and secondary autoimmuune neutropenia (pan/san) are well described entities. several autoimmune neutropenias do not fit the criteria of either pan or san showing peculiar characteristics mainly for older age at onset and/or for duration of the disease; moreover they are not associated , at least at the beginning, with autoimmune markers/diseases aim of the study: to describe a cohort of subjects affected with autoimmune neutropenia, defined as "atypical" (aan), registered in the italian neutropenia registry (inr) and to compare these data with those from subjects diagnosed with pan still in the inr. patient and methods: subjects with neutropenia and positivity of indirect antibodies against neutrophils (registered in the inr from to ) lasting for more than years, or diagnosed after years of age ( up to y), without any associated autoimmune, signs/markers were considered eligible for the present study. results: data from patients were collected: / subjects ( %) were defined as aan and / ( %) as pan. among aan affected patients %, were "long lasting" aan, while % were defined as "late onset" aan .the degree of neutropenia in aan group was mild in %, moderate % and severe in % of the subjects . leukopenia at onset was a common hall mark seen in % of aan patients (median values /mm³ ; range - /mm³ ) especially in the "late onset" aan if compared with pan and " long lasting" one ( p= . ). as for clinical features, almost half of the aan cohort suffered from recurrent or "significative infections", while severe episodes (namely sepsis, meningitis , osteomyelitis , pneumonia, deep abscess or flemmon) were shown in % being more frequent, but non significantly higher than those reported in the pan group ( % )( p=ns) interestingly, recurrent apthae were significantly more seen in the "late onset" aan group if compared with the "long lasting" aan (p= . ). during follow up, markers and/or symptoms of autoimmunity appeared in % of the aan cohort, being another element of peculiarity in respect to pan (p < . ). as for immunological pattern in aan, immunoglobulin values were lower than the references for age in %,while were above them in % of the cohort . lymphocytes subsets evaluation showed decreased value of cd +cd + cells in % of cases, followed by depletion of cd -cd +cd + subtype in %, cd +cd + in % of cases and cd + cd + in % . preliminary study on b memory and t-reg cells values, showed a quantitative deficiency respectively of in % and % of the studied subjects. mutation analysis performed by ngs in % of the subjects identified pathogenic variants of : taci ( ), tinf ( ) and lrba ( ) .comparison between pan and aan is detailed in table . conclusions atypical neutropenia in childhood is a disorder which show many difference with pan; indeed appears an epiphenomenon of a complex immunological disturbances rather than a disease itself. occasionally mutations of genes of immunodeficiency/disimmunity can be demonstrated abstract/case report text background: medications treating ra typically include systemic corticosteroids used to treat inflammation flares, and disease modifying therapies (dmards). traditional dmards include methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine. recently, biologic/immuneresponse modifiers have come to the forefront for overall therapeutic benefit, however, an unfortunate side-effect may be the risk of increased immunosuppression. this study seeks to determine the occurrence rates of immune deficiencies among patients initiating ra therapies. methods: using the pharmetrics plus commercial claims database from - , ra patients (icd- and - codes: m , m ) over the age of were indexed on their first use of a new biologic therapy. all patients were required to have enrollment six-month pre and one-year post index. cohorts of patients were grouped by medication: methotrexate, adalimumab, etanercept, and rituximab. ra patients receiving adalimumab, etanercept, and rituximab were allowed concomitant use of methotrexate, but could not use any other biologic medications in the post period. a minimal adherence of % was required of all biologic treated ra patients. an additional cohort of ra patients untreated with biologic therapies was indexed on their first ra diagnosis within the time window and used as a control. ra patients with comorbid conditions who would also require biologic treatment were excluded including crohn's disease and ulcerative colitis. between group comparisons were made with the no treatment group as the referent. to account for differences in age, gender, and elixhauser comorbidity conditions patients in each cohort were matched : to the rituximab group. results: , ra patients met inclusion criteria: , in the methotrexate group, , receiving etanercept, , receiving adalimumab, and receiving rituximab. a total of , in the treated groups and , in the no biologic treatment group. demographic information including age and gender were significantly different but numerically similar between the groups, with rituximab group having the highest proportion of female patients but limited dispersion with the lowest proportion being in the etanercept group. healthcare utilization metrics highlighted a significantly higher average number of office visits ( . , sd: . vs no treatment . , sd: . , p < . ) and a higher proportion of rituximab patients being hospitalized ( . % vs no treatment . %, p < . ). the diagnosis of immune deficiency was highest among the rituximab group with . % followed by methotrexate . %, adalimumab . %, etanercept . %, and no treatment . %. after matching, similar rates were seen for healthcare utilization to the pre-match results. the post-match odds of being diagnosed with immune deficiency were significantly greater for the rituximab group (or . , ci: . - . ) than the no treatment group. conclusions: the purpose of dmards is to modulate the immune system and decrease autoimmunity in ra. however, this treatment may lead to significant immunosuppression. this study suggests that treatment with certain biologic/immune-response modifier therapies may be associated with higher rates of healthcare utilization. in particular, the increased post-treatment diagnostic coding of immune deficiency demonstrates the heightened awareness among healthcare providers of the chronic immunosuppressive potential of rituximab. evaluation of potential secondary immunodeficiency pre-and post-dmard use should be incorporated into routine practice. abstract/case report text introduction: autoimmune lymphoproliferative syndrome (alps) is a rare inherited disorder of lymphocyte homeostasis due to a fasmediated apoptosis and characterized by non-infectious and nonmalignant lymphoproliferation, autoimmunity, and secondary malignancies (national institute of health criteria). in spite of recent progress, one third of alps patients still remain gene orphan and they have been previously categorized as alps-u. in some cases, patients fitting alps diagnostic criteria have been shown to carry mutation on genes involved in other immune-dysregulation syndromes. aims: the aim of this study is to compare the clinical and immunological features, and the outcome of a cohort of alps patients with mutations on the typical causing genes (fas, fasl, fadd and casp )-here defined as alps-g -vs the ones without a molecular diagnosis or carrying mutations on other genes (both defined as alps-u). patients and methods the demographic, clinical, biochemical, genetic informations and details about treatment are derived from the alps italian network. search of mutations was performed with sanger pcr and/or next generation sequencing techniques (extended to immunodeficiency genes panel). results: alps patients were registered in our data base; the genetic analysis was performed in subjects ( %): / pts ( %) were alps-g and the remaining ( %) alps-u. six-teen out of ( %) alps-u patients resulted to carry mutations on other genes (lrba, stat +cecr, ctla , baffr, taci, nmlrc , ikbkg, gaucher), and the remaining ( %) were negative. the alps-u subjects showed a more complex phenotype compared to the alps-g group, which was characterized by multi-organ involvement (p= . ) and positivity of autoimmune markers (p= . ). (table ). cytopenia affecting one or more haematopoietic lineages was present in both groups ( % and %) with no significant difference, apart from lymphocytopenia that was more frequent in alps-u group (p= . ) ( table ) . as for lymphocyte subets and immunoglobulin dosage no differences were shown within the two groups. vitamin b and il- were more frequently raised in alps-g group (p= . , p= . ) (table ). four out of ( %) patients did not require any treatment. first-line treatment (steroid or intravenous immunoglobulins) controlled the disease only in / ( %) cases. the response rate to second line therapy -micofenolate mofetile (mmf) or rapamycin-was % and % in alps-g and alsp-u group, respectively. moreover, target therapies or drug combinations were more commonly applied in alps-u subjects (p= . ) ( table ) . conclusions: our study showed that alps-u subjects, despite the alps phenotype, represent distinct clinical entities and that genes associated with other immune-dysregulation syndromes are frequently represented in this group ( / , %). the identification of such disorders is crucial for the management of second-line treatment and/or the administration of target therapies abstract/case report text background: we have shown previously that allergic reactivity to ovalbumin (ova) could be regulated in mice following perturbation of immune networks using combinations of an immune ig along with antiidiotypic ig. we have explored features of this regulation including: its persistence after cessation of administration of combined igs; the ability of heterologous igs to produce immunoregulation; a role for treg induction in regulation; and the ability to attenuate responses in mice presensitized to an allergic stimulus. methods: balb/c mice were sensitized to ova. mice also received weekly injections of immune ig or anti-idiotype ig (at separate sites) from either homologous (mouse) or heterologous (human) sources. in the latter case pooled ivig (given im, hence hereafter imig) was used as a source of anti-idiotype ig, and human anti-tet as immune ig. injections of the ig were given from the time of ova sensitization (to attenuate development of immunity), or after pre-sensitization of mice (to attenuate existing allergic responses). all mice were assayed for development of ova-specific serum ige and igg, as well as the production of ova-induced il- , il- , il- , il- and il- in splenocytes cultured for hrs. in studies examining possible mechanism(s) responsible for inhibition of immunity mice received, in addition to the ig treatments described, infusion of depleting anti-cd , and/or anti-cd antibodies, or a mab to tnfsfr , known to expand tregs implicated in regulation of allo immunity. results: combinations of both heterologous and homologous immune igs and anti-idiotype igs attenuated ova allergic responses in both naïve and pre-sensitized mice. this attenuation persisted in mice greater than weeks after cessation of treatment with the igs used. finally, depletion of either cd or cd cells ameliorated the suppressive effect seen, while the combination of anti-cd and anti-cd essentially abolished suppression. suppression was further enhanced by anti-tnfsfr mab. conclusions: we conclude that the combine ig treatment protocols used produced a long-lasting suppression of allergic immunity, even in pre-sensitized animals. the effects seem to depend upon induction and expansion of tregs and represents a novel approach to treatment of allergic disease in humans and other animals. abstract/case report text background wiskott-aldrich syndrome protein (wasp) is found in the cytoplasm of hematopoietic cells but can transit to t lymphocyte nuclei at distinct developmental timepoints. wasp deficiency is a rare, x-linked combined immunodeficiency disease. affected patients display qualitative but not quantitative t cell defects. we report two immune deficient subjects with nearly identical exon frameshift mutations in was, the gene encoding wasp. one subject lacked circulating t cells, the other possessed several distinct cd t cell populations each expressing quantitatively different amounts of wasp. objective to determine how similar was mutations can cause scid in one person and generate b and t cells with heterogenous wasp expression in another. methods to identify somatic was mutations, we deeply sequenced was exons, introns, promoters and ' untranslated regions at , read depth in genomic dna from various b and t cell populations of each subject and their unaffected relatives. we confirmed genomic variants were transcribed and translated by sequencing was transcripts and analyzing wasp in primary cell lysates, both fractionated and not. to model our subjects' diseases we transfected primary cells and cell lines with mutant was transcripts and then measured viability and nuclear localization via confocal microscopy. results deep sequencing of genomic dna revealed all of subject one's cells carried the same germline exon frameshift was mutation. the mutation was incorporated into subject one's was transcripts and translated into a truncated form of wasp, which was relegated primarily to the cell nucleus. subject two possessed three distinct cd t cell subsets that each carried either the germline exon frameshift was mutation or a variety of somatic mutations that circumvented frameshift wasp expression. evasion strategies included exon skipping, adoption of a cryptic exon splice site and reversion to wild type amino acid sequence. subject two incorporated somatic mutations into was transcripts which encoded either stable near full-length proteins or unstable non full-length ones. subject one's sister and subject two's mother, who both carried the germline exon frameshift mutation, produced only wild type transcripts and proteins. conclusion we report two patients with was mutations encoding truncated wasp. if expressed, truncated wasp localized to the cell nucleus, and this was associated with t cell developmental arrest and severe combined immune deficiency. if, through a variety of epigenetic and somatic strategies, t cells could avoid expression of truncated wasp, they would survive but display phenotypical abnormalities and functional defects. patients with pidds show a higher susceptibility to hematopoietic malignancies, in particular to non-hodgkin lymphomas (nhl) that, generally, account for approximately - % of paediatric cancers and their incidence increases with age. recently new gene defects responsible for pidds with lymphoproliferation as a key clinical sign have been identified. our goal is to investigate possible immune-mediated mechanisms underlying malignant lymphoproliferation in children who did not show other typical symptoms of pidds. we retrospectively selected and reviewed the clinical history of nine patients with nhl ( burkitt lymphoma, large b cell lymphoma and lymphoblastic tcell lymphoma). immunophenotyping and exome analysis of known pidds genes were performed after lymphoma remission. six out of nine patients showed a mild hypogammaglobulinemia at time of presentation, not noticed before. moreover, one patient had history of recurrent respiratory infections, one of hematologic autoimmunity and two of nine were ebv-positive at diagnosis. preliminary results show an aberrant b cell phenotype in four patients; exome analysis reveals a novel heterozygous genetic variation in ikzf gene in one patient with burkitt lymphoma and autoimmune cytopenia was identified. concerning the remaining patients, further studies are ongoing. a detailed review of clinical history of paediatric patients affected from nhl as well as an impaired immunophenotyping can be important indicators of immune-mediated disorder underlying lymphoproliferation and helpful signs of possible pidds that should promptly be investigated by genetic analysis. this will allow an appropriate diagnosis and disease management. abstract/case report text introduction: caspase activation and recruitment domain (card ) encodes a scaffold protein that links antigen receptor activation to intracellular signaling. dominant heterozygous loss of function (lof) mutations in card cause a syndrome of severe atopic dermatitis, elevated ige, and allergic disease. atopic dermatitis can be difficult to control leading to substantial morbidity. dupilumab is a humanized monoclonal antibody that blocks il- and il- signaling approved for treatment of refractory atopic dermatitis. we present a case of a -year-old female with card deficiency successfully treated with dupilumab. case: a -year-old puerto rican female with history of recurrent sinopulmonary infections with episodes of pneumonia, moderate persistent asthma, food allergies, recurrent skin boils, and severe atopic dermatitis was referred for further management and evaluation for autosomal dominant hyper-ige syndrome (ad-hies). her atopic dermatitis was refractory to conventional therapy with topical corticosteroids, twicedaily emollient use, and bleach baths; it was also refractory to immunosuppression with mycophenolate mofetil and cyclosporine. on exam the patient exhibited coarse facial features and a high palate. she had eczematous lesions on the face, trunk, and extremities (scorad ). laboratory evaluation showed: eosinophilia ( cells/ul), elevated ige (> ku/l), low igm ( mg/dl), and elevated iga ( mg/dl). lymphocyte subsets and mitogen response were normal but antigeninduced proliferation was abnormal. autosomal dominant hyper ige score was indicating a high likelihood of ad-hies. no mutations in stat were identified and th cell expression was elevated. dedicator of cytokinesis (dock ) deficiency was also considered but dock protein expression was normal. further genetic testing revealed an base pair deletion in card (c. _ del) predicted to be pathogenic. the combination of the patient's phenotype and large deletion was consistent with card deficiency. despite continued immunosuppression with cyclosporine and aggressive skin care, the patient's atopic dermatitis was still severe and poorly controlled. off label (patient < years) treatment with subcutaneous dupilumab mg every weeks was initiated. at last follow-up, months after dupilumab start, the patient had substantial improvement in dermatitis with clear skin on the face, trunk, and extremities (scorad ). cyclosporine was discontinued and topical medications were applied less frequently. discussion: hypomorphic heterozygous dominant negative loss of function mutations in card have recently been associated with severe atopic dermatitis and allergic disease. treatment of atopic dermatitis in card deficiency remains challenging, but dupilumab appears to be an effective alternative to refractory disease. longer follow-up and a larger cohort of card -lof patients treated with dupilumab are necessary to understand the long-term efficacy and safety for use of dupilumab in these patients. abstract/case report text purpose: the micromilieu within premalignant respiratory papillomas supports persistent hpv / infection and disease recurrence in recurrent respiratory papillomatosis (rrp). these patients show polarized (th -/treg) adaptive immunity in papillomas and blood, enriched immature langerhans cell (ilc) numbers, and overexpressed cox /pge in the upper airway. to better understand the adaptive and innate dysregulation in rrp, we studied blood-derived monocytes, ilcs, and tissue-derived ilcs from rrp patients and controls. experimental design: monocyte subpopulations were isolated, differentiated into ilcs, activated, and then assessed by flow cytometry. monocytes were induced to differentiate into ilcs with/ without added pge , and then activated by il- γ, pge , pge +il γ, or lps. ilc cd expression was identified by flow cytometry. monocyte-derived ilcs, papilloma, foreskin, and abdomen skin ilcs, were also analyzed by qpcr for select chemokine/cytokine mrna expression after isolation, hrs later in culture, and again after poly(i:c) or tnfα stimulation. results: the three monocyte sub-populations differed between patients and controls, and patients' monocytes generated fewer ilcs. classical monocytes generated most, but not all ilcs. pge levels were higher in rrp plasma, and added pge reduced control, but not patients' monocyte-ilc differentiation. pge had no effect on ilc maturation identified by cd expression. papilloma-derived ilcs expressed low ccl- , and high ccl- mrna and were unresponsive to poly(i:c) or tnfα. tissuespecific cytokine/chemokine responses between ilcs from papillomas, foreskin and abdominal skin differed. only papilloma ilcs expressed il- γ after isolation, and they up-regulated ccl mrna hrs later without further stimulation. conclusions: monocyte/ilc innate immunity is impaired in rrp, in part due to increased pge exposure. the immunosuppressive papilloma micromilieu likely alters ilc responses that skew, hpv / -specific th /treg adaptive immunity in rrp. abstract/case report text introduction: familial mediterranean fever is a hereditary auto inflammatory disorder that typically manifests with recurrent fevers, abdominal pain and in some patients there is an associated with amyloidosis leading to eventual renal failure. while there are several common mutations in the mefv gene that when homozygous give these classic symptoms, patients with atypical mutations or heterozygous mutations often have a different clinical course. we present identical twin siblings with compound heterozygous mefv mutations but differing clinical phenotypes. case description: the index patient is a year old girl, conceived via ivf, who began having fevers at age . . her fevers occurred every weeks for months before she was referred to immunology for evaluation. her parents describe her as happy and otherwise not ill appearing during these episodes. genetic testing for familial mediterranean fever revealed compound heterozygous e q and p s mutations in the mefv genes. initiation of colchicine therapy in the affected sibling has resulted in a complete resolution of her symptoms. a trial off colchicine resulted in return of cyclic fevers. her identical twin sister was also tested, and carries the same mutation, but is still asymptomatic. this created great concern amongst their parents who had genetic testing prior to undergoing ivf that revealed no parental mutations in mefv. in consultation with genetics the mother was tested again through the same laboratory that had performed testing on the children. this revealed an identical mutation in mom who is also asymptomatic. conclusions: although classic homozygous mefv mutations have resulted in well described fever syndromes, there is considerably less data on heterozygous and compound heterozygous mefv mutations. in these two identical siblings only one patient has a classic manifestation of familial mediterranean fever. while it is possible that the other twin will develop similar symptoms later on in life, it is also possible that another factor is necessary to trigger symptoms in this unusual genetic presentation of fmf. in addition this case highlights the importance of understanding the testing method used by the laboratory performing the genetic testing. while the mother was initially reported as negative the laboratory that performed her testing only tested for the most common mefv mutations. more complete testing, that included the entire gene sequence, revealed that she did contain an mefv mutation in e q, which although more rare is thought to be pathologic when combined when combined with a second mutation. abstract/case report text there are several lines of evidence that link the pi k/akt/mtor signaling pathway to primary immunodeficiencies. hyperactivation of the pi k/akt/mtor/s k signaling pathway in immune cells can be the consequence of dominant gain-of-function mutations in the genes encoding for pi kδ that cause the activated pi kδ syndrome (apds). patients with these mutations may develop immunodeficiency and immune dysregulation as well as neurodevelopmental delay and growth retardation. in addition, mutations of genes within the pi k-akt-mtor pathway were also known to cause megalencephaly and segmental cortical dysplasia. mutations in akt , a member of the akt family of proteins and a downstream effector of pi k-mediated signaling, was shown to be associated with autosomal dominant megalencephalyassociated syndromes. here we describe a year old girl, born to consanguineous healthy parents, who presented with megalencephaly, developmental delay, hypotonia, cervical lymphadenopathy and hepatosplenomegaly. the patient had recurrent hospital and icu admissions for idiopathic thrombocytopenia (treated with ivig), recurrent laryngitis, recurrent peritonsillar abscess, preorbital cellulitis, conjunctivitis with purulent discharge, otitis media, pneumonia with pleural effusion (required drainage), metapneumovirus pneumonia with respiratory failure, recurrent skin cellulitis, and abscesses that grew mrsa (required drainage). in addition, the patient is known to have asthma and allergic rhinitis. mri of the brain showed megalencephaly, ventriculomegally, thin and dysplastic corpus callosum, a normal cerebellum, and myelination appropriate for age. immunoglobulin levels, lymphocyte subsets and the oxidative burst test were all within normal limits. cmvand ebv were not detected. bacterial cultures grew mrsa (skin), strept. pneumoniae, h. influenzae, and e. coli (urine). extensive metabolic workup was done, which was inconclusive (metabolic/mitochondrial diseases). whole exome sequencing identified an akt variant c. g>a; p.(asp asn) in exon . the variant was identified in the patient but not in the parents and it was confirmed by sanger sequencing. further molecular testing concluded that the variant is caused by a de novo mutation during early development. although pathogenic variants in akt gene were shown to be associated with megaloencephaly-associated syndromes, no associations with immune deficiency have be reported. functional studies will be pursued to confirm the link between the clinical phenotype and the identified variant in the akt gene. complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed 'cbm-opathies') result in broad clinical manifestations ranging from combined immunodeficiency (cid) to atopic disease or lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card mutation (c. c>t; p.r *) causing complete card deficiency. results: we recently identified an -month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a -year-old boy with a history of severe pulmonary infections with bronchiectasis (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. testing of both patients demonstrated absent tregs, elevated naïve b cells with absent memory b cells, and panhypogammaglobulinemia. next generation sequencing revealed that both patients were homozygous for the same novel variant of card (c. c>t; p.r *), which rendered card protein undetectable by immunoblot. card deficiency was confirmed by stimulating patient b cells with phorbol -myristate acetate (pma) and ionomycin and immunoblotting for signalling proteins in both the nf-κb (ikkα/β, iκbα, p ) and mapk (mek / , mkk , jnk / , erk / ) pathways as well as cleavage substrates of the malt paracaspase (relb, cyld, bcl , hoil ). nf-κb and jnk activation were completely absent and m a lt p a r a c a p a s e a c t i v i t y w a s l o s t . f u r t h e r m o r e , c oimmunoprecipitation experiments revealed that card was required for optimal malt association with bcl in response to stimulation. to define the impact of card deficiency on the b cell transcriptome, rna-seq experiments were performed. this revealed an inability to upregulate critical genes involved in immunity and tolerance (e.g. cd lg, ctla , il , il ), decreased enrichment in cytokine pathways (e.g. ifn-α, il- , tgf-β), and decreased enrichment in malt -dependent genes. furthermore, rna-seq confirmed the developmental block observed in patient b cells and suggested that b cells were halted at the centroblast to centrocyte transition. both patients ultimately underwent hematopoietic stem cell transplantation (hsct), which restored lymphocyte signalling and activation as measured by nf-κb, jnk, and malt paracaspase substrate cleavage. conclusions: we have presented the most comprehensive clinical and molecular characterization of human card deficiency to date. these two cases highlight the crucial role of card in regulating b cell development, function, and humoral responses, as confirmed by signalling and transcriptomic analyses. furthermore, hsct is potentially helpful for these patients as assays performed on post-transplant cells demonstrated restored signalling and activation. abstract/case report text introduction: primary immune deficiencies (pid) can have a significant impact on the quality of life of patients and their families. as more patients with pid are surviving to adulthood, the need to monitor them closely and ensure they are transitioned appropriately is even more crucial. we compared the perspectives of pediatric and adult immunologists toward the transition of patients with pid at our institution. methods: pediatric allergy/immunology providers at lurie children's hospital and adult allergy/immunology physicians at northwestern university both in chicago, il completed respective surveys anonymously (www.surveymonkey.com). questions were derived from the validated 'attitude' and 'quartt' instruments for transition. respondents were asked to rate their level of agreement on a -point likert scale, ranging from strongly disagree to strongly agree. results: overall, pediatric and adult providers participated (response rate . %). of total respondents, % thought the transition process should be initiated at age - . about % of the adult immunologists selected and older, whereas pediatric providers would begin earlier; . % of pediatric providers note they would initiate transition at age - . both pediatric and adult immunologists agreed that patients should be transferred when the provider felt they were ready ( %) and when they were in stable condition ( %). both adult and pediatric immunologists selected transfer of complete medical file as a preferred communication method for transition. other strategies preferred by adult providers were a referral letter with brief summary of medical history ( . %) and staff meeting with pediatric and adult immunologists ( . %), whereas pediatric providers would prefer a joint outpatient dedicated transition clinic ( . %). pediatric and adult immunologists, patient, parent, and transition liaison were considered the most important active participants in the transition process. the most prominent barriers to a formal transition were unavailability of a transition coordinator or nurse specialists ( %) or of all disciplines of the interdisciplinary team ( %), and limited time ( %). on the other hand, limited demand (too few patients) was strongly rejected as a barrier. all participants agreed during transition patients should be educated about medications and their side effects, their condition and related potential future complications, and symptoms that require seeking health care. over % of participants also agreed that education about how to set up ivig and further insurance needs were important. all participants agreed that the transition process should include assistance on how to promote the patients' independence and self-management skills, medication management/adherence, and understanding of immunoglobulin replacement and side effects. other important transition components included knowing how frequently lab draws are required for monitoring ( %) and having a written individualized transition plan ( %). pediatric providers also thought having an email or telephone help line would be beneficial. conclusion: this study adds to the growing body of literature examining attitudes of immunologists toward transition, and it highlights important transition components and barriers. further work is ongoing to determine the transition needs identified by patients and parents and define markers for successful transfer in order to build a transition policy at our institution specific to immunodeficiency patients. abstract/case report text introduction: bronchiectasis (bq) is an abnormal and irreversible dilatation of bronchi secondary to repeated cycles of airway infection and inflammation. predominantly antibody deficiency is the main group of primary immunodeficiencies (pid) in adults and had been reported up % of subjects with non-cystic fibrosis bronchiectasis (ncfb). hypergammaglobulinemia (igg level higher than , mg/dl) had been observed in . % of ncfb cases (retrospective data). diagnostic delay and inappropriate management of patients with predominantly antibody deficiency can lead to irreversible lung damage or even death from serious infections. the effect of hypergammaglobulinemia on ncfb is unknown. here we present the frequency of immunoglobulin abnormalities (pad and hyperigg) in adults with ncfb in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include patients with ncfb. women and men > and < years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of ncfb cases have been included in the study. the mean age was . years ( - years) with a female:male ratio : . moderate-severe dyspnea was observed in / cases (medical research council -mrcdyspnea scale to ). recurrent pneumonia was found in / cases ( %). the main etiologies of bronchiectasis were: post-infection / ( %); idiopathic / ( %); autoimmunity / ( %); primary immunodeficiency / ( %) asthma / ( %); copd / ( . %); primary ciliary diskinesia / ( %); reflux / ( %) and others. primary immunodeficiencies, . % of ncfb cases, were classified as: predominantly antibody deficiency ( cases) including cvid cases, igm deficiency cases, igg subclasses deficiency case, selective iga deficiency case and hypogamaglobulinemia case. combined immunodeficiency (dock deficiency) case. interestingly igg hypergammaglobulinemia was observed in / cases ( . %) suggesting humoral immune response deregulation. conclusion to the best of our knowledge this is the first prospective study evaluating the etiology of non-cystic fibrosis bronchiectasis (ncfb) in colombia. hypergammaglobulinemia and predominantly antibody deficiencies affect % of adults with ncfb in colombia. our study reinforced the necessity to evaluate humoral immune response in patients with bronchiectasis. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt - /iir-col-bxlt- . abstract/case report text background: early-onset inflammatory bowel disease (eoibd) is defined as ibd diagnosis in children less than years of age. the occurrence of autoimmune disease in children (where it is relatively rare, compared to adults) may be caused by a highrisk predisposition gene (monogenic disorders). mayo clinic children's center has a unique care model, where a patient who is referred for eoibd meets with a team of physicians, including gastroenterology, immunology, genetics, and nutrition. we describe our experience of our eoibd clinic from an immunologic perspective. methods: we conducted a retrospective cohort study through emr chart review of pediatric patients who were referred to our eoibd program ( - ). first diagnosis of ibd under the age of was the inclusion criteria. we assessed the presentation, clinical correlates, and immunologic evaluation. approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: pediatric patients met the inclusion criteria, with ( %) males and ( %) females. the median age of ibd diagnosis was years ( - years range). median values and the distribution of variables used in the nutritional and immune evaluation were assessed (fig ) . nutritional assessment was remarkable for low to low normal hemoglobin and ferritin levels. vitamin d and albumin levels were overall within the normal range. growth parameters indicated that the median bmi percentile was ( - ). with immune and genetic screening, one patient was found to have x linked chronic granulomatous disease (cgd). immune evaluation of other patients was overall within normal limits. fecal calprotectin served a reliable non-invasive biomarker for inflammation with the median being . ( . - . ). of these patients underwent gi pathogen panel testing of which ( %) tested negative, four ( %) tested positive for c. diff, and two ( %) others to shiga toxin-producing e. coli. it was also noted during the chart review that most patients had poor disease control despite undergoing treatment with various anti-inflammatory and immunosuppressive drugs. the patient diagnosed with cgd underwent bone marrow transplantation. a higher proportion of patients referred to our program in recent years underwent a more comprehensive multispecialty evaluation. conclusion: awareness of monogenic causes of inflammatory disorders in children has increased in recent years. it is also important to rule out intestinal infections that can act as ibd mimic. identifying monogenic disorders and other ibd mimics helps with targeted therapy and symptom improvement in these patients who have a difficult-to-treat disease. the group of children with eoibd, regardless of whether there is an inborn error of immunity, suffers from very high morbidity and a high burden of disease. comprehensive immune-nutrition assessment of eoibd patients paves way for further in-depth immunogenic assessments and allows for global management. abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) affecting the nadph oxidase system in phagocytes resulting in increased susceptibility to catalase-positive organisms. holland presented the first report of dual impact of cgd and hiv in a patient with disseminated nocardiosis. because their cgd patient admitted to history of iv drug use, he was frequently screened for hiv. case: a -year-old african american male with known cgd tested positive for hiv by western blot in the ed in when he presented with complaints of intermittent fever and cervical lymphadenopathy. his cgd was diagnosed by nbt blood testing at years of age. he had frequent skin infections and fever prior to diagnosis. clinically, he did so well that his cgd diagnosis was questioned by his immunologists. however, cgd was confirmed by additional abnormal nbt tests and, ultimately, dhr flow cytometry testing. during his second infectious disease consultation for hiv, at age , he disclosed that he was bisexual. previously, the patient was screened for hiv and hiv antibodies in due to anal fissure. he was screened again in for marked cervical and supraclavicular lymphadenopathy. his cd + t cell absolute count was noted to be low ( /mm ) in at age . until , his prior hiv screenings were negative. during his cgd treatment course as an adult, he was known to be variably adherent with administration of interferon gamma due to adverse effects, particularly pain at the site of injection and malaise. at the time of his positive hiv western blot in , his cd + t cell count was / mm . after starting hiv antiretroviral treatment, his viral load became undetectable. at age , he had burkholderia cepacia pyelonephritis resulting in left nephrectomy. sepsis from b. cepacia was fatal (positive blood cultures without known primary source) in at age . his recent viral load was still undetectable and cd + count was /mm . summary: our case reveals the complexities of treating a patient with both primary and acquired immune deficiencies. it illustrates the importance of taking a thorough social and sexual history starting in adolescence, including those patients with pid. patients with pid should be followed closely by a primary care physician, in addition to an allergist-immunologist and infectious diseases specialist, to ensure age appropriate medical and developmental screening. the recognition of pids is improving due to better screening, awareness, and treatment. currently, the rate of hiv infection is highest among young homosexual african american males. it remains important to understand the epidemiology of primary and acquired immunodeficiencies to best identify those at highest risk. ( ) submission id# phosphatase and tensin homolog (pten) hamartoma tumor syndrome identified by newborn tcell receptor excision circle screening for severe combined immunodeficiency δ) subunits that are critical for cellular signaling. heterozygous gain-offunction (gof) mutations in pik cd (encoding p δ) result in activated pi k δ syndrome (apds ), while heterozygous loss-of-function (lof) mutations in pik r (encoding p α) result in activated pi k δ syndrome (apds ). given its role as a negative regulator of the pi k signaling pathway, heterozygous lof mutations in pten (encoding phosphatase and tensin homolog, pten) result in a clinical phenotype that approximates that of apds /apds and is therefore referred to as activated pi k δ syndrome-like (apds-l). however, sequelae of heterozygous pten lof mutations extend beyond the immune system and include a group of disorders collectively known as pten hamartoma tumor syndrome (phts). although severe t cell lymphopenia at birth would be unexpected in apds , apds , or apds-l, below normal t cell receptor excision circle (trec) counts have been reported in apds , but only in individuals outside of the neonatal period. herein, we describe an infant girl with a low trec count at birth who was found to have phts. case description: a -day-old girl, born at a gestational age of weeks, was found to have a low trec count of /microliter (normal => ). a second trec count obtained at weeks of age resulted as /microliter. arguing against a diagnosis of severe combined immunodeficiency (scid), flow cytometric analyses performed at weeks of age revealed only a modestly diminished cd + t cell count ( /microliter; cd + and cd +) with a normal percentage of naïve and memory cd + t cells ( % and %, respectively). by months of age, her cd + t cell count dropped to /microliter, which was accompanied by a significantly decreased percentage of naïve cd + t cells ( %). sequencing and deletion/duplication analysis was pursued via a commercially available -gene panel aimed at genetically defined primary immunodeficiency (pid), in which no clearly pathogenic mutations were identified. over the following months, the patient was noted to have macrocephaly, tall stature ( th percentile), axial hypotonia, and gross motor delays. sequencing and deletion/duplication analysis was then pursued via a commercially available -gene panel aimed at genetically defined macrocephaly and overgrowth syndromes, in which a hemizygous pathogenic mutation in pten (c. a>g, p.gln arg) was identified. subsequent flow cytometric analyses demonstrated findings characteristic of apds-l, including expanded transitional and cd lo b cells, decreased isotype switched memory b cells, increased effector memory t cells, a lowered threshold for intracellular calcium mobilization upon b cell receptor engagement, and increased basal akt (protein kinase b) and s (ribosomal protein s ) signaling. discussion: we report the first case of phts identified by newborn trec screening for scid. as pten is not included in most commercially available, scid-or pid-tailored gene panels, phts would be missed by conventional genetic testing. therefore, analysis for variants in pten should be considered in neonates with low trec counts, macrocephaly, developmental delay, and other suggestive sequelae. abstract/case report text primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory syndrome affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, inducing extreme inflammation and massive tissue infiltration by activated t cells and macrophages. standard chemotherapy-based treatment regimens are toxic and induce remission in only % of patients. to this day, hsct is the only available curative treatment, but the inability to efficiently control the inflammation in many patients prior to transplantation often leads to graft failure, with transplant-related mortality around %. thus, the development of new, more potent and less toxic anti-inflammatory regimens would be a major advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting several jak-dependent cytokines will be more effective than monotherapy to reduce the life-threatening symptoms induced by this pathology. using a perforin-deficient (pko) mouse model, we first tested the effects of blocking antibodies against ifnγ, the dominant cytokine secreted during hlh, in combination with antibodies targeting other highly elevated cytokines, such as il- and il- , on the manifestations of the disease. we found that anti-il- r and anti-il- antibodies, when used in combination with anti-ifnγ antibodies, did not significantly improve the symptoms of hlh compare to anti-ifnγ antibodies alone. further, we found that targeting the jak-stat signaling pathway with ruxolitinib, a specific inhibitor of jak and jak , molecules downstream of ifnγ and il- , but not il- signaling, was as beneficial as anti-ifnγ monotherapy. next, we tested the efficacy of ruxolitinib in combination with anti-il- antibody, as this later cytokine is not jak-dependent and was shown to drive macrophage activation syndrome in other contexts. unfortunately, this combination did not result in better symptom resolution than the use of ruxolitinib only. in contrast, combination therapy using ruxolitinib and anti-ifnγ antibodies showed a striking synergistic effect on the resolution of most disease manifestations, to such an extent that our pko mice presented a clinical phenotype indistinguishable than that of a c bl control mice. our findings demonstrate that jak-dependent cytokines are the main cytokines driving the progression of hlh in pko mice. collectively, our results suggest that anti-ifnγ antibodies and ruxolitinib, although effective independently, should be used in combination to more efficiently suppress hlh progression. these results are particularly relevant since the emapalumab, an anti-ifnγ monoclonal antibody was recently approved by the fda for the treatment of hlh while ruxolitinib will soon be in clinical trials for this indication. this project was supported by funds from the fondation de cancérologie charles bruneau and the canadian institutes of health research (mop- ). abstract/case report text introduction: transcription factor (tcf ), also known as transcription factor e -alpha (e a), is a helix-loop-helix transcription factor which plays a critical role in lymphopoiesis. tcf is required for b and t lymphocyte development. defects in tcf have been associated with agammaglobulinemia , autosomal dominant, characterized by low levels of immunoglobulin and early onset recurrent bacterial infections. deletion or diminished activity of tcf may also play a role in lymphoid malignancies. runs of homozygosity (roh) are contiguous stretches of homozygous genotypes at consecutive polymorphic dna marker positions. roh are important reservoirs of homozygous deleterious variation. the homozygosity heterogeneous hmm (h m ) algorithm was specifically developed for analyzing whole exome sequencing (wes) data. the branch point sequence (bps) is an essential splicing signal located - bases upstream of splice acceptor sites. while bps variants are rare, they may result in aberrant pre-mrna splicing and genetic disorders. these variants may be overlooked by standard wes analysis methods because they are intronic and the mammalian bps is a degenerate motif. objective: describe the method used to identify a bps variant in consanguineous brothers with immunodeficiency, including early onset recurrent infections and b-all, hypogammaglobulinemia, t and nk lymphocytosis, low b cells, and low naïve t cells. methods: wes was performed for all family members. data were analyzed using standard read mapping, variant calling and annotation methods. roh were analyzed using the h m algorithm (magi et al. ). roh from the siblings was intersected (bedtools) and the output was submitted to the genomic oligoarray and snp array evaluation tool (v . ). variants were confirmed by sanger sequencing. results: no candidate disease variants were detected in the coding regions, ' or ' splice sites, or utrs for both brothers; however, analysis of intersected roh revealed a homozygous tcf intronic variant within a putative bps (tcf c. - a>t). sanger sequencing of the mutant cdna revealed activation of a cryptic splice site. heritability for routine childhood vaccines has been shown to range from - %. the genetic component of vaccine response suggests we should be able to predict vaccine response in infants with biomarkers. methods: multi-center study of infants born vaginally at full term and followed through months of life. cord blood was collected at birth & peripheral blood was collected at and months of life. six-month collection was weeks post administration of routine vaccinations, while -month collection was immediately prior to receiving the -month booster vaccines. b cell subsets were analyzed with flow cytometry. vaccine titers and cytokines were measured via multi-plex elisa. study was irb approved. results: our data confirmed the immaturity of the newborn humoral immune system with a lower overall b-cell abundance, a predominance of naïve b cells, an inability to class-switch and produce igg or iga, and a th bias. maturation was observed over the first year with increasing overall b-cell abundance, frequency of memory b-cells producing iga, igg, and igm, and frequency of plasmablasts. scd levels also increased throughout the first year due to microbial translocation reflecting establishment of the microbiome. conversely, cord blood contained high levels of baff, april, scd l, il- , and il- , with levels decreasing thereafter. all infants displayed evidence of humoral immune system activation after getting -month vaccines. total plasmablast levels peaked weeks after receipt of month immunizations. a decrease in total plasmablasts was evident between and months, although levels remained above those at birth, corresponding with the need for -month booster vaccinations to maintain long-lasting immunity. il- and ifn-gamma had a significant positive correlation with memory b cells and plasmablasts at subsequent time points suggesting that these cytokines play a role in b cell differentiation and vaccine response. baff and april cytokines were elevated at birth, consistent with germinal center formation and underwent a compensatory decrease thereafter. april & scd levels in cord blood significantly correlated with higher tetanus titers at months suggesting that vaccine response may be predicted by cytokine biomarkers at birth. response to vaccines was also dynamic with il- levels being significantly correlated with tetanus titers at weeks after receipt of month immunizations. conversely, scd l levels did not correspond to b cell development consistent with a known b cell hyporesponsiveness to cd l in infants. conclusion: humoral immune development is both predictable and dynamic. biomarkers in the cord blood, produced by the infant, are predictors of b cell development and vaccine response in infancy. background: adult-onset immunodeficiency with anti-ifnɣ autoantibodies is a newly described immunodeficiency syndrome characterized by disseminated nontuberculous mycobacterial and other opportunistic infections in previously healthy middle-aged individuals typically from southeast asia. it is caused by the presence of autoantibodies directed against the cytokine ifnɣ, which is required for intracellular pathogen killing by macrophages as well as phosphorylation of the transcription factor stat , which is involved in cell survival gene expression. successful treatment of the immunodeficiency has been described in prior case reports with immunomodulatory therapies, including rituximab. however, there are no standard recommendations for dosing or timing of these agents, or recommendations for longterm monitoring of disease activity. case presentation: a -year-old laotian woman with a history of type diabetes and possible prior hepatitis c infection presented to the immunology clinic for evaluation of immunodeficiency. in the two years prior to presentation, the patient was diagnosed with mycobacterium avium infection involving the parotid gland and lymph nodes of the neck, mycobacterium avium complex bacteremia, histoplasma capsulatum involving the lymph nodes of the neck, and leukocytoclastic vasculitis of the lower extremities. as a child and young adult, she had no severe or recurrent illnesses and did not suffer from any chronic disease. preliminary immunologic testing demonstrated normal t, b, and nk cell subsets, elevated immunoglobulin g, a, and m levels, and protective titers to tetanus, diphtheria, and / pneumococcal serotypes. measurement of anti-ifnɣ autoantibodies was positive, which led to the diagnosis of adult-onset immunodeficiency with anti-ifnɣ autoantibodies. the patient was treated with four doses of monthly rituximab with resolution of the anti-ifnɣ autoantibodies, restoration of normal stat phosphorylation, and depletion of cd -positive b cells. after a -year period of being lost to follow-up, during which she continued to receive rituximab every months at the direction of a local provider, the patient re-presented to the immunology clinic to re-establish care. at that time, the patient had no evidence of anti-ifnɣ autoantibodies based on titers and normal stat phosphorylation. cd -positive b cells remained depleted. the patient also confirmed subjective clinical improvement and denied any interim infectious complications. conclusion: this case provides an example of successful treatment of a patient with adult-onset immunodeficiency with anti-ifnɣ autoantibodies with rituximab. it also highlights the utility of ifnɣ functional testing with stat phosphorylation, which may be used to monitor disease activity and to make decisions about ongoing immunomodulatory treatment. it is necessary to monitor additional immunological markers to refine the diagnosis of a secondary post-lt immunodeficiency to take preventive measures before infections occur. we sequentially measured t lymphocytes and antibodymediated immunity in a -year-old male receiving a lung transplant for idiopathic pulmonary fibrosis to determine which immune indicators could improve the identification of a secondary immunodeficiency. methods: t and b cell numbers, igm, igg, iga, ige and igg subclasses and specific antibodies to s. pneumonia capsular polysaccharides were assessed over a months-long post lt period. the clinical progress, infections and pulmonary function were monitored prior to transplantation and at regular intervals thereafter. results: the patient had progressive idiopathic pulmonary fibrosis starting with an episode of pulmonary hypersensitivity years earlier. he developed increasing respiratory failure progressing to complete dependency requiring a lt in june . he was treated with prednisone, mg/ day continuously for months, then decreased to mg/day. other immunosupressants included mycophenolic acid and tacrolimus and on/ off antibiotics that eventually led to severe tendinitis at - months post lt. at ½-month post lt he developed an early onset bronchiolitis obliterans syndrome (bos) that was controlled by increasing the prednisone dose. sequential immunologic evaluation showed his igg dropping from , mg/dl to after two weeks and then remaining stable at that level for the rest of the observation period. igm and iga had minor variations and ige remained very low. igg fell from - mg/ml pre-lt to - in weeks and remained stable at that level thereafter. antibodies against s. pneumoniae polysaccharides started high, between - ·g/ml for all serotypes and fell rapidly in the first weeks post lt, then continued a steady decline with > % serotypes falling < . ·g/ml at months. twelve of pneumococcal serotype antibodies increased above . ·g/ ml after igg replacement at months. cd t lymphocytes decreased from ± , cells/ul to - at month, remaining at that number after that. cd /th cells increased from - cells/ul to at months when prednisone was tapered down and then decreased to after increasing the prednisone dose again. this decrease coincided with a reduction in bos manifestations. conclusions: immune monitoring revealed an independent decrease of immunoglobulins with a stronger decrease in igg and specific pneumococcal antibodies. the role of th cell increase in developing bos needs further investigation. stat is a frequent target of cancer therapies due to its role in certain malignancies for cell proliferation and metastasis. with lof stat , decreased incidence of some cancers may be expected, however increased rates of lymphoma are described. we sought to describe the incidence and spectrum of malignancy in our relatively large lof stat cohort. methods: we performed a retrospective analysis of lof stat patients evaluated at the nih clinical center to determine the type of malignancies diagnosed, treatments received, and outcomes following therapy. results: a total of patients with malignancies were identified (cancer incidence %). six patients ( %) were diagnosed with non-hodgkin lymphoma (nhl); with diffuse large b-cell lymphoma (dlbcl) and with burkitt lymphoma (bl) with age at diagnosis ranging from years to years with median age of years. pathology staining for ebv was available in four patients; all of whom were negative by eber. all dlbcl patients received da-epoch-r for - cycles, and all achieved complete remission. five of patients with lymphoma are alive and disease-free. one patient died of heart failure years post chemotherapy without disease relapse. two patients were diagnosed with papillary thyroid carcinoma at ages and , one of whom was subsequently diagnosed with nhl. two other patients were diagnosed with basal cell carcinoma of the skin at ages and ; both of whom had prior voriconazole exposure. conclusion: malignancy, most commonly nhl, occurs in patients with lof stat mutations. nhl should be considered in patients with progressive lymphadenopathy, and thyroid carcinoma should be considered in patients with thyroid nodules. patients treated with voriconazole are at an increased risk of skin cancer and require careful skin monitoring. as survival increases, it will be important to monitor the incidence of malignancies diagnosed, as it is possible that decreased stat signaling may prove to be protective of some cancers, such as colon and breast carcinoma, in which increased stat signaling is implicated in pathogenesis. abstract/case report text introduction: recurrent pneumonia is defined as or more episodes of pneumonia in one year or more than pneumonias throughout life (with radiological resolution between episodes). in retrospective studies, up to % of adult subjects with recurrent pneumonia coursed with primary immunodeficiencies (pid). prospective studies evaluating the etiology of recurrent pneumonia are scarce. diagnostic delay and inappropriate management of patients with pid (predominantly antibody deficiency for example) could lead to irreversible lung damage or even death from serious infections. here we present the frequency of primary immunodeficiencies in adults with recurrent pneumonia in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include patients with recurrent pneumonia. women and men > and < years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of recurrent pneumonia cases have been included in the study. the mean age was . years ( - years) with a female:male ratio : . moderate-severe dyspnea was observed in / cases (medical research council -mrcdyspnea scale to ). non cystic fibrosis bronchiectasis was found in / cases ( %). the main etiologies of recurrent pneumonia were: primary immunodeficiency / ( %); asthma / ( . %); autoimmunity / ( . %); primary ciliary diskinesia / ( . %) and others. hypergammaglobulinemia represented / ( %) of cases. primary immunodeficiencies, % of recurrent pneumonia cases, were classified as: predominantly antibody deficiency ( cases) including cvid cases, igm deficiency cases, selective iga deficiency cases, igg subclasses deficiency case, hypogamaglobulinemia case and agammaglobulinemia case. combined immunodeficiency: dock deficiency case and ataxia telangiectasia case. conclusion: to the best of our knowledge this is the first prospective study evaluating the etiology of recurrent pneumonia in colombia. predominantly antibody deficiencies and igg hypergammaglobulinemia affect % of adults with recurrent pneumonia in colombia. this study allows us diagnosed more than new cases of adult onset pid. immunological evaluation is critical in the assessment of patients with recurrent pneumonia. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt - /iir-col-bxlt- . abstract/case report text introduction: lysinuric protein intolerance (lpi) is an autosomal recessive metabolic disorder due to pathogenic mutations in slc a . it is distinguished by decreased plasma concentrations and increased urinary excretion of lysine, arginine and ornithine and can present with multiorgan involvement and a spectrum of immune deficiency. we present a five-year-old female with lpi, early-onset juvenile systemic lupus erythematous (sle), hemophagocytic lymphohistiocytosis (hlh), and granulomatous skin lesions that were positive for vaccine-strain rubella. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, autoantibodies, metabolic studies, and genetic evaluation by next generation and whole exome sequencing. results: a five-years-old female of mixed native american and african american race presented at years of age with severe failure to thrive, history of recurrent fevers, joint swelling, recurrent skin lesions, severe anemia and neutropenia, and hypergammaglobulinemia. upon further evaluation, she demonstrated hyperferritinemia and ana, rnp, smith, and ss-a autoantibodies and was diagnosed with early-onset juvenile sle. laboratory immune evaluation revealed age-appropriate lymphocyte subpopulations and lymphocyte proliferative responses to mitogens and antigens, markedly elevated igg, iga, igm with no associated monoclonality, and protective tetanus and pneumococcal titers. given her severe clinical manifestations at an early age, concern for immunodeficiency prompted further genetic evaluation with next generation dclre c sequencing, which was negative, and whole exome sequencing, which revealed two heterozygous mutations in slc a , consistent with lpi. laboratory metabolic evaluation was also consistent with a diagnosis of lpi. she continued to experience recurrent cutaneous lesions on her upper and lower extremities. biopsy findings were consistent with a granulomatous lesion and subsequently identified by the cdc to have vaccine-strain rubella infection. due to recurrent pneumonia and concern for pulmonary alveolar proteinosis (pap), pulmonology was consulted and eventually confirmed pap, and she has required home oxygen supplementation. given her history of recurrent infections and vaccine-strain rubella infection, supplemental ivig was initiated. her sle has been fairly refractory to medical management, including systemic corticosteroids, mycophenolate, rituximab, and cyclosporine. she recently developed hlh at years of age and is currently maintained on canakinumab, mycophenolate, and systemic corticosteroids, yet continues to have sle and pap that has been difficult to control. conclusion: the range of clinical and immunologic findings in lysinuric protein intolerance has varied widely in the literature. our patient presented with early-onset juvenile sle and did not develop hlh and pap until years after initial presentation. despite relatively normal cellular immunity by laboratory evaluation, our patient was identified to have chronic infection with vaccine-strain rubella virus, indicating severe t cell dysfunction, and poses challenges for future immunomodulatory treatment. introduction: x-linked immunodeficiency with magnesium defect, ebv infection, and neoplasia (xmen) disease is caused by lossof-function (lof) mutations in the magnesium transporter (magt ) gene. it is a rare x-linked combined immunodeficiency and selective congenital disorder of glycosylation. clinical manifestations include chronic ebv viremia, recurrent bacterial and viral infections, lymphadenopathy, splenomegaly, autoimmunity, liver and central nervous system (cns) abnormalities. magt deficiency was first noted to result in chronic ebv infection and an increased susceptibility to ebv+ lymphomas. we recently recognized merkel cell carcinoma at a very young age in two xmen patients, leading to our review of the malignancies in this cohort. methods: we reviewed the records of male patients ( seen at the nih) with confirmed hemizygous lof mutations in magt for diagnosis of malignancy, therapy, and outcome. results: we identified malignancy in patients of with magt deficiency ( %). four patients had hodgkin's lymphoma (hl) (ages - years), three had non-hodgkins lymphoma (nhl) (ages - years), one had kaposi sarcoma ( years) , one patient developed eber-negative liposarcoma (age years) after receiving chemo and radiotherapy for severe lymphoproliferative disease (lpd) at age years and two had merkel cell carcinoma at exceedingly young ages ( and years). all patients had chronic ebv viremia. they all received treatment according to established protocols. currently, all except for three patients are alive and in remission, including one post-hsct. overall malignancy survival of %. it is important to note that three patients who did not have malignancy had ebv lpd so severe that it warranted treatment with a malignancy protocol, with one mistaken as having lymphoma. conclusion: xmen immune deficiency, an x-linked glycosylation disorder, is a multisystem disease associated with increased susceptibility to malignancies. initially, ebv driven lymphoproliferation and lymphoma was described with xmen; however, with increasing diagnoses, more malignancies are being recognized. all the recognized malignancies are associated, at least in part, with dnaviruses, including ebv, hhv- , and merkel cell virus. understanding the clinical phenotype and pathogenesis of this disease will improve monitoring and early diagnosis of malignancies for patients with magt deficiency. abstract/case report text background: primary immune deficiencies (pid) constitute a heterogeneous group of over individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build dozens or thousands of decision trees randomly to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of pid. methods: with a local database of patients with suspected iei, we built a decision tree using c . dtc, and a random forest on python . (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term "immunodeficiency" in their electronic medical records, and then hand-picking cases in which a pid had been confirmed or ruled out. it consisted of patients, of which had been diagnosed with iei. we first split the dataset randomly into training ( %) and testing ( %) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set through cross-validation. results: accuracy was greater than % for the dataset (pid/not). . for the dtc with levels. the attribute with the lowest gini coefficient was low iga ( . ). accuracy for the random forest classifier was . with trees. feature importance was highest for lung infection ( . ), high igg ( . ), low iga ( . ), skin infection ( . ), no isolate ( . ), and allergy ( . ); it was lowest for consanguinity, high igm, central nervous system infection, parasites and no infections. during the random generation of trees, accuracy reached up to %. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot, currently under construction, that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical "red flags". we also have preliminary results of this process applied to a usidnet database with over , patients, and are also working on multinomial logistic regression and naïve bayesian classifiers for this and other databases. abstract/case report text objectives: acute viral respiratory infections (avri) are associated with significant healthcare resource use and cost. the use of intravenous immunoglobulin (ivig) may be an effective treatment for immunosuppressed patients and reduce overall healthcare resource utilization. the goal of this study was to assess hospital resource utilization associated with ivig use among patients hospitalized for avri. methods: using data from the - premier hospital database, we identified patients hospitalized with a diagnosis of avri [respiratory syncytial virus (rsv), parainfluenza virus, rhinovirus, or metapneumovirus], and who had an immune deficiency (chemotherapy treatment, transplant, primary immunodeficiency disorder (pidd), specific antibody deficiency, other immunodeficiency, or disorders of the immune or lymphatic systems). patients receiving ivig within the first hours were compared to patients who did not receive ivig at all. due to the nature of the need to better understand the treatment effect associated with ivig, we used an inverse probability weight-based regression model. since there were substantially more controls than cases, we randomly drew , controls. a logistic regression model was developed to adjust for factors associated with the probability of ivig use within hours of admission. this propensity score was then used to weigh subsequent models to assess length of stay (total and icu) using negative binomial models and logistic regression for inpatient death. results: a sample of , immunocompromised inpatients were identified, receiving ivig within the first hours of admission and , who did not receive ivig. the ivig group was older (mean age vs , p < . ), had more antiviral use ( % vs %, p < . ), and had less cancer ( % vs %, p < . ). after adjustment for immunity type (transplant, cancer), rsv, pidd, age, prednisone, antiviral use, ribavirin use, urban hospital setting, teaching status, intubation and lung disease, patients with ivig use had . less days of hospitalization (p= . ) and . less days in the icu (p= . ) than non ivig users. conclusions: this data analysis suggests that hospital length of stay and icu length of stay were significantly shorter for immunocompromised patients hospitalized for acute viral respiratory infections who were administered ivig within the first hours of admission, as compared to patients who did not receive ivig. it is possible that ivig use may have an impact on hospital resource utilization and costs. future prospective studies would help further assess the role of ivig in patients hospitalized with acute viral respiratory infections. associate professor/yale university abstract/case report text background: cd ligand deficiency is an x-linked combined immunodeficiency associated with opportunistic infections and increased risk of malignancies. expansion of memory cd + t-cells with senescent features is known to be associated with chronic immune stimulation including aging, chronic infection and malignancy. cd + t-cell characteristics of cd l deficient (cd ld) patients in relation to their clinical history have not been described. objective: we studied correlation between cd + t-cell senescence with clinical histories of cd ld patients. methods: we analyzed the frequency and phenotypic characteristics of peripheral cd + t-cell subsets in four cd ld patients ( , , and years old (yo)) and healthy controls (hcs). t cell excision circle (trec) counts and telomere lengths of the patients and hcs were measured using quantitative pcr. in-depth analysis of cd + t-cells of the yo patient and hcs was done using high-dimensional cytometer time of flight analysis (cytof). results: three patients ( , and yo) with histories of recurrent infections and poor compliance with immunoglobulin therapy (ivig) showed an increased frequency of effector memory cd + t-cells with the senescent phenotype compared to age matched hcs. whereas yo patient with excellent ivig compliance starting at infancy did not show any senescence phenotypes of the cd + t-cells. the telomere length and trec count of each patient correlated with the degree of cd + t-cell senescence and their current ages, respectively. in-depth analysis showed similar expression patterns of molecules related to senescence and cytotoxicity in cd + t-cells including cd , t-bet, eomes, granzyme b and perforin in the yo patient and mid-elderly hcs. conclusion: our findings suggest that prompt diagnosis and compliance with ivig starting at the infancy may prevent early onset cd + t-cell senescence in cd l deficiency. abstract/case report text introduction: immunoglobulin g -related disease (igg -rd) is an immune-mediated fibroinflammatory condition that affects multiple organs. when igg -rd is found in the ocular adnexa, the term "igg related ophthalmic disease (igg -rod)" is used. objective: our case describes a patient with igg -rod without systemic involvement. case: mr. x is a -year-old male with a pmh of cml (on imatinib) and allergic rhinitis who presented to clinic with orbital swelling for twenty years. his swelling had always been responsive to steroids, but would return once steroids were tapered. patient was diagnosed with biopsy proven cml in and is currently taking imatinib. because his peri-orbital edema persisted, a right lacrimal gland biopsy was done which showed "marked lymphocytic infiltrate of soft tissue with lymphoid follicles, many plasma cells, and eosinophils. no atypical histiocytes." flow cytometry was negative for malignancy. results: crp . mg/l. esr mm/hr. igg elevated at mg/dl. ct chest from and ct chest, abdomen, pelvis from were without fibrotic changes. assessment: when diagnosing igg -rd, we categorize diagnosis into three levels (possible, probable, or definite) by three criteria (clinical manifestation, elevated serum igg , and histopathology). this is detailed as follows: clinical exam showing organ specific swelling or masses, elevated serum igg (> mg/dl), and histopathology with either lymphocyte and plasmacyte infiltration and fibrosis or infiltration of igg + plasma cells (ratio of igg +/igg+ cells ≧ % and ≧ igg + plasma cells per high power field). not all these components are required for diagnosis, but meeting histopathologic criteria makes diagnosis more probable. our patient's disease was localized to his eye, and patients with igg -rod have unique diagnostic criteria. these criteria are similar to the criteria for igg -rd, but emphasize enlargement of the ocular adnexa, less frequent fibrosis, and ≧ igg + plasma cells per high power field. our patient's histopathology revealed a lymphoplasmacytic infiltrate, but lacked storiform fibrosis or obliterative phlebitis. his serum igg level was mg/dl, and his biopsy was positive for an igg +/igg+ ratio of % and more than igg + plasma cells per high power field. based on this, he meets criteria for igg -rod. conclusion: igg -rod is a rare condition that is usually associated with systemic organ involvement. our case is unique, as no systemic disease has been detected. we also suspect our patient has been living with igg -rod for several years, as his orbital swelling began in high school. it is important to note that he has been on imatinib, a tyrosine kinase inhibitor, for treatment of his cml. imatinib inhibits c-abl and platelet-derived growth factor receptor, tyrosine kinases involved in profibrotic pathways. patient's lack of fibrosis could also be due to his longstanding use of this drug. it is also possible that he has a rare form of igg -rod without systemic involvement. a limited number of such cases have been reported, but no consensus has been made on why disease course was localized. our patient was started on rituximab, and his serum igg decreased to mg/dl after the first cycle. we hope his disease achieves remission. informed consent: informed consent was obtained from all individual participants included in the study. abstract/case report text platelet abnormalities with eosinophilia and immune-mediated inflammatory disease (plteid) is a recently discovered combined immunodeficiency with inflammatory and allergic manifestations with few cases reported. we describe a female patient with compound heterozygous mutation in arpc b gene with suggestive clinical findings of plteid. a -year-old girl presented with chronic diarrhea since neonatal period, with bloody stools and failure to thrive. she also presented atopic dermatitis, recurrent cutaneous and mucosal ulcers, recurrent respiratory infections ( episodes of otitis media, pneumonias) and many episodes of mucocutaneous candidiasis. family history revealed a sibling deceased in the second month of life, who presented a similar clinical picture and a paternal uncle and second degree cousin that died in the first year of life. there is no history of consanguinity. laboratory evaluation revealed peripheral eosinophilia ( /mm ), normal platelet numbers with low platelet volume ( , fl -reference value , - , fl), normal igm levels with elevated igg ( mg/dl-rv - mg/dl), iga ( mg/dl -rv - mg/dl) and ige ( iu/ml -rv a) associated with plteid. t h e i n f a n t r e c e i v e s a n t i m i c r o b i a l p r o p h y l a x i s w i t h sulfamethoxazole-trimethoprim and fluconazole, intravenous immunoglobulin replacement and was referred to hematopoietic stem cell transplantation (hsct). this case was the first one described in brazil and highlights the importance of seeking for a genetic diagnosis in patients with complex clinical phenotypes. precise diagnosis can impact on treatment approach. live vaccines are generally contraindicated in patients with combined immunodeficiency (cid). however, in less severe cid, such as partial dgs, those vaccines can be considered depending on the immunologic status of the patient. there are recommendations regarding to measles, mumps, rubella (mmr) and varicella vaccines, but yellow fever vaccine (yfv) is generally contraindicated in this population. considering the severity of the yellow fever disease and the absence of specific treatment, the use of this vaccine is an important topic for debate in cases of patients from endemic areas. objective: this study aimed to describe the use of yfv and other live attenuated vaccines in patients with dgs, associating it with their immunological profiles and the presence of adverse effects. methods: retrospective study of medical records of patients with dgs confirmed by mlpa or fish, followed in a pediatric reference center for primary immunodeficiencies between and . collected data included: demographic characteristics, medical history, history of immunization with live vaccines, postvaccination adverse reactions and immunological profile, including immunoglobulins levels, serologic vaccination responses, lymphocyte immunophenotyping, lymphocyte proliferation responses to mitogens and prophylactic treatments (antibiotic or immunoglobulins). results: thirty-five patients with confirmed dgs and median age of years ( - y) were included ( m: f). thirty-three children ( %) received mmr vaccine: nine presented t lymphopenia. two of the patients had cd < , one of them with normal mitogenic proliferation response and the other was not tested. three of the patients had low immunoglobulins levels ( / low igg, / low igm and / low iga), and one of them received intravenous immunoglobulin (ivig). twentynine of had normal serologic vaccination responses. adverse effect was only reported by one patient, who had one episode of fever after the administration of all vaccines. yellow fever vaccine was administrated to children ( %): had t cell l y m p h o p e n i a ( b u t c d > ) , a n d a n o t h e r p a t i e n t h a d hypogammaglobulinemia and received ivig and prophylactic antibiotics. twelve of showed adequate serologic responses to mmr and hepatitis b. only patient reported mild reaction (tremors) two days after the yfv administration. the same patient had normal t cells, immunoglobulins and vaccine responses. twenty patients ( %) received bacillus calmette-guerin vaccine (bcg), ( %) received oral polio, ( %) rotavirus and ( %) received varicella vaccine. no severe adverse events were documented in any patient that received live vaccines, and no patient developed measles, mumps, rubella or yellow fever diseases as a consequence of administration of the vaccine. conclusions: in this cohort of pediatric patients with dgs, yfvand other live vaccines were well tolerated, and no severe adverse events were reported, suggesting that widespread contraindication of yfv may endanger unvaccinated patients with less severe phenotype living in endemic areas. immunological evaluation and individualized decisions are always recommended, and further studies are needed to assess the safety of the yfv in this pediatric population. abstract/case report text introduction: lad-i is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from itgb gene mutations encoding for the β -integrin component, cd . severe lad-i (i.e., cd expression on < % of neutrophils) is characterized by recurrent serious infections, impaired wound healing, and childhood mortality. although allogeneic hematopoietic stem cell transplant (allohsct) is potentially curative, its utilization and efficacy are limited by hla-matched donor availability and risk of graftversus-host disease (gvhd). rp-l - (clinical trials.gov # nct ) is a phase / open-label clinical trial evaluating the safety and efficacy of autologous cd + cells transduced with a lentiviral vector (lv) carrying the itgb gene encoding for cd (chim-cd -wpre) in severe lad-i. methods: pediatric patients ≥ months old with severe lad-i (demonstrated by cd expression on < % neutrophils and at least one prior significant bacterial or fungal infection) are eligible. peripheral blood (pb) hematopoietic stem cells are collected via apheresis after mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. cd + hspcs are selected, transduced with chim-cd -wpre lv, and cryopreserved. myeloablative conditioning with busulfan (therapeutic drug monitoring (tdm) dosing with adjustments to enable target area under the curve (auc)) is administered over days, followed by infusion of the thawed investigational drug product (rp-l ). patients are followed for safety assessments including replication competent lentivirus (rcl) and insertion site analysis (isa), and for efficacysurvival to age ( months) and at least -year post-infusion without allohsct, increase in neutrophil cd expression, pb vector copy number (vcn), decrease in infections and/or hospitalizations, and resolution of skin or periodontal abnormalities. results: an initial lad-i patient (age years) with recurrent severe infections and documented itgb mutations has been treated as of november . baseline cd , cd a, and cd b expression were < %. mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target auc. investigational product was comprised of . x e cd + cells/kg with vcn of . copies/cell (liquid culture), and was infused without complications. no serious treatment-emergent adverse events were reported. neutrophil engraftment ( consecutive days of anc ≥ ) was observed days post-infusion. pb pmn cd expression months posttreatment was . % with comparable cd a and cd b expression levels; pb cd (myeloid) vcn at . months was . . safety and efficacy data months post-treatment will be available at the time of presentation, in addition to preliminary data regarding a potential additional patient. conclusion: preliminary evidence demonstrates that rp-l enables itgb genetic correction with robust cd /cd neutrophil expression in this frequently fatal primary immunodeficiency. abstract/case report text introduction: the complement system plays an integral role in the innate immune system and links innate and adaptive immunity. complement deficiencies, hereditary or acquired, are rare. acquired deficiencies are more prevalent, occurring in nephrotic syndrome, reduced hepatic synthesis or transiently in sepsis/viremia. they are also seen in the presence of autoantibodies known to cause depletion of complement factors, such as c nephritic factor (c nef). c deficiency is associated with infection susceptibility, particularly to encapsulated bacteria, and immune complex disease. case description: a year old male was evaluated for recurrent infection. in childhood, he had recurrent sinusitis, otitis media requiring tympanostomy tube placement and persistent pharyngitis despite tonsillectomy. as a teenager, he developed glomerulonephritis, progressing to end stage renal disease and requiring transplant at age . the kidney allograft failed years later, with biopsy demonstrating recurrent glomerulonephritis. the patient was transitioned to peritoneal dialysis and later hemodialysis, due to recurrent pd-related infections. his adult course was complicated by recurrent methicillin sensitive staphylococcal aureus (mssa) catheter and soft tissue infections (cellulitis and abscess), sinusitis, sepsis (streptococcal, mssa and tularemia), multifocal pneumonia and a left below knee amputation for osteomyelitis that required revision surgery. patient reported other autoimmune phenomena including a presumptive diagnosis of vasculitis and possible lupus-like syndrome. the constellation of recurrent infections and autoimmune features was most concerning for an early complement deficiency. prior work up was notable for low c , ch and ah with normal c , factor h and factor i. extensive laboratory work up revealed normal c q, c level and function, serum immunoglobulins, vaccine titers, factor b and factor d levels. atypical hus (ahus) panel revealed a heterozygous silent variant in exon of cfh and a heterozygous polymorphism within an intron in mcp/cd , seen with increased prevalence in the patient population with ahus. wes was notable for a variant of uncertain significance in the vcl gene only. c level and function were markedly decreased, alongside low ch and ah . both sc b- level and c nephritic factor were elevated. a diagnosis of acquired c deficiency due to c nef was made and patient was started on bactrim prophylaxis. he has remained free of serious infection since starting antibiotic prophylaxis. discussion: c nef stabilizes the alternative pathway c convertase, c bbb, increasing its half-life and blocking dissociation. this leads to unregulated consumption of c with subsequent deficiency. c nef has been associated with c glomerulopathy, infection and partial lipodystrophy. however, there is marked heterogeneity in clinical phenotypes with reported asymptomatic individuals. our patient's glomerulonephritis likely represents c glomerulopathy. case reports and series of successful treatment of c glomerulopathy with rituximab and eculizumab have not commented on immune outcomes beyond the kidney. other potential therapeutic strategies include plasma cell depletion with either bortezomib or daratumumab. further study is needed to evaluate these therapies influence on both reversal of c depletion and overall impact on immune function in the setting of c nef. abstract/case report text introduction: patients with heterozygous signal transducer and activator of transcription (stat ) gain of function (gof) pathogenic variants exhibit an array of phenotypes including susceptibility to viral, bacterial, fungal and mycobacterial infections, autoimmunity, and cancer predisposition. progressive disseminated histoplasmosis (pdh) is well-described to affect infants. however, no reports have evaluated underlying monogenic immune dysregulation in previously healthy infants presenting with pdh. we report an infant who presented with pdh and associated hemophagocytic lymphohistiocytosis (hlh) leading to the diagnosis of a heterozygous stat gof mutation. case report: a previously healthy -month old male presented with persistent fever, pancytopenia, transaminitis, elevated ferritin, hepatosplenomegaly and coagulopathy. his clinical and laboratory evaluations were concerning for hlh syndrome. he had no prior history of immune hyperactivation or atypical infections. secondary causes of hlh were investigated, and patient was diagnosed with pdh based on marked histoplasma antigenemia. targeted genetic testing did not reveal a genetic etiology of familial hlh. he was successfully treated with a pulse and taper of dexamethasone as well as liposomal amphotericin b with transition to itraconazole. immunologic evaluation at the time of initial presentation demonstrated increased mean channel fluorescence for both perforin and granzyme noted in his nk cells. his nk function was decreased; however, he had a normal cd a degranulation assay. his b-cell panel demonstrated low non-switched memory b-cells, low switched memory b-cells and low total memory b-cells. given his extreme immune activation with histoplasmosis, abnormal immunologic testing, and persistent lymphopenia despite resolution of his infection, a primary immunodeficiency next generation sequencing panel was sent. the results demonstrated a pathogenic variant in stat (c. c>t; p.ala val). this single nucleotide variant has been previously shown to be pathogenic (clinvar). abstract/case report text introduction: cytotoxic t lymphocyte antigen- (ctla- ) is known to have an important role as a negative regulator of immune responses, participating in the control of regulatory t cells and effector t cells. in mice its absence is associated with fatal autoimmunity and several ctla- mutations, leading to low or absent ctla- expression, have been shown in humans to be associated with a phenotype that includes hypogammaglobulinemia (with recurrent respiratory infections) and several manifestations of autoimmunity (enteropathy, granulomatous lymphocytic interstitial lung disease, organ infiltration, splenomegaly, autoimmune cytopenias, lymphadenopathy, amongst others), in an autosomal dominant mode of transmission. one of the published mutations, c.c t, that results in an alanine to valine substitution (p.a v), with a highly conserved alanine at that position, had a cadd score of and was associated with the phenotype above, and was shown to be associated with a low expression of ctla- on regulatory t cells and with low ctla- function (reduction of ctla- -mediated transendocytosis). methods: after irb approval, we searched for ctla- mutations present in the biome biobank· biorepository, containing whole exome sequencing data on patients, with data obtained using illumina· v hiseq sequencing platform. sifting through all the ctla mutations in the data, we identified four patients with the c.c t mutation described above. extensive chart review of the four patients was performed. results: four patients were found with the ctla- c.c t mutation. none of them had any of the described phenotypical characteristics of ctla- deficiency. patient is a -year-old male with history of coronary artery disease, atrial fibrillation, stroke, hypertension, brain aneurysm, chronic kidney disease, gout and depression. patient is a -year-old female with history of morbid obesity. patient is a -year-old female with history of hypertension, obesity, pre-diabetes, dyslipidemia and iron deficiency anemia. patient is a -year-old female with history of peripheral artery disease, hypertension, dyslipidemia, chronic kidney disease and lung cancer. conclusion: prior literature has attempted to characterize the clinical penetrance of ctla- mutations, suggesting it to be around %, with that number applying to different mutations in ctla- mutation carriers. we screened a large biorepository of more than thousand patients for ctla- patients and identified four patients that carry one of the best described ctla- mutations, previously validated from a functional standpoint and associated with a severe phenotype. none of the four patients demonstrated any of the previously described phenotypical characteristics, and all four have ages above the median age of onset of years. with the increasing use and broad population application of genetic studies, it is crucial to define the value of identifying presumed pathogenic variants in the absence of the adequate phenotype, with all the prognostic, therapeutic and ethical considerations it may imply. prior case reports of pil patients with b-cell malignancies have discussed treatment regimens with chemotherapy, radiation, and/or surgery, but neither the use nor the outcomes of allogeneic hematopoietic stem cell transplantation (hsct) in the management of recurring b-cell malignancies have been readily reported. case description: a -year-old man with pil and an accompanying history of lymphopenia, hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia was diagnosed with diffuse large b-cell lymphoma (dlbcl) of the liver following a preceding history of burkitt lymphoma of the ileum at years of age and dlbcl of the liver at years of age, in which each malignancy was genetically distinct. in addition, the patient had a history of benign nodular adenomatoid hyperplasia of the thyroid at years of age that required a hemi-thyroidectomy. treatment considerations for the patient included chimeric antigen receptor t-cell therapy, autologous hsct, and allogeneic hsct, in which allogeneic hsct was ultimately pursued. prior to hsct, the patient was lymphopenic ( cells/microliter) with significant t-cell lymphopenia ( cells/microliter) and an increased proportion of memory t-cells ( % of his cd + t cells were cd ro+), as well as hypogammaglobulinemic (igg mg/dl; iga mg/dl; igm mg/dl). immediately following treatment of his dlbcl with rituximab, ifosfamide, carboplatin, and etoposide, the patient underwent a matched-related sibling donor hsct with a preparative regimen of busulfan, thiotepa, and fludarabine. now months status-post hsct, the patient has maintained full-donor chimerism and has no evidence of graft-versus-host disease or malignancy. as expected, hsct has not corrected abnormalities in certain parameters associated with his pil, as he continues to display significant hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia, but he has an improved lymphocyte count ( , cells/microliter). discussion: there is no definitive or curative treatment for pil; furthermore, the genetic etiology of pil remains unknown. supportive regimens to help mitigate or offset manifestations of pil exist, such as adherence to a low-fat diet with medium-chain triglyceride supplementation, but there are no therapies available to prevent or reduce the risk of developing bcell malignancies in this patient population. although previous case reports have detailed successful treatment of b-cell malignancies in pil patients with chemotherapy, radiation, and/or surgery, there are no published consensus guidelines regarding management of b-cell malignancies in the setting of pil, especially if recurrent in nature. for non-pil patients with chemotherapy-refractory disease, or recurrent disease following autologous hsct, allogeneic hsct is a potentially curative option. herein, we describe a pil patient with a history of multiple b-cell malignancies who underwent a successful allogeneic hsct, indicating that allogeneic hsct may be an effective treatment option for similarly affected patients. abstract/case report text introduction diarrhea in young infants is common and generally self-limited. in persistent cases, the differential diagnosis is broad and includes infections, food protein-induced allergic proctocolitis, congenital diarrheas and enteropathies. in addition to monogenic inflammatory bowel diseases, many cellular, humoral, and combined immunodeficiencies should be considered, including but not limited to cvid, ipex and ipex-like phenotypes, lad, dyskeratosis congenita, intestinal lymphangiectasia, omenn syndrome, cartilage hair hypoplasia, cgd, il- axis defects, aid deficiency and wiskott-aldrich syndrome. case presentation a full term infant born after an uncomplicated pregnancy to nonconsanguineous honduran parents presented with non-bloody, non-bilious vomiting and dehydration at days of life. the infant later developed frequent loose stools, some of which were bloody, and failure to thrive. his family and prior medical history, including newborn screen, were normal. an extensive workup was initiated which showed: -persistent and severe anemia with a hemoglobin nadir of . mg/dl -hypoalbuminemia requiring multiple infusions -elevated alpha- -antitrypsin and calprotectin level in stool -profound hypogammaglobulinemia with normal iga, igm and ige for age -normal gross and histologic findings on esophagogastroduodenoscopies and colonoscopies besides a gastric ulcer thought not be the cause of his anemia -normal abdominal imaging including ultrasound, ct angiography and mri -no source of bleeding on meckel scan or exploratory laparotomy -normal dhr assay, g pd level and positive myeloperoxidase stain -immunophenotyping showing t cell lymphocytosis affecting cd + more than cd + compartment, with normal lymphocyte proliferation to mitogens -normal sweat chloride level genetic testing was initiated with a targeted immunodeficiency panel which showed variants of unknown significance in adar, dock , lyst, ptprc and tbx genes, none of which adequately explained his presentation. whole exome sequencing showed that he was a compound heterozygote in the dgat gene. a pathogenic variant c. + t>c (ivs + t>c) was inherited from the father and a likely pathogenic variant c. g>c (p.r p) was inherited from the mother. patient was diagnosed with dgat deficiency, an inborn error of lipid metabolism resulting in protein-losing enteropathy (ple). under gastroenterology's guidance, a low fat diet was initiated, resulting in rapid improvement in stool consistency, weight gain, albumin level and stool alpha- -antitrypsin level. he remains on subcutaneous immunoglobulin replacement therapy for ongoing hypogammaglobulinemia. conclusion: protein-losing enteropathies commonly present with intractable diarrhea and significant laboratory derangements due to malabsorption including hypogammaglobulinemia. as a result of these findings and since many of the etiologies are immunologic in origin, immunologists are an integral part of the evaluation of such cases. in cases where immune system interrogation reveal normal results, genetic testing is crucial in guiding the diagnosis. in our case, whole exome sequencing not only provided the diagnosis but also characterized a variant that was previously of unknown significance as likely pathogenic. dietary management provided rapid improvement in growth and nutritional status. ongoing monitoring will reveal if this management also assists in igg level maintenance and hematologic abnormalities or if even more stringent control of dietary fat will be required abstract/case report text background: foxp gene mutations are associated with immune dysregulation polyendocrinopathy x-linked (ipex) syndrome, a rare xlinked monogenic disease of immune dysregulation and autoimmunity. the classic presentation consists of severe enteropathy, dermatitis, and endocrinopathies (commonly early onset insulin dependent diabetes mellitus). clinical presentation and severity can be variable even in family members with the identical variant. we present a patient with ipex symptomatology and a hemizygous variant in the polyadenylation (polya) signal of foxp that is classified as a variant of uncertain significance (vus). this specific variant was reported in a single case study in which the patient improved after hematopoietic stem cell transplantation (hsct). case presentation: a month-old ex -week gestation boy was admitted with lethargy, hypovolemia, electrolyte disturbances, and acute kidney injury. he developed persistent diarrhea and vomiting after receiving rotavirus vaccine. his family history is significant for early deaths of three maternal unclesone stillborn, one death at months and another at years from unknown gastrointestinal problems. he demonstrated peripheral eosinophilia (to . k/ul), elevated ige, and anemia requiring multiple transfusions. he developed severe enteropathy with hypoproteinemia requiring total parenteral nutrition, continual albumin infusions and maintenance of npo. he had generalized edema, respiratory distress requiring high flow nasal cannula, and repeatedly spiked fevers with negative infectious evaluation. acute kidney injury improved but renal ultrasound showed persistent nephrocalcinosis. endoscopy yielded biopsies demonstrating duodenitis with severe villous atrophy, scanty isolated intraepithelial eosinophils and neutrophils, a few crypts with mucin, reactive epithelial changes and increased lamina propria eosinophils. colon biopsies showed mucosa with focally increased lamina propria eosinophils with scanty neutrophils and surface epithelium without cryptitis. esophagitis with reactive epithelial changes, spongiosis, and many intraepithelial eosinophils was also present. the patient's lymphocytes showed unremarkable proliferation to pha and pwm. cd + cd + t cells demonstrated intracellular foxp expression by flow cytometry. a commercially-available immunodeficiency targeted panel revealed that he was hemizygous for a vus in foxp (exon , c.* a>g non coding). this variant is also referred to as an aauaaa>aauaag or aataaa>aataag change in the polya site. he was also heterozygous for vus at these additional loci: cd a c. c>t abstract/case report text introduction: implantation of allogeneic cultured thymus, partially depleted ex vivo of t cells, can result in naïve t cell development in patients with complete digeorge syndrome (dgs). in a few patients, early and transient skin rash, often characterized as "atypical dgs" or late autoimmune manifestations have been reported following implantation. here we describe a patient with complete dgs who developed immune reconstitution inflammatory syndrome (iris) or atypical dgs following thymus implantation. case description: a female patient was diagnosed at birth with complete dgs due to absent t cell receptor excision circles (trec), hypoplastic thymus, profound hypocalcemia with hypoparathyroidism and cardiac defects. the patient also had microretrognathia, oral motor dysfunction, sialorrhea, recurrent aspirations and reflux requiring a gastro-jejunum feeding tube, low-set ears with right ear microotia, semicircular canals atresia, alopecia and mal-rotated kidneys. prior to thymus implantation, the patient was thriving, had no skin rash, no eosinophilia and no t cells. detailed genetic analyses, did not reveal a cause for her syndrome. at months of age pulmonary aspergillosis was diagnosed presumptively. at months of age the patient received an allogeneic t-cell depleted thymus implant from a male donor, without prior conditioning or post-implantation immune suppressive medications. the procedure was uneventful and the patient returned home after days. results: four months after implantation, a pruritic maculopapular rash appeared on the head and trunk that spread to the extremities including the palms and soles. there was no lymphadenopathy or splenomegaly. an infectious etiology could not be found. eosinophilia and an increase in liver enzymes were noted. there was an increase of cd + and cd + t cells with predominantly memory phenotype, which had been undetectable month earlier. analysis of t cell diversity showed a restricted repertoire with expansion of two v-beta families. there was no evidence of donor cells to suggest graft versus host disease. skin biopsy showed minimal superficial perivascular inflammatory infiltrate composed mainly of cd + histiocytes and rare cd + t cells. the patient was treated with prednisone and cyclosporine. a liver biopsy was performed weeks after initiation of treatment that showed moderate and diffuse peri-portal ductular reaction but no duct associated lymphocytic infiltrate or significant duct epithelial injury or ductopenia. the skin rash rapidly resolved with desquamation, while the liver enzyme abnormalities persisted for two more months. cyclosporine and prednisone were weaned over months. t cell numbers, their response to stimulation and diversity have since normalized, as well as trec and naïve t cell production. the patient is producing appropriate antibodies to protein and polysaccharide vaccines. sixteen months after implantation the patient developed grave's disease with markedly elevated free-t , undetectable tsh and elevated antibodies to the thyroid receptor, which rapidly normalized with ongoing methimazole treatment. the patient is currently months after the implantation and is free of infections, thriving and developing appropriately. conclusions: this patient developed atypical dgs or iris, often associated with autologous and allogeneic hematopoietic stem cell transplants, organ transplants or effective treatment of hiv, after successful thymus implantation for complete dgs. abstract/case report text congenital disorders of glycosylation are a rare group of genetic disorders due to defects in protein glycosylation. phosphoglucomutase (pgm ) is an enzyme necessary for the synthesis of uridine diphosphate n-acetylglucosamine, an important precursor for protein glycosylation. patients with autosomal recessive pgm deficiency have a multisystemic disorder characterized by a neurologic impairment and clinical features classically observed in autosomal dominant hyper-ige syndrome due to stat mutations; including recurrent pneumonias, skin abscesses, elevated levels of ige, and abnormalities in connective tissues and bones. we hypothesized that gp , a highly glycosylated protein and coreceptor of the cytokine il- , would be weakly expressed on pgm deficient cells, due to impaired glycosylation. we studied pgm -deficient patients from kindreds and showed that il- -driven stat phosphorylation was impaired in their pbmcs and ebv-transformed b cells. accordingly, the induction of socs target gene was significantly decreased. in contrast, the patients had normal stat phosphorylation and socs induction downstream of il- , a cytokine whose signaling is independent of gp . flow cytometry and immunoblotting showed significantly lower gp expression in peripheral t-cells and ebv-transformed b cells from pgm -deficient patients compared to healthy donors. we did also show that in vitro inhibition of n-glycosylation, using tunicamycin in ebv-transformed b cell line from healthy donor, alters gp -mediated signaling. collectively, our findings demonstrate that defective glycosylation in pgm -deficient patients results in reduced expression of gp and consequently, impaired gp dependent stat phosphorylation and defective il- signaling. this may account for the overlapping clinical features shared by pgm and stat deficient patients. abstract/case report text introduction: there are no known effective therapeutic modalities for patients hospitalized with moderate to severe acute viral respiratory infections, and treatment is primarily supportive. intravenous immunoglobulin (ivig) has been reported in limited cases to be used in this setting, especially in immunocompromised patients. the primary objective of this retrospective study is to compare clinical and economic outcomes among immunocompromised patients hospitalized with viral respiratory infections who received ivig to those who did not receive ivig at a large academic center hospital. methods: we performed a double-center, retrospective cohort study of all immunocompromised patients who were hospitalized for acute documented respiratory viral infections between and . we divided patients into two groups: those who received ivig therapy for respiratory infections, and those who did not receive ivig therapy. data on age, gender, immune status, viral type, immunosuppression type, respiratory support, microbiological data, length of hospital stay (los), icu los, as well as death and readmission rates were extracted from medical records. in order to adjust for severity bias typically present in observational data such as these, we employed inverse probability weighting (ipw) using all collected baseline covariates. outcomes (death, length of stay in hospital and icu, readmission) were examined using a series of logistic and poisson regression models adjusting for baseline covariates and employing ipw. results: a total of individual hospital admissions were analyzed; patients received ivig and did not receive ivig. there were no significant differences between the two groups in terms of mean age, gender . average age was . , % were female, . % were transplant patients of which . % had lung transplant, . % had liver transplant, . % had bone marrow transplants (bmt), . % had kidney transplant, . % had heart transplant and . % had both solid organ and bmt. . % of patients had a hematologic malignancy, and . % had a primary immunodeficiency. the most common isolated respiratory virus w a s r h i n o v i r u s ( . % ) , f o l l o w e d b y r s v ( . % ) , parainfluenza ( . %) and metapneumovirus ( . %). overall, the use of ivig as associated with a significantly shorter icu length-of-stay, with an (or=- . , p= . ), and a higher hospital readmission rate. in the sub-analysis of patients who received ivig within the first hours of hospitalization (n= ), ivig use was associated with a significantly shorter icu los (or=- . , p= . ), significantly shorter overall hospital los (or=- . , p= . ), and no significant change in readmission rate. conclusions: to our knowledge, this is the first retrospective cohort analysis evaluating the effect of ivig in immunocompromised patients hospitalized with respiratory viral infections. the results suggest that immunocompromised patients receiving ivig may have a shorter hospital and icu los, especially if ivig is provided within the first hours of admission. this may result in reduced healthcare costs. this study is limited by its retrospective nature, and the potential bias that patients treated with ivig are sicker to start with. future prospective studies are suggested to further evaluate these findings. ( , ) . the most common precipitant in children is medication, followed by infection ( , ) . although a clear association between mycoplasma pneumoniae and sjs has been established, there is a scarcity of literature exploring the role of this infection in recurrent sjs in children ( ) ( ) ( ) . case presentation: a -year-old female with prior history of sjs was admitted for mucosal and skin lesions in the setting of community acquired pneumonia. her past medical history included sjs with eye involvement, secondary to mycoplasma pneumoniae (ig m positive), occurring five years prior to this admission. she also had frequent episodes of acute otitis media and sinusitis in early childhood. family history was negative for immunodeficiency. her clinical presentation included respiratory symptoms and fever for days treated with ceftriaxone, followed by cefdinir and levofloxacin. her fever improved the day prior to admission, but she developed conjunctival injection, ocular pain, and ulcerative lesions in her mouth and nares. on physical examination, she had low grade fever with mucosal lesions including conjunctival erythema with serous discharge, painful blisters and denudated skin in lips, perioral area, nares, tongue and oropharynx. initial testing included negative blood hsv pcr, blood culture, rapid antigen testing for group a streptococcus and influenza a/b, and elevated crp in . mg/dl and esr mm/hr. right lower lobe p n e u m o n i a w a s c o n f i r m e d w i t h a c h e s t r a d i o g r a p h . nasopharyngeal pcr and serum igm were positive for mycoplasma pneumoniae. she had a mildly elevated anticardiolipin igm ( mpl), a mildly decreased c ( mg/dl) and a negative ana. she was diagnosed with recurrent sjs secondary to mycoplasma pneumonia infection. she completed treatment with levofloxacin for mycoplasma pneumonia, and received cyclosporine and high-dose methylprednisolone. she had bilateral amniotic membrane transplantation to prevent corneal ulceration . she was discharged after clinical improvement, and recurrent oral lesions were noted at followup. immunological work up as an outpatient revealed normal serum immunoglobulins, normal lymphocyte subsets and low pneumococcal titers with adequate response post-vaccination. sjs secondary to mycoplasma pneumonia infection has predominance of mucosal involvement over rash, which was observed in our patient ( , ) . some case series reported a recurrence of sjs up to % within a -year follow up. almost half of patients with recurrent sjs developed multiple sequelae ( , ) . early diagnosis of sjs, especially in those with prior history of sjs, helps to provide appropriate supportive care, monitoring of complications and treatment of possible superinfections ( , ) . conclusions: there is limited information in the literature regarding the role of mycoplasma pneumoniae associated recurrent sjs in children. it is possible that these episodes are triggered by and/or immune predisposition. there is ongoing discussion regarding whether these clinical presentation should be labeled sjs secondary to mycoplasma pneumonia infection or, depending of the skin involvement, m. pneumonia-associated mucositis (mpam) and m. pneumonia-induced rash and mucositis (mirm) ( , , ). mycoplasma should be treated appropriately in patients with recurrent sjs. abstract/case report text background: growing access to genetic testing has facilitated the genetic evaluation of primary immunodeficiencies but has also greatly increased the number of variants of uncertain significance (vus) encountered in clinical practice. interpreting the significance of vus requires multiple lines of evidence. w e d e s c r i b e a n e u t r o p e n i c i n d e x p a t i e n t w i t h hypogammaglobulinemia, unusual hpv susceptibility, and dual heterozygous pathogenic loss-of-function nfkb and heterozygous missense cxcr vus. family analysis showed the nfkb variant was inherited from his mother, while the novel cxcr variant was present in his father and sister. all four patients presented with recurrent infections, warts, and hypogammaglobulinemia. (figure ) the nf-κb gene encodes p /p transcription factor of the canonical nf-κb pathway, the most common autosomal dominant monogenic cause of common variable immunodeficiency (cvid). cxcr is a g-protein-coupled chemokine receptor with cxcl as cognate ligand. autosomal dominant pathogenic gain-offunction cxcr variants lead to impaired receptor downregulation and retention of neutrophils and other leukocytes in the bone marrow defining whim (warts, hypogammaglobinemia, infections, and myelokathexis) syndrome. all cxcr pathogenic variants truncate the carboxyl-tail of the cxcr receptor, a region responsible for receptor internalization, with the exception of one missense non-truncating variant p.e k. case series: the proband (p ) is a -year-old male with history of recurrent bacterial respiratory tract infections, warts, moderate neutropenia, thrombocytopenia and hypogammaglobulinemia requiring immunoglobulin replacement therapy (igrt). bone marrow biopsy didn't show myelokathexis. next-generation panel sequencing identified a novel heterozygous missense cxcr (c. c>a, p.s y) vus. the serine residue is highly conserved up to zebrafish. this variant was present in heterozygous form in two cases in gnomad database ( , alleles). additional whole-exome sequencing revealed a heterozygous pathogenic nfkb variant (c. dup, pa sfs* ) located in the nterminal rel homology domain, consistent with nfkb loss-offunction. both, the patient's sister (p ) and their father (p ), carry the heterozygous cxcr vus but not the pathogenic nfkb variant, and have history of warts, hypogammaglobinemia, and recurrent infections. the hpv susceptibility is particularly striking in p manifesting by genital warts and hpv-positive oropharyngeal cancer. bone marrow evaluation didn't identify myelokathexis in p (p is pending). the mother of the index case (p ) has cvid requiring igrt and immunomodulation. she shares the nfkb variant with p but is negative for the cxcr vus. extensive t and b cell phenotyping revealed low class-switched memory b cell count ( - counts/ul) in all subjects, and loss of transitional and mature naïve b cells in p and p with nfkb variant. proband b cells showed the highest tendency for apoptosis ( - %) within the family. we describe members of a family with similar presentation (infections, hypogammaglobinemia, warts), however variable combination of nfkb and cxcr variants, where either genetic defect or their combination could explain the clinical phenotype. biochemical consequence of our novel cxcr variant is pending. as the proband showed the most severe immune phenotype and neutropenia, we hypothesize that cxcr has a synergistic effect on nkfb loss-of-function. the contribution of cxcr vus of the clinical phenotype of the two other family members is yet to be determined. background: the yield of diagnosis by exome sequencing for some primary immunodeficiencies (pid) has been less than the typical diagnostic rate for clinical exome analysis (~ - %). the relatively low diagnostic rates for certain subtypes of the pids may be attributed to variable expressivity and/or an incomplete understanding of the genetic basis, among others. additionally the extent of multiple diagnoses and phenotypyic expansion in pid is not well explored. cohorts with highresolution clinical and genetic data are instrumental for exploring these questions. we evaluated the use of human phenotype ontology (hpo)annotated datasets to systematically address the prevalence of these issues using a cohort of individuals with pid who participated in research exome sequencing at the niaid. results: we generated a phenotype dataset of individuals with pids by annotating the clinical features of these subjects obtained from electronic health records (ehr) with hpo terms. exome sequencing of these individuals identified probands with a pathogenic or likely pathogenic (p/lp) variant in a gene associated with the respective clinical presentation. we identified probands where the same gene harbored a p/lp variant in at least three unrelated individuals. we used the clinical and genetic data of individuals in the following areas: ) we identified p/lp variants in aire, pik cd, nlrp , fas, ctla , gata , cybb, stat and tnfrsf b in at least ten patients that explained their clinical presentations. this dataset allowed us to characterize variable expressivity of diseases associated with these genes by capturing the variability in the observed hpo terms among probands with p/lp variants in the same gene. dimensional reduction of clinical features of probands allowed us to cluster patients sharing similar phenotypic profiles. we found clinical presentation of individuals with monoallelic p/ lp variants in aire were relatively less variable and clustered more compactly compared to that of individuals with gata variants. ) the extent of multiple diagnoses in pid is not well explored. the benchmark cohort we developed allowed us to identify candidates for multiple diagnosis or phenotype expansion by comparing the phenotype profile of each patient expressed in hpo terms to the hpo terms typically observed for a given pid. for example, we identified gain-of-function pathogenic variant in pik cd in a patient that explained the clinical features of the pid observed in this patient. however, the patient also displayed developmental delay, congenital hemiplegia, cerebral palsy and absent speech. these features are not known to be associated with pik cd variants, making this individual a candidate for > genetic diagnoses. conclusions: we developed a benchmark dataset where clinical features of patients were described using hpo terms. this dataset allowed us to quantify variable expressivity for certain pid subtypes and to systematically identify potential candidates for multiple diagnosis or phenotypic expansion. abstract/case report text warts, hypogammaglobulinemia, recurrent infections and myelokathexis syndrome is a rare combined immunodeficiency due to autosomal dominant gain-of-function mutations of cxcr chemokine receptor. the late diagnosis of whim syndrome in two ukrainian adolescents highlights the diagnostic challenges in this disease. patient , year-old girl, had recurrent pneumonia since the first year of age; overall she had episodes of pneumonia. she has suffered from chronic bronchitis for last several years. she had recurrent otitis media and chronic pyelonephritis. neutropenia was revealed when she was year old. during episodes of bacterial infections she occasionally had normal value of neutrophils. the girl does not receive any treatment. patient , year-old boy, had three episodes of pneumonia when he was , and year old. others symptoms include recurrent herpetic infection, warts on the hands. since years of age he has haven persistent low neutrophil counts. the child was followed by hematologist and since years of age he has received g-csf ( mg/kg) twice a month. both children have leukopenia - cells/mm , neutropenia - - cells/mm , lymphopenia - cells/mm , low number of bcells - - cells/mm . hypogammaglobulinemia was not prominent in both children, they have slightly decreased level of igg ( , g/l), normal level of igm ( , - , g/l), patient has low level of iga , g/l. patient does not have protective level of antibodies to diphtheria and tetanus anatoxin, and anti-hbs antibodies were absent despite complete immunization. bone marrow aspirate revealed hypercellular marrow with granulocytic hyperplasia which was characterized by hypersegmented nuclei and cytoplasmic vacuolization of neutrophils. on molecular analysis of cxcr , heterozygous mutation c. c>t (p.arg *), known as r x mutation, was detected in both patients, confirming the diagnosis of whim syndrome. replacement therapy with intravenous immunoglobulin was started in both children together with antibacterial prophylaxis and g-scf. vaccination with -valent vaccine against hpv infection was recommended for both patients. whim syndrome is very rare immunodeficiency but may be underdiagnosed. the awareness about rare forms primary immunodeficiency is very important in clinical practice for early diagnosis and treatment. methods: clinical providers recruited from nicer institutions electively completed web-based survey questions related to provider characteristics as well as initial diagnostic evaluation of itp, aiha, ain and es via securequestionpro® software. likert scales ranging from ("rarely" < %), ("sometimes" to %), ("half the time" % to %), ("frequently" to %), and ("almost always" to %) were used to ascertain frequency of evaluation for each diagnostic study. statistical analysis and plotting was done using rv . . . plots were created using packages ggplot , v . . and ggiraphextra v . . . mean likert scale scores were calculated for each study for each suspected disease and plotted on radar charts. results: the survey was completed by providers, including hematology/oncology ( . %), rheumatology ( . %), allergy/ immunology ( . %) and other sub-specialties ( . %). a slight majority of physicians ( %) were fellows or within years of graduation; physician extenders and clinical pharmacists were also respondents. the majority ( . %) of respondents indicated that ≤ new immune-mediated cytopenia patients were seen at their institution annually. the vast majority of respondents ( . %) reported evaluating ≤ new es patients per year at their institution with % evaluating ≤ cases annually. collated data from all respondents showed that in all disease states, the primary evaluation was focused on peripheral destruction mechanisms; the majority of patients are only "sometimes" or "rarely" evaluated for bone marrow failure syndromes, connective tissue disease, immunodeficiency and non-malignant lymphoproliferative disorders, but when done were more likely in es ( figure ). evaluations were biased by sub-specialty with higher degrees of connective tissue focus by rheumatology and immunodeficiencies by allergy/immunology (table ) . genetic sequencing was "frequently" or "almost always" sent in . % of itp, . % of aiha, . % of ain and . % of es p a t i e n t s . p e r s o n a l o r f a m i l y h i s t o r y o f a u t o i m m u n e / hyperinflammatory disease, malignancy or cytopenias most strongly influenced the decision to send genetic testing. lack of insurance coverage/negative financial impact on the patient and concerns about the inability to resolve variants of uncertain significance were the biggest barriers for obtaining genetic testing. conclusions: current practices in the evaluation of immunecytopenias are heterogeneous by sub-specialty and globally limited in scope with few patients being evaluated for underlying etiologies. in particular, despite a known high frequency of pathogenic variants in es, less than a third of patients are undergoing sequencing, highlighting a need to reduce barriers to genetic testing. development of a consensus guideline with multi-disciplinary engagement to harmonize an optimal evaluation for patients with immune-mediated cytopenias is needed. interferon regulatory factor- (irf ) binding protein- (irf bp ) was originally identified as a transcriptional co-repressor of irf ( ). mutated irf bp was identified in a -member family with recurrent sinopulmonary infections, progressive hypogammaglobulinemia, and poor response to protein vaccines ( ) . we have now identified additional families ( subjects) with irf bp mutations. clinical histories show an expanded phenotype with / having chronic gastrointestinal disease; with gastrointestinal manifestations as the initial clinical complaint. five had granulomata in liver(x ), spleen, lung(x ) and gastrointestinal tract. five out of six tested had poor pneumococcal vaccine responses and four patients reported viral infections including varicella zoster(x ), influenza a and sapovirus. irf bp is a amino acid protein containing a highly conserved cterminal protein-protein interaction ring domain (rd). constraint metrics from gnomad indicate mild tolerance to missense changes and intolerance to loss-of-function alleles. we identified categories of mutations: rd mutation or deletion (n= patients), null alleles (n= ) and non-rd missense changes (n= ). functional studies assessing the ability to affect nfatdriven luciferase expression were performed. rd mutations ( / ) had more profound loss-of-repression than wild-type, while missense changes had lesser, but still measurable effects. further, mutation categories and functional studies correlated with clinical phenotypes. of patients with rd mutations, / had infections as presenting symptoms, / tested had hypogammaglobulinemia and / were diagnosed with cvid. one patient with a missense rd mutation had only an infectious phenotype (pulmonary mycobacterium avium) with slight decrease in immunoglobulins; in functional studies this mutation had the least effect of the rd mutations. haploinsufficient patients reported respiratory infections ( / ), recurrent urinary tract infections ( / ), gastrointestinal disease ( / ) and hypogammaglobulinemia ( / ). in contrast, / patients with non-rd missense changes presented with gastrointestinal complaints while only patients had infections (recurrent bronchitis, shingles). gi disease prevalence is consistent with high levels of irf bp expression in the colonic crypt cells (human protein atlas). to confirm this, immunohistochemical staining of colon biopsies from two patients was performed, identifying epithelial and glandular cells of the colon. irf bp is involved in multiple processes, including the negative regulation of nfat signaling( ), tcr signaling( ), inflammatory macrophages ( ) , and pd-l transcription ( ) . interaction with the glucocorticoid receptor affecting anti-inflammatory and metabolic transcription ( ) has also been reported. these observations highlight the irf bp response to type-i interferons (irf ) and tcr stimulation (nfat), regulation of inflammatory macrophages and co-regulation of glucocorticoid receptor mediated signaling. the expanding role of irf bp in multiple biologic systems correlates with the broad clinical presentation we observed in our patients. further studies utilizing irf bp mutation knock-in mice will help characterize the gastrointestinal, lung and immune pathology seen in our cohort. abstract/case report text common variable immunodeficiency (cvid) is a disorder of antibody deficiency arising from over genetic lesions. the clinical presentation of patients with cvid varies from recurrent, severe infections to autoimmunity. the immune dysregulation in cvid is especially difficult to treat and the lifespans of patients suffering from autoimmunity are much shorter than those without such complications. unfortunately, we have no way to identify which patients fall into which categories, or even know how many sub-categories of cvid there are. therefore, the field requires a method to classify patients into categories to precisely recognize and aggressively treat the more severe phenotypes. we address this goal by integrating analyses of patient exomes with analyses of cellular signaling. by analyzing stimulation assays with phospho-protein mass cytometry and high-dimensional data analytics, we aimed to elucidate signaling and phenotyping deficiencies in patients with cvid. importantly, our panel identifies all circulating immune cell subsets in whole blood. in eosinophils, we found amplified responses of pp , pstat , and cleaved caspase- in response to tlr / stimulation. we found additional amplified responses of pstat and pstat in cd lo monocytes. this finding suggests a previously unidentified role for eosinophils and cd lo monocytes to contribute to the pathophysiology of cvid. we found abormal numbers of memory b cell counts, total switched b cell counts, and igm+, cd + b cell (plasmablasts) counts between cvid patients and healthy controls. cd expression on b cells was significantly reduced in cvid patients as well. these b cell results mirror findings from prior, seminal studies on cvid. notably, we have found higher pd- expression in the effector cd t cells of patients. integrating phenotype data, genetic analysis, and mass cytometry data will provide a deeper understanding of each patient's phenotype and how the are clustered. we also expect that a better understanding of alterations in the exomes and functions of the circulating immune cells of cvid patients will lead to new therapeutic approaches. abstract/case report text objectives: primary atopic disorders are monogenic disorders leading to profoundly dysregulated allergic responses. studying patients with these disorders has been instrumental in expanding our understanding of the pathogenesis of allergic inflammation with therapeutic implications for common polygenic versions of allergic disease. clinical findings: we have identified a now -year old boy who presented with severe eczema, extremely high blood eosinophil counts ( . x cells/l, normal range: - . x cells/l) after birth and very high serum ige levels ( υg/l, normal range: - ug/l) since birth. known allergic disorders and parasitic infections are ruled out. given the extreme phenotype, whole exome sequencing was performed on the trio of patient and parents, and the patient was found to have a homozygous mutation in the evolutionarily conserved fibronectin iii domain of the osmr gene (c. t>a, p.v d) (figure ). osmr encodes oncostatin m receptor-beta, a component of both the osm type ii receptor and the il receptor, and is important for keratinocyte cell proliferation, differentiation, apoptosis and inflammation. mutations in osmr have been reported in association with familial primary localized cutaneous amyloidosis, however this condition was ruled out in this patient through skin biopsy which showed no amyloid deposits. methods and results: we modelled the c. t>a osmr mutation in hek cells and observed a loss of expression of the osmr receptor on the cell surface (with normal intracellular protein levels). this observation was mirrored in primary fibroblasts obtained from the patient. signal transduction through phosphorylation of stat and stat and gene expression (il and ccl measured via qpcr) was absent after stimulation with osm in patient fibroblasts. these signaling defects were rescued using a lenti-viral transduction approach to introduce the wild-type (wt) osmr gene. whole transcriptome analysis using rna sequencing confirmed that osm mediated jak-stat signalling pathways were deficient in the patient fibroblasts and were rescued after lenti-viral transduction of wt osmr. rna sequencing analysis also suggested significantly enhanced expression of genes in the nf-κb signalling pathway (e.g.: il and cxcl ) and decreased expression of genes in the tgf-β signalling pathway (e.g.: smad and smad ) in patient fibroblasts at baseline. this was also rescued upon lentiviral transduction. conclusion and future directions: our findings shed light into the disease mechanism of a novel primary atopic disorder, caused by a homozygous missense mutation in osmr. abstract/case report text -year-old caucasian female presented to immunology clinic with hypereosinophilia, eosinophilic esophagitis, peptic ulcer disease, severe gi bleeds, and chronic hepatitis. healthy throughout childhood, with minimal infectious history. in adolescence developed chronic severe myalgias and nsaid overuse, to which the peptic ulcer disease and bleeding were attributed. parents healthy and non-consanguineous. son with severe bleeding episodes and small stature. on exam she weighed lb, bmi . sclerae anicteric. tongue deeply furrowed. cervical nodes palpable. heart and lung exam normal. no hepatosplenomegaly. no clubbing of the digits or edema. skin was clear. wbc , /ul, eosinophils /ul, hemoglobin g/dl, normal platelet count. however, platelet aggregation testing abnormal. bone marrow normocellular, and flow cytometric and molecular analysis did not show hematolymphoid malignancy, primary hypereosinophilic syndrome, or systemic mastocytosis. lymph node biopsies did not show lymphoma or aberrant t cell populations. noted to have chronically elevated creatine phosphokinase, ranging from - u/l over two years at our institution. deltoid muscle biopsy showed non-specific myelopathic changes. an adult dystrophy immunostaining panel was normal. ultrastructure examination showed no abnormal storage material. a genetic panel for metabolic myopathies failed to reveal a cause. total igg, iga and igm normal. ige elevated at ku/l, and igg subclasses showed igg elevated at mg/dl. flow cytometry showed normal t, b and natural killer cell numbers. normal proportions of naïve, mature and activated t cells. vaccine response assessment was normal. evaluation for autoimmune/rheumatologic diseases was negative. liver biopsy demonstrated findings consistent with primary or secondary sclerosing cholangitis (without increased igg staining). given her inflammatory phenotype, additional genetic analysis was sent, assessing for primary immunologic disorders. this identified heterozygous variants of uncertain significance in ctla (c. t>a; ps t), zap (c. c>g; p.d e), and stim (c. t>c; p.l s). analysis of the ctla variant in vitro revealed that it was expressed normally. foxp expressing regulatory t cells were present in normal proportions in vivo and appeared phenotypically normal. this variant was found in her unaffected father. the zap variant is present in population databases (rs , exac . %), and was felt unlikely to be clinically relevant. the stim l s variant, although not shown previously in human patients, has been previously shown in vitro to be a gain of function mutation [ ] [ ] [ ] . furthermore, familial analysis revealed that this was a de novo mutation arising in the patient, and present in her son. humans with other gain of function mutations in stim and the orai channel it activates have overlapping syndromes including storkmorken syndrome, tubular aggregate myopathy and york platelet syndrome, characterized by chronic myopathy and platelet aggregation defects [ ] . the stim l s mutation is predicted to cause constitutive stim activation and calcium influx and likely provides an explanation for the patient's chronic myopathy and abnormal platelet aggregation. neither eosinophilic disease, nor cholangitis, have been described previously in stim gain of function-related diseases. it is unclear whether these issues are related to this novel stim mutation, or to other genetic or environmental influences. treatment of diseases caused by overactive crac channels is challenging as no pharmacologic inhibitors are yet clinically available. nomid/cinca syndrome is one of the periodic syndromes associated with cryopyridines. it is a defect in the innate immune system causing excessive activation of the inflammasome, with consequent il- secretion and neutrophil recruitment. clinically, damage occurs to organs such as the skin (neutrophilic urticaria), central nervous system (meningitis and deafness) and joint (arthritis). levy et al. ( ) evaluated a large series of patients and median onset age was . years, while the median age at diagnosis was years, although the symptoms initiate in the first days of life. treatment includes corticosteroids, which act by nonspecifically blocking all inflammatory cytokines, or by blocking il- specifically. if early diagnosis and treatment of the disease is not made, natural evolution leads to motor and adaptive disability and death in % of cases already in adolescence due to infection, neurological complications or secondary amyloidosis. we report a -month-old male child from nonconsanguineous parents who presented shortly after birth, multiple scaling and erythematous lesions throughout the body, evolving with following symptoms: abdominal abscess, hepatitis, meningitis and pioarthritis. laboratory tests showed elevation of inflammatory tests (esr, crp, amyloid protein a) and leukocytosis. the diagnosis was suspected at the nursery where the patient remained hospitalized for days. a personalized multigene panel was requested. it was identified the variant p.gly val, heterozygous for nlrp gene, not described in the literature, confirming the diagnosis of cinca/nomid syndrome. after discharge, it was introduced prednisolone ( , mg/kg/day) and antiinterleukin- (il- ). after the second dose, skin lesions and joint edema regressed, weight gain, and neuropsychomotor development improved. this case reports a very early diagnosis of nomid/cinca syndrome. it warns neonatologists and pediatricians about the need of precocious recognition of the syndrome, probably improving the prognosis of the patient. professor/university center health abc abstract/case report text background: leprosy affects more than , people worldwide. brazil represents the rd. country in the world in leprosy frequency and maranhão state is an hyperendemic region. the city of imperatriz (ma) stands out as a reference center in the care of these patients. according to few reports, lectin pathway of complement system may play a role in susceptibility to leprosy. mannose binding lectin (mbl) and ficolins (fcns) recognize patterns of sugars and acetylated residues (pamp), respectively, in a wide variety of pathogens, including m. leprae. high levels of ficolins and mbl may act unfavorably promoting the spread of m. leprae. the present study evaluated the role of ficolin and mbl in m.leprae patients and contacts. methods: a cross-sectional case-control analytical study was carried out, evaluating clinical and epidemiological data and serum levels of mbl and fcn (elisa) from july to april . the study was approved by ethics committee and informed consent forms were signed before sample collection. data analysis was performed using the spss . for windows statistics program. results: we evaluated serum samples ( patients and healthy family contacts), . % were female, % under years old, % african-brazilian, % of the families had more than contacts at home. clinical data showed multibacillary forms in . %; dimorphic ( %) and virchowian clinical forms ( . %), up to affected nerves in ( . %) and more than lesions in ( . %). it was observed that ( . %) had a reaction, being type ( %) more predominant. disability grade was found in patients ( . %). in children under years, . % were multibacillary, . % dimorphic and % undetermined; ( . %) also had reactions, % type reaction and degree of disability in . % of children with the disease. the evaluation of serum fcn and mbl levels for the patients (n = ) and contacts (n = ) were . ng/ml and . ng/ml, (p = . ), and . ng/ml (p) and . ng/ml (c) (p = . ), respectively. there were lower values of fcn in patients with type reaction (sudden and intense inflammatory processes) versus no reaction ( . ng/ml vs . ng/ml) (p = . ) and in patients with disability grade (severe sequelae) versus disability grade ( . ng/ml vs . ng/ml) (p = . ). higher fcn values was observed in patients with no disability ( . ng/ml) (p = ). mbl concentrations were higher for patients above years in comparison with patients below that age ( . ng/ml vs . ng/ml)(p = . )) and correlated with the occurrence of a multibacillary clinical form. conclusions: mbl and fcn levels were not different in the patients and contacts of m. leprae, nevertheless the presence of severe forms with sequelae (reaction type and disability grade ) were associated with lower levels of fcn . in addition, it is possible that lower mbl levels could influence the higher frequency of multibacillary disease below years old. abstract/case report text introduction: hyper ige syndrome (hies) is a primary immunodeficiency characterized by elevated ige levels. symptoms can range from severe eczema, recurrent skin infections or pneumonias, and typical dysmorphic facies. there have been wide non-immunologic presentations in patients with hies, including retained primary teeth, scoliosis, craniosynostosis, arterial aneurysms and joint hyperextensibility. an association between hies and autoimmune hemolytic anemia (aiha) has further been described in the literature. however, there have been no reported cases of hies in association with iron deficiency anemia and concurrent pica. we present a unique case of a patient with a history of eczema, recurrent skin infections and pica found to have hies and iron deficiency anemia. case presentation: a -year-old boy with a history of allergic rhinitis presented to the allergy & immunology clinic for evaluation of chronic eczema and recurrent skin infections. the patient had a history of multiple hospitalizations requiring intravenous antibiotics for cellulitis and superinfected eczema since he was an infant. symptoms were refractory to the use of multiple skin barrier ointments and oral antihistamines. his mother further noted that for the past two months prior to initial evaluation, he developed a fixation with eating crayons, baby powder and chewing on drywall. physical exam was notable for a dysmorphic face, broad based nose, pale nasal mucosa with ample clear discharge, high-arched palate and lower incisor supernumerary teeth. his skin was characterized by generalized dryness, lichenification and scaly desquamation with boils on extensor surfaces of knees and elbows. initial screening for hies via t-helper functional assay was consistent with decreased expression of il- . genetic testing revealed stat s g missense pathogenic variant consistent with hies. cbc was also notable for decreased hemoglobin at . g/l and mcv of fl. patient was diagnosed with concurrent hies and pica in the setting of iron deficiency anemia. iron supplementation was started and patient's pica improved. discussion and conclusion: our patient with hies had a peculiar initial presentation with the classic signs and symptoms of hies and pica. the diagnosis of hies can often be delayed due to the wide range of clinical presentations. to our knowledge, the association of hies with iron deficiency anemia and pica has been underreported in literature. screening for anemia should be considered when evaluating patients with hies in order to rule out comorbid iron deficiency anemia which can be easily treated with iron supplementation. abstract/case report text introduction: common variable immunodeficiency is a primary immunodeficiency with variable and diverse phenotypic presentations. the two main phenotypes include a group which primarily exhibits recurrent infections and a group with or without infections and primarily inflammatory and autoimmune complications. the latter, may lead to a delay in diagnosis and is associated with poorer outcomes and higher morbidity and mortality. ( ) another group of patients present with t-cell defects, lung disease, autoimmunity, and infections and may be diagnosed as having cvid but instead can have mutations in lrba or pi kinase. this subset of patients has been referred to as "cvid-like" in the literature. ( ) case presentation: patient is an year old female who initially presented to an outside facility due to days of fatigue, fever, and abdominal pain. upon presentation, she was found to have massive splenomegaly, hepatomegaly, and an abnormal chest x-ray showing mediastinal lymphadenopathy and pleural effusion. laboratory results demonstrated pancytopenia, hypogammaglobulinemia, and low b cells, t cells, and nk cells via flow cytometry. she was transferred to our institution for further work up. she did not have any prior history of recurrent infections, asthma/lung disease, or autoimmune conditions. initial ct of the chest was consistent with granulomatous lymphocytic interstitial lung disease. patient was diagnosed with common variable immunodeficiency with granulomatous lymphocytic interstitial lung disease and was treated initially with high dose ivig, corticosteroid taper, rituximab, and imuran. she had interval worsening of pft and lung disease as shown by ct scan. genetic panel for cvid and related conditions revealed variants of unknown significance. one heterozygous mutation in blnk gene (c. g>a) and one heterozygous mutation in lrba gene (c. g>a). she was started on infliximab with plans to repeat ct scan in months. discussion: mutations in both blnk and lrba have been associated with primary immunodeficiency. mutations in blnk, which is located on chromosome , have been associated with autosomal recessive agammaglobulinemia. homozygous or compound heterozygous mutations in lrba on chromosome , can lead to lrba deficiency which encompasses a wide range of clinical presentations including hypogammaglobulinemia, autoimmune disease, inflammatory bowel disease, antibody deficiency, organomegaly, and recurrent infections. ( ) without genetic testing, the clinical presentation can be difficult to distinguish from common variable immunodeficiency. the patient presented has clinical features that can be seen with mutations in both blnk and lrba, however she is heterozygous for both mutations. further analysis, including measurement of lrba protein expression, is needed to further define her underlying immunodeficiency so appropriate treatment can be administered. abstract/case report text a month-old, previously healthy, unvaccinated male presented with one week of diarrhea and cough and was admitted for dehydration and hypoxemia. his mother and sister both had a history of incontinenti pigmenti (ip). on physical exam, he was alert, afebrile, with tachypnea and subcostal retractions. enterovirus/rhinovirus and parainfluenza were detected, but he became progressively hypoxemic and eventually required intubation and high-frequency oscillatory ventilation. chest x-ray showed multifocal bilateral airspace opacities. empiric treatment for pjp with trimethoprim/ sulfamethoxazole and glucocorticoids was started. tracheal aspirate pcr confirmed p. jiroveci. hiv rna pcr was negative. ivig was started due to suspicion for primary immunodeficiency. although his respiratory status gradually improved, he subsequently developed multiple skin lesions. skin biopsy grew mycobacterium szulgai. m. szulgai osteomyelitis of the right fibula and the left nasal bone was also detected, indicating hematogenous spread of the infection. he was started on four-drug anti-mycobacterial therapy and interferon-gamma (actimmune) at doses ranging from μg/m^ three times weekly to μg/m^ qod. immune work-up revealed t-cell lymphopenia [cd +/cd + /μl ( - , /μl) and cd +/cd + /μl ( - , /μl)] with an abnormally increased proportion of memory cd t-cells compared to naïve cells for age. b-cell numbers were normal, and nk cells were decreased [cd +cd +/cd - /μl ( - /μl)]. nk cell lytic function by k lysis was normal, whereas cd a degranulation was decreased. the serum igm level was normal [ mg/dl ( - mg/dl) whereas iga [ mg/dl ( - mg/dl)] and igg [ mg/dl ( - mg/dl)] were elevated. mononuclear cell cytokine response to ligands for tlr -tlr , tlr -tlr , tlr , tlr , and tlr -tlr was normal. dna sequencing revealed a novel nonsense mutation in exon of the ikbkg (p.gln ter (q x) (cag>tag): c. c>t, confirming the diagnosis of nemo deficiency, which was suspected based on the infectious disease presentation and the maternal history of ip. the diagnosis was further supported by signs of ectodermal dysplasia of teeth that appeared starting at months of age. he underwent hsct using bone marrow from a / matched unrelated donor after conditioning with atg, busulfan, fludarabine and rituximab. actimmune therapy was continued until days prior to transplant. for gvhd prophylaxis, he received tacrolimus and low-dose methotrexate. he achieved full donor chimerism post-transplant and has had no significant gvhd. interesting features of this case include the prominence of ip in mother and sister, which is usually due to female heterozygosity for an ikbkg null allele. such null alleles when inherited by the male fetus are embryonic lethal. our patient's nonsense mutation would be expected to result in severely impaired ikbkg protein expression and function. however, the fact that he had was born at term and initially was healthy coupled with his preservation of normal tlr function suggests that his ikbkg allele is likely to be a hypomorphic mutation. studies are in progress using ebv-transformed b-cell lines from the patient to evaluate ikbkg expression and function. also of interest, our patient was able to tolerate relatively high doses of interferon-gamma therapy without inflammatory side effects or an adverse impact on engraftment or gvhd. abstract/case report text background: primary atopic disorders are caused by genetic mutations that skew the immune system towards severe allergic disease. germline gain-of-function (gof) mutations in jak are a newly described monogenic cause of severe atopy, with affected patients demonstrating profound eosinophilia and allergic inflammation. our initial report of this novel condition identified a dramatic clinical response to the combined jak / inhibitor ruxolitinib. we aimed to determine the long-term clinical response to ruxolitinib in patients carrying a germline jak gof mutation, and to characterize the effect of enhanced jak signaling on t lymphocyte effector functions and hematopoiesis. methods: clinical outcomes were evaluated in two pediatric patients carrying the c. c>a (p.a d) gof mutation in jak after . years of ruxolitinib treatment. t cell phenotyping was performed using extracellular surface marker and intracellular cytokine staining by flow cytometry, and by gene expression signature profiling of rna sequencing data. to evaluate the effect of enhanced jak activity on myelopoeisis, we reprogrammed jak gof patient-derived peripheral blood mononuclear cells into induced pluripotent stem cells (ipsc) and performed directed myeloid differentiation. rna sequencing was performed on rna collected during ipsc myeloid differentiation and from whole blood of affected patients before and after ruxolitinib treatment. results: long-term use of ruxolitinib was associated with improved growth, reduced eosinophilia, and control of allergic inflammation without significant infectious complications, however, anemia represented a dose-limiting adverse effect. t cell immunophenotypic analysis revealed severe t helper (th) cell skewing towards a th phenotype preruxolitinib treatment, in keeping with the allergic clinical manifestations. analysis of myeloid differentiation revealed an increased myeloid to erythroid ratio in colonies derived from jak gof ipscs compared to controls. rna sequencing analysis of jak gof human whole blood and ipscs compared to controls revealed upregulation of cytokine and cytokine receptor genes implicated in allergic inflammation and early eosinophil precursor commitment, including csf- and the interleukin- receptor. reactome pathway analysis of genes upregulated in both jak gof ipsc and whole blood compared to controls showed enrichment of several pathways including interferon alpha/beta, interleukin- /- and interleukin- signaling. conclusions: this work demonstrates a critical role for jak in atopic immune dysregulation, specifically driving a th phenotype and eosinophilia. combined jak / inhibition can reverse much of the allergic inflammation, with dramatic clinical effects. this has important implications for our understanding of the pathogenesis and potential therapeutic targets for early life allergic immune dysregulation. had severe combined immunodeficiency (scid) and/or severe disease in association with their combined immunodeficiency (cid) necessitating haematopoietic stem cell transplantation (hsct). we present clinical and laboratory features of new zealand patients from the same family with a novel heterozygous missense variant in rac [c. t>g, p.ile ser (i s)]. the index patient (p -age y, m) has a history of infectious gastroenteritis, staphylococcal aureus conjunctivitis, recurrent otitis media and recurrent herpes simplex virus (hsv)- cutaneous infections. his siblings (p age y, m; p -age y, f) and his mother (p age y, f) all have a history of recurrent viral (hsv- ) and bacterial (staphylococcal aureus, streptococcal pyogenes) cutaneous infections and/or recurrent sinopulmonary infections that respond to empiric antimicrobial therapy. their neutrophils all had enhanced superoxide production in response to stimulation by fmlp and pma as compared to healthy controls'. these findings suggest that rac i s is an activating mutation causing notable abnormalities in neutrophil morphology and nadph oxidase activation similar to other recently reported mutations. this novel mutation expands the phenotypic spectrum of rac activating mutations. clinical management of affected patients needs to be tailored to their phenotype and disease severity. background and aims: heterozygous mutations in cytotoxic tlymphocyte antigen- (ctla ) are associated with recurrent infections, lymphoproliferation, autoimmunity and lymphocytic infiltration of target organs. disease penetrance can be highly variable even among related family members carrying the same ctla mutation. our evaluation of a subset of the ctla patient cohort followed at the national institutes of health (nih) revealed that % of ctla mutation carriers have gastrointestinal (gi) manifestations which include diarrhea and diffuse lymphocytic enteropathy. our aim was to determine whether the intestinal microbiome, metagenome and metabolome could distinguish patients with ctla haploinsufficiency (ctla -h) based on disease severity, and the presence or absence of gi manifestations. methods: clinical metadata and fecal samples were collected from healthy individuals (n= ) and patients with ctla -h (n= ). patients with ctla -h were classified as having minimal (n= , only endocrine and/or dermatological manifestations) or systemic disease (n= , hematological and multi-organ involvement). they were further classified based on whether they had a history of enteropathy (n= ) or active gi disease ( < bowel movements per day and/or blood or mucus in stool) at time of sampling (n= ). metabolomic profiling (using a panel of metabolites) and s rrna gene sequencing (v region) was performed on fecal samples (total samples: ; number of reads/sample: , to , ; median: , ). a subset of samples were subjected to shotgun metagenomic sequencing based on findings from the s rrna gene sequencing analysis. results: all patients with ctla -h and a history of enteropathy or active gi disease also had systemic disease. fecal samples from patients with a history of enteropathy had a distinct microbial community structure (fig. ) which was significantly less diverse (fig. ) compared to healthy individuals and patients with minimal vs. systemic ctla -h. patients with a history of enteropathy had significantly higher relative abundance of bacterial taxa including shigella-escherichia (fig. ) . shotgun metagenomic sequencing confirmed that samples from patients with a history of enteropathy were dominated by subsets of identified escherichia coli strains, all of which share genes coding for specific types of virulence factors such as curli fibers (facilitate uptake into host cells), flagellar proteins (increase motility) and enterobactins (increase bacterial iron transport). meanwhile, samples from patients without active gi disease at the time of collection were enriched for several taxa including bacteroides nordii and akkermansia muciniphila compared to patients with ctla -h and active gi disease (fig. ) . metabolomic analyses showed that asparagine, -hydroxybutyrate, cytosine and cystine were enriched in samples with abundant e. coli, whereas samples without e. coli were enriched in metabolites involved in pyrimidine (holm p= . ), purine (holm p= . ), and alanine/aspartate/glutamate metabolism (holm p= . ) (fig. ) . conclusions: fecal samples from ctla -h patients with a history of enteropathy were heavily colonized with e. coli strains that are associated with a specific metabolomic profile and that share virulence factor genes that may facilitate host invasion. these data suggest that the microbiome and metabolome can distinguish patients with ctla -h and gi disease, and support the potential use of antibiotics or even antimetabolites to treat ctla -hrelated enteropathy. the dna polymerase delta (pol δ) complex is essential for leading and lagging dna strand synthesis. its catalytic subunit (pold ), carries both polymerase and exonuclease activities and plays a crucial role in dna replication and repair. heterozygous pold mutations have been associated with inherited colorectal cancer and mandibular hypoplasia, deafness, progeroid features and lipodystrophy (mdpl) syndrome. more recently a biallelic loss of function mutation in pold (p.r c) that impairs the stability of the pol δ complex, has been reported in related subjects with recurrent infections, deafness and combined immunodeficiency (cid) with t-cell lymphopenia, cd + t cell oligoclonality but preserved b cell proliferation. we report here a second family in which a novel biallelic missense mutation in pold gene was associated with cid. the proband is a -year-old boy born to consanguineous pakistani parents. since infancy he suffered from failure to thrive and recurrent infections, including episodes of pneumonias, multiple otitis media, sinusitis, recurrent cellulitis at the g tube site, bk viruria and shingles. live and dead vaccines were well tolerated. at years of age sensorineural hearing loss together with profound leukopenia (anc cell/μl, alc cells/μl) and hypogammaglobulinemia ( mg/ dl) were identified. intermittent ivig replacement and antimicrobial prophylaxis were initiated. immunophenotyping at years of age showed severe t cell lymphopenia ( cd + cells/μl, cd + cells/μl, cd + cells/μl, figure . volcano plot of metabolites present in fecal samples enriched with e. coli vs. samples without e. coli. c e l l s / μ l , c d + c d h i f o x p + c e l l s / μ l ) , a n d hypogammaglobulinemia (igm mg/dl, igg mg/dl, iga < ). physical exam was remarkable for multiple acquired nevi in the groin area, teeth abnormalities and global developmental delay. whole exome sequencing analysis revealed a homozygous pold missense variant (nm_ c. c>g, p.q e) absent in public databases (cadd score of ). parents were heterozygous. tcr-vβ family expression was normal in both cd + and cd + t cells, but the proportion of t cells expressing vα . (encoded by the distal trav - gene) was markedly reduced (less than %), consistent with impaired vdj recombination at the tra locus and/or with defective thymocyte survival. constitutive expression of γh ax was observed in t and nk cells after h and h of culture in unirradiated conditions. at h post-irradiation ( gy), reduced levels of p-atm were detected in t and nk cells, and lack of atm, smc and h ax phosphorylation was observed in a subset of b cells, suggesting inability of these cells to mount an effective dna repair response. bone marrow examination showed normal trilineage hematopoiesis but decreased proportion of cd -cd + mature b cells and increased proportion of pre-b cells. conclusion: we report the second mutation associated with autosomal recessive pold deficiency. our findings broaden the understanding of the mechanisms underlying the immune defect in this disease to include b cell maturation arrest in the bone marrow and a dna repair defect that may support the generation of a restricted tcr repertoire in the thymus and increased malignancy risk. abstract/case report text following allogeneic hematopoietic cell transplantation (hct) for scid, the development of a diverse t cell repertoire is essential for optimal immune recovery. high-throughput sequencing (hts) of the trb repertoire is the best tool for the evaluation of clonotype dynamics during immune reconstitution as compared to cdr spectratyping and staining of vβ families. we investigated whether longitudinal hts analysis of trb would accurately assess development of tcr repertoire diversity over time and reflect the quality of t cell reconstitution following hct for scid. we wanted to study the effect of conditioning regimen, scid genotype, donor type on tcr diversity post hct. we hypothesized that repertoire diversity may represent an early biomarker to predict long-term immune reconstitution vs. need for a second intervention. we assessed if the trb repertoire post-hct carried a molecular signature of selfreactivity. methods: the composition and diversity of trb repertoire of scid infants, pre-hct and at d, and mo and yearly posttreatment(s) was studied by hts. median time of follow-up was mo. subjects were part of a prospective study of scid by the primary immunodeficiency treatment consortium. equal amounts of total rna extracted from peripheral blood was used as template to semi-quantitatively amplify trb rearrangements. the vdj statistics file (past program) was used to calculate a shannon entropy (h) index of repertoire diversity and simpson ( -d) index of repertoire clonality. results: trb sequence analysis of scid patients showed poor diversity at baseline, followed by improvement to normal complexity (h index > . ) after hct. similar kinetics of development of trb diversity were seen in patients with il rg, jak , and il r defects (n= ) as in those with rag and artemis defects (n= ). in the latter group, however, hct with no conditioning or immune suppression only was associated with persistently lower diversity than hct with conditioning (p < . ), a difference not found in the il rg/jak /il r group (fig. ) . hct from a matched donor ( / conditioned) correlated with higher diversity than hct from a mismatched donor ( / conditioned) (p= . ). having > cd + t cells/ul at mo post-hct correlated with higher trb diversity at and mo post-hct (p < . ). the trb repertoire d post-hct was enriched for the presence of central cysteines at the apex of the cdr (p < . ), a biomarker of self-reactivity ( fig. ). an h-index of . or lower at d after hct predicted need for second intervention (hct or gt) (fig. ) . conclusions: analysis of trb diversity allows for detailed assessment of development of a diverse t cell repertoire following cellular therapies for scid and confirms the need for patienttailored treatment strategies based on scid genotype. t-cell repertoire d post-hct is characterized by a molecular signature that may contribute to the increased rate of autoimmunity early post-transplant. furthermore analysis of trb diversity at d post-hct may identify patients at risk for failure of sustained immune reconstitution, thus prompting a second intervention without delay. abstract/case report text background atopic dermatitis is a chronic, multifactorial, relapsing inflammatory skin condition which is one of the main known health problem worldwide. atopic dermatitis lesions are frequently colonized by staphylococcus aureus and staphylococcus epidermidis. their susceptibility to form biofilms, ability to form adhesive skin colonies which lead to extremely resistant to antibiotics and immune responses. formation of skin biofilm resulted in complex bacterial communities that have unique effects on human keratinocytes, mouse fibroblasts and host immunity. aims: the aims of this study to confirm the specificity of s. aureus or its secreted factors in induction of pro-inflammatory cytokines il- , tslp and toxicity on human keratinocytes and mouse fibroblast. the second aim to study the inhibitory effect of co-culture of s. epidermidis with s. aureus in term of production of pro-inflammatory cytokines and toxicity. method and materials: human epidermal keratinocytes and mouse embryonic fibroblasts cell lines from t were used as a control strain to examine production of inflammatory response (il- and tslp) and cell death induced by s. aureus in the presence and absence of s. epidermidis. tslp and il- were detected by elisa and the apoptosis of s. aureus and s. epidermidis on these cells was evaluated by flow cytometry. result: recent findings propose the important role of skin biofilms in the pathogenesis of atopic dermatitis. s. aureus have been found to induce secretion of pro-inflammatory cytokines and cause apoptosis of human keratinocytes and mouse fibroblasts. presence of s. epidermidis as skin biofilm found to protects the human keratinocytes and mouse fibroblasts from induction of proinflammatory cytokines and cytotoxicity. conclusions and future work: s. aureus are essential in production of inflammatory response and cell death of mouse fibroblasts and human keratinocytes. future work will be carried out to identify the soluble factors that responsible in induction of pro-inflammatory cytokines. in addition, more studies are needed to be able to understand the mechanism by how s. epidermidis reduce the induction and cytotoxicity caused by s. aureus. j clin immunol in adult patients, in whom arbitrarily defined diagnostic criteria for antibody deficiency syndromes are not fulfilled, is subject to interpretation and decision differences reported by immunologists world-wide. in this study, we explored whether training in one particular program would decrease the variability in diagnostic and treatment approaches seen in the responses to two nationwide questionnaires in the uk and the usa. methods: a -minute online survey originally administered to a cross-sectional sample of us allergists/immunologists (usa/i) in january, , was also answered by a/i subspecialists who had trained in the last years at the louisiana state university health science center allergy immunology training program in new orleans (laa/i). respondents were asked questions on patient assessment, antibiotic use, initial igrt, and immune response assessment in decisionmaking to prescribe igrt. usa/i participants were recruited from the dynata physician professional panel. laa/i participants were recruited by the louisiana primary immunodeficiency network (lapin). results: overall, laa/i had consensus responses to the various practice questions close to % of the time, but outliers were always present, as was also observed in the usa/i. there was a higher frequency in the reported care of patients as described in the questionnaire by laa/i. over % of laa/i assessed vaccine responses prior to commencing igg replacement vs only % of usa/i p < . . all la a/i used the pneumococcal vaccine for assessment purposes while few used tetanus and hemophilus influenza, and none used meningitis or salmonella vaccines. these vaccines were still used by some of usa/i. a high level of concordance was observed among all respondents in that only few regarded pneumococcal antibody testing as the definitive test to commence igrt. high resolution chest ct scan was used more often by laa/i before starting igrt. assessment of effectiveness of igrt was decided after only months by more usa/i, vs laa/i, who tended to wait months to decide to continue or discontinue igrt. conclusions: all a/i responders saw a significant number of patients who do not conform to strict diagnostic criteria for antibody deficiency syndromes. there is diversity in the approach of usa allergists/ immunologists in determining the indication for igrt for non-classical antibody deficient patients. laa/i responses made it obvious that post graduate influences always play a role in shaping the way a/i practice evolves after graduation. drawing on clinician experiences through questionnaires offers a valid contribution to developing consent approaches to improve patients' clinical conditions. diagnostic criteria and treatment guidelines would benefit from practice-based realistic recommendations based on a/i experience. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is a rare genetic disorder secondary to a defective fas-mediated apoptotic pathway of mature lymphocytes. it is characterized by chronic nonmalignant lymphoproliferation in the form of lymphadenopathy and/or splenomegaly, autoimmune manifestations such as cytopenias, increased risk of lymphoma, and expansion of tcrαβ+ cd -/cd -(dnt)t-cells. germline or somatic pathogenic variants in fas, fasl, and casp are well described genetic defects associated with alps. the definitive diagnosis for alps, based on the revised nih diagnostic criteria, include both required criteria (chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, or both and elevated tcrαβ+ dnt t-cells) and one of the primary accessory criteria (defective lymphocyte apoptosis or mutation in the genes mentioned above). patients who do not meet the current diagnostic criteria are considered for alps-related disorders. case presentation: we report a -year-old male who presented with recurrent infections, splenomegaly and chronic lymphadenopathy since month of age. due to its chronicity he was evaluated by multiple specialists for malignant and infectious causes. hematological workup including bone marrow biopsy was unremarkable except for an elevated ldh level. infectious workup identified a past cmv infection. clinical course is pertinent for chronic splenomegaly which was identified incidentally at . years of age during an evaluation for intussusception. family history is pertinent for a father with recurrent infections, paternal grandmother with thrombocytopenia of unknown cause requiring platelet transfusions, and paternal cousin with neutropenia. there is no family history of lymphomas. history of chronic lymphoproliferation and recurrent infections prompted an evaluation for lymphoproliferative disorder. full immune workup was notable for elevated plasma il- and il- , normal immunoglobulin levels, lymphocytes subsets, vitamin b level, soluble fasl, and relative frequency (%) but borderline increased absolute count of tcrαβ+ (dn) t-cells. in addition, he was noted to have presence of anti-platelet antibodies, poor lymphocyte proliferation to antigens, and low pneumococcal antibody titers. genetic testing with a pid gene panel identified a likely pathogenic heterozygous variant in prf c. del (p.his thrfs* ), a heterozygous variants of uncertain significance in casp c. c>t (p.pro leu) and stim c. a>g (p.thr ala). the casp variant is present in alleles in gnomad ( k total allele count) and reported deleterious by sift. discussion: unlike the typical alps presentation, characterized by dominantly lymphoproliferation and autoimmunity, our patient's clinical phenotype is striking for recurrent infections, abnormal t-cell function, and poor antibody response. our patient does not the meet diagnostic criteria for alps due to normal relative frequency of dn t-cells. however, presence of elevated of il- , il- , platelet autoantibodies raise concern for alps-related disorder. in addition, family history of recurrent infections and cytopenias raises concern for familial autoimmunity and alpslike phenotype. although casp is associated with autosomal dominant and autosomal recessive alps, the role of this vus is yet to be determined. conclusion: we continue to investigate the pathogenicity of our novel casp vus. further studies include pedigree analysis, fas apoptosis assay and apoptosis pathway testing to assess for the etiology of this alps-related disorder. (word count , max ) abstract/case report text rationale: ocrelizumab is a recombinant anti-cd monoclonal antibody, which binds to a different, but overlapping cd epitope than rituximab. there have been increasing reports evaluating hypogammaglobulinemia and morbidity and mortality in patients receiving rituximab, but there is a paucity of data on hypogammaglobulinemia in patients treated with ocrelizumab. methods: we performed a retrospective review of patients who received ocrelizumab in our healthcare system. we evaluated the demographics, indication for ocrelizumab, frequency of immunologic evaluation, and h y p o g a m m a g l o b u l i n e m i a p r e -a n d p o s t -o c r e l i z u m a b . hypogammaglobulinemia was stratified as mild (igg < mg/ dl or less than lab reference range), moderate (igg < mg/ dl) or severe (igg < mg/dl). results: we identified patients who received ocrelizumab for multiple sclerosis (average number of ocrelizumab cycles = ; range - cycles). there were ( %) female patients, with a mean age of years old (range - ; standard deviation ± t-cells. tnfα, ifnγ, and il- were not statistically different between cgd patients and healthy controls. tnfα, ifny, il- , and il- a expression in patients with cgd who had active colitis or history of colitis were increased as compared to cgd patients without a history of colitis but did not reach statistical significance. in two patients, il- a expression that was elevated pre-hct normalized post-hct. discussion: the mechanism for increased susceptibility to inflammatory disorders in patients with cgd has not been well elucidated. our results agree with previous studies demonstrating increased il- and il- a production from cd + t-cells in patients with cgd indicating a proinflammatory state in these patients at baseline. also, there appears to be an increase in tnfα, ifny, il- , and il- a expression from cd + t-cells that correlates with presence of inflammatory disease vs. those without inflammatory disease indicating that these cytokine perturbations may be able to serve as biomarkers of disease activity. a larger sample size with prospective collection will be analyzed in the future. abstract/case report text background: glucose- -phosphatase catalytic subunit (g pc ) deficiency, is characterized by severe congenital neutropenia, recurrent bacterial infections, mild intermittent thrombocytopenia and a high incidence of congenital cardiac and uro-genital defects. we report the case of a -yo male with chronic neutropenia and thrombocytopenia, who was found to have homozygous pathogenic variants in g pc (c. del, p.phe serfs* ). unique to this case is the patient's long history of misdiagnosis of evans syndrome (chronic autoimmune neutropenia with thrombocytopenia). case presentation: a -yo male with reported diagnosis of chronic autoimmune neutropenia and thrombocytopenia since age was referred to our a b clinic with concern for an underlying immune dysregulation syndrome. he had a history of oral ulcers, gingivitis, recurrent bacterial infections (otitis media, pneumonia, skin abscess) concomitant with severe neutropenia ( < cells/ul), for which he had received treatment with systemic steroids and g-csf since he was years old. he also had history of asthma and short stature, thought to be secondary to his chronic systemic steroid use. we present the first chilean patient with stat gof immunedysregulation . moreover, to our knowledge this is the first stat gof patient presenting with lymphomatoid granulomatosis. this is a severe pulmonary disease in which primary immunodeficiencies including stat gof should be considered in the differential. in this case rituximab successfully resolved pulmonary nodules and respiratory symptoms. there was persistence of mild hepatosplenomegaly but otherwise clinical stability and monitored expectantly until the age of . at that time he presented with fever, left knee and ankle arthritis. he underwent arthrocentesis of the left knee and left ankle, both aspirates were sterile, with notable leukocytosis with heavy neutrophilic predominance. an extensive rheumatologic and infectious workup was non-diagnostic. both sil- r and il- were elevated, , units/ml ( < ) and pg/ml ( < ), respectively. he was treated with systemic corticosteroids, ultimately arthritis resolved after months. at age he presented for the first time with periorbital pain and conjunctival injection of the left eye that persisted after minor trauma. he was found to have nongranulomatous uveitis, which responded ultimately to systemic corticosteroid. he then presented at age with fever and right knee and great toe arthritis. again he underwent arthrocentesis which revealed aseptic arthritis, and at that time was started on anakinra (anti il- β) and prednisone. there was clinical improvement over several weeks followed by return of right knee arthritis, coupled with onset of symptomatic uveitis of the left eye. despite systemic corticosteroids and anakinra and il- blockade, the patient was again admitted shortly thereafter to the hospital with arthritis, fevers, rash and abdominal pain. there was concern for evolving hlh and the patient was ultimately transferred to cincinnati children's for further evaluation and treatment. pertinent inflammatory biomarkers at that time included sil- r of , units/ml and il- level of , pg/ml. in addition to anakinra and systemic corticosteroids, the patient was started on tadekinig alfa (recombinant human il- binding protein) as part of a prospective study. hlh flare ultimately resolved without use of antineoplastic agents, and the patient was discharged home. soluble il- r levels since normalized, and il- levels decreased to less than pg/ml. the patient has been doing well on anakinra and tadekinig alfa, though continues to experience mild to moderate right knee effusion. this case suggests il- inhibition may be an effective therapeutic approach for patients with xiap deficiency. in the absence of neurological involvement and infectious trigger, ruxolitinib was initiated at a dose of mg/m /day, in combination with dexamethasone ( mg/m /day). this treatment led to rapid normalization of the neutropenia ( hours), complete resolution of the splenomegaly ( days) and disappearance of hlh biological markers (triglycerides levels in week, activated hla-dr+ cd t cells in weeks, fibrinogen levels in month), without the need for etoposide or serotherapy. dexamethasone was weaned every two-weeks and stopped after weeks. ruxolitinib was well-tolerated with no side effects. while in complete remission of her hlh, the patient then received alemtuzumab ( . mg/kg total dose) and a fludarabine-based myeloablative conditioning regimen. ruxolitinib was weaned over one week, and a / unrelated transplant was performed with success. the immediate posttransplant period was complicated by a veno-occlusive disease that responded rapidly to defibrotide and a corticosteroidresistant skin and ocular graft-vs-host disease (gvhd) despite a prophylaxis with ciclosporine and mycophenolate mofetil. gvhd was controlled by the reintroduction of ruxolitinib. at months post-hsct, her chimerism is % donor. to our knowledge, this case is the first description of a patient with primary hlh successfully treated in first intent by a combination of dexamethasone and ruxolitinib prior to hsct. our observation suggests that this targeted and less-toxic treatment regimen, that does not include etoposide nor high-dose alemtuzumab, is effective, well-tolerated and could be used in first intent to treat primary hlh. abstract/case report text presentation a -year-old girl, presented in ambulatory consultation, with a -year history of recurrent fever, influenza-like symptoms (sore throat, malaise), associated with self-limited painful genital ulcers (just within the period of fever). the first episode was characterized for an fournier's infection, requiring in-hospital treatment, multiple surgical procedures, antibiotics and hyperbaric oxygen therapy. after that catastrophic debut, she was diagnosed approximately episodes per year pharyngitis (with fever, malaise and sore throat) treated with corticoids, antibiotics and topic medication. the last year, noticed that every episode of fever (total of ) were associated with one or several genital lesions. the patient had no relevant medical history, she didn't receive long-term medication, she received all immunizations, she was sexually inactive, and hadn't apply any topic medication or product on the vulva, there was no trauma history, psychological medical history or sexual abuse. episodic gynecologic examination showed her labia minor several lesions, fibrinous, soft ulcerations on their inner aspect, these lesions had a symmetrical appearance, known as kissing lesions; no vulvar swelling, vaginal discharge or lymphangitis were noticed. there were no other skin or mucous membrane lesions (figure ). investigations viral (hiv, hbv, hcv, ebv, cmv) and treponemal (tpha-vdrl) serologies were negatives. erythrocyte sedimentation rate (esr) and pcr analysis within ulcers episodes were positive. specific antibodies (cardiolipin, anti ro/ss-a, anti la/ss-b, anti ccp, anti ena, anti gliadin, anti tpo anti tpo) serologies were negative. otherwise, important elevation immunoglobulin d was observed ( , mg/dl, twice the normal value): mild elevations of immunoglobulin m and immunoglobulin a were observed. serum subtypes of immunoglobulin g and immunoglobulin e were normal. leukocytosis with monocytes elevation and an increase of lymphocytes b were present. (table ). discussion lipschütz ulcers are uncommon and an often unknown entity for physicians, but it is important to recognize and include it in the differential diagnosis of vulvar ulcerations. this condition is characterised by self-limited painful ulcerations of the vulva or lower vagina in adolescent or young women, non-sexually transmitted, and usually preceded by influenza or mononucleosis-like symptoms. hyperimmunoglobulin d syndrome (hids) is characterized for unremitting fever lasting four to seven days and the presence of palpable tender lymphadenopathy, splenomegaly, arthralgia/arthritis, abdominal pain, and mucocutaneous manifestations. laboratory findings suggestive of hids include elevated age-specific serum immunoglobulin d (igd) and/or immunoglobulin a (iga) levels, elevation of acute phase reactants, and urinary excretion of mevalonic acid during, but not between, attacks. the diagnosis is established if an elevated age-specific level of igd is detected. iga levels are typically measured at the same time but are not required for diagnosis. elevated serum igd is not specific for hids and can occur in patients with certain neoplastic, infectious, heritable, and idiopathic disorders. in the present case report, the patient was treated with colchicine, with favorable evolution and free from new events. levels of ig d, platelets and monocytes remain high. we describe a young female patient presenting recurrent lipschütz ulcers, fever and elevation of serum immunoglobulin d, suggesting that hids could be associated with genitalia ulcers. ( ), and transmission in vivo in extremely low birth weight infants was considered ( ) . based on these considerations, the risk/benefit was considered favorable for restarting pasteurized donor breastmilk feeds. given his small size and young age, we had significant concerns about using ganciclovir prophylaxis and opted to hold this and monitor weekly cmv pcr. the child has been titrated up on these feeds, is gaining weight appropriately, and has had weekly cmv pcrs which are negative x since restarting donor breastmilk. t cells, last checked at weeks of age ( weeks gestational age) remain essentially absent. given the lower risk of nec in premature infants with breastmilk-based enteral feeds, a broader, multi-institutional study is warranted to best examine the safety of pasteurized donor breastmilk in infants with scid and complete digeorge syndrome. transfected with a reporter plasmid (for luciferase), wt or mutant-stat plasmids. nk cell cytotoxicity was measured by cr release assay. we used multiparametric immune profiling to dissect the effect of stat -gof mutations on nk cell developmental phenotype. results: similar to our previous studies, we observed higher levels of stat phosphorylation after two hours of stimulation from the dbd mutation compared to the ccd mutations. the stat activity assay confirmed gain of function observed by flow cytometry, but this activity was higher in k q mutant and d e mutant (ccd-closer to dbd) than v i mutant. all patients demonstrated low nk cell lytic unit compared to healthy donors. interestingly, we observed a correlation between low lytic unit and lower numbers of cd dim perforin + cd + nk cells; much lower in patient with k q mutation. stat -gof patients showed a significant decrease in total nk cell numbers and impaired nk cell maturation was characterized by low expression of cd , and higher levels of immature nk cell markers (cd , nkg a, cd b). conclusions: these data suggest that impairment of nk cell function is affected by the location of the stat mutation and continues to be the case in novel mutations identified. the identification the genotype/ phenotype correlation in the spectrum of the nk cell defect in stat gain-of-function mutants may help to better understand the molecular basis for stat activation and/or function to predict clinical manifestations of disease and ultimately treatment regimens. mutation specific analysis after an amniocentesis showed that one twin was a carrier (l m) and the other was a compound heterozygote (r c and l m). after birth, twin a had a mildly low erythrocyte ada level ( . nmol/h/mg; normal range + ) with normal metabolites and a normal immunophenotype, similar to both parents. twin b showed a normal absolute lymphocyte count and mitogen proliferation, normal t lymphocyte subsets, mildly low b and nk cells with % naïve t cells and a normal trec assay. erythrocyte ada levels were absent in peripheral blood, with mildly elevated metabolites [daxp= . μmol/ml rbc (normal < . ) and %axp= . (normal < . )]. weekly recombinant ada enzyme replacement therapy (ert) was started at week of life with subsequent normalization of the metabolites by week . absolute lymphocyte, t cell subsets were normal at birth but continued to rise slightly above normal range after starting ert. b and nk cell counts were mildly low at birth but normalized by week . genetic testing confirmed the prenatal genotypes in the twin girls. the patient is now months old and doing well with no history of infections. her twin was not an hla match and family is currently awaiting gene therapy approval. discussion: ada deficient patients show substantial clinical and metabolic heterogeneity that tends to correlate with the genotype but phenotypic discordance occurs even within the same genotype. we describe an infant with prenatally diagnosed compound heterozygous mutations in the ada gene (grade-i: r c and grade-ii: l m). ada alleles are graded from -iv with increasing ada expression and decreasing severity respectively. there are reports of children with grade i/iii allele combinations with delayed, late and partial phenotypes. two siblings have been reported with l m allele (grade iii) in combination with a different grade i allele (r q), presenting with combined immunodeficiency at and months. the specific allele combination from our patient has not been previously reported, however, we expected that the grade-i allele likely would be more deleterious than the grade-iii allele. in our case, predicting a future phenotype remains a challenge, creating a dilemma regarding management strategies. however, with only mild metabolite elevations in our patient after birth, we may speculate whether the prenatal diagnosis with early ert precluded the development of a full immunophenotype and it remains to be seen whether nonimmune sequeli may be prevented. conclusion: children with compound heterozygous mutations in the ada gene can pose diagnostic and therapeutic challenges, especially due to the associated metabolic and clinical phenotypic variability. early recognition and treatment may potentially alter long-term morbidity and mortality. (ipex)-like phenotype. immunodeficiency is often combined with impairment of the humoral and cellular compartments. hematopoietic cell transplant (hct) can resolve disease-related manifestations in stat -gof, but overall survival is poor and there is a high rate of secondary graft loss in transplanted patients. jakinibs are a class of medications that block cytokine-induced jak/stat activation. ruxolitinib preferentially inhibits jak and jak and has been used as precision-directed therapy for treatment of stat -gof related manifestations with success in stabilizing and in some cases reversing organ-specific manifestations. the utility and safety of jakinibs for long term treatment of stat -gof and in the prevention of disease-related manifestations is not known. as such, hct is often pursued for patients once disease-related manifestations are controlled with jakinibs. we present a patient with stat -gof mutation with gradual secondary graft loss following hct years ago, that has had continued disease progression despite chronic ruxolitinib treatment. case presentation this is a years-old male diagnosed with a de novo heterozygous stat mutation (c. a>g/a) at age , years following hct for ipex-like disease. he has been treated with ruxolitinib for the last three years. this patient initially presented at months of age with wasting enteropathy, failure to thrive, early-onset type diabetes and hypothyroidism. he had frequent upper respiratory infections during childhood including mycobacterium fortuitum mediastinal lymphadenitis. at years he underwent / matched, unrelated bone marrow transplant following reduced-intensity conditioning. mixed donor chimerism was present in the first days following hct, and he continued to have a slow progressive decline of donor chimerism with full graft loss ( % whole blood donor chimerism) by age . at age , enteropathy returned leading to cachexia and tpn dependence. concurrently, he had recurrent upper respiratory tract infections, lymphopenia, and hypogammaglobulinemia. imaging showed bronchiectasis and lung function was consistent with obstructive lung disease (fev : . l fvc: . l dlco: . ml/min/mmhg). initiation of ruxolitinib at age resolved his enteropathy with discontinuation of tpn and > -pound weight gain. enteropathy has not returned. pulmonary clearance measures have also been employed. dlco initially improved (dlco: . ml/min/mmhg) but obstructive lung pattern continued (fev : . l fvc: . l). after initial improvement, dlco began to decline. over the last years and despite treatment with ruxolitinib, lung function has deteriorated with worsened fev ( . l), fvc ( . l), and dlco ( . ml/min/mmhg). with this progressive decline, the family is now pursuing second hct. discussion jakinibs apply precision-directed therapy for immune dysregulatory features of stat -gof. their use leads to substantial disease control and clinical improvement but does not prevent disease progression. jakinibs should be used as a bridge to definitive therapy with hct in patients with stat -gof mutation. a recent large registry study showed a higher risk of infections (hr . , % c.i . - . ) in children with thymectomy as compared to surgery controls, in addition to demonstrating differences in the risk of cancers, autoimmunity and atopy. limited small studies have described some risk factors for altered immune consequences; however, specific predictors of infections among children with congenital heart disease (chd) undergoing thymectomies have not been systematically assessed. among children with chd and thymectomy, we sought to characterize children with and without reported infections within years postthymectomy and identify predictors of bacterial and viral infections. methods: using a retrospective chart review (institutional irb approved) from / / and / / , we identified children with chd that underwent thymectomy and excluded any known conditions associated with immunodeficiency and those with less than -month follow-up post-thymectomy. first absolute lymphocyte count (alc) after thymectomy was stratified using a cutoff at % of the lower limit of age-adjusted normal values (alc value < % vs alc value > % of the lower limit of age-adjusted normal levels). we sought to assess predictors of reported bacterial (positive blood, cerebrospinal fluid, respiratory cultures and chest-x-ray confirmed pneumonia) and viral infections (positive viral pcr tests) within years postthymectomy. results: we identified children with chd who had thymectomies, of which, % ( / ) were male. the median age at thymectomy was months (interquartile range months- . years); % ( / ) underwent a complete thymectomy; and % ( / ) developed a chylothorax within week post-thymectomy. a substantial proportion of children had an alc below % of the lower limit of age-adjusted normal levels after thymectomy ( % [ / ] pre-thymectomy vs % [ / ] postthymectomy). among children with chd post-thymectomy, % ( / ) and % ( / ) reported bacterial and viral infections within years, respectively. children with post-thymectomy alc values below % of the lower limit of age-adjusted normal levels had higher odds of reported bacterial (or . , % c.i . - . , p= . ) and viral (or . , % c.i . - . , p= . ) infections post-thymectomy as compared to those with an alc greater than % of the lower limit of ageadjusted normal levels (multivariate logistic regression). there was no association with the type of thymectomy (partial vs complete), age at thymectomy, weight at thymectomy, sex or prematurity. conclusions: among children with congenital heart disease with no known immunodeficiency undergoing thymectomy, alc below % of age-adjusted normal levels post-thymectomy may be associated with higher odds of bacterial and viral infections. a retrospective study design with a small sample size poses several limitations; however, this study suggests that post-thymectomy absolute lymphocyte values may be a potentially useful marker to identify higher risk patients in this population. radiological assessments esp. in the ct chest is commonly performed, but has associated radiation exposure and pulmonary function testing, at times, maybe insensitive to small changes in lung pathophysiology. many pids may have overlapping features with short telomere syndromes (sts) a, which are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems, unified by a high cell turnover in these organs. clinical assessment of ageappropriate telomere length (tl) is performed using flow cytometry & fluorescence in-situ hybridization (flowfish). methods: we retrospectively analyzed telomere lengths in lymphocytes and granulocytes using the flow cytometry and fish method .flowfish testing was done at reference laboratories in johns hopkins university (jhu, usa).approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: patients were included in our analysis with females ( %) and males ( %).the median lymphocyte count of our cohort was . ( . - . ).the telomere length was strongly associated with the presence of lung disease (p= . *) and the presence of interstitial lung disease closely paralleled the changes in telomere length (delta-as compared to age adjusted normal percentiles lengths). shorter lymphocytic telomere length was associated with more severe reduction on total lung capacity (tlc; p= . *). conclusion: shorter lymphocytic telomere length served as a reliable biomarker for interstitial lung disease in pid patients. this may open up newer avenues for assessment of aging pathways in pid and may offer the option of using senolytic therapies in pids. mutations in the il- receptor common gamma chain gene (il rg) result in x-linked severe combined immunodeficiency (scid). the common gamma chain is shared by il- , il- , il- , il- , il- and il- receptors. x-linked scid typically presents with low or absent t and nk cells and normal or elevated numbers of b cells. we report a case of x-linked scid with elevated b and nk cell numbers (t-b+nk+). the male patient had an abnormal newborn screen for scid in north carolina. lymphocyte enumeration performed at days of life showed cd + cells/mm , b cells/mm , and nk cells/mm . he had no naïve t cells. repeat lymphocyte enumeration two weeks later showed that the cd + count had increased to /mm . only . % ( cells/mm ) were cd ra+ naïve t cells. he continued to have elevated b cell and nk cell numbers. chimerism studies revealed the presence of % female cells in mitogen-stimulated pbmc by fluorescence in situ hybridization, indicating the presence of transplacentally transferred maternal cells. lymphocyte proliferation responses to pha and cona mitogen stimulation were very low (less than % of normal). immunoglobulin levels were igg mg/dl, igm mg/dl, and undetectable iga and ige. genetic studies revealed a missense mutation in il rg, c. c>t, resulting in an amino acid substitution (p.ala val) in the extracellular domain. family testing showed that the patient's mother was a carrier for this variant. the father and the two healthy older brothers did not have this variant. of note, the family history was significant for lateral maternal male early deaths. at weeks of age, the patient received an unfractionated bone marrow transplant from his hla-identical brother without conditioning or gvhd prophylaxis. at the time of this report's submission, he is weeks post-transplantation and has had successful engraftment (whole blood-cd + fraction was composed of > % donor cells). he also now has normal t cell proliferation in response to mitogens and normal levels of all immunoglobulins. genetic defects that cause primary immunodeficiency can have variable phenotypic presentations. the patient's phenotype was atypical in that he had elevated nk cell numbers. to further evaluate these cells, we checked for stat phosphorylation following il- stimulation of abstract/case report text background: stat gain-of-function (gof) mutations cause a multisystem disease of early onset autoimmunity and lymphoproliferation, severe post-natal growth restriction, and recurrent and/or invasive infections. treatment of the autoimmune and auto-inflammatory features of stat gof patients relies heavily on immunosuppression and is often challenging. the full scope of phenotypes, treatments and outcomes may be broader when analyzing a substantially larger cohort than those already reported. methods: we gathered and analyzed data on patients from centers world-wide with confirmed gof mutations in stat . retrospective chart reviews were performed in accordance with all local ethics and irb committees to determine clinical manifestations, immunophenotype, treatment regimens, success of treatment methods, and overall survival. funcitonal transcriptional activity was assessed by luciferase reporter assay on each individual mutation. results: fifty-nine individual mutations were identified and all conferred gof by a validated luciferase assay. there were mutations in the nterminal domain, in the coiled-coil domain, in the dna binding domain, in the sh domain, and in the transactivation domain with the overwhelming majority being missense mutations. median age at presentation was approximately years; % of subjects are male and % are female. immunodysregulatory features presented in all patients. autoimmune cytopenias were the most common occurring in % of subjects (n= ), followed by lymphoproliferation in % (n= ) with increased frequencies of double negative (cd -cd -)t cells being found in % of of patients tested, enteropathy in % (n= ), endocrinopathy in % (n= ), interstitial lung disease in % (n= ), dermatitis in % (n= ), and inflammatory brain disease in . % (n= ). growth failure was present in % (n= ) with half of those patients having concurrent enteropathy. infections were reported in % of the cohort to include recurrent and/or invasive viral, bacterial, opportunistic, fungal, and mycobacterial infections. prominent abnormalities of immunophenotyping included t cell ( %) and b cell ( %) lymphopenia with reduced t cell proliferation in response to mitogens or antigens in % of those evaluated patients. fifty-nine percent of the patients hypogammaglobulinemia while % exhibited poor specific antibody responses to recall antigens. overall survival was % at data collection.treatment of stat gof patients often included multiple agents: ivig , chronic and pulse steroids, mtor inhibitors, calcineurin inhibitors, rituximab, mycophenolate mofetil, alemtuzumab, tocilizumab, and jakinibs. those started on jak inhibition showed improvement in clinical symptoms and, to date, there are stat gof patients on targeted jak inhibition. thus far, patients have undergone bone marrow transplant with a % survival rate. discussion: stat gof mutations were first reported in to cause a heterogeneous syndrome of autoimmunity and lymphoproliferation with immunodeficiency and infection susceptibility. earlier treatment with targeted therapy such as jak inhibitors has led to reduced disease morbidity. we report the largest cohort of stat gof patients collected through a multi-national collaboration of the longitudinal data and natural history of stat gof disease. understanding the heterogeneity of presentation and key features that will lead to proper diagnosis and early treatment in an effort to prevent long term disease associated sequelae. we present the case of month old male with a novel heterozygous mutation in tcf and two previously unreported phenotypes: ) absent circulating cd + b cells yet preserved immunoglobulin synthesis and vaccine responses and ) significant thrombocytopenia that improved with immunosuppression. there is also a striking family history of two half-sisters who died during early infancy with similar clinical and lab findings and the same genetic change. the infant boy was born at term with respiratory failure and generalized rash. at birth he had thrombocytopenia ( k/ul) and lymphopenia ( / ul). initial absolute cd + t cell count was low ( /ul), yet he had normal thymic output and proliferative responses to mitogens ruling out scid. cd + b cells were < /ul and bone marrow biopsy revealed decreased hematagones, yet he had a normal igm level ( . mg/dl) elevated iga ( mg/dl), and elevated ige ( iu/ml). igg levels were initially obscured by maternal igg and ivig; in turn he made positive titers to diphtheria and tetanus vaccination. the infant has maintained his own igg production. rapid genome sequencing revealed a heterozygous predicted deleterious vous in tcf , in the second transactivation domain (c. c>t, p.pro ser). the same change in tcf was identified in the deceased half-sisters as well as the father: all infants had different mothers, suggesting autosomal dominant inheritance. the sisters had similarly severe thrombocytopenia and absent circulating b cells; their causes of death were not completely understood. the year-old father has normal platelet levels, very low cd + b cells ( /ul), elevated igg ( , mg/dl) and ige ( , iu/ml), and normal levels of iga and igm. the father also has an elevated number of cd + t cells ( , /ul) with an increased percentage of t cells expressing hla-dr ( %). in the months after birth, the infant boy continued to require frequent platelet transfusions. despite the persistent t lymphopenia, there was evidence of increased t cell activation with elevated levels of soluble il- r ( pg/ml) and increased percentage of cells expressing hla-dr ( %) cd ( %), cd ( %), cd ( %), and cd ( %). a -day trial of prednisone was associated with an increase in his platelet count to > k/ul. he was switched to rapamycin as a steroid-sparing agent, and his platelet count has remained > k/ul for several weeks without transfusions. interestingly, his b cell counts also improved after the steroid trial ( /ul) and his absolute lymphocyte count is normalizing on rapamycin. a potential mechanism could be rapamycin decreasing t cellmediated destruction of platelets or b cells. reassessments of t cell activation markers and b cell phenotyping while on rapamycin will be done in the future. in contrast to multiple published cases of tcf mutations associated with complete agammaglobulinemia and absent b cells, we present a case of an infant with absent b cells yet preserved humoral function as well as severe thrombocytopenia responsive to rapamycin. in collaboration with colleagues at nih, studies are underway to understand whether/how the unique change in tcf is related to either phenotype described above. abstract/case report text background: childhood-onset, chronic, multi-system inflammatory diseases are increasingly being characterized as monogenic inborn errors of immunity. arpc b deficiency is a recently described, rare combined immunodeficiency characterized by recurrent/severe infections, a variety of autoimmune manifestations and platelet defects. we describe a case of arpc b deficiency identified in an adult patient with recurrent ulcers/ bechet-like disease, non-malignant lymphoproliferation and intermittent microthrombocytopenia. patient case: at year of age, our female patient was diagnosed with behcet disease based on a history of bloody stools at months, oral ulcers at months and vulvar lesions at year. she underwent rheumatology evaluations for inflammatory arthritis, episcleritis, eczema, vasculitic ulcerating nodules of the trunk, perineum and extremities, and verrucae forming flat plaques similar to epidermodysplasia verruciformis without a unifying diagnosis. other infections include otitis media, sinusitis, pseudomonas ecthyma gangrenosum, cervical lymphadenitis, and pneumonia. at years old, the patient was referred to our immuno-hematology comprehensive program clinic with a concern for malignancy versus a primary immune regulatory disorder (pird). she had a -month history of drenching night sweats, urticarial plaques, edema in her extremities and diffuse cervical, axillary and inguinal lymphadenopathy. past complete blood counts showed intermittent mild microthrombocytopenia. lymph node biopsies were negative for a neoplastic process but identified plasmacytosis, including focally increased iga-kappa+ plasma cells. expert review of the lymph node biopsy, and further evaluation excluded multicentric castleman disease. consideration was also given to autoimmunune lymphoproliferative syndrome (alps)-like disorders; however, her alps flow cytometry panel was nondiagnostic. her basic immune evaluation showed severe t cell lymphopenia (cd + cells/ cm, cd + cells/cm, cd + cells/cm) with adequate b and nk cells, normal lymphocyte proliferation to pha and pwm, and dysgammaglobulinemia with igg g/dl, iga g/dl, igm g/ dl and ige g/dl. due to concern of an underlying pird, a primary immunodeficiency panel was sent for gene analysis with negative results. however, trio clinical exome sequencing identified biallelic variants in the gene arpc b. one allele has a truncating, nonsense pathogenic variant in exon denoted as c. g>t, p.glu ter. the other allele has a likely pathogenic variant in intron denoted as c. - a>g, resulting in disruption of the canonical splice acceptor for exon . this is predicted to cause exon skipping, with an in-frame deletion of amino acids coded by exon . conclusion: this case highlights the value for evaluation for pirds in patients presenting with behcet-like disease, particularly in the context of other autoimmune manifestations and/or microthrombocytopenia. it also underscores that patients with arpc b deficiency may present with chronic non-malignant lymphoproliferation. moreover, this patient emphasizes the value of exhaustive genetic testing for complex immunologic phenotypes. abstract/case report text lipoyltransferase gene defect is associated with severe mitochondrial dysfunction disrupting lipoic acid biogenesis. clinical manifestations associated with early seizures, hypotonia, cardiomyopathy and pulmonary hypertension and encephalopathy. early neonatal death due to sepsis and cardiovascular collapse is commonly seen. the patient is a week preemie male with congenital heart disease who developed severe intractable lactic acidosis on day of life with increased excretion on organic acids of -methyl- , dihydroxybutyric acid. a mitochondrial disorder , echs or hibch deficiency was suspected. at mo of age the patient was admitted for apneic spells and respiratory compromise. he was found to have elevated crp associated with rhinovirus infection and gram-negative bacteremia. due to the history of failure to thrive and sepsis, immunology was consulted. immunologic work up indicated normal b, t and nk cells with normal dhr, but showed agammaglobulinemia. the patient was started on ivig and whole exome sequencing was done. molecular analysis showed compound heterozygote mutations in the lipt gene: c. g>a (p.arg gln) and c. t>g (pval gly). subsequent biochemical analysis also showed biochemical abnormalities consistent with lipt defect. lipoyltransferase is an enzyme involved in activation of a number of enzymes requiring lipoic acid. it is involved in lipoic acid synthesis. lipoic acid is required for the activity of pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. the literature indicates that most patients with lipt defect have a severe, often fatal course. the patient is now almost years old and has stable clinical course without any major infections. he certainly has significant hypotonia and developmental delay. in conclusion, we are presenting the first case of lipt gene mutations associated with agammaglobulinemia who responded well to ig supplementation therapy. our immunologic findings in this case highlights the importance of immunodeficiency work up in challenging cases. as we see more cases lipt gene mutations, we will better understand the clinical spectrum. abstract/case report text a now -year-old male was initially evaluated for concerns regarding food allergy, eczema, food protein-induced enterocolitis syndrome, and failure to thrive. he had reactions of varying severity to multiple foods. these usually involved immediate urticaria or prolonged vomiting, diarrhea, and abdominal pain. ige and skin prick testing was performed to suspected foods and was positive to milk, egg, pork, wheat, peanut, pecan, coconut and corn. these foods had historically caused reproducible immediate symptoms. testing was negative to other suspected foods. he developed an oral aversion and extremely restricted diet. symptoms of abdominal pain, hematochezia, rashes, arthralgias, headaches, fatigue, dyspnea, and palpitations increased. urticaria and severe abdominal pain with vomiting and diarrhea continued intermittently without identifiable triggers on a restricted diet. laboratory markers demonstrated elevated inflammatory markers, anemia, iron deficiency, vitamin b deficiency, and vitamin c deficiency (scurvy). gastroenterology work up did not identify any pathology. gastrointestinal symptoms did not respond to treatment with multiple gerd medications or oral steroids. baseline tryptase was elevated. low histamine diet was initiated and repeat tryptase remained elevated. fractionated tryptase revealed normal mature (beta) tryptase with elevated total tryptase, negative genetics for c-kit mutation, normal urine prostaglandins. family members had tryptase levels drawn. one parent and sibling had elevated tryptase levels, while the other parent's tryptase was normal. hereditary alpha tryptasemia syndrome is defined by elevated blood tryptase levels and symptoms involving multiple organ symptoms. patients with elevated tryptase levels without symptoms are defined as having hereditary alpha tryptase trait. there is significant variability regarding which patients are symptomatic. organ symptoms that may be involved include skin, gastrointestinal, neurologic, connective tissue, cardiac, neuropsychiatric. severe allergic reactions such as anaphylaxis can occur. increased blood levels of the protein tryptase are caused by extra copies of the alpha tryptase gene (tpsab ). treatment is usually directed at specific symptoms, antihistamines, and mast cell stabilizers. research continues into additional treatment options. this patient was started on cromolyn and long-acting antihistamine. his gastrointestinal symptoms and rash/urticaria improved, and he began tolerating a small, but increased, variety of foods. the majority of his constitutional symptoms of fatigue, arthralgias, weakness resolved as he began gaining weight, and hemoglobin, vitamin c and b normalized. his sibling was evaluated and noted to have food allergy, asthma, abdominal pain, gerd, and eczema. she was also started on cromolyn and antihistamines which improved her gastrointestinal symptoms. parent with elevated tryptase was recommended to be evaluated further with allergist. this is an example of a patient with elevated tryptase and multiple organ system involvement. some of his signs and symptoms responded to mast cell stabilizing and antihistamine medications. patients with history of recurrent episodes of allergic reactions to foods and multiple constitutional symptoms would benefit from baseline tryptase levels. family members should also be tested if the patient has elevated tryptase. multiple studies have been published looking at the rates of scid in the united states. the estimated rate of scid prior to screening was per live births. post screening implementation, on average rates of scid were found to be closer to in live births. results : development of a t cell receptor excision circles (trec) scid screen in alberta involved the screening of anonymous term neonates using quantitative pcr for trecs. the cycle threshold for the control gene, rnasep, was set at . as % of our population had a cycle threshold < . ( % ci [ . , . ]). from those bloods spots with adequate dna, a final trec cut off of was chosen, as it would give an accuracy of . %, and fairly low false positive rate of . % ( % ci [ . , . ]). since starting a population based screen for scid in june of , we have identified cases of scid and cases of low trec not caused by scid. to date we have detected one case of reticular dysgenesis, cases of ada scid and one case of x-linked scid. other causes of lymphopenia in the neonatal period detected with abnormal trecs include one syndrome associated with variably affected cellular immunity (charge) and cases of secondary lymphopenia including four cases of prematurity, three cases of diaphragmatic hernia or gastroschisis, four patients with underlying cardiac disease, and one patient with severe hydrops. discussion : canada has multiple unique populations with increased risk of scid. the estimated rate of scid in canada prior to implementation of a population based screen was . per live births. the rate within canada's first nations, métis and inuit populations is . per live births. prior to scid screening, alberta had cases of scid identified between - with an estimated rate of per live births. to date, our screen in alberta has identified cases of scid with a rate of per live births which is significantly higher than previously estimated. given that early diagnosis and definitive management through bone marrow transplant or gene therapy has been shown to reduce mortality this screen will help reduce morbidity and mortality in this vulnerable population. abstract/case report text introduction: human herpesvirus (hhv- ) has the ability to integrate its genome into host telomeres. if this integration occurs in gametes, then the virus can be genetically transmitted and offspring will carry a copy of chromosomally integrated hhv- (cihhv- ) in each somatic cell. this can lead to false attribution of infectious and non-infectious presentations of hhv- , and make the diagnosis of active hhv- infection difficult. we present the case of a patient with meningoencephalitis attributed to hhv- and persistently elevated blood levels of hhv- by pcr concerning for primary immunodeficiency who was discovered to have cihhv- . case description: a -year-old female who carried a past diagnosis of hhv- meningoencephalitis was seen in immunology clinic for follow up of persistently elevated levels of hhv- dna in her blood by pcr. she was born at weeks and as an infant had failure to thrive (ftt), anemia and a varicella like rash after varicella immunization. at the age of , she received flumist vaccine and developed a fever the following day. over the next few days, she developed lethargy, altered mental status, headache, photophobia, seizure, papular rash and oral ulcers. she was admitted to the hospital and csf studies were consistent with viral meningoencephalitis ( wbc, l, m, rbc) although hsv, cmv, ebv and enterovirus were negative. she was treated for presumed hsv encephalitis with days of iv acyclovir and days of high dose steroids. one week after discharge, she again developed papular rash on feet, headache, oral ulcers and lethargy. she was admitted and csf studies this time showed only wbc but positive hhv- . blood and skin swab were also positive for hhv- . immunology was consulted while admitted and work up for primary immunodeficiency was initiated. her work up was normal including responses to vaccine titers, complement studies, moderate in t cells, monocytes, neutrophils, lung and muscle, and low in skin, liver, heart and kidney tissues. analysis of each individual gene expression level by nanostring in comparison with healthy controls demonstrated significantly higher levels of the following irgs: ddx , epsti , gbp , ifi , isg , ly e, oas , oas , oas , rsad , rtp and socs . whole blood rna-seq was performed in patients and pathway analysis with the differentially upregulated genes demonstrated an enrichment of intraluminal vesicle formation and negative regulation of apoptotic signaling pathways. stimulation of pbmcs with the tlr ligands poly i:c, odn, and lps induced a fold increase in ifi and a -fold increase in ifna and ifnb transcription compared to baseline. one patient had constitutive upregulation of stat and stat in monocytes and of stat and stat in t cells. conclusion: we describe a novel immunedysregulatory disease caused by de novo truncating variants in samd l that presents similar to candle with neutrophilic pannicultis and points to an important role of samd l on regulation of adaptive and innate immune responses. acknowledgements: this work was supported by the nih irp of niaid abstract/case report text introduction: ataxia telangiectasia (at) is caused by a defect in the atm gene which is responsible for repair of damaged dna. it is a rare, devastating neurodegenerative disease that results in ataxia and telangiectasias, particularly of the sclera and skin. those with at are at increased risk for immunodeficiency and cancer. the immunodeficiency is variable and may result in deficiency in humoral and cellular immunity in some patients. here we present a patient with ataxia telangiectasia and hypogammaglobulinemia on immunoglobulin therapy who developed recurrent urinary tract infections (uti) and sepsis. case presentation: the patient is a -year-old female with ataxia telangiectasia and hypogammaglobulinemia who presented with three episodes of uti, one of which resulted in prolonged hospitalization due to sepsis and acute kidney injury (aki). she presented with days of flank and back pain and was hospitalized for days for e coli uti. she improved on iv antibiotics and was discharged home to complete treatment with oral ciprofloxacin. due to persistent emesis, she was readmitted weeks later with urosepsis and aki with a creatinine of . mg/dl, over times her baseline creatinine. after additional antibiotics and iv fluids, she improved clinically, renal function normalized and she was discharged home. renal ultrasound was unremarkable with no anatomical abnormalities. she was relatively healthy prior to this with only one episode of bacterial pneumonia in . she receives weekly subcutaneous immunoglobulin therapy dosed at mg/kg with normal igg levels ( , , mg/ dl). at baseline, she had high igm ( mg/dl) and low iga ( < mg/ dl) levels, as well as decreased t cells but normal nk and b cells ( cells/ul cd +, cells/ul cd +, cells/ul cd +, cells/ul cd +cd +, and cells/ul cd +). she has hyperglycemia (on metformin), hypertriglyceridemia (on atorvastatin) and hypertension (on losartan). she is thin and wheelchair bound with bilateral telangiectasias to the sclera, neck, and chest. she has occasional eye bleeding and epistaxis, presumably from her telangiectasias. she has good hygiene and good adherence to medications. she voids voluntarily, has no indwelling urinary catheter and is not sexually active. discussion: patients with ataxia telangiectasia may have frequent viral and bacterial infections, most frequently upper and lower respiratory tract infections, as well as wart and skin infections. based on our review, this is the first reported case of ataxia telangiectasia with hypogammaglobulinemia on immunoglobulin therapy with recurrent utis complicated by urosepsis and aki. despite adequate igg levels on immunoglobulin therapy, our patient continued with recurrent utis. it is uncertain whether her non-ambulatory status, hyperglycemia or related immunodeficiency are the causes for her increased susceptibility to utis. the literature reports patients with at and bladder wall telangiectasias can result in significant hematuria, and perhaps this may be a source of entry for bacteria and consequent development of uti. this suggests that patients with ataxia telangiectasia and recurrent utis may benefit from renal ultrasound and possible cystoscopy to better visualize telangiectasias. we recommend consideration of workup for recurrent utis in patients with ataxia telangiectasia. abstract/case report text objectives: patients with partial rag deficiency frequently present with humoral autoimmunity suggesting breach in tolerance mechanisms and subsequent expansion of autoreactive b cell clones. here we aim to trace polyreactive b cells and their descendants at b cell developmental stages through our in-house bioinformatic pipeline, immchaintracer (ict). methods: the b cell receptor (bcr) was expressed as monoclonal antibodies from single sorted mature naive b cells (n= - per donor) from patients with hypomorphic rag deficiency and healthy donors. xthe recombinant monoclonal antibodies were screened for polyreactivity (dsdna, insulin, lps and ifnα) by elisa. in parallel, igh repertoires were deep sequenced from sorted mature naïve, activated naïve and memory b-cell compartments. our in-house assembled bioinformatic pipeline called immchaintracer (ict) was applied to track down the descendants of cloned autoreactive igh sequences in repertoires of subsets above. results: igh sequences (n= including polyreactive, nonpolyreactive clones) from mature naive b cell from six patients with partial rag deficiency and healthy donors (n= including polyreactive, non-polyreactive) were analyzed with our novel inhouse bioinformatic approach to track lineage fate in repertoire at specific developmental stages. interestingly, . % of the patients' sequences and their descendants were identified in their mature naive, activated naive or memory b cell repertoires, while none of the analyzed healthy donor clones were found at later subsets. furthermore, genealogical analyses of related clones revealed lineage expansion and progressive positive antigen selection of the autoreactive clones in the patients. conclusions our findings demonstrate that peripheral tolerance checkpoint is broken in hypomorphic rag patients. our novel method enables tracing the fate of autoreactive naive b cells in the effector repertoires. we have shown that impaired b cell tolerance allows the expansion and combination of sirolimus and rituximab therapy controlled his autoantibody production which was an important goal for his autoimmune condition. we present a new treatment approach for anti-nmda receptor encephalitis. rituximab was tried on these cases before but the combination of sirolimus and rituximab therapy was never given before. we now recommended that on refractory cases of anti-nmda receptor encephalitis, combination of rituximab and sirolimus therapy can be tried. ( ) submission id# mosaic variants in immune function genes identified through exome sequencing stable with no interim infections and is doing well on thyroid hormone replacement. the current plan is for the patient to undergo a stem cell transplant for the arpc b deficiency as he is at high risk for recurrent infections and severe disease. although this gene mutation is rare, review of the current literature describes patients with this condition that have undergone stem cell transplant and have done well. at this time, this seems to be the best option for management, and it may potentially be curative. abstract/case report text introduction: common variable immunodeficiency (cvid) is a disorder characterized by impaired immunoglobulin production and frequent or recurrent infections, but also associated with an increased risk for developing malignancies such as lymphomas. although intravenous and subcutaneous immunoglobin g replacement has been successful in reducing the number of bacterial infections and prolonging survival, it fails to address other complications that arise from this disorder. we report a case of a patient with cvid who developed mycosis fungoides (mf). mf is a rare form of cutaneous t-cell lymphoma, occurring in about in , to , individuals, lack of treatment could potentially be fatal. case presentation: a -year-old caucasian woman with a history of ulcerative colitis, allergic rhino-conjunctivitis and tonsillectomy was referred to the immunology clinic for evaluation of low serum immunoglobulins. there was no family history of infections or immune deficiencies, but paternal grandfather had colon cancer and maternal grandmother had lung cancer. the patient reported frequent episodes of bronchitis, and sinus infections. immunizations were up to date for her age. medications included azathioprine, cetirizine, fluticasone nasal spray, hyoscyamine, montelukast, lactobacillus, omeprazole, and olopatadine ophthalmic solution. no history of frequent use of systemic steroids. initial serum immunoglobulins revealed normal ige and igm but low iga ( mg/dl, normal range - mg/dl) and low igg ( mg/dl, normal range - mg/dl). cbc with differential, lymphocyte subsets, c and c levels were normal. while she had adequate protective titers against haemophilus influenza type b, diphtheria and tetanus, titers against pneumococcus were < % protective and she failed to mount an adequate response to pneumococcal polysaccharide vaccine. given her diagnosis of cvid, she started scig ( mg/kg every weeks). a year later, she developed a bilateral nonpruritic rash in the abdomen and upper trunk. initial skin biopsy suggested a drug reaction. a subsequent biopsy revealed a superficial perivascular lymphoid infiltrate with focal epidermotropism and positive t cell receptor gamma gene rearrangement, consistent with mf. testing for cell t receptor beta gene rearrangement was negative. while mf treatment consisted of triamcinolone . % ointment, treatment for ulcerative colitis transitioned from azathioprine to vedolizumab. conclusions: cutaneous t cell lymphomas, although uncommon, can be seen in cvid. there are several reasons for the increased risk of lymphoma in cvid. the role of chronic infections and the development of lymphoma as of yet, is not clear. skin reactions to scig products in the areas of infusion are relatively common and resolve promptly. high index of suspicion is crucial in obtaining tissue sample to confirm or rule out malignancy therefore avoiding delaying proper treatment. abstract/case report text patients with lipopolysaccharide responsive beige-like anchor protein (lrba) deficiency present with a plethora of immune related defects including a defective humoral response characterized by low numbers of switched memory b cells and plasma cells, as well as an impaired production of antibodies, leading to recurrent infections. however, the molecular mechanisms behind the defective b cell response remain unknown. to gain better insights into the possible roles of lrba in b cell physiology, we screened for lrba-interacting proteins using computational predictions. twenty-seven proteins involved in vesicle trafficking and autophagy were identified as potential lrba-interacting partners. to validate those potential lrba interactions, we performed coimmunoprecipitations and proximity ligation assays (pla), finding that endogenous lrba interacts with the phosphoinositide kinase regulatory subunit (pik r ) in b cells. pik r (aka vps ) is the regulatory subunit of vps , the catalytic subunit of the pi k-iii complex, which acts as a positive regulator of autophagy by producing phosphatidyl inositol- phosphate (pi( )p). autophagy is a catabolic mechanism essential for cell survival and plasma cell differentiation. in fact, we observed that reduced lrba impaired the production of pi( )p upon autophagy induction. in addition, we observed in both lrba-deficient hela and b cells reduced mobility, abnormal accumulation and increased size of autophaghosomes, accompanied by an atypical lysosomal positioning. these abnormalities are due to a blockade of the autophagosome-lysosome fusion, as detected by reduced lc -ii lipidation upon autophagy induction in the presence of lysosome inhibitors. interestingly, lrba-deficient hela and b cells exhibited enhanced activity of mammalian target of rapamycin complex (mtorc ) signaling, a key suppressor of autophagy whose activation possibly contributes to defective autophagy. taken together, b lymphocytes lacking lrba can form autophagosomes but they fail to fuse with lysosomes. thus, we propose a role of lrba at late stages of autophagy through the binding to pik r . abstract/case report text apds caused by gain-of-function mutations (gof) in the genes (pik cd and pik r ), encoding for the p δ and p subunits of phosphoinositide -kinase δ (pi kδ), results in hyperactivation of the pi k/akt/mtor/s k pathway and lead to immune dysregulation, lymphoproliferation and immunodeficiency. apds manifests with respiratory tract infections, bronchiectasis, susceptibility to herpes group viruses, autoimmunity, cytopenia, lymphoproliferation and lymphoma. gastrointestinal system manifestations include enteropathy, colitis, and liver disease. eosinophilic esophagitis (eoe) or eosinophilic gastrointestinal disease (egd) have been under diagnosed in reported apds cohorts. objectives: to review the incidence, demographics and relevant clinical data for eosinophilic gastrointestinal disease in a single center apds cohort. methods: review of clinical and laboratory findings from apds patients followed at the nih clinical center, from to . results: patients were either historically diagnosed or actively studied at our center for egd. incidence of all egd is % in our cohort and all patients had mutations in pik cd, none in pik r . most patients also had multiple gi manifestations. conclusion: immunopathology and genetic predisposition leading to eoe is complex. eosinophilic gi disease including eoe appears to represent significant gi pathology in apds. this implies that activation of pi k pathway may be directly involved in the etiology of eoe. abstract/case report text introduction: dominant negative mutations in stat (lof stat ; job's syndrome) cause a primary immune deficiency characterized by eczema, recurrent skin and lung infections, and connective tissues and skeletal abnormalities. over the last several years, vascular abnormalities causing tortuous and aneurysmal middle-sized arteries have increasingly been recognized. our institution has been imaging prospectively the coronary and cerebral arteries since - for brain imaging and from - for heart imaging. the purpose of this review is to provide an update on the extent of clinical manifestations noted in hies (continued) pain improved but rapidly worsened with tapering of steroids. the pet/ ct at that time, showed extensive hypermetabolic areas in lymph nodes, femurs, left acetabulum, left pubic ramus, right ischium, sacrum, both iliac bones, eight rib, t and t , both clavicles, both humeral, manubrium, and extension into musculature. initial biopsies were culture negative, s was positive for mac. she was referred to the national institutes of health where she was diagnosed with autoantibodies to interferon-γ . prior to referral, she initially was treated with azithromycin, ethambutol (need to discontinue due adverse effects), amikacin, rifampicin, linezolid, with not no evidence of clinical response. she underwent debridement of epidural anterior abscess to t . surgery involved t - laminectomy, and curettage on several bones. she presented unable to walk secondary to pain and neuropathies. initial laboratory: crp: mg/l; wc . ; hgb: . g/dl; ana : . (strongly positive) cd : ul her first course of rituximab consisted of doses of gm at d , , and monthly thereafter for a total of doses with clinical and radiographic improvement. she was maintained on optimal antibiotics therapymeropenem, rifampin, azithromycin, moxifloxacin, and clofazimine. two years after she completed rituximab, she presented to her home hospital with increased left hip pain and biopsy grew mac. retreatment with rituximab failed to show clinical improvement. her medical regimen was augmented with tedizolid and bedaquiline, however no iv antibiotics were added. rituximab was reinitiated, after the progression of symptoms despite treatment with rituximab; bortezomib, was trialed using the schedule based on the multiple myeloma literature. she completed full cycles (two at the nih, three at home). she subsequently has had clinical improvement and is working again and has not had progressive neurologic decline. the titers of antibodies to interferon-· did not follow the clinical improvement. however, we plan to keep her cd + cells zero and continue the bortezomib given her clinical and radiologic improvement. abstract/case report text for patients with primary immunodeficiencies (pid), finding a genetically defined diagnosis can be critical for prognosis, treatment, and counseling. however, for many patients, determining a genetic etiology remains elusive despite routine gene panel and exome sequencing because of an inability to resolve variants of uncertain significance (vus). crispr-based genome editing could be used to address this need by introducing patient-derived vus into primary human immune cells for further study; however, existing techniques are limited by poor efficiency. we recently developed novel non-viral techniques for large gene editing in primary human t cells and hematopoietic stem cells (hscs). we achieve up to fold greater efficiency than existing tools using crispr cas ribonucleoprotein nanoparticles that are non-covalently linked to homology directed repair (hdr) template dna. whereas mutation analysis has previously been limited to expensive and time-consuming animal models and transformed cell lines, we now have the ability to rapidly recreate any mutation in the native gene locus in otherwise healthy primary human cells. we demonstrate this ability by using our technique to knock-in well characterized loss-of-function mutations in jak and il rg and gain-of-function mutations in jak that are known to cause severe combined immunodeficiency and tumor growth, respectively. we show that these recreated mutations have the expected effects on t cell proliferation and intracellular signal transduction in the setting of il- stimulation. we then use our technique to investigate a prototype case of an adult patient with the unusual combination of common variable immunodeficiency, inflammatory arthritis and uveitis, and neutrophilic urticaria. genetic testing in this patient had previously revealed heterozygous coding vus's in four genes previously associated with a pid disease, including jak , but none of the specific variants have been previously reported. it is thus not clear which mutation (if not more than one) causes this patient's dysregulated cell activation, which limits targeted treatment options with kinase inhibitors or future gene or cell therapy. by knocking our patient's jak variant into primary human t cells and comparing these cells to those carrying wild-type, known loss-of-function, and known gain-of-function mutations, we are able to rapidly characterize the functional impact of our patient's variant and isolate its effect on his complex phenotype. this in vitro genetic engineering approach thus allows patient-specific vus to be modeled directly in primary human immune cells with a rapid turnaround time that is relevant for clinical applications, including molecular diagnosis and screening of pharmacologic or gene therapies. further, similar strategies could be leveraged as a potential basis for future gene correction therapy. abstract/case report text definition : "at variants" comprise a heterogeneous group characterized by the later onset of clinical symptoms, a slower progression, a prolonged lifespan compared to most patients with at and decreased levels of chromosomal instability and cellular radiosensitivity. in these patients, telangiectasia and / or immunodeficiency may be absent, while neurological features are present.( ). material and methods :a years old girl, born out of a non-consanguineous parents with clinical picture consisting in progressive alteration of the march ( months), associated to exotropia, no weight gain or height, alteration of balance while she is sitting, she walks by herself. she has acute bronchiolitis. results and discussion: brain magnetic resonance without alterations. abdominal ultrasonography and cpk normal. ophthalmologist assessment found exotropia, he did not find telangiectasia. motor and sensitive neuro-conduction were reported normal. alpha fetoprotein ng / ml increased. karyotype xx, non-structural alterations. normal auditory-visual evoked potentials. whole exome sequencing (wes): identified the small homozygous pathogenically deletion ¬c. + _ + del taag; p.? have a spelling effect in the splicing. it is not present in the population database or not as a known variant in function mutation of smad has been shown to increase smad phosphorylation in the nucleus in fibroblasts. further research is needed to examine the role of this mutation in t and b lymphocytes, given the interesting immunological phenotype of this patient. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. outbreak of mycoplasma pneumoniae-associated stevens-johnson syndrome characterization of children. with recurrent episodes of stevens johnson syndrome recurrent stevens-johnson syndrome secondary to mycoplasma pneumoniae infection recurrence and outcomes of stevens-johnson syndrome and toxic epidermal necrolysis in children syndrome after mycoplasma pneumoniae infection a review of causes of steven-johnson syndrome and toxic epidermal necrolysis in children submission id# expanded phenotype and genotype-phenotype correlations bsc(hons) msc bmbs mrcp dominant activating rac mutation with lymphopenia, immunodeficiency, and cytoskeletal defects an autosomal dominant scid form due to a gain of function mutation in the rac gene national institute of allergy and infectious diseases (niaid) professor biochemistry/molecular pharmacolog/nyu langone health canada head of the ircm bioinformatics core facility/montreal clinical research institute (ircm) associate investigator/national human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) translational and functional genomics branch/chief and senior investigator of the translational and functionanational human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) formula versus donor breast milk for feeding preterm or low birth weight infants cytomegalovirus (cmv) inacƟvaƟon in breast milk: reassessment of pasteurizaƟon and freeze-thawing prevenƟon of cytomegalovirus transmission via breast milk in extremely low birth weight infants submission id# the costa rican registry for primary immunodeficiencies department chief/department of pediatric immunology and rheumatology, national children´s hospital "dr. carlos sáenz herrera national children´s hospital "dr. carlos sáenz herrera" cid % (n= ), cid with syndromic features % (n= , at ), antibody deficiencies % (n= , xla ) ada deficiency, cd l-, flh- (syntaxin deficiency), ipex and osteopetrosis. nineteen patients developed malignancies, mainly non-hodgkin lymphomas among at patients conclusions: during the study period, pid were registered, mainly at and osteopetrosis cases. scid represented % and xla % of patients allergy and immunology/ department of pediatric immuunology and texas children's hospital director immunogenetics program/department of immunology, allergy and rheumatology at baylor college of medicine associate professor, director food allergy program/department of pediatric immuunology shearer center for human immunobiology associate professor/department of pediatric immuunology, allergy, and retrovirology granuloma faciale: successful treatment of nine cases with a combination of cryotherapy and intralesional corticosteroid injection intralesional steroid injection: a novel method to treat the symptoms of idiopathic granulomatous mastitis long-term effectiveness of intralesional triamcinolone acetonide therapy in orofacial granulomatosis: an observational cohort study sarcoidosis in the periocular scar as the first finding of systemic sarcoidosis: clinical-radiological characteristics a case of scar sarcoidosis of the eyelid submission id# null mutations: a gene dosage effect shubham goel, phd , hye sun kuehn clinical centre, nih, staff scientist/immunology service certified molecular geneticist/national institute of allergy and infectious diseases, national institutes of health genetic counselor/national institute of allergy and infectious diseases, national institutes of health division of intramural research; chief, immunopathogenesis section/laboratory of consultant/university hospital freiburg center for pediatrics and adolescent medicine cci -center for chronic immunodeficiency medical director professor/division of blood and marrow transplantation and cellular therapies new south wales, australia. professor of medicine and pediatrics/icahn school of medicine at mount sinai senior investigator/laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health functional nk assay, t/b/nk panel, tlr assay phd staff scientist/translational autoinflammatory disease section, lcim, niaid, nih, bethesda, md research fellow/translational autoinflammatory disease section post-baccalaureate fellow/translational autoinflammatory disease section pediatric rheumatologist/department of pediatric infectious diseases and immunology shupyk national medical academy for postgraduate education alder hey children's nhs foundation trust hospital, liverpool, uk pediatric rheumatologist/department of pediatrics dra. liliana bezrodnik y equipo dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños hospital de niños ricardo gutiérrez pathology department/children's hospital of philadelphia, philadelphia, pa post-doctoral fellow/translational autoinflammatory disease section results: we identified patients with de novo frameshift variants in samd l (c. dela, p.i lfs* reduced toxicity allogeneic hct with a busulfan, fludarabine regimen: a promising approach for non-cgd primary immune deficiencies requiring myeloablation? phd , blachy davila saldana college of medicine assistant professor/division of bone marrow transplant, immunedysregulation and immuno-hematology program, aflac cancer and blood disorders center instructor of pediatrics/division of bone marrow transplantation and immune deficiency, cincinnati children's division of bone marrow transplantation and immune deficiency, cincinnati children's national institutes of health certified molecular geneticist/national institute of allergy and infectious diseases genetic counselor/national institute of allergy and infectious diseases, national institutes of health operations manager and genetic counselor/national institute of allergy and infectious diseases collaborative bioinformatics resource/national institute of allergy and infectious diseases genome in a bottle analysis team/genome in a bottle consortium division of intramural research (dir), national institute of allergy and infectious diseases (niaid) medical genomics and metabolic genetics branch/national human genome research institute, national institutes of health table clinical and demographic characteristics, severity measures, and previous treatments for the pid cohort geographic region, n (%) north central , ( . ) ( . ) , ( . ) northeast , ( . ) ( . ) , ( . ) south ) ( . ) , ( . ) high-potency oral antibiotics , ( . ) ( . ) , ( . ) systemic high-dose corticosteroids copd, chronic obstructive pulmonary disease cd / cd : . , lymphocytes b , natural killer cell/mm . conclusions: although the atm activity corresponding to this new splicing mutation is unknown, it is presumed that it has some residual function, since splicing mutations is associated with better neurological prognosis have been reported (pidd) . at the immunodeficiencies research unit we discuss and pursue the molecular and genetic diagnoses for patients with suspected pid from all around the country. starting this year, we are processing and analyzing our own patients' wes results at the unit. evaluating the diagnostic yield of wes, as a measure of effectiveness or quality control, may result in process optimization and perhaps allow for better patient selection and resource allocation. objective: to describe and characterize our patients with suspected pidd whose dna samples were sent out for whole-exome sequencing; to analyze and compare our wes diagnostic yield after the first batches of patients; to identify patient attributes that may predict a positive diagnostic wes result. methods: genomic dna was obtained from whole-blood samples of patients with suspected pidd from hospitals in mexico city, monterrey and puebla. wes was performed using a ng sequencer (illumina hiseq) in new jersey (admera health, llc), with % coverage and a x depth of the idt xgen library, human genome version (december ). two fastq files for each patient sample were transferred back to our unit, where the bioinformatic workflow was completed. we used galaxy in the cloud for quality control, mapping & alignment, and detection of variants; variant effect predictor to process, map, annotate and filter variants; and igv (broad institute) and genome browser (ucsc) for visualization. we defined diagnostic yield as the proportion of patients with a genetic diagnosis after analysis of their wes results. we performed multivariate logistic regression, tree partitioning algorithm and linear abstract/case report text a -year-old male with x-linked chronic granulomatous disease (cgd) complicated by severe perianal disease and proctocolitis presented with two weeks of open draining lesions on the thighs, bilateral inguinal regions, and gluteal cleft. the wounds became excruciating and prevented normal ambulation. the patient was admitted for iv antimicrobial therapy, local wound care and systemic steroids. wound cultures, throughout his course, yielded growth of klebsiella pneumonia, candida parapsilosis, malassezia globose, escherichia coli, enterococcus faecalis and staphylococcus epidermidis allowing for directed antibiotic and antifungal therapies. despite improvement, the wounds persisted after several weeks of treatment. faced with recalcitrant cutaneous lesions despite aggressive systemic and topical therapies, we looked to alternative options. noting that other granulomatous diseases show response with intralesional corticosteroid therapy, we considered this for our patient ( , ) . for example, patients with idiopathic granulomatous cheilitis had a complete response after three monthly injections of intralesional corticosteroids ( ) . sarcoidosis patients also improve with intra-granuloma corticosteroid injection ( , ) . our patient received mg triamcinolone acetonide injections two separate occasions, administered in multiple open lesions at eight-week intervals. the cutaneous lesion improvement was gradual and complete resolution of the first open wound was noted fifty-two days from initial steroid injection. to our knowledge, intralesional glucocorticoid therapy has not previously been used to treat cutaneous disease in cgd patients. we are reporting the first cgd patient with successful lesion resolution following steroid injection as part of therapy. as such, we believe this case is significant and suggests that direct lesion injection with glucocorticoids can add to treatment options for cgd patients with recalcitrant cutaneous disease. serum igg, alone or in combination with iga and/or igm, were reduced in all patients. t-follicular helper cells were reduced only in patients carrying biallelic mutations (a.ii. , a.ii. and a.ii. ) but not in any patients with monoallelic tcf null mutation. t cell enumeration and function by means of proliferation was normal in all mutation+ individuals. no mutated (truncated) protein expression was detected from patients with either biallelic or monoallelic tcf null mutations. however, wildtype tcf protein was detectable in about half amount in heterozygous patients. cdna data showed either / or / wt/mutated transcripts ratios in homozygous or heterozygous individuals, respectively, suggesting mutated proteins instability; and all together, protein haploinsufficiency for the heterozygous cases. ex-vivo, cd l and il -induced plasmablast differentiation was found to be reduced in / patients tested ( biallelic patient a.ii. and monoallelic patients a.i. , b.i. and c.i. ). moreover, decreased igg, iga and igm production in vitro correlated with reduced plasmablast cell differentiation.in conclusion, all individuals carrying either mono-or biallelic null mutations have immunological penetrance of the b cell defect. however, while clinical penetrance was complete in patients with biallelic mutation, it was partial for those with monoallelic tcf null mutation suggesting a gene dosage effect for clinical penetrance. in addition, our study emphasizes that tcf is relevant to the plasmablast differentiation process as well as for ig production. further studies are being conducted to evaluate the individual roles of e and e on the immune and clinical features. background: heterozygous gain-of-function (gof) mutations in the stat gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections and autoimmune disease. furthermore, the nk defect we characterized illustrated an immature cd dim nk cell subset with decreased expression of cd , perforin, cd , and impaired cytotoxic capacity associated with increased susceptibility to viral infections observed in these patients. methods: in this study, we evaluated patients with novel stat mutations (d e mutation, located in coiled-coil domain and k q mutation, located in dna-binding domain). a third patient with the previously reported v i mutation (ccd) was also recruited for this study. in vitro, pbmcs from these patients were stimulated with ifn-α for , , and minutes and levels of phospho-stat on cd dim nk cell subset were measured by flow cytometry. the stat activity (firefly and renilla luciferase activities) was evaluated in u a-stat deficient cells abstract/case report text background: t cell lymphopenia associated with genetic syndromes can be identified with low t cell receptor excision circle (trecs) up on the newborn screen for severe combined immune deficiency (scid). jacobsen syndrome (js), q terminal deletion, is a rare genetic disorder seen in / , births characterized by facial dysmorphisms, platelet abnormalities, neurologic complications, immune system abnormalities including t and b cell defects. we report an infant with js found to have low trecs on nbs and review the immune phenotypes of our cohort of patients with js. methods: a retrospective chart review of all patients with js seen by the allergy immunology service at a large tertiary referral center from / / - / / was performed in accordance with irb standards. result: the index patient had two newborn screens hours after birth and two weeks later in accordance with texas state law. the first nbs resulted normal trecs while the second nbs had low trecs. a third nbs was done per protocol and again showed low trecs. subsequently, lymphocyte subsets at months of age showed severe t-cell lymphopenia: cd cells/ dl ( %), cd cells/dl ( %), cd cells/dl, and low recent thymic emigrants (cd +cd ra+ccr +cd +) cells/dl ( %) with normal lymphocyte mitogen proliferation. a chromosomal microarray (cma) revealed a q deletion known to cause js.over the five year study period we evaluated seven patients with js referred to our center. the majority of patients ( %) presented to clinic with history of recurrent infections including recurrent pneumonia, sinusitis, otitis media, skin abscesses and warts. t-cell lymphopenia was found in of ( %), / ( %) had abnormal lymphocyte proliferation (mitogens and antigens) and met criteria for pjp prophylaxis. in addition, / ( %) had antibody deficiency requiring igg replacement therapy. of the cases reviewed, only patients were born during the period of time that texas was performing the nbs. conclusion: jacobsen syndrome can present with a spectrum of immune defects most notably t cell lymphopenia and antibody deficiency. these patients can present at birth with low trecs. this cohort analysis highlights the importance of considering chromosomal genetic syndromes with features of primary immunodeficiency in evaluating patients with low trecs. further evaluation of larger cohorts gathered from neurology or genetics clinics at multiple centers would be helpful for future study in identifying those who need close immunology care. abstract/case report text introduction: recognized as sentinels of the immune system, mast cells (mcs) and dendritic cells (dcs) are both derived from hematopoietic/ progenitor stem cells in bone marrow (bm). the crosstalk and direct contact between these cells have been well documented and play an important role in modulating immune response. we showed that presence/absence of il- directs cell fate; whether progenitor cells will be differentiated into mcs or dcs. we also report an easy method in which in vitro generation of dcs is possible without external induction of gm-csf or il- . material-methods: to produce mcs and dcs in vitro, briefly bm samples were collected from patients with idiopathic thrombocytopenic purpura., bm mononuclear cells (mncs) were seperated by ficoll gradient, and seeded in plates with imdm medium containing fbs %, pen/ strep, and a little amount of methocult, and incubated in oc, % co (day ). the treatments on the following days were as follows: day , imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml); day , imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) + il- ( ng/ml). on day , groups were formed: group i: imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml) + il- ( ng/ml), group ii: imdm (fbs %) + scf ( ng/ml) + il- ( ng/ml), group iii: dmem + fbs %. the cultures were evaluated every days under an inverted microscope. verification of mcs was performed by toluidin blue and tryptaseimmunoflorescence staining. macrophages were verified by cd- immunoflorescence staining. dendritic cells of different stages of abstract/case report text introduction a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or anti-thymocyte globulin (gungor et al.) was efficacious in patients undergoing allogeneic hct for cgd. we report our experience with a similar approach for patients with non-cgd primary immune deficiencies needing a reduced toxicity myeloablative approach. methods we retrospectively reviewed records of consecutive patients who underwent allogeneic hct for primary immune deficiencies with a preparative regimen containing busulfan, fludarabine and alemtuzumab or anti-thymocyte globulin(atg), at three transplant centers between - . busulfan was given either every hours over days with target auc of to μmol/min (based on q hr. dosing) or twice daily over days with target auc of to μmol/min (based on q hr. dosing) or once daily over days with a target auc of - μmol/min (based on q hr. dosing). fludarabine mg/m to mg/ m was given divided over - days. serotherapy included alemtuzumab . - . mg/kg or atg . mg/kg given divided over days. gvhd prophylaxis consisted of cyclosporine and mycophenolate mofetil. results forty patients (was= , hlh= , cd l deficiency= , ipex/veoibd= , scn= , ifngr def./cid/x-scid/msn / lad= ) received busulfan, fludarabine and alemtuzumab or atg for allogeneic hct (first hct in patients and second hct in the patients with hlh). median age was . years (range, . years - . years). patients received a graft from an hla-matched related (n= ), unrelated (n= ), or single allele mismatched related or unrelated donor (n= ). all except one patient engrafted at a median of days (range, - days). one patient developed veno-occlusive disease and two patients developed diffuse alveolar hemorrhage. notably, it was the second transplant for all patients. eight patients ( %) developed grade - acute gvhd and patients ( %) developed chronic gvhd. one patient developed primary graft failure and two patients secondary graft failure. nineteen patients ( %) maintained full donor (> %) chimerism following allogeneic hct. twenty patients ( %) developed mixed chimerism, predominantly in the t-cell lineage, but t-cell donor chimerism progressively increased post-hct. at -year post-transplant, of patients ( %) with mixed chimerism had donor myeloid chimerism > % and t-cell chimerism > %. two patients underwent a second transplant for graft failure. there were deaths in the cohort. overall survival was %( of ) and event free survival was %( of ) at year. conclusion our experience suggests that a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or atg as serotherapy offers a promising approach with low toxicity, durable myeloid engraftment, low incidence of grade - gvhd and excellent survival and can be considered for a variety of primary immune deficiencies where myeloablative hct is desired. abstract/case report text this is an -year-old patient who has the diagnoses of anti-nmda receptor encephalitis with associated catatonia. the patient has history of a pineal gland germinoma diagnosed in august after months of double vision. however, after several months, the patient developed difficulty in sleeping, anxiety and nightmares. the patient presented to emergency department in february with personality changes. he was diagnosed with nmda encephalitis based on the clinical findings as well as presence of elevated serum and csf anti-nmda antibodies. the patient was initially treated with high dose systemic steroids with poor response. due to the worsening of his clinical condition, he was started on plasmapheresis, but had poor response to this therapy as well. these treatments are known standard treatment options for this condition. during his hospital stay, different therapies were discussed. cyclophosphamide was one of the treatment options, but because of the side effect profile and severe toxicity, we recommended different treatment modality. we started the patient on rituximab as well as sirolimus therapy to suppress both t and b-cell responses. after receiving two doses of rituximab in addition to daily sirolimus, the patient showed improvement of his symptoms. and declining nmda receptor antibody titers. this treatment plan was chosen for autoantibody mediated encephalomyelitis due to the fact that rituximab has inhibitory effect on naive b cells, but not on the proliferation of memory b cells and sirolimus has profoundly inhibiting role on memory b cells as well as a t-cell responses. a mosaic gene variant is one which is present in some, but not all, cells within an individual. they are increasingly associated with a number of diseases, including a number of primary immunodeficiency disease (pid). here we systematically analyzed mosaic variants in known or putative immunodeficiency genes from exome sequencing data from individuals. lofreq was used to identify mosaic variants with a variant allele fraction (vaf) of . - . ( . is the vaf for a non-mosaic, heterozygous variant). we removed variants from extreme read-depth (> standard deviations), unmappable, repeat-rich, and duplicated genomic regions. the average number of detected variants per mb was . and the total number of genomic locations with variants was , . mosaic variants were underrepresented in exonic regions, suggesting that coding variants may be deleterious. more mosaic variants were detected in saliva exomes compared to peripheral blood exomes (p-value < x - ), suggesting tissue-specific mosaic variants in buccal epithelial cells and white blood cells of saliva. to understand the clinical relevance of these findings, the variants were further filtered to include only nonsynonymous variants found in fewer than % of samples, leaving a total of , variant locations. of the remaining variant locations, had a pathogenic assertion in the human gene mutation database, and were in international union of immunological societies pid genes. further variant interpretations and clinical correlations are underway. these data suggest that mosaic variants in pid genes are common, vary by location of collection, and may have clinical diagnostic relevance. abstract/case report text introduction/background: the arpc b (actin related protein / complex subunit b) gene is a protein coding gene prominently expressed in blood cells and is necessary for the assembly and maintenance of the human actin-related protein / complex (arp / ). actin polymerization plays a central role in many immune functions including proliferation and differentiation of immune cells, migration, intercellular and intracellular signaling and activation of both innate and adaptive immune responses. defects in the actin cytoskeleton affect hematopoietic cells in the bone marrow and the immune response giving rise to a distinct primary immune deficiency, which is phenotypically similar to wiskott-aldrich syndrome (was). arpc b deficiency clinically presents as a severe multisystem disease which includes platelet abnormalities, recurrent infections, failure to thrive, inflammatory changes in the intestine, eczema, cutaneous vasculitis, eosinophilia, and elevated inflammatory markers. arpc b deficiency is rare and has only recently been described in the literature. here we present a clinical case of a patient found to have a pathogenic arpc b mutation via whole exome sequencing. case report: here we describe a month old somalian boy who presented to the immunology team at months of age with hematemesis, hematochezia, melena, failure to thrive, atopic dermatitis, hypothyroidism, autoimmune thrombocytopenia and recurrent infections concerning for a primary immune deficiency. family history was notable for parental consanguinity and an older sibling with a similar presentation of hemorrhagic gastroenteritis who died in kenya around months of age due to complications of his symptoms. the initial primary immunodeficiency evaluation revealed normal inflammatory makers, normal igg and protective vaccine (prevnar and tetanus) response. iga and ige were elevated at mg/dl and . ku/l respectively. flow cytometry was remarkable for t cell lymphopenia (cd , cd , and cd ) with reduced naïve cd and cd t cells. b and nk cell count were normal. alps panel and was protein expression was unremarkable. whole exome sequencing was performed and revealed homozygotic mutation of arpc b c. t>c, ivs + t>c which was predicted to be a pathological variant. subsequently, dhr flow cytometry with fmlp showed significant increase of dhp fluorescent (mfi . when compared to control mfi . ) consistent with findings from other arpc b deficiency patients. at the time of the most recent clinic visit, the patient has remained abstract/case report text introduction: activation of the nod-like receptor family cardcontaining protein (nlrc ) leads to the formation of an inflammasome. the inflammasome, a large cytosolic multiprotein complex of innate immunity, promotes proteolytic cleavage, maturation, and secretion of pro-inflammatory cytokines, including il- and il- . a gain-of-function mutation (gof) in the gene encoding nlrc or nlrc -inflammasomopathy is characterized by hyperinflammation with persistent elevated il- , infantile enterocolitis, and early-onset macrophage activation syndrome (mas). objectives: to describe phenotypic variation among three siblings with a novel nlrc gof variant and to expand our current understanding of the clinical manifestations of the disease methods: clinical and laboratory features were studied in three siblings of a hispanic family with a novel nlrc gof variant. results: a novel variant, c. g>a (p.arg gln) on the nlrc gene, was identified in a -year-old male with recurrent febrile episodes since one year of age. his laboratory findings showed highly elevated esr, crp, il- , and fecal calprotectin. his endoscopic finding was unremarkable. the recurrent fever partially responded to canakinumab. a -year-old sister with ileocolonic crohn's disease for two years was found with the same nlrc variant and highly elevated il- . crohn's disease was well controlled after adding infliximab infusion to methotrexate therapy. a -year-old sister, who has been asymptomatic and healthy, was tested with the same positive nlrc variant and highly elevated il- . the nlrc variant is inherited from their father, who currently has a diagnosis of psoriasis vulgaris. the il- levels of the three siblings show in the figure patients with flh- develop the classic hlh phenotype early in life, with periods of remission. the pathogenesis is associated with a defect of perforin-dependent cytotoxicity. t-ctl and nk cells fail to remove abnormal cells, consequently, an uncontrolled proliferation and activation of cd + t cells and macrophages develops and generates an inflammatory cytokine storm and a solid organs infiltration. mortality of fhl- is very high without treatment; allogeneic hsct is the only curative therapy. we report the outcome of a child with fhl- , four years after his treatment with an allogeneic hsct using an hla haploidentical donor. case report: a boy born in december , previously healthy, the only child of parents without consanguinity. he has a normal family and perinatal history. when he is years old, he begins suffering from with fever, arthralgia, hepatosplenomegaly and pancytopenia. he meets the diagnostic criteria for hemophagocytic lymphohistiocytosis (hlh) and received treatment with iv methylprednisolone and entered in remission of its hlh. six months later, he restarts his clinical picture of hlh and j clin immunol receives treatment with the hlh- protocol, which leads to remission. ayear later he presents a new episode of hlh that responds to ivig, cyclosporin a and dexamethasone. the possibility of primary hlh is then suggested. when he is years old, the molecular diagnosis is made with the identification of a mutation in the stx gene, the case was classified as fhl- due to syntaxin deficiency. in october , a hsct was performed. at the age of , his clinical status is evaluated and laboratory studies show that his immunodeficiency due to syntaxin deficiency was cured. his -year-old mother, hla haploidentical was used as a donor and a protocol developed for the treatment of osteopetrosis was applied. the hsct protocol did not use a tcell depleted graft. hstc conditioning was done with melphalan days - and - , fludarabine days - to - , anti-thymocyte globulin days - and - , and cyclophosphamide day - . (figure) . clinical and demographic characteristics, including the use of a novel claims-based weighted algorithm (risk vital sign; rvs) and those initiating ivig and scig, were described. stratified analysis based on pid diagnosis codes was performed. probability of receiving available ig treatments based on baseline characteristics was evaluated by logistic regression and propensity score methods. results: selected clinical and demographic characteristics, severity measures, and previous treatments between the overall pid population (n= , ), pid patients initiating scig (n= , ), and pid patients initiating ivig (n= , ) are presented in the table. patient characteristics and previous treatments tended to be stable, although hypertension, obesity, and corticosteroid use increased during the study period. new ig users tended to be older and female, with increased depression, dyslipidemia, and hypertension than all pid patients. new scig users had more diagnoses of respiratory (e.g., asthma, copd) and inflammatory (e.g., arthritis, fibromyalgia, inflammatory bowel disease) comorbidities and less cancer than all pid patients. new scig users compared with new ivig users had increased asthma and copd, fibromyalgia, and inflammatory bowel disease and decreased cancer and peripheral vascular disease. previous corticosteroid use was higher in ig users than all pid patients. among scig users, prior pid treatments of iv antibiotics, and oral high potency antibiotics were similar to all pid patients. ivig users had higher iv antibiotic and antifungal use. rvs was initially developed to identify patients likely to have undiagnosed pid. this analysis applied rvs to patients diagnosed with pid to assess severity. rvs based on -year history in the overall pid cohort was predominantly low, with only . % of patients scoring in the medium and high ranges. rvs was increased in incident ig users, with ( . % medium/high for scig; . % medium/high for ivig). in other markers of severity, scig users had more sinusitis and ivig had more pneumonia than all pid. ig users had fewer abscesses, cellulitis, and otitis media than the full pid cohort. conclusions: this exploratory analysis showed a trend toward increased hypertension, inflammatory and respiratory comorbidity, higher rvs, and previous corticosteroid treatment in patients initiating on ig compared with all pid patients. results could be confounded based on pid diagnosis codes used and warrants further research. author disclosures: mp, cas, and zh are employees and stockholders of the takeda group of companies. jo is a consultant to takeda. jbl is an employee of rti health solutions, an organization funded by takeda to conduct this research. mer was an employee of rti health solutions at the time this research was conducted.presenting author: colin anderson-smits submission topic: immunoglobulin replacement therapy patients in a larger cohort group and to determine if there are patterns of disease not previously reported. methods: we performed a retrospective chart review of patients with lof stat (n= ) followed at the national institutes of health. we specifically looked at tortuosity, aneurysms, and dilation of both coronary and cerebral arteries. epidemiologic information, stat mutation, co-morbidities, and laboratory information were reviewed along with imaging studies, specifically brain mri, brain mra, heart mri, and coronary ct. results: most recently, patients with hies are found to have vascular abnormalities including tortuosity, dilatation, narrowing, and aneurysms of middle sized, cerebral, and coronary arteries. in an effort to determine the extent of vascular involvement in addition to miscellaneous organ involvement, we are reviewing a cohort of patients with hies who were evaluated at the nih. of these patients, are women and are men. of these patients, two have passed away due to vascular events leading to their deaths. there are four patients under the age of , patients between the ages of and , patients between the ages of and , and three patients above the age of (age range - , mean age . ).of the patients, five of these patients were found to have abnormal brain mri/mra at an approximate rate of . %. two of these patients were found to have at least one cranial aneurysm, two of these patients were found to have a level of narrowing or stenosis, and one patient was found to have dilatation.in terms of coronary abnormalities, of the ( . %) patients were noted to have at least one coronary abnormality including dilatation, aneurysm, or tortuosity on heart mri or coronary ct. eight patients ( . %) were found to have dilatation of which four patients were female and patients were male. of the patients, ten patients ( . %) were found to have at least one aneurysm. there were patients ( %) that were found to have at least mild tortuosity. conclusion: vascular abnormalities in our lof stat patients occurred at an exceedingly high rate-cerebral and coronary artery, . % and . % respectively. due to this, patient's with lof stat should be considered for screening with brain and heart imaging. currently, there are no guidelines which outline the appropriate timeline for screening in these patients however following these patients over time will allow us to determine the most appropriate interval for imaging follow up. abstract/case report text year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis and several micro nodules, chronic diarrhea without diagnosis (colonoscopy with mild colitis, without cmv. no bacterias no parasits were found in stools), mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was followed up in other center and in she was diagnosed with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but with low adherence to it. she did not have referred history of lymphoproliferation nor significant viral infections. she had a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid diagnosis, she deceased at years old due to pulmonary infection. one years old son has anemia. her other daughter and son are healthy. objective: to describe unique management of recurrent viral respiratory infections in a qds patient. case: -year-old male with a history of qds complicated by truncus arteriosus, vsd, hypoparathyroidism, asthma, and autism was followed for a history of "frequent pneumonias." he had daily rhinitis and a history of frequent otitis media which resolved after tympanostomy tube placement at age y. however, over the next two years, he had admissions with various respiratory viral infections resulting in respiratory distress and prolonged oxygen need. viruses detected during these separate admissions varied and included parainfluenza , metapneumovirus, rsv, b and coronavirus nl , coronavirus hku , and rhino/enterovirus ( times total). he was previously on a prophylactic course of antibiotics which made no difference in his symptoms. laboratory evaluation showed protective adaptive immunity with normal immunoglobulin numbers for age (igg mg/dl), along with normal b and t cell numbers. he had protective pneumococcal titers and mounted a normal mitogen response. while his nk cell numbers were normal, his nk t cells were low. his tlr functioning also appeared normal. in order to decrease his overall illness burden and to keep him out of the hospital, ivig infusions (~ mg/kg) were initiated monthly. shortly after initiation of treatment, his nasal purulence and drainage resolved and his family noted that he became more active and playful. treatment was continued for months, during which time he had only one episode of influenza infection needing inpatient management. he has been off of ivig for months without recurrence of his viral infections. conclusion: in this patient, severe recurrent viral respiratory infections despite apparently normal adaptive and cellular immunity presents a unique management dilemma. this was not an issue of recurrent bacterial infections as prophylaxis did not make a difference in the frequency of his infections. his nk t cells were low, which could have contributed to his frequent viral infections as nkt cells are known to play a role in viral immunity. the successful use of ivig treatment in his case points to a different use for ivig, namely for the anti-respiratory virus antibodies which are presumably contained within the formulation.given this finding, it may be prudent to consider ivig in management of qds patients, even with normal immune evaluation, in order to decrease risks of complications associated with severe and recurrent viral respiratory infections. abstract/case report text introduction: smad is a critical downstream signaling molecule for transforming growth factor-β (tgf-β) and bone morphogenic protein (nmp ). initially, smad , also known as dpc deleted in pancreatic cancer locus , was described as a tumor suppressor gene, and somatic deletion of the smad is seen in % of pancreatic carcinomas. subsequently, a germline point mutation in the smad gene (p. i v) was reported to cause myhre syndrome (mim# ). myhre syndrome is an autosomal dominant disease characterized by cognitive impairment, hearing loss, and musculoskeletal anomalies. the immunological phenotype of these patients has not been previously described, despite the critical role of tgf-β in regulating t cell response and the prevention of excessive inflammation. case report: a -year-old boy with myhre syndrome was referred to immunology clinic for evaluation of recurrent ear infections. he developed acute otitis media infections as an infant and had tympanostomy tubes placed at one year of life. he also had a recurrent sinus infections. at two years of age, he was diagnosed with autism and sensory neuronal hearing loss. brain mri showed a mildly hypoplastic pituitary gland, and a thickened corpus callosum with decreased myelination. given these findings, whole-genome sequencing was performed, which revealed a heterozygous de novo mutation in smad (p.i v / c. a>g) consistent with the diagnosis of myhre syndrome. through age , he was in the th percentile for height until he was started on growth hormone, which he responded to robustly. he is now he is in the th percentile for height. he had adenoidectomy due to sleep-disordered breathing at the age of years. he is maintained on montelukast and inhaled corticosteroids for treatment of rhinitis and mild persistent asthma. he is on atenolol for the treatment of primary hypertension. on physical exam, he has facial dysmorphisms, thickened skin, and contraction of the fingers consistent with myhre syndrome. immunologic elevation showed significant hypogammaglobulinemia (igg mg/dl), low iga ( mg/dl) and normal igm mg/dl. igg subclasses showed low igg and igg at mg/dl and mg/dl respectively. although he was fully vaccinated, his tetanus antibody was low at . iu/ml. however, this improved after repeat vaccination to . iu/ml. total t and b lymphocyte counts were normal; however, his memory cd and cd t cells were low for age at . % and . %, respectively. additionally, his switched memory b cell count was low at . %. conclusion: smad gain of function (myhre syndrome) can lead to impaired memory t and b cell formation with significant hypogammaglobulinemia and low iga. although the patient was able to respond to protein vaccination (tetanus), it is not clear if he will be able to maintain a long-term response. in a previous study, a similar gain of key: cord- -ecq ye authors: rath, barbara; conrad, tim; myles, puja; alchikh, maren; ma, xiaolin; hoppe, christian; tief, franziska; chen, xi; obermeier, patrick; kisler, bron; schweiger, brunhilde title: influenza and other respiratory viruses: standardizing disease severity in surveillance and clinical trials date: - - journal: expert rev anti infect ther doi: . / . . sha: doc_id: cord_uid: ecq ye introduction: influenza-like illness is a leading cause of hospitalization in children. disease burden due to influenza and other respiratory viral infections is reported on a population level, but clinical scores measuring individual changes in disease severity are urgently needed. areas covered: we present a composite clinical score allowing individual patient data analyses of disease severity based on systematic literature review and who-criteria for uncomplicated and complicated disease. the -item vivi disease severity score showed a normal distribution in a pediatric cohort of children aged – years (mean age . ; s.d. . ; range: to . ). expert commentary: the vivi score was correlated with risk of antibiotic use as well as need for hospitalization and intensive care. the vivi score was used to track children with influenza, respiratory syncytial virus, human metapneumovirus, human rhinovirus, and adenovirus infections and is fully compliant with regulatory data standards. the vivi disease severity score mobile application allows physicians to measure disease severity at the point-of care thereby taking clinical trials to the next level. influenza-like illness (ili) and acute respiratory infections (ari) in children are common. the clinical presentation may range from subtle to severe symptoms requiring advanced medical care [ , ] . the wide spectrum of disease presentations and the role of risk factors (rfs) in terms of disease severity are poorly understood. laboratory diagnostics are not usually ordered in routine care [ ] [ ] [ ] [ ] . surveillance programs should rely on laboratory-confirmed cases rather than clinical suspicion to solve the denominator problem. this will allow the timely detection of virus-specific seasonality in a given (sub)population [ ] . an even greater challenge will present itself when investigators wish to determine the impact of different respiratory viruses on disease burden [ ] . a deeper understanding of disease severity in relation to specific respiratory viruses will help in the monitoring of the real-world impact of 'natural' or untreated disease as well as preventive measures and therapeutic interventions such as vaccines and antivirals. the timely detection of seasonality will help with the targeted and costeffective use of viral diagnostics in hospital-based surveillance settings. ideally, viral diagnostics should be aligned with simultaneous standardized disease severity assessments. standardized measures of disease severity are urgently needed for clinical trials of vaccines and antivirals currently in development for ari caused by influenza (flu), respiratory syncytial virus (rsv), human metapneumovirus (hmpv), adenovirus (adv), or human rhinovirus (hrv) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, it would be desirable to assess, at the point of care, which patients are suffering from severe disease in relation to their perceived rf profile, and to use such point-of-care assessments to individualize the use of anti-infective therapy. experience during the recent influenza pandemic has shown that influenza disease severity appears rather unpredictable, especially in young patients. whilst the majority of adults with severe disease did have previously identifiable rfs, the majority of children affected by severe disease did not [ , ] . the expected or perceived risk of severe outcomes may also influence a physician's decision to test a patient for influenza and other respiratory viruses [ ] . there is little consensus on which symptoms should trigger a physician's suspicion, and local practices differ significantly from site to site and from season to season [ , [ ] [ ] [ ] [ ] . comprehensive reviews of the published literature and disease severity measurements used in clinical trials and surveillance systems are lacking. the numerous observational studies and clinical trials assessing the prevention and treatment influenza and other respiratory viruses have been rather inconsistent. commonly used indicators of disease severity such as 'hospitalization,' a diagnosis of 'pneumonia,' and other adverse outcomes including mortality are known to be highly dependent on the studied population, the medical setting, the choice of data sources, and the availability of resources [ ] . head-to-head comparisons and meta-analyses comparing different preventive and treatment interventions will require universally accepted disease severity measurements. sentinel surveillance systems tend to focus on private practices and laboratory testing based on clinical suspicion on behalf of primary care providers working at surveillance sites [ ] . with children being the most prominent transmitters of influenza, pediatric emergency rooms and large tertiary care hospitals are ideal sites to monitor seasonality covering the entire spectrum of clinical presentations [ ] [ ] [ ] . to create a model system free of selection bias, a perennial quality management (qm) program was instituted at a large pediatric academic center in collaboration with the national reference centre for influenza and other respiratory viruses [ ] [ ] [ ] [ ] [ ] . the specific aims of the presented analyses are ( ) to develop a standardized approach to measuring ili disease severity based on literature review and who guidelines and ( ) to apply new mathematical models to the real-time surveillance of ili in large tertiary care centers. ( ) studies were not one of the following: randomized clinical trials, non-randomized clinical trials, observational studies, or epidemiological studies; and ( ) studies lacked any clinical criterion for disease severity. animal studies, adult studies, meta-analysis, and review papers were also excluded. based on the systematic literature review, the vivi disease severity score was developed as a -item weighed clinical composite score, according to who-criteria of uncomplicated and complicated disease [ ] . the vivi disease severity score is comprised of items describing signs and symptoms of uncomplicated disease (disease severity, uncomplicated: dsu, weighed single-fold) reflecting 'regular' ili activity, whereas the items describing parameters consistent with complicated disease (disease severity, complicated: dsc, weighed threefold) indicate high-impact clinical presentations in the target population (textbox ). the vivi disease severity score was subsequently user tested as a web-user interface as well as a mobile application for tablet computers, to be used at the point of care. based on the most commonly cited rfs for severe disease in the pediatric or adolescent age group, a simple rf score was composed [ ] [ ] [ ] [ ] . the vivi risk factor score (textbox ) was implemented on the same mobile application to allow the reporting of disease severity in relation to previously identifiable rfs in the individual patient. the consultation index is an epidemiological indicator reported weekly by the national reference centre for influenza and other respiratory viruses and the influenza working group, based on the proportion of ari at representative sentinel practices across the country [ ] . the consultation index represents a timely indicator of any deviation from a baseline rate of ari cases presenting to the respective sentinel practices. a 'normal ari activity' is assumed if the consultation index remains below . increased activities are typically measured during the winter months, when seasonal viruses circulate in the community. fluctuations in ari activity as measured by the consultation index represent a useful indicator of disease burden based on actual case numbers. reporting of the number of cases, however, does not reveal information on disease severity with each individual case. by plotting the consultation index with the corresponding average vivi disease severity score in the same graph, we obtain a comprehensive picture of ari disease burden that is based on both actual case numbers and case severity. figure (a) illustrates that disease severity does not always follow the peaks and troughs of case numbers as measured using the consultation index [ ] . the vivi score and the consultation index are therefore measuring opposing end points; one is based on individual disease severity per patient (vivi disease severity score) and the other serves as an epidemiological indicator of ari activity and the overall disease burden within the national surveillance system (consultation index). the vivi disease severity and risk factor score were user tested in the context of a qm program for children with ili at a large pediatric hospital in germany as described previously [ ] [ ] [ ] [ ] [ ] . according to the standard operating procedures, patients with a physician diagnosis of ili and/or fulfilling predefined case criteria (body temperature ≥ °c and ≥ respiratory symptom) admitted to the emergency department (ed) or pediatric inpatient wards, participated in the qm program [ ] [ ] [ ] [ ] [ ] . independent of routine clinical care, a specifically trained qm team obtained nasopharyngeal samples and performed standardized clinical assessments using the vivi disease severity score in line with who criteria for uncomplicated and complicated influenza [ , , ] . the vivi disease severity score was recorded at the first consultation with patients participating in the qm program. physicians in routine care were blinded to the results of the clinical assessments by qm staff, and they were unaware of the vivi disease severity scores assigned by the qm team. qm staff on the other hand assessed patients prior to allocation and treatment decisions on behalf of the clinical team in routine care [ ] . nasopharyngeal specimens were delivered to the national reference centre for influenza and other respiratory viruses for individual rt-pcr testing influenza virus a and b, rsv, hmpv, hrv, and adv as described below. from december until april , a total of children aged - years participated in the qm program. the qm program included both in-and outpatients to represent the broadest possible spectrum of disease severity. from to , all patients presenting the ed were screened for ili criteria once weekly, regardless of whether they were subsequently admitted to the hospital or not. from onward, daily screenings of all inpatients were added (including weekends and holidays). the qm team performed the disease severity assessments independently and the results remained unknown to the routine staff. hence, the data acquired by the qm team did not have any influence on treatment or hospitalization decisions. also, the treating physician did not know the result of the rt-pcr testing when deciding on neuraminidase inhibitor treatment. patients with laboratory-confirmed influenza infection were invited to participate in follow-up assessments whenever feasible. follow-up visits in the qm program were voluntary and scheduled according to the parent's preferences. during follow-up visits, the vivi disease severity score assessment was repeated and recorded by the qm team using the same procedure as during the initial assessment. nasopharyngeal samples were repeated and sent for analogous rt-pcr testing [ ] . the qm program was approved by the institutional review board (ea / / ). informed consent procedures were waived for enhanced quality of care and infection control [ ] [ ] [ ] [ ] [ ] ]. nasopharyngeal swabs were washed out in a total volume of ml of cell culture medium either individually or pooled per patient. rna was extracted from µl of patient specimen using the magattract viral rna m kit (qiagen, hilden, germany) and eluted in µl elution buffer. alternatively, rna was extracted using the magna pure dna and viral na small volume kit (roche deutschland holding gmbh, mannheim, germany) from µl specimen with an elution volume of µl. a volume of µl of extracted rna was subjected to cdna synthesis applying u m-mlv reverse transcriptase (invitrogen, karlsruhe, germany) in a total volume of µl. all cdna samples were analyzed by rt-pcr for the presence of each of the pathogens influenza virus a and b, rsv, hmpv, hrv, and adv as published previously [ ] [ ] [ ] [ ] [ ] . a descriptive analysis of the study sample was performed by calculating proportions and summarizing continuous variables using mean (standard deviation and range) and median (interquartile range). histograms and box plots were used to illustrate the distribution of vivi disease severity scores. correlations between the vivi disease severity score and the consultation index were assessed using scatter plots and pearson's correlation coefficient. the mean difference in vivi disease severity scores was compared across patient and clinical characteristics. statistical significance was assessed using the t-test or the chisquared test as appropriate. to test whether patients with elevated vivi disease severity scores also had elevated rf scores, we performed correlation analysis using pearson's correlation coefficient. these analyses were conducted using stata version (statacorp lp, texas, usa). we further performed regression analyses to identify a set of influential rfs that could model a linear correlation: vivi disease severity score = w × rf , w × rf , . . ., w n × rf n. here, w i is the respective weight factor for feature i in the regression model [ ] . in a subset of patients with laboratory-confirmed influenza infection during the / and / winter seasons, the vivi disease severity score was also used to follow patients longitudinally with respect to viral load and disease severity over time [ ] . to assess the relationship between vivi disease severity score and virus load, we performed pearson correlation analyses for all records, for which more than two followup time point with virology and vivi disease severity scores was available. decision tree analysis [ ] was performed to study the relationship between subgroups with a strong positive and negative correlation between disease severity and virus load. as an objective and data-driven measure to detect seasonality of respiratory viral infections in acute care settings, we introduced time series analysis with change point (cp) detection. the goal of cp detection algorithms is to identify changes in the dynamical behavior within a time series [ ] . the main difference to a statistically oriented analysis is that it assumes that an intrinsic dynamics model generates the data. cp detection therefore identifies those time points, when time series trends start differing significantly from previous data. this procedure allows identification of critical time points when weekly average numbers of laboratory-confirmed influenza infections start to increase (or decrease) compared to preceding weeks. for further detail on cp detection, please refer to the supplemental data. in this paper, we used the cp detection approach to analyze the qm dataset, which allowed computing averages of target variables assigned to respective calendar weeks (such as average rates of laboratory-confirmed influenza infections per calendar week, average disease severity per calendar week, etc.). for the detection of seasonal patterns, we used the following three-step algorithm. ( ) the data were clustered using k-means clustering [ ] into potential seasons. we used k = to model two main seasons (high and low) and a transition between those seasons. ( ) we assigned a preliminary cp to a week w t , if the cluster assignment c(w t ) to the respective week differed from the cluster assignment to the preceding week, i.e. if c(w t ) ≠ c(w t− ). ( ) finally, we computed a list of preliminary cps that would split the dataset into time frames tf . . .tf n , where each time frame t i was defined to lie between two consecutive cps. we then checked for each preliminary cp, whether the values before and after the cp (for the two time frames tf i− . . .tf i ) differed significantly (p < . ) based on a t-test. this procedure ensures that two regions separated by a cp are indeed different. all preliminary cps fulfilling the above criteria were reported. the systematic literature search yielded potentially relevant articles. among these, articles were excluded based on the criteria mentioned above. an additional studies lacked specific criteria for disease severity. finally, a total number of eligible articles were identified, the characteristics of which are summarized in textbox . it became evident that several clinical parameters were shared by multiple studies, as for example hospitalization, oxygen requirement, labored breathing, (p)icu admission, mortality, feeding problems/dehydration/vomiting, fever, wheezing or abnormal breath sounds, etc. all of these commonly used criteria were included in the vivi disease severity score (see also: tief et al. [ ] , in textbox ) except for mortality, which is usually recorded separately in hospital records. the vivi disease severity score was validated in the full qm cohort comprised of patients aged - years (mean: . years; sd: . ; range: - . years). a percentage of . of the qm program participants was under the age of year, . % were aged - years, . % were in the age group - years, and . % were aged - years. a total of ( . %) of the participants were male. a total of ( . %) participants were prescribed antibiotics while only ( . %) were prescribed antivirals in hospital. at presentation, ( . %) were assessed as being in need of hospitalization with ( . %) being in need of intensive care (including assisted ventilation and extracorporeal membrane oxygenation). with regard to viral etiology, in decreasing order of frequency, we identified rhinovirus ( . %), rsv ( . %), adv ( . %), a(h n ) influenza virus ( . %), metapneumovirus ( . %), a(h n ) influenza virus ( . %), influenza b viruses of the victoria-lineage influenza ( . %), and type b viruses of the yamagata-lineage ( . %). in . % of the cases, there was more than concurrent viral infection. table summarizes the findings from the rf assessment exercise carried out as part of the quality monitoring and table summarizes the clinical symptoms at presentation. a total of patients ( . %) had chest-radiography in the ed. chest radiography findings showed that ( . %) had pneumonia, ( . %) had bronchitis, ( . %) had bronchiectasis, ( . %) had bronchiolitis, and ( . %) had other non-pneumonia abnormalities. one hundred and nineteen ( . %) had a lumbar puncture done in the ed and ( . %) had cerebrospinal fluid (csf) chemistry done. sixty-nine ( . %) had csf cultures done with only four ( . %) sample positive for bacteria ( bacillus species, staphylococcus epidermidis, staphylococcus hominis, and unspecified bacteria positive). no cases of streptococcus pneumoniae were identified on culture. during hospitalization, ( . %) had a lumbar puncture and ( . %) had csf chemistry and culture done. four ( . %) samples were positive for bacteria including escherichia coli, micrococcus, staphyococcus epidermidis and streptococcus salivarius, streptococcus mitis/oralis as well as enterovirus in two cases. no cases of streptococcus pneumoniae were identified on culture. a total of ( . %) had a chest radiograph during their inpatient stay. inpatient chest radiography findings showed that ( . %) had pneumonia, ( . %) had bronchitis, ( . %) had bronchiolitis, ( . %) had bronchiectasis, and ( . %) had other non-pneumonia abnormalities. in total, ( . %) of the study participants had been diagnosed with pneumonia on chest radiography at some point during hospitalization. there were two ( . %) deaths recorded in the emergency room. one of the deaths was attributed to encephalitis and sepsis following infection. the cause of death in the second patient was related to serious underlying cardiac disease in a young infant. the vivi disease severity scores showed a normal distribution with a mean score of . (sd: . ; range - ) at initial assessment ( figure ). the vivi disease severity score was significantly higher in patients with the need for hospitalization (mean difference [ % ci]: − . [− . to − . ]; p < . ), with a need for critical care facilities (mean difference [ % ci]: − . [− . to − . ]; p < . ) as well as in those with signs of primary or secondary bacterial lower respiratory tract infections (mean difference [ % ci]: − . [− . to − . ]; p < . ). the median risk factor score in this cohort was (iqr: - ); the median rf score was . (sd: . ) and scores ranged from to . the cp analysis was applied to detect seasonal patterns for each virus detected in the qm cohort. we define a virus to be seasonal if it is not present during the whole year. with this definition, we found that influenza viruses (figure ), as well as rsv and hmpv, showed a strong seasonal behavior ( figure ) with predominance during the northern hemisphere winter months. adv and hrv were 'rapid cyclers' with frequent and brief peaks throughout the year ( figure ). the cp method showed that in a hospital-based syndromic surveillance system, seasons can be detected and defined in real time for each of the respiratory viruses. during the post-pandemic / season for example, influenza a(h n )pdm viruses continued to predominate in the qm cohort. influenza a(h n ) viruses, on the other hand, were absent during the / and / seasons but replaced pandemic h n strains during the subsequent season (see figure ). also, differentiation of influenza b lineages revealed that influenza b yamagata and victoria viruses did not always circulate annually but instead showed alternating patterns. the cp analysis was also used to identify fluctuations in average disease severity per calendar week in the qm program ( figure ). the initial period until summer of , when the qm program was restricted to once-weekly screening of in-and outpatients in the ed, is visually separated from the full surveillance phase beginning with the / winter season, when daily screenings of all inpatients hospitalized with suspected ili fever defined as >= °c. * includes the following items: supplemental oxygen and clinical parameters, such as: oxygen saturation (o sat), o sat-to-fio ratio (o sat/ fio ), or hypoxia. there was a small but significant difference in vivi disease severity scores between those subjects where no viral etiology could be detected (mean vivi disease severity score: . ; sd . ), those identified with a single viral infection (mean vivi disease severity score: . ; sd . ) and those with more than concurrent viral infection (mean vivi disease severity score: . ; sd . ); p (anova) < . . for each patient in the qm cohort, we computed the overall vivi disease severity scores as well as the component of the dsu and dsc symptom category, respectively (table ) . average disease severity with different respiratory viral infections revealed that rsv induced the highest level of disease severity followed by hmpv, influenza a(h n ), and hrv infections. disease severity with adv, influenza b, and influenza a (h n )pdm viruses remained below average ( table ). the vivi disease severity score distributions including viral coinfections are displayed in figure . to illustrate the comparison, we computed the average vivi disease severity score per calendar week and compared to the consultation index during the same week ( figure (a) ). as expected, no significant correlation was observed between weekly vivi disease severity scores and the consultation index (pearson's correlation coefficient r = . ; p = . ) during corresponding weeks, indicating disease severity and case numbers are not linked. to allow visual interpretation, we also plotted the weekly average vivi disease severity score (in the ed prior to october and in ed and inpatient units thereafter) against the time course of the respective seasonal viruses. the results are shown in figure (b). the viruses circulating (represented in % of all qm patients tested: y axis) are shown in relationship to the average vivi disease severity score during the respective calendar week. some viruses peaked simultaneously with the average disease severity but a cumulative effect was more common. the effect of viruses prevalent during the summer months was more pronounced when inpatients were included in the qm program, thus including severe cases requiring hospital admission. the pearson's correlation coefficient r was . indicating a statistically significant but weak positive correlation between vivi disease severity and risk factor scores (p < . ). the distribution of vivi disease severity scores by different rfs is illustrated in table . to evaluate which of the rfs as defined by who [ ] (textbox ) had the highest impact on disease severity (i.e. vivi disease severity score), we performed regression analysis as follows: vivi disease severity score = w × rfs , w × rf , . . ., w n × rf n. regression analysis revealed that there was no specific set of variables that could be used to model this relationship significantly well. the best subset of rf variables was 'rf : infant < years of age,' 'rf : cardiac condition,' 'rf : pulmonary condition,' 'rf : obesity,' and 'rf : diabetes' together yielded a r goodness-of-fit of . . using all rf variables yielded a r of . . to further explore the relationship between age and rf, we studied median and mean vivi disease severity score in infants in children below years of age, and in children aged years and above (table ) . we then performed pearson correlation to test for a potential relationship between vivi disease severity score and patient age. the pearson collation revealed r = − . , suggesting that there is in fact no significant correlation between the vivi disease severity score and patient age. an increasing vivi disease severity score indicates increasing disease severity. the key aspect of the vivi disease severity score is that it provides data standardization across the full spectrum of severity as well as comparison within a cohort, and between different seasons or sites. in the future, this may allow the comparison of various treatment decisions in clinical trials and observational settings. as described above, physicians in routine care were unaware of the results of vivi disease severity score assessments by qm staff and reversely, qm staff were unaware of treatment decisions when assessing a patient. analysis of vivi disease severity score results revealed that disease severity in patients (with any virus) who were prescribed neuraminidase inhibitors was considering the variability in disease presentations and courses of illness with influenza and other respiratory viral infections in children, the vivi disease severity score is not intended to be validated against future clinical events or outcomes. to assess whether the vivi disease severity score could be used to standardize consecutive follow-up visits in clinical trials, a total number of qm patients with influenza diagnoses were followed longitudinally with virology (pcr) and disease severity assessments over time. the overall pearson correlation between vivi disease severity score and virus load (using cycle threshold = ct values) over time was . . a closer look at the correlation histogram ( figure ) revealed three major subgroups: the largest group of patients can be categorized as having a moderate to strong positive correlation (r ≥ . ) between disease severity and viral load over time; a second group of patients showed a strong negative correlation (r ≤ − . ). a third group of patients showed a weak (positive or negative) correlation (− . < r < . ). preliminary decision tree analysis of these groups suggested that a vivi disease severity score below and the rf 'infant below years of age' were connected to a negative correlation between virus load and disease severity. respiratory infections are among the most common reasons for children to be admitted to pediatric hospitals. hospitalbased surveillance of respiratory viral infections is of great value to understand the full disease spectrum, from mild symptoms to serious presentations. children are the most avid transmitters of respiratory viral infections, and the information gained from syndromic surveillance in children's hospitals can complement decentralized sentinel surveillance systems in a meaningful way [ ] . with the advent of rapid diagnostics and mobile health applications, it has now become possible to monitor virological and clinical end points in real time [ ] [ ] [ ] [ ] [ ] [ ] [ ] . traditionally, disease activity is monitored based on epidemiological parameters such as ili or ari incidence, hospitalization rates, or mortality [ , ] . the consultation index was developed by the robert koch institute and has proven to be a sophisticated epidemiological tool to assess background ari activity at representative sentinel practices. fluctuations in ari activity in private practices represent a useful indicator of disease burden based on actual case numbers. reporting of the number of cases, however, does not reveal information on disease severity with each individual case [ ] . the vivi disease severity score aims to fill this gap. the vivi disease severity score is a -item weighed clinical composite score consisting of dsu items reflecting 'regular' ili activity and dsc items indicating 'high-impact' clinical presentations in the target population [ ] . the vivi disease severity score opens avenues to new individual patient data (ipd) analyses, for example to identify clinically relevant seasonal patterns of disease severity linked to different viral diagnoses confirmed in the same group of patients. the vivi disease severity score also allows consistent measurements of disease severity when following individual patients over time, as would be the case in clinical trials [ ] . follow-up assessments are useful whenever standardized severity data need to be recaptured over time. when frequent 'snap shots' of disease severity are combined with virology data, it may be possible to generate a 'moving image' with interesting new applications in clinical research. the introduction of standardized disease severity scores will facilitate headto-head comparisons and the 'meta-analyzability' of clinical trials and observational studies. full compliance of the vivi disease severity score mobile application with clinical data interchange standards consortium (cdisc) standards further expands data interoperability and compliance with reporting formats to regulatory agencies [ ] [ ] [ ] [ ] [ ] [ ] . it is important to note the scope of the proposed disease severity measure. this expert review does not intend to raise expectations that a disease severity score could or should be used to predict future events or physician behavior. instead, statistically significant mean differences are highlighted in bold (t-test p value < . ). *the interpretation of this risk factor was limited or **very limited by a low (*n < ) or very low (**n < ) prevalence rate in the qm population (see also table ). we introduce a simple -item weighted clinical composite score allowing the assessor to translate the current condition of the patient into a two-digit number, which allows comparison of one patient to another, regardless of the setting. to this end, the paper provides the descriptive account of how a standardized score can be utilized to assess the relationships observed between the score and various (independent) treatment and management decisions for readers to draw their own conclusions about how they may in turn use the vivi disease severity score in clinical practice or research. this expert review also introduces time series analysis with change point detection as a mathematical model applied, for the first time, to determining the timing and seasonality or respiratory viruses circulating in a hospital ad emergency room. while the observation that several viruses may circulate in a seasonal pattern is not new, the authors demonstrate that purely data-inherent definitions of seasonality could be an interesting addition to traditionally used methods. comparisons of average vivi disease severity score in the hospital system with the consultation index in the same calendar week (i.e. simultaneous ari consultations in sentinel practices) revealed that the two parameters are intrinsically different. a 'heavy' season with a high frequency of ari consultations is not the same as a 'light' season with fewer but more severe cases. the individual assessments in the qm cohort detected fluctuations in disease severity at a time when increases in overall ari incidence in the general population were not evident. especially during atypical influenza seasons with unusually few or unusually severe cases, the monitoring of disease severity in addition to incidence rates will provide important complementary information. standardized disease severity assessments also enable the cross-cohort comparison of disease burden between different viral pathogens. it is not surprising that rsv was identified as a key contributor to disease severity in a tertiary children's hospital, followed by hmpv disease. a better understanding of the real-world impact of different respiratory viruses on child health will help in the prioritization of drug and vaccine development. the development of the vivi disease severity score is based on a systematic review of the published literature. the review showed that severity assessments have been inconsistent. four clinical management parameters were used commonly as indicators of disease severity: hospitalization, intensive care treatment, oxygen supplementation, and mechanical ventilation (both invasive or noninvasive). the availability of any such measure, however, is highly dependent on the setting. the vivi disease severity score therefore uses the 'need for hospitalization' or 'need for icu admission' (as determined by the assessor) instead. if the assessor determines that a patient would benefit from any such measures, the item can be scored regardless of the availability of icu or hospital beds at the respective time or location. to ensure inter-rater consistency, the qm team was specifically trained to apply established who definitions and standard criteria for the assessment of each aspect of the vivi disease severity score. for example, fever was defined according to marcy et al. [ ] and acute lower respiratory tract infection as per roth et al. [ ] . for use in multicenter settings, the vivi disease severity score app will include help menus in the user interface to ensure that assessors are aware of the same criteria and age-appropriate values. acknowledging that the content, structure, and quality of standardized data are of paramount importance, the development team worked closely with the cdisc to ensure full compliance of terminologies and data elements with industry and regulatory guidance. the literature review showed that grading severity is not the same as predicting severity. especially in young children, disease severity will fluctuate over time, until the episode is resolved eventually. the course of illness may or may not be linear. the vivi disease severity score is designed to help the physician measure and monitor the situation ad hoc, or repeatedly over time, but not to predict the future of the patient. several scores have been designed, not to measure severity ad hoc, but to predict the likelihood of fatal outcomes in the future as is the case with the respiratory index of severity in children [ ] , the pediatric index of mortality score (pims) [ ] , and the pediatric risk of mortality score (prims) [ , ] . these latter two scores (pims and prims) were specific to rsv infections [ ] in infants [ , ] . the kristjansson clinical respiratory score for rsv infections in children [ ] was designed to include children beyond the infant age group. the index of severity was studied in bocavirus infections in infants and children < years [ ] , as was the symptom score for coronavirus infections in children [ ] . very few scores were developed to measure disease severity regardless of the type of respiratory virus causing the disease. the clinical severity score was used to monitor rsv, hrv, and hmpv infections in children < years [ , , ] . the systematic literature review, updated in , confirmed that vivi disease severity score was the only score covering all pediatric age groups and any respiratory virus encompassing any of the key parameters outlined in the published literature to date [ , ] . the vivi disease severity score was also the only composite score that has been validated in a prospective cohort of more than children and adolescents from to years, yielding a normal distribution. regular severity assessments over time can be combined with cp detection methodology to detect of significant changes in disease severity in cohorts. hospital surveillance will thus become feasible in real time, as rapid-turnaround diagnostic tests are evolving [ ] [ ] [ ] [ ] . the use of rapid diagnostic tests can then be targeted according to the local surveillance information. bioinformatics analyses and machine learning algorithms may provide new avenues for the identification of virus-specific seasonality patterns [ ] . during past influenza seasons, differences in the composition of subtypes and disease severity have been significant. the linkage of simultaneous virus surveillance with point-of-care disease severity assessments will advance the understanding of local epidemiology. local epidemiology is key to understanding the impact of different strains on different populations. in north america, influenza a h n viruses reappeared year sooner than in europe, i.e. in / [ ] followed by an unusually 'light' season with few or late cases during the winter of / [ ] . public health agencies in the uk reported a particularly 'severe' season in / [ , ] , whereas australia reported increased rates of severe influenza disease in [ ] [ ] [ ] , similar to mexico during / . classically, seasons have been regarded as 'severe' when coinciding with high overall case numbers, hospitalization rates, or mortality [ , ] . in the future, it will be important to distinguish the impact of fluctuations in influenza (sub)types on disease severity in specific patient groups, based on ipd. the use of standardized measures of severity may also be helpful in the study of medical decision-making and diagnostic algorithms. physicians in routine care often report that their decision to order virus diagnostics is often dependent on a variety of factors such as levels of training, media attention [ , ] , specific requests by patients or parents, 'typical' versus 'atypical' disease presentations, availability and cost of diagnostic tests, insurance status of the patient, time constraints, etc. [ ] . the same applies to the decision to hospitalize a patient. it is safe to assume that testing and rates of hospitalization are not the same at the beginning, peak, and end of an influenza season. standardized disease severity scores may allow hospitals to set objective thresholds for diagnostic testing or admission decisions, depending on local conditions and epidemiology. when population-based indicators are used instead of individual clinical outcome parameters, considerable bias may be introduced due to differences in patient reporting, access to health care [ ] as well as physician awareness and reimbursement [ ] creating challenges in global surveillance systems [ , ] . some surveillance programs use retrospective chart reviews and icd coding. icd codes, however, do not always distinguish between laboratory-confirmed cases and clinical diagnoses [ ] . interpersonal variability and the unpredictable nature of respiratory viral infections pose a challenge to surveillance and preparedness programs [ ] . influenza seasons in particular vary with respect to case numbers and disease severity attributable to various viral subtypes and population strata [ , , , ] . the prospective monitoring of disease severity associated with laboratory-confirmed diagnoses will help to delineate vulnerable subpopulations expressing disease severity differently compared to the population average. real-time surveillance of disease severity may provide public health stakeholders with crucial information to adjust the allocation of hospital beds and resources [ ] . the introduction of ipd disease severity assessments in a hospital-based surveillance system facilitates the timely identification of abnormal patterns of disease severity, i.e. though network analysis [ ] or during time periods when the overall ili disease severity is different from previous seasons or the rest of the year. importantly, fluctuations in disease severity measured by the vivi disease severity score are independent of incidence-based surveillance indices. traditional disease severity estimates have focused on extreme presentations such as mortality rates [ , ] or icu admission [ ] but were not designed to monitor the full spectrum of mild-to-severe disease presentations. additional granularity will be required for clinical trials. when the vivi disease severity score was used to follow patients longitudinally, disease severity was measured consistently from the time of initial presentation until resolution of symptoms. the vivi disease severity score has also been used to measure of subtle changes in disease severity in icu patients requiring organ replacement therapy [ ] . once standardized scores are used consistently, this will open the path to headto-head comparisons of antivirals and vaccines and to prospective ipd meta-analyses. it will be important to investigate the complex relationship between virus load and disease severity and expected outcomes, which would provide important clues for clinical trial design [ ] . patients showing atypical patterns of disease severity for example (such as a negative correlation between virus load and disease severity) may represent individuals where antivirals do not exert the desired effect. additional analyses are underway to understand this relationship better. standardized disease severity measures will facilitate biomarkers studies and the identification of viral and host factors associated with severe outcomes [ ] . a precision medicine approach would lead to individualized risk communication strategies to improve the acceptance of vaccines and antivirals where they are most effective. the low uptake in influenza vaccines in the qm cohort indicates that significant numbers of symptomatic influenza cases might have been prevented through immunization [ ] . the presented work has several limitations: the current experience with the vivi disease severity score is based on a single-center tertiary care setting. additional decentralized studies will be needed to validate the vivi disease severity score in international settings and in private practice networks, where severity may be lower. further studies are planned in adults and the elderly, including the development of a compatible score for patient-reported outcomes. it is possible that different populations yield different results, but standardization is the prerequisite to study any such difference. mobile applications will be particularly useful in low-resource settings, where disease severity may be higher and decisions have to be taken instantly. finally, the effect of antiviral treatment or vaccine prevention on disease severity could not be assessed due to a minimal use of neuraminidase inhibitors and influenza vaccines in the current setting [ ] . the only conclusion that can be drawn is that physicians in this setting hardly ever used antivirals but were more likely to prescribe antibiotics if a patient appeared severely ill, as expressed in significantly higher vivi disease severity scores [ ] . it will be interesting to study decision-making processes and the impact of different forms of medical interventions on disease severity in a variety of settings in the future. at this point, the majority of sentinel surveillance systems are laboratory based yielding limited clinical information but important data with respect to the evolution of influenza viruses, subtypes, resistance, seasonality, and transmissibility. it would be of great benefit to monitor disease severity individually, along with regional and geographic differences in virus circulation, using standardized disease severity measurements such as the vivi disease severity score. our contributions are the following: (a) the design of a hospital-based surveillance program and a unique qm cohort of more than children, where an independent qm team monitored patients daily using standardized clinical assessments and virology at the national reference centre for influenza and other respiratory viruses. (b) a novel disease severity score (the vivi disease severity score) and mobile application to detect specific changes in ipd and the individual course of illness in pediatric clinical trials and observational settings. the presented tools are in line with the priorities issued by regulatory agencies with regards to data standardization and the development of clinical outcome measures for the development of new antivirals. with composite disease severity scores, the focus will shift from virological to clinical end points, and the impact of therapeutic interventions on the quality of disease presentations. only the systematic unbiased and prospective assessment of all cases, whether mild or severe, throughout several seasons, will provide objective insight into the actual disease burden with influenza and other respiratory viruses. mobile health technologies enable new precision medicine approaches not only in clinical trials but also in routine patient care. individualized disease severity assessments in children with influenza and other respiratory viruses will allow the physician to communicate better with the parent or patient, providing the current status as a validated measure of disease severity compared to similar age and population strata. in patents receiving antiviral therapy, progress can be measured and communicated accordingly and again, individually. most importantly, with the availability of validated disease severity measures and standardized datasets, the physician will be able to determine which patients may be 'lagging behind' in their response to therapeutic interventions. a better understanding of the complex relationship between virus load and disease severity in children with different respiratory viruses will provide important clues for a personalized approach to antiviral therapy. biomarker analyses linked to standardized disease severity assessments will help to elucidate why some patients improve rapidly as soon as virus loads decline, whereas a smaller group of patients does not improve as expected. this latter subgroup of patients may benefit from different therapeutic approaches, for example immunomodulation. precision medicine tools such as the vivi disease severity score mobile application will provide important tools for the objective evaluation of new antivirals for soon-to-be treatable respiratory viruses. • regulatory agencies and public health stakeholders have repeatedly called for international consensus on disease severity measures in influenza and other respiratory viruses. • this need has become imminent with the rapid development of new anti-infective therapies for respiratory viral infections in children and adults. • the challenge of data standardization is greatest in infants and young children, who may present with subtle and atypical symptoms. • based on a systematic review of the literature we developed a -item composite clinical score (the vivi disease severity score) for the immediate measurement of disease severity with acute reparatory infections the point-of-care. • the vivi disease severity score was made available as a web-user interface and mobile application for validation in a quality management program including more than children - years of age. • linking standardized diseases severity scores with rapid diagnostics will allow the instantaneous monitoring of incidence rates of acute respiratory viral infections along with the severity of each case. • with this comprehensive manuscript, we are providing insight into the future of observational studies and clinical trials of antivirals for soon-to-be-treatable acute respiratory diseases in children. • the reader is guided through novel analytic approaches that have become possible through rigorously standardized individual patient-data (ipd) analyses of disease severity. br wrote the initial draft of the manuscript. ma, ft, xc and po were in charge of data aggregation, acquisition and qc/qa, and provided important input into the manuscript. xm conducted and interpreted the systematic literature review. bk and ch were in charge of data standardization, ch provided database maintenance and management. bs designed and supervised the laboratory analyses. tc, pm conducted the data analysis. br designed the qm program and the vivi disease severity score and supervised the project. all authors take responsibility for the integrity of the data and the accuracy of the data analysis. all authors have seen and approved the final version of the manuscript. the authors would like to express their gratitude to the team at the robert koch institute for providing virology testing in-kind and to the vienna vaccine safety initiative for providing the vivi disease severity score and mobile application. tc was funded by the german ministry of research and education (bmbf) project grant fo (forschungscampus modal). the authors would also like to express their thanks to members of the vivi think tank for 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tropism as a possible contributor to influenza infection severity increasing the intent to receive a pandemic influenza vaccination: testing the impact of theory-based messages influenza antiviral prescribing practices during the - and - influenza seasons in the setting of increased resistance to oseltamivir among circulating influenza viruses key: cord- - gl km authors: ather, amber; patel, biraj; ruparel, nikita b.; diogenes, anibal; hargreaves, kenneth m. title: reply to: "coronavirus disease : implications for clinical dental care" date: - - journal: j endod doi: . /j.joen. . . sha: doc_id: cord_uid: gl km nan we would like to thank drs. jadhav and mittal for their letter to the editor in which they have put forward their concerns and suggestions regarding the manuscript titled 'coronavirus disease : implications for clinical dental care' ( ). we appreciate their insight and would like to take the opportunity to comment on their suggestions. we strongly agree with the concerns expressed by the authors regarding asymptomatic carriers of coronavirus disease (covid- ) and the potential transmission to dental health care professionals. the authors raise an important point about the use of pulse oximetry to detect low blood oxygen saturation levels in asymptomatic carriers. however, it is noteworthy that pulse oximeter detects hypoxemia in contrast to "hypoxia" as mentioned by the authors. the term hypoxia denotes reduced tissue level oxygenation and is not synonymous with hypoxemia (reduced blood oxygen saturation) ( ) . in agreement with the authors, oxygen saturation measurements are a vital screening tool to identify potential asymptomatic patients and should be incorporated into routine dental clinical practice. notably, the potential importance of silent hypoxemia in covid- patients has become evident only more recently ( ) . thus, at the time of this manuscript submission, we refrained from making recommendations without proper scientific citable evidence. as more is known about covid- , we believe that readers should maintain a pragmatic approach incorporating new peer-reviewed evidence in their practice. we appreciate the authors for elaborating on the outline and basic design considerations for negative-pressure rooms/ airborne infection isolation rooms for dental practices. given the surge in airborne infections over the past few decades, and the fact that only %- % of rooms are equipped with negative pressure in approximately half of urban hospitals ( ), efforts could be directed towards creating more negative-pressure rooms for health care providers. as mentioned by the authors, negative-pressure rooms should be built following stringent guidelines. in addition, these isolation rooms require continuous monitoring to measure pressure differential and proper training of staff to operate and check these rooms for "leaks". covid- ): implications for clinical dental care hypoxemia vs. hypoxia covid- with silent hypoxemia. covid- med stille hypoksemi implementing a negative-pressure isolation ward for a surge in airborne infectious patients tuberculosis infection among health care workers in montreal key: cord- -ctvnmjsl authors: mboowa, gerald; sserwadda, ivan; amujal, marion; namatovu, norah title: human genomic loci important in common infectious diseases: role of high-throughput sequencing and genome-wide association studies date: - - journal: can j infect dis med microbiol doi: . / / sha: doc_id: cord_uid: ctvnmjsl hiv/aids, tuberculosis (tb), and malaria are major global public health threats that undermine development in many resource-poor settings. recently, the notion that positive selection during epidemics or longer periods of exposure to common infectious diseases may have had a major effect in modifying the constitution of the human genome is being interrogated at a large scale in many populations around the world. this positive selection from infectious diseases increases power to detect associations in genome-wide association studies (gwass). high-throughput sequencing (hts) has transformed both the management of infectious diseases and continues to enable large-scale functional characterization of host resistance/susceptibility alleles and loci; a paradigm shift from single candidate gene studies. application of genome sequencing technologies and genomics has enabled us to interrogate the host-pathogen interface for improving human health. human populations are constantly locked in evolutionary arms races with pathogens; therefore, identification of common infectious disease-associated genomic variants/markers is important in therapeutic, vaccine development, and screening susceptible individuals in a population. this review describes a range of host-pathogen genomic loci that have been associated with disease susceptibility and resistant patterns in the era of hts. we further highlight potential opportunities for these genetic markers. hiv/aids, tuberculosis, and malaria are major global public health threats causing substantial morbidity, mortality, negative socioeconomic impact, and human suffering [ ] . whole-genome sequencing (wgs) of hosts and their cognate pathogens has transformed our understanding of the contribution of genomics in infectious disease processes. as the antibiotic era changed our understanding of infections so has the genomic revolution. e host-pathogen coevolutionary "arms race" is a phenomenon that has been described to result from interaction within host innate, adaptive immune responses, exposure to antibiotics, and competition with commensal microbiota [ , ] . when positive selection in one geographical region causes large allele frequency differences between populations than those expected for neutrally evolving alleles, high frequency of the derived allele (i.e., when a new allele increases to a frequency higher than that expected under genetic drift) [ ] and hts technologies are harnessed and used to decipher the nature and dynamics of these signals. e dynamics of host-pathogen interactions (e.g., length of exposure, geographical spread, morbidity, mortality, and cooccurring environmental events) influence the genetic architecture of resistance variants in modern populations [ ] . e application of genomic sequencebased approaches to understand the host-pathogen interface continues to provide us with important clues for our survival. human genetic variation is a major determinant of genetic susceptibility to many common infectious diseases [ ] . malaria, hiv/aids, and tuberculosis are some of the common infectious diseases in which a range of genetic susceptibilities and resistant conferring loci have been identified using both traditional molecular-based approaches and hts technologies. hts has enabled us to identify genomic signatures from these interactions. ese markers identified through infectious disease genome-wide association studies (gwass) have considerable significances that can be exploited to understand host protective mechanisms against pathogens and identify new molecular targets for diagnostic, prophylactic, and therapeutic interventions (table ). in the past, candidate gene studies have been used to identify disease susceptibility genetic loci for many major human infections. but with the advent of hts approaches, many new loci have been and continue to be identified in diverse populations. a paradigm shift from candidate gene studies to gwas and hts has ushered in a "big data" genomic revolution era enabling us to redefine the genomic architecture of host-pathogen disease susceptibility. both exome and whole-genome sequencing approaches are proving more successful and affordable. host genetics influence clinical course of infectious diseases as well as genetic variation of the pathogens determining their survival in presence of selective pressure from the host and environment (antimicrobials) and identify genetic markers of drugresistant pathogens and parasites. furthermore, hts has given us unprecedented resolution of understanding the role of host genetics to infectious diseases susceptibility. is genomic revolution has generated data informative for understanding the frequency of many genetic traits, including those that cause disease susceptibility in african populations and populations of recent african descent [ ] . pathogens have always been a major cause of human mortality, so they impose strong selective pressure on the human genome [ , ] . hts applied to screening populations of host immune-specific cells and their respective pathogens can highlight the host-pathogen unique genetic signatures important in host-pathogen coevolution, profiling immunological history, pathogen-induced immunodominance genetic patterns, predicting clinical outcomes of common infections (such as hiv/aids disease progression phenotypes like long-term nonprogressors and rapid progressors, as well as highly exposed persistently seronegative group), rapid diagnosis plus screening outbreaks involving risk group highly infectious pathogens, and genetic characterization of live-attenuated vaccine vectors (figures (a) and (b)). gwass demand recruitment of large well-phenotyped clinical cohorts within appropriate study designs and settings. however, they are very expensive, and therefore few funding agencies are able to finance such studies yet they may offer unique opportunities to unveil genetically important signals. currently, there are a growing number of communicable disease-specific research initiatives that are specifically interested in looking at the stages of the infections and gwas of disease progression. a classic example is the collaborative african genomics network (cafgen), a h africa-funded consortium probing host genetic factors that are important to the progression of hiv and hiv-tb infection in sub-saharan african children (https://www.h africa.org/consortium/projects/ projects/ -cafgen). cafgen is specifically looking at both rapid and long-term hiv/aids progression status while infectious diseases account for million deaths per year worldwide, and disproportionately affect the young, elderly people, and the poorest sections of society making them a high priority [ ] . e world health organization in estimated the global mortality for tuberculosis, hiv/aids, and malaria to be at . million with most deaths occurring in sub-saharan africa. is region has continued to lead in both prevalence and incidence of these major infectious killer diseases [ ] . research investment in infectious diseases was poorly matched. data show that funding does not correspond closely with burden [ ] . review of findings to date suggests that the genetic architecture of infectious disease susceptibility may be importantly different from that of noninfectious diseases [ ] . other authors have extensively reviewed the genetic susceptibility to diseases [ , , , , [ ] [ ] [ ] . e ancient biological "arms race" between microbial pathogens and humans has shaped genetic variation in modern populations, and this has important implications for the growing field of medical genomics [ ] . as humans migrated throughout the world, populations encountered distinct pathogens, and natural selection increased the prevalence of alleles that are advantageous in the new ecosystems in both host and pathogens [ ] . temporal patterns supporting figure : (a) pipeline for interrogation of pathogen genomes using high-throughput sequencing and computational approaches. dna extraction for hts can be done from either direct clinical specimen of individuals who are suspected to be infected with the disease or from enriched/isolated cultures. quality control and read preprocessing are critical steps in the analysis of datasets generated from highthroughput sequencing technologies. fastqc is an example of a tool for general quality assessment of hts data from all technologies. genomes can be recreated with no prior knowledge using de novo sequence assembly as well as recreating the genome using prior knowledge based on a reference genome-alignment/mapping. e former is necessary for novel genomes and where the sequenced genome differs from reference. sequence data analysis is important in infectious disease outbreak investigations, molecular typing, antimicrobial drug resistance, transmission, surveillance, and microbial evolution. (b) pipeline for interrogation of host genomes using high-throughput sequencing and computational approaches. for a given infectious disease in a population, an appropriate study design is determined and host dna is collected from cases (exposed to pathogen and infected) and controls (exposed to pathogen and uninfected). hts of dna from both cases and control is performed. quality control (qc) procedures vary in different pipelines. ese include qc on individuals for missingness, gender checks, duplicates and cryptic relatedness, population outliers, heterozygosity and inbreeding, qc on snps for missingness, minor allele frequency, and hardy-weinberg equilibrium. many of these are computationally intensive, operationally challenging, and constantly evolving. genome-wide association studies (gwass) involving case-control studies compare the frequencies of common genetic variants, assume an appropriate statistical model, and account for multiple testing correction threshold to identify susceptibility and protective polymorphisms in the population. evidence of host-pathogen coevolution have been reported [ ] . common infectious diseases have shown geographical disparities, for example, mycobacterium tuberculosis lineage comprises globally distributed and geographically restricted sublineages [ ] . molecular epidemiological studies show that, with the exception of sub-saharan africa, almost all hiv- subtypes, circulating recombinant forms, and several unique recombinant forms have been detected, but there is a specific geographic distribution pattern for hiv- subtypes [ ] [ ] [ ] . hiv diversity plays a central role in the hiv pandemic [ ] and has significant implications for diagnosis, vaccine development, and clinical management of patients [ ] . high levels of plasmodium falciparum malaria endemicity are common in africa [ ] . formal characterization of disease-causing agents has always been fundamental to understanding the evolution of pathogens and the epidemiology of infectious disease. linking this information with temporal, spatial, and clinical data can bring understanding of evolution, geographical spread, and disease associations for the pathogen, providing vital information for identifying sources of infection as well as designing interventions to prevent and treat disease [ ] . new sequencing technology has enabled the identification of thousands of single nucleotide polymorphisms in the exome, and many computational and statistical approaches to identify disease association signals have emerged [ ] . foremost is a better understanding of disease pathogenesis and resistance in the expectation that this will lead in time to improved interventions such as better drugs or vaccines to prevent or attenuate the great global burden of infectious disease morbidity and mortality. with over million deaths annually from infectious diseases and the threat of new epidemics and pandemics, this is a very high priority [ ] . during the past decade, we have also witnessed the emergence of many new pathogens not previously detected in humans, such as the avian influenza virus, severe acute respiratory syndrome (sars), and ebola [ ] . now that the scientific community has access to complete genomes of infectious diseases though application of hts, priority should be the dissection of host-pathogen interactions through development of powerful computational tools. hts has profoundly altered our understanding of human diversity and disease. e interaction between hosts and pathogens is a coevolution process which may simply be described as "shooting a moving target." hts and computational modelling tools will offer potential to understand this interaction leading to better vaccine designs and therapeutic targets. sanger dna sequencing is limited in throughput and high cost as compared to hts platforms, which differ in their details but typically follow a similar general paradigm: template preparation, clonal amplification, followed by cyclical rounds of massively parallel sequencing [ ] . nanopore-based sequencing approaches such as single molecule, real-time (smrt) sequencing technologies have been developed and consistently produce some of the longest average read lengths compared to hts. smrt sequencing is particularly useful for projects involving de novo assembly of small bacterial and viral genomes as well as large genome finishing [ ] . many important sequencing platforms are reviewed in great depth [ , ] . computational tools are an important integral part of genomics. new computational methods are constantly being developed to collect, process, and extract useful biological information from a variety of samples and complex datasets [ ] . e scale and complexity of genomic data is everexpanding, requiring biologists to apply increasingly more sophisticated computational tools in the generation, analyses, interpretation, and storage of this data. e data are generated in different sizes, formats, and structures requiring a wide range of tools to manipulate. despite the level of specialization needed in bioinformatics, it is important that life-scientists have a good understanding of it for a correct experimental design which allows them to reveal the information in a metagenome [ ] . hts technologies are generating an astonishing amount of unprecedented information in the history of biology which has spurred a biomedical big data to knowledge (b d k) revolution. us, a new exhilarating rapidly evolving scientific field, bioinformatics (biology meets computer programming), has recently emerged and uses novel computational approaches to help solve important biological problems. bioinformatics is a set of activities: data acquisition, database development, data analysis, data integration, and analysis of integrated data. e majority of available bioinformatics software requires some knowledge of the text-based command line of the unix or linux operating systems, allowing custom programming scripts and pipelines to automate data manipulation and analysis in a single step [ ] . although bioinformatics tools/software are both "open sources" and commercially available, clinicians have limited bioinformatics knowledge [ ] [ ] [ ] [ ] . it is clear that user-friendly bioinformatics pipelines are key to facilitating more widespread use of wgs, with more widespread bioinformatics expertise [ ] . positive selection (also known as darwinian selection) in genes and genomes can point to the evolutionary basis for differences among species and among races within a species [ ] . many other aspects of human biology not necessarily related to the "branding" of our species, for instance, hostpathogen interactions, reproduction, dietary adaptation, and physical appearance, have also been the substrate of varying levels of positive selection. comparative genetics/genomics studies in recent years have uncovered a growing list of genes that might have experienced positive selection during the evolution of humans and/or primates [ ] . microbial genome evolution is shaped by a variety of selective pressures. understanding how these processes occur can help to address important problems in microbiology by explaining observed differences in phenotypes, including virulence and resistance to antibiotics. greater access to whole-genome sequencing provides microbiologists with the opportunity to perform large-scale analyses of selection in novel settings such as within individual hosts [ ] . identification of positive selection genetic signatures in the genomes can help us to understand the kinetics and directions of continuing host-pathogen coadaptation and impact on their diagnosis, transmission, fitness, immunogenicity, and pathogenicity. given the potential for strong selective pressure, that genetic programs controlling hostpathogen interactions in humans and other species are littered with signatures of positive selection [ ] . e high mortality and widespread impact of malaria has resulted in this disease being the strongest evolutionary selective force in recent human history, and the genes that confer resistance to malaria provide some of the best-known case studies of strong positive selection in modern humans [ ] . a number of specific genomic variants including β-globin locus, g pd deficiency, duffy, ovalocytosis, abo, and human leukocyte antigen confer resistance to malaria in the human host. elevated frequencies of hemoglobinopathies such as thalassemia and sickle cell disease, which are caused by mutations at the β-globin locus, are maintained via balancing selection ("the malarial hypothesis") [ ] [ ] [ ] . is hypothesis suggests that some human diseases such as thalassemia are polymorphisms which provide heterozygote advantage because of the trade-offs between the advantages of resistance to malaria and negative effects due to the disease [ ] , where the hemoglobin s (hbs) homozygote disadvantage is recompensed through the malaria resistance of the heterozygote (hbas) in regions of malaria endemicity [ , ] . e ∆ mutation at the ccr locus is a wellstudied example of natural selection acting in humans. e mutation is found principally in europe and western asia, with higher frequencies generally in the north. homozygous carriers of the ∆ mutation are resistant to hiv- infection because the mutation prevents functional expression of the ccr chemokine receptor normally used by hiv- to enter cd + t cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . host genetic factors play important roles in susceptibility to tuberculosis infection, and different gene polymorphisms in different ethnicity and genetic backgrounds may lead to different effects on tuberculosis risk [ ] . polymorphisms in natural resistance-associated macrophage protein (nramp ), toll-like receptor (tlr ), interleukin- (il- ), tumor necrosis factor-alpha (tnf-α), interleukin- receptor antagonist (il- ra), il- , vitamin d receptor (vdr), dendritic cell-specific icam- grabbing nonintegrin (dc-sign), monocyte chemoattractant protein- (mcp- ), nucleotide oligomerization binding domain (nod ), interferon-gamma (ifn-c), inducible nitric oxide synthase (inos), mannose-binding lectin (mbl), and surfactant proteins a (sp-as) genes have been variably associated with tuberculosis infection, and there is strong evidence indicating that host genetic factors play critical roles in tuberculosis susceptibility, severity, and development among different populations [ ] [ ] [ ] . several nramp polymorphisms were significantly associated with ptb in african and asian populations, but not in populations of european descent [ , ] . gwass are based on the "common disease, common variant" hypothesis, and they have been performed largely using single nucleotide polymorphism (snp) arrays that focus canadian journal of infectious diseases and medical microbiology only on common genetic polymorphisms (for which the minor allele frequency is > %) [ , [ ] [ ] [ ] . gwas approach has potential to provide candidates for the development of control measures against infectious diseases in humans [ ] . for over years, candidate gene studies have been used to identify loci for many major causes of human infectious mortality, including malaria, tuberculosis, and hiv- [ ] . e first successful gwas was published in ushering genome-wide approaches that have identified loci in diverse populations. common genetic variants have also been demonstrated to regulate susceptibility/resistance to infectious diseases, for example, the ccr ∆ polymorphism that modulates hiv/aids disease progression [ ] . genome-wide association study approaches are being increasingly utilized to define genetic variants underlying susceptibility to major infectious diseases [ ] . infectious diseases follow a series of stages right from acquisition, disease development, rate of progression, convalescence, and asymptomatic carrier state. erefore, every stage will be influenced by one or a set of mutations in a population or individual. different populations will have mutations that affect diseases at different stages. is is where gwas has and will continue to play an important role in identifying these mutations. a recent study suggested that host genetic risk in tb is depended upon the pathogen's genetic background and demonstrated the importance of analyzing the interaction between host and pathogen genomes in tb [ ] . studies are exploiting unique designs like extreme phenotype designs to identify complex trait genomic loci, while others have identified genetic associations of infectious diseases by integrating estimation of population admixture events to detect disease susceptibility loci after teasing out different ancestries and allelic, genotypic, or haplotype risk ratios. a genomic database for all mutations identified to be conferring resistance to infectious diseases in different populations is a vital product of more than years of gwas. more studies should be appropriately designed to identify new potential infectious disease resistance-conferring mutations in human hosts. we searched the pubmed database for studies published since january using the terms "malaria," "tuberculosis," "hiv/aids," "genome-wide association." search terms included combinations of ((disease-query[title] and genome-wide association[title])) where a disease-query was either a communicable or noncommunicable disease. two search restrictions were set: publication date set (from / / ) and species (humans). figure indicates more gwass carried out in noncommunicable diseases than infectious diseases. ere is an urgent need for a major increase in funding for communicable disease control in the developing world and for more balanced allocation of the resources already provided [ ] . genomics and whole-genome sequencing have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology [ ] . human genetics is an indispensable tool for enhancing the understanding of the molecular basis of many common diseases [ ] . over , snps have been associated with a variety of human traits and complex diseases [ ] . we envisage that with the reducing costs, hts and genomics will become an indispensable component of every healthcare system. hts and computational tools offer a potential to stratify populations for risk of infectious disease based on genomic profiling thereby prioritizing interventions such as vaccines and therapeutics to the "most-at-risk" populations since there is no "one-size-fits-all" approach to treating infectious diseases. with the growing number of sequencing facilities on every continent, the future will offer a less costly approach that will integrate a genomic profile in routine patient management, improving management of diseases, and therapeutic development. it is less likely to find a population or individual who carries mutations conferring resistance to an infectious disease at every stage of the infection. different individuals have different mutations that offer resistance to different stages of the infections. a genomic catalogue of these mutations identified through hts, computational tools, and gwas now combined with the rapidly growing genomeediting technology known as crispr/cas will enable introduction of an array of disease-stage-specific resistanceconferring mutations. is will offer interventions at all levels of the disease process unlike traditional vaccines. disclosure e views expressed in this publication are those of the authors and not necessarily those of the aas, nepad agency, wellcome trust, or the united kingdom government. e authors declare that they have no conflicts of interest. e global fight against hiv/aids, tuberculosis, and malaria: current status and future perspectives evolution of bacterial pathogens within the human host arms races between and within species natural selection and infectious disease in human 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ccr gene -bp deletion in europe global distribution of the ccr gene -basepair deletion distribution of the ccr gene -basepair deletion in west europe. a hypothesis about the possible dispersion of the mutation by the vikings in historical times frequencies of base pair deletion of the (Δ ) allele of the ccr hiv- co-receptor gene in caucasians: a comparative analysis more about the viking hypothesis of origin of the Δ mutation in the ccr gene conferring resistance to hiv- infection host genetic effect on tuberculosis susceptibility in chinese uyghur nramp , vdr, hla-drb , and hla-dqb gene polymorphisms in susceptibility to tuberculosis among the chinese kazakh population: a casecontrol study innate immune gene polymorphisms in tuberculosis host genome polymorphisms and tuberculosis infection: what we have to say? slc a (formerly nramp ) gene polymorphisms and tuberculosis susceptibility: a meta-analysis human genetics of tuberculosis: a long and winding road variation across the allele frequency spectrum genetic heterogeneity in human disease uncovering the roles of rare variants in common disease through whole-genome sequencing dual effect of ccr Δ gene deletion in hiv- -infected patients pathogen lineagebased genome-wide association study identified cd as susceptible locus in tuberculosis what did we learn from the genome-wide association study for tuberculosis susceptibility? is work was supported through the deltas africa initiative (grant no. del- - ) to thrive- (the training health researchers into vocational excellence in east africa). e deltas africa initiative is an independent funding scheme of the african academy of sciences' (aas) alliance for accelerating excellence in science in africa (aesa) and supported by the new partnership for africa's development planning and coordinating agency (nepad agency) with funding from the wellcome trust (grant no. /z/ /z) and the uk government. all authors participated in writing the manuscript. ey further reviewed and approved the final manuscript. key: cord- -xscwk r authors: chassagnon, guillaume; vakalopoulou, maria; battistella, enzo; christodoulidis, stergios; hoang-thi, trieu-nghi; dangeard, severine; deutsch, eric; andre, fabrice; guillo, enora; halm, nara; hajj, stefany el; bompard, florian; neveu, sophie; hani, chahinez; saab, ines; campredon, aliénor; koulakian, hasmik; bennani, souhail; freche, gael; barat, maxime; lombard, aurelien; fournier, laure; monnier, hippolyte; grand, téodor; gregory, jules; nguyen, yann; khalil, antoine; mahdjoub, elyas; brillet, pierre-yves; tran ba, stéphane; bousson, valérie; mekki, ahmed; carlier, robert-yves; revel, marie-pierre; paragios, nikos title: ai-driven quantification, staging and outcome prediction of covid- pneumonia date: - - journal: med image anal doi: . /j.media. . sha: doc_id: cord_uid: xscwk r coronavirus disease (covid- ) emerged in and disseminated around the world rapidly. computed tomography (ct) imaging has been proven to be an important tool for screening, disease quantification and staging. the latter is of extreme importance for organizational anticipation (availability of intensive care unit beds, patient management planning) as well as to accelerate drug development through rapid, reproducible and quantified assessment of treatment response. even if currently there are no specific guidelines for the staging of the patients, ct together with some clinical and biological biomarkers are used. in this study, we collected a multi-center cohort and we investigated the use of medical imaging and artificial intelligence for disease quantification, staging and outcome prediction. our approach relies on automatic deep learning-based disease quantification using an ensemble of architectures, and a data-driven consensus for the staging and outcome prediction of the patients fusing imaging biomarkers with clinical and biological attributes. highly promising results on multiple external/independent evaluation cohorts as well as comparisons with expert human readers demonstrate the potentials of our approach. • a covid- -specific holistic, highly compact multi-omics signature integrating imaging/clinical/ biological data and associated comorbidities for automatic patient staging is presented and evaluated. • short and long-term prognosis for clinical resources optimization offering alternative/complementary means to facilitate triage for covid- • clinically-relevant quantification and staging tool validated by comparison with clinical experts is reported. coronavirus disease (covid- ) emerged in and disseminated around the world rapidly. computed tomography (ct) imaging has been proven to be an important tool for screening, disease quantification and staging. the latter is of extreme importance for organizational anticipation (availability of intensive care unit beds, patient management planning) as well as to accelerate drug development through rapid, reproducible and quantified assessment of treatment response. even if currently there are no specific guidelines for the staging of the patients, ct together with some clinical and biological biomarkers are used. in this study, we collected a multi-center cohort and we investigated the use of medical imaging and artificial intelligence for disease quantification, staging and outcome prediction. our approach relies on automatic deep learning-based disease quantification using an ensemble of architectures, and a datadriven consensus for the staging and outcome prediction of the patients fusing imaging biomarkers with clinical and biological attributes. highly promising results on multiple external/independent evaluation cohorts as well as comparisons with expert human readers demonstrate the potentials of our approach. c elsevier b. v. all rights reserved. preprint submitted to medical image analysis october , china (zhu et al., ) caused by the sars-cov- virus, and it could lead to respiratory failure due to severe viral pneumonia . the disease spread worldwide leading the world health organization to declare it as a pandemic in march . one of the important actions to handle the pandemic is the fast and robust use of imaging along with clinical and biological comorbidities for the quantification and staging of patients upon their hospital admission. being able to identify patients that need intubation upon admission is very important and essential for the management of a hospital's resources and the most optimal management of patients. moreover, a robust staging of the patients could also facilitate proper selection of patients for different treatments, reducing the unnecessary use of the hospital's intensive care units. to the best of our knowledge, currently the staging of the patients is mainly based on clinical and biological biomarkers such as age, sex and other comorbidities li et al., a; yuan et al., ; tang et al., ; onder et al., ; guo et al., ; terpos et al., ) , while the role of imaging is mainly focusing on an estimation of the disease extent from ct scans. this estimation is mainly done by medical experts and hence suffers from inter-and intra-observer variability. in this study, we investigated an automatic method ( to the best of our knowledge this is among a few systematic efforts to quantify disease extent, to discover low dimensional and interpretable imaging biomarkers and to integrate them to clinical variables into short and long term prognosis of covid- patients. the paper is organized as follows: we first review related work mainly focusing on interstitial lung diseases (ilds) diseases, which is followed by a description of all the components and implementation details of our method. we then present the acquired multi-center dataset, the evaluation setting, and the results of our experiments. furthermore, we discuss in detail similarities and differences of our method with other recently proposed methods for quantification and staging of covid- . lastly, we present possible directions for future research. in this section, we provide a short review of previous studies on quantification of ilds since covid- and ilds share a lot of similarities due to their diffuse pathological manifestations, such as ground glass opacities, band consolidations, and reticulations. furthermore, we elaborate on studies that tackle severity or treatment response for such types of disease. there are numerous studies proposed the last years on automatic quantification of ild diseases using ct scans. the main goal of these studies is to develop models that are able to identify one or more types of different pathological lung tissue in ild cases (such as ground glass, consolidation, honeycombing, etc) and successfully separate them from the healthy tissue. initial efforts were mainly based on classification schemes. in particular, small patches including only a single tissue type were extracted and described using a number of handcrafted features focusing mainly on texture, then these features were used to train different machine learning classifiers (gangeh et al., ; huber et al., ) . following recent advances in deep learning and especially the success of convolutional neural networks (cnns), researchers have recently employed such tools also in thoracic imaging tasks (chassagnon et al., ) , with ild quantification being among them. the main advantage of cnns is their ability to generate features automatically from the input, and create meaningful representations for the studied per time problems. in particular, a patch-based framework using a convolutional architecture is presented in for the automatic quantification of different ild patterns. similarly, in (gao et al., ) a patch-based approach is adapted to classify them in different ild patterns. even if the method reported higher performance than other methods based on handcrafted features, the use of patches, besides being time consuming and ineffi-cient, does not exploit the texture of the entire lung. many of the already proposed cnns have further been adapted to perform the task of semantic segmentation in an endto-end fashion instead of only image classification. semantic segmentation refers to the task of infering a class for each of the pixels of an image instead of a single class for an image. such models can be found in literature both in d (badrinarayanan et al., ; ronneberger et al., ) and d (Ç içek et al., ) and have also been used for ild quantification. the authors of present the coupling of d fully convolutional networks with deformable registration for the automatic quantification of systemic sclerosis disease. moreover, in (anthimopoulos et al., ) staging of patients with ilds is very important as it could greatly help clinicians with their daily practice, while choosing treatment options (kolb and collard, ) . there have been a number of studies recently that try to identify and extract biomarkers from ct scans and associate them with the severity and treatment of ild patients. these biomarkers are usually enhanced with clinical and physiological information to provide a scoring system as survival predictor. among the variety of biomarkers, disease extent is one of the most powerful ones providing strong associations with severity and mortality (cottin and brown, ; tomassetti et al., ) . visual scoring of the disease extent on ct can be time-consuming (robbie et al., ) highlighting the need for tools for automatic disease quantification. moreover, except the disease extent, the location of the disease is also very important for the staging. in (depeursinge et al., ; christe et al., ) the quantification of the disease is performed on different lung regions providing descriptive information about the severity of the ild patients. a variety of works report that radiomics, quantitative features extracted from the images, provide valuable information about the severity and response to treatment for different diseases including cancer (sun et al., ) . these features could also provide very good tools for monitoring disease progression and therapeutic response (wu et al., ) . in particular, in (bocchino et al., ) intensity-based characteristics such as skewness and kurtosis were used together with disease extent to distinguish between systemic sclerosis patients with and without ild diseases. moreover, in (lafata et al., ) a variety of image radiomics and their relationship with the pulmonary function were investigated. their results indicate that high-throughput radiomics data extracted from the lungs may be associated with pulmonary function as measured by common pft metrics. in this section, we describe our ai driven scheme for the quantification of ct scans for patients suffering from covid- pneumonia. furthermore, we provide a method for the automatic selection and combination of multi-modal variables towards a holistic signature designed for the covid- triage. on the basis of this interpretable, clinically relevant signature we develop advanced machine learning techniques integrating multi-modal data for severity assessment and short/long term outcome prediction. our method endows robustness, good generalization properties, explainability and establishes causality with known clinical covid- confounding factors. in the following parts of this section, we provide details for all the different components of the system. segmentation of the heart and breast were extracted by using the software art-plan (therapanacea, paris, france). art-plan is a ce-marked solution for automatic annotation of organs, harnessing a combination of anatomically preserving and deep learning concepts. the segmentation of lungs was also performed using art-plan software, but the models used were re-trained using covid- patients in order to address proper segmentation of diseased lungs. in particular, the existing lung models, providing segmentation of left and right lungs, were re- for the registration of the ct scans to the templates, an elastic registration framework based on markov random fields was used, providing the optimal displacements for each template (ferrante et al., ) . in particular, the registration is performed by a non-linear transformer t , corresponding to the operator that optimizes in the continuous domain Ω the following energy, where ρ j corresponds to the different similarity metrics (sum of absolute difference, normalised cross correlation, etc) used to compare the source d volume to the target anatomy, w j are linear constraints factorizing the importance of the different metric functions and ψp¨q is a penalty function acting on the spatial derivatives of the transformation. concerning the details of the architecture, in our experiments each c i consists of a segnet (badrinarayanan et al., ) based architecture. more specifically, for the covide d models the ct scans were separated on the axial view. each network included convolutional blocks, each one containing two conv-bn-relu layer successions. maxpooling layers were also distributed at the end of each convolutional block for the encoding part. upsampling operators were used on the decoding part to restore the spatial resolution of the slices together with the same successions of layers. to fully exploit the d nature of our dataset, the second component of our proposed covidenet is based on a d fully convolutional network similar to d-unet (Ç içek et al., ) . in order to train this model, d sub-volumes of the ct scan that fully included without any downsampling either the left or right lung were extracted. the corresponding sub-volumes were also extracted from the ground truth annotation masks. to this end, we trained the model with the ct scan sub-volume as input and the annotation as target. as far as the architecture is concerned, the model consisted of five blocks with a down-sampling operation applied every two consequent conv d-bn-relu layers. additionally, five decoding blocks were utilized for the decoding path, were at each block a transpose convolution was performed in order to up-sample the input. skip connections were also employed between the encoding and decoding paths. the dimensions of the input that corresponded to the spatial dimensions of the ct scan and consequently the spatial dimensions of the features maps were not bound to some fixed dimension in order to feed the entire left/ right lung volumes. as such, d volumes of arbitrary spatial dimensions could be fed to the network and thus the batch size was fixed to . in order to combine disease extent with disease characteristics and patients commodities, we investigate a variety of imaging characteristics extracted using disease, cardiac and lung segmentations. these imaging characteristics (radiomics) were then combined with meaningful clinical and biological indicators that have been reported to be associated prognosis of covid- . patient charts were reviewed to assess short term ( days after the chest ct) and long term prognosis ( days after the chest ct). for the staging task, patients were divided in groups: those who died, or required mechanical ventilation either at the initial or at a subsequent admission as severe cases (s), and the rest as non-severe cases (ns). for the prognosis task, three distinct subpopulations were defined: those who had a short term negative (sd = short-term deceased) outcome (deceased within days after admission), those who didn't re- cover (ld= long-term deceased) within days after the chest ct (either died after day or still intubated at day ) and those who recovered (lr= long-term recovered). the last two groups formed the short intubated (si) group of patients. radiomics features were extracted from the ct scans using the previously described segmentations of the disease, lung and heart. as a preprocessing step, all images were resampled by cubic interpolation to obtain isometric voxels with sizes of mm. subsequently, disease, lung and heart masks were used using all the calculated attributes (clinical, biological, imaging) we constructed a high dimensional space of size , including clinical/biological variables. a min-max normalization of the attributes was performed by calculating the minimum and maximum values for the training and validation cohorts. the same values were also applied on the test set. to prevent overfitting and discover the most informative and robust attributes for the staging and prognosis of the patients we propose a robust biomarker selection process. feature selection is very important for classification tasks and has been used widely in literature especially for radiomics (sun et al., ) . first, the training data set was subdivided into training and validation on the principle of %- % maintaining the distribution of classes between the two subsets identical to the observed one. to perform features selection, we have created subdivisions on this basis and evaluated variety of classical machine learning -using the entire feature space -classifiers such as regarding imaging features, we identified the following features as more important for the prognosis of the covid- patients. these features include both first and second order statistics together with some shape features. length on the axis. the selected disease area features capture both disease extent and disease textural heterogeneity. disease textural heterogeneity is associated with lesions, the presence of which generates imaging pattern more complex than pure ground glass opacities usually found in mild disease. the selected lung features capture the dispersion and heterogeneity of lung densities, both of which may reflect the presence of an underlying airway disease such as emphysema but also the presence of sub-radiological disease. lastly, the selected heart features can be seen as a surrogate for cardiomegaly and coronary calcifications. the staging/prognosis component was addressed using an ensemble learning approach. similarly to the biomarker extraction, the training data set was subdivided into training and validation sets on the principle of %- %. this subdivision was performed such that the distribution of classes between the two subsets was identical to the observed one. we have used -fold cross validation on this basis and evaluated the average performance of the following supervised clas- to perform the short-term deceased (sd), long-term deceased (ld), long term recovered (lr) classification task, a sd/si (si: intubated at days) classifier and a ld/lr clas-sifier was applied in a hierarchical way, performing first the short-term staging and then the long-term prognosis for patients classified as in need of mechanical ventilation support. more specifically, a majority voting method was applied to classify patients into sd and si cases. then, another hierarchical structure was applied on the cases predicted as si only to classify them into the ones who didn't recover within `days of mechanical ventilation (ld) and the ones who recovered with days on mechanical ventilation (lr). in order to train all the models, each ct scan was normalized by cropping the hounsfield units in the range r´ , s. a variety of hyperparameters including loss functions, learning rates, optimizers had been tested and in this section we report the ones with the best performance for each compo- this retrospective multi-center study was approved by our in- (table ). in addition to the ct examination -when available -patient sex, age, and body mass index (bmi), blood pressure and diabetes, lymphocyte count, crp level and d-dimer level were also collected (table ) . for short-term outcome assessment, patients were divided into groups: those who died or were intubated in the days following the ct scan composed the severe short-term outcome subgroup, while the others composed the non-severe short-term outcome subgroup. for long-term outcome, medical records were reviewed from may th to may th, to determine if patients died or had been intubated during the period of at least one month following the ct examination. the data associated with each patient (holistic profiling), as well as the corresponding outcomes both in terms of severity assessment as well as in terms of final outcome and readers assessment will be made publicly available. imaging (reader c ) were asked to perform a triage (severe versus non-severe cases) and for the severe cases (short-term deceased versus short-term intubated) prognosis process to predict the short-term outcome. the dice similarity score (dsc) was calculated to assess the similarity between the manual segmentations of each ct exam of the test dataset and between manual and automated segmentations. the hausdorff distance (hd) was also calcu- for the stratification of the dataset into the different categories, classic machine learning metrics, namely balanced accuracy, weighted precision, and weighted specificity and sensitivity were utilized. the evaluation of covidenet together with its components and the comparison with the independent experts is summarised in table p= . ). as shown in figure correlation to disease extent from manual segmentations was better when using covi-denet (r " . , p ă . ) compared to covid d (r " . , p ă . ) or covid d (r " . , p ă . ) which oversegmented the disease. examples of disease segmentations are presented in figure . one can observe that the segmentations provided by covidenet are very close to the ones generated by the experts. in particular, the algorithm detects the diseased regions even in the case that they are relatively small capturing all the different opacities of covid- such as ground glass and consolidation. the holistic covid- pneumonia signature is presented in (table ) s/ns outcomes had a balanced accuracy of % (vs % for human readers consensus), a weighted precision of % (vs %), a weighted sensitivity of % (vs %) and specificity of % (vs %) and outperformed the consensus of human readers (figure , table ). our method successfully predicted % of the severe/critical cases opposed to only % for the consensus reader. the superiorty of our approach is also indicated by the higher auc reported ( . ), in comparison with the one achieved by the different readers ( . ). severe cases as depicted in figure referred to diabetic men, with higher level of volume/heterogeneity of disease and c-reactive protein levels. moreover, as indicated in figure the non-uniformity on glrlm for both lung and disease together with the disease extent seems to contribute considerable to the classification of the patients to ns versus s cases. the covid- pneumonia pandemic spiked hospitalizations, while exerting extreme pressure on intensive care units. in the absence of a cure, staging and prognosis is crucial for clinical decision-making for resource management and experimental outcome assessment, in a pandemic context. our objective was to predict patient outcomes prior to mechanical ventilation support. the proposed ensemble classifier aiming to predict the sd/(ld or lr) had a balanced accuracy of % (vs % for human readers consensus), a weighted precision of % (vs %), a weighted sensitivity of % (vs %) and specificity of % (vs %) and outperformed consensus of human readers (table ). our method for prognosis of sd/ ld/ lr had a balanced accuracy of %, a weighted precision of %, a weighted sensitivity of % and specificity of % to provide full prognosis (figure ). concerning the performance of our method for the classification of ld and lr patients (ta-ble ), our ensemble classifier reports a balance accuracy of %, a weighted precision of % a weighted sensitivity of % and a weighted specificity of %. as indicated also in figure the performance of our method reach an auc of . for the sd, a . for the lr and . for the ld classes. moreover, the age, hbp and lung non uniformity on the glszm seems to associate better for this task. moreover, in order to assess the impact of each feature category on the implemented models we performed an ablation study by successively removing one category of features from the categories defined for each classification task. results are presented in table . the feature categories were identified as follows: a) d : disease extent, b) d : disease variables that are shape/geometry related, c) d : disease variables that are tissue/texture, d) o : heart/lungs variables that are shape/geometry related, e) o : heart/lungs variables that are tissue/texture, f) b : age, gender, biologi- cal/obesity/diabetes/fat/high blood pressure. one can observe that the clinical only category contributes a lot for the separation of sd/ld/lr while for the ns/s cases their contribution is marginal, in contrary to the other imaging characteristics. ai-enhanced imaging, clinical and biological information proved capable to identify patients with severe short/long-term outcomes, bolstering healthcare resources under the extreme pressure of the current covid- pandemic. the information obtained from our ai staging and prognosis could be used as an additional element at admission to assist decision making. variety of studies have reported the use of deep learning for the diagnosis and quantification of covid- with ct scans. in particular, studies have already reported on deep learning diagnosing covid- pneumonia on chest cts. in (li et al., b ) the authors proposed the use of a deep learning architec-ture based on resnet for the diagnosis of covid- reporting very high performances, while they investigated the attention maps produced from their network. a very similar method is presented in (mei et al., ) reporting the use of deep learning on covid- diagnosis. moreover, in the authors propose the use of a unet architecture for the quan- table . ity to regress the proposed scores. finally, recently (tilborghs et al., ) presents a comparable study of deep learning based methods for the automatic quantification of covid- . assessing the severity of covid- patients is also a very quickly evolving topic in the medical community with some methods being currently under review. extracting valuable information from the imaging using recent advances is very important and could potentially facilitate the clinical practice. starting with, disease extent is known to be associated with severity (li et al., a; yuan et al., ) erogeneous lesions than pure ground glass opacities observable in mild cases. in (li et al., c) , the authors proposed the use of siamese networks for the severity assessment of covid- directly from ct scans. in (bai et al., ) , the authors proposed a deep learning pipeline based on lstms using d ct slices and a fusion of imaging and clinical information to as- (lassau et al., ) proposed the assessment of severity using a deep learning tool achieving an auc of . on a completely independent cohort, with low sensitivity. again, even if we can not perform a direct comparison our method reports similar performance in a completely independent cohort, while it is based on interpretable features extracted from different regions. finally, in (he et al., ) a d deep learning based approach using a multi-task learning is presented in order to separate covid- patients to severe and non severe cases. to the best of our knowledge this study is the first to have developed a robust, holistic covid- multi-omics signature for disease staging and prognosis demonstrating an equivalent/superior-to-human-reader performance on a multicentric data set. our approach complied appropriate data collection and methodological testing requirements beyond the existing literature (mei et al., heterogeneity of lung densities, reflecting the presence of an underlying airway disease such as emphysema and the presence of sub-radiological disease. among clinical variables, a higher crp level, lymphopenia and a higher prevalence of hypertension and diabetes were associated with a poorer outcome, consistent with previous reports guo et al., ; terpos et al., ) . interestingly, age was less predictive of disease severity than of poor outcome in severe patients. this is linked to the fewer therapeutic possibilities for these generally more fragile patients. lastly, the average body mass index (bmi) in both non-severe and severe groups corresponded to overweight. despite being correlated with bmi, the fat ratio measured on the ct scanner was only weakly associated with outcome. several studies have reported obesity to be associated with severe outcomes chaganti et al., ) and an editorial described the measurement of anthropometric characteristics as crucial to better estimate the risk of complications (stefan et al., ) . however a meta-analysis showed that whereas being associated with an increased risk of covid- pneumonia, obesity was paradoxically associated with reduced pneumonia mortality (wynants et al., ) . overall, the combination of clinical, biological and imaging features demonstrates their complementary value for staging and prognosis. in conclusion, we show that the combination of chest ct and artificial intelligence can provide tools for fast, accurate and precise disease extent quantification as well as the identifica- tensorflow: large-scale machine learning on heterogeneous distributed systems lung pattern classification for interstitial lung diseases using a deep convolutional neural network semantic segmentation of pathological lung tissue with dilated fully convolutional networks segnet: a deep convolutional encoder-decoder architecture for image segmentation classification of interstitial lung abnormality patterns with an ensemble of deep convolutional neural networks performance of a new quantitative computed tomography index for interstitial lung disease assessment in systemic sclerosis quantification of tomographic patterns associated with covid- from chest ct deep learning: definition and perspectives for thoracic imaging computer-aided 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tumour-infiltrating cd cells and response to anti-pd- or anti-pd-l immunotherapy: an imaging biomarker, retrospective multicohort study abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia hematological findings and complications of covid- comparative study of deep learning methods for the automatic segmentation of lung, lesion and lesion type in ct scans of covid- patients staging systems and disease severity assessment in interstitial lung diseases. current opinion in pulmonary medicine atlasnet: multi-atlas non-linear deep networks for medical image segmentation computational radiomics system to decode the radiographic phenotype computed tomographic biomarkers in idiopathic pulmonary fibrosis. the future of quantitative analysis prediction models for diagnosis and prognosis of covid- infection: systematic review and critical appraisal association of radiologic findings with mortality of patients infected with novel coronavirus in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study a novel coronavirus from patients with pneumonia in china writing -review & editing, eric deutsch : validation, writing -review & editing, fabrice andre: validation, writing -review & editing, enora guillo: data curation, writing -review & editing, nara halm: data curation, writing -review & editing, stefany el hajj: data curation we thank mihir sahasrabudhe and norbert bus for their valu- the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -vk lt x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: - - journal: animal models for the study of human disease doi: . /b - - - - . - sha: doc_id: cord_uid: vk lt x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., ) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi. norwalk virus, gii. snow mountain virus, and gii. hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately , deaths annually (patel et al., ) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., ; lutgehetmann et al., ; turcios-ruiz et al., ) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally - days, with symptoms persisting for - days (koopmans and duizer, ) . viral shedding can range from to days in healthy individuals (atmar et al., ) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., ; rockx et al., ) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase (fut ) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., ) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, ; becker et al., ; koopmans and duizer, ) . vomiting results in airborne dissemination of the virus with areas of . m being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to days (makison booth, ; tung-thompson et al., ) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., ; marks et al., ) . viral titers in emesis and fecal suspensions are as high as . × and . × ges (genomic equivalent copies per milliliter), respectively and the % infectious dose is ges (atmar et al., ) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., ; moe et al., ) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., ) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost % in human volunteers (aliabadi et al., ; atmar et al., ) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., ; tacket et al., ) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., ) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., ) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., ) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., ) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., ) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within - days postinfection and lasted anywhere from days to weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., ) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever days postinfection. viral shedding was detected for days. the immune system produced antibodies that dropped in concentration within days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., ) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., ) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., (hsu et al., , khan et al., ) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in % of the animals and virus was detected in the stool of % of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., ) . calves are naturally infected with bovine noroviruses (scipioni et al., ) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease - h postinfection. recovery of virus was achieved after . and h postinfection (otto et al., ) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at %- % followed by weev and lastly veev at less than % (ayers et al., ; griffin, ; steele and twenhafel, ) . there are currently no licensed vaccines or therapies but a recent phase clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in % of the subjects (hannaman et al., ) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., ) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, ) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., ; morgan, ) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., ) . after subq inoculation with weev, suckling mice started to show signs of disease by h and died within h (aguilar, ) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day - postinfection. mice were severely ill by day and appeared hunched and dehydrated. death occurred between days and with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, ; monath et al., ) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only % fatality in mice regardless of the amount of virus (liu et al., ) . comparing susceptibility of inbred and outbred strains revealed that cd- , balb/c, a/j, and c bl mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to weeks old with cns involvement and lethality (blakely et al., ) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, ) . virus begins to replicate in the draining lymph nodes at h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., ; steele et al., ) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., ; steele and twenhafel, ; steele et al., ) . in challenge of c h/hen mice with high dose veev caused high morbidity and mortality (julander et al., b) . viral titers in brain peaked on day postchallenge and remained elevated until animals succumbed on day - postchallenge. protein cytokine array performed on brains of infected mice showed elevated il- a, il- b, il- , il- , mcp- , ifnγ, mip- a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., a) . additionally, a veev vaccine inactivated with , -iodonaphthyl azide v protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., ) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., ; roy et al., ) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., ) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., ) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., ) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, ) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., ) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., ) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day - , followed by death on day - (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, ) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., ) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, ) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, ) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., ) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between - days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, ) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., ) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., ) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at - h after infection and lasted less than h. secondary fever generally began on day and lasted - days (gleiser et al., ) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., ) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., ) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., ) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., ) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized - days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, ) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in (charrel et al., ; rezza et al., ) . the incubation period following a mosquito bite is - days, leading to a self-limiting acute phase that lasts - days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, ) . individuals typically develop a stooped posture due to the pain. for approximately % of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of and years old have an increased risk of developing neurological manifestations (arpino et al., ) . there is currently no approved vaccine or antiviral. wild-type c bl/ adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day , and all succumbed by day , whereas % of -day-old mice were able to recover from infection. by days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., ; ziegler et al., ) . an adult mouse model has been developed by injection of the ventral side of the footpad of c bl/ j mice. viremia lasted - days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., ) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., ) . icr and cd- mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, and % for newborn cd- and icr mice, respectively. the remaining mice fully recovered within weeks after infection (ziegler et al., ) . a drawback of both the ifnα/βr and cd- mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., ) . a chronic infection model was developed using recombinant activating gene (rag −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia - days postinfection, which was detectable up to days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at - days postinfection. despite minimal footpad swelling on day postinfection, on day there was severe muscle damage noted at necropsy, which resolved by day (seymour et al., ) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within day postinfection with clearance following from day to . histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., ) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., ) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed - days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., ; messaoudi et al., ) . fever began at - days postinfection and persisted for - days and - days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., ; labadie et al., ; messaoudi et al., ) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline - days postexposure (chen et al., ) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., ) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., ) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, ) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, ) . it is estimated that there are , deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, ) . there are four distinct serotypes of denv, numbered - , which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, ) . incubation can range from to days, with the average being - days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., ) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, ) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., ) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., ) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit - pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c bl/ are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., ; paes et al., ; shresta et al., ) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c bl/ souza et al., ) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv introduced into ag mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., ) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., ) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., ) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., ; parida et al., ; zhao et al., a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., ) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., ; lin et al., ) . df symptoms developed after infection in nod/scid/il rγko mice engrafted with cd + human progenitor cells (mota and rico-hesse, ) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv (kuruvilla et al., ) . a passaged clinical isolate of denv was used to create a model in immunocompetent adult mice. ip injection in c bl/ j and balb/c caused lethality by day - postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day . this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., ) . vascular leakage was also observed when c bl/ were inoculated with denv (st john et al., ) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv . infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with - being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day . this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., ) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., ) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., ) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., ) . a robust antibody response was observed in multiple studies (marchette et al., ; onlamoon et al., ) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., (omatsu et al., , . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., ) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was -to -fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., ) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in uganda in (smithburn et al., . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early s (melnick et al., ; taylor et al., ) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, ) . the virus first reached the western hemisphere in the summer of , during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, ; lanciotti et al., ) . since , the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from to days (monath and tsai, ) . the mortality rate following neuroinvasive disease ranges from % to % (asnis et al., ; hayes, ; hubalek and halouzka, ; komar, ) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from % to %. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., ) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., ; pletnev et al., ) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., ) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., ) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and % mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., ; johnston et al., ) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., ; cunha et al., ; diamond et al., ; fratkin et al., ) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., ) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first days, became lethargic at approximately day , and developed neurologic symptoms between days and . many of the severely affected animals died - days after infection. viremia was detected in the hamsters within h after infection and persisted for - days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. ( ) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of . the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, ) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., ) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., ) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, ) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., ; d'ortenzio et al., ; hills et al., ; mccarthy, ) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, ) . a mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight days postinfection and met euthanasia criteria by day . microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day (dowall et al., ) . footpad inoculation of the virus leads to a fatal disease in ag mice by day postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., ) . ag mice were also observed to develop neurologic disease by day postexposure (rossi et al., ) . immunocompetent mice are resistant to infection via the subq route (rossi et al., ) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day . viral rna was detected in five out of nine placentas on day postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day postexposure in addition to cortical thinning (cugola et al., ; li et al., a) . ifnar −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., ) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov- e and hcov-nl . four are beta coronavirueses: hcov-oc , hcov-hku , hcov-sars, and mers-cov. hcov- e and hcov-oc were first detected in the s from the nasal passages of humans with the "common cold" (gaunt et al., ) . hcov-nl , which was first isolated in , causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., ) . hcov-hku , first isolated in , has been identified more sporadically but also causes respiratory infections (lau et al., ) . a significant portion of common cold infections in humans are caused by coronaviruses. in and , two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly and % fatality, respectively (assiri et al., ; peiris et al., ) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in as it spread throughout countries in a period of months, with confirmed infections and deaths (roberts and subbarao, ; world health organization, ) . no additional cases of community acquired sars-cov infection have been reported since . the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., ) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., ; rabenau et al., ; rota et al., ) . sars-cov infection has a % case fatality with the majority of cases in people over the age of (peiris et al., ; wang et al., ) . after an incubation period of - days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., ) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., ) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, ; perlman and dandekar, ) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, ) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., ) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, ) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼ %), viral replication, and pathology (roberts et al., ) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., ) . young, -to -week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day and clearance by day postexposure (mcauliffe et al., ) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c bl/ (b ), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day and clearance by day (glass et al., ) . in contrast, balb/c mice - months of age show weight loss, hunched posture, dehydration, and ruffled fur on day - postexposure (bisht et al., ) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. s mice and b mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than year (table . ). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day postexposure. beige, cd −/− , and rag −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., ) . stat ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme (ace ) and cd l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., ) . the variations in the ace sequence across animal species could partially explain the differences in infection severity (li et al., b; sutton and subbarao, ) . since mice in particular have a greater number of sequence differences in ace , transgenic mice were created that express human ace (mccray et al., ; netland et al., ; yang et al., ) . unlike other murine models of sars-cov, mice expressing hace had up to % mortality, with severity correlating to the level of hace expression (tseng et al., ) . with high levels of hace expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of tcid , along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., ) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., ) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., ) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., ) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day postexposure as well as mild alveolar damage in %- % of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., ; ter meulen et al., ) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., ; mcauliffe et al., ; rowe et al., ) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., ) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., ) . viral replication has been detected for up to days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., ; roberts et al., ) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, ) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, ; weiss and scott, ) . as such, immune mice had th type immunopathology upon sars-cov challenge (tseng et al., ) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., ) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., ) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from serial passages in the lungs of young balb/c mice (mccray et al., ; roberts et al., ; rockx et al., ) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., ; omrani et al., ) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october , there were confirmed cases and deaths (li et al., b) . bats serve as the likely natural reservoir since virus with % nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., ) . spread to humans likely comes from infected dromedary camels (adney et al., ; azhar et al., ) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp ), also known as cd (raj et al., ) . the expression and distribution of dpp in the human respiratory tract has recently been well characterized (meyerholz et al., ) . interestingly, dpp expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp (sutton and subbarao, ). in contrast the rhesus macaque and common marmoset have complete homology to human dpp , allowing productive mers-cov infection to occur falzarano et al., ; munster et al., ; yao et al., ) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., ) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp . one approach utilized an adenovirus vector expressing hdpp to transduce mice (zhao et al., b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp resulted in id and ld values of < and tcid , respectively (tao et al., ) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp under either a surfactant protein c or cytokeratin promoter (li et al., b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., ) . common marmosets infected with . × tcid (emc- ) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., ) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days and . viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least tcid (emc- ) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., ) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., ) . this is despite the finding that both species have complete homology with humans within the dpp domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, ) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from % to % in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., ) . marburg virus (marv) first emerged in in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over , suspected, probable and confirmed cases and over , deaths. bundibugyo virus (bdbv) first emerged in in bundibugyo, uganda with confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., ; wauquier et al., ) . approximately % of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table . ). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., ; smither et al., a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type interferon response (bray, ) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., ) . marv angola was particularly resistant to adaptation, but after serial passages in scid mice, infection caused severe disease in balb/c and c bl/ mice when administered in or ip (qiu et al., ) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., ; zumbrun et al., a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd , was particularly susceptible to airborne ma-ebov, with % lethality to low or high doses (approximately or pfu) ( zumbrun et al., a) . these mice developed weight loss of greater than % and succumbed to infection between days and postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged - weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately . µm and a geometric standard deviation (gsd) of approximately . for min. another approach uses immunodeficient mouse strains, such as scid, stat ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, ; lever et al., ; zumbrun et al., a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., ) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., (warfield et al., , ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., ) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il- receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., ) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., ; lever et al., ) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c and c (npc and npc ). npc (−/−) mice were fully susceptible to infection with ebov but npc (−/−) mice were completely resistant (herbert et al., ) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, ; herbert et al., ) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, ; herbert et al., ; tsuda et al., ) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male -to -week-old syrian hamsters which are infected with ld of ma-ebov. virus is present in tissues and blood collected on day and all animals succumbed to the disease by day . infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., ) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., ; connolly et al., ; twenhafel et al., ; zumbrun et al., c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., ) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., ) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses ( or pfu target doses) but lethality drops with low (less than pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., c) . weight loss of between % and % is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day , occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., ) . as such, guinea pigs have been used to establish transmission models (wong et al., a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., ) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., ) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., ; carrion et al., ; davis et al., ; hensley et al., a; reed et al., ; zumbrun et al., b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" ebov strain (marzi et al., ) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the epidemic compared to the outbreak suggest that ebov-makona is less virulent. the large number of cases in the - ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., ) . prominent features of the filovirus infections in nhps are onset of fever by day postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., ) . a cytokine storm occurs with rises in ifnγ, tnf, il- , and ccl (fernando et al., ) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th response (connor et al., ) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., ) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., ; twenhafel et al., ) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., ) . lymphocytolysis and lymphoid depletion are also observed (alves et al., ) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., ) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., b) . marmosets are similarly susceptible to infection with ebov- kikwit (smither et al., ) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., ) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., ) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., ) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is - days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., ) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., ) . nipah virus has an incubation period of days to weeks (goh et al., ) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within - h (lo and rota, ). survivors of infection typically make a full recovery; however, % suffer permanent sequelae, including persistent convulsions (tan and chua, ) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within - days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., ; rockx et al., ; wong et al., ) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., ; williamson et al., ) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with % mortality. clinical signs were apparent - days postinfection with death occurring within days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for - days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., ) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., ; pallister et al., ) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within - days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is - days and leads to respiratory and neurological symptoms (mungall et al., ; johnston et al., ; westbury et al., ) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only % of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., ) . agms are very consistent model of both viruses. it inoculation of the viruses results in % mortality, and death within . and - days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., ) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., ; li et al., ; middleton et al., ) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in % of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., ) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within days (marsh et al., ; williamson et al., ) . animals died within days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., ; wong et al., ) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., ) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., ) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., ) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within - days postinfection (tanimura et al., ) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of million children under the age of , which in turn results in million hospitalizations and approximately , deaths (nair et al., ) . within the united states, hospital costs alone amount to over million dollars per year (paramore et al., ) . outbreaks are common in the winter (yusuf et al., ) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is - days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, ) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., ) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., ) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are %- % with the greatest burden of disease seen in - month olds (ruuskanen and ogra, ). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between . % and . % upon infection (anderson et al., ) . although there are almost rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, ; higgins et al., ; kim and chang, ) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., ) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., ; stark et al., ) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., ). age does not affect primary disease manifestations (graham et al., ) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., ; van schaik et al., ) . virus was detected as early as day and reached maximum titer at day postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., ) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., ; niewiesk and prince, ) . viral replication is -to -fold greater in the cotton rat model than mouse model (wyde et al., ) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., ; prince et al., ) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the th day, dropping to below the levels of detection by day . the histopathology of the lungs appears similar to that of humans after infection (piazza et al., ) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th response in cotton rats, whereas humans tend to have a response skewed toward th (culley et al., ; dakhama et al., ; ripple et al., ) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., ) . ferrets infected by it were found to have detectable rsv in throat swabs up to day postinfection, and positive qpcr up to day . immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., ) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., ) . bonnet monkeys were developed an inflammatory response by day with viral rna detected in both bronchial and alveolar cells (simoes et al., ) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., ; teng et al., ) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., ) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., ; sow et al., ) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within days. rsv replication peaked at days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., ; scheerlinck et al., ) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; ha and na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within days (taubenberger and morens, ) . the rate of infection is estimated at % in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately , people in the united states die each year from seasonal influenza (dushoff et al., ) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., b) . influenza virus replicates in the upper and lower airways, peaking at approximately -h postexposure. infection can be more severe in infants and children under the age of , people over the age of , or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, ; glezen, ) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼ % of all influenza associated fatalities (alonso et al., ; ison and lee, ; speshock et al., ) . death, often due to ards can occur as early as days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, ) . the h n avian strain of influenza, has lethality rates of around ∼ % (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., ; wang et al., ) . h n is another avian influenza a strain that infected more than people and was implicated in deaths. approximately % of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., ) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. ( ) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, ). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, ) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h n for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, ; hagenaars et al., ; ortigoza et al., ) . inoculation is by the in route, utilizing approximately µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of < µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c bl/ j mice. males and females have equivalent disease but influenza is generally more infectious in younger -to -week-old ( - g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx is defective in most inbred mouse strains (staeheli and haller, ) . mice with the knocked-in mx gene have a -fold higher ld- for an influenza a strain (pr ) than wildtype background c bl/ mice (grimm et al., ) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as h postexposure, but most mortality occurs from to days postexposure accompanied by decreases in rectal temperature (sidwell and smee, ). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., ) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha- -acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa , predominates over sa , which is why h , h , and h subtypes usually need to be adapted to mice and h n , h , h , and h viruses do not require adaptation (o'donnell and subbarao, ). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h n and the pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., ; taubenberger, ) . h n infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., ; gubareva et al., ; lu et al., ) . as in humans, the virulence of h n is attributable to damage caused by an overactive host immune response. additionally, mice infected with the h n influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., ) . reassortment influenza viruses of the h n virus and a low-pathogenicity avian h n virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., ) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to weeks. at least one transmission model has also been developed in mice. with h n influenza, transmission rates of up to % among cagemates can be achieved after infection by the aerosol route and cocaging after h (schulman, ). rats (f and sd) inoculated with rat-adapted h n developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., ) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., ) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., ) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., ; ottolini et al., ) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at h postexposure. virus was cleared from the lung by day and from the nares by day , but animals had bronchial and alveolar damage, and pneumonia for up to weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., ; maines et al., ; mclaren and butchko, ) . like humans, ferrets exclusively express neu ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., ) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., ) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa , and sa , ) within the airways similar to that of humans (van riel et al., ) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., ; lambkin et al., ; maines et al., ) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of . - . ml containing approximately - egg id dropwise to each nostril. however, a larger inoculum volume of . ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., ) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., ) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., ) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts - days for mild to moderately virulent strains (maher and destefano, ) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h n strains without adaptation (zitzow et al., ) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., ) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., ) . nonadapted h n , h n , and h n have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., ) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h n influenza infection was developed (paquette et al., ) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., ) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., ) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., ) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h n -infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., ; zitzow et al., ) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., ) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with pandemic h n influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., ) . a superinfection model resembling that of mice has been developed by in instillation of influenza in -to -week-old ferrets followed by in inoculation of s. pneumonia days later (peltola et al., ) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h n (enserink, ; fouchier et al., ; herfst et al., ; oh et al., ) . in general, some subtypes, such as the h n , can transmit easily through aerosol and respiratory droplets (munster et al., ) . of concern, h n isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., ; richard et al., ; zhang et al., ) . h n became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., ) . transmission occurs more readily at the height of pyrexia, but for the h n in particular, can occur before fever is detected (roberts et al., ) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., ) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., ) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h n vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., ) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., ) . h n and h n infection of cynos is very similar to humans (rimmelzwaan et al., ) . cynos develop fever and ards upon in inoculation of h n with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously × pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h n results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the h n caused a very high mortality rate due to an aberrant immune response and ards and had more than % lethality (humans only had a %- % lethality) (kobasa et al., ) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h n (o'donnell and subbarao, ) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., ) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., ) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h n (isoda et al., ; lipatov et al., ) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., ) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h n with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., ; rimmelzwaan et al., ) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after - days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, ) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., ; mundel and gear, ) . after -to -day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, ; laughlin et al., ; peters and linthicum, ) . depending on the severity of the disease when the symptoms start, %- % of the hospitalized patients might die in - days or - days after the disease onset (ikegami and makino, ) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., ) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, and c bl/ were exposed to rvfv via parental or aerosol routes of infection (ross et al., ) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, ; smith et al., ) . mice start to show signs of decreased activity and ruffled fur by day - postexposure. immediately following these signs, they become lethargic and generally die - days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, ) . increased viremia and tissue tropism were reported in mice with (smith et al., ) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., ; reed et al., ) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher , buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, ; peters and slone, ) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. ( ) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., ) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., ) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day post-subq inoculation and the disease was uniformly lethal by day - postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., ) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., ) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. ( ) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days and postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, ) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts - days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, ; swanepoel et al., ) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., ; nalca and whitehouse, ; shepherd et al., ; smirnova, ) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day postinfection (tignor and hanham, ) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day ) and the brain (day ). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., ; bereczky et al., ) . bente et al. infected stat knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in - days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., ) . similar observations were made in this model as in the stat knockout mouse model. animals were moribund and died - days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl , ccl increased dramatically day postchallenge and gm-csf, il- a, il- b, il- , il- , il- p , il- , il- , cxcl , ccl , ccl , and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t- (favipiravir) successfully (oestereich et al., ) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., ; canakoglu et al., , p. ) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, ) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. ( ) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days and after inoculation, only the baboon serum had neutralizing antibody activity on day postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., ) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., (blagoveshchenskaya et al., ). shepherd et al. ( infected species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, ) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., ) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, ) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., ) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, ) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, ) . incubation lasts - weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately % patients. overall mortality rate is %. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, ) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., ; stephen et al., ) . the first report of hps in maine was recently documented (centers for disease control and prevention, ). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., ) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of %. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, ) . incubation typically occurs - days following exposure (young et al., ) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., ) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within days postinfection. clinical signs did not appear until h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day and lasted up to days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., ) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., ) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, ) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., ; wichmann et al., ) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., ) . in a study of panel of laboratory strains of mice, c bl/ mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within - h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., ) . in a recent study, -weekold icr mice was exposed to htnv strain aa via the subq route (seto et al., ) . mice started to show signs of disease by day postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., ; mcelroy et al., ) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., ; sironen et al., ) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within days postinfection. four of six aerosol exposed monkeys and of iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., ) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed - days prior to respiratory signs of the disease that were observed on days - postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, ) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, ) . this virus is responsible for , - , infections per year, leading to approximately deaths (khan et al., ) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., ) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, ) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in % of individuals. the incubation period is from to days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about %; however, of those admitted to the hospital it is between % and %. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within days (mccormick et al., a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., ) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., ) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., ) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., ) . within this model, the nhp becomes viremic after - days. clinical manifestations were present by day and death typically occurred within - days (lukashevich et al., ; rodas et al., ) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., ; gunther et al., ) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day and viremia achieved by day . liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., ) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., ) . stat knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., ) . guinea pig inbred strain was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., ) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., ; qian et al., ) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., ; katz and starr, ) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within - days. virus was first detected at days, and reached maximum titers within days. neurological symptoms began to appear at the same time, and all animals died by day . pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., ) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, ; gowen et al., ; smee et al., ) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects . % of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately . million deaths per year with over , being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv- and hiv- , with hiv- having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv- (de cock et al., ) . hiv targets t-helper cells (cd +), macrophages, dendritic cells (fields et al., ) . acute infection occurs - weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd + t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd + t-cells decline to less than cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., ) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, ) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., ; melkus et al., ) . a number of different humanized mouse models have been developed for the study of hiv, including rag −/− γc −/− , rag −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., ; li et al., ; shimizu et al., ) . cd + human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., ; watanabe et al., ) . hiv- infection by ip injection can be successful with as little as % peripheral blood engraftment (berges et al., ) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv- viremia occurs within approximately days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., ) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., ; neff et al., ) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, ) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd -and human ccr -expressing transgenic luciferase reporter mouse to study hiv- pseudovirus entry (gruell et al., ) . hiv- transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., ; reid et al., ) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv- tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, ) . an adaptation of hiv- was obtained by four passages in pigtailed macaques transiently depleted of cd (+) cells during acute infection (hatziioannou et al., ) . the resulting disease has several similarities to aids in humans, such as depletion of cd (+) t-cells (kimata, ) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, ; walker et al., ) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., ; miller et al., ; stone et al., ) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., ) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., ) . studies are performed in bsl- animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., ) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with tcid of the highly pathogenic siv/ deltab induces aids in most macaques within - months (mean of months) (fuller et al., ) . peak viremia occurs around week . aids in such models is often defined as cd + t-cells that have dropped to less than % of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., ; moldt et al., ; reynolds et al., ) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv- laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv- and is more closely related to hiv- . however, the polymerase region of siv is % homologous to that of hiv- and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv- rt is usually put into siv for such studies (uberla et al., ) . sivmac is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., ) . nhps infected with sivmac have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd + t-cell depletion. additionally, sivmac utilizes the cxcr chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., ) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., ) . one disadvantage of the highly virulent env-shiv (shiv- . p), is that it uses the cxcr coreceptor. of note, env-shivs that do use the cxcr coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., ) . simian-tropic (st) hiv- contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for months, followed by clearance (haigwood, ) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., ) . upon vaginal instillation of tcid of shiv- p , peak viremia was seen around days postexposure with greater than copies/ml and dropping thereafter to a constant level of rna copies/ml at days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac (barouch et al., ) . an example of an intrarectal model utilized juvenile ( -year-old) pigtailed macaques, challenged intrarectally with tcid s of siv mne to study the pathogenesis related to the virulence factor, vpx (belshan et al., ) . here, viremia peaked at approximately days with more than copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult ( -to -year-old) male cynomolgus macaques were intravenously inoculated with aid s of sivmac and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., ) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, ) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., ) . pregnant female pigtailed macaques were infected during the second trimester with mid shiv-sf p by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., ) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., ) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., ) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, ) . there are over human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv- , hpv- , hpv- , hpv- , and hpv- place individuals at high risk for cervical and other cancers. up to % of head and neck cancers are caused by hpv- , particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., ; brandsma, ; campo, ) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., ) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., ; oosterhuis et al., ) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e , e , e , or the entire early region of hpv- from the keratin promoter which is only active in the basal cells of the mouse epithelium (chow, ) . cancers in these models develop upon extended estrogen exposure (maufort et al., ; ocadiz-delgado et al., ; stelzer et al., ; thomas et al., ) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e develop invasive cervical squamous carcinomas (bulut and uren, ) . the tumors occur within months approximately % of the time. another model uses c bl/ mice expressing the hpv -e transgene which are then treated topically with , -dimethylbenz(a)anthracene (dmba) (de azambuja et al., ) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., ; siolas and hannon, ) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., ) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., ) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within - weeks postexposure in experimental settings (johnston et al., ) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv- and hpv- make this a useful model (nicholls et al., ) . these lesions typically spontaneously regress - weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged weeks to years, with no history of copv, are typically used for these studies. infection is achieved by application of a µl droplet of virus extract to multiple . cm scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., ) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, ) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, ) . bpv- infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv- and is associated with the development of cervical cancer ( ostrow et al., ; wood et al., ) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, ; khodakevich et al., ) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in . no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of , a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among reported cases, human cases were laboratory confirmed during an outbreak (nalca et al., ; sejvar et al., ) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, ) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, ) . after a -to -dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, ; jeézek and fenner, ) . a typical maculopapular rash follows the prodromal period, generally lasting - days. the average size of the skin lesions are . - cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically - weeks. the fatality rate is % among the unvaccinated population and death generally occurs during the nd week of the disease (jeézek and fenner, ; nalca et al., ) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table . ). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat -deficient c bl/ mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., ; falendysz et al., ; hutson et al., ; osorio et al., ; sbrana et al., ; schultz et al., ; sergeev et al., ; stabenow et al., ; tesh et al., ; xiao et al., ) . cast/eij mice, one of the inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a fold higher susceptibility to mpxv when compared to in route (americo et al., ) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day postinfection (osorio et al., ) . similarly, c bl/ stat −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality days postexposure. recently sergeev et al. ( ) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused % mortality in days postexposure and days postexposure, respectively, in ground squirrels (tesh et al., ) . the disease progressed very quickly and most of the animals were lethargic and moribund by day postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in - days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. ( ) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels - days postexposure. mortality was observed in % of the animals - days postexposure (sergeev et al., ) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., ; keckler et al., ; smith et al., ; xiao et al., ) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and % mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., ) . hutson et al. ( ) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., ) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., ) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of and pfu provided % mortality with a mean time to death of days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., ) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., ) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., ; johnson et al., ; nalca et al., ; stittelaar et al., ; zaucha et al., ) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day postexposure (nalca et al., ) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., ) . whole blood and throat swabs had viral loads peak around day , and in survivors, gradually decrease until day postexposure. since doses of × pfu, × pfu, or × pfu resulted in lethality for % of the animals, whereas a dose of × pfu resulted in % lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., ) . tree et al. ( ) showed that ifnγ, il- rα, and il- increased dramatically on day postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., ) . studies showed that a × pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received pfu of mpxv intratracheally (stittelaar et al., ) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately -fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., ) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over million people chronically infected and , cases per year of liver cancer due to infection (lee, ) . the virus can naturally infect both humans and chimpanzees (guha et al., ) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., ) . the age at which one is infected dictates the risk of developing chronic disease (hyams, ) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, ) . after this acute phase, lifetime immunity is achieved (wright and lau, ) . of those infected, less than % will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is times more likely to develop in a chronically infected individual than a noncarrier (beasley, ) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, ). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., ; . it was found that %- % of all wild-caught animals from africa are positive for hbv antigen ( lander et al., ) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, ) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., ) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., ) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., ) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., ; yang et al., ) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., ) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, ) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, ) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., ) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., ) . within a population, %- % of all neonatal woodchucks are susceptible to chronic infection (cote et al., ) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., ) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, ) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, ) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., ) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, ) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., ) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., ) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., ) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., ) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv- pathogenesis and for designing pediatric hiv- prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis morbidity and mortality among patients with respiratory syncytial virus infection: a -year retrospective review chikungunya and the nervous system: what we do and do not know the west nile virus outbreak of in new york: the flushing hospital experience hospital outbreak of middle east respiratory syndrome coronavirus diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses norovirus vaccine against experimental human norwalk virus illness determination of the % human infectious dose for norwalk virus an epizootic attributable to western equine encephalitis virus infection in emus in texas evidence for camel-to-human transmission of mers coronavirus integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics disseminated and sustained hiv infection in cd + cord blood cell-transplanted rag −/− gamma c −/− mice choice of inbred rat strain impacts lethality and disease course after respiratory infection with rift valley fever virus rift valley fever: an uninvited zoonosis in the arabian peninsula recombinant norwalk virus-like particles given orally to volunteers: phase i study tropism of dengue virus in mice and humans defined by viral nonstructural protein -specific immunostaining lethal antibody enhancement of dengue disease in mice is prevented by fc modification animal models for the study of influenza pathogenesis and therapy effect of oral gavage treatment with znal and other metallo-ion formulations on influenza a h n and h n virus infections in mice macaque studies of vaccine and microbicide combinations for preventing hiv- sexual transmission early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus hepatitis b virus. the major etiology of hepatocellular carcinoma transmission of norwalk virus during football game vpx is critical for sivmne infection of pigtail macaques experimental respiratory syncytial virus infection of four species of primates pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat- knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice the utility of the new generation of humanized mice to study hiv- infection: transmission, prevention, pathogenesis, and treatment hiv- infection and cd t cell depletion in the humanized rag −/− gamma c −/− (rag-hu) mouse model bacterial infections in pigs experimentally infected with nipah virus evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development genetic basis of attenuation of dengue virus type small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells chimpanzees as an animal model for human norovirus infection and vaccine development a simple technique for infection of mosquitoes with viruses; transmission of zika virus human papillomavirus research: do we still need animal models? human papillomavirus in cervical cancer development of a hamster model for chikungunya virus infection and pathogenesis a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection the cotton rat model of respiratory viral infections correlates of immunity to filovirus infection filovirus vaccines induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice animal models of human-papillomavirus-associated oncogenesis interferon alpha/beta receptor-deficient mice as a model for ebola virus disease zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease effectiveness of influenza vaccination the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis the confirmation and maintenance of smallpox eradication a lethal disease model for hantavirus pulmonary syndrome in immunosuppressed syrian hamsters infected with sin nombre virus nonhuman primate models of chikungunya virus infection and disease tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates pediatric norovirus diarrhea in nicaragua efficacy assessment of a cell-mediated immunity hiv- vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial variation between strains of hamsters in the lethality of pichinde virus infections hepatitis b viruses and hepatocellular carcinoma generation of k -e /n betacat double transgenic mice as a model of cervical cancer limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody a novel vaccine against crimean-congo haemorrhagic fever protects % of animals against lethal challenge in a mouse model interleukin- beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c bl/ mice animal models of papillomavirus pathogenesis immunization of knock-out alpha/beta interferon receptor mice against high lethal dose of crimean-congo hemorrhagic fever virus with a cell culture based vaccine characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues a small nonhuman primate model for filovirus-induced disease severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus outbreak of acute illness-southwestern united states outbreak of west nile-like viral encephalitis-new york, . mmwr morb. mortal in vitro whole-virus binding of a norovirus genogroup ii genotype strain to cells of the lamina propria and brunner's glands in the human duodenum animal models for studying dengue pathogenesis and therapy us doctors investigate more than possible cases of monkeypox mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse chikungunya outbreaks-the globalization of vectorborne diseases inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs the immunobiology of sars* induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein norovirus infection as a cause of diarrhea-associated benign infantile seizures comparative pathogenesis of epidemic and enzootic chikungunya viruses in a pregnant rhesus macaque model development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs model systems to study the life cycle of human papillomaviruses and hpv-associated cancers primary severe acute respiratory syndrome coronavirus i nfection limits replication but not lung inflammation upon homologous rechallenge viral and host factors in human respiratory syncytial virus pathogenesis pathogenesis of pichinde virus infection in strain guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pathogenesis of experimental ebola virus infection in guinea pigs transcriptional profiling of the immune response to marburg virus infection the use of a neonatal mouse model to study respiratory syncytial virus infections a model of denv- infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection a mouse model for chikungunya: young age and inefficient type-i interferon signaling are risk factors for severe disease mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice comparison of the pathogenesis of the angola and ravn strains of marburg virus in the outbred guinea pig model the brazilian zika virus strain causes birth defects in experimental models age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood lassa fever encephalopathy: clinical and laboratory findings profound and prolonged lymphocytopenia with west nile encephalitis first complete genome sequence of zika virus (flaviviridae, flavivirus) from an autochthonous transmission in brazil viral haemorrhagic fevers caused by lassa, ebola, and marburg viruses the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il- production mouse models of hepatitis b and delta virus infection [experimental inhalation infection of monkeys of the macacus cynomolgus and macacus rhesus species with the virus kinetic profile of influenza virus infection in three rat strains pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells validation of an hpv -mediated carcinogenesis mouse model middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques selection of unadapted, pathogenic shivs encoding newly transmitted hiv- envelope proteins b-cells and the use of non-human primates for evaluation of hiv vaccine candidates antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv- in humanized blt mice innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus rift valley fever virus encephalitis is associated with an ineffective systemic immune response and activated t cell infiltration into the cns in an immunocompetent mouse model evidence of sexual transmission of zika virus a susceptible mouse model for zika virus infection identification of a novel coronavirus in patients with severe acute respiratory syndrome subclinical infection without encephalitis in mice following intranasal exposure to nipah virus-malaysia and nipah virus-bangladesh nonhuman primate models of encephalitic alphavirus infection: historical review and future perspectives mortality due to influenza in the united states-an annualized regression approach using multiple-cause mortality data distinct pathogenesis of hong kong-origin h n viruses in mice compared to that of other highly pathogenic h avian influenza viruses postexposure antibody prophylaxis protects nonhuman primates from filovirus disease comparative live bioluminescence imaging of monkeypox virus dissemination in a wild-derived inbred mouse (mus musculus castaneus) and outbred african dormouse (graphiurus kelleni) siv-induced impairment of neurovascular repair: a potential role for vegf smallpox vaccine does not protect macaques with aids from a lethal monkeypox virus challenge influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats an outbreak of viral gastroenteritis following environmental contamination at a concert hall natural history of aerosol exposure with marburg virus in rhesus macaques experimental congo virus (ib-an ) infection in primates further assessment of monkeypox virus infection in gambian pouched rats (cricetomys gambianus) using in vivo bioluminescent imaging respiratory syncytial virus infection in elderly and high-risk adults infection with mers-cov causes lethal pneumonia in the common marmoset immune response to marburg virus angola infection in nonhuman primates fields' virology the susceptibility of rats to rift valley fever in relation to age henipavirus susceptibility to environmental variables pause on avian flu transmission research aetiology: koch's postulates fulfilled for sars virus spinal cord neuropathology in human west nile virus infection therapeutic dna vaccine induces broad t cell responses in the gut and sustained protection from viral rebound and aids in siv-infected rhesus macaques hepatitis b virus infection-natural history and clinical consequences biological heterogeneity, including systemic replication in mice, of h n influenza a virus isolates from humans in hong kong chikungunya virus arthritis in adult wild-type mice epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method development of an acute and highly pathogenic nonhuman primate model of nipah virus infection animal models of hepatitis delta virus infection and disease hepadnavirusinduced liver cancer in woodchucks experimental infection and natural contact exposure of dogs with avian influenza virus (h n ). emerg megaribavirin aerosol for the treatment of influenza a virus infections in mice discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice studies on the virus of venezuelan equine encephalomyelitis. i. modification by cortisone of the response of the central nervous system of macaca mulatta serious morbidity and mortality associated with influenza epidemics a novel respiratory model of infection with monkeypox virus in cynomolgus macaques clinical features of nipah virus encephalitis among pig farmers in malaysia monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses interferon alfacon- protects hamsters from lethal pichinde virus infection primary respiratory syncytial virus infection in mice pneumonitis and multiorgan system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area-puerto rico acute and chronic airway disease after human respiratory syncytial virus infection in cotton rats (sigmodon hispidus) alphaviruses replication fitness determines high virulence of influenza a virus in mice carrying functional mx resistance gene antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv- infection in a transgenic mouse model characterization of influenza a/hongkong/ / (h n ) virus in a mouse model and protective effect of zanamivir on h n infection in mice epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the st century cell culture and animal models of viral hepatitis. part i: hepatitis b expression of the hepatitis delta virus large and small antigens in transgenic mice acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum , -iodonaphthyl azide-inactivated v protects against aerosol challenge with virulent venezuelan equine encephalitis virus dengue: an update pegylated interferonalpha protects type pneumocytes against sars coronavirus infection in macaques asymptomatic middle east respiratory syndrome coronavirus infection in rabbits head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model update on animal models for hiv research norovirus disease in the united states. emerg cardiopulmonary manifestations of hantavirus pulmonary syndrome serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus hendra virus infection in a veterinarian a phase clinical trial of a dna vaccine for venezuelan equine encephalitis delivered by intramuscular or intradermal electroporation deaths from norovirus among the elderly, england and wales. emerg aerosolized rift valley fever virus causes fatal encephalitis in african green monkeys and common marmosets hiv- -induced aids in monkeys west nile fever short communication: viremic control is independent of repeated low-dose shivsf p exposures pathogenesis of marburg hemorrhagic fever in cynomolgus macaques pathogenesis of lassa fever in cynomolgus macaques niemann-pick c is essential for ebolavirus replication and pathogenesis in vivo airborne transmission of influenza a/ h n virus between ferrets animal models in hiv- protection and therapy advances in rsv vaccine research and development-a global agenda transmission of zika virus through sexual contact with travelers to areas of ongoing transmissioncontinental united states establishment of a patient-derived orthotopic xenograft (pdox) model of her- -positive cervical cancer expressing the clinical metastatic pattern resolution of primary severe acute respiratory syndromeassociated coronavirus infection requires stat mucosal immunity and vaccines nipah virus outbreak with person-to-person transmission in a district of bangladesh eastern equine encephalitis virus in mice i: clinical course and outcome are dependent on route of exposure the lesions of experimental equine morbillivirus disease in cats and guinea pigs a lethal disease model for hantavirus pulmonary syndrome hantaan/ andes virus dna vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates persistent infection with and serologic cross-reactivity of three novel murine noroviruses molecular characterization of three novel murine noroviruses identification of hepatitis b virus indigenous to chimpanzees manifestation of thrombocytopenia in dengue- -virusinfected mice transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice west nile fever-a reemerging mosquito-borne viral disease in europe. emerg live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus clinical characteristics of human monkeypox, and risk factors for severe disease shiv- i and passaged progeny viruses encoding r hiv- clade c env cause aids in rhesus monkeys norwalk virus infection associates with secretor status genotyped from sera a prairie dog animal model of systemic orthopoxvirus disease using west african and congo basin strains of monkeypox virus risks of chronicity following acute hepatitis b virus infection: a review the pathogenesis of rift valley fever experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/ / (h n ) in different species of birds and mammals respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice a multimeric l vaccine for prevention of animal papillomavirus infections lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin pathogenesis of lassa virus infection in guinea pigs perinatal transmission of shiv-sf p in macaca nemestrina human monkeypox and other poxvirus infections of man cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route phenotypic changes in langerhans' cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes detailed analysis of the african green monkey model of nipah virus disease experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera treatment of venezuelan equine encephalitis virus infection with (-)-carbodine c h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection blt humanized mice as a small animal model of hiv infection stat -dependent innate immunity to a norwalk-like virus pichinde virus infection in strain guinea pigs reduces intestinal protein reflection coefficient with compensation establishment of the black-tailed prairie dog (cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high-and low-dose monkeypox virus challenges low-dose rectal inoculation of rhesus macaques by sivsme or sivmac recapitulates human mucosal infection by hiv- new opportunities for field research on the pathogenesis and treatment of lassa fever gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease isolation of monkeypox virus from wild squirrel infected in nature in hot pursuit of the first vaccine against respiratory syncytial virus pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine the severe pathogenicity of alveolar macrophage-depleted ferrets infected with pandemic h n influenza virus mouse adaptation of influenza b virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets stepping toward a macaque model of hiv- induced vomiting as a symptom and transmission risk in norovirus illness: evidence from human challenge studies wild-type puumala hantavirus infection induces cytokines, c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the influenza virus adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs west nile viral encephalitis foodborne viruses: an emerging problem low pathogenic avian influenza a(h n ) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies filoviruses: a compendium of years of epidemiological, clinical, and laboratory studies pathology of human influenza a (h n ) virus infection in cynomolgus macaques (macaca fascicularis) dengue virus infection and immune response in humanized rag (-/-)gamma(c) (-/-) (rag-hu) mice chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages viral hepatitis b strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus hku and other coronavirus infections in hong kong epidemic rift valley fever in egypt: observations of the spectrum of human illness hantaviruses. a short review hepatitis b virus infection quantitative measurement of influenza virus replication using consecutive bronchoalveolar lavage in the lower respiratory tract of a ferret model characterization of the activity of '-c-methylcytidine against dengue virus replication diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of hiv sequencing, annotation, and characterization of the influenza ferret infectome lethality and pathogenesis of airborne infection with filoviruses in a alpha/beta −/− interferon receptor-deficient mice experimental inoculation study indicates swine as a potential host for hendra virus early initiation of antiretroviral therapy can functionally control productive hiv- infection in humanized-blt mice zika virus disrupts neural progenitor development and leads to microcephaly in mice middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys study of dengue virus infection in scid mice engrafted with human k cells a comparative study of the pathogenesis of western equine and eastern equine encephalomyelitis viral infections in mice by intracerebral and subcutaneous inoculations hydrodynamics-based transfection in animals by systemic administration of plasmid dna the emergence of nipah virus, a highly pathogenic paramyxovirus the guinea pig as a transmission model for human influenza viruses transmission in the guinea pig model a mouse model for the evaluation of pathogenesis and immunity to influenza a (h n ) viruses isolated 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mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord- -b s stvz authors: guimarães, luísa eça; baker, britain; perricone, carlo; shoenfeld, yehuda title: vaccines, adjuvants and autoimmunity date: - - journal: pharmacological research doi: . /j.phrs. . . sha: doc_id: cord_uid: b s stvz abstract vaccines and autoimmunity are linked fields. vaccine efficacy is based on whether host immune response against an antigen can elicit a memory t-cell response over time. although the described side effects thus far have been mostly transient and acute, vaccines are able to elicit the immune system towards an autoimmune reaction. the diagnosis of a definite autoimmune disease and the occurrence of fatal outcome post-vaccination have been less frequently reported. since vaccines are given to previously healthy hosts, who may have never developed the disease had they not been immunized, adverse events should be carefully accessed and evaluated even if they represent a limited number of occurrences. in this review of the literature, there is evidence of vaccine-induced autoimmunity and adjuvant-induced autoimmunity in both experimental models as well as human patients. adjuvants and infectious agents may exert their immune-enhancing effects through various functional activities, encompassed by the adjuvant effect. these mechanisms are shared by different conditions triggered by adjuvants leading to the autoimmune/inflammatory syndrome induced by adjuvants (asia syndrome). in conclusion, there are several case reports of autoimmune diseases following vaccines, however, due to the limited number of cases, the different classifications of symptoms and the long latency period of the diseases, every attempt for an epidemiological study has so far failed to deliver a connection. despite this, efforts to unveil the connection between the triggering of the immune system by adjuvants and the development of autoimmune conditions should be undertaken. vaccinomics is a field that may bring to light novel customized, personalized treatment approaches in the future. vaccines have been a preventive treatment option available for over years. they have been proven to be effective in preventing infections that previously had high morbidity and mortality. an example of this is the eradication of small pox, which was mainly attributed to successful vaccination programs. preventing a high burden disease has since proven to be a cost effective measure and, as such, vaccines have become a part of multiple national health programs. these promising results led to the development of more and more vaccines and to the study of its applicability in other fields such as cancer prevention and treatment. vaccines are drugs administered to healthy individuals, and much like other drugs, vaccines are associated with adverse events. usually the described adverse events are transient and acute, but may rarely present with hypersensitivity and induction of autoimmunity that may be severe and fatal. these adverse events play an important role in the life of the vaccinated patients. immune mediated diseases arise from various different sources; these include environmental, genetic, hormonal and immune defects. the combination of these defects can be described as the mosaic of autoimmunity [ ] . patient background can be used as a clue to determine the response that may be elicited following drug administration. it has been proven that infectious agents may elicit an autoimmune disease in a prone subject through various mechanisms, including, but not limited to, molecular mimicry, epitope spreading and polyclonal activation [ ] . scientific findings suggest that autoimmunity may be triggered by vaccine adjuvants, of which aluminum compounds (aluminum hydroxide and phosphate) have been the most studied and the most widely used. adjuvants are molecules, which, in combination with antigens, enhance immunological response. this enables an easier and more effective recognition of "non self", which in turn permits the triggering of adaptive and innate immune responses [ ] . recently a new syndrome was described: "autoimmune/inflammatory syndrome induced by adjuvants" (asia). this embodies a spectrum of reactions, which are usually mild but may also be severe. these reactions are attributed to adjuvant stimulation, which can include chronic exposure to silicone, tetramethylpentadecane, pristane, aluminum, infectious components and other adjuvants. all of these environmental factors have been found to induce autoimmunity and inflammatory manifestations by themselves both in animal models and in humans. the mechanisms of this disease will be described in further detail [ ] . this review will focus on general mechanism of vaccines, adjuvant-induced autoimmunity, and on vaccines and the specific autoimmune diseases that they may trigger. adjuvants approved to date for human vaccines are: aluminum, mf in some viral vaccines, mpl, as , as b and as a against viral and parasitic infections, virosomes for hepatitis b virus (hbv), human papilloma virus (hpv), hepatitis a virus (hav), and cholera toxin for cholera. adjuvants may be composed of several different compounds. currently, oil based adjuvants, virosomes, toll-like receptors (tlrs) related adjuvants, mpl, adjuvants made of unmethylated cpg dinucleotides and tuftsin have all been described. it is of great interest the understanding of the mechanisms related to the adjuvant effect, as well as to aluminum salts. aluminum acts through multiple pathways, which do not depend solely on tlrs signaling. each of these pathways leads to an enhanced host immune response [ ] . there are many oil based adjuvants. one is incomplete freund adjuvant (ifa), which contains water in oil emulsion. another is complete freund adjuvant (cfa), which is the same as ifa, except that it also contains killed mycobacteria in addition to water in oil emulsion. usually, cfa is used for primary vaccination and ifa for boosting. recent oil based adjuvants that have been developed are mf (novartis ® ), as (glaxosmithkline ® ), advax tm which are based on inulin compounds (vaxine tm pty) and qs- /iscoms, which are immune stimulating complexes composed of cholesterol and phospholipid with or without antigen (table ) . virosomes are adjuvants that contain a membrane-bound hemagglutinin and neuraminidase obtained from the influenza virus. both components facilitate the uptake into antigen presenting cells (apc) and mimic the natural immune response [ ] . leucocyte membranes have membrane bound pattern recognition receptors (prrs) called tlrs, which are responsible for detecting most (although certainly not all) antigen-mediated infections. their activation leads to adaptive immune responses. for this reason, many adjuvants that are used today are directed to prrs. these adjuvants are called tlrs related adjuvants [ ] . mpl is a series of 'monophosphoryl lipid a obtained from the purification of a modified lipopolysaccharide (lps) of salmonella minnesota. bacterial deoxyribonucleic acid (dna) is immunostimulatory due to unmethylated cpg dinucleotides. vertebrate dna has relatively low amounts of unmethylated cpg compared to bacterial dna. the adjuvant effect of cpg is enhanced when conjugated to protein antigens. this adjuvant is being tested in vaccines directed at infectious agents, allergens and tumor cells [ ] [ ] [ ] . another type of adjuvant is tuftsin. tuftsin is an auto adjuvant, which is a natural self-immunostimulating tetrapeptide (thr-lys-pro-arg). this tetrapeptide is a fraction of the igg heavy chain molecule produced by enzymatic cleavage in the spleen [ ] . its functions include: binding to receptors on neutrophils and macrophages to stimulate their phagocytic activity, increasing tumor necrosis factor alpha (tnf␣) release from human kupffer cells enhancing secretion of il by activating macrophages, activation of macrophages expressing nitric oxide (no) synthase to produce no and enhancement of murine natural cell mediated cytotoxicity in vitro [ ] [ ] [ ] . in summary, it is an adjuvant with minor side effects with a promising effect in restoring innate immune mediated response. adjuvants may exert their immune enhancing effects according to five immune functional activities: . translocation of antigens to the lymph nodes where they can be recognized by t cells. . antigen protection enabling longer exposure. . enhanced local reaction at the injection site. . induction of the release of inflammatory cytokines. . interaction with prrs, specifically tlrs [ ] . a adjuvant effect the term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigenspecific immune response in vivo. the microbial components of adjuvants activate apcs to produce pro-inflammatory cytokines ("non-specific" signal ) and to up-regulate molecules essential for antigen presentation. these molecules include major histocompatibility complex (mhc) class ii (antigen-specific signal ) and b - / . these innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of t cells [ ] . in accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [ ] . it has been shown that auto-reactive t-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [ ] . infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [ ] . an example of this is the causality between viral infection and myocarditis. half the cases of myocarditis are preceded by an acute viral infection. infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [ ] . it has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. an example of this is that bladder administration of bcg (bacille calmette-guérin) has been shown to be effective against superficial bladder cancer development [ ] . it can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". prr-pamp (pattern recognition receptor-pathogen-associated molecular patterns) interactions activate the apcs to promote antigen-specific lymphocytic responses [ ] . the definition of pamps has now broadened, in that the recognized structures do not need to be pathogens. thus the concept of "microbe-associated molecular patterns" (mamps) and of "danger/damage-associated molecular patterns" (damps) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [ ] . tlrs are single-transmembrane prrs localized on cell surface and endosomal membranes. from all the prrs, these are the most studied. tlrs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. the interaction between the four known tlrs adapters: myd , tirap/mal, tram and trif, in tlr signaling, shape the innate immune response. besides prrs the innate immune system also detects proteolytic enzymes generated during infection [ ] . merging the response to different prrs signaling may be the pathway for developing customized responses to different aggressions [ ] . b experimental models of adjuvants many animals have been used in experimental models of adjuvant-related autoimmune conditions [ ] . these include primates, salmons, rabbits and swine; however, the most common are murine models. murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( table ). an interesting model is that described by lujan et al. the authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. an excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. this was suggested to be part of the spectrum of asia syndrome. moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [ ] . c toxicity of aluminum adjuvants aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (bbb) and of eliciting immune inflammatory responses. these are probably the reasons why aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [ , ] . aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. it has been described as a neurotoxin because even when a relatively small amount of aluminium reaches the brain [ ] , is can act as a genotoxin [ ] , a prooxidant [ ] , it can be proinflammatory [ ] , act as an immunotoxin [ ] and also as an endocrine disruptor [ ] . aluminum interferes with many essential cellular processes. memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. moreover, alzheimer's [ ] , amyotrophic lateral sclerosis, parkinsonism dementia [ ] , multiple sclerosis [ ] , and neurological impairments in children have been linked to aluminum neurotoxicity [ ] . brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [ ] . aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. one of the most significant is the activation of the nlrp inflammasome pathway [ ] . nlpr activation has been shown to trigger type diabetes. by using nlpr knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [ ] . on the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines il- ␤ and il- are strongly implicated in the development of several central nervous system (cns) disorders [ ] . the vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. upper limits set by us food and drug administrations (fda) for aluminum in vaccines are set at no more than g/dose. these values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [ ] . the quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the fda. however the scientific basis for this recommendation does not take into account aluminum persistence in the body. the concern about aluminum in dietary intake has been reinforced by the food and agriculture (fao) who expert committee, which lowered the provisional tolerable weekly intake of aluminum from mg/kg/bw ( mg/week, for an average kg human) to mg/kg/bw ( mg/week) [ ] . the amount of dietary intake of aluminum has risen in urban societies to up to mg/day considering the widespread use of processed convenience foods. however, only about . % of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [ ] . absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. moreover, the presence of aluminum in breast tissue was associated with breast cancer [ ] . aluminum compounds persist for up to - years post vaccination in human body. this fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [ ] . the clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: . it can persist in the body. . it can trigger pathological immunological responses. . it can pass through the bbb into the cns where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. there is a link between allergies and autoimmunity since both are the result of an abnormal immune response [ , ] . metals such as mercury, aluminum, nickel and gold are known to induce immunotoxic effects in humans. the immunologic effects of these metals include immunomodulation, allergies and autoimmunity. they may act either as immunosuppressants or as immune adjuvants. metals bind firmly to cells and proteins and thus have the ability to modify autologous epitopes (hapetenization). t-cells then recognize the proteins as foreign and trigger an autoimmune response [ ] . hypersensitivity caused by metals may be referred to as type iv delayed hypersensitivity. the reaction is considered delayed because the first symptoms appear - h after exposure, because it is mostly t-cell mediated and the gold standard for diagnosis of delayed type hypersensitivity is patch testing [ ] . in mercury-sensitized patients, even mercury concentrations within the normal range might provoke neuroallergic reactions in the brain [ ] . identifying metal sensitivity and removal of the sensitizing metals, such as dental amalgam, have been proved successful by showing symptom improvement in patients with previous autoimmune diseases. these diseases included fibromyalgia, autoimmune thyroid diseases and orofacial granulomatosis [ ] [ ] [ ] [ ] (table ). the timeline regarding the field of vaccinology has been divided in two generations, the first regarding the administration of inactivated pathogens in whole or live attenuated forms (e.g., bacillus calmette guerin (bcg), plague, pertussis, polio, rabies, and small- pox) and the second regarding vaccines assembled from purified microbial cell components, also referred as subunit vaccines (e.g., polysaccharides, or protein antigens) [ ] . this latter approach there are obstacles to conventional vaccine development methods such as non-cultivable in vitro pathogens (e.g., hepatitis c, papilloma virus types and , and mycobacterium leprae), antigen hypervariability (e.g., serogroup b meningococcus, gonococcus, malaria), opportunistic pathogens (e.g., staphylococcus aureus) and rapid evolving pathogens such as human immunodeficiency virus (hiv) [ ] . vaccine research gained a new perspective as the genomics field emerged over the last decades. bacterial genomes have been sequenced and analyzed making it possible to choose the best candidate vaccine antigens by using the concept of reverse vaccinology [ ] . the main known factors influencing the observed heterogeneity for immune responses induced by vaccines are gender, age, ethnicity, co-morbidity, immune system, and genetic background. the interaction between genetic and environmental components will dictate the response to vaccines. studying the vaccine and the host will enable the development of customized treatment options. the combination of genetics, epidemiology and genomics in vaccine design has been denominated "vaccinomics" [ ] . the importance of genetic influence is supported by twins and siblings studies, which show familial aggregation. this suggests that genomics is crucial in inter-individual variations in vaccine immune responses [ ] . both human leukocyte antigen (hla) and non-hla gene markers have been identified as markers for immune response to vaccines. multiple studies have shown connections between hla gene polymorphisms and non-responsiveness to the hbv vaccine [ ] . hla region is divided in three sub regions: class i is associated with the induction and maintenance of cell-mediated immune response, class ii is associated with presentation of exogenous antigens to helper t cd + cells and class iii, where immune non hla related genes are located. normal human tissue has at least hla antigens, and although new recombinant haplotypes may occur, it is inherited mostly intact from progenitors [ ] . hla allelic differences are associated with different responses to vaccines, either by hyper or hypo responsiveness. we can infer that a similar response may be associated with different safety in relation to the development of autoimmune reactions to vaccines, particularly in the patients with genetic predisposition to an enhanced response to vaccine inoculation [ ] . furthermore, patients that share the same hla, for instance siblings, have been diagnosed with asia following similar environmental stimuli [ , ] . autoantibodies help to diagnose certain autoimmune diseases, however, they can also be found in healthy individuals. thus, autoimmune diseases cannot be diagnosed based solely on antibody detection [ ] . inoculation of vaccines triggers autoimmune responses that result in the development of autoantibodies. many studies have been carried out in animals, healthy subjects and patients with autoimmune diseases to understand if this development is of clinical significance [ ] [ ] [ ] [ ] . a difference in eliciting the production of autoantibodies in healthy humans has been observed between adjuvanted and non-adjuvanted influenza vaccines [ ] . the annual influenza vaccine has been the most heavily researched vaccine, along with hpv and pneumococcal vaccines as far as their relationship with patients who have previously been diagnosed with an autoimmune disease [ ] [ ] [ ] . autoantibody induction after hpv vaccination was also shown in adolescent girls with systemic lupus erythematosus (sle) [ ] . although induction of autoantibodies was proven following vaccine administration, there have been no proven relation with disease diagnosis in either of the specific groups studied so far [ , ] . it has been widely demonstrated that autoantibodies can develop years before the manifestation of a full-blown autoimmune disease [ ] . moreover, the development of a specific autoantibody is also genetically determined, and the link between genetic, autoantibodies and vaccines may become an even more intriguing area of research [ ] . silicones are synthetic polymers that can be used as fluids, emulsions, resins and elastomers making them useful in diverse fields. they were thought to be biologically inert substances and were incorporated in a multitude of medical devices such as joint implants, artificial heart valves, catheters, drains and shunts. of all the silicone-containing products, the most famous are most likely breast implants. silicon is one of the substances suspected to induce asia [ ] . it is currently believed that exposure alone is not enough to trigger the disease but that it requires the presence of additional risk factors (e.g., genetic susceptibility, other environmental factors) [ ] . silicone exerts local tissue reactions. some of these reactions are considered para-physiological, such as capsular tissue formation around an implant. other reactions are viewed as abnormal, like when capsular contractures and allergic reactions to silicone or platinum (catalyst used in silicone polymerization found in minute concentrations in implants) occur [ ] . cutaneous exposure to silicone with cosmetics or baby bottles could potentially sensitize patients [ ] . there is also a systemic component of silicone exposure related to diffusion of silicone through the elastomer envelope, commonly termed "bleeding". it may arouse systemic effects as it degrades and fragments in tissue, it can also spread throughout the body and lead to the development of cancer or autoimmune phenomena [ ] . patients with ruptured implants complain more frequently of pain and chronic fatigue when compared to patients with intact implants [ ] . anti-silicone antibodies were found to be present in human sera more frequently in patients who have undergone silicone breast implants, however, their pathological significance remains uncertain [ ] . the same was seen for other antibodies such as autoantibodies directed against dsdna, ssdna, ssb/la, silicone and collagen ii, which were found to be present in increased levels in patients after exposure to silicone [ ] . it has also been shown that the formation of autoantibodies is directly related to implant duration. several autoimmune diseases have been linked to silicone exposure including rheumatoid arthritis (ra), systemic lupus erythematosus (sle), polymyositis, systemic sclerosis (ssc) and fibromyalgia. although asia symptoms may arise years after the onset of exposure to silicone implants [ ] , most of the follow-up periods are short and concluding evidence is yet to come regarding this causality. there have been published case reports, epidemiologic and research studies that suggest a connection between several vaccines and certain autoimmune conditions, notwithstanding that, overall the benefits of vaccination outweigh the risks. thrombocytopenia has been reported as the main adverse event following mmr vaccine. after mmr vaccine the onset of immune thrombocytopenic purpura (itp) usually occurred within weeks at a risk rate of : , - , mmr vaccine doses, while the incidence of itp following infections is : for measles and : for rubella [ ] . as the risk of thrombocytopenia is higher in patients who experience natural infection with measles, mumps or rubella than in those receiving the vaccine, vaccination is encouraged. arthralgia complaints have also been reported and they may present as transient arthralgia, acute arthritis and rarely chronic arthritis [ ] . some risk factors have been found to be associated with the development of arthritis in vaccinated patients such as: female gender, older age, prior seronegativity and specific hla alleles [ ] . yf vaccine is only advisable to people in, or going to endemic areas. the risk of developing yf vaccine-associated neurologic disease (yel-and) is inversely proportional to age [ ] . this is why children aged < months cannot be vaccinated and < months, except during epidemics [ ] . vaccination is not advisable to people > years because of possible higher risk of severe adverse effects (saes) even though the incidence remains low [ ] . besides being a vaccine for mycobacterium tuberculosis (tb), the bcg has proved effective as immunotherapy for bladder cancer. although the mechanism is yet to be fully understood, it is thought that bcg binds to fibronectin forming complexes that enable the recognition as "non-self" by the innate immune response of th cells. ultimately the pathways result in the apoptosis of tumor cells [ ] . because of its effect in treating non-muscle-invasive urothelial carcinoma, as well as superficial bladder tumors, it was expected that bcg could play a role in treating other types of cancer, despite data having not corroborated this hypothesis so far. adverse events vary according to the site and method of administration. intradermal administration of bcg has been reported to elicit arthritis [ ] , dermatomyositis [ ] and takayasu's arteritis (ta) [ ] among others. intravesical treatment for bladder cancer can cause reactive arthritis (rea) [ ] . the risk relies on a systemic reaction composed of an early infective phase (pcr positive and response to anti-tb treatment) and a late hypersensitivity reaction [ ] . hbv is a dna virus of the hepadnaviridae family, responsible for acute and chronic liver disease. hbv vaccines are considered the first efficient vaccines against a major human cancer. hbv vaccines have reduced the risk of developing chronic infection and they also have proved to reduce the incidence of liver cancer in children [ ] . the vaccine has been associated mainly with autoimmune neuromuscular disorders. they include, but are not limited to: optic neuritis, guillain-barre syndrome (gbs), myelitis and multiple sclerosis (ms), systemic lupus erythematosus (sle), arthritis, vasculitis, antiphospholipid syndrome (aps) and myopathy [ ] . hbv vaccine is the most common immunization associated with acute myelitis. there are studies that indicate that the pathogenicity behind such vaccine and autoimmunity might be based on cross-reactivity between hbv antigen (hbsag) epitopes, yeast antigens, as well as other adjuvants contained in the vaccine itself [ ] . up to % of cervical cancer deaths, occur in developing countries that lack the ability to fully implement the papanicolau (pap) screening programs. hpv poses a special challenge in vaccine safety. hpv is necessary for the development of cervical cancer. however, most women infected with hpv will not develop the disease since % of infections will resolve within a year and up to % within years without specific treatment. over the course of decades, cancer may result in a small proportion of the remaining infected women. death rate from cervical cancer in - year old girls is zero and long-term benefits are yet to be proven. in this specific case, short term risks to healthy subjects can prove to pose a heavier burden than cervical cancer [ ] . there are at least types of hpv strains, of which have been pathologically associated with cancer. two vaccines, gardasil tm and cervarix tm , are commercially available against hpv. both contain the l capsid proteins of several hpv strains as antigens. gardasil tm contains serotypes , , , . these antigens are combined with aluminum (al) hydroxyphosphate sulphate as an adjuvant. cervarix tm contains a combination of the oil-based adjuvant monophosphoryl lipid a (mpl) and al hydroxide (aso ) as adjuvant and is directed at strains and [ ] . there have been several reports of post-licensure adverse events, some of which have even been fatal [ ] . compared to other vaccines, an unusually high proportion of adverse drug reactions has been reported associated with hpv vaccines [ ] . in , australia reported an annual adr rate of . / , , the highest since . this increase was almost entirely due to adrs reported following the commencement of the national hpv vaccination program for females aged - years in april ( out of a total of adrs records). the numbers only decreased after the cessation of the catch-up schedule. although the percentage of convulsions attributable to the hpv vaccine decreased, the overall report remained comparable between and ( % and % respectively). these reports do not prove the association, but show that there is a higher frequency of adrs related to hpv vaccines reported worldwide, and that they fit a consistent pattern (i.e., nervous system-related disorders rank the highest in frequency) that deserves further investigation [ ] [ ] [ ] . indeed, several autoimmune diseases have been linked to hpv immunization. examples include gbs, ms, acute disseminated encephalomyelitis (adem), transverse myelitis (tm), postural orthostatic tachycardia syndrome (pots), sle, primary ovarian failure (pof), pancreatitis, vasculitis, immune thrombocytopenic purpura (itp) and autoimmune hepatitis (ah) [ ] . influenza is an acute viral infection that affects the respiratory tract and is caused by influenza type a-c viruses of the orthomyxoviridae family [ ] . h n mortality rates in the outbreak showed high risk in those aged years and older, presence of chronic diseases and delayed admission. risk of infection was lower in those who had been vaccinated for seasonal influenza with / trivalent inactivated vaccine [ ] . studies have demonstrated that influenza vaccine is safe and immunogenic in patients with sle or rheumatoid arthritis (ra), diminishing the risk of respiratory infections [ ] . it has been shown that adjuvanted vaccine had more local reactions but did not increase systemic adverse reactions [ ] . molecular mimicry has been suggested as a mechanism to explain an autoimmune response following influenza vaccination. however, a causal relationship between influenza vaccines and induction of autoimmune diseases remains unproved [ ] . diseases or symptoms reported after influenza vaccination include mostly neurological syndromes such as gbs [ref] . nonetheless, influenza vaccines should be recommended for patients with ms, because influenza infection is associated with increased risk of exacerbations. that being said, influenza vaccinations showed increased risk of autoimmune responses suggestive of asia [ ] , vasculitis [ ] and aps [ ] among others. meningococcal disease is caused by neisseria meningitidis. one of the following five serogroups causes almost every invasive disease: a-c, y, and w- . vaccines available so far for its prevention encompass either pure polysaccharide vaccines that use purified bacterial capsular polysaccharides as antigens, or protein/polysaccharide conjugate vaccines, which use the polysaccharide molecule plus diphtheria or tetanus toxoid as tcell-stimulating antigens. n. meningitidis serogroup b (menb) menb glycoconjugate vaccines are not immunogenic and hence, vaccine design has focused on sub-capsular antigens [ ] . menb capsular polysaccharide is composed of a linear homopolymer of ␣( → ) n-acetyl-neuroaminic acid (polysialic acid; psa). menb psa and psa found on neural cell adhesion molecules are structurally identical. as a result of this, it has been proposed that infection with menb or vaccination with psa may be associated with subsequent autoimmune or neurological disease [ ] . no evidence of increased autoimmunity was found to be associated with meningococcal serogroup b infection [ ] . regarding vaccination, the inoculation does not cause autoimmune diseases but may unmask autoimmune phenomena in genetically predisposed individuals. local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines. the intramuscular administration of the conjugate vaccine (versus subcutaneous for that of polysaccharide) may, in part, explain the higher reactivity [ ] . diseases previously associated with meningococcal vaccines are gbs [ ] , henoch-schönlein purpura (hsp) [ ] and bullous pemphigoid (bp) [ ] . streptococcus pneumoniae (pneumococcus) is the main cause of bacterial community-acquired pneumonia and meningitis in western countries, as well as the cause of more than , children deaths in developing countries [ , ] . there are three anti-pneumococcal vaccines commercially available. two of these are conjugated to a protein carrier (pcv and pcv ) and one is not conjugated (ppv ). ppv was licensed in and consists of the capsular polysaccharides of twentythree different streptococcus pneumoniae serotypes ( - , b, f, , n, v, a, a, f, , b, f, c, f, a, , f, f, and f). it does not elicit immunological memory because the immune response it triggers is t-cell independent. it is usually administered to the elderly (above years), as it is believed to be less effective in children. pcv is composed of the most frequent serotypes , b, v, , c, f, and f. pcv is directed at serotypes , - , a, b, f, v, , c, a, f, and f. contrary to ppv both pcv and pcv have an aluminum adjuvant in their composition that elicits a t-cell mediated response [ ] . ever since vaccines were introduced in the healthcare system, prevalence, fatality and admissions for invasive pneumococcal disease have decreased significantly [ ] . vaccine adverse events vary depending on whether the vaccine is adjuvanted or not. in a non adjuvanted vaccine, local reactions are present in % of people vaccinated intra muscularly and in % of those immunized sub-cutaneously [ ] . in conjugated vaccines, this percentage rises to % [ ] . systemic reactions such as fever, irritability, decreased appetite and sleep disturbances occur in - % of recipients of pcv or ppv. symptoms like arthralgia, arthritis, myalgia, paresthesia and fatigue are more frequent in patients post ppv. this may be related to the fact that the vaccines are administered to different age groups. autoimmune risk following ppv vaccine is very low. only case reports were found after ppv vaccine. six of these referred to reactivation of a previous autoimmune disorder. studies directed to access vaccine safety in subjects with autoimmune diseases showed immunization was safe [ , ] . tetanus toxoid (tt) is a potent exotoxin produced by the bacteria clostridium tetani. the toxin has a predominant effect on inhibitory neurons, inhibiting release of ␥-aminobutiric acid (gaba). when spinal inhibitory interneurons are affected the symptoms appear [ ] . the vaccine against c. tetani contains deactivated tetanus toxoid plus an adjuvant (usually aluminium hydroxide). the most studied and prevalent disease associated with tt is antiphospholipid syndrome (aps), but cns complications have also been reported such as optic neuritis, acute myelitis and encephalomyelitis [ ] . in mice, the immune response to tt depends on genetic background and to the specific adjuvant used for immunization. naive balb/c mice, immunized with tt, developed antibodies directed to tt, dsdna and ␤ gpi and were extremely sick [ ] . aps is an autoimmune disease characterized by the occurrence of thrombotic events. patients suffering from this condition have recurrent fetal loss, thromboembolic phenomena, thrombocytopenia as well as neurological, cardiac and dermatological involvement [ ] . the serological marker of aps is the presence of antiphospholipid antibodies (apl), which bind negatively charged phospholipids, platelets and endothelial cells mainly through the plasma protein beta- -glycoprotein-i (b gpi). the presence of igg and igm anti-cardiolipin antibodies (acl) and lupus anticoagulant is associated with thrombosis in patients with aps [ ] . ␤ gpi was identified as the most important antigen in aps. ␤ gpi has several properties in vitro which define it as an anticoagulant (e.g., inhibition of prothrombinase activity, adenosine diphosphate-induced platelet aggregation, platelet factor ix production) [ ] . passive transfer of anti-␤ gpi antibodies induce experimental aps in naïve mice and thrombus formation in ex vivo model [ ] . evidence suggests that the molecular mimicry mechanism between ␤ gpi and tt is one of the possible causes for aps. besides tt, aps has also been reported following hbv and influenza virus vaccination, although data are scarce [ , ] . sle is a multisystem autoimmune disease characterized by the production of a variety of autoantibodies. igg isotype antibodies to double-stranded dna (dsdna) are thought to be diagnostic markers and their presence correlates with disease pathogenesis. several factors including genetic, hormonal, environmental and immune defects are involved in the induction of autoantibodies in this disease [ ] . post vaccination manifestations of sle or lupus like syndrome have been reported and range from autoantibody induction to full blown clinical disease. reports have been published associating sle to hbv, mmr, dtp, hpv, influenza, bcg, pneumococcal and small pox vaccinations [ ] . vaccination in sle diagnosed patients is associated with disease exacerbation and decreased antibody response, which may be due to the underlying disease and the frequent use of immunosuppressive drugs [ ] . a temporal link between sle and hbv vaccination is the only relation that has been demonstrated [ ] . several studies have demonstrated an increased prevalence of hpv in individuals with lupus compared to the general population, which has increased awareness for the need to vaccinate this high-risk population [ ] . to do so, the association between immunization with hpv vaccines and sle like symptoms, as well as the higher incidence of flares in known lupus patients must be taken into account. vasculitis is the name given to a group of autoimmune mediated diseases, which involve blood vessels of different types and sizes. they can be categorized according to several disease features indluding: the type of vessel affected, organ distribution, genetic predisposition and clinical manifestation [ ] . so far, cases of large vessel vasculitis have been detected. this includes cases of giant cell arteritis (gca) following influenza vaccination, cases of takayasu disease (td), and one case of large cell arteritis involving subclavian and renal arteries following hbv vaccines. two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (pmr)-like illness [ ] . one case of polyarteritis nodosa (pan) following the administration of tetanus and bcg vaccine is described. all other cases of pan in adults follow the administration of hbv vaccine [ ] [ ] [ ] . case reports of medium vessels vasculitis -both polyarteritis nodosa and kawasaki disease (kd) -have also been published in pediatric patients. kd has been described one day after the second dose of hbv vaccine and following yellow fever vaccine [ , ] . two cases of pediatric patients with pan have been reported two months after receiving the hbv vaccine [ , ] . eosinophilic granulomatosis with polyangiitis (egpa) after tetanus vaccination [ ] and following hbv vaccine [ ] have been reported. there are also cases of microscopic polyangiitis (mpa) and cases of granulomatosis with polyangiitis (gpa) following influenza vaccines in the literature [ , ] . henoch schönlein purpura (hsp) is the most common vasculitis of childhood. it is generally benign and self-limited. it is mediated by iga immune complex deposition in various tissues as well as in small-sized blood vessels. genetic risk factors play an important role in the pathogenesis of the disease: it is associated with hla-drb* , and . hsp was associated with seasonal influenza, influenza a (h n ), pneumococcal and meningococcal disease, hepatitis a virus (hav), hbv, anti-human papilloma virus (hpv) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [ , [ ] [ ] [ ] . leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [ ] , hav vaccine [ ] , hbv vaccine [ ] , pneumococcal vaccine [ ] , varicella [ ] , rubella, smallpox [ ] and the anthrax vaccine [ ] . dermal vasculitis with pan uveitis has also been described following mmr vaccine [ ] . ra is the most prevalent chronic inflammatory arthritis affecting the synovial membrane of multiple diarthrodial joints. although its etiology has not been completely clarified, deregulation of the immune system is evident with a preponderance of inflammatory cytokines and immune cells within the joints. ra has an estimated heritability of %, leaving a substantial proportion of risk to environmental factors. immunizations have previously been proposed as potential environmental triggers for ra. in the norfolk arthritis register database, of the first patients reported receiving a tetanus vaccination within weeks prior to the onset of arthritis. similarly, a transient rise in rf titer was recorded in out of military recruits - weeks after receiving concomitant immunization against tetanus, typhoid, paratyphoid, mumps, diphtheria, polio and smallpox. however, only showed a persistent elevation in titer and none developed arthritis [ ] . several mechanisms have been proposed to explain the putative association between vaccination and the initiation of ra, the most prominent of which are molecular mimicry and non-specific immune system activation [ ] . vaccines who have been associated with ra include rubella vaccine in which reactive arthritis occurs in % of recipients. controlled studies failed to show persistent arthritis or arthralgia in these patients [ ] . patients following hbv vaccine showed an increase of arthritis in a vaers study, but this was not seen in a large retrospective epidemiological study [ ] . data so far suggest that vaccines carry an insignificant role in the pathogenesis of ra. several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as tnf, aiming to induce high titers of endogenous neutralizing anticytokine antibodies with the goal of breaking natural th tolerance to auto antigens. other cytokines, namely il- il- , mif, rantes, il- , mcp- are also being tested [ ] . another vaccine related therapy uses autologous t cell lines to induce a specific immune response by the host's t cells directed against the autoimmune (vaccine) t cells [ ] . this strategy has been successful in mouse models and has shown encouraging results in a small pilot study of ra patients, where patients showed a clinical response, defined by acr improvement criteria [ ] . uctd is a clinical condition characterized by signs, symptoms and laboratory tests suggestive of a systemic autoimmune disease but that does not fulfill the criteria for any defined connective tissue disease (ctd). such patients with clinical manifestations suggestive of systemic connective tissue disease but not fulfilling any existing criteria are quite frequent: - % of the patients initially asking for a rheumatologic evaluation may at least temporarily be diagnosed as affected by 'undefined' or 'undifferentiated' connective tissue disease. comparing studies on these diseases is unfeasible because of the inexistence of defined criteria for diagnosis [ ] . within years of follow-up, patients usually evolve to defined ctds, which include sle, systemic sclerosis (ssc), primary sjögren's syndrome (pss), mixed connective tissue disease (mctd), systemic vasculitis, poly-dermatomyositis (pm/dm) and ra. maintaining an undefined profile for years makes evolving into ctds less probable and the diagnosis of "stable uctd" reliable [ ] . disease etiology is a concern and it has been associated with vitamin d deficiency and silicone implants, both of which lead to an imbalance in proinflammatory and anti-inflammatory cytokines [ ] . vaccines have also been associated with this disease, namely the hbv vaccine [ ] . etiopathogenesis of uctd is unknown and it has been suggested it might fall on asia spectrum since symptomatic similarities are striking and uctd etiopathogenesis has been associated with adjuvants [ ] . aa is an autoimmune disease, characterized by one or more well demarcated oval and round non-cicatricial patches of hair loss. the disease may affect any hair bearing part of the body and has a great impact on a patient's self-esteem and quality of life. depending on ethnicity and location, aa is the most prevalent skin disease. aa prevalence varies and is estimated to be between . - . % in the united states and . % in singapore [ , ] . as with any other autoimmune disease, the development of aa encompasses genetic and environmental factors. environmental factors associated with aa development are emotional and/or physical stress, infections and vaccines [ ] . secondary syphilis is one of the most well studied examples, however epstein barr virus [ ] and herpes zoster [ ] infections have also been related to the development of the disease. as far as vaccines go, hbv vaccine has been associated with aa development. in one study of patients, developed aa after vaccination with hbv vaccine. of those patients, were rechallenged, and the reappearance of disease was witnessed [ ] . in mice this association failed to be established [ ] . one case of aa was witnessed following tetanus toxoid, as well as two case reports following hpv and mmr vaccine [ ] [ ] [ ] . itp is an autoimmune disease defined by a platelet count of less than platelets/l without overlapping diseases. it can present with or without anti-platelet-antibodies. thrombocytopenia is relatively common and the overall probability of developing itp was , % in a cohort of patients. it was also found that % of patients developed an overlapping aid other than itp [ ] . the etiology of the disease is yet to be fully understood but it has been detected following infectious diseases, such as helicobacter pylori, hepatitis c virus (hcv), novel influenza a infection, rotavirus infection and human immunodeficiency virus (hiv) [ ] . itp onset has also been reported, although rarely, as a severe adverse event following vaccine administration. this was more often observed after measles-mumps-rubella (mmr), hepatitis a and b, diphtheria-tetanus-acellular pertussis (dtap), and varicella vaccinations [ ] . molecular mimicry has been suggested as a possible mechanism for the development of itp, namely following helicobacter pylori infection. its eradication has been shown to increase platelet count and diminish the levels of anti-caga antibody in a subset of h. pylori infected subjects with itp [ ] . these data point towards a beneficial role of h. pylori eradication in chronic itp. two cases of itp following anti-rabies vaccine have been reported and one after hpv vaccine. reactivation of itp was reported two weeks after a tick-borne encephalitis vaccination [ ] . the most consistent association with itp is with the mmr vaccine [ ] . however, it should be emphasized that the number of cases are fewer than expected without vaccination. t d is due to antigen specific reactions against insulin producing beta cells of the pancreas. much like other autoimmune diseases, t d results from a combination of genetic, environmental, hormonal and immunological factors. environmental factors such as pathogens, diet, toxins, stress and vaccines are believed to be involved in the beginning of the autoimmune process [ ] . although the mechanisms by which viral infections cause autoimmune diabetes have not been fully clarified, there is some evidence to suggest a role for natural infections in the pathogenesis of t d mellitus in susceptible individuals [ ] . it has been hypothesized that vaccination could trigger t d in susceptible individuals. although post-vaccination t d may be biologically plausible, cumulative evidence has not supported an increased risk of t d following any vaccine [ ] . several experimental data have suggested that, depending on the timing, vaccination might exert a protecting or aggravating effect on the occurrence of diabetes [ ] . a study suggests that haemophillus influenza type b vaccine might be a risk factor in the induction of islet cell and anti-gad antibodies measured at one year of age [ ] but there are previous studies that show no association between hib and t d [ ] . in a cohort of american military officers diagnosed with t d, there was no association found between vaccination and t d diagnosis [ ] . available data about a relation between the mumps vaccine and t d are still incomplete and their interpretation is difficult because of miscellaneous confounding factors associated with the development of t d [ ] . association between hemagglutinin neuraminidase (h n ) vaccines and t d is so far unproven [ ] . in humans, it has been hypothesized that early-age bcg vaccination is associated with the risk of t d. the few studies conducted to date provided no consistent evidence of an association. there are, however, studies showing a possible temporary boost of the immune function after vaccination [ ] . studies also show that among bcg-vaccinated children who test positive for islet autoantibodies, there is a higher cumulative risk of t d [ ] . in animal experiments it has been observed that bcg seems to have a protective effect against diabetes, however researchers have yet to translate this benefit to humans [ ] . in all, studies results do not support any strong association between vaccination and t d. narcolepsy is a sleep disorder described as excessive sleepiness with abnormal sleep pattern characterized by uncontrollable rapid eye movement (rem) events which occur at any time during the day. these event and may or may not be accompanied by a loss of muscle tone (cataplexy) [ ] . a plethora of data indicates that narcolepsy is caused by the lack of orexin (also known as hypocretin), an important neurotransmitter, which is involved in the regulation of the sleep cycle. in narcolepsy patients, a loss of orexin producing neurons in the hypothalamus and low levels of orexin in the cerebrospinal fluid (csf) has been reported [ ] . narcolepsy has been shown to have an autoimmune background. antibodies against tribbles (trib ) have been found in these patients, which may be related to the pathogenesis of disease. an experimental model of narcolepsy in mice has been made by passive transfer of total igg from narcolepsy patients into the animal's brains through intra ventricular injection [ ] . environmental factors like influenza a virus and streptococcal infections have been associated with disease onset. interestingly, fever by itself without the diagnosis of an infectious etiology was found to be a risk factor for narcolepsy [ ] . several groups have studied and found an increase in the incidence of narcolepsy diagnosis following the introduction of influenza vaccination, specifically, aso -adjuvanted pandemrix tm vaccine. this association was shown in finland especially in [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] year-olds, but also in case reports from other countries [ ] . other studies failed to find an association. the actual infection with h n has been associated with disease development in china, however no such relationship has been noted in europe [ ] . the above-mentioned associations are specifically related to the aso -adjuvanted pandemrix tm vaccine. the same association has not been reported for other h n adjuvanted or non-adjuvanted vaccines. the major difference between the aso and the mf adjuvants is the presence of the ␣-tocopherol. ␣-tocopherol is unique in that it can achieve the highest and longest antibody response by producing an enhanced antigenspecific adaptive immune response. in vitro it was shown that ␣-tocopherol could increase the production of orexin as well as increase the proteosome activity. this increased production of orexin fragments may facilitate antigen presentation to mhc class ii, thus triggering an autoimmune process [ ] . all these data together support the relationship between the h n pandemrix tm vaccine and the development of narcolepsy. gluten induced enteropathy, gluten sensitive enteropathy, or more commonly called celiac disease (cd) is a life-long autoimmune condition mainly of the gastrointestinal tract, specifically affecting the small intestine. the abnormal immune response crates autoantigens which are directed towards tissue transglutaminase (ttg). the two main autoantibodies and the most widespread serological markers to screen for the disease are anti ttg and anti endomysium. two additional auto-antibodies, namely: anti deaminated gliadin peptide and anti-neoepitope ttg were found recently to be reliable for cd screening as well [ ] . cd is an autoimmune disease induced by well-known nutritional environmental factors. the non-dietary ones are less studied and established. several infectious disease have been linked to its development, the so-called infectome [ ] . a clear cause-effect relation is yet to be established for most of the pathogens associated with cd. what has been shown, however, is that in countries with low economic status, inferior hygiene conditions and higher infectious load, cd prevalence is lower [ ] . an epidemiologic relationship was established in between rotavirus infection and cd. data showed that in genetically predisposed individuals, rotavirus infection was related to childhood cd development [ ] . in subsequent research studies, a celiac peptide was recognized and proved to share homology with rotavirus major neutralizing protein vp and with the cd autoantigen ttg. the antibodies directed against the viral protein vp were shown to predict the onset of cd and induce typical features of cd in the intestinal epithelial cell-line t [ ] . it has also been suggested that rotavirus vaccine alters b and t behavior, as the percentage of b-cells was higher in the vaccinated infants [ ] . rotavirus vaccine as an inducer of cd is still in discussion and warrants further study. pmr is an autoimmune inflammatory rheumatic disease characterized by raised inflammatory markers with pain and morning stiffness of shoulders and pelvic girdles and synovitis of proximal joints and extra-articular synovial structures. its diagnosis is clinical and it is typically a disease of the elderly occurring mainly in subjects above . etiopathogenesis of pmr remains unknown, but genetic and environmental factors play a role [ ] . a close temporal relationship has been ascertained concerning epidemics of mycoplasma pneumoniae, chlamydia pneumonia, parvovirus b and peaks of cases of pmr and giant cell arteritis, however this is not clearly proven [ ] . cases of pmr following vaccination have rarely been reported. however, it is believed that post vaccination pmr may be underreported due to its symptomatic similarities with the transient effects of vaccines, namely: arthralgia, myalgia and low-grade fever. this leads to failure in establishing a chronological relationship when the disease is diagnosed. most of the reported cases are associated with seasonal influenza vaccine (inf-v). often, the time interval between vaccine administration and symptoms onset varies from one day, to three months. three cases were reported with associated giant cell arthritis. a case report of relapsing pmr after four years of remission following tetanus vaccination has also been reported [ , ] . acute disseminated encephalomyelitis (adem) is an inflammatory demyelinating disease of the central nervous system (cns). adem is usually poly-symptomatic with encephalopathy (i.e., behavioral change or altered level of consciousness). it affects mostly children and young adults and has higher prevalence in males. its incidence is . - . per per year [ ] . although there is no concrete evidence of a clear pathogenic association, adem has been associated with immunization or previous viral infection. post-vaccination adem accounts for only - percent of all cases, while post-infectious adem accounts for percent of all cases of adem [ ] . the hypothesis that better describes these associations is molecular mimicry. t-cells targeting human herpesvirus- (hhv- ), coronavirus, influenza virus and epstein-barr virus (ebv) have been shown to cross-react with myelin basic protein (mbp) antigens. anti-mbp t-cells were detected in patients following vaccination with simple rabies vaccine [ ] [ ] [ ] . in a post experimental therapy for alzheimer's disease with a vaccine that contained aggregates of synthetic a␤ fragments of amyloid precursor protein, adem was shown to develop in mice [ ] . the experimental model of ms, eae mice, may be induced with injection of a␤ , but only when the latter is administered together with the complete freund's adjuvant [ ] . this observation points to the importance and central role of the adjuvants in induction of adem and autoimmunity in general [ ] . the overall incidence of post vaccination adem is estimated to be . - . per and a higher risk has been reported following immunization against measles. other vaccines accountable for post-vaccination adem include vaccines against the varicella zoster, the rubella, the smallpox and the influenza viruses [ ] . surprisingly, certain vaccines such as anti-tetanus vaccine were shown to have a negative correlation with adem (statistically significant decreased risk) [ ] . hbv immunization has been studied as a possible cause for adem but was later associated with clinically isolated syndrome (cis) (a first time occurring demyelinating episode that may, or not develop to ms) and complete conversion to ms [ ] . as far as case reports are concerned, adem was associated with vaccination with influenza, hepatitis a and b, mmr, hpv and tetanus [ , , ] . bullous dermatoses are characterized by the presence of blisters and autoantibodies against structural components of the skin: desmosomal proteins (in pemphigus), adhesion molecules of the dermal-epidermal junction (in pemphigoid diseases), and epidermal/ tissue transglutaminase (in dermatitis herpetiformis). the most frequent autoimmune bullous diseases are bullous pemphigoid (bp) and pemphigus vulgaris (pv). bp is more frequently observed in the elderly, while the age of onset of pv is between and years. neither of the diseases have any gender preference [ ] . bp and pv etiology is, so far, poorly understood. both diseases have been associated with various environmental factors, which include emotional and/or physical stress, infections and vaccinations [ ] . genetic predisposition has also been studied with overexpression of certain hla class ii alleles. these include hla-dqb * , drb * , drb * , and dqb * . these alleles have been found to be more prevalent in bp patients than in the general population [ ] . pv is associated with certain hla class ii loci such as hla-dr and hladr alleles (drb * and drb * , which is prevalent in ashkenazi jews, iranian and sardinian patients). other loci include drb * (common among japanese and italian patients) and two dqb alleles (dqb * and dqb * ), which are strongly associated with pv. bp and pv patients' sera were found to have significantly higher prevalence of antibodies to hepatitis b virus, hepatitis c virus, helicobacter pylori, toxoplasma gondii and cytomegalovirus [ ] . as far as vaccination is concerned, bp developed in patients following influenza, diphtheria, tetanus, pertussis, hepatitis b, bcg, polio and herpes zoster vaccines [ , , ] furthermore, reactivation of bp following influenza vaccination was reported in one case report [ ] . new onset pv was associated with: influenza vaccine, hepatitis b vaccine, anthrax vaccine, typhoid booster and rabies vaccination. in addition, exacerbation of pv after vaccination was also reported following influenza vaccine and tetanus vaccine [ ] . iim compose a group of skeletal muscles diseases in which myositis without a recognized cause occurs. iim is usually subdivided in entities: dermatomyositis (dm), polymyositis (pm), inclusion body myositis (sibm) non-specific myositis (nsm) and immune mediated necrotizing myopathy (iam) [ ] . iim prevalence is around . × − cases, with a bimodal age of distribution that peaks in childhood and again between and years. dm is the most common inflammatory myopathy while pm is the least frequent. despite exhibiting similar clinical symptoms, the subsets of iim exhibit significant immunopathological variation. dm begins with the activation of the complement and formation of membrane attack complexes (mac). in pm and sibm the fundamental process is related to cd + t cells mediated cytotoxicity [ ] . it is unclear what breaks the tolerance and drives the immune response to induce iim. so far, dm, pm and sibm have been linked to vaccination. several cases have been reported in the literature associating different vaccines with the development of idiopathic inflammatory myopathies. cases of iim had been reported to vaers database up to june . out of these cases, were classified as pm, as dm and an only one as a sibm. dm has been reported after almost any vaccine, however only a few studies have attempted to clarify the possible relationship between dm and vaccination. pm is a frequent misdiagnosed disorder. some reports have associated previous immunization, especially hepatitis b vaccine with pm [ ] . despite being recently differentiated from other iim, sibm has already been related to hbv vaccine [ ] . some vaccines associated with myositis are mmr vaccine, smallpox vac-cine, poliomyelitis (ipv), diphtheria and tetanus toxoid, influenza, hpv and bcg [ ] . fms is an entity that is related to the inability of the cns to modulate pain. the conditioned pain modulation process in the cns appears to be compromised among many fms patients, which might explain the enhanced pain sensation experienced by these patients [ ] . the etiology of fms is yet to be unveiled. genetic predisposition, physical trauma (particularly to the cervical spine), emotional stress (to various stressors) as well as a variety of infections have been linked with fms. vaccines have been associated with the triggering of fms namely rubella and lyme disease vaccines [ ] . there are several reports of fibromyalgia-like disease after vaccination, specifically hpv (martinez-lavin journal of clinical rheumatology ). the medical community and regulatory agencies should be aware of these possible adverse effects aiming at defining their magnitude. chronic fatigue syndrome (cfs) is a disease characterized by disabling fatigue, headaches, concentration difficulties and memory deficits ( %). other symptoms such as sore throat ( %), tender lymph nodes ( %), skeletal muscle pain and feverishness ( %), sleep disruption ( %), psychiatric problems ( %) and rapid pulse ( %) are often observed. it more frequently affects women and has a prevalence of . - . % [ ] . although disease etiology is still unknown, there are several pathogens, such as epstein-barr virus (ebv), which have been associated with cfs. patients often have higher titers of igm to the ebv viral capsid antigen. cytomegalovirus and human herpes virus antibodies were also detected more often in cfs patients, although other reports failed to replicate these results. parvovirus b infection has also been suggested as a trigger to cfs [ ] [ ] [ ] . vaccine inoculation has also been appointed as a probable cause. vaccinations against rubella, q fever and hepatitis b were found to be associated with higher risk of developing cfs while meningococcal vaccine, poliovirus and influenza vaccine were not. surprisingly, staphylococcus toxoid vaccine appeared to have a protective effect [ , , ] . defined in by shoenfeld and agmon-levin asia syndrome is characterized by hyperactive immune response to adjuvants [ ] . as previously stated, asia incorporates four known medical conditions: siliconosis, gws, mmf, and post-vaccination phenomena [ ] . recently, the sick building syndrome (sbs) was proposed as a candidate for the asia spectrum [ ] . all of these diseases satisfy several criteria for fms and seid [ ] . a macrophagic myofasciitis (mmf) mmf has been described as an emerging condition of unknown cause characterized by a pathognomonic lesion in muscle biopsy mixing large macrophages with submicron to micron-sized agglomerates of nanocrystals in their cytoplasm and lymphocytic infiltrates. these lesions were related to aluminum deposits in muscle following immunization with aluminum containing vaccines [ ] . mmf lesion is now universally recognized as indicative of a long-lasting persistence of aluminum adjuvant at the site of prior intramuscular immunization. the long-lasting mmf lesion should be considered as a biomarker of aluminum bio persistence in a given individual. patients with mmf have higher reported myalgia with incidence being up to %. its etiology is not clear but genuine muscle weakness is rare and the diagnosis of fibromyalgia is also rare. higher prevalence of chronic fatigue syndrome (cfs) in patients with mmf has been reported as well. cognitive impairment has been associated with mmf: in one series of mmf patients, up to % had attention and memory complaints and neuropsychological tests were abnormal in % [ ] . b gulf war syndrome (gws) gws is a clinical entity specifically related to a certain time and place in history. it was described among veterans of the military conflict occurring in - in the persian gulf. the syndrome is characterized by chronic fatigue, musculoskeletal symptoms, malaise and cognitive impairment. it clinically overlaps with post traumatic stress disorder (ptsd), fms, cfs and other functional disorders [ ] . the unique conditions that have been associated so far with disease development are the exposure to extreme climate in the persian gulf, exposure to various chemicals (pesticides, depleted uranium), stress provoked by prolonged waiting without actual combat and the intense exposure to vaccinations of the soldiers for fear of biological weaponry [ ] . comparing gulf war veterans and veterans of the bosnian conflict, multiple vaccinations administered to servicemen in the gulf war was identified as a unique exposure [ ] . the mechanism through which vaccination exposure may lead to the development of functional symptoms is not completely understood. the possibility that a shift from th to th type reactions could be of pathogenic significance was raised and is supported by an increased frequency of allergic reactions, low natural killer cell activity and low levels of interferon ␥ and il- in these patients [ ] . one study with gws patients showed a connection between anti-squalene antibodies and symptoms development. this was refuted by a larger study that found no association between antisqualene antibodies and chronic multi-symptom illness [ ] . c asia registry a registry is a collection of data related to patients with the same specific characteristic. it is often the first approach in the study of an area of inquiry. in rare diseases, registries are often the way to get a sufficiently sized sample of patients which can be used either for epidemiological or research purposes. asia syndrome may be underreported because of unawareness and failure to connect the syndrome with the exposure. this registry was created to fully understand the clinical aspects of disease and compare patients from all over the world in order to have fully validated criteria for disease diagnosis and also to define demographic and environmental history of disease. the asia syndrome registry website can be found on the following link: https://ontocrf.costisa.com/en/web/asia. only cases reported by physicians are accepted. to make an informed decision in medicine, there is always a need to weigh the pros and cons. ards may play an important role in deciding whether vaccination is or is not appropriate to a patient. in these cases, patients are immunosuppressed on account of their diagnosis and even more so if they are under specific immunomodelatory medication [ ] . if the efficacy of vaccination is reduced, there is a potential for development of disease flares following vaccination. in the case of live vaccines, its inoculation may even be enough to trigger disease in the host. for these specific reasons, live vaccines are generally contraindicated in patients receiving immunosuppressant medication. there is a need for screening and treatment of latent tuberculosis infection (ltbi) before starting anti-tnf-alpha therapy. the same is true for vaccination. preferably, even recommended vaccination (see table ) should be administered before the initiation of disease-modifying anti-rheumatic drugs (dmards) because these may reduce vaccine efficacy [ ] . immunosuppression equals high risk of infection and lower vaccine efficacy. taking into account safety concerns and efficacy, the eular recommendations for immunizations in aiird patients are: • assess vaccination status in initial investigation. • administer vaccines in a stable disease phase. • live attenuated vaccines are to be avoided especially if immunosuppressive agents are being administered. bcg is not recommended. • administer vaccines ideally before starting dmards and anti-tnf␣ agents. • influenza and -valent polysaccharide pneumococcal vaccination is recommended. • tetanus toxoid vaccination is recommended following recommendations of general population, in case of major and/or contaminated wounds in patients receiving rituximab in the previous weeks tetanus ig is indicated. • hpv and herpes zoster should be considered. • in hyposplenic/asplenic patients, influenza, pneumococcal, haemophilus influenza b and meningococcal c are advisable. • hepatitis a and b is recommended in patients at risk. • travel patients should be immunized according to general population guidelines except for live attenuated vaccines, which are to be avoided [ ] . vaccines have many beneficial effects in combating infectious diseases and preventing mortality and morbidity. they have also proved to be effective cancer treatments by immunomodulation, as demonstrated by the intravesical administration of bcg to treat superficial bladder cancer [ ] . vaccines are however, linked to autoimmunity. beneficial outcomes, like the adjuvant effect are based on immunity triggering and enhanced immunity mechanisms. these same responses account for autoimmunity exertion. vaccines induce the production of autoantibodies, but their pathologic effect is yet to be unveiled. although vaccines are widely considered safe, there are subjects with predispositions to whom vaccines pose a bigger threat. an example is the fact that animal models with autoimmune predispositions develop autoimmune disease following adjuvant exposure. as many as % of recipients of aluminum containing adjuvants may be sensitized to future exposure [ ] . silicon-induced inflammatory fibro proliferative response is irrefutable and well documented. the presence of anti-silicone antibodies and silicone-associated autoimmune phenomena seems very plausible. asia syndrome and aluminum safety studies show that the use of aluminum containing "placebo" in control groups in vaccine safety studies should be carefully evaluated. new studies must be performed using a proper placebo to adequately test vaccine safety. another evident failure in vaccine safety studies are the short-term periods which are evaluated. continued immune system activation has been observed to be a potential mechanism of disease. a disease which is poorly understood so far. vaccine recommendations should be reassessed frequently in different subsets of the population. this does not invalidate the need for vaccines, however, the lower the possibility of exerting adverse events, the easier it will be for the potential benefits to outweigh the risks. vaccinomics represents a major breakthrough in vaccine development and can lead to the development of targeted vaccines to peptides most likely to be immunogenic [ ] . a predictable response to vaccine can be achieved by differentiating the host variability. this can be achieved namely in genetics and pathogen variability. developing a vaccine accordingly will lead to increased specificity in treatment and 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aluminium in children after the use of adsorbed vaccines from a single manufacturer key: cord- -og sg qw authors: howell, gareth j.; holloway, zoe g.; cobbold, christian; monaco, anthony p.; ponnambalam, sreenivasan title: cell biology of membrane trafficking in human disease date: - - journal: int rev cytol doi: . /s - ( ) - sha: doc_id: cord_uid: og sg qw understanding the molecular and cellular mechanisms underlying membrane traffic pathways is crucial to the treatment and cure of human disease. various human diseases caused by changes in cellular homeostasis arise through a single gene mutation(s) resulting in compromised membrane trafficking. many pathogenic agents such as viruses, bacteria, or parasites have evolved mechanisms to subvert the host cell response to infection, or have hijacked cellular mechanisms to proliferate and ensure pathogen survival. understanding the consequence of genetic mutations or pathogenic infection on membrane traffic has also enabled greater understanding of the interactions between organisms and the surrounding environment. this review focuses on human genetic defects and molecular mechanisms that underlie eukaryote exocytosis and endocytosis and current and future prospects for alleviation of a variety of human diseases. the human cell is a complex network of membranes and protein enclosed in a membrane lipid bilayer. the interactions within and associated with such biomembrane bilayers have profound consequences for the organism as a whole; a single defect in just of the potential - , gene products made by each cell can cause devastating, if not fatal, evects for the whole organism. in addition to this, humans pass genetic information onto their ovspring and, with it, any genetic mutations or polymorphisms. it is believed that at least in people have, or will eventually develop, a disease caused by mutation or variation at the gene level. understanding how genetic mutations increase risk for human disease is critical in our understanding and treatment of the majority of human ailments that are caused by interactions between the organism and the environment. this review focuses on the research undertaken in the past years relating to the molecular mechanisms that underlie membrane traycking within eukaryotic cells. we address mechanisms and factors that control protein progression through the secretory and internalization pathways and highlight key human diseases that illuminate mechanisms of membrane traycking. in addition, current and future strategies for therapeutic intervention in such genetic disorders are considered. common to all eukaryotic cells is the presence of multiple biomembrane lipid bilayer compartments, or organelles, which are maintained by specific protein-protein and protein-lipid interactions. such interactions are maintained within each compartment in spite of continuous traycking of membrane-bound and soluble components to diverent intracellular locations, and for secretion from the cell. in the majority of cases, this transfer of material occurs through vesicular movement: fission, docking, and fusion of membrane bilayer-enclosed intermediates occurs between donor and acceptor compartments (palade, ) . proteins, including membrane-bound receptors, secreted enzymes, and antibodies, begin their journey by entering the early secretory pathway at the endoplasmic reticulum (er). from here they are transported through the golgi apparatus and finally distributed to their final destination such as other intracellular organelles, the plasma membrane, or the extracellular environment. but how does a specific protein ''know'' how to reach a specific cellular destination when hundreds of newly synthesized, diverent molecules require specific transport and targeting? many of these transport intermediates or vesicles, whether derived from the er, other internal organelles, or the plasma membrane, are ''coated'' with unique protein complexes, tethering factors, and regulatory factors that ensure correct targeting to an acceptor compartment. vesicle coat proteins, such as the clathrin or coat protein (cop) complexes, are relatively well studied. such complexes are assembled onto the cytoplasmic face of donor compartments to facilitate the fission of transport intermediates. allied with these coat proteins are diverent molecules that mediate recognition of cytoplasmic motifs in cargo proteins either directly (e.g., transmembrane proteins) or indirectly (e.g., soluble secreted enzymes). the snare hypothesis is central to our understanding of vesicular targeting to intracellular compartments (rothman, ; sollner et al., ) . initially uncovered in a screen for intra-golgi transport docking and fusion regulators, the snare (soluble n-ethylmaleimide-sensitive fusion attachment protein receptor) proteins have been found to regulate diverent membrane interactions in all eukaryotes via a highly conserved mechanism for membrane traycking based on accessory docking and fusion regulators. snare proteins are present on both the vesicle (vesicular or v-snare) and the acceptor (target or t-snare) and comprise coiled-coil domains that assemble to facilitate vesicle docking and membrane fusion (bennett, ; pelham, ) . in conjunction with snare proteins, small ras-related rab gtpases are implicated in further ensuring the fidelity of vesicle docking and fusion (olkkonen and stenmark, ) . these -to -kda proteins are gtphydrolyzing enzymes that act to recruit diverent proteins or evectors to membranes in a gtp/gdp-regulated manner (collins, ) . rab gtpase activity and protein conformation are regulated by interaction with soluble and membrane-bound proteins; such regulators can also tether vesicles to acceptor membranes and mediate intracellular signaling. a. early secretory pathway the endoplasmic reticulum (er) is the first stage of quality control along the secretory pathway. proteins destined for secretion (e.g., hormones), the plasma membrane (e.g., membrane-bound receptors), or other intracellular membrane compartments such as the lysosome (e.g., lysosomal proteases) emphysema and liver cirrhosis (perlmutter, ) endophillin ii clathrin-coated pit formation leukemia (dreyling et al., ; jones et al., ; narita et al., ; tebar et al., ) alzheimer's disease presenilin presenilin -involved in cleavage and trafficking of amyloid precursor protein to plasma membrane neurodegenerative disorder (uemura et al., ) tau tau -microtubular stability through formation of aggregates autosomal dominant polycystic kidney disease (adpkd) polycystin- or causes a defect in e-cadherin assembly and basolateral trafficking renal cysts in kidney and other tissues leading to endstage renal failure (charron et al., ) (continued ) autosomal dominant retinitis pigmentosa rhodopsin inhibited interaction of rhodopsin and arf , leading to inhibited post-golgi delivery to rod outer segment narrowing of visual fields, night blindness (deretic et al., ) autosomal dominant ventricular tachycardia cardiac arrhythmia, hyperthermia (yano et al., ) autosomal recessive primary hyperoxaluria mistargeting of peroxisomal proteins to mitochondria kidney disease (danpure, ) ab-lipoproteinaemia mtp er retention thus preventing apob secretion vascular disease (sharp et al., ) batten's disease cln -cln group of gene products implicated in regulating the processing and targeting of lysosomal and synaptic proteins neurological disease (pearce, ) breast cancer caveolin- deletion or dominant negative mutation of caveolin- promotes tumor progression breast cancer (bouras et al., ; williams and lisanti, ) (hayasaka et al., ; matsuyama et al., ) chediak-higashi syndrome (chs) chs /lyst lyst involved in regulation of protein secretion from lysosomes -enlarged lysosomes partial albinism, recurrent bacterial infections, impaired chemotaxis and abnormal natural killer cell function (shiflett et al., ; ward et al., ) choroideremia (chm) rab escort protein (rep ) rab a remains cytosolic due to defective geranylgeranyl modification in chm lymphoblasts x-linked form of retinal degeneration combined factors v and viii deficiency ergic- /p c-type lectin er retention and defective secretion of factors v and viii blood disease congenital finnish nephritic syndrome nephrin (nphs ), podocin (nphs ) er retention kidney inflammation (kestila et al., ; kramer -zucker et al., ) pancreatic atp-sensitive potassium channel (k-atp) er or golgi retention of k-atp due to mutations in its sulfonylurea- (sur ) subunit excess insulin leading to hypoglycaemia (dunne et al., ; yan et al., ) congenital hypothyroid goiter thyroglobulin er storage disease. thyroglobulin is misfolded and accumulates in er constipation, large tongue, swelling around the eyes, failure to suckle, mental retardation (hishinuma et al., ; kim and arvan, ) (fan et al., ; garman and garboczi, ) familial hemophagocytic lymphoschistiocytosis (fhl) perforin -defective ctl (cytotoxic t lymphocytes) mediated killing immunodeficiency (feldmann et al., ; stepp et al., ) munc (hogg et al ., ; mathew et al., ) three forms of menkes disease can arise from diverent mutations in the atp a gene: premature stop codons, deletions, or splicing defects. these can prevent atp a function and/or traycking. classical menkes disease is the most common and fatality usually results by the age of years. in two other nonfatal forms of menkes, mild and occipital horn syndrome, atp a maintains the ability to transport copper ions across intracellular membranes, although traycking to the plasma membrane can be compromised (la fontaine et al., ) . atp a is ubiquitously expressed and is the major copper transporter in cells of the intestine, kidney, and brain. in the liver, however, the major copper transporter is the wilson's disease protein, atp b (bull et al., ) . this second p-type atpase shares strong similarity with atp a and also translocates copper ions across membranes. although these gene products share functional similarities, mutations in atp b result in copper accumulation in the liver and brain. familial hypercholesterolemia is an autosomal dominantly inherited disease caused by mutations in the low-density lipoprotein receptor (ldlr), leading to premature atherosclerosis and coronary heart disease. in healthy individuals, the ldlr is expressed on the surface of cells, where it binds circulating ldl particles and promotes uptake and cellular metabolism of its constituents, which includes cholesterol. in these patients, ldlr alleles display amino acid substitutions (cassanelli et al., ; jensen et al., ) , truncations (lehrm an et al ., ) , or mis sense mutat ions ( leiter sdorf et al ., ) , which can result in er retention and degradation. the point mutation at residue of the insulin receptor compromises the ability of the receptor to dimerize correctly within the er, therefore leading to er retention. decreased plasma membrane levels of insulin receptor cause inhibited insulin binding after stimulus by a meal, and subsequent elevations in plasma glucose levels. this then leads to type ii diabetes mellitus (kadowaki et al., ) . a number of human diseases can induce the er stress response. here, the mutant protein is retained within the er, resulting in either dilation of the organelle, such as in congenital hyperthyroidism (medeiros-neto et al., ) and hypofibrinogenemia (callea et al., ) , or chronic er stress as is the case for hereditary emphysema (perlmutter, ) . in pelizaeus-merzbacher disease, an x-linked leukodystrophy disease, er accumulation of proteolipid protein (plp) results in oligodendrocyte apoptosis (gow et al., ) and the subsequent disruption of white matter formation in the brain observed in humans and mouse models. plp is a central nervous system protein that is the major component of myelin and, when expressed in cultured fibroblasts, is localized to the plasma membrane (gow et al., ) . the link between plp and the er stress response provides a tool for elucidating the cellular response to misfolded protein accumulation . accumulation of proteins within the er, leading to blockage of protein secretion, is an unwanted cellular property and mechanisms have evolved to overcome such events. this disposal of unwanted proteins is termed er-associated degradation (erad) (fig. ) . as recently as the early s, it was still believed that aberrant proteins were degraded within the er (fra and sitia, ) ; however, current models suggest that aberrant er-retained proteins actually undergo retrotranslocation and subsequent degradation in the cytoplasm. retrotranslocation has been proposed to occur through the same ''pore'' used to translocate nascent proteins into the er lumen during translation, namely the sec translocon (biederer et al., ; rö misch, ) . various yeast and mammalian proteins have been shown to be retrotranslocated from the er and degraded within the cytoplasm in a proteasomedependent manner, including the budding yeast proteins carboxypeptidase y, and a mutant pro-a-factor. when a mammalian protein such as cftr is expressed in budding yeast, it matures relatively slowly within the yeast er, leading to retrotranslocation to the cytoplasm and degradation (ward et al., ) . a further example is that of a -antitrypsin deficiency. a -antitrypsin is responsible for inactivating the enzyme elastase produced by lung neutrophils. in this inherited disease, a mutated form of a -antitrypsin is retrotranslocated and degraded in proteasomes, leading to retention of active elastase in lung tissues and thus is a cause of lung emphysema (rutishauser and spiess, ) . however, retrotranslocation and proteasomal degradation may not be functionally coupled processes. pharmacological inhibitors that cause proteasome inactivation lead to egress of molecules such as mhc class i (wiertz et al., a,b) , ribophorin (de virgilio et al., ) , and carboxypeptidase y (biederer et al., ) from the er to the cell cytoplasm. in contrast, inhibition of protein ubiquitination results in the retention of such molecules within the er. schmitz et al. ( ) suggest that two distinct proteasome-regulated pathways mediate degradation of retrotranslocated b-amyloid precursor protein. interestingly, endocytosed toxins that target key cytosolic factors appear to use the erad pathway to move out of the er and into the cytosol (deeks et al., ; hazes and read, ) . cholera and ricin toxins are routed from the cell surface through the golgi apparatus and to the er before being retrotranslocated into the cell cytosol. it is believed that the unusually low lysine content of these protein toxins prevents subsequent er-associated ubiquitination for degradation by the cytosolic proteasome. protein cargo is shuttled between the er and golgi within vesicular intermediates or -nm-diameter spherical vesicles containing coat protein complexes fig. quality control of protein assembly within the endoplasmic reticulum. proteins destined for the secretory pathway (this example shows a transmembrane protein) are cotranslationally translocated from the ribosome into the lumen of the endoplasmic reticulum (er) through a portal referred to as the sec translocon. as the newly synthesized protein enters the er, quality control mechanisms in the form of protein chaperones bind to it and fold it to its correct conformation. further processing occurs through interactions with other chaperones before the successfully folded protein is loaded into copii-coated vesicles and shuttled from the er to the golgi apparatus. however, if the protein carries a mutation that causes it to take on an aberrant conformation the er chaperones will trigger a misfolded protein response. this has two outcomes: either the chaperones will remain bound to the misfolded protein, preventing its escape from the organelle (er retention), or the protein will be ubiquitinated and retrotranslocated through the sec complex for proteasomal degradation in the cell cytoplasm. a number of human genetic diseases are a result of key proteins failing to trayc through the secretory pathway and as a consequence are retained or degraded in this manner. such as copi or copii. initially discovered in mammals and yeast (kaiser and schekman, ; malhotra et al., ; novick et al., ; rothman and wieland, ) , cop complexes are required for the formation of vesicles at the er, er-golgi intermediate compartment (ergic), and golgi apparatus. cop recruitment to membranes facilitates the specific capture, packaging, transport, and delivery of membrane-bound and soluble protein cargo to an acceptor compartment. copii recruitment to sites on the smooth er initiates the formation of anterograde (forward) transport vesicles. these copii vesicles move from the er to the ergic, or vesicular tubular clusters (vtcs). from here, copi-coated vesicles are thought to mediate the continued anterograde movement from the ergic to the cis face of the golgi apparatus (scales et al., ) . the sar p gtpase regulates copii vesicle formation via interaction with the sec p guanine exchange factor (gef). sec p-mediated activation of sar p to a gtp-bound form leads to recruitment of the sec p-sec p heterodimer to membranes; this also initiates protein cargo selection within the er and recruitment of v-snares such as bet p and bos p. binding of sec p-sec p mediates further recruitment of the sec p-sec p complex. this copii complex then acts as a protein scavold that causes deformation of the membrane, resulting in vesicular fission, with anterograde movement of protein cargo-containing copii vesicles to the ergic. copii docking at an acceptor compartment is thought to trigger sec p function, causing a conformational change in sar p and gtp hydrolysis and dissociation or uncoating of the copii complex. thus copii vesicle docking and fusion with an acceptor compartment are mediated by cognate v-snare/ t-snare interactions (kirchhausen, ; kuehn et al., ; matsuoka et al., ; tang et al., ) . a severe hereditary bleeding disorder called combined deficiency of factor v factor viii (f f d) highlights the functional importance of traycking between the er and ergic. some f f d patients are deficient in the ergic-localized ergic- (lman ) protein and display defective secretion of the factor v and viii clotting factors. ergic- is a mannosebinding lectin that acts as a ''cargo receptor'' and recycles between the er and ergic (neerman-arbez et al., ; . however, % of f f d patients show normal levels of ergic- /lman , but are deficient in an associated protein, mcfd , another ergic resident that interacts with ergic- /lman in a calcium-dependent manner . small intestinal cells called enterocytes absorb fats and fat-soluble vitamins from food in the form of fatty acids and monoglycerides. the fats enter the lumenal surface of absorptive enterocytes by free divusion across their membranes, and emerge from the basolateral surface as particulate structures referred to as chylomicrons. formation of chylomicrons occurs within the er and golgi apparatus by vesicular transport before being traycked from the golgi to the plasma membrane. chylomicron retention disease (cmrd), anderson disease, and a neuromuscular disorder, cmrd associated with marinesco-sjö gren syndrome (cmrd-mss) , are examples of inherited diseases that result in compromised fat absorption, low blood cholesterol, and severely depleted blood chylomicron levels. jones et al. ( ) identified eight mutations in the sar p gene product and copii component associated with these lipid absorption diseases, thus strongly implicating a role for the copii vesicular transport system in the movement of dietary fats from the intestine to the circulating bloodstream. copii mediates anterograde trayc from the er to the golgi apparatus; however, copi vesicles appear to function primarily in the retrograde (backward) transfer of proteins from the golgi and ergic back to the er. this retrograde trayc is necessary for recovering escaped er resident proteins, coat and snare proteins that have arrived at the ergic and golgi from copii vesicles, or glycosylation enzymes that have been incorrectly modified (duden, ; lee et al., ) . the golgi-associated copi coatomer is a complex of seven polypeptides: a-, b-, b -, g-, d-, e-, and z-cop gene products, which interact with the donor membrane to form copi vesicles. vesicle formation is triggered by the gtpase adp-ribosylation factor (arf ), which recruits copi coatomer to the donor membrane. transmembrane proteins containing cytoplasmic lysine-based motifs such as kkxx or kxkxx, or soluble proteins containing the c-terminal kdel motif, are recycled by copi-coated vesicles from the golgi apparatus back to the er. the kdel motif, present in soluble er chaperones such as bip and protein disulfide isomerase, is recognized by the membrane-bound kdel receptor (majoul et al., ) . in both cases, cytoplasmic motifs in these transmembrane proteins are recognized and bound by copi coatomer, promoting inclusion into vesicles destined for the er. actin microfilaments are also involved in this retrograde transport step (valderrama et al., ) . this golgi-er step is regulated by the gtpase cdc and n-wasp protein (luna et al., ) , factors previously implicated in actin-linked processes at the plasma membrane. live imaging of cells expressing an engineered fluorescent and temperaturesensitive vesicular stomatitis virus g-glycoprotein (ts vsvg) demonstrated sequential action of copii-and copi-coated vesicles (scales et al., ) . vsvg accumulated in structures close to the er that contained intermediate compartment resident proteins. these structures then matured into vesicles that contained copi proteins. stephens et al. ( ) showed that this ''segregation'' between copii and copi vesicles occurred at a location in close proximity to exit sites on er membranes. a cop-independent mechanism has also been implicated in retrograde trayc between the golgi apparatus and the er. the rab gtpase is implicated in regulating the movement of bacterial shiga toxin b fragment (stb) via a retrograde step from the golgi apparatus to the er. expression of a dominant-negative gdp-bound form of rab inhibited stb retrograde movement, whereas copi transport was unavected (white et al., ) . the golgi apparatus is composed of flattened cisternae and membrane compartments that are closely juxtaposed in a stack-like appearance. in mammalian cells these stacks are positioned end-to-end, forming a ribbonlike structure near the nucleus (barr and warren, ) . the golgi apparatus is a highly dynamic organelle sited at the hub of the secretory pathway with key processing and sorting functions. the golgi is a polarized structure with proteins and lipids from the er received at the cis side, followed by the medial and trans subcompartments, where further glycosylation modifications occur; the trans-golgi network (tgn) is the final subcompartment where sorting and packaging events take place. the golgi apparatus also sorts proteins and lipids bound on a retrograde pathway from the cis-golgi back to the er. in addition, proteins can also return to the tgn from the endomembrane/lysosomal system (fig. ) . controversy exists regarding the mechanism for anterograde movement of cargo proteins within the golgi apparatus. the golgi apparatus contains secretory proteins that can vary in physical size, from relatively small polypeptides to large, bulky multisubunit complexes; all need to reach the tgn for final sorting into transport intermediates. there are also resident glycosylation enzymes that have spatially restricted functions within the golgi, that is, enzymes that function within specific subcompartments to ensure the correct addition or trimming of n-and o-linked sugars on secreted proteins as they progress through the pathway. this raises a key question: how do protein and lipid cargo move through the golgi apparatus while resident enzymes retain their localization? we know that many golgi enzymes contain transmembrane golgi localization signals that mediate targeting to a specific compartment (munro, ) . two models have been proposed: the cisternal maturation model and the vesicular transport model (elsner et al., ; storrie et al., ) . briefly, the cisternal maturation model suggests that large proteins or aggregates remain within a single golgi cisterna, which matures through the retrograde transfer of resident enzymes via copi vesicles. in contrast, the vesicular transport model proposes that newly synthesized protein is traycked from cisterna to cisterna via copi-coated vesicles that sequentially bud ov membranes and fuse with the next subcompartment. in either case, copi-coated vesicles play a central role in intra-golgi the secretory pathway and vesicular traycking. protein enters the secretory pathway at the endoplasmic reticulum (er) and is traycked in copii-coated vesicular structures to the intermediate compartment (ergic/vtc), from which copi-coated vesicles carry it to the cis face of the golgi. cargo protein (c) continues along the secretory pathway through the golgi apparatus to the trans-golgi network (tgn). retention signals in er resident proteins (r) ensure they undergo retrograde traycking from the golgi in copi vesicles. retrograde transport of transport. a number of snare proteins, such as membrin, rbet , gs , and syntaxin- , have also been localized to the golgi apparatus and are required for intra-golgi transport and homeostasis (nichols and pelham, ) . golgi-tethering molecules called golgins and golgi reassembly stacking proteins (grasps) belong to a family of regulatory factors involved in golgi maintenance and vesicular transport. the reader is pointed to an indepth review that covers golgins in more detail (short et al., ) . in brief, the golgins can be anchored to golgi membranes through various mechanisms and contain characteristic coiled-coil domains that extend from the membranes as a rod-like structure (burkhard et al., ) . golgins such as giantin and golgin- are securely anchored to the membrane via a transmembrane domain near their c terminus. electrostatic or ionic interactions mediate the attachment of other golgins to membranes. for example, proteins of the grasp family (grasp and grasp ) bind to gm and golgin- to recruit these factors to the cis and medial golgi membranes, respectively. moreover, a large number of golgins are recruited to membranes via interactions with the rab, arf, and arl (arf-like) gtpases. vesicular and cis-golgi membrane recruitment of golgin p is regulated by rab , whereas membrane attachment of yeast golgin rud p is regulated by arf p. golgin- binds to membranes by interaction with arl p, a member of a new class of arf-like gtpases termed arls (short et al., ) . interestingly, autoantibodies directed against giantin, golgin- , golgin- , gm , and golgin- golgins and grasps are present in patients with autoimmune conditions such as sjö gren's syndrome and systemic lupus erythematosus. in sjö gren's syndrome, moisture-producing glands are targeted by the autoimmune response, resulting in dry eyes and mouth (lichtenfeld et al., ) . systemic lupus erythematosus is a chronic rheumatic condition that avects joints and muscles, causing skin rash and kidney problems. sjö gren's syndrome patients can also simultaneously display both rheumatoid arthritis and systemic lupus erythematosus. golgi biogenesis requires golgin function at diverent stages during cell division. mammalian p is crucial for maintenance of the stacked nature of the golgi cisternae (puthenveedu and linstedt, ) . during mitosis, the golgi stack disperses into clustered vesicles. these vesicles then fuse in the daughter cells to form new cisternae, alignment and stacking of which result in the formation of a fully functional organelle. grasp tethers have been proposed to hold cisternae in close proximity through interactions with p and gm (shorter and warren, ) . the golgin p is also involved in tethering copi vesicles to golgi membranes (sonnichsen et al., ) and may be needed for snare complex assembly (shorter et al., ) . the budding yeast p homolog (uso p) tethers copii-coated vesicles to golgi membranes during anterograde transport from er exit sites to the cis-golgi (barlowe, ; cao et al., ; sapperstein et al., ) . mammalian p is also essential for the tethering of transport vesicles to the cis-golgi (alvarez et al., ) and during intra-golgi transport (seemann et al., ; waters et al., ) . golgins such as golgin- are implicated in the regulation of golgi structure and the formation of the golgi ribbon (diao et al., ) . golgin- may function as a tethering molecule in retrograde trayc from the endosome to the tgn (lu et al., ) . moreover golgins are also implicated as tethering components between the cytoskeleton and the golgi apparatus (short et al., ) . the trans-golgi network (tgn) is the final golgi subcompartment where secreted proteins are sorted, packaged, and directed to their final destination. traycking from the tgn can occur in either a constitutive or regulated manner. constitutive transport is the continuous release of protein from the trans-golgi network. regulated secretion occurs in response to extracellular stimuli such as secretagogues, metal ions, hormones, or growth factors, which trigger the docking and fusion of secretory granules or vesicles with the plasma membrane. various mechanisms control the traycking of proteins from the tgn by the formation and delivery of membrane-derived transport vesicles to the plasma membrane, endosomes, or lysosomal structures (ponnambalam and baldwin, ) . the expression of inactive (dominant-negative) protein kinase d isoforms in tumor lines (liljedahl et al., ) , polarized canine kidney cells (yeaman et al., ) , and mouse fibroblasts (prigozhina and waterman-storer, ) has been shown to inhibit vesicle fission (release) from the tgn. vesicle release is modulated by this family of kinases in response to cellular diacylglycerol (baron and malhotra, ) and binding to an as yet unknown evector protein on the cytoplasmic face of the tgn (van lint et al., ) . in addition, the cdc gtpase is linked to actin remodeling and has been shown to inhibit the exit of basolateral targeted proteins in polarized cells (kroschewski et al., ; musch et al., ) and copper-regulated protein transport (cobbold et al., ) . copper is an essential element and cofactor required for functionality of many secreted enzymes (cuproenzymes). at steady state, atp a (menkes howell et al. disease copper transporter; section iii.a. ) resides in the tgn, where it provides newly synthesized cuproenzymes such as lysyl oxidase with copper ions as they traverse the secretory pathway. when intracellular copper ion levels rise, atp a responds to this environmental danger by redistributing to the plasma membrane in a cdc -regulated manner (cobbold et al., ) . here, atp a acts as a copper ezux pump to remove copper ions from the cytoplasm to maintain homeostatic function and prevent toxicity. when copper levels are reduced, atp a recycles back to the tgn. this endocytic internalization and sorting event is independent of both clathrin and caveolae (cobbold et al., ) , although relying on a cytoplasmic dileucine motif present in the atp a c-terminus petris and mercer, ) . dent's disease, an x-linked kidney disorder that presents with hypercalciuria, nephrocalcinosis (kidney stone formation), and progressive renal failure, is caused by missense, nonsense, and deletion mutations within the endosomal clc- voltage-gated chloride channel. clc- is a member of a large family of voltage-gated chloride channels that have a diverse array of cellular functions including membrane excitability, transepithelial ion transport, and cell volume regulation (thakker, ) . when expressed in xenopus oocytes, a number of missense mutations in the clc- gene localized the channel to the golgi apparatus and showed reduced conductance and significantly reduced plasma membrane (pm) localization (ludwig et al., ) . similarly, expression of mutant clc- alleles in cultured cells revealed an approximate -fold increase in golgi retention (carr et al., ) . a. receptor-mediated endocytosis clathrin-coated vesicles (ccvs) are a route for protein internalization conserved from yeast to humans. roth and porter ( ) first observed this process in mosquito oocytes and these vesicles have subsequently become one of the best characterized membrane transport steps in eukaryotes. clathrin is one of the principal proteins involved in this transport step and, in combination with more than clathrin-associated factors, this unique structural component forms transport vesicles on the cytoplasmic face of the tgn, endosomes, and the plasma membrane. clathrin-coated vesicles bud from their donor membranes and are directed to target membranes by associated proteins and factors. this highly conserved -kda clathrin complex comprises heavy ( kda) and light ( kda) chain proteins that are assembled into a three-legged structure called a triskelion. triskelions can be polymerized by accessory factors into striking lattice-like ''cages'' comprising pentagons and hexagons, resembling a soccer ball structure or buckminsterfullerene. clathrin cages are - nm in diameter; significantly larger than copi or copii vesicles (ccvs). clathrin-coated vesicles are believed to assemble through a sequence of events that can be designated as activation, cargo capture, coat assembly, scission, movement, and vesicle uncoating (kirchhausen, ) . members of a class of clathrin-associated factor termed adaptor protein (ap) complexes are recruited to donor membranes through interactions with a docking complex, which then further interacts with motifs within the cytoplasmic tail of cargo proteins, resulting in ''cargo capture.'' this leads to clathrin cage assembly and the concomitant polymerization of the clathrin triskelion and resultant deformation of the donor membrane. scission, or vesicle release from the plasma membrane, is believed to occur through the action of the gtpase dynamin and other accessory proteins, such as amphiphysin (wigge et al., ) . in the fruit fly drosophila melanogaster, a dynamin gene mutation (shibire) causes temperature-sensitive paralysis. this is likely due to a block in the endocytic uptake of synaptic vesicle proteins at the plasma membrane, leading to a block in recycling and reformation of competent synaptic vesicles at nerve terminals (chen et al., ; koenig and ikeda, ; kosaka and ikeda, ; van der bliek and meyerowitz, ) . the expression of a dominant-negative gdp-bound dynamin mutant, k a, results in compromised ccv formation (herskovits et al., ; van der bliek et al., ) and inhibition of clathrin-mediated internalization of the glucose transporter glut (al-hasani et al., ) , human immunodeficiency virus (hiv) (daecke et al., ) , and influenza virus (roy et al., ) . the scission function of dynamin is assisted by specific lipid-modifying enzymes such as endophilin, synaptojanin, and phospholipase d (bi et al., ; havner et al., ; ringstad et al., ; schmidt et al., ; woscholski et al., ) . finally, ccv uncoating at the target membrane occurs through the actions of the heat shock protein hsc (schlossman et al., ) and auxilin (ungewickell et al., ) . sorting of proteins from donor to target membranes involves the recognition of cytoplasmic sequences in membrane proteins by clathrin-associated ap complexes. four adaptor protein complexes (ap -ap ), each comprising four diverent subunits, have been identified (robinson, ) . the ap complex is involved in clathrin-coated vesicle formation at the tgn for transport to late endosomes; evidence has also implicated a role for this complex in a tgn-to-plasma membrane step (folsch et al., ) . ap is the best-studied of the four complexes and mediates internalization of transmembrane howell et al. receptors at the plasma membrane via clathrin-coated vesicles. the ap complex is involved in traycking from early endosomes to either late endosomes or lysosome-related organelles such as melanosomes, platelet-dense bodies, and antigen-processing compartments. finally, the ap complex was the last to be cloned (dell'angelica et al., a; hirst et al., ; ) . in contrast to ap -ap , ap does not possess the b ''ear'' domain (see below), which allows interaction with clathrin and other cytosolic factors such as eps and auxilin (lundmark and carlsson, ) . by electron microscopy, ap has been localized to vesicles at the tgn, plasma membrane, and early endosomes, although there is debate as to whether these vesicles are clathrin-coated (barois and bakke, ; hirst et al., ) . interestingly, ap and ap may function independently of clathrin (hirst et al., ; vowels and payne, ) , suggesting the existence of another, as yet unidentified, coat protein that is analogous to clathrin. all four ap complexes comprise two large -kda subunits: a b subunit (b -b ) plus a g (ap ), a (ap ), d (ap ), or e (ap ) subunit. in addition, each ap complex contains a -kda subunit (m -m ) and a small -kda subunit (s -s ). ap , - , and - contain two carboxyl ''ear'' domains connected to the head of each large -kda subunit by a flexible hinge of approximately - residues. importantly, the ear domain of the b subunit and the hinge domains of the g and a subunits have been shown to bind clathrin (goodman and keen, ; morgan et al., ; owen et al., ) , and consensus sequences in the hinge domains of b and b have clathrin-binding properties (dell'angelica et al., ) . the b and m subunits of the ap complex interact with motifs present in the cytoplasmic domains of transmembrane proteins to mediate cargo recruitment into clathrin-coated vesicles. such motifs include npxy, yxxØ, and dileucine-based sequences (Ø represents a bulky hydrophobic amino acid). one such motif, npxy, is present in key cellular receptors such as low density lipoprotein receptor (ldlr), epidermal growth factor receptor (egfr or erb ), and insulin receptor, and mediates endocytosis and sorting. importantly, the jd mutation (y c) in ldlr lies within this key motif and causes familial hypercholesterolemia (knoblauch et al., ) . the second tyrosinebased motif, yxxØ, mediates plasma membrane internalization, lysosomal targeting, and basolateral targeting of cargo. this motif is found in lysosomal residents such as lamp- and - , cd , the recycling transferrin receptor (tfr), and tgn-associated recycling membrane proteins, furin and tgn . di-leucine motifs present on transmembrane transporters such as glut (glucose transporter), atp a, and mannose- -phosphate receptors (m pr) can fall into two categories: [de]xxx[li] and dxxll related motifs. the [de]xxx[li] motif is associated with proteins internalized from the plasma membrane and targeted to lysosomes, while dxxll motif is found in transmembrane proteins that shuttle between the tgn and endosomal system (bonifacino and traub, ) . another class of clathrin-associated factor is the golgi-localized, g-earcontaining, arf-binding proteins (ggas) found on the tgn and postulated to interact with ap to mediate transport of m pr (section v.b) to endosomes (doray et al., ) . ggas can act as multifunctional adaptors that link transmembrane proteins, arf gtpases, clathrin and accessory proteins at sites of ccv formation (robinson and bonifacino, ) . the disease oculocerebrorenal syndrome of lowe (ocrl) is an x-linked disorder caused by mutations in the ocrl gene (lowe, ) . the gene product is an inositol -phosphatase that catalyzes the removal of the phosphate from this position on the inositol moiety. the preferred ocrl substrate is pi( , )p , a phosphoinositide shown to be important in endocytosis because of its central role in recruiting accessory proteins to ccvs (padron et al., ) . ocrl has been localized to clathrin-coated vesicles associated with endosomal and tgn membranes (choudhury et al., ) . this is not surprising as ocrl interacts with clathrin and promotes its assembly into clathrin lattices and cages (choudhury et al., ; ungewickell et al., ) . ocrl also interacts with the rac gtpase that regulates actin dynamics, possibly via a gtpase activation domain to accelerate gtp hydrolysis (faucherre et al., ) . although the exact function of ocrl is still unclear, the disease phenotype hints to ocrl function in membrane traycking. ocrl mutations can cause loss of protein expression and phosphatase activity. rnai-mediated inhibition of ocrl expression in cultured human cells results in partial redistribution of a cation-independent mannose- -phosphate receptor and a tgn recycling protein (tgn ) to early endosomes (choudhury et al., ) . this suggests that loss of ocrl perturbs endosome-to-tgn vesicle transport, suggesting a functional requirement for this membrane trayc step. it is possible that ocrl plays a role in anterograde traycking from the tgn-to-endosomes as well, since ocrl is abundantly present on tgn-associated clathrin buds destined for the endocytic pathway. ocrl disease symptoms include congenital cataracts, mental retardation, and renal tubular dysfunction (lowe et al., ) . renal failure in ocrl patients is probably partly caused by defects in solute and protein readsorption in kidney proximal tubules. this is likely due to missorting of megalin and cubilin, cell surface receptors involved in kidney solute uptake. in ocrl patients plasma membrane shedding of these receptors is reduced (norden et al., ) , indicating ocrl regulation of either receptor traycking from the tgn-to-plasma membrane or recycling from plasma membrane-to-tgn. paraneoplastic stiv-person syndrome (sps) is a neurological autoimmune disease characterized by severe muscle stivness and spasms, and often has secondary symptoms including diabetes, epilepsy, and breast cancer. autoantibodies are produced against the clathrin-associated regulator, amphiphysin i (de camilli et al., ) , a protein shown to bind dynamin in nerve terminals (david et al., ) and which is implicated in regulating the endocytosis of neuronal synaptic vesicle components (burns, ) . in support of this hypothesis, sommer et al. ( ) showed that sps-like symptoms could be triggered in rats injected with anti-amphiphysin antibodies from a human sps patient. genetic translocations leading to the formation of hybrid clathrin-accessory proteins can lead to other forms of acute myeloid leukemia, lymphoblastic leukemia and acute megakaryoblastic leukemia (dreyling et al., ; jones et al., ; narita et al., ; tebar et al., ) . in these diseases, an aberrant hybrid protein consisting of the putative transcription factor af and the clathrin accessory protein calm (clathrin assembly lymphoid myeloid leukemia protein) is formed because of a partial inversion of the af gene on chromosome (salmon-nguyen et al., ) . finally, in hermansky-pudlak syndrome (hps) type , a condition that results in partial albinism and prolonged bleeding, mutations have been found in the b a gene that encodes a subunit of the ap adaptor complex (dell'angelica et al., b) . hps is discussed in more detail in section v.c. originally identified more than years ago (palade, ; yamada, ) , caveolae are flask-shaped invaginations of approximately - nm in diameter at the plasma membrane. these plasma membrane profiles are related to lipid rafts and contain unique mixtures of gpi-anchored proteins, transmembrane proteins, signaling factors and lipids, such as cholesterol. caveolae are believed to mediate the uptake of small solutes, regulate protein traycking (hommelgaard et al., ; tagawa et al., ) , transcytosis (transport across endothelial cells) (simionescu et al., ) , signal transduction (insel et al., ; lisanti et al., ; ostrom and insel, ) and cholesterol homeostasis (fielding and fielding, ) . however, their exact role in the internalization of membrane proteins and soluble protein ligands is controversial. caveolin- , also known as vip , is a structural component essential for the formation and stability of caveolae (kurzchalia et al., ; rothberg et al., ) . of the three members of the caveolin gene family (caveolin- , - , and - ) tang et al., ) , caveolin- and - are abundant in a wide variety of cell types including endothelial cells, adipocytes, alveolar type i pneumocytes, and smooth muscle cells (williams and lisanti, ) , whereas caveolin- is a muscle-specific isoform expressed in striated muscle cells such as cardiac and skeletal myocytes (cohen et al., ; tang et al., ) . caveolin- and - are both able to induce formation of caveolae at the plasma membrane (galbiati et al., ; li et al., ) . however, caveolin- requires the presence of caveolin- for expression, membrane localization, and formation of caveolae (razani et al., ) . caveolae are absent from cells that lack caveolin- but can be induced by ectopic expression of the gene (fra et al., ) . caveolins adopt a hairpinlike structure that inserts into the membrane such that the n and c termini are cytoplasmic. caveolins can polymerize to form a striated coat surrounding an invagination site . caveolin- can bind cholesterol (murata et al., ) , which is enriched within both caveolae and lipid rafts (sargiacomo et al., ) ; this may explain why caveolae have been considered a subset of lipid rafts. however, caveolae and lipid rafts are considered to be independent entities as some proteins can be found in one but not the other (liu et al., ) . certain ligands can internalize via a lipid raftdependent but clathrin-independent mechanism in cells that lack caveolae (lamaze et al., ) . a large pool of the plasma membrane caveolar vesicles cluster into dense grape-like structures where individual caveolae appear stacked on top of each another (thomsen et al., ) . these structures are intimately associated with the actin cytoskeleton (stahlhut and van deurs, ) ; caveolaassociated proteins are also implicated in regulating plasma membrane dynamics and cellular movement. a small pool of ''transport-competent'' caveolar vesicles may undergo short-range constitutive fusion and budding cycles just under the plasma membrane . caveolae and caveolins can also be detected at the tgn (dupree et al., ; kurzchalia et al., ) and may form stable ''platforms'' for the movement of proteins and lipids from the tgn to the plasma membrane (tagawa et al., ) . the caveolar pathway can be hijacked and used by pathogens or toxins to gain entry into the cell. viruses such as polyomavirus, echovirus , and simian virus (sv ) use caveolae to internalize viral particles. these viruses cluster lipid rafts and sequester them into caveolae through interactions with raft components such as integrins and glycosphingolipids ; in the case of sv , the virus binds to the raft component ganglioside gm (tsai et al., ) . tagawa et al. ( ) have shown that sv can trigger the long-range movement of transport-competent caveolar vesicles. moreover, cell infection with sv more than doubles the number of caveolae capable of undergoing viral internalization and long-range traycking. caveolae contain much of the molecular machinery required for ''classical'' vesicle fission, docking, and fusion, for example, snare proteins, monomeric and trimeric gtpases, annexins ii and vi, n-ethylmaleimide (nem)sensitive fusion protein (nsf), and atpases (schnitzer et al., ) . caveolae also contain the dynamin gtpases, which can be transiently recruited to sv -loaded caveolae and implicated in membrane scission (henley et al., ; oh et al., ; pelkmans and helenius, ) . internalized caveoladerived vesicles move to an endocytic compartment termed the ''caveosome'' and eventually arrive at the early endosome. after fusion with the target compartment, caveolae do not disassemble but maintain their integrity in the membrane, preserving their compartmentalization and retaining their lipid and protein components (pelkmans et al., ) . the fate of internalized sv viruses after reaching the caveosome eventually results in arrival at the smooth er (pelkmans et al., ) . interestingly, mutations in caveolin have been implicated in muscular dystrophy and cardiovascular disease, and mutations causing the downregulation of caveolin have been linked to the progression of various human carcinomas; it is therefore possible that caveolins may have a tumor suppressor role. the caveolin- and caveolin- genes are located on human q . near the microsatellite repeat marker d s . this region is commonly deleted in various cancers (engelman et al., ) , hinting that caveolin gene deletion may be advantageous for tumor progression. in one report, the caveolin- p l mutation was present in % of breast cancer patients studied (hayashi et al., ) . the p l mutation was also linked to the metastatic potential of tumors and disease prognosis. the caveolin- p l mutation also conferred increased cell migration and altered morphology. caveolin- protein levels can be reduced or absent from a number of human breast cancer cell lines compared with normal mammary cells (lee et al., ) . similarly, silent and missense mutations in caveolin- have also been associated with oral carcinomas (han et al., ) . caveolin- , and to a lesser extent caveolin- , gene expression is downregulated in some cases of thyroid carcinoma (aldred et al., ) . although it remains unclear as to why the loss of caveolin causes cell proliferation diseases such as cancer, one can speculate on the role of caveolin in regulating signaling pathways. in endothelial cells, which have a high abundance of caveolin, the key vascular endothelial growth factor receptor (vegfr ) has been shown to be inactive when localized to caveolae (labrecque et al., ) . this receptor tyrosine kinase modulates the endothelial response to the key vegf-a cytokine and controls angiogenesis and new blood vessel formation, thus regulating neovascularization and tumor growth (neufeld et al., ) . similarly, platelet-derived growth factor (pdgf) receptor tyrosine kinase activity is reduced when associated with caveolae (yamamoto et al., ) . in addition to vegfr and pdgfr, a number of g protein-coupled receptors (gpcrs) have been shown to interact with caveola-associated factors (insel et al., ) . gpcrs are a large family of transmembrane receptors involved in a variety of signal transduction events. these receptors are activated by a range of ligands, including hormones and peptides, and have been linked to a number of cancers such as cell biology of membrane trafficking in human disease thyroid, lung, and gastric. the presence of a number of gpcrs in caveolae suggests that these plasma membrane structures may interact with gpcrs and modulate their signaling potential. lisanti and others (li et al., a) have shown that caveolin- interacts solely with inactive forms of g-protein a subunits, lending credence to the negative regulation hypothesis caused by the association of caveolae with transmembrane signaling receptors. a number of mutations in muscle-specific caveolin- have been associated with four distinct but related autosomal dominant muscle disease phenotypes (woodman et al., ) : limb girdle muscular dystrophy type c (minetti et al., ) , rippling muscle disease, hyperckemia (persistently elevated levels of serum creatine kinase), and distal myopathy. some mutations cause aberrant retention of caveolin- in the golgi and subsequent degradation; other mutations may cause mutant caveolin- to act in a dominant-negative manner by forming unstable aggregates with wild-type caveolin- (galbiati et al., ; sotgia et al., a,b) . hypertrophic cardiomyopathy (hcm) patients have a caveolin- t s mutation that reduces plasma membrane levels (hayashi et al., ) . caveolin gene knockout mice are providing insights into protein function in diverent human diseases. for example, lack of caveolins can cause diabetes, atherosclerosis, and cardiomyopathies in mouse models (cohen et al., ; williams and lisanti, ) . however, such phenotypes have yet to be linked to caveolin dysfunction in humans. phagocytosis is a process used by white blood cells such as macrophages, neutrophils, and dendrites to ingest large particulate material into specialized vesicles called phagosomes. these professional phagocytes are paramount in the defense against infection as they engulf and ingest whole microorganisms such as bacteria. they also use this route for ''mopping up'' apoptotic debris or senescent cells from tissues. in contrast to constitutive pinocytic transport, phagocytosis is regulated by cell surface-localized fc receptor (fcr) contact or interaction with complement-or antibody-coated particles which results in clustering of fcr on the cell surface, a step important for subsequent intracellular signaling and cellular activation (daeron, ) . polymorphisms in leukocyte-specific fcg receptors may contribute to autoimmune diseases such as guillain-barré syndrome or rheumatoid arthritis, and enhanced susceptibility to infection (van sorge et al., ) . fc-mediated binding can trigger a complex signaling response involving extrusion of fine plasma membrane projections (pseudopodia) from the macrophage to surround and engulf the pathogen, forming a phagosome. the signaling response is reviewed in greater detail elsewhere (bokoch, ; chimini and chavrier, ; niedergang and chavrier, ) . in brief, the activation of tyrosine kinases and rho gtpases is triggered through fcr signaling. the rac and cdc gtpases, in conjunction with the downstream evector wasp, mediate remodeling of the actin cytoskeleton, leading to pseudopodium formation and phagosome closure (castellano et al., ; chimini and chavrier, ) . in contrast, the complement mediated-uptake of opsonized particles divers such that they appear to ''fall'' into the cell in a process that requires rho, but not rac or cdc (bokoch, ) . phagocytosis, although designed to destroy pathogens, can paradoxically be used as a route of entry by pathogens such as mycobacterium (m. leprae and m. tuberculosis) or leishmania (nguyen and pieters, ; scott et al., ) . normally, internalized pathogens are destroyed successfully through phagosome maturation into lysosomes and subsequent degradation. mycobacterium can evade host degradation by secreting a soluble serine/threonine protein kinase g molecule into the phagosome. this molecule initiates a signaling response that interferes with phagosome-lysosome fusion, and promotes intracellular pathogen survival (walburger et al., ) . furthermore, phagosome maturation is compromised by a pathogen induced block of p map (mitogen-activated protein) kinase recruitment to the tethering molecule early endosome antigen (fratti et al., ) . the leishmania protozoan parasite, which is transmitted to humans by sand flies, produces a membrane molecule called a lipophosphoglycan, which is inserted into the lipid bilayer of the phagosome in infected macrophages. this lipophosphoglycan is thought to modulate intracellular signaling pathways, resulting in a less fusogenic phagosome and preventing maturation; this would facilitate pathogen replication and disease progression (lodge and descoteaux, ) . molecules internalized from the cell surface by receptor-mediated endocytosis and clathrin-coated vesicles are delivered to the early endosome for sorting. molecules such as low-density lipoprotein receptor (ldlr) and transferrin receptor (tfr) are eyciently recycled between the early endosome and the plasma membrane. however, after ligand-mediated activation (fig. ) , receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are sorted along the endocytic pathway for degradation. early endosomes are thought to be formed through the fusion of internalized vesicles and recruitment of specific proteins and lipids. one key protein traycking through the endosomal-lysosomal system. cell surface receptors are internalized through clathrin-coated vesicles (ccvs) at the plasma membrane. in the cell cytoplasm, ccvs shed their coat components and fuse to produce endosomes. internalized receptors are either recycled from sorting endosomes (housekeeping receptors, e.g., transferrin receptor) or targeted for degradation within the lysosome (signaling receptors, e.g., growth factor receptors) after movement through the late endosome and multivesicular body (mvb) compartments. endosomal regulator is the ubiquitously expressed rab a gtpase. rab a is present on the cytosolic face of the plasma membrane, vesicles, and tubular endosomal profiles (chavrier et al., ) . a number of rab a-associated evector proteins regulate endosomal fusion and mediate protein cargo movement and endosomal sorting (zerial and mcbride, ) . such evector proteins, including , are clustered on the cytosolic face of the early endosome and stabilize the gtp-bound rab a in an activated state (horiuchi et al., ) . gtp-bound rab a directly binds to early endosome antigen eea to regulate vesicular and endosomal tethering. eea contains a c-terminal rab a-binding domain, and a phosphatidylinositol -phosphate-binding zinc finger domain referred to as an fyve (conserved in fab , yotb, vac , and eea ) domain (gaullier et al., ; stenmark et al., ) . overexpression of wild-type rab a, or a constitutively active rab a mutant, causes endosome enlargement and defective traycking through this compartment, whereas expression of a constitutively inactive rab a mutant leads to formation of small endosomes and decreased endocytosis (bucci et al., ) . a family of evector proteins that accelerate gtpase hydrolysis (rabgaps) have been identified: rabgap- binds to rab a and regulates traycking through the endocytic pathway . the importance of rab a activity is further illustrated in the genetic disorder tuberous sclerosis (ts), a disease that causes tumors in the brain, eyes, heart, kidneys, lungs, and skin. ts arises when the tumor suppressor gene, tuberous sclerosis complex (tsc), is absent; introduction of the wild-type tsc gene into an animal model or cultured cells results in tumor suppression and reduced cellular proliferation (kobayashi et al., ; yeung et al., ) . interestingly, the tsc gene product (tuberin) is implicated in regulating gtp/gdp exchange on rab a, thus regulating traycking through this endosome system (xiao et al., ) . in chronic myelomonocytic leukemia (cmml) a genetic translocation causes fusion of rab a evector rabaptin- and the pdgfbr (magnusson et al., ) . this chromosomal translocation results in enhanced cellular proliferation by compromising endosomal fusion and traycking, and thus regulation of growth factor degradation. it is likely that this aberrant gene product is not degraded and triggers sustained intracellular signaling, leading to cell proliferation and tumor progression in a subset of lymphoid cells. recycling from endosomes back to the cell surface is often used by receptors that internalize nutrients such as lipoproteins and ions. receptor recycling rather than degradation conserves receptor functionality and nutrient uptake and reduces energy expenditure in the synthesis of new receptors (mukherjee et al., ) . genetic screens in the nematode caenorhabditis elegans identified rme- and delineated a new family of conserved class of eps homology (eh) domain proteins . both the worm and mouse homologs of rme- are associated with the endosomal compartment: a dominant-negative rme- g r mutant had little evect on receptor-mediated endocytosis but had a substantial evect on endosomal recycling, suggesting a functional role in this step . although information is currently limited, a number of neurological diseases are associated with dysfunction of early endosomal proteins. in some cases of demyelinating polyneuropathy, characterized by progressive weakening and sensory dysfunction of the legs and arms, eea autoantibodies have been detected (selak et al., ) . a number of disorders, from muscular dystrophy to rheumatoid arthritis, reveal the presence of circulating anti-eea antibodies. interestingly, eea epitopes recognized by such autoantibodies varied from patient to patient (selak et al., ) . autoantibodies against eea have also been detected in cases of subacute cutaneous systemic lupus erythematosus (scle), characterized by the appearance of an unsightly red rash, often occurring after sun exposure (mu et al., ) . lysosomes are terminal, membrane-enclosed degradative compartments that interact with other organelles through vesicular transport originating from the secretory, endocytic, and autophagic pathways. this organelle stores various proteases, lipases, hydrolases, and degradative enzymes within an acidic environment that maximizes enzymatic activity and degradation. resident lysosomal membrane proteins, integral proteins, and glycoproteins are targeted to the organelle via the endosome. lysosomal proteases such as cathepsin d are processed in the golgi apparatus to add a mannose -phosphate (m p) moiety to n-linked sugars. the m p moiety is recognized by plasma membrane or tgn-resident mannose -phosphate receptors (m prs) and sorted to the late endosome and eventually the lysosome. here, the acidic ph (ph < . ) results in receptor-ligand disassociation and recycling of the m pr to the tgn. fusion between the endosome and preexisting primary lysosomes allows the delivery of lysosomal resident proteins. the importance of m p-mediated targeting of lysosomal proteins is highlighted in the human neurological disorder, i-cell disease (mucolipidosis ii), where lysosomal enzymes are secreted from cells rather than targeted to the lysosome. the defect in i-cell disease involves lack of m p moiety addition as a result of mutations to the n-acetylglucosamine- -phosphotransferase enzyme usually present within the golgi apparatus (ben-yoseph et al., ) . how lysosomes are formed is still unclear (luzio et al., ) . three mechanisms have been proposed to explain lysosomal biogenesis: vesicular transport between late endosomes and preformed primary lysosomes (griyths and gruenberg, ), early endosomal ''maturation'' to lysosomes (murphy, ) , or the current favored model of ''kiss-and-run,'' in which transient interactions between endosomes and lysosomes transfer endosomal contents to the latter compartment (duclos et al., ; storrie and desjardins, ) . late endosome and lysosome interactions in the kiss-and-run model are thought to be regulated by the rab gtpase, which is present on late endosomes; a vps complex, homologous to budding yeast vacuole fusion regulators, is also implicated in sorting and delivery to lysosomes (seals et al., ) . the mammalian form of the vps complex interacts with syntaxin- , a t-snare that is concerned in regulating membrane dynamics along this route (kim et al., ) . danon disease is caused by point mutations in, or complete absence of lysosome-associated membrane protein (lamp ) or complete absence of this protein: changes which result in cardiomyopathy, myopathy, and mental retardation. in danon disease patients and lamp -deficient mice, autophagic vacuoles accumulate within the cytoplasm; these vacuoles arise via intracellular engulfment of old membranes to form an autophagosome, thus sequestering membranes and proteins for eventual degradation (shintani and klionsky, ) . autophagosomes fuse with lysosomes, leading to degradation for provision of molecules for cellular homeostasis. the accumulation of autophagic vacuoles in lamp -deficient cells suggests that lamp mediates interactions between autophagosomes and lysosomes. this pathway is commonly activated during conditions of cellular stress such as starvation or pathogenic infection (kirkegaard et al., ) . lysosomal storage diseases are caused through insufficient degradation of targeted components within lysosomes, leading to substrate accumulation and lysosome enlargement. more than lysosomal storage diseases have been documented and generally manifest themselves as neurological disorders; disease severity correlates with the levels of lysosomal enzyme activity. niemann-pick disease is a neurodegenerative condition caused by sphingomyelin accumulation in reticuloendothelial cells and ganglion neurons, leading to cell death. it is classified into five types (a-e), each distinguished by either clinical severity or age-related disease phenotype. niemann-pick type a (npa) is most common, with death occurring before years of age. npa patients have point mutations in the smpd gene that encodes a lysosomal sphingomyelinase (levran et al., ; takahashi et al., ) . interestingly, in npc patients, endocytosed ldl particles are not fully degraded in lysosomes, leading to defects in cholesterol metabolism (li et al., a) . the npc disease is caused by mutations in the npc gene, which encodes a lysosomal resident protein with similarity to sterol-sensing enzymes and proteins (scott and ioannou, ) . fabry disease is an x-linked condition caused by changes in lysosomal a-galactosidase activity resulting in glycosphingolipid accumulation within vascular endothelial lysosomes. this leads to angiokeratomas (a wart-like thickening of the skin), progressive renal impairment, cardiomyopathy, and cerebrovascular disease. mutations in the a-galactosidase a gene can also show reduced enzymatic activity of the encoded protein and retention within the endoplasmic reticulum (yasuda et al., ) . receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are degraded by lysosomes after ligand binding and receptor activation. egfr lysosomal targeting is dependent on ligand-stimulated ubiquitination of the cytoplasmic domain. binding of egf to egfr causes downstream signaling, clathrin-mediated internalization, and traycking to endosomes. internalized receptor-ligand complexes are sorted to the late endosome or multivesicular bodies (mvbs), which eventually deliver their contents to the lysosome (katzmann et al., ) . whereas other receptors such as tfr are recycled to the plasma membrane, egfr is moved through the endosome-lysosome system by a ubiquitin-dependent sorting and recognition system. these include the hrs/stam heterodimer and tsg (bilodeau et al., ) present on endosomal membranes. the tsg tumor suppressor gene is mutated in nearly % of breast cancer patients and encodes a membrane-associated protein (lee and feinberg, ) . this factor participates in the sorting of ubiquitinated proteins on the endosome, but its exact function is not clear. in some specialized cells, such as cytotoxic t lymphocytes (ctls), platelets, and melanocytes, regulated secretion can be routed through compartments other than the tgn. such cells have evolved mechanisms whereby modified or secretory lysosomes release their contents at the plasma membrane in response to extracellular stimuli. these secretory lysosomes (sls) share lysosomal characteristics such as acid ph and lamp (lysosome-associated membrane proteins) residents but also contain unique markers such as tyrosinase, present in melanosomes. the ctl secretory lysosomes contain unique components such as perforin and granzymes required for triggering apoptosis in target cells. on ctl contact with a target cell, sls trayc toward the immunological synapse formed between the ctl and target cell. a signal then causes sl fusion with the ctl plasma membrane (stinchcombe et al., ) , and release of sl contents and subsequent target cell death. a number of autosomal genetic diseases causing immunodeficiency and albinism involve defects in regulated lysosomal secretion (stinchcombe et al., ) . in the rare, fatal disease familial hemophagocytic lymphohistiocytosis (fhl) sls congregate at the plasma membrane in ctls, where they can dock but cannot fuse with the membrane. in one group of fhl patients, this disease is due to a mutation in the gene encoding munc - ; this is closely related to the neuronal munc - gene product that is involved in snare complex formation in neuronal cells (feldmann et al., ) . assembly of this neuronal syntaxin- , snap- , and synaptobrevin complex is regulated by munc - , which binds and locks syntaxin- (t-snare) in a closed, inactive conformation, thus preventing it from interacting with snap- (yang et al., ) . however, munc - (sassa et al., ) and rim (rab a-interacting protein evector) (koushika et al., ) can compete with munc - and displace it from syntaxin- . this reinforces the syntaxin- open conformation and allows snare complex formation to occur. munc - may act as a conformational switch to promote t-snare into an ''open'' state, thus allowing formation of the snare complex that mediates synaptic vesicle docking and fusion. from observations of fhl patients, one speculation is that munc - has a role similar to that of munc - in regulating snare complex formation for sl docking and fusion in ctls (yang et al., ) . chediak-higashi syndrome (chs) is a key example of a disease avecting sl function in ctls, with patients displaying hypopigmentation (stinchcombe et al., ) . chs patients have genetic mutations in the lyst or chs gene (barbosa et al., ; perou et al., ) and produce ctls containing strikingly enlarged sls that are able to polarize to the immunological synapse but are unable to fuse with the pm. this suggests a role for the chs gene product in regulating membrane docking and fusion . overexpression of chs leads to the presence of small lysosomes, indicating increased lysosomal fission (ward et al., ) . in addition, chs interacts with snare proteins, further indicating a role in sl fusion (tchernev et al., ) . griscelli syndrome patients also display defects in sl dynamics within ctls and exhibit hypopigmentation and silvery hair. in melanocytes, cells responsible for pigment storage and production, rab a is required to recruit melanophilin to pigment granules called melanosomes (sls). melanophilin binds the myosin motor protein myosin va and regulates melanosome movement along actin cables to the plasma membrane strom et al., ; wu et al., ) . in type griscelli syndrome patients, rab a gtpase is missing or defective, whereas in type griscelli syndrome patients the myosin va motor protein is absent. these defects are also evident in mouse models such as ashen (rab a defective), dilate (myosin va defective), and leaden (melanophilin defective). in both the human griscelli syndromes and the mouse models, melanosomes are clustered in a perinuclear location, a defect attributed to rab a dysfunction (wilson et al., ; wu et al., ) . interestingly, ctls isolated from type griscelli syndrome patients and ashen mice (rab a deficient) are unable to kill target cells, whereas type griscelli syndrome patient and dilate mouse ctls are functional. this suggests that rab a interacts with diverent evectors to induce sl fusion with the plasma membrane in diverent cell types (haddad et al., ) . hermansky-pudlak syndrome (hps) is a fourth example of sl dysfunction and is characterized by oculocutaneous albinism, ceroid deposition, and excessive prolonged bleeding (hermansky and pudlak, ; swank et al., ) . however, hps cannot be viewed as a single disease but a group of at least seven autosomal genetic disorders. each of the seven subgroups (hps - ) is due to mutations in individual genes, most of which encode components of multisubunit protein complexes involved in vesicle traycking , whereas the function of others remains unclear. three of these complexes, termed blocs (biogenesis of lysosome-related organelle complexes), play a role in regulating traycking involved in platelet and melanosome secretion, but their exact functions are unclear (di pietro and dell'angelica, ) . in hps patients a nonsense mutation in the gene encoding the b a subunit of the ap adaptor protein prevents expression of this subunit (huizing et al., ) . as previously mentioned, ap is involved in the recruitment of transmembrane proteins into vesicles at the early endosome for delivery to the lysosome (peden et al., ) . in melanocytes derived from hps patients, the tyrosinase that catalyzes the formation of melanin is not transported to maturing melanosomes (huizing et al., ) . this leads to the characteristic pattern of albinism seen in patients with this condition. furthermore, patients with hps display an impaired ctl response and immune response. ctls from hps patients have lytic granules that cannot move in an oriented fashion toward the microtubule-organizing center; therefore, when ctls are stimulated by contact with target cells, the lytic granules are not targeted to the immunological synapse for cell killing . studies on the cell biology of hiv infection have suggested the existence of a viral secretory compartment. work by marsh and others pelchen-matthews et al., ) has localized viral envelope (gp ) and matrix proteins (p ) to tetraspanin-positive endosome-related organelles in infected macrophages and dendritic cells. these viral secretory compartments move from an intracellular localization to an infectious synapse when infected macrophages or dendritic cells form an immunological synapse with activated t cells. this may be one mechanism for subsequent viral infection of cd -positive t cells, thus causing the impaired immune response seen in patients with acquired immunodeficiency syndrome (aids). cells require a highly organized framework or cytoskeleton to station and move membrane organelles within three-dimensional space. components of the cytoskeleton can guide organelles or vesicles to specific destinations within the cell. the microtubule cytoskeleton is commonly associated with the directional movement of intracellular transport vesicles or intermediates. in contrast, actin has been envisioned to have a structural role in determining cell shape, plasma membrane dynamics, and cell locomotion. however, evidence points to a role for actin in regulated traycking from the tgn (allan et al., ; badizadegan et al., ; cobbold et al., ) and endocytosis (ascough, ; engqvist-goldstein and drubin, ) . the cytoskeleton is a dynamic structure likened to a collapsible scavold that can be rapidly disassembled and reconstituted depending on cellular requirements. actin or tubulin polymerization (elongation) and depolymerization (breakdown) rely on the controlled addition or removal of monomers in a polarized and energy-dependent manner. protofilaments in either structure are both polarized, with the plus end growing at a faster rate. actin cables are each composed of two parallel protofilaments that twist around each other, whereas microtubules are composed of a hollow cylindrical structure comprising parallel protofilaments. actin nucleation is an initial step required for elongation involving formation of a stable trimer subunit base for protofilament elongation. a heptameric complex termed arp / (actin-related protein) binds to the ends and sides of actin filaments to nucleate and further accelerate the growth of the actin network (millard et al., ) . the function of the arp / complex can be regulated by membrane-associated rho gtpases. these regulators, which include cdc and various rac isoforms, act as molecular switches that cycle between an active gtp-bound state and an inactive gdp-bound state. cdc regulates arp / indirectly through its downstream target wiskott-aldrich syndrome protein (wasp), which binds directly to the arp / complex (jave and hall, ) . patients with x-linked wiskott-aldrich syndrome display mutations in the wasp gene and have thrombocytopenia (reduced platelet count), eczema, recurrent infections, hematologic malignancy, and autoimmune disorders (lemahieu et al., ) . approximately disease mutations in wasp have been reported, which lead to defective control of wasp in actin polymerization and severe disease phenotypes (burns et al., ) . wasp expression is restricted to hematopoietic cells, although the ubiquitously expressed n-wasp is present in various cells and tissues (burns et al., ) . the actin network is important for the formation of immunological synapses between cytotoxic t lymphocytes (ctls) and their targets, as well as t lymphocytes and antigen-presenting cells such as macrophages. the formation of the immunological synapse in ctls is essential for the transport, docking, and fusion of sls and subsequent destruction of the target cell as described above. defective wasp inhibits the formation of the immunological synapse and t cell activation (notarangelo and ochs, ) , probably causing the immunological deficiencies observed in wiskott-aldrich syndrome patients. wasp deficiency in t lymphocytes also avects the regulation and composition of lipid rafts (dupre et al., ) , indicating that the cell biology of membrane trafficking in human disease formation of the immunological synapse is dependent on lipid rafts, wasp, and actin dynamics. motor proteins provide the physical force to move membrane vesicles along the polymerized cytoskeletal filaments via atp-dependent hydrolysis. actinbased motor proteins belong to the myosin superfamily. the myosin va gene is mutated in a small number of patients with griscelli syndrome (bahadoran et al., ; pastural et al., ) (other traycking mutations contributing to griscelli syndrome are discussed in section v.c). mutations in the myosin viia gene can cause usher's syndrome, resulting in blindness and deafness. intracellular transport is probably compromised in usher syndrome patients; the mouse shaker model has a mutant myosin viia gene, displaying defective melanosome transport in retinal pigment epithelial cells (liu et al., ) and altered distribution in photoreceptor cells (richardson et al., ) . microtubule motor proteins, which actively move vesicles along the microtubules, and microtubule-associated proteins (maps), serve as docking molecules to bind cargo to motor proteins (gerdes and katsanis, ) . microtubule motor proteins belong to either the kinesin or dynein families. with the exception of the c-terminal kinesins, kinesin-based motors generally transport cargo toward the plus end of the microtubule, whereas the dyneins are minus end-directed motors. long-range vesicular transport is particularly important in neurons, where axons can reach up to m in length. newly synthesized lipids, and secreted or membrane-associated proteins, are made in the cell body; long-range and directional transport is crucial for replenishing the constituents of the presynaptic cleft (at the terminal end of the axon) with synaptic vesicles and plasma membrane receptors (holzbaur, ) . a number of human neurological diseases are linked to mutations in microtubule motors and associated proteins. lissencephaly, a greek term meaning ''smooth brain,'' causes severe brain malformation resulting in mental retardation and epilepsy. one of the genes mutated in the disease is lis (originally identified in miller-dieker syndrome patients with lissencephaly) (reiner et al., ) . the lis protein regulates microtubule motor function by binding dynein and p glued , a component of the dynactin complex that binds to and activates dynein (smith et al., ) . it is proposed that lis regulates retrograde axonal transport. another gene mutated in some patients with lissencephaly is doublecortin, a microtubule-associated protein that binds tubulin and stabilizes microtubules (horesh et al., ; moores et al., ) . the kif b kinesin regulates transport of synaptic vesicle precursors along the neuronal axon. patients with charcot-marie-tooth disease type a display neuronal axonal degeneration due to a loss-of-function mutation in the motor domain of kif b . in alzheimer's disease, a classical sign is hyperphosphorylated aggregates of the microtubule-associated protein, tau, in neuronal cells and tissues. tau protein can influence vesicular transport (ebneth et al., ) by regulating the attachment of motors to microtubules (trinczek et al., ) . one theory is that the tau protein can interfere with kinesin-dependent transport by blocking motor access to microtubules, thus slowing or preventing vesicle movement along axons (mandelkow et al., ) . moreover, an early sign of alzheimer's disease is the loss of synapses and retrograde degeneration of neurons, complemented by a breakdown in intracellular transport. the disruption of microtubule-mediated vesicular traycking may also be a causative factor of the neurodegenerative phenotype of huntington's disease. this disease is caused by expansion of polyglutamine repeats occurring in the brain-enriched protein huntingtin (htt). it has been demonstrated that htt enhances vesicular transport of brain-derived neurotrophic factor (bdnf) along microtubules (gauthier et al., ) . htt is localized in the cytoplasm and is associated with vesicular and microtubule-based trayc through its ability to bind huntingtin-associated protein (hap ) (li et al., b) , a protein that has aynity for the dynactin p glued subunit (engelender et al., ) . dysfunctional polyq-htt associated with the disease state may disrupt the transport of bdnf by binding and blocking the hap /dynactinmediated delivery of bdnf vesicles along microtubules (gauthier et al., ) . this is further supported by the finding that bdnf levels are decreased in brains of huntington's disease patients (ferrer et al., ) . the current treatment of genetic disorders involves addressing the symptoms rather than the cause. to that end, many mild forms of disorders such as niemann-pick disease, and familial hypercholesterolemia, can be controlled by diet regimens and lifestyle changes. in contrast, a life-threatening disease such as cystic fibrosis requires extensive physiotherapy and pulmonary exercise to loosen and prevent mucus accumulation within the lungs. new antidementia drugs are increasingly successful in treating neurological disorders such as alzheimer's disease. drugs such as galantamine, donepezil, rivastigmine, and memantine target the posttranslational processing of bapp to reduce amyloid deposits (prasher, ) . in familial hypercholesterolemia, statin treatment is a common strategy for reducing plasma ldl and cholesterol levels by targeting hmg-coa reductase, the rate-limiting enzyme in cellular cholesterol biosynthesis. furthermore, less commonly used ldl-lowering drugs such as probucol have shown some success in (buckley et al., ) . the administration of adrenalin receptor antagonists (b-blockers) to patients with the cardiac condition long-qt syndrome reduces arrhythmia risk. enzyme replacement therapy (ert) has been carried out in patients with fabry's disease, a lysosomal storage disease. patients are given recombinant lysosomal a-galactosidase (mignani and cagnoli, ) to reduce the risks of strokes and kidney failure associated with the condition. finally, organ transplantation is occasionally carried out for some disease states: for example, bone marrow transplants for chediak-higashi syndrome patients (liang et al., ) and rab a-defective patients with griscelli syndrome (schuster et al., ) and wiskott-aldrich syndrome (filipovich et al., ) . however, although transplant operations can be successful in alleviating the immunological issues associated with these diseases, it does not address problems associated with the nervous system or pigmentation. b. gene therapy: the next generation of medical treatment? completion of the human genome sequencing project has given science the ability to track gene(s) responsible for potentially any genetic disorder and, as a consequence, to allow these genes to be corrected in patients. this is the goal of gene therapy research. of course, gene therapy has a fundamental limitation: it is only really suitable for single-gene defect diseases, and multigenic or chromosomal defects will be beyond the ability of the technique because of the complex nature of the disease. however, there are more than single gene defects that cause human disease, so there are many diseases requiring such approaches. the history of gene therapy is discussed in more detail by russell ( ) and scollay ( ) . much evort has been made in developing techniques that allow successful replacement or augmentation of defective genes. gene therapy is performed by introducing a gene vehicle directly into the patient (in vivo) or by removing cells from a patient, introducing the gene into these cells in culture, and replacing the cells back in the patient (ex vivo). most studies have focused on the use of viral vectors as delivery vehicles. retroviruses are potentially the best gene delivery system (kurian et al., ) . these rna viruses are able to infect a great many cell types and replicate by inserting their viral genes into the genome of the host. the host cellular machinery is then modulated to produce and assemble viral particles. in gene therapy, retroviruses could be used to express the target gene to be replaced but be modified to prevent viral disease (hiv, which is the causative agent of aids, is also a retrovirus). the principal drawback of a retrovirus vector is the possibility that genomic integration could elevate oncogene expression, thus causing cancer. therefore, the majority of clinical trials using retrovirus vectors have been performed ex vivo. a ''successful'' gene therapy experiment was exemplified in the case of a -year-old female patient lacking adenosine deaminase (ada), which results in severely compromised immunodeficiency (ada-scid) and dysfunctional t cells (blaese et al., ) . in this case, a retroviral vector was used to deliver the coding sequence for ada into cells, resulting in successful expression of this enzyme in hitherto defective cells. although successful, it is uncertain whether enzyme replacement treatment (recombinant ada injections) also influenced the patient outcome. adenovirus (adv) (mcconnell and imperiale, ) is a dna virus and key gene therapy vehicle that maintains the viral genome as a separate transmissible episome within the nuclei of infected host cells. the use of attenuated or inactivated adv for human gene delivery has attracted much interest. the advantages of adv gene transfer are that its genome can easily be manipulated and recombinant virus can be grown to high titers in vitro with eycient transduction of target cells in vitro or in vivo. as adv can evectively infect nondividing cells such as lung pulmonary tissues it is a popular vector of choice for gene therapy to treat cystic fibrosis (cf) patients. although there are promising studies (zabner et al., ) , failures have also been noted (knowles et al., ) . a major disadvantage of an adv-based approach is the triggering of a strong host immune response to the virus, which becomes a serious problem in subsequent long-term delivery of recombinant virus for disease alleviation. one approach to circumventing such an issue is to use a viral delivery system that produces a low host immune response such as the adeno-associated virus (aav) (flotte, ) . aav is a nonpathogenic virus that requires coinfection with a helper virus to replicate. however, aav has broad host cell specificity and is diycult to grow in large quantities, probably because of its reliance on a helper virus. finally, nonviral methods are increasingly available for the delivery of dna constructs directly into cells and tissues. these are often lipid-based reagents (e.g., liposomes) that bind to the plasmid dna and fuse with the plasma membrane, thus enabling cytosolic delivery of the gene. the plasmid dna would then be transported into the host nucleus by endogenous cellular machinery. this type of gene delivery can only be performed ex vivo and can be limited by the poor dna transfection eyciency of primary cells or tissues. this type of method, however, is a potentially useful method for delivering genes into progenitors or precursors (e.g., stem cells) before cellular cell biology of membrane trafficking in human disease diverentiation and tissue formation within a particular microenvironment in the body (mendell et al., ) . numerous disease states are caused by protein misfolding within the er, leading to degradat ion (table i ; section s iii. a. an d iii.a. ). one strategy would be to promote the correct protein conformation in a mutant gene product either chemically or pharmacologically. a number of membrane-divusible chemical and pharmacological ''chaperones'' have been identified that could avect protein folding in cells. chemical chaperones such as glycerol and trimethylamine n-oxide (tmao) can restore the wild-type traycking and activity of cftrÁf in cultured epithelial cells (brown et al., ) , and porcine kidney epithelial cells expressing cftrÁf and treated with dimethyl sulfoxide (dmso) increased plasma membrane levels of the channel protein (bebok et al., ) . loo et al. ( ) have demonstrated that a novel quinazoline derivative specific for cftr will rescue the defective traycking of cftrÁf in cultured cells. cell surface levels of a water channel, aquaporin- , can be enhanced with dmso (tamarappoo and verkman, ) . defects in this gene can result in x-linked nephrogenic diabetes insipidus, a condition in which patients are unable to concentrate their urine because of an inability to reabsorb water from the kidneys into the blood. although chemical chaperones are somewhat nonspecific in their action (the protein folding of the whole cell is avected and not just the target protein), pharmacological chaperones can be tailored to individual proteins. for example, the compound sr a is a nonpeptide vasopressin v receptor antagonist (morello et al., ; robert et al., ) . patients with a mutant vasopressin v receptor can also display nephrogenic diabetes insipidus. on treatment, the cell-permeant sr a compound would act as a chaperone and accompany the mutant v receptor to the cell surface to rescue correct functionality. geldanamycin, a naturally occurring antifungal agent, has potential as an anticancer drug (beliakov and whitesell, ; miyata, ) . geldanamycin interacts with and inhibits activity of the heat shock protein and chaperone hsp , a cytosolic cellular stress protein that supports the correct folding, stability, and function of ''client'' proteins. many hsp client proteins are implicated in cell cycle progression, proliferation, and angiogenesis (whitesell and lindquist, ) . the erbb tyrosine kinase complex is implicated in regulation and development of epithelial breast tumors and is an hsp client. inhibition of hsp action by geldanamycin results in degradation of both erbb and downstream signaling evectors, resulting in reduced cellular growth and tumor formation (citri et al., ) . a number of cell-permeable peptide sequences found in viruses and host proteins have been discovered that mediate the delivery of cargo (proteins, drugs, plasmid dna, oligonucleotides) directly into cells (brooks et al., ; gupta et al., ; schwartz and zhang, ) . such peptide sequences could be fused or attached to recombinant or engineered proteins and administered to patients to complement defects of a particular gene product. for example, the drosophila melanogaster antennapedia homeodomain (antp) transcription factor contains a short -residue sequence that mediates protein translocation across biological membrane bilayers in an energy-independent manner (derossi et al., ; joliot et al., ) . other sources of cell membrane-permeable proteins have been uncovered in viruses. the hiv- replication protein tat contains a basic, arginine-and lysine-rich peptide sequence (residues - ) that modulates the translocation of exogenous tat across the plasma membrane in a number of cell types, and is able to activate intracellular genes controlled by an hiv promoter (frankel and pabo, ; mann and frankel, ) . this basic -residue sequence can internalize conjugated b-galactosidase and horseradish peroxidase (fawell et al., ) as well as a fab antibody fragment (anderson et al., ) . the major structural protein of herpesvirus (hsv- ), vp , can trayc between cells (elliott and o'hare, ) , whereas the pres- domain of hepatitis b virus surface antigen acts as a shuttle for peptides and functional proteins (such as egfp) in hepatocytes and other cells (oess and hildt, ) , suggesting further the existence of naturally occurring peptide sequences that may act as drug delivery vectors. finally, a ''synthetic'' amphipathic peptide, fluos-klalklalkalkaalkla-nh , has been shown to be internalized in mast and endothelial cells (oehlke et al., ) . the employment of small molecular inhibitors as a method of treating human disease has moved at exponential pace. a number of compounds have been synthesized or isolated from nonhuman organisms that directly avect cellular function and have been used in research on a variety of diseases. plant-and microorganism-derived polyhydroxylated alkaloids referred to as iminosugars have been used in the treatment of patients with gaucher disease (cox et al., ) . gaucher disease type i and type ii is a lysosomal storage disorder caused by a mutation in the gene encoding the cell biology of membrane trafficking in human disease acid b-glucosidase (gba) enzyme and results in the accumulation of toxic glucosylceramide in a patient's spleen, liver, and bones; manifesting itself in enlargement of these organs, as well as heart and lung disease. iminosugars act on glycosylating enzymes present within the er and golgi and inhibit their ability to transfer sugar moieties onto proteins. one member of the iminosugar family, n-butyldeoxynojirimycin (nb-dnj; also called miglustat or zavesca) inhibits the enzyme important in the maturation of the gba substrate glucocerebroside, namely ceramide glucosyltransferase (cgt) (butters et al., ) . inhibition of cgt has resulted in significantly reduced levels of glucocerebroside in the liver and spleen of patient in clinical trials (cox et al., ) . however, nearly % of patients in the trials displayed osmotic diarrhea as a side evect of the treatment. in a mouse model for human tay-sachs disease, which is caused by a mutation in the gene encoding hexosamidase a, levels of the harmful glycophospholipid gm were significantly reduced on treatment with nb-dnj (platt et al., ) . in addition to the treatment of lysosomal storage diseases, an nb-dnj derivative called miglitol has been used in the treatment of diabetes mellitus, resulting in reduced activity of the sucrose-isomaltase enzyme complex and reduction of carbohydrate digestion (mitrakou et al., ) . a major aspect of human disease is the production and subsequent degradation of misfolded proteins, either by the proteasome or within the lysosome. lysosomotropic agents such as chloroquine cause an increase in the intralumenal ph of endosomes and lysosomes, reducing lysosomal protease activities and the traycking of proteins through the endosome-lysosome system. a number of proteasome inhibitors such as mg , lactacystin, and alln can specifically inhibit the activity of a range of serine and cysteine proteases and chymotrypsin-like enzymes (kisselev and goldberg, ) . proteasome inhibition has been linked with a number of aspects of human disease. treatment of endothelial cells with proteasome inhibitors resulted in apoptosis of proliferating cells (drexler et al., ) and inhibition of plasminogen activator levels; this factor promotes angiogenesis and new blood vessel sprouting (oikawa et al., ) . however, inhibiting proteasome function has broad cytotoxic and apoptotic evects in cells and tissues. chemotherapeutic agents targeting signaling pathways are currently of much interest in relation to cancer therapeutics. cellular proliferation can be regulated by growth factor binding to a cell surface receptor and signaling through either the mitogen-activated protein kinase (mapk) or phosphoinositide- -kinase (pi k) cascades. activation of these pathways induces the expression of oncogenes such as c-jun and c-fos and inhibits apoptosis through a sequence of protein phosphorylation events. shelton et al. ( ) demonstrated that inhibition of the mapk pathway with small molecule inhibitors specific for raf- or mek reduced cell proliferation and induced apoptosis in conditionally transformed hematopoietic cells. however, such pathways also regulate other cellular functions besides proliferation or apoptosis and there are likely to be consequences for cellular homeostasis. structural studies are important in the development of new small molecule inhibitors that target specific enzymes and regulators. c-akt (pkb) is a serine/ threonine protein kinase required for survival and proliferation in many human cancers and its structure has been elucidated (kumar and madison, ; yang et al ., ; and refer ences therei n). chem otherape utic agents have been consequently designed that inhibit c-akt activity; molecules such as h- target the atp-binding pocket in c-akt (kumar and madison, ) . compounds that bind specifically to c-akt isoforms or target specific domains within the kinase have been reported (barnett et al., ) , but there are no reports of clinical trials with such compounds (kumar and madison, ) . finally, small molecule inhibitors are being developed to target the posttranslational processing of proteins or peptides implicated in human disease. the enzyme that catalyzes the initial steps in b-amyloid synthesis, g-secretase, is an attractive target for prevention of amyloid deposits in alzheimer's disease patients (churcher and beher, ) . such small molecule inhibitors could also be used to treat pathogenic infections such as those caused by severe acute respiratory syndrome (sars), influenza, hiv, or hepatitis c viruses. attractive targets are virus-encoded or host proteases required for processing of viral proteins to generate infectious virus particles from the host cell. in the case of the sars virus, a viral chymotrypsin-like cysteine protease is responsible for processing sars viral proteins required for viral replication. inhibition of this protease would evectively inhibit viral replication. a molecule referred to as cs was found to inhibit the replication of human sars with no toxic evect on normal cells (dooley et al., ) . much work is also being carried out in targeting host proteases required for the processing of hiv envelope glycoproteins by the biosynthetic secretory pathway to generate viral gp and gp polypeptides. the completion of the human genome sequencing project has led to the prediction that a large number of diseases will be identified and understood at the gene level . as noted in this review, a number of examples exist in which a single gene mutation can have devastating evects on human function. at present, the symptoms of some mild forms of genetic diseases can be modulated through diet or drug regimens, and some success has been achieved with organ transplantation. gene therapy has attracted much attention but has suvered setbacks due to viral toxicity issues. an alternative strategy is the use of small molecule therapeutics, which cell biology of membrane trafficking in human disease may override specific defects or target specific pathways to compensate for gene defect(s). in addition, our understanding of how we respond at a genetic level to pathological infection will enable us to design evective drug strategies to presently chronic infections. in essence, understanding the cell biological basis for human diseases will enable us to design evective methods to deliver therapeutic strategies to patients. caveolin- and caveolin- , together with three bone morphogenetic protein-related genes, may encode novel tumor suppressors down-regulated in sporadic follicular thyroid carcinogenesis endocytosis of the glucose transporter glut is mediated by the gtpase dynamin motoring around the golgi the p -interactive proteins gm and giantin participate in endoplasmic reticulum-golgi trayc cftr and chaperones: processing and degradation tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide endocytosis: actin in the driving seat traycking of cholera toxin-ganglioside gm complex into golgi and induction of toxicity depend on actin cytoskeleton comment on elejalde syndrome and relationship with griscelli syndrome identification of the homologous beige and chediak-higashi syndrome genes coupled er to golgi transport reconstituted with purified cytosolic proteins the akt/pkb family of protein kinases: a review of small molecule inhibitors and progress towards target validation the adaptor protein ap- as a component of the clathrin coat machinery: a morphological study role of diacylglycerol in pkd recruitment to the tgn and protein transport to the plasma membrane loss of function associated with novel mutations of the scn a gene in patients with brugada syndrome disassembly and reassembly of the golgi apparatus activation of deltaf cftr in an epithelial monolayer hsp : an emerging target for breast cancer therapy snares and the specificity of transport vesicle targeting mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase altered molecular size of n-acetylglucosamine -phosphotransferase in i-cell disease and pseudo-hurler polydystrophy congenital and acquired neutropenia the role of regulated cftr traycking in epithelial secretion phosphatidic acid formation by phospholipase d is required for transport from the endoplasmic reticulum to the golgi complex degradation of subunits of the sec p complex, an integral component of the er membrane, by the ubiquitin-proteasome pathway role of cue p in ubiquitination and degradation at the er surface the vps p hse p complex binds ubiquitin and mediates endosomal protein sorting t lymphocyte-directed gene therapy for ada-scid: initial trial results after years regulation of innate immunity by rho gtpases signals for sorting of transmembrane proteins to endosomes and lysosomes caveolin- in breast cancer tat peptide-mediated cellular delivery: back to basics chemical chaperones correct the mutant phenotype of the delta f cystic fibrosis transmembrane conductance regulator protein the small gtpase rab functions as a regulatory factor in the early endocytic pathway probucol. a reappraisal of its pharmacological properties and therapeutic use in hypercholesterolaemia the wilson disease gene is a putative copper transporting p-type atpase similar to the menkes gene coiled coils: a highly versatile protein folding motif mechanisms of waspmediated hematologic and immunologic disease a step forward for stiv-person syndrome small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders hepatic endoplasmic reticulum storage diseases initial docking of er-derived vesicles requires uso p and ypt p but is independent of snare proteins a role for cbs domain in traycking of chloride channel clc- a ''de novo'' point mutation of the low-density lipoprotein receptor gene in an italian subject with primary hypercholesterolemia actin dynamics during phagocytosis adpkd: a human disease altering golgi function and basolateral exocytosis in renal epithelia localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments multiple forms of dynamin are encoded by shibire, a drosophila gene involved in endocytosis function of rho family proteins in actin dynamics during phagocytosis and engulfment lowe syndrome protein ocrl interacts with clathrin and regulates protein traycking between endosomes and the trans-golgi network gamma-secretase as a therapeutic target for the treatment of alzheimer's disease the achilles heel of erbb- /her : regulation by the hsp chaperone machine and potential for pharmacological intervention lytic granules, secretory lysosomes and disease adaptor protein -dependent microtubule-mediated movement of lytic granules to the immunological synapse novel membrane trayc steps regulate the exocytosis of the menkes disease atpase the menkes disease atpase (atp a) is internalized via a rac -regulated, clathrin-and caveolae-independent pathway actin and microtubule regulation of trans-golgi network architecture, and copper-dependent protein transport to the cell surface role of caveolae and caveolins in health and disease the human genome project: lessons from large-scale biology rab and arf gtpase regulation of exocytosis novel oral treatment of gaucher's disease with n-butyldeoxynojirimycin (ogt ) to decrease substrate biosynthesis copper transporting p-type atpases and human disease defective intracellular transport and processing of oa is a major cause of ocular albinism type involvement of clathrin-mediated endocytosis in human immunodeficiency virus type entry fc receptor biology the molecular basis of alanine: glyoxylate aminotransferase mistargeting: the most common single cause of primary hyperoxaluria type a role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals the synaptic vesicle-associated protein amphiphysin is the -kd autoantigen of stiv-man syndrome with breast cancer a novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation the low lysine content of ricin a chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol low-density lipoprotein receptor-its structure, function, and mutations association of the ap- adaptor complex with clathrin ap- , a novel protein complex related to clathrin adaptors altered traycking of lysosomal proteins in hermansky-pudlak syndrome due to mutations in the beta a subunit of the ap- adaptor rhodopsin c terminus, the site of mutations causing retinal disease, regulates traycking by binding to adp-ribosylation factor (arf ) the third helix of the antennapedia homeodomain translocates through biological membranes rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces rab prenylation and platelet synthesis ubiquitination is required for the retrotranslocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome mutations in the mdr gene cause progressive familial intrahepatic cholestasis the coiled-coil membrane protein golgin- is a novel rab evector required for golgi ribbon formation the cell biology of hermansky-pudlak syndrome: recent advances from genome to drug lead: identification of a small-molecule inhibitor of the sars virus cooperation of ggas and ap- in packaging mprs at the trans-golgi network inhibition of proteasome function induces programmed cell death in proliferating endothelial cells the t( ; )(p ;q ) in the u cell line results in the fusion of the af gene and calm, encoding a new member of the ap- clathrin assembly protein family remodeling of endosomes during lysosome biogenesis involves ''kiss and run'' fusion events regulated by rab er-to-golgi transport: cop i and cop ii function (review) hyperinsulinism in infancy: from basic science to clinical disease wiskott-aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation caveolae and sorting in the trans-golgi network of epithelial cells overexpression of tau protein inhibits kinesin-dependent traycking of vesicles, mitochondria, and endoplasmic reticulum: implications for alzheimer's disease a novel gene for autosomal dominant stargardt-like macular dystrophy with homology to the sur protein family intercellular traycking and protein delivery by a herpesvirus structural protein cisternal maturation and vesicle transport: join the band wagon! (review) huntingtin-associated protein (hap ) interacts with the p glued subunit of dynactin genes encoding human caveolin- and - are co-localized to the d s locus ( q . ), a known fragile site (fra g) that is frequently deleted in human cancers actin assembly and endocytosis: from yeast to mammals accelerated transport and maturation of lysosomal alpha-galactosidase a in fabry lymphoblasts by an enzyme inhibitor lowe syndrome protein ocrl interacts with rac gtpase in the trans-golgi network tat-mediated delivery of heterologous proteins into cells munc - is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (fhl ) brain-derived neurotrophic factor in huntington disease caveolae and intracellular traycking of cholesterol impact of donor type on outcome of bone marrow transplantation for wiskott-aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program adeno-associated virus-mediated gene transfer for lung diseases the ap- a and ap- b clathrin adaptor complexes define biochemically and functionally distinct membrane domains the endoplasmic reticulum as a site of protein degradation de novo formation of caveolae in lymphocytes by expression of vip -caveolin identification of a di-leucine motif within the c terminus domain of the menkes disease protein that mediates endocytosis from the plasma membrane cellular uptake of the tat protein from human immunodeficiency virus induction of p mitogen-activated protein kinase reduces early endosome autoantigen (eea ) recruitment to phagosomal membranes caveolin- null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities phenotypic behavior of caveolin- mutations that cause autosomal dominant limb girdle muscular dystrophy (lgmd- c). retention of lgmd- c caveolin- mutants within the golgi complex hiv- traycking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse structural basis of fabry disease fyve fingers bind ptdins huntingtin controls neurotrophic support and survival of neurons by enhancing bdnf vesicular transport along microtubules microtubule transport defects in neurological and ciliary disease wilson disease the alpha chain of the ap- adaptor is a clathrin binding subunit intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein disrupted proteolipid protein traycking results in oligodendrocyte apoptosis in an animal model of pelizaeus-merzbacher disease evidence that rme- , a conserved c. elegans eh-domain protein, functions in endocytic recycling the arguments for pre-existing early and late endosomes intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides a gtpase-activating protein controls rab function in endocytic traycking defective granule exocytosis in rab a-deficient lymphocytes from ashen mice synaptojanin : localization on coated endocytic intermediates in nerve terminals and interaction of its kda isoform with eps mutation and aberrant expression of caveolin- in human oral squamous cell carcinomas and oral cancer cell lines traycking, turnover and membrane topology of prp charcot-marie-tooth neuropathy type b is associated with mutations of the myelin p gene invasion activating caveolin- mutation in human scirrhous breast cancers identification and functional analysis of a caveolin- mutation associated with familial hypertrophic cardiomyopathy accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells the delta f mutation shortens the biochemical half-life of plasma membrane cftr in polarized epithelial cells dynaminmediated internalization of caveolae albinism associated with hemorrhagic diathesis and unusual pigmented reticular cells in the bone marrow: report of two cases with histochemical studies evects of mutant rat dynamin on endocytosis characterization of a fourth adaptor-related protein complex missense mutation (c r) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport a novel leukocyte adhesion deficiency caused by expressed but nonfunctional beta integrins mac- and lfa- caveolae: stable membrane domains with a potential for internalization doublecortin, a stabilizer of microtubules a novel rab gdp/gtp exchange factor complexed to rabaptin- links nucleotide exchange to evector recruitment and function hereditary neutropenia: dogs explain human neutrophil elastase mutations ap- mediates tyrosinase but not trp- traycking in human melanocytes nonsense mutations in adtb a cause complete deficiency of the {beta} a subunit of adaptor complex- and severe hermansky-pudlak syndrome type the leaden gene product is required with rab a to recruit myosin va to melanosomes in melanocytes caveolae and lipid rafts: g protein-coupled receptor signaling microdomains in cardiac myocytes rho gtpases: biochemistry and biology a common w s mutation in the ldl receptor gene of danish patients with familial hypercholesterolemia encodes a transport-defective protein antennapedia homeobox peptide regulates neural morphogenesis identification and molecular characterisation of a calm-af fusion in acute megakaryoblastic leukaemia mutations in a sar gtpase of copii vesicles are associated with lipid absorption disorders substitution of arginine for histidine at position in the alpha-subunit of the human insulin receptor. a mutation that impairs receptor dimerization and transport of receptors to the cell surface distinct sets of sec genes govern transport vesicle formation and fusion early in the secretory pathway metabolic and molecular bases of menkes disease and occipital horn syndrome receptor downregulation and multivesicular-body sorting stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls positionally cloned gene for a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome endocrinopathies in the family of endoplasmic reticulum (er) storage diseases: disorders of protein traycking and the role of er molecular chaperones a conditional mutation avecting localization of the menkes disease copper atpase. suppression by copper supplementation molecular characterization of mammalian homologues of class c vps proteins that interact with syntaxin- three ways to make a vesicle cellular autophagy: surrender, avoidance and subversion by microorganisms proteasome inhibitors: from research tools to drug candidates a cholesterol-lowering gene maps to chromosome q a controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis a germline insertion in the tuberous sclerosis (tsc ) gene gives rise to the eker rat model of dominantly inherited cancer disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in drosophila a post-docking role for active zone protein rim hiv interaction with endosomes in macrophages and dendritic cells organization of the pronephric filtration apparatus in zebrafish requires nephrin, podocin and the ferm domain protein mosaic eyes cdc controls secretory and endocytic transport to the basolateral plasma membrane of mdck cells copii-cargo interactions direct protein sorting into er-derived transport vesicles akt crystal structure and akt-specific inhibitors defective human ether-a-go-go-related gene traycking linked to an endoplasmic reticulum retention signal in the c terminus retroviral vectors vip , a -kd membrane protein is an integral component of trans-golginetwork-derived transport vesicles regulation of vascular endothelial growth factor receptor- activity by caveolin- and plasma membrane cholesterol intracellular localization and loss of copper responsiveness of mnk, the murine homologue of the menkes protein, in cells from blotchy (mo blo) and brindled (mo br) mouse mutants interleukin receptors and detergent-resistant membrane domains define a clathrinindependent endocytic pathway aberrant splicing but not mutations of tsg in human breast cancer tumor cell growth inhibition by caveolin re-expression in human breast cancer cells bi-directional protein transport between the er and golgi a missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in sephardic jews novel mutations in the wiskott-aldrich syndrome protein gene and their evects on transcriptional, translational, and clinical phenotypes niemann-pick disease: a frequent missense mutation in the acid sphingomyelinase gene of ashkenazi jewish type a and b patients evidence for a regulated interaction between heterotrimeric g proteins and caveolin a huntingtin-associated protein enriched in brain with implications for pathology expression and characterization of recombinant caveolin hermansky-pudlak syndrome type (hps- ) results from mutant dysbindin molecular, anatomical, and biochemical events associated with neurodegeneration in mice with niemann-pick type c disease the listeria protein internalin b mimics hepatocyte growth factor-induced receptor traycking bone marrow transplantation from an hla-matched unrelated donor for treatment of chediak-higashi syndrome familial sjogren's syndrome with associated primary salivary gland lymphoma protein kinase d regulates the fission of cell surface destined transport carriers from the trans-golgi network rme- regulates the distribution and function of the endocytic recycling compartment in mammalian cells caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains mutant myosin viia causes defective melanosome distribution in the rpe of shaker- mice rescue of deltaf and other misprocessed cftr mutants by a novel quinazoline compound organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation; a clinical entity structure and function of the lowe syndrome protein ocrl autoantigen golgin- , an evector of arl gtpase, participates in trayc from the endosome to the trans-golgi network functional evaluation of dent's disease-causing mutations: implications for clc- channel traycking and internalization regulation of protein transport from the golgi complex to the endoplasmic reticulum by cdc and n-wasp the beta-appendages of the four adaptor-protein (ap) complexes: structure and binding properties, and identification of sorting nexin as an accessory protein to ap- function and regulation of the mammalian coppertransporting atpases: insights from biochemical and cell biological approaches membrane dynamics and the biogenesis of lysosomes rabaptin- is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia kdel-cargo regulates interactions between proteins involved in copi vesicle trayc: measurements in living cells using fret purification of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack clogging of axons by tau, inhibition of axonal trayc and starvation of synapses endocytosis and targeting of exogenous hiv- tat protein a novel point mutation in cd causing the expression of dysfunctional cd /cd leucocyte integrins in a patient with leucocyte adhesion deficiency (lad) copii-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes altered traycking and adhesion function of mpz mutations and phenotypes of charcot-marie-tooth disease b biology of adenovirus and its use as a vector for gene therapy the putative tumor suppressors ext and ext form a stable complex that accumulates in the golgi apparatus and catalyzes the synthesis of heparan sulfate congenital hypothyroid goiter with deficient thyroglobulin. identification of an endoplasmic reticulum storage disease with induction of molecular chaperones mutations in rab a cause griscelli syndrome associated with haemophagocytic syndrome myoblast transfer in the treatment of duchenne's muscular dystrophy enzyme replacement therapy in fabry's disease: recent advances and clinical applications signalling to actin assembly via the wasp (wiskott-aldrich syndrome protein)-family proteins and the arp / complex mutations in the caveolin- gene cause autosomal dominant limb-girdle muscular dystrophy long-term evectiveness of a new alpha-glucosidase inhibitor (bay m -miglitol) in insulin-treated type diabetes mellitus genetic disorders avecting proteins of iron and copper metabolism: clinical implications hsp inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents mechanism of microtubule stabilization by doublecortin identification and functional analysis of two novel mutations in the multidrug resistance protein gene in israeli patients with dubin-johnson syndrome pharmacological chaperones: a new twist on receptor folding a conserved clathrin assembly motif essential for synaptic vesicle endocytosis eea , an early endosome-associated protein. eea is a conserved alpha-helical peripheral membrane protein flanked by cysteine ''fingers'' and contains a calmodulin-binding iq motif localization of proteins to the golgi apparatus vip /caveolin is a cholesterol-binding protein maturation models for endosome and lysosome biogenesis cdc regulates the exit of apical and basolateral proteins from the trans-golgi network consistent detection of calm-af chimaeric transcripts in haematological malignancies with t( ; )(p ;q ) and identification of novel transcripts molecular analysis of the ergic- gene in families with combined factor v-factor viii deficiency vascular endothelial growth factor (vegf) and its receptors the trojan horse: survival tactics of pathogenic mycobacteria in macrophages snares and membrane fusion in the golgi apparatus mutations in the er-golgi intermediate compartment protein ergic- cause combined deficiency of coagulation factors v and viii regulation of phagocytosis by rho gtpases urinary megalin deficiency implicates abnormal tubular endocytic function in fanconi syndrome wiskott-aldrich syndrome: a model for defective actin reorganization, cell traycking and synapse formation identification of complementation groups required for post-translational events in the yeast secretory pathway cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically novel cell permeable motif derived from the pres -domain of hepatitis-b virus surface antigens dynamin at the neck of caveolae mediates their budding to form transport vesicles by gtp-driven fission from the plasma membrane of endothelium the proteasome is involved in angiogenesis role of rab gtpases in membrane trayc the evolving role of lipid rafts and caveolae in g proteincoupled receptor signaling: implications for molecular pharmacology the structure and function of the beta -adaptin appendage domain phosphatidylinositol phosphate -kinase i{beta} recruits ap- to the plasma membrane and regulates rates of constitutive endocytosis fine structure of blood capillaries intracellular aspects of the process of protein synthesis two genes are responsible for griscelli syndrome at the same q locus localization and processing of cln , the protein associated to batten disease: where is it and what does it do? localization of the ap- adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins infectious hiv- assembles in late endosomes in primary macrophages snares and the specificity of transport vesicle targeting caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane trayc alpha -antitrypsin deficiency: liver disease associated with retention of a mutant secretory glycoprotein in the endoplasmic reticulum alpha- -antitrypsin deficiency: diagnosis and treatment identification of the murine beige gene by yac complementation and positional cloning the menkes protein (atp a; mnk) cycles via the plasma membrane both in basal and elevated extracellular copper using a c-terminal dileucine endocytic signal participation of the endoplasmic reticulum chaperone calnexin (p , ip ) in the biogenesis of the cystic fibrosis transmembrane conductance regulator prevention of lysosomal storage in tay-sachs mice treated with n-butyldeoxynojirimycin correction of a mineralization defect by overexpression of a wild-type cdna for col a in marrow stromal cells (mscs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects constitutive protein secretion from the trans-golgi network to the plasma membrane review of donepezil, rivastigmine, galantamine and memantine for the treatment of dementia in alzheimer's disease in adults with down syndrome: implications for the intellectual disability population protein kinase d-mediated anterograde membrane traycking is required for fibroblast motility gene replacement reveals that p /snare interactions are essential for golgi biogenesis constitutive skipping of alternatively spliced exon in the atp a gene abolishes golgi localization of the menkes protein and produces the occipital horn syndrome traycking and folding defects in hereditary spherocytosis mutants of the human red cell anion exchanger caveolin- -deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae isolation of a miller-dicker lissencephaly gene containing g protein [beta]-subunit-like repeats myosin viia is required for aminoglycoside accumulation in cochlear hair cells endophilin/sh p is required for the transition from early to late stages in clathrinmediated synaptic vesicle endocytosis mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin v b/v receptor by a pharmacological chaperone adaptor-related proteins adaptable adaptors for coated vesicles surfing the sec channel: bidirectional protein translocation across the er membrane yolk protein uptake in the oocyte of the mosquito aedes aegypti caveolin, a protein component of caveolae membrane coats mechanisms of intracellular protein transport protein sorting by transport vesicles early stages of influenza virus entry into mv- lung cells: involvement of dynamin science medicine, and the future. gene therapy endoplasmic reticulum storage diseases calm-af fusion gene in leukemias: simple and inversion-associated translocation ( ; ) assembly of the er to golgi snare complex requires uso p signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in mdck cells regulation of the unc- -caenorhabditis elegans syntaxin complex by unc- visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi identification, sequence, and expression of caveolin- defines a caveolin gene family an enzyme that removes clathrin coats: purification of an uncoating atpase endophilin i mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid endoplasmic reticulumlocalized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including vamp, nsf, snap, annexins, and gtpases griscelli syndrome: report of the first peripheral blood stem cell transplant and the role of mutations in the rab a gene as an indication for bmt peptide-mediated cellular delivery gene therapy: a brief overview of the past, present, and future phagosome maturation: a few bugs in the system the npc protein: structure implies function rab gtpases, intracellular trayc and disease a ypt/rab evector complex containing the sec homolog vps p is required for homotypic vacuole fusion the role of the tethering proteins p and gm in transport through the golgi apparatus in vivo early endosome antigen. : an autoantigen associated with neurological diseases identification of the b-cell epitopes of the early endosome antigen (eea ) cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia diverential evects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation chediak-higashi syndrome: a rare disorder of lysosomes and lysosome related organelles autophagy in health and disease: a double-edged sword golgins and gtpases, giving identity and structure to the golgi apparatus a role for the vesicle tethering protein, p , in the postmitotic stacking of reassembling golgi cisternae in a cell-free system sequential tethering of golgins and catalysis of snarepin assembly by the vesicle-tethering protein p transcytosis of plasma macromolecules in endothelial cells: a cell biological survey regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian lis snap receptors implicated in vesicle targeting and fusion paraneoplastic stiv-person syndrome: passive transfer to rats by means of igg antibodies to amphiphysin a role for giantin in docking copi vesicles to golgi membranes phenotypic behavior of caveolin- r q, a mutant associated with hyperckemia, distal myopathy, and rippling muscle disease 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family expressed predominantly in muscle copii and exit from the endoplasmic reticulum identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor that prevents normal traycking and function of katp channels mutations of the pds gene, encoding pendrin, are associated with protein mislocalization and loss of iodide ezux: implications for thyroid dysfunction in pendred syndrome the chediak-higashi protein interacts with snare complex and signal transduction proteins clathrin assembly lymphoid myeloid leukemia (calm) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated trayc how peroxisomes arise chloride channels cough up caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic traycking tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles gangliosides are receptors for murine polyoma virus and sv protein traycking and alzheimer's disease role of auxilin in uncoating clathrincoated vesicles the inositol polyphosphate -phosphatase ocrl associates with endosomes that are partially coated with clathrin actin microfilaments facilitate the retrograde transport from the golgi complex to the endoplasmic reticulum in mammalian cells dynamin-like protein encoded by the drosophila shibire gene associated with vesicular trayc protein kinase d: an intracellular trayc regulator on the move fcgammar polymorphisms: implications for function, disease susceptibility and immunotherapy a dileucine-like sorting signal directs transport into an ap- -dependent, clathrin-independent pathway to the yeast vacuole protein kinase g from pathogenic mycobacteria promotes survival within macrophages degradation of cftr by the ubiquitinproteasome pathway use of expression constructs to dissect the functional domains of the chs/beige protein: identification of multiple phenotypes a novel -kd peripheral membrane protein is required for intercisternal transport in the golgi stack rab coordinates a novel golgi to er retrograde transport pathway in live cells hsp and the chaperoning of cancer sec -mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction the human cytomegalovirus us gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol amphiphysin heterodimers: potential role in clathrin-mediated endocytosis the caveolin genes: from cell biology to medicine a mutation in rab a causes the vesicle transport defects observed in ashen mice caveolinopathies: mutations in caveolin- cause four distinct autosomal dominant muscle diseases four contiguous amino acid substitutions, identified in patients with laron syndrome, diverently avect the binding aynity and intracellular traycking of the growth hormone receptor synaptojanin is the major constitutively active phosphatidylinositol- , , -trisphosphate -phosphatase in rodent brain rab a enables myosin va-dependent melanosome capture by recruiting the myosin to the organelle identification of an organelle receptor for myosin-va the tuberous sclerosis gene product, tuberin, functions as a rab gtpase activating protein (gap) in modulating endocytosis the fine structure of the gall bladder epithelium of the mouse caveolin is an inhibitor of platelet-derived growth factor receptor signaling sulfonylureas correct traycking defects of atp-sensitive potassium channels caused by mutations in the sulfonylurea receptor nsec binds a closed conformation of syntaxin a abnormal ryanodine receptor function in heart failure fabry disease: characterization of alpha-galactosidase a double mutations and the d y plasma enzyme pseudodeficiency allele protein kinase d regulates basolateral membrane protein exit from trans-golgi network predisposition to renal carcinoma in the eker rat is determined by germ-line mutation of the tuberous sclerosis (tsc ) gene adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis rab proteins as membrane organizers bleeding due to disruption of a cargo-specific er-to-golgi transport complex charcot-marie-tooth disease type a caused by mutation in a microtubule motor kif b the lebanese allele at the low density lipoprotein receptor locus. nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum sucrase-isomaltase deficiency in humans. diverent mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme surfing the sec channel: bidirectional protein translocation across the er membrane crystal structure of an activated akt/protein kinase b ternary complex with gsk -peptide and amp-pnp key: cord- - f xbq authors: kaneko, kazunari; akagawa, shohei; akagawa, yuko; kimata, takahisa; tsuji, shoji title: our evolving understanding of kawasaki disease pathogenesis: role of the gut microbiota date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: f xbq kawasaki disease (kd) was first described by dr. tomisaku kawasaki in . the etiology of kd has been studied comprehensively but remains largely unknown. the disease seems to result from the interplay of genetic and environmental susceptibility factors with infectious triggers, followed by a subsequent abnormal immune response characterized by increased levels of inflammatory cytokines and chemokines during the acute phase. evidence has mounted to suggest that an imbalance between t helper cells (th s) and regulatory t cells (tregs) is associated with aberrant immune responses in kd. recent advances in culture-independent techniques for detection and identification of intestinal commensal bacteria enabled the discovery that th and treg differentiation are regulated by short chain fatty acids (scfas), in particular butyrate, produced by the gut microbiota. this finding provided a mechanistic link between dysbiosis, defined as changes in the composition of the gut microbiota, and various inflammatory diseases. on this basis, we propose that dysbiosis, with reduced production of scfas leading to imbalances of th s/tregs, could be involved in the etiology of kd. a pilot study supported this hypothesis, as only fecal concentrations of butyrate were significantly reduced in kd patients among scfas. this evolving perspective prompted us to undertake metagenomic analyses of bacterial dna from the feces of kd patients who were antibiotic-naïve at diagnosis. simultaneous measurements of th s/tregs in peripheral blood and scfa concentrations in feces would provide valuable information regarding the association between dysbiosis and dysregulated immune responses in kd. kawasaki disease (kd), named after dr. tomisaku kawasaki deceased june th, , mainly affects young children between the ages of months and years ( ). kd is characterized by persistent fever, bilateral conjunctival congestion, changes of the lips and oral cavity, polymorphous exanthema, changes of peripheral extremities, and acute non-purulent cervical lymphadenopathy ( , ) . although kd was originally reported to be self-limiting and benign ( ) , it is now recognized as a systemic vasculitis with a specific predilection for forming coronary artery lesions. these develop in up to % of children with kd who are not treated with intravenous immunoglobulin ( ) . coronary artery lesions associated with kd are the most common causes of pediatric heart disease in developed countries. the incidence of kd in the japanese population continues to increase and reached cases per , children aged ≤ years per year in ( ) . despite extensive ongoing research into the etiology of kd, the underlying mechanisms of this enigmatic vasculitis are not fully understood ( , ) . the current paradigm of kd pathogenesis is that the disease results from a pathologically amplified immune response against infectious agent(s) in a genetically and environmentally susceptible child ( ) . this paradigm is based on the following observations. first, there is clinical overlap between kd and infectious diseases such as adenovirus and streptococcosis. second, seasonal clustering of kd in the winter and spring mimics that of several viral diseases ( ) . third, temporal clusters of epidemics have been reported in japan, the us, canada, and finland ( ) . moreover, an outbreak in japan began in tokyo and spread throughout the country over a period of months ( ). finally, low incidence in the first months of life suggests at least partial protection from trans-placental antibodies ( ) . the low incidence of kd in schoolchildren indicates a potential role of common antigen(s) that most children encounter uneventfully in early childhood and against which they mount an appropriate and protective immune response ( ) . however, efforts to find a single unifying microbiological cause of kd have been, to date, unsuccessful. standard microbiological techniques, molecular methods and serological investigations have all failed to identify an etiological agent. it has long been held that infection by one or more widely distributed microorganism(s) might elicit dysregulated immune responses in genetically susceptible children resulting in kd. candidate pathogens include epstein-barr virus ( , ) , human herpes virus ( ), human immunodeficiency virus ( ) , human adenovirus ( ) ( ) , and candida spp. ( ) . candida albicans (c. albicans) has recently drawn attention, as administration of caws (water-soluble extracellular polysaccharide from culture supernatants of c. albicans) induces coronary arteritis similar to kd in mice ( ) . several reports suggested that c. albicans plays an important role as an infectious trigger of kd ( , , ) . considering the recent pandemic of coronavirus disease (covid- ), potential links between kd and covid- are also deserving of mention ( ) ( ) ( ) ( ) . clusters of children presenting with kd-like symptoms have been documented in the uk, us, france, and italy, and some of them were confirmed to have covid- . it appears that hyperinflammation associated with covid- could act as primer for kd development in individuals having genetically or environmentally determined predisposition. however, the specific mechanism is not yet defined ( ) . the higher incidence of kd in asian countries suggests a genetic predisposition for acquiring the disease ( ) . increased risk in family members of kd patients in japanese populations ( ) also hint at genetically determined susceptibility. recent advances have been made in identifying disease-susceptibility genes from genome-wide association studies. candidate genes contributing to kd susceptibility include b-lymphoid kinase (blk) ( ) epidemiologic studies have extensively searched for environmental factors that may explain variation and seasonality in kd incidence. surveys from the us and japan have demonstrated that higher precipitation and lower temperatures were associated with higher incidence of kd ( , ) . an investigation of the role of the early social environment in kd susceptibility in a japanese population found that higher household income, smaller family size, and urbanization were associated with increased kd incidence ( ) . this study, however, did not find a significant association between absence of infectious exposures during early life and kd. the prominent role played by the immune system in kd has been confirmed by many studies demonstrating activation of neutrophils and other immune cells as well as overproduction of inflammatory cytokines and chemokines such as tumor necrosis factor-α, interleukin (il)- , il- , il- , and il- , and monocyte chemotactic protein- ( ). levels of inflammatory cytokines and chemokines are reported to be elevated during the acute phase of kd. however, the mechanisms responsible for abnormal immune responses and overexpression of inflammatory cytokines remain unclear. several lines of evidence have revealed decreased numbers of regulatory t cells (tregs) and imbalances between t helper cells (th s) and tregs in acute kd ( ) ( ) ( ) . jia et al. elegantly demonstrated that th proportions and cytokine (il- , il- , and il- ) levels were significantly increased, while treg proportions and expression of treg transcription factors (e.g., foxp ) were significantly decreased in patients with acute kd ( ) . they concluded that th expansion and treg depletion were characteristic of acute kd. furthermore, recent studies suggested that treatment efficacy was associated with decreased th proportions and increased treg proportions, with both returning closer to a normal range ( , ) . thus, th /treg imbalances may contribute to exaggerated immune responses in kd patients. growing evidence suggests that disturbances within intestinal communities of commensal bacteria may lead to illness through aberrant immune system development. while the study of gut microbiology related to human health has a centennial history, recent technological advances have enabled us to explore this field in a more sophisticated manner. current approaches rely primarily on culture-independent methods such as amplification of conserved regions of the s rrna gene present in all bacteria ( ) . studies using these techniques have demonstrated that an adult humans harbor trillion gut bacteria comprising more than , different species and approximately species per person per fecal sample ( ) . development of the gut microbiota, defined as its colonization by microorganisms, might begin not at birth but in utero. however, the existence of viable bacteria in the womb was recently questioned ( ) ( ) ( ) . the maternal microbiota provides the first microbial inoculum, and from birth, microbial diversity increases and converges toward an adultlike microbiome within the first - years of life ( ) . the composition of the microbiota in childhood depends on various factors including sanitation, mode of delivery, maturity at birth, infant diet, antibiotic use during infancy, immunizations, and environmental factors such as geography or diet ( , , , ) . ethnic and genetic factors may also give rise to dysbiosis ( , ) . the factors that can alter the microbiome are being studied as potential drivers of changing trends in non-communicable diseases. dysbiosis, defined as changes in the composition of the gut microbiota, may be associated with several clinical conditions including obesity and metabolic diseases ( ), cancer ( ), autoimmune diseases ( ), allergy ( ), chronic inflammatory bowel diseases ( , ) , chronic kidney diseases ( ) , and autisticspectrum disorders ( ) . we also found that dysbiosis was present in children with idiopathic nephrotic syndrome ( ) . although research on the mechanism(s) through which dysbiosis impairs human health has just begun, regulation of the gut immune system by microbiota is believed to be involved. experiments with germ-free animals (deficient in commensal microbiota including gut microbiota) have demonstrated that microbial colonization promotes anatomical development of the intestinal epithelium, increases epithelial cell turnover rates, and initiates the maturation of gut-associated lymphoid tissue ( ) . both tregs and th s have received much attention in terms of their role in the gut immune system and its regulation by microbiota ( ) . tregs were originally identified as cd -positive, cd -positive and foxp -positive t cells that exerted inhibitory control over immune responses ( ) , maintained tolerance to self-antigens, and prevented autoimmune disease ( ) . there are several immunosuppressive mechanisms mediated by tregs, including secretion of immunosuppressive cytokines such as il- and tgf-β, functional modification or killing of antigenpresenting cells, and cell contact-dependent suppression via cytotoxic t-lymphocyte-associated protein ( ) . tregs mainly arise from naïve t cell precursors following stimulation by short chain fatty acids (scfas) such as acetate, propionate or butyrate produced by the gut microbiota ( , ) . previous studies demonstrated that members of the genus clostridium were potent inducers of treg differentiation through butyrate production ( , ) and that reduced luminal concentrations of scfas resulted in impaired development of intestinal tregs in germ-free mice ( , ) . in these mice, reconstitution with commensal bacteria or administration of scfas, especially butyrate, restored treg frequency ( ) , supporting the role of bacterial metabolites in treg development. therefore, it has been proposed that a decrease in the relative abundance of butyrateproducing microbes may disrupt mucosal immune homeostasis ( ) . the gut microbiota also plays a crucial role in the induction of effector t cell responses in the intestine. th s are a subtype of cd -positive t cells specialized for mounting immune responses against fungi and some extracellular bacteria. in addition to il- a, th s produce il- f, il- , il- , and il- ( ) . because il- is a potent proinflammatory cytokine that amplifies ongoing inflammation, aberrant regulation of th s contributes to development of inflammatory and autoimmune disorders. in germ-free mice, the number of th s was markedly decreased. however, the th compartment was restored by reconstitution with conventional microbiota ( ), thus indicating a crucial role for gut microbes in th development. among commensal bacteria, segmented filamentous bacteria (sfb) are one of the most potent inducers of th s. colonization of mice by sfb causes abundant accumulation of th s in the small intestine via enhanced production of serum amyloid a ( , ) . the existence of human commensal bacteria equivalent to sfb in rodents is probable because mixtures of bacterial strains isolated from fecal samples ulcerative colitis patients could induce th development ( ) . the gut, the largest interface between microbial factors and the host, contains the largest proportion of bacteria and the largest amount of lymphoid tissue in the body. thus, it was hypothesized that the intestinal environment might be reshaped in patients with kd. indeed, kd patients frequently exhibit gastrointestinal symptoms and complications ( ) . the contribution of the gut microbiota to kd has been evaluated in limited numbers of small cohorts using culture-based methods. several studies have been performed to identify the causative microbial agent(s) of kd at disease onset. takeshita et al. showed that the gut microbiomes of kd patients were distinguished by a lack of lactobacilli during the acute phase ( ), while nagata et al. isolated both hsp -producing gram-negative bacteria and gram-positive cocci capable of inducing vβ t cell expansion from kd patients ( ) . these results indicated that distinctive gut microbes might be involved in the pathophysiology of kd. however, because these studies of the microbiomes of kd patients were carried out using culture-based methods, microorganisms that cannot be cultured, which constitute more than half of the human gut microbiome, would have been overlooked. unlike culture methods, metagenomic analyses can reveal the composition of the intestinal microbiota irrespective of the ability to culture microbes. kinumaki of a metagenomic analysis of feces using culture-independent methods ( ) . they collected paired fecal samples from children with kd during the acute and non-acute phases and demonstrated that streptococcus spp., including s. pneumoniae, s. pseudopneumoniae, s. mitis, s. oralis, s. gordonii, and s. sanguinis, were more abundant during the acute phase while the genera ruminococcus, roseburia and faecalibacterium were less abundant. unfortunately, more than half of subjects were treated empirically with antibiotics during the early phase of kd when the fecal samples were collected because clinical and laboratory findings often do not fulfill the diagnostic criteria of kd ( ), but are instead suggestive of bacterial infections ( ) . as antibiotic administration rapidly perturbs the gut microbiota ( ), these results may reflect the effects of antibiotic therapy on the gut microbiota and not dysbiosis associated with kd. here, we would like to focus on a novel viewpoint on the role of the gut microbiota in kd: dysbiosis, defined as changes in the composition of the gut microbiota and caused by various prenatal and postnatal factors that are not necessarily infectious agent(s), might contribute to genetically and environmentally determined predilection for kd. this perspective is illustrated in figure and can be explained as follows: [ ] various factors during the in utero and postnatal period drive dysbiosis in young children; [ ] dysbiosis results in reduced intestinal production of scfas including butyrate; [ ] reduced levels of scfas in the gut cause an imbalance of th s/tregs; and [ ] individuals with th /treg imbalances develop hypercytokinemia triggered by ubiquitous infectious agents(s), followed by kd (figure ) . recent observations revealed that viral respiratory infections may alter microbial growth in the gut leading to dysbiosis ( ) . in addition, the gut microbiota has been shown to play an important role in regulating the generation of virus-specific cd -positive and cd -positive t cells and antibody responses following influenza virus infection ( ) . therefore, we hypothesize that young children with dysbiosis are prone to a vicious cycle of hypercytokinemia following infection by viruses. this paradigm for the pathogenesis of kd contrasts with previous hypotheses, which focused on specific microorganisms, toxins, or pathogen-associated molecular patterns ( ) ( ) ( ) . as butyrate has been reported to limit th differentiation and promote treg development ( ) ( ) ( ) , we hypothesize that kd-associated dysbiosis might be characterized by lower abundance of butyrate-producing bacteria. interestingly, the genera roseburia and faecalibacterium, which were reported to be less abundant in patients with acute kd by kinumaki et al. ( ) , are butyrate-producing bacteria ( , ) . furthermore, the strong association between kd and allergic diseases ( ) ( ) ( ) in which dysbiosis plays an important role ( ) also supports this perspective. recent observations of a potential association between previous antibiotic therapy and development of kd also supports our hypothesis ( ) . in this study, the median interval between the final dose of antibiotics and the onset of kd was . months, which was insufficient time for restoration of the gut microbiota and complete resolution of dysbiosis caused by antibiotic use ( ) . to confirm our hypothesis, further investigations involving metagenomic analysis of bacterial dna from the feces of a larger number of antibiotic-naïve patients with kd is clearly needed. it would be worthwhile to compare the proportions of specific microbial species such as butyrate-producing bacteria. simultaneous analysis of th /treg ratios in peripheral blood and measurement of fecal butyrate concentrations, reflecting the intestinal production of scfas, would also be helpful. we have just begun a project to test our hypothesis with approval from our institutional ethics committee (approval no. ) and parental informed consent. fecal samples will be collected not only from kd patients and healthy children but also from controls with viral infections. this will allow us to specifically characterize dysbiosis in kd because recent observations suggested that viral infection itself may cause dysbiosis ( ) . the results of our pilot study of four acute kd patients (median age . years, range . - . years; boys and girl) and four healthy children (median age . years, range . - . years, boys and girl) supports our perspective as fecal butyrate concentrations were significantly lower in kd patients (p < . , mann-whitney u test). in contrast, fecal concentrations of acetate, lactate, and propionate did not differ between kd patients and healthy children (figure ) . the kd patients studied had a median body mass index of . (range: . - . ), median maximal body temperatures of . • c (range: . - . • c), and median maximal c-reactive protein levels of mg/l (range: - mg/l). all kd patients presented with typical clinical features and fulfilled the diagnostic criteria ( ). none reported diarrhea or constipation and none received antibiotics. fecal samples were provided before intravenous immunoglobulin administration. no cases were complicated by coronary artery lesions. what factors perturb the gut microbiota and cause dysbiosis in young children? as shown in figure , both prenatal and postnatal conditions affect the establishment of the intestinal microbiota in infancy. factors that influence the initial colonization of the gut by microbes include maternal factors such as the maternal gut microbiota, vaginal infection or periodontitis as well as postnatal factors such as cesarean delivery, formula feeding, excessive antibiotic use, host genetics, and the environment ( ). interestingly, formula feeding ( ) and social environment factors such as higher household income, smaller family size, and urbanization were associated with both increased risk of dysbiosis and increased kd incidence ( ) . in addition, the peak age of kd onset ranging from months to years corresponds to the critical period for establishment of the gut microbiota during the first , days of life ( ) . we believe that dysbiosis underlies kd and could contribute to genetically and environmentally determined predilections for kd. therefore, kd might be included in the growing list of dysbiosis-associated conditions. if our perspective is confirmed, it would be valuable to investigate whether supplying probiotics starting at birth could reduce the risk of kd in infancy. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by ethics committee of kansai medical university. written informed 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previous antibiotic use and the development of kawasaki disease: a matched-pair casecontrol study the pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep s rrna sequencing the microbiome in early life: implications for health outcomes breastfeeding and risk of kawasaki disease: a nationwide longitudinal survey in japan shaping microbiota during the first days of life we thank edanz group (https://en-author-services.edanzgroup. com/) for editing a draft of this manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © kaneko, akagawa, akagawa, kimata and tsuji. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - brblfq authors: mao, liang title: modeling triple-diffusions of infectious diseases, information, and preventive behaviors through a metropolitan social network—an agent-based simulation date: - - journal: appl geogr doi: . /j.apgeog. . . sha: doc_id: cord_uid: brblfq a typical epidemic often involves the transmission of a disease, the flow of information regarding the disease, and the spread of human preventive behaviors against the disease. these three processes diffuse simultaneously through human social networks, and interact with one another, forming negative and positive feedback loops in the complex human-disease systems. few studies, however, have been devoted to coupling all the three diffusions together and representing their interactions. to fill the knowledge gap, this article proposes a spatially explicit agent-based model to simulate a triple-diffusion process in a metropolitan area of million people. the individual-based approach, network model, behavioral theories, and stochastic processes are used to formulate the three diffusions and integrate them together. compared to the observed facts, the model results reasonably replicate the trends of influenza spread and information propagation. the model thus could be a valid and effective tool to evaluate information/behavior-based intervention strategies. besides its implications to the public health, the research findings also contribute to network modeling, systems science, and medical geography. recent outbreaks of infectious diseases, such as the h n flu, bird flu, and severe acute respiratory syndrome (sars), have brought images of empty streets and people wearing face masks to television screens and web pages, as fear of unknown diseases swept around the globe (funk, salathé, & jansen, ) . these images depict three basic components of epidemics, namely infectious diseases, information about diseases, and human preventive behavior against diseases. from a perspective of diffusion theory, each of the three components can be viewed as a spreading process throughout a population. the disease could be transmitted through person-to-person contact, the information is circulated by communication channels, and the preventive behavior can spread via the 'social contagion' process, such as the observational learning. the interactions among these three diffusion processes shape the scale and dynamics of epidemics (funk & jansen, ; lau et al., ; mao & yang, ) . mathematical and computational models have been extensively used by health policy makers to predict and control disease epidemics. a majority of existing models have been focused on the diffusion of diseases alone, assuming a 'passive' population that would not respond to diseases (bian et al., ; eubank et al., ; longini, halloran, nizam, & yang, ) . this is rarely the case because it is natural for people to protect themselves when realizing disease risks (eames, tilston, brooks-pollock, & edmunds, ; ferguson, ) . to improve, there has been much recent interest in modeling two diffusion processes in an epidemic, either a behavior-disease diffusion (house, ; mao & bian, ; vardavas, breban, & blower, ) , or an information (awareness)-disease diffusion (funk, gilad, watkins, & jansen, ; kiss, cassell, recker, & simon, ) . these 'dual-diffusion' models have made a remarkable progress toward the reality, but none of them consider all the three diffusion processes together. the third diffusion process has often been neglected or simplified. in the current literature, few modeling efforts have been devoted to explicitly representing all the three components, their spreading processes, and interactions. the lack of such models prevents researchers from unveiling a full picture of an epidemic, and inevitably introduces biases into the deep understanding on human-disease systems. for epidemiologists, it is of difficulty to explore how one diffusion process influences the other two, and what key factors govern the three diffusion processes. without a complete model, health policy makers would not be able to systematically evaluate social-network interventions for disease control, such as mass-media campaigns and behavior promotion strategies. as in the age of information, the fusion of diseasebehavior-information in epidemic modeling becomes a pressing task in public health. to fill the knowledge deficit, this research proposes a conceptual framework to integrate the three diffusion processes, and develops a triple-diffusion model in a realistic urban area. following sections discuss the conceptualization, formulation, and implementation of the model, and evaluate the simulation results. the proposed model conceptualizes a typical epidemic as one network structure, three paralleling diffusion processes, and three external factors (fig. ) . first, the contacts among individuals form a network structure as a basis for diffusion and interaction. second, infectious diseases are transmitted through direct contacts among individuals (the middle layer). disease control strategies, such as vaccination program, case treatment and isolation, pose external effects on the disease diffusion. third, the diffusion of diseases prompts the "word-of-mouth" discussion among individuals, which disseminates the information concerning diseases and prevention (the upper layer). the outbreak of diseases may also stimulate various mass media, such as tv, newspapers, and radio, to propagate relevant information, thus accelerating the diffusion of information. fourth, people being informed start to consider and make a decision toward the adoption of preventive behaviors. the adoptive behavior of individuals also influences their network neighbors to adopt, widely known as the "social contagion" effects (the lower layer). the diffusion of preventive behaviors, in turn, limits the dispersion of diseases and speeds the diffusion of information. behavioral interventions, as an external factor, can be implemented by health agencies to promote preventive behaviors, such as educational, incentive and role-model strategies. during an epidemic, these three diffusion processes interact with one another and form negative/positive feedbacks loops in the human-disease system, shown as arrows between layers in fig. . manipulated by the three external factors, these three diffusion processes, hereinafter named as the triple-diffusion process, determine the spatial and temporal dynamics of an epidemic. the conceptual model is formulated by an agent-based approach, which has gained its momentum in epidemic modeling during the last decade huang, sun, hsieh, & lin, ) . different from classic population-based models, each individual in a population is a basic modeling unit, associated with a number of attributes and events that change the attributes. to represent the contact network, individuals are modeled as nodes and are linked to one another through their daily contacts (as network ties). the individualized contacts are assumed to take place during three time periods in a day at four types of locations (mao & bian, ) , namely the daytime at workplaces, the nighttime at homes, and the pastime at service places or neighbor households (fig. ) . individuals travel between the three time periods and the four types of locations to carry out their daily activities, thus having contact with different groups of individuals and exposing themselves to disease infection. these contacts link all individuals into a population-wide network. two types of individual contacts are modeled in terms of the contact duration and closeness. one type is the close contacts (solid-line ovals in fig. ) that happen at homes (with family members), workplaces (with co-workers), and neighbor households (with friends). this type of contacts last for sufficient time to enable disease transmission. the other type refers to the occasional contacts (dash-line ovals in fig. ) that only happen at service places (with clerks and other consumers). in this case, an individual encounters only a limited number of individuals for a short time period, and thus the contact is less effective for infection. the diffusion of infectious diseases is formulated following the concept of classic susceptible-infectious-recovered (sir) model (anderson & may, ) . each individual possesses a serial of infection states and events as shown in fig. (the red dash-line box, in the web version). the progress of an infectious disease starts with a "susceptible" individual, who may receive infectious agents if having contact with an infectious neighbor in the network. the receipt event triggers a "latent" state, during which the disease agents develop internally in the body and are not emitted. the end of latent period initiates an "infectious" state, in which this individual is able to infect other susceptible neighbors and sustain the cascade of infection in the network. during the infectious period, individual may manifest disease symptoms ("symptomatic") or not ("asymptomatic"). for either state, this individual remains infectious but would be unaware of the infection if asymptomatic. after the infectious period, this individual gets recovered and is assumed to be immune to infection during this epidemic. two disease events connect the disease diffusion with other diffusions. first, the event of symptom manifestation will motivate individuals to discuss disease information, and prompt their social contacts to adopt preventive behavior by posing infection risks. the second event is the receipt of disease agents, which is affected by the diffusion of preventive behavior. specifically, the adoption of preventive behavior reduces the probability of disease transmission p, as specified in equation ( ): and e prevention are three model parameters varying in [ , ]. e contact indicates the effectiveness of a contact to transmit diseases, dependent on the physical closeness of the contact. its value can be calibrated based on the observed characteristics of a disease, such as the basic reproductive number r . i age is an age-specific infection rate, specifying the likelihood of receiving disease agents by age group, such as children, adults and seniors. e prevention indicates the efficacy of a preventive behavior in reducing infection. the parameterization is discussed later in the model implementation (simulating the diffusion of disease section and simulating the diffusion of preventive behavior section) when a specific disease and a specific preventive behavior are selected. regarding to the diffusion of information (blue dash-line box in fig. , in the web version), individuals are initially "unaware" of the disease, but can be "informed" through two channels: the word-ofmouth discussion and the mass media. the former circulates the information locally through the contact network, while the latter disseminates the information globally in the population, both modeled as probabilistic events. first, an informed or symptomatic individual will discuss the disease with each network neighbor at a rate g discussion , as formulated in equation ( ): where t is the current time step and t is the starting time of being informed or manifesting symptoms. the discussion rate g discussion decays nonlinearly as time proceeds, i.e., an individual is more likely to talk about the disease within a few days after being informed or feeling sick. turning to the mass media, the probability of an individual being informed g mass is formulated as a function of total symptomatic case number n s (t) at time step t (equation ( )): the more individuals get sick during the time t, the higher the intensity of mass-media propagation, and thus the greater chance for an individual being informed. the constant b is a scaling parameter that controls the intensity of mass-media propagation, and a small b results in a large g mass . a mass-media campaign then can be modeled by varying the b, the timing of campaign (when to start), and the frequency of campaign (time intervals between two broadcastings). once informed by either the discussion or the mass media, individuals will become decision makers toward the adoption of preventive behavior. in such a manner, the diffusion of information is coupled with the diffusion of disease and that of preventive behavior. individuals being informed start to evaluate and make a decision toward the adoption of preventive behavior (green dash-line box in fig. , in the web version). the decision depends on individuals' own characteristics and inter-personal influence from their social networks. this research uses a threshold behavioral model (granovetter & soong, ) to formulate the decision process. specifically, each individual has two adoption states, and the change of state is calculated based on equation ( ) for a given time step t, individual i will evaluate the proportion of adopters in i's personal network, as the peer pressure of adoption a i (t). once the peer pressure reaches a threshold t p;i (called the threshold of adoption pressure), an individual will decide to adopt. meanwhile, individual i also evaluates the proportion of symptomatic individuals in the personal network, as the perceive risks of infection m i (t). if the perceived risk exceeds another threshold t r;i (termed the threshold of infection risk), an individual will also adopt. the individualized thresholds (t p;i and t r;i ) reflect personal characteristics of individuals, while the events of evaluation represent the inter-personal influence between individuals. in such a way, the diffusion of disease elevates the perceived risks of individuals, and stimulates them to adopt preventive behavior. in turn, the adoption of preventive behavior impedes the diffusion of disease, forming a negative feedback loop in the human-disease system. the proposed triple-diffusion model is implemented in the greater buffalo metropolitan area, ny, usa, with a population of , (according to census ) . each individual is programmed into a software agent with attributes and events (table ) . besides a unique identifier, each individual has groups of attributes, including the network, demographic, spatiotemporal, infection, adoption, and information attributes. the events change the values of corresponding attributes. the social network is realized by a previously developed algorithm that assigns values to the demographic and network attributes of individuals (mao & bian, ) . the value assignment involves a large amount of geo-referenced data, including census data, business location data, land parcel data, transportation network, and results of a household travel survey. statistical distributions derived from these datasets, such as distributions of family size, workplace size, and household daily trips, are used to ensure the validity of value assignments. to differentiate the weekdays and weekends, individuals are not assigned to work (or schools) at weekends except those who work in service-oriented businesses (such as restaurants and grocery stores). those who do not work during the weekends would have increased trips to service-oriented businesses. the completion of assignments forms three linked populations, including a nighttime population at homes, a daytime population at workplaces, and a pastime population at service places or neighbor households. the three populations represent the same set of individuals, but at different locations and time periods of a day. individuals have contact with a number of other individuals at a same time period and same location, forming a spatio-temporally varying network. the simulated network has an average of . daily contacts per person, consistent with the observed number ( . ) from empirical studies (beutels, shkedy, aerts, & van damme, ; edmunds, ; fu, ) . the seasonal influenza is selected as an example due to its natural history has been well understood. a number of influenza parameters are either adopted or calibrated from existing literature as shown in table . the product of i age and e contact determines the transmission probability through one contact (equation ( )), which is used to simulate individuals' transition from susceptible to latent state as a stochastic branching process. the latent, incubation, and infectious periods control the sequential transitions from latent to infectious, symptomatic, and recovered states. two groups of parameters are set to simulate the word-ofmouth discussion and the mass-media effects, respectively. the parameter values are calibrated from the model evaluation later but are reported here. for the word-of-mouth discussion, the initial discussion rate g discussion ( ) in equation ( ) is set to . , and then the g discussion (t) is updated as the time goes by. for the mass media, the scaling parameter b in equation ( ) is specified as , based on which the probability of being informed by mass media g mass can be computed at every time step. the mass-media campaign is assumed to be triggered when the total symptomatic individuals exceed & of the total population, and the frequency of broadcasting follows a weekly basis. with these two probabilities, a monte-carlo simulation is used to determine whether an unaware individual will be informed or not at each time step. the use of flu prophylaxis (e.g., oseltamivir) is taken as a typical example of preventive behavior, because its efficacy is more conclusive than other preventive behaviors, such as hand washing and facemask wearing. three parameters are specified to simulate the behavioral diffusion and couple it with the diffusion of influenza. first, the model assumes that symptomatic individuals has a % likelihood to adopt flu prophylaxis to mitigate the symptoms and reduce their infectivity (mcisaac, levine, & goel, ) . second, the preventive efficacy of flu prophylaxis (e prevention in equation ( )) is set to % and % for susceptible and infectious individuals, respectively, indicating that their likelihood of being infected or infecting others can be reduced by such amount (hayden, ; longini et al., ) . third, the two adoptive thresholds of individuals t p;i and t r;i (in equation ( )) are generated from their statistical distributions using a monte-carlo method. those statistical distributions were derived based on a health behavioral survey, whose details are provided in the supplementary document. each individual is assigned to random numbers from those statistical distributions as their adoptive thresholds. for each time step, the model computes the peer pressure and perceived risk for every informed individual, and updates his/her adoption state using the threshold model. the triple-diffusion model is simulated over days, covering a general flu season (from december to may). at the beginning of simulation, all individuals are assigned susceptible and unaware states. to consider a background immunity before the epidemic, the model randomly selects . % of seniors, . % of adults, and . % of children according to the national immunization coverage (euler et al., ; molinari et al., ) , and directly moves them to the adopted and recovered states. all unselected individuals are set as non-adopters. to initialize the disease diffusion, five infectious individuals are randomly introduced into the study area at the first day. the simulation takes a tri-daily time step and runs the three diffusion processes concurrently in each time step. to stabilize the final outcomes, the model has been implemented by realizations. in each realization, the background immunity, the first five infectious individuals, their contacts, and the infection, awareness, and adoption of these contacts are randomized. the final outcomes are three diffusion curves, namely the epidemic curve (the weekly number of new cases), the adoption curve (the weekly number of new adopters), and the awareness curve (the weekly number of newly informed), all averaged from model realizations. two independent data sources are used to evaluate the model results and calibrate model parameters. one is the weekly reports of laboratory confirmed specimens in the e in buffalo, ny, issued by the new york state department of health (nysdoh, ) . the simulated epidemic curve is compared to the weekly reported data to show the validity of modeling disease diffusion. the other data source is the weekly statistics from google flu trends of the study area (google, ) , which summarizes the number of online flu-related inquires as a tool for monitoring influenza outbreaks (ginsberg et al., ) . relevant to this research, the weekly flu trend data could be a reliable representation to the real diffusion of influenza-related information (fenichel, kuminoff, & chowell, ) , and is compared to the simulated awareness curve from the model. model result evaluation fig. displays the simulated weekly number of newly infected individuals, compared to the actual number of weekly labconfirmed cases during the e influenza season. the shape and peak time of the predicted curve correspond well with those of the reported epidemic, although the magnitude of simulated cases is much larger than the reported data. the first possible reason is that many sick people may choose self-care instead of seeing a doctor, and thus cannot be reported. second, for those who seek healthcare, only a small portion of their specimens were submitted for laboratory testing. therefore, the number of influenza cases is often highly under-reported, and a complete data is rather difficult to collect. the laboratory data, so far, is the best available touchstone for model validation. in this sense, the model performs well in predicting the trend, and at least allows the estimate of a worse case result. fig. compares the simulated weekly number of newly informed people to the excessive weekly google flu search statistics that indicate the amount of online searching behavior relevant to influenza epidemic during the e flu season. the excessive weekly search statistic (the left y axis) is the difference between the observed statistics and its long-term average ( ), which removes searches of influenza in a normal day but not caused by the epidemic. the temporal course of the information diffusion is well predicted. since the two measurements are not in a same unit (primary and secondary axis in fig. ), their magnitudes are not comparable but are highly correlated. to my knowledge, both comparisons show a level of consistency that has rarely been achieved by other epidemic models (ferguson et al., ; funk et al., ; kiss et al., ; vardavas et al., ) . a majority of previous models cannot validate themselves by observed facts, particularly for the information diffusion. the triple-diffusion model, thus, could provide a reliable foundation to devise much-needed control and intervention strategies for infectious diseases, such as behavior promotion strategies and massmedia campaigns. fig. shows how the diffusion of influenza motivates people to adopt preventive behavior. the two diffusion processes take a similar shape, but there is a time lag about week between their peak times. as the number of influenza cases rises, individuals perceive increasing risks, which motivate them to adopt preventive behavior. their adoptive behavior further influences surrounding individuals to adopt. the time lag between the epidemic and adoption curves is possibly the time individuals need to be informed and take preventive actions. fig. also suggests that monitoring the real-time flu prophylaxis sales could detect the epidemic peak about week ahead of the traditional disease surveillance networks, such as the cdc sentinel network, which take up to weeks to collect, process, and report disease cases registered at health centers. the diffusions of influenza and its related information also take a similar bell-shape, but the information peaked approximately one week earlier than the disease (fig. ) . a possible reason is the wide coverage of modern mass media over the population, enabling a faster spread of information than the disease. at the beginning of the epidemic, only a few influenza cases occurred and a vast majority of people were unaware of the disease. as the diffusion of influenza took off (january nde th), individuals started to notice the disease problem and discuss with each other. when the epidemic became more sensational and drawn attention from the mass media (january the th), the awareness curve climbed steeply and reached the peak in four weeks. fig. implies that overseeing the diffusion of disease information, such as the google flu trend, could warn the public e week earlier before the epidemic peak actually occurs. in addition to forecasting the timeline of the three diffusion processes, this spatially explicit model allows the prediction of geographic distributions over time. fig. maps the spatial distributions of simulated infection (square), adoption (circle), and awareness (triangle) are mapped. for the purpose of clarity, only the downtown area is presented. the spatial distributions on days , , and are displayed in order to present different stages of the epidemic. on day the epidemic is on the rise, day is around the peak time, and on day , the epidemic is in decline. still in its infancy, the triple-diffusion model has several limitations in its design and implementation. first, the contact network used in the model could be further refined into three different but partially overlapping networks, namely an infection network, information network, and influential network, each channels a diffusion process. admittedly, the model would be more realistic, but building these networks requires extensive social survey to collect relevant data, which is often costly and time consuming. recent work on extracting social networks from facebook and twitter may be a promising method to address this issue (lewis, kaufman, gonzalez, wimmer, & christakis, ) . second, the effects of mass media is formulated as a simple formula, but could be more complicated if further considering various types of media and their corresponding coverage. probably, a sophisticated function could depict the human communications better, such as an exponential decay or a power-law decay function. life style data of individuals may be helpful to identify their preferred media and delineate the media coverage. third, the discussion rate is assumed homogenous over the study area. this rate may vary between age groups, occupations, and personalities. a health behavior survey may need to estimate discussion rates for different groups of individuals. fourth, there would be a certain amount of uncertainty if the model was used to predict the future influenza outbreaks. if new census data and travel survey data were filled in, this model could reasonably predict the timeline and scale of a future outbreak of seasonal influenza. however, for a pandemic influenza, such as the new h n , most of the model parameters should be adjusted to account for the highly infectious virus, the faster circulation of information, and possibly distinct response of individuals toward preventive behaviors. all these limitations warrant a future study. after all, the goal of modeling is not to predict what exactly happen during an epidemic, but rather to observe how the epidemic may proceed and encourage appropriate questions. in this sense, the model results provide valuable knowledge regarding city-wide epidemics. this article presents an original triple-diffusion model for epidemiology, and discusses its conceptual framework and design. the conceptual framework integrates three interactive processes: the diffusion of influenza, the diffusion of information, and that of preventive behavior, upon a human social network. the agentbased approach, network model, theories from epidemiology, information and behavioral sciences are used to formulate the conceptual framework into a working model. for illustration purposes, the model is implemented in an urbanized area with a large population. compared to the reported data, the proposed model reasonably replicates the observed trends of influenza infection and online query frequency. the model, thus, could be a valid and effective tool for exploring various control policies. there are two key contributions of this research to the literature body of network diffusion theory, public health, and agent-based modeling. first, the proposed triple-diffusion framework is a significant advancement to previous disease-only models and dualdiffusion models, such as bian and mao's work. the fusion of disease, information, and human behavior allows a more comprehensive d cubic view of the human-disease system, which can only be studied from a d planar perspective before. the increase of one dimension exposes much more details of an epidemic and thus enables a deeper understanding of this complex system, for example, the interactive mechanisms among the three diffusion processes. the proposed modeling framework can flexibly accommodate the mobile phone tracking data and the latest census data to improve the accuracy of modeled daytime and nighttime populations. the online social networking data (from facebook and twitter) can also be included to modify the way of communications between individuals, as well as the personal influence between them. second, this model can be further developed into a virtual platform for health decision makers to test disease control policies in many other metropolitan areas. particularly, since the model explicitly represents the diffusion of information and human preventive behavior, it permits a systematic evaluation of disease control policies that have not been well studied before, such as the mass-media campaigns and behavioral incentive strategies. the evaluation results will enrich the family of disease control polices, and help the public health overcome the socio-economic challenges posed by potential influenza outbreaks. infectious diseases of humans: dynamics and control social mixing patterns for transmission models of close contact infections: exploring self-evaluation and diary-based data collection through a web-based interface modeling individual vulnerability to communicable diseases: a 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framework for epidemic modeling visits by adults to family physicians for the common cold transmissibility of pandemic influenza the annual impact of seasonal influenza in the us: measuring disease burden and costs can influenza epidemics be prevented by voluntary vaccination supplementary data related to this article can be found at http:// dx.doi.org/ . /j.apgeog. . . . key: cord- -x i x v authors: gensini, gian franco; yacoub, magdi h.; conti, andrea a. title: the concept of quarantine in history: from plague to sars date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: x i x v the concept of ‘quarantine’ is embedded in health practices, attracting heightened interest during episodes of epidemics. the term is strictly related to plague and dates back to , when the rector of the seaport of ragusa (then belonging to the venetian republic) officially issued a -day isolation period for ships, that became days for land travellers. during the next years similar laws were introduced in italian and in french ports, and they gradually acquired other connotations with respect to their original implementation. measures analogous to those employed against the plague have been adopted to fight against the disease termed the great white plague, i.e. tuberculosis, and in recent times various countries have set up official entities for the identification and control of infections. even more recently ( ) the proposal of the constitution of a new european monitoring, regulatory and research institution has been made, since the already available system of surveillance has found an enormous challenge in the global emergency of the severe acute respiratory syndrome (sars). in the absence of a targeted vaccine, general preventive interventions have to be relied upon, including high healthcare surveillance and public information. quarantine has, therefore, had a rebound of celebrity and updated evidence strongly suggests that its basic concept is still fully valid. the concept of 'quarantine' is radically embedded in local and global health practices and culture, attracting heightened interest during episodes of perceived or actual epidemics. the term, however, evokes a variety of emotions, such as fear, resentment, acceptance, curiosity and perplexity, reactions often to be associated with a lack of knowledge about the origins, meaning, and relevance of quarantine itself. historically quarantine has been defined as the detention and segregation of subjects suspected to carry a contagious disease. more recently, the term quarantine has come to indicate a period of isolation imposed on persons, animals or things that might spread a contagious pathology. nowadays the word quarantine should be used to refer to compulsory physical separation (including restriction of movement) of groups of healthy individuals who have been potentially exposed to a contagious disease. the term 'isolation' must be kept separate from the term quarantine, since the former denotes the separation and confinement of subjects already known to be infected with a contagious disease to prevent them from transmitting disease to other people; the latter, essentially the same procedures but with suspected transmitters of disease. from ancient times different populations have adopted varying strategies to prevent and contain disease. one of these is exactly what we would now call isolation. the old testament evidences how individuals affected by diseases were separated from others, and people with leprosy, as leviticus informs, had to live isolated all their lives. in the new testament, too, leprosy continues to be considered a reason for social discrimination, and is represented as curable only through the phenomenon of a divine intervention. the isolation, temporary or otherwise, of sick people has thus always been extensively used as one of the approaches to limit the spread of disease. another strategy was the establishment of a time limit to the manifestation of diseases. in the v century b.c. hippocratic teaching had established that an acute illness only manifested itself within forty days. the case of plague was representative with respect to this; since a disease manifesting itself after days could not be acute, but chronic, it could not be plague. in the ancient past the term pestis (plague) was used in a broad way to indicate every epidemic characterised by high mortality, and magical practices were implemented to fight different diseases since the idea of preventive instruments (such as quarantine) was still not present. with regard to the real plague (the disease caused by yersinia pestis), one may remember the first great pandemic wave of the greek -roman period, and the recurrent epidemics throughout europe in the vi and vii centuries a.d. against acute, fatal diseases such as bubonic plague attempts were made by healthy communities to prevent entry of goods and people from infected communities. in the vii century a.d. armed guards were stationed between plague-stricken provence and the diocese of cahors. particularly virulent was the impact of the disease on the whole of europe in the middle of the xiv century, when the plague spread from southern europe to germany and russia, causing the death of more than % of the european population. medieval laws, renaissance health achievements and xvi -xviii centuries overview the concept of (modern) preventive quarantine is strictly related to plague and dates back to , when the rector of the seaport of ragusa, today called dubrovnik (croatia), officially issued the socalled 'trentina' (an italian word derived from 'trenta', that is, the number ), a -day isolation period. ships coming from infected or suspected to be infected sites were to stay at anchor for thirty days before docking. this same period of time became days for land travellers, probably because the shorter period was not considered sufficient to prevent the spread of disease, and precisely from the italian number forty ('quaranta') comes the term quarantine. furthermore, the chief physician of ragusa, jacob of padua, also advised establishing a place outside the city walls for the treatment of sick (or suspected to be infected) citizens. the imposition to remain - days in an isolated site was determined not only by health reasons, but also by economic necessity, since the quality and safety of the trade network needed to be protected from the black death. the attention dedicated by the ragusan rulers to the plague was, therefore, responsible for the creation of the first 'official' quarantining as a legal system aimed at defending both health and commercial aspects. the following were the main tenets of the law of ragusa: visitors from areas where plague was endemic would not be admitted into ragusa until they had remained in isolation for a month; whoever did not observe this law would be fined and subjected to a month of isolation; no one from ragusa was allowed to go to the isolation area; people not assigned by the great council to care for quarantined persons were not allowed to bring food to isolated people. in venice set up one of the first known 'lazaretto' (quarantine station) on an island near the city, and the venetian system became a model for other european countries. during the next years similar laws were introduced in italian ports (pisa) and in french ones (marseilles), and they gradually acquired other connotations with respect to their original implementation in the context of the middle ages. one such connotation was the institution of a social body to provide the necessary isolation structures (dispositions, facilities, implementation of the laws themselves); another, of more intellectual and medical content, was the gradual acquisition of the essential mechanisms of contagion. in effect, even during the early renaissance, physicians did not have a clear idea of infectiousness, though many waves of epidemics had succeeded one another in the course of the previous centuries. it was only during the xvi century that girolamo fracastoro defined and empowered the concept itself, through the hypothesis that small particles were able to transmit disease. this led the medical profession to integrate previously adopted remedies, simple and insufficient, with more precise quarantine interventions (even if not at an international level) that, however, became the remote bases for modern epidemiology and health sciences. in the xvi century the quarantine system was expanded through the introduction of bills of health, a type of certification that the last port visited by travellers was free from disease. a clean bill, with the visa of the consul of the country of arrival, entitled the ship to the use of the port without quarantine. in the course of the xviii century the practice of quarantine had become, on the one hand a notable nuisance, and on the other, a source of abuse. with regard to the former point, the periods of quarantine were variable across different countries, so that there was no certainty concerning the time needed to implement the quarantine itself. as consequence, not only delay, but perplexity was caused to travellers. with regard to the latter question, instances of bureaucratic and restrictive implementation of quarantine regulations were rife, and the disinfection of correspondence was used as an excuse for political espionage. the upshot of this diffused dissatisfaction with quarantine measures was the emergence of the awareness of the need for a shared standardisation, which, in turn, led to the call for xix century international conferences. from a scientific-epidemiological point of view the concept of quarantine had come to be defined quite precisely in the course of the xix century, but the contemporaneous health organisation was not systematic and capillary enough to confront bursts of epidemics across europe in an organic way. the mid-xix century cholera epidemics, for example, evidenced the scantiness of international uniformity in quarantine practices. even if france had proposed, already in , a meeting for the discussion of the international standardisation of quarantine, it was only in that the first international sanitary conference took place in paris. collaboration at the international level was hard to achieve since quarantine policies mirrored not only health organisation views, but also national trade protection issues that varied from state to state. open negotiation on quarantine was strongly limited by economic and political agendas, as documented by the rome conference, where a proposal regarding the inspection of quarantine of ships from india, using the suez canal, produced a violent discussion between britain and france based not on health questions, as much as on the extent of british dominance over the canal. with regard to the united states of america, protection against imported pathologies had always been retained a local issue, and so handled by the single states. only sporadic attempts had been performed to impose quarantine requirements until repeated and serious yellow fever epidemics led to the passing of federal quarantine legislation by congress in , a set of laws that paved the way for federal involvement in quarantine activities. in the arrival of cholera from abroad prompted a reinterpretation of these laws so as to endow the federal government with more authority in the imposition of quarantine requirements. it was only in that, after a number of conferences held in the second half of the xix century, an agreement was achieved both in europe and in the united states, concerning the notification of disease and other issues. after this achievement conventions and regulations began to be ratified regarding, in particular, relevant principles for the standardisation of quarantine measures. in the united states, as local authorities realised the benefits of federal involvement, local quarantine stations were gradually turned over to the government; in europe established periods of detention were fixed with special reference to cholera, yellow fever and plague. it is interesting to observe how measures analogous to those employed against the plague have been adopted to fight against the disease that, not by chance, has been termed the great white plague: we refer to tuberculosis (tb). before the tubercle bacillus was recognised as the causative agent of the disease, sanatoria had been set up as the only remedy for sufferers from tuberculosis; this may be considered as an application of the broad concept of 'preventive-therapeutic' quarantine. sanatoria constituted a simple and inexpensive tool to break the chain of transmission of the disease, since they guaranteed isolation. they, therefore, had a precise role in controlling tuberculosis, and it is for this reason that, between and , sanatoria spread across the whole of europe and north america. even during the s, although streptomycin was already on the market ( ), tb hospitals were considered important for tuberculosis therapy as sites dedicated to the isolation of tb patients, as recommended by quarantine practice. in the scenario of contagious diseases of the past, the so-called 'health officers' deriving partly from medieval and renaissance predecessors and partly from figures created by the schools of hygiene, acquired fundamental importance. among their various functions were those of furnishing the single national health systems with appropriate corporate entities and legislative organisms, as well as obviously caring for the health of the whole population. in many european countries, including italy, these 'officers' represented, even in the second half of the xx century, the basis of all public health organisation devoted to the monitoring and control of infectious diseases. in the first years of the xx century, a deep medicalization of quarantine measures occured. in the term 'lazaretto' (used especially for plague) was substituted by that of 'health station', since in europe, particularly in france and in italy, the distinction among sick, suspectedly sick, and healthy people, began to acquire a real medical value. four years later an international office of public health was established, and more than twenty nations adhered to it in less than years. variola and typhus were added to the three (plague, cholera and yellow fever) historical quarantining diseases in , and years later this same international office imposed a set of quarantine rules targeted to all kinds of travellers (by land, sea and air). when the world health organisation replaced the international office of public health the expression 'quarantining diseases' disappeared, and pathologies controlled by international health laws (such as plague, cholera and yellow fever) or pathologies under surveillance (such as poliomyelitis, recurrent fever and typhus) appeared. in the face of this resurgence of attention towards infectious diseases, tuberculosis was again made the object of specific measures, which, however, served to monitor and control other diseases. consequent to the high transmission and seriousness of tuberculosis in the europe of the nineteenth century, various countries set up official entities for the identification and control of infections. in the united kingdom a government-funded agency, the medical research council (mrc), was created in in the hope of finding scientific solutions to the illness. its activity was specifically directed to research. with reference to the other side of the atlantic, in the twentieth century ( ) quarantine measures became the task of the national communicable disease centre, at present called the centre for disease control (cdc) and prevention, an organisation, already equipped, in the sixties, with more than quarantine stations located at every port and international airport, and, in the seventies, shifting its field of action from routine inspection to problem management, intervention and regulation. more recently ( ) the proposal of the constitution of a new european monitoring, regulatory and research institution was made, since the already available system of surveillance, set up in europe to control the onset of epidemics, came up against an enormous challenge in the global emergency of the severe acute respiratory syndrome (sars). in the absence of a targeted vaccine, general preventive interventions had to be relied upon, including high healthcare surveillance and public information. quarantine has, therefore, had a rebound of celebrity, as witnessed by the 'fact sheets' prepared and published by the cdc, in which one may read that 'quarantine is medically very effective in protecting the public from disease'. the 'modern' quarantine for sars is a -day period (the incubation period of sars is in fact - days) and, like the quarantines of the past, has been applied to persons who have been exposed to the disease and who may be infected, while, once again, isolation has been implemented to separate healthy people from sick ones. as mentioned above, the health emergency of sars has constituted a real challenge for health systems. however, it has also put into discussion the real effectiveness of quarantine measures, for, precisely as for every other health intervention, quarantine has limits of application of which the medical and social community should be perfectly aware. a recent paper proposing a compartmental model for the geographical spread of infectious diseases shows how this scheme may be adopted to address the effectiveness of human quarantine. the model itself was applied to data deriving from a canadian historical record regarding the time period of the so-called spanish influenza pandemic ( - ) . information on the daily mobility patterns of subjects engaged in the fur trade throughout central canada before, during and after the epidemic were used to establish whether rates of travel were affected by informal quarantine policies, and then the same methodology was adopted to analyse the impact of observed differences in travel on the diffusion of the epidemic. this same model has suggested that quarantine effectiveness varies depending on when the limitation on travel between communities is applied, and on how long it lasts. an operative template of such a type appears particularly interesting from our historical-scientific point of view since it links historical features to current scientific epidemiological evidence. similar to other effective health measures, quarantine is not a panacea, and has its limits. this is highlighted by the recent risk of bioterrorism, where a potentially large and diverse number of agents may be implicated. in addition, other recent epidemics, such as the acquired immuno deficiency syndrome (aids), cannot be considered quarantine-able not only because of medical but also because of ethical and legal issues. however, good quality evidence overall suggests that the basic concept of quarantine is still fully valid, and that the implementation of correct quarantine procedures must be tailored according to single health, social and geographical conditions. , , large-scale quarantine following biological terrorism in the united states the origin of quarantine quarantine and isolation. th ed. the new encyclopaedia britannica the black death past and present. . some historical problems a state of deference: ragusa/dubrovnik in the medieval centuries storia della medicina dall'antichità a oggi trade and health policies in ragusa-dubrovnik until the age of george armmenius-baglivi history of the concept of quarantine plague, policy, saints and terrorists: a historical survey a short history of quarantine (victor c. vaughan) politics of quarantine in the th century international law and infectious diseases the evolution of the concept of 'fever' in the history of medicine: from pathological picture per se to clinical epiphenomenon (and vice versa) compliance, coercion, and compassion: moral dimensions of the return of tuberculosis international sanitary regulations. rd annotated ed. world health organization guideline on management of severe acute respiratory syndrome (sars) severe acute respiratory syndrome. fact sheet: isolation and quarantine simulating the effect of quarantine on the spread of the - flu in central canada plague as a biological weapon: medical and public health management. working group on civilian biodefense quarantine and the problem of aids some historical comments on quarantine: part one some historical comments on quarantine: part two the authors would like to thank professor luisa camaiora, b.a., m.phil., for her correction of the english. key: cord- -vq uhj i authors: da silva, severino jefferson ribeiro; silva, caroline targino alves da; guarines, klarissa miranda; mendes, renata pessôa germano; pardee, keith; kohl, alain; pena, lindomar title: clinical and laboratory diagnosis of sars-cov- , the virus causing covid- date: - - journal: acs infect dis doi: . /acsinfecdis. c sha: doc_id: cord_uid: vq uhj i [image: see text] in december , a novel beta (β) coronavirus eventually named sars-cov- emerged in wuhan, hubei province, china, causing an outbreak of severe and even fatal pneumonia in humans. the virus has spread very rapidly to many countries across the world, resulting in the world health organization (who) to declare a pandemic on march , . clinically, the diagnosis of this unprecedented illness, called coronavirus disease- (covid- ), becomes difficult because it shares many symptoms with other respiratory pathogens, including influenza and parainfluenza viruses. therefore, laboratory diagnosis is crucial for the clinical management of patients and the implementation of disease control strategies to contain sars-cov- at clinical and population level. here, we summarize the main clinical and imaging findings of covid- patients and discuss the advances, features, advantages, and limitations of different laboratory methods used for sars-cov- diagnosis. t he recent emergence of a novel coronavirus in the human population has caused dramatic and unprecedented impact of the economy and prompted mobilization of public health authorities around the world to counter the rapid spread of the virus. coronaviruses (covs) are members of the coronaviridae family and are an important group of viruses that infect a large number of animals including mammalian and avian species. the coronavirinae subfamily is divided into four genera based on genetic features: alphacoronavirus (α-covs), betacoronavirus (β-covs), gammacoronavirus (γ-covs), and deltacoronavirus (δ-covs). the α-covs (hcov- e and hcov-nl ) and β-covs (hcov-oc and hcov-hku ) cause human infection and have been associated with mild respiratory diseases. in the st century, however, three β-covs have emerged from animal reservoirs to cause severe disease in humans: severe acute respiratory syndrome coronavirus (sars-cov), the middle east respiratory syndrome coronavirus (mers-cov), and the pandemic severe acute respiratory syndrome coronavirus (sars-cov- ). , the genome of covs consists of a single-stranded positive sense (+ssrna) of around kb in size. the genomic rna is capped at the ′ end and has a poly(a) tail at the ′ end, allowing it to act as an mrna for translation of the replicase polyproteins. , the ′ terminal region of the genome encodes a polyprotein that is cleaved into nonstructural proteins involved in the transcription and replication process, and the ′ terminal region encodes viral structural proteins. in december , the world was on alert due to a cluster of severe pneumonia cases of unknown origin in wuhan, hubei province, china. this outbreak was epidemiologically linked to a wholesale animal and seafood market where live and freshly slaughtered animals were kept and sold. of the initial patients hospitalized with pneumonia, two-thirds had a history of direct exposure to this market. on the basis of the clinical presentation and the link with the animal market, similar to sars epidemiology, a cov was suspected as the causative agent and therefore pan-cov pcr primers were used to test the samples followed by sequencing. the causative agent was identified as a novel cov, eventually named sars-cov- , and the respiratory syndrome associated with the infection was designated as coronavirus disease- (covid- ) by the world health organization (who). the sars-cov- genome has about % sequence identity to sars-cov (with whom it is classified into the species severe acute respiratory syndrome-related coronavirus) and % to mers-cov. the most closely related virus to sars-cov- found so far is a cov isolated from bats, named ragt cov, whose nucleotide identity is %, suggesting that sars-cov- is also of bat origin. however, it is not clear whether sars-cov- jumped to humans directly from bats or through an intermediate host. the rapidly increasing numbers of covid- prompted who to declare first a public health emergency of international concern (pheic) on january , and then a pandemic on march , . as of july , , more than million cases of covid- and deaths have been reported in countries and territories around the world. most of the cases have been reported by the usa, followed by brazil, india, russia, south africa, mexico, and peru. different from the other highly pathogenic covs, sars-cov- has acquired the ability to establish sustained humanto-human transmission. its basic reproductive number (r ), i.e., the number of secondary infections generated from one infected individual, is estimated to be between . and . , with a mean of . . ultimately, this metric will require further investigations and may vary across settings and locations. on the basis of the travel history and symptom onset of patients in china, the mean incubation period of covid- has been calculated to be . days, ranging from to up to days. clinically, the spectrum of covid- manifestations ranges from asymptomatic and mild to severe infections requiring oxygen therapy and ventilation support. , , since its emergence, a wide variety of methods have been developed for the purpose of the rapid and accurate diagnosis of covid- . on the basis of clinical criteria alone, sars-cov- cannot be reliably distinguished from infections with other pathogens that cause similar symptoms, including influenza, seasonal cov, adenovirus, bocavirus, human metapneumovirus, parainfluenza, respiratory syncytial virus rhinovirus, bordetella pertussis, legionella pneumophila, myco-plasma pneumonia, , and even the mosquito borne dengue virus. in this context, the laboratory-based diagnosis assumes a role for the clinical management of patients and the implementation of disease control measures. here, we review the clinical features, laboratory methods, and imaging findings that are used for covid- diagnosis. in addition, we explore the next steps of the methods under development for covid- diagnosis. a rapid presumptive diagnosis based on clinical assessment and epidemiological characteristics is critical to ensuring appropriate patient care and controlling viral transmission, thus contributing to disease control. as mentioned and as with other respiratory viral infections, the signs and symptoms of covid- are nonspecific and the clinical spectrum of disease can range from no symptoms to severe pneumonia and death. asymptomatic infection has been reported in many settings, but some patients develop clinical disease at a later stage of infection. the proportion of truly asymptomatic infections is unclear, but some estimates indicated that up to % of infections do not result in overt clinical signs of disease. a recent mathematical model suggested that undocumented infections might be major drivers of sars-cov- spread in the world. according to who criteria, a person is suspected of being infected with covid- in three scenarios: (i) a patient with acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath) and a history of travel to or residence in a location reporting community transmission of covid- disease during the days prior to symptom onset; (ii) a patient with any acute respiratory illness who has been in contact with a confirmed or probable covid- case in the last days prior to symptom onset; and (iii) a patient with severe acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath, and requiring hospitalization) and in the absence of an alternative diagnosis that fully explains the clinical presentation. if a patient then tests positive with a laboratory diagnostic, then the infection becomes a confirmed overall, % of laboratory-confirmed covid- patients develop mild to moderate disease, which includes nonpneumonia and pneumonia cases, % have severe disease (dyspnea, tachypnea, blood oxygen saturation ≤ %, and pao /fio ratio of % of the lung field within − h), and % of cases reach critical state (respiratory failure, septic shock, and/or multiple organ dysfunction/failure). risk factors for severe covid- include age ≥ years and people with preexisting concurrent conditions such as cardiovascular disease, hypertension, diabetes, chronic respiratory disease, immunodeficiencies, cancer, and obesity. − accurate estimates of the covid- case fatality rate are still lacking, but it is believed to be around % and can be as high as . % in patients > years of age. however, this number is dependent on the level of testing in countries, testing accuracy, and death-reporting policies. interestingly, men have a much higher risk of death than women. , table summarizes the clinical signs and symptoms of covid- patients seen in several studies. in general, the most common clinical manifestations are fever, dry cough, fatigue, sputum production, dyspnea, sore throat, headache, myalgia or arthralgia, and chills ( figure ). less common symptoms include nausea or vomiting, nasal congestion, diarrhea, hemoptysis, and conjunctival congestion. in children, sars-cov- infection is generally mild and in some cases asymptomatic; however, when presented, the main symptoms include fever (about %), cough ( %), and pharyngeal erythema ( . %). more recently, several studies have been suggested that covid- infection was associated with cutaneous manifestations in patients. − major manifestations observed in covid- patients including different types of lesions such as purpuric, papulovesicular, livedoid, urticarial, maculopapular, and thromboticischemic. a recent study in european covid- patients reported that . and . % of patients had olfactory and gustatory dysfunctions, respectively. these disorders persisted after the resolution of other symptoms. finally, a single retrospective case series in wuhan, china, reported neurologic manifestations in of hospitalized patients ( . %) with a laboratory-confirmed diagnosis of covid- . these symptoms were more common in severe cases, along with acute cerebrovascular events, headache, dizziness, and impaired consciousness. it is unclear whether the neurologic manifestations were caused by sars-cov- directly or by pulmonary disease or other organ damage indirectly or by cytokines. since this study included only hospitalized patients in a single location, the true percentage of neurological manifestations in covid- needs further evaluation. imaging techniques such as chest x-rays, pulmonary computed tomography (ct) scans, and lung ultrasounds are important tools in the early diagnosis of pneumonia in patients with covid- . although chest x-rays are less expensive and more convenient for follow up in pneumonia cases, the technique has low-resolution and projection overlapping, which could lead to many false-negative covid- cases. reports indicate that covid- patients submitted to chest ct scans on admission presented abnormal results (about %), showing bilateral multiple ground-glass and patchy opacity. a clinical laboratory findings and biomarkers in covid- patients. several studies have reported hematologic and blood chemistry alterations in patients infected by sars-cov- . , major laboratory findings in covid- patients identified by meta-analysis include leukopenia, leukocytosis, decreased albumin levels, increased levels of creactive protein, lactate dehydrogenase (ldh), creatinine kinase, and biliarubin, and a high erythrocyte sedimentation rate (esr). increased levels of creatine kinase and lactate dehydrogenase were associated with myalgia. a growing body of evidence suggests that sars-cov- infection can trigger the overproduction of cytokines in some patients, known as a cytokine storm, which is associated with poor outcomes. , − as for other severe viral infections, the exacerbated production of proinflammatory cytokines may be involved in some of the pathophysiology of covid- , including pulmonary edema and lung failure and damage to the liver, heart, and kidneys. compared to healthy adults, covid- patients had higher levels of il- β, il- ra, il- , il- , il- , il- , basic fgf, g-csf, gm-csf, ifn-γ, ip- , mcp- , mip- a, mip- b, pdgf, tnf-α, and vegf. serum biomarkers associated with severe disease included il- , il- , il- , g-csf, ip- , mcp- , mip- a, and tnf-α. a recent retrospective study of confirmed covid- cases ( fatal and discharged cases) in wuhan, china, identified several serological markers that were more elevated in lethal cases than in survivors: elevated ferritin, il- , myoglobin, creactive protein, and cardiac troponin. together, these findings suggest that covid- mortality might be due to infection-driven hyperinflammation. diagnostic virology of covid- . laboratory virology tests are essential for a correct diagnosis and the population level prevalence of covid- , given the number of asymptomatic cases or nonspecific clinical symptoms. results from these tests guide clinicians and health officials in the management, control, and prevention of covid- . several analytical parameters are used to determine the performance of covid- assays, including clinical sensitivity, clinical specificity, positive predictive value (ppv), negative predictive value (npv), and overall accuracy. the confirmation of a sars-cov- infection in the laboratory can be achieved by direct and indirect virology methods. while direct detection is more specific, indirect methods allow a greater opportunity for virus detection after the acute phase of the disease. in direct tests, the clinical sample is examined directly for the presence of particles, virus antigens, or viral nucleic acids, whereas indirect methods detect the serological response against the infection (figure ). samples. the world health organization (who) recommends that all procedures performed with covid- be done only by trained professionals and should take place in a laboratory with an appropriate level of biosafety. nonpropagative diagnostic assays, such nucleic acid amplification tests (naats), sequencing, and some serological tests (e.g., elisa type assays) can be performed in biosafety level (bsl- ) laboratories provided that the initial processing (before virus inactivation) of samples takes place in a validated biological safety cabinet. however, procedures that involve propagative virus work, such as virus culture, isolation, or neutralization assays, should be performed in laboratories equivalent to bsl- using validated practices. the work team must use appropriate personal protective equipment including eye protection (goggles or a disposable face shield), a respirator or facemask, a long-sleeved gown, and gloves and follow all standard operating procedures regarding sample collection and handling. respirators that offer a higher level of protection such as n respirators or powered air-purifying respirators should be used instead of a face mask when performing an aerosol-generating procedure. samples from patients with a suspected or confirmed case must be transported to un , a code referring to "biological substance category b"; however, virus-infected cultures or isolates must be transported as category a -un , "an infectious substance that affects humans". , specimens for diagnosis. choosing the correct sample for diagnosis tests is an essential step in a reliable diagnosis. sars-cov- infection can be detected in a variety of clinical specimens, such as nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, sputum, tracheal aspirates, and bronchoalveolar lavage. − the median duration of sars-cov- shedding in respiratory samples is (irq, − ) days in survivors, but shedding can last for up to days. given the invasiveness and requirements for equipment and skilled labor, the collection of specimens other than sputum from the lower respiratory tract should be considered only in special situations. a nasopharyngeal swab collected with a fiberplastic shaft swab is the preferred choice for swab-based sars-cov- testing because it provides reliable results while not being too invasive. calcium alginate swabs and wooden shaft swabs are not recommended because they may contain substances that inactivate some viruses and inhibit pcr testing. when collection with a nasopharyngeal swab is not possible, oropharyngeal swabs, nasal midturbinate swabs, or anterior nares (nasal swab) swabs are also acceptable alternatives. swabs should be placed immediately after collection into sterile tubes containing to ml of a viral transport medium to preserve viral integrity. upon collection and with the appropriate storage medium, specimens can be stored at − °c for up to h. in cases of delayed testing or shipping, samples should be stored at − °c or below. inadequate sample collection, handling, and storage are important variables that may result in false-negative test results. in addition to respiratory tract specimens, sars-cov- can also be detected in other samples such as stool, anal swabs, and blood but not in urine. , , sars-cov- detection in blood is not frequent and is associated with disease severity. , the detection rate of sars-cov- rna in fecal specimens is similar to that of pharyngeal swabs; , however, importantly, fecal viral shedding appears to occur for a longer period of acs infectious diseases pubs.acs.org/journal/aidcbc review time. sars-cov- detection in stool is not associated with the presence of gastrointestinal symptoms or the severity of illness. − despite high virus rna levels in feces, successful isolation of sars-cov- from patient stool has been reported. the isolation of sars-cov- from urine and ocular secretions from infected patient has been described. , viral shedding in human breast milk and semen has been detected, , while in tears it has either been undetected or acs infectious diseases pubs.acs.org/journal/aidcbc review detected at very low frequency. serum and plasma samples are used for serological assays, especially week postinfection. the median seroconversion time for total anti-sars-cov- antibodies, igm and then igg, were days , , and , respectively. virus isolation. sars-cov- was first isolated from bronchoalveolar lavage from a patient with pneumonia in vero e and huh cells. the viral identity was confirmed by immunofluorescence using the now-known cross-reactive anti-sarsr-cov rp n antibody, rt-qpcr, and metagenomics sequencing. the cellular infectivity of the isolated virus was confirmed by virus neutralization assay using sera from convalescing patients. culture-based methods for sars-cov- detection have been used in research and public health laboratories in different parts of the world, but virus isolation is not recommended as a routine diagnostic procedure because it has low sensitivity, it is time-consuming, and it requires bsl- containment. sars-cov- is also culturable in several cell lines, including human airway epithelial, , vero e , vero ccl- , and huh- cells. , , , vero e cells express high levels of angiotensin-converting enzyme (ace ), which has been identified as a key a cell receptor for sars-cov- infection. similar to sars-cov- and mers-cov, sars-cov- isolation is enhanced in an engineered vero e that expresses tmprss (transmembrane serine protease ) levels that are -fold higher than in human normal lung tissue and other human cell lines, suggesting that tmprss protease is important for sars-cov- infection. recently, zang and colleagues demonstrated that tmprss and tmprss promote sars-cov- entry into enterocytes. harcourt and co-workers studied the susceptibility of several cell lines such as vero ccl- , vero e , hek- t, a , and huh- as well as the big brown bat kidney cell line (efk b) to support the productive replication of sars-cov- . no cytopathic effect (cpe) was observed in any of the cell lines except in vero e and vero ccl cells, in which the virus grew to > pfu/ml at h postinfection. huh- and hek- t cells showed only modest viral replication, whereas no virus replication was detected in either a or efk b cells. sars-cov- produced distinct plaques in vero e cells, but plaques in vero ccl- cells were not as clear, suggesting that vero e cells might be the best choice for the propagation, quantification, and study of plaque phenotypes of different sars-cov- strains. however, should virus isolation be attempted, vero e cells clearly stand out as a cell line that supports infection and virus production. a recent cross-sectional study used sars-cov- rt-qpcr confirmed specimens and evaluated their ability to infect vero cell lines. for the study, patient samples were incubated on vero cells (ccl- ) during days, and then the cytopathic effect was evaluated. of these, samples ( . %) demonstrated viral growth, but there was no growth in samples with a ct value > , which suggests using samples with low ct values for successful viral isolation. electron microscopy. human cov was first isolated in from a patient with a common cold. later, the virus was named "coronavirus" due to the virus' appearance under the electron microscope that resembles the solar corona. cov particles are pleomorphic, and their surfaces are covered with a distinct layer of projections, which corresponds to the long spike protein. given the virus' distinctive morphology, electron microscopy has been used to observe and identify virus particles after isolation in culture systems in the initial outbreak in wuhan and subsequently by others. , , the first attempts to recover sars-cov- virions with the characteristic fringe of surface spike proteins from the first covid- case in australia failed, but adding trypsin to the cell culture medium immediately allowed the visualization of the virus with characteristic cov morphology, with particles of − nm diameter displaying prominent spikes ( − nm) on the surface. real-time rt-qpcr. the gold standard diagnostic test for sars-cov- infection is viral rna detection by reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) given its sensitivity, specificity, and speed. rt-qpcr shows better performance than serology because it can identify positive cases in the early stage of infection, even during the incubation period of the disease and after symptoms have disappeared. many laboratory-developed tests based on the rt-qpcr assay have been published or are in development for sars-cov- detection. some of these developed assays are specific to the pandemic strain of the virus, and others also recognize genetically similar strains such as sars-cov. molecular targets for sars-cov- rt-qpcr include the genes that encode the nucleocapsid (n), envelope (e), spike (s), and rna-dependent rna polymerase proteins given their high degree of genetic conservation (figure ) . , most assays use two targets in the viral genome, but in the event of reagent shortages, rt-qpcr screening with only one set of primers instead of two may be considered after thorough validation in the individual laboratory. performing a sample preheating step instead of rna extraction can also be considered during a shortage of viral nucleic acid extraction kits. in a study with oropharyngeal swab samples ( positive and negative for sars-cov- ) in denmark, it was found that heating for min at °c resulted in a sensitivity, specificity, and accuracy of . , . , and . %, respectively, as compared with magna pure rna-extracted samples. although some laboratories have recommended heat inactivation of the virus ( − °c for − min) before rna extraction to protect laboratory personnel, this procedure adversely affects the efficacy of rt-qpcr for sars-cov- detection. high rates of false-negative sars-cov- rt-qpcr results and prolonged nucleic acid conversion have been reported in some studies, which have implications for returning to normal activities and covid- control. − in addition to in-house tests, many molecular assay kits have been approved by the food and drug administration's (fda) and have been made commercially available for the detection and amplification of the sars-cov- rna genome (https:// www.fda.gov/medical-devices/emergency-situations-medicaldevices/emergency-use-authorizations). however, diagnostic laboratories should rigorously validate these commercial kits before routine use since the analytical sensitivity of some commercial rt-qpcr tests differs substantially, which could lead to false-negative results. two of the most widely used assays in the western world have been developed by charite, hospital germany and the u.s. cdc. nalla and co-workers compared the performance of the charité(n, polymerase, and e targets) and the cdc (n , n , and n targets) primer/probe sets side-by-side using clinical samples. it was found that all assays were highly specific to sars-cov- , with no cross-reactivity with other respiratory viruses. the cdc n and the charitéhospital e-gene primer/probe sets performed equally well and were the most sensitive assays for detecting sars-cov- . we anticipate acs infectious diseases pubs.acs.org/journal/aidcbc review that this list will grow as several new rt-qpcr assays are being developed to promote the accurate and rapid detection of sars-cov- . the results demonstrated that the median duration between the onset of symptoms to nucleic acid conversion was days (iqr, − ) and that the longest duration was days after the onset of symptoms. it was found that higher viral loads (inversely proportional of the ct value) can be observed in upper respiratory samples soon after symptom onset and that the peak occurs within the first week of illness onset of covid- confirmed patients. , in another study, the authors evaluated the viral shedding patterns demonstrated in patients with mild and severe covid- disease using specimens from patients, including individuals classified as mild cases and classified as severe cases. the results showed that the viral load of severe cases was around times higher when compared with that of mild cases, indicating that higher viral loads might be associated with severe clinical findings. in addition, it was revealed that the ct values of severe cases were significantly lower than those of mild cases in infected patients at the time of admission and that early viral clearance was observed in patients classified as mild cases ( days after onset). sars-cov- -positive rt-qpcr detection has also been demonstrated even after the resolution of covid- symptoms. , for this reason, the cdc has recommended obtaining at least two negative upper respiratory tract samples, collected in intervals of h or longer, to document sars-cov- clearance. on the other hand, the viral shedding in asymptomatic patients with covid- also has been investigated. , zhou and co-workers analyzed the viral shedding pattern in patients that were confirmed to have covid- using rt-qpcr but were asymptomatic during admission to the hospital. subsequently, the study divided the patients into groups: patients who remained asymptomatic during hospitalization (aps) and patients who presented symptoms after admission to the hospital (apis). the results demonstrated that the median ct value of aps ( . , interquartile range [iqr] . − . ) was higher than when compared to apis ( . , iqr . − . ), showing a lower viral load in aps. also, it was found that the duration of viral shedding remained similar in the two patient groups, which reflects the possibility of sars-cov- transmission in the community during the asymptomatic period. in addition, the study findings revealed that the viral load of aps peaked during the first week after admission to the hospital while that of apis peaked during the second week of covid- infection. these findings alert professionals and health authorities to the possibility of transmission during the asymptomatic period of patients with covid- disease. however, further scientific studies with more patients are required to elucidate the real role of asymptomatic patients in the transmission chain. reverse transcription loop-mediated isothermal amplification (rt-lamp). the recent increase in the number of covid- cases in the world has encouraged a global effort to develop point-of-care platforms for diagnosing sars-cov- . reverse transcription loop-mediated isothermal amplification (rt-lamp) is perhaps one of the most promising platforms for rapid development and accessible sars-cov- testing and has many advantages, compared to rt-qpcr, such as high specificity and sensitivity, simple operation, rapid amplification, and low cost,. , rt-lamp assays have been developed for other covs of the same genus (betacoronavirus), including sars-cov , and mers-cov. , not surprisingly, several studies have already demonstrated the use of rt-lamp for sars-cov- detection. − crispr/cas-based diagnostic methods. the clustered regularly interspaced short palindromic repeats (crispr)/cas machinery has recently been adapted as a poc tool for the rapid detection of nucleic acids (dna or rna). , overall, this crispr machinery is programmed to cleave specific sequences in the dna/rna target where the results can be easily observed by combination with a lateral-flow strip. initially, zhang's team developed a crispr/cas-based platform called specific high-sensitivity enzymatic reporter unlocking (sherlock) that, combined with isothermal preamplification to detect strains of single-strand rna viruses, identifies mutations and human genotype dna, and distinguishes pathogenic bacteria. , more recently, using the same knowledge, they adapted a protocol using the sherlock system for sars-cov- detection. on the other hand, mammoth bioscience company developed another platform based on the crispr/cas system named the endonuclease-targeted crispr trans reporter (de-tectr) to detect any rna or dna target, which has now been used to detect the sars-cov- rna genome from respiratory swab rna extracts. the suitability of detectr technology for the detection of sars-cov- was evaluated using patient specimens, including patients with covid- infection and patients with other viral respiratory infections, and then compared with the cdc rt-qpcr as a reference method to confirm covid- infection. the sars-cov- detectr test had % positive predictive agreement and % negative predictive agreement when compared with rt-qpcr results. despite these promising results, crispr/cas-based diagnostic methods are not widely used by diagnostic laboratories and need further implementation. taken together, these results highlight the great potential of crispr-based diagnostic methods as a rapid, specific, portable, and accurate detection platform for the detection of the sars-cov- genome in patient samples. sensors. sensors represent another alternative detection method with rapid and high throughput. since the emergence of sars-cov- , several research groups from different parts of the world have also focused on alternative sensing modalities based on sensors for the diagnosis of sars-cov- that reduce the use of expensive laboratory equipment, trained laboratory personnel, and extensive sample preparation and provide fast and accurate results. in this context, qiu and co-workers developed a dual-functional plasmonic biosensor combining the plasmonic photothermal (ppt) effect and localized surface plasmon resonance (lspr) to detect sars-cov- . in another study, the authors used a field-effect transistor (fet)based sensor to detect sars-cov- from patient samples. in general, these recent research findings are based on technologies previously used for the detection of other viral pathogens that now can be adapted for the detection of sars-cov- . for instance, due to its high sensitivity and specificity, simple operation, low cost, and other advantages, the sensors using programmable biomolecular components named the toehold switch developed in response to previous ebola and zika outbreaks represent another powerful platform for detecting the sars-cov- genome. − genome sequencing. whole genome sequencing was used to identify potential etiological agents involved in the index cases of the covid- pandemic in wuhan. in addition to unequivocally confirming the diagnosis of a sars-cov- infection, regular sequencing of a percentage of patient samples from clinical cases can be used to monitor changes in the viral genome over time and trace transmission patterns. for this purpose, several sequencing protocols based on sanger and next-generation sequencing (ngs) are being applied to rapidly generate the genome sequences. , , lu and colleagues used a combination of sanger, illumina, and oxford nanopore minion sequencing technologies to obtain the whole-genome sequences of sars-cov- from six patient specimens from wuhan, china. holshue and colleagues reported the first case of the novel coronavirus in the united states and used a combination of the sanger method, illumina, and minion to generate the whole-genome sequences. whole-genome sequencing using minion technology coupled with phylogenetic analyses and travel history allowed the identification of sars-cov- entry routes into latin america during its emergence in brazil. as of july , , genome sequences had been deposited in genbank (https://www.ncbi. nlm.nih.gov/genbank/sars-cov- -seqs/), including complete genome sequences from different countries around the world and partial genome sequences. sars-cov- has evolved continuously since its emergence. the binding regions of primers and probes should be monitored continuously for matching to the virus genome as more sequence information becomes available. in this scenario, artesi and colleagues reported that changes within the sars-cov- primer binding region negatively affected the performance of commercial rt-qpcr assays (unpublished data, https://www.medrxiv.org/content/ . / . . . v ). in another study, rana and co-workers analyzed the whole genome sequence of patients with covid- and found variations in the primer and probe binding sites that could produce false-negative results. a comprehensive bioinformatics analysis of sars-cov- sequences from around the world identified the presence of mutations/ mismatches in the primer/probe binding regions of assays out of rt-qpcr assays studied, including the charite-orf b reverse primer and the us-cdc-n- probe. however, it should be noted that except at the ′ end, rt-qpcr can tolerate mismatches at the ′ extremity or in the middle of a primer. thus, oligonucleotide binding regions should be continuously monitored by bioinformatics and also by wet-laboratory experiments in order to identify changes that may influence rt-qpcr performance. , , taken together, whole genome sequencing and bioinformatics will be important in global efforts to combat the pandemic, with the resulting analyses being used to guide a wide range of studies, including diagnostics, , , molecular epidemiology studies, viral evolution, − elucidatation of possible hosts of the virus, , − identification of targets for drugs and vaccines, , molecular determinants involved in virulence and pathogenicity, , and factors related to the host's immune response to the virus. , serology. serological methods are being increasingly used and can be used for diagnosis, contact tracing, and herd immunity assessment, and vaccine efficacy evaluation. seroepidemiologic studies can assist in the investigation of the ongoing pandemic and the retrospective assessment to determine the attack rate or the progress of the pandemic through antibody detection. these studies can assist health authorities and governments in making sound decisions with respect to the implementation of public health measures during the course of the current pandemic. population-based serosurveys for sars-cov- has been applied in several sites to assess the burden of covid- and more accurately determine sars-cov- prevalence and transmission dynamics. − moreover, serology can also be useful in situations where rt-qpcr is negative and there is a strong epidemiological link to covid- infection. in these cases, paired serum samples collected in the acute and convalescent phase can be of diagnostic value. the adaptive immune response to sars-cov- infection has been studied, , , , despite several knowledge gaps in this area. long and co-workers studied the antibody responses to sars-cov- in patients with covid- in china. seroconversion for igm and igg occurred simultaneously or sequentially, and the median day of seroconversion for both immunoglobulins was days after covid- onset. the seroconversion rate for igg reached % days after symptom onset. in a related work, zhao and colleagues showed that the median seroconversion times for total antibodies, igm, and igg were , , and days after sars-cov- infection. severe patients tend to have higher antibodies titers than patients with mild covid- . , to investigate the antibody responses to sars-cov- in convalescing individuals, robbiani and co-workers analyzed plasma samples collected an average of days after the onset of symptoms from convalescing individuals. most convalescent plasmas obtained from recovered patients did not contain high concentrations of neutralizing activity. a related study with convalescing patients detected serum neutralizing antibodies in out of patients, and there was a strong correlation among neutralization antibody titers, the numbers of virus-specific t cells, and anti-s-rbd igg but not with anti-np igg. notably, the antibody response and viral clearance can be delayed in immune-compromised individuals and people posteriorly infected with sars-cov- . in the past few months, a variety of serological tests have been designed to detect sars-cov- , mainly enzyme-linked immunosorbent assays (elisa), immunofluorescence assays (ifa), chemiluminescence enzyme immunoassays (clia), and lateral flow assays (lfa). of these serological methods, elisa, clia, and lfa are used for the first-line screening of patient samples with covid- . most serological assays are based on the sars-cov- nucleocapsid protein (n), transmembrane spike protein (s), or s receptor-binding domain (rbd) because of their high antigenicity. − as mentioned before, sars-cov- enters host cells using its spike protein and represents the main target of neutralizing antibodies produced by the host. in this context, several studies have demonstrated that serological tests using the s antigen are more sensitive than those using n-antigen-based antibody assays. instead of a single antigen, some companies have combined n-and s-based antigens to develop serological assays for sars-cov- detection. cross-reactivity between sars-cov- and sars-cov- has been reported against the n protein but not against the s subunit of the s protein. cross-reactive antibodies between sars-cov- and sars-acs infectious diseases pubs.acs.org/journal/aidcbc review cov- have been detected in both the rbd region located in the s subunit and also in non-rbd regions. anderson and colleagues analyzed patient samples with sars-cov- and sars-cov- infections and showed that there is no detectable cross-neutralization by sars-cov- patient serum against sars-cov- , despite significant cross-reactivity between the n proteins from the two viruses many elisa-based assays have been developed to evaluate the human antibody response (iga, igm, and igg) against sars-cov- . , − cross reactivity to other coronaviruses has been reported, and therefore test results should be interpreted in light of the local epidemiological scenario. for example, it has been shown that igm elisa allows a higher detection efficacy than rt-qpcr after . days of symptom onset, and the combined use of igm elisa and rt-qpcr increases the positive detection rate to . % when compared with a single rt-qpcr test ( . %). the median time of appearance of igm and iga antibodies in plasma was days, while igg was detected days after symptom onset. in the same study, sars-cov- n elisa did not show any cross-reactivity with other covs, with the exception of sars-cov- , a virus that has not been in circulation since the to outbreak. perera and co-workers developed an elisa based on the receptor-binding domain (rbd) of the sars-cov- s protein to detect igg and igm antibodies in human sera and compared the elisa results with confirmatory microneutralization and % plaque reduction neutralization tests (prnt ). they found that prnt were more sensitive than microneutralization to detecting antibodies against sars-cov- , indicating that elisa should be used for screening and prnt should be used for confirmation in large-scale seroepidemiological studies. a magnetic chemiluminescence enzyme immunoassay (mclia) has also been developed for virus-specific antibody detection. in a study with covid- patients, sera from these patients did not cross react with the sars-cov spike antigen, but some cross-reactivity was found with the sars-cov nucleocapsid antigen. many lateral flow assays (lfa) have been developed to detect sars-cov- − and have obtained emergency use authorization from the fda (https://www.fda.gov/medicaldevices/emergency-situations-medical-devices/emergency-useauthorizations). a combined igm/igg assay seems to be a better choice in terms of performance and sensitivity to either antibody alone (igm or igg). recently, li and colleagues developed a rapid lfa to simultaneously detect igm and igg antibodies against sars-cov- within min in human blood. the assay uses the receptor binding domain of the sars-cov- s protein as the antigen. they validated the assay with blood samples previously screened by rt-qpcr, including positive and negative samples. the assay had a sensitivity of . % and a specificity of . % compared to rt-qpcr. in addition, several studies have been used to evaluate the clinical sensitivity of serological tests with covid- patient samples collected on different days after the onset of symptoms. − pan and colleagues evaluated a commercial lateral flow immunochromatographic test targeting viral igm or the igg antibody and compared it with rt-qpcr. the sensitivity of the assay for sars-cov- detection was . % in early-stage patients ( − days after onset), . % in intermediate-stage patients ( − days after onset), and . % in late-stage patients (more than days after onset). in similar study, tang and co-workers compared the diagnostic performance of two sars-cov- commercial serologic tests using specimens from pcr-confirmed sars-cov- patients and control specimens from different days after disease onset (< , − , − , and ≥ days). both assays had poor diagnostic sensitivity during the first days of symptoms, generating a high rate of false-negative results. the time of sampling since the onset of symptoms must be taken into account when performing the serological diagnostic test, and false-negative results can lead to the false assumption that a person is not infected, consequently bringing about serious challenges and implications for the spread or containment of the disease. most patients will seroconvert only in the second week after infection, and thus negative serological test results obtained during the first days of the disease should be interpreted with caution. as opposed to classical serum neutralization assays such as the microneutralization and plaque reduction neutralization test (prnt), pseudovirus neutralization assays for sars-cov- can be performed in biosafety level facilities. pseudovirusbased neutralizing assays for sars-cov- have been developed using vesicular stomatitis virus (vsv) and lentiviral pseudovirus systems. , such systems will facilitate the study of covid- humoral immunity and the development and evaluation of vaccines and therapeutics against sars-cov- once the reliability and cross reactivity have been assessed. antigen detection tests represent another alternative to detecting sars-cov- . antigen detection assays are developed to directly detect viral particles in biological samples such as nasopharyngeal secretions from infected patients. in this context, some rapid antigen assays have been proposed to detect sars-cov- . , − a fluorescence lfa antigen test based on the n protein has been developed and validated using nasopharyngeal and oropharyngeal samples from suspected covid- cases ( positive by rt-qpcr). compared with rt-qpcr, the test achieved a sensitivity of . % and % specificity. however, some commercially available point-of-care tests have very low sensitivity, which hinders their use in covid- diagnosis. , for the validation of serological tests, robust test validation with adequate clinical samples representative of a real-world scenario (e.g., timing from disease onset, different disease severities, different geographical locations, and patient age) is of paramount importance. particular attention should be paid to the low specificity of some assays and the high possibility of false-positive results. a low specificity of serological assays may have important consequences in terms of both diagnosis and population surveillance of covid- patients at the individual level and population level. , at the individual level, the risks posed by false-positive results can be of great concern. for instance, people who have never been infected may return to work or travel because they are considered to be immune to sars-cov- infection. at the population level, false-positive results may increase the prevalence of covid- disease and provide a distorted picture of the higher population immunity and lower mortality rate than what is actually happening, which can negatively affect seroepidemiological surveillance studies. thus, the independent validation of covid- serological tests using samples from different stages of the disease is urgently needed to encourage health care professionals and governments to make sound decisions. differential diagnosis. since the clinical manifestations of covid- are similar to those of other respiratory diseases, differential diagnosis is of paramount importance in assisting physicians in the therapeutic management of patients and health officials in establishing disease control policies. in an effort to do so, respiratory pathogens in patients suspected of covid- in italy have been investigated. here, researchers tested the nasopharyngeal swabs of suspected causes using sars-cov- rt-qpcr and a commercial multiplex respiratory cartridge that detects and differentiates viral and bacterial pathogens. only patients were confirmed to be infected with sars-cov- , and ( . %) were positive for other respiratory pathogens, including ( . %) positive for influenza a and ( . %) positive for influenza b. other pathogens detected in the patient samples were common cold cov (h-cov e, h-cov nl , and h-cov hku ), rhinovirus, enterovirus, metapneumovirus, adenovirus, mycoplasma pneumoniae, streptococcus pneumoniae, and legionella pneumophila. these results highlight the diagnostic differential and demonstrate the importance of using a spectrum of molecular kits for the rapid detection of respiratory pathogens in order to improve the clinical management and treatment of patients infected with covid- . in another study, yan and co-workers reported the cases of two patients in singapore who initially had moderate symptoms including myalgia, a mild cough, and diarrhea and presented with thrombocytopenia and a normal chest radiograph. dengue was suspected, and a rapid serological test for dengue produced false-positive results. as patient symptoms worsened, they were later diagnosed with covid- by rt-qpcr. taken together, these findings suggest that special attention is needed in the differential diagnosis between arboviruses and sars-cov- infection, especially in countries where is there a circulation of denv, zikv, and chikv. moreover, coinfection with sars-cov- and other respiratory viruses, such as common cold cov, influenza, and metapneumovirus, have been reported, highlighting the need for repeat testing based on clinical indications. − the rapid spread of sars-cov- around the world, with mounting cases of fatal pneumonia and the economic crisis, is a global concern. diagnostics represent one of the most powerful tools in mitigating these effects until a vaccine can be established. this needed is further highlighted by overlapping symptoms from other pathogens that can confound diagnosis based only on clinical criteria. as we have reviewed, many approaches have been established for the diagnosis of sars-cov- , and we anticipate the coming months to bring about many more innovative strategies. critically, the development of inexpensive point-of-care diagnostic platforms will accelerate the global response to the current pandemic, especially in countries with health systems devoid of adequate laboratory infrastructure. it will be important that widespread diagnostic development and validation continue in the coming months and that the large-scale implementation of these sars-cov- diagnostic platforms is successful as this pandemic evolves. reliable, easy-to-use assays will be absolutely critical, especially as the disease moves through low-and middle-income countries. 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coronavirus disease clinical performance of different sars-cov- igg antibody tests evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov- antibodies establishment and validation of a pseudovirus neutralization assay for sars-cov- characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov evaluation of novel antigen-based rapid detection test for the diagnosis of sars-cov- in respiratory samples evaluation of rapid antigen test for detection of sars-cov- virus development of a portable, ultra-rapid and ultra-sensitive cell-based biosensor for the direct detection of the sars-cov- s microfluidic immunoassays for sensitive and simultaneous detection of igg/igm/antigen of sars-cov- within min ) diagnostic accuracy of serological tests for covid- : systematic review and metaanalysis coinfection with covid- and coronavirus hku -the critical need for repeat testing if clinically indicated coinfection of influenza virus and severe acute respiratory syndrome coronavirus (sars-cov- ) coinfection with sars-cov- and human metapneumovirus sars-cov- and influenza virus co-infection epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in emerging novel coronavirus ( -ncov) pneumonia china: a descriptive, cross-sectional, multicenter study prevalence and clinical presentation of health care workers with symptoms of coronavirus disease in dutch hospitals during an early phase of the pandemic clinical and virologic characteristics of the first patients with coronavirus disease (covid- ) in the united states clinical features, laboratory characteristics, and outcomes of patients hospitalized with coronavirus disease (covid- ): early report from the united states key: cord- - a n dc authors: charitos, ioannis a; ballini, andrea; bottalico, lucrezia; cantore, stefania; passarelli, pier carmine; inchingolo, francesco; d'addona, antonio; santacroce, luigi title: special features of sars-cov- in daily practice date: - - journal: world j clin cases doi: . /wjcc.v .i . sha: doc_id: cord_uid: a n dc the severe acute respiratory syndrome-coronavirus- (commonly known as sars-cov- ) is a novel coronavirus (designated as -ncov), which was isolated for the first time after the chinese health authorities reported a cluster of pneumonia cases in wuhan, china in december . optimal management of the coronavirus disease- disease is evolving quickly and treatment guidelines, based on scientific evidence and experts’ opinions with clinical experience, are constantly being updated. on january , , the world health organization declared the sars-cov- outbreak as a "public health emergency of international concern". the total lack of immune protection brought about a severe spread of the contagion all over the world. for this reason, diagnostic tools, patient management and therapeutic approaches have been tested along the way, in the desperate race to break free from the widespread infection and its fatal respiratory complications. current medical knowledge and research on severe and critical patients’ management and experimental treatments are still evolving, but several protocols on minimizing risk of infection among the general population, patients and healthcare workers have been approved and diffused by international health authorities. the severe acute respiratory syndrome-(sars-) coronavirus (cov- ), officially named as "coronavirus disease- (covid- )" by the world health organization (who) on february , , is actually the novel coronavirus responsible for one of the most severe worldwide pandemics in recent history [ ] . on january , , the chinese center for disease control and prevention (cdc) reported and confirmed that a cluster of cases of acute pneumonia in people associated with the huanan seafood wholesale market in wuhan, a city in the hubei province of china, were caused by a novel coronavirus, -ncov [ ] [ ] [ ] . from that day, the epidemic spread throughout china, being followed by a rapidly increasing number of cases in other countries throughout the world, with a high morbidity and mortality ratio [ , ] . on march, , the director-general of the who declared covid- a global pandemic [ ] . the coronavirus subfamily, nidovirales order, includes four genera: alpha, beta, gamma, and delta coronaviruses. they are medium-sized, enveloped, positivestranded rna viruses which replicate using a nested set of mrnas [ , ] . their genetic material represents the largest known viral rna genomes (between - kb). the host-derived membrane surrounds the genome, encased in a nucleocapsid, and contains glycoprotein spikes. viral rna replication by rna polymerase occurs in the host cytoplasm. the coronaviruses genome encodes four or five structural proteins [ ] . coronaviruses are very common among birds and mammals, especially in bats, pigs and feline, which represent the major hosts [ , ] . the human coronaviruses (hcovs) number seven. there are five non-sars september , volume issue coronavirus serotypes (two beta coronaviruses -hcov-oc and hcov-hku and two alpha coronaviruses -hcov- e and hcov-nl ) [ ] and a novel coronavirus, the middle east respiratory syndrome coronavirus (mers-cov), that emerged in [ ] . they are community-acquired viruses that continually circulate through the human population, causing asymptomatic infections, accounting for % to % of overall colds and upper respiratory tract infections during winter in adults and some proportion of lower respiratory illness in children [ , ] . in contrast, the last two beta coronaviruses -sars-cov and sars-cov- -jumped to the human population in and respectively, causing acute pneumonia, with a higher mortality rate. respiratory coronaviruses probably spread in a fashion similar to that of the rhinoviruses, via direct contact with infected secretions or large aerosol droplets [ ] [ ] [ ] [ ] . protein-protein binding assays have confirmed that angiotensin-converting enzyme (ace ) is most likely to be the cell receptor through which the virus invades the host cell [ , ] . the scientific community is currently trying to identify the source of the infection, which is still uncertain. according to recent lines of evidence, in late , someone at the huanan seafood market in wuhan was infected with sars-cov- , but specific animal associations have not been confirmed. the viral infectious disease then spread from that first cluster in the capital of china's hubei province to a pandemic. some argue that the involved animals would be bats and pangolins [ ] . the spread of covid- caused by the sars-cov- outbreak has been growing since its first identification in december . on may , , the who's coronavirus disease situation report counted confirmed cases globally since the beginning of the global pandemic and a total of deaths all over the world [ ] . actually, the case fatality rate of the ongoing covid pandemic (the ratio between confirmed deaths and confirmed cases) is . % for the world and . % for italy [ , ] . as a comparison for this global value with the case fatality rate of other coronavirus outbreaks, it was % for sars-cov and % for mers-cov [ ] , instead and approximately between . %- . % for the seasonal flu [ ] [ ] [ ] [ ] . covid- is a new disease and there is limited information regarding risk factors for developing a severe case. what we know is that the covid- pandemic has shown an opposite behaviour than that of other global infectious diseases. indeed, for many other viral and bacterial diseases, such as the previous 'spanish flu' pandemic in and malaria (which is still endemic in many areas of the world), the majority of deaths were young and children; for covid- cases, the elderly are at the greatest risk of dying if infected with the virus. yet, old age is not an isolated risk factor for developing a severe acute viral pneumonia by covid- . based on currently available information, older adults, but also people of any age, who have serious underlying medical conditions might be at higher risk for severe illness. in fact, analysing the case fatality rate of each condition shows that those with an underlying health condition have a higher percentage than those without. more than % of people with a cardiovascular disease, more than % of people with diabetes, % with chronic respiratory disease or moderate to severe asthma, % with hypertension, and more than % with cancer who were diagnosed with covid- have died [ ] . other important risk factors for developing severe complications of covid- disease are related to immunocompromised status due to congenital and acquired immune-deficiencies (i.e., cancer treatment, bone marrow or organ transplantation, autoimmune deficiency syndrome, prolonged use of corticosteroids and immunosuppressive drugs), severe obesity (body mass index of or higher), hypertension, liver disease, chronic kidney disease undergoing dialysis, and cerebrovascular diseases [ , [ ] [ ] [ ] . a possible reason why the elderly are most at risk of dying from covid- might be the fact that they are also most likely to have underlying health conditions. italy and other european countries emerged early on as the countries with the largest outbreaks of the novel sars-cov- outside asia. several prevention and containment measures have been applied worldwide to contain the covid- disease. in italy, the particular seriousness of covid- disease regarding morbidity and mortality and the enormous overload of intensive care units (icus) brought about the italian government's establishment of a series of decree laws [ ] , from february to may , to apply strict and extensive containment measures in all of the italian territory. most non-essential commercial activities were temporarily stopped. the decrees also restricted movements within the regional territory and beyond it, except for proven work reasons, absolute urgency, or for health reasons. but, first of all, to limit the spread of the contagion, careful personal hygiene measures were highly recommended, such as frequent hand-washing, interpersonal safety distancing of at least m to avoid close contacts with potentially infected people, the mandatory use of masks in public closed spaces, and the constant sanitization of public spaces. thanks to all these measures and practices, on may , , the italian government declared initiation of the so-called "phase two" [ ] [ ] [ ] , a period of greater freedom but always with strong recommendations of respecting constant hygiene measures and being on alert for eventual rise in new cases. social distancing is fundamental to prevent the inter-human spread of covid- though flügge's drops, produced as a result breathing, talking, sneezing, or coughing and able to contaminate surfaces. according to the latest release from the national health commission, known as the prc, sars-cov- is believed to be transmitted mostly through respiratory droplets and close contacts. prolonged exposure to high concentrations of aerosols may facilitate transmission. spread is also possible through the conjunctiva. it will be very important during the summer of to better investigate the possible role of air conditioning systems in increasing the virus circulation. finally, an observational study found that sars-cov- does not seem to be present in breast milk and its transmission may take place through respiratory droplets rather than the milk; for this reason, it is not recommended to interrupt breastfeeding [ , ] . sars-cov- persistence (and infectivity) on different surfaces (liquid, solid, or gaseous) is still debated. the % tissue culture infective dose (tcid ) is the measure of infectious virus titre. this endpoint dilution assay quantifies the amount of virus required to kill % of infected hosts or to produce a cytopathic effect in % of inoculated tissue culture cells. a recent experiment performed using aerosols (< µm) containing sars-cov- ( . tcid per ml) showed a reduction from . to . tcid per l of air after h, a reduction from . to . tcid per ml on plastic after h, and the same reduction after h on stainless steel [ ] . these results show a persistence of sars-cov- for many hours on surfaces in experimental conditions, but further peer-reviewed investigation on this topic is needed because it represents an environmental and public health problem concerning hospitals (especially in covid departments), schools, offices, and every day public places. furthermore, previous studies have found that air pollution is a risk factor for respiratory infection, by carrying microorganisms, but it also causes oxidative, proinflammatory and immunological damage to the lungs. various recent studies have explored the relationship between ambient air pollutants and covid- infection. most of these have shown a relationship between long-term exposure to air pollution in cities and risk of infection [ ] [ ] [ ] [ ] . another study investigated in the united states whether long-term average exposure to fine particulate matter (pm . ) is associated with an increased risk of covid- death. it was found that an increase of only μg/m in pm . increases vulnerability to experiencing the most severe covid- outcomes, with statistically significant evidence that this exposure is associated with a % increase in the covid- mortality rate [ ] . however, caution should be used in translating high values of conventional aerosol metrics, such as pm . and pm concentrations, in a mortality predictive factor, because many biases may interfere in a real-life situation, relating to different temperature, humidity, and ultraviolet radiation. we can interpret these data considering air pollution as an additional risk factor for covid- disease, which might contribute to increasing the vulnerability and the clinical outcome, probably also through a previous increase in heart diseases, lung problems and cancer. this could also partially explain the prevalence of the infection in the most industrialized cities of the world, northern italy included, and the effect of national lockdown. finally, it could provide implications for the control and prevention of this novel disease and underscores the importance of continuing to enforce existing air pollution regulations to protect human health both during and after the covid- crisis. as the clinical spectrum of covid- ranges widely from asymptomatic cases to severe pneumonia with a high risk of mortality, there is a need for more research to identify the earliest markers of disease severity. the incubation period for covid- is currently estimated to be between d and d. most infected people develop mild to moderate illness and recover without hospitalization. in the early phases of the disease, clinical manifestations are very unspecific, so differential diagnosis should include other infectious viral diseases that appear with the same symptoms, such as influenza and parainfluenza, the common cold caused by rhinovirus, and those caused by human metapneumovirus, human respiratory syncytial virus and adenoviruses, but also with non-infectious (e.g., vasculitis, dermatomyositis) common respiratory disorders. the most common symptoms of covid- infection are fever (not very responsive to antipyretics), dry cough, dyspnoea and increased respiratory rate (in more fragile patients, the dyspnoea may appear at the onset of symptoms, while in younger subjects without other comorbidities it may appear later), myalgia, and intense fatigue [ ] [ ] [ ] [ ] . clinical studies have shown an incidence rate of diarrhoea ranging from % to %. it may precede or trail the respiratory symptoms [ ] . increasing evidence indicates possible faecal-oral transmission [ ] [ ] [ ] . other gastrointestinal symptoms are nausea and vomiting, abdominal pain, and loss of appetite [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (figure ). less common symptoms include sore throat, headache, nasal congestion, hyposmia/anosmia, ageusia, diffuse aches and pains, and conjunctivitis [ ] . the most common skin manifestations associated with covid- infection include a maculopapular or papulovesicular rash, urticarious lesions, painful acral red purple papules, livedo reticularis, and petechial lesions. the most common areas involved are the trunk, hands and feet, with little itching experienced. these symptoms usually present before the onset of respiratory symptoms and spontaneously disappear within d in all patients. there is no demonstrated correlation, in the majority of the studies, between skin lesions and covid- severity [ ] [ ] [ ] . an italian paper reported skin manifestations of covid- in young patients, of whom were asymptomatic and potentially contagious. these lesions began as erythematous-violaceous patches in the acral sites and slowly evolved to purpuric and then to ulcero-necrotic lesions, followed by a complete "restitutio ad integrum" of tissues. burning and itching were present with some of the lesions [ ] . data provided by the who health emergency dashboard (may , , . am cest) indicated confirmed cases worldwide since the beginning of the epidemic, and in the last h. in total, cases were fatal, with in the last h [ ] . looking at these data, the global situation still appears to be in a dramatic evolution. trying to investigate the reasons why, in some patients, the covid- infection rapidly evolves into a severe acute respiratory syndrome, has led to multiple organ dysfunction and even death being a focus of primary importance. the largest cohort of patients with covid- from china (more than , ) showed that illness severity can range from mild ( %) to severe and critical ( % and % respectively), as shown in table [ ] . mild covid- illness is characterized by a lower respiratory disease, evidenced by clinical assessment or imaging, and with blood o saturation level of > % on room air at sea level. these patients should be admitted to a healthcare facility for close observation. covid- severe illness is defined by a blood o saturation level of ≤ % on room air at sea level, respiratory rate of > , arterial partial pressure of o /fraction of inspiration o of ≤ mmhg, or lung infiltrates of > %. these patients may experience rapid clinical deterioration into a critical disease state. the clinical picture of critical patients with severe inflammatory-induced lung disease and with sepsis or septic shock needing intensive care support and mechanical ventilation is characterized by a wide range of signs and symptoms of life-threatening multiorgan dysfunction or failure, including dyspnoea, tachypnoea (respiratory rate of > /min), tachycardia, chest pain or tightness, hypoxemia, virus-induced distributive shock, cardiac dysfunction, elevations in multiple inflammatory cytokines, renal impairment with oliguria, altered mental status, functional alterations of organs expressed as laboratory data of hyperbilirubinemia, acidosis [serum lactate level > mmol/l ( mg/dl)], coagulopathy, and thrombocytopenia. moreover, an exacerbation of underlying comorbidities is often possible [ ] [ ] [ ] [ ] [ ] [ ] [ ] . old-age patients with pre-existing comorbidities or dyspnoea should be hospitalized and closely monitored, especially at - wk after symptom onset. in fact, as already mentioned, in patients with other pre-existing diseases, covid- may be fatal [ , [ ] [ ] [ ] . the sequential organ failure assessment (commonly known as sofa) score is used for the evaluation of multiorgan damage and to predict icu mortality risk based on lab results and clinical data [ , ] , as well as for validation in a paediatric version [ ] . a kawasaki-like disease -a vasculitis for which diagnosis is based on the presence of persistent fever, polymorphic rash, lymphadenopathy, conjunctival injection, changes to the mucosa, swollen extremities and with coronary artery aneurysms as its main complication -has been described in children infected with covid- , with a monthly incidence much more higher than observed for kawasaki disease across the previous years. there was also a high proportion of shock in those children presenting with hypotension and requiring fluid resuscitation and some needing inotropic support. it is still uncertain, however, if this emerging phenomenon is a kawasaki disease type, with sars-cov- as the triggering agent, or if it represents an emerging kawasaki-like disease characterized by multisystem inflammation [ ] [ ] [ ] . current studies are investigating the relationship between different variables and the risk of death of covid- patients hospitalized for pneumonia. the pulmonary imaging techniques for diagnosis of covid- lung damage include an initial evaluation with chest x-ray, ultrasound, and, if indicated, computed tomography. electrocardiogram should be performed if indicated, especially in patients with september , volume issue cardiovascular risk factors. laboratory testing includes a complete blood count with differential and a metabolic profile, including liver and renal function tests. a chinese sex-, age-and comorbid illness-matched case-control study identified lymphopenia (cd + cd + tcells ≤ cells· μl - ) and cardiac troponin i value of ≥ . ng· ml - as negative prognostic factors associated with an increase in risk of mortality from covid- pneumonia [ ] . many studies have designed different increased laboratory results as early predictors of critical illness, such as leucocytosis with agranulocytosis, elevated lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bilirubin, creatine phosphokinase, myoglobin and cytokines [i.e., interleukin (il)- , il- , il- , granulocyte colony-stimulating factor, interferon gamma-induced protein , monocyte chemoattractant protein- , macrophage inflammatory protein- alpha, and tumour necrosis factor-alpha]. although, these findings remain to be validated by further studies. measurements of inflammatory markers such as c-reactive protein, ddimer, and ferritin, while not part of standard care, may have prognostic value [ ] [ ] [ ] . during infectious disease outbreaks, triage is particularly important to separate patients likely to be infected with the pathogen of concern. the cdc has released comprehensive guidelines for management of patients with covid- , including those who are critically ill [ ] . it would be very important to make hotlines available that patients can call to notify the facility that they are seeking care and which can be used as telephone consultation for patients to determine the need to visit a healthcare facility; through this, patients could be informed, before arriving for triage, of preventive measures to take as they come to the facility (e.g., wearing a mask and having tissues to cover cough or sneeze). moreover, healthcare facilities should consider telemedicine (e.g., cell phone videoconference or teleconference) to provide clinical support without direct contact with the patient [ ] . emergency departments should have a "respiratory waiting area" for patients coming in with respiratory symptoms (suspected covid- patients), with clear signage at the entrance, physical barriers (e.g., glass or plastic screens) installed to limit close contact between registration desk personnel and potentially infectious patients. a facemask should be given to patients with respiratory symptoms as soon as possible, if they do not already have one. the number of accompanying family members in the waiting area should be limited. dedicated clinical staff (e.g., physicians or nurses) should be assigned for physical evaluation of patients presenting with respiratory symptoms at triage. these staff should be trained on triage procedures, covid- case definition, and appropriate personal protective equipment (commonly referred to as ppe) use (e.g., mask, eye protection, gown, and gloves) [ ] . all the hospital staff (healthcare workers, lab technician, cleaners) and visitors must protect themselves and others by correctly using the ppe and respecting standard precautions, which will include performing hand hygiene frequently (with an alcohol-based hand rub if your hands are not visibly dirty or with soap and water if hands are dirty), contact and droplet precautions, selection of ppe-based risk assessment (e.g., n respirators or powered, air-purifying respirators rather than a surgical mask, eye protection such as face shield or goggles, gowns, simple gloves or heavy-duty gloves, and boots or closed work-shoes), cleaning, disinfection and injection safety practices, and single-patient dedicated medical equipment (e.g., stethoscopes, blood pressure cuffs, and thermometers) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the confirmed cases must be hospitalized, possibly in single, isolated rooms with negative air pressure as well as a dedicated bathroom and anteroom. if not possible, the confirmed cases must be, in any case, hospitalized in a single room with a dedicated bathroom and transferred as soon as possible to a safe structure. confirmed covid- patients may be hosted in the same room. if available, airborne infection isolation rooms should be used. covid- patients are often very complex and require a multidisciplinary medical team which includes at least the following specialists: emergency doctor, pulmonologist, infectious disease specialist, critical care physician, and medical laboratory technician [ ] [ ] [ ] . a first approach during triage is using the quick-sofa (commonly referred to as the qsofa) for a rapid identification of high-risk septic covid- patients [ , ] . the american disease cdc recommends some criteria priorities for testing patients with suspected covid- , as shown in september , volume issue table . for initial diagnostic testing for sars-cov- , the cdc recommends collecting and testing an upper respiratory specimen (nasopharyngeal/oropharyngeal swab collected by a healthcare professional) for a rapid molecular in vitro diagnostic test, utilizing an isothermal nucleic acid amplification technology intended for the qualitative detection of nucleic acid from the sars-cov- viral rna (real-time reverse transcriptasepolymerase chain reaction) [ , ] . testing lower respiratory tract specimens is also an option. for patients who develop a productive cough, sputum should be collected and tested for sars-cov- . the induction of sputum is not recommended. when under certain clinical circumstances (e.g., those receiving invasive mechanical ventilation), a lower respiratory tract aspirate or bronco-alveolar lavage sample should be collected and tested as a lower respiratory tract specimen [ , ] . plasma cells take at least - d to develop antibodies against covid- . for this reason, serological tests are not sensitive enough to accurately diagnose a recent infection, even in symptomatic patients. clinical recovery has been correlated with the detection of igm and igg antibodies, which signal the development of immunity. actually, there are no data concerning the possibility of reinfection after recovery from covid- . viral rna shedding declines with resolution of symptoms and may continue for days to weeks. however, the detection of rna during convalescence does not necessarily indicate the presence of viable infectious virus [ ] [ ] [ ] . optimal management of covid- is evolving quickly and treatment guidelines based on scientific evidence and experts' opinions with clinical experience are updated frequently. until now, there are no food and drug administration (fda)-approved drugs for covid- and no vaccine is currently available (even if there are many experimental trials, such as the vaccine promoted by the united states' biotech firm moderna); hence, infected people primarily rely on symptomatic treatment and supportive care [ ] . meanwhile, an array of drugs approved for other indications as well as multiple investigational agents are being studied for the treatment of covid- in several hundred clinical trials all over the world. patients with severe infection are currently being treated with o therapy. patients with viral pneumonia, hypoxemic respiratory failure/acute respiratory distress syndrome, sepsis and septic shock, cardiomyopathy and arrhythmia, and/or acute kidney injury often require noninvasive or mechanical ventilation and support in the icu. hemodynamic support is essential for managing septic shock. hospitalization is also fundamental for the management of complications from prolonged hospitalization itself, including secondary bacterial infections, thromboembolism, gastrointestinal bleeding, and critical illness polyneuropathy/myopathy [ ] [ ] [ ] [ ] . at present, the national institutes of health covid- treatment guidelines do not recommend the use of any agents for pre-exposure prophylaxis and post-exposure prophylaxis against sars-cov- outside of the setting of a clinical trial, because no drug has actually been proven to be safe and effective for treating covid- [ ] [ ] . moreover, no specific treatment is also recommended for persons with suspected or confirmed asymptomatic or pre-symptomatic covid- infection. to help reduce fever and diffuse aches related to covid- infection, either acetaminophen or ibuprofen can be prudently used, without exceeding the recommended dose per day of mg [ , ] . most of the recommendations for the management of severely and critically ill patients with covid- are extrapolated from experience with other lifethreatening infections and they do not deviate substantially from the management of other patients with severe diseases; although, special precautions in this infectious disease are required. these measures include high-flow nasal oxygen and noninvasive ventilation in non-severe forms of respiratory failure, while intubation and protective mechanical ventilation are required in severe forms. prone position ventilation and extracorporeal membrane oxygenation have been used many times for very acute patients with refractory hypoxemia despite lung-protective ventilation [ ] . systemic corticosteroids and inappropriate administration of antibiotics are not recommended for the viral pneumonia's treatment, although some centres recommend it but only in case of evidence of bacterial infection [ , , ] . although no antiviral treatments have been approved, several approaches have been proposed to limit viral reproduction, particularly drugs that have been used to treat malaria and autoimmune diseases and already used against past outbreaks, including those of sars-cov and mers-cov. these include antiviral drugs, such as lopinavir, ritonavir, nelfinavir, and remdesivir. the last one is an inhibitor of rna polymerase with in vitro activity against multiple rna viruses [ ] . alpha-interferon ( e.g., million units by aerosol inhalation twice per day), chloroquine ( mg every h), and hydroxychloroquine ( mg every h) are also used. chloroquine was introduced in clinical practice in to treat malaria, while hydroxychloroquine was introduced in and prescribed for the treatment of systemic lupus erythematosus [ ] . the efficacy for systemic lupus erythematosus is based on the capability of this drug to inhibit toll-like receptor signalling and to reduce cytokine production, especially that of il- and il- . starting from this consideration, hydroxychloroquine, and thereafter chloroquine, has been proposed as a helpful treatment for covid- patients, in which some reports showed a direct antiviral effect in vitro due to an interference with ace- receptors [ ] . chloroquine and hydroxychloroquine, which are not fda approved for covid- , are available from the strategic national stockpile for hospitalized adults and adolescents (weighing ≥ kg) under an emergency use authorization [ , , ] . there has been supposition (never proven) that azithromycin may help to reduce the overactive immune response to the sars-cov- infection that otherwise causes inflammatory damage. unfortunately, the most recent human studies suggest no benefit and a strong statement was released, advising against the use of the combination of hydroxychloroquine and azithromycin, underlying the higher risk of death due to lethal heart arrhythmias with both hydroxychloroquine and azithromycin are used alone and especially when used in combination. in fact, it is well known that hydroxychloroquine and, moreover, chloroquine may have several side effects on the extrapyramidal, cardiovascular and digestive systems, which are more severe if associated with other medications (i.e. haemolysis with dapsone, severe hypoglycaemic effects with anti-diabetics, qt elongation, or torsades de pointes with ciprofloxacin and other antimicrobials, etc.) [ ] [ ] [ ] . however, to date, this issue remains controversial [ ] . tocilizumab, a humanized igg monoclonal antibody directed against the il- receptor and commonly used in the treatment of rheumatoid arthritis, has already been used in a rhesus macaque model of mers-cov infection and is currently being used in experimentations against covid- . two chinese large randomized clinical trials, which enrolled over patients and other studies which are also underway in european countries and in the united states are likely to definitively answer the question of whether the drug is effective in treating covid- , and so it could be approved for use and produced in large amounts [ ] . september , volume issue on march, , the fda allowed convalescent plasma from recovered patients to be used in patients with serious or immediately life-threatening covid- infections. these antibody-containing plasma, in many cases, showed the intended effect in fighting the illness, shortening the length or reducing the severity of the disease, but this treatment is still considered experimental and more randomized, controlled studies must be done to test its efficacy and safety. it has been estimated that herd immunity against covid- (that is, an indirect protection given by recovered patients to those who are not immune to the disease) is around % to . % [ ] . moreover, covid- patients show a contemporary hypercoagulation and hypofibrinolytic state due to dysregulation of the coagulation and fibrinolytic systems, with elevated d-dimer and fibrinogen and deposition of fibrin in the air spaces and lung parenchyma caused by the activated tissue factor exposure on damaged alveolar endothelial cells and on the surface of leucocytes. the patients also show significantly elevated levels of plasminogen activator inhibitor released from lung epithelium and endothelial cells. prophylaxis treatment with low molecular weight heparin is considered important to limit covid- patients' coagulopathy, but, at the same time, it is fundamental to degrade pre-existing fibrin in the lung by promoting local fibrinolysis with tissue-type plasminogen activator as intravenous thrombolytic treatment. its nebulizer form is currently in phase ii clinical trial and may provide a targeted approach in covid- patients to degrade fibrin and improve oxygenation in critically ill patients [ ] . new research fields are concerned with the use of immuno-enhancers (interferons, thymosin α- , thymopentin, levamisole, cyclosporine a), vitamins (a, b, c, d, e), alipoic acids, minerals (selenium, zinc, iron), omega- polyunsaturated fatty acids, nacetylcysteine and d-ribose-l-cysteine, probiotics, and the intravenous infusion of allogeneic expanded umbilical cord mesenchymal stem cells that show antiviral and antimicrobial properties, and which must be deepened. the italian college of anesthesia, analgesia, resuscitation and intensive care have reported guidelines to use these cells in covid- patients, in the hope of decreasing the number of patients going to the icu, and also getting them out of icu relatively quickly [ , ] . despite this, there are insufficient data to recommend either for or against the use of any antiviral or immunomodulatory therapy in patients with covid- who have mild, moderate, severe, or critical illness [ ] . researchers are carrying out incessant efforts towards understanding these topics, including on translational regenerative approaches, such as mesenchymal stem cells [ , ] . the sars-cov- pandemic is a serious health problem and a challenge of global concern. during these months, we've had to learn, step-by-step, all about this novel coronavirus, ranging from its origins (which are still uncertain) to its mode of transmission, identifying people most at risk, and searching for old and new strategies which could help patients in fighting against this invisible enemy. still, despite this, even more studies are needed to provide more effective preventive measures and treatment policies and to determine what is the proper social behaviour in public places and the rules of conduct for healthcare professionals' management and resource planning; ultimately, the collective knowledge will lay a solid foundation for winning the battle against this epidemic. world health organization. pneumonia of unknown cause -china rapid progression to acute respiratory distress syndrome: review of current understanding of critical illness from covid- infection what we know so far: covid- current clinical knowledge and research diarrhea during covid- infection: pathogenesis, epidemiology, prevention, and management the occurrence of diarrhea in covid- patients covid- and the digestive system coronavirus disease (covid- ) pandemic burst and its relevant consequences in dental practice comment to the article "olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid- ): a multicenter european study calzavara-pinton p. varicella-like exanthem as a specific 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respiratory syndrome coronavirus breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies treatment of covid- : old tricks for new challenges response to the editorial "covid- in patients with cardiovascular diseases": covid- treatment with hydroxychloroquine or chloroquine and azithromycin: a potential risk of torsades de pointes off-label" use of hydroxychloroquine, azithromycin lopinavir-ritonavir and chloroquine in covid- : a survey of cardiac adverse drug reactions by the french network of pharmacovigilance centers qt prolongation, torsades de pointes, and sudden death with short courses of chloroquine or hydroxychloroquine as used in covid- : a systematic review covid- : researchers raise concerns over chloroquine study that halted who trial prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection covid- : herd immunity and convalescent plasma transfer therapy fibrinolytic abnormalities in acute respiratory distress syndrome (ards) and versatility of thrombolytic drugs to treat covid- n-acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why a successful history: probiotics and their potential as antimicrobials current status of potential therapeutic candidates for the covid- crisis mesenchymal stem cells as promoters, enhancers, and playmakers of the translational regenerative medicine expanded umbilical cord mesenchymal stem cells (uc-mscs) as a therapeutic strategy in managing critically ill covid- patients: the case for compassionate use key: cord- - stbjl authors: liu, tianyuan; nogueira, leandro balzano; lleo, ana; conesa, ana title: transcriptional differences for covid- disease map genes between males and females indicate a different basal immunophenotype relevant to the disease date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: stbjl worldwide covid- epidemiology data indicate clear differences in disease incidence among sex and age groups. specifically, male patients are at a higher death risk than females. however, whether this difference is the consequence of a pre-existing sex-bias in immune genes or a differential response to the virus has not been studied yet. we created decovid, an r shiny app that combines gene expression data of different human tissue from the genotype-tissue expression (gtex) project and the covid- disease map gene collection to explore basal gene expression differences across healthy demographic groups. we used this app to study differential gene expression between men and women for covid- associated genes. we identified that healthy women present higher levels in the expression of interferon genes and the jak-stat pathway leading to cell survival. acknowledges that a vaccine solution for the general population might not be ready before mid or late . at the same time, very recent re-infection cases suggest that immunity may not always persist, and active disease surveillance of past cases may be required. as the second wave of the pandemic steadily progresses in europe and the us, possibly with new variants of the virus and affecting a more diverse and younger population, novel or improved treatment targets and options are likely to remain as important for the management of the disease. one of the most intriguing aspects of covid- is the different grade of severity it affects different people. although the acknowledged risk factors such as pre-existing conditions such as cardiovascular disease (zheng et al., ; d. wang et al., ; chen et al., ) , diabetes (apicella et al., ; fang et al., ; guo et al., , ) , obesity (simonnet et al., ) , age (liu et al., ; cdc covid- response team, ; litcovid) and sex (higher mortality in men than women (gebhard et al., ; jin et al., ) suggest a relationship between the physical condition and disease progression, the precise pathways of this relationship have not been clearly established yet. sex-associated differences between males and females that have been proposed to affect covid- incidence include lifestyle (e.g., smoking and drinking) and mental health. the smoking population of males is higher than females, and smoking may be a risk factor for severe covid- (cai, , ; jamal et al., ; grundy et al., ) . in contrast, men differ from women during the pandemic in sleeping and signs of depression (c. . a recent study showed that male and female covid- patients differed in their immune response, with the first showing a stronger cytokine response while the second is having a higher t-cell activation pattern (takahashi et al., ) . interestingly, the pattern of covid- risk factors is not fully shared with other similar recent pandemics such as sars, mers, h n . for example, sars patients were more frequent among the healthy young people (yin and wunderink, ) . age and complications, but not gender, was the most significant risk factors for mortality in the arab and south korean studies of mers (park et al., ) . several studies on the h n influenza revealed the younger age, chronic conditions, and female sex as risk factors of the disease (viasus et al., ) . this suggests that the observed covid- risk factors, especially those associated with age and sex groups, might be the result of specific interactions of the sars-cov- with intrinsic physiological characteristics, including the basal immunophenotype, of these population groups. however, which specific immunological characteristics imprint an existing condition that how this relates to covid- remains to be explored. the covid- disease map is a valuable resource to investigate the molecular responses to sars -cov- infection and understand the biological pathways leading to the severe manifestations of the disease. this database also creates an opportunity for interrogating existing molecular data on differences associated with covid- risk factors in the general population. in this paper, we present the decovid app, a shiny app, to explore basal expression level differences in covid- disease map genes between men and women and different age groups. we used data from the gtex database, which contains rna-seq profiles for hundreds of demographically diverse healthy individuals in multiple tissues and allows us to interrogate covid- genes globally or individually. we used this resource to study basal expression differences in covid- genes between man and woman and found that a different immunological state is prevalent in both genders. interestingly some of these differential pathways have been shown to be relevant for disease severity progression and be characteristic of the sex-biased immune response to the virus, providing ground for hypotheses on the molecular basis of the covid- sex-bias. we anticipate that the decovid app could be a useful tool for researchers to explore the molecular etiology of covid- demographic differences. the decovid shiny app combines a selection of human tissue specific gtex data with the covid- disease map database to allow quick exploration of basal gene expression values and differences in the healthy human population for genes described to be important for covid- . we included data from blood, lungs, heart, kidney, stomach, and brain for being tissues described to be affected by sars-cov- infection. the sample size for different tissues is different in the gtex datasets. the largest sample size is the whole blood with samples, while the smallest dataset is the stomach tissue with samples (figure a) . the total number of collected covid- disease map is genes , . a gene ontology (go) term enrichment analysis indicates that genes related to cytokines, response to virus, innate immune response, transcriptional regulation, and kinase activity populate this collection (figure b) . the decovid app is a user-friendly implementation to allow an analysis of these data for researchers without strong bioinformatics skills (figure ) . extensive documentation and video tutorials are provided to the facility for a quick start. users should indicate the demographic factor for differential expression analysis. in case age is selected, the age threshold value to classify samples as old or young should be specified. once a significance p.value and a fold-change threshold are provided, the app computes differential expression and provides results as a list of genes and explore the distribution of gene expression values for specific genes of the covid- disease map collection or investigate the enrichment of specific immune responserelated functions among genes with sex or age expression biases. screenshot of the decovid app showing on the left dialog panels for input parameters (contrast type and significance thresholds), and on the right results panels with list of differentially expressed genes, gene-specific expression plots and enrichment analysis. using decovid, we analyzed the extent of gene expression differences among demographic groups across different covid- affected tissues (table ) . the analysis revealed a wide-range of significant gene expression differences for covid- genes between when comparing sexes and age groups for all evaluated tissues. differences where lower when imposing a fold-change threshold of . for the magnitude of the mean expression level. whole-blood were the tissue with the highest number of expression differences for covid- genes followed by the brain. from this simple analysis, we concluded that the covid- disease map represents a disease signature with significant differences across demographic groups that may be worth exploring for hypothesizing on the molecular basis for differences in disease incidence among these groups. decovid identified basal sex-biased expression differences in immune-response genes associated to differential disease incidence between man and women since covid- fatality rates are consistently higher in men than women in many different countries worldwide (supplementary figure ) , we hypothesized that sex is a pre-existing condition that influences the severity of the disease and that the study of basal differences in expression differences in covid- relevant genes in healthy individuals could reveal insights into the molecular basis of these differences. we used the decovid app to explore these differences and concentrated on the blood tissue for capturing systemic immune responses. a total of genes (p.value < . and fold change > . ) were differentially expressed between men and women in blood samples, regardless of the age group (figure a) . we found high expression in females of genes involved in the innate immune response, particularly type i interferons, such as ifna , ifna , ifne, ifit , and ifit , anti-apoptotic regulators such showed that female patients have a high t-cell level (takahashi et al., ) , which were postulated as a critical factor for the differential fatality incidence between sexes. therefore, with a high level of baseline expression of type interferons, female patients are more likely to maintain a high t-cell level in the early stage, which might lead to a milder evolution of the disease. here we speculate that these innate immunity pathways may play a crucial role in the a b early control of respiratory coronavirus infection (figure b) . moreover, data indicate higher expression in women of jak, multiple members of the stat family, and cell survival genes such as mcl and bcl a. it has been shown that the jak-stat signaling pathway mediates the synthesis of anti-apoptotic regulators such as mcl , bcl a, and bcl l upon induction of high type interferon levels (sepúlveda et al., ) , agreeing with the observed gene expression phenotype in females. finally, we detected significant increased tumor necrosis factor tnfsf b expression in females. this gene stimulates the synthesis of chemokines ccl and cxcl through nfkb subunit pathway, leading to b-cell homing through the synthesis of ccl (lópez-giral et al., ) , lymphopoiesis through the synthesis of cxcl (silva et al., ; sen, ) , and development and survival through the synthesis of tnfsf b (caamaño et al., ; gerondakis and siebenlist, ) . since b-cells are part of the humoral immunity component of the adaptive immune system and are responsible for mediating the production of antigen-specific immunoglobulins (ig) directed against invasive pathogens, higher levels of these chemokines in females might lead to a faster response against a potential viral infection. in males, in turn, the mentioned processes were comparatively downregulated, while the only process upregulated compared to females were those related to the expression of pro-inflammatory cytokines (figure b) , particularly il , il and il . also, chemokine cxcl levels are higher in men, which supports the hypothesis of a first stage of male response against a viral infection being more related to inflammation and th cell differentiation processes. here we present the decovid app as a resource to explore sex and age differential expression patterns in the healthy population for genes described to be involved in covid- disease pathways. the gtex data, used in this work has been recently mined for sex-specific expression differences concluding that these are tissue-specific and generally small, also observed in our analyses (oliva et al., ) . this study focused on the sex-specific genetic effects on gene expression associated to complex genetic traits. in our application we repurpose the valuable gtex database for the study of covid- relevant pathways while providing a mechanistic model to interpret differential expression results. we used decovid to investigate the gene expression differences between men and woman that could explain differences in disease severity among these demographic groups, with men being more likely to have a fatal outcome than women. we found that two major immune response pathways were differentially expressed between men and women in the healthy population. women showed higher expression of interferon genes while men have higher levels of pro-inflammatory th cytokine genes. these two pathways have been proposed to be critical factors for fatal response to the virus infection, with patients that present an interferon-mediated response having a better prognosis than those responding with a massive cytokine activation (blanco-melo et al., , ; hadjadj et al., ) . in agreement, animal models of sars-cov- and mers-cov infection showed that failure to elicit an early type interferon response correlates with the disease severity (channappanavar et al., ) . perhaps more importantly, these models demonstrate that timing is key, as type interferons are protective at the early stages of a viral disease. on the other hand, clinical studies showed a differences in the immunophenotype between female and male covid- patients with women having a more robust t-cells activation while male patients presenting a higher plasma level of pro-inflammatory cytokines (takahashi et al., ) . in this study we showed that these immune system differences, which are critical to covid- progression, represent a sex-associated pre-existing condition that is likely to create a differential predisposition to disease outcome between men and women and may explain the observed sex-bias in covid- mortality. future studies should address whether these gene expression patterns translate into functional differences in patients infected with sars-cov- , whether they have prognosis value and of if they present any relationship with hormonal levels, which have also been linked to covid- severity (strope et al., ; channappanavar et al., ; scully et al., ) . all together our results show that decovid is a useful resource to investigate the status of covid- genes across demographic groups and human tissues and postulate on the differential pathways to disease operating in each case. we used rna-seq data from the genotype-tissue expression project (gtex) that contains data from tissue sources obtained from deceased but considered healthy individuals from both sexes and a wide range of ages (lonsdale et al., ) . gene read counts matrix and the annotation files from the gtex portal (https://www.gtexportal.org/home/datasets). in other to provide a homogenous set of human tissue reference samples, gtex data were analyzed for possible biases, and samples belong to individuals with a "ventilator case" label as a cause of death were discarded as they clearly segregated from remaining samples on a principal component analysis plot (supplementary figure ) . the covid- disease map genes were downloaded from https://github.com/wikipathways/cord- and available on march th, . the list is mined from pmc papers in covid- open research dataset (cord- ) using machine learning approaches, so they are positively related to the covid- disease process (lu . the decovid software is a shiny app written in r with a user-friendly interface. the app can be installed through a docker image or directly download from github (see supplementary materials for installation). decovid already has integrated all essential data from gtex, and no additional downloads are necessary. differential gene expression is calculated using edger (robinson et al., ) and multiple testing correction is applied following the benjamini and hochberg(bh)method (benjamini and hochberg, ) . results are provided either as lists of differentially expressed genes, heatmaps of sex and age mean expression values, and gene-specific boxplots showing the distribution of expression values across demographic groups. go enrichment analysis of significant gene sets is provided through the cluster profile r package and uses the list of covid- disease map as a reference set (yu et al., ) . covid- in people with diabetes: understanding the reasons for worse outcomes covid- disease map, building a computational repository of sars-cov- virus-host interaction mechanisms mers transmission and risk factors: a systematic review edger: a bioconductor package for differential expression analysis of digital gene expression data considering how biological sex impacts immune responses and covid- outcomes control of b lymphocyte apoptosis by the transcription factor nf-kappab bcl- expression is mainly regulated by jak/stat pathway in human cd + hematopoietic cells the tlr -trif pathway protects against h n influenza virus infection transcription factors of the alternative nf-κb pathway are required for germinal center b-cell development high prevalence of obesity in severe acute respiratory syndrome coronavirus- (sars-cov- ) requiring invasive mechanical ventilation sexx matters in infectious disease pathogenesis are sex discordant outcomes in covid- related to sex hormones? sex differences in immune responses that underlie covid- disease outcomes factors associated with severe disease in hospitalized adults with pandemic (h n ) in spain immediate psychological responses and associated factors during the initial stage of the coronavirus disease (covid- ) epidemic among the general population in china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan mers, sars and other coronaviruses as causes of pneumonia key: cord- - qhumjas authors: brown, lisa; murray, virginia title: examining the relationship between infectious diseases and flooding in europe: a systematic literature review and summary of possible public health interventions date: - - journal: disaster health doi: . /dish. sha: doc_id: cord_uid: qhumjas introduction many infectious diseases are sensitive to climatic changes; specifically, flooding. this systematic literature review aimed to strengthen the quality and completeness of evidence on infectious diseases following flooding, relevant to europe. methods a systematic literature review from – was performed. focused searches of the following databases were conducted: medline, scopus, pubmed, cochrane library, and evidence aid. personal communications with key informants were also reviewed. results thirty-eight studies met the inclusion criteria. evidence suggested that water-borne, rodent-borne, and vector-borne diseases have been associated with flooding in europe, although at a lower incidence than developing countries. conclusion disease surveillance and early warning systems, coupled with effective prevention and response capabilities, can reduce current and future vulnerability to infectious diseases following flooding. defining what constitutes a flood can be quite complex as floods can take many forms; therefore, no universal definition exists. generally and in the context of this review, a flood is defined as the overflow of areas that are not normally submerged with water or a stream that has broken its normal confines or has accumulated due to lack of drainage. overall, different flood characteristics affect the severity of the flood event; specifically, regularity, speed of onset, velocity of flow, and depth of water. quantifying the level of flooding has proven to be difficult; however, the emergency events database (em-dat) provides information about flood events and the impact of floods. for a flood to be classified as a disaster or flood event by em-dat one of the criteria must be fulfilled: either ten or more people killed; or more people affected; declaration of a state emergency; and/or call for international assistance. em-dat defines a flood as a significant rise in water level in a stream, lake, reservoir, or coastal region and includes general river floods, flash floods, and storm surges or coastal flooding. flood disasters hit some european regions very frequently, and in some circumstances every year. in europe from - , flash floods and general floods were reported by em-dat. in terms of the number of people affected, out of the most important floods ever recorded in europe occurred during the - decade. a study concluded a rising number in flood disasters from - in the european union (eu). according to frei et al. there has been a significant trend toward increased intense winter rainfall events in europe. other studies do not find a rising incidence of flooding. for example mudelsee et al. examined river flood patterns in central europe, and despite the occurrence of two flood events exceeding the -year flood level in and , found no increased trend in extreme flood frequency over recent decades. analyzing the more frequent, small-magnitude flood events as well as high-magnitude floods can make it easier to detect shifting trends in flood frequency. flood trend analysis is essential to understand future flood risk and vulnerability. • what evidence-based public health interventions are used to minimize infectious disease incidence following flooding. • knowledge gaps and issues for further research. the initial search generated , relevant articles. after reviewing the abstracts, full-text articles were examined in more detail for eligibility. of these articles, peer-reviewed articles were found to fit the inclusion criteria. increased infectious disease transmission and outbreaks following global flood events have been documented ( table ). the study design and main results of all papers found meeting the inclusion criteria are listed in detail in appendices a-d. some articles and gray literature not meeting the specific inclusion criteria were incorporated into the conceptual framework to give a better contextual outline. water-borne outbreaks are an acute aftermath of flood disasters, mainly as a result of contaminated drinking water supply. intense precipitation can mobilize pathogens in the environment and transport them into the aquatic environment, increasing the microbiological agents on surface water. [ ] [ ] [ ] [ ] chen et al. found extreme torrential rain (> mm) was a significant risk factor for enteroviruses (rr = . ; % ci . - . ) and bacillary dysentery (rr = . ; % ci . - . ). globally, water-borne epidemics have shown an increasing trend from - which coincides with the increasing number of flood events. according to a global systematic literature review performed by cann et al. the most common water-borne pathogens to be identified following flooding were vibrio spp. the most common water-borne pathogens associated with heavy rainfall were campylobacter, followed by vibrio spp. appendices a, b list published studies which have reported post-flood increases in cholera, cryptosporidiosis, non-specific diarrhea, rotavirus, and typhoid and paratyphoid. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] several studies have implicated excess rainfall in water-borne disease outbreaks because of the transportation of bacteria, parasites, and viruses into water systems. marcheggiani et al. showed a potential association between flood events and a range of water-borne infectious diseases in italy; including, legionellosis, salmonellosis, hepatitis a, and infectious diarrhea. reacher et al. performed a historical cohort study following a severe flood in in lewes, both climatic and non-climatic impacts, such as land-use dynamics, are expected to influence future flooding in europe. although considerable limitations remain in the ability to make robust projections of changes in flood size and frequency due to climate change, common projections appear to be emerging. according to the latest intergovernmental panel on climate change's (ipcc) srex report there is a - % probability that the intensity of heavy precipitation and the proportion of total rainfall will increase particularly in northern mid-latitudes and high latitudes of europe. the highest total precipitation increases are projected to occur during the winter months. although the ipcc states a general decrease in mean precipitation in the southern european region, rainfall may become more irregular and intense. however there remains low confidence in projections of changes in riverine floods. climate change is likely to increase the frequency of storm surges and coastal flooding due to rise in sea levels, and threaten an additional . million people per year in europe by the s. overall, changes in the climate that may affect the transmission of infectious diseases include temperature, humidity, altered rainfall, and sea-level rise. flooding can have a range of health impacts but this review focused solely on infectious diseases. the diseases most likely to be affected by flooding are those that require a vehicle for transfer from host to host (water-borne) or a host/vector as part of its life cycle (vector-borne). flood-affected areas serve as ideal breeding grounds for pathogens and may alter vector breeding grounds and zoonotic reservoirs. , where infectious disease transmission is endemic, it can present a major public health concern following flooding. the risk of infectious diseases following flooding is exacerbated by the fact many factors work together to increase incidence. the significance of the association between precipitation and disease is potentially amplified when considering the effects of global climate change and land use changes. flooding can alter the equilibrium of the environment and may affect the incidence and geographic range of climate-sensitive infectious diseases. a better understanding of the associations and underlying mechanisms of infectious disease outbreaks following flooding will help support evidence-based flood policies and mitigation strategies. this systematic literature review aimed to identify and examine the relationship between infectious disease incidence and flooding in order to gain a better understanding of: outcome (combined with or) amoebiasis, bacillary dysentery, burul*, campylo*, chikungunya, cholera, communicable disease*, contamination, crypto*, dengue, dengue virus, dermatitis, diarrhea*, diarrhea*, disease*, disease vector*, disease outbreak*, epidemic*, enteric fever, escherichia coli, gastrointestinal, giardia*, hanta virus infections, health, health effect*, health impact*, hemorrhagic fever, hepatitis a, hepatitis e, illness, infectio*, infectious disease*, japanese encephalitis, legionellosis, leptospirosis, lyme disease, lymphatic filariasis, malaria, morbidity, mosquito*, norovirus, naeg*, outbreak*, onchocerciasis, physical health, plague, pollut*, public health, q fever, risk factor*, rodent*, rodentborne, rodent-borne, rodent related, rodent-related, salmonellosis, sars virus, severe acute respiratory syndrome, shigellosis, schistosomiasis, tick*, tick-borne encephalitis, tularaemia, tularemia, typhoid, water, waterborne, water-borne, water related, water-related, west nile fever, vector*, vectorborne, vector-borne, vector related, vector-related, yellow fever, yersini* risk, rising temperatures, overcrowding, poor sanitation, poor health care, poverty, and an abundance of rats and other animal reservoirs. rodent-borne pathogens can be indirectly affected by ecological determinants of food sources which have an effect on the size of rodent populations. for example, lack of garbage management and collection following flooding where rubbish is left on the streets contributes to an increased rodent population. appendices a, c summarize the key studies assessing the relationship between flooding and rodent-borne diseases. outbreaks of leptospirosis were observed in the czech republic following floods in and . , the rate of serologically confirmed cases of leptospirosis was three times higher than usual at . cases/ , inhabitants (average incidence rate was . cases/ , inhabitants). the first leptospirosis outbreak in austria in july , involved four athletes who swam in recreational waters during a triathlon. heavy rains had preceded the triathlon ( mm). this outbreak demonstrates a risk of contracting leptospirosis in recreational waters, especially after heavy rainfall. in marseilles, france the incidence of leptospirosis identified in the laboratory increased significantly between january and july (p < . ). between and , the rate of leptospirosis incidence in southern france was very low, . cases/ , inhabitants. in , this incidence increased to england. the risk of gastroenteritis was significantly associated with depth of flooding in people whose households were flooded (rr = . ; % ci . - . ; p for trend by flood depth = . ). additionally, an outbreak of norovirus in american tourists was linked to direct exposure to floodwater contaminated with raw sewage in germany. earlier research has shown an association between waterborne diseases and flooding in high-income countries. from - , more than half of the water-borne disease outbreaks in the united states were preceded by heavy rainfall (p = . ). research from finland found that water-borne disease outbreaks from - were associated with un-disinfected groundwater contaminated by floodwaters and surface runoff. surveys in high-income countries where individuals reported their own symptoms have indicated an increase in water-borne diseases following flooding. , [ ] [ ] [ ] rodent-borne rodent-borne diseases are climate sensitive and may increase during heavy rainfall and flooding because of altered patterns of human-pathogen-rodent contact. flooding and heavy rainfall have been associated with numerous outbreaks of leptospirosis from a wide-range of countries around the world. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] areas at the highest risk for leptospirosis outbreaks are those where multiple risk factors are likely to coexist; such as, increased flooding ahern et al. reviewed earlier studies addressing flood-associated outbreaks of leptospirosis from a wide-range of countries: argentina, brazil, cuba, india, korea, mexico, nicaragua, portugal, and puerto rico. in in the krasnodar territory in russia, a severe outbreak of leptospirosis took place in connection with a high flood. sanders et al. stated that flooding after heavy rain favors leptospires. it prevents animal urine from being absorbed into the soil or evaporating; therefore leptospires may pass directly into the surface water or persist in mud. the evidence of this review, supported by several other reviews, suggests the association between leptospirosis and flooding is fairly robust even in high-income countries. vector-borne precipitation changes are known to effect the reproduction, development, behavior, and population dynamics of arthropod vectors, their pathogens, and non-human vertebrate reservoirs. mosquito-borne infections tend to increase with warming and certain changes in rainfall patterns. vector-borne diseases are unlikely to be a problem during the onset phase of the flood, as many vector breeding habitats are expected to . cases/ , inhabitants. the first three autochthonous cases identified in marseilles (october ) were preceded by heavy rainfall. the study showed the first autochthonous case was identified after a period of flooding preceded by heavy rainfall over several days ( . mm/day; . mm/day; mm/ day with an episode of mm/ hr). similarly, the other two autochthonous cases occurred during a period of high rainfall ( . - . mm). pellizzer et al. performed a sero-epidemiological study to evaluate the risk of leptospirosis in a population in northeast italy exposed to a severe flood event. this area is endemic for leptospirosis and exhibits and average of cases/ , inhabitants. seven out of subjects exposed to floodwaters exhibited anti-leptospira specific igm antibodies and five were confirmed positive by micro-agglutination test. re-testing a few months later found significant antibody titers greater than against serovar copenhangeni in three cases ( . % seroconversion rate). overall, the rate for seroconversion for leptospirosis appeared to be low, and while flooding appeared to be the sole risk factor, confirmation was not possible due to a lack of a control group. ahmed et al. pakistan, cross-sectional study- , flood affected individuals interviewed to determine frequency of infectious diseases. gastrointestinal ( %), skin and soft tissue infection ( %), conjunctivitis ( %), ear, nose and throat infection ( %), respiratory tract infection ( %), suspected malaria ( %). no comparative data before flooding. bich et al. vietnam, cross-sectional study-rural and urban districts interviewed within mo after flood about social, economic, and health impacts. in each district, a flooded commune and a less affected commune (control commune) were selected. no statistically significant differences in proportion of dengue cases in flood affected and less affected communes. higher proportions of pink eye and dermatitis in severely flood affected communes. in flood affected communes, / urban cases (p < . ) and / rural cases (p < . ) contracted pink eye after flood. in flood affected communes, / urban cases and / (p < . ) rural cases contracted dermatitis after flood. chen et al. taiwan, - routine data-analysis of a database integrating daily precipitation and temperature and an infectious disease case registry. heavy precipitation ( - mm) a significant risk factor for enteroviruses (rr = . ; % ci . - . ) and dengue fever (rr = . ; % ci . - . ). extreme torrential rain (> mm) a significant risk factor for enteroviruses (rr = . ; % ci . - . ) and bacillary dysentery (rr = . ; % ci . - . ). associations between precipitation levels and enterovirus infections, japanese encephalitis (p < . ), and stronger linear relationships between precipitation and bacillary dysentery, dengue fever, leptospirosis (p < . ). marcheggiani et al. italy, - routine data-national statistics collected by italian ministry of health. association between hepatitis a, salmonellosis, infectious diarrhea, leptospirosis, cutaneous and visceral leishmaniasis, legionellosis and flood events from - seemed to exist. milojevic et al. bangladesh, controlled interrupted time series-diarrheal incidence of a cohort of , residents classified as flooded or non-flooded in . after fully controlling pre-flood rate differences and seasonality, no clear evidence of excesses mortality or diarrhea risk during/ after flooding. no evidence of excess risk from acute respiratory illnesses during flood but moderate increase in risk mo after flood (rr = . ; % ci . - . ). su et al. taiwan, routine data-to clarify association between leptospirosis and melioidosis epidemics and flooding. positive correlation for leptospirosis (r = . ; p < . ) and for melioidosis (r = . ; p < . ) with cumulative rainfall. increase in melioidosis cases significantly associated with > mm/day (p < . ). number of leptospirosis cases positively correlated with -h cumulative rainfall (r = . ; p = . ). appendix b. studies assessing the relationship between infectious diseases and flooding: water-borne apisarnthanarak et al. thailand, case report- melioidosis patients located through active case surveillance. cases reported excess flooding of homes and had traditional risk factors for melioidosis. all cases survived. auld et al. canada, outbreak investigation-e. coli o :h and campylobacter outbreak. outbreak occurred several days after heavy rainfall ( -d accumulation - mm). heavy rainfall hypothesized as a causative factor of the outbreak. carrel et al. bangladesh , - longitudinal study- -y data cluster analysis of health surveillance and geographic information system to investigate temporal and spatial distribution of cholera following flood protection interventions. , confirmed cholera cases. two clusters of lower than expected cases, clusters of higher than expected cases found (p < . ). following flood protection interventions, overall decrease in cholera incidence, differences in the geography of high vs. low spatial clusters of cholera, and shifts in location of unusually high spatio-temporal cholera clusters. harris et al. bangladesh hashizume et al. bangladesh, routine data-number of observed cases of cholera and non-cholera diarrhea per week during flood and post-flood periods compared with expected numbers. during flooding, cholera cases . times higher ( % ci - ) and non-cholera cases . times higher ( % ci . - . ) than expected. post-flood period, cholera cases . times higher ( % ci . - . ) and non-cholera cases . times higher ( % ci . - . ). ko et al. taiwan, routine data-melioidosis outbreak. melioidosis cases identified following flooding. onset within d. qadri et al. bangladesh, routine data-diarrheal stools collected from patients during flooding. of stool specimens tested, positive for v. cholerae o ( . %), for shigella spp ( . %), for salmonella spp ( . %). reacher et al. england, historical cohort study-post-flooding survey interview. flooding associated with significant increase in risk of gastroenteritis with depth of flooding (rr = . ; % ci . - . p = . , p for trend by flood depth = . ). schwartz et al. bangladesh , , , routine data-diarrheal patients during the , , and floods compared with non-flood periods. cross-sectional study- , individuals provided flood survey health data. house/yard flooding signficantly associated with gastrointestinal illness (incidence rate ratio = . ; % ci . - . ). volume issue appendix c. studies assessing the relationship between infectious diseases and flooding: rodent-borne amilasan et al. the philippines, hospital-based investigation-investigating risk factors for leptospirosis mortality following flooding. prospective surveillance and retrospective data collection. outbreak of leptospirosis cases, cases died. patients predominately young and male. delayed initiation of treatment, older age, jaundice, anuria, hemoptysis increased risk for death. bhardwaj et al. india, case-control study-identifying risk factors for leptospirosis during flooding. confirmed cases and age and sex matched fever and healthy controls given a questionnaire. chiu et al. taiwan, - routine data-analyze characteristics of patients with laboratory-diagnosed leptospirosis and correlate onset of symptoms with exposure to floodwater. patients identified with history of contact with contaminated soil/water. / patients ( %) suffered from leptospirosis after typhoon. dechet et al. guyana, routine data-laboratory testing on suspected leptospirosis hospitalizations and deaths. confirmed outbreak of leptospirosis after severe flooding. sero-epidemiogical study-evaluated leptospirosis risk in flood-exposed population. / patients exposed to floodwaters exhibited anti-leptospira specific igm antibodies and confirmed positive. re-testing months later found significant antibody titers > against serovar copenhangeni in cases ( . % seroconversion rate). flooding appeared to be sole risk factor, verification not possible due to lack of control group. radl et al. austria, outbreak investigation-leptospirosis. st documented outbreak of leptospirosis in austria. four serologically confirmed cases, all triathlon athletes. triathlon preceded by heavy rainfall ( mm (appendices a, d) older studies have shown associations. in romania, flooded basements were a significant risk factor for wnv in apartment dwellers (p = . ). in , heavy rains in moravia, czech republic resulted in flooding, and mosquito populations in the area amplified immediately. wnv activity was reported in the area. hubálek and halouzka stated environmental factors such as flooding can facilitate the re-emergence of wnv. be overwhelmed by the flood waters. while flooding may initially wash out vector populations, they return when the waters recede. receding flood water can provide ideal breeding habitats. therefore, vector-borne diseases are likely to have mid-term to long-term impacts on health following flooding (fig. ) . vector-borne virus outbreaks are strictly determined by the presence of the pathogen and particular competent disease vectors. the current and future establishment of exotic mosquito species in europe is a cause for serious concern, as the newly introduced species may already be disease vectors or could potentially become vectors. west nile virus (wnv) emerged in europe after heavy rains and flooding, with outbreaks in romania in - , the czech republic in , and italy in . the flood in the czech republic resulted in mass mosquito breeding with a biting frequency peaking at bites per person per minute. specimens from flood-affected residents were examined serologically for mosquito-borne viruses. paired serum samples showed one tahyna virus infection among residents. jiménez-sastré et al. sampled dwellings in tabasco, mexico, post-flood for dengue fever cases and found the geographical distribution of dengue fever cases was associated with the proximity of two permanent bodies of water. chen et al. found heavy precipitation was a significant risk factor for dengue fever (rr = . ; %; ci . - . ). additionally, more non-european appendix d. studies assessing the relationship between infectious diseases and flooding: vector-borne hassan et al. sudan, outbreak investigation-rift valley fever. confirmed human cases including deaths. outbreak followed heavy rainfall with severe flooding. hubalek et al. czech republic, routine data-specimens from residents in flooded area examined serologically for mosquito-borne viruses. antibodies detected after flood for tahyna, sindbis, and batai viruses, with only activity found for tahyna virus among residents. jiménez-sastré et al. mexico, cross-sectional study-convenience sampling of dengue fever in flooded colonies. cases with positive serology of igg ( . %) and cases of positive igm ( . %). geographical distribution associated with proximity to permanent water bodies. tong et al. australia , - routine data-assessment of variability in environmental and vector factors on ross river virus transmission. wu et al. china, - longitudinal study-review of retrospective data to determine intermediate host snail dispersal patterns and acute and chronic infections of schistosomiasis after floods. average number of acute schistosomiasis cases recorded in flood years . times higher than in years with little to no flooding. re-emerging and new snail infested areas in flood years on average . and . times larger than in years with normal water levels. flooding of marshlands identified as main driver for vector dispersal. responsible for local disease transmission, the factors that influence transmission, location of breeding ground, and which measures of control should be implemented. local destruction of breeding sites after flooding has receded is extremely effective, so individuals should remove unused vessels and stagnant water when possible. water storage containers need to be covered to protect from disease vectors, such as egg-laying female mosquitoes. individuals can protect themselves against mosquito bites by using repellents during biting hours, mosquito nets, and screens in doors and windows. individual and community awareness and participation is essential for successfully reducing the risk of infectious diseases following flooding. understanding the social and cultural influences on response behavior in the time of a flood emergency is crucial to inform the design and targeting of warnings and health education messages. some studies showed the frequency of infectious diseases can increase in the weeks to months after flooding, and figure illustrates when infectious disease outbreaks following flood events are likely to occur. however, there remains scientific uncertainty about the strength of association between infectious disease incidence and flooding. floods can cause population displacement and changes in population density, raise concern about waste management and the availability of clean water, as well as affect the availability and access to healthcare services. all of these are risk factors for an infectious disease outbreak. kouadio et al. and watson et al. suggested that unless there is a substantial population displacement, there is minimal risk of infectious disease transmission and outbreaks following flooding. overall, the risk of infectious disease following flooding is context-specific, differs between countries, and is dependent upon a number of synergistic factors. outbreaks of leptospirosis and diarrheal diseases following flooding have been documented in europe , , , , , , , [ ] [ ] [ ] but the evidence of increased incidence of vector-borne diseases following flooding is lacking because the time lag before onset can be several months. past studies have indicated possible associations between vector-borne diseases and flooding in europe. , [ ] [ ] [ ] european residents may be exposed to these risks while traveling. foreign relief workers can potentially introduce infectious diseases into an area affected by flooding and these workers may be susceptible to endemic diseases that are more prevalent because of the flood. surveillance in flood-affected areas is fundamental to understanding the impact of flooding on infectious disease incidence. surveillance and early warning systems may reduce current and future vulnerability. a comprehensive risk assessment could help determine priority diseases for inclusion in the enhanced surveillance system and prioritize prevention and control measures. in addition to surveillance and early warning systems to detect epidemic-prone diseases, assuring access to clean water, proper sanitation, adequate shelter, and primary healthcare services is essential. despite a considerable amount of research on the relationship between infectious diseases and flooding, globally and in summary of possible public health interventions public health interventions include those made before, during, and after flooding to reduce vulnerability to infectious diseases. interventions need to take place at a variety of levels: individual, household, community, regional, national, and international. the public health measures cited in the literature to reduce the risk of infectious diseases as a result of flooding focus on: risk assessments, enhanced surveillance systems, and specific prevention and control measures depending upon the type of infectious disease risk. , a rapid disease risk assessment should be conducted by a representative multi-agency group within the first week of the flood including: data on the flooded region and displaced persons, the main disease threats for the enhanced surveillance system, baseline data collection, and identification of priority interventions. , during a flood event, hand-held devices that allow workers to enter and analyze data in the field can assist the rapid risk assessment. existing disease surveillance systems can be enhanced to target specific diseases or syndromes and to support timely response actions to reduce disease impact and risk of transmission. , public health teams need to establish adequate disease surveillance systems which take into account the inherent disruption of the public health infrastructure that may occur during flooding. an enhanced surveillance system should be adaptable and context-specific, monitor key epidemiological data and compare with baseline data, monitor vulnerable groups, identify any emerging outbreaks, and result in timely public health action. in high-income countries, risk assessments and surveillance systems need to be very refined to detect small differences from baseline incidence data. prevention of infectious diseases following flooding involves maintenance of health services, provision of shelter, clean water supplies, proper sanitation, regular and adequate food supply, and in some cases mass vaccination campaigns and control of disease vectors. , water and sanitation are vital elements in the transmission of water-borne diseases; hence, providing clean drinking water is a priority in the initial days following flooding. clasen et al. found that household interventions were more effective in preventing diarrhea than interventions at the water-source. interventions at the household level reviewed included: chlorination, filtration, solar disinfection, and combined flocculation and disinfection. ejemot-nwadiaro et al. found hand-washing interventions can reduce diarrhea episodes by one-third. rodent control is another prevention measure that needs to be considered during flooding. the local rodent species and their behaviors should be identified, water and food storage containers should be rodent-proofed, and solid waste should be properly stored, collected, and disposed. , according to bhardwaj et al. prompt and vigilant fever surveillance activities in pre-flooding preparedness plans, rodent control programs, and improvement of environmental sanitary conditions may help greatly reduce leptospirosis incidence. vector control can reduce disease transmission by rendering the environment unfavorable for the survival, development, and reproduction of the vector. , establishing surveillance for the introduction of new vector species could contribute substantially to vector-control. an expert should identify vectors • papers on unrelated subject areas; such as, biochemistry, molecular biology, and genetics. personal communications between key informants were conducted in conjunction with the literature review. the context of the questions included the current state of knowledge of the association between flooding and infectious diseases and potential solutions to mitigate the risks. because flooding is a natural disaster and cannot be induced experimentally, the research evidence was unlikely to be the considered 'gold standard' of a systematic literature review or a randomized controlled trial. most of the data were observational, and because of the insufficient numbers of similar studies and variations in outcome reporting, no studies were excluded on the basis of study quality. a formal assessment of bias was not possible for each individual study. it is important for health officials and the public to understand that exacerbation of disease risk factors contribute to infectious disease outbreaks following flooding. population and individual vulnerability and resilience factors can worsen or mitigate infectious diseases following flooding. the community needs to be aware of actions that can facilitate or prevent infectious disease. to mitigate infectious disease risk following flooding, those involved in flood planning, response, and recovery should be aware of the results of this systematic literature review. if climate change causes more floods, then the future health burden of infectious diseases from floods could increase. in europe, maintenance and continuous adaptation and improvement of public health measures is important to sustain the low risk of infectious disease outbreaks following floods. presently, there are clear research needs to improve the understanding of the association between infectious diseases and flooding: • more robust epidemiological studies on infectious diseases covering the pre-, mid-, and post-flood periods. • further research assessing the effectiveness of public health interventions minimizing risk from infectious diseases following flooding. • investigation of infectious disease incidence following smaller flood events. • analysis of the differences between summer and winter flooding on infectious disease incidence. • analysis of the differences between flash and riverine flooding on infectious disease incidence. no potential conflicts of interest were disclosed. this work was carried out within public health england's department of extreme events and health protection, funded partly by the eu project "public health adaptation strategies europe, the body of information still remains fragmentary. many studies attempted to collect data retrospectively, had methodological shortcomings, lacked longitudinal data/baseline health data, control groups for comparison, and measures of clear disease outcomes. the studies included in this review were mainly observational studies with widely varying quality levels and study designs. because it is unethical to conduct experimental studies on this topic, rigorous observational studies must be continue to be undertaken. observational studies can present particular challenges because of the unpredictability of the timing and location of floods. reporting and recall bias was very likely in many studies. additionally, many studies relied on data from disease surveillance systems. obtaining relevant disease surveillance data pre-, mid-, and post-flooding is frequently challenging. population displacement can distort the rates of comparison for infectious disease incidence. the quality and robustness of disease surveillance systems can vary from country to country, and a country with a weak disease surveillance system will probably lack pre-flood baseline data. flood damage to pre-existing public health infrastructure can exacerbate weaknesses in a disease surveillance system. furthermore, it is difficult to attribute an increase in infectious disease incidence solely to a flood event, and therefore this issue may be under-investigated and under-reported. finally, this systematic review is not entirely exhaustive, and there may be many other reports in gray literature, but the quality is likely to be lower than the peer-reviewed published reports identified. the search strategy used was adapted from two studies ( table ) . , all papers with the specified search terms in their titles, abstracts, or keywords were searched for. focused searches of the following databases were conducted: medline, scopus, and pubmed. the cochrane database of systematic reviews was searched for further existing epidemiological reviews, as well as evidence aid. further relevant articles were identified manually from cited references from each selected full-text paper. data from gray literature were not systematically searched, but sources and advice from key experts were discussed in the accompanying text. inclusion criteria • papers published from january to september . ahern et al. included studies associated with infectious disease incidence following flooding up to . • epidemiological studies. • studies conducted in any country, because europe experiences a wide range of climate and geographical variation. • papers in all languages with english abstracts. • all papers where an explicit link is studied between flooding as an exposure and an infectious disease as an outcome. exclusion criteria • papers concerned primarily with mental health effects, flood-related injuries, population displacement, economic costs, and disruption of food supplies. a role of high impact weather events in waterborne disease outbreaks in canada risk factors for typhoid and paratyphoid fever in jakarta, indonesia health impacts of flooding in lewes: a comparison of reported gastrointestinal and other illness and mental health in flooded and non-flooded households outbreak of norovirus infection associated with contaminated flood water extreme precipitation linked to waterborne disease outbreaks. sciencedaily did a severe flood in the midwest cause an increase in the incidence of gastrointestinal symptoms? waterborne epidemics in finland in - a case control study to explore the risk factors for acquisition of leptospirosis in surat city, after flood resurgence of field fever in a temperate country: an epidemic of leptospirosis among seasonal strawberry harvesters in germany in leptospirosis on oahu: an outbreak associated with flooding of a university campus leptospirosis following a flood in the veneto area later leptospirosis after flood in tabasco, mexico strikes, flooding, rats, and leptospirosis in marseille, france climate change, flooding, urbanisation and leptospirosis: fuelling the fire? infectious diseases following natural disasters: prevention and control measures infectious diseases that pose specific challenges after natural disasters: a review global health impacts of floods: epidemiologic evidence climate change and infectious diseases in europe extreme water-related weather events and waterborne disease risks of water-borne disease outbreaks after extreme events influence of environmental factors and human activity on the presence of salmonella serovars in a marine environment systematic review of waterborne disease outbreaks following extreme water events effects of extreme precipitation to the distribution of infectious diseases in taiwan heavy rainfall and waterborne disease outbreaks: the walkerton example factors determining vulnerability to diarrhoea during and after severe floods in bangladesh medicaid outpatient utilization for waterborne pathogenic illness following hurricane floyd flood hazards and health: responding to present and future risks. tyndall centre for climate change research global trends in waterrelated disasters: an insight for policy-makers. the united nations world water assessment program. international center for water hazard and risk management building human resilience: the role of public health preparedness and response as an adaptation to climate change health impacts of floods in europe: data gaps and needs from a spatial perspective major flood disasters in europe future change of precipitation extremes in europe: intercomparison of scenarios from regional climate models no upward trends in the occurrence of extreme floods in central europe summary for policymakers: managing the risks of extreme events and disasters to advance climate change adaptation protecting health in europe from climate change disease emergence from global climate and land use change infectious diseases in the aftermath of monsoon flooding in pakistan infectious diseases of severe weather-related and flood-related natural disasters we would like to acknowledge carla stanke world health organization regional office for king's college london); and brittany scheckelhoff epidemics after natural disasters the threat of communicable diseases following natural disasters: a public health response frequency of infectious diseases among flood affected people at district rajanpur, pakistan flood-associated melioidosis in a non-endemic region of thailand impacts of flood on health: epidemiologic evidence from hanoi spatiotemporal clustering of cholera: the impact of flood control in matlab shifting prevalence of major diarrheal pathogens in patients seeking hospital care during floods in melioidosis outbreak after typhoon, southern taiwan health effects of flooding in rural bangladesh enterotoxigenic escherichia coli and vibrio cholerae diarrhea flooding and communicable disease fact sheet mosquitoborne viruses geographic distribution of dengue fever cases in flooded zones from villahermosa, tabasco, in rift valley fever outbreak in sudan climatic, high tide, and vector variables and the transmission of ross rover virus effect of floods on the transmission of schistosomiasis in the yangtze river valley, people's republic of china risk factors for west nile virus infection and meningoencephalitis, romania west nile fever in czechland nile fever--a reemerging mosquito-borne viral disease in europe public health guide in emergencies communicable disease control in emergencies: a field manual communication, data sharing, and collaboration, at the disaster site. comput in civil eng interventions to improve water quality for preventing diarrhea typhoon-related leptospirosis and melioidosis longitudinal epidemiology of leptospirosis in the czech republic monitoring of the epidemiological situation in flooded areas of the czech republic in year outbreak of leptospirosis among triathlon participants in langau leptospirosis outbreak following severe flooding: a rapid assessment and mass prophylaxis campaign leptospirosis in mumbai: postdeluge outbreak leptospirosis following a major flood in central queensland outbreak of leptospirosis after flood, the philippines leptospirosis after typhoon in taiwan anti-epidemic provision for the population in emergency situations in the krasnodar territory increase of leptospirosis in dengue-negative patients after a hurricane in puerto rico in the health impacts of floods. in: [few r and matthies f] flood hazards and health: responding to present and future risks. tyndall centre for climate change research a review of the invasive mosquitoes in europe: ecology, public health risks, and control options key: cord- -cv mkpzd authors: kim, jung heon; bae, wonjun; kim, jiyeon; hwang, eung soo title: an urgent need for global preparedness against the reemergence of “forgotten” infectious diseases in korea date: - - journal: j korean med sci doi: . /jkms. . .e sha: doc_id: cord_uid: cv mkpzd nan the authors have no potential conflicts of interest to disclose. . hav infection was not an important pathogen until the s- s because almost all persons were infected in early ages with low sequelae by poor hygienic environment. they had immunity to hav and there was no need for vaccination at those time. however rapid increases of hav from , cases in to , cases in were reported with an outbreak with , cases in . some of hav cases were imported from overseas by abroad travelers. what's next? measles, pertussis, and more? we should not make these infectious diseases as "never-ending stories," and comprehensive global preparedness for preventing outbreaks is needed urgently. we already know that vaccine program pursues high rate of vaccination. according to the report from korean centers for disease control and prevention (kcdc) in , vaccination rates between and -year-old children for national essential vaccination were over %; bcg . %, hepb . %, dtap . %, ipv . %, mmr . %, var . %, je . %. the vaccination rate in nip was reported in young ages but there is little nation-wide survey data of their appropriateness of immune formation to prevent infection after vaccination. we do not know why mumps and varicella are continuously and increasingly prevalent in spite of high vaccination rates. is it from a vaccine procedure failure or genetic changes of causative agents? it is impossible to answer this question immediately because there is little basic background data in korea. we habitually adopt the data of infection status, seroepidemiological data from other advanced countries when an outbreak occurs without our own continuous and routine investment for the basic infection data. if they were utilized without global understanding or analyzed without the comparison with our own data, it would not always help to resolve our need because the situation of each country is different from each other. we already know changes much in genetic characteristics of many causative pathogens, nutrition, individual and herd immunity, ways of domestic and international transportation, socioeconomical and environmental situation from the past ones. therefore, it is imperative that a global and comprehensive preparedness mechanism be implemented, not only for the emerging infectious diseases but also re-emerging, "forgotten" ones. the kcdc is the sole entity responsible for the control of legal reporting of communicable diseases in the country, but it has limited resources and faces an uphill battle in realizing complete preparedness for all infectious diseases. we have already learned a difficult lesson from the dearth of infectious diseases specialists during the influenza epidemic in and the middle east respiratory syndrome-related coronavirus (mers-cov) outbreak in . it is true that, in response to these crises, the korean government has reformed the structure of kcdc and increased its workforce to more effectively control emerging infectious diseases. however, old-fashioned infectious diseases, scarlet fever, mumps, hepatitis a, varicella, and zoster are in resurgence. it is necessary to gain a comprehensive understanding of the characteristics of pathogens, hygiene levels, immunity status and changes in each age group, environmental alterations, dietary nutrition, vaccine supply, treatment modalities, international relationship of diseases, so on. in order to make and keep korea safe from infectious diseases, we must expend every effort to go beyond the current fragmented approaches to institute a more balanced framework predicated on a mutually-reinforcing, cross-sectoral network of stakeholders. this would necessitate, inter alia, strengthening the planning and implementation capacities of korean government including kcdc, enhancing the participation of regional government bodies, supporting academic research at universities, infectious disease institutes and related entities. references . ministry of health and welfare national childhood vaccination coverage among children aged years in korea the research was performed using literature and services at the seoul national university college of medicine. key: cord- -cq tj authors: bavishi, chirag; bonow, robert o.; trivedi, vrinda; abbott, j. dawn; messerli, franz h.; bhatt, deepak l. title: acute myocardial injury in patients hospitalized with covid- infection: a review date: - - journal: prog cardiovasc dis doi: . /j.pcad. . . sha: doc_id: cord_uid: cq tj the coronavirus disease (covid- ) is now a global pandemic with millions affected and millions more at risk for contracting the infection. the covid- virus, sars-cov- , affects multiple organ systems particularly the lungs and heart. elevation of cardiac biomarkers, particularly high-sensitivity troponin and/or creatine kinase mb, is common in patients with covid- infection. in our review of clinical studies, we found that in studies including , patients, the weighted pooled prevalence of acute myocardial injury was % (ranged from % to % depending on the criteria used). the plausible mechanisms of myocardial injury include, ) hyperinflammation and cytokine storm mediated through pathologic t-cells and monocytes leading to myocarditis, ) respiratory failure and hypoxemia resulting in damage to cardiac myocytes, ) down regulation of ace expression and subsequent protective signaling pathways in cardiac myocytes, ) hypercoagulability and development of coronary microvascular thrombosis, ) diffuse endothelial injury and ‘endotheliitis’ in several organs including heart, and, ) inflammation and/or stress causing coronary plaque rupture or supply-demand mismatch leading to myocardial ischemia/infarction. cardiac biomarkers can be used to aid in diagnosis as well as risk stratification. in patients with elevated hs-troponin, clinical context is important and myocarditis as well as stress induced cardiomyopathy should be considered in the differential, along with type i and type ii myocardial infarction. irrespective of etiology, patients with acute myocardial injury should be prioritized for treatment. clinical decisions including interventions should be individualized and carefully tailored after thorough review of risks/benefits. given the complex interplay of sars-cov- with the cardiovascular system, further investigation into potential mechanisms is needed to guide effective therapies. randomized trials are urgently needed to investigate treatment modalities to reduce the incidence and mortality associated with covid- related acute myocardial injury. particularly the lungs and heart. the cardiac manifestations of the infection place an overwhelmed health care system under considerable stress due to the substantial resources and potential intensive care support required for these patients. in this concise review, we will focus on acute myocardial injury in covid- infection, its prevalence, plausible pathophysiologic mechanisms, guidance on the use of cardiac biomarkers, and general management strategies. elevation of cardiac biomarkers, particularly high-sensitivity cardiac troponin (hs-troponin) and/or creatinine kinase mb, is a marker of myocardial injury. elevation of cardiac biomarkers is common in patients with covid- infection. in our review of clinical studies with at least covid- patients (published until may th , ), we found that in studies( - ) including , patients, the overall prevalence of acute myocardial injury ranged from % to % depending on the criteria used. (table ). the overall crude prevalence of acute myocardial injury was . % ( , / ). using meta-analytic approach ( ) , the overall weighted pooled prevalence estimate of acute myocardial injury was found to be % ( % confidence interval j o u r n a l p r e -p r o o f lactate dehydrogenase. in another seminal study of covid- patients by li et al.( ) markers of cellular and immune dysregulation were found to be associated with myocardial injury. on multivariate adjusted analysis, age, wbc count, neutrophil percentage, lymphocyte percentage, cd + t cell counts, cd + t cell counts, cd + t cell counts, cd +cd + nk cell counts, hs-c-reactive protein, and procalcitonin were independently associated with myocardial injury in patients with covid- . it appears that the magnitude of elevation of cardiac troponin may be associated with severity of disease and prognosis ( ) . shi et al. ( ) studied patients with confirmed covid- , the prevalence of myocardial injury defined as hs-troponin i above the th percentile was . %. both, ck-mb > . ng/ml (hazards ratio, . , p< . ) and cardiac troponin i > . ng/ml (hazards ratio, . , p= . ) were found to be independently associated with increased inhospital mortality. in a prospective cohort study by du et al( ) of patients with covid- pneumonia, troponin i ≥ . ng/ml was independently associated with mortality in addition to age ≥ years, pre-existing cardiovascular (cv) or cerebrovascular diseases and cd +cd + tcells ≤ cells/μl. further large-scale prospective studies are needed to thoroughly investigate these findings. j o u r n a l p r e -p r o o f colleagues including consecutive hospitalized patients with covid- , the incidence of arrythmia was found to be %. in a recent study by goyal et al( ) , examining the clinical characteristics of first consecutive patients with covid- admitted in hospitals in manhattan, . % of patients had a cardiac arrythmia during hospitalization. although the type of arrythmia was not described in these reports, both tachy-and brady-arrythmias can occur. in another study( ) of covid- patients by guo et al, the overall incidence of ventricular tachycardia/fibrillation was . % and was notably more common in patients with myocardial injury compared with those without ( . % vs . %, p< . ). in the study of covid- patients by zhou et al,( ) hf was observed in . % of patients, however, the etiology of heart failure was not reported. acute myocardial injury, arrythmia, and hf can manifest either alone or can occur in combination based on the clinical course. the covid- virus (sars-cov- ), uses the angiotensin converting enzyme (ace) for entry into target cells. ace is predominantly expressed by epithelial cells of the lung, intestine, kidney, heart, and blood vessels. while ace cleaves angiotensin i to angiotensin ii and leads to vasoconstrictive, pro-inflammatory, and pro-oxidative effects through the angiotensin ii receptor type (at- ) receptor, ace leads to anti-inflammatory, anti-oxidative and vasodilatory effects through the angiotensin - -mas receptor complex. the protective effect of ace in lung is well defined, and therefore down regulation of ace due to viral binding to this receptor plays a key role in acute lung injury and acute respiratory distress syndrome. our understanding of the pathophysiology of covid- and host immune responses is still evolving, however, immunemediated inflammation plays a key role in the pathogenesis of covid- ( , ) . on one hand, table . treatment protocols on in-patient management of covid- from several us hospitals have included hs-troponin as a part of the routine laboratory assessment ( , ) . in patients with elevated hs-troponin, clinical context is important and myocarditis as well as stress induced cardiomyopathy should be considered in the differential, along with type i and type ii mi. evaluation of nt-probnp should be considered if clinically indicated. covid- induced myocarditis has been reported in case reports from china and elsewhere that usually requires aggressive management ( ) ( ) ( ) ( ) . new onset cardiomyopathy and arrythmias with elevated troponins should raise the suspicion of myocarditis. the initial diagnostic modality includes echocardiography and right and left cardiac catheterization with placement of a pulmonary artery catheter for continuous hemodynamic monitoring in critically ill shock patients. cardiac mri and endomyocardial biopsy are more definite tests. however, in the face of covid- pandemic, when resources are limited and one of the primary goals is to minimize health care personnel's exposure, imaging modalities may not be readily available. conceivably, cardiac biomarkers can be used to aid in diagnosis as well as risk stratification. given the complex interplay of sars-cov- with the cv system, further investigation into potential mechanisms is needed to guide effective therapies. epidemiological studies and randomized trials are urgently needed to investigate treatment modalities regulating immune function and inhibiting inflammatory responses to reduce the incidence and mortality associated with covid- related acute myocardial injury. figure shows prevalence estimates of acute myocardial injury (boxes) with % confidence limits (bars) for each study selected; pooled prevalence estimate is represented by diamond in this forest plot. clinical stages are based on national institute of health treatment guidelines ( ) . acute myocardial injury is typically seen in advanced stages of disease and is associated with worse prognosis. clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in analysis of myocardial injury in patients with covid- and association between concomitant cardiovascular diseases and severity of covid- clinical course and risk factors for mortality of adult inpatients with 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for coronavirus disease (covid- ) covid- and renin angiotensin blockers: current evidence and recommendations wang y et al key: cord- - u e authors: kasai, takeshi; nakatani, hiroki; takeuchi, tsutomu; crump, andy title: research and control of parasitic diseases in japan: current position and future perspectives date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: u e between and , japan eliminated several major parasitic diseases. in , the japanese hashimoto initiative was the first global programme to target parasitic diseases. thereafter, japan expanded its international cooperation to cover infectious diseases through integrated development programmes to improve health, to alleviate poverty and to help to achieve the millennium development goals of the united nations. parasite control remains a major component of all subsequent operations. opportunities to build upon past successes in order to improve the situation in the developing world – in addition to tackling emerging national threats – are promising. substantial challenges remain and japan has introduced major national reforms to try to overcome them. between and , japan eliminated several major parasitic diseases. in , the japanese hashimoto initiative was the first global programme to target parasitic diseases. thereafter, japan expanded its international cooperation to cover infectious diseases through integrated development programmes to improve health, to alleviate poverty and to help to achieve the millennium development goals of the united nations. parasite control remains a major component of all subsequent operations. opportunities to build upon past successes in order to improve the situation in the developing world -in addition to tackling emerging national threats -are promising. substantial challenges remain and japan has introduced major national reforms to try to overcome them. setting the scene japan occupies a unique position with respect to infectious disease research and control. japan has been confronting problems that are faced by wealthy nations and problems that persistently threaten poor, developing countries because the japanese islands extend from almost the arctic north to the southern tropical okinawa islands and because the nation recently rose quickly from relative poverty to a position among the wealthiest countries. japan also has the invaluable experience of successfully driving the health of its citizens from a postwar low point to the elevated levels seen today. a major factor in this accomplishment was the control of parasitic diseases. today, the country faces new challenges because the profile of parasitic diseases has shifted from the high prevalence of soil-transmitted parasitoses, seen in the s, to epidemics of previously rare diseases and imported and opportunistic infections. novel parasites, especially in immunocompromized hosts, often cause serious manifestations and present problems with diagnosis and treatment. they also necessitate individual clinical interventions rather than the mass control operations that succeeded in the past. for a better understanding of the current position and future prospects it is important to bear past experience in mind. stemming from japanese advances in the s, health-improvement initiatives and control programmes that involve mass screening and treatment were implemented that now enable japanese nationals to enjoy one of the healthiest lifestyles in the world. in the space of two decades, nationwide, interdisciplinary, multisector publichealth campaigns against parasitic diseases led to the control of ascariasis, trichuriasis, hookworm and other soil-transmitted parasitoses and the eradication of other major diseases such as malaria, schistosomiasis and lymphatic filariasis [ ] [ ] [ ] [ ] [ ] [ ] . parasite control proved to be a leading component of successful postwar development in japan. for example, in terms of the causal factors behind the rapid declines in mortality seen between and , the control and decline of parasitic diseases was second only to advances in combating diseases of the circulatory system [ ] . consequently, during the to years after the war, parasite research and parasite-control programmes were priorities. parasitology departments were among the most important in universities and research institutes and there was close integration between research and field control programmes. in , only % of the population had a safe water supply and outbreaks of waterborne diseases were frequent [ ] . enactment of the water supply law in led to the swift introduction of proper water and sanitation systems ( figure ), which had a rapid and notable impact ( table ) . as coverage expanded, water-borne infectious diseases declined markedly. application of knowledge about the environment, hygiene and disease linkages, together with national advances in science, technology and medicine (i.e. vaccines, drugs and diagnostics such as the kato-katz thick smear test for schistosomiasis), proved crucial. the government also cooperated with numerous nongovernmental organizations (ngos) that were established nationwide and were devoted to the control of parasitic diseases. many researchers were also actively involved in field control programmes that operated in the vicinity of their research facilities. collaboration between the education and health sectors was also pivotal. nationwide, schools were one of the few postwar public infrastructures that remained functional. deworming interventions in schools were combined with health education, which was provided by schoolteachers and supported by researchers. those involved in deworming programmes recognized the invaluable nature of linking different local-level resources, especially schools and community health centres. they also experienced the impact of 'community empowerment' [ , ] . the rapid socioeconomic development in japan, of which the traditional 'village living' philosophy was a driving force, saw the nation evolve from being one that received overseas development assistance (oda) in the early- s to donor nation status by the late- s. the initial focus of japan was to concentrate on helping neighbouring countries in asia but in the new millennium and with globalization increasing that view has expanded to encompass a global assistance focus, with special attention currently being shown to africa. the unique oda philosophy in japan, which is based on the overriding concept of self help and human security, contributes to overseas development by exporting its achievements in disease control and in shaping support for disease research and control activities (http:// www.mofa.go.jp/policy/oda/cooperation/anniv /pamphlet/ contents.html). based on its successes in controlling several major parasitic diseases and its desire to help other nations achieve similar success, japan proposed a global parasite-control initiative at the group of seven (g ) summit in denver, usa, which emphasized the necessity of international cooperation to control parasitic diseases. a report entitled 'global parasite control for the st century' submitted at the group of eight (g ) summit in birmingham, uk, specified the intention by japan to help developing countries to improve their ability to control parasitic diseases and also persuaded the g nations to embrace the proposal [ ] [ ] [ ] . the so-called 'hashimoto initiative' (hi), named after its architect and the japanese prime minister at the time, hashimoto, represented an unprecedented step in japanese oda history. in japan, the prime minister or parliament can influence oda policy but, usually, the bureaucracy or civil servants have been the de facto operator of oda policy [ ] . thus, the hi was unique. hashimoto fervently desired to contribute to the health of people in less-developed counties by transferring japanese experience, especially in disease research and control. he envisioned support for japanese experts who engage in such work at centres that were established abroad as a contribution not only to global health but also as a means to maintain the skills and expertise of parasitology researchers in japan. three years after the hi was proposed, and mindful of major changes regarding the global disease-control scene, japan expanded the scope of the target infectious diseases and announced the more expansive okinawa infectious disease initiative (idi) in [ , ] . the idi aimed to provide $ billion over a five year period. parasitic diseases were retained as a primary target in what was planned as a comprehensive and cohesive range of measures to further social and economic development in recipient countries (http://www.mofa.go.jp/mofaj/gaiko/summit/ko_ / outline/jp/overview.html). actual spending exceeded expectations and totalled >$ billion in four years ( figure ). the idi incorporated several major strategies, including promotion of research and of public-health activities. this was the first time that research was clearly defined as a matter for action in a health-related oda policy document. it quickly became apparent, however, that the shortage of experienced personnel in parasitic-disease control was a crucial issue. as a result, plans were put in place for the development of a global network of research institutions to target infectious and parasitic diseases, to promote research on infectious and parasitic diseases in impoverished countries with a focus on benefiting the poor, and promotion of international cooperation in vaccine research and development [ ] . these centres were expected to provide technical support to field control programmes using japanese oda and would mimic what had happened during the successful national control efforts in japan. japan also had a leading role in the development of the global fund to fight aids, tuberculosis and malaria (gfatm: http://www.theglobalfund.org/en/). spurred by the idi, gfatm was established in after discussion at subsequent g summits and a united nations (un: http://www.un.org) general assembly special session on hiv/aids. the then prime minister, koizumi, expressed strong commitment to the global endeavour at a symposium to commemorate the fifth anniversary of the kyushu-okinawa summit and announced an additional contribution of $ million (http://www.mofa.go.jp/policy/ health_c/gfatm/address .html). in the year , all un member states adopted the millennium declaration [ ] , which put poverty reduction at the centre of concerns and incorporated specific, measurable development goals to be achieved by . these became known as the millennium development goals (mdgs) [ ] . the mdgs comprise eight goals, three of which refer explicitly to health. at the high-level forum on health mdgs in asia and the pacific, japan stated that achieving the health mdgs is a crucial step that is necessary to attain all of the eight mdgs (http://www.mofa.go.jp/policy/oda/ category/health/state .html). in response to the adoption of the mdgs, following the completion of the idi in march , japan expanded the idi into a comprehensive, multisectoral health and development initiative (hdi) [ ] , which was designed to continue and enhance active support in the health-related sector through oda with emphasis on accomplishing the mdgs, particularly in africa [ ] . in the hi, school-based deworming of intestinal helminths was chosen as a main focus for international disease-control programmes. the direct beneficial effects of deworming are well documented and include improved nutritional status, increased weight gain [ ] , better micronutrient uptake, improved cognitive development of children [ ] , improved physical fitness and appetite [ ] and increased school attendance [ ] . however, the reason that school-based deworming was selected for particular attention was not only the direct effects but also the indirect socioeconomic and health benefits that would be bestowed on communities [ , ] . the concepts inherent in the hi remain major pillars of the new hdi. japan also adopted a strong research component within the hdi and included 'strengthening research capacity on the ground and paying due respect to local condition' as the underlying basic policy. crucially, this reflects the japanese self-help philosophy. the hdi emphasizes the importance of understanding local culture, traditions and norms towards ill health and seeks to strengthen research capacity at the field level, in particular those research activities that are designed to facilitate and improve project implementation. in japan, the parasitic-disease research and control community underwent some fundamental changes as a result of its success [ ] . after the eradication of key parasitoses from japan and effective control being established over many others, parasitic diseases were no longer viewed as a subject of major concern. consequently, extensive and well-supported research groups began to lose their resources. they became divided into two groups: those that maintained their interest in disease-control programmes and applied research turned their attention to tackling parasitic diseases outside japan, and those which kept their focus on japan and, instead, concentrated on basic laboratory research on other diseases. this exacerbated the disconnect between research and control. the potential threat from infectious diseases, including those that are newly emerging and some that were never eradicated from japan (coupled with increasing international trade and travel), mean that japan must remain continually alert and prepared. this was exemplified by the recent outbreaks of severe acute respiratory syndrome (sars) and avian flu, which focused attention on how japan is able to respond to infectious disease threats to its inhabitants, in addition to maintaining its responsibility to protect its less well-off neighbours. this was indicated by the creation of a regional stockpile of tamiflu to help combat an outbreak of avian flu, should it arise [ ] . thus, the disconnect between research and control of parasitic diseases, coupled with the rapidly shrinking cadre of skills and expertise capable of dealing effectively with parasitic-disease matters [including the relatively sudden disappearance of community-based health centres ( figure )] has been a major concern in japan. consequently, the government has taken substantial measures to rectify the problem. the hi conceptualized the future of japanese parasitic-disease research and control worldwide. to remain a leader within the hdi, parasitic-disease control programmes will continuously need to work with japanese oda policy makers and position themselves as one of the best means of attaining the mdg targets. one major drawback is that the japanese experience, whereby parasitic-disease control contributed substantially to overall social development, remains primarily an anecdotal record and is not documented in health-system research terms. it is, therefore, important to monitor and evaluate ongoing programmes in order to collect the data and present the results in a more scientific manner. the research agenda that is set by parasitic-disease control is not complete [ ] and an additional body of knowledge and action that takes the functioning of the health system into account must complement the disease-control approach. recognition of the importance of health-system research, in conjunction with attaining the mdgs, has been increasing [ ] . the japanese ministry of health, labour and welfare organized a symposium for health-system development in at which the importance of the linkage between the healthsystem research agenda and a disease-specific generated research agenda was confirmed [ ] . it is reported that undernutrition is the underlying cause of a substantial proportion of all child death [ ] and programmes that are aimed at controlling soil-transmitted helminths have already demonstrated their positive impact on nutrition. therefore, it is important to maintain the integration of efforts to control parasitic diseases with all ongoing healthsystem activities and improvement efforts, nationally and internationally. in japan today, parasitoses are less of a public-health dilemma and more a problem for individuals, with the primary difficulty being one of diagnosis rather than treatment. commonly used parasitological tests are not effective for some of the new diseases and many clinicians are untrained or inexperienced in dealing with them [ ] . moreover, the successful control of parasites has led to a vacuum of interest and attention, with parasitology departments having disappeared from medical schools and the number of staff in the departments that remain has declined substantially. in the late- s, of the medical and veterinary schools in japan, only had either a parasitology or tropical medicine department [ ] . japan needs to solve this dearth of national experts in parasitic-disease research and control -it needs to refocus and rejuvenate its research base. the gaps between applied research, field control and basic research also need to be bridged. the japanese research community has historically benefited enormously from working in close collaboration with control programmes to overcome parasitic diseases. since parasitic diseases became rare and of minor importance in japan, the research community has been diluted and crucial experience and knowledge has been condensed, inherited by a declining number of individuals. it is important to establish collaboration and coordination between groups, in the short-term to overcome the problem of the lack of human resources and in the long-term to promote and improve parasitic-disease research and control. it is important that japan is able to interface and work with the international community and particularly with specialists from countries where parasitic-disease control projects take place. research priorities for health and development in wealthy and resource-poor countries are not the same [ ] . basic research in japan might not necessarily focus on needs that are deemed a priority elsewhere. overall, the crucial factor is that the application of medical science in developing countries should be a joint effort between scientific institutions in countries from the south (i.e. resource-poor) and the north (i.e. wealthy) [ ] . irrespective of a national or global focus, there must also be an underlying principle of engaging affected communities themselves and of them having a pivotal role in developing interventions jointly with ngos, industry and local and central health departments. furthermore, it is imperative to ensure that research is cohesively and continually integrated into control initiatives, as was the case in japan and elsewhere. an example is the community-directed treatment system being used by the african programme for onchocerciasis control (http://www.apoc.bf) to combat onchocerciasis in africa [ ] . japan is facing the (re)emergence of several water-and food-borne parasitic diseases and parasitic zoonoses. this is because of increasing international trade, faster transport, demographic changes and increasing immigration and tourism [ ] . in addition, there are now cases of imported diseases such as malaria and cysticercosis [ ] and the northernmost hokkaido island prefecture is experiencing an epidemic of alveolar echinococcus [ ] . as part of a major rejuvenation of research and development work in science and technology, which began in , the government has been taking active steps to boost national capacity in all aspects of disease research and control. the fundamental reforms and almost complete restructuring of the science and technology system in japan has caused a paradigm shift from 'science, technology and society' to 'science and technology for society' [ ] . there has also been an increase in funding that is devoted to parasitic and infectious diseases. between and , $ - million annually was being devoted to parasitology research from the ministry of education, culture, sports, science and technology (mext). overseas collaboration is also expanding, with the national institute for science and technology policy reporting that % of all scientific publications in arose from international collaborations, compared with only % in . in , mext allocated $ million to inaugurate a new programme to intensify research into measures for coping with infectious diseases and to create centres of excellence (coe) nationally and internationally. so far, at least three coe have been created overseas and four in japan, all coordinated through the newly established center of review trends in parasitology vol. no. research network for infectious diseases (http://www. riken.go.jp/engn/index.html). the third japanese science and technology basic plan ( - ) is designed to promote science and technology that provides an immediate, exact solution to socially essential issues, including international bioterrorism and emerging or re-emerging infectious diseases, such as sars and avian flu, that threaten global safety and security. in addition, human resources will be developed in order to respond to new needs and to contribute towards combating infectious diseases and bioterrorism, natural sciences, humanities and social science and the burgeoning bioinformatics and technology disciplines (http://www .cao.go.jp/ cstp/english/basic/index.html). these activities should promote ongoing international collaboration in basic research, including genomics work. in addition, the activities should facilitate the participation of japan-based basic research groups in field control and applied-research programmes in developing countries that are supported by japanese oda and technical cooperation. despite the challenges, the opportunities for further advancements in parasitic research and control are good, with renewed interest and entrepreneurship, the development of new techniques, tools and delivery systems and deployment of substantial amounts of funding. knowledge drives productivity, economic growth and health; however, unless all stakeholders put a concerted effort into resolving the challenges, the opportunities might fade and the unique and privileged acquired knowledge and experiences of japan could be lost. the analysis and opinions presented in this article represent the personal views of the authors and do not represent the official position of the ministry of health, labour and welfare or the government of japan. the authors declare no conflict of interest. filariasis in asian region retrospective review of the progress in parasitology in japan: the past and the present parasitology and parasitic diseases in japan control of parasitoses in japan historical aspects for the control of soiltransmitted helminthiasis increasing parasitic 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evolution (n y) doi: . /s - - - sha: doc_id: cord_uid: tcuv v modern evolutionary research has much to contribute to medical research and health care practices. conversely, evolutionary biologists are tapping into the rapidly expanding databases of medical genomic information to further their research. these two fields, which have historically functioned in almost complete isolation, are finding mutual benefit in the exchange of information. the long-term benefits of this synthesis of two major areas of research include improved health care. recently, efforts to catalyze this relationship have brought together evolutionary biologists, medical practitioners, anthropologists, and ethicists to lay the groundwork for further collaboration and exploration. the range of overlap is surprisingly broad and potentially invaluable. evolution has not traditionally been considered to be an important aspect of medicine, and medical practitioners and researchers have not traditionally approached their work from the perspective offered by evolutionary biology. medical practice tends to focus on the immediate, or proximate, causes of disease, including genetics and lifestyle, to identify a potential treatment. an evolutionary viewpoint pushes the focus out farther to look at long-term ecological relationships, including symbiotic bacteria, parasites and pathogens, historical lifestyles, and the genetics of populations. conversely, evolutionary biologists have not traditionally paid much attention to the medical implications or applications of their findings, with a few exceptions such as the study of emerging infectious diseases. other evolutionary studies have seemed far removed from contemporary human health issues. recently, however, these traditions have been giving way to a new approach, as researchers and medical practitioners discover connections based on evolutionary biology that are leading them to new conclusions about their respective fields. evolution is providing clues about puzzling medical results, and studies of human health are giving us new information about the rate and driving forces of evolution. initiatives such as the human genome project and the haplotype map have provided new tools for studying evolution and human health. genes are important, environment is important, the interaction between the two is important-but so is human evolution, as it exerts an ongoing influence on characteristics of the human population. in may , evolutionary and medical researchers, doctors, ethicists, and anthropologists met at the national evolutionary synthesis center to catalyze the emerging scientific discipline known as evolutionary medicine. although the participants work on different aspects of human health, they all realize the value of evolution in their work. as the title of the meeting-evolution in contemporary human populations: medical, genetic and behavioral implications (govindaraju )-suggests, this group of scientists clearly share the belief that evolution is going on in contemporary human populations. in fact, a recent study presents evidence that evolution may have been occurring even faster in human populations than expected over the past , years (hawks et al. ) . the field of medicine has developed to address the wide array of environmental and lifestyle-induced diseases. over time, doctors have learned more about the relationship between environment and disease, including factors such as hygiene and lifestyle. more recently, genomic studies have opened the door to studying genetic differences between populations, bringing us the possibility of more effective individualized medicine. all in all, medicine seems to be making great progress in preventing, treating, and curing disease without including evolutionary information. so what does understanding our evolutionary history add to our ability to improve human health? it has been shown that human evolution, in the form of natural selection for particular traits, can occur over the short term-just a few generations, "the blink of an eye to evolutionary biologists," according to yale university evolutionary biologist stephen stearns, one of the nescent meeting organizers. continued lactose tolerance in adults is an example of a trait that has evolved in certain populations within the last , years, since the domestication of milkproducing livestock (tishkoff et al. ; swallow ) . "if a doctor can become aware of how all these different traits actually interact with each other, then you begin to see your individual patient as something that isn't just the symptom sitting in front of you in the office that day," stearns says. "if you treat that symptom, then the perspective of evolutionary biology will tell you what all of the possible associated reactions are. the point is, they're a bit broader, and they may contain more surprises than a strictly medical orientation would give you. evolution gives you a broader view of the whole way an organism reacts, and why it does sobecause it's been shaped by evolution in certain ways." psychiatrist randolph nesse of the university of michigan, a strong advocate of evolutionary medicine, agrees: "once a doctor starts understanding how the body came to be, instead of seeing the body as a machine-which it's not-he or she starts seeing the body as a product of natural selection, where everything in the body is pretty good, because it's got to be, and nothing is perfect….this perspective is needed for literally every aspect of the body that's vulnerable to disease. why hasn't natural selection made it better? there are always good reasons." among those reasons is the idea that evolution involves an array of trade-offs, compromises in selection that favor one trait at the inevitable expense of another. stearns, nesse, and david haig of harvard university eloquently explain the importance of evolutionary trade-offs in the initial chapter of evolution in health and disease (stearns and koella ): no trait is perfect. every trait could be better, but making it better would make something else worse. our vision could be as acute as that of an eagle, but the price would be a decreased capacity to detect color, depth, and movement in a wide field of vision. if the bones in our wrists were thicker they would not break so readily, but we would not be able to rotate our wrists in the wonderful motion that makes throwing efficient. it the stomach made less acid we would be less prone to ulcers, but more prone to gi infections. every trait requires analysis of the trade-offs that limit its perfection. consideration of these compromises that have evolved over time can guide medical decisions about altering our physiology and behaviors. the evolutionary trade-off between survival and reproductive success of the individual is an interesting example. reproduction is expensive, on many fronts. just looking at mating success, high testosterone levels may provide a man with a competitive advantage in securing a mate, but will also decrease his resistance to pathogens and parasites (muehlenbein and bribiescas ) . a woman who has regular and frequent menstrual cycles has more opportunities to become pregnant, but is also at increased risk of cancer (strassmann ) . exposure to higher hormone levels, either incidentally or by design, can have unexpected side effects because of this evolutionary trade-off. reproductive success is ultimately the more important factor in evolution, as one's evolutionary fitness is measured by the number of offspring that survive to reproductive age themselves. the compromise results from the fact that we produce only one or two offspring at a time, and human offspring require extended parental care. humans who threw everything into mating but did not survive long enough to raise the few resulting offspring would be unsuccessful because their offspring would not survive either. radically changing traits that have been shaped over time runs the very real risk of tipping the balance in unexpected, and potentially unpleasant, directions. homo sapiens has been around for about , years, and both fossils and genomic studies present evidence of ongoing evolution in h. sapiens. as a species, h. sapiens has experienced many lifestyle changes-some generated by culture, others by new or changing environments. anthropology can provide valuable insights into our past, and this information can improve our application of evolutionary medicine (trevathan ) . we learn from studies of ancient societies, but also from the few remaining traditional hunter-gatherer societies and by comparisons between different societies. a major shift in lifestyle was the development of agriculture. agriculture led to many changes, including dietary alterations and exposure to zoonoses from domesticated animals. agriculture required groups to abandon a mobile hunter-gatherer lifestyle and live in sedentary populations, such as farming villages. ultimately, agriculture provided the ability to support large urban populations. sedentary groups encounter issues such as accumulation of wastes and transmission of epidemic diseases (armelagos et al. ) , issues that increase with population size. even more recently, increases in expected life span and change in diets have led to a rise in the frequency of chronic diseases (armelagos et al. ; leonard ). these changes in the environment and the increase in population size have the potential to create a surge in the rate of evolutionary change (hawks et al. ). clinicians and biomedical researchers need to incorporate an understanding of this mismatch to modernity into their explorations of pathophysiology. in simple terms, many aspects of our modern environment and lifestyle, including diet, exercise, exposure to chemicals, hygienic practices and circumstances, and a variety of other elements, are mismatched to our bodies' evolutionary state. for example, if-thanks to our prehistoric ancestors-our physiology is evolutionarily hardwired for low caloric intake and intense physical activity, our rich modern diets and sedentary lifestyles mismatch our innate metabolism (neel ; wang and mariman ) . evolutionary medicine tells us that many of today's so-called "diseases of civilization" that are so highly prevalent and growing rapidly in incidence, such as diabetes, obesity, and cardiovascular disease, may well be fueled by a metabolic mismatch (leonard ) . this phenomenon can be readily observed in contemporary populations that have undergone a "nutrition transition" in which rapid socioeconomic, demographic, and technological changes have brought profound changes in diet and activity patterns, often resulting in sudden and widespread increases in metabolic disorders. for example, a recent paper examined the changes in diet and health in the rapidly urbanizing black south african population. the study found that the change in lifestyle was accompanied not only by an improvement in micronutrients but also by an increase in obesity and chronic diseases (vorster et al. ). characterizing such mismatches through the use of today's analytical tools promises to shed important new light on the etiology of these and many other common, complex disorders (e.g., autoimmune, inflammatory, and mental/behavioral conditions) and offers an innovative approach to the identification of novel interventions and therapies. continuing to look at mismatches between what we are prepared for evolutionarily and what we experience with a modern lifestyle, we find puzzling results that require an understanding of our evolutionary history to solve. for example, recent studies have demonstrated a connection between decreased intestinal parasite load and increased incidence of autoimmune diseases such as asthma (lau and matricardi ) . in one study, children who had been dewormed showed an increase in the occurrence of allergic reactions to house mites. why? what was the link between removing worms and developing allergies? one proposed explanation is the "hygiene hypothesis" (strachan ) , which takes an evolutionary view and considers that the historically "normal" condition for humans is a constant barrage of parasites and that the immune response evolved in response to these conditions. an over-reactive immune system is a recognized cause of allergies, and it turns out that the presence of helminthes, common intestinal parasites, actually increases regulation and control of the immune response. when these children were dewormed, regulation of their immune response decreased, allowing an allergic response to house mites. an interesting application of this research is the experimental use of intestinal parasites in the treatment of autoimmune diseases such as irritable bowl syndrome, crohn's disease, and multiple sclerosis (wickelgren ) . evolution can also shed light on normal physiological responses such as vomiting and fever and guide medical interventions (nesse and williams ) . in terms of evolution, a trait will be selected for if it provides some advantage. an example is "morning sickness"-the nausea and vomiting suffered by % of pregnant women (badell et al. ) . nausea and vomiting are commonly experienced in the first and second trimesters, and generally, foods such as meat, fish, poultry, and eggs elicit the strongest responses. clearly, feeling nauseated and vomiting frequently are unpleasant experiences at the very least, and hospitalization for rehydration is frequently required. occasionally, more serious complications arise. however, there is significant evidence that this physiological process is associated with successful pregnancies (weigel and weigel ) . one tested hypothesis posits that this response is beneficial to both mother and embryo because it reduces the mother's intake of, and embryo's exposure to, potentially toxic chemicals and pathogens during key developmental stages (flaxman and sherman ) . in addition, the foods most commonly avoided are animal products which tend to harbor more pathogens than plant foods. the immune system of pregnant women is depressed to avoid rejection of the fetus, so both mother and embryo are at higher risk from pathogens. the benefits of this response may well justify the discomfort and risks-or at least provide some psychological comfort to suffering women. evolution is not something that happened long ago and then stopped. it remains an active force in the modern world not only in human populations but also in the pathogens and parasites with which we live. one need look no farther than the local news or health clinic for proof. the evolution of antibiotic-resistant strains of tuberculosis made the news in spring when a man with extremely drugresistant (xdr) tb made multiple international flights (cnn ) . fortunately, no one else was infected in this instance, but the recent evolution of drug-resistant tb has revived old fears, especially in places where the complicated medical treatment required for these strains is not available. evolutionary medicine can help physicians take into account the effects of people's changing environments and lifestyles. human behaviors such as conflict, poverty, and urbanization provide opportunities for infectious diseases to spread (halstead ) . today, more people live in areas of high population density-a change from the more traditional small community. high population density urban centers are ideal for transmission of pathogens, particularly when coupled with high rates of poverty (armelagos et al. ) . old antagonists are not the only concern. behaviors that bring humans into contact with pathogen reservoirs in wildlife, such as logging and the exotic animal and bush meat trades, also create opportunities for the transmission of "zoonoses"-pathogens normally found in animal hosts but capable of infecting humans (patz et al. ) . for example, logging brings roads to new areas, providing easy access for local inhabitants who come to hunt for food. in the process, hunters either become prey themselves as they are infected by novel pathogens or bring the pathogens back to populated areas in the form of bush meat (chomel et al. ). sometimes, the infection is relatively benignfor example, children often pick up worms from household pets-but sometimes, the transfer is deadly. the ancestor of the human immunodeficiency virus (hiv) is the simian immunodeficiency virus, which is found in a wide range of primate species and rarely causes disease in the primate host (hahn et al. ) despite the fact that hiv is devastating in humans. taking an evolutionary approach to medicine can help us deal with novel pathogens by increasing our understanding of the pathogen and facilitating the development of appropriate responses. when the mysterious severe acute respiratory syndrome (sars) virus emerged on the world scene, researchers quickly gathered information about its physical structure, life cycle, and genetics. by comparing its characteristics with those of known viruses, researchers were able to quickly identify the novel sars virus as a coronavirus (who ) . that crucial piece of information allowed health care professionals to develop appropriate diagnostic tools, preventive measures, and treatments based on knowledge of how other coronaviruses behave. additionally, the information about viral type allowed field researchers to identify the source of the infection based on known hosts of coronaviruses. civet cats, sold live in meat markets, were frequently found to be infected with a sars virus. further research indicated that horseshoe bats, which were also sold in these markets, had a more ancestral variant of the virus (britigan ; lau et al. ; wendong et al. ) . the virus was also found to be more widespread in bats. the civet cats probably act as an intermediate host, or "amplifier." understanding the lifestyle and environment in which the disease arose helped to rapidly control it, preventing a feared worldwide epidemic. recently, new technologies such as improved genome sequencing and microarrays are generating huge quantities of genomic information. bioinformatic tools for analysis of this ever-expanding volume of raw data are developing apace, and genomics, the study of whole genomes, has become an independent biological discipline. the medically relevant value of this data is the potential to identify the genes underlying disease for the purpose of developing treatment or even prevention. evolutionary medicine plays a critical role in the interpretation and application of this information, since human evolutionary history has shaped the genome. humans as a species are relatively young with a mere , -year history. the pattern of our relatively recent global dispersal from our home in africa is reflected in our genes. different populations demonstrate our common ancestry and recent isolation during dispersal (kidd and kidd ) . interpretation of genomic data must take our evolutionary history into account to yield useful applications. one of the most informative analytical approaches is comparison between sequences, either of small genomic regions or entire genomes. by examining differences and similarities between the genomes of two organisms, researchers learn about ancient genes that have deep evolutionary roots and about genetic differences between organisms that make them unique. in comparing the human genome with those of closely related primates, we learn what minor differences may be important keys to our human condition. comparisons between the human genome and more distantly related organisms provide information about ancient genes that play important roles in fundamental processes such as development and physiology. this can be done on various levels. microarrays allow comparisons between sets of genes or two entire genomes. bioinformatic tools allow comparisons of multiple genes, sections of genomes, or entire genomes. a new method that exploits both microarray technology and genome sequencing is the genome-wide association (gwa) study. in a gwa, a microarray of all of the known human single nucleotide polymorphisms (which are simply a change in one nucleotide) is screened for differences between diseased and healthy individuals. to give the study statistical significance, samples from thousands of individuals are used. in a recent gwa, researchers identified genomic polymorphisms potentially associated with seven different diseases (wellcome ). this information can be used to guide future research on the genetics of these diseases. the gwa is yet another method based on enormous data sets that may lead to significant advances in medicine. resources to promote this kind of megaanalysis include the human genome project and the hapmap, which uses collections of single-nucleotide differences as markers for larger genetic regions. the hapmap is important because genomes of any two humans differ only by . %. researchers can use the general markers from the hapmap to narrow down their searches to areas of variation. an important key to drawing useful information from genomic data is the ability to link a genotype to a phenotype. this is a difficult problem for several reasons: genomic data are not usually collected from a specific individual with an extensive health history; family medical histories are rarely complete; environmental conditions vary between populations, etc. one approach to deal with this issue is the use of large, long-term studies of specific populations. the framingham heart study (fhs) is the quintessential large cohort, long-term study. initiated in to investigate the causes of heart disease and stroke, the project collected data on more than , people living in framingham, massachusetts. in addition to birth, marriage, reproduction, and death data, the participants were given physical exams and lifestyle interviews. followup physicals were given every years. adult children of the original cohort and the children's spouses, as well as the original cohort's grandchildren, also participated in the study. this study allowed researchers to identify the major environmental risk factors for cardiovascular disease, such as smoking and high blood pressure. because of the extensive data collected for the project and the virtually unprecedented length of the study, the fhs is incredibly valuable to medical researchers. the data are now being mined by groups researching other diseases such as obesity and eye and lung diseases. other studies of large or multigenerational cohorts are also under way using different populations. diddahally govindaraju is the director of the fhs genetics laboratory at the boston university school of medicine and was one of the organizers of the nescent meeting. he is enthusiastic about the concept of applying evolutionary principles to cohort data such as that generated over nearly six decades in the fhs. "i was essentially sitting on a wealth of medical data that has been collected for the last years," he says. "having been raised on evolutionary biological thought in the first part of my professional life, i looked at this and realized that the doctors were not looking at these data from an evolutionary biology perspective." dr. govindaraju and his colleagues have organized a working group to explore the fhs from an evolutionary viewpoint. the group proposes to look for microevolutionary changes in the study population and to use the extensive medical data to correlate differences in genomic inheritance and phenotypic outcomes. given the vast quantities of data available because of the genomics revolution and multi-generational, longitudinal clinical cohorts such as the framingham heart study, it has become increasingly clear that the analysis of new and existing data from an evolutionary perspective promises to yield important insights into long-standing questions about human physiology and pathophysiology. combined with the development of new technologies that have given us entire genomes and the tools with which to study them, these vast data sets have the potential to launch an evolutionary medicine revolution. it should come as no surprise that evolutionary medicine comes with a unique set of ethical issues. a field that encompasses so many aspects of who we are as individuals and has the power to provide better health to so many people also has the potential to be abused in a variety of ways. positive applications such as personalized medicine, in which an individual would receive the most beneficial medical treatment based on his or her genomic makeup, hold great promise for improving the efficacy of medical care. however, it is no great stretch to imagine that genetic information about individuals could be misused outside of the medical realm. communication of complex evolutionary medicine concepts to the general public will be fraught with cultural perspectives, biases, and historical antagonism, and miscommunication could have serious consequences for individuals and society. privacy concerns are a major issue. when so much data, including genetic information, are being gathered on so many people, how can a participating individual's privacy be protected while simultaneously making the information as widely available as possible (nhgri ) ? also, the use of particular populations in studies is a double-edged sword. in some cases, researchers can learn a great deal by studying a particular group whose members share a lifestyle or ancestry. but how can that group be certain that the information will not be used against them, by insurance companies, for example? on the other hand, if beneficial information is gleaned from one group, how is that benefit made available to other groups who were not studied? another important issue to consider is the concept of race (kittles and weiss ) . particularly in the usa, the sensitive nature of this issue cannot be overstated. the genetic concept of race and the social concept of race are not the same. in colloquial use, the term race designates a group of people based on their appearance and culture. among the research community the definition of race has not been consistent or well defined. scientifically, race correlates roughly with shared genetic ancestry usually based on geographic proximity, but even these parameters fail to clearly delineate groups. there is some debate as to whether race is a legitimate biological concept at all. social concepts of race also influence how research is done by biasing selection of populations to be sampled, methodologies, and other aspects of research. paradoxically, some diseases are associated with socially defined races, such as tay-sachs disease in the ashkenazi jews, making it necessary to take into account these social groupings in conducting research. the exciting potential of evolutionary medicine to yield important new insights into long-standing questions about human physiology and pathophysiology was summed up by stearns: "when you see how evolution illuminates those questions, you see there's the potential for a new way of looking at them to save literally hundreds of millions of lives. that is an enormous payoff, and it's the kind of thing that people in medical research have felt all of their lives, but it's a new motivation for evolutionary biologists, and it's a revelation to them to realize that they could bring that to the table." evolutionary, historical, and political economic perspectives on health and disease treatment options for nausea and vomiting during pregnancy journal watch infectious diseases exotic pets, and emerging zoonoses man knew he had tb before flying to europe morning sickness: a mechanism for protecting mother and embryo evolution in contemporary human populations: medical aids as a zoonosis: scientific and public health implications human factors in emerging infectious diseases recent acceleration of human adaptive evolution human genetic variation of medical significance race, ancestry, and genes: implications for defining disease risk worms, asthma, and the hygiene hypothesis severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats lifestyle, diet and disease: comparative perspectives on the determinants of chronic health risks testosterone-mediated immune functions and male life histories genetic discrimination diabete mellitus: a "thrifty" genotype rendered detrimental by "progress evolution and the origins of disease members of the working group on land use change disease emergence. unhealthy landscapes: policy recommendations on land use change and infectious disease emergence (eds) evolution in health and disease hay fever, hygiene, and household size menstrual cycling and breast cancer: an evolutionary perspective genetics of lactase persistence and lactose intolerance convergent adaptation of human lactase persistence in africa and europe evolutionary medicine the nutrition and health transition in the north west province of south africa: a review of the thusa (transition and health during urbanization of south africans) study insulin resistance in an energy-centered perspective nausea and vomiting of early pregnancy and pregnancy outcome. a meta-analytical review wellcome trust case control consortium. genome-wide association study of , cases of seven common disease and , shared controls bats are natural reservoirs of sars-like coronaviruses can worms tame the immune system coronavirus never before seen in humans is the cause of sars acknowledgments the authors would like to thank the organizers and participants of the evolution in contemporary human populations: medical, genetic and behavioral implications meeting at nescent for providing access to the meeting and supporting information, and reviewers for their helpful comments. key: cord- -sbppgkza authors: donohoe, holly; pennington-gray, lori; omodior, oghenekaro title: lyme disease: current issues, implications, and recommendations for tourism management date: - - journal: tour manag doi: . /j.tourman. . . sha: doc_id: cord_uid: sbppgkza lyme disease is a bacterial infection spread through the bite of an infected tick. in the last few decades, the number and spatial reach of new cases has increased globally and in the united states, lyme disease is now the most commonly reported vector-borne disease. despite this evolving public health crisis, there has been little-to-no discussion of the implications for tourism supply and demand. this paper reviews the scientific literature to identify lyme disease risk factors and the implications for tourism management are discussed. the major contribution of this paper is a set of recommendations for tourism managers who may be tasked with mitigating the risks for visitors and employees as well as the potential impacts of lyme disease on destination sustainability. lyme disease is the world's fastest growing vector-borne zoonotic disease with cases reported in over countries and endemic foci in north america, europe, and asia (who, ) . the world health organization (who, ) reports that the risk is generally low except for those travelling to rural areas, particularly campers, hikers, and workers in countries or areas at risk. however, this assessment may give a false sense of security for people working or travelling in areas where the risk of lyme disease is uncertain and it may breed complacency amongst those responsible for occupational, travel, or public health. the northeastern united states is traditionally defined as the endemic global center for lyme disease and the public health risk is highest in this area. in , systematic surveillance for lyme disease was initiated by the u.s. centers for disease control and prevention (cdc, a), with states reporting cases. in a standardized case definition was approved and by , the number of cases reported had increased over percent (cdc, b; ciesielski et al., ) . the cdc ( a) states that only percent of lyme disease cases are being recorded which translates into approximately , estimated cases in the united states each year (fig. ) . a majority ( percent) have been concentrated in the northeast, yet cases have been reported in every state and in many countries around the world and their geospatial analysis reveals that lyme disease has extended well beyond traditionally defined endemic areas (cdc, b; diuk-wasser et al., ) . lyme disease case rates have been increasing exponentially at the global scale while in the united states it has become the number one and most medically significant vector-borne infectious disease (abbott, ; piesman & eisen, ) . while it is possible to surmise that better diagnostics and increased reporting are responsible for case rate trends in the united states and other parts of the world, case rates vary because of social, environmental, and economic variables such as climate and ecological change, tick and host species range expansion, human demographics and behaviour, land use patterns, clinical practice, disease reporting standards, surveillance technologies, and true incidence (daniels, falco, schwartz, varde, & robbins, ; grenfell & harwood, ; horowitz et al., ) . lyme disease is caused by borrelia burgdoferi e a bacterial pathogen transmitted to humans through the bite of an infected tick. the pathogen is sylvatic; meaning that it cycles between wild animal hosts and vectors. humans are not the primary host and as such are usually incidental or dead-end hosts. although the symptoms have been observed since the th century, it has only been or so years since the etiological agent and the primary vector(s) for lyme disease were recognized and their medical significance reported. the infection is characterized by a distinctive bull's eye rash that occurs in e percent of patients (cdc, ) . from the early symptoms of fever, headache, muscle and joint pain to the more serious symptoms such as cognitive impairment, cardiac abnormalities, arthritis, and paralysis, the impacts on individual health are noteworthy (who, ) . diagnosis is completed through a clinical examination and confirmed through serological tests and treatment consists of oral or intravenous antibiotics. currently, there is no vaccine available for human use but several are available for veterinary use (willadsen, ) . although the medical and scientific communities have been pursuing the problem for years, tourism has been slow to recognize that lyme disease can affect both tourism supply and demand. in the supply context, outdoor workers in endemic areas are at increased risk because of their frequent exposure to ticks and tick habitat (piacentino & schwartz, ) . antecedent studies have established that outdoor workers are up to times more likely to be infected than the general population (bowen, schulze, hayne, & parkin, ; smith et al., ) . given that a proportion of individuals employed in the tourism, parks, and outdoor recreation sectors are required to spend some to all of their time working outdoors, the potential threat to individual and industry health is cause for concern. for those infected, days to weeks of sick time may be required to complete treatment and the medical costs can be significant. in addition to the social costs, the u.s. department of labor reports that workplace illnesses have a major impact on an employer's economic sustainability and profit margin. the annual number of nonfatal illnesses has been estimated at , with direct cost of $ billion and indirect costs of approximately $ billion (leigh, ) . direct costs include workers' compensation payments, and it is estimated that employers pay $ billion per week for direct workers' compensation costs (u.s. department of labor, ) . workers' compensation covers less than percent of these costs, so all members of society share the economic burden. indirect costs include but are not limited to training replacement employees, implementation of corrective measures, lost productivity, and costs associated with lower employee morale and absenteeism. the direct medical costs associated with lyme disease in the united states are estimated at . billion dollars annually but the true cost of lyme disease is the profound impacts on personal health and well-being (maes, lecomte, & ray, ; magnarelli, ; zhang et al., ) . from a demand perspective, the who reports that the risk to those travelling to endemic areas of the world is generally low but nonetheless, they warn travellers to avoid tick-infested areas and tick exposure on their dedicated lyme disease webpage for international travellers (http://www.who.int/ith/diseases/lyme/en). when outdoor activities are planned during a trip to an endemic area for tick-borne disease, a tourist is at risk and the who's assessment can be misleading (falco & fish, ; hayes, ; raoult et al., ; smith et al., ) . their risk can be magnified because tourists may not have all of the necessary information or access to resources that would enable them to make informed decisions regarding lyme disease prevention before and during travel (jonas, mansfeld, paz, & potasman, ; kelly-hope, purdie, & kay, ) . for tourists who are aware of health risks before choosing a destination, s€ onmez and graefe ( ) and kozak, crotts, and law ( ) report that perceived risk can be a strong predictor for avoiding certain regions or changing travel plans. dixon et al. ( ) report that the macroeconomic consequences of a health threat or crisis are highly sensitive to demand-side effects such as the reductions in international tourism and leisure activities that result from elevated risk perceptions. for example, during the severe acute respiratory syndrome (sars) pandemic, traveller's health risk perceptions had a significant effect on international tourism demand. over cases and deaths in countries were reported in with a majority of cases occurring in asia (who, ) . the initial spread of sars was exponential with predictive models showing that if uncontrolled, a majority of people would become infected wherever it was introduced (dye, ) . air travel to areas affected by the advisories decreased dramatically during the epidemic (kuo, chen, tseng, ju, & huang, ; zeng, carter, & de lacy, ) . in east asia, tourist arrivals dropped by percent during the month of april compared to the same period in , with china, hong kong, singapore, and vietnam reporting the greatest losses (pine & mckercher, ; wilder-smith, ) . growth of the broader travel and tourism economy, which measures visitor spending and capital investment around the world, slowed to . percent down from percent in previous years and international arrivals fells . percent (world travel and tourism council, ) . while lyme disease is certainly not a pandemic and it cannot be spread person to person, like sars and other infectious disease risks, it can prompt fear and anxiety that most certainly can affect a traveller's perceptions and by extension; it can affect their travel choices and behaviours (birnbrauer, pennington-gray, & donohoe, ). herrington's ( ) national survey of american risk perceptions regarding ticks and lyme disease found perceived risk to be a predictor of behavioural change. those who had seen ticks, had heard about lyme disease, were concerned about being bitten, or knew someone who had lyme disease were more likely to engage in tick-bite prevention such as avoiding tick habitat and wearing insect repellent. brewer et al.'s ( ) research confirms that higher risk judgements encourage people to engage in protective behaviour, indicating a causal relationship between perceived risk and behaviour modification. although empirical research has yet to confirm as much, lyme disease beliefs and attitudes have likely been having an impact on the decision-making processes and behaviours of outdoor workers, recreationists and travellers (hanson & edelman, ) . knowing that lyme disease poses a real and growing threat to the tourism industry, the purpose of this paper is to first, review the scientific literature to identify lyme disease risk factors and second, to critically assess the implications for tourism management. a search for scholarly journal articles about lyme disease was performed using pubmed, proquest global, and google scholar on july , . the search parameters were narrowed to identify articles where "lyme disease" occurred in the title then additional words were added to winnow down the results to relevant articles: "travel", "tourism", "recreation(al)", "park(s)", "occupation(al)", "risk", and "prevention" (table ) . no temporal restrictions were used. after removing duplicates, unique scholarly articles were identified and reviewed. the search for articles with "lyme disease" and "travel" or "tourism" resulted in articles in all three search engines. the fact that lyme disease has not been examined in the context of tourism and travel is no surprise because the tourism domain has not traditionally focused the research lens on health risks. there is a research and knowledge gap regarding health risk generally and lyme disease specifically, as well as their potential and real impacts on the tourism industry. it should be noted that a similar pubmed search using "tick-borne disease" and "tourism" yielded papers dealing with other tick-borne diseases that occur independent of or in conjunction with lyme disease. anaplasma, babesia, bartonella, ehrlichia, and rickettsia for example, may be just as significant as lyme disease in some parts of the world yet they are equally underappreciated in the travel and tourism context. these papers were consulted in the preparation of this paper and they are also included in the analysis that follows. when "recreation/al" and "parks" was added to the search, articles were identified, of which were case or exploratory studies of ixodes scapularis tick populations and their hosts in select recreational parks in canada (morshed, scott, fernando, mann, & durden, ; scott et al., ) , italy (curioni et al., ) , and the united states (daniels et al., ; falco & fish, ; lane et al., ) . two articles surveyed parks visitors (hallman, weinstein, kadakia, & chess, ) and recreational destination workers (rees & axford, ) about their lyme disease knowledge, tickexposure, tick-bite prevention behaviours and/or clinical history with the disease. of the articles where "occupation(al)" or "work(er)" appeared, the majority were seroprevalence studies of individuals working in the forestry, parks, land management, and agricultural sectors in various locations around the world (e.g. cisak, w ojcik-fatla, zają c, sroka, & dutkiewicz, ; goldstein et al., ; guy, bateman, martyn, heckels, & lawton, ; nakama, muramatsu, uchikawa, & yamagishi, ; smith et al., ; stanchi & balague, ) . one study from the united states reported the results of a survey of workers regarding their knowledge of lyme disease and their behaviour regarding tick-bite prevention and one article provided a comprehensive review of the occupational risks (piacentino & schwartz, ) . over articles were found that contained "lyme disease" and "risk" in the title. a majority of these articles assumed a natural science perspective (i.e. epidemiology, medicine, microbiology, and zoology) and reported on the spatial and temporal factors affecting ticks, the causative agent e the borrelia pathogen(s), host species, environment, and human exposure (e.g. allan, keesing, & ostfield, ; glass et al., ; guerra et al., ; maher, nicholson, donnelly, & matyas, ; ogden et al., ) . a minority of the papers focused on behavioural risks and a review of these papers revealed a consensus that human exposure to ticks is the central risk factor for infection (e.g. diuk-wasser et al., ; fish, ) while knowledge and prevention act as risk mediators (e.g. mckenna, faustini, nowakowski, & wormser, ; smith, wileyto, hopkins, cherry, & maher, ) . articles that contained the key word "prevention" did so primarily as a recommendation for public agencies responsible for reducing the burden of infection. piesman and eisen ( ) and poland ( ) provide a review of lyme and tick-borne disease prevention strategies and mowbray, amlôt, and &rubin ( ) provide a review of education and communication interventions to prevent tick-borne disease. a critical synthesis of this body of research was completed to identify lyme disease risk factors as they have potential implications for tourism management. it is the aforementioned factors e exposure, knowledge, and personal protection behaviour -that comprise the discussion in the sections that follow. . . tick exposure i. scapularis or the blacklegged tick is the primary vector for lyme disease in the united states. in the united states and other areas of the world, dozens of tick species have been implicated in the spread of lyme and lyme-like borrelia pathogens to humans and animals (e.g. barbour, maupin, teltow, carter, & piesman, ; burgdorfer, ; clark, ; clark, leydet, & hartman, ; harrison et al., ; mather & mather, ; nakao, miyamoto, & fukunaga, ; piesman & sinsky, ; piesman, clark, dolan, happ, & burkot, ) . epidemiological studies have explored the relationship between ticks, climate, seasonality, host species, and disease risk in a variety of local or regional contexts (e.g. estrada-peña, ; sumilo et al., ; wielinga et al., ) . predictive models have been developed using remote sensing, satellite imagery, geographical information systems, and advanced statistical analysis (e.g. daniels et al., ; diuk-wasser et al., ; werden et al., ) . the research indicates that vegetation and climate are the two central predictors of suitable/unsuitable habitats for tick species and it is therefore possible to identify high-risk areas and potential emergent areas for lyme disease. the areas with the highest risk for human exposure are those that contain suitable tick habitat, ticks capable of transmitting the lyme disease pathogen, and have high host activity (gray et al., ; schulze, jordan, & hung, ) . empirical approaches have laid the groundwork for a better understanding of the relationship between environmental factors and tick distribution. however, they fail to account for the vital role that human behaviour plays in the disease transmission process. the global increase in interaction between humans and wildlife through population growth, economic development, and an increasingly mobile society that brings people into endemic areas for work and leisure is amplifying personal risk (kollaritsch et al., ; quine et al., ; rizzolli, hauffe, vourc'h, neteler, & rosa, ) . the rapid expansion of the tourism industry and air travel is of concern as the importation of pathogens and vectors by travellers (purposefully or not) and specifically ticks and tick-borne disease pathogens is a real threat to human and ecological health (e.g. daugschies, ; hall, ; holzer, ; schuster et al., ) . european case reports demonstrate statistical relationships between outdoor recreation in tick-infested forests and tick-borne disease infection (e.g. daniel, danielov a, k rí z, jirsa, & no zi cka, ; kahl & radda, ; sumilo et al., ) . early case studies in the united states failed to show a significant increase in risk associated with outdoor recreation (e.g. bowen et al., ; ciesielski et al., ; falco & fish, ) but smith et al.'s ( ) research found that persons who had spent more than h per week in outdoor activities in endemic areas were . times more likely to test positive for lyme disease. since the s, a small body of literature has evaluated the occupational risk for lyme disease. in new york, schwartz and goldstein et al. ( ) confirmed that lyme disease is a hazard of outdoor work and a study in new jersey found that outdoor workers were nearly five times more likely to have contracted lyme disease than indoor workers (bowen et al., ) . in the broader international and tickborne disease contexts, the research shows similar relationships between tick exposure and infection. in lithuania, motiejunas, bunikis, barbour, & sadziene ( ) report that those at highest risk for a tick-borne disease (established through seropositivity testing) are outdoor workers ( percent tested positive/n ¼ ). recent case studies in europe (e.g. bartosik, sitarz, szyma nska, & buczek, ; bochnickova & szilagyiova, ; franke, hildebrandt, meier, straube, & dorn, ; jameson & medlock, ) confirm that ticks, lyme and other tick-borne disease pathogens are common across europe and they pose a significant occupational risk to outdoor workers. two studies in poland involving serological testing of both ticks and humans confirmed that those who perform work in forest environments are regularly exposed to tick-borne disease pathogens and as such, are at a higher risk of infection. two dutch studies of lyme disease seroprevalence in outdoor workers showed a statistically significant risk (or, . ; percent ci, . e . and or, . ; percent ci, . e . ) relative to the control group (kuiper et al., ; van charante, groen, mulder, rijpkema, & osterhaus, ) . in , piacentino and schwartz published a meta-analysis of articles on the occupational risk of lyme disease and report that the scientific evidence demonstrates that those who work outdoors in endemic areas are at an increased risk for contracting lyme disease because of their exposure to ticks. it is evident in this body of literature that exposure to ticks is the single most significant human risk factor and the first line of defence against tick bites should be avoidance of these areas completely or seasonally (during periods of high tick activity), or where appropriate, control of the tick population. given the geospatial expansion of tick species, increasing human mobility and encroachment into tick habitat, and the attendant expectation that future interactions between humans and ticks are likely to increase, vigilance and on-going monitoring is required as new and extant tick-borne pathogens pose potential threats to public health. lyme disease risk is magnified when individuals are not aware of the risks, do not mitigate risks, are not familiar with infection signs and symptoms, and do not know where to seek information or support . it is widely accepted that knowledge plays an important role in mediating lyme disease risk as it is a pivotal precursor to preventative behaviour (piacentino & schwartz, ; poland, ). herrington's ( ) national survey found that a majority of americans know about ticks and the risks they present to human health. in the tourism and recreation context, hallman et al. ( ) found that over percent of people surveyed at three state parks in new jersey reported knowing about lyme disease and could name at least one bite prevention strategy (table ). in recreational and agricultural settings in poland, bartosik et al. ( ) found that workers do not know about the health risks associated with ticks and they are not protecting themselves against tick bites while at work. in the forestry sector, cisak et al. ( ) found that workers possess only basic tick-borne disease knowledge. in france, a study of nearly outdoor workers revealed that a majority would like to have access to educational materials so that they can make informed decisions about tick-bite prevention (thorin et al., ) . it is difficult to draw conclusions on the basis of the limited study of lyme and associated tick-borne disease knowledge, but the literature suggests that knowledge varies geographically and although it has not yet been correlated, it may be that knowledge is higher in areas where lyme and/or other tick-borne diseases are endemic and therefore the risk is higher and investments in educational interventions may be greater. in the latter case, the science appears more developed and it suggests that educational interventions do influence an individual's lyme disease knowledge. for example, gould et al. ( ) evaluated resident behaviours following an intensive community-wide education program and found that percent were aware of the risks and were taking steps to prevent tick exposure. gray et al. ( ) assessed knowledge of lyme disease among a sample of students before and after an educational intervention and found that general knowledge of ticks and lyme disease improved. jenks and trapasso ( ) found that knowledge of tick removal and the signs and symptoms of infection improved following a one-on-one educational intervention by a physician. maher et al. ( ) measured the impact of a lyme disease education program for children in the united states and found that participants had more knowledge of ticks and more confidence in their ability to do a tick check. fox ( ) reports that individuals were more knowledgeable about wearing protective clothing following an educational intervention while knowledge of other personal protection measures was not significantly changed. this body of work provides empirical evidence that the communication of objective and clear messages about tick-borne disease and tick exposure can be effective for increasing public knowledge as well as an individual's ability to make informed decisions about personal protection. concomitantly, prevention investments specifically targeted at increasing public knowledge are an important risk mediator because knowledge can empower the individual to choose to engage in personal protection behaviours. not knowing about lyme disease is most certainly a risk factor for those living or visiting endemic areas for work or leisure. it is widely accepted in the health risk literature that behaviour plays an important role in minimizing disease risk . behavioural modification related to lifestyle, hygiene, and personal protection is the primary method for the prevention of infectious diseases where prophylactics are unavailable. for example, noroviruses are the most common cause of epidemic gastroenteritis on cruise ships and they are responsible for at least percent of all gastroenteritis outbreaks worldwide (goodgame, ; hall et al., ) . hand hygiene (washing with soap and water) is the single most important method for preventing infection and controlling transmission (hall et al., ) . to prevent mosquito-borne infectious diseases such as dengue fever and west nile virus, personal protection through the use of insect repellents and the avoidance of outdoor activity during peak mosquito feeding times is recommended in conjunction with communitybased chemical and biological control investments (renganathan et al., ) . in the case of malaria, the use of insecticide-treated bed nets has been shown to minimize human-vector contact and the incidence of infection by up to percent (choi et al., ) . in the absence of a lyme disease vaccine for human use, the u.s. centers for disease control ( a) recommends personal protection measures for avoiding tick bites and infection: ( ) avoid tick areas; ( ) avoid direct contact with ticks; and ( ) remove ticks from your body (table ). if tick habitats cannot be avoided, there are simple things that individuals can do to minimize the risk for tick bites and pathogen exposure (see table ). wearing protective clothing mechanically decreases direct skin exposure and wearing light-coloured clothing makes it more likely that the tick will be detected and removed before it finds a feeding site (garcia-alvarez, palomar, & oteo, ) . repellents containing deet (n, n-diethylmeta-toluamide) applied to skin and permethrin-based insecticides applied to clothing have been shown to effectively decrease the risk of tick bites and they are reasonably safe to use (patey, ; piesman & eisen, ) . recently, vaughn et al. ( ) evaluated the protective effectiveness of factory-based and longlasting permethrin (insecticide) treated uniforms among a cohort of outdoor workers in north carolina. the incidence of workrelated tick bites reported by the treatment group was significantly lower than the control group and the treated uniform prevented % of tick bites. the results indicate that long-lasting permethrin impregnated uniforms can be highly effective for deterring tick bites for up to one year. if a tick bite should occur, the tick should be removed using the proper technique of grabbing the tick close to the skin and pulling gently upward with tweezers (tick removal guide: http://www.cdc. gov/ticks/removing_a_tick.html). the tick should be saved in a sealed container and provided to a physician for testing when the victim seeks medical attention (due, fox, medlock, pietzsch, & logan, ; garcia-alvarez et al., ) . in this regard, knowing the early signs and symptoms of infection are important for early diagnosis and treatment (symptom guide: http://www.cdc.gov/ ticks/symptoms.html). it is important to note that not all bites will result in infection because not all lyme disease transmissioncapable ticks carry the pathogen. and, the public health risk for lyme disease is affected by spatiotemporal patterns of the tick vector, its life stage, preferred host species (i.e. mice, deer), as well as seasonality, climate, and a range of other environmental factors (adelson et al., ; diuk-wasser et al., ; ostfeld et al., ; qui, dykhuizen, acosta, & luft, ; schulze, jordan, schulze, mixson, & papero, ) . it is equally important to note that while numerous personal protection strategies have been recommended by public health authorities (table ) , they vary in cost, acceptability, and effectiveness, and their uptake has been universally poor (corapi et al., ) . research in areas where lyme disease is endemic has demonstrated that despite adequate knowledge about its symptoms and transmission, individuals are not taking action to protect themselves from the risk of infection. in the tourism and recreation context for example, hallman et al. ( ) reported that over percent of visitors surveyed in new jersey state parks could name at least one tick bite prevention measure but a majority ( percent) took no precautions. shadick, daltroy, phillips, liang, & liang ( ) study of residents and visitors to martha's vineyard in massachusetts found that knowledge about lyme disease alone was not a predictor for protective behaviours and visitors were less likely to protect themselves than residents. a similar study conducted ten years later found that visitors to nantucket (located next to martha's vineyard) were significantly more likely to take precautions when visiting if they had been exposed to a lyme disease education program while on the ferry to this island destination . in the united kingdom, a study of early lyme disease patients found that participants were more likely to perform tick checks, monitor for symptoms, and seek medical attention after a countryside visit (i.e.) rather than take precautions before and during the potential exposure (marcu, barnett, uzzell, vasileiou, & susan, ) . those who regularly frequented the countryside were the least likely to perform during-visit precautions (marcu et al., ). herrington's ( ) national survey of americans found that despite knowing about lyme disease risk, a majority ( percent) of those surveyed were not taking action to prevent tick bites. conversely, gould et al.'s ( ) study of knowledge, attitudes, and behaviours of connecticut residents e a highly endemic area of the united states, found that percent knew some to a lot about lyme disease, percent felt they were very or somewhat likely to get lyme disease in the coming year, percent used personal protective behaviours to prevent lyme disease, and percent reported using environmental tick controls (e.g. pesticide application on personal property). in the occupational context, "adherence to national institute for occupational safety and health-recommended tick bite prevention methods is poor" in the united states and the same is suggested in the literature for other parts of the world (vaughn et al., : ) . in poland for example, cisak et al. ( ) found less than percent of the forestry workers they surveyed were practising tick-bite prevention. bartosik et al. ( ) found that high-risk workers do not possess knowledge of the potential consequences of a tick bite, are unlikely to take protective measures beyond insect repellent application, and those from urban areas were less likely to protect themselves. the mixed results from the study of residents, visitors, and outdoor workers suggest that a variety of factors are influencing the adoption of personal protection and that more research is needed to better understand how they vary spatially, temporally, socioeconomically, or otherwise (bayles, evans, & allan, ) . a limited number of controlled trials have attempted to empirically measure the relationship between knowledge, tick-bite prevention, and seropositivity (laboratory confirmed measure of infection). daltroy et al. ( ) conducted a randomized trial of a lyme disease educational program whereby approximately , visitors to nantucket -a high-risk area in massachusetts, were provided with educational materials before arriving. after their visit, study participants reported their tick-bite prevention while in nantucket and provided a blood sample for serological screening. the study found that visitors who were knowledgeable about lyme disease were significantly more likely to practice tick-bite prevention during their visit and were also significantly less likely to be infected with lyme disease during their stay. at months post visit, participants self-reported that they had been infected with lyme disease and of these were confirmed by physicians. the analysis showed lower rates of self-reported illness among those who received educational materials and long-term visitors (> weeks) were more likely to have lower relative risk than short-term visitors. similarly, malouin et al. ( ) assessed the impact of a lyme disease educational campaign in maryland and found a significant difference between the intervention and non-intervention control group in terms of knowledge and the self-reported use of prevention methods but no significant serological difference between groups was observed. in their study of outdoor workers in new york state, schwartz and goldstein ( ) found that when workers were knowledgeable and they practised personal protection, the occupational risk for lyme disease infection was significantly diminished (as measured by tick exposure odds ratios). a recent study looked at a variety of environmental and behavioural/ personal risk factors for seropositivity and found that exposure and age were associated with positive lyme serology and wearing protective clothing was significantly associated with negative serology (finch et al., ) . this lean body of literature suggests that taking action to prevent tick bites before, during, and after visiting a natural and/or known tick area (see table ) can be effective for preventing lyme disease and that knowledge is a precursor to the adoption of personal protection behaviours (beaujean, bults, van steenbergen, & voeten, ) . this finding is congruent with theoretical explanations found in the health education literature. the health belief model was one of the first theories of health behaviour and it remains one of the most widely recognized and applied in the field. the model suggests that an individual's readiness to take action, is influenced by their beliefs about their susceptibility to a health threat, perceptions of the benefits of taking action to prevent it versus the perceived barriers to taking action, and confidence in their ability to take action (self-efficacy) (rosenstock, ) . this model has been used to better understand an individual's decisionmaking and behaviours related to infectious disease risk (e.g. glanz, rimer, & viswanath, ; harrison, mullen, & green, ; janz & becker, ) and recently it has been shown to be useful for understanding the grey area between tick-borne disease knowledge and personal protection behaviour (e.g. bayles et al., ; beaujean, et al., ) . in the same vein, the theory of planned behaviour posits that behaviour can be deliberative and planned and that an individual's attitude toward a behaviour, the perceived control they have over a behaviour, as well as normative beliefs and subjective norms act to shape an individual's behaviours (ajzen, (ajzen, , . theory of reasoned action suggests that a person's behaviour is determined by his/her intention to perform the behaviour and that this intention is, in turn, a function of his/her attitude toward the behaviour and his/her subjective norm (fishbein & ajzen, ) . the difference between these two theories being that the former suggests that attitude and control are predictors of behavioural intention and action while the later suggests behavioural intention is the most important determinant of behaviour (madden, ellen, & ajzen, ) . while knowledge about a health threat is a precursor to an individual's transition from knowledge to action, there are numerous theories and models that posit a variety of factors to explain the adoption [or not] of health risk prevention behaviours. concomitantly, it must be acknowledged that theories such as these do not capture or model the complexity of behavioural change in the context of health and wellbeing. if it were a simple equation (e.g. increased knowledge of health risk ¼ behavioural change), then nearly everyone would lead healthy lifestyles, they would protect themselves against health risks, and many illnesses would be non-existent. however, it is not as simple as a linear or causal relationship and there are many factors that affect health behaviour. for example, marcu et al. ( ) and beaujean et al. ( ) report that individuals don't comply with tick bite prevention recommendations because they interfere with their enjoyment of the outdoors (e.g., they refuse to wear long clothes on a hot day), they believe the risk of tick bites is low (perceived susceptibility), they do not believe that the recommendations are effective (e.g., insect repellent does not always prevent bites), and they are not confident in their ability to identify a tick or a tick bite. in this case, an improved understanding of the individuals and segments at risk for lyme and other tick-borne diseases as well as the factors that do or do not influence their behaviours is needed to inform the development of targeted and tailored public health interventions (bayles et al., ; beaujean et al., ; mowbray et al., ) . the epidemiological evidence is strongly indicative of a causeeffect relationship between tick exposure and lyme disease infection. although the science is embryonic when it comes to the relationship between knowledge, personal protection behaviour, and lyme disease risk, it certainly suggests that these factors can act as risk moderators and mediators. short of major changes in tick ecology, vaccine availability, tick control, and human behaviour, predictive models suggest that lyme disease will continue to be a public health threat in an increasing number of areas around the world. when we examine this issue through a tourism lens, it is clear that lyme disease is posing a real and growing threat to both supply and demand in endemic and emergent areas of the world. decisions regarding the primary prevention of vector-borne diseases such as lyme disease are as a matter of course, made at the national, state, or regional level and do not involve tourism industry stakeholders but should. in the past, a plethora of methods for large-scale lyme disease prevention including vaccines, biological and chemical tick control methods have been adopted by government agencies at a variety of scales (garcia-alvarez et al., ) . in , the united states federal drug administration (fda) approved the lymerix™ vaccine and the cdc advisory committee on immunization practices (acip) recommended vaccination for those living in high risk areas (nigrovic & thompson, ) . although it was shown to reduce new infections in vaccinated adults by percent, the manufacturer voluntarily withdrew the product from the market in amidst reports of side-effects, a government investigation, a class-action lawsuit, negative media coverage, and declining sales (nigrovic & thompson, ) . in the absence of a human vaccine, a variety of chemicals have been used to reduce the overall tick population but the effect has proven short-lived, their use impractical on a largescale basis, and chemical resistance has been observed in tick species (george, ) . wildlife management has been explored as a less-toxic alternative but attempts to limit the primary host population through hunting, landscape modifications, and movement restrictions (e.g. fencing) have produced mixed results and they too are impractical on a large-scale basis (heymann, ; piesman & eisen, ) . while these interventions may have blunted the tick population and pathogen lifecycle, "they have clearly not been sufficient to lower the number of [lyme] cases, and the epidemic continues to gain momentum" (hayes & piesman, : . given that these efforts have not yet achieved the desired end, willadsen ( : ) argues that "the achievement of the full potential of vaccination, the application of biocontrol agents and the coordinated management of the existing technologies all pose challenging research problems." with an eye to the future, scholars are expressing hope that the development and application of new scientific tools and technologies (i.e. molecular techniques) will have the capacity to revolutionize tick control methods and policies into the future or better yet, they will manifest an effective human vaccine (garcia-alvarez et al., ; sonenshine, kocan, & de la fuente, ; willadsen, ) . in the absence of such a vaccine or other suitable prophylactic and effective tick control methods, piesman and eisen ( : ) argue that lyme disease education is the single most important area in which public health agencies, employers, and other stakeholders can invest to reduce the burden of infection. they argue that the next logical step in prevention is to "ensure ready access to objective information empowering the individuals de facto responsible for control of ticks and tick-borne diseases to make rational and informed decisions regarding their personal risk of exposure to tick-borne pathogens and to take appropriate actions to mitigate risk of tick bites and pathogen exposure." in the united states for example, the cdc has an active tick-borne disease prevention program with a dedicated focus on lyme disease and many state health agencies have developed similar public health education programs. however, they fail to account for the unique characteristics of tourists who may not be familiar with the local area and health risks, and they are unlikely to know where to seek local health risk information. the implications are such that the tourism industry and the tourists upon which it depends are left vulnerable to the real and perceived risk of lyme disease. perhaps it is best that disease prevention be left in the hands of public health experts, but this does not negate the role that tourism bodies can play, in partnership with public health and other organizations, to address the growing lyme disease problem in many parts of the world. it is recommended that tourism industry associations, destination management organizations (dmo), and convention and visitors bureaus (cvb) for example, represent the industry's unique interests, characteristics, needs, and concerns in the public health decision-making forum at national, state, and regional levels (e.g. form a task group). and it is important that these same industry stakeholders assume a leadership role in the dissemination and operationalization of any public health interventions that affect those providing travel and tourism services and those consuming them. when considering the day-to-day management of tourism in high-risk destinations and emergent areas for lyme disease, parsons ( ) and pennington-gray et al. ( ) recommend that managers take a proactive rather than reactive management approach to potential health threats so that they can mitigate the risks and deal with the threat more effectively. ritchie ( ) and pennington-gray, thapa, kyriaki, cahyanto, and mclaughlin ( ) recommend the development of a multi-phase management plan to help destinations and/or attractions, accommodations, and other servicing businesses avoid or limit the severity of the threat's impact on supply and demand. the plan should address: ( ) how to prevent the threat, ( ) what to do in the event of, and ( ) how to recover. in the later cases, generalizable tourism crisis management guidance is available in the literature (e.g. faulkner, ; ritchie, ; young & montgomery, ) . in the specific lyme disease context, it is strongly recommended that tourism managers become informed and take action to protect visitors and employees from the risk of acquiring lyme and other tick-borne diseases. public health authorities should be consulted about the risks for tick-borne disease in the area and any potential "hotspots" for high tick activity and disease risk should be identified and marked. for example, brochures could be provided to visitors upon entry into state or national parks and warning signs could be posted at trail heads, picnic areas, or other high human activity areas where the risk for a tick encounter exists. similarly, park managers could provide brochures and workshops to employees so that they can be made aware of the risks and the ways to protect themselves and park visitors. in endemic areas, travellers arriving by air or ferry or other could be provided with an informative pamphlet in arrival, departure, or visitors centres. public health authorities and local pest management experts should be consulted when making tick control decisions at tourism attractions so as to avoid costly, illegal, or harmful mistakes. where it is feasible or appropriate, tourism managers should seek out a biological tick control expert to recommend strategies for tick and/or wildlife control (e.g. deer fencing). for example, removing leaf litter and cutting back tall grass and brush in high human activity areas is a simple and low cost way to reduce ticks and tick encounters in tourism destinations. it is also recommended that tourism management stakeholders seek out educational materials from public health authorities and make them available to employees and visitors. in the united states, the cdc has a free toolkit that includes downloadable fact sheets in english, spanish, and portuguese and pamphlets and trail signs can be ordered free of charge (http://www.cdc.gov/lyme/toolkit/index. html). in michigan, the public health authority has made available a downloadable fact sheet entitled "preventing lyme disease in recreational camp settings" that provides recommendations for staff and visitor protection (http://www.michigan.gov/documents/ emergingdiseases/camp_guidelines_ _ .pdf). the california department of public health has developed an interactive website to provide information about infected ticks as part of its statewide vector-borne disease surveillance program. the data on the map represents ticks collected and tested by the california department of public health as well as vector control and public health agencies in the state (http://cdphgis.maps.arcgis.com/apps/socialmedia/ index.html?appid¼ d fb d f d a acb d ). this website is a useful model for developing publicly available risk maps for tick-borne diseases in other areas of the world. tourism managers can provide invaluable information to surveillance programs such as the one in california by reporting tick encounters and providing ticks collected in and around tourism attractions to the local authorities for testing. this information is valuable because it can inform public health authorities about emerging outbreaks and it is a pragmatic way in which the tourism industry can take an active role in the monitoring of tick-borne diseases on a local-to-global scale. the world health organization ( ) published the international travel and health book and it contains authoritative information about the geographical distribution, risk for travellers, and recommended precautions for lyme disease and a myriad of other health risks (http://www.who.int/ith/en/). on a biannual basis, the cdc (cdc & brunette, ) publishes health information for international travel or the "yellow book" as it is commonly known (http://www.cdc.gov/features/yellowbook). while it is written primarily for health practitioners, international corporations, volunteer organizations, travel industry professionals, and travellers often refer to this book for recommendations for addressing health risks and international travel. making the aforementioned resources available to employees and visitors by providing copies onsite or sharing the web links to these multimedia resources would enable access to authoritative information before, during, and after travel. tourism managers can use existent resources such as the ones mentioned and/or they can invest in the development of their own communication plan and tools so as to account for the unique risks, visitor or employee demographics, or other conditions specific to their site. for example, rocky mountain national park developed a downloadable brochure that details the health risks and unique ecology associated with ticks in the park (http://www. nps.gov/romo/planyourvisit/upload/ticks_ .pdf). it is also recommended that an occupational risk workshop or training module be offered so as to improve employee knowledge of the risks and the action they can take to prevent the disease and educate visitors. it must be stressed that regardless of the intervention, whether it be tick control or educational programs, tourism management stakeholders should consult and collaborate with travel health experts and public health authorities if we are to make any gains in tackling this health and safety issue. there is a role for tourism and it is recommended that tourism stakeholders partner with appropriate agencies and organizations, become active in local surveillance efforts, and co-develop educational or other interventions. this breadth of expertise and perspective is required for addressing and yielding positive results with respect to the complexity that is lyme and tick-borne disease control. this paper has critically reviewed and synthesized the literature concerned with lyme disease and three human risk factors have been identified. exposure was confirmed to be the only prognostic factor for infection while knowledge and personal protection behaviour were identified as mediating risk factors. the review noted that the research concerned with these risk factors is lean albeit evolving and there is much to be learned from all relevant scientific domains from anthropology, to entomology, to pathology, to zoology. by extension, there is a lot of work required to translate science into real-world solutions. piesman and eisen ( : ) state this future research need succinctly: "academic research on tick-borne diseases must be brought into the real world and effective methods for the prevention of tick-borne disease must be made cheap, easy, and safe." research funding is best spent developing a vaccine but until such a vaccine is deemed safe for widespread use or an effective tick control method is developed, research concerned with the aforementioned risk factors is needed to better inform public health interventions targeting the minutiae of human behaviour. in the tourism context, we must first expand beyond our geographical understanding so that we can better understand the spatial reach of lyme disease and destinations at-risk. risk maps are commonly used as a decision support tool by public health agencies, the medical community, and those who are tasked with protecting individual or community health. while the cdc ( ) makes an updated national risk map (based on surveillance data) available every few years, there is much for the tourism industry to learn from the fine-scale surveillance and mapping of disease epidemiology, tick populations, human behaviour, and the environment. as previously stated, tourism managers can play an active role in disease surveillance by monitoring and reporting what is happening on the ground at tourism attractions and destinations. in the global context, understanding has been constrained by research primarily focused on north america and europe. future research should focus on other endemic and emergent areas of the world. specific attention should be paid to destinations where nature-based activities are the primary touristic attraction as the potential impact of lyme and other tick-borne diseases could be significant for destination sustainability. research is also needed to better document and predict the scale and scope of the lyme disease risk associated with human activities and behaviours. in completing this review, it is apparent that behavioural research is an important but inadequately studied aspect of lyme disease prevention. activity-based risk research has focused on outdoor activities generally and outdoor work and outdoor recreation specifically. future research should focus on identifying differences, if at all, between different kinds of outdoor work and/or outdoor recreation activities. insight into these differences would certainly help tourism managers categorize the risks and plan accordingly. decisions concerning personal protection against lyme disease are made at the individual level yet little is known about perceptions of risk and other potential catalysts for protective behaviour. future research should focus on identifying unknown factors and better understanding known factors as well as their influence on an individual's choice to engage in protective behaviour. in the tourism context, the catalyst(s) may be different between the general population, tourists, and outdoor workers and this certainly requires research investments if the industry is to plan a response. future research should also be concerned with improving our very limited understanding of the impacts that lyme disease is having on the tourism industry. based on this review, we can surmise that it is having an impact on employee health, travel choices, and the economic sustainability of tourism in endemic areas but empirical research is needed to identify the scale and scope of past, present, and future impacts. theoretically driven research is needed to improve our knowledge of the relationships between public health, tourism, and the natural environment so that tourism management stakeholders can be empowered to be active agents in evolving and transdisciplinary efforts to prevent, manage, and recover from lyme disease outbreaks. holly donohoe, ph.d. is an assistant professor in the department of tourism, recreation and sport management at the university of florida. central to her research are critical examinations of tourism planning, management and marketing while evaluation science and its use of select methods and techniques for risk, impact, and policy analysis are considered her realm of expertise. current research includes projects related to vector-borne diseases and their impact on the tourism industry. studies range from baseline studies of public awareness, the analysis of public health and tourism crisis management policy frameworks, to the assessment of occupational risk for tourism a nd re creation prof essionals in at-risk environments. dr. lori pennington-gray is the director of the tourism crisis management institute at the university of florida. she received her ph.d from michigan state university ( ), her ms from the pennsylvania state university ( ) and her bs from waterloo university in canada ( ) . she has expertise in tourism marketing, planning and development, policy and crisis management. she has been involved with a number of tourism studies globally and has worked with a number of countries on tourism policy initiatives. dr. pennington-gray has published more than refereed articles, has brought in more than $ m in external research dollars and made over presentations. dr. pennington-gray teaches both undergraduate and graduate students the concepts of tourism policy, planning, marketing and crisis management. oghenekaro omodior, is a doctoral student in the department of tourism, recreation and sports management at the university of florida. he received his undergraduate training in medical laboratory sciences and a master of science degree in clinical biochemistry from olabisi onabanjo university in nigeria and masters of public health from the university of south carolina. karo's research is concerned with spatial patterns and public health impacts of vector-borne and emerging infectious diseases on tourism and outdoor recreation. he is a fellow 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uniforms: a randomizedcontrolled trial for tick bite prevention tick control: thoughts on a research agenda geography, deer, and host biodiversity shape the pattern of lyme disease emergence in the thousand islands archipelago of ontario longitudinal analysis of tick densities and borrelia, anaplasma, and ehrlichia infections of ixodes ricinus ticks in different habitat areas in the netherlands international travel and health international travel and health: lyme borreliosis (lyme disease crisis management and its impact on destination marketing: a guide for convention and visitors bureaus short-term perturbations and tourism effects: the case of sars in china economic impacts of lyme disease key: cord- -a vlkwce authors: brough, helen a.; kalayci, omer; sediva, anna; untersmayr, eva; munblit, daniel; rodriguez del rio, pablo; vazquez‐ortiz, marta; arasi, stefania; alvaro‐lozano, montserrat; tsabouri, sophia; galli, elena; beken, burcin; eigenmann, philippe a. title: managing childhood allergies and immunodeficiencies during respiratory virus epidemics – the covid‐ pandemic: a statement from the eaaci‐section on pediatrics date: - - journal: pediatr allergy immunol doi: . /pai. sha: doc_id: cord_uid: a vlkwce while the world is facing an unprecedented pandemic with covid‐ , patients with chronic diseases need special attention and if warranted adaptation of their regular treatment plan. in children, allergy and asthma are among the most prevalent non‐communicable chronic diseases, and healthcare providers taking care of these patients need guidance. at the current stage of knowledge, children have less severe symptoms of covid‐ , and severe asthma and immunodeficiency are classified as risk factors. in addition, there is no evidence that currently available asthma and allergy treatments, including antihistamines, corticosteroids, and bronchodilators, increase the risk of severe disease from covid‐ . most countries affected by covid‐ have opted for nationwide confinement, which means that communication with the primary clinician is often performed by telemedicine. optimal disease control of allergic, asthmatic, and immunodeficient children should be sought according to usual treatment guidelines. this statement of the eaaci section on pediatrics puts forward six recommendations for the management of childhood allergies and immunodeficiencies based on six underlying facts and existing evidence. viruses interact with most living organisms, mostly in a symbiotic way. nevertheless, this equilibrium can be disturbed in many occasions and lead to outbreaks of disease. in the case of the delicate interaction between viruses and human beings, many significant epidemic outbreaks of viral disease are linked to respiratory symptoms. while all humans are at risk of being infected regardless of age, gender, or health, vulnerable populations at higher risk of developing a severe form of covid- disease have been identified. among them, older age is a major risk factor. since the beginning of the pandemic, it has become rapidly apparent that children less often present symptoms and that these are less often severe. the only systematic review bringing together data on children has been published recently and concluded that children account for %- % of the diagnosed covid- cases often have milder disease than adults, and mortality rates are extremely low. however, with hundreds of papers published on covid- in the past few months, the problem of reporting the same patients in different manuscripts was raised ; duplicate reporting may lead to inaccurate scientific record and thus potentially misleading results in any systematic review and meta-analysis. the first reliable data came from more than . case reports reviewed by the chinese center for disease control and prevention showing that less than % of cases were younger than years of age. however, as discussed in the paper and outlined in an accompanying editorial one should bear in mind that in this study testing for other viruses was not standardized, and two-thirds of cases were control of allergic, asthmatic, and immunodeficient children should be sought according to usual treatment guidelines. this statement of the eaaci section on pediatrics puts forward six recommendations for the management of childhood allergies and immunodeficiencies based on six underlying facts and existing evidence. allergy, asthma, biologics, children, coronavirus, corticosteroids, covid- , immunodeficiency, sars-cov- , treatment clinically diagnosed, but not virologically confirmed. in addition, more of the severe and critical cases were in the suspected than the confirmed group which suggests that some suspected cases might be caused by other respiratory infections. the cdc in the united states recently published a review of laboratory-confirmed covid- in children between february and april , . among ( . %) reported cases for which age was known, ( . %) were among children aged < years. the majority of pediatric cases ( %) were male, and male predominance persisted even in infants. among pediatric cases with information on underlying conditions, % (n = ) had at least one underlying condition, most commonly chronic lung disease (including asthma) (n = ), followed by cardiovascular disease (n = ), and immunosuppression (n = ). information on hospitalization status for children was available for only ( %) cases. infants accounted for the highest percentage of hospitalizations; of the infants with known hospitalization status, % (n = ) were hospitalized, including five who were admitted to intensive care. there was little variation in the percentage hospitalized among children > year of age. limitations of this study included substantial missing data; thus, statistical comparisons were not possible and need to be interpreted with caution. however, similarly to the wuhan review of pediatric cases, children were less severely affected than adults, and infants were more likely to be hospitalized. of note, given the higher percentage of asymptomatic children in the population, the real percentage of pediatric subjects with severe or critical disease may be even lower. the reason for the relatively milder clinical presentation in children is mostly unknown. as outlined in the chinese study, several factors in relation to the exposure and host factors may account for this observation; in addition, since the children's immune system is still developing, it may react to pathogens differently than do adults. angiotensin-converting enzyme ii (ace- ) which acts as the receptor for coronaviruses may be structurally and functionally less mature in the airways of children. [ ] [ ] [ ] as children attend daycare/ schools, they are exposed to a variety of viruses including other types of coronaviruses, and they may be better equipped with general antiviral defense mechanisms of the immune system. another potential hypothesis suggested that the simultaneous presence of other viruses in the respiratory tract mucosa, which is very typical for young children, may limit the growth of sars-cov- by direct virus-to-virus interactions and competition. similar to the observation made in children with sars, the children with covid- infection may not be mounting a generalized cytokine storm but rather may be responding with elevation of specific cytokines. in a recent study with clusters of adults with pneumonia cases in wuhan, china, the cytokine profile associated with covid- disease severity was characterized by increased interleukin (il)- , il- , granulocyte colony-stimulating factor, interferon-γ-inducible protein , monocyte chemoattractant protein , macrophage inflammatory protein -α, and tumor necrosis factor-α. it is also possible that children have less severe disease because they have fewer chronic health conditions. it is important to note that although children have less severe covid- disease, they are still able to pass on the virus, even while asymptomatic. in addition to age, chronic health conditions such as renal failure, diabetes, hypertension, and heart disease are major risk factors for developing more severe symptoms of covid- . patients with asthma (particularly severe or uncontrolled asthma) and immunodeficiency have also been classified to be at increased risk of developing more severe covid- , based more on common sense rather than mounting evidence. however, recently, the cdc in the united states released a morbidity and mortality weekly report which suggested that adults with a history of asthma were more likely to be hospitalized with covid- ; those hospitalized with covid- had a higher rate of a history of asthma ( . %) than the general population ( . %). while public policy is majorly focusing on "flattening the curve," that is, preventing a too rapid spread of covid- and on providing adequate health care for patients with severe respiratory symptoms in this very aggressive disease, many health providers are faced with questions about safe management of their patients with chronic health conditions. many elective face-to-face hospital services have been discontinued, in favor of remote consultations that have also been substantially scaled back, which provides its own challenges. the care of children with allergies or immune conditions is being adapted to the current situation, with more remote working and providing guidance to children to reduce likelihood of infection in children who would be deemed at higher risk of severe covid- disease. guidance is strongly needed on how to manage children with allergic diseases during the pandemic, particularly, as the pandemic is hitting the northern hemisphere during the tree and grass pollen pollination season. how to run a clinic under the condition of the covid- pandemic is reviewed in the eaaci position paper. although the current covid- pandemic may fade away and hopefully eventually a vaccine may be available, it is unavoidable that new respiratory viruses will appear and that similar questions will arise again in the future. hopefully, these recommendations will be helpful also in future similar events. it became rapidly evident that severe covid- infections were particularly prevalent in specific risk groups. a retrospective cohort involving adults hospitalized in wuhan at the early onset of the disease identified pre-existing comorbidities in % of the patients. hypertension was most common followed by diabetes, and by coronary heart disease. chronic obstructive lung disease was a pre-existing condition in only patients with of them having a fatal outcome due to covid- . asthma or allergy was not mentioned as a risk factor in this population. older age was associated with increased odds of in-hospital death; this observation has now been confirmed during the course of the disease in europe and north america. evaluating asthma and allergy more specifically as potential risk factors, a retrospective study based on electronic medical records from hospitalized covid- adult patients investigated pre-existing asthma or allergic diseases. in this adult population, drug hypersensitivity was self-reported in . % and urticaria in . %. however, asthma or other allergic diseases were not reported by any of the patients. even though there are no data specifically addressing this question, cdc states that people of all ages with chronic lung disease including moderate to severe asthma are listed as having high risk. pre-existing allergies have not been classified as risk a factor. concerns have also been raised regarding a more severe course of there are no data regarding whether the treatment with inhaled corticosteroids (ics) modifies the susceptibility to or severity of covid- . previous studies have shown that ics especially when used at high doses may be associated with an increased risk of pneumonia in adult patients with chronic obstructive pulmonary disease. in this regard, one should keep in mind that children have different phenotypes of asthma than adults. in children, however, a recent meta-analysis has shown that regular use of ics may not increase the risk of pneumonia or other respiratory infections in children with asthma. early childhood is a time associated with frequent infections due to common respiratory viruses such as respiratory syncytial virus (rsv) or rhinovirus. in children with increased bronchial hyperactivity, recurrent viral infections predispose to episodes of bronchoconstriction. in order to prevent this, these patients are frequently treated with ics. during such treatments, increased severity or frequency of viral infection has not been observed. since asthma itself may be a risk factor for the severity of covid- since primary immunodeficiencies (pid) are congenital disorders, patients with pid might represent a potential group-at-risk in the current pandemic of covid- . from the very onset of the pandemic, a special focus was given to this patient group predisposed to infections with respiratory viruses. an international consensus recently summarized how to best manage patients with pid during the pandemic. according to current knowledge in april , it is not yet known whether any specific form of immunodeficiency poses a particular threat to patients. as a joint project, question- the current covid- pandemic might also pose a risk to pediatric patients with secondary immunodeficiencies, such as patients on immunosuppressive therapy for autoimmune or severe allergic diseases. it is recommended to continue the treatment, including immunosuppressants. in the case a secondary deficiency is treated by immunoglobulin substitution, continuation is recommended. the use of convalescent plasma might be considered in these cases in the future, when this now emerging covid- treatment option is scientifically established. precautionary recommendations for patients with immunodeficiencies follow the national guidelines for the general population and include strict hygiene and social distancing measures to limit exposure. a general consensus has been reached to continue established therapies for the immune disorder, even immunosuppressive therapy for autoimmune complications of the underlying disease. however, the current pandemic poses an exceptional challenge and safety concern for patients treated with cellular therapies, not only limited to the field of pid. the main drawback to define tailored safety recommendations for patients with immunodeficiencies arises from our lack of knowledge regarding immune mechanisms during covid- . cytotoxic lymphocytes, essential to control viral infections, were described to be markedly decreased in total cell numbers, and nk and cd + t cell function was exhausted in patients with covid- infection. moreover, toll-like receptor activation associated with alpha-interferon and tnf-alpha as well as il- and il- production seem to play an essential role in the control of the viral infection. to increase our knowledge in disease mechanisms, we need to learn from clinical and immunologic characteristics of patients with severe in contrast to moderate disease. in children and young adults, covid- mainly occurs mildly and without life-threatening complications. if covid- causes severe to lethal disease as observed in sporadic cases in these age groups without other comorbidities, it is tempting to speculate that severity is due to a defect in defense against the infection. therefore, a targeted search for possible monogenic immunodeficiencies by next generation sequencing and further advanced methods was launched in these patients, which will greatly advance our understanding of immune protection against covid- . there has been no scientific evidence that allergy treatments either increase susceptibility to sars-cov- or the severity of covid- disease. pediatric allergists should treat patients with allergic asthma, allergic rhinitis, or other allergy conditions according to usual guidelines, without restricting the use of any specific medication. one exception to this is the advice to withhold biologics during acute covid disease. in addition, there is also reason to believe that proper treatment of these diseases might prevent unnecessary visits to physicians and hospitals and thus reduce the risk of being exposed to the sars-cov- virus. of note, the global initiative for asthma (gina) recommends avoiding the use of nebulizers for asthma attacks due to the increased risk of disseminating covid- (to other patients and to physicians, nurses, and other personnel); thus, pressurized metered-dose inhaler (pmdi) via a spacer is the preferred 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infection clinical and immunologic features in severe and moderate coronavirus disease parenting in a time of covid- shielding advice for very high-risk groups american college of allergy, asthma & immunology position paper on the use of telemedicine for allergists managing childhood allergies and immunodeficiencies during respiratory virus epidemics -the covid- pandemic: a statement from the eaaci-section on pediatrics key: cord- -sbz uk h authors: bonam, srinivasa reddy; wang, fengjuan; muller, sylviane title: lysosomes as a therapeutic target date: - - journal: nat rev drug discov doi: . /s - - - sha: doc_id: cord_uid: sbz uk h lysosomes are membrane-bound organelles with roles in processes involved in degrading and recycling cellular waste, cellular signalling and energy metabolism. defects in genes encoding lysosomal proteins cause lysosomal storage disorders, in which enzyme replacement therapy has proved successful. growing evidence also implicates roles for lysosomal dysfunction in more common diseases including inflammatory and autoimmune disorders, neurodegenerative diseases, cancer and metabolic disorders. with a focus on lysosomal dysfunction in autoimmune disorders and neurodegenerative diseases — including lupus, rheumatoid arthritis, multiple sclerosis, alzheimer disease and parkinson disease — this review critically analyses progress and opportunities for therapeutically targeting lysosomal proteins and processes, particularly with small molecules and peptide drugs. discovered in the s by christian de duve, lyso somes are membrane bound vesicles containing numerous hydrolytic enzymes that can break down biological polymers such as proteins, lipids, nucleic acids and polysaccharides , . lysosomes have long been known to have a key role in the degradation and recycling of extracellular material via endocytosis and phagocytosis, and intracellular material via autophagy (reviewed elsewhere - ) (fig. ). the products of lyso somal degradation through these processes can be trafficked to the golgi apparatus for reuse or for release from the cell through lysosomal exocytosis, which is important in immune system processes. in addition, it has become clear more recently that lysosomes have an important role in other cellular processes including nutrient sensing and the control of energy metabolism , - (fig. ) . alterations in lysosomal functions, either in the fusion processes involved in the general pathways mentioned above or related to the function of lyso somal enzymes and non enzymatic proteins, can result in broad detrimental effects, including failure to clear potentially toxic cellular waste, inflammation, apopto sis and dysregulation of cellular signalling . such defects have been implicated in many diseases, ranging from rare lysosomal storage disorders (lsds), which are caused by the dysfunction of particular lysosomal proteins, to more common autoimmune and neurodegenerative dis orders , , . despite some limitations, impressive results have been achieved in treating several lsds through enzyme replacement therapy (ert). in addition, sub stantial efforts have been focused on therapeutically targeting the autophagy processes upstream of lyso somes [ ] [ ] [ ] [ ] . however, there has so far been less attention on investigating the potential to directly target lysosomes with small molecules and peptide drugs. nevertheless, with recent advances in understand ing of lysosomal function and dysfunction in dis eases, promising novel opportunities for therapeutic intervention through targeting lysosomes specifically are beginning to emerge. this review will provide a brief overview of lysosomal biogenesis, structure and function, and describe the role of lysosomal dysfunc tion in lsds as well as other, more common diseases. specifically, the article will focus on organ specific and non organspecific autoimmune diseases, including lupus, rheumatoid arthritis (ra) and multiple sclerosis (ms), as these have not been extensively reviewed elsewhere, but will also briefly highlight neurodegen erative disorders such as alzheimer disease (ad) and parkinson disease (pd), to further illustrate the breadth and nature of the emerging therapeutic opportunities. the current 'toolbox' of pharmacological agents that modulate lysosomal functions and emerging novel tar gets and strategies in this set of indications will be high lighted. it should be noted that therapeutic approaches to treat inflammatory and autoimmune diseases aim to inhibit the deleterious excessive lysosomal activity, whereas lysosomal activation would be the goal in the treatment of neurodegenerative diseases. although beyond the scope of this review, such approaches may have applications in other diseases in which lysosomes may play a role, including cancer, metabolic diseases and ageing (reviewed elsewhere , ). the formation of mature lysosomes is a complex process, which involves the fusion of late endosomes that contain material taken up at the cell surface with transport ves icles that bud from the trans golgi network , , . these vesicles contain nearly different hydrolytic enzymes (grouped into nucleases, proteases, phosphatases, lipases, endocytosis a vesicle-mediated process by which cells engulf membrane and extracellular materials. several endocytic pathwaysphagocytosis, pinocytosis and receptor-mediated endocytosis -utilize different mechanisms to internalize material. clathrinmediated endocytosis is the major endocytic pathway in mammalian cells. fig. | the central position of lysosomes at the crossroads of major autophagic pathways. a | functional lysosomes are involved in the degradation (endocytic and autophagic) and regulation of exogenous and endogenous cellular material, including recycling processes. extracellular material endocytosed by the endosomes and intracellular cargo internalized by the autophagosomes fuse with lysosomes for degradation, which produces energy (atp production) and source molecules for the macromolecules. mechanistic target of rapamycin complex (mtorc ) plays a key role in lysosomal nutrient sensing signals (lysosome-to-nucleus axis) to regulate energy metabolism. factors such as energy levels, type of ph, ion channel regulation and others decide the fate of the catabolic process. during lysosomal exocytosis, the lysosomal content favours plasma membrane (pm) repair, bone resorption, immune response and elimination of pathogenic stores. b | the lysosome is the ultimate cell compartment that digests unwanted protein materials generated by macroautophagy , microautophagy (pathways during which the cytoplasmic material is trapped in the lysosome by a process of membrane invagination) and chaperone-mediated autophagy (cma). in general, lipid droplets (lds) are degraded by lipophagy , a subtype of macroautophagy , which is activated by cytosolic lipases. cma has also been demonstrated to participate in the degradation of lds in which perilipin (plin / ) proteins are phosphorylated (p) by amp-activated protein kinase (ampk) with the help of the hspa chaperone. mechanistic target of rapamycin complex (mtorc ) and akt (also known as protein kinase b) are negative regulators of cma , where they exert their effect on the translocation complex of cma. in situations of starvation, negative regulators are controlled by pleckstrin homology domain and leucine-rich repeat protein phosphatase (phlpp). lysosomal stability effects the transcription factor eb (tfeb) translation to the nucleus in which tfeb binds to the coordinated lysosomal expression and regulation (clear) motifs to regulate the transcription of genes. ef a, elongation factor a; lys, lysosome; rac , ras-related c botulinum toxin substrate . an endocytic process by which certain cells called phagocytes (for example, macrophages) internalize large particles (> . µm) such as bacteria, other microorganisms, foreign particles or aged red blood cells, for example, to form a phagosome. a vital, finely-regulated and evolutionarily-conserved intracellular pathway that continuously degrades, recycles and clears unnecessary or dysfunctional cellular components. autophagy is crucial for cell adaptation to the environment and to maintain cell homeostasis, especially under stress conditions. cytosolic apparatus, meant for the regulation of proteins (modification, storing and transportation) and some forms of lipids to the other cytosolic compartments via the trans-golgi network or outside the cell. a process of the secretory pathway in which lysosomes are fused with the plasma membrane and empty their contents outside the cell. this process plays an important role in plasma membrane repair, bone resorption, immune response and elimination of pathogenic stores (mainly in lysosomal storage disorders). (lsds) . a group of heterogeneous disorders caused by defects in the lysosomal enzymes leading to the accumulation of unmodified or unprocessed components in the lysosomes, which ultimately influence other vital pathways in the cells. lsds implicate various vital systems of the human body including the skeleton, brain, skin, heart and central nervous system, which are connected with different metabolic pathways. sulfatases and others), which are synthesized in the endo plasmic reticulum and delivered to the transport vesicles via diverse systems, such as mannose phosphate tags that are recognized by mannose phosphate receptors (mprs) at the membrane , or glucocerebrosidase (gcase) that is transported to lysosomes by lysosomal integral membrane protein , an ubiquitously expressed type iii transmembrane glycoprotein mainly located in endosomes and lysosomes . mature lysosomes have an acidic internal ph, at which the lysosomal hydrolases are active, and a lin ing known as a glycocalyx that protects the internal lysosomal perimeter from the acidic environment of the lumen , , . this acidic environment is maintained through the activity of a vacuolar type proton adeno sine triphosphatase (v atpase), which harnesses energy from hydrolysing atp to drive the translocation of protons through a v membrane domain (reviewed elsewhere , ). other key lysosomal proteins include struc tural proteins such as lysosomeassociated membrane protein (lamp ); proteins involved in trafficking and fusion, such as soluble n ethylmaleimidesensitive factor attachment protein receptors (snares) and rab gtpases; transporters such as lamp a, which has a key role in chaperone-mediated autophagy (cma); and ion channels such as the chloride channel clc and the cation channel mucolipin , a member of the transient receptor potential (trp) family that is also known as trpml (refs , ). most of the proteins are deliv ered through the clathrin adaptor protein alkaline phosphatase (alp) pathway, but some proteins are translocated through the lysosome associatedprotein transmembrane , a protein that is preferentially expressed in immune cells , . although the concept still remains controversial, two lysosome species -conventional or secretoryare often distinguished based on their physical, bio chemical and functional properties. catabolism is the main function of conventional lysosomes, and several other lysosome related organelles (lros), such as mel anosomes, the late endosomal major histocompatibility complex class ii (mhcii) compartment (miic), lytic granules from neutrophils, eosinophils, basophils, mast cells, cd + t cells and platelets, complement these functions , [ ] [ ] [ ] [ ] [ ] . many of the lros act as professional secretory organelles. lros share with lysosomes the majority of typical characteristics (acidic environment, lysosomal transmembrane proteins, fusion property to phagosomes and others), in addition to particular properties resulting from their specific cargoes (for example, melanosomes contain melanosome specific transmembrane glycoprotein, and natural killer cells and cd + t cells contain perforins and granzymes). the detailed mechanisms of biogenesis and secretion of lros remain unclear, although it is known that genetic defects in lros are involved in rare autosomal recessive disorders characterized by reduced pigmentation, such as chediak-higashi disease and hermansky-pudlak syndrome . secretory lysosomes contain many more proteins in addition to those contained in conventional lysosomes, and they participate in multiple cell func tions such as plasma membrane repair, tissue and bone regeneration, apoptotic cell death, cholesterol homeostasis, pathogen defence and cell signalling . lysosomal biogenesis and function are regulated by the basic helix-loop-helix leucine zipper transcription factor eb (tfeb) and the coordinated lysosomal expres sion and regulation (clear) network , , (fig. ) . for example, autophagy, a crucial process in immunity and autoimmunity , is transcriptionally regulated by tfeb . interestingly, lysosomal exocytosis, which is important in many immune functions, also depends on tfeb activation , . moreover, it has been demonstrated that tfeb orchestrates lysosomal ca + signalling . the fact that multiple lysosomal processes are dependent on tfeb activation strengthens its role as a master regulator in lysosomal functions. like other transcription factors, tfeb undergoes phosphorylation and dephosphory lation via different cytosolic and lysosomal pathways (fig. ) , processes regulated by mechanistic target of rapamycin complex (mtorc ), a master controller of cell growth , . lysosomes are at the crossroads of various degrada tive pathways, including endocytosis (phagocytosis) and autophagy (fig. ). three main forms of autophagy have been described: macroautophagy (the most extensively characterized form), microautophagy and cma. at the initiation of macroautophagy, a double membrane sequestering compartment termed the phagophore, which contains cytoplasmic material, is formed and matures into a vesicle called the autophagosome. the cargo is degraded into vacuoles issued from the fusion of autophagic vesicles and lysosomes (called autolyso somes), and the resulting short products are released back into the cytosol for reuse or, according to some times contested observations, possibly dispatched into the miic for ultimate processing and mhcii molecule loading for presentation to cd + t cells , . in contrast to macroautophagy, microautophagy is characterized by direct lysosomal engulfment of cytosolic material into lysosomes, via the formation of characteristic invag inations of the lysosomal membrane. the third major form of autophagy is cma, which involves the recog nition of substrate proteins containing a kferq like motif by a hspa /hsc containing complex (fig. b) . in cma, two proteins have a key role: hspa ensures the selectivity of proteins, which will be degraded via the cma pathway; and lamp a translocates the tar geted cytosolic proteins across the lysosomal mem brane (reviewed elsewhere ). the terminal step of autophagy is called autophagic lysosome reformation, in which tubular proto lysosomes are extruded from autolysosomes (containing lysosomal membrane com ponents) and mature into functional lysosomes . this step is not solely a lysosomal biogenesis process; it also includes a series of elements that are tightly correlated with the regulation of autophagy . in combination with autophagy, lysosomes are involved in both innate and adaptive immune func tions, including foreign material recognition (bacterial, parasitic and viral), activation of pattern recognition receptors (such as toll like receptors (tlrs) and nucleo tide oligomerization domain like receptor), antigen processing and presentation, especially in the context multiple sclerosis (ms) . a demyelinating disease in which the myelin sheaths wrapped around nerve fibres in the central nervous system are progressively destroyed by immune cells and possibly also by autoantibodies. parkinson disease (pd) . a neurodegenerative disorder with symptoms including slowness of movement and a loss of fine motor control, owing to the degeneration of dopamineproducing neurons in the substantia nigra. (cma). a selective autophagy pathway in which proteins that contain a signal kferq-like sequence are targeted by hsap /hsc chaperones and translocated into lysosomes via lamp a. transcription factor eb (tfeb). a protein that plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. it controls lysosomal function via the coordinated lysosomal expression and regulation (clear) gene network (including genes coding for hydrolases, lysosomal membrane proteins and the proton pump v-atpase complex), and additional lysosome-related processes such as autophagy, endocytosis and exocytosis. a finely-regulated process during which the cell forms a double-membrane sequestering compartment named the phagophore, which matures into the autophagosome. a double membrane-bound vesicle, which encloses cellular constituents and fuses with lysosomes to form phagolysosomes where the engulfed material is digested or degraded and either released extracellularly via exocytosis or released intracellularly to undergo further processing. of mhcii molecules, t cell homeostasis, antibody pro duction and induction of various immune signals (co stimulation and cytokine secretion) . besides being a degradative organelle, the lysosome has recently been recognized as a cellular signalling platform , . it plays an important role in nutrient sensing through mtorc and other additional protein complexes, or the so called 'lysosome nutrient sensing machinery' . the discovery of a stress induced lysosome tonucleus signalling mech anism through tfeb further supports the key role of lysosomes in cellular signalling . the lysosome occupies a central position in the main tenance of cellular homeostasis, being involved in the exclusion of infectious agents from penetrating host tis sue and concomitantly promoting immune regulation. lysosomes must therefore be able to respond quickly, with increased or decreased functions, to various metabolic conditions aimed at protecting cells from death or damage. lysosomes are very diverse in size and shape. for reasons that are not totally understood -pos sibly according to their position in the cytosol and/or their composition -some lysosomes in a single cell are more prone to act and defend cells. given the wide range of functions of lysosomes in all metabolic compartments of the cell, any dysregulation of their activity could lead to the impairment of various elements of the cellular metabolic machinery (including the transport and bio genesis of sugar (glycolysis), lipids, proteins and nucleic acids) and of metabolic pathways, phagocytosis, endo cytosis and autophagy. although the underlying mech anisms are far from being fully deciphered, it has been seen that lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative dis eases. dysfunctions of lysosomes can affect the proper activity of other organelles such as peroxisomes and after their synthesis in the rough endoplasmic reticulum (rer), the substrates (cargo) that are intended to be degraded through the endo-lysosomal pathway are transported to lysosomes via the trans-golgi network (tgn). among the key enzymatic systems that are involved in the lysosomal enzyme transportation of cargos from golgi to lysosomes, the best studied is the mannose- -phosphate (m p) receptor (mpr) system, which binds newly synthesized lysosomal hydrolases in the tgn and delivers them to pre-lysosomal compartments. a few components synthesized in the late golgi compartment are delivered directly to lysosomes via the -alkaline phosphatase (alp) pathway. lysosomal components, such as enzymes (lytic enzymes and kinases), membrane-bound proteins/complexes (mechanistic target of rapamycin (mtor)), transporters and ion channels (vacuolar-type proton adenosine triphosphatase (v-atpase), trpml and osteopetrosis associated transmembrane protein (ostm )) and chaperone-mediated transportation are the best-known targeting sites for lysosomal dysfunction. as depicted in the figure, many pharmacological antagonists and agonists exert activities that potentially correct lysosomal dysfunction and therefore represent potential effective pharmacological tools. clear , coordinated lysosomal expression and regulation; cq, chloroquine; hcq, hydroxychloroquine; mtorc , mtor complex ; ptdins( , )p , phosphatidylinositol- , -bisphosphate; raptor , regulatory-associated protein of mtor; ser , smooth endoplasmic reticulum; tfeb, transcription factor eb. www.nature.com/nrd mitochondria, leading to excessive production of reac tive oxygen species with pathological features associated with ageing, cancer, chronic inflammation, neurological diseases, male infertility and infections. such dysregulation is thus central to lsds, and also implicated in a wide range of other disorders, includ ing autoimmune and neurological disorders, in which the autophagy-lysosomal network under the control of tfeb has attracted considerable attention. lsds are a heterogeneous group of about inherited metabolic disorders, which have an incidence of ~ in , live births . these disorders and their treatment have been reviewed extensively elsewhere , , and so will only be covered relatively briefly here. the mutations responsible for most lsds have been largely elucidated (tables , ), and many result in the dysfunction of a particular lysosomal hydrolase, leading to the accumu lation of the substrate of that hydrolase. for example, in gaucher disease, the sphingolipid glucocerebroside accumulates in cells (particularly macrophages) and organs, including the liver and spleen, owing to defi ciency in the enzyme gcase , . in certain lsds, the resultant pathology can be explained by the nature of molecules that accumulate (tables , ). thus, the abun dance of cerebrosides and gangliosides that deposit in the central nervous system (cns) of patients with sphingo lipid storage disorders, such as type ii (acute infantile neuronopathic) gaucher disease, underlies the severe neurological symptoms of such disorders , . in patients with pompe disease, which is caused by α glucosidase deficiency, the high levels of non degraded glycogen that accumulate in muscles could explain the observed myopathy , . however, how the undegraded material accumulates and causes the observed cellular and organ pathology in many other lsds remains unclear. the accumulation of such undigested macromol ecules or monomers in lsds instigates the formation of secondary products, which ultimately escape from the endosomal-autophagic-lysosomal pathways , and lead to multiple consequences that affect most organs, including the brain, liver, spleen, heart, eyes, muscles and bone (table ) . most, if not all, organelles are altered in lsds, including endosomes, autophagosomes and lysosomes, and their functions in lysosomal formation/ reformation and fusion of endosomes or autophago somes to lysosomes are abnormal. alterations in several autophagy processes have also been described in lsds. thus, deregulated mitophagy, which results in the accu mulation of damaged mitochondria, occurs in lsds, leading to major inflammatory consequences in specific tissues , . perturbations in mitochondrial dynamics are frequently observed, which have been linked to the increased production of reactive oxygen species, atp production and ca + imbalance. in lsds, reduced macro autophagy activity (with a decreased autophagic flux) rather than hyperactive autophagy processes, as seen in numerous autoimmune diseases, seems to be responsible for the accumulation of non degraded cytoplasmic pro teins such as α synuclein, huntingtin (htt) and others . mucolipidosis type iv (table ) , a disease characterized by severe neurological and ophthalmological abnormal ities, is caused by mutations in the mcoln gene and is inherited in an autosomal recessive manner. this gene encodes a non selective cation channel, mucolipin , which has recently been shown to be required for effi cient fusion of both late endosomes and autophagosomes with lysosomes , . impaired autophagosome degrada tion results in the accumulation of autophagosomes in lsds . microautophagy processes that do not involve de novo synthesis of nascent vacuoles also appear to be impaired in lsds, and were notably revealed in primary myoblasts from patients with the muscle wasting con dition pompe disease . finally, defective cma compo nents, such as lamp a, could also lead to lysosomal dysfunction. for example, mutations in the lamp gene have been claimed to cause danon disease (inherited in an x linked dominant pattern) . further investigations are needed to support this assertion. lysosomes are involved in pathways central to the immune system, including the degradation of intra cellular and extracellular material, plasma membrane repair, cell death signalling, cell homeostasis and death. although the direct involvement of lysosomes in immunity is far from fully understood, it has long been expected that lysosome dysfunction will have a major impact in immune diseases (table ) . strikingly, however, this field has not been extensively explored. however, elevated levels of lysosomal enzyme activity have been reported to occur in several autoimmune diseases, such as ra, systemic lupus erythematosus (sle), dermatomyositis and psoriasis , , , , [ ] [ ] [ ] [ ] . as discussed, autophagosomes formed during the autophagy process must fuse with lysosomes to generate mitophagy a key process that selectively disrupts damaged mitochondria by autolysosomal degradation, preventing excessive reactive oxygen species and activation of cell death. (htt). discovered in , htt is a protein of kda that is widely expressed within the central nervous system. its structure has been elucidated recently by cryo-electron microscopy. the protein is essential for embryonic development and neurogenesis. it is involved in transcription, vesicle transport, protein trafficking, endocytosis and autophagy. (sle). a chronic, relapsingremitting autoinflammatory syndrome that has multiple and heterogeneous symptoms, including arthralgia, swollen joints, fever, fatigue, chest pain, kidney inflammation, cardiovascular disease and neuropsychiatric complications. its aetiology is mostly unknown. reduction of α-d-mannosidases causes reduced lysosomal breakdown of mannose-based oligosaccharides in many tissues inherited lsd characterized by immune deficiency (susceptibility to infections including pulmonary infections), facial and skeletal abnormalities, hearing impairment and intellectual deficit fabry disease α-galactosidase reduced lysosomal metabolism of α-galactosyl lipids, globotriaosylceramides, causes vascular diseases (cardio, cerebro and renal diseases) in patients , gaucher disease (types , and ) β-gcase accumulation of glucosylceramides in leukocytes (especially in macrophages) leads to abnormalities in the visceral organs (type ) and neurological defects in both children and adults (types and ) , gm gangliosidosis β-galactosidase abnormal lysosomal storage of gm -ganglioside (oligosaccharides) causes skeletal manifestations and neurological impairment in humans , krabbe disease (globoid cell leukodystrophy) defects in the galactocerebrosidase provoke accumulation of galactosylceramide and galactosylsphingosine (psychosine). patients' brain histology shows myelin loss, neuroinflammation and axonal degeneration arylsulfatase a or saposin-b (activator protein; rare cases) defects in the enzymes lead to the accumulation of sulfogalactosylceramide in major organs. it affects the different age groups of humans with development signs and symptoms of the disease , mucopolysaccharidoses enzymes involved in mucopolysaccharide catabolism accumulation of mucopolysaccharides within lysosomes leads to skeletal and joint abnormalities in humans , multiple sulfatase deficiency sumf (formylglycinegenerating enzyme needed to activate sulfatases) abnormal accumulation of multiple, including sulfated, glycosaminoglycans causes neurodegeneration and psychomotor retardation in humans pompe disease α-glucosidase accumulated undegraded glycogen in the muscles and peripheral nerves was observed in humans , sandhoff disease β-hexosaminidase a and b enzyme defects cause gm -ganglioside accumulation in lysosomes, which induces nervous system damage in humans , mucolipidosis (type ii and iii) n-acetyl glucosamine phosphoryl transferase α/β enzyme deficiency results in accumulation of unphosphorylated glycoproteins, which causes motor function and neurological disorders in humans mucolipidosis iv mucolipin-i defects in this lysosomal membrane protein (ca + channel) cause accumulation of mucopolysaccharides and lipids, thereby resulting in hepatosplenomegaly , dysmorphic features and neurological disorders in humans cystinosis cystinosin (cysteine transporter) defects in this lysosomal transporter, cystinosin, cause accumulation of cystine in different organs, first in kidneys and later in other organs in humans , danon disease l amp defects in l amp (especially l amp b) cause accumulation of glycogen and other autophagic components in cardiomyocytes of humans, which results in cardiac diseases l amp b is highly expressed in the brain, cardiac and skeletal muscles defective autophagy processes observed in sgs of mrl/lpr mice crohn's disease abnormal lysosomal ph deregulation of proton-sensing g protein-coupled receptor (gpr ) was observed in both mice and human www.nature.com/nrd peptide epitopes for further processing, clear possibly deleterious apoptotic debris, fuel the amino acid pool and produce energy (fig. ). any deviation in this com plex processing will affect crucial immune cell functions, such as the control of cytokine release, autoimmune cell anergy and programmed cell death of type i (apop tosis) and type ii (autophagy). secretory lysosomes regulate the release of both pro inflammatory and anti inflammatory cytokines, in a process that is depen dent on the type of stimulation. in addition, lysosomes degrade glucocorticoid receptors, which are essential to bind glucocorticoids, although the reasons are not known . in this complex system, lysosomes execute anti inflammatory action via the phospholipase a and cyclooxygenase pathways, and also induce inflam mation through the il β-caspase pathway. in both conditions (pro inflammatory and anti inflammatory), lysosomes act as indirect precursors for autoimmunity. however, induction and suppression of inflammatory signals are stimulus dependent . lysosomal cathepsins have a central role in degrading biological macromolecules in the lysosomes and in the immune response. there are approximately members in this large protease family, most of which are endo peptidases that can cleave peptide bonds of their protein substrates , . cathepsins a and g are serine proteases, cathepsins d and e are aspartic proteases and cathep sins b, c, f, h, k, l, o, s, v, x and w are cysteine pro teases. for example, cathepsin s is responsible for the degradation of antigens (and autoantigens) in antigen presenting cells (dendritic cells, macrophages and b cells), and is therefore involved at an upstream level in the presentation of mhcii-(auto)antigenic peptide complexes to cd + t cells . cathepsin l preferentially cleaves peptide bonds with aromatic residues in the p position and hydrophobic residues in the p position. it is central in antigen processing, bone resorption, tumour invasion and metastasis, and turnover of intra cellular and secreted proteins involved in growth regu lation. cathepsin l deficient mice display less adipose tissue, lower serum glucose and insulin levels, more insulin receptor subunits, more glucose transporter type and more fibronectin than wild type controls . cathepsin g is primarily known for its function in killing and digestion of engulfed pathogens . it is also involved in connective tissue remodelling at sites of inflammation . anti neutrophil cytoplasmic antibodies reacting with cathepsin g have been identified in some patients with sle . abnormal antigen processing and presentation is known to be one of the upstream events that perturb immune responses in sle . because this process is mediated through lysosomes, it was rational to speculate that lyso somal functions could be altered in lupus. interestingly, hypotheses were raised in the s on the 'lysosomal fragility' in lupus, but without much further pursuit . the composition and fluidity of the lysosomal mem brane are effectively crucial in the regulation of lyso somal fusion with other vesicular organelles and for lysosomal uptake of macromolecules. the integrity of the lysosomal membrane also ensures the prevention of release of lysosomal enzymes into the cytoplasm. some lysosomal enzymes released from 'fragile' lysosomes were regarded potentially harmful in lupus . autoimmune diseases (cont.) lysosomes are abnormal in splenic b cells from fas deficient murphy roths large (mrl)/lpr mice, a mouse model of lupus, compared with b cells from healthy cba/j mice . tfeb expression was increased, indicating an enhanced biogenesis of lysosomes, and the lysosomal volume was raised. the expression levels of lamp and cathepsin d were also increased. these results reinforce previous data showing that the expres sion and activity of some lysosomal enzymes (such as cathepsins s, l and b) that play important roles in antigen processing are altered in lupus and other autoimmune diseases , . substantial variations of the acidic endo lysosomal ph also occur in mrl/lpr mice, being raised by ph units in splenic b cells , . this ph change could dramat ically influence the activity of soluble lysosomal hydro lases (such as cathepsins) as well as lysosomal membrane proteins (such as lamps) that are critical for lysosome activity. ph may also affect the elimination of immune complexes that accumulate in lupus as a result of deficits in complement, lower expression of scavenger receptors, increased expression of fcγ receptors and other rea sons . these immune complexes, which contain non selective igg antibodies or autoantibodies associated with autoantigen (including some apoptotic debris), can initiate inflammation of tissues once deposited (for example, in the kidneys and the skin) and generate a cas cade of deleterious effects, such as the release of harmful cytokines and chemokines . recent studies have highlighted the key role of mam malian target of rapamycin complex (mtorc ) in the disruption of lysosome acidification that occurs in this process . in normal conditions, the regulation of lyso somal acidification requires cleavage of the rab small gtpase rab a, occurring on the surface of phago cytic vesicles by locally activated caspase . this finely regu lated process requires the association of cofilin with actin that surrounds the vesicle and recruits caspase , which then activates caspase (ref. ). in lupus prone macro phages, chronically active mtorc enhances cofilin phosphorylation, thereby hampering its association with actin and affecting the downstream cascade of events leading to the appropriate acidification of lysosomes . the importance of mtorc and mtorc has been established earlier in lupus t cells, and in particular, in this context, mtorc activity was increased whereas mtorc activity was reduced . in addition, lysosomal cathepsin k was seen to contrib ute to the pathological events that develop in fas lpr mice, another model of lupus disease, in part through its activity in tlr proteolytic processing and subsequent effects on regulatory t cells. cathepsin k deficiency in fas lpr mice reduced all kidney pathological manifesta tions (glomerulus and tubulointerstitial scores, glo merulus complement c fraction and igg deposition, chemokine expression and macrophage infiltration) and decreased the levels of potentially pathogenic serum autoantibodies . in line with these internal alterations of lysosomes, notably those related to cathepsin functioning, deregu lation of autophagy has been reported to contribute to lupus pathology , - . autophagy failures have been described in the lymphocytes of mrl/lpr mice and (nzbxnzw)f mice , , , (two spontaneous murine models of systemic autoimmunity of distinct genetic ori gins and that display different mhc haplotypes) as well as in t and b lymphocytes of patients with sle , , . murine and human t cells from the peripheral blood showed a significant accumulation of autophagic vac uoles compared with normal . the underlying reasons for the dysfunctions in autophagy observed in lupus are not clearly understood, but several indepen dent investigations have identified risk loci spanning autophagy linked genes in patients with lupus - . recent studies have demonstrated an increase in the level of macroautophagy in salivary gland t lymphocytes and in tears and conjunctival epithelial cells of patients with primary sjögren's syndrome (sjs) , . alteration of cma activity was also recently found to occur in the salivary glands of mrl/lpr mice that develop a second ary sjs like disease . lysosomes, which as discussed are mechanistically involved at the downstream level of both macroautophagy and cma, were found to be altered in salivary glands. flow cytometry analyses revealed that the mean ph of acidic vesicles in mrl/ lpr salivary glands was significantly higher compared with those in mouse control glands and the atp con tent was significantly diminished in mrl/lpr salivary gland cells . furthermore, amounts of several leukocyte glycosidases and proteases were revealed to be increased in leukocytes of patients with sjs in comparison with healthy controls . notably, raised levels of the lyso somal enzymes glucosidase, β glucuronidase and dipeptidyl peptidase i are involved in the tissue injury in sjs . increased expression of lacrimal gland cathepsin s was also reported, which may have application as a diag nostic tool in sjs . two members of the ras oncogene family, rab d and rab , were found to be implicated in the regulation of cathepsin s secretion levels in sjs . in vitro studies on lacrimal gland acinar cells suggested further that secreted ifnγ from acinar cells increases cathepsin s expression and that ifnγ stimulated the mhcii mediated antigen presentation in ocular pathogenesis of sjs . lysosomal cathepsins have important roles in the induc tion and diagnosis of ra, and levels of several cathepsins (b, d, g, k, l and s) that are present in the serum and synovial fluid of patients have been proposed as a basis for ra diagnosis [ ] [ ] [ ] [ ] [ ] [ ] . cathepsin s and cathepsin l are highly expressed in synovial macrophages and thymic cortical cells. they each exert essential roles in the posi tive selection of t cells and antigen presentation, respec tively, and participate in the local inflammation and matrix degradation that occurs in joints . cathepsin b is involved in collagen degradation, which leads to joint destruction in ra , . expression of cathepsin g, which participates in joint inflammation through its chemoattractant activity, has been shown to be raised in the synovial fluid of patients with ra when compared with individuals with osteoarthritis . autoantibodies www.nature.com/nrd reacting with cathepsin g were also identified in patients with ra . compared with patients with osteoarthritis, cathepsin k expression was found to be elevated in ra , and genetic deletion of this particular cathepsin was shown to reduce inflammation and bone erosion in ra conditions via tlr mediation . neurological autoimmune diseases ms, myasthenia gravis, guillain-barré syndrome, chronic inflammatory demyelinating polyneuropathy (cidp), neuromyelitis optica and neuropsychiatric lupus are neurological diseases induced by abnormal auto immunity , [ ] [ ] [ ] [ ] [ ] . neurological autoimmunity against various proteins, such as myelin in ms or n methyl d aspartate receptor in neuropsychiatric lupus , , , can affect various structures within the cns and peripheral nervous system, with diverse consequences. although the exact cause of amyotrophic lateral sclerosis (als) still remains unknown, studies support the existence of auto immune mechanisms, and als is therefore also included in this section. indeed, autoantibodies against ganglio side gm and gd a, sulfoglucuronylparagloboside, neurofilament proteins, fas/cd and voltage gated ca + channels have all been reported in patients with als (reviewed elsewhere ) . in general, the origin of the breakdown in immune tolerance that occurs in this set of neurological diseases is not known. only recently have investigations discov ered that autophagy processes are altered in some of these diseases , , [ ] [ ] [ ] [ ] [ ] . in ms and in experimental auto immune encephalomyelitis, an experimental model of ms, upregulation of the protein kinase mtor has been described, and treatment with rapamycin/sirolimus (an immunosuppressant that inhibits mtor and conse quently stimulates macroautophagy) ameliorates some clinical and histological signs of the disease . increased levels of macroautophagy markers were measured in the blood and brain of patients with ms , . however, impaired macroautophagy was found in the spinal cord of experimental autoimmune encephalomyelitis mice . in a rat model mimicking human cidp, both macro autophagy and cma processes were found to be hyper activated in lymphatic system cells and non neuronal cells (sciatic nerves) of peripheral nervous system cells . in als, current data are conflicted . some data suggest an activation of macroautophagy processes with an accumulation of autophagosomes in brain tissues of patients with als, or an increase of autophagic vacuoles, aggregated ubiquitin and sod proteins associated with map lc b ii in motor neurons of mice developing an als like disease , . in contrast, other data suggest a reduction of autophagy activity , . mutations in sqstm , valosin containing protein, dynactin (a pro tein complex that activates the dynein motor protein, enabling intracellular transport) and rab (a member of small gtpases that is important in the process of endosomes and autophagosomes maturation) have also been described in als [ ] [ ] [ ] [ ] . further studies are required to better understand the type and extent of autophagy dysfunction in this family of complex diseases. there are only a few published studies on lyso somal dys function in neurological autoimmune diseases (table ) . these notably include lysosome fragility, which was observed in patients with ms in the white matter of cerebral tissue, an area of the cns that is mainly made up of myelinated axons . lysosome fragility was also suspected in sle (see above) and other rheumatic auto immune diseases, albeit in other organs , , . as noted above, significant variations in lysosomal ph have been measured in autoimmune conditions such as lupus and sjs, but to our knowledge such studies conducted in the brain or elements of the peripheral nervous system of patients or animal models with neurological autoimmune diseases have not been published . in cidp, it has been shown that schwann cells dedif ferentiate into immature states and that these dediffer entiated cells activate lysosomal and proteasomal protein degradation systems , . based on these observations, schwann cells have been claimed to actively participate in demyelinating processes via this dedifferentiation process, but the mechanism involved remains unde fined . in the rat model of cidp mentioned above, it was shown that lamp a expression was drastically increased in the sciatic nerve macrophages and reduced macroautophagy was observed in schwann cells and macrophages . in ms, studies conducted on white matter demon strated that lysosomes are involved in myelin sheath degeneration as well as in the fragmented protein forma tion. lysosomal swelling was observed near the degen erated materials of astrocytes , and an accumulation of lipids was found . it has been hypothesized that lyso somal swelling/permeabilization might cause the release of hydrolases in the cytosol, where they affect native proteins . in als, patients also show dysfunctions in the endo/ lysosomal pathways, which affect both lower and upper motor neurons (table ) . cathepsin b was particularly found to be involved in the motor neuron degeneration, whereas cathepsins h, l and d were not significantly affected . a cdna microarray analysis on post mortem spinal cord specimens of four sporadic patients with als revealed major changes in the expression of mrna in genes including increased expression of cathepsins b and d . several disease causing mutations in genes related to autophagy have been identified, such as sod , tdp , fus, ubqln , optn, sqstm and c orf (refs , ), but none of them code for lyso somal proteins. so, a crucial remaining issue is to clearly determine whether the lysosomal abnormalities that are observed are linked to intrinsic defaults of lysosomes or result from upstream dysregulation in autophagosome formation and fusion , , . insufficient clearance of neurotoxic proteins by the autophagy-lysosomal network has been implicated in numerous neurodegenerative disorders . in disorders such as ad, huntington disease (hd) and pd, modi fied or misfolded proteins abnormally accumulate in specific regions of the brain. accumulation of aggregated proteins is also seen in als (see above). these abnor mal proteins form deposits in intracellular inclusions or extracellular aggregates, which are characteristic for caused by antibodies targeting the muscle acetylcholine receptor or other neuromuscular junction proteins such as musclespecific kinase. these antibodies compromise communication between nerves and muscles, leading to muscular weakness and fatigue. chronic inflammatory demyelinating polyneuropathy (cidp). a progressive autoimmune disorder in which peripheral nerves (roots and trunks) and brachial plexuses are damaged owing to demyelination. it causes muscle weakness, sensory loss and reduced reflexes. also known as devic's syndrome, this disease is characterized by an inflammation and demyelination of the optic nerve (optic neuritis) and the spinal cord (myelitis). antibodies reacting with aquaporin- water channels in the brains of patients are implicated in neuromyelitis optica. (als). also known as motor neuron disease, this disease generally starts with muscle twitching and weakness in a limb, or slurred speech. it can affect control of the muscles needed to move, speak, eat and breathe, and can be fatal. nature reviews | drug discovery each disease [ ] [ ] [ ] . although there has been substantial research in this field, it is still unclear why sophisti cated 'quality control' systems, such as the lysosomeautophagosome system in particular, fail in certain cir cumstances to protect the brain against such protein accumulation . in ad, one of the most common neurodegenerative disorders, some alterations in the endo/lysosomal path ways have been described (reviewed elsewhere , ). the amyloid precursor protein (app) is cleaved by β and γ secretases into amyloid β peptide (aβ) fragments, particularly aβ and aβ (ref. ). these fragments are found in the amyloid plaques that are one of the hallmarks of ad (the other being neurofibrillary tangles containing phosphorylated tau), and have been widely considered to have an important role in ad pathogen esis , . cell based experiments have demonstrated that lysosomal cathepsins have a role in the generation of aβ peptides (through cathepsins d and e) and the degrada tion of aβ peptides (by cathepsin b) . lysosomal dys function has been observed in patients with ad , , and accumulation of the aβ fragment in neuronal cells was shown to lead to lysosomal membrane alter ations, which cause neuronal cell death . in this context, it is noteworthy that inhibition of cathepsin d, which is involved in the lysosomal dysfunction and notably in the cleavage of the tau protein into tangle like fragments, diminishes its hyperphosphorylation in the brain of patients with ad . in addition, patients with ad with an inherited form of the disease may carry mutations in the presenilin proteins (psen and psen ), app or apolipoprotein e, resulting in increased production of the longer form of the aβ fragment (reviewed else where ). mutation of psen , for instance, leads to direct disruption of the lysosomal acidification due to impaired delivery of the v a subunit of v atpase, a proton pump responsible for controlling the intracellu lar and extracellular ph of cells. the acidification defi cit causes excessive release of lysosomal ca + through trpml channels, which has numerous deleterious effects . these findings strongly support the hypothesis that dysfunction of endo/lysosomal pathways is pivotal in ad. approximately % of patients with pd have a family history of the disorder, although the underlying molec ular mechanisms remain unclear. in the context of lysosomal dysfunction, it is notable that the most com mon of the known pd genetic mutations are in gba (encoding the lysosomal β gcase) -the same gene that underlies gaucher disease -which are present in up to % of patients with pd in the united states . gba mutations are also associated with dementia with lewy bodies . several other genes linked to pd are directly or indirectly related to the endo/lysosomal machinery, such as mutations in snca (coding for α synuclein) , . a hallmark of pd is the presence in neurons of protein inclusions called lewy bodies, which are mainly com posed of fibrillar α synuclein. the α synuclein protein is normally degraded by the lysosomes through the cma pathway, but macro aggregates of α synuclein mutants, which display a longer half life compared with the non aggregated wild type protein, are not degraded by this pathway and, rather, would be degraded via the macro autophagy pathway [ ] [ ] [ ] [ ] . it was further shown that the mutant proteins bind to lamp a and inhibit the translocation of other substrates and, therefore, their final degradation . biochemical analyses suggest that α synuclein is mainly degraded by lysosomal proteases and notably by cathepsin d, rather than by non lysosomal proteases (for example, calpain i) , . accumulation of α synuclein was observed in cathepsin d deficient mice, whereas, conversely, the accumulation of α synuclein aggregates was reduced in transgenic mice that over expressed this cathepsin, resulting in protection of dopaminergic neuronal cells from damage . hd is a rare autosomal dominant neurodegener ative disease caused by an aberrant expansion of cag trinucleotide repeats within exon of the htt gene, which results in the production of aggregation prone htt mutants (mhtt) that are detrimental to neu rons , . whereas htt has a protective role against neuronal apoptosis, accumulation of mhtt, however, induces pathophysiological consequences including lysosomal and autophagy dysfunctions. thus, mhtt perturbs post golgi trafficking to lysosomal compart ments by delocalizing the optineurin/rab complex, which, in turn, affects lysosomal function . excessive mhtt induces accumulation of clathrin adaptor com plex in the golgi and an increase of clathrin coated vesicles in the vicinity of golgi cisternae . the activity of several cathepsins such as b, d, e, l and z has also been linked to hd , , , [ ] [ ] [ ] . cathepsin d is respon sible for full degradation of htt but is less efficient at degrading mhtt, which is processed by cathepsin l , . cathepsin z also cleaves htt and elongated poly glutamine tracts , . thus, lysosomal modulators acting on cathepsin activity might have beneficial effects in the treatment of hd. notably, hyperexpression of cathepsin d (and cathepsin b) was shown to protect primary neurons against mhtt toxicity . alterations in macro autophagy, mitophagy and cma have also been implicated in hd , . cma activity was increased in response to macroautophagy failure in the early stages of hd , a result supported by the findings that hspa and lamp a have important roles in the clearance of htt and that shrna mediated silencing of lamp a increased the aggregation of mhtt . other studies focusing on the htt secretory pathway revealed that mhtt secretion is mediated by the ca + dependent lyso somal exostosis mechanism via the synaptotagmin sensor in neuro a cells . the extracellular release of mhtt was efficiently inhibited by the phosphoinositide kinase and sphingomyelinase inhibitors ly and gw . hd dependent perinuclear localization of lysosomes was also demonstrated . increasing evidence thus implicates lysosomal (and autophagy) dysfunction in the pathogenesis of neuro degenerative disorders , , , , , , . tfeb has received particular attention in this regard [ ] [ ] [ ] , with recent data suggesting that tfeb is selectively lost in patients with ad (as well as als) . increasing tfeb activity might therefore prevent neuronal death and restore neuro nal function in certain neurodegenerative diseases, including pd . a major microtubuleassociated protein of a mature neuron. hyperphosphorylated tau accumulates with ubiquitin in ageing neurons as the neurofibrillary tangles that were identified in numerous neurodegenerative diseases called tauopathies that include alzheimer disease. lysosomes as therapeutic targets given the evidence discussed above, the various lyso somal pathways and their components could represent potential pharmacological targets for a wide range of diseases. when considering lysosomes as targets, it is important to note the need for specificity; that is, agents that will not target all lysosomes, but will specifically tar get those lysosomes/lysosomal proteins that are defective in certain organs, tissues or cells. in addition, inhibitors or activators of lysosomal components may be required, depending on the disease context. there has been considerable interest in therapeuti cally targeting different autophagy pathways, including lysosome dependent pathways, and progress in the discovery and development of small molecules and biologics that target these processes has been reviewed extensively , [ ] [ ] [ ] [ ] , . however, very few therapies that specifically target lysosomal components have so far been generated and found to be effective in clinical trials, with one general exception -the development of erts and small molecule drugs for lsds (box ). this topic has recently been comprehensively reviewed and so will not be discussed in depth here. it is important to target lysosomes and not the whole autophagy process for several reasons. first, regard ing safety, the integral role of lysosomes in several key physio logical processes means that therapeutic windows for pharmacological intervention with unacceptable side effects may be limited. for example, azithromycin, an antibiotic with anti inflammatory properties that is used in the treatment of patients with chronic inflammatory lung diseases such as cystic fibrosis, was found to block autophagy in macrophages, inhibiting intracellular kill ing of mycobacteria within them and, thereby, increasing the risk of mycobacterial infection . second, in some diseases, autophagy may be enhanced in certain tissues or organs but compromised in others, for example in the spleen and salivary glands of mrl/lpr mice . this phe nomenon makes it highly challenging to identify a single drug able to correct a failure, unless a cell specific target ing molecule could be incorporated into the autophagy activator/inhibitor to enable tissue specificity . again, the precise targeting of lysosomes in specialized cells may circumvent the complexity of dysregulation mecha nisms of autophagy processes in pathophysiological settings , , , . as indicated, the current arsenal of lysosome specific targeted drugs is small. in fact, many drugs claimed to target lysosomal components have also been found to be capable of interacting with several non lysosomal receptors, limiting their efficacy and safety . one exam ple is provided by chloroquine (cq), a aminoquino line compound, and its derivative hydroxychloroquine (hcq), which are widely prescribed to patients with rheumatic diseases, and historically also for the prophy laxis and treatment of malaria (fig. ) . cq and hcq are lysosomotropic agents and as such they raise intralyso somal ph, thereby affecting overall lysosomal function and impairing autophagic protein degradation (fig. ) . although the mechanism of action of these agents is not fully elucidated, it is well established that cq and hcq display pleiotropic activity [ ] [ ] [ ] and have impor tant deleterious properties. in certain settings, they have been claimed to operate by interacting directly with tlr ligands and not through an effect on the lysosomal ph, for example . toxicity of cq/hcq, in particular in the eye (cornea and macula) and the occurrence of cardio myopathies , remains a major limitation. the observed ocular toxicity is related to the total cumulative dose rather than the daily dose; therefore, it becomes a serious potential problem in the cases of long term use. several hcq analogues and mimics have been designed that aim to retain the therapeutic activity without secondary effects , . furthermore, most, if not all, of the small molecules that have so far been identified and investigated as mod ulators of autophagy and/or lysosomal functions exhibit complex pleiotropic properties affecting the overall function of lysosomes, and also different autophagy pathways (for example, mtor dependent and mtor independent pathways), as well as other quality control mechanisms that affect the cell life/death balance. as dis cussed below, several widely used molecules exert dual, sometimes opposite, effects on upstream and down stream molecular events of the autophagy-lysosomal network. several robust assays to characterize autophagy acti vators and inhibitors, as well as lysosomal effectors, are currently available and validated (table ) . however, each assay has inherent biases, and so it is necessary to use several independent, in vitro and in vivo approaches to ascertain the reactivity and specificity of novel molecules able to modulate these pathways (box ). in this regard, the tremendous work in recent years to establish international guidelines for standardizing research in autophagy -and, in particular, to propose relevant methodologies for monitoring autophagy that are accepted by the whole community -is unique , . a better definition of terms and concepts has also box | enzyme replacement therapies for lysosomal storage disorders enzyme replacement therapy (ert) for lysosomal storage disorders (lsd) involves administration of a functional version of the defective enzyme in the particular lsd. following administration, the enzyme is delivered to the target cells (typically mediated by mannose or mannose- -phosphate receptors), where it breaks down its substrate in lysosomes, thereby ameliorating the lsd . the approach was pioneered with the use of glucocerebrosidase (gcase) purified from placentae in the s to treat patients with gaucher disease, and a recombinant version of gcase was then introduced in the s . following the success of this approach in treating gaucher disease, other recombinant enzymes have been approved for other lsds, including fabry disease, mucopolysaccharidosis (mps) i, mps ii, mps vi and pompe disease (table ) , and many further erts for other lsds are in clinical trials . although ert has provided an effective treatment for patients with some lsds, it has limitations. recombinant enzymes administered by intravenous injection are not able to cross the blood-brain barrier, and so are not effective for central nervous system manifestations of lsds . low expression of the receptors that mediate delivery on the cell surface of target cells can also be a challenge for the effectiveness of ert for some lsds . for example, in pompe disease, the level of expression of mannose receptors on skeletal muscle cells is low, necessitating high doses of ert to achieve a therapeutic effect . numerous developments are being studied to address such limitations, with a focus on enzyme modifications that enable better access of enzymes to their receptors and on nanomaterials that enable safe and efficient delivery of enzymes via intra-cerebroventricular/intrathecal administration , , , . been adopted by the community, leading to much eas ier understanding between researchers worldwide . these guidelines and definitions should be used by investigators evaluating new molecules designed to selectively target key steps of autophagy or developing new high throughput screening methods for autophagy modulating pharmacological molecules. however, even the more sophisticated and detailed assays will not reca pitulate the full complexity of integrated living systems, which can only be established in clinical trials. the pipeline of specific agonists and antagonists of autophagic activity is currently small, particularly for cma (tables , ; figs , ). however, high throughput screening programmes to identify such small molecules are ongoing, which should yield additional therapeutic targets and useful tools. small molecules that specifically target lysosomes are even rarer (table ; fig. ). small molecule drugs developed specifically for particular lsds, including substrate reduction therapies and small molecule chaperones, have reached the market, but other small molecule candidates for more common diseases are at an earlier stage of development. these molecules that more specifically act on lysosomes, some of which have been discovered by high throughput screening, mostly target lamp a, various lysosomal enzymes such as cathepsins, acid sphingomyelinase, α galactosidase a and acid β glucocerebrosidase, and chaperones such as hspa and β nacetyl hexosaminidase. although not solely present in lysosomes, v atpase, a proton pump responsible for controlling the intracellular and extracel lular ph of cells, and trpml , a cation channel located within endosomal and lysosomal membranes, are also pertinent targets. below and in table , we summarize the availabil ity of pharmacological tool compounds and progress in drug development, where applicable, for each broad target class. in addition to erts for lsds (box ), drug discovery programmes have also focused on alter native small molecule based approaches, which may be particularly relevant for lsds that affect the cns, due to the lack of blood-brain barrier penetration by erts . small molecules used in substrate reduction thera pies prevent the accumulation of substrates of the defec tive enzymes in lsds by inhibiting enzymes involved in substrate production . miglustat was the first such drug to be approved in the early s by the us food and drug administration and the european medicines agency for gaucher disease and in for niemann-pick disease type c in europe. this iminosugar inhibits glucosylceramide synthase (gcs), which catalyses the initial step in formation of many glycosphingolipids. within cells, glycosphingolipids tend to localize to the outer leaflet of the plasma membrane; they cycle within the cell through endocytic pathways that involve the lysosome. inhibition of gcs therefore reduces the deleterious accumulation of glycosphingolipids within lysosomes with potential therapeutic benefits in dis eases like lsds. miglustat also inhibits disaccharidases in the gastrointestinal tract, resulting in diarrhoea as a side effect . eliglustat, another gcs inhibitor that does not penetrate the cns, was also approved for gaucher disease in . other gcs inhibitors in clinical devel opment include lucerastat, a miglustat analogue with an improved safety profile that is currently in a phase iii trial for fabry disease (fd) , , and ibiglustat, which penetrates the cns. the latter is in clinical development for fd (phase ii), for gaucher disease type (phase ii) and for patients with pd who carry a mutation in gba (phase ii). recent findings generated in a small number of patients have suggested a possible link between pd and fd , which also exists between patients with pd and gaucher disease who have gba mutations (see above). finally, genistein, a pleotropic natural product that inhibits kinases involved in the regulation of proteo glycan biosynthesis and also affects tfeb function, is in a phase iii trial for sanfilippo syndrome . substrate mimetics that inhibit lysosomal enzymes have also been found to stabilize mutated enzymes in lsds, thereby leading to restoration of some enzyme activity when suitable subinhibitory concentrations are used, as the enzyme remains stable and functional after dissociation of the inhibitor , . the pioneering exam ple of this approach is migalastat, described above, that binds to the active site of α galactosidase a, which is mutated in fd, and stabilizes the mutant enzyme. other examples of this strategy include afegostat in gaucher disease (which failed in a phase ii clinical trial in due to lack of efficacy), pyrimethamine in sandhoff disease and tay-sachs disease, and ambroxol in gaucher disease with neurological symptoms (table ). agents that are at earlier developmental stages include n octylβ valienamine, a competitive inhibitor of βglucosidase, for gaucher disease; n acetylcysteine for pompe disease; α lobeline, , , trihydroxyisoflavone and azasugar in krabbe disease; and n octyl epi β valienamine and n, s(n′butyliminomethylidene) thio deoxygalactonojirimycin indicated in gm gangliosidosis . the chemical structures of these pharmacological chaperones have been described recently , . finally, an alternative strategy for stabiliz ing mutant enzymes, by binding away from the active site, is also being investigated. a promising example of this approach is ncgc , a non inhibitory small molecule chaperone of gcase discovered by screening for molecules that improved the activity of the mutant enzyme , . treatment with ncgc reduced lyso somal substrate storage and αsynuclein levels in dopamin ergic neurons derived from induced pluripotent stem cells from patients with gaucher disease with parkinson ism , . further testing of ncgc in patients with pd and gba mutations is awaited. although promis ing, conflicting viewpoints still remain on the strength of such small molecule based approaches, primarily because these compounds bind to the catalytic site of enzymes, which may be a risk at high concentrations if they inhibit rather than increase activity , . more clinical trials are therefore required in order to analyse the robustness of this approach. cathepsin modulators. robust genetic and pharma cological preclinical investigations have consistently showed that regulating cathepsin activity can favour ably improve pathological features in certain auto immune and inflammatory diseases. inhibitors of several cathepsins (b, d, l, k and s) have been described , and their activity has been evaluated in rheumatic auto immune diseases (such as sle, ra and sjs) and neuro degenerative disorders, notably in ad (table ) . selective inhibition of cathepsin s with a potent active site inhibitor known as ro (roche) mitigated disease in mrl/lpr lupus prone mice, by reducing prim ing of t and b cells by dendritic cells, and plasma cell generation . promising data have also been generated in murine models, in the context of diabetic nephrop athy and cardiovascular diseases . further studies based on cathepsin s inhibitors should evaluate the clinical safety and utility of treating patients affected by autoimmune and inflammatory diseases . cathepsin k, which is highly expressed by osteoclasts and very effi ciently degrades type i collagen, the major component of the organic bone matrix, is also a potential target for modulating lysosomal dysfunction in some of the dis orders discussed above, such as sle . yet further investi gations with selective cathepsin k inhibitors are required to determine whether this targeted strategy might apply in sle and other inflammatory conditions in which articular manifestations are a major component (ra, ankylosing spondylitis, psoriatic arthritis and others). it should be noted, however, that various cathepsin k inhibitors have been pursued for postmenopausal osteo porosis, including odanacatib (merck) which reached phase iii trials . although odanacatib was effective, its development was discontinued in due to an increased risk of stroke in treated patients. other cathep sin inhibitors and their context of clinical evaluation are listed in table . despite multiple efforts to develop selective pharma cologic cathepsin modulators, important concerns still fluorescence measurement (flow cytometry or fluorescence microscopy) of cellular staining of acidotropic dyes, such as lysotracker dyes , simple to use but is not quantitative as stated by the manufacturer; can be adapted to clinical trial settings western blot and fluorescence imaging of lysosomal markers such as l amp , l amp etc. , simple but does not provide information on subcell populations limited usage in primary cells as they are hard to transfect bsa , bovine serum albumin; l amp, lysosome-associated membrane protein; n/a , not available; qpcr , quantitative pcr; tfeb, transcription factor eb. www.nature.com/nrd remain with regard to off target effects due to activity against other cathepsins or towards cathepsins present at non relevant or unwanted sites. nonetheless, the under lying biology and clinical effects of certain cathepsin inhibitors or activators remain of considerable interest and could guide future therapeutic approaches. as reported below, v atpase, a multisubunit atp driven proton pump, is best known for its role in acidification of endosomes and lysosomes. regulating the function of v atpase may impact lyso somal activity and, hence, the acidification of spe cialized cells and diverse signalling pathways, such as autophagy. v atpase inhibitors like bafilomycin a and concanamycin a are non selective compounds (table ; fig. ) that inhibit both mammalian and non mammalian v atpases, which control the lysosomal ph of acidic vesicles via a manner that is not fully under stood (fig. ) . through this mechanism, bafilomycin a inhibits autophagic flux by preventing the acidifi cation of endosomes and lysosomes . bafilomycin and cq also affect mitochondrial functions, as discovered recently using intact neurons . benzolactoneenamides (salicylihalamide a, lobatamides and oximidines; than bafilomycin a and concanamycin a, but also much less potent. further investigations into v atpase regulation of signalling pathways are needed to identify specific and safe molecules that regulate this vital proton pump . ion channel modulators. as discussed above, lysosomal ion channels are master elements of lysosome activity and, thereby, of cell homeostasis. in the family of trp channels, trml is essential, being widely expressed in late endosomes and lysosomes, and preferentially associ ates with lamp in the lysosomal membrane , , . genetic mutations leading to inactivation of trpml cause a rare genetic disorder called mucolipidosis type iv (mliv). pharmacological activation of trpml ameliorated some lysosomal functions that are clas sically associated with mliv, npcs and certain lsds (tables , ; fig. ). thus, the small molecule sf (fig. ) , which was identified in a screen for trpml acti vators, was defined as an activator of both trpml and trpml (ref. ), and displayed an additive effect in combination with the endogenous activator phospha tidylinositol , bisphosphate (ptdins( , )p ) , . an analogue of sf , in which chlorine on the thiophene had been replaced by a methyl group, showed greater efficacy on trpml activation , . another molecule called ml sa (figs , ) , acting as a mucolipin synthetic agonist, also showed an additive effect with endogenous ptdins( , )p on trpml channels . it is important to note that in neurological diseases, as well as in other indications in which lysosomal acidification is defec tive (see above), interfering with trml may have contraindications. a central modulator of lysosomes is the lipid kinase fyve finger containing phosphoinositide kinase (pikfyve), which converts phosphatidylinositol phosphate into ptdins( , )p . the latter regulates ca + release from the lysosome lumen and is required for acidification by v atpase. inactivation of pikfyve leads to many patho physiological problems including neurodegeneration and immune dysfunction, mostly related to impaired autophagic flux and alteration of lysosomes (trafficking, ca + transport, biogenesis and swelling) . the small molecule apilimod ( fig. ; table ) was originally identi fied as an inhibitor of tlr induced il and il , and later found to be a highly specific inhibitor of pikfyve . apilimod was evaluated in clinical trials involving sev eral hundred patients with t helper and t helper cell mediated inflammatory diseases such as crohn's dis ease, ra and psoriasis , . it was well tolerated in more than human subjects (normal healthy volunteers and patients with inflammatory disease), but the clinical trials did not meet their primary endpoints and further development was abandoned. apilimod is currently being evaluated in a clinical trial (nct ) aimed at defining a maximum tolerated dose in patients with b cell non hodgkin lymphoma and monitoring safety, pharmacokinetics, pharmacodynamics and prelimi nary efficacy . ym is another selective inhibi tor of pikfyve (table ; fig. ). this inhibitor contains a fyve type zinc finger domain. ym was found to significantly reduce the survival of primary mouse hippo campal neurons in culture and reversibly impair endo somal trafficking in nih t cells, mimicking the effect produced by depleting pikfyve with small interfering rna. it was also found to block retroviral exit by budding several parameters have been used to evaluate lysosomal functions (table ) . alteration of lysosomal volume is an important sign of lysosomal dysfunction; it has been observed in various diseases, such as autoimmune syndromes, cancers and lysosomal storage diseases . it can be measured by staining cells with acidotropic dyes such as lysotracker dyes and immunoblot of lysosomal membrane proteins such as lysosome-associated membrane protein (lamp ). variation of lysosomal volume is often related to changes in lysosomal biogenesis, which can be assessed by the expression level and cellular location of transcription factor eb (tfeb). however, precise determination of lysosomal functions relies on measurement of lysosomal luminal ph and degradation activity. several fluorescence probes that measure lysosomal ph (table ) are commercially available. abnormal lysosomal ph affects lysosomal degradation activity, which can be followed, for example, by detecting the degradation of endocytosed fluorescence dq-bsa . in complement, the activity of specific enzymes, such as cathepsins b, d and l, can be tested using commercially available kits. other lysosomal parameters can be evaluated to deepen the examination of lysosomal status, including lysosomal membrane stability and integrity and lysosomal ca + ion signalling, for example (table ) . lysosomal function is essentially linked with autophagy activity as autophagy is a lysosomal-dependent degradation pathway. thus, a series of methods routinely applied for assessing macroautophagy in mouse models and patients with autoimmune diseases is summarized . to ascertain the extent of autophagy defects, a combination of techniques, such as western blot and flow cytometry, measurement of autophagy makers, fluorescent imaging and electron microscopy, in the presence and absence of lysosomal protease inhibitors, is recommended. several review articles have described reliable methods dedicated to the measurement of chaperone-mediated autophagy (cma) activity [ ] [ ] [ ] . increased expression levels of lamp a and hspa , two key players in cma, have been shown to occur in a mouse model of lupus . however, it should be noted that increased expression levels of hspa and lamp a starting from a total lysate is only indicative of cma upregulation; this test is not sufficient to allow any firm conclusion, and it is necessary to examine their expression levels in purified lysosomes or in lysosome-enriched fractions. nature reviews | drug discovery chaperone modulators. molecules targeting chaperone proteins involved in lysosomal function have also been designed for potential therapeutic applications. one of these molecules is ver , a small molecule inhib itor of hspa , a key element of cma , . ver binds to the nucleotide binding domain of hspa and hsp , and acts as an atp competitive inhibitor of atpase and chaperone activity. in a mouse model of ad ( xfad mice), intraperitoneal treatment with ver reduced the two main pathological fea tures of ad (amyloid plaques and paired helical filament tau accumulation) and improved object recognition, location and episodic like memory . another molecule, the mer phosphopeptide p (table ; fig. ), was also shown to interact with hspa (fig. ) and lodge in the hspa nucleotide binding domain , . p and ver , however, do not have the same mechanism of action, and their effects were not additive . p is a phosphorylated analogue of a nominal peptide that was initially spotted in a cellular screening assay using overlapping peptides covering the whole spliceosomal u k protein and cd + t cells collected from the lymph nodes of lupus prone mrl/lpr mice . p peptide enters b cells via a clathrin coat dependent endocytosis process to reach early endosomes and then late endosomes/lysosomes . it affects cma that is hyperactivated in lupus, likely by hampering the cma mediating chaperone hspa (ref. ). p peptide reduces the excessive expression of hspa and lamp a observed in lupus mice, alters the (auto)antigen presentation by mhcii molecules in the miic compartment and, consequently, attenuates the activation of autoreactive t cells . a significant diminution of mhc molecule expression at the surface of antigen presenting cells was measured in mice that received the p peptide intravenously and on patient's peripheral cells treated ex vivo with the peptide , , . as a downstream consequence, the activation of auto reactive b cells and their differentiation into autoantibody secreting cells is repressed , . t cells from patients with lupus are no longer responders ex vivo to peptides encompassing cd + t cell epitopes . the effect of p on cma was demonstrated in vitro, using a fibroblast cell line that stably expresses a cma reporter , . p , which selectively targets the cma/lysosome process and has no effect on mitophagy , has been evaluated in murine models mimicking other rheumatic diseases with very promising results, notably in mice developing sjs features , in mice with neuropsychiatric lupus symp toms and in rats that develop a cidp like disease with disturbance of both cma and macroautophagy in sciatic nerves . in clinical trials that included patients with sle, p formulated in mannitol was found to be safe and non immunogenic after several subcutaneous admin istrations of peptide , , . p showed significant efficacy in a multicentre, double blind, phase ii trial . this peptide is currently being evaluated in phase iii trials in the united states, europe and mauritius. in con tinuation, an open label trial including several hundred patients with lupus worldwide is planned. another peptide has been discovered that, in con trast to p , activates cma . this mer peptide called humanin was originally identified from sur viving neurons in patients with ad, and was found to directly enhance cma activity by increasing substrate binding and translocation into lysosomes. humanin interacts with hsp and stabilizes the binding of this chaperone to cma cargos as they bind to the lyso somal membrane. these results are important as humanin had been shown to possess some cardioprotective and neuro protective properties in diseases such as ad, cardio vascular disease, stroke, myocardial infarction, diabetes and cancer . in addition to the targets discussed above, there are a few emerging potential lysosomal therapeutic targets for which there is strong biological validation, but not yet any small molecules in development that target them. an example with likely pharmacological tractability is a lysosomal k + channel called tmem , which is impor tant for maintaining the membrane potential and ph stability in lysosomes . deficiency in tmem may play a critical role in pd pathogenicity . importantly, the structure of tmem has been recently refined . another target for which ligands have not yet been validated is the kcnq / channel (also named m channel or kv . / . channel). it has been shown in npc disease that reduced cholesterol efflux from lyso somes aberrantly modifies neuronal firing patterns . this disruption of lysosomal cholesterol efflux with decreases in ptdins( , )p dependent kcnq / chan nel activity may lead to the aberrant neuronal activity. the cholesterol transporter and ptdins( , )p floppase, abca , is responsible for the decline in ptdins( , )p that consequently modifies the electrical properties of npc disease neurons. dysfunction in the activity of kcnq / or altered levels of ptdins( , )p , due notably to genetic mutations, might also be involved in other neuropathies (for example, some forms of epilepsy, hd, pd, ad, als and friedrich ataxia). although further experiments are needed to validate the link discovered between hyperexcitability and cell death in npc disease and other neurodegenerative diseases, small molecules such as retigabine, an anti convulsant drug that keeps kcnq / channels open, might rep resent important therapeutic tools , . other channel opener ligands of kcnq /q include ica and its derivatives. another promising therapeutic target is sphingo myelin phosphodiesterase (smpd ). defects in the gene encoding smpd cause niemann-pick disease type a and type b. smpd converts sphingomyelin to ceramide, and also has phospholipase c activity. reduced activity of acid sphingomyelinase, associated with a marked decrease in lysosomal stability, has been described in patients with niemann-pick disease, a phenotype that was corrected by treating cells with recombinant hsp . finally, as lamp a, a specific lysosomal protein that displays a decisive role in cma, has been shown to be overexpressed in certain pathological settings such as certain cancers and inflammatory diseases (autoimmune or non autoimmune), downregulating its expression www.nature.com/nrd might be therapeutically beneficial , . as mentioned above, however, in other indications there is a defect in lamp a. the latter can be due to reduced stabil ity of the cma receptor and not to decreased de novo synthesis (for example, in ageing) or can result from aggregation to the lysosomal membrane of pathogenic proteins such as α synuclein, ubiquitin carboxy terminal hydrolase l (a deubiquitinating enzyme) and mutant tau, known to amass in neurodegenerative disorders (see above). targeting lamp a therefore remains a challenge, although several strategies may be envisaged, for example by controlling de novo synthesis, by ham pering its multimerization into lysosomes (possibly via hsp and/or other chaperones) or by regulating the degradation rate of lamp a monomers (for reuse) into lysosomes. challenges and outlook current research into lysosomal function and dysfunc tion is revealing novel roles of lysosomes in disease pathogenesis and highlighting new opportunities to treat such lysosomal and autophagy related diseases. as in the case of autophagy modulation , , , lysosomal activation or inhibition must be investigated with cau tion, as lysosomal activity can be abnormally reduced or enhanced in some organs or tissues and not in others, and, at another scale, lysosome activity can be altered in certain lysosomes and not in others within the same cell. biodistribution studies in vivo must be undertaken to avoid accumulation of pharmaceuticals in healthy organs or tissues. there is an obvious requirement for safety, to ensure that a drug used as a lysosome modu lator for a particular type of lysosomal disease does not increase vulnerability to another disease. there is still much to be learned about the intimate working of lysosomes. this is due to the abundance of constitutive elements that comprise these vesicles, the added complexity resulting from their plasticity (ion channels and transporters, acidification and swell ing) and the vast amount of proteins and peptides that are translocated into lysosomes and digested by lytic enzymes. sensitive analysis methods have allowed important information to be generated about lyso somal membrane proteins, a large majority of which are transporters . however, many questions remain related to how their expression is regulated and how they reg ulate their translocator and chaperoning activities. for example, certain cells only contain so called secretory lysosomes (as in cytotoxic t cells), whereas other cell subsets contain both conventional and secretory lyso somes (as in platelets). considering the large family of endo lysosomal vesicles, the whole notion of 'secretory' and 'conventional' lysosomes remains a matter of debate. in many instances, lysosomes act as a basal cell metab olism organelle; whereas in other cases, they assist in the regulation of homeostasis through unconventional secretory pathways, known as lysosomal exocytosis, and different signalling mechanisms. although several assays used to measure the activ ity of lysosomes have been validated worldwide (box ; table ), they have their limitations, including issues associated with reliability, performance and sensitivity, notably in vivo. another level of complexity comes from the inherent organelle heterogeneity, which is an issue of tremendous importance. unfortunately, with the tools and equipment we have in hand today, it is virtually impossible to examine what happens in the lysosomes of an individual patient. the introduction of micro fluidic single cell analysis technologies has enabled cellular populations to be characterized and huge advances to be performed. however, the level of precision has not yet been achieved at the level of lysosomes ( . - . μm). we know that lysosomes are heterogeneous in nature, composition and activity even in 'normal' settings; they are not all equally competent for autophagy or any other types of activity. currently, this is obviously the focus of intense research. although a certain number of preclinical studies involving lysosomal regulators have been conducted over the years, only a small number of lysosome targeted therapeutics have so far moved into clinical develop ment. one of the biggest advances in developing such strategies would be the identification of a genetic sig nature that would allow those patients most likely to respond to a specific therapy to be selected. however, at this stage of our knowledge of specific lysosome directed drugs and intrinsic lysosomal failures, genetic features that might predict potential responders are still lacking (with the exception of lsds). further investi gations are required to achieve this level of knowledge, which obviously will also depend on the type of disease, heterogeneity and frequency. another issue associated with the development of lysosometargeted therapeutics relates to delivery. the use of nanovectors represents an attractive delivery method, owing, in particular, to their unique ability to penetrate across cell barriers and, via the endo lysosomal pathway, to preferentially home in on orga nelles such as lysosomes. several nanoscale galenic forms have been developed to serve as vectors or car riers of proteins, peptides or nucleic acids, and a vast literature describes the many advantages of using such nano structures in nanomedicine. however, safety is a concern as some carbon nanostructures have been claimed to induce nanotoxicity, accompanied by the induction of autophagy and lysosomal dysfunction [ ] [ ] [ ] [ ] (reviewed elsewhere [ ] [ ] [ ] [ ] . the purpose of this review is to gain awareness of the importance of lysosomes in disease, and to encour age the development of novel lysosomal targeted drugs. however, more research is needed to characterize com ponents that are specifically linked to the lysosome, such as lamp a and hspa , and to more clearly define their specific involvement in lysosome biogenesis and metabolism. special attention should be given to the mode of administration of lysosome targeted medica tions in order to minimize toxicity and promote specific targeting. it is our hope that a large field of therapeutic applications could emerge from such investigations, encompassing rare and common autoimmune, neuro degenerative and metabolic diseases, as well as cancer, senescence and ageing. nature reviews | drug discovery tissue fractionation studies. . intracellular distribution patterns of enzymes in rat-liver tissue lysosomal membrane 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adopts a novel tetrameric architecture niemann-pick type c disease reveals a link between lysosomal cholesterol and ptdins( , )p that regulates neuronal excitability functional upregulation of the m-current by retigabine contrasts hyperexcitability and excitotoxicity on rat hypoglossal motoneurons hsp stabilizes lysosomes and reverts niemann-pick disease-associated lysosomal pathology chaperone-mediated autophagy is required for tumor growth restoration of chaperonemediated autophagy in aging liver improves cellular maintenance and hepatic function role for nanomaterialautophagy interaction in neurodegenerative disease a functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through akt-tsc -mtor signaling tuning cell autophagy by diversifying carbon nanotube surface chemistry silica nanoparticles enhance autophagic activity, disturb endothelial cell homeostasis and impair angiogenesis autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticle-induced autophagy in biomedical research berberine as a potential autophagy modulator the authors apologize to all those whose work is not cited due to space limitations. they gratefully acknowledge hélène jeltsch-david for critically reading the manuscript. this research was funded by the french centre national de la recherche scientifique, région alsace, the laboratory of excellence medalis (anr- -labx- ), initiative of excellence (idex), strasbourg university, and immupharma france. s.m. is grateful to the university of strasbourg institute for advanced study (usias) for funding f.w., and acknowledges the support of the transautophagy cost action (ca ), the club francophone de l'autophagie (cfatg) and the european regional development fund of the european union in the framework of the interreg v upper rhine programme. all authors made substantial, direct and intellectual contribution to the work and approved it for publication. s.m. discloses the following conflicts of interest: research funding (paid to institution) and a past consultant for immupharma; co-inventor of cnrs-immupharma patents on p peptide; owns immupharma shares. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. s.r.b. and f.w. declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.www.nature.com/nrd key: cord- -jpzvjl i authors: schmidt, robert e. title: types of renal disease in avian species date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: jpzvjl i renal disease in birds is frequently encountered. like most other animals, birds are susceptible to a full spectrum of renal insults,such as toxins, tumors, infections, and degenerative conditions. accurate diagnosis of renal disease is based on a complete history,physical examination, and laboratory evaluation of the patient. because it is often required for a more definitive diagnosis, special attention is given to histopathologic evaluation of renal tissue,whether through a premortem biopsy or collection at gross necropsy. bacteria found in tubules and occasionally in the interstitium. as nephritis becomes chronic, tubular necrosis, cyst formation, distortion, and interstitial fibrosis with mononuclear cell infiltration become evident. initial hematogenous lesions may be present in glomeruli. organisms associated with necrosis and a pleocellular inflammatory infiltrate may be seen. necrosis of the tubular epithelium is sometimes prominent, but inflammation may be minimal or nonexistent in cases of bacterial nephritis. distal collecting tubules and cortical collecting ducts are primarily affected by bacterial infections. subacute ascending infections have a marked inflammatory response with heterophils in the lumen of the tubules. tubulointerstitial lesions are locally extensive. with severe locally extensive lesions, it may be difficult to determine whether the infection began in the tubules and involved the interstitium or vice versa. a wide range of gram-positive and gram-negative bacteria are known to cause kidney disease, either as an ascending infection or as part of a systemic disease. staphylococci and streptococci are common pathogens [ , ] . other bacteria that can affect the kidney include members of enterobacteriaceae, listeria sp, erysipelothrix rhusiophathiae, and pasteurella sp [ ] . mycobacterial and chlamydophila psittaci infections are generally systemic infections. they may cause lesions in the kidney [ ] but often do not. mycobacterial lesions are similar to those found in other tissues. renal lesions caused by c psittaci are characterized by interstitial inflammation composed primarily of histiocytes, plasma cells, and lymphocytes, with intracytoplasmic organisms seen in histiocytes in some cases. any bacterial organism associated with sepsis may be found in avian renal tissue. fungal infection of the kidney occurs either as an extension of a fungal infection of the abdominal air sacs [ , ] or as a component of fungemia where a fungus has invaded a vessel, resulting in fungal thrombosis of blood vessels. fungal hyphae in the lesion give it specificity. isospora sp and eimeria sp are found in the kidneys of nearly all species of wild ducks and geese [ ] [ ] [ ] [ ] [ ] , as well as in other avian species [ ] [ ] [ ] . organisms are predominantly found in the epithelium of perilobular collecting ducts and the medullary collecting tract. eimeria truncata may cause a more severe disease in juvenile waterfowl. organisms are found in tubular epithelial cells or free in the lumen associated with inflammatory cells and necrotic debris. cryptosporidial infection of the kidney of birds is reported [ ] [ ] [ ] [ ] . kidneys appear swollen and pale. slight proliferation of tubular epithelial cells may be seen, and organisms are present on their surface. encephalitozoon hellem is a potential cause of renal disease. lesions are most commonly seen in lovebirds and budgerigars but have been reported in other psittacine birds [ , ] . gross changes may be absent, or small pale foci may be present in the renal parenchyma. histologically, small protozoal organisms are present in cells and may be free in necrotic foci and the lumen of the renal tubules. systemic sarcosporidial infection may lead to interstitial nephritis with an infiltrate that is primarily lymphoplasmacytic [ ] . organisms are usually not seen. schizonts of leukocytozoon can also be found in the kidney [ ] . trematodes may be incidental findings or lead to clinical renal disease in some birds. these infections are most common in waterfowl. the flukes are found in collecting tubules in the medullary cone [ , ] . severe infections result in obstruction of the tubules, variable inflammation and necrosis, and secondary dilatation proximal to the obstruction. visceral larval migrans due to baylisascaris procyonis may lead to necrotic tracts and larvae within the kidneys [ ] . adenovirus infection of the kidney is seen in a variety of birds [ , ] . grossly there may be some nonspecific renal enlargement. microscopic lesions are usually minimal, ranging from mild interstitial mononuclear cell infiltration to tubular epithelial cell vacuolation and necrosis. scattered tubular epithelial cells have karyomegalic nuclei containing large, darkly eosinophilic or basophilic inclusion bodies. polyomavirus infection may be acute and cause the kidneys to be slightly swollen. virus may be recovered from the kidney [ ] . histologically, renal tubular epithelial cells may have karyomegaly, with affected nuclei containing clear or amophophilic, slightly granular inclusion bodies. these may be differentiated from those of adenovirus by their tinctorial properties. both primary and secondary lesions may occur in nonbudgerigar psittacine birds with avian polyomavirus disease [ , ] . intranuclear inclusion bodies and accompanying karyomegaly are commonly seen in mesangial cells. glomeruli will appear swollen. as many as % of these birds will develop a secondary glomerulopathy. this lesion is caused by the deposition of dense aggregates of immune complexes. finches with polyomavirus infection may have both renal tubular epithelial and mesangial karyomegaly with intranuclear inclusion bodies. nonsuppurative inflammation may be present in the renal interstitium in other viral infections, including reovirus [ ] and paramyxovirus [ , ] . paramyxovirus- in pigeons may cause an interstitial lymphoplasmacytic nephritis and tubular necrosis, with granular and hyaline casts present in the tubules. west nile virus causes a variable lymphocytic interstitial nephritis as part of generalized disease [ ] . other viruses that may cause renal disease include coronavirus, togavirus, and influenza a virus [ ] . except as a sequela to polyomavirus infection (discussed in the previous section), immune complex glomerulopathies are infrequently documented in birds, but autoimmune glomerulonephritis has been produced [ ] [ ] [ ] . in chronic cases, there may be proliferation of parietal epithelium and glomerular crescent formation. eventually glomerular shrinkage, fibrous connective tissue proliferation, and sclerosis occur. avian renal disease has a wide variety of noninfectious causes. many of these result in chronic disease with a common gross and histologic appearance. dehydration results in reduced urine flow and sludging of the urate crystals within the tubules. gross lesions are characterized by multifocal white to yellow-white foci or streaks that represent urate deposits. the gross appearance is similar to that of mineralization. microscopically, urates are dissolved during the fixation process but leave behind needle-shaped and amorphous spaces surrounded by an eosinophilic protein matrix [ , ] . disorders of protein metabolism may lead to elevation of uric acid; however, whether this in turn may result in urate deposition has not been established [ ] [ ] [ ] . other nutritional problems may result in renal disease. metastatic mineralization of the kidney is a common lesion in nestling parrots and to a lesser extent in adult birds. a nutritional imbalance is suspected. renal lipidosis may be secondary to a high-fat diet or chronic hepatic disease. on a gross level the kidneys are pale, and microscopically there is fat in tubular epithelial cells. lipid-containing macrophages are usually present in glomerular capillaries. vitamin a deficiency leads to squamous metaplasia of the epithelium of the ureters and collecting ducts, which in advanced cases results in the transformation of the ureteral epithelium to a keratinized epithelium [ , [ ] [ ] [ ] [ ] [ ] . high-cholesterol diets have also been associated with diffuse renal disease, including proliferative glomerulopathy, periglomerular fibrosis, multifocal interstitial nephritis, and lipid-laden cells within the glomeruli of pigeons [ ] . renal amyloidosis is most frequently observed in waterfowl and small passerine birds. multiple organs in addition to the kidney are generally involved. grossly the kidneys may be enlarged, pale, and somewhat friable. histologically, the amyloid is eosinophilic or amphophilic and may be deposited in glomerular or tubular basement membranes and the walls of renal arteries and arterioles [ , , ] . iron storage disease primarily affects the liver, but iron pigment is also seen in renal tubular cells in many affected birds. the iron does not cause an inflammatory or degenerative response [ , ] . renal disease and lesion formation may be secondary to a wide variety of conditions [ ] [ ] [ ] [ ] . ischemic (hemoglobinuric, myoglobinuric) nephrosis is secondary to a number of problems. hemoglobinuric nephrosis is infrequent because of the rarity of hemolytic disease in birds. myoglobinuric nephrosis may be a sequela to exertional rhabdomyolysis or severe crushing injury to muscle [ ] . in both cases, the kidneys may be dark brown. tubular degeneration and lumenal accumulation of amorphous eosinophilic material resembling myoglobin are seen microscopically in proximal convoluted tubules, and eosinophilic casts are noted in collecting tubules. history is important in making a diagnosis of toxic nephropathy, because most renal toxins cause similar gross and histologic lesions; therefore, a definitive causative diagnosis is often not possible based on gross and histopathologic changes alone. sometimes the tentative diagnosis is made by excluding all other possibilities [ ] . vitamin d -and vitamin d analogue-based rodenticides are toxic in birds. these rodenticides cause increased intestinal absorption of calcium and hypercalcemia. decreased urinary calcium excretion may also occur. calcium is deposited in soft tissues, including the kidney [ ] . gentamicin sulfate and amikacin are two aminoglycoside antibiotics that are commonly used in birds. gentamicin sulfate is more nephrotoxic than amikacin. aminoglycoside toxicity results in kidney enlargement and changes resembling those seen with other causes of renal failure [ ] . lead and zinc toxicity both may cause acute tubular necrosis [ ] [ ] [ ] [ ] [ ] . gross changes vary from none to swollen, pale kidneys. cadmium, mercury, and arsenic are also nephrotoxic [ ] . several mycotoxins, including oosporein, citrinin, and ochratoxin, have been shown to cause disease in poultry or domestic waterfowl [ ] [ ] [ ] . aflatoxins may also be a problem; they can cause degeneration of the proximal convoluted tubules and thickening of glomerular basement membranes [ , ] . excessive salt ingestion leads to renal problems that result in urate deposition and gross and histologic lesions [ , ] . a variety of other nephrotoxins have been reported to affect birds [ , , , ] . acute renal hypoxia/ischemia is usually related to a localized or generalized vascular problem. the results are tubular necrosis, protein leakage, and urate deposition. lesions are similar to the various problems discussed under metabolic disorders, and differential diagnoses include many of these conditions. renal hemorrhage may be secondary to trauma, ischemia, or a variety of primary disease conditions. the hemorrhage may be visible grossly and may affect both interstitium and tubules. the end result of many of these conditions is chronic or end-stage renal disease with severe fibrosis [ ] . renal tumors are reported in many species of birds but are particularly common in the budgerigar [ ] [ ] [ ] . renal carcinoma is the most common tumor of the kidney; however, adenoma, nephroblastoma, cystadenoma, fibrosarcoma, lymphosarcoma, and other neoplasms are also reported in the avian kidney [ ] . the most common presenting sign of renal neoplasia is unilateral or bilateral lameness or paralysis [ ] . these symptoms result from compression of the lumbar and sacral nerve plexi as they pass through or dorsal to the kidney, respectively, or from tumor growth into and adjacent to the synsacrum. skeletal muscle atrophy and osteopenia may also be seen. metastasis is occasionally reported [ ] . embryonal nephromas (nephroblastomas) are most commonly reported in chickens but are also found in psittacine and small passerine birds. they are usually unilateral but may be bilateral and are grossly similar to carcinomas [ ] . lymphosarcoma may be isolated to the kidney but is usually a part of generalized neoplastic disease. grossly, the kidneys are pale, mottled, and moderately firm with nodular or diffuse cell infiltration. myeloproliferative disease and histiocytosis are also reported in the avian kidney [ ] [ ] [ ] . other primary sarcomas are possible but are rarely reported, and metastatic sarcomas 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enteritis first observed in china a novel polyomavirus (goose hemorrhagic polyomavirus) is the agent of hemorrhagic nephritis enteritis of geese membranous glomerulopathy as an indicator of avian polyomavirus infection in psittaciformes characterization of the avian polyomavirus-associated glomerulopathy of nestling parrots a comparative study of avian reovirus pathogenicity: virus spread and replication and induction of lesions avian paramyxovirus type infections in racing pigeons in california. i. clinical signs, pathology, and serology paramyxovirus- infection (newcastle disease) of pigeons west nile virus infection in birds and mammals avian viruses: function and control. lake worth (fl): wingers publishing ultrastructural studies of experimental autoimmune glomerulonephritis in normal and bursectomized chickens experimental autoimmune glomerulonephritis in chickens new avian model of experimental glomerulonephritis consistent with mediation by cellular immunity. nonhumorally 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captive gentoo penguins (pygoscelis papua papua) an epizootic of lead poisoning in greater flamingos (phoenicopterus ruber roseus) in spain zinc toxicosis as a cause of sudden death in orangebellied parrots (neophema chrysogaster) toxicity of aspergillus ochraceus contaminated wheat and different chemical forms of ochratoxin a in broiler chicks avian gout caused by oosporein, a mycotoxin produced by chaetomium trilaterale oosporein toxicosis in the turkey poult ultrastructure of hepatic and renal lesions in chickens fed aflatoxin suspected sodium toxicity in hand-reared great blue heron (ardea herodia) chicks salt toxicosis in commercial turkeys the ultrastructure of nephrocalcinosis induced in chicks by cestrum diurnum leaves acute , -dichlorophenoxyacetic acid intoxication in broiler chicks use of carboplatin in the treatment of renal adenocarcinoma in a budgerigar neoplasms in budgerigars (melopsittacus undulatus): clinical, pathomorphological and serological findings with special consideration of kidney tumours adenocarcinoma of the kidney in two budgerigars right leg muscle atrophy and osteopenia caused by renal adenocarcinoma in a cockatiel (melopsittacus undulatus) metastatic renal carcinoma in an african grey parrot (psittacus erithacus erithacus) lymphosarcoma in a juvenile blue and gold macaw (ara araruana) and a mature canary (seriunus canaries) multicentric histiocytosis in young chickens. gross and light microscopic pathology morphologic diagnosis of avian neoplasms key: cord- - g lio l authors: keesing, felicia; belden, lisa k.; daszak, peter; dobson, andrew; harvell, c. drew; holt, robert d.; hudson, peter; jolles, anna; jones, kate e.; mitchell, charles e.; myers, samuel s.; bogich, tiffany; ostfeld, richard s. title: impacts of biodiversity on the emergence and transmission of infectious diseases date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: g lio l current unprecedented declines in biodiversity reduce the ability of ecological communities to provide many fundamental ecosystem services. here we evaluate evidence that reduced biodiversity affects the transmission of infectious diseases of humans, other animals and plants. in principle, loss of biodiversity could either increase or decrease disease transmission. however, mounting evidence indicates that biodiversity loss frequently increases disease transmission. in contrast, areas of naturally high biodiversity may serve as a source pool for new pathogens. overall, despite many remaining questions, current evidence indicates that preserving intact ecosystems and their endemic biodiversity should generally reduce the prevalence of infectious diseases. supplementary information: the online version of this article (doi: . /nature ) contains supplementary material, which is available to authorized users. i n june , a new organization, the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes)patterned after the intergovernmental panel on climate change (ipcc)-was established to assess changes to the diversity of life on the earth and how these changes will affect human well-being . human well-being would be adversely affected by biodiversity losses if ecosystems with reduced biodiversity are less able to provide the ecosystem services-such as carbon sequestration, nutrient cycling and resistance to drought-on which humans rely. in recent years, a consensus has emerged that ecosystem functions decline as biodiversity is lost . here we examine how biodiversity affects the transmission and emergence of infectious diseases and evaluate the evidence that reduced disease transmission is an important ecosystem service provided by high biodiversity. biodiversity encompasses the diversity of genes, species and ecosystems. increases in human populations have resulted in an unprecedented and precipitous loss of biodiversity . current extinction rates are estimated to be at least - , times background extinction rates and future extinction rates (over the next years) are estimated to be to times present extinction rates . a large proportion of species in all assessed taxa are currently threatened with extinction ( % of birds, % of mammals, % of amphibians; % of gymnosperms; % of corals ) and the best estimate of population trends of birds, mammals, amphibians, reptiles and fish indicates that since global population sizes have declined by almost % (ref. ) . global and local extinction rates of some taxa, particularly microbes, have not been well characterized. for the many organisms that are symbionts of other organisms, extinction of their hosts can cause their extinction too . collectively, these declines and extinctions are caused by changing the earth's ecosystems to meet growing demands for food, fresh water, fibre, timber and fuel, and by climate change. changes in biodiversity have the potential to affect the risk of infectious disease exposure in plants and animals-including humansbecause infectious diseases by definition involve interactions among species. at a minimum, these species include a host and a pathogen; often many more species are involved, including additional hosts, vectors and other organisms with which these species interact. intriguingly, biodiversity may play a dual role in the emergence and transmission of infectious diseases. on the one hand, high biodiversity may provide a larger potential source of novel pathogens, but on the other hand, biodiversity can reduce further pathogen transmission for both longestablished and newly emerging diseases. we first review the effects of biodiversity on the transmission of established diseases and then turn to disease emergence. transmission of pathogens between species biodiversity loss might affect disease transmission through several mechanisms (box ). if the effect of each species on pathogen transmission were entirely idiosyncratic, one would expect that diversity declines would be equally likely to cause a decrease or an increase in disease transmission in the remaining species. however, in recent years, a consistent picture has emerged-biodiversity loss tends to increase pathogen transmission and disease incidence. this pattern occurs across ecological systems that vary in type of pathogen, host, ecosystem and transmission mode ( table ) . as an example, west nile virus is a mosquito-transmitted virus for which several species of passerine birds act as hosts. three recent studies detected strong correlations between low bird diversity and increased human risk or incidence of west nile encephalitis in the united states - . communities with low avian diversity tend to be dominated by species that amplify the virus, inducing high infection prevalence in mosquitoes and people, while communities with high avian diversity contain many species that are less competent hosts. for hantavirus pulmonary syndrome, a directly transmitted zoonotic disease, correlational and experimental studies have shown that a lower diversity of small mammals increases the prevalence of hantaviruses in their hosts, thereby increasing risk to humans (box ). diversity has a similar effect for plant diseases, with species losses increasing the transmission of two fungal rust pathogens that infect perennial rye grass and other plant species . recent attention has focused on assessing the mechanisms by which reduced biodiversity increases pathogen transmission (box ). biodiversity loss can clearly increase transmission if it reduces predation and competition on reservoir hosts, thereby increasing their density. however, controversy has centred around whether the loss of species can increase transmission in other ways . this is because field studies like those on west nile virus, hantaviruses and rye grass have typically not controlled for changes in host density that can result from changes in 'species richness' (the number of species present in a community, which is a measure of taxonomic diversity). as a consequence, it has been difficult to separate the effects of higher density from those of reduced diversity. recent experiments confirm that increases in disease transmission can occur when species richness declines even if host density stays constant. one of the best examples comes from a study of schistosoma mansoni, a trematode that causes schistosomiasis in humans. the parasite alternately infects snails and humans via free-living infectious stages. host snails were placed in tanks at a constant density either alone or with one or two other species of non-host snails and then exposed to the parasite . in single-species treatments, host snails were % more likely to be infected because parasites in multi-species treatments often ended up in dead-end hosts. increased parasite-host encounter rates caused by reduced diversity are sufficient to increase disease transmission for schistosoma. the loss of species can increase encounter rates between pathogens and hosts, as in the schistosoma example, when the lost species are not hosts for the pathogen. but if the lost species are indeed hosts capable of transmission, this declining diversity could also reduce the total number of hosts, thereby decreasing transmission if all else remains equal , . certainly reductions in the number of hosts can reduce the number of vectors and also their infection prevalence , , but empirical examples are relatively rare, in part because the issue has been neglected, and also because all else rarely remains equal. for example, the loss of hosts can cause compensatory increases in the abundances of other hosts, such that total host abundance changes little relative to total host abundance in more diverse communities. even when total host abundance does decline in less diverse systems, differences in host quality among species can alter simple correlations between host abundance and infection risk . pathogen transmission is not always a function of host density. for example, the number of infectious bites delivered by highly mobile vectors like mosquitoes can be independent of the density of the host population . transmission of directly transmitted pathogens like hantaviruses can also be independent of host density if transmission involves behavioural encounters, for example, aggressive interactions between rodents, and if the frequency of these encounters does not vary much with host density , . in systems like these, the loss of host species can actually increase transmission if the lost hosts are suboptimal for parasite development and reproduction; this is because these suboptimal hosts absorb pathogens but are poor at transmitting them. in sum, reducing biodiversity can increase disease transmission when the lost species are either not hosts for the pathogen or are suboptimal ones. for pathogens for which transmission is a function of host density, loss of diversity is most likely to increase transmission if the loss causes an increase in the density of competent hosts. the number and diversity of examples of pathogens for which species loss leads to increases in total transmission suggests that these conditions are frequently met (table ) . additional studies in other disease systems would better establish the generality of these relationships. the loss of particular species in a community clearly has the potential to increase disease transmission. but does reducing diversity itself increase transmission, or is increased transmission the consequence of the removal of particular species? the answer depends on how species composition changes as richness changes , . for example, if those host species most responsible for amplifying the pathogen tend to persist or even thrive as biodiversity is lost, then disease risk will consistently increase as biodiversity declines. on the other hand, if amplifying species tend to disappear as biodiversity declines, then biodiversity loss will tend to reduce disease risk. these hypothetical possibilities indicate the importance of understanding both the non-random sequences by which species are lost from communities, and whether the species that tend to occur only in more species-rich communities tend to amplify or buffer pathogen transmission. in several case studies, the species most likely to be lost from ecological communities as diversity declines are those most likely to reduce pathogen transmission. in the lyme disease system of eastern north america, for example, the white-footed mouse is simultaneously the most abundant host species, the most competent host for the lyme bacterium, and the highest-quality host for immature tick vectors the loss of biodiversity can affect the transmission of infectious diseases by changing: ( ) the abundance of the host or vector. for plants, seeding experimental fields with plant species that are not hosts for fungal pathogens decreased threefold the pathogen load of species that are hosts, apparently by reducing host density through competition . on the other hand, a greater diversity of host species can sometimes increase pathogen transmission by increasing the abundance of vectors . ( ) the behaviour of the host, vector or parasite. in a more diverse community, one of the parasitic worms that causes schistosomiasis (which infects million people worldwide) is more likely to end up in an unsuitable intermediate host. this can reduce the probability of subsequent infection of humans by - % (ref. ) . for hantavirus in utah, usa, rodent hosts on more diverse plots are more likely to come in contact with heterospecific mammals and less likely to come in contact with conspecifics, reducing the probability of transmission of the virus . in principle, higher diversity could influence behaviours with a resulting increase in disease transmission or could alter the evolutionary dynamics of virulence and transmission pathways. ( ) the condition of the host or vector. in experimental rice fields in china, rice plants in genetically diverse mixtures had drier leaves because the mixture changed microclimatic conditions . as a consequence, infection with rice blast fungus was less prevalent in diverse fields. genetically diverse plantings can also lead to induced resistance in host plants because they are exposed to similar pathogens that are specialists on the other cultivars . for some disease systems (for example, lyme disease), multiple mechanisms operate in concert, leading to a compounding effect of biodiversity loss on increased disease transmission (table ) . . ticks that attempt to feed on virginia opossums are likely to be groomed off and killed. green-andyellow circles show the mean number of ticks per hectare fed by mice or opossums; yellow shading shows the proportion of ticks infected after feeding. blue circles show the mean number of ticks per hectare groomed off and killed. ticks that feed on mice are highly likely to become infected with the bacterium that causes lyme disease, whereas those that feed on opossums are not. case study of hantavirus pulmonary syndrome hantaviruses are a group of negative-stranded rna viruses associated with murid rodents. they can cause severe morbidity and mortality in humans, with case-fatality rates near % (ref. ) . infected rodents shed hantavirus in saliva, urine and faeces; transmission to humans occurs through inhalation of aerosolized excreta as well as through rodent bites . the risk of human exposure increases as the density and infection prevalence of rodent reservoirs increase . in a field study in oregon, usa, the only variable significantly linked to infection prevalence in deer mouse host populations was mammalian species diversity, with the prevalence of the hantavirus sin nombre virus rising from % to % as diversity declined. deer mouse population density was not statistically associated with sin nombre virus infection prevalence, suggesting that high diversity reduced intraspecific encounters rather than host abundance . a study in utah, usa , also found a negative correlation between small-mammal diversity and sin nombre virus infection prevalence in deer mice. as in oregon, high diversity reduced infection prevalence apparently by reducing intraspecific encounters rather than by reducing host density, a result supported by experiments . the conclusions of these studies were supported by an experimental study of hantaviruses in small mammal communities of panamá . in replicated plots, small-mammal diversity was reduced by trapping and removing species that are not hosts for the virus; infection prevalence in hosts was compared on manipulated and unmanipulated plots (box figure) . experimentally reduced small-mammal diversity caused an increase in the density of host species and also in seroconversion rates and seroprevalence within hosts (box figure) . review research (fig. ) . as a consequence, this host species infects a high proportion of the ticks within forest communities. the white-footed mouse is also an ecologically resilient species, present in both species-rich and speciespoor communities . in contrast, virginia opossums are poor hosts for the pathogen, kill the vast majority of ticks that attempt to feed on them, and are absent from many low-diversity forest fragments and degraded forests where mice are abundant , . therefore, as biodiversity is lost, the host with a strong buffering effect-the opossum-disappears, while the host with a strong amplifying effect-the mouse-remains. the primary hosts for the pathogens that cause west nile encephalitis, hantavirus pulmonary syndrome, and bartonellosis also appear to be resilient species that increase in abundance as biodiversity is lost , , . whether an organism's host competence and its resilience to factors that reduce biodiversity are causally related is an unresolved but critical issue. traits that make a host resilient to biodiversity loss may also make them susceptible to pathogen infection and transmission. such a relationship would explain the frequency with which the link between diversity loss and disease transmission has been observed in nature ( table ). for plants, species that are fast-growing and nutrient-rich with relatively high metabolic rates-characteristics of 'weedy' speciescan be more competent hosts for arthropod vectors and plant pathogens than those with less weedy traits . plants with these weedy traits are also more likely to become more abundant when plant diversity declines . consequently, the very species that have traits permitting persistence in degraded and species-poor ecosystems are also more likely to carry high pathogen and vector burdens. a similar pattern may occur in vertebrates-resilience in the face of disturbances that cause biodiversity loss, such as habitat destruction and fragmentation, is facilitated by lifehistory features such as high reproductive output and intrinsic rates of increase . vertebrates with these features tend to invest minimally in some aspects of adaptive immunity [ ] [ ] [ ] ; we hypothesize that this may make them more competent hosts for pathogens and vectors. understanding the interrelationships among pathogen transmission, biodiversity loss and interspecific differences in immune function is an important area for future research. such studies would illuminate how frequently resilient species are also those that increase pathogen transmission, and might provide general rules about the impact of biodiversity loss on disease transmission. could changes in biodiversity within the bodies of organisms also alter pathogen transmission? recent improvements in the ability of researchers to detect unculturable microbial species have allowed documentation of the tremendous diversity of microbes upon and within plants and animals. in human bodies, for example, % of all cells are microbial . a number of studies have begun to show links between diseases and the diversity of an organism's 'microbiome'. changes in the composition of microbiomes are frequently associated with infection and disease. for example, corals suffering from white plague disease have microbial communities distinctly different from those in healthy corals . in humans, bacterial vaginosis results from changes in the composition of the vaginal microbial community , and this in turn increases the risk of hiv infection . although changes in microbial species composition associated with infection are welldocumented, few studies have investigated the effects of changes in diversity itself. in a recent investigation, patients with recurrent episodes of infection caused by the bacterium clostridium difficile had significantly lower diversity of intestinal microbes than did control patients . correlational studies such as these, though intriguing, make it difficult to determine whether changes in microbial communities are the cause or the consequence of infections. but some experimental studies clearly demonstrate that increasing microbial biodiversity can protect against infection. for example, children with a history of ear infections given a mixture of five strains of streptococcus were less likely to develop subsequent infections compared to a control group . similarly, reducing microbial diversity within a host can increase transmission. when mice with persistent infections of c. difficile were treated with antibiotics that reduced the diversity of intestinal microbes, they began shedding c. difficile spores at high rates . in some of these examples, a rich microbial community appears to regulate the abundance of endemic microbial species that can become pathogenic when overly abundant . in other cases, high microbial species diversity can prevent colonization by invasive pathogenic species. for example, the more diverse the microbiome surrounding the roots of wheat plants, the more protected the plants were against invasion by the pathogenic bacterium pseudomonas aeruginosa . similarly, piglets raised in natural environments supporting a high diversity of microbes were more resistant to invasion by pathogenic gut microbes than those raised in more sterile environments . the effects of microbial diversity within and upon host bodies show intriguing similarities to the effects of macroscopic species diversity on disease transmission in aquatic and terrestrial ecosystems. further exploration of these similarities, and particularly the specific mechanisms operating within hosts, is a critical research frontier because changes in microbial diversity might accompany biodiversity loss in their hosts. for pathogens already established within ecological communities, we have shown that biodiversity loss frequently increases the rate of transmission. but what role, if any, does biodiversity have in the processes by which new pathogens emerge? between and , over emerging disease events were identified in humans around the world . concomitantly, other emerging infectious diseases also appeared in wildlife, domesticated animals, and crop and wild plants. emerging infectious diseases include those in which the pathogen has evolved into a new strain within the same host species, for example, through the evolution of drug resistance (methicillin-resistant staphylococcus aureus or mrsa) or switched to new host species (for example, human immunodeficiency virus or hiv, severe acute respiratory syndrome or sars). in some cases, the switch to new host species is accompanied by a change in geographic range (for example, west nile virus in the americas). for pathogens that establish in new species, the emergence process involves multiple steps, including the initial invasion into the new host ('spillover'), the production of transmission stages within the new host, and the establishment of the pathogen in the host population as a whole , . the effect of biodiversity may vary for each of these steps. for the initial invasion, biodiversity may act as a source pool. this hypothesis is supported by surveys of emerging diseases of humans: most are zoonotic-jumping to humans from other vertebrate animals . in one recent analysis, the probability of emergence of pathogens from wildlife to humans was positively correlated with mammalian wildlife species richness when data were corrected for reporting bias . other environmental and socioeconomic factors that bring humans into closer contact with potentially new pathogens (for example, forest clearing for agriculture, wildlife hunting) may also contribute to this pattern. indeed, almost half of the zoonotic diseases that have emerged in humans since resulted from changes in land use, from changes in agricultural or other food production practices, or from wildlife hunting (fig. ) . these human activities increase rates of contact between humans and animals, which may be a critical factor underlying spillover. once spillover of the pathogen into a new host has occurred, high densities of that host species may facilitate pathogen establishment and transmission within the new host . for example, nipah virus spilled over from wild fruit bats to domestic pigs in malaysia; high densities of pigs in local farms appear to have facilitated establishment of pig-to-pig transmission, and the pathogen then spilled over from pigs to humans . such high densities of domesticated species are almost always associated with low biodiversity. in contrast to emergence through host-switching, % of emergence events between and arose through the evolution of drug resistance . for these cases, biodiversity of microbial communities within hosts may have a protective effect; human use of antibiotics is research review thought to select for resistant microbes by eliminating the great diversity of non-resistant microbial strains and species that suppress resistant strains in the absence of antibiotics. investigations using recent advances in microbial detection support this idea , . thus, reduced microbial diversity may be an important underlying cause of the emergence of drug-resistant pathogens; this too requires further investigation. the addition of particular species-for example, natural enemies or competitors-can reduce the impacts of established pathogens. for example, experimental addition of a naturally occurring bacterium, janthinobacterium lividum, to the skin of the endangered frog rana mucosa eliminated frog mortality from experimental infection with chytridiomycosis, which is devastating amphibian populations worldwide . for corals, application of phages isolated from natural communities can control the spread of bacterial infections . the growing interest in 'probiotics' for humans and harvested species provides another example of this approach . more broadly, biodiversity itself seems to protect organisms, including humans, from transmission of infectious diseases in many cases (table ) . preserving biodiversity in these cases, and perhaps generally, may reduce the incidence of established pathogens. to preserve high diversity in nature, conservation scientists have developed robust methods that reflect the key principle that larger areas sustain larger numbers of species . methods of conserving microbial diversity within and upon bodies or in the environment are less well developed, but avoiding the overuse of antimicrobial compounds is essential. critically, future research on the relationship between biodiversity and disease must avoid conflating the effects of biogeographic patterns of biodiversity (for example, higher diversity in lower latitudes) with those of anthropogenic reductions in extant biodiversity, because policy and management options can far more readily affect the latter than the former. for emerging diseases, the observation that a more diverse microbiome within a host suppresses strains that are resistant to antimicrobial compounds suggests that avoiding the over-use of these compounds in medicine and agriculture can prevent the emergence of resistant strains. for pathogens that emerge by switching host species, three management approaches are warranted. first, potential emergence 'hotspots' could be predictable on the basis of land-use change and underlying biodiversity patterns; these areas should be targeted for surveillance of endemic wildlife pathogens that have the potential to jump host species , . second, preserving and protecting intact habitats in these hotspots provides a simple, direct way of reducing human-animal contact and reduces the likelihood of emergence of new pathogens, although methods for achieving reduced contact are not always straightforward . and third, to reduce the probability that pathogens become established and transmissible within a new host population once spillover occurs, the husbandry of high-density monocultures of domestic animals, particularly in areas at high risk of spillover, should be subject both to more intensive surveillance and to measures that reduce contact between wildlife and livestock. managing potential emergence hotspots by attempting to eliminate them is likely to backfire because the species most resilient to habitat destruction and degradation may be those that amplify pathogen transmission. despite many recent advances in our understanding of biodiversity and disease, much remains to be learned. first, we must increase the number of disease systems for which we understand the effects of biodiversity loss on disease transmission across a range of spatial and temporal scales. we must also focus on how to implement specific policies informed by this science. future research, for example, should monitor changes in epidemiology in regions in which conservation measures are imposed compared to reference sites. a major challenge will be to untangle the complex ways in which other global anthropogenic trends-such as climate change, biotic exchange, nutrient pollution, armed conflict and economic collapse-interact with biodiversity loss to influence disease dynamics, and which of these trends have the greatest impacts on human well-being. despite remaining questions, connections between biodiversity and disease are now sufficiently clear to increase the urgency of local, regional, and global efforts to preserve natural ecosystems and the biodiversity they contain. globally, almost half of these diseases resulted from changes in land use, changes in agricultural and other food production practices, or through wildlife hunting, which suggests that contact rates between humans and other animals are an important underlying cause of zoonotic disease emergence. 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ecological determinants of american crow mortality due to west nile virus during its north american sweep effects of species diversity on disease risk effects of grassland plant species diversity, abundance, and composition on foliar fungal disease zooprophylaxis or zoopotentiation: the outcome of introducing animals on vector transmission is highly dependent on the mosquito mortality while searching comparison of four species of snails as potential decoys to intercept schistosome miracidia panicle blast and canopy moisture in rice cultivar mixtures use of multiline cultivars and cultivar mixtures for disease management hantavirus pulmonary syndrome in five pediatric patients-four states the ecology and evolutionary history of an emergent disease: hantavirus pulmonary syndrome author information reprints and permissions information is available at www.nature.com/reprints. the authors declare no competing financial interests. readers are welcome to comment on the online version of this article at www.nature.com/nature. correspondence and requests for materials should be addressed to f.k. (keesing@bard.edu). supplementary information is linked to the online version of the paper at www.nature.com/nature. key: cord- -w bjtq authors: lennox, angela m. title: care of the geriatric rabbit date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: w bjtq the average life span reported in laboratory and lay literature for the domestic rabbit is to years. the author and other veterinarians are now regularly seeing rabbits living to or years, the oldest reported in the author's practice being years. rabbits are herbivorous prey species with continually growing (elodont) teeth. this feature allows the geriatric rabbit to possess teeth that are essentially “new”, a distinct advantage over geriatric carnivores. expanded longevity, while generally desirable, necessarily accompanies an increase in geriatric disorders. this article examines the spectrum of disease that can affect the geriatric rabbit as well as crucial factors concerning the clinical management of the animal up to the end of its life. an improved understanding of geriatric disorders in pet rabbits allows early recognition and the opportunity to improve quality of life. animals, for example, feeding of diets with excessive vitamin d and calcium. other causes include urolithiasis, bacterial infections, neoplasia, and nephrocalcinosis. encephalitozoon cuniculi (ecun) has been demonstrated to produce mild to severe interstitial nephritis. a recent study of histopathologic examinations of pet rabbits that died or were euthanized showed that of rabbits with ecun spores detected in the brain, also had interstitial nephritis; . % of renal lesions were described as chronic, whereas the rest were acute. however, the same study showed that the degree of severity of lesions did not always correlate with the degree of clinical symptoms. antemortem diagnosis of ecun as a cause of renal failure in rabbits is difficult, but can be supported with serology and protein electrophoresis. management of acute renal failure is similar to that for other species. management of chronic renal failure is supportive. some cases benefit from long-term administration of subcutaneous fluids and hand feeding. the gif tube implant kit is a silicone catheter designed for long-term implantation in the subcutis for administration of fluids (gif-tube, practivet, phoenix, az). though designed for dogs and cats, the author and others have used this product in rabbits with chronic renal failure requiring longer-term at-home subcutaneous administration of fluid (see hospice and end-oflife issues later in this article) (fig. ) . the manufacturer provides a demonstration cd of implantation instructions for practitioners and at-home use for owners. erythropoietin has been administered to rabbits with secondary anemia. the author has encountered numerous rabbits experiencing good quality of life for many months, despite muscle wasting and persistently elevated blood urea nitrogen and creatinine. cardiovascular disease is gaining recognition in exotic companion mammals as the level of veterinary care increases and patients age. the rabbit is a model for atherosclerosis in humans, as lesions are readily produced by feeding a diet high in fat. however, little is known about naturally occurring disease in the pet rabbit. the thoracic cavity and lungs of the rabbit are exceptionally small in comparison with those of other similar-sized animals. severe reduction in pulmonary mass (as in mediastinal tumors) does not seem to produce symptoms until late in the course of the disease. therefore, it is likely the rabbit may not exhibit cardiac-related respiratory symptoms until late in the course of the disease. symptoms can include depression, exercise intolerance, and increased respiratory rate and effort, which in the author's experience may be absent at rest and significantly worse with exercise. diagnosis of heart disease is enhanced with radiography and cardiac ultrasonography, which is important to better characterize heart disease and determine treatment options. normal parameters for echocardiography have been described. cardiac conditions reported in the rabbit include valvular disease (endocardiosis) and cardiomyopathy. several diseases have been reported to cause cardiomyopathy in the laboratory rabbit, but incidence in pet rabbits is unknown, and likely to be low. these include nutritional deficiencies (vitamin e deficiency), viral (coronavirus), bacterial (salmonellosis or pasteurellosis), or protozoal (ecun) infections, and toxins. , excessive stress may produce heart disease. stress causes catecholamine release in rabbits, and when sustained can result in coronary vessel constriction with ischemic cardiomyopathy. treatment of congestive heart failure relies on use of drugs traditionally used in canine medicine, and are based on anecdotal reports of success. drugs used by the author for management of cardiac disease are listed in table . drug dosages should be adapted to clinical response and improvement of echocardiographic results. the use of taurine has been shown to improve cardiovascular function in laboratory rabbits with induced cardiac failure (see table ). vascular disease can produce hypertension. indirect blood pressure is determined using an ultrasonic doppler and pediatric cuff (width %- % of the diameter of the limb circumference) usually placed proximal to the carpus. acquiring an audible pulse with the doppler requires significant practice; initial difficulty encountered should not discourage the practitioner from developing this skill. the author and others normally note blood pressures in normal patients between and mm hg using this method. osteoarthritis and vertebral spondylosis are commonly encountered in older rabbits. associated signs and symptoms include reluctance to move, urine/fecal staining due to inability to clean and properly direct the urine stream, and lameness. the rabbit is a laboratory model for trauma-induced arthritis and response to drug therapy. in rabbits whose legs were immobilized with plaster casts, those receiving intra-articular injections of . ml hyaluronic acid showed significant reduction of cartilage degeneration when compared with rabbits receiving a saline injection. similar effects (retardation of progression of osteoarthritis) were seen in rabbits undergoing anterior cruciate ligament transection that were injected with an intra-articular mixture of glucose or dextrose, amino acids, and ascorbic acid times weekly. other drugs shown to reduce degenerative changes in rabbit osteoarthritis models include intra-articular administered sodium hyaluronate and orally administered glucosamine hydrochloride, mg by mouth daily. [ ] [ ] [ ] several studies exist on the use of joint health products in dogs, and include glucosamine, chondroitin, p fp (indian and javanese tumeric extract), green-lipped mussels, and u- fatty acids. results are variable; however, a systematic review of the literature showed that there is moderate evidence that some joint health products (jhps), including green-lipped mussels products, p fp, a combination of chondroitin sulfate, glucosamine hydrochloride, and manganese ascorbate, provide some benefit. the author is unaware of studies on the effects of jhps in rabbits with naturally occurring disease. product recommendation can be difficult, and dosing is extrapolated from other species. the acclaim system has been proposed as a means for veterinarians to evaluate the label claims and quality of specific jhps ( table ) . housing should be optimized for rabbits with spondylosis or joint disease. nonslip, soft surfaces are beneficial, as the force produced by hopping is much higher than that produced by walking. lowering one side of the litter box allows easier access. owners must be instructed to clean the perineum daily (see later discussion on perineal dermatitis). dental disease is not necessarily associated with aging in rabbits. many older rabbits manage to avoid acquired dental disease and possess essentially normal, continually renewing teeth well into old age. other older rabbits may develop varying patterns of dental disease apparently related to slowing or cessation of tooth growth, which may be due to attrition of the alveolus. severe acquired dental disease, however, is primarily a disease of younger rabbits; in these patients, evidence of dental disease is often apparent before years of age. accurate diagnosis and excellent owner compliance can result in adequate disease management and acquisition of normal expected life span; in many cases these patients eventually die of diseases unrelated to those of dentition. the author and others frequently see geriatric rabbits that have had years of regular dental care. in some cases all teeth are eventually lost, and patients survive with good to excellent quality of life on a diet of liquid critical care (oxbow animal health, murdoch, ne). diagnosis and treatment of dental disease is presented in great detail elsewhere. several practitioners have reported unilateral or bilateral abduction of the thoracic limbs in older, often larger breed rabbits (fig. ) . the condition seems to be associated with muscle wasting and is generally progressive. housing on nonslick surfaces is helpful. though not a primary disease disorder, muscle wasting and moderate to severe weight loss is a common feature in aged rabbits. causes are varied, and can include chronic renal failure and acquired dental disease. a thorough approach is required to discover the underlying etiology. supplemental feeding is often beneficial. critical care can be offered in a dish or via syringe feeding. the manufacturer provides detailed instructions for product use. fig. . older rabbit with a history of severe acquired dental disease, retrobulbar abscess, and enucleation of the right eye. the rabbit survived with good quality of life for several years on oxbow critical care and soaked pellets. worsening splay leg of the thoracic limbs resulted in decreased mobility. the owner used towels and a stuffed animal to support the pet during brief hospice care at home before ultimately choosing euthanasia. age-related ocular lesions, including cataract formation, are common in rabbits. current literature suggests a high percentage of ocular lesions may be caused by ecun. a study describing histologic features of ecun-induced ocular lesions in rabbits demonstrated intraocular locally extensive pyogranulomatous infiltration of the posterior chamber with disruption of the anterior lens capsule. spores were identified via immunohistochemical staining in all cases. another study described ocular lesions as phacoclastic uveitis. all samples from abnormal eyes (n ) obtained via enucleation or phacoemulsification were positive for ecun via the polymerase chain reaction. stenosis of the ear canal is a common finding in aged lop-eared rabbits. in the author's experience, all older lop-eared rabbits have some degree of stenosis, which predisposes to otitis. disease is difficult to manage medically due to abnormal anatomy. lateral ear canal resection, ostectomy, and total ear canal ablation may be required in selected cases. pododermatitis can be encountered in rabbits of any age, and is often a result of improper husbandry (wire-bottom cages, inadequate cleaning); however, any condition impairing ability to groom normally and exposure to urine/feces due to decreased mobility can contribute. inability to groom also leads to perineal accumulation of feces and urine and dermatitis, or scalding. it is important to address the underlying cause of decreased mobility. pododermatitis is treated with local wound management (debridement, flushing with or without bandaging), housing on soft surfaces, and antibiotics plus analgesics. underlying immunocompromise and wasting can delay or prevent healing in some cases. severe cases with pus and involvement of tendons, ligaments, and joints may require surgical intervention, including amputation, which should be considered carefully in the geriatric rabbit. perineal dermatitis is treated the same as any other infected wound, with careful clipping of the hair, cleansing and application of antibiotics, and use of soothing ointments or products designed to promote healing once infection has resolved and granulation has begun. some elderly rabbits benefit from regular preventative shaving of the perineum in order to allow the owner to cleanse more effectively. the author prefers dilute chlorhexidine and application of silver sulfadiazine cream (ssd, par pharmaceutical, shreveport, la), zinc products (zn derm, addison biological laboratory inc, fayette, mo), or healing products (heal-x soother plus cream or spray, zoological education network, lake worth, fl). neoplasia occurs in rabbits, and incidence of all neoplasms increases with age. the most commonly encountered neoplasm is uterine adenocarcinoma. uterine adenocarcinoma can produce few signs until the disease is advanced. the most commonly reported sign is hematuria, and uterine masses are often palpable. the abnormal uterus may be palpable, and metastasis, especially to liver and lungs, can occur in some cases. the most common clinical presentation of thymoma is bilateral exophthalmos, which may worsen with exercise or excitation. respiratory symptoms usually occur later in the course of the disease. treatment options include surgery and radiation. in human medicine, drug dosages are often adjusted for geriatric patients due to assumed decrease in renal and hepatic function. although specific recommendations for geriatric rabbits are unavailable, drug dosage modification should be considered, especially when organ disease has been positively identified. chronic renal failure impacts drug metabolism; therefore, adjustments in drug dosages should be considered in patients with chronic renal failure. in humans, various strategies for estimation of glomerular filtration rate (gfr) are used for ''adjusting the dosage of medications excreted by glomerular filtration.'' for example, in human patients with impaired clearance, dosage of butorphanol is adjusted to half the normal dose. although estimation of gfr is unavailable for rabbit patients, serious consideration should be given to lowering dosages of all drugs, in particular, any drugs metabolized by glomerular filtration in rabbits with suspected renal insufficiency. drugs more commonly used in rabbits metabolized at least partially via glomerular filtration include sulfonamides such as trimethoprim sulfamethoxazole. while procaine penicillin is excreted via tubular secretion, impaired renal function causes delayed excretion in humans and other species. meloxicam is metabolized by the liver. use is not recommended in humans and tested animal patients that are dehydrated, or have liver or renal disease (due to reduction of blood flow to the kidneys). human anesthesiologists routinely modify protocols and drug dosages for the elderly. a geriatric rabbit otherwise in good health may not necessarily represent a serious anesthetic risk; however, clinicians must assume some alteration of cardiovascular and renal health with advancing age. certain drugs should be avoided in older patients. medetomidine (pfizer animal health, new york, ny) is listed frequently as a choice for pre-anesthesia and anesthesia in the rabbit. a few studies have demonstrated use in rabbits, but use in elderly patients has not been investigated. it should be noted that medetomidine and dexmedetomidine are contraindicated in dogs or cats with cardiovascular disease, respiratory disease, liver or kidney disease, or in any debilitated patient. therefore, use in the geriatric rabbit should be avoided or used cautiously. the author and others prefer a balanced approach to sedation and anesthesia for all exotic animal patients, including pre-anesthesia, analgesia, local/regional analgesia, and anesthesia if required. use of a balanced approach allows for reduction of any single agent, including inhalant agents, thus increasing patient safety. many brief procedures producing minimal discomfort can be accomplished with sedation only, for example, radiography, placement of an intravenous catheter, and clipping or minor wound care. in geriatric patients, dosages of all drugs are reduced depending on patient condition. for geriatric patients in which the need for sedation/anesthesia for diagnostic or therapeutic procedures outweigh the risks, the author has had great success with the combination of midazolam with an opioid (butorphanol, buprenorphine, hydromorphone, fentanyl), with the addition of low-dose ketamine if required ( table ). the addition of local analgesia with lidocaine, mg/kg as a local or regional block is care of the geriatric rabbit extremely beneficial. when additional anesthesia is required, inhalant isoflurane or sevoflurane can be added at minimal effective concentrations. an alternative is the use of injectable etomidate combined with midazolam. etomidate is commonly used in geriatric and high-risk human patients, and is apparently unaffected by impaired renal function. etomidate must be injected intravenously, and combined with a benzodiazepine to prevent temporary tetany and seizures. onset of action is immediate, and duration is approximately to minutes in rabbits and other mammals. the author's experience with this drug has been overwhelmingly positive, even in patients with advanced disease conditions. however, death occurred in a single geriatric rabbit with suspected advanced cardiac disease. significant attention has been directed toward hospice, or palliative end-of-life care for companion animals. a recent internet search revealed numerous businesses offering hospice services for traditional pets. the concept of hospice care can be applied to aged rabbits as well. in general, the hospice setting should be clean, quiet, and stress-free. if possible, ill or dying rabbits should not be separated from bonded companions, and the author is unaware of situations in which bullying or harassment has occurred. bedding should provide traction, be soft, absorbent, and easy to clean. hospital absorbent pads are ideal (wings maxima disposable underpads, tyco healthcare) (fig. ) . food and water bowls and bottles are placed within easy reach. caloric and fluid needs can be supplied through syringe feeding of critical care formula as per manufacturer's instructions. nasogastric tube feeding can be considered for those rabbits unable or unwilling to eat; however, stress associated with this procedure should strongly be considered. nasogastric feeding should be considered a temporary measure only. some rabbits benefit from administration of fluids, either subcutaneously or intravenously. willing owners can be instructed to do either in the home setting. in selected cases, an implanted subcutaneous catheter is beneficial (see earlier section on chronic renal failure). daily care also includes gentle cleaning of the perineum to prevent scalding. in rabbits with chronic epiphora, accumulations of secretions should be removed frequently to avoid dermatitis. rabbits unwilling or unable to groom may benefit from shaving of the hair of the perineum in order to reduce fecal and urine accumulation and allow owners to more effectively cleanse the area. analgesia for hospice patients should emphasize optimal pain control, with lesser regard for untoward systemic effects. choosing the time for euthanasia is difficult in most circumstances, and is particularly complicated in rabbits due to their inherent tendency to hide signs of illness. a significant change in routine, and unwillingness to eat or accept treats or groom are clear indications of distress in rabbits. in situations whereby their condition is unlikely to improve, euthanasia should be considered. humane, stress-free euthanasia requires careful planning and implementation, especially when the owners wish to be present. the american veterinary medical association guidelines on euthanasia for rabbits suggest the following: barbiturates, co , co, or potassium chloride in conjunction with general anesthesia. other listed acceptable techniques such as cervical dislocation are unacceptable to most veterinary staff and owners. the author prefers the following technique, especially when owners wish to be present: induction with medetomidine at mg/kg, and ketamine, mg/kg administered intramuscularly. owners are encouraged to hold their pet during induction as comfort level allows. smaller doses may be repeated if necessary. once deep anesthesia is achieved with no response to toe pinch, euthanasia solution is administered intravenously or by cardiac puncture. owners should be given options regarding disposition of the pet after euthanasia. frequently chosen options include home burial (local ordinances permitting), and private or group cremation. most small animal cremation companies are willing to provide services to owners of rabbits and other exotic pets (fig. ) . rabbit and rodent dentistry ferrets, rabbits and rodents, clinical medicine and surgery encephalitozoonosis in pet rabbits (oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation application of elisa and protein electrophoresis in the diagnosis of encephalitozoon cuniculi infection in rabbits critical care of the rabbit nikolaj nikolajewitsch antischkow established the cholesterolfed rabbit as a model for atherosclerosis research cardiovascular physiology and diseases of the rabbit textbook of 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of thymoma in rabbits estimating glomerular filtration rate with a new equation:application to pharmacy and drug dosing pharmacokinetis of meloxicam in rabbits after single and repeat oral dosing a comparison of ketamine-midazolam and ketamine-medetomidine combinations for induction of anesthesia in rabbits available at: www.drugs.com/vet/ dexdomitor.html it's great to sedate available at: www.drugs.com/vet/ etomidate.html available at: www.avma.org/issues/animal_welfare/euthanasia gloucester (uk): british small animal medical association exotic animal formulary beneficial effect of taurine in rabbits with chronic congestive heart failure key: cord- -gu nnv d authors: chandran, uma; mehendale, neelay; patil, saniya; chaguturu, rathnam; patwardhan, bhushan title: chapter network pharmacology date: - - journal: innovative approaches in drug discovery doi: . /b - - - - . - sha: doc_id: cord_uid: gu nnv d abstract the one-drug/one-target/one-disease approach to drug discovery is presently facing many challenges of safety, efficacy, and sustainability. network biology and polypharmacology approaches gained appreciation recently as methods for omics data integration and multitarget drug development, respectively. the combination of these two approaches created a novel paradigm called network pharmacology (np) that looks at the effect of drugs on both the interactome and the diseasome level. ayurveda, the traditional system of indian medicine, uses intelligent formulations containing multiple ingredients and multiple bioactive compounds; however, the scientific rationale and mechanisms remain largely unexplored. np approaches can serve as a valuable tool for evidence-based ayurveda to understand the medicines’ putative actions, indications, and mechanisms. this chapter discusses np and its potential to explore traditional medicine systems to overcome the drug discovery impasse. drug discovery, the process by which new candidate medications are discovered, initially began with random searching of therapeutic agents from plants, animals, and naturally occurring minerals (burger, ) . for this, they depended on the materia medica that was established by medicine men and priests from that era. this was followed by the origin of classical pharmacology in which the desirable therapeutic effects of small molecules were tested on intact cells or whole organisms. later, the advent of human genome sequencing revolutionized the drug discovery process that developed into targetÀbased drug discovery, also known as reverse pharmacology. this relies on the hypothesis that the modulation of the activity of a specific protein will have therapeutic effects. the protein that the drug binds to or interacts with is also referred to as a "target." in this reductionist approach, small molecules from a chemical library are screened for their effect on the target's known or predicted function (hacker et al., ) . once the small molecule is selected for a particular target, further modifications are carried out at the atomic level to ameliorate the lock-and-key interactions. this one-drug/onetarget/one-therapeutic approach was followed for the last several decades. the information technology revolution at the end of th century metamorphosed the drug discovery process as well (clark and pickett, ) . advancements in omics technologies during this time were used to develop strategies for different phases of drug research (buriani et al., ) . computational power was implemented in the discovery process for predicting a drug-likeness of newly designed or discovered compounds and ligandprotein docking for predicting the binding affinity of a small molecule with a protein three-dimensional structure. in silico tools were developed to predict other pharmacological properties of the drug molecules such as absorption, distribution, metabolism, excretion, and toxicity-abbreviated together as admet (van de waterbeemd and gifford, ; clark and grootenhuis, ) . the technological advancements triggered discovery efforts in a direction to discover more specific magic bullets that were completely against the holistic approach of traditional medicine. this magic bullet approach is currently in decline phase. the major limitations of this drug discovery approach are side effects and the inability to tackle multifactorial diseases. this is mainly due to the linearity of this approach. during the peak, historical time of drug discovery and development of natural productsÀbased drugs had played a significant role due to their superior chemical diversity and safety over synthetic compound libraries (zimmermann et al., ) . currently, it is estimated that more than one hundred new, natural productÀbased leads are in clinical development (harvey, ) . many active compounds (bioactives) from traditional medicine sources could serve as good starting compounds and scaffolds for rational drug design. natural products normally act through modulation of multiple targets rather than a single, highly specific target. but in drug discovery and development, technology was used to synthesize highly specific mono-targeted molecules that mimic the bioactives from natural compounds rather than understanding the rationale behind their synergistic action and developing methods to isolate the bioactives from natural resources. researchers understand that most diseases are due to dysfunction of multiple proteins. thus, it is important to address multiple targets emanating from a syndrome-related, metabolic cascade, so that holistic management can be effectively achieved. therefore, it is necessary to shift the strategy from one that focuses on a single-target, new chemical entity to one of a multiple-target, synergistic, formulation-discovery approach . this tempted the research world to go back and extensively explore natural sources, where modern pharmacology had begun. this renewed research focus indicates the need to rediscover the drug discovery process by integrating traditional knowledge with state-of-the-art technologies (patwardhan, a) . a new discipline called network pharmacology (np) has emerged which attempts to understand drug actions and interactions with multiple targets (hopkins, ) . it uses computational power to systematically catalogue the molecular interactions of a drug molecule in a living cell. np appeared as an important tool in understanding the underlying complex relationships between botanical formula and the whole body berger and iyengar, ) . it also attempts to discover new drug leads and targets and to repurpose existing drug molecules for different therapeutic conditions by allowing an unbiased investigation of potential target spaces (kibble et al., ) . however, these efforts require some guidance for selecting the right type of targets and new scaffolds of drug molecules. traditional knowledge can play a vital role in this process of formulation discovery and repurposing existing drugs. by combining advances in systems biology and np, it might be possible to rationally design the next generation of promiscuous drugs (cho et al., ; hopkins, ; ellingson et al., ) . np analysis not only opens up new therapeutic options, but it also aims to improve the safety and efficacy of existing medications. the postgenomic era witnessed a rapid development of computational biology techniques to analyze and explore existing biological data. the key aim of the postgenomic biomedical research was to systematically catalogue all molecules and their interactions within a living cell. it is essential to understand how these molecules and the interactions among them determine the function of this immensely complex machinery, both in isolation and when surrounded by other cells. this led to the emergence and advancement of network biology, which indicates that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biology and disease pathologies in the st century (barabási and oltvai, ) . during the first decade of the st century, several approaches for biological network construction were put forward that used computational methods, and literature mining especially, to understand the relation between disease phenotypes and genotypes. as a consequence, lmma (literature mining and microarray analysis), a novel approach to reconstructing gene networks by combining literature mining and microarray analysis, was proposed (li et al., ; huang and li, ) . with this, a global network was first derived using the literatureÀbased, cooccurrence method and then refined using microarray data. the lmma biological network approach enables researchers to keep themselves up to date with relevant literature on specialized biological topics and to make sense of the relevant large-scale microarray dataset. also, lmma serves as a useful tool for constructing specific biological network and experimental design. lmmaÀlike representations enable a systemic recognition for the specific diseases in the context of complex gene interactions and are helpful for studying the regulation of various complex biological, physiological, and pathological systems. the significance of accumulated-data integration was appreciated by pharmacologists, and they began to look beyond the classic lock-and-key concept as a far more intricate picture of drug action became clear in the postgenomic era. the global mapping of pharmacological space uncovered promiscuity, the specific binding of a chemical to more than one target (paolini et al., ) . as there can be multiple keys for a single lock, in the same way, a single key can fit into multiple locks. similarly, a ligand might interact with many targets and a target may accommodate different types of ligands. this is referred to as "polypharmacology." the concept of network biology was used to integrate data from drugbank (re and valentini, ) and omim (hamosh et al., ) , an online catalog of human genes and genetic disorders to understand the industry trends, the properties of drug targets, and to study how drug targets are related to disease-gene products. in this way, when the first drug-target network was constructed, isolated and bipartite nodes were expected based on the existed one-drug/one-target/onedisease approach. rather, the authors observed a rich network of polypharmacology interactions between drugs and their targets (yildirim et al., ). an overabundance of "follow-on" drugs that are drugs that target already targeted proteins was observed. this suggested a need to upgrade the singletarget single-drug paradigm, as single-protein single-function relations are limited to accurately describing the reality of cellular processes. advances in systems biology led to the realization that complex diseases cannot be effectively treated by intervention at single proteins. this made the drug researchers accept the concept of polypharmacology which they previously thought as an undesirable property that needs to be removed or reduced to produce clean drugs acting on single-targets. according to network biology, simultaneous modulation of multiple targets is required for modifying phenotypes. developing methods to aid polypharmacology can help to improve efficacy and predict unwanted off-target effects. hopkins (hopkins, (hopkins, , observed that network biology and polypharmacology can illuminate the understanding of drug action. he introduced the term "network pharmacology." this distinctive new approach to drug discovery can enable the paradigm shift from highly specific magic bulletÀbased drug discovery to multitargeted drug discovery. np has the potential to provide new treatments to multigenic complex diseases and can lead to the development of e-therapeutics where the ligand formulation can be customized for each complex indication under every disease type. this can be expanded in the future and lead to customized and personalized therapeutics. integration of network biology and polypharmacology can tackle two major sources of attrition in drug development such as efficacy and toxicity. also, this integration holds the promise of expanding the current opportunity space for druggable targets. hopkins proposed np as the next paradigm in drug discovery. polypharmacology expands the space in drug discovery approach. hopkins had suggested three strategies to the designers of multitarget therapies: the first was to prescribe multiple individual medications as a multidrug combination cocktail. patient compliance and the danger of drugÀdrug interactions would be the expected drawbacks of this method. the second proposition was the development of multicomponent drug formulations. the change in metabolism, bioavailability, and pharmacokinetics of formulation as well as safety would be the major concerns of this approach. the third strategy was to design a single compound with selective polypharmacology. according to hopkins, the third method is advantageous, as it would ease the dosing studies. also, the regulatory barriers for the single compound are fewer compared to a formulation. an excellent example of this is metformin, the first-line drug for type ii diabetes that has been found to have cancerinhibiting properties (leung et al., ) . the following years witnessed the application research of np by integrating network biology and polypharmacology. a computational framework, based on a regression model that integrates human proteinÀprotein interactions, disease phenotype similarities, and known geneÀphenotype associations to capture the complex relationships between phenotypes and genotypes, has been proposed. this was based on the assumption that phenotypically similar diseases are caused by functionally related genes. a tool named cipher (correlating protein interaction network and phenotype network to predict disease genes) has been developed that predicts and prioritizes disease-causing genes (wu et al., ) . cipher helps to uncover known disease genes and predict novel susceptibility candidates. another application of this study is to predict a human disease landscape that can be exploited to study the related genes for related phenotypes that will be clustered together in a molecular interaction network. this will facilitate the discovery of disease genes and help to analyze the cooperativity among genes. later, cipher-hit, a hitting-time-based method to measure global closeness between two nodes of a heterogeneous network, was developed (yao et al., ) . a phenotypeÀgenotype network can be explored using this method for detecting the genes related to a particular phenotype. a net-workÀbased gene clustering and extension were used to identify responsive gene modules in a conditionÀspecific gene network aimed to provide useful resources to understand physiological responses (gu et al., ) . np was also used to develop mirnaÀbased biomarkers (lu et al., ) . for this, a network of mirna and their targets was constructed and further refined to study the data for specific diseases. this process integrated with literature mining was useful to develop potent mirna markers for diseases. np was also used to develop a drug geneÀdisease comodule (zhao and li, ) . initially, a drug-disease network was constructed by information gathered from databases followed by the integration of gene data. the gene closeness was studied by developing a mathematical model. this network inferred the association of multiple genes for most of the diseases and target sharing of drugs and diseases. these kinds of networks give insight into new drug-disease associations and their molecular connections. during the progression period of network biology, natural products were gaining importance in the chemical space of drug discovery, as these have been economically designed and synthesized by nature for the benefit of evolution (wetzel et al., ) . researchers began analyzing the logic behind traditional medicine systems and devised computational ways to ease the analysis. a comprehensive herbal medicine information system that was developed integrates information of more than anticancer herbal recipes that have been used for the treatment of different types of cancer in the clinic, individual ingredients, and small organic molecules isolated from herbal medicines (fang et al., ) . this system, which was developed using an oracle database and internet technology, facilitates and promotes scientific research in herbal medicine. this was followed by the development of many databases that serve as a source of botanical information and a powerful tool that provides a bridge between traditional medicines and modern molecular biology. these kinds of databases and tools made the researchers conceive the idea of np of botanicals and their formulations to understand the underlying mechanisms of traditional medicines. we refer to such networks as "ethnopharmacological networks" and the technique as "network ethnopharmacology (nep)" (patwardhan and chandran, ) . shao li pioneered this endeavor and proposed this network as a tool to explain the zheng (syndrome of traditional chinese medicine (tcm)) and the multiple-targets' mechanism of tcm (li, ) . li et al. tried to provide a molecular basis for -year-old concept of zheng using a neuro-endocrine-immune (nei) network . zheng is the basic unit and key concept in tcm theory. it is also used as a guideline in disease classification in tcm. the hot (hans zheng in mandarin) and cold (re zheng) are the two statuses of zheng which therapeutically directs the use of herbs in tcm. chinese herbs are classified as hotÀcooling and are used to remedy hot zheng and coldÀwarming herbs that are used to remedy cold zheng. according to the authors, hormones may be related to hot zheng, immune factors may be related to cold zheng, and they may be interconnected by neurotransmitters. this study provides a methodical approach to understand tcm within the framework of modern science. later they reconstructed the nei network by adding multilayer information including data available on the kegg database related to signal transduction, metabolic pathways, proteinÀprotein interactions, transcription factor, and micro rna regulations. they also connected drugs and diseases through multilayered interactions. the study of cold zheng emphasized its relation to energy metabolism, which is tightly correlated with the genes of neurotransmitters, hormones, and cytokines in the nei interaction network ma et al., ) . another database, tcmgenedit, provides information about tcms, genes, diseases, tcm effects, and tcm ingredients mined from a vast amount of biomedical literature. this would facilitate clinical research and elucidate the possible therapeutic mechanisms of tcms and gene regulations (fang et al., ) . to study the combination rule of tcm formulae, an herb network was created using collaterals-related formulae . they developed a distance-based, mutual-information model (dmim) to uncover the combination rule. dmim uses mutual-information entropy and "between herb distance" to measure the tendency of two herbs to form an herb pair. they experimentally evaluated the combination of a few herbs for angiogenesis. understanding the combination rule of herbs in formulae will help the modernization of traditional medicine and also help to develop a new formulae based on the current requirement. a network targetÀbased paradigm was proposed for the first time to understand the synergistic combinations , and an algorithm termed "nims" (network tar-getÀbased identification of a multicomponent synergy) was also developed. this was a step that facilitated the development of multicomponent therapeutics using traditional wisdom. an innovative way to study the molecular mechanism of tcm was proposed during this time by integrating the tcm experimental data with microarray gene expression data (wen et al., ) . as a demonstrative example, si-wu-tang's formula was studied. rather than uncovering the molecular mechanism of action, this method would help to identify new health benefits of tcms. the initial years of the second decade of the st century witnessed the network ethnopharmacological exploration of tcm formulations. the scope of this new area attracted scientists, and they hoped nep could provide insight into multicompound drug discoveries that could help overcome the current impasse in drug discovery (patwardhan, b; . nep was used to study the antiinflammatory mechanism of qingfei xiaoyan, a tcm . the predicted results were used to design experiments and analyze the data. experimental confirmation of the predicted results provides an effective strategy for the study of traditional medicines. the potential of tcm formulations as multiple compound drug candidates has been studied using tcm formulations based np. tcm formulations studied in this way are listed in table . . construction of a database containing , natural product structures, followed by their docking to target proteins of fda-approved drugs, shows the amount of space shared in the chemical space between natural products and fda drugs (gu et al., a) . molecular-docking technique plays a major role in np. the interaction of bioactives with molecular targets can be analyzed by this technique. molecular dockingÀbased nep can be a useful tool to computationally elucidate the combinatorial effects of traditional medicine to intervene disease networks (gu et al., c ). an approach that combines np and pharmacokinetics has been proposed to study the material basis of tcm formulations (pei et al., ) . this can be extrapolated to study other traditional medicine formulations as well. in cancer research, numerous natural products have been demonstrated to have anticancer potential. natural products are gaining attraction in anticancer research, as they show a favorable profile in terms of absorption and metabolism in the body with low toxicity. in a study all of the known bioactives were docked for their property to interact with cancer targets (luo et al., ) . it was inferred that many bioactives are targeting multiple ejiao slurry regulates cancer cell differentiation, growth, proliferation, and apoptosis, and shows an adjuvant therapeutic effect that enriches the blood and increases immunity xu et al. ( b) xiao-chaihu decoction and da chaihu-decoction xchd treats diseases accompanying symptoms of alternating fever and chills, no desire for food or drink, and dry pharynx, while dchd treats those with symptoms of fullness, pain in abdomen, and constipation. dragon's blood used in colitis and acts through interaction with putative targets xu et al. ( a) protein targets and thus are linked to many types of cancers. np coupled to sophisticated spectroscopical analysis such as ultra-performance liquid chromatographyÀelectrospray, ionizationÀtandem mass spectroscopy (uplc-esi-ms/ms) is a useful approach to study the absolute molecular mechanism of action of botanical formulations based on their constituent bioactives (xu et al., a) . bioactiveÀtarget analysis has shown that some of the botanical formulations are more effective than their corresponding marketed drugÀtarget interactions . this indicates the potential of np to better understand the power of botanical formulations and to develop efficient and economical treatment options. the holistic approach of botanical formulations can be better explained by np. a study has reported this property by exemplifying a tcm formulation against viral infectious disease . not only does the formulation target the proteins in the viral infection cycle, but it also regulates the proteins of the host defense system; thus, it acts in a very distinctive manner. this unique property of formulations is highly efficient for strengthening the broad and nonspecific antipathogenic actions. thus, network-based, multitarget drugs can be developed by testing the efficacy of the formulation, identifying, and isolating the major bioactives and redeveloping a multicomponent therapeutic using the major bioactives based on synergism (leung et al., ) . np also serves to document and analyze the clinical prescriptions of traditional medicine practitioners . a traditional medicine network that links bioactives to clinical symptoms through targets and diseases is a novel way to explore the basic principles of traditional medicines (luo et al., ) . the network-based approaches provide a systematic platform for the study of multicomponent traditional medicine and has applications for its beneficial modernization. this platform not only recovers traditional knowledge, but it also provide new findings that can be used for resolving current problems in the drug industry . this section explains a handful of ethnopharmacological networks that were developed to understand the scientific rationale of traditional medicine. dragon's blood (db) tablets, which are made of resins from dracaena spp., daemonorops spp., croton spp., and pterocarpus spp., is an effective tcm for the treatment of colitis. in a study, an np-based approach was adopted to provide new insights relating to the active constituents and molecular mechanisms underlying the effects of db (xu et al., a) . the constituent chemicals of the formulation were identified using an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry method. the known targets of those identified compounds were mined from literature and putative targets that were predicted with the help of computational tools. the compounds were further screened for bioavailability followed by the systematic analysis of the known and putative targets for colitis. the network evaluation revealed the mechanism of action of db bioactives for colitis through the modulation of the proteins of the nod-like receptor signaling pathway (fig. . ) . the antioxidant mechanism of zhi-zi-da-huang decoction as an approach to treat alcohol liver disease was elucidated using np an and feng, ). an endothelial cell proliferation assay was performed for an antiangiogenic alkaloid, sinomenine, to validate the network targetÀbased identification of multicomponent synergy (nims) predictions. the study was aimed at evaluating the synergistic relationship between different pairs of therapeutics, and sinomenine was found to have a maximum inhibition rate with matrine, both through the network and in vitro studies. the discovery of bioactives and elucidation of the mechanism of action of the herbal formulae, qing-luo-yin and the liu-wei-di-huang pill, using np, has given insight to the design validation experiments that accelerated the process of drug discovery . validation experiments based on the network findings regarding cold zheng and hot zheng on a rat model of collagenÀinduced arthritis showed that the cold zhengÀoriented herbs tend to affect the hub nodes in the cold zheng network, and the hot zheng-oriented herbs tend to affect the hub nodes in the hot zheng network . np was used to explain the addition and subtraction theory of tcm. two decoctions: xiao chaihu and da chaihu were studied using np approach to investigate this theory. according to the addition and subtraction theory, the addition or removal of one or more ingredients from a traditional formulation resulted in a modified formula that plays a vital role in individualized medicine. compounds from additive herbs were observed to be more efficient on diseaseÀassociated targets (fig. . ) . these additive compounds were found to act on diseases through drug targets (li et al., a) . experimental verification of the antithrombotic network of fufang xueshuantong (fxst) capsule was done through in vivo studies on lipopoly-saccharideÀinduced disseminated intravascular coagulation (dic) rat model. it was successfully shown that fxst significantly improves the activation of the coagulation system through targets from four herbs (sheng et al., ) . np analysis of the bushenhuoxue formula showed that six components-rhein, tanshinone iia, curcumin, quercetin and calycosin-acted through targets for the treatment of chronic kidney disease. these predictions were validated using unilateral ureteral obstruction models, and it was observed that even though the individual botanicals showed a significant decrease in creatinine levels, the combination showed lower blood creatinine and urea nitrogen levels (shi et al., ) . the antidiabetic effects of ge-gen-qin-lian decoction were investigated using an insulin secretion assay, and an insulinÀresistance model using of the ingredients showed antidiabetic activity using np studies (li et al., b) . to confirm the predictions of the network of liu-wei-di-huang pill, four proteins-pparg, rara, ccr , and esr -that denote different functions and are targeted by different groups of ingredients were chosen. the interactions between various bioactives and their effect on the expression of the proteins showed that the np approach can accurately predict these interactions, giving hints regarding the mechanism of action of the compounds (liang et al., ) . experimental results confirmed that the core ingredients in modified simiaowan, obtained through network analysis, significantly increased huvec viability and attenuated the expression of icam- and proved to be effective in gout treatment (zhao et al., ) . the role of anthraquinone and flavanols (catechin and epicatechin) in the therapeutic potential of rhubarb in renal interstitial fibrosis was examined using network analysis and by conventional assessment involving serum biochemistry, histopathological, and immunohistochemical assays (xiang et al., ) . in silico analysis and experimental validation demonstrated that compound / of fructus schisandrae chinensis targets gba /shbg . np is a valuable method to study the synergistic effects of bioactives of traditional medicine formulation. this was experimentally shown on the sendeng- formulation for rheumatoid arthritis (fig. . ). data and network analysis have shown that the formulation acts synergistically through nine categories of targets (zi and yu, ) . another network that studied three botanicals, salviae miltiorrhizae, ligusticum chuanxiong, and panax notoginseng for coronary artery disease (cad), displayed their mode of action through targets, out of which are common among the botanicals (fig. . ) . these common targets are associated with thrombosis, dyslipidemia, vasoconstriction, and inflammation . this gives insight to how these botanicals are managing cad. another approach using np is the construction of networks based on experimental data followed by literature mining. this method is very effective for large space data analysis, which will help to derive the mechanism of action of the formulation. a network of qishenyiqi formulation having cardioprotective effects, constructed based on the microarray data and the published literature, showed that main compounds were found to act through pathways, out of which are immune and inflammation-related (li et al., c) . the mechanism of action for the bushen zhuanggu formulation was proposed based on lc-ms/ms standardization, pharmacokinetic analysis, and np (pei et al., ) . the efficacy of shenmai injection was evaluated using a rat model of myocardial infarction, genome-wide transcriptomic experiment, and then followed by a np analysis. the overall trends in the ejection fraction and fractional shortening were consistent with the networkÀrecovery index (nri) from the network . in order to develop an ethnopharmacological network, exploring the existing databases to gather information regarding bioactives and targets is the first step. further information such as target-related diseases, tissue distribution and pathways are also to be collected depending on the type of study that is going to be undertaken. the universal natural products database (unpd) (gu et al., a ) is one of the major databases that provides bioactives information. other databases that provide information regarding bioactives include cvdhd (gu et al., b) , tcmsp (ru et al., ) , tcm@taiwan (sanderson, ) , supernatural (banerjee et al., ) , and dr. dukes's phytochemical and ethnobotanical database (duke and beckstrom-sternberg, ) . the molecular structures of bioactives are usually stored as "sd" files and chemical information as smiles and inchkeys in these databases. any of these file formats can be used as inputs to identify the targets in protein information databases. binding database or "binding db" (liu et al., ) and chembl (bento et al., ) are databases for predicting target proteins. binding db searches the exact or similar compounds in the database and retrieves the target information of those compounds. the similarity search gives the structurally similar compounds with respect to the degree of similarity as scores to the queried structure. the information regarding both annotated and predicted targets can be collected in this way. this database is connected to numerous databases, and these connections can be used to extract further information regarding the targets. the important databases linked to binding db are uniprot (bairoch et al., ) , which gives information related to proteins and genes; reactome, a curated pathway database (croft et al., ) ; and the kyoto encyclopedia of genes and genomes (kegg), a knowledge base for systematic analysis of gene functions and pathways (ogata et al., ) . therapeutic targets database (ttd) (zhu et al., ) gives fully referenced information of targeted diseases of proteins, their pathway information, and the corresponding drug directed to each target. disease and gene annotation (dga), a database that provides a comprehensive and integrative annotation of human genes in disease networks, is useful in identifying the disease type that each indication belongs to (peng et al., ) . the human protein atlas (hpa) database (pontén et al., ) is an open database showing the spatial distribution of proteins in different normal human tissues. the information of the distribution of proteins in tissues can be gathered from hpa. the database also gives information regarding subcellular localization and protein class. an overall review of the methods to implement np for herbs and herbal formulations is also available, including a systematic review of the databases that one could use for the same (kibble et al., ; lagunin et al., ) . integration of knowledge bases helps data gathering for network pharmacological studies, and its knowledge base shows the inter-relationships among these databases (fig. . ) . the counts of entities, such as bioactives, targets, and diseases, can vary based on the knowledge bases that are relied on for data collection. an integration of knowledge bases can overcome this limitation. another factor that affects the counts of these entities is the time frame for data collection. this change occurs due to the ongoing, periodic updates of the databases. a network is the schematic representation of the interaction among various entities called nodes. in pharmacological networks, the nodes include bioactives, targets, tissue, tissue types, disease, disease types, and pathways. these nodes are connected by lines termed edges, which represent the relationship between them (morris et al., ) . building a network involves two opposite approaches: a bottom-up approach on the basis of established biological knowledge and a top-down approach starting with the statistical analysis of available data. at a more detailed level, there are several ways to build and illustrate a biological network. perhaps the most versatile and general way is the de novo assembly of a network from direct experimental or computational interactions, e.g., chemical/gene/protein screens. networks encompassing biologically relevant nodes (genes, proteins, metabolites), their connections (biochemical and regulatory), and modules (pathways and functional units) give an authentic idea of the real biological phenomena (xu and qu, ) . cytoscape, a java-based open source software platform (shannon et al., ) , is a useful tool for visualizing molecular interaction networks and integrating them with any type of attribute data. in addition to the basic set of features for data integration, analysis, and visualization, additional features are available in the form of apps, including network and molecular profiling analysis and links with other databases. in addition to cytoscape, a number of visualization tools are available. visual network pharmacology (vnp) , which is specially designed to visualize the complex relationships among diseases, targets, and drugs, mainly contains three functional modules: drug-centric, target-centric, and disease-centric vnp. this disease-target-drug database documents known connections among diseases, targets, and the usfda-approved drugs. users can search the database using disease, target, or drug name strings; chemical structures and substructures; or protein sequence similarity, and then obtain an online interactive network view of the retrieved records. in the obtained network view, each node is a disease, target, or drug, and each edge is a known connection between two of them. the connectivity map, or the cmap tool, allows the user to compare gene-expression profiles. the similarities or differences in the signature transcriptional expression profile and the small molecule transcriptional response profile may lead to the discovery of the mode of action of the small molecule. the response profile is also compared to response profiles of drugs in the cmap database with respect to the similarity of transcriptional responses. a network is constructed and the drugs that appear closest to the small molecule are selected to have better insight into the mode of action. other software, such as gephi, an exploration platform for networks and complex systems, and cell illustrator, a java-based tool specialized in biological processes and systems, can also be used for building networks . ayurveda, the indian traditional medicine, offers many sophisticated formulations that have been used for hundreds of years. the traditional knowledge digital library (tkdl, http://www.tkdl.res.in) contains more than , classical ayurveda formulations. approximately of these are popularly used at the community level and also as over-the-counter products. some of these drugs continue to be used as home remedies for preventive and primary health care in india. until recently, no research was carried out to explore ayurvedic wisdom using np despite ayurveda holding a rich knowledge of traditional medicine equal to or greater than tcm. our group examined the use of np to study ayurvedic formulations with the wellknown ayurvedic formulation triphala as a demonstrable example (chandran et al., a, b) . in this chapter, we demonstrate the application of np in understanding and exploring the traditional wisdom with triphala as a model. triphala is one of the most popular and widely used ayurvedic formulations. triphala contains fruits of three myrobalans: emblica officinalis (eo; amalaki) also known as phyllanthus emblica; terminalia bellerica (tb; vibhitaka); and terminalia chebula (tc; haritaki). triphala is the drug of choice for the treatment of several diseases, especially those of metabolism, dental, and skin conditions, and treatment of cancer (baliga, ) . it has a very good effect on the health of heart, skin, eyes, and helps to delay degenerative changes, such as cataracts (gupta et al., ) . triphala can be used as an inexpensive and nontoxic natural product for the prevention and treatment of diseases where vascular endothelial growth factor aÀinduced angiogenesis is involved . the presence of numerous polyphenolic compounds empowers it with a broad antimicrobial spectrum (sharma, ) . triphala is a constituent of about ayurveda formulations and it can be used for several diseases. triphala combats degenerative and metabolic disorders possibly through lipid peroxide inhibition and free radical scavenging (sabu and kuttan, ) . in a phase i clinical trial on healthy volunteers, immunostimulatory effects of triphala on cytotoxic t cells and natural killer cells have been reported (phetkate et al., ) . triphala is shown to induce apoptosis in tumor cells of the human pancreas, in both in vitro and in vivo models (shi et al., ) . although the anticancer properties of triphala have been studied, the exact mechanism of action is still not known. the beneficial role of triphala in disease management of proliferative vitreoretinopathy has also been reported (sivasankar et al., ) . one of the key ingredients of triphala is amalaki. some studies have already shown the beneficial effect of amalaki rasayana to suppress neurodegeneration in fly models of huntington's and alzheimer's diseases (dwivedi et al., (dwivedi et al., , . triphala is an effective medicine to balance all three dosha. it is considered as a good rejuvenator rasayana, which facilitates nourishment to all tissues, or dhatu. here we demonstrate the multidimensional properties of triphala using human proteome, diseasome, and microbial proteome targeting networks. the botanicals of triphala-eo, tb, and tc-contain , , and bioactives, respectively, according to unpd data collected during june . of these, a few bioactives are common among the three botanicals. thus, triphala formulation as a whole contains bioactives. out of these, bioactives were score- , based on binding db search carried out during june . eo, tb, and tc contain , , and score- bioactives, respectively ( fig. . ). the score- bioactives that are common among three plants are chebulanin, ellagic acid, gallussaeure, , -digalloyl-beta-d-glucopiranoside, methyl gallate, and tannic acid. this bioactive information is the basic step toward constructing human proteome and microbial proteome targeting networks. thirty-six score- bioactives of triphala are shown to interact with human protein targets in combinations (fig. . ) . quercetin, ellagic acid, , , , , -pentagalloylglucose and , , , -tetrakis-(o-galloyl)-beta-d-glucose are the four bioactives that interact with the maximum number of targets: , , and , respectively. the other major bioactives that have multitargeting property include catechin; epicatechin; gallocatechin; kaempferol; and trans- , ', ', , -pentahydroxylflavane. the major protein targets of triphala include alkaline phosphatase (alpl); carbonic anhydrase (ca ); coagulation factor x (f ), dna repair protein rad homolog (rad ); gstm protein (gstm ); beta-secretase (bace ); plasminogen activator inhibitor (serpine ), prothrombin (f ); regulator of g-protein signaling (rgs) , , and , tissue-type plasminogen activator (plat); and tyrosineprotein phosphatase nonreceptor type (ptpn ). the targets of triphala are associated with disease types, which include disease indications (fig. . ) . the major disease types in which triphala targets are associated include cancers, cardiovascular diseases, nervous system diseases, and metabolic diseases. analysis of existing data indicates that targets of triphala bioactives are involved in the different types of cancers making it the largest group of diseases, involving triphala targets. this linkage is through the interaction of bioactives and target proteins in different bioactiveÀtarget combinations. the types of cancers which are networked by triphala include pancreatic, prostate, breast, lung, colorectal and gastric cancers, tumors, and more. quercetin, ellagic acid, prodelphinidin a , and , , -benzenetriol are the important bioactives; and rad , bace , f , mmp , igf r, and egfr are the important targets that play a role in cancer. triphala shows links to indications of cardiovascular diseases through bioactives and targets. the cardiovascular diseases that are covered in the triphala network include atherosclerosis, myocardial ischemia, infarction, cerebral vasospasm, thrombosis, and hypertension. the bioactives playing a major role in cardiovascular diseases are quercetin, , , , , -pentagalloyoglucose, , , , -tetrakis-(o-galloyl)-beta-d-glucose, bellericagenin a , and prodelphinidin a , whereas the targets playing an important role are serpine , f , f , and fabp . triphala's network to nervous system disorders contains diseases in which the significant ones are alzheimer's disease, parkinson's disease, diabetic neuropathy, and retinopathy. in this subnetwork, bioactives interact with targets through different interactions. quercetin, , , , , -pentagalloyoglucose, , , , -tetrakis-(o-galloyl)-beta-d-glucose, and epigallocatechin- -gallate are the most networked bioactives whereas the most networked targets are bace , serpine , plat, aldr, ca . the association of triphala with metabolic disorders is determined by six bioactives that interact with seven targets. the major metabolic diseases come in this link are obesity, diabetic complications, noninsulin-dependent diabetes, hypercholesterolemia, hyperlipidemia, and more. the bioactives having more interactions with targets are ellagic acid, quercetin, and bellericagenin a , whereas the highly networked targets are igf r, fabp , aldr, and akr b . triphala bioactives are also linked to targets of other diseases comprising autoimmune diseases, ulcerative colitis, mccuneÀalbright syndrome, psoriasis, gout, osteoarthritis, endometriosis, lung fibrosis, glomerulonephritis, and more. the proteome-targeting network of triphala, thus, shows its ability to synergistically modulate targets that are associated with disease indications. this data is generated with the available information that included only one-fifth of the total number of bioactives. further logical analysis and experimental studies based on the network result are needed to explore the in-depth mechanism of action of triphala. for researchers in this area, these kind of networks can give an immense amount of information that can be developed further to reveal the real mystery behind the actions of traditional medicine. triphala is also referred to as a "tridoshic rasayana," as it balances the three constitutional elements of life. it tonifies the gastrointestinal tract, improves digestion, and is known to exhibit antiviral, antibacterial, antifungal, and antiallergic properties (sharma, ; amala and jeyaraj, ; sumathi and parvathi, ) . triphala mashi (mashi: black ash) was found to have nonspecific antimicrobial activity, as it showed a dose-dependent inhibition of gram-positive and gram-negative bacteria (biradar et al., ) . hydroalcoholic, aqueous, and ether extracts of the three fruits of triphala were reported to show antibacterial activity against uropathogens with a maximum drug efficacy recorded by the alcoholic extract (bag et al., ; prasad et al., ) . the methanolic extract of triphala showed the presence of active compounds using gc-ms and also showed potent antibacterial and antifungal activity (amala and jeyaraj, ) . triphala has been well studied for its antimicrobial activity against gram-positive bacteria, gram-negative bacteria, fungal species, and different strains of salmonella typhi (amala and jeyaraj, ; sumathi and parvathi, ; gautam et al., ; srikumar et al., ) . triphala showed significant antimicrobial activity against enterococcus faecalis and streptococcus mutans grown on tooth substrate thereby making it a suitable agent for prevention of dental plaque (prabhakar et al., (prabhakar et al., , . the application of triphala in commercial antimicrobial agents has been explored. a significant reduction in the colony forming units of oral streptococci was observed after % triphala was incorporated in a mouthwash formulation (srinagesh et al., ). an ointment prepared from triphala ( % (w/w)) showed significant antibacterial and wound healing activity in rats infected with staphylococcus aureus, pseudomonas aeruginosa, and streptococcus pyogenes (kumar et al., ) . the antiinfective network of triphala sheds light on the efficacy of the formulation in the simultaneous targeting of multiple microorganisms. also, this network provides information regarding some novel bioactiveÀtarget combinations that can be explored to combat the problem of multidrug resistance. among the bioactives of triphala, score- bioactives target microbial proteins of microorganisms. the botanicals of triphala-eo, tb, and tccontain , , and score bioactives respectively which showed interactions with microbial proteins. they act through modulation of targets which are associated with diseases such as leishmaniasis, malaria, tuberculosis, hepatitis c, acquired immunodeficiency syndrome (aids), cervical cancer, candidiasis, luminous vibriosis, yersiniosis, skin and respiratory infections, severe acute respiratory syndrome (sars), avian viral infection, bacteremia, sleeping sickness, and anthrax ( fig. . ). the microorganisms captured in the triphala antiinfective network includes candida albicans, hepatitis c virus, human immunodeficiency virus , human papillomavirus type , human sars coronavirus leishmania amazonensis, mycobacterium tuberculosis, staphylococcus aureus, plasmodium falciparum, and yersinia enterocolitica. in mycobacterium tuberculosis, dtdp- -dehydrorhamnose , -epimerase rmlc is one of the four enzymes involved in the synthesis of dtdp-l-rhamnose, a precursor of l-rhamnose (giraud et al., ) . the network shows that triphala has the potential to modulate the protein through four bioactives such as punicalins, terflavin b, -o-(s)-flavogallonyl- -o-galloylbeta-d-glucopyranose, and , -o-(s,s)-gallagyl-alpha/beta-d-glucopyranose. research on new therapeutics that target the mycobacterial cell wall is in progress. rhamnosyl residues play a structural role in the mycobacterial cell wall by acting as a linker connecting arabinogalactin polymer to peptidoglycan and are not found in humans, which gives them a degree of therapeutic potential (ma et al., ) . triphala can be considered in this line to develop novel antimycobacterial drugs. the network shows the potential of gallussaeure and -galloylgallic acid to modulate human immunodeficiency virus type reverse transcriptase. inhibition of human immunodeficiency virus at the initial stage itself is crucial and thus, targeting human immunodeficiency virus type reverse transcriptase, at the preinitiation stage is considered to be an effective therapy. protein e of human papillomavirus (hpv ) prevents apoptosis of figure . the microbial proteomeÀtargeting network of triphala. dark green verus are the botanicals of triphala and oval green nodes are the score bioactives. targets, diseases, and microorganisms are represented by blue diamond nodes, red triangle nodes, and pink octagon nodes, respectively. infected cells by binding to fadd and caspase and hence being targeted for development of antiviral drugs (yuan et al., ) . kaempferol of triphala is found to target protein e of hpv , which is a potential mechanism to control the replication of the virus. the network also shows triphala's potential to act on plasmodium falciparum. enoyl-acyl carrier protein reductase (enr) has been investigated as an attractive target due to its important role in membrane construction and energy production in plasmodium falciparum (nicola et al., ) while the parasite interacts with human erythrocyte spectrin and other membrane proteins through protein m aspartyl aminopeptidase (lauterbach and coetzer, ) . trans- , ', ', , -pentahydroxylflavane, epigallocatechin, and epicatechin can modulate both while epigallocatechin -gallate can regulate enoyl-acyl carrier protein reductase and, quercetin and vanillic acid can act on m aspartyl aminopeptidase. epigallocatechin -gallate can also target -oxoacyl-acyl-carrier protein reductase which is a potent therapeutic target because of its role in type ii fatty acid synthase pathway of plasmodium falciparum (karmodiya and surolia, ) . epigallocatechin -gallate and quercetin are the bioactives that have shown maximum antimicrobial targets interaction. while epigallocatechin -gallate shows interaction with -oxoacyl-(acyl-carrier protein) reductase, cpg dna methylase, enoyl-acyl-carrier protein reductase, glucose- phosphate -dehydrogenase, hepatitis c virus serine protease, ns /ns a and yoph of plasmodium falciparum, saccharomyces cerevisiae, and spiroplasma monobiae; quercetin acts on c-like proteinase ( cl-pro), arginase, beta-lactamase ampc, glutathione reductase, m aspartyl aminopeptidase, malate dehydrogenase and tyrosine-protein kinase transforming protein fps of escherichia coli, fujinami sarcoma virus, human sars coronavirus (sars-cov), leishmania amazonensis, plasmodium falciparum, saccharomyces cerevisiae, and thermus thermophiles. np has gained impetus as a novel paradigm for drug discovery. this approach using in silico data is fast becoming popular due to its cost efficiency and comparably good predictability. thus, network analysis has various applications and promising future prospects with regard to the process of drug discovery and development. np has proven to be a boon for drug research, and that helps in the revival of traditional knowledge. albeit there are a few limitations of using np for nep studies. this is because the bioactives form the foundation of any traditional medicine network. . absorption, distribution, metabolism, excretion, and toxic effects (admet) parameters associated with the bioactives/formulation when they are administered in the form of the medicine need to be considered in order to extrapolate in silico and cheminformatics data to in vitro and in vivo models. in silico tools that offer the prediction of these parameters can be depended on for this. but traditional medicines are generally accompanied by a vehicle for delivery of the medicine. these vehicles, normally various solvents-water, milk, lemon juice, butter, ghee (clarified butter), honey-that alter the solubility of the bioactives, play a role in regulating admet parameters. experimental validation studies are required to evaluate this principle of traditional medicine. . target identification usually relies on a single or a few databases due to the limited availability of databases with free access. this can occasionally give incomplete results. also, there may be novel targets waiting to be discovered that could be a part of the mechanism of action of the bioactives. to deal with this discrepancy in the network, multiple databases should be considered for target identification. integration of databases serving similar functions can also be a solution for this problem. in addition to this, experimental validation of the target molecules using proteinÀprotein interaction studies or gene expression studies will provide concrete testimony to the network predictions. . a number of traditional medicines act through multiple bioactives and targets. synergy in botanical drugs helps to balance out the extreme pharmacological effects that individual bioactives may have. the interactions of bioactives with various target proteins, their absorption into the body after possible enzyme degradation, their transport, and finally their physiological effect are a crucial part of traditional medicine (gilbert and alves, ) . however, in vitro assays or in silico tools are unable to give a clear idea as to the complete and exact interactions in a living organism. np is only the cardinal step toward understanding the mechanism of bioactives/formulations. but this gives an overview of the action of traditional medicine which can be used to design in vivo experiments and clinical trials. this saves time and cost of research and inventions. . it is observed that formulations are working by simultaneous modulation of multiple targets. this modulation includes activation of some targets and inhibition of other. in order to understand this complex synergistic activity of formulation, investigative studies regarding the interactions of ligands with targets are to be carried out. this can be achieved by implementing high-throughput omics studies based on the network data. network pharmacological analysis presents an immense scope for exploring traditional knowledge to find solutions for the current problems challenging the drug discovery industry. nep can also play a key role in new drug discovery, drug repurposing, and rational formulation discovery. many of the bioactiveÀtarget combinations have been experimentally studied. the data synthesis using np provides information regarding the mode of action of traditional medicine formulations, based on their constituent bioactives. this is a kind of reverse approach to deduce the molecular mechanism of action of formulations using modern, integrated technologies. the current network analysis is based on the studies that have been conducted and the literature that is available. hence, the data is inconclusive as a number of studies are still underway and novel data is being generated continuously. despite its limitations, this still is a favorable approach, as it gives insight into the hidden knowledge of our ancient traditional medicine wisdom. np aids the logical analysis of this wisdom that can be utilized to understand the knowledge as well as to invent novel solutions for current pharmacological problems. determination of antibacterial, antifungal, bioactive constituents of triphala by ft-ir and gc-ms analysis antibacterial potential of hydroalcoholic extracts of triphala 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comprehensive pathway network analyses of co-operative transitions in plasmodium falciparum beta-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding network pharmacology applications to map the unexplored target space and therapeutic potential of natural products triphala promotes healing of infected full-thickness dermal wound chemo-and bioinformatics resources for in silico drug discovery from medicinal plants beyond their traditional use: a critical review network-based drug discovery by integrating systems biology and computational technologies systems pharmacologybased approach for dissecting the addition and subtraction theory of traditional chinese medicine: an example using xiao-chaihu-decoction and da-chaihu-decoction a network pharmacology approach to determine active compounds and action mechanisms of ge-gen-qin-lian decoction for treatment of type diabetes traditional chinese medicinebased network pharmacology could lead to new 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action herb network analysis for a famous tcm doctor' s prescriptions on treatment of rheumatoid arthritis. evidence-based complement a novel network pharmacology approach to analyse traditional herbal formulae: the liu-wei-di-huang pill as a case study bindingdb: a web-accessible database of experimentally determined protein-ligand binding affinities network pharmacology study on major active compounds of siwu decoction analogous formulae for treating primary dysmenorrhea of gynecology blood stasis syndrome computational pharmacological comparison of salvia miltiorrhiza and panax notoginseng used in the therapy of cardiovascular diseases triphala and its active constituent chebulinic acid are natural inhibitors of vascular endothelial growth factor-a mediated angiogenesis computational identification of potential microrna network biomarkers for the progression stages of gastric cancer systems pharmacology strategies for anticancer drug discovery based on natural products multiscale modeling of druginduced effects of reduning injection on human disease: from drug molecules to clinical symptoms of disease drug targeting mycobacterium tuberculosis cell wall synthesis: genetics of dtdp-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dtdp-glucose to dtdp-rhamnose bridging the gap between traditional chinese medicine and systems biology: the connection of cold syndrome and nei network analysis and visualization of biological networks with cytoscape discovery of novel inhibitors targeting enoyl-acyl carrier protein reductase in plasmodium falciparum by structure-based virtual screening global mapping of pharmacological space rediscovering drug discovery network ethnopharmacology approaches for formulation discovery integrative approaches for health: biomedical research, ayurveda and yoga material basis of chinese herbal formulas explored by combining pharmacokinetics with network pharmacology the disease and gene annotations (dga): an annotation resource for human disease significant increase in cytotoxic t lymphocytes and natural killer cells by triphala: a clinical phase i study. evid. based complement alternat the human protein atlas as a proteomic resource for biomarker discovery evaluation of antimicrobial efficacy of herbal alternatives (triphala and green tea polyphenols), mtad, and % sodium hypochlorite against enterococcus faecalis biofilm formed on tooth substrate: an in vitro study evaluation of antimicrobial efficacy of triphala (an indian ayurvedic herbal formulation) and . % chlorhexidine against streptococcus mutans biofilm formed on tooth substrate: an in vitro study potent growth suppressive activity of curcumin in human breast cancer cells: modulation of wnt/beta-catenin signaling tcmsp: a database of systems pharmacology for drug discovery from herbal medicines anti-diabetic activity of medicinal plants and its relationship with their antioxidant property databases aim to bridge the east-west divide of drug discovery cytoscape: a software environment for integrated models of biomolecular interaction networks network pharmacology analyses of the antithrombotic pharmacological mechanism of fufang xueshuantong capsule with experimental support using disseminated intravascular coagulation rats a network pharmacology approach to understanding the mechanisms of action of traditional medicine: bushenhuoxue formula for treatment of chronic kidney disease triphala inhibits both in vitro and in vivo xenograft growth of pancreatic tumor cells by inducing apoptosis aqueous and alcoholic extracts of triphala and their active compounds chebulagic acid and chebulinic acid prevented epithelial to mesenchymal transition in retinal pigment epithelial cells, by inhibiting smad- phosphorylation evaluation of the growth inhibitory activities of triphala against common bacterial isolates from hiv infected patients antibacterial efficacy of triphala against oral 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identifying roles of "jun-chen-zuo-shi" component herbs of qishenyiqi formula in treating acute myocardial ischemia by network pharmacology network-based global inference of human disease genes the study on the material basis and the mechanism for anti-renal interstitial fibrosis efficacy of rhubarb through integration of metabonomics and network pharmacology a systems biology-based approach to uncovering the molecular mechanisms underlying the effects of dragon's blood tablet in colitis, involving the integration of chemical analysis, adme prediction, and network pharmacology study on action mechanism of adjuvant therapeutic effect compound ejiao slurry in treating cancers based on network pharmacology alternative medicine. intech network pharmacological research of volatile oil from zhike chuanbei pipa dropping pills in treatment of airway inflammation navigating traditional chinese medicine network pharmacology and computational tools modularity-based credible prediction of disease genes and detection of disease subtypes on the phenotype-gene heterogeneous network small molecule inhibitors of the hpv -e interaction with caspase an integrative platform of tcm network pharmacology and its application on a herbal formula network understanding of herb medicine via rapid identification of ingredient-target interactions dbnei . : building multilayer network for drug-neidisease systems pharmacology dissection of the anti-inflammatory mechanism for the medicinal herb folium eriobotryae network pharmacology study on the mechanism of traditional chinese medicine for upper respiratory tract infection a network pharmacology approach to determine active ingredients and rationality of herb combinations of modifiedsimiaowan for treatment of gout a co-module approach for elucidating drug-disease associations and revealing their molecular basis deciphering the underlying mechanisms of diesun miaofang in traumatic injury from a systems pharmacology perspective network pharmacology-based prediction of the multi-target capabilities of the compounds in taohong siwu decoction, and their application in osteoarthritis a network-based analysis of the types of coronary artery disease from traditional chinese medicine perspective: potential for therapeutics and drug discovery therapeutic target database update : a resource for facilitating target-oriented drug discovery multi-target therapeutics: when the whole is greater than the sum of the parts studying traditional medicine that would hopefully get resolved in the future. the major limitations and possible solutions are listed: . nep currently relies on various databases for literature and bioactive mining. databases, though curated, may show discrepancies due to numerous sources of information, theoretical, and experimental data. moreover, the botanicals that undergo certain preparatory procedures during the formulation of the medicine may have its constituents that have chemically changed due to the procedures; like boiling, acid/ alkali reactions, interactions between the bioactives, etc. a way to navigate around this problem is to make use of modern, high-throughput chemical identification techniques like ultra-performance liquid chromatogra-phyÀelectrospray ionizationÀtandem mass spectroscopy (uplc-esi-ms/ms). this technique will help to identify the exact bioactives or the chemical constituents of the formulation, and will enrich the subsequent key: cord- -itdx wqi authors: white, alexandre i r title: historical linkages: epidemic threat, economic risk, and xenophobia date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: itdx wqi nan the art of medicine historical linkages: epidemic threat, economic risk, and xenophobia as a historian and medical sociologist, i have been studying the histories of international responses to epidemic events and what they can tell us about the nature of power, economics, and geopolitics. a historical understanding of the international regulations for containing the spread of infectious diseases reveals a particular focus on controls that have protected north american and european interests. in the past months, there have been xenophobic attacks on people of asian descent connected to coronavirus disease and precipitous losses in global stock exchanges and risk of recession. most reports have treated these as separate phenomena: considering one to be a cultural consequence of epidemic fears run rampant and the other to be the impact of the pandemic on global trade. yet if one pauses to consider the history of the global management of pandemic disease threats, epidemics and global commerce have been inextricably related. part of this history is the role of xenophobic responses to infectious disease threats. the xenophobia that has occurred in relation to the covid- pandemic can be situated in a longer history that dates back to th-century epidemics and the first international conventions on controlling the spread of infectious diseases. while quarantine, cordon sanitaire, and other social distancing practices date back to th-century europe and earlier, by the th century the spread of epidemic diseases emerged as a problem that required an international, coordinated response. european colonial expansion brought smallpox and other diseases to the americas and africa from the time of columbus to the s. these epidemics wrought widespread devastation for indigenous peoples. simultaneously, europeans encountered new diseases in the tropics. colonisation brought a particular encounter with diseases capable of harming europeans. the napoleonic wars were global in nature and also revealed the vulnerability of european powers to diseases emerging from their colonial domains, and the capacity of these diseases to emerge in europe. by the end of the th century, however, the preexisting forms of ad-hoc and uncoordinated quarantine of ships at port by european powers was being tested, especially in the mediterranean. epidemics of plague and cholera that would claim hundreds of thousands of lives in europe-while claiming far more in india and elsewhere-became a concern. but quarantines were costly, and were also an effective tactic for imposing trade tariffs and enacting trade wars under the guise of public health. a new system was needed to better manage the spread of infectious disease. from to , conferences were held to standardise international regulations for the establishment of quarantine and the sanitary management of plague, cholera, and yellow fever. in , the first international sanitary conventions were adopted, codifying the first agreements for the prevention of the international spread of infectious diseases. these conventions aimed to maximise protection from disease with minimum effects on trade and travel. plague, cholera, and yellow fever, became the focus of massive international concern due to their threat to continental europe and the economic threats the diseases posed to global trade. the early international sanitary conventions did not police the spread of these three diseases from europe to other countries or focus on any diseases endemic to europe. the threat of diseases emerging from colonial sites that could disturb systems of trade and travel led to aggressive control of these diseases in sites of epidemic outbreak and aggressive scrutiny of those people deemed to be responsible for disease spread. the importance of colonial trade from asia led to the rise of a particular scrutiny and bias against people of asian descent-especially chinese migrants and indian muslims travelling around the world. in the eyes of colonial health officials and the drafters of the first international sanitary conventions, the spread of cholera and plague was an economic, epidemic, and political risk to the long-term stability of the global economy. the particular anxieties over the threat of plague being spread by the free travel of colonised populations drove the colonial administrators in ceylon (now sri lanka) to prophesise the potential collapse of the tea industry-and by extension their entire colony. because trade with europe was so crucial to the colony, in the late th century the colonial administrators endeavoured to sacrifice all trade with india rather than risk the threat of plague arriving with migrant workers from the subcontinent. in one letter between colonial administrators, it was suggested, in a derogatory way, that if even a single person from india or east asia entered ceylon without being exposed to sanitary surveillance "there would have been great peril to the colony for these coolies being free immediately on landing (in ceylon) to spread over the island would scatter the seeds of disease as they went". such xenophobic sentiments were shared elsewhere. the heightened scrutiny and bias against non-europeans who were blamed for spreading disease have historically resulted in aggressive racist and xenophobic responses carried out in the name of health controls. in in cape town, south africa, an epidemic of bubonic plague resulted in the quarantine and forced removal of most of the city's black african population to a racially segregated quarantine camp. this camp and practice of eviction can be viewed as part of the blueprint for future forced removals and a precursor to racially segregated south african townships before and during apartheid. similar scrutiny was a feature of the policing of the hajj. under the international sanitary conventions from to , muslim pilgrims travelling from india were perceived in europe as a threat because of their potential to meet and spread disease to european muslims during the hajj, who would then return to europe by passage through the suez canal. quarantines and controls were enacted for muslims pilgrims who travelled both from india to mecca and back to europe after the pilgrimage. the disease surveillance and sanitary system that governed the hajj has historically been one of the largest of its kind in the world. concerns about the economic risks of disease spread were not limited to european empires, and neither were the xenophobic practices associated with those concerns. the usa has a history of anti-chinese sentiment in response to epidemics. historian james mohr has described how in honolulu, doctors, colonial administrators, and the general us colonial population lamented the outbreak of bubonic plague in because it prompted fears that the city would become associated with asia, where plague was then present. as plague spread in honolulu and countries around the world closed their borders or quarantined all vessels arriving from its port, the honolulu city administrators embarked on a full quarantine of the city's chinatown, allowing no one to leave. these quarantines imposed considerable hardships on those within, limiting employment, movement, and access to supplies. the area of quarantine encompassed chinese and non-us properties immediately near the harbour, but avoided buildings and businesses that were owned by white americans and immediately connected to sites of quarantine. ultimately, the public health authorities burned contaminated buildings, but fires spread beyond their control and consumed most of chinatown in flames. similar anti-chinese responses occurred in san francisco during the plague epidemic of - , when chinese-specific quarantines were enacted. my own research suggests that the concern for the trading relationships central to us economic growth were pivotal to us congress endorsing the creation of who. in a report accompanying the resolution that ultimately heralded us support for who, it stated that: "particularly in our shrinking world, the spread of disease via airplane or other swift transport across national boundaries gives rise to ever present danger. thus to protect ourselves that we must help wipe out disease everywhere…the records of our export trade show that countries with relatively high living standards buy most of our goods. if the rest of the world continues in illhealth and abject poverty our own economy will suffer." in , the un and world health assembly transferred responsibility for the international sanitary conventions to who in its charter. the international sanitary conventions were reformed and ultimately renamed under who to the international health regulations in , which were revised to their current form in . more recently, nations have aligned infectious disease control policy alongside concerns for national security. in the current pandemic of covid- , we also see the links between epidemic risk, xenophobic responses, and the global economy. verbal and physical attacks on people of asian descent and descriptions of the disease as "the chinese virus" are all connected in this long legacy of associating epidemic disease threat and trade with the movement of asian peoples. we have seen huge sell-offs on asian stock markets and distinct drops in share prices in european and us financial markets. what was once an initial economic concern for global trade as it related to china has now had effects on all scales of the economy from small businesses to the fortune and potentially on a scale we have not seen since the worst financial crises of the th century. when we think about the framing of disease threats, we must recognise that the history of international infectious disease control has largely been shaped by a distinctly european perspective, prioritising epidemic threats that arose from colonial (or now post-colonial) sites that threatened to spread disease and affect trade. covid- is a serious and dangerous pandemic, but we must ask ourselves who our responses are designed to protect and who are they meant to vilify? in a pandemic, the best responses are those that protect all members of the population. a eurocentric or us-centric view that excludes or stereotypes others will do much more harm than good. as the epicentre of the epidemic shifts for now to europe and the usa and as global responses intensify, we should be prepared for more economic risk and confront racist or xenophobic responses for what they are-bigoted opinions with no basis in public health or facts. center for medical humanities and social medicine, johns hopkins university and johns hopkins school of medicine, baltimore, md - , usa alexandrewhite@jhu.edu plague and fire: battling black death and the burning of honolulu's chinatown contagion: how commerce has spread disease global risks, divergent pandemics: contrasting responses to bubonic plague and smallpox in cape town security, disease, commerce: ideologies of postcolonial global health the evolution, etiology and eventualities of the global health security regime two regimes of global health the politics of securing borders and the identities of disease campbell l. chinese in uk report "shocking" levels of racism after coronavirus outbreak. the guardian, feb , tavernise s, oppel jr ra. spit on, yelled at, attacked: chinese-americans fear for their safety.the key: cord- -i jh bcn authors: zanetti, a. r.; zappa, a. title: emerging and re‐emerging infections at the turn of the millennium date: - - journal: haemophilia doi: . /j. - . . .x sha: doc_id: cord_uid: i jh bcn summary. after world war ii, mankind believed that infectious diseases were on the way to being defeated. unfortunately, they still are the second worldwide cause of death. globalization changes promote the emergence of new infections and pandemics; international deliveries and travelling facilitate the dissemination of infectious agents; man‐induced environmental changes create new opportunities for contacts between species, leading to infections in aberrant hosts, including man; global warming enables insects, a major vector of pathogens, to thrive in more countries. the main pandemics have been caused by viruses, such as hiv and novel subtypes of influenza viruses. in addition, prion proteins are a threat. the transmission of the creutzfeld jakob disease variant through blood transfusion and the recent discovery of prion protein in the spleen of a haemophilia patient are a matter of further concern. the end of the war against infectious diseases is not in sight. mankind’s battle with pathogens has lasted millennia and is destined to continue. throughout history man has periodically been the victim of epidemics: we have historical reports that vividly report these calamities, such as 'the plague of justininan' [ ] dating back to the roman empire, 'the black death' in the middle ages [ ] , the 'spanish flu' in [ ] , the emergence of acquired immunodeficiency syndrome (aids) in the early s and the more recent severe acute respiratory syndrome (sars) at the beginning of the xxi century [ ] . after world war ii, the extensive knowledge on infectious agents and their hosts, environmental factors involved in their transmission, improvements in hygiene, healthcare and socioeconomic status and, last but not least, the discovery of effective antimicrobial therapy and the availability of vaccines, lulled man into believing that 'the war against infectious diseases had been won' and that epidemics were a phenomenon of the past [ , ] . nothing could have been further from the truth. at the beginning of the third millennium, infectious diseases still are the second cause of death in the world (the first in developing countries). fifteen million humans die every year of infectious diseases, the five 'big killers' being aids, tuberculosis, malaria, respiratory infections and infantile diarrhoea [ , ] . in the last decades, a number of new pathogens responsible for emerging infectious diseases, such as avian and swine flu, aids, sars, west nile, ebola and variant of creutzfeldt-jacob disease (vcjd) have been identified and other infectious diseases are re-emerging after a period of quiescence, such as malaria and tuberculosis caused by multidrug resistant strains [ , , , , ] . the picture is compounded by the so-called 'deliberately emerging' pathogens, such as anthrax and smallpox -biological weapons that humans might plan to spread deliberately as an act of bioterrorism [ , , ] . in the world of globalization, regular exchanges of enormous amounts of goods throughout the world and low-cost frequent travelling, whereby hundred thousands of people move from one side of the earth to the other within a matter of hours, facilitate the dissemination of infectious agents (microbial traffic). infectious diseases that once upon a time would have remained confined to their ecological niche can now potentially spread rapidly to every corner of the earth before preventive measures can be implemented [ , ] . what is more, a number of other factors promote not only the dissemination but also the emergence of new infectious diseases: intensive farming and breeding associated with crowding promote the development of foci of infection; global warming has modified the climate, making insects, a major vector of pathogens, able to thrive in countries where the climate was previously hostile; the exploitation of natural resources has produced environmental changes that create opportunities for new contacts between species leading to emergence of infections in new hosts. the ability of an infectious agent to mutate and to jump the barrier species from animals to humans contributes to the emergence of zoonotic diseases. indeed, most of the outbreaks in the last years have been caused by infectious agents normally hosted by wild animals (reservoir hosts). following the loss of their normal habitat (e.g., climate changes, ecological changes in land use), these animals can move closer to farms where they infect livestock (spillover hosts). in parts of the world characterized by crowding and low hygiene standards, the livestock then infect man. in some cases, man results to be a 'dead-end' host (aberrant host), who does not transmit the infection any further. for instance, in the s, an unexplained epidemic of acute cardiopulmonary syndrome occurred unexpectedly in a navajo tribe living on the border of the four corners region (new mexico, arizona, colorado, utah) of the southern-western united states [ ] . this potentially deadly disease caused by an unknown virus (sin nombre virus or 'nameless virus') subsequently identified as a member of the hantavirus genus (bunyaviridae family) is transmitted from mice to humans by inhalation of aerosolized virus-contaminated rodent excreta (saliva, urine, droppings). during the navajo outbreaks, the wet and mild conditions as a result of changes in climate caused by el niñ o favoured an abundance of mice food allowing a significant increase in the deer mouse population (peromyscus mariculatus), the natural reservoir of this virus [ ] . the migration of infected rodents near human settlements created the ideal conditions for the outbreak onset. the deforestation that occurred in australia and malaysia in the late s forced fruit bats (flying foxes), the reservoir of nipah and hendra viruses (henipavirus genus, paramyxoviridae family), to move closer to human dwellings creating the conditions for the emergence of focal outbreaks of encephalitis with high fatality rate in humans [ ] . in this case, viruses were transmitted from bats to livestock animals (pigs and horses) and from these to humans. in other cases, however, zoonotic transmission is followed by viral adaptation to the new host that makes human-to-human transmission feasible. this is the case of several mutated viruses [ , , ] . most of the emerging infections are caused by viruses, which, according to nobel prize winner peter medawar, are 'bad news inside a protein envelope'. the 'bad' news consists of strands of dna or rna that are able to penetrate mammalian cells, obliging them to reproduce the viral components instead of their own. the bad news can get even worse as viruses adapt continually to the environment by mutation, recombination or gene reassortment [ , ] . the two key features of intracellular replication and genetic evolution make viral infections particularly difficult to combat: currently available anti-viral agents block viral replication by various mechanisms, but hardly remove dormant viruses that have already penetrated inside host cells and the protection conferred by vaccination can be made fruitless by genetic evolution of the pathogen, which may enable it to escape previously induced immunity. an example that shows why zoonotic transmission must be avoided is the infection by hiv. its natural reservoir is a non-human primate, called pan troglodytes troglodytes, in which it is fairly harmless. man became probably infected through contacts with infected blood during hunting and slaughtering processes ('bush meat', hunter's cut) about - years ago [ ] . the infection remained confined to areas of africa up to about years ago when international travelling and exchanges became more widespread. infected subjects moved from africa to haiti; from there, the infection then spread, as a result of sex tourism, first to gay communities in cities of north america (los angeles, san francisco, new york) and then to europe. at first, it spread in high-risk groups, such as male homosexuals, intravenous drug abusers and recipients of multiple transfusions; then, it extended to the general population. the outcome is the current pandemic: in , who estimated that million adults and children were living with hiv infection/aids worldwide, that more than million people have died of the disease so far and that the number of deaths in the last year amounted to million [ ] . another well known example of recent worldwide pandemic is the sars. this emerging airborne viral disease had a great impact on account of its rapid international spread under favourable conditions created by our highly mobile, closely interconnected world. the origin of sars was in areas of dense population in southern china, where humans and animals are packed side by side providing ideal conditions for a new virus to emerge by jumping across species. the natural reservoir of sars virus (sars-cov) is the horseshoe bat, the spillover host is the masked palm civet or raccoon dog; man is an aberrant host [ ] . the pandemic originated in guangdong, in southern china, where families often cohabit with the animals they rear. the promiscuity enabled the sars-cov to skip across species and find a new host -man. it has been documented how the first identified infected human (index case) unintentionally infected another humans in the metropole hotel in hong kong, who then travelled to five different countries (germany, canada, thailand, vietnam and singapore), giving rise to new foci of infection. this ultimately led to a worldwide epidemic affecting more than people, with a death rate of . % [ ] . this event showed how modern transport systems can enable an emerging infection in one part of the earth to spread rapidly throughout the globe. however, it also showed how international healthcare co-operation can achieve control of a rising pandemic within less than a year. however, not all viruses behave this way. some viruses adopt the so called 'hit and run' strategy as they cause recurrent, geographically confined, usually self-limiting outbreaks. this is the case of ebola and marburg viruses, both members of the family filoviridae, causing severe and often fatal haemorrhagic fevers in humans and non-human primates. in , transmission of ebola virus from its natural reservoir, which is still unknown, to man resulted in two simultaneous but separate, explosive outbreaks of human disease caused by two distinct virus subtypes in zaire and sudan with a case-fatality rate of approximately % and % respectively. the disease was self-limited both in space and time: it remained confined to well-defined geographical areas and mysteriously resolved [ ] . since then, recurrent outbreaks have been reported in african countries including sudan, gabon, the democratic republic of congo, republic of congo and uganda. a variant of the african ebola virus, the reston virus, was first identified in in reston, virginia, usa and in in italy (siena) in colonies of cynomolgus macaques imported from the philippines [ ] . despite its high pathogenicity in non-human primates, this virus does not seem to cause disease in humans. the infection by the marburg virus followed a similar course: in , german laboratory workers were infected by african green monkeys belonging to the cercopithecus aethiops species imported from uganda and developed fatal haemorrhagic fever [ , ] . west-nile, dengue and chikungunya viruses: vector-borne throughout the world arthropod-borne viruses are very common (more than ) and more than cause diseases in humans in more than countries world-wide, with a disease burden as high as a hundred million cases every year. ecological factors, such as new routings of long-distance bird migrations and the increased long-distance air travel facilitate the movement of infected persons, livestock and exotic arthropod vectors around the world contributing to the spread of vector-borne diseases from the original niches to new geographical areas. changing in public health policy, insecticide and drug resistance are additional factors which can contribute to the emergence or resurgence of vector-borne infectious diseases [ ] . an example is the west nile virus (wnv) infection, originally confined to africa (along the nile), southern europe, parts of the middle east and india, which has recently modified its epidemiological picture. in , wnv was firstly imported into the new york area and, thanks to the presence of specific vectors (culex mosquito) and natural susceptible hosts (crows, jays), the infection rapidly spread coast to coast. man is a dead-end incidental host: it has now been estimated that - million people have been infected by wnv in north america. west nile (wn) is a potentially serious illness whose symptoms may range from mild to severe. in about % of cases, infection is asymptomatic and asymptomatic carriers may be infectious via blood transfusion or through organ and tissues donations [ ] . in , there were documented cases of wnv transfusion-transmitted infections, which induced the usa blood collection agencies (bcas) to implement blood screening for this virus [ ] . from june to september , several hundred potentially infectious components were identified by the tests. in the last years, outbreaks of wnv have been reported in some european countries both in horses and humans. in september-october , the first two cases of wnv neuroinvasive disease have been reported in northern italy [ ] . to prevent infection through blood donations by infected donors during the viremic peak, screening programmes have been introduced during the seasonal mosquito activity in the affected area. other examples of vector-borne diseases that have extended elsewhere in view of the climate changes and global warming is the re-emergence of dengue virus (dv) infection transmitted by aedes aegypti mosquito in tropical and subtropical countries. the susceptibility of aedes albopictus also known as 'tiger' mosquito (a mosquito of asian origin currently widespread in countries with a temperate climate) to serve as a new vector of dv gives new opportunities to the virus to extend its area of spread. the tiger mosquito which is the vector of chikungunya virus (chikv), a virus that caused several epidemics in africa, southeast asia, the western pacific and india, was first introduced in italy in the early s by importing used tyres. in , the chikv transmitted by this mosquito caused the first autochthonous epidemic outbreak in europe, in the north-east of italy (emilia romagna region) which involved more than cases [ , ] . this prompted the national blood centre and regional health authorities to take specific precautionary measures to ensure the safety of blood supply. influenza a is an airborne virus that repeatedly produces epidemics every year during the cold season. its hosts include birds (natural reservoir) and a number of mammals, including whales, seals, horses, pigs and man. it is particularly able to adapt to new hosts, thanks to its unique capacity for genetic variation: its surface proteins are able to mutate up to % of their amino acid sequence without losing their capacity to infect mammalian cells and its genome is made of eight rna segments that are genetically independent and that can therefore randomly reassort, forming antigenically novel subtypes [ ] . such novel strains have caused a number of dangerous pandemics in the past, such as the spanish flu in , asian flu in and hong-kong flu in . currently, avian flu is a real threat caused by mutated strain h n , which continues to spread in humans. the cumulative number of human cases of avian influenza h n reported by who by august amounted to cases and deaths [ ] . the issue is whether this virus or other avian viruses (h n , h n ) will cause a novel pandemic or not [ ] . to cause a pandemic, three characteristics are required: (i) the ability of the virus to replicate in a human host, (ii) the population has to be susceptible to it, and (iii) human-to-human transmission has to be possible. at present, for h n , the first two features are satisfied, but not the third. however, it cannot be excluded that it will develop the third feature in the future. on the contrary, human-to-human transmission has been proven for the novel swine flu caused by the influenza a (h n ) virus (s-oiv) isolated for the first time in march-april in mexico [ ] . this novel strain is a cause for concern as it has resulted from the unique reassortment of genetic elements of influenza viruses that normally infect three different kinds of host -swine, birds and human beings. the study of its structure has revealed that it differs considerably from its predecessors, isolated in human beings: its h antigen, which enables it to bind and infect cells of different species, has changed in terms of amino acid sequence alignment by approximately % and its n antigen, which enables it to digest sialic acids (neuraminidase activity), ensuring better access and spread in the organism, has changed by about %. this means that exposure to its predecessors may not provide any immunity to this unique strain and the whole population on earth is theoretically at risk [ ] . indeed, in april , who declared a phase- alert indicating that a pandemic was imminent and that it was urgent to finalize the organization, communication and implementation of the pandemic preparedness plans. on june , who then announced the beginning of a flu pandemic with a total of cases worldwide and deaths. only one month later, on july , , who reported that the number of infections had increased up to nearly with deaths. laboratory-confirmed cases as officially reported to who as of august amount to with deaths. given that countries are no longer to test and report individuals, we can estimate that the number of cases reported actually understates the true number of cases. at the time of writing (mid-august ), who considers the pandemic to be moderate. the speed at which the infection is spreading is such that infection of millions of people worldwide is considered to be inevitable. however, symptoms are mild and most people recover from the infection without the need for hospitalization or medical care. the rate of severe infections is currently similar to what is generally seen during local seasonal influenza. however, monitoring is essential as it is not possible to predict how the pandemic will evolve. h n might disappear spontaneously, cause a mild pandemic or a severe pandemic after virus mutation/re-assortment. concerning blood transfusions, the risk of direct transmission of influenza via blood or blood products is extremely low. the major issue will be a temporary loss of blood donors during the pandemic peak, with impact on blood supply, as donors may not attend blood collection services on account of illness. an atypical infectious agent is the prion protein, a conformational variant of a naturally occurring protein that cannot be removed by normal degradation processes and accumulates forming fibrous aggregates that are believed to be involved in the pathogenesis of the disease [ ] . the new prion disease that emerged in man in the uk in was a food-borne infection caused by modern intensive meat production based on 'industrial cannibalization': a prion disease that has been known to affect sheep and goats for more than years (scrapies) had been transmitted to cattle via inadequately sterilized fodder of animal origin, giving rise to an epidemic of bovine spongiform encephalopathy (bse), also called 'mad cow disease', which in turn was then transmitted to man via meat consumption. prion-infected humans develop a variant of classic sporadic creutzfeld-jakob disease (vcjd), an invariably fatal neurodegenerative disease. it is not known what proportion of prion-infected humans develop symptomatic disease. also, the mean latency period from prion infection to the development of overt disease remains unknown. over the period - , a total of cases were reported, nearly all in the uk and attributed to dietary exposure. possible transmission of vcjd by blood transfusion has been documented in the uk [ ] . the emergence of prion disease has led to a reduction in the number of donors as people with a history of visiting the uk during the cattle bse epidemic are excluded. recently, the prion protein has been found in the spleen during the postmortem examination of a year-old patient suffering from haemophilia who died of causes unrelated to vcjd. indeed, this patient did not have any neurological symptoms before death [ ] . of some concern is the fact that in , this patient received a batch of factor viii manufactured using plasma from a donor who developed symptoms of vcjv months after donation. although the mode of transmission has not been confirmed, the uk health protection agency together with the haemophilia centre doctor's organization alerted haemophiliacs about this finding. a related alert had been made a few years earlier: in patients with bleeding disorders who had been treated with plasma-derivatives made with blood collected in uk between and were told that they were potentially at risk of vcjd and excluded from donating organs. however, although patients exposed to contaminated products in the past may be at risk for vcjd, this does not mean that currently available plasma-derived products (manufactured with uk free plasma) still carry such risk. [ ] . an important treatment option for patients with bleeding disorder is the administration of recombinant factors viii, which is not derived from plasma and undergoes several processing steps to remove any contaminating viruses and prions [ ] . the development of new antimicrobial drugs and antibiotics together with the availability of safe and effective vaccines led to a spirit of optimism towards the definitive control and prevention of several infectious diseases. man may have won some battles against infectious diseases, but the war is still raging and the end is not in sight. in general, there is no way to predict when or where the next pathogens will emerge. emerging and resurging diseases are common in areas rich in wild animals and where natural and social environmental factors, including man's activities, increase the opportunities for the pathogens to extend their ecological niches and to be transferred to and from humans and other animals. the continual emerging of new 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further challenge to blood system infection with chikungunya virus in italy: an outbreak in a temperate region the chikungunya epidemic in italy and its repercussion on the blood system influenza virus: a master of metamorphosis world health organization. cumulative number of confirmed human cases of avian influenza a/(h n ) reported to who avian influenza virus (h n ): a threat to human health outbreak of swine-origin influenza a (hinl) virus infection -mexico towards a sane and rational approach to management of influenza h n transmissible spongiform encephalopathies: the story of a pathogenic protein possible transmission of variant creutzfeldt-jacob disease by blood transfusion preclinical vcjd after blood transfusion in a prnp codon heterozygous patient signs of variant creutzfeldt-jakob disease in a patient with hemophilia: fviii concentrates most likely cause pathogen safety of manufacturing processes for biological products: special emphasis on kogenate bayer the authors stated that they had no interests which might be perceived as posing a conflict or bias. key: cord- - ugdxbmy authors: laskar, rezwanuzzaman; ali, safdar title: mutational analysis and assessment of its impact on proteins of sars-cov- genomes from india date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ugdxbmy the ongoing global pandemic of sars-cov- implies a corresponding accumulation of mutations. herein the mutational status of genomes from india along with their impact on proteins was ascertained. after excluding gaps and ambiguous sequences, a total of variable sites ( parsimony informative and singleton) were observed. the most prevalent reference nucleotide was c ( ) and substituted one was t ( ). nsp had the highest incidence of sites followed by s protein ( sites), nsp b ( sites) and orf a ( sites). the average number of mutations per sample for males and females was . and . respectively suggesting a higher contribution of mutations from females. non-uniform geographical distribution of mutations implied by odisha ( samples, mutations) and tamil nadu ( samples, mutations) suggests that sequences in some regions are mutating faster than others. there were mutations ( ‘neutral’ and ‘disease’) affecting amino acid sequence. nsp has a maximum of ‘disease’ variants followed by s protein and orf a with each. further, constitution of ‘disease’ mutations in genomes from asymptomatic people was mere % but those from deceased patients was over three folds higher at % indicating contribution of these mutations to the pathophysiology of the sars-cov- . the ongoing covid- global pandemic began from wuhan, china and has devastated millions of lives, economies and even nations as a whole. the first reported case was in created a scare as they had a relatively higher mortality rate. however, sars-cov- is by far the most contagious one [ ] [ ] [ ] [ ] . the higher incidence of viral infections would imply a faster evolution process for sars-cov- [ ] . this is so because more the virus replicates, higher are the chances of it accumulating mutations with the possibility of it leading to altered dynamics of its virulence, pathogenesis and interactions with host. the changes may not be necessarily favoring the virus; however, the unpredictability demands caution. the sars-cov- genome encodes for non-structural proteins in addition to the replicase polyprotein, the spike (s) glycoprotein, envelope (e), membrane (m), nucleocapsid (n) and other accessory proteins [ ] . the impact of mutations in all the regions of the genome needs to be assessed to understand viral evolution. with a definitive possibility of india becoming the most affected country by sars-cov- in near future and the demographic burden involved, its pertinent to be analyze the accumulating variations in the genome accounting for possible changes in protein and their potential to alter the virus in any manner. on th june, we retrieved fasta sequence congregations from india along their rational meta data from gisaid epicov server to construct the phylo-geo-network and analyze the haplogroups along with their geographical distribution across different states of india [ ] . herein we extend our study using the same congregation of sequences to analyze the nature and composition of the observed mutations and their impact on proteins of sars-cov- . gisaid epicov is an open access repository of genomic and epidemiologic information about novel corona viruses from across the world from wherein sequences were extracted and alignment performed as previously reported [ ] . briefly, fasta sequence congregations along their rational meta data from gisaid epicov (www.epicov.org) server. for mutational profile analysis with clinical correlation, we selected genomes of deceased patients from existing congregation. however, there were just two genomes for asymptomatic patients in the congregations. so, on . . , we downloaded fasta sequences with patient status from the same server and selected genomes from asymptomatic patients. as the data filter for genome extraction, we used hcov- as a virus name, human as a host, india as a location and complete sequence with high coverage. details of the asymptomatic samples are given in supplementary file . the sequences thus extracted were analyzed with nc_ . from wuhan, china as reference. mega(v. ) is a multithreaded tool for molecular and evolutionary analysis. multiple sequence alignment (msa) of the extracted sequences [ ] was initially visualized by this software then the variable sites are exported into spreadsheets with or without missing/ambiguous and gap sites along their respective positions [ ] . using this software, we estimate the mcl (maximum composite likelihood) nucleotide substitution pattern and tajima's neutrality test to understand transition transversion bias and nucleotide diversity [ , ] . piro iglsf is a matlab-based simulation software, we used this for the identify the location of mutated nucleotide position on specific gene [ ] . coronavirus typing tool of genome detective (v. . ) and covid- genome annotator of coronapp are webtools for analysis of protein and nucleotide mutation [ , ] . we used these tools for annotation, identification and classification of mutated protein followed by verification and validation of the positions with the mutated nucleotide sites by the output of mega. the nucleotide similarity percentage was validated by ncbi blast (blast.ncbi.nlm.nih.gov) to investigate the sequence diversity. sift, provean and ws-snps&go are the prediction tools which report positive or negative impact of variants on protein phenotype. the assessments are focused upon scores using several algorithms. it is expected that a sift score of < . is diseased ("affect protein function"), and that > . is neutral ("tolerated"). this is stated that a provean score of < − . is diseased ("deleterious"), and > − . is neutral. ws-snps&go 's phd-snp method is estimated to be > . mutation in the probability of disease, and < . is neutral [ ] [ ] [ ] . composition and distribution of variable sites (table ) and its negative value indicated the significance of these variable sites. however, excluding the gaps and ambiguous sequences reduced this percentage cover to . % encompassing variable sites which we have used for subsequent analyses reported in this study. this included parsimony informative (pi) sites and singleton sites (snp: single nucleotide polymorphism). the pi sites are those whose incidence was observed in multiple samples whereas singleton sites had a restricted single sample incidence. the distribution of these sites according to various substitutions, protein localizations and impact therein has been summarized in figure , supplementary file . as evident therein, c→t ( sites) forms the most prevalent mutation in both pi and singleton sites and g→t ( sites) comes a distant second. the common aspect of two most prevalent mutations is "t" being the substituted nucleotide. further, there were two multi-variable (mv) sites each in pi and singleton category wherein two separate mutations were observed at the same site in different samples. the details of observed mv sites have been summarized in table . the distribution of the variable sites across proteins of sars-cov- in a non-uniform manner is reflective of the differential contributions of proteins in evolution. as per our data, nsp had the maximum of variable sites followed by s protein ( sites), nsp b ( sites) and orf a ( sites) ( figure ; supplementary file ). these four proteins account for over half of the total variable sites of the genome and may be considered as drivers of genomic evolution for sars-cov- . the mutations of s protein have been the focus for multiple research groups owing to its plausible impact on viral entry to the host cell but the mutations elsewhere may be equally relevant as the viral genome is known to harbor only what's essential [ ] [ ] [ ] . we believe a holistic approach is required to understand the evolution as more often than not the selection advantage being offered by any mutation is a chance event and can be from any part of the genome. in terms of the impact of these variable sites on amino acid sequence of the viral proteins we classified them into four categories. first, the sites located in the extragenic region and hence no influence on the coding proteins. there were such variable sites localized to the utr regions ( in 'utr and in 'utr). secondly, snp-silent included those variable sites wherein the nucleotide change was leaving the amino acid sequenced unaltered. a total of such sites were distributed across the genome. thirdly, the variable sites which were leading to the introduction of a stop codon were referred to as snp-stop and there were such sites in our study. lastly, the variable sites which were affecting the protein sequence are referred as snp in the study and there were such sites (supplementary file ). the prevalence and distribution of these sites has been summarized in figure and results of the prediction of their impact on protein has been discussed later. in order to understand the underlying dynamics of substitutions, we performed the maximum composite likelihood estimate of nucleotide substitution as shown in table we thereon looked at these variations in combination with their prevalence across samples. the most prevalent nucleotide at the variable sites in reference sequence was c ( ) followed by g ( ) whereas t was by far the predominantly substituted nucleotide ( , %). also, the other three nucleotides had an almost equal representation in substitutions (a- , g- , t- ). this biased prevalence was not restricted to the alignment but was also getting translated to population incidence. there was a total of mutations with c as reference nucleotide and mutations with t as substituted nucleotide across studied genomes. the composition of variable sites, their substitutions and prevalence across samples has been summarized in figure and supplementary file . evidently, any particular mutation may be incident across multiple samples and a single sample can harbor multiple mutations. a cumulative number for the same has been referred to as "sum of mutation incidence" herein and thereafter in this study. we subsequently analyzed the patient's dataset with reference to age and gender for the incidence of mutations. however, since patients' data wasn't cumulatively available, the data for this aspect isn't exhaustive but representative for samples ( females and males). the patients whose genomes were used in the study and age was known were classified into seven categories from infancy to over years. the maximum number of patients for both males and females belonged to mature adulthood category of to years with and samples respectively (figure , supplementary file ) . this adheres to the fact that the older population is at a greater risk for infection owing to a possibly weaker immune system and other physiological conditions. the simple question of whether or not age and gender are associated with accumulation of genome variations has a not so simple answer. the overall average number of mutations per sample was . and the corresponding values for males and females separately was . and . respectively. thus, women were contributing more to the mutational accumulation as compared to males. the individual mutational load for different age groups in males and females has been represented in figure . evidently, women are contributing more to the mutational load except for three age groups; - years, - years and - years. the highest difference on the basis of gender is for - years ( . ) but since there was just one female sample in that age group, it can't be emphasized much in isolation but the overall pattern does seem relevant. this is more so because, in terms of incidence, males are almost . times of the females but in terms of variations, fewer females are contributing more to the mutational load. possibly, the virus is behaving differently depending on gender. the mutational distribution across different states of india was subsequently ascertained. generally speaking, more the virus replicates more should be the accumulated variations. the fact that the samples used in the study aren't uniformly distributed across states provides for an intriguing template for analysis. the number of samples and the mutations therein for different states has been summarized in figure however, we can surely say that some sequences are mutating more than the others but whether the geographical location is playing a role needs to be ascertained. a total of snps which were present which were altering the amino acid sequence. their details and positions have been summarized in table and supplementary file . we also ascertained the prevalence of these variants across samples. the most incident variant q h localized to orf a was present in samples followed by a d in nsp present in samples. amongst the silent snps, y y in m protein was present in samples followed by d d in s protein with incidences. the overall data for variants present in genomes or more has been summarized in figure a . conversely, we also assessed the accumulation of variations in a given genome as summarized in figure b . interestingly, one sample (genome id ) had highest incidence of mutations while samples harbored just a single mutation. there were samples with no mutations and with more than one mutation. to account for these, the sum of mutation incidence has been used in this study as explained above. the impact of mutations on proteins was predicted through three different tools; sift, provean, ws-snps&go; which classified the mutations as "neutral/tolerated" or "disease/affect protein function/deleterious". for the sake of simplicity, we have referred the results from all sites as neutral and disease. though the prediction outcomes of the three tools were not in sync for all sites but since the classification of outcomes were on similar lines, the results can be represented in a binary manner with four categories. first two categories represent wherein the three tools have the same prediction; either all predicting a site to be "neutral" or "disease". the other two categories represent deviation between prediction outcomes. they are "disease by one, neutral by two" and "disease by two and neutral by one". for comparison between variants, any mutation predicted as disease by two or three tools are considered as disease and mutations predicted as neutral by two or three tools are considered as neutral. the distribution of disease and neutral variants across the different genes of sars-cov- has been shown in table and supplementary file . these could be analyzed in three aspects. first, in terms of overall incidence. the maximum variants affecting protein sequence were present in nsp ( ) followed by spike (s) protein with variants. secondly, if we focus only on variants with predicted outcome as "disease" then nsp has a maximum of such variants followed by s protein and orf a with variants each. thirdly, we looked at those proteins which had more disease variants as compared to neutral. there were five such proteins namely: nsp , nsp , nsp , orf a, e, orf a. of these nsp had just two variants and both of them were predicted as disease by all three tools. others had differential bias towards disease variants. thus, we can say that though some regions of the genome have more variations but mostly neutral while others with fewer variations are more impactful in terms of their predicted impact due to more disease variants. conversely, mutations in some proteins can be relatively better tolerated by the viral genome. the overall protein prediction outcomes of the genomes have been summarized in figure . there were total of mutations ( %) and mutations ( %) which are predicted to be neutral and disease respectively by at least two tools. these predictions suggest that even though mutations are accumulating in sars-cov- , they are predominantly neutral. this is the possible reason that no major virulence or physiological deviations have been observed so far. in order to further assess impact of these variations we compared their prevalence across samples which were asymptomatic with those wherein the patient died. the idea was that if predictions are true, then asymptomatic samples should have more of neutral mutations whereas deceased ones should have more of disease mutations. the present congregation of samples in the study had just asymptomatic samples and deceased. thereon, we included mutations with those of deceased samples. their comparative data has been shown in table . the p value therein represents the probability that a given variant chosen at random to be neutral or disease. taking the threshold as common prediction by at least two tools the data gives interesting insights. as shown and previously mentioned, for the original congregation of samples, % mutations were neutral (p value . ) and % were disease the mutational accumulation in sars-cov- genomes is a multifactorial event with some areas of genome more prone to mutations, selective mutations being more prevalent, nonlinear assimilation of mutations across various states and differential correlation between mutational impact on proteins and physiological state. age and gender specific bias in incidence of mutations was observed. the asymptomatic samples had higher occurrence of neutral variants while deceased samples had relatively higher incidence of disease variants. a cross-linking of mutational dynamics and patient history will provide for better correlation and understanding of the variations in sars-cov- genomes. ; d= ) a t, t i, y c, e d, p s, g v, s i, p l, m i, h y, a t, s i, t k, t i, m i, v a, s g, t i, g s, t i, t a, t i, n t, k r, s f, k e, g e, p s, l f, a v, v i, t i, t i, l v, p l, h y, a v, s f, ; d= ) l f, n y, e d, a s, s f, g s, q r, t i, t i, e q, a s, t i, e d, t i, v i, q h, a s, t s, g v, t i, a s, i k, s p, d y, a v h q, p l t i, f c, l p, t k, v f, d y, c f g c a t a v s f i n c f a s, t i, h y, v l, g v, k r, k n, g v, q k orf a (n= ; d= ) v l, g v, s a, v f, t i l f, s f, s l, t i i t, l f, t i i t, l f, l f, q h severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a major outbreak of severe acute respiratory syndrome in hong kong a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster epitope-based chimeric peptide vaccine design against s, m and e proteins of sars-cov- etiologic agent of global pandemic covid- : an in silico approach identification of a novel coronavirus causing severe pneumonia in human: a descriptive study phylo-geo-network and haplogroup analysis of novel coronavirus (ncov- ) genomes from india molecular evolutionary genetics analysis across computing platforms statistical method for testing the neutral mutation hypothesis by dna polymorphism prospects for inferring very large phylogenies by using the neighbor-joining method microsatellite diversity, complexity, and host range of mycobacteriophage genomes of the siphoviridae family identification from high-throughput sequencing data coronapp : a web application to annotate and monitor sars-cov- mutations sift web server: predicting effects of amino acid substitutions on proteins predicting the functional effect of amino acid substitutions and indels : predicting stability changes upon mutation from the protein sequence or structure missense mutations in sars-cov genomes from indian patients geographic and genomic distribution of sars-cov- mutations mechanisms of viral mutation the authors thank the department of biological sciences, aliah university, kolkata, india for all the financial and infrastructural support provided. authors acknowledge all the authors associated with originating and submitting laboratories of the sequences from gisaid's epiflu™ (www.gisaid.org) database on which this research is based. the authors declare they have no competing interests. not applicable. all data pertaining to the study has been provided as supplementary material of the manuscript. rl: methodology, investigation, formal analysis and validation sa: conceptualization, supervision and writing. key: cord- -yf kt e authors: mejia, rojelio; hotez, peter; bottazzi, maria elena title: global covid- efforts as the platform to achieving the sustainable development goals date: - - journal: curr trop med rep doi: . /s - - -y sha: doc_id: cord_uid: yf kt e purpose of review: in this commentary, we summarize and put into perspective the recent information that highlights the associations between coronavirus disease and poverty. we also bring attention to another dimension that will most likely exacerbate the severity and long-term sequelae of covid- in impoverished populations, that is, the comorbidities and the presence of tropical infections. recent findings: during this first half of , the covid- pandemic has emerged as a poverty-related neglected disease on at least two fronts. first, is its significant impact in low-income neighborhoods in the usa, the epicenter of the pandemic. second, is its emergence in poor urban areas of south america, and now in asia and africa. in both fronts, the pandemic is contributing heavily towards the loss of public health gains that we managed to achieve globally during the last two decades. specifically, any advances made as part of the united nations millennium development goals (united nations, ) is eroding, and for the first time, the number of people entering extreme poverty is increasing. adding to this descent into poverty are new disruptions in ongoing disease control programs, routine vaccination strategies, and a reduction of capacity building efforts globally. therefore, and as highlighted by many others, we support the notion that a way forward to eliminate this coronavirus pandemic should include linking covid- control to other tropical or poverty-related diseases. summary: covid- is slowing or reversing global health and development gains. to be successful and achieve the global goals including the control of pandemics such as the one seen from the covid- , we must rely on strong leadership leading to impactful public policies and global collaborations, including global covid- vaccinations, and potentially linking them to programs for childhood and adult vaccinations and programs for malaria, tuberculosis, hiv/aids, and neglected tropical disease treatments. opportunities also include the creation of unique research opportunities and funding models and increase science engagement for international diplomacy. this can only be done with a better understanding of the relationships between coronavirus disease, poverty, and tropical diseases. the recent global pandemic caused by the novel severe acute respiratory syndrome coronavirus (sars-cov- ) and its associated coronavirus disease [ ] has affected almost every country in the world [ ] . the world health organization is closely tracking the rapid spread of covid- throughout the world, with daily situation reports [ ] . table shows a summary of the who's report of the total number of covid- cases and the total number of covid- deaths by region, taken from the last day of each month(march through july ). increasingly, there is a recognition that covid- is striking people living in poverty. this is happening on two fronts. beginning with new orleans in the spring of , covid- has caused significant devastation in low-income neighborhoods in us metro areas. african-american, latino, and native american populations living in these areas disproportionately account for the deaths and hospitalizations from covid- [ ] . in addition, now, covid- is affecting latin american nations, especially in poor urban areas-the favelas-of brazilian metro areas, and increasingly india and adjacent countries in south asia, and africa. covid- has spread into these regions and populations aided by weakened health systems with inadequate public health interventions leading to an increase in numbers of cases. we are already starting to see significant levels of mortality in brazilian cities, with the expectation that large metro areas and mega-cities in india and africa will follow. the initial entry of covid- into the southern hemisphere is happening in central and south america. overwhelmingly, brazil is the majorly affected country, especially in the favelas, the densely crowded slums of cities such as sao paulo, rio de janeiro, and salvador de bahia, but especially the northern tropical cities of belem, fortaleza, and manaus. urban areas of chile, peru, and ecuador are also affected, and covid- is expected to become one of the most widespread virus infections in latin america (table ) . we also see significant levels of illness in mesoamerica, referring to mexico and central america. currently, mexico has the highest case fatality rates in the americas [ ] . the caribbean is also beginning to see significant levels of infection with trinidad and tobago exceeded only by mexico for case fatality rates, according to the pan american health organization. for the past two decades, these countries within the americas have confronted many health challenges mixed together with other political and socioeconomic hurdles [ ] . beyond efforts to implement public health control, there are concerns that affected populations will experience long-term disabilities due to pulmonary scarring, cardiovascular complications, and neurologic deficits [ ] , as well as other clinical manifestations still being discovered [ ] . accordingly, these virus-related morbidities will add to the many already found in these populations already affected by non-communicable diseases functioning as major risk factors for covid- , as well as other infections, including the neglected tropical diseases (ntds). a recent article in the public library of sciences neglected tropical diseases journal provides a complete description and list of what are the ntds predominantly all found in these regions [ ] . now, covid- is accelerating in india and elsewhere in south asia. estimates indicate that india could eventually exceed the usa or brazil in terms of the total number of cases and deaths. there is an urgency to better understand the geographic distribution of india's covid- epidemic, especially in terms of specifically affected urban areas. covid- is also now affecting sub-saharan africa, beginning with south africa and egypt as the most affected countries. however, it is uncertain whether the relatively low number of cases across the region mostly reflects underreporting. for example, tanzania has made claims to be unaffected by covid- without external validation [ ] . the usa like many of the ntds, covid- has disproportionally targeted the most impoverished populations in high-income regions around the world [ ] . the concept of blue marble health refers to the "poorest of the rich" in nations such as the usa [ ] . a recent us study revealed that in the early stages of the pandemic, the populations living in the most underserved communities had higher rates of deaths due to covid- , compared with affluent locations [ ] . initial thoughts hypothesized that these findings could be primarily due to a lack of access to testing in these communities. but it also gave clarity that these underserved communities found themselves at a serious disadvantage since the start of the pandemic. social distancing is typically difficult in crowded households in low-income neighborhoods, where many do not have the opportunities to work from home. these poor populations generally have pre-existing health conditions due to inadequate access to high-quality healthcare. among them are high rates of obesity, diabetes, and cardiovascular disease. as is expected, of course, this also occurs in many areas around the world, especially in populations living in low-and middle-income countries (lmics). in brazil, for instance, a study evaluating the vulnerabilities and deprivations of their populations has identified several main factors contributing to this effect: hygiene, shelter, physical distance, and health recovery capacity [ ] . the basis by which poverty functions as a risk factor for covid- requires further investigation. sars-cov- uses a respiratory route of infection, associated with droplet contact and possibly airborne transmission, and therefore, population density is known to have large adverse effects [ ] . it is very common that those who fall within low socioeconomic strata and/or live below the poverty level [ ] generally have several generations living under a single household. therefore, living in close quarters with multiple inhabitants means direct contact with commonly used items; it can increase the risk of being exposed to the virus. in urban settings, additional factors that can lead to overcrowding may be the result of high rental costs, leading to families having to live in multi-family buildings and apartments [ ] . the economic downturn due to social distancing and decreased consumption of products and services has further disproportionally affected the poor. people living in poverty generally have less access to health communication on cable news networks or post-secondary education, working hourly positions that are more at the mercy of recessions. since they do not maintain a standard salary, many people in poverty need to continue working hourly jobs and place themselves at risk from public exposure with covid- . other factors can influence covid- morbidity and death, including age and non-communicable comorbidities such as diabetes, pulmonary, heart, or other organ-related diseases [ , ] . this, in turn, can feedback and influence the high levels of transmission of sars-cov- seen in these populations contributing to the observed epidemiology of covid- within these populations [ ] . individuals with low socioeconomics also have higher comorbidities that come from poor nutrition and the lack of access to primary medical services. health care is difficult to receive for people living below the poverty level. in both urban and rural settings, there is both a lack of accessible clinics for primary care and also a lack of insurance or the capacity to pay for preventive medical care. higher rates of smoking and alcohol consumption are also prevalent in both urban and rural people living in poverty. obesity, for instance, is associated with low-income populations, usually from consuming foods high in fats and carbohydrates that are still affordable. diabetes is also a significant problem that can be related to obesity. diabetes can severely impact the immune response of a person. all these health comorbidities can be the difference between asymptomatic covid- infection and intubated in the intensive care unit. there is increasing evidence that tropical diseases are also causative for propagating poverty in those infected [ ] . in the usa, an urban study in new york city reported a direct correlation with more toxocara parasite in city parks from poor neighborhoods compared with rich boroughs [ ] . another study in a clinic that serves low-income patients located in washington d.c. was found to have endemic levels of a potentially dangerous parasite called strongyloides stercoralis in this low-income population [ ] . a third study done in chicago found evidence of both toxocara and strongyloides in this urban population [ ] . major us cities had high levels of covid- infections and a higher prevalence in those living below the poverty line [ ] . for rural areas in the usa, there are studies in alabama that show hookworm (necator americanus), and strongyloides stercoralis is still present and transmitting in the population of poor rural alabama [ ] . in texas, a study detected . % of people infected with strongyloides stercoralis from a poor area outside of austin, tx, that has limited sanitation facilities [ ] . covid- has also been prevalent in the southern usa [ ] . given the dire public health crisis created by the emergence of covid- , it is anticipated that global resources will be committed to disease control and prevention. as for funds for the covid- vaccination scale, it will be interesting to look at opportunities to bundle these efforts for other tropical infections. for example, adult vaccinations for covid- could be linked to scale-up for other adult vaccines, including influenza. as new vaccines for schistosomiasis and chagas disease enter advanced development, there may be opportunities to link these as well [ ] . outside of vaccines, we will need to look at bundling covid- vaccines with mass treatments for malaria, tuberculosis, and hiv/aids, as well as largescale programs for neglected tropical diseases. until then, there are concerns that calls for social distancing might interrupt existing programs. there remains the possibility that we might see a resurgence in hiv/aids, tuberculosis, and malaria, and neglected tropical diseases. we have also seen how childhood vaccination programs for measles and other conditions were interrupted in the usa during the spring of [ ] . we may face similar interruptions in global child vaccination programs led by gavi, the vaccine alliance. the poor living in urban or rural areas of high-income countries and the most impoverished living in lmics have increased risk for both covid- and tropical diseases. these combinations disproportionally affect the working poor, both in health and economically, as to suffer long-term setbacks from missing work. the year is notable for the extraordinary global spread of covid- and the interruptions in key public health programs for both neglected diseases and vaccine-preventable diseases. we face the potential for a slowing, halting, or even reversal of all of the amazing gains since the launch of the millennium development goals. given the anticipated support for covid- vaccinations globally, it would seem wise to look at how some of the lapsed interventions could be resurrected and bundled. conflict of interest maria elena bottazzi and peter hotez are inventors of a low-cost covid- vaccine for global health which was recently licensed by baylor college of medicine to a commercial third party for scale up and production. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. naming the coronavirus disease (covid- ) and the virus that causes it the impact of covid- and strategies for mitigation and suppression in low-and middle-income countries world health organization. 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poverty and covid- : risk factors and deprivations in brazil. researchgate racial, economic and health inequality and covid- infection in the united states office of the assistant secretary for planning and evaluation. poverty guidelines spatial disparities in coronavirus incidence and mortality in the united states: an ecological analysis as of comorbidities and multi-organ injuries in the treatment of covid- covid- )-united states human intestinal parasite burden and poor sanitation in rural alabama toxocara species environmental contamination of public spaces strongyloidiasis in latin american immigrants: a pilot study parasitic infections represent a significant health threat among recent immigrants in chicago clinical characteristics of covid- in new york city prevalence of intestinal parasites in a low-income texas community running the gauntlet": formidable challenges in advancing neglected tropical diseases vaccines from development through licensure, and a "call to action effects of the covid- pandemic on routine pediatric vaccine ordering and administration-united states about the sustainable development goals publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -apb l tq authors: ward, m.p.; kelman, m. title: companion animal disease surveillance: a new solution to an old problem? date: - - journal: spat spatiotemporal epidemiol doi: . /j.sste. . . sha: doc_id: cord_uid: apb l tq infectious disease surveillance in companion animals has a long history. however, it has mostly taken the form of ad hoc surveys, or has focused on adverse reactions to pharmaceuticals. in a blue ribbon panel was convened by the u.s. white house office of science and technology policy to discuss the potential utility of a national companion animal health surveillance system. such a system could provide fundamental information about disease occurrence, transmission and risk factors; and could facilitate industry-supported pharmaco-epidemiological studies and post-market surveillance. disease watchdog, a prospective national disease surveillance project, was officially launched in january to capture data on diseases in dogs and cats throughout australia. participation is encouraged by providing registrants real-time disease maps and material for improved communication between veterinarians and clients. from january to mid-november , an estimated % of veterinary clinics australia-wide joined the project. over disease cases – including canine parvovirus (cpv), canine distemper, canine hepatitis, feline calicivirus, feline herpesvirus, and tick paralysis – were reported. in new south wales alone, cpv cases in dogs were reported from postcode locations. new south wales data was scanned using the space–time permutation test. up to clusters (p < . ) were identified, occurring in all months except march. the greatest number of clusters (n = ) were identified in april. the most likely cluster was identified in western sydney, where cases of cpv were reported from a postcode in february. although the project is still in its infancy, already new information on disease distribution has been produced. disease information generated could facilitate targeted control and prevention programs. surveillance of infectious diseases in companion animals has a long history. this surveillance has mostly taken the form of ad hoc surveys, or has focused on adverse reactions to vaccines and other pharmaceuticals. most of these activities have not included an ongoing measurement of the population using comparable methods, so strictly do not meet our current understanding of disease surveillance. a notably exception is surveillance for rabies, for which passive surveillance systems exist (for example, see blanton et al., ; tenzin et al., tenzin et al., , . an example of using secondary data sources for surveillance is research on the recent epidemic of canine leptospirosis in north america (for details, see ward et al., ward et al., , a ward, a,b) . this research program was initiated using data from the veterinary medical database (vmdb). the vmdb was established in north america in by the national cancer institute to collect, store, and retrieve veterinary clinical information (hayes et al., ; priester, ) . it contains a standardized abstract of every animal examined at participating veterinary teaching hospitals in the united states and canada. at the time of initiation of this study, veterinary teaching hospitals in the united states and canada had contributed data (ward et al., ) . results showed a clear trend in increasing diagnoses of canine leptospirosis, suggesting this to be a re-emerging zoonosis. however, because the vmdb is a secondary data source, data analysis was limited to those covariates recorded in the vmdb, and results might be subject to diagnostic bias and might not necessarily represent the target population. for spatial analyses (ward, a) , research was limited to the location of the hospitals reporting the cases. later, data generated within a specific veterinary teaching hospital data management system was integrated (using a geographic information system) with data generated from a state veterinary laboratory accession management system to conduct more focused research on spatial distribution and risk factors (ward et al., a,b) . this allowed recommendations to be made to reduce the impact of this epidemic of leptospirosis. however, integrating databases and data cleaning, error checking and validation consumed a considerable amount of time, and the system could not identify new trends in near-real time. a critical gap identified was database integration and analytical methodologies that could be applied routinely. most research in the area of companion animal disease surveillance has focused on systems to detect adverse vaccine and other drug reactions. for example, moore et al. ( a) developed a surveillance system that utilized electronic medical records from a large corporate small animal general practice in the united states, in which each of more than hospital locations in this system used the same computerized medical record system. records of dogs receiving a range of vaccines (including for bordetella, coronavirus, multivalent distemper-adenovirus-parainfluenza-parvovirus-leptospirosis, giardia and rabies) were extracted and searched for adverse reactions. using the collective data recording of these hospitals, adverse events were identified during a -year period, representing , , doses of vaccine administered to , , dogs. the use of veterinary practice databases to complement spontaneous reporting systems for vaccine safety is one example of recent advances in companion animal disease surveillance (moore et al., b) . this system has also been used as a research tool to identify space-time clusters of adverse events associated with canine rabies vaccine (moore et al., ) . in september , a blue ribbon panel was convened on behalf of the u.s. white house office of science and technology policy to discuss the potential utility and possible strategies for design and implementation of a national companion animal health surveillance system (stone and hautala, ) . a goal of such a system is improved veterinary care, and it could be also used to develop evidence-based veterinary practices. data derived from a surveillance system could provide fundamental information regarding disease incidence, prevalence, transmission, and risk factors. such an enhanced understanding of disease epidemiology would enable practitioners to evaluate the effectiveness of treatments and intervention programmes. such a system could also facilitate industry-supported pharmaco-epidemiological studies and post-market surveillance, which would enable the development of safer and more effective veterinary products. as stated by the blue ribbon panel, a companion animal surveillance system should ideally be a public/private partnership involving representatives from the veterinary community and the veterinary products industry who are involved in the design of the system in order to promote compliance and leverage resources. in january , virbac animal health in australia launched disease watch-dog, a prospective national disease surveillance project, to capture data on communicable disease cases and outbreaks, with the ultimate aim of the reduction and control infectious diseases in cats and dogs. this project is unique as it involves a growing number of participants, and as well as gathering data, encourages participation by providing benefits to registrants including real-time maps of disease occurrences and outbreaks, material for improved communication between veterinarians and clients, and empowerment of staff. from january to mid-november, an estimated % of veterinary clinics australia-wide have joined the project, with > cases of disease reported. while the project is still in its infancy, the analysis of initial data is demonstrating some important findings with respect to disease distributions in space and time and risk factors, and there is considerable potential to gather vital information that could lead to targeted vaccination and treatment efforts which could be the key to the control of various diseases. in this paper we report details of this surveillance tool and illustrate its utility with a case study of canine parvovirus (cpv) occurrence and distribution during a -month period in the state of new south wales. the purpose of this paper is to demonstrate the feasibility of surveillance in pet animal populations, and to highlight the value of spatial analysis of such surveillance data. disease watchdog was created out of the need for better epidemiological data on current and emerging companion animal diseases. despite significant advances in technology, veterinary science is lacking recent studies on the occurrence and distribution of diseases that affect the lives of millions of animals worldwide. incidence of common infectious diseases such as canine parvovirus (cpv), canine distemper virus (cdv), feline calicivirus (fcv), feline rhinotracheitis virus (fvr), feline immunodeficiency virus (fiv), and feline leukemia virus (felv) are largely unknown. with comprehensive data and a better understanding of the epidemiology of these diseases, solutions to problems such as how to formulate best practice vaccination protocols during disease outbreaks or how to strategically address endemic disease with preventive and treatment techniques can be proposed. as a national disease surveillance system, disease watchdog was developed to gather epidemiological data and to provide real-time mapping that demonstrates disease occurrences at suburb level. data needs were based on the following considerations: fields in which data could be recorded were then assembled into a web-based system and the website interface carefully designed to maximize compliance from users. estimated time to enter a case of disease is currently s. features to improve compliance include a ''litter'' checkbox where multiple affected littermates can be simultaneously entered into the database, reducing repetitive data entry. a chart can be downloaded from the website so that users can record cases on paper as an interim measure, then enter cases in a batch if this is easier for the user. to maximize accuracy, user access to submit data is limited to registered veterinarians and veterinary nursing staff of veterinary clinics. veterinary clinics register for access to the disease watchdog site at www.diseasewatchdog.org and once approval is granted, an email provides username and password. there is no charge for using disease watchdog. once a month, all registered users are sent an email reminder to enter cases for the previous month if they have not already done so. the monthly email also provides an opportunity to communicate with users, updating them on improvements to the system and how best to use the program. when a user registers, a pack of resources is sent to the clinic to which they belong, to help communicate their involvement to their staff and clients. an emphasis on empowerment of staff to be involved in the program aims at encouraging participation and collegiality. the message behind the program is that together, the veterinary profession has the power to control diseases through participation in the program, data collection, analysis and then strategic disease prevention and treatment. a benefit for veterinary clinics of participating in disease watchdog is the ability to view maps of disease cases and outbreaks in real-time. data entered via the website is immediately visible on the disease maps that any registered participant can view. maps can then be used to show clients where at-risk areas for disease exist, highlight the importance of disease prevention, and to monitor disease outbreaks in their local area. measurement of disease cases and recording of outbreaks also provides strong data that can also be communicated through channels such as the australian veterinary association's media office, to allow alerts to be transmitted to the general public via local and national media. never before in australia has accurate recording of disease outbreaks been available on this scale for this number of diseases. a feature of disease watchdog is the role of disease surveillance champions. these are individuals within veterinary practices that are responsible for data entry. these individuals routinely update data within the system, for their practice, on a monthly basis. they act as the primary contact point within each practice, and promote the advantages of the disease watchdog surveillance system to colleagues and clients. disease watchdog was upgraded to version . in september , partly in response to suggestions and ideas for improvements from contributors. this upgrade provided both contributors and analysts with a number of additional features and benefits for better searching and display of disease cases and outbreaks, and improved recording of information. some of the important news features include: . allowing the graphing feature to be controlled by disease surveillance champions. this feature displays disease cases and outbreaks -enabling the diseases, locations and timeframes that are displayed to be personally selected, for better representation of disease outbreaks relevant to a local community. disease watchdog users can adjust the graph that displays on the home page, to suit the needs of their individual clinic. the graph of current outbreaks of disease defaults to the last months. . a new facility to search by radius around a suburb, providing more relevant information on the diseases in the user's area and allowing improved mapping. this facility could also be useful for research studies to identify risk factors for disease occurrence. . a feature to allow multiple diseases to be displayed by assigning different colors to each disease, so that multiple diseases can be shown simultaneously. more information on diseases in a suburb can be obtained by the user simply by clicking on a colored flag placed at the suburb location. displaying multiple diseases allows practices and their clients to visualize which diseases are prevalent in their specific area. . tick paralysis data has been added to disease watch-dog. tick paralysis is an acute, progressive ascending motor paralysis. it is caused by a salivary neurotoxin produced by some species of ticks. in australia ixodes holocyclus is the principle cause of cases of tick paralysis in domestic animals. this tick species can be found on native animals along the east coast of australia. reported distribution of tick paralysis has previously been mostly anecdotal. inclusion of tick paralysis in disease watchdog allows for the first time the spatial and temporal distribution of this important disease to be monitored in australia. recording tick paralysis cases will enable veterinarians to alert clients to the dangers of ticks in specific areas, and also whether tick paralysis is seasonal in their area and the importance of tick prevention and awareness. . two more infectious diseases were added to the disease watchdog list; the distribution of felv and feline infectious peritonitis (fip) can now be monitored. these diseases were chosen as the next additions because of their apparent lower prevalence, but higher mortality. felv is caused by a retrovirus that can be transmitted between cats via saliva or nasal secretions, and can be fatal. fip, caused by a coronavirus, is incurable and generally fatal. other diseases will be added to disease watchdog in the future, based on the perceived health importance and ability of veterinarians to detect and diagnose the health condition. the development and maintenance of disease watch-dog to date has been funded entirely by virbac animal health. awareness of the disease watchdog initiative is being achieved within the veterinary community via the support of the australian veterinary association (which represents most veterinarians within australia), idexx laboratories (a company that develops and markets diagnostics within australia; http://www.idexx.com.au/), vetnostics (part of a network of pathology laboratories operating throughout australia; http://www.vetnostics.com.au/), and gribbles veterinary (a veterinary pathology organization providing veterinary diagnostic and analytical services throughout australia and new zealand; http://www.gribblesvets.com.au/info/general/home/get/ / /). between january and november , registrants joined disease watchdog across australia (a small number are non-veterinary registrants and do not record disease cases), representing approximately % of veterinary clinics australia-wide. based on estimates of veterinary clinic numbers across the states of australia, participation levels range from % in victoria ( ) to % in new south wales ( ). to demonstrate the utility of the disease watchdog system, data analysis focused on reports of cpv in the state of new south wales. cpv is an infectious disease of dogs caused by cpv type . it is highly contagious and is spread via direct contact or indirectly via fecal contaminated environments and fomites. this disease is often diagnosed in puppies unprotected by maternal antibodies or vaccination. although cpv first appeared in the late s as a pandemic (johnson and spradbrow, ; walker et al., ) and other epidemics have been reported (sabine et al., ) , the spatial distribution, seasonality and risk factors for cpv are not well understood. in a questionnaire survey of veterinary practitioners in australia and new zealand seeking details of their experience with cpv infections in , sabine et al. ( ) found that explosive outbreaks of disease had occurred in most parts of australia in that year. no obvious pattern of spread over the continent could be detected. an overall mortality rate of % was estimated. in a serological survey, smith et al. ( ) found that cpv was generally a disease of dogs less than months of age. the current analysis focuses on the spatial distribution of this disease, as a case study of the application of the disease watchdog surveillance system. data extracted from disease watchdog included date of diagnosis and the residential postcode. for the purposes of demonstration, data selected was restricted to reported cases occurring between january and september, in the state of new south wales. all data was joined to a postcode shapefile (gda , new south wales lambert conformal conic projection) in arcgis v. . (esri inc., redlands ca). the lambert conformal conic projection system is one of the best for 'middle' latitudes. the state of new south wales lies between latitudes and °s. spatial relationships were measured on the scale of meters. only cases which had valid data entered for location (postcode) and diagnosis date (day, month) were included in spatial analyses. for all postcodes included in the study, the postcode centroid was calculated (arctools, arcgis . ). most postcode polygons in new south wales are relatively small: % of the postcodes cover a land area of km . data were scanned using the space-time permutation test (satscan v. . kulldorff m. and information management services, inc. satscantm v . : software for the spatial and space-time scan statistics. http://www.satscan.org/, ). the permutation test was used because the number of dogs at-risk of cpv per postcode is unknown. however, it was assumed that during the -month study period the distribution of the population of dogs would have remained stable across postcodes. future research using disease watchdog will include estimation of the dog and cat population at-risk within suburbs and postcodes. this might be achieved by annual surveys of disease watchdog participants, or by estimations based on reported disease cases. based on a maximum period of infectiousness for cpv of up to - weeks (goddard and leisewitz, ) , the temporal scanning window was restricted to days, with windows of , , and days used. the spatial scanning window was restricted to km, with windows of , , and km used in a series of separate analyses. the analytical approach, recognizing that it was a demonstration of the application of the disease watchdog surveillance system, was exploratory and attempted to provide insights into the epidemiology of cpv in the new south wales dog population -a subject on which no peer-reviewed information has previously been published. the selection of scanning windows was motivated by an assumed level of clustering that might occur within an area covered by up to - postcodes (in urban and semi-urban areas of new south wales) within a relatively short timeframe, i.e., potential local outbreaks of cpv. an additional exploratory analysis was performed using the scan statistic bernoulli model. for these analyses, cases were reports of cpv in which the affected dogs had been vaccinated and controls were those dogs diagnosed with cpv that reportedly had not been vaccinated. the results of such an analysis might reveal clusters of cpv disease associated with vaccine failure. in all scan statistic analyses, permutations were performed and significant (p < . ) clusters were identified and mapped. clusters were interpreted based on the ratio of observed to expected cases occurring within the cluster. data was exported from disease watchdog, processed within arcgis v. . (esri inc., redlands ca) and then exported to satscan v. (kulldorff m. and information management services, inc. satscan™ v . : software for the spatial and space-time scan statistics. http:// www.satscan.org/, ) for analysis. between january and september, , there were reports submitted to disease watchdog, representing disease cases. only one case of fip, and two cases each of cdv and chv, were reported. a total of and cases of fcv and fhv were reported, respectively. tick paralysis was added to disease watchdog in september, and cases were reported during this month. all reports contained valid data for diagnosis and postcode. during the study period, cases of cpv were reported from new south wales ( cases/ reports), queensland ( / ). victoria ( / ), western australia ( / ), the northern territory ( / ), south australia ( / ) and tasmania ( / ). in new south wales, most cpv reports ( ; %) consisted of only one case. reports with , , , , and cases were made on , , , , and occasions. most of the cases were male ( ; %). a total of breeds were reported to be affected. the following breeds accounted for > % of cases: australian cattle dog ( ; %), staffordshire terrier ( ; %), jack russell terrier ( ; %), bull terrier ( ; %), mastiff ( ; %), maltese ( ; %) and fox terrier ( ; %). the mean ( % confidence interval) and median (interquartile range) ages were ( - ) and ( - ) months, respectively. the minimum age of cases was months. the most common methods of diagnosing cpv were the elisa snap ( ; %) and clinical signs ( ; %). immunofluorescence and pcr were rarely used ( and , respectively). outcome was reported for cases, with treatment ongoing at the time of reporting for a further cases. of those cases with a final outcome reported, ( %) died, ( %) were euthanized, and ( %) recovered. vaccination status was reported for cases, of which ( %) cases were vaccinated. no significant association was found between survival status (recovered versus died or euthanized) and gender (male versus female, odds ratio (or) . ; p = . ), method of diagnosis (clinical versus test, or . ; p = . ) or vaccination status (unvaccinated versus vaccinated, or . ; p = . and unknown versus vaccinated, or . ; p = . ). dogs that survived were older ( weeks) than those that died ( weeks), p = . . the greatest number of cases of cpv were reported in the months of february and april ( % each). half of all cases were reported between march and june. the data entry screen in disease watchdog (www.diseasewatchdog.org) is shown in fig. . examples of the reports that can be generated and viewed on the disease watchdog website are shown in fig. . reports include histograms showing the frequencies of diseases reported during a given period, and maps of the distribution of cases. an example of the spatial search capacity of disease watchdog is shown in fig. . cpv cases were reported from postcodes. only one case was reported from postcodes; or more cases were reported from the following postcodes: , , , , , , , , (fig. ) . only of these ( and ) were in areas of substantial human population. regardless of the size of the scanning window used (all combinations of , , and km and , , and days), the most likely cluster was identified in western sydney, in which clinics reported cases of cpv on february (observed Ä expected . ). depending on the scanning window used, between and significant (p < . ) additional clusters were detected. at larger spatial scanning windows, fewer clusters were detected. the size of the temporal scanning window had little influence on the number of clusters detected. clusters were detected in every month except march. the most clusters were identified in april. twelve of these clusters were only of one day duration; the other clusters lasted from to days (median days). the clusters (fig. ) were distributed throughout most of the state, excluding the southeast and the far west. all but two of these clusters were located outside the metropolitan area of sydney. using reports of cpv in which the affected dogs had been vaccinated as cases and controls as those dogs diagnosed with cpv that reportedly had not been vaccinated, the same location in western sydney was identified as the primary cluster, although the observed Ä expected ratio was less ( . ). in contrast, in this analysis no significant (p < . ) secondary clusters were detected. surveillance of small animal populations for infectious diseases has been rare. almost all examples of surveillance systems focus on the detection of rabies cases. for example, in the united states the system for rabies surveillance is well-developed and based on the reporting of cases (usually accompanied by submission of diagnostic samples) to the centres for disease control and prevention. however, even in this system, the focus is cases in wildlife, rather than companion animals (for example, in approximately % of the cases were in wildlife, and % were in domestic animals; blanton et al., ) . other studies have used a surveillance-like design. for example, biggeri et al. ( ) used a -stage sampling design, with first stage transects, to study the risk of dog parasitic infections in the city of naples, italy, during - . this system focused on zoonotic parasitic diseases, including infections with trichuris, isospora, toxascaris, ancylostoma and toxocara. however, the study apparently was driven by a research goal and not as an ongoing effort, thus, not strictly matching the definition of surveillance. other initiatives have used existing hospital management systems as a secondary data source for monitoring diseases in companion animal populations. for example, moore et al. ( ) used databases complied by banfield, the pet hospital corporation in the united states to describe several diseases. development of a disease surveillance system for companion animal disease as a primary objective apparently has not been successfully undertaken previously. the blue ribbon panel convened on behalf of the u.s. white house office of science and technology policy in to discuss the potential utility and possible strategies for design and implementation of a national companion animal health surveillance system (stone and hautala, ) stressed the importance of consultation with end-users: ''the type of information collected, the frequency with which it is collected, and the format in which it is disseminated should be determined in accordance with user goals''. disease watchdog is unique in its flexibility and ability to be modified in response to suggestions by those who use the system. for example, the upgrade of the system in september included several features identified as important by end-users, such as graphing and displays capabilities, and the addition of new diseases such as tick paralysis. the blue ribbon panel also identified four fundamental principles relevant to the design of a companion animal surveillance system: syndromic versus disease-specific surveillance; the ability to leverage existing systems; predefined response protocols; and integration with other health surveillance systems. syndromic surveillance involves collection of information on clinical syndromestwo or more characteristic clinical signs. disease watchdog is based on the diagnosis and reporting of specific disease, rather than syndromes. expansion of disease watchdog to include syndromes (for example, vomiting and diarrhea) could be implemented with comparative ease. however, such a modification would need to be driven by the endusers, which are primarily veterinary practices. in the case of cpv, a substantial proportion ( %) of cases was diagnosed using the snap elisa. in this situation, reporting cases based on syndrome might not provide additional benefit to the end-user. in contrast, cases of tick paralysis are generally diagnosed based on clinical signs (for exam-ple, between and september the reported cases of tick paralysis were diagnosed based on clinical presentation ( %), tick found ( %) or tick crater only found ( %)). tick paralysis was added to disease watchdog in september in response to requests from end-users of the system. for an applied surveillance system such as disease watchdog, the usefulness of syndromic surveillance needs to be assessed in partnership with the enduser. acute cpv enteritis can reportedly occur in dogs of any breed, age, or sex. however, puppies between weeks and months of age appear to be more susceptible (goddard and leisewitz, ). in the current study the median (interquartile range) age of reported cases was ( - ) months. this finding was unexpected, based on previous statements regarding the age pre-disposition for cpv. of those cases with reported vaccination status, only % of cases were vaccinated. this might, in part, explain a higher age of cases than expected. however, the common belief amongst veterinarians that cpv is a disease of puppies may need revision, following further research using data generated by disease watchdog. some breeds -mostly large breeds such as rottweiler, doberman pinscher, labrador retriever and german shepherd dog -have been reported to be more at risk for severe cpv enteritis (goddard and leisewitz, ) . none of these breeds were commonly reported to be cases in the current study. however, the proportion of a breed in a population -and the proportion that are presented at veterinary clinics -needs to be taken into account before breed predisposition can be determined. goddard and leisewitz ( ) state that the risk of cpv in sexually intact males is twice that of sexually intact females. in the current study more reported cases were male than female, but the difference was small ( % versus %). half of all cases were reported between march and june, which represents the autumn months. generally cpv is considered to peak in summer (goddard and leisewitz, ) . as more data is accumulated in disease watchdog, a more accurate description of seasonality can be made. many clusters of cpv were identified during the short -month study period in new south wales. such a spatial distribution of cases is likely to indicate that local factors are important, generating local epidemics. follow-up studies, using larger datasets, might be able to identify some of these factors. based on preliminary analysis, apparently vaccination practice is unlikely to explain most of these clusters. there are three specific design elements for a companion animal surveillance system (stone and hautala, ) : target population; data collection, analysis and standards; and mechanisms for dissemination of results. the system must have a clearly defined target population. this is necessary so that the disease frequency estimated can be referred to a defined population. in disease watchdog, the target population is comprised of veterinary practices that report disease cases. this is a subset (currently nearly onethird) of all practices in australia, the reference population. 'all practices in australia' is a well-defined population, since practices need to be licensed by the relevant state or territory authority where they operate. a current gap in disease watchdog is that the population of dogs and cats (and their demographics -such as age, breed and gender distributions) -is unknown. such data -when available -is for specific locations only (toribio et al., ) . this information could potentially be collected within the disease watchdog system, for example by having contributors annually describe their practice caseload during the previous months, or by estimating the likely population at-risk based on the proportions of cases reported, and potentially informed by known covariates (for example human population size and socioeconomic status) within the practice area. variable disease watch-dog adoption proportions for different australian statescurrently ranging from % in victoria to % in new south wales -means that inferring the impact of disease in different areas needs to be done with caution. a companion animal disease surveillance system should collect a comprehensive data set comprising both clinical and epidemiological data (stone and hautala, ) . disease watchdog collects spatial information via the postcode of the owner's address. it is assumed that this is the area in which the animal has spent most time during the short period preceding disease diagnosis, and assumed to be the area that other animals are at risk of the same disease. the actual date of diagnosis is reported in disease watchdog. diseases currently reported in this system can be diagnosed on the basis of clinical presentation, diagnostic test (including elisa snap, immunofluorescence, pcr) or other. other information collected includes animal name, suburb, state, postcode, species and breed, age (years/months/weeks), sex, neuter status, whether a litter was affected and if so, the number of animals in the litter and the total number affected, case outcome (recovered, died, euthanized, treatment ongoing) and vaccination status and date. a unique case identification number is generated by the system, and the case is linked to the clinic name and the veterinarian's name. finally, within a surveillance system, each user should define triggers for implementing responses and identify data that will support specific responses (stone and hautala, ) . within disease watchdog, the response to perceived clusters of disease rests with the end-user, the veterinary clinic. a feature of this system is the ability for practices to conduct spatial searches within their local area. this allows veterinarians and veterinary assistants to promote disease control and prevention based on empirical data. this might be one reason why the adoption of disease watchdog has been high during its first months of operation. two tasks that could facilitate major advances in the surveillance of companion animal populations are standardization of data captured within such systems, and the development of a set of core analytical tools. a current barrier to disease surveillance is an inability to rapidly and validly integrate different surveillance systems. the barrier is usually a lack of consistency in the scale (spatial, temporal) at which data is recorded, a lack of standard case definitions (e.g. method of diagnosis, method of measurement) and differences in which auxiliary data is recorded (e.g. animal data such as age, gender, breed and population demographic data -the 'population at-risk'). identifying key surveillance systems and key individuals, an agreement on standards that should be used could be reached. an analogy is the recent development of the reflect statement: methods and processes of creating reporting fig. . the distribution of spatio-temporal clusters of cases of canine parvovirus reported in the disease watchdog surveillance system between january and september in new south wales, australia. clusters were detected using the scan statistic permutation test, with spatial scanning windows up to km radius and temporal scanning windows up to days in length. guidelines for randomized control trials for livestock and food safety (o'connor et al., ) . an array of analytical tools are available for detecting trends and clusters in disease surveillance data (for example, see ward and carpenter, a,b; ward, ) . there is a lack of consistency regarding which tools should be applied routinely, and also a lack of knowledge of the performance of the different tools applied to different types of surveillance data. as above, agreement is needed in order to correctly evaluate the results of analysis of surveillance data. to our knowledge, results of the analysis of the spatial distribution of cpv cases have not been published in the peer-reviewed literature. a likely barrier is the lack of large, good quality surveillance datasets. disease watch-dog fills that gap: in new south wales alone, cases were reported during just months. considering the lack of knowledge of spatial distribution of cpv and the lack of knowledge of the population at-risk within each postcode, we used the permutation model. the model does not require any assumptions to be made with respect to how the population at-risk is characterized. the only assumption is that the population at-risk remains relatively stabile during the study period. as more analyses are undertaken of the spatial distribution of diseases in pet populations, analytical approaches are likely to be refined. during a short period of operation, disease watchdog has been adopted by nearly one-third of australian veterinary clinics. epidemiologic data, including location and date, are providing new insights into the distribution of diseases of dogs and cats in australia. ability of end users to produce disease maps is one likely explanation of its success to date. disease mapping in veterinary epidemiology: a bayesian geostatistical approach rabies surveillance in the united states during canine parvovirus the veterinary medical data program (vmdp): past, present, and future isolation from dogs with severe enteritis of a parvovirus related to feline panleucopaenia virus a space-time cluster of adverse events associated with canine rabies vaccine postmarketing surveillance for dog and cat vaccines: new resources in changing times adverse events diagnosed within three days of vaccine administration in pet dogs the reflect statement: methods and processes of creating reporting guidelines for randomized control trials for livestock and food safety multiple primary tumours in domestic animals. a preliminary view with particular emphasis on tumors in dogs canine parvovirus infection in australia during canine parvovirus-five years later serological observations on the epidemiology of parvovirus enteritis of dogs meeting report: panel on the potential utility and strategies for design and implementation of a national companion animal infectious disease surveillance system reemergence of rabies in chhukha district re-emerging rabies in dogs and other domestic animals in eastern bhutan demographics and husbandry of pet cats living in sydney, australia: results of cross-sectional survey of pet ownership a serological survey of canine parvovirus infection in new south wales clustering of leptospirosis among dogs in the united states and canada seasonality of canine leptospirosis in the united states and canada and its association with rainfall spatio-temporal analysis of infectious disease outbreaks in veterinary medicine: clusters, hotspots and foci analysis of time-space clustering in veterinary epidemiology techniques for analysis of disease clustering in space and in time in veterinary epidemiology prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) serovar-specific incidence of and risk factors for leptospirosis among dogs presented to a veterinary teaching hospital geographical risk factors for leptospirosis among indiana dogs key: cord- -mrrctuxt authors: freeman, hugh james title: review: adult celiac disease and the severe “flat” small bowel biopsy lesion date: journal: dig dis sci doi: . /b:ddas. . .d sha: doc_id: cord_uid: mrrctuxt classification of architectural changes in the small intestinal biopsy may be clinically useful to define the cause of diarrhea or suspected malabsorption, especially in adults. pathologic changes may include severe (flat) or variably severe (mild or moderate) abnormalities. for some disorders, small bowel biopsy findings may be very distinctive and lead to a specific diagnosis. for others, like adult celiac disease, biopsy changes are less specific. indeed, it is becoming increasingly appreciated that several conditions can produce similar histopathologic changes. serological assays, including endomysial antibodies and tissue transglutaminase antibodies, may be very useful tools for screening and case finding in clinical practice. however, demonstration of characteristic changes in the small intestinal biopsy is critical, along with a gluten-free diet response. to reveal a specific cause. while detailed lists of tests and algorithms appear in medical texts, these are costly and time-consuming. moreover, some function tests are patient-dependent and based on timed collections that are difficult to do with desired laboratory precision. often, some can be circumvented by small bowel biopsy or done later if additional documentation is necessary (e.g., fecal protein loss studies, -hr fecal fat measurements, barium radiographs). some patients with malabsorption may have no significant diarrhea. instead, weight loss or anemia associated with iron deficiency may lead to a biopsy to exclude a small intestinal cause. or, there may be other unexplained abnormal chemistries (e.g., low serum carotene, folate, or proteins, particularly albumin). finally, a positive screening celiac antibody blood test (e.g., antibodies to endomysium or tissue transglutaminase) should lead to biopsy to confirm the serologically based suspicion of celiac disease and define pathological changes in the small bowel before treatment ( ) . even a normal small bowel biopsy may be useful, in most instances, to exclude structural small bowel causes of diarrhea, particularly those with diffuse and severe changes in the proximal small intestine, such as classic celiac disease. normal or abnormal biopsy appearances, freeman however, should be interpreted cautiously depending on the biopsy site. for example, endoscopic specimens from the proximal duodenum may normally appear to have shorter villi with "pseudoflattening" over regions of brunner's glands (i.e., brunner's gland artifact) but crypt hyperplasia is not present. in addition, biopsies showing histologic evidence of apparent "duodenitis" or nonspecific inflammation in the duodenal bulb may indicate peptic-related disease. indeed, a broader spectrum of disease might be considered (e.g., crohn's disease) if similar changes are found in more distal small intestine. for this reason, screening biopsies (even if the duodenal mucosa is visually normal) to exclude a proximal small bowel mucosal cause for diarrhea should be done distal to the duodenal bulb. biopsy interpretive artifacts may result from biopsy handling. technical trauma may result from the biopsy instrument (i.e., biopsy crush artifact) or during transfer of the biopsy from forceps to filter paper or mesh. poorly oriented specimens may lead to tangential sections outside of the core of the paraffin-embedded tissue block. endoscopic pinch biopsies are now most commonly submitted to hospital pathology laboratories, but these are smaller than suction biopsies and more difficult to accurately orient for serial sectioning. however, clinical experience has demonstrated that acceptable biopsy material may be obtained for routine diagnostic purposes ( , ) . usually, two or three biopsies with regular-sized forceps from the second or third portion of the duodenum are sufficient. in special circumstances (e.g., recurrent symptoms in established celiac disease), more biopsies from multiple sites along the length of the small intestine (using a longer instrument) may be required. for example, in celiac disease and suspected lymphoma, only of small bowel biopsies proved to be positive for malignant cells ( ) . for clinical purposes, abnormal small bowel biopsies may be classified based on the degree of disturbed architecture together with specific diagnostic features ( ). multiple biopsies from separate sites are most useful since some disorders reveal only focal changes (e.g., crohn's disease), while others may show diffuse, but more variable severe abnormalities (e.g., giardiasis). sampling of normal-and abnormal-appearing small bowel mucosa should be done, especially if erosive or ulcerative macroscopic changes are endoscopically evident, since some pathologic features may be easier for the pathologist to appreciate in biopsies from less inflamed or less reactive epithelium. for clinicians, there may be limited time to examine biopsies, so the single most important imperative is clear communication of the clinical problem to the patholo-gist. good-quality biopsies should be submitted and their sites precisely defined to optimise histologic evaluation. a clinically relevant pathological interpretation based on the degree of severity of architectural disturbance may be very helpful and can be especially valuable for clinical management ( , ) . during endoscopy, macroscopic changes may be appreciated. in celiac disease, for example, absence of normal structures or a smooth tubular mucosal surface has been described ( ) . also, a so-called "scalloped" or "ridged" endoscopic appearance has been noted ( ) . these macroscopic changes are not specific to celiac disease, however, and should never be relied on for definitive diagnosis. indeed, in most patients with celiac disease, no significant endoscopic alteration is recorded despite the presence of severely abnormal histologic abnormalities. often, changes of celiac disease may be reported later by the pathologist even if the disease was not clinically or endoscopically suspected. even very experienced endoscopists estimated that celiac disease was diagnosed "unexpectedly" in over % simply because screening biopsies were done from normal duodenal mucosa ( ) . a visually normal endoscopic appearance alone (without biopsies) is simply not sufficient to exclude small bowel mucosal disease, including celiac disease. diarrhea, weight loss, and malabsorption of a broad range of nutrients occur in most patients with classic celiac disease. in addition, significant histopathologic changes are usually present in the proximal small bowel, the so-called severe "flat" mucosal lesion (figures and ) . indeed, the most common cause of this biopsy abnormality is untreated celiac disease ( ). these changes appear to be similar to those described as the so-called flat destructive or marsh type lesion ( , ) . several features should be present. villi are absent or rudimentary. increased lamina propria lymphoid cell elements and increased intraepithelial lymphocytes are seen and the surface epithelium appears more cuboidal (rather than columnar). crypt cell hyperplasia (not hypoplasia) with an increase in the crypt epithelial cell mitotic index (i.e., the number of mitoses per crypt epithelial cell) is present. in celiac disease, treated with a strict gluten-free diet, these changes revert toward normal ( figure ). longterm studies have shown that biopsies from comparable proximal small bowel sampling sites will eventually show improvement. villi reappear and crypt mitotic indices normalize. surface epithelial cells become more columnar. the cellularity of the lamina propria diminishes and the absolute numbers of intraepithelial lymphocytes fall. changes also occur in another dimension-along the length of the small intestine. these may not be so well appreciated in the modern era since endoscopic biopsies are usually obtained under direct vision from the proximal small intestine alone (rather than hydraulic suction biopsies from multiple sites in more distal jejunum and ileum). the extent and severity of these abnormalities through the length of the small intestine seem to correlate better with the patient's clinical status than changes seen with repeated sampling from similar proximal small intestinal mucosal sites. in celiac disease with clinically significant malabsorption, small bowel changes may be severe, with architectural changes extending beyond the proximal jejunum. farther along the small intestine into the ileum, less severe, often patchy, rather than diffuse, architectural changes may be present. possibly, this is because with clinically more severe disease, longer lengths of small intestine are exposed to higher concentrations of dietary gluten. or, alternatively, some pathological changes, particularly in more distal small intestinal sites, may be more "indirect" and driven by recirculating memory t cells ( ) . even in the most distal ileum, however, earlier human studies demonstrated that the mucosa was very sensitive to gluten-containing peptides infused experimentally through a long small bowel tube ( ) . this proximal-todistal gradient in severity of mucosal disease makes it more difficult to detect changes of celiac disease with ileal biopsies (obtained at colonoscopy), and, more importantly, this gradient is altered with a gluten-free diet. with more extensive small bowel disease, diarrhea and malabsorption of many nutrients result. after treatment with a gluten-free diet, clinical improvement occurs, with resolution of diarrhea and weight gain, reflecting improved absorption along the length of the intestinal tract. concomitant with these clinical improvements, it appears that resolution of the abnormal histologic changes occurs in a distal-to-proximal direction. conceptually, this is potentially very important in the clinical and histologic assessment of the response to a gluten-free diet. prolonged periods of gluten restriction may be required, even up to several months or years after diarrhea resolves, to show normalization of biopsies from the most proximal small intestine ( ) . repeated biopsies from the same proximal small intestinal sites after only a few weeks on a strict gluten-free diet may not be sufficient to show a convincing histologic response, even if the patient is clinically improved (i.e., resolution of diarrhea and weight gain). a clinical diagnosis of classic celiac disease, therefore, requires that two criteria be fulfilled: first, typical biopsy changes of untreated celiac disease in the proximal small bowel should be present; and second, improvement should occur with dietary gluten restriction. most often in adults, the clinical improvement may be sufficient to be convinced that celiac disease is responsible, especially if diarrhea resolves and weight gain results. in some patients with few clinical symptoms, however, repeated biopsies may be needed to show histologic normalization of the small bowel mucosa. serologic screening tests (e.g., endomysial antibodies or tissue transglutaminase) are inadequate to diagnose celiac disease or even define compliance to a gluten-free diet ( ) ( ) ( ) . false-negative serological tests occur. some are due to serum iga deficiency, occasionally associated with celiac disease ( ). any adult with chronic diarrhea or malabsorption suspected to be caused by celiac disease should have a small bowel biopsy to exclude the disorder. on the other hand, if one or more serological screening tests are positive, a biopsy should be done to determine if the test was correct since false-positive serological results also occur ( ). recently, even strongly positive tissue transglutaminase antibody assays were recorded using a commercially available test kit in patients with no other disease detected and entirely normal small bowel biopsies ( ) as well as in a patient with a severe flat lesion not histologically responsive to gluten restriction ( ) . thus, positive serological results, even if accompanied by biopsy changes consistent with untreated celiac disease, are not sufficient to diagnose celiac disease. a convincing response to a gluten-free diet must still be documented. in some patients with diarrhea or malabsorption, only mild or moderate degrees of altered villous architecture are present ( ). in the mild lesion, villi remain unaltered or only minimally altered in size. the epithelium, however, is very abnormal, with loss of polarity and a marked increase in intraepithelial lymphocytes. sometimes, there may also be an accompanying increase in lamina propria cellularity. these changes appear to be similar to those classified elsewhere as an infiltrative or marsh lesion ( ) . in the moderate lesion, there is also a definite change in villous architecture. these findings may be classified as an infiltrative hyperplastic or marsh lesion ( ) . often, these variably severe (i.e., mild or moderate) changes are not specific and may be detected in a number of disorders, including celiac disease, and with the previously documented glutensensitive small intestinal changes of dermatitis herpetiformis ( ) ( ) ( ) or intestinal lymphoma ( , ) . similar histologic changes have also been reported in some asymptomatic first-degree relatives of celiac disease patients ( , ) and, therefore, may, in some instances, represent part of the pathological spectrum of celiac disease ( ) . histologic changes less severe than those typical of celiac disease, however, should prompt investigation for a nonceliac cause, especially to exclude infectious agents (e.g., giardiasis, cryptosporidiosis). in a recent report ( ) , however, intraepithelial lymphocytosis with normal small bowel mucosal architecture was recorded in of patients. in these, a favorable clinical response to a gluten-free diet was described in (about %). although the changes in these patients may have been related to gluten sensitivity, repeated histological studies were not recorded. others have also suggested that celiac disease might be diagnosed without typical villous structural change, using immunohistochemical markers to label intraepithelial lymphocytes ( ) ( ) ( ) . additional studies will be required to confirm these intriguing observations. detection of celiac disease may be delayed, even into later adult years ( ) . in some with clinically silent celiac disease, only very limited morphologic changes may be detected, with only mild or moderately severe abnormalities in villous structure, whereas others may have severe histologic changes but limited to the most proximal small intestine. as a result of this limited involvement of the most proximal small intestine, only isolated deficiencies of specific nutrients, such as iron, may be present with little or no clinically detectable diarrhea or weight loss. a number of disorders have now been recognized as a possible clue to this form of clinically unsuspected or occult celiac disease including isolated iron, folic acid, or calcium deficiencies ( ) , dermatitis herpetiformis ( ) ( ) ( ) , autoimmune types of thyroid disease (i.e., hashimoto's thyroiditis) ( ), pediatric insulin-dependent diabetes mellitus ( ) , small intestinal adenocarcinoma ( ) , and some lymphomas ( , ) , particularly, although not exclusively, t-cell forms of lymphoma in intestinal ( ) and nonintestinal sites, including the liver ( ), spleen ( ) , and thyroid gland ( ) . rarely, the clinical presentation may be dramatic, with free intestinal perforation due to a complicating malignant lymphoma ( , ) . in addition, celiac disease has been detected in patients with an initial diagnosis of a microscopic form of colitis, such as lymphocytic or collagenous colitis ( , ) , lymphocytic gastritis ( ) , and lymphocytic sclerosing cholangitis ( ) . interestingly, determination of endomysial antibodies or tissue transglutaminase antibodies did not increase detection of celiac disease in small numbers of serologically screened patients with either lymphocytic or collagenous colitis ( ). in addition, feeding high-gluten-containing diets to two patients with lymphocytic colitis did not elicit small intestinal changes of celiac disease ( ) . in contrast, a recent prospective biopsy evaluation of consecutive collagenous colitis patients revealed that over % also had underlying and unrecognized celiac disease ( ) . another category of clinically silent celiac disease is latent celiac disease, reported in patients with dermatitis herpetiformis or small intestinal lymphoma ( , ) . in this condition, histologic assessment of the small bowel architecture is initially normal. following a high-glutencontaining diet, however, histologic changes of variable severity may be induced, indicating that the small intestinal mucosa in these possibly genetically predisposed patients is gluten-sensitive, as reflected in this latent small intestinal mucosal lesion. these changes in the small intestinal mucosa do not occur in otherwise normal volunteers fed high-gluten-containing diets. demonstration of histologic improvement in these gluten-induced changes in the small intestinal mucosa with a gluten-free diet in patients with latent celiac disease was also documented ( , ) . not infrequently, biopsies with minimal changes are labeled compatible with celiac disease. in this setting, a number of issues immediately result. a patient having florid symptoms or laboratory test abnormalities suggestive of malabsorption with celiac disease should have more than just mild nonspecific changes in small bowel biopsies. if only minimal morphologic changes are present, there are at least two possibilities: first, another disorder may be causing the symptoms, and, second, normal mucosa is being erroneously diagnosed as mild chronic inflammation. the consequences of a false-positive diagnosis of celiac disease are not minimal. major disruptions in culinary lifestyle may result and there are implications related to a number of possible associated conditions (including lymphoma). rarely, additional studies may be required. some patients with only mild changes in villous architecture could still have celiac disease ( , ) . a trial of gluten-free diet followed by a high-gluten diet challenge might be done with biopsies to determine if the mucosa is gluten-sensitive. a final diagnosis of celiac disease should only be made with certainty if the mucosal abnormalities can be shown to be gluten-sensitive. there also seem to be some patients with symptoms attributed to gluten ingestion (or, more often, ingestion of grains such as wheat) without abnormal small bowel biopsy changes. often, despite reintroduction of dietary gluten, even for prolonged periods, repeated small bowel biopsies are normal. likely, some form of functional disorder is present. without evidence of gluten-sensitive mucosal changes, a diagnosis of celiac disease cannot be established. in some patients with well-defined celiac disease and well-documented histologic improvement on a gluten-free diet, clinical symptoms of diarrhea or malabsorption may recur. these may be associated with recurrent and severe histologic changes usually seen in untreated celiac disease. in most, poor compliance with a strict gluten-free diet or inadvertent ingestion of dietary gluten can be documented, sometimes from a ubiquitous source, such as pill capsules or communion wafers. sometimes, evaluation by an astute dietitian or even hospitalization may be required to identify the source of gluten. in others, another cause for diarrhea or weight loss may develop (e.g., infectious diarrhea). for these celiac disease patients, treatment of the infection often will resolve the recurrent symptoms. alternatively, some may develop histologic changes in the small intestine directly related to malabsorption of a specific nutrient and superimposed on the changes of celiac disease. this might result in refractory clinical symptoms (e.g., zinc deficiency associated with ongoing malabsorption) or independent morphologic changes, in-cluding macrocytic epithelial cells and crypt epithelial hypoplasia (e.g., folic acid deficiency). in others, a related cause, such as pancreatic exocrine insufficiency with pancreatic calcification, may develop in long-standing celiac disease with long-standing malnutrition ( ) . sometimes, reevaluation of the original diagnosis is necessary to ensure that the correct diagnosis was initially established. finally, some may develop diarrhea caused by a recognized association of celiac disease, such as collagenous or lymphocytic colitis, or a serious complication, such as lymphoma ( ) . rarely, an unusual disorder, collagenous sprue may occur. this was originally described in a patient with celiac disease ( ) . in most patients, severe panmalabsorption with diarrhea, weight loss, and marked nutritional and electrolyte disturbances may develop. many of these patients may eventually require ongoing nutritional support to survive. interestingly, iga endomysial antibodies have been detected in collagenous sprue, providing additional evidence of a direct link with preexisting celiac disease ( ) . finally, as in celiac disease, rare collagenous sprue patients have also been recently reported to develop lymphomas ( , ) . in a small number of patients with well-documented celiac disease, no specific cause for refractory symptoms appears evident. some, but not all, of these patients may have an unusual and poorly understood syndrome characterized by recurrent or persistent small bowel histologic changes of variable severity, splenic hypofunction, and a peculiar form of pathologic cavitation of mesenteric lymph nodes ( ) . some of these refractory patients eventually develop or are found to have a concomitant intestinal lymphoma ( ) . if rebiopsy is done in patients with refractory or recurrent symptoms, the pathologist should examine biopsies for evidence of collagenous sprue or a lymphoma. pretreatment biopsies should also be reviewed to determine if originally there was truly convincing improvement on a gluten-free diet. without evidence for biopsy improvement with gluten restriction, other causes for diarrhea and/or malabsorption should be considered, since celiac disease may not even be present. detection of a persistently severe flat lesion in duodenum and jejunum, however, may not mean that refractory celiac disease is necessarily present since histologically severe changes may persist for prolonged periods even after more distal intestinal mucosa has improved. similarly, in patients with celiac disease, the appearance of iron deficiency may not necessarily reflect refractory celiac disease due to impaired iron absorption in the proximal small intestine. other causes, including superimposed occult blood loss, may still require exclusion. the terms unclassified sprue or sprue-like intestinal disease have been used to describe patients who may have a severe (flat) or variably severe mucosal lesion but have not been shown to respond to a gluten-free diet. this possibly represents a heterogeneous group of small intestinal disorders, a "wastebasket group" with no specific cause. some possibly represent the atrophic hypoplastic or marsh lesion described elsewhere ( ) . most remain severely symptomatic with malabsorption and profound wasting in spite of a gluten-free diet. some could have a "clinically resistant form" of celiac disease, whereas others eventually prove to have a difficult-to-diagnose intestinal lymphoma. in patients recently reported with the label of "refractory sprue," an abnormal subset of intraepithelial lymphocytes was described with morphologically normal, but phenotypically abnormal lymphocytes ( ) . most died with uncontrolled malabsorption despite steroid therapy and parenteral nutrition. in a subsequent report by the same group ( ) , partial trisomy of the q region was recorded. additional studies are needed to determine if this intriguing observation will prove to be a specific prognostic marker of possible lymphoma development. another usual disorder, originally described in children ( ), with associated enterocyte or goblet cell antibodies, has been recently described in adults ( ) ( ) ( ) ( ) . pathologically, this so-called autoimmune enteropathy is a form of unclassified enteropathy and fails to respond to a glutenfree diet. some studies have suggested that this intriguing intestinal disorder has a very distinct pathogenesis ( ) . other causes of a severe (flat) or variably severe small intestinal biopsy lesion may be associated with diarrhea or malabsorption. table lists some of these causes with their treatment, if available. although some believe that oats may be consumed safely by celiac patients ( ), even for prolonged periods ( ), a recent report has documented that oats alone may have induced villous atrophy ( ) . certain infections have very distinctive findings that may lead to their recognition (e.g., giardiasis). in some, treatment with a specific antimicrobial agent may cause rapid symptom resolution and complete normalization of biopsy changes. often, pathologic changes are present but no specific infectious agent can be detected. in children, for example, severe changes may occur, possibly related to a viral agent or an escherichia coli infection, that may spontaneously resolve without treatment ( ) ( ) ( ) . more often, however, especially in adults, severity of architectural changes in the small intestine is variable and limited. in giardiasis, for example, only % of small bowel biopsies were severely abnormal. most biopsies either were normal or showed only mild to moderate changes. a number of other protozoan agents may cause small intestinal inflammation. detection of mature adult organisms, their trophozoites, or some intracellular component of the life cycle within the surface epithelium or on the epithelial surface may lead to a specific diagnosis. isospora belli ( , ) , cryptosporidium parvum ( , ) , cyclospora cayetanensis ( ) , and the microsporidiosis agents (i.e., enterocytozoon bieneusi and encepalitozoon intestinalis), may be seen in small bowel biopsies ( ) , and in some patients, specific treatment may resolve the diarrhea or malabsorption. in others, if there is no immunologic compromise, spontaneous resolution of the infection may occur. most often, however, these infectious agents are detected in patients with acquired immune deficiency syndromes, following transplantation or after hiv infection ( ) . often, in this setting, the presence of multiple agents makes definition of the precise cause of pathologic changes in the small intestine impossible. parasites may also cause altered architecture and small bowel lesions with a morphologically distinctive agent (e.g., strongyloides stercoralis, hookworm, schistosoma or capillaria species). in some patients, there may be morphologic similarities to eosinophilic gastroenteritis, a diagnosis that can only be established after parasitic disease has been excluded. viral agents, like cytomegalovirus, have been detected in small intestine, particularly from immunocompromised patients ( ) . often, it is not clear if the viral agent is the cause of the diarrhea or the intestinal pathologic changes. similarly, hiv-infected patients may develop a wide range of mucosal changes from severe (flat) to variably severe changes; in these patients, it is not known if the viral agent per se directly causes the pathologic abnormalities or if changes are indirect, possibly related to some immunologic alteration, malnutrition, or another infection ( ) . still, other viral agents, including the sars coronavirus ( ), appear to cause no histological alteration in the small intestine, even though scattered viral particles may be detected on the surface of microvilli of the enterocyte. fungal organisms have also been found in the small intestine, including candida species and histoplasmosis. patients are usually immunocompromised ( ) . in addition, stasis in the small intestine or bacterial contamination (i.e., bacterial overgrowth) related to altered small intestinal motility may induce some nonspecific changes ( ) . possibly, the most distinctive pathologic changes of any small intestinal bacterial infection are associated with mycobacterium avium-intracellulare in aids ( , ) . severe or variably severe architectural changes occur, with clusters of foamy macrophages containing acid-fast organisms expanding the lamina propria region. these changes are reminiscent of whipple's disease with distended lacteals caused by periodic acid-schiff (pas)positive macrophages. in whipple's disease, malabsorption, arthritis, lymphadenopathy, hyperpigmented skin, and neurologic changes develop. foamy macrophages with the whipple's agent are present; however, acid-fast stains are negative ( , ) . other disorders may cause changes that are associated with diarrhea in adults. although only limited architectural changes may be evident, some very distinctive histopathologic changes may permit a specific diagnosis. these include crohn's disease ( , ) , intestinal lymphoma, eosinophilic gastroenteritis, lymphangiectasia ( ) , macroglobulinemia ( ) and amyloidosis ( ), abetalipoproteinemia ( ) , some lipid storage disorders, including fabry's disease ( ) , radiation injury, and drug-induced small intestinal disease, such as triparanol, neomycin, busulfan, methotrexate, and some nonsteroidal antiinflammatory drugs, i.e., sulindac ( ). recently, similar changes in the small intestine have been recorded with the use of azathioprine ( ) . gastrointestinal pathology and its clinical implications biopsy diagnosis of the digestive tract mucosal biopsy of the gastrointestinal tract serological testing for screening in adult celiac disease comparison of suction capsule and endoscopic biopsy of the small bowel mucosa small bowel biopsy for malabsorption. comparison of the diagnostic adequacy of endoscopic forceps and capsule biopsy specimens small bowel malignant lymphoma complicating celiac sprue and the mesenteric lymph node cavitation syndrome mucosal biopsy techniques and interaction with the pathologist scalloped valvulae conniventes: an endoscopic marker of celiac sprue survey of gastroenterologists on the diagnosis and treatment of adult patients with celiac disease in british columbia the natural history of gluten sensitivity: defining, refining and re-defining mucosal pathology in gluten sensitivity t-cell mediated intestinal injury studies on celiac sprue. iv. the response of the whole length of the small bowel to a gluten-free diet non-alcoholic chronic pancreatitis with pancreatic calcification-presenting manifestation of occult celiac disease reliability of antitransglutaminase antibodies as predictors of gluten-free diet compliance in adult celiac disease strongly positive tissue transglutaminase antibody assays without celiac disease lack of usefulness of antitransglutaminase antibodies in assessing histologic recovery after gluten-free diet in celiac disease the small intestinal mucosa in dermatitis herpetiformis. i. severity and distribution of the small intestinal lesion and associated malabsorption the small intestinal mucosa in dermatitis herpetiformis. ii. relationship of the small intestinal lesion to gluten patchiness and duodenal-jejunal variation of the mucosal abnormality in celiac disease and dermatitis herpetiformis primary abdominal lymphoma-presenting manifestation of celiac sprue or complicating dermatitis herpetiformis multifocal small bowel lymphoma and latent celiac sprue studies on the familial nature of celiac sprue using biopsy of the small intestine a family study of celiac disease clinical and pathological spectrum of celiac disease-active, silent, latent, potential significance of intraepithelial lymphocytosis in small bowel biopsy samples with normal mucosal architecture intestinal antibody patterns of celiac disease: association with gamma/delta t cell receptor expression by intraepithelial lymphocytes, and other indices of potential celiac disease intraepithelial lymphocytes in celiac disease clinical spectrum of biopsy-defined celiac disease in the elderly adult celiac sprue-changes in the pattern of clinical recognition celiac associated autoimmune thyroid disease. a study of patients with overt hypothyroidism high prevalence of celiac disease in patients with type diabetes detected by antibodies to endomysium and tissue transglutaminase occult celiac disease in an octogenarian presenting with a small intestinal adenocarcinoma malignant histiocytosis of the intestine-a t-cell lymphoma fulminant liver failure with necrotizing foci in the liver, spleen and lymph nodes in celiac disease due to malignant lymphoma t-cell lymphoma of the thyroid gland in celiac disease free perforation due to lymphoma in biopsy-defined or suspected celiac disease collagenous colitis associated with small intestinal villous atrophy colonic lymphocytosis in patients with celiac sprue lymphocytic gastritis in patients with celiac sprue or sprue-like intestinal disease occult celiac disease associated with lymphocytic sclerosing cholangitis prevalence of celiac disease in collagenous and lymphocytic colitis failure of added dietary gluten to induce small intestinal histopathological changes in patients with watery diarrhea and lymphocytic colitis collagenous colitis as the presenting manifestation of biopsy-defined celiac disease latent celiac sprue is a raised intraepithelial lymphocyte count with normal duodenal villous architecture clinically relevant? lymphoproliferative and intestinal malignancies in patients with biopsy-defined celiac disease collagenous sprue-an unrecognised type of malabsorption hyposplenism, anti-endomysial antibodies and lymphocytic colitis in collagenous sprue the histologic spectrum and clinical outcome of refractory and unclassified sprue collagenous sprue associated with an extensive t-cell lymphoma cavitation of mesenteric lymph nodes, splenic atrophy, and a flat small intestinal mucosa abnormal intestinal intraepithelial lymphocytes in refractory sprue recurrent partial trisomy q -q in clonal intraepithelial lymphocytes in refractory celiac sprue flat small intestine mucosa and autoantibodies against gut epithelium autoimmune enteropathy and villous atrophy in adults autoimmune enteropathy in an adult with autoimmune multisystem involvement intestinal goblet cell autoantibody associated enteropathy autoimmune enteropathy and colitis in an adult patient mechanisms of villous atrophy in autoimmune enteropathy and celiac disease a comparison of diets with and without oats in celiac disease no harm from five year ingestion of oats in celiac disease oats induced villous atrophy in celiac disease virus particles in epithelial cells of duodenal mucosa from children with non-bacterial gastroenteritis immunofluorescent demonstration of enteropathic e. coli in tissues of infants dying from enteritis the mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis human coccidiosis, a possible cause of malabsorption-the life cycle in small bowel mucosal biopsies as a diagnostic feature chronic intestinal coccidiosis in man-intestinal morphology and response to treatment cryptosporidial enteritis in a patient with congenital hypogammaglobulinemia acute enterocolitis in a human being infected with the protozoan cryptosporidium light and electron microscopic identification of cyclospora species in the small intestine-evidence of the presence of asexual life cycle in human host light microscopic diagnosis of microsporidiosis in patients with aids small intestinal pathogens in aids cytomegalovirus infection of the gastrointestinal tract in a patient with late onset immunodeficiency syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection candidiasis of the duodenum and jejunum small intestine bacterial overgrowth intestinal infection with mycobacterium avium in acquired immune deficiency syndrome (aids)-histological and clinical comparison with whipple's disease aids with mycobacterium aviumintracellulare lesions resembling those of whipple's disease whipple's disease granulomatous (crohn's) disease of the upper gastrointestinal tract-a study of patients with mucosal granulomas small intestinal biopsy in a patient with crohn's disease of the duodenum. the spectrum of abnormal findings in the absence of granulomas chylous ascites, intestinal lymphangiectasia and the "yellow nail" syndrome intestinal involvement in waldenstrom's macroglobulinemia intestinal malabsorption-first manifestation of waldenstrom's macroglobulinemia the jejunal mucosa in two cases of abetalipoproteinemia pathophysiologic and ultrastructural basis for intestinal symptoms in fabry's disease sulindac associated small bowel lesion severe villus atrophy and chronic malabsorption induced by azathioprine key: cord- -ld bux h authors: eslick, guy d. title: future perspectives on infections associated with gastrointestinal tract diseases date: - - journal: infect dis clin north am doi: . /j.idc. . . sha: doc_id: cord_uid: ld bux h there are a vast number of infectious agents that are associated with gastrointestinal (gi) tract diseases. the epidemiology of gi diseases is changing, with a greater number of conditions increasing in incidence. challenges exist with establishing cause-and-effect relationships because of the ubiquitous nature of these organisms and the milieu in which they exist. advances in technology should provide novel methods for identifying and diagnosing these organisms and the relationship they have with a specific digestive disease. prevalence were observed for certain gi tract diseases, including gastroesophageal reflux disease (gerd) with an increase of per , population, hepatitis c with per , population, chronic constipation with per , population, intestinal infections with per , population, and pancreatitis with per , population. the prevalence of digestive diseases around the world is enormous and varies from country to country ( table ) . worldwide there has been a dynamic shift in the epidemiology of gi tract diseases, with some diseases such as peptic ulcer decreasing dramatically since the discovery of helicobacter pylori infection and a larger number of conditions increasing, such as gerd, nonalcoholic fatty liver disease, diverticular disease, barrett esophagus, cholelithiasis, alcoholic liver disease, hepatitis c, chronic pancreatitis, esophageal cancer and colorectal cancer. [ ] [ ] [ ] [ ] [ ] [ ] in conjunction with this increasing incidence of digestive diseases are the re-emergence of certain infectious agents (box ) (eg, cholera) and the identification of new agents (eg, h pylori, laribacter, campylobacter concisus), which are associated with gi tract diseases. since the discovery of h pylori there has been an enormous interest in the relationship between microorganisms and gi tract diseases, including cancers. one of the main issues associated with infections and disease is determining the relationship of the cause and effect. the landmark article by sir austin bradford hill in titled the environment and disease: association or causation? became widely known as the bradford hill's criteria. there were criteria that were required to be met to determine a cause-and-effect relationship (box ). it is usually difficult to meet all these criteria, particularly when trying to find the cause-and-effect relationships between organisms in the small intestine or colon because of the large number of organisms living in these environments. even for h pylori infection and the relationship with gastric cancer, although it is currently the only bacterium classified as a class i carcinogen, the evidence supporting this relationship is not complete in terms of bradford hill's criteria. there are a large number of organisms believed to be responsible for diseases of the digestive system. some of these organisms are true pathogens, whereas others are merely commensal in nature and are unlikely to ever produce any pathologic condition. table shows the various types of microbes that are associated with diseases of the gi tract covered in this issue; it is by no means all-inclusive but provides the current magnitude of an ever-increasing field of research. at present, some of these diseases are only associated with a single group of organisms (eg, irritable bowel syndrome), whereas other diseases are affected by all groups of organisms (eg, appendicitis). there are a variety of methodological and technical issues related to infectious agents and their role in digestive diseases. for diseases of the colon, the major limitation remains the inability to completely identify these organisms. identification of bacteria was mainly conducted using culture-based methods. now, the focus in identification of bacteria is increasingly based on using molecular techniques. many of these techniques allow the detection and identification of viable but nonculturable cells that are metabolically active but not reproducing. gene sequencing using single-stranded rna has been a key method in being able to elucidate multitudes of organisms that remain unknown. at present, there are approximately bacterial species, and this number is estimated as just the tip of the iceberg. the development of molecular studies of the emergence and/or spread of antimicrobial resistance genes within pathogen populations methods offers great promise not only in research and development but also in the diagnostic setting (eg, stool samples) ( table ) . , clearly, metagenomics, in which genetic material is directly retrieved from environmental sources, will play a critical role in the future development of determining infectious agents of the gi tract. the use of high-throughput technology has already produced important findings in relation to the gi tract microflora, including the differences between adults and children, with numerous uncultured organisms being the crux of the normal human adult gut flora which remain stable but other organisms change depending on environmental and genetic factors, whereas in infants there appear to be a constant transformation of organisms over time (figs. and ) . there have been several new detection methods developed, with some of these using nanoscale electrochemical detectors and others using dna sensors (extrachromosomal dna). the use of stable-isotope probing is also being investigated, but even this technique has limitations. although these technologies are increasing the understanding of the gut microflora, there remains large gaps of knowledge regarding the metabolic functions of these organisms and the relationship they have with human gi disease. these will be extremely fruitful areas of research and development in the coming years. research on therapeutic approaches that target resistance mechanisms modification of existing antimicrobials to overcome emergent resistance antimicrobial research, as related to engineered threats and naturally occurring drugresistant pathogens, focused on the development of broad-spectrum antimicrobials the burden of digestive diseases in the united states. us department of health and human services cancer statistics changing trends in gastrointestinal disease in the asia-pacific region the burden of gastrointestinal and liver diseases burden of digestive diseases in the united states part i: overall and upper gastrointestinal diseases burden of digestive diseases in the united states part ii: lower gastrointestinal diseases burden of digestive diseases in the united states part iii: liver, biliary tract, and pancreas the burden of gastrointestinal disease: implications for the provision of care in the uk emerging infections of the gastrointestinal tract the environment and disease: association or causation? high-throughput diversity and functionality analysis of the gastrointestinal tract microbiota advances in nucleic acid-based diagnostics of bacterial infections electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of dna tools for the tract: understanding the functionality of the gastrointestinal tract key: cord- -dao kx authors: rife, brittany d; mavian, carla; chen, xinguang; ciccozzi, massimo; salemi, marco; min, jae; prosperi, mattia cf title: phylodynamic applications in (st) century global infectious disease research date: - - journal: glob health res policy doi: . /s - - -y sha: doc_id: cord_uid: dao kx background: phylodynamics, the study of the interaction between epidemiological and pathogen evolutionary processes within and among populations, was originally defined in the context of rapidly evolving viruses and used to characterize transmission dynamics. the concept of phylodynamics has evolved since the early (st) century, extending its reach to slower-evolving pathogens, including bacteria and fungi, and to the identification of influential factors in disease spread and pathogen population dynamics. results: the phylodynamic approach has now become a fundamental building block for the development of comparative phylogenetic tools capable of incorporating epidemiological surveillance data with molecular sequences into a single statistical framework. these innovative tools have greatly enhanced scientific investigations of the temporal and geographical origins, evolutionary history, and ecological risk factors associated with the growth and spread of viruses such as human immunodeficiency virus (hiv), zika, and dengue and bacteria such as methicillin-resistant staphylococcus aureus. conclusions: capitalizing on an extensive review of the literature, we discuss the evolution of the field of infectious disease epidemiology and recent accomplishments, highlighting the advancements in phylodynamics, as well as the challenges and limitations currently facing researchers studying emerging pathogen epidemics across the globe. electronic supplementary material: the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. globalization has dramatically changed the way in which pathogens spread among human populations and enter new ecosystems [ , ] . through migration, travel, trade, and various other channels, humans have and will continue to intentionally or unintentionally introduce new organisms into virgin ecosystems with potentially catastrophic consequences [ ] . humans are not the only culprits, however; global climate pattern changes can alter local ecosystems, creating favorable conditions for the rapid spread of previously overlooked or even undiscovered organisms among humans, giving rise to unexpected epidemics [ , ] . recent years have been marked by global epidemics of ebola, dengue, and zika, derived from pathogens previously restricted to local outbreaks [ ] . according to the world health organization, more than one and a half billion people are currently awaiting treatment for neglected tropical diseases with similar potential for global spread, for which we have limited knowledge of etiology and treatment options [ ] . this lack of knowledge further limits our ability to investigate the putative role of these pathogens in future epidemics or even pandemics. epidemiological strategies have been and still are the first line of defense against an outbreak or epidemic. despite conventionality, traditional epidemiological methods for the analysis of global infectious diseases are subject to errors from various sources (fig. ) and are thus often inadequate to investigate the epidemiology of an infectious disease. putative outbreak investigations typically ensue following case notification of one of the diseases recognized by local and global public health organizations. trained investigators subsequently collect data on cases and diagnoses to establish a disease cluster. during active surveillance, more cases may be detected through outreach to healthcare facilities and nearby health departments. relevant case contacts, such as family, friends, and partners, are also sought to provide details on demographics, clinical diagnoses, and other potential risk factors associated with the spread of the disease [ ] . however, the lack of infrastructure, trained personnel, and resources in low-and middleincome countries are prohibitive against field epidemiology investigations, as contact tracing and surveillance both require systematic, unbiased, and detailed investigations. the reconstruction and interpretation of transmission networks are often very sensitive to response, selection, and recall biases and are strictly limited by surveillance data collected in many regions with diverse socioeconomic and cultural backgrounds [ ] [ ] [ ] . in addition, even with a highly effective surveillance system, environmental, zoonotic, and vector-borne transmission dynamics confound analysis by shadowing alternative (i.e., not human-to-human) routes of disease acquisition. furthermore, routine analyses of pathogen subtype and drug resistance are conducted only in a subset of developed nations, wherein variation in screening assays and protocols and therapy regimens increases the discordance in surveillance [ , ] . despite the limitations to traditional infectious disease epidemiology, major advances in study designs and methods for epidemiological data analysis have been made over the past decade for a multifaceted investigation of the complexity of disease at both the individual and population levels [ , ] . however, many challenges for infectious disease research remain salient in contemporary molecular epidemiology, such as the incorporation of intra-and inter-host pathogen population characteristics as influential factors of transmission. combating current and future emerging pathogens with potential for global spread requires innovative conceptual frameworks, new analytical tools, and advanced training in broad areas of research related to infectious diseases [ ] [ ] [ ] . an expanded multi-disciplinary approach posits advancement in infectious disease epidemiology research and control in an era of economic and health globalization [ , , , ] . fortunately, recent developments in phylogenetic methods have made possible the ability to detect evolutionary patterns of a pathogen over a natural timescale (months-years) and allow for researchers to assess the pathogen's ecological history imprinted within the underlying phylogeny. when reconstructed within the coalescent framework, and assuming a clock-like rate of evolution, the evolutionary history of a pathogen can provide valuable information as to the origin and timing of major population changes [ ] . phylogenetic methods also provide key information as to the evolution of both genotypic and phenotypic characteristics, such as subtype and drug resistance (fig. ) . even though phylogenetic methods are also limited in certain areas, such as restriction of analysis to only the infected population, a significant subset of these limitations can be overcome by complementary use of data from surveillance (both disease and syndromic) and monitoring [ ] (fig. ) . by integrating phylogenetic methods with traditional epidemiological methods, researchers are able to infer relationships between surveillance data and patterns in pathogen population dynamics, such as genetic diversity, selective pressure, and spatiotemporal distribution. systematic investigation of these relationships, or phylodynamics [ ] , offers a unique perspective on infectious disease epidemiology, enabling researchers to better understand the impact of evolution on, for example, spatiotemporal dispersion among host populations and transmission among network contacts, and vice versa [ , ] . the study of the interconnectedness of these pathogen characteristics was previously limited by the cost and timescale of the generation of molecular data. recent decades have been characterized by technology with the ability to rapidly generate serial molecular data from identifiable sources for which we can obtain detailed relevant information through epidemiological surveillance, allowing for the merging of phylodynamics and epidemiology, or evolutionary epidemiology [ , ] . hence, progress in the field of molecular evolution has provided the opportunity for real-time assessment of the patterns associated with local, national, and global outbreaks [ ] , cross-species transmission events and characteristics [ ] , and the effectiveness of treatment strategies on current [ ] and recurring epidemics [ ] . these assessments are essential for monitoring outbreaks and predicting/preventing pandemic inception, a good example being the recent study of middle east respiratory syndrome coronavirus global transmission [ ] (additional file (video s )). but has the, field of evolutionary epidemiology quite reached its full potential? in this article, we systematically discuss how the application of phylodynamic methods has and will continue to impact epidemiological research and global public health to understand and control infectious diseases locally and across the globe. in a strict sense, the concept of phylodynamics is anything but new. the phylogenetic tree reconstructed by haeckel in using phenotypic traits [ ] was used to explain the distribution of the earliest humansthe "twelve races of man"-across the globe and the location of the "centre of creation." this incorporation of both spatial information and phylogenetic relationships in the inference of population distributions and diversity among geographical locations is a branch of phylodynamics, often referred to as phylogeography. since then, the progression of genetic sequencing technology as well as geographical information systems (gis) has enabled evolutionary biologists to gain a higher resolution view of infectious disease dynamics. the st century, in particular, has witnessed unparalleled advances in methods and techniques for molecular sequence data generation and analyses. however, the relationship of progress and perfection is far from linear, along with its relationship to navigational ease. for example, phylodynamic inference has transitioned into a highly statistics-focused process with the corresponding challenges, including informative samples that can significantly affect the accuracy of results [ ] [ ] [ ] . several research groups [ , ] have reviewed and/or demonstrated the impact of neglecting critical quality control steps on obtaining reliable inferences using the recently developed phylodynamic frameworks, particularly with high throughput, or next-generation, sequencing (ngs) data. some important steps include ensuring uniform spatial and temporal sampling [ ] , sufficient time duration between consecutive sample collections for observing measurable evolution [ ] , coverage of deep sequencing, and consideration of genomic recombination [ ] . the reliance on phylodynamic methods for estimating a pathogen's population-level characteristics (e.g., effective population size) and their relationships with epidemiological data suffers from a high costincreasing the number of inference models, and thus parameters associated with these models, requires an even greater increase in the information content, or phylogenetic resolution, of the sequence alignment and associated phenotypic data. low coverage [ ] and the presence of organism-or sequencing-mediated recombination [ ] , can skew estimates of the evolutionary rate and even impact the underlying tree topology, particularly when dealing with priors in the bayesian statistical framework commonly used for phylodynamic inference. programs such as splitstree [ ] can take as input a nucleotide alignment and output a network in which the dual origins of recombinant sequences are displayed in a phylogeneticlike context. however, network-reconstructing programs have difficulty distinguishing actual recombination events from phylogenetic uncertainty, and branch lengths do not usually reflect true evolutionary distances [ ] . despite much work ongoing in this area, there are currently no broadly applicable methods that are able to reconstruct phylogenetic network graphs that explicitly depict recombination and allow for phylodynamic inference. although the bayesian framework has shown to be fairly robust with the inclusion of recombinant sequences in large population studies [ ] , the inclusion threshold has not been thoroughly investigated and is likely dependent on a number of factors, such as sample size and sequence length. recombinant sequences are thus usually removed prior to analysis; however, the ability to incorporate recombinant sequences is imperative given our knowledge of the role of recombination in virus adaptation [ ] , for example. more details on methods that can potentially account for recombination, applicable to a variety of pathogens, are discussed by martin, lemey, & posada [ ] . while the traditional realm of phylogenetics has focused on rapidly evolving viruses, the development of whole-genome sequencing (wgs) has made possible the expansion of phylodynamic methods to the analysis of slower-evolving microorganisms, such as bacteria, fungi, and other cell-based pathogens. wgs has widened the range of measurably evolving pathogens, allowing for the identification of sparse, genetically variable sites, referred to as single nucleotide polymorphisms (snps), among populations sampled at different time points. the use of wgs in phylogenetics is highly beneficial not only in resolving relationships for slower-evolving organisms but also in reconstructing a more accurate evolutionary history (phylogeny) of an organism, rather than the genealogy (single gene), which can differ significantly from the phylogeny due to the presence of selective pressure or even genetic composition [ ] . however, as with phylodynamic analysis of rapidly evolving viruses, wgs analysis of cell-based pathogens comes with its own challenges, as discussed in detail elsewhere [ ] . implementation of phylodynamic and/or phylogeographic analysis has transitioned over the last two decades from maximum likelihood to the bayesian framework. this framework provides a more statistical approach for testing specific evolutionary hypotheses by considering the uncertainty in evolutionary and epidemiological parameter estimation. given surveillance data (e.g., the duration of infection) and the specification of an epidemiological mathematical model, bayesian phylogenetic reconstruction can also be used to estimate epidemiological parameters that might otherwise be difficult to quantify [ ] . for example, during the early stage of an epidemic, wherein the pathogen population is growing exponentially, the rate of exponential growth can be estimated from the phylogeny using a coalescent model that describes the waiting time for individual coalescent events of evolutionary lineages. this rate estimate can be combined with knowledge of the duration of infection for a particular pathogen to estimate the basic reproduction number, r (e.g., [ ] ), as well as the prevalence of infection and number of infected hosts. transmission dynamics can similarly be inferred following the early exponential growth of the pathogen, during which the pathogen has become endemic. estimation of these parameters is described more thoroughly in volz et al. [ ] . with the expansion of phylodynamic methods to global epidemics, theoretical studies have found that inferences of infection dynamics within the coalescent framework are limited by the assumption of a freely mixing population [ ] . this assumption is often violated with the inclusion of several isolated geographical areas with single or few pathogen introductions. without considering this factor, population structure within a phylogeny can severely bias inferences of the evolutionary history and associated epidemiological parameters [ , ] . to overcome this limitation, software packages such as beast (bayesian evolutionary analysis sampling trees) [ ] [ ] [ ] have recently developed algorithms that allow for the integration of coalescent, mathematical, and spatial diffusion models [ ] [ ] [ ] [ ] [ ] [ ] . more importantly, beast readily implements a comparative phylogenetic approach, which incorporates parameterization of phenotypic trait evolution to identify predictors of population dynamics and spatial spread, all of which are estimated/assessed simultaneously during reconstruction of the evolutionary history [ , ] . statistical evaluation of the risk factors for pathogen population growth and spread can be performed concurrently with the assessment of phylogenetic resolution within the data [ ] , discussed above as a challenge to complex phylodynamic analyses. for example, in the absence of strong phylogenetic resolution, bayesian statistics are more sensitive to long-branch attraction bias [ , ] , wherein rapidly evolving lineages appear to be closely related, regardless of their true evolutionary relationships. this phenomenon, therefore, influences inferences of spatiotemporal spread of the studied pathogen, as well as estimation of the relationship of pathogen population behavior with potential risk factors, such as climate change, host and/or vector distribution, accessibility and so on. the influence of low-resolution molecular data on the reliability of phylodynamic inferences highlights the importance of the implementation of the method described by vrancken et al. [ ] , or even a priori estimation of the phylogenetic and temporal resolution (sufficient time between sampling) [ , ] . unlike other phylogenetic frameworks, bayesian inference enables utilization of prior knowledge in the form of prior distributions (in combination with information provided by the data); however, abuse of prior knowledge is possible and can lead to incorrect conclusions. even within the bayesian school of thought, scientists do not always agree with regard to the specification of prior distributions under certain conditions. the incorporation of prior information is, however, intuitively appealing, as it allows one to rationalize the probability of an estimate based on previous knowledge of the typical behavior of the parameter among populations of the organism under study. but what can we do if we have no knowledge regarding a particular organism or population? this has become a more pertinent issue recently with the increasing rate of discovery, facilitated by ngs, of organisms for which we have limited prior knowledge, such as novel viruses and bacteria, [ ] . one of the advantages of the bayesian phylodynamic approach is the ability to test multiple hypotheses regarding the evolution or epidemiological models used to describe infectious disease behavior, but because of the intricate relationship of these models, reliable inferences require testing of all combinations of the individual proposed models. although often neglected due to computational complexity, improved estimates of marginal likelihoods used for statistical model comparison have been demonstrated with less computational effort [ ] . additionally, if we know that we know nothing about the parameter in question, then, in fact, we know something. referred to as the "objective bayesian" approach, this ideal allows researchers to alter a normally "subjective" prior to create one that is minimally informative. this term is used because the impact of this type of prior on parameter estimation can be controlled to a minimum, allowing the data to dominate the analytical process and conclusions drawn [ ] . although similarly appealing, this approach can be particularly problematic with small datasets [ ] or biased datasets, such as the exclusion of potential intermediate sampling locations [ ] . the expanding volume of sequence data and increasing efforts to combine epidemiological and laboratory data in open access locations can help to improve evolutionary estimates. additionally, the growing availability of data and collaboration can accelerate our understanding of the emergence and spread of infectious diseases through coordinated efforts by multidisciplinary researchers across various institutions and public health organizations. more detail on the benefits of open access databases and data sharing in the context of phylogenetic epidemiology is reviewed in [ ] and [ ] . combining pathogen genetic data with host population information (e.g., population density and air traffic) in a statistical framework is critical for the reliable assessment of factors potentially associated with pathogen population dynamics and geographic spread. the comparative phylogenetic approach described above [ ] was used recently to identify potential determinants of the dengue virus (denv) introduction to and spread within brazil. results from nunes et al. [ ] suggested that for three denv serotypes, the establishment of new lineages in brazil had been occurring within to -year intervals since their primary introduction in , most likely from the caribbean. additionally, they observed that aerial transportation of humans and/or vector mosquitoes, rather than distances between geographical locations or mosquito (particularly aedes aegypti) infestation rates, were likely responsible. the study by nunes et al. marked one of the first uses of the comparative phylogenetic approach for vector-borne tropical diseases and implies the need for a similar approach in future studies aimed at investigating transmission patterns of a broad range of emerging vector-borne viruses. for example, this approach will allow researchers to determine if specific universal factors, such as vector species, are predictive of global transmission route or if health policy and prevention strategies tailored specifically to the pathogen, irrespective of the vector, are required for effective control. with the development of molecular clock models for serially sampled data [ ] , phylogenetic analyses have helped to uncover the timing of transmission events and epidemiological origins. moreover, when paired with comparative phylogeographic models, researchers have been able to identify risk factors most likely associated with these particular events. since the inception of the zika virus (zikv) pandemic around may of in brazil [ ] , phylogeneticists and epidemiologists have sought to reveal mechanisms by which zikv has spread and the factors fueling the wide geographical leaps. a full-genome phylogeographic analysis of zikv isolates collected during - revealed very intricate spatiotemporal transmission patterns across africa prior to the introduction into asia [ ] . from its origin in uganda, two independent transmission events appeared to play a role in the spread of zikv from east africa to the west circa : the first involved the introduction of zikv to côte d'ivoire with subsequent spread to senegal, and the second involved the spread of the virus from nigeria to west africa. results from spatiotemporal analysis demonstrated that uganda was the hub of the african epidemic as well as the common ancestor of the malaysian lineages sampled during the outbreak [ ] . following the emergence and rapid spread of zikv in brazil and other south american countries [ ] , faria's group sought to further characterize the spatiotemporal dynamics of zikv following introduction into this region [ ] . in addition to sequencing data, air traffic data for visitors to brazil from other countries associated with major social events during - were included to test different hypotheses of airline-mediated introduction of zikv in brazil. the results linked the origin of the brazilian epidemic to a single introduction of zikv estimated to occur between may and december , consistent with the confederations cup event, but predating the first reported cases in french polynesia. although these findings are of great value and importance to public health organizations, the authors drew an additional, and similarly valuable conclusion-large-scale patterns in human (and mosquito) mobility extending beyond air traffic data will provide more useful and testable hypotheses about disease emergence and spread than ad hoc hypotheses focused on specific events. this conclusion further supports the proposal for greater availability of epidemiological data among the scientific community. understanding both the rapid spread of the virus throughout south and central america and the caribbean as well as the initial emergence of the virus from the ugandan zika forest in the early s is important for application to the control of future outbreaks, but increasing data may not be the only answer. moreover, several different risk factors are likely responsible for these two migration events. therefore, a more comprehensive approach that allows for the analysis of multiple potential factors and their distinct contribution to independent migration events without the loss of information (i.e., use of data that span the entire evolutionary history) is imperative for fully understanding a global epidemic from beginning to present. a combined approach to understanding the emergence and expansion of an epidemiologically diverse viral population: hiv crf _ag in the congo river basin although viral spread is often attributed to human mobility [ ] , factors such as population growth and accessibility can also play an important role, as with the emergence of human immunodeficiency virus type (hiv- ) group m subtypes a and d in east africa [ ] and circulating recombinant form (crf) _ag in regions of the congo river basin (crb) [ ] . the democratic republic of congo (drc) has been reported to be the source of hiv- group m diversity [ ] [ ] [ ] ; however, the epidemiological heterogeneity of crf _ag within surrounding regions comprising the crb had remained a mystery since its discovery in [ ] , with prevalence ranging from virtual non-existence [ ] [ ] [ ] [ ] [ ] [ ] to accounting for as high as % of infections [ ] , depending on the geographical location. the region with the highest proportion of crf _ag infections, cameroon [ , ] , has been characterized by a rapidly growing infected population ( . % in to % in [ ] ), of which the majority ( %) is caused by this clade. using both molecular sequence data and unaids surveillance data [ ] , the spatiotemporal origin of crf _ag was estimated to occur in the drc in the early s ( ) ( ) ( ) ( ) , with the rapid viral population growth in cameroon following a chance exportation event out of drc. although similar phylodynamic techniques as described above for other viral species were used to infer the spatial origins of crf _ag, the timing of the origin of this viral clade was inferred using both coalescent analysis of molecular sequence data and prevalence information [ , ] . coalescent models allow for estimation of the effective population size (ne), of fundamental importance to infectious disease epidemiology, as it describes the level of genetic diversity within a population over the course of its evolutionary history. during the exponential growth period of an epidemic, the change in ne has been shown to linearly correlate with prevalence of infection [ , ] and can, therefore, be used to estimate the latter, as mentioned above, but also, when combined, faria et al. [ ] were able to show that fitting of ne and prior prevalence data can narrow the uncertainty of the temporal origin estimates by over % as compared to coalescent estimates alone. furthermore, surveillance data was recently used during simultaneous phylodynamic coalescent estimation to identify factors associated with ne dynamics throughout the entire evolutionary history of the cameroonian sequences [ ] , revealing that changes in ne were more reflective of incidence dynamics rather than prevalence, consistent with previous mathematical modeling [ , ] . although associations between ne and potentially related factors are frequently assessed, statistical analysis of these has until recently been primarily limited to post hoc examination (e.g., [ , ] ), which ignores uncertainty in demographic reconstruction, as discussed above. simultaneous implementation of evolutionary reconstruction and estimation of the relationship of covariate data with ne will be available in the newest version of beast v [ ] . although this tool has obvious implications for global assessment of factors contributing to the growth and dynamics of an epidemic, similar applications of this method to other data sets has suggested that reduced molecular data relative to covariate data may result in an impact of inclusion of the data on ne estimates. this finding posits a potential concern for convenience sequence sampling, as factors that are not responsible but are represented by large amounts of data may influence ne estimates, resulting in unreliable population dynamic inferences. as mentioned above, care is needed to ensure sufficient sampling and an appropriate sampling strategy for reliable reconstruction of the evolutionary and epidemiological history of the infectious organism of interest. traditional phylodynamic analysis applied to nosocomial outbreaks has been successfully used in the past to identify the likely source; however, the inclusion of extensive patient data, such as treatment regimens, admission and discharge dates, and length of stay, can improve not only phylogenetic estimates but also the translation of the interpretation to public health policy. epidemiological and genomic data on methicillin-resistant staphylococcus aureus (mrsa) infections were recently utilized by azarian and colleagues to reconstruct mrsa transmission and to estimate possible community and hospital acquisitions [ ] . findings from this study revealed that as high as % of the mrsa colonization within the hospital's neonatal intensive care unit (nicu) was acquired within the nicu itself. these findings indicated that current, standard prevention efforts were insufficient in preventing an outbreak, calling for the improvement of current care or alternative implementation strategies. the earlier uses of phylodynamic methods focused primarily on the molecular evolution of rapidly evolving viruses, greatly advancing the fields of virus vaccine and treatment strategies [ ] . on the other hand, epidemiological approaches have focused on influential factors related to social, economic, and behavioral patterns. integrating the phylodynamics and epidemiology approaches into a single analytical framework, referred to as evolutionary epidemiology [ , ] , represents one of the most powerful multi-disciplinary platforms. examples discussed herein of the adoption of an integrative and multifactorial mindset reveal the potential for accelerating our understanding of the emergence and spread of global infectious diseases, presently expanded to include bacterial and other cell-based pathogens. however, although a highly evolved analytical platform and an improved understanding of the translation of molecular evolutionary patterns to infection and transmission dynamics have aided in facilitating this transition, several challenges still remain. the st century has witnessed a major shift in breadth of scientific knowledge at the level of the individual researcher, requiring more focused training (e.g., molecular mechanisms) and greater collaborative efforts; meanwhile, a consensus of commonality and crossdisciplinary understanding is necessary for globalization of not only the economy, but also public health. this kind of understanding can be better achieved through interdisciplinary instruction on the theoretical and application skills related to both phylogenetics and epidemiology during early education. if successfully achieved, this combined training, in addition to access to modern ngs technology, such as handheld sequencers, would increase the mobility of labs and researchers, expanding the concept of lab-based research. mobilized labs would, in turn, reduce our current reliance on few major public health organizations and the impact of limited resources on sampling and surveillance in developing countries. increasing mobility is nevertheless inconsequential without the cooperative sharing of genomic and epidemiological information. although data are typically readily available to the public following peer-reviewed publication, the median review time of manuscripts submitted to, for example, nature is days [ ] , this in addition to the time required for thorough analysis of the original data. this timeline seems quite long in retrospect of the "spanish flu," which spread to one-third of the global population in a relatively brief -month period [ ] . data sharing prior to publication, even if only among a proportion of consenting institutions, may accelerate the process of dissemination of research findings to public health decision makers and practitioners, and its practice is not entirely unheard of. an excellent example of this type of collaboration is the "nextstrain" project (http://www.next strain.org/). nextstrain is a publicly available repository currently comprised of evolutionary datasets for ebola, zika, and avian and seasonal influenza viruses contributed by research groups from all over the world for the purpose of real-time tracking of viral epidemics. similar projects have also recently developed in other research fields. modeled after the stand up to cancer initiative, the synodos collaborative funded by the children's tumor foundation in partnership with sage bionetworks brings together a consortium of multidisciplinary researchers, who have agreed to the sharing of data and relevant information, as well as results [ ] . the ultimate goal of this cooperation is to accelerate the drug discovery process, which is highly applicable to global infectious disease research. without a similar collaborative approach to synodos, the preparedness of the global reaction to rising epidemics is at risk. recent years have been marked by local outbreaks across vast geographical regions within a timespan of months to years. hence, both the rapid dissemination of data and results and the rapid response of government and public health organizations are required for the effective prevention of a global epidemic, or pandemic. additionally, with the type of results, particularly risk factors, that are generated using this multifaceted approach (e.g., both human population and pathogen molecular characteristics), the question then arises of how organizations will actually utilize this information for treatment and prevention strategies. moreover, as the techniques and methods advance, are the infrastructures in place for global cooperation and immediate response following the presentation of a potentially more complex story? although gaps remain in current evolutionary modeling capabilities when used with epidemiological surveillance data, it is only a matter of time before the challenges described herein and elsewhere are met with more realistic models that capture the complexity of infectious disease transmission. furthermore, theoretical research in the field of infectious disease phylodynamics is still growing. consequently, there is a need for a review of the more recently developed methods 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prevalence of diverse hiv- strains was stable in cameroonian blood donors from two independent epidemics of hiv in maryland world health organization. who | epidemiological fact sheets on hiv and aids epidemic dynamics revealed in dengue evolution viral phylodynamics and the search for an 'effective number of infections' understanding past population dynamics: bayesian coalescent-based modeling with covariates a high-resolution genetic signature of demographic and spatial expansion in epizootic rabies virus genomic epidemiology of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit does it take too long to publish research? influenza: the mother of all pandemics children's tumor foundation announces historic new initiative in neurofibromatosis research the "virogenesis" project receives funding from the european union's horizon research and innovation program under grant agreement no. . not applicable.availability of data and materials not applicable. authors' contributions bdr and cm contributed to the writing of the manuscript and creating of figures and additional material. bdr, cm, xc, mc, ms, jm, and mp discussed the contents of the manuscript and contributed to editing and revision. all authors read and approved the final manuscript. no financial or non-financial competing interest existed for any one author during the writing of this manuscript. not applicable. reported studies do not involve human participants, human data or human tissue. submit your next manuscript to biomed central and we will help you at every step: key: cord- -ys n authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ys n today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid- s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, ; lyon et al., ; morse, a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, ; morse, ; silver, ) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., ) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture , years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., ) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~ %), m.m. musculus (~ %), and m.m. castaneus (~ %) (wade and daly, ; wade et al., ) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., ) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over years bp. by the s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., ; yu et al., ) , and a common y chromosome contributed by m.m. musculus (bishop et al., ) through its contribution to the genome of m.m. molossinus (nagamine et al., ) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c bl/ mice contain minor genetic elements derived from m. spretus (hardies et al., ) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in (haldane et al., ) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table . ) (lyon et al., ) . mice have pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, ) . for more information on comparative genomics, see chapter animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h ) on chromosome . the h complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h alleles, termed h haplotypes. for example, the balb h haplotype is h d and the c bl h haplotype is h b . the international immunogenetics (imgt) information system provides details on h haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h complex (stuart, ) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., ) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c bl/ j mouse in by the international mouse genome sequencing consortium (waterston et al., ) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c bl/ n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., ; skarnes et al., ) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., ) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table . ). the first inbred strain (dba) was developed by c.c. little in , with the subsequent creation of over inbred strains and stocks of mice (festing, ) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., ; silver, ) . inbred mouse lines are termed strains, and are achieved by or more brother × sister (filial; f) generations (table . ). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after f generations. most commonly used inbred mouse strains represent or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f , if branches have separated for generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, ) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c bl/ j, s /svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly % of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, ; welsh et al., ) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, ) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in embryonic stem cells, with backcrossing onto c bl/ inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, ) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of breeding pairs, e.g., heterozygosity will decrease at % per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + generation. each mated pair in the nth generation contributes one female and one male to the n + generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in , and are therefore quite homogeneous genetically (chia et al., ) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd allele (serfilippi et al., b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., ) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c bl/ beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, ) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., ) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, ; justice et al., justice et al., , . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, ) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, ) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream ′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., ) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_ _ sanger) (nagy, ) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of mice and early gene transfer experiments in the late s and early s led to the development of es cell lines derived from several different strains. this early emphasis on teratomas prompted creation of 'better' mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the mouse (simpson et al., ; threadgill et al., ) . this realization gave rise to the need to revise mouse nomenclature (festing et al., ) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but es cell lines have been commonly used. recent international knockout mouse program efforts use c bl/ n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_ _sanger). for this reason, most es cell lines are xy, which favors male chimerism. if the es cells are of (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c bl/ ) and backcrossed for n generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c bl/ n es cells, so that chimeric males are bred directly with c bl/ mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., ) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, ; peng et al., ) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., ) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., ; wijshake et al., ; gaj et al., ) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok (cathomen and joung, ; joung and sander, ) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, ) . talens function similarly, but are composed of tandem repeats of - amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions and (joung and sander, ) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok , produce dsb (hsu et al., ) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., ; wijshake et al., ) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., ; wijshake et al., ) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., ; wijshake et al., ) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., ) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., ; wijshake et al., ) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., ; sander et al., ; bae et al., ; reyon et al., ; herscovitch et al., ; wolfson, ) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than separate outbred stocks and traditional inbred strains, often with multiple substrains (table . ). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd ), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c bl/ j mouse serves as an example. the inbred strain c bl originated from abbie lathrop's female (and male ) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c bl mice split into major substrains, e.g., c bl/ and c bl/ . substrains are identified by appending a forward slash (/) after the inbred strain name. since , uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c bl/ j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c bl/ mice is c bl/ n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c bl/ j substrains (such as c bl/ jjci and c bl/ jjmsslc), and a number of c bl/ n substrains (such as c bl/ njci, c bl/ ncrlcrlj, dba/ j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c h/hesn c bl/ j-tyrc- j /+ co-isogenic segregating inbred mutant strain carrying the albino j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h complex was transferred from c bl/ j (b ) to the akr background b .cba-d mit -d mit congenic strain in which the chromosomal segment between d mit and d mit was transferred from cba to b b .cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b and are carried in coupling, i.e., on the same chromosome b .cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd- /ty recombinant inbred (ri) strain number in a set of ri strains derived from a c bl/ j (b) female mated to a dba/ j (d) male and made by taylor (ty) recombinant congenic (rc) strain number in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c bl/ ntac). significant differences may exist among these substrains (mekada et al., ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c bl/ ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the ' ' in c bl/ , the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm unc is a targeted mutation (tm), first line ( ) congenic mice are often derived from es cells, backcrossed onto a background strain, such as c bl/ . under such circumstances, when the backcross generation is at n , the '.' symbol is used between the background inbred strain and the donor strain (e.g., c bl/ n. p /olahsd-abc tm zzz , abbreviated as b . -abc tm zzz . when backcrossing is incomplete but at the n generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b ; -abc tm zzz . if the background strain is mixed genetic origin, it is designated stock. -abc tm zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b .cg-abc tm zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il rgtm wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in -derived es cells, followed by intercrossing the chimeric mice with c bl/ or other background strains of mouse. when an f hybrid is created, the female partner is listed first, e.g., a c bl/ j × s /svpas hybrid would be designated: c bl/ j s /svpasf . ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c bl/ jxdba/ j- , c bl/ jxdba/ j- , etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc- , cc- , etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table . ), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c bl/ j s /svpasf mice would be b f and c bl/ jxdba/ j- ri mice would be bxd- . parental order is an important consideration in nomenclature, as a b mouse is genetically different from a b mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c bl/ j mice, so that c bl/ j beige mice, which are co-isogenic with c bl/ j mice, are designated c bl/ j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb ) led mice are inbred fvb/n mice in which the rat erb gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb ), the first line ( ) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., ) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., ) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, ) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table . ). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table . . nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., ; knapka, ) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about - g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. . and . ). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft ( ) . knapka ( ) . b linoleic acid: . % is adequate. c john and bell ( ) . d theuer ( ) . e knapka et al. ( ). f nutrition ( . g hurley and bell ( ) . h pleasants et al. ( ) . nutrients, and contaminants must be kept to a minimum (see also chapter ). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., ) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. ( ) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink - ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, nd edition, aclam series published by academic press. normative data on the mouse are presented in table . , and clinical chemistry reference ranges are summarized in table . . mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about kcal/m per h to maintain body temperature for each °c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water ( . days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from °c to °c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of °c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about . - . °c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at °c were shown to expend more energy compared with mice housed at intermediate ( °c) and a higher temperature ( °c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., ) . in contrast, other studies report that mice in a t a range of - °c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, ) (fig. . loeb and quimby ( ) . a mouse at rest uses about . ml o /g/h, which is about times more o /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o capacity; a slight shift in the o -dissociation curve, enabling o to be unloaded in the tissue capillaries at a high p o ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about μm in diameter, or about half the size of glomeruli in rats. there are, however, . times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table . ). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to mosm/l, whereas humans can concentrate to a maximum of mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about : . . mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter ). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid- s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, ) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in , the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., ) (fig. . ) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf present at - cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at - cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf ), l. salivarius (strain asf ), and bacteroides distasonis (strain asf ), based on phenotypic criteria. s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., ) . the s rrna sequence of strain asf is essentially identical to the s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf is a parabacteroides sp. the spiral-shaped strain, strain asf , is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., ) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf -eubacterium plexicaudatium; asf -firmicutes bacterium; asf and asf -clostridium sp.) (fig. . ) . the s rrna sequence information was determined by dewhirst et al. ( ) and draft genome sequences for each member of asf were recently published (wannemuehler et al., ) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. . ) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l section of the gi tract bacteroides sp. asf the sections are taken from the esophagus (esop.) (section e ), stomach (sections s and s ), small intestine (sections i to i ), ileocecal junction and apical cecum (sections c and c , respectively), and colon (sections l to l ). from sarma-rupavtarm et al. ( ). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be % larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd + and cd + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between and days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, ) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., ) . normal hematologic values are listed in table . . bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, ) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. . ) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from to mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from to /min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c t l s c , with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c bl/ j mouse (sidman et al., ) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., ) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. . ) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. . ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. . ). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, ) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/ / j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day in the male and day in the female. ovarian follicle development begins at weeks of age and matures by days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as - days. puberty is slightly later in the male (up to weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every - days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table . ). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. . ) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about - h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook ( ) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table . ) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within days with a second estrus in about days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. . ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place ( ) testis, ( ) head of epididymitis, ( ) caudal epididymitis, ( ) vas deferens, ( ) testicular vein, ( ) ampullary gland, ( ) seminal vesicle, ( ) anterior prostate, ( ) ureter, ( ) bladder, ( ) ventral prostate, ( ') dorsal prostate, ( ) urethra, ( ) bulbourethral muscle, ( ) ischiocavernosus, ( ) bulbourethral gland, ( ) diverticulum of bulbourethral gland, ( ) penis, ( ) preputial gland, ( ) glans penis, ( ) prepuce, ( ) testicular artery, and ( ) vas deferens artery. adapted from (komarek, ) . fallopian tube, ( ) uterine horn, ( ) endometrium, ( ) cervix, ( ) vagina, ( ) vaginal vestibulum, ( ) clitoris, ( ) clitoral gland, ( ) urethra, ( ) bladder, ( ) medial ligament of bladder, ( ) lateral ligament of bladder, ( ) left ureter, ( ') right ureter, ( ) mesovarium, ( ) mesometrium, ( ) ovarian artery, ( ) uterine horn artery, and ( ) ovarian artery and vein. adapted from (komarek, ) . adapted from komarek ( ) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for - h after ovulation. gestation is usually - days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to - days in certain inbred strains. the effective reproductive life of some inbred strains approaches years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by months of age. average litter size is strain dependent and commonly ranges from to pups. maternal care can account for about % of the variation in body weight of neonatal mice. nursing females usually lactate for weeks. milk production increases up to days postpartum and then declines until weaning at days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., ) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, ) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table . ). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, ; rodriguez and boehm, ) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. ( ) . c ferrero et al. ( ) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., ; sturm et al., ) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., ; chamero et al., ; liberles and buck, ; restrepo et al., ) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome contains a cluster of mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts ( - mg/ml in urine) and are potent allergens for humans, particularly mus m (ag or ma ), which is encoded by the mup gene (sharrow et al., ) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, ) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, ) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, ) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., ) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, ) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., ) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, ; van loo et al., ) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley ) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, ) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr (recognizing lps), tlr / (ss and dsrna) and tlr (dna), nod-like receptors (nod / ), and rig-like receptors (rig-i, mda- ) among others (takeuchi and akira, ) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) , and , which include natural killer (nk) cells (spits et al., ) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b- lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd or cd . cd + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg , igg a/c , igg b , and igg . polymorphisms exist on the ig locus such that some strains of mice produce the igg a subtype (e.g., balb/c), whereas others produce igg c (e.g., c bl/ ) (zhang et al., ) . additional allelic polymorphisms of the locus also exist. for example, balb/c and sv mice express the igh-a allotype, whereas c bl/ mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c bl/ , which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c bl/ mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg a/c is dominant, whereas in extracellular bacterial infections igg dominates the response. igg b and igg are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table . ). these include the large family of interleukins (ils, currently il- to il- ), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., ) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr - /cxcl - and ccr - /ccl - (allen et al., ) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd t cells towards a particular effector response. for example, secretion of il- in response to activation of tlr or will result in the induction of interferongamma (ifn-γ) production by cd t cells, whereas il- and tgf-β production by dc will induce cd t cells to secrete il- (kara et al., ) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., ) . the classic t h /t h dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg a/c, whereas production of il- leads to the secretion of igg . interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd t cells and are induced by the same signals and transcriptional regulators (spits et al., ) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, ) . while initial studies identified two particular antagonistic effector response types (termed t h and t h and classified by t-cell production of ifn-γ and il- , respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., ; sica and mantovani, ; spits et al., ) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table . ). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il- rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd + stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, ) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., ) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table . ) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice ( %) with that of a gene knockout ( %) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd . /cdc . or cd . /cd . (thy . /thy . ). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd or cd as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., ) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter . there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., ) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, ; pritchett-corning et al., ) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter ). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in (marchal, as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih- , washington university, st. louis , beijing , ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c- , hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., a) . during natural epizootics, it was observed that a, bc, dba/ , dba/ , and cba strains developed acute fatal infections, whereas c bl/ mice were resistant to severe disease (briody, ) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/ , and c h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c bl/ mice were highly resistant to lethal infection (bhatt and jacoby, c; wallace and buller, ) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., a; jacoby et al., ) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner ( ) and esteban and buller ( ) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. . ). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., ; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about weeks, virus has been found in scabs and/or feces for up to weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. . ) (fenner, b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, ) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. . ) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, ; jacoby and bhatt, ) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within - days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. . ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs - days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., ) . ectv infection also can be detected by ifa (buller et al., ) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by - days, but not in infected mice (bhatt and jacoby, a) (fig. . ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, , ) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and t cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus ) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd + t cells in mice inoculated within approximately week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes - weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis - days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., ) (pritchett-corning et al., ) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus , but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small ( -kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns- and ns- , which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp- and vp- , are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a cells), c rat glial cells, sv (simian virus )-transformed human newborn kidney ( k cells), t-cell lymphomas (el ), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b- -prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., ; segovia et al., ) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h h -igh null mice (naugler et al., ) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., ; livingston et al., ) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than weeks (smith, ; smith and paturzo, ). infection appears to last less than month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., b) . dba/ mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c bl/ neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., ; naugler et al., ; segovia et al., ) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., ) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp ) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns can be used to detect antibody to both groups, but is less sensitive than vp assays (livingston et al., ) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp or all rodent parvovirus group specific ns exons (besselsen, ; besselsen et al., ) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., ) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., ; livingston and riley, ; pritchett-corning et al., ) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd + t-cell clone designated l and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin (il- ) (mckisic et al., ) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l ) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv- , mpv- , and mpv- . mpv- includes a number of closely related variants, including mpv- a, mpv- b, and mpv- c. in addition, a hamster parvovirus isolate is closely related to mpv- , which is infectious to mice and likely to be of mouse origin (besselsen et al., ; christie et al., ) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., ) . immunologic perturbations are the most likely signs of infection (mckisic et al., ) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b- -prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for weeks, with transmission to sentinels exposed during the first weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., ) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to weeks, whereas transmission by mice inoculated as weanlings occurred during the first weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., ) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., ) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. . ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. . ) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., ) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., ) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., ) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav- or fl) and murine mastadenovirus b (with the representative strain being mav- or k ). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav- can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within days. mav- virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav- . epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav- riley, , pritchett-corning et al., ) . transmission occurs by ingestion. adult mice experimentally infected with mav- may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav- excrete virus in feces for at least weeks but eventually recover. athymic mice can shed mav- for at least weeks and episodically for at least months. pathology infection with mav- causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., ) . infection with mav- produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. . ) . inclusions are easier to detect in infant mice than in adults. infection of c.b- -prkdc scid mice with mav- results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., ) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav- neutralizes both strains, but antiserum to mav- neutralizes mav- weakly at best. therefore, mav- antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav- infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav- infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., ; sebesteny et al., ) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to months in cba mice inoculated with virus as adults (berke and dalianis, ) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c h, c , cba, swr, and others being most susceptible, and c bl/ being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, ) followed by viremic dissemination and acute, lethal disease. tumors appear - months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c bl/ -scid mice (szomolanyi-tsuda et al., ) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, ; pritchett-corning et al., ) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., ) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., ) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f /f -positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c , akr, c h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv- n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv- homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels ( - times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, conventional unit being equivalent to . international units (iu). normal plasma levels are - iu, whereas in ldv infection, levels as high as iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, serum samples were tested by serum ldh enzyme assays, among which % were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., ) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, ) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning - days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are - months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, ; pritchett-corning et al., ) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n- ) cells, bhk- cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter ). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus . viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, ; pritchett-corning et al., ) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly %, and mortality can vary from % to %, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c bl/ mice are highly resistant to clinically apparent infection, whereas dba/ mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice ( - weeks old) and is associated with seroconversion within - days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. . ) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. . ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. . ) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., ) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to weeks, with peak titers occurring at about days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk- cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of - weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., ) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., ) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, ; pritchett-corning et al., ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung - days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within weeks after exposure to virus and are largely resolved by weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. . ) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk- cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., ) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., ) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus , and , which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk- cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus infection in contemporary mouse colonies is rare (livingston and riley, ; pritchett-corning et al., ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus results in a similar distribution but significantly milder lesions compared to reovirus . in contrast, reovirus is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, ; pritchett-corning laboratory animal medicine et al., ) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. . ) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for - weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv ), mhv- , mhv- , mhv-s, and mhv-a . numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc . a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt- cells derived from a rectal carcinoma in a c bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. . ) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. . ) . epizootics of enterotropic mhv have been known to result in % mortality among neonatal mice in a breeding colony. older mice ( - weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, ; pritchett-corning et al., ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. ( ) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within - weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/ mice are highly susceptible to mhv- and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., ) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. . ) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. . ) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. . ) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure . necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. . ) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. . ) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. . and . ). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over °c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk- cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at . - . % of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. . ) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, ; pritchett-corning et al., ) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over months of age. however, neurologic infection can persist in the brain and spinal cord for at least year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in , and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., ; pritchett-corning et al., ) . over mnv isolates have been found in mouse research colonies around the world, which display nearly % genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., ) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw . cell line, as well as a microglial cell line (wobus et al., ) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., ) . experimental infection of and c h mice with mnv- caused mild diarrhea (kahan et al., ) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., ) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c bl/ j, hsd:icr, and jcl:icr and c.b- -prkdc scid mice, with fecal shedding for at least - days (goto et al., ; hsu et al., ; thackray et al., ) . although not clinically ill, rag null mice are unable to clear infection . comparative studies with mnv- and mnv- have shown differences in virus replication and shedding (kahan et al., ) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., ; kahan et al., ; wobus et al., ) . pathology naturally and experimentally infected stat or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., ; ward et al., ) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat null mice (karst et al., ) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., ) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., ) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., ) . embryo transfer and cesarean rederivation are also effective (goto et al., ; perdue et al., ) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., ; compton, ) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., ; leduc et al., ; tsai et al., ) . over cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., ; kawamata et al., ; lloyd et al., ; umenai et al., ) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., ) , infection of wild m. musculus has been documented in the united states (baek et al., ) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., ) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd + t cell (taruishi et al., ) and nk function as demonstrated in human studies (braun et al., ) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately % of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl , musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c h, grs) that carry mtv or mtv loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop % prevalence of thymic lymphoma between and months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to % of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg- gs, c bl -timp- −/− , and upa-nog), but also in immuncompetent strains (b j, icr, bash , and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. ( ) detected high viral load (up to genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., ) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., ) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., ) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, ) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., ) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about . % of conventionally housed mouse colonies in the united states and . % in europe (pritchett-corning et al., ) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c bl background appear to be resistant to pathogenic infection, whereas balb/c, c h, dba/ , swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., ; lai et al., ) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. . ) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. . ) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., ) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., ) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., ) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., ; sun et al., ) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., ) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., ) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, ) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., ) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after - h of rolling, animals become comatose and usually die within h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., ; percy and barthold, ) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within - days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every h, beginning at h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter ). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., ; pritchett-corning et al., ) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. . ) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., ) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. . ) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine ( mg/l) in drinking water may eradicate infection (matsushita and suzuki, ) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., ) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain ), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, ; schauer et al., ) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., ) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. ( ) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., ; collins et al., ) . host genotype can influence the course and severity of disease (barthold et al., ) . for example, dba, nih swiss, and c bl mice are relatively resistant to mortality, whereas c h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, ; newman et al., ) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for - weeks in surviving animals ( fig. . ) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. . ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., ) . barthold et al. ( ) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, ; silverman et al., ) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, ) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., ) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, ) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for - h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., ) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., a; percy and barthold, ) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. . ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., ; boot et al., a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., ) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., ; dole et al., ) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., ) . enrofloxacin in the drinking water at mg/kg daily for days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after days of treatment there were no detectable carriers when the colony was screened weeks later (matsumiya and lavoie, ) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c bl/ ) stimulated transcription of multiple proinflammatory cytokines for at least days with residual elevation detectable days later (patten et al., ) . pioneering studies conducted in the s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , ward et al., ) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. . ). subsequently, it was associated with inflammatory bowel disease in several murine models (table . ) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. . ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, ; fox et al., ) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , ward et al., ; suerbaum et al., ) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, ) . to date, the genus includes formally named helicobacter spp. assigned on the basis of s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere ( % co , % n , % h ). there are currently formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. ( ) tcrα, β mutants defective t-receptors typhlocolitis chin et al. ( ) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. ( ), shomer et al. ( c bl/il- −/−c lacks il- typhlocolitis burich et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) c blrag mice infected with h. bilis also developed ibd (shomer et al., ) . c ibd also developed in c bl/il- −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., ) ; also ibd produced with h. trogontum (whary et al., ) and h. cinaedi (shen et al., ) . d h. bilis produces ibd (maggio-price et al., and colon cancer (maggio-price et al., ) . have been formally named (except for h. rappini) (fox and lee, ; franklin et al., ; whary and fox, ) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., ) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., ) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., ; fox et al., b; taylor et al., ; lofgren et al., ) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., ; knutson et al., ; shomer et al., ; erdman et al., b; nguyen et al., ) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table . ). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. . ) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b c f mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b c f mice. in this context, a/jcr, c h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c bl/ mice are resistant (ward et al., ) . the finding of severe liver disease and tumor induction in b c f mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., ; fox and lee, ; ihrig et al., ; franklin, ; hillhouse et al., ) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., a; shames et al., ; beckwith et al., ) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a . -μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at . μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, ) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., ) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., ) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il- −/− and scid mice (franklin et al., (franklin et al., , fox et al., ) . decreased reproductive efficiency has been reported in il knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., ) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for weeks (del carmen martino-cardona et al., ) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, ) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., ) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., ) . h. hepaticus infections provoke a strong th proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b c f , ab f , b af , and carko mice (hailey et al., ; fox et al., a; garcia et al., garcia et al., , h. salmonellosis (ganaway, ; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately serovars and are a major cause of food-borne illness in humans (fookes et al., ) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., ) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il- , il- β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, ) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, ) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., e; percy and barthold, ) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, ) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., ) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., ; weisbroth, ; percy and barthold, ) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., ; scanziani et al., ; dole et al., ) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c bl/ mice (mcwilliams et al., ) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. . ) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., ) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., ) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., ) . antibiotic treatment has been unrewarding (burr et al., ) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., ) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c bl/ , c h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. . ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, ) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., ) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., ) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, ) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., ; arthur et al., ) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b c f mice (davis et al., ; rao et al., ) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep- and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., ) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c h/hej and nmri-foxn (nu) mice (bleich et al., ) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il rg tm wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., ) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap /bi/ ) associated with frequent recurrences and more severe clinical disease (abou chakra et al., ; mcfarland, ; kuijper et al., ) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., ) , guinea pigs (lowe et al., ) , rabbits (thilsted et al., ; ryden et al., ) , prairie dogs (muller et al., ) , ostriches (frazier et al., ) , and horses (diab et al., ) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at - °c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, ) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than h postingestion. when c bl mice were challenged with cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by h postchallenge (chen et al., ) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., ) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of mg vancomycin for days to experimentally infected gnotobiotic mice caused a -to -log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., ) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., ) . a c bl mouse model of recurrence/relapse cdi has been reported (sun et al., ) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., ) . a recent study in c bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., ) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring - µm in length and . - . µm in diameter (murray et al., ) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., ) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., ) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, ; rozengurt and sanchez) . disease has been observed in mice of both sexes, from to days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, ) , type b (rozengurt and sanchez, ) , and type d (clapp and graham, ) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., ) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of mg/l for weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, ) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, ; mcgorum et al., ) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, ) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in -to -week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, ; ganaway, ; ganaway et al., ; percy and barthold, ) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., ; kawamura et al., ) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c bl/ mice are more resistant than dba/ mice to tyzzer's disease (waggie et al., ) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., ) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., ) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. . ) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., ) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c bl/ mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, ) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., ) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter ). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, ) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in , p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, ) . it was renamed as a new species, p. carinii, when observed in a rat in (delanoë, p. and delanoë, m. ) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., ; stringer et al., ; wakefield et al., ) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., ; frenkel, ) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., ; shultz and sidman, ; walzer et al., ; weir et al., ) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., ) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., ) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, ; cushion, ) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. . ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. . ) (roths et al., ) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened -to -mm cysts in affected parenchyma (fig. . ) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., ) . pcr can be used to detect active infection (gigliotti et al., a; reddy et al., ) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., ; roths et al., ) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature ( - °c), and growth is observed at - days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the s found the rates exceeding %. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., ) . c h/he mice are particularly susceptible to giardiasis, whereas balb/c and c bl/ mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, ) . c bl/ females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, ) and by pcr (mahbubani et al., ) . treatment, prevention, and control murine giardiasis can be treated by the addition of . % dimetridazole to drinking water for days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., ) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at - weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., ) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded % among domestic mouse colonies in the mid- s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., ; brett and cox, ) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., ) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding . % dimetridazole to drinking water for days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., ) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, ; taylor et al., ) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, ) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., ) . adults are extremely small ( - mm) and have eggs with prominent polar filaments and rostellar hooks (fig. . ) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen - days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in weeks. the cycle from ingestion to patency takes - days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel ( . % in the diet for days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter ). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, ) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from . to . mm in length, and male worms are smaller ( . - . mm). eggs are flattened on one side and have pointed ends (fig. . ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than % of barrier colonies and about % of conventional colonies were affected (jacoby and lindsey, ; carty, ) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in - days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within h. worms remain in the cecum for - days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., ) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to -weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., ) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., ) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., ; le blanc et al., ; lipman et al., ; flynn et al., ; wescott, ; zenner, ) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., ; skopets et al., ; toth et al., ) . fenbendazole diets can be fed with week on/ week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., ; gadad et al., ; landin et al., ) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are . - . mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. . ) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for - days. they move to the proximal colon about weeks after infection of the host. because the life cycle is - days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in - weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately - days. mature females inhabit the large intestine, where they survive from to days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require - days at °c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a, ,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a, ,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table . . these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, ; carty, ) reported mite infestations in % of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in % koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., ) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., ) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. . ) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. . ) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. . ) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., ) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., ) , especially those involving skin, and has been shown to elevate serum ige (morita et al., ) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in days and are usually attached to the middle third of the hair shaft. the life cycle may range from to days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within - days of birth, and parasites may live for - days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in - days. larval forms last about days, followed by nymphal forms on day . adults appear by day and lay eggs within h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in - weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to days. infestation may result in hypersensitivity dermatitis, to which c bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, ; fox, ) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter ). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, ) . primary amyloidosis is common among aging mice (lipman et al., ) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c bl mice. other strains, such as balb/c and c h are highly resistant to amyloidosis (percy and barthold, ) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., ) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. . ) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c h, and especially dba mice (eaton et al., ; brownstein, ; brunnert et al., ) . it is found in the myocardium of the left ventricle ( fig. . ) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, ) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc gene. knockout abcc −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., ) . other inbred mouse strains, including kk/hlj and s /svimj, also develop vibrissae dermal mineralization and have an snp (rs ) in the abcc gene associated with low levels of abcc protein expression in the liver. dba/ j and c h/hej mice have the same polymorphism and low abcc protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., ) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within - h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv- (pirofski et al., ) . deficiencies (tobin et al., ) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out - years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, ) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, ) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, ) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c bl/ background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at % of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, ) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, ; ward, ) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c bl/ and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. . ) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., ) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, ) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c bl/ mice free of known pathogens (feinstein et al., ) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. . ). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c black mice (koch and gowen, ) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c black mice (pierro and spiggle, ) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day of gestation (koch and gowen, ) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., ) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. . ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., ) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., ) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., ) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., ; percy and barthold, ) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (< % in -year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b c f mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., ) . myocardial and epicardial mineralization is described above (section iii,b, ). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/ and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., ) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c bl, s /svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym and/or ym chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., ) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., ) , chemical injury (yadav et al., ) , and gastritis and gastric tumors by helicobacter infection (fox et al., ) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c bl/ is normal and is melanosis caused by melanin deposition (weissman, ) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table . ). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches %. nzb and nzb × nzw f hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as months of age, but clinical signs and severe disease are not present until - months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than months is %. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b c f mice from national toxicology program -year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., ) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., ) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b c f mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c bl/ mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., ) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of . months (range, - months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as %) developmental defects in inbred and congenic strains of c bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., ) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., a) . other strains such as c h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, ) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., ) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., (jones et al., - maronpot et al., ; percy and barthold, ) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of - %. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr , tlr , and tlr can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., ) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c bl/ and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu. , blocking erythroid cell differentiation, and in conjunction with the loss of p tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., ) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., ) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c h, a, and dba/ , have a high natural prevalence of mammary tumors. other strains, such as balb/c, c bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c h mice. if surgery is done in adult mice - months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. . ) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c bl apc +/− female mice infected with h. hepaticus (rao et al., ) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., ) . the prevalence of spontaneous liver tumors in b c f hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., ) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., ) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than % of these tumors are pulmonary adenomas that arise either from type pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than strains and substrains, tissues and organs, over , tumor frequency records, and nearly histopathological images and descriptions. risk factors for recurrence, complications and mortality in clostridium difficile infection: a systematic review detection of 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astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg a and igg c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas mediated genome engineering key: cord- -cog nma authors: watkins, kevin title: emerging infectious diseases: a review date: - - journal: curr emerg hosp med rep doi: . /s - - - sha: doc_id: cord_uid: cog nma purpose of review: this review highlights some of the recent concerning emerging infectious diseases, a number of them specifically that the world health organization has categorized as priorities for research. recent findings: emerging and reemerging infectious diseases account for significant losses in not only human life, but also financially. there are a number of contributing factors, most commonly surrounding human behavior, that lead to disease emergence. zoonoses are the most common type of infection, specifically from viral pathogens. the most recent emerging diseases in the usa are emergomyces canadensis, the heartland virus, and the bourbon virus. summary: in addition to the aforementioned pathogens, the severe acute respiratory syndrome, middle east respiratory syndrome, nipah virus, new delhi metallo-ß-lactamase- enterobacteriaceae, rift valley fever virus, and crimean-congo hemorrhagic fever virus are reviewed. these pathogens are very concerning with a high risk for potential epidemic, ultimately causing both significant mortality and financial costs. research should be focused on monitoring, prevention, and treatment of these diseases. in , an expert committee that produced the institute of medicine report on emerging infections defined them as "new, reemerging, or drug-resistant infections whose incidence in humans has increased within the past two decades or whose incidence threatens to increase in the near future." additionally, six major contributors to these diseases were presented and included changes in human demographics and behavior, advances in technology and changes in industry practices, economic development and changes in land-use patterns, dramatic increases in volume and speed of international travel and commerce, microbial adaptation and change, and breakdown of public health capacity [ ] . a common theme to recent emerging infectious diseases is that the majority are of animal origin [ ] . in fact, some vector-borne pathogens that are projected to be introduced into new regions include rift valley fever and japanese encephalitis viruses in the americas and crimean-congo hemorrhagic fever virus in eurasia [ ] . additionally, the majority of them have been viral. in addition to the cost of human life, emerging or reemerging infectious diseases can be very costly financially. the recent zika outbreak is estimated to have cost $ . billion in economic losses in [ ] . the world health organization has prioritized a number of infectious diseases as requiring urgent need for research and development given the concern for potential of severe outbreaks. this article reviews the majority of emerging infections on this list, a few others that have emerged in the usa as well, and those that have not recently emerged but that the who has prioritized as having a high likelihood of causing outbreaks in the future. in , an outbreak in southern china of atypical pneumonia of undetermined etiology spread to neighboring countries and eventually across the world. the coronavirus that caused the severe acute respiratory syndrome (sars) was then discovered [ ] . hong kong and beijing were the most severely affected cities with estimated costs in the far east of this article is part of the topical collection on infectious disease * kevin watkins kwatkin @gmail.com approximately $ billion by may alone [ ] . over probable cases were reported in countries with a mortality rate of about % by the end of the epidemic in july . sars reemerged in small scales from the end of to . it is a positive-sense rna coronavirus whose mean incubation period is days [ ] . the clinical presentation varied by patient age: children developed typical mild upper respiratory tract infection while teenagers and adults developed a more severe but predictable course [ , ] . phase i of the course was associated with increasing viral load with flu-like symptoms. phase ii was characterized by recurrence of fever with hypoxemia and progression of pneumonia. about % would require supplementary oxygen and approximately % would require intubation due to the development of acute respiratory distress syndrome (ards). watery diarrhea was a prominent extrapulmonary manifestation. additionally, hepatitis was a common complication and patients occasionally developed neurologic manifestations including status epilepticus. the clinical worsening in phase ii is thought to be immune-mediated. laboratory findings include lymphopenia, elevated aminotransferases, elevated lactate dehydrogenase (ldh), and creatine kinase (ck), with results consistent with disseminated intravascular coagulopathy. progression from unilateral opacities to bilateral involvement was found on imaging. sars is detected in respiratory secretions, urine, and feces [ ] . however, the most notable finding is diffuse alveolar damage [ ] . advanced age, severe hepatitis, high initial ldh, high neutrophilia on presentation, diabetes mellitus, low cd and cd counts, and high initial viral load are all prognostic factors for severe disease. some survivors have shown persistent lung function abnormalities. it is spread by close contact via droplet transmission, though there is some evidence that it might follow airborne transmission [ ] . the virus has a high rate of infecting healthcare workers, accounting for approximately % of cases [ ] . the original outbreak was thought to have started from the handling of wild animals, particularly the civet cat and raccoon dog [ , ] . if suspected, a patient should be placed in respiratory isolation. a single test result, either positive or negative, is insufficient to make conclusions about a patient's diagnosis as both false positives and false negatives occur. it is initially tested for via serology or reverse-transcriptase-polymerase chain reaction (rt-pcr). however, two specimens obtained from either different sources or from the same source on different occasions confirm the diagnosis. specimens can be obtained from a nasopharyngeal swab or stool. additionally, seroconversion is another diagnostic method. it is vital to identify patients with sars, as isolation within days of illness significantly reduces secondary transmission [ ] . although ribavirin was used in the initial outbreak, it has not been shown to have in vitro activity against the virus. corticosteroids should be used only in the late-phase for rescue purposes as it may prolong viremia if given early [ ] . most of the other agents that have shown promise, including monoclonal antibodies, nitric oxide, protease inhibitors, and interferons have not yet shown activity in humans and there have been very few animal studies [ , ] . part of the concern about the potential for future epidemics lies in the fact that the family of viruses from which sars comes is notorious for frequent mutations [ ] . additionally, there has been decreased worldwide attention, leading to decreased funding for research, as there have been no reported cases of sars since [ ] . the middle east respiratory syndrome coronavirus (mers-cov) was first identified in . it is a ß coronavirus that is enveloped with a positive-sense rna genome. the clinical features may include flu-like symptoms, gastrointestinal symptoms, severe pneumonia with acute respiratory distress syndrome, septic shock, disseminated intravascular coagulopathy, and multi-organ failure [ ] . lab findings may include lymphopenia, thrombocytopenia, elevated ldh levels, and elevated aminotransferases [ , ] . however, approximately % of cases had no or mild symptoms while almost % had severe symptoms [ ••] . the incubation period is - days. children and younger adults seem to be less susceptible [ ] . a large proportion of the confirmed pediatric cases have been asymptomatic and have been acquired via household contacts [ ] . primary infection results from contact with a dromedary camel or its products, though human-to-human transmission is possible. cases acquired via human transmission tend to be more mild and the number of infected contacts tends to be limited. in fact, the majority of secondary cases have been associated with healthcare settings, where the majority of outbreaks have occurred and about % of cases have been healthcare workers [ ] . with the exception of south korea, outbreaks have been limited to the middle east where dromedary camels are found [ ] . in the south korean outbreak, there were cases and deaths [ ••] . cases have been imported to europe, asia, africa, and the usa [ table ]. the cases in the usa were detected promptly, so no secondary cases occurred. both cases occurred in may and were healthcare workers in saudi arabia [ ] . as of july , there have been laboratory-confirmed cases, with the majority of these in saudi arabia. the reproduction number is less than with significant heterogeneity [ ••, ] . in the south korean outbreak, "spreaders" had significantly higher rates of underlying pulmonary disease. most transmissions occurred early in the disease course, during days - after symptom onset and were associated with patients going to multiple healthcare facilities before diagnosis [ ] . human-to-human transmission seems to require close contact, though has not been sustained. most cases requiring hospitalization are those with chronic comorbidities, such as obesity, diabetes, end-stage renal disease, or chronic lung disease [ ] . the mortality rate is about % [ ••] . among those who were critically ill, patients frequently had underlying chronic disease, and extrapulmonary manifestations were common [ ] . mers can be detected in respiratory tract secretions with bronchoalveolar lavage specimens providing a better yield than nasopharyngeal swabs; however, serum samples may also be obtained [ ] . the centers for disease control and prevention (cdc) recommends evaluation for patients with respiratory symptoms and travel to an affected country within days or for those who are in a cluster of patients with severe acute respiratory disease in which mers-cov is being evaluated [ ] . testing should not be performed before h of exposure [ ] . healthcare workers with exposure during aerosol-generating procedures are at high risk for transmission; because of this, the cdc recommends airborne and contact precautions for suspected or known cases [ ] . patients suspected to have mers should be admitted if they have shortness of breath, pneumonia, or hypoxemia. otherwise, they may be cared for at home in isolation; however, these cases should be carefully selected for potential of spread [ ] . treatment is primarily supportive with mechanical ventilation and renal replacement therapy as needed as there are no specific antivirals that have been developed [ ] . a number of studies have been performed with a variety of medications, including interferons (ifn), ribavirin, protease inhibitors, mycophenolic acid, cyclosporin a, chloroquine, nitazoxanide, antibiotics, fusion inhibitors, and steroids. perhaps the most promising human studies have involved a combination of ribavirin and interferons, though overall results have been mixed. some have shown no benefit, but have been critiqued that treatment was too late. other studies have shown improved -day survival but not at days, and others have shown improved survival at both [ , , ] . a small retrospective study had all eight patients who received combination therapy with ifn-ß and mycophenolic acid survive, though they had lower acute physiology and chronic health evaluation ii scores compared to patients who received other agents [ , ] . two new viruses that have recently been discovered in the usa are the heartland and bourbon viruses. the heartland virus is a member of the bunyaviridae family in the genus phlebovirus with single-stranded, negative-sense rna [ ] . the lone star tick (amblyomma americanum) transmits the disease [ ] . it was first discovered in from a farmer in missouri. clinical features include flu-like symptoms, leukopenia, thrombocytopenia, and mild transaminitis and develop within days of tick bite. six additional cases were identified during - ; four of these patients were hospitalized and one patient with multiple underlying diseases died. five patients were in missouri and one was in tennessee [ , ] . the deceased patient had developed hypoxemic respiratory failure with severe thrombocytopenia, acute kidney injury, and upper gastrointestinal bleeding and his family ultimately opted to transition to comfort measures. his clinical course was consistent with severe infection from ehrlichia chaffeensis except that he did not improve with doxycycline [ ] . diagnosis should be suspected when a patient does not improve with doxycycline therapy and can be made with detection of viral rna by rt-pcr [ •]. the bourbon virus is in the genus thogotovirus within the family orthomyxoviridae and is pleomorphic with an rna genome. the case patient was from bourbon county, kansas, in . within several days of tick bite, he developed flu-like symptoms. later findings included leukopenia, thrombocytopenia, hyponatremia, and mild transaminitis; the case patient then developed congestive heart failure and later died after withdrawal of care [ ] . a study of tick populations in missouri revealed that amblyomma americanum ticks were carriers of the virus, though transmission from these ticks is not certain [ ] . little is known otherwise about the disease as confirmed cases have been scarce. nipah encephalitis was first noted in an outbreak of fatal encephalitis starting in in malaysia. it is an rna virus from the family paramyxoviridae, genus henipavirus. the outbreak initially involved pig-farming villages but spread to other areas [ ] . since discovery, outbreaks have been recognized almost yearly in bangladesh and occasionally in india [ ] . it is characterized by a high attack rate with neurologic features at presentation. the strain in malaysia rarely displays respiratory symptoms, though the more concerning bangladeshi strain often has a respiratory component [ , ] . prodromal symptoms consisting of sore throat, myalgias, fever, headache, and vomiting are common with the ultimate development of altered mental status and potentially seizures and coma. the majority of patients show brainstem dysfunction with pinpoint pupils, dysautonomia, and abnormal vestibulo-ocular reflex. myoclonus may also be present and characteristically involves the diaphragm and anterior neck muscles. patients commonly develop thrombocytopenia and abnormal liver function tests. cerebral spinal fluid analysis yields normal glucose with elevated white cell counts and protein typical of viral infections. diagnosis can be made by rt-pcr. most patients with encephalitis had abnormal electroencephalograms and magnetic resonance imaging studies (mri). mris commonly show small discrete hyperintense lesions in the subcortical and deep white matter [ ] . pathophysiologically, nipah virus causes a widespread vasculitis most commonly involving the brain and lungs [ ] . mortality rates have been quoted to be between and %. the majority of people infected develop severe disease. neuropsychiatric sequelae are not uncommon (one third of survivors) with persistent cognitive impairment, cerebellar signs, and peripheral nerve lesions [ , ] . relapse and late-onset encephalitis may also occur months or years after the acute illness, though rates are rare (about %). additionally, relapse or late-onset disease has a lower mortality rate of about % with minimal brainstem involvement. transmission can consist of consumption of food contaminated by bat saliva (raw sap is common), contact with infected animals, or human-to-human spread [ , ] . sustained human-to-human transmission beyond generations has not been recognized and the reproductive number has averaged . . those that are at greatest risk of infection are providers of fatally infected patients with prominent respiratory secretions. however, most patients do not transmit infection to anyone [ ] . bats of the family pteropodidae are the natural reservoirs, though numerous mammals can become infected. treatment consists of supportive care. treatment with ribavirin was tried during a malaysian outbreak with % reduction in mortality; however, this was not a controlled clinical trial and did not reach statistical significance [ ] . the concern about a potential for pandemic arises from the fact that it is an rna virus and the bangladeshi strains have high genetic variability with potential for an increase in the reproductive number [ ] . an experimental human monoclonal antibody (m . ) has shown promise in nonhuman studies [ ] . another emerging infectious disease is the dimorphic fungus, emergomyces canadensis. although other emergomyces species have been discovered throughout europe and africa, e. canadensis was only recently discovered in north america [ ] . emergomyces was previously named "emmonsia-like" pathogens. there are numerous species of emergomyces, with the most common being e. africanus that causes infections in those with hiv in south africa. no animal infections have been documented [ ] . there have been four cases noted so far in immunocompromised patients that developed systemic disease with fungemia. patients commonly had pneumonia as well as skin lesions. it is suspected that inhalational infection occurs and that geographic range involves central and western north america [ ] . the diagnosis is made via histopathology or culture of affected tissue [ ] . limited susceptibility testing has shown susceptibility to itraconazole and amphotericin b. treatment should follow typical guidelines for dimorphic fungal infections in immunocompromised hosts with amphotericin b for - weeks followed by itraconazole for months [ ] . one of the most concerning emerging infectious diseases is the n e w d e l h i m e t a l l o -ß -l a c t a m a s e - ( n d m - ) enterobacteriaceae. these "superbugs" seemed to have originated in the indian subcontinent and cases were first reported in , though retrospective analysis shows its presence as early as . the ndm- gene is carried on a plasmid, bla ndm- , that is easily spread among bacteria [ ] . although carbapenamases are not new, the level of promiscuity of ndm- is higher than others [ ] . additionally, studies have shown both a high level of inter-lineage and inter-species gene transfer though the dominant mechanism seems to be the latter. it is most commonly found among escherichia coli and klebsiella pneumoniae, but has also been found within all species of enterobacteriaceae as well as acinetobacter baumanii and pseudomonas species [ ] . although gram-negative bacteria have typically been treated with ß-lactams, fluoroquinolones, and aminoglycosides, ndm-positive pathogens contain up to antibiotic resistance genes and are only susceptible to tigecycline and colistin, with the exception of a few strains sensitive to aztreonam and certain aminoglycosides [ ] . multiple variants have been reported (i.e., ndm- ). ndm- is widely found throughout both hospital strains as well as community strains in india, which is consistent with the rates of extendedspectrum ß-lactamase (esbl) of % in both the community and hospital setting [ , ] . it is likely that the overuse of antibiotics led to excretion into the waste water systems, ultimately resulting in selection-pressure for multidrug-resistant organisms [ , ] . in fact, some estimate that at least million indian residents have gut flora consisting of ndm- containing bacteria [ ] . ndm- has spread to numerous other countries; it is believed that the medical tourism industry propagated its dissemination. in fact, seven out of eight tourists to india were colonized with esbl-positive bacteria upon return, which they did not have upon departure [ ] . the balkans have been another area with high rates of ndm-positive bacteria, which has now spread to all continents except south america and antarctica [ fig. from ref- erence ] ]. ndm-positive bacteria have even now been found in companion animals in the usa [ ] . adding to the concern is that there are few antimicrobials in the research pipeline with activity against gram-negative pathogens. rift valley fever virus (rvfv) is a single-stranded rna virus of the genus phlebovirus, family bunyaviridae. the majority of infected individuals are asymptomatic. most patients who are symptomatic have a self-limiting febrile illness; however, a small minority develop severe disease with acute hepatitis, renal failure, and hemorrhagic manifestations. those who survive this phase often develop neurologic symptoms consisting of vision loss and encephalitis. long-term sequelae may develop with blindness, hemiparesis, quadriparesis, incontinence, hallucinations, or coma. the fatality rate is - %, though patients with hemorrhagic disease have a fatality rate as high as %. diagnosis is made by detection of viral rna and immunoglobulin serology, particularly during the acute febrile stage [ ] . it is often transmitted by mosquitoes, typically of the aedes genus, though exposure to infected animal tissue or consumption of their products can also cause disease [ ] . rvfv is endemic in sub-saharan africa with periodic outbreaks after heavy rainfall and flooding. it has spread to the middle east and the french island of mayotte. outbreaks often have significant effects on both humans and livestock. characteristics of an rvfv outbreak include the sudden increase in abortions and mortality of young animals with the appearance of disease in humans. models have been developed with successful predictions of outbreaks using satellite imagery of sea surface temperatures, rainfall, and vegetation. given this spread out of africa, there is concern about it reaching the mediterranean basin and europe. a number of competent vectors for rvfv have been identified in europe. it has additionally been isolated from other hematophagous arthropods including ticks and sand flies, though their significance is not currently understood. there is currently no specific treatment, though a vaccine, tsi-gsd- , has provided protection following primary inoculation and single boost schedule [ ] . crimean-congo hemorrhagic fever virus (cchfv) produced its first major known outbreak from to in the crimean peninsula, though it has likely been causing outbreaks for centuries. it was not identified until . as such, it has not recently emerged but is listed as a priority agent for research and development by who due to its potential for spreading and causing more severe outbreaks. cchfv is a member of the bunyaviridae family, genus nairovirus. it has many aliases, including asian ebola, karakhalak (black death), khunymuny (nose bleeding), and khungribta (blood taking). cchfv derives its name from outbreaks both in crimea and the belgian congo and has been reported throughout many parts of africa, the middle east, europe, and asia [ fig. from reference ]. crimean-congo hemorrhagic fever (cchf) is the most widespread tick-borne viral infection of humans [ ] . it is a negative-sense rna virus transmitted by numerous tick species; because of this, agricultural workers are most commonly affected [ , ] . additionally, human-to-human transmission may occur via contact with skin, mucous membranes, or body fluids of infected patients [ ] . standard barrier precautions are recommended for patients with suspected disease [ ] . domestic livestock are the main reservoirs. outbreaks have been increasing in recent years. likely targets of the virus are endothelial cells, hepatic cells, and kupffer cells. the clinical course of cchf follows phases consisting of incubation, prehemorrhagic, hemorrhagic, and convalescence [ ] . there is a spectrum of severity from a mild, febrile illness to hemorrhagic shock with multiorgan failure [ ] . the incubation period varies by method of acquisition; it typically takes - days by tick bite and - days by exposure to infected patients. the prehemorrhagic stage is nonspecific with flulike symptoms. the hemorrhagic stage follows and develops within - days of symptoms in severe disease [ ] . one differentiating feature is that severely ill patients often develop a pattern of large ecchymosis not seen in other hemorrhagic fevers [ ] . laboratory findings include leukopenia, thrombocytopenia, elevated aminotransferases, elevated ck and ldh, fig. distribution of crimean-congo hemorrhagic fever virus and prolonged prothrombin time and activated partial thromboplastin time. convalescence usually occurs - days after onset of illness [ ] . diagnosis is commonly made via detection of viral rna with rt-pcr or serology. there has traditionally been no specific treatment for cchf, though ribavirin has demonstrated both in vitro and in vivo activity [ ] . furthermore, both the cdc and who recommend use of ribavirin [ •] . a report from turkey claimed that a combination of methylprednisolone, intravenous immunoglobulin (ivig), and freshfrozen plasma was beneficial, but there was no control group for comparison [ ] . ivig and methylprednisolone may be beneficial by improving thrombocytopenia [ •, ] . two vaccines have been developed, though they have not been through randomized clinical trials [ ] . cchf has never been demonstrated in vaccinated individuals and the bulgarian ministry of health has reported a fourfold decrease in cases since vaccine implementation [ , ] . ribavirin might be beneficial for post-exposure prophylaxis [ ] . mortality rates have varied between and %, with worsening prognosis if the disease is acquired nosocomially, with higher levels of aminotransferases, higher viremia, or with coagulopathy [ , ] . long-term sequelae have been documented but are rarely permanent and include impaired vision and other neurologic symptoms [ •] . cchf has epidemic potential with high rates of mortality, nosocomial infection, and difficulty with prevention and treatment [ ] . additionally, it is the most genetically diverse of the arboviruses [ ] . attempts to control cchf via eradication of tick vectors have proven inefficient and domestic animals are asymptomatic even when highly viremic [ • ]. infectious diseases promise to be one of the biggest challenges in the coming decades, with new ones emerging unpredictably. successful clinical outcomes require a proactive approach with research and development, as human behavior contributes to new infections emerging, particularly with zoonoses. although this strategy would prove costly, this article has demonstrated a few examples of how much an emerging infectious disease can cost during an outbreak. this article has reviewed a number of emerging infections that the who has categorized as priorities for research and development due to their potential for epidemics or even a pandemic. additionally, it has reviewed a concerning drug-resistance mechanism that threatens to provide bacteria with resistance to all antibiotics. finally, a few infectious diseases that have emerged in the usa have been covered, though little is known about them, with a paucity of cases to date. conflict of interest the author declares that he has no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. emerging infectious diseases: a cdc perspective emerging infectious diseases in : years after the institute of medicine report emerging infectious disease: a proactive approach a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome (sars): a review of the history, epidemiology, prevention, and concerns for the future severe acute respiratory syndrome and coronavirus severe acute respiratory syndrome (sars) severe acute respiratory syndrome and influenza: virus incursions from southern china who guidelines for the global surveillance of severe acute respiratory syndrome (sars) anti-sars coronavirus agents: a patent review ( -present) middle east respiratory syndrome coronavirus: a comprehensive review middle east respiratory syndrome: what clinicians need to know middle east respiratory syndrome coronavirus: update for clinicians this article gives the majority of a summary of perhaps most significant recent emerging infectious disease in terms of both mortality and number of cases middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia risk factors for transmission of middle east respiratory syndrome coronavirus infection during the outbreak in south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus a review of treatment modalities for middle east respiratory syndrome treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia a new phlebovirus associated with severe febrile illness in missouri notes from the field: heartland virus disease-united states heartland virus associated death in tennessee update and commentary on four emerging tick-borne infections novel thogotovirus associated with febrile illness and death bourbon virus in field-collected ticks nipah encephalitis-an update the pandemic potential of nipah virus therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody emergomyces canadensis, a dimorphic fungus causing fatal systemic human disease in north america emergomyces: a new genus of dimorphic fungal pathogens causing disseminated disease among immunocompromised persons globally global spread of antibiotic resistance: the example of new delhi metallo-ß-lactamase (ndm)-mediated carbapenem resistance the new medical challenge: why ndm- ? why indian? emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study the emergence of pan-resistant gramnegative pathogens merits a rapid global political response rift valley fever virus: a review of diagnosis and vaccination, and implications for emergence in europe risk factors associated with human rift valley fever virus infection: systematic review and meta-analysis crimean-congo hemorrhagic fever crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity crimean-congo haemorrhagic fever virus: past, present and future insights for animal modelling and medical countermeasures recent advances in research on crimean-congo hemorrhagic fever key: cord- -m nb kf authors: vignier, nicolas; bouchaud, olivier title: travel, migration and emerging infectious diseases date: - - journal: ejifcc doi: nan sha: doc_id: cord_uid: m nb kf emerging infectious diseases (eid) threaten public health and are sustained by increasing global commerce, travel and disruption of ecological systems. travelers could play a role in importing eids and could be a sentinel of major epidemics. in connection with the extension of poverty, urbanization, extensive livestock rearing and globalization, we could be exposed to a third epidemiological transition characterized by zoonotic diseases and infections with multidrug-resistant bacteria. the risk appears low for emerging infectious diseases, or very low for high-risk emerging infectious diseases, but higher for multidrug-resistant enterobacteriaceae carriage with possibly limited consequences. the role played by migrants is weaker than imagined. immigrants don’t play the role of sentinel epidemic so far. they could play a role in importing multidrug-resistant enterobacteriaceae, but it is poorly evaluated. emerging infectious diseases (eid) threaten public health and are sustained by increasing global commerce, travel and disruption of ecological systems. travelers could play a role in importing eids and could be a sentinel of major epidemics. in connection with the extension of poverty, urbanization, extensive livestock rearing and globalization, we could be exposed to a third epidemiological transition characterized by zoonotic diseases and infections with multidrugresistant bacteria. the risk appears low for emerging infectious diseases, or very low for high-risk emerging infectious diseases, but higher for multidrug-resistant enterobacteriaceae carriage with possibly limited consequences. the role played by migrants is weaker than imagined. immigrants don't play the role of sentinel epidemic so far. they could play a role in importing multidrug-resistant enterobacteriaceae, but it is poorly evaluated. emerging infectious diseases (eids) have led to cooperation between countries, the first international epidemic response conference in and the establishment of who in . eids are diseases that have appeared recently or that have recently increased in frequency, geographical distribution or both ( ) . since the end of the th century, there has been a constant stream of newly identified pathogens and an increasing occurrence of pandemic threats to global health ( ) . eids threaten public health and are sustained by increasing global commerce, travel and disruption of ecological systems and in particular urbanization. urbanization is characterized by rapid intensification of agriculture, socioeconomic change, and ecological fragmentation, which can have profound impacts on the epidemiology of infectious disease ( ). however, their interactions with travel and migrations are less well known. travelers could play a role in importing eids and could be a sentinel of major epidemics. in france, there are more than million travelers every year, . million of which are destined for areas at high risk for health. there are several modes of travel: tourist, business or visiting friends and relatives. trips can be very short or extended in time. infectious diseases are rare health events, with the exception of common infectious diseases such as traveler's diarrhea and are a single cause of death, far behind accidents and cardiovascular disease ( ) . the risk of emerging infections such as dengue in a risk zone was estimated at % for one month of travel ( ). we have seen (re-)emergence of diseases imported by travellers in europe, such as chikungunya and dengue in france and itay, and malaria in greece ( ) ( ) ( ) . apart from these examples, these are rare situations. however, with global travel growth, the risk could become more tangible ( ) . a particular concern is that of multidrug resistant enterobacteriaceae (mre) carriage. mre acquisition is very frequent among travellers to tropical regions ( ) . the acquisition was higher in asia ( %) than in sub-saharan africa ( %) or latin america ( %). however, the same study showed that mre carriage was limited in time and disappeared after a few months. migration is a global phenomenon that influences the health of individuals and populations over the course of their lives ( ) . migrants are special travellers who, in most case, do not migrate by choice. migrants are considered at higher risk for a range of health problems including infectious diseases as hiv, hepatitis b, tuberculosis, schistosomiasis and malaria ( , ) . this higher risk is partly due to poor socioeconomic conditions and, in some countries, is due to the lack of rights to health coverage for undocumented migrants ( ) ( ) ( ) ( ) . existing evidence from different european countries highlights the difficulties to access health services that migrants are facing ( ) ( ) ( ) ( ) . these infectious diseases unequally expose the majority nicolas vignier, olivier bouchaud travel, migration and emerging infectious diseases population, from none at all (e.g., malaria) to a little (e.g., tuberculosis). one can take the examples of epidemics of middle east respiratory syndrome coronavirus -merscov-and ebola, for which no secondary case has been reported in france. among the published studies on migrants and infectious diseases, the majority were non-emergent diseases with the exception of mdr tuberculosis and multidrug-resistant bacteria ( , ) . in connection with the increased use of antibiotics in low-resource countries, there is a worrying increase in the prevalence of multidrug-resistant bacteria ( , ) . this increase could lead to an increased risk for migrants and their relatives, but there are few data on this point ( ) . the risk seems particularly increased when they return home to visit friends and relatives ( ) . while antimicrobial resistance is of concern, the prospects for pandemic spread of a bacterial or fungal emerging pathogen by migrants seem less likely ( ) . endemic disease, as tuberculosis, impose a far higher public health burden than epidemic disease ( ) . denmark experienced an increase in the incidence of tuberculosis in the s in relation to the increase in the number of cases among migrants ( ) . the rate of tuberculosis in france is times higher among immigrants than in the majority population. refugees and asylum seekers may have a heightened risk of mdr-tb infection and worse outcomes but the data remains poor ( ) . thus, there is little evidence to support the theories by which migrants would expose the host population to significant infectious risk. however, human diseases acquire a social status based on their perceived risk that determines their acceptability ( ) . in a study that we conducted with a number of doctors in france (infectious diseases and general practitioners), they were asked if firsttime migrant people represent a vector of infectious diseases different from the majority population: % answered no, % yes but weakly, % yes but moderately, % yes significantly and % did not know. thereby, apart from infections such as tuberculosis and multidrug-resistant bacteria, the introduction of eids into human populations seems to be more often a consequence of economic development that brings zoonotic reservoirs in closer proximity to people. indeed, most pandemic threats are caused by viruses from either zoonotic sources or vectorborne sources ( ) . there is a need for rapid diagnosis of eids. systems biology approaches can lead to a greater understanding of eids pathogenesis and facilitate the evaluation of newly developed vaccine-induced immunity in a timely manner ( , ) . close collaboration is therefore needed between specialists in tropical medicine, in public health, immunologists and biologists to anticipate the risk of eids in order to achieve the sustainable development goals established by the united nations in ( ). the who established a department of pandemic and epidemic diseases in to better prepare for and respond to eids. in conclusion, in connection with the extension of poverty, urbanization, extensive livestock rearing and globalization, we could be exposed to a third epidemiological transition characterized by zoonotic diseases and infections with multidrugresistant bacteria ( ) . the risk appears low for eids, or very low for high-risk eids, but higher for mre carriage with possibly limited consequences. the role played by migrants is weaker than imagined (except for tuberculosis). immigrants don't play the role of nicolas vignier, olivier bouchaud travel, migration and emerging infectious diseases sentinel epidemic so far. they could play a role in importing mre, but it is poorly evaluated. opportunities and challenges in modeling emerging infectious diseases the perpetual challenge of infectious diseases identification of a novel coronavirus in patients with severe acute respiratory syndrome probable person-toperson transmission of avian influenza a (h n ) urbanization and disease emergence: dynamics at the wildlife-livestock-human interface overseas fatalities of united states citizen travelers: an analysis of deaths related to international travel health risks among travelers--need for regular updates prevention of malaria resurgence in greece through the association of mass drug administration (mda) to immigrants from malaria-endemic regions and standard control measures autochthonous case of dengue in france madrid: untwo high rate of acquisition but short duration of carriage of multidrug-resistant enterobacteriaceae after travel to the tropics migration and health: a framework for st century policy-making public health aspects of migrant health: a review of the evidence on health status for labour migrants in the european region. who health evidence network synthesis reports public health aspects of migrant health: a review of the evidence on health status for undocumented migrants in the european region. who health evidence network synthesis reports was access to health care easy for immigrants in spain? the perspectives of health personnel in catalonia and andalusia. health policy review: immigrants and health care access, quality, and cost undocumented migrants in canada: a scope literature review on health, access to services, and working conditions. j immigr minor health access to health insurance coverage among sub-saharan african migrants living in france: results of the anrs-parcours study migration and health in an increasingly diverse potential barriers to the use of health services among ethnic minorities: a review health care for undocumented migrants: european approaches. issue brief (commonw fund) immigrants' access to ambulatory care in france. questions d'économie de la santé screening for infectious diseases among newly arrived migrants: experiences and practices in non-eu countries of the mediterranean basin and black sea how do economic crises affect nicolas vignier, olivier bouchaud travel, migration and emerging infectious diseases migrants' risk of infectious disease? a systematic-narrative review access to effective antimicrobials: a worldwide challenge fecal colonization with extended-spectrum beta-lactamase-producing enterobacteriaceae and risk factors among healthy individuals: a systematic review and metaanalysis unusual microorganisms and antimicrobial resistances in a group of syrian migrants: sentinel surveillance data from an asylum seekers centre in italy patients hospitalized abroad as importers of multiresistant bacteria-a crosssectional study emerging viral diseases from a vaccinology perspective: preparing for the next pandemic when an emerging disease becomes endemic migration, refugees, and health risks multidrug-resistant tuberculosis and migration to europe systems biology-based platforms to accelerate research of emerging infectious diseases the evolution of disease: anthropological perspectives on epidemiologic transitions key: cord- -c gmu authors: davis-wurzler, gina m. title: current vaccination strategies in puppies and kittens date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: c gmu motivation in writing this article stems from many things: a lack of time spent in the veterinary curriculum discussing vaccines, a growing concern(by the general public and the veterinary community) regarding adverse reactions associated with vaccines, and a desire to prevent a recurrence of preventable infectious diseases resulting from a fear-driven cessation of vaccine administration. the objectives of this article are to present a basic review of immunology as related to vaccines, to discuss general guidelines for pediatric vaccines in canine and feline patients,and to offer suggestions as to how we can most positively influence our patients' health from the first visit. has a minimal effect in these species. it also occurs during initial suckling through the ingestion of colostrum and lactation, which have more significant effects in these species [ ] . this maternal immunity does provide initial protection against many pathogens but, of course, is dependent on the health and immune status of the mother as well as the health of the fetus and neonate. although this may result in temporary protection for the infant, in the long term, it may be deleterious to that individual's health by essentially keeping that animal naive to different antigens (eg, maternal antibody interference with vaccination of the neonate). maternal or passive immunization is effective in protecting neonates for the first several weeks of life but begins to decline and lose the ability to protect against diseases rapidly as the maternal antibodies are degraded through natural catabolic processes. between the ages of and weeks, depending on multiple factors (including the species; amount of maternal antibody produced, transferred, and absorbed; and individual health status of the neonate), most puppies and kittens have maternal antibody levels below protective levels. if present at high enough levels, however, maternal antibodies can interfere with the neonate's ability to respond to vaccination, because the circulating maternal antibody within the puppy or kitten may effectively respond to and neutralize the vaccine antigen or render it ineffective by preventing recognition of the antigen by the immune system [ ] . this is one reason for multiple sequential vaccines in kittens less than weeks of age and puppies less than weeks of age. maternal antibodies can interfere with immunization, although the level of maternal antibody present may not be protective against pathogens. a functioning immune system is composed of multiple parts. innate immunity is the oldest (evolutionarily), least specific, and most immediate (in terms of response to potential invaders and/or pathogens) form of immunity. macrophages, neutrophils, dendritic cells, and natural killer (nk) cells, combined with numerous products produced by these cells, comprise the innate immune system. examples of some of the chemical components produced and released by these cells in response to microbial invasion include lysozyme, complement, and various cytokines, such as tumor necrosis factor-a and interleukins, as well as various vasoactive molecules, such as histamine [ ] . active immunization is the process of the individual responding to an antigenic stimulus appropriately by natural infection or vaccination. active immunization is processed through the acquired immune system. the two main types of acquired immunity are cell-mediated immunity and antibody, or humoral, immunity. cell-mediated immunity is predominantly directed against pathogens that typically are obligate intracellular organisms. examples include viruses, some obligate intracellular bacteria, some fungi, and protozoa. t lymphocytes are the predominant effector cells and are dependent on foreign protein (antigen) being presented to them before they can take effect against the pathogens; thus, multiple cell types are involved in forming cell-mediated immunity. antibody or humoral immunity is predominantly directed against pathogens that can survive outside the host or at least survive extracellularly. examples include most bacteria, fungi, protozoa, and helminths. multiple cells act in concert to confer humoral immunity as well, but the primary effector cell is the b lymphocyte [ ] . kittens and puppies have varying degrees of ability to respond to antigens, whether attributable to natural or vaccine exposure, based on antigen load, route of exposure, antigenic virulence, genetics of the individual animal, and levels of persistent maternal immunity. in naive animals whose maternal immunity has declined sufficiently so as not to interfere with an immune response, the first vaccine should stimulate a primary immune response. this initial exposure and recognition process and the ability to produce antibody to respond to the antigen typically take to days. subsequent exposures to the same antigen elicit a stronger response; a greater amount of antibody is produced, and the subsequent response is faster. this is known as the secondary, or anamnestic, immune response. although multiple cell lines are involved in this response, subsets of t and b lymphocytes known as memory cells preserve the host's ability to recognize and respond to antigens to which the animal has previously been exposed [ ] . to design, recommend, and actuate an effective plan for each patient, a practitioner must have familiarity with multiple variables. those variables include the duration of protection conferred on the neonate by the mother, the typical length of time maternal antibody may persist and pose interference with the young animal's ability to respond fully to a vaccine, and the length of time needed for an appropriate response. in addition, knowledge of the various diseases that pose risks to pediatric patients and of safe efficacious vaccines available is critical. in essence, we must assess each patient as an individual within the population to provide optimal wellness over the lifetime of each individual as well as the population. this rationale has led to the concept of core and noncore vaccines, two terms commonly used when discussing vaccination within the veterinary field. criteria for assigning vaccines into these categories as well as a third category, ''generally not recommended,'' are based on the following factors [ ] [ ] [ ] [ ] [ ] : . morbidity and mortality associated with the specific disease (does the organism cause serious illness, or does it cause a mild transient disease that may pose only minimal risk to the individual or population?) . prevalence or incidence rate of the disease (although a specific disease may not commonly be seen, the organism is ubiquitous in the environment and therefore poses a risk to the individual or population) . risk of the individual for exposure to the disease (eg, indoor-only animal versus free-roaming individual, regional variations of occurrence) . efficacy of the vaccine (does the vaccine prevent infection or simply ameliorate some signs or length of the disease?) . risks associated with administering the vaccine (are the risks associated with that vaccine greater than the risk of the disease?) . potential for zoonotic disease . route of infection or transmissibility when these criteria are assessed, general guidelines may be generated for the individual practitioner as well as for the veterinary community at large. again, guidelines are not to be thought of as absolutes, nor are they to be used to establish standard of care. they are, simply stated, tools for each of us to use to promote optimal wellness for our patients when considering all factors affecting the individual's health (environmental, organismal [pathogen and host], owner concerns, and current vaccine technologies) [ ] [ ] [ ] [ ] [ ] . there are multiple vaccines available for our canine and feline patients; yet, most vaccines fall within three basic categories. assignment of vaccine products (which are considered biologic agents rather than drugs and are therefore assessed and approved by the us department of agriculture [usda] rather than the us food and drug administration [fda]) into these categories is based on how the product is created. simply stated, modified-live vaccines (ml) are vaccines created by altering (attenuating) the pathogen in some way so that it is no longer able to cause serious or clinical disease in the targeted species. modified-live virus vaccines (mlv) are therefore vaccines containing live but avirulent virus. killed vaccines are vaccines produced by inactivating the pathogen completely, rendering it incapable of reproducing and thereby unable to cause disease. the third category of vaccines consists of recombinant vaccines. there are multiple types of recombinant vaccines, and this category itself has three subcategories. these vaccines use genetic technologies to introduce genetic material directly into the host (no vector is used [eg, purified subunit vaccines, type i recombinant]), alter the genetic material to change its virulence (gene deletion, type ii recombinant), or incorporate genetic material from the desired pathogen into an attenuated vector organism (eg, feline recombinant rabies, type iii recombinant) [ , ] . within the near future, multiple new technologies are likely to provide us with even more choices, hopefully providing our patients with better protection against disease with minimal vaccine-associated risks. for a comparison between vaccine types, the reader is referred to table . vaccines are available in single-dose and multiple-dose (tank) vials. the use of single-dose vial vaccines is highly recommended in these species. conversely, the use of multiple-dose vials is discouraged because of the increased risk of contamination and the inability to ensure consistent levels of antigen and adjuvant in individual doses from a single vial [ , ] . multivalent vaccines are not recommended in cats other than the core feline vaccine designed to protect against feline panleukopenia, feline herpesvirus i (fhv-i), and feline calicivirus (fcv). because of increased inflammation at the site of multivalent vaccines, all other vaccines should be given as a separate vaccine at the indicated site (see discussion on feline core and noncore vaccines) [ , ] . allowing vaccines to acclimate to room temperature before administration, particularly in cats, is recommended, because the administration of cold vaccines was found to have an increased association with tumorigenesis in cats [ ] . the practitioner is advised always to follow manufacturer's directions for dose and route of administration. using a topical product parenterally or splitting doses should never be done. administration of a modified-live bacterin vaccine designed for topical administration yet administered parenterally may have serious and potentially fatal consequences (fig. ) . a full dose is required to stimulate the immune system; there is no medical basis for giving a smaller dose to a toy-breed dog, and this practice could lead to vaccine failure in that animal. if done with a rabies vaccine, the practitioner is not following federal requirements, which carries potential legal implications [ , ] . the interval between various vaccines, whether using the same product serially in the initial series or using different products in an adult animal, should never be less than to weeks. interference between the first product administered and a second vaccine product may lead to failure to respond to that second vaccine optimally. the exact mechanism of this interference is unknown but may be associated with interferon produced by cells processing an ml agent or by transient immunosuppression by an ml agent. multiple vaccines administered at the same time do not seem to elicit this interference and is therefore an acceptable practice [ , ] . the reader is referred to tables and for a comparison between pediatric canine and feline core, noncore, and generally not recommended vaccines. the diseases that fall within this category carry high rates of morbidity or mortality; they are of public health concern, are readily transmissible, or may be ubiquitous in the environment. in addition, safe efficacious vaccines are available and provide sterile immunity (prevent infection) or confer a high degree of protection (do not prevent infection but may confer protection, such that the animal does not develop clinical signs of disease) [ , ] . essentially, the vaccines that fall within this category are recommended for each individual within the population regardless of that animal's lifestyle or locale. canine distemper virus (cdv), an enveloped morbillivirus, has been well controlled because of widespread vaccination programs over the past several decades. the disease still persists, however, and in addition to high virulence, it is readily transmissible. infection with the virus causes respiratory, gastrointestinal, and neurologic signs and is often fatal [ ] . the distemper vaccine is commonly administered as part of a multivalent product. the general recommendation is to use a modified-live or recombinant, multivalent product beginning at to weeks of age and to give serial vaccines every to weeks until the puppy has reached to weeks of age [ , , ] . currently, there is one product that contains modified-live distemper virus, canine adenovirus type ii (cav-ii), and canine parvovirus (cpv) (continuum; intervet, millsboro, delaware), but there are numerous vaccines with high safety and efficacy records that also include canine parainfluenza virus. there is also a vaccine that combines a recombinant canarypox-vectored cdv with modified-live adenovirus ii, parainfluenza, and parvovirus (recombitek; merial ltd, duluth, georgia), and this vaccine seems to be less affected by maternal antibody interference than mlv vaccines [ ] . other studies support the improved ability of recombinant vaccines to overcome maternal antibody interference as compared with mlv vaccines [ , ] . most puppies receive two or three distemper vaccinations, depending on the age when they are first presented to the veterinarian. it is, however, the interval between or the ''timing'' of the vaccinations rather than the ''number'' that is important. serial vaccinations help to increase the likelihood of a complete response of the patient and thereby decrease the risk of vaccine failure that may occur when only one vaccine is administered. in addition, by eliciting a secondary immune response, they may help to increase the level of circulating antibody and decrease the lag time between exposure to an antigen and achievement of maximal antibody level [ ] . potential causes for vaccine failure include an mlv vaccine that was improperly stored and therefore lost its efficacy, the vaccine was improperly administered (wrong route or accidental loss of vaccine onto the skin of the patient), the patient's immune system did not respond (the immune system may have been responding to another antigenic challenge, or the vaccine may have been given too soon after a previous vaccine), and maternal interference [ ] . in theory, if a puppy were kept ''sequestered'' from exposure to this virus, one mlv distemper vaccine administered after weeks of age would confer protection for at least year [ , ] . in reality, however, most pet owners are not inclined to isolate their puppies for the first months of life, nor should they. early socialization is an important part of families bonding with their puppies. exposure to various people, other dogs, and new places helps to decrease behavior problems in young adult and mature dogs [ ] . as long as the last distemper vaccine is administered after weeks of age, the puppy should be able to mount a strong active response and fully overcome any residual maternal antibody. the current recommendation is to have the puppy return year later (when approximately months old) for administration of another dose of distemper vaccine. after the first ''annual'' vaccination, triennial immunization is recommended for mlv vaccines [ , ] . annual revaccination is currently recommended if the recombinant product is used [ , ] . there are two types of adenovirus that cause disease in our canine patients. canine adenovirus type i (cav-i), a nonenveloped virus in the family adenoviridae, causes the potentially fatal disease infectious canine hepatitis. clinical signs include fever, depression, vomiting and diarrhea, and potential abbreviations: ab, antibody; felv, feline leukemia virus; fvr, feline viral rhinotracheitis; mlv, modified-live virus; pma, persistent maternal antibodies. a because of increased susceptibility for infection in kittens, vaccination against felv is strongly recommended for all kittens. in single-cat households, households with known negative viral status of all cats, and households with indoor only cats, the practitioner may elect to consider this a noncore vaccine. petechiation and ecchymotic hemorrhage secondary to hepatic dysfunction. in addition, uveitis and renal disease are associated with infection with this virus. cav-ii causes respiratory tract disease. cav-i is associated with severe potentially fatal disease, and protection against this disease is recommended. transmission is via the oronasal route and exposure to infected secretions. cav-ii infection typically results in mild self-limiting disease and is therefore considered to be a noncore disease; however, the mlv product designed for prevention of cav-i has been associated with adverse effects, such as uveitis and corneal edema (an arthus reaction, similar to effects caused by natural infection) [ , ] . the current recommendation is to use the cav-ii mlv because it stimulates the immune system to protect against cav-i and cav-ii without the associated adverse reaction caused by the type i vaccine [ , , ] . a modified-live adeno-type ii virus is typically included in a multivalent injection (as mentioned previously) and is therefore usually administered at intervals of to weeks, beginning between and weeks of age and ending between and weeks of age. a vaccination year later is recommended before instituting triennial vaccinations. canine parvovirus cpv is a nonenveloped type parvovirus. the predominant form currently causing infection in the united states is type b, but other subtypes exist and cause disease elsewhere [ ] . because the virus is nonenveloped, it may exist (outside a host) under certain environmental conditions and is somewhat resistant to many disinfectants. transmission is via the fecal-oral route, and clinical signs include lethargy, anorexia, pyrexia, vomiting, and diarrhea (typically hemorrhagic). young animals seem to be at highest risk for developing severe life-threatening disease. the current recommendation for vaccination is to use a multivalent mlv beginning at to weeks of age and to repeat the vaccine at intervals as stated previously (every to weeks until the puppy is to weeks of age). there has been some concern that certain breeds may be at increased risk for contracting and developing severe parvoviral disease (eg, doberman pinschers, rottweilers), but it is generally agreed that these breeds mount an appropriate response to a quality product if the last vaccine is given between and weeks of age [ , ] . recent studies using mlv cpv b strains showed a higher antibody response to cpv and cpv b and that the cpv b strain vaccines were better able to overcome maternal antibody interference than the cpv strain vaccines used [ , ] . an alternative would be to use serial vaccinations of killed virus if the practitioner were concerned about the potential for vaccine-induced clinical disease in one of the breeds believed to be more susceptible to this virus; however, these vaccines are less immunogenic [ ] . immunization year after completing the initial puppy series is recommended, with subsequent triennial vaccinations [ , , ] . rabies virus, an enveloped virus in the rhabdoviridae family, is capable of infecting all mammals [ ] . because it is an enveloped virus, it is not stable in the environment and is readily inactivated by most common disinfectants. the virus is transmitted through infected saliva, most commonly from a bite by an infected animal. clinical signs range from anxiety or other vague behavior changes to pica, dysphagia, photophobia, and paralysis. because of the zoonotic potential and implications regarding public health, canine vaccination programs are strongly regulated and enforced. the current recommendation is to vaccinate puppies using a killed virus vaccine at a minimum of or weeks of age. state regulations vary as to the minimum age for canine rabies vaccination; in california, the legal minimum age of canine vaccination against rabies is weeks. a second rabies vaccine (killed product) is administered year later and then annually or triennially thereafter depending on local regulations [ , ] . it is the practitioner's professional responsibility to acquire knowledge of and maintain adherence to regional laws regarding rabies vaccination frequency [ ] . vaccines in the noncore category have limited efficacy, or the organism causing disease is not readily transmissible or may have limited geographic distribution or prevalence. additionally, the diseases these vaccines are designed to prevent may be so mild or self-limiting that the risk associated with administering the vaccines may be greater than that of the actual disease. finally, some vaccines may interfere with common screening methods for disease detection and are therefore not recommended unless absolutely warranted for a specific individual. it is the burden of the practitioner, along with the pet owner, to make decisions regarding which, if any, of the noncore vaccines should be administered to a puppy [ ] [ ] [ ] [ ] . a bacterial pathogen that causes acute hepatic and renal disease, leptospirosis is typically transmitted through urine of infected animals (reservoir hosts include dogs, rats, wildlife, and livestock) and in contaminated water. there are at least two different species (leptospira interrogans and leptospira kirschneri) that can infect dogs, with multiple serovars (variants of the same species) of l interrogans causing disease in dogs [ ] . although these organisms have the potential to cause serious disease, dogs are not likely to be at risk in a mostly urban and controlled environment (eg, housed in a fenced yard with no exposure to wildlife or livestock). a dog that frequents rural environments or has exposure to waterways or livestock is definitely at risk of infection, however, and should therefore be protected against the disease. again, the initial puppy appointments should involve a thorough history and include the owner's plans for that dog's future use. if an owner brings a labrador retriever puppy to the veterinarian for ''whatever vaccines it needs,'' it is up to the practitioner to ask: ''will it be a hunting dog, will it be used in field trials, or will it be exposed to wildlife and waterways?'' the border collie that lives on a working sheep ranch surely should be vaccinated as well. conversely, a long-haired miniature dachshund that spends its days on its owner's lap is at minimal risk of exposure; therefore, vaccination is most likely not warranted. in essence, the regional distribution, seasonality (increased prevalence during and immediately after the rainy season), and lifestyle of the puppy should factor into the decision as to whether the puppy should be vaccinated. if the decision is made to vaccinate against leptospirosis, the general recommendation is to wait until the puppy is at least weeks of age; at that time, a killed or purified subunit vaccine is administered. infection is serovar specific, and no cross-protection is seen between different serovars; therefore, vaccination with those serovars known to cause disease in a given region is recommended. currently, there is one killed purified subunit vaccine (leptovax; fort dodge animal health, overland park, kansas) that contains four serovars (icterohaemorrhagiae, canicola, pomona, and grippotyphosa); however, the duration of immunity against grippotyphosa and pomona is unknown, and there are no vaccines available for autumnalis or bratislava. an initial series of two to three vaccinations should be administered monthly and repeated at least annually thereafter as long as exposure to the agent exists [ ] . the recommendation to wait until the puppy is at least weeks old before administering the leptospirosis vaccine is based on the increased potential for adverse events associated with this vaccine and to increase the likelihood of a complete immune response minimizing ineffective vaccinations [ , ] . bordetella bronchiseptica is a bacterial agent that causes infectious tracheobronchitis. infection with this agent may occur in concert with other agents infecting the respiratory tract (eg, canine parainfluenza virus, cav-ii). transmission occurs via direct contact or through aerosolized microdroplets from infected dogs and is most likely to occur under crowded conditions, such as boarding and grooming facilities and dog show venues. the current recommendation is to vaccinate puppies at risk a minimum of week before potential exposure with a combination vaccine containing an avirulent live bacterin for b bronchiseptica and a modified-live canine parainfluenza virus. although the vaccine can be administered to puppies as young as to weeks of age, it is generally not indicated unless the puppy is in a kennel environment [ ] . many organized obedience classes commonly require proof of vaccination against bordetella at the time of enrollment or before beginning the course. the general consensus is that intranasal vaccines are superior to parenteral vaccines because they stimulate rapid local immunity [ , ] . if the puppy is intermittently exposed throughout the year (eg, traveling to shows or boarding or grooming facilities), the vaccine should be administered every months. as stated previously, parainfluenza may occur in concert with other respiratory tract agents. the vaccine recommendations are as stated for b bronchiseptica if indicated. there are multiple products available, but the product currently recommended is the combination intranasal vaccine containing a modified-live parainfluenza virus with an attenuated b bronchiseptica bacterin. for optimal protection, the vaccine should be administered every months to annually if indicated. alternatively, many multivalent products containing modified-live cdv, cav ii, cpv, and parainfluenza are available and appropriate for use [ ] . borrelia burgdorferi is a vector-borne spirochete bacterium responsible for lyme disease (borreliosis). transmission occurs when an infected tick (various species within the ixodes genera, also referred to as ''hard ticks'') bites and remains attached to a host, in this case, a puppy. direct horizontal transmission is not likely to occur; therefore, the risk to human beings and other pets is thought to be minimal. if a puppy has a significant burden with infected ticks, this, of course, increases the exposure to others in the household; however, because ticks typically do not reattach once they have taken a complete meal, the risk is thought to be quite small unless appropriate tick control is not instituted [ ] . vaccination to protect against lyme disease is controversial, because the duration of immunity and degree of protection provided by vaccination are unknown and vaccination with some vaccines interferes with standard screening diagnostics [ ] . therefore, vaccination against lyme disease is warranted only if a puppy is at high risk for tick exposure and only if it lives in a borrelia endemic area. there are killed and recombinant (outer surface protein a [ospa] subunit) vaccines available for use against b burgdorferi, and if vaccination is deemed warranted, the current recommendation is to use one of the subunit vaccines before exposure to ticks. the vaccine can be given as early as weeks of age and should be repeated to weeks later [ ] . the best prophylaxis is likely achieved by using appropriate tick prevention, such as fipronil with methoprene spray or spot-on products (frontline top spot; merial ltd, iselin, new jersey), amitraz collars (preventic collar; virbac, fort worth, texas), or an imidacloprid/permethrin topical product (canine advantix; bayer animal health, shawnee mission, kansas) [ , ] . these products should be chosen and recommended carefully by the veterinarian based on household situations, owner concerns, and age of the puppy. this virus, also a morbillivirus, can stimulate an immune response that is crossprotective against cdv. the indication for using this vaccine is for puppies that may have maternal antibody to distemper virus sufficient to cause interference with distemper vaccination but not adequate to protect against infection. if indicated (see discussion of special circumstances), a single vaccination with an mlv vaccine should be given intramuscularly as early as weeks of age. subsequent immunizations with mlv cdv vaccines should be given serially as recommended previously (see section on cdv) [ , , ] . canine measles vaccines should never be administered to female puppies older than weeks of age because they may develop an acquired immune response to the virus. this could be problematic if a female puppy vaccinated against measles at weeks of age, for example, later became pregnant. if the dog developed antibodies to the measles virus and maintained immunologic memory, it would confer measles antibody to puppies via passive transfer, thus rendering measles vaccination in those puppies ineffective. a more appropriate alternative to administering a measles vaccine to a young puppy thought to be at risk for infection but too young to receive an mlv cdv vaccine would be to use a recombinant cdv vaccine, thereby decreasing the likelihood of maternal interference [ ] an enveloped virus belonging to the family coronaviridae, this virus is transmitted via the fecal-oral route. vaccination against this disease is generally not recommended, because the vaccines provide questionable protection and the actual prevalence of the disease is unknown. those most likely to be infected and develop clinical disease are neonates less than weeks of age. clinical signs may include diarrhea, possibly hemorrhagic but typically self-limiting. the general recommendation is to vaccinate puppies against cpv (as recommended previously), because this practice seems to confer protection against coronavirus in addition to preventing infection with cpv- [ , ] . this protozoal parasite causes diarrhea in canine and feline patients as well as in many other mammalian species, including human beings. transmission is via the fecal-oral route, with animals contracting the agent from contaminated feces or water. there is a killed vaccine available; however, vaccination against this agent is typically not recommended, because most animals are not at risk to contract the parasite, the vaccine does not prevent infection (it may ameliorate clinical signs and decrease cyst shedding), and the disease is readily amenable to therapy (fenbendazole, albendazole, and metronidazole are off-label uses but commonly accepted as standard of care). because puppies should be prophylactically dewormed at regular intervals, it is unwarranted to use this vaccine even if the disease is suspected, because a standard anthelmintic dose of fenbendazole given for several days should resolve the infection [ , , , ] . a vaccine designed to protect against envenomation by crotalus atrox, the western diamondback rattlesnake, was released onto the market recently. the original provisional licensure was granted to provide possible protection against that single species of snake and was granted for use only in california. recently, the company was granted extended provisional licensure for multiple states and has extended their claim for potential protection against multiple species of members of the crotalidae (pit vipers). at this time, no challenge studies have been performed in the canine species to validate efficacy claims; all claims are based on antibody titer to the venom component included in the vaccine, to murid challenge studies, and to anecdotal reports of protection of naturally occurring envenomation [ ] . the manufacturer does not claim that vaccination with this product completely protects against the effects of envenomation; rather, the manufacturer claims that it may slow the onset of clinical signs and decrease the severity of signs. immediate veterinary care is still the ''gold standard'' for any snakebite. because of the great potential for variability in envenomation (site of bite on an animal, size of the snake, amount of venom injected into an animal, and species of snake), field observations and anecdotal reports of protection are difficult to substantiate. challenge studies done under controlled conditions are likely necessary to validate the efficacy of this product. at present, because of the preceding statements, this vaccine is not recommended for general use. aversion training and keeping dogs out of areas known to favor rattlesnake habitation as well as immediate veterinary evaluation and care are still the standard recommendations for preventing and treating disease associated with rattlesnake envenomation. canine adenovirus type i as stated in the canine core vaccine section, cav-i causes serious disease in dogs; however, the use of the cav-i vaccine is associated with a high incidence of adverse events. vaccination with the cav-ii vaccine induces an immune response that is protective against cav-i and cav-ii without the adverse effects. the recommendation is to use the cav-ii vaccine as part of the canine core vaccination program; the cav-i vaccine should not be used [ ] . feline panleukopenia, a nonenveloped parvovirus closely related to cpv, causes serious and often fatal disease in kittens. transmission typically occurs from direct contact with infected animals, although in utero infection and fomite transmission also occur. clinical signs typically include pyrexia, anorexia, lethargy, and vomiting and diarrhea. kittens may be immunosuppressed subsequent to pancytopenia associated with this viral infection. kittens infected in utero may exhibit cerebellar disease. prevention is achieved using mlv vaccines beginning between and weeks of age. the standard recommendation is to use a parenteral product (as opposed to intranasal products, which have a higher incidence of postvaccinal viral shedding and potential for clinical disease induced by the more virulent viruses in these vaccines) [ , , , ] . as is the case for canine cdv, cav, and cpv, the core feline diseases, with the exception of rabies, are typically administered in a multivalent product in a series. there are numerous vaccine products containing feline panleukopenia virus, herpesvirus i, and calicivirus (see additional discussion). the current recommendation is to choose an mlv nonadjuvanted product from a reputable manufacturer. vaccines are administered subcutaneously in the right thoracic limb and given every to weeks until the kitten is to weeks of age. repeat administration is recommended year later before instituting a triennial schedule [ , , ] . feline herpesvirus i fhv-i, also known as feline viral rhinotracheitis virus, is an enveloped virus causing respiratory tract disease in cats. clinical signs include sneezing, nasal congestion and discharge, conjunctivitis, and ocular discharge. in addition, kittens may exhibit pyrexia, anorexia, and lethargy along with oral and/or lingual ulcerations and associated hypersalivation. in some cases, ulcerative crusting dermatitis that may mimic other dermatologic disease occurs [ ] . the virus typically causes upper respiratory disease, but the lower respiratory tract may become involved, especially in neonates or debilitated animals. infection with this virus is lifelong, although many cats ''recover'' and do not show clinical signs. cats infected with fhv-i may have recurrent ''outbreaks,'' especially in times of stress or if their immunity is otherwise compromised. cats may persistently shed the virus and act as a source of infection in shelters, catteries, and multiple-cat households. therefore, prevention before exposure is key in controlling this disease [ , ] . vaccination with an mlv or recombinant vaccine beginning as early as to weeks of age is recommended. this is commonly administered as part of a multivalent product and is given subcutaneously in the right thoracic limb. the current recommendation is for kittens to receive a second vaccination weeks later. the last vaccine in the series should be given when kittens are at least weeks old. the vaccine should be given year later before beginning the triennial schedule [ , ] . feline calicivirus fcv causes respiratory tract disease in kittens and cats. because it is a nonenveloped virus, it is more resistant to disinfectants and may therefore persist in the environment. signs are similar to those associated with fhv-i, but lameness and stomatitis are also commonly seen. transmission of fhv-i and fcv is through direct contact, exposure to contaminated secretions, aerosolization, and fomites [ , ] . a newer highly virulent strain of fcv was recently identified and carries a high incidence of mortality. transmission is through direct contact or via fomites. prior vaccination against fcv does not seem to be protective against this strain, and adult cats seem to be more severely affected than kittens [ , ] . the current recommendation is as previously discussed for panleukopenia and fhv-i: administering an mlv or recombinant virus parenteral vaccine beginning at or weeks of age, with a subsequent dose of vaccine weeks later (last vaccination should be when the kitten is at least weeks old). a booster vaccination should be administered year later and then every years [ , ] . as stated previously, rabies virus affects all mammals; in this country, most documented rabies cases in pet animals occur in cats [ ] . because of the significant risk to pets, wildlife, and human beings, vaccination against rabies virus is highly recommended for all kittens and cats, even those kept inside [ , ] . local requirements vary, but the general recommendation is that all kittens should be vaccinated beginning at weeks of age with the recombinant rabies vaccine designed for use in cats [ ] . this product uses gene-splicing technology: reverse transcriptase is applied to rabies viral rna to create complementary dna. the segment of rabies virus dna that codes (a codon) for the immunogenic protein associated with the virus (glycoprotein g) is then spliced from the rabies dna and inserted into a canarypox virus. the canarypox virus is attenuated and nonpathogenic to mammalian cells and therefore carries no potential to cause disease in this species. because the vaccine is essentially a modified-live product, the canarypox virus can enter cells, delivering the codon for rabies virus glycoprotein g to its targeted site. once inside the cell, the canarypox virus is unable to replicate, but the rabies glycoprotein g codon is preserved, leading the host cell to express the glycoprotein on its surface. this stimulates cell-mediated and humoral immune responses. in addition to the benefit of stimulating both types of immunity, the fact that no adjuvant is needed is beneficial [ ] . rabies vaccines should be administered subcutaneously in the right pelvic limb as distally as is reasonably possible; the level of the stifle is acceptable, and areas distal to the tarsus are not appropriate. currently, there is only a recombinant rabies vaccine approved for use in cats (purevax feline rabies vaccine; merial ltd, duluth, georgia). the current usda approval and label state that this product should be administered annually. there are multiple killed virus rabies vaccines approved for use in cats, with initial vaccination occurring at weeks of age and a subsequent vaccination year later. these products are highly efficacious but may carry an increased association with the development of fibrosarcoma formation because they contain adjuvants [ , , , , ] . because regulations vary depending on the state or region, the veterinary practitioner must be familiar with local laws regarding rabies vaccination in this species [ ] . feline leukemia virus (felv) is a retrovirus affecting cats of any age, but kittens and juvenile cats seem to be most susceptible to infection [ ] . clinical signs are numerous and nonspecific, and they include pyrexia, failure to thrive, and chronic or recurrent respiratory tract and gastrointestinal disease. infection in kittens occurs via vertical transmission from the queen to the fetus but may also spread horizontally from the queen to the kitten during lactation and grooming. transmission also occurs through direct and usually prolonged contact with other infected cats from behaviors like grooming and sharing food and water bowls as well as litter boxes. viral screening using an elisa test designed to detect antigenemia should be performed on all kittens, even if their owners plan to house them strictly indoors. because the elisa test detects antigen, maternal antibody and vaccination do not interfere with test results. therefore, kittens of any age may be tested [ ] . if a kitten is antigen-negative, the current recommendation is to administer a recombinant vaccine on the second visit. a second dose of vaccine should be administered weeks later, followed by vaccination year after administration of the last felv vaccine in the kitten [ , ] . the recommended site for administration of any felv vaccine is the left pelvic limb as distally as is reasonably possible [ ] . currently, there is only one recombinant felv vaccine available (purevax recombinant leukemia vaccine; merial ltd, duluth, georgia). this vaccine is administered with a needle-free high-pressure device that deposits the vaccine in skin, subcutaneous, and muscle tissues (vetjet delivery system, merial ltd, duluth, georgia, which is manufactured by bioject, tualatin, oregon) [ ] . there are killed virus vaccines that are efficacious; however, because they contain killed virus, they require an adjuvant to maximize the host's immune response. because of documented associations between adjuvants and the formation of fibrosarcomas, the use of adjuvants in cats should be avoided when adjuvantfree products with comparable efficacy are available. the most recent findings linking injections of any type with fibrosarcoma formation in this species further support the use of a needle-free system as a viable means of vaccine delivery [ , , ] . under the current vaccine guidelines released in in the american veterinary medical association council on biologic and therapeutic agents' report on cat and dog vaccines, vaccination against felv is only indicated if a kitten or cat is allowed to go outside or if the kitten or cat lives with an felv-positive cat. because kittens are most vulnerable to infection and may have exposure if outdoors and because immunity increases with age, it is rational to vaccinate all kittens against this disease with a repeat vaccination year later. subsequent to that, if the cat is housed strictly indoors and does not live with an infected (felv-positive) cat, additional vaccinations are not indicated [ ] . chlamydophila felis, formerly known as chlamydia psittaci, is a bacterium that causes upper respiratory tract disease in kittens and cats. the most common sign is conjunctivitis, but sneezing and nasal discharge may also be present. transmission is typically through direct contact with infected cats. kittens are most commonly affected but usually recover fully with appropriate antibiotic therapy-topical oxytetracycline (ophthalmic ointment) or systemic tetracycline or doxycycline. vaccination against this agent typically does not prevent infection but may prevent clinical signs of disease. because the vaccine does not fully prevent infection and carries an association with adverse events that may be greater than the actual disease, routine vaccination of household pets with this product is generally not recommended. it may be of use in some environments in which the risk of infection is high, however, such as shelters or catteries [ , ] . if vaccination is deemed appropriate by the practitioner, an attenuated parenteral vaccine can be given to kittens beginning at weeks of age, with a second dose given to weeks later [ ] . this bacterial agent causes respiratory tract disease in cats, and cats affected by stress, poor nutrition, or overcrowding seem to be more susceptible. although many infected kittens show mild self-limiting disease with signs that include pyrexia, sneezing, and nasal and ocular discharge, bronchopneumonia has been documented. there is a topical modified-live bacterin vaccine designed for use in this species, but it is generally not recommended for routine use. if the practitioner thinks protection against b bronchiseptica is warranted based on the kitten's risk of exposure (eg, attends cat shows, goes to a boarding facility), administration of the vaccine designed for use in cats may be considered [ ] . a single dose of the ml intranasal vaccine can be given to kittens as young as weeks of age [ ] . the product designed for use in dogs should not be used in cats. there are multiple vaccines in addition to those described and recommended previously; however, many of these diseases pose a minimal risk to most of the feline population or the vaccines are minimally efficacious at preventing infection or disease and therefore are generally not recommended. additional reasons not to use some of these products are vaccine interference with screening tests and adverse events associated with some vaccines. a retrovirus, feline immunodeficiency virus (fiv) primarily affects cats by compromising their immune system, leaving them vulnerable to opportunistic infections. in addition to immunosuppression, with most of the effect targeted against the cell-mediated (t-cell) immune response, infection with fiv carries an increased risk for development of certain types of neoplasia, with b-cell lymphoma being the most common. transmission occurs most commonly from breeding and fighting. the virus is not spread through casual contact between housemates not engaging in the behaviors stated previously, nor is it spread through casual encounters between nonbreeding and nonfighting cats outside. naturally occurring infection of kittens from queens is rare; however, kittens can become fiv antibody-positive via passive transfer from ingestion of colostrum of fiv-positive queens or queens previously vaccinated against fiv [ ] . fiv antibody levels acquired from maternal transfer in kittens that are actually negative for fiv virus decline over the first several months of life. the standard screening test for fiv is an elisa test designed to detect fiv antibody. the elisa was designed to detect antibody rather than antigen, because infected cats produce high levels of circulating antibody in contrast to low levels of circulating virus [ ] . because kittens may have circulating fiv antibody, although actually being negative for fiv antigen, it is generally not recommended to test kittens less than months of age. if a kitten is tested and a positive result is obtained, the test result should be repeated with a different methodology (western blot or polymerase chain reaction [pcr] ) and should be repeated once the kitten is older than months of age [ ] . if a kitten is truly not infected, the maternal antibody wanes by months of age, leading to seroconversion. if, however, a kitten or cat remains seropositive, the recommendation is made to keep the cat indoors only from that point on so as to prevent infection of other cats and to decrease exposure to potential environmental pathogens. fiv-infected cats can live for years; unless otherwise indicated by concurrent disease, euthanasia is generally not indicated for most owned pets. there is a killed fiv vaccine available, but the efficacy of this product is still unknown. there are five known subtypes of fiv virus, and the vaccine has been formulated to protect against subtypes a and d; however, the predominant subtype infecting cats in north america and europe seems to be subtype b. it is unknown if cross-protection exists between the different subtypes [ ] . because the vaccine elicits a strong antibody response, vaccinated kittens and cats become seropositive on elisa and western blot tests, because both tests detect antibody. a pcr test is available but is currently only performed at certain laboratories. because of the increased technologic needs and increased costs of this test, it is not considered the standard screening test. if done under specific conditions, it can detect virus and therefore may be of benefit in differentiating between cats with viremia (truly infected cats) and kittens or cats with circulating antibody attributable to maternal transfer or vaccination. because of the nature of transmission of the virus and interference with the standard screening methods for infection, vaccination against fiv is not currently recommended. keeping cats indoors if possible, neutering all cats going outside, and preventing exposure to stray or feral cats that may be more likely to engage in fighting behaviors remain the gold standards for preventing this disease [ , ] . the disease feline infectious peritonitis (fip) is caused by a member of the coronaviridae. feline enteric coronavirus (fecv) and fip virus are two phenotypes of the same virus. fecv transmission occurs through the fecal-oral route, where it typically infects the intestinal epithelium, but the organism can be transmitted via fomites and persists for long periods in the environment. most cats infected with fecv do not show clinical signs of disease or may have transient diarrhea; some persistently shed the virus in their feces [ ] . fecv can, however, undergo random mutations within a host, creating fip virus, although the virus does not mutate to this form in most cats and most cats do not develop fip. the fip virus enters and replicates within macrophages, where it can then be disseminated throughout the body. clinical signs are numerous but commonly include weight loss, failure to thrive, diarrhea, pyrexia, and chronic respiratory tract disease. two main types of the disease exist, the dry (noneffusive) and the wet (effusive) forms. both are ultimately fatal diseases [ ] . although there is a vaccine available, efficacy and indication for use are believed to be minimal, if at all [ ] . the current recommendation is not to use this vaccine based on efficacy concerns and the minimal risk of infection in most kittens and cats. infection with fecv and mutation with subsequent development of disease occur most commonly in multiple-cat households (five or more), catteries, and shelters. the standard screening test for fip is a serologic indirect immunofluorescent antibody (ifa) test designed to detect antibody. this test may be of some value, but results need to be interpreted with caution and concomitantly with signalment, clinical signs, and other laboratory data. prior vaccination against fip yields positive ifa results, further posing potential complications in routine screening of this disease. in general, kittens are most vulnerable to this disease, with greater than % of cats with fip being less than years of age [ ] . prevention is directed toward decreasing stress in kittens and cats in multiple-cat households and preventing exposure of naive kittens and cats in environments known to have high endemic levels of fecv and at depopulating catteries known to have high prevalence rates of fecv and fip [ , ] . because of the complexity of this disease and the limited space and objectives of this discussion, readers are encouraged to review the texts by greene [ ] and ettinger and feldman [ ] for a more comprehensive review of this disease. as discussed in the canine section, vaccination with this product does not prevent infection but may decrease fecal shedding of infective cysts. because vaccination does not prevent infection and the organism is readily treated with fenbendazole or metronidazole, routine use of this product in cats is generally not recommended [ ] . vaccines are potent biologic agents designed to prevent disease. any foreign product administered to an animal has the potential to be associated with an unexpected response by that animal. although vaccines must meet usda requirements for safety, efficacy, potency, and purity, there still exists the potential for adverse events with products that have met those standards. veterinarians should always report adverse events related to vaccination to the vaccine manufacturer. some adverse events are more likely to occur with certain agents, whereas others seem to have an increased rate of occurrence in certain breeds. still others may be idiosyncratic and are not predictable. the following is offered as a brief overview of some types of adverse events associated with vaccination and to offer suggestions as to how a practitioner might best respond to and prevent those events from recurring. the reactions seen most commonly are local inflammation at the site of the injection or general malaise, pyrexia, and anorexia for to days after vaccination [ ] . most of these reactions are self-limiting and require nothing more than monitoring by the animal owner. it is appropriate for the practitioner to note any reaction, along with a description of signs exhibited, in the medical record and to offer supportive care if indicated. in some instances, administration of an ml causes transient mild clinical disease. supportive care and isolation from unvaccinated animals are recommended, because the vaccinated animal showing clinical disease sheds the vaccinal organism and is potentially infectious to other animals [ ] . contact information for vaccine manufacturers, support agencies, and disease-reporting organizations is included in table . feline injection site sarcomas, also known as feline vaccine-associated sarcomas or fibrosarcomas, develop secondary to local inflammation of injection sites. there is an increased risk for development of these tumors associated with adjuvants and certain repositol agents, such as long-acting penicillin and corticosteroid injections [ ] . measures to prevent these tumors are aimed at decreasing the local inflammatory response by avoiding the use of adjuvants in this species and administering only those vaccines indicated for the individual animal. multiple vaccines should not be administered in one site because this may increase the amount of inflammation in that site. following the recommended sites for injection is strongly recommended (see individual vaccine sections for specific sites) [ , ] . there are specific guidelines as to how a practitioner should proceed if a cat develops a swelling at the site of a vaccination or injection. the practitioner is advised to monitor the patient closely, documenting three-dimensional measurements and temporal association if a mass or swelling develops at the site of a vaccination. the ''three-two-one rule'' developed by the feline vaccine-associated sarcoma task force should be closely applied. ''three'' refers to persistence of the mass for months or longer, ''two'' refers to a size of cm or greater, and ''one'' applies if the mass increases in size after month. if any of these criteria are met, the mass should be biopsied using wedge technique or needle biopsy, allowing for complete resection of the biopsy margins in the future and subsequent referral to an oncologist or surgical oncologist if fibrosarcoma is confirmed. fine needle aspiration is not recommended for evaluation of potential injection site sarcomas [ , ] . most vaccine manufacturers have programs established to help defray the medical and surgical costs associated with these tumors, and the practitioner is advised always to notify the vaccine manufacturer whenever an adverse event is seen. type i hypersensitivity type i hypersensitivity, also known as immediate hypersensitivity and, in some cases, anaphylaxis, is mediated by ige antibody. the host's immune system may react to anything contained within the vaccine product, including cellular products used for culture, adjuvant, preservative, and the antigen itself, and such a reaction typically occurs within to hours after the administration of a vaccine. in the dog, the most common signs are angioedema (fig. ) , urticaria, and pruritus, but symptoms may progress to respiratory distress and fulminant vascular collapse (anaphylaxis). in the cat, the acute onset of vomiting and diarrhea with associated hypovolemia and respiratory and vascular shock may be seen [ ] . if an animal develops any of these signs within the first several hours after vaccination, it should be presented to the veterinarian immediately for emergency medical care and support. it is not the goal of this review to offer therapies for shock; thus, the reader is referred to emergency veterinary literature for recommended therapies. the point here is to advise the practitioner to proceed with caution when using vaccines that may have a higher incidence of these reactions or in breeds that may be at increased risk for immediate hypersensitivity. the increased association between leptospirosis vaccines and type i reactions is well documented, and there are reports that toy breeds, particularly miniature dachshunds, may be at increased risk for type i reactions associated with leptospirosis vaccination [ ] . if an animal does have a type i reaction to a vaccine, the signs exhibited by the patient, interval between vaccine administration and onset of signs, and therapeutics administered should be well documented in the medical record as well as plans for future vaccination of that patient. ideally, once an animal has this type of reaction to a vaccine, that product should not be used again in that patient. all subsequent vaccines should be administered after a complete physical examination, and the vaccine should be given early in the day to allow monitoring of the patient in the hospital for several hours; however, if this is not possible, the patient should remain in the veterinary hospital for monitoring for at least minutes, followed by subsequent monitoring by the owner at home for several hours. pretreatment with diphenhydramine is an option; it is given parenterally (subcutaneous or intramuscular route) at a dose of . - . mg/kg to minutes before vaccination if hypersensitivity is a concern. administration of corticosteroids concurrently with vaccination to prevent a hypersensitivity reaction is neither appropriate nor recommended because of potential immunosuppression and vaccine interference, however [ , ] . the patient's medical record should be identified, outside and inside, to prevent future accidental readministration of that product. advising the owner that the patient should never receive that product again is important. type ii hypersensitivity reactions (autoimmune reactions) are suspected to occur in dogs secondary to vaccine administration. although this theory is yet unproven, there are reports of dogs developing immune-mediated thrombocytopenia and immune-mediated hemolytic anemia temporally associated with recent vaccination. if a dog develops either of these conditions within to months after vaccine administration, the practitioner would be advised to consider the risk/ benefit ratio of subsequent use of that product in that patient [ , ] . type iii hypersensitivity reactions are immune complex reactions. examples include the anterior uveitis associated with the use of the cav-i vaccine and table the complement-mediated rabies vaccine-induced vasculitis-dermatitis seen in dogs. other examples include glomerulonephritis and polyarthritis. antihistamine administered at the time of vaccine does nothing to prevent the reaction, nor is it recommended to administer corticosteroids concurrently with vaccination. once an animal has had this type of reaction, subsequent use of that product should be avoided in that patient [ , ] . type iv hypersensitivity reactions are cell-mediated responses occurring locally or systemically. examples include sterile granulomas at the sites of vaccine administration or polyradiculoneuritis. many sterile granulomas resolve without any intervention; however, for more severe reactions, the practitioner is referred to various medical texts for recommendations [ , ] . the previous discussion applies mainly to puppies and kittens owned by individuals. puppies and kittens housed in shelters face unique challenges, as do orphaned animals. these animals may not have received colostrum, and it is more likely that their mothers were not adequately vaccinated. the implications are that these animals are less likely to have received maternal antibodies, leaving them more vulnerable in the earliest stages of life. in addition, they frequently are malnourished, have an increased parasite burden, and are placed in crowded environments, possibly with high numbers of endemic pathogens. the american animal hospital association task force is currently developing recommendations specifically designed for puppies in these environments. in general, neonates that may not have received colostrum or are housed under these conditions may be vaccinated at an earlier age and ideally should be vaccinated before or at the time of entry into the shelter. the recombinant distemper vaccine could be given in this circumstance and should be administered . vaccination against additional diseases (canine and feline upper respiratory diseases) is indicated as well (see previous vaccine sections). husbandry is extremely important in these animals: providing proper nutrition, anthelmintics, and clean and dry housing is paramount. in general, these animals are special subsets of the general population facing challenges that most young animals do not experience. fiscal considerations and overall population health applies in these cases much more so than to individual client-owned pets. vaccines are perhaps one of the practitioner's greatest tools in preventing disease and maintaining individual and population health. they are to be used with forethought based on the risk of disease to the population and the individual balanced with assessment of the risks associated with individual vaccines. it is the practitioner's role to educate pet owners regarding actual risks associated with undervaccination and overvaccination. the goal is to reach the highest level of overall animal health with the minimum number of adverse events based on scientific and epidemiologic merit. immunity in the fetus and newborn the defense of the body b cells and their response to antigen report of the american animal hospital association (aaha) canine vaccine task force: executive summary and canine vaccine guidelines and recommendations avma council on biologic and therapeutic agents' report on cat and dog vaccines vaccines and vaccinations: guidelines vs. reality vaccines and vaccinations: the strategic issues report of the american association of feline practitioners and academy of feline medicine advisory panel on feline vaccines infectious diseases of the dog and cat in: veterinary immunology an introduction. th edition. philadelphia: saunders elsevier multicenter case-control study of risk factors associated with development of vaccine-associated sarcomas in cats philadelphia: saunders elsevier canine viral disease canine vaccination protection of dogs against canine distemper by vaccination with a canarypox virus recombinant expressing canine distemper virus fusion and hemagglutinin glycoproteins vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection using recombinant attenuated poxvirus vaccines bsava manual of canine and feline behavioural medicine. quedgeley, gloucester (united kingdom): british small animal veterinary association evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges infectious canine hepatitis and canine acidophil cell hepatitis infectious diseases of the dog and cat seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines canine parvovirus (cpv) vaccination: comparison of neutralizing antibody responses in pups after inoculation with cpv or cpv b modified live virus vaccine duration of serologic response to five viral antigens in dogs national association of state public health veterinarians. compendium of animal rabies prevention and control prevalence of and risk factors for leptospirosis among dogs in the united states and canada: cases ( - ) leptospirosis: a re-emerging zoonotic disease canine vaccination infectious diseases of the dog and cat canine borreliosis diseases caused by systemic bacterial infections infectious diseases of the dog and cat impact of giardia vaccination on asymptomatic giardia infections in dogs at a research facility enteric protozoal infections, giardiasis presented at the dr. ross o. mosier th annual western veterinary conference use of serologic tests to predict resistance to feline herpesvirus , feline calicivirus, and feline parvovirus infection in cats other feline viral diseases infectious diseases of the dog and cat update on feline calicivirus: new trends an outbreak of virulent systemic feline calicivirus disease rabies surveillance in the united states during epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats feline leukemia virus report of the american association of feline practitioners and academy of feline medicine advisory panel on feline retrovirus testing and management comparison of the safety and efficacy of a recombinant feline leukemia virus (felv) vaccine delivered transdermally and an inactivated felv vaccine delivered subcutaneously chlamydial infections textbook of veterinary internal medicine feline immunodeficiency virus infection feline immunodeficiency virus infection and related diseases infectious diseases of the dog and cat feline infectious peritonitis and feline enteric coronavirus infectious diseases of the dog and cat textbook of veterinary internal medicine vaccine-associated feline sarcoma task force. vaccine-associated feline sarcomas vaccine-associated feline sarcoma task force. the current understanding and management of vaccine-associated sarcomas in cats plumb's veterinary drug handbook vaccine-associated adverse events immune complexes and type iii hypersensitivity key: cord- -nnm n a authors: varadé, jezabel; magadán, susana; gonzález-fernández, África title: human immunology and immunotherapy: main achievements and challenges date: - - journal: cell mol immunol doi: . /s - - - sha: doc_id: cord_uid: nnm n a the immune system is a fascinating world of cells, soluble factors, interacting cells, and tissues, all of which are interconnected. the highly complex nature of the immune system makes it difficult to view it as a whole, but researchers are now trying to put all the pieces of the puzzle together to obtain a more complete picture. the development of new specialized equipment and immunological techniques, genetic approaches, animal models, and a long list of monoclonal antibodies, among many other factors, are improving our knowledge of this sophisticated system. the different types of cell subsets, soluble factors, membrane molecules, and cell functionalities are some aspects that we are starting to understand, together with their roles in health, aging, and illness. this knowledge is filling many of the gaps, and in some cases, it has led to changes in our previous assumptions; e.g., adaptive immune cells were previously thought to be unique memory cells until trained innate immunity was observed, and several innate immune cells with features similar to those of cytokine-secreting t cells have been discovered. moreover, we have improved our knowledge not only regarding immune-mediated illnesses and how the immune system works and interacts with other systems and components (such as the microbiome) but also in terms of ways to manipulate this system through immunotherapy. the development of different types of immunotherapies, including vaccines (prophylactic and therapeutic), and the use of pathogens, monoclonal antibodies, recombinant proteins, cytokines, and cellular immunotherapies, are changing the way in which we approach many diseases, especially cancer. the knowledge of human immunology has improved exponentially in recent years, and more advances in the near future are certainly imminent. the immune system is extremely complex, but we are now developing new tools and skills to study it. several factors have been involved in these advancements, and the most important ones include the development of thousands of different monoclonal antibodies that allow the identification of a large variety of cell subpopulations and the functional analysis of immune cells. these tools, together with new and sophisticated technologies, such as single-cell analysis, imaging techniques, omics (including massive dna-rna sequencing, proteomics, and metabolomics data and new tools for processing these data, such as artificial intelligence and machine learning approaches, mathematical modeling, etc.), newly designed animal models (using conventional transgenic/knockout/knock-in mice or new technologies such as crispr-cas (clustered regularly interspaced short palindromic repeats-crispr-associated protein ), are increasing our knowledge about how our immune system functions. the study of the interaction between the immune system and other systems, such as the nervous and endocrine systems or the microbiome, in several illnesses has produced interesting results with important clinical applications. all of these advances can be applied to several immunemediated pathologies, but overall, the success achieved with some types of immunotherapies in recent years is revealing new ways to explore and manipulate the immune system for our benefit. writing a review about human immunology is a significant challenge, but we have attempted to bring together recent knowledge about the immune system, immune-mediated illnesses and types of immunotherapies. the last two decades have witnessed a major revolution in the field of immunology. the traditional classification of the immune system into two different arms, namely, innate and adaptive components that collaborate to respond to foreign antigens or to perform self-/nonself-discrimination, has become much more complex. the development and application of new technologies have provided new findings and created a new landscape in which the immune system establishes cross talk, not only between immune components but also with commensal microorganisms , and other important systems, such as the endocrine and nervous systems [ ] [ ] [ ] . these developments have forced immunologists to reformulate the immunological architecture that confers protection, which has made the study of the immune system especially attractive. moreover, these advances have led to an increased interest in better understanding, managing, and manipulating the immune response in both health and disease. the characterization of new immune cell subsets has been a constant feature in the immunology field. this evolution is clearly reflected in the discovery of an innate counterpart of t lymphocytes, collectively named innate lymphoid cells (ilcs) , and in the identification of different types of effector cd and regulatory t cells . innate lymphoid cells (ilcs). ilcs are lymphocytes, but in contrast to adaptive immune cells, they can colonize lymphoid and barrier tissue sites during fetal development, do not undergo somatic recombination and do not express antigen-specific receptors , . in addition to lymphoid organs, ilcs are enriched in barrier tissues, such as the gastrointestinal tract, airways, and skin , . these innate cells have been considered to be tissue-resident cells, but recent studies suggest that ilcs can migrate through the lymphatic system during homeostasis or enter into the circulation upon infection and inflammation , . currently, five different ilcs are defined on the basis of their transcription factor expression, different cytokine production and/or developmental patterns : natural killer (nk) cells (discussed below), lymphoid tissue inducer cells (ltis) and three subsets of helper-like ilcs (ilc s, ilc s, and ilc s), which are considered to be the innate counterparts of t helper (th) , th , and th cells, respectively. the main focus of this review is ilcs. ilc s are dependent on the t-box transcription factor t-bet and produce interferon gamma (inf-γ), but they differ in the expression of eomesodermin transcription factor . ilc s express cd in humans and cd r in mice, but the natural cytotoxicity receptor nkp (also known as ncr ) is expressed in both species , . ilc s constitute the most homogeneous class of ilcs; they are dependent on gata and rorα, and they produce type cytokines, mainly interleukin (il- ) and il- . ilc s are involved in immune responses to parasite infection, and in humans, they express chemoattractant receptor-homologous molecule expressed in t h cells (crth ) and high levels of cd , whereas most mouse ilc s express st (a member of the il- receptor family) , . the development and function of ilc s depend on the transcription factor rorγt. both human and mouse ilc s can produce granulocyte macrophage colony-stimulating factor (gm-csf), il- , and/or il- , . in humans, two major ilc subsets can be distinguished on the basis of the expression of the natural cytotoxicity receptor nkp (also known as ncr ) , . both types can produce il- , but the production of il- is mainly confined to nkp + ilc s. extensive research has focused on deciphering the role of ilcs to ensure the maintenance of tissue homeostasis and immune protection , . ilcs express particular sets of receptors in a tissuespecific manner, and these allow the detection of host-derived signals (including those from alarmins, neuronal mediators, microbia, and the diet) . the integration of these endogenous signals is essential for the maintenance of tissue homeostasis, but dysregulation of ilc responses leads to inflammation and disorder , . ilc are mainly involved in early protection against viruses and bacteria , , but their response to dysregulated local proinflammatory cytokine production in adipose tissues leads to the development of metabolic disorders and obesity . il- and il- produced by ilc s induce goblet cell differentiation and the recruitment of eosinophils, basophils, and mast cells , which are involved in protection against infection by helminths and viruses, but when uncontrolled, these cells drive allergic responses and metabolic disorders. moreover, the depletion of ilc s in animal models suggests a role for these cells in atopic dermatitis and asthma . ilc s are abundant in mucosal tissues, and ncr + ilc s have been proven to be essential for regulating the balance between commensal and pathogenic bacteria through the production of il- . in contrast, ncr − ilc s can promote colitis in a model of inflammatory bowel disease . the lack of immunodeficiency in ilc-deficient patients led to the proposal that ilcs are dispensable in the presence of functional t cells and b cells . however, recent studies support the idea that ilcs cannot be considered to have functions that only duplicate those of the adaptive immune system. in addition to those showing the essential role of lti cells in the formation of secondary lymphoid organs during embryogenesis and the postnatal development of intestinal lymphoid clusters, recent studies also provide evidence that subsets of ilcs express multiple factors that modulate the adaptive immune response in health and disease , . in particular, ilc s and ilc s modulate the t-cell response. studies in mice suggest that in healthy intestine, ilc s express major histocompatibility complex (mhc) class ii molecules but lack the expression of costimulatory molecules; therefore, they inhibit microbiota-specific t-cell responses, thus preventing intestinal inflammation . it seems that the interaction between ilc s and tfh cells limits il- secretion and the production of iga by mucosal b cells . studies with murine models have significantly contributed to the classification and understanding of the role of ilcs in the immune system, especially since similarities have been observed between ilcs identified in mice and humans . however, the differences between these two species present real challenges , because human ilcs have unique attributes that are only now being elucidated, with further work required in this exciting field. the roles of ilcs in immunity and their cross talk with other components of the immune response await further analysis. detailed coverage of this topic is beyond the scope of this review, and we refer the reader to recent reviews that provide more information on the biology of human and mouse , ilcs. t cells and plasticity. t cells are categorized as tα/β and tγ/δ cells, depending on the type of t-cell receptor (tcr) that they express . human tγ/δ cells, similar to their murine counterparts, are a minor population ( - % of nucleated cells) in peripheral blood, but are especially abundant in barrier tissues such as the epidermis [ ] [ ] [ ] . the three main subsets of t cells carrying α/β receptor are the cd +t helper cells and cd +cytotoxic and cd + cd + regulatory t cells . new effector cd + helper t-cell subsets (initially classified as th and th ) , have been recently described, and at least six human th cell subsets have been identified to date: th , th , th , tfh, th , and th cells , . all of these cells recognize foreign peptides presented by class ii mhc molecules on antigenpresenting cells (dendritic cells, macrophages, and b lymphocytes). th cells are required to activate macrophages and cellmediated immunity to kill intracellular pathogens , whereas th cells are important in facilitating eosinophils to fight against parasitic helminths and b cells for antibody production and antibody class-switching to generate iga or ige . th cells are required to mobilize neutrophils for the clearance of fungi and extracellular bacteria, and they are also involved in mucosal protection . th and th cells are also involved in mucosal immunity; th cells protect against parasites , , and th cells prevent microbial translocation across epithelial surfaces and promote wound healing , . as mentioned in the introduction to ilcs, studies on human th cells isolated from lymphoid organs and blood samples, along with recent observations on the developmental mechanism of distinct th cell subsets, have revealed both similarities and differences of human and mouse th cells , , . tfh cells are very important for germinal center reactions, antibody class switching, affinity maturation, and the development of high affinity antibodies and memory b cells , . at the surface marker level, tfh cells are generally characterized by the expression of cxcr , the chemokine receptor for cxcl , which is highly expressed on b-cell follicles for expressing inducible t-cell costimulator (icos) and programmed death protein (pd- ) , , which enable their involvement in the interaction of tfh cells and b cells . the definition of a given t cell lineage is based on its ability to sense different inductive cytokines, to produce particular cytokines or to express a lineage-specifying transcription factor. th cells produce ifn-γ and express t-bet ; th cells are characterized by il- , il- , and il- production and gata- expression , ; ptregs, which are induced in the periphery from naïve precursors, produce tgf-β and express foxp (tr cells are il- -secreting tregs that do not express foxp ) . th cells produce il- a, il- f, and il- and express rorγt , , and tfh cells produce il- and il- and express the bcl transcription factor. in addition, th cells, which produce il- and express the aryl hydrocarbon receptor (ahr) , , and th cells, are characterized by the expression of il- and the transcription factor pu. . additional levels of regulation, such as the differential expression of micrornas, long noncoding rnas (lncrnas), and protein stability and function, have been found to control various aspects of th cell differentiation and effector function , . cd + cytotoxic t cells express the dimeric cd marker and have specific lytic capacity to target cells through several mechanisms, including the release of cytotoxic granules, secretion of cytokine tumor necrosis factor alpha (tnfa) and interferon gamma, and the induction of cell death through the interactions of fas and the fas ligand , . their tcrs are restricted to interactions with peptides presented by class i mhcs. regulatory t cells (tregs) include thymically derived and peripherally induced regulatory t cells (ttregs and ptregs, respectively), and they produce either il , tgf-beta, il- or combinations of these proteins . ttregs express the transcription factor foxp and secrete il and tgf-β; ptregs, which are induced in the periphery from naïve precursors, can also be subdivided into il- -induced tregs [tr cells] (which secrete large amounts of il- and moderate levels of tgfβ), th cells (which produce il- and tgf-β), and tgfβ-induced tregs [itregs], which may or may not express foxp . moreover, new subsets of regulatory t cells have been described. they include follicular regulatory t cells (which express foxp and bcl- and cxcr ), which modulate the function of tfh cells and fine-tune the germinal center response [ ] [ ] [ ] , and a il- dependent regulatory population of cells (referred to as itr cells), which show potent suppressive potential in several mouse disease models . other regulatory populations have also been described, including bregs and cd + tregs, which are the analogous counterparts of tregs [ ] [ ] [ ] . recent studies have revealed the capacity of differentiated t cells, particularly th cell and ptreg subsets, to change their phenotype in response to changing contexts [ ] [ ] [ ] [ ] [ ] . becattini et al. found that human memory cd t cells primed in vivo by pathogens (e.g., candida albicans and mycobacterium tuberculosis) or vaccines (tetanus toxoid) are highly heterogeneous, both at the population and clonal levels. with respect to studies on human arthritis, nistala et al. proposed that th cells are recruited to the joint and converted to th / or th cells in response to local il- levels. this plasticity has also been observed with in vitro assays under conditions that mimic a disease site, namely, low tgf-β and high il- levels . these results are inconsistent with the original idea of th lineage stability and provide new possibilities for disease treatment aimed at inducing particular th subsets to modulate the immune response against pathogens or to control detrimental immunity , , . trained and adaptive immune memory other classical concepts in fundamental immunology, such as immune memory, are also changing. the specificity and the capacity to generate long-lived memory cells are two properties that have been classically used to distinguish innate immunity from adaptive immunity. adaptive immunity is clearly based on the specific recognition of antigenic determinants by somatically diversified receptors (b cell and t cell receptors (bcr and tcrs, respectively)) and on its capacity to respond more effectively to restimulation with the same antigen. in contrast, innate immune responses have traditionally been considered nonspecific and without the capacity to adapt . however, the discovery of germline-encoded pattern recognition receptors (prrs) and the "trained innate" immunity (or innate immune memory) have provoked a shift in our understanding of the immune response. in , medzhitov et al. demonstrated that pattern recognition receptors (prrs) expressed on innate cells recognize invariant molecular structures expressed by invading pathogens . after the interaction, prrs trigger the expression of costimulatory molecules and activate important signaling pathways to induce the activation of innate and adaptive immune cells. prrs mainly belong to four families: toll-like receptors (tlrs), nod-like receptors (nlrs), c-type lectin receptors (clrs), and peptidoglycan recognition proteins (pgrps) , . the profiles of prrs expressed by innate cells can lead to partially specific recognition of a type of microorganism; e.g., innate cells can distinguish between gramnegative and gram-positive bacteria and modulate the immune response based on this recognition, although they cannot differentiate between bacterial species . the idea that only jawed vertebrates developed immunological memory has also been challenged by the observation of resistance to reinfection in organisms that lack an adaptive immune response, such as plants and invertebrates , . recent studies have shown that monocytes and macrophages exposed to candida albicans or β-glucans exhibited an enhanced secondary response . in addition, immunization of mice with bacillus calmette-guérin (bcg, the tuberculosis vaccine) induces t cellindependent protection against secondary infections by candida albicans, schistosoma mansoni or influenza virus [ ] [ ] [ ] [ ] . thus, organisms are protected not only against the original microorganism but also to unrelated pathogens. the mechanisms underlying the establishment of this innate immune memory differ from those involved in adaptive immune memory . after infection or vaccination, innate immune cells (such as monocytes and macrophages) display long-term functional changes through epigenetic and metabolic reprogramming, including histone acetylation, methylation and modulation of noncoding rnas [ ] [ ] [ ] . in turn, the faster and more pronounced reactivity of adaptive immune cells (t and b lymphocytes) upon reinfection is characterized by permanent changes in the genome of cells, such as mutations, gene rearrangement, clonal expansions, as well as epigenetic modifications, all of which ensure a more persistent effect than is endowed by trained immunity , , . other cells for which immunological memory has been described include tγ/δ cells and innate lymphoid cells . recently, some authors have proposed that nk cells are also capable of immunological memory [ ] [ ] [ ] [ ] . antigen-specific recall responses by human nk cells were observed by nikzad et al. in humanized mice and in varicella zoster virus (vzv)-exposed adult human volunteers, in which cytotoxic nk cells were recruited to sites of an vzv test antigen challenge on the skin. sensitization with haptens using mice lacking t cells and b cells led to the generation of hapten-specific memory nk cells . the recall response persisted for more than four months after priming, and was adoptively transferred to naïve mice . interestingly, nk cells exhibit memory that is not only specific to a given virus, such as cytomegalovirus , , but that is also induced in the absence of a defined antigen , . furthermore, new studies suggest that trained immunity is not a phenomenon that is restricted to immune cells, because epithelial stem cells also retain memory of previous inflammatory challenges by displaying an enhanced wound-healing capacity upon skin damage . given the data outlined above, immunological memory is now recognized to be highly diverse and not restricted to b cell-or t cell-mediated adaptive immunity. much remains to be learned in this field, but the different manifestations of immunological memory described above offer an important basis for clinical applications, such as the development of novel vaccination strategies or new therapies for pathological situations in which immunological memory can be detrimental, such as allergies or autoimmune diseases , , . interaction of the immune system and the microbiome the immune system has evolved in the presence of commensal microorganisms that colonize barrier surfaces of vertebrates and invertebrates , . the cross talk between the natural host microbiome and immune system is particularly interesting in the gastrointestinal tract, where the density and diversity of indigenous bacteria, viruses and fungi are greatest compared to those of other anatomical sites . in the literature, reports of observed changes in microbial community composition during diseases are diverse and include those in inflammatory bowel disease (ibd), obesity, metabolic syndrome, and multiple sclerosis [ ] [ ] [ ] [ ] [ ] . however, the microbiome can be influenced by different factors, such as the specific niche that it occupies, diet, stress, environmental factors, and host genetics, and a specific correlation does not necessarily infer causation. the presence of these commensals in mucosal tissues has been known since before metchnikoff, but the current knowledge on the role of the microbiome in shaping the immune system throughout life came mostly from the development of next-generation sequencing (in particular, the reduction in the cost of s ribosomal rna gene sequencing) and the use of germ-free animal models, which can be colonized even with human microbiota . germ-free mice are characterized by atrophy of peyer's patches with few germinal centers and isolated lymphoid follicles, a lower number of b, t, and dendritic cells and a decreased level of immunoglobulins, particularly iga and igg . these effects are observed at the mucosal and systemic levels, and they can be reversed within weeks after the colonization of germ-free mice with commensal bacteria . moreover, colonization with commensal bacteroides fragilis revealed the immunomodulatory effect of bacterial polysaccharides in restoring systemic cells and the differentiation of cd + t cells into regulatory t cells (foxp + tregs), which in turn favor mucosal immunomodulation . the induction of th cell maturation by segmented filamentous bacteria has also been reported . these important examples emphasize the major roles of the commensal microbiome in the maturation of mucus-associated lymphoid tissue and the systemic immune system. the development of new technologies to better track the locations and activities of distinct microbial populations is essential to elucidate host-microbe interactions, through which other systems, such as the nervous system, seem to play important roles , - . the better characterization of some immune cell subsets, trained immunity, and host-microbiome interactions provides a few very good examples that prove the maturation of immunology in the last few decades. in this sense, studies with mouse models have significantly contributed to the increase in our fundamental knowledge; however, the differences between murine and human immunology are notable, and conclusions drawn from mouse studies are sometimes not fully translated to humans . if we want to fully exploit the power of the immune system for human health, greater effort is required for understanding human immunology. immunologists, in cooperation with experts from other fields, have developed a variety of protocols and tools to achieve greater selectivity in the identification and analysis of human cell subsets, types of cytokines and receptors, chemokines, etc. these tools range from biological approaches that rely on next-generation sequencing, mass spectrometry, and bioinformatics to immune monitoring technologies based on multiparameter flow cytometry and single-cell gene expression analysis. although not without limitations, these techniques provide a much better picture of the whole immune system than individual and independent approaches. immune-mediated illnesses comprise a wide variety of diseases characterized by the dysregulation of a normal immune response. most of these illnesses are complex disorders believed to arise from a combination of genetic and environmental factors . infectious diseases infectious diseases are caused by pathogens (viruses, bacteria, fungi or parasites that infect the host body), and they remain a leading cause of mortality worldwide. prominent examples include illnesses produced by mycobacterium tuberculosis, human immunodeficiency virus (hiv), plasmodium falciparum or the current coronavirus disease (covid- ) outbreak caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), which has already infected millions of people and produced thousands of deaths in many countries. for a number of years, many people believed koch's postulates, which implied that virulence traits reside solely in the pathogen. however, recent advances in molecular biology have shown that host genes play major roles in infection, together with a wide range of environmental variables . to date, six gene products endowing infectious disease susceptibility have been validated in the literature: ( ) hemoglobin subunit beta; ( ) band -anion transport protein; ( ) duffy antigen/ receptor, which is associated with plasmodium spp. infections; ( ) the prion protein associated with creutzfeldt-jakob disease; ( ) fucosyltransferase and , which is associated with norwalk virus infections; and ( ) c-c motif chemokine receptor (ccr ) coreceptor, encoded by an immune-related gene and leads to the impairment of the entry of the human immunodeficiency virus (hiv) into helper t cells, thus avoiding/decreasing the progression to acquired immunodeficiency syndrome . another gene associated with infectious disease and the immune system is the natural-resistance-associated macrophage protein (nramp ), which encodes an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. it is a susceptibility locus for increased ratios of infection with leishmania spp. parasites and certain strains of salmonella spp., mycobacterium bovis and mycobacterium tuberculosis , . in addition, it has been suggested that functional variants of immunoglobulin fc gamma riia (cd ) are related to the development of invasive encapsulated bacterial infections . moreover, because of recently acquired genomic data, new human polymorphisms have been discovered, some of which play roles in changing immunoglobulin levels, seroconversion rates or the intensity of antigen-specific immune responses. in addition, they also contribute to human susceptibility to infection by viruses such as influenza, rhinovirus and respiratory syncytial virus . these polymorphisms are mapped within the mhc (hla-dqb * , hla-drβ , or hla-dpβ ), natural killer cell immunoglobulin-like receptors and (kir dl and kir ds ) and natural killer lectinlike receptor d (kldr- ) . several recent studies available as preprints have analyzed certain genes that may explain the differences in the variable expression of and susceptibility to covid- by patients, either by affecting the host receptor for the virus (angiotensin i converting enzyme (ace- )) , immune genes (tlr and others) or blood groups (group o seems to be the most protective) , and more extensive omics studies are now underway with larger numbers of patients. in , the physician paul ehrlich first used the term "horror autotoxicus" to describe the way autoimmunity contradicts the natural aversion to self-injury ("living with the enemy", reviewed in ). currently, according to the american autoimmune related disorders association, more than autoimmune diseases have been identified. historically, these diseases were considered to be rare, but current epidemiological data have shown that they affect approximately - % of the population worldwide. some of the most common autoimmune diseases include type diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease (https://www.aarda.org/diseaselist/). although significant progress has been made in understanding the mechanisms of autoimmune diseases and the nature of selftolerance, these disease remain major burdens on health systems around the world. autoimmune diseases arise when the immune system attacks normal components of the body . the concept of immune tolerance is defined as the ability of the immune system to prevent the targeting of self-molecules, self-cells or self-tissues. on the other hand, the failure to distinguish self from nonself is often termed a break of tolerance, and it is the basis for an autoimmune disease . what are the mechanisms that lead to a break in tolerance? autoimmune diseases are complex disorders that are believed to arise from a combination of genetic (mutations and higher inheritance frequency of some types of major histocompatibility complex alleles), epidemiological (age and sex) and environmental (infections, microbiota, tobacco, chemicals and pharmaceutical drugs). factors these factors trigger a break in self-tolerance with the activation of self-reactive lymphocytes through several mechanisms, such as molecular mimicry, the overexpression and abnormal expression of mhc class ii molecules in peripheral tissues, thymic aging, and immunodeficiencies (discussed below) and many others. some lymphocytes escape control due to polymorphisms in several genes that affect the routes of lymphocyte activation. other causes may include defective antigen presentation by some mhc variants with specific polymorphisms. therefore, the self-reactive lymphocytes that have escaped control and react against self-constituents initiate the autoimmune process . although a large number of genome-wide association studies (gwas) have led to the identification of hundreds of polymorphisms associated with the development of different autoimmune diseases, it has proven difficult to define the role of most of these polymorphisms in the breakdown of tolerance to a selfantigen [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is worth highlighting, however, that the mhc remains the main genetic factor associated with human autoimmunity , . other gene variants identified are common to many autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type i diabetes, ulcerative colitis, autoimmune hepatitis and numerous other autoimmune diseases. for example, the protein tyrosine phosphatase nonreceptor type (ptpn ) gene encodes a protein that inhibits t-cell activation in the adaptive immune system, whereas it promotes myeloid cell activation; interferon regulatory factor -transportin (irf -tnpo ) is involved in the accumulation of lymphocytes within lymphoid organs and failed elimination of autoreactive naïve t cells; btb domain and cnc homolog (bach ) has a critical role in immunoglobulin class-switching recombination, somatic hypermutation of immunoglobulin encoding genes and the activation of tissue macrophages. a more complete list of genes associated with autoimmunity can be found in the review by wang et al. researchers are currently looking for the missing heritability in autoimmune diseases by focusing on the study of methylome profiles, genetic cargos in extracellular vesicles, genetic alterations, and ways in which the microbiome may affect these diseases. immune-mediated rejection of tissue allografts was first described in by the british immunologist peter medawar , . only three years later, george snell described the mhc, which carries the histocompatibility genes, and one decade later, jean dausset described the human leukocyte antigen (hla); each of these scientists was recognized with the nobel prize in physiology and medicine . since its discovery, mhc has emerged as the most polymorphic gene locus in eukaryotes with hla and related alleles, more than nucleotide variants reported in the individual-participant data-international immunogenetics/ human leukocyte antigen (ipd-imgt/hla) work group database (https://www.ebi.ac.uk/ipd/imgt/hla/), release . . , / / . although the main barrier for long-term organ and tissue grafting is driven by hla incompatibilities, other important players play roles in transplant rejection. in particular, minor histocompatibility antigens, which are peptides derived from allelic variants of normal cellular proteins, presented by class i or ii mhc antigens induce cellular immune responses in hla-matched individuals who lack the same allelic variant . natural killer (nk) cells also play important roles in transplantation through their killer cell immunoglobulin-like receptors (kirs), which are receptors for hla class i molecules. nk cells expressing an inhibitory kir-binding self-hla can be activated when exposed to allografts that lack a ligand for the inhibitory receptor . the locus that codifies these receptors displays a considerable degree of polymorphism, with alleles reported in the individual-participant data-international/killer cell immunoglobulin-like receptors (ipd/kir) work group database, release . . , / / . more recently, we have begun to appreciate the importance of non-hla genetic factors in the development of transplant rejection; examples include polymorphisms in the genes encoding cytokines, such as tumor necrosis factors (tnf), interleukins (il- , il- and il- ), interferon gamma (ifn-γ), and transforming growth factor-β (tgf-β ). other genes encode pathogen recognition receptors, with nucleotide-binding oligomerization domaincontaining (nod (card )) being the most widely studied, although conclusive data have not been obtained to date . primary immunodeficiencies (pids) comprise a heterogeneous group of more than genetic disorders that result in defects in the immune response . pids are considered mendelian disorders because they are mainly autosomal recessive disorders that often display incomplete penetrance, which affects the severity and onset of the disease. with the exception of immunoglobulin a (iga) deficiency, pids are considered to be rare disorders, as their prevalence worldwide ranges from to among , people . unsurprisingly, these types of diseases are not uncommon in highly consanguineous populations such as those in the middle east/northern africa (mena) region. the incidence of consanguinity marriage in these areas ranges between and %, which leads to a unique population in which autosomal recessive diseases arise, with the prevalence of pid in these countries as high as in , people . although more than genes have been described for pids, approximately % of the causal genes remain unknown, and next-generation sequencing studies performed in mena populations are contributing to the search for currently unknown genes that cause pids . a complete and updated list of pid-causing genes and diseases can be found at the european society for immunodeficiencies (esid) webpage (https://esid.org) . clinical manifestations of pids are highly variable; many disorders involve an increased susceptibility to several types of infections, but some patients develop autoimmune diseases. patients usually present recurrent sinus or ear infections or pneumonia within a one-year period; other indicators are failure to thrive, poor response to prolonged use of antibiotics, and persistent thrush or skin abscesses . depending on the affected pathway, pids are associated with varying levels of severity, times of onset, and risks of infection by certain groups of microorganisms. according to the international union of immunological societies (iuis) (https://iuis.org/ committees/iei/), inborn errors of immunity can be classified as follows: (a) immunodeficiencies that affect cellular and humoral immunity; (b) combined immunodeficiency (cid) with associated or syndromic features; (c) predominant antibody deficiencies; (d) diseases of immune dysregulation; (e) congenital defects of phagocyte number, function, or both; (f) defects in intrinsic and innate immunity; (g) autoinflammatory disorders; (h) complement deficiencies; and (i) phenocopies of a pid , . however, pids are broadly classified as follows according to the component of the immune system affected: • t-cell immunodeficiency, e.g., defects in the ifn-γ/il- pathway and mutations in the autoimmune regulator (aire) gene. • b-cell (antibody-mediated) immunodeficiency: gamma-globulinemia, x-linked common variable immunodeficiency (cvid), selective iga deficiency, specific antibody deficiency, and igg subclass deficiency. • combined immunodeficiency: wiskott-aldrich syndrome, ataxia telangiectasia, digeorge syndrome and severe combined immunodeficiency (scid). • phagocyte defects: chronic granulomatous disease, hyperimmunoglobulin e (ige) syndrome and leukocyte adhesion deficiency. • complement defects (deficiency in early, late or regulatory complement components) . autoinflammatory diseases systemic autoinflammatory diseases (aids) are characterized by recurrent acute inflammatory episodes secondary to a dysregulated inflammatory process that typically develops during childhood, with recurrent episodes of fever, rashes, and disease-specific patterns of organ inflammation. genetically speaking, these are hereditary disorders, andto date, more than genes (table ) have been identified as causes of aids, which can be grouped according to the pathway that is altered . ( ) inflammasome. the inflammasome is a multiprotein intracellular complex that detects pathogenic microorganisms and stressors and activates the highly proinflammatory cytokines il- β and il- . genes affected in this group are mefv (mediterranean fever pyrin innate immunity regulator), which is related to familial mediterranean fever (fmf); nlrc (nlr family card domaincontaining ); nlrp (nlr family pyrin domain-containing ) and wdr (wd repeat domain ) . ( ) type-i interferon (ifn)-mediated disorders. these disorders are characterized by the upregulated expression of genes induced by ifn. the gain of function by variants of tmem (transmembrane protein ) is the core manifestation of this disorder group, but other genes have been identified, including ddx (dexd/h-box helicase ), dnase (lysosomal deoxyribonuclease ), pola (dna polymerase alpha subunit) and usp (ubiquitin-specific peptidase ) , . ( ) ubiquitination disorders. ubiquitination is a process that marks proteins for degradation via the proteasome, which is required for the processing of intracellular antigens (such as virus proteins or mutated tumor proteins) and their presentation by class i hla molecules. ubiquitination involves three main steps: activation, conjugation and ligation, which are performed by ubiquitin-activating enzymes (e s), ubiquitin-conjugating enzymes (e s), and ubiquitin ligases (e s). ubiquitination disorders are caused by variants of the psmb , psmb , psma and psm genes (proteasome s subunit beta , subunit beta , subunit alpha and subunit alpha , respectively), affecting the proteasome subunits, proteasome maturation protein gene (pomp) and/or proteasome assembly chaperone (psmg ), by encoding proteasome assembly molecules . in addition, other genes in this group, such as otulin (otu deubiquitinase with linear linkage specificity), encode ubiquitin peptidases, i.e., proteins involved in ubiquitination assembly complexes, such as hoil- (hemeoxidized irp ubiquitin ligase ) and hoip (nhp -like protein homolog). finally, the loss of function due to variants of the tnfaip (tnf-alpha-induced protein , also known as a ) gene, which encodes a protein with ubiquitin ligase and ubiquitinase activity, has also been described . ( ) inflammatory or innate immune regulators. a large number of genes have been found to affect the pathways/ mechanisms involved in macrophage and b-cell differentiation and lymph node development, among many functions. genes in this group include ada (adenosine deaminase ), tnfrsf a (tnf receptor superfamily member a), adgre (adhesion g protein-coupled receptor e ), trnt (trna nucleotidyltransferase ), lacc (laccase domain-containing ) and ap s (adaptor related protein complex subunit sigma ) . allergy allergic diseases can be termed complex diseases that involve both genetic and environmental factors, and they influence not only the development of ige-mediated sensitivity in the case of hypersensitivity type i allergies but also the subsequent development of clinical symptoms in a range of tissues, including skin, nose, and lung tissue . since the first report of a link between chromosome q and atopy in , knowledge about the common risk variants for allergic diseases has increased exponentially, mainly because of gwas. most allergic diseases have allergy-related traits such as asthma, with the strongest association mapped to chromosome q . however, the disease-associated gene at this locus remains unclear; one of the candidate genes is ormdl (sphingolipid biosynthesis regulator ) due to its role in sphingolipid synthesis and the regulation of eosinophils. other genes associated with asthma are interleukin (il ) and its receptor, il rl (interleukin receptor-like ), hla region, smad (sma-and mad-related protein ) and il rb (interleukin receptor subunit beta) . as asthma and other allergic-associated traits could be present in patients without allergies, some researchers performed gwas analysis on cohorts of patients who had high levels of allergenspecific immunoglobulin e (ige) or a positive skin prick test. as a were identified, and the strongest association was on chromosome q . this locus has been associated with two genes: c orf (emsy transcriptional repressor, brca interacting), a potential regulator of interferon-stimulated gene, and lrrc (leucine rich repeat-containing ), which is involved in transforming growth factor beta (tgfβ)-signaling in t regulatory cells. the rest of the associated loci involved in the pathogenesis of allergy highlight the importance of the th responses (stat (signal transducer and activator of transcription ), tslp (thymic stromal lymphopoietin), bcl (b-cell lymphoma protein), il rl (interleukin receptor-like ), il (interleukin ), gata (transacting t-cell-specific transcription factor binding protein )); innate immunity (tlr / / (toll-like receptor / / )); tgfβ-signaling (lrrc (leucine rich repeat-containing ), smad (mothers against decapentaplegic homolog )); t-cell (il (interleukin ), ptger (prostaglandin e receptor )) and t regulatory box (lrrc (leucine rich repeat-containing ), il- , nfatc (nuclear factor of activated t cells ), foxa (forkhead box a )) . in the last two years, researchers have focused on epigenomewide association study (ewas) of allergy processes. the epigenetic landscape is specific for a given cell; thus, ewas requires careful selection of the relevant cell type for a given biomedical condition. for allergies, ewas has mainly been performed on nasal mucosal cells and whole blood (although the result was later normalized by the number of circulating eosinophils). nasal mucosal cells comprise cd + t cells, cd + t cells, myeloid cells, innate lymphoid cells, b cells, double-negative t cells, granulocytes, cd + cells, and plasma cell populations . in all of these studies, cpg-associated regions were identified, from which the smad gene, coding for an important regulator of t-cell differentiation, was replicated in three independent cohorts . of all of the genes in whole blood identified using ewas, only the acot (acyl-coa thioesterase ), epx (eosinophil peroxidase), gja (gap junction protein alpha ) and mettl (methyltransferase-like ) genes were confirmed in the nasal cell populations . in , ehrlich proposed the idea that mutant cells arise continuously and that the immune system scans for and eradicates these mutant cells before they manifest clinically . however, immune surveillance remained a controversial topic until its acceptance in the s . immune surveillance is the recognition and elimination of cancerous cells by lymphocytes, which act as sentinels that recognize transformed cells. ultimately, during tumor progression, cancer cells show low immunogenicity and resistance to immune effector cells, thus expanding and escaping immune control. the way in which cancer cells modify the immune system has been called immune editing . the key of immunosurveillance is cancerous cell expression of tumor antigens that can activate various immune cell phenotypes; for simplicity, any overexpressed, mutated, dysregulated, or rearranged gene product expressed by a cancerous cell may be considered a tumor antigen. it is critical to consider that most of these proteins, except those derived from virus-infected cancer cells, are primarily self-proteins, but they are expressed with mutation(s) or minor changes in their antigenic structure . one mechanism by which cancer cells escape from immune recognition is antigenic modulation. for example, the loss of mhc class i molecule expression leads to aberrant antigen masking, which is one of the mechanisms described for tumor cells that escape specific antitumor t-cell immune responses . in addition, the mhc-peptide-t cell receptor complex elicited by a tumor antigen shows weak stability, since high-affinity t-cells tend to be rendered tolerant to these antigens . another mechanism is the direct inhibition induced by cancer cells due to their interaction with surface regulatory molecules, table . also called checkpoint molecules. these molecules include programmed cell death- (pd ) and cytotoxic t-lymphocyteassociated protein (ctla- ), which induce the inhibition of host t cells. although these checkpoints usually help conventional immune responses control immune activation, they can also be used by tumor cells to inhibit antitumoral t-cell responses . pd ) is a transmembrane protein expressed on t, b, and nk cells, and it binds to pd ligands (pd-l and pd-l ) on target cells. when it binds to its ligand on tumor cells, pd inhibits tumor cell apoptosis, causes peripheral effector t-cell exhaustion, and promotes the conversion of effector t cells into regulatory t cells , . ctla is also a physiological negative regulator of t-cell activation. the interaction with cd /cd in the tumor leads to the inhibition of t-cell function and suppressed effector activity . knowledge of these two checkpoint inhibitors has opened the door to new antitumoral therapeutic approaches, such as the use of monoclonal antibodies that block the aforementioned interactions (anti-pd , anti-pd-l , or anti-ctla- ), which are called checkpoint inhibitors . in addition, tumor cells create an inhibitory microenvironment around them. malignant cells can recruit other cells, such as immune cells and fibroblasts, which can be corrupted by tumor cells. the interaction between tumor and nontumor cells creates the tumor microenvironment, which is mostly driven by the dynamics of the tumor promoting the proliferation/expansion of cancer cells. for example, tumor and stromal cells release multiple factors, such as the chemokine ccl (c-c motif chemokine ligand ), which inhibits effector t-cell functions and attracts tregs to the microenvironment . tumor cells use different mechanisms to promote cancer progression and further metastasis. the complete immunological eradication of cancer is the goal of antitumoral immunotherapy and is discussed later in this review. immunosenescence and inflammaging aging is accompanied by the decline and dysregulation of immune efficacy, which results in an increased vulnerability to infectious diseases, diminished responses to vaccination, and reduced tumor clearance. immune alterations mainly manifest as a reduction in the number of naïve peripheral blood cells and a relative increase in some types of memory cells . natural aging causes progressive atrophy of the thymus, which is called thymic involution. the endpoint is a significant decrease in naïve t cells, which reduces the diversity of the t-cell antigen receptor (tcr) repertoire and culminates in disrupted t-cell homeostasis . the cellular and molecular hallmarks of aging have been described as genomic instability, telomere attrition, epigenetic alterations, sarcopenia, changes in intracellular communications, cellular senescence, immunosenescence and mitochondrial dysfunction . the process of aging alters the innate and adaptive immune systems. in terms of innate immunity, aging results in a decreased number of circulating monocytes and dendritic cells, reduced phagocytic properties of macrophages and neutrophils, and impaired antigen presentation by dendritic cells . as mentioned above, aging also generates a reduction in the t-cell and b-cell receptor repertoire due to the accumulation of senescent or exhausted lymphocytes, together with a decrease in the number of circulating naïve t and b cells , . on the other hand, nk cell cytotoxicity is maintained in centenarians, and an increase in the number of these cells is observed in healthy aging people . moreover, cd + t cells exhibit cytotoxic features in centenarians; this is an acquired characteristic for cd + t cells that usually have helper, but not cytotoxic functions under physiological conditions . in addition to these features, chronic inflammation is considered the key that underlies the phenomenon called 'inflammaging', which is related to elevated self-reactivity and results in the typical chronic low-grade, systemic inflammatory phenotype observed in the elderly in the absence of acute infection. currently, it is believed that self-reactive t cells are the main contributors to this process. it has been proposed that this basal inflammatory state contributes to the development of some diseases, such as type ii diabetes, alzheimer's disease and atherosclerosis . understanding the mechanisms of age-related disorders in immune regulation is important for identifying more efficient strategies of immune rejuvenation and for the effective induction of vaccination-mediated immunity in older individuals . immunotherapy includes the use of certain components of the immune system (antibodies, cells, cytokines, etc.) for the treatment of various cancers and autoimmune diseases and the manipulation of the immune system through vaccines for the prevention and treatment of infectious and allergic diseases (fig. ) . immunotherapy using microorganisms or their components in vaccines was first practiced centuries ago; soluble substances such as poly-and monoclonal antibodies, as well as cytokines, have been used for many years, but recently, cellular immunotherapy has emerged in clinical practice. although immunotherapy can be used for many diseases (infections, autoimmune diseases, macular degeneration, allergic diseases, etc.), it is being used most expansively in the cancer field. the main goal is to destroy the tumor, either directly or indirectly (by enhancing the patient's immune system), while offering greater specificity and fewer side effects than conferred by conventional therapies. pathogens and vaccines for infectious diseases immunotherapy associated with pathogens was first linked to the prevention of infectious diseases, starting from variolization (in the x century), followed by edward jenner's vaccination against smallpox (in the xviii century) and subsequently many other preventive vaccines for infectious diseases. the great advances in the knowledge about infectious diseases took place in the nineteenth century, but the xx and xxi centuries are clearly the vaccination centuries, as many new successful vaccines (with attenuated or dead pathogens, subunits, recombinant proteins, carbohydrates or dna) introduced against a variety of pathogens. more recently, and with the increased knowledge of the human microbiome, the use of microorganisms in therapy has seen a resurgence. some intestinal infections, such as those produced by clostridium difficile, can be cured with the transfer of intestinal bacteria from healthy people (feces transplantation) . numerous other attempts to use microorganisms to cure inflammatory illnesses (crohn's disease, ulcerative colitis, etc.) have met with limited success , which indicates that this type of therapy is much more complex than initially anticipated. as a consequence, many more studies are required to ensure that this approach can be used for curative immunotherapy. researchers are also working on genetically modified or artificial bacteria (e.g., based on salmonella enterica, listeria monocytogenes or lactobacillus lactis), but only limited effects have been observed to date . oncolytic viruses (ovs). although the use of bacteria in antitumoral therapy has been largely restricted, the use of therapeutic viruses is increasing. virus-based therapy was introduced in the s with the use of adenovirus, but only in recent years has it been used in practice in the clinic. oncologic viruses have the capacity to attack tumor cells in a preferential manner and induce immunogenic cell death (icd) and host antitumor immunity (fig. ) . the first virus approved for use in therapy was a recombinant oncolytic adenovirus named h , which was licensed in by the china food and drug administration (cfda) for treating head and neck carcinoma in combination with chemotherapy . ten years later, the oncolytic attenuated-modified virus herpes simplex i-talimogene laherparepvec (t-vec, imlygic®) was approved by both european (emea) and american (fda) agencies for the treatment of melanoma . the virus is modified by the insertion of human gm-csf and deletion of the icp gene. since the approval of t-vec, a new era has dawned on the use of ovs in cancer therapy , . currently, oncolytic viruses from the adenoviridae, herpesviridae, picornaviridae, reoviridae and poxviridae families are in different phases of clinical studies for several types of tumors , . for example, reovirus against brain tumors (alone or combined with other therapies) or maraba virus against triple-negative breast tumors , offer some hope to patients with these types of cancer. viral sequences can be modified by genetic engineering techniques, thus making the virus more prone to infect some cells and enhancing viral infiltration and tumor tropism. combinations with other components (immunomodulators, drugs, and cytokines) are also being explored to suppress antiviral immunity and enhance antitumoral cytotoxicity . vaccines for cancer prevention. it is clear that certain viruses and bacteria play roles in cancer development. viruses such as genital herpes, hepatitis b, epstein barr or human papilloma and bacteria such as helicobacter pylori have been associated with cancers of the uterus and liver, in burkitt's lymphoma, and oral/genital and stomach cancers, respectively . therefore, immunization against these pathogens offer protection not only from infection but also from cancer. therapeutic vaccines. once an illness has developed, the intention of a therapeutic vaccine is to eliminate or decrease its pathology. thus, vaccines are used for cases of allergies, cancers and autoimmune diseases. allergy (type ): allergen-specific immunotherapy (ait) aims to modulate the immune system against an allergen, thus modifying the natural course of the allergic disease and conferring longlasting benefits . the basic ait involves the introduction of repeated doses of allergen (either injectable or sublingual allergen extract tablets) and often in escalating doses in a controlled manner, followed by a maintenance phase. in cases for which long-lasting tolerance is acquired, therapy may be discontinued. allergen extracts can be obtained from different sources, such as cat hair and pelt, mites, different types of pollen, venom protein, foods, etc. allergy vaccines are currently the only effective therapy that can stop the progression of the illness because treatment with anti-inflammatory drugs, such as anti-histaminic drugs or corticoids, mitigates the symptoms of the allergic processes but does not modify the natural course of the disease , . ait has been shown to induce the activation of antigen-specific tregs and il- -producing bregs (br ) subtype cells, which is combined with anergy caused by th cells and the production of allergen-specific igg antibodies that can compete with ige for binding to allergens . in the past, most vaccines were developed using natural allergen extracts. however, significant progress has been made in recent years to correctly characterize the allergen at the molecular level, and some of these allergens are now being produced by recombinant technologies, nucleic acid-based strategies, or synthetic peptide chemistry . cancer: another therapeutic approach for vaccines is in the field of cancer. therapeutic cancer vaccines that contain self-or nonself-patient tumor lysates, viral vectors, mutated tumor proteins or peptides, among other types administered in the presence of adjuvants can activate the immune system to induce antitumoral responses . the goal is to activate the th and tc cell compartments to expand specific cytotoxic t and nk cells directed against tumor cells. some vaccines are more immunogenic than others, and this effect can be related to several factors, such as the types/numbers of genetic mutations in the tumor, expression of neoantigens, production of viral proteins, an immunosuppressive environment, lack of expression of histocompatibility complex molecules, etc., which together may explain the large variability in tumor elimination . therapeutic cancer vaccines are generally very safe, and major secondary effects have not been observed, although large differences in patient responses are detected. moreover, this strategy may be used in conjunction with other complementary therapies , such as monoclonal antibodies, chemotherapy or cellular therapy , . several patients are currently taking part in clinical trials and are receiving therapeutic cancer vaccines against different types of tumors, such as lung (clinicaltrials.gov identifier: nct ), prostate (clinicaltrials. gov identifier: nct ) or pancreas (clinicaltrials.gov identifier: nct ), using individual or combined therapies. autoimmunity: in the case of therapeutic vaccines for autoimmune diseases, such as multiple sclerosis, diabetes, myasthenia gravis or guillain barré syndrome, the intention is to induce tolerance to self-antigens through the activation of regulatory cells (tregs and bregs) and tolerogenic dendritic cells, thus avoiding the immune response to self-components . due to the large variety of autoimmune diseases, the different etiologies and extensive variability, even in the same type of disease, designing a vaccine that can be useful for a wide range of patients is very difficult. however, several researchers are obtaining good results in animal models with nanostructures and peptides that induce specific tolerance, and it is predicted that, in the near future, these types of therapies will be applied to patients suffering from autoimmune diseases (reviewed by serra and santamaria ). polyclonal antibodies (pabs)-serotherapy the discovery of antibodies by dr. e. von behring and kitasato at the end of the xix century highlighted the potential of antibodies to neutralize tetanus and diphtheria toxins. this discovery opened the way to exploring the potential clinical applications of conventional antiserum-containing polyclonal antibodies from immunized animals/humans . this "serotherapy" was initiated by dr. roux and dr. yersin, who used antidiphtheria serum to treat several children . after this initial success, the use of serotherapy was increased for use against diphtheria and other diseases but also led to the identification of problems, such as immunogenicity with the formation of immune complexes (arthus reaction), the variability and limitation of the antibody batches, the content of a mixture of classes and subclasses of antibodies with different biological activities, and their temporal effects. for all of these reasons, therapy with polyclonal antibodies was very much restricted to special cases, such as the use of gamma-globulins for the prevention of rhesus (rh) maternal-fetal incompatibility and tetanus or snake venom toxicity . with the identification of gamma-globulin-deficient patients by dr. bruton in , the use of immunoglobulins as therapeutic molecules for the treatment of humoral immunodeficiencies was initiated. however, some problems were encountered in the initial phases, mostly related to the serum preparation and aggregation/ fragmentation of antibodies. since their initial use, several efforts have been made to avoid impurities and to improve the purification process, and several commercial products are now available (as intravenous or subcutaneous preparations). currently, many patients with humoral immunodeficiencies are successfully being treated to prevent them from catching infectious diseases. more recently, the therapeutic applications of immunoglobulins have expanded to other diseases, such as against covid- caused by sars-cov- infection (see below), autoimmune disorders and kawasaki syndrome in children . the beneficial effects seem to be mediated by several immunological mechanisms, including viral neutralization, inhibition of inflammatory cells and activation of immune regulators . monoclonal antibodies (mabs) the development of monoclonal antibodies (mabs) by c. milstein and g. köhler in (nobel prize winners for physiology/ medicine in ) changed medicine and immunology completely, along with many other disciplines. monoclonal antibodies are produced from the fusion of two cells to generate a hybrid cell or hybridoma with two characteristics, i.e., the production of one specific antibody and immortality. dr. milstein is considered to be the father of modern immunology for his crucial contribution . the development of many different mabs has enabled the identification of new molecules and the development of more accurate diagnostic approaches; specific, fast and inexpensive technologies; processes for the purification/concentration of compounds; and better and more specific therapy. mabs can now be used against specific targets according to the concept of the "magic bullet", a term coined by dr. paul ehrlich at the beginning of the xx century (reviewed in ref. ). numerous different mouse and rat mabs were produced against several molecules, but due to their murine origin, patients treated with these mabs suffered from hypersensitivity and immune responses , . thus, most mabs currently used in clinical applications are linked to radioactive elements and used for diagnostic purposes (table ) . in an effort to avoid immunogenicity, mabs were subsequently modified by genetic engineering approaches to carry mostly sequences of human origin. several research groups and companies developed chimeric and humanized mabs ( table ) , and these mabs included additional modifications, such as changes in the carbohydrates (glycosylation) and/or antibody regions, with the aim of improving their therapeutic action [ ] [ ] [ ] [ ] [ ] . moreover, fragments of recombinant antibodies (fabs, single-chain fvs, different v regions, fusion proteins, smaller antibodies, etc.) increased the variability of these potential therapeutic agents. the generation of fully human mabs took more time due to technical difficulties and ethical issues; therefore, researchers sought alternative methods to conventional approaches, such as the development of transgenic animals carrying human immunoglobulin genes using minilocus vectors, artificial yeast/human chromosomes or p vectors. the generation of knockout mice (in which mice lack ig genes) and further crosses with transgenic mice carrying human antibody sequences led to the generation of mouse strains that were able to produce fully human mabs , . other initiatives, such as the generation of immunodeficient mice in which human bone marrow or libraries of recombinant phages carrying human variable genes were reconstituted, allowed the development of more fully human antibodies ( table ) . sir greg winter, nobel prize winner in chemistry in , , became the pioneer of mab humanization through the genetic engineering of an antibody (campath ), later developing a fully human antibody (antitumor necrosis factor alfa, tnf-a) using recombinant phage technology , , . several companies are currently developing human antibodies using these and new technologies (reviewed in , , , ) . since , the list of approved mabs for human therapy has continued to increase (table ) , and many more mabs are in clinical trials [ ] [ ] [ ] . the versatility of mabs is based on a different mechanism of action : human immunology and immunotherapy: main achievements and challenges j varadé et al. -neutralization/blocking of soluble elements. for example, the neutralization of cytokines (tnf-α) and growth factors (vascular endothelium growth factor) prevents the exhibition of their effects, i.e., inflammatory and angiogenic effects, respectively , . -complement activation. igg/igm antibodies activate the complement cascade by the classical route, which leads to the death of the target cell , . -cytotoxicity mediated by nk cells. nk cells can facilitate mab killing of target cells. the mab, after binding to a target cell, can attach to fc receptors on the surface of nk cells to trigger the release of granzymes and perforin, thus inducing cell target death , . -induction of cell death by apoptosis. certain mabs directed against some membrane molecules can directly activate apoptosis . -blocking activation signals. antibodies can block some membrane receptors and avoid cell activation/proliferation activation/proliferation , . -carriers of toxins, pro-drugs, enzymes, and radioactive elements. mabs are able to concentrate select compounds around target cells, providing a much more selective therapy than conventional chemo-or radiotherapy . -check point inhibitors. leading to a recent revolution in cancer therapy, the identification of several inhibitory molecules can be blocked by mabs, thus leading to the activation and proliferation of antitumoral t cells. molecules such as ctla- and pd and its ligand pdl- , maintain immune cells under controlled conditions. however, it is possible to reactivate the antitumoral immune responses by blocking some of these molecules with mabs, either directed to only one of them or by using various antibodies in combination (for example, against ctla- and pd ) . the results obtained with these therapeutic mabs against checkpoint inhibitors in some types of cancer have been amazing. for their contribution to the understanding of the roles of ctla- and pd- , the swedish academy gave the nobel prize in to dr. j.p. allison and dr. t. honjo, respectively . however, this therapy is not efficacious in all types of cancers for several reasons, such as the expression of these and other checkpoint inhibitors in immune cells, the number of antitumoral cells in each patient, an immunosuppressant microenvironment, the rate of cancer mutations, and the expression of histocompatibility molecules. there is a large list of recombinant proteins that are currently being used for human therapy, including interleukin (il- ), interferons (ifns) and gm-csf. il- was identified in as a growth factor for t lymphocytes, and soon after dr. rosenberg started to use it in antitumoral therapy , . years later, in , il- was approved by the fda for patients with metastatic renal cancer and in for the treatment of metastatic melanoma . interferon (ifn) was described in by isaacs and lindenmann . the interferon family is the largest family of cytokines and is classified into three different types (i, ii, and iii). type i ifns (including ifn-α and ifn-β) exhibit several molecular actions that may be very relevant for use in therapy for a range of pathologies (such as autoimmune diseases and cancers) . in , the fda approved human ifn-α a and ifn-α b for patients with hairy cell leukemia and later on for patients with multiple sclerosis. since their initial use, these interferon species have been approved for many other diseases, including chronic hepatitis b and c, lymphoma, advanced melanoma, and as adjuvants together with other therapies for several types of cancers , . another cytokine is gm-csf, which activates the production of granulocytes and monocytes from bone marrow myeloid progenitors and has shown adjuvant antitumoral effects , . other cytokines, such as il- , il- , il- , il- , il- , and il- , , are being tested in several clinical trials, and it is expected that some of them, either alone or in combination, can be used in future antitumoral therapy. other recombinant proteins are already on the market, some of which are derived from antibodies, with some advantages such as small size, low immunogenicity and general ease of production. examples are etanercept and abatacept (ctla- ig), which were approved by the european medicines agency in and , respectively. the former is a chimeric protein that carries the external portion of the tumor necrosis factor (tnf) receptor linked to the igg fc region, which captures soluble tnf to block its inflammatory effects . the latter example is a fusion protein that combines the extracellular portion of human ctla- and igg fc. abatacept is a competitive inhibitor that blocks t-cell activation and can be used in the treatment of inflammatory illnesses such as rheumatoid arthritis . natural killer (nk) and lymphokine-activated killer (lak) cells. natural killer (nk) cells were described in the s based on their capacity to eliminate tumor cells without prior sensitization, with differences observed compared with specific cytotoxic t cells (which are activated based on the recognition of the target cells) , . in , piontek et al. reported that nk cells have the ability to preferentially kill cells that had lost the expression of the major histocompatibility complex class i molecules , . lymphokine-activated killer (lak) cells are a heterogeneous population that includes not only nk but also nkt and t cells, which can be generated in an in vitro culture of peripheral blood mononuclear cells (pbmcs) in the presence of il- . dr. rosenberg and collaborators carried out studies using these cells in the presence of il- (reviewed by rosemberg ). these lak cells showed good antitumoral responses in % of the melanoma patients who received them as therapy . however, secondary effects such as liver toxicity and the expansion of the treg population limited their therapeutic effect. researchers started to design new recombinant il- with some mutations to avoid the activation of tregs , with linking it to polyethylene glycol (peg) to increase its half-life and efficacy . another cytokine described later, il- , showed similarities to il- in many respects , and it had some unique advantages, such as the capacity to activate nk and cytotoxic t cells (tc) but not tregs. il- is being used in different versions (alone, as a heterodimer with receptor il- /il ra or il rα igfc, or in an agonist complex with alt- ) and in combination with other therapies in several clinical trials (examples: nct , nct , and nct ). more recently, researchers have focused their attention on other cytokines and combinations (such as il- , il- , and il- ) , which are able to activate nk cells in vitro and induce a good responses in animal models. in some human clinical trials, remission has been observed for patients with acute myeloid leukemia , , which broadens the options for the use of nk cells in the treatment of this pathology. the properties of nk cells reveal their versatility as treatments against tumors. nk cells are able to kill tumors through several mechanisms, including receptor-mediated cytotoxicity, antibodydependent cell-mediated cytotoxicity (adcc) and death receptormediated apoptosis, but they also secrete cytokines such as interferon gamma that enhance the antitumoral adaptive immune response. nk cell adoptive transfer (either autologous or allogenic nks) is currently being tested in clinical trials for hematological diseases and solid tumors, and numerous research groups have recognized their potential in other situations, such as transplant rejection and pregnancy. nk cell lines, memory-like nk cells and stem cell-derived nk cells are additional types of cells that can be used for tumor immunotherapy . regarding other cellular therapies, nk cells as substitutes for t cells for use upon transformation with an chimeric antibody receptor (car) are being explored (see below). dendritic cells. paul langerhans identified dendritic cells in human skin in , but these cells were not named until by dr. ralph m. steinman (nobel prize in ) and dr. zanvil a. cohn, who chose the term because the cell morphology, with long extensions, resembles that of neuronal dendrites . in humans, dendritic cells are obtained from different sources that vary in origin, maturation state and tissue distribution (skin, lymphoid tissue, circulating cells). among the main types of dendritic cells, plasmocytoids are conventional myeloid dc and dc , pre-dc and monocyte-derived dendritic cells. in the epidermis, there are three types: langerhans cells (lc), monocyte-derived lc-like cells and inflammatory dendritic epidermal cells (idecs) . as indicated above, dcs are antigenpresenting cells and are the only cells that are able to activate naïve t lymphocytes. a subpopulation of dcs also carries out a process known as cross-presentation. in this way, they facilitate the activation of both helper and cytotoxic t lymphocytes . in addition to their participation in the immune response, they can be used in antitumoral therapeutic vaccines , . it is possible to generate a type of blood monocyte-derived dendritic cell in the presence of a mixture of cytokines in culture -a process that induces their subsequent maturation and activation in the presence of tumor antigens (cell lysates, recombinant or purified antigens, peptides, rna, dna, and viral vectors ). these cells can also be obtained from bone marrow hematopoietic cd + progenitor cells . other sources, such as circulating or skin dendritic cells, are relatively scarce and are therefore not usually used. after their differentiation and activation in vitro , , dcs are exposed to tumor antigens and infused back into the patient (either by blood infusion or injected into areas near the lymph nodes or even directly into them) to reach the secondary lymphoid organs as soon as possible, at which point they can present antigens to the t cells. this approach is a type of individualized therapy and is therefore expensive. the first approved vaccine in which autologous dendritic cells were used was sipuleucel-t (provenge) , which was a treatment for prostate cancer refractory to hormonal treatment. immunotherapy with dendritic cells is currently being tested in more than clinical trials for various tumors: brain, pancreas, mesothelioma, melanoma and many others (clinicaltrials.gov identifiers: nct , nct , nct , and nct , respectively). the data indicate that the therapy is well tolerated and has led to increased patient survival in some trials. furthermore, complete cure and partial remission outcomes have also been observed. the lack of efficacy on other tests was probably due to the presence of immunosuppressive factors in the tumor environment. another therapeutic use of dendritic cells involves their induction of immunosuppression both in transplants and in autoimmune diseases . in an autoimmune pathology such as multiple sclerosis, the intention is to achieve stable tolerogenic dendritic cells that can act against some autoantigens (such as myelin peptides) in the presence of vitamin d , dexamethasone, or other agents . phase i clinical trials have generally shown good tolerance to this therapy without serious adverse effects . however, greater control of this treatment is necessary in several respects to obtain the best therapeutic results ; e.g., the human immunology and immunotherapy: main achievements and challenges j varadé et al. type of dendritic cells and ex vivo differentiation, the antigens used, and the injection route are important considerations. gamma/delta t cells (tγ/δ). human t cells expressing γ/δ tcr cells have interesting properties, including the capacity to kill a broad range of tumor cells. the advantages of these cells in cancer therapy are based on their independence from mhc expression on tumor cells and that their relative insensitivity to some inhibitor molecules (such as pd- ). the initial clinical application, with the adoptive transfer of autologous vδ + cells after ex vivo expansion, showed only sporadic responses , and different exploratory studies are currently being carried out to increase their clinical therapeutic use. allogeneic vδ + cells are also being explored in cancer therapy; e.g., they are being used against refractory hematological malignancies and advanced cholangiocarcinoma . although the basis of immune regulation was suggested centuries ago, regulatory t cells were described by sakaguchi et al. as cd + cd + natural regulatory t cells that expressed the forkhead box p transcription factor (foxp ) . later, induced or adaptive regulatory t cells were also identified, including different subsets that carry several phenotypic markers and express various cytokine secretion profiles . all of these factors play crucial roles in the maintenance of immunological self-tolerance by suppressing autoreactive t cells. the manipulation of tregs to achieve therapeutic outcomes is a field of great interest, because of both their expansion and activation in diseases, such as allergic and autoimmune diseases, and as a potential targets for cancer immunotherapy . tumor-infiltrating lymphocytes (tils). lymphocytes that infiltrate solid tumors are called tumor-infiltrating lymphocytes (tils). in , thomas and burnet proposed that the immune system performs tumor immune vigilance, with lymphocytes as sentinel cells leading to the elimination of somatic cells transformed by spontaneous mutations , . since the end of the s, dr. rosenberg has been trying to prove and improve the effective use of tils. the process starts with surgery and the isolation of tils from a tumor, followed by til activation in culture in the presence of cytokines, cellular expansion and, finally reinfusion into the patient. since its inception, this therapy has been improved markedly, with an increase in optimal responses from less than % to the current - %, in some cases. these higher success rates are due, in particular, to the prior preparation of the patient, including the depletion of lymphoid tissues, to avoid an expansion of regulatory cells , myeloid suppressor cells and other cells that can compete with the transferred tils. currently, there are more than trials in which tils are being used alone or in combination with other immunotherapies on several tumors, such as melanoma, metastatic colorectal cancer, glioblastoma, pancreatic cancer, hepatobiliary cancer, ovarian cancer and breast cancer. this individualized therapy has limitations; it can only be used on solid tumors, and the number and specificity of the tils and the type of tumor and microenvironment make standardizing this therapy difficult. chimeric antigen receptor (car). since tils include a variety of t lymphocytes with different specificities, the next step was to obtain t cells of a single type (monoclonal cells) carrying a clonal receptor capable of recognizing tumor antigens. this effort was carried out for the first time in mice and subsequently, in , in humans with a transgenic tcr against the mart- melanoma antigen , . these types of receptors are known as ttcrs, but their ability to recognize antigens is restricted since they can only identify the peptides presented by antigen-presenting cells on self-histocompatibility molecules. this situation changed because of one of the latest revolutions in antitumor therapy, the development of t lymphocytes that carry a chimeric antigen receptor (car) based on a specific antibody directed to a target surface molecule , . these modified t cells can directly recognize tumor cells without required antigen processing or presentation by professional antigen-presenting cells. moreover, the car includes all of the necessary elements for intracellular signaling and activation of helper and cytotoxic t lymphocytes. car therapy was developed by one of its pioneers, dr. carl june at the university of pennsylvania in the united states , who used modified t lymphocytes that carried a chimeric antigen receptor to target cd + leukemic b cells. after interacting with cd + cells, these modified car t cells were activated and able to proliferate and exert cytotoxic functions against target cells. in this case, both tumor and healthy b cells were affected. although bone marrow continues to produce b lymphocytes, in cases of severe b lymphopenia, it is possible to provide exogenous immunoglobulins periodically. the whole process of the current car t-cell therapy begins with blood donation, from which lymphocytes are purified and genetically modified in vitro by a viral vector, which carries the genes coding for the chimeric antigen receptor. the cells are expanded in the presence of cytokines in culture and are subsequently reinfused into the patient. this type of cellular immunotherapy is individualized for each patient, with his/her car t cells ultimately destroying the tumor. since the first generation of cars appeared, namely, a chimeric receptor composed of an anti-cd -specific single-chain variable fragment linked to a transmembrane domain and intracellular signaling domain of the t cell receptor (cd ζ chain), researchers started to modify the original design. new generations of cars, including the cd ζ subunit together with other signaling domains, such as cd , cd , cd ( - bb), cd , and icos, or combinations (cd ζ, cd , and cd ) , have been developed in the second and third generations of cars to improve several aspects, such as the activation, proliferation and survival of car t cells. the fourth generation of cars show improved the antitumoral effects by carrying additional molecules (such as cytokines or drugs), improvements to the safety of car tcell therapy through the use of suicide genes and many new designs, such as dual cars or the so-called split universal and programmable (supra) car system . in addition to t cells, other types of cells, such as nk cells, are now being explored for use in antitumoral responses . in an effort to avoid using personalized treatment, researchers are now working on universal cars that may be used on many different patients without inducing the problem of rejection [ ] [ ] [ ] [ ] . the encouraging results obtained with this therapy have led to interest from companies, and some commercialized examples are available, although many more "in-house" or academia-produced cars are in clinical trials. car t-cell therapy was initially designed for use against hematological cancers (leukemia and lymphomas), but many new opportunities have been opened for its use against solid tumors , infectious diseases (such as hiv) , allotransplantation, autoimmune diseases and severe allergies . china and the usa are the leading countries in producing car t-cell therapy, and numerous clinical trials are underway. immunotherapy for covid- patients coronavirus disease (covid- ), which is produced by severe acute respiratory syndrome coronavirus (sars-cov- ), affects millions of people in many countries. most of the infected patients ( - %) are asymptomatic or have mild symptoms, but the disease in some patients progresses to a moderate or severe illness that requires hospitalization in intensive care units because of respiratory distress, multiorgan failure, and/or other pathologies, and more than one-half million fatal cases have been human immunology and immunotherapy: main achievements and challenges j varadé et al. reported worldwide. the most vulnerable population includes aging patients and those with comorbidities such as hypertension, diabetes and cardiovascular diseases. there are several aspects of the covid- pathogeny that suggest an overreaction of the immune system in severely ill patients, with increased levels of inflammatory cytokines such as il- , il- and others (creating the so-called "cytokine storm"), together with blood lymphopenia and cd t cell and nk cell exhaustion. special therapies have not yet been identified to cure these patients, and preventive vaccines are not yet available, but some immunotherapies have been proposed as adjunct therapies, and some of these are currently in different phases of clinical trials . the immunotherapeutic strategies include the following: future challenges in immunotherapy immunotherapy has been used for centuries, but only in recent years has this area expanded rapidly in several respects, mostly by the use of soluble elements (monoclonal antibodies and cytokines) and, more recently, with immune cells (cellular immunotherapy). there are many fields in which immunotherapy faces a range of challenges: vaccines. . researchers are working on reducing the number of injections by employing a combination of vaccines. several current vaccines contain components from - pathogens in a single injection, and these are able to provide adequate protection against all of these pathogens . . researchers are developing more stable and durable vaccines. improvements in the half-lives of vaccines, for example, by lyophilization, while maintaining immunogenicity is expected to reduce current problems, especially those involved in the transportation of vaccines to remote areas . in this respect, nanotechnology can help in the design of more stable vaccines that lead to slow antigen release and improved immunogenicity . . researchers are working on vaccines that confer protection against all serotypes of a specific pathogen (universal). this outcome is especially important for pathogens with high variability (such as the influenza virus). researchers are designing vaccines that can protect against several variants by using common regions that can induce protective immune responses to all or most of the variants . . researchers are developing alternative routes of administration (e.g., oral, inhaled, intranasal, skin, rectum, vagina) as substitutes for intramuscular or subcutaneous injections. one of the problems to be solved is the immune tolerance developed to elements delivered by the oral route, but some vaccines are already effectively administered by this route (such as the oral polio, cholera, typhoid fever and rotavirus vaccines). the intranasal route has also proven effective for some vaccines (nasal influenza vaccine), and vaccines administered through other routes are under investigation. . researchers are seeking the early protection of newborns . newborns are very susceptible to infections due to their immature immune system . moreover, the protection exerted by maternal antibodies transferred through the placenta during pregnancy against some pathogens interferes with the development of the newborn's own immune response. greater knowledge on ways to activate the immature immune system early will enable the development of vaccines for newborns. moreover, immunization of pregnant women may help to enhance neonatal protection against several pathogens . . researchers are developing new and more effective vaccines. this effort is crucial for very prevalent pathogens such as mycobacteria tuberculosis, hiv virus or plasmodium falciparum. although there are treatments against these pathogens, most are not curative-as in the case of hiv; prevention is the best way to stop their spread. . researchers are working to address emerging pandemics. in the case of new pathogens, such as sars-cov- , which has produced a recent global outbreak, effective vaccines are urgently required . new technologies for vaccine formulations and routes of administration, the identification of immune-related factors of protection and modifications to the governmental regulatory and approval process for vaccines for emerging pathogens are challenges that must be faced to achieve a rapid vaccination procedure for outbreaks. hundreds of vaccines against sars-cov- (using different strategies such as live attenuated or inactivated pathogens, viral vector-based, viral rna, dna, recombinant proteins, peptides, etc.) are now under development, and some are in clinical trials. however, the need to develop a new vaccine in a short period of time should not negate the main principles of vaccination use: safety and immune protection. . researchers are working on genetic (rna, dna) vaccines because they have great advantages, including no requirement for growing a pathogen. genetic vaccines can be obtained in a much shorter time, with much faster and safer production processes, and can be transported much more easily. however, the immunogenicity of these vaccines must be improved, and other problems need be avoided, such as the potential deleterious effects of integrating vaccine sequences into cells . . researchers are developing safer and more powerful adjuvants. many years ago, the only adjuvant authorized for vaccines was aluminum hydroxide (alum), but currently, several adjuvants are on the market . the use of ligands that activate the innate immune response, such as those linked to tlrs or nanostructures with adjuvant effects, is currently under study. . researchers are boosting trained immunity, a new concept related to the innate immune memory-like described for nk cells (expansion) and macrophages (epigenetic modifications). knowledge of how to handle trained immunity will enable better vaccine design and more effective secondary responses . . researchers are seeking to eradicate diseases from the earth. the greatest challenge, eradicating disease is possible in the short term for some pathogens, such as poliovirus. very few cases of polio have been recently reported, and these reports came from only three countries; therefore, it is feasible that this disease can be eradicated in the near future. human immunology and immunotherapy: main achievements and challenges j varadé et al. . advances are challenged by the anti-vaccine movement. paradoxically, there are people who doubt the beneficial effects of vaccines, and they are putting the health of their own children and society in general at risk . the effectiveness of community protection conferred through vaccinated people is disrupted by decreased numbers of immunized persons. this lesser coverage enables pathogens to infect the most susceptible people, such as small children, elderly patients and those who cannot be vaccinated due to certain pathologies or because they are undergoing immunosuppression treatment. thus, news about the return of illnesses that were nearly forgotten, such as tetanus in italy (the first case in years), the death of a child in catalonia from diphtheria, or the exponential increase of measles cases (already counted in the thousands) worldwide , should make parents think carefully about the risks of not protecting children by vaccines. the world health organization (https://www.who.int/topics/vaccines/en/) argues that anti-vaccine movements can roll back all the achievements thus far in this field and have cited this issue as one of the main challenges to be resolved. addressing the antivaccine movement requires a coordinated effort of professionals to inform parents adequately and perhaps other types of coercive measures that some countries are already applying (financial fines, denial of access to public assistance in childcare units, removal of authorization to travel/live in some countries, new laws, and so on). antibodies and cytokines. immunotherapy with monoclonal antibodies has been a true revolution for many pathologies, as has the use of certain cytokines and recombinant fusion proteins. it is therefore predicted that these approaches may have a bright future, and regulatory agencies are expected to authorize many more mab-based therapies in the coming years, especially given the good results obtained in clinical trials. complete antibodies or those modified to increase their functionality or decrease their immunogenicity, combinations of antibodies and cytokines, antibody fragments, etc., are only some of the many possibilities for this type of product, which will expand the range of therapeutic options. one of the main problems regarding the use of antibodies in therapy, especially in cancer, is based on their often unpredictable efficacy. large variability in terms of remission and durable clinical benefits between patients is observed (for example, in the antitumoral responses by antibodies directed to the checkpoint inhibitors). thus, the main challenge is to understand the situations in which an antibody will have the desired effect. it is crucial to find validated biomarkers (with predictive and/or prognostic value) that can help to stratify or select patients for the best immunotherapy. a better understanding is also required for tumor heterogeneity, resistance to some drugs and immunosuppressive microenvironments . an in-depth immunological study, together with a personalized approach, is certainly the way to improve the success of these types of therapies. in combination with conventional therapies (radiotherapy, chemotherapy, and surgery), other immunotherapeutic drugs or cellular immunotherapies can also help to maximize the efficacy of this immunotherapy, but increases in toxicity will be another challenge to face. pathogens. the use of oncolytic viruses (ovs), bacteriophages that selectively infect bacteria, modified pathogens for vaccines or for antitumor immuno-activation, and the manipulation/ modification of the microbiota are some of the therapies that are being considered. ovs are designed to kill tumor cells and to activate the immune system against those cells. however, many of ovs have shown limited therapeutic effects when applied in monotherapy; therefore, much more work is required to improve their systemic antitumor effects and avoid the attenuation of the virus, which limits the viral replication. several obstacles, such as low viral delivery and spread, resistance to therapy and antiviral immunity, have been observed . thus, the main challenges with oncolytic viruses are addressed by improving their antitumoral efficacy, including the optimization of viral delivery, the development of ovs engineered to activate the immune system (e.g., by releasing cytokines), and their use as adjuvant therapies or in combination with other immunotherapeutic agents, such as immunomodulators . regarding gut microbiota manipulation as a therapeutic approach, fecal microbiota transplantation is an effective therapy for recurrent clostridium difficile infection and is now being investigated for other indications, such as inflammatory bowel disease and cancer. some of the challenges facing microbiome transplantation are the lack of precise knowledge about the complete microbiome and the mechanisms of action involved in its therapeutic capacity, the large variability of its effectiveness and the external factors that affect it. more studies are centered on understanding how to manipulate bacterial colonies, the discovery of therapeutic molecules, nutrient competitions, etc., that are required for successful application. the best type of therapy (either individual or the combination of bacteria) is also under debate, along with how to reach the market by translating this individualized therapy into commercial scale products. the safety and potential adverse long-term effects are also being assessed. other components (nanomaterials and small molecules). nanomaterials. to obtain approval for the use of other elements from incipient fields, such as the use of different types of nanostructures, either alone or in combination with other immunotherapies, it is important to resolve certain issues. in the case of nanoparticle use, a better understanding of the interaction between nanomaterials and biological media; nanoparticle biodistribution, metabolism and biocompatibility; and the reproducibility of the synthesis and scaled up production of nanomaterials are among the issues to address. small molecules. a greater knowledge of several molecules involved in the immune system has led to the development of new therapeutic agents, which have been synthesized by traditional chemistry and block or activate intracellular signaling. the low cost of production of these molecules, along with the scaling and reproducibility of small-molecule batches, has attracted the attention of pharmaceutical companies interested in a whole set of new immunomodulatory drugs. a better understanding of the mechanism of action of small-moleculebased drugs and proof that they offer higher efficacy than existing therapies, either in monotherapy or in combination therapy, are challenges that face those seeking to engineer new types of targeting molecules. cellular immunotherapy. to date, cellular immunotherapy has been an individualized therapy with high production costs, and it requires the involvement of multidisciplinary groups in hospitals. a real challenge in the field of cellular immunotherapy is the acquisition of universal off-the-shelf cell therapies to replace those currently made to order in a very personalized manner. the development of universal cells, for example, in the case of car tcell therapy, would increase the number of patients who could benefit from this treatment at thus reduce the costs. other challenging aspects of cellular immunotherapy are the life-threatening toxicity of induced and their lack of effect on solid tumors, which is mostly due to the immunosuppressive tumor microenvironment. this approach requires new strategies to overcome these difficulties. in addition to cancer, cellular immunotherapy has a long history of use against autoimmunity, infectious diseases, allergies and transplantation rejection. it is also important to find biomarkers for prognosis/prediction that can help to optimize this method. other therapies that involve the use of activated nk cells, tumor-infiltrating lymphocytes, vaccination with dendritic cells, etc., are having partial clinical success. similar to other treatments, these approaches require further study, but it is feasible that they may become reality in the near future. greater knowledge of the immune system, especially concerning the variety of cellular and humoral components and the close regulation among them, the interaction with other systems or with elements such as the microbiota, will allow the development of new types of therapies that may be safer, more effective and specific but with much lower toxicity than found in current therapies. this long journey has been possible due to the efforts of numerous researchers (throughout the centuries), who have contributed with their work, creativity, successes and failures to advance our knowledge of the immune system, cellular components, membrane markers, interactions, signaling pathways and many more aspects. this great combined effort has paved 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study and conceived the project, and all the authors participated in writing the paper. competing interests: a.g-f is a copromoter of the spin-off nanoimmunotech. there are no competing financial interests in relation to the work described. key: cord- -oecpqf j authors: nan title: aspho abstracts date: - - journal: pediatr blood cancer doi: . /pbc. sha: doc_id: cord_uid: oecpqf j nan myelodysplastic syndrome (mds) and frequently arise in the context of inherited bone marrow failure (bmf) syndromes, such as shwachman diamond syndrome (sds). monosomy /del( q) is associated with high grade mds and propensity to progress to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited bmf. development of non-transplant strategies to treat bone marrow failure without simultaneously stimulating outgrowth of malignant clones remains a major challenge. objectives: the aim of this study is to investigate the molecular consequences of del( q) in the context of bmf with the goal of developing more effective treatments. design/method: to study the biological and molecular consequences of monosomy/del( q) in bmf, induced pluripotent stem cells were generated from sds patients (sds-ipsc) . a deletion of the mds-associated region of the long arm of chromosome was then introduced using a previously published modified cre-lox approach. results: the sds ipsc phenocopied bone marrow failure with slow proliferation and impaired hematopoietic differentiation. we next explored whether deletion of q conferred a relative fitness advantage within the context of bone marrow failure. proliferation of the sds-del( q) ipscs was reduced below that of both the isogenic sds ipscs and normal controls without an increase in cell death. sds-del( q) demonstrated reduced hematopoietic differentiation compared with isogenic sds cells. these data demonstrate that deletion of q fails to confer a relative growth advantage relative to isogenic sds ipscs and results in further impairment of hematopoiesis. to gain insight into the mechanisms of del q-associated clonal evolution in sds, we performed rna sequencing (rnaseq) of sds+/-del( q) ipsc. expression of tgf pathways and their downstream targets were reduced in sds-del( q) ipscs compared to isogenic sds ipsc. single cell rnaseq analysis of primary sds bone marrow cells confirmed that the tgf pathway is hyperactivated in sds. western blot analysis showed increased phospho-smad levels in sds ipscs compared to sds-del( q) and normal controls, while total levels of smad were unchanged. pharmacological targeting of tfg with small molecule inhibitors resulted in selective improvement of sds hematopoietic colony formation and myeloid differentiation without stimulating outgrowth of the isogenic sds-del( q) cells or normal controls. these results demonstrate that del( q) reverses the tgf pathway hyperactivation of sds. furthermore, inhibition of tgf selectively rescues hematopoiesis in sds but not in isogenic del q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del q clonal outgrowth. background: standard therapy of medulloblastoma consists of treatment with alkylating agents and radiation after surgical resection. although a statistically significant increase in survival is reported with this regimen, / rd recur and become resistant this class of agents ultimately leading to mortality. large numbers of somatic mutations were observed in recurrent medulloblastoma (rm) after alkylating agent and radiation treatment. high mutation rates in tumors can have twofold effect; ) a large number of non-synonymous mutations that have no role as drivers can still cause functional tumor antigens increasing the neoantigen burden and immunogenicity. moreover, ) such tumors can gain mutations in canonical or non-canonical dna repair pathways leading to a gain in the number of mutations as seen in case of glioblastoma, this can lead to even higher accelerated mutational rate. evidences suggest that high mutational load can cause higher neoantigen burden thereby making the tumor more susceptible to immune checkpoint inhibition. we propose that post therapy recurrent medulloblastoma gain mutational signature and immunophenotype of malignancies demonstrating clinical response to immune checkpoint therapy. objectives: ) rm has molecular signatures identical to tumors with high immunogenicity and clinical response to immune check point inhibition. ) rm has the immune inflammatory phenotype; harboring high percentage of tumor infiltrating lymphocytes (tils), macrophages and monocytes. design/method: to test our hypothesis, we downloaded the raw bam files of previously published data from international cancer genome consortium (icgc) . this set of about matched primaries and recurrent medulloblastoma cases forms our discovery cohort. we have called somatic variants using the gatk pipeline by the broad institute. to validate our key findings, we have procured human medulloblastoma specimens and are conducting whole exome sequencing. the primary assays utilized to assess immunogenicity are immunohistochemical (ihc) staining of formalin fixed and embedded recurrent medulloblastoma tissue to identify tils, tumor associated macrophages and other markers. mg/m had dlts of dyspnea (grade )/hypoxia (grade ) but no dlts were observed in any other cohort. adverse events were generally mild to moderate, consistent with the safety profile observed in adults. across the desc cohorts, plasma concentrations were dose-proportional and steady state concentrations were lower on day vs. day . mean systemic exposure in the mg/m cohort was ∼ -fold greater compared with the adult rp d of mg bid. a pk:pd relationship between tazemetostat exposure and h k me levels in peripheral blood monocytes and granulocytes was observed in the desc phase. consistent and significant post-dose reductions in h k me occurred at doses ≥ mg/m . further analysis of twelve patients treated at the rp d confirmed that h k me inhibition was maximally inhibited. doses - mg/m showed confirmed objective responses (cr/pr) per recist/rano in patients with es (n = ), chordoma (n = ), and atrt (n = ). background: previous studies established that the platelet/ fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (nk) cells. however, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. objectives: determine the role of fibrin polymerization and fibrin(ogen) engagement of integrins iib and m in metastasis. design/method: we performed experimental and spontaneous metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. results: expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin m (fib - a) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)inflammatory cell interactions mediated by m in metastasis. to directly determine the importance of thrombinmediated fibrin polymerization in metastasis, we analyzed metastatic potential in fibaek mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a mutation in the a chain thrombin cleavage site. metastatic potential in fibaek mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymerization in the metastatic process. however, the fibaek mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains significant prometastatic properties. in order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and fib Δ mice, carrying a form of fibrinogen lacking the chain binding motif for the platelet integrin iib . surprisingly, this mutation had no impact on metastatic potential. together, these studies suggest fibrinogen plays a multifaceted role in metastasis. fibrin(ogen)-leukocyte interactions mediated by m appear to have a role in metastasis. previous studies showed that macrophages promote the metastatic potential of circulating tumor cells, which may represent at least one important m expressing cell type whose prometastatic behavior is influenced by fibrin(ogen) interactions. these studies show that thrombin-mediated fibrin polymerization promotes metastasis, but soluble fibrinogen retains some significant prometastatic capacity. surprisingly, loss of the fibrinogen chain iib binding motif had no impact on metastasis. given the established importance of platelets in metastasis, these findings suggest that fibrin (ogen) is capable of platelet stabilization through mechanism(s) independent of this iib binding motif. platelets may bind polymerized fibrin at other sites, and/or fibrin interactions with other matrix proteins capable of binding iib are sufficient to support platelet functions required for metastasis. the role of platelets in hemostasis and thrombosis is well defined, but it is becoming increasingly evident that platelets also assist in host defense and inflammation. platelets participate in the innate immune system through direct antimicrobial activity and interactions with effector cells (chapman , garlanda , kapur ). in the adaptive immune system, platelets recruit and costimulate t-cells, and promote b-cell differentiation and antibody class switching (kapur , morrell ). the question remains: which mechanisms influence platelet immune function and are they developmentally regulated? preliminary studies in the palis lab have revealed significant dif-ferences in embryonic versus adult platelet gene expression, including regulators of immune and inflammatory responses such as beta -microglobulin (b m) and major histocompatibility complex class i (mhc ). mhc is expressed on all cell surfaces except red blood cells and its molecular chaperone b m is a marker of inflammation highly expressed in platelet alpha granules (zufferey ). preliminary data from the morrell lab reveals a mass release of b m during platelet activation, which drives monocyte differentiation to an inflammatory phenotype through tgfb receptor signaling. we therefore sought to determine whether developmental changes in platelet b m expression mediate differences in platelet-mediated monocyte activation. with trilineage hematopoiesis with a predominance of early myeloid precursors, with full maturation. microarray, elane and sbds sequencing and deletion/duplication analyses were negative. immunologic evaluation was significant for agammaglobulinemia and an absence of memory (cd +cd +) b cells. a gene primary immunodeficiency panel revealed two variants of unknown significance-c. g>a and c. g>t in dnmt b; one previously reported in association with icf . parental testing demonstrated parental heterozygosity. centromeric instability was confirmed in mitogen stimulated lymphocytes showing characteristic, multibranched chromosomes containing at least arms of chromosome and joined near the centromere. decondensation of the qh and qh regions and triradial configuration of chromosome was noted, and a diagnosis of icf syndrome was made. the patient was started on monthly intravenous immunoglobulin (ivig). prophylaxis for pneumocystis jiroveci pneumonia and respiratory syncytial virus was initiated. a / matched sibling hsct is being planned. demonstrated the diagnosis of high grade osteosarcoma. the patient was started on multi-agent chemotherapy with planned a whole femur prosthesis at time of local control. cases of osteosarcoma have been described in the literature in patients with nf (median age; years, range - years) with slightly male predominance ( cases). the femur was the most common site of involvement ( cases). four patients died of metastatic disease despite surgery and multi-agent chemotherapy. conclusion: nf represents a major risk factor for development of malignancy and uncommonly osteosarcoma in adolescents and adults. we report a rare case of an extensive involvement of osteosarcoma of the left femur in a child with known diagnosis nf . this presentation should alert the pediatric oncologists to monitor for bone tumors in patients with nf by physical exam and detailed medical history. hasbro children's hospital, providence, rhode island, united states background: dysautonomia is a paraneoplastic syndrome most commonly described in adult malignancies. despite current therapies aimed at symptoms management, it is often debilitating. we present a case of a -year-old girl who initially presented with autonomic dysfunction and was subsequently found to have hodgkin lymphoma. objectives: describe hodgkin lymphoma presenting with dysautonomia and discuss symptom management with rituximab design/method: case report a year-old-girl presented with severe symptoms of orthostatic hypotension necessitating prone positioning to prevent syncopal episodes. additionally, she reported anhidrosis, xerostomia, urinary retention, and constipation. she had unmanageable peripheral neuropathic pain despite multiple analgesia medications. initially, it was suspected that her symptoms were caused by an atypical presentation of guillain-barre syndrome. she was treated with intravenous immunoglobulin g, without response. due to a suspicion of a paraneoplastic syndrome a positron emission test/cat scan (pet/ct) was performed and revealed widespread fdg-avid nodal and splenic disease. pathology from a thoracoscopic biopsy of a mediastinal lymph node demonstrated classical hodgkin lymphoma. she was classified as stage ivb. a paraneoplastic panel obtained during the first cycle of chemotherapy revealed elevated anti-amphiphysin antibodies and glutamic acid decarboxylase (gad) antibodies. therapy was initiated with abe-pc (doxorubicin, bleomycin, etoposide, prednisone, cyclophosphamide) ; vincristine was held given her significant neuropathy. due to persistence of autonomic symptoms following her first cycle and presence of antiamphiphysin and gad antibodies, rituximab was incorporated into her treatment. following two cycles abe-pc, she had a rapid early response by fdg-pet/ct. she completed an additional three cycles of abd-pc. end of therapy imaging demonstrated complete response with a single persistent mildly fdg-pet avid lymph node (deauville ) and her antibodies were negative. she continues treatment of maintenance rituximab with significant improvement, but not resolution, of her orthostatic hypotension. at this time, the patient can ambulate with assistance. constipation and urinary retention have fully resolved and, her peripheral neuropathy, xerostomia, anhidrosis have improved. conclusion: this is rare case of a pediatric hodgkin lymphoma patient developing dysautonomia associated with antiamphiphysin and glutamic acid decarboxylase antibodies and subsequently managed with chemotherapy and rituximab. clinicians should be suspicious of a paraneoplastic syndrome when a neurologic disorder fails to improve with standard treatment. results: labs obtained at an outside hospital one month prior to presentation showed absolute neutrophil count (anc) and hemoglobin . g/dl. she presented to our institution with days of fever, hepatomegaly cm below costal margin, a white plaque on her tongue, and circumferential perianal ulceration. labs were significant for anc and hemoglobin . g/dl. anti-granulocyte antibody testing was positive. bone marrow biopsy showed arrest of neutrophil maturation. after initiation of filgrastim ( . mcg/kg/day), her anc increased to > and repeat bone marrow biopsy demonstrated left shifted myelopoiesis. biopsy of her oral lesion demonstrated invasive actinomyces prompting a prolonged course of antibiotics. biopsies of her oral and anal lesions were reported as myeloid sarcoma without mll rearrangement. chemotherapy was not initiated due to complete resolution of both lesions within weeks of initiating filgrastim and appropriate antibiotic coverage. she has not developed any further lesions concerning for malignancy. testing for common genes associated with severe congenital neutropenia and autoimmune lymphoproliferative syndrome was negative. her immunoglobulin levels and the measurement of age-appropriate vaccine responses were normal. after her lymphocyte subpopulation analysis indicated a selective deficiency in cd positive t-lymphocytes (absolute cd cell count ), the severe combined immunodeficiency panel from genedx showed compound heterozygous mutations in results: a male infant was born with a large thigh mass. the child was clinically well aside from restricted movement of affected leg. mri showed mass expanding into pelvis without other lesions. an interventional-radiology guided core biopsy of the mass was reported as high-grade spindle cell sarcoma without etv rearrangement. surgery was deferred because of concern that it would result in excessive morbidity. the mass was treated with vincristine and dactinomycin per infantile fibrosarcoma protocols. after months of therapy, no significant change in size of the mass was noted on physical exam or imaging. repeat biopsy was obtained to confirm diagnosis and allow for expanded tumor testing. this biopsy showed triphasic distribution of adipose, fibrous and mesenchymal tissue consistent with fhi with rare sarcomatous foci. additional chemotherapy was deferred and the child was followed clinically. his tumor has remained approximately the same size and still unresectable. next generation sequencing of tumor utilizing panel based technology revealed braf-erc fusion consistent with braf activating mutation. this mutation was confirmed by fluorescent in situ hybridization (fish) probe for braf. braf and mek inhibitors have been pursued as treatments to decrease size of tumor and allow for resection. conclusion: braf mutations have been characterized in a variety of malignancies. inhibition of braf and downstream signaling components has produced promising results in a variety of patients. this is the first case report of a braf mutation in a fhi. although management of fhi is typically surgical, this does suggest a potential therapeutic target and may allow for improved surgical outcomes especially in cases where up-front surgery would result in unacceptable morbidity. genetic sequencing of fhi and other rare tumors is an important tool and has the potential to identify mutations amenable to targeted therapies. background: icf is a rare autosomal recessive disorder characterized by hypo-or agammaglobulinemia and often opportunistic infections suggesting t-cell dysfunction. it is further categorized into subtypes - based on mutations in dna methylation. mutations in the helicase-lymphoid specific (hells) gene, which is required for t-cell proliferation and participates in de novo dna methylation, are characteristic of icf type (icf ). of approximately reported cases of icf, less than percent are characterized as icf . while malignancy has been reported in icf (angiosarcoma, acute lymphoblastic leukemia), and icf (hodgkin lymphoma), here we describe the diagnosis and management of an icf patient with neuroblastoma and neutropenia, which has not been previously described. objectives: describe a novel phenotype and mutation of icf and its management to further expand our understanding of this disease. results: a month ex- week premature male with bronchopulmonary disease and failure to thrive presented with acute respiratory failure in the setting of recent viral bronchiolitis with associated chronic diarrhea. he was subsequently diagnosed with multiple infections including pjp pneumonia, norovirus, parainfluenza, rhinovirus, and pseudemonal cellulitis. he presented with profound neutropenia and agammaglobulinemia with presence of b and t cells on lymphocyte phenotyping. ct revealed a paraspinal mass that was mibg-avid on further study, strongly suggesting neuroblastoma. bone marrow was normocellular and negative for malignancy, however revealed marked granulocytic hypoplasia and maturation arrest concerning for severe congenital or, less likely, immune-mediated neutropenia. metastatic workup was negative. whole exome sequencing revealed a homozygous variant of unknown significance (c. t>c) in the hells gene, portending a working diagnosis of icf syndrome. immunoglobulin supplementation, pentamidine prophylaxis, and g-csf were initiated. he was able discontinue g-csf after months of treatment. his neuroblastoma, initially categorized as l , met criteria for observation. however, followup mri revealed interval growth nearing the spinal canal. he underwent tumor resection, confirming mycn non-amplified, favorable histology neuroblastoma. after infectious prophylaxis and immunologic support were initiated, he incurred two other hospitalizations, the first for g-tube cellulitis and the second for parainfluenza respiratory illness. he now has stable neutrophil counts off g-csf and remains in remission from neuroblastoma. current plan is to proceed with bone marrow transplantation for immunodeficiency. conclusion: icf has not previously been described with neutropenia or neuroblastoma. this report not only describes a novel mutation and phenotype of icf and the management thereof, but also reveals the potential curative role of bone marrow transplantation in such disease. staten island university hospital -northwell health, staten island, new york, united states background: desmoid tumors are rare tumors that arise from highly differentiated fibroblasts. they occur in isolation or as part of the disease spectrum of familial adenomatous polyposis (fap) . fap mutations between codons - typically correlate with increased extraintestinal disease such as desmoid tumors and upper gastrointestinal polyps. we describe a patient with a large intra-abdominal desmoid tumor who is heterozygous for a c. c>t (p.arg cys) apc gene mutation. we are not aware of any other patients reported with this germline apc mutation presenting with a desmoid tumor. objectives: to discuss a novel apc mutation and the presentation of a rare case. design/method: review of clinical presentation, genetic analysis and management of a rare tumor. a -year-old female with no significant medical history presented with abdominal asymmetry and intermittent pain. she reported urinary urgency, shortness of breath, early satiety, decreased appetite and a -pound weight loss over the course of months. ct scan of the abdomen demonstrated a × cm abdominal tumor abutting the local organs but no presence of bowel obstruction. a biopsy revealed a spindle cell neoplasm favoring fibromatosis. there was no known family history of fap, colon cancer, or desmoid tumors. apc gene mutation analysis demonstrated a c. c>t (p.arg cys) heterozygous gene variant. due to size and location of the tumor, it was initially deemed unresectable. the patient was started on a course of monthly liposomal doxorubicin. she tolerated the initial cycles well and interval ct after cycles of chemotherapy revealed a % decrease in tumor volume. variability exists in phenotypic presentation with regards to the location of the afp mutation locus. while fap mutations associated with desmoid tumors typically have changes in the - codon region, our patient presented with a heterozygous mutation resulting in a missense mutation at codon . due to the change in polarity and size, the mutation is not considered to be of conservative nature. we are only aware of one other report of this mutation, which occurred in an individual with a personal and family history of colon cancer. we are not aware of any patients with desmoid tumors who also have this germline apc gene mutation. our case report highlights an apc gene mutation that is not well-described; we are not aware of any other cases of this mutation reported in patients with desmoid tumors. future evaluation and tracking of this mutation may lead to the determination of further clinical significance. background: over time, advanced care planning for location of death has been associated with increased deaths at home rather than in the hospital. in some cases, however, complex management and symptom control can prevent families from achieving their goal of keeping their child out of the hospital and at home at the end of life. ascites is a sequelae of many conditions including malignancy that might lead to significant morbidity. increasingly, interventional procedures are being utilized. peritoneovenous "denver" shunts are placed internally with one end in the peritoneal space and the other buried within a major vessel such as the svc. a one-way valve and pump buried under the skin allows the patient to pump fluid from the peritoneal to the vascular space. the shunt is used frequently in adults, but has not seen much use in pediatric oncology patients. objectives: to describe a case of a terminally ill patient with refractory wilms tumor with ivc involvement who received symptomatic relief with denver shunt placement. results: an -year-old female was diagnosed with relapsed, refractory, metastatic wilms tumor with pulmonary and hepatic involvement, with tumor extension to the hepatic veins and ivc. multiple chemotherapeutic regimens and palliative radiation to the ivc were administered, but her disease continued to progress, leading to pressure on the portal vein and portal hypertension. the resulting ascites was causing the patient significant pain and was difficult to manage. the patient's code status was changed to dnr/dni after discussion with her mother, who identified a desire to have the child die at home as comfortably as possible. a peritoneovenous shunt was placed in order to control the patient's pain and avoid frequent medical procedures and therapies. despite initial anxiety, the patient was able to utilize the pump and achieve significant improvement in her ascites and pain. she was able to spend the remaining six weeks of her life at home. ascites is a common phenomenon of end stage disease. peritoneovenous shunts are a treatment modality that may be considered to allow for pain control at the end of life for pediatric oncology patients with ascites. the procedure is relatively low risk, allows for self-control of the pump to maintain comfort, and is easy enough to use by the patient or family. background: extraneural metastases (enm) from pediatric glioblastoma multiforme (gbm) are rare, with an estimated frequency of . %. etiologic factors include multiple neurosurgical procedures and sarcomatous dedifferentiation. their occurrence can seriously affect the patient's quality of life and survival. while enms have been well documented in adults, pediatric cases have not been previously summarized. a year old male with a cerebral gbm developed extension of disease outside of the neuraxis approximately months post initial presentation and at the time of disease progression. metastases included exracranial temporal lesions, cervical and mediastinal lymph nodes and s of s bilateral lung nodules. a large pleural-based soft tissue metastatic focus was identified on imaging when the patient presented with respiratory distress secondary to a right tension pneumothorax, which was recognized and managed promptly. we summarize the main reported cases in literature to better define risk factors for and evaluate the proposed mechanisms underlying these systemic metastases. design/method: we performed a literature review on the pubmed database using the terms gbm and enm. patients under years of age who met the weiss criteria for the diagnosis of enm from primary cns tumors were included. results: our patient fulfilled two of the three weiss criteria with confirmed gbm at the primary site with all enm in the temporal soft tissue and cervical lymph nodes displaying histopathologic features similar to the primary cns tumor. the intrathoracic adenopathy and lung nodules detected upon chest imaging during workup for respiratory distress were assumed to represent additional metastatic foci. our literature review identified pediatric patients with enm from gbm with a median age of years (range . - years) and a slight female predominance ( % females vs. % males). the most common sites of metastases reported were pleura/lungs, bones, lymph nodes and liver. in of patients, metastases were associated with csf shunting. conclusion: pediatric oncologists should have an increased index of suspicion when caring for patients with gbm, particularly those who have undergone shunting procedures and present with systemic symptoms including bony pain, respiratory changes, transaminitis or cytopenias which should prompt timely investigation for enm. although enm of cns tumors carry very poor prognosis, their diagnosis has potential therapeutic importance because treatment of metastatic lesions may alleviate symptoms and improve the quality of life. additional studies may be warranted to evaluate the incidence of enm that can provide valuable insight into the pathogenesis and biology of high-grade gliomas. nicklaus children's hospital, miami, florida, united states background: sinusoidal obstruction syndrome (sos) has been reported in patients undergoing intensive chemotherapy and as a complication post-hematopoietic stem cell transplan-tation. sos may be complicated by portal hypertension, hepatorenal disease or multi-organ failure. however, despite treatment, there may be further potential complications that can be anticipated in patients with history sos. we report two patients with history of sos presented with post-procedural bleeding after gastric tube placement. we believe that their presentations may be associated to their previous diagnosis of sos. design/method: pubmed search was done with search for terminology including "sinusoidal obstruction syndrome" "defibrotide", and "bleeding". papers relevant to our cases were selected for literature review. results: case : a year-old female with history of desmosplastic medulloblastoma status-post resection and intensive chemotherapy was diagnosed with sos one month after her second part of planned tandem transplant. she was managed with paracentesis and defibrotide. due to malnourishment, patient had a gastric tube placement months after she completed therapy and had an episode of upper gastrointestinal bleeding postoperatively from the g tube site. case : similarly, a year-old male diagnosed with anaplastic medulloblastoma status post resection and adjuvant multiagent chemotherapy. his treatment course was complicated with sos after the second cycle of induction chemotherapy which responded to -day course of defibrotide. likewise, the patient had a major bleeding event from the g-tube site approximately two months after sos diagnosis. defibrotide was discontinued in both cases before g-tube placement. both patients had no previous history of bleeding disorders or relevant family history. in addition, comprehensive laboratory evaluations were within normal limits before both procedures. in sos, there is blockage of fluid out of the liver that leads to congestion, ascites, ischemia of the liver, and post-sinusoidal portal hypertension. two related causes of sos should be considered as an explanation for g-tube bleeding. similar patients should have close monitoring postoperatively or if possible surgical intervention should be delayed until the sos process has been evolved. nicklaus children's hospital, miami, florida, united states background: the development of treatment related acute myeloid leukemia (t-aml) and myelodysplastic syndromes (t-mds) is a potential complication after cytotoxic chemotherapy or radiation therapy. the incidence of development of t-aml/t-mds varies from - % depending on the treatment regimen used. cutaneous myeloid sarcoma (ms) is a common presentation of extramedullary leukemia and usually occurs in the setting of aml. we report a rare case of cutaneous ms in an adolescent female after successful treatment for ovarian yolk sac tumor (yst) stage i with bep (bleomycin, etoposide and cisplatin) therapy. the ms was managed only with biopsy and close observation. design/method: a pubmed search was conducted for queries including t-aml/t-mds, cytotoxic agents, cutaneous myeloid sarcoma, regression. relevant papers were selected for literature review. a year-old female was diagnosed with a left ovarian yolk sac tumor, for which she underwent left salpingooophorectomy and successfully completed cycles of bep over months. during routine follow-up months after initiation of treatment for ovarian yst, she was noted to have a small, non-tender, indurated nodule on the left side of her upper back approximately cm in diameter. punch biopsy of the skin nodule was performed and pathology was positive for cutaneous myeloid sarcoma. at the time of next follow-up less than one month later, the skin lesion had resolved. two subsequent bone marrow aspirates were performed one month apart and were negative for leukemic involvement or mds. examinations and work-up including whole body pet with ct scan were negative for evidence of disease. although cutaneous ms can be regarded as the herald of systemic myeloid disease rather than a localized process, our patient was monitored periodically with physical exam and laboratory evaluations. she remains free of disease more than four years after the presentation of cutaneous ms without any further treatment. spontaneous regression ms has been previously reported. the authors would like to stress that a conservative approach with close observation could be an option in cutaneous ms even with history of chemotherapy exposure. nesreen ali, iman sidhom, sonia soliman, sherine salem national cancer institute, cairouniversity, egypt children cancer hospital egypt, egypt background: acute leukemia is the commonest malignancy in childhood. the coincidental occurrence of leukemia with hemophilia is extremely rare. hemophilia is a congenital rare x linked bleeding disorder. the main complication of the two diseases is bleeding diathesis which may be lifethreatening due to many factors, deficiency of coagulation factors in hemophilic patients, thrombocytopenia from disease and chemotherapy in leukemic patients, certain cytotoxic drugs such as asparaginase which may result in coagulation disorders and infection which may lead to disseminated intravascular coagulation. objectives: reporting such a case is imperative to set up treatment guidelines for prevention of bleeding and to optimize the therapeutic approach for these patients. design/method: seventeen years old boy, presented to children cancer hospital egypt in june with pallor and multiple ecchymoses.he was diagnosed with precursor b acute lymphoblastic leukemia, cerebrospinal fluid (csf) was free, the chromosomal analysis revealed hypodiploidy , xy. he had moderate type of hemophilia a since birth, factor viii level was . % at time of diagnosis, coagulation profile revealed prolonged partial thromboplastin time (normal - ), factor viii was low %, prothrombin concentration and prothrombin time were normal % and seconds, virology screening for hepatitis b core igg/igm, hbs ag, hiv and hc igg /igm were negative.the patient started induction total xv sjcrh protocol, factor viii unit/kg was given at presentation before doing bone marrow aspiration(bma), csf and as a prophylactic before intramuscular asparaginase injection, intrathecal and bma. it was given immediately within hours before the procedures and platelets transfusion was given regularly to maintain platelets count about , . the minimal residual disease by flow cytometry was . % and . % at d and d induction. results: our patient received his induction and reintensification chemotherapy without any major bleeding event which reveals the success of our guidelines for the prevention of bleeding. he developed very early relapse at w maintenance by the same clone. he received salvage chemotherapy but didn't achieve remission and died out of disease and resistant clone. the development of leukemia on top of hemophilia is a major problem. bleeding complication during chemotherapy can be prevented by regular prophylactic factor viii and platelets concentrate transfusion with good supportive care. life threating bleeding complication may be correlated with the severity of hemophilia. we need to collect data about the biology of leukemic cells, complications, and cause of death to optimize care for these patients. background: mucoepidermoid carcinoma (mec) is a rare malignancy that arises from exocrine glands in the upper aerodigestive tract and tracheobronchial tree. conventionally, mec diagnosis is based on histology, with prognosis based on the extent of resection and detection of metastases. mec is characterized by a translocation of chromosomes q and p resulting in a fusion between the mect and maml genes, that occurs in - % of cases. this fusion transcript has been recognized to have a favorable impact on disease features and prognosis of mec. however, recent studies indicate that high grade mec can have mect -maml fusion positivity and multiple other genomic imbalances that have not been studied in much detail. owing to the rarity of mec tumors, more definitive data related to the clinical and prognostic significance of these molecular markers are limited. objectives: . identify the presence or absence of mect -maml fusion in the tissue of our patient. . analyze the incidence of the fusion in mec cases in children and young adults retrieved from the iowa cancer registry. . determine if fusion status correlates with clinical, pathological and outcome data in our cohort. design/method: we describe the case of a year-old caucasian male who presented with recurrent pneumonia, persistent cough and radiographic evidence of right lobar collapse. bronchoscopy revealed an endobronchial lesion and the patient underwent right upper lobe sleeve resection. pathology report was consistent with low grade muco-epidermoid carcinoma. we retrieved archived formalin-fixed paraffinembedded (ffpe) specimens of pediatric and young adult mec cases (ages - ) reported in iowa from - using the iowa cancer registry. testing for the mect -maml fusion in the index case and ffpe specimens will be done using a custom-designed laboratory validated next generation sequencing (ngs) assay with the ability to detect novel fusion partners. clinical, pathological and outcome data (age, sex, tumor site, tumor size, nodal metastases, clinical stage, histologic grade, treatment and follow up) will be analyzed to correlate with fusion status. the mect -maml fusion tested positive in our index patient. we will obtain irb approval to test for the fusion in the archived ffpe specimens and correlate clinical, pathological and outcome data. conclusion: mect -maml fusion is a frequent event in mec that has prognostic and potential therapeutic applications in adults. the results of this study may enlighten the clinical management of mec in children and young adults. children 's mercy hospital, kansas city, missouri, united states background: mutations in the samd gene are associated with a rare syndrome comprising of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (mirage syndrome). diagnosis is made through exome sequencing. in the largest reported case series, of eleven patients diagnosed with mirage syndrome, two developed loss of chromosome . given the potent growth restricting activity of samd mutants, the loss of chromosome is considered the first documentation of adaptation by aneuploidy mechanisms in humans and led to myelodysplastic syndrome (mds), with deaths occurring from related complications at and years of age. objectives: to report a case of mirage syndrome with congenital thrombocytopenia progressing to bone marrow failure, managed uniquely with bone marrow transplantation. results: male born at weeks gestation with prenatal diagnosis of iugr, two vessel cord, oligohydramnios was found to have ambiguous genitalia, adrenal insufficiency, partial panhypopituitarism and congenital thrombocytopenia with bone marrow showing absence of megakaryocytic precursors. severe thrombocytopenia was present from birth. bone marrow evaluation demonstrated a hypocellular marrow with markedly reduced megakaryocytic and myeloid precursors and no evidence of myelodysplasia. he required gastric tube placement for failure to thrive, had a laryngeal cleft repaired and developed focal segmental glomerulosclerosis. mpl gene testing for congenital amegakaryocytic thrombocytopenia was negative. testing for fanconi anemia, shwachman-diamond syndrome and dyskeratosis congenita was also negative. approximately % of cells had loss of heterozygosity on chromosome q. exome sequencing showed that he is heterozygous for a de novo gain of function variant, c. g>a (p.arg gln), identified in the samd gene, confirmed by sanger sequencing and consistent with a diagnosis of mirage syndrome. at years of age, he developed pancytopenia requiring frequent transfusions with platelets and packed red blood cells. he underwent a successful bone marrow transplant at years of age without significant complications, and remains transfusion independent without cytopenias greater than months from bone marrow transplantation. conclusion: it is imperative to pursue work up for persistent congenital thrombocytopenia in a stepwise multidisciplinary manner. to the best of our knowledge, this is the first case of mirage syndrome associated bone marrow failure treated with bone marrow transplant. due to the individual rarity of mirage syndrome and pediatric myelodysplastic syndrome, it is important to maintain an index of suspicion given their association and explore bone marrow transplant as a therapeutic option. results: the patient demonstrated disease regression, initially, and continued without disease progression for months. the regimen has been well tolerated with only minimal side effects of dry skin (ctcae grade ) and a transient episode of brief erythrodysesthesia (ctacae grade ) that resolved spontaneously. the combination of sorafenib and capecitabine was effective and well tolerated in this adolescent patient with fl-hcc. our observations, although in a single patient, lend support for further testing of this novel oral chemotherapy regimen in patients with fl-hcc, a disease for which there is no effective standard chemotherapy approach. background: epstein-barr virus (ebv) is a ubiquitous virus associated with a broad range of malignancies due to its oncogenic potential. history of organ or bone marrow transplantation, immunosuppressive therapy, and primary or acquired immunodeficiency syndromes increases the risk of ebvassociated tumors. epstein-barr virus associated smooth muscle tumors (ebv-smt) are unique and rare neoplasms typically discovered in immunocompromised patients. most information related to pathogenesis and therapeutic options is limited to case reports and case series of adult patients. there are several gene expression pathways that ebv utilizes, the most notable of which is the mammalian target of rapamycin (mtor) pathway. the mtor pathway performs a key role through integrating various cell growth signals and factors to regulate protein synthesis and metabolism related to smooth muscle proliferation. sirolimus is an immune modulating therapy that targets the mtor pathway to block activation of lymphocytes. objectives: several case reports have demonstrated shortterm clinical remission of ebv-smt in adult patients with the use of sirolimus. we report the first case of long-term background: bilateral neuroblastoma is characterized as neuroblastoma arising in both adrenal glands, a rare presentation with little data on its genetic make-up. a two-monthold patient was diagnosed with bilateral neuroblastoma in our clinic. her risk assignment was based on biopsy of the left adrenal lesion, which showed mycn amplification, an unfavorable genetic marker. treatment regimen was intensified accordingly and after courses of chemotherapy tumors were excised. patient went on to receive a stem cell transplant and immunotherapy. with no knowledge of genetic similarity between the two tumors it is unclear whether biopsy of the right lesion would have yielded similar results or whether bilateral biopsies are needed for risk assessment of bilateral neuroblastoma. objectives: utilize whole exome sequencing (wes) to characterize the genomic signature of bilateral adrenal neuroblastomas excised following chemotherapy treatment. design/method: paraffin-embedded samples from left (l) and right (r) tumors underwent wes at the broad institute. we analyzed resulting data including somatic variant calls, indel mutations, and copy number variants (cnvs) using ingenuity software to evaluate and compare differences between the two tumor samples. preliminary analysis of the data shows important descriptive information on the two tumor samples. out of somatic mutations in the r tumor cells and mutations in the l tumor cells, only two common somatic mutations were present. out of cnv calls in the r tumor and in the l tumor, cnvs were common between the two tumors, or % of each tumor's cnv calls. there was a fold higher frequency in gains versus losses. the median size of the common cnvs was , (range to , , bp). cancerrelated genes with increased copy numbers included transcription factors, receptors for signal transduction pathways, and histone methylation proteins. conclusion: preliminary analysis of the wes results of the two adrenal tumors show some genomic divergence. because the tumor tissue was exposed to chemotherapy prior to excision it is difficult to determine whether genomic divergence is a result of independently originated tumors or subsequent adaptation to chemotherapy of a clonal cell population. the high number of common cnvs in the two tumors points to a common cell of origin, however the low number of common somatic mutations does not fit that picture. a future study to help elucidate the question will be wes of the original biopsy tissue to provide information on tumor mutations prior to the effects of chemotherapy. baylor college of medicine, houston, texas, united states background: although there has been significant improvement in the overall survival rates of children with cancer many children will still die from their illness or complications secondary to treatment. research surrounding the deaths of children who succumb to their disease is warranted to ensure we are providing the best care possible for these patients. objectives: this case series aims to explore pediatric cancer deaths by focusing on perhaps the most extreme cases of high intensity end of life care. we explore those patients whom we know are dying or our very likely to die as evidence by their do not resuscitate (dnr) orders. in all of these cases despite the patients very grim prognosis, their great likelihood of death and limitations placed of resuscitation methods all patients continued end of life care in the pediatric intensive care unit (picu). the primary medical records of all children with a cancer diagnosis who died between february , and january , in the picu with a dnr order seven days or earlier prior to death. each medical history included disease-directed treatment history and response with particular attention to the events surrounding the terminal admission. results: eight patients met criteria for this study representing . % of all cancer patients who died during this time period and . % of those who died in the icu. the average time between dnr and death is . days ( days - days). the average length of terminal admission was . days ( day - days). the average time between diagnosis and dnr is . months ( months - months). the average time between diagnosis and death is . months ( months - months). conclusion: these cases highlight the journey that patients, families and providers endure leading up to death. medical care is complex, there are very few absolutes that are encountered when caring for patients and decisions around limiting or withdrawing medical care are made in a context of the prior journey. . these cases help to understand the complexity of death and how two seemingly opposite ideals can be congruent in the event of an anticipated death. most of these cases show the need for improved anticipatory guidance surrounding death and greater consideration for de-escalation of care when death is expected. the hospital for sick children, toronto, ontario, canada background: rhabdomyosarcoma (rms) is the most common soft tissue sarcoma in children, with embryonal (erms) and alveolar (arms) representing the most common subtypes. arms tumors are associated with inferior outcome when compared to erms, and they are characterized in about % of the cases by a t ( ; ) or t( ; ) chromosomal translocation with creation of a pax -foxo or pax -foxo fusion gene, respectively. it is increasingly clear that the pax-foxo fusion status is an important poor prognostic factor, thus the histological classification tends to be replaced by the fusion status, particularly in terms of risk stratifica-tion in contrast to arms, there are no recurrent chromosome alterations in erms; however, there are multiple numerical chromosome changes that are frequent in these tumours: gain of chromosome , , and have been found in to % of emrs karyotypes. moreover, erms tumors show frequently allelic loss, the .p . chromosomal region being the most frequently involved. recently, novel gene fusions have been described also in erms tumours. these fusions involved mainly the ncoa and or the vggl genes. the rearrangement partners are variable, and include, i.e. pax ( q ), srf ( q ) and tead ( p ). objectives: to present a patient who died as a consequence of brain metastases while on therapy in the setting of an foxo negative rms and the identification of a new translocation t( ; )(q ;q ). design/method: case report and retrospective review of the literature. we report a case of pelvic embryonal rhabdomyosarcoma in a -month old boy. he was treated as per cog arst intermediate risk group, but unfortunately was found to have a large cerebellar tumour during the course of his chemotherapy treatment and he subsequently passed away. a novel translocation between chromosomes and was observed in of metaphase cells by g-band analysis in the autopsy sample of the brain lesion. breakpoints of the translocation were estimated to be at q and q . there were no additional clonal chromosome abnormalities in the tumour cells. conclusion: erms tumors with fusion genes involved have been exclusively described in patients less than months of age; they seem to be associated with spindle cell histology and, a favorable outcome. in our patient, a novel ( ; ) translocation was found and clinically, the patient had a dismal outcome. further studies are indicated to inquire whether this finding is of significance in term of prognosis for these patients. children 's national medical center, washington, district of columbia, united states background: iatrogenic immunodeficiency-associated lymphoproliferative disorders (lpds) are a group of lymphoid s of s proliferations or lymphomas that are well known to be associated with an immunosuppressed state. these disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or ptld), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. these disorders are strongly associated with infection by the epstein-barr virus (ebv) as a result of impaired immune function in the immunosuppressed state. while this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on antileukemia chemotherapy are lacking. background: atypical teratoid/rhabdoid tumor (at/rt) of the central nervous system (cns) in children younger than years old has a prevalence of % to % and accounts for . % of all pediatric cns tumors. only - % of patients have leptomeningeal dissemination. rhabdomyosarcoma is the most common soft tissue tumor in childhood, but represent only - % of all pediatric cancers. rarely, it can metastasize or even directly extend into the cns, but typically, cases of cns involvement arise either from parameningeal areas or other primary sites. primary spinal or meningeal rhabdomyosarcoma is extremely rare. objectives: our objective is to describe two unique cns malignancies presenting as rare, primary leptomeningeal disease. design/method: case a -month-old female presented with vomiting, fatigue and listlessness, despite a normal head ct and brain mri. csf showed hypoglycorrhachia and mild pleocytosis. ceftriaxone was started, but she developed nuchal rigidity and cranial nerve vii palsy. repeat brain mri showed evolving leptomeningeal enhancement concerning for meningitis. she gradually developed worsening opisthotonus and ultimately a brain biopsy of the temporal lobe was consistent with at/rt. case a -year-old male presented with new generalized tonic-clonic seizure activity and intermittent headaches with photophobia, phonophobia, and vomiting. brain mri was significant for enhancement of interpenducular and suprasellar cisterns extending to the optic nerves and chiasm most consistent with meningitis. neurosurgery ultimately placed a lumbar drain for hydrocephalus, and a tissue biopsy demonstrated primary meningeal rhabdomyosacroma. results: in case , our patient's temporal lobe biopsy demonstrated grade iv malignant tumor cells consistent with atypical teratoid/rhabdoid tumor. fish demonstrated a homozygous deletion of smarcb ( q . ). she was started on chemotherapy per the dana farber at/rt protocol but ultimately was discharged home on hospice. in case , our patient's lumbar arachnoid biopsy demonstrated cellular tumor consistent with group iiia embryonal rhabdomyosarcoma. immunostaining was positive for cd , desmin, myogenin, and myo-d with neural markers ema and gfap highlighting the meninges but without a neural component to the tumor. he completed craniospinal radiation to gy total with lumbar boost to . gy total. he is currently receiving chemotherapy per arst protocol. conclusion: these two cases are particularly instructive because of their similar initial presentations and neuroimaging, but with very different and unique diagnoses. university of iowa, iowa city, iowa, united states background: ebf -pdgfrb fusion causes ph-like b-cell acute lymphoblastic leukemia (b-all), which has a philadelphia positive phenotype without the bcr-abl translocation. this is one of several mutations associated with ph-like b-all and leads to downstream overexpression of tyrosine kinase. ebf -pdgfrb fusion accounts for about % of children with ph-like b-all. patients with ph-like b-all previously had poorer outcomes with conventional chemotherapy. the addition of tyrosine kinase inhibitors (tki), like imatinib, has improved the outcome for many patients predicted to have tki sensitive mutations. objectives: to review clinical characteristics and outcomes of two cases of ph-like b-all at the university of iowa stead family children's hospital and to compare these outcomes to similar cases reported in the literature. design/method: a retrospective chart review was performed for two cases of ph-like b-all diagnosed and treated at the university of iowa stead family children's hospital. results: both patients were males diagnosed at years of age with high wbc count ( , and , ) and positive for ebf -pdgfrb gene fusion. patient (pt ) was cns b at presentation while patient (pt ) was cns negative; neither had testicular involvement. both started treatment according to cog protocol aall . peripheral blasts cleared by induction day for pt and induction day for pt . at end of induction, pt had m bone marrow and pt had m bone marrow but mrd %. dasatinib was started induction day for pt and induction day for pt . pt was still not in remission at end of consolidation; bone marrow cell culture for tki resistance showed best response to dasatinib. pt proceeded to anti-cd car t-cell therapy followed by tbi-based matched unrelated donor bone marrow transplant. pt had negative mrd at the end of consolidation and continues chemotherapy according to aall , dasatinib arm. both patients are currently clinically well. our patients had the same tyrosine kinase gene fusion and similar initial clinical courses. while both patients had persistent disease at end of induction, pt had almost % blasts while pt had significant reduction of disease burden before starting tki. pt showed good response with the addition of dasatinib while pt did not. these findings suggest that response to conventional chemotherapy may potentiate the effect of tki and may predict overall outcome. there are likely additional factors which must be taken into account when determining response to tki for patients with ph-like b-all which have not yet been identified. background: medulloblastoma is the most common malignant brain tumor of childhood. classically, medulloblastoma presents as a well-defined mass lesion in the cerebellum, with a high rate of metastatic dissemination. primary leptomeningeal medulloblastoma (plmb) is an exceedingly rare type of medulloblastoma presentation with a dismal prognosis in which patients present with isolated leptomeningeal disease without an associated mass. to our knowledge, only three pediatric and three adult cases of plmb (ages - years) have been reported, all of which died within months of diagnosis. this is the first case of plmb to report a molecular classification. objectives: to report the case of a pediatric patient with plmb in which histopathologic and molecular characterization was performed and to describe the patient's treatment and clinical course. design/method: retrospective review of the patient's electronic medical record and review of the literature. a -year-old boy presented with headache, vomiting, diplopia, and fatigue. physical examination revealed upward gaze palsy, left-sided extremity and facial weakness, and ataxia. magnetic resonance imaging (mri) of the brain revealed diffuse cerebellar leptomeningeal enhancement and edema without an identifiable mass and moderate hydrocephalus. mri of the spine and cerebral spinal fluid analysis were normal. a diagnosis of cerebellitis was rendered, and the patient underwent placement of a ventriculoperitoneal shunt. an extensive infectious, neurologic, rheumatologic, and oncologic workup did not identify an etiology. empiric antibiotics, high-dose steroids, and intravenous immunoglobulin therapy yielded minimal improvement. two months later, repeat mri of the brain performed for declining mental status demonstrated progressive thickening of cerebellar leptomeningeal disease. a suboccipital craniectomy with decompression and cerebellar biopsy were performed. pathologic examination revealed a diagnosis of plmb, classic histology, non-wnt/non-shh, without gain/amplification of myc/mycn, and p wild type pattern. craniospinal radiation to cgy with a cgy boost to the posterior fossa was delivered with concurrent carboplatin/vincristine over six weeks. two months following chemoradiation, mri of s of s the brain demonstrates significantly reduced pathological leptomeningeal enhancement of the cerebellum, and the patient is awaiting initiation of systemic chemotherapy while recovering from a surgical wound infection. conclusion: plmb is extremely rare but should be considered in patients with cerebellitis and diffuse leptomeningeal involvement who are refractory to medical management or in whom an etiology has not been identified. cerebellar biopsy is recommended early to enable timely treatment and improved outcomes. molecular classification should be performed in cases of plmb to further characterize this disease, inform treatment decisions, and improve clinical outcomes. background: primary intracerebral osteosarcoma is extremely rare and limited to case reports. ptpn gain of function is associated with noonan syndrome, which has increased risk of multiple cancer types including brain tumors, but osteosarcoma has never been described. ptpn mutations have been reported in many cancers as both oncogenes and tumor suppressors, however no ptpn mutations have been described in osteosarcoma. pdgfr-a is a growth factor receptor whose activation is implicated in several malignancies. pdgfr-a and ptpn concurrent mutations are described in glioblastoma. there is no known link between holoprosencephaly, noonan syndrome, and osteosarcoma. we report a case of multifocal intracerebral osteosarcoma in a child with lobar holoprosencephaly and chronic subdural hemorrhage and discuss the genetic changes found in the tumor. design/method: a seven-year-old caucasian female, with a known diagnosis of lobar holoprosencephaly, chronic subdural hemorrhage and well controlled seizure disorder presented with status epilepticus shortly after completing antibiotic therapy for infection of subdural hematoma. mri showed diffuse dural thickening with mass lesions in the frontal lobe, temporal lobe, and the parasagittal region, the largest of which was contiguous with the subdural space but none of the lesions were associated with bone on mri or by direct neurosurgical visualization. tissue obtained for concern for recurrent infec-tion resulted in a diagnosis of high grade osteosarcoma. dna analysis was performed to help guide treatment choice. results: standard metastatic work-up was negative for skeletal primary tumor or metastatic lesions outside of the brain. she was treated with high dose methotrexate for two cycles per modified aost . despite maximal supportive care, she quickly developed rapid tumor growth as well as intratumoral hemorrhage with resultant herniation and death from respiratory failure just three months after diagnosis. tumor gene sequencing discovered three mutations with described roles in cancer: pdgfra d >vr, kdm a loss of exons - , and ptpn a v. conclusion: to our knowledge, primary multifocal extraosseus intracerebral osteosarcoma has not been previously described. despite known cns penetration of high dose methotrexate, this tumor proved resistant and aggressive. holoprosencephaly is associated with a multitude of known genetic drivers, but none are found in this case. furthermore, the genetic changes in this tumor are not typical for osteosarcoma. pdgfr-a over-expression is described in osteosarcoma, but is not clearly correlated with worse overall survival. further research is required to determine the role of ptpn in osteosarcoma. background: anaplastic lymphoma kinase (alk) encodes a receptor tyrosine kinase whose activation induces pathways associated with cell proliferation, angiogenesis, and cell survival. alk rearrangements are rare in neuroblastoma, while alk mutations and gene amplification occur more frequently. alk mutations have been found to be associated with increased alk protein expression that is associated with a worse prognosis. alk is commonly mutated in neuroblastoma at three hotspots (f , r , and f ). the eml -alk rearrangement has mostly been associated with lung adenocarcinomas, with only a few cases of non-lung cancers found. it has never been reported in neuroblastoma. multimodal therapy and to report the successful management of treatment related iron overload. results: a -year old male presented with abdominal swelling and ct showed a right kidney mass and bilateral lung nodules. he underwent right radical nephrectomy with lymph node sampling. pathology was reviewed centrally and revealed wilms tumor with diffuse anaplasia with rhabdomyosarcoma arising within the stromal component and of nodes positive. he received adjuvant intensive chemotherapy and radiation to the hemiabdomen and whole lungs. the -week chemotherapy regimen was vincristine, doxorubicin, cyclophosphamide (per cog arst ) alternating with carboplatin and etoposide (per cog aren revised uh- ). treatment was complicated by multiple episodes of fever and neutropenia and anorexia requiring g-tube placement. post-therapy, he had persistent neutropenia and thrombocytopenia without related complications. every months for evaluations he underwent a bone marrow which revealed normocellular marrow with maturing trilineage hematopoiesis. evaluation for a bone marrow failure syndrome was unrevealing. starting at months into therapy and all posttherapy imaging showed splenomegaly. he received units of packed red blood cells through the duration of therapy. he was diagnosed with iron overload based on serum ferritin and imaging, including t *mri. he received therapeutic phlebotomy for years with normalization of serum iron studies, t * of the heart, and liver iron concentration. he is more than years from completing therapy with no evidence of recurrent disease. asymptomatic cytopenias persist and he has no evidence of iron overload. conclusion: though a rare development, clonal sarcomatous transformation can occur in wilms tumor. our patient's tumor was successfully treated with intensive multimodal therapy targeting the diffusely anaplastic wilms and the rhabdomyosarcomatous component. treatment-related iron overload in a pediatric patient with a solid tumor was successfully treated with phlebotomy. consideration should be given to screen patients with solid tumors who receive multiple packed red cell transfusions for iron overload at the completion of cancer therapy. primary children's hospital, university of utah, salt lake city, utah, united states background: malignant solid tumors are less frequently encountered in infants. primitive myxoid mesenchymal tumors of infancy (pmmti) are a myofibroblastic malignancy and cases are rarely reported in the literature. cure is achieved in the majority of cases with surgical resection, however treatment for unresectable cases remains an enigma. recently published literature postulates that the newly discovered bcor duplication found in pmmti is tumorigenic via an epigenetic pathway. this molecular signature resembles that of clear cell sarcoma of the kidney (ccsk) and the growing number of bcor mutated sarcomas. a similar chemotherapeutic backbone and local control used for ccsk, has been proposed for the unresectable subset of pmmti. utilizing this approach a month-old with relapsed disease has remained disease free for months. however, given the rarity of this disease and the lack of published literature, there is no known standard of care treatment for unresectable and/or recurrent ppmti. we report a case of unresectable recurrent pmmti, a rare infant tumor, with less than cases reported. design/method: medical record, radiological studies, pathology and literature was reviewed. results: our patient is a now month-old female who presented with constipation and lower extremity weakness in the first weeks of life. an mri demonstrated a large lumbar epidural mass with spinal cord impingement. given prolonged (> days) neurological symptoms and location, emergent chemotherapy was initiated. biopsy showed a bcor positive, primitive myxoid mesenchymal tumor of infancy (pmmti). she was treated with ifosfamide, carboplatin and etoposide, and demonstrated clinical and radiographic response. we gave two additional cycles of cyclophosphamide, carboplatin and etoposide until surgical resection was feasible followed by two post-surgical cycles of chemotherapy. unfortunately, four month post-therapy mri demonstrated two new lesions; an unresectable paraspinal soft tissue mass and a left iliopsoas groove mass. given bcor association and reported successful therapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide, we elected to incorporate vinca-alkaloid and anthracycline into her regimen. she is being treated with vdc/ie with plan for radiation consolidation. conclusion: pmmti is a locally aggressive tumor, for which surgical resection is curative. for those not amendable to resection, best care practices are still being determined. we report a case of pmmti initially responsive to chemotherapy, but not curative. this is the second case to conclusively demonstrate chemo-responsiveness. bcor mutation seems to be a common feature of this cancer; its role in the pathogenesis and as a target is an area of investigation. medical college of wisconsin, milwaukee, wisconsin, united states background: atypical teratoid/rhabdoid tumors (atrt) are central nervous system (cns) tumors that most commonly occur in very young children. there is no widely accepted standard of care for atrt patients, and while survival rates are improving they are historically poor. patients with metastatic disease to the spine at diagnosis have a worse prognosis, and for patients > years old, the presence of metastatic disease often results in the use of craniospinal radiation. the importance of correctly identifying metastatic disease at diagnosis aids in decision making and can have both prognostic and therapeutic implications. mr imaging at diagnosis is used to identify metastatic disease; however, here we present a case of diffuse leptomeningeal enhancement that spontaneously resolved after resection of a primary supratentorial atrt. objectives: to describe the resolution of diffuse leptomeningeal enhancement after resection of a primary atrt tumor in a -month-old prior to any adjuvant therapy. results: a -month-old male presented with a month history of vomiting and weight loss, regression of gross motor developmental milestones, and left hemiparesis. a brain mri demonstrated a × . × . cm solid and cystic right atrial mass with diffusion restriction and post-contrast enhancement. smooth diffuse enhancement was noted along the surface of the brainstem and within the interpeduncular fossa. a spine mri demonstrated diffuse circumferential post-contrast enhancement along the surface of the entire spinal cord. the patient underwent a successful near total surgical resection of the primary mass. pathology confirmed the loss of ini- staining in tumor cells, consistent with a diagnosis of atrt. no immediate adjuvant radiation or chemotherapy was given. repeat imaging was completed days after resection. brain mr demonstrated expected post-operative changes within the surgical cavity without definitive residual mass or leptomeningeal enhancement. spine mr demonstrated complete resolution of the previously seen circumferential enhance-ment along the entire spinal cord. csf evaluation at that time was negative for tumor cells. after recovery from surgery, chemotherapy treatment was initiated. conclusion: leptomeningeal enhancement at the time of diagnosis of atrt has historically been considered clear evidence of metastatic disease. this case raises questions about the previously accepted etiology of these imaging changes and suggests that widespread leptomeningeal enhancement should be carefully interpreted in future patients with similar imaging findings. in this setting, clinicians should consider repeat imaging following primary surgical resection in order to provide appropriate prognostic information and inform therapeutic decisions. poster # primary ewings sarcoma of cervical cord mimicking cauda equina syndrome sucharita bhaumik, joshua chan nyu winthrop hospital, mineola, new york, united states background: ewing's sarcoma (es) is a malignant primary bone tumor usually involving long bones. primary es of spine is quite uncommon ( . %) and its location in the cervical spine is even more rare. cauda equina syndrome (ces) is symptoms due to damage to the bundle of nerves below the end of the spinal cord known as the cauda equina (low back pain, radiating shooting pain down the legs, paraplegia, and loss of bowel or bladder control). it often occurs with lesions of lumbosacral spine. treatment with high-dose steroids may provide pain relief and improved neurologic function (by reducing edema) while awaiting diagnostic studies objectives: to demonstrate an unusual clinical presentation and emergent management of cervical es presenting with ces like symptoms. : year old male presented with a left sided posterior neck mass. soon after, he developed weakness of left arm, urinary and stool retention and inability to walk or bear weight in both legs. on physical exam a left tempero-occipital × cm fixed, non-tender, non-fluctuant mass was noted as well as motor and sensory impairment of left upper extremity, bilateral spastic paraplegia and loss of sphincter control. mri cervical spine showed a left cervical tumor with moth eaten appearance involving the vertebral bodies of c -c , adjacent muscles, displacing vital structures of the neck and compressing the cervical spinal cord. the thoracic and lumbosacral spine had no disease involvement. due to rapidly worsening spinal cord compression he was emergently treated with high dose steroids. he gained back all function in his extremities and regained bowel and bladder control. this eliminated need for urgent neurosurgical intervention. results: biopsy of the neck mass showed small blue round cells consistent with es with ewsr gene rearrangement. staging work up revealed no additional metastatic involvement. he then initiated treatment for localized es with systemic chemotherapy and radiotherapy and has had excellent response to treatment so far. conclusion: this is the first known case of non metastatic primary cervical es mimicking ces where an acutely enlarging mass presented with rapidly progressive neurologic deficits due to compression of anterior spinothalamic tract. in these unusual presentations of ces without lumbodorsal involvement it is important to consider cervical lesions. early rapid steroid initiation should be considered while awaiting biopsy results to prevent worsening cord compression followed by es focused treatments. this increases the chance of a successful outcome. the initial improvement with steroids may confuse the tumor with being a lymphoma children 's mercy hospital, kansas city, missouri, united states background: von willebrand disease (vwd) is a relatively common bleeding disorder with a high degree of genotypic and phenotypic variation. bleeding is usually mucocutaneous but can be severe and include muscle and joint bleeds especially in type vwd patients. most common bleeding management consists of desmopressin, anti-fibrinolytics, and/or plasma-derived antihemophilic factor/von willebrand factor (ahf/vwf) complex. a recombinant vwf has become available in the last few years. anaphylaxis and inhibitor development in vwd are rare. objectives: to describe the rare clinical manifestation of anaphylaxis to factor concentrate in a patient with severe type vwd. results: a -year-old female with severe type vwd [baseline vwag %, activity < %, factor viii (fviii) %] originally presented with heavy menstrual bleeding (hmb) leading to anemia requiring blood transfusion. she underwent placement of a levonorgestrel-releasing intrauterine device (lngiud) and began norethindrone. her hmb continued despite the lngiud and an increase in norethindrone dosing. plasma derived ahf/vwf complex was administered, which she had previously received. following the infusion, the patient developed anaphylaxis with hives, wheezing, tachycardia, and itching requiring doses of diphenhydramine and dose of hydrocortisone with resolution of symptoms. subsequently, she received recombinant vwf without incident. however, due to her low fviii level, she also required treatment with a full length recombinant fviii product. she again developed hives and itching after this infusion. she has since received recombinant vwf with recombinant fviii/fc fusion protein without further allergic reaction. there was no evidence of an inhibitor with her most recent post-infusion vwf level was %, factor viii %. conclusion: anaphylaxis to plasma derived factor products has been documented far less frequently within the vwd population compared to those with hemophilia and is typically seen in those with large gene deletions, usually with type disease. therefore, similar type vwd patients with severe disease may benefit from gene sequencing. it is unclear in this patient's case to which aspect of her treatment she is allergic, as she reacted to plasma-derived ahf/vwf and full length recombinant fviii, but not recombinant vwf or recombinant fviii/fc fusion protein. we hypothesize that she may be allergic to an epitope in the fviii b domain, or that the presence of fc fusion may have had a protective effect. further investigation including genetic analysis is planned. nodules. biopsies were consistent with neuroendocrine carcinoma, large cell type (g ). next generation sequencing revealed a khdrbs -braf fusion. he received conventional cytotoxic chemotherapy regimens both with cisplatin/doxorubicin, capecitabine/temozolomide, and doxorubicin/etoposide, but achieved a minimal response followed by rapid disease progression, massive ascites, and renal failure secondary to bilateral ureteral obstruction. results: based on his prior genomic testing, therapy with single agent mek inhibitor (trametinib) was initiated. this produced a rapid, dramatic response with greatly reduced disease burden at all sites, resolution of ascites and return to completely normal activity within months. this response lasted for approximately months before the tumor again progressed. further therapy with an erk inhibitor was ineffective, and the patient expired from progressive disease. located on the chromosome q , the braf oncogene, as part of the ras/mapk pathway, is involved in cellular proliferation, differentiation, migration, and apoptosis. braf mutations are recognized in a wide range of adult malignancies: thyroid cancers, non-small cell lung cancer, cholangiocarcinoma, ovarian cancers, and multiple myeloma. braf mutations have also been described in adult neuroendocrine carcinoma of the colon. trametinib is a highly specific inhibitor of mek /mek , a downstream mediator in the braf pathway. it has demonstrated activity in a number of tumors including advanced melanoma and gliomas. trametinib was chosen for this patient based on his atypical braf fusion. we believe this is the first documented case of its successful use in neuroendocrine carcinoma in the pediatric population. conclusion: this case demonstrates the presence of braf fusion in a case of pediatric neuroendocrine carcinoma and significant response to single agent mek inhibition in this context. this cases raises the question as to whether the combination of a targeted inhibitor, in addition to either conventional chemotherapy or other braf inhibitors, might offer a better approach to therapy than current treatment options. albany medical center, albany, new york, united states background: warm autoimmune hemolytic anemia (waiha) is characterized by autoantibody, and occasional complement binding of protein antigens, on the surface of red blood cells at temperatures ≥ oc resulting in targeted destruction. we describe the case of a year old male with a history of evan's syndrome, poor immune response to vaccines and lymphoid hyperplasia, presenting with altered mental status and severe anemia, found to have a warm igg pan agglutinin with evidence of both intra and extravascular hemolysis. his course was complicated by respiratory failure requiring intubation, pulmonary emboli, enterococcus bacteremia and hypertension. he received multiple transfusions with only transient increases in hemoglobin. the aiha was refractory to multiple rounds of treatment with high dose steroids, ivig, rituximab, cyclophosphamide, bortezomib, plasma exchange and mycophenolate mofetil (mmf). objectives: given the refractory nature of our patient's aiha the decision was made to trial eculizumab, a monoclonal antibody targeting c complement, preventing its cleavage and activation, and shown to be effective in treatment of atypical hemolytic uremic syndrome and hemolysis due to an igm cold agglutinin. prior to eculizumab infusion, ch and sc b- assays were significantly elevated. design/method: the patient was given two doses of eculizimab days apart. results: his hemoglobin steadily rose independent of red cell transfusions with a corresponding decrease in reticulocyte count, ldh and ch levels. the patient has remained stable with a normal hemoglobin ( - g/dl) on maintenance steroids and mmf. although we cannot definitively conclude that eculizumab directly caused his recovery, the clinical course post-eculizumab suggests this may be an efficacious treatment for aiha. genetic testing showed monoallelic frameshift mutation of the nfkb gene and monoallelic missense mutation of the dock gene. given the role of nfkb in both immunodeficiency and autoimmunity, it is thought that the patient's phenotype is due to nfkb haploinsufficiency and he is currently considering hematopoietic stem cell transplant. st. joseph's regional medical center, paterson, new jersey, united states background: heterozygous -thalassemia typically manifests as thalassemia minor, characterized by mild microcytic hypochromic anemia with minimal clinical ramifications. coinheritance of -globin gene triplication has been reported to exacerbate the clinical and hematological phenotype ofthalassemia trait, due to increase in the alpha/non-alpha-chain imbalance. reported phenotypes range from asymptomatic thalassemia minor to moderate thalassemia intermedia, usually diagnosed in adulthood without transfusion dependence. this combination has been described in mediterranean, european and asian populations, but rarely reported in hispanics. objectives: to report two cases of unusually severethalassemia intermedia in hispanic patients with heterozygosity for triplicated -globin gene and a ( )-thalassemia allele. results: case : sixteen-month-old male of mexican descent presented with persistent microcytic anemia and jaundice. peripheral smear showed nucleated rbcs with basophilic stippling and target cells. hemoglobin electrophoresis revealed: hba- %, hbf- %, hba - . %. -globin gene testing revealed heterozygosity for ( ) mutation (ivsi-i, g→a). given the unusually severe anemia, -gene testing was performed which showed -globin gene(anti . ) triplication ( / ). at four years, he had splenomegaly and bilateral maxillary prominence. head ct showed irregular contour of the parieto-occipital region due to medullary expansion. due to significant persistent anemia ( - g/dl) and progressive bony deformities of the skull, patient began chronic transfusions at age eight after family declined splenectomy.case : fifteen-year-old female, of peruvian and honduran descent, presented for evaluation prior to cholecystectomy for gallstones and recurrent ruq pain. father had known thalassemia trait. her hb was . g/dl with hypochromia, microcytosis, and target cells. electrophoresis indicated -thalassemia trait (hba- %, hba - . %, hbf- . %), confirmed by gene testing (heterozygous for a ( ) mutation in codon c>t). given jaundice and gallstones, -globin gene analysis was ordered showing triplication ( / ). ruq pain resolved post-cholecystectomy, but she developed persistent painful splenomegaly. she began hydroxyurea to increase gamma-globin production and decrease excess alpha chains, but it was discontinued due to hematological toxicity. due to recurrent luq pain and progressive splenomegaly, she underwent laparoscopic splenectomy at age with resolution of symptoms and improved hemoglobin. conclusion: -globin gene testing should be considered in -thalassemia carriers with an atypical clinical presentation including hispanic patients. the wide variability in the phenotypic expression of (anti . ) mutation andthalassemia trait suggest interplay of other genetic factors which remain undefined. the clinically significant presentation amongst certain subjects, as in our two cases, makes it imperative to identify these factors to aid in phenotype prediction and genetic counseling. ashley bonheur, shivakumar subramaniyam, jogarao vedula, sucharita bhaumik nyu winthrop hospital, mineola, new york, united states background: wilms tumor (wt) is one of the most common solid malignant neoplasms in children. a diverse range of genes and mechanisms are implicated in wt pathogenesis. predisposing syndromes result from a disruption of wt gene, crucial for renal and gonadal embryogenesis. another gene is wt gene locus at p , an area of imprinting. the p tumor suppressor gene on chromosome p . is seen in patients with anaplastic histology. in addition to these genes, whole and partial chromosome gains of q, , q, , , & and losses of p, p, q, q, as well as loss of heterozygosity (loh) are commonly seen. some genetic markers appear to be predictive of outcome and are now incorporated into the assigning of risk-directed therapy. patients with loh at chromosome p and q are treated with more intensive chemotherapy, as they have been associated with increased risk of relapse and mortality. objectives: to describe a new complex translocation involving chromosome , , and in a case of pediatric wt. design/method: a four-year old female presented with abdominal pain and emesis. on exam, patient had a firm and large abdominal mass. radiologic studies revealed a complex lobulated right renal mass. right radical nephrectomy was performed. histopathologic studies showed wt with triphasic histologic features with blastema predominance, invasion of the lymphovascular and perinephric adipose tissues, perinephric lymph node involvement and no anaplasia. chest ct scan showed bilateral lung metastases. tumor cytogenetics showed an abnormal karyotype, a complex translocation of , , and . the rearrangement occurred due to translocation between chromosomal bands q and q , with an insertion of q - on the q region. pcr based genotyping using microsatellite markers additionally identified loh for chromosome p and q . the patient was treated for high risk stage iv wilms tumor with favorable histology and received intensive chemotherapy and radiation therapy to the flank and the lungs. she is now in remission months after, with no evidence of recurrence on surveillance scans. complex translocations associated with wt have not been rigorously studied. a question for further study is whether there is any relationship between recurrence potential with a complex translocation compared to common chromosomal abnormalities. further knowledge of the molecular pathology and genetic changes in wt will help the development of new targeted therapies, as well as new biomarkers to aid diagnosis, risk stratification, and monitoring of treatment and relapse. results: a week-old girl was referred for evaluation of an abnormal newborn screen. mother was a known carrier of hb khartoum trait while father was a known carrier of thalassemia trait. patient's hemoglobin quantification performed by capillary zone electrophoresis showed hbf %, hb variant %, and no detectable hba. the hb variant ran in the d zone, a pattern consistent with mother's hb. alkaline agarose gel electrophoresis banding pattern showed f/s. acid agarose gel electrophoresis pattern showed v/f. later testing revealed abnormal isopropanol stability with + precipitation at minutes. this electrophoresis pattern is consistent with the pattern previously reported of hb khartoum. clinically, the patient is a healthy, active child whom we have followed for two years. she has not had any significant anemia outside of her physiologic nadir. she has not had any hemolytic episodes, and her bilirubin levels have always been within the normal range conclusion: to the best of our knowledge, this is the only reported case of hb khartoum/ thalassemia. the proline to arginine substitution of hb khartoum introduces a charged group on the chain at the site of contact. the resulting unstable chains can dissociate into monomers and favor the formation of methemoglobin, leading to hemoglobin instability. we had wondered if this unstable hemoglobin might result in clinical hemolysis when challenged with oxidative stress, such as in periods of infection. however, in the two years we have followed this patient, she has never had a hemolytic episode. at two years of age, she has hbf . %, hb khartoum . %, and hba . %. whether hbf elevation is protective from oxidative stress remains to be determined as we continue to follow this child. university of puerto rico -medical science campus, san juan, puerto rico, united states background: gm gangliosidosis is a lysosomal disorder caused by -galactosidase deficiency due to mutations in the glb gene. it is a rare autosomal recessive neurodegenerative disorder with an incidence of about : , - : , live births worldwide. this neurological disorder has three clinical forms. gm type , or infantile form is characterized by psychomotor regression by the age of months, visceromegaly (hepatosplenomegaly), macular cherry red spot, facial and skeletal abnormalities, seizures, and profound intellectual disability. we present a -year-old female with gm type and acute lymphocytic leukemia (all). design/method: she was diagnosed with gm type at the st months of age and family history was remarkable for an older sister with gm type . diagnostic studies reveal homozygous exon of the glb gene for a sequence variant defined as c. c>t, predicted to an amino acid substitution p.aarg cs. results: patient presented to our hospital with petechiae in lower extremities, pallor and intermittent tracheal bleeding. physical examination shows a hemodynamically stable girl that is chronically ill dependent of mechanical ventilation, severe mental retardation and scatter petechiae at upper and lower extremities. laboratory workup revealed severe normocytic anemia (hgb: . g/dl) with immature peripheral cells and thrombocytopenia ( × /l). serum chemistry revealed increase ldh ( u/l), increase hepatic enzymes (ast: u/l), normal uric acid level. there was no evidence coagulopathy. chest x ray was unremarkable except for evidence of chronic pulmonary illness. abdominal sonogram hepatosplenomegaly. during hospitalization, bone marrow aspirate and biopsy was performed which was diagnostic of b cell acute lymphoblastic leukemia (all) with . % lymphoblast and orderly myeloid/erythroid maturation. flow cytometry: % b lymphoblast with aberrant phenotype c/w b-acute lymphoblastic leukemia. karyotype revealed hyperdiploid female of favorable prognosis. cytogenetic by fish: hyperdiploid all with extra copies of runx and igh (no bcr-abl translocation). family was oriented about the new diagnosis and the dismal prognosis in conjunction to her primary condition. parents agree on no chemotherapy treatment for all with only supportive treatment. to this date, there is no evidence in literature that has previously described association of gm and leukemia. life expectancy of patient's primary condition is null therefore, correlation with leukemia might not be a coincidental finding. this patient opens the possibility of malignancy as part of gm type thus, malignancy diagnosis should be considered as part of their medical lifetime course. university of south florida, tampa, florida, united states background: hematological manifestations related to hiv infection are not uncommon, with thrombocytopenia having an estimated prevalence of - %. the pathophysiology is likely multifactorial. studies suggest that the primary mechanism may be immunologic resulting in accelerated platelet destruction. additional theories suggest that infection of megakaryocytes may also play a role causing inadequate platelet production. treatment of hiv-related thrombocytopenia is challenging. first-line treatments include initiation and optimization of antiretroviral therapies, immunoglobulin (ivig), and glucocorticoids. however, this approach is not effective in all patients and second line treatment options are less well studied, particularly in the pediatric population. objectives: we aim to present and discuss the case of a year old patient with perinatally acquired hiv- infection and persistent thrombocytopenia who, after failing first line therapies, showed normalization of platelet count on the novel thrombopoietin receptor agonist, eltrombopag. design/method: a retrospective chart review of the case patient's medical record was conducted. additionally, a thorough literature review was performed on this topic including the pathophysiology of hiv related thrombocytopenia and its treatment modalities. the patient required monthly ivig infusions for about year, but did not show a sustained response, often with platelet count dropping to less than , in between infusions. after initiation of mg eltrombopag daily the patient showed a sustained increase in platelet count (range , - , ). during a brief week lapse in eltrombopag treatment his platelet count dropped to , . upon re-initiation of therapy his count increased to , . the patient has remained asymptomatic, off of ivig for over one year, with undetectable hiv viral load and greater than cd t cell counts. no side effects or grade laboratory abnormalities were reported. conclusion: treatment of hiv-related thrombocytopenia can be challenging. first line therapies, including ivig and glucocorticoids, are not effective in all patients. several other treatment modalities have been utilized, including anti-d immunoglobulin, dapsone, danazol, interferon alfa, vincristine, thrombopoetic growth factors including romiplostim and eltrombopag, or splenectomy, but these are less well studied. this represents the first reported case of a pediatric patient with hiv who showed a positive response to eltrombopag with a sustained improvement in platelet count and no adverse effects from treatment. eltrombopag may be a safe alternative to first line therapies in those patients with hiv and refractory thrombocytopenia, however additional studies are needed. university of illinois college of medicine at peoria, peoria, illinois, united states background: achromobacter xylosoxidans is a gram negative rod with peritrichous flagella which causes rare opportunistic infections most commonly encountered by immunocompromised patients. it is primarily associated with uncomplicated bacteremia, cather-associated infections, and pneumonia. most reports of bacteremia associated with a. xylosoxidans are nosocomial, associated with neoplasm, and occurring mainly in adults. most reported infections with a. xylosoxidans in children are associated with cystic fibrosis. there are very few reported cases of septic shock from a. xylosoxidans bacteremia and pneumonia in the pediatric oncology population. objectives: to describe a rare case of a. xylosoxidans septic shock in a pediatric patient with relapsed neuroblastoma results: a -year old boy with history of stage iv highrisk neuroblastoma underwent standard frontline therapy with chemotherapy, hematopoietic stem cell transplant, radiation therapy, and immunotherapy, followed by a dfmo trial for maintenance. his -month follow-up scans demonstrated relapse and he was subsequently treated with additional chemotherapy, surgical resection, and mibg therapy, crizotinib for an eml -alk fusion and finally ifosfamide, carboplatin and etoposide (ice). he developed neutropenic fevers and was started on cefepime, vancomycin and fluconazole. blood cultures were initially negative. on the th day of fever, his previously scheduled pet scan was performed during hospitalization and showed new pulmonary opacities. he did not have respiratory symptoms, but therapy was escalated to meropenem, vancomycin and amphotericin. emergent bronchoscopy was performed the same day, with all bacterial and fungal cultures remaining negative. overnight, he developed tachypnea and saturations in the upper s, requiring nasal cannula. ir-guided lung biopsy was performed the next day, a flexible bronchoscopy was done to remove blood clots in the airway, the patient was placed on a ventilator, femoral lines were placed, granulocytes ordered and pressors were started for deterioration to presumed septic shock. arterial and femoral lines were placed but patient continued to have hemodynamic instability on multiple pressors. the following day, blood and respiratory cultures returned positive for results: at days after the start of iti, the inhibitor was < . bu and continued undetectable months after initiation of iti therapy. in this patient, iti with high-dose plasma-derived factor viii and von willebrand factor (vwf) complex was well tolerated and effective. genetic analysis confirmed a large factor viii gene duplication of exons to . we believe our patient developed inhibitor so quickly ( exposure days) due to the possibility of this mutation causing a frameshift that introduces a premature termination codon. this might be functionally similar to a deletion in the factor viii gene which poses the highest risk for inhibitor development in patients with severe hemophilia a. this variant has only been identified previously in two unrelated patients diagnosed with severe hemophilia a. this duplication is not listed in dbsnp variant database, nor observed in the general population database. our case proves the effectiveness of this method for patients with severe hemophilia a and an inhibitor. it also shows that more research is needed to identify patients at risk for inhibitor development. background: mercaptopurine ( -mp) is a prodrug that is a core component of maintenance chemotherapy for patients with a diagnosis of acute lymphoblastic leukemia (all). suppression of the neutrophil count is used to demonstrate adequate dosing of -mp during this phase of therapy. bone marrow suppression is mediated by the active metabolite -thioguanine ( -tgn), whereas the metabolite -methylmercaptopurine nucleotides ( -mmpn) has been shown to cause hepatotoxicity. allopurinol has been used infrequently in all maintenance therapy in the setting of skewed metabolism when adequate myelosuppression is difficult to achieve due to excessive hepatic toxicity. when given in combination with allopurinol a reduced dose of -mp may result in both increased -tgn levels and decreased -mmpn levels. objectives: describe the characteristics and clinical course of patients treated with allopurinol and reduced dose -mp during maintenance chemotherapy for all. we performed a retrospective chart review of patients at aflac cancer and blood disorders center of children's healthcare of atlanta with new diagnoses of b or t-cell all who received allopurinol during maintenance chemotherapy. we identified eleven patients with b-cell or tcell all who received allopurinol adjunctive therapy during maintenance chemotherapy at a single institution between - . these patients received adjunctive allopurinol for - weeks (median weeks) with reduced -mp ( - % of full dose). all ten patients with genetic testing for thiopurine s-methyltransferase (tpmt) had wildtype genotype associated with normal enzyme levels. indications for allopurinol use were most commonly unfavorable -mp metabolite levels, transaminitis (n = ), pancreatitis (n = ) and hyperbilirubinemia (n = ). favorable metabolite shift was achieved in all patients. liver enzymes improved in of patients with transaminitis after initiation of allopurinol/reduced -mp. three patients who experienced pancreatitis during maintenance did not have recurrence after initiation of allopurinol ( of these patients previously reported). six patients developed pancytopenia while on allopurinol, and two of those patients developed pancytopenia severe enough to require allopurinol cessation. four patients developed isolated anemia (hgb < . g/dl) without thrombocytopenia or severe neutropenia. no patient has experienced a recurrence of leukemia. overall, treatment with allopurinol and reduced dose -mp was successful in producing a favorable -mp metabolite distribution and reducing toxicity. therapy was generally tolerated; however a major and notable side effect was pancytopenia, in two cases severe enough to stop allopurinol treatment. anemia may be more prominent with allopurinol usage. allopurinol effect is variable among individual patients despite normal tpmt genotypes. baylor college of medicine, houston, texas, united states background: congenital sideroblastic anemia, b-cell immunodeficiency, periodic fevers and developmental delay syndrome (sifd) is a rare inherited sideroblastic anemia syndrome, first described in with clinically similar cases. genetic variations of trnt were identified as causative. objectives: to present an unusual presentation of a patient with sifd complicated by diagnosis of concomitant alpha thalassemia trait. design/method: retrospective chart review. a five month old male infant was referred to our hematology center for evaluation of elevated hemoglobin barts identified on newborn screen. despite numerous attempts, blood work was unable to be collected. at seven months of age he had microcytic anemia (hemoglobin . g/dl, mean corpuscular volume fl) more severe than what would be expected with alpha thalassemia trait. no variant hemoglobin was identified with isoelectric focusing or high performance liquid chromatography. by nine months of age he developed growth failure, intermittent emesis with fevers, developmental delays (predominantly gross motor), hearing loss, a disproportionally large head and coarse, thinning hair. over the next ten months, he was seen by numerous specialists for seemingly unconnected problems including sensorineural hearing loss, elevated liver enzymes and growth hormone deficiency. alpha globin analysis revealed deletion of two alpha globin genes. at months of age, he was admitted with one week of fevers, jaundice, and emesis. peripheral blood smear showed microcytic hypochromic anemia with marked anisopoikilocytosis including target cells, elliptocytes, tear drops, spherocytes, poikilocytes, marked polychromasia, and coarse basophilic stippling. given the inconsistency of his laboratory findings with the diagnosis of alpha thalassemia trait and clinical syndromic findings, bone marrow biopsy was performed which revealed rare ringed sideroblasts. one month later whole exome sequencing revealed trnt splicing variant c. - c>g and novel missense variant c. a>t consistent with sifd. hemoglobin barts on newborn screen with moderate to severe microcytic anemia directed initial diagnostic work-up towards variant alpha thalassemia. as additional medical conditions developed the focus shifted to a unifying syndrome. compared to previously described cases, our patient was diagnosed at an older age, presented with anemia rather than episodes of febrile illnesses, and had rare sideroblasts on bone marrow examination. diagnosis in this case led to identification of the novel c. a>t variant in his sister who had similar, but milder, features. sifd is a rare disease with variable phenotypic severity making diagnosis challenging without high index of suspicion which is crucial for appropriate management. wiseman, blood, . chakraborty, blood, background: cholelithiasis is uncommon in childhood. cholelithiasis is known to occur more frequently in children with predispositions, including female sex, obesity, parenteral nutrition, previous abdominal surgery, use of oral contraceptives, family history of gallstones, chronic hemolytic anemias, hepatobiliary disease, or exposure to specific drugs. although there have been occasional case reports linking cholelithiasis to childhood leukemia or leukemia therapy, the prevalence and risk factors of cholelithiasis in patients with childhood leukemia remain unclear. objectives: to estimate the prevalence of cholelithiasis in patients diagnosed with childhood acute lymphoblastic leukemia (all), and to evaluate possible risk factors for the development of cholelithiasis in patients with childhood all. we performed a computer-assisted review of the electronic medical records of patients diagnosed for b or t-cell all at children's healthcare of atlanta in the period from to . patients with diagnoses of cholelithiasis, cholecystitis or who had a cholecystectomy were identified. possible risk factors of age, sex, bmi, history of abdominal surgery and parenteral nutrition use were abstracted. patients with underlying chronic hemolytic anemia or pre-existing gallbladder disease were excluded. results: seventeen cases of cholelithiasis and cases of cholecystitis without documented cholelithiasis were identified. among patients with cholelithiasis, were female. median age at diagnosis of cholelithiasis was . (range . - . ) years. seven patients had no symptoms referable to cholelithiasis at the time of diagnosis. the median age of leukemia diagnosis among these patients was . (range . - . ) years. the median interval from diagnosis of leukemia to gallbladder disease was . years. four patients had bmi over the th percentile for age. two patients had a prior history of intraabdominal surgery. no patient received oral contraceptive pills. six patients received parenteral nutrition for more than days. there was no documented family history of cholelithiasis. seven patients did not receive any cholelithiasis directed therapy. two patients were managed with medical management only, with endoscopic retrograde cholangiopancreatogram with stone extraction, and with cholecystectomy. our study estimates the prevalence of cholelithiasis in childhood lymphoblastic leukemia to be . %, higher than the reported prevalence in the general pediatric population of . - . %. although our cohort size is small, it appears that all therapy and supportive care modalities associated with all are likely to play a larger role in the development of cholelithiasis than known predisposing factors in the general population. further studies are warranted. background: an uncommon side effect of intravenous immunoglobulin (ivig) administration is clinically apparent, sometimes severe hemolysis. we describe a severe case of coombs-positive hemolytic anemia secondary to ivig administration. ivig is a blood derivative manufactured from pools of , to , individual plasma donations. ivig is not abo-type restricted, so anti-a, anti-b and anti-a,b isoagglutinins are detectable. objectives: to describe a rare but serious type of transfusion reaction leading to gross hemolysis after ivig administration. results: a -year-old male with a past medical history of obstructive sleep apnea and obesity was admitted to the pediatric intensive care unit for adenoviral pneumonia and subsequent respiratory failure requiring mechanical ventilation. he had a complex hospital course with many complications including acute respiratory distress syndrome (ards), septic-shock, and coombs-positive hemolytic anemia. the patient was treated with commercial ivig (baxter/baxalta) -mg/kg daily for five days. he had two isolated episodes of severe hemolysis in relation to ivig administration requiring multiple transfusions of packed red blood cells (prbc). examination of pre-transfusion peripheral blood smear showed spherocytosis with rouleaux formation and large clumped rbc aggregates. the patient's blood type was classified as blood group a, rh-negative and his initial prbc transfusions were of this type. subsequently, the patient's coombs test was found to be positive using polyspecific and anti-igg typing sera. the patient's antibody screen against reagent group o screening cells was negative ruling out autoimmune hemolytic anemia. however, type specific anti-a antibodies were detected in his plasma as well as the acid eludate prepared from the coombs-positive red blood cells. it was concluded that the patient's hemolysis was due to anti-a antibodies presumed to arise from ivig. the patient's rbc transfusions were changed to o-negative blood and the hemolytic process resolved. the patient ultimately died due to complications of ards. although hemolysis is a known side effect of ivig, it is rarely considered when deciding to administer ivig. in addition, it has rarely been described in the pediatric population. ivig is used in the treatment of a growing number of medical conditions. due to the critical nature of many of these patients, hemolysis secondary to ivig may not be considered and continued blood transfusions with the patient's specific blood type may be used. it is crucial to remember that severe hemolysis can occur from ivig, and the importance of transfusing with blood group o, rh-negative blood when applicable. university of maryland medical center, children's hospital, baltimore, maryland, united states background: coagulopathy is a well-described complication of acute promyelocytic leukemia (apml), and remains a leading cause in induction failure. with treatment, coagulopathy associated with apml has been shown to rapidly improve. multiple organ dysfunction syndrome (mods) in apml, including acute respiratory distress syndrome (ards), has been associated with infection, traumatic injury, malignant infiltration, and cytokine release syndrome. when mechanical ventilation is no longer sufficient, extracorporeal membrane oxygenation (ecmo) can be considered; however, coagulopathy, severe end-organ damage, and malignancy are all relative contraindications to initiation of treatment. we report the case of a -year-old female presenting in respiratory failure, disseminated intravascular coagulopathy (dic), with intracranial hemorrhage, and mods, diagnosed with apml, successfully treated with ecmo therapy. design/method: retrospective case analysis and literature review. our patient, a -year-old female was admitted in respiratory failure and altered mental status, following a fall shortly prior to presentation. initial laboratory values were notable for pancytopenia, dic, and acute renal failure. a non-contrast head ct showed left temporal lobe intraparenchymal hemorrhage. she was diagnosed with apml by peripheral smear, later confirmed by fish for t( : ), and was started immediately on high-risk induction chemotherapy as per cog protocol aaml , including all-trans retinoic acid, arsenic trioxide, idarubicin, and dexamethasone. cvvhd was required for acute renal failure. despite maximal respiratory support, she remained hypoxemic, with oxygenation index of , pao /fio ratio of . ecmo was initiated hours after start of induction, hours after admission. coagulopathy resolved on day of induction, ecmo was discontinued after days, mechanical ventilation and cvvhd were stopped after days and she continued to improve, eventually achieving remission with few neurologic side effects. despite relative contraindications to ecmo, this patient was successfully treated with ecmo without significant neurologic side effects. the correction of her coagulopathy was multifactorial: ) restoration of adequate oxygen delivery via ecmo improving endothelial function; ) successful organ support to allow sufficient response to induction chemotherapy with atra leading to the terminal differentiation of leukemic blasts; ) complement and contact system activation through contact with ecmo circuitry. this case illustrates that ecmo can still be considered in patients despite coagulopathy and end organ damage. sinai hospital of baltimore, baltimore, maryland, united states background: primary polycythemia vera is an extremely rare diagnosis in the pediatric patient and is defined by a marked elevation of red blood cells due to erythropoietin-independent mechanisms. presentations of this disorder range from the asymptomatic person to severe thrombotic events, such as budd-chiari syndrome or cerebrovascular stroke. mutations in the jak gene are found in adult and pediatric patients with polycythemia vera; however, the jak v f mutation is less commonly identified in pediatric patients. we describe an otherwise healthy -year-old female who presented with a significantly elevated total erythrocyte count, hemoglobin, and platelets, incidentally discovered upon routine annual blood work obtained by her pediatrician. design/method: this is a report and discussion of a rare case. demonstrated cellular marrow with trilineage hematopoiesis and no dysplasia. cytogenetics were not assessed. his hemoglobin and platelet count recovered but leukopenia and neutropenia persisted. follow-up evaluation at three months revealed fevers, ongoing cytopenias, a one-month of a nodular skin rash on the trunk and extremities resembling erythema nodosum, and hepatitis (peak alt and ast of , and , , respectively). following clinical evaluation, a skin biopsy was performed and was remarkable for atypical lymphocytes within the subcutis with t-cell markers, a high ki- , and positive tia- , perforin, and -f immunoperoxidase stains. negative stains for cd , cd , and ebv were noted. these results are consistent with sptcl. additional evaluation did not support a diagnosis of hlh. a staging evaluation was performed. pet-ct showed widespread hypermetabolic subcutaneous activity in the legs, trunk and skull and diffuse marrow hyperplasia. bone marrow demonstrated involvement with precursor b-cell acute lymphoblastic leukemia, with a mll gene rearranagement. his skin biopsy was retrospectively stained with tdt, cd , pax- , cd a, and cd with negative results, and a blood smear taken at the time of the skin biopsy did not demonstrate leukemic cells. conclusion: this is the first report of a patient with sptcl having a synchronous malignancy. the patient is doing well, currently in the maintenance phase of treatment for his all, and his skin disease has resolved on pet-ct. while it is possible that his presentation was a function of chance, the possibility of an underlying immune dysfunction or cancer predisposition warrants further investigation. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hereditary xerocytosis (hx) is a rare red blood cell (rbc) dehydration disorder, characterized by variable hemolysis and propensity to iron overload. hx is often misdiagnosed as hereditary spherocytosis (hs). while splenectomy is curative for hs, it is relatively contraindicated in hx due to a substantial thromboembolism risk, signifying the importance of delineating these diseases. blood smear abnormalities are variable and often insufficient to make an accurate diagnosis. osmotic-gradient ektacytometry and genetic confirmation are critical in distinguishing these overlapping disorders. objectives: describe a family with hx, initially misdiagnosed as hs. discuss the importance of distinguishing these disorders and the utility of ektacytometry in making this distinction. design/method: a -year-old caucasian male was diagnosed with hs after presenting with prolonged neonatal jaundice starting on the first day of life. he described mild scleral icterus and history of intermittent jaundice and dark urine, without need for transfusions. his father, paternal uncle and paternal grandmother were all diagnosed with hs during childhood and underwent cholecystectomy. additionally, his father underwent splenectomy for abdominal pain. the child's blood counts revealed compensated anemia (hb . gm/dl) and reticulocytosis (arc × /mcl) with increased mcv ( . fl) and mchc ( . gm/dl). blood smear showed increased polychromasia and poikilocytosis with rare spherocytes and few stomatocytes. while the child had normal ferritin, his father had iron overload (ferritin ng/ml) despite no prior transfusions. osmotic-gradient ektacytometry profile of the child and father's rbcs showed a characteristic left-shifted, bell-shaped curve with decreased omin and ohyp, diagnostic of hx. the family is currently undergoing genetic studies. despite clinical similarities between hs and hx, distinguishing these diseases has significant management implications. hx is a disorder of rbc permeability, causing shortened rbc survival. stomatocytes on blood smear can raise suspicion for hx, but are insufficient to make an accurate diagnosis. identifying characteristic biomechanical membrane properties using osmotic-gradient ektacytometry is the gold standard for clinical diagnosis, which can then be confirmed by molecular studies. hs and hx can be easily and reliably distinguished using ektacytometry, as both disorders have very distinctive curves representing different rbc deformability patterns. after hx diagnosis was made, we counseled the family against splenectomy, as the risk of thromboembolism is significantly increased in hx compared to hs, and the father was diagnosed with iron overload. conclusion: hx is commonly misdiagnosed as hs. this case highlights the importance of making this distinction, and the utility of osmotic-gradient ektacytometry in reliably distinguishing these conditions. penn state health children's hospital, hershey, pennsylvania, united states background: relapsed acute myeloid leukemia (aml) presenting as an isolated central nervous system myeloid sarcoma (cns ms) is very rare and its treatment is not well-defined. thiotepa, vinorelbine, topotecan and clofarabine (tvtc) has been successful for re-induction therapy to induce remission prior to hematopoietic stem cell transplant (hsct). objectives: to describe our experience in utilizing tvtc therapy in two children with no extramedullary disease at initial diagnosis who presented with relapsed aml as intracranial myeloid sarcomas. results: case : month-old female was diagnosed with flt negative aml and completed treatment per the children's oncology group (cog) aaml study on the low risk arm without bortezomib. cerebral spinal fluid (csf) negative at diagnosis. fish testing positive for tcf gene deletion of unknown significance. mrd was undetectable after induction i and remained undetectable after each cycle. nine months off therapy, recurrent headaches prompted mri imaging which revealed two posterior fossa masses. csf and bone marrow testing were negative. stereotactic biopsy of the larger mass confirmed recurrence of aml. patient underwent two cycles of tvtc with a total of seven doses of intrathecal cytarabine with almost near resolution of the cns ms. completed cranial radiation and proceeded to allogeneic stem cell transplant with unrelated cord marrow donor and is disease free at approximately day + .case : year-old female diagnosed with flt and mll negative aml and completed treatment per cog aaml study on the low risk arm without bortezomib. csf negative at diagnosis. mrd was undetectable after induction i and completed therapy without complications. two months off therapy, a retrospective analysis of her diagnostic bone marrow by the cytogenetic laboratory to test a new panel identifying novel q partners revealed a cryptic insertional : (mllt /mll(kmt a) translocation. at four months off therapy, acute mental status changes prompted mri imaging which revealed two intracranial ms and lumbar spine involvement. resection of the larger lesion for symptomatic relief confirmed the mllt /mll(kmt a) fusion. csf positive for blasts and marrow negative for relapsed disease. patient completed two cycles of tvtc with a total of seven doses of it cytarabine with near resolution of cns disease (only mm contrast enhancement in the medulla). she received craniospinal radiation and is awaiting improvement in her cardiac function before proceeding to hsct. conclusion: tvtc is a successful reinduction regimen for relapsed aml with cns ms prior to hsct. background: acute severe anemia can be a life-threatening medical condition. the differential is quite broad for possible etiologies of acute severe anemia, including autoimmune hemolytic anemia (aiha) and atypical hemolytic uremic syndrome (ahus). autoimmune hemolytic anemia is an antibody-mediated process that targets the protein antigens located on the surface of red blood cells. treatment options for aiha include corticosteroids, with up to % of patients being responsive, with some requiring splenectomy. atypical hemolytic uremic syndrome is a medical urgency, defined as the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. the etiology is usually due to genetic causes, or less commonly, due to autoantibodies or idiopathic reasons. prognosis is very poor. objectives: differentiating between autoimmune hemolytic anemia and atypical hemolytic uremic syndrome can be a time-sensitive diagnostic dilemma while the patient is in critical condition, but this important delineation can vastly alter therapeutic options. design/method: here we discuss two cases highlighting the diagnostic workup involved in differentiating between atypical hemolytic uremic syndrome and autoimmune hemolytic anemia. patient a is a -year-old male who presented in extremis with severe anemia, uremic encephalopathy, and severe acute renal injury requiring hemodialysis and multiple blood transfusions. patient b is a -month-old male, who also presented in extremis with respiratory failure secondary to adenovirus/rhinovirus/enterovirus, with acute progressive renal failure and microangiopathic hemolytic anemia, requiring hemodialysis and cardiorespiratory support. : patient a underwent a full hematologic and infectious disease workup. subsequent laboratory studies confirmed enteropathogenic e.coli (epec) in the patient's stool; blood cultures remained negative. renal biopsy results were consistent pigment nephropathy. bloodwork indicated positive direct coombs. patient a was ultimately treated with steroids mg/kg/day, with significant improvement. patient b also included a full hematologic work-up, including adamts activity and ahus genetic panel, as well as full infectious disease work-up. subsequent laboratory test-ing revealed blood cultures growing streptococcus pneumoniae, with adamts activity at % (adult ref range: >/ = %), and normal complement levels. imaging findings also supported diagnosis of ahus. the management of a critically ill patient with acute severe anemia requires a thorough hematologic and infectious disease work-up. while molecular and genetic are helpful in definitive diagnosis of ahus, the utility of such results is limited by time. overlapping clinical presentation of a patient in extremis due to acute severe hemolytic anemia with progressive renal failure presents a rather broad differential, with time-sensitive treatment and prognostic implications. the favorable response to steroids delineates aiha from hus. background: d- -hydroxyglutaric aciduria (d- -hga) is a rare metabolic disorder characterized by developmental delay, hypotonia, and bi-allelic mutations in d- hydroxyglutarate dehydrogenase (d hgdh) or isocitrate dehydrogenase (idh ). metaphyseal chondromatosis with d- -hydroxyglutaric aciduria (mc-hga) is a type of d- -hga that has been previously reported in seven patients (omim ; pmid ), three of whom had somatic mosaicism for r variants in isocitrate dehydrogenase (idh ). we describe a -year-old boy with mc-hga who subsequently developed acute myeloid leukemia (aml) and was found to have a r variant in idh in a leukemic bone marrow sample. we report the first case of aml with this metabolic disorder. design/method: a -year-old hispanic boy presented with short stature, developmental delay, abnormal skin pigmentation, and unilateral congenital cataract. workup revealed multiple skeletal enchondromatosis and elevated urine d- -hydroxyglutaric acid levels. he was diagnosed with mc-hga. no pathogenic variants in d hgdh, idh and idh were identified in peripheral blood. germline testing with biopsies of skin lesions was declined by the family. two years later, he presented with streptococcal sepsis and pancytopenia. blasts were noted on peripheral smear. bone marrow morphology was consistent with acute myelomonocytic leukemia (∼ % blasts). chromosome analysis showed normal xy, and molecular testing by pyrosequencing idh and idh revealed a r c variant in idh ( % mosaicism). the patient is being treated as per the cog study aaml . end of induction i bone marrow aspirate was hemodiluted, but there was no obvious residual disease by flow cytometry ( . - . % sensitivity) or morphology. the previously identified idh variant was no longer detectable (limit of detection < %). although targeted therapy for aml with idh mutation is currently in phase i clinical trials in adults, there is no safety or efficacy data for using idh inhibitors in children. treatment with ivosidenib is therefore not currently an option for our patient. conclusion: this is the first case of aml reported with this rare metabolic disorder. somatic r variants in idh have been identified in three other mc-hga cases. this same mutation leads to the accumulation of d- -hydroxyglutarate in gliomas and aml. without any confirmed germline mutation or somatic mosaicism testing of multiple specimen sources, we can only speculate that the patient has an underlying somatic idh mutation associated with mc-hga which subsequently led to leukemogenesis. we present the first case of this association, to increase index of suspicion for development of aml in children with metabolic disorders associated with variants in idh . background: congenital combined deficiency of the vitamin k-dependent coagulating factors (vkcfd) is a rare heterogeneous autosomal recessive bleeding disorder. vkcfd is caused by mutations in the genes of either gamma-glutamyl carboxylase (ggcx) or vitamin k epoxide reductase complex (vkorc), which are responsible for the gammacarboxylation of vitamin k dependent proteins (vkdps) allowing for their activation. the clinical presentation ranges from no bleeding to intracranial hemorrhage. to date, vkcfd has been reported in few patients worldwide. objectives: we report a case of a girl with novel homozygous mutation of the ggcx gene, highlighting her clinical and biochemical characteristics with a review of the literature. a -month-old girl of consanguineous emirati parents, presented to our hospital with a history of bleeding from puncture site after receiving her second-month vaccine. that was associated with episodes of mild mucosal bleeding. review of systems was negative for jaundice, steatorrhea and failure to thrive and physical exam was unremarkable. investigations revealed markedly prolonged pt and aptt with high inr. fibrinogen, hemoglobin and platelets were always normal. activities of vitamin k-dependent factors including fii, fvii, fix, fx, protein c and s were all low. a measurement of proteins induced by vitamin k absence (pivka-ii) was done and came very high. this was associated with a mild elevation in liver enzymes but normal liver function test. the picture was supporting vitamin k deficiency, and as a result, she was started on oral vitamin k supplements of mg/day. she responded partially to vitamin k and required higher doses to stabilize her inr. after excluding acquired causes and due to her requirement of high doses of vitamin k, a mutation in either ggcx or vkorc genes was suspected. genetic analysis was conducted for her which revealed a novel missense homozygous mutation in the ggcx gene (c. a>t) confirming the diagnosis of combined deficiency of vitamin k-dependent clotting factors type . the asymptomatic parents were both heterozygous for the same mutation. results: she is currently stable on mg/day of vitamin k supplements. conclusion: vkcfd is a rare bleeding disorder with an overall good prognosis due to the availability of several effective therapeutic options. the function of the mutated gene is unknown. our patient demonstrated a partial response to vitamin k supplements suggesting presence of a residual carboxylation capacity and a possible role of this gene in the enzymesubstrate interactions. university of alabama at birmingham, birmingham, alabama, united states s of s background: gata is a zinc finger transcription factor that plays a critical role in the regulation of hematopoiesis and lymphatic angiogenesis. mutations leading to gata deficiency (gd) have been linked to a variety of clinical conditions. patients with gd have a striking predisposition to develop myelodysplastic syndrome (mds), acute myeloid leukemia (aml), or chronic myelomonocytic leukemia (cmml). acute lymphoblastic leukemia (all) has not been associated with gd, although the association of bcell all and gd has been previously reported. objectives: to describe a unique association of gata deficiency and t-cell all in a young child. results: an -year-old female presented with a one-week history of fever and malaise. she had a significant past medical history of verruca plantaris and self-resolving leukopenia associated with febrile illnesses. significant family history included sister with neutropenia and human papilloma virus (hpv) infection, and mother with neutropenia, monocytopenia, atypical mycobacterial infections, and hpv infection. peripheral blood revealed hemoglobin . g/dl, hematocrit . %, platelets , /ul, and white blood cell , /ul (neutrophils /ul, lymphocytes /ul, monocytes /ul). patient underwent a bone marrow biopsy demonstrating lymphoblast infiltration. flow cytometry analysis demonstrated monoclonal lymphoid blast population that co-expressed cd , cd , cd , nuclear tdt, cd , however, lacked expression of cd , cd , cd , cd , hla-dr, or myeloperoxidase. findings were consistent with tcell all with aberrant myeloid markers. cytogenetics analysis revealed ,xx,dic( ; )(p . ;p . ). patient began treatment as per children's oncology group aall and achieved remission at the end of induction. course of therapy was complicated by episodes of fever, reciprocating junctional tachycardia, asparaginase-associated thrombosis, viral meningitis, recurrent episodes of verruca plantaris, and resistant streptococcus pneumoniae or haemophilus parainfluenza infections causing chronic cough. later, she was also found to have low igm levels; after completion of therapy, she developed monocytopenia. lymphocyte subset panel revealed absent b cells, decreased number of natural killer (nk) cells, and cd /cd inversion. further work-up included gata sequence analysis that showed heterozygous nonsense mutation (c. c > t/c; reference nm_ ) likely resulting in gata haploinsufficiency. patient continues to be in remission, is receiving monthly immunoglobulin replacement and is on azithromycin for atypical mycobacterial prophylaxis. surveillance bone marrow biopsies have shown no evidence of mds or leukemia, however, have demonstrated persistent hypocellularity. the possibility of undergoing an allogeneic bone marrow transplant is actively being discussed given its curative potential. clinicians should be aware that t-cell all may be associated with gata deficiency. cincinnati children's hospital medical center, cincinnati, ohio, united states background: treatment for severe hemophilia a is centered on factor viii (fviii) replacement therapy. development of an alloantibody (inhibitor) against fviii is a significant treatment complication occurring in as many as - % of patients. high titer inhibitors render treatment with factor viii ineffective, necessitating the use of bypass agents that may not achieve hemostasis with the same efficacy. considering the substantial ramifications of inhibitor development on treatment, eradication of inhibitors is of great importance to achieve adequate hemostasis in this patient population. desensitization by immune tolerance induction (iti) is the primary method of inhibitor elimination. however, not all patients respond to iti. immunomodulation may be considered as the next line of therapy, although controversy remains in regards to agent selection and use. objectives: there is incomplete data on the use of immunomodulation therapy for inhibitor eradication in severe hemophilia a. we present a case of a pediatric patient with severe hemophilia a and high titer inhibitor who failed initial iti therapy to better illustrate potential treatment options for the future. design/method: a retrospective chart review was performed on a patient with severe hemophilia a at cincinnati children's hospital medical center. results: an -year-old caucasian male with severe hemophilia a secondary to intron inversion, was initially diagnosed following extensive bleeding after circumcision at birth. he was identified as having an inhibitor ( bethesda units (bu)) at months of age after exposure days of treatment. he failed multiple attempts of iti, with recombinant and plasma-derived (pd) fviii. he was advanced to immunomodulation therapy in combination with pdfviii, however demonstrated anaphylaxis to rituximab and ofatumumab. he underwent tolerization to rituximab, and received a six month course with a partial response (nadir of . bu). months following last dose of rituximab, a rising inhibitor titer ( . bu) was found. mycophenolate mofetil (mmf) was initiated with subsequent inhibitor stabilization and a decreasing titer ( . bu) over the course of the following year. mmf has been well tolerated without major side effects or infection throughout therapy. conclusion: development of an inhibitor against fviii is a considerable complication in patients with severe hemophilia a. use of immunomodulatory therapies following iti failure remains controversial. mmf has not been well studied in this patient population. we report a case of a patient who is being successfully treated with mmf with minimal side effects. further prospective studies should be considered to further define the role of mmf immunomodulation therapy. background: down syndrome (ds) children with aml (ds aml) have higher cure rates than their non-ds counterparts. outcomes for refractory/relapsed cases, however, remain dismal. somatic mutations of the gene encoding the transcription factor gata in ds aml patients are responsible for the observed hypersensitivity of ds aml blasts to cytosine arabinoside (ara-c). in view of excellent survival rates (approaching %) of ds aml patients, the ongoing children's oncology group (cog) aaml study seeks to determine the feasibility of treating standard risk (minimal residual disease/mrd negative) ds aml patients using a reduced dose ( -fold decrease) ara-c backbone. although results from japanese trials with this approach are promising, north american and european data are conflicting. although chromosome rearrangements in ds aml do not appear to carry the same adverse prognostic significance as in non-ds aml, monosomy in ds aml patients has been associated with a moderately worse outcome. isochromosome q, however, is rare and has only been reported in previous cases of ds aml. objectives: to report our institutional experience of very early relapse involving cases of ds aml patients treated per the reduced dose ara-c arm ( . g/m ) of the aaml study. design/method: we hereby report the disease course and cytogenetics of the above ds aml patients. : patient is a month old caucasian female who had gata mutation negative aml. patient is a -year old caucasian male whose chromosomal analysis revealed isochromosome q ( copies of the long arm of chromosome ). both patients achieved negative mrd (< . %) after induction i chemotherapy with thioguanine, low-dose ara-c and daunorubicin and proceeded per the reduced dose ara-c arm of aaml . patient relapsed immediately after completion of chemotherapy. salvage chemotherapy with mitoxantrone/high dose ara-c (hidac) failed to induce a second remission and the patient subsequently died of disease. patient relapsed within months from end of therapy. the patient underwent salvage chemotherapy utilizing a hidac backbone and remains in disease remission. the noted very early relapse following a reduced dose ara-c regimen in our above ds aml children suggests that testing for gata mutation and chromosome rearrangements may play a useful role in the development of future risk-stratified treatment strategies for ds aml. university of rochester, rochester, new york, united states background: in developed countries in the st century, severe nutritional deficiency is not an often considered differential diagnosis of unexplained childhood anemia. aside from iron deficiency anemia, vitamin deficiency severe enough to impact hematopoiesis is uncommon in the general pediatric population. here we present the unique case of a -monthold infant who presented with intermittent emesis, failure to thrive (ftt), developmental delay, macrocytic anemia, and neutropenia which was initially concerning for a congenital bone marrow failure syndrome. instead, she was discovered to have an underlying, potentially familial deficiency of b . objectives: . to describe the unique case of an infant with b deficiency. . to outline the importance of including b deficiency in the differential diagnosis of unexplained megaloblastic anemia in children. a -month-old exclusively breastfed infant presented for gastroenterology evaluation due to persistent emesis and poor weight gain over the course of months. her history was notable for delayed developmental s of s milestones and hypoactivity. marked pallor prompted hematologic evaluation, which revealed concern for macrocytic anemia (hemoglobin . g/dl, mcv ), reticulocytopenia ( . × ^ / l), and neutropenia (anc . × ^ /l). an otherwise reassuring physical examination and laboratory evaluation was notable only for the discovery of an undetectable b level and marked hyperhomocysteinemia ( mol/l). her hemoglobin (hgb) continued to decline (to . g/dl) over the first few days after presentation, and she required red blood cell (rbc) transfusion. within only a few days of initiation, daily cyanocobalamin injections resulted in a robust reticulocytosis response, improved hgb, immediate normalization in the neutrophil count, and resolution of hyperhomocysteinemia. additional history and laboratory evaluation from the patient's mother revealed a concurrent, asymptomatic maternal b deficiency as well as a history of a need for b supplementation in the maternal grandfather, raising concern for an inherited etiology. despite the rarity of vitamin-deficient hematologic abnormalities in the general pediatric population, b deficiency should be considered as a potential cause of an otherwise unexplained megaloblastic anemia, especially in the setting of concurrent ftt and neurodevelopmental delay. a detailed family history should be obtained in such cases and may have helped to prevent this patient's clinical sequelae had the deficiency been discovered sooner. our patient has experienced a favorable clinical response to b supplementation, attesting to the importance of vitamin b in early childhood growth and development. background: peg-asparaginase is universally utilized in the treatment of pediatric acute lymphoblastic leukemia (all). despite its high efficacy in this disease, it is associated with hypersensitivity and allergy in - % of patients. protracted anaphylaxis has been described in circumstances such as severe food allergy with ongoing allergen exposure; however, it has not yet been described in relation to peg-asparaginase. we describe the first reported case of protracted anaphylaxis after peg-asparaginase administration, provide guidance as to time course and management of protracted anaphylaxis, as well as evidence that erwinia asparaginase may be safely administered even in this high risk population. objectives: to provide guidance regarding the duration, course and management of protracted, severe anaphylaxis after peg-asparaginase therapy. a year old male with very high risk all presented for consolidation therapy with peg-asparaginase (intramuscular) and vincristine. one hour after administration, he developed generalized hives and angioedema, for which he was given diphenhydramine. he then quickly developed progressive hives, angioedema, subjective throat and chest tightness, and wheezing. he was treated with diphenhydramine, epinephrine, albuterol, and methylprednisolone with resolution of symptoms. one hour later, symptoms recurred and the patient became hypotensive; he was retreated with methylprednisolone and epinephrine, and was transferred to the pediatric intensive care unit (picu). in the picu, he was placed on an epinephrine drip, and continued on methylprednisolone, diphenhydramine, cetirizine, albuterol, and ranitidine. the epinephrine drip was successfully discontinued after hours, and his other medications were gradually weaned over the course of two weeks. of note, the patient did have st segment changes in his electrocardiogram during the first hours of anaphylaxis. these were associated with normal ventricular function as per echocardiogram, and resolved within one week. this patient has subsequently tolerated multiple doses of erwinia asparaginase (intramuscular) without premedication. this patient was acutely managed in the pediatric intensive care unit with steroids, anti-histamines, and continuous infusion epinephrine. symptoms consistent with severe anaphylaxis including hives, angioedema, throat and chest tightness, wheezing, and hypotension persisted for a total of four days before finally resolving. he has thus far tolerated multiple doses of erwinia asparaginase without any symptoms of allergy, hypersensitivity, or anaphylaxis. protracted severe anaphylaxis after peg-asparaginase therapy can be successfully managed with multi-agent therapy, including antihistamines, steroids, and continuous infusion epinephrine. re-challenge with an alternate form of asparaginase may be tolerated, even in a patient with protracted anaphylaxis to peg-asparaginase. ucsf benioff children's hospital oakland, oakland, california, united states background: vincristine (vcr) is widely used in pediatric cancers. unlike most cytotoxic agents, hematopoietic toxicity is uncommon. vcr-induced anemia has been observed but its mechanism has not been well studied. vinca alkaloid-induced membrane changes were seen in early studies of hereditary spherocytosis (hs) and anecdotal cases suggest vcr may increase hemolysis in such patients. here we describe a case involving severe vcr-induced anemia in a patient with hs and an explanation as to the mechanism. objectives: to describe the mechanism of vcr-induced anemia in hs. design/method: case report. a year-old female with hs was diagnosed with t-lymphoblastic lymphoma. she had required packed red blood cell (prbc) transfusions as a neonate and thereafter had done well without episodes of acute hemolysis or aplasia. complete blood counts (cbc's) demonstrated a compensated hemolysis, and she did not require further transfusions until she commenced chemotherapy. by the start of maintenance she had received many more prbc transfusions than the average patient. intermittent drops in hemoglobin (hb) did not correlate with any particular agent, and she had stable, mild splenomegaly. a clear pattern emerged during maintenance. her hb was - g/dl at monthly clinic visits, when she received vcr, intermittent intrathecal methotrexate, and corticosteroids. within - days, her hb dropped to . ± . g/dl, and reticulocyte count decreased from . to . ± . %. transfusion at day corrected hb, and the reticulocytes and hb returned to baseline. white blood cell and platelet counts did not change after vcr. blood samples from pre, immediately post, and days post vcr were analyzed and rbc characteristics and markers of hemolysis were not significantly different. ektacytometry showed identical curves, indicating no change in rbc deformability. in vitro incubation of patient blood samples with vcr also did not affect the osmotic deformability, confirming that a change in rbc rigidity was unlikely the reason for the drop in hb. these data indicate that a dysregulation of erythropoiesis was responsible for the anemia after vcr, rather than damage of peripheral rbc's. in most patients, maintenance therapy for lymphoblastic lymphoma does not cause severe anemia, likely because a temporary reduction in erythropoiesis in patients with a normal rbc survival and low reticulocyte count is not noticed. however, in a patient with decreased rbc survival and a brisk reticulocytosis, a disruption in rbc generation is more apparent. in conclusion, vcr administration to patients with an rbc disorder warrants close observation for potentially severe vcr-induced anemia. background: the addition of tyrosine kinase inhibitors (tki) to conventional chemotherapy has improved outcomes for pediatric patients with philadelphia chromosome-positive (ph+) acute lymphoblastic leukemia (all), however there remains an increased risk of relapse compared to other types of childhood all. typically, in relapsed disease the philadelphia chromosome persists and several mechanisms of resistance involving acquired mutations of the bcr-abl chimeric oncoprotein have been reported. objectives: describe a unique case of a pediatric patient with ph+ b-precursor all relapsing with b-precursor all without the philadelphia chromosome. results: an -year-old boy was diagnosed with ph+ bprecursor all with the presence of the t( ; )/bcr-abl translocation by cytogenetics and fluorescence in situ hybridization (fish), respectively. additional abnormalities included gains of runx and loss of one copy of etv . a remission bone marrow with negative minimal residual disease (mrd) was achieved at the end of induction with dasatinib and the esphall chemotherapy backbone. duration of tki therapy was two years post diagnosis. nearly one year after the completion of therapy, cytopenias prompted a bone marrow investigation. relapsed b-precursor all was established by immunophenotyping, however fish analysis did not identify the bcr-abl rearrangement. moreover, quantitative reverse transcriptase pcr was negative for the bcr-abl fusion transcript. again fish analysis of the bone marrow revealed multiple additional copies of runx and mono-allelic loss of etv , similar to the initial diagnostic sample. the patient was re-induced per aall anticipating a ph+ all relapse. however, with confirmation of the loss of the ph+ clone, tki therapy was not re-initiated. due to positive mrd of . % at the end of re-induction therapy, the patient was salvaged with blinatumomab therapy and subsequently underwent an allogenic stem cell transplant with a sibling donor. conclusion: this is the first known report of a pediatric patient with ph+ b-precursor all who developed recurrent b-precursor all without the philadelphia chromosome. the persistent findings of gain of runx and loss of etv makes it unlikely that a second unrelated b-precursor all developed following successful treatment of the original disease. this case highlights the possibility of a genetically distinct subclone present at the onset of disease that shared abnormalities of runx and etv but did not contain the philadelphia chromosome. nevertheless, the subclone harbored leukemogenic potential in the absence bcr-abl expression. it is plausible that the predominant clone present at diagnosis was effectively treated with dasatinib and extinguished, but the bcr-abl -negative clone persisted in the face of tki therapy. background: ligneous conjunctivitis is a rare form of pseudomembranous conjunctivitis that develops specifically in patients with type plasminogen deficiency. lack of plasmin activity in those patients result in defective fibrinolysis and formation of fibrin-rich membranous material/ masses that develops on the palpebral conjunctiva as well as other sites in the body.current management involve surgical excision of the masses that is usually complicated by multiple recurrences. recently, use of topical plasminogen concentrates helped delaying recurrence, but currently, those concentrates are not commercially available. we report on a -year-old omani girl, with hypoplasminogenemia who required optimization of plasminogen level at the time of surgery to delay/ prevent recurrence. objectives: case report on the peri-operative use of ffp versus cryopricipitate transfusion as an alternative replacement of plasminogen during surgical excision of ligneous conjunctivitis. design/method: pharmacokinetic study was performed to assess plasminogen recovery after ffp ( ml/kg) and precipitate ( bag/ kg) transfusion results: plasminogen levels remained subnormal after either ffp or cryoprecipitate administration. with ffp, the maximum concentration reached was almost % of normal. although half-life of plasminogen is known to be - . days, the patient seemed to have a high catabolic rate after receiv-ing cryoprecipitate, with plasminogen levels reaching basal levels within hours. because of the better recovery profile with ffp, we opted to give ffp before and after surgery. peri-operative management included ffp transfusion at ml/kg/ hours one day before and for days post operatively, followed by ml/kg once daily from day - , then ml/kg on th post-operative day. topical treatment was initiated using antibiotic and steroids ed on the day of surgery, followed by heparin ed on the second day. on follow up, she used topical heparin, cyclosporine, prednisolone, and topical lubricant eye drops for variable duration. clinical picture remained stable for almost year post operatively, when she started to develop recurrence of ligneous lesions again. background: ponatinib (inclusig®, ariad pharmaceutical) is a rd generation multi-targeted tyrosine kinase inhibitor (tki) approved for treatment of adults with chronic myeloid leukemia (cml) and philadelphia chromosomepositive acute lymphoblastic leukemia (ph+ all) resistant to or intolerant of other tkis. ponatinib has numerous drug-drug interactions and a black box warning for associated serious adverse vascular events and hepatotoxicity. for this reason, ponatinib use has been confined to specific high-risk populations. however, in patients who prove refractory to other therapies, the potential benefits of ponatinib may outweigh risks. to date, ponatinib has not been studied in the pediatric/adolescent and young adult (aya) population. furthermore, literature describing the use of ponatinib alone or in combination with other agents in pediatric oncology patients is scarce. objectives: to describe a single institutional experience using ponatinib in the pediatric patients with ph+ all. design/method: two cases of ponatinib use in pediatric ph+ patients resistant to other tkis were identified at our institution and are described. peripheral blood samples obtained from both patients identified bcr-abl p fusion transcripts and sanger sequencing was used to identify resistant mutations. results: our first case is a -year-old female who received upfront multi-agent chemotherapy plus dasatinib for ph+ all. relapse was confirmed on end-of-therapy bone marrow evaluation, thus bcr-abl mutation testing was performed and revealed a t i mutation. ponatinib was initiated then discontinued after one week due to clinically significant fluid retention with peripheral edema and bilateral pleural/pericardial effusions. the second case is a lateadolescent female with ph+ all who relapsed -years after stem cell transplant (sct). following relapse, tki therapy included both imatinib and dasatinib. due to persistence of bcr-abl fusion transcript despite tki therapy she was switched to ponatinib. shortly following initiation of ponatinib she developed a diffuse, maculopapular rash, which persisted despite dose reduction, resulting in ultimate discontinuation of the drug. bcr-abl mutation testing identified f l and f v resistance-conferring mutations. to date, there is scant existing literature detailing the use of ponatinib in pediatric patients. appropriate dosing is undefined and side effect profile not well described, particularly when used concurrently with other chemotherapeutic agents. thus, this case series reporting the response to and toxicity of ponatinib in pediatric ph+ all patients has important clinical implications. additionally, this is the first report of a pediatric ph+ all patient with documented t i mutation underscoring the importance of bcr-abl mutational testing, particularly at the time of relapse. cooper university hospital, camden, new jersey, united states background: myh -related disorder is a rare autosomal dominant disease, encompassing several subtypes: may hegglin anomaly, epstein syndrome, fechtner's syndrome, and sebastian syndrome. heterozygous mutations are seen in the gene encoding non-muscle myosin heavy chain iia (nmmhc-iia) which is involved in cell motility as well as functions to maintain cellular shape and integrity. the presentation of myh -rd is mainly characterized by macrothrombocytopenia, but various related expressions exist: nephritis often leading to renal failure, cataracts and sensorineural deafness ( ). a -year-old girl with history of extensive dental caries, hyperactivity, and speech delay due to suspected hearing loss was incidentally found to have thrombocytopenia at the time of genetic evaluation. she did not have any bruising or excessive bleeding. she did not respond to observation, immunoglobulins, or steroid therapy. her platelet count remained persistently low ( - k/ul). she underwent extensive evaluation to rule out platelet disorder vs. coagulation defect. her peripheral smear showed enlarged platelets by giemsa stain but no inclusion bodies were noted in granulocytes. her platelet aggregation and platelet surface glycoprotein by flow cytometry were negative. her coagulation profile was also normal. objectives: this case report summarizes the complexity in diagnosing myh -rd in a pediatric patient. design/method: since a unifying diagnosis for her clinical presentation was not apparent, whole exome sequencing (wes) was undertaken. results: wes revealed the r c heterozygous pathogenic variant, located in exon in the myh gene. myh gene alteration explained the patient's clinical features of macrothrombocytopenia and hearing loss. this mutation was paternally inherited, and her father demonstrates mosaicism. he was asymptomatic with normal platelet count but his morphology showed enlarged platelets with no inclusion bodies in granulocytes. when dealing with patients who have mild or no symptoms of bleeding diathesis but evidence of persistent macrothrombocytopenia, considering a platelet disorder belonging to myh -rd can help delineate certain predisposing syndromes and guide clinical management. patients are likely to benefit from early genetic testing while receiving supportive therapy. wes can highlight syndromes and provide information on recurrence risk for families. the renal and hearing abnormalities are indistinguishable between epstein and fechtner's syndromes, but the pathogenic variants differ ( ). the genotype-phenotype correlation implies that our patient may have either syndrome, although clinical features compatible with nephritis have yet to manifest. patients should be monitored closely for long-term progression of myh disease, and treatments should be initiated accordingly. we present an -year old female evaluated by genetics at birth due to prenatal microcephaly. chromosomes and microarray were normal. at age she developed standard risk pre-b-cell acute lymphoblastic leukemia (all). she completed treatment in and has been doing well in the interim, remaining in complete clinical remission. during and after treatment she exhibited developmental delay and neurocognitive deficits. at age her height and weight were at or below the th centile and head circumference was below the nd centile (approximately standard deviations below the mean and corresponding to the th centile for a -month-old girl). bone age was appropriate. she had a distinctive triangular face with micrognathia and a pointed nose resembling a seckel-like syndrome. the patient also had clinodactyly of the th toes, zygodactylous triradius involving the nd and rd left toes, tendency to sydney line in the right palm and a radial loop in the left middle finger. the patient's unique clinical presentation prompted a more thorough genetic evaluation, which led to a novel finding we feel is clinically significant with regard to the development of malignancy. design/method: whole exome sequencing (wes) was performed on the patient as well as her biological parents (trio). a de novo heterozygous mutation in the gene pcdh with potential relation to the phenotype was discovered. this c. dupa variant causes a frameshift starting with codon asparagine , changing this amino acid to a lysine residue and creating a premature stop codon at position of the new reading frame denoted p.asn lysfsx . this variant is predicted to cause loss of normal protein function via protein truncation or nonsense-mediated mrna decay. conclusion: pcdh is a member of the protocadherins family which is important in cell-to-cell adhesion and synaptic function in the central nervous system and is highly expressed in areas of the brain involved in higher cortical function and speech. aberrant expression of protocadherins has been associated with the development of malignancies in many organ systems. with regards to leukemia, the methylation status of this gene at diagnosis has been implicated in the prognosis of all and could be used as a biomarker to predict relapse. this patient's de novo mutation and clinical presentation are unique to what has been previously presented in the literature. we feel that this mutation is a clinically significant finding that may shed light on the role of this gene in the development of hematopoeitic malignancies. background: acquired hemophilia a (aha) is an uncommon and potentially life-threatening hemorrhagic disease characterized by sudden onset of bleeding in patients with neither personal nor family history of bleeding dyscrasia. it is usually seen in adults with autoimmune diseases, solid tumors, lymphoproliferative diseases, pregnancy or during the postpartum period; occurrence in the pediatric population has rarely been reported. we report a case of an otherwise healthy teenager who was found to have aha when he presented with acute onset of atraumatic soft tissue hematoma. results: a -year old male of middle eastern descent with history of congenital absence of the right external ear, but otherwise in good general health, presented to our emergency department with a three day history of progressive worsening of right lower leg pain, swelling, and paresthesia, without preceding history of trauma. evaluation by the pediatric orthopedics service documented significantly elevated compartment pressures, necessitating immediate four-compartment fasciotomy. pre-operative labs were significant for prolonged activated partial thromboplastin time (aptt) of . ( . - . ) seconds with normal prothrombin time (pt) and international normalized ratio (inr). ptt did not correct on mixing studies, suggesting the presence of a circulating anticoagulant. factors xii and xi were in the normal range; factor ix was elevated, ( - ). factor viii level was % and fviii inhibitor level was . bethesda units (< . ), confirming the diagnosis of aha. work up for autoimmune disease was negative. his bleeding and surgical hemostasis were managed with recombinant factor vii (novoseven) mcg/kg every hours for hours post operatively, with gradual interval prolongation. factor viii antibody eradication was managed with prednisone mg/kg/day. factor viii and inhibitor levels normalized by day of hospitalization. recombinant factor vii was discontinued; steroids were gradually tapered and discontinued at discharge (hospital day ). conclusion: acquired hemophilia is likely an underdiagnosed condition in pediatrics. while it is typically seen in adults with underlying autoimmune disease, solid tumors, lymphoproliferative disease, or during pregnancy or the postpartum period, pediatric cases may have no identifiable etiology. this case highlights the importance of considering this diagnosis in any patient with unexplained bleeding regardless of their age, so as to intervene early and prevent adverse consequences. university of oklahoma, oklahoma city, oklahoma, united states background: myeloid neoplasms associated with eosinophilia is a rare subtype of chronic leukemia characterized by clonal eosinophilia. the true incidence is unknown due to its rarity and possible classification as idiopathic hypereosinophilia syndrome. the most common chromosomal aberrations involve platelet-derived growth factor receptors (pdgfrs). we report one such rare case in a pediatric patient. most of the pediatric management of this entity is derived from adult case reports and case series. objectives: to describe a case of chronic leukemia presenting as eosinophilia results: a previously healthy year old caucasian male presented with a several week history of migrating joint pain, splenomegaly, and abnormal blood counts with leukocytosis, thrombocytopenia and absolute eosinophilia. white blood cell differential showed myeloid precursors suggestive of chronic myeloid leukemia. bone marrow evaluation showed % blasts and % eosinophils. bcr-abl testing was negative, ruling out cml. fish analysis for eosinophilic clonality revealed deletion of chic gene, resulting in fip l /pdgfra fusion gene, diagnostic for myeloid neoplasm with eosinophilia associated with pdgfr abnormalities. treatment was started with tyrosine kinase inhibitor (tki), imatinib mg daily. within months, fish analysis for fusion gene was negative. after approximately months of daily imatinib, he was switched to maintenance dose of mg weekly. he is approximately months since diagnosis and doing well on maintenance imatinib. in , the who revised its classification of some chronic eosinophilic leukemias to myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement of pdgfra, pdgfrb, fgfr . the most common abnormality is the fip l /pdgfra fusion gene. other less common abnormalities include fusion genes kif b-pdgfra and etv -pdgfrb and point mutations in pdgfra . some features of chronic eosinophilic leukemia include absolute eosinophilia, splenomegaly, elevated vitamin b and tryptase levels, and organ damage from eosinophil infiltrates and cytokine release. patients with rearrangements or mutations involving pdgfra are usually very responsive to imatinib. starting doses have not been well studied or established. experts recommend co-administration of corticosteroids during the first few days of imatinib therapy in patients with a history of cardiac involvement and/or elevated serum troponin levels to prevent myocardial necrosis, a rare complication of imatinib therapy in eosinophilic patients. fortunately our patient did not have cardiac involvement and to date has not exhibited signs of chronic tki toxicity. conclusion: myeloid neoplasms with eosinophilia constitute a rare form of chronic leukemias. they are often associated with pdgfr abnormalities and are usually very responsive to tyrosine kinase inhibitor therapy. walter reed national military medical center, bethesda, maryland, united states background: germline samd l mutation is a rare cause of constitutional bone marrow failure with a unique propensity for clonal evolution to monosomy and mds. objectives: previous case series have demonstrated diverse clinical outcomes in patients with a germline samd l mutation. our case presents a novel samd l mutation (p.val leu). additionally, the case highlights the challenges in clinical decision making for a patient with a gene mutation that is known for clonal evolution towards monosomy with risk of progression to myeloid malignancy, but also known for self-correction through uniparental disomy or inactivating mutations which results in disease remission. design/method: a retrospective chart review and review of the literature was performed. dna was isolated from peripheral blood and used for whole exome sequencing. a peripheral blood sample from the patient's mother and father showed no samd l mutation. skin biopsies of the patient and parents were evaluated for uniparental disomy or new mutations. to determine the pathogenicity of this novel mutation, the specific samd l mutant dna was transfected into the human embryonic kidney cell line to assess its role in inhibiting cell proliferation. our patient presented at months of age with pancytopenia and hypocellular bone marrow in the setting of s of s sepsis. he had evidence of dysfunctional immune activation with hemophagocytosis and elevated soluble il with simultaneous severe hypogammaglobulinemia. analysis of the peripheral blood showed no increase in chromosomal breakage, normal telomere length, and normal flow cytometry. gene testing for primary hemophagocytic lymphohistiocytosis and inherited bone marrow failure were negative. after the patient recovered from his presenting illness, a repeat bone marrow biopsy demonstrated improved cellularity with myelodysplasia and cytogenetics significant for monsomy .whole exome testing demonstrated a novel samd l mutation. the patient continued to require intermittent ivig and failed to demonstrate appropriate leukocytosis with intermittent infections. on repeat bone marrow evaluation over the course of months, the patient demonstrated no evidence of evolution towards self-correction and had a persistent monosomy clone. the patient is scheduled to undergo a matched unrelated donor bone marrow transplant. our case highlights the unique clinical picture associated with constitutional marrow failure and clonal evolution secondary to a novel samd l mutation which is thought to cause pancytopenia by inhibiting cellular proliferation and often results in the development of monosomy which rescues hematopoiesis but with a risk for malignancy. background: notable labs developed a flow cytometricbased assay with a custom robotic platform to test fdaapproved drugs for anti-cancer activity against individual patient's tumor cells. this personalized assay is a potential method for identifying novel agents and drug combinations to treat aml patients who have failed standard therapies. objectives: to present the case of a teen who underwent successful treatment of relapsed aml post-sct with bortezomib, panobinostat, and dexamethasone-a regimen selected based upon results of notable lab testing. results: a -year-old male with m -aml had an isolated bone marrow relapse months after completion of scheduled therapy. at relapse, his aml was flt -itd positive. he achieved a second remission with negative mrd and underwent matched sibling donor bmt after busulfan/cyclophosphamide conditioning. bma performed on day + was mrd positive ( . %). repeat bma done on day + showed . % mrd. he started sorafenib on day + . he received donor lymphocyte infusion (dli) on day + , then received cycles of azacitadine (aza) followed by dli. marrow mrd by flow after sorafenib alone, sorafenib with dli, and sorafenib with aza/dli were %, . %, and . %, respectively. treatment was complicated by varicella meningitis, grade i skin agvhd, febrile neutropenia and c. difficile colitis, and metapneumovirus pneumonia. despite extremely low levels of leukemia (marrow mrd . %), notable lab testing performed on the patient's leukemia cells from marrow collected after aza/dli/sorafenib revealed sensitivity of his leukemic blasts to a combination of bortezomib, panobinostat, and dexamethasone. because of prolonged cytopenias, multiple infectious complications, and persistently positive mrd, he discontinued aza/dli/sorafenib and on day + started bortezomib . mg/m iv on days , , , and ; panobinostat mg po on days , , , , , ; and dexamethasone mg po on days , , , , , , , and . chemotherapy cycle started days later. he tolerated treatment without side effects and with resolution of rash and cytopenias. he achieved full donor chimerism, negative flt -itd, and complete remission by morphology and flow after two cycles. notable lab testing is a powerful tool for evaluating the sensitivity of small populations of leukemic blasts to novel drug therapy. results from notable lab testing may serve as a useful guide for treatment selection after failure of standard aml therapy. this patient achieved morphologic and mrd remission post-sct with bortezomib, panobinostat, and dexamethasone-a regimen predicted to be efficacious based upon notable lab results. maria ahmad-nabi, christine knoll, sanjay shah, esteban gomez, lori wagner phoenix children's hospital, phoenix, arizona, united states background: development of inhibitors in patients with factor ix deficiency (fixd) is a well-recognized complication occurring in - % of patients. within this subset a small percentage can develop anaphylaxis to factor. desensitization with cyclophosphamide, an alkylating agent used in the management of various oncologic malignancies, and reported for use in factor viii desensitization has been previously unreported for use in desensitization in patients with fixd. rituximab, an anti-cd antibody, however has been used. objectives: to induce immune tolerance (it) in patients with inhibitors to factor ix with either novel or under reported methods using cyclophosphamide and/or rituximab. we report a case series of patients at phoenix children's hospital with fixd who achieved it with cyclophosphamide and/or rituximab. results: patient one was a year old male with severe fixd, who at the time of desensitization had inhibitor levels of bu. he was desensitized with cyclophosphamide, then admitted for infusion of recombinant factor ix. he experienced a few minor symptoms of intolerance including an urticarial rash which was self-limited, and hemarthrosis of the right elbow on day which responded to novo . he tolerated the remainder of his infusion without issues. he continued recombinant factor ix daily, and returned to clinic for monthly cyclophosphamide for months. he did develop urticaria with hemarthrosis and spontaneous muscle bleeds which were tempered with zantac, zyrtec, solumedrol, and benadryl. he remained without a recurrence of inhibitors, however did have intermittent hemarthrosis of his ankles thereafter requiring prophylactic twice daily dosing recombinant factor ix. patient two was a year old male with severe fixd and a family history of anaphylaxis to factor causing early death in all male relatives with the disease. he had never received factor ix and did not have a detectable inhibitor prior to desensitization. he successfully underwent desensitization to recombinant factor ix with rituximab in the icu, and returned to clinic for weekly infusions x . he experienced no adverse reactions concerning for anaphylaxis. he continued to tolerate factor ix products without evidence of intolerance, development of inhibitors, and continues on as prophylactic dosing of recombinant factor ix every other day. our experience at a single institution proves cyclophosphamide as a novel agent for inducing it in those with fixd and anaphylaxis. it also provides further evidence that rituximab can desensitize patients with severe fixd. differences include longer duration for cyclophosphamide therapy ( months vs month). background: cartilage-hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with defective cell-mediated immunity caused by mutations in the ribonuclease mitochondrial rna processing (rmrp) gene. cancer incidence is -fold higher in patients with chh than in the general population, especially non-hodgkin lymphoma. the use of rituximab, an anti cd antibody, results in decreased host b-cell number and impaired humoral function for - months. the safety of rituximab in pediatric patients with cancer and immunodeficiency is not well documented. a diagnosis of underlying immunodeficiency may discourage physicians from using rituximab due to the risk of severe bacterial infection or viral re-activation. objectives: to report a case of burkitt lymphoma in a young adult female with chh and defective cellular immunity successfully treated with rituximab. results: an -year old amish female with disproportionate short stature presented to our center for management of stage iv biopsy proven burkitt lymphoma with myc rearrangement. she had presented a week earlier with cervical, occipital, and submandibular lymphadenopathy, splenomegaly; fevers, night sweats, and weight loss for - weeks. on exam, her height was three feet associated with brachydactyly, mild bowing of the legs, normal size head without frontal bossing, fine and sparse hair. she had normal intelligence. her pattern of dysmorphisms was suggestive of chh (genetic testing not performed at time of diagnosis). pet-ct scan showed stage iv disease with involvement of cervical lymph nodes, spleen, iliac bone and bone marrow. treatment with standardintensity fab/lmb therapy (group c) with the addition of rituximab was initiated. she had an incomplete response to cop (∼ % reduction of tumoral masses) but achieved complete remission after copadam . her course was complicated with severe varicella zoster but she completed therapy and remains in complete disease remission for months after treatment completion. genetic testing subsequently performed proved homozygosity for chh with a n. a>g variant. she had no other opportunistic infections during or after therapy. conclusion: the use of rituximab was both safe and beneficial in our patient despite defective cell mediated immunity secondary to chh suggesting that rituximab may be safe to use in patients with cellular immune deficiencies. background: hemophilia a and b are bleeding disorders characterized by deficiency in factor viii or ix, respectively. spontaneous or provoked hemarthrosis is a known complication of hemophilia. repetitive episodes of hemarthrosis can lead to debilitating hemophilic arthropathy. lyme disease is a tick-born infection which is endemic to increasing parts of the united states. chronic lyme disease, the phase in which lyme arthritis typically develops, occurs months to years after initial infection and is characterized by swelling of one or more large joints generally in the absence of systemic symptoms. objectives: review cases of hemophilia a and b patients with episodes of provoked hemarthrosis refractory to intensive recombinant factor replacement therapy found to have concurrent lyme arthritis. design/method: we report two clinical cases and review relevant literature. results: first, we report a year-old male with moderate hemophilia a with a provoked knee hemarthrosis which failed to improve despite months of intense factor replacement therapy requiring multiple hospitalizations. factor replacement regimens included twice daily standard half-life recombinant factor viii products or daily to every other day extended half-life recombinant factor viii products with trough levels aimed as high as - %. factor viii pk studies were obtained for dosing, to confirm adherence, and to evaluate for subclinical inhibitors (inhibitor testing was negative). given protracted symptoms additional workup for hemarthrosis was pursed. lyme titers were positive for ( )igg, though negative for igm. he was treated with days of doxycycline during which time hemarthrosis greatly improved on examination and imaging, and he was able to recover function through physical therapy. second, we report a year-old male with moderate hemophilia b who required multiple hospital admissions for a provoked knee hemarthro-sis with no improvement in symptoms despite weeks of daily or twice daily factor replacement with standard halflife recombinant factor ix products aiming for % correction. we performed inhibitor testing (which was negative) and pk studies to assess for non-detectable inhibitors, dosing and adherence. lyme testing was positive for ( )igg, though negative for igm. he was treated with amoxicillin for days during which time hemarthrosis significantly improved on examination and imaging. diagnosis and follow-up imaging studies for both patients included mri and serial bedside ultrasounds performed as per uc san diego school of medicine mskus guidelines. background: relapse/refractory aml following allogeneic hematopoietic stem cell transplant (hsct) holds a high mortality rate. current relapse/refractory therapy modalities for younger patients may include re-induction with a clofarabinebased regimen followed by second allogeneic hsct. even for patients who undergo second hsct, the five-year survival rate is dismal. new therapies, including small molecule inhibitors, are being studied in the post-hsct relapse setting or those unfit for hsct with promising results. venetoclax is a small molecule inhibitor that has received breakthrough designation for aml treatment in elderly patients objectives: to report a young adult aml patient with relapse post hsct who was successfully re-induced with topotecan, vinorelbine, thiotepa, clofarabine (tvtc) and has sustained remission with venetoclax maintenance therapy. this approach appears to be unique in terms of reported literature. results: our patient is now a -year-old female noted to have mll rearranged aml at initial diagnosis when she was years old. she underwent chemotherapy consisting of cytarabine/daunorubicin according to standard + . due to persistent disease, she was re-induced with g-csf, clofarabine, and high-dose cytarabine (gclac) which put her in cr. her course was complicated by sepsis, colitis, gastrointestinal bleed, deep venous thrombosis, and transfusionassociated circulatory overload. given her co-morbidities, she received another cycle of clofarabine/cytarabine, and then proceeded to reduced intensity allogeneic hsct, according to bmt ctn . the patient tolerated hsct well and experienced no transplant-related complications, including no acute or chronic gvhd. unfortunately, she relapsed about month's post-hsct. initial salvage therapy consisted of another course of g-clac, but due to persistent disease the decision was made to re-induce her with topotecan, vinorelbine, thiotepa, and clofarabine (tvtc). during this time however, she was found to have extensive infection with a fusarium species requiring a course of anti-fungal therapy. bone marrow evaluation showed no residual disease with an mrd of < . %. once the absolute neutrophil count recovered, the patient was started on single-agent venetoclax for maintenance therapy, which has been well-tolerated. she remains in morphologic remission for over months. we describe herein a young adult with multiply relapsed aml wherein tvtc re-induction, followed by maintenance with venetoclax were safely used in the post-hsct setting. venetoclax therapy in the relapsed aml setting warrants further study. background: vitamin b deficiency is uncommon in children in developed countries, especially in the absence of risk factors like malabsorption or inadequate dietary intake. it often presents with non-specific symptoms and signs and can elude diagnosis. the recognition and treatment of vitamin b deficiency is critical as it can lead to bone marrow failure as well as severe neurological and developmental problems in children. to increase index of suspicion of vitamin b deficiency anemia in children. we report a rare case of vita-min b deficiency anemia in a child who presented with a severe macrocytic anemia, with signs of hemolysis and concern of malignancy. design/method: an almost three-year-old previously healthy girl presented with a few day history of fever, emesis, fatigue and pallor. she had no dysmorphic features, hepatosplenomegaly or lymphadenopathy on exam, growth and development were normal. laboratory findings showed severe macrocytic anemia (hemoglobin . grams/dl; mcv . fl) with reticulocytopenia. signs of intravascular hemolysis were present with elevated lactate dehydrogenase ( , units/l) and haptoglobin below assay limit. immune-mediated hemolysis was ruled out. initial picture of a hemolytic anemia was compounded by other findings of moderate neutropenia, mild thrombocytopenia and peripheral smear showing occasional blasts. further workup was done with a broad differential diagnosis that included leukemias, hemolytic anemias, bone marrow failure syndromes, and specific deficiencies. results: workup revealed abnormally low vitamin b levels along with significantly elevated homocysteine and methylmalonic acid levels indicating functional vitamin b deficiency. bone marrow evaluation showed megaloblastic anemia and dyserythropoiesis consistent with vitamin b deficiency, and ruled out leukemia. vitamin b deficiency can cause a hemolytic anemia like picture secondary to intramedullary hemolysis due to ineffective erythropoiesis. myeloid precursors are also affected which can lead to neutropenia, thrombocytopenia, and abnormal peripheral blood cells. in our patient, initial symptomatic anemia was treated with blood transfusion, followed by intramuscular vitamin b injections with normalizing lab values. so far, workup for an etiology for vitamin b deficiency is negative except for an equivocal range of anti-parietal cell antibodies raising concerns for pernicious anemia; however it is rare in this age group. another rare condition is an inborn error of the cobalamin transporter. she is currently on oral vitamin b supplementation and further workup will be planned based on response. conclusion: this case highlights the importance of early consideration and thorough evaluation of vitamin b deficiency in children with unclear etiology of anemia, so that prompt treatment can be initiated. memorial hospital/ university of miami, miami, florida, united states background: despite great success in the treatment of acute lymphoblastic leukemia (all), the outcomes for patients with relapsed all remain poor. prognostic indicators include timing and site of relapse. blinatumomab, is the first agent in its class that simultaneously binds cd -positive cytotoxic t cells to cd -positive b cells resulting in lysis of malignant cells. however, mechanisms of leukemia resistance to blinatumomab are unclear. objectives: to describe a case with multiple sites of extramedullary (em) relapse during blinatumomab therapy. results: a -year-old hispanic male with philadelphia positive, cd -positive b-precursor cell all refractory to chemotherapy, had failed a bone marrow (bm) and was placed on blinatumomab and imatinib. he achieved minimal residual disease (mrd)-negative systemic remission, but during his fifth cycle developed bilateral periorbital masses. biopsies confirmed cd -negative isolated em relapsed disease, which was treated with radiation therapy (rt). there was notable resolution of em disease and he continued systemic therapy. subsequently, he presented with a painful left scapular swelling. imaging showed muscle and lung parenchymal em relapse with cd -positivity confirmed on histology. he continued on blinatumomab with localized rt while awaiting car-t cell therapy. his bm mrd remained negative until he developed systemic mrd-positivity with cd -positive blasts following the sixth cycle. primary resistance to blinatumomab is poorly understood. it is proposed that expansion of cd -negative clones or downregulation of cd following blinatumomab may play a role. this was observed in our patient's periorbital relapse; but subsequent em and systemic relapses were cd -positive, consistent with the co-existence of multiple clones in relapsed all. it has also been postulated that em relapse could be linked to the failure of blinatumomab or t cells to migrate to em sites of disease or drug inactivation by the microenvironment. the second em relapse in our patient, with cd -positive disease suggests this as a possible mechanism of relapse. this was reported in patients with cd positive non-hodgkin lymphoma (nhl), and higher doses of blinatumomab however, have shown promising results in this population. despite blinatumomab's effectiveness in inducing remissions in patients with refractory/relapsed all, it appears to have limitations in patients with em disease. these may arise either from the multiclonality associated with relapsed all or due to the emergence of resistance to blinatumomab, including failure to migrate to em sites. background: cyclic neutropenia is a rare hereditary disorder, characterized by recurrent neutropenia, cycling at about week intervals, with variable associated symptoms including oral ulcers and fever. there are reported cases of cyclic neutropenia associated with chronic inflammation leading to development of reactive aa amyloidosis. one patient also presented with amyloid goiter. we report a new case of cyclic neutropenia with associated renal and thyroid amyloid. design/method: a -year-old female presented with a month history of thyromegaly, and recurrent aphthous ulcers associated with fevers. laboratory workup showed severe neutropenia, anemia, azotemia, and abnormal thyroid function, with an absolute neutrophil count - / l, hemoglobin - . g/dl, serum creatinine - . mg/dl, and uric acid - . mg/dl. thyroid stimulating hormone was elevated - . iu/ml, and normal free t . urinalysis showed + protein, + blood, and - urine red blood cells/hpf. chest radiograph showed mild narrowing of the trachea from thyroid compression. bone marrow biopsy showed a hypocellular marrow, with tri-lineage hematopoiesis, left shifted myeloid maturation with very rare mature neutrophils. both renal biopsy and thyroid fine needle aspiration revealed abundant amyloid. of note, her father had aa amyloidosis, resulting in end-stage renal disease (esrd) requiring hemodialysis, and recurrent aphthous ulcers. the family history suggested a familial predisposition. genetic testing revealed a pathogenic elane c. a>t gene mutation with autosomal dominant inheritance confirming the diagnosis of cyclic neutropenia. we treated our patient with daily granulocyte colony stimulating factor to reduce the burden of chronic inflammation induced by cyclic neutropenia, and to preserve renal and other end organ function affected by further amyloid deposition. results: proband with elane gene mutation positive cyclic neutropenia, amyloidosis of thyroid and kidney, with a positive paternal history of aa amyloidosis resulting in esrd. cyclic neutropenia may result in chronic inflammatory states leading to secondary amyloidosis. university of kentucky, lexington, kentucky, united states background: overall survival of burkitt lymphoma (bl), regardless of stage, is greater than % in the pediatric population when treated with multi-agent chemotherapy. adenovirus is a common, usually self-limited infection within the pediatric population; however, findings can vary within an immunocompromised host. hepatitis is a rare complication, with very few reports of radiologic findings in this patient population. we discuss a three year old male with history of bl who presented with clinical and radiographic evidence of relapse but was found to have adenovirus hepatitis. design/method: a case report of a patient with bl in complete remission after completion of standard of care chemotherapy, who presented with return of high fever, elevated ldh, transaminitis and hepatic lesions. we describe the hepatic imaging and pathology consistent with adenovirus hepatitis in this immunocompromised host. our patient presented at three years old with a six week history of worsening abdominal pain and fevers. he was found to have a right sided pleural effusion, multiple lesions of the liver, and diffuse abdominal lymphadenopathy; biopsy of lymph tissue was consistent with bl. he completed therapy per anhl arm b and was in a complete remission at the end of planned therapy. one month after completion of therapy, he returned with high fever, abdominal pain and transaminitis, similar to his initial presentation. ct scan showed multiple hypodense discrete lesions throughout the liver and re-accumulation of right sided pleural effusion. ldh peaked at u/l (uln u/l). uric acid remained within normal limits. bilirubin peaked at . mg/dl, conjugated . mg/dl. liver biopsy was performed, showing smudgy nuclei with immunohistochemical staining positive for adenovirus. there was no evidence of lymphomatous involvement. resolution of hepatic lesions and transaminitis, with normalization of ldh and fever, occurred with symptomatic treatment alone. adenovirus is known to cause systemic disease in immunocompromised patients and rarely hepatitis. no pediatric patients with discrete hepatic lesions secondary to adenovirus have been reported in the literature. three cases of discrete hepatic lesions have been reported in adult immunocompromised patients, two with fatal fulminant liver failure and one who required cidofovir. this case demonstrates that a common pediatric viral infection can present with lesions concerning for metastatic disease in a pediatric lymphoma patient. prompt diagnosis is vital in the management of these patients when recurrent lymphoma is in the differential. background: heparin induced thrombocytopenia (hit) is an immunologic process in which antibodies bind a heparin complex and cause a paradoxical hypercoagulable state. ramifications of this process may include a multitude of thrombotic events and bleeding complications secondary to platelet consumption. in our patient, hit manifested as increased bruising, an acute decrease in platelet count, and continual clotting of her crrt circuit. hit, although rare in pediatrics, should be included in the differential for children with thrombocytopenia who have received heparin products. to present a unique case report of a critically ill pediatric patient who developed hit in the presence of multiorgan system failure and to discuss the challenges encountered with identification of an alternative anti-coagulant. results: a yo obese, caucasian female child presented to our facility with bilateral pulmonary emboli (of unclear etiology). initially, she was started on a continuous heparin infusion, but was transitioned to enoxaparin within days without issue. five days after enoxaparin was initiated, the patient developed acute kidney injury (evidenced by increasing creatinine) attributable to her biventricular heart failure. due to her need for continuous renal replacement therapy (crrt), she was transitioned back to a continuous heparin infusion. whereas her initial platelet count on transition was normal, she developed severe thrombocytopenia ( , ul) within hours. due to intermediate risk but low suspicion for hit, pf antibodies were sent which were positive. after much discussion, she was transitioned to an argatroban infusion which was titrated according to ptt levels. within hours, her platelet count normalized. at discharge, she was prescribed apixaban for anti-coagulant management. conclusion: hit is an uncommon presentation in the pediatric population. given its rarity, there is often a delay in diagnosis which increases risk of complications such as bleeding, stroke, and limb ischemia. even if the diagnosis is suspected or proven, there may be challenges in initiating alternative agents as limited data exists on pediatric options. as argatroban remains the treatment of choice for patients with hit, experience in pediatric patients is limited, and dosing recommendations have been extrapolated from adult studies. anecdotal data exists for use of bivalirudin in children, although studies, primarily, focus on use in specific cardiac cases. in our patient's case, choice was further complicated by renal failure. this case study highlights the need for further research regarding the identification of a secondary anti-coagulant agent for use in pediatric patients with hit. background: subcutaneous panniculitis-like t-cell lymphoma (sptl) is a rare form of non-hodgkin's lymphoma characterized by infiltration of cytotoxic t-cells into subcutaneous tissue. sptl occurs in both adults and children and can present in both patient populations as either alpha/beta or gamma/delta subtypes. patients with the gamma-delta phenotype have an overall poorer survival, although the exact etiology is unclear. interestingly, both subtypes of sptl can present with secondary hemophagocytic lymphohistiocytosis (hlh), and this is associated with a worse prognosis. currently, there are no standardized treatment protocols for sptl, and clinical management includes watchful waiting, corticosteroids/immunosuppression, chemotherapy, and stem cell transplant. the primary objective was to compare how two patients with the same diagnosis responded acutely to therapy. we performed a retrospective chart review of two pediatric patients at our institution who were diagnosed with alpha/beta sptl and secondary hlh. we examined each presentation, treatment course, and outcome. we then completed a brief review of the current literature describing treatment of and outcomes for sptl with secondary hlh. results: these two patients presented in a similar manner with signs and symptoms of hlh. each was then subse-quently diagnosed with alpha/beta sptl after biopsy of cutaneous nodules and each had diffuse disease, as measured by pet. however, they demonstrated vastly different acute responses to therapy. one patient was pre-treated with systemic glucocorticoids before receiving definitive chemotherapy and tolerated therapy well as an outpatient. the other patient started systemic chemotherapy without steroid pretreatment and developed severe cytokine storm characterized by hypotension, cardiac dysfunction, multi-organ failure and cytokine elevation. both patients achieved complete remission (cr) after treatment with chop chemotherapy and remain disease-free - months off therapy. in patients presenting with sptl and secondary hlh, we propose that initial treatment with antiinflammatory or anti-cytokine therapy can decrease, or even prevent, the possibility of life threatening cytokine release as a result of cytotoxic chemotherapy. background: congenital dyserythropoietic anemia type ii (cda ii) is a rare autosomal recessive disorder, rarely presenting in the neonatal period. iron overload often occurs as a late sequela of ineffective erythropoiesis and intramedullary hemolysis. objectives: to report the novel use of iron chelation in an infant with cda ii associated with severe iron overload. the patient is a -month-old, former -week infant with prenatal non-immune hydrops and transfusion-dependent fetal anemia who presented with persistent anemia, reticulocytopenia, hyperbilirubinemia, liver dysfunction, and hyperferritinemia. his initial ferritin was . ng/ml, tibc ug/dl, and transferrin mg/dl. his bone marrow biopsy showed trilineage hematopoiesis and erythroid dyspoiesis characterized by binucleation of late-stage precursors. genetic testing revealed a compound heterozygous missense mutation and splice site mutation in the sec b gene, confirming the diagnosis of cda ii. initial liver biopsy revealed mild portal fibrous expansion, and abundant hepatic iron deposition. his ferritin continued to increase, peaking at , ng/ml, along with liver enzymes peaking at an alanine aminotransferase (alt) of u/l and aspartate aminotransferase (ast) of u/l. ferriscan showed an elevated estimated liver concentration of . mg/g dry tissue. repeat liver biopsy months later showed giant cell hepatitis with worsening mild portal fibrosis and hemosiderosis. additionally, tissue liver iron concentration was mcg/g dry weight. cardiac t * mri revealed mild cardiac iron deposition. given his significant degree of iron overload, deferoxamine was used to reduce hemosiderosis and liver morbidity in preparation for bone marrow transplantation. the patient received deferoxamine mg/kg/day iv x days/week for three months, without any clinically significant adverse events. blood counts and hepatic and renal function were monitored weekly without any abnormalities. growth parameters and liver enzymes significantly improved while receiving chelation therapy. as a noninvasive, cost-effective method, serum ferritin levels were monitored monthly to gauge response to treatment. despite receiving blood transfusions every - weeks, serum ferritin decreased to ng/ml and liver enzymes decreased to alt u/l and ast u/l prior to bone marrow transplantation. we report the use of deferoxamine in a patient with cda ii less than years of age, for treatment of iron overload. our patient tolerated deferoxamine well without significant adverse events or organ toxicity. deferoxamine may be a well-tolerated method of reducing iron burden in young patients with iron-loading pathologies. background: low grade gliomas with kiaa- -braf fusions typically have a favorable prognosis with infrequent rates of high grade transformation, low rates of metastasis and even lower rates of extra cns metastasis. while highgrade transformation has been reported for tumors with braf v e mutations and cdkn a deletions, it has not been pre-viously reported in gliomas with kiaa- -braf fusions. while there are case reports of high-grade cns malignancies metastasizing through a ventriculo-peritoneal (vp) shunt, low-grade gliomas metastasizing in this manner are extremely rare. objectives: to describe a unique case of peritoneal tumor dissemination of a braf fusion positive high grade neuroepithelial tumor in a child with a vp shunt placed for multifocal braf fusion positive low grade astrocytomas results: an eight-year-old male was initially diagnosed with multifocal low-grade astrocytomas of the hypothalamus and c -c spinal cord. initial testing revealed the kiaa- -braf fusion, but no cdkn a or braf v e mutation. initial surgical management included a vp shunt and resection of the cervical spinal lesion. he received vincristine and carboplatin, followed by transition to vinblastine given new thoracic metastatic lesions after months of therapy. at months after diagnosis, scans were concerning for diffuse leptomeningeal progressive disease and new intracranial lesions, necessitating craniospinal radiation. following a near cr, he presented months later with acute onset of abdominal pain. a ct scan revealed peri-renal and perirectal soft tissue masses, confirmed by exploratory laparotomy to be peritoneal tumor dissemination of high grade neuroepithelial tumor. a kiaa -braf fusion was noted and confirmed by rt-pcr, identical to that seen in the original cns tumors. additional findings included deletion of chromosome p (without q loss) and heterozygous and homozygous deletion of cdkn a found by fish. brisk mitotic activity justified a high-grade designation. salvage chemotherapy consisted of cycles of ice with subsequent resolution of pet-avid disease and only minimal peri-nephric tissue remaining. given the favorable response, surgical resection and multiple tissue biopsies were performed which documented no residual active disease. the shunt was revised and he started trametinib for maintenance. we present a unique case of peritoneal dissemination of high grade neuroepitheial tumors with the same kiaa- -braf fusion as multifocal low grade astrocytomas in a child with a vp shunt. this raises suspicion for tumor metastasis and transformation to a higher grade malignancy versus two distinct diseases, which may be indicative of an underlying cancer predisposition. texas children's hospital, houston, texas, united states background: polycythemia is a common referral to hematology. it is important to evaluate for a high oxygen affinity hemoglobinopathy, ensuring appropriate testing is performed for early diagnosis and avoidance of additional tests and procedures. a year old mexican female presented with an elevated hemoglobin and hematocrit, symptoms of plethora of her hands and feet, chest pain, palpitations, and fatigue. further confounding the picture, she also had significant menorrhagia and iron deficiency. she was diagnosed with the rare high oxygen affinity hemoglobin new mexico variant, only previously described once in the literature in a year old black boy. objectives: the patient initially presented at age with a hemoglobin of . g/dl and a hematocrit of . %. initial work up consisted of a hemoglobin electrophoresis which diagnosed sickle cell trait, a co-oximetry panel which was normal, and erythropoietin level of mu/ml, also normal. she was then lost to follow up and re-referred at age . she is a competitive basketball athlete, and at that time, she presented with a hemoglobin of . g/dl, and hematocrit of %. erythropoietin level continued to be normal at mu/ml. design/method: cardiology was consulted regarding chest pain and palpitations with a normal evaluation. chest x-ray was also normal. a bone marrow aspirate and biopsy was performed with results significant for mild erythroid hyperplasia and mild reticulin fibrosis. jak mutation, von hippel lindau, bpgm, and hereditary erythrocytosis mutations including phd , hif a, and epor mutation analysis were sent, all of which were normal. testing to mayo clinic for p rbc oxygen dissociation returned low at mmhg ( - mmhg normal range) and subsequently a hemoglobin electrophoresis identified a hemoglobin variant leading to beta globin gene sequencing. results: patient found to be heterozygous for hemoglobin new mexico, with . % hb new mexico and . % hba, and . % hba . there was no evidence of hbs. when evaluating patients with polycythemia, maintaining a high index of suspicion for high affinity hemoglobinopathies may eliminate further unnecessary and invasive testing for patients. caution should be used when using hemoglobin electrophoresis testing since hb new mexico is known to migrate similarly to hbs on hplc with minimal change that may not be detected in regular laboratories. most high affinity hemoglobinopathies are reported to not have significant symptoms. in this case, our patient complains of fatigue, occasional palpitations and plethora of hands and feet. we will need to further follow this patient for possible attributable symptomatology. divya keerthy, simone chang, warren alperstein, patricia delgado, claudia rojas, ofelia alvarez, matteo trucco university of miami jackson memorial hospital, miami, florida, united states background: improved technology is enabling detection of previously unidentified translocations and mutations in otherwise unclassified sarcomas. one such mutation is the bcl- co-repressor -internal tandem duplication (bcor-itd) allowing for the new classification of bcor positive undifferentiated round cell sarcomas (urcs). this sarcoma has a similar appearance to clear cell sarcoma of the kidney (ccsk), potentially representing an extra-renal manifestation of this tumor, but their clinical pathologic features are not identical. objectives: this case highlights how recombinant polymerase chain reaction (rt-pcr) and bcor immunohistochemical staining can ease the diagnosis of this rare sarcoma. results: a month-old female presented for right sided pre-septal cellulitis and a temporal subcutaneous mass. the detection of multiple other subcutaneous nodules on exam raised the concern for malignancy and she was admitted for evaluation. she had two subcutaneous masses on her abdomen, with more cutaneous masses on her legs, back, shoulder, cheek and submandibular areas. she lacked spontaneous lower limb movement and had bilateral clonus. imaging confirmed multiple masses throughout the body including paravertebral area from t to l , bilateral adrenal glands, left kidney and muscles of upper and lower extremities. initial differential included neuroblastoma, infantile myofibromatosis, rhabdomyosarcoma or atypical presentation of a renal tumor. however, synaptophysin and chromogranin stains were negative. with standard immunohistochemistry, the tumor could be only broadly classified as "undifferentiated sarcoma" maintaining the diagnostic challenge. using rt-pcr in the setting of a morphologically primitive round cell neoplasm with strong bcor expression, two external institutes simultaneously diagnosed the tumor as bcor-urcs. the primary lesion is unknown but potentially may have arose from the kidney. bcor-urcs has a heterogeneous histology with tumor cells appearing monomorphic in nests of - cells separated by septa with uniform nuclei. there is frequently an "orphan annie eye" appearance and sparse cytoplasm to the cells. diagnosis cannot be made solely on evaluation of this nonspecific histology. rt-pcr uses the genetic abnormality in undifferentiated sarcomas to narrow the differential and bcor immunohistochemical staining provides further context. bcor has significant diagnostic value given its sensitivity and specificity in urcs. another potential marker includes ywhae-nutm b fusions, which occur in smaller subset of cases, but requires further study. rt-pcr has helped further classify tumors leading to the diagnosis of a rare undifferentiated sarcoma with bcor overexpression. while this technology is beneficial, its availability is limited. if accessibility improves, earlier identification and treatment may be possible maximizing the chance for a positive outcome. background: hematohidrosis is a rare condition that mimics bleeding disorders. cases present with oozing blood tinged fluid from various sites like eyes, ears, nose, skin, etc. reported causes of this condition were stress or fear, physical activity, psychological disorders. the condition is self-limited and don't affect the general condition of the patients, but it may contributes to psychosocial problems and may increases their stress and anxiety. so this condition needs to be promptly treated. to test the response of this disease and the associated headache to propranolol treatment. design/method: our case female patient years old st offspring of non consanguineous marriage, was admitted with recurrent episodes of oozing blood tinged fluid from eyes, ears and nose months before admission, about . - ml from each orifice, lasted - minutes and subsided spontaneously. it could involve the sites simultaneously or - sites. the number of attacks was - times per day then gradually increased to - times per day. later on the patient developed a bleeding attack from umbilicus. these attacks were aggravated by stress and physical activity and decreased with rest and sleep. the condition was associated with severe headache involving the whole head, throbbing in nature of gradual onset, increased by physical activity and relieved by analgesics. the condition was not associated with vomiting, blurring or diminution of vision, ocular pain, eye discoloration. no earache, tinnitus or diminution of hearing. there was no other form of discharge from eyes, ears or nose. no history of ecchymotic patches, bleeding from other orifices or blood product transfusion. no history of trauma, drug intake, fever or rash. no symptoms of other system affection. past history of recurrent attacks of epistaxis and two operations were done that passed without remarkable bleeding. no similar condition in the family physical examination was free, no evidence of psychological problems. complete blood count, coagulation profile, platelets function, factor and c.t brain were normal. oozing fluid from the patient was analyzed showed the same components as blood. results: our case started oral propranolol . mg/kg/day based on its use in similar cases in literature. the frequency of attacks and headache reduced then stopped after months of treatment and didn't recur after stoppage of propranolol. propranolol can treat this condition successfully. further investigations are needed to determine the link between this condition and severe headache our case was suffering from. background: wilms tumor is the most common renal solid tumors of childhood and is derived from primitive metanephric cells located in the kidney. primary extra-renal wilms tumors (erwt) are extremely rare, estimated to comprise . - % of all wilms tumors. despite similar histologic appearance intrarenal and erwts differ in embryologic tissues of origin. erwts arise from the more primitive mesonephric or pronephric origin and, therefore, can develop anywhere along the craniocaudal migration pathway of these primitive tissues, most often retroperitoneal, inguinal/genital, lumbosacral/pelvic and mediastinal. these tumors are typically staged and treated per national wilms tumor study (nwts) guidelines, and, by definition, are stage ii or greater due to location beyond the kidney borders. based on the cases reported in the literature, outcomes for erwt are comparable to renal wilms tumors with an % local recurrence rate and an % two-year event-free survival. we report the first case of a stage iii testicular extrarenal wilms tumor in an -month-old male with an intrabdominal undescended testis who underwent complete surgical excision followed by chemotherapy and inguinal radiation. results: a full term -month old male underwent orchipexy for an undescended left testicle. the testicle was noted to be grossly abnormal with a pea-sized thickened tissue adherent to the upper pole and a separate mass outside of the scrotum on the superior epididymis. both masses were removed, and s of s pathology demonstrated wilms tumor with favorable histology and negative margins. ct imaging of the chest, abdomen and pelvis were negative for a primary renal tumor, local residual disease, pathologic lymph node enlargement or distant metastases. the tumor was classified per nwts as stage iii due to tumor removal in multiple pieces. the patient completed dd- a treatment with vincristine, doxorubicin and dactinomycin per aren with cgy left inguinal radiation. he is currently months off therapy without clinical or radiographic evidence of recurrent disease. primary erwt is an extremely rare malignant neoplasm associated with challenges in diagnosis, staging and treatment. based on the cases reported in the literature, outcomes are similar to that of intrarenal wilms tumor. there are four pediatric paratesticular wilms tumors reported in the literature and, to the best of our knowledge, this is the first case of stage iii testicular wilms tumor successfully treated with dd- a chemotherapy and radiation. in erwt, nwts guidelines for staging and treatment should be applied with evaluation of both kidneys to exclude an intrarenal primary tumor. background: patient is a yo f, with esrd secondary to atypical hus versus ttp, who presented with thrombotic microangiopathy, aki, thrombocytopenia and anemia after a living unrelated donor kidney transplant. patient initially had downtrending creatinine. on post-op day , hematology was consulted for an increasing ldh and drop in platelets. peripheral smear was notable for an absence of schistocytes. yet, biopsy of the kidney revealed microthrombi. the patient was diagnosed with a thrombotic microangiopathy. plasmapharesis was initiated on day # , at which time ms r was noted to have significantly elevated creatinine. plasmapharesis did not yield any correction in labs and significant bruising developed. patient was started on eculizimab; plasmapharesis was stopped. shortly after, creatinine, anemia and thrombocytopenia corrected to levels at which she was discharged. overall, patient was found to have progressive anemia, thrombocytopenia, an increasing creatinine and ldh ( s) concerning for atypical hus, despite absence of schistocytes on peripheral smear. she responded well to eculizimab, with correction of hematologic changes during induction. she was discharged on eculizimab and continued to respond with normalizing platelet counts and hemoglobin. the differential in light of patient's thrombotic microangiopathy and thrombocytope-nia also included ttp. yet, adamts remained normal. dic was unlikely given normal fibrinogen level and d-dimer. objectives: presentations of atypical hus vs ttp. discuss eculizumab as a treatment of atypical hus. highlight atypical presentations of illness in transplant patients. results: despite absence of schistocytes by smear, pt was diagnosed with atypical hus based on presentation and after failing plasmapharesis, she responded well to eculizumab. though her presentation was abnormal, her response to this antibody that blocks the complement cascade suggests that she was experiencing a complement-mediated process. there are rare documented cases in the literature of atypical hus without schistocytes. hemolytic uremic syndrome (hus) is characterized by hemolytic anemia, thrombocytopenia and acute kidney injury. atypical hus is a diagnosis of exclusion, not due common etiologies such as shiga toxin. among atypical causes are complement-mediated forms, caused by an antibody to complement factor. in addition to plasmapharesis, renal transplant and supportive care, the mainstay of treatment for atypical hus is eculizumab (an antibody that blocks the complement terminal cascade). this case describes a patient unique in that, she was diagnosed with atypical hus without any schistocytes by smear. secondly, she responded to eculizumab, with unremarkable gene studies. finally, this case highlights that transplant patients often have unique presentations. nicklaus children's hospital, miami, florida, united states background: synovial sarcoma is a spindle cell tumor categorized as a soft tissue sarcoma. the chromosomal translocation t(x; ) leading to the ss -ssx fusion protein is unique to this sarcoma. it is a slow growing tumor with common recurrences and often, at presentation, with evidence of metastatic disease. if resection is not feasible, then neoadjuvant with adjuvant chemotherapy is recommended. metastasis carries an unfavorable prognosis given synovial sarcoma historically does not respond well to chemotherapy. trabectedin is a well-tolerated alkylating agent currently indicated for the treatment of liposarcoma and leiomyosarcoma. we present a -year-old male with metastatic synovial sarcoma to the lungs that progressed and was refractory to chemotherapy. he was administered trabectedin as a form of palliative chemotherapy, with significant clinical and radiographic response. design/method: pubmed search was done with search for terminology including "synovial sarcoma" and "trabectedin". papers relevant to our case were selected for literature review. a -year-old male patient presented with a large right axillary mass. initial imaging showed a heterogeneous multiseptated mass invading the subscapularis and teres major muscles along with innumerable lung nodules. biopsy confirmed diagnosis of monophasic synovial sarcoma. the patient was started on protocol arst with ifosfomide, mesna, doxorubicin. he completed cycles followed by radical resection and sessions of radiation. due to progression of disease multiple chemotherapy regimens were tried including topotecan and cyclophosphamide, protocol advl with lorvotuzumab, and pazopanib. imaging of the chest continued to show significant progression of metastasis. the patient's clinical status deteriorated with worsening respiratory status, requiring l of oxygen therapy, and inability to ambulate. he was started on trabectedin . mg/m for palliative care. after cycles of treatment patient was no longer requiring oxygen and was ambulating without assistance. radiological imaging showed significant reduction in number and size of lung nodules. trabectedin is a recently approved alkylating agent for the management of sarcomas resistant to first line treatment. response in synovial sarcoma is scarcely documented in the pediatric population. epidemiology places the most common age group in the young adults and children. our case opens the doors to further consideration of the use of trabectedin in the pediatric patient with metastatic synovial sarcoma. background: gata is an x-linked gene that plays critical role in hematopoiesis. mutations of gata gene can be associated to various blood disorders including diamond blackfan anemia, cytopenia, congenital dyserythropoietc anemia and acute megakaryoblastic leukemia. we report a patient with macrocytic anemia and platelet dysfunction who carries a novel gata mutation that has not been reported. results: a now -month-old male with complex medical history including prematurity at weeks, dysmorphic features, global developmental delay, hyperinsulinism, hypogonadotropic hypogonadism, growth hormone deficiency, micropenis, failure to thrive, patent ductus arteriosus status post ligation, and severe hypotonia, was referred to hematology at months old for resolved, transient thrombocytopenia and macrocytic anemia since month of age. chromosomal microarray showed chromosome deletion of q . , which is the rps gene. he doesn't have a family history of diamond blackfan anemia (dba), despite mom having the same rps mutation. he was then diagnosed with dba. his lab workup showed mild macrocytic anemia (hgb . g/dl, mcv fl), normal to inappropriately low reticulocyte count, normal white blood cell and platelet counts, hgf %, erythroid ada . eu/gm hgb (elevated). he has abnormal pfa- , with prolonged closure time of both adp and epinephrine. he had low von willebrand antigen and ristocetin cofactor activity. he has severe pancreatic insufficiency. bone marrow biopsy showed normocellular marrow with trilineage hematopoietic maturation, without ringed sideroblasts. since mother has the same rps gene mutation, maternal labs were done and showed no evidence of macroytosis or anemia. the diagnosis of dba was questioned. whole exome sequencing did not identify any pathogenic sequence changes in the coding regions of rps gene, but detected a gata mutation r w, which was reported variant of uncertain significance. his mother shares the same mutation and is asymptomatic, but she may not be affected since gata iis xlinked. his father doesn't harbor the gata mutation. conclusion: gata gene encodes zinc finger dna binding hematopoietic transcription factor, which is important during erythroid differentiation. gata mutation r w has not been reported in literature and is a novel variant of gata mutation, which might be contributing to this patient's clinical picture. further studies are warranted to confirm gata mutation r w to be a pathogenic sequence change. alexander boucher, tomoyuki mizuno, alexander vinks, greg tiao, stuart goldstein, james geller cincinnati children's hospital medical center, cincinnati, ohio, united states background: hepatoblastoma (hb), the most common pediatric primary hepatic malignancy, can be associated with specific congenital syndromes. recently, chronic kidney disease and genitourinary anomalies have been linked to hb. cisplatin is a key chemotherapeutic agent in treating hb but its renal clearance and toxicity profile can limit its use for those with end-stage renal disease (esrd). objectives: using an institutional case series, we present data using cisplatin for hb in dialysis-dependent esrd and define recommended dosing for future use. design/method: a chart review of patients with concurrent hb and esrd on dialysis treated with cisplatin at our institution was undertaken. demographic data, diagnostic history, tumor pathology, alpha fetoprotein (afp), hearing assessments, dosing schema, treatment outcomes, and therapyrelated toxicities were reviewed. total cisplatin levels were collected at time points within days after each infusion. free cisplatin levels were also collected for infusions, as were dialysate cisplatin levels. pk parameters were generated using bayesian estimation with a published population pk model as a priori information. results: three patients meeting these criteria were identified. each had "low risk" (non-metastatic resectable) disease at presentation and underwent upfront resections. all had congenital renal anomalies with esrd prior to their hb diagnosis. all cisplatin infusions were given over hours, followed hours later by hemodialysis. patients and received cisplatin at % of children's oncology group's ahep weight-based dosing ( . mg/kg). patient received % of ahep body surface area-based dosing ( mg/m ) during cycle but required a second dose reduction ( mg/m ) for cycle due to prolonged cisplatin exposure (total area under the curve mg⋅h/l; average for all seven evaluable cycles mg⋅h/l) and early sensorineural hearing loss at - hz. no other hearing loss in any patient was identified; mild toxicities also included grade - emesis and grade neutropenia and thrombocytopenia. the median (range) of clearance, volume of distribution at steady-state, and elimination half-life at terminal phase for total platinum were . ( . - . ) l/hour/ kg, . ( . - . ) l/ kg and ( - ) hours, respectively. patients and received cycles with rapid afp normalization. patient required an additional cycles, for a likely second primary hb year after initial therapy. cisplatin can be used successfully in pediatric patients with esrd on hemodialysis to treat hb with minimal morbidity using % standard mg/kg-based dosing ( . mg/kg), achieving pharmacologically appropriate cisplatin exposures. background: treatment for immune thrombocytopenia (itp) has been grouped into rescue and maintenance therapy and often is reserved for patients with bleeding, severe thrombocytopenia, or for improvement in quality of life. splenectomy is considered one of the more invasive but definitive treatments with success rates of - %. treatment of itp can be more difficult in the setting of previous treatment with immune modulation or when the patient is immunocompromised and not a candidate for splenectomy. objectives: present an interesting case of a patient with an autoimmune disease that presented with severe thrombocytopenia, un-responsive to rescue therapy, and requiring emergent splenectomy in the setting of acute intracranial hemorrhage (ich). a year old female with a history of juvenile dermatomyositis presented with a fine purpuric rash on her extremities, wet purpura, and a platelet count of k/ l. bone marrow evaluation at that time was consistent with itp. she was on cyclosporine and plaquenil for dermatomyositis. platelets failed to increase after three doses of intravenous immunoglobulin and high dose steroids. following a two week course of oral prednisone and eltrombopag, she presented with persistent severe thrombocytopenia of k/ l, anemia of . g/dl, and a lower gi bleed. she was started on amicar, novo-seven, rituximab, and given platelet transfusions with no improvement in bleeding. subsequently, she developed a subdural hematoma with midline shift. surgery performed an emergent open splenectomy with concurrent continuous platelet transfusion. results: she was monitored closely post operatively and, due to ich, transfused to maintain platelets greater than k/ l. by week post-op she had normal platelet counts off transfusions. all medications were stopped within three days of discharge. she represented eight days later with abdominal pain and thrombocytosis and was found to have a portal vein, splenic vein and mesenteric vein thrombosis. she was started on lovenox therapy and admitted for monitoring due to her history of ich. it is unknown whether our patient's underlying immune dysregulation and history of treatment with immunosuppressive medications may have contributed to her unresponsiveness to multiple therapeutic agents. in addition, her significant bleeding did not allow us to fully evaluate her response to second tier therapy. this adds to the scarcity of literature of itp response in pediatric patients with autoimmune disease, and may support more aggressive therapy upfront in these patients. background: multivisceral organ transplantation involves concurrent transplantation of the stomach, pancreas, liver, and intestine with splenectomy, and has been classically used in the pediatric population for infants with intestinal failure from disorders affecting foregut integrity. while there is some data demonstrating its efficacy in adults with low-grade abdominal malignancies, it has not been traditionally used for hepatocellular carcinoma treatment. to describe a unique pediatric case of multivisceral organ transplantation as definitive therapy for refractory fibrolamellar hepatocellular carcinoma in an adolescent male. a year old male presents with a history of fibrolamellar hepatocellular carcinoma, tumor invasion of the portal vein, severe portal hypertension complicated by bleeding esophageal varices and hypersplenism. he had two treatments with yttrium- radioembolization, without significant response. he completed six cycles of traditional chemotherapy in combination with sorafenib with resolution of petavidity, but minimal decrease in tumor size and continued portal hypertension. since his disease remained relatively stable for over years, he was evaluated and listed for multivisceral organ transplantation. at approximately years and months after diagnosis, he underwent en bloc liver, pancreas, stomach, small bowel, and colon transplant with splenectomy. a single lymph node was positive for malignancy at the time of resection. in addition to expected post-transplant complications, he also developed skin only acute graft versus host disease at weeks after transplant, treated successfully with a thymoglobulin course. he clinically improved and was back to his baseline activity level, on full oral feedings within months post-transplantation. at three and six month post-transplantation, there is no concern for relapsed hepatocellular carcinoma on comprehensive imaging and evaluation. he is maintained on protocol immunosuppression and posttransplant support. we present the first known case of successful multivisceral organ transplantation in the treatment of refractory pediatric fibrolamellar hepatocellular carcinoma. background: hematohidrosis is a rare disorder that presents with spontaneous excretion of whole blood from intact skin or mucosa. diagnosis is based on clinical observation of the occurrence with the proven presence of erythrocytes and other blood components, without other abnormalities to account for the phenomenon. the existing literature is scarce and consists of primarily case studies. most reports describe bleeding from facial sites around the eyes, ears, and nose. the available literature suggests anxiety and physical or emotional stress reactions as the most common inciting events. little evidence exists regarding the ideal therapeutic approach, however propranolol has been used successfully to reduce bleeding frequency and severity in multiple case reports. a specific genetic etiology has not been elucidated, and no familial cases have previously been reported. we present a pair of half-siblings, both of whom presented with spontaneous cutaneous and mucosal bleeding before two years of age, and report on preliminary results of propranolol therapy. tanzania. at months of age, he became ill and developed spontaneous bleeding from his ears, nose, and scalp. he continued to have frequent bleeding episodes, usually related to illness or physical distress. a bleeding diathesis work-up was unremarkable, however some episodes were severe enough to require transfusions. the patient was subsequently diagnosed with hiv and hepatitis b, presumably acquired via unscreened blood product transfusions. patient b is an infant female born to the same mother as patient a, with a different father. she was healthy until two months of age when she developed spontaneous bleeding from the hairline, eyelids, ears and genital/rectal area. bleeding episodes were nearly always associated with irritability and crying. extensive coagulation workup was unremarkable. results: propranolol therapy was started in both patients, titrated to a goal of mg/kg/day. in both patients, the frequency and duration of bleeding episodes significantly improved. patient b continues to have milder occasional bleeding episodes from her eyes, ears and scalp but has significantly less discomfort and irritability during the episodes. conclusion: to our knowledge, there are no prior reports involving two related patients with hematohidrosis. this case series suggests that there may be a genetic predisposition which has yet to be identified. propranolol has shown effectiveness in reducing symptom frequency and severity. background: gliomas are the most common central nervous system tumors in children. they are classified into different grades based on genotype (idh, braf, tsc, etc.). lowgrade gliomas such as oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas are classified as grades i and ii. of the molecular level alterations this case report focuses on the braf v e mutation. braf is a member of the raf family of serine/threonine protein kinases and it plays an important role in cell survival, proliferation and terminal differentiation. objectives: here we discuss two cases where dabrafenib, a braf kinase inhibitor, was utilized in the management of gliomas. the cases focus on the use of dabrafenib late versus early in disease course. design/method: patient jl is a year old female who was diagnosed with a low-grade glioneuronal tumor (c -t with a metastatic lesion to the brain) in . jl was treated with chemotherapy, radiation, and surgical resection. despite treatment, the patient's disease progressed. she developed lower extremity dysfunction, urinary incontinence, poor truncal control, and hydrocephalus. dabrafenib was started after the braf v e mutation was confirmed. patient lg is a year old female who presented in november with left facial and upper extremity weakness. ct and mri scans demonstrated a mixed solid and cystic lesion extending from the optic chiasm and hypothalamus to the right thalamus and posterior basal ganglia with additional involvement of the right cerebral peduncle. neurosurgical intervention was undertaken and dabrafenib was started after the braf v e mutation was confirmed. results: patient jl's mri scans have demonstrated improvement of the spine with diminished areas of enhancement along thecal margins, decreased volume and enhancement within the trigeminal plate cistern and resolution of ependymal enhancement within the right ventricle. the patient's most recent mri exhibits no disease progression in head or spine. jl has shown improvement clinically since starting dabrafenib. patient lg has shown improvement in strength and recent mri of the brain has shown resolution of enhancement along surgical resection margins, decreased hyperintensity along the inferomedial aspect of the right basal ganglia and no new enhancements. conclusion: low grade gliomas can alter a person's quality of life and even lead to life threatening complications. often the standard chemotherapy, radiation and surgery don't prevent these complications. genetic analysis can help clinicians target therapy towards certain mutations such as braf v e. dabrafenib has shown to decrease tumor burden, early utilization as therapy can help prevent morbidity and mortality. children's hospital of pittsburgh of upmc, pittsburgh, pennsylvania, united states background: copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. copper requirements in children over the age of are mg/day, which is readily acquired in a typical diet. copper deficiency is known to occur in patients with the rare x-linked mutation and in older individuals with gastrointestinal bypass surgery; however, it is rarely reported in other conditions. objectives: to highlight individuals with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency, thus a high index of suspicion must exist in order to diagnose the disorder. design/method: a y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with slowly progressive fatigue, weakness, gait instability, and weight loss. his longstanding feeding difficulties were refractory to intensive feeding programs. his daily diet consisted of - oz of milk and - individual servings of butterscotch pudding ( - calories/day, . mg iron/day). initial complete blood count demonstrated white blood cell count of . , absolute neutrophil count of , hemoglobin of . , mean corpuscular volume of < , reticulocyte count of . , platelet count of . review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis and ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. iron studies demonstrated ferritin of , total iron binding capacity of , and % iron saturation. he had no evidence of b , folate deficiency or blood loss. additional evaluation revealed a serum copper level of (range - ), and cerulosplasmin of . (range - ). results: once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. he received iv copper mcg/kg/day x days then weekly intravenous infusions. given his malnutrition, a gtube was placed to begin oral copper repletion and enteral nutrition. within weeks his copper level improved as well as his blood counts. unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although, he has made gains in his gross and fine motor abilities. conclusion: acquired copper deficiency in the pediatric population is a rare event but given the hematologic and neurologic consequences, prompt recognition and treatment is important. this patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, b and folate. background: lymphoepithelioma-like thymic carcinoma (lelc) is a rare, aggressive neoplasm with a high rate of invasion, metastasis and recurrence. there are no known curative therapies for metastatic lelc. we report the case of a -year-old male who presented with metastatic ebv positive lelc. sites of disease included a large primary anterior mediastinal mass and metastases to hilar lymph nodes, lungs and liver. he was initially treated with cisplatin and fluorouracil followed by mediastinal radiation. he had a partial response to therapy but his end of therapy scans showed disease progression in lungs, liver, and hilar, supraclavicular and axillary lymph nodes. objectives: molecularly targeted therapies tailored to the patient's genetic profile offer a novel approach to obtain improved survival outcomes. design/method: the patient enrolled on a precision medicine trial, nmtrc : molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancer (nct ). in this study, tumor/normal whole exome sequencing and tumor rna sequencing were performed and a molecular report detailing the results of genomic and gene expression analysis was generated. a treatment plan was designed within a molecular tumor board comprising oncologists, pharmacists, genomicists, and molecular biologists with domain expertise. results: exome sequencing revealed somatic coding point mutations and no structural mutations (focal copy number changes or translocations). candidate somatic driver mutations included tp s x and r w as well as kit n k. both genes have been previously implicated in thymic carcinoma. rna expression analysis demonstrated aberrant activation of biological pathways, including overexpression of kit, hdac , and , tyms, and dhfr. the molecular tumor board selected the combination of pemetrexed ( mg/m ) on day of a day cycle, imatinib ( mg daily), and vorinostat ( mg days - , - , and - ) . on day of cycle , he was admitted with a herpes zoster infection and imatinib was discontinued in order to reduce risk of herpes zoster recurrence. imaging after cycles showed a complete metabolic response on f- fdg pet and a partial response by ct size criteria. as of december , the patient had received cycles of pemetrexed and vorinostat. scans in december showed an increase in the size and metabolic activity of two right lower lobe pulmonary nodules. there were no new sites of disease and imatinib was re-started. background: systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organ systems and is associated with many different autoantibodies. patients can present with vague constitutional symptoms including fever, rash, fatigue, and weight loss. some of the various hematologic manifestations of sle include anemia of chronic disease, leukopenia, autoimmune hemolytic anemia (aiha), and idiopathic thrombocytopenic purpura (itp). these can be the presenting signs of sle. evans syndrome (es), a disease characterized by itp and aiha, is a rare hematologic manifestation of sle. neurofibromatosis (nf ) is a relatively common neurocutaneous disorder. these patients are at risk of developing benign and malignant tumors. its association with autoimmune disorders, including sle, remains rare. objectives: there are few cases in the literature that have patients with the combination of sle and nf . this is the only case that has a patient with sle, nf , and es. results: a -year-old caucasian female presents with two months of vaginal bleeding, weight loss and petechiae. her exam is remarkable for petechiae and café au lait macules. laboratory findings show severe anemia and thrombocytopenia. she receives blood and platelet transfusions during stabilization, and a bone marrow aspirate is performed to rule out a malignancy which is negative. based on the presence of thrombocytopenia and a positive coombs test, an autoimmune process such as es is considered. screening tests for sle reveal positive antinuclear and anti-double stranded dna antibodies as well as low complement. she receives intravenous immunoglobulin and methylprednisolone and eventually her vaginal bleeding slows and her counts recover. she begins sle therapy with hydroxychloroquine and azathioprine. due to the presence of café au lait macules on her exam, a genetics evaluation is performed and the patient is also diagnosed with nf . to date, there are seven cases of sle with nf reported in the literature, only two of which are pediatric cases. there are no reports of the combination of sle, nf , and es. conclusion: es is a rare hematologic manifestation of sle but can be the initial presentation of this disease. one large study estimates % of childhood-onset sle cases are observed to have es. screening for sle should be considered in all es patients even in the absence of typical clinical findings. association of nf and sle has been rarely described. whether this association reflects a causal relationship or is coincidental needs more investigation. (lube, ped blood & cancer, ) . university of california san diego, la jolla, california, united states background: high grade glioma (hgg) has poor outcomes in adults and children. extraneural metastases are very rare in hgg, and poorly characterized with only a few small case series in adults and only isolated case reports in pediatrics. no genomic data has previously been published for any children with hgg who develop extraneural metastases. objectives: our objective is to describe the natural history of two children with hgg and bony extraneural metastases, comparing their clinical characteristics as well as whole exome sequencing data for both tumors. this information would suggest similar patients should be monitored closely for extraneural metastasis and may benefit from more systemic therapy. design/method: we present a case series of two patients who presented with hgg and had development of bony metastases less than six months after initial diagnosis. both patients had molecular profiling with whole exome sequencing (wes). the first patient was an -year-old male with a tumor found in the left lateral ventricle invading into the fornices, hypothalamus, and left midbrain, who had subtotal resection. bony metastasis were found at . months after diagnosis, and he died months after diagnosis. he initially received radiation, followed by nivolimuab. the second was a -year-old female with a tectal/pineal tumor and multiple spinal cord metastases, who had subtotal resection. she developed bony metastasis at . months after diagnosis and died months after diagnosis. her histologic diagnosis was pineoblastoma, revised to hgg after whole exome sequencing. she received craniospinal radiation followed by chemotherapy per acns (cisplatin, vincristine, and cyclophosphamide) for cycles. when she failed to respond satisfactorily to this therapy, wes of tumor was performed and the findings were consistent with hgg. treatment was transitioned to temodar and lomustine after hgg diagnosis was given. she had ongoing progressive disease despite this therapy as well as trials of nivolumab, everolimus, and vorinostat. neither patient had extraneural metastasis at presentation. in both tumors, whole exome sequencing identified the h f a k m mutation. both tumors also had additional known mutations associated with hgg but no other overlapping mutations. this case series represents the first description of the genetic alterations of pediatric hgg patients who developed extraneural metastases. while h f a k m is a common mutation in pediatric midline hgg, especially dipg, and is associated with more aggressive disease, there has not been an association with extraneural metastasis prior to this series. background: deferiprone-induced agranulocytosis is a well -known albeit rare side effect of the drug. incidence of agranulocytosis varies from . - . %, while milder neutropenia is reported in . % of patients treated with deferiprone. deferasirox is unknown to cause such a complication. clinical trials and post marketing side effect monitoring studied possible correlations between different risk factors and development of agranulocytosis. unfortunately, no studies directly addressed a special risk in a community with background of ethnic neutropenia, like oman. objectives: to report on the incidence of neutropenia among omani children with b thalassemia using different iron chelators design/method: a retrospective study conducted on patients < year-old with b thalassemia treated with different iron chelators. electronic patients records were reviewed to detect episodes of neutropenia either mild (anc . -< . /cmm), moderate (anc . -< ), severe < . , or agranulocytosis anc = ). data were collected including sex, age, personal or family history of ethnic neutropenia, iron chelating agent, infective complications, management and outcome. detailed clinical, laboratory ± radiological information were reported for patients who developed life-threatening agranulocytosis. among young patients with b thalassemia, treated between - in squh, neutropenia, was reported in patients ( . %).severe neutropenia was encountered on occasions in patients ( / : . %) ( on deferiprone including episodes of agranulocytosis, on defersirox, on combined chelation, and off chelation). moderate neutropenia was encountered in patients ( / : . %), on occasions: deferiprone ( ), deferasirox ( ), combined chelation ( ), and episodes off chelation. mild neutropenia was more prevalent, encountered in patients ( . %) on occasions ( on deferiprone, on defersirox, on combined chelation, and off chelation) of patients exposed to deferiprone, patients had neutropenia ( %), higher than previously reported. deferiproneinduced agranulocytosis was encountered in patients ( / = . %). three of them had life threatening complications. one patient developed pneumonia complicated by rupture of pulmonary artery aneurysm-massive hemoptysis, who recovered fully after catheter embolization. the second had facial cellulitis and treatment with gcsf was complicated by frequent ventricular extrasystoles. the third had sepsis, disseminated herpes simplex and required admission to icu for inotropic support. in a community with background ethnic neutropenia, neutropenia is more common to be encountered among thalassemic patients, both on and off chelation therapy. careful monitoring of anc and rational choice/modification of chelating agents is required for optimal management of iron overload and to avoid life threatening complications. objectives: this case control study aimed to evaluate the systolic and diastolic cardiac function in groups of children with ti: non transfused group and a group that received early regular blood transfusion comparing them to healthy controls. design/method: thirteen regularly transfused patients with ti with a mean age of . + . years were compared with eight patients who are non-transfused or minimally transfused (< rbcs transfusion/year); mean age . + . years and healthy controls with a mean age of . ± . years. clinical parameters and standard echocardiographic and tissue doppler imaging (tdi) were compared. results: young non-transfused ti patients had a statistically significant higher peak late diastolic velocity of the left ventricular inflow doppler, a mitral valve a wave duration over the pulmonary vein a wave duration ratio and the pulmonary s of s vein s/d velocities ratio compared to the transfused group with p values of . , . , . respectively. in addition, they have a lower e/a ratio of the mitral valve inflow and a larger left atrial to aortic diameter ratio compared to the control group with p values of . and . respectively. the diameters of the right and left outflow tract were significantly larger in the non transfused group with a trend to have a higher cardiac index compare to the transfused group. systolic function was similar in the studied groups and none of the patients had evidence of pulmonary hypertension. young patients with ti who are receiving early regular blood transfusion have normal systolic function. diastolic function assessment revealed indicators of an abnormal relaxation of the left ventricle in the non transfused group which indicate diastolic dysfunction. the abnormalities affected multiple diastolic function parameters which give an indication that the changes are clinically significant. a statistically significant increase in the diameters of the outflow tracts are likely attributed to high cardiac output status in nontransfused ti patients as they had a trend to have a higher cardiac index. these findings support the early commencing of regular blood transfusion therapy for ti patients to prevent serious cardiac complications in adult life. background: in the -week sustain study, crizanlizumab . mg/kg significantly reduced the frequency of scpcs versus placebo ( . vs . , p = . ) and increased the time to first on-treatment scpc ( . vs . months, p = . ) in patients with sickle cell disease (scd). to evaluate time to first scpc in sustain study subgroups and the likelihood of not experiencing scpc for the duration of the trial using post hoc analyses. design/method: sustain was a randomized, double-blind, placebo-controlled, phase study (nct ). inclusion criteria were: scd patients aged - years; - scpcs in previous months; concomitant hydroxyurea use permitted if ≥ months and stable dose for ≥ months. patients were randomized : : to receive intravenous crizanlizumab . mg/kg, . mg/kg, or placebo. study treatments were administered on days and , then every weeks to week , with the final assessment at week . median time to first scpc after first dose was summarized for crizanlizumab . mg/kg or placebo in these subgroups: - or - scpcs in previous months; scd genotype; and hydroxyurea use at baseline. hazard ratios (hrs) for crizanlizumab . mg/kg versus placebo were calculated based on cox regression analysis, with treatment as a covariate. descriptive statistics were used to summarize the frequency of patients who were scpc event-free for the duration of the study by prior scpc events, scd genotype, and hydroxyurea use at baseline. : patients received crizanlizumab . mg/kg and received placebo. there was a meaningful delay in time to first scpc with crizanlizumab . mg/kg versus placebo observed in the entire study population. the effect was present in both scpc subgroups, and the largest treatment difference was observed in hbss scd versus other genotypes ( . vs . months; hr: . ). in patients taking hydroxyurea who experienced - scpcs in the previous year, time to first onstudy scpc was longer with crizanlizumab . mg/kg versus placebo ( . vs . months; hr: . ). a greater proportion of patients treated with crizanlizumab . mg/kg were scpc event-free versus placebo in each of the analyzed subgroups. one third of patients who were taking hydroxyurea and treated with crizanlizumab . mg/kg were scpc event-free during the study versus . % with placebo, possibly suggesting an additive effect. with crizanlizumab . mg/kg, there was a clinically meaningful delay in time to first scpc and an increased likelihood of being scpc-free versus placebo in all subgroups investigated. cincinnati children's hospital medical center, cincinnati, ohio, united states background: shwachman-diamond syndrome (sds) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (mds) and acute myeloid leukemia (aml). objectives: this multi-institutional retrospective study investigated clinical features, treatment, and outcomes of sds patients who developed mds or aml by central pathology review. design/method: nine individuals presented with aml ( male, female), mds-eb / ( males, females, with mds ( male and female), and one male with isolated persistent somatic tp mutation. one mds-eb and mds patient progressed to aml. median age (years) at diagnosis of mds was (range . - ), mds-eb / was (range . - ) and aml was . (range . - ). complex cytogenetics were noted in / aml cases, with one having normal cytogenetics. complex clonal cytogenetic abnormalities were noted in of mds-eb /eb patients and clonal abnormalities in of mds patients. follow up was available for aml patients; are deceased. received chemotherapy with intent to proceed to hematopoietic stem cell transplant (hsct). four failed to achieve remission and died with disease without proceeding to transplant. one patient proceeded to hsct without prior chemotherapy. four of six transplanted subjects died with relapsed disease. treatment related mortality was largely infectious or gvhd. the sole surviving aml patient had normal cytogenetics, achieved remission with chemotherapy and underwent hscts with separate stem cell infusions due to two primary graft failures. he remains alive in remission more than years after diagnosis. of the mds-eb / patients, underwent ric hsct, three of whom are alive, one died of infection. the fifth patient has stable disease on continued decitabine monotherapy for . years. of mds patients with treatment data, had upfront hsct therapy, upfront chemotherapy and had no therapy. three patients required ≥ hscts all due to graft failure. follow up is available for , of whom are deceased, with relapsed disease. treatment related mortality was largely infectious or graft failure. one individual died of hepatic failure unrelated to mds. seven mds patients are alive in remission. in summary, prognosis is poor for patients with sds who develop aml due to resistant disease and treatment-related complications. better markers for risk stratification are needed to identify patients who would benefit from early transplant. novel therapeutic strategies are urgently needed to improve outcomes of sds patients with mds or aml. background: unlike primary myelofibrosis (pmf) in adults, which is associated with somatic mutations in jak , mpl, or calr, myelofibrosis in children is rare and the underlying genetic mechanisms remain elusive. here we describe families with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis (cmtfm) due to germline mutations in the megakaryocyte-specific immune receptor tyrosine-based inhibitory motif (itim) receptor g b-b. objectives: to characterize the clinical phenotype, histological features and identify the causative gene for cmtfm. we performed affymetrix snp . genotyping on the index family to identify shared regions of homozygosity by descent. whole exome sequencing (ws) was performed on all three pedigrees to identify potentially causative mutations. we studied affected children from families, with macrothrombocytopenia, anemia, mild leukocytosis and a distinctive pattern of bone marrow (bm) fibrosis centered around clusters of atypical megakaryocytes. affected children had mild to moderate bleeding symptoms and required platelet and red cell transfusions. none showed evidence of extramedullary hematopoiesis, and all were negative for mutations in jak , mpl, and calr. snp genotyping identified multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (mhc) on chromosome p (lod = . ). we focused on this region because affected individuals in two families shared a common homozygous human leukocyte antigen (hla) type and had congenital adrenal hyperplasia (cah) due to -hydroxylase (cyp a ) mutation; the cyp a and hla loci are located at p . and p . - p . . wes revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor g b-b, encoded within the candidate linkage region. we identified two distinct g b-b frameshift mutations (c. _ + dup; p. fs and c. inst; p. fs) in individuals within these three families. no other mutations that segregated with the phenotype were identified. to validate g b-b as a potential disease-causing gene, we evaluated g b-b expression in bm biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody. g b-b was strongly s of s and selectively expressed in megakaryocytes of control samples, but completely absent in clinically affected individuals. a murine knockout that lacks g b-b has a strikingly similar phenotype with macrothrombocytopenia, myelofibrosis and aberrant platelet production and function, further affirming the causality of g b-b mutations. we showed that autosomal recessive loss-offunction mutations in g b-b cause cmtfm, uncovering the molecular basis of this rare disease. loss of g b-b-dependent inhibition of megakaryocyte activation likely underlies the distinctive focal myelofibrotic phenotype and might be important in other forms of marrow fibrosis. cardinal glennon, saint louis, missouri, united states background: intrauterine transfusion is the method of choice for management of fetal anemia due to red blood cell alloimmunization. despite the decrease in prevalence of anemia due to rhesus d alloimmunization with prophylactic administration of anti-rhd immunoglobulin in rh d negative patients, maternal red red blood cell alloimmunization with other type of red blood cell antigens remains an important cause of fetal anemia. newborn who received intrauterine transfusion for hemolytic disease may have prolonged postnatal transfusion requirement. objectives: -to evaluate clinical outcome of fetuses and newborns who received intrauterine transfusions. -to determine the need of packed red blood cell transfusions until months of age. we conducted a retrospective case series study of all intrauterine transfusions due to anemia secondary to red blood cell alloimmunization performed in our regional center ssm in st louis missouri, between april and january . we evaluated the indications, diagnosis, gestational age, and frequency of intrauterine transfusions, along with the infant's gestational age at birth, duration of admission, timing of blood transfusion and monitoring of hemoglobin. results: intrauterine transfusions were performed in patients. the most common causes of alloimmunization were due to d antibodies (n = , %) and kell antibodies (n = , . %). the median gestational age of the first intrauterine transfusion was . weeks, and the median pre-transfusion hemoglobin was . g/dl. the gestational age at the first intrauterine transfusions was found to be significantly correlated with the number of postnatal transfusions (r = . . p = . ). the median gestational age at birth was found to be weeks ( . - . weeks), with a hemoglobin of . ( . - . ). in our population, patients ( %) received postnatal transfusions, of which were during the first weeks of life, and close monitoring follow up with a hematologist was established in patients at their discharge from the nursery/nicu. one neonatal death occurred and severe morbidity due to severe anemia occurred in one infant. despite the continuing risk factor for persistent anemia, only patients had follow up hemoglobin monitored by their primary care provider. conclusion: infants with anemia due to red blood cell alloimmunization treated with intrauterine transfusion should be monitored closely via regular complete blood count for persistent anemia due to suppression of fetal erythropoiesis. sebastian hesse, piotr grabowski, juri rappsilber, christoph klein dr. von hauner childen's hospital, lmu university hospital, munich, munich, germany background: neutrophil granulocytes are the most abundant leukocytes in the peripheral blood. validated diagnostic options for these cells are limited, leaving many patients with functional neutrophil defects without a defined diagnosis. objectives: here we evaluate proteomics as a new diagnostic tool to investigate defects of neutrophil granulocytes. we analyzed neutrophil granulocytes from children with severe congenital neutropenia (scn) associated with elane mutations, children with chronic granulomatous disease (cgd) with cyba ( ) or cybb ( ) mutations and children with leukocyte adhesion deficiency (lad) due to itgb mutations. in addition we collected samples of children with genetically undetermined neutrophil defects. neutrophils from healthy individuals served as controls. cells were isolated from fresh venous blood using negative selection (purity > %). whole cell proteome analysis was done by data-independent acquisition. showed a correlation coefficient of ∼ . . principal component analysis demonstrated unequivocal separation of the proteome of healthy and diseased cells. differential expression analysis showed minimal proteome aberrations in lad with deficiency in cell surface receptors and upregulation of alpl (total downregulated proteins: / total upregulated proteins: ). analysis of neutrophils from cgd patients also showed limited proteome aberration. cyba and cybb were both diminished independent of genotype, whereas protein clusters around a stat / centered network were increased (total down: / up: ). neutrophils with elane mutations showed the gravest proteome disturbance (total down: / up: ) with an upregulated translational apparatus (srpdependent ribosomes and protein folding complexes) and increased mitochondrial proteins. proteins of each granule subset were dysregulated and metabolic pathways upregulated. a detailed analysis of the proteome from patients with genetically undefined diseases is currently ongoing. one patient with clinical phenotype of cgd was found to have no mutations of nadph oxidase members in whole exome sequencing but critically low levels of ncf on protein level. heterozygosity mapping showed autozytocity in the ncf region warranting current efforts to sequence promoters and intronic regions of the gene. mass spectrometry based proteomics promises exciting new insights into monogenic disease of neutrophil granulocytes and may offer new diagnostic options, in particular in synergy with genome sequencing. by virtue of our international care-for-rare alliance, open to new partners, we hope that our proteome focus may lead to better delineation of as yet unknown disease of neutrophil granulocytes. background: warm autoimmune hemolytic anemia (aiha) is an igg mediated disease. although it can be post-viral, it is often idiopathic and can also be a forme fruste for malignancy or an autoimmune disease. initial management includes steroids. it often relapses on steroid wean and can be refractory to the use of second line treatment such as rituximab. objectives: abatacept (ctla- -ig fusion protein, ctla- mimetic) has been used to ameliorate autoimmune manifestation associated with ctla- haploinsufficiency. we used abatacept as a novel therapeutic agent to manage patients with refractory aiha. design/method: a retrospective case series of two patients at phoenix children's hospital with severe refractory aiha. results: patient , a previously healthy year old female, presented with weeks of icterus, fatigue, and hemoglobinuria. spleen was enlarged cm below the costal margin. laboratory evaluation demonstrated: hemoglobin . g/dl, mild leukopenia /microliter, platelets , /microliter, reticulocytosis . %, positive direct coombs' test, mycoplasma igm and igg positive. bone marrow evaluation showed a hypercellular marrow. she continued to need packed red blood cell (prbc) transfusions despite receiving high dose steroids, ivig and rituximab from may-july . in august, she started sirolimus decreasing her transfusion requirement. after starting abatacept ( mg/kg/dose bi-monthly for three doses and then monthly) in october, she maintained hemoglobin of - g/dl without transfusion. patient , a previously healthy month old male, presented with one week of progressive fatigue, jaundice, and poor feeding. splenomegaly was absent. laboratory evaluation revealed hemoglobin . g/dl, leukocytosis , /microliter, platelets , /microliter, reticulocytosis . %, negative direct coombs' test, and non-specific reactivity on antibody screen. evaluation for inherited hemolytic anemia including a next generation sequencing panel was negative. further evaluation by blood bank showed + positive coombs' for c d due to a warm antibody. cold agglutinin disease was ruled out. bone marrow evaluation was normal. he received high dose ivig as a steroid sparing agent but continued to require prbc transfusions weekly. when prednisone did not seem to slow down hemolysis, treatment with abatacept was initiated and he has not required transfusions for two months. steroids are being weaned. we present successful treatment of two refractory aiha cases with abatacept. patient is steroid and transfusion free and continues on monthly abatacept and sirolimus. patient is also transfusion free and continues on a steroid taper. ctla- is crucial for suppressive function of treg cells. abatacept by binding to cd / seems to enhance treg activity ameliorating autoimmune hemolysis. children's minnesota, minneapolis, minnesota, united states s of s background: transfusional iron overload is common in patients receiving chronic red cell transfusions. as a result, iron chelation is required to minimize toxicity from iron overload. chelation with a single agent can be inadequate at controlling or reducing iron burden. when combination therapy is required deferoxamine may be added to oral chelation. deferoxamine is generally given subcutaneous over - hours for - days a week at - mg/kg/day. many patients struggle to remain compliant with this schedule which has prompted trials of intravenous high-dose (hd) deferoxamine. prior reports of short-term hd deferoxamine have shown minimal side effects however, prolonged use of hd deferoxamine has known toxicity. when compliance is a concern, our center has used hd deferoxamine infusions at mg/kg/hr x hours every to weeks. objectives: evaluate the safety and efficacy of hd deferoxamine at our institution to help guide future therapy. design/method: a retrospective review was completed of patients previously treated with hd deferoxamine between april and september at children's minnesota. final sample included patients ages to years with underlying diagnosis of thalassemia ( ) and diamond-blackfan anemia ( ). deferoxamine infusions were given for hours every - days with a mean length of treatment of days. results: all patients were on combination therapy with deferasirox, however deferasirox was held during deferoxamine infusion. mean pre-deferoxamine liver iron concentration (lic) was . mg/g and mean post lic was . mg/g (p = . ). ferritin mean pre-deferoxamine was ng/ml compared with mean post ng/ml (p = . ). two patients had possible allergy, leading to deferoxamine discontinuation. one patient developed hives, eye swelling and cough while the other had emesis and cough. another patient experienced facial nerve palsy of unclear etiology, which did not recur with resumption of deferoxamine. no respiratory complications were seen. results showed significant decrease in iron burden following combination therapy with high dose deferoxamine and deferasirox. no significant pulmonary, liver, renal, vision, or hearing toxicities were observed. three patients reported reactions to deferoxamine infusions. however, one of these was able to successfully continue deferoxamine without further incident. short-term, hd deferoxamine was effective at reducing lic in combination with oral chelation but requires further evaluation to assess for potential increased risk of toxicity. short-term hd deferoxamine may be considered in the setting of poor compliance of subcutaneous administration or inadequate chelation with single agent therapy. further studies are needed to clarify ideal dosing, timing and risk of toxicity. background: immune thrombocytopenia (itp) is the most common cause of symptomatic thrombocytopenia in childhood but remains a diagnosis of exclusion warranting further evaluation if atypical findings are present. two male children ( months and years old) with newly diagnosed immune thrombocytopenia (itp) were found on initial evaluation to have persistent elevations of lactate dehydrogenase (ldh), alanine aminotransferase (alt), and aspartate aminotransferase (ast). these serum enzyme abnormalities cannot be attributed to itp. in the setting of thrombocytopenia, elevated transaminases and ldh create diagnostic complexity for the hematology/oncology provider as their elevation raises concern for malignancy, hemolytic disease, and other systemic diseases. to raise awareness about an unexpected pattern of duchenne muscular dystrophy in patients undergoing evaluation for itp. to expand the differential of a hematologist/oncologist when abnormal labs support a nonhematologic diagnosis design/method: this case-series of two patients with their clinical and laboratory findings were discovered with retrospective chart review. results: after a thorough evaluation for hemolytic anemias, liver disease and infectious etiologies was negative, bone marrow and liver biopsies were considered. eventually, both children were found to have severely elevated serum creatine kinase (ck). skeletal muscle has the highest concentration of ck of any tissue. thus, significant ck elevation is almost exclusively attributable to muscle injury and is the most sensitive and specific enzyme for diagnosis of muscle disease. referral to a neuromuscular specialist and further genetic testing confirmed the diagnosis of duchenne muscular dystrophy in both children allowing initiation of appropriate interventions. to date, there is no clear genetic predisposition to itp in patients with muscular dystrophy although further investigation may be needed. hematology/oncology providers should consider obtaining a serum ck to rule out muscle disease in any male child with unexplained elevations of serum ldh and/or aminotransferases, as it provides an easy and inexpensive, non-invasive approach to screening. additionally, clinical history and physical examination can aid in the diagnosis of muscular dystrophy, with gross motor delay, abnormal muscle bulk, gower's sign, and proximal muscle weakness all possible findings. objectives: to identify the range of cbcs in patients with ds without infections, hematologic or immune disorders and to create more accurate reference ranges for total white blood count; hemoglobin; hematocrit; mcv; platelet count and absolute neutrophils (anc), lymphocytes, monocytes, eosinophils, and basophils. design/method: a retrospective investigation of healthy pediatric patients with ds who received a cbc between and as part of their medical care at a single, large, pediatric teaching hospital. the study group consisted of children with ds (male = , . %; mean age = . years, sd = . ) at time of blood draw. initially children were reviewed for possible participation in the study; however, patients were excluded due to not meeting the study's inclusion criteria. descriptive statistics were performed on demographic and clinical characteristics. kruskal-wallis h tests, anova, and t-tests were run to determine the significant associations between independent means. results: a significant difference in absolute neutrophils between racial groups, f( , . ) = . , p = . , was observed. there was an increase in anc from . +/- . with african americans to . +/- . in the other racial groups and to . +/- . with caucasians. differences were also found in anc in hispanics/latinos versus non-hispanic/latinos. the results were higher in non-hispanics and latinos, a significant difference of -. ( % ci, -. to -. ), t( ) = . , p = . . preliminary kruskal-wallis h tests run determined that there were significant differences between age groups for total white blood cell, hemoglobin, hematocrit, platelets, lymphocytes and anc. further studies are being run to evaluate in which age groups these differences lie and create reference ranges by age, race and sex. conclusion: among patients with ds, there are differences between racial groups and age groups. this data has been compared to previously established reference ranges for cbcs, but we are currently establishing healthy cbc controls which we will use to validate the reference ranges. these ranges will be published to help guide providers in workup and management of patients with ds. background: transfusion is a critical part of the care provided in the neonatal intensive care unit, but it is not without risks. low birth weight and premature infants can become anaemic from an immature haematopoietic system and frequent phlebotomy. these infants often receive multiple red blood cell transfusions. identifying infants more likely to require such intervention is important in ensuring the appropriate usage of this scarce resource. to determine whether birth weight, gestational age, gender, length of stay and mode of delivery can predict red cell concentrate (rcc) transfusion, units required, donor exposure and time to exposure. design/method: a retrospective chart review of all infants born below weeks gestation and/or birth weight less than , g who received a red blood cell transfusion between july and july in the cork university maternity hospital neonatal unit. results: infants met the inclusion criteria, ( . %) received a rcc transfusion. our study showed lower gestational age (p< . ) and lower birth weight (p< . ) infants are more likely to be transfused. donor exposure increases with a lower birth weight (p = . ). multivariate analysis showed infants with a lower gestational age (or - . per day; p< . ); lower birth weight (or - . per g; p< . ) and a longer length of stay (or . per day; p< . ) are more likely to receive a higher number of rcc transfusions. the time to first rcc transfusion is shorter in those with lower birth weight (or . per g; p< . ) and lower gestational age (or . per day; p< . ). gender and mode of delivery were not found to be predictors of red blood cell transfusion in this study. conclusion: low birth weight and premature infants are more likely to receive a rcc transfusion during admission to the neonatal unit. our study highlights predictors of rcc transfusion, donor exposure and time to transfusion. these can be used in identifying at risk infants, counselling parents and in anticipating transfusion requirements. emily southard, r. grant rowe, david williams, akiko shimamura, taizo nakano children's hospital colorado, aurora, colorado, united states background: the mecom locus encodes transcription factors that regulate hematopoietic stem cell self-renewal and maintenance. overexpression of mecom has been noted in - % of acute myeloid leukemia, several solid tumors, and denotes a poor prognosis. mutations that reduce mecom expression or that disrupt protein function, however, have been implicated in the development of bone marrow failure (bmf) through undefined pathways. an association between mecom mutations and radioulnar synostosis with amegakaryocytic thrombocytopenia (rusat) syndrome has been reported, however further characterization of this phenotype has yet to be explored. to characterize the phenotypic spectrum of a cohort of pediatric patients with novel mecom mutations. we performed a retrospective review of five patients with mecom mutations who were referred to hematology at children's hospital colorado or boston children's hospital. clinical, laboratory, and genetic data was collected on subjects and available family members. results: four of subjects were identified in infancy presenting with congenital cytopenias or physical dysmorphisms that prompted broad genetic screening. platforms for genetic detection included microarray, targeted genetic panels, and whole exome sequencing. three of subjects with cytopenias presented with congenital thrombocytopenia, of whom rapidly progressed to severe aplastic anemia. four of subjects presented with congenital anomalies, of whom demonstrated radioulnar synostosis. additional dysmorphic features identified include craniofacial (low set ears x ), cardiac (pda x , vsd x , aortic root dilation x ), pulmonary (pulmonary hypertension x , arteriovenous malformations x ), and developmental delay. one subject presented at age years with acute pancytopenia, hypocellular marrow, no dysmorphisms, and a mecom variant of unknown signif-icance. the identified mecom mutations include one . mb deletion involving several genes including mecom, one variant affecting a splice acceptor consensus sequence predicted to disrupt splicing, and three novel missense mutations, tyr cys, arg thr, and tyr cys, all of which were absent from public databases and were predicted in silico to be deleterious. we describe the phenotypic spectrum of patients with novel mecom variants. a subset of patients lacked radio-ulnar synostosis and had presence of additional systemic anomalies, demonstrating a varied clinical phenotype that is not isolated to rusat syndrome. a centralized publically accessible database to share clinically annotated mecom variants, together with analysis by experts in mecom function would advance our understanding of the clinical interpretation of mecom variants. mecom should be considered in the differential diagnosis of bone marrow failure and we advocate for the inclusion of mecom in targeted sequencing panels. cairo university, cairo, egypt background: beta thalassemia is regarded as a serious public health problem in the mediterranean region, southeast asia, and the middle east. however, very few studies have been conducted to assess the quality of life (qol) among thalassemia major patients. objectives: to assess the quality of life among b-thalassemia major patients using short form (sf)- questionnaire and to determine the factors associated with their quality of life. design/method: a cross-sectional study was conducted among thalassemia major patients who were attending the hematology outpatient clinic at cairo university hospital, during the study period. data were collected between october and march . the quality of life was assessed for patients aged ≥ years. the mean age of the studied group was . ± . years. the majority ( . %) had one monthly blood transfusion. the mean total score of sf- was . ± . . general health perception domain was the most affected domain with mean score, while vitality was the least affected one. there was no statistically significant difference between males and females regarding different quality domains except for vitality where the mean score was significantly higher in males than females (p = . ). age at onset of disease, and at first blood transfusion were the most documented factors positively correlated with the quality of life among the enrolled thalassemia patients. conclusion: the quality of life in thalassemia major patient was found to be compromised. all thalassemia patients should undergo assessment of the quality of life so that interventions focusing on the affected domains can be implemented. background: international adoption of children with special needs has become more prevalent in recent years leading to tremendous growth in the number of u.s. thalassemia patients adopted from foreign countries. currently % of the , thalassemia patients registered in the cooley's anemia foundation (caf) patient database have been adopted from foreign countries, primarily china. as this population continues to grow, further information is needed in order to provide these families with best supportive care. the primary goal of this study is to characterize the socio-demographics and health statuses of adopted children with thalassemia and their families. a secondary goal is to describe adoptive families' motivations, experiences, challenges, and support resources. design/method: a redcap survey was accessed by families of adopted children with thalassemia through the caf website and caf social media from january to august . following a four-question screen, eligible subjects were directed to complete an adoption questionnaire. families who had at least one adopted child with thalassemia receiving care at a participating thalassemia treatment center or hematology office in the u.s. were considered eligible. descriptive statistics were analyzed using sas . . respondents who were ineligible or who provided incomplete data were removed from the dataset prior to analysis. of survey respondents, qualified and completed the survey. these households had adopted a total of children with thalassemia ( . % male), most from china ( . %), where they had been living in orphanages ( . %). legal guardians identified primarily as christian ( . %). the majority had completed post-secondary education ( . %) with reported household incomes greater than $ , ( . %). most adoptive families were connected to an adoption group or community including online groups, local support groups, and adoption networks ( . %). commonly cited challenges were: ) volume of frequent medical appointments, ) insufficient support from their local care centers, and ) financial burdens. the reality of care for the population of adopted patients with thalassemia in the u.s does not seem to match the expectations set by their providers. we are hopeful this data will be used to assist adoptive families navigating the complexities of thalassemia care. the findings suggest that this population would benefit from additional outreach, education, guidance, and advocacy resources -especially in the early stages of adoption and during initiation of post-adoption medical care. background: in many higher-income countries, thalassemia major has become a chronic disorder; many outcomes are different in emerging countries with more limited resources. most analyzes of health-related quality of life (qofl) in thalassemia have been conducted in high-income settings. objectives: to assess the impact of health status on qofl in thalassemia patients in an emerging country. we assessed qofl in randomly-selected patients ( thalassemia major; with hemoglobin e thalassemia; five thalassemia intermedia) at the national thalassemia center in kurunegala, sri lanka where approximately patients are managed. treatment is free, but compared to north america/europe, access to tertiary staff and other resources are limited. overall, control of body iron as estimated by serum ferritin concentration (mean± sem, ± g/l) was not optimal in many patients. to understand the impact of health status on qofl, we used the sf v health survey, analyzing scores of physical function, pain, general health, social functioning, emotional and mental health, to generate overall physical and mental component scores. results: compared to reports from higher-income countries (american journal of hematology ; : - ), physical function scores (mean±sd, . ± . ) were similar in sri lankan patients; indeed, in three categories (physical role, social function, emotional role), sri lankan scores were slightly higher. by contrast, compared to scores from higherincome settings, those estimating bodily pain, general health, and mental health were significantly lower, resulting overall in a significantly lower physical component score in sri lankan patients. male sri lankan patients reported higher scores than females, and somewhat surprisingly, in four categories (physical function, physical role, social function and emotional role) reported higher scores than those obtained in higher-income settings. lower scores in physical functioning, leading to an overall lower physical component score, were recorded by females. patients with hemoglobin e thalassemia reported generally poorer qofl than those with thalassemia major. the lack of differences in qofl in patients with "high" and "low" hemoglobins was likely related to low pre-transfusion hbs (mean±sem, . ± . g/dl) in nearly all patients. these early data in a small cohort of thalassemia patients in an emerging setting suggest that in many patients bodily pain, reduced mental health, and poorer views of general health affect overall qofl. prospective studies in larger cohorts including evaluation of adequacy of transfusions and chelation therapy, complications, and overall accessibility of care may guide approaches to improve qofl in lower-income settings of thalassemia care. geetanjali bora, anand prakash dubey, tarun sekhri, mammen puliyel, aparna roy maulana azad medical college, new delhi, delhi, india background: in the last two decades, the presence of osteopenia has been described in optimally treated patients with transfusion dependent thalassemia, the pathogenesis of which seems to differ from osteopenia in non-transfused patients. the prevalence rate of low bone mineral density (bmd) in pediatric population is highly variable amongst studies done worldwide. furthermore, the role of metabolic and endocrine factors in determining bone mass in this population is not well understood. objectives: to assess bmd in subjects with transfusion dependent beta thalassemia by dual-energy-x rayabsorptiometry and find its co-relation with clinical, biochemical and hematological parameters. design/method: this is a comparative cross-sectional study and includes patients with transfusion dependent beta thalassemia between ages to years enrolled from a thalassemia day care center in the year - . at the time of enrollment age, sex, bmi z scores, pubertal staging, duration and type of chelation therapy were noted. enrolled subjects were scanned for bmd at lumbar spine l - and left femoral neck using dexa scan. the bmd was expressed in mean values and z scores. age, bmi, ethnicity and gender matched historic controls were used to generate z scores. ml of pre transfusion fasting venous blood samples were obtained to test for serum calcium, phosphate, alkaline phosphatase, pth, thyroid function panel, serum ferritin and serum igf- levels. mean values for pretransfusion hemoglobin and serum ferritin over last months were calculated. results: total no of subjects , median age . years, male ( %), female ( %), ethnicity % asian, bmi < rd centile ( %), pre pubertal %, all receiving transfusion and chelation therapy. prevalence of low (z score < - sd) and very low (< - . sd) bmd was %, % at l -l respectively and %, % at left femoral neck respectively. there was trend of lower bmd z scores with advancing age. statistically significant co-relation (p value < . ) was found between low bmd and low mean pretransfusion hemoglobin, serum phosphate, igf - and vitamin d levels conclusion: a sizable proportion of children and adolescents with transfusion dependent thalassemia have suboptimal bone mineral density and this decline may start as early as - years of age despite being on transfusion regimen highlighting the importance of yearly dexa screening and optimization of pre-transfusion hemoglobin, vitamin d and igf levels. vanderbilt university medical center, nashville, tennessee, united states background: it is well described that iron deficiency anemia (ida) can co-present with thrombocytosis or thrombocytopenia, though cases of thrombocytopenia are less frequent than thrombocytosis. prior reports of thrombocytopenia have included adult and pediatric patients with menorrhagia ( - ), menorrhagia due to uterine fibroids ( ), or other gynecologic abnormalities ( ). our cases highlight the pattern of ida, thrombocytopenia, and menorrhagia in the setting of significant menstrual clotting without observed gynecologic abnormalities in african-american adolescents. objectives: to describe the clinical course of three adolescent females with severe ida, menorrhagia, and thrombocytopenia. results: our cases included three female african-american patients ages - who presented with severe anemia and concurrent thrombocytopenia in the setting of menorrhagia. all three patients reported heavy and prolonged menstrual cycle bleeding with significant clots. two of the three were admitted for transfusions at presentation and noted to have significant menstrual bleeding with continued blood loss requiring additional transfusions until bleeding was controlled with estrogen therapy. these two patients were evaluated with pelvic ultrasounds revealing a prominent endometrium in both patients and hyperechoic material consistent with a clot in one patient. average hemoglobin on presentation was . gm/dl ( . - . ), average platelet count was , /mcl ( , - , ), and average mcv was ( - ). all had severe iron deficiency with an average ferritin of ng/ml ( - ) subsequently treated with oral iron. one patient had a prior history of ida that required transfusion and had subsequent normalization of her complete blood count. two patients had subsequent thrombocytosis before normalization of their platelet counts. two patients received platelet transfusions: one due to recent neurosurgical intervention with a higher goal platelet count and the other to help control menstrual bleeding after a nadir platelet count of , . a review of the clinical history and red cell indices pointed to ida and ongoing blood loss from menorrhagia as the reason for the bicytopenias. the thrombocytopenia in these cases may have been exacerbated by consumption of platelets in the significant clots all three patients reported. it is reasonable to treat with iron supplementation and supportive care which may include transfusions or management of menorrhagia with oral contraceptives or other hormonal methods. background: sickle cell disease is one of the most common inherited red blood cell disorders, yet many are not aware of their carrier status. the american college of obstetricians and gynecologists' guidelines recommend that pregnant women of african, mediterranean and southeast asian descent be screened for hemoglobinopathies with a cbc and hemoglobin electrophoresis . however, adherence to this practice and frequency of improper screening with sickledex is unknown. proper screening and counseling can impact families' knowledge, allowing for establishing relationships with pediatric hematology providers earlier. objectives: we sought to assess prenatal hemoglobinopathy screening practice patterns and methods of obstetrics & gynecology (obgyn) and family medicine providers in the nyc regional area. design/method: a cross-sectional electronic survey was administered to obgyn and family medicine practitioners from four nyc institutions. questions focused on prenatal hemoglobinopathy screening practices using case scenarios with variations on parental trait status and ethnicities. chisquare analyses were used to compare the two provider groups on categorical variables. there were total responses; surveys were complete, of which were obgyn and family medicine providers. respondents were mainly from academic medical centers, with the majority being faculty ( % of the obgyns and % of family medicine). no significant difference was found in frequencies of screening patients with a positive family history of a hemoglobinopathy. when asked about screening practices for patients without a personal/family history of a hemoglobinopathy, % of obgyns versus % of family medicine providers "always" screened for hemoglobinpathies (p = . ). when analyzed by ethnic background, there were significant differences by group in screening patients of white ( % vs %), black ( % vs %), mediterranean ( % vs %), and asian descent ( % vs %) (p≤ . for all). however, in cases where the hemoglobinopathy carrier status of both parents was known, there was no difference in screening with a hemoglobin electrophoresis. furthermore, > % of all respondents use sickledex for screening in the case scenarios. conclusion: this pilot survey highlights a difference in the methods and likelihood of prenatal hemoglobinopathy screening based on the type of prenatal care provider. screening differences can lead to variations in prenatal guidance, diagnostic procedures, informed decision-making and knowledge of families referred to pediatric hematology clinics. this is the first study analyzing prenatal screening for hemoglobinopathies in obgyn and family medicine. improving prenatal screening practices by collaborating with hematologists may decrease health care disparities and allow for earlier relationship building with pediatric hematology. . acog, opinion# , poster # hermansky-pudlak syndrome: spectrum in oman background: hermansky-pudlak syndrome (hps) is a rare autosomal recessive disorder, characterized by the triad of oculocutaneous albinism, a hemorrhagic diathesis resulting from storage pool-deficient platelets, and accumulation of ceroid/lipofuscin-like material in various tissues. before , nine different types of hermansky-pudlak syndrome were identified, which can be distinguished by their signs and symptoms and underlying genetic cause. in , a tenth type was defined based on mutations in the ap d gene. hps type is characterized in addition by severe neutropenia and recurrent sinopulmonary infection. the disease is more common in puerto rico, and this is the first report from oman. to describe the clinical, laboratory and genetic characteristics of hps sub-types in oman, including the first cases of hps type . design/method: this is a retrospective study, including cases with hps that had been suspected clinically and confirmed through genetic mutation analysis. clinical data included sex, age at presentation, initial clinical presentation (skin, eyes, development, neurological involvement, bleeding tendency, recurrent infections) and course of disease. laboratory data (complete blood counts, platelet and absolute neutrophil counts, coagulation screening, platelet function tests by platelet function analyzer, and platelet aggregation studies using different agonist had been recorded. pcr and next generation sequencing for genetic confirmation by testing mutations in hps , ap b , hps , hps , hps , hps , dtnbp ,, bloc s , bloc s genes had been done. results: seven omani cases with hps have been identified ( males and females). their age ranged between (at birth) to years. two patients had hps type , patient had type , while the other cases had hps type . no other sub-types were encountered in oman. all patients were products of consanguineous marriage. one patient had adrenal hge, while the others had mild hemorrhagic phenotype, characterized by recurrent bruising and mild epistaxis. laboratory testing confirmed variable platelet aggregation defects with different platelet agonists. all patients had characteristic hypopigmentation, iris transillumination, nystagmus, and foveal hypoplasia. both patients with hps type had the same homozygous mutation in the ap b gene (c. _ delta), and presented with severe neutropenia. early diagnosis and initiation of gcsf on one of them improved outcome and prevented the development of complications. late diagnosis in the other patient resulted in the development of bronchiectasis as a result of recurrent sinopulmonary infections. background: sickle cell disease (scd), a genetic disorder characterized by defective sickle hemoglobin (hbs), triggers red blood cell sickling, hemolysis, vaso-occlusion, and inflammation. ischemic injury from scd starts in infancy and accumulates over a lifetime, causing pain, fatigue, and progressive end-organ damage that culminates in early mortality. voxelotor (gbt ) is an oral, once-daily therapy that modulates hemoglobin's oxygen affinity, thereby inhibiting hemoglobin polymerization. objectives: to assess the safety, pharmacokinetics, and efficacy of voxelotor in pediatric patients with scd. design/method: this ongoing study is being conducted in parts: part a: a single dose of voxelotor mg in pediatric and adolescent patients; part b: multiple doses of voxelotor mg/d or mg/d for weeks in adolescents. part b's primary objective is to assess the effect of voxelotor on modifying anemia. secondary objectives include measuring other markers of disease modification, such as hemolysis; daily scd symptoms, using a patient-reported outcome (pro) measure; and safety. results: as of november , , patients ( females) had received voxelotor mg and patients ( females) had received voxelotor for ≥ weeks. the median age for the patients was years, % were receiving hydroxyurea (hu), and % had ≥ painful crises in the past year. data for hemolysis measures are available for patients who received voxelotor for weeks. six of the patients achieved a hemoglobin (hb) response of > g/dl increase. laboratory markers of hemolysis improved concordantly; the median reductions in reticulocytes and indirect bilirubin were % and %, respectively. ten of patients showed reduction in total symptom scores (tss) at week , with a % median reduction in tss from baseline. there were no treatmentrelated serious adverse events (aes) or drug discontinuations due to aes. voxelotor mg for weeks in adolescents with scd, the majority receiving hu, demonstrated consistent, sustained efficacy on hb levels and measures of hemolysis; > % of patients showed a > g/dl improvement in hb. improvement in tss in mildly symptomatic patients suggests that the pro is sensitive to treatment effect and supports use in the ongoing hope phase study. voxelotor's reassuring safety profile is consistent with results in adults. these interim results support ongoing clinical evaluation of voxelotor as a potential disease-modifying therapy for adults and children with scd. supported by global blood therapeutics. background: acute kidney injury (aki) is a common complication in sickle cell disease (scd), and a potential risk factor for sickle nephropathy. aki is associated with acute decline in hemoglobin (hb) during vaso-occlusive pain crisis and acute chest syndrome (acs). it is unclear which pathologic factor plays a stronger role in aki development during hb drop: increase in free heme during vaso-occlusive events secondary to hemolysis or hb decline itself. objectives: to investigate if hb decline alone is associated with aki, we tested if the renal function of patients with scd worsened during parvovirus b -induced transient aplastic crisis (tac), in the absence of accentuated hemolysis. design/method: with irb approval, a retrospective study of patients who had laboratory confirmed parvovirus-b was conducted. serum creatinine (scr), both during and within months from the tac event, was collected. comparisons of the clinical and laboratory characteristics were analyzed using the wilcoxon test for continuous variables. aki was defined as an increase in scr by ≥ . mg/dl or a % increase in scr from baseline. to evaluate differences in change in hb on aki risk, changes in scr during tac were compared to those during pain crisis or acs admissions by fitting a generalized linear mixed model for binary outcome. a comparative sample of acs events and vaso-occlusive pain crisis were used to estimate rates of aki according to hb levels. results: three ( %) of the patients with scd developed aki during tac. no association was identified between change in hb from baseline to tac event (p = . ). no cases of aki were identified until hb decreased < . g/dl or the change in hb was ≥ . g/dl from baseline. next, we developed a model to evaluate the impact of change in hb from baseline for patients admitted with tac, pain crisis or acs on aki. with a g/dl decrease in admission hb from baseline, patients with tac had a % probability of developing aki, while acute chest syndrome and pain crisis would have a % and % probability, respectively. our data suggest that aki is still prevalent during parvovirus b -induced tac. however, the risk of aki during a tac event is and times lower than that from severe anemia induced by acute chest syndrome and vasoocclusive pain events, respectively. hemolysis-induced anemia during scd crisis appears to have a more significant role in the development of aki as compared to agenerative anemia. background: the natural history of hemoglobin e beta thalassemia (hbethal), the commonest form of severe beta thalassemia worldwide, has been examined in very few longterm studies. previously, we reported findings in hbethal patients in sri lanka. objectives: to evaluate longterm requirements for transfusion and splenectomy, complications and death in hbethal patients. design/method: all available patients were reviewed - times annually over years. results: patients ( %) died, aged (mean ± sem) . ± . years; the (known) causes commonly included iron overload ( ) and infection ( ); patients surviving patients are aged . ± . years. of patients originally classified by severity (group the mildest, and group the most severe, phenotypes), ( %) were assessed as mild (groups and ), of whom transfusions had been discontinued in . ultimately, / ( %) resumed transfusions, often following shifts to increasingly severe phenotypes including increasing intolerance to anemia. age at resumption of transfusions (following a transfusion-free interval of . ± . years) was . ± . years; in the more severe groups and , regular transfusions were stopped in / patients and resumed in / ( %), at younger ages ( . ± . years) and after shorter transfusion-free periods ( . ± . years) than in "milder" patients. mid-parental height (mph) was ultimately achieved in %. patients ( %) were splenectomized; updated analysis of responses to splenectomy (originally "group " patients), showed that splenectomy (at . ± . years) was followed by an extended, but impermanent, transfusion-free interval ( . ± . years); % patients resumed transfusions, usually related to exercise intolerance or poor growth. in groups and , complications of anemia and ineffective erythropoiesis, including leg ulcers (in % and %) and gallstones ( % and %), were more frequent than in groups and ; fractures were observed ( - %) across all groups, except for regularly-transfused group patients ( %). pulmonary artery pressures > mm were recorded in % patients. evaluation of patients with hbethal requires observations over years, without which definition of patients as "mild" or "severe" may be misleading. while in many patients transfusions may be withheld or reduced in frequency, troublesome complications may surface with advancing age even in "milder" patients. although individual consideration of transfusion requirements is critical, the availability of effective chelation, where this can be provided without prohibitive cost, may alter the balance of risks and benefits of regular transfusions in hbethal. (premawardhena a. lancet ). background: social determinants of health (sdh) are environmental and socioeconomic factors, such as access to food and housing that affect health outcomes. pediatricians are increasingly screening for sdh as part of primary care visits, however less is known about screening for sdh in pediatric hematology. evidence suggests that sdh play a role in disease severity for children with scd, who face significant socio-economic and racial disparities. the goal of our quality improvement (qi) project was to increase the percentage of patients with scd who were connected to community resources for unmet social needs. design/method: we based our intervention on the successful implementation of wecare in our institution's pediatric primary care clinic. eligible patients were identified at the start of each clinic session. on arrival the parent was given a self-reported screening tool for six sdh (childcare, education, employment, food, utilities and housing). results were entered in the electronic health record by the physician or social worker who then printed a pre-existing resource list for patients with a positive screen. we used a series of plan-do-study-act (pdsa) cycles to study tests of change. we tracked process measures (percentage of patients screened, percentage of patients with an unmet social need who received a resource sheet), outcome measures (percentage of patients with an unmet social need who connected with a community resource) and balancing measures (staff, patient and provider satisfaction). run charts were reviewed weekly and then monthly to inform further tests of change. examples of pdsa cycles include who gave the paper survey to patients (social worker or physician versus medical assistant) and length of time between surveys ( to months). results: between august and december screening rates improved from % to %. of the patients screened, % report at least one unmet social need; of those % received a targeted list of community resources in the first month of the project, and % in the fifth month. finally, % of patients reached by phone had connected with a community resource within weeks of the clinic visit. we have successfully implemented universal screening for sdh for patients with scd in our urban pediatric hematology clinic without requiring extra staff. next steps include further pdsa cycles to connect more patients to appropriate resources, and tracking improvement in health care utilization outcomes from addressing sdh in this vulnerable patient population. background: the clinical manifestations of sickle cell disease (scd), chronic hemolytic anemia, and vaso-occlusion occur as a direct result of sickle hemoglobin (hbs) polymerization. voxelotor (gbt ) is a first-in-class, oral, oncedaily investigational agent designed to modulate hemoglobin's oxygen affinity in a targeted approach to inhibit hbs polymerization. objectives: to examine the pharmacokinetics (pk), safety, and dosing of voxelotor in children (aged - years) and adolescents (aged - years) with scd from part a of the gbt - study. design/method: gbt - is an ongoing, open-label, phase a study in patients aged - years with scd (sickle cell anemia or sickle beta zero thalassemia). part a of this study (the focus of this abstract) is examining pk of singledose ( mg) voxelotor. pk samples to measure whole blood and plasma voxelotor concentrations were collected up to days following single-dose administration. separate population pk (ppk) models were developed to describe the concentration versus time profiles of voxelotor in whole blood and plasma using nonlinear mixed effects modeling (non-mem, version . ). ppk modeling and physiologically based pk (pbpk) modeling were used to simulate voxelotor pk parameters and support dose selection for future evaluation in younger children. : part a included adolescents ( females; median age years [range - ]) and children ( females; median age . years [range - ]). mean weight was . kg (range - kg) and . kg (range - kg) in adolescents and children, respectively. voxelotor was well tolerated with no drugrelated grade ≥ adverse events (ae) or serious aes. a compartment model with first-order absorption best described the pk of voxelotor (and was the same model structure used for adults with scd). voxelotor pk exposures in adolescents were comparable to those observed in adults, but higher exposures were observed in children. ppk and pbpk modeling support the use of a weight-based dosing strategy in younger children (aged < years) in future trials. adult voxelotor doses can be used in adolescents. however, based on higher pk exposures, a lower weight-based dosing strategy is recommended in children. ppk and pbpk modeling provides an innovative approach to minimize experimental dosing in children and accelerate dose selection of voxelotor in ongoing and future clinical studies. this abstract is supported by global blood therapeutics. background: hydroxyurea (hu) reduces rates of acute complications, and improves long term outcomes in patients with sickle cell disease (scd) and is now fda approved for children. through previous work we have increased the number of eligible patients on hu in our clinic, however accessing a compounding pharmacy remained a significant barrier to hu adherence for infants and children who cannot swallow capsules. objectives: the objective of our quality improvement project was to improve adherence to hu among pediatric patients with scd at our urban safety net hospital by addressing barriers to obtaining liquid hu. design/method: to begin we met with the leadership of our outpatient pharmacy which offers mail order delivery. however, like most retail pharmacies, they do not have the necessary protective equipment to compound liquid hu. through a series of discussions, we began a unique partnership with our institution's inpatient chemotherapy pharmacy who compounds the liquid hu and delivers it to the outpatient s of s pharmacy, who then dispenses liquid hu to families. using a series of plan-do-study-act (pdsa) cycles we tracked adherence by calculating the medication possession ratio (mpr), defined as the percentage of days in a given period of time that each patient had their medication on hand. the mpr for liquid hu mpr among enrolled patients was tracked by pharmacy staff and reviewed monthly. additional pdsa cycles included adding automatic refills and reminder calls by pharmacy staff and improving communication about delivery. we also tracked patient satisfaction. results: between march and december , a total of thirty pediatric patients were enrolled in our program for on-site compounding and free mail order delivery of liquid hu. mpr for liquid hu is currently . % among enrolled patients, significantly higher than the mpr of % reported in the literature, and has risen steadily since the beginning of the project. families are highly satisfied with the program, specifically appreciating the convenience of mail order delivery, saving on delivery fees, and reminder calls when refills were due. by compounding and dispensing liquid hu directly from our institution's outpatient pharmacy we have significantly improved adherence to this hu therapy in our high-risk population. next steps include analysis of change in clinical outcomes for patients enrolled in this program. as adherence to hydroxyurea is associated with decreased acute care utilization and cost, programs such as ours could play a crucial role in reducing the excessive costs and ed utilization among this patient population. background: experience with the iron-chelator deferasirox is reported widely in higher-income settings. by contrast, real-life experiences in emerging countries are infrequently reported. objectives: to evaluate, in a non-trial setting, the real-life response to deferasirox in an emerging country. design/method: in sri lanka's national thalassemia center which manages patients without tertiary staff, quantitative evaluations of body iron or estimates of extra-hepatic iron, the records of patients who began deferasirox in / were retrospectively reviewed. results: baseline assessments (mean±sem) indicated substantial iron loading [serum ferritin (sf) , ± ug/l; serum alt ± . u/l (normal ≤ u/l)]. deferasirox was introduced at low doses ( . ± . mg/kg/day); many patients started at < mg/kg and, after months, doses remained ≤ mg/kg/day in % patients. after months, sf in % patients remained > , ug/l; only by months had (mean) sf declined to < , ug/l ( ± ; p< . ). similarly, mean alt normalized (to ± u/l) only by months. death and complications were not systematically recorded by staff who had been charged, without provision of additional resources, with the introduction of this new drug in hundreds of patients. these results contrast to those in sri lanka's tertiary thalassemia center where, in patients following the introduction of deferasirox ± . mg/kg/day, sf declined rapidly, even in relatively less ironloaded patients (from , ± to , ± g/l after months; p = . ). these findings underscore the importance, during the implementation of new drug regimens in lowerincome centers with marginal resources, for investments in methods to quantitate body iron burden, hands-on educational initiatives to guide day-to-day management by competent but non-expert staff, and data systems to record efficacy, effectiveness, toxicity and compliance. such investment is critical to optimising therapy and improving complications in thalassemia patients worldwide: even in sri lanka, where resources directed to thalassemia management are greater than in most of asia, results in the oldest living cohort (born - ) indicate under-treatment [elevated iron burdens (sf , ± ug/l) and high prevalences of diabetes ( %) and hypothyroidism ( %)]. even in a younger cohort (born - ) which has benefitted from improved treatments, the prevalence of many complications exceeds those reported from high-income settings. over the next decade, and two decades after the who declaration that the impact of thalassemia on global mortality and morbidity is underrecognized, increased investments by governmental and nongovernmental sources will be necessary to improve outcomes for asian patients with thalassemia. background: a major barrier to success in hydroxyurea (hu) treatment of patients with sickle cell disease (scd) is non-adherence. objectives: to optimize hu adherence in patients with scd. design/method: a care model was designed by the sickle cell (qi) team at children's hospital to improve hu adherence among scd patients. the original model included bimonthly family phone contact, monthly dispensing pharmacy phone contact and lab monitoring. adherence measures included obtaining hu from pharmacy monthly, completion of monthly labs, hb f percentage and mcv, and mtd achievement. from / - / , several pdsa cycles refined our care model. a one-year follow-up survey gathered feedback on the care model. the first-year data involved ∼ patients. the biggest improvements resulted from making pharmacy calls before patient/family calls, shipping liquid hu to outlying patients, and tracking call time/content. the qi goal was % hu adherence by / . the % baseline adherence rate increased to % by / , and has remained in that range. the completion rate of patient/parent phone calls increased from % the first month to % at six months. pharmacy prescription pick-up has increased from % to % per month. lack of liquid hu availability was overcome by shipping the medication to the patient's home. parental hesitance to share information by phone, especially with qi team members with whom they had no established relationship, was overcome by having the longtime sickle cell nurse do many of the early calls. however, survey feedback showed families became comfortable with several clinic personnel calling. the calls gave families the opportunity to ask questions about their child and/or get additional information about scd. the calls also provided an opportunity for seasonal flu shot or tcd testing reminders. the surveys gave information on the optimal time of day to reach each family, providing individualization and further increasing the percentage of completed calls. two families surveyed said they no longer needed two calls a month because they were now able to remember to pick up hu, administer it, and get labs on their own. this qi project has not only improved hu adherence, but also fostered health education/counseling, increased patient/parent satisfaction, and enhanced service utilization. medical team member and patient/family comments demonstrate that it has helped build relationships and trust between families and the medical care system. based on survey feedback, we will further individualize care to increase adherence rate and sustain improvements. cincinnati children's hospital medical center, cincinnati, ohio, united states background: the thalassemias are a heterogeneous group of genetic blood disorders caused by mutations that decrease or eliminate the synthesis of the -and/or -globin subunits of hemoglobin. the phenotype of thalassemia depends on the interaction of the -and -globin gene clusters, because both loci determine the -/ -chain balance. for example, a -thalassemia phenotype can be more severe than expected when coinherited with -globin gene triplication (copy number gain), which exacerbates the -/ -globin imbalance. objectives: describe four individuals with an incorrect diagnosis of -thalassemia trait who were later properly diagnosed by comprehensive genetic testing to have -thalassemia intermedia caused by heterozygous -thalassemia mutations coinherited with triplicated -globin loci. design/method: sequence analysis of the -globin (hba /hba ) and -globin (hbb) genes, and copy number variation analysis of the -and -globin gene clusters by multiplex ligand-dependent probe amplification. results: four unrelated individuals of northern european ancestry were evaluated for signs and symptoms not explained by a diagnosis of -thalassemia trait (previously made by a pediatric hematologist), including growth delay, splenomegaly, moderate anemia, marked elevation of hemoglobin f, thalassemic facies, reticulocytosis, and/or indirect hyperbilirubinemia. genetic testing revealed that all were heterozygous ( / ) for the same, single -globin mutation [hbb.c. c>t (p.q *)] and also heterozygous for an -globin triplication ( / anti- . ). their previous diagnoses of thalassemia trait had been made by complete blood counts, hemoglobin electrophoresis, and/or sequence analysis of the -globin genes only. these individuals' phenotypes ranged from moderate anemia only to multiple stigmata of thalassemia, demonstrating the phenotypic variation of a thalassemia genotype. correct diagnosis was made at an average age of . years. a trial of chronic transfusions was initiated for one patient for growth failure. all were educated about the potential for exacerbations of anemia, gallstones, osteoporosis, and iron overload (even without transfusions). parental genetic testing was recommended to assess reproductive risk, because inheritance of this complex genotype can be apparently autosomal dominant. conclusion: heterozygosity for a -thalassemia mutation does not necessarily indicate -thalassemia minor or "trait". when coinherited with -globin gene triplication, a symptomatic form of -thalassemia can occur. correct and timely diagnosis of thalassemia requires careful consideration of the degree of anemia and examination for organomegaly, bony changes, and jaundice. sequence analysis and copy number variation analysis of both the -and -globin gene clusters is key. hematologists need to be aware of this diagnostic possibility and how to test for it to prevent inaccurate or delayed diagnosis. background: the burden of healthcare costs for sickle cell disease (scd) is nationally estimated at over $ billion. the major components of these costs are inpatient and emergency center (ec) visits, many of which are potentially avoidable. in several chronic conditions, a subset of patients account for most of the avoidable encounters. identifying these patients is the first step in targeted care delivery. objectives: to measure and analyze scd patient utilization patterns in the ec and inpatient at texas children's hospital (tch). we identified all individuals under years old with any encounter at tch associated with an international classification of disease (icd)- or code for scd, including hgb ss, hgb sc, and hgb s/beta thalassemia. for each patient, we identified all inpatient and ec encounters in the days prior to their most recent encounter. finally, each encounter was classified as associated with pain, acute chest syndrome (acs), or "other" using an algorithm of discharge diagnosis codes and pharmaceutical delivery. the total number of scd-associated ec and inpatient encounters over the prior year was calculated for each patient. we stratified each patient according to their utilization patterns: low ( - encounters), intermediate ( - encounters), and high (≥ encounters). we identified unique patients with scd that had at least one encounter from july until june . there were , scd-related encounters in the days prior to their most recent encounter. most ( %, n = ) patients exhibited low-utilization patterns and % (n = ) were intermediate. finally, a small subset ( %, n = ) demonstrated high-utilization patterns and accounted for % of all encounters. high-utilization was associated with older age and public payment mechanisms. pain encounters were predominantly in pre-adolescents and teenagers with high-and intermediate-utilization patterns. acs was most frequent in pre-teens and younger teens in the intermediate-utilization group. finally, the youngest-aged high and intermediate users presented for other reasons such as febrile episodes and splenic sequestration. our findings reflect national trends in that a significant portion of encounters are attributed to a small subset of patients exhibiting a high-or "super-" utilization pattern. at our institution, scd super-utilization is associated with older age and pain. we also identified a group of infants and toddlers with frequent encounters for fever. to comprehensively address this burden, it will be important to design interventions targeted toward age and specific medical needs. background: background: the rarity of diamond blackfan anemia (dba) has hindered describing the spectrum of disease, identifying predictive correlations, and guiding datadriven recommendations. long-term toxicities from steroid or transfusion therapy that start in childhood remain the major clinical problems in patients with dba who do not receive stem cell transplant. objectives: objective: to define the dba patient population at st. jude children's research hospital including treatment responses and toxicities to help inform recommendations on treatment and monitoring. design/method: method: medical records were reviewed for all patients with dba treated at st. jude between and for diagnostic testing, treatment types and regimens, and outcomes. two-sample t-test or wilcoxon rank sum test was used to compare continuous variables in two groups depending on the normality of the data tested by shapiro-wilk test. results: a total of patients with dba were identified with a median age of . years (range months - years) at last follow up. a ribosomal protein gene mutation was identified in / patients ( %) with an rps mutation / ( %). thirteen different congenital malformations were described in / patients ( %). fourteen of twenty ( %) patients treated with corticosteroids had an initial response and of those achieved full remission. three patients became steroid-refractory and were unable to wean to an acceptable dose. five of twenty patients continue on lower-dose steroids. five patients currently require no therapy. univariate analysis revealed no statistically significant genetic predictors of response or remission, however, / rpl patients responded to steroids with / ( %) in long-term remission. ten patients are maintained on chronic transfusions and have undergone successful hematopoietic stem cell transplant. nineteen of treated patients ( %) had a treatment-related toxicity. patients on steroids were more likely to have short stature than patients on transfusions or in remission (p = . ). severe bone mineral density deficit occurred in / ( %) patients, in before age years. eight patients had hepatic iron overload, in one documented by age years. other severe toxicities included restrictive cardiomyopathy from iron overload, pathologic fracture, diabetes mellitus, and premature ovarian failure in one patient each. this genotypically and phenotypically heterogeneous dba cohort had a high rate of treatment-related toxicities, notably growth retardation, bone density loss, and hepatic iron overload even in very young children. these findings underscore the need for early standardized monitoring. background: patients with sickle cell disease (scd) face worsening morbidity and mortality between ages and , when they must transition from pediatric to adult healthcare.( ) an effective curriculum addressing disease knowledge, educational and vocational skills, self-efficacy, and social supports is critical to a successful transition. traditional didactic approaches have not led to durable knowledge retention. ( ) technology-based methods have been attempted, but the best educational approach remains unknown. objectives: . to understand how adolescent and young adult (aya) patients with scd view existing transition education. . to include patient preferences in improving our transition curriculum. we developed a qualitative survey to assess patient views of existing approaches for learning about scd and their opinions about preferred transition topics. thirty patients with scd aged to years old were recruited between january and december . responses were managed using redcap electronic data tools hosted at the university of rochester.( , ) qualitative and quantitative data analyses were performed, including independent t-testing to compare responses between age groups. results: approximately % of subjects were under years of age, while % were or older. seventy-one percent had a computer, and . % had a cell phone, with most reporting daily use. subjects reported greatest satisfaction with learning from their doctor during clinic visits ( . % agree or strongly agree) and websites on a cell phone ( . % agree or strongly agree); the least popular methods were online chat rooms and microsoft® powerpoint presentations. satisfaction was similar across age groups. recommended transition topics were viewed positively, with subjects ranking highest understanding their bloodwork ( . % agree or strongly agree) and understanding laws protecting students with chronic disease ( . % agree or strongly agree). older subjects ( - years old) agreed more strongly with learning about opioid addiction and understanding differences between adult and pediatric doctors than did younger subjects ( - years old) (p < . ). this pilot study was successful in helping us to understand the educational needs of aya patients with scd. preliminary data underscore the importance of education provided by the pediatric hematologist. our results also suggest that the optimal use of technology-based methods requires further investigation and that tailoring transition education by age group may be useful. background: similar to patients with transfusion-dependent beta-thalassemias (tdt-beta), survivors of hemoglobin barts hydrops fetalis (homozygous alpha- -thalassemia, tdtalpha) will require lifelong transfusions of erythrocytes. we have previously shown that a transfusion strategy that is based on the guidelines developed for tdt-beta (conventional transfusion) is suboptimal for these patients owing to the differences in the pathophysiology of anemia in the two conditions: in tdt-alpha, conventional transfusion strategy will lead to a gradual increase in non-functional hbh with subsequent tissue hypoxia and hemolysis. an aggressive transfusion strategy that was based on reduction of hbh and increase in "functional" hemoglobin level resulted in improvement of tissue oxygenation and reduction of hemolysis but was associated with significant increase in transfusional iron burden [amid et al, blood ] . objectives: to define the optimal chronic blood transfusion targets for hbh% and functional hemoglobin in patients with tdt-alpha. design/method: following research ethics board approval, longitudinal data of patients with tdt-alpha ( males, median age . ( . - . ) were retrospectively collected. variables of interest included total pre-transfusion hemoglobin, hbh%, and "functional" hemoglobin [measured as total hemoglobin x ( -hbh/ )]. outcome variables were lactate dehydrogenase (ldh, marker of hemolysis), and soluble transferrin receptor (str, marker of erythropoiesis). hemoglobin analysis was done using high-performance liquid chromatography and capillary zone electrophoresis. we examined the association of "functional" hemoglobin with str, and hbh% with ldh, using repeated-measures anova to adjust for the effect of multiple testing. we constructed receiver operating characteristic curve and calculated the area under the curve to define the best cut-off values for variables of interests. there was a strong association between functional hb and str, as well as hbh and ldh. the optimal cut-off for "functional" hemoglobin that was associated with str < . mg/l was g/l (auc = . , sensitivity and specificity of . % and % respectively). the optimal cut-off for hbh to supress ldh to < u/l was % (auc = . , sensitivity and specificity of . % and % respectively). the optimal pre-transfusion hbh% for reduction of hemolysis was % and the optimal "functional" hemoglobin to adequately supress erythropoiesis was g/l. to meet these hbh% and functional hb targets by simple blood transfusions, patients with tdt-alpha would require a hypertransfusion regimen with a minimum pre-transfusion total hb of g/l and consequently high transfusional iron burden. an alternative approach using exchange transfusion to reduce hbh% and improve functional hemoglobin would be associated with less volume of transfusion and potentially better long-term outcome. hospital sacre coeur, milot, haiti background: initial results of work developing a pediatric sickle cell disease (scd) clinic at the hôpital sacré coeur (hsc) in milot, northern haiti were presented at aspho . the purpose of this clinic is for a pediatrician with a special interest in scd to provide scd care, advising on trait and managing disease with penicillin prophylaxis (pcn) and hydroxyurea therapy (hu) for select patients. this clinic was started in collaboration with a us based hematologist and support from yale-new haven hospital. objectives: to describe the success and challenges of providing pcn and hu in the scd clinic at hsc through a review of patient records. design/method: since this clinic's inception, a database of patients, with basic clinical information has been kept and made accessible, through 'drop-box', to the us hematologist. the records of those that presented to the clinic were reviewed. the hemoglobin diagnosis was made either by clinical history and sickle cell prep or by hemoglobin electrophoresis through alpha laboratory, port-au-prince, haiti. results: ninety-nine individuals were seen in the first years of the program. fifty-six underwent a hemoglobin electrophoresis. of these , are ≤ years old. thirty-two were started on pcn vk, of which / ( %) were ≤ years old. eleven patients were started hu therapy. all patients on hu have shown progressive increases in hemoglobin. there have been no clinical complications of hu therapy. none of the patients taking hu have required hospitalization or transfusion in . three patients (not on hu) were hospitalized in for complications of scd (osteomyelitis, pain). in , with less than half the numbers in the program, there were admissions for severe anemia, pain, stroke and splenic sequestration. with ongoing external support and a local reputation for excellence in sickle cell care, the clinic at hsc has been able to expand services and improve the health of a growing number of patients with scd. early data suggests that pcn and hu therapies are helping to reduce complications and improve quality of life. challenges to date have included lack of funding for transportation to clinics, for hospitalizations and to cover the cost of electrophoreses. at the same time as continuing providing excellent care and gathering data, it is crucial to explore opportunities for collaboration and cooperation in ways that will assure that the clinic can become independently sustainable while continuing to improve the quality of life for the individuals it serves. background: ykl- is an inflammatory glycoprotein expressed by infiltrating macrophages in various inflammatory conditions. it has been found to be elevated in patients with different pathological conditions like acute and chronic inflammations, increased remodeling of the extracellular matrix (ecm), development of fibrosis and cancer. several studies have found elevated ykl- concentrations in sera of patients with liver diseases such as hepatic fibrosis by hepatitis c virus. it has been suggested that ykl- concentrations reflect the degree of liver fibrosis. to evaluate serum ykl- levels in patients with -thalassemia and its relation to viral hepatitis, liver stiffness as assessed by transient elastography (fibroscan, fs) and hepatic iron concentration. design/method: a prospective study included patients with -tm ( males and females) with mean age . ± . years (range: - years). serum ferritin level, liver enzymes (alt and ast), hbs ag, anti hcv ab and serum ykl- using elisa kit were evaluated. all patients were subjected to liver mri t * to detect liver iron content by the sequence and transient elastography (fibroscan, fs) to assess degree of liver stiffness. results: mean fibroscan value was ( . ± . ) kpa with a median . (range . to ) kpa. ( %) patients were categorized as f - and ( %) were stage f - , ( %) patients had severe fibrosis. their median serum ferritin was ng∖ml, with ( %) patients had values exceeding g/l. median cardiac t * was . with patients had values below ms, and the median lic was . mg/g dw with patients showed readings above mg/g dw. nyl- was evaluated as a marker of inflammation and liver fibrosis and showed mean value . (± . ) pg/ml, and range from to pg/ml. mean ykl- was significantly higher among males (p = . ), patients on chelation therapy (p = . ), patients on dfs (p≤ . ), in those with abnormal liver enzymes, splenectomised patients, patients with hbv sero-positivity, those with moderate elevation of t * and patients with high grades of liver fibrosis (p< . ). ykl- showed positive correlation with the rate of transfusion, lic, ferritin, alt and ast but negative correlation with weight, height and t *. roc curve analysis revealed that the cutoff value of ykl- at pg/ml could differentiate -tm patients with and without viral hepatitis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). roc curve analysis revealed that the cutoff value of ykl- at pg/ml could detect -tm patients with liver cirrhosis with . % sensitivity and specificity of . %, area under the curve (auc) . , positive predictive value . and negative predictive value . (p< . ). conclusion: serum ykl- levels are elevated in patients with -thalassemia and can detect patients with active viral hepatitis and liver stiffness. background: the most common splenic complication in pediatric patients with sickle cell disease (scd) is acute splenic sequestration (ass), which has often been managed with splenectomy. although splenectomy has been a treatment of choice for years, long-term vascular complications have not been thoroughly evaluated. pulmonary hypertension (phtn) is a severe complication of scd. in adults with scd, phtn has been associated with a -month mortality rate of approximately %. it has been reported that splenectomized patients with hemolytic disorders are at even greater risk of phtn. several medications exist to treat phtn, but with few studies of their efficacy or toxicities in patients with scd. additionally, these patients are often treated with either chronic prbc transfusions or hydroxyurea (hu) to raise hemoglobin, reduce hemolysis, and prevent vaso-occlusive events. objectives: to evaluate effect of chronic prbc or hu vs. no intervention, on tricuspid regurgitant jet velocities (trv) in pediatric patients with scd and history of splenectomy. design/method: retrospective chart review of splenectomized patients with hbss followed at marian anderson center at st. christopher's hospital for children, philadelphia, between and . we analyzed trvs ( hu, prbc, and from control group receiving neither treatment) from patients ( hu, prbc, neither). mean age at echo was . +/- . . data was analyzed with linear correlations and analysis of variance (anova), including the post hoc test of least significant difference (lsd) for all pairs of treatment groups. results: trv was not significantly correlated with age at time of assessment or with time between splenectomy and trv. univariate anova among groups yielded trv means of: . +/- . cm/s (hu), . +/- . (prbc), . +/- . (neither). we found a notable difference as the mean of the hu group was almost cm/s lower than the others, but no overall statistically significant association for any of the groups exists. however, when we performed post hoc tests to adjust for multiple comparisons and looked at all pairings within the anova, we found that the lsd between the hu and the prbc groups was statistically significant (p = . ), and that a trend exists between the hu group and the neither treatment group (p = . ). our data suggests that treatment with hu is correlated with a reduction in trv in pediatric patients with scd who underwent splenectomy. given these promising results, we believe our data warrants further study with larger treatment groups. nancy olivieri, gaurav sharma, susmita nath, rajib de, tuphan kanti dolai, prakas kumar mandal, abhijit phukan, amir sabouhanian, robert yamashita, angela allen, david weatherall, prantar chakrabarti background: hemoglobin e thalassemia (hbethal), which accounts for % of all severe beta thalassemia worldwide, has an estimated prevalence of . / , in west bengal, from which little information about clinical findings has been reported. objectives: to document clinical and laboratory findings in patients with hbethal, ultimately to improve resources for clinical management. design/method: we reviewed records from: a database recording patient names; clinic charts; "special" charts containing additional details; and, in transfused patients, transfusion day-care records. additionally, because in india's public hospitals original lab/imaging reports are commonly retained at home, % of families were interviewed to provide additional information. we excluded records of patients aged < years and patients aged < years who had not been reviewed since . results: while at least one visit had been recorded in , hbethal patients at nrs hospital, most patients are not regularly reviewed there. we examined charts [ ( %) aged ≥ years; ( %) aged - years; % male], representing approximately % of regularly-reviewed patients. most families ( . %) reported monthly incomes (< , indian rupees), below the monthly cost of living ( , rupees) in kolkata. mean (±sem) hemoglobin was . ± . g/dl. % patients were receiving eight or more transfusions per year; from , % had been treated with deferasirox, . ± . mg/kg/day. iron control estimated by serum ferritin concentration ( . ± g/l) was highly variable. a total of % patients were splenectomized. a substantial obstacle to documenting complications was the lack of recording, in any of the five sources, of many relevant parameters: for example, the status of sexual maturation (normal, delayed, or absent) was documented in less than %, and measurements of fasting blood glucose in less than %, of records. where recorded, complication rates were high: delayed/abnormal sexual maturation was recorded in % patients aged > years; in the patients aged > years and those aged - years, respectively, hypothyroidism was recorded in % and %, and elevated serum alt in % and %. in most evaluable patients > years, height was measured between the rd- th percentiles. cardiac findings, rarely documented, included pulmonary hypertension and reduced left ventricular ejection fractions in a few patients. despite dedicated attention to many aspects of thalassemia care, insufficient documentation limited a clear understanding of the current morbidity in hbethal patients. investment in personnel and technology will be critical to record relevant information, ultimately to improve clinical management, over the next decade. children's hospital of richmond at vcu health, richmond, virginia, united states background: sepsis is a common cause of death in children with sickle cell disease (scd). recommendations for care of fever in children with scd include immediate medical evaluation including blood culture and initiation of broad-spectrum antibiotic therapy. the increasing availability of pcr-based respiratory pathogen panels (rpp) provide the opportunity to rapidly identify viral causes of fever. the role for rpps in identifying the source of fever in children with scd and how it affects provider practice is not well studied. ( ) to determine the epidemiology of respiratory virus-associated fever in children with scd and ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all children with scd seen in our emergency department (ed) with temperature ≥ . oc at home or in the ed from january , , through september , , as well as, all febrile children for whom rpps were sent since the introduction of rpps april . we reviewed the results of blood cultures, rpps, chest radiographs, and ed notes and discharge summaries to identify sources of infections. independent t test and chi-square analysis were used as appropriate to compare results using spss©. overall, the rate of bacteremia was %. there were no cases of bacteremia among children with positive rpps. % of children with negative rpps had true bacteremia. a positive rpp did not reduce the likelihood of bacteremia (p . ). patients with bacteremia had higher presenting temperatures than those without bacteremia ( . oc vs . oc, p . ). the most common rpp findings were rhinovirus/enterovirus ( %), human metapneumovirus ( %), and influenza a ( %). sending an rpp did not affect admission rate ( % and % respectively, p . ); however, likelihood of admission was lower in patients with positive rpps ( % vs %, or . [ . - . ], p . ). length of stay (los) was shorter in patients for whom an rpp was not sent ( . vs . days, p . ). as previously reported, bacteremia in febrile children with scd is very low, but remains a serious concern, particularly in the setting of high fever (> oc). a positive rpp did not reduce the odds of bacteremia, but did have a sta-tistically significant impact on both admission rate and los. more work is needed to understand how rpp results impact provider decision-making and care for children with scd. cincinnati children's hospital medical center, cincinnati, ohio, united states background: diffuse myocardial fibrosis is a common, if not defining, feature of the heart in sickle cell anemia (sca) that is strongly associated with diastolic dysfunction. we found diffuse myocardial fibrosis in every patient in a sca cohort (n = ) ranging in age from to years (niss ). the treatment and prevention of this complication of sca has not been studied before. objectives: because diffuse myocardial fibrosis must begin in early childhood, we hypothesized that early initiation and uninterrupted use of disease-modifying therapy for sca can prevent it. design/method: we use cardiac magnetic resonance imaging (cmr) to measure the myocardial extracellular volume fraction (ecv) to quantify diffuse myocardial fibrosis in individuals with sca who have been treated, uninterrupted, with hydroxyurea or chronic transfusion therapy since ≤ years of age. two comparison groups were used: individuals with sca who have not been treated with disease-modifying therapy since ≤ years of age (n = ) and controls without sca (n = ). results: we studied individuals ( m/ f) with a mean age of . years (range - ). mean age at the start of diseasemodifying therapy was . ± . years (range - ). only had evidence of mild diffuse myocardial fibrosis (ecv . ); the other had no detectable diffuse fibrosis (all had ecv < . , the upper limit of normal). mean ecv was . ± . , which was significantly lower than the ecv of individuals with sca who have not received early uninterrupted therapy ( . ± . ; p = . ) and not statistically different from normal controls ( . ± . ; p = . ). none had macroscopic fibrosis by late gadolinium enhancement or evidence of myocardial hemosiderosis by t * imaging. no patient had diastolic dysfunction by echocardiographic classification, right heart catheterization, or both. disease-modifying therapy for sca can prevent diffuse myocardial fibrosis, and possibly diastolic dysfunction, if started in early childhood. prospective trials of disease-modifying and anti-fibrotic therapy are planned to prevent diffuse myocardial fibrosis, which can be monitored noninvasively by cmr, and improve outcomes in sca. (niss, blood, ) . background: a statewide sickle cell surveillance system (sscss) was developed with the goal of determining the prevalence of sickle cell disease (scd) in indiana and the level of care that patients receive throughout the state. persons with scd are at high risk of infection, especially with encapsulated organisms, as well as at increased complications from influenza. utilizing sscss data, the relationship between vaccination status and mortality was explored. to determine if vaccination status is associated with mortality in persons with scd. the project was granted a waiver of consent by the st. vincent irb. death certificates were obtained to identify cause of death. deceased patients (cases) were matched by age, gender, and sickle genotype to living patients (controls). vaccination data were collected from the medical record and the children and hoosier immunization registry program (chirp) through the date of death for each case. cases and controls were assigned a point for completion of the pneumococcus, meningococcus and haemophilus influenza type b (hib) vaccine series and one point if the influenza vaccine was given within a year prior to death of the cases [max vaccine status score (vss): ]. total points were compared between the cases and controls. two tailed t-tests to compare means of continuous data and wilcoxon signed-rank test to compare ordinal data. one thousand forty-eight individuals were included in the sscss. six hundred and seven ( . %) were seen at one institution and included in this analysis (mean age = years). thirty-three of the ( . %) were deceased at the time of analysis. six point one ( . )% of controls and . % of cases received a vss of . the mean vss for cases was . ± . and . ± . for controls. thirty point three ( . ) % of controls had a vss of one or more, compared to % of cases (p = . ). patients who died of infection [streptococ-cus (n = ), pseudomonas (n = ) and unidentified organisms (n = )] were not up to date on vaccination against encapsulated organisms, but two had received the influenza vaccine in the year prior to death. in this sample, mortality occurred exclusively among adult patients, which is consistent with current patterns in developed countries. among these adults, vss and mortality rates were not related. limitations to the study include small sample size and potential incompleteness of vaccine records. vaccination rates and other standard of care indicators should be explored in a larger cohort of patients to determine associations with mortality. background: sickle cell disease (scd) is a genetic disorder resulting in acute and chronic complications, including delayed puberty. delayed puberty can have adverse physical and psychosocial effects on affected children and families. there are no published reports from ghana on pubertal timing in children with scd. the aim of this cross-sectional study was to describe pubertal changes in children with scd at korle bu teaching hospital (kbth), accra, and compare these findings to those in a control group without scd. design/method: children with scd and children with hb aa, ages - years, were consecutively recruited and matched for age, sex and socioeconomic status. investigator-administered questionnaires were used to obtain demographic data for all participants and information on menarche (girls only). pubertal status was assessed by physical examination using tanner staging. testicular volumes were determined in boys using a prader orchidometer. body mass index (bmi) and socioeconomic status (ses) of participants were analyzed to determine if there were any associations with tanner stage. of the with scd, ( . %) were hb ss and ( . %) hb sc. females comprised . % (cases and controls). mean age at onset of breast development was significantly delayed in girls with scd ( . ± . years) compared to controls ( . ± . years) but there was no significant age difference at onset of pubic hair development. mean age at menarche was significantly delayed in girls with hb ss ( . ± . years) and hb sc ( . ± . years), compared to those with hb aa ( . ± . years). in boys, the mean ages at onset of puberty were significantly delayed in those with scd ( . ± . years, for genital development and . ± . years, for pubic hair development), compared to those without scd ( . ± . years and . ± . years, respectively). mean testicular volumes were significantly lower in cases compared to controls, across all age ranges (p< . ). mean bmi in both cases and controls were similar at onset of breast development in girls. however, in boys with and without scd, mean bmi values were significantly different at pubertal onset. in univariate analysis, ses was not associated with tanner stage for both genital and breast development. mean ages at pubertal onset were significantly delayed in children with scd. longitudinal studies are needed to further characterize any associations with bmi and determine potentially modifiable risk factors affecting pubertal onset in scd. background: sickle-cell disease (scd) is a life-threatening genetic disorder associated with multiple chronic and acute complications. specific monitoring and treatment for children is a major part of the medical focus, but there remains a lack of real-world evidence of the disease burden and practice patterns among the pediatric scd population. objectives: to examine the clinical burden and management of scd among pediatric patients. design/method: a retrospective claims study was conducted using the medicaid analytic extracts database from jan - dec . pediatric patients (aged < years) with scd were identified using icd- -cm diagnosis codes ( . - . , . - . ). the first observed scd diagnosis during the identification period was designated as the index date. patients were required to have continuous medical and pharmacy benefits for at least months pre-and months post-index period. patient data were assessed until the earliest occurrence of the following events: disenrollment, death, or the end of the study period. patient demographic and baseline clinical characteristics, clinical outcomes (mortality, incidence of pain crisis, complications), scd management, and healthcare utilization were examined. all variables were analyzed descriptively. results: a total of , patients met the study inclusion criteria, with a mean age of . years. most patients were black ( . %) and had a charlson comorbidity index score of ( . %). mortality during follow-up was . in personyears, and the event rate of pain crisis in the inpatient setting was . in person-years. the three most common complications after pain crisis (highest rates in person-years) were fever ( . ), infectious and parasitic diseases ( . ), and asthma ( . ). rates of life-threatening complications were also examined in person-years, including acute chest syndrome ( . ), stroke ( . ), splenic sequestration ( . ), pulmonary hypertension ( . ), and pulmonary embolism ( . ). . % of patients were prescribed antibiotics during the one-year post-index period. other frequent medications utilized among children were folic acid ( . %), nonsteroidal anti-inflammatory drugs ( . %), opioids ( . %), and hydroxyurea ( . %). . % of patients had a blood transfusion within one year post-index date. patients had frequent health care utilizations in the inpatient ( visit), emergency room ( visits), office ( visits), and pharmacy ( visits) settings during the one-year follow-up period. pediatric scd patients are burdened with a high rate of complications including pain crisis. in addition, patients utilized a substantial amount of health care resources including outpatient office care and acute care visits. background: novel use of hydroxyurea in an african region with malaria (noharm, nct ) is a randomized controlled trial of hydroxyurea for very young children with sickle cell anemia living in uganda. during year , study participants received blinded study treatment of hydroxyurea or placebo; those receiving hydroxyurea had no increased risk of malaria, but had both laboratory and clinical benefits. during year , all study participants received openlabel hydroxyurea treatment. to assess the effects of open-label hydroxyurea treatment in a very young population of children with sickle s of s cell anemia living in uganda. study endpoints included the rates and severity of malaria infections, clinical sickle-related events, and laboratory effects. design/method: all children in the noharm trial were enrolled at mulago hospital sickle cell clinic in kampala uganda. during year , all children received open-label fixeddose hydroxyurea ( mg/kg/day) for months, after previously receiving either hydroxyurea or placebo for months. results: a total of children entered year of the noharm trial and received fixed-dose hydroxyurea, including males and females, at an average age of . ± . years. among children previously on placebo, there were malaria events in children, including with severity grade ≥ , and three deaths (two acute chest syndrome, one sepsis). clinical adverse event rates dropped from . to . per patient year, and hospitalizations were reduced from to . expected hematological benefits of increased hemoglobin, mcv, and fetal hemoglobin, along with decreased neutrophils and reticulocytes, were rapidly achieved. laboratory adverse events were infrequent at . events per patient-year, and only half of those were dose-limiting hematological toxicities. among children previously on hydroxyurea, there were malaria events in children, including with severity grade ≥ , and two deaths (one acute chest syndrome, one sepsis). clinical adverse event rates and hospitalizations were maintained at low rates, the hematological benefits of hydroxyurea continued throughout the extended treatment period, and dose-limiting toxicities remained infrequent. fixed-dose hydroxyurea treatment of young children with sickle cell anemia living in uganda is associated with no increased risk for malaria. clinical and laboratory benefits occur, including children previously on placebo who crossed-over to hydroxyurea treatment. future studies should focus on the optimal dosing and monitoring strategies, in an effort to determine the overall feasibility and safety of introducing hydroxyurea therapy across sub-saharan africa. background: acute chest syndrome (acs) is the second most common cause of hospitalization in patients with sickle cell disease and is a leading cause of morbidity and mortality. in mid- , an algorithm was implemented at cohen children's medical center to initiate transfusions within four hours of diagnosis of acs in order to improve patient outcomes. objectives: the aim of this project was to analyze the effect of early blood transfusion on the outcomes of patients with acs. we focused on the number of total transfusions, need for exchange transfusion, need for intensive care unit (icu) stay, and length of hospitalization. design/method: a retrospective chart review was completed on patients admitted to ccmc with a primary diagnosis of sickle cell disease and a secondary diagnosis of either acs or pneumonia during the years of - . data from the three years directly prior to implementation of the algorithm was compared to data from the three years directly after implementation of the algorithm. a total of patients were analyzed, of which belonged to the pre-algorithm group and to the postalgorithm group. patients from the post-algorithm group had a higher incidence of transfusions ( % with a mean transfusion number of . pre versus % with a mean of . post) as well as exchange transfusion ( % pre versus % post). the post-algorithm group had a shorter overall length of stay (mean of . days pre versus . days post). while the overall percentage of patients requiring an icu admission was similar in each group ( % pre versus % post), the post-protocol group had a lower likelihood of requiring an icu admission for reasons outside of line placement for exchange transfusion, most commonly for icu-level respiratory support ( % pre versus % post). despite a higher total number of transfusions, early recognition and transfusion for acs can lead to decreased lengths of hospitalization as well as decreased need for icu-level respiratory support. further studies comparing different center's clinical practice guidelines are necessary to improve the standard of care. background: novel use of hydroxyurea in an african region with malaria (noharm) was the first placebocontrolled randomized clinical trial of hydroxyurea in sub-saharan africa. in noharm, young children with sca received either hydroxyurea or placebo during year , followed by open-label hydroxyurea for all study participants during year . an ancillary noharm project was designed to determine if hydroxyurea treatment lowers transcranial doppler (tcd) velocities and possibly reduces stroke risk in this very young cohort. objectives: to perform tcd screening on the noharm cohort, measuring the time-averaged mean velocity (tamv) at the end of both year and year . we hypothesized that the maximum tamv would be lower for noharm study participants receiving hydroxyurea compared to those receiving placebo, and that key clinical and laboratory parameters would also influence tcd velocities. design/method: all children enrolled in noharm were eligible to undergo tcd examination at two study time points: month - when they were completing the blinded treatment phase, and again at month - at the end of the open-label treatment phase. tcd measurements included tamv readings from the main intracranial arteries: middle cerebral artery, distal internal carotid artery, and bifurcation on tcd. all tcd examinations were scored and classified as normal (less than cm/sec), conditional ( - cm/sec) or abnormal (greater than or equal to cm/sec), with higher scores correlating to greater risk of stroke. results: at the end of year , tcd exams were conducted of which were suitable for analysis ( hydroxyurea, placebo). based on the maximum tamv, the median velocity was cm/sec (iqr - ) for children on hydroxyurea and cm/sec (iqr - ) on placebo, p = . . maximum tamv values had negative correlations with hemoglobin concentration (- . ), fetal hemoglobin (- . ), and oxygen saturation (- . ); positive correlations were noted with age ( . ) and absolute neutrophil count ( . ). at the end of year , tcd exams were conducted and all were suitable for analysis; the median velocity was cm/sec on open-label hydroxyurea treatment, regardless of previous blinded treatment. all correlations with tamv were maintained except for age. conclusion: compared to placebo, hydroxyurea treatment for young children with sca living in uganda was associated with lower tcd velocities, which have been correlated in other studies with lower risk of primary stroke. tcd velocities were correlated with hematological and clinical parameters that can be improved by hydroxyurea therapy. children's hospital of richmond at virginia commonwealth university, richmond, virginia, united states background: acute chest syndrome (acs), defined by respiratory symptoms and a new pulmonary infiltrate, is a serious complication of sickle cell disease (scd). acs can occur during hospitalization for non-pulmonary conditions, such as a vaso-occlusive crisis or after surgery. nih clinical practice guidelines encourage incentive spirometry (is) which decreases the incidence of acs. it is additionally widely accepted that early, frequent ambulation in post-operative and pneumonia patients decreases the length of stay (los). to decrease acs events in children with scd at our children's hospital, we aimed for is use in % of ageappropriate pediatric sickle cell admissions. design/method: a multidisciplinary team examined inpatient acs prevention practices, including is, at children's hospital of richmond. key drivers were identified, including educational awareness of patients and healthcare staff, order placement, and documentation. we aimed for all scd patients ≥ months of age hospitalized with any admission diagnosis to participate in is with the use of a traditional incentive spirometer or similar age-and ability-appropriate devices (e.g. positive expiratory pressure devices, bubbles, and pinwheels). we secondarily aimed to increase activity events, specifically ambulation and out of bed time. educational and outreach tools included patient informational brochure and incentive program, and staff informational sessions and reference materials at workstations. a disease-specific order set was implemented including desired is and activity orders. data were collected prospectively may through november , during which pdsa cycles were conducted. admissions during the corresponding months of the previous year were reviewed for comparison. independent t-test analysis was performed using graftpad prism statistical analysis software. results: improvements reaching statistical significance included increase in is order placement from % to % of admissions (p < . ), and admissions with documented is use increased from % to % (p < . ). los decreased from a mean of . days to . days (p . ). post-admission development of acs also decreased from % to % of admissions, but did not reach statistical significance (p . ). there was an additional increase in appropriate activity order placement and documentation of activity events. conclusion: improving education and outreach to patients and staff, including implementation of a disease-specific order set, can improve is use and activity events. the decline seen in incidence of acs development during hospitalization, though not statistically significant, and the decreased los are encouraging, and efforts continue to improve on these trends. background: painful vaso-occlusive crises (voc) are a frequent and debilitating complication of sickle cell disease (scd) and are thought to occur due to progressive blockage of the microvasculature with rigid sickle shaped red blood cells. any trigger that decreases the microvascular blood flow (mbf) can promote entrapment of sickled cells in the microvasculature and progression to voc. exposure to cold wind and changes in weather are common triggers of voc and are associated with increased frequency of hospitalizations for pain in patients with scd. there is limited experimental data on the physiologic effects of these factors on peripheral perfusion in scd. to study the effect of graded thermal stimuli on the peripheral mbf in scd. design/method: scd and control (healthy or sickle trait) subjects aging to years were exposed to their individual threshold temperatures for heat and cold detection, heat and cold pain via tsa-ii thermode that was placed on the thenar eminence. mbf was measured on the contralateral thumb using photo-plethysmography (ppg). the vasoconstriction response within the complex ppg signal was detected using cross-correlation technique. mean mbf was derived from the ppg amplitude during each of these stimuli and compared to baseline mbf. cross correlation analysis showed that cold pain caused significant vasoconstriction response in % of the subjects, followed by heat pain ( %), cold detection ( %) and heat detection ( %).there was a significant drop in the mbf during cold pain (p < . ), heat pain (p < . ), heat detection (p = . ) and cold detection (p = . ) when compared to baseline mbf, with cold pain causing the greatest drop in mbf. thermal sensitivity and mbf responses were comparable between scd and controls. conclusion: exposure to graded thermal stimuli causes a progressive drop in mbf with exposure to cold pain eliciting the strongest vasoconstriction response. vasoconstriction occurred in the contralateral hand at an average of seconds after the stimuli, suggesting a neurally mediated mechanism. although there was no significant difference in vasoconstriction responses between scd and controls, the drop in mbf in patients with sickle cell disease can increase the likelihood of entrapment of the sickled red blood cells, leading to vaso-occlusion. these findings are consistent with extensive reports in literature that exposure to cold weather is associated with a higher frequency of voc. this suggests that neurally mediated vasoconstriction is likely an important factor in the pathophysiology behind cold exposure leading to voc in scd. background: vaso-occlusive crisis (voc) is a major cause of hospital admissions in children with sickle cell disease (scd). although the use of clinical biomarkers in voc has been studied, especially with regards to acute chest syndrome (acs), there is less data regarding overall voc severity prediction. in addition new biomarkers such as platelet to lymphocyte ratio (plr), neutrophil to lymphocyte ratio (nlr), and lymphocyte to monocyte ratio (lmr) have been little studied with regards to scd. objectives: to identify whether admission laboratory values, changes from well baseline laboratory values, and new biomarkers such as plr, nlr, and lmr could predict severity of vaso-occlusive crisis in children with sickle cell disease admitted with voc. design/method: this was a retrospective single center observational study of admissions of voc in children aged - years with hbss or hbs-b thal from september to november excluding those on hyper-transfusion protocol or having an admission diagnosis of acs. univariate analysis was done using student's t-test, mann-whitney non parametric test, or fischer's exact test as appropriate depending on the distribution between admission laboratory data of complete blood count (cbc), reticulocyte count, comprehensive metabolic panel, lactate dehydrogenase (ldh), change from well baseline cbc values within months previously, plr, nlr, lmr, and the development of complicated voc. complicated voc was defined as the development of secondary acute chest syndrome, prolonged admission duration > days ( hours), requirement of blood transfusion, and readmission within days. results: a total of admissions were studied. fifty-nine ( . %) were female. of the , ( . %) were complicated with no significant differences in sex (p . ) or age (p . ). univariate analysis revealed significant elevations in total bilirubin (p . ), ldh (p . ), and platelet count (p . ) in those with complicated voc. there is also significant difference in the percentage change of platelet count from baseline with greater decline in uncomplicated voc (p . ). there were no significant differences in plr (p . ), nlr (p . ), or lmr (p . ). conclusion: elevations in total bilirubin, ldh, and platelet count in admission laboratory values are associated with developing complicated voc. in addition, those with complicated voc present with significantly less decline in platelet count from baseline well cbc. plr, nlr, and lmr do not seem to be useful predictive biomarkers for severity of voc. background: sickle cell disease (scd) causes health problems of varying frequency and severity. the only validated biomarker for children with scd is transcranial doppler. if reliable predictors existed for scd severity, children with scd could be treated according to risk category. many patients with scd face psychosocial or economic hardships, but these factors have not been evaluated as risk markers for medical or functional severity of scd. objectives: the goal of this project was to develop and stratify a preliminary list of psychosocial risk factors for health outcomes that could be used as scd severity predictors. st. vincent institutional review board. a list of potential psychosocial risk factors for adverse health outcomes was compiled based on assessment materials utilized by the sickle safe program (indiana's hemoglobinopathy newborn screening follow-up program). this list of items was distributed to child abuse prevention ( ) and scd ( ) experts, who ranked each item on a likert scale of (least important) to (most important). mean scores were calculated using spss version ; assessments were retrospectively analyzed to determine psychosocial risk factor frequency. risk factors occurring in ≥ % of homes were considered high frequency events. overall, there was high agreement among experts on the risk factors that were considered the most important predictors of severe scd outcomes. the risk factor with the highest frequency ( %) was eligibility for public assistance programs. fifteen risk factors were rated ≥ by the experts. four ( . %) were high frequency events occurring in ≥ % of homes: a child with hbss or hbs thalassemia not taking hydroxyurea ( %); parent report that they had treated a fever (> ®f) at home in the past months ( %); tobacco use by someone in the household ( %); and the family reporting significant psychosocial stressors in the past year ( %). tobacco use in the home was significantly correlated with several other risk factors (smoking during pregnancy [r = . ], other health concerns in the child [r = . ], and child having health insurance [r = - . ]), suggesting that it is part of a constellation of health risk. in general, the risk factors that were rated as most important for health outcomes occurred less frequently in the sample. this study represents important progress toward identifying a group of psychosocial risk factors for scd severity, which is a necessary first step for future investigation of empirical relationships between candidate risk factors and scd outcomes. unitversity of cartagena, cartagena, bolivar, colombia s of s background: sickle cell disease is an autosomal recessive disorder characterized by a mutation in the -globin chain, which produces hbs. acute and chronic complications as aplastic crisis, acute chest syndrome, priapism, stroke, leg ulcers and primary/secondary prevention of stroke can be treated with simple transfusion or exchange transfusion. the latter offers advantages as lower iron overload, post-treatment hbs goal control, lower viscosity and improved microvascular circulation. but it is not a widely-used option because is associated with technical difficulties. objectives: standardization of a new partial exchange transfusion protocol in a group of patients with sickle cell disease, within the framework of a chronic transfusion program. design/method: this is a prospective descriptive study, which included patients under years with sickle cell disease ( hbss, hbs-tal), with indication of partial exchange transfusion in a chronic transfusion program, according to the institutional protocol; patients who fulfilled the inclusion criteria were enrolled in the study between february and december . a registry of the medical and technical complications was made in each of the procedures. a database was constructed in excel, and the graph-pad prism® version oc software was used for statistical analysis. the sequence is as follows: isovolemic phlebotomy and transfusion of packed red cells. depending of the recent hemoglobin level ( hrs), we do the phlebotomy there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg; isovolemic solution (ns , %) there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg and packed red cell transfusion there: hb: - . : cc/kg, hb: - . : cc/kg, hb> : cc/kg. the safety of this exchange transfusion protocol was analyzed in patients with sickle cell disease ( procedures). there were no differences in the sex distribution, and the median age was years. % of the population was homozygous. the indication of transfusion was . %( / ) primary stroke prevention, . %( / ) secondary stroke prevention and . %( / ) was other reason. a low percentage of complications was found ( . %); of which, those of medical origin (hypotension and nausea/vomiting) were only presented in . % of the total procedures. the standardization of this protocol was safe and its use could be extended to other low-income centers that treat patients with sickle cell disease that need chronic transfusion program including patient with hemoglobin level until gr/dl. we suggest do studies for measure the security and efficacy of this protocol in patients with acute complications. background: clinical trials that aim to achieve pain reduction have challenges achieving clinical endpoints as pain has no quantifiable biomarkers and may be unrelated to scd. furthermore, the threshold of seeking medical care differs between patients and vocs that occur at home are missed. we present a non-interventional, longitudinal study to identify vocs in patients with scd. objectives: to examine the longitudinal relationship between pros and biomarkers in subjects with scd before, during, and after a self-reported voc event, in order to build a model of in-home and clinical voc and to collect longitudinal pros and biomarker data from subjects that span voc events in the home, clinic and the hospital. design/method: longitudinal measures of pain, fatigue, function, activity, and biomarkers from scd patients in steady state and voc were studied over a six month period. patients self-reported pain, fatigue, function, and medication use using a novel epro tool. voc was reported in real-time, triggering a mobile phlebotomy team. blood was collected sequentially after self-reported voc (at home or hospital). blood samples were drawn two days after resolution of voc, as reported by the patient. during non-voc periods, blood was drawn every weeks to establish a baseline. biomarkers included leukocyte-platelet aggregates and circulating microparticles, cell and soluble adhesion molecules, cytokines, inflammatory mediators and coagulation factors. patients wore an actigraphy device to track sleep and activity and rest. results: twenty-seven of thirty-five patients experienced a total of days with voc > hr, of which only days resulted in healthcare utilization. voc days had significantly higher pain and fatigue scores. voc days were associated with significantly decreased functional scores, with significantly greater decreases during vocs requiring medical contact compared to at-home vocs. different activity profiles were identified for non-voc, at-home voc and medical contact voc days by actigraphy monitoring. at-home voc days exhibited increased daytime resting compared to non-voc days. medical contact vocs had decreased average and peak activity, and increased daytime resting compared to non-voc days. a sleep fragmentation index trended up for both at-home ( %) and medical contact voc days ( %). significant changes during voc days were observed in: c-reactive protein ( % increase), nucleated rbc ( % increase), monocyte-platelet aggregates ( % increase) and neutrophil-platelet aggregates ( % increase), interleukin- ( % increase), interleukin- ( % increase) and tnfalpha ( % increase). the identification and assessment of at-home vocs through use of epros, actigraphy and biomarkers is feasible as demonstrated by this innovative at-home study design. background: risk-stratifying sickle cell disease (scd) patients and demonstrating response to disease-modifying therapies is challenging due to the phenotypical heterogeneity of scd. a pathogenic role for procoagulant von willebrand factor (vwf) via excess vwf high molecular weight multimers (hmwm) has been proposed, with variable reports of increased vwf and hmwm in crisis vs. steady-state in adults, but less so for vwf in children with scd. moreover, vwf and multimers have not been studied in sickle trait. objectives: our pilot study evaluated the potential for vwf antigen (vwf:ag) and hmwm on densitometric tracings to serve as biomarkers for disease severity or treatment response in children and young adults with scd compared to sickle trait (hbas) siblings. design/method: we evaluated vwf:ag, vwf multimers and retrospective clinical data from hbss, hbsc and hbas subjects at steady state. one hbsc subject also had a crisis sample. median scd age was years ( . - . years). % were female. scd severity was judged by annual vasoocclusive and acute chest events, or stroke/elevated tcd. eight of ( hbss and hbsc) took hydroxyurea. four hbss subjects had severe scd, all of whom were chronically transfused. results: mean vwf:ag (normal - iu/dl) was higher for hbss ( +/- . ) and severe hbss ( +/- . ) compared to hbsc ( +/- . , p = . and . , respectively); however, lacked statistical significance when compared to hbas ( +/- . , p = . and . , respectively). vwf:ag was elevated in / ( %) steady-state, including / ( %) with "severe" disease on chronic transfusion and / ( %) taking hydroxyurea, in hbsc crisis but no hbsc / ( %) at baseline. vwf:ag was high in / ( %) hbas siblings. four ( %) had increased hmwm at baseline: hbss/severe disease/chronic transfusion, hbss/hydroxyurea and hbsc untreated. hmwm were increased only during vaso-occlusive crisis in hydroxyureatreated hbsc subject. no ultra-large hmwm were observed. in this preliminary study, in young scd subjects, vwf:ag trended higher in hbss vs. hbsc and in severe hbss participants at a single time-point, but serial evaluations at baseline, in crisis and with optimized diseasemodifying therapy are needed to determine the potential of vwf:ag and hmwm as biomarkers for severity or treatment response. surprisingly, vwf:ag was high in some sickle trait subjects. since hbas is associated with some health challenges such as increased thrombosis risk, further examination of vwf and endothelial dysfunction in sickle trait may provide novel insights into its role as a biomarker. background: the national heart lung & blood institute(nhlbi) guidelines for acute management of voe recommends rapid evaluation and treatment of pain, including administration of a parenteral opioid within -minutes of triage or -minutes from registration, pain reassessment & repeat opioid delivery within - -minutes. inf use has been increasing in peds due to its rapid onset and ease of administration. objectives: to evaluate ped utilization of inf & its effect on intravenous (iv) opioid administration and pain control for the treatment of voe. design/method: a retrospective review of emr was performed on children with scd± years presenting to a ped with voe (pain scores on a - scale) from jan-june . variables studied were median time (iqr, %ci) from ped arrival to first-parenteral-opioid-administration, time-to-first-iv-opioid, first & final pain score, disposition and readmission rate. time-to-first-iv-opioid was also compared to historical data (jan-dec ,n = ) prior to inf protocol initiation. . additionally, % patients received iv opioids within minutes of ed arrival in the inf+iv opioid vs. % in the iv opioids alone group (p< . ). no differences in -hour-returnrates were found in any of the groups, including inf alone group. conclusion: use of inf in the ped for voe is an excellent strategy to shorten time-to-first-parenteral-opioidadministration, improve pain scores & improve adherence to the nhlbi guidelines. however we had distinct unexpected findings: ( ) delays in iv opioid delivery after inf use & ( ) inf alone appeared to provide sufficient pain control without iv opioids for disposition home in % of voe patients. whether the latter reflects insufficient pain management or that there is a milder subgroup for whom inf alone is sufficient, requires further investigation. this study illustrates our experience with a ped-based inf protocol in terms of unanticipated delays in iv opioids and also discharges after inf alone. efforts are underway to further improve use of inf in voe management. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: folate supplementation is commonly included as standard management in patients with sickle cell disease. however, clear evidence supporting the clinical benefits of this practice is lacking. a single study demonstrated improvement on the occurrence of repeat dactylitis at a higher dose of folic acid. to compare clinical outcomes in pediatric patients with sickle cell disease treated with folate supplementation versus those who were not. design/method: this study was a retrospective chart review that included patients to years old with sickle cell disease type ss and s followed at st. christopher's hospital for children. data collected included information about folate supplementation, red cell indices and the presence or absence of clinical outcomes including vaso-occlusive crisis requiring hospitalization in the last six months, acute chest syndrome, infections, asthma, sleep apnea, nephropathy, cerebral vascular disease, stroke and avascular necrosis. analysis of variance (anova) was used to evaluate mean differences between age, number of infections, number of voc events, hemoglobin, reticulocyte count, and mean corpuscular volumes. additionally, chi square analysis was implemented to evaluate differences in folate and non-folate groups for left ventricular remodeling (lvr), sickle cell nephropathy, asthma, obstructive sleep apnea (osa), nocturnal hypoxia, and avascular necrosis (avn). mean differences between the folate and non-folate groups were compared for patients on and off hydroxyurea therapy. one hundred and seven patients met inclusion criteria following review of clinical data. of the patients included in the study, patients were found to be taking folate ( %), while patients were not ( %). statistical analysis showed that there were no significant differences in the incidence of clinical outcomes between patients on folate versus those who were not on folate. of the patients who were not on hydroxyurea, hemoglobin levels were significantly higher in patients on folate versus those who were not (p = . ), but not significantly different for the patients on hydroxyurea. this study suggests that folate supplementation makes no significant impact on the red blood cell indices of anemia nor on the incidence of adverse clinical outcomes in children with sickle cell disease. however, a larger prospective study is needed to guide future considerations for folate supplementation in sickle cell patients in the clinical setting. background: tanzania ranks rd globally for the number of infants born annually with sickle cell disease (scd) but lacks a national newborn screening program. the prevalence of sickle cell trait (sct) and scd is highest in the northwestern regions around lake victoria served by bugando medical centre (bmc) a teaching and consultancy hospital in mwanza. bmc also houses the hiv early infant diagnosis (eid) laboratory that tests dried blood spots (dbs) from hivexposed infants. dbs can be tested for hiv and then retested for sickle cell trait and disease. to determine the prevalence of sickle trait and disease by region and district in northwestern tanzania using existing public health infrastructure. secondary objectives explored associations between sct, scd, malaria and hiv. design/method: the tanzania sickle surveillance study (ts ) is a prospective year-long cross-sectional study of hivexposed infants born in northwestern tanzania, whose dbs collected by the eid program are tested at bmc and available for further testing of sct and scd. samples from children ≤ months of age were tested by isoelectric focusing (ief) and scored independently by two tanzanian staff as normal, sct, scd, variant, or uninterpretable. dbs samples scored as disease or variant were repeated. over the course of months, ief gels have been run. a total of , dbs samples have been scored, including , from children less than -months old. the overall prevalence of sct is . % and the prevalence of scd is . %, along with . % hemoglobin variants. quality of the laboratory results is extremely high, with only . % dbs samples yielding an uninterpretable result. geospatial mapping of the first , samples revealed a regional scd prevalence ranging from . % up to . % among the regions served by bmc. the prevalence of sct and scd is very high in northwestern tanzania. geospatial mapping will identify high prevalence areas where targeted newborn screening can be started using existing public health infrastructure with minimal start-up cost and training. further data will enhance the accuracy of the map to the district level. background: pediatric patients with sickle cell disease (scd) could develop obstructive, restrictive or mixed abnormalities of pulmonary function (pf). several publications report progressive worsening of pf over time, which could lead to severe morbidity in adult patients with sickle cell disease. in adults with sickle cell anemia up to - % of mortality is related to lung disease. early intervention aimed at improvement of lung function could significantly decrease morbidity and possibly improve life expectancy. among disease modifying approaches commonly used in scd are hydroxyurea (hu) and chronic prbc transfusions. both interventions lead to increase of hemoglobin, decrease of hbs fraction, leading to decreased hemolysis. reports of effect of hu on pulmonary function are conflicting with some suggesting no effect and others proposing a slower decline of pulmonary function. the goal of our study is to evaluate effect of disease modifying therapies, like hu and chronic prbc on change of pulmonary function in pediatric patients with sickle cell disease. design/method: this study utilized a retrospective chart review of children with scd who had multiple pfts. we analyzed pfts from patients done during clinic visits. scd patients were divided into three treatment groups: hydroxyurea, chronic transfusions or neither. data was analyzed with linear correlations and analysis of variance (anova). comparison were made between the three groups specifically observing the changes in absolute numbers on pfts over time using the first and last pft the patient had. results: there were a total of patients with multiple pfts (ranging from - ); control ( ), hydroxyurea ( ) and chronic transfusion ( ). the mean changes of the control, and hydroxyurea for the pft parameters fev (- . the chronic transfusion group demonstrated a small improvement in pfts over time for fev ( . ), fvc ( . ), fef - ( . ), however there was a decline in fev /fvc (- . ). however, there was no statistically significant (p-value < . ) in the difference in any pfts parameters between any of the groups. in children with scd there is a decline of pf parameters over time. although no significant differences were seen between the three groups it appears chronic transfusion may improve or limit the decline in pfts. larger studies need to be done to evaluate difference in pf decline in patients with scd patients. background: the use of mobile technology in health care has been a growing trend. patients with chronic diseases such as sickle cell disease (scd) require close monitoring to provide appropriate treatment recommendations and avoid complications. we conducted a feasibility study for patients with scd hospitalized for pain using our self-developed mobile application (tru-pain: technology resources to better understand pain) and a wearable activity tracker. subjective symptoms such as pain and objective data such as heart rate (hr) were measured. we aimed to ) correlate nursing recordings with mobile technology recordings; ) get feedback from patients about usability. design/method: we enrolled patients with scd > years old and < hours from admission for uncomplicated vasoocclusive crisis, excluding patients admitted to icu. patients were given an ipad and a wearable device. they were instructed to record in the application at least once per day and to keep the wearable on, removing only to charge. prior to discharge, patients completed a feasibility questionnaire. we enrolled patients, % females, median age . (range to ) who were admitted for a median days (range to ) for uncomplicated pain crisis. patients used the application throughout hospitalization and made one entry/day (range to ). pain scores recorded via tru-pain correlated well (r = . , p< . ) with pain scores recorded in emr. there was an average of , data points recorded per day, by the wearable, with a maximum of , data points/day. the median amount of hours of wearable data per day was . (maximum of . ). the hr recorded via the wearable correlated significantly with the hr recorded in emr (r = . , p-value < . ). as for usability, % of patients indicated never having a problem with the technology, % found tru-pain 'very easy' or 'somewhat easy' to use, and % were 'very satisfied' with their participation in the study, indicating that it helped them track their pain. our pilot study during hospitalization shows strong potential for using tru-pain for patients with scd. pain data from application and hr from wearable correlated well to the emr data. according to the feedback received, our application was easy to use and helped patients track their pain. despite limitations of battery life, the use of wearable technology is feasible, providing additional data such as activity. we are optimistic that we can continue to improve our tru-pain system to help improve care in patients with scd. background: hydroxyurea, chronic blood transfusion, and bone marrow transplantation can reduce complications, and improve survival in sickle cell disease (scd), but are associated with a significant decisional dilemma because of the inherent risk-benefit tradeoffs, and the lack of comparative studies. these treatments are underutilized leading to avoidable morbidity and premature mortality. there is a need for tools to provide patients high-quality information about their treatment options, the associated risks, and benefits, help them clarify their values, and allow them to share in the process of informed medical decision making. objectives: to develop a health literacy sensitive, web-based, decision aid (ptda) to help patients with scd make informed choices about treatments, and to estimate in a randomized clinical trial the acceptability and effectiveness of the ptda in improving patient knowledge, involvement in decisionmaking and decision-making quality. design/method: we conducted qualitative interviews of scd patients, caregivers, stakeholders, and healthcare providers for a decisional needs assessment to identify decisional conflict, knowledge, expectations, values, support, resources, decision types, timing, stages, and learning, and personal clinical characteristics, and to guide the development of a ptda. transcripts were coded using qsr nvivo . stakeholders completed alpha and beta testing of ptda. we conducted a randomized clinical trial of adults, and of caregivers of pediatric patients to evaluate the comparative efficacy of the ptda, vs. standard of care. results: ptda (www.sickleoptions.org) was developed per decisional needs described by stakeholders and finalized following alpha testing, and beta testing by and stakeholders respectively. in a randomized trial of subjects considering various treatment options, qualitative interviews revealed a high level of usability, acceptability, and utility in education, values clarification, and preparedness for decision making of the ptda. a median % rated the acceptability of ptda as good or excellent and provided narrative comments endorsing the acceptability, ease of use, and utility in preparation for decision making. the ptda met international standards for content, development process, and efficacy with the exception of having a full range of positive and negative experiences in patient stories. compared to baseline ptda group had statistically significant improvement in preparedness for decision making (p = . ) and informed subscale of decisional conflict (p = . ) but not for decisional self-efficacy, knowledge, choice predisposition, or stages of decision-making. a ptda for patients with scd developed following extensive engagement of key stakeholders was found to be acceptable, useful, easy to use, to improve preparedness for decision making, and decrease decisional conflict. background: painful vaso-occlusive crisis (voc) accounts for the majority of emergency department (ed) visits and hos-pitalizations in sickle cell disease (scd). we are interested in studying mental stress and associated autonomic nervous system (ans) imbalance that cause vaso-constriction as possible triggers of scd pain. to this end, we developed a mobile phone application (app) to record daily pain frequency and intensity as clinical endpoints that might be predicted by ans parameters measured in the laboratory. in particular, we think that the aura may represent ans instability that precedes or even triggers change in blood flow and voc. objectives: to assess the feasibility of using an app to evaluate frequency and severity of voc and its potential association with mental stress and presence of aura. design/method: an app was developed for both ios and android systems to allow patients to track pain, stress, and aura. the idea was to create an app that was easy to use with the intent to only capture pain episodes, rather than detailed description of the pain. all scd patients were eligible and a parent version was available for younger children. de-identified data was automatically transferred to a hipaa compliant database via a cloud-based server interfaced to the main research project database. a feedback questionnaire was implemented after at least a month of utilization to assess usability. of the scd patients enrolled, participants utilized the app and of the participants that provided feedback indicated the app was easy to navigate. the mean pain scale was out of (standard deviation . ) for those that entered they had pain that day. although the mean stress level was out of , there was a statistically significant correlation between increasing stress levels and increasing pain scores (p < . ). aura was reported by patients, with patients reporting more than episodes. moreover, on days aura was present there was greater incidence that pain was present as well (p < . ). however, there was no statistically significant association between pain intensity and presence of an aura (p = . ). conclusion: consistent with prior research, reported pain intensity is significantly associated with reported stress intensity. although there was an association between presence of aura and pain, it did not seem to correlate with pain intensity. this uniquely designed app can monitor scd pain clinically and help understand the role of sickle dysautonomia in the genesis of scd pain. university of florida college of medicine, gainesville, florida, united states background: evidenced-based guidelines recommend the emergent evaluation of fever in children with sickle cell disease (scd). as the prevalence of bacteremia has decreased, outpatient management has become more common. however, fever can sometimes herald other complications of scd, such as acute chest syndrome, vaso-occlusive pain crisis, splenic sequestration, or aplastic crisis. institutional practices regarding fever management in scd remain variable, and little is known about the clinical outcomes of children hospitalized for uncomplicated fever. objectives: the primary objective was to determine the rate of bacteremia or scd-related complications per febrile episode in children with scd admitted to a single institution between january and june for uncomplicated fever. this was a retrospective cohort study of febrile patients up to years of age with scd, any genotype, admitted to the university of florida during the defined study period. eligible patients were identified by a database search using admitting diagnosis codes for scd and fever based on the international classification of diseases th and th revisions. encounters were manually reviewed to confirm eligibility. patients were excluded if they had other indications for hospitalization apparent at the time of admission, such as an acute vaso-occlusive episode requiring parental narcotics, asthma exacerbation, or additional complications of scd. the database search identified encounters, of which were excluded based on confounding indications for hospitalization. sixty-three eligible patients accounted for hospitalizations. the median age was years (range weeks- years); . % were male. mean duration of hospitalization was . days (range - days). eight positive blood cultures were identified; six of these were classified as contaminants. bacteremia or the development of a scd-related complication was identified in ( . %) admissions. these included acute chest syndrome (n = ), bacteremia (n = ), splenic sequestration (n = ), and red cell transfusion (n = ). exploratory analyses of potential predictors of bacteremia or scd-related complications showed no association with the presenting white blood cell count or degree of fever (p = . ). of the patients classified as having a scd-related complication, % had hemoglobin ss disease and % had at least one prior documented complication. % of the patients transfused had at least one prior transfusion. conclusion: while improvements in preventative care have substantially lowered rates of bacteremia in children with scd, fever warrants careful evaluation for other acute scdrelated complications. providers should consider inpatient observation in select cases. additional studies are warranted to define subsets of patients suitable for outpatient fever management. background: children with sickle cell disease (scd) exhibit lower neurocognitive functioning than healthy peers, even in the absence of stroke. among the domains commonly affected, working memory (wm) seems particularly affected by disease processes and wm deficits have significant implications for academic achievement and disease selfmanagement. few interventions to improve working memory in pediatric scd have been evaluated. to determine the effects of cogmed, a homebased computerized wm training intervention, in children with scd using a randomized controlled trial design. design/method: participants (ages - ) with scd completed a baseline neuropsychological assessment and those with wm deficits were randomized to either begin cogmed immediately or enter an -week waitlist. cogmed is a homebased intervention completed on an ipad that consists of increasingly challenging exercises targeting visual-spatial and verbal wm, practiced over sessions. at the end of training, participants completed a post-intervention neuropsychological assessment, including tests of visual-spatial and verbal wm from the wechsler intelligence scale for children-fifth edition (wisc-v). results: ninety-one participants (m age = . , sd = . ; % female; % hbss) enrolled in the study; % (n = ) exhibited wm deficits and were randomized to either begin cogmed immediately or wait - weeks before starting cogmed. among those that have received the intervention and reached the end of their training period (n = ), participants ( %) completed at least cogmed sessions, ( %) finished at least sessions, and finished at least sessions ( %). the mean number of completed cogmed sessions was . (sd = . ). paired samples t-tests revealed significant improvements on the working memory index (t[ ] = - . , p = . ) and on the digit span (t[ ] = - . , p = . ), and spatial span-backward (t[ ] = - . , p = . ) subtests. improvements were especially pronounced for participants completing at least sessions. partial correlations controlling for respective baseline scores indicated that the number of cogmed sessions completed was positively correlated with post-test scores on digit span (r = . , p = . ) and spatial span-backward (r = . , p = . ) subtests. among participants who completed at least cogmed sessions, % scored in the average range or higher on the working memory index at the post-intervention assessment, compared to % at baseline. results support the efficacy of cogmed in producing significant improvements in wm. a dose-effect was observed such that participants who completed more cogmed sessions had greater improvements in wm. home-based cognitive training programs may ameliorate scd-related wm deficits but methods for motivating and supporting patients as they complete home-based interventions are needed to enhance adherence and effectiveness. background: sickle cell disease is associated with myriad complications that lead to significant morbidity and early mortality. hydroxyurea has been used successfully to reduce the incidence of these complications and has led to significant improvements in quality and duration of life. at children's minnesota we recommend hydroxyurea in all patients with hb ss/s thalassemia as early as months of age with a goal of starting all patients before months of age. objectives: the purpose of this study was to evaluate the use of hydroxyurea therapy in young patients with sickle cell disease, with particular attention to those children less than one year of age. design/method: a retrospective chart review was conducted on patients less than years of age with sickle cell disease who began hydroxyurea therapy between january , and december , . the study population was divided into three cohorts based upon age at hydroxyurea initiation: cohort ( - year), cohort ( - years), and cohort ( - years). outcomes included laboratory data, clinical events (hospitalization, dactylitis, pain crisis, transfusion, splenic sequestration, acute chest syndrome), and toxicity occurring in the first years of life. results: a total of patients were included in cohorts (n = , mean age . months), (n = , mean age . months), and (n = , mean age . months). patients in cohort had higher hemoglobin (p = . ) and mcv (p = . ) and lower absolute reticulocyte count (p = . ) when compared to cohort . the wbc (p = . , < . ) and anc (p = . , . ) were significantly lower compared to both older cohorts. however, no patient had therapy held because of neutropenia. the mean baseline hemoglobin f in cohort was . % compared to . % and . % in cohorts and respectively (p = . , p< . ). the mean duration of therapy in cohort was . months, compared to . months in cohort (p = . ) and . months in cohort (p = . ). during this time, hb f levels remained higher in cohort (mean . %) compared to cohorts and (mean . %, p = . and mean . %, p = . ). patients in cohort experienced fewer hospitalizations (p = . ), pain crises (p = . ), and transfusions (p = . ). there was no difference in toxicity between groups. hydroxyurea was used safely in infants to months of age and resulted in more robust hematologic responses and a decrease in sickle-related complications when compared with patients starting hydroxyurea later in life. children's national health system, washington, district of columbia, united states background: children with sickle cell disease (scd) have a significantly greater risk of silent or overt cerebral infarction than the general population. infarcts are associated with declines in cognitive functioning and academic achievement. while infarcts are reliably identified using mri, scans are expensive and occasionally necessitate sedation. moreover, mri's are not recommended for routine monitoring of cerebral infarcts. additional tools are needed for discriminating the presence of a cerebral infarct that are brief, noninvasive, inexpensive, and repeatable. objectives: to evaluate differences in performance on cogstate, a computerized neurocognitive assessment, in patients with scd with and without history of cerebral infarct. design/method: participants included children with scd ages - (m = . , sd = . ; % female; % s of s hbss) enrolled in a cognitive intervention trial. participants completed the cogstate pediatric battery, which measures processing speed, sustained attention, verbal learning, working memory, and executive functioning. history of silent or overt infarct was determined via health record review. participants also completed measures of intelligence (iq) and math fluency. results: participants' standard scores across most neurocognitive measures were lower than expected compared to the standardization sample (mean iq = . , sd = . ). thirty percent of participants (n = ) had a documented history of cerebral infarct. participants with a history of cerebral infarct scored lower on cogstate tasks measuring sustained attention (t[ ] = . , p = . ) and executive functioning (t[ ] = . , p = . ), as well as on a measure of math fluency (t[ ] = . , p = . ). receiver operating characteristic (roc) analyses demonstrated that the cogstate task measuring sustained attention was a fair discriminant of patients with and without a history of infarct (auc = . , ci = . - . , p = . ), whereas iq score was not (auc = . , ci = . - . , p = . ). cogstate processing speed and sustained attention tasks fairly discriminated between patients with at least average or below average intelligence (auc = . , ci = . - . , p = . and auc = . , ci = . - . , p = . , respectively). finally, the cogstate processing speed task was good at discriminating between at least average or below average math fluency (auc = . , ci = . - . , p< . ). multiple tasks in the cogstate pediatric battery appear to adequately identify patients with a history of cerebral infarcts. in addition, cogstate tasks appear to be fair predictors of impairments in iq and academic achievement outcomes. cogstate is inexpensive and can be easily administered in a medical setting with minimal training in approximately minutes. results support the potential for cogstate to be used as a screening tool for medical and neuropsychological abnormalities in children with scd. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: cardiovascular disease contributes to the morbidity and mortality of patients with sickle cell disease (scd). hydroxyurea therapy in scd has known clinical efficacy including improving anemia, decreasing episodes of vasoocclusive crisis and acute chest syndrome, and decreasing mortality. effect of hydroxyurea on cardiac function in children with scd is not well studied. an earlier study suggested the protective effect of hydroxyurea on left ventricular (lv) hypertrophy in scd. we hypothesized that hydroxyurea use would be associated with decreased lv remodeling and improved cardiac function. we aimed to evaluate the association between hydroxyurea use and lv remodeling and cardiac dysfunction in children with scd. design/method: we completed a retrospective study of patients with scd who were to years old, followed at st. christopher's hospital for children and had an echocardiogram completed in the past months. data collected included gender, bmi, scd genotype, hydroxyurea use, chronic transfusion use, and d and doppler echocardiographic parameters. cardiac structure, geometry, systolic function, and diastolic function echocardiogram parameters were included. analysis of variance (anova) tests were performed to assess for statistical significance of differences in cardiac parameters between patients with and without hydroxyurea use. analysis of covariance (ancova) tests were performed to control for age. results: demographic and echocardiogram data was collected on all patients who met inclusion criteria. of the patients included, ( %) were on hydroxyurea therapy. patients on hydroxyurea had significantly lower mean relative wall thickness (p = . ) and significantly higher mean peak early lv filling velocities (p = . ) and peak early lv filling/septal annuli early peak (e/ea) velocities (p = . ); however, only the e/ea velocities remained significant when controlling for age (p = . ). mean peak early lv filling velocities approached significance when controlling for age (p = . ). hydroxyurea therapy resulted in a significantly higher e/ea velocity, suggesting that these patients had worse diastolic function. it is possible that the patients initiated on hydroxyurea already had worse disease manifestations than those not on hydroxyurea, possibly accounting for the decreased diastolic function. when controlling for age, hydroxyurea use did not result in significant differences in cardiac structure parameters, systolic function parameters or cardiac geometry. prospective studies and larger sample size are needed to validate our findings, examine for additional statistically significant differences, and develop preventive strategies for cardiovascular disease in children with scd. background: acute chest syndrome (acs) is now the leading cause of death in children with sickle cell disease; mortality in the u.s. is reported to be - % and is mostly due to respiratory failure. early transfusion improves clinical outcomes. although patients with concurrent asthma are considered at increased risk for poor outcomes, risk factors for respiratory failure in pediatric acs have not been well-defined. to determine whether specific epidemiological and clinical features of children hospitalized with acs are predictive of the need for mechanical ventilation. design/method: data from the kids' inpatient database were reviewed to identify patients age < years with a discharge diagnosis of acs for the years , , , and . outcomes were defined by the international classification of diseases, ninth revision, clinical modification code. data were weighted to estimate total annual hospitalizations according to hospital characteristics in the united states. trends in healthcare costs, length of hospital stay, transfusion, and mechanical ventilation use were analyzed using multivariable linear regression. in addition, multivariable logistic regression was used to ascertain specific clinical or epidemiologic factors associated with mechanical ventilation use after adjusting for patient and hospital characteristics. the total hospitalizations for acs were , in ; , in ; , in ; and , in . reported use of mechanical ventilation ranged from . % to . % and was associated with non-black compared to black children (or, . ; %ci, . to . ) and the fall season (or, . ; %ci, . to . ), but not with age, preexisting asthma or hb-genotype. comorbidities of obesity (or, . ; %ci, . to . ), obstructive sleep apnea (or, . ; %ci, . to . ) and heart disease (or, . ; %ci, . to . ) were associated with mechanical ventilation use. the use of simple and exchange transfusion during all acs admissions ranged from . % to . % and . % to . %, respectively. among pediatric acs patients, those with obesity, obstructive sleep apnea or heart disease were at increased risk for respiratory failure and might benefit from early intervention (e.g., transfusion). surprisingly, asthma in children with acs does not appear to be a distinct risk factor for respiratory failure, and further studies are needed to clarify whether differences in treatment approach (e.g., addition of corticosteroids, bronchodilators) might impact on acs progression and/or severity even in high risk patients without asthma. objectives: to compare pulmonary functions between aa and k children with scd and to assess if a high hb f level contributes to better function. design/method: a cross sectional study was done on children with scd (hb ss disease) followed in comprehensive sickle cell programs. aa patients were followed at brookdale hospital, ny and k patients were followed in mubarak hospital, kuwait. children between the ages of and years who had pulmonary function tests (pft) done as a routine screening were enrolled. pft was done using spirometer and plethysmography. patients with congenital or anatomical lung abnormality, heart disease, pulmonary disease such as acute chest syndrome, acute asthma or pneumonia within weeks were excluded. results: there were children ( in each group) with scd,. restrictive pattern on pft was seen in / ( %) of aa vs. / ( %) of k (p> . ). obstructive pattern was seen in / ( %) of aa vs. / ( %) of the k group (p> . ). in both groups, children ( %) had normal pft. three/ ( %) in the aa group had a hb f> % as compared to / ( %) in the k group (p< . ). abnormal pft was noted in / children ( %) in each group. hbf was > % in / ( %) in the aa group vs. / ( %) in the s of s k group (p< . ). in patients with abnormal pft, mean hbf was . ± . in aa group, compared to . ± in k group (p< . ). conclusion: abnormal pft is highly prevalent among children with scd in both groups. aa children are more likely to have restrictive disease and k to have an obstructive pattern. level of hbf did not seem to protect k patients from abnormalities on pft. this finding should emphasize the importance of performing pft as part of the initial evaluation of all children with scd. background: sickle cell disease (scd) is a life-threatening disease with varied clinical spectrum and severity leading to premature death. there is a lack of validated prognostic marker in scd. recent evidence suggests that inflammation and platelet adhesion plays a critical role in the pathophysiology of vaso-occlusion in scd. elevated mean platelet volume(mpv) values are associated with a higher degree of inflammation in many disease states but it's effect on sickle cell disease or it's severity is unknown. objectives: to analyze the role of mpv in predicting disease severity/mortality in pediatric patients with scd. design/method: this is a single center retrospective study and included patients with sickle cell disease between months and years of age during a -year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . demographic information, lab data and clinical information including acute chest syndrome (acs), priapism, transfusions, sepsis, pain crisis, avascular necrosis were collected. all laboratory data were collected in steady state with no crisis in the recent past months. the disease severity score/probability of death was calculated using a validated model to predict risk of death in sickle cell disease (sebastiani et al. blood ) . pearson test was used to analyze correlation between mpv and probability of death. results: total no. of patients = ; male ( . %); female ( . %). median age is . years. all patients were of african-american origin. disease severity, hb ss - ( %); hb sc - ( . %) and sickle-beta thalassemia ( . %). patients on hydroxyurea has significantly lower mpv, p = . and this is independent of hb f levels. mpv has a significant positive correlation with the probability of death, p = . and correlation coefficient, r = . . on subgroup analysis, the correlation is even more significant in the age group between and years, p = . , r = . . using linear regression model, with probability of death as a dependent variable and hydroxyurea, mpv as independent variables, mpv maintains a significant association with probability of death (p = . ). conclusion: mpv is an independent biomarker predicting disease severity and probability of death in pediatric patients with sickle cell disease. hydroxyurea a known disease ameliorating agent is associated with lower mpv values. this effect is independent of the levels of fetal hemoglobin and may be due to anti-inflammatory effect of hydroxyurea or effect on the platelets. background: major success with initial qi projects by the sickle cell care team at children's hospital has precipitated ongoing inclusion of the qi approach to many other aspects of patient care. objectives: to optimize scd patient care utilizing qi processes. design/method: success of the scd qi team's initial project on transcranial doppler studies (tcds) and a second more complex project on hydroxyurea (hu) adherence, led to additional projects on completion of key immunizations, rbc phenotyping, and vitamin d level testing. using similar processes and principles from the hu adherence project, plan-do-study-act (pdsa) cycles were used to conduct smallscale tests of change. patient chart prep sheets, created for bi-monthly pre-appointment chart prep meetings, were significantly modified to include these focused care qi objectives. because of difficulty with emr database capability, data collected from the emr was tracked in excel spreadsheets or other unique tracking vehicles for the various parameters. for example, due to the clinic's diffuse, geographically scattered population, many separate non-shared primary care emrs, and lack of a mandatory state immunization registry; immunization records needed to be retrieved from pcps, outlying hospitals, public health departments, and fqhcs, and added to the emr and excel database. starting in / , all such data was collected and updated monthly. in one year's time ( - ) , the average immunization completion rate for seven key immunizations (pcv , pcv , hepatitis a, hepatitis b, meningococcal a, meningococcal b, and hpv) has increased by %. the biggest improvements were a % and % increase in completion for meningococcal a and meningococcal b, respectively. completion rate for rbc phenotyping rose from . % to . %. patients with at least one vitamin d lab test increased from . % to . %. since starting the tcd project in , the percent of patients who have completed their annual tcd has gone from a baseline of % to a sustained value of > %. conclusion: these qi projects have not only increased adherence to national recommendations for care of scd patients, they have helped establish a scd clinic methodology to create and implement sustainable processes. having the focused care initiatives prominently displayed on the patients' chart prep sheet serve as a reminder to medical team members to check the status of that item. this methodology is currently being used to formulate additional qi projects on annual renal function parameters and specialty visits, such as annual eye and dental exams. background: dominican republic has a high burden of sickle cell disease, and - % of children with homozygous hbss (sickle cell anemia, sca) will develop primary stroke. transcranial doppler (tcd) ultrasonography is an effective screening tool for primary stroke risk, but is not routinely available in dominican republic. hydroxyurea and blood transfusions are available, but no prospective screening and treatment program for stroke prevention has been implemented to date. ( ) to screen a large cohort of children with sca living in dominican republic, using tcd to identify elevated stroke risk; ( ) to determine the effects of treatments for stroke prevention (hydroxyurea for conditional velocities and transfusions for abnormal velocities). we hypothesized that both hydroxyurea and blood transfusions will decrease elevated tcd velocities and help prevent primary stroke. design/method: stroke avoidance for children with república dominicana (sacred, nct ) features a research partnership between cincinnati children's hospital and robert reid cabral children's hospital in dominican republic. the protocol, consent forms, and redcap database were prepared collaboratively and translated into spanish, and then irb approval was obtained at both institutions. in the initial prospective phase, children receive tcd screening over a -month period; those with conditional tcd velocities (maximum time-averaged velocity - cm/sec) receive fixed-dose hydroxyurea at mg/kg/day, followed by dose escalation to maximum tolerated dose, while those with abnormal tcd velocities (≥ cm/sec) receive monthly transfusions for stroke prevention. results: a total of children were enrolled in sacred, with an average age of . ± . years. initial tcd screening revealed ( . %) normal, ( . %) conditional, ( . %) abnormal, and ( . %) inadequate velocities. among children ( males, females, average age . ± . years) who initiated hydroxyurea at mg/kg/day for conditional tcd velocities, completed six months of treatment with expected hematological benefits including significant increases in hemoglobin concentration ( . to . g/dl) and fetal hemoglobin ( . to . %). no clinical strokes have occurred in the treatment group. repeat tcd examination after -months of hydroxyurea treatment revealed % ( / ) with previous conditional velocities had normal tcd velocities. the prevalence of conditional tcd velocities in the dominican republic is high, indicating an elevated stroke risk among children with sca. hydroxyurea treatment is associated with improved hematological parameters, lower tcd velocities, and probable decreased stroke risk. sacred is an important prospective and collaborative research trial providing epidemiological data regarding tcd screening, stroke risk, and hydroxyurea effects among children with sca. background: red blood cell aggregation is a rheologic property that explains the shear-thinning behavior of blood. at lower shear rate blood flow, red cells tend to aggregate, s of s whereas in higher shear rate blood flow, these aggregates are dispersed. this property is especially important in the venous system, where low shear rate blood flow predominates. there is inconsistent data in the literature concerning aggregation and aggregability in sickle cell disease (scd). objectives: because the lorrca and myrenne instruments have been shown to be similarly effective methodologies in red cell aggregation measurements, we aimed to determine whether the measurement of aggregation indices in scd, by myrenne and by lorrca, is consistent in our lab. design/method: we measured aggregation in blood samples corrected to % hematocrit. aggregability was measured using kda dextran in the myrenne but not the lor-rca. aggregation index using lorrca was measured in patients with scd and healthy subjects enrolled in a study of blood flow between and . aggregation and aggregability using the myrenne was measured in patients with scd and healthy subjects enrolled in a separate study of blood flow between and . results: using lorrca, we found that aggregation index in patients with scd was less than that of healthy subjects (p< . ). in the myrenne, aggregation at stasis was slightly higher in patients with scd compared to healthy subjects (p = . ) but aggregation at low shear rotation was not different. aggregability was higher in the patients with scd compared to healthy subjects at both stasis and low shear rotation (p< . ). red cell aggregation is an important determinant of low shear blood flow. deoxygenated venous blood is particularly important to low shear blood flow in patients with sickle cell disease. we found that two different aggregometers predict different aggregation results for scd. it is unclear why there is a systematic difference between the two methods, but there are some possibilities. first, the syllectogram in the lorrca is generated by the backscatter of light from the laser, while the myrenne measures transmitted light. second, the distance between the bob and cup in the lorrca is microns, while the gap between plates in the myrenne is microns, which might affect the disaggregation of red cells. further work is needed to understand the differences in red cell aggregation and aggregability when using these instruments, particularly when using aggregation as a predictor of blood flow and tissue perfusion. background: children with sickle cell disease (scd) are at risk of acute splenic sequestration crisis (assc). assc is a life-threatening complication characterized by splenomegaly, pain and severe anemia. assc most often occurs in young children with the most severe forms of scd and one-third of patients will have more than one episode. treatment is based primarily on expert opinion and includes blood transfusion and surgical splenectomy. objectives: we plan to assess the clinical practice patterns of physicians treating children with assc. design/method: a survey study was performed. the survey included six scenarios of severe scd with variation in age, hydroxyurea-use, and episode number of assc; questions focused on the acute and chronic management of assc. the survey was disseminated on three occasions over a six-month period, using an online survey tool, surveymonkey, to pediatric hematologist-oncologists participating in the american society of pediatric hematology-oncology hemoglobinopathy special interest group. the survey had a response rate of % ( / ). most respondents were recent graduates ( %; / ) practicing in academic urban centers with greater than sickle-cell patients. seventy-nine percent ( / ) recommended hydroxyurea initiation in - m/o with severe scd. prophylactic penicillin after surgical splenectomy was continued by % ( / ) after years. for the acute management of assc results did not vary despite patient age, hydroxyurea use, and the number of previous assc episodes. simple transfusion was preferred by % ( / ), with % ( / ) recommending slow transfusion and % ( / ) recommending routine simple transfusion. for the chronic management of assc, results varied based on patient age and the number of previous assc episodes. for a m/o after the first episode, % ( / ) recommended observation and % ( / ) hydroxyurea initiation. for a m/o with any prior episode of assc, % ( / ) recommended chronic transfusion therapy and % ( / ) surgical referral for splenectomy. for a y/o after the first episode, % ( / ) recommended surgical splenectomy and % ( / ) increasing hydroxyurea dose. for a y/o with any prior assc episode, % ( / ) recommended referral for surgical splenectomy. in this survey, we found most providers continue to recommend simple transfusions for assc and surgical splenectomy after two episodes. the majority of providers continue to delay referral for surgical splenectomy until age two, but earlier referral in children under two and use of chronic transfusion therapy were also reported. variability in chronic management highlights the need for further research of splenic sequestration. background: developing therapies for sickle cell disease (scd) is challenging in part because the accepted endpoint, vaso-occlusive crisis (voc), occurs infrequently, does not measure full disease burden, and is a measure of healthcare utilization. in phase / studies of patients with scd, voxelotor (gbt ) has demonstrated increased hemoglobin (hb) levels and reduced hemolysis and has been safe and welltolerated. voxelotor is being evaluated in the ongoing hope phase trial. objectives: to report the innovative phase / hope trial design with novel primary and secondary outcomes to accelerate drug development. design/method: hope (nct ) is a phase , randomized, placebo-controlled, multicenter study of oral voxelotor in patients with scd (aged - years) with baseline hb . - . g/dl and - episodes of voc in the prior year. to accelerate clinical trials to support drug development, the study combines a phase exploratory, dose-selection phase (group ) with a pivotal phase (groups / ). patients in group will be randomized : : to voxelotor or mg/day or placebo. analysis for dose selection will occur when the final patient has received weeks of treatment. group will continue enrollment with randomization : : until dose selection based on analysis of the group cohort. group will allow for a seamless transition into group , which will randomize patients : to the selected dose or placebo. the final data analysis set will include group patients who received placebo or the selected dose and all group patients. the primary endpoint is an objective laboratory measure and surrogate of clinical benefit, increase in hb > g/dl, from baseline to weeks based on voxelotor mechanism of action (inhibition of hb polymerization). this trial is the first to use a patient-reported outcome (pro), the -item sickle cell disease severity measure, as a secondary endpoint. this novel electronic pro, developed specifically for the hope study following fda guidance, will evaluate changes in scd symptom exacerbation and total symptom score from baseline to weeks. additional secondary endpoints include measures of hemolysis, rates of voc, transfusions, and opioid use. the study was designed to enable selection of pro-defined symptom exacerbations or traditionally defined voc as the key secondary endpoint after the group analysis. results: this study is ongoing. the hope trial, expected to complete enrollment by late , will evaluate the efficacy and safety of voxelotor compared with placebo in patients with scd. supported by global blood therapeutics. background: inflammation, coagulation activation, oxidative stress and blood cell adhesion are elements of sickle cell disease (scd) pathophysiology. patients with scd have low levels of the omega- fatty docosahexaenoic acid (dha) and eicosatetraenoic acid (epa) in plasma and blood cell membranes. dha is a bioactive fatty acid with anti-inflammatory, anti-blood cell adhesion and anti-oxidant properties. altemi-atm is a novel dha ethyl ester formulation with a proprietary delivery platform (advanced lipid technology® (alt®)) that enhances oral dha bioavailability. the scot trial investigated the effects of altemiatm in children with scd. objectives: to demonstrate the effects of altemiatm on blood cell membrane omega- index and selected biomarkers of inflammation, coagulation, adhesion and haemolysis associated with scd. s of s design/method: children with scd, aged - years (n = ), were enrolled. subjects were randomized to receive either placebo or one of three daily oral doses of altemiatm ( - , - or - mg/kg/day dha) for two months. the effects of altemiatm on red blood cell (rbc), white blood cell and platelet membrane omega- fatty acids index (total dha + epa levels) were assessed after four weeks of treatment. the effects of altemiatm on markers of inflammation, adhesion, coagulation, and hemolysis were assessed after eight weeks of treatment. cell membrane dha and epa concentration was determined by using lc-ms/ms method. the percent changes from baseline on blood cell membrane omega- index and select scd biomarkers were compared between the three dose groups and placebo using a mixed-model repeatedmeasures (mmrm) analysis with baseline blood cell membrane omega- index, hydroxyurea use, and treatment as fixed effects and patient as a random effect. after four weeks of treatment, blood cell membrane dha and epa levels were significantly increased in all altemiatm doses (p< . ). after eight weeks of treatment, significant reductions were observed in se-selectin (p = . ), and d-dimer (p = . ) in patients exposed to altemiatm dose level vs. placebo. hemoglobin was significantly increased at altemiatm dose level versus placebo. plasma high-sensitivity c-reactive protein, lactate dehydrogenase, soluble vascular cell adhesion molecule- and white blood cell count showed improvement after weeks of treatment in all three altemiatm doses levels but did not reach significance. conclusion: treatment with altemiatm enriches dha and epa in blood cell membranes of patients with scd and improves select sickle cell disease biomarkers of blood cell adhesion and thrombin generation. these findings provide insight into the mechanisms of action of altemiatm in sickle cell disease. brown university -hasbro children's hospital, providence, rhode island, united states background: despite clinical advances in the treatment of sickle cell disease (scd) in pediatric and young adult patients, pain remains a significant source of disease-related morbidity. physical therapy has been shown to be useful for the treatment of pain in children and young adults with various chronic illnesses of which pain is a significant component, however no data exists regarding potential benefits of physical therapy in pediatric and young adult patients with scd. objectives: to query healthcare providers and others involved in the care of pediatric and young adult scd patients regarding possible benefits of and barriers to physical therapy as a potential treatment modality. design/method: we conducted a web-based survey of healthcare providers within the new england pediatric sickle cell consortium (nepscc) in an attempt to identify potential benefits of and barriers to outpatient physical therapy in this patient population. results: nearly % of survey participants felt that physical therapy had the potential to be "somewhat beneficial" or "very beneficial" in pediatric and young adult patients with scd. a majority of physicians reported having referred patients with scd for physical therapy in the past. the most frequently identified perceived potential benefits included improved functional mobility, improvement of chronic pain symptoms, decreased use of opiates, improved mood symptoms, improved acute pain symptoms, and improved adherence with medications and clinic visits. significant perceived barriers identified included lack of transportation, time constraints, patient lack of understanding, and difficulty with insurance coverage. our study indicates that healthcare providers have an overwhelmingly positive view of the use of physical therapy in the management of pediatric and young adult patients with scd. significant barriers exist which need to be addressed. future research should focus on patient and parent perspectives regarding physical therapy, as well as a randomized controlled trial of a physical therapy intervention in this patient population. background: vitamin-d deficiency is fast becoming increasingly recognized in patients with sickle cell disease (scd). while it is estimated that these patients are five times more likely to develop vitamin-d deficiency, the exact clinical significance of this is largely unknown. given that this deficiency can be inexpensively and easily treated, our study sought to establish the prevalence of vitamin-d deficiency in our patient population and its relationship with disease severity. objectives: to estimate the prevalence of vitamin-d deficiency in patients with scd in our institution and to analyze their disease severity in relation to their vitamin-d level. design/method: through retrospective chart review we analyzed subjects that represent a cohort of patients followed at the adult and pediatric hematology services at university of miami with known diagnosis of scd that had a vitamin-d level drawn between january st, and august st, . we conducted a cross-sectional study and recorded the first vitamin-d level during this period. patient demographics, medical and social history information were collected along with laboratory data. the number of admissions for vaso-occlusive crisis (voc) and acute chest syndrome within one year preceding the collection the vitamin-d level was also recorded. results: a total of charts were reviewed, adult charts and pediatric charts. after exclusion, patients were enrolled. subclinical vitamin-d deficiency is only evident on laboratory blood testing of vitamin-d ( -hydroxy) and according to this laboratory result patients were classified as sufficient (≥ ng/ml), insufficient (< to ng/ml) and deficient (< ng/ml). out of the cases, . % ( / ) were deficient, . % ( / ) were insufficient and . % ( / ) were optimal. after statistical analysis two negative correlations were identified, increasing vitamin-d levels with decreasing white blood cell count (ci %- . (- . , - . )and decreasing incidence voc (ci %- . (- . , - . ). conclusion: this study confirms that there is a significant prevalence of vitamin-d deficiency in patients with scd. furthermore, the results of this investigation proved that vitamin-d deficiency is associated with acute pain and leukocytosis in patients with scd. given the multitude of confounding factors that affect vitamin-d absorption and intake, multivariate analyses are required to truly further investigate this relationship. texas children's hospital, houston, texas, united states background: hemophagocytic lymphohistiocytosis (hlh) is a rare but life-threatening condition of hyper-inflammation that is characterized by splenomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, hemophagocytosis and coagulopathy. although timely diagnosis is imperative, it is often challenging as these individual signs and symptoms may occur in a variety of clinical conditions. to report a case of undiagnosed sickle cell anemia presenting with severe ebv viremia and associated hemophagocytic lymphohistiocytosis results: a -month-old previously healthy male presented with respiratory distress, increased fatigue, and a focal seizure following a two-week history of cough and lowgrade fevers. physical exam was consistent with hypovolemic shock and revealed significant splenomegaly. laboratory testing revealed severe hypoglycemia, acidosis and electrolyte disturbances including hyperkalemia, hyperphosphatemia, and hyperuricemia. labs showed a leukocytosis (wbc , ), severely low hemoglobin ( . ), and platelets of , . coagulation testing revealed prolonged pt/inr and ptt, hypofibrinogenemia and a highly elevated d-dimer. additional workup was completed to determine etiology of acute presentation, given broad differential diagnosis. infectious studies were consistent with an acute ebv infection (plasma ebv pcr > , ). elevated levels of soluble il- and ferritin completed / criteria for the diagnosis of hlh. bone marrow evaluation showed trilineage hematopoiesis with no abnormal blast population or hemophagocytosis. results from hemoglobin electrophoresis sent from the initial cbc sample were notable for hbs . %, hbf . %, and hba of %, confirming the diagnosis of sickle cell disease. the patient was started on hydroxyurea and penicillin and splenomegaly resolved. with supportive care, he demonstrated gradual improvement in symptoms and laboratory abnormalities, including normalization of soluble il- , ferritin, cd , il- levels, immunoglobulins, and declining ebv titers. nk cell function has remained abnormally low, not eliminating the possibility of acquired hlh despite spontaneous improvement. conclusion: splenic sequestration associated with sickle cell disease in combination with acute infectious mononucleosis could have explained many of the presenting symptoms including anemia, thrombocytopenia, and splenomegaly. however, it does not explain the unusually high ebv titer and degree of inflammation meeting diagnostic criteria for hlh, which raises concern for an underlying immunologic abnormality such as x-linked lymphoproliferative disorder (xlp). although testing for xlp was negative, he will require s of s continued monitoring in the future for signs of relapse. this case illustrates the complexity of diagnosing lymphohistiocytic disorders and the significant overlap in presentation between these disorders and other medical conditions. background: vaso-occlusive crisis (voc) is one of the most distressing occurrences in patients with sickle cell disease (scd). patient controlled analgesia (pca) is recommended by nih and expert opinions favor its early use. we aim to review the use of pca in patients with voc and to evaluate if its early use is associated with faster pain control and reduced length of stay (los). design/method: this retrospective single center study included all pediatric patients admitted and treated with pca for a severe voc from to . "early" use was defined as start of pca within hours of arrival in the emergency department (ed) and "late" use after hours. time to reach adequate analgesia was defined as oucher, verbal scale or faces pain scale < / obtained twice consecutively in a -hours interval. time to reach adequate analgesia and los were compared between early-pca and late-pca groups. results: a total of patients presented episodes of voc treated with pca during the study. sixty-one episodes ( %) were treated with early-pca and ( %) with late-pca. both groups were comparable in terms of age ( . vs . years old), gender ( . % female vs . %), hemoglobin phenotype ( . % hbss vs . %), but median pain score at admission was higher in early-pca than in late-pca ( / vs / , median difference ( % ci , ). early-pca was associated with a median reduction in los of . days ( % ci . , . ) (median early-pca los . vs late-pca . days). time to reach analgesia could be evaluated only in a subset of patients ( in early-pca and in late-pca group). although time to reach adequate analgesia tended to be shorter in the early-pca group, it was not statistically different: median . hours vs . hours, difference of . ( % ci - . , . ). side effects were observed during ( . %) pca treatments ( / ( . %) episodes in early-pca, / ( . %) in late-pca group) among which ( . %) were significant adverse events. these were observed in patients who required interventions: desaturations requiring oxygen without intubation, neurologic abnormalities (hallucinations, visual abnormalities, no stroke), urinary retentions. conclusion: early use of pca for severe voc was associated with a reduced length of hospital stay despite that these patients had higher pain score on admission. prospective studies are needed to support these positive outcomes. background: acute chest syndrome is one of the leading causes of death in children with sickle cell disease - . while the cause of acute chest syndrome most commonly is not identified, fat embolism and infectious causes are believed to be most common. with an extremely high mortality rate, rapid identification and initiation of therapy is essential for survival. case presentation: we describe the case of an -year-old female with sickle cell sc disease who was admitted for vasoocclusive pain crisis and quickly progressed to multi-system organ failure due to fat embolism syndrome and parvovirus b infection objectives: the case highlights the presentation and diagnosis so other providers can optimize outcomes for those with this under-recognized syndrome design/method: her parvovirus studies returned after days which showed: parvovirus b dna pcr detected; parvo igg . (positive > . ); and igm . (positive > . ). the patient experienced an approximately . g/dl drop in hemoglobin( . to . g/dl/ hrs) with progressive thrombocytopenia (from , to , /ul) and a peripheral smear showed microcytic,normochromic red cells with nucleated rbcs and occasional nuclear budding, slight polychromasia, schistocytes, and polymorphic cells with toxic granules that suggested leukoerythroblastosis. she was emergently transferred to the regional quaternary care hospital for ongoing ecmo therapy where she experienced a change in her pupillary exam prompting a stat ct scan that showed severe, diffuse cerebral edema with transtentorial herniation. the decision was made to withdraw life-sustaining therapies and her family refused a post-mortem autopsy examination. fat embolism syndrome is a severe and uncommonly recognized complication of sickle cell disease, seen most commonly in those with a non-ss phenotype and previous mild disease course who present with severe, unrelenting vaso-occlusive pain episode and/or acute chest syndrome that progresses to respiratory distress with altered mental status and cutaneous changes. rapid identification and initiation of exchange transfusion therapy should be initiated with clinical suspicion because of the extremely high mortality rate. although previously considered rare, it needs to be considered in the differential diagnosis of more commonly encountered complications of sickle cell disease. background: patients with sickle cell disease (scd) experience vaso-occlusive crisis (voc), which results in extreme pain, often requiring opioids and admission. genetic and environmental factors affect the frequency and severity of these episodes. previous research has born conflicting evidence on whether environmental temperature is contributory. edmonton, alberta is the northern most city with a population over a million in north america. there is an increasing sickle cell population which is exposed to extreme winter conditions. this provides a suitable population and atmosphere to study the influence on cold external temperatures in scd. this study sought to identify if pediatric patients with scd, experience greater morbidity in cold external temperatures. board approved retrospective case control series. patients were identified through a clinical database, and emergency visit, phone call and admission data was collected over a fiveyear period. the average, minimum and change in temperature on day of presentation, and hours prior, was collected from the government of alberta, and was statistically analyzed using descriptive statistics, to determine the relation to vaso-occlusive events. results: one-hundred and eighteen patients were identified, and voc events reviewed. the mean patient age was . years of age with a range from . - years old. the female to male ratio was equivalent with female ( . %) and male ( . %) voc events. eight records ( %) had docu-mented cold exposures. the analysis between the temperature and the frequency of events did not yield significant correlation. average and minimum temperature on day of admission had the largest percentage of voc events occur at mild temperatures, from - . to • c and - . to respectively. change in temperature on day of admission, and hours had the largest percentage of voc events at a mild to moderate change in temperature of - degrees. data at & hours prior to admission showed similar results. secondary data analysis accounting for the lower proportion of extreme weather days in comparison to moderate temperate days showed no significant impact. there was no correlation of average, minimum or change in temperature on day of admission, or hours prior. multiple cofounding factors likely contribute to these results. as it was a retrospective study many confounding and precipitant factors may not be recorded or identified. a prospective study to better record specific cold exposure is warranted. children's national health system, washington, district of columbia, united states background: achieving optimal anticoagulation with unfractionated heparin (ufh) in pediatric patients receiving extracorporeal membrane oxygenation (ecmo) is often challenging due to antithrombin (at)-mediated heparin resistance (hr). intermittent at dosing during pediatric ecmo support does not maintain adequate at levels. continuous at infusion (cati) presents an alternative strategy to achieving consistent goal at levels and optimizing heparinization. however, cati during pediatric ecmo has not been adequately studied. objectives: to describe our center's experience with an ecmo cati protocol. design/method: in , we modified our ecmo anticoagulation protocols to include ufh titration according to anti-factor xa (anti-fxa) levels and cati in patients with at-mediated hr. the cati rate was calculated using baseline and goal at levels while accounting for the circuit volume. cati was administered with ufh into the circuit via a s of s y-infusion set. at and anti-fxa levels were monitored every hours. recombinant at (r-at) concentrate was used at our center until with subsequent transition to a plasmaderived at (pd-at) concentrate. due to the longer half-life of pd-at concentrate, the protocol was modified so cati is stopped once target at and anti-fxa levels are achieved. we conducted a retrospective study of all patients who received cati during ecmo support at our center. data are reported as median and interquartile range and compared using the mann-whitney u test. two-tailed p-value < . was considered statistically significant. since , patients [ males, age month ( . - )] on ecmo support received catis ( rat, pd-at) per our protocol ( patients received pd-at infusions during one ecmo run). the duration of cati was hours ( - ). cati administration led to significant increases in at and anti-fxa levels from baseline of % ( - ) and . units/ml ( . - . ) to the first level within goal of % ( - ) and . units/ml ( . - . ), respectively (p< . ). the respective times to achieve goal at and anti-fxa levels were hours ( - ) and hours ( - ). the respective peak at and anti-fxa levels were % ( - ) and . units/ml ( . - . ). during cati, no patient required circuit change, patient developed cannula thrombosis and patients experienced non-fatal major bleeding. conclusion: cati in pediatric patients receiving ecmo support with close monitoring of at and anti-fxa levels was associated with significant rapid increase in at, optimization of heparin effect, and reduction in thrombotic complications without increase in major bleeding compared to prior reports. a prospective study of this at dosing strategy is warranted. children's hospital of orange county, orange, california, united states background: inherited factor xiii (f ) deficiency is a rare bleeding disorder with wide heterogeneity in clinical manifestations ranging from mild bruising, and mucosal and umbilical stump bleeding to spontaneous, severe intracranial bleeding. the bleeding phenotype is influenced not just by zygosity of the fxiii mutation alone, but also by co-inheritance of variants in other clotting protein genes that also play a major role in clot formation and stability. we present a series of three siblings found with f a gene variant and platelet dysfunction linked to bleeding phenotype. design/method: retrospective chart review of the index case, coagulation studies and whole gene sequencing. the index patient presented at two years of age with a subdural hematoma after a fall, requiring emergent craniotomy. a week after initial evacuation, she re-bled, prompting an extensive work-up for potential bleeding disorders, including f activity, von willebrand profile, comprehensive fibrinolysis panel, pai- antigen level, platelet mapping thromboelastogram (plt-teg), and f genetic analysis. the patient's identical twin and older sibling, who had symptoms of bruising, underwent a similar evaluation. the index patient demonstrated consistently low f activity ( - %), and platelet function testing revealed decreased response to adp agonists. the twin and older sibling had normal f levels, and only slightly decreased response to adp in platelet studies. whole gene analysis of f and other genes on our next generation panel, revealed several intronic deletions in the index patient that were not shared by her siblings, which likely account for her decrease in circulating f levels. her symptoms have responded well to monthly treatment with factor concentrate. all three children shared the f variant, pro leu, previously described as a risk factor for intracranial hemorrhage. the f mutation, pro leu, has been associated with intracranial hemorrhage in young women, but the presence of the variant alone may not be enough to cause a severe bleeding phenotype. family studies identified novel deletions in the index patient which may account for her decreased f levels, which would have been overlooked with standard sequencing. future studies, including evaluation of 'platelet' f levels, should be performed when platelet dysfunction is detected. further laboratory and clinical evaluation is required to delineate the long term implications of the interaction of even mild f deficiency if present with additional clotting disorders such as the platelet function defect in these siblings. background: acquired hemolytic anemia can occur due to mechanical shearing of red blood cells and is classically seen in patients with prosthetic heart valves. there are reports of this same traumatic effect with other repairs, including annuloplasty. following valvular procedures flow disturbances can exist across the valve that lead to shear stress and hemolysis. although von willebrand disease (vwd) is typically seen due to an inherited disorder in the pediatric population, flow disturbances in the setting of valve abnormalities can lead to acquired von willebrand syndrome (avws). von willebrand factor multimers become unfolded and elongated in the setting of shear stress resulting in increased susceptibility to cleavage by adamsts- . specifically, loss of high molecular weight multimers (hmwms) can lead to a syndrome akin to type a vwd. objectives: to describe a case of mechanical hemolysis with acquired type a vwd design/method: a -month-old girl with history of hypoplastic left heart syndrome and severe tricuspid valve insufficiency underwent norwood procedure, blalok-taussig shunt placement and subsequently a bidirectional glenn and tricuspid valve annuloplasty. during the following month she requires weekly red blood cell (rbc) transfusions due to intermittent anemia. she also experienced bloody stools and dark urine. laboratory evaluation was notable for normocytic anemia, reticulocytosis, elevated lactate dehydrogenase, and low haptoglobin consistent with hemolytic process. immune-mediated hemolysis from transfusion reaction or presence of autoimmune or alloimmune antibodies testing was negative. to investigate gi bleeding, work up for vwd revealed normal vw activity and antigen but with loss of high molecular weight multimers consistent with acquired type a vwd. in consultation with cardiology, it was felt her tricuspid valve insufficiency jet could be leading to mechanical hemolysis and avws. a repeat echo showed persistent moderate tricuspid insufficiency but no other significant changes. due to the patient's continued need for weekly rbc transfusions she was subsequently trialed on pentoxifylline which is used in adult patients to decrease blood viscosity and increase erythrocyte flexibility in patients with mechanical hemolysis. her transfusion needs remained the same and the medication was discontinued after two weeks. she required one transfusion a week later but no transfusions since that time. although not commonly seen in pediatric patients, the diagnosis of mechanical hemolysis accompanied by avws should be pursued in a patient with congenital heart disease with significant anemia and/or bleeding. the work up in these patients is difficult as echocardiograms can be inconclusive thus an extensive hematologic evaluation is usually necessary. objectives: our aim was to assess incidence of and potential risk factors for central line-related dvt at our institution between - . additionally, our goal was to analyze if that incidence differed between the three central line types and identification of line-specific risks. design/method: a retrospective chart review of central line placements in pediatric patients at cleveland clinic between - was conducted. data included demographics, potential risk factors, line characteristics and any related thrombotic events. the study cohort consisted of lines in pediatric patients aged - years of age. there were . thrombi ( % ci . - . ) per , line days. statistically significant risk factors for thrombus include diagnosis group (liquid tumor highest rate of %, solid tumor lowest at %), type of line (picc %, broviac %, and mediport %), location of line, greater number of lines per patient, peg asparaginase ( % vs %), sepsis, and history of procoagulant state. line characteristics such as lumen size and number of lumens were not identified as a significant risk. there was a significantly higher rate of thrombus in than in the previous years when pooled ( % in vs . % from - , p = . ). the incidence of dvt in pediatric patients at our institution was highest with broviac lines, and significant risk factors in our patient population included liquid tumor, femoral vein location, peg asparaginase, sepsis, and history of a procoagulant state. the incidence of thrombi was highest in , and therefore highlights the urgent need for improvement in nationwide hospital practices to minimize risk of thrombi formation and early detection in the higher-risk s of s populations. there is still much to be learned regarding the characteristics specific to different central lines, which would influence thrombi formation. nyu winthrop hospital, mineola, new york, united states background: pediatric immune thrombocytopenic purpura (itp) is an autoimmune disorder with platelet counts < causing increased risk for significant hemorrhage. there is increased immunologic platelet destruction due to production of specific autoantibodies along with inhibition of platelet production. few randomized trials exist to guide management and ultimately each patient requires an individualized treatment plan. itp may be acute (diagnosis to m) or chronic (> months). one of the treatments of chronic itp is laparoscopic splenectomy (ls), which is very well tolerated. a rare complication of ls is splenosis, an autotransplantation or implantation of ectopic splenic tissue within the abdominal cavity or in any other unusual body compartment. splenosis is sometimes associated with relapsed itp due to preserved immune activity. the usual management of symptomatic splenosis is surgical resection. objectives: to describe medical management in a young patient with itp relapsed due to extensive unresectable splenosis following ls design/method: our patient was originally diagnosed at years with itp and was treated with ls at years of age for chronic severe thrombocytopenia and persistent bleeding not responding to first line therapies. she tolerated it well and had a complete response (cr) defined as a platelet count of > measured on occasions > days apart and absence of bleeding. she maintained a normal platelet count for twelve years after which she relapsed (loss of response after cr) with severe thrombocytopenia and hematuria necessitating high dose steroids. ct scans showed multiple wellcircumscribed soft tissue masses in the left lower quadrant adjacent to uterus and left ovary, involving left omentum and the anterior abdominal wall partly. findings were confirmed by damaged rbc nuclear scan to be splenosis. during laparoscopy the splenosis lesions were deemed too extensive and were not resected completely to avoid postoperative morbidity. she was started on sirolimus around the same time for treatment of her relapsed itp and steroids were weaned off. results: eight months since beginning sirolimus with therapeutic levels she remains in cr with no bleeding and has not required any steroids, immunoglobulins or anti d immunoglobulin. conclusion: sirolimus is a safe and effective steroid-sparing agent in treatment of chronic itp. this is the first instance of a patient with poorly resectable splenosis responding well to medications for itp. more data is needed regarding the longterm efficacy of such an intervention and whether it will eliminate the need for a second surgery in relapsed itp patients with extensive splenosis. background: storage pool disorders affecting platelets result in bleeding symptoms related to a deficiency or defect in alpha granules or delta granules. in delta-storage pool disorders (dspd,) there is a deficiency of the delta granules and their constituents, which results in the inability of platelets to properly activate as well as lack of proper constriction of blood vessels during bleeding episodes. amongst patients with dspd, females most commonly present with menorrhagia, while males tend to present with epistaxis and easy bruising. the international society on thrombosis and hemostasis (isth) developed a screening bleeding assessment tool (bat) for mild bleeding disorders, shown to be a validated tool in children. diagnosis of dspd is classically made with a platelet electron microscopy (pem) value < . delta granules per platelet (dg/pl), but recently lower diagnostic thresholds of dg/pl or even . dg/pl have been suggested. objectives: evaluate the correlation between pem and bleeding scores, and also examine various cut-off values used to diagnose and risk stratify patients with dspd. design/method: retrospective chart review of pediatric patients followed by hematology with a diagnosis of dspd was performed. clinicians obtained bleeding scores for each patient as standard of care in the hemostasis clinic. quartile ranges were established to appropriate three stages of severity based upon bleeding scores. statistical analysis was performed using software r and exploratory data analysis to evaluate for a correlation. results: amongst all patients, the average bat score was . and pem was . dg/pl. the average bleeding score for pem between . dg/pl and dg/pl was . , while the average bleeding score for pem below dg/pl was . . the correlation coefficient between pem and bleeding scores is . . using a threshold of dg/pl, % of patients would have met diagnostic criteria. quartile ranges for the bleeding scores are as follows: st quartile was - , nd quartile was - , and rd quartile was > . conclusion: patients with a more marked granule deficiency do not exhibit a more severe bleeding phenotype, suggesting proper platelet function is not solely determined by granule quantity in these patients. bleeding severity may be more appropriately assessed with bleeding scores rather than pem values, and using quartile ranges may aide in risk stratification and therapeutic interventions for dspd patients. further work remains to determine the optimal diagnostic threshold of pem dspd in pediatric populations. texas children's hospital, houston, texas, united states background: warfarin management has many challenging aspects including pharmacogenomics, food and drug interactions, lack of standardized dosing, patient compliance, tracking lab results from multiple lab locations, and the potential for significant bleeding or thrombotic complications. a literature review revealed limited data highlighting anticoagulation monitoring workflow and emr documentation and specifically, no data in the pediatric population. historically, the texas children's hospital cardiology and hematology centers were each documenting anticoagulation data within the epic tm system differently. epic's tm original design for anticoagulation documenting resulted in the necessity to duplicate documentation in order to see at-a-glance critical anticoagulation monitoring information. objectives: the objective of this project was to standardize inr documentation across departments to reduce the risk of patient safety events and improve workflow. design/method: a workgroup assembled consisting of nurses from the cardiology and hematology departments, along with staff members from the epic tm is support group. the workgroup identified current documentation practices, available epic tm tools, and brainstormed ideas to streamline and improve both documentation with the current epic tm tools. physician partners were identified in cardiology, hematology and coagulation laboratory to gain their input. a new anti-coag (ac) encounter was developed and first made available in an epic tm practice environment, then once approved, epic tm written education and training session were completed by both departments' staff. results: surveys were sent to health care providers in the cardiology and hematology centers prior to the new ac encounter, and also to health care providers six months after implementing the ac encounter. six responses were received for each survey. the pre-implementation survey showed the most problematic part of the documentation system for anticoagulation was no single place in the emr to find a complete anticoagulation picture. post ac encounter implementation survey results revealed more health care providers using the epic tm inr reminder pool, less time needed to compile a report of three months of anticoagulation information, less time needed to document individual encounters, less locations needed to document ac information and decreased amount of types of documentation used. standardized ac encounters improves workflow with less time needed to document and compile information, less types of documentation utilized and easier access to patients ac information. next steps include retrospective review of patients' inr time in therapeutic range to determine if there was an impact on patient compliance and continue to evaluate and modify the ac encounter to enhance user friendliness. caitlin tydings, jennifer meldau, christine guelcher, carole hennessey, eena kapoor, michael guerrera, yaser diab s of s children's national health system, washington, district of columbia, united states background: venous anatomic abnormalities (vaas) are considered a risk factor for developing deep vein thromboses (dvts) that occur as a result of significant alterations in venous blood flow. identification of predisposing vaas can be challenging. hence, diagnosis can be delayed or overlooked especially in pediatric patients. dvts in children or adolescents with predisposing vaas have been only described in sporadic case reports and small case series. objectives: to describe characteristics and outcomes of dvts in pediatric patients with underlying vaa treated at our center. design/method: we conducted a retrospective chart review of all pediatric patients with objectively confirmed extremity dvt treated at our institution over a -year period from to and identified all patients with underlying vaas. patients were managed according to standardized institutional protocols based on published guidelines. post-thrombotic syndrome (pts) was assessed at our center using the manco-johnson instrument. relevant data were collected and summarized using descriptive statistics. during the study period, of pediatric patients ( %) [ females, median age years (range - )] diagnosed with extremity dvt at our center were found to have an underlying vaa. vaas included may-thurner anomaly ( patients), venous thoracic outlet obstruction ( patients) and inferior vena cava (ivc) atresia ( patients). additional provoking factors were identified in patients at time of presentation. dvt locations included upper extremity veins ( patients), lower extremity veins ( patients) and lower extremity veins and ivc ( patients). the majority of dvts [ patients, ( %)] were completely occlusive. high risk thrombophilia (defined as inherited deficiency of antithrombin, protein c, or protein s, or antiphospholipid antibody syndrome) was present in patients ( %). all patients were treated with therapeutic anticoagulation with patients continuing indefinite anticoagulation. endovascular interventions were performed in patients and included percutaneous pharmacomechanical thrombectomy and/or catheter-directed thrombolysis ( patients), balloon angioplasty ( patients) and stent angioplasty ( patients). surgical interventions included thoracic decompressive surgery ( patients) and surgical thrombectomy ( patient vvas represent an important risk factor for developing extensive extremity dvt in adolescents. this special population is at risk for short-term and long-term com-plications. early identification and correction of vaas may improve outcomes. however, multicenter, prospective studies are needed for developing optimal evidence-based treatment approaches. alexander glaros, roland chu, sureyya savasan, meera chitlur, madhvi rajpurkar, yaddanapudi ravindranath children's hospital of michigan, detroit, michigan, united states background: acute budd-chiari syndrome (bcs) is a rare thrombotic emergency in children, and etiologies/treatment are less well-defined than in adults. in adults, a systematic approach including anticoagulation, relief of venous obstruction, and treatment of the underlying cause has proven successful. more recently treatment has tilted towards aggressive surgical interventions, which carry significant risk and are often not feasible. objectives: review our experience with three different patients with bcs and suggest a mechanistic based approach to treatment. the records of three patients with bcs were reviewed and their presentations, etiologies, treatment, and outcomes were reported. results: patient a was a -year-old female with paroxysmal nocturnal hemoglobinuria who presented with recurrent worsening abdominal pain over several months. narrowing of inferior vena cava (ivc) and hepatic veins was noted on imaging. liver transplant was not considered surgically feasible. she was treated with eculizumab, steroids, and anticoagulation with restoration of hepatic venous flow in weeks. patient b was a -year-old male with several weeks of right upper quadrant pain, fatigue, and pre-syncopal episodes, with a history of blunt abdominal trauma from football scrimmage weeks earlier. he was found to have near complete occlusion of the ivc and hepatic veins. liver transplant was not considered feasible. he was successfully treated with anticoagulation alone. patient c was a -yearold male with acute myeloid leukemia in induction cycle who developed severe pancytopenia; typhlitis was diagnosed and managed medically. days later he acutely decompensated, arrested, and was placed on extra corporeal membrane oxygenation, and imaging showed complete occlusion of the portal vein, hepatic veins, and ivc to the level of the atrium, with bilateral pulmonary emboli. emergency liver transplant or catheter based interventions was deemed not feasible. treatment with eculizumab was considered for presumed inflammation induced complement activation (c mg/dl [normal - ]; ch was u/ml [normal - ]) as a trigger for thrombosis, but the patient progressed quickly and died before it could be initiated. our experience with bcs shows that invasive interventional options and liver transplant may not be feasible in most patients for multiple reasons. rapid diagnosis and aggressive etiology-based medical management are paramount to successful treatment of this rare complication. eculizumab may be considered in treating bcs with complement activation not only due to innate disorders, but also secondary to acute inflammation when proper laboratory evidence is present. background: platelet aggregation studies are the gold standard for the diagnosis of platelet function defects during the evaluation of a patient with bleeding problems. the platelet aggregation test measures how well platelets clot in response to different concentrations of epinephrine, adenosine diphosphate (adp), collagen, arachidonic acid and ristocetin. because platelet function defects are often under-recognized and under-diagnosed in the pediatric patient, the true incidence is unknown. we report our experience in the diagnosis of platelet defects at our institution over a -year period in order to add some clarity to the limited pediatric data available. objectives: our primary objective is to document correlations/trends between less well-known platelet function abnormalities and clinically significant bleeding at our institution over a -year period. design/method: after appropriate irb approval obtained, we performed a retrospective chart review of all children who had platelet aggregation testing done from to . data collected included demographics (age, sex, race), personal and family history of bleeding, screening for coagulation defects and platelet aggregation test results. symptoms examined in our data were limited to epistaxis and heavy menstrual periods. for each of these symptoms, results were further analyzed to those with abnormal responses to adp and epinephrine. patients with existing bleeding diagnoses and those with incomplete medical records were excluded. we identified patients. of the patients with epistaxis, % had abnormal platelet aggregation testing while only % of those with heavy menstrual periods had abnormal results. within our population, abnormal platelet function assay (pfa- ) results or race did not appear to correlate with abnormal platelet aggregation testing. in the cases of epistaxis, sex was also noncontributory. our preliminary results suggest that platelet aggregation testing was more useful in predicting platelet defects in those with a clinical bleeding history of epistaxis as opposed to heavy menstrual periods. for other presenting symptoms, platelet aggregation testing did not offer diagnostic benefit. abnormal response to adp in the platelet aggregation test was the most common finding in our population; the clinical significance of which is not well understood. going forward, we plan to document whether abnormal results correlated significantly with the subsequent final diagnoses of our patients. background: decision making for severe hemophilia a in previously untreated patients (pups) has recently become a significant ethical debate. recombinant factor viii (rfviii) products previously were recommended to avoid transmission of blood borne pathogens associated with plasma-derived fviii (pdfviii) products. however, the increased incidence of fviii alloantibody inhibitors with rfviii products compared to pdfviii products has challenged this former standard of care. despite the support of the medical and scientific advisory council, recommendations considering pdfviii products for a pup remains controversial. design/method: we used a modified utilitarian approach involving clinical, public health, and research ethics. shared decision making permeates the framework to maximize understanding, minimize bias, respect informed consent or dissent, and provide care that aligns with patient and family values when medically and practically feasible. the framework has three tiers. first, it evaluates whether resources are scarce or abundant for equitable resource allocation. if fviii products are scarce, we s of s recommend developing a central supply for emergency use and then evaluating the needs of the severe hemophilia a patients. prioritization of who receives the factor products would be decided by a designated team based on the availability of the factor products and clinical scenarios, with no preference given to those on research trials. however, if resources are abundant, treatment for acute bleeding and standard of care prophylaxis measures, including primary prophylaxis, could continue. the second tier accounts for whether there is a new infectious epidemic or concern where a pathogen cannot be eliminated. if there is, healthcare and public health workers may limit the use of pdfviii products. if not, pdfviii and rfviii products are to be equally considered. the third tier evaluates whether the clinical scenario is emergent or not. if there is acute, emergent bleeding, the immediately available resource should be used, along with bypassing and/or adjuvant resources as needed until the bleeding has resolved or improved. to align with patient and family preferences, attempts to have both pdfviii and rfviii products available at similar costs in institutions would be ideal. this ethical framework endeavors to balance autonomy, beneficence, nonmaleficence and justice in helping guide discussions among providers, pups with severe hemophilia a, and their families. disclaimer: findings and conclusions are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention, emory university, or children's healthcare of atlanta. background: von willebrand disease (vwd) is a common bleeding disorder which affects up to % of the population without gender predilection. bleeding associated with this condition results from a deficiency or abnormality in von willebrand factor interfering with formation of primary hemostasis. ehlers-danlos syndrome (eds) is a group of rare inherited connective tissue disorders which may have an associated bleeding manifestation without abnormalities in coagulation testing. bleeding symptoms reported in eds result from capillary and tissue fragility. joint hypermobility syndrome (jhs) is an inherited condition which is nearly indistinguishable from eds iii. reports of coinheritance of vwd and eds or jhs are infrequent. the objective of this retrospective study was to review patients with coexisting vwd and eds or jhs at the indiana hemophilia and thrombosis center in order to describe the type and severity of bleeding symptoms, physical examination findings, and pertinent laboratory data. design/method: the electronic medical record database of the indiana hemophilia and thrombosis center was queried for patients with a diagnosis of vwd and one of the following descriptors: hypermobility syndrome, hypermobility, hypermobile joints, or ehlers-danlos syndrome. the records of identified patients were reviewed for demographics, type and severity of bleeding symptoms, beighton scores (bs), vwd antigen, ristocetin cofactor, factor viii levels, vwd multimer pattern, vwd subtype, genetic testing for eds, and family history of eds. results: a total of patients with dual diagnoses of vwd and eds and patients with vwd and hypermobility were identified with this query. two patients had completed genetic testing for eds, and one had a col a gene mutation identified. significant bleeding symptoms in the vwd and eds group included hematuria and postoperative hemorrhage. two of these patients had delayed wound healing postoperatively. seven of the patients identified to have type i vwd and jhs had moderately severe and somewhat unusual bleeding episodes reported including hematuria, hematemesis, and hemoptysis; of these patients had significant perioperative bleeding. females composed % of the vwd and eds group and % of the vwd and jhs group. conclusion: coinheritance of vwd and eds is an uncommon phenomenon. patients with vwd and eds or jhs may have atypical and moderately severe bleeding, especially with procedural intervention. incorporation of bs into the assessment of patients with bleeding disorders is useful to identify potential inherited collagen disorders, as diagnosis of these conditions may impact clinical management. in the year-long phase ii study (ro fd ), / khe patients responded. patients were followed for years after study completion, collecting data on growth and development, complications of therapy, unexpected toxicities, and need for continuing sirolimus. objectives: after study therapy treatment of one year, objectives include: . assess long term toxicity over the - year period after study therapy completion . assess unexpected toxicity . assess overall condition of the patient . assess need for restart or continuation of sirolimus therapy design/method: prospective follow-up of patients with a diagnosis of khe from institutions. inclusion criteria: follow-up for - years post-study. results: follow-up included data at year (n = ) and - . year (n = ) time points. average age at the start of treatment was months. of patients were available for follow up. four patients are no longer on sirolimus: one patient completed study therapy and remains off treatment (ot) ( years), required years of treatment and is now . years ot and required an additional treatment course prior to successful discontinuation now and months ot. of the patients still on sirolimus, all restarted medication for symptoms of pain, swelling and/or edema interfering with quality of life and have made an average of . attempts to discontinue sirolimus. no patient had reoccurrence of kmp. all patients had improvement in clinical and radiologic appearance of khe but all have residual lesions noted on imaging and/or clinical exam. no unexpected toxicity, growth delay, developmental issues or other long term toxicity of sirolimus was noted. conclusion: this is the first prospective data on long-term follow up of khe patients treated with sirolimus. although numbers are small, sirolimus is well tolerated; however, over half the patients were still on medication at - year follow up. this stresses the need for continued long term follow up in these young patients and investigation of the mechanism of sirolimus effect. nationwide children's hospital, columbus, ohio, united states background: recent studies have identified that adult persons with hemophilia (pwh) have a higher prevalence of hypertension and renal disease than the general population. while hematuria is a known complication of hemophilia a and b (ha, hb), its long-term impact on pwh is not currently known. by annually screening our patients with urinalysis, our pediatric center identified that just under half of our patients demonstrated hematuria over a four-year period. motivated by a desire to identify early markers of hypertension and renal disease, we sought to determine if this finding is reflected in the pediatric hemophilia population as a whole. objectives: establish the population-wide prevalence of hematuria in pediatric pwh. design/method: we used the pediatric health information system (phis) database, which contains clinical and resource utilization data for inpatients from hospitals nationwide, to analyze the prevalence of hematuria, hypertension, renal disease and related diagnosis codes in pediatric pwh who were admitted from january to september . results: during the five-year period, , unique pediatric pwh accounted for , admissions. while the majority of admissions were for bleeding or infectious concerns, ( . %) patients had an affiliated admission code for hematuria. for admissions as a whole, the median age was years with % of those admitted being infants, % toddlers, % children, % adolescents, % older than . we identified % of admissions were for ha with the remaining % were for hb. there were ( %) admits in which a bypassing agent was administered. the median length of stay for persons with hematuria was days compared to days for nonhematuria/other bleeding. there were ( . %) admissions with hypertension reported; though, only patients received an antihypertensive medication during that admission. additionally, only ( . %) admissions reported a diagnosis code of renal disease. our study demonstrated that pediatric pwh are experiencing hematuria. in general, only patients with persistent hematuria require hospital admission so we suspect this data underrepresents the numbers of pwh experiencing hematuria that is managed in the outpatient setting. we also suspect that hypertension is grossly underreported and undertreated in pediatric pwh. additionally, there are a low number of patients experiencing renal disease requiring hospital admission among this cohort. given that there is little research into the long-term impact of hematuria in hemophilia, we feel these findings support the need for further vigilance of our pediatric pwh. background: gla and gsd can aggressively destroy bone, with significant impact on morbidity and mortality. the mtor inhibitor, sirolimus has been shown to be effective in the treatment of these diseases. based on the addition of mtor inhibition to bisphosphonate therapy in metastatic cancer therapy, regimens have been used for refractory or high risk gla and gsd but there is heterogeneity of diagnosis, and variability of drug regimens and assessment of effectiveness. objectives: . assess the variability of clinical features of gla and gsd . assess the heterogeneity of diagnosis . assess drug regimens and response assessment across multiple institutions design/method: we conducted a retrospective review from institutions of cases of gla and gsd treated with sirolimus and a bisphosphonate for at least months with assessment of clinical features, treatment protocols, response regimens and side effects. results: patients included gla (n = ) and gsd (n = ). the average age at diagnosis was years. clinical features included effusions: gla (n = ), soft tissue lymphatic malformations: gla (n = ), gsd (n = ), multiple splenic lesions: gla (n = ), and soft tissue swelling at the site of bony lesion: gsd (n = ). the presenting symptom in patients was pain with patients (gla) presenting with shortness of breath. fracture was noted in patients: gla ( ), gsd ( ). diagnostic and/or response imaging included mri, ct, bone scan, skeletal survey and dexa scan. treatment consisted of: initial sirolimus use with the addition of bisphosphonate secondary to worsening disease (n = ), initial therapy with other agents (interferon, chemotherapeutic agents, radiation) and change to sirolimus and bisphosphonate secondary to toxicity (n = ), sirolimus and bisphosphonates (n = ) and sirolimus, bisphosphonates and interferon (n = ). seventeen patients had stable disease and patients had improvement of disease. sirolimus protocol was standard; however, bisphosphonate protocol varied in dosing and frequency. side effects were tolerable and expected with no grade iii or iv toxicity. sirolimus and bisphosphonates are a safe and effective therapy for gsd and gla. a consistent medication regimen, redefined response and an improved radiologic classification will be important for the development of a prospective clinical trial. background: hemophilia a is a bleeding disorder from the deficiency of clotting factor viii. the most significant sequelae of hemophilia a is the tendency to develop hemarthrosis that incites joint destruction. the prevalence of overweight and obesity has been increasing in the general and hemophilia population and leads to several morbidities including arthropathy. this is a particular concern for hemophilia a as arthropathy is a consequence of joint bleeding. objectives: the purpose of this study was to detect the relation between body mass index (bmi) and joint health endpoints in a pediatric hemophilia population. design/method: participants in this study included patients from the hemostasis and thrombosis center at children's hospital los angeles. participants were pre-screened and approached for this study during routine follow-up appointments. patients aged - years old who have been diagnosed with hemophilia a, including mild, moderate, and severe, qualified for the study. informed consent was obtained from the patients or parents before enrollment. joint health was objectively measured by physical therapists from children's hospital los angeles using the hemophilia joint health score (hjhs). an hjhs total score is calculated by assessing: swelling, duration of swelling, muscle atrophy, crepitus on motion, flexion loss, extension loss, joint pain, and muscle strength in major joints. subjective data was also obtained by patients recording their annual bleed rate within the past year. of the patients, ( %) were normal weight, ( %) overweight, and ( %) obese. we used chi-square analysis to compare joint scores across bmi classifications (chi square = . , df = , p-value = . ). although, this did not approach statistical significance, the average hjhs score in patients who had a hjhs > shows an increasing trend among bmi classifications: . in normal bmi patients, . in overweight bmi patients, and . in obese bmi patients. the average number of annual bleeds in those with positive values show: in normal bmi patients, in overweight bmi patients, and in obese bmi patients. although a positive effect of adiposity was found in the joints of hemophilia a pediatric patients, the effect shows there was not enough evidence to conclude a difference. future studies are needed to address whether obesity has an effect on hemophilia and to determine whether overweight/obesity can lead to further complications in hemophilic joints. background: stagnant blood flow in slow-flow vascular malformations (vm), particularly in their venous components, can lead to localized intravascular coagulation (lic) that is characterized by elevated d-dimer levels, low fibrinogen and decreased platelet count this coagulation derangement can lead to localized thrombosis or bleeding which can result in pain, functional limitations, and possible progression to disseminated intravascular coagulopathy (dic). the treatment of vm and their associated coagulopathy has proven difficult. patients with complex vm are frequently managed with sirolimus, an mtor inhibitor, and have clinical benefits, including reduction of pain and improvement in functional impairment. it is possible that some of these improvements from sirolimus could be secondary to improvement in the coexisting lic. objectives: this study assessed the use of sirolimus to manage the coagulopathy seen in slow-flow vm. design/method: we reviewed charts of patients with vm who are followed in the vascular anomalies center at arkansas children's hospital and were started on sirolimus. efficacy was objectively assessed through improvement of ddimer, fibrinogen and platelet count. three sets of lab values (pre-sirolimus, - months post-sirolimus, and most recent) were obtained for each patient when available. we identified a total of patients who had been prescribed sirolimus. eighteen were excluded based on underlying condition other than slow-flow vascular malformation and for inadequate medical records. a total of patients ( combined vascular, venous) were included in the study. all had elevated d-dimer levels (mean . mcg/ml feu, median . mcg/ml feu, range ( . - . )) prior to treatment. two patients had an associated low fibrinogen (below mg/dl), indicating severe lic. with treatment, ( . %) patients showed an overall decrease in d-dimer levels with an average decrease of . mcg/ml feu between pre-and post-sirolimus labs, and an average decrease of . mcg/ml feu between pre-sirolimus and most recent values. the two patients with low fibrinogen prior to treatment showed a decrease in d-dimer levels (mean decrease of . mcg/ml feu) and an increase and normalization in fibrinogen (mean increase . mg/dl) after beginning sirolimus. no patient had thrombocytopenia. we report that treatment with sirolimus was effective in improving coagulopathy associated with slowflow vm as evidenced by decreased d-dimer levels and increased fibrinogen and/or platelets. long-term use of this medication in this population may decrease the bleeding and thrombotic complications that these patients experience, especially following invasive vascular procedures. background: safety and efficacy of bay - , a sitespecifically pegylated b-domain-deleted recombinant factor viii, in previously treated adolescents and adults aged - years with severe hemophilia a was demonstrated in the phase / protect viii study and ongoing extension. objectives: this subanalysis examines the efficacy and safety of bay - in adolescents in protect viii and the ongoing extension study (data cutoff, january ). design/method: in protect viii, patients (including adolescents) received bay - on demand or as prophylaxis for weeks. prophylaxis regimens for weeks - were twice-weekly ( - iu/kg), every- -days ( - iu/kg), or once-weekly ( iu/kg) infusions based on bleeding during a -week run-in period of iu/kg twice-weekly prophylaxis. patients continued their prophylaxis regimens in the extension or changed regimens at any time. results: twelve patients aged - years were included in the protect viii intent-to-treat population; s of s additional patient discontinued after dose (included in safety population). for patients receiving prophylaxis before study enrollment, median (range) number of total and joint bleeds in the months before study entry was . ( - ) and . ( - ), respectively. ten patients ( . %) had target joints at baseline (median [range], [ - ] per patient). during weeks - of protect viii for the entire time patients remained on their designated prophylaxis dosing frequency, the median (quartile [q] ; q ) annualized bleeding rate (abr) for patients receiving twice-weekly (n = ), every- -days (n = ), and once-weekly prophylaxis (n = ) was ( ; . ), . ( ; . ), and . ( ; . ), respectively (overall prophylaxis [n = ], . [ . ; . ]). two patients switched from once-weekly to twice-weekly (n = ) or every- -days prophylaxis (n = ), and number of bleeds decreased from to in one patient and to in the other. all patients from the main study continued in the extension; mean abr in the extension was . and varied by dosing regimen (twice weekly [n = ], . ; every days [n = ], . ; once weekly [n = ], . ). two patients changed from every- -days to once-weekly prophylaxis during extension (mean abr, . ). one patient had a nonneutralizing antibody to bay - at baseline; end-of-study titers were negative. no patient developed anti-peg antibodies or factor viii inhibitors or experienced a serious adverse event related to bay - during the main study or extension. in previously treated adolescents with severe hemophilia a, bay - prophylaxis was effective in prevention of bleeds, with less bleeding overall versus prestudy, and was generally well tolerated. funded by bayer. cincinnati children's hospital medical center, cincinnati, ohio, united states background: vascular malformations (vms) consist of a heterogeneous group of congenital disorders characterized by the abnormal development of blood and/or lymphatic vessels, which cause a broad spectrum of clinical manifestations. although considered benign, vms are frequently associated with cutaneous complications that can cause significant morbidity such as nodular overgrowth, skin thickening, pruritus, oozing or bleeding of lymphatic blebs and secondary infection. oral sirolimus has shown to be effective in the treatment of complicated vascular malformations but has known side effects and need for frequent laboratory monitoring. currently, there are limited studies on the use of topical sirolimus for the treatment of cutaneous manifestations of vascular malformations. objectives: to evaluate the efficacy and safety of topical sirolimus in vms with cutaneous complications and propose indications for use. design/method: this is a retrospective review of medical records of patients with vascular malformations treated with topical sirolimus from january to december . response was determined by subjective and objective improvement. results: twenty-four patients, ( %) females and ( %) males, with vascular malformations and cutaneous manifestations were treated with topical sirolimus. age ranged from - years. indications for treatment were: blebs ( %, n = ) causing either leaking, bleeding, pain, pruritus, swelling or recurrent infection; nodular overgrowth % (n = ); pyogenic granuloma % (n = ); bleeding % (n = ) and cosmetic % (n = ). treatment course ranged from - months. no major side effects were reported. one patient reported burning and itching sensation. regarding clinical response: % (n = ) patients had improvement in cutaneous lesions; % (n = ) had a stable lesions; and % (n = ) stopped treatment due to side effects. for prior/concomitant treatment: % (n = ) had prior surgery, laser or sclerotherapy; % (n = ) had concomitant oral sirolimus. of the patients not receiving concomitant systemic sirolimus, only % (n = / ) had been on oral sirolimus. of these patients, % (n = / ) had a very good response to topical treatment. : topical sirolimus appears to be beneficial and well-tolerated with a minimal side effect profile for the treatment of cutaneous manifestations of vascular malformations as a single agent or as adjuvant therapy with systemic sirolimus when symptoms are not adequately controlled. further studies are needed to prospectively analyze efficacy and safety of topical sirolimus in this patient population. objectives: to evaluate the safety and efficacy of long-term romiplostim in children with itp. design/method: all patients received weekly sc romiplostim from - g/kg to target platelet counts of - × ( )/l. median (min-max) treatment for the patients was ( - ) weeks for a total of patient-years, or . years per patient. at baseline, median (min-max) age was ( - ) years; % were female; . % had prior splenectomy. median (min-max) average weekly dose was . ( . - . ) g/kg, including escalation to a stable dose; patients started on g/kg. reasons for discontinuing romiplostim (n = , %) included consent withdrawn (n = ), required other therapy (n = ), and ae (n = ) (asthenia, headache, dehydration, and vomiting in one patient and anxiety in the other; none treatment related). fifty four serious aes occurred in patients but were treatment related in one (concurrent grade thrombocytopenia, grade epistaxis, and grade anemia). anti-romiplostim neutralizing antibodies were detected in one patient who discontinued to receive other therapy; antibodies were absent on retesting. from week on, median platelet counts remained > × ( )/l; median platelet counts were > × ( )/l from weeks - . nearly all ( %, / ) patients had ≥ platelet response (platelet counts ≥ × ( )/l, excluding ≤ weeks after rescue medication). most ( %, / ) patients had a platelet response ≥ % of the time and % ( / ) did ≥ % of the time. sixty ( %) patients (or caregivers) self-administered romiplostim. fifteen ( %) patients had treatment-free periods of platelet counts ≥ × ( )/l for ≥ weeks (ie, remission); these patients ( girls, boys) had had itp for a median (min-max) of . ( . - ) years, none had prior splenectomy, and had received romiplostim for . ( . - ) years. all had platelet counts > × ( )/l for ≥ months and / for ≥ months; the median (min-max) duration of being ≥ × ( )/l was ( - ) weeks. of baseline characteristics such as sex, platelet counts, itp duration, and number of past itp treatments ( , , , > ), only age < years was predictive of developing treatment-free periods ≥ weeks (p = . ). in this seven-year open-label extension, > % of children with itp achieved a platelet response and romiplostim was well tolerated. importantly, % of patients were able to discontinue all itp medications for ≥ months. funded by amgen inc. background: sirolimus is an immunosuppressive drug that is widely used in solid organ and bone marrow transplantation, and more recently for the treatment of vascular and lymphatic anomalies. sirolimus has been associated with decreased immunity in the transplant setting in patients that have received other immunosuppressive drugs or were immunosuppressed from previous chemotherapy. the effects of sirolimus on the immune system in chemotherapy naïve children who have not received other immunosuppressive agents are not well understood, and there is variability in the approach to fever and pcp prophylaxis. to understand the effects of sirolimus on the immune system of patients with non-complicated vascular or lymphatic anomalies by evaluating anc, alc prior to and after sirolimus therapy. design/method: multi-institutional retrospective review was done to include patients with non-complicated vascular or lymphatic anomalies. those with effusions/ascites, multiorgan involvement, or history of vascular-anomaly-related infections prior to treatment were excluded. results: twenty patients with kaposiform hemangioendothelioma (n = ), generalized lymphatic anomaly (n = ), cloves syndrome ( ), and simple vascular malformation (n = ) were included. age at initiation of sirolimus treatment ranged from . - years. male to female ratio was : . sirolimus was initiated due to extensive disease, lack of response to steroids or bisphosphonates, pain, dment, lymphatic drainage, and prevention of ongoing overgrowth. prior to the start of sirolimus (sir- ) the mean anc was and alc was . the target level of sirolimus varied by indication and patient, and ranged from to . after the st steady state level, month after sirolimus (sir- ) the mean anc decreased to and alc was . at months after sirolimus (sir- ) the mean anc was and alc was . the first sirolimus levels (sir- ) mean was . ; and sir- level was . . nine patients were placed on pcp prophylaxis at the start of sirolimus. none of these patients had an infectious complication while on sirolimus at a median f/u of months. one patient had mild neutropenia (anc > ) which normalized after discontinuation of pjp prophylaxis. conclusion: in this small cohort of patients we found that the anc and alc level in patients with non-complicated vascular or lymphatic anomalies at sir- was not different from the sir- or sir- . prospective studies that specifically track anc, alc, igg, and lymphocyte function should be conducted to better understand the effects of sirolimus in the immune system. this data will allow for uniform recommendations regarding prophylaxis and management of febrile episodes. background: acute infections and the associated systemic inflammation can increase the risk of venous thromboembolism (vte) and in certain well-defined clinical scenarios may be the primary trigger of vte in pediatric patients. pediatric data on vte in the setting of acute infection are sparse. objectives: to describe characteristics and outcomes of vte in pediatric patients with acute infections. we conducted a retrospective chart review of all pediatric patients with objectively confirmed vte treated at our institution since and identified all patients in whom an acute infection was identified as a vte trigger. patients were managed according to standardized institutional protocols based on published guidelines. relevant demographic, clinical and laboratory data were collected and summarized using descriptive statistics. since , acute infection was identified as a trigger in of vtes ( %) diagnosed at our center. the median age at time of vte diagnosis in this group was . years (interquartile range . - ). males were more commonly affected than females, representing % of cases. neonatal vte events accounted for % of cases. sepsis was the most common acute infection to be identified as a vte trigger [ / cases ( %)]. most vte events ( %) associated with acute infections were considered hospital-associated vtes. at time of vte diagnosis, % of patients were critically ill. extensive vte (defined as completely occlusive thrombosis involving > venous segment) occurred in % of patients. acute infection was deemed to be the primary trigger for vte in / patients ( %). infection-associated vtes in this cohort included cerebral sinus venous thrombosis due to sinus or cns infection ( patients, %), septic throm-bophlebitis ( patients, %), lemierre's or lemierre's-like syndrome ( patients, %) and osteomyelitis-associated deep vein thrombosis ( patients, %). systemic anticoagulation was prescribed in / patients ( %). anticoagulationrelated major bleeding occurred in / patients ( %). vte complications included vte recurrence ( patients, %), vte progression ( patient), acute pulmonary embolism ( patients) and arterial ischemic stroke ( patients). our study indicates that acute infection is a common risk factor for pediatric vte, especially in critically ill children, and can be the primary trigger in a significant proportion of vte cases associated with acute infections. anticoagulation appeared to be overall safe in this population and was associated with low rates of serious vte-related acute complications. however, our study also suggests that this population may be at increased risk for vte recurrence and anticoagulation-related major bleeding. background: epithelioid hemangiomas (eh) are rare benign vascular tumors that occur in soft tissues and bone and present between the third and sixth decades of life. a subset ( %) of eh harbor fos rearrangement. eh has been described in children, but little is known about the long-term outcomes of pediatric eh. the main objective is to obtain data to be used for improved understanding of this rare disease in order to provide standardization of care and development of future research studies. board-approved retrospective review of clinical, pathologic, and radiographic characteristics, and treatment outcomes in patients diagnosed with eh between and . results: eight patients were male; mean age at diagnosis was . years (range: - ). lesions involved the lower extremities (n = ), cranium (n = ), pelvis (n = ), and spine (n = ). multifocal disease was identified in five patients. the most common presentations involved significant localized pain and neurologic symptoms: headache, cranial nerve injury, loss of consciousness. radiographic studies identified variable features, such as multifocal lytic bony lesions with sclerotic margins, enhancing soft tissue component, and surrounding inflammatory edema. histologically, all specimens were composed of vascular channels lined by epithelioid endothelial cells without significant cytologic atypia; solid cellular areas (n = ). endothelial cells were positive for cd and egr, and negative for camta . fos rearrangement was assessed in only one specimen and detected. mean follow-up time was days (range: - ). patients were treated with surgical resection, intravascular embolization, bisphosphonates, propranolol, interferon, and sirolimus. one patient treated with interferon and one with sirolimus exhibited partial response for mean follow-up of . days. although eh is a benign neoplasm, it is difficult to manage without standard protocols and portends considerable morbidity. our findings suggest medical management, particularly sirolimus, may benefit these patients; however, long-term follow-up is needed in treated children. novel fos inhibitors are in development and may benefit patients with fos rearrangement. penn state health children's hospital, hershey, pennsylvania, united states background: central venous catheters (cvc) are often required in critical care settings in order to provide a secure point of access for life sustaining care. clinical studies identify cvc presence as the single most important risk factor for deep vein thrombosis (dvt) in children. venous thromboembolic event (vte) incidence rates in critically ill children with a cvc range from . - % and . - . per catheter days depending on the population studied. per institutional protocol, the penn state health children's hospital picu (hershey, pa) utilizes a low dose continuous infusion of unfractionated heparin (ldufh) at units/kg/hr as prophylaxis against cvc-related vte and to maintain line patency. the efficacy of this approach has never been evaluated. to determine if ldufh for prophylaxis results in lower incidence of cvc-related vte, catheter dysfunction and central line associated blood stream infection (clabsi) without increasing morbidities. to determine if the incidence of catheter related vte is lower than historical published data, a retrospective chart review was conducted utilizing the institutional electronic medical record for all patients in , aged - . years, who had a cvc during a picu admission. secondary objectives such as the incidence of catheter dysfunction, clabsi, and any associated bleeding complications are also being analyzed. results: interim data analysis revealed cvcs ( nontunneled cvc, totally implantable devices, tunneled lines, peripherally inserted central catheters [picc] ) in total patients with a median age of . years. overall vte incidence was . % ( / ) with vtes associated with non-tunneled cvc and with piccs. sixty one percent of non-tunneled cvcs received ldufh and % ( / ) of the patients with vtes associated with non-tunneled cvcs did receive ldufh prophylaxis. vte incidence rate of nontunneled cvcs with ldufh was . % ( / ) and . per picu catheter days. the only other vte events identified within our study cohort were in the picc group where two patients experienced vte, one of which was receiving ldufh. clabsi incidence was . % ( non-tunneled cvc, tunnel cvc, picc). no major bleeding complications were associated with ldufh. preliminary data demonstrates ldufh is efficacious in preventing cvc-related vte in comparison to published reports. further analysis will compare another similar sized and acuity level picu which does not practice the same method. background: fibroadipose vascular anomaly (fava) is a rare, challenging disorder associated with pik ca mutations. fava often causes painful replacement of muscle and soft tissues with fibrotic and adipose tissue and is associated with ectatic draining veins. treatments for focal lesions are surgical excision, cryoablation or sclerotherapy and the role of medical therapy is unclear. some fava lesions are too extensive or directly involve neurovascular structure, resulting in refractory pain. objectives: to retrospectively evaluate the efficacy of sirolimus in patient with residual symptoms after procedural therapies for fava design/method: retrospective review of individual cases from institutions of fava refractory to other therapies treated with sirolimus for at least months. cases were s of s identified by polling member of the aspho vascular anomalies special interest group. results: all seven patients report improvement on sirolimus therapy. all patients had received prior procedures, including sclerotherapy ( patients), cryoablation ( patients) and/or resection ( patients). mean age at sirolimus initiation was y (range - y). mean length of therapy is . months (range - months). six patients were treated with bid dosing and one adult received daily dosing. goals of sirolimus were improvement in pain or musculoskeletal dysfunction. pain and function improved in all patients, including discontinuation of narcotic use and resumption of participation in sports. time to symptom improvement ranged from - weeks. in four patients for whom dose was lowered, pain recurred in all four and responded to restarting or increasing sirolimus dose. while all patients do not have pre-and postsirolimus imaging, decrease in fava lesion size is seen in cases with available imaging. sirolimus side effects are similar to prior reports, most commonly mouth sores, elevated lipids and acne. we report the first known data supporting a role of sirolimus in refractory fava cases. sirolimus is welltolerated and initial improvement is rapid, within weeks of initiation. whether sirolimus has a role in upfront therapy to reduce lesion size prior to procedures deserves further study. objectives: to assess platelet responses in children with itp receiving romiplostim. design/method: eligible children had itp for ≥ months, ≥ prior therapy, and screening platelet counts ≤ × ( )/l or uncontrolled bleeding. weekly dosing was from - g/kg to target platelet counts of - × ( )/l. bone marrow biopsies were evaluated in europe at baseline and after or years (cohorts and ). as of mar , patients received ≥ dose. at baseline, median (min-max) age was ( - ) years, itp duration was . ( . - . ) years, and platelet count was ( - ) × ( )/l; patients ( %) had had prior splenectomy. the median (q , q ) % time with a platelet response (platelet count ≥ × ( )/l, no rescue medications past weeks) in months - was % ( %, %) (primary endpoint). over the course of the study, % ( / ) of patients had a platelet response. four patients maintained platelet counts ≥ × ( )/l with no itp medications for ≥ weeks. median (min-max) treatment duration was ( - ) weeks for patient-years in total. median (min-max) average weekly romiplostim dose over the course of the study was . ( . - . ) g/kg; the median dose was g/kg at year (n = ) and g/kg at years (n = ). most ( %) patients initiated self-administration. sixty-four patients ( %) discontinued treatment, most frequently for lack of efficacy (n = ), patient request (n = ), and adverse event (ae) (n = ). fortyone ( %) patients had serious aes (saes) including epistaxis ( %) and decreased platelet count ( %). five patients had treatment-related saes: headaches, abdominal pain, and each of presyncope and neutralizing antibodies (ab). there were cases of neutralizing ab to romiplostim (of patients tested), but none to tpo; / had continued elevated platelet counts and in / cases ab were not found on retesting. for cohort , of patients with baseline bone marrow biopsies, had evaluable on-study biopsies scheduled for year; patient had an increase from grade to . there were no findings of collagen or abnormalities. in this interim datacut of a romiplostim openlabel study in children with itp, % of children had a platelet response. overall, the median dose was . g/kg; the median romiplostim dose over time reached g/kg. no new safety signals were observed over patient-years. funded by amgen inc. background: hepatic hemangiomas are benign vascular tumors without a medical home, managed by multiple specialties. the diagnosis has been assigned historically to various vascular lesions affecting the liver with completely different clinical presentations, resulting in difficult standardized management. objectives: the consensus steering committee identified an acute need of clear definitions and evaluation guidelines using the updated international society for the study of vascular anomalies (issva) classification. the goal was to formulate recommendations that will be adopted by all specialties involved in the care of children with hepatic hemangiomas. design/method: we used a rigorous, transparent consensus protocol, with input from multiple pediatric experts in vascular anomalies from hematology-oncology, surgery, pathology, radiology and gastroenterology. in the first section, we precisely define the subtypes of hepatic hemangiomas seen in children (congenital and infantile) using clinical course, histology and radiologic characteristics. inclusion and exclusion limits to the diagnosis are noted. the following two sections describe these subtypes in further detail, including complications to be considered during monitoring and respectively recommended screening evaluations. conclusion: while institutional variations may exist for specific clinical details, a clear understanding of the diagnosis of hepatic hemangiomas affecting the pediatric population and the possible complications that require screening during the monitoring period should be standard. as patients with hepatic hemangiomas are managed by different medical and surgical specialties, a multidisciplinary consensus based on current literature, on the data extracted from the liver hemangioma registry and on expert opinion was required and was accomplished by this manuscript. objectives: to investigate the association between routine prophylaxis with bay - and bleeding outcomes after adjusting for key patient and pharmacokinetic (pk) characteristics. design/method: the leopold kids study evaluated safety and efficacy of bay - prophylaxis in previously treated boys aged ≤ years with severe hemophilia a. patients received bay - - iu/kg x/wk (n = ) or > x/wk (n = ) and were followed up for - months. prophylaxis dose and frequency were assigned by investigators. pk parameters, including area under the curve (auc), half-life, and clearance, were derived from a population pk model and reflect predicted pk values with a -iu/kg dose. patient characteristics were compared between the x/wk and > x/wk groups using wilcoxon rank sum or chi-square tests. negative binomial regression was used to model the association between prophylaxis frequency and annualized bleeding rate (abr) for total bleeds, first without adjustment and then adjusting for age, pk parameters, and bleed history. results: mean ± sd age for patients in this analysis was . ± . years. patients receiving prophylaxis x/wk had more bleeding episodes in the months before study entry (mean ± sd, . ± . [median, . ] for x/wk vs . ± . [ . ] for > x/wk; p = . ) and were more likely to have been treated on demand ( % vs %; p = . ). pk parameters were similar between the x/wk and > x/wk groups. without adjustments, abr during the study was % higher in the x/wk group compared with the > x/wk group (rate ratio [rr], . ; % ci, . - . ; p = . ). abr was % lower in the x/wk group (rr, . ; % ci, . - . ; p = . ) after adjusting for age, auc, and number of bleeds in the prior months. conclusion: abr was numerically lower but not significantly different between the x/wk and > x/wk groups after adjusting for age and pk parameters. these findings suggest that even among patient groups that are homogeneous with respect to age, pk, and bleed history, further individualization of bay - prophylaxis based on other characteristics may help reduce bleeding episodes even at a lower treatment frequency. larger real-world studies are needed to verify these findings. funded by bayer. stanford, palo alto, california, united states s of s background: vascular malformations may be of lymphatic, arterial, venous or capillary endothelial origin. they may be simple or complex, with complex malformations being a combination soft tissue and skeletal overgrowth. although likely present at birth, these malformations often become symptomatic with puberty or infection, and range from little or no clinical impact to life threatening symptoms. in malformations primarily of venous origin, pain may be significant and hypothesized to be caused by phlebolith development (intra-malformation thrombi), inflammation, consumptive coagulopathy, vascular engorgement, and endothelial proliferation. anti-angiogenic and anti-platelet therapies have been reported to relieve pain. however, the use of anticoagulation for pain is not well described. objectives: to report clinical features and outcomes of patients with vascular malformations of venous origin treated with anticoagulation for pain. we performed a retrospective review of patients with vascular malformations followed by the hematology service between january and december who were treated for pain with anticoagulation. pain relief was determined both by wong-baker pain scales and patient report. clinical data were extracted from electronic medical records. we identified five patients with venous malformations (vm) who had received anticoagulation for pain. four patients were female and median age was years old (range to years old) at time of initiation of anticoagulation. all five patients had vm of the extremity, two with vm of the lower extremity, and three patients had vm of the upper extremity. two patients had concomitant coagulopathy and demonstrated decreased d-dimer after initiation of anticoagulation. four patients received enoxaparin, and one adult patient received rivaroxaban. all patients reported improvement in pain after administration of anticoagulation. one patient exhibited mild epistaxis and bruising at the injection site. there was no significant bleeding or other complications. pain is a significant complication in patients with venous malformations. our case series suggests that anticoagulation is a safe and effective therapy for pain relief in this population. further investigation is indicated to compare the effect of anticoagulation to other therapeutic interventions such sclerotherapy, surgery, and sirolimus in the treatment of pain associated with venous malformation. maria ahmad-nabi, christine knoll, sanjay shah, lucia mirea phoenix children's hospital, phoenix, arizona, united states background: estimates of the incidence of dvt in patients with osteomyelitis range widely from %- %, however risk factors and outcomes of dvt in this cohort have not been thoroughly established. objectives: this study aims to estimate the incidence of dvt in patients with osteomyelitis, and to assess risk factors and outcomes of dvt in this cohort. design/method: after irb approval, a retrospective chart review was conducted for patients aged - years seen at phoenix children's hospital between - with icd / codes for osteomyelitis. exclusion criteria included chronic recurrent multifocal osteomyelitis, and chronic dvt. demographics, clinical factors and outcomes were compared between osteomyelitis patients with and without dvt using the fisher-exact and wilcoxon-rank sum tests, as appropriate for the data distribution. results: a total of study subjects with osteomyelitis had a mean (standard deviation) age of . ( . ) years. dvt was present in ( % of ) patients, and ( %), ( %) and ( %) patients received anticoagulation for < , - and ≥ weeks, respectively. patients with vs without dvt were more likely to be male ( % vs %; p-value = . ), and had significantly higher rates of bacteremia ( % vs %; p-value = . ). rates of central lines were comparable between dvt and non-dvt patients ( % vs %; p-value = . ); however patients with dvt vs without dvt had significantly longer mean length of stay ( vs days; p-value < . ) and higher rates of icu admission ( % vs %; p-value < . ). the incidence of dvt among osteomyelitis pediatric patients was estimated at %, with risk increased by male sex and bacteremia. patients with dvt had significantly higher rates of icu admission and longer length of hospital stay. many of these patients had standard practice management of their dvt with - weeks of anticoagulation. our data highlights the need for recognition of high risk patients, and the need for future efforts targeting dvt prophylaxis. baylor college of medicine, houston, texas, united states background: lymphatic malformations (lm) frequently occur in the head and neck and can often be disfiguring and even life-threatening. management options include observation, surgery, sclerotherapy, and sirolimus. the optimal sequence of therapeutic interventions has not been determined due to the lack of comparative clinical trials or established guidelines. thus, prenatal planning with a multidisciplinary team is beneficial. we present a case series of ten children with head and neck lms evaluated in at our multidisciplinary vascular anomalies center. a chart review was performed to assess treatment modalities and recent trends. results: seven of patients ( %) with head and neck lms were diagnosed prenatally. six patients required an ex utero intrapartum treatment procedure. all patients were started on sirolimus at a median age of . months (range days - years). four patients most recently started on sirolimus were less than months of age at the time of initiation. six patients underwent partial excision of lm during the first year of life; none of whom received sirolimus prior to surgery. sirolimus was discontinued in one patient given chronic clostridium difficile infections, and non-compliance in another patient. five patients received sclerotherapy. tracheostomy was necessary in six patients; one patient was de-cannulated after months on sirolimus. all patients have had radiographic and clinical improvement of lm with varying treatment modalities. current clinical observations show improved response with sirolimus and demonstrate tolerability of sirolimus at a young age. conclusion: treatment of pediatric head and neck lms is challenging and a multidisciplinary approach is necessary. as the majority of patients are diagnosed prenatally, prenatal planning and discussion of potential use of sirolimus is beneficial. availability of vascular anomalies experts in the prenatal/neonatal period offers the best management results, and early initiation of sirolimus should be considered for complex lesions. long-term follow up is warranted to investigate the efficacy and timing of treatment options. yale school of medicine, new haven, connecticut, united states background: to mitigate transfusion of pathogencontaminated platelets, amotosalen, a synthetic psoralen compound, is added to sdp components. exposure to uv-a light activates amotosalen and crosslinks dna/rna base pairs, preventing replication of a broad spectrum of viral, bacterial, and other pathogens that may contaminate platelets. pr-sdps were fda approved for clinical use with no age restrictions in . we initiated use of pr-sdps in november of for all patients. we retrospectively analyzed usage of pr-sdp vs conventional (non-pr) platelets (cp) in neonatal and pediatric patients with thrombocytopenia to compare hemostatic efficacy and the incidence of transfusion reactions (tr) for these products, after one year of a dual platelet inventory. design/method: since pr-sdp were fda-licensed, no irb approval was required; pr-sdp and cp were both considered standard of care. we evaluated transfusions for all pediatric patients age - years who received any platelet transfusion between november and november . we determined the volume (mean ml ± sd) of each type of platelet component transfused, the number of platelet transfusion episodes, and reported trs based on cdc hemovigilance guidelines. a subgroup analysis was performed for thrombocytopenic neonates ( - months). results: patients - years who received only cps (n = ) received a total of , ml of platelets ( ± ml/patient) over transfusions ( . ± . episodes/patient). for comparison, in patients who received only pr-sdp, a total of , ml of platelets ( ± ml/patient, p = . ) were infused over transfusions ( . ± . episodes/patient, p = . ). for neonates ( - months, n = ) who received only cps, , ml of cps ( ± ml/ patient) were transfused over episodes ( . ± . episodes/patient). for comparison, those who received only pr-sdp (n = ), received , ml of pr-sdp ( ± ml/patient, p = . ), transfused over episodes ( . ± . episodes/patient, p = . ). for all recipients - years (n = ), including additional patients who received both cp and pr-sdp, there were three reported allergic trs over transfusion episodes, while no allergic reactions were reported with pr-sdp transfusions. one febrile tr was reported to cp transfusion, while three were reported for pr-sdp. in conclusion, pr-sdps, in our pediatric population age - years, were comparable to cp products in regards to volume and episodes of platelet transfusions, and incidence/type of transfusion reactions. pr-sdp were safe and effective for use in this pediatric patient population. background: vascular anomalies are classified as either vascular tumors or vascular malformations. fibro-adipose vascular anomaly (fava) is a newly described entity which presents with distinct clinical, radiographic and histopathologic findings. we present a case in which the diagnosis of fava was complicated by a persistent low platelet count secondary to immune thrombocytopenia (itp). to describe a challenging diagnosis of a novel vascular anomaly (fava) complicated by severe thrombocytopenia. a year old male presented to hospital with bruising and left thigh pain related to a remote sports injury. blood work revealed a platelet count of × /l, but with an otherwise normal complete blood count. the following were also normal: aptt and fibrinogen; d dimer levels were slightly increased. he was treated with one dose of ivig ( . mg/kg) for presumed itp and responded well with his platelet count increasing to × /l. he returned to hospital weeks later with recurrent thrombocytopenia and worsening leg pain. an ultrasound of the left thigh revealed a . cm x . cm x . cm lesion within the vastus medialis. the diagnosis of an intramuscular hematoma secondary to persistent thrombocytopenia was made. the patient presented with multiple episodes of thrombocytopenia over the next several months. his itp did not respond to oral prednisone ( mg/day for days). he continued to have short-lived responses to ivig requiring infusions every other week as his platelet count would fall below × /l. his leg pain progressed, restricting him to a wheelchair. further imaging by mri brought into question the diagnosis of a hematoma and a biopsy of the thigh lesion was performed. the results were consistent with a diagnosis of fava; this was subsequently excised. conclusion: this is a unique case where a vascular anomaly was misdiagnosed as a hematoma due to a patient's persistent thrombocytopenia and history of an injury. fava is a newer entity which, unlike other vascular anomalies, has not been linked to thrombocytopenia or a localized consumptive coagulopathy. after excision of the fava, the patient's chronic pain, and mobility resolved, though his itp persisted. objectives: this preliminary, exploratory analysis of realworld administrative data was conducted to determine units dispensed and factor replacement product-related direct expenditures associated with a currently marketed shl or ehl rfix product. design/method: de-identified claims data from the commercially available truven health marketscan® research u.s. claims database were used to identify direct expenditures and number of international units (ius) dispensed for all patients aged - years with a diagnosis code of icd- . /icd- d who used nonacog alfa or eftrenonacog alfa during the study period (june , to july , ). reference weight measurements from the centers for disease control and prevention national center for health statistics' (cdc nchs) anthropometric data were used to estimate product dispensation on an iu per kg basis. the nonacog alfa and eftrenonacog groups comprised and patients, respectively. the median [iqr] age in the two groups was . [ . ] and . [ . ] years, respectively. while of the patients in the eftrenonacog alfa group had > calendar quarter of available data, only of the patients in the nonacog alfa group had > available quarter. the median rfix product dispensation per quarter was , ius (iqr, , ius) in the nonacog alfa group and , ius (iqr, , ius) in the eftrenonacog alfa group. incorporating attributed weight values, the median rfix product iu dispensation per kg per week was . iu/kg/wk (iqr, . iu/kg/wk- . iu/kg/wk) in the nonacog alfa group, and . iu/kg/wk (iqr, . - . iu/kg/wk) in the eftrenonacog alfa group. applying wac prices (eftrenonacog alfa = $ . /iu; nonacog alfa = $ . /iu), the calculated estimates of $/kg/week were $ and $ in the nonacog alfa and eftranonacog alfa groups, respectively. conclusion: preliminary real-world data derived from a large u.s. claims database revealed differences in product dispensation and factor product-related expenditures among pediatric patients with any severity of hemophilia b to whom an shl or ehl rfix product was prescribed. refinements of these data, potentially to exclude instances of sporadic usage, may shed light on real-world dispensation of rfix products among pediatric hemophilia b patients. background: vascular malformations can be classified as simple (including capillary, venous, lymphatic, arteriovenous), combined, malformations of major named vessels or associated with other anomalies. multiple modalities including laser treatments, sclerotherapy, embolization, surgery and pharmacological intervention (with mtor inhibitors like sirolimus) have been used for treatment of vascular malformations. these interventions have been used alone or in combination with varied outcomes. we present our institution's experience with a multimodal approach to simple and combined vascular malformations. design/method: we performed a retrospective chart review of patients with vascular malformations who were referred to our center for an interventional radiology evaluation from june -july . we included patients (age at presentation: months - years), referred initially for interventional radiology procedures (irp) for vascular malformations. all patients had symptoms of pain and/or swelling/deformity. diagnosis of was based on vascular imaging (doppler ultrasound, mri/a/v). nine patients had venous malformations (vm), five had macrocystic lymphatic malformations (lm), six had lymphatic-venous malformations (lvm), and two arteriovenous malformations (avm). patients initially underwent interventional radiology procedures. all the vm patients responded to sclerotherapy alone. three patients with lm responded to sclerotherapy alone, remainder required surgical intervention. one avm patient responded well to embolization, the other needed surgical resection after embolization. four lvm patients underwent irp with minimal improvement in symptoms ( - procedures attempted), surgical resection was attempted in patients with poor response and patients were started on sirolimus ( . mg/m /dose twice a day). all lvm patients started on sirolimus have responded well (decreased pain and swelling); time to initial symptom response ranged from weeks - month from starting medication. in this case series, patients with simple vm responded well to sclerotherapy alone, avm and lm patients needed irp and/or surgery for complete response. complex lvm did not respond well to surgery or irp; . % had improvement in clinical symptoms with addition of sirolimus to the treatment regimen. response to various modalities of treatment varied based on the type of vascular malformation. a multidisciplinary approach to management of vascular malformations is essential to provide multimodal therapeutic options for rapid symptom relief and improve the quality of life of these fragile patients, especially those with complex malformations. background: von willebrand disease (vwd) is the most common bleeding disorder in humans, affecting ∼ % of the united states' population. desmopressin (ddavp) is a longacting vasopressin analog that induces vasoconstriction and release of vwf. ddavp is used in patients with vwd and as a surgical prophylaxis, but carries anti-diuretic properties. to avoid electrolyte imbalance and hyponatremia, fluid restrictions are recommended in the hours post-ddavp administration. objectives: this study sought to examine perioperative practices and outcomes following ddavp administration and a fluid restriction protocol in a population of pediatric patients with von willebrand disease. design/method: a retrospective chart review was conducted for patients with von willebrand disease who underwent surgical procedures at children's hospital of pittsburgh of upmc between january , and december , . patient age, sex, weight, diagnosis, surgical procedure, total fluids administered, and post-operative sodium level were recorded. the primary outcomes noted were the proportion of patients exceeding % of the recommended fluid consumption for the -and -hour periods post-ddavp s of s administration, as defined by local guidelines. secondary outcomes were the presence of any bleeding requiring an er visit or readmission or hyponatremic seizures within hours of ddavp administration. results: data was compiled for patients ( females, males). the mean age was . years (sd . years), median age was years (range to years). procedures included dental ( ), otolaryngology ( ), orthopedics ( ), gastrointestinal ( ), plastics ( ), neurosurgery ( ), ophthalmology ( ), dermatology ( ), general surgery ( ) and gynecology ( ). % of patients exceeded % of the fluid volume recommended for the first -hour period post-ddavp administration while still in the surgical setting. no patients exceeded % of the fluid volume recommended for the total -hour period post-ddavp administration. post-operative sodium levels were obtained in only of patients. no patients returned to the er or were admitted for bleeding in the hours post-ddavp administration. no patients returned to the er or were admitted for hyponatremia or seizures in the hours post-ddavp administration. maintenance of a fluid restriction protocol effectively deterred negative outcomes in this cohort. however, a significant fluid volume was administered in nearly a third of patients despite the restrictions. given the risk of hyponatremia, and limited compliance with fluid restrictions, postoperative sodium levels should be recorded in following ddavp administration to assess the possibility of a hyponatremia and to reinforce the importance of fluid restrictions and their communication. results: a male fetus required in utero insertion of a pleuroamniotic shunt for bilateral pleural effusions diagnosed antenatally by ultrasound. shortly after delivery at term, he developed respiratory distress and was found to have reaccumulation of the pleural effusions. blood work on day of life showed a platelet count of , / l, which then decreased precipitously. he demonstrated schistocytes on blood-smear, signs of consumptive coagulopathy with hypofibrinogenemia and high d-dimers, and compensatory reticulocytosis. he required multiple transfusions and admissions to the intensive care unit for respiratory support. investigations ruled out congenital ttp, neonatal alloimmune thrombocytopenia, and noonan syndrome. given high clinical suspicion for an underlying vascular lesion causing kmp, a full body mri without contrast was undertaken. this showed a focal area of suspicious signal intensity in the upper paraspinal musculature. an ultrasound and mri with contrast demonstrated an extensive infiltrative vascular lesion involving the paraspinal musculature, prevertebral space, posterior extrapleural space, mediastinum, and neck. the child was commenced on prednisone ( mg/kg/day) and rapamycin ( . mg/m twice/day). there was no clinical or laboratory improvement after one month. a biopsy was performed which confirmed khe. in the second month of rapamycin therapy, the platelet count gradually normalized and the patient was discharged from hospital at . -months of life. prednisone was weaned off at . months of life. a repeat mri at months showed significant reduction in the khe. he is now almost years into therapy and doing well. conclusion: this is a unique case of khe with kmp that initially presented with extensive and recurrent pleural and pericardial effusions. this case demonstrates the importance of suspecting an underlying vascular malformation in the presence of kmp. our patient had a delayed but overall good response to rapamycin. further studies investigating duration of rapamycin therapy is key for the optimal management of these patients. rosa diaz, donald mahoney, lakshmi srivaths, donald yee texas children's hospital, houston, texas, united states background: since von willebrand disease (vwd) is the most common inherited bleeding disorder, it must co-exist with other less common bleeding disorders in some dually affected patients. however, reports of combined deficiencies in factor viii (fviii) and von willebrand factor (vwf) are rare. objectives: to study the prevalence and bleeding phenotype of combined deficiencies of fviii and vwf in males with hemophilia a in a hemophilia treatment center. design/method: we retrospectively reviewed the electronic medical records of males with hemophilia a followed at our institution during the past years. the primary and secondary outcomes for the study were ( ) the prevalence of combined fviii and vwf deficiencies and ( ) the bleeding phenotype of these patients. we identified vwf deficiencies in % (n = ) of the patients with hemophilia a. most (n = , %) patients were tested for vwf deficiency as part of the initial hemostatic evaluation, but one-third were tested due to clinical concern for inadequate response to fviii concentrate. the median duration of follow up was . years (range . to . ). patients were referred to our clinic at a median age of months (range to years) for evaluation of easy bruising (n = , %), mucosal (n = , %) and surgical bleeding (n = , %). primary diagnoses included with severe, moderate and mild discrepant hemophilia a. secondary diagnoses included with low vwf activity, type vwd and with type unclassified. patients experienced episodes of musculoskeletal (n = , %), mucocutaneous (n = , %) and cns bleeding (n = , %). a total of patients received factor prophylaxis. half of the patients were initially treated with fviii concentrates but subsequently changed to combined fviii/vwf products due to the frequency of breakthrough bleeding despite good compliance. all patients are on combined fviii/vwf products at the time of this review. a total of ( %) of this cohort developed chronic joint disease manifest as decreased range of motion and/or abnormal mri findings. combined deficiencies of fviii and vwf were present in % of our center's hemophilia patients. these patients exhibited a severe bleeding phenotype as evidenced by the high frequency of hemarthrosis, need for prophylaxis and high prevalence of chronic joint disease. while the optimal treatment strategy remains to be elucidated, early recognition of a combined deficiency may have important clinical implications, particularly in patients who demonstrate a suboptimal response to fviii concentrate alone. background: childhood neutropenia is heterogeneous and may be congenital or acquired. cerebral cavernous malformation (ccm ) is a neurovascular malformation disorder where lesions consist of low flow, dilated capillary endothelial channels with increased permeability, predisposing to hemorrhage and thrombosis. programmed cell death protein (pdcd ) activity has been implicated in glia and neuron migration, and recently linked to the dysregulation of the actin and microtubule cytoskeleton, thereby affecting cellular morphology and migration. variants of pdcd encoding pdcd have been associated with ccm . ccm causes a greater and earlier disease burden than other ccms, with % presenting younger than years. some patients have associated extra-neuronal manifestations, suggesting that pdcd plays a role in other tissues. we describe a patient with significant blood cytopenias associated with ccm . design/method: retrospective chart review to obtain patient data. results: an -month old female presented with seizure and was found to have multiple intracranial cystic lesions and abscesses due to s. pneumonia serotype f. during her treatment, she developed anemia (hemoglobin . - . g/dl), thrombocytopenia (platelets , - , cells/l), and profound neutropenia (absolute neutrophil counts of zero). initial bone marrow evaluation revealed a normocellular marrow but with marked granulocytic hypoplasia and % hematogones on flow cytometry. florescent in situ hybridization excluded cytogenetic changes characteristic of myelodysplastic syndrome. further evaluation included testing for neutrophil antibodies, chromosome breakage, and telomere length and results were normal. whole exome sequencing excluded mutations affecting congenital neutropenia genes, but detected a de novo pdcd variant (c. + g>a), thereby diagnosing ccm . the neutropenia has responded well to granulocyte colony stimulation factor (gcsf), which is still needed at months of age. moreover, the thrombocytopenia has progressed, requiring periodic platelet transfusions. over time, the bone marrow hematogone population has decreased to % at months of age, though the granulocytic hypoplasia persists. conclusion: our case describes the first patient with neutropenia and thrombocytopenia associated with ccm . we hypothesize the pdcd variant is the etiology of bone marrow dysfunction due to its role in actin and microtubule cytoskeleton formation, akin to the pathophysiology of xlinked neutropenia. supportive features of an underlying genetic cause of marrow dysfunction include the persistence of cytopenias beyond infection resolution as well as presence of hematogones. hematogones were previously reported to occur in patients with other congenital neutropenia disorders, indicating they could be a feature of congenital neutropenia and may be reactive to surrounding cell apoptosis. further testing of pdcd role in hematopoiesis should be explored. background: - % of adult women will suffer from heavy menstrual bleeding (hmb) during their lifetime. % of women with inherited bleeding disorders suffer from hmb. there is a paucity of data about hmb among adolescents and young adults (aya), a population in which hmb may have large social and educational effects. objectives: to study the social and academic implications of hmb in an aya population. design/method: this is a questionnaire based survey conducted in a medium-sized city in california. we recruited females - years of age from one high school and from local university. the questionnaire was set up in research electronic data capture (redcap) at our institute which allowed us to obtain objective data about the respondents' menstrual cycles. a link was sent to the high school students via their online portal schoolloop and to the university students via social media and word of mouth. data was collected over weeks from may to august . we received replies, some were not complete. using regression analysis, data was analyzed from respondents in the age group of - (with a mean age of ) years. we developed a composite score for hmb based on factors including saturation levels, number of pads, duration of bleeding, soaking of a pad within two hours, passage of clots, size and number of clots, and gushing sensation. we conducted statistical analysis of the drivers and implications of hmb based on the composite score. results indicate that having a relative with hmb, having other bleeding problems, and having anemia are drivers of higher hmb score. the results also indicate that hmb adversely affects quality of life as measured by participation in sports, social activities, after-school activities, tiredness, absenteeism, and gpa. hmb is also associated with increased rates of anemia and use of anti-depressants. hmb-driven anemia further adversely affects gpa. under-represented minorities are more likely to have a higher hmb score, as well as an increased adverse effect of hmb on gpa. the results suggest that the social costs of hmb are pervasive in the aya population, and especially pronounced among minorities. a relative with hmb is a significant driver of heavy menstrual bleeding. a hemostatic screen should be included when assessing the aya population with hmb. johns hopkins all children 's hospital, st. petersburg, florida, united states background: propranolol is a non-cardioselective beta blocker medication frequently prescribed for hemangiomas and hyperthyroidism. propranolol inhibits types i and ii iodothyronine deiodinases, enzymes that convert bioinactive thyroxine (t ) into bioactive triiodothyronine (t ). hypothyroidism is a well-recognized complication of diffuse hepatic hemangiomas that produce type iii deiodinase, an enzyme that converts t into bioinactive reverse t and t into diiodothyronine. thyroxine is typically selected for replacement in this population, even though doses up to % above physiologic may be necessary. we hypothesized that low dose, nearly physiologic t would be safer and equally effective because it bypasses propranolol's impact on the pituitarythyroid axis. we report an infant with diffuse hepatic hemangiomatosis and acquired hypothyroidism successfully treated with propranolol, prednisone, and triiodothyronine. design/method: a mo healthy female presented with abdominal distension, poor oral intake, and hepatomegaly. mri confirmed diffuse hepatic hemangiomatosis, the largest lesion measuring . cm by . cm. thyrotropin (tsh) was elevated at . (reference range* . - mcgiu/ml), total t # (rr - ng/dl), and total t ^ . (rr - mcg/dl). treatment was started with prednisone ( mg/kg/day) for three weeks, propranolol ( mg/kg/day) and t ( . mcg/kg/day). the t dose was slowly titrated to a maximum of . mcg/kg/day. thyroid hormone levels rapidly improved on t replacement. after two weeks, the tsh was . , tt , and tt . . after eight months, the tsh was . , tt , and tt . . at twelve months, the tsh dropped to . , tt , and tt . , suggesting decreased tumor production of type iii iodothyronine deiodinase. liver mri confirmed fewer hemangiomas, largest being . cm by . cm. the patient's t dose was reduced. both propranolol and t were discontinued after twenty-four months of treatment. one year off all therapy, this child has normal growth and development, only two < . cm hepatic hemangiomas and no evidence of hypothyroidism (tsh . ; tt ; tt . ). conclusion: t at near physiologic doses corrects the consumptive hypothyroidism associated with diffuse hepatic hemangiomas. t replacement is preferable to thyroxine due to its lower risk of rebound hyperthyroidism as the hemangiomas involute and type iii deiodinase production declines. there are two prior case reports describing t use without t , one employing propranolol and the other utilizing steroids for hemangioma management. this is the first case report with long term follow-up of a child treated with multimodal therapy including propranolol, prednisone, and triiodothyronine. *rr = reference range; #tt = total t ;^tt = total t background: multifocal lymphangioendotheliomatosis with thrombocytopenia (mlt) is a rare congenital disorder first described in that is characterized by multiple vascular abnormalities commonly involving the skin and gastrointestinal tract as well as consumptive coagulopathy often resulting in gi bleeding in infancy( ). to describe an unusual presentation and successful management of mlt in a neonate. design/method: baby h was born at full term after a pregnancy complicated by maternal sinus venous thrombosis requiring anticoagulation beginning at weeks. at birth, she was diagnosed with multiple hemangiomas based on clinical exam. at two weeks of age, she developed melena and hematemesis. cbc revealed platelet count of and she was referred to the ed. abdominal ultrasound was concerning for abnormal hepatic waveform; cxr showed multiple pulmonary nodules. workup revealed no other lesions and no further hematologic abnormalities. biopsy of presumed hemangioma ultimately revealed a smooth muscle-lined vascular proliferation without glut- immunoreactivity, consistent with mlt. her early course was complicated by an acute hemodynamically significant gi bleed; esophagogastroduodenoscopy identified six bleeding vascular malformations within the stomach that were injected with epinephrine and sclerosed with successful hemostasis. she received multiple prbc and platelet transfusions. central access was obtained and she was started on oral sirolimus based on previous reports of successful use in management of vascular malformations given its antiangiogenic and immunosuppressive effects ( ). she has tolerated it well with no evidence of toxicity and has achieved a partial response with stable of hemoglobin > and platelet count > . cutaneous lesions have diminished in intensity and she has had no further signs of gi bleeding. she receives pentamidine for pcp prophylaxis. she continues to have appropriate growth and development. we describe here an unusual presentation of an already rare disease. while cutaneous and gi lesions are typical of mlt, pulmonary involvement is not well-described in the literature. early identification of tissue-based diagnosis enabled timely stabilization and treatment of the patient. five months later, she continues to tolerate sirolimus and has shown significant response with diminished coloration of cutaneous lesions, stable blood counts, and no further bleeding. mlt is a relatively newly-recognized disorder with significant phenotypic variability. given that bleeding secondary to a kasabach-merritt-type consumptive thrombocytopenia is the major cause of morbidity and mortality in the first year of life in children with mlt, it is essential to recognize the diagnosis and initiate appropriate treatment as early as possible. north, arch background: patients with generalized joint hypermobility (jhm) may experience easy bruising or bleeding given the association between these symptoms and abnormalities in collagen, a required component of primary hemostasis. heavy menstrual bleeding (hmb) is a common initial presentation for females with underlying hemostatic defects and may be the sole manifestation of a bleeding disorder. however, limited reports describe jhm as a cause of hmb, leading to under recognition. objectives: to describe the clinical characteristics and management of young women presenting with hmb in the setting of jhm. design/method: this study utilized our hmb research registry. we included subjects - years, seen in the nationwide children's young women's hematology clinic between february and november with both hmb and jhm. medical records were retrospectively reviewed for history of presentation, menorrhagia impact questionnaire (miq): a validated quality-of-life tool for females with hmb, medication profiles and relevant laboratory studies. results: twenty-five patients met inclusion criteria (median age years, range - ) with an average beighton score of . (range to ). participants presented an average of . years (range months to years) after menarche despite % of patients reporting heavy to very heavy menses since menarche. according to the miq responses, most participants expressed hmb-associated limitations in physical activities ( %), social activities ( %), and work or school activities ( %). of the participants, % reported bleeding symptoms in addition to hmb, most commonly easy bruising ( %), epistaxis ( %) and cutaneous bleeding ( %). forty percent of young women presented with anemia due to chronic blood loss. results of hemostatic testing were unremarkable, with the exception of one patient who was also found to have type von willebrand disease. additionally, % of females reported arthralgia, with knees and ankles the most commonly affected joints. at time of presentation, % of participants reported failure of initial therapies and most patients ( %) were managed long-term with oral hormone therapy. in a small population of young women found to have jhm who initially presented with hmb, patients were likely to have prior bleeding symptoms as well as substantial delays from menarche to timing of presentation at our young women's hematology clinic despite limitations in activities of daily life. greater awareness of the associations between bleeding symptoms and jhm, despite typically normal hemostatic laboratory results, is necessary so that patients can more easily be identified and receive appropriate therapy. the objective is to determine the impact of cl care practices involving the home environment on ambulatory clabsi rates. design/method: information for the pi was collected through a comprehensive survey that was completed annually by the ccbdn member hospitals. responses to the questions about cl care practices involving the home environment were selected from the pi for . ambulatory clabsi rates and ambulatory total bloodstream infection (bsi) rates were obtained from another ccbdn database. the proportion of hospitals that did or did not employ a particular cl care practice was tallied. the mean ambulatory clabsi rate and mean ambulatory total bsi rate of the hospitals that did or did not employ a particular cl care practice were compared using generalized linear model techniques assuming an underlying negative binomial distribution. results: twenty-five hospitals submitted responses to the questions about cl care practices involving the home environment. one hospital was excluded for lack of bsi data. sixty-three percent of the hospitals programmatically educated parents about all aspects of the cl care bundle. the mean ambulatory clabsi rate for the hospitals that educated parents was significantly lower than that of the hospitals that did not ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory total bsi rate was also significantly lower ( . infections/ cl days vs. . infections/ cl days; p = . ). the mean ambulatory clabsi rates and mean ambulatory total bsi rates were not significantly different for the other cl care practices. conclusion: an analysis of cl care practices involving the home environment reveals that parental education of all aspects of the cl care bundle is associated with a lower ambulatory clabsi rate and lower ambulatory total bsi rate. this finding highlights the importance of systematically teaching family members the proper method of handling cl. background: children undergoing chemotherapy are at a high risk for developing nausea. dr. amy baxter in collaboration with pediatric oncology patients and nurses, developed and validated a pictorial nausea rating scale for children aged - years, called the baxter retching faces (barf) nausea scale. staff nurses at a large, academic, pediatric hospital located within washington, d.c., have identified variability in nursing assessment and documentation of chemotherapy induced nausea and vomiting (cinv) in pediatric oncology patients. the purpose of this quality improvement project was to utilize the barf scale to standardize assessment and documentation of nausea in pediatric oncology patients receiving chemotherapy. the primary aims of this project were to: assess feasibility of the barf scale in clinical practice; increase nursing knowledge about cinv through education sessions; increase documentation of nausea assessments through the use of the scale. the secondary aim of this project was to: increase the recognition of nausea through the use of a standardized assessment tool. design/method: the pdsa model was used to guide the design and implementation plan. in the first phase of the project data was collected to identify the prevalence of nausea in patients admitted for chemotherapy in the prior three months. education sessions discussing cinv and the utilization of the barf scale were conducted. pre and post assessment of nurses' knowledge of cinv and documentation were assessed. in the second phase the barf scale was implemented into practice. nurses were asked to utilize the barf scale to assess and document nausea scores in patients, aged to years, receiving chemotherapy. at the end of the implementation period nurses were surveyed about the feasibility of the scale. post data was collected to identify the prevalence of nausea documented in the electronic health record. this project was undertaken as a quality improvement initiative at children's national and it does not constitute as human subjects research. as such it was not under the oversight of the institutional review board. results: all data has been collected; however complete data analysis will be conducted in the upcoming weeks. background: sickle cell disease (scd) is the most common inherited blood disorder in the united states (us); however, there are few quality measurements to evaluate scd practice. in , the nhlbi published guidelines that include two key interventions for children with sickle cell anemia (sca): the use of transcranial doppler (tcd) screening for stroke prevention and hydroxyurea (hu) to prevent scd pain crisis. we conducted a national survey of scd management sent to providers in over institutions in the us to better assess knowledge of the guidelines and barriers to hu counseling and tcd screening guideline implementation. it was hypothesized that the barriers to tcd screening are different than barriers to hu counseling and prescribing. a -question anonymous survey was sent to providers by mail (follow-up by email). survey themes included nhlbi guidelines knowledge and comfort with understanding and implementing both tcd screening and hu use. the response rate was % ( / ) however one survey was incomplete. thus, were analyzed in the final data set. all of the respondents are in active practice, % s of s in academics and all care for children with scd. the majority of providers ( %) felt "very" or "extremely" confident in their knowledge of tcd screening and interpretation. similarly, % of providers felt "very" or "extremely" familiar with hu dosing and management. for tcd screening, % of providers estimated their screening rates were > % and % providers felt their annual screening rates were - %. the two biggest barriers to tcd screening noted by providers (of moderate to extreme significance) included: lack of support staff ( %) and lack of time during a patient visit ( %). regarding hu prescribing practices, % of providers offered hu to at least % of children with sca over nine months of age. the biggest barrier to hu prescribing noted by % of providers was concerns about patient adherence or access to the medication. only % providers felt that lack of support staff was a moderately significant barrier to hu prescribing. the pediatric scd providers surveyed all have access to the nhlbi guidelines. despite widespread guideline knowledge, there are different barriers for tcd screening versus hu prescribing, which prevent optimal implementation. as a result, although both recommendations are from the same nhlbi guideline, they likely will require different implementation strategies (systems-based interventions for tcd screening; interventions to improve patient adherence for hu counseling) to improve outcomes. background: invasive fungal disease (ifd) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation. the current diagnostic 'gold standard' of ifd remains culture of infected tissue obtained by biopsy. noninvasive biomarker testing for galactomannan or , -beta-d-glucan (bg) can have low sensitivity and does not provide species-level identification. nextgeneration sequencing (ngs) of cell-free plasma is a promis-ing noninvasive approach to providing species-level identification of ifd via a blood test and can further guide specific treatment. objectives: describe the incidence of positivity for fungal specific pathogens on ngs analysis in a high-risk immunocompromised pediatric population and correlate results with other 'standard' infectious studies if performed. design/method: immunocompromised pediatric patients with suspected ifd were enrolled and plasma was collected at time of enrollment. ngs was performed on extracted dna in cell-free plasma (karius, redwood city, ca). after removing human reads, remaining sequences were aligned to a curated database including pathogens. organisms present at a significance-level above a predefined threshold were reported. results: twenty-seven samples from enrolled patients have been processed thus far. of these subjects, were enrolled for prolonged febrile neutropenia (≥ hours) despite broad-spectrum antibiotics, for recrudescent febrile neutropenia, for abnormal imaging, and with other findings. after evaluation of routine studies performed, patients met criteria for proven ifd, for probable ifd, and for possible ifd using eortc/msg guidelines. the ngs plasma test identified the same pathogen as cultured from infected tissue or blood in % ( / ) of the proven cases. in the probable cases, pneumocystis jirovecii was identified in a patient with a positive bg ( pg/ml) and pneumonia. among the possible cases, toxoplasma gondii was detected in a patient with prolonged febrile neutropenia and lung imaging suggestive of ifd. additionally, candida glabrata was isolated in a patient with prolonged febrile neutropenia but no other criteria for ifd. numerous pathogens were also identified that could explain the above clinical parameters, including hsv , cmv, vzv, hhv , ebv, bk polyoma virus, and ureaplasma parvum. the cell-free plasma ngs test can detect invasive fungal infections from blood. the test identified fungi from proven ifd, detected pathogens in both probable and possible ifd cases, and is a useful diagnostic tool in the evaluation of ifd. supplies and sample shipment and processing supported by karius, inc. baylor college of medicine, texas children's hospital, houston, texas, united states background: practicing medicine is a lifelong learning process. as noted in the institute of medicine's seminal report, 'to err is human,' adverse outcomes do not typically result from individual recklessness; rather, they result from faulty systems, processes, or conditions that provide an environment conducive to making a mistake, or failing to prevent one. learning to systematically review errors and translate lessons learned into quality improvement (qi) initiatives is a critical component of practice-based learning and improvement for practitioners at all career levels. objectives: to develop a methodical, self-reflective and nonthreatening approach to incident analysis and translation of lessons learned into qi initiatives. design/method: we used a validated, structured case audit approach, modified from szostek et al: ) review all documentation relating to the case and identify all health care providers involved; ) interview stakeholders, including those who directly provided and supported care; ) use a qi tool to conduct a root-cause analysis; ) identify a systems issue that contributed to the outcome; and ) propose systems-level interventions and prioritize initiatives based on effort-yield projections. results: pdsa cycle : plan: establish a committee to ) identify potential cases, ) triage cases for conference presentation, ) determine timing and frequency of conferences, ) develop a training manual, ) record identified qi initiatives. do: we established a quarterly section-wide meeting to which all members of the pediatric hematology/oncology service are invited, including administrative and nursing leadership. we developed a training manual and structured presentation template. prioritized cases were discussed in advance during multidisciplinary case review sessions, and presented by senior fellows who were instructed to focus discussion on potential opportunities for qi. study: we identified cases, meeting criteria for mmi presentation. qi initiatives identified from this conference resulted in a number of systemic practice changes; however, we encountered challenges to sustaining these changes over time. act: objectives for the next pdsa cycle are to ) establish a method for tracking the adherence to recommended changes in practice, ) maximize sustainability by integrating qi initiatives into institutional qi leadership and practice standardization committees. we have successfully implemented an mmi conference that meets out of institute of medicine quality domains: safety, effectiveness, patient-centeredness, timeliness, and efficiency. a standardized, consistent approach to mmi presentations that includes identification of contributing factors and specific qi implications has the potential for improving both provider education and patient care/safety. johns hopkins university, baltimore, maryland, united states background: receiving a cancer diagnosis is a life-changing event for patients and caregivers, although little is known about the experience. while some oncologists receive dedicated training in delivering this bad news, the initial conversation is often with a primary pediatrician, and these providers often feel they do not receive adequate training in the communication of a cancer diagnosis. objectives: our objectives were two-fold: first, to better define the experiences of caregivers/patients when told of a cancer diagnosis, and to query how caregivers/patients believe providers can improve the disclosure of this bad news. secondly, to assess what, if any, training primary pediatricians received in this skill, and to assess how comfortable providers in various settings and stages of training are with communicating cancer diagnoses. design/method: from november - , semistructured, in-depth interviews were conducted with pediatric oncology patients and caregivers of patients (n = ) diagnosed in the past year regarding their experiences receiving the diagnosis at our institution. in addition, pediatric residents (n = ), outpatient pediatric primary care physicians and pediatric emergency medicine physicians (n = ) were interviewed regarding their experiences delivering cancer diagnoses. interviews were analyzed following principles of thematic analysis. interviewers with patients and caregivers had two common themes: ) all emphasized their wish for direct and thorough information; ) both patients and caregivers emphasized the gratitude they felt for physicians who gave them hope by emphasizing the good prognosis of their child's cancer. lack of training in this area, as well as lack of comfort delivering this news was common will all providers. additionally, providers report variable approaches to giving bad news, including ) whether to tell caregivers separately or tell the child and parents together, and ) whether to give favorable prognostic information. additionally, attending physicians also differed significantly in their approaches to teaching residents. while some believed residents should give the news to gain experience, others felt that this is not appropriate if residents are inexperienced. only one resident reported ever receiving feedback on his communication skills in this type of discussion. conclusion: we plan to build on these interviews to develop a national survey of patients, caregivers, and providers to better understand the issues surrounding this discussion. we will use the findings to develop a communication curriculum for pediatric residents, focusing on the discussions that occur in the outpatient setting by primary pediatricians. background: human papilloma virus (hpv), common in both females and males, is responsible for pathologies ranging from benign genital warts to cervical and penile cancer. hpv strains and are responsible for , malignancies each year in the united states, and one third of them arise in men. pharmaceutical companies have now developed a vaccine that will help prevent the virus-associated malignancies. the cdc initially recommended that females ages - years receive the vaccine series, then starting in they expanded the eligibility to males ages - years. despite being widely available and highly publicized, only % of eligible females receive the full vaccine series. objectives: this study aims to assess the knowledge of hpv, the attitudes towards the hpv vaccine, and identify barriers preventing its full utilization. once identified, we aim to overcome the barrier(s) in order to improve vaccination rates in eligible adolescents. we distributed a standardized questionnaire to the parents of eligible female and male patients in our pediatric hematology-oncology clinic. it assessed the parents' knowledge of hpv and the vaccine, their views of the vaccine, and reasons why they may oppose it. results: approximately % of parents claim they have been educated about hpv, mostly by their primary care physician. however, % did not know what disorders hpv caused; % felt the vaccine should not be added to the typical vaccine schedule; % of parents do not intend to vaccinate their child. of those that opposed the vaccine, one-third were concerned about potential side effects and nearly % feel they do not have enough information. additionally, % of parents are not aware that the vaccine is available at their child's doctor and only % of parents have discussed the hpv vaccine with their child's doctor. the largest barrier to the utilization of the hpv vaccine that we have identified appears to be lack of educa-tion. as a result, we have begun distributing the cdc's hpv and vaccine patient guide to our patients' families as an intervention. we are currently in the process of re-administering our survey to these families after implementing the intervention to assess its success in increasing both knowledge and utilization of the hpv vaccine. cancer institute, chennai, chennai, tamilnadu, india background: rasburicase is a recombinant urate oxidase enzyme approved for use in tumor lysis syndrome (tls) and it acts by reducing serum uric acid levels. using rasburicase at the recommended dose of . mg/kg/day for days is expensive and it is not known whether this extended schedule is clinically beneficial compared to a single fixed dose of . mg. the aim of the present study was to evaluate the efficacy of single dose rasburicase . mg in prevention and management of tls. design/method: rasburicase is available as single use . mg vial. at our institution a single dose of rasburicase . mg irrespective of bodyweight has been used in adults and in children a dose of . mg/kg (maximum . mg) has been used since for prevention and management of tls and subsequent doses are given based on biochemical response and clinical condition. we retrospectively analysed the case records of patients who had received rasburicase from january to january . the study included patients with hematological malignancies who received rasburicase. children accounted for . % (n = ) patients and males comprised % (n = ). rasburicase was used prophylactically in ( . %) patients, for laboratory tls in patients ( . %) and for clinical tls in ( . %) patients. single fixed dose rasburicase prevented laboratory/clinical tls in % of the prophylactic group and prevented clinical tls in % of the laboratory tls group. none of the patients in prophylactic and laboratory tls group developed clinical tls. however, majority of the patients with clinical tls required more than one dose rasburicase. single dose of . mg ( vial) rasburicase is efficient in preventing and managing laboratory tls and is economically viable in resource constrained settings. nicole wood, lauren amos, nicholas clark, chris klockau, karen lewing, alan gamis children's mercy kansas city, kansas city, missouri, united states background: medication reconciliation for newly diagnosed oncology patients is complicated and cumbersome. these patients are often admitted on no medications, and leave on multiple. chemotherapy and supportive medications are crucial. despite numerous individuals overseeing this process, prescribing errors or omissions still occur. when reviewing the literature, improvement occurs when there is an interprofessional and standardized process to medication reconciliation. objectives: this project's aim was to improve the accuracy of the discharge medication reconciliation process from % to % from february -august . the process measure was the percentage of patients discharged with an accurate checklist. additional time for staff spent in completing the checklist and avoiding an increased error rate by changing the prescribing process were followed as balancing measures. we created a discharge medication checklist which included a list of required home medications prescribed by the resident, ideally hours prior to discharge. it required fellow or attending review and pharmacy to review the list and educate the family. checklists were collected monthly and reviewed against the electronic medical record (emr) for accuracy. results: six pdsa cycles were completed. there were errors during the data collection time frame. in pdsa cycle , a patient received acetaminophen for pain control which is avoided at home. in addition, this patient received diphenhydramine instead of ondansetron, which is preferred as an antiemetic. in pdsa cycle , a patient with a pending diagnosis was sent home with acetaminophen. of note, this patient did not have a checklist completed upon discharge. this project provides a novel and important method to standardize the discharge medication reconciliation process in a complex patient population. it clarifies which types of medications these patients need, provides pharmacy teaching to families which was not done previously, and prescribes discharge medications to families sooner. after the first medication reconciliation error, the checklist was revised. no further errors were made following revision, with the exception of one patient without a completed checklist at dis-charge. our accuracy rate increased from % at baseline to % following implementation. we are in the process of making the checklist electronic and accessible in the emr. in the interim between the end of data collection and implementation into the emr, a leukemia patient was sent home without an epinephrine pen, further demonstrating the importance of this standardized discharge process. for this reason, we have re-instituted the checklist until the electronic version is available. background: survivors of pediatric cancer are at risk of losing pre-existing protective antibodies to vaccine preventable diseases. in a prior study, % of children < years lost humoral immunity to measles as a result of chemotherapy induced alterations in immune system. measles in recipients of immunosuppressive chemotherapy has mortality rates up to %. because of volitional vaccine refusal, there has been a dramatic increase in measles infection from cases in to in , including several statewide outbreaks. small pediatric oncology practices frequently share floor/clinic space with the general pediatric patients putting them at risk for measles since virulence starts hours prior to symptoms. there is no standard protocol for revaccinating post-chemotherapy patients. to assess measles risk based on serial humoral immune status in a cohort of pediatric oncology patients receiving intensive chemotherapy design/method: patients < years age with known vaccination status receiving intensive chemotherapy between july -june at our institution's pediatric oncology practice were included in this prospective study. serial measles igg antibodies were measured at diagnosis, months and months after initiation of chemotherapy using elisa. measles immunity was defined per lab standards. a comparison of pre-chemotherapy and serial post-chemotherapy immunization titers was made for all patients by diagnosis. the study population consisted of children ( male); patients had all, non-hodgkin lymphoma, sarcoma and other solid tumors. two patients ( . %), both unvaccinated had non-protective measles antibody levels at s of s baseline. of the remaining patients, . % patients ( leukemia, lymphoma and sarcoma) lost protective antibody titers at months after initiation of chemotherapy and . % ( leukemia, lymphoma and sarcoma) at months after initiation of therapy. % of the remaining patients who retained measles antibody titers within protective range at months also demonstrated a steady decline in antibody titers at and months from therapy initiation. the loss of protective measles humoral immunity occurred significantly more often in patients with leukemia compared to other malignancies. oncology patients in our practice undergoing intensive chemotherapy demonstrated progressive waning of protective measles igg titers. our data suggests that it should be standard practice to check all patients for measles humoral immunity prior to starting chemotherapy and at completion. larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients, an intervention that is easily applicable and of low cost. background: the accurate determination of glomerular filtration rate (gfr) is important to screen for acute kidney injury, to dose chemo-therapy, and to identify risk for chronic kidney disease.being correlated with inulin clearance, measured gfr by iohexol plasma disappearance (igfr) is a new gold standard for measurement of gfr in pediatric cohort studies. igfr is based on the clearance of an exogenous marker and is unaffected by endogenous compounds or a patient's muscle mass. we compared igfr with -hour urine creatinine clearance ( crcl) and gfr estimating equations using serum creatinine (scr) and serum cystatin c (cystc) in pediatric patients with cancer. we recruited participants who were ages to yrs, continent of urine, and diagnosed with a malignancy in the past years. eligible subjects had stable kidney function for at least two weeks prior to the assessment of igfr. consented subjects had baseline assessments including height, weight and vital signs. blood samples were obtained for serum chemistry, and time zero iohexol. igfr determined by ml iohexol solution infused over - minutes followed by ml of sterile saline. blood was drawn at , , and minutes.at the same time of igfr, the crcl was collected. igfr was calculated using a two-compartment model and area under the curve. we compared igfr to published gfr equations (schwartz et al, kidney int ). results: ten subjects ( female/ male) agreed to participate. the distribution of diagnoses for the subjects: all = , lymphoma = , brain tumors = and hepatocellular carcinoma = . six patients were off therapy. the lower gfrs are noted in patients who had malignancies other than leukemia, likely due to the use of cisplatin based therapy. the average igfr was ml/min/ . m^ whereas crcl was . ml/min/ . m^ ; demonstrating the crcl overestimates gfr compared to igfr. comparing igfr to univariate equations using scr, cystc, and the multivariate equation with both, the univariate cystc equation correlated well with igfr; the others overestimated igfr. we found that crcl overestimated igfr. the univariate cystc equation better correlated to igfr than equations with scr. the poor performance of scr based methods to assess gfr might be due to decreased muscle mass and inadequate nutritional status. creatinine-based determinations of gfr alone, may not be accurate in this population. further study is needed to determine if igfr should be a standard of care to assess gfr in children with cancer particularly who are receiving nephrotoxic medications and incontinent of urine. background: pediatric oncology patients undergoing chemotherapy through indwelling venous catheters are at increased risk for severe sepsis especially when neutropenic due to chemotherapy. rapid triage and early recognition are essential because delayed initiation of antibiotics and fluids in these patients or delayed transfer to higher level of care after initial stabilization is associated with poor clinical outcome. our pediatric oncology out-patient clinic is designated as an article unit whereby the providers can initiate and give treatment such as intravenous fluid, antibiotics, chemotherapy and blood products. objectives: global aim-optimize management of early sepsis and decreased morbidity, mortality and hospital length of stay in the high risk pediatric oncology patients. smart aim-improve timely management with initiation of fluids and antibiotics and transfer of septic patients to higher levels of care by % in months in above patients design/method: multidisciplinary team with physicians and nurses was created. retropective chart review of sepsis patients treated at the clinic from april to october was done using an audit sheet to identify the barriers in the delivery of care. three patients were identified and data analyzed prior to intervention; two were analyzed post interventions. a key driver diagram was created by the group to drive intervention. a process map was designed to identify the different steps in the care of these patients to pinpoint areas needing improvement. different timed data points were used starting from time of arrival to clinic, time to antibiotics and fluids and time to transfer to higher level of care. rapid pdsa cycles were done to improve the processes and delivery of care. run charts were created. there was an improvement close to the goal of % for all data points used. pdsa cycles for improvement included conducting frequent mock codes with appropriate feedback real time coaching and process planning with nursing staff. we partnered with pharmacy for close loop communication with clinic staff and we improved communication between physicans at different levels. conclusion: sepsis in neutropenic pediatric oncology patients is deadly and can be reversed with timely management at different levels. given the promising results of the above project, we want re-inforcement of the processes to be a part of the daily practice of first line clinical staff. eventually we will extend the principles learnt in management and triage of sepsis to other outpatient emergencies chemotherapy related anaphylaxis background: chemotherapy-induced nausea and vomiting (cinv) is a common side effect in children receiving antineoplastic chemotherapy. recommended prophylactic antiemetic medications are based on the classification of chemotherapy emetogenicity. however, despite appropriate use of these antiemetic agents, some patients will still experience nausea and/or vomiting. children's oncology group clinical practice guidelines recommend the addition of olanzapine to prophylactic regimens for management of breakthrough cinv. objectives: our pediatric hematology oncology center implemented a quality improvement (qi) project aimed to increase the use of olanzapine in pediatric cancer patients years of age and older receiving moderately or highly emetogenic chemotherapy and experiencing breakthrough cinv over a month period. design/method: this qi project was conducted utilizing plan-do-study-act (pdsa) cycles. for the first pdsa cycle, baseline data was collected through chart review to determine the rate of olanzapine use for breakthrough cinv over a month period from july to december . breakthrough cinv was defined as use of or more doses of antiemetic agents other than those given for cinv prophylaxis. guidelines for treatment of breakthrough cinv were reviewed with pediatric hematology/oncology attending physicians and fellows. flyers were created that listed chemotherapy regimens considered moderately and highly emetogenic. if a patient experienced breakthrough cinv, a flyer was to be placed in the patient's roadmap binder to signal olanzapine should be added to the next chemotherapy block. data was collected over a month period in september following this first intervention. the second pdsa cycle consisted of didactic education and training of pediatric oncology nurses as well as pediatric residents regarding the addition of olanzapine for breakthrough cinv. rates of olanzapine use were then collected from october through november . results: olanzapine use increased from . % at baseline to . % after the first pdsa cycle ( = . , p = . ). after the second pdsa cycle, olanzapine use increased another . % to . % ( = . , p = . ). the administration of olanzapine was successfully increased by modifying patients' roadmaps after patients experienced breakthrough cinv as well as with education and training of pediatric oncology staff, fellows, residents, and nurses. background: venous thromboembolism (vte) is increasingly affecting children. according to an administrative database study, there was a % increase in the incidence of vte among children admitted to free-standing children's hospitals in the united states from to . risk factors for hospital-acquired vte are well-known and well-studied in adults, with evidence-based preventative measures available. similar guidelines are lacking for children. objectives: there is an ongoing national-initiative to develop and institute methods for screening and preventing hospitalacquired vte in children. in / , nationwide children's hospital instituted an electronic screening form required for all patients admitted ≥ hours. patients were scored and riskstratified based on eight risk-categories. a summated score was used to determine the vte risk level, and used to make prophylaxis recommendations for patients ≥ years; as well as patients ≥ years who were admitted to an intensive care (icu), surgical, or trauma unit. the purpose of this irb exempt, quality improvement initiative was to retrospectively review our experience with this risk-stratification tool. results: hospital-acquired vte events occurred in unique subjects. median age at vte diagnosis was years. only ( %) vte occurred in children ≥ years of age. ( %) vte were deep vein thrombosis (dvt), and ( . %) involved pulmonary embolism. vte was most common in subspecialty units including the pediatric and cardiac icus ( . %); neonatal icu, ( . %); and hematologyoncology, ( . %). ( %) vte were associated with central venous catheters (cvc) and events ( %) were associated with altered mobility. congenital heart disease/heart failure was the most common chronic medical condition associated with vte ( ( . %) events); whereas infection and trauma/surgery were the most common acute medical conditions associated with vte ( ( . %) and ( %) events, respectively). during ( %) events, subjects scored a summated score ≥ . in summary, in this single institution, prospectively maintained database, cvc remains the most common risk factor for vte, followed by cardiac disease, infection and trauma/surgery. most subjects who developed vte scored high (score ≥ ) on our screening tool. only a small proportion of vte occurred in patients older than years and thus eligible for thromboprophylaxis. our results indicate that future vte prevention endeavors should include these age groups in addition to exploring more aggressive prophylactic modalities including pharmacological prophylaxis. background: pediatric fellows are required to have active engagement in quality improvement (qi) activities, and yet a national acgme review found most trainees had "limited knowledge of qi methods" and "limited participation in interprofessional qi teams". the twenty fellows in our pediatric hematology/oncology training program identified blood culture utilization as their qi priority. our institution recently introduced a hospital-wide decision algorithm to guide providers regarding when to obtain blood cultures. there is often a low threshold to obtain blood cultures in immunocompromised pediatric oncology patients, but these are often low-yield or result in falsepositives. our fellows spearheaded a project to implement the algorithm in the inpatient pediatric oncology population and improve the proportion of appropriately drawn blood cultures. we investigated how appropriately the algorithm was being utilized on the inpatient pediatric oncology floor prior to and after several educational steps aimed at disseminating the algorithm to members of the care team. our primary endpoint was to quantify the proportion of culture episodes drawn "inappropriately", with a goal of reducing inappropriate episodes to ≤ %. the algorithm was initially introduced to the nursing staff and residents covering the twenty-bed inpatient unit in september . qi project planning took place with upper level fellows in january . fellows and faculty received intensive training on the algorithm in july-august . we then conducted a retrospective chart review of blood culture episodes drawn between august and november . upper level fellows scored ∼ culture episodes as to whether the decision to culture and number of cultures drawn were "appropriate" or "inappropriate", and catalogued the indications for culture episodes and if applicable, why the episode was found to be inappropriate. additionally, fellows discussed inappropriate culture episodes with the team onservice, to provide direct feedback on where the algorithm failed. results: between august -december on average cultures/ patient-days were drawn. forty-nine percent of culture episodes were inappropriate. from january -october , following targeted education on the algorithm, the rate of blood cultures drawn decreased to cultures/ patient-days. the average proportion of inappropriate culture episodes fell to . %, representing a % decrease in inappropriate culture utilization. correct application of a decision algorithm for blood culture utilization can reduce total cultures drawn on an inpatient pediatric oncology unit. fellow-led education of the multi-disciplinary team decreases the rate of inappropriate culture episodes as well as provides active engagement in qi. background: inadequate understanding of sickle cell disease (scd) is common and can affect patients' compliance and therefore their morbidity and mortality, especially after transition to adult care. optimal clinical care for scd includes disease education, which can be difficult given the breadth of possible topics and limited time in clinic. it is unclear how best to provide personalized, efficient education for adolescents with scd. this quality improvement (qi) study aimed to implement a questionnaire-based system to improve patients' knowledge of their scd and documentation of education by the nurse or physician. the study objective was to improve provider documentation and patient knowledge about their scd by identifying patients' gaps in comprehension. by january , the study aimed to increase education documentation from % to %. by april , the study aimed to increase use of a smart phrase for education documentation from % to %. by june , the study aimed to increase patients' knowledge about their disease by %. design/method: twenty-one scd patients enrolled on an irb approved qi study, with twenty active patients. our comprehensive team generated a questionnaire with knowledgebased questions for two age groups: - and - years old. at each comprehensive visit, a questionnaire was distributed, with at least -month intervals. the provider scored questionnaires and reviewed two educational topics, with wrong answers taking priority. plan-do-study-act (pdsa) cycles included pdsa# : patients completed questionnaire. pdsa# : a smart phrase addressing questionnaire topics was created and shared with providers. pdsa# : patients received education handouts during clinic education. documentation in clinic notes was the process measure and questionnaire scores was the outcome measure. results: pdsa# is complete, pdsa# has four patients remaining, and pdsa# is ongoing. due to variable visit frequency, there are multiple concurrent cycles. after pdsa# , free text documentation was completed an average of % over the course of months. after pdsa # documentation increased to % within months and questionnaire scores increased from an average of % to %. of the questions that patients got wrong on their first visit, they were significantly more likely to improve on retesting if the topic was taught to them than if it was not addressed ( % vs. %, p = . ). we are currently completing pdsa# and collection of post pdsa# data. questionnaire-based scd education coupled with standardized smart phrases improves patients' scd knowledge and documentation by providers. further improvement in knowledge is expected with the addition of handouts. background: exposure to suffering can have a profound impact on the wellness of caregivers, often referred to as the "cost of caring". this cost is especially high in pediatric hematology/oncology. repeated exposure to suffering has the potential to negatively impact resilience and increases the risk of burnout, thus impacting quality of care and patient satisfaction. we have developed a peer support team utilizing the critical incident stress management (cism) model. this model has been successfully used in other professions that frequently face traumatic events such as fire fighters, police and emergency medical technicians. the h.o.p.e.s. team (helping our peers endure stress) consists of volunteer multidisciplinary staff members who have received training to provide both group and peer support following any 'critical incident' that may impact one or more staff members. we hypothesize that implementation of the h.o.p.e.s. team will improve staff resilience, decrease overall rates of burnout and improve compassion satisfaction. s of s design/method: we are using both empiric metrics and anecdotal reports to assess the impact of the h.o.p.e.s. team. prior to the activation of the team, all pediatric hematology/oncology clinical staff members were surveyed using validated tools to assess their levels of resilience, burnout, secondary trauma and compassion satisfaction (proqolv and brief resilience scale). they were also asked to rate the number of times they had experienced critical incidents, as well as their perceived level of distress after dealing with traumatic events. after the h.o.p.e.s. team has been functional for months, we will send the same survey to staff members to measure changes, paying special attention to resilience and rates of burnout and compassion satisfaction. results: enthusiasm for development of the team has been high. of people approached to volunteer their time to participate in the multidisciplinary team agreed, including attending physicians, fellows, nurses, nurse practitioners, child life specialists, social workers, clergy and psychologists. all volunteers participated in a -day training conducted by an instructor from the international critical incident stress foundation. engagement in the first staff survey has been high, with of responding to date. data collection is ongoing. clinical staff in pediatric hematology/oncology may be particularly vulnerable to burnout and decreased resilience by repeatedly witnessing suffering and trauma. peer support interventions following critical incidents may lead to increased resilience and compassion satisfaction while decreasing rates of burnout. enthusiasm for the development of a peer support team has been high. background: monthly blood transfusions are an indicated therapy for pediatric patients with sickle cell disease with certain complications. maximizing transfusion efficiency in a busy infusion clinic requires: ensuring that appropriate blood units are available in the hospital blood bank; laboratory specimens are obtained from patients in advance; and coordination of clinic appointment and nursing availability. we sought to improve clinic efficiency through identifying ways to better communicate with patients/families regarding upcoming laboratory and transfusion appointments, and to assess the efficacy of implementing a web-based personalized text reminder (pinger.com). we measured the baseline frequency with which transfusion appointments were missed by families, moved to later within the week, or delayed due to late labs. a convenience sample of patients receiving monthly transfusions received a questionnaire about patient/parent preferences for appointment reminders and barriers to keeping appointments. those patients/parents who did not opt-out of an additional text reminder received personalized texts from their care team reminding them of lab and transfusion appointments. rates of missed/moved/delayed appointments were compared between the group receiving the additional text messages and the group only receiving standard, hospitalgenerated appointment reminders (telephone call). results: forty-one families ( patients) responded to the survey, capturing information on % of patients receiving chronic transfusion therapy. thirteen families ( %) declined the additional text reminders. families reported a preference for text reminders ( %), more often than email ( %) or telephone ( %), and % of families wanted to receive reminders for both transfusion and laboratory appointments. the majority ( %) of families reported competing work/life priorities as the reason for missed/late appointments. other families noted transportation/travel ( %), fear/illness/pain ( %), and lack of reminders ( %) as the reason for missed appointments. at baseline (twelve weeks), . % of appointments were missed on a weekly basis (range - of available per week), . % were moved, and % of appointments were delayed. during our intervention period (twelve weeks), % were missed, . % were moved, and . % were delayed (combined, both groups). there was no difference in missed ( . % texted vs . % standard), moved ( . % texted vs . % standard) or delayed ( . % text vs . % standard) appointments. though families at our center reported a preference for a text-based reminder, personalized text reminders for appointments did not improve clinic efficiency as measured by missed, moved or delayed transfusion appointments. there was no improvement in appointment adherence in the group receiving personalized texts in addition to standard hospital reminders. university of utah, salt lake city, utah, united states background: childhood cancer outcomes have improved significantly, in large part due to multi-institution collaborative clinical trials run by the children's oncology group (cog). approximately half of eligible children with cancer will enroll on a therapeutic trial, but little is known about the factors affecting caregiver decision-making regarding enrollment or how well the required elements of informed consent are conveyed during the consent process. objectives: . assess coverage of ten of the required elements of informed consent for cog therapeutic trials. . describe factors affecting caregiver decision-making regarding therapeutic trial enrollment. we surveyed families of children who were offered enrollment onto a phase cog therapeutic study for an initial cancer diagnosis in the previous months. fisher's exact or wilcoxon rank-sum tests were utilized to compare demographic and other motivating factors related to enrollment decision-making. results: seventy participants were surveyed. regarding of the basic required elements of informed consent, % knew the trial involved research, % knew consent was required, % knew the enrollment length for the trial, % knew they could continue care independent of enrollment, % knew who to contact with questions, % knew there were options besides enrollment, % knew they could withdraw at any time, % knew the information was confidential, % knew there were risks associated with the trial, and % knew there were benefits. of all participants, % (n = / ) enrolled onto a therapeutic study. among enrollees, % (n = / ) of the primary caregivers had completed college compared to % (n = / ) of those not enrolled (p = . ). when asked about factors impacting their decision, % (n = / ) of those enrolled said they felt there were no risks or did not know if there were risks associated with the study compared to % (n = / ) of those choosing not to enroll (p = . ). of those enrolled, % (n = / ) reported the physician recommendation "somewhat" or "strongly" affected their decision to enroll compared to % (n = / ) of those not enrolling (p = . ). of those who enrolled, % (n = / ) reported feeling pressured to enroll while % (n = / ) of those not enrolled reported pressure (p = . ). of enrollees, % (n = / ) reported they did not have enough time to decide compared to % (n = / ) of those not enrolled (p = . ). failure to convey all required elements of informed consent highlights possible deficiencies in the consent process for cog therapeutic trials. caregivers' perception of being pressured and lack of time to make an informed decision may impact clinical trial enrollment. background: abnormal uterine bleeding (aub) is a frequent adolescent gynecologic complaint. however, limited research exists to guide management, and acute care varies. we sought to improve emergency care for adolescents with aub by developing a clinical effectiveness guideline (ceg) and assessing its impact on quality of care. design/method: a stakeholder engagement group consisting of members from the departments of hematology/oncology, adolescent medicine, general pediatrics, and emergency medicine designed a ceg algorithm for emergency aub management. pediatric residents received ceg training and their knowledge and attitudes were assessed using pre and post intervention surveys. icd- and codes identified electronic health record data for patients presenting to the pediatric emergency department (ed) for aub months before and after ceg implementation. pre-pubertal patients and those with vaginal bleeding from trauma were excluded. a weighted, -point scoring system consisting of prioritized aspects of history, laboratory studies and management was developed to quantify the quality of care provided. t-test, chi square test, wilcoxon rank sum test, and a run chart were used for analysis. of the patients identified, met inclusion criteria. there were % of patients currently using some form of contraception, while . % had bleeding related to a current or recent pregnancy. median aub quality care scores were pre-and post-intervention (p = . ). run chart data showed no shifts or trends (overall median score, -points). both pre and post-implementation, points were deducted most frequently for not assessing personal/family clotting disorder history and inappropriate use/dosing of oral contraceptives. we successfully designed and implemented a ceg and educational intervention for aub management in a pediatric ed. these data suggest our ceg may be an effective tool to improve emergency aub care for adolescents, though additional cycles are needed. background: high-dose methotrexate (hd-mtx) is a common chemotherapy administered inpatient at most centers. its administration is particularly susceptible to error due to the need for frequent drug levels with resulting changes in supportive care. errors can prolong patient stay and cause patient harm. objectives: global aim-to reduce the length of stay (los) of hd-mtx admissions. smart aims-to increase the percentage of patients whose pre-hydration fluids are started by am from % to % by / / , and to increase the percentage of patients who receive hd-mtx by pm from % to % by / / . we used rapid process improvement methods to target earlier methotrexate administration. a key driver of prolonged los was hypothesized to be drug levels returning overnight rather than in the day time due to delayed hd-mtx start. changes implemented have included scheduling hd-mtx patients as the first patients of the day for their exam in clinic and scheduling labs to pass for hd-mtx on the day prior to admission. there are ongoing pdsa cycles to change the location of pre-hydration start from the inpatient room to the clinic exam room in order to meet hd-mtx administration time goals. we are piloting two different education materials to improve patient experience. one explains hd-mtx levels in a red/yellow/green stoplight format and the other reminds patients how to prepare for the admission. other interventions regarding how we test urine ph and safety checks in the ordering process for history of delayed clearance are in the planning stage. the project is ongoing, but as of / / , we start methotrexate by pm % of the time which is improved from a baseline of %. when the project was started, pre-hydration was never started before am. now, fluids are started by am % of the time. pdsa cycles are ongoing and we have yet to sustain reductions in los, but some months have shown decreased los by as much as hours from baseline measurements. rapid cycle improvement can be utilized to decrease los hd-mtx admissions. this has important financial implications as well as the potential to reduce secondary harm from unnecessary time in the hospital. pediatric cancer centers should schedule hd-mtx admissions first thing in the morning so that data regarding kidney injury and drug clearance can be interpreted by the day team and children are not cleared for discharge in the middle of the night. background: education and training for interdisciplinary pediatric oncology providers requires training in principles of palliative and end-of-life (eol) care. the experiences of bereaved parents can inform and enhance palliative care educational curricula in uniquely powerful and valuable ways. the objective of this study is to present an innovative palliative care educational program for oncology providers facilitated by trained bereaved parents who serve as volunteer educators in local and national palliative care educational forums and to describe how incorporation of bereaved parents in these educational forums affects participant comfort with communication and management of children at the eol. design/method: survey tools were adapted to determine how bereaved parent educators affected participant experiences in different educational forums: institutional seminars on pediatric palliative and eol care, role-play based communication training sessions, and an international symposium on pediatric palliative oncology. pre-and post-session surveys with incorporation of retrospective pre-program assessment item to control for response shift were used in the evaluation of institutional seminars and communication training sessions. results from feedback surveys sent to all attendees were used to appraise the participants experience in the international oncology symposium. results: involvement of trained parent educators across diverse, interdisciplinary educational forums improved attendee comfort in communicating with, and caring for, patients and families with serious illness. importantly, parent educators also derive benefit from educational with interdisciplinary clinicians. integration of bereaved parents into palliative and eol care education is an innovative and effective model that benefits both interdisciplinary clinicians and bereaved parents. background: poorly controlled chemotherapy-induced nausea and vomiting (cinv) significantly impairs patients' quality of life and contributes to ongoing medical costs through increased length of stay in the hospital or readmissions and outpatient visits for control of nausea, vomiting or dehydration. lack of adherence to national evidenced-based guidelines that dictate antiemetic prescribing for variably emetogenic chemotherapy leaves patients vulnerable to increased cinv and its ensuing complications. objectives: to review our institution's antiemetic prescribing practices and their consistency with the antiemesis guidelines from the national comprehensive cancer network (nccn) and children's oncology group (cog)-endorsed supportive care guidelines and to further develop tools to increase adherence to these national-based guidelines to improve control of cinv. we performed a retrospective chart review of inpatient chemotherapy encounters. we evaluated emetogenicty of chemotherapy (high, medium, low), initial antiemetic regimen ordered, number of as needed medications required and adherence to national evidenced based guidelines tailored to each level of emetogenicity in the prescription of antiemetics. results: fifty-five total inpatient chemotherapy encounters were reviewed over months. eighteen of these encounters were considered to have been highly emetogenic chemotherapy (hec) with the remaining of these considered to be moderately emetogenic. only out of hec encounters completely included all guideline-recommended agents. there was a demonstrable lack of consistency across providers with dosing of aprepitant and most as needed medications. there was significant variation in order of first, second and third line anti-emetics ordered -with lorazepam and promethazine being used most frequently. with an aim of improving antiemetic prescribing practices for our patients, we are currently rebuilding chemotherapy treatment plans in our electronic medical record to incorporate antiemetic drug order sets that follow evidenced-based guidelines for variably emetogenic chemotherapy. this will be used in conjunction with an education initiative about best practices in supportive care for all prescribers of antiemetics. review of our department's recent inpatient chemotherapy encounters show we are falling short in following nationally recommended standards for appropriate antiemetic coverage during chemotherapy. identification of these deficiencies allows for implementation of quality initiatives to improve prescriber adherence to evidenced-based guidelines for better control of cinv. background: there are currently no consensus guidelines for the management of pediatric oncology patients presenting with fever without neutropenia. historically, these patients had been treated similarly to neutropenic patients with empiric antibiotics. while there has been a shift towards reducing unnecessary empiric treatment, there has been limited research into the outcomes associated with withholding empiric iv antibiotics in this patient population. we assessed the safety and efficacy of our institution's current protocol of observing well-appearing patients who present with fever without neutropenia and compared the outcomes of the patients who did and did not receive empiric iv antibiotics. design/method: this was a prospective, single-institution cohort study. patients were included if they were currently undergoing chemotherapy for an oncologic diagnosis and presented initially as an outpatient with fever and nonneutropenia (defined as anc ≥ cells/mm ). for each episode we recorded lab and blood culture results, signs and symptoms of initial presentation, and clinical outcomes, including antibiotic administration and hospital admission. results: a total of episodes of well-appearing patients with fever without neutropenia were identified. compliance with the institutional protocol was high; . % of patients were observed without receiving empiric iv antibiotics. the majority of patients were discharged home and there were no serious complications or infectious deaths. the incidence of positive blood cultures was low ( . % including several likely contaminants), despite the presence of central venous catheters in the majority ( . %) of patients. there were no significant differences in age, oncologic diagnosis, central s of s line access, anc value, or incidence of bacteremia between patients who did and did not receive empiric iv antibiotics. patients who were admitted to the hospital were significantly more likely to have received iv antibiotics (p < . ) despite documentation of a reassuring exam. however, admitted patients who initially received iv antibiotics were just as likely to discharge within hours compared to patients who were observed. we propose that empiric iv antibiotic administration in febrile, non-neutropenic, otherwise well-appearing patients is unnecessary. our study demonstrated no adverse consequences of observation and no significant differences in clinical outcomes between patients who did and did not receive iv antibiotics aside from rate of hospitalization. this supports the practice of observation without empiric antibiotics for such patients. background: children with hepatoblastoma (hb) undergo repetitive computed tomography (ct) scans to determine response to treatment and assess for relapse. this imaging exposes children to radiation, anesthesia, and imposes financial and emotional burden. objectives: review our institutional experience to determine if afp measurements are sufficient to assess response to treatment and detect relapse. we conducted a retrospective chart review of all patients diagnosed with hb at our institution between - . data collected included serum afp, total number and type of imaging studies during and post treatment, and how relapse or progressive disease was detected. results: thirty-one patients were diagnosed with afp positive hb. during therapy, ct scans were performed: to assess for response to therapy or surgical planning (average scans/patient) and due to concern for progression with rising afp. off therapy, surveillance ct scans were performed (average of . scans/patient) and ( %) included the chest in patients with no lung metastasis at diagnosis. relapsed patients averaged . surveillance scans, . of which were done before relapse was noted on imaging. there were no cases of radiographic evidence of relapse without a prior increase in afp. during treatment, response to therapy based on imaging correlated with a decline in afp in all patients, arguing that repetitive scans are not needed in this setting unless required for surgical planning. only of scans performed during off therapy surveillance displayed evidence of relapse, all of which were preceded by rise in afp. our study represents the largest cohort of hb patients. prior studies suggest similar results, but included fewer patients, lower stage of disease and less than years of surveillance monitoring. at our institution, the cost of a ct c/a/p is $ , with reimbursement varying from - %. in comparison, the cost of an afp measurement is $ . . many scans also require anesthesia and result in emotional toil for families concerned about this procedure as well as the results. thus, afp demonstrates greater sensitivity, with significant cost savings and decreased emotional burden, and should be used for monitoring both during and off therapy, replacing routine serial imaging. background: we observed that our practice of drawing daily blood cultures in hospitalized patients with fever and neutropenia was wasteful; it resulted in excessive negative cultures that did not add to patient care. the smart aim of this quality improvement project was to reduce the number of negative blood cultures drawn on hospitalized patients with fever and neutropenia by % in months. design/method: after reviewing published evidence suggesting drawing daily blood cultures in febrile neutropenic patients was unnecessary, a new blood culture guideline was implemented: cultures were drawn at presentation for fever with neutropenia and, if negative at hours, repeat cultures were not drawn except for clinical change, new fever after being afebrile > hours, or antimicrobials were being changed/broadened. to impact key drivers, we educated staff and changed blood culture order sets to require providers to select a reason for ordering the culture and to eliminate a nursing order to draw daily cultures with fever. we compared the number of blood cultures drawn per central linedays (/ -cld) and the proportion of positive versus negative cultures pre-guideline (july -may ) and postguideline (june -december ). we calculated the cost savings from reducing cultures. to assess patient safety, potential septic events without a corresponding positive blood culture were reviewed. data were analyzed by service (oncology and stem cell transplant). a chi-square test was used to compare rates. in stem cell transplant patients, pre vs. postguideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). in oncology patients, pre vs. post-guideline, there were vs. total cultures drawn/ -cld; vs. positive ( % decrease, p = . ) and vs. negative cultures/ -cld ( % decrease, p< . ). the decreased positive culture rate among oncology patients may be due to decreased culture contaminants and/or the effect of a concurrent initiative to decrease clabsi in that group. there were safety concerns; however, chart review concluded that the guideline did not lead to missed infections in these patients. for the first months of the guideline, the total cost savings in blood cultures was $ , . . the implementation of our new blood culture guideline successfully led to a substantial reduction in the collection of negative cultures and a cost savings without compromising the detection of bacteremia in hospitalized pediatric patients with fever and neutropenia. background: there are various evidence-based guidelines for treatment of adult cancers, such as the nccn guidelines. previously, care was standardized for most new diagnosis pediatric cancer patients through enrollment on a clinical trial. with decreasing clinical trial availability and enrollment and few, if any, evidence-based guidelines for pediatric cancer, care standardization is challenging for pediatric cancers. objectives: to assess consistency of care, as determined by plan of treatment by diagnosis, for pediatric patients receiving chemotherapy for newly diagnosed cancer at a single center. design/method: patients with a new cancer diagnosis at a large, tertiary care pediatric oncology center in calendar year were identified through reports from the chemotherapy order entry (coe) system. reports included diagnosis (recorded through standardized options) and the plan of treatment. chart review was used to exclude patients who started treatment elsewhere and patients being treated for relapse, to clarify diagnosis if the standardized options in coe were unclear, and to clarify treatment plan if needed. data was entered and analyzed in a redcap database. specific diagnoses were clustered into higher level disease groups and the distribution of treatment plans for patients within each was determined. this project was deemed exempt from irb approval for human subject research as a qualifying quality improvement project. of the patients with a first chemotherapy order in , were excluded due to one or more reasons: stem cell transplant ( ), transfer of care ( ), relapse ( ), and other ( ). an additional patients were excluded because < patients/year/diagnosis. there was no cns tumor disease group with > patients. thus, patients with hematologic malignancies or non-cns solid tumors are the focus of this analysis. for patients with intermediate risk rhabdomyosarcoma, the plan of treatment was the standard arm of a cog protocol, arst for patients and arst for subsequent patient after protocol activation. for all other diseases including lymphoblastic leukemia/lymphoma (excluding infants), classical hodgkin lymphoma, aml (excluding trisomy and apml), stage iii/iv burkitt lymphoma/diffuse large b-cell lymphoma, posttransplant lymphoproliferative disease, wilms tumor, rhabdomyosarcoma, ewings sarcoma, osteosarcoma, neuroblastoma, and retinoblastoma, only one treatment plan per risk category was used. conclusion: this analysis demonstrates highly consistent chemotherapy treatment at a single center for patients with hematologic malignancies and non-cns solid tumors. next steps include exploring strategies to group diagnoses for cns tumors and assessing the quality of evidence supporting the treatments given. background: rapid initiation of empiric antibiotics in patients with fever and neutropenia has been shown to reduce morbidity and mortality. current practice guidelines call for the initiation of antibiotics in these patients within sixty minutes and time-to-antibiotic (tta) has been suggested as a quality-of-care measure. many institutions, including our own, face barriers to meeting this time limit. objectives: utilizing a quality improvement model, determine barriers and implement an intervention to reduce the time-to-antibiotics for pediatric febrile patients with suspected neutropenia who present to the emergency department (ed) at our institution. we have identified and implemented an intervention utilizing the plan-do-study-act model for quality improvement. a twelve-month retrospective review was conducted to evaluate the efficacy of the current practice algorithm at our large, academic tertiary-care hospital. subjects identified were pediatric oncology patients undergoing active chemotherapy who presented to the ed with febrile neutropenia. we identified two specific barriers, triage level assignments and delay in ordering antibiotics. to address these barriers, we have created a wallet sized "fever card" that patients were instructed to show upon arrive to the ed. in collaboration with the ed staff, efforts were also made to educate all pediatric staff on the use of the fever card. post-intervention data collection is currently underway and pre-and post-intervention antibiotic delivery times will be compared. the pre-intervention cohort consisted of thirty-three encounters with a mean time-to-antibiotic delivery of minutes, or seventy-five minutes greater than the accepted standard of care. only one patient received antibiotics within sixty minutes of arrival. post-intervention data collection is currently underway. since identifying two barriers to meeting the standard of care at our institution, we have implemented a quality improvement measure that empowers patient families to direct appropriate triage in the ed as well as simplifying the treatment protocol for ed providers. we expect to identify an improvement in time-to-antibiotics from the pre-intervention to the post-intervention period. background: sickle cell disease (scd) is a genetic disorder in which sickle hemoglobin (hbs) triggers multiple downstream effects, including red cell sickling, hemolysis, vaso-occlusion, and inflammation. scd, a lifelong disease initiated at birth with injury that accumulates over time, causes significant end-organ damage and clinical complications that are undertreated and associated with early death. homozygous mutation (hbss) causes the severe form of scd. individuals with scd are at increased risk of infection, stroke, and retinopathy. clinical guidelines for pediatric patients with scd recommend prophylactic penicillin use (ages - ), annual screening for stroke with transcranial doppler (tcd) imaging (ages - ), and annual ophthalmology exams to assess for retinopathy (ages ≥ ). there are limited real-world data on implementation of these nhlbi-based recommendations. objectives: to describe utilization of penicillin, tcd screening, and ophthalmology care in children with hbss disease. medicaid administrative claims databases were used to identify us patients aged - years at first indication of hbss recorded in each calendar year from to . patients were required to have medical and pharmacy benefits for the calendar year in which they were identified and for months prior to their first recorded hbss indication. prior year utilization of penicillin, tcds, and ophthalmologist visits was measured for each annual cohort. annual cohorts included - commercial (mean age . years, % female) and - medicaid (mean age . years, % female) patients with hbss disease. fewer than half of all patients had received a tcd scan in the previous year, with similar rates seen across all age groups for both payers. ophthalmologist visits increased as patients aged, and while patients aged - years had the highest proportion with an ophthalmologist visit in both payer populations, the overall implementation remained low. in contrast to the low use of tcd and ophthalmology visits, penicillin use was highest in the - year age group: > % use in any given year for both payers. conclusion: although our data demonstrated high penicillin use in the - year age group, consistent with guidelines there is an opportunity to improve implementation of other guidelines-based recommended screening. for example, tcd screening can identify children at risk of scd-related stroke in order to initiate preventive therapies. further research to understand potential barriers to proper screening and to evaluate strategies to improve awareness, adherence, and implementation of recommended screenings in children with scd is warranted. supported by global blood therapeutics. background: childhood cancer therapy has improved where there are many long-term survivors. while psychosocial difficulties in pediatric cancer survivors are recognized, the prevalence of these problems at initial survivorship presentation is unclear. objectives: to examine the prevalence of overall internalizing symptoms (e.g., depression/anxiety) in pediatric cancer survivors presenting to a survivorship clinic and to examine how this is mitigated by receiving psychological services and by evidence of parental depression/anxiety. design/method: pediatric cancer survivors attending their first visit at the reach for survivorship clinic at vanderbilt (ages - ) were included. survivors' parents ( % female) completed the child behavior checklist (cbcl), beck depression inventory-ii, and beck anxiety inventory. survivors > years completed a self-report. the wilcoxon rank-sum and pearson's test were used for univariate analyses. the effect size and % confidence intervals (ci) estimated from the multivariable linear regressions were reported. results: childhood cancer survivors a median of years old and . years off therapy were included. thirty one survivors ( %) showed at least borderline clinical internalizing problems (t score > ) on the cbcl, but only of these patients ( %) reported receiving psychological services. nine other survivors with normal t score ≤ also reported receiving psychological services. parental depressive and anxiety symptoms were correlated to the parental report of survivor overall internalizing symptoms (spearman = . , p = < . and = . , p = < . respectively), however they were not correlated to survivor selfreports. furthermore, parents with mild to severe depressive symptoms or mild to severe anxiety symptoms were more likely to rate their child as having higher overall internalizing symptoms (p = . ; p = . , respectively). multivariable linear regression showed that when adjusted for age, gender, cancer diagnosis and time off treatment, reported utilization of psychological services ( = . , % ci [ . , . ],p = . ), and parent depressive symptoms ( = . , [. , . ],p< . ) were significantly associated with child overall internalizing symptoms. in an otherwise identical alternate model substituting parental anxiety for parental depression, parental anxiety was also a significant risk factor ( = . , [. , . ], p< . ). alternatively, parent anxiety/depressive symptoms were not significantly associated with child self-report of internalizing symptoms. childhood cancer survivors have an elevated prevalence of experiencing internalizing symptoms but seldom report receiving psychological services. childhood cancer survivors' parents with anxious/depressed symptoms are more likely to rate their children as having more internalizing problems, compared to patient self-reports. ongoing longitudinal analyses will help clarify the best timing for potential interventions. background: life expectancy for adults with sickle cell disease (scd) has remained unchanged over the past years despite improvements in pediatric scd survival. at greatest risk are the adolescents and young adults (ayas) transitioning from pediatric to adult care. allen county ranks rd in scd incidence among the counties in indiana, and has board certified pediatric hematologist-oncologists. when children "age out" of the pediatric system, there are few providers knowledgeable about managing adults with scd in the region. a novel partnership between hematologists and the family medicine residency program in allen county was initiated to educate family medicine residents (fps) about scd, hydroxyurea (hu), and management of scd-related complications with the goal to increase the number of knowledgeable providers to care for adults with scd. to determine the effectiveness of online learning modules in educating fps about hu, best practices for aya scd care and transition. three online learning modules about scd (comprehensive care of ayas with scd, hu, best practices in aya transition) were developed and cme-accredited. electronic pre-and post-tests were distributed to fps with five questions for each module covering: contraception; screening tests; hu indications, dosing and monitoring; developmental milestones and scd knowledge assessments. the st vincent irb reviewed the protocol and granted a waiver of consent. results: twenty-six fps ( %) completed the pre-and posttests. over two-thirds correctly identified the clinical benefits of hu on both assessments. knowledge about the rationale for hu therapy increased after the completion of the hu module ( % correct on pre-test vs. % on post-test, p = . ). the proportion of correct responses increased for all comprehensive aya scd care post-test questions, but only the leading cause of death and the priapism-related questions reached statistical significance ( % vs. %, p = . ; % vs. %, p = . , respectively). the proportion of correct responses for of the transition-focused questions was unchanged ( % for both), while the proportion of correct post-test responses on the self-care assessment question significantly increased ( % vs. %, p = . ). after module completion, fps were able to correctly identify common scd complications and why hu is an effective treatment for individuals with scd. the best practices of transition clinic module may need modification to improve physician understanding of the intricacies in establishing and maintaining a scd transition clinic. overall, online training is effective at educating fps and could be used to increase the number of providers knowledgeable about scd care. background: survival rates for pediatric hodgkin lymphoma (hl) exceed % with contemporary therapy. studies of pediatric hl survivors treated in the s- s have shown increased risk for treatment-related chronic health conditions. risk-adapted therapy, including tailored radiotherapy, has been developed to reduce long-term morbidity while maintaining excellent survival. little is known about chronic conditions associated with contemporary therapy presenting during the first years from therapy completion (early outcomes). objectives: to analyze survival and early outcomes of pediatric hl patients treated with contemporary therapy. we conducted a retrospective review of hl patients diagnosed < years of age at our institution from - . three-year overall (os) and event-free (efs) survival were calculated with kaplan meier statistics using sas . . results of standardized screening for targeted toxicities that developed between - years from therapy completion were identified and graded per ctcae criteria. censoring occurred at date of death, years from therapy completion, or december , . data from the last collection point were used for prevalence calculations in cases with multiple evaluations. we identified patients ( % male; % non-hispanic white; mean age at diagnosis . ± . years) with a median time since therapy completion of . years (range . - . ). initial treatment included: ( %) chemotherapy only and ( %) multimodality treatment. all patients received anthracyclines (median dose mg/m ) and % received alkylating agents (median cyclophosphamide equivalent dose [ced] mg/m ). the -year os was % with an efs of % ( % chemotherapy only, % multimodality treatment; p = . ). patients with relapsed/refractory disease received salvage treatment including chemotherapy only (n = ), multimodality therapy (n = ), or multimodality treatment including stem cell transplant (autologous n = ; autologous+allogeneic n = ). no patients developed thyroid dysfunction, cardiac dysfunction, subsequent neoplasm, or male gonadal dysfunction during the study period. pulmonary dysfunction was limited to ctcae grade . anti-mullerian hormone (amh) below the normal range was found in / pubertal females who received ced ≥ mg/m compared to / females who received ced < mg/m . two of the females with low amh also had follicle stimulating hormone > iu/ml. this study is the first to evaluate early outcomes in pediatric hl survivors. the results indicate contemporary chemotherapy and a lower rate of radiotherapy utilization lead to excellent -year survival rates with minimal early toxicities. females exposed to ced ≥ mg/m are at increased risk for gonadal dysfunction and should be prioritized for fertility preservation approaches prior to initiation of cancer therapy. background: cancer is one of the leading disease-related causes of death among individuals aged < years in the united states. recent evaluations of national trends of pediatric cancer used data from before , or covered ≤ % of the us population. objectives: this study describes pediatric cancer incidence rates and trends by using the most recent and comprehensive cancer registry data available in the us. design/method: data from us cancer statistics were used to evaluate cancer incidence rates and trends among individuals aged < years during - . data were from states and covered % of the us population. we assessed trends by calculating average annual percent change (aapc) in rates using joinpoint regression. rates and trends were stratified by sex, age, race/ethnicity, us census region, county-based economic status, and county-based rural/urban classification, and cancer type, as grouped by the international classification of childhood cancer (iccc). we identified , cases of pediatric cancer during - . the overall cancer incidence rate was . per million; incidence rates were highest for leukemia ( . ), brain tumors ( . ), and lymphoma ( . ). rates were highest among males, aged - years, non-hispanic whites, the northeast us census region, the top % of counties by economic status, and metropolitan counties. the overall pediatric cancer incidence rate increased (aapc = . , % ci, . - . ) during - and contained no joinpoints. rates increased in each stratum of sex, age, race/ethnicity (except non-hispanic american indian/alaska native), region, economic status, and rural/urban classification. rates were stable for most individual cancer types, but increased for non-hodgkin lymphomas except burkitt lymphoma (iccc group ii(b), aapc = . , % ci, . - . ), central nervous system neoplasms (group iii, aapc = . , % ci, . - . ), renal tumors (group vi, aapc = . , % ci, . - . ), hepatic tumors (group vii, aapc = . , % ci, . - . ), and thyroid carcinomas (group xi(b), aapc = . , % ci, . - . ). rates of malignant melanoma decreased (group xi(d), aapc = - . , % ci, - . -- . ). this study documents increased rates of pediatric cancer during - , in each of the demographic variables examined. increased overall rates of hepatic cancer and decreased rates of melanoma are novel findings using data since . next steps in addressing changing rates could include investigation of diagnostic and reporting standards, host biologic factors, environmental exposures, or potential interventions for reducing cancer risk. increasing pediatric cancer incidence rates may necessitate changes related to treatment and survivorship care capacity. background: while childhood cancer treatment modalities have improved, the delayed effects of cancer treatment continue to compromise the quality of life in survivors. metabolic syndrome (ms) is diagnosed based on the presence of three of the following findings -obesity, dyslipidemia, hypertension and insulin resistance per the world health organization (who) criteria. the increased risk of ms among childhood cancer survivors was first reported in the 's and is known to increase the incidence of cardiovascular disease in these individuals. objectives: assess the frequency of ms in childhood cancer survivors at our institution. . we conducted a retrospective chart review on pediatric cancer survivors, - years of age, who had been treated at sri ramachandra medical institute and research foundation between august and august . patients who received at least one year of treatment with s of s chemotherapy and/or radiation and surgery were included. medical history, family history of diabetes, cardiovascular diseases, and hypercholesterolemia, tanner staging, weight for height (< y per who criteria), bmi (> y per indian academy of pediatrics iap), blood pressure (nhlbi criteria), fasting blood sugar levels and lipid profile were obtained from the charts. statistical analysis of the data was done using ibm spss statistical software (version ). results: patients were studied, . % were male. . % were under years of age, . % between - years and . % above years. leukemia survivors comprised . % of the sample and non-leukemic's were . %. . % were treated with chemotherapy alone, . % with radiotherapy and chemotherapy, and . % underwent surgery with radiotherapy and chemotherapy. hypertension was found in . % of the study group, dyslipidemia in %, impaired fasting blood glucose in . % and . % were found to be obese. % of the study group was diagnosed with ms based on who criteria. conclusion: % of our study population was found to have ms per who criteria. individual metabolic complications were detected in % of the population. acute lymphoblastic leukemia (all) survivors appeared to be at high risk in our population. ms has been known to increase cardiovascular complications in cancer survivors. a multidisciplinary team approach to management of these patients is important to closely monitor and manage the long-term complications related to ms such as type diabetes and atherosclerosis. such an approach is essential to decrease long term morbidity and mortality from ms in this vulnerable population. background: the -year survival rate for childhood cancer exceeds %. however, up to % of these children require admission to the pediatric intensive care unit (picu) within three years of diagnosis. these children account for approximately % of all picu deaths, with mortality being higher for those post-hematopoietic stem cell transplant (hsct). national guidelines recommend that providers share informa-tion regarding prognosis and treatment options within the first hours of icu admission. these prognostic goals of care conversations (pgocc) are critical to the care of children with malignancies, a subpopulation at risk for increased mortality. to determine the frequency of pgocc as well as describe differences in patient characteristics and critical care therapies by pgocc status. design/method: a retrospective cohort study was conducted using the university of michigan virtual picu system database. picu admissions lasting longer than hours for patients ages to years between july , and june , with an oncologic diagnosis and/or hsct were identified. data on pgocc, patient demographics, diagnoses, picu interventions, and outcomes were recorded and compared between children with pgocc and those without using chi square test for categorical variables and kruskal-wallis test for continuous data. of picu admissions, % were male; the mean age was . years. the leading diagnoses were acute lymphoblastic leukemia ( %), acute myeloid leukemia ( %), lymphoma ( %), neuroblastoma ( %), and brain tumors ( %), and % of patients were post-hsct. pgocc was documented in ( %) patients. in comparison with patients who did not have a pgocc, children with a pgocc were more likely to be readmitted to the picu ( % vs. %, p < . ) and more likely to have had relapse of disease ( % vs. %, p< . ). patients with a pgocc had higher severity of illness scores (p = . ), higher use of non-invasive ( . % vs. . %, p = . ) and invasive conventional ventilation ( . % vs. . %, p< . ), and high frequency ventilation ( . % vs. . %, p < . ). also, patients with pgocc were more likely to receive continuous renal replacement therapy ( . % vs. . %, p< . ), arterial catheterization ( . % vs. . %, p< . ), and cardiopulmonary resuscitation ( . % vs. . %, p< . ). in only in critically ill children with hematologic-oncologic disease is pgocc held. children with pgocc were sicker and received more critical care interventions. future research is needed to evaluate the content of pgocc. background: central nervous system (cns) tumors and autism spectrum disorder (asd) represent significant disease cohorts in the pediatric population. asd diagnoses in children have a prevalence of %, in every children in the united states. additionally, more than , cns tumors are reported in children age to years in the united states with brain tumors being the most common solid tumor and the leading cause of death among all childhood cancers. the genetic etiology of autism and cns tumors is complex. specific gene alterations present in certain cancers have similarly been described and suspected to play a role in asd subtypes. targeted therapy panels, like foundation one (fo), have been beneficial in guiding treatment for some cancers based on distinct gene alterations. given the genetic overlap, the potential for therapeutic benefit and crossover from such actionable gene target panels merit further exploration in asd and cns tumors. we aim to identify and describe genetic alterations with known actionable targets in cancer therapy from fo as potential diagnostic, therapeutic and research targets for neurodevelopmental diseases. we plan to discuss the common genetic alterations between our cancers and neurodevelopmental diseases described in the literature. fo data was extracted and compared to the literature. each reported gene alteration from fo plus the keywords "autism", "psych" were used on pubmed to search for a suspected association if any with a neurodevelopmental disorder. results: twenty-one patients representing a cohort of six unique (astrocytoma-five, ependymoma-six, gbm-four, glioma-three, nerve sheath tumor-one, etmr-two) cns tumors were investigated. fo produced eighty total with sixty unique gene alterations. thirty-one ( %) of these yielded at least one published, suspected association to a neurodevelopmental disorder. the most common gene alterations were tp -four, cdkn a/b-five and braf-four. the main functional categories were cellular: proliferation, structure, differentiation and degradation; chromatin modeling; histone transcriptional modification; dna methylation and repair; strna; and neural signaling. sixty unique gene alterations were found in our cns tumor set using foundation one. thirty-one ( %) of these discrete alterations paired with at least one description in the literature as having been similarly altered in an asd subtype. many of these alterations have actionable targeted therapies presented through foundation one for our cns tumors and may be a relevant guide in the future of targeted therapy and research in asd subtypes. monoclonal antibody therapy usage is associated with significantly improved survival in b-cell nhl aya patients. although the usage has increased in the aya population from to , the magnitude of the increase is low. factors that affect the use of mab include race and insurance s of s type. further research is warranted to identify why privately insured patients are less likely to receive these drugs. background: prevention of chemotherapy-induced nausea and vomiting (cinv) remains a challenge despite advances in pharmacotherapy and the development of cinv clinical practice guidelines by the pediatric oncology group of ontario (pogo) that have been endorsed by the children's oncology group. achieving control of cinv in pediatrics further is complicated by the difficulty young children have vocalizing their symptoms. use of a validated nausea-assessment tool in conjunction with improved adherence to evidence-based guidelines may result in better quantification of symptoms and reduction of both nausea severity and vomiting frequency for pediatric patients undergoing chemotherapy. the pediatric nausea assessment tool (penat) has been validated for children ages - , and its integration into clinical practice may help optimize cinv control. objectives: this single-institution study sought to improve control of cinv in patients admitted for chemotherapy by standardizing the antiemetic regimens prescribed by all providers according to an institutional cinv algorithm developed from the pogo guidelines. we hypothesized that treatment using a standardized guideline would improve cinv control in patients admitted for chemotherapy. a baseline cohort of admissions for chemotherapy completed penat assessments and cinv diaries prior to receiving chemotherapy, four times daily during each admission, and daily for days following completion of chemotherapy from may , to january , . providers then were provided an institutional cinv treatment algorithm based on the pogo guidelines and received education at departmental meetings on appropriate implementation of this algorithm. a second cohort of admissions completed penat assessments and cinv diaries in a similar fashion from july , to december , . results: complete control of vomiting markedly improved following cinv guideline implementation ( % vs %, p <. ) with treatment failure also significantly reduced ( % vs %, p <. ). after controlling for the degree of emetogenicity of chemotherapy received, a patient was . times more likely to vomit prior to guideline implementation (or . , ci . - . ). there was no difference in nausea control, even after adjusting for the emetogenicity of chemotherapy. conclusion: control of chemotherapy-induced vomiting (civ) improved following widespread implementation of an institutional cinv treatment algorithm at a single institution. the severity of nausea reported remained unchanged which may reflect the difficulty of assessing nausea or an inadequate sample size. future research may focus on cinv treatment management through the use of guidelines specifically for breakthrough cinv and delayed cinv. background: aspho's professional development committee (pdc) recognized pediatric hematologists-oncologists (phos) serving in the united states (us) military have unique professional development needs that may not be addressed by aspho or a similar professional society. these individuals may also encounter challenges when transitioning to a civilian career. however, barriers to professional development have not been systematically characterized. the objectives were to characterize the number of phos with current or prior military service (mphos) and to identify any unmet professional development needs. design/method: a working group consisting of pdc members and both senior and early career mphos was formed. initial comments were solicited by email from known mphos regarding potential gaps in professional development and interest in working with aspho to improve support of mphos. a survey was developed and piloted with four members of the advisory group, questions were revised based on their feedback, and a final version was distributed via the aspho website and online community forum. targeted emails were sent to mphos identified through aspho and military databases. eligibility to complete the survey included ) completion of a fellowship in pediatric hematologyoncology, and ) current or prior service as an active duty military provider. quantitative and qualitative information were collected, including demographic data and perceived barriers to professional development. responses were summarized using descriptive statistics. results: sixty-five mphos were identified and surveys were completed for a % response rate. respondents were engaged in a variety of professional activities; % were male, % were serving active duty commitments, and % felt there were professional development gaps. areas of concern were categorized into nine themes with the most concerning being ) limited civilian knowledge of mpho practices ( % of participants), ) inability to attend professional society meetings ( %), and possibility of deployment ( %). participants expressed a desire for educational products to meet their specific needs and for networking opportunities with civilian colleagues. qualitative analyses identified concerns about low patient numbers and practice size. a subset of mphos perceive significant gaps in professional development. additional research is needed to better define areas for intervention, but many of the concerns align with those of similarly sized civilian programs and may be addressed through professional society networking opportunities, such as an aspho special interest group. background: infertility is an established cause of distress and has a negative impact on quality of life among childhood cancer survivors. the american society of clinical oncology has established guidelines on fertility counseling for individuals of reproductive age diagnosed with cancer, with the goal of improving reproductive and psychosocial outcomes. studies have shown that instituting a fertility team that can provide counseling and discuss fertility preservation (fp) options results in improved patient satisfaction in patients with cancer. objectives: the goal of this study was to examine predictors of referrals to the multidisciplinary fertility team, and documented fp interventions among these patients. design/method: an irb-approved retrospective medical record review was performed at a large pediatric academic center. all patients with new cancer diagnoses receiving chemotherapy were included from january (when the fertility team was established) to present. a standardized abstraction form was used to collect information about: age at diagnosis, gender, cancer type, whether a fertility consult was placed, and documented fp interventions. data were summarized descriptively and comparisons were made using nonparametric statistical methods. results: patients met inclusion criteria, of which ( %) were male. cancer types were as follows: leukemia/lymphoma, cns tumors, sarcomas, embryonal tumors, and langerhan's cell histiocytosis (lch). the mean age was . years, (range < - years). overall, % of all patients had a consultation with the fertility team. patients were significantly less likely to have a fertility consult if they were younger (p< . ). further, there were differences in the consultation rate between diagnoses, with % of sarcoma patients completing a consult, compared to % of those with cns tumors, % of those with embryonal tumor, % of those with leukemia/lymphoma and none of the patients with lch. our findings show that many children, adolescents, and young adults newly diagnosed with cancer are still not receiving fertility counseling despite: ) an expanding body of literature supporting the need to provide this counseling, ) guidelines published by several organizations recommending discussions about infertility risk and fp options, and ) presence of a multidisciplinary fertility team. specific strategies need to be developed to improve access for younger children, and for disease groups in whom fertility consults are underutilized, such as youth with cns tumors, embryonal tumors, and leukemia/lymphoma. background: socioeconomic status (ses) has on impact on overall survival in the pediatric oncology population. unfortunately, data are insufficiently detailed to explain the mechanism behind this phenomenon. how parents handle the health management demands placed on them at the time of a child's cancer diagnosis may represent a point of differentiation in health outcomes. objectives: determine the association between socioeconomic factors, cancer literacy, and parents' understanding of home emergency management and their responses to instances of pain, nausea, and fever. in a prospective observational study of parents whose children were newly diagnosed with cancer, we obtained demographic information and, using a validated instrument, (dumenci, ) we evaluated cancer literacy. we tested understanding of the education parents received about home emergency management with a -item multiple-choice vignette-based questionnaire focused on actions needed in home scenarios. we then followed parents' actual behavior through periodic phone calls assessing instances of nausea, pain, and fever and their responses to these episodes. results: preliminary analysis of participants showed an average score of on the -item parental understanding questionnaire (range - ). variables associated with increased score were college-level education by . points ( % ci [. to . ]), private insurance by . points [. to . ] and adequate cancer literacy by . points [. to . ]. actual behavior reported by families indicated that married parents and those with income above $ , were less likely to treat instances of pain by % ( % ci [ to ]) and % [ . to ], respectively. white parents, those with college-level education, and those with adequate cancer literacy were less likely to treat instances of nausea by % [ to ], % [ to ] and % [ to ], respectively. no associations were found between socioeconomic markers and parental responses to instances of fever. our findings suggest an association between demographic and socioeconomic markers and improved parental understanding of home emergency management. paradoxically, the same markers show a decrease in treatment response to pain and nausea. larger prospective studies are needed to link this behavior pattern to health outcomes, and help inform the extent of ses impact on home emergency management. emory university/children's heathcare of atlanta, atlanta, georgia, united states background: cardiovascular disease is a leading cause of morbidity and mortality in childhood cancer survivors (ccs). previous research showed wide practice variation in referral patterns to cardiology from the survivor clinic and in recommendations from cardiologists about the need for further testing or exercise restrictions. to develop a cardio-oncology algorithm in order to standardize referrals to cardiology and provide guidelines for cardiologists evaluating pediatric ccs. design/method: survivorship and cardiology experts developed a weighted scoring system for pediatric ccs who received cardiotoxic therapy based on time since treatment and risk factors identified by the children's oncology group (cog) and american heart association (aha). the cardiooncology algorithm assigned a score of - . the score range was categorized to guide cardiology referral: screening echo only ( - ), consider cardiology referral ( - ), recommend cardiology referral ( - ), and regular cardiology follow-up (≥ ). the algorithm also provides recommendations to cardiologists for screening and exercise modifications based on the score. after establishment of the algorithm, a convenience sample of institutional survivor clinic patient charts were retrospectively reviewed from the first month of each quarter from april -march to validate the algorithm, evaluate referral patterns to cardiology, and assess cardiology recommendations. the retrospective chart review evaluated patients ( % male; % non-hispanic white; % leukemia survivors; median age at diagnosis . years [range - . ]; median time off-therapy . years [range . - . ]). patients ( %) received anthracyclines (median dose mg/m , range - ) and ( %) received cardiac radiation. assigned cardio-oncology scores resulted in: % echo only, % consider cardiology referral, % recommend cardiology referral, and % regular cardiology followup. when evaluating detection rates of late effects by cardiooncology score, survivors ( %) had an abnormal echo: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. assessing referral patterns prior to initiation of the algorithm revealed forty-two survivors ( %) referred to cardiology: / echo only, / consider referral, / recommend referral, and / regular cardiology follow-up. of the patients seen by a cardiologist at our institution, had further diagnostic testing ordered (i.e., stress test) and received exercise restrictions. a cardio-oncology algorithm and guidelines will standardize cardiac care for survivors by assigning a score to guide referral and cardiology practice after referral. prospective clinical use has begun and review will occur in one year to determine changes in detection rates of cardiac late effects, referrals, and recommendations from cardiologists. oregon health and science university, portland, oregon, united states background: delirium affects - % of patients (pts) in pediatric intensive care units (picu) and is associated with increased length of stay, decreased attention in school, and post-traumatic stress disorder. the diagnostic and statistical manual of mental disorders (dsm v) defines delirium as a "disturbance of consciousness […] with reduced ability to focus, sustain or shift attention" due to an underlying medical condition. despite the medical complexity of the hospitalized pho population, there are no published prospective studies looking at delirium in these pts. hypothesizing that delirium is under recognized in the pho population, we designed a year-long prospective study using a validated screening tool to determine the frequency of delirium in hospitalized pho pts and to identify associated clinical factors. design/method: baseline frequency of pts with symptoms suggestive of delirium was determined through retrospective chart review using a data mining program of electronic medical records (emr). for the prospective study, pho and picu nurses were trained to use the cornell assessment for pediatric delirium and to record scores within the emr on all pho pts once every -hour shift. predetermined demographic and clinical variables were entered daily into a red-cap database on all hospitalized pho pts. results: baseline frequency of delirium, without active screening, was determined to be . % of hospitalized pho pts. in the first months of the prospective study, consecutive admissions occurred among unique pho pts: oncology, hematology, and stem cell transplant pts. pts had at least positive delirium screen, for a prevalence per admission of . %. statistically significant variables associated with delirium, at p < . by univariate logistical regression, included prolonged length of stay, pt location (picu vs pho unit), and fever. adjusting for length of stay, administration of benzodiazepines and opiates were also significantly associated with delirium, p = . and . , respectively. on average, nurses completed delirium screening in % of each pts' -hour shifts. study accrual ends in jan and final data analyses will be reported in the abstract presentation. conclusion: delirium does occur in the pho hospitalized population and screening by trained nursing staff is feasible. pts at highest risk appear to be pts with prolonged hospital stays, picu admissions, or frequent use of benzodiazepines/opioids. routine screening should improve our recognition of delirium and allow us to promptly intervene, or prevent delirium in an effort to avoid potential acute and long term consequences. background: with high survival rates for children and adolescents with hodgkin lymphoma (hl), treatment regimens are now designed to maximize cure while decreasing risk of long-term health outcomes associated with chemotherapy and radiation therapy. within contemporary treatment regimens, the comparison of toxicities experienced by patients receiving chemotherapy plus radiotherapy (crt) versus only chemotherapy (co) has not been studied extensively. objectives: this study examines select self-reported adverse health outcomes in survivors of contemporarily-treated pediatric hl to better understand the balance between efficacy and toxicity associated with chemotherapy and radiation therapy. (cog) ahod that evaluated a response-based treatment paradigm in pediatric hl. patient who received initial chemotherapy were randomized based on early response to continued chemotherapy, chemotherapy plus radiotherapy or augmented chemotherapy plus radiotherapy. patients completed self-report questionnaires on health problems at , , , and years following therapy. we examined selected patient-reported pulmonary, gastrointestinal (gi), cardiac and endocrine outcomes. kaplan-meier survival curves were used to determine probability of survival without the selected adverse health outcome. log-rank tests were used to compare the co versus the crt group. results: a total of , enrolled patients, patients in the co group and patients in the crt group, completed , questionnaires at a median of . years after s of s completion of therapy (q , q : . , . ) which were analyzed. the cumulative -year incidence of endocrine dysfunction was significantly greater in the crt group versus those in the co group ( % versus %; p< . ), driven by the incidence of hypothyroidism ( % versus %; p< . ). there were no significant differences in cardiac ( % versus %; p = . ), pulmonary ( % versus % p = . ), and gastrointestinal dysfunction ( % versus %; p = . ) between the co and crt patients. conclusion: this study demonstrates low cumulative incidence overall of organ dysfunction early post completion of contemporary therapy for hl. the addition of radiation therapy significantly increased risk for hypothyroidism, but with no higher risk noted for cardiac, pulmonary or gi dysfunction. limitations include self-report status, potential selection bias, and relatively short latency period following end of therapy. longer follow-up is needed to determine more delayed risks for organ dysfunction in order to best define the balance between therapeutic efficacy and long-term adverse health outcomes related to chemotherapy and/or radiation therapy. background: identification of an organism via bronchoalveolar lavage (bal) or respiratory tract biopsy (rtb) has historically been considered the gold standard for diagnosis of invasive fungal infection (ifi); however, data previously published by our group showed that these procedures infrequently lead to a change in management in children with an oncological diagnosis or undergoing hematopoietic stem cell transplant (hsct). there is also a paucity of data on the cost of ifi in this population. to compare the costs of work-up and management of pulmonary ifi diagnosed based on ct scan alone versus ct scan or chest x-ray prompting a bal or rtb. design/method: we collected cost data on patients at ann & robert h. lurie children's hospital of chicago undergoing chemotherapy or within months of hsct who were suspected of having an ifi between and . in order to include sufficient time to account for post-procedure compli-cations but avoid including costs unrelated to ifi, data were included for days from the day of their diagnostic scan or procedure. cost data was available for of the patients previously studied. thirty-six of these patients were diagnosed with suspected ifi based on ct only and patients underwent bal or rtb. when evaluating specific costs, inpatient beds costs were higher in the bal and rtb group (median $ , versus $ , , p = . ), yet there was only a trend towards higher costs for antifungal agents (median $ , versus $ , , p = . ) and respiratory support (median $ versus $ , p = . ). many of the initial ct scans were not captured in the -day evaluation period for the bal or rtb group based on the study design; however, even when accounting for ct scans up to a week prior these procedures, the total cost of ct scans was higher in the ct only group (median $ versus $ , p = . ), as they had more scans. despite this, total costs were significantly higher for patients who underwent bal or rtb versus ct scan only (median $ , versus $ , , p < . ). combined with our previous data that bal and rtb infrequently leads to a change in management in children with an oncological diagnosis or undergoing hsct suspected to have an ifi, the significantly higher costs associated with these procedures makes these invasive diagnostic techniques even less desirable. batra, pediatr blood cancer, . background: while infants > months of age with acute lymphoblastic leukemia (all) have a poor prognosis, infants with acute myeloid leukemia (aml) fare better despite more intensive therapy. there are limited data on this difference, particularly differences in supportive care requirements during induction therapy for infants. objectives: to compare induction mortality and resource utilization in infants relative to non-infants aged < years, separately for all and aml. design/method: we used previously established cohorts of children treated for new onset all or aml at children's hospitals in the us contributing to the pediatric health information system. patients with down syndrome were excluded. follow-up started on the first day of induction chemotherapy and continued until the earliest of: days after commencement of chemotherapy, start of the subsequent course, or death. high acuity of presentation, defined as icu requirements involving or more organ systems within the first hours following initial admission were compared using log binomial regression. -day inpatient mortality was compared using cox regression. resource utilization rates (days of use per inpatient days) were compared using poisson regression. results: a total of all ( infants, non-infants) and aml ( infants, non-infants) were included in the analyses. infants were more likely to present with high acuity compared to non-infants for both all ( % and %, rr = . , % ci: . , . ; p< . ) and aml ( % vs %; rr = . , % ci: . , . ; p = . ). infants with all had higher inpatient mortality compared to non-infants even after accounting for differences in acuity of presentation ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ). in contrast, inpatient mortality was more similar for infants and noninfants with aml ( . % vs . %, adjusted hr = . % ci: . , . ; p = . ) and comparable to rates among infants with all. infants with all and aml had higher rates of utilization of fresh frozen plasma, cryoprecipitate, diuretics, supplemental oxygen, and ventilation relative to non-infants. infants with all also had higher rates of total parenteral nutrition, ecmo, and patient controlled analgesics compared to noninfants. infants with all experienced significantly higher induction mortality compared to noninfants, a difference not entirely explained by acuity at presentation. differences in ru among infants may reflect higher presentation acuity and greater treatment related toxicity. further work is needed to elucidate the contribution of treatment related toxicity to early mortality in infants with all. background: fever in a child with cancer is a medical emergency due to the significant risk of a serious bacterial infection. many attempts have been made to risk stratify these patients. the respiratory pathogen panel (rpp) is a panel of polymerase chain reaction tests that identify seventeen common respiratory viruses and three bacterial infections. samples are taken via nasopharyngeal swab. rpps are frequently sent, but we do not have data to determine whether a positive result can lead to stratification to a lower risk of bacterial infection. ( ) to determine the epidemiology of respiratory virus-associated fever in pediatric oncology patients ( ) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all pediatric oncology patients seen in our emergency department (ed) with fever from the introduction of the rpp in april to september , . we reviewed the results of blood cultures, rpp, chest radiographs, and discharge summaries to identify sources of infection. we also identified the patients' cancer diagnosis, age, absolute neutrophil count (anc), and absolute lymphocyte count (alc). results: positive rpps were found among pediatric oncology patients who presented to the ed with fever. the most common positive rpp findings were rhinovirus/enterovirus (rev) ( %), parainfluenza ( %), influenza ( %), coronavirus ( %), and polyviral ( %). among patients with a positive rpp, % had bacteremia compared to % bacteremia among all pediatric oncology patients with fever (or . [ . - . ], p . ). all cases of bacteremia were associated with rev. there was no bacteremia identified in patients with rpps positive for other viruses (or . [ . - . ], p . ). rev positivity did not confer a lower risk of bacteremia than rpp negative patients ], p . ). anc (p = . ) and alc (p = . ) less than , and number of patients with severe neutropenia (p = . ) were not statistically different between the rev and non-rev positive rpp groups. rpps positive for viruses other than rev reduced the likelihood of bacteremia in febrile pediatric oncology patients in the ed setting. patients with bacteremia may have concurrent infection with rev. a larger study is warranted to determine if positive rpp results can inform clinical management of a child with febrile neutropenia. emily mueller, anneli cochrane, seethal jacob, aaron carroll s of s background: the usage of mobile health (mhealth), which refers to the application of mobile or wireless communication technologies to health and healthcare, has grown exponentially in recent years. mhealth tools have been used by caregivers of other vulnerable populations, but little has been focused on caregivers of children with cancer. objectives: to conduct a survey to understand the mobile technology usage, barriers, and desired mhealth tools by caregivers of children with cancer. we conducted a mailed cross-sectional paper survey of caregivers of all children who were diagnosed with cancer at riley hospital for children between june, and june, . the survey contained questions, both fixed and open-ended, in both english and spanish. up to three rounds of surveys were sent to those who did not respond. of the respondents, they were primarily parents ( . %), median age was . years (range - ), and most were white ( . %) and non-hispanic/latino ( . %). the top three annual household income brackets included $ , to $ , ( . %), $ , to $ , ( . %) and under $ , ( . %). the majority had an education: . % college graduates, % graduate degree, and . % high school education or ged. nearly all respondents owned a smart phone ( . %) and . % owned a tablet. the majority used an ios operating system ( . %), while . % reported use of a device with an android operating system. all caregivers reported use of at least one mobile website/app regularly for their personal use. while . % of respondents reported no barriers to mobile technology use, the top barrier selected was "data limitations" ( . %). overall, . % wanted at least one medical managementrelated website/app: medical knowledge ( . %), healthcare symptom tracking/management ( . %), and medication reminders ( %). healthcare system-related desires were high, as . % wanted access to their child's medical record and . % wanted a website/app to facilitate better communication with medical providers. there were no significant associations between socioeconomic status (income or education) with barriers or types of websites/apps desired by caregivers. since the vast majority of caregivers use mobile technology with minimal barriers, future research should focus on designing an mhealth tool to address the medical management needs by caregivers of children with cancer. by supporting caregivers through this type of mhealth tool, it could positively impact patient clinical outcomes through greater adherence to medications and treatment protocols. background: in children with fever and neutropenia, early initiation of targeted antibiotic therapy improves outcomes, yet there are no standards for choice of empiric antibiotics. in our institution implemented an early empiric ceftriaxone (eec) protocol to reduce time to antibiotic administration in febrile hematology-oncology patients who are potentially neutropenic when the absolute neutrophil count is not yet know. ceftriaxone is given immediately after obtaining blood for culture and lab studies. in patients found to be neutropenic, ceftriaxone is discontinued and cefepime is initiated. the purpose of this retrospective study was to evaluate our eec protocol in neutropenic patients by assessing ceftriaxone sensitivity of positive blood cultures and comparing rates of adverse outcomes with a cohort of patients treated prior to implementation of the protocol. we are now conducting a prospective study to more thoroughly investigate antibiotic sensitivities of organisms isolated from blood cultures of neutropenic patients. design/method: hematology-oncology patients with at least one positive blood culture between january and december were identified. patient demographics, neutrophil count, antibiotic treatment, isolated organisms and sensitivities, and adverse outcomes (increased respiratory support, hypotension requiring intervention, and icu admission) were obtained by retrospective chart review. fisher exact test was used to compare dichotomous variables between patient groups. we are now prospectively identifying febrile neutropenic patients with positive blood cultures and performing antibiotic sensitivity testing to several antibiotics commonly used as empiric therapy for febrile neutropenia. results: retrospectively, we identified neutropenic patients with a total of bacterial isolates from blood cultures. of organisms isolated, were tested for sensitivity to ceftriaxone and ( %) were not sensitive, / ( %) of gram-positive cultures and / ( %) of gram-negative cultures. ten of ( %) eec patients had an adverse outcome versus / ( %) of non-eec patients (p = . ). notably, % of eec patients required icu admission versus % of non-eec patients (p = . ). thus far our data obtained prospectively is revealing similar rates of ceftriaxone resistance with / cultures not sensitive to ceftriaxone ( %, ci . %- . %). in our retrospective study, no statistically significant difference was seen in overall adverse outcome rate between the two cohorts, though icu admission rates were significantly higher in eec patients. ceftriaxone resistance rates were high in tested isolates, which is further supported by preliminary data from our ongoing prospective study. given these data, eec may not be effective at improving outcomes in febrile neutropenic pediatric hematology-oncology patients. background: approximately in children diagnosed with cancer will die of their disease, despite advances in treatment. results: two focus groups of six parents each met in june . the parents were predominantly female ( female, male) and had lost their children an average of . years prior (range - . years). two parents were in the same family. nearly all patients were offered palliative care ( / ), all were offered hospice and most died at home ( at home, in the icu). parent discussion uncovered six broad themes: beneficial provider qualities, optimal communication, helpful systematic supports, struggles to feel like a good parent, struggles with a loss of control and unmet needs. parents appreciated providers who were consistent, reliable and honest. parents desired communication that was sensitive to the needs of the patient and family with a balance of hope and realism. parents appreciated the tangible supports pro-vided by social work and the emotional support of child life both for the patient and their siblings. some parents struggled to define and advocate for their child's quality of life, especially when it led to disagreeing with the medical team. several parents expressed frustration with unfamiliar caregivers in the hospital, especially trainees. they expressed a strong desire for more anticipatory guidance about the end of life including how to discuss it with their children. they also wished for a cancer-specific support group for bereaved parents. conclusion: bereaved parents of pediatric oncology patients in our focus groups appreciated consistent, reliable providers who communicated with a balance of realism and hope. they appreciated the tangible and emotional support they received and wanted more anticipatory guidance at the end of their child's life. these results can help guide clinical care, especially in communities without strong palliative care support. further research is needed to develop interventions to improve end of life care. background: clinical trials involving human subjects depend on informed consent (ic) to ensure ethical protections for participants. parents of children with cancer often lack full understanding of the basic elements of ic for clinical trials. additionally, the stress of their child's cancer diagnosis may affect their decision-making capabilities. this is especially problematic as these children rely on parents to fully comprehend clinical trials and weigh their benefits and risks. physician communication is critical for effective family-centered care. the acgme mandates that training programs teach and assess trainees' communication skills. however, there are currently no published curricula aimed at training pediatric hematology/oncology fellows to deliver ic effectively for cancer clinical trials. to develop and pilot-test a simulation-based curriculum to enhance communication skills of pediatric s of s hematology/oncology fellows in the delivery of ic for cancer clinical trials. we developed, tested, and implemented the curriculum from to in two phases. in phase- , we reviewed literature on simulation-based curricula and completed a needs assessment to create a clinical scenario and full curriculum using standardized patients. using miller's pyramid model, fellows' assessments included: immediate de-brief, surveys to assess pre/post confidence and knowledge of the basic ic elements ("knows" and "knows how"), and -degree summative assessments compiled from fellow self-assessments, faculty, and standardized patients ("shows how"). after initial testing and refinements done with fellow, in phase- , we implemented the curriculum with our fellows. likert scale ( strongly disagree- strongly agree) and basic p values are reported. results: fellows gave high mean ratings for training relevance ( . ) and standardized patients' preparedness ( ). almost all ( . ) reported they have used the knowledge gained in their clinical practice. increase in self-reported confidence (pre/post) was noted in all domains: general -describing possible benefits of the clinical trial . / vs. . / (p = . ), risks and potential side effects . / vs. . / (p = . ), and explaining alternatives . / vs. . / (p = . ); research -discussing purpose of the clinical trial . / vs. . / (p = . ), and randomization . / vs. . / (p = . ); and family-centered -addressing emotions during ic . / vs. . / (p = . ), and delivering bad news . / vs. . / (p = . ). summative evaluation mean ratings for all fellows were . (range . - . ). our novel simulated-based ic curriculum, significantly increased fellows' self-reported confidence and skills during ic delivery. importantly, our ic curriculum addressed not just research-related content but also management of parental emotional needs during the ic discussion. next phase includes kirkpatrick model program evaluation and dissemination across other training programs in our institution. national kaohsiung normal university, kaohsiung, taiwan, province of china background: taiwan's childhood cancer foundation reported in that the -year survival rate of childhood cancer was %. as a result, many childhood cancer survivors were back in school after treatment. however, childhood cancer survivors' educational outcomes suffered because of their long-term absence from school and late effects of cancer and cancer treatment. a few school reentry protocols have been developed by the nursing professionals in taiwan to facilitate students' return to school but remained experimental in nature and hardly accessible. parents, students, and teachers were left to their own devices to make individual school reentry plans. objectives: this study aimed to examine and uncover the commonalities among three middle school students' successful school reentry experiences from their teachers' perspectives and to analyze the factors contributing to their success. design/method: this is a qualitative interview study. indepth semi-structured interviews were conducted with three middle school teachers in december about their perceptions, observations, and experiences working with adolescent childhood cancer survivors. the students were two boys with leukemia and one girl with bone cancer. they were diagnosed in the first year of middle school when they were - years old and returned to school for the third and the final year. these students met the following criteria for successful school reentry: regular school attendance, average/above average academic performance, friendship maintenance, and high school diploma. the theme -bring the class to the hospital was found to be the key to the adolescents' successful return to school. without a prescribed school reentry protocol and in the face of limited bedside education services, the homeroom teachers, as links between school, home, and hospital, brought the class to their hospitalized students. they doubled as bedside teachers conducting lessons at the hospital or students' homes, became friends with the parents, witnessed firsthand the students' pain and triumph during treatment, brought the students back to school for visits and celebrations, delivered the classmates' wishes and news to the students, encouraged and welcomed classmates' visits to the hospital, and, together with parents and other teachers, developed flexible school reentry schedules for the students. this on-going study demonstrated the critical roles and functions of homeroom teachers in successfully bringing the students back to school during and/or after cancer treatment. further analysis will be focused on how and why these three homeroom teachers were able to carry out this unexpected task on top of their already full workload. jennifer kesselheim, shicheng weng, victoria allen, collaborative group fellowship program directors dana-farber/boston children's cancer and blood disorders center, boston, massachusetts, united states background: a novel, -module, case-based curriculum entitled "humanism and professionalism for pediatric hematology-oncology" (hp-pho) aims to foster pho fellows' reflection on grief and loss, competing demands of fellowship, difficult relationships with patients and families, and physician well-being and burnout. in small group facilitated sessions, fellows work to identify coping strategies and explore how the challenges of fellowship influence both their own doctoring and the patient experience. objectives: to administer the hp-pho curriculum in a prospective, cluster-randomized trial, measuring whether exposure to this educational intervention, compared to standard conditions, fosters humanism and professionalism and improves satisfaction with training. design/method: pho fellowship programs (n = ) were cluster-randomized to deliver usual training in humanism and professionalism (control) or the novel curriculum (intervention) during the - academic year. the primary outcome measure was the pediatric hematology-oncology self-assessment in humanism (phosah). secondary measures included a -point satisfaction scale, the maslach burnout inventory (mbi), the patient-provider orientation scale, and the empowerment at work scale. participating fellows were pre-tested in summer and post-tested in spring . a change score was calculated for each study instrument. we compared each outcome between arms using mixed effect models adjusted for pre-test score as a fixed effect and site as a random effect. results: randomization yielded intervention and control fellows. the two arms did not significantly differ in distribution of fellow age, gender, or post-graduate year. the intervention sites successfully administered of ( %) modules. change scores on the phosah were not significantly different between the control and intervention arms (adjusted mean difference = . ; % confidence interval [ci] - . , . ; p = . ). compared to the control arm, fellows' exposed to the curriculum gave significantly higher ratings on several items within the satisfaction scale including satisfaction with their training on "physician burnout" (adjusted mean difference = . ; % ci . , . ; p< . ), "physician depression" (adjusted mean difference = . ; % ci . , . ; p< . ), "balancing professional duties and personal life" (adjusted mean difference = . ; % ci . , . ; p = . ), and "humanism overall" (adjusted mean difference = . ; % ci . , . ; p = . ). change scores on other secondary measures were not significantly different between study arms. conclusion: exposure to the hp-pho curriculum did not alter fellows' self-assessed humanism and professionalism. however, the curriculum proved feasible to administer and intervention fellows expressed higher levels of satisfaction in their humanism training, indicating the curriculum's positive impact both for fellows and their learning environment. background: recent work has documented significant levels of unmet needs among adolescents and young adults with cancer, particularly psychosocial challenges during the transition to adulthood, (e.g., abrupt disruption to school and social life, and social isolation). given that adolescents and young adults drive mobile app use, a mobile-phone may be an ideal way to deliver a psychosocial intervention to adolescents and young adults with cancer. to use a patient-centered approach to inform a mobile-based mindfulness and social support intervention for adolescent and young adult patients with cancer. design/method: participants were ten aya with sarcoma ( % female; % adolescents); parents of the five adolescents, and six healthcare providers (n = ). formative research involved three steps: ( ) in-depth interviews were conducted with ten aya with sarcoma; parents of the five adolescents, and six healthcare providers (n = ). ( ) adaptations were made to an existing mindfulness app which offers a program for youth. modifications included creating a -week "mindfulness for resilience in illness" program, with relaxation exercises, and the addition of videos featuring two sarcoma survivors as program hosts. content was informed by the mindfulness curriculum for adolescents, learning to breathe. ( ) a private facebook usability group was organized to (i) elicit beliefs about the mindfulness app and potential future enhancements, and (ii) promote social support. results of the in-depth interviews revealed themes around adolescents' functioning and coping, including body image concerns; recurrence-related anxiety; anger over loss; and being overwhelmed by medical information. themes from the interviews were incorporated into a demonstration version of the mobile app. a patient-centered approach is widely recommended in the development of mobile-based health behavior change interventions and may be a useful way to inform development of a mobile-based mindfulness and social support intervention for adolescents and young adults with cancer. background: medical trainees consistently report suboptimal instruction and poor self-confidence in communication skills. despite these deficits, few training programs provide comprehensive pediatric-specific communication education, particularly in the provision of "bad news." an in-depth survey to examine the historical experience and communication needs of pediatric fellows was conducted at a large academic pediatric center as the first step towards the development of a comprehensive communication curriculum. to determine the previous educational and clinical experiences of pediatric subspecialty fellows, assess their levels of comfort in the context of various communication topics, and query potential modalities and topics for future communication training. design/method: the needs assessment survey was developed using previously developed and validated questions and review of the literature. the survey was reviewed by internal and external pediatric oncology and palliative experts and pre-tested with a subset of trainees to enhance content validity. results: thirty-two out of a total of fellows completed the survey ( % completion rate), of which % were pediatric hematology-oncology or subspecialty fellows. most fellows had participated in previous teaching sessions ( %), including those involving role play or simulation ( %). however, few fellows had received feedback from senior clinicians on their communication skills ( % of fellows had received feedback ≤ times). on a scale of -x, with indicating "not well prepared," the mean score for of communication items was < . fellows felt least prepared to lead discussions around informed consent for experimental therapies, end of life care, and autopsy. fellows indicated that didactic educational sessions and additional coursework were less useful strategies for improving their communication skills, whereas small group role play sessions with faculty and/or bereaved parent educators were most useful. fellows' overall communication preparedness score was not correlated with post-graduate year but was positively associated with the number of times they previously had delivered bad news to patients and families. fellows requested additional training on many topics, with greatest interest in learning skills to optimize communication with an angry patient or family. additional topic requests included placing limitations on resuscitation, withdrawing/withholding further therapy, and ageappropriate inclusion of patients in difficult discussions. despite self-report of prior communication skills training, pediatric subspecialty fellows felt underprepared to participate in difficult discussions with patients and families. learners identified role-playing and coaching with real-time feedback from other physicians and bereaved parents as more useful training strategies as compared to didactic sessions. background: when children die of cancer, parents must adjust to their child's absence amidst the lingering turmoil of what preceded their death: witnessing their child undergo painful treatments, making difficult decisions, and anticipating a devastating loss, all the while hoping for a recovery. adjustment to a child's death, as depicted by current bereavement literature, necessitates making meaning of one's loss. professional care staff can help parents make sense of their child's illness, and in turn, of their own parental experience during treatment. however, the extent to which relationships with professional care team members influence parents' ability to make sense of, and successfully cope with, their loss has not been examined. objectives: to examine how bereaved parents' interactions with their deceased child's pediatric oncology professional care team have impacted their grief symptoms design/method: to better understand how interactions with professional care staff relate to parents' grief outcomes, we conducted a mixed-methods study examining staff impact on parental grief. thirty participants whose children died of cancer one to three years ago completed an in-depth interview and psychometrically validated surveys measuring meaningmaking, depression, and grief symptoms. results: correlational analyses of the measures found that an increase in meaning making was associated with lower depressive and grief symptoms. a content analysis of the interviews found that many participants regarded staff "like family," had on-going relationships with staff after their child died, and described various ways staff interactions during treatment and after the child's death helped them make sense of their loss. in particular, participants described how interactions with staff have helped them find benefits in their loss and learn to create a new relationship with their child despite their physical absence. quantifying the interview data and statistically analyzing it along with the measures found that participants' increased frequency of describing staff's positive impact on their grief correlated with higher meaning-making scores and lower grief symptom scores. our study found that bereaved parents who lost their children to cancer were articulate in sharing their experiences of staff engagement and communication during treatment, offering numerous examples of how staff aided them in making meaning of their loss that were reliably associated with their subsequent grief. we hope the results of this mixed methods research encourage further study of the importance of staff interaction with families during the critical period of their children's care, and the lasting impact this can have regardless of the treatment outcome. memorial sloan kettering cancer center, new york, new york, united states background: although resiliency has been recognized as necessary for healthcare professionals, trainees feel unprepared for the emotional challenges inherent in caring for sick and dying patients. compounded by long hours, challenging work environments, and lack of formal training on handling emotionally difficult situations, many institutions are recognizing the need for interventions to reduce trainee distress. the goals of this fellow-led quality improvement initiative were: ) to determine whether there is a need for emotional support amongst pediatric hematology and oncology fellows, ) to provide formal resiliency and debriefing sessions, and ) to measure feasibility, acceptability and effectiveness of implemented curriculum. design/method: an anonymous survey to determine need for resiliency and debriefing sessions following a traumatic event was distributed to active pediatric hematology & oncology fellows at memorial sloan kettering cancer center in january . once need was established, an intervention consisting of a formal curriculum was developed and initiated in june , involving: ) scheduled and ad hoc debriefing sessions in response to traumatic events (including patient death, codes, interpersonal conflicts, end-of-life care); led by a psychiatrist and social worker with fellows and a pediatric oncologist mentor in attendance, and ) a resiliency didactic curriculum, led by a palliative medicine specialist, focused on skills such as contesting cognitive distortions and mindfulness. the effectiveness of these sessions will be measured using follow-up anonymous surveys at months (currently underway) and months post-initiation of intervention. the initial survey demonstrated most trainees ( / ) were present at or more deaths during their training, while less than half of respondents had attended a post-event debriefing session. % of respondents felt there was not sufficient emotional support from the institution for physicians caring for dying patients. a separate pre-intervention survey found all respondents ( / ) expressed a need for regular debriefings, and nearly all anticipated that they would benefit from such debriefings. concerns identified by trainees that would preclude participation in the curriculum included preference to deal with emotional situations privately and time constraints. trainees identified a need for formal debriefings and resiliency skill development. the program was easily implemented, and is both feasible and acceptable with good attendance. feedback received at the -month mark will determine deficits and possible improvements to the curriculum. the -month survey will measure effectiveness of the program and whether it should be continued. background: acute kidney injury (aki) is a common but under-recognized complication among patients with leukemia. it is associated with prolonged hospital stays, increased mortality, progression to chronic kidney disease, and delays or changes in cancer therapy which may affect a patient's prognosis. however, data on aki in pediatric patients with cancer is still lacking overall. we investigated the incidence of aki in patients who were newly diagnosed with all at our center from january to september . we performed a retrospective chart review of all patients who were newly diagnosed with all from neonate to years in our facility. we determined the incidence of aki in our population using the kidney disease: improving global outcomes (kdigo) diagnostic criteria. we also assessed for nephrotoxic exposures, nci all risk stratification and risk of aki, and tumor lysis syndrome (tls). we identified patients diagnosed during the study period who met inclusion criteria. median follow-up time was . months (range . - . ). the cohort was predominantly male ( . %) and hispanic ( . %). our analysis showed . % had aki by kdigo criteria ( % grade , . % grade , and % grade ), . % had aki on presentation, and % had multiple aki episodes during the study period. older age and longer length of hospitalization were associated with aki (p = . and p = . , respectively). there was no association between aki and nci all risk classification, contrast exposure, hyponatremia, elevated white blood cell count, uric acid levels, antimicrobial therapy, or diuretic use in this study. conclusion: aki was a common finding in our study population. the majority had grade aki by kdigo criteria. however, aki was associated with older age and a longer length of stay. further study is needed to determine the short-and long-term impact of aki on pediatric patients with all. st. jude children's research hospital, memphis, tennessee, united states background: in some regions, the availability of trained pediatric oncologists is a limiting barrier for the care of children with cancer. in , the unidad nacional de oncología pediátrica (unop) and the universidad francisco marroquín school of medicine in guatemala established a pediatric hematology/oncology fellowship program sponsored by st jude children's research hospital to provide central america and the caribbean with well-trained specialists. a systematic analysis of the impact of fellowship programs in pediatric oncology has never been done, especially in the context of a regional education program. objectives: this study sought to analyze the impact of the unop fellowship program based on the regional number of providers, pediatric cancer centers and patient volume. in addition, it sought to characterize the jobs and scientific output of the graduates. the impact will be evaluated in the context of a cost analysis. to define the volume of providers, pediatric cancer centers and patients, the directors of pediatric cancer centers in central america were sent an online survey to obtain these data. all the centers contacted maintain an updated hospital-based patient registry. in addition, the graduates of the fellowship program were also sent an online survey, asking about their job at graduation, current role and scientific productivity. the cost analysis will include assessment of direct costs including salaries and stipends for away rotations, as well as the indirect costs of faculty time spent teaching. since the establishment of the unop fellowship program, the region has more providers for pediatric cancer (p< . ) and centers treat a larger volume of patients (p< . ). two new centers have opened with graduates of the program. all but one graduate practice pediatric oncology ( / ) and the majority do it in their country of origin ( / ). no graduate practices outside of this region. almost half of the graduates ( %) hold a leadership role at their institution. the majority of their time is spent in the public sector (> %). the majority of graduates participate in clinical research ( %) and have participated in the creation or implementation of therapeutic protocols ( %). on average, the graduates have published peer-reviewed articles since completion of training. the unop fellowship program has had a favorable impact on pediatric cancer care in the region, contributing to the capacity to treat a larger volume of patients. graduates practice pediatric oncology in the region in the public sector, frequently hold leadership roles and are scientifically productive. background: abandonment of treatment is a major cause of treatment failure and poor survival in children with cancer in low-and middle-income countries. the incidence of abandonment in peru has not been reported. objectives: the aim of this study was to examine the prevalence and associated factors of treatment abandonment in pediatric patients with cancer of peru. we retrospectively reviewed the sociodemographic and clinical data of children referred between january and december to the two main tertiary centers for childhood cancer, located in lima, peru. definition of treatment abandonment was used from the siop (international society of paediatric oncology) podc (paediatric oncology in developing countries) abandonment of treatment working group recommendation. results: data of children diagnosed with malignant solid tumors and lymphomas were analyzed, of which ( . %) abandoned treatment. univariate logistic regression analysis showed significant higher abandonment rates in children living outside the capital city, lima (p< . ); prolonged travel time to a tertiary center (> hours; or . , p = . ); living in a rural setting (or . ; p< . ) and lack of parental formal job (or . ; p = . ). according to cancer diagnosis, children with retinoblastoma were more likely to abandon compared with other solid tumors. in multivariate regression analyses, rural origin and lack of formal parental employment were independently predictive of abandonment. conclusion: treatment abandonment prevalence in our country is high and closely related to socio-demographical factors. treatment outcomes could be substantially improved by strategies that help prevent abandonment of therapy based on these results. st. jude children's research hospital, memphis, tennessee, united states background: to improve the quality of a pediatric hematology/oncology fellowship program, a systematic assessment must be performed that can evaluate its current state and identify areas of opportunity, as well as modifications over time. unfortunately, widely agreed-upon metrics of quality for pediatric hematology/oncology fellowship programs currently do not exist. this is particularly important in this field due to the global shortage of specialists. for this reason, an assessment instrument that is applicable throughout the world must be created. objectives: the st. jude global education program assessment tool (epat) is a novel instrument that seeks to evaluate pediatric hematology/oncology fellowship programs around the world in systematic and objective way. epat will help determine key performance indexes that are relevant for quality education in pediatric hematology/oncology fellowship programs and establish the framework for improvement. design/method: firstly, key domains to be evaluated for program assessment were identified a priori based on the continuum of pediatric hematology/oncology fellowship programs in the context of geography and educational structure. subsequently, questions were formulated to evaluate these key domains, seeking to assess elements involved in ensuring competence in clinical practice, academic productivity and regional impact. due to the novelty of this tool and the lack of defined metrics of quality, epat relies on expert opinion in a two-step process: internally in the department of global pediatric medicine at st. jude children's research hospital and, subsequently, from a panel of experts in global pediatric oncology and medical education from around the world. ten key domains were identified to evaluate all aspects relevant to training programs around the world, regardless of educational and geographic context. questions have been created to assess these domains and, to make epat quantitative, these have assigned weights with a value reflective of their relative importance. this grading system allows for a score in each key domain, permitting monitoring of changes over time. epat is currently at the stage of external expert review, and subsequently will be piloted in five fellowship programs around the world to provide different geographical and patient care contexts for its validation. once epat is finalized, it will be distributed to pediatric hematology/oncology fellowship programs around the world to be applied. epat proposes a novel strategy to assess training programs in a systematic way that includes all aspects relevant for a training program in a global context. this tool will help guide improvements in pediatric hematology/oncology fellowship programs and assure a well-trained workforce. background: with the improvement in pediatric oncology patient survival and outcomes in the past several decades, monitoring for recurrence and long-term effects of therapy has become even more important. the utilization of personalized treatment summaries and survivorship care plans (scps) is one way to communicate this information with patients and families. the american college of surgeons commission on cancer (coc) created a standard regarding provision of scps to % of eligible patients by december , as a metric for accreditation of all cancer centers. the standard applies to all patients with stage i, ii, and iii cancer diagnoses and requires creation of the scp within one year of diagnosis or six months of completing treatment. during implementation at our pediatric cancer center, we identified barriers to use of the guidelines in the childhood cancer setting. objectives: define eligibility for an scp for pediatric oncology patients to include all patients with curative intent and to deliver scps within six months of finishing therapy. design/method: using chart review and a cancer center registry query, we identified childhood cancer patients potentially eligible for an scp by collecting stage, goal of therapy, and dates of treatment. all patients with curative intent were deemed eligible for an scp regardless of stage i-iv. patients being followed in the oncology clinic for posttreatment surveillance and care were included even if they had received an scp in the survivorship program or were greater than six months off therapy at time of implementation. as expected in the pediatric oncology population, acute lymphoblastic leukemia (all) was the most common diagnosis comprising . % of patients. all is stratified into risk groups instead of surgical staging categories, and treatment duration is greater than one year, unlike many adult-onset malignancies. these differences required interpretation of the guidelines to apply to our pediatric population for all and other pediatric diagnoses with non-surgically based staging. our pediatric oncology clinic has to date provided scps to of eligible patients by adapting the guidelines to focus on patients with curative intent to receive an scp by six months off therapy. cancer staging guidelines and goals for curative intent as well as lengths of treatment vary between the pediatric and adult populations. the coc guidelines require adaptation for optimal applicability to the pediatric oncology population. background: education in communication for fellows in fields that require difficult discussions with families are few in nature. adult learning pedagogies such as role play are under-utilized in medical education, and have been shown to be as effective as traditional teaching methods such as lecture. an -module course for fellows in hematology/oncology, hospice and palliative medicine, radiation oncology, and pediatric hematology/oncology was implemented in january/february . fellows participated in the program. topics covered including fundamentals of communication, coping and spirituality, delivery of bad news, communicating with families, sexual dysfunction during treatment, palliative care/death and dying, and burnout. objectives: overall goal of this course is to foster holistic physicians who views their patients as people with cancer, not cancer patients, and physicians that can communicate effectively with their patients throughout the disease continuum. by the end of the course, learners should be able to practice the fundamental principles of good communication. design/method: fellows initially participated in a pre-course osce to establish baseline skills. osce was facilitated by the center for learning and innovation at northwell, and included actors portraying a pediatric patient and family member to whom the fellow had to break bad news. two months later, the course was carried out over the span of eight weeks and included didactic sessions followed by minutes of role play scenarios. five of the eight modules included role play, with faculty members serving as simulated patients. after the course, a second breaking bad news osce was held. both osces were filmed, and feedback was given by the on-site actors. additionally, faculty members were given access to the videos in an on-line format and were given an evaluation tool to assess the fellows' performance pre-and post-intervention. fellows were given subjective surveys pre-and post-course as well. results: subjective data from participants showed a noticeable increase in comfort level in all areas on the pre-and post-course survey. data obtained from osce videos showed improvement in communication skills as assessed by sps and faculty members using a new evaluation tool developed by faculty. initial first-run data shows that this course is successful in improving communication skills as well as increasing fellows' comfort level across several domains of communication. future directions for our course include improving and validating our assessment tool, expanding our topic base to include more aya and pediatric scenarios, faculty development for improved role play, and investigating impact on practice after course completion. background: acute lymphoblastic leukemia (all) is the most common form of childhood cancer with approximately children diagnosed each year. survival rates have improved significantly over the past several years. children with all are at risk for developing musculoskeletal complications during and after completion of treatment, which can contribute to impaired activity, elevated body mass index (bmi), and risk for complications. interventions involving physical activity could improve musculoskeletal strength as well as overall health in these children. the aims of this study are to examine the feasibility of a directed physical activity program for children with newly diagnosed all during the initial intensive phase of therapy and to evaluate the overall health and quality of life of children participating in the directed physical activity program. design/method: all subjects will receive education materials about the importance and safety of physical activity and a nutrition handout. all subjects will also participate in the directed physical activity program under the supervision of a trained physical therapist for at least minutes every week for weeks. the program will entail four stations including a cardiovascular, balance/proprioception, strength and flexibility, and coordination and cardio. feasibility will be assessed by tracking the participation rate throughout the study period. other assessments will be made at study entry, at the end of weeks of physical activity initiative and months after completion of the intervention. assessments include overall strength and flexibility, weight, height, bmi, blood pressure and performance scores. descriptive statistics will be used for this study. results: a total of patients, male and female, enrolled in the study over a . month period. patient ages ranged from - years. half of the patients enrolled have completed the week program and all patients had stability or improvement of their physical functioning scores. further data collection and analysis is ongoing. patients in the early intensive phase of all therapy are at risk for complications that can affect their physical functioning. a directed physical activity protocol may improve their overall physical functioning. patients may not need specific physical therapy; however a directed physical activity program appears to be beneficial for these patients. the main roadblocks to successful completion of the program were difficulty with scheduling, strain on the parents and patient from treatment, unplanned admissions for fever, as well as nausea and fatigue at time of visit. albany medical center, albany, new york, united states background: communication skills are a core competency highlighted by the acgme. increasing resident confidence in delivering difficult news has been shown to lead to more s of s effective communication. currently, the majority of residency programs lack formal training in communication skills. our objective was to demonstrate feasibility and efficacy of integrating a standardized-patient based training program for communication skills into the curriculum of pediatric residents design/method: to date, pediatric and medicine/pediatric residents have participated in the program during the intern year. the program consists of three, two-hour long sessions, in which each resident is given several opportunities to act out case scenarios with a standardized patient. scenarios included informing a parent of their child's new cancer diagnosis and disclosure of a positive hiv test to a teenager. residents received post hoc peer to peer, and preceptor to learner feedback. pre and post-program surveys were completed by residents. results: following course completion residents reported an increase in confidence in multiple areas of communication including giving a difficult diagnosis (p< . ), discussing a poor prognosis (p< . ), responding to different patient/family member emotional responses i.e. crying or anger (p< . ), and organizing vital information to be relayed (p< . ). in conclusion, communication skills training of pediatric residents is feasible and provides a platform for developing valuable skills not taught elsewhere within the curriculum. background: for children with cancer, transitioning back to school during or after treatment can be challenging. literature supports the need for school re-entry programs to ease this transition. however, these programs vary widely among pediatric cancer institutions with little data addressing their program components. data from this study provides information on current school re-entry programs across these institutions. objectives: one objective of this study was to assess for correlation between the presence of a school re-entry program and other factors, such as geographic location and institution size. a second objective was to establish a list of differences between institutions' school re-entry program components. finally, we aimed to describe current school reentry practices, as well as program benefits and perceived areas for improvement. states with membership in the children's oncology group were offered enrollment in this study. a member of each institution was invited to participate in a survey established by the research team. this person was closely associated with the institution's school re-entry practices. each interview queried institution demographics, as well as program components (e.g., participants, target audience, resources). comment was also collected on program benefits and potential for improvements. analysis of transcripts was performed using pearson's correlation to assess for relationships between institution size, geographic location, and program presence. grounded theory was used for analysis of benefits and improvements. results: thirty-nine of forty-one pediatric institutions who were offered enrollment participated in this study. twentynine institutions ( %) indicated the presence of a school reentry program, and ten ( %) stated they had none. no correlation was found between institution size and the presence of a school re-entry program (p = . , ns). there was also no correlation found between institution location and the presence of a school re-entry program (p = . , ns). a major theme surrounding the benefits of having a program included education for the returning student's peers. for those with programs, perceived improvements included increasing staffing and the ability to offer more services. the results do not support the hypothesis that the presence of a school re-entry program is influenced by the size and geographic location of the treating institution. however, data seem to suggest that available staffing may influence the presence of a program. future studies are needed to address other potential influences, as well as to take an evidence-based approach to determine the effectiveness of the interventions present in these programs. cohen children's medical center/ zucker school of medicine at hofstra-northwell, new hyde park, new york, united states background: genetics/genomics is evolving at an extremely rapid pace. current advances lead to individual algorithms toward disease treatment for each disease with multiple branch points. fellows learn only a fraction of the knowledge and there is no formal approach to teaching critical analysis of information and application algorithms toward disease. additionally, as knowledge evolves extremely rapidly, any approach must teach self-acquisition and application of evolving discoveries. objectives: to create, implement and evaluate a novel curriculum for genetics/genomics targeted toward pediatric hematology/oncology fellows design/method: the curriculum includes four components: ) genetic and genomic medical knowledge, with one initial team-based learning session and weekly online multiple choice questions; ) essential pathways, which will teach molecular pathways common in oncogenesis and relevant to targeted therapy in microteaching sessions with using auditory, visual and tactile learning; ) knowledge acquisition and clinical judgment, to allow learners to gain experience into researching data available, then developing and prioritizing potential treatment plans using problem-based learning sessions in which they will stage a patient, research treatment options, prioritize and present findings; and ) synthesis to demonstrate independent ability to research and recommend therapy through an independent project in which the learner, given a case, will present the case and research findings, genetics/genomics, molecular pathways and make recommendations for therapy in molecular tumor board for faculty and fellows. to evaluate, we plan to recruit to institutions, match for size of programs and implement in half and evaluate nd and rd year fellows in both groups by mcq exam and satisfaction surveys. the creation of a multi-module, adult-learning based curriculum for genetics and genomics in pediatric oncology is feasible. implementation and evaluation are necessary to demonstrate efficacy. background: neuroblastoma is the most common extracranial solid tumor in children. chimeric anti-gd antibody ch . (dinutuximab) therapy has improved the survival of children with newly diagnosed high-risk, neuroblastoma patients as well at the time of first relapse/progression. acute neuropathic pain is a well-documented side effect of dinutuximab administration. however, additional adverse effects including sensorimotor neuropathy, ocular symptoms, and behavioral changes have been described. the incidence and severity of these effects are currently not well-documented in pediatric patients. with improved long term survival of patients receiving this modality, it is important to look for the potential late effects of dinutuximab. objectives: to determine the incidence and severity of neurologic, ophthalmologic, or behavioral changes after dinutuximab administration at our institution. we performed a retrospective chart review using our electronic medical record. we included all patients with high-risk neuroblastoma between the ages of and years at our institution diagnosed between and who received dinutuximab. patients with history of opsoclonus-myoclonus syndrome or gross sensorimotor neuropathy prior to receiving dinutuximab were excluded. we examined clinical documentation for subjective reports and objective exam findings of neurologic, ophthalmologic, or behavioral changes. we also looked for referrals made to neurology, ophthalmology, physical medicine & rehabilitation (pm&r), and psychology. : twenty-two patients met inclusion criteria. at the time of chart review, patients were alive and were deceased. eighteen patients received dinutuximab per anbl ; patients received dinutuximab per anbl . of these patients, patients reported symptoms of interest and reported multiple symptoms. six patients reported symptoms that began at least months after completing dinutuximab. nine patients had objective findings on exam, including decreased deep tendon reflexes, abnormal pupils, and nearsightedness. for patients, referrals were made to ophthalmology, pm&r for neuropsychologic testing, or neurology. two patients who reported symptoms of interest were not referred to a specialist. conclusion: neurologic, ophthalmologic, and behavioral symptoms were commonly reported and demonstrated on exam among pediatric patients with high-risk neuroblastoma who received dinutuximab. it is important to identify these effects so that appropriate specialist referrals can be placed for adequate management of these changes. we recognize that these symptoms may not be solely due to dinutuximab as these patients receive other agents including opioids, so a prospective trial is needed to further evaluate the long-term effects of dinutuximab and to determine how best to screen for these effects. akron children's hospital, akron, ohio, united states background: pediatric cancer is the leading cause of diseaserelated death in children in the united states (u.s.). in , over fifteen thousand children were diagnosed with cancer in the u.s. this population is at high risk for malnutrition due to the multimodal therapies they receive: surgery, chemotherapy, radiation therapy, antibody therapy, and/or bone marrow transplant. adverse effects of these therapies include taste changes, loss of appetite, diarrhea, vomiting, and/or mucositis, making it difficult for the children to be able to consume adequate amounts of nutrition during therapy. there is no "gold standard" measurement tool for identifying patients at risk for malnutrition. nutritional status is not frequently evaluated as a component of clinical trials. assessment of anthropometric measurements (weight, height, z-scores) at diagnosis, as well as over the duration of treatment, can assist in the early identification of malnutrition. the incidence and prevalence of malnutrition in this population is unknown at akron children's hospital. the purpose of this study is to describe the nutritional status and provision of nutritional support therapies in pediatric patients during their first year post new oncologic diagnosis. objectives: identify the incidence and prevalence of malnutrition across oncologic diagnostic categories over the first twelve months post diagnosis. we performed a retrospective records review of all patients newly diagnosed with cancer in at akron children's hospital. demographic and anthropometric data was collected at time of diagnosis and nutritional status categorized by z score. anthropometric and nutrition support data was then collected every two months for the first year after diagnosis along with incidence of unplanned inpatient admissions. results: a total of patients were included in the analysis, with . % malnourished at time of diagnosis; . % developed malnutrition the first year. patients with solid tumors represented % of patients with pre-existing or acquired malnutrition. overall, % of patients received at least one nutritional support modality. patients with pre-existing or acquired malnutrition had a non-significant increase in unplanned admissions (p = . ). our study demonstrated that patients with solid tumors were found to be at increased risk of pre-existing and acquired malnutrition, followed by leukemias, and experienced higher incidence of unplanned admissions in the time period observed. prospective, multi-center replication of this study, including detailed collection of nutrition therapies is recommended to guide development of diagnosis specific nutrition support guidelines. background: pediatric and young adult oncology patients treated with intense chemotherapy have a high incidence of transfusional iron overload. iron deposition can lead to heart failure/arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. however, there is a paucity of data regarding recommendations for management of transfusional iron overload in these cancer survivors. consequently, long-term complications of transfusional iron overload specific to these patients have not been assessed. objectives: to assess screening and phlebotomy-based treatment algorithms for this population. design/method: a retrospective chart review of pediatric and young adults who completed oncology management, had iron overload, and initiated phlebotomy treatment was conducted. tiered screening occurred in patients that received at least packed red blood cell (prbc) transfusions. patients were recommended for evaluation and possible phlebotomy if: ( ) liver iron concentration (lic) > mg of iron/gram dry weight liver tissue by ferriscan and/or ( ) cardiac mri t * < ms. during phlebotomy, iron status was assessed quarterly and phlebotomy discontinued with lic < or normalization of ferritin/imaging lic verification. descriptive statistics were employed to report the characteristics of the study population. spearman correlations were utilized to describe associations between transfusions, lic, ferritin, iron saturation and number of phlebotomy sessions. results: twenty five survivors underwent phlebotomy. the mean age was . years (sd . ) and ( %) were female. oncologic diagnoses: all ( %), aml ( %), nhl ( %), ewing sarcoma ( %), osteosarcoma ( %), neuroblastoma ( %) and cns ( %). patients received a median of . (iqr - ) transfusions. median number of phlebotomy sessions was (iqr - ) over . years (iqr . - . ). prior to phlebotomy, median lic was . mg/g (iqr . - . ) and ferritin was . ng/ml (iqr - ) . no patients demonstrated abnormal cardiac t * mri (n = ). ( %) patients completed phlebotomy. one discontinued due to poor vascular access. no patients developed iron deficiency. lic was reduced by a median of . mg/g (iqr . - . ) and ferritin by ng/ml . correlation between number of transfusions and phlebotomy sessions was poor (r = . ). conclusion: management guidelines are lacking for transfusional iron overload in pediatric and young adult survivors of cancer. we demonstrate a phlebotomy algorithm that is effective and tolerated. correlation between number of transfusions received and phlebotomy treatments was poor, necessitating serial assessments. using this management algorithm, prospective studies can evaluate the effect of iron removal on iron overload complications in this patient population. penn state children's hospital, hershey, pennsylvania, united states background: cancer therapy leads to an impaired immune system that takes time to recover. it is important to ensure that these survivors have adequate immunity to prevent common yet potentially severe childhood illnesses. no validated guidelines currently exist for surveillance testing or re-immunization in this population. retrospective analysis involving a small cohort of pediatric cancer patients treated at penn state children's hospital showed % of patients screened for varicella immunity after therapy completion did not have adequate disease titers. to determine the proportion of pediatric cancer survivors who have lost humoral immunity to previously received vaccines; to determine the rate of response to single dose boosters or full vaccine series in seronegative subjects after one booster. design/method: pediatric cancer survivors treated at the children's hospital who are at least months from completion of cancer therapy are prospectively tested for antibody levels to hepatitis b, tetanus, varicella, measles, and strains of pneumococcus ( , b, v, c, f, and f). samples are analyzed by the cdc for measles and varicella avidity. seronegative subjects by commercial studies, are eligible to receive booster vaccines. titers are rechecked at least weeks after boosters to re-evaluate immunity; if still seronegative, subjects will receive the entire vaccine series. titers are finally tested at least weeks after the final dose of the vaccine series. immunity analyzed after therapy, after boosters, and after vaccine series. results: of pediatric cancers survivors who completed therapy, % were non-immune to hepatitis b, % nonimmune to > % of pneumococcal strains tested, % nonimmune to measles, % non-immune to varicella, and % non-immune to tetanus. of subjects who received mmr vaccine after therapy and prior to study enrollment did not have protective antibodies to measles. of the subjects who received varicella vaccine after end of therapy and prior to study enrollment, did not maintain protective antibody levels. cdc results for measles and varicella are pending, as well as repeat studies after vaccine boosters and series. conclusion: a significant percentage of pediatric cancer survivors do not retain immunity to hepatitis b, pneumococcus, measles, and varicella. after one booster, a high percentage of subjects did not develop protective immunity to varicella. only subject did not have immunity to tetanus, which is consistent with the high immunogenicity of tetanus toxoid. formal guidelines are needed to protect this population from vaccine-preventable illness post-therapy. children's hospital of richmond at virginia commonwealth university health system, richmond, virginia, united states background: childhood cancer survivors are at risk for being overweight. diet and physical exercise are important in maintaining a healthy lifestyle and weight; however, it has been reported that cancer survivors are less active than their peers. one reason for this may be that there are no clearly established risk-based exercise recommendations for cancer survivors. another reason may be that providers tend to focus s of s recommendations for exercise more towards patients who are overweight. objectives: to describe changes in physical fitness of childhood cancer survivors who exercise. design/method: 'moving forward' is a wellness and physical fitness program that the center for care beyond the cure at chor offers in partnership with the ask childhood cancer foundation and the ymca. the program is available for any childhood cancer survivor between y and y age, being seen at our center. survivors define their fitness or wellness goals and then work with a trainer once a week (at least) for min sessions throughout the year to achieve these goals. baseline and ongoing measurements for core strength, endurance, overall strength and balance were collected. the average of each of the parameters of all participants were compared from the beginning to the end of the program. over the year, there was a % increase in endurance as measured by the average of the miles walked in minutes, % increase in core strength as measured by the average number of sit-ups in secs, an % and % increase in overall strength as measured by the average weight lifted by leg press and the average weight lifted by chest press, and a % increase in balance as measured by the average number of seconds balancing on a single leg. in addition, each child had actually gained weight in the process with an approximately % increase in the average of the weights of all children. there are benefits to regular exercise beyond weight control, and improvements in physical fitness can be seen even without weight loss. regular physical exercise results in improved physical fitness and should be universally advocated to all patients. determining insulin resistance, measuring changes in fatigue and wellness perception following exercise are future directions that we intend to explore. dana-farber cancer institute, boston, massachusetts, united states background: improvements in adolescent and young adult cancer patient (aya) survival rates and quality of life outcomes have lagged behind those of children and older adults, highlighting a need for research targeting this unique population. current literature supports the value of strong ayaclinician communication, notably in facilitating therapeutic alliance, however little is known about aya communication priorities during cancer care and barriers to optimal ayaclinician communication. objectives: to explore aya and oncology clinician communication priorities and to identify barriers and facilitators to aya-oncology clinician communication. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years) from a single large academic institution and oncology clinicians (physicians and nurse practitioners) from academic institutions in the northeastern united states. interviews were conducted in english by phone or in person. all interviews were audio-recorded and transcribed verbatim. analyses were aided by nvivo software. ayas identified a wide range of topics as important to discuss with clinicians. the most frequently identified topics were ) side effects of treatment (with an emphasis on physical appearance and function, n = ), ) social issues (including friendship, family, and school, n = ), ) looking ahead to the future (n = ), and ) sexual & reproductive health (including future fertility, contraception, and romantic relationships, n = ). clinicians prioritized ) cancer treatment and side effects (n = ), ) emotional and psychological health (n = ), and ) sexual and reproductive health with a focus on fertility risk and fertility preservation (n = ). aya reported facilitators to good communication including an open and long-established relationship with the clinician (n = ) and clinician engagement in age-appropriate and patient-directed conversations (n = ). barriers included parental presence during visits (n = ). clinicians reported barriers including ) clinician discomfort (not feeling wellequipped to discuss psychosocial topics such as sexual health, spirituality, and relationships with peers, n = ), ) presence of parents/family (n = ), and ) perceived patient discomfort discussing specific topics (such as sexual health, n = ). clinicians acknowledged the need for collaborative efforts with additional team members (i.e. nurses, psychosocial providers) to assist in meeting aya communication needs. conclusion: aya and clinician-reported communication priorities are largely aligned. however, ayas emphasize some topics, such as social function, appearance, and sexual health that are not highly prioritized by clinicians, which may result in gaps in care for ayas in treatment and in survivorship. these data identify opportunities for intervention, including clinician education, patient and family education, clinic-based intervention, and systems-based changes that can be developed and tested. background: primary care physicians (pcps) cite lack of knowledge and inadequate communication with the oncology team as major barriers to providing recommended surveillance for late effects of treatment to childhood cancer survivors. a standardized telephone handoff to pcps posttherapy is a potential strategy to increase survivorship care by pcps through interactive communication. to determine the feasibility of a structured telephone communication using the situation, background, assessment, and recommendation (sbar) communication tool delivered by a trained oncology nurse to increase pcp knowledge and willingness to provide survivorship care. design/method: from / / to / / , a registered nurse expert in childhood cancer survivorship attempted to contact by telephone the pcps of the most recent patients attending yale's childhood cancer survivorship clinic that were < years old, english-speaking, and ≥ years posttreatment. all pcps had been previously sent an individualized survivorship care plan (scp) that listed the patient's previous treatment history and recommended surveillance tests. upon successful contact and after confirming receipt of the scp, the nurse explained the definition of late effects, description of patient's diagnosis and treatment history, and associated potential late complications and schedule of recommended surveillance tests. the pcp was also asked about his/her ability and willingness to provide needed surveillance for late effects in the future. overall, of pcps were successfully contacted with a median of phone call (range: - ) that lasted a median of minutes (range: - ) after a median of business day (range: - ). no pcps ended the call mid-conversation. all pcps were receptive and expressed appreciation for the call. twenty-five of ( %) pcps expressed an understand-ing of the material discussed and endorsed belief in their ability and willingness to provide late effects surveillance for their patients. no pcps questioned discussing their patient's care with a nurse versus a physician. interactive, structured communications between nurses and pcps by telephone are feasible and are associated with high-levels of pcp confidence in providing survivorship care. background: childhood cancer (cc) admissions account for % of non-newborn pediatric hospitalizations. these hospitalizations are longer and more expensive than other hospitalizations. admission payer (medicaid or commercial) reflects both health policy and sociodemographic status. the objective of this study was to determine if length of stay (los) or cost of cc admissions differed by payer. we used the kids inpatient database, a sampling of all pediatric hospital discharges in the united states. analysis for this study was limited to admissions containing a cancer diagnosis in any discharge icd- codes. admissions were further subcategorized by discharge codes according to diagnosis (leukemia, lymphoma, solid tumor and brain tumor) and reason for admission (chemotherapy, procedure, infection, non-infectious toxicity or "other"). charges were converted to costs using cost-to-charge ratios. multivariable linear regression models were performed to control for age, gender, race, reason for admission, and diagnosis. results: there were , weighted admissions for children with a cancer diagnosis in . of these admissions, . % had medicaid, . % had commercial insurance, and less than % had other payers. the mean los for medicaid admissions was . days ( % ci . - . ), compared with . days ( % ci . - . ) for commercial insurance. surgical admissions accounted for the largest difference in length of stay with medicaid admissions being . days longer than those covered by commercial insurance ( . days vs . days), however, the difference was significantly different for all reasons for admission. in multivariable analysis admissions associated with commercial insurance were % shorter s of s (p< . ), accounting for approximately one hospital day, than admissions associated with medicaid after controlling for other variables including race. the mean overall cost for medicaid admissions was $ , ( % ci - ), compared with $ , ( % ci - ) for commercial insurance. in the multivariable model, cost was collinear with race. conclusion: los and cost of admissions associated with medicaid differed from those associate with commercial payers. medicaid admissions were % longer on average than commercial insurance, accounting for a difference in length of stay of approximately one day although the difference varied with the reason for hospitalization (chemotherapy, surgical procedure, infection, other toxicity, other). costs of admissions were not independent of race. further investigation into potential explanations for this difference including differential access to home care needs, outpatient reimbursement differences, social indications for prolonged hospitalization, and provider biases, is warranted. background: pediatric cancer is a major cause of morbidity and mortality among children surpassed only by accidents. despite improved outcomes in high income countries (hic) survival rates remain poor in the developing word. there are various diagnostic and therapeutic limitations contributing significantly for the survival gap. the main objective of the study is to to evaluate the outcomes of pediatric cancer in armenia and identify diagnostic and therapeutic limitations in the country. we conducted a retrospective study among (≤ years old) children with cancer (solid tumors and hematological malignancies), who were diagnosed and treated at the clinic of chemotherapy of muratsan hospital complex of yerevan state medical university between and . those patients, who didn't receive chemotherapy for any reason were not included in the study cohort. epidemiological, social, medical information was collected through the patient charts review. this included patient age at diagnosis, sex, place of residence (city vs village), the educational level and employment status of parents, type of cancer, stage, presentation of symptoms, first medical specialty consulted and the time consulted, initial work-up, the type of treatment received, information on the diagnosis/treatment received abroad. results: at our clinic during the mentioned period of time the majority of patients presented with hematologic malignancies- %. ( . %) patients had information on diagnosis delay. average delay in diagnosis was about days. in % of cases the first contact with "healthcare system" was through pediatrician, and in % with surgeon. out of relapsed patients received salvage treatment in armenia and abroad. from those who stayed for treatment in armenia patients survived. majority of relapsed patients had acute lymphoblastic leukemia. from leukemia patients immunophenotyping and cytogenetics were available for ( . %) patients; the majority of missing cases were between and , when these diagnostic modalities were not available or affordable in the country. ( %) patients received part of diagnosis and/or treatment abroad. the most frequent reason for going abroad was bone marrow transplantation, otherwise none available in armenia. out of patients were lost to follow-up, patients had a fatal outcome. patients were in remission at a median follow up of . years. conclusion: unavailability of cancer registry and several essential diagnostic/treatment modalities, luck of multidisciplinary care and palliative support, high rate of out-of-pocket expenses were among the main challenges of pediatric cancer care in armenia. background: adverse drug reactions (adrs) are increasingly recognized as important and sometimes irreversible complications of cancer treatment. anthracyclines and cisplatin are effective chemotherapeutic agents, but their use can be limited by cardiotoxicity (anthracyclines) and ototoxicity (cisplatin) in up to % of patients. genetic variants that can be used to predict who is most at risk of developing these adrs have been discovered and replicated. objectives: to create pharmacogenetic risk prediction models for anthracycline and cisplatin toxicities and discuss results with oncologists to facilitate incorporation into treatment decision-making when appropriate. design/method: risk prediction models were developed from the linear regression of strongly-predictive genomic variants (odds ratios ≥ ) discovered and replicated in at least three patient populations. these models were used to assess an individual patient's genomic risk of developing cardiotoxicity from anthracyclines or hearing loss from cisplatin. risk results were returned to oncologists showing where the specific patient's genetic risk of toxicity lies on a continuum between the lowest and highest risk groups across all studied patients using a multi-gene model. interviews were conducted with patients, families, and oncologists to determine how results were valued and utilized. results: patients have been genotyped and had their genetic risk results returned to their oncologists. the first patients have been characterized to determine the impact these test results have had on their clinical care. results were described as being useful in decision-making by patients and/or oncologists in % of cases. additionally, for patients in the most extreme risk groups (highest and lowest risk), a change in treatment plan was ordered % of the time for cisplatin patients and % of the time for anthracycline patients. this included increased cardiac and audiological monitoring, the addition of a protective agent, or choosing an alternative treatment protocol if the risk outweighed the benefits of remaining on the current treatment plan. in interviews, patients indicated that they felt more involved in decision making, and felt reassured by understanding their genetic risk of toxicities. genetic risk prediction models for anthracycline cardiotoxicity and cisplatin ototoxicity were highly utilized by patients and oncologists in decision-making. results were found to be an important tool for informing patients of the risk of adrs during cancer treatment, and resulted in patients and their families feeling more involved in decision-making. background: childhood cancer survivors are at increased risk of developing executive dysfunction, and low socioe-conomic status (ses) has been identified as one of the mediators of executive functioning. previous studies have used traditional measures of ses, such as parents' education level, family annual income and occupation. but more recently, area based socioeconomic measures like block group poverty status are deemed to be more useful in monitoring of social inequalities in health in the united states. block groups are statistical divisions of census tracts and generally contain between and , people. the current study aims to understand the association of block group poverty status (percentage of households in family's block group of residence living below the federal poverty level) with executive functioning among cancer survivor children. design/method: we used a retrospective cohort of childhood cancer survivors. relevant information was collected from the medical record, administrative data sets and parent-filled surveys. address information was geocoded using arcgis . to obtain data on the block group poverty status. a priori cut-points were set to represent block groups with families living below poverty level at %, . % to . %, and ≥ . %. executive functioning were assessed through a parent-rated instrument, the behavior rating inventory of executive functions (brief). multiple linear regressions were used to determine the relationship between block group poverty status and the brief scores. results: data was examined from families of childhood cancer survivors, ranging in age from to years. in this sample, . % families reported an annual income <$ , , . % reported income between $ , and $ , while . % reported annual income ≥$ , . primary care giver of . % of cancer survivors had more than more high school education, and . %, . % and . %, of families were living in a block groups with %, . - . % and ≥ % poor households respectively. block group poverty level was not significantly associated with annual income levels (spearman's rho = . , p = . ), or parental education level (spearman's rho = - . , p = . ). in a step-wise multiple linear regression, there was no statistically significant association seen between block group poverty status and executive functioning after adjusting for co-variables in the final model. future prospective study with a bigger sample size, longer follow up period and more robust measures of the executive functioning like a clinician administered test are needed to understand the effect of block group poverty status on executive functioning. to d completion was . days (range - ). all parents strongly agreed/agreed that d was helpful and would recommend d participation to another family. ten parents ( %) reported time spent on d was "just right." no parent felt more worried due to the intervention, though parent found d participation stressful. this interim analysis suggests that parents have a favorable d experience and recommend the intervention. to date, < % of enrolled parents fail to participate. d shows promise as an acceptable interdisciplinary communication intervention targeted to the early treatment period for childhood cancer. children 's hospital and research center oakland, oakland, california, united states background: screening echocardiograms are recommended by children's oncology group (cog) guidelines to assess for anthracycline-induced left ventricular (lv) systolic dysfunction. the yield of screening echocardiograms during chemotherapy and in the immediate post-therapy period is uncertain. objectives: to assess the incidence of lv dysfunction detected by screening echocardiograms during chemotherapy and in the immediate post-therapy period, defined as - months off-therapy. design/method: children diagnosed with cancer between january -march who received anthracycline chemotherapy were identified. echocardiograms were performed as per protocol, institutional and cog guidelines, and were reviewed retrospectively. lv dysfunction was defined as fractional shortening (fs) < % or ejection fraction (ef) < % ( ) results: in this cohort (n = , median age years), the most common diagnosis was all ( . %), followed by aml ( . %). of echocardiograms, ( . %) were performed during treatment and in the immediate posttreatment period. thirty-eight ( . %) patients had a > % decrease in fs compared to their pre-treatment echocardiograms. none of these patients required any treatment modification or cardiac medications. only patient ( . %) had echocardiogram-proven lv dysfunction discovered on a screening echocardiogram during her treatment course. she eventually died due to multi-organ failure following septic shock. this patient was receiving treatment for aml and had received mg/m of doxorubicin-equivalent anthracyclines at the time of the abnormal echocardiogram. one patient with metastatic ewing sarcoma had borderline lv dysfunction with a fs of % detected a month before completion of therapy. she had received mg/m of doxorubicin equivalent anthracyclines at the time of the abnormal echocardiogram. she did not require any therapy modification or additional cardiac medications. serial echocardiograms done on this patient have shown stable ventricular function. no off-therapy screening echocardiograms identified lv dysfunction. in our experience, the yield of echocardiograms to detect anthracycline-related cardiac dysfunction during treatment and in the immediate post-therapy period is very low. one patient developed lv dysfunction during treatment and one had borderline fs, while no lv dysfunction was identified within months of completing chemotherapy. though fs decreased in % of patients, none required intervention. further study is needed to optimize the use of echocardiography screening in children treated with anthracyclines. references: . landier w et al. jco . background: platinum-based chemotherapy increases the risk of sensorineural hearing loss in children with cancer. little is known about the impact of hearing loss on cognitive and emotional functioning in survivors. to determine the association of severe/profound hearing loss after platinum-based chemotherapy with ) cognitive impairment and ) emotional distress (i.e. anxiety and/or depression). cross-sectional study of all patients attending yale's childhood cancer survivorship clinic ≥ years off therapy for cancer diagnosed at < years and treated with cisplatin and/or carboplatin, but with no history of cns tumor, cranial radiation, congenital hearing loss, or developmental delay. hearing loss severity and hearing aid data were abstracted from audiograms and detailed clinical history. cognitive impairment was defined as behavior rating inventory of executive function t score ≥ , assessment by neuropsychologist, and/or history of special education. emotional distress was determined by brief symptom inventory t score ≥ (global or two subscales) or behavioral and emotional screening system t score ≥ , psychologist interview, and/or history of psychotropic medication/psychotherapy. the most recent available patient data were used. logistic regression with sas software, version . was performed. results: overall, patients ( % female, % white) met eligibility criteria with a median age of . years (iqr = . ) at diagnosis and . years at evaluation (iqr = . ) after a diagnosis of sarcoma ( %), neuroblastoma ( %), or other ( %) for which % received cisplatin and % received carboplatin. fifteen patients ( %) had severe/profound hearing loss in at least one ear. patients with severe/profound hearing loss had a significantly increased risk of cognitive impairment (or = . ; % ci = . - . ), but not emotional distress, compared to patients without severe/profound hearing loss. there was no significant association between age at diagnosis, current age, time since diagnosis, sex, race, ethnicity, or diagnosis with either cognitive impairment or emotional distress. similarly, there was no significant interaction between ) age at diagnosis and hearing loss or ) sex and hearing loss with either cognitive impairment or emotional distress. ten of the ( %) patients with severe/profound hearing loss in at least one ear were recommended hearing aids, of which ( %) reported compliance most of the time. we conclude that severe/profound hearing loss is significantly associated with cognitive impairment, but not emotional distress, in childhood cancer survivors. our data supports the need for interventions to improve hearing in these patients, including compliance with hearing aids. background: who grade anaplastic astrocytoma is a high grade glioma dependent on vascular endothelial s of s growth factor (vegf) mediated angiogenesis for its growth and infiltration. bevacizumab is a recombinant humanized monoclonal antibody which binds vegf-a and inhibits angiogenesis. common adverse effects of bevacizumab are hypertension, proteinuria, thrombosis and bleeding. while animal model based studies have shown that bevacizumab may impair ovarian function the effects of bevacizumab therapy on human fertility are not clear. since the physiology of pregnancy involves neovascularization/angiogenesis it is recommended that conception be avoided for at least months following exposure to bevacizumab. to describe the course of a young adult who became pregnant after receiving bevacizumab and radiation therapy for treatment of an anaplastic astrocytoma. a year old woman diagnosed with a localized hemispheric who anaplastic astrocytoma was treated with chemotherapy and radiation (temozolomide/ . gy) followed by cycles of bi-weekly bevacizumab/temozolomide. patient opted not to pursue fertility preservation prior to initiation treatment. she experienced bevacizumab-associated proteinuria and hypertension during treatment but received all protocol mandated doses (cumulative doses: bevacizumab = mg/kg; temozolomide = . gm/m ). she had a spontaneous unassisted pregnancy months after completing treatment. her pregnancy was uneventful and she was normotensive throughout. fetal ultrasonography at , , , weeks revealed no abnormality of the brain, heart, great vessels, kidney, extremities, placenta and umbilical cord. at weeks she delivered a female infant via cesarean section (birth weight: grams, apgars: and ) excessive post-partum hemorrhage was not reported. placenta was bi-lobed and weighed g. histological analysis revealed normal placental villous development and maturation and two small infarcts. conclusion: exposure to bevacizumab in our patient had no detrimental effect on fertility and on placental/fetal vascular development. we hope this report will add to the existing data on the effects of bevacizumab therapy on fertility. children's healthcare of atlanta, emory university school of medicine, atlanta, georgia, united states background: reports of malnutrition incidence and prevalence in young cancer patients are variable and not well established. previous research suggests children, especially less than years old, treated with intensive cancer-directed therapy are at higher risk for malnutrition. however, no standardized assessment has been used to evaluate risk in this population. objectives: we aim to assess the trends of weight-for-age for patients following cancer diagnosis. this study will be the first to use a standardized measure of treatment intensity (intensity treatment rating scale, itr- ) and will assist in targeting interventions for identification and treatment of malnutrition. design/method: this observational, retrospective study obtained data through the center's pediatric cancer registry and electronic medical record. patients were classified by tumor type (brain or non-brain tumor) and treatment intensity (itr- ). itr- incorporates diagnosis, chemotherapy, radiation, and surgery, beginning with lowest intensity ( ) to highest intensity ( ). inclusion criteria included new cancer diagnosis - at less than years old, with weight obtained and available within days of therapy start date. incomplete data, alternate growth charts, or treatment intensity of , were excluded. weight was obtained at start of therapy and through years after treatment initiation (approximately days) and converted to z-scores adjusted for age and sex. weight trajectories were modeled using generalized linear mixed models with subject-specific random intercepts and spline functions. separate functions were constructed for subgroups of interest (tumor type and itr). results: there were patients included: patients with brain tumors ( . %) and with non-brain tumors ( . %). of included patients, had treatment intensity of ( . %), of ( . %) and of ( . %). over the observation period, , valid weights were recorded. at initiation of treatment, no difference existed between z-score by tumor type (p = . ) or by intensity ( vs. , p = . ; vs. , p = . ; vs. , p = . ). tumor type did not affect z-score through the follow up period. z-scores were higher for intensity rating vs. and vs. (p = < . and p = . respectively) at days after the start of treatment and persisted through days (p = . and p< . respectively). higher treatment intensity is associated with decline in z-score and failure to return to baseline. future directions include further analysis on specific risk factors and timing of weight loss, longer-term follow-up of weight trends, and targeted interventions for identification, prevention, and treatment of malnutrition. objectives: asses the pt requirements for bleeding episodes in a prospective cohort of pcp using a < × e threshold compared to a < × e /l threshold in a historical cohort. we collected pt data in all pcps treated at our center between january/ through december/ . diagnosis, prescription for pt (prophylaxis vs bleeding disorder), plt count and transfused units were assessed for each pt. pcps treated from january/ through june received prophylactic pt with a < × e threshold (cohort a), and pts treated from july/ through december/ received prophylactic pt with a < × e threshold. pts done for procedures and pts with concomitant hemorrhagic pathology were excluded. we compared the number of pts prescribed as prophylaxis vs bleeding episode between cohorts. data analyzed: graphpad prims . ®. statistical analysis: percentages with confidence interval (ci); t-student test (parametric variables) and mann-whitney test (nonparametric variables). statistical significance: p< . . we reviewed pts ( in cohort a, cohort b) in patients. % had acute leukemia, % received and auto or allo hsct. diagnoses and the proportion of patients undergoing hsct was comparable in both cohorts. the average number of pts per patient was , in cohort a and , in cohort b (p = ns), but a significant difference was found when hsct patients were excluded from this comparison ( , pt per patient in cohort a vs , in cohort b, p = , ), which resulted in an estimated , % reduction in pts prescription. furthermore ( , %) pts were prescribed for bleeding episodes in cohort a versus ( , %) in cohort b (p = ns). patients receiving hsct in the entire group ver-sus those not receiving hsct had similar pt requirements for bleeding episodes ( % vs , % p = ns) conclusion: a < × e plt count threshold for prophylactic pts is safe in pcp in chemotherapy and hsct. it can result in a significant reduction in pt usage. key words: platelets, transfusions, prophylaxis, cancer, childhood. ucsf benioff children's hospital oakland, oakland, california, united states background: transition of care for adolescent and young adult (aya) survivors of childhood cancer from pediatric to adult-oriented long-term follow-up (ltfu) is complex. loss to follow-up is common, and little is known about the success rates among different models. the survivors of childhood cancer program (sccp) at ucsf benioff children's hospital oakland employs a community-based model for transitional care. our multidisciplinary team provides aya survivors a comprehensive treatment summary and recommendations, then facilitates transition to primary care or adult oncology ltfu programs. evaluate the success rate for transition of care among aya survivors of childhood cancer in our ltfu program, and identify barriers to successful transition. design/method: aya patients seen from november to august in the sccp with intent to transition were asked by email or telephone if they had followed up with their designated provider. the primary outcome was successful transition, defined as establishing care within months of their visit. patients were also asked about barriers to transition and to rate the new provider's familiarity with their cancer history and ltfu needs. results: transition was intended for patients. eightyseven were contacted and responded. of these, ( %) successfully transitioned, while ( %) were lost to followup. ages ranged from to years, at to years since completion of therapy. ten ( %) transitioned to a primary care provider, ( %) to an adult oncology ltfu program, and ( %) to a pediatrician. patients rated their new provider's knowledge above average ( . ) on a -point scale from poor ( ) to excellent ( ). survivors lost to follow up indicated the following barriers to transition: loss/change of insurance ( ), inability to find a provider ( ), too busy/forgot ( ), problems with transportation ( ), concerns about cost/copay ( ), and s of s other ( ). twelve patients requested further assistance with transition. conclusion: two-thirds of responding patients successfully transitioned. more work is needed to overcome various barriers to transition for one third of aya survivors. albany medical center, albany, new york, united states background: the transition from active treatment, to offtherapy follow-up, is a stressful event for parents of children with cancer. the psychosocial needs of parents after therapy have received limited attention in the united states with only published quantitative studies, the largest with parents. we have secured funding for and recruited a transition care coordinator (tcc) to investigate this further. objectives: our objective is to assess and screen parents at the end of their child's treatment, and to develop interventions to support parents during this time and thereafter. design/method: after informed consent, a standardized questionnaire, the psychosocial assessment tool (pat . ), was administered to parents at end of therapy (t ), months later (t ) and year later (t ). the tcc provided "universal" intervention to all families with an end of therapy binder containing a treatment summary, follow-up roadmaps, information on late effects, and survivor scholarships. based on their pat . scores, some parents were provided intervention specific to symptoms (targeted intervention for scores - . ) or referred to a behavioral health specialist through the clinic social worker for counseling (for scores > ). results: analysis of pat data showed that % of parents (n = ) scored in the targeted or clinical ranges; % of parents scored in those ranges at pat . significant gender differences were revealed with the mean score for men of . and for women of . . this was confirmed by showing statistical significance (p = . ) when analysis was conducted for only a subgroup of data composed of couples (n = ). analysis of pat data by couples (n = ) showed the mean score for men was . and for women was . (p = . ). gender differences were most apparent in caregiver stress reaction questions that focused on ptsd symptoms. when the subgroup of couples' scores (n = ) for caregiver stress reaction at pat was analyzed, there was a significant difference (p = . ) in caregiver stress reaction with a mean of . for men versus . for women. [note: subcategory scores range from to ]. this study was initiated in october using a tcc and the pat . screening tool. the results suggest greater stress on mothers after therapy, with a substantial proportion of parents having symptoms of ptsd after therapy. background: hodgkin lymphoma (hl) is a common childhood cancer characterized by an inflammatory microenvironment. chemotherapy and radiation may exacerbate this inflammation and contribute to the development of late effects (pneumonitis or pulmonary fibrosis). in a heterogeneous cohort of childhood cancer survivors exposed to pulmonarytoxic therapy, no association between pro-inflammatory cytokines and late pulmonary dysfunction was observed. our objective was to test this association in a relatively uniform cohort of survivors of hl, given the well-recognized proinflammatory background of this disease. objectives: to characterize off-therapy pulmonary function in survivors of hl treated with contemporary therapy, and to investigate its association with persistent systemic inflammation. design/method: blood samples, clinical data, and pulmonary function tests were obtained from survivors of hl ≥ months off therapy. lung function score (lfs), a validated method for assessing degree of pulmonary dysfunction on a scale of i to iv, was determined from diffusion capacity and forced expiratory volume in one second (fev ). for a control group, blood samples from patients with benign, noninflammatory hematologic conditions were used. plasma concentrations of inflammatory cytokines were measured on a luminex platform (emd millipore). associations between clinical features or cytokine levels and lfs i (normal) vs. ii-iv were evaluated using logistic regression or wilcoxon rank sum tests, respectively. results: of survivors (mean age at diagnosis: years, range: - ; mean time off therapy: . years, range: . - ), % were categorized as lfs ii (mild dysfunction), % as lfs iii (moderate dysfunction), and no survivors as lfs iv (severe dysfunction). higher lfs was associated with female sex (p = . ) but not other demographic, disease, or treatment factors. forty-eight survivors had blood samples collected at a mean age of . years (range: - ) with a mean time since treatment completion of . years (range: . - . ). of controls, the mean age at time of blood collection was years (range: - ). survivors did not have significantly elevated cytokine levels compared to controls. female survivors of hl ≥ months off therapy are at increased risk of pulmonary dysfunction. neither evidence for pulmonary dysfunction, as measured by lfs, nor duration of time off therapy were related to systemic inflammation in this study. pulmonary function deterioration and clinical pulmonary symptoms are rarely observed immediately following therapy but increase over time. future studies may consider exploring the contribution of systemic inflammation to pulmonary late effects in survivors farther off therapy, when risk for this late effect is greater. background: thyroid carcinoma is a very rare tumor in pediatrics, accounting for . - % of childhood carcinomas in the united states and europe. we aim to detect the risk of second malignancies among pediatric thyroid cancer survivors. the cohort analysis consisted of pediatric cancer patients aged less than years diagnosed with a primary thyroid cancer and identified by site code icd- - : c , reported to a seer database between and . they were followed up by death or the end of the study period (december , ) . out of patients diagnosed primarily with thyroid carcinoma, there were patients who had incidences of subsequent malignancies. the mean age of patients at initial diagnosis of thyroid cancer was years. females ( . %) had significantly higher incidence of second malignancies (sm) than males ( . %). the overall standardized incidence ratio (sir) of sm in thyroid pediatric patients was higher than expected (sir = . ). some specific sites showed significantly higher incidences: salivary gland (sir = . ), gum and other mouth (sir = . ) and kidney (sir = . ). the overall risk of sm in patients received radioactive iodine was higher than expected (sir = . ). the cumulative inci-dence of sms from the initial diagnosis of thyroid cancer was calculated with the survival methodology of competing risk, death treated as a competing event. cumulative incidence of sm was . % [ % ci ( . , . %)] at years and substantially expanded after years, reaching . % [ % ci ( . , . %)] at years. the cumulative incidence of each tumor type at years was . % [ % ci ( . , . %)] for breast cancer, . % [ % ci ( . , . %)] for salivary gland, . % [ % ci ( . , . %)] for each one of kidney and cervix uteri and . % [ % ci ( , . %)] for each one of ovary and melanoma of the skin. cumulative incidence of sm was stratified based on race, gender and radiotherapy exposure, but there was no statistical difference in each of them. conclusion: race, gender, histological subtypes, and radioactive iodine may play an important role as prognostic factors for developing sm among pediatric thyroid cancer survivors. identification of underlying mechanisms that raise the risk of sm is important for both treatment and follow-up strategy. background: the ethical practice of informed consent requires it be both voluntary and understood by the research participant. in pediatric oncology, parents must undergo informed consent to enroll their child with cancer into clinical trials, but often it can be difficult to understand especially for parents with low english proficiency. previous research has shown that parents of children with cancer have difficulty understanding voluntariness, and that parental satisfaction with informed consent does not always correlate with adequate comprehension. objectives: to examine socio-demographic and contextual correlates of comprehension of informed consent, voluntariness, and satisfaction in parents who consented to participation of their child in a cancer clinical trial. we focused on characterizing differences between non-hispanics and hispanics, the fastest growing ethnic group in the u.s. design/method: parents/guardians (n = ) of children aged - years with newly diagnosed cancer, who had consented to participation of their child in a clinical trial for cancer treatment at rady children's hospital-san diego were s of s prospectively recruited. parents completed questionnaires assessing comprehension, voluntariness, satisfaction, health literacy, socio-demographics, and acculturation level, if hispanic. comprehension was surveyed at baseline and longitudinally at months. comprehension, voluntariness and satisfaction outcomes were analyzed by socio-demographics, health literacy, and acculturation level using logistic regression. results: of the participants surveyed, ( . %) were hispanic and ( . %) were non-hispanic. we found that higher health literacy was associated with greater objective comprehension (p< . ), voluntariness (p< . ), socioeconomic status (p< . ), and acculturation (p< . ). hispanics reported lower objective comprehension (p = . ), voluntariness (p = . ), health literacy (p< . ) and ses (p = . ) compared to non-hispanics. spanish-speakers reported lower voluntariness (p = . ), health literacy (p< . ), and acculturation (p< . ) compared to englishspeakers. at the -month follow-up, comprehension in hispanics significantly improved (p = . ) compared to their baseline comprehension. satisfaction was moderately high across all subgroups and was not significantly impacted by socio-demographics, health literacy, or acculturation. in this study, with equivalent numbers of hispanic and non-hispanic participants, we found that hispanic and spanish-speaking parents of children with newly diagnosed cancer had inadequate informed consent comprehension, voluntariness and health literacy despite high satisfaction. our study suggests that hispanics and individuals with limited english proficiency are not making truly informed decisions for their child with cancer. to ensure the ethical practice of research in pediatric oncology, the informed consent and decision-making process must be improved with culturally and linguistically interventions for these underserved populations. memorial sloan kettering cancer center, new york, new york, united states background: pediatric oncology patients undergo repeated bone marrow aspirations and biopsies (bma/bx). these potentially painful procedures can exacerbate anxiety and distress. standard practice at memorial sloan kettering (msk) department of pediatrics is to use propofol, which has amnestic but no analgesic properties. we sought to evaluate whether the addition of local anesthetic would improve patient experience with bma/bx. the purpose of reppair: reducing procedural pain and improving recovery of quality of life (qol) (nct ) is to evaluate the efficacy of local anesthesia with ropivacaine in reducing procedural pain and improving post-procedure qol in pediatric neuroblastoma patients undergoing bma/bx with general anesthesia. reppair is a prospective, randomized, crossover clinical trial that opened for enrollment october . eligible patients were - years old with neuroblastoma. participants were observed on trial for two sequential bm procedures; one procedure with intervention a: propofol alone (pa), and the other with intervention b: propofol plus ropivacaine (p+r). participants were randomized to intervention sequence ab or ba and were blinded to the order of interventions. participants and recovery room (rr) nurses, who were also blinded, followed a standardized postprocedure pain management algorithm. the primary endpoint was percentage of participants requiring opioid analgesia in the hours post-procedure. secondary endpoints included total opioid in hours, non-opioid analgesia use, pain scores, time to first opioid, and short-term qol. qol was assessed by a parent-proxy metric that evaluated pain interference with sleep, physical, emotional, and social recovery. as of january , patients were assessed for eligibility and patients were randomized ( have completed both procedures). for the primary endpoint, a slightly higher proportion of participants required opioid for pa than p+r ( % versus %, p = . ). pain scores in the rr were significantly higher for pa than p+r (median [ th, th percentile]: [ , ] versus [ , ], p = . ). there were no statistically significant differences in total opioid or non-opioid analgesia, -and -hour pain scores, median time to first opioid, or pain interference scores. there were no adverse events. conclusion: preliminary findings of the reppair trial suggest that local anesthesia does not reduce the need for opioid analgesia or improve short-term qol in pediatric patients undergoing bma/bx with general anesthesia. local anesthesia did improve pain scores in the immediate recovery period. final results of this study will help establish evidence-based guidelines and optimize the experience of pediatric patients with bone marrow procedures at our center. background: children with advanced cancer experience a range of symptoms throughout treatment or at end of life, some of which are poorly controlled. minimizing suffering, including effective symptom management, in children with advanced cancer is a central value for pediatric oncology clinicians. patient-reported outcomes have been used in symptomrelated research in pediatric oncology patients; however the majority of literature specific to symptoms during palliative care and end of life for children and adolescents with advanced cancer is based primarily upon medical record reviews and to a lesser extent, patient self-report. the purpose of this study was to prospectively describe symptom frequency, severity, and level of distress in children/adolescents with advanced cancer using patient selfreport and parent proxy. design/method: a prospective cohort design was used for this study. five pediatric oncology institutions from across the united states participated. children and adolescents were eligible to participate if they were - years of age, englishspeaking, and had a diagnosis of advanced cancer, defined as a -week history of progressive, recurrent, or non-responsive disease or a decision not to pursue curative-focused therapy. a modified version of the memorial symptom assessment scale (msas) was used to measure symptom frequency, severity, and level of distress and was administered to child/parent dyads electronically via smartphones every two weeks. information regarding disease status and cancer treatment was collected concurrently. data was analyzed using descriptive statistics and univariate logistic regression analysis. results: a total of children and adolescents and parents participated in the study. the median age of child participants was years, with half being male. the median age of parents was years. the child participants had a variety of primary diagnosis, including: leukemia/lymphoma (n = , %), solid tumor (n = , %), and brain tumor (n = , %). the most frequently reported symptoms by children with advanced cancer and parents were pain (n = / , . %), lack of energy (n = / , . %), and nausea (n = / , . %). presence of disease (p = < . ), recent disease progression (p = . ), and receiving cancer therapy (p = . ) were significant factors on the presence of pain. high intensity cancer therapy was a significant factor on pain frequency (p = . ) and level of distress (p = . ). it is feasible to collect data prospectively in children with advanced cancer regarding symptom frequency, severity, and level distress. clinicians' increased understanding of the symptom experience may promote communication with children and adolescents and timely intervention. more research is needed to understand symptom clusters in children with advanced cancer. vanderbilt children's hospital, nashville, tennessee, united states background: febrile neutropenia (fn) is a frequent occurrence in children undergoing chemotherapy. though guidelines recommend adding a second antibiotic to broad-spectrum antipseudomonal coverage in specific scenarios, augmenting empiric therapy with a second antibiotic is common practice. additional empiric antibiotic (aea) use increases the risk of antibiotic toxicity and future antimicrobial resistance. data clarifying the indications for aea are limited in pediatric patients. objectives: to identify risk factors for gram-positive (gp) and gram-negative (gn) bacteremia in patients presenting with fn to determine situations in which aea use is warranted. design/method: a retrospective chart review was conducted of pediatric severe fn with absolute neutrophil count < / l occurring at a single institution between and . potential a priori risk factors based on clinical reasons for antibiotic expansion were chills, hypotension, mucositis, skin or soft tissue infections (sstis), recent administration of highdose cytarabine (hdac), and a diagnosis of acute myeloid leukemia (aml). potential factors for gn bacteremia were chills, hypotension, mucositis, and abdominal pain. the association between each potential risk factor and gp or gn s of s bacteremia was identified. logistic regression was used for multi-variable analysis. the review yielded episodes. gp bacteremia was isolated in cases ( . %) and gn bacteremia in episodes ( . %). in multivariable analysis, hypotension (or . ( % ci . , . ), p = . ) and sstis (or . ( . , . ) , p = . ) were independently associated with increased risk of gp bacteremia, while mucositis (p = . ), recent administration of hdac (p = . ) and chills (p = . ) were not. ten patients with aml didn't receive hdac, thus the association between aml and gp bacteremia could not be reliably estimated. hypotension (or . ( . , . ), p< . ) and chills (or . ( . , . ), p< . ) were independently associated with a higher risk of gn bacteremia, while mucositis (p = . ) and abdominal pain (p = . ) were not. of the gn infections, ( %) were resistant to cefepime, the empiric agent of choice at our institution. patients with fn with sstis, hypotension, or recent hdac had increased risk of gp bacteremia indicating potential benefit of empiric vancomycin in these settings, while mucositis and chills were not associated with gp bacteremia. hypotension and chills were associated with gn bacteremia, potentially warranting empiric antibiotic expansion, while mucositis and abdominal pain were not. identifying specific indications for aea use in pediatric severe fn use may improve antimicrobial utilization, decrease unnecessary antibiotic use, and improve patient outcomes. background: for children/young adults with incurable high grade gliomas (hggs), like diffuse intrinsic pontine glioma (dipg) or glioblastoma multiforme (gbm), oncologists endeavor to align therapy with patient/family goals of care, but may be influenced by providers' preferences or limited resources. ethical challenges can arise around the perceived purpose, risks and benefits of therapy options, provider conflicts of interest, access to care, deciding decisional priority between patients and families, and conflicts around end-oflife care. objectives: evaluate factors that play into longitudinal decision making for children and young adults with hggs, their families and oncologists using a qualitative approach with ethnographic elements. design/method: eligible patients were aged - with dipg, gbm, or secondary hgg. patient exclusions included: non-english speaking, in state custody, death prior to diagnosis, seen by oncology once, or an oncologist declined participation. key decision making visits (e.g. mri reviews) were serially audio-recorded, along with subsequent : semistructured interviews with patients and/or parents about the decision making process. field notes from clinician meetings, chart notes, and oncologist questionnaires were obtained. discussions and interviews were transcribed and independently coded by three investigators. inter-rater reliability was assessed during code book development. discrepancies were discussed until consensus met. constant comparison analysis with maxqda software continued until thematic saturation. results: twenty-two of eligible patients were approached; agreed to participate. one withdrew upon transferring care. mean age was . years (sd . ); % male, % caucasian, % african american, % hispanic, and % asian. four encounters, ( . hours), were recorded on average per patient. parent/patient interview themes included: ) hope (for a cure, prolonged life, and quality of life), ) importance of physician recommendations, ) importance of support systems (family, community, social media), ) food (as cancer etiology, intervention) ) finances (personal, research funding), ) communication (with medical providers, family, community), ) death, and ) god (beliefs, prayer, existential questions). oncologists desired prolonged quality of life, while patients/families transitioned to that hope from hope for a cure. decisions made in the setting of hggs are multi-factorial, ultimately reflecting the competing values of decision makers. optimism about treatment efficacy is held in tension with poor prognosis, allowing for functional hope. acknowledging patients' and families' shifting hopes allows for changes in goals of care and shared decision making. future work is needed to ) develop preference tools for pediatric patients and families to inform medical providers and ) provide training in communication and shared decision making with oncologists. emory university, atlanta, georgia, united states background: bone marrow transplantation (bmt) is a potentially curative but underutilized treatment for scd. our previous work has shown that there is variation in physician philosophy and practice in considering bmt as a treatment option for patients with scd, and physicians may not discuss this with patients and families as a potential treatment option. in a randomized clinical trial to test the effectiveness of a decision aid for disease modifying therapies for sickle cell disease, adult patients with scd as well as caregivers of adult/pediatric patients were interviewed about how they seek or have sought information related to scd, made decisions about treatments for scd, and identified a treatment option they were interested in learning more about using the decision aid tool. we performed a secondary analysis of these baseline data to understand patient information needs and attitudes regarding bmt as a treatment option for scd. the goals of this analyses was to understand patient and caregivers' attitudes and perceived information needs regarding bmt as a treatment option for scd. we performed an analysis of baseline interviews from caregivers of patients with scd or adult patients from a randomized control trial for a decision aid tool for scd. of the interviews belonged to caregivers of patients with scd. in addition to reviewing interviews for discussion of bmt, we interrogated for mention of terms such as 'bone marrow transplant' or 'cure' or 'stem cell transplant'. interviews were coded using nvivo and analyzed for emerging themes. results: of the baseline interviews, interviews met selection criteria. thirteen of the interviews were with caregivers of pediatric patients, and the remainder were with adult patients, including young adult patients with scd. the majority of participants want to learn about bmt or curative options. in many participants, this was expressed despite knowledge that they were not a likely candidate for transplant. desired information about bmt included eligibility, benefits, risks, long-term effects, quality of life and financial aspects related to bmt. of the patients who discussed how they learnt about bmt, approximately half mentioned that their healthcare provider had not previously mentioned this to them. we then examined knowledge of bmt and attitudes with demographic and clinical variables. patients and caregivers of pediatric patients with scd want to learn about bmt as a treatment option. healthcare providers should consider discussing bmt with their patients with scd. natasha frederick, anna revette, alexis michaud, jennifer mack, sharon bober dana-farber cancer institute, boston, massachusetts, united states background: adolescents and young adults (ayas) consistently identify the need for improved patient-clinician communication on sexual and reproductive health (srh) issues. however, oncology clinicians do not routinely integrate srh conversations with ayas through disease treatment and survivorship. little is known about why these conversations do not take place. objectives: explore aya perceptions of and receptiveness to srh communication with oncology clinicians and to identify barriers and facilitators to these conversations. design/method: semi-structured interviews were held with aya cancer patients and survivors (ages - years, men, women). twelve participants were on active treatment and were within years of treatment completion. interviews were conducted in english by phone or in person. the interview transcript underwent pre-testing with ayas. all interviews were audio-recorded and transcribed verbatim. transcripts were analyzed and summarized by two trained qualitative researchers according to standard comprehensive thematic qualitative analysis methods. analyses were aided by nvivo software. results: ayas perceived existing srh communication between ayas and oncology providers as inadequate. all ayas reported a need for improved srh communication with oncology providers, and three key areas of need emerged: ) general education; ) addressing specific srh issues experienced during treatment and survivorship; and ) understanding the long-term impact of cancer and treatment on srh. ayas felt that current srh discussions are limited and too narrow in scope and scale. ayas reported that most srh conversations focus exclusively on fertility (n = ), usually taking place at the start of treatment. other additional yet limited communication reported was about sexual activity (n = ), contraception (n = ), sexual function (n = ). no ayas reported conversations about potential treatment complications related to sexuality other than infertility. key barriers to srh conversations include patient discomfort initiating conversation (n = ) and presence of family members (n = ), with additional reported barriers including perceived provider discomfort (n = ), lack of rapport with provider (n = ), and age/gender differences (n = ). ayas felt that s of s communication tools such as handouts, brochures, and websites would be helpful facilitators to direct communication from the oncology clinician, and wanted conversations to start before treatment initiation and to continue through treatment and survivorship conclusion: ayas identify a key role for pediatric oncology providers in srh care from diagnosis through survivorship, however multiple barriers interfere with discussions about srh on a regular basis. identified barriers suggest that future efforts should focus on provider education and training in srh and srh-related communication in order to optimize care provided to this unique patient population. background: peripherally inserted central venous catheters (picc) provide secure vascular access in pediatric patients for the delivery of necessary therapies. the ease of placement in the inpatient and outpatient settings has expanded their utilization. however, recent data analyses show a significant increase in venous thromboembolism (vte) risk with the use of picc lines. with its rising use, modifiable risk factors need to be understood for preventative measures. objectives: in this study we aim to understand patient and catheter specific characteristics in relation to the development of vte. design/method: with irb approval, a retrospective interrogation of the electronic medical record and a picc database, at rainbow babies and children's hospital, was completed. the study cohort contained patients < years of age who had a picc line placed between january of and december of . data collected included indication for line placement, line dwell time, location of insertion including blood vessel and extremity, number of attempts at line placement, lumen size and indwelling line length. in addition, we collected number of days to vte formation, associated symptoms and location of vte. chi-squared analyses and fischer's exact test were used where appropriate for statistical analysis. we analyzed ( neonatal) newly placed picc lines. fifty line-associated vte events were found, for an incidence of . %. all vte occurred with the placement of the first picc line. intravenous therapies were the most common reason for line placement. no statistical significance was found between various indications for placement. the most common symptom of vte manifestation was extremity swelling, follow by extremity pain. right extremity picc was found to have a higher incidence of vte. larger catheter lumen sizes (> french) had a higher incidence of vte. we found a mean time of . days to vte detection. we were unable to find any clinical, patient or line specific factors leading to increased vte formation after statistical analysis. special consideration should be given to the duration of picc line use as this may reduce the incidence and comorbities associated with vte. there is still much to be understood about catheter associated vte formation as our analyses indicates the need for prospective data collection on a larger scale in hopes to create guidelines related to catheter use in pediatrics. background: the decision to transfuse a patient is a complex one and is never based solely on a number; however, certain hemoglobin or platelet count thresholds have been proposed in aiding physicians make transfusion decisions. in our hospital, the thresholds for packed red blood cell (prbc) and platelet transfusion in pediatric oncology patients are hemoglobin levels below . g/dl and platelet counts below , /mm (< , for brain tumors), respectively. recently, these thresholds have been questioned and we were asked whether we could safely lower the thresholds to < . g/dl of hemoglobin and < , /mm platelet count objectives: to investigate platelet and hemoglobin transfusion thresholds for oncology patients at children hospital of michigan design/method: retrospective chart review over a -month period, examining platelet and hemoglobin pretransfusion levels for each prbc and platelet transfusion given to oncology patients results: over the course of months, eligible oncology patients (median age years) received transfusions ( prbc transfusions and platelet transfusions). the mean pretransfusion hemoglobin level was . ± . g/dl (range . - . ) (n = ) for total prbc transfusions and this was not different among disease categories (p = . ). patients who had anemia symptoms and signs (n = ) had a slightly lower hemoglobin level compared to those who did not (n = ): . ± . vs . ± . g/dl (p = . ). the mean pretransfusion platelet count was , ± , /mm (range , - , ) for total platelet transfusions (n = ); , ± , /mm in patients with brain tumors (n = ); , ± , in patients with leukemia (n = ); and , ± , in patients with solid tumors (n = ). the mean pretransfusion platelet count was significantly higher in transfusions for brain tumors compared to that in the other disease groups (p< . for both). the mean pretransfusion platelet count was not different among those patients who had bleeding/bruising symptoms ( , ± , , n = ) versus those who did not ( , ± , , n = ) (p = . ). the bleeding/bruising rate was slightly but insignificantly higher in those who had platelet counts < , vs those who had ≥ , ( . % vs . %, p = . ). since most patients develop symptoms of anemia at hemoglobin above g/dl and about / of patients develop bleeding/bruising symptoms at platelet counts above , /mm , our current policy so far reflects a safe threshold for transfusion, and further lowering of the thresholds should be investigated in prospective studies. background: renal impairment is an important complication of childhood cancer and its treatment. serum creatinine level is frequently used as a screening test to monitor renal function; however, patients can have significantly decreased glomerular filtration rate (gfr) with normal serum creatinine. to determine the prevalence of chronic kidney disease (ckd) among children with cancer diagnosis, based on calculated gfr. to compare the difference between using serum creatinine value alone versus gfr in detecting ckd. design/method: retrospective review of medical records of patients, age - years, diagnosed between / - / with solid tumors were analyzed. serum creatinine and calculated gfr using schwartz formula were recorded. ckd as classified by the foundation of kidney disease and outcome quality initiative was used: ckd stage : gfr ( to ml/min per . m ) ckd stage : gfr ( to ml/min per . m ) statistical analysis using spss software v. . chi-squared test for proportions within group, and pearson chi-squared and fisher exact tests for statistical differences between groups. p-value < . was considered to indicate significance results: out of the records reviewed, ( %) were males and ( %) females, with mean age of . ± . years. ( . %) patients received one or more of nephrotoxic chemotherapy drugs; cisplatinum, carboplatinum, or ifosphamide mainly in the non-wilms solid tumors group ( . %) compared to ( . %) in the wilms tumor (wt) group. based on calculated gfr (by schwartz formula) ckd stage /or was diagnosed in ( %) patients with overwhelming majority ( %) were in the mild stage ckd, only ( . %) of those patients had abnormally high serum creatinine levels (p = . ). . % of patients who received nephrotoxic chemotherapy developed ckd, compared to . % in those who did not receive it, (p = . ). despite that only / ( %) of wt group patients received nephrotoxic chemotherapy, yet this group had higher percentage of ckd ( . %) compared to non-wt group ( . %) p = . . significantly lower mean gfr . ± was noticed in the wt group compared to . ± in non-wt group (p = . ) conclusion: high prevalence of mild ckd was found among solid tumor patients. using serum creatinine alone as measure of renal function significantly under estimates renal impairment in those patients. early identification of ckd is easily achieved by using calculated gfr, which can helps providers and care givers to avoid potential nephrotoxic antibiotics, contrast media, nsaids and dehydration that may further deteriorate renal function the university of texas southwestern medical center, dallas, texas, united states background: children with down syndrome (ds) have increased risk of developing leukemia. pediatric patients with ds-associated acute lymphoblastic leukemia (ds-all) are known to have significant toxicities with reinduction chemotherapy and historically poor outcomes with stem cell transplant (sct). anti-cd chimeric antigen receptor (car) t-cell therapy, tisagenlecleucel, demonstrated high rates of durable complete remission (cr) and a manageable safety profile in children with r/r b-cell acute lymphoblastic leukemia (b-all). objectives: characterize the efficacy and safety of tisagenlecleucel in pediatric/young adults with ds-all. design/method: pooled data from single-arm, multicenter, phase trials of tisagenlecleucel in pediatric/young-adult patients with r/r b-all (eliana, nct ; ensign, nct ) were analyzed. eight patients with ds-all were enrolled (data cutoff: eliana, november ; ensign, february ). seven were infused with tisagenlecleucel; patient died from all progression and intracranial hemorrhage before infusion. no manufacturing issues occurred during production. / infused patients were male, / had prior sct (age range, - years). / patients achieved cr or cr with incomplete blood count recovery (cri) by day (d) (cr+cri, %); died before d and was not evaluable. analysis of minimal residual disease was negative in bone marrow in responding patients. two patients had cd negative relapses at and months. ongoing remissions in patients without relapse ranged from to months. the safety profile (n = ) appears similar to that in patients without ds in the same trials (n = ). grade (g) / cytokine release syndrome occurred in % ( / ) of patients with ds and in % without ds. rates of other g / adverse events of special interest did not appear to favor a consistent trend between patients with/without ds (febrile neutropenia: % vs %; neurological events: % vs %; tumor lysis syndrome: % vs %). g / infections were not observed in patients with ds ( % vs %). one patient died after infusion due to intracranial parenchymal hemorrhage on d associated with ongoing coagulopathy. time and extent of tisagenlecleucel expansion and long-term persistence were similar between groups. conclusion: this is the first analysis of car t-cell therapy in pediatric patients with r/r b-all and ds. these data suggest that toxicities appear similar to those in patients with b-all without ds, remission rates in ds-all are high, and longterm outcomes with sustained persistence appear promising. further exploration of tisagenlecleucel as an alternative to sct in children with r/r ds-all is warranted. sponsored by novartis. background: hispanic adolescence and young adults are twice as likely to develop acute lymphoblastic leukemia (all) with high risk features as non-hispanic whites. they also have poor prognosis and % higher death rate. b-all with crlf overexpression caused by genetic alteration of the cytokine receptor, crlf is five times more common in this subgroup. approximately % of crlf b-all cases also have ikzf genetic alterations. ikaros is involved in transcriptional regulation of several important genes involved in leukemogenesis. overexpressed casein kinase ii (ck ) impairs functions of ikaros. objectives: understand the molecular mechanisms that regulate crlf expression in crlf b-all. here we present evidence that ikaros-mediated repression of crlf transcription in b-all in hispanic children is regulated by ck . design/method: primary b-all patient samples from hispanic children were used. ikaros retroviral transduction, ikaros shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: ikaros binding to promoter of crlf was confirmed using quantitative chip. functional experiments such as overexpression of ikaros in b-all primary cells results in transcriptional repression of crlf whereas ikaros silencing using shrna resulted in increased transcription. these results suggest that ikaros negatively regulates crlf expression. molecular inhibition of ck with shrna targeting the ck catalytic subunit, as well as pharmacological targeting of ck with cx resulted in transcriptional repression of crlf . ck inhibition was associated with increased ikaros dnabinding to the promoter of crlf . however, the ability of cx to repress crlf is lost or severely reduced, in cells with shrna silencing of ikaros, as compared to cells with intact ikaros. moreover, similar results were noted following treatment with cx in leukemia cells obtained from high risk b-all patients with deletion of one ikzf allele. ikaros binds poorly to promoters of crlf gene in these cells. treatment with cx restores ikaros dnabinding to the promoters of crlf , which is associated with its strong repression. serial qchip analysis of the epigenetic signature at the crlf promoter showed that increased ikaros binding to the crlf promoter, following ck inhibition, is associated with enrichment for the h k me histone modification, which is a marker of repressive chromatin. results demonstrate that crlf expression is epigenetically regulated by the ck -ikaros axis .cx show antileukemic effect via restoration of ikaros tumor suppressor function, resulting in crlf repression suggesting advantage of using ck inhibitors as potential therapeutic approach in crlf altered b-all. results: hypodiploid all (modal chromosome number < and/or di < . ) was identified in patients ( . % of all patients; . % of nci standard risk (sr) and . % of nci high risk (hr)), who were removed from frontline protocol therapy post-induction. overall -year efs and os were . %± . % and . %± . %. transplant status was retrospectively available for / ( %), of whom underwent hsct in cr . five-year efs with hsct was . %± . % vs. . %± . % without (p = . ). -year os with and without hsct was . %± . % vs. . %± . % (p = . ). when corrected for the median time to hsct ( days), there were no significant differences in -year efs or os rates with and without hsct: . %± . % and . %± . % vs. . %± . % and . %± . %. no nci risk group or mrd subset benefitted significantly from cr hsct. sr patients (n = ) had -year efs and os of . ± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. hr patients (n = ) had -year efs and os of . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. for those with end-induction mrd < . % (n = ), -year efs and os were . %± . % and . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. end-induction mrd-positive patients (n = ) fared poorly with both year efs and os of . %± . % with hsct (n = ) vs. . %± . % and . %± . % without. multivariate regression analysis including nci risk group, mrd, and cr hsct, showed only mrd negativity was significantly associated with efs (hr . , p< . ) and os (hr . , p< . ). patients with hypodiploid all fare poorly, particularly those with end-induction mrd ≥ . %. while cr hsct is a standard treatment approach, it does not confer significant benefit. we were unable to assess bridging therapy prior to hsct, and comparator groups are small. taken together, however, new strategies are urgently needed for these patients. background: ras-pathway mutations are known to play a pivotal role in a significant proportion of myeloid malignancies, including upwards of % of pediatric aml cases. ras-pathway mutations in myeloid malignancy commonly co-occur with mutations of epigenetic regulators, suggesting cooperative leukemogenesis. among the epigenetic modifiers most frequently mutated in myeloid malignancy are regulators of dna methylation. this indicates that the alteration of dna methylation contributes to leukemogenesis. the ten-eleven translocation (tet ) is an epigenetic regulator that plays an important role in regulation of dna methylation through its action of hydroxylation of -methylcytosine, which ultimately leads to passive de-methylation of dna cytosines. in myeloid malignancy, loss of function tet mutation is one of the most frequently co-occurring lesions in ras mutated malignancy. how specifically the altered methylation patterns in ras-pathway driven diseases promotes leukemogenesis is unclear. objectives: we hypothesize in mice with a ras-pathway mutation, that when an epigenetic modifier co-occurs, such as loss of function of tet , this primes stem cells and/or early differentiating progenitors for transformation by preventing the repression of stem cell self-renewal genes, inhibiting differentiation, enhancing ras signaling and leading to leukemogenesis. we have generated a novel murine model with constitutive deletion of tet (tet -/-) combined with an inducible activating krasg d mutation (krasg d/wt). mice have been tracked for evidence of hematologic malignancies and compared to mice with corresponding single genetic lesions. cooperative leukemogenesis will be demonstrated by decreased latency to disease onset, impact on malignancy lineage, in addition to investigating mechanistically through which pathways leukemogenesis may be promoted. results: krasg d/wt/ tet -/-mice demonstrate statistically significant differences in peripheral white blood cell count, hemoglobin, and platelet levels as early as -weeks post ras-pathway activation. peripheral cell lineage analysis demonstrates early skewing toward myeloid differentiation and marked splenomegaly in mice harboring both genetic lesions compared to wild type or mice with single genetic lesions. phospho-flow cytometric analysis reveals increased perk and ps activation in krasg d/wt/ tet -/-sca- enriched bone marrow cells compared to either genetic lesion alone. our study utilizing a murine model to examine how in ras-pathway mutations the addition of a co-occurring epigenetic lesion demonstrates that these lesions appear to cooperate to promote early myeloid differentiation with attendant changes in signaling pathways. this exploration to elucidate the mechanics of ras-pathway mediated disease lay the foundation for identification of patients who may benefit from existing therapies, such as dmtis, or identify new signaling targets for therapeutic exploration. background: the humoral immunogenicity of car , a chimeric antigen receptor (car) with a murine scfv domain developed for treatment with tisagenlecleucel in relapsed/refractory (r/r) pediatric/young-adult acute lymphoblastic leukemia (all), was evaluated in studies. little is known about the presence/impact of preexisting/treatmentinduced anti-murine car (mcar ) antibodies in patients treated with car therapy. objectives: patients from eliana (nct ; n = ) and ensign (nct ; n = ) were evaluated before and after tisagenlecleucel infusion to determine the impact of anti-mcar antibodies on cellular kinetics, efficacy, and safety. design/method: anti-mcar antibodies were determined by flow cytometry and reported as median fluorescence intensity. assay validation included evaluation of the interferences of intravenous immunoglobulin (ivig) treatment with the anti-mcar antibody assay. impact of preexisting and treatment-induced immunogenicity on cellular kinetics, efficacy, and safety was determined. treatment-induced immunogenicity was defined by a positive increase in anti-mcar antibody levels over baseline and was assessed by calculating the fold-change between preexisting (ie, baseline) and postinfusion levels. results: % of patients displayed preexisting anti-mcar antibodies; a similar incidence was detected in healthy volunteer samples during method validation. % of patients developed treatment-induced anti-mcar antibodies. no relationship was identified between tisagenlecleucel expansion (auc - d) and preexisting/treatment-induced anti-mcar antibodies (r < . and r = . , respectively); similar results were seen for cmax. presence of treatment-induced anti-mcar antibodies did not appear to impact transgene persistence or response. kaplan-meier estimates showed that preexisting/treatment-induced anti-mcar antibodies did not appear to impact duration of response or event-free survival. strip plots showed consistent levels of preexisting/treatment-induced anti-mcar antibodies across patients with safety events, including cytokine release syndrome, neutropenia, thrombocytopenia, and neurological events. there was no apparent relationship between treatment-induced anti-mcar antibodies and b-cell recovery categories (≤ months, > and ≤ months, > months, and ongoing sustained aplasia). no association existed between time of b-cell recovery and presence of treatment-induced anti-mcar antibodies. b-cell aplasia requiring ivig occurred following tisagenlecleucel in the majority of patients. the tisagenlecleucel concentration-time profiles in patients with treatment-induced anti-mcar antibodies were categorized by time following ivig administration. time of ivig administration had no impact on in vivo transgene expansion and persistence. we report the first comprehensive assessment of the impact of anti-mcar antibodies on clinical endpoints with car therapy. pediatric/young-adult patients with r/r all had a high frequency of baseline anti-mcar antibodies, and preexisting/treatment-induced anti-mcar antibodies did not impact the cellular kinetics, safety, and efficacy of tisagenlecleucel. cell-mediated immunity studies are ongoing. sponsored by novartis. background: adoptive immunotherapy, using cd engager (cd -eng) t-cells, has shown success in preclinical studies, recognizing and killing acute myeloid leukemia (aml) blasts in vitro and in vivo. cd -eng t-cells secrete bispecific molecules that recognize cd (t-cells) and cd (aml blasts), and are able to direct transduced t-cells and recruit bystander t-cells to kill cd -positive blasts. however, cd -engs do not provide costimulation and have not shown the capability for sequential killing of targets in vitro. we are seeking to improve the expansion, persistence and sequential killing capabilities of cd -engs by genetically modifying these cells with an inducible costimulatory molecule, which can be activated by a chemical inducer of dimerization (cid). we generated a retroviral vector encoding cd -eng and the inducible costimulatory molecule myd .cd linked by a a sequence (cd -eng. a.imc). cd -eng and cd -eng.imc t-cells were generated by retroviral transduction, and their effector function was compared with and without cid. we used flow cytometric analysis to assess transduction efficiency, chromium release assays to evaluate cytolytic activity, and elisa to determine cytokine production. we successfully generated cd -eng.imc tcells and achieved a mean initial transduction efficiency of % that was maintained above % throughout our study period. cd -eng.imc t-cells +/-cid and cd -eng t-cells readily killed cd -positive aml blasts (molm and kg a) in cytotoxicity assays when compared to the cd -negative control (k ). in co-culture assays, cd -eng.imc t-cells secreted increased il- and ifn-gamma in the presence of cid and cd -positive targets (kg a and molm ) when compared to co-culture with cd -positive targets in the absence of cid. in addition, cd -eng.imc t-cells displayed enhanced sequential killing capabilities and ifn-gamma secretion when stimulated weekly with cid and tumor cells at a : ratio when compared to cd -eng t-cells. conclusion: cd -eng.imc t-cells are able to recognize and kill cd -positive aml blasts in an antigen dependent manner. cd -eng.imc t-cells have improved effector function in the presence of cid as judged by cytokine production and their ability to sequentially kill cd -positive target cells. thus, inducible myd and cd costimulation is a promising strategy to improve the effector function of cd -eng t-cells, and warrants further active exploration in preclinical studies. background: eliana (nct ; n = ) is a pivotal multicenter study testing the efficacy of tisagenlecleucel, anti-cd car-t, in children/young adults with r/r b-all. tocilizumab (toci) has been used for management of moderate/severe (grade / ) crs in ≈ % of patients treated with tisagenlecleucel at equivalent doses used in approved nononcological pediatric indications (< kg received mg/kg; ≥ kg received mg/kg [ mg max dose]).( ) crs onset, as graded by the penn grading scale, generally occurred at a median of days (range, - ) after infusion, requiring administration of - toci doses in some patients via a protocol-specific treatment algorithm. toci is a humanized monoclonal antibody that inhibits il- receptor (il- r) signaling. the pharmacokinetics (pk) and pharmacodynamics (pd) of toci in pediatric patients with b-all with carassociated crs have not previously been described. objectives: characterize toci pk/pd for crs management following tisagenlecleucel infusion and describe its impact on cellular kinetics. design/method: toci pk and levels of soluble il- r (sil- r) were determined from serum and quantified using validated assays. maximum toci concentration (cmax) was derived using noncompartmental methods. sil- r, proinflammatory cytokines, and crs resolution time were characterized to describe toci pd. summary statistics and graphical analyses of tisagenlecleucel exposure by number of doses were performed to describe the impact of toci on tisagenlecleucel kinetics in patients responding to tisagenlecleucel infusion. : / patients with crs received the first toci dose at a median of days (range, - ) after crs onset. seventeen patients received dose (range, . - mg/kg); received doses ( - mg/kg); received doses ( - mg/kg), per the crs treatment algorithm. first-dose mean cmax (sd) was ≈ ( . ) g/ml; second dose, ≈ ( ) g/ml. individual patient pd concentration-time profiles showed increased sil- r levels after the first toci dose which remained elevated following the second dose. following toci administration, median time to crs resolution (including fever resolution) was days (range, - ). crs onset coincided with tisagenlecleucel expansion, followed by a peak in serum cytokines, including il- . the geometric mean auc - day and cmax of tisagenlecleucel transgene (by pcr) were % and % higher in tisagenlecleucel-responding toci-treated patients. conclusion: crs symptoms resolved within a median of days after toci administration. toci levels achieved in patients with b-all were similar to reported pediatric nononcological indications (tocilizumab label) and resulted in concentration/time-dependent sil- r increases. transgene continued to expand and persist following toci administration. these data support treatment with toci for crs management. ( ) buechner, eha, . sponsored by novartis. background: in acute myeloid leukemia (aml), mesenchymal stem and stromal cells (mscs) in the bone marrow microenvironment contribute to extrinsically mediated chemo-resistance and are therefore important potential therapeutic targets. the study of patient-derived mscs is at a competitive disadvantage, however, because traditional means of isolating mscs from a bone marrow aspirate interferes with isolating the more highly prioritized leukemic cells. many opportunities to study mscs are therefore missed. objectives: to develop a novel method of isolating mscs using the otherwise discarded portion of a bone marrow aspirate, thereby de-coupling the isolation of primary mscs from the isolation of leukemia cells. design/method: aml patient bone marrow aspirates were obtained prospectively from the children's oncology group. healthy patient marrow was purchased. experimental mscs were isolated from the bottom-most layer (rbc-layer) produced by density-gradient separation of a bone marrow aspirate, which is typically discarded. control mscs were isolated from the buffy coat (mnc layer). non-adherent cells were removed after hours, and adherent cells were cultured at % co with mem-alpha containing % fbs. growth curves were obtained by seeding -well plates with , cells per well. cells were stained using oil red o to observe adipocyte differentiation. results: rbc-layer mscs grow successfully following overnight shipment of the aspirate. identical to mnc-layer mscs, rbc-layer mscs exhibit a fibroblastic morphology and are adherent to plastic. rbc-layer mscs persist in culture up to passages before senescence. they exhibit a slower growth curve relative to mnc-layer mscs, but their overall doubling time is similar at approximately hours. surprisingly, mscs from the rbc-layer exhibit adipocyte differentiation on stimulation, revealing their stem-cell like qualities. we present a method of isolating mscs from the discarded portion of a bone marrow aspirate that does not interfere with the isolation of leukemia cells from the same patient. this portion of the aspirate can be shipped, or can sit for at least hours, without sacrificing its mscs. rbclayer mscs are nearly identical to mscs obtained conventionally. perhaps most importantly, rbc-layer mscs retain a stem-cell like capacity, showing them to be a highly valuable cell population in aml research. future plans include investigating potential selective enrichment of stem-cell mscs in the rbc-layer, which could explain the unexpected difference in growth kinetics. aml researchers now have the opportunity to study this exciting component of the bone marrow microenvironment without sacrificing valuable leukemic cells in the process. background: neutropenia is one of the most frequent side effect of chemotherapy associated with an increase in the risk of infection, especially in the cases when the depth and duration of neutropenia are extended. some genes, as variations of darc, gsdma and cxcl are known to influence white blood cell and neutrophil counts. our previous study conducted in children with acute lymphoblastic leukemia (all), showed that polymorphisms in these genes might play a role in the onset of chemotherapy complications during consolidation and maintenance treatment. objectives: in order to support our previous finding, we have expanded the study to the induction period in a cohort of all children treated at the sainte-justine university health center between july and july . design/method: previous associated single nucleotide polymorphisms (snps) in darc, gsdma and cxcl genes were analyzed for an association with the complications occurring during induction including the duration of low neutrophil count (pnn) and low absolute phagocyte count (apc), proven infections and delay between induction and consolidation phases. results: significant effect was found for all studied polymorphims. minor alleles of darc rs , cxcl rs and gsdma rs were all associated with higher risk of complications during induction treatment, whereas that of darc rs (particularly gg genotype) had a protective effect. the gg genotype of rs was associated with a lower risk of post-induction delay (p = . or = . , %ci . - . ), less frequent febrile episodes (p = . ) and lower number of days with apc/pnn count reduction (p = . for apc< . and p = . for pnn< . ). in contrast, the minor t allele of another darc polymorphism (rs ), was associated with longer apc/pnn count reduction (p = . for apc< . and p = . for pnn < . ), as it was the tt genotype of gsdma rs (p = . for apc< . and p = . for pnn< . ). the patients with the gsdma rs had also a higher risk of documented febrile episodes (p = . or = . %ci - . ). the aa genotype of rs cxcl was associated with a higher risk of post-induction delay due to infection (p = . , or = . , % ci . - . ). conclusion: this complementary study confirmed our previous results, showing overall that variations in darc, gsdma and cxcl genes influence the onset of chemotherapy complications in pediatric all, regardless of treatment phases. these polymorphisms might be useful pharmacogenetics markers possibly guiding an adjustment of chemotherapy intensity. background: pediatric acute myeloid leukemia (aml) has a poor survival rate of about % and there is an urgent need for newer targeted therapies. car t-cell based therapies are effective against all but similar therapies against aml are still under development. recent clinical trials have highlighted the concerns about toxicity and therapy related deaths from car t-cells. antigen selection is the key factor determining the specificity, efficacy and toxicity of car t-cells. while contemporary adoptive t-cell therapies use monoclonal antibodies against tumor associated antigens we employed the naturally occurring flt ligand (fl) to target aml cells expressing flt receptors. flt receptor is expressed on multipotent and myelomonocytic progenitors as well as myeloid leukemia cells. to generate fl containing chimeric tlymphocytes designated flcar t-cells and to evaluate their efficacy against aml cells. design/method: flcar was constructed by fusing the coding sequences of the human fl, cd costimulatory domain, and cd -zeta chain (intracellular region) in series. it was then cloned into the phiv-egfp lentiviral vector for expression in cell lines and primary t cells obtained from healthy donors. the empty phiv-egfp vector was used as a negative control. flcar was expressed on both cd + and cd + t-lymphocytes, confirmed by western blot. cell cytoxicity was evaluated by co-culturing flcar t-cells and aml cells followed by flow cytometric analyses. cytokine production was assessed by analyzing expression of interleukin- using quantitative rt-pcr. results: flcar t-cells were generated from cd + jurkat and cd + tk- cell lines with up to % lentiviral transduction efficiency. the efficiency for primary t cells was lower ( - %). flcar was expressed as a ∼ kda protein in cells and was partially phosphorylated on tyrosine. the expression of flcar on lymphocytes lead to increased basal il- expression in the cells. this was further augmented (by > folds) upon co-incubating flcar t-cells with flt expressing target cells. jurkat cells, tk- cells and primary human t cells expressing flcar suppressed the growth of flt -expressing aml cell lines and primary aml cells in vitro. notably, flcar t-cells generated from healthy donors caused strong inhibition of aml cells even at a lower transduction efficiency. in vivo experiments using nsg-sgm mice xenografted with human aml cells are underway. our data demonstrate that flcar can be effectively expressed on t-lymphocytes and mediate potent cytotoxicity against flt -expressing aml cells in vitro. being a completely human derived chimeric protein, it represents a promising candidate for further therapeutic development. holly pacenta, kelly sullivan, ahwan pandey, kelly maloney, joaquin espinosa children's hospital colorado, denver, colorado, united states background: individuals with down syndrome (ds) have a -fold higher risk of developing acute lymphoblastic leukemia (all) than the typical population. there are several important differences between all in individuals with ds (ds-all) and all in individuals without ds (nds-all): first, patients with ds-all have a lower percentage of favorable cytogenetic features compared to nds-all. second, patients with ds-all are more likely to have activating mutations in jak , crlf overexpression, and ikzf deletions. despite these clear genotypic differences, this knowledge has not yet been exploited for therapeutic purposes in ds-all. when outcomes for ds-all are compared to nds-all with similar cytogenetic features, the survival rates are similar. however, individuals with ds-all have an increased risk of treatment-related mortality (trm). current therapy for ds-all is similar to that for nds-all, with the exception of small changes to decrease toxicities that are more prevalent in ds-all. it was recently identified that interferon signaling is constitutively activated in healthy individuals with t . we hypothesize that aberrant interferon signaling could play a role in the unique leukemias observed in ds patients. objectives: to identify differences in gene expression and intracellular signaling cascades that are unique to individuals with ds-all, relative to both nds-all and healthy individuals with ds that can be exploited for therapeutic use. design/method: bone marrow samples were obtained from ds-all patients and matched nds-all patients based on clinical characteristics and genetic features. rna sequencing of these samples was performed and a total of samples were used for the transcriptome analysis ( ds-all vs. nds-all). the differential expression data was generated by deseq and analyzed using ingenuity pathway analysis. the analysis revealed that the chromosome genes that have been implicated in leukemogenesis are not differentially expressed in the ds-all samples, relative to nds-all. an inflammatory signature was identified, which included interferon gamma as an upstream regulator with predicted activation in ds-all. this finding is consistent with prior observations from healthy individuals with ds. other examples of results with potentially actionable targets include the upregulation of several genes in the ras pathway and genes involved in histone methylation. the increased interferon signaling seen in healthy individuals with ds was also identified in ds-all. this may contribute to the development of mutations in inflammatory pathways such as jak and crlf in ds-all. targeting these common pathways with small molecule inhibitors may have a therapeutic benefit in ds-all. cincinnati children's hospital medical center, cincinnati, ohio, united states background: next-generation sequencing (ngs) guides precision medicine approaches in oncology using therapies targeting molecular alterations found within an individual cancer. increased availability of ngs coupled with a proliferation of targeted drugs in development heightens the need for reliable pre-clinical animal models. here we report a patientderived xenograft (pdx) system with integrated molecular profiling for pre-clinical testing of conventional cytotoxic and novel targeted agents. objectives: to utilize ngs from patients with pediatric leukemia to guide rational pre-clinical trials in pdx leukemia avatars, and to determine pdx mice tolerance of and response to cytotoxic and targeted therapies. pediatric acute lymphoblastic leukemia (all) samples were obtained in adherence to an irb-approved protocol and xenografted into nod/rag/interleukin- (il- )rg (nrg) mice. ngs was performed clinically using the foundationone® heme panel. a de novo all sample bearing mutations involving jak , crlf , ntrk , cdkn a/b, ptpn and wt was used for pre-clinical testing. thirty-seven nrg mice were transplanted with million patient cells/mouse via iv injection. standard -drug induction chemotherapy was administered consisting of vincristine, dexamethasone, pegaspargase, and daunorubicin [vxpd, n = mice], in comparison to vehicle control [n = ]. parallel pdx cohorts were treated with single agent targeted therapies based on ngs findings, including ruxolitinib [n = ], crizotinib [n = ] and loxo- [n = ]. the four-week treatment period began on day + from transplant after confirmation of engraftment. following completion of therapy, residual disease burden was analyzed by flow cytometry (hcd +, mcd -cells) in the bone marrow [bm] . to date, pdx models have been established using over thirty ngs-profiled pediatric all samples, including six samples bearing philadelphia (ph) chromosome or phlike mutations. pre-clinical testing was performed in a repre- conclusion: ngs reveals concomitant mutations in ph-like all that may represent additional targets for therapy, or predict tyrosine kinase inhibitor (tki) resistance. we show that all xenograft nrg mice can tolerate a -week multi-agent cytotoxic chemotherapy induction regimen, as well as rational targeted agents, and serve as a robust pre-clinical model for precision medicine trials. background: osteonecrosis is a well-characterized all therapeutic toxicity attributed to glucocorticoids, asparaginase, and methotrexate that disproportionately affects adolescents. in ccg- , alternate-week dexamethasone during double delayed intensification (di) reduced osteonecrosis vs continuous dexamethasone with single di in rapid early responders (rer) ≥ y. to compare efs and os between hr-all patients with vs without osteonecrosis. design/method: hr-all patients - y on aall ( - ) received cog augmented therapy with a × randomization to: ( ) induction dexamethasone ( mg/m d - ) vs prednisone ( mg/m d - ), and ( ) interim maintenance (im) high-dose methotrexate (hdm) vs escalating-dose methotrexate/pegaspargase (ema). rer received single, and slow early responders (ser) double, im/di. initially, all received monthly dexamethasone maintenance pulses, patients ≥ y received di alternate-week dexamethasone, and patients ≤ y received di continuous s of s dexamethasone. there were osteonecrosis-related amendments: after / all patients ≥ y received di alternateweek dexamethasone; after / all patients ≥ y were assigned to induction prednisone, and all patients received di alternate-week dexamethasone and maintenance prednisone pulses. results: osteonecrosis was confirmed in / patients. the y cumulative incidence (ci) was . % overall and increased with age: - y . %, - y . % (alternateweek dexamethasone . % vs continuous dexamethasone . %; p< . ), ≥ y . % (p< . ). among randomized rer patients ≥ y, ci differed by glucocorticoid (dexamethasone . % vs prednisone . %; p = . ) but not methotrexate assignment (hdm . % vs ema . %; p = . ). among randomized ser patients ≥ y, ci was . % with no difference by regimen. results were similar for patients ≥ y. in the entire study population, patients with osteonecrosis had superior y efs ( . % vs . %; p< . ) and os ( . % vs . %; p< . ) than those without osteonecrosis. y efs was significantly higher among randomized patients ≥ y with vs without osteonecrosis ( . % vs . %; p< . ); this finding was present in different age ranges (≥ y, ≥ y, ≥ y) and rer/ser subsets within each, especially in the ≥ y rer ( . % vs . %; p = . ) and ser ( . % vs . %; p< . ) cohorts. across groups, asparaginase allergy was significantly associated with reduced osteonecrosis risk (≥ y: hr . ; p = . ). patients who develop osteonecrosis have significantly increased efs and os, suggesting host differences that increase sensitivity to develop osteonecrosis and render all cells more chemo-responsive. pennsylvania state university, hershey, pennsylvania, united states background: cdc (cell division cycle protein ) belongs to rho family of small gtpases in ras-oncogene superfamily. pro-oncogenic role of overexpressed cdc in ras driven solid tumors are well known. however, role of cdc in leukemia is yet to be established. ikzf encodes ikaros protein which has important role in regulation of lymphoid development and tumor suppression in leukemia. casein kinase ii (ck ) oncogene is overexpressed in leukemia. ck impairs ikaros function which can be restored by using ck inhibitors. objectives: to investigate role of cdc in leukemia and regulation of cdc by ikaros and ck in b-cell acute lymphoblastic leukemia (b-all). shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: cdc is identified as one of the ikaros target genes by analysis of genome-wide dna binding of ikaros using chip-seq and qchip in b-all primary cells. expression of cdc was also noted to be higher in all patient samples compared to normal bone marrow. functional experiments showed that ikaros overexpression via retroviral transduction results in transcriptional repression of cdc . ikaros silencing using shrna resulted in increased expression of cdc . these data suggest that ikaros negatively regulates transcription and expression of cdc . ck directly phosphorylates ikaros and impairs its function as transcription factor. we noted that molecular inhibition of ck via sirna as well as treatment with specific ck inhibitor, cx also decreases expression of cdc . treatment with cx of primary b-all with ikaros haploinsufficiency restores ikaros binding to cdc promoter and represses cdc expression. however, this effect is evident only in presence of ikaros. treatment with cx in ikaros silenced (ikaros shrna) cells showed no change in expression of cdc . these results emphasizes the importance of ikaros in regulating cdc expression. furthermore, we analyzed the changes in epigenetic signature at the cdc promoter following treatment with cx . results show that loss of histone marker of open chromatin (h k ac) and increased histone marker for repressive chromatin (h k me ), at the cdc promoter. these data suggest that ikaros transcriptionally represses cdc via chromatin remodeling. a specific cdc inhibitor, ml showed cytotoxic effects on primary b-all cells. conclusion: cdc may have important role in hematologic malignancies. expression of cdc in b cell all is regulated by ikaros and ck . these results suggest that targeting cdc could be a potential therapeutic strategy in leukemia. caitlyn duffy, laura hall, justin godown, koyama tatsuki, scott borinstein monroe carell jr. children's hospital at vanderbilt, nashville, tennessee, united states background: systemic corticosteroids are widely used as treatment of acute lymphoblastic leukemia (all) and lymphoblastic lymphoma. there are anecdotal reports of bradycardia in pediatric patients receiving corticosteroids, but a more extensive analysis of this effect is needed. objectives: the aim of this study was to describe the incidence, severity, and timing of steroid-induced bradycardia and document any adverse events associated with bradycardia. design/method: we performed a retrospective review of all newly diagnosed patients at our center ( - ) with all/lymphoblastic lymphoma who received corticosteroids (dexamethasone - mg/m /dose or prednisone mg/m /dose) during induction chemotherapy. patients were excluded if they had a pre-existing cardiac abnormality or if they received prior corticosteroids. the average hour heart rate (hr) was assessed for the period prior to initiating steroid therapy and for the hour period surrounding the nadir following steroid administration. the degree and time of steroid induced bradycardia was assessed. adverse patient events and concomitant medication use was documented to identify other contributing factors to bradycardia. a total of children ( females, males, months- years) were included in the analysis with demonstrating a decrease in mean hr following steroid administration. median hr decrease was . beats per minute (quartiles . - ) from prior to initiating steroids to surrounding nadir. sixty one percent developed bradycardia less than or equal to the st percentile for their age range. nadir occurred doses (range - ) into treatment, which corresponded to hours ( - ) after initiation of therapy. of patients who experienced bradycardia, % were associated with dexamethasone rather than prednisone. hr nadir was not associated with other vital sign abnormalities. after completion of induction chemotherapy, % of patients had documented resolution of bradycardia with hr greater than the th percentile for age. it was observed that the children who continued to have relatively low hr were often younger ( months- years old). examination of nadir hr during subsequent hospitalizations in which steroids were not being administered (excluding hr during procedural sedation) did not demonstrate a significant incidence of bradycardia. concomitant opioid, beta-blocker, or other medication exposure did not contribute to the incidence of bradycardia. corticosteroid-induced bradycardia is extremely common in children, teenagers, and young adults with all receiving induction chemotherapy. bradycardia was not associated with clinical adverse events and resolved after completion of corticosteroid treatment. therefore, further cardiac assessment may not be warranted in the presence of bradycardia suspected to be secondary to steroid administration. baylor college of medicine, houston, texas, united states background: survival in newly diagnosed pediatric acute myeloid leukemia (aml) is approximately %; however survival falls dramatically if a patient relapses. currently, approximately one-third of patients with pediatric aml relapse on standard chemotherapy regimens. aml cells are exposed to proteotoxic stress at baseline due to their rapid and inefficient metabolism; proteotoxic stress increases after chemotherapy due to accumulation of reactive oxygen species resulting in misfolded proteins. this leads to activation of cell stress pathways, such as the unfolded protein response (upr) in the endoplasmic reticulum. because an activated upr can make cells more sensitive to proteotoxic stress, we hypothesize that upr activation correlates with response to chemotherapy. objectives: determine the status of upr in pediatric aml and its correlation with chemosensitivity; design/method: peripheral blood samples from pediatric patients with aml were collected at the start of induction chemotherapy, - hours (h) and h post initiation of systemic chemotherapy. tumor cells were sorted from peripheral blood mononuclear cells. expression of upr proteins was determined by chemiluminescence using an automated capillary electrophoresis system. clinical correlations were performed using an annotated database. we measured five upr proteins: grp (glucose regulated protein kda), phospho-eif , inositol-requiring enzyme (ire ) and activating transcription factor (atf ). patients with aml had - times higher expression of upr proteins (except atf ) at baseline than normal controls. grp -the key upr driver-had the highest level of protein expression in myeloid blasts. there was a wide variability in the level of baseline upr expression. eight out of samples expressed > fold increase in grp above those with the lowest grp levels. similarly, and patients respectively, had a > fold increase in peif and ire , compared to patients with low basal expression of these upr proteins. in our limited sample set, there was a trend towards lower overall survival (os) and event-free survival in patients with low baseline grp and ire . conclusion: upr has a variable expression at baseline in pediatric aml, with a trend towards lower os in patients with a low basal grp and low ire expression, suggesting less chemosensitivity in this subgroup. conversely, it is possible that blasts with an upregulated upr prior to chemotherapy manage proteotoxic stress less effectively, having faster apoptosis and hence a better response to chemotherapy in patients with a high basal upr. we are currently expanding our findings in a larger cohort of patients enrolled in the children's oncology group aaml protocol. background: children with newly diagnosed acute lymphoblastic leukemia (all) undergo chest x-ray (cxr) evaluation during initial diagnostic workup to ensure safe airway management. however, to our knowledge, no systematic assessment of cxr findings has been reported. objectives: to evaluate cxr findings at diagnosis of all and their associations with clinical characteristics. we reviewed the cxr findings at diagnosis of all in patients treated on the total xv and xvi protocols at st. jude children's research hospital. findings were evaluated for associations with clinical characteristics at presentation, and the clinical management of mediastinal masses was reviewed. mediastinal masses were seen in ( . %) of patients evaluated and were more common in older patients (mean age, . years) than in younger patients (mean age, . years) (p = . ), in males than in females (p = . ), and in patients with t-all than in those with b-all (p< . ). also associated with mediastinal masses were a higher white blood cell count (wbc) at diagnosis (mean, . × /l) (vs. a lower wbc; mean, . × /l) (p< . ), cns involvement (vs. no involvement) (p = . ), and standard/high-risk disease (vs. low-risk disease) (p< . ). other cxr findings included pulmonary opacity ( patients [ . %]), bronchial/perihilar thickening ( patients [ . %]), cardiomegaly ( patients [ . %]), and osteopenia/fracture/periosteal lesions ( patients [ . %]). pulmonary opacity was more common in younger patients (mean age, . years) than in older patients (mean age, . years) (p = . ) and in those with t-all (vs. b-all) (p = . ). bronchial/perihilar thickening, cardiomegaly, and osteopenia/fracture/periosteal lesions were also more common in younger patients than in older ones (p< . , p = . , and p< . , respectively) and in those with low-risk disease (versus standard/high-risk disease) (p< . , p = . , and p = . , respectively). of the patients with a mediastinal mass on cxr, underwent a confirmatory chest ct scan, and ( . %) were confirmed to have a mediastinal mass. notably, patients ( . %) had airway compression, and compression of venous structures was identified in of patients ( . %) who received iv contrast. the clinical course was evaluated for patients with mediastinal masses detected by cxr. fifty patients ( . %) required icu admission (mean stay, . days). general anesthesia was used for only patients ( . %), and patients ( . %) had a less invasive peripherally inserted central catheter. no deaths occurred in the acute phase. conclusion: cxr at the time of all diagnosis can detect various intrathoracic lesions and is helpful in planning initial diagnostic workup and management. background: mertk is a receptor tyrosine kinase that is aberrantly expressed in % of pediatric primary aml samples. mertk inhibition with the small molecule tyrosine kinase inhibitor (tki) mrx- decreases tumor burden and prolongs survival in aml xenografts. while treatment with mrx- reduces leukemia in the peripheral blood, it is less effective in the bone marrow, suggesting a role for the marrow microenvironment in therapeutic resistance. the jak/stat pathway has been implicated as a mediator of bone marrow derived resistance to tkis and inhibitors of this pathway are in clinical development for the treatment of aml. to determine the role of the bone marrow stromal niche in mediating resistance to mertk inhibition and to evaluate the efficacy of combined mertk and jak/stat inhibition. design/method: aml cell lines were cultured with or without the hs stromal cell line or hs conditioned medium, then treated with mrx- +/-the jak/stat inhibitor ruxolitinib, or control. induction of apoptosis and cell cycle arrest in aml cells was measured by flow cytometry. expression of h ax and total and phosphorylated stat were determined by immunoblot. results: co-culture with stromal cells significantly reduced aml cell death and g /m phase arrest in response to treatment with nm mrx- compared to no co-culture (cell death: . % versus . %, p< . ; g /m arrest: . % versus . %, p< . ). g /m arrest was accompanied by an increase in h ax expression which was similarly abrogated in co-culture. conditioned medium did not provide protection from mrx- induced apoptosis, g /m arrest, or h ax induction. mrx- inhibited stat phosphorylation but direct co-culture and conditioned medium potently increased basal stat phosphorylation which was not inhibited by mrx- . to determine whether the observed induction of stat phosphorylation was functionally relevant, cocultures were treated with both mrx- and ruxolitinib. while ruxolitinib potently inhibited the phosphorylation of stat in the presence of co-culture, combination treatment did not overcome stromal mediated protection from mrx- induced apoptosis. similarly, the addition of exogenous gm-csf induced stat phosphorylation but did not yield protection from mrx- functional effects in the absence of co-culture. together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from mrx- induced apoptosis, cell cycle alterations, and dna damage. while co-culture potently induces phosphorylation of stat in leukemia cells, this is neither necessary nor sufficient for stromal-cell mediated protection from mertk inhibition and combined treatment with jak/stat inhibitors is unlikely to be therapeutically efficacious. background: mercaptopurine ( -mp) is an immunosuppressive thiopurine drug that is a key component of acute lymphoblastic leukemia (all) treatment. -mp is metabolized into -thioguanine ( -tgn), which is responsible for anti-leukemic effects, as well as -methylmercaptopurine nucleotides ( -mmpn/ -mmp), which are associated with hepatotoxicity. some patients preferentially metabolize -mp to -mmpn/ -mmp, increasing their risk for hepatotoxicity and potentially reducing anti-leukemic effects. hepatotoxicity can cause interruptions or delays in therapy that may jeopardize cure rates. allopurinol has been increasingly used in patients with inflammatory bowel disease (ibd) to shunt -mp metabolism toward -tgn and away from -mmpn to minimize hepatotoxicity and preserve therapeutic effects. objectives: this retrospective chart review expands upon our previously published case series of three patients with all in whom allopurinol was successfully used to redirect -mp metabolism. twelve additional patients have subsequently received allopurinol and -mp combination therapy at texas children's hospital. data from this larger patient sample, with longer follow up, is being analyzed to increase knowledge of the effectiveness and longitudinal effects of adding allopurinol to -mp to reduce risk of hepatotoxicity. design/method: data were abstracted from the electronic medical records of patients with all treated at texas children's hospital from to present, who had been found to have evidence of altered -mp metabolism and in whom allopurinol was added to -mp therapy due to concern for risk or recurrence of hepatotoxicity. metabolite levels, -mp dose, and alanine transaminase (alt) prior to initiation of allopurinol and approximately weeks later were compared. wilcoxon signed-rank test was applied for statistical analysis. : after the addition of allopurinol, patients experienced a significant decrease in mean levels of -mmpn (p = . ), correlating with a significant decrease in mean alt (p = . ). with the initiation of allopurinol, the mean -mp dose was decreased from to mg/m /day over an -week period. mean -tgn levels increased (p = . ). in follow up beyond weeks, no patients had further holds in -mp due to hepatotoxicity. addition of allopurinol appears to shift metabolism from -mmpn toward -tgn, with increases in mean -tgn levels despite a decrease in mean -mp dose. this may limit negative side effects, thus resulting in fewer gaps in therapy and possible improved outcomes. further analysis of -mp dose titration and effects on anc over time as well as effects on overall survival is ongoing. prospective background: alterations in epigenetic patterning are a fundamental feature in acute myeloid leukemia (aml). treatment with dna methyltransferase inhibitors (dnmti) yields responses in aml, but the molecular mechanisms underlying this effect are poorly understood. in prior work, we demonstrated induction of genes involved in the pirna rna (piwi) silencing pathway as a common gene feature of aml cell lines treated with decitabine. the piwi pathway is an rna silencing system, distinct from classical small rna transcriptional silencing, responsible for transposon-silencing in gametogenesis; emerging data suggest a role for this system in somatic cells. based on these data, we postulate that piwi induction plays a crucial role in aml recovery following demethylation and that disruption of this pathway would modulate response and/or recovery from decitabine treatment. to assess the effect contribution of the pirna pathway response following dnmti treatment in aml. design/method: to choose target genes in the pirna pathway for disruption, molm cells were first treated with escalating doses of decitabine. using quantitative rt-pcr, the dose-dependent expression of several pirna-associated genes were analyzed. two genes, mael and piwil , were selected for disruption experiments based on preliminary data suggesting decitabine dose-dependent responses. molm cells were transduced with shrna targeting these genes using a lentivirus delivery system with selection in puromycin. knockdown efficiency was assessed by rt-qpcr. to determine how gene disruption affected cell growth, knockdown cells were treated with decitabine nm. proliferation was assessed by celltiter glo assay following decitabine treatment. clonogenic potential was assessed by colony forming assays of transduced cells after treatment with decitabine at nm and nm. results: following decitabine exposure in molm , there was a markedly increased expression of mael and piwil compared to untreated cells ( : and : , respectively) . thus, these were the candidate genes chosen for disruption. of mael shrna constructs, two resulted in a % relative expression of mael compared to controls. of the piwil shrna constructs, the best knockdown showed % relative expression. there were no significant differences in proliferation or clonogenicity of stably selected mael or piwil knock-down molm cells following decitabine treatment. using gene knockdown procedures, mael and piwl do not appear to have a marked effect on growth and response to decitabine treatment in molm . however, these results may be limited by inefficient knockdown using shrna targeting methods. further work using a cas /crispr based inactivation of these genes is ongoing. children cancer hospital cairo, egypt background: hypodiploidy < chromosomes is very uncommon and have particularly poor outcomes in childhood acute lymphoblastic leukemia (all). it is subdivided into: near-haploid ( - chromosomes), lowhypodiploid ( - chromosomes) and high-hypodiploid ( - chromosomes). to determine if minimal residual disease (mrd) can identify a group of patients with better prognosis in the hypodploid population who can be treated with intensive chemotherapy alone. design/method: a retrospective study that included all patients under age of diagnosed as hypodiploid b-precursor all during the period between january -december and treated at children's cancer hospital egypt on sjcrh total study-xv for ir/hr all. sixteen patients had < chromosomes ( nearhaploid and low-hypodiploid), constituting % of all pediatric patients with b-precursor all during the study period. patients with near-haploid all had a median age of years (range - ), initial leukocyte count (wbc) median of . × /l (range . - . ), ( . %) were males and / ( . %) had hr-nci criteria. four patients ( . %) are alive in complete remission(cr) (range - months, median ), one died in induction and ( . %) had hematological relapse (range . - months, median ). patients with low-hypodiploid all had significantly older age (median years, range - ), median wbc . × /l (range . - . ), / ( . %) were males. one patient ( . %) is alive in cr, one died in induction, one failed to achieve cr post-induction and patients( %) had hematological relapse (range . - . months, median . ). mrd< . % by flow-cytometry on day- and end of induction was achieved in / ( . %) and / patients( %) with near-haploid, compared with / ( . %) and / patients( %) with low-hypodiploid; respectively (p = . , p = . ;respectively). allogeneic transplantation was performed during initial remission only in mrd negative patients (one relapsed and one is in cr) and in the patient with induction failure (relapsed post-transplant). five of the total six patients who had negative mrd on day- and end of induction are alive in cr ( / with chemotherapy alone). all patients with negative mrd at end of induction but with mrd levels≥ . % on day- (range . - . %) relapsed as well as all patients with detectable mrd at the end of induction. the difference in relapse was statistically significant in relation to negative-mrd on day- (p = . ), but not at end of induction(p = . ). conclusion: children with hypodiploid all and negative mrd on day- of induction are highly curable with intensive chemotherapy alone, while patients with negative mrd at the end of induction and detectable mrd on day- had dismal outcome. background: overall survival in pediatric acute myeloid leukemia (aml) has plateaued between - %, with death during induction chemotherapy seen in - % of patients. respiratory complications contribute to morbidity and mortality in pediatric aml induction, however the incidence, patterns, and predictors of respiratory adverse events (aes) during this period are unknown. to estimate the incidence of respiratory aes during induction therapy for de novo pediatric aml, to characterize and grade these respiratory aes, and to identify predictors of respiratory ae development. we conducted a retrospective longitudinal study from presentation to day in institutional de novo pediatric aml patients (≤ years) between march and december . outcomes included any nci ctcae grade - respiratory ae or death from another cause. demographic, disease, and treatment-related data were abstracted. the most specific, best-fitting ctcae category and grade for each ae was determined. descriptive statistics, survival analysis, multivariable logistic regression analysis, and time-toevent distributions were performed (sas v . , cary, nc) . among eligible patients, . % (n = ) experienced discrete respiratory aes. incidence of grade - aes was . % (n = ). a bimodal time-to-event distribution demonstrated peaks at treatment days and . induction death occurred in . % (n = ) including deaths from respiratory failure associated with disseminated fungal disease. in univariate analysis, those experiencing aes differed significantly in regards to older age at diagnosis (p< . ), higher initial wbc (p = . ), higher initial peripheral blast percentage (p = . ), coagulopathy at diagnosis (pt (p = . ), d-dimer (p = . )), fluid overload status (p< . ), occurrence of infection (p = . ), and occurrence of tumor lysis syndrome (tls) (p = . ). patients with hyperleukocytosis (p = . ), fluid overload (p< . ), and fab m morphology (p = . ) each had a significantly decreased probability of completing the follow up period without experiencing a respiratory ae. on multivariable analysis, fluid overload (aor . [ % ci: . - . ) and older age (aor . [ % ci: . - . ) were significantly associated with ae occurrence when gender, hyperleukocytosis, tls, and infection status were held constant. we describe a high incidence of respiratory aes during pediatric aml induction. fluid overload and older age at diagnosis are independently associated with ae development when controlling for other proposed risk factors. interventions focused on conservative fluid management and offset of fluid overload should be explored in newly diagnosed pediatric aml in an effort to reduce respiratory complications during induction. overall, all survival rates are outstanding and have continued to improve with risk-adapted therapy. the most striking improvement occurred in t-all where -year os rates now exceed % and parallel b-all. survival improvements, however, have not been observed uniformly across all subgroups. while the gap in outcome differences narrowed among blacks, outcomes for hispanics have remained static. further, no improvements in survival were observed in infants or ayas and new treatment approaches have been implemented for these populations. background: acute myeloid leukemia (aml) accounts for approximately % of new childhood leukemia cases. chest x-ray (cxr) is performed in all newly diagnosed aml cases to evaluate the safety of airway management for anesthesia during diagnostic procedures; however, cxr results in pediatric patients with aml have not been described. objectives: the primary objective was to evaluate cxr findings at diagnosis in patients with aml. the secondary objectives included assessing associations between cxr findings and clinical characteristics, with the overall goal of aiding in the evaluation of the use of cxrs as an initial diagnostic study in pediatric patients with aml. design/method: cxr findings and clinical characteristics were evaluated in patients with newly diagnosed aml who were enrolled in one of three protocols at st. jude children's research hospital (aml , aml , and aml ). the findings were categorized based on radiologic reports. further, the associations of these findings and clinical characteristics were evaluated. we evaluated cxr findings in a total of patients: from aml ; from aml ; and from aml . common cxr findings were pulmonary opacity (n = , . %), bronchial/perihilar thickening (n = , . %), splenomegaly (n = , . %), mediastinal mass and lymph nodes (n = , . %), pleural effusion/thickening (n = , . %), demineralization/fracture/periosteal lesions (n = , . %), scoliosis (n = , . %), and granulomatous disease (n = , . %). three cxr findings were associated with younger age at diagnosis: pulmonary opacity (median age, . years in patients with positive findings vs. . years in those with negative findings, p< . ), bronchial/perihilar thickening (median age, . years vs. . years, p< . ), and demineralization/fracture/periosteal lesions (median age; . years vs. . years, p = . ). two cxr findings were associated with older age at diagnosis: scoliosis (median age, . years vs. . years, p< . ) and granulomatous disease (median age, . years vs. . years, p = . ). higher white blood cell counts (wbcs) at diagnosis were associated with cxrs showing pulmonary opacity (median wbc; . × ^ /l vs. . × ^ /l, p = . ) or splenomegaly (median wbc; . × ^ /l vs. . × ^ /l, p = . ). french-american-british (fab) m /m subtypes were more frequently associated with pulmonary opacity compared with others (p< . ). we did not find significant differences between female and male patients. conclusion: cxr in patients with newly diagnosed aml showed a variety of thoracic, abdominal, and bony lesions that are important for the initial evaluation and management. pulmonary opacity was the most common finding and was frequently seen in patients who were younger or had higher wbcs at diagnosis or fab m /m . background: children diagnosed with acute lymphoblastic leukemia (all) require a central venous catheter (cvc) to administer chemotherapy safely. both external and internal cvcs carry risks of complications including thrombosis, infection, and possible replacement. internal catheters, such as a port, are generally used for the majority of patients for the duration of treatment since therapy lasts for several years. many institutions place a port at the time of diagnosis. other institutions prefer to start induction therapy via placement of a peripherally inserted central catheter (picc) and defer port placement until the completion of induction therapy due to concerns of increased risk of infectious complications with port placement. objectives: to compare rates of common cvc associated complications by type of cvc placed at start of induction therapy in children treated for newly diagnosed all at the jimmy everest center (jec) at the university of oklahoma health sciences center. design/method: a retrospective chart review analyzed data from newly diagnosed all patients treated at the jec between - . data was collected on complications including thrombosis, bacteremia, insertion site infection, cvc malfunction and need for removal. data collection began at the start of induction and was completed at the end of induction therapy. statistical analysis used a univariate and multivariate logistic regression model to compare complication rates between those who had a port versus those who had a picc placed at start of induction. results: data was collected on patients. fifty-six patients had a port placed at start of therapy while had a picc placed. fourteen percent of patients had a cvc associated complication. univariate analysis showed no statistically significant difference in rates of cvc associated complications between the groups (port %, picc . % p = . ). the rates of hospitalization for cvc associated complications were similar between both groups (port %, picc % p = . ). rates of cvc removal were also similar between both groups (port %, picc % p = . ). multivariate model that included baseline patient characteristics including type of all, patient body surface area, gender, ethnicity and age continued to demonstrate no significant difference in cvc associated complications between both groups. conclusion: this single institution study showed that there was no significant difference in cvc associated complications between port and picc line placement at the start of childhood all induction therapy. port placement can be considered as a safe option at the start of induction therapy. complete remission [cr] or cr with incomplete blood count recovery [cri]) within treatment cycles - . interim data are reported (nct ). results: seventeen patients were enrolled and received ≥ dose of lenalidomide; median age was years (range - ); patients were female. patients received median prior regimens (range - ). nine patients had previously undergone bone marrow transplantation (bmt). four patients had relapsed aml and were refractory to immediate prior treatment. median duration of study treatment was weeks (range - ); patients completed a median of treatment cycle (range - ). all patients were evaluable for primary outcome; achieved morphologic cri after cycles (no patients achieved cr). the responder was a -year-old male with history of r/r aml after first-and second-line treatment, bmt, and salvage chemotherapy. at baseline, he had a complex cytogenetic karyotype (monoallelic − q . , − q, − q . , − p ) with no identifiable molecular mutation; he was also positive for del( q) (− q , − q ). his post-treatment karyotype showed no abnormalities. sixteen patients experienced treatment failure; due to resistant disease, of indeterminate cause, and had treatment failure before a post-baseline assessment was performed. all patients experienced ≥ grade - treatment-emergent adverse event (teae). the most commonly reported were thrombocytopenia (n = ), anemia (n = ), febrile neutropenia (n = ), and hypokalemia (n = ). fifteen patients experienced ≥ teae related to lenalidomide. all patients discontinued treatment; remain in follow-up. the study is now closed to enrollment. ten patients died on study: during treatment, during follow-up. all deaths were attributed to aml or complications due to aml. conclusion: third-line lenalidomide monotherapy was associated with clinical response in of pediatric patients with r/r aml; however, treatment exposure was limited. safety data are consistent with the known profile of lenalidomide. lenalidomide was not an efficacious treatment for r/r pediatric aml. funding: celgene corporation, summit, nj, usa. cook children's medical center, fort worth, texas, united states background: it is well documented that pediatric patients with acute lymphoblastic leukemia (all) often experience significant weight gain during induction therapy and later struggle with obesity. however, some patients experience unintended weight loss during induction therapy; since this issue is not well reported, it often goes undertreated. although malnutrition is reported to be associated with decreased survival, increased risk of infection, and loss of lean body mass, there remains a scarcity of in-depth analysis of prevalence and risk factors that contribute to this problem. our study attempts to address this critical yet unmet need. objectives: our aim was to identify the clinical risk factors and outcomes associated with weight loss during induction therapy for pediatric all. design/method: this was a retrospective chart review of patients between and years of age diagnosed with all at cook children's medical center from / / to / / . for each patient, we collected height, weight, age, body mass index (bmi) z-scores at diagnosis and end of induction therapy, risk stratification, and whether consolidation was delayed. patients with a bmi > th percentile at diagnosis were categorized as being overweight or obese. using logistic regression analyses, we examined which variables predicted whether the patient had an increase or decrease in bmi z-score throughout induction. a critical alpha level of . indicated statistical significance. results: ninety-six patients met our inclusion criteria. of these, % experienced a decrease in bmi during induction therapy. compared to patients whose bmi increased during induction, patients with a decrease in bmi were more likely to be overweight or obese at diagnosis ( % vs. %; p< . ), to be ≥ years of age ( % vs. %; p< . ), to have a high-or very-high-risk stratification ( % vs. %; p< . ), and to experience a delay in the start of consolidation therapy ( % vs. %; p< . ). conclusion: this research highlights a risk not previously identified in the literature that may impact outcomes. patients treated on high-or very-high-risk protocols, who are overweight or obese at diagnosis, and who are ≥ years of age at diagnosis should be monitored closely for weight loss during induction therapy. patients who experience weight loss should receive prompt intervention. it is our hope that this information can be used for future prospective studies and to help develop evidence-based guidelines. background: p abnormalities have been observed in some patients with hematologic malignancies. loss of p function as a tumor suppressor gene in the chromosome plays an important role for development of leukemia. these patients usually have poor outcome due to the chemotherapy and are associated with poor prognosis. objectives: this study aimed to identify frequency of p abnormalities between iranian children and adult patients with aml (acute myeloid leukemia) malignancy. design/method: the p abnormalities were analyzed via bone marrow karyotyping and fish method in acute myeloid leukemia patients. in this study, p abnormalities were observed in ( %) patients out of diagnosed cases. a significant strong correlation between p abnormalities and other high risk factors (poor risk cytogenetic) were observed. from patients with aml malignancy ( p abnormalities), ( %) patients have complex karyotype, ( %) patients monosomal karyotype and ( %) patients have monosomal karyotype accompanied with a complex karyotype. overall, p abnormalities are independent risk factor in acute myeloid leukemia and evaluation of these abnormalities by fish or other complementary techniques prior to treatment, might help for better risk stratification of high risk aml patients. background: hepatotoxicity in treatment of acute lymphoblastic leukemia (all) is well studied and transiently affects most patients receiving antimetabolite therapy. rarely, patients develop liver injury severe or prolonged enough to undergo a liver biopsy. little is known about how these patients differ from patients that develop transient hepatotoxicity. we sought to describe disease and treatment characteristics for all patients that developed hepatotoxicity severe enough to undergo liver biopsy. we also looked for pre-dictive factors for liver biopsy, including signs of early hepatic injury from the initial treatment protocol. design/method: pathology reports of all patients from the liver biopsy database at children's healthcare of atlanta were collected. controls were matched : for age, all subtype, and treatment protocol. demographics, treatment protocols, and overall outcomes were collected through the electronic health record. hepatic lab results for transaminases, coagulation, and albumin were collected for induction, consolidation, interim maintenance, delayed intensification, and maintenance. results: sixteen patients diagnosed between - (median age at diagnosis years, range - ; % male; % pre-b all) were included in the case series. the median time from diagnosis to liver biopsy was . years (range - ). eight patients ( %) were in maintenance at the time of biopsy; none had active disease. eight ( %) were postbone marrow transplant. biopsy results included: steatosis ( ), acute inflammatory/infectious ( ), liver infiltration ( ), fibrosis ( ) and graft-vs-host disease (gvhd) ( ). six patients were deceased; -year all-cause mortality from diagnosis was %. thiopurine methyltransferase (tpmt) status was known in % cases and % controls. all cases had intermediate or wildtype status, which did not differ from controls (p > . ). patients requiring liver biopsy did not have evidence of acute hepatotoxicity (ast/alt > × normal values) during their initial treatment protocol. hepatotoxicity requiring liver biopsy is a rare outcome of all treatment. these patients had elevated rates of relapse, bmt, and -year all-cause mortality, suggestive of a more severe disease process. however, it is difficult to sort out the temporality of relapse, bmt, and hepatoxicity requiring biopsy in this limited sample. additionally, patients with bmt preceding liver biopsy have other confounding factors that makes them difficult to include in the analysis. finally, our limited descriptive data show no notable correlation between early hepatotoxicity and later indication for liver biopsy. future cohort or case-control studies with larger sample sizes are required to further explore early predictive factors for severe hepatotoxicity requiring liver biopsy. nathan gossai, joanna perkins, michael richards, yoav messinger, bruce bostrom background: the majority of chemotherapeutic agents used to treat hodgkin lymphoma are teratogenic. pregnancy screening prior to the start of chemotherapy is supported by clinical guidelines and baseline testing is a standard component in therapeutic trials. there is limited data available on the incidence of pregnancy screening prior to the start of hodgkin therapy but previous studies suggest that pregnancy screening, especially at pediatric institutions, is not consistently completed. objectives: the objective of this study is to evaluate the incidence of pregnancy screening and contraceptive counseling prior to the start of therapy in females diagnosed with hodgkin lymphoma. design/method: a retrospective chart review was performed for all female patients newly diagnosed with hodgkin lymphoma from to at the hospital for sick children in toronto, ontario. all patients who were intended to receive multi-agent chemotherapy were included, regardless of age. data collected included demographic and disease information, chemotherapy regimen and enrollment on clinical trial. all pregnancy testing within two weeks prior to the start of therapy was captured, as well as type of pregnancy test performed, documentation of menstrual status, contraceptive counseling and contraceptive provision. univariate and multivariate analyses were used to describe factors influencing the incidence of pregnancy testing. results: a total of female patients with newly diagnosed hodgkin lymphoma between the ages of and years were identified. sixty patients ( %) had pregnancy testing done prior to the start of therapy. testing modalities included serum and urine screens as well as quantitative beta-hcg measures. older age (p = . ), documentation of menstrual status at diagnosis (p = . ) and diagnosis between and (p = . ) were associated with higher incidence of screening. enrollment on a therapeutic trial was not associated with a higher incidence of screening (p = . ). contraceptive counseling was documented for patients ( %) and patients ( %) were prescribed contraceptive medications during therapy. pre-chemotherapy pregnancy testing was completed on % of females with newly diagnosed hodgkin lymphoma. improvement is required and interventions, including clarification of institutional standards, modification of chemotherapy order sets and staff education, are planned. (rao et al., cancer, ) . university of louisville, louisville, kentucky, united states background: granulocytic sarcomas (also known as chloromas or leukemia cutis) were first described by a. burns in . they are solid tumors comprised of immature granulocytic cells and represent extramedullary manifestations of underlying leukemia. chloromas are most commonly associated with acute myeloid leukemia. they may arise in other myeloproliferative disorders but are rarely seen in b or t cell acute lymphoblastic leukemia (all). objectives: although patients with all rarely have chloromas, it should remain on the differential for patients with unusual swelling or masses. design/method: we present a case series of two patients from our institution diagnosed with b cell all who had a chloroma as the presenting symptom. the first patient is a yo who presented to his primary provider with nasal congestion and a one-week history of bilateral eye swelling and was referred to an allergist when the symptoms did not resolve with anti-histamines. his review of systems was otherwise negative. he was referred urgently to ent two months later for a × cm mass palpated along the medial border of the left eye. an mri showed a left facial mass surrounding the zygoma and extending into the anterior inferior left orbit. biopsy revealed b cell acute lymphoblastic lymphoma, and bone marrow aspirate and biopsy confirmed the diagnosis as b cell all. the second patient is a yo who presented to his primary doctor for rapid growth of a scalp nodule that had been present for about months. he was referred to dermatology and treated for a supposed kerion from tinea capitis. the lesion continued to grow and became more irritated with this treatment. punch biopsy revealed a complicated phenotype of lymphoblastic lymphoma. however, after a lymph node biopsy and bone marrow aspirate and biopsy, the diagnosis was confirmed as b all. his only other positive point on review of systems was a questionably pathologic -pound weight loss and an area of matted cervical lymph nodes. for both of our patients, the chloromas completely disappeared during induction therapy. it is worth noting that both of these patients presented with the chloroma as the only symptom of the underlying leukemia. this led to initial misdiagnosis and delay in identifying their leukemia. therefore, while it is very rare for a patient with b all to present with a chloroma, our experience shows that all should be on the differential for patients presenting with unusual swelling or masses. background: hodgkin lymphoma (hl) is a lymphoproliferative neoplasm that commonly presents with history of adenopathy and a predictable pattern of disease involvement with or without systemic symptoms of fever and/or weight loss. in the hands of an experienced oncologist the diagnosis of hl is usually not a challenge. occasionally a diagnostic challenge is presented by a patient who has an atypical presentation which is suggestive of an alternative diagnosis. we describe a case series of patients diagnosed with hl whose initial clinical presentations lead to a diagnosis different form hl. honduras, nicaragua and the united states. results: six pediatric oncology centers from the american continent conducted a retrospective review of patients diagnosed with hl since . patients that had an initial presentation not suggestive of hl or who were initially diagnosed with a disease other that hl were included for a total of patients. argentina n = , guatemala n = , honduras n = , nicaragua n = , united states n = . five patients were female and male. patient's ages ranged from to years. most patients (n = ) were older than years. three patients ( %) presented with non-immune cytopenias without overt lymphadenopathy, of those one had active hemophagocytic syndrome. five patients ( %) were suspected to have localized solid tumors: ewing sarcoma n = , rhabdomyosarcoma n = , hepatocellular carcinoma n = , and soft tissue tumor of the cheek n = . two ( %) metastatic solid malignancy as they presented with disseminated pulmonary nodules. five ( %) with autoimmune disorders: hashimoto thyroiditis n = , autoimmune hemolytic anemia n = , nephrotic syndrome n = . ten ( %) with chronic infectious processes: brucella n = , tonsillar abscess n = , splenic abscess n = , and tuberculosis (tb) n = . patients with suspected tuberculosis were diagnosed outside of the united states. six of patients were ultimately diagnosed as having both tb and hl. seventeen patients had ann-arbor stage iii or iv, seven patient had stage ii with either b symptoms or bulky disease. patients were treated with various chemotherapy regimens according to the treating center: abvd, abve-pc oepa-copdac, avpc, beacopp. two patients had recurrent disease, one died of disease progression and one died from causes not related to hl conclusion: a small proportion of hl patients have atypical or unusual presentations. hl should be included in the differential diagnosis of solid tumors, autoimmune disorders, infections or cytopenias. the most common atypical presentation is an infectious process. background: acute lymphoblastic leukemia (all) represents the largest group of pediatric malignancies. the high cure rate of childhood all represents one of the most remarkable success stories in the war on cancer. in a lower middle income country (lmic) like the philippines, we reviewed the five year survival in a tertiary referral center. objectives: this retrospective cohort study aims to determine the survival of children - years old with all treated at a tertiary referral center for childhood cancer in the philippines from january to december . design/method: this is a retrospective cohort study that reviewed medical charts of newly diagnosed all ages to years old from january to december . a total of subjects were included in the study. the year overall survival (os) and event free survival (efs) were . % and . %, respectively. the year os for standard risk all was . % and for high risk patients was %. the year os for the patients on remission was . % and for those who relapsed was . %. univariate and multivariate by cox proportional hazards regression revealed wbc count at diagnosis, risk classification, immunophenotyping, and development of relapse showed significant prognostic impact for mortality. age and gender were reported with no prognostic significance. the -year os and efs were lower compared to developed countries but are comparable with other lmics. the prognostic factors for relapse and mortality were compatible with the literature. overall, the adopted treatment protocols for childhood all in this institution showed acceptable results. relapse has a significant prognostic impact for mortality. development of accessibility to care, increase awareness, early detection and resources at hand should be achieved. improvement in the follow up protocol to prevent delays in the treatment, patient education to prevent non-compliance and psychosocial support, to developed better supportive care, and expand facilities should be given emphasis to further improve survival and prevent relapse. objectives: here, we seek to further characterize this entity by describing the pathologic and clinical features of pediatric cases of burkitt-like lymphoma with q aberration. we collected pathologic and clinical data from the medical record on all pediatric high grade b-cell lymphoma (hgbcl) cases diagnosed at our institution over a -year period ( - ) . for those cases classified as neither burkitt lymphoma nor diffuse large b-cell lymphoma (dlbcl), fish for myc, bcl- and bcl- , as well as array comparative genomic hybridization (acgh), were performed. we identified cases of hgbcl, including cases of burkitt lymphoma presenting as purely leukemic phase. of the hgbcl cases, had burkitt lymphoma as defined by myc rearrangements, and had dlbcl. collectively, the majority of these patients had primary disease outside of the head/neck, and most patients presented with advanced stage (iii-iv) disease. of the remaining cases, q aberration was identified in cases using acgh. all cases histologically and immunophenotypically resembled burkitt lymphoma but lacked myc rearrangement, instead showing proximal gains in q -q and telomeric losses in q . qter. all cases involved primary disease in the cervical lymph node and/or tonsil. three of these cases were localized (stage ii), and the fourth case involved a few metabolically active but non-enlarged lymph nodes in the chest and abdomen (stage iii). all patients achieved complete remission with standard therapy for mature b-cell lymphoma, and were alive with no clinical evidence of disease at a median follow-up of months. although the number is small, our results suggest that the majority of non-burkitt, non-dlbcl cases of pediatric hgbcl carry q aberrations. in addition, patients with q aberrations appear to be more likely to present with lower stage disease, thus requiring less intensive therapy, and also tend to have primary disease in the head/neck. these findings further support the classification of burkitt-like lymphoma with q aberration as a distinct pathologic and clinical entity, and we propose that all pediatric non-burkitt, non-dlbcl cases of hgbcl regularly undergo further workup for possible q aberrations. marie claire milady auguste, joseph bernard st damien hospital, port-au-prince, port-au-prince, haiti background: hodgkin lymphoma (hl) and non-hodgkin lymphoma (nhl) account for % of cancers in the united states pediatric population ( , ). in central america and the caribbean, they are in second position among all types of pediatric cancers ( ). a previous study on pediatric cancers in haiti showed that the lymphomas were in fifth place after the leukemias, wilms tumor, retinoblastoma and the sarcomas ( ). the main objective of this study is to present the epidemiological profile of lymphomas managed at a haitian pediatric hospital. design/method: this is a retrospective study conducted on the cases of lymphoma diagnosed and managed at st damien hospital from january to december . key variables such as age, gender, stage at diagnosis, histopathological types and outcome were collected to present the characteristics of this retrospective cohort. of the cases of cancer diagnosed during the study period, ( . %) had the diagnosis of lymphoma. the sex ratio was . ( males for females) and the average age was . years [ - years]. there were cases of hl ( . %) and cases of nhl ( . %). . % of the patients were diagnosed at stages iii and iv. among the hl cases, ( . %) were nodular sclerosis lymphoma, ( . %) with mixed cellularity and ( . %) with lymphocytic predominance. for the nhl cases, ( . %) were burkitt's lymphoma and ( . %) lymphoblastic t-cell lymphoma. among the patients for who immunohistochemistry was found, the cases of hl were cd -positive and out of cases of nhl were cd -negative. only patient was hiv-positive, and patients had a confirmed exposure to epstein-barr virus. patients ( . %) were lost to follow-up, ( . %) were in remission, ( %) relapsed, ( . %) were still in treatment and ( %) were deceased. university of chicago, chicago, illinois, united states background: due to the adoption of risk-adapted therapy, pediatric and adolescent acute lymphoblastic leukemia (all) is associated with high cure rates. despite excellent outcomes in most children, patients with certain blast cytogenetic features do not fare as well. furthermore, african american, native american, and hispanic patients have worse outcomes than caucasian patients. while the outcome discrepancies are certainly multifactorial, and blast cytogenetics are related to age, it remains unclear whether ethnicity and blast cytogenetics correlate. the diverse patient population at the university of chicago provides an opportunity to evaluate for such a correlation. objectives: to describe cytogenetic findings in a racially and ethnically diverse population of patients of all age groups diagnosed with all at university of chicago from to and determine if there is a correlation between race/ethnicity and blast cytogenetics. results: a total of newly diagnosed patients with all between the ages of - from - were included in this study. of those, patients ( . %) had b-all, had t-all ( . %), one had early t-cell precursor all and one had mixed phenotype all (b/t). caucasians accounted for % of patients, african americans (aa) %, hispanics . %, asians . %, and % were of other races. age distribution had a bimodal pattern, with a peak in incidence at and another at years of age, consistent with published data. cytogenetic categories included: t( ; )(p ;q ), q rearrangements (kmt a), iamp , t( ; )(q ;p . ), t( ; ) (q ;q ), hypodiploidy, hyperdiploidy and double trisomy of chromosomes and . aa and hispanic patients with b-all presented more frequently between the ages of - years compared to caucasians (p = . and . , respectively). in aa patients, t( ; ) (q ;p . ) was overrepresented (p = . when compared to caucasians), and was mainly observed in patients between - years. caucasian patients were more likely than non-caucasians to have hyperdiploidy (p = . ), especially in patients aged - years. the rate of t( ; )(q ;p . ) was significantly higher in aa patients in our cohort, in particular in patients between the ages of - years. hyperdiploidy was more likely in caucasians aged - years. these findings may suggest that varying blast cytogenetics could contribute to outcome differences between races. ahmed elgammal, yasser elborai, mohamed fawzy, asmaa salama, eman d el-desouky, lobna shalaby national cancer institute, cairo, cairo, egypt background: hodgkin lymphoma (hl) in children is one of the malignancies that have a high chance of cure. stage iv hl remains a challenge for getting good clinical outcome as in other stages. many treatment protocols used to give combination chemotherapy while combined modality treatment is the mainstay in other treatment protocols. objectives: we aimed in to assess the outcome using consolidation radiotherapy to chemotherapy (combined modality treatment) versus combination chemotherapy alone in treatment of stage iv hl. design/method: we included patients with stage iv hl and whose data were retrieved from the medical records of the pediatric oncology department, national cancer institute, cairo university, egypt from till june and were followed till august . treatment was either to give cycles of abvd (adriamycin, bleomycin, vinblastine, dacarbazine) only or to give cycles of abvd followed by consolidation radiotherapy. the study included cases; were males and were females. mean age was . years ranging from to years. the histopathology subtype was nodular sclerosis in the majority of cases ( cases) followed by mixed cellularity ( cases) then only one case of lymphocyte rich. nine cases were initially bulky while cases were not. constitutional manifestations were present in cases while it was absent in cases. bone marrow was involved in only cases. radiotherapy was given after completion of chemotherapy to cases while cases received chemotherapy only. the -year overall survival for patients who received radiotherapy was superior to those who received chemotherapy alone; % versus . % respectively with statistical significance (p = . ). the -year progression free survival was also higher with radiotherapy than others; % versus . % (p = . ). patients with stage iv hl who received consolidation radiotherapy apparently had a better outcome than those who received chemotherapy only. this suggests that radiotherapy contributes significantly with chemotherapy to the cure rate for those patients. the feinstein institute for medical research, manhasset, new york, united states background: microrna (mirnas) are short non-coding rnas that play a decisive role in cancer biology, including leukemia. exosomes are microvesicles ( - nm) produced by most cells in biological fluids. exosomes represent the fingerprint of the parental tumor and are loaded with bioactive markers such as mirnas, which may regulate tumor growth. exosomal cargo can be transferred into target cells changing their biological properties. our study investigates a functional role for exosomal mir- a in pediatric acute lymphoid leukemia (p-all). objectives: / to demonstrate that p-all exosomes induce cell proliferation / to confirm that exosome-induced cell proliferation is disease-stage specific / to analyze exosomal mir- a expression profiles in p-all / to authenticate that inhibition of exosomal mir- a reduces leukemia proliferation design/method: exosomes were isolated by ultracentrifugation from healthy donors (hd) & p-all serum and conditioned medium (cm) of sup-b , jm , and cl- (control) human cell lines. cell lines were exposed to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion for hrs in triplicates. proliferation was assessed by microscopic cell counting and confirmed by gene expression for proliferation, pro-survival and pro-apoptotic genes. mirna profiling was performed with the human cancer pathway finder microarray (qiagen). silencing of exosomal mir a was carried out by a mir- a inhibitor (qiagen), utilizing exo-fecttm exosome transfection reagent (sbi, system biosciences). further, exosomal mir- a silencing was confirmed by q-pcr. cellular uptake of texred-sirna (sbi, system biosciences) was confirmed by flow cytometry. transfer of exosomal mir a to the target cells was evaluated by q-pcr. we elucidated that cm-derived exosomes from sup-b and jm cell lines induce cell proliferation in sup-b , jm (autocrine and paracrine) and cl- cells (paracrine) (p< . ). serum p-all exosomes promote paracrine cell proliferation in all cell lines compared to hdderived exosomes (p< . ). heatmap analysis of mirna profiles of leukemia exosomes (all cell lines and p-all) identified mir- a significantly upregulated in leukemia exosomes compared to controls. mir- a was also upregulated in all cell lines after exposure to leukemia exosomes that induced proliferation. moreover, exosomal mir- a inhibition reduces leukemic proliferation in pediatric all. our data suggest that all exosomes induce cell proliferation of leukemic cell lines in both paracrine and autocrine fashion. exosomes regulate these phenomena in a highly orchestrated way, by transfer of functional exosomal mirnas such as mir- a. the results of this study suggest s of s that exosomal mir- a inhibition can act as a novel way for growth-suppression of pediatric leukemia. results: a total of disease sites were detected at pet/ct, while sites were detected at contrast-enhanced ct and bone marrow biopsy (bmb). pet/ct showed improved detection of nodal lesions (p < . ) (kappa value = . ), extranodal lesions (p < . ) (kappa value = . ) and bone marrow (p < . ) (kappa value = . ) compared to contrast enhanced ct and bmb. pet/ct had upstaged cases ( %) and down-staged cases ( . %) (p < . ) (kappa value = . ). among the upstaged cases, patients ( . %) were upstaged from stage ii to iii, based on residual in pet/ct not seen in contrast enhanced ct after abdominal mass excision. four patients ( . %) were upstaged from stage iii to iv based on bone marrow uptake in fdg-pet without positivity in bma or bmb.regarding response assessment, sensitivity was % for pet and % for contrastenhanced ct (p = . ). specificity was % for pet and % for ct (p< . ). positive predictive value for pet was %, while was % for ct scan (p< . ). negative predictive value for both pet and ct was % (p = . ). five patients had nd biopsy to confirm viability of the residual lesions, lesions were negative in pathological examination (all of them were metabolic negative in pet/ct; deauville score below ). one lesion was positive in pathological examination (was positive in pet/ct; deauville score of ). conclusion: pet/ct detected additional sites compared with contrast-enhanced ct and resulted in changing stage of disease. pet scan is significantly more specific than ct in the management of children with burkitt lymphoma. background: deep sequencing of the immunoglobulin heavy chain (igh) locus indicates that each b all is composed of innumerable subclones. in many cases, subclones exhibit differing phenotypic qualities. however, it remains unclear whether subclones demonstrate distinct tissue distribution within a patient. objectives: . to quantify the extent of clonal heterogeneity in diagnostic b all specimens; . to identify variability in clonal composition between bone marrow (bm) and peripheral blood (pb) disease sites. design/method: igh sequencing was performed on purified dna from pairs of matched bm and pb patient specimens. multiplex pcr was used to globally amplify the igh locus; next generation sequencing (ngs) was performed using illu-mina® miseq. index clones (defined as ≥ % of all sequence reads in a specimen) and their subclone progeny (defined by shared nucleotide bases immediately upstream of a common jh, or n_jx) were identified using igblast-determined vh and jh alignments (http://www.ncbi.nlm.nih.gov/igblast/) and an established in-house computational pipeline. results: up to index clones per specimen were discovered in of the samples. in the remaining ( bm/pb pairs), pair did not reveal a clonal igh and was eliminated from analysis; in the other, clone frequency did not reach the % index threshold, but predominant clonal precursors were inferred by the prevalence of their subclone progeny. subclone counts ranged from to , per index clone. a combined , subclones derived from pb index clones were observed; in contrast, bm index clones gave rise to only subclones. subclone heterogeneity was observed between all paired specimens. in bm/pb pairs, index clones existed in equivalent proportions between disease sites. in contrast, bm/pb pair demonstrated high-frequency index clones in the bm ( . % & . %) with limited representation of these clones in the pb ( . % & . %, respectively); in this case, the most prevalent clone in the pb ( . %) matched the least frequent index clone in the bm ( . %). similarly, another pair showed a predominant index clone in the pb ( . %) which was below index threshold ( . %) in the bm. in paired patient specimens, index clone predominance was discovered to be overtly distinct between bm and pb. among all pairs, the extent of subclone progeny derived from each index clone showed marked variability, with far higher subclone frequency in the pb than in the bm. our data indicate that b all clonal composition differs between disease sites. valley children's healthcare, madera, california, united states background: tuberculosis (tb) presenting with hodgkin lymphoma (hl) is rare. their coexistence could lead to delay in diagnosis of both tb and hodgkin lymphoma due to the similarities in signs and symptoms of presentation. most cases have been reported in the adult literature. we describe a case series of children that were suspected to have tb and were found to have coexisting tb and hl. results: a retrospective review of hl patients in guatemala and argentina over six years, uncovered patients with simultaneous diagnosis of tb and hl. eight patients were from guatemala (incidence of . %) and from argentina (incidence of . %). there were females and males. age ranged from - years (mean . years, media years). nine patients were suspected to have tb at presentation by the referring physician. two patients were found to have tb at the time of relapse through routine tissue culture. initial systemic symptoms included fever (n = ), weight loss (n = ), and night sweats (n = ). six patients had a second systemic symptom in addition to fever. time for referral to oncology center ranged from weeks to months. nine patients were diagnosed with tb and hl through a tissue cultures and with serum quantiferon. one patient was found to have hl without tb. two patients had no systemic symptoms and the diagnosis of tb came to light through routine tissue culture. five patients had stage iiib and ivb, two stage iia and one iib at diagnosis. hl treatment was given according to the insti-tutional standards depending on stage and risk with abvd, oepa/copdac +/-radiation therapy, and ice for relapse. five patients started anti tb treatment (isoniazid, rifampin, pyrazinamide +/-ethambutol for months followed by isoniazid and rifampin for - weeks) simultaneously with chemotherapy, and three others after completing cycles. the two relapsed patients started tb treatment after cycles of chemotherapy. seven patients are alive and have been followed for months - years. one patient died during therapy, another died for causes not related to tb or hl and one is currently receiving treatment. conclusion: tuberculosis can coexist with hl. in areas were the prevalence of tb is high, microbiology investigations of biopsy specimen should be strongly considered. therapy for tb can be given simultaneously with chemotherapy. coexistence of tb and hl does not appear to affect outcomes. the children's hospital affiliated to the capital institute of pediatrics, united states background: the pi k/akt signaling pathway plays a central role in cell growth, proliferation and survival in physiological conditions. this signal pathway is considered to be an innovative targeted therapy of cancer, and its abnormal activation has been proved to be related to t-cell acute lymphoblastic leukemia (t-all). despite improved treatment strategies, such as multi-drug combination, high-dose chemotherapy and all kinds of application and popularization of hematopoietic stem cell transplantation, children with drug resistance or relapse t-all are still rather worse and its overall outcome and prognosis are much poorer than the more common b-lineage all. objectives: to explore the relationship between the pi k/akt pathway and the pediatric t-all, so as to probe the exact molecular mechanisms of t-all and provide more directions for its treatment. design/method: cases of new or recurrent acute t lymphocyte leukemia children with clinical information were collected in the children's hospital affiliated to the capital institute of pediatrics from dec. to oct. , with age and gender matched healty children as control (all was informed consent). the expressions of key genes in pi k pathway were s of s analyzed by western blot rt-pcr analysis, the pi k enzyme activities were detected by elisa,and the ccrf -cem's proliferation and its apoptosis were tested by mtt and flow cytometry technology on t-all cell lines ccrf-cem in different treatment group. the results of t-all children in clinical showed that pi k protein and gene expression level were higher apparent than the control group (p< . ), and pi k enzyme activity increased as well (p< . ); pi k inhibitor ly made a significant inhibition of cell proliferation and promoted cell apoptosis. ly also enhanced the effectiveness of clinical commonly used chemotherapeutic drug dnr. in combination ly and dnr treatment group cell viability dramatically declined, apoptosis and the apoptosis relation protein casepase expression in t-all patients was obviously higher than the control and the single drug group; pi k/akt signaling pathway related proteins and gene expression level, pi k, akt, gsk transcription in ccrf-cem were significantly higher than the control (p< . ), while pten transcription was significantly lower than the control (p< . ). the abnormal activation of pi k/akt signaling pathway might play an important role in pediatric t-all patients, especially in the cell proliferation or apoptosis. the results might provide new train of thought and direction in targeted suppress this signal pathway or in combination with other chemotherapy drugs therapy in looking for the more effective and less cytotoxic treatment of pediatric t-all. cleveland clinic children's hospital, cleveland, ohio, united states background: non-hodgkin lymphomas (nhls) are a heterogeneous group of lymphoproliferative diseases which comprise % of all childhood malignancies. nhls can be divided in to b cell lymphomas and t cell/natural killer (nk) cell lymphomas depending on immunophenotype, molecular biology, and clinical response to treatment. although nk/t cell lymphomas occurring in childhood and adolescence comprise a small portion of all lymphomas, they present many diagnostic and therapeutic challenges. the role of angiogenesis in lymphoma pathogenesis is becoming more evident. high molecular weight kininogen (hk) is a central compo-nent of the kallikrein-kinin system. it has been previously reported that cleaved hk (hka) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in matrigel plug and corneal angiogenesis models. however, the role of endogenous kininogen in regulation of angiogenesis is in tumor microenvironment is unknown. objectives: to elaborate the role of hk in lymphoma angiogenesis, we used a murine t-cell lymphoma model and compared angiogenesis and tumor growth between wild-type and kininogen deficient (mkng -/-) mice. we also evaluated the effect of hka on lymphoma cell proliferation. design/method: el- murine t-cell lymphoma cells ( × ^ ) were implanted into wild-type and mkng -/-mice. tumor size was measured using calipers and tumor volume was calculated using the formula volume = length × width^ × . . seventeen days after cell implantation, tumors were harvested and processed by immunoblotting and immunofluorescent staining. cell proliferation assays (mts) were performed to investigate any possible inhibitory effect of hka on el- cell growth, with human umbilical vein endothelial cells (huvec) were used as a positive control. results: el- lymphomas grew more rapidly and to larger sizes in mkng -/-mice compared to wild-type mice, with significant differences apparent by day after tumor implantation (p< . ). by day , the volume of tumors in mkng -/-mice was approximately . -fold larger than in wild-type mice (mean volume ± standard deviation; ± vs. ± mm , respectively, p< . ). mts assays showed that hka does not directly inhibit the proliferation of el- cells in vitro, though it does significantly impair the viability of ecs studied simultaneously. conclusion: these findings suggest that hk is an important endogenous regulator of angiogenesis and tumor growth in this t-cell lymphoma model, and suggests that hka specifically modulates endothelial proliferation in tumor microenvironment. further work is needed to understand the mechanisms underlying these findings and provide future anti-angiogenic approaches to increase the therapeutic options for patients with nhl. bruce bostrom, jack knudson, nathan gossai, joanna perkins, michael richards, jawhar rawwas, susan sencer, julie chu, nancy mcallister, yoav messinger children's minnesota, minneapolis, minnesota, united states background: osteonecrosis causes significant pain and morbidity in older patients treated for acute lymphoblastic leukemia. besides altering the schedule of dexamethasone in delayed intensification there is no other intervention known to reduce the incidence of symptomatic osteonecrosis. pamidronate has been shown to reduce bone pain from osteonecrosis but not to prevent joint collapse when advanced. objectives: to compare the incidence of symptomatic osteonecrosis in patients who received prophylactic pamidronate compared with concurrent controls. to describe any increase in side effects from the use of pamidronate. design/method: patients age to years at time of all diagnosis were given intravenous pamidronate monthly for one year at the discretion of the primary oncologist starting in the first year of therapy. concurrent controls were patients age to who did not receive pamidronate. all patients were treated according to the concurrent cog protocols and received intermittent dexamethasone during delayed intensification. patients with bcr-abl all were excluded as the use of imatinib may increase the risk of osteonecrosis. imaging was only done if osteonecrosis was suspect based on clinical symptoms. patients were censored at the time of relapse. data were analyzed as of / / . this retrospective study was approved by the children's minnesota irb. of the patients evaluated % were male and % female, % had b-cell and % t-cell. the median followup is . years with a range of . to years. pamidronate was given to patients with developing symptomatic osteonecrosis. there were concurrent controls with developing osteonecrosis. there was no significant difference in the leukemia lineage, gender distribution or body mass index (bmi) at diagnosis between groups. for all patients the median bmi was with a range of to . the age at diagnosis was significantly higher in the pamidronate group with a median of . years vs. . in the controls (p = . ). by kaplan-meier analyses the incidence of symptomatic osteonecrosis was significantly lower in the pamidronate group at % vs. % in controls. the log-rank p-value was . and the breslow p-value, which is more sensitive to early events, was . . there were no untoward side-effects from pamidronate. pamidronate infusions significantly reduced the incidence of symptomatic osteonecrosis in patients over the age of compared to concurrent controls who did not receive pamidronate. arahana awasthi, dina edani, janet ayello, christian klein, mitchell cairo new york medical college, valhalla, new york, united states background: mature b-nhl, including bl and pmbl express cd +/cd b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ % yr. efs, cairo et al. blood. ; gerrard/cairo et al., blood, , goldman/cairo et al. leukemia, . pv has been demonstrated to possess significant preclinical activity against indolent cd b+nhl (polson et al. can. res. ). we previously observed that obinutuzumab (anti-cd mab) significantly enhanced cell death and increased overall survival against bl (awasthi/cairo et al., bjh ) in xenografted nsg mice. however, additive/synergistic effects of pv with obinutuzumab against mature pmbl/bl are unknown. to determine the efficacy of the pv or obinutuzumab/rtx alone or in combination against pmbl and rituximab (rtx) sensitive/resistant bl cell lines. design/method: raji rh (provided by m. barth, md, roswell park cancer institute) and raji/ karpas p (atcc, usa) were cultured in rpmi. tumor cells were incubated with pv, and/or anti-cd b, mmae (generously supplied by genentech inc.) with obinutuzumab /rituximab ( ug/ml) for hr with nk cells at : e: t ratio and cytotoxicity was determined by delfia cytotoxicity assay. six to week old female nsg (nod.cg-prkdcscid il rgtm wjl/szj), were divided into groups: pbs, isotype control, pv, anticd b mab and mmae ( mg/kg). mice were xenografted with intravenous injections of luc+ bl and pmbl cells and tumor burden was monitored by ivis spectrum system. results: os of mice receiving pv alone was significantly increased compared to anticd b ab or isotype control in raji ( . vs. vs. . our preliminary data indicates that pv significantly increased survival in bl and pmbl nsg xenografts compared to anti-cd b ab alone. furthermore, pv in combination with obinutuzumab significantly enhances in-vitro cytotoxicity in bl and pmbl compared to obinutuzumab or pv alone. results: maximal grades (g) / , , and crs occurred in , , and patients, respectively. median lowest fibrinogen levels were . , . , and . g/l in patients with maximal g - , , and crs, respectively. %, %, and % of patients with maximal g - , , and crs had lowest reported fibrinogen levels of ≥ to < . g/l. eight patients (all with g crs) had very low fibrinogen levels (< g/l), which occurred before (n = ) or during (n = ) maximal crs grade or at time of improvement (n = ). no patients with maximal g - crs had < g/l fibrinogen levels. at the onset of < g/l fibrinogen levels, patient had concurrent g , and had g - increased international normalized ratio and activated partial thromboplastin. cryoprecipitate was the primary treatment in the us, and fibrinogen concentrate (fc) guidelines for tisagenlecleucel-associated coagulopathy were developed for other countries because administration of fresh frozen plasma can be problematic. fc was available at / sites for infused patients: / (g crs) and / (g - crs). cryoprecipitate was available at / sites for infused patients: / (g crs), / (g crs), and / (g - crs). risk of bleeding increases in pediatric patients with comorbid thrombocytopenia and anticoagulant treatments. / patients had g / decreased platelets within day of < g/l fibrinogen levels. fatal case of intraparenchymal cranial hemorrhage occurred during resolving crs with g hypofibrinogenemia, ongoing thrombocytopenia, and continuous veno-venous hemofiltration with citrate. hypofibrinogenemia was observed more frequently in patients with higher crs grades during/when crs was improving or resolving. fc and cryoprecipitate treatment guidelines were developed. frequent monitoring and fibrinogen replacement are needed in patients with g / crs. sponsored by novartis. its prolonged cns half-life, may allow a reduction in the number of intrathecal injections. objectives: to safely reduce the burden of therapy by reducing the number of it injections and reducing the total dose of doxorubicin with the addition of liposomal cytarabine and rituximab. design/method: patients ( - years) with cd + b-nhl with fab group b good risk (=stage i/ii and stage iii with ldh < xuln), fab group b intermediate risk (=stage iii ldh ≥ xuln and stage iv {bm blasts < %}) and fab group c high risk were eligible. patients received fab backbone therapy with the addition of six rituximab ( mg/m ) doses; two doses prior to each of two induction courses and one dose prior to each of two consolidation courses. cumulative doxorubicin was reduced from to mg/m in gr patients. after systemic methotrexate clearance, patients received age based dosing of it liposomal cytarabine. it injections were reduced from nine to five. the primary outcome is safety and toxic deaths among evaluable patients with an estimated -year survival above %, monitored by an independent dsmb. results: to date, evaluable patients, fab group b and group c ( cns positive), median age years (range - ), males, burkitt/ dlbcl with gr, ir and hr have enrolled. there has been one grade anaphylactic reaction to rituximab and one grade facial nerve palsy. no other serious adverse events were attributable to protocol therapy. there has been death from progressive disease and relapse at a median follow up of months. efs and os are % and %, respectively. our initial results show excellent efs and os, consistent with published standard of care outcomes, with the addition of rituximab and intrathecal liposomal cytarabine despite the reductions in therapy. further enrollment is ongoing and continued long term outcomes are needed to confirm early results. future randomized studies are needed to examine both short term (mucositis, infections, hospitalization days) and long term (late cardiac toxicity) endpoints. . goldman etal, leukemia, . cairo etal, jco st. jude children's research hospital, memphis, tennessee, united states background: bereaved parents identify significant spiritual needs around time of death and throughout their bereavement journeys. spirituality has been identified as a primary means by which bereaved parents can find meaning in their losses, and this ability to find meaning is associated with lower maladaptive grief symptoms. the use of spiritual coping strategies has been associated with improved coping and mental health outcomes among bereaved parents. objectives: to better understand how bereaved parents' experiences with spirituality throughout bereavement effects objective measures of grief, depression, and meaning-making. design/method: thirty participants whose children died of progressive cancer or related complications one to three years prior to participation completed an in-depth semi-structured telephone interview about their experiences with grief. participants were prompted to describe the impact of their spirituality on their bereavement processes. additionally, participants completed surveys related to grief (prolonged grief disorder questionnaire, pg- ), depression (beck depression inventory, bdi), and meaning-making (integration of stressful life experiences scale, isles). results were analyzed using a mixed methods approach including semantic content analysis of qualitative content and kruskal-wallis h test and post-hoc analyses of quantitative data. results: correlation analyses demonstrated significant differences between participants with positive and negative spiritual experiences of bereavement. participants with negative experiences of bereavement had a statistically significant increase in scores on the pg- compared to those with positive spiritual experiences signifying greater symptoms of prolonged grief. participants with negative spiritual experiences with grief had significantly lower scores on the isles, suggesting a lesser degree of adaptive integration of their losses. there were no significant differences in depression scores between groups. conclusion: bereaved parents that have a negative spiritual experience of bereavement are at increased risk for prolonged grief symptoms and are less likely to find meaning in their children's deaths than bereaved parents that describe a positive spiritual experience of bereavement. providers should consider exploration of spiritual beliefs and provision of spiritual care for parents of children facing life-limiting illnesses during treatment and bereavement. background: langerhans cell histiocytosis (lch) is an inflammatory myeloid neoplasia characterized by frequent relapse, with treatment failure associated with higher risk of death and neurodegenerative disease (lch-nd). activating somatic mutations in mapk pathway genes have been identified in almost all cases, with braf-v e in approximately % of lesions. targeted therapies have been successful in treating other refractory cancers with braf v e mutations (such as melanoma). given the central role of mapk pathway activation in lch, mapk pathway inhibition may be an effective therapeutic strategy for children with lch. objectives: the purpose of this study was to report the efficacy and toxicity profile of a retrospective cohort of patients with lch treated with mapk pathway inhibitors. design/method: medical records from pediatric patients with lch (systemic and/or lch-associated neurodegeneration) who were treated with a mapk pathway inhibitor were retrospectively reviewed from five institutions. all patients had failed at least one prior systemic therapy and had a proven mapk pathway mutation. results: all patients in this series were less than years old (median = . years; range: - years) with a median of three prior treatments (range: - ). at the time of initial mapk inhibitor use, nine of the patients had lch-nd diagnosed clinically and/or by radiographic imaging; the remaining three patients had systemic disease. patients were treated for a median of months (range: - months) with various reasons for discontinuation. three patients received combination mapk inhibitor therapies and three patients received other concurrent lch-directed therapies. four of the twelve patients had a grade or toxicity reported and three of these patients required dose reduction in order to be able to successfully resume therapy. overall survival was % with median month follow-up (range: - months) with only one patient achieving transient complete response. the remaining ten patients had partial response or stable disease and four of these patients developed progressive disease while on therapy. conclusion: mapk pathway inhibitors may be a relatively safe salvage therapy for refractory systemic lch and lch-nd but the efficacy and durability of this strategy remains to be defined. combination with cytotoxic chemotherapies may be required in order to eradicate the disease-causing cell. future prospective trials of mapk pathway inhibitors for patients with refractory lch are needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. cincinnati children's hospital medical center, cincinnati, ohio, united states background: medication adherence during maintenance therapy has been shown to have a direct relationship with disease relapse in pediatric leukemia. previous research determined that patients who are ≤ % adherent to mercaptopurine ( mp) have a greater risk for relapse. the primary aim of the present study is to examine the relationship between metabolite profiles of mp with behavioral adherence rates obtained via electronic monitoring at , , and days. it is hypothesized that patients demonstrating low levels of thioguanine (tgn) and methylated mercaptopurine (mmp) will have lower behavioral adherence rates prior to the blood draw. design/method: in a multisite, prospective study of patients ages - years diagnosed with acute lymphoblastic leukemia (all) or lymphoblastic lymphoma (lbl), mp adherence was measured across months of maintenance therapy using behavioral adherence (electronic monitoring) and pharmacological (metabolites) measures of mp. mp is metabolized into mmp and tgn. cluster analysis was used to generate three mutually-exclusive profiles of mp adherence. behavioral adherence rates were calculated for , , and days prior to the blood draw. results: this study identified three metabolite profiles of mp across months. previous research indicated that low levels of both metabolites suggest nonadherence to medication. low levels of one metabolite with high levels of another metabolite indicate adherence to mp. in this study, . % of the low tgn-low mmp group had -day behavioral adherence rates ≥ % (mean = %); . % had adherence rates < % (mean = . %). in the high tgn-low mmp group, . % had a mean -day adherence of %; . % had adherence rates < % (mean = . %). the low tgn-high mmp group had % of patients with a mean -day adherence level of %; % had adherence rates < % (m = . %). at and -days, to % of patients in the low tgn-low mmp group had adherence rates < %. conclusion: these findings suggest that electronic monitoring and metabolite concentrations can be used to monitor mp medication adherence during maintenance therapy. it is notable that there is a sub-sample of pediatric patients who are identified as being nonadherent to mp based on electronic monitoring, however, metabolite levels indicate adherence to mp. similarly, a sub-sample of patients were identified as being adherent based on electronic monitoring, but metabolite profiles indicated sub-therapeutic levels of mp. our findings underscore the clinical significance of using both objective measures of medication adherence to inform clinical decision making. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hemophagocytic lymphohistiocytosis (hlh) is a life-threatening hyperinflammatory syndrome characterized by non-remitting fevers, rash, hepatosplenomegaly, cytopenias, liver dysfunction and coagulopathy, and can include central nervous system involvement. several genetic diseases cause hlh by impairing normal lymphocyte or macrophage function. the hlh panel at the cincinnati children's genetics laboratories includes genes associated with hlh and other lymphoproliferative diseases, including the genes that cause primary hlh (prf , unc d, stxbp , stx , rab a), x-linked lymphoproliferative diseases (sh d a, xiap), itk deficiency (itk), hermansky-pudlak syndrome types and (ap b and bloc s ), chediak-higashi syndrome (lyst), cd deficiency (cd ), xmen syndrome (magt ) and lysinuric protein intolerance (slc a ). deletion/duplication analysis is available as a reflex test for all genes, as copy number variations (cnvs) are not directly assessed by sequencing. objectives: the prevalence of cnvs among large groups of patients with hlh in north america is unknown. we assessed the frequency of cnvs in the genes on the hlh panel through a retrospective review of orders for deletion/duplication analysis performed after next-generation or sanger sequencing: orders for all genes on the panel, and orders of - genes from the panel. deletion/duplication analysis was performed on a custom × k microarray annotated against ncbi build (ucsc hg , march ). deletion/duplication analysis resulted in a confirmatory diagnosis in of cases ( . %). pathogenic or likely pathogenic cnvs were most common in the three x-linked genes: sh d a ( deletions), xiap ( deletions, duplication), and magt ( deletions). hemizygous deletions in xlinked genes in male patients were typically suspected after amplification failure during previous sequencing. of the autosomal recessive genes, pathogenic cnvs were observed once in each of three genes: rab a (heterozygous), lyst (heterozygous), and stxbp (homozygous). in the two heterozygous cases, a second change was not identified by sequencing, so deletion/duplication analysis did not offer a confirmatory diagnosis. in patients, deletion/duplication analysis was performed after a pathogenic or likely pathogenic variant was identified in an autosomal recessive gene during sequencing; however, in no case was a second mutation uncovered by cnv analysis. we recommend that deletion/duplication analysis be routinely performed in all male patients with hlh who lack a genetic diagnosis after sequencing of hlh-associated genes, especially if any regions failed to amplify. deletion/duplication analysis may be performed in female patients after sequencing if a genetic form of hlh is highly suspected, but the yield is expected to be low. cleveland clinic children's hospital, cleveland, ohio, united states background: the development of post-transplant neoplasia, typically from lymphoproliferative disease (ptld), is a severe complication in transplant recipients and affects approximately % of pediatric solid organ recipients. rates of lymphoma in adult heart transplantation patients are comparatively low, at less two percent at ten years. there are few published reports of the long-term outcomes of neoplasia after pediatric heart transplantation. we aimed to identify the subsequent malignancies that occurred in pediatric heart transplantation patients in a large single institution, and describe their treatment and subsequent clinical course. we performed a retrospective chart review of all pediatric heart transplant recipients followed at the cleveland clinic children's hospital from january to october . we excluded patients who died within days of heart transplantation. we reviewed in depth the history and clinical course of subjects who developed neoplasms. results: between and , patients underwent heart transplantation and survived at least days post transplantation. nine patients ( . %) developed a subsequent malignancy. in this case series, the median age at heart transplant was years old and the median time to develop neoplasia was . months. primary neoplasia included monomorphic ptld ( ), polymorphic ptld ( ), burkitt lymphoma ( ), hodgkin's lymphoma ( ), plasmacytoma-like lymphoma ( ) and epstein-barr virus-associated smooth muscle tumor (ebv-smt) ( ). one patient with hodgkin lymphoma subsequently developed monomorphic ptld, one patient with polymorphic ptld subsequently developed ebv-smt and later, an undifferentiated gastric cancer. one patient with monomorphic ptld developed an ebv-smt. evidence of epstein-barr virus was present in six of nine patients at diagnosis of first malignancy. four of nine patients received reduction in immunosuppression as a primary intervention for the initial malignancy, with two complete responses (cr), one partial response, and one with progressive disease. five patients were treated with chemotherapy, with four cr and one with progressive disease. three patients died of malignancy (recurrent ebv-smt, undifferentiated gastric cancer, and monomorphic ptld post-hodgkin disease) and two patients died of other transplant related complications. conclusion: secondary malignancies represent a significant disease burden to survivors of cardiac transplantation. as expected, much of the malignancy burden is driven by ebv. despite aggressive histology, many malignancies can be successfully cured in this setting with a multidisciplinary approach. stanford university school of medicine, palo alto, california, united states background: current treatment of langerhans cell histiocytosis (lch) is based on extent of organ system involvement and if high risk systems are affected. gastrointestinal (gi) involvement is diagnosed in about % of lch patients, and classically presents in children under years of age with malabsorption, failure to thrive, bloody diarrhea and anemia. although the gi system is considered standard risk, a mortality rate over % occurring within years of diagnosis has been reported. this study was performed due to this discrepancy and the limited number of published cases. objectives: to review the clinical course and outcomes of patients diagnosed with gi lch. design/method: a retrospective chart review of patients with histologically confirmed gi lch diagnosed in the last years identified from the bass center histiocytosis clinical database was performed. two other pediatric hematology/oncology centers (ucsf benioff children's hospital oakland and san francisco) were queried for additional cases. results: four patients with biopsy proven gi lch [ subjects ( . %) from database records and l from center queries] were identified. failure to thrive, hypoalbuminemia, bloody diarrhea and rash were the most common presenting symptoms. lch of the skin was found in all patients. risk organ systems were involved in patients. of note, subjects were of african racial background. the median age at diagnosis was . months ( . months to years), mean albumin . g/dl ( . - . g/dl), mean esr of mm/hr ( - mm/hr). all patients initially received combination therapy per lchiii protocol (vinblastine, prednisone, and mercaptopurine). two patients had recurrent disease and received second line therapy (cytarabine, cda, and local radiation therapy). all patients are alive without active disease at last follow-up ( to months after completion of therapy). a systematic approach to evaluate gi involvement should be performed in children diagnosed with lch. from our experience, combination chemotherapy for patients with lch involving the gi tract is an effective intervention for active disease. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bhatia indicated that rates of mp adherence ≥ % have better clinical outcomes. those with adherence rates ≤ % have an increased risk for disease relapse. the present study investigated patterns of mp medication adherence using group-based trajectory modeling in a large sample of pediatric patients. to describe patterns of behavioral adherence during the maintenance phase of therapy for a cohort of pediatric patients ages - years who were diagnosed with acute lymphoblastic leukemia or lymphoblastic lymphoma (n = ). previous research has documented the relationship between optimal levels of medication adherence with positive health outcomes. it was hypothesized that three groups would be identified: optimal adherence, deteriorating adherence, and chronic nonadherence. it was hypothesized that patients in the optimal adherence group would have adherence rates ≥ %. those with poor adherence would have adherence rates ≤ %. design/method: the present study was a longitudinal, multisite study investigating adherence to -mercaptopurine in a pediatric cohort of patients using electronic monitoring devices. daily adherence rates (electronic monitoring of mp) were examined across -months. health outcomes were measured at quarterly intervals through medical chart reviews. results: unconditional growth curve modeling indicated that the mean percentage of behavioral adherence was . % at baseline and declined to . % at -months. three trajectories of mp behavioral adherence were identified: ) optimal adherence ( % of patients): averaging % behavioral adherence across months; ) moderate adherence ( %): relatively stable nonadherence with rates of % across months; and, ) chronically nonadherent ( %): adherence decreased from % to %. with respect to patterns of medication adherence and relationship to clinically-relevant health outcomes, there were no significant differences in health outcomes between patients in the adherent versus nonadherent trajectories, including mean absolute neutrophil counts (anc), risk for infection as measured by anc, healthcare utilization, or risk for disease relapse. although longitudinal patterns of mp behavioral adherence were not related to health outcomes, it is notable that only % of the current sample had adherence rates ≥ %. in fact, % of the current sample demonstrated adherence rates ≤ %. our findings are important for development of future adherence promotion studies in pediatric cancer. our findings underscore the relative significance of tailoring adherence promotion interventions to subgroups of patients, including those with problematic patterns of adherence. patients who demonstrate adequate levels of adherence could still benefit from less intensive, preventative interventions to sustain and improve adherence. sophie gatineau-sailliant, pascale grimard, marie-claude miron, guy grimard, anne-sophie carret, jean-marie leclerc chu sainte-justine, montreal, quebec, canada background: vertebral involvement in langerhans cell histiocytosis (lch) is still a subject of interest, due to its low frequency and the absence of management's guidelines. objectives: to provide additional information on presentation, treatment and morbidity of pediatric lch vertebral lesions, we report cases of children with vertebral lesion of biopsy-proven lch, between january st and december st , at sainte-justine university health center (montreal, quebec, canada). we conducted a retrospective study by reviewing charts and imaging of vertebral lch in a population of children (median age of . years at lch diagnosis), followed for a median duration of months. symptoms at presentation, treatment modalities and morbidities were collected. results: vertebral lesions were present at lch diagnosis in of cases. they were usually diagnosed secondary to back pain in of cases and were asymptomatic in only one case. despite an epidural extension in of cases, no child developed neurological symptoms. lesions frequently involved vertebral body ( of cases) and were rarely unstable ( of cases). out of vertebral lesions, most of them had a dorsal localization ( of lesions) and of patients had lch in multiple vertebrae. at diagnosis, median vertebral height loss was . % compared to % at last imaging control. most used imaging modalities were pet-scan and plain x-rays. treatments were diverse and consisted in chemotherapy in all children but three and bisphosphonates in only cases. radiation therapy was not used in any patient. six out patients did benefit of an orthosis. a lch recurrence was observed in patients and involved vertebrae in cases. one patient with treatment-resistant lch disease had relapses, and required multiple lines of treatment. all children were alive and disease-free at their last follow-up, patients having radiological vertebral sequelae and only had clinical sequelae. our study is consistent with the epidemiological data described in larger cohorts of children with vertebral lesions of lch and the favorable prognosis associated with such lesions. nevertheless, aggressive treatment and long term follow-up seemed to be essential as recurrences are s of s not rare and spontaneous bone regeneration often incomplete. plain x-rays appears to be a good follow-up tool for vertebral lesions as it allows reliable measures, less exposure to radiation at lower cost. national cancer institue, giza, giza, egypt background: acute lymphoblastic leukemia (all) is the most common type of childhood cancer and also the most complicated in the treatment, so it requires many interventions for both treatment and to alleviate suffer form side effects. pancreatitis is one of the toxicities, which is more common in all as it appears in about % of the patients. it occurs in many drug combinations which induce pre-pancreatitis and even direct destruction of pancreatic tissues. pancreatitis can be induced by many drugs used in the treatment such as chemotherapeutic agents or supportive treatment. lasparaginase is the backbone drug of the treatment of all in which to doses are required to achieve complete remission status in the induction phase of treatment and to doses in the maintenance phase.it is an enzyme that destructs the l-asparagine amino acid into aspartic acid and ammonia thus deplete the asparagine from the extracellular matrix . many drugs are investigated for their effect on treatment of induced pancreatitis such as interleukin- , nsaid as antiinflammatory, glycerin tri nitrates as improvement of microcirculation, tnf-alpha antibody, paf inhibitor as specific anti-inflammatory and low molecular weight heparin .none of the drugs was investigated for their ability to prevent the occurrence of pancreatitis. objectives: this study was designed to evaluate the protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis design/method: acute pancreatitis was induced in rats by intra-muscular injection of l-asparaginase ( i.u/kg) given daily for five days. enoxaparin was given subcutaneous ( i.u/kg) and diclofenac was given intra-peritoneal ( mg/kg) daily for five days. then, markers of pancreatic injury, lipids, immune cell infiltration and oxidative stress were analyzed with histo-pathological examination of the pancreatic tissue results: during acute pancreatitis, oxidative stress markers were significantly changed as indicated by reduced tis-sue glutathione and increased malondialdehyde levels. this was accompanied with significant increase in immune cells infiltration as indicated by high levels of myeloperoxidase and pro-inflammatory cytokine tnf-alpha. triglyceride only showed increase level. treatment with enoxaparin and/or diclofenac restored levels of biochemical markers including serum alpha-amylase, reduced glutathione, malondialdehyde, pro-inflammatory cytokine tnf-alpha, myeloperoxidase and triglyceride. histological injuries of pancreatic tissues as vacuolation and necrosis of epithelial lining pancreatic acini, inflammatory cells infiltration and focal pancreatic hemorrhage were also reduced by treatment with enoxaparin and/or diclofenac. the present study emphasizes the potential protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis background: rosai dorfman disease (rdd), or sinus histiocytosis with massive lymphadenopathy (shml), is a rare condition of immune dysregulation of unknown etiology arising from the massive accumulation of non-langerhans type histiocytic cells inside lymph nodes. the disease classically presents as bulky, painless lymphadenopathy often associated with infection showing distension of lymph node sinuses by abundant histiocytic cells (cd a(-), s- (+)/cd (+)). in some cases, the disease can be self-limiting, but in cases with a prolonged chronic course of exacerbations and remissions, those with extranodal involvement, or disease that threatens vitals structures, treatment may be necessary. there is no treatment consensus. to describe a case of life-threatening, unresectable, recurrent rdd successfully treated with langerhans cell histiocytosis (lch) -inspired therapy. design/method: we compared this case to the current literature on chemotherapeutic treatments for rdd. we searched pubmed, ovid, and google scholar for similar cases. we believe this to be the first reported case of using lch therapy to successfully treat rdd. an -year-old male presented to an outside hospital with two years of massive neck swelling causing torticollis. biopsy confirmed rdd. he was intermittently treated with courses of antibiotics with partial response. surgical removal of the affected lymph nodes was unsuccessful due to proximity to the spinal cord. two years later, the patient presented to our institution. he was initially treated with prednisone with a fast tapering dose, but after a second relapse the decision was made to try chemotherapy following the lch- protocol of weekly vinblastine ( mg/m ), -mp ( mg/m ), and high dose steroid bursts. he experienced two additional relapses off therapy at ages and years old, including cmv(+) associated septic shock and cytokine storm requiring rapid response, picu admission, and ionotropic support. this last episode was treated with a more prolonged induction and maintenance therapy. an extended and slowly tapered maintenance therapy regimen of . years of daily -mp, monthly vinblastine and steroids with a slowly tapered dose during his fourth remission has resulted in -months of continuous complete remission-the longest stretch of his life. no similar cases were found. literature search demonstrated no consensus regarding the most effective treatment of rdd, with no previous cases being successfully treated following lch chemotherapy protocols. we hypothesize that the multi-agent relatively mild lch- therapy mitigates the immune dysregulation of rdd. this case suggests that lch- therapy can be used to treat cases of rdd that is not amendable to surgery or observation. nicklaus children's hospital, miami, florida, united states background: central venous catheters (cvc) are necessary in the management of patients with malignancies, especially children. patients with acute leukemia (al) have higher rates of central line associated complications such as bloodstream infections compared with other malignancies. objectives: to examine the choice of placement of cvc and the differences in outcome between peripherally inserted central catheters (picc) and ports in patients with leukemia during induction. design/method: retrospective chart review of patients with newly diagnosed leukemia at nicklaus children's hospital between and . results: ninety four patients with a new diagnosis of leukemia undergoing induction chemotherapy were identified. the average age was . years. overall, ( . %) patients had a port placed and ( . %) had a picc placed. the decision for picc or port was subjective and physician based. the main outcome measures were local inflammation/infection, bacteremia, thrombophlebitis, blocked catheter and premature removal. the most common complication was bacteremia ( . %). in a multiple logistic regression analysis for predicting whether patients had at least one complication, results showed that having at least one complication is . times the odds in patients with aml compared to patients with all (p = . ). when comparing picc vs. ports, patients with picc had more frequent episodes of blocked catheters ( . %) and premature removal ( . %) compared to the patients with ports ( . % and . %) (p = . and p = . respectively) during induction. local inflammation, bacteremia and thrombophlebitis were not statistically different (p = . , p = . and p = . respectively). the most common place for port placement was the right subclavian vein ( %). there was no significant association between port location and having at least one complication (p = . ). acute lymphocytic leukemia subgroup analysis: fourteen patients ( %) in the picc group had at least one complication and ( %) in the port group but that was not statistically significant (p = . ). our series showed a higher incidence of blocked catheters and premature removals with picc compared to ports in patients with leukemia during induction. the choice of placement of picc vs port was subjective and physician based. patients with all, despite receiving steroids and asparaginase during induction, did not show a statistically significant increase risk in thrombosis or infection but larger numbers may be needed in future studies. university of california, san francisco, san francisco, california, united states background: hemophagocytic lymphohistiocytosis (hlh) is classically a disorder of young children meeting systemic hyperinflammation criteria. presentation in late adolescence is uncommon. furthermore, though cns signs occur in - % of cases, initial isolated neurologic presentation is rare, frequently resembling encephalitis or demyelinating disorders. these cns signs can be isolated or precede systemic disease, delaying hlh diagnosis. hlh declaring in adolescence with predominant psychiatric features has not been well documented. objectives: to describe a case of cns hlh presenting with neuropsychiatric features in absence of classic hlh criteria. design/method: retrospective review of clinical, radiologic, histologic, immunophenotypic, and molecular features of a patient with cns hlh. a -year-old female presented with acute-onset headaches following nine months of progressive anxiety, short-term memory loss, emotional lability, perceptual disturbances, and hypomania. brain mri demonstrated numerous enhancing t hyperintense supratentorial and infratentorial white matter lesions in the left thalamus and caudate head. brain biopsy showed histiocyte-rich inflammation and associated demyelination. extensive evaluation including universal microbial pcr failed to reveal underlying infection or malignancy. past medical history was notable for presumptive pulmonary sarcoidosis diagnosed months prior with progressive respiratory failure with associated granulomatous pulmonary nodules which responded to systemic immunosuppression. at presentation of her neuropsychiatric symptoms, she had normal sil- r, ferritin, fibrinogen, and triglycerides. there was no pancytopenia, coagulopathy, bone marrow hemophagocytosis, fevers, or splenomegaly. given the possibility of partial immune suppression of systemic symptoms and the prominent neurologic symptoms, hlh screening labs were sent and notable for decreased natural killer and cytotoxic t lymphocyte function, normal granzyme expression and cd a mobilization, and absent perforin expression. genetic testing confirmed compound heterozygous mutations in prf (c. g>a, c. a>c) and familial hlh type . she was treated with low-dose dexamethasone and intrathecal chemotherapy per hlh- . due to lack of evidence of systemic inflammation, vp- and high-dose steroids were held. within one week of initiating therapy, she had decreased anxiety and improved cognition, with sustained, incremental neuropsychiatric improvement with additional intrathecal treatments. she tolerated dexamethasone tapering without symptom flare. mri also demonstrated parenchymal lesion improvement. for definitive treatment, she underwent unrelated allogeneic hematopoietic cell transplantation and remains at neurologic baseline as of eight months post-transplant with ongoing imaging improvement. conclusion: this case of familial hlh with compound heterozygous perforin mutations in an adolescent with isolated neuropsychiatric symptoms illustrates that cns hlh may be an underrecognized phenomenon in absence of systemic signs. standard hlh therapy may effectively reverse these symptoms with associated radiologic responses. rush university children's hospital, chicago, illinois, united states background: posterior reversible encephalopathy syndrome (pres), a recognized complication of pediatric leukemia treatment has been reported in up to % patients in various series. hypertension, chemotherapy and cortical spreading depression have been implicated in the pathophysiology. due to the combinations used, it is difficult to identify the offending drug, several have been implicated. since delay of chemotherapeutic treatment in children with high risk leukemia is unfavorable, it is important to recognize the characteristic radiologic findings, manage appropriately and reintroduce the treatment as soon as possible. pharmacoethnicity is now recognized as an important factor for variation in neurotoxicity in children with all. ethnic differences in reported pres events in pediatric patients with all has not been well described in literature. to describe the factors associated with pres in a cohort of high risk pediatric all patients at a single institution. design/method: a total of children with an average age of years ( - years) diagnosed with all between - were retrospectively reviewed for the occurrence of pres. various demographic factors, therapy received, clinical features, radiology related findings and management were reviewed. a search for all published articles on pres in leukemia was conducted using pubmed databases. results: five ( %) children (average age . years) developed pres during days - of induction. % of the patients that developed and % of those that did not develop pres were hispanic. all the patients that developed pres and % of those that did not were diagnosed with high risk all. all patients received vincristine, % received daunomycin and intrathecal methotrexate and % received asparaginase in the week prior to the event. mri findings confirmed pres in all patients with no evidence of methotrexate related leukoencephalopathy or leukemia. at the time of pres all patients were in remission based on mrd and spinal fluid cytology. two-thirds of the patients had seizures and hypertension at the time of the event with no prior history of either. all patients had complete recovery of normal mental status after resolution of pres. a higher incidence of pres than previously reported was noted in our series. hispanic ethnicity, high-risk all and exposure to vincristine, daunomycin and intrathecal methotrexate in induction were associated with pres in our cohort. a new association that emerged was that of hispanic ethnicity with pres .larger studies to understand the importance of pharmacoethnicity in pres may help in individualization of chemotherapy based on ethnic differences. children's hospital of illinois, peoria, illinois, united states background: hyper ige syndrome is a primary immunodeficiency characterized by susceptibility to skin and lung infections as well as increased propensity for malignancy. hemophagocytic lymphohistiocytosis (hlh) is a syndrome characterized by overwhelming activation of t lymphocytes and macrophages occurring as either primary hlh caused by genetic abnormalities or secondary hlh associated with infectious, malignant, metabolic, or immunodeficiency causes. we describe the first case to our knowledge of hlh in a patient with hyper ige syndrome. to describe a case of hlh in a pediatric patient with hyper ige syndrome. results: a -year old caucasian male with known autosomal dominant hyper ige syndrome (stat mutation) was transferred to the pediatric intensive care unit secondary to concern for septic shock. the patient had persistent slow bleeding from oral lesions and central catheter sites despite the addition of aminocaproic acid and recombinant factor viia. he also required numerous blood product transfusions sec-ondary to anemia and thrombocytopenia. clinical suspicion was high for hlh and the patient met criteria for diagnosis of hlh with the following: ferritin > , ng/ml, triglycerides mg/dl, decreased nk cell function with the sample only containing % nk cells, elevated soluble il- receptor at u/ml, splenomegaly, and fever. infectious workup was remarkable for a positive ebv qpcr with , copies/ml suggestive of ebv driven secondary hlh. familial hlh testing was unable to be completed. therapy was initiated based upon the hlh- study. the addition of ruxolitinib and anakinra were considered but the patient declined rapidly prior to treatment. ct of the head was concerning for a stroke with signs of edema and increased intracranial pressure likely leading to the development of symptoms consistent with brain stem herniation. the decision was then made to withdraw care. conclusion: to our knowledge, this is the first report of hlh in a patient with hyper ige syndrome. diagnosing hlh requires a high index of suspicion in critically ill patients, and prompt initiation of therapy is essential. this challenging case of hlh in a patient with hyper ige syndrome highlights the diagnostic challenge, variable presentation, and need for effective therapy in this vulnerable patient population. background: adolescents and young adults (ayas) with cancer are at risk for psycho-social as well as physical symptom burden during cancer therapy. the purpose of this study is to explore psychological and physical symptoms endorsed by aya while receiving therapy for cancer design/method: surveys were given in both inpatient and outpatient settings during cancer therapy. symptom screening in pediatrics tool (sspedi) and memorial symptom assessment scale (msas). symptoms severity was rated by teens on a point likert scale. spss , used for statistical analysis. results: : a total of aya on cancer therapy (age range - . years) % female, % male, . % acute leukemia, . % solid tumors, and . % diagnosis was not reported. % of aya on cancer therapy reported at least or more symptoms, % reported > symptoms cluster. of the physical symptoms that were reported as most distressing to the teens, mouth sores and headaches were the top causes. of the physical symptoms that were most frequently endorsed; fatigue was on the top ( %), followed by change in appetite %, vomiting %, and pain %., the least was bowel habit changes. aya rated sadness as the most frequent psychological symptom %, followed by feeling angry %, and scared %. statistically significant difference was noticed based on gender difference with more females reported symptoms (p = . ), while type of cancer (acute leukemia versus solid tumors) was not statistically different. conclusion: aya with cancer reported multiple physical and psychological symptoms with significant distress. females seem to report more symptoms compared to males. screening aya for cancer therapy related symptoms is feasible during routine visits and adds important information about the aya well-being. background: sinus histiocytosis with massive lymphadenopathy (shml), also known as rosai-dorfman disease, is a rare histiocytic proliferative disorder of unknown etiology. many treatment modalities have been employed; however, no uniform guidelines exist. objectives: literature review of treatment options for shml. design/method: chart review was performed on pediatric patients diagnosed with shml at the children's hospital at montefiore between and after irb approval. inclusion criteria included children between the ages of and years with shml. exclusion criteria included children with cutaneous shml. four cases of shml seen at montefiore are described. a comprehensive review of the literature identified additional cases published between and . manuscripts that did not include the treatment modality or outcome were excluded. results: many of the patients with shml responded to observation alone. of patients, patients were observed, with ( %) having resolution of disease, five having stable disease, and five being lost to follow-up. one patient received subsequent systemic therapy. surgical management was con-ducted upfront in patients. of those, ( %) had resolution of disease, one had stable disease, and one had recurrence with no further therapy noted. of the remaining nine patients, % were successfully treated with systemic therapy, consisting of either steroids ( ) or steroids and chemotherapy ( ). systemic therapy was used as first-line therapy in patients. steroids alone or in conjunction with chemotherapy resulted in resolution of disease in / and / patients ( / , %), respectively, with four patients having stable and three with progressive disease. chemotherapy without steroids resulted in resolution of or stable disease in / patients. radiation was ineffective. conclusion: shml is a rare disease with no published guidelines for treatment. from the results of the cases and a detailed review of the literature, it can be suggested that observation may be considered as first line management in patients providing there are no significant symptoms. for patients who are symptomatic or have significant progression, surgery may be considered. in patients with recurrence or refractory disease, steroids and/or chemotherapy may be used. the presence of nodal or extra-nodal disease did not seem to have a significant impact on the course of treatment. given the rarity of the disease, it is difficult to conduct a randomized control trial. further work, involving collaboration between centers and cooperation with the international rare histiocytic disorders registry would be helpful. boston children's hospital, boston, massechusettes, united states background: increasing census and intensified work compression on the inpatient oncology service at our institution was identified as leading to resident dissatisfaction, impaired resident learning and decreased perceived quality of patient care. objectives: to evaluate the impact of a redesign of a pediatric inpatient hematologic malignancy (ihm) service on resident perceptions of the educational value of the rotation and safety of patient care. design/method: during the - academic year, we initiated a bundled intervention on the ihm service. modifications included ) decreased patient volume: the ihm service was divided into two teams, utilizing an extra attending -a teaching service consisting of residents and fellows and a team comprised of nurse practitioners. ) intentional patient team assignment: patients were deliberately assigned to a care team based on educational opportunities and provider skill sets. ) intentional attending faculty selection: attending faculty with deeper clinical and teaching experience were selected to supervise on the teaching team. ) increased weekend staffing. after completing the service, junior residents completed an electronic survey to evaluate their perceptions of the educational value of the rotation, as well as their ability to deliver safe care while on the rotation. fisher's exact tests were used to compare responses from residents in who experienced the redesign to residents in , whose experience results: survey completion rates were % ( / ) in and % ( / ) in . intervention residents were significantly more likely than comparison group residents to choose the answers "very good" or "excellent" to describe both the overall quality of the rotation ( % intervention vs. % comparison, p< . ) and the educational experience on rounds ( % intervention vs. % comparison, p< . ). intervention residents also reported caring for fewer average primary patients daily on weekdays as compared to comparison residents ( . vs . patients, p< . , % ci - . to - . ). furthermore, intervention residents were more likely than comparison residents to "agree" or "strongly agree" that they could provide safe patient care on weekend days ( % intervention vs. % comparison, p< . ) and on nights ( % intervention vs. % comparison, p< . ) while on the oncology service. a redesign initiative of an oncology service with the development of a new teaching service led to improved resident perceptions of the educational value of the rotation and ability to provide safe care to patients. this approach could be useful to other services and institutions to promote similar outcomes in resident education and patient care. background: alk-positive histiocytosis is a rare histiocytic proliferative disorder that has been reported in three infants presenting primarily with hepatosplenomegaly, anemia, and thrombocytopenia. given the rarity of this disease, there are no standard treatment algorithms for this diagnosis and the disease course and outcomes remain largely unknown. the published series describes treatment ranging from monitoring alone to multi-drug chemotherapy regimens. there was ulti-mately resolution of presenting symptoms in all three cases despite varying treatment strategies. objectives: to report a newly diagnosed case of alkpositive histiocytosis that was treated with a novel approach using cytarabine monotherapy. results: a full term male infant presented at birth with difficulty feeding and hyperbilirubinemia. over the first few weeks of his life, he subsequently developed thrombocytopenia, transaminitis, and profound hypoalbuminemia. by six weeks of life, he was experiencing significant abdominal ascites requiring repeat paracenteses, massive hepatosplenomegaly, respiratory distress secondary to abdominal distension, anemia, and coagulopathy. he underwent numerous diagnostic tests, including a liver biopsy followed by a bone marrow biopsy that showed alk-positive histiocytic infiltrates in both sites. treatment was initiated with cytarabine mg/kg/day x days, repeating every weeks. throughout his course of five cycles of treatment, he experienced intermittent fevers and mild nausea with no other adverse events. by the end of five cycles, his hepatosplenomegaly resolved, his blood counts normalized, he demonstrated weight gain on oral feeds, and his liver enzymes normalized. he is currently months post completion of therapy and remains well with a normal physical exam and laboratory values. conclusion: treatment of alk-positive histiocytosis with lose dose cytarabine resulted in complete resolution of our patient's symptoms with minimal treatment related adverse effects, and few long-term treatment related risks. given the rarity of the diagnosis, the reporting of effective novel treatment options is important for future patient care. background: adult patients with melanoma or lung cancer harboring braf v e have benefitted from the development and subsequent approval of specific braf inhibitors. as such, delineating the subset of similarly targetable pediatric oncology patients may spur development and rational use of these inhibitors in children. importantly, other point mutations and fusions of braf may also be targetable in s of s children analogous to recent emerging data in adult cancer patients. objectives: to define the genomic landscape of known and novel braf alterations and raf fusions in pediatric malignancies and report index cases with clinical response to braf or mek inhibitors. design/method: dna was extracted from microns of ffpe sections of , tumors from pediatric (< years of age) oncology patients, and cgp was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of x for up to cancer-related genes plus introns from genes frequently rearranged in cancer. genomic alterations (ga) included base substitutions, indels, copy number alterations and fusions/rearrangements. a total of ( . %) braf-altered pediatric malignancies were identified. ( . %) harbored a single kinaseactivating braf short variant, indel, or fusion. an alteration resulting in reduced braf kinase activity was identified in ( . %) tumors while ( . %) tumors harbored multiple braf alterations, of which contained at least a single activating short variant. the remaining tumors ( . %) contained functionally uncharacterized variants. kinaseactivating braf alterations were identified in diverse tumor spectra comprised of brain tumors ( . %; subtypes), carcinomas ( . %; subtypes, with melanoma constituting % of cases), hematological malignancies ( . %; subtypes), sarcomas ( . %; subtypes), and extracranial embryonal tumors ( . %; subtypes). seventy-two ( . % of braf-altered cases) braf fusions were identified, ( . %) of which were kiaa -braf; involved the novel fusion partners: stard nl and khdrbs . seven ( . %) raf fusionpositive cases, predominantly brain tumors ( ), were identified; involved the novel fusion partners: tmf and sox . index cases of response to therapy of intracranial tumors will be presented. we describe a population of pediatric patients with targetable braf alterations predominantly enriched in primary intracranial tumors, but spanning diverse solid tumor types and hematologic malignancies. we additionally report a cohort of raf fusion-positive patients. an index case and multiple previous reports suggest raf or mek inhibitors may benefit pediatric patients with either intracranial or extracranial disease, and development of such drugs in pediatric indications is strongly warranted. background: diffuse midline gliomas (dmg) with h k m mutation, including diffuse intrinsic pontine glioma (dipg), are the leading cause of brain tumor-related deaths in children. there are no effective therapeutic strategies and the median survival remains dismal. genomic studies have identified a recurrent mutation in the majority of dmgs involving a lysine to methionine substitution (k m) in histones . and . , resulting in changes in the epigenetic landscape that dysregulate gene expression and promote gliomagenesis. panobinostat, a multiple histone deacetylase (hdac) inhibitor, was found to be one of the most effective agents against dipg patient-derived cell cultures and xenograft models in previous studies and is presently in clinical trial for dipg. hdac inhibition with panobinostat may also exhibit activity against h k m+ diffuse midline gliomas of the thalamus and spinal cord. to evaluate the effect of panobinostat as a single agent against patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures and in an orthotopic xenograft murine model of h k m+ spinal cord glioma. design/method: patient-derived thalamic and spinal cord h k m+ diffuse midline glioma cell cultures were treated with single agent panobinostat at a range of concentrations. cell viability was evaluated using the celltiter-glo assay. panobinostat was systemically administered to orthotopic xenograft murine models of luciferase-expressing spinal cord h k m+ diffuse midline glioma. response to panobinostat was evaluated with ivis in vivo imaging. results: hdac inhibition with panobinostat significantly decreases cell proliferation with an ic of nm and nm in the spinal cord and thalamic glioma patient-derived cell cultures respectively. panobinostat slowed tumor growth in murine models of spinal cord glioma by . -fold in the brain (p = . , n = ) and -fold in the spinal cord (p = . , n = ) when compared to vehicle controls after week of administration. panobinostat is in clinical trials for dipg. this study suggests that hdac inhibition with panobinostat may also be beneficial for patients with thalamic and spinal cord diffuse midline glioma h k m mutants. background: brain tumors are the most common solid tumor of childhood and the leading cause of childhood cancer deaths. while medulloblastoma is the most common malignant brain tumor of childhood with a -year survival - %, children with high-grade gliomas (hggs) such as glioblastoma multiforme (gbm) fare much worse with a -year survival of - %. implicated in this poor outcome is the presence of treatment resistant brain tumor stem-like cells. gbm stem-like cells (gscs) have been implicated in tumor growth, treatment resistance and patient relapse, making them a key therapeutic priority. antipsychotic drugs (apds) have been used for decades in various psychiatric clinical settings and are associated with a lower incidence of cancer, including malignant brain tumors. currently, atypical apds are being evaluated for their potential to alleviate cancer and treatment induced side effects. furthermore these drugs may have direct anti-tumor effects, potentially via inhibition of dopamine d receptors (drd ). objectives: determine the anti-cancer effects of atypical apds on gbm stem-like cells design/method: the anti-cancer effects of apds (quetiapine and risperidone) were evaluated on gbm stem-like cell lines developed in our laboratory (glio and ) and the group medulloblastoma cell line hdmbo . cell proliferation/viability was determined using trypan blue exclusion and mts assays. the effect of apds on cancer stem cell self-renewal was determined by neurosphere assay. receptor expression and apds effect on cell cycle proteins were examined by western blot analysis. results: western blot analysis of gscs and hdmbo demonstrated robust drd expression indicating a viable therapeutic target. both apds induced dose dependent cell death of all cell lines tested. treatment with only um of either apd for days significantly reduced cell proliferation by % (hdmbo ) and - % (gscs). consistent with these findings, we observed an increase in cell cycle inhibitors p and p . furthermore at day both apds induced a robust increase in gsc death, approximately % compared to only % in non-treated controls. lastly, um apds significantly reduced gsc neurosphere formation compared to untreated controls by up to % suggesting inhibition of gbm stem cell self-renewal. our data indicates that clinically relevant concentrations (low micromolar) of these apds induce anticancer effects in both gscs, which are enriched with tumor initiation/propagation properties, and in the group (myc amplified) medulloblastoma cell line. these apds represent strong candidates as potential adjuvant therapies for the treatment of these brain tumors. background: while the poor prognosis for high risk neuroblastoma (hrnb) underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. the identification of agents that target specific molecular pathways associated with the development or progression of diseases holds promise. dfmo, an inhibitor of odc, has been shown to decrease lin and mycn and target cancer stem cells in preclinical studies. currently % of patients undergoing immunotherapy relapse. dfmo is in studies to prevent relapse after immunotherapy and may be helpful during immunotherapy as well. the hypotheses for this study were that: ) the incorporation of a targeted therapy, selected based upon upfront tumor genomic interrogation, into standard induction chemotherapy for hrnb is safe, feasible and may increase the pr/cr/vgpr response rate at the end of induction therapy; and ) the addition of dfmo as maintenance during immunotherapy is safe and feasible and may decrease the relapse rate for hrnb. a multicenter feasibility pilot trial in subjects with newly diagnosed hrnb within the beat childhood cancer consortium. at diagnosis, patients' tumors underwent dna exome and rna sequencing which were analyzed within a molecular tumor board to identify the single best drug of targeted agents to be added to cycles - of induction chemotherapy. after consolidation with asct and radiation, the patients received dfmo along with standard dinutuximab and retinoic acid and dfmo for years after immunotherapy. patients were evaluated for additional toxicities with the addition of targeted agents and dfmo in addition to induction response. results: the pilot study of eligible patients has shown this process to be feasible. all patients have completed induction portions of the study. the combination of targeted agent with chemotherapy was shown to be safe without any unexpected toxicities. delays between induction cycles were < weeks and related to surgery, infection, or thrombocytopenia. the induction response demonstrated % cr/vgpr/pr rate, which suggests improvement over historical %. in addition, patients were eligible for the combination of dfmo with dinutuximab and retinoic acid was well tolerated and safe without additional toxicities due to dfmo. the pilot study of patients has shown the process of genomic sequencing and addition of a targeted agent to upfront chemotherapy and addition of dfmo to dinutuximab and retinoic acid maintenance therapy in newly diagnosed hrnb patients and is feasible and safe without any unexpected toxicities. background: identifying sub-populations of medulloblastoma tumors with stem cell-like properties holds promise for reducing disease recurrence, but there is no known unifying marker of medulloblastoma cancer stem cells. the granulocyte stimulating factor receptor (gcsf-r or cd ) is well understood in the context of hematopoiesis, but its role in solid tumor pathogenesis is less clear. neuroblastoma and melanoma subpopulations expressing gcsf-r have cancer stem cell properties of chemoresistance and increased tumorigenicity, and are enriched in tumors after chemotherapy. gcsf-r activation leads to signaling through the jak-stat pathway, suggesting a potential therapeutic target. we hypothesized that a subpopulation of medulloblastoma cells would express the gcsf-r and that this subpopulation would demonstrate chemoresistance and response to inhibitors of the jak/stat pathway. objectives: our objective was to identify a subpopulation of medulloblastoma cells expressing the gcsf-r and determine their relative growth rates, tumorigenicity, and responses to chemotherapy and jak/stat inhibition. design/method: medulloblastoma cell lines were sorted via flow cytometry for gcsf-r surface expression. subpopulations of gcsf-r-positive and -negative medulloblastoma cells were then monitored for growth by continuous live cell imaging. responses to chemotherapy were measured in subpopulations of gcsf-r-positive and -negative medulloblastoma cells using continuous live cell imaging to measure percent cell confluence and cell viability assays. ic values were calculated for each cell line and each agent. parental medulloblastoma cell lines and isolated gcsf-r-positive and -negative subpopulations were also treated with the jak / inhibitor ruxolitinib and growth rates, viability, and ic values were calculated. results: gcsf-r surface expression was identified on . - . % of medulloblastoma cell lines. isolated gcsf-r positive cells demonstrate a slower growth rate compared to gcsf-rnegative or parental unsorted medulloblastoma cells. gcsf-r positive cells are more resistant in vitro to vincristine, etoposide, and carboplatin, when compared to the gcsf-r negative population and an unsorted population of the same cell line. ruxolitinib is cytotoxic to medulloblastoma cells in vitro, with higher ic values noted in gcsf-r positive cells compared to unsorted and gcsf-r negative cells. we show that a subpopulation of gcsf-r positive cells are present in multiple medulloblastoma cell lines via flow cytometry, and that isolated gcsf-r-positive cells have a slower growth rate than gcsf-r-negative or unsorted populations. we also show that ruxolitinib has in vitro activity against medulloblastoma cell lines. we propose that jak inhibition may represent an adjunct therapy targeting overall tumor burden and specifically targeting the gcsf-r-positive subpopulation of medulloblastoma cells that may drive tumor recurrence. we investigated the efficacy of intensified adjuvant chemotherapy in osteosarcoma patients. design/method: we retrospectively analyzed the medical records of children with osteosarcoma treated at asan medical center between and . all patients received a -drug induction consisting of cycles of cisplatin and doxorubicin along with cycles of methotrexate (map), and proceeded to surgical resection. adjuvant ct was map or map with the additional ifosfamide and etoposide (mapie), and mapie was mainly considered for poor responders (tumor necrosis below %) or patients with metastases. results: among patients, patients had metastases at diagnosis. surgery was conducted in patients who responded to induction ct, and showed over % tumor necrosis. among patients who proceeded to adjuvant ct, and patients received to map and mapie protocols. with a median follow-up of months, the -year overall survival (os) and event-free survival (efs) rates of all patients were % and . %. of those patients, patients recurred, and of them died of disease progression. relapsed patients received salvage ct and/or surgery, and were rescued after autologous stem cell transplantation (sct). three patients developed treatment-related acute myeloid leukemia, and they are alive after allogeneic sct. according to the response to neoadjuvant ct, the os rates of good responders (n = ) and poor responders (n = ) were % and . % (p = . ), and efs rates were . % and . % (p = . ). of the poor responders, patients received map as adjuvant ct, and the other received mapie. the os rates of map and mapie group were . % and . % (p = . ), and efs rates were . % and . % (p = . ), respectively. when patients were classified into three groups: . localized disease & necrosis ≥ % (n = ), . localized disease & necrosis < % (n = ), . metastatic disease (n = ), survival rates were in the order of group > > (os = %: . %: . %, efs = . %: . %: %). in each group, intensified adjuvant ct by mapie did not improve survival outcomes. conclusion: initial metastatic disease and poor histological response to neoadjuvant ct were major risk factors for poor survival in osteosarcoma patients. we found that adding ifosfamide and etoposide to map did not improve survival outcomes of patients with adverse risk factors. more effective adjuvant therapy for these patients is needed. background: circulating cell-free dna (cfdna) that shed from tumors into circulation have been used for noninvasive molecular profiling in adult cancers but little is known about its utility in pediatric cancers. pediatric patients with metastatic and refractory solid tumors are known to have poor survival rates, and a key challenge in their management is obtaining biopsy samples especially at times when disease is widely spread or the patient is physically unfit for sampling. the development of a noninvasive profiling strategy is critical for optimizing molecularly guided therapy and assessing response to treatment. in this study, we want to determine the utility of cfdna to noninvasively analyze the molecular profiles of pediatric solid tumors such as neuroblastoma (nb), osteosarcoma (os), and wilms tumor (wt). design/method: tumor, plasma, and matched controls were collected from patients with nb, wt, and os, at diagnosis or time of disease progression. cfdna was extracted from the plasma and analyzed through multiple methodologies including a targeted next generation sequencing panels and shallow whole genome sequencing (swgs). results: fifteen nb patients, os patients, and wt patients had tumor molecular profiles known from different targeted next-generation sequencing platforms. in the cfdna of / nb patients, somatic mutations and copy number alterations previously reported in the tumors were detected, including recurrent nb drivers such as mycn amplification, alk, and atrx mutations. mutations not detected in the original tumor were also found in / nb patients including nras, mll , arid b, some of which are potentially actionable. in os, mutations known from the tumor were found in the cfdna of of patients, including atrx and notch mutations, as well as copy number alterations such as cdk amplification, which has targetable therapeutics available. of the two wt patients analyzed, cfdna revealed the same mutations as tumor in one patient, however in a cohort of patients where tumor was not available, cfdna revealed recurrent driver mutations such as amer , dicer . it is feasible to noninvasively identify somatic mutations and copy number alterations in cfdna of patients with pediatric solid tumors. establishing a platform using cfdna to identify molecular profiles of these tumors can serve as a powerful tool for guiding treatment and monitoring response to treatment. background: despite multi-modality therapy, the prognosis for patients with metastatic osteosarcoma remains poor necessitating development of novel targeted therapies. immunotherapy can be exploited to target osteosarcoma with exquisite specificity but remains limited by insufficient tumor specific targets. objectives: to overcome the dearth in tumor specific antigens, we have explored the use of tumor derived mrna (representing a tumor specific transcriptome) for development of personalized nanoparticle vaccines. design/method: rna-nanoparticles (rna-nps) can be amplified from limited amounts of biopsied tissue for induction of tumor specific t cells against osteosarcoma. since local vaccination strategies are mired by poor overall immunogenicity, we assessed the feasibility, immunogenicity and antitumor activity of intravenously administered rna-nps (tumor mrna complexed to dotap nanoliposomes) in pre-clinical murine and canine tumor models. we identified a clinically translatable np formulation for the delivery of rna to antigen presenting cells (apcs) that induces in vivo gene expression and preserves rna stability over time. tumor derived rna-nps induced antigen specific t cell immunity and mediated anti-tumor efficacy in several pre-clinical solid tumor models (i.e. b f , kr b). when administered intravenously, rna-nps increased expression of co-stimulatory molecules (i.e. cd , cd , cd , ccr ) and pd-l on cd c+ cells throughout reticuloendothelial organs (i.e. spleen, liver, bone marrow) and within the tumor microenvironment; this phenotype was strictly dependent on type i interferon. targeted inhibition of type i interferon signaling (via infar mabs) abrogated anti-tumor efficacy mediated by rna-nps. we enhanced the immunogenicity of this platform by simply combining mrnas encoding for immunomodulatory molecules (i.e. hcv-pamps, gm-csf) or by combining rna-nps with immune checkpoint inhibitors. addition of checkpoint inhibitors (pd-l mabs) to rna-nps increased tumor infiltrating lymphocytes, and intratumoral mhc class i/ii expression, and mediated synergistic anti-tumor activity in settings where pd- or pd-l inhibition alone did not confer therapeutic benefit. we then explored the feasibility of rna-nps in a large animal osteosarcoma model. in ongoing studies for canines with osteosarcomas, we have shown that sufficient amounts of rna can be extracted, amplified, and manufactured into personalized rna-np vaccines. conclusion: rna-nps reprogram systemic immunity and mediate anti-tumor activity providing near immediate immune induction without the complexity of cellular immunotherapy. the immune correlate of preclinical response to rna-nps is hallmarked by interferon dependent pd-l expression on activated apcs (cd c+ mhcii+ cd + cells). based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of rna-nps targeting canine osteosarcoma before first in-human evaluation. background: ewing sarcoma is an aggressive bone tumor affecting mainly adolescent and young adults. treatments are based on compressed schedule chemotherapy combined with local control (surgery and/or radiation). prognosis is poorer for patients with metastatic disease, older age and central primaries. survival when disease recurs within two years of diagnosis is < %. the ews-fli fusion gene t( ; ) (q ; q ) has been well characterized as a dominant ews driver-gene. the most common variation is ews exon with fli exon ( % of fusion positive patients). we designed a novel pbi-shrna tm ews/fli type lpx which has demonstrated, safety and efficacy in animal model (rao et all). the pbi-shrna strategy silences target gene expression by concurrently inducing translational repression and p-body sequestration as well as post-transcriptional mrna cleavage. to determine the safety and maximum tolerated dose of intravenous administration of pbi-shrna tm ews/fli type lipoplex in patients advanced ews. design/method: phase i study × escalation cohort. testing pbi-shrna tm ews/fli type lpx (starting iv dose of . mg/kg) on patients (≥ age ) with advanced ewing's sarcoma, all with a type translocation. intravenous infusion was given twice a week for weeks with the following escalation schema: % → % → % → % → %. required kps > % and adequate organ function. cytokines induction pre and post-infusion was analyzed (il- , il- , tnf-alpha, il ra). first cohort of patients has been enrolled (ages between - years). three relapsed patients had > lines of therapy and patient had refractory disease, patients received a complete cycle of pbi-shrna tm ews/fli type lpx with twice a week infusions. a total of doses were given. the most prominent related toxicity has been hematological, patient developed transient g neutropenia, another patient developed g anemia that required prbc transfusion, and of note this patient had significant bone and bone marrow involvement. one patient only received two lpx infusions; she developed a fatal rsv pneumonia. other reported grade toxicity includes fatigue and headache. evaluable patients (n ) had stable disease between and months before progression. one patient had sustained response for month before progression, two patients are still alive. our preliminary experience supports the safety and potential efficacy of pbi-shrna tm ews/fli type lpx as novel treatment for advanced ews with limited toxicity. il- increase correlates with higher bi-shrnai ews/fli lpx infusion rate and clinical symptoms. further clinical testing is indicated. background: as more children with cns malignancies (bt) are surviving, the late effects of the therapies they receive are better described. studies show that radiation therapy is particularly harmful to neurocognitive functioning, specifically processing speed, working memory, and attention span. these deficits have negative effects on quality of life, especially in academic and professional settings. a large proportion of s of s adult survivors of bt are unable to reach adult milestones such as living on their own, holding a steady job, and getting married. proton beam radiation therapy (pbrt), is touted for the potential to have fewer and less severe side effects than traditional photon radiation therapy (xrt). because of the properties of protons, the amount of damaging energy released in non-target healthy tissue is reduced when compared to xrt. although a study comparing iq testing between pbrt and xrt found no difference between the two therapies, no studies have compared the specific neurocognitive domains. it would be valuable to evaluate full neurocognitive testing scores (nct) since the specific domains, particularly processing speed (psi), appear to be most vulnerable to radiation therapy. objectives: our primary aim was to assess differences in psi for patients with bt who underwent pbrt versus xrt. a secondary aim was to assess differences in iq (fsiq) and working memory (wmi). we retrospectively evaluated all patients treated for bt at the jimmy everest cancer center within the past years who received rt and had nct post radiation. we examined the full nct results for both subsets of participants to evaluate differences in the specific domains of processing speed, working memory, and iq by measuring percentiles scored in these domains. objectives: we report our experience on imaging children with mm treated uniformly on an institutional melanoma trial. we retrospectively reviewed the clinical and imaging findings of patients with ajcc stage iic-iv cutaneous mm treated on our institutional mel protocol. brain mri/ct, pet/ct, ct chest, abdomen, and pelvis (ctcap) were performed at diagnosis in all patients. on treatment, stratum a patients (peg-interferon; ajcc iic, iiia, iiib) (n = ) had the same imaging repeated every months; stratum b (peg-interferon and temozolomide; unresectable measurable disease metastatic, or recurrent) (n = ) had pet scans every months and brain imaging every months; those in stratum b (peg-interferon and temozolomide; unresectable non-measurable, metastatic, or recurrent) (n = ) had the same imaging performed every months. off therapy all patients continued same imaging every months for years. results: there were patients ( female; median age years). eleven had spitzoid and conventional melanoma. primary sites included head/neck (n = ), trunk (n = ), and extremities (n = ). patients with spitzoid melanoma had imaging studies ( pet, ctcap, ct chest, ct brain, and mri brain) with a median of , , , and studies/patient respectively. median cost per patient was $ , . thirteen studies ( . %) showed suspicious lesions with additional scans and diagnostic biopsies of which one only was positive stratum a with tert promoter mutation and died from disease). for conventional mm, studies ( pet, ctcap, ct chest, ct brain, and mri brain) were performed with a median of , . , , , studies/patient respectively. median cost per patient was $ , . twenty ( %) showed suspicious lesions with additional scans and diagnostic biopsies; four were positive (two at diagnosis); both died of disease; the other two recurred locoregionally and were detected clinically; both are alive and disease free; one patient had diffuse metastases and died shortly after enrollment. after a median follow up of . years (range . - . ) patients are alive and disease free. children with spitzoid melanoma should have minimal imaging at diagnosis and follow-up given the low risk of recurrence and low yield and high cost of aggressive imaging protocols. patients with conventional mm should be imaged according to the adult guidelines. nationwide children's hospital, columbus, ohio, united states background: the role of infections in the long term outcome of patients with bone tumors is controversial. two retrospective studies have shown increased survival in osteosarcoma patients who had a post-operative wound infection, while another showed no changes in overall survival. to determine the relationship between wound infections and/or bloodstream infection (bsi) on survival in pediatric and young adult patients with osteosarcoma and ewing sarcoma treated at a tertiary children's hospital. design/method: a retrospective chart review was performed for patients with diagnosis of osteosarcoma or ewing sarcoma from - . patients received standard chemotherapy regimens for their disease type and stage. local control included surgical resection and/or radiation therapy. presence of infection was determined by bsi or wound cultures while receiving treatment for primary tumor. the median age of patients was (range - years) at diagnosis. % had a diagnosis of osteosarcoma and % had ewing sarcoma. of these, % of patients developed an infection during treatment; % had bsi, % had wound infections, and % had both. patients with bsi had a year os of . %, compared to % in those without bsi (p = . ). those with both bsi and wound infections had the poorest overall survival of %, compared to . % for patients without any infection. patients with wound infections alone had a year os of . %, compared to % of patients without a wound infection. our analysis revealed decreased os in patients with bsi; however, this could be due to other confounding factors in the presence of bsi. those with bsi or bsi and wound infections had the poorest survival. wound infections without bsi were associated with a slight increase in survival; however, this study was limited by the number of patients that had local wound infections. with the use of newer surgical techniques, availability of antimicrobials and routine use of prophylactic antibiotics, the incidence of infections while undergoing treatment is low. however, the importance of this clinical observation indicates a likely enhanced immune system associated with infection, supporting the role of immunotherapy for treatment of these aggressive tumors. background: hypoalbuminemia is a well-recognized effect of cancer and other chronic illnesses and is often regarded as a marker of malnutrition. in adults, hypoalbuminemia has been associated with adverse outcomes in patients with cancers of the lung, pelvis, head and neck, gastrointestinal tract, and bone marrow, as well as in some pediatric patients with ewing sarcoma and hodgkin lymphoma. hypoalbuminemia has not been well studied in children with cancer. to determine the incidence of hypoalbuminemia (using age-specific references) in children with cancer receiving chemotherapy at baseline (prior to starting chemotherapy) and to determine whether hypoalbuminemia is associated with inferior -year overall survival. design/method: we performed a single institution, irbapproved, retrospective review of pediatric oncology patients diagnosed between and . five-year survival was estimated using the kaplan-meier method; groups were compared using cox regression. we identified pediatric patients with a first diagnosis of cancer, brain tumor, or other condition possibly requiring chemotherapy. of these patients, were excluded for reasons including not receiving chemotherapy and missing data, leaving patients who had a serum albumin level within days prior to starting chemotherapy. the mean age was . years (sd . years); % were male; % were non-hispanic. the most common diagnosis was acute lymphoblastic leukemia ( of ; %). one hundred thirty nine of ( %) had hypoalbuminemia prior to starting chemotherapy. there was no statistically significant difference in -year overall survival between those with and without hypoalbuminemia ( % vs. %, respectively; hazard ratio . , % c.i. . - . ). conclusion: hypoalbuminemia at baseline in pediatric oncology patients requiring chemotherapy is common (one in five), and was not associated with inferior -year overall survival in this cohort. leptomeningeal metastases at diagnosis. standard treatment for completely resected, non-anaplastic supratentorial ependymomas is close observation. treatment for anaplastic or incompletely resected non-anaplastic ependymomas is maximal safe surgical resection followed by focal radiation. however, up to % of localized ependymomas recur. the role of chemotherapy in treating ependymomas is under investigation. extraneural metastases of anaplastic ependymomas have rarely been reported and the outcome is dismal. objectives: to report extraneural cervical node metastases of a non-anaplastic ependymoma and successful treatment with surgical resection, radiation, and systemic chemotherapy. design/method: retrospective review of patient medical records, including radiographic imaging and tumor tissue pathology, and comprehensive literature review. results: a previously healthy -year-old girl underwent gross total resection (gtr) of an isolated right parietal lobe ependymoma (who grade ii). at age years, magnetic resonance imaging (mri) revealed an isolated localized recurrence. she underwent gtr followed by observation. at age years, she again experienced isolated localized recurrence and underwent gtr followed by . gy focal conformal photon radiation. at each recurrence, pathology revealed a non-anaplastic ependymoma, and cerebral spinal fluid (csf) cytopathology and spine mri were negative. at age years, she developed an enlarged right posterior cervical chain lymph node. subsequent mri revealed a large rim-enhancing, t hyperintense lymph node and multiple abnormally enhancing regional nodes consistent with metastases. biopsy revealed a non-anaplastic ependymoma. mri of the brain and spine, computed tomography of the chest, abdomen, and pelvis, and csf and marrow evaluations were unremarkable. chemotherapy according to acns was initiated. mri after course demonstrated significant node size reduction. she underwent right neck node dissection. only one right level ii lymph node showed metastases. she was treated with . gy irradiation to the neck and additional courses of chemotherapy. she remains in remission months and months after diagnosis of metastatic disease and end of therapy, respectively. literature review reveals rare reports of extraneural metastatic disease of anaplastic ependymomas to bone, lung, or liver, and only involving lymph nodes, all associated with a poor outcome despite multimodal therapy. to our knowledge, this is the first report of extraneural metastases of a non-anaplastic ependymoma. extraneural metastases should be considered in children previously treated for non-anaplastic ependymomas who experience systemic symptoms, even in absence of cns relapse. multimodal treatment offers potential long-term disease control with acceptable toxicity. arun gurunathan, joel sorger, andrew trout, joseph pressey, rajaram nagarajan, brian turpin cincinnati children's hospital medical center, cincinnati, ohio, united states background: pigmented villonodular synovitis (pvns) is a benign neoplasm of the synovium. standard treatment is surgery, but post-operative recurrence rate is as high as %. radiation therapy can be used for local control, but is associated with late effects. while pvns is rarely fatal, aggressive disease and/or extensive surgery can result in substantial functional impairment. colony stimulating factor- (csf ) overexpression, often due to chromosomal translocation involving csf , drives pvns through recruitment of synovial-like mononuclear cells expressing the csf -receptor. tyrosine kinase inhibitors such as imatinib are active against the csf -receptor, and have shown benefit in the post-surgical relapse setting. however, questions remain regarding the broader application of imatinib and regarding optimal response assessment. to present three patients with pvns, each with different clinical scenarios, who demonstrate clinical response to imatinib monitored by changes in metabolic activity (maximum suv) on pet/ct. results: three patients with pvns demonstrate pet/ct response to imatinib, guiding management of their challenging clinical scenarios. patient is a year-old female with left hip pvns and high grade articular cartilage loss, with decrease in metabolic activity (suvmax . to . in months) on neoadjuvant imatinib, enabling total hip replacement surgery planning. patient is a year-old female with left knee pvns with recurrences after synovectomies, spared subsequent surgical control attempts after clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). patient is a year-old male with right knee pvns that recurred after total knee replacement, now with clinical improvement correlating with pet/ct response to imatinib (suvmax . to . in months). all patients would have been characterized as stable disease by response evaluation criteria in solid tumors (recist). in each of these patients, imatinib has been tolerated well, with no therapy interruptions and absent or easily managed side effects (one patient takes dronabinol for decreased appetite, one patient takes prn immodium for diarrhea). all patients are currently still taking imatinib, with therapy length ranging from five to eleven months. in our series of three patients with pvns, imatinib shows promise for disease management in neoadjuvant and adjuvant settings with a tolerable side effect profile. imatinib should be considered in the treatment of pvns to spare surgical and radiotherapy related morbidity, and treatment effect can be monitored by pet/ct. background: metastatic rhabdomyosarcoma (rms) carries a poor prognosis with three-year event free survival rates ranging between %- % (depending on oberlin risk factors) due to the lack of significantly effective breakthroughs in the recent past. there is an urgent and unmet need for new treatment strategies against this disease. metastatic rms cell lines exhibit increased expression of the erm family membrane-cytoskeleton linker protein ezrin. knockdown of ezrin expression using sirnas decreases the metastatic potential of these cells, whereas forced expression of ezrin results in increased degree of metastasis. the activity of ezrin is controlled by its phosphorylation at the threonine (thr ) residue at the c-terminus of the protein, suggesting that alteration of ezrin phosphorylation may control rms growth and metastasis. our goal was to determine if pharmacological inhibition of thr phosphorylation in ezrin affects the growth, survival and metastasis in rms in vitro as well as in vivo. design/method: rms cell lines representative of the alveolar and embryonal histological subtypes were used. rms cells were treated with a small molecule inhibitor of ezrin, nsc , which specifically dephosphorylates ezrin at the thr residue. baseline expression of ezrin and perm levels as well as the effect of nsc on perm levels in the rms cell lines was determined by western blotting of cell lysates. viability of cells was assessed by trypan blue exclusion, and morphology visualized by bright field microscopy. the extent of apoptosis was detected by imaging caspase / activation using fluorescent microscopy. motility of rms cells was examined by performing a wound-healing assay. subcutaneous and orthotopic xenografts were established in nsg mice using rd cells (embryonal rms). mice harbor-ing xenografts were treated with intraperitoneal injections of nsc or dmso. results: ezrin is constitutively phosphorylated at the thr residue in a majority of the rms cell lines examined. nsc dephosphorylates ezrin at the thr residue in these cell lines. treatment with nsc inhibits growth, induces apoptosis and inhibits the migration of rms cell lines in vitro. further, treatment of nsg mice bearing subcutaneous or orthotopic embryonal rhabdomyosarcoma xenografts with nsc significantly impedes tumor progression without any obvious adverse effects. our findings suggest that dephosphorylation of ezrin at the threonine residue may have the potential to be a novel therapeutic strategy for rms patients. all india institute of medical sciences, new delhi, new delhi, delhi, india background: the role of laparoscopy in the management of pediatric intra-abdominal solid tumors is yet to be established. the safety of laparoscopic management of pediatric intra-abdominal tumors is still questionable. we study the results of the initial case series of pediatric intraabdominal tumors managed laparoscopically at our institute from july onwards. design/method: total children ( -males, females) who presented to us with pediatric intra-abdominal tumors were included. the tumors included wilms tumor (n = ), neuroblastoma(n = ), adrenal cortical tumor(n = ), ovarian teratoma(n = ).children were between months - years and received neo-adjuvant chemotherapy. a -port laparoscopic nephrectomy and lymph node sampling for wilms tumor and adrenalectomy for adrenal tumors was performed. the tumors were removed in-toto with no rupture (except in one). specimens were retrieved through a lumbar incision (n = ) or an inguinal incision(n = ). all the children are under regular follow up. two children with wilms tumor had recurrence. the neuroblastoma child underwent open surgery for recurrence later. conclusion: laparoscopy/laparoscopic assisted removal of pediatric intra abdominal tumor is a feasible and safe option. it has the advantage of less postoperative pain, shorter hospital stay and a better cosmetic result. proper patient selection, port placement and laparoscopic experience are contributory. background: targeting of proteins and cell surface antigens specific to cancer cells with monoclonal antibodies has proven to be an effective form of treatment in many forms of cancer. gd is a cell surface disialoganglioside that is expressed on the cell surface of some normal tissues including nerve cells, melanocytes, and mesenchymal stromal cells and is overexpressed in some pediatric cancers like neuroblastoma and osteosarcoma. dinutuiximab is a chimeric monoclonal antibody that is fda approved for the treatment of patients with high risk neuroblastoma and under investigation for the treatment of relapsed osteosarcoma. little is known about the patterns of gd expression in other pediatric malignancies. objectives: we sought to describe the patterns of gd expression in the following pediatric sarcomas: synovial sarcoma, rhabdomyosarcoma and ewing sarcoma. design/method: synovial sarcoma (n = ), rhabdomyosarcoma (n = ) and ewing's sarcomas (n = ) formalin fixed, paraffin embedded cores were obtained from the seattle children's research institute tissue microarray (tma) biorepository. tma blocks consisting of melanoma cores stained with and without gd antibody were used as positive and negative controls, respectively. slides were incubated with anti-ganglioside gd antibody clone q (ab from abcam) diluted : in % normal goat serum and % bsa in tbs overnight at ˚c. the negative control of human melanoma section was incubated in % normal goat serum and % bsa in tbs without primary antibody. the expression of gd was indicated by characteristic brown diaminobenzidine staining. the intensity and location of tissue staining were assessed and compared to positive and negative controls. staining was considered positive (+++) if the intensity of the staining was consistent with that of the positive control with - % of cells staining positive. classification of intermediate gd expression (++) was assigned to slides in which - % of cells stained positive. slides were classified as sporadic staining (+) if - % of cells stained positive. tissue was considered (-) if there was complete absence of staining, similar to the negative control. objectives: to evaluate the clinical presentation, management and treatment outcomes of children with malignant germ cell tumor at our institute design/method: a prospective study was conducted from june to dec in the department of pediatric surgery in a tertiary care institute in a developing country. all patients were evaluated for local disease and metastatic disease by imaging and tumor markers. risk stratified chemotherapy was used with low risk tumor receiving no chemotherapy, intermediate risk: courses of peb chemotherapy and high risk: courses of peb + courses of pe. upfront resection of the primary or the residual disease after neoadjuvant chemotherapy if feasible was performed. follow up was done with monthly tumor markers for months and imaging studies every - months for initial years. five year overall survival and disease free survival was calculated. results: during the study we treated children who formed the study group. of these ( %) were gonadal ( ; % testicular and ; % ovarian) and the remaining ( %) were extragonadal with sacrococcygeal (sct) being the most common site ( %). one hundred and thirteen children ( %) presented to us primarily while the remaining had received treatment elsewhere. stage or stage disease at presentation was present in ( %) children. recurrence was noted in ( %) patients. respectively. patients with testicular mgct and children with age - years and males had significantly poor rfs rates. conclusion: patients with mgct should be staged correctly and adjuvant chemotherapy is advisable to all patients except stage i endermal sinus tumor of testis. awareness regarding the same is still lacking in our country. meticulous follow up is needed as more than % of will recur. cure rates are dismal in children with recurrent mgct especially those who are not chemotherapy naïve. nemours children's specialty care, jacksonville, florida, united states background: radiotherapy for pediatric head and neck tumors often results in mucositis, limiting oral intake and compromising patients' nutritional status. this may be reduced through the improved conformality offered by proton therapy. despite widespread use of enteral tube feeding through a percutaneous gastrostomy (peg) or nasogastric tube (ngt), there is little data available regarding overall incidence of ngt/peg placement and perspectives of pediatric patients and caregivers. objectives: to (a) estimate the need for ngt/peg support and (b) characterize patient and caregiver perceptions surrounding enteral feeding in children with head and neck tumors undergoing proton therapy. design/method: dependent on development stage, patient (n = ) or parents (n = ) filled out a series of customized surveys according to a prospective irb approved study. seventythree percent of patients also received concurrent chemotherapy. questions addressed their current feeding route and perception, for example, "what aspect(s) of tube feedings are beneficial to you?" and "what aspect(s) of tube feeding worry or scare you?" fifty-five surveys were distributed before and after radiation, and with any change in feeding route. results: at the start of proton therapy, patient had a ngt and patients had peg. of these, patients ( %) had a ngt/peg in place exclusively for the administration of medication; only patient ( %) needed a ngt/peg for nutrition. in those patients without ngt/peg, % would "consider" enteral feeds. in patients without ngt/peg, the most commonly cited benefit was "maximizing my nutrition" ( %) and the most common negative aspect was "fear" of tube placement ( % of patients). all sub-populations ( % of patients) cited change in appearance as a negative aspect. in patients without ngt/peg at the start of proton therapy, % of patients/caregivers felt enteral feeding to be "unnecessary," and % of these patients would not "consider" ngt/peg even if their "physician advised it." over the course of proton therapy, the patients/caregivers who deemed enteral feeding "unnecessary" decreased from % to %. at completion of treatment, patients ( %) were using a ngt/peg tube for nutritional support but only one ( %) patient relied exclusively on their enteral feeds. two patients (without ngt/peg) ( %) required parenteral support. our data does not support prophylactic placement of ngt/peg in of children with head and neck tumors undergoing proton therapy. ongoing research is needed to identify which patients will need ngt or peg to supplement their diet. in this cohort, anticipatory counseling should focus on pain, cosmesis, and utility. children's national medical center, washington, district of columbia, united states background: ovarian sex cord-stromal tumors (osct) are rare neoplasms that typically present with signs/symptoms of an adnexal mass and signs of hormonal production approximately % of ovarian sex cord-stromal tumors in children are sertoli-leydig cell tumors (slct) with median age of presentation years overall. to our knowledge the youngest reported case in the literature describes a -month old female in china with a slct that was treated with oophorectomy alone. some studies have found an association in families between pleuopulmonary blastoma and osct with a germline mutation leading to dicer syndrome, which has been associated with a younger age at diagnosis. , objectives: to describe an unusual case presentation of slct in an infant results: -month old, twin female, ex- week premature infant presented to the emergency department on multiple occasions for abdominal distention and feeding intolerance initially thought to be related to previous omphalocele repair and umbilical hernia. an ultrasound demonstrated an × cm mass arising from the right ovary with large volume ascites. she required admission to the intensive care unit due to s of s respiratory distress from her significant ascites. serum tumor marker including hcg, afp and ldh were negative. patient underwent right oophorectomy with tumor capsule noted to be open at time of surgery. further imaging post operatively demonstrated no other sites of disease. the patient was classified as figo stage ic due to the presence of her significant abdominal ascites that was presumed to be malignant pre-operative tumor rupture. the pathological diagnosis was challenging and eventually resulted as a mixed germ cell sex cord stromal tumor with pattern of sertoli cell tumor with neuroendocrine differentiation. based on the staging of figo ic with pre-operative rupture, the decision was made to treat with a standard platinum based regimen as there is a higher incidence of relapse in stage ic patients when compared to ia treated with observation alone. our patient tolerated four cycles of chemotherapy well and end of therapy scans showed no evidence of disease. interestingly, her dicer mutation genetics performed by ion torrent tm next generation sequencing was negative in germline and tumor studies. to our knowledge, our patient is the youngest described with slct. she will continue to be followed with serial imaging alone as she had no evidence of elevated tumor markers at diagnosis. , due to young age and unusual diagnosis, she was referred to cancer genetics team. background: approximately % of patients with wilms tumor (wt) have metastatic disease at diagnosis and often have a grave prognosis. limited cell lines are available for the study of metastatic wt and long-term passaged cell lines do not always recapitulate the human condition. focal adhesion kinase (fak) is a non-receptor tyrosine kinase that controls cellular pathways involved in the tumorigenesis of pediatric renal tumors. using a novel patient-derived xenograft (pdx) model from a patient's primary wt (coa ) and matched isogenic metastatic wt (coa ), we previously demonstrated that fak is expressed and its inhibition led to decreased tumorigenicity of both the primary and metastatic pdxs. kinomic profiling is an innovative, high-throughput method used to investigate kinase signaling to identify potential therapeutic targets. to date, the kinomic profile of primary and metastatic wt has not been examined. objectives: investigate baseline kinomic differences between primary and metastatic wt and evaluate kinases upstream and downstream of fak as potential targetable therapies. design/method: cells from coa and coa were treated with pf- , (pf), a small molecule fak inhibitor. protein from cell lysates of treated and untreated coa and coa were combined with kinase buffer, atp, and fluorescently labeled antibodies and loaded into a phosphotyrosine kinase or serine-threonine kinase pam-chip® per the uab kinome core protocol. phosphopeptide substrate analysis with the pamstation® kinomics workstation (pamgene® international), pamchip® protocol using evolve software, and bionavigator v. . were used to analyze kinases upstream and downstream of fak. the primary wt had increased epha , ror sgk and decreased pdgfrb relative to the paired metastatic wt at baseline. treatment with pf increased ron, pdgfrb, p s kb, mak, camk g, vacamkl, camk d, ck a and pskh in the primary wt. treatment with pf decreased tnk , lmr , cck , epha , pdk , sgk , lkb and increased pskh in the paired metastatic wt. primary wt displayed a different kinomic profile compared to metastatic wt in a matched isogenic pdx model. these data reveal that alternative therapies to specifically target metastases are needed. furthermore, fak inhibition resulted in diverse kinomic alterations between primary and metastatic wt. inhibitors targeting many of these pathways, such as pdgfrb inhibitors, are currently available and potentially could be combined with fak inhibitors in the treatment of wt. the results of the current study indicate that kinases upstream and downstream of fak in primary and metastatic wt warrant further investigation. background: use of high-dose methotrexate (hd-mtx, g/m^ ) is a mainstay of standard therapy for pediatric osteosarcoma (os) in north america. in pediatric os, there is a narrow therapeutic window for hd-mtx, with decreased tumor response rate with mtx concentrations < m and decreased survival due to severe toxicity with concentrations > m. risk factors for hd-mtx toxicity have been defined in adults, including body mass index (bmi) and male gender, but such studies have not been conducted in children. we sought to examine the relationship between mtx levels and toxicities during hd-mtx infusion for pedi-atric os, thereby identifying risk factors for increased toxicity and providing a framework for therapeutic drug monitoring. design/method: this retrospective chart review included patients treated at texas children's hospital with hd-mtx as first-line therapy for os from - . data abstracted from electronic records included patient characteristics, bmi and body surface area (bsa), baseline and post-treatment laboratory values, mtx levels and hours after dose given ( h, h), hour mtx cleared (mtx < . um), grade / mucositis, myleosuppression, persistent lft elevation (ctace v . ), and % tumor necrosis. correlation between h mtx level and other covariates was summarized using descriptive statistics. we reviewed hd-mtx infusions corresponding to patients. bmi was found to significantly impact h mtx level (p< . ). female gender was also significantly associated with higher h mtx level (p< . ). percent necrosis (available in patients) was associated with h mtx levels at near-statistical significance (p = . ). h mtx level was not found to contribute to toxicities or associate significantly with mtx clearance. analysis in a larger cohort is ongoing. we have identified at least one patient factor (bmi) that significantly impacts h mtx levels and is of potential use for future modeling, as current models incorporate bsa only. our findings concord with studies in adult os in that bmi significantly impacts h mtx level but diverge in that female gender is associated with higher h levels. importantly, these data support targeting h mtx levels to ensure that minimum concentration for adequate tumor necrosis is reached. these results do not suggest that monitoring h levels would prevent toxicities, thus necessitating further characterization of any intrinsic patient factors that associate with toxicity. overall, our definition of the clinical factors that associate with h mtx levels contributes to a framework for therapeutic drug monitoring in pediatric os. children 's mercy hospital kansas city, kansas city, missouri, united states background: post consolidation immunotherapy with dinutuximab, aldesleukin (il- ), granulocyte macrophage colony stimulating factor (gmcsf) and isotretinoin is standard of care for children with high risk neuroblastoma. dinutuximab is combined in alternating cycles with s of s gmcsf or il , followed by a th cycle with isotretinoin alone. il- is administered as a hour continuous infusion on days - at miu/m /day followed by a higher infusion dose, . miu/m /day, in combination with dinutuximab on days - of cycles and . the miu/m /day dose may be administered inpatient or in the ambulatory setting. objectives: to retrospectively compare the incidence of inpatient and outpatient side effects and complications associated with low dose ( miu) il to provide the tolerability data necessary to evaluate these venues for future administration options. design/method: this study was a descriptive, singlecentered definitive study utilizing a retrospective convenience sample population of children with high risk neuroblastoma who received low dose il either as an inpatient or an outpatient without exclusion from may to june . subjects were identified by a tumor registry query post irb approval. electronic and paper medical records were reviewed for the dates and location of the infusions, the home health company used if applicable and all documentation regarding clinical status, side effects and toxicity. demographics was limited to age and gender. results: infusion venue was chosen by provider preference. twenty-six infusions, inpatient and outpatient via separate home health companies were all administered in entirety and without interruption. there were males and females ranging from - years of age. two children received a single outpatient infusion due to intolerance of il when combined with dinutuximab and received therapy in both settings. fever, inpatient and outpatient was the only common side effect. no source of infection was ever identified. there was one incidence of diarrhea and one patient with pruritus in both the outpatient and inpatient settings respectively. no planned outpatient infusions required subsequent admission however the outpatient fever did necessitate an er evaluation. conclusion: low dose il can successfully be administered outpatient. the medication has minimal side effects with fever occurring in %, none of which were associated with infection. no outpatient infusion required a subsequent admission. no patients who received cycle infusions outpatient opted to receive the next cycle inpatient. baylor college of medicine, houston, texas, united states background: metastatic ewing sarcoma (es) has an extremely poor overall survival, necessitating investigations into molecular mechanisms to identify novel targets and develop new therapies. we previously performed an in vivo study, using our mouse model, designed to provide insights into transcriptomic and proteomic signatures for metastatic es to identify potential therapeutic targets. comparing profiles of primary tumors to corresponding metastatic lesions, we identified aberrant expression of integrin ß (itgb ) and downstream activation of integrin-linked kinase (ilk) in metastatic lesions compared to primary tumors, implicating this pathway as a key regulator in the ability of es to establish and enhance metastasis. our hypothesis is that upregulation of itgb and its downstream signaling events play a key role in es metastasis and are viable therapeutic targets. objectives: to investigate the role of itgb and its downstream signaling pathways in driving the establishment and enhancement of metastasis in es and to investigate this pathway as a potential therapeutic target. to investigate the role of itgb and ilk in es metastasis, we used sirna to knock down itgb and ilk expression in established es cell lines and then performed functional assays in vitro, including cell proliferation and invasion/migration assays. we also tested inhibition of this itgb signaling pathway using available small molecule inhibitors targeting itgb , ilk and the downstream target ap- , using cilengitide, compound and sr , respectively. we are currently using these small molecule inhibitors as treatment in vivo and assessing rates of metastatic tumor formation. we generated stable itgb and ilk overexpression and knockdown cell lines, which we are using for similar in vitro and in vivo investigations. knockdown of itgb and ilk in our sirna cell lines resulted in decreased cell proliferation and decreased invasion and migration compared to controls. we also found significantly decreased cell proliferation using each of the small molecule inhibitors in vitro. our preliminary studies using compound in vivo established a safety profile and dose escalation is underway to assess the effectiveness of inhibiting es metastasis. these results support our hypothesis that itgb and its downstream signaling events play a key role in the ability of es to establish metastatic foci and may serve as a potential therapeutic target. we continue to investigate this pathway in vitro. we are also using our small molecule inhibitors and itgb and ilk overexpression and knockdown approaches to study these effects on metastatic tumor development in vivo using our mouse model. background: neuroblastoma (nbl) is characterized by phenotypic heterogeneity. outcome is excellent for patients with low-(lr) and intermediate-risk (ir) disease, whereas only % of high-risk (hr) patients will survive. -hydroxymethylcytosine ( hmc) is an epigenetic marker of active gene transcription, and hmc profiles are prognostic in many types of adult cancers. we hypothesized that hmc profiles will serve as robust biomarkers in children with nbl tumors, refining current risk stratification. objectives: analyze genome-wide hmc in nbl tumors and correlate hmc deposition with chromosomal copy number and gene expression. design/method: hmc was quantified by nano-hmc-seal-seq from the dna extracted from hr, ir and lr nbl tumors. read counts and clinical data were analyzed with deseq to identify genes with differential hmc patterns between risk groups. chromosomal copy number was assessed by chromosomal microarray analysis (cma) in a subset of samples ( lr and hr). expression of genes located on chromosome p was evaluated using publically available microarrays (e-mtab- ) of hr nbl tumors with known p loh status. results: globally, lr tumors had more hmc peaks ( , ) than ir ( , , p = . ) tumors, or hr tumors ( , , p = . ). , genes had different patterns of hmc deposition in hr versus lr tumors. ( %) of these genes mapped to chromosome p and had decreased hmc in hr versus lr tumors (padj < . ). in the cma analysis p deletion was detected in of the tumors tested. in the tumors with p loss, genes that map to p showed decreased hmc deposition compared to the hr tumors without p loss (p< . ). further, compared to the tumors without p loss, the expression of of the p genes was decreased (p< × - ), including chd , camta , and arid a, known and proposed tumor suppressor genes in nbl. conclusion: different patterns of hmc accumulation are associated with neuroblastoma risk classification. nano-hmc-seal-seq is sensitive to copy number variations and has the potential to identify these changes in patient tumors. our results suggest that hmc deposition contributes to the silencing of tumor suppressor genes in p and may also regulate the transcription of other genes that drive tumor phenotype. background: metastatic osteosarcoma has a -year survival rate of - %. pulmonary metastases remain a major treatment challenge in osteosarcoma. current treatment with conventional chemotherapy shows inadequate activity towards metastases and has toxic systemic side effects. chloroquine is a widely used anti-malarial drug and has been shown to have promising anti-cancer and anti-metastatic activity. polymeric drugs have been shown to have multiple advantages over their small molecular parent drugs, including enhancing the therapeutic efficacy, an improved pharmacokinetics profile and decreased systemic toxicity. we hypothesized that by developing chloroquine into a polymeric drug and combining it with conventional chemotherapy it will improve the treatment of metastatic osteosarcoma. objectives: to identify the optimal combination of polymeric chloroquine (pcq) with conventional chemotherapy active in osteosarcoma as a new means of treating metastatic disease in a murine osteosarcoma model. we synthesized and developed pcq and evaluated its anti-invasive activity using an osteosarcoma cell migration and invasion assay. we evaluated the efficacy of cell killing using combination drug therapies with pcq and a panel of conventional chemotherapy agents (doxorubicin, docetaxel, cisplatin and paclitaxel) using celltiter blue cell viability assay. to develop the murine osteosarcoma model, we intravenously injected luciferase-expressing human osteosarcoma cells b into nsg mice. we administered the drug combination that showed the strongest in vitro synergy to the mice and evaluated their anti-cancer and anti-metastatic effects in vivo. tumor growth and suppression were evaluated using whole body bioluminescence imaging. results: we successfully synthesized pcq that contains . % chloroquine with a molecular weight of . kd. pcq was also found to decrease the toxicity of the parent chloroquine. pcq showed strong inhibition of osteosarcoma cell migration with % inhibition compared to % by chloroquine. we screened the combination drug therapies and found the combination of pcq and doxorubicin to show the strongest synergism. the pcq/doxorubicin combination is currently being evaluated in the murine model. combination drug therapy using pcq and doxorubicin showed synergistic cell killing and inhibition of cell migration in vitro. the combination represents a promising treatment strategy for pulmonary metastatic osteosarcoma. emory university/children's healthcare of atlanta, atlanta, georgia, united states background: survival for relapsed high-risk neuroblastoma (rnb) is < %, underscoring the critical need for novel therapies. rnbs have increased ras/raf/mapk mutations and increased yes-associated protein (yap) transcriptional activity. yap is a transcriptional co-activator that binds with tea-domain (tead) transcription factors to regulate cellular proliferation, self-renewal, and survival. we found that shrna inhibition of yap decreases nb cell proliferation and sensitizes ras-mutated nbs to mek inhibitors, supporting yap as a tractable therapeutic target. verteporfin (vp), a photodynamic drug used for macular degeneration, is the only drug found to inhibit yap expression or yap:tead binding to kill tumor-derived cells. peptide is a mer yap peptidomimetic that also disrupts yap:tead interactions. we sought to determine whether these compounds are potent in nb via yap direct effects. design/method: yap expressing (nlf, sk-n-as) or yap null (ngp, lan , sk-n-as-shyap) human-derived nbs were incubated with vp, with and without direct light exposure, or with peptide . celltiter-glo and immunoblots were used to assess for cell death and yap-downstream protein expression, respectively. results: without direct light exposure, vp inhibits yap expression at nm dosing, yet no nb cell death was observed at equal or higher concentrations. egfr and erk / were inhibited along with yap, confirming yap/ras pathway coregulation. when vp was exposed to direct incandescent light for minutes, > % nb cell death occurred in all nbs tested, even those lacking yap. peptide caused no cell death or yap inhibition up to um. neuroblastomas are resistant to vp at doses sufficient to inhibit yap expression. in macular degeneration, light-activated vp produces reactive oxygen species, which we hypothesize is the off target mechanism killing nbs independent of yap. given the off target effects and the need for light activation, vp is not an ideal preclinical or clinical yap inhibitor. accordingly, peptide has poor cell permeability and low tead affinity, leading to its lack of efficacy. given the relevance of yap in rnb and other cancers, we are chemically optimizing a yap peptidomimetic with enhanced permeability, nuclear localization, and tead affinity to create a bonafide yap inhibitor for preclinical and clinical application. kayeleigh higgerson, aaron sugalski, rajiv rajani, josefine heim-hall, jaclyn hung, anne-marie langevin ut health san antonio, san antonio, texas, united states background: osteosarcoma is the most common bone malignancy in children, adolescents, and young adults. most study cohorts have to % hispanic patients that encompass many different hispanic backgrounds. the university of texas health science center at san antonio (uthscsa) sarcoma team serves a latino population that is predominantly mexican american, thus providing a unique opportunity for evaluation this population. this study expands on previous data collected from january to december from the same institution, providing increased insight into outcomes of mexican american children, adolescents, and young adults with osteosarcoma. objectives: to further understanding of osteosarcoma in latino children, adolescents and young adults. design/method: a retrospective analysis of demographics, tumor characteristics, response to treatment, and survival outcome of all localized osteosarcoma of the extremity patients below years of age diagnosed and treated by the uthscsa sarcoma team between january and june was performed. results: in our original cohort from january to december , we observed a significantly decreased -year eventfree survival (efs) in patients diagnosed before age (preadolescent) relative to patients diagnosed between ages and ( % vs. %, p< . ). patients had a -year overall survival (os) and event-free survival of % and % respectively. in our expanded cohort from january to june we evaluated sixty-six patients with a median age of (range, to y) with localized high-grade osteosarcoma of the extremity. the expanded cohort was % mexican american, with a median follow-up of months (range, to ). the analysis of our expanded cohort is ongoing and we postulate that the findings will hold true, as we increase the cohort size and length of follow-up. conclusion: analysis of our previous cohort, predominantly of mexican american ethnicity, showed that preadolescent patients had an increased rate of relapse when compared with previous large studies. we also showed a trend towards decreased efs for the entire cohort. we hypothesize that we will further validate these findings with this expanded cohort and this will support further investigation into potential causes of poor outcome in this vulnerable latino population. background: neuroblastoma in infants has the potential to regress or mature spontaneously. growing literature showed that some cases subjected to initial observation didn't show inferior outcome compared to actively treated similar categories. objectives: we investigated whether early active treatment can be safely avoided/deferred in selected favorable cases at the children's cancer hospital-egypt (cche). design/method: patients enrolled on the watch and see strategy (w&s) at cche had small primary tumor; inss stage - , uncomplicated stage s or stage infants (< days). tissue biopsy was not mandatory for infants below months of age with localized adrenal mass (stage - ). on progression, immediate intervention took place according to stage and risk of disease after biological characterization. results: thirty four nbl patients were enrolled on w&s strategy; m/f: . / . eighteen patients had stage s disease, patients had stage - and were stage . primary adrenal site was reported in patients ( . %), patients ( . %) had small mass measuring ≤ cm in its largest diameter. the -year os & efs were . ± . % and . ± %, respectively, with months median follow-up (range: - months). spontaneous total/near total resolution of mass occurred in / patients ( %). median time to eliciting regression was . months (range: . - . months), and . months (range: - months) till complete resolution. only / patients ( . %) witnessed progression ( local, distant and combined local and distant progression); median time to progression was months (range: - months) with / deaths after starting chemotherapy. watch and see strategy is a safe approach in localized and uncomplicated stage s neuroblastoma. progressive cases could be rescued. baylor college of medicine, houston, texas, united states background: ga- dotatate binds to somatostatin receptor expressed in neuroendocrine tumors (nets). it was approved by fda in for use with pet/ct scan for localization of somatostatin receptor positive nets in adult and pediatric patients. pediatric approval was based mainly on extrapolation of data from adults. objectives: to describe the use of ga- dotatate pet/ct scan in children with neuroendocrine tumors and compare with other imaging modalities. design/method: patients with nets enrolled in texas children's rare tumor registry between february and october were reviewed and those patients who underwent ga- scan were included. results: four patients with nets underwent ga- scans without any adverse reactions. first patient was a -yearold female with small bowel net with multiple liver metastases. mri abdomen and fdg pet at diagnosis showed s of s multiple liver metastases but could not identify the primary lesion. ga- scan was able to accurately identify the enlarged lymph nodes in the small bowel and was better than fdg pet in delineating the liver metastases. second patient was a -year-old female with recurrent small bowel net with liver, lung and paraspinal metastases. the lesions were initially detected by ct scan. octreotide scan failed to show any uptake in the identified lesions while ga- was taken up by the liver lesions, lung lesions > cm in size and the paraspinal lesion. third patient is an year-old male with pancreatic net with peripancreatic lymphadenopathy, multiple liver metastases and cardiophrenic lymph node involvement. the primary lesion in the pancreas could not be identified by ct scan, ct angiogram, mibg scan, or octreotide scan. in addition, there was uncertainty about involvement of the enlarged cardiophrenic lymph node. in addition to clearly identifying the primary lesion, ga- scan was able to detect multiple peripancreatic lymph nodes not detected by other scans and revealed uptake in the cardiophrenic lymph node confirming its involvement by the tumor. fourth patient is a -year-old female with malignant abdominal paraganglioma with solitary lung metastasis. both mibg scan and ga- scan were able to identify the primary lesion. ga- scan was performed after the lung metastasis was removed and thus its ability to detect it could not be confirmed. background: neuroblastoma is the most common extracranial solid tumor of childhood, with overall survival for high-risk patients (hrnbl) near %. the outcomes of hrnbl have improved with high dose chemotherapy followed by autologous stem cell rescue (abmt). data about factors influencing the rate of hematopoietic recovery following abmt in hrnbl is lacking in the literature. our objective was to identify factors influencing the rate of hematopoietic recovery following abmt in hrnbl. design/method: this was a retrospective chart review of patients with hrnbl treated at texas children's hospital from to . neutrophil engraftment was considered the first of three consecutive days with post-transplant neutrophil count greater than cells/ul. red blood cell and platelet engraftment were considered at a hemoglobin greater than g/dl and platelets greater than , /ul three days after the last transfusion. race and conditioning regimen were analyzed using one-way anova; amount of infused cells was analyzed using pearson correlation coefficients; chemotherapy delay and bone marrow (bm) involvement after cycle of induction chemotherapy were analyzed using independent sample t-tests. the study included males and females with a median age at diagnosis of . years. thirtyeight patients were caucasian, african-american, hispanic, asian, and did not have race documented. the mean dose of infused cd + cells was . × ^ cells/kg. forty-five patients received conditioning therapy with carboplatin/etoposide/melphalan (cem), received busulfan/melphalan (bu/mel), and received thiotepa/cyclophosphamide (thiotepa/cpm). the conditioning regimen administered was significant (p = . ) for time to engraftment of neutrophils, with bu/mel at . days, cem at . days, and thiotepa/cpm at days. a delay of chemotherapy during induction (n = ) was significant (p = . ) for time to platelet engraftment of greater than , /ul and trended towards significance (p = . ) for time to neutrophil engraftment. bm involvement at diagnosis and after cycle of induction was not significant for time to engraftment. dose of stem cells infused was the only variable significant for hemoglobin engraftment. background: osteosarcoma (os) is the most prevalent aggressive primary malignancy of the bone affecting children and young adults. approximately % to % of patients have metastatic disease at initial presentation, and % of those patients have isolated pulmonary metastases. although overall survival in patients with os has improved with advances in therapy, there have been no significant improvements in survival outcome in patients with metastatic disease. recent studies suggest that tumor-associated vascular cell adhesion molecule (tvcam- or cd ) plays a critical role in the metastatic progression of various tumors. indirect evidence from these studies suggest that vcam- / integrin signaling promotes tumor survival and metastatic progression by changing the tumor niche and associated immune response. to determine if interfering vcam- / signaling between pulmonary metastatic osteosarcoma (pos) and macrophages (macs) by down-regulating vcam- , depleting macs or blocking vcam- / signaling will reduce pos and improve overall disease-free survival. design/method: we used a pair of spontaneous, high-grade murine os cell lines from balb/c mouse (h- d), k and k m (derived from in vivo k metastasis). we used lentiviral shrnas to knockdown vcam- mrna and protein expression in k m (vcam- kd). we introduced luciferase into k , k m and various k m shrna cell lines to follow lung metastasis by bioluminescence (bli). we depleted macs by intranasal administration of liposomal clodronate formulation. we tested the ability of k and k m supernatants to polarize m macs into m or m phenotype in vitro. we also administered anti- monoclonal antibody (anti- mab) intranasally to assess the outcome of functional blockade of vcam- / signaling. results: k m over-expressed vcam- compared to k . mac depletion in k m -bearing animals exhibited reduced pos. weekly administration of anti- mab resulted in % tumor-free rescue among mice with established k m pos. interestingly, supernatant from k m but not k preferentially induced m -like macs, suggesting a novel integrin-mediated mechanism of m differentiation. validation data with additional os cell lines will be presented. despite aggressive multimodal therapy, overall outcome for patients with pos remains dismal at - %. for this reason, novel and directed therapy approaches are desperately needed. molecular targeted approaches for therapy are challenging, due to the complex genetic heterogeneity of os. immune-modifying therapy is a promising new alternative approach for pos. university of chicago, chicago, illinois, united states background: only half of all patients diagnosed with high-risk neuroblastoma achieve long-term survival. imetaiodobenzylguanidine (mibg) scans are routinely used to evaluate disease at diagnosis and following treatment, and the extent of disease is quantified using the curie scoring system. a previous study by yanik et al., has shown that for high-risk patients with mycn non-ampliified tumors, scores less than versus greater than following cycles chemotherapy are associated of superior survival, whereas scores less than versus greater than were prognostic in patients with mycn-amplified tumors. however, the prognostic significance of specific sites of metastatic disease at diagnosis is not known. to determine if site of metastatic disease determined by i-metaiodobenzylguanidine (mibg) imaging in high-risk patients at the time of diagnosis was associated with outcome design/method: we performed a retrospective chart review of high-risk neuroblastoma patients treated at comer children's hospital and lurie children's hospital in chicago between and with positive mibg scans at the time of diagnosis. we collected imaging data as well as other clinical data including bone marrow status. sites of disease were defined as curie regions with any positive value. kaplan-meier analysis was performed to evaluate the association with disease sites and survival. pearson correlation coefficients were calculated to compare bone marrow disease to sites of positivity on mibg scan. the cohort consisted of high-risk patients. had skull disease, and had pelvic disease. the presence of mibg positive disease in the skull and in the pelvis trended toward worse efs. efs at years for patients with disease in the skull at diagnosis was ± % and for patients without skull disease was ± % (p = . ). efs at years for patients with and without pelvic disease was ± % and ± % (p = . ). consistent with prior data, we found that the presence of bone marrow disease was associated with worse survival with year efs of ± % and ± % with and without marrow disease at diagnosis (p = . ). there is the highest correlation between pelvic disease on mibg scan and bone marrow disease with pearson coefficient . . pelvic disease noted on mibg scan likely reflects underlying bone marrow disease. in patients with high-risk neuroblastoma, skull disease and pelvic disease on mibg scan at diagnosis may predict worse event free survival. background: osteosarcoma is one of the deadliest cancers in the pediatric population with little progress in morbidity and recurrence rates since the 's. oncolytic herpes simplex- virus (ohsv) is an attenuated virus that has shown encouraging results against certain solid tumors. programmed cell death protein (pd)- -mediated t cell suppression via engagement of its ligand, pd-l , is also of particular interest due to recent successes in selected cancers, especially those with high genetic mutational loads. most pediatric cancers do not have a wide variety of mutations; however, osteosarcoma has a chaotic genome, prone to genetic mutations. it has been shown through numerous other studies that pd- inhibition alone is not sufficient to result in statistically significant tumor growth delays in osteosarcoma models and patients. we hypothesize the addition of ohsv therapy as an immunologic stimulus to pd- inhibition is efficacious for osteosarcoma. ( ) to determine whether ohsv therapy enhances response to pd- inhibition in immunocompetent murine models of osteosarcoma and ( ) to quantify and characterize the anti-tumor t-cells infiltration after treatment with ohsv and pd- inhibition individually and in combination. we utilized an immunocompetent transplantable murine model using a cell line derived from a spontaneous metastatic osteosarcoma (k m , balb/c background). we transplanted established tumor wedges subcutaneously and monitored tumor volume by caliper measurement. once tumors reached - mm , we administered intratumoral injections of hsv ( × plaque-forming units) every other day for a total of injections. we then gave intraperitoneal injections of ug anti-pd- or control antibody twice weekly, up to weeks, starting from the last dose of virus treatment. we monitored tumor growth via calipers twice weekly until tumors reached mm or cm diameter. we quantified and characterized innate and adaptive immune cell infiltrates in tumors using flow analysis. we found significantly prolonged survival with our combination therapy group compared to all other groups. we found that anti-pd- by itself had little impact on t cell recruitment while the combination group had higher influx of cd + cells with a reduced amount of t-regulatory cells (cd +foxp +cd +). we also found an increase in cd + effector memory cells. osteosarcoma is a deadly cancer with therapeutics remaining unchanged for the last years. here, we describe prolonged murine survival after treatment with combination of pd- inhibition and ohsv injection. the combination treatment changed the microenvironment to be more inflammatory. our data support further preclinical and clinical studies. background: neuroblastoma is the second most common cause of cancer related death in children. treatment for high-risk neuroblastoma has improved significantly over the past twenty years, however cure rates remain below %. immunotherapy has emerged as an effective therapy for neuroblastoma, however new modalities and targets are needed to improve outcomes. objectives: our lab has developed a chimeric antigen receptor (car) that targets b -h (cd ), an immune checkpoint molecule overexpressed on many cancers, including neuroblastoma. we hypothesized that b -h would be a good target for car based immunotherapy for neuroblastoma. design/method: neuroblastoma tissue microarrays of primary patient samples were screened for b -h expression by immunohistochemistry and cell lines were screened using flow cytometry. b -h car t cells were tested in vitro by measuring tumor cell killing and cytokine production after coculture with tumor cell lines and in vivo in an orthotopic model of neuroblastoma. results: b -h expression was detected by ihc on % of the screened neuroblastoma patient samples. b -h was expressed at high levels ( + or +) in more than half of these samples ( %). almost all cell lines screened were homogeneously positive for b -h by flow cytometry. retrovirally transduced b -h . - bb. car t cells were cocultured with three b -h positive neuroblastoma cell lines (sk-n-be , kcnr, and chla ) and robust tumor cell killing was demonstrated using an incucyte assay. supernatant from the co-cultures was harvested after hours and both interferon gamma and il- production were detected by elisa.in an orthotopic subrenal capsule xenograft model of neuroblastoma, mice treated with b -h car t cells show significant reductions in tumor growth and prolonged survival compared to those treated with untransduced control t cells. however, the treatment is not always curative.b -h car t cells express high levels of exhaustion markers (pd , tim , and lag ) when compared to cd car controls. in order to overcome inhibition from exhaustion, b -h car t cells were co-cultured with neuroblastoma cell lines and pd- blocking antibody. nivolumab significantly increased the production of il- and interferon-gamma by b -h car t cells. further studies are underway to determine if b -h car t cell activity is enhanced in vivo by treating animals with pd- blockade along with car t cells. conclusion: b -h is expressed on a majority of neuroblastoma samples and appears to be a promising candidate for car t cell therapy. b -h car t cells demonstrate activity against neuroblastoma xenografts that may be enhanced by the addition of pd inhibitors. helen devos children's hospital, michigan state university, grand rapids, michigan, united states background: osteosarcoma is the most common bone tumor in children. it is often metastatic at diagnosis and in this scenario less than % of children survive. polyamines, small molecules found in all cells, are involved in many cell processes including cell cycle regulation, immune modulation, cell signaling and apoptosis. they are also involved in tumor development, invasion and metastasis. in neuroblastoma, inhibition of the polyamine biosynthesis pathway with odc inhibitor alpha-difluoromethylornithine (dfmo) results in decreased cell proliferation and differentiation. these finding have led to multiple phase i and phase ii multicenter clinical trials in pediatric neuroblastoma patients. dfmo is an attractive drug as it is oral, well-tolerated, can be given for prolonged periods and is already used in pediatric patients. the polyamine pathway has not been evaluated in osteosarcoma. objectives: evaluate effect of inhibition of polyamine biosynthesis with dfmo on osteosarcoma proliferation and cell differentiation. design/method: up to three osteosarcoma cell lines were used: mg- , u- os and saos- . cells were exposed to mm dfmo for days with replacement of media and dfmo on day . intracellular polyamine levels were measured by high performance liquid chromatography (hplc). cell numbers were obtained with a hemocytometer using trypan blue. flow cytometry cell cycle distribution (facs) and propidium iodide were used to evaluate for cell cycle arrest. the protein expression of several osteosarcoma differentiation markers was measured by sds-page and western blot using differentiation specific antibodies. a bioluminescent cell viability assay was used to measure cell recovery over several days after dfmo was removed and replaced with standard media. results: dfmo exposure resulted in significantly decreased cell proliferation in all cell lines. after treatment, intracellular spermidine levels were nearly eliminated in all cells. cell cycle arrest at g was observed in u- os. cell differentiation was most pronounced in mg- and u- os cells as determined by increased osteopontin levels. remarkably, cell proliferation continued to be suppressed for several days after removal of dfmo. conclusion: based on our findings dfmo is a promising new adjunct to the current osteosarcoma therapy for high risk patients. it is a well-tolerated oral drug that is currently in phase ii clinical trials in pediatric neuroblastoma patients as a maintenance therapy. the same type of regimen may also improve outcomes in metastatic or recurrent osteosarcoma patients for whom there have been essentially no medical advances in the last years. background: recent studies demonstrate that lower levels of the ews-fli fusion oncoprotein are associated with enhanced metastatic capability in ewing sarcoma. the nf-kb transcription factor is a critical mediator of cxcr and cxcr -driven metastasis in multiple cancers, and increased cxcr and cxcr expression have each been associated with increased metastasis and poor prognosis in ewing sarcoma. we thus sought to investigate the impact of ews-fli on cxcr /cxcr -dependent nf-kb signaling in ewing sarcoma. objectives: the goals of this study are ) to determine the impact of cxcr /cxcr signaling on metastasis-associated nf-kb target gene expression in ewing sarcoma and then ) to investigate how the ews-fli fusion oncoprotein modulates this response . design/method: we utilized multiple ewing sarcoma cells lines including a , chla , chla , tc and tc . cxcr /cxcr cell surface expression was determined by flow cytometry. ews-fli level was modulated using sirna and expression levels were confirmed by western blot and rt-pcr. p dna binding was measured via elisa. nf-kb target gene expression was assessed via rt-pcr. results: consistent with ihc analysis of primary and metastatic patient tumor samples, the paired primary and metastatic ewing sarcoma cell lines chla and chla showed dramatic differences in cxcr and cxcr expression, with the metastatic chla line demonstrating much higher expression of both receptors. other cell lines (nonpaired) showed variable cxcr /cxcr expression. genetic knock-out of cxcr lead to significant decrease in expression of both cxcl /sdf- and il- , two nf-kb transcriptional targets known to play a key role in tumor metastasis. knock-out of cxcr did not alter endogenous ews-fli mrna levels. conversely, lowering the level of ews-fli using sirna lead to enhanced nf-kb signaling, indicated by an increase in p dna binding. consistent with this observation, treating ewing cell lines with ews-fli sirna also resulted in significantly increased nf-kb target gene expression compared to control cells and target gene expression was then further enhanced upon cxcr /cxcr receptor stimulation with the receptor ligand cxcl /sdf- . our findings indicate that the ews-fli oncoprotein negatively modulates cxcr /cxcr -dependent nf-kb signaling. this suggests that ews-fli low, cxcr /cxcr high cells, which are associated with enhanced metastasis and poor prognosis, would be anticipated to exhibit enhanced expression of key nf-kb target genes. importantly, the nf-kb pathway is a druggable target that could potentially serve as an "achilles heel" in this subset of high risk tumors. current work is evaluating nf-kb inhibition as an approach to treating metastatic and refractory ewing sarcoma. background: acute graft versus host disease (agvhd) is a major cause of morbidity and mortality following allogeneic bone marrow transplant (bmt) in pediatric patients. gastrointestinal (gi) agvhd is the most serious manifestation. recently, decreased paneth cell (pc) in a predominantly adult cohort was shown to correlate with agvhd clinical grading and response to treatment. we aim to demonstrate the relationship between pc counts and gi agvhd stage and response to therapy. design/method: charts of patients who underwent endoscopy following bmt between - were reviewed. for repeated biopsies during the course of agvhd, only the first was included for analysis. one pathologist retrospectively reviewed the biopsies and counted pcs in high powered fields; the average pc count was analyzed. twenty-six percent of biopsies were reviewed by a second blinded pathologist. statistical associations between pc counts and day (d ) response, agvhd stage, and other study covariates of interest were gauged using general linear regression. agreement in pathologist pc counts was quantified by intraclass correlation (icc). the research was approved by the children's healthcare of atlanta irb. results: seventy-eight biopsies were included in the analysis. mean age at transplant was . years ± . (range: months - years). most patients underwent transplant for hematologic malignancies ( , %). the majority of transplants used a matched unrelated donor graft -including cords ( , %) and myeloablative conditioning regimens ( , %) - % received total body irradiation. of these, % were diagnosed clinically with gi agvhd (stage , %; stage , %; stage , %; stage , %). icc showed good agreement ( . ) between the pathologists. mean pc was . for patients with no gut agvhd, . for stage , . for stage , . for stage and . for stage (p = . ). on multivariate analysis pc was strongly associated with gi agvhd stage (p< . ) after controlling for age, preparative regimen intensity, and diagnosis (malignant vs. non-malignant). mean pc counts were significantly lower in patients with no response to steroid therapy at d (complete response (mean . ) vs. persistent disease ( . ) vs. partial response ( . ) (p = . )). patients diagnosed with gi agvhd with pc counts less than had a higher risk of mortality (hr . , % ci: . , . ; p = . ). lower pc count correlated with stage gi agvhd, refractory disease at d , and mortality. incorporating pc count in pathology review during gi agvhd work-up may help in agvhd risk stratification. background: there have been increasing discussions pressuring health care teams and institutions for potentially bearing the cost of clostridium difficile infections (cdi) as a health care-associated infection in the recent years. the pediatric oncology patient population, though small, accounts for significant portion of all cdi with - -fold increased risk. hematopoietic stem cell transplant (hsct) recipients constitute a unique subset with distinct risk factors, such as severe immune deficiency state and graft versus host disease (gvhd). although there is ample data on cdi in adult hsct recipients, reports on pediatric experience are limited. objectives: to evaluate the incidence and patterns of cdi among pediatric hematology, oncology and hsct inpatients at our institution. a retrospective review of all clostridium difficile (cd) stool tests performed using toxin enzyme immunoassay and later, polymerase chain reaction targeting toxin genes between and in a large, urban academic children's hospital was performed. the data were analyzed for hematology, oncology, hsct inpatient population and all the other cases separately and statistical comparisons were performed. results: a total of samples were submitted to the microbiology laboratory for cd testing during the study period. while hematology patients constituted . %, oncology . %, hsct . % and others . % of the cases on whom cd testing was done; per patient average test number was . , . , . , and . , respectively. of all the cd tests per-formed, . % were positive. test positivity was higher in hsct ( . %) and oncology ( . %) cases tested compared with hematology ( . %) and other cases ( . %) with statistical significance (p< . ). overall recurrence rate was . %; hsct patients had the highest recurrence with a rate of % followed by oncology ( . %), hematology ( . %) and other ( . %) cases, again reaching statistical significance (p< . ). again, hsct patients had the highest average number of recurrences at . ( - ) followed by oncology . ( - ), general . ( - ) and hematology . ( - ) groups. there was no seasonal variability in the incidence of cdi among populations analyzed. prolonged hospital stay/antibiotic use and persistent diarrhea due to gvhd are the likely reasons for higher rate of cd testing in hsct as a result of increased monitoring and thus might have even caused underrepresentation of positive cd test frequency. higher incidence and frequencies of recurrence underscores the inevitable nature of cdi in hsct population as a consequence of the current therapies and may lead to future radical treatment approaches like fecal implantation. background: viral infections remain a challenge to treat post hct in children, and significantly contribute to morbidity and mortality. virus specific t cells (vsts) have shown tremendous clinical efficacy in treating viral infections post-hct, with minimal toxicity and long term efficacy. we have used donor-derived vsts in individual patients, however not all donors are agreeable to the process, and numerous patients may benefit from vsts who do not have an identified donor/have other disease indications objectives: we sought to actively build a third-party vst bank, for "off the shelf" use in eligible patients. design/method: vsts targeting cmv, adenovirus and ebv were manufactured using one of techniques. initially ebv transformed b cells were genetically modified with an ad f pp vector and used as antigen presenting cells (apc) to stimulate and expand ebv, ad and cmvpp specific t cells. more recently, vsts were expanded using s of s apc pulsed with commercially available peptide pools (pep-mixes) to expand ebv/cmv/ad specific t cells. products were entered into the "bank" via two mechanisms: a) left over products from our "donor-derived" protocol when patients no longer required vsts or were not at risk of developing viral infections, or b) by targeting regular blood donors based on their hla typing to ensure an appropriate mix of high frequency hla types for optimal patient matching and antigen presentation based on current knowledge of antigen presentation. results: a total of products are currently in the thirdparty vst bank ready for use. twenty seven of these are from our donor derived protocol, and three from targeted donors. all vst products met safety and in vitro efficacy testing. thirteen vst infusions have been given to patients. eleven infusions have been given for cmv and two for adenovirus. five out of seven patients responded to thirdparty vst infusions, with a median of vst infusions per patient (range - ). the median hla matching was out of per patient (range to ) no patients experienced adverse reactions, gvhd or other toxicity related to the vst infusion. a third-party vst bank is feasible and produces clinically appropriate vsts for use in patients with viral infections. hla typing and matching of vst products is essential to reduce toxicity and promote appropriate antigen presentation and expansion of vsts in vivo. further work is underway to further characterize the vsts using epitope mapping to better define the hla restriction and immunogenicity of each vst product. akron children's hospital, akron, ohio, united states background: acute graft-versus-host disease (agvhd) is a well-known complication of hematopoietic stem cell transplant (hsct) and a major cause of post-transplant related morbidity and mortality. first line therapy of agvhd involves corticosteroids and calcineurin inhibition. in patients with severe refractory gvhd, mortality can reach up to %. currently, there is no standard of care for the treatment of steroid refractory agvhd. many centers have looked at the use of antibody mediated control of agvhd to competitively inhibit the inflammatory cascade. basiliximab, a chimeric monoclonal antibody against the t-cell il- receptor, has been used in adults with steroid refractory agvhd. patients receiving this medication have demonstrated complete and partial responses to therapy with minimal toxicities. objectives: report the successful use of basiliximab in the treatment of agvhd in a -year-old following matched unrelated (mud) hsct. design/method: a -year-old male underwent mud transplant for high risk aml with monosomy . conditioning regimen included busulfan, fludarabine and equine atg. his clinical course was complicated by fever, mucositis and agvhd (stage skin; stage gi-biopsy proven). gvhd prophylaxis included tacrolimus and methotrexate, however with progressive skin rash, diarrhea, and early satiety, gvhd treatment with corticosteroids was initiated. as the patient continued to have worsening symptoms, basiliximab therapy was started. the patient received doses ( mg) iv basiliximab on two consecutive days and then received weekly therapy for a total of doses leading to initial improvement. the patient further developed acute on chronic gvhd on day + , and subsequently received a second course of basiliximab. after initial administration of basiliximab, the patient had near complete resolution of symptoms. however, with a small wean in his tacrolimus dose, the patient experienced another skin gvhd flare prompting the second basiliximab course. the patient was subsequently weaned off all immunosuppression by day + . the only acute complication the patient experienced while receiving basiliximab was right toe paronychia and asymptomatic low ebv titer. the patient is currently off all immunosuppression at the time of report without evidence of cgvhd. conclusion: this single case report, in a young pediatric patient, demonstrates the use of basiliximab may be a safe and efficacious treatment for pediatric patients with agvhd. university of california, san diego, la jolla, california, united states background: clinical outcomes after allogeneic hematopoietic stem cell transplantation (hsct) depend on restoration of t lymphocyte populations. association between recovery of cd +foxp + regulatory t cells (tregs) and protection from chronic graft versus host disease (cgvhd) has been described in adult hsct. in adults, t cell recovery is driven by expansion of donor t cells and treg reconstitution is hypothesized to result from peripheral conversion. restoration of t cells in pediatric patients has a larger contribution from thymopoiesis, however, the relationship between thymopoiesis and treg recovery is undefined. objectives: we hypothesized that effective thymopoiesis is important for restoration of treg populations and protection from cgvhd in pediatric hsct patients. design/method: we performed longitudinal flow cytometry of peripheral blood t cells from pediatric hsct patients and age-matched healthy donors. laboratory data were correlated with clinical outcomes to evaluate impact. recovery of tregs occurred in / ( . %) patients by post-transplant day . day treg frequency in patients that developed cgvhd ( . ± . % of cd + t cells) was reduced compared to cgvhd-free patients ( . ± . %). failure to restore tregs to > . % of cd + cells by day was associated with increased risk of cgvhd in the first year post-hsct (rr = . , p = . ). a majority ( . ± . %) of tregs from patients recovering the peripheral treg compartment expressed helios, a marker of thymic-derived tregs; only . ± . % of tregs expressed helios in patients failing to restore adequate tregs. this prompted examining the relationship between defects in thymopoiesis and inability to restore tregs. we evaluated thymic function by flow cytometry quantification of cd ra+cd +ptk + recent thymic emigrant (rte) cd + cells (confirmed by qpcr for trec content). most ( / , . %) hsct patients had detectable rtes by day post-hsct. thymic production of rtes was persistently absent in patients that developed cgvhd (< / ^ cd + cells in / patients), compared to cgvhd-free patients ( / patients > rte/ ^ cd + cells by day , average . ± . / ^ cd + cells). post-hsct thymic activity as measured by rte enumeration correlated with treg restoration; / ( %) rte+ patients restored tregs, compared to / ( %) of rte-patients. conclusion: failure to restore tregs after allogeneic hsct results in increased risk for cgvhd. in pediatric patients thymic generation of new t cells is an important contributor to restoration of the treg compartment. this data supports further investigation into mechanisms impairing post-hsct thymopoiesis and suggests peripheral blood tregs may be a prognostic biomarker for cgvhd. background: haploidentical stem cell transplantation (haplo sct) is riddled with unique challenges. objectives: we present our experience in the use of haplo sct with post-transplant cyclophosphamide (ptcy) and the adaptations required for each disorder for optimal outcome. design/method: we performed a retrospective study at the pediatric blood and marrow transplant unit, apollo cancer institutes, chennai, india. children up to years of age, diagnosed to have benign disorders and underwent haplo sct with ptcy from to july were included. results: ptcy was used in i.e. % haplo transplants for children with benign disorders. the underlying conditions included fanconi anemia , severe aplastic anemia , mds , jmml , hemoglobinopathy , prca , xld and primary immunedeficiency disorders (pid) . source of stem cells was peripheral blood in %, bone marrow in %. conditioning included fludarabine with treosulphan or cyclophosphamide for pids and aplastic anemia respectively. neutrophil engraftment by day+ - with a durable graft was noted in % transplants with graft versus host disease in %, cmv reactivation in %. mortality rate was % with infants less than months of age developing severe fatal cytokine release syndrome. the median follow up is year with years being the longest. no significant late effects have been noted with chronic skin gvhd in children. survival rate was superior among children with pids with survival of % in this group. haplo sct with ptcy is a feasible and costeffective option for cure in children with life-threatening benign disorders with no compatible family or matched unrelated donor. careful patient selection, reducing cyclophosphamide related free radical toxicity with the use of n acetylcysteine, limiting t cell numbers by capping cd at × /kg, post-transplant viral monitoring protocols are required to reduce morbidity and mortality. we have been working on universal access to care for children from s of s all socioeconomic background and incorporating innovations to reduce the cost of hsct without compromising outcomes. haploidentical hsct using tcr / depletion costs usd as compared to ptcy priced at usd. children with severe aplastic anemia and pids can be transplanted using reduced intensity conditioning and ptcy. in hemoglobinopathies, pretransplant immunosuppression is required to prevent graft rejection. graft versus host disease remains the main cause of mortality in children with fanconi anemia. mortality in infants less than months after ptcy has been high, tcr / depletion would be superior in this cohort. cincinnati children's hospital medical center, cincinnati, ohio, united states background: fanconi anemia (fa) is a congenital bone marrow failure syndrome with hsct the only curative option for associated bone marrow failure. patients with fa undergoing hsct may experience increased toxicity related to either their underlying disease, or the effects of medications, resulting in the inability to tolerate prophylactic medications or sideeffects from anti-microbial therapy. objectives: we postulated that increased cd cell dose would be associated with a rapid immune reconstitution and therefore early withdrawal of anti-infective prophylactic medications. design/method: patients with fa transplanted at cchmc from an unrelated donor had peripheral blood stem cell grafts collected and cd selection performed. where possible, patients had serial measurements of their immune system performed at varying intervals post hsct. we defined immune reconstitution as normalization of lymphocyte subsets-cd , cd , cd and cd cells, as well as a normal response to mitogen stimulation including phytohemagglutinin, concanavalin a and pokeweed. the first measurement of either normal cell number or mitogen response was recorded for each patient. results: a total of patients underwent hsct for fa at cchmc between and . patient demographics included a median age of years at hsct, the vast majority of patients having a fully matched or one anti-gen mismatched donor, and the majority of patients transplanted for bone marrow failure. there was a statistically significantly decreased time post-transplant to immune cell recovery in patients receiving > × /kg cd cells (median . ) compared to those receiving < × /kg cd cells (median . ). the median time to normalization of cd count was days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg), cd count days (cd count > /kg) versus days (cd count < /kg) and cd count days (cd count > /kg) versus days (cd count < /kg). time to normalization of mitogen response was decreased posttransplant in those patients receiving increased cd cell dose at time of transplant, though this was not significant, reflecting low number of patients with evaluable responses. no patients in either group experienced gvhd or graft failure. patients with fa who are transplanted with higher cd cell doses have quicker immune reconstitution than those who receive lower cell doses. along with benefit to patients including less risk of infection and early termination of immune-prophylaxis medications, this supports the use of high dose cd selected grafts in this vulnerable population. background: parvovirus b (pvb ) infection after transplantation was first reported in . since then, numerous cases of pvb infections after hematopoietic stem cell transplantation (hsct) and solid organ transplantation (sot) have been reported. most report anemia as the predominant clinical manifestation. however, pvb has been associated with pancytopenia, hepatitis, myocarditis, and allograft rejection. we present a patient with acute lymphoblastic leukemia who developed bone pain and pancytopenia following hsct in the setting of pvb infection. to describe an unusual presentation of pvb in a patient with acute lymphoblastic leukemia following hsct. design/method: a search of the english-language medical literature was performed using pubmed and medline databases. a review of the patient's medical history was performed. a year old male with relapsed b-cell all and history of "fifth disease" in infancy presented four months after hsct with focal left arm pain and difficulties fully extending the arm. bone mri showed enhancement of the medullary space centered within incomplete transverse cortical fracture interpreted as pathologic fracture due to neoplastic involvement of the ulna with no history of inciting injury. subsequently, peripheral blood counts decreased from low normal values to wbc . k/microl, anc /microl, plt k/microl, and hemoglobin . g/dl. the patient's chimerism remained % donor. a bone marrow biopsy and aspirate were performed to assess for recurrent leukemia given persistence of bone pain and developing pancytopenia. marrow findings included morphologic cytopathic effects with erythroid precursors and strong parvovirus staining with no signs of red cell aplasia or recurrent b-cell disease by morphology or flow cytometry. pvb was detected in blood by pcr and immunoglobulins with resolution of cytopenia and bone pain. this case highlights an unusual constellation of symptoms following hsct in a child with all. unexplained bone pain and medullary infiltrates with pancytopenia suggestive of recurrent leukemia were likely triggered by pvb infection. the question remains if he had reactivation of pvb , a primary infection by a new strain, or the virus was aquired through stem cells. bone biopsy could not be justified in light of clinical improvement. so far, bone lesions have only been described with congenital pvb infection. pvb appears to be uncommon after hsct, with a review of literature yielding pediatric cases. however, it may be underestimated due to lack of routine screening. our patient's presentation supports that evaluating for pvb may be warranted in hsct patients presenting with symptoms suggestive of relapsed leukemia. background: cardiac injury may occur during hematopoietic stem cell transplant (hsct) in pediatric patients and can be asymptomatic for many years. recommendations for screening are available for patients who received anthracyclines or chest irradiation, but no guidelines exist for unexposed longterm survivors. we sought to define the prevalence of echocardiographic abnormalities in long-term survivors of pediatric hsct and determine the need for screening in asymptomatic patients. design/method: we analyzed echocardiograms performed on long-term survivors (≥ five years) who underwent hsct at cincinnati children's hospital between and . we analyzed echocardiograms for left ventricular ejection fraction (ef), end-diastolic dimension (lvedd), septal thickness, posterior wall thickness, and global longitudinal strain (gls). we normalized linear measurements for age and patient body surface area. we included for further analysis patients who had echocardiogram obtained for routine surveillance. results: a total of patients underwent hsct and were alive more than years after transplant in , with having an echocardiogram obtained ≥ five years postinfusion. those with an echocardiogram were transplanted more recently (median vs. ). however, no difference between screened and unscreened individuals was noted for age at transplant, sex, transplant indication, anthracycline exposure, chest irradiation, or cyclophosphamide based preparative regimen. indications for echocardiograms included: cardiac symptoms ( . %), congenital cardiac anomalies ( . %), hypertension ( . %), known cardiac or pulmonary disease ( . %), routine post-hsct surveillance ( . %), and unknown ( . %). the mean time post-hsct was . years. among routine surveillance echocardiograms, the mean ef z-score was - . . mean lvedd zscore was - . , mean septal thickness z-score - . , mean posterior wall thickness z-score - . , and mean gls - . %. for patients that had echocardiogram performed for routine surveillance, / patients ( . %) had ef measured, and / ( . %) had ef z-scores ≤ - . (abnormally low). patients exposed to anthracyclines had a mean z-score ef of - . vs. unexposed patients - . (p = . ). among individuals who received neither anthracyclines nor tbi only / ( . %) was found to have an abnormal ef, . % (z-score - . ) or gls (- . %). only one patient who had a normal ejection fraction (z-score - . , ef . %) had an abnormal gls, - . % (normal ≤ - . ). long-term survivors of pediatric hsct who are asymptomatic and did not receive radiation or anthracyclines likely do not require surveillance echocardiograms, unless indicated by clinical symptoms. patients exposed to anthracyclines or tbi require close echocardiographic s of s screening and clinical monitoring for the development of cardiac complications. duke children's hospital, durham, north carolina, united states background: children undergoing pediatric blood and marrow transplants (pbmt) experience significant symptom distress. mobile health (mhealth) technologies can be leveraged to collect and monitor patient generated health data, and subsequently enhance our understanding of pbmt symptom clusters, patterns, and trajectories. better understanding of symptom complexity can foster development of precision health strategies to improve patient outcomes. however, limited research exists in integrating mhealth technology into pbmt management. we aimed to explore the feasibility, acceptability, and usability of using a pbmt specific mobile application to collect and monitor symptoms and wearable technology (apple watch) to measure objective data such as heart rate (hr) and activity. design/method: an exploratory mixed method design began in october to monitor pbmt symptoms for patients using real-time data from: ) a self-developed mhealth application (app) to collect subjective symptom data; and ) apple watch to collect physiologic measures such as heart rate and number of daily steps. data is collected pre-transplant through days. acceptability will be assessed through satisfaction surveys at study completion. we have enrolled patients to date who are all currently using the app and watch. patients' average frequency of daily charting in the app %. the wearable average daily recorded measurements are for hr and for step count. most common symptoms recorded within the app include fatigue and pain. we have noted trends in data including a decrease in activity following transplant and gvhd and an increase following engraftment. patients have stated "the app is helpful to keep track of how my pain is doing day to day" and "i try to take more steps each day than the day before". patients often remove the watch for charging, then forget to put it back on, but consistently put it on upon reminder. finally, parents often were required to make app entries with patients too sick to record. we continue to enroll patients with enthusiasm from both patients and parents to use mhealth during pbmt. preliminary findings suggest feasibility of using the mhealth devices is strongly correlated to the patient's post-transplant stage and is facilitated by caregiver participation with device management (charging devices, reminders to wear watch and record in app). patients reported satisfaction and ease of use with devices, but found it difficult to keep up with charging and charting. these findings indicate using mobile devices may be useful methods to collect patient generated health data. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bacterial bloodstream infections (bsi) are a common complication following hematopoietic stem cell transplantation (hsct) in both pediatric and adult populations, and are associated with poor outcomes. there is limited data describing the outcomes and characteristics of patients who develop three or more bsi after hsct. objectives: to describe the characteristics and outcomes of pediatric patients who develop three or more blood stream infections in the first-year post hsct. design/method: we performed a retrospective chart review of consecutive patients who underwent hsct at our institution from through to compile this case series. data were collected through the first year post-hsct including: patient demographics, underlying disease and therapy characteristics; and transplant complications such as thrombotic microangiopathy (tma), graft versus host disease (gvhd) and overall survival. bsis were classified according to current center of disease control guidelines. results: of patients, ( %) developed or more bsi in the first-year post transplant (total bsi cases = including all patients). of the cases, the majority underwent allogeneic hsct (n = / ; %). most cases were from unrelated donor (n = / , %). more than half of patients had grade - gvhd (n = / , %). sixteen ( %) had tma. of these cases, tma preceded the first bsi in n = / ( %). the majority of bsis were classified as central line-associated bloodstream infections (clabsis, n = / , %), followed by mucosal barrier injury laboratory-confirmed bloodstream infections (n = / , %) and secondary bsi (n = / , %). the majority of isolated organisms ( %) were associated with mucosal barrier injury pathogens. one-year overall survival in the cohort was % (n = / ). pediatric patients undergoing hsct who develop or more bsis in the first-year post transplant demonstrated an increased rate of tma compared to the overall institutional incidence of roughly %. tma diagnosis preceded the first bsi in over half of patients, suggesting that tma may predispose to recurrent bsi. improved strategies for early detection and treatment of tma as well as prevention of clabsis may help reduce the number of bsis ultimately leading to decreased morbidity and mortality in this patient population. background: in neutropenic pediatric patients, infection remains a significant cause of morbidity and mortality. while granulocyte transfusions have been utilized for decades to treat infections, including in the pediatric population, the efficacy of this intervention remains poorly described. previous guidelines have primarily utilized information from adult populations. furthermore, recruitment of donors typically involves friends or relatives of the patient with periodic involvement of community donors. the use of a readily available local donor population to improve availability has yet to be well described. as the immunocompromised population is particularly susceptible to worsening infection and clinical deterioration, the ability to rapidly harvest and deliver granulocytes warrants further investigation. to investigate the efficacy, safety, and outcomes of severely immunocompromised patients receiving granulocyte transfusions from a local altruistic granulocyte program in a pediatric tertiary care center. design/method: a retrospective review was performed to evaluate the context for receiving a transfusion as well as primary outcomes including infection clearance, survival to discharge, and overall mortality. the indiana blood bank assisted with timing the interval from initial order placement to onset of first granulocyte infusion. results: among the patient population reviewed, patients received separate granulocyte regimens. ages ranged from - years with a mean neutrophil count of at time of first transfusion. indications for transfusions included bacteremia (n = ), fungal pneumonia (n = ), and fungemia (n = ). primary outcomes included clearing infection ( %) and surviving to discharge ( %). the median time from initial order placement to infusion was hours, although there was no significant difference between responders who cleared the infection and non-responders who did not. however, additional investigation found that ward patients had a % chance of surviving to discharge while patients in the icu at time of initial transfusion had a % chance of survival to discharge. the readily available granulocyte transfusion program allows patients to quickly receive therapy in neutropenic settings. this is beneficial for patients as transfusion prior to clinical decompensation correlates with increased likelihood of infection clearance, and subsequently improved mortality. further investigation is needed, likely as a prospective study, to better explore circumstances that are beneficial for granulocyte transfusions. background: donor lymphocyte infusions (dli) are composed of immune cells to treat relapse after hematopoietic cell transplantation (hct). to date, data regarding its efficacy is limited in pediatric populations. furthermore, while outcomes related to cd content have been characterized, to our knowledge, the relationship between outcomes and other cellular content in dli has never been reported. objectives: determine whether the primary hematological malignancy, presence/absence of graft-versus-host disease s of s (gvhd), and unique phenotypic content of each dli impact overall survival (os) in pediatric patients with hematological malignancies. design/method: irb-approved, retrospective study investigating all consecutive dlis given to patients at the children's hospital of wisconsin. analyses were conducted using mann-whitney, fisher's exact, and chi-square. from from - patients ≤ years old with hematologic malignancies [myeloid (aml/ mds/cml/jmml),n = ; lymphoid (all),n = ] underwent dlis ( %% ≥ dlis). the median time between hct and dli was . (range, . - . ) years. there were significant differences between the lymphoid and myeloid groups, respectively, in regard to median age at hct ( . vs . yrs, p = . ) and at first dli ( vs years, p = . ). ultimately, there were no statistically significant differences in gvhd or os in products with either higher or lower cd , cd , cd , cd , or cd cellular content. however, the median cd /kg content was more than double in the patients who developed gvhd as compared to patients who exhibited no gvhd after dli ( . × vs . × , p = . ). patients receiving one dli had a -year os of ± % vs those receiving + dli of ± % (p = . ). with a median follow-up of . (range, . - . ) years, the year estimated os of patients in the lymphoid group was higher at ± % vs ± % in the myeloid group, although not significant (p = . ). our results indicate a survival benefit when using dli in a subset of patients who relapse after hct. unlike adult studies demonstrating little effect of dli in lymphoid diseases, many children with all achieved durable remission. while our analysis did not demonstrate that dli cellular content had a statistically significant effect on gvhd or os, it is possible that differences could be found if a larger population and more targeted cell doses were studied. more data will be needed to further define these relationships and identify patients who stand to benefit most. cincinnati children's hospital medical center, cincinnati, ohio, united states background: many arabic speaking muslim parents of children requiring bone marrow transplantation (bmt) receive medical care in the united states. providers may not understand the impact of islamic parents' religious beliefs and practices on their health care experience. objectives: to explore how islamic parents used religion in decision making and to understand the impact of their religious beliefs and practices on their overall health care experience. design/method: we used grounded theory, an inductive method gathering data from interviews and analyzing text, to identify core themes. ten caregivers of bmt children from middle eastern countries were interviewed by an arabicspeaking provider; interviews were coded by an interdisciplinary team. we identified key themes: . patience is a core belief in islam. patience results from the acceptance of allah's will. behaviors showing patience include praying rather than questioning and crying. . al qur'an provides comfort, healing, and protection. families listen to recitations of al qur'an in the patient's room because they feel that this practice not only comforts them but promotes healing as well. for some, certain portions of the qur'an were especially meaningful such as surat al-baqara, which explains that while we may think something is bad for us, allah will know it is good for us. . religious care in the medical center helped families feel respected. religious care in the medical center included interactions with chaplains, who were understood to be "religion experts," and provision of space for prayer and religious resources. . seeking religious consultation. religious consultation from imams or religious scholars (muftis or sheikhs) provides interpretations of the qur'an applied to the family's specific situation helps families make difficult decisions and follow allah's plan. . muslim beliefs guided decision making; muslim practices brought comfort, strength, and peace. drawn from the parents' understanding of islam. parents who addressed this topic said they would only do what islam allowed. they did indicate that most aspects of healthcare were understood to be allowed within islam. additionally, muslim practices of prayer, reading/listening to qur'an, and giving alms all provided comfort, strength and peace. we identified several recurring themes through our interviews that allowed us to understand how families use their muslim faith to deal with their children's illnesses and how it influences their decision making. we believe this better understanding will allow for more informed conversations about patients' health care and decision making, and shows respect for religious beliefs and practices. nemours/dupont hospital for children, wilmington, delaware, united states background: virtually all children will be infected with human herpesvirus (hhv- ) by the age of two. hhv- reactivation after stem cell transplantation causes multiorgan toxicities, including encephalitis, with inflammation and destruction of the temporal lobes and hippocampi, memory loss, and seizures. catatonia is characterized by posturing, immobility, mutism, and autonomic instability, and it's associated with various psychiatric and medical conditions. we describe a patient with hhv- encephalitis and unusual neurologic sequelae, including cognitive and neurobehavioral dysfunction and catatonia, which may impact our understanding of the pathophysiology of hhv- reactivation encephalitis. objectives: describe a case of hhv encephalitis with practice implications for stem cell transplantation. results: our patient was diagnosed with acute myeloid leukemia at age . within years, he relapsed and received two stem cell transplants. on the th day after his second transplant, he developed hyponatremia and refractory seizures. brain mri showed edema in the medial right temporal lobe with linear ischemic change. eeg showed diffuse encephalopathy. cerebrospinal fluid (csf) demonstrated white blood cells, red blood cells, and hhv- by pcr. his prophylactic antiviral was switched to foscarnet and ganciclovir. repeat mri showed abnormal signals in bilateral medial temporal lobes and the right insula. three months later he developed episodes of diaphoresis, hypothermia, agitation, mutism, and unusual posturing, recurring almost daily, recognized as catatonia. mri showed improvement of the abnormalities in the bilateral medial temporal lobes and hippocampi. eegs showed diffuse slowing. after months of antiviral therapy, csf was negative for hhv- . over the ensuing years, he had numerous episodes of diaphoresis, hypertension, hypothermia, pruritis, confusion, agitation, cogwheel rigidity, and bizarre posturing. dopamine blocking agents did not help. clonazepam helped reduce their frequency, and hot showers helped break acute episodes. further mris showed generalized cortical volume loss. he suffered from depression and severely impaired sleep and cognitive function. we describe a novel, debilitating outcome of hhv encephalitis which may provide diagnostic considerations as we continue to improve our understanding of the breadth of possible neurologic sequelae in transplant patients. hhv- is understood to infect and destroy the temporal lobes and hippocampi, but our patient's autonomic dysfunction indicate involvement of the hypothalamus and basal ganglia. antidopaminergic agents may worsen catatonia, and they were not effective for our patient. treatment of catatonia includes benzodiazepines; electroconvulsive therapy was not attempted in this case but may also be useful. background: epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld) is a lifethreatening complication in patients following hematopoietic stem cell transplantation, with a frequency estimated at . % and a cumulative incidence of mortality estimated as high as %. studies of ebv have hypothesized that the tonsils are critical for propagating this infection, as tonsillar epithelial cells have been shown to be the site of primary viral infection and continued viral shedding; however, to date no studies have been performed assessing the role of tonsillectomy in patients with ebv ptld. objectives: identify patients with localized ebv ptld treated with tonsillectomy to identify prognostic factors that may be able to help guide future treatment decisions. design/method: patients treated at memorial sloan kettering cancer center who had received hematopoietic stem cell transplantation and had billing codes for both ebv and tonsillectomy were eligible for inclusion in this study. a retrospective chart review was performed, assessing patient demographics, transplant characteristics, laboratory values, tonsillar pathology, and clinical course. any patient who did not have unilateral or bilateral tonsillectomy performed or who had non-localized disease (defined as disease involvement outside of the oropharynx and neck) was subsequently immunodeficiency; % (n = / ) fanconi anemia (fa); % (n = / ) hemoglobinopathy; % (n = / ) non-fa marrow failure and % (n = / ) a metabolic disorder. seventy one percent (n = / ) had normal amh for age pre-transplant, % (n = / ) had low amh for age pre-transplant; of these, % (n = / ) had an oncologic diagnosis; % (n = / ) had fa; % (n = / ) had previously treated hlh; % (n = / ) had non-fa marrow failure; one had a metabolic disorder and one a hemoglobinopathy. of the patients with post-transplant amh measurement % (n = / ) had low levels. of the patients with previously normal pre-transplant amh % (n = / ) underwent myeloablative conditioning (mac) regimen with a % (n = / ) having low amh levels post-transplant compared to %(n = / ) who underwent reduced intensity conditioning (ric) regimen with % (n = / ) having low amh levels post-transplant (p . ). fifteen percent (n = / ) had low levels pre-transplant and underwent mac regimen with % (n = / ) remaining low; % of these patients (n = / ) had fa. nine percent (n = / ) had low levels and underwent a ric regimen with % (n = / ) of amh levels remaining low; % (n = / ) of these patients had hlh treated prior to transplant. conclusion: amh levels can be used for detection of premature ovarian failure and fertility counseling. there is a higher risk of premature ovarian failure with mac regimens and prior chemotherapy vs ric regimens. follow up of this cohort will provide more information to understand the effects of hsct in ovarian function and the usefulness of amh as a predictor of fertility potential. background: there are no proven strategies to prevent blood stream infections (bsi) secondary to oral mucosal barrier injury after hematopoietic stem cell transplant (hsct). additionally, we recently reported progressive gingivitis and dental plaque accumulation in hsct recipients despite our current oral standard of care (three times daily oral rinse). xylitol is a non-fermentable sugar alcohol that reduces dental caries, plaque accumulation, and oral disease progression by inhibiting bacterial growth. we hypothesized that the addition of xylitol to standard oral care will decrease dental plaque accumulation, gingivitis and bacteremia from oral flora. objectives: identify a clinically effective strategy to improve oral health and prevent bsi secondary to bacterial translocation through the oral mucosa in patients undergoing hsct. we are conducting a prospective randomized control study to test our hypothesis. those in the intervention arm receive our current standard of care (three times daily oral rinse) in addition to daily xylitol wipes; controls receive oral standard of care alone. oral exams are performed at baseline and weekly for the first days post hsct. metagenomic shotgun sequencing (mss) of gingival samples is performed at all time points to evaluate microbiome diversity and pathogenic bacterial load. finally, we performed whole genome sequencing of pathogenic bacterial isolates causing bacteremia to assess for genetic relatedness to corresponding strains present within the patient's oral microbiome preceding the infection. : preliminary interim analysis of patients demonstrates improved oral health in patients receiving xylitol (n = ) over those receiving standard of care (n = ), measured by the oral hygiene index (p = . ) and gingivitis index (p = . ). in the nine patients having complete oral mss analysis, xylitol appeared to be associated with decreased streptococcus mitis/oralis domination in the oral microbiome. finally, patients receiving xylitol had no incidence of streptococcus mitis/oralis bacteremia through the first days compared to three patients ( %) in standard of care arm. interestingly, streptococcus mitis/oralis comprised % of the oral microbiome in one child who subsequently developed a streptococcus mitis/oralis bsi. we expect to complete this study in the next months (n = ). the addition of xylitol to oral standard care appears to decrease dental plaque and gingivitis in patients undergoing hsct. xylitol may also impede streptococcus mitis/oralis dominance in the oral microbiome with potential reduction in blood stream infections. (range: - days). twenty-one mdli ( %) were administered because of lymphopenia, fourteen of them ( %) in patients with concomitant viral/opportunistic infections. mixed chimerism/graft failure was the motive of % of the mdli (n = ) and six ( %) were administered to accelerate immune reconstitution. all infusions were well tolerated without appearance or worsening of gvhd. an increase in t-cell counts was observed following six mdli ( . %), although it was a transitory response ( - weeks) in five cases. viral/opportunistic infections were controlled in five cases ( . %), requiring a median of mdli to achieve this response. none of the mdli administered in cases of mixed chimerism/graft failure were effective in reverting this situation. our preliminary data suggests that mdli, is a safe adoptive immunotherapy strategy even with high dose of t-cells without infusion side effects or gvhd complications. some efficacy has been observed in patients with lymphopenia and opportunistic infections, with no positive results in patients with mixed chimerism/graft failure, up to date. however, to determine the real efficacy of this strategy, prospective studies are required. jun zhao, kristen beebe, lucia mirea, alexandra walsh, shane lipskind, alexander, ngwube phoenix children's hospital, phoenix, arizona, united states background: male adolescents undergoing myeloablative hematopoietic stem cell transplantation (hsct) develop infertility with impaired spermatogenesis with reported rates ranging from % to %. in nonmalignant diseases, myeloablative regimens have been replaced with reduced intensity conditioning (ric) with the hopes of better survival rate, less organ toxicity and improved quality of life. despite the increased use of ric regimens for hsct, the effects of ric on fertility remain unknown. objectives: to assess fertility following ric hsct in young adult males. we assessed gonadal function and semen characteristics in adolescent males (> years) who received a single ric hsct at phoenix children's hospital for nonmalignant diseases during - . male patients who were a minimum of year from ric hsct and had postpubertal development at tanner stage iii or above were eligible for this study. gonadal status was assessed by measuring fsh, lh, testosterone, and inhibin b levels, and semen anal-yses assessed fertility indicators (semen volume, sperm concentration, motility, viability, forward progression, morphology, and total count). results: hormone levels and semen analysis have been obtained for patients thus far. the median time between transplant and semen analysis was years. post hsct, ( %) patients showed abnormally elevated lh levels, but fsh, testosterone (total and free), and inhibin b levels were within normal range for all patients. sperm morphology and viability testing were not able to be performed due to low concentrations and volumes. as a result, the total motile sperm count, the most useful estimate for fertile potential, is essentially for all patients. conclusion: recruitment is ongoing, but so far our limited results suggest that ric hsct may have detrimental longterm effects on male fertility. a multi-institutional trial may be appropriate due to small patient numbers at each institution. we are currently exploring options to expand to other centers. further consideration is warranted regarding decisions made by providers, ways to improve anticipatory counseling provided to patients and their families prior to transplant, and how to augment the preventive care of these patients in longterm follow-up. currently all male patients being considered for ric transplant should be counseled to sperm bank prior to transplant. background: a previous systematic literature review identified all published studies of defibrotide treatment for patients of all ages with vod/sos. to assess day+ survival for defibrotidetreated pediatric patients (≤ or ≤ years, per study) all patients exhibited infectious complications with at least viral infection. four patients also had bacterial infections. of note, no patient developed evidence of fungal infections. conclusion: early institution of ecp in patients with high risk acute gvhd (grade - ) was very effective at treating agvhd, allowed for an aggressive steroid taper and contributed to excellent overall survival rates ( %). infectious complications were primarily viral and bacterial, with no fungal infections in this very high risk population. background: vod/sos is a life-threatening complication of hsct conditioning. vod/sos with multi-organ dysfunction (mod) may be associated with > % mortality. defibrotide is approved to treat hepatic vod/sos with renal/pulmonary dysfunction post-hsct in the us and severe hepatic vod/sos post-hsct patients aged > month in the eu. there are few published data on survival of neuroblastoma patients with vod/sos post-hsct. objectives: to report day+ survival and safety post hoc for patients with neuroblastoma and vod/sos post-hsct in the defibrotide t-ind trial. design/method: vod/sos was diagnosed by baltimore or modified seattle criteria or biopsy, with/without mod, after hsct or chemotherapy. defibrotide treatment ( mg/kg/day) was recommended for ≥ days. this post hoc analysis is based on adult and pediatric patients receiving ≥ dose of defibrotide, including with mod. results: among patients with neuroblastoma, developed vod/sos after hsct. for these post-hsct patients, . % were male and . % were female, median age was years (range - years): . % aged - months, . % - years, . % - years, and patient > years. day+ survival data were available for / of these neuroblastoma patients ( with mod and without mod); had autologous and had allogeneic transplants. kaplan-meier estimated day+ survival for the neuroblastoma group was . % ( % confidence interval [ci] , . %- . %). for the mod and no mod subgroups, kaplan-meier estimated day+ survival was . % ( % ci, . %- . %) and . % ( % ci, . %- . %), respectively. in the overall t-ind hsct population aged ≤ years (n = ) and pediatric autologous hsct subgroup (n = ), kaplan-meier estimated day+ survival was . % and . %, respectively. treatment emergent adverse events (teaes) occurred in . % (n = / ), with serious teaes in . % ( / ; most common: multi-organ failure, . % [ / ]). teaes lead to treatment discontinuation in . % (n = ; most common: pulmonary hemorrhage, n = ); death occurred in . % (n = ; > %: multi-organ failure, . %; vod/sos, . %). treatment-related adverse events, as assessed by investigators, occurred in . % (n = ; most common: pulmonary hemorrhage, . %). this post hoc analysis found kaplan-meier estimated day+ survival of . % in patients with neuroblastoma and vod/sos post-hsct, which was consistent with outcomes in pediatric patients after autologous hsct. the safety profile of defibrotide in neuroblastoma patients was consistent with the overall hsct population in this study and other defibrotide studies in pediatric patients. cincinnati children's hospital medical center, cincinnati, ohio, united states background: blood stream infections occur in nearly % of patients undergoing hematopoietic stem cell transplant (hsct) and fever is often the first symptom. timely administration of antibiotics is associated with improved outcomes, thus, early recognition of fever is paramount. current standard of care (soc) includes episodic monitoring of temperature in hospitalized patients, which may delay fever detection. therefore, continuous real-time body temperature measurement may detect fever prior to the current soc. temptraq is a food and drug administration cleared class ii medical device and consists of a soft, comfortable, disposable patch that results: of patients, were started on a pca in the days post hct. % were male with median age of y. % had all, and % aml. matched related donors were used in % and % received tbi. pca was initiated median d+ . oral mucositis alone was the most common indication ( %). a majority of patients were started on hydromorphone ( %); % started on morphine and % started on fentanyl. % started on continuous infusion. pca was used for a median of days (range - days). median pain score was highest d+ of pca use, however, there was inconsistency in charting of numerical pain scores. on d+ , patients had insufficient data to determine efficacy of pain control; of the remaining patients, % had good pain control while % had moderate and % had poor pain control using our devised scale. the most common toxicity observed was respiratory depression (∼ %), however, etiology was often multifactorial and not due to opiates alone. analysis is ongoing to assess variables predicting pca use as well as efficacy of pain control and correlation between current reporting scales and patient perception. conclusion: pca use is common in pediatric hct yet pain control remains inadequate. there's a need for better evaluation of pca management, especially uniform assessment of pain, thereby improving quality of life post hct. children's national health system, washington, district of columbia, united states background: actinomycosis is a rare invasive anaerobic gram-positive bacterial disease caused by actinomyces spp. that may colonize the oropathynx, gastrointestinal tract and urogenitial tract and can lead to abscesses. respiratory tract actinomycosis is characterized by pulmonary cavities, nodules, consolidations and pleural effusions. although actinomyces are nearly always sensitive to penicillin they are frequently resistant to cephalosporins and variable sensitives to fluoroquinolones. although rare in children, immunosuppressed patients are at increased risk for actinomycosis. to describe a case of next-generation sequencing identification of actinomycosis. a -year-old male with a history of very high risk b-cell acute lymphoblastic leukemia who was months status post a / matched unrelated donor bone marrow transplant complicated by prolonged fevers, persistent weight loss, and splenic lesions, treated with posaconazole and levofloxacin developed fever and cough in the setting of neutropenia. blood cultures demonstrated staphylococcus epidermidis. ct showed micronodules and effusion not consistent with s. epi, prompting bronchoscopy. all bacterial cultures were negative. patient was prescribed a three-week course of vancomycin with rapid improvement. design/method: s next generation sequencing (ngs) from bronchoalveolar levage sample was performed at the university of washington laboratory results: ngs assay from bronchoalveolar lavage showed major abundance of actinomyces most closely related to meyeri or oodontolyticus. demonstrated actinomyces. the patient was started on a six month course of amoxicillin with continued clinical improvement. in retrospect, the splenic nodules that were presumed fungal disease were likely actinomycosis, partially treated with levofloxacin. this case highlights the potential utility of ngs in the diagnosis of rare diseases in immunocompromised patients. actinomycosis was only demonstrated through ngs and led to a change in treatment regimen and durable clinical improvement. because actinomyces often mimics malignancy, tuberculosis or nocardiosis, the use of this novel test both targeted appropriate therapy and reduced the exposure to unnecessary medications to treat the differential diagnosis. finally, we highlight that actinomyces should be considered in patients who present with unexplained fevers, weight loss, and night sweats. haneen shalabi, cynthia delbrook, maryalice stetler-stevenson, constance yuan, bonnie yates, terry j. fry, nirali n. shah center for cancer research, national cancer institute, national institute of health, bethesda, maryland, united states background: car-t therapy, while effective, may not be durable for all, and antigen negative escape is a growing problem. hct, in relapsed/refractory all, can be curative, particularly for those in an mrd negative remission. we demonstrated that cd directed car-t therapy effectively rendered patients into mrd negative remissions (by flow cytometry) and the leukemia free survival post-hct was high . in pastorek, jesssica bruce, michael a. pulsipher, chloe anthias, peter bader, andre willasch, jennifer sees, jennifer hoag, wendy pelletier, brent logan, pintip chitphakdithai, lori wiener university of pittsburgh, pittsburgh, pennsylvania, united states background: more than , pediatric hscts are performed in north american and europe each year. the ethics of exposing a healthy child to donation procedures which have some risks and no direct medical benefits continue to be a topic of debate. pediatric donors may experience psychological distress and poorer quality-of-life during and after donation compared to healthy controls. although there are fact/jacie requirements related to the management of pediatric donors, it is unclear what standardized practices exist for psychosocial assessment/management of this group. objectives: to describe transplant center practices for psychosocial evaluation/ management of pediatric donors (< years) and to examine differences in practices by location (cibmtr/ebmt) and number of harvests (volume). design/method: data were collected via a single crosssectional survey distributed electronically to cibmtr and ebmt centers between / / and / / . : / ( %) of cibmtr and / ( %) of ebmt centers completed the survey. most centers had written eligibility guidelines for pediatric donors ( %). most also had a process for ensuring that donors were freely assenting to donate ( %), managed by a transplant physician ( %). a single physician often jointly managed donor/recipient care ( %). half of centers had a pediatric donor advocate ( %), who was most often a physician ( %) or social worker ( %). cost was the largest barrier to having a donor advocate ( %). most centers performed psychosocial screening of donors ( %) but rarely declined donors based on psychosocial concerns ( %). less than half of centers provided post-donation psychosocial follow-up ( %). comparisons by center location indicated that ebmt centers were more likely to have a physician doing joint donor/recipient care ( % vs. %; p = . ), less likely to have a psychosocial assessment policy ( % vs. %; p = . ), less likely to have a donor advocate ( % vs. %; p = . ), but marginally more likely to do post-donation psychosocial follow-up ( % vs. %; p = . ). large volume centers were more likely to have a psychosocial assessment policy than their medium/smaller counterparts ( % vs. %, %; p = . ) â€"there were no other differences on key psychosocial management variables by volume. although most centers have written guidelines for pediatric donor eligibility and mechanisms for ensuring assent, substantial numbers of donors do not undergo psychosocial assessment, are jointly managed with the recipient by a single physician without an assigned donor advocate, and do not receive psychosocial follow-up. the field would benefit from guideline development for the psychosocial management of pediatric donors. background: germline mutations in samd and samd l genes cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy) and ataxia-pancytopenia syndromes, respectively, and are associated with chromosome deletions, mds and bone marrow failure (bmf). there are limited data on outcomes of hct in these patients. to describe outcomes of allogeneic hct in patients with hematologic disorders associated with samd /samd l mutations. results: seven patients underwent allogeneic hct for primary mds (n = ), congenital amegakaryocytic thrombocytopenia (camt)(n = ), and dyskeratosis congenita (n = ). retrospective exome sequencing revealed gain-of-function mutations in samd (n = ) or samd l (n = ) genes. constitutional mosaic monosomy was present in cases. two samd patients had features of mirage syndrome. unusual findings of panhypopituitarism, laryngeal cleft, and glomerulosclerosis were noted in one case. in another case with a samd mutation hypospadias & bifid scrotum were the only findings. the remaining patients had no phenotypic abnormalities. median age at hct was y (range: . - . ). patients received transplants from bone marrow (matched unrelated (n = ) & hla identical sibling (n = )), or unrelated cord blood (ucb) (n = ). five mds patients received myeloablative s of s conditioning (busulfan-based (n = ) or tbi-based (n = )); patients (mds (n = ); camt (n = )) received reducedintensity conditioning (ric) (fludarabine, cyclophosphamide, with ratg or alemtuzumab). syndrome-related comorbidities (diarrhea, infections, malnutrition, electrolyte imbalance, lung disease and hypoxia) were present in both patients with mirage syndrome. one patient with a familial samd l mutation, mds and morbid obesity failed to engraft following ric double ucbt. she died one year later from refractory aml. all other patients achieved neutrophil and platelet engraftment, at a median (range) of ( - ) and ( - ) days, respectively. posttransplant complications included severe hypertension (n = ), pericardial effusions (n = ), veno-occlusive disease of liver (n = ), and recurrent aspiration pneumonias (n = ). one patient developed grade iii agvhd which resolved with treatment. one patient developed mild skin cgvhd and suffers from chronic lung disease. all surviving patients had resolution of hematological disorder and sustained peripheral blood donor chimerism ( - %). overall survival was % with a median follow-up of years (range: . - . y). patients with hematological disorders associated with germline samd /samd l mutations tolerated transplant conditioning without unusual, or unexpectedly severe toxicities. allogeneic hct led to successful resolution of mds or bmf, with excellent overall survival. more data is needed to refine transplant approaches in samd /samd l patients with significant comorbidities, and develop guidelines for their long-term follow-up. shyamli singla, tiffany simms-waldrip, andrew y. koh, victor m. aquino background: steroid-refractory acute graft versus host disease (agvhd) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation (hsct). basiliximab (anti-il -r monoclonal antibody) as a single agent or in combination infliximab (anti-tnf-monoclonal antibody) has demonstrated efficacy in adult cohorts with steroid-refractory agvhd, but has not been well studied in the pediatric population. we adopted the use of basiliximab and infliximab as our institutional standard of care for steroid-refractory agvhd in pediatric hsct patients. to determine the response and survival of hsct children who received basiliximab and infliximab for the treatment of steroid-refractory agvhd. design/method: we retrospectively reviewed children who received basiliximab and infliximab for steroid-refractory agvhd refractory between september and december . complete response (cr) was defined as resolution of all clinical signs of agvhd. partial response (pr) was defined as at least one grade reduction in one target organ (e.g. skin, gut or liver) without increased grade in another target organ. no response was defined as either no improvement or progressive worsening of agvhd in at least one organ. baseline demographics, transplant details, laboratory findings, and treatment outcomes were also evaluated. results: of the evaluable hsct patients, children (median age yrs, range mo- yrs) with steroid-refractory agvhd received combination monoclonal antibody (mab) therapy. the median time from the start of steroid therapy to initiation of mab was days. the overall glucksberg grade of agvhd at the time of initiating mab therapy was grade i (n = , . %) ii (n = ; %), iii (n = ; %) or iv (n = ; %). the overall response rate was %, with ( %) patients achieving cr, ( . %) patients achieving pr, and ( . %) patients with no response at days following the start of mab therapy. the median overall survival was , , and days for patients who exhibited cr, pr, and no response, respectively. the overall survival at year following start of mab therapy was %. background: the role of high dose chemotherapy (hdc) and autologous stem cell rescue (ascr) in patients with high risk (advanced metastatic or relapsed) soft tissue sarcomas is controversial. despite multimodal chemotherapy, radiotherapy, and local control measure advancements, prognosis of patients with advanced metastatic or unresectable and relapsed sarcomas remains poor, with less than % years disease free survival. objectives: to determine if consolidation with myeloablative hdc and ascr improves relapse free (rfs) and overall survival (os) outcomes in a high risk patient subgroup. we performed retrospective review of all high risk soft tissue sarcoma patients who underwent hdc and ascr at the children's hospital at montefiore, bronx, ny between october and january . the protocol was approved by albert einstein college of medicine institutional review board. results: patients ( primary metastatic high risk disease, relapsed or recurrent disease) received hdc with ascr. primary diagnoses were rhabdomyosarcoma (rms) (n = , alveolar histology), primary site nasopharynx (n = ) and lower extremity (n = ). ewing's sarcoma (ews) (n = ), axial site (pelvic) in patients ( %). median age years (range - years), ( %) were male. all patients were in complete metabolic remission before transplant. median pre transplant comorbidity index was (range - ). patients ( rms and ews) received conditioning with carboplatin, etoposide and melphalan. remaining patients with ews received conditioning with busulfan, melphalan and topotecan. all patients received peripheral blood mobilized hematopoietic stem cell transplantation. stem cell mobilization achieved with high dose filgrastim in all patients except one who required addition of plerixafor. median cd +/kg s of s recipient body weight cell dose infused was . × ^ (range . - . × ^ ). median times to neutrophil and platelet (> , / l) engraftment were (range - ) and ( - ) days respectively. patients ( %) developed bk viuria (one with grade iii hemorrhagic cystitis); ( %) developed cmv viremia; and one patient ( %) had asymptomatic ebv viremia. there was no graft failure, sinusoidal obstruction syndrome or transplant related mortality. median follow up post-transplant was days (range - days). year probability of os and rfs were % and % respectively. hdc with ascr is a promising therapeutic strategy to consolidate remission and improve survival in select high risk soft tissue sarcoma patient subgroups. prospective clinical trials will inform the impact of disease status prior to hdc and ascr on outcome, optimal conditioning and long term relapse free and overall survival. background: absence of minimal residual disease is paramount for cure of pediatric acute lymphoblastic leukemia (all). the testis may harbor occult leukemia and this disease may result in treatment failure. objectives: the purpose of this study was to assess the longterm outcomes of boys with or without testicular leukemia pre-hematopoietic stem cell transplantation (hsct). design/method: retrospective analysis of boys with high-risk de novo ( with hypodiploidy all) or recurrent/refractory all was conducted. flow cytometry of bone marrow mononuclear cells was used to determine remission status. testicular evaluations were performed by physical examination and wedge biopsy pre-hsct. the median age at time of transplant was . years. all patients were in remission by flow cytometry of bone marrow mononuclear cells at the time of transplant and none had evidence of clinically apparent testicular disease. testicular leukemia was detected in patient and he underwent bilateral orchiectomy. he developed acute graft versus host disease (gvhd) of the duodenum and sigmoid colon which resolved, and the leukemia remains in second complete remission and he is free of hsct-related morbidity . months post-hsct. of the patients without testicular leukemia died a median of . months (range, . to . ) post-hsct ( with adenovirus infection and each with thrombotic microangiopathy and aspergillus pneumonia); experienced infection (staphylococcus species, corynebacterium, enterococcus, klebsiella, citrobacter, e. coli, epstein barr virus, adenovirus, bk virus, human herpesvirus- , candida albicans, fusarium, aspergillus, yeast, and other fungus); experienced gvhd ( of the gi tract, of the skin, of the liver, of the eyes, of the mouth, and of the lungs); and developed a second neoplasia (right lower leg leiomyosarcoma). one patient developed bone marrow minimal residual disease ( . % phenotypically abnormal cells detected months after / matched sibling hsct). reinduction therapy comprised weekly doses of rituxan, courses of blinatumomab and donor lymphocyte infusions with il- . two subsequent bone marrow evaluations were minimal residual disease negative. thirteen months post-hsct residual disease recurred ( . %) and he will receive inotumumab. overall median survival post-transplant of the boys is . months (range, . to . ) and of the surviving boys is . months (range, to . ). conclusion: testicular biopsy can detect occult leukemia pre-hsct. testicular leukemia pre-hsct does not appear to increase the risk of subsequent relapse or other hsct-related adverse events compared to those without it. yaya chu, nang kham su, sarah alter, emily k. jeng, peter r. rhode, mathew barth, dean a. lee, hing c. wong, mitchell s. cairo new york medical college, valhalla, new york, united states background: rituximab has been widely used in frontline treatment of b-nhl including burkitt lymphoma (bl), however, some patients retreated with rituximab relapse, which limit patient treatment options. novel therapies are desperately needed for relapsed/refractory b-nhl patients. several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (car), as well as second-generation anti-cd antibodies. nature killer (nk) cells play important roles in the rejection of tumors. however, nk therapy is limited by small numbers of active nk cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of nk cell immunosurveillance. our group has successfully expanded functional and active peripheral blood nk cells (expbnk). b t m was generated by fusing alt- , an il- superagonist, to four single-chains of rituximab. b t m displayed tri-specific binding activity through its recognition of the cd molecule on tumor cells, activated nk cells to enhance adcc, and induced apoptosis of b-lymphoma cells. objectives: to examine if b t m significantly enhances the cytotoxicity of expbnk against rituximab-sensitive and -resistant bl cells. design/method: expbnks were expanded with lethally irradiated k -mbil - bbl and isolated using miltenyi nk cell isolation kit. alt- and b t m were generously provided by altor bioscience. nk receptors expression and cytotoxicity were examined as we previous described. ifng and granzyme b levels were examined by elisa assays. equal doses of rituximab, alt- , rituximab+alt- , obinutuzumab (obinu) were used for comparison. igg was used as controls. anti-cd car expbnk cells were generated as we previously described by mrna electroporation. rituximab-sensitive raji andresistant bl cells raji- r and raji- rh, were used as target cells. results: b t m significantly enhanced expbnk cytotoxicity against rituximab-sensitive raji cells, rituximab-resistant raji- r cells and resistant raji- rh cells compared to the controls igg, rituximab, alt- , rituximab+alt- , obinu (p< . , e:t = : ). furthermore, we confirmed the enhanced cytotoxicity by measuring ifn-g and granzyme b production. b t m significantly enhanced ifn-g and granzyme b production from expbnk against raji, raji- r and raji- rh compared to igg (p< . ), rituximab (p< . ), alt- (p< . ), rituximab+alt- (p< . ), and obinutuzumab (p< . ). when compared to anti-cd car expbnk cells, b t m + expbnk had the similar cytotoxicity against raji, raji- r and raji- rh as anti-cd car expbnk cells did (p> . ). conclusion: b t m significantly enhanced expbnk activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant bl cells. the in vivo functions of b t m with expbnk against rituximab-sensitive and -resistant bl cells using humanized nsg models are under investigation. background: cardiac dysfunction, including left ventricular systolic dysfunction (lvsd), is a known complication in stem cell transplant (sct) survivors. while detection of lvsd by echocardiography is important in this population, there has been minimal research to determine if subclinical cardiac dysfunction exists in sct patients. cardiopulmonary exercise testing (cpet) is a valuable tool to assess cardiac function, and to determine how the heart responds to the stress of exercise. no studies have been performed to determine if sct patients with normal lvsd on standard echocardiography may have abnormal cpet. to determine the feasibility of cpet, as well as additional echocardiographic parameters, to detect dysfunction in sct patients with a normal ejection fraction on echocardiogram. design/method: we performed a cross-sectional analysis of sct survivors who were at least years post sct, years of age or older and with an ejection fraction > % (low end of normal range) on echocardiogram. we assessed the exercise capacity of all patients with cpet, and sub-clinical cardiac dysfunction through tissue doppler and strain analysis from the echocardiogram. results: seven patients ( male) have qualified and completed this study so far with an average age of . ± . years. the median time from transplant is . ± . years. all seven patients had a normal ejection fraction, however four patients had abnormalities on their cpet. these abnormalities included abnormal predicted peak oxygen consumption (vo ) ( %± . , normal > %) (the best predictor of functional capacity), predicted oxygen pulse ( %± . , normal > %) (measure of cardiac stroke volume) and ventilatory efficiency (ve/vco slope) ( ± . , normal < ). submaximal exercise data, used when patients are unable to complete a maximal effort test, demonstrated low-normal predicted vo at anaerobic threshold ( . %± . %, normal > % of was . days while patients who received autologous infusions had a mean number of days to engraftment of . . engraftment after hsct needs to be prompt to minimize duration of neutropenia and maximize survival rates . our data demonstrates that the infusion of hematopoietic stem cell products with a syringe or iv pump is an effective method of delivery for stem cell products and does not delay the time to engraftment. the median days to neutrophil engraftment was . days. this is comparable to data from the nmdp, which reports engraftment occurs within - days. the main limitation to this study was its small sample size due to the number of transplants done at our center. however, it does provide evidence to support that infusion of stem cell products via pump mechanism is a safe alternative to the infusion by gravity method in the process of the hematopoietic stem cell administration. johns hopkins all children 's hospital, st. petersburg, florida, united states background: leukemic relapse remains the most common cause of treatment failure after allogeneic hematopoietic cell transplant (allohct) for myeloid malignancies. most children who relapse post-allohct will die of their disease, making interventions to minimize this risk a high priority. objectives: to evaluate the safety and efficacy of posttransplant azacitidine for relapse prevention in children undergoing allohct for myeloid malignancy. design/method: we retrospectively reviewed the charts of children undergoing allohct for myeloid malignancies between february and november at johns hopkins all children's hospital. results: during the study period, children (ages to years, median ) underwent allohct for myeloid malignancies: de novo acute myeloid leukemia (aml), ; mixed phenotype acute leukemia, ; treatment-related aml, ; juvenile myelomonocytic leukemia with aml transformation, ; and myelodysplasia/aml, . thirteen were in first complete remission, were in cr or greater. most patients ( / ) received fludarabine/melphalan/thiotepa conditioning; received hla-identical related or unrelated donors, and received haploidentical bone marrow grafts with post-transplant cyclophosphamide. three patients never received planned azacitidine ( early relapse; early trm), leaving evaluable patients. azacitidine ( mg/m /dose for days, in -day cycles for up to cycles) was started at a median of days post-transplant (range - ). two-thirds ( / ) of patients received eight or more cycles. of five patients who stopped therapy early, only one was due to toxicity; other reasons included severe gvhd ( ), parental preference ( ), and relapse ( ). cycle delays occurred in patients, with a median cycles delayed per patient, mostly for mild myelosuppression with early cycles. no patient required blood product transfusion during therapy, but g-csf was used in three patients to maintain anc> / l. dose-modifications were made in patients (renal tubular acidosis, acute kidney injury, and myelosuppression). there were relapses ( %), two of which occurred in patients in cr , for a relapse incidence of % in patients in cr , with a median follow-up of months (range . to ). no patients who received azacitidine died of transplant-related mortality. conclusion: administration of azacitidine in children undergoing allohct for myeloid malignancies is safe and feasible, with most patients successfully receiving all planned cycles. toxicity was acceptable and there was no trm or secondary graft failure. despite the limitations of a small cohort, relapse incidence-particularly in patients transplanted in cr suggests a potential benefit in disease control that warrants investigation in follow-up studies. background: despite significant improvements in the success rate of hematopoietic cell transplantation (hct), graft failure remains an important complication in patients transplanted for severe aplastic anemia (saa). second allogeneic hct can salvage patients, but -year overall survival (os) rates have been reported as low as % . objectives: identify patients who developed dropping donor chimerism, graft rejection, and/or graft failure after first hct for saa, necessitating additional hcts or cellular boosts (defined as stem cell products infused without preceding chemotherapy), and evaluate treatment-related complications and os. with vod/sos with and without multi-organ dysfunction (mod) pubmed and embase databases were searched for "defibrotide and retrospective chart reviews; excluded publication types were: case reports (< cases); meta-analyses; reviews; animal, modeling, pharmacokinetic, chromatography, and adult-only studies; guidelines; articles; and letters. resulting reports were screened for exclusion criteria. full-text articles were then reviewed for eligibility. study characteristics of selected publications were summarized, and publications were categorized by patients' mod status. when necessary, additional data tables were requested. a random effects model was used for pooling data for efficacy. interstudy heterogeneity was assessed with cochran's q-test. percentage of total variation across studies due to heterogeneity (i ) was evaluated we quantified ∼ proteins in each sample. reproducibility for one donor at different time points children 's minnesota, minneapolis, minnesota, united states background: pediatric and young adult hodgkin lymphoma (hl) has five-year survival rates > %. chemotherapy required to achieve this rate is associated with a lifetime risk of cardiac deaths, second malignancies, pulmonary disease and infertility. as effective salvage therapy exists, outcomes may be improved by de-intensifying initial therapy to lessen toxicity.objectives: we piloted a regimen in low and intermediate risk hl patients using agents without known association to significant late effects. this retrospective chart review was approved by children's minnesota irb.design/method: the bvg(p) regimen incorporated bortezomib ( . mg/m day , , , ); vinorelbine ( mg/m day , ); gemcitabine ( mg/m day , ) every days and prednisone ( mg/m /dose bid x days). we treated newly diagnosed patients, ages - years, with non-bulk stage iia (n = ) or iib (n = ) hl. two patients received bvg and received bvgp with the addition of prednisone.results: newly diagnosed patients were all pet negative after the first or second cycle and remained pet negative at end of therapy, cycles. nausea was well controlled with -ht antagonists and scopolamine. pegfilgrastim was not necessary due to the high absolute neutrophil count nadir [median . and minimum . × /l]. there were no episodes of febrile neutropenia, infection or transfusion need. no patients experienced alopecia. one patient developed sensory neuropathy after the eighth dose of bortezomib that was controlled with gabapentin and a switch to subcutaneous bortezomib administration. of the five newly diagnosed patients, four remain in remission at , , , days; relapsed at previous disease sites at days and subsequently achieved remission with bvgp with the addition of brentuximab. this series provides early evidence to stimulate expansion of this pilot experience and subsequent multiinstitutional study leading to a randomized trial of bvgp and current chemotherapy for low and intermediate hl. st jude affiliate clinic at st francis hospital, tulsa, oklahoma, united states background: symptoms suggestive of morning hypoglycemia has been noticed in children receiving all chemotherapy. only few small studies looked at this therapy related complication. factors increase risk of hypoglycemia in all patients include accelerated starvation, steroid induced adrenal suppression, mercaptopurine therapy and prolonged fasting for procedures.objectives: to study the prevalence and risk factors for hypoglycemia during all therapy design/method: medical records of of children (up to years old) treated for all between - ( patients) were studied for evidence of morning hypoglycemia defined as blood sugar (bs) < mg/dl. statistical mean differences between the subgroups were analyzed with spss using a nonparametric mann-whitney u test.results: fifty two percent ( %) of patients developed hypoglycemia during all treatment, with an average of . episodes/patient. % were males and % females. almost / ( %) of patients with hypoglycemia were in maintenance phase of therapy. % of hypoglycemic episodes occurred in % of patients. majority of hypoglycemic episodes ( . %) occurred on the day of procedure when patients were fasting overnight. . % of hypoglycemic episodes occurred in children ≤ years, with . % in ≤ years. patients who developed hypoglycemia were significantly younger (mean age at time of diagnosis of all was . ± . at the hypoglycemia group versus the non-hypoglycemia ( . ± . ) p< . . no statistically significant difference was found regarding sex, or tpmt genotype. % of hypoglycemic children-all < years of age-presented with life threatening hypoglycemia symptoms including seizure and loss of consciousness. this study showed high prevalence of hypoglycemia during childhood all therapy. younger age, especially ≤ years, is associated with higher risk of hypoglycemia as well as life-threatening episodes. to decrease fasting hypoglycemia during therapy for childhood all, we recommend that children under the age of years receive bed time snack high in proteins and complex carbohydrates, and to get them up early the day of procedure to take clear sugary drink. hospital for sick children, toronto, ontario, canada ann & robert h. lurie children's hospital of chicago, chicago, illinois, united states background: childhood brain tumors are the most common solid malignancy and the leading cause of cancer-related mortality in children. the most aggressive type of pediatric central nervous system (cns) tumors is diffuse intrinsic pontine glioma (dipg). despite decades of clinical trials, there has been no substantial improvement with respect to therapeutic outcomes with most children eventually succumbing to the disease. research on adult high-grade gliomas has shown a targetable pathway through the inflammationinduced expression of indoleamine , dioxygenase (ido ) and its recognized ability to suppress the anti-tumor immune response. a limited understanding into the role of ido in pediatric central nervous system tumors serves as the foundation of this research project. furthermore, the integration of nanotechnology is a fundamental step for the investigation and targeting of ido . spherical nucleic acids (snas) composed of nanoparticles have been shown to transverse cellular membranes, exhibit stability in physiological environments, escape from degradation, and create precise targeting in brain tumors.objectives: the purpose of our project is to delineate the role of ido in pediatric dipg, and develop small inhibitory (si)rna oligonucleotides and snas aimed at therapeutically inhibiting the gene expression of immunosuppressive ido . our specific aims are to: ( ) confirm the gene expression ido in different human dipg cell lines; ( ) generate and characterize sirna oligonucleotides targeting human ido in vitro; and ( ) generate and characterize gold nanoparticles for targeted inhibition of ido .design/method: unique patient-derived dipg cell lines were grown in culture, stimulated with increasing concentrations of the proinflammatory cytokine, ifn , and analyzed for mrna levels. sirna specific to ido was transfected into cells. sna generation is in progress.results: ido is expressed in multiple human pediatric dipg cell lines. sirna targeting ido among exons and results in a significant decrease in overall ido expression by dipg cells. sna generation for targeting ido with improved penetration & stability is ongoing, with preliminary results demonstrating a robust ability to inhibit ido expression. the grim prognosis of children with dipg, the lack of effective therapies, and the expression of ido by human dipg cells emphasize the importance of developing the treatment capability to inhibit ido gene expression, as a excluded from this study. the remaining patients were analyzed using descriptive statistics.results: a total of patients meeting inclusion criteria were identified. of these, patients ( . %) received tonsillectomy alone, ( . %) underwent tonsillectomy and decreased immunosuppression, ( . %) received tonsillectomy and rituximab, and another ( . %) received tonsillectomy with additional therapy (including ebv-specific cytotoxic tlymphocytes, donor leukocyte infusion, and chemotherapy). of the patients who received tonsillectomy with or without a decrease in immunosuppression, all were diagnosed with high-grade lymphoma and achieved clinical remission following tonsillectomy with no evidence of relapse to date. on further analysis looking at ptld risk factors, all patients were under years of age, all received t-cell depleted grafts, and none had significant graft-versus-host disease (gvhd) at the time of ptld diagnosis. we have identified a population of patients with localized ebv ptld that achieved clinical remission with no evidence of recurrence following tonsillectomy, suggesting that tonsillectomy alone may be an adequate treatment for localized ebv ptld in a specific subgroup of patients. further analysis is needed to identify characteristics of this subgroup to determine which patients would be most likely to respond to this treatment. university of rochester, rochester, new york, united states background: malignant central nervous system (cns) tumors in young children have a poor prognosis and pose a significant therapeutic challenge. consolidation therapy with carboplatin and thiotepa was piloted in ccg- , cog acns , and cog acns with the goals of intensifying therapy and omitting or delaying radiation.objectives: to document outcomes for patients undergoing carboplatin/thiotepa consolidation with autologous stem cell rescue (ascr) and to demonstrate the feasibility and toxicity of this regimen.design/method: patients up to years old (median age: months) with malignant cns tumors treated at the university of rochester from - with at least one cycle of carboplatin ( mg/kg/day x days) and thiotepa ( mg/kg x days) followed by peripheral blood ascr were included in retrospective analysis. data were recorded on time to engraftment (defined by absolute neutrophil count (anc) recovery to > . × ^ /l), length of hospitalization, toxicity with each consolidation cycle, progression free survival (pfs) and overall survival (os). stem cell harvest data were also collected.results: eleven patients with malignant cns tumors ( atypical teratoid/rhabdoid tumor, primitive neuroectodermal tumor, glioblastoma multiforme, and pineoblastoma) received a total of cycles of carboplatin/thiotepa. of these, underwent stem cell harvest at our institution, with complications limited to procedure-related hypotension for patient with known autonomic instability, and catheter-associated deep vein thrombosis (dvt) for patient. four patients were in complete remission (cr) /status-post gross total resection, was in cr , and had residual tumor at the time of consolidation. nine patients received planned consolidation cycles, patient (of ) planned cycles, and patient of an anticipated cycles thus far. average time to engraftment for these cycles was . (+/- . ) days, with a mean hospital length of stay of (+/- . ) days. fever occurred in of cycles ( %); infectious toxicity included documented bacterial infection in cases (enterococcus faecalis bacteremia in , klebsiella pneumoniae in ). there were no regimenrelated deaths. with a mean follow-up of months, survivors have not yet completed all therapies, and patients have relapsed ( have died of disease). of the survivors, have been disease-free for > months. background: autologous hematopoietic stem cell transplantation (auto-hsct) has resulted in improved survival for patients with high-risk neuroblastoma. treatment intensification is however associated with greater complications. data on early infectious complications in low-and-middle income countries are limited.objectives: to review the early infectious complications following auto-hsct in patients with high-risk neuroblastoma.design/method: a retrospective chart review of pediatric patients with high-risk neuroblastoma who underwent auto-hsct at the american university of beirut medical center between and was conducted. infectious complications during the first days post-transplant were reviewed.results: forty-three patients ( males and females) with a median age at diagnosis of . years [range: . - . ] years underwent auto-hsct during the above-mentioned period. conditioning regimen consisted of melphalan, etoposide and carboplatin. all patients received antiviral and antifungal prophylaxis. median time for neutrophil engraftment was days [range: - ]. bacteremia and clostridium difficile infections occurred in ( %) and ( %) patients respectively. seven ( %) patients developed enterocolitis diagnosed by imaging, were adenovirus induced. cmv viremia was diagnosed in ( %) patients, of whom required treatment. varicella zoster reactivation, parvovirus viremia, toxoplasmosis encephalitis, bk virus cystitis ( patients) and central nervous system ebv related post-transplant lymphoproliferative disorder were diagnosed in different patients. there was no invasive fungal infection. sixteen ( %) patients have died, of whom died in the early post-transplant period, due to disease progression and ( . %) due to infectious complications. among the patients who died due to infection, developed toxoplasmosis encephalitis, developed severe enterocolitis, of which were adenovirus related. the mean igg level within one week post-transplant was lower in patients with clinically significant viral infection compared to others ( vs . mg/dl, p: . ). the mean igg level at the time of clinically significant bacterial infection was lower in infected patients compared to others ( . vs . mg/dl, p: . ). neither absolute lymphocyte count nor absolute neutrophil count at day post-transplant affected the incidence of clinically significant infections. our results show that the rate of infections during the early post auto-hsct period is higher than what has been described in developed countries and has a significant impact on mortality. prevention, early detection and improvement in the treatment is required to improve outcome. university of miami, miami, florida, united states background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many malignant and non-malignant (bone marrow failure, immunodeficiency, or metabolic diseases) in pediatrics. despite advances in medicine, graft-versus-host-disease (gvhd) remains a significant cause of non-relapsed morbidity and mortality, specifically in those with malignant diseases.objectives: to highlight the complexity to acute gvhd management and seldom-described treatment approach. a year male with a history of high risk acute myeloid leukemia (aml) due to failed induction therapy. he received a matched ( / ) unrelated donor hsctmarrow product-conditioned with busulfan, fludarabine, and anti-thymoglobulin (atg). his post-transplant course was complicated by hhv- viremia, pres (prompting a change from prograf to cyclosporine), mucositis, and grade iii acute gvhd (skin s , gut s , liver s ) around post transplant day , which later morphed to ocular involvement by d+ . he was started on mg/kg steroids with good response but flared up with each attempt to taper steroid dose. a course of rituximab and later atg were tried without success in weaning off steroids. switching cyclosporine to sirolimus did not provide any additional benefit either. extracorporeal photopheresis (ecp) was started times a week. he initially responded well, yet was not able to wean off steroids. in addition, he developed a flare when ecp session was reduced to days per week. ecp was therefore increased to days per week, which appeared to stabilize skin lesions. a trial of weekly methotrexate was attempted to wean off steroids and photopheresis, which provided no response. finally, a trial of bortezomib on days , , , and of a day cycle as published in a case series of multiple myeloma patients who developed post hsct gvhd. skin lesions improved remarkably however dose had to be reduced due to related pancytopenia. given the response to therapy, he was continued on a weekly dose of bortezomib, receiving a total doses, which has permitted the slow taper of prednisone that has since been discontinued without a major flare. he however is currently maintained on ecp times per week, which is now been slowly withdrawn.conclusion: management of acute gvhd in pediatric patients after hsct can be challenging with no definite options for those who fail steroids or become steroid dependent after initial response. in these situations, bortezomib could be a valid therapeutic option. background: neuroblastoma (nbl) is the second most common solid tumor in children and despite recent treatment advances, overall survival for high risk nbl remains < %. the addition of immunotherapy has improved survival and includes anti-gd antibody therapy. the success of antibody therapy in neuroblastoma is primarily due to natural killer (nk) cell mediated antibody dependent cellular cytotoxicity. we previously demonstrated that nk cells from patients with high risk nbl can be successfully isolated and expanded to large numbers and exhibit potent anti-tumor effects against nbl ( ). thus, infusions of autologous expanded nk cells in high risk nbl in combination with anti-gd antibody are being studied in clinical trials. toll-like receptors (tlr) present on the surface of leukocytes are responsible for pathogen recognition, and activation of these receptors stimulate the production of cytokines that critically link innate and adaptive immune responses. the tlr agonist, poly(ic) is a synthetic analog of dsrna that has previously been shown to directly stimulate cytokine production and improve cytotoxicity in primary nk cells through activation of genes regulated by interferon-response elements (ire) ( ). we hypothesized that ex vivo activation of tlr pathways in nk cells during our normal -day expansion using k feeder cells expressing membrane bound il- would enhance their function.design/method: nk cells were isolated from peripheral blood mononuclear cells and expanded with our previously described expansion protocol in media containing il- and ug/ml poly(ic) ( ). at the end of the -day expansion, nk cells expanded with poly(ic) were compared to controls using a calcein cytotoxicity assay to measure cytotoxicity against high risk neuroblastoma and cytometric bead array to measure cytokine production. : surprisingly, the addition of poly(ic) during nk cell expansion did not improve proliferation, cytokine production or cytotoxicity compared to our standard expansion method. rnaseq demonstrated that our standard expansion method results in a modest decrease in tlr expression at the transcriptional level, but significant upregulation of several ireregulated genes. we conclude that either our standard approach interferes with tlr signaling or saturates the innate immune response pathway such that co-stimulation with poly ic does not produce an additive effect. we are performing expression analysis on nk cells receiving poly(ic) during expansion to further explore this hypothesis. background: gonadal dysfunction leading to infertility is a complication after hematopoietic stem cell transplant (hsct). anti-müllerian hormone (amh) is a marker of ovarian reserve; it is not controlled by gonadotropins and has minimal inter-cycle variations, therefore, it can be used as a marker of ovarian reserve and aid in fertility counseling.objectives: assess ovarian reserve in hsct patients utilizing amh levels. background: tgf beta is an immune suppressive cytokine frequently elevated in the tumor microenvironment causing tumor immune evasion. acute tgf beta treatment potently inhibits nk cell cytotoxicity, cytokine secretion, and proliferation. however, tumor infiltrating nk cells receive chronic inhibitory tgf beta signals in conjunction with activating signals from tumor cells. objectives: to this end, we hypothesized that long-term tgf beta-cultured nk cells would induce functional and phenotypical changes on nk cells that differ from short-term tgf beta treatment.design/method: to explore this, primary human nk cells were cultured with the leukemia cell line, k , alone or with exogenous tgf beta for weeks. : surprisingly, nk cells cultured in tgf beta proliferated faster, and upon challenge with a variety of cell line targets they secreted much greater quantities of ifnÎ ( -to -fold increase against / cell lines) and tnf ( -to -fold increase against / cell lines). further, the high cytokine secretion induced in these nk cells was no longer inhibited by adding additional tgf beta. degranulation was also increased ( / cell lines), however cytotoxicity was not enhanced in a -hour cytotoxicity assay. after resting in il- , the cytokine hypersecretion of tgf betacultured nk cells was maintained for several weeks suggesting this functional change might involve cellular reprogramming. we investigated the mechanism behind these functional changes and profiled genes involved in tgf beta signaling. we found significant reduction of smad transcription which corresponded to a striking decrease in smad chromatin accessibility. we also found significantly increased smad and decreased tgfbr expression. phenotypic analysis revealed that tgf beta also induced remodeling of the nk receptor repertoire with decreased nkp , cd , and klrg and upregulation of trail. the functional consequences of these tgf beta-induced changes on in vitro and in vivo nk cell function are currently under investigation. background: the use of t-cell depleted grafts in haploidentical stem cell transplantation (hsct) has been associated with a delay in early t-cell recovery which increases the risk of viral infections, relapse or graft rejection. conventional donor lymphocyte infusion (dli) after hsct transplantation is effective but conditioned because of a high prevalence of gvhd. the infusion of selected lymphocyte subpopulations with low aloreactivity is emerging as an effective strategy to rectify this issue. the depletion of cd ra+ naive lymphocytes, preserving cd ro+ memory t-cells, could provide a safe source of functional lymphocytes with anti-infection, antileukemic and anti-rejection properties, and lower rates of adverse effects. our objective is to present data of patients that have received cd ro+ memory t-cells dli (mdli) and assess its safety and outcome. we present data of mdli performed after hsct in cases of mixed chimerism, persistent lymphopenia, viral/opportunistic infections or as a strategy to accelerate immune reconstitution.results: fifteen patients with diagnosis of all (n = ), aml (n = ), mds (n = ), saa (n = ), sideroblastic anemia (n = ) and cgd (n = ), received mdli after hsct. a total of forty-three mdli were infused. the median dose of cd ro+ memory t-cells infused was . × /kg (range: . × - . × /kg), with a median dose of cd ra+ naive t-cells of . × /kg (range: - . × /kg). the mdli were infused at a median of seventy-seven days after hsct (range: - days), with a median interval between mdli of thirty-four days results: eight published studies reported survival outcomes for pediatric vod/sos patients (n = ), across all defibrotide doses. estimated day+ survival ( % confidence interval) was % ( %- %). for vod/sos with mod, studies were identified (n = ) with pooled estimated day+ survival of % ( %- %). only one openlabel expanded-access study, the treatment-ind, reported outcomes separately for pediatric vod/sos patients without mod (n = patients aged ≤ years). the day+ kaplan-meier estimated survival for those patients was % ( %- %). safety results were not pooled due to differences in reporting methodology; however, study results were consistent with the safety profile of the phase historicallycontrolled trial in vod/sos patients with mod ( % pediatric), in which / defibrotide-treated patients and all controls experienced ≥ ae. hypotension was the most frequent ae ( %, defibrotide; %, controls); common hemorrhagic aes (ie, pulmonary alveolar and gastrointestinal hemorrhage) occurred in % of defibrotide-treated patients and % of controls. in this pooled analysis of studies with defibrotide-treated pediatric patients with vod/sos, estimated day + survival was % (without mod, %; with mod, %). safety results in individual studies were generally consistent with the known safety profile of defibrotide. taken together, these results show a largely consistent defibrotide treatment effect in pediatric patients treated with defibrotide for vod/sos, with or without mod. results: six patients met inclusion/exclusion criteria. all patients were started on ecp while concurrently receiving . to mg/kg steroid therapy for agvhd plus a calcineurin inhibitor. patients had initiation of ecp within a maximum of weeks from initial diagnosis of agvhd (range - days). patients had grade - agvhd ( / patients with grade ) with skin, liver, and gi gvhd represented. patients received ei-ecp - times per week for the first weeks and then had ei-ecp frequency tapered based on initial response.after weeks of therapy patient had a decrease in overall gvhd grade by grade. all patients were able to have steroids tapered, with doses decreased by an average of % ( % - % decrease).at weeks of therapy, one patient with grade agvhd died of mof associated with infections. three patients had complete resolution of agvhd and patients decreased by grade. steroid doses were decreased by an average of % ( % - % decrease). continuously measures axillary temperature and wirelessly transmits real time-time data. the primary aim of the study was to evaluate the feasibility, safety and tolerability of continuous temperature monitoring in hsct patients using temptraq. we are performing a prospective observational study of pediatric patients ( - years of age) undergoing hsct at cincinnati children's hospital in cincinnati, ohio. enrolled patients wore a temptraq patch for days. a - rating scale survey was completed by the parent/guardian at the end of the study to determine tolerability, ease of use, satisfaction and desire for future use in the inpatient and outpatient setting. temperature data from the temptraq patch was compared to the standard episodic temperature monitoring to determine detection of febrile episodes. seven of ten patients have completed screening. we anticipate completion of the study in early february. the temptraq patch was well tolerated by study subjects (mean tolerability rating of . / ). one patient developed skin breakdown at the site of the temptraq patch attributed to recent thiotepa. the patch was easy to apply with an easy of application rating of . / . parents were overall satisfied (rating . / ) and would like to use the temptraq patches in future hospitalizations (rating . / ) and at home (rating . / ). temptraq patch identified fever (≥ . • f) in patients. the fever was never detected by episodic monitoring (soc) in patients and significantly delayed in the other patients (> hours). temptraq was well tolerated in pediatric hsct patients. timely fever detection was improved in temptraq over the current soc. background: serotherapy is commonly used in patients undergoing hematopoietic stem cell transplant (hsct) to reduce the incidences of engraftment failure and graft versus host disease. however, one well-known side effect is fever. as children undergoing hsct have compromised immune defenses, fever may also be an early indicator of bloodstream infection, which would warrant prompt use of broad-spectrum antibiotics. in a subset of patients with serotherapy-associated fever, antibiotics, which may induce antibiotic resistance and increase costs, may be unnecessary. we aimed to determine the incidence and characteristics of serotherapy-related fever, as well as the likelihood of concomitant bacteremia, in our institutional experience. a -year retrospective chart review was conducted of pediatric patients who received serotherapy as part of hsct conditioning at the university of minnesota. one-hundred sixty eight consecutive hsct patients who received serotherapy -either atg (n = ) or alentuzumab (n = ) -were identified. the median age at hsct was -years (range, . - years). a total of patients ( %) developed fever while on serotherapy (atg = , alentuzumab = ). one-hundred sixteen patients presented fever following the first infusion, and the median onset of fever was hours after commencing infusion (range, . - hours). fever resolved at a median hours (range, - hours). one hundred and fourteen patients ( %) underwent blood cultures. only seven patient were not started on ( %) empiric antibiotics, while % (n = ) were on antibiotic treatment prior to serotherapy for previously known or suspected infections. nine patients ( % of febrile patients, % of all patients) had positive blood cultures (atg = ; alentuzumab = ). no infection-associated deaths were observed.conclusion: while fever is common during serotherapy conditioning in children undergoing sct, episodes of concomitant bloodstream infection are rare. ongoing analysis identified potential risk factors for bacteremia as recent history of infection, first episode of fever following second or subsequent infusions, and previous central line placement. further analysis is being conducted to identify subgroups of patients for whom close monitoring alone may be safe. background: hsct is potentially curative for caya with high-risk leukemias; however, most lack an hla-matched aspho abstracts related donor. the risk of gvhd is increased with unrelated (urd) or partially matched related (pmrd) donors. selective t-cell depletion based on the elimination of t cells carrying and chains of the t-cell receptor may greatly reduce the gvhd risks, while allowing the maintenance of mature donor-derived alloreactive nk cells and / (+) t cells, which may augment the anti-leukemia effect.objectives: this is a prospective study of caya with acute leukemia who underwent hsct with mmrd or urds and tcr / /cd depletion. outcomes included engraftment, toxicities, viral reactivation, and relapse.design/method: this study included caya with acute leukemia transplanted between october and may . all received a myeloablative preparative regimen with targeted busulfan (n = ) or tbi ( cgy/ fractions) (n = ), with thiotepa ( mg/kg) and cyclophosphamide ( mg/kg). atg ( mg/kg x ) was given to those receiving haploidentical grafts and to the first who received urd grafts. immune suppression was not given post-hsct. the stem cell source was mobilized peripheral blood stem cells (pscs), which then underwent tcr / /cd depletion utilizing the clinimacs device under gmp conditions in the chop cellular immunotherapy lab.results: median age was (range . - . ). diagnoses included all ( -b-cell, -t-cell) and aml ( ; -secondary aml). urd were used for ; were / allele matched and were / matched. haploidentical donors were used for . median cd (+) dose - . × , / (+) cd (+) cells - . × , and b cells - . × . all patients achieved an anc at a median of d+ ( - ), and % had platelet engraftment at median d+ ( - ). nine patients ( %) developed acute gvhd (all skin, grades i-iv). five developed chronic gvhd (skin, gut, lung): limited in , extensive in . viral reactivations included: adenovirus ( , %), bk virus ( , %), cmv ( , %), and hhv ( , %). nine ( %) patients relapsed at a median of days (range - ) post-hsct, including aml patients ( . %) and all patients ( . %). transplant-related mortality was %; causes included sepsis ( ) and ards ( ). os was %; efs was % (gvhd-free efs %, lfs %). hsct with tcr / /cd depletion demonstrates excellent engraftment kinetics with limited gvhd without immune suppression. elimination of post-hsct immunosuppression may offer an excellent platform to augment anti-leukemic immune therapy or to enhance immune reconstitution. background: hematopoietic cell transplantation (hct) is the only curative treatment available for patients with sickle cell disease (scd). low bone mineral density (bmd) has been described in scd, but little is known about the impact of curative hct on this outcome. to determine the prevalence of low bmd and variables associated with low bmd in scd patients after hct. we conducted a retrospective chart review of scd patients who underwent hct at children's healthcare of atlanta (choa) between / and / and survived ≥ year post-hct. transplant characteristics, post-hct dual-energy x-ray absorptiometry (dexa) scan results, vitamin d levels, graft-versus-host-disease (gvhd) status, and fsh levels were reviewed. for patients - years of age, height corrected z-scores were calculated using a nihvalidated calculator, with t-scores used for older patients. bmd was categorized as low if between - and - sd below the mean and clinically significantly low if >- sd, in accordance with the children's oncology group long-term follow-up guidelines. vitamin d levels < ng/mol were considered deficient, and fsh levels > miu/ml suggestive of premature ovarian failure. fisher's exact test was used to compare variables in those with normal versus abnormal dexa scan results, with p< . considered significant.results: hct was performed on patients with scd, with surviving ≥ year post-hct. dexa scans were obtained in patients ( % female), with mean time from hct to dexa scan being years ( . - . years) and mean age at time of dexa . years ( . - . years). patients with and without dexa scans did not differ by sex, donor source, age at transplant, or vitamin d status. low bmd was noted in patients ( . %), with these patients more likely to be > years (pubertal; . versus . %, p = . ). acute gvhd was more common in patients with low bmd ( . versus . %), but not statistically significant (p = . ). clinically significant low bmd was noted in patients ( . % of those with dexa scans). these patients were older ( . years at testing), were more likely to be male ( . %), and all had acute and chronic gvhd, while none had evidence of gonadal failure.conclusion: clinically significant low bmd is uncommon after hsct for scd. patients at risk for low bmd include older patients and likely those with gvhd. this preliminary data suggests routine dexas may not be indicated for all patients who undergo hct for scd, but further data is needed. background: causes of renal dysfunction after hematopoietic cell transplantation (hct) include damage from radiation, nephrotoxic medications, graft vs. host disease (gvhd), hepatorenal syndrome, viral infections, or transplant associated microangiopathy. we sought to investigate the incidence of, and risk factors for, acute kidney injury in pediatric hct patients and associated risk with mortality.design/method: data from patients who underwent hct between and at a single institution were sequentially retrospectively captured on irb approved protocol. acute kidney injury (aki) was defined at multiple time points post-hct using the standardized criteria: kidney disease: improving global outcomes (kdigo). interval differences between values were analyzed using wilcoxon rank sum testing and categorical variables were analyzed using chi-square analysis.results: ninety-eight patients were included in the study: allogeneic (n = ) and autologous (n = ), mean age . years, of whom % were african american, % asian, % caucasian, % latino, and % mixed race. forty-seven percent of patients developed aki within the first years of hct. increased risk for aki was associated with a lower pre-transplant creatinine level (p = . ), abnormal pretransplant bun (p = . ) and an unrelated donor (p = . ) while preparative regimen intensity, race, or primary disease were not. twenty-six percent of patients developed aki within days of hct. of those with aki, % were exposed to either cidofovir, aminoglycosides, and/or ambisome for at least days versus % without aki and % were exposed to vancomycin compared to % without aki. evaluating outcomes at year after hct, of those with stage aki: % had reduced gfr and % died, while % had reduced gfr and % had died for patients with aki stage or . the absence of aki by day was associated with % reduced gfr and % death at -year after hct. overall, those with aki at any time in the first year post-hct had a . fold increased risk of death compared to those without. for patients who required renal replacement therapy (rrt, n = ), the risk of death was . fold greater compared to those who did not. in the % of patients who survived rrt, both recovered renal function within years.conclusion: acute kidney injury is common after pediatric hct, and may be associated with low creatinine, abnormal bun, unrelated donor pre-hct, and renal toxic medications. early-onset aki post hct is associated with an increased risk of mortality. these data should be validated in a larger prospective study but may offer opportunities to intervene and enhance outcomes. background: myeloablative hematopoietic stem cell transplant (hct) for pediatric malignant disease is associated with significant morbidity with % patients experiencing mucositis. patient controlled analgesia (pca) utilizing opioids is an effective strategy for pain management. we sought to describe and analyze pca use in d+ days post myeloablative hct for malignancies at lurie children's hospital of chicago from - .design/method: utilizing retrospective chart review, pca details were collected: indication, initiation day, pca duration, team managing pca (anesthesia or palliative), medication and dose in morphine equivalents, and pca toxicities. efficacy of pca was evaluated on pca day + , + , + , + using demands %, maximum pain score (rflacc, faces, vas) and subjective patient, parent and/or pain team perception of pain control. we devised a scale based on the above to designate pain control as good, moderate or poor. variables being analyzed include recipient age, sex, donor type, source, diagnosis, tbi use, gvhd/trm. this analysis, we analyze the depth of remission, car-t persistence, and post-transplant gvhd on our phase i anti-cd car-t protocol (nct ) to better understand the role of car-t in the peri-hct setting.design/method: children and young adults with relapsed/refractory cd + all treated on our phase i anti-cd car-t protocol were analyzed. mrd was assessed by flow cytometry (fc) in all, with pcr-based mrd analysis using igh or tcr testing assessed in select patients. hcts were performed at each patient's local institution based on standard of care and included varying conditioning regimens, donor types, stem cell source, and gvhd prophylaxis.results: on our cd car trial, patients were treated, the majority of patients (n = ) having relapsed following a prior hct. / patients ( %) attained a cr, of whom were mrd negative by fc. concurrent pcr based mrd analysis available in patients demonstrated that all patients achieved pcr based negativity. in , this was simultaneous with the month mrd negative fc, and in , pcr negativity was achieved over time (fc remained negative). patients proceeded to hct at a median time of days (range: - days) post-car-t, which was a first hct in . these two patients remain in an mrd negative cr, year post-car-t. no patients developed acute or chronic gvhd. car persistence was seen in patients who had detectable car-t cells on the pre-hct marrow suggesting the possibility of ongoing anti-leukemia surveillance prior to initiation of the conditioning regimen.conclusion: by inducing pcr negativity, car-t therapy may have a synergistic role with hct to improve leukemia free survival, prior to emergence of antigen negative leukemia, without an increased risk of gvhd. while the sample size is small, car-t therapy may offer an effective bridge to hct, particularly for those who are pcr negative, and those who have not had a previous transplant. given the underlying risk of hct related trm, pre-hct car may potentially allow for hct conditioning de-intensification as it may not be needed to eradicate residual disease. lee dw, ash abstract , background: post-transplant lymphoproliferative disease (ptld) is a complication after solid organ transplantation (sot) that is frequently due to epstein -barr virus (ebv) as a decrease in ebv-specific t cell immunity due to immune suppression allows for uncontrolled proliferation of ebv-infected b cells. outcomes for ptld are suboptimal with relapse rates approaching %. however, ebv-infected b cells in ptld express the ebv antigens lmp and lmp that can be targeted with immune therapy.objectives: we hypothesize that third party "off the shelf" lmp-specific t cell products may improve outcomes and decrease associated co-morbidities for patients with ptld by not only target the lymphoproliferating ebv-infected b cells but also restoring ebv-specific immunity.design/method: lmp-specific t cells (lmp-tcs) are manufactured from eligible donors with a broad range of hla types in our gmp facility to be used in a children's oncology group (cog) trial (anhl ) for patients with ptld after sot. lmp-tc products are manufactured from healthy donors using autologous monocytes and lymphoblastoid cell lines (lcl) transduced with an adenoviral vector expressing Δlmp and lmp as antigen presenting cells. lmp-tc products undergo comprehensive characterization by ifn-elispot assay to determine lmpspecific epitopes, class i and/or ii response, and hla restriction to guide selection of lmp-tc product for each patient.results: thus far, lmp-tc products have been manufactured. lmp-tcs were active against lmp (mean: sfu/ × ^ cells; range: - ), lmp ( ; - ), and lcl ( ; - ) as determined by ifn-elispot assay. at the time of cryopreservation, the lmp-tc products comprised a mean of % cd + t-cells, % cd + t-cells, and % nk cells. no b cells or monocytes were detected in the final products. thus far, we have identified novel lmp epitopes (lmp specific: n = ; lmp specific: n = ). approximately % of the lmp-tc products have lmp-specific activity through multiple hla alleles, and % have a mixed class i and class ii response. conclusion: thus, lmp-specific t cell products can be expanded from healthy donors to creat a third party bank, and identifying epitopes and hla alleles with lmp activity will facilitate selecting the most appropriate product for patients. while lmp-specific t cells have previously demonstrated safety and efficacy in phase i studies, anhl is the first trial using cellular therapy within a cooperative group setting. children's cancer hospital at the university of texas md anderson cancer center, houston, texas, united states background: in , the united states food and drug administration (fda) approved the first chimeric antigen receptor t cell (car-t) therapy; tisagenlecleucel. this cd -directed genetically modified autologous t cell immunotherapy has shown response rates of almost % among children and young adults with b-cell precursor acute lymphoblastic leukemia (all) that are refractory or in second or later relapse. cytokine release syndrome (crs) and car-t cell related encephalopathy syndrome (cres) are well described toxicities associated with car-t therapy. crs is a systemic inflammatory response and is typically characterized by fever, hypoxia, tachycardia, hypotension and multi-organ toxicity. cres may occur concurrently or following crs, or without any associated crs symptoms and is characterized by encephalopathy, delirium, seizures and rarely cerebral edema. almost half of patients who receive tisagenlecleucel may require pediatric intensive care unit (picu) support. crs and cres are generally reversible but may be associated with fatal outcomes. pediatric specific management guidelines, comprehensive training of multidisciplinary staff, effective communication and phased infrastructure ensure that adequate resources are available to facilitate early diagnosis and appropriate management of pediatric patients with crs and cres and allow for optimal patient outcomes and accreditation by the foundation for accreditation of cellular therapy (fact).objectives: develop a comprehensive program to ensure safe administration of immune effector cell (iec) therapy to pediatric patients.design/method: an inter-disciplinary pediatric cartox (car t cell therapy associated toxicity) committee consisting of cell therapy and picu physicians, neurologists, fellows, nursing leadership, advanced practice practitioners, pharmacists, registered nurses and social workers was created to monitor patient toxicity and establish specific clinical guidelines and diagnostic and treatments algorithms for pediatric patients receiving iec therapy. educational modules were developed as (i) live in-services and (ii) an online module with a competency based assessment. electronic medical record (emr) order sets and documentation and warning systems were also developed by the committee. the pediatric cartox committee developed a diagnostic and treatment algorithm for patients receiving iec therapy. emr orders and flowsheets were developed to support adherence to the algorithm. inter-disciplinary staff training and competency assessments were closely tracked. almost % of identified staff have completed training and achieved competency including, pediatric cell therapy staff, emergency center, picu, outpatient clinic/triage, neurology and sub-specialty staff and nocturnalists.conclusion: an inter-disciplinary approach can assist in institutional readiness for an iec program, promote quality assurance and perhaps fact iec accreditation. future directions include a program for ongoing staff competency assessments. predicted peak vo ) and abnormal oxygen uptake efficiency slope at the anaerobic threshold ( . ± . . , normal ± ). additionally, on echocardiogram three patients had evidence of diastolic dysfunction as evidenced by an elevated e/a ratio ( . ± . ) on tissue doppler. three patients demonstrated depressed longitudinal peak systolic strain (- . ± . ), indicating dysfunction not captured by ejection fraction. in this feasibility study, sct patients without evidence of lvsd on standard measures by resting echocardiogram can demonstrate abnormal exercise capacity. additionally, they can demonstrate systolic and diastolic dysfunction by measures not always included in standard echocardiography. these data suggest the need for a more thorough screening of survivors, and will be further validated as additional patients are recruited for this study. background: in hematopoietic transplantation, the t lymphocytes of the inoculum play a determining role in promoting hematopoiesis, transferring immunity to pathogens and acting as mediators of the graft-versus-leukemia effect (gvl). however, they are also responsible for graft-versus-host disease (gvhd), the main cause of post-transplant morbidity and mortality. the depletion of cd ra lymphocytes, by eliminating naive t lymphocytes from the inoculum, aims to conserve the gvl without producing gvhd.design/method: since april , patients ( boys and girls), with a median age of years, have undergone an allogeneic hematopoietic transplant from an hla donor identical with cd ra/cd depletion. the indication for transplant was: acute lymphoblastic leukemia ( ), acute myeloblastic leukemia ( ), myelodysplasia ( ) and medullary aplasia ( ). the donor was familiar in cases and unrelated in . the conditioning regimen was with fludarabine, busulfan and thiotepa. the median of cd + cells infused was . × / kg. on the day , + and + a programmed infusion of × / kg lymphocytes cd ra-was performed.results: all the patients grafted with a median leukocyte (> . × / l) and platelet (> × / l) engraftment time of and days, respectively. only one patient has developed acute gvhd grade i and no patient has developed chronic gvhd. immune reconstitution was early and rapid in all t cell subsets no patient has relapsed so far and only patient with myelodysplasia has developed an aml. she has received a nd transplant and has died of relapse. there was no case of toxic mortality. the event-free survival (sle) was ± % with a median follow-up of months. at present, patients are alive, out of immunosuppressive treatment and doing well. allogeneic transplantation with cd lymphocytes ra depletion resulted on very encouraging results, with a very low incidence of acute and chronic gvhd, but preserving the gvl effect by infusing cd ra-donor lymphocytes. miami children's health system, miami, florida, united states background: hematopoietic stem cell transplantation (hsct) using autologous or allogeneic progenitor cells is a potentially curative treatment for patients with high-risk malignancies and nonmalignant conditions. the american society for blood and marrow transplantation developed a task force to establish consensus guidelines for defining patient care in hsct and advocated for further studies to delineate safe procedural steps as an increasing amount of hsct are being offered to patients. there is limited evidence to support engraftment in recipients who receive their infusions via iv or syringe pump. we present novel data from patients who achieved neutrophil engraftment following hsct by a pump mechanism.objectives: to provide evidence supporting the use of pump (intravenous or syringe) infusion method in hematopoietic stem cell transplantations.design/method: a retrospective review was completed for patients who underwent hsct between and . inclusion criteria included patients who had received hematopoietic stem cell transplants between and and who were ages months to years old. the main outcome measure was days to neutrophil engraftment (defined as the first of three consecutive days with an anc > × /l).results: among patients who received infusion of hematopoietic stem cell products via pump mechanism, patients ( . %) received autologous products and ( . %) received stem cells from allogeneic donors. neutrophil engraftment (anc > × /l) occurred in a median of . days after stem cell infusion. the mean number of days to engraftment for patients who received allogeneic infusions s of s design/method: a retrospective chart review was performed at the children's hospital of wisconsin. statistical analyses included kaplan-meier estimate for os, mann-whitney test for comparing outcomes between subjects, and descriptive analyses.results: from - , patients with a median age of . ( . - . ) years at st hct were identified. patients were conditioned with cy/atg (n = ), cy/flu/atg (n = ), or cy/flu/atg/tbi (n = ) and received marrow (n = ) or cord blood (n = ) with median cd /kg dose of . ( . - . ) × . two patients developed grade i acute graftversus-host disease (gvhd); none developed chronic gvhd. due to dropping chimerism, graft rejection, or graft failure, nd hct (n = ) or boost (n = ) was offered. the median cd chimerism prior to hct/boost was ( - )%. median time between st hct and nd hct or boost was days ( days- . years). in patients receiving nd hct, used the same donor, of which used the same stem cell source (marrow) and switched to peripheral blood stem cells (pbsc). in patients who switched donors, used pbsc and used cord blood. most patients receiving nd hct underwent a uniform conditioning regimen of cy /flu /equine atg/ gy tli (n = ) or cy /flu /rabbit atg/ gy tbi (n = ); one received cy/atg. acute and chronic gvhd (limited seen in %) developed in % and % of patients, respectively. four patients required additional boosts and additional hct. after final intervention, cd and whole-blood chimerism at last follow-up was between - % (n = ) and - % (n = ), respectively. with a median follow-up of . ( . - . ) years, of patients are alive with an estimated -year os of . ± . %, having performance status ≥ % (n = ) or % (n = ). one patient developed chronic extensive gvhd and died of fungal infection . years after nd hct. our single-center experience demonstrates excellent ability to salvage patients who develop graft failure after initial hct. transplant-related complications such as gvhd and infections remain significant concerns. key: cord- -ol buwld authors: gonzales, natalia m.; howell, viive m.; smith, clare m. title: th international mammalian genome conference meeting report date: - - journal: mamm genome doi: . /s - - - sha: doc_id: cord_uid: ol buwld nan during november - , , the th annual international mammalian genome conference (imgc) attracted researchers from all over the world to yokohama, japan to discuss the latest advances, tools, and techniques in mammalian genetics. organized by piero carninci (riken) and the international mammalian genome society (imgs; www.imgs.org), the meeting brought together scientists from countries, not to mention hundreds of online participants that followed the live tweeting of talks (#imgc ). social media has become an integral part of the imgc, complementing the scientific content and facilitating ongoing discussions between scientists around the globe. the conference opened with a bioinformatics workshop, guided tours of riken laboratories, and a trainee symposium, giving ph.d. students and early-career postdoctoral researchers an opportunity to share their works in a collegiate and mentoring setting and vie for the chance to present at the main meeting. participants were officially welcomed to yokohama at the evening reception, where old friends and new were met over a smorgasbord of local cuisine. the main meeting was divided into sessions showcasing the wide-ranging research interests of imgs members. these included human disease models and immunology, neuroscience, development and stem cells, genomics and computational analysis, epigenomics and noncoding rnas, advances in genome editing, and largescale resources. the verne chapman lecture was given by professor john mattick, director of the garvan institute of medical research in sydney, and the inaugural darla miller distinguished service lectureship was awarded to janan eppig, professor at the jackson laboratory and a pioneer of the mouse genome informatics (mgi) program project. several poster sessions, a mentor lunch for trainees and workshops on bioinformatics, systems genetics, scientific literature curation, gene enrichment analysis, and fantom were also featured in the meeting program, which culminated in a feast of epic proportions. abstracts from the meeting are available at www.imgc .jp. the imgc has a strong reputation for advocating new scientific talent, epitomized by the trainee scholarships awarded for conference travel, the mentor-trainee lunch, presentation awards, and numerous opportunities to present work and receive feedback. the trainee symposium is an integral part of the meeting, featuring oral presentations from graduate students and postdoctoral researchers. the wide variety of topics presented exemplified the diversity and utility of mammalian model systems for both clinical and basic research. xenograft mouse models were utilized by hiroyuki yoda (ts- ; chiba cancer centre research institute) and takahiro inoue (ts- ; chiba cancer centre research institute) to demonstrate the in vivo anti-tumor effects of novel alkylating agents targeting the oncogenes mycn in neuroblastoma, and kras g d/v in colorectal cancer, respectively. the power of forward genetics was demonstrated by lisa gralinski (ts- ; university of north carolina) using the precollaborative cross to identify sars-coronavirus susceptibility loci and irina treise (ts- ; german mouse clinic), exploiting n-ethyl-n-nitrosourea (enu) mutagenesis to uncover molecular mechanisms of immunodeficiency. the sanger knockout mouse resources were highlighted by kifaythullah liakath-ali (ts- ; kings college london), who conducted a phenogenomics screen to investigate the genetic basis of skin phenotypes. gennadiy tenin (ts- ; university of manchester) followed up on human genomewide association studies (gwas) of the congenital heart defect tetralogy of fallot by developing an in vitro mouse heart culture for testing candidate genes by sirna knockdown. ximena ibarra-soria (ts- ; wellcome trust sanger institute) took us on a journey through the mouse olfactory system, demonstrating that novel olfactory receptor genes can be identified, characterized, and distinguished from environmental regulators despite the system's overwhelming complexity. several speakers focused on behavioral traits, including yuki matsumoto (ts- ; national institute of genetics), who identified a locus on chromosome associated with mouse tameness in wild-derived heterogeneous mouse stocks, and akira tanave (ts- ; national institute of genetics) who is exploring the etiology of anxiety and stress by studying strain differences between wild-derived msm mice and c bl/ mice. guzel gazizova (ts- ; kazan federal university) introduced the dormouse as a genetic model, discussing how transcriptome-level differences between two genera of dormice can be analyzed to yield insights into the evolution of hibernation and to clarify the status of the dormouse in the mammalian phylogeny. belinda goldie (ts- ; kyoto university) took us further into the world of gene expression, demonstrating the importance of extending micro-rna (mirna) analysis beyond evolutionarily conserved targets when exploring gene regulation of human neuronal synapses. hazuki takahashi (ts- ; riken) demonstrated a high-throughput system to optimize antisense long-noncoding rnas that increase translation of target mrnas, and riti roy (ts- ; university of western australia) examined expression profiles of receptors and ligands in cell lines profiled in the fantom project and tumors profiled by the cancer genome atlas (tcga) to understand how cancer cells communicate. mice were not the only model system represented by trainee symposium talks. pavel mazin (ts- ; skolkovo institute of science and technology) examined brain rna-seq data from humans, chimpanzees, and macaques to identify species and age-related differences in alternative splicing patterns. pavel prosselkov (ts- ; riken) branched from the mammalian tree, using the sea squirt to investigate the role of gene paralogs in cognition. finally, brandon velie (ts- ; swedish university of agricultural sciences) showcased the horse, an underutilized organism in mammalian genetics, as a model for identifying genetic factors related to locomotion, allergic diseases, and eczema. ximena ibarra-soria, akira tanave, hazuki takahashi, and irina treise were named as the lorraine flaherty awardees for their talks during the trainee symposium ( fig. ; table ) and received the opportunity to present their work at the main conference. several plenary sessions focused on a range of mammalian tools and resources for modeling human disease. the session showcased tools such as recombinant inbred lines (rils), outbred populations, classic crosses, and enu mutagenesis to yield new understanding and identify candidate genes for disease susceptibility, while knockout and patient-derived xenograft mice enabled further mechanistic insight. the session featured many talks utilizing the phenotypic diversity and genetic mapping power of diversity outbred (do) mice and the collaborative cross (cc), community resources * years in the making. fernando pardo- umass medical school) showed that resistance loci underlying tuberculosis pathogenesis could be mapped in * cc lines and * lines of the incipient c bl/ dba/ (bxd) cross. interestingly, bacterial modules could be mapped onto the host genome to understand how both host and pathogen genomes determine disease outcome. the mapping resolution of the do population was also highlighted in cancer, with nigel crawford (o- ; national institutes of health, bethesda, md) using quantitative trait locus (qtl) mapping in tramp j:do f males to identify metastasis susceptibility loci for prostate cancer. the power of the enu approach was demonstrated by several talks. gaetan burgio (o- ; australian national university) identified host factors altering malaria infection outcomes that could be targeted as a novel host-directed antimalarial therapy, and kart tomberg (o- ; university of michigan) mapped thrombosis modifier genes by bulk exome sequencing mice from a sensitized enu suppressor screen. both talks featured the use of gene editing candidate mutations with crispr/cas to validate causative alleles. the impact of mouse models on precision oncology was showcased by carol bult (o- ; the jackson laboratory), who discussed how patient-derived xenograft models can provide a platform for testing therapeutic options to guide treatments for breast and other cancers (fig. ) . kate ackerman (o- ; university of rochester) used inducible wt creert to determine the impact of loss of ctnnb at different time-points, concluding that b-catenin is critical for diaphragm development during a defined window of time. han kyu lee (o- ; duke university) analyzed polymorphisms among the ancestral haplotypes of inbred mouse strains to map loci for ischemic stroke outcomes, validating the interleukin receptor as a candidate by analysis of gene expression patterns and knockout models. other features of this session included a gwas of aerobic capacity in rats segregated on running ability by yu wang german center for neurodegenerative diseases tuebingen) conducted a massive forward genetic screen using human exome data, followed by systematic rnai screens in worms, flies, and human cell lines to identify genes and pathways involved in parkinson's disease. this year's imgc included a mini-session that sought to address the question of whether or not the mouse is still relevant as a model for human disease. while this particular audience needed no convincing of the fundamental scientific understanding gained through use of the mouse as a model, tsuyoshi miyakawa (o- ; fujita health university) gave a thought-provoking narration of his lab's response to a controversial publication claiming otherwise. his careful consideration of arguments made from both sides of the controversy emphasized the importance of understanding the experimental design and methods for analyzing a study before interpreting its results. miyakawa compared his re-analysis of mouse genomic data from seok et al. ( ) with the original study, which had concluded that genomic responses in mouse models poorly mimic human inflammatory diseases. miyakawa's group drew the opposite conclusion from the data, demonstrating that responses in mouse models greatly mimic human inflammatory diseases (takao and miyakawa ) . alterations to the seok et al. ( ) analysis included comparing genes that exist in both mouse and human, not just human disease genes that lack rodent homologs. he emphasized the need to define appropriate phenotypes and choose appropriate statistical methods. ultimately, he concluded, the % overlap in gene expression between mouse and human does not mean that the mouse is a bad model; instead, the overlapping % probably contain the genes that are the most important for disease. the panel responses also emphasized other advantages of mouse models, including but not limited to access to tissue, ability to measure responses at different time-points on the same background and the capacity to do epigenetic studies. the symposium also raised a conundrum of the scientific review process by highlighting ways in which the design and methods chosen to address a question can either obscure or reveal scientific truths, inciting a thoughtful series of questions about training and the process of peer review. how can we ensure that future generations of biologists are adequately trained to evaluate statistical methods? when the same data can be analyzed to show very different results, potentially affecting funding decisions on models, is the ultimate onus for publication on the journal or expert scientific reviewers? these questions were discussed in the open forum and reflect a larger conversation taking place within the wider scientific community, where they will undoubtedly continue to be discussed. this plenary session encompassed the use of mouse embryonic stem cells (mescs), gene expression analysis, and recent advances in genome engineering to address fundamental questions about development and degenerative disease. anne czechanski (o- ; the jackson laboratory) described how her experience deriving novel pluripotent mescs with the inhibitor cocktail i led to the unexpected observation that female cell lines experience a higher rate of attrition than male cell lines. future transcriptional profiling may uncover why the combination of x chromosome dosage and i culture conditions leads to attrition of female lines and will have important implications for those using mescs. sandra richardson (o- ; university of queensland) used retrotransposon capture sequencing (rc-seq) to deduce the timing and frequency of retrotransposon insertions in multigeneration c bl/ pedigrees. she presented data showing retrotransposition in the early embryo resulting in somatic and germline genetic mosaicism, adding to the evidence for retrotransposition as an important source of genetic diversity. patrizia rizzu (o- ; german center for neurodegenerative diseases) shared how she used fantom data to understand how a hexanucleotide repeat expansion influences the transcriptional profile of c orf , a gene involved in neurodegenerative disease. yasuhide furuta (o- ; riken) derived compound mutant mice from mescs containing multiple targeted mutations in the fibroblast growth factor (fgf) signaling system to study the role of fgf family genes in eye development. due to tight linkage between the genes of interest and reduced fertility in fgf mutant lines, it was previously impossible to generate compound mutants from an experimental cross. however, the development of crispr/cas allowed furuta's group to measure the extent of functional redundancy within the fgf pathway and uncover novel roles for its constituents. gabriela sanchez-andrade (o- ; wellcome trust sanger institute), recipient of this year's verne chapman young investigator award (table ) , closed the session with a discussion of her efforts to identify new biomarkers of neurodegenerative disease in a mouse model of frontotemporal dementia and parkinsonism (ftdp) with severe olfactory deficits. olfactory dysfunction is one of the earliest and most common symptoms of neurodegenerative disease in humans, yet the underlying molecular mechanisms are unknown. sanchez-andrade's impressive analysis of the olfactory epithelium measured differential expression and protein dynamics in wild-type and transgenic mice to identify a set of candidate genes correlated with onset of ftdp. furthermore, she demonstrated that similar changes in the expression of these genes occur in other brain regions relevant to ftdp pathology, highlighting the potential of her approach to identify additional biomarkers of human disease. the imgc featured three sessions on genomics and computational analysis. although the selected talks were diverse in both content and approach, each of them touched upon at least one of the following themes: advances in omic technology, molecular mechanisms underlying complex traits, and the relationship between genomic architecture and mammalian evolution. speakers in the first session discussed their experiences with single-cell rna-seq to address a series of questions that remain integral to the imgc each year; namely, what tools and technologies are driving our field forward? what exciting possibilities do they create, and what obstacles do we face in using these methods and interpreting their results? anna mantoski (o- ; roslin institute) shared her solutions to some of the analytical puzzles that technical variability can create for single-cell sequencing experiments and described how studying differences in gene in describing cap analysis of gene expression (cage), a method for capturing single-cell transcriptomes, charles plessy (o- ; riken) highlighted many of the challenges familiar to researchers using rna-seq and shared how his strategy of combining cage with techniques including ''pseudo-random'' primers to remove rrna, molecular tagging, and fragmentation can improve the quality of single-cell data. anton kratz (o- ; riken) further emphasized cage's potential as he described how his group applied translating ribosome affinity purification (trap), which can isolate the ribosome-associated transcriptome (the translatome) to purkinje dendrites to measure transcription at the subcellular level and identify biomarkers for specific cell types. much of the remaining work sought to address broad evolutionary questions dealing with the relationship between biological mechanisms, complex traits, and genomic architecture. martin taylor (o- ; university of edinburgh) discussed how the distribution of replicationassociated polymorphisms in mouse and human genomes may be explained in part by patterns of transcription factor binding and chromatin accessibility in the paternal germline. satoshi oota (o- ; riken) used enu mutagenesis in the mouse to observe evolution in real time, which allowed gaining insight into how the distribution of gc content has evolved in mammalian genomes. andrew morgan (o- ; university of north carolina chapel hill) explored the functional and evolutionary impacts of large copy number variations at a specific locus in the mouse genome. another prominent theme among the research featured in the sessions on genomics and computational analysis was the relationship between genotypes and quantitative phenotypes at both the level of the cell and the organism. jason lin (o- ; chiba cancer center research institute) discussed how a new class of molecules that combine the histone deactylase inhibitor saha and dna-binding pyrrole-imidazole polyamide (saha-pip) can induce epigenetic reprogramming and regulate pluripotency. steven munger (o- ; the jackson laboratory) used a system's genetics approach in the do to resolve the conflict between the expectation of high mrna-protein expression correlation from the central dogma of biology and recent observations suggesting a weak correlation. his work showed that while the levels of many proteins are regulated by nearby variants that influence mrna expression levels (cis-eqtl), the relative stoichiometry of proteins in stable partnerships and complexes is a key posttranslational regulator of protein abundance that can act to buffer individual member proteins against cis-acting transcriptional variation. robert young (o- ; university of edinburgh) presented evidence of divergence in the patterns of promoter gain and loss in humans and mice, offering insight into the evolutionary processes that contribute to gene expression and phenotypic diversity in both species. peter williamson (o- ; university of sydney) used a panel of inbred mouse strains to identify qtl related to metabolism, body composition, lactation, and other complex traits. a common approach featured at the imgc each year is the use of the mouse as a model for understanding how biological processes influence and respond to changes in the mammalian genomic landscape. notably, this year's sessions on genomics and computational analysis also included speakers that used a variety of other organisms in their research. in addition to the several talks mentioned above, we heard from david beier (o- ; seattle children's research institute) who analyzed the genomewide distribution of nonsense mutations in the exomes of individuals without severe mendelian disorders. he found that the strength of heterozygote selection correlated with the likelihood of recessive lethality and that many genes with established roles in developmental diseases had high heterozygote selection. martin frith (o- ; national institute of advanced industrial science and technology, tokyo) used sequence data from humans, chimps, orangutans, and dogs to classify different types of human chromosomal rearrangements, which occur in slowly evolving regions. finally, isaac adeyemi babarinde (o- ; national institute of genetics) introduced the audience to the capybara (the world's largest living rodent) and shared how he used its genome to understand the relationship between mutation rate and body size in rodents. for the first time at an imgc, two plenary sessions were devoted to epigenomics and noncoding rnas. this highlights the increasing awareness of looking beyond the traditional gene-protein relationship for understanding transcriptional control in both normal and diseased states in mammals. the influence of parental diet was featured in two presentations. joseph nadeau (o- ; pacific northwest research institute) illustrated that both folate supplementation and mutations in rna modulating genes such as dnd , a cf, and apobec bias fertilization toward wildtype genotypes. johannes beckers (o- ; helmholtz zentrum, munich) reported the epigenetic inheritance of an acquired metabolic disorder. he showed that a parental high-fat diet increased offspring susceptibility to obesity and type diabetes. both speakers stressed the importance of understanding the underlying mechanisms related to diet for guiding future public health policies. different aspects of genomic location as a determinant of transcriptional regulation were also discussed in the first plenary session. through determination of the three-dimensional configuration of x chromosomes in different tissues and cells, christine disteche (o- ; university of washington) demonstrated that genes that escape x inactivation preferentially localize at the periphery of the nucleus. she also showed that the expressed alleles of imprinted genes have greater chromatin contacts than the silent alleles, suggesting that these expressed regions are under greater organizational constraints. reanalysis of the publicly available encode data led siddharth sethi mrc harwell) to the discovery that transcription factor-binding sites are highly enriched in dnase hypersensitive sites compared to promoter and enhancer regions. as such, dnase hypersensitivity data could be used as an alternative method for discovering enriched regulatory motifs with the aim of improving understanding phenotypic variability. the second plenary session continued the theme of using genomewide data to understand transcriptional control by different noncoding elements-promoters, enhancers, microrna, and l -transposable elements. to dissect the relationship between dynamic changes in mrna and enhancer rna, erik arner (o- ; riken) measured the activities of promoters and enhancers over time in a number of cell types following different biological stimuli. he presented data supporting enhancer transcription as the earliest event in successive waves of transcriptional change. this phenomenon was observed in multiple biological systems, suggesting this may be a general feature of mammalian transcriptional regulation, contrary to models showing coexpressions of enhancers and promoters. albin sandilan (o- ; university of copenhagen) provided a clinical application, presenting rna-seq data profiling colon specimens from patients with inflammatory bowel disease. a promoter set was identified, which could accurately distinguish between the primary disease subtypes: crohn's disease and ulcerative colitis. moreover, , active enhancer regions were identified with subsets induced in general inflammation or specifically in one subtype. these enhancers had links to both known and novel genes involved in the pathogenesis of inflammatory bowel disease. michiel de hoon (o- ; riken) turned attention toward the role of mirna in gene regulation. he presented the results from a collaboration spanning centers internationally, which analyzed deep, sequencing data of paired small rna and cage libraries across a wide range of cell types. this analysis revealed two classes of mirna: celltype-specific mirnas and ubiquitous mirnas. cell-typespecific mirnas are highly expressed in only a few cell types and may act as buffers of gene expression. ubiquitous mirnas are expressed in most cell types, but depleted in particular cell types and may be important in preventing inappropriate activation of transcriptional programs. valerio orlando (o- ; ircss fondazione santa lucia) presented the last talk in the plenary session focusing on the epigenetic role of retrotransposable elements, specifically long interspersed nuclear elements (l ). analysis of l transcription followed that of enhancer elements and myogenic program in normal muscle cells but was absent in duchenne muscular dystrophy (dmd)-affected muscle cells. pharmacological rescue of the dmd phenotype by histone deacetylase inhibitors or gene therapy was accompanied by normal l expression. he proposed that deregulation of l mobilization is a key trait in loss of cell identity and disease. genome editing using crispr/cas technology has taken the scientific world by storm, allowing rapid and efficient editing in eukaryotic cells. the in vitro and in vivo applications of crispr/cas genome editing was a strong theme at this year's meeting, with every plenary session including talks that made use of this technology, as well as the plenary session being completely devoted specifically to advances in genome editing. marie-christine birling (o- ; institut clinique de la souris) kicked off the session demonstrating crispr/cas genome editing in rats. she described her group's effort to generate alleles with precise gene deletions and duplications of a -mb region by means of two different guide rnas on both sides of the target region. kazuto yoshimi (o- ; national institute of genetics) then combined crispr/cas ''scissors'' with single-stranded oligodeoxyribonucleotides as the ''paste'' mechanism to ligate the cut sites for efficient replacement of rat genes with human genes. finally, dave bergstrom (o- ; jackson laboratory) presented his lab's modified crispr approach to enable rapid ''humanizing'' of large segments of the mouse genome, giving the example of replacement of a mouse tumor suppressor gene with kb of the orthologous human gene. inbred strains with whole-genome illumina sequencing and discussed the challenges of making alignments in complex regions. attendees were treated to the test site launch of the latest data available through the uscd genome browser (http://www.hgwdev-mus-strain.sdsc.edu/cgi-bin/hggateway). the verne chapman lecture titled ''the hidden layer of regulatory rna in mammalian genome biology'' was delivered by eminent molecular biologist john mattick (o- ; garvan institute of medical research). mattick is known for his research in revealing the central role of nonprotein-coding dna in the production of regulatory nonprotein-coding rnas (ncrnas) and his efforts to understand how ncrna regulation contributes to the staggering level of phenotypic complexity observed throughout the animal kingdom. he began his lecture with the quote ''are we letting a philosophy of the protein-coding gene control (our) reasoning? what then is the philosophy of the gene?'' he pointed out that this quote was not from this year or even this century-it was in fact a concern attributed to nobel laureate barbara mcclintock years ago. he reminded us that the mammalian genome contains only * , protein-coding genes, the same number as in simple nematodes. on the other hand, the extent of nonprotein-coding dna increases with increasing developmental and cognitive complexity, reaching . % in humans. he guided us through the events leading up to his discovery and the explosion of studies that followed, peppering his tale with vivid descriptions of the findings and figures that have influenced him over the years. he shifted effortlessly between concrete descriptions of data and philosophical speculations on the evolution of paradigm shifts, the mysteries of biological complexity, and the silent assumptions that inform (and occasionally hinder) scientific practice. he entertained the audience with his encyclopedic knowledge of molecular genetics, framing his research within a larger historical context that served to complement data-driven descriptions of his and others' work establishing ncrnas as prominent regulators of development, cognition, and disease in the mammalian genome. mattick also elaborated on the relevance of ncrnas to incipient areas of research and technology development in epigenomics and neurobiology, creating a memorable and thought-provoking experience for both experienced investigators and trainees (fig. ) . this year there were two additional keynote lectures, both showcasing scientific advancements using induced pluripotent stem cells (ipscs). masayo takahashi (o- ; riken center for developmental biology) provided a historical perspective of ipscs, from basic research to clinical application. it is remarkable that the field has gone from the invention of ipsc technology to the clinic in only years. dr. takahashi presented the first human application of ipsc-derived targeting age-related macular degeneration, a retinal disease. she described an impressive panel of experimental validations for retinal pigment epithelial cell sheets derived from ipscs, including whole genome, genotyping arrays, methylome, and single-cell analysis. next, her group hopes not only to treat the retinal epithelium but also to use ipscs to derive photoreceptors to completely restore vision in patients. the talk discussed the use of allogenic as well as autologous ipscs and treated the audience to an early view of potentially revolutionary treatments utilizing ipscs, highlighting the steadily growing wave of interest in this technology. hideyuki okano (o- ; keio university graduate school of medicine) then went on to discuss the use of ipscs in the study and treatment of neurological disorders. he described work to establish ipscs from patients with psychiatric disorders and characterize their pathophysiology. in a quest to investigate human psychiatric and neurological disorders more effectively, hideyuki then presented his group's impressive work generating transgenic marmoset models of parkinson's disease. the marmosets express human synuclein and recapitulate typical human diseases including sleep disturbances, lewy bodies, tremors, and gait abnormalities. in summary, the meeting showcased a variety of cutting edge mammalian genetic approaches, concepts, and results-from large-scale mapping to single gene approaches and everything in between. the meeting ended in a spectacular fashion, with attendees treated to the spa and fig. a spectacular finale for an amazing conference massage facilities at the yokohama minatomirai manyo club, donning kimonos and yakutas for a traditional banquet (fig. ) . attendees were entertained by local performers before awards were made to acknowledge exceptional trainee oral and poster presentations, and to thank the outgoing imgs secretariat and nominations and elections committee. it is certainly the first and only imgs meeting to date where ongoing scientific collaborations were made and discussed in a traditional japanese onsen: a most fitting end to a wonderful meeting. we eagerly await the next imgc, which will be part of mouse genetics at the allied genetics conference (tagc) in orlando, florida in july . that conference will bring the mouse genetics community together with yeast, ciliate, c. elegans, drosophila, and zebrafish communities to highlight the importance of model systems in understanding and translating fundamental advances in genetics. updates about tagc and the following imgc in heidelberg, germany in can be found at www.imgs. org. follow us on twitter (https://www.twitter.com/imgs_ news) or facebook (https://www.facebook.com/mamma lian.genome/). genomic responses in mouse models poorly mimic human inflammatory diseases genomic responses in mouse models greatly mimic human inflammatory diseases key: cord- -e ih fkx authors: rahman, md. siddikur; peeri, noah c.; shrestha, nistha; zaki, rafdzah; haque, ubydul; hamid, siti hafizah ab title: defending against the novel coronavirus (covid- ) outbreak: how can the internet of things (iot) help to save the world? date: - - journal: health policy technol doi: . /j.hlpt. . . sha: doc_id: cord_uid: e ih fkx • iot within infectious disease epidemiology is an emerging field of research, however the ubiquitous availability of smart technologies, as well as increased risks of infectious disease spread through the globalization and interconnectedness of the world necessitates its use for predicting, preventing and controlling emerging infectious diseases; • considering the present situation in china, iot based smart disease surveillance systems have the potential to be a major breakthrough in efforts to control the current pandemic. with much of the infrastructure itself in place already (i.e. smartphones, wearable technologies, internet access) the role this technology can have in limiting the spread of the pandemic involves only the collection and analysis of data already gathered. • more research must be carried out for the development of automated and effective alert systems to provide early and timely detection of outbreaks of such diseases in order to reduce morbidity mortality and prevent global spread. the outbreak of a new coronavirus (covid- ) that originated in wuhan, china, seems to be unstoppable. the virus has already infected more than , people around the world, with at least , deaths as of march , . , the outbreak of pneumonia of unknown aetiology started in wuhan, hubei province in china , linked to the huanan seafood market. the virus is of the genus betacoronavirus and is related to the viruses that cause middle eastern respiratory syndrome (mers) and severe acute respiratory syndrome (sars). the who was first alerted on the st of december ,although several cases of pneumonia of unknown aetiology were detected much earlier (dec , ). this delay in declaring the occurrence of an outbreak and failure to alert international authorities in a timely manner contributed to the uncontrolled spread of the disease. this pandemicis now the focus of global attention. in an effort to mitigate the spread of the virus, china expanded a lockdown of the focal city wuhan that included cities, and encompassed million individuals. experts initially raised concerns regarding the sustainability of this effort and have warned that the country was at risk for a repeat of an epidemic similar to the sars epidemic. , at this time it appears that the pandemic is under control in most of china, although criticism remains regarding the use of what some have termed "draconian" measures for stifling its spread. the world is now struggling to control the unprecedented spread of the virus which includes a record number of morbidities and mortalities. since there is no specific treatment for coronaviruses, and efforts to contain the spread have thus far failed , there is an urgent need for global surveillance of individuals with active covid- infection. the inclusion of an integrated digital disease surveillance system may be crucial to the control of this disease. a wealth of new technologies in the form of the internet of things (iot) is gaining growing global attention and becoming increasingly available for predicting, preventing and monitoring emerging infectious diseases. the iot refers to an interconnected web of smart devices, sensors, and individuals through which data can be collected in its raw form and transmitted through the internet to be analyzed for patterns or trends. state-of-the-art iot-enabled health monitoring systems (health monitoring systems) provide real-time surveillance through the use of wearable health-monitoring devices, cloud-based remote health testing, and artificial intelligence (ai). these monitoring systems utilize, in real-time, social media, public data, and health data, combined with the use of supervised, unsupervised, and machine learning. when ai and machine learning merge with distributed cloud, practical blockchain, system software automation, and ai speech recognition, the health monitoring systems enable the creation of a reliable remote monitoring system between patient and doctor. features of this iot-enabled health monitoring systems include online triage, ai secure chat, and telehealth. these technologies are now easily accessed via simple user interfaces on reliable mobile apps and webbased systems due to lightweight application programing interfaces (apis) and edge computing ability. with the rise of these technologies, data privacy has become an increasing concern specifically regarding the potential for data misuse and abuse. a new field within information technology has emerged, termed digital ethics. this branch of ethics is the study of moral problems relating to data and information, algorithms, and corresponding practices and infrastructures, described in detail elsewhere. therefore, hospital and immigration must be ready to share critical information such as data of an increased number of patients with high fever and people movement going in and out of the country to the iot system to allow them to be analyzed in real time. in addition, all related equipments, especially edge servers and cloud servers with a g network, need to be installed to ensure fast transaction to all devices accessed by the computation engines and the different levels of end users. the new wave of digitizing medical records has caused a paradigm shift in the healthcare industry. the industry is witnessing an increase in the sheer volume of data in terms of complexity and diversity. big data is emerging as a plausible solution with the promise to transform the healthcare industry. a paradigm shift from reactive to proactive healthcare may result in an overall decrease in healthcare costs and eventually lead to economic growth. while the healthcare industry harnesses the power of big data, security and privacy issues are becoming increasingly important as emerging threats and vulnerabilities grow. when dealing with healthcare monitoring, privacy and data security should be carefully considered. developers can help to integrate security into devices, applications, and systems. with regard to data sharing, developers can use a client-server model, in which the server shares a certain type of information with clients while keeping other information protected by appropriate credentials. iot within infectious disease epidemiology is an emerging field, however, the ubiquitous availability of smart technologies, as well as increased risks of infectious disease spread through the globalization and interconnectedness of the world necessitates its use for predicting, preventing and controlling emerging infectious diseases. web-based surveillance tools and epidemic intelligence methods have recently emerged in several countries to facilitate risk assessment and timely outbreak detection, however widespread use of the available technologies is lacking. due, in part, to the rapid growth of the chinese economy and globalization, iot-enabled health monitoring in a global healthcare infrastructure would provide targeted information for health officials, and has the potential to improve efforts to locate, contain, and prevent infectious diseases. it may help to quickly diagnose infected patients and accurately predict the possible spread of a disease to other locations utilizing travel data. ultimately, an iot based surveillance system might help reconstruct the progression of an outbreak and stabilize the economy of the source country rather than having to lock down major cities, borders, and businesses. web-based tools for surveillance of the flu virus (influenza) have been utilized. for example, google flu trends (gft) was tracking health-related search engine queries in order to monitor, in real-time, influenza activity. however, it is important to note that gft was discontinued due to concerns regarding data inaccuracy. the flaw in gft highlights a commonplace issue in big data analysis (and any data analysis), overfitting of data to a small number of cases. the failure of gft emphasizes the utilization of other real-time health data for predicting trends in infectious diseases. a more critical evaluation of the uses of iot in surveillance must be addressed, as well as a deeper look into the privacy concerns that its use brings. in addition to web-based tools for surveillance, event-based iot tracking collects and sends raw data from a multitude of informal sources (news articles, social media posts, internet queries) in an attempt to detect events with potential epidemic spread faster than traditional more conservative methods. this has led to advances in infectious disease modeling and pathogen discovery and diagnostics (rapid molecular identification of pathogens). mobile computing in the form of mobile health (m-health) can also increase the efficiency of a healthcare system by involving various services, applications, third party apis, and mobile sensors not otherwise used for health-related purposes. safety and health monitoring applications such as wearable iot enables real-time health monitoring and would be beneficial for improving global health. these technologies could reduce gaps in monitoring systems that exist due to a sheer incapacity to monitor such large geographical areas or populations. , a data mining module, which is machine learning-based approaches such as the support vector machine (svm) is also fundamental for detection, forecasting, and diagnosis of potential diseases. these approaches have been applied in the fields of computer science and medical diagnoses but are relatively novel within the field of infectious disease epidemiology. considering the present global situation, iot based smart disease surveillance systems have the potential to be a major breakthrough in efforts to control the current pandemic. with much of the infrastructure itself in place already (i.e. smartphones, wearable technologies, internet access) the role this technology can have in limiting the spread of the epidemic involves only the collection and analysis of data already gathered. the combined role of iot and related new technologies might shape early recognition of outbreaks and prevent the spread of zoonotic infectious diseases including the covid- , if potential steps are taken to enhance and utilize the data. smart disease surveillance systems based on iot would provide simultaneous reporting and monitoring, end-to-end connectivity and affordability, data assortment and analysis, tracking and alerts, as well as options for remote medical assistance to be adopted, to detect and control zoonotic infectious disease outbreaks in china and other affected countries. more research must be carried out for the development of automated and effective alert systems to provide early and timely detection of outbreaks of such diseases in order to reduce morbidity, mortality and prevent global spread. these prompt and effective public health measures need to be taken to avoid the risk of continuing outbreaks and the possibility of a local outbreaks turning into a global pandemic such as this one. mapping the wuhan coronavirus ( -ncov) novel coronavirus ( -ncov) situation reports world health organization (who) world health organization (who). coronavirus epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study china locks down million people as anger grows over virus china expands virus lockdown, encircling million wearable iot enabled real-time health monitoring system new technologies in predicting, preventing and controlling emerging infectious diseases soft ethics, the governance of the digital and the general data protection regulation security for mobile and cloud frontiers in healthcare on secure data management in health-care environment new technologies in predicting, preventing and controlling emerging infectious diseases big data. the parable of google flu: traps in big data analysis internet of things for healthcare using effects of mobile computing: a systematic literature review an internet-of-things (iot) network system for connected safety and health monitoring applications comparative evaluation of the use of artificial neural networks for modelling the epidemiology of schistosomiasis mansoni key: cord- -qilq q h authors: taniguchi, kiyosu; yoshida, makiko; sunagawa, tomimasa; tada, yuki; okabe, nobuhiko title: imported infectious diseases and surveillance in japan date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: qilq q h surveillance of imported infectious diseases is important because of the need for early detection of outbreaks of international concern as well as information of risk to the travelers. this paper attempts to review how the japanese surveillance system deals with imported infectious diseases and reviews the trend of these diseases. the cases of acquired infection overseas were extracted from the surveillance data in – . the incidence and rate of imported cases of a series of infectious diseases with more than one imported case were observed by the year of diagnosis and place of acquired infection. during the period , cases that could be considered to be imported infectious diseases were identified. shigellosis ranked as the most common imported disease, followed by amebiasis, malaria, enterohemorrhagic escherichia coli infection and the acquired immunodeficiency syndrome, typhoid fever, dengue fever, hepatitis a, giardiasis, cholera, and paratyphoid fever. the annual trends of these diseases always fluctuated but not every change was investigated. the study reveals that the situation of imported infectious diseases can be identified in the current japanese surveillance system with epidemiologic features of both temporal and geographic distribution of cases of imported infectious diseases. however, further timely investigation for unusual increase in infectious diseases is needed. because of the current global travel and trade, there is no border for infectious diseases. even in japan, which belongs to a temperate climate zone many tropical infectious diseases are found in the local hospitals. but there have been several case reports describing difficulty of early diagnosis and treatment. e it is important to provide information to travelers on particular risks and to increase protection, as well as information for local clinicians on the current situation of endemicity of infections in the foreign countries in order to facilitate early diagnosis and to avoid nosocomial infection. from the viewpoint of public health, the introduction of new pathogens may result in their establishment in the country. public health surveillance is one of the essential components for infectious disease control and no doubt a starting point for control. because of current circumstances a surveillance system should be designed not only at the national level but also at the global level of infectious disease control. current national epidemiological surveillance for infectious diseases (nesid) in japan requires that all notifiable diseases should be reported with the presumptive place of infection. this report summarizes the data from the nesid from to on the situation of imported infectious diseases in japan. the national epidemiological surveillance for infectious diseases (hereafter referred to as nesid) is conducted based on the law concerning the prevention of infectious diseases and medical care for patients of infections (hereafter referred to as the infectious disease control law) enacted in april . infectious disease surveillance system before then is described elsewhere. infectious diseases included in this law were categorized into iev with specific means for control based upon the public health impact of each disease as shown in table . all physicians must report cases of categories ieiv immediately and va within days after identification to local public health centers which are the primary level institution for disease control and prevention located strategically throughout the nation. local public health centers are expected to enter data into the nationwide electronic surveillance system, which enables data to be shared throughout the system including all local public health centers, local and national governments, quarantine stations, local infectious disease surveillance center, local public health laboratory and central infectious disease surveillance center, which is the infectious disease surveillance center of national institute of infectious diseases. category vb diseases, which include sentinel reporting diseases, should be reported by designated sentinel medical institutions weekly or monthly with the number of clinical cases aggregated by sex and age groups. all reports should be compatible with the reporting criteria which were documented in detail for each disease including clinical and laboratory case definitions for categories va and vb of hospital sentinel reporting disease, and only clinical case definitions for other vb sentinel reporting diseases. cases of category ieva diseases should be reported with sex, age, method of laboratory confirmation, symptoms on diagnosis (descriptive), date of onset, date of consultation, date of diagnosis, estimated date of infection, date of death (if patients died), area of permanent residence (in-country or foreign countries), presumptive place of infection (domestic or foreign countries), contact to the vectors or activities on the fields (yes or no), estimated infection route, another patients in the family members, colleagues, or neighbors (cluster or not). the presumptive place where infection was acquired should be described based on reasonable situation considering travel history and incubation period according to the interview of patients. the cases with the presumptive place of infection in a foreign country (hereafter referred to as imported cases) were extracted from the nesid data from april to march . data in are only available in apriledecember because of the change of the law in april and data in are included until march. finally nine years data are reviewed. annual trend of total, imported, and domestic cases of disease containing one or more imported one are recorded and attributable events and causes are investigated with information in the line listing data and relevant epidemiological reports. incidence rates per , , population are calculated using the census population and imported disease per , , outbound travelers are calculated using the outbound travelers by the japan national tourist organization. in the period observed , cases that could be considered to be imported infectious diseases were identified. these include various infectious diseases as listed in table with reported number of cases (imported, domestic, unknown and total), imported case rate among imported and domestic cases, incidence rate of domestic cases per year per , , population and the incidence rate of imported cases per year per , , outbound travelers. shigellosis ranked as the most common imported infection, followed by amebiasis, malaria, enterohemorrhagic escherichia coli (ehec) infection and the acquired immunodeficiency syndromes (aids), typhoid fever, dengue fever, hepatitis a, giardiasis, cholera, and paratyphoid fever. the rate of imported diseases of malaria, dengue fever and rabies is complete as they are not endemic in japan and over % in coccidioidomycosis, paratyphoid fever, typhoid fever, cholera, shigellosis and echinococcosis (echinococcus granulosus). although coccidioides is not considered to be indigenous, a domestic case is identified with no history of overseas travel. however, this case was a dealer of imported cotton and he may have acquired the infection from fungi attached to the imported cotton. the annual trends of imported diseases always fluctuate because of the local situation and sometimes there is sudden increase because of cluster among the same tour groups. the amebiasis tended to increase recently both in domestic and imported cases. and cases acquired infection through sexual contact represented % of the total cases. there were continuous reports of imported disease of aids, syphilis and hepatitis b. dengue fever is increasing year by year, but malaria is decreasing gradually. typhoid and paratyphoid fever and hepatitis a showed an increase and decrease throughout observation period. although the outbreak among group tours to endemic countries was reported to account for the increase of imported diseases, investigation of attributable events or causes were not always made in a timely manner. retrospective investigation could recognize the increase of cases returning from certain countries, but it was difficult to seek further risk factors because limited information was listed on line. public health surveillance is defined by the world health organization as the ''systematic ongoing collection, collation, and analysis of data and the timely dissemination of information to those who need to know so that action can be taken.'' the basic principle for disease control and prevention is the same no matter where it is acquired. but target groups who need to know differ. the precautionary information should be communicated to travelers with the risk assessed properly. of course rapid detection of cases can lead to rapid response and early containment and finally to prevention of indigenous transmission of exotic pathogens. in this respect, effective infectious disease surveillance is essential. in this study it was not difficult to overview the situation of imported infectious diseases because the current japanese surveillance system requires the presumptive place of infection including the specified country if possible. but there have been no studies on the evaluation of reporting rate. before april when the infectious disease control law was revised, e cases of malaria were reported annually in the framework of the old infectious disease prevention law, but the research group on chemotherapy of tropical diseases reported by their field investigation that approximately patients have been confirmed with malaria annually. it means that cases notified to the ministry of health and welfare were about % in those days. reports of malaria peaked in one year after the new law enactment and steadily decreased to the same level before . on the other hand, dengue fever is increasing. it is not possible to determine whether the reporting rate of malaria has decreased recently or not, it will be necessary to evaluate the surveillance system including assessment of missed opportunity for diagnosis and treatment. it might be better to report febrile illness with travel history abroad for effective detection and evaluation. it is natural that reported imported cases of malaria, dengue fever and rabies are complete. domestic case of coccidioidomycosis is reported to be caused from imported materials. a proportion of typhoid/paratyphoid fever, cholera and shigellosis are acquired inside the country without doubt because there is no travel history abroad. as there is a report of vibrio cholerae from imported food, further is required investigation of the source of infection in each cases. most of the imported cases were reported with the suspected country of infection. analysis using e data showed suspected countries of infection for cholera are india, philippine, and indonesia (in descending order); india, indonesia, and china for shigellosis; india, indonesia and nepal for typhoid/paratyphoid fever; philippine, thailand and india for dengue fever; papua new guinea, nigeria, india and indonesia for malaria. but it depends upon the number of travelers and the local situation in certain countries which might change year by year. more detailed analysis using country specific travelers is necessary. aids, syphilis, hepatitis b, and giardiasis are part of imported infectious diseases. as they have the unique feature as sexually transmitted diseases (std), it might be better to handle these separately. but it is important to monitor imported std because they could increase local infection rates, and to provide information for travelers. in the current study, it was noted that unusual increases of reported imported infectious disease were not fully investigated for attributable events or causes in a timely manner although several events affecting the number of reports were identified. retrospective analysis can provide the country of infection, but more timely information is necessary for travelers. the capacity of timely investigation and risk assessment should be enhanced further. the results of investigation of an outbreak among a tour group sharing common source of infection or cluster in time and of travel place of individual tourists not related each other will be reflected or involved in a local epidemic, which can be linked to international investigation and control activities. under the current circumstances of pandemic alert, the timely sharing of imported infectious disease at the global level will also be necessary. difficulty of proper diagnosis for an imported vivax malaria patient from africa plasmodium vivax malaria with clinical presentation mimicking acute type idiopathic thrombocytopenic purpura clinical characteristics of imported malaria in japan: analysis at a referral hospital overview of infectious disease surveillance system in japan case definitions for reporting in compliance with infectious disease law anonymous. imported mycoses in japan. iasr: e . available from anonymous. cholera e , japan. iasr: e . available from iasr: e . available from enterohemorrhagic escherichia coli infection as of typhoid fever outbreak among group tour member to bangladesh cholera enterotoxin production in vibrio cholerae strains isolated from the environment and from humans in japan the author has no conflict of interest. key: cord- - me xh authors: wang, wei; chen, jin; sheng, hui-feng; wang, na-na; yang, pin; zhou, xiao-nong; bergquist, robert title: infectiousdiseases ofpoverty, the first five years date: - - journal: infect dis poverty doi: . /s - - - sha: doc_id: cord_uid: me xh although the focus in the area of health research may be shifting from infectious to non-communicable diseases, the infectious diseases of poverty remain a major burden of disease of global health concern. a global platform to communicate and share the research on these diseases is needed to facilitate the translation of knowledge into effective approaches and tools for their elimination. based on the “one health, one world” mission, a new, open-access journal, infectious diseases of poverty (idp), was launched by biomed central in partnership with the national institute of parasitic diseases (nipd), chinese center for disease control and prevention (china cdc) on october , . its aim is to identify and assess research and information gaps that hinder progress towards new interventions for a particular public health problem in the developing world. from the inaugural idp issue of october , , a total of manuscripts have been published over the following five years. apart from a small number of editorials, opinions, commentaries and letters to the editor, the predominant types of publications are research articles ( . %) and scoping reviews ( . %). a total of contributing authors divided between affiliations across countries, territories and regions produced these publications. the journal is indexed in major international biomedical databases, including web of science, pubmed, scopus and embase. in , it was assigned its first impact factor ( . ), which is now . . during the past five years, idp has received manuscripts from countries, territories and regions across six continents with an annual acceptance rate of all contributions maintained at less than %. content analysis shows that neglected tropical diseases (ntds), followed by the “big three” (hiv/aids, malaria and tuberculosis) and infectious diseases in general comprise % of all publications. in addition, a series of thematic issues, covering publications in all, was published as separate parts of the first five volumes. these publications were cited times, which equals an average of . times per publication. the current challenge is to identify cutting-edge research topics and attract and to publish first-rate publications leading to increasing importance and impact of the journal in its field. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. the global burden of disease today is less due to infectious diseases than non-communicable diseases, with chronic disorders such as heart disease, stroke and cancer currently being the leading causes of morbidity and mortality worldwide [ ] . however, infections remain a major global health concern, particularly in the developing world [ ] , where the human immunodeficiency virus and the acquired immune deficiency syndrome (hiv/aids), viral hepatitis, tuberculosis and malaria, still kill millions of people around the globe every year [ , ] . parasitic diseases, e.g., malaria and schistosomiasis, do not always remain limited to their age-old geographic distributions complicating control efforts and challenging the progress towards their elimination [ ] [ ] [ ] [ ] . additionally, emerging and re-emerging infectious diseases like the severe acute respiratory syndrome (sars), influenza, ebola virus disease (evd), dengue, middle east respiratory syndrome (mers) and zika virus disease threaten human health and global security [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . infectious diseases are inextricably linked to poverty in a vicious cycle [ ] . these diseases, characterized by high morbidity and mortality that mainly occur in resource-limited areas, belong by definition to the group of diseases that are more prevalent among poor and vulnerable populations [ ] . they rank within the top ten ailments with respect to years lost due to ill-health, disability or early death, expressed as disability-adjusted life years (dalys) [ ] . in a global perspective, this leads to huge economic losses, both for the individual and society, and a global platform to communicate and share the research on the infectious diseases of poverty was felt to be needed to facilitate the translation of knowledge into effective approaches and tools for the elimination of these diseases. on october , , infectious diseases of poverty (idp) was launched as a new, open-access (oa) journal on infectious diseases by biomed central, in partnership with the national institute of parasitic diseases (nipd), chinese center for disease control and prevention (cdc). its preferential aims are to identify and assess research and information gaps that hinder progress towards new interventions for public health problems, in particular those connected with poverty in the developing world. based on the "one health, one world" mission mentioned in the global report on research for the infectious diseases of poverty [ ] , the journal publishes work on topics and approaches that address essential public health questions related to this issue. along with the actions proposed by this report, idp, aims to achieve the goal of improving research capacity and create a better environment for the research on the infectious diseases of poverty [ , ] . the purpose of this article is to review the publication activities of idp in its first five years by comparing the record with other journals with a similar focus in order to understand the current trends of research in publications on infectious diseases. we performed an in-depth bibliometric analysis of all publications during the first five volumes covering the period from october , to october , through a joint search using the publication name "infectious diseases of poverty" in pubmed, web of science (wos) (formerly isi web of knowledge) and the journal's website. we also compared with two other journals in this area, international journal of infectious diseases and bmc infectious diseases. the publication data and metrics of idp, international journal of infectious diseases and bmc infectious diseases were collected using these journals' homepages, submission systems and the wos citation database as of th february, . we used ordinary descriptive statistics to analyse the performance of idp during its first five years supported by biomed central's databases [ ] . idp is currently managed by an editor-in-chief and two managing editors, one based in china and the other in france. they perform the bulk of the day-to-day activities, including communication with authors, choosing and contacting referees for peer review, and making the final decisions with regard to publication or rejection of manuscripts. this core team is supported by five deputy editors and associate editors from asia, africa, europe, north america, south america and oceania. to assure quality and impact from the start, idp appointed an international editorial board of well-known scientists with expertise within the fields of infectious diseases, parasitic diseases, social sciences and economy. the current editorial board consists of members, based in uk (n = ), usa (n = ), china (n = ), australia (n = ), switzerland (n = ), brazil (n = ), cameroon (n = ), india (n = ), nigeria (n = ), japan (n = ), ghana (n = ), belgium (n = ), thailand (n = ), greece (n = ), democratic republic of congo (n = ), south africa (n = ), kenya (n = ) and senegal (n = ). from the inaugural issue of october, up to and including october, , a total of manuscripts were published in idp. the annual publication record is presented in fig. . out of all publications, research articles ( . %) and scoping reviews ( . %) dominated with the remaining publication being editorials, opinion articles and letters to the editor (fig. ) . a total of contributing authors, divided between affiliations across countries, territories and, regions produced these publications during the five-year period (tables , and ). the publications included countries, territories and regions from the developing world ( %) [ ] . the journal is indexed in major international biomedical databases, including wos, science citation index expanded, medline, doaj, pubmed, scopus and embase. in , it was assigned the first impact factor ( . ), which is now . (fig. ) . up to th february , the publications in the first five idp volumes were cited times in total, i.e. a mean citation of . times per paper. table presents the most highly cited publications, which includes five reviews, two editorials, one opinion article, one research article and one letter to the editor. interestingly, the most highly cited publication in idp is an editorial published in [ ] , which may be explained by the fact that it dealt with surveillance and response defining this approach as a research priority during the stage moving towards elimination of tropical diseases, which received much global attention [ ] [ ] [ ] [ ] [ ] . the importance and significance of the surveillance and response approach are also highlighted by two other highly cited publications in idp [ , ] . we refer here to two reviews, one discussing gap analysis of the chinese three major tropical diseases (schistosomiasis, malaria and echinococcosis), and the other assessing the morbidity due to schistosomiasis japonica in china [ , ] . in addition, an editorial opinion focusing on the chinese schistosomiasis control and health systems and a research article reporting on babesia microti and plasmodium co-infections along the china-myanmar border were also commonly cited [ , ] . among three other publications, which have been cited times and more, are two reviews describing global epidemiology: the burden of infectious diseases of poverty [ ] and that of clonorchiasis [ ] , together with an editorial introducing the mission, aims and scope of idp [ ] . the number of articles of the publications accessed through internet reached a total of accesses up to th february, , with a mean of . times per publication. table summarizes the five most highly accessed publications, which have been accessed more than times. they include three research articles, one focusing on the interplay between infectious disease emergence and global climate change [ ] , one dealing with the risk factors of malaria among pregnant women in nigeria [ ] and one discussing the reaction of chinese social media to the outbreaks caused by the mers coronavirus and avian influenza a (h n ) [ ] . two reviews, one on tuberculosis [ ] and the other on malaria and other vector-borne protozoan diseases [ ] were also often downloaded. interestingly, the most highly accessed publication is a research article exploring the potential correlation between the emergence of infectious diseases and global climate change, perhaps explained by the currently worldwide focus on a large number of emerging and re-emerging infectious diseases in this context [ , ] . during the -year period investigated, countries idp received manuscripts from countries, territories and regions across six continents ( fig. ) that included from the developing world ( %) [ ] . the annual acceptance rate of all submitted manuscripts received was maintained at less than % throughout the period analysed (fig. ). content analysis showed that neglected tropical diseases (ntds), followed by the "big three" (hiv/aids, malaria and tuberculosis) and other infectious diseases, were in the focus of the publications, which consisted of % of total publications (fig. ). idp is dedicated to communicate global health concerns on finding ways for poverty alleviation and to publish papers dealing with the following topics: ( ) approaches addressing essential public health questions related to infectious diseases of poverty; ( ) multi-disciplinary concerns of infectious disease of poverty, such as the biology of pathogens, vectors, diagnosis, surveillance and response, treatment and case management, epidemiology including ecohealth issues and modelling, zoonoses and animal reservoirs, control strategies and implementation of new technologies; and ( ) trans-disciplinary or multi-sectoral effects involving health systems, environmental management and innovative technology. a total of thematic series were published in the first five volumes of idp with - issues appearing each year ( table ). the thematic series were issued to allow the journal to remain in the frontline in the field of swiss tropical public health institute university of basel the aga khan university fudan university the world's currently biggest donor for scientific research, the bill and melinda gates foundation (bmgf) demands that the outcomes of what it supports must be freely available to all. this is set to change scientific research publications at its core, since this requirement bars publication in 'premier-league' journals such as nature and science. increasingly, scientific journals are accepting the oa mandate and idp wisely decided to follow suit when its first volume appeared more than five years ago. as an international, peer-reviewed oa journal, idp has its own specific characters as follows: . the journal provides the abstracts in the six official languages of the united nations (un), which enables clear and convenient communication of public health concerns related to the infectious diseases of poverty [ ] . . fast-track publication is provided for articles of exceptional public health importance and urgency with online publication completed within a month of submission. . the journal focuses on publication of original and empirical trans-disciplinary research for the control of infectious diseases which predominantly affect poor populations. free preprint repository, biorxiv (http://biorxiv.org/), is another revolution quietly taking place and gradually found more and more useful for scientists as it establishes priority and exposure, while, on the other hand, there is no peer-review. this important development must be taken under consideration in the publication world and scientific journals, including, idp, need to find a way to participate in this expansion of how new findings are brought into the public sphere. in , a thematic series issue entitled "ecohealth and eids -dynamics between environmental change, development and eids in asia", which was supported by the international development research centre (idrc) of canada and published with the aim to showcase and encourage trans-disciplinary ecohealth research in asia and beyond. the publications in this thematic series were cited times, with a mean of . times per publication. it attracted downloads and reads in total, with a mean of per publication. since the launch and continuing, idp aims to identify and assess research and information gaps that hinder progress towards new interventions for the particular public health problem of poverty and infections in the developing world. in its first five volumes, the journal published studies from developing economies ( %) and attracted contributions from developing economies ( %); however, there are still more than % of the global developing economies that have not yet contributed a paper to the journal. further work to attract manuscripts from the developing world is urgently needed. in addition, % ( / ) of all manuscripts in the first five years were submitted by african scientists; however, even if only slightly more than % ( / ) of these were finally published, this indicates a strong african interest in publishing with idp as well as a trend of improving quality of research conducted in africa. the journal was assigned a first impact factor of . in ; however, this metric decreased to . in , which can be explained by the large rise in the number the journal may seem less attractive for contributions pertaining to high-impact topics in infectious disease research compared to international journal of infectious diseases and bmc infectious diseases, which may be explained by the fact that it is a relatively new journal, which has not yet garnered an adequate international following. publication of 'hot' research topics and results changing the direction of research should be strived for and this can be achieved through invited manuscripts and thematic series. in the future, work attracting high-quality papers from europe and usa in addition to papers from the developing world, should be strengthened. during the past five years, idp has been strengthening its academic impact; however, its international impact remains to be enhanced relative to other leading journals in infectious diseases. the following priority has been recommended by its editorial board: . invitation of contributions from global health policy makers and high-impact scientists; . publication of thematic series pertaining to global 'hot' topics in public health, emerging or re-emerging diseases and the outcome of international major projects on infectious diseases; finally, the question of a paper's true value must be mentioned as it is at the heart of the collaboration between author(s) and journal. that question extends naturally to the question of what the true value of a scientific journal is. this question has been approached in various ways, not the least by awarding impact factors. this first attempt is still the most utilized metric for assessing a journal's merit, but new developments are telling us that a faster and wider range of researchrelated metrics are needed. the number of clicks, downloads and reads are already providing valuable insights into how published content is being used and shared by fellow scientists. a multitude of commercial companies are already active in this field, for example, altmetric (https://www.altmetric.com/), plum analytics (http:// plumanalytics.com/) and researchgate (https://www.researchgate.net/). meta (http://www.meta.com) has gone one step further by using algorithms recognizing the unique features characterizing widely cited papers, and the company claims that this can predict a paper's future impact before its publication. more than million papers and million researchers have already been profiled by the company (https://en.wikipedia.org/wiki/ meta) to support this claim. idp has achieved its preliminary goal to become a platform for the identification of research and information gaps that hinder progress towards new interventions for public health problem connected to poverty in the developing world. however, there is still a long way to go to further advance research and evidence-building based on improved public health interventions in poor settings. it is believed that idp can significantly facilitate progress towards the elimination of infectious diseases related to poverty. in this process, idp will not only be judged by the quality of the articles published, but also regarding how it fits into the new world of merit assessment. global, regional, and national age-sex specific all-cause and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study global burden, distribution, and interventions for infectious diseases of poverty global, regional, and national incidence and mortality for hiv, tuberculosis, and 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public and media concern justified in regions currently unaffected? combating infectious diseases of poverty: a year on social sciences research on infectious diseases of poverty: too little and too late? global report for research on infectious diseases of poverty surveillance and response systems for elimination of tropical diseases: summary of a thematic series in infectious diseases of poverty elimination of tropical disease through surveillance and response surveillance-response systems: the key to elimination of tropical diseases surveillance and response: tools and approaches for the elimination stage of neglected tropical diseases planning an integrated disease surveillance and response system: a matrix of skills and activities malaria elimination: surveillance and response reaching the surveillanceresponse stage of schistosomiasis control in the people's republic of china: a modelling approach need of surveillance response systems to combat ebola outbreaks and other emerging infectious diseases in african countries research gaps for three main tropical diseases in the people's republic of china assessment of morbidity due to schistosoma japonicum infection in china schistosomiasis control and the health system in p.r. china co-infections with babesia microti and plasmodium parasites along the china-myanmar border the global epidemiology of clonorchiasis and its relation with cholangiocarcinoma prioritizing research for "one health -one world infectious disease emergence and global change: thinking systemically in a shrinking world factors associated with risk of malaria infection among pregnant women in community based interventions for the prevention and control of tuberculosis control of malaria and other vector-borne protozoan diseases in the tropics: enduring challenges despite considerable progress and achievements global climate change and infectious diseases global climate change and infectious diseases we would like to thank the staff from biomed central for their kind provision of some data. authors' contributions ww and py conceived and designed the study. ww, jc, nnw and hfs collected and analyzed the data. ww prepared the first version of the manuscript. py, xnz and rb provided valuable comments on the manuscript. ww and py revised and finalized the manuscript. all authors read and approved the final version of the manuscript. submit your next manuscript to biomed central and we will help you at every step: key: cord- -j tvmvd authors: batsukh, zayat; tsolmon, b.; otgonbaatar, dashdavaa; undraa, baatar; dolgorkhand, adyadorj; ariuntuya, ochirpurev title: one health in mongolia date: - - journal: one health: the human-animal-environment interfaces in emerging infectious diseases doi: . / _ _ sha: doc_id: cord_uid: j tvmvd the asia pacific strategy for emerging diseases (apsed) requires collaboration, consensus, and partnership across all the different actors and sectors involved in different aspects of emerging disease. guided by apsed, mongolia has established a functional coordination mechanism between the animal and human health sectors. surveillance, information exchange and risk assessment, risk reduction, and coordinated response capacity and collaborative research have been identified as the four pillars of the zoonoses framework. intersectoral collaboration has been clearly shown to be a crucial tool in the prevention and control of emerging zoonotic diseases. a “one health” strategy has been implemented under the concept of ‘healthy animal-healthy food-healthy people’. an intersectoral coordination mechanism established between the veterinary and public health sectors has expanded its function to incorporate more work on food safety, emergency management, and effects of climate change on zoonotic diseases. its membership includes the human health sector, the veterinary sector, the national emergency management agency, the environment sector, emergency management and inspection authorities, and the world health organization (who). the main outputs of the coordination mechanism have been strengthened surveillance and response activities and laboratory capacities. the coordination mechanism has also strengthened the surveillance and response capacity of neglected zoonotic diseases, such as brucellosis, anthrax, and tick-borne diseases. through regular meetings and brainstorming sessions, both sectors have developed joint operational plans, a long-term risk reduction plan – , initiated a prioritization exercise and risk assessment for zoonotic diseases, and reviewed and revised standards, procedures, and communication strategies. in , a list of experts on major zoonoses were identified from different sectors and formed into a taskforce to identify the focal points for rabies, brucellosis, and vector-borne diseases. as a result, disease control strategies are now linked to scientific research and epidemiological expertise. mechanism established between the veterinary and public health sectors has expanded its function to incorporate more work on food safety, emergency management, and effects of climate change on zoonotic diseases. its membership includes the human health sector, the veterinary sector, the national emergency management agency, the environment sector, emergency management and inspection authorities, and the world health organization (who). the main outputs of the coordination mechanism have been strengthened surveillance and response activities and laboratory capacities. the coordination mechanism has also strengthened the surveillance and response capacity of neglected zoonotic diseases, such as brucellosis, anthrax, and tick-borne diseases. through regular meetings and brainstorming sessions, both sectors have developed joint operational plans, a long-term risk reduction plan - , initiated a prioritization exercise and risk assessment for zoonotic diseases, and reviewed and revised standards, procedures, and communication strategies. in , a list of experts on major zoonoses were identified from different sectors and formed into a taskforce to identify the focal points for rabies, brucellosis, and vector-borne diseases. as a result, disease control strategies are now linked to scientific research and epidemiological expertise. mongolia is a landlocked country in east and central asia, situated between and bordering china and russia, and with a population of . million as of . the country has the lowest population density in the world, one person per . km . mining and agriculture, and are the two main sectors of the mongolian economy. for centuries, the mongolians have been engaged in animal husbandry, raising horses, sheep, goats, cattle, and camels. agriculture, primarily herding, is the traditional basis of the mongolian economy, contributing about % of gdp and providing % of national employment. livestock husbandry is the main economic pillar, vital for public good, and the significant source of export income. due to increasing urbanization and socioeconomic development of country in recent years, migration from rural to urban and suburban areas has been increasing. in , only . % of the population resided in rural areas. approximately, % of the population is nomadic or seminomadic. administratively, mongolia is divided into provinces, and the capital city, ulaanbaatar. mongolia has an extreme continental climate with long, cold winters and short summers, during which most precipitation falls. the temperature is as low as - to - °c in the winter and can reach °to °c in the summer. global climate change is believed to have had an influence on the climate; the annual average climate temperature has risen by . °c over the last years, and in the last years, the temperature has risen faster and the rainfall has decreased in mongolian forest-steppe regions. due to environmental and human impacts in the last few years many rivers, streams, and lakes have dried, pasture growth has decreased by - %, pasture plant species numbers have reduced and it has resulted in an increase in land degradation and desertification. natural disasters such as drought, heavy snowfall, flood, snowstorms, windstorms, extreme cold and hot temperatures, and earthquakes recurrently occur throughout the year. mongolia is very dependent on nature and climate due to its traditional nomadic lifestyle throughout four seasons of the year. the large herder population has a greater chance of zoonotic infections. as the mongolian economy is heavily reliant on herding and agriculture, the harsh winters and periodic droughts have adverse effects on livestock and agriculture, and also on the health status of the population. the livestock population was . millions as of , down from . million at the end of . pig and poultry population are not prominent. endemic zoonotic diseases such as brucellosis, anthrax, rabies, plague, and tick-borne diseases create important public health problems. in recent years, endemic zoonoses have expanded and outbreaks of number of transboundary diseases have emerged in both animals and humans. climate change and extreme weather conditions have had an adverse effect on biodiversity, distribution of animals, and microflora, which can lead to the emergence of zoonotic agents and create favorable conditions for disease outbreaks. over bacterial and viral and parasitic zoonotic diseases were reported in animals. six out of diseases listed as transmissible diseases notifiable to the oie were reported in mongolia, and four diseases have a potential risk for further spread. the significance of zoonoses is increasing due to improved animal husbandry practices, climate change, desertification, and developments in the mining sector. in spite of the progress achieved, anthrax, brucellosis, tick-borne diseases, and rabies still constitute a threat to human health and welfare. the the overall vision of the coordination committee is to have ''strong human and animal health sectors, together with emergency response and national inspection agencies working in partnership toward the attainment of a healthier community''. the coordination committee has responsibility for developing joint policy on the prevention and control of priority zoonotic diseases; for approving action plans produced by a technical working group; for making recommendations on risk assessment, early warning and response activities during outbreaks; for reviewing and revising zoonotic diseases standard operational procedures (sops) and guidelines to reflect intersectoral collaboration; for providing methodological assistance to improve the capacity of professional institutions at the national and subnational level; for coordinating cooperation among different sectors in carrying out early detection and response functions; and for monitoring and evaluating overall zoonotic disease prevention and control. the director-general of the national centre for zoonotic diseases in the moh serves as secretariat, and is responsible for routine coordination and management. before the establishment of the coordination committee, moh and mofali developed a written memorandum of understanding (mou) to conduct joint surveys on zoonotic diseases in - . both sectors exchanged annual statistical reports and conducted joint serological surveys. the results of the survey helped define the distribution of major zoonoses which are important to both animal and human health. the surveys identified new diseases in mongolia, such as tick-borne encephalitis, west nile fever, lyme disease, rickettsia, and q fever. the joint survey promoted collaboration between two sectors. the new diseases have been added to the list of notifiable diseases to reflect current threats. however, most of the activities were aimed at gathering information about zoonotic pathogens only. notable changes observed in the two sectors during the survey were transferred by the joint task to surveillance with ongoing and systematic collection of information in order to define the extent of disease problem, and to disseminate this information to improve public health awareness, early warning, diagnosis, prevention, and control. the first meeting of the intersectoral coordination committee took place in march , and was attended by its members, the secretariat, the technical working group and evaluation team, as well as by representatives from who and fao. the outcome of the meeting was discussion of the draft joint operational plan. the first activity was to map existing capacity and surveillance systems, and response and risk reduction measures in both the animal and human health sectors. based on the results of this assessment, an operational plan of action was developed to address the gaps and to improve zoonose control strategies. quarterly meetings have been held and priorities set for actions and interventions. regular meetings between veterinary and public health professionals proved to be an important activity to improve and stimulate intersectoral cooperation. during times of emergencies, both sides communicated frequently and joint technical working group meetings were conducted. a good example of this is the brainstorming joint response review meeting of veterinary and human health authorities in september following the outbreak of anthrax in animals and humans. all meetings are organized in cooperation with the who and other international organizations. the cost of organizing joint meetings and conferences was paid back by the harmonization of legislation, joint planning, and sharing of resources. this included sharing information and surveillance data and cooperation at the local level in outbreak response. this cooperation has been tested during real time outbreaks and the lessons learned from those exercises used to improve the rapid response measures. the coordination committee organized the first national conference on zoonoses in june . the participants were professionals from both the human and veterinary sectors at national and subnational levels. this was the first ever joint meeting between two sectors at a professional level. the meeting reviewed results of joint assessment on existing capacity and system for surveillance and response in the following areas: • human resources • response capacity • information and surveillance • laboratory • logistics and supplies. after the national conference, the intersectoral coordination mechanism was formally set up at all levels in mongolia. at the community level, social awareness, public education, and media play an important role. it has also enabled the use of better risk communication and health education strategies at the community level. risk communication and promotion of programs directed primarily at occupational risk groups and school children were implemented with assistance from local government. at the national level, the coordination mechanism was aimed at improving information exchange, expertise sharing, mutual technical support, and harmonization of legislation. in , a joint strategy for long-term risk reduction of priority zoonotic diseases for - was developed by the ministries of health and of food and agriculture. information sharing, surveillance, risk assessment, and risk reduction the intersectoral coordinating committee on zoonoses carried out a prioritization exercise and risk assessment of zoonotic diseases in january . these included endemic zoonoses reported in humans, zoonoses reported in animals, vector-borne diseases, and diseases at risk of being imported. a total of zoonoses were identified that are important for both animal and human health sectors. the technical working group that consisted of veterinary, public health, laboratory, research institute, and academic personnel held a series of discussions and conducted detailed risk assessments. who's prioritization tool as well as other countries' methodologies and tools were adopted for this prioritization exercise. the priority diseases, namely, plague, avian influenza, anthrax, brucellosis, rabies, tickborne encephalitis, echinococcosis, and tularemia were defined as diseases that required a coordinated surveillance and response. endemic diseases like brucellosis and anthrax, which have been listed by who as ''neglected'' were identified as priority diseases by moh and mofa. the exercise specially defined malaria, dengue fever, glanders, toxoplasmosis, west nile fever, japanese encephalitis, hemorrhagic fever with renal syndrome, and cryptosporidiosis as diseases that should be targeted for collaborative research. the coordination committee developed sops for information sharing, surveillance, and response for the priority diseases such as avian and pandemic influenza, anthrax, tick-borne diseases, rabies, brucellosis, plague, and some parasitic diseases. the veterinary and health sectors routinely cross-notify and exchange information, based on the sops. in addition to surveillance data, both sectors should exchange outbreak information within h, and laboratory data and event information (immunization, cluster of cases, livestock abortion, sudden death of animals, survey results, food-borne disease) on a monthly basis. weekly disease information has been shared with moh, mofa, who, fao, and other partners through an electronic newsletter since march . mongolia has one of the highest incidences of human brucellosis in the world. national brucellosis surveillance was established in the , and a test-and-slaughter strategy commenced in . the government implemented a vaccination strategy from to . as a result, the prevalence of animal brucellosis has decreased from to . %. however, in the s human brucellosis re-emerged following transition to free market economy, collapse of systems that were responsible to public health issues and lack of resources to continue surveillance accordingly. in , a new vaccination strategy was introduced with the aim of eradicating the disease by , but attempts to control the disease have been unsuccessful because of inconsistent strategies with respect to vaccination of livestock and the detection and elimination of infected animals from the herd. the seroprevalence of brucellosis in humans, livestock, and dogs was investigated as a pilot project in sukhbaatar and zavkhan province with support from swiss development agency. the results of the study by veterinary and medical epidemiologists served as a baseline for assessing and monitoring the effectiveness of a conjunctival vaccination campaign in . in addition, the conjunctival vaccine campaign has assisted the development of new strategy for national brucellosis control and for livestock export. despite the increase in the number of registered animal brucellosis cases, the moh did not report an increase in the number of human brucellosis. in mongolia, the disease incidence is largely unknown because many cases are missed due to a lack of diagnostic facilities at the subnational level. only - % of cases of acute human brucellosis are reported, and it is estimated that less than one in cases are reported indicating a significant under-reporting. animal sector surveillance data helped the human health sector to review surveillance and laboratory practice to improve reporting. brucellosis is identified as one of priority zoonoses for both animal and human health sector. in , animal and human sector have started baseline prevalence survey. over , serum samples from five major species of animals and , serum samples from human were collected and laboratory investigation were carried out, following oie recommendations. a mass vaccination campaign has been implemented with the aim of controlling and eradicating animal brucellosis by . the country was divided into three sectors and . million animals were vaccinated in in st sector, with a future plan to vaccinate animals in remaining two sectors, and then to provide annual vaccination of newborns. in response to growing burden of anthrax in the mongolia, a technical working group has developed a strategy for the prevention and control of human and animal anthrax. this is the first risk reduction disease strategy that has been prepared with involvement of human, animal, emergence management, inspection agency, food safety and intelligence authorities, and with international partners. the strategy has been based on global best practice and experience gained over the past years of responding to outbreaks as well as sporadic cases of anthrax. a gis-based risk map has been developed for anthrax to provide a common platform. in addition, a joint technical working group has been established with professionals from the institute of veterinary medicine, the national centre for zoonotic diseases, the central veterinary diagnostic laboratory to act as a professional advisory, and technical implementation body to develop methodological recommendations and policy documents for approval by relevant authorities. in response to increasing numbers of rabies cases in wildlife, the veterinary and public health sectors have combined with local government over the past years to conduct community education and awareness activities in schools, workplaces, and among the general population. on world rabies day , the moh organized a rabies awareness and prevention campaign and conducted training for healthcare workers, veterinarians, school doctors. the moh also distributed brochures and posters for children, parents, and dog owners on rabies prevention, and video spots and cartoons were produced and broadcasted by media. the veterinary sector also initiated dog vaccination, and stray street dogs were destroyed in four districts. an avian influenza surveillance program has been established in wild birds in order to provide an early warning system and to improve the existing surveillance network. the surveillance team consisted of representatives from the veterinary, health, environment, inspection, and other related institutions, and was a good example of multisectoral cooperation. the two human and animal sectors have developed an epidemiological atlas of zoonotic diseases in mongolia, . the atlas contains approximately maps that illustrate the distribution of major or rare and neglected zoonotic diseases. every map contains key information about the infectious agent including: icd- code, epidemiology, epizootiology, climate data, vegetation, transmission, incubation period, clinical findings, therapeutic options, and key references. in addition, the atlas includes population density, livestock density, antibiotic use, immunization coverage, and other relevant factors and will be regularly updated. it will be made available online by . the use of gis tools and geo-referenced, subnational level epidemiological data allowed the production of maps that improve spatial quality of previous maps. it was shown that diseases such as brucellosis, glanders, and bovine leucosis in animals have been introduced into previously unaffected areas by cattle movement. the atlas will lay the basis for novel, evidence-based methodologies to estimate the population at risk and burden of disease, ultimately leading to more targeted interventions. the atlas has also helped to streamline field data collection. joint risk assessment and investigations have been conducted after cross-notification of outbreaks of foot-and-mouth disease, newcastle disease, human and animal anthrax, rabies, and avian influenza in wild birds. during outbreaks of anthrax, a rapid response team consisting of veterinarians, medical epidemiologist, inspectors and emergency officers, implemented quarantine and movement restrictions, and developed risk maps using gis. animal vaccinations, enhanced surveillance in the food market, and health education and communication activities has led to effective outbreak response. the subclinical, gastrointestinal form of anthrax was identified for the first time by the rapid response team. existing rapid response infrastructure has been improved into multisectoral joint rapid response teams that operate at the district and provincial levels; rapid response teams have been trained and established in provinces. working together has made it possible to prevent zoonotic diseases, not merely to react to them once they have occurred. laboratory integration, surveillance activities, and recognition of the importance of risk assessment have also increased. under the apsed framework, communication and cooperation of veterinary and human health laboratories have increased significantly in the last years. laboratories share information, experience, diagnostic kits, laboratory specimens and lab equipment for surveillance, response, and research activities. health laboratories have benefited from more advanced laboratory resources of veterinary laboratories, including personnel. during an unusual outbreak of human anthrax in , the veterinary laboratory assisted in validating results and undertook confirmation tests. subnational veterinary laboratories in all provinces have been equipped with pcr equipment and reagents. the veterinary laboratory also supported laboratory diagnosis of a rabies outbreak in uvurkhangai province and in an unusual anthrax outbreak in khovd province. following annual serological surveys, the analysis of the laboratory findings was carried out jointly by laboratory staff from the veterinary and health laboratories, and the methodologies used in both sectors were reviewed and experiences shared. as a result of human and animal sector collaboration, the diagnostic capacity of human health laboratories has been improved significantly. new advanced methods and techniques for isolation, identification, and confirmation of zoonotic viral and parasitic pathogens have been introduced at the national level. a number of commercially available diagnostic kits have been introduced for diagnosis at the nrcidnf and the number of diseases diagnosed by molecular assays has increased to . serological and molecular diagnostic tools have become available for the diagnosis of tick-borne encephalitis, lyme disease, and rickettsia which had previously been diagnosed only by clinical presentation. however, hantavirus, west nile virus, japanese encephalitis virus, crimean congo hemorrhagic fever virus, dengue virus, and many others cannot be diagnosed due to technical limitations, and thus the true burden and epidemiology of these diseases in mongolia is still unknown. in addition to the collaboration with veterinary laboratories, training in advanced countries is seen as important for increase capacity at the laboratory diagnostic level. since , over professionals have been trained in laboratory biosafety in russian federation, kazakhstan, people's republic of china, germany, and japan. approximately % of the trained lab professionals were from provincial veterinary and medical diagnostic laboratories. as a result of collaborative molecular biology research with foreign colleagues from various countries including russia, china, the usa, germany, and japan, various techniques such as clustered regularly interspaced short palindromic repeats (crispr), duplex polymerase chain reaction (pcr), variable number tandem repeats (vntr), multiple loci vntr analysis (mlva), have been introduced to research and diagnostic laboratories for animal and human diseases, and have determined unique and specific genes of y.pestis, b.anthracis, rabies virus, tick-borne encephalitis virus, and some species of rickettsia. in addition, hantavirus, west nile virus, anaplasmosis, erlichiosis, and toxoplasmosis were newly identified using these techniques. several complications still exist that constrain sharing of resources between human and animal diagnostic laboratories and the biggest challenge for the intersectoral coordination committee on zoonoses will be to change the legal and ethical environment. mongolia is planning to establish a laboratory network between public health, clinical, veterinary, and food laboratories in - . lessons learned from managing previous outbreaks highlighted the importance of advocacy and public education. a communication and behavior change strategy was reviewed by the coordinating committee meeting in . it emphasizes the need for advocacy and a public education campaign targeted at high-risk groups. a proactive approach in building effective communication with media was also stressed. endemic zoonoses such as plague, anthrax, and vector-borne diseases occur regularly due to a lack of public awareness, and there is a high infectivity rate of brucellosis among herdsmen and veterinarians. unsafe cultural traditions are widespread among the general population, such as consumption of raw milk, undercooked sheep liver, and sour cream made from raw milk. public health education programs need to be aimed at specific community groups, school children, and occupational groups, taking into account culture, beliefs, traditions, educational level, social status, occupation, and age. an involvement of community and local government in health education through health education in schools and in the workplace has proved to be effective. health messages on how to prevent infection with tick-borne diseases and the production of leaflets and posters were distributed before the tick season. in addition, a monthly press conference has been initiated by the moh to ensure important public health messages are widely disseminated; the first press conference held on march advocated a one world, one health approach to public health. regular awareness programs are conducted by state veterinary and animal breeding department, institute of veterinary medicine, and the moh through tv programs, brochures, video spots, cartoons for children, and press conferences. training materials and courses for risk reduction measures and interventions were developed for anthrax, plague, tick-borne diseases, brucellosis, and avian influenza collaboratively by animal and human health sectors. joint staff training activities and short training courses on mosquito biology and surveillance, risk assessment of common zoonotic diseases, data management, database design, vector-borne diseases have been conducted for medical and zoonotic epidemiologists, biologists, laboratory staff, and meteorologists. tick-borne diseases such as tick-borne encephalitis, lyme disease, and rickettsia are a growing concern in mongolia, as their prevalence continues to increase with expansion into new areas. pastoral animal husbandry, climate change, desertification, development of mining sector, new tick species, and vector distribution in mongolia combine to create an important public health problem. to mitigate these risks, a korean international cooperation agency (koica) funded project has supported vector surveillance, climatic monitoring and community education to high-risk population. this initiative is multisectoral, and is bringing together people with different backgrounds and sectors. at the regional level, emerging diseases surveillance and response (esr) and malaria, vector-borne and parasitic disease (mvd) units are working together. climate change studies are complex and require multisectoral collaboration. building on the achievements of the intersectoral coordination mechanism, a comprehensive surveillance system for vector-borne diseases has been established. surveillance procedures have been developed for anaplasmosis, q fever, tickborne encephalitis, tickborne boreliosis, rickettsia, and erlichiosis. tick distribution and species are monitored in relation with microclimate and human infections. erlichiosis and anaplasmosis, toxoplasmosis, and crimean congo hemorrhagic fever infections were identified for the first time in humans, and anaplasmosis platys was identified for the first time in ticks. the veterinary laboratory is undertaking genetic studies on ticks. correlation of infected tick density with variations in human incidence and climate determinants has helped to identify factors associated with disease transmission. risk maps on tick prevalence, density, biotype, climate data, and vegetation has provided useful public health information for early warning. increased risk communication and staff training has resulted in improved protective behavior of the nomadic population. the national center for zoonotic diseases has established good collaboration and partnerships with many international organizations and institutions from various countries including china, kazakhstan, russia, japan, switzerland, the usa, and germany. epidemiologists interested in zoonoses have been cooperating with chinese academy of inspection and quarantine since on collaborative research directed at understanding the natural foci and the conditions affecting disease incidence each side of the border of both countries. this collaborative research has also enhanced laboratory capacity, including a substantial donation of virology laboratory equipment to the ncidnf by the chinese academy of inspection and quarantine. the laboratory will be basis for conducting cross-border surveillance, on-the-job training of laboratory staff, and confirmation of events and diseases of public health importance. the ncidnf conducts collaborative research on plague and tick-borne diseases with the bundeswehr institute of microbiology of munich. both institutions carry out annual joint field investigations and expeditions. the results from these studies have been published and presented at an international zoonoses conference held in mongolia. extensive research and cross border surveillance of bacterial, parasitic, and viral diseases have been conducted in collaboration with gamalei institute of epidemiology and microbiology, and natural foci of leptospirosis, cryptosporidiosis, and toxoplasmosis have been detected for the first time in mongolia. studies on the molecular biology of plague, tick-borne diseases, and other emerging diseases have been undertaken by veterinary and public health specialists with colleagues from the university of florida. an important part of the collaboration with the university of florida is a 'one health' training program which started in , and which attracted a number of staff members from the institute of veterinary medicine and the moh. it is hoped that the course may attract the us and international students. the curriculum will include studies in environmental health, modern laboratory techniques, epidemiology, biostatistics, food safety, climate change, gis, toxicology, and zoonotic infections research. apsed has facilitated an intersectoral coordination mechanism between human health and other sectors. however, although ongoing risk assessments are conducted during outbreaks, there has been no comprehensive cross-sectoral risk assessment for all priority zoonoses. evidence-based decision making and response, and utilization of risk assessment findings, need to be further improved. it has also been realized that an enabling legal environment is critical for effective control of zoonoses. the annual intersectoral simulation exercise has been a useful way to review response capability, and to update and revise the coordinated response guidelines. subnational level planning and information sharing between veterinary and health epidemiologists, however, is still weak. at the local level, the involvement of the veterinary health departments is crucial for effective monitoring of instances of zoonotic disease in wild and domestic animals. there is also need to improve both the health laboratory capacity and in epidemiological capacity in the animal sector. during the annual review meeting in , the need for developing and implementing a common monitoring and evaluation framework was highlighted, and poor coordination and confusion over roles and responsibilities among veterinary, health and inspection agencies on food safety, and import and export control need to be addressed. financial contribution is crucial for the success of zoonoses control in the country, so that effort from mofali and moh is requested to have more efficient way to raise the fund and harmonize international donor recourse, by drawing attention of potential donors for the activity in the zoonoses field. we believe a good foundation has been established for a coordination mechanism between the veterinary and public health sectors, and the generic capacity for zoonoses control and prevention has improved considerably. in addition, the zoonoses coordination framework has attracted more resources from international partners and allowed pooling of resources. thus while an important process has started, there is still much to do to reduce the risk of zoonotic diseases in mongolia. editorial addition asia pacific strategy for emerging diseases (apsed) and its role in responding to zoonotic disease threats. john s mackenzie, curtin university, perth, and the burnet institute, melbourne apsed was developed in as a joint initiative by the south-east asian (searo) and western pacific regional offices (wpro) of the who to meet the challenges of emerging diseases that pose serious threats to regional and global health security (who ) . apsed provided a common strategic framework for countries and areas of the two regions to strengthen their capacity to manage and respond to emerging diseases including epidemic-prone diseases, and to develop the capability to comply with the core capacity requirements of the new international health regulations ( ) . it had the support of all regional countries ( in searo and in wpro), and thus represented countries with a combined population of . billion people, more than half of the world's population. the development of apsed was greatly influenced by several major emerging zoonotic disease events in the asia pacific region, and especially by the emergence of severe acute respiratory syndrome (sars) and highly pathogenic avian influenza h n (hpai), as well as the initial outbreak and continued recurrences of nipah virus. during the first years of the strategy, the two regions experienced a number of infectious disease threats including the establishment of hpai as an endemic disease, the rapid global spread of pandemic influenza h n , and a large number of other acute events with significant public health impact. taken together, these provided important lessons in pandemic response and demonstrated the need to further strengthen public health emergency preparedness and improve monitoring and evaluation. apsed ( ) recognized that many emerging diseases were zoonoses, and that an important component of the strategy was the development of plans to detect, manage, and respond to infectious diseases at the human-animal interface. during the first years of the strategy, a guide was developed in collaboration with colleagues from the world organization for animal health (oie) and the food and agriculture organization of the united nations (fao) entitled 'zoonotic diseases: a guide to establishing collaboration between animal and human health sectors at the country level' to assist countries with their planning (who ). thus, considerable progress was made in the two regions toward strengthening core capacities needed to prevent, detect and respond to threats posed by emerging diseases, and has provided a good foundation for expanding the scope of apsed. this led to a biregional consultation to explore how to take the strategy forward for the next years, resulting in the development of apsed ( ) (who ). the new strategy has expanded to eight focus areas, including zoonoses, with a strong statement recognizing the importance of zoonotic diseases and with an undertaking to continue working in collaboration with fao and oie and other partners … 'to contribute to the concept of ''one health''', and acknowledging that reducing the risk of transmission of zoonotic diseases requires close collaboration between and links with the food safety, environment, and wildlife sectors. it also states that the experience and lessons learned with hpai (h n ) provide a good foundation to consolidate and strengthen national and regional coordination mechanisms for surveillance information-sharing and coordinated responses by human and animal health sectors. in response to the strategy, a number of countries in the regions have developed plans to coordinate and collaborate between their human and animal sectors, and in some instances, also their environmental sectors, through a 'one health' approach. mongolia is one such example, and the description of their plans and activities clearly demonstrate how they are building a sustainable and collaborative approach toward managing zoonotic diseases, and developing the capacity to diagnose and respond to new emerging disease threats-a good example of operationalising 'one health' at the national level. other examples are given in the chapters by dr g gongal and dr b coughlan. zoonotic diseases: a guide to establishing collaboration between animal and human health sectors at the country level. world health organization regional office for south-east asia, new delhi, and the regional office for the western pacific key: cord- -rvjvuxor authors: liu, chen-hsuan; hsu, yung-hsiang title: the role of comparative pathology in the investigation of zoonoses date: - - journal: tzu chi medical journal doi: . /s - ( ) - sha: doc_id: cord_uid: rvjvuxor abstract emerging and re-emerging zoonoses have raised great concerns in both human and animal health worldwide in the past years. rudolph virchow proposed a “one medicine” discipline and emphasized the importance of cooperation years ago. in the face of emerging threats from unpredictable zoonoses, human medicine and veterinary medicine should not be separate and independent sciences. anatomic pathologists who are capable of analyzing and interpreting anatomical manifestations of diseases to obtain a definite diagnosis or exclude a wide variety of diseases play an important role in the diagnostic team. although disease-associated microbes are numerous, morphologic patterns of tissue reaction caused by microbes are limited. therefore, the interactions between microbes and host determine the histological changes in the target tissues. the contributions of anatomic pathology, with its use of morphologic similarities and special techniques, are important in zoonosis diagnosis. this can be seen in retrospective case studies of recent zoonoses such as multinucleated syncytial giant cells in severe acute respiratory syndrome and mouse hepatitis virus infection, syncytial cells in henipahvirus infection and paramyxovirus, neuronal vacuolation in bovine spongiform encephalopathy and variant creutzfeldt-jakob disease, and streptococcus suis type meningitis. in taiwan, the chinese society for comparative pathology, which was established in , provides for this interaction. interlaboratory cooperation plays an important role in the diagnosis, surveillance, and control of emerging and re-emerging zoonoses. emerging and re-emerging diseases have had a devastating impact on both human and animal health worldwide in the past years, and zoonotic diseases have raised particular attention (table ) . it is estimated that three-fourths of emerging diseases are zoonotic [ , ] . facing the invasion of so many unpredictable pathogens and the emergence of bioterrorism as a global threat, a fast and definite diagnosis has become more important to eradicate or control the spread of diseases. among diagnostic tools, anatomic pathology is effective in recognizing or excluding infectious diseases based on morphological changes in organs in affected species [ ] [ ] [ ] . human and veterinary pathologists are familiar with their own fields. however, german physician rudolph virchow ( - ), a towering figure in th medicine and pathology, stated, "between animal and human medicine, there is no dividing line-nor should there be" while working on the zoonosis of trichinosis and hydatid disease [ , ] . this emphasis based on diseases across species lines established the concept of "one medicine" and led to the development of comparative medicine and comparative pathology. the diagnosis of infectious diseases, especially zoonosis, requires the collaborative efforts of physicians, epidemiologists, microbiologists, parasitologists, and pathologists, as well as veterinarians to seek causal agents. anatomic pathologists are capable of analyzing and interpreting anatomical or functional manifestations of diseases from autopsy, biopsy and cytology materials using routine processes and advanced diagnostic skills. they can provide important clues to the diagnostic team in characterization of diseases [ , , ] . years of experience have shown that data obtained from animal necropsies can be useful in determining the cause of death, which allows comparison with human counterpart diseases. histopathological and cytopathological observations often allow for establishment of a definite diagnosis or exclusion of a wide variety of diseases. compared with other diagnostic methods such as culture, immunodiagnosis and molecular biology, anatomic pathology is safe, economical, fast and reliable. it can help provide a disease diagnosis using formalin-fixed tissues, and direct visualization of lesions with fewer falsenegative results. although disease-associated microbes are numerous, morphologic patterns of tissue reaction caused by microbes are limited. therefore, the interactions between microbes and host determine the histologic changes in the target tissues. this allows anatomic pathologists to analyze rare or unknown pathogens and quickly report their findings to a diagnostic team, which gathers information from the pathological, microbiological, immunological and molecular aspects to confirm disease. in pathological study, a routine hematoxylin and eosin (h&e) stained section can directly visualize some infectious agents or their products (e.g. bacterial clumps, most protozoans, all helminthes and viral inclusion bodies) within lesions ( it is very difficult to predict when and where the next zoonosis will emerge and the ultimate outcome depends on myriad interconnected factors. thus, rapid and efficient collaboration based on the one medicine concept is necessary. comparative pathology particularly focuses on comparison of the disease granulomatous inflammation toxocara spp. migration absence/minimal reaction trichinellosis (fig. ) , toxocara spp. migration prions vacuolation of neurons and neuropil scrapie (fig. ) , bovine spongiform encephalopathy, variant creutzfeldt-jakob disease (fig. technique use acid-fast stain mycobacteria (fig. ) , nocardia, cryptosporidium brown-brenn stain gram +, gram -grocott's methenamine silver stain fungi immunohistochemistry antigen detection (fig. ) in situ hybridization nucleic acid detection (fig. ) in situ polymerase chain reaction nucleic acid detection mucicarmine stain cryptococci (fig. ) periodic acid-schiff stain fungi (fig. ) , amebae transmission electron microscopy ultrastructure of target cells or microbes (fig. ) process in humans and other animals. to solve the complexities of emergence and re-emergence of zoonoses, effective and integrated collaboration between human and veterinary pathology is needed. with the use of morphologic features and auxiliary special techniques, anatomic pathologists contribute greatly to the diagnosis and consequent management of zoonoses (table ) . retrospective case studies of recently occurring zoonoses reflect the importance of diagnostic pathology with its use of morphologic similarities in disease diagnosis. in , an outbreak of severe acute respiratory syndrome (sars) occurred in several countries and caused human deaths. the cause of the disease has been attributed to a coronavirus. coronaviruses in animal species have been well known for years. in coronavirus mouse hepatitis virus (mhv), multinucleated syncytial giant cells (fig. ) in lesions of the liver and colon share common features with one of the characteristic lesions of sars (fig. ) [ , ] . further study indicated that intranasal infection of a/j mice with mhv strain produced pulmonary pathologic features of sars, including interstitial pneumonitis, dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death in all animals [ , ] . outbreaks of equine hendra virus infection in australia in and nipah virus infection in malaysia in both caused mortalities in humans, horses and pigs. the two viruses are now classified as a new genus henipahvirus in the family paramyxoviridae. in past years, paramyxoviruses have caused devastating mortality in humans and major economic loss from animal outbreaks, such as that seen in measles in humans, canine distemper in canine species, and rinderpest in cattle, as well as newcastle disease in poultry. the most commonly affected organs are those in the respiratory, gastrointestinal and nervous systems. among the lesions, syncytial cells throughout the epithelium and in the vascular endothelium are a characteristic diagnostic feature [ ] . examples of scrapie in sheep versus kuru, and bovine spongiform encephalopathy in cattle versus variant creutzfeldt-jakob disease show common features of vacuolation of neuronal cell bodies and processes, spongiform changes in neuropil, no inflammatory infiltrates, and astrogliosis in the central nervous system [ ] . important morphologic observations from veterinary and human diagnostic pathologists provide valuable information in the exploration of mysterious diseases, such as prion diseases. streptococcus suis type infection is a reemerging zoonosis and is transmitted from pigs to humans through close contact. an infection in sichuan, china, in caused cases of human disease and deaths. purulent meningitis was the most common finding in both human and pig cases [ , ] . routine h&e sections plus immunohistochemical staining provided rapid diagnosis and contributed greatly to the treatment of the disease. humans and other animals live in an unprecedented era in which emerging and re-emerging zoonoses can occur anywhere and anytime and the consequences can be severe. to control and prevent zoonotic diseases, human medicine and veterinary medicine should not be separate and independent sciences. through the one medicine discipline, anatomic pathologists contribute valuable information to the diagnostic team by interpreting and analyzing morphologic changes. in taiwan, this interaction was established through the chinese society for comparative pathology in . in , proceedings from the conferences of the society were published, and work continues in this area. we believe that comparative pathology will play an important role in the diagnosis, surveillance and control of unpredictable emerging and re-emerging zoonoses. emerging zoonotic epidemics in the interconnected global community a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply infectious disease pathology pathology and emerging infections-quo vadimus? the importance of the autopsy in emerging and reemerging infectious diseases avian influenza: virchow's reminder interrelations of human and veterinary medicine; discussion of some aspects of comparative dermatology will pathologists play as important a role in the future as they have in the past against the challenge of infectious diseases the emerging role of pathology in factious diseases intestinal cryptosporidiosis of pigs in taiwan cryptosporidiosis in tanzanian goat kids: scanning and transmission electron microscopic observations infectious diseases transmission and scanning electron microscopy of host cell entry by toxoplasma gondii detection of streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs a non-biotin polymerized horseradish-peroxidase method for the immunohistochemical diagnosis of canine distemper immunohistochemical diagnosis of mouse hepatitis virus and mycoplasma pulmonis infection with murine antiserum pulmonary pathological features in coronavirus associated with severe acute respiratory syndrome (sars) murine hepatitis virus strain produces a clinically relevant model of severe acute respiratory syndrome in a/j mice nervous system pathology and molecular diagnosis of transmissible spongiform encephalopathy streptococcal diseases streptococcal toxic shock syndrome caused by streptococcus suis serotype we are extremely grateful to dr chin-cheng lee of shin kong wu-ho-su memorial hospital, dr cheng-hsiang hsiao of national taiwan university hospital, dr lin-lin chueh of national taiwan university, and dr chung-tiang liang of the national laboratory animal center for their kindness in providing the valuable photos in this article. key: cord- -sxt lw l authors: belz, katie title: rabbit hemorrhagic disease date: - - journal: seminars in avian and exotic pet medicine doi: . /j.saep. . . sha: doc_id: cord_uid: sxt lw l abstract rabbit hemorrhagic disease (rhd) is a rapidly lethal infectious viral disease of the european rabbit (oryctolagus cuniculus) characterized by high mortality rates, acute hepatic necrosis, and disseminated intravascular coagulation. although this disease is considered enzootic in europe and parts of asia, it is rarely seen in the western hemisphere since its eradication from mexico in . in recent years, three cases of rhd have been identified in the united states. due to the quick action of veterinarians these cases were confined and controlled before the disease could spread. r abbits are raised for food, for pets, and as show animals worldwide. rabbits are particularly important in asia and central europe, where small-scale rabbitries are common and an integrated part of the culture. in this article an emerging disease, rabbit hemorrhagic disease (rhd), is described. rhd already has had a major global impact and is a real threat to the rabbit industry and pet rabbits in the americas. rabbit hemorrhagic disease was first recognized in china in . the epicenter of the outbreak occurred in a group of commercially bred angora rabbits recently imported from germany to jiangsu province, china. these rabbits soon exhibited a contagious rapidly fatal disease that spread over , square kilometers in months killing , rabbits. this disease was variously called "x-disease of rabbits," "rabbit viral sudden death," "picornavirus hemorrhagic fever in rabbits," "hemorrhagic septicemia syndrome in rabbits," and "infectious necrotic hepatitis of leporidae." today, this disease is known as rabbit hemorrhagic disease (rhd), viral hemorrhagic disease of rabbits, or rabbit calicivirus disease (rcd). since , rhd has disseminated widely and has been reported in over countries from asia, africa, europe, and the americas. [ ] [ ] [ ] [ ] disease rhd is caused by the rabbit hemorrhagic disease virus (rhdv). rhdv is a member of the family caliciviridae, genus lagovirus, and is closely related but distinct from the european brown hare syndrome virus, which causes similar symptoms and disease as rhdv in hares. rhdv is readily disseminated. virus is shed in the feces and nasal secretions of infected rabbits. both ingested and inhaled virus can result in infection. the virus is stable in the environment. this allows rhdv to be spread by contact with contaminated caging, shavings, bowls, feedstuffs, and other fomites. the virus can also be passively transported by flies over short distances and can also travel by movement of people, equipment, wild or domestic animals, pelts, and rabbit carcasses. the incubation period for rhdv is short, only to hours. rhdv is highly infectious and highly virulent. as a result, morbidity and mortality rates may reach % to %. death usually occurs between and days postinfection, although some rabbits may live for several days before they die. the disease is confined to rabbits over months of age. it is believed maternal immunity protects the infant rabbits from this disease. as the maternal immunity declines at months of age, the rabbits become susceptible to rhdv. [ ] [ ] [ ] three forms of the disease are recognized depending on past history of the disease in the affected rabbit population. a peracute form of the disease occurs in naïve rabbit populations. in this form of the disease, rabbits die suddenly, exhibiting no or very few signs. the acute form of the rhd is seen in rabbit populations where the disease is enzootic. these rabbits typically show some signs of disease before death. a subacute form of the disease is uncommon and occurs in the later stages of an epizootic. in this form of the disease, affected rabbits are clinically ill, but many survive. [ ] [ ] [ ] several clinical signs have been observed in rabbits both experimentally infected with rhdv and those that were naturally infected (fig ) . not all clinical signs were present in every animal. animals may have a temperature above °f ( °c), show rapid respiration, cyanosis, and become anorexic and recumbent. severe diarrhea is common. in the late stages of the disease, various neurological signs are apparent, including lateral recumbency, paddling, ataxia, and terminally frenetic behavior sometimes accompanied by squealing. opisthotonos has been observed in many animals, and % of infected rabbits have a bloody foamy discharge from their nose and, less frequently, from the vagina (fig ) . hematological findings in rabbits with the acute form of the disease include lymphopenia, thrombocytopenia, and alterations in the coagulation panel. liver enzymes also are expected to be elevated. , typically, rabbits dying with rhd are in good body condition and have recently ingested food. rhd causes a severe diffuse necrotic hepatitis and disseminated intravascular coagulation. the liver may be pale, yellow, gray, friable, or congested and have a distinct lobular pattern. multifocal petechial hemorrhages occur in the liver, lungs, kidneys and heart (figs and ) . the spleen is often dark and engorged. pneumotracheitis and tracheal hemorrhage are common, and jaundice has been occasionally noted. lesions of the digestive tract are usually absent, although in some outbreaks, a catarrhal enteritis is a feature of rhd. in cases of acute rhd the characteristic hemorrhages might not be present. [ ] [ ] [ ] microscopic lesions of the liver are characterized by an acute diffuse hepatic necrosis and hemorrhage with minimal inflammation. round eosinophilic inclusion bodies are seen in hepatocytes. lymphoid necrosis in the spleen and lymph nodes is another characteristic lesion of rhd. glomeronephritis, encephalomyelitis, and enteritis also may be seen histologically. - figure . rabbit with rabbit hemorrhagic disease. this animal is severely depressed and poorly responsive. in a sudden outbreak of rabbit hemorrhagic disease, the duration of signs in rabbits before death is usually less than a day (photograph kindly provided by elizabeth morales salinas). a presumptive diagnosis can be made on client history, clinical signs, and postmortem findings ( table ). in north america and mexico, if this disease is suspected, a regulatory veterinarian should be immediately contacted. at specialized facilities, a diagnosis can be confirmed by electron microscopy of fixed tissues or homogenates of fresh tissue, immunofluorescense, a hemaglutination assay, or a viral antigen detection elisa. a complete set of tissues should be formalin-fixed for histopathology. small intestine, lung, liver, spleen, and kidney samples should be saved fresh for virus detection. eradication is currently considered the best response to this disease when it is introduced to a country that does not have rhd. when infected rabbits are identified, all in contact rabbits are destroyed. the carcasses, all wooden caging and housing, and all organic material is buried. metal cages and food bowels and glass water bottles are cleaned and then disinfected with % sodium hypochlorite solution. buildings that housed rabbits are power washed and then disinfected with sodium hypochlorite solution. soil that may have been in contact with rabbits, rabbit feces or urine is treated with the sodium hypochlorite solution and then calcium hypochlorite (lime). in the united states, sentinel rabbits were housed on the facilities and monitored for disease for month before the facility was allowed to be repopulated. treatment is only an option in countries were this disease is enzootic. because of the vigor and speed with which this disease kills rabbits, most rabbits are dead before any care can be administered. if rabbits are exhibiting clinical signs, isolation, supportive care, and symptomatic treatment is all that can be done. in areas were rhd is enzootic, cleanliness is vital. the virus is inactivated with a % sodium hypochlorite solution. rabbits should be housed indoors if possible. stray animals should be kept away from rabbit housing and flying insects kept to a minimum. access to the rabbitry should be limited, and caretakers should not have contact with other rabbits from other facilities. maximizing sanitation within the rabbitry, especially cleaning and disinfecting cages between rabbits, may reduce the chance of disease transmission. new stock should be quarantined in a separate facility for months, as rabbits that have survived rhd shed virus in their feces for up to months. , immunization vaccines against rhd are available in countries with enzootic rhd. there is a -month and a -year vaccine available. the vaccines are relatively safe, although illness, infections at injection sites, and death have been documented. rhd vaccines are not available in the united states and canada at this time. the vaccine currently being used in australia is cylap hvd (cyanamid, spain) and is a killed vaccine. it is safe to consume a rabbit vaccinated against rhd. rabbits can be immunized at weeks of age or older. if the rabbit is less then weeks old at first vaccine, a second immunization should be given in weeks. animals demonstrating any signs of ill health should not be immunized. the americas were free of rhd until december of when it is thought that a shipment of -this is the most common disease seen in clinical practice. -signs can range from soft stool and brown watery diarrhea with or without mucous or blood, to enterotoxemia, sepsis. severely affected rabbits will become anorectic and depressed, and die. -rabbits become hypothermic and moribund and die after - hours. occasionally a chronic form of the disease is seen with rabbits ittermitedly having diarrhea, anorexia, and weight loss. -post mortem findings include petechial and ecchymotic hemorrhages on the serosal surface of the cecum, sometimes including the appendix and colon. -gas may also be present in the small intestines, cecum, and colon. there have been three outbreaks of rhd in the united states. the first was an isolated rabbitry in iowa in . this premise was depopulated and additional cases were not found. the source of this outbreak was never determined. the second outbreak originated in utah in , but was first recognized in an illinois facility that had received rabbits from the utah source. both facilities were depopulated and the disease eradicated. the source of this disease was not confirmed; however, individuals on the utah facility had a history of traveling to countries where this disease was enzootic and may have brought the virus back on their clothing. the third outbreak occurred in a zoo in new york state. the veterinarian for the zoo suspected rhd and immediate action kept the disease from spreading from the zoo. the source of this outbreak is thought to be imported rabbit meat. rhd is a disease with a potentially devastating impact on the rabbit industry in countries where this disease has previously been excluded. as the economy becomes increasingly global and rabbit products from rhd-infected countries are imported into rhd-free countries, the chance for outbreaks increases. veterinarians are the first line of defense against rhd and are vital to its control. if rhd is suspected at your clinic, you should immediately contact local or national regulatory authorities. outbreak of rabbit hemorrhagic disease in domestic lagomorphs viral hemorrhagic disease of rabbits and human health rabbit haemorrhagic disease: the new scourge of oryctolagus cuniculus molecular epidemiology of rabbit haemorrhagic disease virus world organization for animal health ministry of agriculture and forestry: background information about rcd vaccine biological control of the rabbit in australia: lessons not learned? rabbit haemorrhagic disease virus: a calicivirus with differences ferrets, rabbits, and rodents clinical medicine and surgery key: cord- -gioqkeda authors: rosenthal, joshua title: climate change and the geographic distribution of infectious diseases date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: gioqkeda our ability to predict the effects of climate change on the spread of infectious diseases is in its infancy. numerous, and in some cases conflicting, predictions have been developed, principally based on models of biological processes or mapping of current and historical disease statistics. current debates on whether climate change, relative to socioeconomic determinants, will be a major influence on human disease distributions are useful to help identify research needs but are probably artificially polarized. we have at least identified many of the critical geophysical constraints, transport opportunities, biotic requirements for some disease systems, and some of the socioeconomic factors that govern the process of migration and establishment of parasites and pathogens. furthermore, we are beginning to develop a mechanistic understanding of many of these variables at specific sites. better predictive understanding will emerge in the coming years from analyses regarding how these variables interact with each other. scientists and public health officials have observed and mapped the geographical incidence of infectious diseases in relation to weather and climate for hundreds of years, and formally for at least a half century (reisen, ) . in recent years, this work has accelerated significantly in the context of predictions for global climate change (ipcc, b) . the development of and response to the ipcc predictions of probable increases of vector-borne and diarrheal diseases during coming decades (ipcc, a) has catalyzed a great deal of research, analysis, and speculation regarding the nature, magnitude, and extent of possible changes. numerous, and in some cases conflicting, predictions have been developed regarding the frequency, severity, and duration of epidemics that may emerge. with respect to the biogeographical focus of this issue, the central question is whether pathogens and parasites that are currently restricted to tropics and lower latitudes where the world's greatest biodiversity lies move toward poles (mostly north) and upward in altitude. perhaps the more controversial topic today is the corollary to this question-how much will future ranges of diseases that that do move be constrained by socioeconomic conditions, including our capacity to control them (hay et al., ; patz et al., ; lafferty, ) ? until very recently, climate projections for the coming decades have been limited to very coarse scale projections using global circulation models (gcms) at the level of continents and gross latitudinal and altitudinal changes (fowler et al., ) . downscaling such climate models is still in its infancy and only beginning to be useful for ecological and public health research at the regional, national, and sub-national scale for which most species distribution studies are conducted and validated (beaumont et al., ) . as such, epidemiologists, ecologists, geographers, and others are grappling with the implications of global climate projections for infectious diseases using a variety of approaches. these include biological process models, statistical geospatial analysis of current and historical prevalence and incidence, and macroeconomic demographic models, all coupled with climate analyses and projections. although we are still at an early stage in our ability to make predictions for these extraordinarily complex phenomena, we are beginning to see some general patterns with regard to the important geophysical factors that govern biological basis for distribution change, the role of transport of disease, vectors and hosts, the biotic assemblages that influence establishment, and the socioeconomic conditions that constrain or enhance these dynamics. underlying most predictions for climate change effects on parasite and pathogen distribution are the physiological factors that regulate survivorship, reproduction, and transmission, and their interaction with extrinsic environmental changes associated with climate: precipitation, humidity, air and water temperature, principally. under moderate greenhouse gas emission scenarios, gcm models project during the next century approximately - °c average warming and greater precipitation at higher latitudes, with decreased precipitation at lower latitudes, and increases in heavy precipitation events in many regions (ipcc, b) . biological process models are an important component of the discussion. biological models estimate the responsiveness and thresholds of parasites, pathogens, and vectors to temperature and precipitation. these models can be used to estimate survivorship, transmissibility, and reproduction, including estimates of r -the basic reproductive ratio-under different climate scenarios (see lafferty, for review). based largely on studies of vector and/or parasite development, warming and increases in humidity are predicted to open up new zones for malaria in africa (epstein et al., ; martens, ) , parasitic nematodes in the arctic (kutz et al., ) , west nile virus (reisen et al., ) , lyme disease in north america (ogden et al., ) , and schistosomiasis in china (zhou et al., ) . historical analyses of climate patterns coupled with these biological process models provide additional explanatory power. for example, observed altitudinal increases in falciparum malaria in the east african highlands during the past years have been associated with increasing temperatures and are consistent with models of anopheline mosquito vector development . process models also suggest, as one would expect, that in some cases, the same climate trends may reduce survivorship or transmission of pathogens, parasites, and their vectors at the warmer boundaries of their current ranges (lafferty, ) . that is because future temperatures may exceed the biological thresholds for survival or transmission at these boundaries. more analysis of biological maxima and minima of pathogens, parasites, and their vectors, including their diurnal fluctuation thresholds, in concert with downscaled climate models or historical records will be helpful in this regard. paaijmans et al. ( ) analyzed development time of plasmodium falciparum parasites in relation to that of its vector under a range of diurnal temperature fluctuations. they found that at the higher mean temperatures (> °c) associated with endemic malaria transmission in africa effects of global warming on malaria dynamics may be overestimated. however, at lower mean temperatures associated with epidemic outbreaks (< °c), such as those found in the kenyan highlands, real increases in r with a couple degrees of warming are biologically possible, and consistent with epidemiological trends during the past years in that region. similarly, changes in seasonality, especially intensity of rainfall, has been shown to be important in several diseases (koelle et al., ; altizer et al., ; pascual et al., ) , particularly cholera dynamics. more frequent heavy rainfall events are predicted to occur in some areas, although the confidence levels and scaling of these future precipitation models may still be insufficient to support geographically specific predictions. whereas vector-borne diseases, and to a lesser extent diarrheal diseases, have been the principal focus of discussion, directly transmitted diseases, such as influenza and cold viruses, are likely to change as well. recent laboratory work (shaman and kohn, ) on the persistence and transmission efficiency of influenza has shown the virus to be constrained markedly by absolute humidity. more recent epidemiological analysis support this finding (shaman et al., ) and suggest that the marked seasonality and generally predictable geographical trajectory of seasonal influenza likely reflect, at least in part, climate variables. analyses of biological processes in relation to climaterelevant geophysical parameters establish the potential for climate change effects. however, they are insufficient to explain the highly complex dynamics of infectious disease transmission and establishment, including biological, climatological and socioeconomic considerations. a frequently used alternative approach to predicting biogeographic spread is based on mapping the statistics of current and historical disease or vector prevalence or transmission coupled with climate projections, often based on gcms (rogers and randolph, ; mabaso et al., ; ebi et al., ) . more recent statistical mapping (also called niche modeling) approaches have been able to use downscaled climate models (de la rocque, ) in their projections. several have predicted northward shifting zones of infection with modest impacts on overall extent of distribution (rogers and randolph, ; hay et al., ; nakazawa et al., ) . although essential, many of these statistical mapping projects make their own set of assumptions. particularly problematic are those that assume that today's public health and socioeconomic conditions, including surveillance and control capabilities, populations affected, and other ecological and economic conditions, will be similar to future conditions at new locations. one significant and long-presumed constraint on longrange biogeographical spread of invasive species, including pathogens, parasites, vectors, and hosts, has been the role of spatial distance on the ability to arrive at a new hospitable site (macarthur and wilson, ; reperant, ) . this is, of course, less of a constraint today and for the foreseeable future due to a diversity of globalized transport opportunities. specifically, agriculture trade (arzt et al., ) , livestock movement (fevre et al., ) , wildlife trade (karesh et al., ) , human travel and migration (mac-pherson et al., ) , ship ballast water (aguirre-macedo et al., ) , auto tire trade (hawley et al., ) , and the global transport of desert dust containing microbiota regularly move pathogens, vectors, and infected hosts all around the earth. at small and intermediate scales, distance is still a very important determinant of disease movement. for example, triatomine vector dispersal of chagas disease in argentina at a scale of meters to a few kilometers (zu dohna et al., ) or movement of african trypanosomiasis at a scale of tens to hundreds of kilometers via animal migrations (batchelor et al., ) are clearly constrained by distance. of course, if pathogens establish in a new zone and the process is allowed to continue incrementally year after year, diseases can and have moved across countries in a decade without the aid of ''modern transportation.'' however, at continental and intercontinental scales, movement of many diseases in a warming world is unlikely to have a linear relationship to distance because of the numerous means of long distance transport operating today. because infectious diseases are components of species networks, simply arriving in a new location may be insufficient for establishment and creation of endemic lifecycles. a number of studies have shown that emerging and reemerging diseases, particularly those in the tropics, are predominantly zoonotic and/or vector-borne diseases (daszak et al., ; woolhouse and gowtage-sequeria, ; wolfe et al., ) . the principal ecological determinant for successful establishment for zoonotic and vector-borne disease success in a decadal time frame is probably a broad host range (woolhouse and gowtage-sequeria, ; daszak et al., ) . in recent years, a number of vector-borne viral diseases have migrated across or between continents through their ability to acquire new vectors (west nile, chikungunya) or utilize diverse and widely dispersed domestic (rift valley) or native (west nile) animal hosts (gould and higgs, ). importantly, the capacity to acquire new vector and host species often is facilitated by the capacity of the pathogen to evolve mutant strains that are efficiently acquired and transmitted by this new species. parasitic and infectious diseases with long, specialized, and/or especially complex life cycles involving multiple host species would logically be more challenged to establish themselves in new habitats by the need to find appropriate hosts and vectors. for example, the highly pathogenic alveolar echinococcosis (fox tapeworm) has a northern distribution that is influenced by climate but constrained by landscape variables that determine host availability (danson et al., ; mas-coma et al., ) . egg survival is dependent on ground moisture levels, but intermediate stages survive in rodents, and its definitive hosts are typically foxes, coyotes, and dogs. at large spatial scales across china and inner mongolia, a. echinococcus distribution has become focal due to the interaction of the requirements for a physical environment that is moist and cool enough to allow for egg survival and for the grassland landscapes that support both high densities of small mammals and canine hosts (giraudoux et al., ) . high levels of forest cover or cleared agricultural landscapes reduce intermediate and/or definitive host densities. it follows that for long-lived, host-specialized parasites, such as echinococcus, it is likely that long time periods are required for the evolutionary changes required to enable dispersal to zones with insufficient host densities of its original species. however, some historical and paleontological studies of parasites suggest that host-switching behavior in parasites in response to ecological changes has been more common than generally thought and global climate changes are likely to produce broadening of parasite host ranges even for long-lived, nominally specialized parasites (brooks and hoberg, ) . by contrast, water-borne diarrheal diseases often are predicted to increase with climate change. biogeographically restrictive interaction of diarrhea-causing pathogens with new hosts or other pathogens has received little attention to date in the context of climate change. an exception is a series of papers by colwell and workers on associations of vibrio cholera with copepods in zooplankton (gil et al., ; constantin de magny et al., ) . for directly transmitted human diseases, the sorts of species network challenges may be even less of a constraint. humans are most everywhere, highly mobile, and share most diseases readily. the principal biological barriers are host immune systems. largely for these reasons we see rapid cross-national and continental movement and invasion by new viruses, such as seasonal influenza, sars, and other coronaviruses. although much of the climate change and disease discussion has focused on vector-borne diseases (lafferty, ) , the ''biotic simplicity'' of directly transmitted diseases, in concert with the climate sensitivity of viruses as discussed earlier, might argue that we should pay more attention to them in climate change discussions. although there has been little serious discussion that climate change will not impact infectious disease distributions, a view expounded by a number of authors (reiter, ; zell et al., ; lafferty, ) discounts the relative role of climate change compared with other major influences on human disease. these factors include efficacy of control measures, such as vector control, sanitation, vaccination, and chemotherapy (hay et al., ) , as well as land use, urbanization, travel, wealth, and social behavior (gubler et al., ; sumilo et al., ) . supporting this view, statistical mapping analyses (rogers and randolph, ; hay et al., ) indicate that the spatial extent and number of countries reporting endemic malaria has shrunk toward the tropics from a much wider range during the twentieth century. the authors attribute this principally to better control of vectors and disease. further support can be seen in the rapid reemergence of tick-borne encephalitis in central and eastern european states after the collapse of the soviet union (sumilo et al., ) . statistical comparisons of results of extensive socioeconomic surveys and public health data across five former soviet states indicate that various socioeconomic, land use, and public health programs can explain most of the variation across countries in the spread of tick-borne encephalitis during this period. in this analysis, climate variables were not assessed, but significant unemployment, reversion of agricultural lands, reduced pesticide use, reduced chemotherapy, and individual exposure through increased field collection of wild foods appear to have contributed to rapid increases during the early s. the interaction of climate change, infectious diseases, socioeconomic conditions, and behavior is going to be increasingly important in the coming years and represents an exciting and complex area for research (patz et al., ) . for example, several authors have predicted mass migrations in response to climate change, particularly in response to worsening droughts in sub-saharan africa (ramin and mcmichael, ) (ipcc, a) . if borne out, it is likely that many diseases will travel with migrants, and in some cases will establish new infectious species or strains with significant effects on receiver populations (macpherson et al., ) . similarly, availability of clean water and hygienic behavior are principal determinants of diarrheal disease outbreaks, and clean water will be increasingly in short supply in much of africa and parts of southeast asia in the coming decades due to overexploitation and climate change (ipcc, a) . in an analysis of frequency all cause diarrhea in children younger than age years in low-and middle-income countries, lloyd et al. ( ) found that low mean monthly rainfall during the course of a year was significantly associated with disease. surprisingly, neither temperature nor simple measures of socioeconomic development (gdp or gcp) were significant in this analysis of a disease type mostly managed by traditional public health measures, such as sanitation and education. diarrheal diseases are produced by a diversity of bacterial, viral, and protozoan organisms. strain diversity within a species is high locally (e.g., jiang et al., ) around the world, and migration to new zones has significant implications for biologically naïve host populations. finally, given the importance and complexity of socioeconomic conditions on diseases, a variety of approaches and disciplines will be necessary to assess their effects on pathogen and parasite biogeography (parkes et al., ) . macroeconomic and demographic modeling may offer underexploited utility in this regard. a recent paper by tang et al. ( ) is instructive; they set out to disentangle the causality of a ''climate-income trap model for human health'' by examining national life expectancy statistics ( african, non-african countries) in a crosssectional analysis. using a series of regression models and variation partitioning analyses to separate pure and combined effects of income and climate their findings support the hypotheses that: ) income can moderate the adverse effect of climate on life expectancy (le); ) as income grows climate will have less of an effect on le; and ) climate has a larger effect on le in african countries than in non-african countries, and further that for african countries, climate has had a larger effect on le than wealth. debates over the impact of climate change on infectious disease emergence and migration are certain to continue in the years to come. accumulation and validation of biological process models, and ''ground truthing'' these with statistical and mapping approaches help provide us greater clarity and ensure that we are asking the right questions. macroeconomic and demographic approaches may help us address some of the big questions related to the role of disease management capability. critical to a greater understanding of these dynamics and more predictive power is greater availability of downscaled general circulation models to scales that are more relevant to community and landscape biology and public health. i have not addressed time scales in any depth here, but they are extremely important aspects of biogeographic changes in relation to climate. longitudinal studies on the multi-decadal time frames that climate change presents are a major challenge. but the time required for evolution of host range, for population dynamics of hosts and parasites to stabilize, and for medical and public health systems to adapt will all have major roles in determining biogeographic patterns of disease, and will all operate relatively independently. creative approaches are needed to address this problem. debates about whether socioeconomic conditions or climate will be more important are probably more polarized than is productive. both are clearly important. both of these complex suites of variables are interacting with each other, and may do so in different ways for different diseases in different places. however, understanding these disease forcing factors and their interactions with each other are crucial to our ability to predict and prevent the most severe health outcomes. the public health and medical community has made enormous strides in control of infectious diseases during the past half-century. widespread availability of antimicrobial drugs, vector-control systems, diagnostics, vaccines, and increasingly sophisticated predictive models represent a powerful set of tools to protect public health from emerging diseases. however, it would be unwise to discount the potential importance of climate change, land use impacts, and other ecological determinants of future disease. historically, ecological factors, including climate, have been extremely important in determining both the range and severity of diseases. these factors are now aggravated by the near disappearance of antimicrobial drug development in the industrial pharmaceutical sector, dramatic reductions in training and support for vector control scientists and implementers, persistent scientific and financial challenges in developing new drugs and vaccines for even the most widespread diseases (e.g., hiv, malaria, tb), and medical workforce shortages in the poorest parts of the world. as our scientific capabilities to describe and model diseases and disease processes expands rapidly, it will be important to ensure that we understand the changing nature of our planet and continually adapt our systems and invest in them to meet the challenges. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any 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in the continental united states socio-economic factors in the differential upsurge of tick-borne encephalitis in central and eastern europe the income-climate trap of health development: a comparative analysis of african and non-african countries origins of major human infectious diseases host range and emerging and reemerging pathogens impact of global warming on viral diseases: what is the evidence? potential impact of climate change on schistosomiasis transmission in china spatial re-establishment dynamics of local populations of vectors of chagas disease key: cord- -odmnvv m authors: darcel, c. title: reflections on scrapie and related disorders, with consideration of the possibility of a viral aetiology date: journal: vet res commun doi: . /bf sha: doc_id: cord_uid: odmnvv m the transmissible spongiform encephalopathies of domesticated animals, scrapie in-sheep and bovine spongiform encephalopathy (bse), and transmissible mink encephalopathy are more than a scientific curiosity; under certain circumstances their impact on commercial activities can be calamitous. knowledge of their causation and pathogenesis is still rudimentary, but many consider than an unconventional agent, the prion (a brain protein, prp), that is not associated with nucleic acid is involved in both. others believe that conventional viruses, which replicate by virtue of their nucleic acid-defined genes, are involved in the causation and progression of the encephalopathies but that technical problems have prevented their identification. others postulate even more exotic causative agents. while this paper will particularly address the possibility of a viral aetiology for these diseases, it is also emphasized that our knowledge of the state of the immune system in animals with encephalopathy needs broadening. there are remarkable gaps in our knowledge of the histopathology of these diseases, particularly the nature of the characteristic vacuoles. much further work is needed on the biochemical changes in the brain and the serum, particularly of the latter as it could lead to an additional means of recognizing clinical cases without waiting for the animal to die with subsequent examination of the brain for characteristic lesions and the presence of protease-k-resistant prp. in the transmissible spongiform encephalopathies (kimberlin, ) , a variety of neurological signs follow non-inflammatory, vacuolar, degeneration in the brain and spinal cord. accumulations of amyloid and other proteinaceous fibrils occur. these fibrillar accumulations can form plaques similar in appearance to those seen in alzheimer's disease in man. after a very long quiescent latent period following infection, the spongiform encephalopathies progress very slowly over a period of weeks to months. this group of diseases provides a good example of disastrous events in the field of veterinary medicine that have put into question all our current concepts of the genesis of disease. these events have also had the positive effect of focusing attention on enormous gaps in our knowledge and stimulating a great deal of research. reference to older literature on scrapie can be made in books edited by kimberlin ( a) and prusiner and hadlow ( ) . the advent of bse in britain, starting in the s, resulted in the appearance of numerous articles on the significance of the spongiform encephalopathies. these include a number of more recent reviews (little and thorsen, ; chesebro, ; powell, ) . scrapie, a disease of sheep, was the first of these diseases to attract enough attention to stimulate research into its causation and recognition. the disease had been recognized for some time but it did not become the subject of experimental work until the s, when cuiu and chelle, cited by gordon ( ) , reported on its transmission. its accidental transmission during large-scale field trials of vaccination of sheep against louping-ill also gave impetus to research on this disease .. the vaccine had been prepared from formaliuized brain and spleen suspensions from louping-ill-infected yearling sheep (gordon, ) . starting ½ years after vaccination, scrapie started to appear in animals of all breeds vaccinated with one lot of vaccine. this unfortunate experience prompted transmission studies designed to determine the nature of the infective agent and the pathogenesis of the disease in sheep. conclusions drawn from the vaccination trials and transmission experiments were that ~crapie, given by subcutaneous inoculation, had a latent period of years and longer; that the infective agent was resistant to . % formalin; that the disease appeared more quickly and in a higher percentage of recipients following intracerebral than following~subcutaneous injection; and that the causative agent was probably a filtrable virus. a similar disease to scrapie is bovine spongiform encephalopathy (bse), of which the first ca~e of a major enzootic was confirmed in november in england (bradley and matthews, ) . the emergence of this disease was considered to be related to the ingestion of meat and bonemeal derived from ruminant sources following countrywide changes in the rendering process (wilesmith et al, ) . the feeding of ruminant-derived meat and bonemeal to cattle in britain was stopped by law in . since the implementation of this ban, the occurrence of new cases of this disease in cattle has been reduced (wilesmith and ryan, ) . the bse outbreak drew attention to spongiform encephalopathies that occurred around the same time in other bovids, kept in zoological and wildlife park collections in britain cunningham et al, ) . these cases could have developed through the animals being fed similar rations to the cattle population. spongiform encephalopathy has also been found in mule deer and captive elk in north america young, , ; wells and mcgill, ) , evidence that agents causing encephalopathy are present in wild ruminants in that continent. • spongiform encephalopathy has recently been demonstrated in domesticated and wild felids (wells and mcgill, ; willoughby et al, ) . *please see notes section at end of text. transmissible mink encephalopathy (tme) (marsh and hanson, ; marsh, ) , which is rarely observed in ranch-raised animals, is caused by an agent with the same physicochemical properties as the scrapie agent. in the first outbreaks of tme, it could not be excluded that the occurrence of the disease had followed feeding of meat and offal from sheep, but the feeding of sheep meat was not involved in a more recent and major outbreak of tme on a ranch in the united states (marsh et al., ) . three diseases are associated with spongiform encephalopathy in man: kuru, creutzfeldt-jakob disease (cjd) and gerstmann-straiissler-scheinker disease (gssd). kuru, which is very similar to scrapie, is befieved to be associated with funeral practices involving ritualistic cannibalism (gajdusek, ) . a very full account of this and other human spongiform encephalopathies and follow-up experimental work is given by brown and gajdusek ( ) . cjd usually occurs sporadically at a very low incidence but deslys and colleagues ( ) refer to a group of cases of cjd that occurred in france among children treated between january and june with human growth hormone (hgh). the hormone had been extracted from large numbers of cadaveric hypophyses. the cases were confined to individuals with the same genetic status as sporadic cases of cjd, who were homozygous at codon of the prp gene. gssd shows a clinical overlap between kuru and cjd but is familial and related to a point mutation on chromosome , which encodes an amyloidogenic protein prp (brown, ) . as in scrapie and bse, the causative agent of cjd can be transmitted to experimental animals (gibbs et al, ; manuelidis and manuelidis, ; tateishi et al, ) . thus spongiform encephalopathies have been described in several species, and in most of them the disease has been shown to be transmissible to animals of the same species, where this is ethically possible, and to certain laboratory animals. the purpose of this paper is to give a condensed overview of the aetiology, pathology and familial nature of some of the spongiform encephalopathies as well as aspects of diagnosis and serology; it will particularly address the possibility of a viral aetiology for these diseases. there are many difficulties in studying either the natural or experimentally induced diseases: the animals involved, the incubation period required for the emergence of the disease, the innate resistance of a proportion of the population seen as an expression of genetic influences, the differing behaviour of strains of agents isolated from a given species, the symptomatology, the pathology, the uncertain nature of the agent and its means of transmission, the perceived 'lack' of an immunological response or changes in the immune system, and the biological hazards involved in conducting experiments. none of the infective agents that cause the spongiform encephalopathies can be demonstrated except by animal inoculation. sheep were the first experimental animals used for research on this group of diseases (scrapie) and the intracerebral route of inoculation was found to be better than the subcutaneous route for obtaining the maximum incidence of disease. there are ethical, logistical and humanitarian considerations against using large animals for this type of study. the finding that the spongiform encephalopathies were transmissible to certain laboratory animals (mice, hamsters and laboratory primates) was a critically important advance. it then became possible to titrate the infectivity of spleen, brain and other tissues in small laboratory animals, which was essential if any real advance in our understanding of the causative agents was to be obtained. however, a problem with the use of laboratory animals is that they may carry other disease agents in a latent form. the presence of such agents might even determine whether or not scrapie appears following inoculation with infected material and also the progress of the disease. this possibility has attracted attention, and 'caesarian-derived, barrier-maintained colonies have been the rule for most serious work in small laboratory animals. neoplasia, however, remains a problem in some colonies of laboratory rodents' (r.m. barlow, personal communication, ) . the need to hold and observe animals for long periods of time is a great inconvenience in studies on natural and experimental transmission of the spongiform encephalopathies. in bse, for instance, some cases are still emerging that may reflect exposure to the agent in the early s. in ovine experiments, the v -year period of observation noted in the early scottish transmission experiments was not excessive, although enough data can usually be collected within a -year period in sheep. in mice, scrapie infection progresses more quickly, and if - -week-old animals are inoculated intracerebrally with large amounts of the agent most of them will develop the disease within months (chandler, ) ; in hamsters the disease may proceed even more rapidly (kimberlin and walker, ) . contributing to the incubation period is the phenomenon that has been termed the 'zero phase' (dickinson and outram, ) . this is a period after infection when no infectivity can be demonstrated from such organs as the brain, spleen and fiver. the severity and type of lesions vary, and a quantitative assessment of these can be used as a characteristic of the biological behaviour of scrapie isolates. transmission experiments in inbred mice have shown that there are mixtures of agents in a natural case of scrapie in a sheep, and as many as distinct stains have been so identified. at least four murine strains of scrapie have been derived from the 'drowsy goat' source, the result of scrapie transmission experiments in goats (bruce and dickinson, ) . such a spectrum of behaviour by different isolates of field cases would be expected in studies with the spongiform encephalopathies of other species, especially if they truly represent passages of the scrapie agent. the agent causing bse is transmissible to mice and can be orally transmitted to c b mice by feeding bse brain (barlow and middleton, ) . however, the nature and distribution of pathological lesions in experimentally infected mice were similar even when the source of the infection was different species of bovidae , and there was no evidence of strain variation. although transmissible encephalopathy is not observed naturally in pigs, it can be produced experimentally in this species ) by transmission of bse. differently located neural lesions and different signs from those seen in tme were observed in mink receiving brain tissue from bse-infected cattle. transmission was effected both parenterally and orally (robinson et al., ) . in transmission experiments with cjd and kuru, chimpanzees and squirrel monkeys are the most sensitive, and are uniformly susceptible, but the disease has also been transmitted to laboratory animals. guinea-pigs died quite rapidly of hydrocephalus, which did not appear to be due to the presence of any additional conventional virus in the inocuhim. hamsters seemed uniformly susceptible and developed paralysis of the hind limbs and skin hypersensitivity. mice that were infected with guinea-pig-derived cjd developed signs of skin hypersensitivity and locomotor problems after a long latent period (manuelidis and manuelidis, ) . not all transmissible spongiform encephalopathies are due to scrapie-like agents. mice, homozygous for the grey tremor trait, develop a transmissible encephalopathy that is not accompanied with the coat pigmentation, the seizures, tremors and ataxia characteristic of the clinical disease in the donor animals. further, brain homogenates from these mice do not show the presence of proteinase-k-resistant prpsc (bendheim et al, ) . 'most neurological diseases follow the same general sequence of events: excitement -, hyperaesthesia -~ dementia ~ dullness -* coma -, death; incoordination -~ ataxia -, paresis -, paralysis. it is the duration of each phase which varies with the cause and the neural centres critically damaged' (r.m. barlow, personal communication, ) . the signs seen in scrapie, which are not specific, reflect damage to the neural centres, following the two alternative progressions noted above. for instance, with reference to the louping-ill vaccination trials in the s, louping-ill itself is also characterized by muscle tremor and incoordination of the hind limbs. there may be hypersensitivity to noise and touch. unlike scrapie, the disease progresses more rapidly. death follows within - days of signs of developing. some of the signs seen in scrapie show points of resemblance. in scrapie, there are also signs of increased skin reactions to pressure and other stimuli. there is a characteristic 'nibbling' reaction and a pruritus manifested by the sheep rubbing, leading to loss of wool over the rump, thighs and tail base. there is impairment of locomotion, starting with problems in the hind limbs (blood et al., ) . although scrapie is a far more chronic disease than louping-ill, it is curious that variations of hyperaesthesia are observed in both these diseases, with very different aetiologies. in bovine spongiform encephalopathy (bse), hypersensitivity to touch and sound are also common signs, but there are many other subtle changes in behaviour. however, cases of disease with these very signs, but which are not bse in terms of their histopathology, can occur in cattle (jeffrey and wilesmith, ) . it has also been suggested (gibbs et al, ) that bse may have occurred in the united states under the clinical guise of the 'downer cow' syndrome, even though cattle disease due to bse has not been reported in that country. these workers attempted experimental transmission of the scrapie agent in brain homogenate from sheep or goats to cattle. three cattle developed neurological signs - months after inoculation, consisting of progressive difficulty in rising, stiff-legged stilted gait, incoordination, disorientation and terminal recumbency. from onset to terminal signs, the disease lasted - . months. there were resemblances to the 'downer cow' syndrome. while autopsy revealed insufficient changes to confirm a clinical diagnosis of bse, the protease-resistant protein prp - was detected in each of the animals with neurological signs; none was detected in the other animals. if the results of this experiment can be replicated, the consequences are serious. the 'downer cow' is a familiar one to veterinarians and, while the usual aetiology is a ca/p imbalance, successful repetition of this experiment would mean that, where that imbalance cannot be demonstrated, the presence or absence of bse will have to be verified. the signs and symptoms that follow the onset of the human spongiform encephalopathies show some differences between types: in kuru there is a fine tremor of the head, trunk and limbs, together with the onset of a progressive ataxia; in cjd the symptoms are of dementia and, instead of the fine tremor, there is a myoclonic jerking; in gssd there is an overlap with the signs of kuru and cjd and there is also a marked dementia. the type of hypersensitivity associated with scrapie and bse is absent. returning to the pruritus seen in scrapie and bse, there seems to have been no investigation of the cause of the irritation. one must question whether the hyperaesthesia is evidence solely of nerve irritation or whether it is a pointer to a development of the hypersensitivity or skin lesions seen in some virus infections (fenner et al., ) or even related to the local release of histamine or a related compound. there are no immunological tests to aid recognition of the diseased living animal. clinical signs are not sufficiently specific to diagnose disease and diagnosis depends on the examination, by experienced pathologists, of stained sections of brain tissue for characteristic lesions (wells and mcgiu, ) , electron microscopy to detect the characteristic saf fibrils ) and tests to detect prpsc, the prion protein characteristically found in the spongiform encephalopathies (cho, ; rubenstein et al, ) . this latter protein is characteristically resistant to proteinase-k. prpsc contributes to the formation of the amyloid aggregates or plaques seen in the brain of animals with scrapie. prpsc is encoded by the prp gene, which expresses a similar protein prp-c (sensitive to the action of proteinase-k) in uninfected tissues. in the past, one histological characteristic of the brain lesions seen in spongiform encephalopathies drew much interest, this being the occurrence of large vacuoles outside the outer wall of neurons. no good explanation has yet been found for these vacuoles, which invariably appeared empty by both fight and electron microscopy -the larger vacuoles appearing to have formed by a coalescence of smaller vacuoles. although interesting, these vacuoles are not pathognomonic, as similar vacuolation has been seen in the cerebrum of mice infected with a parainfluenza virus (baringer et al, ) and also in lymphoma-prone wild mice (gardner et al., ) . nevertheless, such vacuoles are a good diagnostic aid. as 'empty' inclusions, they presumably represent unstainable material, fluid, gas or volatile hydrocarbon . the possibility of these vacuoles representing unstainable material was addressed many years ago by darcel and colleagues ( ) . these authors were aware of two familial diseases in man: castaigne-lhermite's and wilson's diseases. in both diseases, the patient's behaviour shows some resemblance to certain signs seen with the spongiform encephalopathies, namely a motor disability in the first disease, a progressive rigidity, a coarse tremor of the limbs, and some degree of dementia in the second. in castaigne-lhermite's disease, histological examination of the nervous system reveals the accumulation of glycogen within the nerve cells, varying from small droplets to large masses. in wilson's disease, where the onset is most common in the second decade of life, copper accumulates in large amounts in the corpus striatum and in other tissues. while there are differences in the clinical signs and the histopathology shown by both diseases from those seen in scrapie, it was thought worthwhile to determine the glycogen and copper levels in the brains of sheep with experimentally induced scrapie. the levels of neither copper nor glycogen were elevated compared to the values found in the brain of normal sheep . with respect to the vacuoles representing fluid accumulations, this is judged unlikely because body fluids, even cerebrospinal fluid, contain protein which is stainable by routine histological procedures. we are left with the possibility that these vacuoles result from the accumulation of gas in the form of bubbles or the presence of inclusions of volatile hydrocarbons. the most likely gases to be released would be oxygen or carbon dioxide. the latter would be taken up by the buffering action of the tissues, while oxygen free radicals are released by enzymes such as the o -and h -forming oxidases . in the blood, molecular o would be quickly bound by haemoglobin in the red cells, but most locations in the brain are beyond the 'blood-brain barrier' and, there, oxygen would be poorly soluble and quite toxic. with regard to the possibility of the vacuoles being due to volatile hydrocarbons, both ethane and pentane are released from cells when there is peroxidation of polyunsaturated lipids (miiller and sies, ) . efforts to detect evidence of such peroxidation in brain tissue from cases of transmissible encephalopathy have yet to be made. although hunter ( ) lists a number of changes in the brain with the progress of scrapie, neither this nor a survey of the literature shows any indication that the possibility that these vacuoles are gas inclusions has been vigorously researched. resolution of this problem could have led to a more directed approach to the biochemical changes that occur in the encephalopathies. biochemical changes that have been noted include changes in amino acids in the blood plasma and brain tissue but, except for great increases in the activity of hydrolytic enzymes, evidence of other biochemical alterations in the brain, plasma and urine is scanty (kimberlin, b) . liver function is normal . however, there has been the recent observation in one case of bse of a very high plasma glucose concentration (scott et al, ) . more data are needed as consistently high blood glucose could point to other biochemical investigations that should be made. millson and bountiff ( ) found a group of five glycosidases that showed greatly increased activity in the brain of mice with clinical signs following inoculation of the agent, four of these showing an increase before clinical signs appeared. one wonders whether such increased activity would spill over into the plasma and be usable for an early diagnostic test for the development of a spongiform encephalopathy. scrapie is transmissible, but the causative agent has not been shown to be a virus. one of the difficulties in accepting a conventional virus as the agent has been its resistance to damage with a variety of agencies. in spite of this, a viral aetiology has not yet been excluded. the possibility has recently been advanced that mollicutes (spiroplasmas) are responsible (see below), but most current thinking envisages a different kind of transmissible agent. two such agents have been postulated -'prions' and 'virinos'. the 'priori' was postulated to be an infectious protein that directs its own replication (prusiner, ) . the 'virino' hypothesis suggests the existence of a very small scrapie-specific nucleic acid (dickinson and outram, ) . evidence for the priori and virino and other possible hypotheses for the causation of the encephalopathies will now be considered. the variety in the infective agents that can cause disease is awe-inspiring, particularly with regard to the way they are propagated, retain their characters, and cause problems for the host. with bacteria and viruses, the infective agent carries its own genetic material in the form of either rna or dna, but a decade ago it was suggested that an altered protein can itself transmit the encephalopathies. this is the prion prpsc (prusiner, ) , or scrapie-associated fibril protein (saf), which is present as fibrils or rods, - kda in size, that can be seen by electron microscopy intracellularly and extracellularly in the brain of animals affected by scrapie. prpsc is heterogenous on electrophoresis, although this may be due to different degrees of glycosylation of this sialo-glycoprotein (somerville and ritchie, ). species differences have been found in bovine, murine and ovine prpsc (groschup and pfaff, ) . the homozygous prp genotype has been found to predispose to sporadic cad (palmer et al., ) and a prion protein missense variant is linked to the occurrence of gssd (hsiao et al., ) . the possibility that protein can be infectious in the absence of an association with nucleic acid is intriguing. prion protein and scrapie-associated fibrils are especially characteristic of brain lesions in scrapie (merz et al, ; bolton et al, ) . however, although hamster and mouse spleen contain only low levels of prp mrna , they can contain large amounts of the transmissible agent. this is hardly good evidence that the prion is the infective agent. kitamoto and colleagues ( ) were able to demonstrate a correlation between titres of the cjd agent in the brain and spleens of infected mice and their content of prp, while czub and colleagues ( ) were not able to do so for scrapie. unfortunately, it is difficult to resolve this question, since titration of the scrapie agent is attended with wide errors (marsh et al, ) and the specificity of serological methods for determining saf/prpsc is open to question, since prpsc is produced by the same gene as its homologous normal protein, differing only in its sensitivity to proteinase-k. another fact suggesting that the 'prion' is not the infective agent for scrapie is that prpsc expressed in vitro by a cloned prp gene is ineffective in inducing the disease. when the hamster gene for expressing this protein is introduced into mice, this species now has the susceptibility to scrapie characteristic of hamsters rather than the susceptibility of mice; the transgenic mice still have to be infected with the scrapie agent (scott et al, ) . other evidence indicating that the 'prion' or saf is not the causative agent of scrapie is given by braig and diringer ( ) . finally, bueler and colleagues ( ) showed that mice bred for lack of expression of the prp gene are resistant to the development of scrapie. after the introduction of syrian hamster prp transgenes, these mice became highly susceptible to hamster but not mouse prions. again, it has been found (xi et al, ; demaimay et al., ) that, following treatment of hamsters with amphotericin b or a derivative of this antibiotic, the accumulation of prp was not correlated with replication of the scrapie agent. race and colleagues ( ) described experiments with cloned neuroblastoma cultures with a high titre of the scrapie agent. while these workers acknowledged that a proteinase-k-resistant protein developed with scrapie infection in these experiments, they found no evidence that the pk-resistant form of prp was necessary for infectivity. they concluded that the pk-resistant prp of scrapie arose in vivo as a result of alteration of pk-sensitive prp by changes in the physiological conditions of cells as they degenerate, and that the evidence for the inseparability of the scrapie agent and pk-resistant prp was still circumstantial, a view not shared by bolton and colleagues ( ) . caughey and colleagues ( ) speculated, from further work on these neuroblastoma cultures, that the increased tendency of prp to aggregate is the origin of this resistance to pk. lewin ( ) was ready to accept that the prion is involved in the aetiology of scrapie but only as a carrier of a small protein or peptide that he called a protovirin. he suggested two alternative mechanisms by which such an entity could act either as an rna template or as an extraneous dna operon. it appears that it is very difficult to resolve the problems of whether prpsc is the causal agent of scrapie or a carrier agent, and even whether the causal agent is a protein, by the approaches that have been so far made. whatever the outcome, it is certain that prp plays an important part in the pathogenesis of the transmissible spongiform encephalopathies (weissmann, a,b) . the discussion on the role of prions is taken up again in other sections of this paper. the virino hypothesis suggests that the infectious agent of scrapie is a very small piece of nucleic acid with a behaviour intermediate between that of viruses and that of viroids (dickinson and outram, ) . it has been further suggested that the virino is not translated and is therefore dependent on host-coded protein to form an infectious unit (kimberlin, ) . with the nucleic acid probe techniques now available, it would be thought that virinos would be detectable if they exist, but no report of such work has yet appeared. characteristic features of spongiform encephalopathies include their long latent period for development of overt disease and the need for the presence of 'susceptibility' genes and the prp gene in the prospective host. this does not rule out the involvement of a conventional virus in causation of these diseases. although no such virus has yet been demonstrated in infective extracts, many believe that such viruses may be involved. chesebro ( ) , reviewing the role of prp in the aetiology of scrapie, raised the possibility that a mutation of this gene increases the susceptibility to an unknown but ubiquitous conventional virus. rohwer ( ) summarized in a masterful way why conventional viruses may still be the most likely causes of the spongiform encephalopathies. for example, the long latent period needed for the spongiform encephalopathies to develop is similar to the length of time often required by lentiviruses such as the maedi-visna virus of sheep. however, no connection has been made between such viruses and scrapie. a long latent period for disease to develop is seen with other viruses, for example oncornaviruses. examples are the lymphoid leukosis viruses of chickens; these are noted for their ability to activate the formation of oncogenes. oncogenes are involved in turnout formation because their presence leads to perturbation of the normal cell physiology. scrapie is not characterized by the development of tumours but oncogenes could still be involved, tied as they are to normal cell physiology and to gene changes. studies similar to those now being made in oncology might be most fruitful. references should be made to oncogenes in the human genome, where as much as . - . % may be derived from endogenous sequences. one such sequence is the erv- virus, first recognized by low-stringency hybridization to probes derived from the pol region of the chimpanzee ch endogenous provirus (boyd et al., ) . this work is mentioned as being indicative of a type of approach that should be undertaken on a major scale with bovine and ovine tissues. particular attention should be paid to the possibility of retroviruses akin to the mink-cell focus (mcf) inducing viruses being involved in the pathogenesis of the spongiform encephalopathies . the possible involvement of other retroviruses, particularly the lentiviruses, maedi-visna in sheep and visna-like virus in cattle (evermann, ) should not be overlooked as possible contributors to the development of scrapie and bse, in their respective hosts. attention is also drawn to the mouse hepatitis viruses, coronaviruses that have the ability to enter into recombination with other viruses (luytes et al., ) and are widely present in mouse stocks; there are also neurotropic strains (kyuwa and stohlman, ) . louping-ill virus is a tick-transmitted member of the virus family flaviviridae (fenner et al., ; gao et al., ) that is closely related to other european tick-borne encephalitis viruses (jiaug et al., ) . it was the evaluation of a vaccine designed to prevent disease due to louping-ill that led indirectly to the initial major studies on scrapie (see above). could vaccination against infection with this virus have increased the susceptibility to scrapie? another virus that has been mentioned in the context of scrapie causation is the hepadnavirus. some time ago, carl eklund stated that 'the scrapie agent is not a member of a unique group of pathogens but is a medium-sized virus whose resistance to heat is analogous to that of serum hepatitis virus' (hotchin, ) . this suggestion seems to have attracted little attention, but there is a strong possibility that hepatitis b viruses (tiollais and buendia, ) are more widespread than previously believed. viruses similar to human hepatitis b virus have been found in woodchucks and ground squirrels (minuk et al., ) , while antibody to this virus can be demonstrated in many other animals, including pigs, goats, cattle and especially sheep (hoofnagle et al., ) . of particular interest, with relation to the virino hypothesis of scrapie, is the fact that another agent is associated with hepatitis b virus. this agent is the hepatitis delta virus (hdv). hdv is a small defective viroid-like rna virus (negro et al., ) that requires another virus, hepatitis b virus, as a helper. thus, even if hepatitis b viruses are found in farm animals, the search should not stop there but continue with a search for hdv-like viruses. humphrey-smith and colleagues ( ) noted that the presence of spiroplasma-like inclusions has repeatably been noted in cases of cjd, that spiroplasmas show a number of similarities with the scrapie-associated fibrillar protein (saf), and that lesions minicking spongiform encephalopathy are produced by spiroplasma-infected rodents. spiroplasmas belong to the class mollicutes, order mycoplasmatales. like mycoplasmas, spiroplasmas, some of which are pathogenic for insects and plants, are a family within this order. humphrey-smith and colleagues ( ) suggested that spiroplasmas should not be ignored as a possible cause of spongiform encephalopathies. there are indeed immunological cross-reactions between the proteinaseresistant proteins of spiroplasma mirum and prpsc (bastian et al., ) and there are proteinase-k-resistant proteins in other members of the class mollicutes (butler et al., ) . it would have been thought that articles would have appeared by now listing attempts to demonstrate antibody to s. mirum and other spiroplasmas in the sera of sheep with scrapie and cattle with bse and attempts to correlate the titres against such agents with the onset of signs, but there do not appear to have been any such papers. scrapie is a particularly frightening disease. the transmissible agent is very resistant to heat (mould and dawson, ) and to other agents normally destructive to biological materials and it gives the appearance of an ability to cross species boundaries. the first clue to the resistance of the agent came from the louping-ill vaccination trials in the s (see introduction), where scrapie developed following the inoculation of formalinized tissue. while later shown not to be completely resistant to the action of formalin, the agent's resistance to a number of other disinfectants is unexpectedly high for the viruses and similar agents with which we are familiar. further, studies on the critical size of the agent by x-irradiation and by exclusion chromatography give very different estimates of the limiting size of the agent. x-ray studies suggest a size of . x kda, while the much larger figure of × kda is suggested by chromatographic studies. this difference suggested to hunter ( ) , a source for much data on the physical properties of the scrapie agent, that a two-component system was necessary for scrapie infectivity, a small informational component that provides the radiation target, and a larger component that provides a protective or carrier role. hunter ( ) also indicated that only proteases have much effect on scrapie activity and that, even for this effect to occur, the substantial amounts of lipid associated with brain tissue must be removed. part of the evidence that led to the prion concept was the finding that the proteinase pronase destroyed the activity of the agent. this pointed to a need for a protein component for its infectivity (cho, (cho, , . however, certain tissues contain proteinase inhibitors that can block the effectiveness of proteinases and, in studies on the kind of activity proteinases have on brain extracts, darcel ( ) found that pronase, highly active in control tests on sheep l';aemoglobin, had no proteolytic effect on the hirt supernatant of normal mouse brain. reasons for this lack of activity with brain extracts should be explored and extended to proteinase-k, to which prpsc is more resistant that its homologue in normal mouse brain . the sensitivity to this enzyme was found to va~ between three stains of the mouse-passaged scrapie agent (kascak et al., ) '. without data on what the proteinase-k was really accomplishing, this information cannot be convincing. there will not wittingly be repeats, on any worthwhile scale, of what i have termed the accidental 'experiments'. thus it is useful to look back at them and see whether any information was missed in the earlier analysis. the louping-ill vaccination trial provided much of the information on scrapie that we have today: the long latent period before disease appears, the resistance of the agent and the resistance of much of the population to the development of the disease. rather than implicating the acquisition of infection by ingestion of 'contaminated' feed, that event directly implicated parenteral 'infection'. no possibility should be overlooked in studies of a disease as strange as scrapie. even though louping-ill virus affects the same areas of the nervous system as evidenced by clinical signs, inactivated louping-ill virus seems to have been excluded as a contributing factor to the development of scrapie by the design of the vaccination trials. however, the possibility that the injection of a killed louping-ill virus can potentiate infection of the scrapie agent deserves further study s. other controls in attempting to understand the results of the vaccination trials of the s could be to examine brains of sheep with louping-ill for the presence of protease resistant prpsc and also to test the sera of sheep used in scrapie-transmission experiments for antibodies to a wide spectrum of louping-ill virus isolates. while the above concerns spread of scrapie by parenteral injection, there has been a suggestion that natural transmission may occur from sheep eating dropped placentae (dickinson et al., ) , but this is contraindicated by the fact that ewes usually confine themselves to eating their own placenta. the interpretation that a change in the rendering of sheep offal led to the outbreak of bse is based on statistical evidence and on the conclusion that, in the circumstances at the time, no other explanation was possible. it was also an explanation that could be adopted for political reasons and gave some justification for the control procedures that were adopted for controlling bse. however, it has been pointed out that the human risk from eating mutton and lamb must be negligible since, in a country where scrapie occurs as a disease and where this meat has been eaten for a very long time, the incidence of encephalopathies, such as cjd, that might be expected to follow, is actually very low ( . cases per million) (mathews, ). finally, despite earlier concerns, a connection between the feeding of sheep offal and the incidence of tme has not been proven (see above). avery and colleagues ( ) considered that, if scrapie were a chronic disease caused by an infective agent, the level of gamma-globufin, the serum protein fraction containing antibody, might well be raised. unfortunately, the electrophoretic patterns were similar for the sera of both normal and scrapie-affected animals. nevertheless, despite the 'rather overwhelming evidence for "immunological silence"' in scrapie, stites and colleagues ( ) advanced some evidence for the development of an immunological response. they stressed that the lack of detection of an immune response does not convincingly disprove its existence. whatever the causal agent, if its tropism were entirely neural, then insufficient agent might reach the blood stream to lead to an immunological response. this explanation appears weak, but another possible explanation is that saf is too close to its equivalent produced by the prp gene of normal animals to give a response, although it would be expected that even a small difference would lead to the production of recognizable antibody. a more direct immunological approach would be to look for antibody to other disease entities; for example, with what frequency are antibodies to blv encountered in cattle in cases of bse? with what frequency are maedi-visna antibodies encountered in sheep with scrapie? these viruses are quite prevalent in dairy cattle and in sheep, respectively, and are slow viruses in their own right. both viruses have a dna phase and could well have an association with genes for both susceptibility to the encephalopathies and amyloidal fibrin formation. antibodies to hepadnaviruses, to mcf viruses and to spiroplasma should also be sought. unless there were an associated immunodeficiency, the absence of detectable antibody to these agents would largely remove them from consideration as having primary or secondary roles in the causation of the spongiform encephalopathies. evidence of an immunological deficiency should also be sought using current technology similar to that used by rao and colleagues ( ) to study the immune deficiency produced by the avian retrovirus, avian erythroblastosis virus. stites and colleagues ( ) affirmed that current information could accommodate the possibility that negative regulation or active immunosuppression play an important role in the pathogenesis of scrapie. they noted that mice show a distinct splenomegaly after intracerebral inoculation with the agent, that the agent at first appears in higher concentrations in the spleen than in the brain, and that splenectomy prolongs the incubation period. they also recorded altered responses of spleen cells to mitogens. as in other studies with laboratory animals, they could not entirely rule out the possibility that a passenger virus or microorganism was responsible for these changes. this possibility of the presence of passenger viruses and microorganisms is a constant obstacle to understanding the results of agent transmission experiments in laboratory animals. is spiroplasma mirum, proposed as a possible cause of scrapie, merely a passenger responding to a suppressed immune state in the same way as pneumocystis carini in patients with aids (redfield and burke, ) ? any passenger agents involved in the spongiform encephalopathies might well vary according to the host species. because of the widespread consumption of products obtained from cattle by most of the human population, the recent outbreak of bse has caused great alarm and has been the subject of a considerable correspondence in the british medical press. it is not known whether cattle are the final hosts, nor whether the infection can spread to humans by the consumption of beef, milk or products, such as bovine albumin used in various forms of therapy . with the ability for agents causing the spongiform encephalopathies to cross specimen boundaries, the fear that people might acquire human forms of these diseases from cattle has to be very real. it might be assumed, for instance, that, as a result of the epidemic of bse through which british cattle have passed, slaughterhouse workers will have inadvertently come into contact with preclinical cases (howard and castle, ; deauer and lacey, ; coyle and harvey, ) . scrimgeour and brown ( ) pointed out that the most efficient means for transmission of scrapie, other than the parenteral routes used experimentally, are via the conjunctival and nasal mucosae and cutaneous abrasions. they suggested that slaughterhouse workers would be prone to this type of contact with microbial zoonoses. the wearing of protective clothing by these individuals when they are extracting the cranial contents from cattle was also advised. however, these fears of the human population acquiring bse should be tempered by realism (collee, ) . cjd often occurs in clusters with a history of familial relationships, and these seem to have more relevance to the incidence of the disease than do eating habits (taylor, ) . if bse presents a hazard to the human population, especially to veterinarians or to slaughterhouse personnel, a statistical study of the occurrence of spongiform encephalopathy over the next several years should show whether the incidence of cjd has increased as a result of the intimate exposure to cattle. the potential risks to which the british meat-eating population has been exposed have led to funding of the surveillance of cases of cjd that arise in the united kingdom, with particular reference to the dietary and occupational history and other possible risks . however, fear and devakumar ( ) have questioned the accuracy of such an ongoing survey, given the variety of symptoms exhibited by patients with cjd. a major problem with experimental work on the encephalopathies is the resistance of the agent. in performing experiments on scrapie, etc., great care has to be taken to avoid using contaminated laboratory equipment. illustrations of the care that has to be taken are the technical details given in papers on the oral transmission of bse to mice (barlow and middleton, ) and in the transmission studies with alzheimer's disease (manuelidis and manuelidis, ) . the innate resistance of a proportion of the population to experimental transmission of the spongiform encephalopathies is seen as the result of genetic influences. some animals within a species will prove resistant to experimental infection following infection. however, the degree of resistance will vary according to the route of infection. in most species, the natural route of infection appears to be the oral route and innate resistance is then likely to be more important than when animals are infected intracerebrally. offspring from infected ewes are prone to develop scrapie. this may be evidence of a familial trait and the practice has been to remove affected family lines in attempts to control the disease °. however, it is uncertain whether this predisposition of sheep to scrapie is due to increased exposure at time of parturition or to gene-dependent susceptibility. there are specific genes for susceptibility to scrapie: sip, which controls the incubation period of the disease in sheep, and the sinc gene in mice (bradley and matthews, ; kimberlin, ) . sinc has also been found genetically linked to the prn protein gene (hunter et al., ) . the finding by deslys and colleagues ( ) that all the cases of cjd acquired by injections of human growth hormone in french children occurred in individuals who were homozygous at codon of prp is seen as support for the importance of the prp gene in the development of encephalopathies. a complication in the british outbreak of bse is that the national gene pool among cattle will have changed over the same time-frame as the epidemic owing to the widespread adoption of artificial insemination (ai) and embryo transfer (et). perhaps a new study of the data that have been accumulated on this outbreak might point to certain bulls or certain cows that have had an unexpectedly high incidence of bse in their progeny. if such an effect could be demonstrated, it would mean either that these cases have followed the introduction of genes for susceptibility into previously unaffected herds or that actual infection occurred through the use of these breeding techniques. wrathall and brown ( ) stated that there is no evidence to suggest that vertical transmission of bse occurs and discussed et and ai studies with scrapie-infected sheep. it has been thought that et and ai might provide a means for breaking the disease transmission cycle in scrapie and bse, but such studies will be complicated by the existence of the genes for susceptibility. the research done on the prion theory has greatly expanded our knowledge of these diseases, but the most active promoters of the prion ask us to believe that it, and its gene, can cause the encephalopathies, that it is responsible for the predisposition to and the pathogenesis of these diseases, and that it can ensure its own replication. much more controlled work on the prion is necessary before conclusions of this kind can be justified. for instance, can the material of which prions are a component (saf) act as a viral promoter of other neurotropic viruses? an investigation of such a possibility would seem to be essential in understanding the role that the prion plays. studies with known neurotropic viruses, with the viruses given in the presence and absence of saf, should show whether this material can have a promoter effect. tile quotation at the beginning of this article describes workers in the field of the encephalopathies very well, each interested in their own particular area. in the author's case, the bias is towards viruses being involved in the causation of these diseases. in considering the experimental work that has been done, the differences in the biological activities of the isolates of the transmissible encephalopathies are impressive. if bse were really a manifestation of transmitted scrapie, one would expect to find that its isolates behaved with the variability shown by scrapie isolates. this does not occur. not knowing the cause of the transmissible encephalopathies makes the approach to controlling these diseases very difficult. the outbreak of bse in britain has disrupted the export of pedigree cattle, and there were other ramifications. there is the concern, not substantiated, that once transmitted to a new species, such as bovine, the agent might persist in the new species. there are also fears that the disease might become established in other countries and cross the species boundary to man. the expenditure of money for research on the encephalopathies is therefore justified from the agricultural standpoint alone, and the present paper has tried to suggest areas that are suitable for further exploration. one interpretation of the cause of the outbreaks of scrapie, tme and bse noted in this paper is that material in the feed or in the inoculum triggered the growth of a quiescent but unrecognized virus. is such triggering of a quiescent virus possible? we know that contact with certain compounds can lead to the multiplication of latent oncornaviruses (robinson et al, ) and that corticosteroids can provoke the emergence of viral activity with latent herpesvirus infections (darcel and dorward, ; fenner et al, ) . other mechanisms may also exist for the stimulation of virus growth. it is peculiar how refractory the transmissible spongiform encephalopathies are to investigation, and a real understanding of their causation is still elusive. yet, with research, there is a chance of discovering new types of disease agents and learning much about the nature of important neurological diseases. . autophagic vacuoles in mice, with sequestered cytoplasmic areas containing ribosomes and occasionally mitochondria and small secondary vacuoles, have been described in neuronal perikarya and neuronal processes of mice with experimental scrapie and cjd (liberski et al, ) . there seems to be a renewed interest in these neuropil and neuronal vacuolations in circumscribed areas of the brain, with both scrapie and bse (shinagawa et al, ; sasaki et al, ; wells et al, ; wells and wilesmith, ; liberski, ; scott et al, ) . . further references to lhermite's and wilson's diseases are given in the paper by . to study the possibility that these characteristic vacuoles are due to oxygen would mean the application of a variety of biochemical and histochemical techniques, including a qualitative and quantitative study of the enzymes just mentioned (brunori and rotilio, ) . r.m. barlow (personal communication, ) has suggested that marginal copper deficiency, which is very common in sheep, would affect the superoxide dismutases and facilitate accumulations of oxygen free radicals and toxic peroxides, but the result from studies on copper levels in the brains of sheep with scrapie and those of normal sheep do not support this idea. mcf murine viruses are formed de novo by recombination between exogenous, infectious ecotropic virus and endogenous sequences related to xenotropic virus. members of this group of viruses have been isolated from spontaneous mouse lymphomas and can infect both murine and xenogenic cells (cells of other species). special attention is drawn to work of gardner and colleagues ( ) on a routine retrovirus motor disease. mcf-like viruses have also been recovered from feline (neil et al, ) and avian oncogene-murine chimeric systems (feuer et al, i ) . other references that provide a useful entry to this field include adachi et al. ( ) , chattopadhyay et al ( ) , di fronzo and holland ( ), khan et al ( ) , koch et al ( ) , levy et al. ( ) , makino et al. ( ) , oliff et al. ( ) , quint et al. ( ) and shields et al. ( ) . . if scrapie-infected brain had been used instead of normal brain and loss of activity of the scrapie agent had been observed, then the effect would probably have been due to non-proteolytic factors in the pronase. the suggestion was offered (darcel, ) that the brain extracts contained a proteinase inhibitor. this should be looked for, and, if found, removed from the system before applying the proteinase treatment. these authors' protocols show that brain homogenates were homogenized with a solution containing proteinase-k, and the supernatant resulting from centrifugation was incubated for i hour. the action of the enzyme was then blocked and the solution was then treated with micrococcal nuclease. many other steps followed before titres were determined in mice and hamsters and compared with those in the original homogenates. the name 'berry-dedrick phenomenon' was coined to describe the results that followed the appearance of myxomatosis after inoculation of rabbits with a heat-inactivated preparation of myxoma virus inoculated into rabbits together with live fibroma virus. this was later explained as resulting from the presence of live, residual myxomatosis virus (fenner et al., ) . however, cross-reactivation is a known phenomenon by which infectious virions containing active genes from both viruses can result from genetic recombination between an infectious virus and a related inactivated virus (fenner et al., ) my late colleague robert kasting became ill some years after we concluded our studies of amino acids in scrapie and normal brain. he felt that the signs of neurological disease that he exhibited meant he had acquired scrapie from aerosols produced by grinding the brains. his brain was sent to the rocky mountain laboratory after his death for detailed histology and for transmission experiments. the results did not confirm his suspicions. i . six of the lambs born to recipients of embryos transplanted from donor ewes that had been experimentally infected with scrapie developed this disease (foster et al., ) . no correlation was found between susceptibility and the phenotypes for haemoglobin, blood potassium levels, lactoglobulins, albumin, pre-albumin, esterase, haemoglobin, transferrin, reduced glutathione or a-mannosidase hunter, ) . characterization of the env gene and long terminal repeat of molecularly cloned friend mink cell focus-inducing virus a note on serum proteins in normal and scrapie infected sheep fnrther observations on the neuropathology of experimental scrapie in mouse and hamster oral transmission studies of bse to mice antiserum to scrapie-associated fibril-protein cross-reacts with spiroplasma mirum fibril proteins scrapie agent proteins do not accumulate in grey tumour mice purification of scrapie agents: how far have we come? identification of a protein that purifies with the scrapie prion copurification of sp - and scrapie agent from hamster brain prior to detectable histopathology and clinical disease 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encephalopathy in mice produced by inoculation of scrapie brain characteristics and contributions of defective, ecotropic, and mink-cell focus-inducing viruses involved in a retrovirus-induced immunodeficiency syndrome of mice spongiform encephalopathies: the transmissible agents spongiform encephalopathies: prp and the scrapie agent requirement of a protein component for scrapie infectivity inactivation of the scrapie agent by pronase antibody to a scrapie-associated fibril protein identifies a cellular antigen bovine spongiforna encephalopathy and man bovine spongiform encephalopathy and risk to health transmissible spongiform cncephalopathy in greater kudu (tragelaphus strepsiceos) the major protein of saf is absent from spleen and thus not an essential part of the scrapie agent biochemical aids to the genetic typing of sheep -a further note pronase does not reduce the protein content of hirt supernatant of normal mouse brain potassium and hemoglobin types in sheep with special 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encephalopathy the biology of animal viruses veterinary virology specificity of disease induced by m-mulv chimeric retroviruses containing v-myc or v-src is not determined by the ltr studies on the natural transmission of scrapie in sheep by embryo transfer transmission of bovine spongiform encephalopathy and scrapie to mice unconventional viruses and the origin and disappearance of kurn sequencing and antigenic studies of a norwegian virus isolated from encephalomyelitic sheep confirna the existence of louping-ill virus outside great britain and ireland murine retrovirus motor neuron disease strain variation in the viruses of creutzfeldt-jakob disease and kuru experimental transmission of scrapie to cattle advances in veterinary research: louping-ill, tick-borne fever and scrapie studies on a species-specific epitope in routine, ovine and bovine prion protein antibody to hepatitis b surface antigen in nonprimate animal species scrapie as a slow and latent virus concern about bovine 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extracts of brain, urine and plasma from normal and scrapie sheep expression of mink cell~focus forming murine leukemia virus-related transcripts in akr mice slow virus diseases of animals and man biochemical and behavioural changes in scrapie transmissible encephalopathies in animals characteristics of a short incubation model of scrapie in the golden hamster organ distribution of proteinase-resistant prion protein in humans and mice with creutzfeldt-jakob disease molecular analysis of the envelope gene and long terminal repeat of friend mink cell focus-inducing virus: implications for the functions of these sequences pathogenesis of a neurotropic murine coronavirus, strain jhm in the central nervous system of mice normal expression of polynlorphic endogenous retroviral rna containing segments identical to mink cell focus-forming virus infectious peptides: postulated mechanisms of protovirus replication in scrapie ultrastructural neuropathologic features of bovine spongiform 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hepatocellular carcinoma in the canadian richardson ground squirrel (spermophilus richardsonii) the response in mice to heat treated scrapie agent assay of ethane and pentane from isolated organs and cells basis of hepatitis delta virus disease? feline leukaemia virus: generation of pathogenic and oncogenic variants the envelope gene and long terminal repeat sequences contribute to the pathogenic phenotype of helper-independent friend viruses homozygous prion protein genotype predisposes to sporadic creutzfeldt-jakob disease spongy brain: the mystery of mad cow disease. toronto globe and mail novel proteinaceous infectious particles cause scrapie pathogenesis, immunology, virology and molecular biology of the spongiform ence_phalopathies generation of akr mink cell focus-forming xenotropic-like provirns provides recombinant long terminal repeat sequences analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones immune abnormalities in avian erythroblastosis virns-infected chickens hiv infection: the clinical picture production of unique c-type viruses by chicken cells grown in bromodeoxyuridine experimental infection of mink with bovine spongiform encephalopathy the scrapie agent: 'a virus by any other name detection of scrapie-associated fibril proteins using anti-saf antibody in non-purified tissue preparations spongiform encephalopathy in sheep scrapie: electron microscopic observations bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (prp) and vacuolation in brain transgenic mice expressing hamster priori protein produce species-specific scrapie infectivity and amyloid plaques bovine spongiform encephalopathy in an adult british friesian cow bse and potential risk to slaughtermen calorie restriction suppresses subgenomic mink cytopathic focus-forming routine leukemia virus transcription and frequency of genomic expression while impairing lymphoma formation characterization of the scrapie agent isolated from sheep in japan differential glycosylation of the protein (prp) forming scrapie associated fibrils the immunology of scrapie a transmissible variant of creutzfeldt-jakob disease with kuru plaques bovine spongiform encephalopathy and human health hepatitis b virus the prion's progress a 'unified theory' of priori propagation recently described scrapie-like encephalopathies of animals: case definitions the distribution of neuronal vacuolation in bovine spongiform encephalopathy (bse) is constant a novel progressive spongiform encephalopathy in cattle bovine spongiform encephalopathy: diagnostic significance of vacuolar changes in sgleeted nuclei of the medulla oblongata bovine spongiform encephalopathy: observations on the incidence during bovine spongiform encephalopathy: epidemiological studies on the origin bovine spongiform encephalopathy: case-control studies of calf feeding practices and meat and bone meal inclusion in proprietary concentrates bovine spongiform encephalopathy and risk to health chronic wasting disease of captive mule deer: a spongiform encephalopathy spongiform encephalopathy of rocky mountain elk spongiform encephalopathy in a captive puma (felis concolor) embryo transfer, semen, scrapie amphotericin b treatment dissociates in vitro replication of the scrapie agent from prp accumulation i am deeply appreciative of the help given me by dr h.j. cho and dr r.m. barlow in the preparation of this manuscript. lisa kalischuk-timensen helped collect references by computer searches. this essay is dedicated to the memory of my friend robert kasting, for his assistance many years ago. notes . louping-ill is a tick-borne virus-caused disease of lambs, characterized by incoordination of the hind limbs and by hypersensitivity to noise and touch (blood et al., ) , seen principally in great britain and russia. key: cord- - pbem d authors: jeney, zs.; jeney, galina title: recent achievements in studies on diseases of common carp (cyprinus carpio l.) date: - - journal: aquaculture doi: . / - ( ) -t sha: doc_id: cord_uid: pbem d abstract parasitic, fungal, bacterial and viral diseases of common carp (cyprinus carpio l.) are reviewed. besides a general overview of parasites of carp, swimbladder inflammation, caused by sphaerospora spp., is discussed in detail. saprolegnia spp. is the most important fungal pathogen. aeromonas hydrophila and the atypical aeromonas salmonicida, as well as flexibacter columnaris, are described as the major bacterial pathogens of carp. spring viraemia of carp caused by rhabdovirus carpio is presented as the main viral infection of common carp. details on methods of treatment and prevention are presented together with a description of the given diseases. the role of environmental stress, including “normal” culture practice and pollution, in the outbreak of diseases of common carp is discussed. prospective methods to minimize the risk of diseases as well as their limitations are presented. carps (common carp, indian major carps and chinese carps) are the biggest group of cultured fish. their aquaculture production was mt in . among them common carp (cyprinus carpio l.) is probably the most widely "spread" species. its annual production was mt in (fao, ) . this figure does not contain the production of three countries in asia (bangladesh, india and vietnam), producing mt of freshwater fish, including a large proportion of common carp. in a recent review on aquaculture in asia, new and csavas ( ) gave a corrected figure ( mt) for common carp production in as well as a forecast for asia for the year : mt. common carp is cultured on almost all continents under a wide range of geographic, climatic and technological conditions. as a result, there is a large group of pathogens parasitising on carp andl or causing diseases. this is especially true for parasitic diseases. due to limited space, only selected aspects of diseases of cultured common carp can be reviewed. general overviews of parasitic, fungal, bacterial and viral diseases, as well as environmental stress are followed by a detailed description of the most important diseases. nutritional diseases are not discussed in this review. representatives of almost all major systematic groups of parasites have been found in carp. altogether parasite species were described (landsberg, ) . the most important ones that cause disease are listed in table . as can be seen, with few exceptions they have a world-wide geographical distribution. it should be emphasised that environmental stress appears to play a major role in the development of epizootics caused by parasites of carp. this is especially true for ectoparasitic infestations, where mortality rates are typically high in habitats where fish become overcrowded, such as in overwintering storage ponds or in densely populated growing ponds and reservoirs (paperna, ) . other environmental factors such as inadequate oxygenation, accumulation of metabolites (primarily the highly toxic ammonia), handling, fish concentrations during netting or harvesting etc. may also play a role in the development of epizootics with ectoparasitic protozoa. swimbladder inflammation (sbi) is a common, economically important disease of the common carp. first observed in hungary by szakolczai ( ) among three-summer-old carps, it had long been known in neighbouring countries (hofer, ; roth, ) . here we list the main steps in the long history of this disease. there are divergent opinions on the aetiology of sbi. certain authors have regarded it as a viral disease (arshanica, ; ahne, ; bachmann and ahne, ) , while others (neciporenko et al., ; kanaev et al., ; szakolczai, ; markiewicz, ; mattheis and kulow, ; kocylowski et al., ) have advocated the primary aetiological responsibility of bacteria, because of the isolation of many bacterial strains from carp with sbi. szakolczai ( ) detected bodies reminiscent of unicellular parasites ( coccidia, pleistophora) in squash preparations of affected swimbladder and suggested they might be the causal agent of the disease. kanaev and kuzmin's ( ) report on experimental reproduction of sbi by feeding carp organs containing myxobolus spores agreed well with szakolczai's protozoan hypothesis. otte ( ) implied that the blood flagellate cryptobia cyprini may be responsible for sbi. molt& ( a) observed during studies on carp renal sphaerosporosis that the latter condition was frequently developed by fish with clinical sbi, and he postulated a relationship between the prevalence of sbi, renal sphaerosporosis and the condition caused by the c-blood protozoan originally described by csaba ( ) . waluga and budzynska ( ) detected the developmental stages of sphaerospora carassii, the causal agent of gill sphaerosporosis in carp, in visceral blood vessels, but did not pursue the possible identity of these stages with the c-blood protozoan. indirect proof of a relationship between sbi and renal sphaerosporosis has emerged from grishcenko's ( ) abbrevations: geographical range -c = cosmopolitan; as = asia; e = europa; na= north america; su = former soviet union. gross pathology -dpbc = decaying and puriginaeous branchio-cutaneous; resp = respiratory; so = sensory organs; circ = cicmlatory; vm = viscera-muscular; f = free parasite; c = cystic; dig = digestive. from de kinkelin and hattenberger ( ) and molniir and szakolczai ( ) with modification. observation that hosts with sbi harboured spore-like bodies in the renal tubules, and from a photograph published by schlperclaus ( ) as the image of the swimbladder wall, although it actually shows a renal tubule packed with sphaerospores. kovacs-gayer has regularly detected developmental stages of parasitic protozoa in the wall of the swimbladder of carp fry with sbi (molnar, b; kovacs-gayer et al., ) . dykova and lom ( ) have described the parasite which frequently occurs in the kidney of the common carp and which was formerly regarded as sphaerospora angulata fujita, , as a new species, sphaerospora renicola. since the works of molmir ( ) , kovacs-gayer et al. ( ) , korting ( ) and csaba et al. ( ) led to the identification of parasites designated as k-protozoa causing swimbladder inflammation in the common carp, with the presporonic developmental stages of the kidney parasite sphaerospora renicola dykova and lom , a vast amount of knowledge has accumulated on the complex developmental cycle of sphaerospora-type parasites. it has become known that the early developmental stages described by csaba ( ) and designated as "c blood protozoa" by molnar ( ) and as "ubo" by lom et al. ( ) consistently appear in the blood of carp before renal sphaerosporosis develops. the works of lom et al. ( ) have revealed that the blood stages can also be demonstrated in the blood of other sphaerospora-infected cyprinid fishes. moreover, in the blood of these cyprinids baska and molnar ( ) found circulating forms morphologically resembling the k-stages of the common carp. in common carp, the k-stages were earlier detected first of all in the swimbladder wall (kovacs-gayer et al., ; ksrting, ; csaba et al., ; landsberg, ; odening, ) . the possible occurrence of k-stages in other locations has only been suggested by kbrting ( ) , kijrting et al. ( ) and the ter hofte et al. ( ) ) who found the developmental stages in the integument of the head, in the orbit and in hepatic granules of the common carp. recently, lom et al. ( ) have found sphaerospora stages resembling k-protozoa in the rete mirabile of non-cyprinid fish (gasterosteus a&e&s), although they failed to clearly identify these forms with the developmental stages of s. elegans found in the kidney. molnar ( ) found that s. renicola k-stages, previously considered to be typical swimbladder parasites, are blood stages just like c-parasites; however, they are frequently caught in the capillaries. these stages can also produce lesions in organs other than the swimbladder, and are directly responsible for the exophthalmos regarded as a sign of swimbladder inflammation, as well as for the intraorbital haemorrhages and tissue necrosis. the following detailed information is based on the above literature as well as on the review by molt-i&r and szakolczai ( ) . causative agent. the early developmental stage of sphaerospora renicola, the causative agent of swimbladder inflammation, can be detected in the acute stage from the wall of the swimbladder. in the fibrous connective tissue of infected swimbladder, groups of protozoa with a diameter of - pm can be visualized, that are parasitic cells developing with gemmation, as well as their progenies. in the plasma of the primary developmental form, occurring among the tissues, - secondary forms will develop, and later they will serve as a site for development of - tertiary forms. after destruction of the primary form the so-called tertiary developmental stages, which include one secondary form and two tertiary forms, through the bloodstream will reach the renal tubules where spores of s. renicola will develop from them. following the acute phase of sbi the sphaerosporosis will always be developed in the renal tubules. sbi is always preceded by the appearance of so-called csaba protozoa. these are the earliest developmental stages of s. renicolu; they flow freely in the blood and propagate by endogen gemmation, resulting in daughter cells in each mother cell. the parasitic sbi finishes in weeks, and if no complication occurs, the disease is over. in most cases the facultative bacteria (aeromonas spp., pseudomonas spp.) will cause complications. pathogenesis. on the basis of the observed changes the disease can be divided into stages: ( ) in the first stage the blood vessels are dilated, and beside the hyperaemia only pitechiae can be observed in the swimbladder. ( ) in the second stage the hyperaemia decreases; in the wall of the swimbladder brown or black spots with sharp borders can be seen. ( ) in the third stage the wall of the swimbladder becomes thickened, and an exudate appears as a sign of inflammation. peritonitis, as well as adherence of the peritoneum covering the intestine and the abdomen, is not unusual. ( ) the fourth stage is characterised by exacerbation of the above processes, and certain layers of swimbladder are necrotised. ( ) in the fifth stage cysts are formed in the wall of the swimbladder; the cavity of the swimbladder is filled with serous-purulent matter. inflammation spreads to other organs, and secondary peritonitis develops. the first two stages are typical of parasitic swimbladder inflammation, while in the third the bacterial complication probably plays a role. the fourth and fifth stages appear only when there are complications. clinical symptoms. in the first two stages the fish do not show any typical signs. the changes listed (haemorrhages, hyperaemia) can be detected only at necropsy. in the chronic form (the last three stages) sick individuals loose their balance, and swim on their back or side, or with the head downwards. their caudal fin stands out of the water; from time to time they try to dive but without success. the abdomen of sick fish increases markedly in size. this enlargement expands into the whole abdomen or appears only in the region of the anus, depending on whether both parts or only the second part of the swimbladder is affected. in the course of autopsy the thickened wall of the abdomen can be detected. abdominal organs generally do not show pathological signs. due to functional disturbances of the kidneys some fish will show general oedema, but without haemorhaggic signs referring to spring viraemia. the swimbladder looses its typical form, and either both or only one of the two chambers will enlarge. the glossy silver-white colour of the outer wall becomes mat or whitish grey. on cutting through the wall l- cm thick parts are seen that include holes of different sizes with serous-purulent matter. in the exudate very often green-bean-sized fibrin nodules can be found. the real cavity of the swimbladder is strongly contracted, and contains a minimal amount of a water-like serous exudate. the inner epithelium is healthy, with a glossy silverwhite colour. some lo- % of fish will show clinical signs, but mortality is only occasional. these fish are very sensitive to any kind of handling (harvesting, transport etc.). other fish species kept together with carp (bream, crucian carp, pike-perch) do not show these signs of swimbladder inflammation. in the early stages of the disease protozoa can be easily detected in the blood and swimbladder by impression smears or histological preparations. sphaerospores in the kidney are visualized on native preparations. in chronic forms a diversified bacterial flora can be isolated from the swimbladder. the course of the disease is rapid in fingerlings, the parasitic form easily recovers, the swimbladder regenerates within a few weeks, and only the pigmented spots will indicate the past infection. in the case of complications the greater part of the population will die. in the chronic form, only occasional mortality is observed. treatment. no treatment is available. for prevention of sphaerosporosis fumagillin is the only effective medicament. in the course of development of different forms of swimbladder inflammation l- weeks feeding with medicated food containing . % fumagillin will prevent the development of disease. this very expensive procedure can be avoided by preventing bacterial complication by feeding antibiotics, as well as by destroying the infective spores by drying and disinfecting the ponds. fungal infections of common carp are serious problems of both natural fisheries and intensive aquaculture. fungal infections are also a persistent problem in warmwater hatcheries as any dead eggs quickly become a focus of proliferation which can than spread to adjacent healthy eggs. in spite of their importance our knowledge about the fungal infections is still poor, for two basic reasons: difficult identification of pathogenic fungi (pickering and willoughby, ) and the prolific growth of saprophytic fungi once the fish is dead (willoughby, ) _ the potential fish pathogens of the family saprolegniaceae was reviewed by pickering and willoughby ( ) (table ) . saprolegnia spp., achlya spp. and branchiomyces spp. are probably the major pathogenic fungi in the common carp. here we give a short description of the genus saprolegnia, probably the most, but still insufficiently, studied of these pathogens, as well as saprolegniasis of common carp, the disease caused by them. gill rot, branchiomycosis plehn ( ) causative agent. the genus saprolegnia is characterised by the production of biflagellata zoospores which swim away after emptying of the zoosporangia. new zoosporangia often form in the old ones. saprolegniaparasitica coker (in willoughby's system of classification syn. saprolegnia diclina type ) is most frequently associated with fish, and produces sexual organs only in special cases (oh% and farkas, ) . zoosporangia are - pm long, cylindrical, often irregular in shape, and appear on the nd- rd day of culture. ripening and release of zoospores soon begins. the zoospores are spherical with two flagella, motile and pm in diameter. clinical signs. gross pathology of saprolegnia infection is characterised by conspicuous fungal colonies growing on the body surface of the fish. these cotton-wool-like lesions are normally white in colour, or discoloured by the accumulation of debris between the fungal hyphea or as a result of a simultaneous bacterial infection. the fungus can colonise almost any area of the external surface of the fish. histopathological changes of the saprolegnia infection are characterised as follows. the penetration of fungal hyphae is restricted to the epidermis and dermis. muscular lesions are uncommon but occasionally develop when fungal and bacterial penetration to the muscle surface occurs. in case of small fish, hyphae may invade the deeper tissues of the fish and penetrate the vital organs (bootsma, ) . degenerative changes in the epidermis and dermis include focal oedema and ultimate sloughing of the epidermis (pickering and willoughby, ) . inflammatory responses are normally absent or weakly developed (wolke, ; pickering and richards, ) unless bacterial infection also develops. according to pickering and willoughby ( ) there is no evidence that pathogenic saprolegnia strains produce any toxins that might be transmitted systemically. once infected, fish do not normally recover from saprolegniasis unless treated. death of the fish is due to massive osmoregulatory problems caused by destruction of the superficial tissues over large areas of the body surface although the survival time is influenced by the precise location of damaged tissue. saprolegniasis has been considered to be a secondary condition with fungal hyphae normally colonising existing lesions on the fish. although fungal infections are often secondary to other diseases and to physical damage, there is also evidence that saprolegnia diclina type (syn. s. parasitica) can also act as a primary pathogen on undamaged, healthy fish (pickering and christie, ) . the following conditions can increase the susceptibility of fish to infection: wounds are potential sites for saprolegnia colonisation; physical damage (tiffney, ; egusa, ) ; high stocking density; any environmental conditions and manipulations that result in physical damage; sexual maturation; other infections, especially those that cause ulceration of, or damage to, the superficial tissues of the fish; stress-mediated increase of plasma corticosteroid (nord et a ., ; neish and hughes, ) . infections during all stages of the life cycle of carp and other commercially produced fish species (foster and woodbury, ; scott and o'bier, ; nelson, ; alderman, alderman, , . recently, stabilised chlorine dioxide was suggested as a control agent for fungal infections, first of all saprolegniasis (hiney and smith, ) . general measures to improve water quality should also be considered. according to the most recent review, altogether only bacteria have been reported to be pathogenic for fish, which is surprisingly low considering the large number of fish species and their diverse environments (fryer and rohovec, ) . of these bacteria, were isolated from and associated with diseased carp (table ) . the term "motile aeromonas septicaemia" (mas) is used to describe motile aeromonad infections of warmwater fish, including common carp. the motile aeromonads are characteristically bacteria of fresh water. the motile species are often ubiquitous members of the aquatic ecosystem, but all can be components of the microbial flora of aquatic animals and may be pathogens of poikilotherms, homiotherms and even man (fraire, ; salton and schnick, ) . motile aeromonads show very considerable heterogeneity and their taxonomy is confused. this group comprises a. hydrophila (syn. a. liquefaciens) , a. sobria and a. caviae (syn. a. hydrophila subsp. anaerogenes) . of these a. hydrophila is of great importance for carp culture. according to austin and austin ( )) aeromonas hydrophila may include several distinct species. a. punctata is no longer a recognised species and isolates previously identified as a. punctata would now be classified as a. hydrophila (frerichs, ) . although motile aeromonads may occur in relatively unpolluted water, they are much more abundant in waters with a high organic load (hazen et al., ) . because the "normal" conditions of a carp-rearing pond are close to these, and aeromonas hydrophila is a normal inhabitant, carp are constantly exposed to infection. disease outbreaks are common when carp are under stress from crowding, low oxygen, high temperatures, etc., and significant losses from such outbreaks occur annually. bullock and herman ( ) lnglis and hendrie ( ) clerx ( ) farkas ( ) farkas and oldh ( ) reichenbach-klinke ( ) causative agent. a. hydrophila is characterised by active motility, achieved by means of single polar flagellum. it is a gram-negative bacilli measuring . x . -l pm. a. hydrophila is aerobic and facultatively anaerobic, fermenting carbohydrates with the formation of acid and producing -butanediol. it is cytochrome-oxidase-positive, reduces nitrates, and is resistant to the vibriostat o/ (roberts, ) . as to extracellular production, a. hydrophila produces haemolysin, cytotoxins and enterotoxins (boulanger et al., ; wretlind et al., ; donta and haddow, ) as well as gelatinase, caseinase, elastase, lectithinase and desoxyribonuclease (nord et al., ) . the pathogenicity of a. hydrophila often seems to be associated with stressed or compromised hosts. the principal feature of the pathogenesis is generalised dissemination in the form of a bacteraemia, followed by elaboration of toxins, tissue necrosis and bacterial haemorrhagic septicaemia. in most cases the a. hydrophila infection has a secondary character and the disease is associated with primary viral or parasitic infection, with sudden change in environmental or nutritional status, or with husbandry changes. there is some doubt, therefore, as to whether aeromonas hydrophila should even be considered as a primary pathogen. there is little doubt, however, that once it has invaded a host which has been compromised for whatever reason, it rapidly makes the condition worse and can be responsible for ultimate death. the complexity of the situation makes the diagnosis and treatment difficult. unless the primary cause has been excluded, treatment of aeromonasis will be of little value in the longer term (roberts, ) . clinical signs. clinical signs of the disease are rupture of minor blood vessels and haemorrhages caused by haemolysin, which may be associated with ulcerative skin lesions. the size of external lesions may vary. the ulcers are usually shallow. secondary infection of ulcers by fungi is common. in carp the condition may be pre-acute, with few signs, acute, with the typical syndrome described above, or chronic with large, long-standing ulcers. it is often associated with abdominal oedema or dropsy. internally the principal feature is hyperaemia of the viscera with haemorrhage over mesenteritis and within the visceral and parietal peritoneum. there may be an excess of clear or reddish ascitic fluid in clinical dropsy. the spleen and the kidney are enlarged. treatment. control of a. hydrophila infection should be linked to the control of the underlying factors which have facilitated its invasion of the host. however, when an outbreak occurs, it is very difficult to determine the underlying stress factors or primary infections and it is not possible to change water flows or stocking densities with any ease. thus treatment is limited to the use of antibacterial compounds in the feeds. generally there are two basic problems with the application of medicated feeds. the first is the development of antibiotic resistance (mitchell and plumb, ) . such a plasmid-related drug resistance has been described for most of the antibiotics used to treat aeromonad infections (chang and bolton, ; ansary et al., ) . the second problem is that the efficacy of such treatment could be very low in some cases due to the low appetite of the sick fish. efforts to develop vaccines against mas have not been successful, mainly because of the wide serological diversity among a. hydrophila strains (wolf, ) . ulcerative erythrodermatitis of carp is a subacute to chronic disease which varies in morbidity and mortality, and is generally associated with ulcerative inflammatory lesions in the skin and muscle. before it was considered as the chronic form of disease called "dropsy of carp". fijan et al. ( ) isolated a virus (rhabdooirus carpio) from the acute form of infectious dropsy and differentiated the acute form as "spring viraemia of carp" and the chronic form as "carp erythrodermatitis". bootsma ( ) isolated a bacterium from the ulcerative form. later bootsma and blommaert ( ) classified the bacterium as an atypical aeromonas salmonicida. causative agent. the disease is caused by an atypical aeromonas salmonicida (ce bacterium) which is a gram-negative, non-motile bacterium measuring . - . x . -l . ,um. atypical aeromonas salmonicida from carp belong to the subspecies known as aeromonas salmonicida subspecies nova according to biochemical characteristics (bohm et al., ) and dna/dna re-association studies (belland and trust, ) . the bacterium can be isolated from the hyperaemic surroundings of the ulcers of sick fish in most cases. on blood agar after h it will produce small colonies, under which b haemolysis can be observed after h. the bacterium does not produce pigment. it ferments glucose and maltose with the formation of acid, but without gas production. the ce bacterium is cytochrome-positive and hydrolyses gelatine and casein. it is resistant to penicillin and polymyxin b, but sensitive to medicines like oxytetracycline, neomycin, nitrofurantoin, sumetrolim (sulfamethoxazole + trimethoprim) (molnar and szakolczai, ) . it seems that the ce bacterium is able to infect carp in the region of mechanical injuries, such as wounds caused by bloodsucking parasites. clinical signs. at an early stage, small spherical nodules can be observed on the fins; later at these sites the finrays break down. the scales are surrounded by inflamed rings; afterwards the scales are lost, and the epithelium and corium become necrotized. the resulting ulcers with markedly hyperaemic surroundings often spread into muscle. such ulcers can be found everywhere on the body except the head. histologically, there is a lack of epithelium, and necrosis of the corium and muscle, as well as the presence of large numbers of inflammatory cells, can be observed. inside the ulcer is filled with granular tissue. in internal organs neither macroscopic nor microscopic pathological signs can be seen, but sometimes slight fatty degeneration is visible. at the beginning the appetite of the fish is normal, but later it decreases due to exacerbation of ulceration. the course of the disease is slow, mortality rarely reaching - %. other economic losses are caused by the slow growth of the sick fish. in the presence of favourable environmental conditions the erythrodermatitis is curable. the sites of healed ulcers show strong pigmentation, which differs from healthy skin. treatment. treatment is successful with the use of antibacterial compounds in the feeds. oxytetracycline, neomycin, nitrofurantoin and sumetrolim (sulfamethoxazole + trimethoprim) are usually effective, and although resistance to them was not found in hungary, standard bacteriological and resistance investigations are highly recommended before starting the treatment course (molnar and szakolczai, ) . in prevention of erythrodermatitis of carp, good rearing practice and improvement in environmental conditions (firstly by removing blood-sucking parasites) play the primary role. columnaris disease is distributed worldwide. many species have been effected by columnaris disease, including the common carp. gill necrosis of common carp, caused by unfavourable environmental conditions (ammonia, ph and temperature) and flexibacter columnaris infection, was suggested to count as a separate disease (farkas and & , ) . these authors described the gill necrosis as a three-stage, complex disease. the first stage is initiated and maintained by environmental stress, caused most typically by ammonia intoxication. the second stage starts when f. columnaris invades the damaged gill. the third stage occurs if the fish survives the bacterial invasion. in the present review we give a general description for columnaris disease of common carp. causative agent. flexibacter columnaris syn. cytophaga columnaris is the causative agent of this disease. carp is susceptible to columnaris under environmental conditions favourable to the bacterium and stressful to the fish. outbreaks occur when water temperatures reach °c and above (farkas and oh&, ) . f. columnaris primarily attacks the external tissues, and uninjured tissue also appears to be attacked. the first indication of the infection is generally the appearance of a white spot on some part of the head, gills, fin or body. this is usually surrounded by a zone with a distinct reddish tinge, leading to under-running of adjacent skin. lesions on the end of gills or fins extend principally from the distal end towards the base, and the tissues are eroded and destroyed. lesions are covered with a yellowish white mucoid exudate consisting largely of swarms of f. columnaris (wakabayashi, ) . the bacteria are not usually found systemically until a relatively large amount of external skin or gill damage has taken place; thus it would appear that the bacteria enter the bloodstream through the external lesions and are probably not directly involved in causing death (wood, ) . in wet preparations f. columnaris shows a slow gliding movement and gather into characteristic column-like masses. the filamentous cells on the columns also display an active flexing movement. the cells are gram-negative, slender and rather long bacilli, . - . pm long. the bacteria grows well on low nutrient cytophaga agar, producing pale yellow rhizoid colonies, which can vary markedly in size and shape. the colonies tend to spreading with irregular margins and they adhere to the agar (wakabayashi, ) . according to bernardet and grimont ( ) , growth occurs in cytophaga broth supplemented with . or . % nacl and at lo- "c, strictly aerobic. catalase and cytochrome oxidase are produced; nitrate is reduced to nitrite; hydrogen sulphide is produced. cellulose, carboxymethyl cellulose, chitin, starch, aesculin and agar are not hydrolysed. no acid is produced from carbohydrates in ammonium salt/sugar medium. gelatin, casein and tyrosine are hydrolysed. lysine, arginine and ornithine are not decarboxylated. epizootology. in the epizootology of the disease the following environmental factors play an important role: water temperature, water quality and the density of fish. flexibacter columnaris attacks fish only at comparatively high water temperatures. in experimental infection studies, carried out with different fish species, below the range of . - . "c no effect of bacteria was reported (fish and rucker, ; holt et al., ) . wakabayashi and egusa ( ) studied the effect of water temperature on columnaris disease. no mortality occurred in experimentally infected fish at or °c % of those held at °c died and all of the exposed fish held at - °c died. the mean time to death was . , . , . , . and . days at , , , and "c, respectively. pacha and ordal ( ) reported a clear relationship between the pathogenicity of f. columnaris and the water temperature: high-virulence strains were found to infect fish and produce disease at lower temperature than low-virulence strains. mortalities were produced at . "c by highvirulence strains, whereas low-virulence strains were able to initiate infection only when the temperature was increased to °c. water quality is the other important environmental factor because the long-term survival of the pathogen in pond water is thought to provide a reservoir of infection. fijan ( ) indicated that f. columnaris could persist for longer periods in water of high hardness and organic matter content, but survival time was reduced significantly in water with a ph of . . soft water of about ppm cac , especially when acid or of low organic matter content, did not provide a favourable environment for the organism. chowdhury and wakabayashi ( ) showed that f. columnar-is survived for the longest period in water with hardness of . mg/l cac . arsenic and nitrite ( ppm) enhanced, while copper decreased the f. columnaris infection (macfarlane et al., ; hanson and grizzle, ) . f. columnaris grows very well on the particles derived from the break-up of pelleted diets in water (sugimoto et al., ) . density of fish has a clear effect on the f. columnaris infection: at higher stocking densities, higher rates of infection as well as mortality occur (becker and fujihara, ) . it also seems that crowding not only reduces resistance of fish to f. columnaris but also increases the chances of the organisms coming into contact with the fish. the results of the study carried out by wakabayashi and egusa ( ) suggested that the higher the initial density of f. columnaris or the concentration of fish, the more certainly infection occurred and the earlier mortalities started to occur. treatment. control of columnaris disease should be based firstly on good rearing practice and on improvement of environmental conditions. some of the possible measures have practical implications, such as avoidance of overfeeding and reduction of wild fish in the water supply. others like cooling the water temperature (wood, ) ) or reduction of f. columnaris through adding significant numbers of competitive bacteria (e.g., citrobacter freundii) to susceptible fish ponds (chowdhury and wakabayashi, ) do not sound very practical for a typical carp pond farm. although positive results were published on the feasibility of vaccination against columnaris disease in channel catfish (moore et al., ) , a commercially available vaccine is not likely in the near future. amend ( ) reviewed the chemotherapy of columnaris disease. for systemic infections, sulphonamides or antibiotics are added to the food. sulphamerazine and oxytetracycline are administered therapeutically in the feed in a two-stage regime: mg/kg/day for days followed by - mg/kg/day for days. in the most detailed work on fish viruses and viral diseases viruses that were isolated and the resulting diseases in fish are listed (table ) . two of them are from carp: rhab- fijan et al. ( ) sbchelkunov and shchelkunova ( ) wolf ( ) dovirus carpio (causative agent of spring viraemia of carp) and herpesvirus cyprini (causative agent of carp pox). carp iridovirus is one of the listed viral infections of indeterminate pathogenicity. of viruses that have been visualised but not yet isolated, one is the carp coronavirus (wolf, ) . causative agent. spring viraemia of carp (svc) is an acute haemorrhagic and contagious viral infection of the common carp in europe. fijan et al. ( ) isolated a virus (rhabdouirus carpio) , proved its aetiologicalrole in svc and called the disease "spring viraemia of carp". before this time the disease was known as "infectious dropsy of carp" (idc) and was considered to have either a bacterial or a viral aetiology. two forms of idc became recognised: an acute or ascitic form and a chronic or ulcerative form. fijan et al. ( ) in their already cited work showed that rhabdovirus carpio produced the acute form of what had been termed "idc". the chronic or ulcerative form was later named "carp erythrodermatitis" and was shown to be caused by a non-motile bacterium of the genus aeromonas (bootsma et al., ) . the initial isolation of rhabdovirus carpio was followed by confirming isolations in czechoslovakia (macura et al., ; tesarcik, ) in hungary (bcktsi and szabo, , ) and in great britain (bucke and finlay, ) . during the os, the protozoan aetiology of swimbladder inflammation (sbi) was proved (csaba et al., ; korting, ; kovacs-gayer, ) and the possible role of svcv in sbi was no longer considered. clinical signs. svcv has the characteristics of rhabdoviruses and the agent is pathogenic to carp of all ages. the disease becomes manifest when the water temperatures rise in the springtime. moribund fish show dark colouration, low respiration, exophthalmia, inflamed and oedematous vent, pale gills and haemorrhages in the skin. internally there are frequently haemorrhages in the viscera and airbladder, and peritonitis with serous or haemorrhagic exudate is mostly present in acute cases. a clinical diagnosis of svc is possible if serious mortality occurs among carp during spring or early summer and affected specimens show the key features of the behavioural changes and the above-described signs. for confirmative diagnosis the rhabdouirus carpio should be isolated and identified serologically, or the viral antigen should be identified serologically in tissue sections. epizootology. under natural conditions, most transmission is probably horizontal (wolf, ) ) although vertical transmission may also take place in some situations (b&k&i and csontos, ) . during outbreaks and several months thereafter, virus is shed by faeces and possibly urine (ahne, ; baudouy et al., ; pfeil-putzien, ) . blood-sucking parasites, like the carp louse (argulusfoliaceus) and the leech (piscicola geometru) are vectors for svcv. they both carried the virus passively, and multiplication did not occur (ahne, ) . the virus is transmitted by the water route and the gills were the primary portal of entry for svcv. water temperature plays an important role both in the outbreak of the disease and the immunity acquired. cases of svc occur in ponds in springtime, mostly at water temperatures between and °c and mortality ceases with the increase in water temperature. mortality may sometimes continue up to °c and this temperature was therefore mentioned by fijan ( ) as the upper thermal range of the disease. in experimentally infected carp kept under various controlled temperature conditions baudouy et al. ( ) found the svc mortality to be most severe at temperatures below -l °c; at °c and above the defence mechanisms were able to protect the fish from mortality. according to ahne ( ) and fijan and matasin ( ) the infection of susceptible carp with svcv at °c does not result in mortality: at these and higher temperatures the defence mechanisms are able to restrict virus replication and to eliminate it. treatment. theoretically there are basic control measures for svc: ( ) avoidance of sources of virus; ( ) development of specific-pathogen-free (spf) broodstock; ( ) temperature manipulation (rearing fish above °c) ; ( ) genetic selection for resistance to svc ; ( ) immunoprophilaxis. practically all these control measures have their limitations: no methods to detect carrier state, unpracticality of control, lack of methods of chemical control, and quality problems with the only tested svc vaccine (tesarcik et al., ) . as is known, most of the pathogens of common carp are facultative pathogens. this means that they are almost always present in the water surrounding the fish, but they can snieszko ( ) on the primary role of environmental stress in the outbreak of fish diseases is especially important in warmwater aquaculture, including carp culture, where mainly surface waters are used and the basic water quality parameters are subject to a wide range of fluctuations, as well as where the level of management is generally low. due to this phenomenon it is not surprising that several diseases of carp are typically "stress-mediated" : certain parasite infestations (costiosis, trichodinosis) , bacterial gill disease, flexibacteriosis and spring viraemia of carp. environmental stress factors predisposing to diseases in carp are listed in table . for a better understanding of the role of environmental stress factors in the outbreak of fish diseases it is essential to study the response of the fish organism. in the scheme suggested for the stress response of fish (peters, ; mauzeaud and mauzeaud, ) the most important physiological and biochemical changes occurring at the primary, secondary and tertiary levels were summarised. certain of these changes can also be employed to detect diseases at early stages or sublethal effects of environmental contaminants. a summary of the physiological tests for stress assessments of salmonids was published by wedemeyer and yasutake ( ) . a similar system for tilapia (surotherodon aureus) and silver carp (hypophthalmichthys molitriw) was presented by bejerano ( ) . data are available on the "normal" ranges or values of some of these parameters for common carp (svobodova, ) . the limits of the "normal" values of these parameters in fish can only be overcome by a description of the typical changes under the effect of different environmental factors, compared to the changes in "non-treated" groups. for the assessment of the effects of environmental stress on common carp a system of clinical diagnostic parameters was set up and tested (jeney and jeney, , ; jeney, z. et al., a,b ). using this system, different environmental stresses of both natural and antropogenic origin were investigated. typical changes (increase or decrease, compared to the control) in these parameters are shown in table . as can be seen, most of the natural and anthropogenic environmental factors studied, even though typical for "normal" technological conditions, causing marked changes at all three levels of stress response of the carp. further development of carp culture will be accompanied by several changes that have well-established incidences of pathological conditions: increase in culture densities, frequent degradation of environmental quality, mixing of populations of different origin and manipulations etc. all these factors are increasing the probability of the advent and seriousness of diseases. research on fish pathology has allowed the progressive setting-up of a rather large variety of possible interventions: specific diagnostic methods, sanitary prophylaxis and disinfection, vaccination, chemotherapy, immunomodulation and genetics of disease resistance will continue to supply new possibilities to breeders (fig. ) . however, several constraints often make such interventions difficult and expensive; thus prophylaxis based on strict sanitary isolation, which is relatively easy in terrestrial species, is often more difficult in the aquatic environment where the water, other fish species or even invertebrates or other vertebrates constitute potential contaminating agents which are difficult to eliminate (chevassus and dorson, ; anderson, ) . antibiotic therapy, mostly applied orally, has more and more to cope with resistance in selected bacteria due to repeated treatments. in theory, vaccination is the most valuable approach, but, so far, effective vaccines are available only for a limited range of pathogens and not for those infecting common carp. in the case of viral diseases, attenuated vaccines are not authorised and killed vaccines are expensive to produce. both kinds of vaccines also prevent any serological differentiation between vaccinated and contaminated fishes. research on a recombinant subunit vaccine against spring viraemia of carp aimed at the production of a low-cost, completely safe and effective vaccine could provide a possible solution (rossius and thiry, ) . the small-scale market is a special challenge for any products to be introduced into aquaculture (de kinkelin and michel, ) . these limitations raise a question about the future of chemotherapy. although chemotherapeutic agents are available against a wider range of pathogens than vaccines, they also have limitations in terms of the pathogen groups against which they are effective. chemotherapy is also a palliative rather than a final solution to problems of disease and its use in the aquatic environment is not free from practical problems. for example, attention is being drawn increasingly to the potential hazards that intensive use of drugs could represent for the environment and for human health. use of immunomodulators in fish culture offers a wide range of attractive methods for inducing and building up protection against diseases. immunostimulants may be used alone, inducing elevated activities in the non-specific defence mechanisms (anderson, ; jeney and anderson, ) . during recent years several substances have been tested in carp to enhance non-specific defence mechanisms and to build up protection against a wide range of pathogens. levamisole treatment of carp fingerling stimulated growth and reduced natural mortality due to losses (siwicki and korwin-kossakowski, ). vitamin c is a popular immunostimulant added to diets of certain animals. glucans, long-chain polysaccharides extracted from yeasts, are good stimulators of non-specific defence mechanisms in fish. schizophyllan, scleroglucan, and lentinan were evaluated in carp for their ability to enhance protection against e. tarda and a. hydrophila (yano et al., (yano et al., , . in fish injected with these polysaccharides increased phagocytic activity and protection against the pathogen were observed. research on immunostimulants for use in food fish, including carp is in progress. due to the above-mentioned situation, fish geneticists and pathologists are interested in the search for intrinsic resistance factors protecting the individual during part or the whole of its biological cycle in the course of aquaculture development (chevassus and dorson, ) . promising data are available on selective breeding of carp with high tolerance to particular diseases kirpichnikov, ; sijvcnyi et al., ; houghton et al., ) ) or to stressful environmental conditions ) as well as on selecting for traits which have proven to be genetically correlated to disease resistance: cortisol -in atlantic salmon and rainbow trout (refstie, ; fevolden et al., fevolden et al., , . zellkulturen an verschiedenen siisswasserteleostengeweben und untersuchungen iiber die litiologie der schwimmblasenentztindung der karpfen. thesis uptake and multiplication of spring viremia of carp virus in carp, cypn'nus carpio l aeromonas salmonicidu from salmonids and cyprinids -serological and cultural identification infections with suprolegnia in pike culture studies on the aetiology of carp erythrodermatitis columnaris disease of cultured carp, c.vprinus carpio l.: characteristics of the causative agent atiologie der erythrodermatitis beim karpfen isolation and preliminary identification of the causative agent of carp erythrodermatitis isolation of enterotoxigenic aeromonas from fish identification of spring viraemia in carp (c,vprinus curpio l.) in great britain edwardsiella infections of fishes plasmids and resistance to antimicrobial agents in aeromonas sobria and aeromonas hydrophila clinical isolates genetic resistance to disease in fishes effects of sodium, potassium, calcium and magnesium ions on the survival of flexibacter columnaris in water effects of competitive bacteria on the survival and infectivity of flexibucter columnaris an unidentifiable extracellular sporozoan parasite from the blood of the carp studies into the possible protozooan aetiology of swim bladder inflammation in carp fry les dominantes pathologiques chez la carpe et les cyprinides d'(blevage symposium on fish vaccination sphaerospora renicola nsp., a myxosporean from carp kidney, and its pathogenicity cytotoxic activity of aeromonas hydrophila the existence of primary infectious disease in the so-called "fungus disease" in pond-reared eels fao fisheries circular no. , revision , fidi/c rev. , statistical tables filamentous flauobacterium sp. isolated from fish with gill disease in cold water gill necrosis-a complex disease of carp selection for high and low stress response in atlantic salmon (salmo salar) and rainbow trout (oncorhynchus mykiss) disease resistance in atlantic salmon (salmo salar) selected for high or low responses to stress the survival of chondrococcus columnaris in waters of different quality infectious dropsy in carp -a disease complex spring viraemia of carp: preliminary experiments on vaccination by exposure to virus in water isolation of the viral causative agent from the acute form of infectious dropsy of carp hyperimmunization ofcarp withrhabdouirus carpio some properties of the epithelioma papulosum cyprini (epc) cell line from carp c'yprinus carpio infectious disease of pacific salmon the use of malachite green as a fish fungicide and antiseptic aeromonas hydrophila infection pathology of marine and estuarine organisms the swimbladder inflammation of carps. veterinariya (moscow),~~ nitrite-induced predisposition of channel catfish to flexibacter columnaris infection prevalance and distribution of aeromonas hydrophilu in the united states initial investigations into the fungicidal activity of stabilized chlorine dioxide against saprolegnia in a freshwater hatchery handbuch der fischkrankheiten relation ofwatertemperature to ffexibucter columnaris infection in steelhead trout (salmo gairdneri), coho (uncorhynchus kisutch) and chinook ( . tschawytscha) salmon differences in resistance of carp, cyprmus carpio l., to atypical aeromonns salmonicida pseudomonus and alteromonas infections giucan injection or bath exposure given alone or in combination with a bacterin enhance the non-specific defence mechanisms in rainbow trout (oncorhynchus mykiss) effect of artificial propagation following thermal stress on the activity of serum transaminases (got, gpt) of carp (cyprinus carpio l transaminase enzyme activity of cyprinid fish depending on environmental factors and bacterial infection effect of rhabdovirus infection on selected blood parameters of wells (silurus glanis l.) acute effect of sublethal concentration of nh, on common carp (cyprinus carpio l.). ii. effect of ammonia on got, gpt, gldh enzymes activities and atp level in blood effects of stress factors of fish culture on some biochemical and hysiological parameters of carp studies on the effect of trichlorphon on different biochemical and physiological parameters of common carp (cyprinus carpio) a system of diagnostic parameters for detection of environmental impact of fish primary and secondary stress responses of common carp (cyprinus carpio l.) caused by artificial propagation effect of thermal stress on noradrenaline level in different organs, serum glucose level and serum got, gpt, ldh and cholinesterase activity of common carp (cyprinus carpio l.) thermal pollution influences physiological status of fish. summary of papers of th scientific session on environment analysis propanidid, a new anaesthetic for use in fish propagation cortisol measurements in fish acute effect of sublethal concentration of nh, on common carp (cyprinus carpio l.). i. effect of ammonia on level of adrenaline and noradrenaline in different organs of carp stress sensitivity of four genetically different strains of common carp the swim bladder inflammation of carp and the control measures against the disease. rosselchozisdat (moscow) microflora and parasitofauna of carps diseased in swimbladder inflammation genetic basis of fish selection increase in the resistance of carp to dropsy by means of breeding. ii. the course of the selection and evaluation of the breeding groups selected increasing the resistance of carp to dropsy by breeding. i. methods of breeding for resistance protozooan parasites associated ith swimmbladder inflammation (sbi) in young carp myxosporidien bedingte schwimmenblasenentztindung bei karpfenbrut studies on the etiology and pathogenesis of carp swimbladder inflammation histopathological studies on protozooan swimbladder inflammation o f common carp fry studies on the protozoan aetiology of swim-bladder inflammation in common carp fry swimbladder inflammation and kidney sphaerosporosis in carp (cypn'nus carpio) in israel parasites and associated diseases in fish in warm water aquaculture, with special emphasi on intensification unindentified mobile protozoans from the blood of carp and some unsolved problems of myxosporean life cycles notes on kidney infecting species of the genus sphaerospora fhelohan (myxosporea), including a new species s. gobionis sp.nov., and on myxosporean life cycle stages in the blood of some freshwater fish myxosporean vegetative stages in the choroidal rete mirabile of gesterosteus aculeatus infected with myxobilatus gasterostei and sphaerospora elegans effects of five metals on susceptibility of striped bass to flexibacter columnaris the positive cytopathic effect from case of spring viremia of carp swimbladder inflammation-a new disease of carp die schwimmblasenerkrankung des karpfens adrenergic responses to stress in fish toxicity and efficacy of furanase on channel catfish infected experimentally with aeromonas hydrophila renal sphaerosporosis in the common carp, cyprinus carpio l sphaerosporosis, a new kidney disease of the common carp the occurence of sphaerospora renicola k-stages in the choroidal rete mirabile of the common carp the book of fish diseases attempts to control flexibacter columnaris epizootics in pond-reared channel catfish by vaccination aquafeeds in asia -a regional overview the swimbladder inflammation of carp as the complicaton of dropsy fungal diseases in fish a review of the literature on the use of malachite green in fisheries observations on saprolegniasis of adult sockey salmon characterization of three aeromonas and nine pseudomonas species by extracellular enzymes and haemolysins sphaerospora renicola (myxosporidia) der erreger der protozoaren schwimmblasenentziindung des jungkarpfens effect of temperature, ph, antibiotics, formalin and malacite green on the growth and survival of saprolegnia and achlya parasitic on fish die eitrige schwimmblasenentzlindung des karpfens myxobacterial diseases of salmonids disease caused by parasites in the aquaculture of warm water fish zur interpretation des begriffs new results in the diagnosis of spring viraemia of carp caused by experimental transmission of rhabdouirus carpio with carp louse (argulusfoliaceus) sexual differences in the incidence and severity of ectoparasitic infestation of the brown trout factors influencing the structure, function and biota of the salmonid's epidermis saprolegnia infections of salmonid fish eine neue karpfenkrankheit und ihr erreger: branchiomyces sanguinis genetic differences in stress response in atlantic salmon and rainbow trout some aspects of mycobacterial infections in fish motile aeromonad septicaemia towards a recombinant subunit vaccine against spring viraemia of the carp virus krankheiten der aquarienfische und ihre bekiimpfung aeromonas hydrophila peritonitis aquatic fungi associated with disease fish and fish eggs results of virological studies of gill necrosis diseased carps the influence of levamisole on the growth of carp the effects of environmental stress on outbreaks of infectious diseases of fishes comparative examination of susceptibility of two genotypes of carp (cyprinus catpio l.) to infection with aeromonas salmonicida pathogenic relation between columnaris disease in cultured eel and the formula feeds toxicology of aquatic animals. mzvz csr a cesky rybarsky svaz untersuchungen der schwimmblasenentzllndung bei karpfen anhand von zwei fallen in ungam c-und k-protozoen studies in the relations between air bladder inflammation and infectious dropsy in carps summarized results of pilot vaccination of carp against spring viremia in the the identity of certain species of the saprolegniacea parasitic to fish bacterial disease of fish preliminary experiments on environmental factors influencing the prevalence of columnaris disease sphaerospora invasion as the cause of a massive mortality in carp populations methods for determining the tolerance of fishes to environmental stressors clinical methods for the assessment of the effects of environmental stress of fish health observations of fungal parasites of lake district salmonids fish viruses and fish viral diseases pathology of bacterial and fungal diseases affecting fish diseases of pacific salmon: their prevention and treatment, nd edn. state of washington department of fisheries, hatchery division separation of two haemolysins from aeromonas hydrophila enhancement of resistance of carp cyprinus carpio to experimental edwardsiez[a tarda infection, by some @ , -glucans polysaccharide-induced protection of carp cyprinus carpio l. against bacterial infection the authors express their thanks to dr. kalman molnl and dr. jozsef szakolczai for critical discussions and comments on the manuscript. key: cord- -q d uza authors: von oertzen, tim j title: covid‐ – neurologists stay aware! date: - - journal: eur j neurol doi: . /ene. sha: doc_id: cord_uid: q d uza in december an epidemic outbreak of a new virus disease in the chinese area of wuhan was reported. the new disease caused primarily symptoms of a respiratory tract infection with cough, shortness of breath, and viral pneumonia. the clinical spectrum showed mostly mild symptoms with some patients developing bilateral pneumonia needing partly intensive care treatment and causing death( ). it became apparent, that this new disease was highly contagious, and – intensive care ‐ patients numbers were rapidly rocketing, overwhelming the capacity of local health care facilities. and officially declared the covid- pandemia ( ) . as of today ( th may), official numbers report more than , million cases worldwide, more than , death, and approximately , million patients recovered from the disease. and these numbers are reflecting only those being tested for sars-cov whereas for many reasons the number of worldwide affected people is thought be much higher. this article is protected by copyright. all rights reserved severe affected cases. prospective data collection of large international multicenter cohorts will be necessary to fully understand the disease and its neurological involvement. we neurologists need to stay vigilant and aware on neuro-covid- , taking a joint effort to characterize the disease on scientific grounds. to facilitate this, the european academy of neurology developed the ean neuro-covid registry (energy) for prospective data collection (ean.org). web-editor in chief, european academy of neurology references accepted article clinical features of patients infected with novel coronavirus in wuhan who director-general's opening remarks at the media briefing on covid- - neurologic manifestations of hospitalized patients with coronavirus disease neurological manifestations and neuro-invasive mechanisms of the severe acute respiratory syndrome coronavirus type accepted article key: cord- -vrnxucye authors: argano, christiano; scichilone, nicola; natoli, giuseppe; nobili, alessandro; corazza, gino roberto; mannucci, pier mannuccio; perticone, francesco; corrao, salvatore title: pattern of comorbidities and -year mortality in elderly patients with copd hospitalized in internal medicine wards: data from the reposi registry date: - - journal: intern emerg med doi: . /s - - - sha: doc_id: cord_uid: vrnxucye currently, chronic obstructive pulmonary disease (copd) represents the fourth cause of death worldwide with significant economic burden. comorbidities increase in number and severity with age and are identified as important determinants that influence the prognosis. in this observational study, we retrospectively analyzed data collected from the reposi register. we aimed to investigate comorbidities and outcomes in a cohort of hospitalized elderly patients with the clinical diagnosis of copd. socio-demographic, clinical characteristics and laboratory findings were considered. the association between variables and in-hospital, -month and -year follow-up were analyzed. among in-patients, ( . %) had a diagnosis of copd. patients with copd had more hospitalization, a significant overt cognitive impairment, a clinically significant disability and more depression in comparison with non-copd subjects. copd patients took more drugs, both at admission, in-hospital stay, discharge and -month and -year follow-up. comorbidities were more frequent in copd patients. cerebrovascular disease was an independent predictor of in-hospital mortality. at -month follow-up, male sex and hepatic cirrhosis were independently associated with mortality. ics-laba therapy was predictor of mortality at in-hospital, -month and -year follow-up. this analysis showed the severity of impact of copd and its comorbidities in the real life of internal medicine and geriatric wards. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. chronic obstructive pulmonary disease (copd) represents an important leading cause of morbidity and mortality with high economic and social costs: according to the who, copd is the fourth most common cause of death worldwide, and it is estimated to be the third by ; furthermore, the global burden of copd is expected to increase in the coming years, due to the prevalence of smoking and aging of the world population [ ] . comorbidities are an essential component of copd burden. some of these are related to aging, others may have the same underlying mechanisms (e.g. systemic inflammation) or share common risk factors (e.g. smoking exposure), but all of them are able to afflict prognosis [ ] . some comorbidities occur more frequently in copd patients, independently from pulmonary severity disease [ ] . they increase in number and severity with age and have a major impact on the patient's quality of life, hospitalization and mortality [ ] . in this sense, divo and colleagues identified twelve comorbidities associated with increased mortality [ ] . however, recommendations on management of respiratory diseases are based on evidence from studies with restrictive inclusion criteria or no representative enrollment [ , ] , thus not accounting for complicating effects from coexisting conditions and treatments. therefore, their management and prevention might provide benefit in reducing the global cost load especially since international recommendations on copd management do not systematically include the evaluation of comorbid conditions in the diagnostic approach or in the treatment decisions of the disease, thus focusing on isolated lung impairment rather than multimorbidity. given this background, the aim of this study was to assess comorbidities and outcomes in a cohort of elderly patients with the clinical diagnosis of copd, hospitalized in internal medicine and geriatric wards participating to the reposi (registro per lo studio delle polipatologie e politerapie simi) registry study. retrospectively, we analyzed the collected data within the frame of the reposi project in the recruitment weeks of , , and . reposi is an independent and collaborative register, organized by the italian society of internal medicine (simi) and the mario negri institute for pharmacological research. it involved the creation of a network of internal medicine and geriatric wards that collected information about polytherapy on elderly patients, affected by multiple diseases. patients were eligible for reposi if: ( ) they were admitted to one of the participating internal medicine wards during the index weeks chosen for recruitment (one in february, one in june, one in september, and one in december); ( ) their age was years or older; ( ) they gave informed consent. each ward had to enroll at least ten consecutive eligible patients during each index week recording data on socio-demographic details, the main reason for admission and comorbidities, diagnoses, treatment (including all drugs taken at hospital admission and recommended at discharge), clinical events during hospitalization and outcome. during those weeks, all participating centers had to complete the registration of all patients admitted, indicating those who were consecutively enrolled. for patients who were excluded, the reason had to be given. also, data on mortality or any new hospitalization were collected, with a telephone interview performed by a physician to the patient or his/her relatives, -and months after hospital discharge. then, a final database was created and checked by the mario negri institute for pharmacological research. the project's design is accessible at the related website [ ] . subjects were referred as having copd if a diagnosis of the disease was reported in previous medical charts, or whether the diagnosis was posed at admission, as judged by the clinician. given the nature of the study, the spirometric assessment was judged not to be a pre-requisite to confirm the diagnosis. socio-demographic variables such as age classes, marital status, living arrangement and need for assistance in daily living, were considered along with laboratory findings in patients with copd compared to the ones without it. the following clinical characteristics were evaluated: respiratory and non-respiratory disease distribution at hospital admission (according to international classification of diseases-ninth revision); cognitive status and mood disorders (by the short-blessed-test [sbt] [ ] and the geriatric-depression-scale [gds] [ ] ,respectively; performance in activities of daily living at hospital admission (measured by means of the barthel index [bi] [ ] ; severity and comorbidity index(assessed by the cumulative-illness-rating-scale cirs-s and cirs-c, respectively) [ ] , glomerular filtration rate (using the chronic kidney disease epidemiology collaboration-formula [ ] ), length of hospital stay, drugs prescriptions (at admission, discharge, at and months follow-up), destination at discharge, in-hospital and -month and -year mortality rate. the association between variables and in-hospital, -month and -year mortality was analyzed. quantitative variables were summarized as mean ( % confidence intervals), and categorical variables as percentage. patients with significant disability were selected according to a bi score of ≤ . fisher's exact-test for contingency tables, z test and non-parametric mann-whitney u test were used when appropriate. a multivariate logistic analysis was used to assess the relationship between variables and in-hospital, -month and -year follow-up mortality. variables were chosen according to the hosmer-lemeshow methodology [ ] . after univariate analysis, only variables with a p < . were included in the final model; then, through a backward process, variables were excluded until a significance level of p < . was reached for each variable. the application of hosmer-lemeshow test is a measure of how well the model fits the data without any choice of variables by researcher to put into the multivariate model. a two-tailed p < . was considered statistically significant. stata statistical software , release (stata-corp, college-station, tx-usa) was used for database management and all the analyses. during the recruitment period, out of inpatients were eligible for this analysis ( patients had missed variables); ( . %) presented with a diagnosis of copd. among them, % were male with a mean age of years. table shows the demographic characteristics and modifiable risk factors of the two study groups. interestingly, almost half of the copd in-patients had history of previous hospitalizations compared to only one-third of non-copd inpatients. a significantly higher proportion of copd subjects also showed history of alcohol consumption and were more often morbidly obese. in-patients with copd had a significantly higher cumulative illness rating scale for the evaluation of severity and comorbidity index (p < . for both comparisons). as shown in table , significant overt cognitive impairment was documented in almost half of in-patients with copd, while a quarter needed positioning of urinary catheter. in-patients with clinically significant disability (bi ≤ ) were . % in comparison with individuals without copd ( . %, p = . ). moreover, gds was shown to be more frequently abnormal (mean-score equal to . ). in addition, . % had a probable depression (gds > ) as opposed to non-copd individuals. copd patients took more drugs than those without copd, both at admission, at in-hospital stay, at discharge and at -and -year followup (table ) . overall, disease distribution showed that arterial hypertension, ischemic heart disease, atrial fibrillation, heart failure, chronic renal failure, peripheral artery disease, overt hypertensive heart disease, anemia, rheumatic diseases, prostatic hypertrophy, osteoporosis, pneumonia, gastroesophageal reflux disease, respiratory failure, and cholelithiasis were more frequent in copd patients ( table ) . as shown in table , subjects with copd had significantly longer hospital stay; in addition, higher rates of rehospitalization at -year after discharge were recorded. in-hospital and within -year mortality did not differ between the two groups. however, when we assessed independent predictors of mortality, running univariate analysis (see appendix) and then multivariate analysis ( fig. ) according to hosmer-lemeshow methodology, cerebrovascular disease and current ics-laba therapy were independently associated with in-hospital mortality. at -month follow-up, male gender, hepatic cirrhosis, and ics-laba therapy were predictors of mortality. at -year follow-up, ics-laba therapy was the only predictor of mortality. copd exacerbation did not represent an independent predictor of mortality in older hospitalized people even if % of patients with copd had exacerbation. in this observational study on the reposi registry, we assessed the distribution of comorbidities and the occurrence of outcomes in a population of elderly copd in-patients admitted to the internal medicine and geriatric wards, with the aim to evaluate whether copd subjects behave differently from non-copd individuals. overall, the current findings suggest that copd subjects are at higher risk of death within the first year from admission to the hospital. although comorbidities are increasingly identified as important factors of copd management and outcomes [ ] , studies specifically designed to evaluate the relationships between comorbidities and long-term outcomes in subjects with a diagnosis of copd admitted to an internal medicine ward are scarce [ ] , and this is also true for several chronic diseases [ ] . a recent study showed that the addition of comorbidities to age, bmi, blood markers and indexes such as smoking status, dyspnea assessment, airway obstruction produced a model, known as barc index, that performed better than established index scores in predicting -year mortality [ ] . our analysis showed that copd in-patients are more often older men, smokers or former smokers, and live with their relatives, in agreement with our previous findings [ ] . moreover, copd patients are severe obese fig. multivariate analysis in copd patients according to in-hospital, -month and -year mortality. or odds ratio, % ci % confidence interval, ics/laba inhaled corticosteroids and long-acting beta -agonists in combination, gfr glomerular filtration rate calculated by ckd-epi formula; gfr is referred to values every ml/ min; barthel index is referred to values every points; diastolic blood pressure is referred to values every mmhg. only the final model is shown according to hosmer-lemeshow methodology. for the selection of variables see appendix and statistical analysis section consistently with recent data that seem to confirm that obesity is more common in copd patients compared to subjects who do not have copd [ ] . interestingly, individuals with a diagnosis of copd had more frequent mood changes, indicating higher level of distress, in agreement with those from the nhanes study of . % of subjects with copd suffering from depression [ ] . shane et al. showed that up to % of patients with copd had clinically significant depressive symptoms, a proportion higher than that recorded in other chronic diseases such as stroke, diabetes, coronary heart disease, arthritis, hypertension, and cancer [ ] . similarly, copd patients showed worse cognitive impairment than non-copd patients; in the study by dodd et al., up to % of patients with copd exacerbation had features of cognitive impairment [ ] . a recent systematic review and meta-analyses outlined that one in four subjects with copd has mild cognitive impairment [ ] . in addition to affecting pharmacological treatment, comorbidities may impair the ability to use inhalation devices [ ] ; for example, cognitive impairments affect the ability to properly use the inhaled device, and anxiety and depression can reduce the adherence to treatment. it follows that the choice of the proper inhaler should also take into account the relative contribution of concomitant diseases in affecting the correct use of the device. it is commonly accepted that cognitive impairment and depression lead to progressive disability [ , ] , especially in oldest-old subjects [ , ] , thus potentially affecting short-and long-term outcomes. the current findings also show that the presence of anemia is associated with the frequency of exacerbations and increasing healthcare costs [ , ] . the phenomenon is relevant in clinical practice: indeed, cote et al. found that anemia was present in % of copd inpatients [ ] . the possible mechanism consists in persistent elevated interleukin levels, in particular il- , that interfere with the erythropoietin response [ ] . the current analysis highlighted that copd patients had a worse functional status than patients without copd; this is of clinical importance, given that hospitalized elderly patients affected by pneumonia with a clinically significant disability were already shown to have higher mortality risk [ ] . lanièce et al. found that severe disability was the most important predictor of early re-admission among elderly inpatients [ ] . recent data showed that male gender, previously hospitalized, polypharmacy (more than drugs), lower functional status and frailty, depression, heart diseases, copd, urinary tract infection were associated to a higher risk of hospitalization [ ] . moreover, heart failure, diabetes and stroke were associated with a prolonged hospital stay (> days) in hospitalized copd patients [ ] . the current findings on comorbidities distribution showed a significant prevalence of respiratory failure and respiratory conditions other than copd, as well as cardiovascular diseases, chronic renal failure, prostatic hypertrophy, rheumatic diseases, and gastroesophageal reflux disease. an interesting speculation on these findings comes from the theory of network medicine [ ] , based on which human diseases are not independent of each other, but rather the consequence of different biological processes that interact in this complex network, defined as "diseasome". in this regard, copd is among the best scenario in which multiple factors such as chronic inflammation, aging-related changes, altered immune response, increased oxidative stress, consequences of smoke exposure and physical inactivity are variably interwound. aging per se is characterized by chronic low-grade systemic inflammation, and is associated with multiple chronic conditions, including copd [ , , ] ; interestingly, a relationship among systemic inflammation, comorbidities and copd outcomes has been clearly documented [ ] . of note, ischemic heart disease, heart failure, myocardial infarction, diabetes, lung cancer, osteoporosis, metabolic syndrome, are all characterized by low-grade inflammation and frequently associated with copd [ ] . the question is whether, and to what extent, comorbidities affect mortality independent of lung disease. using data from the multicenter observational study eclipse, agusti and colleagues [ ] proposed the systemic inflammome, a network representation of systemic inflammation in individuals suffering from copd, which may account, in a proportion of subjects who are persistently inflamed, for significantly higher rates of all-cause mortality. the prevalence of comorbidities in patients with copd was assessed by divo and collaborators [ ] , who identified specific comorbidities significantly associated with increased mortality. the relative contribution of each comorbidity to mortality and the relationships among comorbidities led to the so-called "comorbidome". vanfleteren et al. [ ] identified five clusters of comorbidities: ''cardiovascular '', ''cachectic'', ''metabolic'' and ''psychological'' and ''less comorbidity'' . the authors however failed to find any association with mortality. our findings indicate that cerebrovascular disease significantly increased the risk of death during hospitalization. on the other hand, cirrhosis and men gender were significantly associated with -month mortality. these observations are in agreement with kim et al. that found a significant statistically association between copd and increased risk of stroke [ ] , and with divo et al. that found that the risk of death was strongly associated with different comorbidities including liver cirrhosis, suggesting a correlation with lifestyle and social behavior [ ] . these data were also confirmed by baty et al. that found a higher prevalence of alcoholic cirrhosis in their nationwide analysis of hospital admissions for copd in switzerland [ ] . moreover, our results are consistent with previous studies that identified comorbidities that were associated with copd progression and exacerbation frequency, poor quality of life, higher mortality and increase of costs management [ , , ] . the current analysis highlights the role of the ics-laba regular treatment, which was independently associated with in-hospital, -month and -year follow-up mortality. this result was unchanged even if variables such as copd exacerbation, heart failure, atrial fibrillation, ischemic heart disease, oral anticoagulants, anti-platelet drugs had been included into the model. in a recent meta-analysis, horita et al. found that patients treated with laba-lama had fewer exacerbations and a significantly lower risk of developing pneumonia in comparison with ics-laba [ ] . in addition, ernst et al. suggested a limit use of ics and ics-laba in copd patients on the basis of the evidence of adverse effects, especially severe pneumonia, leading to excess mortality [ ] . although the causes of mortality are not known, it cannot be excluded that chronic use of ics was responsible for severe adverse events in compromised subjects. the lack of data on the dosage or the class of corticosteroids does not allow to draw firm conclusions on the contribution of the active drug. similarly, it is plausible to hypothesize that laba variably influenced the outcome. a recent study showed the importance of bi as a strong predictor of -days, -and -month mortality in elderly patients with pneumonia [ ] . simonetti et al. found that pneumonia severity and low functional status are the main factors associated with mortality in elderly people with community acquired pneumonia [ ] . vitacca et al. suggested the utilization of a unique instrument, i.e. the bi-dyspnea, to provide a global assessment of disability evaluating both respiratory and motor impairment [ ] . formiga et al. demonstrated that a better functional status and a lower comorbidity conditions were independent predictors of mortality at -years in -year-old community-dwelling subjects [ ] . in the current study, disability did not enter the multivariate analysis as independent predictor of mortality, although the barthel score suggestive of physical impairment clearly distinguished the copd phenotype (fig. ) . a possible explanation for the apparent discrepancy between studies lies in the lack of information on the lung functional impairment, which may variably affect the ability to interact with daily activities. it is therefore logical to hypothesize that disability is one of the strongest predictors of mortality also in copd. further studies are needed to confirm it. with regard to the protective function of higher glomerular filtration rate, our data are consistent with those of singanayagam et al. who established that chronic renal failure was significantly associated with increased short-term mortality in patients with copd [ ] . a potential explanation lies in the glomerular damage by arterial stiffness along with hypoxic damage to tubules and interstitium as possible mechanisms in the relationship between copd and chronic renal failure [ ] . we found that blood pressure had a protective role regarding in-hospital mortality. our findings are in agreement with previous observations that showed a reverse association between higher blood pressure and mortality in oldest old patients [ , ] . moreover, recent analysis showed that in contrast to the general population, in frail elderly patients increased blood pressure is associated with reduced mortality. a possible explanation is that high blood pressure is necessary to maintain sufficient organ perfusion in a population of older subjects who are likely to have significant vascular damage [ , ] . regarding sex, our results are consistent with a previous study that showed in elderly hospitalized patients a male profile, smokers or former smokers, affected by copd, coronary artery disease and cancer responsible for re-hospitalization and higher mortality [ , ] . this observational study has some limits. first, there was no specific information about how the diagnosis of copd was formulated (gold criteria, radiological criteria), and the severity of copd was not taken into account. given the lack of spirometric confirmation, it cannot be excluded that a proportion of subjects actually suffered from chronic diseases other than copd. however, the observational nature of the design and the exploratory approach limit the weaknesses of the findings. second, the lack of information on the appropriateness of prescriptions, and the opportunity to exclude potential confounders that goes beyond the scope of the reposi study. the major strength of the study is the multicenter design of the reposi register and the large number of participating centers resulting in a comprehensive sample of the elderly patients hospitalized in internal medicine and geriatric wards. in conclusion, this study showed the impact of copd and its comorbidities in the real-world scenario of internal and geriatric wards, identifying factors that are linked with short-and long-term outcomes. the current findings strongly support that the management of copd patients should include identification and treatment of its comorbidities. this approach should be the first step for personalized care based on a multidimensional assessment of elderly patients affected by copd. clinical data monitoring and revision: carlotta franchi, laura cortesi, mauro tettamanti, gabriella miglio (istituto di ricerche farmacologiche mario negri irccs, milano) edoardo alessandro pulixi (azienda ospedaliera della provincia di lecco azienda ospedaliera policlinico sant'orsola-malpighi raffaella arnò (azienda ospedaliera universitaria policlinico s barbara brignolo ottolini (fondazione irccs cà granda ospedale maggiore policlinico costanza caccia dominioni (irccs policlinico san matteo di pavia mosè bartone (ospedale giovanni paolo ii lamezia terme, catanzaro, unità operativa chiara pes, alessandro delitala (azienda ospedaliera-universitaria di sassari policlinico umberto i, sapienza università di roma maria antonietta bleve (azienda ospedaliera "cardinale panico giordano sigon (fondazione irccs cà granda ospedale maggiore policlinico fondazione irccs cà granda ospedale maggiore policlinico giuliana ceriani (fondazione irccs cà granda ospedale maggiore policlinico tiziana tognin (asst di unità operativa complessa di medicina generale cesare poletto (dipartimento di scienze mediche policlinico umberto i, roma, smsc -medicina interna a e malattie metaboliche dell'osso) clinica san carlo casa di cura polispecialistica maria pasquale ospedale di monselice azienda ospedaliera universitaria, udine). nicola passariello, luca rinaldi (presidio medico di marcianise filippo alessandro montalto (azienda ospedaliera universitaria policlinico paolo giaccone azienda ospedaliera per l'emergenza cannizzaro azienda ospedaliera universitaria maggiore della carità azienda ospedaliera universitaria città della salute e della scienza di torino projections of global mortality and burden of disease from to complex chronic comorbidities of copd characterisation of copd heterogeneity in the eclipse cohort comorbidities of copd comorbidities and risk of mortality in patients with chronic obstructive pulmonary disease are asthmatics enrolled in randomized trials representative of real-life outpatients? what proportion of chronic obstructive pulmonary disease outpatients is eligible for inclusion in randomized clinical trials the reposi registry of multimorbidity and polypharmacy validation of a short orientation-memory-concentration test of cognitive impairment depression scales for the elderly: gds, gilleard, zung functional evaluation: the barthel index cumulative illness rating scale a new equation to estimate glomerular filtration rate applied logistic regression, nd edn copd classification models and mortality prediction capacity prevalence of comorbidities in chronic obstructive pulmonary disease patients: a metaanalysis working group on, copd, spanish society of internal medicine et al ( ) comorbidities and short-term prognosis in patients hospitalized for acute exacerbation of copd: the epoc en servicios de medicina interna (esmi) study predicting copd -year mortality using prognostic predictors routinely measured in primary care gender-differences in disease distribution and outcome in hospitalized elderly: data from the reposi study obesity in copd: revealed and unrevealed issues the prevalence of clinically-relevant comorbid conditions in patients with physician-diagnosed copd: a cross-sectional study using data from nhanes - prevalence and risk factors for depressive symptoms in persons with chronic obstructive pulmonary disease cognitive dysfunction in patients hospitalized with acute exacerbation of copd cognitive impairment in chronic obstructive pulmonary disease and chronic heart failure: a systematic review and meta-analysis of observational studies copd in the elderly patient association of comorbidity with disability in older women: the women's health and aging study risk factors for hospital readmissions in elderly patients: a systematic review reposi investigators et al ( ) comorbidity does not mean clinical complexity: evidence from the reposi register unsuspected risk factors of frequent exacerbations requiring hospital admission in chronic obstructive pulmonary disease economic burden of patients with anemia in selected diseases haemoglobin level and its clinical impact in a cohort of patients with copd anemia of chronic disease disability, and not diabetes, is a strong predictor of mortality in oldest old patients hospitalized with pneumonia incidence and main factors associated with early unplanned hospital readmission among french medical inpatients aged and over admitted through emergency units factors associated with accelerated hospitalization and re-hospitalization among medicare home health patients factors associated with a prolonged length of stay after acute exacerbation of chronic obstructive pulmonary disease (aecopd) network medicine: a network-based approach to human disease from copd to chronic systemic inflammatory syndrome? chronic inflammation (inflammaging) and its potential contribution to age-associated diseases evaluation of copd longitudinally to identify predictive surrogate endpoints (eclipse) investigators et al ( ) comorbidity, systemic inflammation and outcomes in the eclipse cohort inflammatory biomarkers and comorbidities in chronic obstructive pulmonary disease evaluation of copd longitudinally to identify predictive surrogate endpoints (eclipse) investigators et al ( ) persistent systemic inflammation is associated with poor clinical outcomes in copd: a novel phenotype clusters of comorbidities based on validated objective measurements and systemic inflammation in patients with chronic obstructive pulmonary disease stroke risk among patients with chronic obstructive pulmonary disease: a systematic review and meta-analysis comorbidities and burden of copd: a population-based case-control study co-morbidity, body mass index and quality of life in copd using the economic burden of copd in the presence of comorbidities long-acting muscarinic antagonist (lama) plus long-acting beta-agonist (laba) versus laba plus inhaled corticosteroid (ics) for stable chronic obstructive pulmonary disease (copd) inhaled corticosteroids in copd: the clinical evidence management of community-acquired pneumonia in older adults development of a barthel index based on dyspnea for patients with respiratory diseases evidence of functional declining and global comorbidity measured at baseline proved to be the strongest predictors for long-term death in elderly community residents aged years: a -year follow-up evaluation, the octabaix study predictors of mortality in hospitalized adults with acute exacerbation of chronic obstructive pulmonary disease renal and hepatobiliary dysfunction in chronic obstructive pulmonary disease the relationship between blood pressure and mortality in the oldest old rethinking the association of high blood pressure with mortality in elderly adults: the impact of frailty relationship between high blood pressure and cardiovascular outcomes in elderly frail patients: a systematic review and meta-analysis relation between blood pressure and mortality risk in an older population: role of chronological and biological age sex-differences in the pattern of comorbidities, functional independence, and mortality in elderly inpatients: evidence from the reposi register conflict of interest the authors report no conflicts of interest. the study including human participants has been performed in accordance with the ethical standards of the declaration of helsinki and its later amendments. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - ddsj authors: gianfrancesco, milena; hyrich, kimme l; al-adely, sarah; carmona, loreto; danila, maria i; gossec, laure; izadi, zara; jacobsohn, lindsay; katz, patricia; lawson-tovey, saskia; mateus, elsa f; rush, stephanie; schmajuk, gabriela; simard, julia; strangfeld, anja; trupin, laura; wysham, katherine d; bhana, suleman; costello, wendy; grainger, rebecca; hausmann, jonathan s; liew, jean w; sirotich, emily; sufka, paul; wallace, zachary s; yazdany, jinoos; machado, pedro m; robinson, philip c title: characteristics associated with hospitalisation for covid- in people with rheumatic disease: data from the covid- global rheumatology alliance physician-reported registry date: - - journal: ann rheum dis doi: . /annrheumdis- - sha: doc_id: cord_uid: ddsj objectives: covid- outcomes in people with rheumatic diseases remain poorly understood. the aim was to examine demographic and clinical factors associated with covid- hospitalisation status in people with rheumatic disease. methods: case series of individuals with rheumatic disease and covid- from the covid- global rheumatology alliance registry: march to april . multivariable logistic regression was used to estimate ors and % cis of hospitalisation. age, sex, smoking status, rheumatic disease diagnosis, comorbidities and rheumatic disease medications taken immediately prior to infection were analysed. results: a total of cases from countries were included. nearly half of the cases were hospitalised ( , %) and ( %) died. in multivariable-adjusted models, prednisone dose ≥ mg/day was associated with higher odds of hospitalisation (or . , % ci . to . ). use of conventional disease-modifying antirheumatic drug (dmard) alone or in combination with biologics/janus kinase inhibitors was not associated with hospitalisation (or . , % ci . to . and or . , % ci . to . , respectively). non-steroidal anti-inflammatory drug (nsaid) use was not associated with hospitalisation status (or . , % ci . to . ). tumour necrosis factor inhibitor (anti-tnf) use was associated with a reduced odds of hospitalisation (or . , % ci . to . ), while no association with antimalarial use (or . , % ci . to . ) was observed. conclusions: we found that glucocorticoid exposure of ≥ mg/day is associated with a higher odds of hospitalisation and anti-tnf with a decreased odds of hospitalisation in patients with rheumatic disease. neither exposure to dmards nor nsaids were associated with increased odds of hospitalisation. epidemiology rheumatic diseases therefore, people with rheumatic disease may be at higher risk for a more severe course with covid- , including hospitalisation, complications and death. importantly, some medications used to treat rheumatic diseases, such as hydroxychloroquine and interleukin- (il- ) inhibitors, are being studied for the prevention and/or treatment of covid- and its complications including cytokine-storm. [ ] [ ] [ ] at present, the implications of covid- for people living with rheumatic diseases remain poorly understood. to address this knowledge gap, a global network of rheumatologists, scientists and patients developed a physician-reported case registry of people with rheumatic diseases diagnosed with covid- . this report aims to ( ) describe the demographic and clinical characteristics of the first patients submitted to the covid- global rheumatology alliance (c -gra) physician registry and ( ) identify factors associated with hospitalisation for covid- in this population. details of the registry design have been described elsewhere. [ ] [ ] [ ] briefly, c -gra data regarding individuals with rheumatic diseases diagnosed with covid- are captured from rheumatology physicians via two parallel international data entry portals for regulatory reasons: one limited to european countries ( eular. org/ eular_ covid _ database. cfm; hosted by the university of manchester, uk) and a second for all other sites ( rheum-covid. org/ provider-global/; hosted by the university of california, san francisco, california, usa). two patients sit on the c -gra steering committee and they contributed to the design of the registry, the questions being asked and the analysis of the results. the c -gra has a patient board, composed entirely of patients. these patients, and others, will be involved in disseminating the results of this analysis once published. no public were involved in the design or analysis of this project. physicians indicated whether the diagnosis of covid- was based on pcr, antibody, metagenomic testing, ct scan, laboratory assay or a presumptive diagnosis based on symptoms only. data elements for this analysis included physician city, state and country. countries were assigned to the six who regions ( www. who. int); the 'americas' was further divided into north and south. case information including age, sex, smoking status, rheumatic disease diagnosis, disease activity and comorbidities was collected. medications prior to covid- were categorised as: conventional synthetic disease-modifying antirheumatic drugs (csdmards; antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, cyclosporine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus); biologic dmards (bdmards; abatacept, belimumab, cd- inhibitors, il- inhibitors, il- inhibitors, il- /il- inhibitors, il- inhibitors, tumour necrosis factor inhibitors (anti-tnf)) and targeted synthetic dmards (tsdmards) namely janus kinase (jak) inhibitors. physicians reported the approximate number of days from symptom onset to symptom resolution or to death. the primary outcome of interest was hospitalisation for covid- . as of april , a total of cases were entered in the registry; hospitalisation status was unknown for four cases and these were excluded from analysis. continuous variables are reported as median (iqr). categorical variables are reported as number and percentage (%). in univariable analyses, differences in demographic and rheumatic disease-specific features according to hospitalisation status were compared using χ tests for categorical variables and mann-whitney u tests for continuous variables. the independent associations between demographic and disease-specific features with the odds of covid- hospitalisation were estimated using multivariable-adjusted logistic regression and reported as or and % cis; covariates included in the model were age group (< years vs > years), sex, rheumatic disease (rheumatoid arthritis (ra), systemic lupus erythematosus (sle), psoriatic arthritis (psa), axial spondyloarthritis (axspa) or other spondyloarthritis, vasculitis and other), key comorbidities (hypertension, lung disease, diabetes, cardiovascular disease and chronic renal insufficiency/end-stage renal disease), smoking status (ever vs never), physician-reported disease activity (remission, minimal/low disease activity, moderate disease activity or severe/high disease activity; or as a binary variable: remission and minimal/low disease activity vs moderate and severe/high disease activity), dmard type (no dmard, csdmard only, b/tsdmard only, csdmard and b/tsdmard combination therapy), non-steroidal anti-inflammatory drugs (nsaid) use (yes vs no) and prednisone-equivalent glucocorticoid use ( mg/ day, - mg/day, ≥ mg/day). categories with cell sizes < by hospitalisation status were collapsed to ensure sufficient power in the adjusted model. for univariable and multivariable models, patients with more than one of the following diseases recorded were classified as follows: sle>ra>psa>vascu-litis>axspa/other spondyloarthritis>other. cardiovascular disease and hypertension were collapsed as a single comorbidity in the regression model due to significant collinearity between the two variables. due to concerns regarding the possibility of confounding by indication, disease activity and prednisoneequivalent glucocorticoid use were analysed by including only one of the variables in the multivariable analysis at a time, and by including both variables in the multivariable analysis at the same time. unknown/missing data ( % smoking status, % nsaids, % glucocorticoids) were treated as a separate category in multivariable models. in exploratory analyses, the independent association between antimalarials and specific b/tsdmard therapies with hospitalisation status was estimated using multivariable logistic regression. to assess the robustness of the results, sensitivity analyses were performed. first, we repeated the above analyses after excluding patients with a 'presumptive diagnosis', meaning that the patient's physician thought he/she had symptoms consistent with the disease, but there was no evidence of the patient having: a) a confirmatory covid test; b) documentation of chest imaging showing bilateral infiltrates in keeping with covid- pneumonia or c) close contact with a known covid- -positive patient. second, we limited the analyses to patients whose covid- outcome was known (resolved/died) or for whom at least > days from symptom onset (or diagnosis date if symptom onset was unknown) had elapsed, as it is unlikely that a patient would be hospitalised > weeks after onset. third, we excluded cases with missing/unknown values within the covariate set included in the multivariable analyses. data were considered statistically significant at p< . . cell counts < are represented by 'n< ' in tables to protect patient anonymity. all analyses were conducted in stata v. . (statacorp). data quality was assessed by two data quality teams (one at the university of manchester, uk and the university of california, san francisco) who also confirmed there were no duplicate entries. due to the deidentified and non-interventional nature of the study, it was determined by the institutional review board that patient consent was not required. c -gra physician registry was determined 'not human subjects research' by the uk health research authority and the university of manchester, as ( ) psoriatic arthritis ( ) axial spondyloarthritis or other spondyloarthritis ( ) vasculitis ( ) sjögren's syndrome ( ) other inflammatory arthritis ( ) inflammatory myopathy ( reported days from onset to resolution or death (n= ), median (iqr) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) n (column %) for categorical variables unless otherwise noted. percentages may not sum to due to rounding. *cases could have more than one disease diagnosis. 'other' rheumatic disease category included (each n< ): undifferentiated connective tissue disease; ocular inflammation; autoinflammatory syndrome; mixed connective tissue disease; antiphospholipid antibody syndrome; calcium pyrophosphate deposition disease; systemic juvenile idiopathic arthritis; juvenile idiopathic arthritis, not systemic; igg -related disease. †chronic obstructive pulmonary disease, asthma, interstitial lung disease or other not specified. ‡csdmard medications included: antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, cyclosporine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus; b/tsdmard included: abatacept, belimumab, cd- inhibitors, il- inhibitors, il- inhibitors, il- /il- inhibitors, il- inhibitors, anti-tnf and janus kinase inhibitors. b/tsdmard, biologic or targeted synthetic dmard; csdmard, conventional synthetic dmard; dmard, disease-modifying antirheumatic drug; il, interleukin; nsaid, non-steroidal anti-inflammatory drug; tnf, tumour necrosis factor. table continued well as under united states federal guidelines assessed by the university of california, san francisco and patient consent was not required. we did not systematically capture how cases were identified before being entered into the registry and therefore we cannot detail this. however, we are aware of a number of large institutions that are systematically collecting all cases in their health system/district and entering them into the registry. the demographic and clinical characteristics of the first cases in the c -gra physician registry are shown in table . the majority of cases in the registry were from north america and europe, female and in the - age range, the countries that the cases were reported from are shown in online supplementary table . the most common rheumatic disease was ra ( , %), followed by sle ( , %) and psa ( , %). the most common comorbidities were hypertension ( , %), lung disease ( , %), diabetes ( , %), cardiovascular disease ( , %) and chronic renal insufficiency/end-stage renal disease ( , %). most cases were never smokers ( , %) and either in remission or had minimal/low disease activity ( , %). five patients were pregnant ( %). nearly half of the cases reported to the registry were hospitalised ( , %), and % ( ) were deceased. covid- diagnoses were predominately made through pcr testing ( , %), followed by laboratory assay of unknown type ( , %), ct scan ( , %) or other ( , %) (individuals could be tested using more than one method). fifty-two ( %) cases had a presumptive diagnosis only (online supplementary table ). the median number of days from covid- symptom onset to resolution or death was (iqr: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . demographic and clinical characteristics stratified by sex are presented in online supplementary table . demographic and clinical characteristics stratified by hospitalisation status are shown in table . differences by age group in hospitalisation status were observed: most hospitalised patients were over age ( %), compared with % of nonhospitalised cases (p< . ). in unadjusted analyses, differences in hospitalisation status by disease revealed a higher percentage of people who were hospitalised had sle and vasculitis ( % and %, respectively) versus those who were not hospitalised ( % and %, respectively), while a lower proportion of patients who were hospitalised had psa and axspa or other spondyloarthritis ( % and %, respectively) compared with those who were epidemiology igg -related disease. ‡csdmard medications included: antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, ciclosporin, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus; b/ tsdmard included: abatacept, belimumab, cd- inhibitors, il- inhibitors, il- inhibitors, il- /il- inhibitors, il- inhibitors, anti-tnf and janus kinase inhibitors. b/tsdmard, biologic or targeted synthetic dmards; csdmard, conventional synthetic dmard; dmard, diseasemodifying antirheumatic drug; il, interleukin; nsaid, non-steroidal anti-inflammatory drugs; tnf, tumour necrosis factor. not ( % and %, respectively). there were more comorbidities among hospitalised cases, including hypertension ( % vs %), lung disease ( % vs %), diabetes ( % vs %), cardiovascular disease ( % vs %) and chronic renal insufficiency/end-stage renal disease ( % vs %) (all p< . ). there was no association between disease activity and hospitalisation status (p= . ). nsaid use was reported less frequently among hospitalised patients than non-hospitalised patients ( % vs %, p= . ), while there was a higher proportion of patients receiving high doses of glucocorticoids among those who were hospitalised than not hospitalised ( % vs % for doses ≥ mg/ day, p= . ). we found no significant difference in hospitalisation status by sex, antimalarial therapy (either monotherapy or in combination with other dmards) or reported days from symptom onset to symptom resolution or death. in a multivariable model, age over years (or= . , % ci . to . ), hypertension/cardiovascular disease (or= . , % ci . to . ), lung disease (or= . , % ci . to . ), diabetes (or= . , % ci . to . ) and chronic renal insufficiency/end-stage renal disease (or= . , % ci . to . ) were associated with higher odds of hospitalisation (all p< . ) (table ) . treatment with b/tsdmard monotherapy just prior to covid- diagnosis was significantly associated with a lower odds of hospitalisation compared with no dmard therapy (or= . , % ci . to . ; p= . ). glucocorticoid therapy at prednisone-equivalent doses ≥ mg/ day, however, was associated with a higher odds of hospitalisation compared with no glucocorticoid therapy (or= . , % ci . to . ; p= . ). neither adding disease activity to the model with glucocorticoids nor replacing glucocorticoids by disease activity changed the direction, strength or significance of the relationship between the various variables and hospitalisation status in a meaningful way (data not shown). further analyses were conducted to examine the independent association of antimalarials and specific b/tsdmards with hospitalisation. a total of % of cases were taking antimalarials before hospitalisation. the largest subgroup of b/tsdmard therapies was anti-tnf medications ( %). we found no significant association between antimalarial therapy and hospitalisation (or= . , % ci . to . ; p= . ) after adjusting for sex, age over years, rheumatic disease, smoking status, comorbidities, other csdmard monotherapy, b/tsdmard monotherapy, csdmard-b/tsdmard combination therapy (excluding antimalarials), nsaid use and glucocorticoid dose. a significant inverse association between any anti-tnf therapy and hospitalisation was found (or= . , % ci . to . ; p= . ), after controlling for sex, age over years, rheumatic disease, smoking, comorbidities, csdmard monotherapy, other b/tsdmard monotherapy, csdmard-b/tsdmard combination therapy (excluding anti-tnf), nsaid use and glucocorticoid dose. small numbers of non-anti-tnf b/tsdmards precluded analysing the association of these individual agents with hospitalisation (online supplementary table ). our findings remained largely unchanged in sensitivity analyses excluding those with a presumptive diagnosis (n= ; online supplementary table ), those with unknown outcomes (n= ; online supplementary table ) and those with missing/unknown values (n= ; online supplementary table ) . this manuscript describes the largest collection of covid- cases among patients with rheumatic diseases, with cases from countries. we identified factors associated with higher odds of covid- hospitalisation, including older age, presence of comorbidities and higher doses of prednisone (≥ mg/ day). we did not see an association between prior nsaid use or antimalarials and hospitalisation for covid- . we did find b/tsdmard monotherapy to be associated with a lower odds of hospitalisation, an effect that was largely driven by anti-tnf adjusted ors from models including all variables shown. *p value for multivariable logistic regression model (see 'methods' section for details). †patients with more than one disease within these five diagnoses were classified as follows: systemic lupus erythematosus>rheumatoid arthritis>psoriatic arthritis>vasculitis>axial/other spondyloarthritis>other. other rheumatic disease category included (each n< ): undifferentiated connective tissue disease; ocular inflammation; autoinflammatory syndrome; mixed connective tissue disease; antiphospholipid antibody syndrome; calcium pyrophosphate deposition disease; systemic juvenile idiopathic arthritis; juvenile idiopathic arthritis, not systemic; igg -related disease. ‡chronic obstructive pulmonary disease, asthma, interstitial lung disease or other not specified. §csdmard medications included: antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, cyclosporine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus; b/tsdmard included: abatacept, belimumab, cd- inhibitors, il- inhibitors, il- inhibitors, il- /il- inhibitors, il- inhibitors, anti-tnf and janus kinase inhibitors. b/tsdmard, biologic or targeted synthetic dmards; csdmard, conventional synthetic dmard; dmard, disease-modifying antirheumatic drug; il, interleukin; nsaid, nonsteroidal anti-inflammatory drug; tnf, tumour necrosis factor. therapies. over half of the reported cases did not require hospitalisation, including many patients receiving b/tsdmards. the rate of hospitalisation was higher than in cohorts of general patients with covid- but this likely reflects the mechanism by which we collected the case information and should not be interpreted as the true rate of hospitalisation among patients with rheumatic disease infected with sars-cov- . prior to this report, there had been several small case series of covid- in patients with rheumatic disease reported from europe. [ ] [ ] [ ] [ ] with few exceptions, prior large descriptive studies of patients with covid- from china, europe and the usa have not included rheumatic disease in their baseline comorbidities. [ ] [ ] [ ] [ ] [ ] [ ] these studies have not allowed for further inference on the characteristics of patients with rheumatic disease and their associations with covid- severity. in accordance with previous studies of covid- in different populations, we found that patients with comorbidities such as hypertension, cardiovascular disease and diabetes had higher odds of hospitalisation. [ ] [ ] [ ] we also found that glucocorticoid use at a prednisone-equivalent dose ≥ mg/day was associated with an increased odds of hospitalisation, which is in agreement with prior studies showing an increased risk of infection with higher dose of glucocorticoids. we did not find a significant association between antimalarial use and hospitalisation in adjusted analyses. the use of hydroxychloroquine for the treatment of covid- , which was based on in vitro studies, has had mixed results. studies from one group suggested a benefit on the surrogate outcome of viral clearance among hospitalised patients, but these studies either had inadequate or no comparator groups. two randomised controlled trials of hydroxychloroquine had conflicting findings. a phase iib randomised controlled trial comparing two doses of chloroquine among patients hospitalised with covid- with historical controls from wuhan detected a negative safety signal-qtc prolongation-but no clinical benefit. finally, two observational studies using propensity score matching to account for confounding by indication have found no significant benefit with either hydroxychloroquine alone or combined with azithromycin on clinical outcomes including mortality ; however, epidemiology these studies were limited by design issues and a high risk of bias due to unmeasured confounding. we also did not detect a significant association between nsaid use and hospitalisation in adjusted analyses. although no prior data in patients with covid- have supported a deleterious effect of nsaids on clinical outcomes, early reports cautioned against the use of nsaids suggesting harm when used during the clinical course of covid- . these observations, while anecdotal, may also relate to confounding by indication, since nsaids are also often sold over-thecounter and may not be documented in hospital records with the same accuracy as prescription medications, leading to a reporting bias. we found a lower odds of hospitalisation with b/tsdmards monotherapy in our primary multivariable analysis, which was driven largely by anti-tnf therapies. the number of cases taking other biologic drugs or jak inhibitors was small, and may have been insufficient to demonstrate other underlying effects if present. although we caution against causal inference regarding drug effects given significant potential for residual confounding in our study, we also note that there is biological plausibility for the potential benefit of biologic medications in treating covid- , as evidenced by those with more severe disease having higher levels of cytokines, including il- and tnf. the use of il- inhibitors is being investigated for covid- , particularly in cases complicated by aberrant inflammatory responses or 'cytokine storm'. this is based on two initial case series of fewer than patients. anti-tnfs have also been suggested as a potential therapy in covid- , but this has been based solely on preclinical data. randomised, placebo-controlled trials are needed to clarify potential benefits or harms of biologic therapies in treating covid- . strengths of our study include the first large analysis of patients with rheumatic diseases and covid- . all case data were entered by rheumatology healthcare providers. the c -gra physician registry includes cases from countries suggesting that our findings are more generalisable than singlecentre or regional studies. the registry collects information on specific rheumatic disease diagnoses, which to date have not been captured in large, published case series of covid- . despite these strengths, there are important limitations to these registry data. the c -gra registry is voluntary and does not capture all cases of covid- in patients with rheumatic disease. this approach to data collection places limitations on causal conclusions and temporal relationships and therefore we can only make limited inferences based on our results. there is selection bias due to several factors, including geographic location, hospitalisation status and disease severity, with the more severe cases most likely to be captured. therefore, the data cannot be used to comment on the incidence of covid- in this patient population or its severity. since the registry's inclusion criteria are restricted to those with rheumatic disease and covid- , this precludes the ability to make comparisons with those who do not have rheumatic disease, or those with rheumatic disease who do not have covid- . although physicians may be contacted for follow-up information for unresolved cases, this is a crosssectional analysis and there is the possibility that some patients may not have progressed to their maximum level of care prior to enrolment. in our dataset, % of cases were unresolved or had an unknown resolution status, although exclusion of these cases in sensitivity analyses did not change our conclusions. furthermore, while we have collected information on medication use prior to covid- diagnosis, we do not have specific data on the duration of treatment, medication dose, or additional historical treatments. at the time of this report, the c -gra databases remain open for further case reports. with additional cases, we will be able to examine more detailed outcomes associated with specific rheumatic diseases and covid- treatments, as well as the outcomes of covid- in people with rheumatic diseases. this series of cases demonstrates that the majority of patients with rheumatic diseases captured in our registry recover from covid- . in some cases, exposure to specific medication classes is associated with lower odds of hospitalisation; however, these findings should be interpreted with caution because of a high risk of bias. results support the guidance issued by the american college of rheumatology and the european league against rheumatism, which suggest continuing rheumatic medications in the absence of covid- infection or sars-cov- exposure. in this series of people with rheumatic disease and covid- , use of dmards did not increase the odds of hospitalisation. as in the general population, people with rheumatic diseases who are older and/or have comorbidities have a higher odds of covid- -related hospitalisation. anti-tnf treatment was associated with reduced odds of hospitalisation while prednisone use ≥ mg/day was associated with a higher odds of hospitalisation. there was no difference in antimalarials, such as hydroxychloroquine, or nsaid use between those who were or were not hospitalised. twitter pedro m machado @pedrommcmachado and philip c robinson @ philipcrobinson the risk of infections associated with rheumatoid arthritis, with its comorbidity and treatment a rush to judgment? rapid reporting and dissemination of results and its consequences regarding the use of hydroxychloroquine for covid- covid- : consider cytokine storm syndromes and immunosuppression baseline use of hydroxychloroquine in systemic lupus erythematosus does not 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hydroxychloroquine usage in united states veterans hospitalized with covid- covid- : european drugs agency to review safety of ibuprofen clinical features of patients infected with novel coronavirus in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study effective treatment of severe covid- patients with tocilizumab tocilizumab treatment in covid- : a single center experience trials of anti-tumour necrosis factor therapy for covid- are urgently needed college of rheumatology guidance for the management of adult patients with rheumatic disease during the covid- pandemic eular provisional recommendations for the management of rheumatic and musculoskeletal diseases in the context of sars-cov- competing interests mg reports grants from national institutes of health, niams, outside the submitted work. klh reports she has received speaker's fees from abbvie and grant income from bms, ucb and pfizer, all unrelated to this manuscript. klh is also supported by the nihr manchester biomedical research centre. sa-a has nothing to disclose. lc has not received fees or personal grants from any laboratory, but her institute works by contract for laboratories among other institutions, such as abbvie spain, eisai, gebro pharma, merck sharp & dohme españa, s.a., novartis farmaceutica, pfizer, roche farma, sanofi aventis, astellas pharma, actelion pharmaceuticals españa, grünenthal gmbh and ucb pharma. md reports no competing interests related to this work. she is supported by grants from the national institute of health, pfizer independent grants for learning and change, genentech, horizon pharma. she has performed consultant work for amgen, novartis, regeneron/sanofi unrelated to this work. lg reports personal consultant fees from abbvie, biogen, celgene, janssen, eli lilly, novartis, pfizer, sanofi-aventis, ucb and grants from eli lilly, mylan, pfizer, all unrelated to this manuscript. em reports that lpcdr received support for specific activities: grants from abbvie, novartis, janssen-cilag, eli lilly portugal, sanofi, grünenthal s.a., msd, celgene, medac, pharmakern, gafpa; grants and non-financial support from pfizer; nonfinancial support from grünenthal gmbh, outside the submitted work. gs reports no competing interests related to this work. her work is supported by grants from the national institutes of health and agency for healthcare research and quality. she leads the data analytic center for the american college of rheumatology, which is unrelated to this work. as reports grants from a consortium of companies (among them abbvie, bms, celltrion, fresenius kabi, eli lilly, mylan, hexal, msd, pfizer, roche, samsung, sanofi-aventis and ucb) supporting the german rabbit register and personal fees from lectures for abbvie, msd, roche, bms, pfizer, outside the submitted work. sb reports no competing interests related to this work. he reports non-branded marketing campaigns for novartis (<us$ ). rg reports non-financial support from pfizer australia, personal fees from pfizer australia, personal fees from cornerstones, personal fees from janssen new zealand, nonfinancial support from janssen australia, personal fees from novartis, outside the submitted work. jsh reports grants from rheumatology research foundation, grants from childhood arthritis and rheumatology research alliance (carra), personal fees from novartis, outside the submitted work. es reports non-financial support from canadian arthritis patient alliance, outside the submitted work. ps reports personal fees from american college of rheumatology/wiley publishing, outside the submitted work. jy reports personal fees from astrazeneca, personal fees from eli lilly, grants from pfizer, outside the submitted work. pm reports personal fees from abbvie, personal fees from eli lilly, personal fees from novartis, personal fees from ucb, outside the submitted work. pr reports personal fees from abbvie, nonfinancial support from bms, personal fees from eli lilly, personal fees from janssen, personal fees from pfizer, personal fees from ucb, non-financial support from roche, personal fees from novartis, outside the submitted work. zi, lj, pk, slt, sr, jfs, lt, kw, wc, jwl and zsw have nothing to disclose. patients and/or the public were involved in the design, conduct, reporting or dissemination plans of this research. refer to the 'methods' section for further details. ethics approval the c -gra physician registry was determined 'not human subjects research' by the uk health research authority and the university of manchester, as well as under united states federal guidelines assessed by the university of california, san francisco and patient consent was not required.provenance and peer review not commissioned; externally peer reviewed. data availability statement request for access to data from the registry should be made to the data access and sharing committee of the covid- global rheumatology alliance.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained.orcid ids laure gossec http:// orcid. org/ - - - x pedro m machado http:// orcid. org/ - - - philip c robinson http:// orcid. org/ - - - key: cord- -txlvla j authors: gonzalez–dunia, daniel; sauder, christian; de la torre, juan carlos title: borna disease virus and the brain date: - - journal: brain res bull doi: . /s - ( ) - sha: doc_id: cord_uid: txlvla j viruses with the ability to establish persistent infection in the central nervous system (cns) can induce progressive neurologic disorders associated with diverse pathological manifestations. clinical, epidemiological, and virological evidence supports the hypothesis that viruses contribute to human mental diseases whose etiology remains elusive. therefore, the investigation of the mechanisms whereby viruses persist in the cns and disturb normal brain function represents an area of research relevant to clinical and basic neurosciences. borna disease virus (bdv) causes cns disease in several vertebrate species characterized by behavioral abnormalities. based on its unique features, bdv represents the prototype of a new virus family. bdv provides an important model for the investigation of the mechanisms and consequences of viral persistence in the cns. the bdv paradigm is amenable to study virus–cell interactions in the cns that can lead to neurodevelopmental abnormalities, immune-mediated damage, as well as alterations in cell differentiated functions that affect brain homeostasis. moreover, seroepidemiological data and recent molecular studies indicate that bdv is associated with certain neuropsychiatric diseases. the potential role of bdv and of other yet to be uncovered bdv-related viruses in human mental health provides additional impetus for the investigation of this novel neurotropic infectious agent. to establish a persistent infection in its host, a virus has to meet two essential requirements. first, it must avoid clearance by the host immune surveillance. second, it must adopt a noncytolytic strategy of multiplication. to achieve these goals, viruses have developed a plethora of strategies [ , ] . the central nervous system (cns) has intrinsic properties that favor the establishment of viral persistence [ ] , namely: (a) the existence of the blood-brain barrier and the paucity of immune elements in the brain, that provide the cns with an immunologically isolated environment; (b) viral replication is often restricted in cells of neuroectodermal origin, thus facilitating persistence; (c) viral spread over long distances within the cns is facilitated by the intricate networks of cell processes and cell interactions. evidence provided by epidemiological and clinical data, together with virological studies, have led to the hypothesis that chronic viral infections of the cns contribute to human mental disorders of unknown etiology. it is therefore of interest to identify relevant infectious agents, as well as to understand the mechanisms by which viruses persist in the cns and interfere with brain functions. borna disease virus causes cns disease in a broad range of vertebrate animal species that is manifested by behavioral abnormalities and diverse pathology. bdv provides an important paradigm for the investigation of the mechanisms and consequences of viral persistence in the cns. studies on this viral system will contribute to the elucidation of immune-mediated pathological events involved in virally induced neurological disease, as well as the mechanisms whereby viruses induce neurobehavioral disturbances in the absence of the hallmarks of cytolysis and inflammation. moreover, serological data and recent molecular epidemiological studies indicate that bdv can infect humans, and is possibly associated with certain neuropsychiatric disorders. based on its unique biological and genetic characteristics, bdv represents the prototype of a new family of viruses. consequently, investigations on bdv may provide new insights into virus-cell interactions in the cns. the prospect of finding other bdv-like viruses, some of them possibly clinically relevant, provides further impetus for the study of this novel neurotropic infectious agent. the purpose of this review is to discuss our present knowledge of bdv persistence in the cns. we will first briefly describe the biology of bdv, with special focus on the recent developments regarding its molecular biology. we will then examine the interaction between bdv and the cns. finally, we will discuss the evidence supporting a possible role of bdv in certain human mental disorders. of borna, in saxony, where many horses from a cavalry regiment died during an epidemic in . early studies showed that bd could be transmitted by using brain homogenates from diseased horses, demonstrating the infectious nature of the disease [ , , , ] . sensitivity to detergents, ultraviolet light, and organic solvents, together with filtration studies suggested that bd was caused by an enveloped virus, the borna disease virus. besides horses and sheep, infections with bdv have been documented in cattle, cats, donkeys, rabbits, and ostriches [ , , , , ] . sporadic cases of bd have also been reported in several other animal species [ ] . moreover, experimental infections have revealed the remarkable wide host range of bdv, which includes birds, rabbits, and rodents, as well as nonhuman primates [ , , , , , , , , , , ] . bdv infection leads, after a variable incubation period, to diverse pathological manifestations that depend on the species, immune status and age of the host, route of infection, and the particular strain of virus used for infection. the adaptation of bdv to small laboratory animals, such as the rabbit and the rat, has facilitated more detailed studies on bdv pathogenesis and the neurobehavioral alterations accompanying the infection. filtration experiments estimated that bdv particles had a size of to nm [ ] . recent electron microscopy studies have shown that preparations of cell-free infectious bdv contain enveloped particles of roughly spherical morphology, approximately nm in diameter [ , ] . intracytoplasmic virus-like particles with similar characteristics have also been observed in thin sections of bdv-infected cells [ ] . all bdv isolates are characterized by being highly neurotropic and by having a noncytolytic strategy of replication [ ] . cells and tissues of nonneural origin exhibit only a low susceptibility to infection. the paucity of cell-free virus associated with bdv infection has long hampered its molecular characterization. the establishment of persistently infected cell lines and the use of molecular genetic approaches has enabled the recent progress in the molecular characterization of this infectious agent. these studies have demonstrated that bdv is a nonsegmented, negativestranded (nns) rna virus [ , , , ] . the cloning and complete sequencing of two bdv isolates has uncovered the genomic organization of bdv [ , , , ] . the genome is about . kb long, with complementary Ј and Ј untranslated regions at its termini (fig. ) . the antigenomic polarity contains information for at least five open reading frames (orf). similarly to other members of the mononegavirales, the genome can be divided in three main gene blocks: block codes for the nucleoprotein and polymerase cofactors, represented by the bvp (orf i) and bvp (orf ii) proteins of bdv; block codes for the matrix and virus envelope proteins, whose likely counterparts in bdv are the bvp (orf iii) and bvp (orf iv) proteins, respectively; and block codes for the viral polymerase, identified as orf v in the bdv genome. the molecular biology of bdv has been the subject of recent reviews [ , , ] and is not the focus of this review. we will only briefly describe here the main features that distinguish the replication and gene expression regulation of the bdv genome. the bdv nucleoprotein (np) bvp is present at high levels in infected cells and tissues. this protein is likely encoded in two isoforms of and kda [ , , ] . this may be related to the presence of two in-frame initiation codons in the bvp gene sequence. the bvp protein is acidic, with a high ser/thr content and is phosphorylated at serine residues [ , , ] . these features are consistent with the phosphoprotein (p) transcriptional activator found in other nns rna viruses. the transcription unit encoding bvp can also direct the synthesis of a polypeptide of kda (bvp ). recent data from our laboratory indicate that bvp is present in infected cells. the orf encoding bvp starts nucleotides upstream from bvp and overlaps, in a different frame, with the first nucleotides of orf ii (fig. ) . the function of bvp is presently unknown. a similar situation has been described for the p gene of sendai virus [ ] and vesicular stomatitis virus [ ] . bdv orf iii (bvp ) likely represents the bdv matrix (m) protein. in contrast to other nns rna viruses, bdv m protein is glycosylated and data suggest that it might be present at the surface of the virion envelope [ ] . orf iv is predicted to encode for a polypeptide of kda (bvp ). sequence features suggest that this protein is a viral surface glycoprotein (gp). recent reports have provided experimental evidence that bvp is involved in virus entry [ , ] . bvp is present as two forms in bdv-infected cells [ ] . one form of approximately kda (gp- ) corresponds to the full-length product encoded by orf iv and accumulates in the endoplasmic reticulum. the molecular weight of this polypeptide, higher than kda, is due to glycosylation. a shorter product of kda (gp- ) corresponds to the c-terminus of gp- and is generated via cleavage by the cellular protease furin [ ] . moreover, gp- is present at the surface of infected cells. both gp- and gp- are associated with infectious virions. these features indicate a novel maturation pathway for a nns rna virus surface gp and, hence, for the assembly of bdv particles [ ] . orf v is capable of encoding a polypeptide with a predicted molecular mass of kda, whose deduced amino acid sequence displays strong homology with the nns rna viral polymerases (l protein family) [ ] . this homology is particularly high in the case of the conserved putative catalytic domain. bdv has the property, unique among known animal nns rna viruses, of a nuclear site for the replication and transcription of its genome [ ] . consistent with this finding, bdv ribonucleoproteins (rnp) are found in the nucleus of persistently infected cells [ ] . as with other nns rna viruses, bdv rnp are infectious upon transfection of susceptible cells [ ] . bdv exhibits a complex transcriptional pattern in infected cells. subgenomic messenger rnas (mrnas) encoding bvp and bvp are monocistronic. in contrast, mrnas encoding the m, gp, and l proteins are polycistronic. in the other nns rna viruses, these polypeptides are usually encoded by monocistronic mrnas. in addition, bdv does not exhibit the configuration of transcription termination signal, intergenic region, and transcription initiation signal that is characteristically present at the gene boundaries of nns rna viruses [ ] . bdv transcription units and transcriptive signals frequently overlap (fig. ) . moreover, bdv utilizes the host splicing machinery to generate some of its mrnas [ , ] . genomic stability is another striking characteristic of bdv. the two bdv complete genomic sequences yet determined have more than % homology at the nucleotide level [ , ] . this is remarkable for rna virus isolates with different origin and passage history in animals and cultured cells. these sequences are also very similar to those obtained from field cases of bd in different animal species [ , ] , as well as to sequences derived from human cases of bdv infection [ , , , ] . this extraordinary sequence conservation, uncommon for an animal nns rna virus, may indicate that there is a prevalent and stable species of bdv in nature, with the ability to infect several animal species, including humans. the reasons for the great stability of the bdv genome are unknown. interestingly, sequence analysis reveals that the l proteins of bdv and sonchus yellow net virus, a plant nucleorhabdovirus that also replicates in the nucleus, are the most distantly related to the l proteins from animal rhabdoviruses, raising intriguing questions about the evolutionary origins of bdv [ ] . gonzalez-dunia, sauder and de la torre bdv is unique not only for its extraordinary wide host range, combined with a remarkable genomic stability, but also in various aspects of its biology. expression of the bdv compact genome is regulated by an overlap of transcription units and transcriptive signals, an overlap of orf, a readthrough of transcription termination signals and rna splicing. the concurrent use of such a diversity of strategies for the regulation of gene expression is unique among known nns rna viruses. taken altogether, this has led to the proposal that bdv represents the prototype of a new taxon of nns rna viruses [ , ] . the portal of entry for bdv infection has not formally been identified. however, field observations have led to the hypothesis that a primary route of infection might be through the nose and the olfactory neuroepithelium [ ] . animals may become infected by contact with saliva, excretions, nasal secretions, or by exposure to contaminated food or water. contact experiments have suggested that healthy carrier animals might represent a source of infection, because the disease can be transmitted between horses and sheep living in the same stable [ ] . oral infection has been achieved in several animal species, suggesting that the virus could also be transmitted by the oral/gastric route [ , ] . finally, although formal evidence is lacking, colostrum and milk could play a role in the infection of the newborn [ ] . experimental infection of the rat has allowed the dissection of the dissemination pathway of bdv [ , ] . after initial replication in the neurons located at the site of entry, bdv likely migrates intraaxonally in an anterograde or retrograde direction towards the cns. the pathway of dissemination of bdv following intranasal inoculation of the rat has been thoroughly analyzed [ , ] . the virus initially replicates in the neuroreceptor cells of the olfactory epithelium. subsequently, the virus gains access to the cells of the main olfactory bulb, including the tufted and mitral cells, as well as the periglomular cells, the granule cells, and the short axon neurons of the internal granular layer of the main olfactory bulb. following invasion of these sites, the virus will reach the efferent targets of the main olfactory bulb, like the anterior olfactory nucleus, the prepiriform cortex, the olfactory tubercle, the olfactory amygdala, and the entorhinal cortex. further spread is then possible into numerous diencephalic and telencephalic areas, through polysynaptic neuronal connections. likewise, experimen- tal infection into the foot pad of a rat results in a retrograde transport of bdv, through the sciatic nerve, and then from neuron to neuron into the cns [ ] . moreover, evidence of oral infection suggests that the nerve endings innervating intestinal regions are suitable for uptake of the virus and its transport to the cns [ ] . alternatively, oral uptake of bdv may also result in the infection of the gut epithelium. the virus could then gain access to the surrounding lymphoid tissue, infect target blood cells, and be transported by the bloodstream into the cns. the location of the initial site of entry will condition the incubation period preceding the onset of overt bd, reflecting the time required for viral transport into the brain. a similar mode of dissemination has been described for rabies virus, another nns rna virus [ ] . both rabies and bdv spread along neuronal chains. the manner in which these chains are utilized is determined by the natural connections of neurons. such a mode of spread is compatible with a synaptic transfer of these viruses. moreover, the lack of detection of mature virus particles at any stage during bdv propagation has led to the hypothesis that viral spread ensues in the form of bare rnp [ , ] . this hypothesis has been further strengthened by the demonstration that rnp are infectious [ ] . within the cns, bdv will exhibit a preferential tropism for the limbic system, including the hippocampus [ , , ] . this area will eventually harbor the highest viral load regardless of the initial mode of infection, even if viral spread is not restricted to the limbic system and will diffuse throughout the cns [ ] . hence, bdv is also found in the hypothalamus, thalamus, and cerebral cortex. viral antigen accumulates in the nucleus, the perikaryon, and processes of the infected neurons [ ] . the nucleolus, however, remains free of antigen. in the nuclei of infected neurons, aggregates of virus-specific material are likely to form the joest-degen inclusion bodies that characterize bdv-infected cells [ ] . the topography of the infection in the hippocampus is particularly interesting. the stratified distribution of viral markers coincides with that of some excitatory amino acids receptors, in particular the glutamate receptors [ ] . also, specific neurotransmitter pathways are affected by bdv infection [ , , ] . this could indicate that these receptors are used by the virus or by viral rnp for their transneuronal transfer. besides neurons, glial cells are also permissive for bdv. virus antigen and rna appear rather early in astrocytes, followed by oligodendrocytes, ependymal cells, as well as schwann cells in the peripheral nervous system [ ] . it is probable that glial cells become secondarily infected after bdv release by the neurons. in agreement with this hypothesis, purkinje cells are the only cells in the cerebellum containing bdv antigen early after infection. later on, glial cells also become infected [ ] . this is also reminiscent of findings described in other biphasic cns diseases caused by rna viruses, such as theiler's virus infection of the mouse. in this case, the acute phase is characterized by an active viral replication in neurons. subsequently, the virus accesses glial cells in the spinal cord, which is required for the establishment of persistence [ , ] . in the late stages of infection, bdv diffuses centrifugally, probably by using an anterograde axonal transport, and viral markers have been detected in peripheral nerves of all tissues and organs. consequently, many tissues and organs become positive for viral markers [ , , ] . these include lachrymal, salivary or sebaceous glands and, as a rule, any ectodermal/epithelial tissue that can be reached by bdv through central or peripheral innervation. however, it is likely that extra neural tissue will become eventually infected only if the virus is delivered through the peripheral axons for a long time. the dissemination of bdv within the host could effectively ensue in the form of rnp. however, horizontal transmission of bdv would likely require shedding of mature, enveloped particles with the ability to initiate infection in a new host. despite numerous attempts, such particles have never been detected at any stage during bdv infection [ ] . therefore, the extremely low levels of infectious virus shed by infected animals may explain the poor rate of contagion of bdv. neuropathogenesis of the natural bdv infection. the neuropathogenesis of naturally occurring bd has been best characterized in the horse, where more than % mortality is observed. there is also increasing evidence that horses can be subclinically infected with bdv [ , , , ] . borna disease is defined as a nonpurulent polioencephalomyelitis [ , , ] . the inflammatory cells form massive perivascular cuffs, as well as more diffuse tissue infiltrates. inflammatory infiltrates are present predominantly in the gray matter, but can also invade the underlying white matter. reactive astrocytosis usually follows the acute phase of the disease. interestingly, despite this strong inflammatory reaction, neuronal degeneration is limited. horses may develop dimness of vision and partial or complete blindness, as a result of the degeneration of the optic nerve and neurons in the retina, likely caused by the lymphocytic infiltration [ , ] . however, bdv may also replicate in the retina without exerting any overt neuronal damage [ ] . the brain regions most affected by the inflammatory response are the olfactory bulb, the hippocampus, and the caudate nucleus [ ] . inflammation is moderate in the spinal cord and absent in the cerebellum [ ] . the colocalization of cell infiltrates with sites of high viral expression suggests that the inflammatory reaction is due to the presence of viral antigen. the neuropathological features of bd in sheep are very similar to those described in horses [ , , ] . symptoms observed during natural bdv infection are summarized in table . neuropathogenesis of the experimental bdv infection. a broad spectrum of animals can be experimentally infected with bdv ( table ). the rat is the most commonly used model for the study of bdv pathogenesis. the age, immune status, and genetics of the rat, as well as viral factors can influence the course of infection [ , , , , , ] . adult rats infected intracerebrally or intranasally with bdv usually develop an immune-mediated biphasic behavioral disease. rats display clinical signs and a histopathological picture very similar to that described for the naturally infected horse [ , , , , , ] . the onset of clinical signs, including movement abnormalities, aggressive and hyperactive behavior, coincides with the appearance of an inflammatory reaction in the brain that reaches its maximum of severity between days and after infection. parts of the limbic system, the cerebral cortex, and the olfactory bulb exhibit the strongest inflammatory reaction, which is limited to the gray matter. some level of inflammation is also observed in the basal ganglia, the diencephalon, and to a lesser degree, in the midbrain and medulla. this extensive inflammatory reaction leads to neuronal destruction that in some cases may cause a hydrocephalus. as with the infection of horses, the cerebellum rarely shows signs of inflammation. high virus replication is observed in the eyes of bdv-infected rats, which is accompanied by retinitis and leads to blindness due to the neuronal loss in the retina. the initial aggressive hyperactive behavior is followed by apathy, somnolence, and depression. eventually, some animals may also develop obesity [ ] . this chronic phase is characterized by a steady decline in the inflammatory reaction, despite continuous high viral load in the cns. to our knowledge, this has gonzalez-dunia, sauder and de la torre not been observed in other viral infections. the mechanisms underlying this delayed immune-tolerance are unknown. it is possible that sustained expression of high levels of viral antigens may result in the exhaustion of high affinity virus-specific t-cells [ , ] . in contrast to the situation depicted above, bdv neonatal infection of the rat causes a life-lasting persistent infection that is characterized by the lack of a cellular immune response to bdv and the absence of clinical signs of bd [ ] . this infection has been designated as a ''persistent, tolerant infection of the newborn'' (pti-nb) rat [ ] . nevertheless, the humoral response to bdv is not significantly impaired in pti-nb rats [ , ] . brains of pti-nb rats harbor a viral load comparable to that found in rats with acute bd [ , , , ] , illustrating the noncytolytic replication of bdv. although pti-nb rats do not show clinical signs, they exhibit distinct deficiencies in emotional and cognitive functions [ , ] , as well as physiological and neurodevelopmental abnormalities [ , ] . hence, the pti-nb rat provides a valuable model to investigate the consequences of bdv infection in the cns without immune-mediated damage (see sections . and . ). bd as an immune-mediated neurological disease. immunological mechanisms frequently contribute to the pathophysiology of virus-induced cns diseases [ ] . experimental bd provides a valuable model to study t-cell-mediated immunopathology in the cns [ , ] . the cell-mediated immune response to bdv plays an essential role in the development of bd. this has been shown by studies on immunocompromised animals. bdv infection of rats treated with cyclophosphamide, cyclosporin a, or of athymic rats does not lead to inflammation and development of disease [ , , ] . however, adoptive transfer of spleen cells from bd rats into bdvinfected immunosuppressed recipients results in inflammatory changes in the brain and concomitant bd [ ] . in contrast, transfer of immune serum does not lead to disease, demonstrating that antibodies are not involved in the immunopathological process induced by bdv [ ] . studies on pti-nb rats provide further evidence for the role of the cell-mediated immune response in the development of bd. bdv replicates in the thymus of pti-nb rats [ ] . as with other viruses, early expression of viral antigens in the thymus can promote clonal deletion of t-cells that would normally respond to the infectious agent [ ] . thus, the lack of bdv-specific precursor t-cells in the spleen and lymph nodes likely explains the absence of an inflammatory reaction in the cns of pti-nb rats. pathogenic role of cd ϩ and cd ϩ t-cells in bd. both cd ϩ and cd ϩ t-cells are present in the cns cell infiltrates and contribute to the immune-mediated pathology associated with bd [ , ] . the role of cd ϩ t-cells in bd was supported by the isolation of a bdv-specific cd ϩ t-cell line with the ability to induce disease upon adoptive transfer into bdv-infected immunosuppressed recipients [ , ] . adoptive transfer of these cd ϩ t-cells into immunocompetent uninfected rats did not cause encephalitis or disease, demonstrating that this bdv-specific tcell line was not encephalitogenic per se. recent evidence indicates that a direct cytotoxic activity of cd ϩ t-cells plays a rather limited, if any, role in bd [ ] . although bdv-specific cd ϩ t-cells can exhibit major histocompatibility complex (mhc) class ii restricted cytotoxic activity in vitro [ , ] , there is not, however, convincing evidence of such an activity in vivo [ , ] . it should be noted that cd ϩ t-cells may acquire cytotoxic activity during in vitro cultivation [ ] . in addition, there is no evidence of mhc class ii expression in neurons, the main cell type destroyed by the inflammatory response [ , , ] . furthermore, cd ϩ t-cells are present predominantly in perivascular cuffs, whereas few reach the brain parenchyma, where the immune-mediated damage occurs [ , ] . besides, adoptive transfer of noncytolytic virus-specific cd ϩ cells into bdv-infected immunosuppressed recipients can lead to the development of bd only if cd ϩ t-cells enter the brain [ ] . development of disease in these recipients can be prevented by treatment with monoclonal antibodies that specifically block cd ϩ t-cell activity [ , ] . conversely, treatment of bdv-infected rats with anti-cd ϩ antibodies does not effectively prevent inflammation and tissue damage in the cns [ ] . taken together, these findings suggest that cd ϩ t-cells contribute to the pathogenesis of bd predominantly as t-helper cells [ , , ] . nevertheless, a potential lytic role of virus-specific cd ϩ t-cells cannot be completely excluded [ ] . in particular, astrocytes may express mhc class ii antigens [ , ] , thus being potential targets for cd ϩ t-cells. cd ϩ t-cells have been shown to play a role in measles-and coronavirus-induced encephalomyelitis [ , ] . however, these cases are considered as examples of virus-induced autoimmunity, illustrated by the ability of these cells to trigger an inflammatory reaction in the cns of uninfected animals. on the other hand, there is ample evidence that cd ϩ t-cells act as effector cells in the immune-mediated pathological reaction observed in bd. elimination, or functional blocking, of cd ϩ t-cells prevents both bdv-induced neurological symptoms and histopathological changes in the brain [ , , , , ] . in addition, lymphocytes isolated from rat brains during acute bd exhibit mhc class i-restricted cytotoxic t-lymphocyte (ctl) activity in vitro [ ] . adoptive transfer of these lymphocytes into immunosuppressed bdv-infected recipients results in the extravasation of cd ϩ t-cells in the brain parenchyma, leading to immunopathological and clinical signs similar to experimental bd [ ] . therefore, neuronal damage seen in bd appears to be mediated by the cytotoxic activity of cd ϩ t-cells present in the brain parenchyma of bdv-infected rats. cd ϩ ctl can destroy cells presenting virus-derived peptides in the context of mhc class i presentation [ ] . evidence suggests that mhc class i antigens are expressed at the surface of astrocytes and neurons in the cns of bdv-infected rats, concomitantly with the peak of cd ϩ t-cell infiltrate in the cns and the onset of neuronal degeneration [ , , ] . under normal conditions, cells of the cns express very low, if any, mhc antigens, but their expression can be upregulated during pathological situations [ , ] . it has been proposed that virally-infected neurons avoid destruction by ctl because of the lack of mhc class i expression [ , ] . however, synaptically silent neurons may express mhc class i molecules [ ] . bdv is one of the rare cases in which it has been shown that a viral infection of the cns can induce neuronal expression of mhc class i in vivo [ , , , , ] . the molecular mechanisms responsible for bdv-induced mhc class i expression by neurons are presently unknown. it is possible that bdv infection of neurons directly causes upregulation of mhc class i expression. alternatively, induction of neuronal mhc class i expression may be due to the action of cytokines secreted by inflammatory cells or virally-infected glial cells [ , , ] . certain cytokines, including interferon (ifn) ␥ or ␤ and tumor necrosis factor ␣ (tnf␣) can upregulate mhc class i expression on neurons in vitro and to some extent in vivo [ , ] . evidence suggests that bdv-infected astrocytes could secrete ifn ␣/␤, which, in turn, could induce mhc class i expression in neurons [ ] . neurons in the cns are terminally differentiated cells without the capacity of proliferation. consequently, destruction of virallyinfected neurons by the immune response can cause an irreversible loss of cells whose functions are required for normal brain performance. the existence of the blood-brain barrier significantly restricts the degree of immunological surveillance and the vigor of the immune response in the cns. however, activated virus-specific ctl can access the brain parenchyma. bdv-mediated induction of mhc class i expression on neurons can then be detrimental for the host by allowing destruction by ctl. furthermore, this bdv-specific ctl activity is unable to control the infection and viral load is not significantly decreased after regression of the immune response. thus, the immune-mediated damage is likely to gonzalez-dunia, sauder and de la torre have more devastating consequences than those directly due to the mere bdv persistence in neurons. role of cytokines in bdv pathogenesis. cytokines and free radicals have recently gained attention as other mediators of immunopathology. there is increasing evidence that certain stimuli, including viral and bacterial infections, can upregulate cytokine expression in the cns, resulting in tissue damage and neurological disease [ , , , ] . along with activated lymphocytes and macrophages present in immune infiltrates, resident cns cells like neurons, astrocytes, and microglia are potential producers of cytokines [ ] . proinflammatory cytokines including il- ␣, tnf␣, and il- can cause neuronal damage [ , ] . levels of il- , tnf␣, and il- ␣ mrnas are significantly increased in bd rat brains and their expression correlates with the degree of inflammation and severity of neurological signs [ ] . moreover, production of ifn ␥ by infiltrating cd ϩ t-cells could induce mhc class ii expression on astrocytes and microglia. this, in turn, would enhance cd ϩ t-cell activity and contribute to the immunopathology of acute bd. it has also been proposed that the decline of inflammation during the chronic phase of bd might be mediated by cytokines such as ifn ␥ [ ] . free radicals such as nitric oxide, generated by the inducible nitric oxide synthase (inos), can also cause direct neuronal damage [ , ] . increased cns expression of inos mrna is observed in rats with bd. levels of inos mrna correlate with the severity of neurological signs and degree of inflammatory lesions [ , ] . the finding of up to -fold increased amounts of nitric oxide in bdv-infected rat brains suggests a possible contribution of nitric oxide to bd pathogenesis [ ] . however, it is also possible that the inos mrna expression in the cns merely reflects a nonspecific activation of microglia, thus being more of a secondary than a primary pathogenic importance. bdv is considered as a noncytolytic virus in vitro, because infected cultured cells do not exhibit impaired growth or survival [ ] . analysis of the situation in animals with bd is complicated by the massive inflammatory response accompanying the infection. thus, it is difficult to distinguish cell and tissue damage directly caused by bdv replication from the damage due to the antiviral immune response. the pti-nb rat model (see above) has provided a system to study virus-induced structural and functional cns alterations without inflammation [ ] . studies on pti-nb rats have suggested that bdv can induce a selective damage on specific neuronal populations [ ] . however, this restricted neuronal degeneration is not considered as a ''classical'' cytopathic effect, but is rather due to an interference of bdv infection with the postnatal development of cns areas that experience extensive postnatal maturation. one of the most noticeable morphological features of the pti-nb rat brain is the considerable cerebellar hypoplasia induced by bdv perinatal infection. also, the dentate gyrus in the hippocampus undergoes progressive degeneration. these traits are accompanied by a prominent and widespread astrocytosis [ ] , defined as an increase in the number and size of cells expressing the glial fibrillary acidic protein (gfap), an astrocyte-specific marker [ ] (see also the increased levels of gfap mrna expression in pti-nb rats shown in fig. ). other perinatal virus infections, including lymphocytic choriomeningitis virus (lcmv), rat parvovirus and reovirus type iii, also induce damage to the cerebellum [ , , ] . the cerebellum developmental arrest has been attributed in these cases to either an immune-mediated, or a virus-induced lysis of the dividing imma-ture cerebellar granule cells. the characteristics of bdv-induced cerebellar hypoplasia have been detailed in a recent report [ ] . the arrest of cerebellar development occurs between and days postinfection. this coincides with a progressive atrophy of the granule cell layers. in contrast to the other viral systems mentioned above, the granule cells are not infected by bdv. purkinje cells are the predominant, if not the only, cell type infected in the cerebellum at the early stages of its development [ ] . therefore, it appears that bdv-induced cerebellar damage is caused by different mechanisms than those proposed with other perinatal virus infections. evidence indicates that purkinje cells play a main role in supporting the multiplication, maturation, and migration of the granular cells [ ] . this role could be altered by bdv infection. the fig. . tissue factor (tf) messenger rna levels are markedly increased in the cns of bdv persistently infected (pti-nb) rats. total rna was isolated from brains of pti-nb rats (lanes to ) and sham infected control rats (lanes and ), and analyzed by northern blot hybridization for the expression of tf and glial fibrillary acidic protein (gfap). viral rna was detected in bdv-infected rats using a bvp cdna probe. levels of tf mrna were normalized to those of the housekeeping gene glyceraldehyde -phosphate dehydrogenase (gapdh). see also [ ] . massive astrocytosis observed in pti-nb rats might also be involved in the cessation of granule cell migration, especially given the key role of astrocytes in providing the physical track upon which the granule cells migrate [ ] , as well as neurotrophic support to surrounding cells [ ] . the hippocampus consistently harbors the highest viral load in pti-nb rat brains [ , , ] . despite considerable replication of bdv in this area, there is little effect on the lifespan of the infected neurons. one notable exception is the dentate gyrus (dg), whose neurons are progressively destroyed [ ] . the dg is formed primarily during the postnatal period. although birth of cells in the dg begins embryonically, the vast majority (Ͼ %) of dg granule neurons are generated after birth [ ] . hence, neuronal multiplication and migration will occur in this area well into adulthood. this represents a unique characteristic for neurons of the mammalian adult brain, that are usually considered as a whole as postmitotically differentiated cells [ ] . dentate gyrus degeneration has also been observed following persistent neonatal infection with lcmv [ ] . it has been shown that degeneration of the dentate gyrus and abnormalities in synaptic function occur after lcmv clearance from the granule cells. electrophysiological measurements demonstrated that these cells exhibit a persistent hyperexcitability. this was due to a decreased inhibition, usually mediated by a subpopulation of gaba interneurons that were affected by the infection [ ] . the mechanisms underlying bdv-induced dg degeneration are presently unknown. given the noncytolytic effect of bdv on other neuronal populations, one could hypothesize that bdvmediated degeneration of dg granule neurons is likely due to either (a) the unique characteristics of these mitotically active neurons, or (b) the disruption of pathway(s) essential for dg maturation [ ] . these questions warrant further investigations, especially considering the importance of the dg and hippocampus in learning and memory. in this respect, bdv represents an important model to investigate the pathogenesis of cns disease linked to abnormal cns development and to better understand the regulation of cns plasticity. the effector mechanisms involved in bdv-induced neuronal damage are still poorly understood. however, bdv infection is not likely to cause direct neuronal destruction but triggers instead a series of secondary events leading to cell damage within the cns. most of the cns alterations induced by bdv infection of immunocompetent adult rats can be attributed to the virus-induced cellular immune response. however, bdv-mediated impairment of brain activity can also occur without the hallmarks of inflammation and widespread cytolysis, owing to the virus' ability to cause changes in neuronal organization and to interfere with brain cell functions required to maintain cns homeostasis. the pti-nb rat offers a unique system to study the mechanisms underlying these virus-cns cell interactions. astrocyte functions are essential to neurons [ ] . it is, therefore important to investigate the consequences on brain function associated with the prominent astrocytosis that characterizes pti-nb rat brains. we have already mentioned the key role of astrocytes in providing a substrate for neuronal migration during brain development. the astrocytic network participates in brain information processing in cooperation with neuronal networks [ ] . astrocytes also play roles in the elimination of neurotoxins and in the production of various cytokines, which can act as neuronal differentiation factors [ ] . cytokines can contribute to the regulation of neuronal neurotransmitter gene expression and, hence, can dramatically alter synaptic function [ , ] . these changes can, in turn, contribute to complex animal behavior as well as to plasticity processes accompanying learning and memory. expression of high levels of proinflammatory cytokines [ ] and of complement cascade components [ ] have been documented in the rat cns during acute bd (see above). these molecules were likely to have been produced by inflammatory cells invading the brain parenchyma. therefore, it was difficult to assess the intrinsic response of resident cns cells. to separate these two events, morimoto et al. used dexamethasone treatment of rats after bdv infection [ ] . this synthetic glucocorticoid effectively prevented inflammation in the cns of bdv-infected rats. however, several proinflammatory cytokines such as tnf␣ and il- were still induced in the brains of dexamethasone-treated rats, suggesting that these might have been produced by cns resident cells. however, dexamethasone treatment of rats per se may have a profound action on the cytokine expression pattern of cns cells [ ] . studies on pti-nb rats may provide valuable information regarding the contribution of cns resident cells to disturbances in cytokine gene expression caused by bdv. perinatal bdv infection of the rat provides a system to dissect the elements involved in bdv-induced alterations of cns homeostasis without inflammation. so far, very few studies have addressed such questions in this model. recently, a bdv-induced upregulation of tissue factor (tf) was demonstrated in the cns of pti-nb rats [ ] . tf, a transmembrane receptor, is the primary initiator of the coagulation protease cascade that results in the generation of the protease thrombin [ ] . besides, tf appears to have pleiotropic roles and has been implicated in signal transduction, angiogenesis and brain function [ , , , ] . in the cns, tf is primarily, if not solely, produced by astrocytes [ ] . tf expression is markedly increased during persistent infection with bdv, both in vivo and in vitro [ ] (fig. ) . this upregulation is due to bdv infection of astrocytes, and is mediated by an increased transcription of the tf gene and a stabilization of tf mrna. the significance of this finding is still a matter of speculation. however, because this phenomenon occurs without hemorrhage, inflammation, or disruption of the blood-brain barrier, it raises interesting questions about the consequences of tf upregulation and suggests that tf may fulfill functions other than hemostasis in the cns. there is increasing evidence that proteins of the coagulation and fibrinolysis systems may function in the cns independent from blood clotting, such as regulating normal brain development and defending the brain against damage caused by stroke, trauma, and other injuries [ ] . any imbalance in the protease activities may contribute to neuronal damage. tf is the primary initiator of the thrombin activation system, a protease that has been implicated in several neurodegenerative diseases, such as alzheimer's disease [ , ] . interestingly, thrombin is predominantly expressed by dopaminergic neurons of the mesencephalon [ ] . therefore, this may suggest a link between tf upregulation and the dopaminergic abnormalities observed in bd rats (see section . ). these findings suggest that upregulation of tf expression due to bdv infection may play an important role in the cns response to this viral assault and perhaps also in the bdvmediated disturbances of cns function. whether cns expression of other genes, including cytokines, is specifically altered by bdv infection remains to be established. behavioral abnormalities caused by bdv infection (table ) were first described in naturally infected horses and sheep [ ] . the acute phase of disease is characterized by movement disorders, accompanied by excitability and hyperactivity. the later stages of the disease are frequently manifested by somnolence, gonzalez-dunia, sauder and de la torre depression, apathy, and anorexia. mortality rates are as high as - % in horses and % in sheep. animals that survive remain chronically infected and recurrent episodes with exacerbation of behavioral alterations may occur [ ] . both host and viral factors influence significantly the behavioral abnormalities associated with bdv infection. for example, hamsters and black-hooded rats do not develop apparent behavioral or neurological symptoms, despite the presence of virus in the cns [ , ] . strain specific differences in the susceptibility to disease have also been observed in mice. thus, although bdv induces encephalitis to a similar degree in mrl/ϩ and mrl/lpr mice, only mrl/ϩ mice develop behavioral abnormalities [ ] . this dissociation between inflammation and symptoms indicating the onset of the disease may reflect differences in the composition and immunological properties of the cellular infiltrate [ ] . the host genetic background likely contributes to these differences. alternatively, host factors could also modulate the vulnerability of brain cells to the inflammatory reaction [ ] . bdv-infected tree shrews illustrate important aspects of the consequences that bdv persistent infection may have in the behavior of highly developed animals [ ] . tree shrews (tupaia glis) are phylogenetically classified at the root of the primates and exhibit a complex behavioral repertoire [ ] . tree shrews inoculated with bdv develop a persistent infection that is frequently associated with behavioral disorders. interestingly, housing conditions have a strong influence on the behavioral manifestations after infection. only a small number of animals kept in isolation show behavioral abnormalities, whereas all paired animals display profound sociosexual behavioral disturbances. these include a tendency to accept partners more quickly, a need for increased body contact, as well as a reversal in the role of the sex partners and an impaired breeding behavior of the females. some animals kept in isolation also show neurological symptoms (table ). in these cases, an initial hyperactive phase, that coincides with the peak of the neurological symptoms, is followed by a phase of decline. all these animals recover, but relapse of clinical symptoms can occur in some cases. it is unknown why clinical symptoms are observed only in some animals, especially because all have comparable viral load in the brain and similar titers of serum antibodies to bdv [ ] . the behavioral alterations exhibited by bdv-infected tree shrews are controlled by the limbic system and can be summarized as a disinhibition toward the environment [ ] . interestingly, evidence indicates that virus-specific lesions in neurons of the limbic system represent a hallmark of bdv infection. behavioral disturbances associated with bdv infection have been thoroughly studied in the rat model. intracerebral inoculation of the adult immunocompetent rat causes a persistent infection that is associated with the previously described immune-mediated biphasic neurobehavioral disease (see section . . ). bd rats develop a progressive movement and behavior disorder, accompanied by hyperactivity. several studies have focused on the pharmacology and neurochemistry of this hyperactive syndrome [ , , ] . based on similarities with the behavioral disturbances caused in rats by neurotoxic or electrolytic lesions in the frontal cortex or its catecholamine afferents [ ] , it has been proposed that bdv-induced disturbances might be due to alterations of the dopamine system. dopaminergic activity is enhanced in the prefrontal cortex of bd rats, revealed by high levels of , -dihydrophenylacetic acid, the primary dopamine metabolite. yet, dopamine receptor density is not affected in the prefrontal cortex [ ] . analysis of dopamine receptor density in rostral striatal areas, such as the nucleus accumbens, revealed a loss of dopamine reuptake sites correlated with decreased numbers of d and d receptors binding sites [ ] . the nucleus accumbens is a primary site of integration between limbic and motor information and has been involved in the control of motor, cognitive, emotional, and reward processes [ ] . thus, abnormalities in the mesocorticolimbic dopaminergic network may constitute the neural substrate of hyperactivity in bd [ ] . furthermore, similarities between these alterations and those observed in manic depressive or schizophrenic patients [ , , ] may be an important pathophysiologic feature linking the bd rat model to human psychiatric diseases. besides dopamine, alterations in other neurotransmitter systems have also been described in bd rats and include changes in mrna levels of cholecystokinin, glutamic acid decarboxylase, and somatostatin [ ] . the behavioral abnormalities observed in bd rats are likely to be a consequence of the inflammatory response. in particular, the high levels of cytokines produced by the immune infiltrates during the acute phase of disease could cause behavioral changes, including hyperactivity and aggressiveness [ , ] . the correlation of cytokine levels in bd rat brains with the degree of inflammatory reaction and severity of neurological symptoms supports this hypothesis. it should be reminded that the hippocampus frequently harbors a high viral load [ , , ] , thus becoming a primary target for the virus-specific inflammatory reaction. this brain structure plays essential roles in governing emotion, learning and memory [ ] . therefore, cytokine-mediated disturbances of hippocampal functions can cause severe behavioral abnormalities [ , ] . the later phase of bd is characterized by the decline of the inflammatory reaction and by symptoms like apathy and somnolence. this likely reflects the neuronal damage caused by the immune response. in contrast to bdv-infected immunocompetent adult rats, pti-nb rats develop a chronic infection with no overt signs of classic bd. however, pti-nb rats exhibit distinct cognitive, behavioral and physiological abnormalities (table ) [ , ] . pti-nb rats show impaired cognitive functions in different learning paradigms, including spatial discrimination and discriminated avoidance tasks [ , ] . results from open field and neophobia tests revealed emotional disturbances in pti-nb rats, manifested as a reduced resting behavior and decreased anxiety. moreover, these animals displayed hyperactivity when placed in a novel environment [ ] . reduced body weight and length were also observed in pti-nb rats, compared to age-and sex-matched uninfected controls. impaired growth did not seem to be related to disturbances in growth hormone physiology, because levels of growth hormone, insulin growth factor type , and glucose were unchanged in pti-nb rats [ ] . these differences in growth appeared only after weaning, suggesting that the feeding behavior might have been affected by perinatal bdv infection. in addition, pti-nb rats exhibited an altered preference for a saline solution when it was paired with either water, saccharin, or quinine solutions [ ] . the mechanisms underlying this altered salt taste preference are unknown. disturbances in the postnatal maturation of the cerebellum and hippocampus induced by bdv, which have been reviewed above, probably contribute to the neurobehavioral abnormalities observed in these animals. clinical, epidemiological, and virological data indicate that viruses can persist in the cns and induce progressive neurological disorders, which are associated with diverse pathological manifestations. these include alterations in behavior and cognition [ , , ] . these findings have led to the hypothesis that viruses can contribute to neuropsychiatric disorders whose etiology remains unknown [ ] . the wide host range of bdv, to-gether with the observation that bdv-infected animals exhibit behavioral disturbances resembling some human mental disorders, prompted studies aimed at determining an association of bdv with these disorders. seroepidemiological studies. seroepidemiological studies conducted in different laboratories over the last years have consistently shown an increased bdv seroprevalence in neuropsychiatric patients (table ) . however, considerable variations in prevalence rates have been described, ranging from . to % in psychiatric patients and to . % in controls (table ) . moreover, follow-up studies on psychiatric patients revealed a high bdv seroprevalence ( . %), reaching up to % among patients with major depression [ ] . increased bdv seroprevalence has also been reported in immunosuppressed hiv-infected patients from europe and thailand [ , ] , individuals with parasitic infections in africa [ ] , as well as patients with neurological disorders [ ] . these serological findings should be cautiously evaluated because of the following reasons: (a) in many cases, only limited information was provided about the composition of the subject group analyzed. thus, factors such as the geographic area, the heterogeneity of diagnoses, and clinical status of patients may have significantly influenced these results. for example, a slightly higher percentage of seropositives is found in patients' sera derived from areas in southern germany, where bdv is endemic in animals [ , , ] (table ) ; (b) differences in the experimental procedures used to detect antibodies to bdv in human sera may also have affected the results. in particular, the immunofluorescence assay used in many of these studies [ ] [ ] [ ] is considered to be unreliable [ , ] . the use of immunofluorescence assays for bdv serology poses difficulties because of the very low bdv antibody titers in human sera [ ] . furthermore, sera from neuropsychiatric patients often contain autoantibodies that react with nuclear structures [ , , ] . thus, the nuclear localization of bdv antigens in infected cells makes necessary the use of appropriate controls to discriminate between bdv-specific staining and nuclear staining due to autoantibodies. increased specificity and sensitivity are now provided by western blot assays that use either bdv-infected cell extracts [ , ] , or recombinantly expressed bdv antigens [ , ] . interestingly, results from studies conducted using this improved serologic test have still shown a significantly higher seroprevalence in neuropsychiatric patients ( . - %) compared to nonpsychiatric control groups ( - . %) [ , , , ] . detection of bdv antigens and rna in human peripheral blood mononuclear cells. the cloning and molecular characterization of bdv [ , ] has allowed the development of specific and sensitive procedures for the detection of bdv rna in biological samples. using reverse transcriptase-pcr (rt-pcr) procedures, bdv rna can be detected in the peripheral blood mononuclear cells (pbmc) of infected rats [ , ] . this finding led to the use of a similar approach to examine the prevalence of bdv in humans. thus, bdv rna was first detected in the pbmc from four out of six hospitalized psychiatric patients (inpatients) [ ] . this initial study has been followed by more comprehensive molecular epidemiological investigations. bdv rna prevalence of % ( of ) [ ] , or . % ( of ) [ ] , has been documented in pbmc of japanese neuropsychiatric inpatients. studies on japanese healthy blood donors revealed a viral prevalence of . % ( of ) [ ] and % ( of ) [ ] . studies on german neuropsychiatric patients revealed a bdv rna prevalence in pbmc of % ( of ), whereas % ( of ) of normal controls were positive for viral rna [ ] . these studies have also shown that patients harboring detectable levels of viral rna in their pbmc are frequently bdv seronegative; conversely, bdv-seropositive individuals have frequently nondetectable levels of viral rna [ , , ] . recently, a high bdv seroprevalence and viral rna in pbmc have been detected in blood samples from japanese patients diagnosed with chronic fatigue syndrome (cfs) [ , ] . bdv antibodies were detected in . % ( of ) cfs patients, but only in three cases of healthy controls. bdv rna was detected in % ( of ) cfs cases and . % ( of ) control cases. patients with cfs frequently exhibit neuropsychiatric symptoms, including severe depression. the etiology of cfs is unknown, but evidence suggests the involvement of viral infections [ ] . thus, these preliminary and intriguing results deserve further investigation. the number and nature of the infected cells in pbmc are still controversial. in persistently infected rats, the prevalence of bdvinfected cells in pbmc has been estimated at per ϫ cells [ ] . the need of highly sensitive rt-pcr procedures to detect bdv rna in pbmc from humans or bdv-infected animals indicates an extremely low viral load in pbmc. however, some investigators have reported that to % of the monocytes of bdv-positive patients express bdv antigens [ , ] . this would suggest that about % of the pbmc are infected with bdv. in this case, even assuming a very low viral load per cell, detection of bdv rna should not require the use of nested rt-pcr. the reasons for this apparent discrepancy remain to be determined. data from the rat model suggest that bdv is present in a very small fraction of circulating stromal cell precursors, rather than in the monocytes [ ] . pbmc could have become infected during passage through the brain, or they may represent primary targets with the ability to spread the infection into the cns, a situation that has been proposed for other neurotropic viruses [ , ] . intravenous injection of rats with cell-free bdv is not followed by the establishment of a persistent infection [ ] . this argues against cells within the pbmc being primary targets for bdv. sequence analysis revealed a high degree of conservation of both inter-and intrapatient bdv sequences, as well as a close genetic relationship between human and animal-derived bdv sequences [ , , ] . this finding is consistent with the remarkable genetic stability of bdv in animals, which has been dealt with in a previous section. however, a higher sequence variability has been reported in human bdv sequences derived from japanese patients' pbmc [ ] . this might reflect strain differences due to geographical location. nevertheless, this higher variability may have been overestimated by the experimental procedures used to determine these sequences (see discussion in [ ] ). the high genetic stability of bdv is an uncommon finding for an rna virus. because the mutation frequencies of rna viruses exceed by more than a million fold those of their eukaryotic hosts, extremely rapid virus evolution is anticipated and frequently observed [ ] . however, rna viruses can also display long-term stasis both in nature and in laboratory conditions, as a result of selection for fit master sequences in rather constant environments [ , ] . the high sensitivity of nested rt-pcr allows the detection of very low levels of target sequences. this method is also prone to artifacts due to inadvertent contaminations with laboratory sources of bdv. moreover, the high degree of bdv sequence conservation proscribes the use of sequence analysis to pinpoint cases of contamination. these concerns can be addressed by following well-established guidelines to avoid contamination problems during rt-pcr assays [ , ] . nevertheless, the low level of viral rna in pbmc, close to the sensitivity threshold of nested rt-pcr, can hamper the reproducibility of results between laborato- gonzalez-dunia, sauder and de la torre ries [ ] . it is worth noting that although the detection of bdv rna in pbmc provides a valuable diagnostic tool, it does not necessarily reflect the viral load in the cns. for example, pti-nb rats harbor extremely low levels of bdv rna in their pbmc, whereas high levels of viral antigen and rna are expressed in the cns [ , ] . isolation of human bdv. recently, bdv has been isolated from pbmc of three german psychiatric inpatients with affective disorders [ ] . the isolation was achieved by cocultivation of pbmc with a human oligodendroglia cell line, followed by serial passages of these cells. rescue of human bdv required the use of pbmc samples with strong viral antigen expression and at least passages of the initial cocultivation [ ] . these requirements to achieve virus isolation may explain why initial attempts to isolate bdv from humans had been unsuccessful [ ] . sequence analysis of the three human bdv isolates revealed a very close relationship with known bdv animal strains [ ] . this high sequence conservation, together with the number of passages necessary to isolate the virus, has raised concerns about possible contamination with laboratory strains and stresses the need of further studies to confirm these findings. detection of bdv antigens and rna in the human brain. given the hypothesis that bdv contributes to human mental disorders, it should be expected that bdv also infects the human cns. some investigators have used rt-pcr to search for bdv sequences in autopsy brain material from cases of schizophrenia and affective disorders [ , ] . these initial studies failed to detect bdvpositive samples. however, a recent study has documented the detection of bdv antigen and rna in human autopsy brain samples [ ] (fig. ) . the initial selection of the cases analyzed in this study was based on histopathological features resembling those observed in chronically bdv-infected animals. in particular, damage to the hippocampus and astrocytosis were used as screening criteria. among autopsy cases examined, five were found with sclerosis of the hippocampus and astrocytosis as the main histopathological findings. these patients presented with a clinical history of mental disorders manifested as severe depression and memory loss. in four of these five patients, immunohistochemistry and in situ hybridization revealed the presence of viral antigen and rna in neurons and astrocytes within the hippocampal formation. in contrast, bdv markers were not found in control cases [ ] . detection of viral rna required the use of nested rt-pcr, suggesting that failure to detect bdv rna in previous studies, using single round rt-pcr, might have been related to lack of sensitivity. these findings demonstrate that bdv can persist in the human cns, providing the basis for more comprehensive studies aimed at determining whether bdv persistence in the cns is associated with specific human mental disorders. bdv rna has also been detected recently in clinical samples of brain tumors [ ] . viral rna was detected in of specimens with grade glioblastomas, but in none of lower grade astrocytoma specimens. patients with malignant brain tumors, especially glioblastoma multiforme, are frequently severely immunosuppressed [ ] . these findings provide additional support to a possible relationship between immunosuppression and levels of bdv expression. further studies are required to clarify this issue. a recent report [ b] has described the detection of bdv rna in postmortem brain samples from individuals with mental disorders. this study reported the detection of bdv p gene transcripts in out of patients with schizophrenia and out of patients with bipolar disorder. the data discussed above provide strong evidence that bdv can infect humans and persist in the cns. the establishment of an association between bdv and certain neuropsychiatric disorders awaits confirmation from more comprehensive molecular epidemiological studies. however, it should be emphasized that finding such an association does not prove the contribution of bdv to the pathophysiology of these mental disorders. in addition, bdv has been associated with both schizophrenia and affective disorders, raising the question of how the same infectious agent could be involved in two different mental disorders. schizophrenia and affective disorders are chronic and complex cns diseases, manifested by multiple signs and symptoms that tend to recur, often in a cyclical fashion [ , ] . epidemiological data indicate that environmental factors, such as stress, contribute to these disorders [ , , , , , ] . there is compelling evidence that viral infection of the brain represents an important risk factor [ ] . moreover, findings in bdv-infected animals suggest that the biology of bdv is compatible with its potential role in these mental disorders [ ] . bdv-infected animals display a diverse range of symptoms, including neurobehavioral alterations with cyclic episodes. the remarkable heterogeneity in disease phenotype associated with bdv infection depends on both host and viral factors, as well as other exogenous factors. thus, stress can contribute to recurrent disease episodes in bdv chronically infected animals [ , ] . neurostructural abnormalities have been associated with schizophrenia and affective disorders. these affect several brain regions but are predominantly localized to medial temporal lobe structures. however, functional alterations suggest that the disease process may also affect other brain areas, involve integrative sensory function and motor coordination [ ] . abnormalities within the hippocampal formation are frequently observed in these disorders [ , , ] . studies using high resolution volumetric magnetic resonance imaging have shown that affective disorders are associated with structural changes in the hippocampus [ ] . it has been suggested that the long-term overexpression of glucocorticoids, frequently seen in patients with major depression, could cause degeneration of hippocampal neurons, which express high levels of glucocorticoids receptors [ ] . in the case of schizophrenia, hippocampal alterations are thought to reflect an altered process of cell migration during brain development, possibly triggered by an early viral infection [ ] . bdv has a predominant tropism for limbic system structures [ , , ] and perinatal infection causes hippocampal damage. interestingly, magnetic resonance imaging studies have suggested that cerebral abnormalities occur more frequently in bdv-seropositive psychiatric patients [ , ] . moreover, pti-nb rats display cerebellar hypoplasia. the cerebellum is linked with limbic structures through multisyn- were analyzed for the expression of bvp antigen and glial fibrillary acidic protein (gfap). both hs and ad cases exhibited a prominent astrocytosis as determined by gfap expression. bvp antigen was detected in the hs case but not in the ad case. see also [ ] . scale bar: m. gonzalez-dunia, sauder and de la torre aptic circuits, which have been implicated in mood regulation [ ] . there is also evidence of a decreased size of the cerebellum in patients with affective disorders [ , , ] . altered neurotransmitter functions affecting mainly catecholamines and especially dopaminergic systems have been implicated in the pathophysiology of human mental diseases [ , , ] . this altered neurotransmission activity is thought to affect the regulation of limbic system functions, which, in turn, can lead to the various endocrine disturbances observed in these disorders [ , ] . bdv infection causes distinct cns dopamine disturbances [ , ] . bdv can also affect other neurotransmitter systems [ ] , or the expression of neurotrophic factors required to maintain brain homeostasis. it is worth noting that bdv infection induces a prominent and chronic astrocytosis that was also observed in the autopsy brain samples from human cases identified as positive for bdv antigen and rna [ ] . bdvinduced disturbances in astrocyte functions may contribute to neuronal dysfunction by affecting complex interactions within neural networks (see section . ). major human mental disorders, including schizophrenia and affective disorders, likely involve a concert of genes with varying penetrance among individuals that interact with exogenous factors to generate a variety of clinical phenotypes [ ] . bdv can represent an exogenous factor contributing to these disorders. viral variants, even single amino acid substitutions, can exhibit remarkable differences in their expression pattern in cells and tissues of the infected host, that are associated with diverse disease phenotypes [ , ] . similarly, genetic differences among individuals play a critical role in the outcome of a viral infection [ ] . thus, it is conceivable that depending on the individual's genetic vulnerability and the properties of specific viral variants, bdv infection could contribute to distinct types of mental disorders. the source of bdv and routes for human infection are also germane to the role of bdv in neuropsychiatric disorders. evidence suggests that the nose is a main site of virus entry in animals [ ] . sporadic evidence has suggested the possibility of transmission from bdv-infected animals to humans [ , ] . the close genetic relationship between bdv sequences derived from humans and animal strains provides support for this hypothesis. in this regard, it is of interest that domestic cats are susceptible to bdv, representing potential carriers of bdv [ , ] . the detection of bdv rna in a significant percentage ( . to %) of blood donors' pbmc raises the concern of a possible transmission by blood transfusion [ ] . as with other neurotropic viruses, a small number of bdv-infected cells present in a blood sample could reach the brain and establish a persistent infection within the cns. depending on the genetic susceptibility of the host and on viral factors, this persistent infection may have diverse clinical consequences, manifested after a variable period of incubation. one should keep in mind that stress, a common finding in psychiatric patients, can induce immunosuppression by altering glucocorticoids levels [ ] . this, in turn, could enhance susceptibility to infectious agents, including bdv. the molecular analysis of human biological samples to detect bdv poses considerable technical difficulties. comprehensive molecular epidemiological studies aimed at determining the prevalence of bdv in neuropsychiatric patients from different geographic areas will help to evaluate the significance of bdv as a potential human pathogen. these studies will require the use of standardized methods for the detection of viral antibodies and rna sequences in human biological samples, as well as uniform criteria for the clinical diagnoses. 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and further information about the genome detection of borna disease virus rna in naturally infected animals by a nested polymerase chain reaction mhc-restricted cytotoxic t cells: studies on the biological role of polymorphic major transplantation antigens determining t-cell restriction-specificity, function, and responsiveness handbuch der viruskrankheiten ii we wish to especially acknowledge beatrice cubitt for critical discussions and for her long standing expert assistance. this work was supported by nih grant ns - . dgd is supported by the institut pasteur and cs is a fellow of the german stipendienprogramm infektionsforschung, dkfz, heidelberg. this is publication -np from the scripps research institute. all authors contributed equally to this review. key: cord- -cb u s s authors: bedford, juliet; farrar, jeremy; ihekweazu, chikwe; kang, gagandeep; koopmans, marion; nkengasong, john title: a new twenty-first century science for effective epidemic response date: - - journal: nature doi: . /s - - -y sha: doc_id: cord_uid: cb u s s with rapidly changing ecology, urbanization, climate change, increased travel and fragile public health systems, epidemics will become more frequent, more complex and harder to prevent and contain. here we argue that our concept of epidemics must evolve from crisis response during discrete outbreaks to an integrated cycle of preparation, response and recovery. this is an opportunity to combine knowledge and skills from all over the world—especially at-risk and affected communities. many disciplines need to be integrated, including not only epidemiology but also social sciences, research and development, diplomacy, logistics and crisis management. this requires a new approach to training tomorrow’s leaders in epidemic prevention and response. when nature published its first issue in , a new understanding of infectious diseases was taking shape. the work of william farr , ignaz semmelweis , louis-rené villermé and others had been published; john snow had traced the source of a cholera epidemic in london (although robert koch had not yet isolated the bacterium that caused it ). the science of epidemiology has described patterns of disease in human populations, investigated the causes of those diseases, evaluated attempts to control them and has been the foundation for public health responses to epidemic infections for over years. despite great technological progress and expansion of the field, the theories and practices of infectious disease epidemiology are struggling to keep pace with the transitional nature of epidemics in the twenty-first century and the breadth of skills needed to respond to them. epidemiological transition theory has focused mostly on the effects of demographic and socioeconomic transitions on well-known preventable infections and a shift from infectious diseases to non-communicable diseases . however, it has become clear that current demographic transitions-driven by population growth, rapid urbanization, deforestation, globalization of travel and trade, climate change and political instability-also have fundamental effects on the dynamics of infectious diseases that are more difficult to predict. the vulnerability of populations to outbreaks of zoonotic diseases such as ebola, middle east respiratory syndrome (mers) and nipah has increased, the rise and spread of drug-resistant infections, marked shifts in the ecology of known vectors (for example, the expanding range of aedes mosquitoes) and massive amplification of transmission through globally connected, high-density urban areas (particularly relevant to ebola, dengue, influenza and severe acute respiratory syndrome-related coronavirus sars-cov). these factors and effects combine and interact, fuelling more-complex epidemics. although rare compared to those diseases that cause the majority of the burden on population health, the nature of such epidemics disrupts health systems, amplifies mistrust among communities and creates high and long-lasting socioeconomic effects, especially in low-and middle-income countries. their increasing frequency demands attention. as the executive director of the health emergencies program at the world health organization (who) has said: "we are entering a very new phase of high-impact epidemics… this is a new normal, i don't expect the frequency of these events to reduce." . we have to act now but act differently: a broader foundation is required, enhancing traditional epidemiology and public health responses with knowledge and skills from a number of areas ( table ) . many of these areas have long been associated with epidemic preparedness and response, but they must now stop being seen as esoteric 'nice things to have', and instead become fully integrated into the critical planning and response to epidemics. this will require considerable changes by the global public health community in the way that we respond to epidemics today and how we prepare for and seek to prevent those of tomorrow. it will mean reshaping the global health architecture of the response to epidemics and transforming how we train new generations of researchers and practitioners for the epidemics of the future . the modern research culture-often shaped by the behaviour of funders-has required many researchers to specialize in narrow fields, with less emphasis on translation than on field-specific innovations. although this siloed landscape has brought major advances in global health, it is not fit for the transitional phase of epidemic diseases: rapidly evolving, high-impact events bring together communities, responders and researchers who do not routinely interact. different assumptions, cultures and practices, each of which may be widely accepted within a particular community, make working together in outbreak situations more challenging. fundamental to success is respect and understanding of the contribution each party brings. in a successfully integrated approach, we each have to realize that our knowledge and skills are a small part of a rapidly expanding toolkit (box ). we need to understand major trends in research and how and when they may influence the response to an epidemic, develop new research to strengthen the support that we can provide across other areas and learn to operate in multi-stakeholder situations-including, at times, as part of a critical debate to bring better practices to the fore. central to this approach must be the communities who are at risk and those affected by epidemics: local people are the first responders to any outbreak and their involvement in the preparation and response activities is essential. from communities, through local and regional health authorities, national public health institutes and international organizations-including many essential partners in sectors beyond public health-the integrated approach must be supported. the who, in particular, has a critical part to play, using its unique mandate not to lead every aspect of preparation, response and recovery, but to change its practices, facilitate integration with and among others, and ensure accountabilities are built in from the bottom to the top. a wave of cholera epidemics across europe in the s and s catalysed a new era of 'infectious disease diplomacy' globally. nations recognized that infections do not stop at borders and that therefore multilateral collaboration is essential to protecting citizens from lethal epidemics. the development of germ theory through the second half of the nineteenth century transformed ideas about the causes of infections, informing scientific research as well as clinical responses. scientific understanding translated into vaccines and antibiotics, while programmes for child health, hygiene, clean water and sanitation became common in the twentieth century. as a result, childhood diseases such as measles and mumps became rare, smallpox was eventually eradicated and polio was eliminated from all but a handful of countries . many people thought that infectious diseases would soon be history. sir frank macfarlane burnet is often cited for his remark in the s that, with the emergence of new diseases being a distant prospect, "the future of infectious diseases will be very dull" . although the focus in high-income nations turned to non-communicable diseases, which constituted a considerable and increasing burden on the health of their citizens, infectious diseases did not disappear. some endemic infections such as malaria and tuberculosis were not susceptible to elimination strategies, and new diseases with epidemic and pandemic potential emerged. ebola virus disease was first identified in the s, hiv/aids in the s, nipah virus in the s, sars and mers at the start of the twenty-first century, and many more have since been identified. far from becoming 'very dull', the field of infectious disease epidemiology has sometimes struggled to adapt: as late as , respected researchers used a nineteenth century 'law' of epidemiology to make predictions about the aids epidemic-these turned out to be vast underestimates . advances in other fields gave epidemiology the chance to evolve. in , when the editors of the international journal of epidemiology provocatively asked whether it was time to 'call it a day' given the putative power of genomics to explain diseases over the capacity of epidemiologists to describe them, their conclusion was that it had the potential to positively transform epidemiology as much as the rise of germ theory a century earlier. at least pathogens that affect humans have been identified as emerging, re-emerging or evolving since the s , while increasing rates of antimicrobial resistance threaten to make formerly controlled infections, such as malaria, untreatable -this also limits our ability to control their epidemic potential. the demographic transition is driving much of this: human society is becoming more urban than rural for the first time in our history, bringing large numbers of people (and often animals) together in densely populated areas . agricultural and forestry practices are changing the relationships between people, animals and our respective habitats . travel is more accessible around the world, advances in computer science and computing speeds have led to a number of applications of artificial intelligence across society . applications in epidemiology include tracking online searches about disease symptoms to aid early detection of epidemics, although more sophisticated methods may be required before artificial intellegence becomes a reliable detection tool . crystallography modern x-ray diffraction and electron microscopy can reveal structures of viruses and antibodies in such detail that it is possible to identify specific sites of vulnerability on the virus. a previous study showed how such techniques identified an antibody that was much more potent against respiratory syncytial virus than the only currently available intervention . developing vaccines for emerging infectious diseases has many challenges, including the time it takes, a limited market and strict regulatory requirements for products that will be given to healthy people . platform technologies use one underlying approach with standardized processes and some antigen-specific optimization to speed up both development and manufacture of vaccines. for example, vector-based platforms combine an antigen, or a gene for an antigenic protein or peptide, in a virus-like particle or liposome. such platform technologies have the potential to deliver vaccines a few months after an emerging pathogen is identified and sequenced, rather than years . review so migration, trade and tourism bring more people into contact and thus affect disease transmission . climate change has many effects on ecosystems and environments, not least in changing the habitats and migratory habits of disease vectors . states with weak health systems are far less likely to cope with or recover from multiple emergent demands without damaging routine services . inequalities , inequities and distrust in national structures and institutions compound people's vulnerabilities . conflict increases the risk of epidemics and makes responding to them close to impossible . since , there have been several outbreaks of ebola (including the two biggest in history), not to mention outbreaks of sars, mers, nipah, influenza a subtype h n , yellow fever, zika and the continued spread of dengue. epidemics overlap and run into each other, yet the world is not currently equipped to cope with this increasing burden of multiple public health emergencies. preparing for epidemics, therefore, requires global health, economic and political systems to be integrated just as much as infectious disease epidemiology, translational research and development, and community engagement. epidemics represent shared risks that cross borders and all of society. health systems, routine care, trust in governments, travel, trade, business-all are disrupted during an epidemic. with such broad risks, the preparation and response must be nationally owned and led, internationally supported and undertaken with a whole-of-society approach. some initiatives have started to build frameworks for this to happen in a coordinated way. for example, the who's pandemic influenza preparedness framework brings together nation states, industry, other stakeholders and the who to implement a global approach to pandemic preparedness and response . a focus must be building coordinated regional and country expertise, resources and capacity through national and regional public health institutions . this brings its own challenges-governance of institutions, leadership, collaborations and interventions have to be impeccable or misconduct can thrive . unwelcome in itself, misuse of funding, resources or people within efforts intended to support an epidemic response will also undermine trust in the organizations that respond to an outbreak and, in turn, prolong the outbreak. key governance components include drafting policies in advance and being willing to implement those policies for data collection and sharing during epidemics. they must be flexible enough to enable affected communities and nations to retain ownership of the response, while drawing on international expertise to find the best possible response. governance should also include processes for vaccine and therapeutic approvals during outbreaks. however, it is clear that the centre of gravity for leadership, governance and implementation must be where the need is greatest if these are to truly deliver. in , julian tudor hart proposed the inverse care law: "the availability of good medical care tends to vary inversely with the need for it in the population served." . an analogue of the inverse care law can be applied to public health and epidemiology. expertise in these fields has traditionally gravitated towards centres of excellence in europe and the united states. of course, high-income countries are not immune to the disruption associated with epidemics, especially in an era of misinformation and growing mistrust in authorities and public health initiatives. however, the centre of gravity must shift so that globally representative distributed networks of collaborating centres can jointly ensure coverage in the regions that urgently need these skills on the ground . international collaborations remain important; however, strengthening epidemiology, public health and laboratory capacity in low-and middleincome countries is essential . collaborative interventions should not be limited to when there is a major outbreak, but be integrated into regular interactions. capacity, resources, expertise and governance can be supported by the increasing role for regional and national centres of disease control. the us centers for disease control (cdc) lends its expertise all around the world in addition to protecting the us population. in , the european cdc started, followed by the china cdc in and by the africa cdc in . although more can be done to improve data sharing and access to laboratories, the networks and connections between these centres have strengthened all of their work, as well as having a positive effect on public health systems in low-and middle-income countries. during the pan-european wave of cholera in the s, there were riots across the continent: doctors, nurses and pharmacists were murdered, hospitals and medical equipment destroyed . similar reports today usually come from communities that have not had positive prior interactions with public health initiatives, and thus the encounter with national or international teams who arrive only in response to a 'new' disease means that trust can never be assumed and has to be earned on both sides. engagement needs to start before an outbreak-ensuring that patients, their families and their communities are at the centre of all public health is essential for the successful prevention and response to epidemics. there is no public health without the support of the community. for example, the early detection of disease events will be improved if more national and regional public health institutions establish community event-based surveillance systems. communities are the first to know when something unusual happens -therefore training and mobilizing community volunteers to report such occurrences is a costeffective way to rapidly detect diseases and contain them at the source. this will also help to sustain engagement between communities and the organizations that respond to outbreaks. furthermore, improved information flow between the community and the public health system should provide a better understanding of local social networks to complement other means of tracking chains of transmission between individuals and places. this can be the community themselves, or it might be veterinarians who see clusters of sick animals, or nurses and doctors who care for patients in primary care-or it may be teams that are often forgotten in public health initiatives, such as those working in critical care facilities; it is striking how the first cases of nipah, sars, mers and influenza a subtype h n were all first identified by clinical teams in critical care facilities. an inclusive, whole-of-society approach is challenging, and the challenges may be magnified in a conflict or post-conflict zone. wars and conflicts not only increase the risk of epidemics as people move to escape violence and health services become harder to maintain , but also make public health responses vulnerable to interruption, thus making them less effective. then, miscommunication, mistrust, disease and violence can fuel each other in a vicious cycle. engaging local communities remains the highest priority, even in unstable contexts such as north kivu and ituri provinces of the democratic republic of the congo (drc) , where an ebola epidemic started in august . it seems inevitable that responding to epidemics in politically unstable environments will become more common, and skilled negotiators and peacekeepers will have to be better integrated in response teams. equally essential, therefore, will be an improved understanding of these challenging operational contexts among affected communities and external responders alike. social scientists have long applied their skills and knowledge in epidemic responses, although their roles have become more visible in recent years . by focusing on communities, social science humanizes the epidemic response , helps to increase understanding of context and may uncover associations between the context or local practices and the risk of transmission. the social science in humanitarian action platform has successfully produced rapid reports and briefings on regions in which an epidemic has been identified, and the global research collaboration for infectious disease preparedness includes a social science research funders' forum to 'propel research in this area' , acknowledging that its integration in the preparation and response to outbreaks is often missing or added as an afterthought to solve a problem that could have been forseen. there is still much to learn about how epidemic responders and social scientists can make the most of each other's expertise and how data from social science can fit into the wider information architecture of epidemic response. as an example, behavioural surveillance will be critical in twenty-first century responses to disease outbreaks . just as behavioural surveillance to improve the understanding of hiv was crucial in identifying high-risk groups for hiv infection, so human behaviours will continue to be important as we respond to future infectious diseases. for instance, the ebola virus outbreak in west africa probably began before december , but it took several months before hospital transmission and traditional burial practices were found to be the leading causes of its rapid spread. the increasing prevalence of mobile phones, wireless internet connectivity and social media activity raises the possibility of using these tools to gather data for epidemiological studies, diagnostics , population mobility during an ebola epidemic or influenza incidence in real time . future developments in predictive technology, machine learning and artificial intelligence will bring more opportunities to move towards 'precision public health' (box ). the use of data from people is becoming strictly controlled, however, and it will be a challenge to persuade countries to invest in a new surveillance system, for example, before its general effectiveness has been demonstrated at a country level . even then, technology-based solutions should be integrated with community-based programmes and other existing epidemic preparedness and response systems because surveillance is more effective when standardized among different countries, districts and communities. to this end, suites of guidance and open-access standardized tools are being developed for reporting cases of disease, as well as consent forms, standard operating procedures and training materials , properly validated diagnostic assays and access to quality-assurance panels in public and veterinary health. the rising trend of engaging citizens in data gathering is also welcome-the use of mosquito-recognition apps enables the collection of data far beyond the capacity of routine mosquito surveillance . this way, citizens feed information into the public health system and the feedback loop offers a fast and direct way to provide citizens with details of potential actions that they can take. as well as potentially supporting diagnosis and surveillance , the fast-developing field of genomic epidemiology can yield information to track the evolution of a virus such as ebola during an epidemic , . there will be times when it can detect outbreaks better than traditional epidemiology, illustrating the need to have these tools available in the same toolbox. during the large lassa fever outbreak in nigeria in , real-time genomic sequencing provided clear evidence that the rapid increase was not due to a single lassa virus variant, nor attributable to sustained human-to-human transmission. rather, the outbreak was characterized by vast viral diversity defined by geography, with major rivers acting as barriers to migration of the rodent reservoir . these findings were crucial in containing the outbreak. developing and sustaining the capacity to conduct real-time sequencing with adequate bioinformatics analyses at regional and national levels will be challenging in low-and middle-income countries. moreover, investments in relatively high-tech capacity (such as real-time sequencing) are competing with other, arguably more fundamental needs, such as equipment and training in primary laboratories. political engagement must be nurtured between epidemics: it is not enough to offer technological and laboratory support during a crisis, even with the promise of building capacity, if the political will is not there. however, with proper preparation, and accessible and trusted data sharing and governance mechanisms, laboratories with limited resources may be able to leap-frog into the twenty-first century , . vaccination is one of the most effective public health interventions and innovative strategies for research and development of vaccines, such as using ring vaccination as a trial design during ebola epidemics since - , must be encouraged. at the start of the - epidemic in west africa, vaccine candidates were already in development, based on a long history of preclinical research, although a lot of work was still required to get clinical trials underway in time to be useful . in , when zika was first internationally recognized as a pathogen that could cause birth defects , there was hardly any research and no vaccines in late-stage development. two-and-a-half years later, results from three phase i clinical trials had been reported , although challenges remained for further development. the lack of a profitable market for such products means that pharmaceutical companies lack the incentives to push this work between epidemics. initiatives such as the coalition for epidemic preparedness innovations are attempting to positively disrupt financing models for vaccines against epidemic diseases , and stockpiles of meningococcal vaccine, yellow fever vaccine and oral cholera vaccine are maintained by the international coordinating group to minimize potential delays due to limited manufacturing capacity . similarly, if investigational treatments or vaccines are to be used as part of the response to an epidemic, ethical protocols for managing informed consent and introducing them in clinical settings must be planned in advance with at-risk communities (box ). trial designs precision medicine refers to the use of genomic sequencing to retrace the specific course of a disease in individual patients, with the aim of being able to choose the best treatment option for each person. in public health, the analogous idea of precisely directing the right intervention to the right population is equally appealing. the potential of such an approach has been illustrated by the identification of two areas in the united states in that were at risk of zika transmission . rather than the whole country, or even only florida, being declared at risk, these two areas each measured less than km , and the response focused only on these specific neighbourhoods. by contrast, a campaign against yellow fever, also in , defined risk 'at the level of entire nations'. a broad interpretation of precision public health incorporates many different types of data to increase the power of epidemiology . such data would not only include genomic information, but also satellite imaging, mobile phone data, social media use data and so on. for example, a study published in combined epidemiological surveillance data, travel surveys, parasite genetics and anonymized mobile phone data to measure the spread of malaria parasites in southeast bangladesh . a retrospective analysis of mobile phone call data in sierra leone from showed how it might have been used to assess the impact of travel restrictions on mobility during the ebola epidemic . the principle of selecting the most relevant information from all available data seems within the scope of good epidemiological practice already. the challenge is recognizing and incorporating new types of data when they become available. should be created as soon as the option becomes viable. the essential consideration is how the resulting data can add to previous trials and influence the approach to trials in future epidemics. for example, research during the - ebola epidemic enabled progress on therapeutic agents that are now being trialled in the ongoing outbreak in drc . scientific progress during and between epidemics must be matched by other workstreams, such as the preparation of supply chain logistics and communication with at-risk populations. plans have to be made for a series of future outbreaks, enabling adaptive, multi-year, multi-country studies . similar plans are needed for continual preclinical research to ensure that future vaccine and therapeutic pipelines will be filled. the term 'one health' is used to acknowledge that human, animal and ecosystem health are tightly interconnected and need to be studied in the context of each other (fig. ) . changes in the environment-whether natural or anthropogenic-affect interactions between pathogens, vectors and hosts in multiple and complex ways, making the emergence or decline of endemic, epidemic and zoonotic diseases difficult to predict, while epidemics of animal diseases can challenge a community's access to food. the fact that pools of viruses, bacteria and parasites are maintained in wild and domesticated animals makes surveillance of potentially zoonotic diseases an intrinsic part of one health epidemic planning. many agencies and nations around the world now use prioritization tools such as those developed by the us cdc or the united nations (un) food and agriculture organization (fao) to identify and prioritize zoonotic diseases of concern. an early precedent was a joint consultation on emerging zoonotic diseases by the who, the fao and the world organisation for animal health in . understanding disease ecology in the zoonotic reservoir could potentially lead to ways to predict the risk of human disease, thus providing the basis for smart early-warning surveillance systems. individual countries with limited resources for epidemiological studies and epidemic preparation and response must decide their own priorities. however, infectious diseases do not respect borders. similarly, the interdisciplinary nature of one health means there are several different lenses through which different sectors assess risks and priorities. for one health approaches to work, these multiple perspectives must be taken into account, whether human health or animal health, ecology or social sciences . epidemics do more than cause death and debilitation: they increase pressure on healthcare systems and healthcare workers and draw resources from services not directly linked to the epidemic. this can leave a legacy of distrust between people, governments and health systems, although more-positive outcomes have been found to strengthen relations between communities and public authorities. the full social and economic costs of the ebola outbreak in west africa have been estimated to be as high as us$ billion when including the effect on health workers, long-term conditions suffered by , ebola survivors, and costs of treatment, infection control, screening and deployment of personnel beyond west africa. as healthcare resources became increasingly allocated to the ebola response, hospital admissions fell and deaths from other diseases rose markedly, adding us$ . billion to the estimated cost. such pressure can be withstood in high-income countries with strong health systems, but in low-income countries the pressure can quickly reach a breaking point. ebola killed almost . % of doctors, nurses and midwives in guinea, . % in sierra leone and just over % in liberia . this is compared to mortality between . % and . % of the whole population of these countries. estimates of the effect of this loss on maternal mortality suggest that thousands more women may have died in childbirth each year since the epidemic ended. beyond the tragic deaths of so many healthcare workers, people were less likely to use health services for children or adults during the epidemic, suggesting decreased trust or even fear of healthcare settings . more recently, in some areas affected by the ebola outbreak in drc, the introduction of free non-ebola healthcare led to unprecedented demand. however, healthcare facilities box in , the prevent project received wellcome funding to provide ethics guidance "at the intersection of pregnancy, vaccines, and emerging and re-emerging epidemic threats" . this was in response to the newly recognized association between infection with zika virus during pregnancy and microcephaly in the newborn. developing a vaccine was an obvious route to explore, but many researchers felt that they could not conduct clinical trials with pregnant women because it is generally assumed that the risk to the woman, the fetus or both outweighs any potential benefit. however, as heyrana et al. argue: "preventing pregnant women from participating in clinical trials is well intentioned but misguided." . prevent rapidly developed guidance for including pregnant women and their babies in zika vaccine research , and has since extended their scope to "a roadmap for the ethically responsible, socially just, and respectful inclusion of the interests of pregnant women in the development and deployment of vaccines against emerging pathogens." . integrating ethics in the preparation and response to epidemics does not close off avenues of research; it opens up possibilities and expedites progress. were not given sufficient additional resources to care for the number of people, which may have contributed to nosocomial infections. survivors, too, need to be cared for long after the epidemic is declared over. a cohort of more than , children is growing up in brazil after being born with microcephaly because their mothers were infected with zika during pregnancy. tracking the development of these children increases understanding of the effects of zika infection and helps to define what medical and social support the affected families may need as many of the children will grow up with severe developmental delays . the challenges posed by twenty-first century epidemics are real and changing: future epidemics will be fuelled by conflict, poverty, climate change, urbanization and the broader demographic transition. in our response we must consider epidemics not as discrete events, but rather as connected cycles for which we can prepare, even if we cannot predict specific outbreaks. the challenge is then to choose the right response at the right scale in the right area at the right time. there needs to be a greater emphasis on absorbing and using positive lessons from each episode and avoiding those that led to negative outcomes . the way that we train practitioners and researchers working in all fields relevant to today's epidemic landscape has to change. a modern approach that is capable of characterizing epidemics and the best ways to control them must go beyond a narrow definition of epidemiology that sustains artificial barriers between disciplines. instead, it must be able to integrate tools and practices from a diverse range of established and emerging scientific, humanistic, political, diplomatic and security fields. we believe that such an approach needs to become the norm for the curriculums of schools of public health around the world. as well as training new generations of epidemiologists so that they have the skills, knowledge and networks to recognize and make use of every tool available to help them to do their work effectively, the entire architecture of the response to epidemics has to be adapted. only then will we be able to maintain the comprehensive and effective response-including prevention and research-needed to stop epidemics and protect people's lives, no matter what the circumstances. celebration: william farr ( - )-an appreciation on the th anniversary of his birth rediscovering ignaz philipp semmelweis ( - ) louis-rene villerme ( - ), a pioneer in social epidemiology: re-analysis of his data on comparative mortality in paris in the early th century john snow's legacy: epidemiology without borders this is a wide-ranging meeting report that places modern epidemiology in the context of the past two hundred years and highlights the importance of bringing in new disciplines, remaining open-minded and using those skills across a wider range of societal issues than are traditionally considered public health robert koch and the cholera vibrio: a centenary epidemiology for the uninitiated the epidemiologic transition. a theory of the epidemiology of population change large ebola outbreaks new normal, says who applied epidemiology and public health: are we training the future generations appropriately? managing epidemics: key facts about major deadly diseases ignaz semmelweis, carl mayrhofer, and the rise of germ theory history of vaccination the global eradication of smallpox from emergence to eradication: the epidemiology of poliomyelitis deconstructed natural history of infectious disease p farr's law applied to aids projections epidemiology-is it time to call it a day? global rise in human infectious disease outbreaks pandemics, public health emergencies and antimicrobial resistance -putting the threat in an epidemiologic and risk analysis context how urbanization affects the epidemiology of emerging infectious diseases microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg travel, migration and emerging infectious diseases understanding the link between malaria risk and climate the ebola outbreak, fragile health systems, and quality as a cure health inequalities and infectious disease epidemics: a challenge for global health security historical parallels, ebola virus disease and cholera: understanding community distrust and social violence with epidemics war and infectious diseases: challenges of the syrian civil war pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who how africa can quell the next disease outbreaks the ability to prevent, detect and respond to any health issues will always depend on the local capacity and although international partners can bring complementary expertise and resources, it is the local capacity that is critical; in this article, the authors argue for national investment in public health, health systems, science and local leadership un health chief orders probe into misconduct the inverse care law agenda setting, research questions and funding for biomedical research has historically been led from northern hemisphere countries in an unequal northern-southern hemisphere relationship science granting councils in sub-saharan africa: trends and tensions international federation of red cross and red crescent societies. community-based surveillance: guiding principles conflict and emerging infectious diseases institutional trust and misinformation in the response to the - ebola outbreak in north kivu, dr congo: a population-based survey application of social science in the response to ebola towards people-centred epidemic preparedness and response: from knowledge to action anthropology in public health emergencies: what is anthropology good for? launching a new era for behavioural surveillance integrated biological-behavioural surveillance in pandemic-threat warning systems taking connected mobile-health diagnostics of infectious diseases to the field population mobility reductions associated with travel restrictions during the ebola epidemic in sierra leone: use of mobile phone data national and local influenza surveillance through twitter: an analysis of the - influenza epidemic social media and internet-based data in global systems for public health surveillance: a systematic review isaric. protocols & data tools enhancing early warning capabilities and capacities for food safety real-time, portable genome sequencing for ebola surveillance infection control in the new age of genomic epidemiology genomic surveillance elucidates ebola virus origin and transmission during the outbreak genetic diversity and evolutionary dynamics of ebola virus in sierra leone genomic analysis of lassa virus during an increase in cases in nigeria the integration of genomics and other types of data into the surveillance, prevention and response of epidemics is critical and can help to transform the ability to enhance public health; although the tools are now available, it will be key to ensure that these new approaches are fully integrated and not seen as esoteric ivory tower research, but instead as an essential component of twenty-first century epidemiology, public health and epidemics-the next generation of leaders need to be efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola Ça suffit!) a seminal study that shows that ring vaccination could be used in the midst of a devastating ebola epidemic and, furthermore, that innovation research can be conducted in an epidemic, trial designs can be adapted without compromising scientific integrity and that ebola can be prevented through vaccination ethical rationale for the ebola "ring vaccination" trial design ebola vaccination in the democratic republic of the congo insights from clinical research completed during the west africa ebola virus disease epidemic gone or forgotten? the rise and fall of zika virus current status of zika vaccine development: zika vaccines advance into clinical evaluation cepi: preparing for the worst the development of global vaccine stockpiles the ebola clinical trials: a precedent for research ethics in disasters it is an ethical imperative to consider and implement research in an epidemic setting as, for many epidemic diseases, it is the only time at which to conduct the research that will inform and improve the lives of the individuals affected during epidemic and to ensure that future generations are better prepared; however, such research is challenging at many levels and it is critical to have an ethical framework that guides the research, places individuals and communites at the heart of the research and facilitates the maximum benefit for the maximum number of people improving vaccine trials in infectious disease emergencies progression of ebola therapeutics during the - outbreak ebola therapies: an unconventionally calculated risk performance of different clinical trial designs to evaluate treatments during an epidemic the one health concept: years old and a long road ahead surveillance of zoonotic infectious disease transmitted by small companion animals prioritizing zoonoses: a proposed one health tool for collaborative decision-making evaluation of the emergency prevention system (empres) programme in food chain crises report of the who/fao/oie joint consultation on emerging zoonotic diseases (who european centre for disease prevention and control. towards one health preparedness the economic and social burden of the ebola outbreak in west africa epidemics cause enormous disruption to countries, regions and the world; however, the focus is often on the epidemic itself, the pathogen and its immediate effect rather than the much broader effect that the epidemic has not only on the healthcare system-which lasts long after the epidemic itself-as routine vaccination programmes often collapse, maternal-child health suffers, and malaria, hiv and tuberculosis clinics and surgery-all aspects of healthcare-are disrupted, but also on the wider society, as mistrust and tension occurs between citizens, authorities and governments, and education, investments, businesses, trade and tourism inevitablely suffer leading to an economic impact that can health-care worker mortality and the legacy of the ebola epidemic patterns of demand for non-ebola health services during and after the ebola outbreak: panel survey evidence from further pieces of evidence in the zika virus and microcephaly puzzle responding to the ebola virus disease outbreak in dr congo: when will we learn from sierra leone? artificial intelligence in medical practice: the question to the answer? artificial intelligence and big data in public health structure of rsv fusion glycoprotein trimer bound to a prefusionspecific neutralizing antibody vaccine platforms: state of the field and looming challenges platform technologies for modern vaccine manufacturing four steps to precision public health precision" public health -between novelty and hype offline: in defence of precision public health mapping imported malaria in bangladesh using parasite genetic and human mobility data pregnant women & vaccines against emerging epidemic threats: ethics guidance for preparedness, research, and response increasing the participation of pregnant women in clinical trials the ethics working group on zikv research & pregnancy. pregnant women & the zika virus vaccine research agenda: ethics guidance on priorities, inclusion, and evidence generation acknowledgements we thank m. regnier at wellcome for editing the manuscript.author contributions all authors developed the scope and focus of the review and contributed to the writing of the manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to j.f. reviewer information nature thanks peter byass, sharon peacock and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. reprints and permissions information is available at http://www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - twnkv authors: perveen, shagufta; orfali, raha; azam, shafiq; aati, hanan y.; bukhari, khulud; bukhari, sara i.; al-taweel, areej title: coronavirus ncovid- : a pandemic disease and the saudi precautions date: - - journal: saudi pharm j doi: . /j.jsps. . . sha: doc_id: cord_uid: twnkv now ncovid- has a foothold in many countries, and the threat of a pandemic situation has risen. recently a novel coronavirus (ncovid- ) has first emerged in china, causing multiple symptoms in humans and closely related to those caused by sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome). the ncovid- has reported in wuhan city of china has recently infected over six million people and at least . million confirmed deaths all over the world, while . million people has recovered from this deadly virus. many instances of this respiratory syndrome coronavirus infection have already reported in more than countries and territories. in contrast, the majority of cases reported in the usa, brazil, russia, spain, uk, italy, france and many more countries. in today's context, the coronavirus is one of the significant issues faced by the world with plenty of cases. in these circumstances, rapid reviews which recommended by who (world health organization), and these recommendations are very significant, helpful and cover current data with different preventive measures developed by the saudi cdc (saudi centre for disease prevention and control). this review article describes the possible modes of transmission so that proper preventive actions should be taking. importantly, this work mentioned the animal reservoir through which may infect humans, and it must be identified to break the transmission chain. in additions, this review paper briefly discussed the spread of the coronavirus in the arabian peninsula and what precaution measures are in place by each country to limit the spreading of this virus. taking into consideration the preventive measures by pharmacists as part of health care professions, however, the number of infected people, especially those with close contact with ncovid- patients, are rise day by day and currently seems unstoppable. ncovid- is the name of a new respiratory disease, abbreviated from coronavirus disease , while n stand for new or novel and the causative agent of this disease is severe acute respiratory syndrome coronavirus (ncovid- ). coronaviruses are a group of family-based viruses contains strains that roots potentially diseases in birds and mammals. it is the vast class of viruses that cause viral respiratory sickness extending from common cold symptoms to more severe lung diseases. in comparison to other members of coronaviruses ,which cause humans respiratory infections, sars-cov (first then it has spread to different countries and territories all over the world, and it seems more deadly. however, ncovid- is a novel strain, not identified in humans previously [ ] . coronaviruses have zoonotic origins from animals such as dromedary camels (camelus dromedarius), civet cat (paguma larvata) and bats [ ] . from their reservoirs, and worlds alarming alert, and the emergency among different countries, especially europe, america and asian countries draw the attention of almost every human across the globe ( figure ). this review paper shows aggregate and consolidates the most critical issues related to the virus outbreaks' origin, sources of virus transmission, signs and symptoms as well as the radiographic features from affected subjects. additionally, it illustrates some preventive measures taken by the saudi government that can control further spread of the sars-cov- . the first coronavirus case reported in china, wuhan city among people who had visited a seafood and meat market, and it was highly suspected to be transmitted from animal to human and then transmitted from human to human or to pets or vice versa. based on different studies, the virus might be originated in bats [ , ] . however, no bats were found for sale purpose at the market, which confirmed that another unknown animal might help in the transmission of the virus to humans. many of these coronaviruses primarily infect the individual workers in direct contact with animals, as reported previously. one study recommended pangolin or snake as maybe the transitional animal as there were sold in the seafood and meat market [ , , ] . many studies explore the potential virus reservoir by comparing the sequence analysis of relative synonymous codon usage (rscu) among different animal species based on the sars-cov- genome sequence. results suggested that sars-cov- is a recombinant virus between the bat coronavirus and unknown origin coronavirus that located within the viral glycoprotein spikes presented on cell surface [ ] . one more research team applied algorithm for analysis of ncovid- nucleotide sequencing to recognize other viruses' origins, and the results appeared that both bats and minks could be the latent host for ncovid- . additionally, a study revealed that ncovid- has most homogeneous genetic information with bat coronavirus and resembling snake codon usage bias [ ] . sars first recognized in guangdong, china, november , and then spread to more than thirty countries which began with cases and deaths. it was the first new epidemic disease in the st century. the host of this epidemic disease was thought to be a civet cat; however, during seven months since the occurrence, it was under controlled [ ] . (table ) [ , ] . spanish influenza was also an epidemic which its outbreak from to , and it was the most devastating epidemic on record, which killed between to million people around the world. several measures applied during that era, such as fresh air, sunlight, standards hygiene and face masks in order to reduce mortality rates. ebola virus was also an epidemic disease which first outbreaks in ebola river, zaire . the - outbreaks were in guinea then rapidly extend to west africa with more than deaths (table ) . all over the world scientists are working to find out the different spreading routes of these viruses. in general, the common way is through respiratory droplets which may transfer from the patient through coughing and sneezing. further tests have also confirmed the presence of these viruses in patients' stool, suggesting that it can be spread through the excretory product. currently, it is entirely unclear whether people can also become infected by touching contaminated surfaces or objects. older people and people with long term medical issues are more susceptible to be infected with this virus. these critical chronic health problems such as lungs diseases, sickle cell disease, liver disease, heart problems, kidney disease, cancer, high blood pressure, hiv and diabetes are more likely to get the risk of serious sickness. pregnant women (current or recent) and the person with weak immunity are also at a higher risk of severe illness due to this virus. additionally, smokers are at great risk of disease because smoking reduces the natural protection against this type of viruses. social distancing or self-quarantine is one of the best tools to mitigate the spread of the ncovid- . in , two different states of america detected a few cases of influenza among civilians of philadelphia and saint louis. both of the cities were planning for a massive military parade, which was the city's largest parade ever. the head of philadelphia city decided to continue the events which were the big reason for quickly spreading the virus and made it a worldwide pandemic. after three days, every bed in philadelphia's hospitals were filled with sick and dying patients, infected by this deadly virus. while the head of the st. louis city decided to cancel its parade, closed all schools, playgrounds, libraries, courtrooms and churches around the entire city; the public gathering was also banned. after a month, more than ten thousand people in philadelphia passed-away due to flu while deaths toll at saint-louis was not exceed cases. these precautions demonstrate the advantage of cancelling events, massgatherings and distancing social dissociation routines. the fatality cases rates are intended by dividing total deaths from a disease by the number of cases. it is expressed as a percentage (%) and used as a measure of disease severity. were died. so, the total cfr due to sars is . % which is higher than the current ncovid- and mers. most ncovid- cases occurred in health care facilities; however, the routes of direct or indirect transmission of these remain unidentified. up till now, not any specific treatment or any vaccine is available. however, serious attempts to develop preventive therapy and different researches in many laboratories are ongoing with the hope of some great findings [ ] . ncovid- has similarities and differences with flu in terms of effects and symptoms. but it is a contagious viruses which can spread rapidly and usually caused high fever and shortness breath (table ) . common symptoms of air pollution/common cold/flu vs ncovid- . allah's messenger ‫)ﷺ(‬ said: '(there is) no 'adwa (no contagious disease is conveyed without allah's permission). nor is there any bad omen (from birds), nor is there any hamah, nor is there any bad omen in the month of safar, and one should run away from the leper as one runs away from a lion.'' [ ] however, it is one of the fundamental facts for preventing these viruses from spreading further. this assessment proved the scientific evidence that supports the restriction on travelling during the expansion of epidemic diseases mentioned in the hadith. carriers of this virus travelled to anywhere without any clinical check-up or incubation stay during the last three months from china, iran and italy to the globe, which made an alarming situation of spread, severity and inaction for the world. shortly after that, more than one hundred twenty thousand cases were reported; among these eighty thousand found only in china. the arabian peninsula is the largest peninsula on earth and located in western of asia continent. can work together to sharing expertise in this field where many infections are zoonotic and mutual advantage to prevent the coronavirus spread [ ] [ ] [ ] . multiple studies demonstrate a normal chest x-ray in ncovid- patients or pneumonic consolidation could be seen. final results of chest ct, depending on the severity and days of illness [ , ] . in most of the cases, it includes bilateral lung involvement, rounded ground-glass opacities and peripheral distribution. while, it may include bronchial wall thickening, consolidation, linear opacities and crazy paving pattern. however, there is no evidence of cavitation, or pleural effusion have found in any patient (figure ) . in one study, of patients ( %) with non-complicated ncovid- pneumonia, in whom the temporal progression of the ct appearances in ncovid- was studied, the severity of lung abnormalities peaked at days post symptom onset, with a gradual tailoff after this time [ ] . in another study of thirty-six patients, hrct showed rapid changes over time with fibrous stripes appearing upon improvement in the disease course [ ] . another study represented different stages of disease processes on chest ct scan from days of symptom onset. accordingly, the disease divided into three categories, the saudi centre for disease prevention and control plays an essential role in preventing the spread of ncovid- by continuous monitoring the number of cases by assisting the risks, develop awareness programs and plans to eradicate ncovid- . the centre develops certain measures for communities, and the public to prevent the disease from spreading in schools, colleges, workplace and mosques (table ). the centre took the responsibility of reducing the death and disability rates by spreading awareness and positive impact on the health behaviours of individuals and societies. also, to enable all society segments to control their health, prevent all causes of ncovid- through the guidance they developed on ncovid- surveillance in healthcare and community settings [ ] . ‫ﺗﻮﻛﻠﻨﺎ‬ ‫ﻭﺗﻄﺒﻴﻖ‬ ‫ﺍﻟﺪﺍﺧﻠﻴﺔ‬ ‫ﻭﺯﺍﺭﺓ‬ table saudi cdc recommended preventive measures for the public and communities. in order to control further spread of the ncovid- virus, people who are infected or suspected to curry the disease should be isolated from others and follow up on their treatment course under restricted infection-controlled precautions. the saudi cdc listed guidelines for standard measures established by the who for ncovid- , which mainly concern on hand, respiratory and food hygiene practice: -frequently hand hygiene using alcohol-based hand rub or detergents and water. -covering mouth and nose are essential when coughing and sneezing. take on consideration to dispose of any used tissue, immediately. -if you have respiratory problems, wear a medical mask and clean your hands after disposing of the face mask. -if you have fever, cough and difficulty breathing, directly seek medical care or contact the saudi moh (ministry of health) hot line and share previous travel history. -when visiting crowded area, maintain social distance (not less than meter) from individuals with respiratory problems (e.g. sneezing and coughing). -before touch your nose, mouth and eyes wash your hands. -when visiting animal and meet markets, especially in places currently recorded cases of a novel coronavirus; avoid direct contact with live animals. the saudi cdc guidelines, mainly concern on health workers who are in close contact with ncovid- patients, according to the who findings on this virus. it transmitted between people through close contact and droplets; not the airborne transmission. therefore, additional precautions for those workers should be taken includes gloves, medical masks, goggles/face shield, gowns, as well as respirators (e.g. n or ffp ) and the way how to put on, how to remove and how to dispose of it properly. pharmacists play a great role in the health society with their different responsibilities. in this review, we explain much more details of measures that should be taken with the pharmacists in saudi arabia. the following are types of pharmacies which have high risk exposure to the virus and certain precautions to be implemented to protect themselves and prevent health care transmission; community pharmacists are the health professionals most dealing with the public. they supply medicines for those with health-related problems, checking the pharmaceutical stocks (medicines, sterilizers and masks…etc). they also provide the patients at the time of dispensing their prescriptions with counselling. they inform the public, health professions and patients. those pharmacists could consider as a link between the general public and health professionals in primary health care. noticeably, the possibility of exposure of community pharmacist to ncovid- exists, since they could interact with suspected patients; therefore, the first-line pharmacist should follow the precautions listed in (table ) to protect them as well. ksa ministries and higher official authorities took immediate and prompt action from the beginning of this pandemic for fighting against the deadly virus through a variety of actions. in the beginning, more than hundred billion riyals sr was provided by the ksa worldwide. also, establishing six regional laboratory all over the ksa to conduct , tests per day and mobile laboratory to conduct , tests per day [ ] . hospital pharmacists are responsible for managing the stocks of medication supply used in the hospital and are accountable for manufacturing, purchasing and quality testing for (table ) . pharmacist and the pharmacy workforce can play an essential role in reducing the transmission of novel ncovid- virus. these precautions could take place by the following: because of the similarity in the symptoms between ncovid- , flu and common cold viruses, which includes fevers, coughing and sometimes severe lung infections, and because that common colds and flu are seasonal, which drop down in summer and spring. many people expressed their hope that the new virus could decrease it spread during spring and summer, and accordingly, the number of infected cases will decrease. however, there is no scientific evidence yet since the virus is still new and early to approve that. if the traveller suffers from symptoms indicating an acute respiratory illness before or after travel, the travellers must seek medical attention and inform the travel history of their health care provider. who standards recommended methods to prevent many diseases by cleaning hands with alcohol or rubbing hands with soap and water, as well as healthy nutritional practices. also, when sneezing or coughing, the nose and mouth should be covered with elbow or tissue and then throw the tissue away immediately, and hands should wash very well. avoid close contact with anyone who suffers from a fever and cough, and any person who suffers from fever, cough and difficulty breathing should seek medical care early and report his previous travel record of those who provide him with medical care. advice to conduct an exit check at international airports and ports in the affected areas, with the aim of early detection of travellers with symptoms for further evaluation and treatment, thus preventing the export of the disease. exit screening includes an examination of signs and symptoms (fever above degrees, coughing), passengers with symptoms of respiratory infection advise to leave the affected areas and directing travellers with symptoms for further medical examination, followed by a test for ncovid- , keeping confirmed cases under isolation and treatment. encourage examination at local airports, railway stations and long-distance bus stations as necessary. this manuscript has discussed various elements regarding the ncovid- infection and its correlation with other coronaviruses diseases, control, and the role of the different approaches in containing and preventing the spread of this disease. saudi governments imposed aggressive actions to control disease spreading. additionally, forcing people to stay at home and by developing certain regulations on ncovid- surveillance in healthcare and community settings through the saudi cdc. pharmacists as members in the health organizations and following the saudi cdc preventive measures is mandatory to protect themselves and others. on the contrary, it should turn into a movement, having supporting organizations worldwide, for better control of emerging diseases, including ncovid- as numbers of nearly contact patients increases day by day. the authors declare they have no conflicts of interest. asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus (sars-cov- ): facts and myths coronaviruses: important emerging human pathogens a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human host and infectivity prediction of wuhan novel coronavirus using deep learning algorithm sars: the first pandemic of the st century infectious diseases transmissible from animals to humans. fourth edition epidemic infections and their relevance to the gulf and other arabian peninsula countries time course of lung changes on chest ct during recovery from clinical characteristics of hospitalized patients with liming xia. initial ct findings and temporal changes in patients with the novel coronavirus pneumonia ( -ncov): a study of patients in wuhan chest ct findings in covid- : relationship to duration of infection. respiratory medicine importance of early precautionary actions in avoiding the spread of covid- : saudi arabia as an example the authors would like to extend their sincere appreciation to the deanship of scientific key: cord- -xit najq authors: van damme, wim; dahake, ritwik; delamou, alexandre; ingelbeen, brecht; wouters, edwin; vanham, guido; van de pas, remco; dossou, jean-paul; ir, por; abimbola, seye; van der borght, stefaan; narayanan, devadasan; bloom, gerald; van engelgem, ian; ag ahmed, mohamed ali; kiendrébéogo, joël arthur; verdonck, kristien; de brouwere, vincent; bello, kéfilath; kloos, helmut; aaby, peter; kalk, andreas; al-awlaqi, sameh; prashanth, ns; muyembe-tamfum, jean-jacques; mbala, placide; ahuka-mundeke, steve; assefa, yibeltal title: the covid- pandemic: diverse contexts; different epidemics—how and why? date: - - journal: bmj glob health doi: . /bmjgh- - sha: doc_id: cord_uid: xit najq it is very exceptional that a new disease becomes a true pandemic. since its emergence in wuhan, china, in late , severe acute respiratory syndrome coronavirus (sars-cov- ), the virus that causes covid- , has spread to nearly all countries of the world in only a few months. however, in different countries, the covid- epidemic takes variable shapes and forms in how it affects communities. until now, the insights gained on covid- have been largely dominated by the covid- epidemics and the lockdowns in china, europe and the usa. but this variety of global trajectories is little described, analysed or understood. in only a few months, an enormous amount of scientific evidence on sars-cov- and covid- has been uncovered (knowns). but important knowledge gaps remain (unknowns). learning from the variety of ways the covid- epidemic is unfolding across the globe can potentially contribute to solving the covid- puzzle. this paper tries to make sense of this variability—by exploring the important role that context plays in these different covid- epidemics; by comparing covid- epidemics with other respiratory diseases, including other coronaviruses that circulate continuously; and by highlighting the critical unknowns and uncertainties that remain. these unknowns and uncertainties require a deeper understanding of the variable trajectories of covid- . unravelling them will be important for discerning potential future scenarios, such as the first wave in virgin territories still untouched by covid- and for future waves elsewhere. late in , a cluster of acute respiratory disease in wuhan, china, was attributed to a new coronavirus, - later named severe acute respiratory syndrome coronavirus (sars-cov- ). it was soon discovered that the virus is easily transmitted, can cause summary box ► severe acute respiratory syndrome coronavirus (sars-cov- ), the virus that causes covid- , has spread to nearly all countries of the world in only a few months. it is unique that an emerging respiratory virus becomes a pandemic, and can continue human-to-human transmission unabated, probably permanently. ► depending on the context, the trajectory and the impact of the covid- epidemic vary widely across affected countries. this is in fact the case with most infectious diseases. ► despite limited initial knowledge on covid- , most societies have deployed draconian measures, including lockdowns, to contain the virus and mitigate its impact. this had variable success, but invariably with profound socioeconomic collateral effects. ► through research and rapid sharing of its findings, progressively more insights on sars-cov- and covid- have been uncovered (knowns), mainly based on evidence from china, europe and the usa; however, important knowledge gaps remain (unknowns). ► the different covid- epidemics and the responses unfolding in the global south are little described, analysed or understood. insights from these less researched contexts are important for discerning potential future scenarios, not only for the first wave in virgin territories still untouched by covid- , but also for future waves. ► more understanding of lived experiences of people in a variety of contexts is necessary to get a full global picture and allow learning from this variety. ► bmj global health and emerging voices for global health have launched a call for such on-the-ground narratives and analyses on the epidemics of, and responses to, covid- . severe disease and can be quite lethal especially in the elderly and those with comorbidities. [ ] [ ] [ ] [ ] the new human disease is called covid- . soon it became clear that its global spread was unstoppable. even with draconian containment measures, such as strict movement restrictions, the so-called lockdown, it spread, and within a few months reached almost all countries and was declared a pandemic by the who. table summarises key events in the unfolding of the covid- pandemic, from december to may . this progression is quite unique. new human pathogens emerge frequently from an animal host, but most cause only a local outbreak. human-to-human transmission stops at some point, and the virus can only re-emerge as a human pathogen from its animal host. only very rarely does an emerging pathogen become a pandemic. over the past decades, a totally new pathogen emerged, caused serious disease, and spread around the globe continuously only once before: the hiv. it seems increasingly likely that sars-cov- transmission will be continuing. all countries are now facing their own 'covid- epidemic'. in only a few months, the scientific community has started to learn the virus's characteristics and its manifestations in different contexts. but we fail to understand fully why the virus spreads at different speeds and affects populations differently. our main objective is to make sense of those different expressions of the covid- pandemic, to understand why covid- follows variable trajectories in ways that are often quite different from the collective image created by the mediatisation of the dramatic covid- epidemics in densely populated areas. we start by exploring the role of context, followed by a brief summary of what is already known at the time of writing about sars-cov- and covid- . we then bmj global health compare these knowns with what is known of some other viral respiratory pathogens and identify the critical unknowns. we also discuss the coping strategies and collective strategies implemented to contain and mitigate the effect of the epidemic. we finally look ahead to potential future scenarios. the unfolding covid- pandemic: importance of context initially, human-to-human transmission was documented in family/friends clusters. [ ] [ ] [ ] [ ] [ ] [ ] progressively, it became clear that superspreading events, typically during social gatherings such as parties, religious services, weddings, sports events and carnival celebrations, have played an important role. [ ] [ ] [ ] [ ] dense transmission has also been documented in hospitals and nursing homes possibly through aerosols. sars-cov- has spread around the world through international travellers. the timing of the introduction of sars-cov- has largely depended on the intensity of connections with locations with ongoing covid- epidemics; thus, it reached big urban centres first and, within these, often the most affluent groups. from there, the virus has spread at variable speeds to other population groups. as of may , the most explosive covid- epidemics observed have been in densely populated areas in temperate climates in relatively affluent countries. the covid- pandemic and the lockdowns have been covered intensively in the media and have shaped our collective image of the covid- epidemic, both in the general public and in the scientific community. the covid- epidemic has spread more slowly and less intensively in rural areas, in africa and the indian subcontinent, and the rural areas of low and lower-middle income countries (lics/lmics). not only the media but also the scientific community has paid much less attention to these realities, emerging later and spreading more slowly. the dominant thinking has been that it is only a question of time before dramatic epidemics occur everywhere. this thinking, spread globally by international public health networks, has been substantiated by predictive mathematical models based largely on data from the epidemics of the global north. however, what has been observed elsewhere is quite different although not necessarily less consequential. the effects of the covid- epidemic manifest in peculiar ways in each context. in the early stages of the covid- epidemic in sub-saharan africa, the virus first affected the urban elites with international connections. from there, it was seeded to other sections of the society more slowly. in contrast, the collateral effects of a lockdown, even partial in many cases, are mostly felt by the urban poor, as 'stay home' orders abruptly intensify hardship for those earning their daily living in the informal urban economy. governments of lics/lmics lack the budgetary space to grant generous benefit packages to counter the socioeconomic consequences. international agencies are very thinly spread, as the pandemic has been concurrent everywhere. donor countries have focused mainly on their own covid- epidemics. the epidemic is thus playing out differently in different contexts. many factors might explain sars-cov- transmission dynamics. climate, population structure, social practices, pre-existing immunity and many other variables that have been explored are summarised in table . although all these variables probably play some role, many uncertainties remain. it is difficult to assess how much these variables influence transmission in different contexts. it is even more difficult to assess how they interact and change over time and influence transmission among different social groups, resulting in the peculiar covid- epidemic in any particular context. we do not attempt to give a complete overview of viruses but select only those viruses that emerged recently and caused epidemics such as ebola, that have obvious similarities in transmission patterns such as influenza and measles, or that are closely related such as other coronaviruses. respiratory viruses such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and avian influenza a and also ebola have originated from animal hosts and caused human diseases (table ) . these viruses do not continuously circulate from human to human. they create an outbreak only when there is interspecies cross-over transmission, most frequently from bats to another animal host. the first human case of a disease from an emerging viral pathogen, the 'index case' or 'patient zero', is invariably someone in close contact with the originating animal host or an intermediary animal host. if this contact occurs in a remote rural community, the spread is usually slow, at low intensity, and could fade out before the pathogen gets a chance to spread to another community. the spread can suddenly intensify if seeded in a densely populated community, frequently in a particular context such as a hospital or during a social event, often referred to as a superspreading event. when the spread reaches a city, it can become a major outbreak, from where it can spread further; this happened with sars-cov in hong kong in and with ebola in conakry, freetown and monrovia in - . but at some stage human-to-human transmission is interrupted and the outbreak stops. only very exceptionally can a new viral pathogen sustain continuous human-to-human transmission. other viral diseases such as measles and influenza are 'old' diseases; they have been studied in great depth. what can we learn from them? measles and influenza: the importance of context it is thought that measles emerged thousands of years ago in the middle east. it is assumed that a cross-over occurred from the rinderpest virus, to become the human measles virus. measles has since spread around the globe in continuous human-to-human transmission. when measles, along with other viruses such as smallpox and influenza, was introduced in the americas by european conquerors, it contributed to a massive die-off of up to % of the original population. the transmission dynamics of sars-cov- can be compared with influenza. influenza typically causes yearly epidemics in temperate climates during winter with less seasonal patterns in tropical or subtropical regions. in hotter climates, such as in sub-saharan africa or south and southeast asia, it is transmitted year round, often not identified as influenza. such different epidemic patterns of influenza are still incompletely understood but thought to be associated with temperature and humidity and human behavioural factors such as indoor crowding. but, in contrast to sars-cov- , the influenza virus is not new. influenza is a very old disease, certainly circulating for several centuries. it has infected most human beings living on the planet already, many of them several times, leaving some immunity but no durable protection. the virus also mutates, giving rise to a new dominant strain every influenza season. influenza is every year a slightly different virus (due to antigenic drift as a result of progressive mutations) with major differences every few decades (antigenic shift as a result of recombination with novel strains). one such antigenic shift resulted in the h n 'spanish' influenza pandemic, which had an estimated case fatality rate (cfr) of %- %, killing millions. box summarises some key facts about h n , including factors thought to be associated with its high cfr. a major difference between covid- and influenza is that sars-cov- is a new pathogen and influenza is not. at the time of writing (may ), sars-cov- has triggered an immune response in over million confirmed infections (and probably in many more), definitely too few to create anything close to herd immunity. calculations using an estimated reproductive number (r ) for sars-cov- suggest that herd immunity would require at least % of the population to have protective immunity (see box ). like covid- , measles and influenza have different epidemic patterns in different contexts. this also is the case for cholera, tuberculosis, hiv/aids and most infectious diseases. the difference in patterns is most pronounced and so is easily understood with vectorborne and water-borne diseases. epidemic patterns are also different for air-borne infections, although they are less easily understood. transmission of respiratory viruses is influenced by factors related to the virus and box pandemic h n influenza, - ► the h n virus probably infected one-third of the world's population at that time (or ~ million people). ► the pandemic had three waves in quick succession; the second wave, in , was worse than the first wave. ► high mortality, especially in younger persons ( - years; ~ % of total deaths) in the pandemic, may have been due to antibody-dependent enhancement and 'cytokine storms'. another possible explanation is that older persons had some protective cross-immunity from previous influenza outbreaks while younger persons did not. ► h n continued to circulate along with seasonal influenza viruses, often recombining to produce more severe local outbreaks, including other pandemics between and , giving it the nickname 'mother of all pandemics'. ► the original h n strain was replaced by a(h n )pdm virus that resulted from an antigenic shift and caused the h n influenza pandemic. ► the h n virus originated in pigs in central mexico in march and was responsible for an estimated deaths worldwide with an estimated cfr< . %. ► during the pandemic, mortality was much lower than in the pandemic. higher mortality in persons younger than years was related to cytokine storms. a role of protective crossimmunity from previous influenza strains in older persons has been suggested. ► after august , the a(h n )pdm virus appeared to have integrated with circulating strains of influenza and continues to cause localised seasonal influenza outbreaks worldwide. box on the use of mathematical models during epidemics a dominant way of studying the transmission dynamics of an infectious disease such as covid- , and predicting the amplitude and peak of the epidemic in a population (city, province, country) and analysing the effect of control measures is using mathematical models. based on available data and several assumptions, a model attempts to predict the course of the epidemic, the expected number of infections, clinical cases and deaths over time. critical is the effective reproductive number (rt). when rt > , the number of cases in a population increases; when rt < , the number of cases decreases. a relatively simple and widely used model is the susceptible-exposed-infectious-recovered model, as used in the two papers recently published in bmj global health on covid- in africa. there are many more types of models, with varying degrees of complexity. the use of such models has strengths and limitations. building a mathematical model implies trade-offs between accuracy, transparency, flexibility and timeliness. a difficulty, in general, is that the parameters on which the model is based, the so-called assumptions are frequently uncertain (table ) and predictions can vary widely if any of the parameters are modestly different. this uncertainty is captured in a sensitivity analysis, leading to various possible quantitative outcomes, usually expressed as a range of plausible possibilities, between 'worst-case' and 'best-case' scenarios. with a new disease such as covid- , certainly at the start of the outbreak, the parameters had to be based on very limited data from a particular context. however, many variables can widely differ across communities as they critically depend on contextual factors (table ) . in mathematical models, all such uncertainties and unknowns are somehow hidden in the complex formulae of the model, as a quasi 'black box'. few people have the knowledge and skill to 'open up the black box'. as uncertainties in covid- are large, the range of possibilities produced by a model is wide, with the worst-case scenario typically predicting catastrophic numbers of cases and deaths. such predictions are often misunderstood by journalists, practitioners and policy-makers, with worst-case estimates getting the most attention, not specifying the huge uncertainties. bmj global health the human host but also by factors related to the natural and human environment (table ) . however, we are quite unable to explain fully which factor has which influence, how these factors vary among different social groups and how interdependent or isolated they are. we are certainly unable to fully model all these variables mathematically to explain the epidemic pattern across a variety of different contexts. too many variables and their interrelations are difficult to quantify, and when all these factors change over time while the pathogen continues to spread in diverse societies, the complexity becomes daunting. understanding transmission dynamics is a bit less daunting for measles, as several variables are well known and rather constant across individuals and contexts. the natural transmission pattern of measles, before the introduction of vaccines, has been well described. measles is mostly a childhood disease, but this is not the case in very remote communities, where measles transmission had been interrupted for extended periods (such as the faroe islands). measles affected all age groups when reaching new territories, causing dramatic first-wave epidemics, a phenomenon called 'virgin soil epidemic'. the latest stages of the global dissemination of measles have been well documented, including in australia, the fiji islands and the arctic countries, where such virgin soil epidemics occurred in the th and the mid- th centuries. fortunately, measles infection creates robust protective immunity and after a first wave becomes a typical childhood disease, affecting only those without any prior immunity. human-to-human transmission of measles virus in a community stops when the virus cannot find new susceptible human hosts and the so-called herd immunity is reached. but transmission of measles continues elsewhere on the planet from where it can be reintroduced a few years later when the population without protective immunity has grown large enough to allow human-to-human transmission again. the epidemic patterns of measles are easily understood as measles is highly infectious, creates disease in almost every infected person and leaves lifelong natural immunity. measles circulation, prior to vaccination, was continuous only in large urban areas with high birth rates. everywhere else reintroduction occurred typically every - ► genetic stability or variability (affecting the potential of long-lasting immunity). ► viral load determines the incubation period with the formula high load ->short incubation period ->high severity. human host ► human susceptibility to the virus; transfer of parental immunity to newborns. ► route and efficiency of human-to-human transmission. ► presence and capacity of asymptomatic carriers to transmit the virus. ► immunity created after infection, its robustness and how long-lasting it is. ► severity and duration of the disease: proportion symptomatic, lethality (cfr). ► pathogenicity and disease spectrum; disease pattern according to age and comorbidities, and related potential to spread. natural environment ► temperature, humidity and seasonal changes in climate affecting the stability and transmission potential of the virus and human susceptibility. ► increasing extreme weather conditions such as droughts and severe storms, as well as global climate change may also affect transmission patterns. ► air pollution may also play a role in the transmission and stability of the virus. human environment/social geography ► demographic variables such as population density, age structure and household composition. ► mixing patterns within households, including bed sleeping patterns, related to housing conditions and hygiene practices. ► house construction with solid walls or permeable walls (thatched walls, straw mats). ► mixing patterns among households related to settlement patterns: social networks, urban-rural differences, working conditions, religious practices and commuting patterns. ► variables related to built environments, road infrastructure and socioeconomic conditions. ► mobility between communities, including international travel. ► crowding institutions: for example, elderly homes, extended families, boarding schools, child institutions, seclusion during tribal ceremonies, hospitals, nursing homes, military barracks and prisons. cfr, case fatality rate. years but sometimes only after or years in isolated rural communities (such as among nomadic groups in the sahel), causing epidemics among all those without acquired immunity and having lost maternal antibodies. these diverse patterns of measles epidemics have been fundamentally changed by variable coverage of measles vaccination. they can still help us make sense of the diversity of covid- epidemics being observed in . measles illustrates convincingly that the transmission pattern of a respiratory virus is strongly influenced by the demographic composition, density and mixing pattern of the population and the connectedness to big urban centres. measles transmission is continuous only in some large urban areas. it presents in short epidemics everywhere else with variable periodicity. this transmission pattern may well be a bit similar for covid- . but it took thousands of years for measles to reach all human communities while sars-cov- spread to all countries in only a few months, despite measles being much more transmissible than sars-cov- . factors such as increased air travel and more dense community structures play bigger roles for sars-cov- than they did for measles. comparison with other pathogenic coronaviruses sars-cov- has many close relatives. six other human coronaviruses (hcovs) are known to infect humans. sars-cov and mers-cov (causing sars and mers, respectively) are very rare and do not continuously circulate among humans. the other four (hcov- e, hcov-oc , hcov-hku and hcov-nl ) cause the common cold or diarrhoea and continuously circulate and mutate frequently. they can cause disease in the same person repeatedly. the typical coronavirus remains localised to the epithelium of the upper respiratory tract, causes mild disease and elicits a poor immune response, hence the high rate of reinfection (in contrast to sars-cov and mers-cov, which go deeper into the lungs and hence are relatively less contagious). there is no cross-immunity between hcov- e and hcov-oc , and new strains arise continually by mutation selection. coping strategies and collective strategies how a virus spreads and its disease progresses depend not only on the variables described above (table ) but also on the human reactions deployed when people are confronted with a disease outbreak or the threat of an outbreak. all these variables combined result in what unfolds as 'the epidemic' and the diverse ways it affects communities. what a population experiences during an epidemic is not fully characterised by the numbers of known infections and deaths at the scale of a country. such numbers hide regional and local differences, especially in large and diverse countries. the epidemic reaches the different geographical areas of a country at different moments and with different intensities. it affects different communities in variable ways, influencing how these communities perceive it and react to it. what constitutes a local covid- epidemic is thus also characterised by the perceptions and the reactions it triggers in the different sections of the society. even before the virus reaches a community, the threat of an epidemic already causes fear, stress and anxiety. consequently, the threat or arrival of the epidemic also triggers responses, early or late, with various degrees of intensity and effectiveness. the response to an epidemic can be divided into individual and household actions (coping strategies), and collectively organised strategies (collective strategies). coping strategies are the actions people and families take when disease threatens and sickness occurs, including the ways they try to protect themselves from contagion. collective strategies are voluntary or mandated measures deployed by organised communities and public authorities in response to an epidemic. these include, among others, isolation of the sick or the healthy, implementation of hygiene practices and physical distancing measures. they can also include mobility restrictions such as quarantine and cordon sanitaire. coping strategies and collective strategies also include treatment of the sick, which critically depends on the availability and effectiveness of diagnostic and therapeutic tools, and performance of the health system. collective strategies also include research being deployed to further scientific insight and the development of diagnostic and therapeutic tools, potentially including a vaccine. implementation of these measures depends not only on resources available but also on the understanding and interpretation of the disease by both the scientific community and the community at large, influenced by the information people receive from scientists, public authorities and the media. this information is interpreted within belief systems and influenced by rumours, increasingly so over social media, including waves of fake news, recently labelled 'infodemics'. coping strategies and collective strategies start immediately, while there are still many unknowns and uncertainties. progressively, as the pandemic unfolds and scientists interpret observations in the laboratory, in the clinic, and in society, more insights are gained and inform the response. table lists measures recommended by the who for preventing transmission and slowing down the covid- epidemic. - 'lockdown' first employed in early in wuhan, china, is the label often given to the bundle of containment and mitigation measures promoted or imposed by public authorities, although the specific measures may vary greatly between countries. in china, lockdown was very strictly applied and enforced. it clearly had an impact, resulting in total interruption of transmission locally. this list or catalogue of measures is quite comprehensive; it includes all measures that at first sight seem to reduce transmission opportunities for a respiratory virus. however, knowledge is lacking about the effectiveness of each measure in different contexts. as a global health bmj global health agency, the who recommends a 'generic catalogue' of measures from which all countries can select an appropriate mix at any one time depending on the phase of the epidemic, categorised in four transmission scenarios (no cases, first cases, first clusters, and community transmission). however, under pressure to act and with little time to consider variable options, public authorities often adopted as 'blueprint' with limited consideration for the socioeconomic context. the initial lockdown in china thus much inspired the collective strategies elsewhere. this has been referred to as 'global mimicry', : the response is somehow partly 'copy/paste' from measures observed previously (strong path dependency). some epidemiologists in northern europe (including the uk, sweden and the netherlands ) pleaded against strict containment measures and proposed that building up herd immunity against sars-cov- might be wiser. towards early april , it became increasingly clear that reaching herd immunity in the short term was illusive. most countries thus backed off from the herd immunity approach to combating covid- and implemented lockdowns. the intensity of the lockdowns has been variable, ranging from very strict ('chinese, wuhan style'), over intermediary ('french/italian/new york city style' and 'hong kong style'), to relaxed ('swedish style'), or piecemeal. the effectiveness of lockdowns largely depends on at what stage of the epidemic they are started, and how intensively they are applied. this is quite variable across countries, depending on the understanding and motivation of the population and their perceived risk ('willingness to adhere'), on the trust they have in government advice ('willingness to comply'), and on the degree of enforcement by public authorities. the feasibility for different population groups to follow these measures depends largely on their socioeconomic and living conditions. it is obviously more difficult for people living in crowded shacks in urban slums to practise physical distancing measures and strict hand hygiene when water is scarce than for people living in wealthier parts of a city. collateral effects of the response every intervention against the covid- epidemic has a certain degree of effect and comes at a cost with collateral effects. each collective strategy ( ) has intended and unintended consequences (some are more or less desirable); ( ) is more or less feasible and/or acceptable in a given context and for certain subgroups in that society; ( ) has a cost, not only in financial terms but in many other ways, such as restrictions on movement and behaviour, stress, uncertainty and others. these costs are more or less acceptable, depending on the perception of the risk and many societal factors; ( ) can be implemented with more or less intensity; and ( ) can be enforced more or less vigorously. the balance between benefit and cost is crucial in judging whether measures are appropriate, which is very context specific. furthermore, benefits and costs are also related to the positionality from which they are analysed: benefits for whom and costs borne by whom? more wealthy societies with strong social safety nets can afford increased temporary unemployment. this is much more consequential in poorer countries, where large proportions of the population live precarious lives and where public authorities cannot implement generous mitigation measures at scale. the adherence to hygiene and distancing measures depends not only on living conditions but also on risk perception and cultural norms. mass masking has been readily accepted in some asian countries, where it was already broadly practised even before the covid- bmj global health epidemic. it remains more controversial in western societies, some of which even have legal bans on veiling in public places. lockdowns are unprecedented and have triggered intensive public debate. not surprisingly, the impact of lighter lockdowns on the transmission is much less impressive; they decrease transmission but do not stop it. quite rapidly, the justification for lockdowns shifted from stopping transmission to 'flattening the curve'. also, once a lockdown is started, rationalised, explained and enforced, it is difficult to decide when to stop it. exit scenarios, usually some form of progressive relaxation, are implemented with the knowledge that transmission will be facilitated again. what we already know the available information on sars-cov- and the spectrum of covid- disease is summarised in tables and . it is increasingly becoming clear that most transmission happens indoors and that superspreading events trigger intensive dissemination. the virology and immunology of sars-cov- / covid- are being studied intensively. this is critical not only to understand what will potentially happen in future waves but also for the development of a vaccine. some scientists and companies are very upbeat about the possibility of producing a vaccine in record time. having a vaccine is one thing, but how effective it is, is quite another. as acquired immunity after a natural infection is probably not very robust (table ), it will also be challenging to trigger robust immunity with a vaccine, but perhaps it is not impossible. many questions remain, some of which are summarised in table . regarding the severity of covid- , initial fears of very high mortality have also lessened. it has progressively become clear that many infections remain asymptomatic, that severe disease is rare in children and young adults, and that mortality is heavily concentrated in the very old and those with comorbidities. table summarises a fuller overview of the present state of knowledge regarding covid- . with covid- epidemics unfolding rapidly, several of the variables in the transmission of sars-cov- and the disease spectrum of covid- could be quantified. this allows for mathematical modelling. several models have been quickly developed, leading to predictions of the speed of transmission and the burden of covid- (box ). predictive models developed by the imperial college ; the center for disease dynamics, economics & policy and johns hopkins university ; the institute for health metrics and evaluation ; harvard university ; and the who, including an 'african model', are a few that are influencing containment strategies around the world. although the covid- pandemic triggered unprecedented research efforts globally, with over scientific papers published between january and april , there are still critical unknowns and many uncertainties. tables and summarise many of the knowns, but their relative importance or weight is not clear. for instance, the virus can spread via droplets, hands, aerosols, fomites and possibly through the environment. however, the relative importance of these in various contexts is much less clear. these factors undoubtedly vary between settings, whether in hospitals, in elderly homes, or at mass events. the weight of the variables also probably differs between the seeding and initial spread in a community and the spread when it suddenly amplifies and intensifies. the importance of each variable probably also depends on climatic conditions, not only outdoors, but also on microclimates indoors, influenced by ventilation and air conditioning and built environments. we summarise the critical unknowns in table along some elements to consider in addressing the unknowns and thoughts on their importance. uncertainty remains, leading to controversy and directly influencing the choice of containment measures. controversy continues regarding when and where lockdown or more selective measures are equally effective with lower societal effects. relationship between the dose of the initial infectious inoculum, transmission dynamics and severity of the covid- disease new evidence is being discovered rapidly. some evidence comes from field observations and ecological studies; other evidence results from scientific experiments or observations in the laboratory and the clinic. sense-making by combining insights from different observations and through the lens of various disciplines can lead to hypotheses that can be tested and verified or refuted. one such hypothesis is that there is a relationship between the dose of virus in the infectious inoculum and the severity of covid- disease. several intriguing observations in the current pandemic could be (partially) explained by such a relationship. we develop this hypothesis in box , as an example of possible further research, to create new insight which may influence control strategies. this viral inoculum theory is consistent with many observations from the early stages of the covid- pandemic, but it is not easy to test scientifically. as covid- is a new disease, we should make a distinction between ( ) the current - 'virgin soil pandemic' caused by sars-cov- , specifically in how it will further spread around the globe in the first wave, and ( ) the potential future transmission in subsequent waves. in some countries, transmission will continue at lower levels. in other countries, such as china, the virus bmj global health may have been eliminated but can be reintroduced in identical or mutated form. for the current first wave, using influenza and the common cold as reasonable comparisons, it is possible that the major epidemics, as witnessed in wuhan, northern italy, or new york, will typically occur in temperate climates in the winter season. some predict that such epidemics will last between and weeks (but this is just a plausible and reasonable comparison in analogy with seasonal influenza). it is possible that in hotter climates the transmission may become continuous, year round at lower levels. it is increasingly clear that hot climate does not exclude superspreading events as observed in guayaquil, ecuador and in various cities in brazil. ventilation, air-conditioning and crowded places may still create favourable environments for intensive transmission. it is also quite possible that the more difficult spread of sars-cov- in such climates may, in certain table knowns, uncertainties and unknowns about severe acute respiratory syndrome coronavirus (sars-cov- ), as of may origin of sars-cov- ► most probably from bats via intermediate animal hosts to index case. all subsequent cases resulted from human-to-human transmission. transmission ► mainly through respiratory droplets from infected persons ; by hands, after contamination at nose, mouth or eyes; also through air on exposure to sneezing or coughing from an infected person at close distance. ► through aerosols, while singing/talking loudly in congregations, groups, parties, karaoke, and so on, especially in poorly ventilated spaces. ► through fomites. ► possibly via faecal-oral route ; detection in sewage. [ ] [ ] [ ] ► related to peak in upper respiratory tract viral load prior to symptom onset in presymptomatic (paucisymptomatic) persons. ► transmission dynamics in asymptomatic persons not fully elucidated although viral shedding occurs. influence of climate and/or air pollution on transmission ► influence of climate on the capacity of the virus to survive outside human body (in air, in droplets, on surfaces, etc.) and to spread has been speculative. ► may spread more readily in milder/colder climate ; although variability of the reproductive number could not be explained by temperature or humidity. ► existing levels of air pollution may play a role; air pollutants, such as particulate matter, nitrogen dioxide and carbon monoxide, are likely a factor facilitating longevity of virus particles. ► elevated exposure to common particulate matter can alter host immunity to respiratory viral infections. immunity-protective antibodies ► igm and iga antibody response - days after onset of symptoms, does not depend on clinical severity, correlates with virus neutralisation; igg is observed ~ days after onset of symptoms, may or may not correspond to protective immunity. whether antibody response is long lasting has remained unclear. ► rechallenge in rhesus macaques showed immunity post primary infection. how protective immunity after first infection is against subsequent infection with an identical or mutated strain has been uncertain. ► incidental reports showed recovered persons positive by real-time pcr, later attributed to testing errors. seroprevalence to sars-cov- ► reported estimates for seroprevalence range between . % and . % ; differences in timing of the serosurvey, the use of assay kits with varying sensitivity/specificity, and different methods for detection may contribute to this large variation. ► seemingly high seroprevalence may be due to cross-reactive epitopes between sars-cov- and other hcovs. ► whether seroprevalence implies immune protection is unclear, yet, some countries have considered use of 'immunity passports'. ► for herd immunity to be effectively achieved, an estimated seroprevalence of % of the population will be required. other studies estimate between . % and % seroprevalence in different countries. communities, be compensated for by human factors such as higher population density, closer human contacts and lesser hygiene (as, for instance, exist in urban slums in mega cities in low income countries). how all this plays out in sub-saharan africa, in its slums and remote areas, is still largely unknown. with sars-cov- , transmission scenarios are mainly based on mathematical models despite their serious limitations (box ). as the virus continues to circulate, it will progressively be less of a 'new disease' during subsequent waves. the immunity caused by the first epidemic will influence how the virus spreads and causes disease. whether later waves will become progressively milder or worse, as observed in the - spanish influenza, is a matter of intense speculation. both views seem plausible and the two are not necessarily mutually exclusive. indeed, immunity should be defined on two levels: individual immunity and herd immunity. individual immunity will dictate how mild or severe the disease will be in subsequent infections. herd immunity could be defined in different communities/regions/ disease spectrum ► many different estimates: ► initially, it was estimated that among infected, % remained asymptomatic, %- % had mild/moderate disease, %- % had severe disease, and %- % became critically ill. - ► very variable estimates for remaining totally asymptomatic (estimated %- % [ ] [ ] [ ] [ ] ). ► what determines that an infection remains asymptomatic? ► quasi-absence of disease in children: why? case fatality rate (cfr) ► initial estimates cfr: %- %; comparisons: influenza . %; common cold: %; sars: %- %; mers: %. ► calculated infection fatality rates (cifr) and calculated cfr (ccfr) on the princess diamond were . % and . %, respectively (for all ages combined), and projected cifr and ccfr for china were between . %- . % and . %- . %, respectively. in gangelt, germany: ccfr of . %. ► cfr is influenced significantly by age; male sex; comorbidities; body mass index and/or fitness; and adequacy of supportive treatment, mainly oxygen therapy. if a vaccine is developed? ► what type of vaccine will it be (live/non-live, classic killed, dna, or recombinant)? ► will it need special manufacture and transport conditions (such as cold chain)? ► how robust will be vaccine-acquired immunity? after how many doses? ► how protective will it be against infection? ► for how long will vaccine-acquired immunity last? and hence: how often will the vaccine have to be administered? only once? or yearly? ► will there be any adverse effects? acquired immunity is not very strong; hence, what is the consequence regarding herd immunity? ► to achieve herd immunity, how efficient will the vaccine need to be? ► what proportion of the population (critical population) will need to be vaccinated? ► how long will it take to effectively vaccinate the critical population? ► will vaccination be acceptable in the population? or will vaccine hesitancy reduce uptake? what are the socioeconomic implications? ► which countries will get the vaccine first (implications for lics/lmics)? ► how expensive will the vaccine be? ► will vaccination be made mandatory, especially for international travel? the various degrees of societal disruption and the collateral effects on other essential health services (eg, reluctance to use health services for other health problems, because of 'corona fear'). our growing knowledge may enable us to progressively improve our response. learning from the variety of ways the covid- epidemic is unfolding across the globe provides important 'ecological evidence' and creates insights into its epidemiology and impacts. until now, the insights gained on covid- have been largely dominated by the covid- epidemics in the global north. more understanding of lived experiences of people in a variety of contexts, where the epidemic is spreading more slowly and with different impacts, is necessary to get a full global picture and allow learning from this variety. this is an important missing piece of the covid- puzzle. bmj global health and emerging voices for global health have launched a call (https:// blogs. bmj. com/ bmjgh/ / / / from-models-to-narratives-andback-a-call-for-on-the-ground-analyses-of-covid- spread-and-response-in-africa/) for such on-the-ground narratives and analyses of the spread of and response to covid- , local narratives and analyses that will hopefully help to further enrich our understanding of how and why the covid- pandemic continues to unfold in multiple local epidemics along diverse trajectories around the globe. table some critical unknowns in sars-cov- transmission which transmission patterns will occur and will human-to-human transmission continue permanently? ► seasonal transmission in temperate climate? ► continuous tides, with ups and downs? ► the experience from china and some other countries showed that 'local elimination' is possible but risk of reintroduction remains. ► increasingly unlikely that elimination everywhere is possible. this will strongly depend on: how strong will the acquired immunity after a first infection with sars-cov- be and how long will it last? ► evidence of acquired immunity against subsequent infections has been limited. ► measurable antibodies have been observed in most persons who have recovered from covid- , and research in animal models has suggested limited possibility of reinfection. ► it is still unclear as to how robust the immunity is and how long it will last. ► debate on use, practicality and ethics of 'immunity passports' for those recovered from covid- has been ongoing. how stable is the virus (mutation) and do the different clades seen worldwide have any effect on the transmission potential/severity of the disease? ► if the virus mutates quickly and different strains develop, then antibodydependent enhancement might be an important risk, as in dengue with its four different strains. if so, then in subsequent waves progressively more severe cases could occur. ► this has been reported for the spanish influenza, where the second and third waves were characterised by a more severe disease pattern. what is the role of children in transmission? ► children have quasi-universally presented less severe disease. however, their susceptibility to infection remains unclear, with large heterogeneity reported between studies. ► their role in transmission has remained unclear, but evidence points to a more modest role in transmission than adults. how significant are asymptomatic carriers in transmission? ► there have been several reports of asymptomatic transmission and estimates based on modelling. ► increasing consensus that asymptomatic carriers play an important role in transmission. box relationship between the dose of the initial infectious inoculum, transmission dynamics and severity of the covid- disease hypothesis: the dose of the virus in the initial inoculum may be a missing link between the variation observed in the transmission dynamics and the spectrum of the covid- disease. it is plausible that: ► viral dose in inoculum is related to severity of disease. ► severity of disease is related to viral shedding and transmission potential. this hypothesis plays out potentially at three levels: ► at individual level: a person infected with a small dose of viral inoculum will on average develop milder disease than a person infected with a high viral inoculum and vice versa. ► at cluster level: a person with asymptomatic infection or mild disease will on average spread lower doses of virus in droplets and aerosols and is less likely to transmit disease; when the person transmits, the newly infected person is more likely to have milder disease than if infected by a severely ill person, who spreads on an average higher doses of virus. this causes clusters and chains of milder cases or of more severe cases. ► at community level: in certain contexts, such as dense urban centres in moderate climates during the season when people live mostly indoors, the potential for intensive transmission and explosive outbreaks is high, especially during indoor superspreading events. in other contexts, such as in rural areas or in regions with hot and humid climate where people live mostly outdoors, intensive transmission and explosive outbreaks are less likely. outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle new-type coronavirus causes pneumonia in wuhan: expert a novel coronavirus from patients with pneumonia in china coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and 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isn't mutating quickly, suggesting a vaccine would offer lasting protection. the washington post genomic epidemiology of novel coronavirus implications of test characteristics and population seroprevalence on 'immune passport' strategies systematic review of covid- in children shows milder cases and a better prognosis than adults susceptibility to and transmission of covid- amongst children and adolescents compared with adults: a systematic review and meta-analysis presumed asymptomatic carrier transmission of covid- transmission of -ncov infection from an asymptomatic contact in germany acknowledgements we would like to thank johan leeuwenburg, piet kager, and luc bonneux for useful comments on a previous draft, the teams of the riposte corona, inrb, kinshasa and the belgian embassy in kinshasa for welcoming and hosting wvd during his unscheduled extended stay in kinshasa during the lockdown, march-june . we are thankful to mrs. ann byers for editing the manuscript at short notice. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.competing interests none declared. provenance and peer review not commissioned; externally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. key: cord- -p pns r authors: malik, yashpal singh; verma, atul; kumar, naveen; deol, pallavi; kumar, deepak; ghosh, souvik; dhama, kuldeep title: biotechnological innovations in farm and pet animal disease diagnosis date: - - journal: genomics and biotechnological advances in veterinary, poultry, and fisheries doi: . /b - - - - . - sha: doc_id: cord_uid: p pns r the application of innovative diagnostic technologies for the detection of animal pathogens at an early stage is essential in restricting the economic loss incurred due to emerging infectious animal diseases. the desirable characteristics of such diagnostic methods are easy to use, cost-effective, highly sensitive, and specific, coupled with the high-throughput detection capabilities. the enzyme-linked immunosorbent assay (elisa) and polymerase chain reaction (pcr) are still the most common assays used for the detection of animal pathogens across the globe. however, utilizing the principles of elisa and pcr, several serological and molecular technologies have been developed to achieve higher sensitivity, rapid, and point-of-care (poc) detection such as lateral flow assays, biosensors, loop-mediated isothermal amplification, recombinase polymerase amplification, and molecular platforms for field-level detection of animal pathogens. furthermore, animal disease diagnostics need to be updated regularly to capture new, emerging and divergent infectious pathogens, and biotechnological innovations are helpful in fulfilling the rising demand for such diagnostics for the welfare of the society. therefore, this chapter primarily describes and discusses in detail the serological, molecular, novel high-throughput, and poc assays to detect pathogens affecting farm and companion animals. livestock, poultry, and aquaculture are among the fastest growing and expanding agriculture sectors to fulfill the need of the growing population of humans. however, the growth in this sector is under the continuous increasing threats of infectious diseases worldwide. this menace is further aggravated by globalization in animal trade for various purposes. the sudden entry of an infectious disease in a new country or geographical location could lead to delayed diagnosis and rapid spread into the susceptible animal population. in response to climate change, vector-borne diseases are also increasing worldwide. to prevent the spread of infectious diseases, one of the basic and critical requirements as prescribed by the world organization of animal health (oie) is the application of rapid, accurate, and highly sensitive identification of infectious agents. though the term "biotechnology" was coined in the year by karl ereky, the tangible biotechnological advancements in improving the human and animal health were started in the late th century. since then, biotechnological applications have been making significant contributions in the development of novel powerful diagnostic assays for the efficient diagnosis and control of animal infectious diseases. importantly, biotechnology has made the availability of pen-side tests for use at the field level to detect the causative infectious agent during a disease outbreak. in this chapter, we primarily describe and discuss the innovative biotechnological advancements made in the animal disease diagnosis in a step-wise manner. the impact of infectious diseases is immense and is felt all across the world. infectious diseases have affected the whole society, economy, and political system. vital sectors are under continuous economic loss and unrelenting development. the infectious diseases have taken a huge physical toll on animals and humans. this has pressed on humanity and has caused substantial economic, social, and mental losses. thus, it is a matter of animal health and economic interest to invest in strategies to give a blow to infectious diseases and put them under control. elimination of the pathogens and/or their vectors from their natural reservoirs would always be a first thought, but the removal is not easy, as they are constantly emerging and it is always very difficult to predict the emergence of infectious agents. evolution of pathogens is putting extra challenges, pressing on humanity to look at the new strategies and forcing the researchers to look for innovative ones. the newly evolved pathogens are always more advanced and deadly from the previous ones and put up a strong resistance. "from the evolutionary perspective, they [viruses and bacteria] are 'the fittest' and the chances are slim that human ingenuity will ever get the better of them." with the increase in the knowledge of infectious diseases and science, the degree of pace in pathogen discovery has increased. to keep pace and for better diagnosis, new tools and techniques need to keep on evolving. this is not only to quickly detect the pathogens, but also to make predictions with probable and possible outbreaks. to understand the scenario and to reach a definite conclusion, knowledge of epidemiology and pathogenic etiology also needs to be studied. this will facilitate in understanding the ancestry of the pathogen, and provide an insight and a mechanism for the epidemic, endemic, and even pandemic outbreaks. this system will also assist in understanding the interface transmission between and the directional flow of the zoonotic infectious diseases. thus, phylogenetic analysis and epidemiology would aim toward strategizing the challenges during pathogen surveillance and discovery. sars, a coronavirus was pandemic in . but epidemiology and microbiology mediated to stem its disastrous results and also the causative agent of sars was identified. bacteria, viruses, and parasites, present in feces, contaminate foodstuffs and cause disease in humans and animals, affecting the social set up and consumer demands. to increase the productivity and for the maintenance of good health of animals, antibiotics are frequently administered resulting in the growth and emergence of antibiotic-resistant bacteria. this further aggravates the condition, and makes the situation more appalling. overseas imported pets were also found to transmit and carry over the diseases to humans (smith et al., ) . even aquaculture is at risk of contaminants with the virus directly affecting the marine lives, as in the case of the new virus discovered in salmon (finstad et al., ) . even honeybees and other pollinators are transmitting pathogens like fungi, bacteria, and viruses through the contaminated food items (cox-foster et al., ) . an array of classical and conventional techniques have been developed and used for the laboratory diagnosis of infectious agents or pathogens. the techniques include serological, cell culture, and electron microscopyÀbased methods, which are either time-consuming or labor-intensive or both. however, with the advancement in the biotechnology field, new and robust diagnostic techniques are continuously evolving and taking over the conventional methods (caliendo et al., ) . presently, molecular detection-based methods such as polymerase chain reaction (pcr) or its variants, and serological methods such as enzyme-linked immunosorbent assay (elisa), are being used worldwide for the accurate diagnosis of many animal diseases. however, point-of-care (poc) and high-throughput novel assays have been developed recently. furthermore, we discuss the pros and cons of frequently used diagnostics assays for animal diseases in accordance with the following sections: serological methods were introduced in the early s for the diagnosis of pathogens. various serological diagnostics have been developed such as complement fixation, counter-immunoelectrophoresis, immunofluorescence in cell culture, elisa, radio immunoassay, immune adherence haemagglutination assay, reverse passive hemagglutination assay, latex agglutination (la), chemiluminescent immunoassay, and immunochromatography test (ict). among these, ict and elisa especially sandwich elisa and competitive elisa are used frequently in the commercial diagnostic kits for animal diseases worldwide. ict assays mostly utilized mammalian igg in commercial diagnostic kits, however, avian igy antibodies, with added advantages over the mammalian igg, have been employed for the detection of norovirus, rotavirus, and astrovirus in the fecal samples with good sensitivity and specificity ranging between % and % (khamrin et al., ) . modifications in elisa format or combinations with other diagnostic methods have proved novel ways to detect pathogens more efficiently and accurately. for example, recently a novel elisa for the detection of group a rotavirus antigen in the fecal samples of multiple host species has been developed . this assay utilizes the potential use of synthetic peptides and is based on the detection of conserved vp protein using anti-recombinant vp antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immune-dominant epitopes within vp protein) antibodies as detector antibodies. another assay, which is simple to perform without the requirement of laboratory facilities, is dot-elisa. a highly sensitive and specific dot-blot assays for rapid detection of staphylococcal enterotoxin-a in food has been reported (singh et al., ) . dot-elisa has been employed in diagnosing various important poultry diseases (alam et al., ; dhama et al., ; he et al., ; majumder et al., ; manoharan et al., ) . immuno-pcr is another powerful assay that has been used for the immunodetection of viral nucleic acids. by combining elisa with pcr, sensitivity of detection can be increased up to times and is especially useful in detecting low quantity viruses in the stool samples (bonot et al., ) . the major advantage of immune-pcr is that several viral nucleic acids can be detected simultaneously. recently, a combination of nanoparticles with the immuno-pcr, also known as nanoparticle amplified immune pcr (npa-ipcr), has been reported which increases the sensitivity -folds compared to elisa and several folds to rt-pcr. antigen detection using an antibody bound to gold nanoparticle cofunctionalized with thiolated dna complementary to a hybridized dna has been developed (perez et al., ) . here, the presence of antigen/virus particles activates the formation of a "sandwich" complex of gold nanoparticle construct, virus, and an antibody functionalized nanoparticles used for extraction. now, this complex is heated to c, thus releasing dna tags followed by the detection through real-time pcr. npa-ipcr offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection. nucleic acid-based detections are used through the amplification methods, hybridization methods, which could be in situ, in vitro, and in vivo. the most common and widely used hybridization-based method is in situ hybridization, which could utilize fluorescent (fish) or chromogenic (cish) molecules. the cish-based assays for the rapid characterization of microorganisms, such as mycobacterium species and the dimorphic fungi in positive culture samples have been described (louro et al., ; scarparo et al., ) . recently, a fish-based assay has been developed for the identification and differentiation of mycobacterium tuberculosis complex from nontuberculous mycobacteria (baliga et al., ) . nucleic acid amplification methods are amongst the best in detecting pathogens with high sensitivity and specificity in the clinical samples. various modifications in nucleic acid amplification methods have provided collectively robust methods to yield better and accurate results. these modifications could be categorized into two amplification methods viz pcr and its variants, and isothermal amplification methods. these are the most common tools used for the pathogen detection worldwide. the three basic variants include ( ) real-time pcr, which is a modified version of conventional pcr, where quantification of dna sequence is possible without any further step of running the amplified product on agarose gel; ( ) multiplex pcr, where multiple sequences can be detected in a single reaction mixture, and ( ) reverse transcriptase pcr (rt-pcr) where rna is transcribed to cdna and this cdna is used in the amplification as template. the real-time pcr can utilize different fluorescence chemistries such as sybr green, taqman, or molecular beacon probes. recently, a taqman real-time rt-pcr assay has been developed for rapid detection and quantification of japanese encephalitis virus in swine blood and mosquito vectors (pantawane et al., ) . in isothermal amplification, a number of target dna copies increase at a constant temperature in just one cycle without the need of a thermocycler. various techniques have been developed using isothermal amplification methods viz nucleic acid sequence-based amplification (nasba), transcription-mediated amplification (tma), signal-mediated amplification of rna technology (smart), strand displacement amplification (sda), rolling circle amplification (rca), loopmediated isothermal amplification (lamp), isothermal multiple displacement amplification (imda), helicase-dependent amplification (hda), circular helicasedependent amplification (chda), single primer isothermal amplification (spia), and strand invasion-based amplification (siba). in all these methods, isothermal temperature amplified products can be visualized on gel through the various structures visible on gel or by the incorporation of dyes in the special structures formed on amplification serving in real-time detection. among all these techniques, lamp is the most widely used isothermal amplification method which is in fact an autocycling strand displacement dna synthesis, which deploys four primers forming a stem-loop dna by self-primed dna synthesis and a dna polymerase with strand displacement activity (malik et al., ; parida et al., ) . recently, an improved strategy using a double-labeled probe to overcome the problem of false positivity of lamp together with target gene real-time quantification is devised for detection of avian orthoreovirus (kumar et al., ) and salmonella spp. (mashooq et al., ) . other potential isothermal amplification techniques are recombinase polymerase amplification (rpa) and nasba. the former has brought a breakthrough in the detection of nucleic acids as it does not require denaturation of the template. rt-rpa is an extension of the above method, in which bacterial rt is used in the amplification of rna. rt-rpa was developed to study the outbreak of footand-mouth disease (fmd) disease in egypt (abd el wahed et al., ) . high degree of fidelity, portability, cost efficiency, simplicity, sensitivity, and tolerance to inhibitors, put this method into the category of resounding techniques, and implementation is quite easy at quarantine stations (moore and jaykus, ); while the latter one requires initial denaturation of the template followed by temperature labile polymerase dependent isothermal amplification and was designed specially to detect rna (compton, ) . a multiplex real-time nucleic acid sequence-based amplification (qnasba) system for the simultaneous detection of rotavirus a, norovirus genogroup ii/astrovirus has recently been developed (mo et al., a) . rt-nasba proved as more efficient than the conventional rt-pcr and taqman rt-pcr assays (mo et al., b) . a microarray is a multiplex lab-on-a-chip test. it is an arrangement of the large amount of biological materials for high-throughput screening on a solid support generally a glass slide, through the detection-based assays. microarray has done wonders in the high-throughput screenings and for the breakthrough causes of the outbreaks. simultaneous detections of coinfections and other more phenomenal changes during the outbreaks are the crucial developments to study the infectious diseases in endemic regions. their easiness has brought the working systems onto the platform on global diagnostics. multiple diagnostics with hybridizing ability put it at more ease to strategize the control management programs. but, this technique comes at high expenditures. data management skills and their interpretations need off to the most important tasks to be worked on. with the advent, new kits for point-of-care detections, bioelectric arrays, and liquid microarrays are in the development process. this would be an easy and an improved hybridization method for individual probe and target combinations with accurate detections. this will reduce the effort from clinical diagnosis to the personal level. these all will help in understanding the proper and common pathogens with scaling down the time. peptide nucleic acids (pnas) are highly versatile synthetic oligonucleotides, in which the native sugar-phosphate backbone of dna is replaced with amino acids. pnas bind to complementary dna strands with higher specificity and strength. furthermore, they are resistant to nucleases and proteases, making them a highly stable diagnostic reagent. the pna-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid (ray and nordén, ) . the potential diverse uses of pna have been exhaustively described in a recent review (gambari, ) . a rapid label-free visual pna-based assay for detection and pathotyping of newcastle disease virus has also been reported (joshi et al., ) . similarly, pna-based beacons have also been used in hiv genotyping with high specificity (zhang and apella, ) . aptamers are artificial nucleic acid ligands that are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. dna aptamers in particular have many advantages over antibodies (brody and larry, ) . aptamers, first reported in , are attracting interest in the areas of diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors (o'sullivan, ) . the aptamers have proved to be potential diagnostic assays, especially in the detection of toxins such as brevetoxin- , potent marine neurotoxins (shimaa et al., ) , marine biotoxinpalytoxin (shunxiang et al., ) , β-bungarotoxin (β-butx), and a neurotoxin from the venom of bungarus multicinctus (ye et al., a) . furthermore, aptamers have been used for the serological detection of mycobacterium bovis (fu et al., ) , cryptosporidium parvum (iqbal et al., ) , and prion disease (saijin et al., ) . biosensors are portable, easy to handle, ultrasensitive, quick, and may be quite specific with less probability of a false positive. biosensors work on various principles viz detecting the changes in the ph, the ion concentrations, mass by specific hybridization, enzymatic reaction, loss of functionality, change in the electrical potential, change in color, and temperature. based on these principles, many biosensors have been devised for the detection of animal pathogens; for example, an extended-gate field-effect transistor for the direct potentiometric serological diagnosis of the bhv- (tarasov et al., ) , nanowire-based immunosensor for bovine viral diarrhea virus (bvdv) (montrose et al., ) , luminescence resonance energy transferÀbased biosensors for the ultrasensitive detection of the h strain (ye et al., b) , quartz crystal microbalance (qcm)Àbased immunosensors to detect h n (li et al., ) , and spectrosenstm optical microchip sensors for foot-and-mouth disease virus (fmdv) (bhatta et al., ) . the limited methods for detection of microbial signatures and the advent of new technology for quick and parallel gene expression capacities have eased in the detection of microbial disease. next-generation sequencing (ngs) is now being increasingly applied in understanding the molecular epidemiology, transmission, and characterization of animal pathogens. instead of gene-by-gene analysis, large deposits of genes available in the clinical sample can be detected in a single test. applications of ngs are considered as more resourceful. thus, it is widely accepted as a diagnostic tool and speedily is being replaced with most other molecular diagnostic technologies and has brought revolution in the diagnosis of pathogens. various modifications and improvements have brought a huge change in the sequencing and identification of genomes. it all started with pyrosequencing on roche , with small read lengths and less efficiency. roche was followed by ion-torrent illumina platform. the ngs has made it possible to sequence the complete viral genomes of many viruses cost-effectively such as including an avian influenza virus (croville et al., ) , classical swine fever virus (leifer et al., ) , and bluetongue viruses (rao et al., ) . recently, nanopore technology, with the promising improvement has brought a wonderful efficiency with emerging science and technology (goodwin et al., ) . nanopore systems can sequence both dna and rna viral genome in real time. this technology is based on the principle that when a strand of dna/rna is allowed to pass through a nanopore, the current is changed as the based a, t, c; and c passes through the pore in different combinations. using these systems, sequencing can be performed on the portable minion device, the benchtop gridion and the high-throughput, high-sample number promethion. recently, nanopore sequencing has proved a revolutionary diagnostic tool in detecting the ebola virus (hoenen et al., ) , influenza viruses (keller et al., ; wang et al., ) and porcine viral enteric disease complexes (theuns et al., ) . overall, the biotechnological innovations have equipped us now to have high-resolution sequencing tools that are revolutionizing the ability of veterinary diagnostic laboratories to detect emerging animal pathogens. with the advent of many biotechnological advances in veterinary diagnostics, point-of-care diagnostics (pocd) are now available for economically important animal diseases. the pocd is basically a simple, rapid, and portable diagnostic device that can be applied at the field level in effective monitoring the disease status. most of the commercially available pocds utilize either antigen/antibody or nucleic acid detection technologies. the former is usually available in the format of lateral flow assays or immunochromatographic strip tests. these assays are simple to use, rapid, inexpensive, disposable, and thus make them the ideal assay for pocd for animal pathogens. the commercially available immunochromatographic strip tests for economically important animal diseases are summarized in table . . these assays are equally sensitive as compared to elisa (ferris et al., ) . furthermore, combining these assays with smartphones has made the increased sensitivity and quick reporting of results possible (yeo et al., ) . therefore, these assays offer a novel herd level surveillance tool, and provide immediate results to the farmers. however, these assays have less analytical sensitivity as compared to nucleic acid-based pocd. the real-time pcr (qpcr) is a well-established tool with high sensitivity of pathogens detection and recently, qpcr has been transitioned into pocd platform. these platforms are fully automated combining nucleic acid extraction, thermal cycling, and reporting of results on-site. for example, minilab (enigma diagnostics) is a platform ( À kg) which can be easily carried to field level and it combines silica paramagnetic-bead-based nucleic acid extraction with lyophilized qpcr reagents in a single cartridge. this platform has been validated for aiv, asfv, csfv, and fmdv (goldenberg and edgeworth, ) . however, this platform is still not available commercially. there are other platforms that do not include nucleic acid extraction step (need to be done separately), such as genesig (primerdesign ltd, united kingdom), genedrive (epistem ltd, manchester, united kingdom), cepheid smartcycler (cepheid), t-cor (tetracore), and r.a.p.i.d. (idaho technologies) (takekawa et al., (takekawa et al., , . the genesig is now supplying lyophilized qpcr assay kits for bovine, equine, porcine, avian, canine, and feline different pathogens. however, these kits are not yet licensed for diagnosis of animal pathogens and are for research purposes only. as per the agreement on trade related aspects of intellectual property rights (trips) under paragraph of article , many countries have excluded diagnostic, therapeutic, and surgical methods of humans or animals from the scope of patentable systems. however, the important patented technologies that are being used in the various formats of diagnostic assays are provided in table . . a high-speed reagent system for qpcr, full velocity technology has been developed by the stratagene which saves time in addition to highly reproducible results. this technology has been used for infectious diseases, cancer, and drug sensitivities testing and already granted five us patents, us , us , us , us , and us . besides, a pocd product, dual path platform (dpp) has been developed by the chembio diagnostic systems, on which tests to detect hiv and syphilis have already been developed. this company in collaboration with national institutes of health and the infectious disease research institute, united states is working constantly to use this platform for the detection of infectious diseases of humans and animals. farm animals reared all over the world for major agricultural and production purposes majorly include cattle, buffalo, sheep, and goats. since the last few decades, a number of infectious diseases have been found associated with farm animals, causing colossal loss to the livestock rearing community and few of them being zoonotic in nature, becoming a problem for the public health. highly contagious livestock diseases such as fmd, hemorrhagic septicemia, peste-des-petits ruminants, and surra cause irreparable economic losses. several other infectious diseases of dairy cows such as bvd, johne's disease, tuberculosis, infectious bovine rhinotracheitis, and liver fluke infestations are generally regarded as being widespread and endemic. the best known and arguably most important discovery of farm animal diseases in the last few decades is bovine spongiform encephalopathy (bse), and others include digital dermatitis, neosporosis and bovine abortion, bovine neonatal pancytopenia, arcanobacterium pluranimalium, and schmallenberg virus. among all, the world organization for animal health classifies fmd and bse as diseases of major interest in cattle. these diseases are known to have a significant effect on dairy production either directly due to death or indirectly due to effects on fertility or milk production, and subsequently, culling. the disease conditions are usually identified based on history and clinical profile of the affected population, but for affirmative diagnosis of the pathogens responsible, the identification of the causal agent is done on the samples or clinical specimens for submission to diagnostic labs. the clinical profiles of several diseases overlap, making diagnosis a little tricky and cumbersome, so initially, the isolation of the infectious agent in pure form using cell culture systems or growth on specific and selective medium became a chosen method for the diagnosis of many pathogenic diseases in farm animals. albeit their usefulness as most sensitive method of detection, they are not used routinely due to time-lapse in confirming illness (bursle and robson, ) . these methods may take hours to several weeks to obtain a confirmatory result. therefore, other approaches based on morphology/biochemical properties of pathogens took the lead and were favored for pathogen detection and identification. several infectious viral disease agents viz astrovirus, adenovirus, rotavirus, etc. were identified through electron microscopy (ong and chandran, ) . but, these also have some drawbacks like more time consuming, less sensitivity, and costly instrumentation. apart from these techniques, approaches like detection of pathogen-specific antibodies or detection of antigenic proteins of pathogens were adopted and categorized under serological assays. serological assays measure antigenÀantibody interactions for diagnostic purposes. these assays are continuously being improved with technologies like rapid strip detection, thus becoming the most preferred tools and are broadly referred to as immunoassays. enzyme immunoassays (elisa) have always been the field applicable diagnostic methods in the detection of various farm animal diseases caused by fmdv, clostridium perfringens, m. bovis, and escherichia coli. hitherto reports have shown the problem of false negative results and cross-reactivity in some of the serological methodologies. innovations including the use of synthetic biology by making highly reactive peptides help to increase the sensitivity and avoid the cross-reactivity issues to some extent. with the more recent advances in diagnostics with the availability of sequences, nucleic acid-based methods have complemented the established techniques as more specific and sensitive methods in detection of pathogens with lesser false positive results in comparison to serological-based methods (bursle and robson, ) . pcr and real-time pcr methods are regularly used in the detection of campylobacter, shigella, bovine respiratory syncytial virus, eimeria, salmonella species, and many other pathogens. likewise, seminested and nested pcr have been developed for the detection of babesia bovis and babesia bigemina. these nucleic acid-based techniques are amongst the standard detection methods and are routinely used for testing in diagnostic laboratories. to improve the efficacy and promote the simplicity, modifications in the form of isothermal amplifications, like lamp and polymerase spiral reaction (psr) have been adopted and are better in the application process, as these are easy to perform, portable, specific, sensitive, and most importantly, quick and cost-effective. lamp is established to be an apex, leading diagnostics for the detection of many farm animal-related diseases like fmd, brucellosis, bovine popular stomatitis, sheep pox, and goat pox (dukes et al., ; song et al., ; zhao et al., ) . likewise, psr has been developed for detection of brucella spp. (das et al., ) , bovine herpesvirus- (malla et al., ) , and canine parvovirus (gupta et al., ) . to further increase the sensitivity and specificity, the combination of elisa and pcr-like immune-pcr, proximity ligation assay, pcr-elisa, have been successfully discovered making the detection -fold more sensitive. the pathogen detected with these combinations includes low pathogenic strains of campylobacter (ding et al., ) . nasba, restriction fragment length polymorphism, amplified fragment length polymorphism, and random amplification of polymorphic dna, are various biotechnological tools that have been further advancing the diagnosis of various infectious diseases. in addition, ngs has brought a revolution in the diagnosis of many pathogens. it appears helpful in the identification of many pathogens, especially viruses in the fecal matter and those that could not be isolated in cell culture system. mining of sequences in samples gives varied genome sequences providing the clues of not only pathogens, but also new strains, genotypes, new viruses, and even the zoonotic efficiencies of the viruses. anis and coworkers demonstrated that targeted bovine ngs is a specific and cost-effective tool for diagnosis of major bovine pathogens in clinical samples (anis et al., ) . even pathogens with low pathogenicity such as bovine enteroviruses (bev), adenoviruses in wild captive animals, and hepatitis e viruses have been revealed in the mining of sequences in the sample. one of the approaches to disease diagnosis is the development of biosensors. assays based on biosensors uses the transducers to convert the biological interaction of pathogen with its specific antibodies to measurable signals. biosensors have been quite useful in the diagnosis in poc detection. biosensors with specific biochemical recognition helped in the identification of e. coli in cattle (dharmasiri et al., ) . colibacillosis is also seen in a variety of farm animals like cattle, pigs, and goats. in c. perfringens detection, epsilon-toxin-specific monoclonal antibody was immobilized onto single-walled carbon nanotubes and adjusted to detect relevant concentrations of toxin in nanomolars and were comparable to elisa-based results. many other methods like mass spectrometry, microarrays, and maldi-tof are also under employment for the detection of many farm animals-associated pathogens, like francisella tularensis, staphylococcus aureus, enterococcus faecalis, e. coli (demirev and fenselau, ; lundquist et al., ; van baar, ) . companion animals are the domesticated animals kept for company of human beings or for utilitarian purposes, that is, guarding, herding, military/police activity. they have grown along with the human civilization and evolution and have developed a good bond with humans. although there is a variety of species which are suitable as companion animals (dogs, cats, rabbit, ferrets, caged birds, fishes, and guinea pigs), dogs and cats are the most common companion species. their physical, behavioral, social, and emotional needs can be easily met at home. on the other hand, dogs and cats play a fundamental role in the life of human beings with many physiological and psychological benefits (wood et al., ) . also, living with companion animals makes human surroundings happier and prosperous. as these animals enrich our lives, it becomes our responsibility to take care of the companion animals and to protect them from any kind of harm. there is a spectrum of infectious diseases that occur in companion animals. lyme disease, psittacosis, hookworms, and salmonella are amongst the most common diseases in pet animals. (lembo et al., ; palatnik-de-sousa et al., ). since these animals share a close environment and are in direct contact with humans, hence they have a potential to spread these infections to human beings. chances of introduction of new diseases also arise on the import of animals from foreign lands. thus, maintenance of strict trade rules and regulations and hygienic conditions becomes a necessity. once the disease occurs, it is important to identify the causative agent to improve the effectiveness of treatment and to control the disease. since the clinical observations are not sufficient and can overlap with other diseases leading to misdiagnosis, several validated laboratory assays are used for confirmatory diagnosis. until years ago, these laboratory tests exploited cell culture (for isolation of specific pathogen) and serological assays (for detection of antibodies generated against a specific pathogen or antigenic proteins). few examples composed of vero cells and recombinant vero-slam cells used for culturing rickettsia rickettsia, and toxoplasma gondii, madin-darby canine kidney cells for canine adenovirus and canine herpesvirus. similarly, serological methods include a long list. immunodiffusion testing is most often used to detect antibodies to fungal pathogens in dogs, such as aspergillus fumigatus, coccidioides immitis, and blastomyces dermatitidis. agglutination tests include the microscopic agglutination test for serologic diagnosis of leptospirosis (agglutination of live leptospires) and the cryptococcal antigen la test (agglutination of antibody-coated latex beads). hemagglutination inhibition is used to determine antibody titers to cpv and canine influenza virus, and it evaluates the ability of serum to inhibit erythrocyte agglutination by these viruses. elisa is commonly used for the detection of feline retroviral, heartworm, giardia, leishmania, and tick-borne infections. indirect ifa for serologic testing in dogs and cats include quantitative serology for some tick-borne infectious diseases (e.g., ehrlichia canis, anaplasma spp.). direct immunofluorescence assay in veterinary medicine include diagnosis of giardia oocysts, felv within monocytes in peripheral blood or bone marrow, or canine distemper virus within epithelial cells from a conjunctival scraping. many of these assays involve the use of polyclonal antibodies, or, more commonly, monoclonal antibodies-dependent diagnosis. with the recent boom in the database of sequences for pathogens, new diagnostic tools like pcr, real-time pcr, and multiplex pcr have almost replaced the established techniques and are adopted as routine diagnostics for testing clinical samples. canine respiratory coronavirus, canine adenovirus- , canine herpesvirus, feline herpesvirus- , canine distemper virus, west nile virus, and encephalitis viruses are some of the examples, which are routinely diagnosed using these techniques. other biotechnological tools cover hybridization assays, pna, nanoparticles-based assays, etc. hybridization-based methods have also been found to be compatible with the diagnosis of many diseases and composed of taqman-based probes, molecular beacons, and fret-based probes. although not yet widely used for veterinary applications, pna probes are now increasingly available to detect target dna. fluorescent pna probes, followed by signal amplification were used to differentiate between m. tuberculosis complex and nontuberculous mycobacterium spp. (zerbi et al., ) . in another example, ngs has also been used for the comparison of the oral microbiome of canines with their owners as they are in direct contact with their pets and many diseases might get transmitted to them (oh et al., ) . gold nanoparticle-based immunochromatographic strip test using a combination of mab and pab was developed as an alternative for on-site and cost-effective diagnosis of cpv infection . another use of biotechnology has been observed for rapid and early detection of cpv using a qcm biosensor (kim et al., ) . also, for genotyping of cpv- , conventional methods are time consuming, therefore, a probe-based duplex fluorescence melting curve analysis (fmca) for genotyping six different cpv- variants (original cpv- , cpv- a, cpv- b, cpv- c, and vaccine strains of cpvpf and cpvint) using only two taqman probes has been developed (liu et al., ) . despite the fact that a wide range of diagnostic tools are available, there is a considerable chance for better advancement in diagnostics, in terms of speed and accuracy, to control and eradicate economically important diseases. in the near future, use of new biotechnological tools like biosensors and nanotechnology will pave the way. further, ngs platforms like minion (a portable, real-time ngs sequencer) coupled with nanopipe analysis are promising tools to perform bacterial and viral disease investigation in low throughput laboratories and specifically in the field (beato et al., ; shabardina et al., ) . although, yet not been adopted for animal disease diagnosis, but novel platforms such as smartphonebased diagnosis (which expands nucleic acid-based detection assays toward pocd) like rt-lamp and fluorescent lateral flow immunoassay (already developed for zika virus and dengue virus) provide exciting opportunities for veterinary diagnostics in the near future (rong et al., ) . biotechnological innovations have brought new generation diagnostic methods for rapid and sensitive diagnosis of various diseases of livestock and pet animals. infectious diseases entail remarkable economic loss, weak food production system, food insecurity, and high maintenance cost of the agriculture sectors including farm animals, poultry, and aquaculture. besides, these diseases carry a huge risk of transmission to humans as sporadic and endemic zoonoses. classical and conventional diagnostic methods are labor intensive, time consuming, less sensitive, and difficult to meet the needs of the emerging pathogen diagnostics. thus, new innovations have to be worked on and need to be practiced. over the long term, innovations will be helping in the diagnosis of pathogens with accurate, sensitive and specific detections. ngs, biosensors, and advanced amplification techniques will persist for longer periods in their constant modified forms. innovations will always be bringing the new applications in the diagnostics for the improved versions of techniques. new technique applications come with the cost and unbroken funding will be putting new prospective techniques into the trials. these techniques should be simplified in the innovations for their easy practices at the field itself, without looking for any skilled personnel/highly equipped laboratories. there is no conflict of interest. a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus dot elisa for newcastle disease, infectious bursal disease and mycoplasmosis evaluation of targeted next-generation sequencing for detection of bovine pathogens in clinical samples rapid method for detecting and differentiating mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with dna probes identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms rapid detection of foot-and-mouth disease virus with optical microchip sensors detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time pcr assay with classical tools aptamers as therapeutic and diagnostic agents non-culture methods for detecting infection better tests, better care: improved diagnostics for infectious diseases a metagenomic survey of microbes in honey bee colony collapse disorder field monitoring of avian influenza viruses: whole-genome sequencing and tracking of neuraminidase evolution using pyrosequencing rapid visual isothermal nucleic 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acids: a review on recent patents and technology transfer the enigma ml fluabÀrsv assay: a fully automated molecular test for the rapid detection of influenza a, b and respiratory syncytial viruses in respiratory specimens polymerase spiral reaction (psr): a novel, visual isothermal amplification method for detection of canine parvovirus genomic dna complementary monoclonal antibody-based dot elisa for universal detection of h avian influenza virus nanopore sequencing as a rapidly deployable ebola outbreak tool detection of cryptosporidium parvum oocysts on fresh produce using dna aptamers rapid label-free visual assay for the detection and quantification of viral rna using peptide nucleic acid (pna) and gold nanoparticles (aunps) direct rna sequencing of the coding complete influenza a virus genome evaluation of a rapid immunochromatography strip test for detection of astrovirus in stool specimens a novel assay for detecting canine parvovirus using a quartz crystal microbalance biosensor peptide-recombinant vp protein based enzyme immunoassay for the detection of group a rotaviruses in multiple host species a double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus approaches to define the viral genetic basis of classical swine fever virus virulence the feasibility of canine rabies elimination in africa: dispelling doubts with data a nanobeads amplified qcm immunosensor for the detection of avian influenza virus h n application of duplex fluorescence melting curve analysis (fmca) to identify canine parvovirus type variants direct identification of mycobacterium avium complex and mycobacterium gordonae from mb/bact bottles using accu probe discrimination of francisella tularensis subspecies using surface enhanced laser desorption ionization mass spectrometry and multivariate data analysis development of recombinant σb protein based dot-elisa for diagnosis of avian reovirus (arv) rapid detection of human rotavirus using nsp gene specific reverse transcription loop-mediated isothermal amplification assay novel polymerase spiral reaction (psr) for rapid visual detection of bovine herpesvirus genomic dna from aborted bovine fetus and semen rapid serological profiling by an immunocomb-based dot-enzyme-linked immunosorbent test for three major poultry diseases development and evaluation of probe based real time loop mediated isothermal amplification for salmonella: a new tool for dna quantification rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification comparative detection of rotavirus rna by conventional rt-pcr, taqman rt-pcr and real-time nucleic acid sequence-based amplification novel single gold nanowire-based electrochemical immunosensor for rapid detection of bovine viral diarrhoea antibodies in serum development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses comparison of the oral microbiomes of canines and their owners using next-generation sequencing identification of gastroenteric viruses by electron microscopy using higher order spectral features aptasensors À the future of biosensing? decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with leishmune in brazilian endemic areas taqman real-time rt-pcr assay for detecting japanese encephalitis virus in swine blood samples and mosquitoes loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction deep sequenc-ing as a method of typing bluetongue virus isolates peptide nucleic acid (pna): its medical and biotechnical applications and promise for the future smartphone-based fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of zika virus nonstructural protein aptamer-based assay for prion diseases diagnostic direct identification of mycobacteria from mb/bact alert d bottles: comparative evaluation of two commercial probe assays nanopipe-a web server for nanopore minion sequencing data analysis development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus aptamer-based competitive electrochemical biosensor for brevetoxin- enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin development and evaluation of simple dotÀblot assays for rapid detection of staphylococcal enterotoxin-a in food establishment of loop-mediated isothermal amplification (lamp) for rapid detection of brucella spp. and application to milk and blood samples field detection of avian influenza virus in wild birds: evaluation of a portable rrtÀpcr system and freeze-dried reagents rapid diagnosis of avian influenza virus in wild birds: use of a portable rrtÀpcr and freeze-dried reagents in the field a potentiometric biosensor for rapid on-site disease diagnostics nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry minion nanopore sequencing of an influenza genome the pet connection: pets as a conduit for social capital? recognition of bungarus multicinctus venom by a dna aptamer against β-bungarotoxin upconversion luminescence resonance energy transfer (lret)-based biosensor for rapid and ultrasensitive detection of avian influenza virus h subtype smartphone-based fluorescent diagnostic system for highly pathogenic h n viruses amplified in situ hybridization with peptide nucleic acid probes for differentiation of mycobacterium tuberculosis complex and non-tuberculous mycobacterium species on formalin-fixed, paraffin embedded archival biopsy and autopsy samples advantages of peptide nucleic acids as diagnostic platforms for detection of nucleic acids in resource-limited settings development of loopmediated isothermal amplification assay for specific and rapid detection of differential goat pox virus and sheep pox virus all the authors of the chapter thank and acknowledge their respective universities and institutes. key: cord- - eycqf o authors: robertson, colin; nelson, trisalyn a.; macnab, ying c.; lawson, andrew b. title: review of methods for space–time disease surveillance date: - - journal: spat spatiotemporal epidemiol doi: . /j.sste. . . sha: doc_id: cord_uid: eycqf o a review of some methods for analysis of space–time disease surveillance data is presented. increasingly, surveillance systems are capturing spatial and temporal data on disease and health outcomes in a variety of public health contexts. a vast and growing suite of methods exists for detection of outbreaks and trends in surveillance data and the selection of appropriate methods in a given surveillance context is not always clear. while most reviews of methods focus on algorithm performance, in practice, a variety of factors determine what methods are appropriate for surveillance. in this review, we focus on the role of contextual factors such as scale, scope, surveillance objective, disease characteristics, and technical issues in relation to commonly used approaches to surveillance. methods are classified as testing-based or model-based approaches. reviewing methods in the context of factors other than algorithm performance highlights important aspects of implementing and selecting appropriate disease surveillance methods. early detection of unusual health events can enable coordinated response and control activities such as travel restrictions, movement bans on animals, and distribution of prophylactics to susceptible members of the population. our experience with severe acute respiratory syndrome (sars), which emerged in southern china in late and spread to over countries in months, indicates the importance of early detection (banos and lacasa, ) . disease surveillance is the principal tool used by the public health community to understand and manage the spread of diseases, and is defined by the world health organization as the ongoing systematic collection, collation, analysis and interpretation of data and dissemination of information in order for action to be taken (world health organization, ) . surveillance systems serve a variety of public health functions (e.g., outbreak detection, control planning) by integrating data representing human and/or animal health with statistical methods (diggle, ) , visualization tools (moore et al., ) , and increasingly, linkage with other geographic datasets within a gis (odiit et al., ) . surveillance systems can be designed to meet a number of public health objectives and each system has different requirements in terms of data, methodology and implementation. outbreak detection is the intended function of many surveillance systems. in syndromic surveillance systems, early-warning signals are provided by analysis of pre-diagnostic data that may be indicative of people's care-seeking behaviour during the early stages of an outbreak. in contrast, systems designed to monitor food and water-borne (e.g., cholera) pathogens are designed for case detection, where one case may trigger a response from public health workers. similarly, where eradication of a disease in an area is a public health objective, surveillance may be designed primarily for case detection. alternatively, where a target disease is endemic to an area, perhaps with seasonal variation in incidence, such as rabies, monitoring space-time trends may be the primary surveillance objective (childs et al., ) . surveillance systems differ with respect to a number of qualities which we term contextual factors. for evaluation of surveillance systems, this is well known, as the evaluative framework set out by the centre for disease control and prevention (cdc) encompasses assessment of simplicity, flexibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness, and stability (buehler et al., ) . selection of appropriate methods for space-time disease surveillance should consider system-specific factors indicative of the context under which they will be used (table ) . these factors are summarized in table , and are the axes along which we will review methods for space-time disease surveillance. there has been rapid expansion in the development of automated disease surveillance systems. following the bioterrorism attacks in the united states, there was expanded interest and funding for the development of electronic surveillance networks capable of detecting a bioterrorist attack. many of these were designed to monitor data that precede diagnoses of a disease (i.e., syndromic surveillance). by may there were an estimated syndromic surveillance systems in development throughout the us (buehler et al., ) . due to the noisy nature of syndromic data, these systems rely heavily on advanced statistical methods for anomaly detection. as data being monitored in syndromic systems precede diagnoses they contain a signal that is further removed from the pathogen than traditional disease surveillance, so in addition to having potential for early warning, there is also greater risk of false alarms (i.e., mistakenly signaling an outbreak) (stoto et al., ) . one example is a national surveillance system called biosense developed by the cdc in the united states. bio-sense is designed to support early detection and situational awareness for bioterrorism attacks and other events of public health concern (bradley et al., ) . data sources used in biosense include veterinary affairs and department of defense facilities, private hospitals, national laboratories, and state surveillance and healthcare systems. the broad mandate and national scope of the system necessitated the use of general statistical methods insensitive to widely varying types, quality, consistency and volume of data. two methods used in biosense are a generalized linear mixed-model which estimates counts of syndrome cases based on location, day of the week and effects due to seasonal variation and holidays. counts are estimated weekly for each syndrome-location combination. a second temporal surveillance approach computed for each syndrome under surveillance is a cumulative sum of counts where events are flagged as unusual if the observed count is two standard deviations above the moving average. the selection of surveillance methods in biosense considered factors associated with heterogeneity of data sources and data volume among others. another example is provided by a state-level disease surveillance system developed for massachusetts called the automated epidemiological geotemporal integrated surveillance (aegis) system, where both time-series modelling and spatial and space-time scan statistics are used (reis et al., ) . the modular design of the system allowed for 'plug-in' capacity so that functionality already implemented in other software (i.e., satscan) could be leveraged. in aegis, daily visit data from emergency department facilities are collected and analyzed. the reduced data volume and greater standardization enable more advanced space-time methods to be used as well as tighter integration with the system's communication and alerting functions (reis et al., ) . decisions on method selection and utilization are based on a variety of factors, yet most reviews of statistical methods for surveillance data compare and describe algorithms from a purely statistical or computational perspective (e.g., buckeridge et al., ; sonesson and bock, ; . the selection of statistical approaches to surveillance for implementation as part of a national surveillance system is greatly impacted by design constraints due to scalability, data quality and data volume whereas the use of surveillance data for a standalone analysis by a local public health worker may be more impacted by software availability, learning curve, and interpretability. selection of appropriate statistical methods is key to enabling a surveillance system to meet its objectives. a frequently cited concern of surveillance systems is how to evaluate whether they are meeting their objectives (reingold, ; sosin and dethomasis, ) . a framework for evaluation developed by the cdc considers outbreak detection a function of timeliness, validity, and data quality (buehler et al., ) . the degree to which these factors contribute to system effectiveness may vary for different surveillance systems, especially where objectives and system experiences differ. for example, newly developed systems in developing countries may place a table contextual factors for evaluation of methods for space-time disease surveillance. the spatial and temporal extent of the system (e.g., local/regional/national/ international) scope the intended target of the system (e.g., single disease/multiple disease, single host/multiple host, known pathogens/unknown pathogens) function the objective(s) of the systems (outbreak detection, outbreak characterization, outbreak control, case detection, situational awareness (mandl et al., ; buehler et al., ) , biosecurity and preparedness (fearnley, ) ) disease characteristics is the pathogen infectious? is this a chronic disease? how does it spread? what is known about the epidemiology of the pathogen? technical the level of technological sophistication in the design of the system and its users (data type and quality, algorithm performance, computing infrastructure and/or reliability, user expertise) greater emphasis on evaluating data quality and representativeness, as little is known about the features of the data streams at early stages of implementation (lescano et al., ) . algorithm performance is usually measured by sensitivity, specificity and timeliness. sensitivity is the probability of an alarm given an outbreak, and specificity is the probability of no alarm when there is no outbreak. timeliness is measured in number of time units to detection, and has been a focus of systems developed for early outbreak detection (wagner et al., ) . the importance of each of these measures of performance need to be evaluated in light of the system's contextual factors outlined in table . our goal in this review of approaches to space-time disease surveillance is to synthesize major surveillance methods in a way that will focus on the feasibility of implementation and highlight contrasts between different methods. first, we aim to place methods in the context of some key aspects of practical implementation. second, we aim to highlight how methods of space-time disease surveillance relate to different surveillance contexts. disease surveillance serves a number of public health functions under varying scenarios and methods need to be tailored and suited to particular contexts. finally, we provide guidance to public health practitioners in understanding methods of space-time disease surveillance. we limit our focus to methods that use data encoded with both spatial and temporal information. this paper is organized as follows. the next section describes space-time disease surveillance. following, is a description of different statistical approaches to spacetime disease surveillance with respect to the contextual factors outlined in table . we conclude with a summary and brief discussion of our review. methods for space-time disease surveillance can address a surveillance objective in a variety of ways. most methods assume a study area made up of smaller, nonoverlapping sub-regions where cases of disease are being monitored. the variable under surveillance is the count of the number of cases. in retrospective analysis, the data are fixed and methods are used to determine whether an outbreak occurred during the study period, or characterize the spatial-temporal trends in disease over the course of the study period (marshall, ) . in the prospective scenario, the objective is to determine whether any single sub-region or collection of sub-regions is undergoing an outbreak (currently), and analysis occurs in an automated, sequential fashion as data accumulate over time. prospective methods require special consideration as data do not form a fixed sample from which to make inferences about (sonesson and bock, ) . parallel surveillance methodologies compute a test statistic separately for each sub-region and signal an alarm if any of sub-regions are significantly anomalous (fig. a) . while in vector accumulation methods, test statistics in a parallel surveillance setting are combined to form one general alarm statistic (fig. b) . conversely, a scalar accumulation approach com-putes one statistic over all sub-regions for each time period (frisen and sonesson, ) (fig. c ). for example, rogerson ( ) used the tango ( ) statistic to monitor changes in spatial point patterns. statistical tests in space-time disease surveillance generally seek to determine whether disease incidence in a spatially and temporally defined subset is unusual compared to the incidence in the study region as a whole. thus, this class of methods is designed to detect clusters of disease in space and time, and suit surveillance systems designed for outbreak detection. most spatial cluster detection methods such as the geographical analysis machine (openshaw et al., ) , density estimation (bithell, ; lawson and williams, ), turnbull's method (turnbull et al., ) , the besag and newell ( ) test, spatial autocorrelation methods such as the gi * (getis and ord, ) , and lisas (anselin, ) , and the spatial scan statistic (kulldorff and nagarwalla, ) are types of statistical tests. the development of methods for space-time cluster detection naturally evolved from these purely spatial methods. we can stratify methods in the statistical test class into three types: tests for space-time interaction, cumulative sum methods, and scan statistics. space-time interaction of disease indicates that the cases cluster such that nearby cases in space occur at about the same time. the form of the null hypotheses is usually conditioned on population, and can factor in risk covariates such as age, occupation, and ethnicity. detecting the presence of space-time interaction can be a step towards determining a possible infectious etiology for new or poorly understood diseases (aldstadt, ) . additionally, non-infectious diseases exhibiting space-time interaction may suggest the presence of an additional causative agent, such as a point source of contamination and/or pollution or an underlying environmental variable. these methods require fixed samples of space-time data representing cases of disease. all tests for space-time interaction consider the number of cases of disease that are related in space-time, and compare this to an expectation under a null hypothesis of no interaction (kulldorff and hjalmars, ) . the knox test ( ) uses a simple test statistic which is the number of case pairs close both in space and in time. this count is compared to the null expectation conditional on the number of pairs close only in space, and the number of pairs close only in time; i.e., the times of occurrence of the cases are independent of case location. a major shortcoming of the knox ( ) method is that the definition of ''closeness" is arbitrary. mantel's ( ) test addresses this by summing across all possible space-time pairs, while diggle et al. ( ) identify clustering at discrete distance bands in the space-time k function. for infectious diseases, it is likely that near space-time pairs are of greater importance, so mantel suggests a reciprocal transformation such that distant pairs are weighted less than near pairs. the mantel test can in fact be used to test for association between any two distance matrices, and is often used by ecologists to test for interaction between space and another distance variable such as genetic similarity (legendre and fortin, ) . the reciprocal transformation used in the mantel statistics assumes a distance decay effect. while this may be appropriate for infectious diseases, for non-infectious diseases or diseases about which little is known, this assumed functional form of disease clustering may be inappropriate. a different approach is taken by jacquez ( ) where relations in space and time are defined by a nearest neighbour relation rather than distance. here, the test statistic is defined by the number of case pairs that are k nearest neighbours in both space and time. when space-time interaction is present, the test statistic is large. another method for testing an infectious etiology hypothesis given by pike and smith ( ) , assesses clustering of cases relative to another control disease, though selection of appropriate controls can be difficult. the scale of the disease surveillance context can impact the selection of space-time interaction tests because these tests are sensitive to changes in the underlying population at risk (population shift bias). therefore, large temporal scales will be more likely to exhibit changes in population structure and introduce population shift bias. an unbiased version of the knox test given by kulldorff and hjalmars ( ) accounts for this by adjusting the statistic by the space-time interaction inherent in the background population. changes in background population over time can be incorporated into all space-time interaction tests using a significance test based on permutations conditioned on population changes. however, this obviously requires data on the population over time which may not always be easy to obtain. space-time interaction tests are univariate and therefore only suitable for testing cases of a single disease. consideration of multiple host diseases is possible, though there is no mechanism to test for interaction or relationships between different host species. another major consideration is the function of the surveillance system or analytic objective. interaction tests can only report the presence or absence of space-time interaction. they give no information about the spatial and temporal trends in cases, nor consider naturally occurring background heterogeneity. a final point is that these tests use case data, and therefore require geo-coded singular event data, making these methods unsuitable when disease data are aggregated to administrative units. cumulative sum methods for space-time surveillance developed out of traditional statistical surveillance applications such as quality control monitoring of industrial manufacturing processes. in cusum analysis, the objective is to detect a change in an underlying process. in application to disease surveillance, the data are in the form of case counts for sub-regions of a larger study area. a running sum of deviations is recalculated at each time period. for a given sub-region, a count y t of cases at time t is monitored as follows where s t is the cumulative sum alarm statistic, k is a parameter which represents the expected count, so that observed counts in exceedence of k are accumulated. at each time period, an alarm is signalled if s t is greater than a threshold parameter h. if a cusum is run long enough, false alarms will occur as exceedences are incrementally accumulated. the false-positive rate is controlled by the expected time it takes for a false alarm to be signalled, termed the in-control average run length, denoted arl . the arl is directly related to the threshold value for h, which can be difficult to specify in practice. high values of h yield long arl and vice versa. in practice, approximations are used to estimate a value for h for a chosen arl (siegmund, ) , though this remains a key issue in cu-sum methods. the basic univariate cusum in ( ) can be extended to incorporate the spatial aspect of surveillance data. in this sense, cusum is a temporal statistical framework around which a space-time statistical test can be built. in an initial spatial extension, rogerson ( ) coupled the (global) tango statistic ( ) for spatial clustering in a cusum framework. for a point pattern of cases of disease, compute the spatial statistic, and use this value of the statistic to condition the expected value at the next time period. observed and expected values are used to derive a z-score which is then monitored as a cusum (rogerson, a) . one scalar approach taken by rogerson ( b) is to monitor only the most unexpected value, or peak, of each time period as a gumbel variate (gumbel distribution is used as a statistical distribution for extreme values). an additional approach is to compute a univariate cusum in a parallel surveillance framework (woodall and ncube, ) . here the threshold parameter h must be adjusted to account for the multiple tests occurring across the study area. yet this approach takes no account of spatial relationships between sub-regions (i.e., spatial autocorrelation). cusum surveillance of multiple sub-regions can be considered a multivariate problem where a vector of differences between the observed and expected counts for each sub-region is accumulated. spatial relationships between sub-regions can be incorporated by explicitly modelling the variance-covariance matrix. rogerson and yamada ( ) demonstrate this approach by monitoring a scalar variable representing the multivariate distance of the accumulated differences between observed and expected over all sub-regions. this is modelled as , and p is a variance-covariance matrix capturing spatial dependence, and s t is a  p vector of differences between observed and expected cases of disease in time t for each p sub-region (rogerson and yamada, ) . cusum methods are attractive for prospective disease surveillance because they offer a temporal statistical framework within which spatial statistics can be integrated. they therefore overcome one of the limitations of traditional spatial analysis applied to surveillance in that repeated testing over time (and space) can be corrected for. a full description of the inferential properties of the cusum framework is given by rogerson ( a) . these methods are therefore most appropriate for long temporal scales, especially when historical data are used to estimate the baseline. multivariate cusum given by rogerson and yamada ( ) is for a singular disease over multiple sub-regions, but could be used to monitor multiple diseases over multiple sub-regions. this may be most applicable in a syndromic surveillance application. the simplicity of univariate cusum makes training and technical expertise less of a factor than the multivariate case. multivariate cusum is also more difficult to interpret and specification of the threshold parameter requires simulation experimentation or a large temporal extent from which to establish a baseline. scan statistics developed originally for temporal clustering by naus ( ) test whether cases of disease in a temporally defined subset exceed the expectation given a null hypothesis of no outbreak. the length of the temporal window is varied systematically in order to detect outbreaks of different lengths. this approach was first extended to spatial cluster detection in the geographical analysis machine (openshaw et al., ) . the spatial approach looks for clusters by scanning over a map of cases of disease using circular search areas of varying radii. kulldorff and nagarwalla ( ) refined spatial scanning with the development of the spatial scan statistic which adjusts for the multiple testing of many circular search areas. the spatial scan statistic overcomes the multiple-testing problem (common to many local spatial analysis methods) by taking the most likely cluster defined by maximizing the likelihood that the cases within the search area are part of a cluster compared to the rest of the study area. significance testing for this one cluster is then assessed via monte carlo randomization. secondary clusters can be assessed in the same way and ranked by p-value. in kulldorff ( ) , the spatial scan statistic is extended to space-time, such that cylindrical search areas are used where the spatial search area is defined by cylinder radius, and the temporal search area is defined by cylinder height. in prospective analysis, candidate cylinders are limited to those that start at any time during the study period and end at the current time period (i.e., alive clusters). significance is determined through randomization and comparing random permutations to the likelihood ratio maximizing cylinder in the observed data. an additional consideration to take account of multiple hypothesis testing over time (correlated sequential tests) is given by including previously tested cylinders (which may be currently 'dead') in the randomization procedure (kulldorff, ) . the space-time scan statistic (kulldorff, ) approaches the surveillance problem in a novel way and aptly handles some key shortcomings of other local methods (multiple testing, locating clusters, pre-specifying cluster size). however, a limitation is that the expectation is conditional on an accurate representation of the underlying population at risk, data which may be hard to obtain. in long term space-time surveillance scenarios, accurate population estimates between decennial censuses are rare or must be interpolated. in syndromic applications, where cases are affected by unknown variations in care-seeking behaviours, the raw population numbers may not accurately reflect the at-risk population. in kulldorff et al. ( ) , the expected value for each unit under surveillance is estimated from historical case data rather than population data. generating the expected value from the history of the process under surveillance is most suitable for real-time prospective surveillance contexts where the current state of the process is of interest. this extension allows the application of the space-time scan statistic in a wider range of surveillance applications. a remaining limitation of the cylindrical space-time scan statistic is the use of circular search area over the map. the power of the scan statistics that use circularbased search areas decline as clusters become more irregular in shape, for example, for cases clustered along a river valley or where disease transmission is linked to the road network. the spatial scan statistic has been extended to detect irregularly-shaped clusters in patil and taillie ( ) and tango and takahashi ( ) . extensions of these approaches to space-time are active areas of research. a space-time version of the tango and takahashi ( ) method uses spatial adjacency of areal units added incrementally up to k nearest neighbour units which are connected through time to form -dimensional prism search areas (takahashi et al., ) . a similar approach is given by costa et al. ( ) . however, these methods are very computationally intensive. scan statistics are one of the most widely used statistical methods for outbreak detection in surveillance systems. space-time scan statistics are able to detect and locate clusters of disease, and can condition expected counts for individual sub-regions on population data or on previous case data, making these methods suitable for implementation where data volume is large. the scope of scan statistics, like most statistical tests, is limited to monitoring case data, either case event point data or counts by sub-region. scan statistics are best served to detect and locate discrete localized outbreaks. secondary clusters can be identified by ranking candidate clusters by their likelihood ratio. yet region-wide outbreaks cannot be detected with scan-statistics because of the assumed form of a cluster as a compact geographical region where cases are greater than expected. novel space-time methods that search for raised incidence via graph-based connectivity may model spatial relationships of disease processes more accurately than circular search areas. however, the computational burden and complexity of these approaches limits their use to expert analysts and researchers. at the root of the problem is a conceptual discrepancy between the definition of a disease outbreak (which disease surveillance systems are often interested in detecting) and a disease cluster (defined by spatial proximity) which is common to all statistical testing methods for space-time surveillance (lawson, ) . model-based approaches to surveillance developed recently as the need emerged to include other variables into the specification of our expectation of disease incidence. for example, we often expect disease prevalence to vary with age, gender, and workplace of the population under surveillance. statistical models allow for these influences to adjust the disease risk through space and time. a second impetus for the development of statistical models for disease surveillance is that a large part of epidemiology concerned with estimating relationships between environmental variables and disease risk (i.e., ecological analysis) provided a methodological basis from which to draw. modelling for space-time disease surveillance is relatively recent, and this is a very active area of statistical surveillance research. again we stratify statistical models into three broad classes: generalized linear mixed models, bayesian models, and models of specific space-time processes. generalized linear mixed models (glmm) offer a regression-based framework to model disease counts or rates using any of the exponential family of statistical distributions. this allows flexibility in the expected distribution of the response variable, as well as flexibility in the relationship between the response and the covariate variables (the link function). one application of this approach to prospective disease surveillance for detection of bioterrorist attacks is given by kleinman et al. ( ) . here, the number of cases of lower respiratory infection syndromes in small geographic areas act as a proxy for possible anthrax inhalation. a glmm approach is used to combine fixed effects for covariate variables (i.e., season, day of the week) with a random effect that accounts for varying baseline risks in different geographic areas. in kleinman et al. ( ) , the logit link function is used in a binomial logistic model to estimate the expected number of cases y it in area i for time t. this is a function of the probability of an individual being a case in area i at time t and the number of people n it in area i at time t. this expectation is conditional on a location specific random effect b i and is then converted to a z-score and evaluated to determine if it is unusual (i.e., an emerging cluster). this approach was extended to a model using poisson random effects in kleinman ( ) . the use of glmm in prospective surveillance has also been suggested for use in west nile virus surveillance due to the ease with which covariates can be included and flexibility in model specification (johnson, ) . the glmm approach has attractive advantages as a flexible modelling tool. particularly, relaxation of distributional assumptions, flexibility in link functions, and the ability to model spatial relationships (at multiple spatial scales) as random effects make glmm useful for prospec-tive space-time disease surveillance. the scale and scope of the surveillance context does not limit a model-based approach, and models may be even more useful when data abnormalities such as time lags occur (as estimates can be based on covariates alone). one feature of glmm that are important for many disease surveillance contexts are the ease with which spatial hierarchies can be incorporated. ecological relationships that are structured hierarchically that impact disease emergence (e.g., climate, vegetation, vector life-cycle development) can be represented and accounted for. further, human drivers of disease emergence (e.g., land-use policies, travel patterns, demographics) are often organized hierarchically through administrative units. in social sciences glmms are often used (i.e., multi-level models) that incorporate these 'contextual effects' on an outcome variable. a further advantage of glmms is their ability to incorporate spatial variation in the underlying population at risk by conditioning the expected value on the random effect component (b i in eq. ( )). where fewer people are present, the expected value is adjusted toward the mean. this can somewhat account for the small-numbers problem of smrs in epidemiology, reducing the likelihood of estimating extremely low expected values in rural areas. bayesian models have been used extensively in disease mapping studies (best et al., ; lawson, ). analysis of disease in a bayesian framework centers around inference on unknown area-specific relative risks. inference on this unknown risk distribution is based on the observed data y and a prior distribution. these are combined via a likelihood function to create a distribution for model parameters which can be sampled for prediction. bayesian models have been applied for retrospective space-time surveillance (e.g., macnab, ) and are now being developed for prospective space-time disease surveillance. the basic bayesian model can incorporate space and time dependencies. in abellan et al. ( ) a model is described where the counts of disease are taken to be binomial distributed, and the next level of the model is composed of a decomposition of the unknown risks into model parameters for general risk, spatial effects, temporal effects, and space-time interaction. estimation requires specifying prior distributions for each of the model components and sampling the posterior distribution via monte carlo markov chain (mcmc) methods. here, the authors describe space-time bayesian models for explanation of overall patterns of disease, speculating on their use in disease surveillance contexts. rodeiro and lawson ( a) offer a similar model based on a poisson distributed disease count. specifically, the counts y i are poisson with mean a function of the expected number of cases e ij in location i at time j and the area-specific relative risk rr ij . similar to abellan et al. ( ) , the log (rr ij ) are decomposed into spatial effects u i , uncorrelated heterogeneity v i , temporal trend t j , and space-time interaction c ij : again, these components need prior distributions specified. for the spatial correlation term, a conditional autoregressive model (car) is suggested for modelling spatial autocorrelation. residuals are then extracted from model predictions for incoming data and can be used to assess how well the data fits the existing model. as discussed in rodeiro and lawson ( a) , monitoring residuals in this way makes the detection of specific types of disease process change feasible by adjusting how residuals are evaluated. while adding to the complexity of the analysis, this may be of great use in a surveillance application. alternative proposals such as bayesian cluster models with ''a priori" cluster component for spatiotemporal disease counts was developed by yan and clayton ( ) . more recently, bayesian and empirical bayes semi-parametric spatiotemporal models with temporal spline smoothing were developed for the analysis of univariate spatiotemporal small area disease and health outcome rates (macnab, a; macnab and gustafson, ; ugarte et al., ) and multivariate spatiotemporal disease and health outcome rates (macnab, b) . tzala and best ( ) also proposed bayesian hierarchical latent factor models for the modelling of multivariate spatiotemporal cancer rates. these spatiotemporal models, with related bayesian and empirical bayes methods of inference, may also be considered for disease surveillance applications. the statistical methodology for applying bayesian models to surveillance in space-time is still being developed, and as such these approaches are suited primarily to researchers. bayesian models are attractive because they allow expert and local knowledge of disease processes to be incorporated via the specification of prior distributions on model parameters. however, this can also be a drawback, as a subjective element is introduced to the model. it is generally recommended that sensitivity analysis be conducted on a variety of candidate priors for model parameters (e.g., macnab and gustafson, ; macnab, a) . these technical aspects of model-fitting require advanced statistical training. a further complexity of bayesian models is estimation. mcmc methods are required for generating the posterior distributions for these types of models and are computationally very demanding (although see rodeiro and lawson, b) . this might negate the use of these approaches in surveillance contexts that require daily refitting of models (i.e., fine temporal resolution), however, monthly or annual model refitting may be possible. as with glmms, bayesian models lend themselves to modelling hierarchical spatial relationships, and this can be important for both ecological and humanmediated drivers of disease emergence. some modelling approaches to surveillance have been designed to model specific types of spatial processes, generally represented as a realization from a statistical distribution. while all models require some distributional assumptions, those considered here purport to associate specific statistical processes with disease processes in the context of surveillance. in held et al. ( ) , a model is based on a poisson branching process whereby outcomes are dependent on both model parameters describing a particular property (e.g., periodicity) and past observed data. spatial and space-time effects can also be included as an ordinary multivariate extension. a useful aspect of this formulation for disease surveillance is the separation of the disease process at time t into two parts: an endemic part v and an epidemic part with conditional rate ky tÀ the endemic component can also be adjusted for seasonality, day of the week effects and other temporal trends. extended to the multivariate case, the model becomes where the endemic rate adjusted by the number of people in area i at time t, and area-specific previous model estimates for the epidemic part. spatial dependence can be incorporated by adding a spatial effects term that accounts for correlated estimates in ky i;tÀ via a weights matrix. however, this type of model yields separate parameters for each geographical unit. a point process methodology for prospective disease surveillance is presented in diggle et al. ( ) . point data representing cases are modelled with separate terms for spatial variation, temporal variation, and residual spacetime variation. the method is local, in the sense that recent cases are used for prediction, producing continuously varying risk surfaces. however, there are also global model parameters which estimate the background variation in space and time estimated from historical data. outbreaks are defined when variation in the residual space-time process exceeds a threshold value c. different values for the threshold parameter are evaluated and exceedence probabilities are mapped. model parameters are fixed allowing the model to be run daily on new data. however, as noted in diggle et al. ( ) , this may fail to capture unknown temporal trends, and periodic refitting may be required. a different approach is given by järpe ( ) , which instead of decomposing the process into separate components, monitors a single parameter of spatial relationships in a surveillance setting. this is similar in spirit to rogerson's work (rogerson, ) monitoring point patterns with spatial statistics, though here a specific underlying process is assumed: the ising model. the ising model represents a binary-state two dimensional lattice (sites coded or ). there are two parameters for the ising model; one governs the overall intensity (probability of a site being a ), and another the spatial interaction (probability of nearby sites being alike). in järpe ( ) , the intensity parameter is assumed equal and unchanging, and the surveillance is performed on the interaction parameter under different lattice sizes and types of change. the interaction parameter is essentially a global measure of spatial autocorrelation. this can then be monitored using temporal surveillance statistics such as cusum. since the properties of the underlying model are known, järpe is able to detect very small changes in spatial autocorrelation which could indicate the shift of a disease from endemic to epidemic. while significant spatial autocorrelation is often present at both endemic and epidemic states, changes in clustering can reveal threshold dynamics of the process in a surveillance setting. this is a common feature of forest insect epidemics (peltonen et al., ) . further, the effect of the lattice size can easily be estimated, and as lattice size is increased, sensitivity to changes in the interaction parameter increases as well. while most methods discussed thus far have been developed with the analysis of aggregated counts of disease in mind, analysis of sites on a lattice may have applicability in certain disease surveillance contexts. for example, square lattices are used for remotely sensed image processing, and surveillance of the presence or absence of a disease in these sampling units using an ising modelbased approach could incorporate remotely sensed environmental covariates (e.g., normalized differential wetness index) as is commonly done for zoonotic disease risk mapping and forecasting (kitron et al., ; rogers et al., ; wilson, ) . however, it is unclear how covariates are included in the ising model. this highlights an important point with model-based approaches to prospective surveillance: the main advantage of models is to incorporate extra information and to estimate smooth relative risks, yet as models grow in complexity they become more difficult to re-fit. this has implications for how suitable models are in different surveillance contexts. where the temporal scale is large, expected counts can be based on observed data rather than using census or other data sources. this is particularly important where diseases follow seasonal trends. with limited temporal data available, estimating model parameters may make be impacted by regular variation in disease occurrence. for surveillance systems monitoring many small areas (i.e., large spatial scale), the held et al. ( ) model would be of limited value as separate parameters need to be estimated for every sampling unit. broad scale patterns over large areas might better captured by the point process approach of diggle et al. ( ) . although here, case event data with fine spatial resolution is required. for all modelling approaches, complex decisions are required such as what covariates to include, how often to re-fit the model, how to test incoming data for fit against the existing model which require advanced statistical knowledge. this limits the applicability of modelling approaches to advanced analysts and researchers except for use in a black-box sense by analysts and public health practitioners. surveillance models can be tailored to detect specific types of disease process changes, such as a regionwide increase, or small changes in spatial autocorrelation suggesting a shift from endemic to epidemic states. however, models also required additional tests to determine if incoming data differ from the expected (i.e., modelled) pattern of cases. thus, in practice surveillance models are best utilized to estimate a realistic relative risk, and can then be combined with statistical tests such as cusum (järpe, ) and scan statistics . research into space-time disease surveillance methods has increased dramatically over the last two decades. many new methods are designed for specific surveillance systems, or are in experimental/developmental stages and not used in practical surveillance. here, we report on some newly developed approaches for public health surveillance to alert readers to the most recent developments in these emerging research areas. while test and model-based approaches to surveillance build on classical statistical methods, many recent spacetime disease surveillance methods have been developed specifically to take advantage of advanced computing power and data sources. these approaches include networks (reis et al., ; wong and moore, ) simulation-based methods such as agent-based models (eubank et al., ) and bootstrap models (kim and o'kelly, ) , and hidden markov models (madigan, ; sun and cai, ; watkins et al., ) . other new methods are designed to address limitations of existing surveillance methods. one problem for most methods of surveillance, is the specification of the null hypothesis, or expected disease prevalence. while expected rates are generally conditional on population data, spatial heterogeneity in the background rates are rarely accounted for. that is, complete spatial randomness (csr) is the underlying null model. goovaerts and jacquez ( ) have used geostatistical approaches, estimating spatial dependence of background rates via the semivariogram, to develop more realistic null models for disease cluster detection. the geostatistical framework has the advantage of estimating spatial dependence from the data, rather than defining it a priori via a spatial weights matrix as is common in disease mapping models. another problem common to most surveillance methods is that maps of disease represent either home address (case events) or small areas (tract counts). unusual clusters on the map imply heightened risk is associated with those locations. however, movement of animals and people decouples the location of diagnosis from disease risk by modifying exposure histories. methods that account for mobility may be an important area for future surveillance, especially in the context of real-time, prospective outbreak detection. the relationship between case, location, and exposure is further complicated by disease latency periods, which gives rise to space-time lags in diagnoses (schaerstrom, ) . this may be most important in the context of retrospective cluster analysis and investigation of possible environmental risk factors. statistical tests have been developed to account for exposure history and mobility for case-control data (jacquez and meliker, ) and case-only data (jacquez et al., ) . kernel-based approaches to risk estimation that incorporate duration at each location have been utilized for amyotrophic lateral sclerosis (sabel et al., ) . the general approach is to model and analyze the space-time path of individuals in the sense of hägerstrand ( ) . as personal location data continues to become ubiquitous due to new technology such as gps-enabled cell phones, surveillance methods that account for individual space-time histories may see more application in public health surveillance. the development of space-time disease surveillance systems holds great potential for improving public health via early warning and monitoring of health. the selection of which method(s) to implement in a given context is dependent on a variety of factors (table ) . this review has demonstrated that there is no best method for all systems. there are many aspects to consider when thinking about methods for space-time disease surveillance. many of the methods described in this review are active areas of research and new methods are constantly being developed. as more data sources become available, this trend is expected to continue, and the methods described here provide a snapshot of options available to public health analysts and researchers. a brief outline of some of the factors reviewed and how they relate to surveillance methods is given below. the spatial scale of the surveillance context is an important factor for selecting appropriate methods. spatial effects (i.e., clustering) are likely only of interest when cases/counts collected over a relatively large, heterogeneous area are being analyzed. over smaller more homogeneous areas, where spatial effects are negligible, temporal surveillance is optimal. when space-time surveillance is warranted, choice of which surveillance approach to use may be impacted by how spatial effects can be incorporated. where spatial scale is small, one would likely focus on either process models or statistical tests which use an underlying distribution for the null hypothesis (i.e., poisson model). the temporal scale of surveillance is also important. large temporal scales can use either testing or modelling methods, and most suit methods where baselines are estimated from previous cases, such as with the space-time permutation scan statistic. short temporal scales are not appropriate for models when diseases have complex day of the week effects or seasonal variation in incidence. scale will also affect the computational burden placed on the system. many approaches reviewed here, particularly statistical tests such as scan statistics, use approximate randomization to generate a distribution of a test statistic under the null hypothesis. methods that utilize randomization procedures, while powerful, impose constraints when applied with large spatial-temporal datasets. most methods are designed for a single disease and all methods are suitable for single host diseases, but finer detail in case distribution may be important for multiple host zoonotic diseases. stratification into separate diseases by host type will result in a loss of information as associations between host types will be lost. as zoonotic diseases make up the majority of emerging infectious diseases (greger, ) , multiple host surveillance methods are required. multivariate tests such as multivariate cusum can be used to monitor multiple signals. modelling approaches can also be used by creating a generalized risk index as the variable under surveillance. multivariate extensions to existing methods can be used to monitor associations between two diseases, for example, human and animal strains of the same pathogen. the objective of surveillance is one of the main drivers of method selection. all statistical tests are commonly used for outbreak detection. in general, modelling approaches are better suited to monitoring space-time trends. for what has been termed situational awareness, multiple signals are usually monitored. this is often the case in large syndromic applications such as biosense and essence. these contexts are best suited to a modelling approach, as often heterogeneity needs to be modelled with covariates. consideration of technical expertise is required for practical disease surveillance. broadly speaking, greater statistical expertise is required for model-based methods than testing (understanding model assumptions, parameterizing models, preparing covariate data, and interpreting output), while testing concepts are generally easier to grasp. however, for epidemiologists already familiar with generalized linear mixed models, some model approaches that incorporated space and time may be quickly attainable, such as that of kleinman et al. 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regression b-spline smoothing in bayesian disease mapping: with an application to patient safety surveillance spatial and syndromic surveillance for public health implementing syndromic surveillance: a practical guide informed by the early experience the detection of disease clustering and a generalized regression approach a review of methods for the statistical analysis of spatial patterns of disease visualization techniques and graphical user interfaces in syndromic surveillance systems. summary from the disease surveillance workshop the distribution of the size of the maximum cluster of points on a line using remote sensing and geographic information systems to identify villages at high risk for rhodesiense sleeping sickness in uganda a mark geographical analysis machine for the automated analysis of point data sets upper level set scan statistics for detecting arbitrarily shaped hotspots spatial synchrony in forest insect outbreak roles of regional stochasticity and dispersal a case-control approach to examine diseases for evidence of contagion, including diseases with long latent periods if syndromic surveillance is the answer, what is the question? aegis: a robust and scalable real-time public health surveillance system monitoring changes in spatio-temporal maps of disease online updating of space-time disease surveillance models via particle filters predicting the distribution of tsetse flies in west africa using temporal fourier processed meteorological satellite data surveillance systems for monitoring the development of spatial patterns a set of associated statistical tests for spatial clustering monitoring spatial maxima monitoring change in spatial patterns of disease: comparing univariate and multivariate cumulative sum approaches spatial clustering of amyotrophic lateral sclerosis in finland at place of birth and place of death apparent and actual disease landscapes. some reflections on the geographical definition of health and disease sequential analysis: tests and confidence intervals a review and discussion of prospective statistical surveillance in public health evaluation challenges for syndromic surveillance-making incremental progress syndromic surveillance: is it worth the effort? large-scale multiple testing under dependence a flexibly shaped space-time scan statistic for disease outbreak detection and monitoring a class of tests for detecting 'general' and 'focused' clustering of rare diseases a flexibly shaped spatial scan statistic for detecting clusters monitoring for clusters in disease: application to leukemia incidence in upstate new york bayesian latent variable modelling of multivariate spatiotemporal variation in cancer mortality spatio-temporal modeling of mortality risks using penalized splines the emerging science of very early detection of disease outbreaks disease surveillance using a hidden markov model emerging and vector-borne diseases: role of high spatial resolution and hyperspectral images in analyses and forecasts multivariate cusum quality-control procedures classical time-series methods for biosurveillance world health organization. global early warning system for major animal diseases, including zoonoses (glews) a review of public health syndromic surveillance systems a cluster model for space-time disease counts this project was supported in part by the teasdale-corti global health research partnership program, national sciences and engineering research council of canada, and geoconnections canada. the authors would like to thank dr. barry boots for direction and suggestions during the starting phase of this research. key: cord- - uoberfu authors: tiwari, bhagyashree; sellamuthu, balasubramanian; drogui, patrick; tyagi, r.d. title: future impacts and trends in treatment of hospital wastewater date: - - journal: current developments in biotechnology and bioengineering doi: . /b - - - - . - sha: doc_id: cord_uid: uoberfu the world’s population growth and economic development result in the increased requirement of land, water, and energy. this increased demand leads to the deforestation, loss in biodiversity, imbalance in agriculture and food supply, climate change, and increase in food and travel trade, which result in emergence and reemergence of infectious diseases. this chapter discussed various emerging infectious diseases and their causative agents (buruli ulcer and bunyvirus). furthermore, this chapter further illustrates the emergence of superbugs and the associated threat due to the presence of pharmaceutical compounds in the environment. the prevalence of pharmaceuticals in the environment exerts ecotoxic effects on living organisms and causes thousands of death every year. the threats associated with the pharmaceutical presence in the environment were briefly discussed in this chapter. finally, this chapter provides the alternative methods to avoid the use of antibiotics and to develop novel treatment technologies (such as phage therapy) to degrade and remove the pharmaceutical compounds. infectious diseases are the second leading cause of death annually across the globe. the causative agent of most emerging infectious diseases is viruses; every year approximately more than two novel viral pathogens are identified, which can cause illness in a human. since the s, an average of infectious diseases has been emerged and known to cause pandemics such as ebola, swine flu, chikungunya, and zika (table À ) [ ] . the emergence of infectious diseases has severe health and socioeconomic impacts; thus the following section discusses the various infectious disease that emerged within the last two decades and presenting few pandemic viral diseases as a suitable example [ , ] . factors for emergence include natural process (evolution of pathogen), infectious agents transfer from vertebrate to mammals, antimicrobial resistance (amr), and climate change. the factors responsible for the emergence of infectious diseases such as ( ) the evolution of new strain, ( ) the introduction of a host to enzootic, ( ) translocation of infected wildlife, ( ) farming practices, and ( ) others were provided. the viral evolution rate occurs through mutation and adaption, which are much faster than any other microscopic organisms that is why a large fraction of emerging and reemerging pathogens are viruses ( %). among these % of emerging viruses, rna viruses are prominent one because of their higher nucleotide substitution rate which enables them to invade and amplify in broad host range [ ] . the environment changes, social condition, and their interaction are the important factors which lead to the viral evolution and ultimately to disease emergence. the decrease in biological diversity due to deforestation, prevalence of micropollutant in natural environment, and climate change triggers the evolution of opportunistic pathogen [ ] . for instance, the outbreak of nipah virus diseases was occurred due anaplasma phagocytophilum [ ] buruli ulcers mycobacterium ulcerans [ ] to the extensive deforestation of a forest of southeast asia [ ] . the deforestation leads to migration of fruit bats for food quest to the agriculture land. these fruit bats are natural reservoir (host) of nipah virus and their migration to cultivable land lead to transmission of nipah virus disease in farm animals and subsequently in humans [ ] . the increase in temperature due to climate change affects the ecology, survival, and behavior of arthropod vectors, which subsequently changes population dynamic with increased disease transmission rate. for example, the rise in temperature from c to c decreases the proliferation time of plasmodium falciparum in anopheline mosquitoes vector from to days. usually, p. falciparum requires days to proliferate in the vector at c. however, the high temperature reduces its proliferation time by increasing egg production, by increasing metabolic rate, and by reducing the larval and pupal period duration [ ] . moreover, the proximity of natural host such as rodents and human is the ease and travel aids for the spread of emerging viruses. the viral infection begins from binding of virus to the receptor of host cell, and during the emergence of new disease, viruses develop either the ability to bind the new receptor or use homologue receptor in new host species. the spread of coronavirus (cov) which causes severe acute respiratory syndrome (sars) in human occurs due to cross-species transmission from raccoon dogs and chinese ferret badgers [ ] . the examination of wild species of cov did not have the sign of sarsÀcov infection but the cov viruses isolated from horseshoe bats have sarsÀcov infection. the cov binds to angiotensin-converting enzyme (ace ) receptor to infect humans. however, in bats, ace viral receptor is not use for infection by cov. later it was studied that the mutation in ace receptor aids the adaptation of cov in human cells. the spread of ebola, marburg, and measles viruses is some of the example of virus evolution and which contributes to the emergence of disease. the virus evolution and adaptation is difficult to predict and thus raises a question how the effect of emerging viral infection can be reduced. this requires a holistic approach to identify the drivers of emergence with effective surveillance. in an increased cases of severe fever with thrombocytopenia syndrome (sfts) were frequently reported in rural areas of china. the associated pathogens of thrombocytopenia and other similar diseases were not detected in majority of the pathogen samples which implies that the pathogen associated with sfts was newly evolved. the emergence of this unknown infection having average fatality rate of % due to organ failure leads to the implementation of enhanced surveillance to identify causative agent of sfts infection [ ] . the surveillance data revealed strain of viruses as causative of sfts infection and regarded as sfts viruses (sftsvs). sftsvs are rna virus with three single-stranded rna genomes. the complete sequencing of sftsv, rna genome reveals that they belong to genus phlebovirus, family bunyaviriade. the sftsv is transmitted in human through infected ticks and transmission of blood and other body fluid of infected person lead to human-to-human transmission of infection. the phlebovirus infection in human results in mild febrile illness. however, the major symptoms of sfts include thrombocytopenia, high fever, leukocytopenia, and lymphadenopathy. currently, it is believed that the vertical transmission of phlebovirus in arthropod vector (ticks such as haemaphysalis longicornis) helps in maintenance cycle of virus and amplification of virus occur in vertebrate host (human). the laboratory diagnosis of body fluid samples of sfts patient showed elevated level of serum and important enzymes and cofactor such as creatine kinase, alanine aminotransferase, lactate dehydrogenase, and aspartate aminotransferase. the sftsv replicates in the spleen of infected patient which increases the number of macrophages and platelets. the in vitro assay revealed that sftsv facilitates the phagocytosis of platelets by adhering on the surface of platelets and ultimately causing thrombocytopenia. the spread of sftsv in china and the detection of sftsv like viruses in other parts of world such as in the united states and europe emphasized on the urgent need of understanding of pathogenesis and transmission cycle of virus which help in the development of efficient vaccine. the changes in the hostÀenvironment lead to the evolution of opportunistic and novel pathogens due to which infectious diseases emerge. the forces which shape the emergence of disease in human are similar to the drivers found in wildlife and domestic animals. these drivers alter the interplay of hosts, environment, and pathogen thereby modulating disease ecology and developing or acclimatizing pathogen in hosts. the drivers alter interaction pattern of pathogenÀhost and environment which to either of three events ( ) altering the genetic trait of pathogen which causes severe disease in same host; ( ) emergence of pathogen in new hosts; and ( ) redistribution of pathogens that result in its establishment in new geographical area. frequent use of antimicrobial compounds and mass rearing of animals result in amr and eventually increase virulence pathogenicity of a pathogen. mass rearing of food animals to meet the demand of growing population results in intensification of genetically similar animals of same sex and age at confined place. this leads to transmission of pathogen with increases population turnover which supports emergence of novel trait. the transmission of highly pathogenic asian avian influenza a (h n hpai) is the well-known example of mass rearing [ ] . the drivers which are responsible for the disease emergence in new host include interspecies contact, wildlife migration, and increase contact between different hosts. the worldwide change in ecological landscape results in close contact between human and animals which eventually lead to transmission of microbial reservoir of animals (birds, rodents, and bats) to human. for instance, the emergence of nipah virus in human was due to transmission of virus from foraging fruit bat to pigs [ ] . the transport of food, live animals, plants with accompany insects for international trade, migration of birds, and wild animals results in geographical jump of pathogens. the bacterium ralstonia solanacearum hitchhikes to the united states via geranium plant which is imported from kenya [ ] . persisters are slow growing or nongrowing organisms which remain in stagnant during the presence of antimicrobial compound but have the capacity to resuscitate and grow under specific conditions. the persisters formation is mediated by variety of stress such as due to nutrient depletion, oxidative stress heat, acidic ph, and the presence of toxic compound (antibiotics). antibiotics such as tetracycline, rifampin, and ciprofloxacin were shown to enrich persisters formation by inhibiting protein synthesis, rna synthesis, or by antioxidative defense [ ] . persisters are the cause of biofilm infection, recurrent infection, and chronic infection and contribute toward prolongation of therapy time (e.g., tuberculosis, lyme disease). the repeated treatment of persistent infection may result in the development of drug-resistance microbes, often seen in the case of tuberculosis. persistent could be pre-or postantibiotic depending on its development in the host. the capacity of formation of persisters varies greatly among the bacterial species. for instance, persisters of mycobacterium tuberculosis are not removed from the host even after chemotherapy, while the single antibiotic treatment is sufficient for curing infection caused by streptococcus pneumoniae. the physical and psychological stress, host immune and hormonal factor, and coinfection also affect the persisters formation in the host. the mechanism behind the persisters formation is not well understood. however, it is believed that epigenetic changes, changes in dna modification, and posttranslational modification induce expression of persisters gene. the genes which involve in persisters formation are rela, sucb, hipa, ubif, and phou. the mutagenesis approach is used to study genes involved in persisters formation; however, the short antibiotic exposure, screening of partial mutant library, and aeration during antibiotic exposure are factors which result in failure of mutagenesis process for identifying persisters genes. zoonotic diseases are vector (mosquito, ticks, and bugs) aided or nonaided disease caused by bacteria, virus, parasites, prions and fungi, and transmitted from animals to human. various modes of transmission are direct contact between infected animal and human, via arthropod vector, consumption of contaminated animal food (meat and pork), and ingestion of aerosolized pathogens present in the environment. in last two decades, many vector borne pathogens spread in new geographical regions. the emergence and reemergence rate of zoonotic infection has increased inescapably due to urbanization, deforestation, climate change, international trade (frequent travel), population movement, and encroachment into animal habitats. the most emerging zoonotic vector borne diseases in the united states and canada include tick borne lyme disease, babesiosis, human granulocytic anaplasmosis and mosquito borne west nile virus (wnv), california serogroup viruses, and cache valley virus [ ] . the change in land use alters the abundance and interaction of vectors and hosts (wildlife and domestic animals) which results in emergence of vectors. for instance, deforestation in amazon and eastern africa enhances the breeding of anopheles' mosquito due to sunlight and standing water. similarly in north america, increased hunting changes the predator community and results in increased abundance of small animals such as mice, chipmunks, and shrew which are main host of spirochete borrelia burgdorferi, causative agent of lyme disease [ ] . socioeconomic changes and human activities are the another factor which governs spread and emergence of pathogen. the lyme disease was reported to occur more in high-income people in europe due to the more recreational activity and living in new homes in broad-leaf woodlands with cooccurrence of wildlife result in frequent exposure to vector. vector born zoonotic disease could be controlled by prompt identification of cause with subsequent action which requires integration of public health officials, researchers, and public [ ] . arbovirus is an acronym for arthropod-borne viruses. dengue virus, wnv, and chikungunya virus are recent examples of arboviruses which are transmitted from the arthropods (ticks, bugs, and mosquito) to vertebrate. arboviruses use arthropods as a vector (carrier) for transmission and do not causes any sickness in them. bunyaviridae, reoviridae, flaviviridae, and togaviridae are the most prevailing arboviruses families that cause diseases in human and animals [ ] . the emergence of arboviruses is governed by three factors, that is, high mutation frequency, varying anthropological behavior, and climate change. they easily adapt new host by altering receptor specificity, antigenicity, environmental conditions, and by efficient transmission. for instance, emergence of chikungunya virus in asia due to mutation in surface protein which increases its reproduction, transmission, and infection efficiency in aedes albopictus. they are able to maintain themselves for years in mosquito eggs or via attaining transstadial stages in ticks. domestic animals, livestock, and human are not important part of arbovirus life cycle because of nonviraemic transmission of arbovirus between ticks without infecting vertebrate host. this feature of arbovirus adds additional limitation for controlling disease emergence [ ] . mosquito eradication, development of live-attenuated vaccines, antiviral drugs, and molecule are few methods used to control and prevent arboviral infection. however, high mutational frequency will result in emergence of new pathogenic arboviruses. since it has been proven by genome sequencing of mosquitoes that they are the carrier of various known and unknown viruses, control of localized arthropod during endemic could be a possible solution for regulating the emergence of arbovirus [ ] . buruli ulcer (bu) is necrotizing skin disease caused by mycobacterium ulcerans recognized as one of most neglected tropical disease by world health organization (who). the bu lead to the formation of skin ulcers which results in osteomyelitis. it causes pandemic in humid tropical and subtopical region, often where humans are in proximity with slow moving or stagnant contaminated water. the transmission of m. ulcerans follows multihost transmission dynamics, that is, multiple hosts of aquatic environment such as scrapers, scavengers, and predators become contaminated with m. ulcerans and result in its passive dissemination via organism-to-organism contact. phylogenetic studies revealed that the m. ulcerans was emerged from species mycobacterium marinum which causes cutaneous disease in human and also shown to infect fish. m. ulcerans are widely distributed in africa; however, the bu cases reported in specific geographical villages. researchers postulates that a specific m. ulcerans strain might have pathogenicity or virulence to cause bu [ ] . the current knowledge lacks the understanding of spatiotemporal distribution of m. ulcerans and required detailed scenario of diversity of m. ulcerans strains existing in environment [ ] . superbug is a term coined for bacterial species which confers resistant toward majority of antibiotics. emergence of superbugs implies the frequent detection of novel bacterial pathogens which caused unrecognized life-threatening infections. the frequent emergence and spread of superbugs worldwide causes a concern that human life is heading back, toward the preantibiotic era, where the entire population of a society was wipe out due to the simple infection. united nation (un) general assembly of addresses the amr [ ] as a health emergency and acknowledge that amr has deleterious effect on human health and sustainable development. who identified a group of amr resistant "priority pathogens" that requires urgent strategic action (table À ) . who categorize these priority pathogens into three categories, that is, critical, high, and medium priority based on the need for new antibiotics. the criteria for prioritization of pathogens were developed using multicriteria decision analysis technique which includes amr pathogens which causes mortality, prevalence of resistance, transmissibility, treatability, healthcare and community burden, -year trend of resistance, preventability in hospital and community settings, and current pipeline [ ] . to fight against increasing prevalence of antibiotic-resistant superbugs and to limit the indiscriminate use of antibiotics, who prepared three groups of antibiotics namely access, watch, and reserve group to ensure appropriate prescription and use. access group comprises of antibiotics which are prescribe as in common infection as first and second choice. the first-choice antibiotics are narrow spectrum with low-resistant potential, whereas the second-choice antibiotics are broad spectrum having higher resistant potential. antibiotics such as β-lactam, chloramphenicol, and clindamycin come under this category. watch group identifies pharmacological antibiotic classes which prescribes as first or second choice but have a limited number of indication. the watch group has higher resistance potential compared with the access group. watch group comprises macrolides, quinolones and fluoroquinolones, and glycopeptides class of antibiotics [ ] . who created reserve group of antibiotics to reserve some antibiotics as the last resort in superbug infection. this group of antibiotics should be recommended as "last resort," in case of life-threatening infection when other alternatives are failed in treatment. reserve groups include eight antibiotic or antibiotic class which are aztreonam, fosfomycin, fourth-generation cephalosporins (cefepime), oxazolidinones (linezolid), fifth-generation cephalosporins (ceftaroline), tigecycline, polymyxins (polymyxin-b, colistin), and daptomycin [ ] . recently, among these eight antibiotic classes, resistant determinant against colistin was detected in people of rural vietnam. the prevalence of colistin resistant escherichia coli in intestine of resident of vietnam was extremely high, that is, approximately %. previously, mutation that causes colistin resistant was not transferable; thus it is not considered as pathogenic, as intestinal e. coli is nonpathogenic. finally, a transmissible colistin resistance gene (mcr) was detected in china, which has the potential to transfer colistin resistant to pathogenic microbes. the prevalence of mcr gene represents a serious concern regarding the emergence of superbugs that are resistant to last resort of antibiotic [ ] . currently, there are only fewer option (such as last resort antibiotics) to tackle antibiotic-resistant superbugs, and the finding of mcr gene indicates toward the needs of innovative research which results in better understanding of superbug emergence and also to research and development on quality, safe, efficacious, and affordable antimicrobial medicines, especially new antibiotics and alternative therapies, vaccines, and diagnostics. the various chapters of this book discussed about the occurrence and prevalence of human and veterinary pharmaceuticals (antibiotics, antidepressant, antidiabetic, radioactive agents, etc.) at different environmental sites. these pharmaceutical compounds are biologically active compounds that are known to have a specific mode of action (moa) in human and animals even at low concentration. due to genetic relatedness (presence of conserved gene) across species, these compounds interact with protein and cell lineage of nontarget organisms (conserved therapeutic drug targets) and elicit a response in their body (targets metabolic pathway, enzyme or mediators of cell signaling molecule). for instance, ibuprofen which is a cyclooxygenase inhibitor was found to interfere with arachidonic acid signaling pathway in marine clams ruditapes philippinarum [ ] . however, the effect of pharmaceuticals may vary from species to species due to difference in gene expression of the same gene across species or due to change in solubility or potency of drugs because of binding of pharmaceuticals with organic molecules present at environmental sites. therefore to understand the environmental risk posed by these contaminants, conceptual model of moa of pharmaceuticals was used. the moa [ ] approach involves the assessment of drug targets and its evolution between mammals and the model species. the conceptual model of moa approach in marine organisms reveals the presence of fluoxetine at environmental concentration was able to control the serotonin signaling pathway, and this alteration in physiological signaling pathway results in defects in reproduction, locomotion, and metabolism of aquatic organisms [ ] . many short-term toxicity studies reported that the drug molecules do not have an acute toxic effect on aquatic organisms because of their presence in low concentration (ng/l to low μg/l), but their constant release and exposure to aquatic biota have long-term chronic effects [ , ] . however, many laboratory studies at high concentration of pharmaceutical compounds report direct lethal effects. tetracycline concentration around À μg/l leads to low periphyton (nematode, bacteria, and algae) concentration in mesocosm stream [ ] . structure disruption in kidney and intestine of rainbow trout and brown trout due to diclofenac ( μg/l) was reported [ ] . prolonged exposure to pharmaceuticals in low concentration leads to the change in species trait and behavior of aquatic organisms. antidepressant and psychiatric drugs such as fluoxetine and oxazepam cause disruption in ecological interaction even in low concentration [ ] . indeed, even by considering that these drugs are diluted after their release, it is evident that they have a toxic effect on the aquatic ecosystem. the widespread occurrences of pharmaceuticals at various environmental sites such as in ocean, river, and groundwater raise a concern regarding the risk associated with human health. for instance, the birth control and growth stimulator agent like estrogen have the potential to cause prostate and breast cancer in humans (if the estrogen concentration is used above the threshold limit). the worldwide discharge of estrogen from livestock and human ranges approximately , and , kg/year, respectively. the us national toxicology program declared estrogen as carcinogen, the no adverse effect concentration of estrogen in human is . mg/day and frequent detection of estrogen in drinking water concentration ranging from . to . ng/l represent a health risk [ ] . estrogen was reported to cause abnormalities in animals such as permanent infertility (clover disease in sheep due to feeding on clover plant which has high phytoestrogen level) [ ] . however, human health risk assessment studies reported no appreciable risk to human due to exposure of pharmaceuticals mixture [ ] . due to emergence of antibiotic-resistant pathogens and unavoidable use of antibiotics, concomitant environmental perturbation caused by climate change might make the earth is not suitable for humans and other livings. thus researchers focus on finding alternative methods to avoid use of antibiotics and developing novel treatment technologies to degrade and remove the pharmaceutical compounds. prediction of emerging signals by theoretical and bioinformatics tool will highly help to alarm the researchers, hospitals, and public about the nearby occurrence of resistant bug. such predictions are underway by monitoring microbial community dynamics in different environmental samples; however, these studies facing enormous challenges in the developmental face [ , À ] . several research studies have been conducted to observe a change in microbial metabolic pathways for a while. such study results could predict microbial interactions and route of evolution, and might apply to precisely pinpoint the emerging organism in the mixed microbial community. these studies are still under research stage, such studies are using genomic, metabolomic, and trancsriptomic tools to predict the bug emergence. recently, geoghegan and holmes [ ] attempted predicting the virus emergence with an interest of biomedicine and preventing unpredicted incidence [ ] . they have monitored the evolution of viruses in short-term period to highlight the cross-species transmission and emergence. they have concluded that predicting emergence requires a new mechanistic and integrated approach, which might permit or stop the emerging viral spread into new hosts [ ] . with the fact that microbial resistant to pharmaceuticals, the treatment options are reduced; however, alternative methods have been explored to protect human and animal from microbial illness. the microbial drug resistance leads to direct and indirect consequences as described by who report [ ] . the severe direct consequences are prolonged illness, impossible to protect the patients undergoing surgery and augmented treatment cost. whereas the indirect impacts of amr are depleting the global economy (due to the loss of productivity by sickness) and higher costs of treatment. thus who proposed a global action plan to assure preventing the transmission of infectious disease and providing complete treatment with the help of safe and effective medicines [ ] . to achieve the global action plan successfully, five objectives have been proposed by who: ( ) to improve awareness and understanding of amr; ( ) to strengthen knowledge through surveillance and research; ( ) to reduce the incidence of infection; ( ) to optimize the use of antimicrobial agents; and ( ) to ensure sustainable investment in countering amr. these objectives are likely to be met through political leaders, member states, the secretariat, and international and national partners across multiple sectors. notably, individual responsibilities like recycling unused medicines, intake of only prescribed medicines, and keeping the environment clean would highly help to achieve the who's global action plan for the betterment of human and animal health. increasing resistance to antibiotics and the emergence of "superbugs" that are resistant to drugs of last resort have highlighted the great need for alternative treatments of bacterial disease. this has led to renewed interest in the potential of phage to treat bacterial pathogens. the term "phage therapy" usually refers to the treatment of bacterial infections with intact phage; however, there are other ways in which phage can be used as antibacterials. phage can be used as "lethal agent delivery systems" to introduce nonspecific or toxic antimicrobials [ , ] , or genes encoding antimicrobials [ ] into selected pathogenic bacteria. in another method (targeted gene transfer), filamentous phage can be modified to carry therapeutic genes and to target cell surface antigens or receptors on mammalian cells, transducing them by receptor-mediated endocytosis [ ] . although phages are not an antimicrobial tool in itself, this approach could be used to deliver antimicrobials to intracellular bacterial pathogens. phage secrets lysins enzymes which lyse the host bacteria. lysins are host specific and use cell wall components that are essential for viability as receptors; therefore bacterial resistance is rare [ ] . initial tests of lysins against clinically relevant gram-positive bacteria, such as methicillin-resistance staphylococcus aureus, look promising [ , ] . the use of multiple phage lysins with different cleavage sites could increase therapeutic effectiveness and further reduce the chance of bacteria developing resistance. lysins can also be used to make bacterial ghost vaccines. to produce better quality of treated sludge, wastewater treatment processes must achieve a minimum log reduction in e. coli loads and a final end product (sludge) with a maximum admissible concentration of e. coli cfu/g [dry solids (ds)] and zero salmonella in g (ds). similarly, united states environmental protection agency regulations are categorized into class a and class b sludges. class a pathogen reduction requirements are more stringent than those of class b sludges which are subject to application restrictions. class a sludges are required to reduce pathogens to below the detectable limit (, most probable number of salmonella, , plaque forming unit of enteric viruses, and , viable helminth ovum per g ds). treatment processes designed to achieve pathogen reduction to a required level can incur substantial capital and operating costs [ ] . development of phage treatment of sludge may provide long-term and cost-effective control of potentially pathogenic bacteria (e.g., e. coli and salmonella). successful phage treatment of wastewater bacterial pathogens would be dependent on the prevalence and diversity of pathogen species within wastewater. it would be virtually impossible to produce phage targeted at all pathogenic serotypes. for example, there is a high diversity of e. coli serotypes [ ] and around known salmonella serotypes [ ] . however, wastewater treatment conditions intrinsically reduce the numbers of some pathogenic bacteria. therefore there is potential for phage treatment to be used successfully in combination with biological sludge stabilization processes to reduce the abundance of specific pathogenic bacterial strains such as e. coli o . indeed, research on phage therapy for reduction of this pathogen in animal reservoirs is already underway [ ] . although phage-derived therapies have several advantages compared with antibiotics, the combined use of phage, or phage lysins, and antibiotics is an attractive treatment regime. indeed, such a product is available commercially in georgia: phagobioderm is a biodegradable polymer composite impregnated with a lytic phage cocktail and ciprofloxacin [ ] . using phage with antibiotics should increase the efficiency of treatment and reduce the emergence of resistant mutants because the surviving bacteria would need to acquire at least two separate resistance mechanisms. to kill the multidrug-resistant bacteria researchers from ibn (institute of bioengineering and nanotechnology), chin et al. [ ] developed a novel synthetic molecule and demonstrated selectively destroying five multidrug-resistant bacteria [ ] . recently polymer-based advanced drug designing to combat multidrug resistances is widely explored, which are described in detail [ , ] . due to climate change, a steady increase in temperature (about . c) was recorded in the past years, which was clearly observed on lands than the ocean in recent days [ ] . however, ice melting in north pole, increase in sea level, and alteration in raining patterns are clear and known effects of examples of global warming. climate change affects the water cycle in the following aspects: ( ) poor water quality, ( ) less availability (quantity for consumption and use), ( ) evaporation leads increases the microbial load (concentration) and toxic ions concentration, ( ) water depletion in animal-farming and agriculture (leads to food scarcity), and ( ) effects on freshwater biodiversity is still undetermined. ultimately, these changes will hugely affect wastewater microbiome (including beneficial and pathogenic microbes), the concentration of toxic pollutants and biological process performance, which was discussed in detail in chapter , treatment of wastewater containing pharmaceuticals: biological treatment. recently, who reported that climate change affects the infectious disease transmission pattern (fig. À ) . various infectious diseases and their agents (viruses, bacteria, protozoa, and multicellular parasites) have adapted (via evolution) humans as a primary host, which raises serious threat mankind to face in near future. due to the negligent, persistent and accidental activities of humans (industrial) raised the environmental pollution (chemicals including pharmaceuticals), which led to inseparable effect like climate change. furthermore, development of drug-resistant organisms and increased pathogen survival rate, only raising panic about the human, animal, and environmental health. thus researchers are continuously searching alternatives to these environmental problems. due to pharmaceuticals release in the environment and climate change, the survival of pathogens prolongs. high load of infectious bacterial strains dwelling in wastewater further favors the transfer of antibiotic-resistance gene. this could be a classical example for multidrug-resistant bacterial strains that are prevalence in tropical countries (like china and india). this draws immediate attention of public, health, and international sectors to fight against, pollution, climate change, and drug resistance. the advancement in medicinal research helps in control and elimination of various infectious diseases such as smallpox. however, emergence and reemergence of infectious diseases due to various factors, that is, natural evolution, climate change, amr, and many more, is a matter of concern. the knowledge about the evolution and life cycle of pathogens (bacteria, virus, or parasite) using omics study (proteome, metabolome, epigenome, and transcriptome) and via next-generation sequencing could help in the prevention and control of infectious diseases. the implantation of ecological approaches, network, and system biology approaches could provide a better understanding of the environmental factors of disease-emergence and drug-resistance mechanisms. the information gained from these approaches could help in assessing the immune mechanisms of pathogen and a developing treatment strategy. review on fate and mechanism of removal of pharmaceutical pollutants from wastewater using biological approach a highly pathogenic new bunyavirus emerged in china emerging infectious diseases of wildlife-threats to biodiversity and human health perspectives of public health laboratories in emerging infectious diseases persisters, persistent infections and the yin-yang model drivers, dynamics, and control of emerging vector-borne zoonotic diseases major emerging vector-borne zoonotic diseases of public health importance in canada global and local environmental changes as drivers of buruli ulcer emergence world health organization, global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, world health organization carbapenem-resistant acinetobacter baumannii: epidemiology, surveillance and management complete genome sequence of pseudomonas aeruginosa pao , an opportunistic pathogen global spread of carbapenemase producing enterobacteriaceae multilevel population genetic analysis of vana and vanb enterococcus faecium causing nosocomial outbreaks in countries pathogenesis of methicillin-resistant staphylococcus aureus infection management of helicobacter pylori infection-the maastricht v/florence consensus report the disease burden associated with campylobacter spp incidence of invasive salmonella disease in sub-saharan africa: a multicentre population-based surveillance study molecular mechanisms of antibiotic resistance serotype distribution of streptococcus pneumoniae causing invasive disease in children in the post-pcv era: a systematic review and meta-analysis epidemiology of invasive haemophilus influenzae disease how do the virulence factors of shigella work together to cause disease? front host range and emerging and reemerging pathogens unhealthy landscapes: policy recommendations on land use change and infectious disease emergence anthropogenic deforestation, el niño and the emergence of nipah virus in malaysia climate change: effects on animal disease systems and implications for surveillance and control pathogen-host-environment interplay and disease emergence factors responsible for the emergence of arboviruses; strategies, challenges and limitations for their control metabolic resource allocation in individual microbes determines ecosystem interactions and spatial dynamics the selection and use of essential medicines: report of the who expert committee, (including the th who model list of essential medicines and the th model list of essential medicines for children wide dissemination of colistin-resistant escherichia coli with the mobile resistance gene mcr in healthy residents in vietnam human pharmaceuticals in the marine environment: focus on exposure and biological effects in animal species health and ecological risk assessment of emerging contaminants (pharmaceuticals, personal care products, and artificial sweeteners) in surface and groundwater (drinking water) in the ganges river basin environmental impact of estrogens on human, animal and plant life: a critical review temporal dynamics of periphyton exposed to tetracycline in stream mesocosms water-borne diclofenac affects kidney and gill integrity and selected immune parameters in brown trout (salmo trutta f. fario) ocean viruses and their effects on microbial communities and biogeochemical cycles what is flux balance analysis? fast automated reconstruction of genome-scale metabolic models for microbial species and communities evolution of bidirectional costly mutualism from byproduct consumption a shared limiting resource leads to competitive exclusion in a cross-feeding system: role of environment for cross-feeder coexistence community structure follows simple assembly rules in microbial microcosms species interactions differ in their genetic robustness the spatial and metabolic basis of colony size variation predicting virus emergence amid evolutionary noise world health organization, global action plan on antimicrobial resistance targeting antibacterial agents by using drug-carrying filamentous bacteriophages use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections evolving phage vectors for cell targeted gene delivery bacteriophage lytic enzymes: novel anti-infectives phage lytic enzymes as therapy for antibiotic-resistant streptococcus pneumoniae infection in a murine sepsis model the recombinant phage lysin lysk has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant staphylococcus aureus a novel sustained-release matrix based on biodegradable poly(ester amide)s and impregnated with bacteriophages and an antibiotic shows promise in management of infected venous stasis ulcers and other poorly healing wounds dictionary of microbiology and molecular biology antigenic formulas of the salmonella serovars bacteriophage-resistance systems in dairy starter strains: molecular analysis to application a macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset antibiotic-containing polymers for localized, sustained drug delivery recent advances in the treatment of pathogenic infections using antibiotics and nano-drug delivery vehicles, drug des van der valk, managing water resources under climate uncertainty key: cord- -ps jyjkl authors: nan title: full issue pdf date: - - journal: jacc: cardiooncology doi: . /s - ( ) - sha: doc_id: cord_uid: ps jyjkl nan disease, coronary artery disease, arrhythmias, and pericardial disease, depending on the exact cardiotoxic agent ( ) . in this state-of-the-art review, we focus on long-term cardiac diseases after treatment for childhood cancer. we discuss the prevalence, risk factors, prevention, prediction, and surveillance of cardiac disease in this population (central illustration). we systematically searched pubmed for studies that described cardiac adverse events in children treated with cardiotoxic cancer treatments. we limited the search to full-text articles written in english and articles published within the last years. we selected articles with a study cohort of which > % were treated for childhood cancer before the age of years. for studies describing the prevalence or cumulative incidence of heart failure, we reviewed articles with a minimum of ccs; a minimum of ccs was required for the other outcomes. studies on primary prevention strategies were identified from previous cochrane searches ( ) ( ) ( ) . based on these criteria, studies were considered to be described in this review ( figure ). the full search strategy is provided in supplemental table . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . anthracyclines are clearly associated with cardiomyocyte damage. although the exact mechanism of anthracyclineinduced cardiotoxicity has not been fully elucidated, early studies indicate that cardiotoxicity through reduction-oxidation reaction cycling and the generation of reactive oxygen species may be the cause. more recently, topoisomerase b has been proposed to be a mediator of doxorubicin-induced cardiac injury ( ) . systolic dysfunction can eventually progress to heart failure. heart failure is one of the most frequent cardiac late effects in ccs ( , ) , and contributes to significant morbidity and non-cancer-related mortality later in life ( , ) . a large cohort from the childhood cancer survivor study investigated the occurrence of heart failure, defined by the common terminology for criteria adverse events as grade to . based on questionnaires concerning long-term ccs, the reported cumulative incidence is . % by years of age ( ) . these results confirmed earlier reports that anthracyclines and chest rt are strongly associated with heart failure ( ) . recently, it has been shown that even low-to-moderate chest rt doses increase the risk of heart failure substantially ( , ) . in the dutch later (late effects after childhood cancer) cohort, feijen et al. ( ) reported a cumulative heart failure incidence of . % years after childhood cancer diagnosis in ccs who received cardiotoxic cancer treatment. higher exposure to mitoxantrone and cyclophosphamide were suggested as novel treatment-related risk factors ( ) . although mitoxantrone has traditionally been classified as an anthracycline, it has been suggested that mitoxantrone results in cardiotoxicity through mechanisms different from anthracyclines ( , ) . mitoxantrone has a nonlinear dose-response relationship with heart failure risk ( , ( ) ( ) ( ) , and compared to doxorubicin, mitoxantrone is times more cardiotoxic. in addition, a younger age at diagnosis and presence of traditional cardiovascular risk factors may play a role in the development of heart failure ( ) . the influence of sex on the development of myocardial dysfunction is still incompletely conclusive ( , , , , ) . coronary artery disease. the risk of coronary artery disease (cad) is substantially increased in ccs. in the childhood cancer survivor study, the cumulative incidence of cad by age years was . % in survivors with and without exposure to cardiotoxic cancer treatments ( ) . this risk is dependent on chest rt dose with no established safe dose; this risk is also higher in males. the cumulative incidence of symptomatic cad at age years the main risk factors for cardiac disease in childhood cancer survivors are anthracyclines, mitoxantrone, and chestdirected radiotherapy dose. primary prevention strategies may reduce the risk of anthracycline-induced cardiomyopathy. there is an increased prevalence of traditional cardiovascular risk factors in childhood cancer survivors; screening and early management are important to modify risk. multivariable risk prediction models may help to individualize prevention and surveillance strategies. increases to % in males exposed to > gy of radiation ( , ) . the st. jude lifetime cohort study detected cad based on either history, electrocardiogram (ecg), or echocardiography in . % of asymptomatic ccs . years after cardiotoxic therapy ( ) . however, evidence from (non)invasive coronary angiography is scarce. a study evaluating computed tomography in asymptomatic hodgkin lymphoma ccs years old or younger (n ¼ ) exposed to chest rt showed coronary artery lesions to be very proximal, placing large portions of the myocardium at risk ( ) . valvular ( , , ) . chest rt has been identified as an important risk factor that increases at higher doses ( ) . other risk factors are treatment with anthracyclines, hypertension, congenital heart disease, and younger age at diagnosis, although these have not been uniformly shown in all studies ( , , ) . mild tricuspid regurgitation was most prevalent in studies describing valvular disease, but this is also very common in the general population ( , , ) . in lymphoma ccs who were exposed to chest rt, valvular heart disease, defined as mild or higher for left sided valves and moderate or higher for right sided valves, was most frequently detected in the aortic and mitral valves ( ) . valvular abnormalities after chest rt are most likely caused by direct irradiation injury to the valve cusps or leaflets, causing thickening, fibrosis, and calcification ( , ) . these processes progress with age and increase in prevalence over time ( , ) . hence, ccs without echocardiographic abnormalities after a short followup period are still at risk of severe valvular heart disease. pericardial disease. besides paraneoplastic and infectious causes, pericardial disease can arise from chest rt. late constrictive pericarditis, in particular, can lead to disabling symptoms and a poor prognosis ( ) . however, data on pericardial disease in ccs are limited. the childhood cancer survivor study showed a -fold higher risk of pericardial disease in all ccs versus siblings ( - year cumulative incidence, . %) and a dose-response relation with chest rt ( ) . a single-center study in ccs older than years after diagnosis (n ¼ , ; % no cardiotoxic therapy), reported symptomatic pericarditis in only ccs ( ) . although the diagnosis of constrictive pericarditis is difficult by echocardiography, thickening of the pericardium as well as hemodynamic consequences (e.g., "septal bounce," abnormal respiratory variations in doppler findings) can be suggestive. upon high clinical suspicion, cardiac computed tomography, magnetic resonance imaging (mri), and/or invasive hemodynamic evaluation may be needed to confirm the diagnosis ( ) . arrhythmias. the prevalence of symptomatic cardiac arrhythmias in long-term ccs is reportedly low ( , , , ) . in , ccs, the cumulative incidence of grade to arrhythmia by years of age was . % ( ) . a subsequent study (n ¼ , ) showed that chest rt > gy, anthracycline dose $ mg/m , dyslipidemia, and hypertension are risk factors for symptomatic arrhythmia ( ) . myocardial fibrosis caused by chest rt may contribute to the occurrence of arrhythmias. other frequently used cancer agents for pediatric cancers such as cisplatin, cyclophosphamide, and tyrosine kinase inhibitors may also be associated with supraventricular and ventricular arrhythmias ( , ) . prolonged qtc interval, which has arrhythmogenic potential, has been shown in ccs who received anthracyclines with and without chest rt ( , ) . also, rhythm disturbances such as premature ectopic beats and atrioventricular blocks have been reported in ccs ( ) ( ) ( ) . the literature on ecg abnormalities in large cohorts of long-term ccs is sparse ( , ) , data on the use of ambulatory ecg monitoring to define the prevalence of brady-and tachyarrhythmias induced by cardiotoxic cancer treatments are needed, but must be carefully weighed against the potential patient burden and clinical significance. childhood cancer survivors preventive measures for cancer treatment-induced cardiotoxicity. as the risk of cardiac disease is high in chest rt and anthracycline-treated survivors, and as omitting or diminishing the use of cardiotoxic treatments is not always possible, prevention is critical ( ) . advanced radiotherapy techniques to minimize exposure to the heart have been developed; the impact of those improvements is reflected by the decrease in cad in more recent treatment eras ( ) . extensive research has been devoted to the identification of possible cardioprotective interventions during anthracycline treatment that do not have negative effects on antitumor efficacy or other noncardiac adverse effects. below we discuss preventive measures that have been studied during anthracycline treatment. we focus primarily on randomized controlled trials (rcts) as they provide the highest level of evidence to answer this type of question. because of developmental changes and the differences in the body composition of children, data from adults cannot be reliably extrapolated to children ( ) . d e x r a z o x a n e . dexrazoxane is one of the most widely investigated cardioprotective pharmacologic interventions. it has been shown in adult cancer patients to prevent clinical and subclinical cardiac damage ( ) . the few published pediatric rcts have included participants diagnosed with leukemia, lymphoma, and sarcoma ( ) ( ) ( ) . these studies suggest that there are no significant differences in clinical heart failure between dexrazoxane and control patients ( , ) , although dexrazoxane might have a protective effect on asymptomatic cardiotoxicity ( , ) . all studies included relatively short-term follow-up, and the impact on outcomes after longer follow-up is yet unknown. currently, dexrazoxane is not routinely used in clinical practice for all children treated with anthracyclines. this might be explained by a concern over interference with antitumor efficacy and the occurrence of secondary malignancies ( ). however, high-quality evidence to support an increased risk of secondary malignancy is lacking. a cochrane systematic review identified no significant differences between treatment groups ( ) , which is in line with more recently published randomized trials ( , ) . a recently published nonrandomized study in pediatric patients with acute myeloid leukemia mortality. an overview of validated prediction models in ccs is provided in supplemental table . heart failure prediction models. practical models to predict heart failure onset before the age of the discriminatory abilities of the model were further shown by a cumulative incidence of heart failure at age years of . % in the low-risk group, whereas this was . % in the high-risk group. importantly, . % of the ccs were at low risk according to the model and thus unlikely to develop heart failure. ( ) . thus, early, at years after diagnosis, cardiovascular risk factors have been shown to provide little incremental information to prediction models for heart failure and ischemic heart disease ( , ) . in a more recent study, diabetes, hypertension, and dyslipidemia were used in the prediction of heart failure and ischemic heart disease in ccs who were , , , or years of age at time of prediction, with relative risks comparable to moderate doses of anthracyclines ( ). cardiovascular risk factors were present in approximately % of the ccs at the age of years and were strong predictors of heart failure and ischemic heart disease. although the discrimination of the prediction models improved with the addition of cardiovascular risk factors, the c statistics were modest for both events ranging from . to . in the derivation cohort with successful replication in the other one-half of the cohort. both the heart failure and the ischemic heart disease predictions models showed good calibration. a small, very-highrisk group was identified with cumulative incidences of heart failure or ischemic heart disease of w % at age years; survivors in this very-high-risk group may benefit from more frequent surveillance and/or early interventions to modify their risk. however, low-risk survivors who may be excluded from further surveillance could not be identified with these models as cumulative incidences of heart failure (w . % to . %) and ischemic heart disease (w % to . %) were still significantly higher compared to siblings at the age of years. in the context of prediction model-guided surveillance, this can be seen as the benefit of early detection of asymptomatic cardiac dysfunction among those who will develop heart failure (true positives) weighted against the potential harm of an unnecessary diagnostic workup and/or treatment in those who will not develop heart failure (false positives). through decision modeling using simulations it has been shown that routine echocardiographic surveillance for asymptomatic cardiomyopathy every years may be more cost-effective, especially in those treated with an anthracycline dose < mg/m ( ). decision modeling provides weaker evidence on the clinical impact compared to an rct, but it requires no follow-up and is less expensive to perform. such analyses could be performed to assess clinical impact and cost-effectiveness before conducting an rct. there are different methods and techniques available to detect anthracycline treatment induced cardiomyopathy. much of the research in detection of cardiac diseases is focused on improving early detection of myocardial dysfunction. we will describe diagnostic methods that have been studied over the past decade in ccs. an abnormal gls in % of the cohort who were exposed to anthracyclines and/or chest rt and had normal lvefs. both cumulative anthracycline dose > mg/m and any cardiac rt dose were associated with an increased risk for abnormal gls ( ) . it is currently unknown whether an abnormal gls is associated with development of an lvef < % or clinical heart failure in ccs. diastolic dysfunction after cardiotoxic cancer treatment has also been described in ccs ( , ) . in the st. jude lifetime cohort, diastolic dysfunction grades to (based on peak mitral flow velocity, mitral septal and lateral early diastolic velocity, and left atrial volume) was detected in % of all ccs who were exposed to cardiotoxic treatment and in . % with normal lvef ( ) . one must be aware of the dif- table ). the current lack of mechanistic insights for ( , ) tyrosine kinase inhibitor- ( ) , and trastuzumab-related cardiotoxicity ( ) . inflammation studied primarily to reduce major adverse cardiovascular events was associated with a significant reduction of lung malignancy ( ). meta-analyses of therapies lowering low-density lipoprotein cholesterol (ldl-c) demonstrated that prevention of cardiovascular events was proportional to the absolute reduction in ldl-c levels with no major safety concerns, specifically no increase in malignancies ( , ) . however, available data from previous ldl-c-lowering clinical trials have not systematically adjudicated data for malignancy, because these were not considered disease-related events ( ) . an increased rate of malignancy associated with the use of the combination of ezetimibe and simvastatin was unexpectedly reported in the seas (simvastatin and ezetimibe in aortic stenosis) trial ( ) , which compared simvastatin-ezetimibe to placebo in , adults with mild-to-moderate aortic stenosis over a median follow-up of . years ( ). ezetimibe is a nonstatin drug that inhibits the intestinal absorption of cholesterol by targeting the transmembrane protein, nieman-pick c -like ( ) . it is recommended for further ldl-c reduction in combination with a statin ( values are median ( th, th percentiles) or n (%). wilcoxon rank-sum test of differences between with and without primary malignancies for continuous variables. chi-square test of frequencies between with and without malignancies for categorical variables. *the no-malignancy group summary statistics are based on patients without malignancy diagnosis prior to death, loss to follow-up or end of the study. acei ¼ angiotensin-converting enzyme inhibitor; acs ¼ acute coronary syndromes; arb ¼ angiotensin receptor blocker; hdl-c ¼ high-density lipoprotein cholesterol; hs-crp ¼ high-sensitive c-reactive protein; iqr ¼ interquartile range; ldl-c ¼ low-density lipoprotein cholesterol; mi ¼ myocardial infarction; trs p ¼ timi risk score for secondary prevention ( ) . of the skin, as well as limiting malignancy events to only those with available pathology reports. we also performed a competing risk analysis integrating allcause death as a competing outcome in the model using fine and gray's method. finally, we also evaluated the hrs in both arms with increasing duration of follow-up as done in previous publications ( , ) . all analyses were performed using sas software, simvastatin/ezetimibe (red) and placebo/ezetimibe (blue). the primary malignancy endpoint was defined as new, relapsing, or progressive malignancy (excluding nonmelanotic skin malignancy). the cumulative incidence plots were presented graphically using kaplan-meier product-limit method. ci ¼ confidence interval; hr ¼ hazard ratio. simvastatin/ezetimibe (red) and placebo/ezetimibe (blue). the cumulative incidence plots were presented graphically using kaplan-meier product-limit method. ci ¼ confidence interval; hr ¼ hazard ratio. table ) . results were similar between treatment groups for deaths due to malignancy by location table . values are n (%). (p > . for each comparison) (supplemental table ). further analyses by gender showed no significant differences between treatment groups for both the primary malignancy endpoint (p > . for each comparison) (supplemental tables and ) and deaths due to malignancy (p > . for each comparison) (supplemental tables and ). finally, we did not observe any differences in the extent of malignancy by treatment arm ( table ) . in subgroup analyses, the rates of the primary malignancy endpoint were similar between treatment groups for each of the pre-specified high-risk subgroups (each p for interaction > . ) ( figure (figure ). during the years of follow-up, there was no divergence in the km curves over time ( figure ) ; the year-by-year hrs for the primary malignancy endpoint comparing the two treatment arms did not demonstrate a progressive trend over time ( table ) . the number of events and the -year kaplan-meier rates are shown. total cholesterol: th percentile ¼ . mg/dl, th percentile ¼ . mg/dl and th percentile ¼ . mg/dl. abbreviations as in figure . some concern has been expressed that statin and other ldl-c-lowering drugs might be carcinogenic and consequently part of the malignancy data was collected retrospectively. fifth, although the analyses of outcomes stratified by subgroups were pre-specified, the power was low in these subgroups. finally, we did not measure phytosterols or other potential protective or tumor-promoting factors in blood to evaluate the effect of ezetimibe on the potential mechanistic pathways related to malignancy. we found that ezetimibe did not increase the rates of malignancy nor deaths due to malignancy in , patients with recent acs treated with simvastatin and followed up for a median of years totaling , patient-years of follow-up. acknowledgment therapies to reduce ldl-c such as statins or ezetimibe have significantly improved cardiovascular outcomes; this benefit has been ascribed to pleiotropic effects beyond lowering ldl-c. however, such pleiotropic mechanisms have raised safety concerns about long-term use. a potential carcinogenic effect has been of particular concern as earlier studies sug- -year risks in cantos were % (range % to %) and % (range % to %) for total cancer; % (range % to %) and % (range % to %) for colorectal cancer; and % (range % to %) and % (range % to %) for lung cancer. conclusions lifetime and -year risk of total, colorectal, and lung cancer can be estimated reasonably well in patients with established cvd with readily available clinical predictors. with additional study, these tools could be used in clinical practice to further aid in the emphasis of healthy lifestyle changes and to guide thresholds for targeted diag- ( ) . for the current study , patients were included, after exclusion of patients younger than years or older than years. detailed descriptions of the ucc-smart cohort and the cantos trial have been published elsewhere ( ) ( ) ( ) . the studies were approved by institutional review boards and all participants provided written informed consent. an overview of eligibility criteria is provided in supplemental table . missing data (per variable # . % for ucc-smart and # . % for cantos) were singly imputed by weighted probability matching using multivariable regression for the baseline and outcome data. complete case analysis yielded similar model coefficients. continuous variables were truncated at the st and th percentile to limit the effect of outliers on the model coefficients (i.e., leverage) ( ) . to prevent overfitting, predictors were preselected based on presence in previously published risk prediction models of multiple cancer types. antiplatelet use (aspirin, p y -adp receptor antagonist, or other, such as dipyridamole) was added as a predictor, due to its inclusion in multiple previously published prediction models for colorectal cancer and due to the common use of antiplatelet therapy in patients with cvd. furthermore, it was required that the variables were readily clinically available, as well as present in the derivation dataset. this led to the following predictors: age, sex, smoking status, weight, height, alcohol use, use of antiplatelet medication, and diabetes mellitus (supplemental table details an overview of predictor selection). in addition, crp was added as a predictor after a literature search for predictors of cancer was performed ( , , , ) . definitions of the predictors in the ucc-smart cohort and cantos trial are provided in supplemental table . was calculated for each following life-year. next, for each subsequent age year the probability of being healthy and alive at the start of that time interval (age year) (e t þ ) was calculated by multiplying the survival probability (e t ) by the event-free survival probability during that year ( À a t À b t ). these steps were repeated from the age at baseline of an individual patient to the maximum age of years, and together these predictions form an individual lifetable ( , ) . the cancer-free life expectancy was determined as the age at which the median estimated cancer-free survival curve is %. for -year and lifetime risk of cancer, the cumulative cause-specific risks were truncated at years after the age at baseline, and at the age of years, respectively. cantos study populations are shown in table ( ), leading to restricted variation in cancer types. our cancer prediction models performed reasonably well, and calibration plots before and after ( ). second, complex models based on machine learning would not be expected to provide improved performance or better prognostication in the current study, consistent with other large-scale external validation studies ( , ) . from a clinical perspective, a key concern should be whether the predictions from the risk calculator are well calibrated ( ) . for example, the -year-old male in the example calculation sheet has a predicted . % risk of lung cancer, while the risk may in fact be . %, or %. this type of miscalibration has been common in earlier evaluations of lung cancer prediction models ( ) . indeed, the investigators state that "calibration is a more clinically relevant for risk ( ) . it was recently rediscovered and presented through a "decision curve" ( , ) , which is unfortunately missing from the current report. future work should consider the clinical decisionmaking perspective more fully, with net benefit in a decision curve as a step towards a more comprehensive cost-effectiveness analysis. in sum, the presented risk calculator is very promising given its high-quality data sources, large values are mean ae sd or %. *p < . across diuretic categories. †defined as nt-probnp > , pg/ml or bnp > pg/ml. ‡defined as troponin-t > . ng/ml or troponin-i > . ng/ml. ace ¼ angiotensin converting enzyme; arb ¼ angiotensin receptor blocker; attr ¼ transthyretin amyloidosis; bmi ¼ body mass index; bnp ¼ b-type natriuretic peptide; dbp ¼ diastolic blood pressure; egfr ¼ glomerular filtration rate; gdmt ¼ guideline directed medical therapy; hf ¼ heart failure; lvef ¼ left ventricular ejection fraction; mra ¼ mineralocorticoid receptor antagonist; nt-probnp ¼ n-terminal pro-b-type natriuretic peptide; nyha ¼ new york heart association; sbp ¼ systolic blood pressure; shfm ¼ seattle heart failure model. table ). figure a) . similarly, for the uk þ diuretic dose þ nyha functional class model, survival was . years, . years, and . years for the low-, intermediate-, and high-risk groups, respectively (log-rank p < . ) ( figure b ). we also tested the ability of the risk scores to predict a combined outcome of mortality or cardiac transplantation ( table ). optimism-adjusted aucs at years ( adding diuretic dose to the uk score increased the auc from . to . and additionally adding nyha functional class, increased it to . . for diuretic dosing, points were assigned for mg/kg, point for > to . mg/kg daily dose, points for > . to mg/kg daily dose, and points for > mg/kg daily dose. *difference in discrimination slopes between models, where the discrimination slope is the difference between the mean predicted probabilities for events and nonevents. †sum of net proportions of persons with and without the event correctly assigned to a different risk category; ranges from À to . ‡similar to the idi except compares difference between median predicted probabilities. idi ¼ integrated discrimination improvement; nri ¼ net reclassification index; other abbreviations as in tables and . for diuretic dosing, points were assigned for mg/kg, point for > to . mg/kg daily dose, points for > . to mg/kg daily dose, and points for > mg/kg daily dose. abbreviations as in tables to . cheng et al. study limitations. our cohort included both wtattr and hattr. it is possible that risk markers may be differentially predictive in these distinct cohorts because natural disease progression is more aggressive with hattr, and there may also be differences between mutation types. of note, the mayo risk score ( ) only the latter remained significant in multivariable analysis. cardiovascular diagnosis usually includes imaging; thus cardiologists and patients have a tendency to focus on those results. although cardiovascular imaging has revolutionized the diagnosis of cardiac amyloidosis, the role in prognosis is less clear. when the addition of imaging variables to the staging systems is considered, the availability, reproducibility, and incremental value must also be considered. in contrast to the current study, nyha functional rodney falk appropriately coined the term "toxicinfiltrative cardiomyopathy" in reference to the toxicity of circulating light chains (al) to cardiomyocytes in al ( ). similar mechanisms of cardiomyocyte toxicity due to ttr oligomeric intermediates have been reported in attr ( , ) . cardiac amyloidosis is not a simple infiltrative con- doacs in cancer such as those related to trial designs or the risk for contamination or crossover between the groups. data synthesis and analysis. we extracted data from the original primary publications ( ) ( ) ( ) ( ) the flowchart describes study search, screening, and selection processes. sabatino et al. were included in the systematic review and metaanalysis ( ) ( ) ( ) ( ) . the study flowchart is described in figure . table . supplemental figure . overall, the risk for selection table . figure d) . overt bleeding that was associated with a decrease in the hemoglobin level of $ g/dl, led to a transfusion of $ u of blood, occurred in a critical site, or contributed to death overt bleeding that did not meet the criteria for major bleeding but was associated with the use of medical intervention, contact with a physician, interruption of the assigned treatment, discomfort, or impairment of activities of daily living. mcbane et al. ( ) ; overt bleeding plus a hemoglobin decrease of $ g/dl or transfusion of $ u of packed red blood cells, or intracranial, intraspinal/epidural, intraocular, retroperitoneal, pericardial, intra-articular, or intramuscular with compartment syndrome, or fatal bleeding overt bleeding not meeting the criteria for major bleeding but associated with medical intervention, an unscheduled contact with the health care team, or temporary anticoagulant cessation gi ¼ gastrointestinal; other abbreviations as in table . sabatino et al. over the past few years, the use of doacs has revo- values are n (%). *basal cell or squamous cell carcinoma of the skin, primary brain tumor or known intracerebral metastases, and acute leukemia were not included in the caravaggio trial. †basal cell or squamous cell carcinoma of the skin were not included in the select-d trial. ‡basal cell or squamous cell carcinoma of the skin were not included in the hokusai vte cancer trial. §other than brain tumors. ǁ data not available, because brain tumors were included under "other tumors". abbreviations as in tables and . the data from the caravaggio trial concerning the risk of major bleeding were particularly noteworthy. it appears that this finding was not related to any differences in the definition of major bleeding, because this was relatively consistent across trials ( table ). in contrast, the differences in reported major bleeding risk might have resulted from the heterogeneity in enrolled populations. patients with primary brain tumors, brain metastases, and acute leukemia were excluded from the caravaggio trial. patients with a recent diagnosis of testicular/retroperitoneal germ cell cancer and orchidectomy # weeks prior scheduled for cisplatin-based chemotherapy or active surveillance were recruited. participants were stratified into groups by management: ) active surveillance for stage low-risk disease; ) to cycles of adjuvant bleomycin, etoposide, and cisplatin (bep) chemotherapy for stage high-risk disease; or ) to cycles bep chemotherapy for metastatic disease. red circles illustrate study assessments. blood samples were simultaneously drawn from each table ) . this remained numerically greater than baseline thereafter, but was not statistically significant. there were trends toward abbreviations as in table . table ) . there was no differences between groups (p ¼ . ). endothelial dysfunction is a key feature in de novo thrombosis and rupture of pre-existing atherosclerotic plaque ( ) . as such, it is an important component to the risk of thrombotic cardiovascular events in the early period following cisplatin-based chemotherapy. in our cell-based study, exposure of haecs to cisplatin was associated with decreased t-pa mrna expression and activation of akt and erk / . in previous work, cisplatin has been shown to reduce endothelial cell survival and induce apoptosis in human dermal microvascular endothelial cells (hmec- ) ( ) and in the vasa nervorum in rats ( ) . these data support the hypothesis that cisplatin induces direct endothelial toxic effects resulting in increased stress kinase signaling and a propensity for thrombosis via reduction in the capacity for endogenous fibrinolysis. although adverse thrombotic effects are most frequently observed in the early period following chemotherapy, cisplatin is detectable in serum for several years after treatment ( ) . this chronic exposure may provoke low-grade endothelial stimulation and a consequent pro-atherogenic environment ( , ) . our subsequent assessment was with forearm venous occlusion plethysmography, the gold standard for assessing endothelial function ( ) . in appears to be the time period of increased cardiovascular risk. while these effects predominantly occur in the early period after treatment, cisplatin is detectable in serum several years after treatment ( ) . it remains possible that chronic exposure to low levels of cisplatin may cause low-grade endothelial stimulation that contributes to the pathophysiology underlying cardiovascular events occurring more than a decade after initial exposure ( , ) . nephrotoxicity is a major, dose-limiting side effect of cisplatin that affects % to % of patients ( , ) . we found evidence of early nephrotoxicity following cancer an acute decrease in fmd was seen as early as with the first cycle of carboplatin and paclitaxel chemotherapy ( ) . in conjunction with the current findings, one would conclude that the common denominator is platinum drugs and that there is no gender-related difference. furthermore, one would conclude that platinum drugs seemingly induce relatively acute changes in endothelial cells that translate into a reduction in flow-mediated vasodilation and alterations in nitric oxide bioavailability. interestingly, a prior study in testicular cancer survivors showed that those patients who were exposed to cisplatin-based chemotherapy nearly to more than years ago had a more severe reduction in fmd and higher levels of circulating endothelial cells than those not exposed ( ) . the fact that cisplatin levels are detectable even nearly years from therapy supports the theory of long-term exposure and endothelial injury ( ) . a difference in vascular reactivity between cisplatin-exposed and cisplatin-nonexposed was not seen in the current study by cameron et al. ( ) . it is important, however, to realize that the vascular response was blunted in both groups, even in comparison with patients with metabolic syndrome (considered to account for the vascular outcomes in testicular cancer survivors as mentioned) ( ) . such results can also not be unequivocally attributed to hypogonadism ( , ) . ( ) ( ) ( ) ( ) . in patients that survive beyond years, mortality rates are to times higher than the general population, corresponding with a % shorter life expectancy regardless of age at transplantation ( ) . the incidence of heart failure (hf) has been reported to be between . % to . % in those who have survived at least years after hsct ( , , , ) , and the risk of cardiovascular (cv) related mortality is to times higher in hsct survivors than in the general population ( were measured on separate occasions to reduce bias. studies followed current guidelines for evaluation of lv function and cardiotoxicity ( , ) . measurements were averaged from consecutive heart cycles. d-lvef was manually traced using the modified simpson's biplane method ( ) . d-lvef was calculated with semiautomated software for endocardial detection, and was subsequently manually adjusted. a d pyramid volume acquisition was obtained from to cardiac cycles, adjusting for depth and sector width ( to ), resulting in an average volume rate of frames/s (range to frames/s). sex-specific cutoffs for d-lvef were used as recommended ( ) . gls ( , , , ) . however, this study cannot discern the precise timing of lvsd onset, and one could potentially speculate that cardiac injury occurred at time of therapy, with a further worsening in lvsd over time. another possible explanation is that young patients are at higher risk of heart disease due to organ immaturity and growth disturbances caused by cardiotoxic therapies ( ) . this may explain the smaller lv size in survivors compared with control subjects, even after consideration for cv disease and confounders. sex has previously been implicated as a risk factor for cardiotoxicity ( , ( ) ( ) ( ) , but this association was not found in our study. cyclophosphamide has historically been noted to have cardiotoxic effects ( ) . however, published data linking alkylating agents to long-term heart disease is scarce. in contrast, anthracycline exposure is known to cause myocyte depletion, and is shown to increase the risk of hf by -fold in long-term ( , , , , ) . in addition to cardiotoxic therapies, we found that traditional cv risk factors have a potential role in modifying the risk for lvsd in long-term survivors of allo-hsct. cv risk factors are commonly reported in hsct survivors, occurring more frequently in allo-hsct compared with autologous-hsct survivors, resulting in a higher prevalence of heart conditions in allo-hsct survivors ( , , ( ) ( ) ( ) . survivors in this study had a high prevalence of cv risk factors, but at a level comparable to other studies ( , , , , ( ) ( ) ( ) . abbreviations as in table . treated with hsct at various ages ( , , , , , ) . the significance of hypertension in contributing to prevailing cardiac disease in hsct survivors has previously been shown ( , ) . however, in our study, given that the onset and duration of hypertension is unknown, it is unclear if the cumulative consequences of hypertension were fully manifested. indeed, the echocardiograms showed little evidence of concentric remodeling beyond a small but significant difference in indexed lv end-diastolic volume. however, hypertension is known to cause myocardial fibrosis that leads to reduced longitudinal shortening of the heart ( ) . this may explain why hypertension was found to be an independent predictor of gls. hypothyroidism has been reported in % of longterm survivors of hsct with busulfan conditioning ( ) . hypothyroidism was found in . % in our cohort, although no association with lvsd was found in our data. dyslipidemia has been reported in % to % of survivors of hsct ( , , , , , ) . in our study, gvhd was found to be a highly prevalent complication of allo-hsct survivors. there is limited evidence that gvhd directly mediates myocardial damage. however, active chronic gvhd values are median ( th, th percentiles), n (%), or mean ae sd. *significant difference between anthracycline $ mg/m and < mg/m in bonferroni post hoc analysis (< . ). †significant difference with both treatment groups (anthracycline $ mg/m and < mg/m ) with no anthracycline group in bonferroni post hoc analysis (< . ). ‡no significant difference between treatment groups in bonferroni post hoc analysis. §echocardiographical parameters are adjusted for covariates of age, heart rate, body mass index, and diastolic blood pressure. ksignificant difference between anthracyclines $ mg/m and no anthracycline in bonferroni post hoc analysis (< . ). ¶ase cube formula ( ) . table . has been shown to be associated with a higher risk of cv death ( ) . it is thought that the chronic inflammation processes instigated by gvhd results in endothelial damage leading to accelerated atherosclerosis ( ) . we did not find gvhd (acute or chronic) to be consistently associated with lv systolic function, and found a very modest association with remodeling ( , ) . a remaining challenge with gls is the lack of consensus in defining absolute cutoffs for dysfunction, variability, and intervendor differences ( ) . the gls cutoff used in our analysis was based on our normal data, using the same equipment and operator. this value (À %) is conservative when compared with a meta-analysis performed by yingchoncharoen et al. ( ) , which found the % ci for normal gls to be À . % to À . % ( ) .  phosphate-buffered saline) and sacrificed weeks later. as observed in previous studies ( , ) , body mass at the time of sacrifice was lower (À %) in the group administered with doxo ( table ) . lower body mass with doxo was associated with lower heart, quadriceps, gastrocnemius, and epididymal white adipose tissue mass, food consumption (À %), and water intake (À %) ( table ). there were no significant differences in the mass of other tissues and organs (supplemental table ). we first sought to establish that total ros bioactivity was increased in huvecs exposed to plasma from doxo-treated mice compared with sham control mice. to do so, we assessed total cellular ros using the cellrox fluorescent probe (thermo fisher [waltham, massachusetts]; catalog# c ) and found that ros bioactivity was greater in huvecs incubated with plasma from doxo-treated mice compared with sham control mice (p < . ) ( figures a and e ). next, we aimed to determine if enhanced mitochondrial ros contributed to the greater total huvec ros induced by doxo administration by assessing mitochondrial ros bioactivity using the mitosox fluorescent probe (thermo fisher, catalog# m ). we observed greater mitochondrial ros in huvecs exposed to plasma obtained from doxo compared with sham-treated animals (p < . ) (figures b and e ). to determine if this greater ros bioactivity was linked to changes in mitochondrial volume, we figure ) . to determine associations of these plasma metabolites with mitochondrialspecific aortic ros, we performed linear regression analyses. we found that plasma diphosphate (p ¼ . ) and lactate (p ¼ . ) tended to be positively associated with aortic mitochondrial ros (supplemental figure ) . a full report of metabolite abundance is provided in supplemental table . dot blot array. to expand on this initial analysis of potential circulating signals influenced by doxo treatment, we next targeted inflammatory pathways, given that doxo administration is associated with higher levels of pro-inflammatory proteins in the circulation ( ) . to address this aim, we assessed plasma concentrations of different cytokines and chemokines via a dot-blot array. we found that all but of these markers did not differ significantly in plasma from doxo-treated mice compared with sham control mice ( table ). the exceptions were the antiinflammatory cytokine interleukin- , which was only slightly (< %), but significantly (p ¼ . ) lower in the doxo mice, and vascular endothelial growth factor-a (vegf-a), which stood out as being % lower in the doxo treated group (p < . ). moreover, plasma vegf-a was inversely associated to accomplish this, we supplemented plasma from table ). doxo chemotherapy has been shown to impair endothelial function (i.e., reduce edd) in conduit arteries of cancer survivors ( ) . however, the underlying mechanisms and associated therapeutic targets have not been established, because mechanism-focused investigations to date have been largely limited to endothelial cell culture models ( , ) . in the present study, we first determined that these findings also are in agreement with previous work reporting excess production of mitochondrial ros and impaired vascular endothelial function in mice with genetic sod insufficiency ( ) . it should be noted that other ros generating pathways may also contribute, including oxido-reductases such as endothelial no synthase or nadph cytochrome p reductase ( ) . another key finding from our experiments was that changes in the composition of the circulating milieu may contribute to doxo treatment-associated anthracycline treatment in these same children ( ) . the decrease in nrg after anthracyclines has also been observed in adults undergoing anthracyclinebased chemotherapy, which is interpretable as a sign of vascular endothelial cell injury ( ). endothelial cell-derived nrg acts through the erbb receptor tyrosine kinase family to regulate the growth and survival of cardiac myocytes, and protects from anthracycline cardiac cytotoxicity ( , ) . mitoq and other coenzyme q preparations are available over the counter, and coenzyme q supplementation has been studied not only as a way to limit injury to the cardiovascular system, but also kidney and reproductive organs ( , ) . these prom- table ). fulfilled criteria for myocarditis was based on european were receiving dexamethasone as palliative treatment for metastatic disease (supplemental table ). and with other noncardiac iraes. crp in patients with no iraes was < mg/l at baseline and between c and c also was not significantly different (p ¼ . ). in this retrospective study of lung cancer patients receiving ici, there was an % incidence of mace to the rural population of eastern nc ( , ) . indeed, higher inflammatory signals and incidence of pneumonitis were also observed in this lung cancer population in comparison to those reported previously ( , ) . furthermore, in the lung cancer patients who received radiation, it has been suggested that the synergistic effect of radiotherapy and immunotherapy for priming of an endogenous antigenspecific immune response may contribute to a higher incidence of mace by t-cell recognition of shared antigens ( ) . values are mean ae sd or n (%). *analysis was not performed secondary to limited sample size. bb ¼ beta-blocker; bnp ¼ brain natriuretic peptide; cad ¼ coronary artery disease; ccb ¼ calcium channel blocker; ckd ¼ chronic kidney disease; copd ¼ chronic obstructive pulmonary disease; crp ¼ c-reactive protein; cva ¼ cerebrovascular disease; dm ¼ diabetes mellitus; egfr ¼ estimated glomerular filtration rate; hld ¼ hyperlipidemia; htn ¼ hypertension; nlr ¼ neutrophil-lymphocyte ratio; raas ¼ renin-angiotensinaldosterone system; wbc ¼ white blood cell count; other abbreviations as in table . timing of the development of mace with a median delay of days and a median of doses from the first ici administration in this study was similar to that previously reported ( , , ) . in view of this timing of onset from earlier studies, there is a general recommendation that baseline and surveillance cardiac testing (echocardiogram, ecg, tni and bnp) be considered during this potential mace window period, noted as after the second and after the fourth ici cycle administration ( ) . most of the present mace cases were not associated with a decrease in ef from baseline, which is consistent with previous studies ( , ) , suggesting that relying solely on ef in ici-treated patients with mace may be limited for the detection of ircs. tni was observed to be mildly elevated at the time of mace. however, elevations in tni have also been observed in cancer patients receiving cancer therapy, including icis without any cardiotoxicities ( , ) , thus suggesting that its utility may also be limited for the detection of irc. as demonstrated by this study, symptoms of irc may be variable, including nonspecific symptoms of our institution that observed a significant increase in crp with nivolumab-related pneumonitis that was mitigated with use of tocilizumab ( ) . similar inflammatory-mediated mechanisms such as interleukin (il)- may be observed in car t cell-related cardiotoxicities ( ) further highlighting and extending the utility of crp in the detection of ircs. another inflammatory marker that has been previously studied in noncardiac iraes and observed to be elevated at the time of irae is nlr ( , ) . nlr is easily obtained, inexpensive, and a routine test, and is calculated from the total white blood cell count that reflects the ratio of the innate (neutrophils) and the adaptive (lymphocyte) immune pathways, whereby an elevation results in an imbalanced toxic inflammatory response with release of cytokines ( ) . we observed an increase in nlr in patients who experi- however, ici treatment can result in immune-related adverse events (iraes) targeting any organ. cardiovascular iraes, particularly myocarditis, have received considerable attention due to their potentially fatal outcome ( , ) . ici were initially tested and approved as single therapy in patients with metastatic melanoma, a cancer type that historically had few treatment options and poor survival ( ) . later, these therapies were tested in patients with lung cancer and renal cell carcinoma ( , ) . in the latter studies, ici were tested either in combination with or following exposure to classic chemotherapies or targeted therapies. as a result, there was a growing need to define the cardiovascular sequelae in lung cancer patients treated with ici, where the cardiovascular risk was complicated by exposure to cardiotoxic nonimmune-based cancer therapies and a high prevalence of conventional cardiovascular risk factors ( ) . there was also a need to identify biomarkers or imaging approaches that screened for early cardiotoxic effects from immunotherapy and to identify patients at risk who would benefit from closer monitoring ( ) . histopathological analysis of the emb specimen showed intense myocardial inflammation comprised of numerous giant cells associated with many mononuclear cells, and prominent myocyte necrosis ( figure ). immunohistochemistry showed that the inflammatory cells were mainly cd positive giant cells and macrophages and cd -positive t-lymphocytes, many of them exhibiting granzyme b, perforin, and tia cytotoxicity markers. pd-l was expressed only by inflammatory cells and not by cardiomyocytes. cd and cd lymphocytes as well as nkp -positive nk cells were poorly represented (not shown). immunohistochemistry for viral protein detection was performed on heart biopsies and showed foci of positive cardiomyocytes surrounded by inflammatory infiltrates, indicating endomyocardial viral protein synthesis activities. steroids were gradually tapered and discontinued at months in light of the viral analysis. cardiac magnetic resonance imaging showed at weeks: ) resolution of myocardial edema (average myocardial t ¼ ms (figure ) , t values of ms, and extravascular volume decreased to %); ) recovery of lvef to %; and ) improvement of right ventricular function with an ejection fraction of % at weeks to % at months follow-up. the cancer had not progressed at months follow-up, and the patient reported normal functional status. ici therapy was not restarted at the time of last follow-up. this case highlights the heterogeneity of pathogenesis of acute myocarditis on ici therapy. we describe here acute myocarditis with long-term ici therapy, secondary to giant cell myocarditis due to enterovirus. giant cell myocarditis is uncommon; its pathogenesis is poorly understood, and prognosis is poor. recent data from an international registry including patients with histologically proven severe myocarditis demonstrated that giant cell myocarditis bears the worst prognosis ( ). the favorable outcomes in our patient, although the number of patients eligible to ici treatment is increasing dramatically, the pathogenesis of myocarditis needs to be further investigated. based on only extensively studied cases ( ) emerging clinical syndromes ( ) . the american heart association (aha) has recently proposed diagnostic criteria for ici myocarditis diagnosis based mainly on clinical characteristics, abnormal biomarkers, and/or cardiac imaging abnormalities ( ) . the recent publication of ici myocarditis definitions by the aha has the value of standardizing criteria to improve reporting. steroids are the first-line front treatment of ici myocarditis, despite poor evidence to support this, followed by other immune modulators, plasma exchange, or even ctla- agonist (abatacept) infusions. in the present study case, steroids were introduced and then discontinued after the confirmation of active viral markers on emb. indeed, the prognosis of t-cell ici myocarditis is partly driven by early initiation of steroid treatment ( ), which should not be held when awaiting histological or viral results. endomyocardial biopsy is recommended whenever possible in fulminant myocarditis ( ) . it is, however, likely to be overlooked in cancer patients. this case report emphasizes the risk for opportunistic viral infection during long-term ici therapy and utility of biopsy. the first mechanism to be considered in the setting of ici myocarditis is t-cell mediated, prompting steroids as a first-line therapy early after hospital admission. endomyocardial biopsy should be performed whenever possible to rule out infectious causes of myocarditis to guide therapy and further our understanding of the potential varying presentations of ici myocarditis. were paramount in preventing discordant care, especially when her clinical status had many rapid changes while she was in the intensive care unit. she completed car t cell therapy and was discharged to acute rehabilitation after a one-month hospitalization. she was unable to be restarted on gdmt due to hypotension. her pa d was mm hg at discharge. the plan was to obtain a positron emission tomography scan after acute rehabilitation to reevaluate her cancer. her initial course at the facility was uncomplicated, and she was able to participate in physical therapy. she had weekly appointments in the oncology clinic and an appointment with the advanced hf team. the pro- patients ( ) . hypotension is typically treated with intravenous fluids to maintain systolic blood pressure > mm hg. third-spacing of fluids with capillary leak is common, and this can lead to noncardiogenic pulmonary edema. a single-center retrospective study demonstrated that % of patients developed the composite outcome of arrhythmias, decompensated hf, or cardiovascular mortality ( ). the cardiac toxicities associated with car t cell therapy have led many centers to view significant cardiovascular disease as a relative contraindication ( ). in of the main clinical trials for car t cell therapy in patients with refractory dlbcl, patients were excluded if their lvef was # % to % ( ). our team decided to proceed with car t cell therapy in the patient described in this report because of her lack of other comorbidities, preserved performance status, and normal invasive hemodynamics. it was also her only treatment option for refractory dlbcl. the cardiomems device-which is typically used to manage outpatients with hf-was an essential component of our patient's medical treatment ( ) . for example, at periods in the patient's hospitalization she was found to have diffuse pulmonary infiltrates, hypoxemia, and hypotension. her pa d was and mm hg the incidence of secondary metastatic tumors to the heart is to times more common than primary malignant tumors, and estimated to be in the range of . % to . % at autopsy series in the general population, and up to . % in patients with known malignancies ( , ) . the incidence of cardiac metastases has increased over the past decades, likely due to improved life expectancy of oncological patients and advances in diagnosis ( , ) . the most common primary malignancies metastasizing to the heart include lung cancer, breast cancer, malignant melanoma, germ cell tumors, and hematological malignancies ( - ). the most frequent mode of metastatic spread of scc of the cervix is via the lymphatics to the para-iliac and para-aortic lymph nodes ( ). hematogenous spread is unusual and most commonly involves the lungs, bone, liver, and brain ( ). cardiac metastases from cervical cancer are exceedingly rare ( ) ( ) ( ) . the vast majority of cases have involved the right heart, presumably due to hematogenous spread of cervical cancer to the inferior vena cava via the uterine veins, coupled with filtration of tumor cells by the lungs. patients with cardiac metastases from cervical cancer present with a myriad of symptoms that depend on tumor location and size, leading to heart failure, arrhythmias, or tamponade ( - ). many cases were diagnosed postmortem. antemortem diagnosis was usually achieved by multimodality imaging including transthoracic and transesophageal echocardiography, ct angiography of the heart, cmr, and f-fdg pet/ct ( - ). tissue diagnosis should be pursued for prognostic implications and optimizing treatment, but may not be feasible. our patient had no symptoms related to the cardiac metastasis. the tumor was discovered by f-fdg pet/ct during the process of initial clinical staging of the disease. f-fdg pet/ct is a particularly useful imaging modality in this setting. together with cmr for detailed tumor characterization, these imaging modalities provide a comprehensive assessment of disease status that is essential for optimal shared decision making ( , ) . the median survival of patients diagnosed with cardiac metastasis from cervical cancer is months ( to months) ( ) ( ) ( ) . the management of these patients should be individualized and discussed within a dedicated multidisciplinary tumor heart team ( - ). a multimodality treatment strategy including chemoradiation, biological therapy, and surgery is necessary to prolong survival and maintain quality of life. in this regard, in a prospective randomized trial, bevacizumab has been shown to improve survival when added to the commonly used platinum-paclitaxel and topotecan-paclitaxel chemotherapy regimens ( ) . of note, treatment with bevacizumab has been shown to be associated with increased incidence of adverse cardiovascular side-effects such as uncontrolled hypertension, thromboembolism, cardiac ischemia, and heart failure ( ) . the decrease in lvef in our patient occurred immediately after resection and remained stable, and our patient did not have severe cardiac limitations during follow-up. her low lvef was readily managed with cardiac medications. near-complete resection of the interventricular septum via a right ventriculotomy parallel to the interventricular septum. reconstruction of the septum using a bovine pericardial patch (asterisk) sewn into the left ventricular side of the septum. lad ¼ left anterior descending coronary artery; other abbreviations as in figure . surgical resection should be considered for isolated cardiac metastasis in patients with favorable response to initial therapy and in whom complete resection is deemed feasible ( - ). our patient had excellent response of the disease in the cervix and of the single pelvic bone metastasis with no evidence of new disease, with the exception of the cardiac metastasis, which remained metabolically active. this is not an infrequent clinical phenomenon in patients with primary or secondary malignant tumors of the heart ( ), presumably due to differential response of the tumor to cancer therapy in different tissue environments ( ) . given the patient's young age, good functional status, imaging suggesting feasibility of complete resection, as well as lack of an alternative effective treatment, we proceeded with surgery followed by intensive chemoradiation and bevacizumab with curative intent. although the pathological examination in our case did not reveal residual tumor in the tissue specimen, surgical resection had an important impact on the design of her concurrent therapy and prognosis. the local recurrence supports the presence of tumor and findings of the pre-operative f-fdg pet/ct, highlighting the potential for false-negative tumor histological examinations following neoadjuvant chemotherapy ( ) . the radiological evidence of local recurrence in the area of slim resection margins-the basal-posterior septum-underscores the technical challenge of achieving complete r resection with clean margins when treating primary or secondary malignant tumors of the heart ( , ). in summary, cardiac metastases from cervical cancer should be included in the differential diagnosis of secondary malignant tumors of the heart. we believe that the patient-specific, multimodality management with curative intent strategized and delivered by an experienced and dedicated multidisciplinary team was key to achieving exceptional survival in our patient. twitter: @ayeletdaniels. distribution in the basal inferolateral and anterolateral free wall of the left ventricle. the etiology of this scar was unclear because the patient had no prior cardiac history, and to our knowledge, ibrutinib had not been known to be associated with myocardial scar. no abnormality was noted in the right ventricular apical region, which was the likely origin of his va. initially, the patient was managed with intravenous amiodarone for recurrent vt. after extensive multidisciplinary discussions among cardiology, electrophysiology and oncology teams, ibrutinib was discontinued due to its potential association with vt. the patient was started on metoprolol, and an implantable cardioverter-defibrillator (icd) was placed for secondary prevention. in terms of af, his cha ds -vasc score was , and anticoagulation was deferred, particularly given the potential bleeding risks with ibrutinib ( ). although it remained uncertain if ibrutinib played an integral role in the pathophysiology of vt, the medication was discontinued with close outpatient cardiology and oncology follow-up. because he had been in remission for several years, he was not started on alternative therapy for wm. on follow-up visits with close surveillance every months with cbc, comprehensive metabolic panel, and igm levels, he continued to be without evidence of symptomatic cytopenias, hyperviscosity, hepatosplenomegaly, lymphadenopathy, neuropathy, amyloidosis, cryoglobulinemia, or cold agglutinemia and did not meet criteria for reinitiation of therapy. on icd interrogation at months after cessation of ibrutinib, there were no further arrhythmias. ibrutinib is an oral, irreversible bruton's tyrosine kinase inhibitor used to treat a broad spectrum of b-cell proliferative disorders, including chronic lymphocytic leukemia (cll) as first-line therapy, mantle cell lymphoma, marginal zone lymphoma, and wm ( ) . cardiovascular toxicities associated with ibrutinib are the association of ibrutinib with vas is becoming increasingly recognized ( , , ) . in randomized controlled trials, the incidence of all-grade vas in patients treated with ibrutinib (n ¼ , ) compared with patients in the control arm (n ¼ ) was . % versus . %, and for grade or greater vas was . % versus % ( ). in analyses from a u.s.-based comprehensive cancer registry cohort, male sex, previous af, hf, coronary artery disease, diabetes, widened qrs, and valvular disease were associated with the development of any arrhythmias (va and supraventricular tachyarrhythmias) ( ). among those without baseline hf or coronary artery disease, the estimated , person-year incidence rate for vas was compared with . among similar nonibrutinibtreated subjects, which suggested an observed versus expected relative risk of . (p < . ). regarding drug dosage, in study in which approximately % of the patients experienced vas, % were taking at least mg of ibrutinib per day, and only % were taking mg or lower per day ( ). among those with ibrutinibassociated vas, the median time-to-event was months (range . to . months) ( ). ibrutinib has been reported to cause polymorphic vt without qtc prolongation as well as in the absence of structural heart disease ( , ) . tomcsányi et al. ( ) reported the case of a -year-old woman on ibrutinib for cll with underlying left bundle branch block and af who experienced ibrutinib-induced polymorphic vt in the absence of other causes ( ) . the initiation of vt was not characterized by short-long-short cycles as is seen in torsade de pointes. in polymorphic vt not related to prolonged qt, an alteration in the cardiac calcium homeostasis associated with ryanodine receptor-calmodulin-dependent protein kinase pathways is suspected. as such, it has been hypothesized that an interaction between these and pi k-akt pathways could potentially lead to polymorphic vt with ibrutinib ( ). although the awareness of ibrutinib-associated va is increasing, there are no published management guidelines. our patient was initially treated with amiodarone, then started on a beta-blocker. in the case described by tomcsányi et al. ( ) , the patient was treated with amiodarone and remained arrhythmia-free for months. amiodarone was thereafter discontinued due to pulmonary toxicity, and discontinuation led to the recurrence of vt. sotalol was found to be ineffective, as were class i antiarrhythmic agents ( ) . as ibrutinib is primarily metabolized by hepatic cytochrome p a (cyp a ), caution must be exercised when used in combination with amiodarone and the calcium-channel blockers diltiazem and verapamil, given that they inhibit cyp a and potentially result in increased serum levels of ibrutinib ( ) . amiodarone may increase the plasma concentration of ibrutinib by -to -fold ( ). ibrutinib interruption or dose adjustment may be appropriate prior to the initiation of amiodarone. furthermore, the class ia antiarrhythmic quinidine and the beta-blockers carvedilol and nadolol also interact with ibrutinib, increasing the serum concentrations of the antiarrhythmic drugs ( ) . according to the ibrutinib package insert, ibrutinib interruption is recommended for any nonhematological toxicity that is grade or greater. once the toxicity has resolved to grade or baseline, ibrutinib may be reintroduced at a low dose ( ) . for severe or refractory arrhythmias, such as seen in our patient, drug discontinuation may be necessary. in general, icd implantation is recommended for the secondary prevention of sudden cardiac death due to life-threatening vt/ventricular fibrillation in patients in whom a completely reversible cause cannot be identified ( ) . our patient had hemodynamically unstable vt episodes requiring resuscitation, and although ibrutinib was felt to be the culprit, the presence of delayed enhancement on cardiac magnetic resonance imaging made it difficult to rule out underlying structural heart disease, such as an infiltrative cardiomyopathy. additionally, an icd is indicated if it is expected to improve overall mortality. because our patient's long-term prognosis from wm was favorable, it was felt that he would benefit from an icd, particularly because ibrutinib or another therapy could be indicated in the future. in patients with cancer whose overall prognosis is poor (i.e., low expectation of survival with an acceptable functional status beyond year), icd therapy is not recommended. as ibrutinib is often used in patients with cll whose prognosis is generally favorable, icds for secondary prevention have a potential for long-term benefit. an additional advantage of an icd is providing prognostic parameters, such as the burden of atrial arrhythmia, nonsustained vt, and treated episodes of vt/ ventricular fibrillation, which could help to risk-stratify patients in the future prior to reintroduction of potentially cardiotoxic drugs. ibrutinib is a bruton's tyrosine kinase inhibitor associated with a well-known side effect of af. reported cases of vas associated with ibrutinib are rare. we present a case of vt storm in a patient receiving ibrutinib for wm. as the recognition of ibrutinib-associated vas is increasing, more data are needed to guide best management strategies for vas triggered by ibrutinib. the following clinical scenarios focused on cancer prevention highlight the value of systematic cardiovascular risk assessment in individuals at high risk of cancer who are treated with cancer risk-reducing therapies. recommendations regarding the evaluation of lipid abnormalities and the role of coronary artery calcium (cac) scan are provided, balancing existing evidence with the need for rigorous data that specifically applies to cancer populations. in terms of additional potential cardiovascular risk factors, brca has been implicated in the repair of dna double-stranded breaks, and its loss-of-function is associated with reduced cardiac performance and accelerated cardiomyocyte death in murine models. human studies investigating the association between brca / mutations and incident cardiovascular disease (cvd) have shown conflicting results, potentially due to variable sample sizes and ethnic-specific differences in the pathogenesis of cvd ( ). bilateral prophylactic oophorectomy is associated with a % reduction in the risk of epithelial ovarian cancer in brca / mutation carriers. in women younger than years of age, the risk of cardiovascular death after surgical oophorectomy is % higher than that of healthy control subjects ( ) . premature menopause was shown to predict future coronary heart disease and stroke in the mesa (multi-ethnic study of atherosclerosis) cohort and represents an important risk-enhancing factor ( ). the patient has a borderline-range ( % to . %) estimated ascvd risk in the presence of a risk-enhancing factor, namely premature menopause. given this, we would advocate for a discussion regarding initiation of moderate-intensity statin therapy. however, the patient has expressed a preference to avoid taking additional medications. in this case, a cac scan could help refine ascvd risk assessment ( ) . an agatston score of $ and/or a cac score $ th percentile for the patient's age, sex, and race would suggest a benefit to statin initiation, although there are no randomized clinical trial data supporting the use of cac in treatment decisions. a cac score of would increase this patient's -year coronary heart disease risk to % using the mesa risk score calculator, and would further support the use of lipid-lowering therapy. nicotine cessation should be a primary strategy to reduce cvd risk in this patient, while the importance of healthy lifestyle habits, normal-range blood pressure and lipid profile, weight loss, and glycemic control should also be emphasized. aspirin and sulindac are commonly used, contemporary pharmacoprophylactic therapies in patients who are at high risk of colorectal cancer, with the former being the preferred drug for individuals with sporadic adenomas and the latter for those with fap. in the late s, celecoxib was approved for use in patients with fap. the risk-reduction benefit appeared to be significant with a % lower colorectal polyp burden rate in fap patients treated with celecoxib and a % lower incidence of duodenal polyps. however, in a large study of colorectal adenoma prevention, long-term use of celecoxib ( or mg twice daily) was associated with a . -fold increased risk of death from cvd, myocardial infarction, stroke, or heart failure ( ). in addition, a meta-analysis of randomized controlled trials showed that celecoxib use was associated with a -fold increased risk of myocardial infarction. a subsequent prospective investigation, assessing the safety of a lower dose of celecoxib ( mg daily), did not demonstrate a statistically significant hazard; as such, the cardiovascular risk mediated by celecoxib remains incompletely understood ( ) . in , due to high cardiovascular event rates, rofecoxib was removed from the market, and a black-box warning was issued for celecoxib the following year. this patient is in an intermediate-risk ascvd category, but has additional factors that increase his likelihood of future cardiovascular events: chronic nonsteroidal anti-inflammatory drug (nsaid) use and a strong family history of premature ascvd. importantly, the presence of diabetes mellitus in an individual to years of age is an indication for the use of moderate-intensity statins regardless of ascvd score, and in an individual it is important to understand that patients with fap who are treated with preventive total colectomy have favorable long-term survival outcomes. the notion that cvd represents an important competing risk to cancer mortality should be conveyed and serve as a motivating factor to introduce lifestyle changes and consideration of pharmacological therapies. consideration of chronic nsaid initiation in this patient, especially in the context of conventional cardiovascular risk factors, should prompt referral to an internist or cardiologist. numerous risk-reducing interventions are utilized by cardiologists and oncologists in patients at risk of developing cardiovascular disease or cancer. some therapies used to lower the incidence of cancer in individuals at increased risk for malignancy (e.g., prophylactic bilateral oophorectomy in carriers of brca / mutations, sulindac in patients with familial adenomatous polyposis) may potentially increase the risk of cardiovascular disease. consideration of such interventions, particularly in individuals with coexisting cardiovascular risk factors or comorbidities, should prompt referral to cardiology for careful risk assessment and modification that integrates traditional risk factors and the effects of cancer risk-reducing therapies. ascvd ¼ atherosclerotic cardiovascular disease; cv ¼ cardiovascular; nsaid ¼ nonsteroidal antiinflammatory drug. importantly, decisions regarding long-term nsaid therapy should balance the reduced polyp and potential colorectal cancer rate with the increased cvd risk. attainment of optimal blood pressure and glycemic control should be targeted. in addition, a cac scan could be considered. if highly abnormal, it could be used to counsel and motivate this patient to further modify his lifestyle ( ) . it would also be reasonable to check a lipoprotein(a) level given the strong family history of premature atherosclerosis. due to the expected long-term use of sulindac, statin therapy and improved lifestyle habits would be appropriate risk-reduction interventions in this case. cardiovascular risk assessment in individuals who are deemed to be at high risk for cancer is distinct from that in patients with an established cancer. specific therapies aimed at reducing the risk of cancer may augment cardiovascular risk and deserve careful consideration. two important tenets of cardiovascular risk assessment include evaluation of lipid abnormalities and understanding the role of cac imaging in refining ascvd risk stratification in this unique population. although more research is needed regarding the value of cac burden in patients with cancer, available evidence suggests that elevated cac scores are predictive of both future cardiovascular events and cancer. in a recent study, cac > , when compared with a score of , was associated with a . -fold increase in the risk of cv death and a % increase in the risk of cancer death ( ). the most common cause of death among individuals with cac was cancer ( %), whereas patients with cac > experienced most of the mortality due to cvd. in a study of patients with locally-advanced nonsmall-cell lung cancer treated with thoracic radiation therapy, an increased cac score, measured from planning radiation therapy computed tomography, was associated with an elevated risk of all-cause mortality (hazard ratio: . ; confidence interval: . to . ; p ¼ . ) ( ) . in a separate analysis of breast cancer patients who had received radiation therapy, higher pre-rt cac scores were associated with a higher likelihood of acute coronary syndrome at years of follow-up (hazard ratio: . ; confidence interval: . to . ; p ¼ . ) ( ) . coronary calcification is therefore a predictor of cvd in the cancer population and further research is needed to better characterize the contribution of cause-specific mortality and cardiovascular events in cancer patients according to cac levels ( ) . last, breast arterial calcifications detected on screening mammogram are an independent marker for the presence of coronary artery disease and may help identify women at higher risk of ascvd. lipid-lowering therapies should be considered in patients with cardiovascular risk factors or disease, as per the standard ascvd risk score. particular medical interventions to decrease the risk of cancer, such as prophylactic oophorectomy or long-term nonsteroidal anti-inflammatory drugs, may increase the risk of ascvd and should be regarded as deleterious factors. in the coming decade, cardio-oncologists may be asked to assess and manage cardiovascular toxicities in individuals who receive cancer risk-reducing interventions but who are free of cancer. given the increasing data supporting an overlap between the molecular and clinical underpinnings of cancer and heart disease, oncologists should consider involving their cardiovascular medicine colleagues for risk assessment and preemptive management. in addition, longitudinal cardiovascular safety evaluations of specific patient subgroups in whom prophylactic anticancer therapies are considered should be performed to provide an evidence-based understanding of the risks and benefits of such therapies. cardiotoxic risk ( ) ( ) ( ) ( ) ; and the intersection between cardiovascular disease and cancer in our patients ( ) ( ) ( ) . in the second quarter of alone, we had nearly , article usage sessions, and each quarter, this number has grown. the work of our authors is being read for utilization in clinical practice and research, which is always the most important metric for a jacc journal. as a physician scientist, i recognize that the choice of "which journal" is not an easy one and requires careful consideration. we remain committed to serving our authors and working tirelessly to ensure that the peer review process is respectful, fair, constructive, and as seamless as possible. our average time to first decision in second quarter was days, and we will strive to maintain this standard. we seek to partner and work collaboratively with our authors to ensure the highest quality contribution to our community. i am grateful to the reviewers, who provide timely, incisive insight and lend their valued expertise to help ensure the quality of our journal. we have had nearly peer reviewers contribute their evalua- over the next year, we will improve access to jacc: cardiooncology through additional platform changes to improve searchability and integration across the jacc journals. clinical, translational, and basic science original research manuscripts will continue to serve as our foundation. as we evaluate each manuscript, we will continue to ask ourselves the following key questions: are the findings valid? is the methodology rigorous? is the topic of clinical importance? are the findings incremental to our current understanding of the topic and do they fulfill an evidence gap? what is the potential clinical impact and the potential for advancing the field? we will continue to publish state-of-the-art reviews and primers, and similarly ask ourselves: is this an authoritative, critical appraisal of the literature? is it comprehensive, yet focused? is this data-driven and accurately reflective of the current evidence? we will look to clinical case challenges to provide evidence-based descriptions of unique cases that thoughtfully illustrate the diagnostic and therapeutic dilemmas that we as clinicians face as we care for our patients ( ) . our viewpoints will continue to express opinion pieces on important and timely topics, and present thought-provoking, community-building, evidence-based perspectives. we will continue to grow our international engagement events, podcasts, live journal clubs, and dynamic case presentations, each occurring at least once per quarter. we also will launch a new "how to" series that will offer practical, evidence- oncologists primarily consulted general cardiology ( %), as compared with cardio-oncology ( %) for evaluation of treatment-related cardiotoxicity. overall, a lack of awareness ( %) and lack of and education in the growing field of cardiooncology. considering these respondents likely represented a more engaged group of practitioners, there was a lack of general cardio-oncology knowledge, uncertainty of local available resources, and low reported rate of cooperation between cardiologists and oncologists. although we were not able to assess the reasoning for these observed practice patterns, as it was beyond the scope of this study, enhanced collaboration between disciplines will be important and necessary to deliver optimal cardiovascular and oncologic care to this patient group. previous studies indicate that approaches vary between cardiologists and oncologists when using cardio-oncology services for the treatment of patients with cancer. peng et al. ( ) reported that most cardiologists ( %) felt that they should monitor for cardiotoxicity even in the absence of symptoms. however, the same study indicated that only . % of oncologists shared this view. furthermore, % of oncologists felt that cardiologists should be involved only when patients developed cardiotoxicities, but only . % of cardiologists agreed with that opinion. most cardiologists believed that access to cardiooncology services would improve prognosis ( . %), whereas only . % of oncologists shared this view. our study did not directly address this same question, but found that only % of oncologists and % of cardiologists indicated that they felt "very comfortable" interacting with their colleagues for comanagement of cancer and heart disease. sadlerd@ccf.org. twitter: @dsadlermd, @floridaacc, @flasco_org, @accintouch, @dr_mike_fradley, @dr_roohikhan. transthyretin amyloid cardiomyopathy: jacc state-of-the-art review natural his a new staging system for cardiac transthyretin amyloidosis cardiovascular disease mortality after chemotherapy or surgery for testicular nonseminoma: a populationbased study testicular cancer: a mechanisms of cisplatin nephrotoxicity white-paper- .pdf a new equation to estimate glomerular filtration rate crosstalk between vascular redox and calcium signaling in hypertension involves trpm (transient receptor potential melastatin ) cation channel marked impairment of protease-activated receptor type -mediated vasodilation and fibrinolysis in cigarette smokers: smoking, thrombin, and vascular responses in vivo intra-arterial substance p mediated vasodilatation in the human forearm: pharmacology, reproducibility and tolerability role of the endothelium in the vascular effects of the thrombin receptor (protease-activated receptor type ) in humans vascular effects of apelin in vivo in man the vasodilator action of nebivolol in forearm vasculature of subjects with essential hypertension fire simulation and cardiovascular health in firefighters vascular damage in testicular cancer patients: a study on endothelial activation by bleomycin and cisplatin in vitro antiangiointravascular ultrasound imaging for coronary thrombosis after cisplatin-based chemotherapy caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis arterial events in cancer patients-the case of acute coronary thrombosis vascular toxic effects of cancer therapies treatmentrelated cardiovascular toxicity in long-term survivors of testicular cancer comprehensive characterisation of the vascular effects of cisplatin-based chemotherapy in patients with testicular cancer impact of platinum-based chemotherapy on the progression of atherosclerosis vascular fingerprint and vascular damage markers associated with vascular events in testicular cancer patients during and after chemotherapy cardiovascular risk in long-term survivors of testicular cancer long-term platinum retention after platinumbased chemotherapy in testicular cancer survivors: a -year follow-up study hematopoietic stem cell transplantation in europe : more than transplants annually reduced mortality after allogeneic hematopoietic-cell transplantation late mortality after allogeneic hematopoietic cell transplantation and functional status of long-term survivors: report from the bone marrow transplant survivor study vascular endothelium as 'novel' target of graft-versus-host disease pericarditis in patients with chronic graft-vs-host disease national institutes of health hematopoietic cell transplantation late effects initiative: the cardiovascular disease and associated risk factors working group report prevalence of outpatient cancer treatment in the united states: estimates from the medical panel expenditures survey (mpes) chemotherapy-induced cardiotoxicity: detection, prevention, and management long-term chemotherapy-related cardiovascular morbidity cardiovascular disease in adult survivors of childhood cancer epirubicin versus doxorubicin: which is the anthracycline of choice for the treatment of breast cancer? gp phox-containing nad(p)h oxidase increases superoxide formation by doxorubicin and nadph enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin redox cycling of anthracyclines by cardiac mitochondria. i. anthracycline radical formation by nadh dehydrogenase adriamycin-induced interference with cardiac mitochondrial calcium homeostasis cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin role of endothelium in doxorubicin-induced cardiomyopathy a critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin chemotherapeutic drugs and mitochondrial dysfunction: focus on doxorubicin, trastuzumab, and sunitinib glabridin prevents doxorubicin-induced cardiotoxicity through gut microbiota modulation and colonic macrophage polarization in mice vegf-b gene therapy inhibits doxorubicin-induced cardiotoxicity by endothelial protection endothelial function in young adult survivors of childhood acute lymphoblastic leukemia cellular senescence promotes adverse effects of chemotherapy and cancer relapse mitochondria-targeted antioxidant (mitoq) ameliorates age-related arterial endothelial dysfunction in mice sex differences in anthracycline-induced cardiotoxicity: the benefits of estrogens shortterm interleukin- treatment improves vascular endothelial function, endurance exercise capacity, and whole-body glucose metabolism in old mice doxorubicin acts through tumor necrosis factor receptor subtype to cause dysfunction of murine skeletal muscle oxidant stress and endothelial cell dysfunction redox regulation of mitochondrial function increased accumulation of doxorubicin and doxorubicinol in cardiac tissue of mice lacking mdr a p-glycoprotein redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses innovative medicine: basic research and development antioxidants and cancer prevention antioxidant supplementation and breast cancer prognosis in postmenopausal women undergoing chemotherapy and radiation therapy endothelial nitric oxide synthase-dependent superoxide generation from adriamycin doxorubicin accumulation in individually electrophoresed organelles doxorubicin induces endotheliotoxicity and mitochondrial dysfunction manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction rapid-onset endothelial dysfunction with adriamycin: evidence for a dysfunctional nitric oxide synthase mechanism of guanine-specific dna damage by oxidative stress and its role in carcinogenesis and aging nuclear factor-{kappa}b activation contributes to vascular endothelial dysfunction via oxidative stress in overweight/obese middle-aged and older humans chronic supplementation with a mitochondrial antioxidant (mitoq) improves vascular function in healthy older adults the mitochondria-targeted anti-oxidant mitoquinone decreases liver damage in a phase ii study of hepatitis c patients a double-blind, placebo-controlled study to assess the mitochondria-targeted antioxidant mitoq as a disease-modifying therapy in parkinson's disease vitamin c mitigates oxidative/nitrosative stress and inflammation in doxorubicin-induced cardiomyopathy protective effects of mito-tempo against doxorubicin cardiotoxicity in mice doxorubicin inactivates myocardial cytochrome c oxidase in rats: cardioprotection by mito-q. biophys the role of endothelins and their receptors in heart failure plasma endothelin- and doxorubicin cardiotoxicity plasma endothelin- as a marker for doxorubicin cardiotoxicity doxorubicin-induced oxidative stress and endothelial dysfunction in conduit arteries is prevented by mitochondrial-specific antioxidant treatment coenzyme q for prevention of anthracycline-induced cardiotoxicity anthracycline causes impaired ventricular-arterial coupling in breast cancer patients after treatment with anthracycline-containing adjuvant chemotherapy serum cardiovascular growth factors during doxorubicin chemotherapy in children with leukemia circulating neuregulin during the transition from stage a to stage b/c heart failure in a breast cancer cohort cardiac endothelial cells regulate ros-induced cardiomyocyte apoptosis through neuregulin- beta/ erbb signaling neuregulin- protects ventricular myocytes from anthracycline-induced apoptosis via erbb -dependent activation of pi -kinase/akt endothelial cells regulate physiological cardiomyocyte growth via vegfr -mediated paracrine signaling protective mechanisms of coenzyme-q may involve up-regulation of testicular p-glycoprotein in doxorubicin-induced toxicity reno-protective efficiency of coenzyme q on adriamycin-induced nephrotoxicity fundamental mechanisms of immune checkpoint blockade therapy immune checkpoint blockade therapy for cancer: an overview of fda-approved immune checkpoint inhibitors immunerelated adverse events associated with immune checkpoint blockade fulminant myocarditis with combination immune checkpoint blockade cardiovascular toxicities associated with immune checkpoint inhibitors: an observational, retrospective, pharmacovigilance study immune checkpoint inhibitors-related cardiotoxicity cardiotoxicity associated with ctla and pd blocking immunotherapy cardiotoxicity of immune checkpoint inhibitors association between immune-related adverse events and clinical efficacy in patients with melanoma treated with nivolumab: a multicenter retrospective study risk factors for immune-related adverse events associated with anti-pd- pembrolizumab healthy north carolina : social determinants of health indicators inflammatory signature difference in rural urban and regional occupational exposure in lung cancer current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-oncology what is the normal value of the neutrophil-to-lymphocyte ratio? immune checkpoint inhibitor-related adverse cardiovascular events in patients with lung cancer tocilizumab for the management of immune mediated adverse events secondary to pd- blockade myocarditis in patients treated with immune checkpoint inhibitors influenza vaccination and myocarditis among patients receiving immune checkpoint inhibitors serial troponin for early detection of nivolumab cardiotoxicity in advanced non-small cell lung cancer patients prognostic value of troponin t in cardiac risk stratification of cancer patients undergoing high-dose chemotherapy cardiovascular events among adults treated with chimeric antigen receptor t-cells (car-t) car t cell-induced cytokine release syndrome is mediated by macrophages and abated by il- blockade vinten-johansen j. involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury impairment of the pd- pathway increases atherosclerotic lesion development and inflammation proatherogenic immune responses are regulated by the pd- /pd-l pathway in mice ctla -igg ameliorates homocysteine-accelerated atherosclerosis by inhibiting t-cell overactivation in apoe-/-mice impact of baseline steroids on efficacy of programmed cell death- and programmed death-ligand blockade in patients with non-small-cell lung cancer trends in stage distribution for patients with nonsmall cell lung cancer: a national cancer database survey pembrolizumab versus chemotherapy for pd-l -positive non-small-cell lung cancer cardiovascular toxicities associated with immune checkpoint inhibitors fulminant myocarditis with combination immune checkpoint blockade improved survival with ipilimumab in patients with metastatic melanoma nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer pembrolizumab plus axitinib versus sunitinib for advanced renal-cell carcinoma cardiovascular toxic effects of targeted cancer therapies management of cardiac disease in cancer patients throughout oncological treatment: esmo consensus recommendations characterization of immune checkpoint inhibitor-related cardiotoxicity in lung cancer patients from a rural setting grounding cardio-oncology in basic and clinical science acc/aha key data elements and definitions for cardiovascular endpoint events in clinical trials: a report of the american college of cardiology/american heart association task force on clinical data standards (writing committee to develop cardiovascular endpoints data standards) myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-oncology pd- modulates radiation-induced cardiac toxicity through cytotoxic t lymphocytes cardiovascular toxicities associated with immune checkpoint inhibitors: an observational, retrospective, pharmacovigilance study key words immune checkpoint inhibitors, inflammatory markers, myocarditis, neutrophil-tolymphocyte ratio management and outcomes of severe aortic stenosis in cancer patients healthcare cost and utilization project (hcup) aha/acc focused update of the aha/acc guideline for the management of patients with valvular heart disease: a report of the american college of cardiology/american heart association task force on clinical practice guidelines transcatheter aortic valve replacement in oncology patients with severe aortic stenosis cardiac complications of thoracic irradiation roadmap for biomarkers of cancer therapy cardiotoxicity early changes in cardiovascular biomarkers with contemporary thoracic radiation therapy for breast cancer, lung cancer, and lymphoma normalization of ejection fraction in subjects with systolic heart failure. is it really normal? a myocardial deformation study assessment of early radiation-induced changes in left ventricular function by myocardial strain imaging after breast radiation therapy late-onset giant cell myocarditis due to enterovirus during treatment with immune checkpoint inhibitors i mmune checkpoint inhibitor (ici) therapy has significantly improved the prognosis of many advanced cancers cardiovascular adverse effects such as myocarditis are uncommon, but bear high mortality rates of nearly % ( ). ici myocarditis typically occurs early, and potential factors associated with ici myocarditis are combination therapy however, the clinical presentation of ici myocarditis is heterogeneous, with preserved left ventricular ejection fraction in one-half of the cases, and high rates of conduction abnormalities and ventricular arrhythmias ( ). ici myocarditis can also occur late, although notably, there is limited information on late-onset myocarditis with long-term ici therapy a -year-old male patient presented with myalgias and dyspnea after the th course of nd-line ici monotherapy (nivolumab [anti-pd- ] mg/kg every weeks) for metastatic renal cell carcinoma. the electrocardiogram showed sinus tachycardia, low qrs voltage, and t-wave inversion in the anterior leads with no st-segment changes. cardiac biomarkers were elevated (troponin i , ng/l, b-type natriuretic peptide ng/l). left heart catheterization revealed normal coronary arteries. cardiac magnetic resonance issn cardiovascular imaging the authors attest they are in compliance with human studies committees and animal welfare regulations of the authors fatal toxic effects associated with immune checkpoint inhibitors: a systematic review and meta-analysis cardiovascular toxicities associated with immune checkpoint inhibitors: an observational, retrospective, pharmacovigilance study clinical features, management, and outcomes of immune checkpoint inhibitorrelated cardiotoxicity myocarditis in patients treated with immune checkpoint inhibitors fulminant myocarditis with combination immune checkpoint blockade fulminant versus acute nonfulminant myocarditis in patients with left ventricular systolic dysfunction comparative immunologic characterization of autoimmune giant cell myocarditis with ipilimumab aberrant pd- ligand expression contributes to the myocardial inflammatory injury caused by coxsackievirus b infection myocarditis in the setting of cancer therapeutics: proposed case definitions for emerging clinical syndromes in cardio-oncology management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy report a -year-old woman with advanced diffuse large b-cell lymphoma (dlbcl) was referred for chimeric antigen receptor (car) t cell therapy. she was diagnosed with dlbcl years prior to presentation and had no significant past medical history. she received a total of cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop), with a lifetime exposure of doxorubicin > mg/m . she underwent an autologous hematopoietic stem cell four years later, her dlbcl recurred, requiring more cycles of r-chop. one month after she completed the last cycle, her transthoracic echocardiogram (tte) demonstrated a left ventricular ejection fraction (lvef) of % to % and global longitudinal strain of À % (normal: À % to À %). two months following therapy, she was admitted to the hospital with acute decompensated heart failure (hf) coronary computed tomography demonstrated mild coronary artery disease, and cardiac magnetic resonance imaging showed no evidence of infiltrative or inflammatory disease. she was presumed to have anthracycline-induced cardiomyopathy and was initiated on furosemide and guideline-directed medical therapy (gdmt), including lisinopril . mg and metoprolol succinate . mg daily. up-titration of gdmt was limited, and spironolactone or eplerenone could not be added, secondary to hypotension. she was referred to a cardio-oncologist for risk stratification prior to car t cell therapy. she had good exercise tolerance, riding a stationary bike for to min at a time. a right heart catheterization (rhc) demonstrated mildly elevated biventricular filling pressures with normal cardiac output (right atrium mm hg, pulmonary artery / / mm hg it was felt that car t cell therapy was her only therapeutic option. multidisciplinary discussions between her hematologist/oncologist, cardio-oncologist, and advanced hf team were held to determine feasibility. derangements in blood pressure and oxygenation, ranging from mild hypotension and hypoxemia to issn dr. bishop has received nonfinancial support from articulatescience llc; and has received personal fees from united healthcare, seattle genetics, celgene, juno therapeutics, novartis, crispr therapeutics, kite pharma, and pharmacyclics. dr. uriel has received grant support from abbott the authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and food and drug administration guidelines, including patient consent where appropriate. for more information chimeric antigen receptor t-cell therapies for lymphoma chimeric antigen receptor t-cell therapy for cancer and heart: jacc council perspectives cardiovascular events among adults treated with chimeric antigen receptor t-cells (car-t) yakoub-agha i. an international survey on the management of patients receiving car t cell therapy for haematological malignancies on behalf of the chronic malignancies working party of ebmt design and rationale of haemodynamic guidance with cardiomems in patients with a left ventricular assist device: the hemo-vad pilot study advances in diagnosis and treatment of metastatic cervical cancer cardiac metastasis in cervical cancer a rare case of cardiac metastatic mass case report of cardiac metastasis from cervical squamous carcinoma and its literature review tumors metastatic to the heart cardiac tumors: jacc cardiooncology state-ofthe-art review surgery for tumors of the heart improved survival with bevacizumab in advanced cervical cancer differential growth and responsiveness to cancer therapy of tumor cells in different environments akim@uchc.edu. twitter: @agnesskim , @hmadgula ibrutinibassociated atrial fibrillation ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia cardiovascular toxicities associated with ibrutinib the risk of atrial fibrillation with ibrutinib use: a systematic review and meta-analysis ventricular arrhythmias and sudden death in patients taking ibrutinib ventricular arrhythmias following ibrutinib initiation for lymphoid malignancies highlights of prescribing information ibrutinib, an approved tyrosine kinase inhibitor as a potential cause of recurrent polymorphic ventricular tachycardia ventricular tachycardia caused by ibrutinib dr. kopecky has served as a consultant for prime therapeutics; has received research support from true health; has served as data safety and monitoring board chair for applied clinical intelligence; has served as a board member for mayo clinic support services; and has served as a task force member for mayo clinic cv p&t. dr. gersh has served as cro for trials involving edwards percutaneous valve devices through baim institute; has served on the dsmb for the reprise study ); has served as a general consultant for myokardia; and has served on the steering committee of the garfield study (thrombosis research institute) risk of cardiovascular disease in women with brca and brca mutations increased cardiovascular mortality after early bilateral oophorectomy early menopause predicts future coronary heart disease and stroke: the multi ethnic study of atherosclerosis association of coronary artery calcium in adults aged to years with incident coronary heart disease and death cardiovascular risk associated with cardiovascular safety of celecoxib, naproxen, or ibuprofen for arthritis coronary artery calcium and the competing long-term risk of cardiovascular vs. cancer mortality: the cac consortium elevated coronary artery calcium quantified by a deep learning model from radiotherapy planning scans predicts mortality in lung cancer is the coronary artery calcium score associated with acute coronary events in breast cancer patients treated with radiotherapy? breast arterial calcium: a game changer in women's cardiovascular health? jacc: cardiooncology: poised to serve a maturing, collaborative field proceedings from the global cardio-oncology summit jacc: cardiooncology videos resiliency and our cardio-oncology community the novel coronavirus disease (covid- ) threat for patients with cardiovascular disease and cancer covid- clinical trials: a primer for the cardiovascular and cardiooncology communities al amyloidosis for the cardiologist and oncologist efficacy of neurohormonal therapies in preventing cardiotoxicity in patients with cancer undergoing chemotherapy safety of continuing trastuzumab despite mild cardiotoxicity: a phase i trial efficacy of dexrazoxane in preventing anthracycline cardiotoxicity in breast cancer repeated remote ischemic conditioning reduces doxorubicin-induced cardiotoxicity osimertinib-induced cardiotoxicity: a retrospective review of the fda adverse events reporting system (faers) increase in blood pressure associated with tyrosine kinase inhibitors targeting vascular endothelial growth factor car t-cell therapy-related cardiovascular outcomes and management: systemic disease or direct cardiotoxicity? cardiovascular safety of degarelix versus leuprolide for advanced prostate cancer: the pronounce trial study design hypertension in cancer patients and survivors: epidemiology, diagnosis, and management increased cancer prevalence in peripartum cardiomyopathy ischemia and bleeding in cancer patients undergoing percutaneous coronary intervention infiltrative lymphoma-associated bradycardia and cardiac conduction abnormalities deaths: leading causes for the economic burden of chronic cardiovascular disease for major insurers health of patients with cancer and cancer survivors an international survey of healthcare providers' knowledge of cardiac complications of cancer treatments practices in management of cancer treatmentrelated cardiovascular toxicity: a cardio-oncology survey acc multistate chapter cardio oncology network to improve physician participation, awareness, and education in cardio oncology myocardial ischemia: leading the differential diagnosis of polymorphic vt in a -year-old man was acute coronary syndrome.heart failure (hf): he did not have symptoms or signs of hyperviscosity syndrome or hf. systemic amyloidosis associated with wm leading to amyloid cardiomyopathy was also considered.drug toxicity: a high index of suspicion was maintained about the potential association between ibrutinib and ventricular arrhythmia (va). electrocardiogram at the time of evaluation by emergency medical services showed af with a ventricular rate of beats/min and nonspecific st/t-wave abnormalities. complete blood count, serum electrolytes, and renal function were within normal limits at the time of admission, and serial serum troponin levels were undetectable. a transthoracic echocardiogram showed normal biventricular size and function with no regional wall motion abnormalities. furthermore, coronary angiography demonstrated very mild luminal irregularities without obstructive disease. in the cardiac intensive care unit, he was noted to have multiple premature ventricular complexes and nonsustained vt with a left bundle branch block morphology and right superior axis, indicating an origin at the right ventricular apex (figure ). he underwent cardiac magnetic resonance imaging, which demonstrated a small area of mid-myocardial delayed enhancement in a nonvascular key: cord- -nzc ir n authors: guo, sun-wei title: china’s “gene war of the century” and its aftermath: the contest goes on date: - - journal: minerva doi: . /s - - - sha: doc_id: cord_uid: nzc ir n following the successful cloning of genes for mostly rare genetic diseases in the early s, there was a nearly universal enthusiasm that similar approaches could be employed to hunt down genes predisposing people to complex diseases. around , several well-funded international gene-hunting teams, enticed by the low cost of collecting biological samples and china’s enormous population, and ushered in by some well-connected chinese intermediaries, came to china to hunt down disease susceptibility genes. this alarmed and, in some cases, enraged many poorly funded chinese scientists, who perceived them as formidable competitors. some depicted foreign gene-hunters as greedy pilferers of the vast chinese genetic gold mine, comparing it to the plundering of national treasures from china by invaders in the past, and called upon the government and their fellow countrymen to rise up and protect china’s genetic gold mine. media uproar ensued, proclaiming the imminent “gene war of the century.” this article chronicles the key events surrounding this “war” and its aftermath, exposes some inherent complexities in identifying susceptibility genes for complex diseases, highlights some issues obscured or completely overlooked in the passionate and patriotic rhetoric, and debunks some misconceptions embedded in this conflict. in addition, it argues that during the entire course of this “war,” the public’s interest went conspicuously unmentioned. finally, it articulates several lessons that can be learned from this conflict, and outlines challenges facing human genetics researchers. around , and amid the talks of hong kong's upcoming return to china and later the asian financial crisis, a recurring topic in the chinese media was the so-called ''gene war of the century'': the lopsided condemnation of foreign scientists coming purportedly to pilfer china's vast genetic resources for a profit. some scientists wrote articles and gave lectures, calling for heightened vigilance for the pilfering act, and proposed that the country protect its precious genetic resources by conducting genetic research on its own. while the public might have been completely flummoxed by some esoteric and arcane jargons, the message was nonetheless loud and clear: the western capitalists were trying to profit from china's unique genetic heritage. in a country with a past history of repeated foreign invasion, defeat, and humiliation, this message struck a tender emotional chord and caused an eruption of national furor. the person who likely triggered, perhaps unintentionally, the first spark of this ''war'' is xiping xu, a chinese expatriate at harvard at the time. despite his repeated proclamation as a staunch and unwavering patriot loyal to his beloved motherland and dedicated to the advancement of china's science and technology, he nonetheless later became embroiled in an avalanche of controversies surrounding the ''gene war.'' he effectively became a lightning rod for all the controversy on genetic resources, intellectual rights, informed consent, and the protection of human research subjects. well over a decade has since passed. what was at stake? did these precious genetic resources actually exist? who was the most likely beneficiary of the gene-hunting endeavor? how did this ''war'' end? who were the winners and losers, if there were any in the first place? what happened after the conflict? as war is invariably a continuation of politics by other means, what was the politics behind it? what happened to the people who were embroiled in the ''war''? the answers to all these questions can be addressed, at least in part, now with the benefit of a / hindsight. war breaks out simply because of irreconcilable conflicts in interest. the ''gene war,'' whether it was real or fictitious, was no exception. this article chronicles the key events surrounding the ''war'' and its aftermath, exposes some inherent complexities in identifying susceptibility genes for complex diseases, highlights some issues obscured or completely overlooked in the passionate and patriotic rhetoric, and debunks some misconceptions embedded in the lopsided condemnations. in addition, it describes how, during the entire course of the ''war'' of intense and often politically charged uproars, the patients' interest was conspicuously unmentioned and likely overlooked. examining the larger issues regarding science and politics, it also argues that the ''war'' and its surrounding events can be best understood through the lens of credibility contest vying for resources. finally, it lists several lessons that can be learned from this conflict, and outlines challenges facing current researchers in human genetics. ''gene war of the century'': the genesis in , the human genome project (hgp) was launched. with a price tag of billion us dollars and a -year timeline, this ambitious megaproject aimed to sequence the entire human genome, with the ultimate goal of ''understand[ing] the human genome'' and ''knowledge of the human as necessary to the continuing progress of medicine and other health sciences as knowledge of human anatomy has been for the present state of medicine.'' supported by the us department of energy (doe) and the us national institute of health (nih), the hgp was the culmination of several years of research building on a series of breathtaking breakthroughs in molecular genetics. considered the ''genetic blueprint for human beings'' and hailed as the ''book of life'' or simply the holy grail, the human genome, when completely sequenced, would purportedly unlock the secrets underlying a plethora of human traits as mundane as facial resemblance between parents and offspring and as complex as human behavior. against this foreground, human genetics research entered a golden age. in , scientists identified (called ''cloned'') the genetic mutation responsible for a rare genetic disease called cystic fibrosis, that is, the gene responsible for the disease was identified with known location and size. in the inaugural year of the hgp, a gene responsible for breast cancer was localized, or ''mapped,'' to chromosome . in the following few years, the genes responsible for huntington's disease, breast cancer ( - % of the cases), alzheimer's disease and other rare genetic diseases often with a tongue-twisting name would be cloned (see table for the timeline of research milestones and events surrounding the ''gene war''). following on the heels of successful cloning of genes for these mostly rare mendelian diseases in the early s, there emerged a nearly universal enthusiasm, hope or even conviction that similar gene-mapping approach could be employed to hunt down susceptibility genes predisposing people to various complex diseases -primarily common chronic diseases such as asthma, diabetes, and cancer that invariably have an elusive pathogenesis and collectively contribute to the major health burdens (lander and schork ; risch and merikangas ) . it was hoped that once genes were identified, the characterization of their functions would not only help better understand genotype-phenotype relationships, but also facilitate the development of specific therapies and preventative measures and the identification of people at increased risk of developing the disease (collins and mckusick ) . it was also hoped that once the risk of particular combinations of genotype and environmental exposure is known, medical interventions, such as lifestyle changes, could then be institutionalized to target high-risk groups or individuals (collins and mckusick ) . some biotech companies quickly saw the potential of enormous business opportunities and joined the fray. human genome sciences, founded in by william a. haseltine, a noted harvard professor and aids researcher, partnered with some genomics companies and soon filed patents on , genes and, in , quadrupled its stock price (zimmer ). other genomics companies followed suit. yet this practice had one problem: most, if not all, patented ''genes,'' in fact, rna transcripts, were merely pieces of cdna without any known functions at the time of filing. to understand what a gene does and how it does, and to establish the causal relationship between a gene and a human disease, let alone treatment, is by no means an easy task, even with modern technology. very often, it is a slow, arduous, painstaking, and imprecise process full of dead-ends and false leads. many other biotech companies and academic scientists took a different approach called ''positional cloning.'' this approach eliminates the need to understand the molecular genetic mechanisms underlying the disease of interest. instead, through the collection of pedigrees enriched with patients having the disease, the existing genetic sign posts (called dna markers, which are scattered around the human genome with known locations and content) would be used to localize the responsible gene in a particular region. this would allow researchers to zero in the gene, identify it, and ultimately figure out its function and its relationship with the disease through extensive lab work. thus, by ''positioning'' the gene, the gene could be cloned and its functions and roles in disease pathogenesis elucidated without any prior knowledge of the possible pathogenesis of the disease. this conceptual simplicity and beauty, coupled with increasingly fast and affordable methods of making genetic signposts (called ''genotyping'') attracted many biomedical scientists and even converted many of them this discovery was later found to hold universally true in all organisms, including humans, and became a corner stone and a principle in genetics. basically, if a trait is determined by a gene, then the gene will tend to go hand in hand with its neighboring signposts when transmitted from parents to offspring-thus the term ''linkage.'' if many relatives in a pedigree having the same trait all carry the same signposts, then there is a chance that the gene responsible for the trait is near to the signposts. although different cross-breeding cannot be made in humans for obvious reasons, this difficulty can be offset by statistical computations. the advent of personal computers in the s coincided with the booming of human genetics and proved to be indispensable in this endeavor. the discovery of various classes of dna markers also facilitated the gene hunting. the approach based on pedigree data is called ''linkage analysis.'' since , scientists found that for common diseases, another approach, called ''association studies,'' can be more powerful in gene hunting. association studies identify disease genes by finding the significant gene frequency differentials between a group of unrelated healthy individuals and another group of unrelated people with the disease of interest. ''gene war of the century'' and its aftermath to human genetics, who were frustrated by the slow, arduous and often fruitless process of finding the cause(s) for disease. thus, in the s hardly a week went by without a news report or announcement of the discovery of genes for some disease, at least in the us. one high-profile study, published in in a prestigious journal, science, reported the localization of a gene in chromosome x q that is responsible for male sexual orientation. this approach has one catch, however. one critical prerequisite for position cloning is the availability of a sufficient number of pedigrees saturated with people having the same disease, along with correct diagnoses or precise measurements of the disease (called ''phenotyping''). once such pedigrees are collected, phenotyping and genotyping can proceed and the positional cloning endeavor can start. since genotyping and phenotyping a large number of people can be costly, there are strong incentives to cut the cost of either process. everything else equal, locations with low costs of acquiring blood samples (for genotyping purposes) and labor (for phenotyping purposes) would be extremely attractive. in , sequana therapeutics, a start-up biotech company located in la jolla, california, announced that it had achieved two significant research milestones related to its asthma gene discovery program. it analyzed dna collected from about people on tristan da cunha, an island in the south atlantic, about , miles from south africa. approximately % of the island residents had asthma, presumably passed on from an original settler generations ago. the announcement prompted cash payments of $ million from boehringer ingelheim, ingelheim, germany, based on a prior agreement. boehringer later paid sequana an additional $ million for its exclusive right to market the drug derived from the putative asthma gene, while sequana retained the exclusive right for developing gene-based diagnostics. sequana announced in late may that it had identified a mutated gene that predisposes people to asthma, a feat hailed by one clinical investigator as ''perhaps this century's most important finding in the etiology of asthma'' (asthma gene discovered. ). early in the same year, sequana announced that it had signed a letter of intent with pe applied biosystems to form a broad-based dna sequencing joint venture in shanghai, china. circa , several well-funded international gene-hunting teams, lured apparently by the low cost of collecting biological samples and the sheer population size and also ushered in by some well-connected chinese scientists working in north america, arrived in china to hunt down susceptibility genes for various complex diseases (shou ) . one biotech company from california announced that it had discovered a large pedigree of hundreds of people enriched with asthma patients in a small village in zhejiang province, china. perhaps the most notable team was one led by dr. xiping xu, who was at that time working at harvard and was well-connected in anhui, china. an anhui native, xu spent the mid- s after high-school as a ''barefoot doctor'' in anhui, providing basic medical care for peasants after a small amount of training at a time when access to medical care in rural china was a luxury. he received his medical degree from anhui medical college (now anhui medical university) in the early s, and was admitted to beijing medical college (now peking university school of medicine) for a student exchange program, a prep program for oversea studies. he went on to get his ph.d. degree in epidemiology in japan, went to harvard to receive his post-doctoral training in epidemiology in respiratory diseases, and received his master's degree in biostatistics from harvard university school of public health (hsph). he stayed on at hsph as a faculty member. even at harvard, xu apparently kept close ties with the anhui medical college, and was involved in several epidemiological studies in the anhui province. when his post-doc supervisor, dr. scott weiss, a harvard university respiratory epidemiologist, told geoffrey duyk, a geneticist who had left harvard to join a biotechnology start-up called millennium pharmaceuticals, that one of his postdoctoral fellows came from the anhui province and had access to a large number of dna samples, they instantly saw the potential and quickly formed a collaborative relationship to discover susceptible genes in complex diseases in anhui (keim ) . a formal partnership between harvard and millennium was established, where xu would direct the collection of a large number of dna samples in anhui, for which millennium would pay the university $ million (keim ) . with tens of thousands of blood samples provided by anhui's villagers, the partnership hoped to identify the susceptibility genes predisposing people to asthma, obesity, miscarriages, schizophrenia and other illnesses. contingent upon its access to the anhui population's dna, millennium also secured sizable capital shortly afterwards from the swedish pharmaceutical company astra ab and from another pharmaceutical giant, hoffmann-la roche. the company's access to dna from the ''large, homogeneous population'' of anhui province was also featured prominently when millennium went public in , raising $ million in its initial public offering (keim ) . leveraging the existing and some preliminary data collected from anhui, xu went on to apply for more funding support from the nih. a search of crisp, a searchable database of nih-funded biomedical research projects (crisp ) using ''xiping xu'' as the principal investigator (pi)'s name reveals that xu received grant on genetics of airway responsiveness and lung function in besides two other nih non-genetics grants, and in he received nih grants on genetics of osteoporosis, airway responsiveness and lung function, nicotine addiction vulnerability, hypertension, and asthma on top of other nih grants (table ) . being a pi almost concurrently on nih grants is remarkable, especially for a junior faculty without much track record, but having nih grant support in diverse research areas ranging from osteoporosis, hypertension, nicotine addiction, and asthma to human reproductive effects due to endocrine disruption or rotating shift work is extraordinary and certainly breathtaking. in all, he received well over million usd in grant support from the nih, the pharmaceuticals industry, the march of dimes, and other funding agencies to investigate genetics of various complex diseases (yang ; keim ) . recognizing his scholarly potential and the growth area he was in, hsph promoted xu to associate professor in and made him the director of the newly established program in population genetics (now disbanded) in hsph. the solid financial backing and extensive connections allowed xu to enlist the enthusiastic support of the local government officials and his alma mater, who helped xu collect thousands of blood samples from rural villagers. a nearly palpable aura of harvard university which xu embodied and was (and still is) revered by many in china as the premium research institution and the most prestigious institution of higher learning also helped. many villagers were illiterate, had no idea what would be done with their samples, and were given merely empty promises of free medical care in exchange for their blood samples. these lapses of oversight, deliberate or otherwise, would return later to haunt xu and his team. the increasing number of scientists like xu with huge financial backing arriving in china to conduct genetic research alarmed many poorly funded chinese scientists, who perceived the situation as a major threat to their profession and a danger of eclipsing their own work. in november , about leading chinese biomedical and genetic researchers gathered in xiang shan, beijing, and held a conference on ''the human genome project and the development strategy for the st -century medicine'' to evaluate and discuss the situation. one incident further fueled the concern shared by all participants. a translated version of a science article was presented at the conference, which stated that xu sought to gain ''access to million chinese through collaboration with six chinese medical centers.'' but in the chinese version, the program became one that would ''take blood samples from million chinese'' (hui and jue ) . this seemingly astronomical figure incensed all conference participants, who were at that time cash-strapped and still playing catch-up with the west. they quickly reached a consensus and soon made it public: ( ) china's genetic resources should not be pilfered by foreigners; ( ) chinese scientists should immediately grasp the opportunity to find disease genes and patent them; ( ) we should educate the people, and raise the awareness and importance of protection of our genetic resources; ( ) we welcome all international collaborations based on fairness and mutual benefits; ( ) through various avenues, the chinese scientists should be vocal about certain views deemed to be harmful to china's genetic research (xiao et al. ) . soon after the xiang shan conference, some scientists published articles depicting foreign gene-hunters as greedy pilferers of the vast chinese genetic gold mine and comparing them to past foreign invaders plundering china's national treasures. they called upon the government and their fellow countrymen to rise up and protect the putative genetic gold mine of the chinese gene pool (fang a, b; lv ) . shortly after, financial details about the millennium-harvard deal based on anhui samples were leaked to the chinese press and caused a media blitz of condemnations. the media called it an imminent ''gene war of the century'' (shou ) . in fact, the notion of foreign capitalists profiting from china's precious genetic resources sparked such a fury that several other genetic research projects unrelated to xu were stalled for a year (hui and jue ) . around the same time, it was rumored that one prominent geneticist, who received his ph.d. from cal-tech in the s for his work on ladybug genetics, yet had no training in either medical genetics or genetic epidemiology, had written a letter to president jiang zemin urging the government to take the matter seriously and to protect china's precious genetic resources. this, along with the media furor, duly alarmed the central government. in , the office for management of china's human genetic resources was quickly established under the auspice of the ministry of science and technology, and charged with overseeing the handling and export of all biological specimens potentially containing genetic materials. soon a de facto law, the interim measures for the administration of human genetic resources, promulgated by the general office of the state council (ministry of science and technology & the ministry of public health, ), was enacted in june . it mandated that genetic resources were not allowed to be taken abroad without explicit permission and observance of due procedures as defined in the interim measures. funding for domestic human genetic research subsequently poured in (swinbanks ), which spurred human genetics research in china (du et al. ) . two genome research centers, one located in beijing and the other in shanghai, were established. while the phrase, ''china's genetic resources,'' has been used widely and extensively, surprisingly no definition has ever been officially provided. some scientists likened china's genetic resources to natural resources, claiming that, as the most populous nation in the world, china has the largest number of ethnic groups and also has the widest and the most complex disease spectrum (xiao et al. ). in addition, with a long documented history and many isolated populations, there were many genetic isolates and thus china has the purest genetic heritage in the world. therefore, china is a ''gene giant'' and ''the new world of genes,'' making many technologically advanced nations envy and salivate (xiao et al. ) . however, the analogy between ''genetic resources'' and natural resources has several problems. granted, the vast population facilitates the recruitment of patients with rare diseases for medical research and the low cost of collecting specimens is conducive to large-scale biomedical research. however, china did not and still does not necessarily have more types of diseases, and even if it did, few people outside china would be interested in finding the genetic causes for diseases that are unique to the chinese population. in fact, for many rare genetic diseases (called ''orphan diseases'') for which susceptibility genes have been cloned, many pharmaceutical companies are often reluctant to invest in drug research and development (r&d) for these diseases due to concerns of profitability. the values of natural resources are determined by their amount, their extractability, and market demand. there are two forms, renewable (such as wind power) and non-renewable (such as fossil fuels). a commodity is considered a natural resource when the primary human activities associated with it are extraction and purification, as opposed to creation. thus, mining, petroleum extraction, fishing etc. are natural resource industry, but agriculture is not. since gene identification requires much more than collecting blood samples and are both labor and knowledge intensive, genetic resources are, by definition, not natural resources. in addition, unlike fossil fuels, genetic resources are not entirely non-renewable, if the disease spectrum remains the same. with dramatic economic and social changes, the living standard in china has risen remarkably in the last years. following these changes, diet and lifestyles also have changed quite dramatically. as a result, the disease spectrum in the chinese has shifted, especially in coastal regions. the incidences of breast cancer, colon cancer, prostate cancer, hypertension, and type diabetes all have risen sharply in the last decade (xiang et al. ) . in large cities such as beijing, childhood obesity is used to be very rare but now it is reported to be in the range of % (and another % of children are overweight). in contrast, incidence of stomach cancer has decreased, especially in those highincidence areas where living standards have been improved. ''gene war of the century'' and its aftermath the rapidly changing disease spectrum means that, firstly, the genetic resources would be forever lost if not used in a timely fashion for gene-hunting purposes. secondly, hunting disease susceptibility genes for a disease that obviously has a strong environmental component was an uncharted territory -no one at the time was absolutely certain how it would turn out. over ten years would elapse before people realized that heritability would vanish even for human traits that are known to be mostly hereditary (maher ) . lastly, the notion of china's genetic resources touched upon some thorny issues. unlike other natural resources, genetic materials, as in blood samples, exist only in the human body, which is technically owned by their hosts, not by the state. if a person strikes a deal with a drug company, or acts simply out of genuine altruism, and is willing to give away his blood sample, does the state have the right to intervene? if so, would such intervention infringe on the donor's human rights? giving away or even selling one's blood sample is certainly different from prostitution or selling one's own body parts. if the state does have the right to intervene, where can we draw the line? unfortunately, such questions were never raised and discussed. for biomedical research, there surely is such thing as a genetic resource. but what is it? what constitutes a genetic resource? contrary to the popular belief, population size and diversity of diseases, in and by themselves, actually do not make china ''an ideal place for gene hunters'' (guo et al. ) . few among the common diseases in china are known to have a hereditary component or to be amenable for positional cloning. in fact, while a small portion of breast cancer cases, for example, may be attributed to gene mutations, the large proportion of common and complex diseases is unlikely due to a few gene mutations or polymorphisms (see below). as demonstrated by an interventional study conducted in finland, by reducing body weight, eating healthy and regularly engaging in physical exercise, the risk for developing type diabetes can be reduced by nearly % (tuomilehto et al. ). the dramatic changes in incidence of various diseases in china clearly show that many complex diseases have a very strong environmental component. indeed, a -year interventional study conducted in da qing, china, shows that, after merely years of lifestyle intervention after recruitment, those in the intervention groups had a % lower incidence over the year period as compared with control participants (li et al. ) , very similar to the finnish results. what constitutes a genetic resource, then? an ideal population for positional cloning and association studies should have a well-enumerated genetic disease positional cloning is a method of gene identification in which a gene for a specific disease is identified. a scientist can know nothing about the disease etiology. but just by collecting family data, dna, and some sleuthing, s/he could roughly localize the putative gene in a chromosomal region (i.e. positioning). then, with other molecular genetic tools, the scientist can then identify the gene from the region-thus the phrase, positional cloning. heritage, such as that of the finns (norio et al. ) , and a relatively welldelineated population genetic structure, such as in finland, where extensive church records often exist that document pedigree information for many populations. the catalogue of the genetic disease heritage would allow for correct specification of genetic models and facilitate gene identification. a well-delineated genetic structure of the population should facilitate fine-mapping (i.e. zero-in) and genetic association studies. in contrast, when the ''gene war'' broke out, the documentation of chinese disease heritage was scant at best, and its research in population genetics and genetic epidemiology lagged far behind that of other developing countries. since genetic epidemiology is itself a new branch of epidemiology, and since the design, execution and analysis of genetic epidemiologic studies require not only the expert knowledge of disease epidemiology but also a good command of statistical genetics, genetic epidemiology in china was in its infancy at the time. consequently, there was little, if any, genetic epidemiologic research in china. as a result, little was known of the mode of inheritance, penetrance, and gene frequency for major complex diseases. even credible estimates of disease incidence/prevalence were hard to find. therefore, the notion that china is ''an ideal place for gene hunters'' is questionable and somewhat dubious. the fact that after well over a decade no genes for any common, chronic disease have been identified in china is a testament to this. while calling for protection of china's genetic resources and equating large number of dna samples to huge commercial profits, virtually no one at the time was even remotely aware that there are actually numerous obstacles to this gene prospecting. first, there were huge financial barriers. hunting susceptibility genes for complex diseases usually requires a large number of blood samples, along with accurate phenotypic data in the first place. while genotyping costs have been reduced substantially, they were still expensive in the late s, especially when the whole genome would be scanned. these procedures alone would require a significant upfront capital investment. and like any other scientific endeavor, the gene hunting expedition would carry inherent risk of failure, lacking any guarantee that the investment would bear any financial rewards. the demand for huge resources, coupled with the uncertainty of yield from the investment, raises the question as to whether these endeavors were actually good investments, especially in a developing nation like china where there were and still are problems in providing affordable and equitable medical care for all its citizens (hsiao ). indeed, lifting living standard for all, improving child and maternal health, and better health education on healthy lifestyles (smoke-cessation, healthy a well-enumerated genetic disease heritage can provide the scientists with basic information about the disease of interest, such as mode of transmission and disease frequency. a well-delineated population genetic structure would come in handy when trying to narrow down the gene in a chromosomal region. penetrance refers to the probability that a person with a certain genotype (a genetic makeup) will develop the disease. ''gene war of the century'' and its aftermath diet, physical exercises etc.) can have a proven improvement of health of the general population. secondly, there were numerous scientific hurdles, some seemingly insurmountable. hunting for complex disease genes was an uncharted territory in , and no one could be reasonably sure as to whether there were susceptibility genes, and, if so, whether they could be identified, especially with reasonable time and resource constraints. even if they could be identified, whether they could fulfill the promises of better diagnosis and treatment is also completely uncertain. the genetic mechanism for sickle cell anemia has been known for well over half a century, for example, but so far no gene-derived therapeutics is available. lastly, there were technical hurdles. to genotype large number of samples with high accuracy and reasonable cost was still a challenge around . methodologically, how to analyze the data for diseases which apparently are also influenced by environmental factors and aging process was, and still is, a serious challenge. in addition, how to handle gene-gene interaction, gene-environment interaction, and variable age of onset posed formidable analytical challenges (di and guo ) . these hurdles were further compounded by the lack of a systematic catalogue and documentation of hereditary diseases in china in terms of disease frequency, mode of transmission and penetrance, the lack of documentation of population genetic structures in china, and the scarcity of well-trained genetic epidemiologists. even though the per-sample cost of sample collection was relatively low, this only advantage rarely offset the other, more fundamental deficiencies, and boded ill for many gene-hunting endeavors. viewing the xiping xu case as the nexus of international, transnational, national, and local interests, sleeboom eloquently provides ten different perspectives representing the views and ideals of different interest groups on xu's genetics research program in china (sleeboom ) . indeed, it is often stated that there are several stakeholders in the putative ''gene war'': chinese scientists, foreign gene hunters, and the chinese government. all of them apparently had a vested interest, mostly financial, in china's genetic resources. however, one critically important stakeholder and a potential beneficiary of this gene prospecting, obscured by the media blitz, were actually the patients of various complex diseases in china and the rest of the world. somehow, their voices were muffled and not heard. indeed, from a patient's perspective, it really does not matter which country finds the genes first and comes up with an efficacious therapeutics as long as s/he can access it at a reasonable cost and within a reasonable timeframe. in the s, china's drug r&d lagged significantly behind the west. most, if not all, drugs and diagnostic kits with proven efficacy used in china today have sickle cell anemia is a life-long blood disorder, characterized by abnormal, sickle-shaped red blood cells that do not have usual elasticity as normal red blood cells do. the disorder is caused by a single mutation in a gene called hemoglobin and manifests excruciating pain and shortened lifespan. it has been known to be an abnormality in the hemoglobin molecule since . been first discovered and developed outside china. in fact, all major pharmaceutical companies have now set up manufacturing facilities in china, and almost all drug companies market their products in china once approved by the state food and drug administration of china, a counterpart of the fda of the us. in fact, anecdotal evidence suggests that, when money is not an issue, many patients in china, especially those with potentially fatal diseases, usually prefer imported drugs or drugs made by foreign companies even though cheaper, domestically made drugs of purportedly equal efficacy are available. thus, one simple fact was overlooked in the entire media blitz: an intellectual right on genes can be profitable only when it has a market. china's market is too big to ignore for any rational pharmaceutical company. and when a drug company sells its gene-derived products to china using materials collected from chinese, some patients may still reap the fruits of gene prospecting. this seemingly obvious fact was completely overlooked at the height of the ''gene war.'' the attention that xu's projects drew was certainly unexpected and was likely distracting to xu's projects. xu vehemently denied that he was exploiting poor villagers in anhui for personal gains. he lamented that ''i came from china, and i love the country. but i have been treated like a traitor'' (hui and jue ) . fearing that the media furor and the lopsided condemnation from scientists in china would torpedo his projects in anhui, xu quickly moved to act. when a letter to the editor appeared in science questioning the validity of the ''gene war'' (guo et al. ) , xu quickly translated it into chinese and circulated it among chinese officials. xu also enlisted the support of several established chinese scholars in the us. he appealed to peng peiyun, who was then the director of the state family planning commission, soliciting support for his projects. he adamantly maintained that he was patriotic, and his projects in anhui and elsewhere in china had already trained chinese scientists and elevated their research capabilities. xu apparently had mastered the finesse of keeping a good relationship with the government officials and adroitly played the card of a patriotic oversea chinese scholar. the official people's daily reported in that ''in the last few years, the chinese biomedical researchers collaborated with the world-famous harvard university on various projects, and achieved exciting results in the pathogenesis of complex chronic diseases…. in particular, the research in genetic epidemiology of asthma and hypertension is now at the forefront in the world'' ([benefiting thousands and thousands of families.] ). in another report, it said that ''harvard's project has so far produced postdocs, doctoral students, visiting scholars, and senior investigators'' ([east and west collaborate for the health of humankind.] ). in xu's hometown, the provincial newspaper reported, after enumerating various human genetics research projects with harvard, that ''these collaborative projects not only injected new vitality to anhui's science and technology but also helped attract investment in the amount of about million ''gene war of the century'' and its aftermath rbm yuan'' ([anhui-native scientist climb peak of human genetics.] ). it remarked that ''these projects helped establish tens of laboratories with advanced instruments, …, and laid the foundation for our nation to catch up and surpass the west in human complex diseases research in the st century'' ([anhui-native scientist climb peak of human genetics.] ). xu's preference for dealing with the high-rank officials, however, went overboard and nearly caused his undoing (see below). as xu's various gene-hunting projects in anhui took off, some disturbing incidents involved in these projects gradually surfaced and leaked to the press. participants were initially promised by the research team that they would get free or reduced-cost medical care, but these promises were never honored. informed consent supposed to be obtained from potential participants actually was not-a flagrant violation of nih regulations. rumors circulated regarding coercion by local officials to participate in the projects, sample mishandling and mix-ups in the lab. prompted by these allegations, a fact-finding team of six people from harvard, including xu and his mentor, scott weiss, arrived in anhui in march to investigate the ethical and scientific integrity of xu's projects, but found no irregularities. five months later, the department of health and human services (dhhs) launched its own investigation of harvard's genetic research in china, based on the complaint of a whistle-blower from hsph alleging violations of us human subject protection regulations (pomfret and nelson ) . in late , the washington post ran a lengthy report detailing the allegations that chinese villagers had not been given low-cost medical care as they were promised in exchange for providing blood samples for xu's us-funded genetic research. the report also included allegations of xu's lapses in informed consent (pomfret and nelson ) . partly because of the controversy surrounding this case and others like it in china, the us embassy in beijing issued an advisory recommending that american scientists stop conducting medical research in extremely poor areas of china like anhui. in march , an investigative report by two xinhua news agency reporters was published in outlook, a major chinese magazine. the report reiterated some of the allegations made in an earlier report published in the washington post and supplemented them with interviews with chinese farmers in an isolated region of the anhui province and their various predicaments. the controversies surrounding xu's anhui projects reached a new crescendo at the symposium on bioethics, biotechnology and biosecurity held in early april , which was sponsored by the united nations educational, scientific, and cultural organization (unesco) and organized by the hangzhou municipal government. xiong lei, the lead reporter of the outlook report, presented her investigation and findings with their interview with chinese rural villagers in anhui. her presentation elaborated various irregularities in xu's projects, including the lack of informed consent, broken promises of providing medical care for those who participated in xu's projects, and xu's taking more blood samples than officially approved. an intense debate ensued, starting with xu's anhui collaborators, who maintained that the rate of getting informed consent was close to %, and that the chinese side did reap benefits from the collaboration with the harvard team. but their presentation was confronted by incensed chinese scientists, who questioned their numbers and practice. xu's legal counsel also made some comments, but the comments were challenged and ridiculed. some scientists expressed grave concerns about the loss of chinese genetic materials to the west, in fear of jeopardizing china's own genetic research. prompted by the outlook report, china's office for management of human genetic resources also launched its own investigation. it soon concluded, in june , that xu's projects did not violate any chinese regulations, and told the us embassy so (pomfret a) . but the controversy took another turn in late june . in a letter to xu dated june , , the dean of the hsph reprimanded him, strongly criticizing him for writing two letters to senior chinese government officials, in which xu urged the government to silence the voice from his detractors and to take actions against a major figure who had criticized his work. defending himself as a patriot, xu's letter suggested that the outlook report had released state secrets to ''foreigners'' (pomfret b) . the dean condemned xu's actions, and warned him that he would ''not receive the continued support of the school for you or your research if you persist in exercising independent action.'' if he continued his campaign against journalists and others who questioned his research, the letter said, xu would face ''appropriate sanction'' (pomfret b ). yet xu's woes, unfortunately, showed no sign of abating. on march , , the federal office for human research protections (ohrp) of the dhhs issued a scathing indictment of the hsph research. the ohrp found that projects in china that hsph was involved, including projects on which xu served as the pi, failed to be approved by institutional review boards (irb); where approval had been granted, significant and unannounced changes were often made. it found that many of the informed consent documents approved by the hsph irb included complex language that would not be comprehensible to all subjects, particularly for rural chinese subjects. hsph was charged with failing to minimize risks to their vulnerable subjects, such as economically or educationally disadvantaged persons. in the end, subjects never even received the free medical care as promised. as a result of the indictment, xu was ordered to suspend all human subject interventions in his active studies pending the outcome of an internal audit. this new development was soon reported dutifully in china's press (xiong and wang ) . on may , , lawrence summers, then the president of harvard university, gave a speech at peking university. when responding to a question from a student in the audience regarding harvard's projects in anhui, summers admitted that some irregularities in the projects were ''wrong.'' xu eventually breathed a sigh of relief when ohrp sent a letter to hsph in early may , stating that all issues raised in the hsph-involved china studies have been either resolved or dissolved because of unsubstantiated allegations (as in alleged forged informed consent documents). consequently, ''there should be no need for further involvement of ohrp'' in these matters. notably, the letter acknowledged that xu had decided not to continue the hypertension study due to ohrp's concern that some of the interventions in the protocol exceeded the level of minimal risk. shortly afterwards, hsph held a press release announcing the ''[c]onclusion of u.s. government's inquiry into hsph genetic research in china. '' soon after the hsph news release, one internet article, by xiong lei, alleged complacency and a likely cover-up on the part of the chinese government. it raised several issues (xiong ) . first, among the harvard projects in china that the ohrp found to have problems, projects were headed by xu. yet officially, only of xu's projects had ever been approved by the government. hence there was a glaring discrepancy. second, the official from the office for management of human genetic resources, who was in charge of the investigation of the allegation of irregularities in xu's projects, told xiong privately that it was not an official investigation. however, the same official then turned around and told the american embassy that no violation was found. it was rather strange that the results of this unofficial investigation, which was shielded from the media and the public, would then be used by the americans to prove that there are no irregularities in these projects. lastly, one peasant in anhui whose ordeal led to the exposure of apparent lapses in informed consent later recanted his story after talking with officials from anhui and the central government. presumably, he changed his story because of pressure he experienced. this changed story explained why the ohrp eventually found no wrong-doings in xu's projects (xiong ) . since xiong's article appeared in a website that is officially blocked in china, it did not cause any stir. in china's news media, the criticism of xu's anhui projects also subsided consequently. xu's woes finally ended. more than a decade has passed since the purported ''gene war.'' despite well over a decade of hard work and well over million us dollars in grant support of various forms, xu's team has so far not cloned a single gene for any complex disease or disorder. in fact, they are not even close. other teams were no luckier. in fact, besides numerous reports of association of diabetes, asthma, and other complex diseases with certain genetic polymorphisms, so far not a single gene has been proven to be chiefly responsible for any of these diseases. most genetic loci identified to be associated with the disease risk confer only miniscule relative risks, ranging from . to . (kraft and hunter ) . even when genetic polymorphisms that are associated with a modest increase in risk are combined, they generally have a low discriminatory and predictive ability (janssens and van duijn ) . a more recent study reports that after examination of genetic variants reportedly linked to heart disease, the variants turned out to have no value in predicting disease among , women who had been followed for over years and that family history had better predictive value (paynter et al. ). for human height, a trait that is known to be mostly hereditary, it is calculated that approximately , single nucleotide polymorphisms that are required to explain % of the population variation (goldstein ). this nearly astronomical number certainly inspires no enthusiasm for conducting large-scale gene hunting projects, and questions their value in genetic screening, genetic testing, and the possibility of developing gene-derived therapy (wade ). the idea that disease genes can be quickly identified, patented, and then quick profits can be made now seems to be too naïve. indeed, years after the first draft of the human genome was completed, medicine has yet to see any large part of the promised benefits (wade ) . even gene patenting, the very topic that made the ''gene war'' so contentious, has begun to encounter resistance (kintisch ). there is indication that, at least in the united states, the status of gene patenting is changing (kintisch ). in fact, the us government recently decided that human and other genes should not be eligible for patents because they are part of nature (pollack ) . this move, viewed as ''a bit of a landmark, kind of a line in the sand,'' followed a surprising ruling made in march , by judge robert w. sweet of the united states district court in manhattan, which ruled the patents held by myriad pharmaceuticals and the university of utah on two genes that predispose women to breast and ovarian cancer invalid (pollack ). on june , , the u.s. supreme court ruled in association for molecular pathology vs. myriad genetics that ''naturally occurring'' dna sequences, but not lab-created synthetic cdnas, are off-limits for patent protection. millennium pharmaceuticals, the initial financial backer of xu's projects, pulled out of anhui early in , with no significant medical or business discoveries to show for its $ . million investment (pomfret and nelson ) . it since has moved into a field of drug r&d that seeks to customize medical treatments for individual patients. it has grown into a successful, billion-dollar enterprise. yet no doubt xu's anhui projects played a crucial role early in its success by securing a much needed infusion of funds (pomfret and nelson ) . for sequana therapeutics, despite its public announcement of the discovery of the asthma gene in , so far there have been no publications from the company regarding the gene. the claim has never been independently verified. the prospect of making diagnostics or therapeutics derived from the putative gene never materialized. it was acquired by arris pharmaceuticals, forming axys pharmaceuticals which later on formed axys advanced technologies, later bought by chemrx. the remains of axys were bought by celera. what used to be sequana therapeutics no longer exists. human genome sciences' stock price reached its peak at $ on january , and went through two splits in . its president and founder haseltine said that his work ''speeds up biological discovery a hundredfold, easily.'' he talked of finding ''the fountain of youth'' in genes in the form of ''cellular replacement'' therapies. the company raised more than $ billion in investments by . in september , the company reported that it had found a way to treat large, painful sores that often plague elderly patients, using a protein spray called repifermin, made by a human gene called keratinocyte growth factor- . in february , however, the company said that it was ending the development of repifermin because it showed no more benefit than a placebo in clinical trials. another initial drug also failed in clinical trials. in late , the company announced haseltine's retirement and named h. thomas watkins the new president and ceo. in , the first draft version of the human genome was published, thanks to collaborative work among geneticists from china, france, germany, japan, united kingdom and united states. in , the completed version of the human genome was published, marking the completion of the hgp. from the first draft of the human genome, it was quickly learned that there are about , genes, less than one quarter of , ''genes'' patented by the human genome sciences. along with this shrinkage in the number of genes, the company stock price also shrank dramatically: the closing price on july , , was $ . , a reduction of . % from its historical high. other genomics companies have not fared much better. iceland-based decode genetics, for example, was founded in to identify human genes associated with common diseases using population studies. its stock price reached $ . on september , , but plummeted to $ . on july , , a reduction of . % in value. its stock was removed from the nasdaq biotechnology index in november . the company's annual report reveals that its net losses were in excess of million us dollars, and that they have never turned a profit. its annual report says that ''decode has recorded substantial operating and net losses over the past years'' and the company filed for chapter bankruptcy in the same year (http://www.decode. com/investors/dcgn-sec-filings.php). it was acquired by amgen at the end of for million usd. on the chinese side, human genetics research moved on with the infusion of research funding from the government. most scientists who participated in the xiang shan symposium established themselves in genetics research. several of them were later elected to the chinese academy of sciences, the most prestigious honor that can be bestowed on to a scientist in china. besides all the trappings and perks, being an academy member also carries far more influence and political clout than its us counterpart. huangmin yang, the most vocal in criticizing xu's projects, went on to establish china's premier genome research center, and his career culminated recently in the completion of the diploid genome of the first asian individual (wang ) , rumored to be the dna extracted from yang himself. the purported ''gene war'' has a profound resonance. even today, over a decade after, the reverberations of the media blitz and the fallout are still palpable: a google search of ''gene war'' or ''genetic resource'' turns up numerous websites still talking about the ''gene war'' or even the purported attempt by the us to wage war against china using ''gene weapons'' (tong ) . the ''war'' also spurred a flurry of research papers in chinese scholarly journals, universally calling for the protection of china's genetic resources and profit-sharing arising from gene research (jia and wang ; jiang and wei ) . xu left harvard in and joined the school of public health, university of illinois in chicago, as a non-tenure track research professor of epidemiology (http:// www.cade.uic.edu/sphapps/faculty_profile/facultyprofile.asp?i=xipingxu), apparently failing to secure a tenured position at harvard. the negative publicity surrounding xu likely made him seem more a liability than an asset to hsph, especially when he and his group had made no important discoveries. in , the epidemiologist-turned genetic epidemiologist went through another metamorphosis and became an entrepreneur. he was the chief technology officer and, as of writing, is now the president of ausa pharmed company in shenzhen, china. in a glowing profile of xu and his company by people's daily, xu is quoted as saying, ''i used to write prescriptions for people in a small village, and now i am writing a big prescription for people all over the world'' (wang ) , apparently referring to the company's blockbuster-to-be drug for stroke prevention, yiye (''yi'' is the pronunciation of the first syllable of enalapril in chinese and ''ye'' is that of folic acid). according to the company's website, the drug, a polypill consisting of a combination of enalapril (an angiotension converting enzyme inhibitor, or acei, used as an anti-hypertensive drug, on which merck had a patent, now expired) and folic acid (a member of the vitamin b family, used to prevent neural tube defects for pregnant women, and, as an auxiliary, to treat hyperhomocysteine and other conditions), is the fruit of extensive research by ausa scientists, ''the only class i cardiovascular drug approved by the state food and drug administration (the us fda counterpart in china) in the last three years with all china-owned intellectual rights, and is the first novel drug in the world that can simultaneously control two risk factors for stroke, hypertension and hyperhomocysteine.'' in , xu was among the first that were granted the ''thousand scholars'' support, a program designed to attract full-professor-level senior professionals from overseas to work in china. this is a title with enormous privileges and perks given to a select group of best scholars recruited from overseas by beijing. on december , , xu was featured in the oriental satellite tv's special program, people in years, a program that profiled prominent people and their achievements in the years of economical reform in china. in the program, xu talked about his early life as a ''barefoot doctor,'' his admission to peking university and then to harvard, and his dream, as a youth, of ''writing big prescriptions for people all over the world.'' he talked about his work in epidemiologic studies of air pollution and health and his new venture in developing drugs for chinese people. he made no mention, however, about his genetics studies and their associated controversies, and showed no trace of bitterness. curiously, the ausa-sponsored clinical trial on the evaluation of yiye in prevention of stroke was registered at the clinical trial registry, www. clinicaltrials.gov, on november , , which coincided with the official approval from the sfda of yiye. the registered trial, china stroke primary prevention trial (csppt), is a phase iv trial (nct ), which compares the efficacy of enalapril and folic acid combination vs. enalapril alone in preventing strokes. as of writing, its status is listed as ''on-going, but is not recruiting participants.'' its estimated completion date is august, . in modern society, science has become a firmly institutionalized social activity, attracting people through offering generally prized opportunities of engaging in socially approved patterns of association with one's fellow and the consequent creation of cultural products esteemed by the group, in addition to economic benefits that science may offer (merton [ ). as merton eloquently put it, ''such group-sanctioned conduct tends to continue unchallenged, with little questioning of its reason for being. institutionalized values are conceived as selfevident and require no vindication'' (merton [ ). in modern science, especially in biomedical research, scientific enquiries often require large amount of resources-expensive instruments and reagents, lab space, and talented and hardworking students. hence the pressure for getting resources is enormous. anything that promises to help ease the pressure is welcome. scholars of sociology of science often view science as agonistic, made up of rival individuals or groups vying to have their scientific ideas or views recognized and/or accepted as valid (greenhalgh ) . science, as a space of maps of culture, is drawn by scientists hoping to have their research accepted as valid and recognized, their practices esteemed and patronized, and their culture sustained as home of objectivity, reason, truth or utility. the maps are then used by all who are unsure about the reality (gieryn ). yet maps of science change over time, as competing cartographers are constantly drawing, erasing, and redrawing the boundaries of science. by doing this, the scientist cartographers claim authority over a particular issue by taking it within their science or turf. thus, vying for acceptance or the valid ''truth'' among scientists is essentially a credibility contest, with winners viewed as the epistemic authority (gieryn ) . the one with the epistemic authority obviously would be the most influential, and their views and voices would be the most visible and vocal when it comes to policy issues (greenhalgh ). gieryn's credibility contest metaphor aptly depicts the quest for epistemic authority in science, it also is applicable, rather fittingly, to the situation when scientists vie for resources from funding agencies. in fact, when the process of deciding who would get resources lacks procedural justice, and when there is a lack of tradition for open and rational debate and of a checks-and-balances system, the credibility contest becomes literally a ''beauty'' contest-the most glamorous, in terms of rank, status, or simply seniority in the administrative ladder, but not necessarily the vision, merit, or talent, would get the resources. in a country where political loyalty and connections are valued far more than scientific merit and talent, the credibility contest further becomes a contest of political correctness or patriotism. coupled with the lack of a clean and efficient government and of transparency and also with the pervasiveness of guanxi or personal connections, this contest might create winners who are not necessarily the scientifically most visionary. as human beings, scientists are also susceptible to all human frailties. aside from questing for epistemic authority, they also compete for resources and influence, and often vie for political clout, credit, fame, and even glamour, especially when such activities help their quest for epistemic authority or increase their professional and personal gains. if there are no set rules of the game with certain procedural justice or a checks-and-balances system in place that can curtail the tendency and channel it into the maximization of the common good, the human frailties, coupled with the lack of proper avenues for open debate, the contest would be an invitation for inefficiency, waste, corruption, and even disaster. winners might take all, but in the long run the bad money drives out the good. the spectacular fiasco in containing the sars epidemic and in sequencing the coronavirus that causes sars are a prime example (enserink ; cao ) . lured mostly by the low cost of collecting large dna samples and the perceived genetic homogeneity, many gene hunters from the west came to china in the hope to identify genes responsible for complex diseases, and some of them may have hoped to get rich in the process. this was mostly accomplished through some wellconnected chinese intermediaries ''as experienced guide,'' as washington post reporter john pomfret put it (pomfret and nelson ) . letting the intermediary do the leg work did spare them from doing the dirty field work but also insulated them from the sentiment from villagers and the scientific establishment in china and prevented from establishing a rapport with these people. from a scientific standpoint, many gene-hunting projects were launched without much understanding of the population genetic structure of the chinese population or foundational genetic epidemiologic data, let alone the appreciation of the inherent risk in this scientific endeavor. there was no plan b that one could have something to fall back on in case what was planned did not pan out. the execution also was fraught with various deficiencies. with little or no oversight, the daily work was left to the hands of not-so-well trained people. and when rumors of irregularities surfaced, inspection was largely perfunctory, nothing more than sugar-coating or bandaging. it would have been a miracle if such projects were ever productive. faced with numerous well-endowed gene hunting teams coming to china, the cash-strapped chinese genetics scientists had every reason to be worried. the taking of large number of dna samples and, worse yet, the tracking down of some large pedigrees with rare genetic diseases would effectively deprive their chance of finding disease genes, outshining them in the genetic research of chinese populations and threatening their careers. providing dna materials without any reasonable share of possible future financial payoff for the people who donated their blood could also be a concern, but it is not clear which was the primary concern. by calling the attention of the central government through evoking nationalism via calling the protection of china's genetic resources, they got the resources and also claimed a territory that would be off-limit to ''foreign devils.'' yet by doing so, no one apparently was aware then of numerous and enormous hurdles to gene prospecting and vastly underestimated its complexity and challenge. by evoking the urgency to protect china's genetic resources, some scientists played the card of nationalism, wining the contest in getting resources through nudging the unsuspecting government to cough up some much needed funds to thwart ''foreign devils''' pilfering act. through the drafting and implementation of the interim measures for the administration of human genetic resources, the domestic scientists effectively enacted an embargo of the transfer of all dna-containing materials from china to the outside by drawing a clearly demarcated boundary. this may explain why dr. xiping xu repeatedly proclaimed, in many public occasions during the entire course of the ''war,'' that he is a patriot. well-connected and clearly a master of nuances of guanxi, he certainly knew the psyche of many chinese and government officials. lapses and missteps aside, he was no match to domestic scientists united in the name of patriotism. yet his biggest deficiency in the credibility contest was attributable to his betting on a wrong horse: many, if not all, of his well-funded projects did not pan out in the end, producing no headline scientific discovery and failing to establish an epistemic authority. while credibility contest to quest for epistemic authority depicts science and its working, the contest for credibility, glamour or patriotism in getting resources as we see in the ''gene war'' may be ultimately detrimental to china's science and technology. today, china's r&d investment, in terms of dollar amount, has increased dramatically as compared with the era of ''gene war.'' it reached a historical high of . billion yuan, or about . billion us dollars, in , amounting to . % of china's gdp. as a result, china's scientific output, measured by the number of papers published in international journals, also has increased remarkably and is reportedly ranked as the second in the world, just behind the us. yet in terms of average number of citations-a rough measure of impact or research quality, china was ranked a distant th . a more disconcerting observation is that the fruit of biomedical research seldom translates into better patient care, better therapeutics, better prognostics, or better prevention. in other words, the vast majority of tax-payers have not received any tangible benefit from the supposedly noble and grandiose scientific endeavor. this situation, if left unchanged, is not going to justify for heavy investment in biomedical research and to win the continued support from tax-payers in the long-run. ultimately, it would raise the issue of sustainability of biomedical research in china. this problem will become all the more acute as china enters into an aging society in which health care cost will surely skyrocket. it should be noted that, at the height of the ''gene war,'' similar concerns were also raised in finland and india. however, few seemed to have framed their concerns at the level of nationalism, and even fewer have gone overboard and demonized, often in passionate and patriotic rhetoric, all foreign-supported gene hunting projects. the near paranoid that instigated towards all foreign-backed gene-hunting project did help to cough up some much needed research funding from the government, but also fermented xenophobia and some utterly unfounded yet sensational non-sense such as ''gene weapons'' (tong ) and tarnished genetic research in china. remarkably, when a book was published in , sensationally claiming that the sars virus was deliberately manufactured by the west based on dna materials smuggled out of china (tong ) , no one stood out and debunked such scientific rubbish. the chinese also carry a burden of humiliation and painful memories of the past as a result of repeated ravages by foreign aggression and exploitation in the last two centuries. consequently, issues that may be remotely related to national sovereignty or foreign exploitation are hot-button ones. minor incidents can be easily escalated into a major event, as evidenced by the calls for boycotts of french carrefour and other foreign retailers in china in response to the disruptions of the olympic torch relay in paris in , and, more recently, by the vandalization of japanese-made cars in many chinese cities at the height of anti-japanese sentiment rekindled by the territorial dispute between china and japan. china's current funding system and the science policy-making also are vulnerable to subterfuge of various kinds, behind which personal gains are often masquerading as patriotism or national interest. as ambrose bierce once defined, patriotism is a ''combustible rubbish ready to the torch of any one ambitious to illuminate his name.'' it is a challenge to weed out charlatans impersonating as patriots, but the best bet would be a transparent system that values merit, talent and vision above cheap patriotic or nationalistic rhetoric. as china is aspiring to be a leader in science and technology, this ''gene war of the century'' and its aftermath, as narrated here, serve as a cautionary tale. it reminds us, first and foremost, how important it is to be clear-headed and not to follow blindly whatever in vogue. very often, what we see is the conspicuous ''me-too'' science, following whatever fashionable. yet the most important ball that all eyes should lay on, i.e. better health for all, is seemingly lost. in the absence of procedural justice in the process of deciding who would get resources, and when there is a lack of tradition for open and rational debate and of a checks-and-balances system, the credibility contest for resources would easily become a ''beauty'' contest. in a country where political loyalty and nationalism are valued more than scientific merit and talent, the credibility contest would further become a contest of political loyalty, political correctness, or patriotism. the news of pilfering of china's genetic resources by foreign companies could easily strike a chord of painful memories of foreign aggression and exploitation in the last two centuries. the isolation from the world community for nearly three decades since one key finding of the hgp is that all human races are . % identical-dna sequence-wise. therefore, racial differences are genetically insignificant. for many genes, it has been established that genetic variations within the same racial group are larger than those among racial groups. thus, scientifically, it is impossible to devise a ''gene weapon'' to target a specific racial or ethnic group. ''gene war of the century'' and its aftermath the founding of the people's republic purportedly due to the prejudice, discrimination, and containment of the western imperialists and capitalists-or so it was told by the state media-also helped foster or reinforce a nearly xenophobia attitude towards anything perceived to be dangerous if coming from outside of china, especially it touches on ideology. thus, a spark of minor incident could easily kindle the fire of tumultuous nationalistic uproar. hence, the ''gene war'' holds a lesson that being seemingly the most patriotic is not an assurance for good science. the mere possession of resources does not guarantee scientific productivity, either. vision and brain are more important when it comes to scientific innovation and discovery. the over-politicizing science will ultimately prove to be detrimental to china's science and technology. we have seen it during china's great cultural revolution, in which nearly everything in science and technology was politicized. but science and technology then were essentially decimated, and the characteristic hallmark was jia, da, kong or falsehood, grandeur, and emptiness. in addition, when political loyalty prevails over talent and vision, some unscrupulous scientists can hijack the value system to their own advantage. and when there is also a lack of avenue for open debate, then one project purported of to be of national importance could be usurped by another with purportedly greater importance. the conflict also reminds us that scientists are no more than human beings and have all the human frailties. personal gains or some ulterior motives can be camouflaged as patriotism or public interest, as shown in the politics of paleoanthropological nationalism in china (sautman ) . as merton succinctly put it, ''any extrinsic reward-fame, money, position-is morally ambiguous and potentially subversive of culturally esteemed values. for as rewards are meted out, they can displace the original motive: concern with recognition can displace concern with advancing knowledge. an excess of incentives can produce distracting conflict'' (merton [ ). without any measures or system to guard against this, the interest of the nation and of the public will suffer. in human genetics, china's premier challenge now remains to be, as was a decade ago, ''to build up a critical mass of highly competent and visionary scientists who will be able to bring chinese genetics into the world community'' (guo et al. ) . the ultimate goal of biomedical research in general and human genetics in particular is to bring tangible benefits and better health to the general public, not merely some ranking of scientific output, for it is the source of gaining sustainable support from the general public and of economic prosperity. science thrives on openness, reason, and the competition of ideas, and it suffers when subjected to political agenda, faux patriotism or nationalism. anhui-native scientist climb peak of human genetics noncoding rna transcription beyond annotated genes implications of the human genome project for medical science the search for genetic variants predisposing women to endometriosis confirmation of susceptibility gene loci on chromosome in northern china han families with type diabetes china's missed chance shiji zhi zhang: jiyin qian duo zhan [war of the century: the war to seize the genes shiji zhi zhang: jiyin qian duo zhan [war of the century: the war to seize the genes asthma gene discovered cultural boundaries of science: credibility on the line common genetic variation and human traits just one child: science and policy in deng's china gene war of the century? 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genome research set to take off in china the last defence: the agony of the loss of chinese genes prevention of type diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance genes show limited value in predicting diseases a decade later, genetic map yields few new cures new drug research in china lift heavy siege. people's daily methods for time trend analysis of cancer incidence rates experimental studies on somatic gene therapy for diabetes i. structuring of recombinant from human insulin gene and mammalian expression vector prc/cmv scramble for chinese genes not ended harvard's genetic research projects in china a ''violation advances in science and progress of humanity: a global perspective on dna the gene puzzle acknowledgments the author would like to thank drs. cong cao, partha majumder, margaret sleeboom-faulkner for their constructive comments on an earlier version of this manuscript. he also thanks dr. yang huanmin for the reading of an earlier draft, two anonymous reviewers and the editors for their helpful comments. thanks also goes to charles guo for his critical reading of and helpful comments on the manuscript. key: cord- -l yyr l authors: grabbe, stephan; beissert, stefan; enk, alexander title: systemic immunosuppression in times of covid‐ : do we need to rethink our standards? date: - - journal: j dtsch dermatol ges doi: . /ddg. sha: doc_id: cord_uid: l yyr l the current sars‐cov‐ pandemic particularly endangers older people with pre‐existing cardiopulmonary and metabolic conditions. however, it is also currently under discussion whether patients under immunosuppressive therapy also have a higher risk of suffering a severe course of the covid‐ disease. in principle though, there is currently no data available for a general reduction or pause of immunosuppression in patients with autoimmune diseases because of the sars‐cov‐ pandemic. however, since there is currently neither an effective therapy nor corresponding vaccination protection, the indication for a prolonged immunosuppressive therapy should be made with special care. in particular, immunotherapeutic agents that produce long‐term effects (e.g., rituximab) should be used with special caution. in contrast, immunomodulating substances that do not suppress antiviral immunity (e.g. systemic immunoglobulins, doxycycline), or that have intrinsic effects on sars‐cov‐ (calcineurin inhibitors, chloroquine, hydroxychloroquine) may be useful alternatives. viruses activate the innate immune system (especially macrophages, dendritic cells and other myeloid cell types) by binding to so-called "pattern recognition" receptors, which include toll-like receptors (tlr) on the cell membrane and in endosomes as well as cytoplasmic inflammasome activators [ ] . these lead to the production of type interferon and a multitude of inflammatory cytokines. in this way, the adaptive immune system (especially t and b cells) is activated, the expansion of virus-recognizing t cells and the formation of neutralizing antibodies is stimulated and the virus, in most cases, eliminated. although this is the normal case also in covid- patients, in individual patients the virus can cause hyperactivation of the immune system, which then triggers the clinical picture of acute respiratory distress syndrome (ards). typical findings in this situation are, in addition to increasing respiratory distress, maximally elevated inflammatory parameters and inflammatory cytokines (especially the current sars-cov- pandemic particularly endangers older people with pre-existing cardiopulmonary and metabolic conditions. however, it is also currently under discussion whether patients under immunosuppressive therapy also have a higher risk of suffering a severe course of the covid- disease. in principle though, there is currently no data available for a general reduction or pause of immunosuppression in patients with autoimmune diseases because of the sars-cov- pandemic. however, since there is currently neither an effective therapy nor corresponding vaccination protection, the indication for a prolonged immunosuppressive therapy should be made with special care. in particular, immunotherapeutic agents that produce longterm effects (e.g., rituximab) should be used with special caution. in contrast, immunomodulating substances that do not suppress antiviral immunity (e.g. systemic immunoglobulins, doxycycline), or that have intrinsic effects on sars-cov- (calcineurin inhibitors, chloroquine, hydroxychloroquine) may be useful alternatives. systemic immunosuppression in times of covid- : do we need to rethink our standards? il- , il- β, il- ) in serum. neutrophilia, lymphopenia as well as the ratio of these leukocyte populations to each other, the extent of the increase in ferritin, crp, il- , d-dimers and fibrinogen and the oxygen saturation parameters (sao / fio ) are suitable prognostic parameters for the course of the disease [ ] [ ] [ ] [ ] [ ] . the laboratory parameters for severe co-vid- disease are very similar to those for hemophagocytic lymphohistiocytosis (hlh), which can occur in the course of hematological neoplasia or as a side effect of immune checkpoint inhibitor or car-t cell therapy [ ] . the severe inflammatory response (hyperinflammation) is the rationale for clinical testing of cytokine antagonists (anti-il- : tocilizumab, il- ra: anakinra, anti-il- β: canakinumab) or for administration of corticosteroids in severe sars-cov- infections. in addition to hyperinflammation, however, most patients suffer from an exhaustion of the adaptive immune system (immune exhaustion) later in the course of the disease, accompanied by increasing lymphopenia and reduced activation of t cells, which can be recognized, for example, by t cell expression of the surface markers pd- and tim- [ , ] . this state of exhaustion of the immune system after hyperactivation, comparable to muscular exhaustion after extensive physical activity, can ultimately lead to a collapse of the antiviral immune response and the death of the patient. for this reason, the early therapeutic administration of corticosteroids in this disease is very controversial, especially as it is also mostly counterproductive in other severe viral infections [ , ] . however, high-dose corticosteroids can be beneficial to hospitalized patients in the later course of the disease [ ] . immunosuppression -a risk for severe forms of covid- disease? due to the complex immune regulation in sars-cov- infections, in which too much immune activation ultimately causes the failure of immune control of the pathogen and inflammatory regulation, the question arises as to how patients who have a therapeutically altered immune system react to this infection. in this regard, initial, still very preliminary findings are already available; several publications unanimously report that the course of the disease is significantly more severe in immunocompromised patients who have undergone a heart or kidney transplant and is fatal in about - % of these patients [ ] [ ] [ ] [ ] [ ] . in contrast, the mortality rate was not significantly increased in patients with chronic inflammatory bowel disease, though it should be noted that only a minority of these patients had taken immunosuppressive drugs in higher doses [ ] . there was also no increased risk of severe disease progression in patients with systemic psoriasis [ , ] . however, an individual case report describes a severe course in a vasculitis patient taking rituximab [ ] . due to their profound and long-term effect on antibody production, the administration of b-cell-depleting anti-cd antibodies may be particularly problematic in this context, though conclusive studies are still lacking. overall, the data to date suggest that high-dose and long-term immunosuppression can worsen the course of covid- and is associated with a higher risk of death. there is thus some evidence that therapeutic immunosuppression might increase the risk of a severe course of sars-cov- infection, at least in multimorbid patients with pre-damaged organs or cardiovascular damage. although di altobrando et al. [ ] did not find indications for a particularly severe course of disease in immunosuppressed patients with bullous autoimmune dermatoses, one should considered whether the indication for a potent immunosuppression in dermatological diseases in times of the corona pandemic needs to be more strictly defined, particularly since bullous autoimmune diseases, collagenoses, drug allergies or vasculitis typically occur in elderly patients with comorbidities which are anyway associated with the risk of a severe course of disease. however, this does not apply equally to all immunosuppressants and immunotherapeutic agents, as the substances have different mechanisms of action and some have little or no suppressive effect on the immune system as a whole. it is known from approval studies and registry data that therapy with most biologicals is not associated with a significantly increased risk of viral diseases. similarly, in a recent meta-analysis, there was no evidence of a severe course of sars-cov- infection under therapy with tnf blockers, il- blockers or il- blockers [ , ] , and the latter two are even used therapeutically to combat sars-cov- induced hyperinflammation. so far, there are also no indications for a disease-worsening effect for other immunosuppressive drugs (methotrexate [mtx], azathioprine, jak-inhibitors, tacrolimus). a report on covid- disease in patients with a range of autoimmune diseases of the skin, joints and intestines treated with various immunosuppressive drugs showed a hospitalization rate comparable to that of covid- patients in the general population [ ] . there is also little data on the effects of long-term glucocorticoid therapy on the course of covid- disease. however, a first study in this area also found no evidence of more severe disease progression in cortisone-pretreated patients, although the dose and duration of steroid therapy were not recorded [ ] . in summary, there is currently no evidence for a generally aggravated course of infection of covid- disease in patients that are under chronic immunosuppression. despite a paucity of data, a general discomfort remains when interfering with the immune system in a situation where antiviral defense is of particular relevance. however, some immunotherapeutic agents appear to be particularly unproblematic for pharmacological reasons alone in the context of a sars-cov- infection. these include in particular chloroquine and hydroxychloroquine. there has already been speculation in the lay press about a possible therapeutic efficacy of chloroquine or hydroxychloroquine in covid- disease, so that these substances are often regarded as uncritical for the long-term treatment of patients with autoimmune diseases. however, borba et al. [ ] report on a randomized clinical trial for the therapy of advanced, severe covid- disease with high-dose chloroquine ( , mg/die), which showed an increased mortality in the chloroquine-treated group. thus, chloroquine (or hydroxychloroquine) therapy is probably not effective or even counterproductive in advanced covid- disease. in contrast, there is at least some pharmacological rationale for a possible prophylactic effectiveness of this substance in protection against sars-cov- infection, since chloroquine inhibits endosomal nadph oxidase and virus uptake into cells [ ] [ ] [ ] . however, in clinical practice, long-term therapeutic use of hydroxychloroquine in patients with systemic lupus erythematosus does not appear to protect against covid- disease or a severe course of the disease [ , ] . thus, there is currently no evidence that therapy with chloroquine or hydroxychloroquine has any negative or protective effects on sars-cov- infection or that it changes the course of infection. cyclosporine also seems to be less problematic, since on the one hand humoral immunity is only slightly affected by this substance and no increased rates of viral infections have been described even with long-term therapy [ ] , and on the other hand because cyclosporine has an intrinsic antiviral activity, which also acts against coronaviridae [ ] . for the treatment of bullous pemphigoid there is, besides the systemic corticosteroids, also the option of a therapy with tetracycline, nicotinamide and topical clobetasol, which in a recently published long-term study showed a survival advantage over systemic steroid treatment for the mostly elderly and multimorbid patients independent of a risk of sars-cov- infection [ ] . in the opinion of the authors, this therapeutic alternative, which is currently not widely used in germany, therefore merits greater consideration at the present time. the administration of intravenous immunoglobulins is a further, particularly interesting alternative to conventional immunosuppressants. this is especially true for all bullous autoimmune skin diseases, but also for most collagenoses, neurological autoimmune diseases, scleromyxedema, severe drug allergies (e.g., toxic epidermal necrolysis) and vasculitis, for which this therapy is a therapeutic alternative according to the european s guideline [ ] . interestingly, there is even evidence that common commercially available immunoglobulin preparations contain antibodies reactive to sars-cov- [ ] , probably due to cross-reactivity with conventional corona viruses with which the donors have had contact. in addition, the successful therapeutic use of immunoglobulins in severe covid- disease has already been reported [ ] . in this respect, therapy with intravenous immunoglobulins may not only improve the underlying disease without having to accept immunosuppression, but may even offer some protection against sars-cov- infections. therefore, the authors recommend that this therapy option should be considered especially in patients with other risk factors for a severe course of sars-cov- infection. the current sars-cov- pandemic presents us with new challenges, not least in the selection of immunotherapeutic agents for the treatment of autoimmune diseases. a differentiated consideration of the various substances appears to be just as necessary as a careful scientific analysis of the course of disease in immunocompromised patients with covid- disease. essentially, there is currently no data available for a general reduction or pause of immunosuppression in patients with autoimmune diseases, since the risk of an insufficient therapy of these mostly severe diseases is clearly higher than that of an aggravated course of covid- disease. nevertheless, due to their pharmacological effects, the indication for a long-term and high-dose systemic steroid therapy as well as for a therapy with anti-cd antibodies should currently be defined with particular care. stephan grabbe, md department of dermatology of university medicine university medical center mainz langenbeckstrasse mainz, germany e-mail: stephan.grabbe@unimedizin-mainz.de pattern recognition receptors and inflammation prompt predicting of early clinical deterioration of moderate-to-severe covid- patients: usefulness of a combined score using il- in a preliminary study all those d-dimers in covid- c-reactive protein level may predict the risk of covid- aggravation. version . open forum infect dis characteristics and prognostic factors of disease severity in patients with covid- : the beijing experience prognostic value of leukocytosis and lymphopenia for coronavirus disease severity the many faces of anti-covid immune response reduction and functional exhaustion of t-cells in patients with coronavirus disease (co-vid- ) high-dimensional immune profiling by mass cytometry revealed immunosuppression and dysfunction of immunity in covid- patients effect of dexamethasone in hospitalized patients with covid- : preliminary report corticosteroids as adjunctive therapy in the treatment of influenza use of corticosteroids in coronavirus disease pneumonia: a systematic review of the literature characteristics and outcomes of recipients of heart transplant with coronavirus disease sars-cov- infection in patients on renal replacement therapy. report of the covid- registry of the spanish society of nephrology (sen) preliminary data on outcomes of sars-cov- infection in a spanish single centre cohort of kidney recipients covid- infection in kidney transplant recipients a single center observational study of the clinical characteristics and short-term outcome of kidney transplant patients admitted for sars-cov pneumonia characteristics and prognosis of patients with inflammatory bowel disease during the sars-cov- pandemic in the basque country (spain) rituximab for granulomatosis with polyangiitis in the pandemic of covid- : lessons from a case with severe pneumonia systemic or biologic treatment in psoriasis patients does not increase the risk of a severe form of covid- the impact of the co-vid- pandemic on patients with chronic plaque psoriasis being treated with biological therapy: the northern italy experience should sars-cov- influence immunosuppressive therapy for autoimmune blistering diseases? associations between immune-suppressive and stimulating drugs and novel covid- -a systematic review of current evidence covid- in immunemediated inflammatory diseases -case series from covid- infection and glucocorticoids: update from the italian society of endocrinology expert opinion on steroid replacement in adrenal insufficiency effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection: a randomized clinical trial characterization of the early steps of human parvovirus b infection hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase clinical course of coronavirus disease (covid- ) in a series of patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine covid- infection in a northern-italian cohort of systemic lupus erythematosus assessed by telemedicine cyclosporine therapy during the covid- pandemic is not a reason for concern why choose cyclosporin a as first-line therapy in co-vid- pneumonia tetracycline, nicotinamide, and lesionally administered clobetasol as a therapeutic option to prednisone in patients with bullous pemphigoid: a comparative, retrospective analysis of patients with long-term follow-up european guidelines (s ) on the use of high-dose intravenous immunoglobulin in dermatology currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus antigens sars-cov- inflammatory syndrome. clinical features and rationale for immunological treatment key: cord- -zim nond authors: proal, amy; marshall, trevor title: myalgic encephalomyelitis/chronic fatigue syndrome in the era of the human microbiome: persistent pathogens drive chronic symptoms by interfering with host metabolism, gene expression, and immunity date: - - journal: front pediatr doi: . /fped. . sha: doc_id: cord_uid: zim nond the illness me/cfs has been repeatedly tied to infectious agents such as epstein barr virus. expanding research on the human microbiome now allows me/cfs-associated pathogens to be studied as interacting members of human microbiome communities. humans harbor these vast ecosystems of bacteria, viruses and fungi in nearly all tissue and blood. most well-studied inflammatory conditions are tied to dysbiosis or imbalance of the human microbiome. while gut microbiome dysbiosis has been identified in me/cfs, microbes and viruses outside the gut can also contribute to the illness. pathobionts, and their associated proteins/metabolites, often control human metabolism and gene expression in a manner that pushes the body toward a state of illness. intracellular pathogens, including many associated with me/cfs, drive microbiome dysbiosis by directly interfering with human transcription, translation, and dna repair processes. molecular mimicry between host and pathogen proteins/metabolites further complicates this interference. other human pathogens disable mitochondria or dysregulate host nervous system signaling. antibodies and/or clonal t cells identified in patients with me/cfs are likely activated in response to these persistent microbiome pathogens. different human pathogens have evolved similar survival mechanisms to disable the host immune response and host metabolic pathways. the metabolic dysfunction driven by these organisms can result in similar clusters of inflammatory symptoms. me/cfs may be driven by this pathogen-induced dysfunction, with the nature of dysbiosis and symptom presentation varying based on a patient's unique infectious and environmental history. under such conditions, patients would benefit from treatments that support the human immune system in an effort to reverse the infectious disease process. toward the end of a career spent studying persistent bacteria in chronic disease, microbiologist gerald domingue wrote, "it is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious disease" ( ) . this thinking greatly applies to the illness me/cfs. me/cfs is characterized by neuroinflammation, severe fatigue, excessive post-exertional exhaustion, disturbed sleep, flu-like episodes, cognitive problems, sensory hypersensitivity, muscle and joint pain, headache, bowel symptoms, and severe impairment of daily functioning ( ) . severely ill individuals are often wheelchair dependent, bedridden, and unable to perform basic tasks of work or daily living. the history of me/cfs strongly suggests that infectious agents play a central role in driving the disease process. these include early associations with epstein barr virus (ebv)/human herpes virus (hhv ), the relapsing-remitting nature of me/cfs symptoms and antibodies/"autoantibodies" detected in patients with the disease ( , ) . a number of me/cfs outbreaks have also been reported, in which numerous people in the same geographical location developed the illness simultaneously ( ) . indeed, many me/cfs patients present with symptoms after suffering from a severe bacterial or viral infection. these infections often correlate with travel to a foreign country or exposure to pollutants or molds, suggesting that such pathogens take advantage of factors that compromise the host immune system. chronic inflammation is a hallmark of persistent infection. reports of cytokine activation in me/cfs clarify that the disease is associated with an inflammatory response ( ) . montoya et al. ( ) found me/cfs cytokine activation increased with disease severity, suggesting patients may struggle with a growing infectious burden over time. other me/cfs research teams have identified various forms of mitochondrial dysfunction in patients with the illness ( ) . there are now dozens of well-characterized mechanisms by which bacteria and viruses dysregulate mitochondrial metabolism ( , ) . many research teams have searched for well-characterized single pathogens in patients with me/cfs. these analyses often reveal elevated titers of igg and igm antibodies toward pathogens such as epstein barr virus, cytomegalovirus, parvovirus and m. pneumonia ( , , ) . several teams have also attempted to identify a single novel pathogen that might drive the entire me/cfs disease process. however, the discovery of the human microbiome now allows single microbes and viruses to be studied as members of complex communities. humans harbor these vast ecosystems of bacteria, viruses and fungi in nearly all tissue and blood ( ) ( ) ( ) ( ) . organisms in the microbiome continually interact with each other, and with the human genome, to regulate host metabolism and gene expression in both health and disease ( , ) . a growing number of inflammatory disease states, including neurological conditions and cancers, are tied to dysbiosis or imbalance of these human microbiome communities ( ) ( ) ( ) ( ) . gut microbiome dysbiosis has been identified in me/cfs ( ) . this dysbiosis is characterized by changes in microbe species composition and/or diversity. pathogens, or groups of pathogens, can promote dysbiosis by altering their gene expression in ways that promote virulence, immunosuppression and dysregulation of host genetic and metabolic pathways ( ) . when seemingly disparate biomedical findings on me/cfs are interpreted through the lens of these microbiome-based paradigms and platforms, a cohesive picture of the me/cfs disease process emerges. me/cfs may be driven by pathogeninduced dysfunction, with resulting microbiome dysbiosis varying based on a patient's unique infectious and environmental history. under such conditions, patients would benefit from treatments that, like those now being developed for cancer, support the human immune system in an effort to reverse the inflammatory disease process. in the usa, me/cfs cases were first formally reported to the cdc in the s ( ) . at the time, human microbes were typically only detected with culture-based laboratory methods. then, around the year , novel genome-based technologies began to revolutionize the field of microbiology ( , ) . these technologies identify microbes based on their dna or rna signatures rather than their ability to grow in the laboratory. the results of these genome-based analyses were remarkable: vast communities of microbes were identified in the human body that had been missed by the older culture-based techniques. these extensive ecosystems of bacteria, viruses, fungi, and archaea are collectively known as the human microbiome ( ) ( ) ( ) . today, so many novel microbes have been identified in homo sapiens that our human cells are equivalent to or even outnumbered by those of our microbial inhabitants ( ) . the tens of millions of unique genes harbored by this microbiome dwarf the ∼ , genes in the human genome ( , ) . for example, just one analysis of the human gut, skin, mouth, and vaginal microbiomes uncovered millions of previously unknown microbial genes ( ) . this has forced science to redefine the human condition. humans are best described as holobionts, in which the microbial genomes and the human genome continually interact to regulate metabolism and immunity ( , ) . early human microbiome studies characterized microbial ecosystems in the gut and on mucosal surfaces. however, the microbiome has now been shown to extend to nearly every human body site. these include the lungs, the bladder, the placenta, the testes, and the uterus [ ( , ( ) ( ) ( ) ( ) ]. jakobsen et al. ( ) found that previously sterile implants removed from joints, bones, pacemakers, and skulls of symptom-free patients were colonized by a range of bacterial and fungal organisms. another study demonstrated the presence of novel tissue specific bacterial dna profiles in a variety of mouse organs including the brain, heart, liver, muscle and adipose tissue ( ) . microbial communities also appear to persist in healthy human blood ( ) ( ) ( ) . a dna virome was recently identified in healthy human blood ( ) . another study reported both bacterial and fungal communities in the blood of healthy subjects. analysis of these organisms was performed by microbial resuscitation of blood culturing and microscopy in addition to next generation dna sequencing ( ) . whittle et al. ( ) recently characterized a human blood microbiome using a range of complementary molecular and classical molecular biology techniques. another study identified a larger amount of bacterial rdna in blood specimens from healthy individuals than in matched reagent controls ( ) . kowarsky et al. ( ) detected over , previously unidentified viruses, bacteria, and fungi in human blood samples obtained from immunocompromised patients. the study almost doubled the total number of anelloviruses found in humans. in order to classify many of these organisms the team was forced to add new branches to the "tree of life." they concluded that the newly discovered microbes "may prove to be the cause of acute or chronic diseases that, to date, have unknown etiology." the microbiome is inherited and evolves with the host. babies are seeded in the womb, during birth, and after birth by extensive microbiome communities in the placenta, the vaginal canal, and breast milk, among other body sites ( , ) . microbes/pathogens acquired from the external environment are further incorporated into the microbiome over time. for example, once acquired, cytomegalovirus persists as a member of the microbiomewith a significant impact on host immunity. brodin et al. ( ) found that the lifelong need for the body to control cmv causes approximately % of all t cells in cmv+ individuals to be directed against the virus. immune cells and associated microbes can travel between the human body and brain via several newly discovered pathways that bypass the classical blood-brain barrier ( ) . benias et al. ( ) documented a previously uncharacterized fluidfilled lattice of collagen bundles that appears to connect all human tissues. this human interstitium drains directly into the lymph nodes. two research teams have demonstrated the existence of a previously undiscovered meningeal lymphatic system ( , , ) . the network's fluid pathways connect the cerebrospinal fluid and cervical lymph nodes directly to the brain. while great progress has been made in characterizing the human microbiome, our understanding of the body's microbial ecosystems is still in its infancy. metagenomic analyses of the microbiome in all body sites regularly identify species or strains of bacteria, archaea, fungi, and/or viruses not previously understood to persist in homo sapiens ( ) . for example, just one study of the bladder microbiome identified previously unidentified viruses in subjects' urine samples ( ) . new strains of known microbes are also regularly identified. for example, as of august , the ncbi database contains ∼ , lactobacillus genomes, with newly characterized lactobacillus genomes added on a weekly basis ( ) . successful identification of these known or novel human organisms hinges on the careful choice of technology and methodology used for detection purposes ( ) . while the majority of human microbiome studies center on bacteria, awareness of viruses, fungi, and archaea has increased in recent years ( ) . for example, manuela et al. ( ) found an almost : ratio of archaeal to bacterial s rrna genes in human appendix and nose samples. identification of this archaeome abundance and diversity required use of a very specific archaea-targeting methodology. viruses are the most abundant life forms on the planet and in the human body, but have been relatively hard to detect until very recently ( ) . viruses that primarily infect bacteria, called bacteriophages, are particularly abundant in human microbiome communities. nguyen et al. ( ) estimate that ∼ billion bacteriophages traffic human tissue and blood on a daily basis. however, the human virome also harbors a plethora of human-associated viruses. newberry et al. ( ) are correct to assert that this human virome is understudied in me/cfs. paez-espino et al. ( ) at the joint genome institute have undertaken a large project called "uncovering the earth's virome." the project's goal is to better identify known and novel viruses in earth's ecosystems including the human body. viral identification requires the use of specific metagenomic tools, pipelines, and annotation platforms, with findings entered in the jgi img/vr database ( ) . the project is progressing at such a rapid pace that viral diversity in img/vr has more than tripled since august ( ), (figure ) . nevertheless, the vast majority of the gene content (over million genes in total) remains unknown or hypothetical. we must subsequently consider the possibility that asyet unidentified microorganisms may contribute to chronic inflammatory conditions like me/cfs. failure to do so would be akin to studying ∼ % of the animals in the rainforest and arriving at firm conclusions about the entire ecosystem based on that information alone. certain members of the human microbiome may also be difficult to detect based on their location and/or lifestyle. for example, chen et al. ( ) found that elevated cytokine expression in response to hsv-infected peripheral nerve ganglia persisted even when the virus entered a latent, non-replicating state. while it is important to pursue identification of novel organisms in me/cfs blood and tissue, ample data already exists on better-studied components of the microbiome. microbial and viral survival strategies, virulence mechanisms and collective behaviors are also characterized by a high degree of functional redundancy ( , ) . we must accept the complexity inherent to the human microbiome and further study these common mechanisms of survival and persistence. we must examine microbe and viral activity, microbe and viral gene expression, and the myriad ways in which the proteins and metabolites created by these organisms interact with the host immune system, the host genome, and each other. the genes of our microbial inhabitants greatly outnumber the ∼ , in the human genome. it follows that the majority of metabolites in homo sapiens are produced or modified by the microbiome. wikoff et al. ( ) found a large effect of the gut microbiome on murine blood metabolites including antioxidants, toxins and amino acids. for example, production of the metabolite indole- -propionic acid was completely dependent on the presence of gut microbes and could be established by colonization with the bacterium clostridium sporogenes. many microbes, viruses and their corresponding proteins/metabolites directly modulate the activity of host metabolic, immune, and neurological pathways. in other words, the human holobiont is controlled by the human genome, our microbial/viral genomes and their respective metabolites working in tandem. a growing number of studies provide examples of this metabolic overlap. while many such studies have been conducted in mice, their general trends carry over to humans. for example, me/cfs is associated with natural killer (nk) cell abnormalities, including reduced natural killer cell activity ( ) . these findings must be interpreted to account for the fact that nk activity is modulated by the bacterial microbiome. one study found that bile acids modified by the gut microbiome impacted liver cell gene expression in a manner that controlled nk cell accumulation and anti-tumor activity ( ) . similarly, a mixture of lactic acid bacteria from kefir increased the cytotoxicity of human nk khyg- cells to human chronic leukemia cells and colorectal tumor cells ( ) . the microbiome and its metabolites also impact the activity of related immune cells. rothhammer et al. ( ) found that tryptophan created by the gut microbiome interacted with the ahr receptor on microglia/astrocytes. subsequent changes in gene expression regulated communication between the two cell types. various forms of autonomic dysfunction are also common in me/cfs ( , ) . it is subsequently important to consider that the microbiome may contribute to host blood pressure regulation. pluznick et al. ( ) found that gut microbiomederived short chain fatty acids such as acetate and propionate travel to the kidneys and blood vessels. there they impacted activity of olfr and gpr , two host receptors that control circulation and blood flow. microbial modulation of host pathways can also drive inflammatory disease processes. pathogens and their associated proteins/metabolites control human metabolism and gene expression in a manner that can push the holobiont toward a state of imbalance and illness. for example, rizzo et al. ( ) found that human herpes viruses a/ b infected nk cells. this infection significantly modified expression of key host mirnas and transcription factors. mycobacterium leprae has been shown to alter human gene expression in a manner that allows it to hijack and reprogram adult schwann cells to a stem-like state ( ) . in a murine model of diabetes, liu et al. ( ) found that eltas, a protein created by s. aureus, prevented insulin from correctly binding its target receptor. this inhibited the phosphorylation of downstream signaling proteins and caused the mice in the study to develop impaired glucose tolerance. the ability of pathogens to interfere with host metabolism is tied to the dynamics of the communities in which they persist. like organisms in any ecosystem, human microbes constantly interact, both directly and indirectly. the proteins and metabolites they create are also in continual interplay. communities of microbes often exhibit synergistic interactions for improved nutrient acquisition, protection from host defenses, and survival in an inflammatory environment ( ) . these include biofilm formation and cooperative signaling via quorum sensing peptides. humphries et al. ( ) recently reported that biofilm bacteria can additionally communicate via ion channel-mediated electrical signaling. even viruses seldom act as single entities. diversity and equilibrium of the bacterial microbiome is regulated by bacteriophage predator-prey dynamics ( ) . pfeiffer and virgin ( ) found that enteric viral virulence is regulated by the activity of neighboring bacteria, fungi, and even helminths. these processes are called "transkingdom interactions." for example, human norovirus can bind carbohydrate histo-blood group antigens present on certain bacterial cells. this facilitates the ability of norovirus to infect human b cells ( ) . interacting microbes may contribute to dysbiosis or imbalance of microbiome communities ( ) . this dysbiosis is characterized by substantial shifts in community structure and diversity. in many cases, pathogens proliferate to inhabit niches once occupied by more innocuous microbes. most well-studied inflammatory disease states are tied to some form of microbiome dysbiosis. these include psoriatic arthritis, systemic lupus erythematosus, type and diabetes, parkinson's disease and a growing number of cancers ( , , ) . the gut microbiome can initiate and promote colorectal cancer at all stages of tumorigenesis by acting as an inducer of dna damage, generating epigenetic changes, regulating cell growth, and modulating host immune responses ( ) . the breast tissue microbiome of women with breast cancer has been shown to differ substantially in composition, virulence and diversity from that of healthy controls ( ) . species composition of the bronchoalveolar microbiome shifted toward a more pathogenic state in patients with sarcoidosis ( ) . alterations in the enteric virome were reported prior to disease onset in children susceptible to developing type diabetes ( ) . several research teams have tied me/cfs to bacterial gut microbiome dysbiosis. giloteaux et al. ( ) found that gut microbiome bacterial diversity was decreased in me/cfs subjects compared to heathy controls. the team also noted increases in certain bacterial species associated with either pro-inflammatory or anti-inflammatory activity. nagy-szakal et al. ( ) also analyzed the me/cfs gut microbiome. the study detected seven gut bacterial species whose relative abundance differed from that of control subjects and were strongly associated with me/cfs. while these findings are of interest, gut microbiome composition is additionally impacted by a host of environmental variables that cause large shifts in the region's microbial ecosystems. these include geographic location, food consumption, and even time of day ( ) . many research teams studying inflammatory conditions have struggled to isolate and/or replicate disease-induced gut microbiome dysbiosis in the face of this "noise." for example, frémont et al. ( ) found that intestinal microbiome species composition differed between patients with me/cfs and healthy controls. however, significant changes in intestinal microbiome composition were also identified between control subjects from norway and controls subjects from belgium. this variation was proposed to arise from differences in diet between the two cultures. studies of the me/cfs blood microbiome may be less subject to this environment-induced variability. furthermore, identification of microbial and/or viral communities in me/cfs blood would allow for a broader picture of possible infectious and inflammatory processes. for example, giloteaux et al. ( ) found that me/cfs subjects had higher levels of bacterial lipopolysaccharides (lps), lps-binding proteins and soluble cd in blood. the team suggested that these inflammatory markers may indicate translocation of gut bacteria into the blood. however, the markers could also reflect the presence of bacteria in the blood itself. the blood microbiome can be characterized if the microbial dna/rna in samples is first separated from that derived from the human genome. olde loohuis et al. ( ) used rna sequencing of reads from whole blood to analyze microbial communities in the blood of almost patients with three neurological conditions: bipolar disorder, schizophrenia, and amyoytrophic lateral sclerosis. the team identified a wide range of bacterial and archaeal phyla in subjects with all three disease states. they observed increased microbial diversity in schizophrenia subjects compared to the two other groups, and replicated the finding in an independent dataset. stephen quake's cell-free dna shotgun sequencing technologies can also characterize bacterial communities in human blood, and can be additionally extended to identify viruses and fungi ( ) . communities of microbes and viruses may also persist in me/cfs brain tissue. readhead et al. ( ) recently detected a range of persistent viruses in the alzheimer's brain. these included herpesviruses, torque teno viruses, adenoviruses, and coronaviruses. the alzheimer's brain has also been shown to harbor bacterial and fungal communities ( , ) . branton et al. ( ) identified hundreds of bacteria and bacteriophage-derived samples in brain tissue removed from patients with epilepsy, and in brain samples obtained from hiv/aids patients after autopsy. in fact, studies of the virome provide significantly extended context on microbiome community dynamics and disease processes. this is because bacteriophages (phages) infect, and subsequently modulate the activity of the bacterial microbiome ( ) . for example, duerkop et al. ( ) characterized the intestinal virome in a model of t-cell-mediated murine colitis. the intestinal phage population changed in colitis, and transitioned from an ordered state to a stochastic dysbiosis. phage populations that expanded during colitis were frequently connected to bacterial hosts that benefit from or are linked to intestinal inflammation. tetz et al. ( ) identified changes in the parkinson's gut bacteriophage community. these included shifts in the phage/bacteria ratio of bacteria known to produce dopamine. species-level studies of the microbiome are also greatly enhanced by analyses that provide further context on disease activity. this is an important consideration because, in theory, the microbiome of a patient with me/cfs could harbor the exact same microbial, viral and/or fungal species as that of a healthy subject. yet many of these organisms could be acting very differently in patients with the disease. species-level analyses of the me/cfs microbiome must subsequently be accompanied by studies that characterize microbial and viral gene expression and/or metabolism. since many of the proteins and metabolites in the human holobiont are microbial in origin, composition of the me/cfs proteome and metabolome change with microbiome species composition. proteome and metabolome analyses additionally reflect microbiome activity. this is because microbes and viruses frequently alter their gene expression in ways that cause them to express different proteins and metabolites over time. the human genome and related epigenetic changes also contribute to the metabolic diversity, although the high level of redundancy between human and microbial metabolites can make the origin of these associations hard to pinpoint. schutzer et al. ( ) demonstrated that the me/cfs cerebrospinal proteome differs substantially from that of healthy controls (figure ) . indeed, of , identified proteins ( . %) were unique to patients with me/cfs, providing strong evidence that me/cfs is indeed characterized by microbiome dysbiosis in tissue and blood. composition of the blood metabolome has also been shown to shift in me/cfs. one such study reported elevated plasma levels of choline, carnitine, and complex lipid metabolites in me/cfs patients ( ) . another analysis demonstrated a sustained hypo-metabolic response in patients with the disease ( ) . this dour-like state can be driven by exposure to adverse environmental conditions, as would be expected if the me/cfs immune system struggles to manage microbiome dysbiosis and associated pathogens. studies of the metabolome are often conducted in an attempt to identify disease-specific biomarkers. however, the metabolome can also be screened for metabolites that directly induce or suppress biological function in patients with a given illness. these studies help dissociate cause from effect and allow for possible modulation of disease phenotype. for example, johnson et al. ( ) investigated the metabolic influence of microbial biofilms on colon cancer tissue and related cancer occurrence. they found that up-regulation of a biofilm-derived polyamide metabolite enhanced both biofilm formation and cancer growth. studies of microbiome activity must account for the fact that pathogens detected in patients with me/cfs are also regularly identified in healthy subjects or in patients with related inflammatory conditions. this is particularly true of studies that have searched for ebv, hhv , cytomegalovirus, and other viruses able to be identified by pcr and/or antibody testing in me/cfs cohorts. this same trend is likely to hold for lessstudied or unidentified human microbes and viruses. while these "overlapping" results are often viewed as problematic, they make sense in light of research that clarifies how differently microbes act depending on host immune status, neighboring species, and a wide range of other variables. for example, susceptibility to hiv infection has been shown to vary based on the species composition and activity of the bacterial vaginal microbiome ( ) . indeed, most human microbes are pathobionts: they can change their gene expression to act as pathogens under conditions of imbalance and immunosuppression ( ) ( ) ( ) . for example, s. pneumoniae can persist as a highly adapted commensal or a virulent pathogen depending on its ability to evade the host immune response ( ) . s. aureus causes a range of illnesses, from skin infections to life-threatening diseases such as endocarditis and meningitis. however, ∼ % of the healthy human population harbors s. aureus as a member of the normal nasal microbiome ( ) . s. aureus virulence in these communities is determined by a number of factors, including the signaling and competitive strategies employed by neighboring microbes. the same is true of escherichia coli (e. coli), which also persists in numerous forms. one study found that "commensal" e. coli could evolve into virulent clones in fewer than generations ( ) . for most microbes, this evolution toward pathogenicity occurs via the acquisition of new genes or alteration of the current genome in a manner that induces gene loss ( ) . for example, loss of muca increases the ability of pseudomonas aeruginosa to evade phagocytosis and resist pulmonary clearance ( ) . it should also be noted that every microbial species represents many different strains, each of which may vary in the set of genes it encodes or in the copy number of such genes. this intra-species variation endows each strain with distinct functional capacities, including differences in virulence, motility, nutrient utilization, and drug resistance ( ). greenblum et al. ( ) identified extensive strainlevel copy-number variation across species in metagenomic samples obtained from patients with irritable bowel syndrome. this was especially true of genes tied to specific community functions, including functions related to community lifestyle. differences in gene copy-number also impacted adaptive functions linked to obesity. yao et al. ( ) found that deletion of a single bacteriodes gene-and the bile salt hydrolase it expresses-altered host metabolism in a manner that impacted weight management, circadian rhythm and immunity. a microbe or a virus' location can also influence its activity. for example, much of the human population harbors hhv- . however, in alzheimer's disease, hhv- a was recently identified in human brain tissue ( ) . there, its activity was shown capable of regulating host molecular, clinical, and neuropathological networks in a manner that can contribute to inflammation and neuronal loss. vanelzakker ( ) has proposed that hhv- may also infect the vagus nerve in me/cfs, resulting in altered gutbrain axis signaling in patients with the illness. the human immune system also plays a central role in determining microbe and viral activity. a robust immune response is often capable of controlling pathogen virulence. however, if pathogens overcome the immune response, or the immune system is suppressed by medications, chemicals, or other environmental factors, pathobionts are more likely to alter their gene expression in a manner that promotes disease. pathobionts can subvert the human immune response by collectively altering their gene expression. yost et al. ( ) performed an excellent gene ontology (go) enrichment analysis of the oral microbiome during periodontal progression. over the two-month study period, changes in the metagenome of non-progressing sites were minor. however, active sites that progressed to periodontitis were characterized by numerous functional genomic signatures. in fact, the team reported a complete rearrangement at the metagenome level between baseline sites that progressed to periodontitis and those that did not. go terms associated with processes including peptidoglycan biosynthesis and potassium transport were highly enriched at baseline sites that later progressed to periodontitis. genes controlling ciliary motility and crispr-associated proteins were also active during initial stages of disease progression. at the breakdown point, active sites expressed genes associated with ferrous iron transport and response to oxidative stress. progression to periodontitis was also correlated with increased expression of putative virulence factors associated with a range of bacterial species. mycoplasma, bacteriophage, and eukaryotic viral activity were higher in progressing sites compared to baseline samples. the team concluded that periodontitis progression is driven by the whole oral microbial community and not just a few select pathogens. in effect, under conditions of increasing inflammation and imbalance, the entire oral community appeared to act together as a pathogen. indeed, groups of bacteria not generally considered pathogens upregulated a large number of the putative virulence factors in active sites. these included veillonella parvula, a microbe almost always associated with dental health. community-wide shifts in microbiome virulence are often driven by dominant pathogens-organisms that become established as central components of the microbiome while suppressing commensal growth and activity ( ) . in other cases, keystone pathogens promote inflammation even when present as quantitatively minor members of the microbiome. for example, p. gingivalis often comprises just . % of periodontal biofilms, yet drives destructive changes in host-microbe interplay by profoundly impairing the innate immune response ( ) . pathogens able to persist inside the cells of the immune system are uniquely positioned to drive inflammatory disease ( ) . indeed, most well-characterized pathogens, including many connected to me/cfs, are capable of intracellular persistence ( ) . by surviving in this fashion, they can directly interfere with human transcription, translation, and dna repair processes (figure ) . pathogens in the cell cytoplasm may further dysregulate the epigenetic environment ( ) . for example, upon infecting a macrophage, mycobacterium tuberculosis alters the expression of human genes ( ) . h. pylori infection predisposes to genomic instability and dna damage, including double strand breaks ( ) . ebv infection of b cells can also promote persistent damage to human dna ( ) . the thousands of metabolites and proteins expressed by intracellular pathogens also interact with the host genome, further modifying human gene expression in a manner that promotes disease. even bacterial quorum sensing peptides can dysregulate human pathway activity. wynendaele et al. ( ) found that quorum sensing molecules created by gramnegative bacteria altered human gene expression in a manner that promoted in vitro angiogeneisis, tumor growth, and neovascularization in colon cancer. intracellular pathogens can also travel between cells via recently characterized tunneling nanotubules (tnts) ( , ) . these cytoplasmic extensions of dendritic cells, glial cells and related human cells allow for the intracellular transfer of micrornas, messenger rnas, prions, viruses, and even whole organelles such as mitochondria ( , ) . for example, hivinduced tunneling nanotubule formation appears to mediate approximately half of hiv virus spread among monocytederived macrophages ( ). dysfunction driven by intracellular infection is compounded by the fact that microbial proteins and metabolites are often identical or similar in structure to those created by their human hosts. the molecular mimicry or sequence homology between these proteins and metabolites makes it increasingly difficult for the human holobiont to recognize "foreign" from "self." for example, altindis et al. ( ) found that viruses carry sequences with significant homology to human insulin-like growth factors (vilps). these vilps can bind human and murine igf- receptors in vitro, resulting in autophosphorylation and downstream signaling. e. coli harbors a large, diverse network of proteins that actively promote endogenous dna damage in cells ( ) . however, at least of these dna-damaging proteins have human homologs that also promote dna damage and mutagenesis in the human host. indeed, redundancy between human and microbial metabolites, proteins, and pathways is so great that the potential for molecular mimicry to contribute to host immune and metabolic dysfunction is semi-infinite. for example, viral vesicles and human extracellular vesicles (evs) share considerable structural and functional similarity ( ) . these similarities are so extensive that it is difficult to distinguish evs from (noninfectious) viruses. many pathogens employ common survival mechanisms to persist in host cells, tissue and blood. the metabolic dysfunction driven by these different microbes and viruses can result in similar clusters of human inflammatory symptoms. the ability of various pathogens to dysregulate activity of the vitamin d nuclear receptor (vdr) is an excellent example of how different microbes can drive similar disease processes. the vdr regulates expression of hundreds of human genes, many of which regulate inflammatory and malignant processes ( , ) . the receptor also controls signaling of tlr and several families of antimicrobial peptides including cathelicidin (ll- ) ( ) . pathogens capable of slowing vdr activity can subsequently facilitate their survival by slowing the innate immune response. pathogens frequently linked to me/cfs or inflammatory disease have evolved to survive in this fashion. vdr activity is downregulated as much as times in epstein barr virus-infected lymphoblastoid cell lines ( ) (figure ) . hiv, m. tuberculosis cytomegalovirus, borrelia burgdorferi and mycobacterium leprae additionally dysregulate vdr activity to various degrees ( , ( ) ( ) ( ) ( ) . the fungus aspergillus fumigatus secretes a gliotoxin that significantly downregulates vdr expression ( ) . because disabling the innate immune system via the vdr pathway is such a logical survival mechanism, other uncharacterized bacteria, viruses or fungi may have also evolved to dysregulate receptor activity. it follows that me/cfs patients with similar symptoms may not always test positive for the same mix of pathogens and/or communities of pathobionts. similarly, composition of the me/cfs microbiome, proteome and metabolome should be expected to differ somewhat from study to study. instead of worrying about these inconsistencies, the me/cfs research community should strive to better characterize even more common mechanisms of pathogen survival and persistence. the ability of pathogens to disable the host immune response extends far beyond the vdr pathway. pushalkar et al. ( ) identified a distinct and abundant pancreatic microbiome associated with progressive pancreatic cancer. this dysbiotic microbiome drove oncogenesis by suppressing macrophage differentiation and t cell activity. another study found that candida albicans's transition from extracellular to intracellular pathogen was accompanied by a coordinated, time-dependent shift in gene expression for both host and fungus ( ) . these gene expression changes led to a gradual decline in proinflammatory cytokine activation by the host immune system. these findings shed light on a recent me/cfs study. hornig et al. ( ) reported distinct alterations in plasma immune signatures-including prominent activation of both pro-and anti-inflammatory cytokines-early in the course of me/cfs. however, these alterations were not observed in subjects with a longer duration of illness. there are a number of explanations for hornig's observations. me/cfs-associated pathogens may gradually alter their gene activity to persist in more latent, intracellular forms less recognized by the host immune system. or, in early-stage me/cfs, the immune system may actively attempt to target a growing infectious burden. over time however, pathogens in the microbiome may disable the immune response to a point where "immune exhaustion" occurs ( , ) . immunopathology and cytokine production would subsequently drop. the resulting disease state could be compared to a garden, in which healthy plants become progressively stifled by others, such as kudzu vine. many research teams are studying how "acute" pathogens (i.e., well-characterized agents associated with known infectious diseases) can cause chronic symptoms by persisting in latent forms. these include zika, borrelia burgdorferi, influenza, and other well-characterized viruses and bacteria ( ) . zika was shown capable of persisting in the cerebrospinal fluid (csf) and lymph nodes of infected rhesus monkeys for months after the virus had been cleared from mucosal secretions, peripheral blood and urine ( ) . viral persistence in both the lymph nodes and csf was correlated with upregulation of genes involved in pro-inflammatory and anti-apoptotic pathways. tens of thousands of ebola survivors have developed chronic symptoms months or years after initial infection ( ) . these "post-ebola syndrome" symptoms include extreme fatigue, severe pain, eye problems, and a host of neurological issues. while the virus is hard to identify in the blood of such patients, ebola has been detected in men's semen years after "recovery" ( ) . another study found that, up to a month after initial infection, influenza viruses regulated the long-term expression of inflammatory and neuron/glia-specific genes in mice ( ) . this was associated with chronic neuroinflammation characterized by an increase in the number of activated microglia and impairment of spatial memory formation. persistent measles virus is associated with conditions such as paget's disease, multiple sclerosis and crohn's disease ( , ) . measles virus rna has been detected in blood, respiratory secretions, urine, and lymphoid tissue for weeks to months after clearance of the "acute" infection ( ) . doi et al. ( ) found that, in vitro, measles virus persistence correlated with viral transition from a lytic to non-lytic mode that allowed the virus to evade the host innate immune response. hundreds of studies demonstrate that acute bacterial pathogens can transition into latent forms that lack a classical cell wall ( ) . these persistent variants are referred to as l-form bacteria. antibiotic use can induce persistent l-form growth. in fact, researchers create l-forms by deliberately culturing classical bacteria in conjunction with the beta-lactam antibiotics ( ) . one microarray analysis of l-form growth revealed upregulation of genes shared in common with persister cells and biofilms ( ) . l-form bacteria have been implicated in dozens of chronic conditions including rheumatoid arthritis, multiple sclerosis and sarcoidosis ( , , ) . a better understanding of these chronic sequelae would greatly benefit the me/cfs community, since a similar chronic progression of symptoms is frequently documented in me/cfs. at different points in history, me/cfs has been called "post-polio syndrome, " "chronic mononucleosis syndrome" and "post-viral syndrome" due to the fact that chronic symptoms are often noted after acute infection with polio virus, epstein barr virus, influenza or a range of other pathogens ( ) ( ) ( ) . chia et al. ( ) found that patients with acute enterovirus infection went on to develop a multitude of chronic symptoms consistent with an me/cfs diagnosis. years after the initial infection, enterovirus protein and rna were still present in these patients' stomach biopsies. it is clear that novel methodologies may be required to best identify pathogens in their latent forms. pathogens that persist inside human immune cells and associated tunneling nanotubuoles have been particularly hard to detect. when persistent zika virus was identified in rhesus monkeys, zikaspecific antibodies were not detected in the csf, despite prolonged and robust responses in peripheral blood. this suggested an additional mechanism for viral persistence in certain "anotomic sanctuaries." modern living has also increased the likelihood that "acute" infections may generate chronic sequelae. before the advent of antibiotics, steroid medications and childhood vaccines, almost half of all children under the age of five died from acute infectious disease ( ) . today, most individuals in first-world countries survive repeated acute infections over the course of decades. while certain dominant or keystone pathogens may be reliably identified in patients with me/cfs, composition of the me/cfs microbiome will likely differ between patients. even in hiv/aids, where an easily detected virus dysregulates immunity, disease symptoms reflect a mix of those driven by hiv, and those driven by "co-infectious" agents able to take advantage of the immunocompromised host ( ) . no two patients with hiv/aids are expected to harbor the same mix of these additional persistent bacteria, fungi, and viruses. the same is true of most cancers, an increasing number of which are now tied to severe microbiome dysbiosis ( ) . it is widely accepted that no two cancers are alike, and any tumor-associated microbiome is expected to differ somewhat among individual study subjects ( ) . this same pattern, in which unique infectious history impacts symptom presentation may also occur in me/cfs. a recent study by brodin et al. ( ) demonstrated the profound impact of infectious history on host immunity. the team performed a systems-level analysis of healthy twins between the ages of and . they measured immune parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that % of these are dominated, and % almost completely determined, by non-heritable environmental influences. many of these parameters became more variable with age, emphasizing the cumulative influence of environmental exposure. the team also calculated how acquisition of just one chronic pathogen-cytomegalovirus (cmv)-conditions the immune response. identical twins discordant for cmv infection showed greatly reduced correlations for many immune cell frequencies, cell signaling responses, and cytokine concentrations. in general, the influence of cmv was so broad that it affected of the measurements dispersed throughout the immune network. these and related findings led brodin and team to conclude that the immune response is "very much shaped by the environment and most likely by the many different microbes an individual encounters in their lifetime." the above suggests that me/cfs may be driven by a process we have termed "successive infection." during successive infection, an "acute" infection or "initial immunosuppressive event" dysregulates the host immune system. this makes it easier for microbes to subvert the immune response by acting as polymicrobial entities. pathobionts alter their gene expression to better promote community-wide virulence. infected human cells fail to correctly express human metabolites in the presence of pathogen-generated proteins, metabolites, and enzymes. dysfunction driven by molecular mimicry increases. certain pathogens may infect mitochondria or central nervous system tissue. intracellular pathogens slow the human immune response, causing the host to more easily acquire other infectious agents. this creates a snowball effect in which the microbiome becomes increasingly dysbiotic as the strength of the immune response weakens over time. eventually, the human host may present with symptoms characteristic of me/cfs or a related inflammatory diagnosis. the unique symptoms any one patient develops are expected to vary based on the species, strain, virulence, and location of the pathogens driving dysbiosis, along with the myriad ways in which the metabolites and proteins created by these organisms cause dysfunction by interacting with those of the host. early childhood infections may predispose to dysbiosis at a later date ( ) . for example, measles depletes host b and t lymphocytes for up to - years after initial infection. this immunosuppression can reset previously acquired immunity and renders the host more susceptible to other pathogens ( ) . in other cases, a toxic environmental exposure, infection during surgery or the difficulty of enduring a traumatic event may weaken the immune response to a point where previously subclinical infections become active. me/cfs outbreaks, in which numerous patients developed the illness at relatively the same time, may well represent this phenomenon at work. the successive infectious process may even begin in the womb. depending on the health of the parent, founding microbiome communities in the placenta, vagina and breast milk may already be dysbiotic. cabrera-rubio et al. ( ) found that the breast milk microbiome of obese mothers tended to contain a different and less diverse bacterial community then that of normal-weight mothers. pathogens in the placental microbiome can alter methylation of human dna in a manner that may negatively impact the later life disease of premature babies ( ) . many aspects of modern living can additionally drive successive infection. antibiotic use disrupts the ecology of the human microbiome ( ) . antibiotic resistance genes from farm animals and produce are regularly transferred into the human food supply ( ) . electromagnetic radiation has been shown to lower immunity ( ) . the immunosuppressive biologics and supplements often prescribed for inflammatory disease further allow pathobionts in the microbiome to proliferate. for example, diaz et al. ( ) found that the salivary bacteriome of patients taking immunosuppressive biologics was more permissive to the growth of oral opportunistic pathogens. modern society also exposes the average person to pathogens our recent ancestors were unlikely to encounter. international airports harbor pathogens from across the globe ( ) . food products, and the microbes they contain, are frequently imported from foreign destinations ( ) . one study found a range of opportunistic pathogens enriched in showerhead biofilms ( ) . numerous pathogens were identified in commonly smoked cigarettes ( ) . many such pathogens are capable of persisting in human microbiome ecosystems, where the host may lack immunity toward their presence. me/cfs is a spectrum disorder with a diagnostic criterion that includes a range of physical and neurological symptoms ( ) . if successive infection contributes to me/cfs, this variability in symptom presentation is expected. furthermore, factoring "unique infectious history" into the disease process helps explain why patients with me/cfs often suffer from a multitude of symptoms not included in the official diagnostic criteria. because patients with me/cfs suffer from such diverse symptoms, it has been argued that they should be grouped into separately studied "subgroups" ( ) . in some cases this makes sense. for example, studies that distinguish earlystage/late-stage me/cfs patients may further elucidate how the immune response is modified by the microbiome over time. however, if successive infection contributes to me/cfs, future research should also focus on better understanding the common pathogenesis shared by all subjects. indeed, successive infection may contribute to other conditions tied to microbiome dysbiosis, persistent infection, and adverse environmental exposure. me/cfs should be studied in concert with these other conditions, which include gulf war syndrome, ehlers-danlos syndrome, chronic lyme disease, and fibromyalgia among others ( ) ( ) ( ) ( ) ( ) . the high levels of comorbidity and symptom overlap between patients with me/cfs and these related inflammatory diagnoses strengthens this assumption. a number of autoantibodies have been detected in patients with me/cfs ( ). this has led some research teams to postulate that me/cfs should be regarded as an "autoimmune" disorder ( ) . however, the classical "theory of autoimmunity" is in the process of being re-evaluated ( , ) . increasing evidence suggests that "autoantibodies" are actually created in response to chronic, persistent microbiome pathogens. under such conditions molecular mimicry or structural homology between pathogen and host proteins can result in "collateral damage" toward human tissue. for example, vojdani et al. ( ) found that different alzheimer's-associated pathogens or their molecules could react with antibodies against amyloid beta via molecular mimicry or the binding of bacterial toxins to amyloid beta. a growing body of research documents "autoantibody" production in response to a range of bacterial, viral and fungal pathogens/pathobionts. these pathogens are not shortterm "triggers" but persist as members of complex microbiome communities. for example, "autoantibody" production was recently tied to microbiome pathobiont enterococcus gallinarum. manfredo et al. ( ) detected e. galinarum in the mesenteric veins, lymph nodes, spleens and livers of mice made genetically prone to autoimmunity. in these mice, the bacterium initiated the production of "autoantibodies, " inflammation and activated t cells. however, this "autoantibody" production stopped when e. gallinarum's growth was suppressed with the antibiotic vancomycin or with an intramuscular vaccine. in addition, e. gallinarum-specific dna was recovered from liver biopsies of human autoimmune patients, and co-cultures with human hepatocytes replicated the murine findings. indeed, many pathogens can drive the activated or clonal t cell expansion often associated with "autoimmune" conditions. for example, tuffs et al. ( ) found that s. aureus endotoxins triggered uncontrolled activation of t cells. this led to a proinflammatory cytokine storm that accounted for both t cell activation and related inflammation. microbes and their metabolites control bidirectional signaling between the gut and the brain via pathways collectively known as the gut-brain axis ( ) . the gut-brain axis involves various afferent and efferent pathways including the vagus nerve, with signaling impacting neural, endocrine, and immune processes. the gut's enteric nervous system contains over million neurons-more than in either the peripheral nervous system or spinal cord ( ) . it follows that gut microbiome dysbiosis may modulate brain activity. for example, sampson et al. ( ) transplanted fecal samples from patients with parkinson's disease into germ-free mice. these mice, and not controls, exhibited physical symptoms associated with parkinson's. however, there is also growing evidence that humans may harbor a brain microbiome. researchers at harvard university are characterizing this ecosystem as part of the ongoing "brain microbiome project." in what marks a major paradigm shift, multiple research teams have shown that both amyloid beta and prion protein (prp) are potent antimicrobial peptides ( , ) . amyloid beta exhibited antimicrobial activity against a range of common microorganisms with a potency equivalent to, and in some cases greater than, cathelicidin (ll- ) ( ). these pathogens included s. typhimurium, candida albicans and, more recently, a number of herpesviruses capable of persisting in the alzheimer's brain ( , ) . the findings strongly suggest that amyloid beta and prp are not useless byproducts of abnormal brain catabolism. rather, they appear to form a mediated response of the innate immune system toward infectious agents in brain tissue ( ) . a number of brain abnormalities have been reported in patents with me/cfs ( ) . these findings must be interpreted in light of these novel infection-based paradigms and in concert with emerging data on the brain microbiome. in a study of aortic aneurysms, gottlieb et al. ( ) reported that bak snp-containing alleles were detected in aortic tissue but not in blood samples from the same patients. more recently ursini et al. ( ) found that schizophrenia gene risk loci that interact with early-life complications are highly expressed in the placenta. however, these loci were differentially expressed in placentas from women who suffered complications during pregnancy. they were also differentially upregulated in placentae from male compared with female offspring. these and related findings strongly suggest that the environment can select for human genome activity. for example, harley et al. ( ) found that in ebv infected cells, ebna and its transcription factors modulated the activity of human genes associated with risk for multiple sclerosis, rheumatoid arthritis, type diabetes and other conditions. in fact, nearly half of systemic lupus erythematosus risk loci were occupied by ebna and co-clustering human transcription factors. studies of the human genome must also account for the full extent of microbial dna and rna in human tissue and blood. if a genomic assembler fails to account for this contamination, chances of a false positive single nucleotide polymorphism (snp) increase significantly during analysis ( ) . contamination with even a small amount of microbial dna/rna-just one or two base pairs of difference-is enough to cause significant statistical errors in this fashion. if me/cfs is driven by successive infection, treatments that support or activate the human immune system could improve microbiome health by allowing patients to better target persistent pathogens. development of such therapies should be a priority for the me/cfs research community. however, most immunostimulative treatments that target pathogens are characterized by immunopathology-a cascade of reactions in which inflammation, cytokine release and endotoxin release are generated as part of the immune system's response to microbial death ( ) ( ) ( ) . the death of intracellular pathogens is particularly difficult for the host to manage, as the body must deal with debris generated from apoptosis. inflammation is also generated in response to bacterial cell wall components including the endotoxin lipopolysaccharide of gram-negative strains ( ) . luckily, immunopathology-generated symptoms are generally temporary in nature, and tend to subside as an increasing number of pathogens are eradicated. temporary immunopathology resulting from antimicrobial treatment has been documented for over a century, with symptoms varying depending on the nature of targeted pathogens. first referred to as the jarisch-herxheimer reaction, the phenomenon was originally observed during treatment of syphilis with mercury and penicillin ( , ) . the jarisch-herxheimer reaction/immunopathology has since been noted in a broad spectrum of chronic inflammatory conditions including tuberculosis and brucellosis ( ) . short-term immunopathology is also a central feature of acute infection. if a patient develops the flu, inflammatory symptoms increase as the immune system releases cytokines and chemokines in response to the infecting virus. hiv/aids patients undergo a form of immunopathology called immune reconstitution inflammatory syndrome (iris) following treatment with combination antiretroviral therapy (art) ( ) . iris occurs as art enables the host immune system to better target pathogens acquired during previous periods of hiv-driven immunosuppression. a range of well-characterized pathogens have been linked to iris including the herpesviruses and m. tuberculosis ( ) . however, the inflammatory reaction is also noted in culture-negative patients, suggesting iris may also involve novel or uncharacterized pathogens ( ) . over the past decade, in concert with our clinical collaborators, we developed an immunostimulative therapy used to treat patients with a range of chronic inflammatory conditions ( ) . treatment centers on the use of a putative vdr agonist in the form of olmesartan medoxomil, with the goal of reactivating components of innate immunity under vdr control. in , we published a series of case histories demonstrating improvement in me/cfs patients administered this treatment ( ) . however, all me/cfs subjects administered the therapy experienced immunopathology and associated inflammatory symptom increases that lasted for many years. as a general trend, patients administered the treatment during earlier stages of disease experienced less immunopathology, emphasizing the need for immunostimulative therapies to be used in a predictive and even preventative fashion. while some me/cfs physicians may feel uneasy about the suffering induced by immunopathology, other research communities have become accustomed to treatments that cause temporary discomfort. novel cancer immunotherapies also generate immunopathology by activating patient t cells. this allows the immune system to better target malignant tumors ( , ) . the resulting "cytokine release syndrome" leads to profound, temporary, symptom increases. however, these increased symptoms are considered acceptable, as patients who endure the reaction are more likely to enter a state of remission. since many forms of cancer are tied to severe microbiome dysbiosis, at least part of the "cytokine release syndrome" may result from the death of persistent pathogens. the history of me/cfs strongly suggests that infectious agents play a central role in driving the disease process. however, the discovery of the human microbiome has revolutionized the manner in which persistent infection and chronic inflammation are understood and studied. humans harbor extensive microbiome communities of bacteria, viruses, and fungi in nearly all tissue and blood. the hundreds of millions of unique genes harbored by this microbiome dwarf the ∼ , in the human genome. humans are best described as holobionts, in which these microbial genomes and the human genome continually interact to regulate host gene expression, metabolism and immunity. many inflammatory disease states, including neurological conditions and cancers, are tied to dysbiosis or imbalance of human microbiome communities in various body sites. while gut microbiome dysbiosis has already been identified in me/cfs, distinct microbial and viral communities may additionally persist in me/cfs blood and brain tissue. possible identification of these microbiomes should be a priority for the me/cfs research community, but analysis requires the use of very specific technologies and methodologies. pathogens and their associated proteins/metabolites control human metabolism and gene expression in a manner that can push the human holobiont toward a state of illness. studies of microbiome dysbiosis in me/cfs must consider this microbe and viral activity. most human microbes can alter their gene expression to act as pathogens under conditions of imbalance and immunosuppression. this pathobiont behavior is further determined by the activity and virulence of neighboring microbes. patients with me/cfs may harbor many of the same microbes and viruses as heathy individuals, yet these pathobionts may act with increased virulence in patients with the illness. intracellular pathogens, including several associated with me/cfs, have been shown to directly interfere with human transcription, translation, and dna repair processes. molecular mimicry between host and pathogen proteins/metabolites further exacerbates this interference. interacting microbes can also drive disease by changing their collective gene expression. other pathogens disable mitochondria, or may infect central nervous system tissue in ways that dysregulate signaling via the gutbrain axis. antibodies and/or clonal t cells identified in patients with me/cfs are likely activated in response to many of these persistent microbiome pathogens. different pathogens have evolved similar survival mechanisms to disable the host immune response and/or host metabolic pathways. the metabolic dysfunction driven by these different microbes can result in similar clusters of inflammatory symptoms. me/cfs may be driven by this pathogen-induced dysfunction, with the nature of dysbiosis and symptom presentation varying based on a patient's unique infectious and environmental history. an initial infection or environmental exposure weakens the host immune system. this makes it easier for pathobionts to subvert the immune response and interfere with host gene expression and metabolism. a snowball effect begins, in which the microbiome becomes increasingly dysbiotic as the strength of the immune response weakens over time. the unique symptoms any one me/cfs patient develops are expected to vary depending on the location, species, strain and virulence of the pathogens driving this dysbiosis. thus, while certain dominant or keystone pathogens may be identified in me/cfs, composition of the me/cfs microbiome, metabolome, and proteome should be expected to differ somewhat among individual patients. these common mechanisms suggest that me/cfs is best studied in concert with other chronic conditions tied to microbiome dysbiosis, persistent infection and adverse environmental exposure. these include fibromyalgia and gulf war syndrome, but also conditions like post-ebola syndrome in which severe chronic symptoms develop after infection with an "acute" infectious agent that is able to persist in latent forms. treatments that support or activate the human immune system could allow me/cfs patients to improve microbiome health by better targeting pathogens over time. like the novel immunotherapies being developed for cancers, these immunostimulative therapies would be expected to generate temporary immunopathology. institutional reviewers hesitant to approve immunopathology-based therapies should consider that me/cfs quality of life is typically very low, with patients demonstrating a substantial increase in mortality from suicide ( ) . it often takes patients years to receive a diagnosis of me/cfs. this delay wastes a valuable period during which the immune system is most responsive to immunostimulatory treatment. patients treated during earlier stage disease are also less likely to experience severe or long-lasting immunopathology. this suggests that immunostimulative therapies should be administered in a predictive and even preventative fashion. in addition, interventions or treatments that might help patients better manage the byproducts of immunopathology (bacterial lps etc.) should become a priority for the research 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infection: a systematic review and meta-analysis immunostimulation in the treatment for chronic fatigue syndrome/myalgic encephalomyelitis immunostimulation in the era of the metagenome cytokine release syndrome mortality of people with chronic fatigue syndrome: a retrospective cohort study in england and wales from the south london and maudsley nhs foundation trust biomedical research centre (slam brc) clinical record interactive search (cris) register the authors would like to acknowledge michael eason kirkpatrick and janet raty for their help with graphic design. they also thank sara nicole azevedo for her help with technical editing of the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © proal and marshall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - xwcxe l authors: di altobrando, ambra; patrizi, annalisa; bardazzi, federico title: should sars‐cov‐ influence immunosuppressive therapy for autoimmune blistering diseases? date: - - journal: j eur acad dermatol venereol doi: . /jdv. sha: doc_id: cord_uid: xwcxe l in this dramatic period where the whole world is affected by the outbreak of coronavirus disease (covid‐ ), scientific data relating to the causative virus sars‐cov‐ as well as the subsequent therapeutic repercussions on the management of other diseases should be divulged in order to share as much information as possible among experts in a timely manner. subsequent therapeutic repercussions on the management of other diseases should be divulged in order to share as much information as possible among experts in a timely manner. regarding autoimmune blistering diseases, it is already widely acknowledged that physicians should search for triggers in all newly identified patients before starting any therapy, including infectious agents. but what about patients already in immunosuppressive therapy for these potentially life-threatening disorders? given the current lack of scientific evidence on the basis of which official recommendations with a high degree of reliability are possible, some indications have been proposed by the international pemphigus and pemphigoid foundation, as the result of expertise and clinical common sense, inspired by a principle of prudence. however, no clear and comprehensive data have been provided on the management of ongoing immunosuppressive therapies in these patients. regarding other inflammatory diseases, the indications of the major scientific societies of dermatologists, rheumatologists and gastroenterologists in italy - , but also the american academy of dermatology association, suggest that if the patient is stable or in good health, the stop of the ongoing biologic therapy is not reasonable/indicated, as the risk of reactivation of the underlying pathology could add an additional risk factor to infections, including covid- . this article is protected by copyright. all rights reserved here we report our experience of around patients suffering from autoimmune bullous diseases and in treatment with immunosuppressive drugs currently referring to our bullous diseases outpatient service, sant'orsola-malpighi university hospital, bologna, italy. about patients are visited per week in the space of one day. since th march , in accordance with the sant'orsola-malpighi hospital directives made following the last decree of the president of the council of ministers (dpcm) of march , (gu serie generale n. del - - ), all the outpatient services, of any priority, first visits or control visits, have been temporary suspended and in place telephone calls have been made to all patients who were scheduled to be visited in our outpatient service in the following weeks to prevent them from leaving their home and crowding the hospital, given that social distancing is one of the most effective safeguards in order to limit the spread of the virus. we therefore held a telephone consultation, checking the health of patients over the last month. in particular, we asked whether patients in immunosuppressive therapy among possible trigger factors of autoimmune blistering diseases, immunization and viral infections are mentioned in the literature, although the underlying immunological mechanism is still unclear. [ ] [ ] [ ] the most acceptable hypothesis involves the possible molecular mimicry existing between viral and epidermal proteins, and over activation of the immune system as a consequence of the viral attack. indeed, in autoimmune blistering disorders, once the autoantibodies bind to this article is protected by copyright. all rights reserved their targets, namely self-structural proteins, several pathways are activated, including complement activation and deposition, and neutrophilic chemotaxis, with the release of proteases and elastases that lead to blister formation and of cytokines such as il- and il- , which recruit additional immune cells. to the best of our knowledge, no studies regarding previous viral outbreaks and the effects of these viruses on autoimmune blistering disease patients have been reported in the literature so far. moreover, the pathogenesis of sars-cov- infection in humans is still unclear, although massive and prolonged chemokine response known as 'cytokine storm' correlating with high morbidity and mortality has been observed in these patients. we hypothesize that the interruption of immunosuppressive therapy in autoimmune blistering disease patients may determine a dysregulation of inflammatory cytokines that not only exacerbates the bullous disease itself but may also be involved in the pathogenesis of the viral infection. therefore, it is likely that the management of the inflammatory processes guaranteed by immunosuppressive therapies not only controls blistering, but also contributes to a less aggressive organic response to sars-cov- . in conclusion, to date there is a lack of direct scientific evidence to support the continuation of immunosuppressive therapies in patients infected with sars-cov- . therefore, it will be crucial for our community to learn of more cases of autoimmune bullous disease patients under immunosuppressive treatment who have developed covid- , in order to better quantify the risk of infection under immunosuppressive therapy. moreover, now more than ever, research into autoimmune blistering diseases should focus attention on emerging safer therapeutic options that decrease the rate of mortality and morbidity as well as the risks connected to the therapy itself. what is novel in the clinical management of pemphigus information for pemphigus and pemphigoid patients related to coronavirus disease ° comunicazione del presidente sir: infezione da covid- accepted article this article is protected by copyright. all rights reserved infezione da coronavirus: vademecum per i pazienti affetti da psoriasi cutanea e/o artropatia psoriasica italian group for the study of inflammatory bowel disease, avviso per i pazienti con mici guidance on the use of biologic agents during covid- outbreak three case reports of post immunization and post viral bullous pemphigoid: looking for the right trigger bullous pemphigoid viruses and pemphigus: an intriguing never-ending story hypothesis for potential pathogenesis of sars-cov- infection--a review of immune changes in patients with viral pneumonia acknowledgments: the patients in this manuscript have given written informed consent to publication of their case details. this article is protected by copyright. all rights reserved key: cord- - uflg oc authors: allen,, koya c. title: tracking the traveler without a passport: perspective on surveillance of imported disease date: - - journal: j travel med doi: . /jtm. sha: doc_id: cord_uid: uflg oc nan s ince the turn of the century, we have experienced increases in a variety of disease threats including bioterrorist attacks, pandemic influenza, emergence of novel diseases, and re-emergence of known pathogens. although every disease scenario had specific criteria surrounding its spread, a common factor in nearly all included travel. it is not a new concept that travel is in itself a vector for disease transmission and in some respects one of the most important factors, given our increasingly global society. the mode of disease transmission, whether vector borne, such as dengue, or airborne, such as influenza, provides us with a clear picture of the necessary mitigation strategies for minimizing transmission; however, our prevention strategies have not advanced to keep in line with the role of travel in the rapid spread of diseases. our borders have significant restrictions for the movement of people and goods, but not for the potential pathogens traveling with them. there is substantial lack of addressing the human role, or more specifically the traveler's role in disease spread. we tend to view the traveler as a victim of diseases present in endemic travel destinations when infection is acquired. we need to evolve our approach to travel health by recognizing the traveler as an important aspect of the disease transmission cycle. this would allow researchers and practitioners to extend prevention strategies beyond endemic regions and the individuals therein, and consider international travel behavior a significant facet of imported disease risk. leggat and franklin address the importance of risk perception in travel behavior and how it influences adherence to pre-travel health recommendations. additionally, rossi and genton discuss the uncertainty of pre-travel risk assessments, and express concern that pre-travel history is not indicative of actual travel behavior. travel health needs a fresh perspective and alternative concept for understanding risk and the intricate link that exists between perceptions, behavior, and the role they play in our capabilities for successful prevention and response to imported diseases. one strategy could be the use of the concept of "cultural embeddedness" (ce), which could aid our understanding of the relationship between travel behavior and imported disease risk. figure exemplifies this concept within the travel health behavior theory "ce and mosquito avoidance practices (map)." cemap explains the influence of the social and physical environment, which influences perceptions, intended and actual international travel behavior for compliance to map. the concept takes into consideration the different levels of influence, which may help or inhibit adherence to pre-travel health recommendations, and inherently influence the risk of acquiring common vector-borne infections such as dengue. although cemap may appear as an idiosyncratic concept of prevention, it can provide a basis for future research and a strategy for practice-based travel medicine utilizing a public health approach. avoidance practices (cemap). the theory of cemap represents the process of cultural embeddedness, which occurs from the planning phase of travel through the travel experience. cemap is an unbounded spiral that continues over the course of each travel experience. this international travel behavior theoretical framework is also grounded in the public health approach and social-ecological framework for health behaviors. travel, including imported middle eastern respiratory syndrome coronavirus (mers-cov) from the arabian peninsula, and imported and consequently local-acquired dengue cases in florida. most recently, the inadvertent spread of chikungunya throughout the caribbean poses a threat for imported cases and introduction to other regions. the main issue is the lack of preemptive strategies using collaborative efforts that can aid in early detection and outbreak prevention. there were great strides in response to sars in , which highlights the impact of international collaboration; yet, an effective response is not exactly primary prevention. nevertheless, similar efforts should become standard procedures for these persistent disease threats that rapidly emerge. admittedly, not every country has the capability to develop programs following broad-based recommendations for prevention and control of diseases such as dengue and chikungunya; however, collaboration between countries and organizations and the use of surveillance tools and networks, such as health-map, geo-sentinel, pro-med, and other more sophisticated aggregate surveillance systems, make it possible for not only outbreak detection but also outbreak prediction. the key is to improve strategies utilizing these tools and protect our borders from infiltration by diseases because once established, we lose our opportunity for prevention and must switch gears to response and control. for dengue, clear guidelines exist for the prevention, detection, control, and response to outbreaks. for example, guidelines include personal protection, community engagement in environmental protection, vector surveillance, risk communication, laboratory detection, and clinical management. broad-based strategies also exist, such as the integrated management strategy for dengue prevention and control (ims-dengue), to provide guidance for program development at the country level. next steps in dengue control must be innovative and specific to the dengue threat within a given locale. lagi and colleagues, for example, conducted a retrospective analysis of confirmed cases to determine the characteristics of imported dengue in italy. they concluded that increased awareness and inclusion of diagnostic methods that aid in early diagnosis could thwart secondary transmission in italy during periods of aedes albopictus activity. in taiwan, a pioneering approach to surveillance included the use of fever screenings. although not % effective, fever screenings can prevent imported cases and partially block secondary transmission within the region. for dengue, we need a robust preemptive strategy for surveillance, which incorporates multiple facets of the transmission cycle. the strategy should include early warning capabilities by identifying imported disease "hotspots," using methodology similar to gardner and colleagues' network-based regression modeling to estimate the relative risk of imported cases. this could assist an active surveillance strategy in high-risk cities and a fever-screening program in high-risk airports. moreover, the strategy should include complementary passive surveillance and vector surveillance and control programs. a robust prevention strategy may sound sexy; however, it is important to note that disease transmission is complex; therefore, disease prevention is also complex. keeping in mind a simplistic view of imported disease consequences-travel-acquired infection, importation, secondary transmission, and subsequent outbreaks-we can then delve into prevention details at each stage. robust surveillance may not be the answer everywhere, but it may be for certain areas. furthermore, importation is not always preventable, so effective response strategies are essential. risk perception and travelers the reliability of pre-travel history to decide on appropriate counseling and vaccinations: a prospective study cultural embeddedness and the international traveler: influences on travel behavior for the prevention of imported dengue world health organization. who statement on the fifth meeting of the ihr emergency committee concerning mers-cov (online). geneva: who. available at tallahassee, fl: florida health chikungunya outbreak in the caribbean region faster … but fast enough? responding to the epidemic of severe acute respiratory syndrome dengue: guidelines for diagnosis, treatment, prevention and control integrated management strategy for dengue prevention and control in the caribbean subregion. (martinique): pan american health organization (us) airport sentinel surveillance and entry quarantine for dengue infections following a fever screening program in taiwan a predictive spatial model to quantify the risk of air-travel-associated dengue importation into the united states and europe the author states that he has no conflicts of interest to declare. key: cord- -tc vulou authors: reavill, drury r.; lennox, angela m. title: disease overview of the urinary tract in exotic companion mammals and tips on clinical management date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: tc vulou diseases of the urinary tract are reviewed, covering infectious (bacterial, viral, parasitic), degenerative, congenital, metabolic, nutritional, neoplastic, obstructive, and toxic causes. some clinical presentations and diagnostic procedures are described for ferrets, rabbits, guinea pigs, hamsters, mice, rats, chinchillas, hedgehogs, and sugar gliders, as well as therapies. the anatomy of the kidney is conserved among mammals. the kidneys are located within the sublumbar peritoneal space, with the right kidney positioned cranially to the left. in female rodents, there is a separate external orifice for the urinary tract and the reproductive tract. in male and female sugar gliders (petaurus breviceps), the urinary tract terminates into a cloaca. male chinchillas (chinchilla lanigera), guinea pigs (cavia porcellus), rats (rattus norvegicus), mice (mus musculus), hamsters, gerbils, and ferrets (mustela putorius furo) have an os penis or baculum. there are a few histologic differences of the kidney, including the number of papilla and length of the nephrons. most differences are related to the natural environment of the animal. these differences are noted between desert-dwelling species requiring optimal ability to concentrate urine, and other species in which water deprivation is not common. of clinical significance is the specific gravity of urine in desertdwelling species, which is generally higher. the most common problems affecting the ferret urinary system described in the literature are aleutian disease of the kidney, renal tumors, renal cysts, urolithiasis, and bacterial cystitis. in one author's database (drr), hydronephrosis, nephritis (all causes), and renal mineralization are the most common lesions of submitted kidneys. aleutian disease is caused by a parvovirus. the lesions are immune mediated and vary in severity. gross lesions may be minimal and nonspecific. emaciation and organ enlargement can be seen. in the kidney, membranous glomerulonephritis is common and a lymphocytic-plasmacytic inflammatory infiltrate is seen in the interstitium. this infection is uncommon in ferrets and the mortality is generally low. systemic coronavirus can also affect the kidneys, and the primary lesion is granulomatous inflammation. in a study showing ultrasonography results in ferrets with systemic coronavirus, nephromegaly was found in out of patients. canine distemper is essentially % fatal. affected ferrets present with neurologic clinical signs, bronchopneumonia, hyperkeratosis of the planum nasale and footpads, and a papular rash starting on the chin. however, the most productive tissues to evaluate for viral inclusions are the urinary bladder, renal pelvis, and biliary epithelium, and in suspected cases these tissues should be submitted (fig. ) . immunohistochemistry can be applied for confirmation. bacterial nephritis and pyelonephritis occur in ferrets and can be severe. these conditions are thought to be caused by ascending infections of the lower urinary tract. a variety of organisms can be the cause. grossly, the kidney may be abscessed and histologically there is necrosis and a neutrophilic infiltrate. organisms may be seen but culture is necessary for a definitive diagnosis. bacterial infection of the kidney, prostate, or urinary bladder can result in inflammatory cells (often undergoing degeneration) and bacteria in the urine. nephrosis is considered a degenerative disease of geriatric ferrets, and is often an incidental finding. nephrosis is characterized by damage of tubular epithelial cells. it is a nonspecific lesion that can have many causes, including ischemia, toxic exposure, and shock. nephrocalcinosis is reported more frequently in european pet ferrets than laboratory ferrets, and lesions include calcium deposition in the renal tubules. the cause of renal mineralization is usually not determined. soft tissue mineralization is usually considered dystrophic (secondary to tissue injury) or metastatic (associated with excessive calcium in the blood), but neither cause is obvious in most cases seen at necropsy. the lesion is usually in the renal papilla. rare cases of lymphomaassociated hypercalcemia have also resulted in renal dystrophic mineralization. the mechanism is most likely azotemia and the nephrotoxic effects of prolonged hypercalcemia. renal cysts are a common and usually incidental finding in the ferret (fig. ) . a -year retrospective analysis of ferrets with cystic kidney disease noted that % had renal cysts, and % primary polycystic disease. cysts may be single or multiple and may be present in the cortex of or both kidneys. it is speculated that acquired cysts arise from gradual distention of the nephron, caused by obstruction by exudates or fibrosis tissue, although the cause is seldom determined. rare cases of true polycystic disease have been reported in the ferret. this congenital lesion may result in markedly enlarged cystic kidneys that fill the posterior abdomen. many small cysts throughout the cortex and medulla characterize polycystic kidneys. another rare congenital lesion is an extramural ectopic ureter. this condition is the most common congenital cause of urinary incontinence in domestic canines. young ferrets present with urinary incontinence and urine scalding around perineal and inguinal areas. the ectopic ureter can be visualized via excretory urography. in case, nephroureterectomy was chosen instead of ureteral transplant. the ureter progressed extramurally to enter the distal urethra, caudal to the urethral sphincter, or vagina (on contrast). the most common renal neoplasm is malignant lymphoma. lymphoma usually involves multiple organs and can be severe in the kidney. grossly, there is variable replacement and distortion of the kidney. histology is typical, with a diffuse monomorphic sheet of lymphoid cells. impression smears can often provide a diagnosis. other neoplasms have been reported, although primary tumors of the urinary tract system are uncommon. from study of ferret tumors, only urinary tumors were recognized. there were renal carcinomas and unspecified renal tumors. another study evaluating ferret tumors identified only renal tumor listed as a sarcoma, not otherwise specified, and lymphoma involving the kidney. one renal adenocarcinoma is described in detail collected from an adult neutered female. this pleomorphic renal adenocarcinoma had multiple metastases to the lung, liver, greater omentum, right renal pelvis, and systemic lymph nodes. the tumor cells were pleomorphic with a large number of giant cells and arranged in tubular and cystic patterns. it was confirmed of renal origin by immunohistochemistry with strong positive cd and cytokeratin. two cases of transitional cell carcinoma arising from the renal pelvis, with completely replacing the kidney, have been reported. the presenting complaint was hematuria. , obstructive disorders urolithiasis the clinical signs of urolithiasis include stranguria and dysuria. uroliths in ferrets commonly consist of sterile struvite or magnesium ammonium phosphate. based on study evaluating cases, neutered males have a significantly increased risk of developing sterile struvite urolithiasis. these uroliths are more likely to be retrieved from the lower urinary tract than from the upper urinary tract. stone formation is suspected to be caused by a vegetable-based protein diet that increases urine ph, leading to struvite stone formation. cystine uroliths are uncommon, with a reported incidence of % in study evaluating cases. again, males are predisposed to urolithiasis, possibly because the distal portion of the urethra has a j-shaped bend with a decreased diameter of the urethra. all cystine uroliths have been found in the lower urinary tract. the uroliths are ovoid and smooth, light yellow to tan, and range in number from to more than . cystine uroliths are composed of nonessential sulfurcontaining amino acid; there may be a familial cause based on studies in other mammals. calcium oxalates are the next most common type of urolith. prostatic disease other causes of urinary obstruction in the male ferret are abnormalities of the prostate caused by adrenocortical disease. males can develop prostatic hyperplasia, cysts, and abscesses. prostatic enlargement is caused by increased levels of the hormones estradiol, testosterone, and -hydroxyprogesterone produced by the neoplastic adrenal cortex from an adrenal gland tumor. some animals develop concurrent urinary tract infection, including small urethral or cystic calculi. urethral blockages can also be caused by sloughed proteinaceous debris from squamous metaplasia of prostatic tissue and by small struvite calculi, which develop from the increase in ph caused by bacterial cystitis. preputial tumors in male ferrets can also result in urethral obstruction. these aggressive tumors are poorly responsive to both surgery and radiation therapy. nevertheless, wide surgical resection has been recommended, although the procedure may necessitate partial or total penile resection (fig. ) . any disease of the kidneys can result in renal insufficiency or failure. symptoms may include polyuria/polydipsia (pu/pd), weight loss, weakness, nausea, and decreased appetite, as can be seen in other traditional pet species. physical examination findings are also consistent and often include dehydration, although hydration status can be challenging to determine by testing skin turgor in patients experiencing rapid weight loss. some clinicians have anecdotally reported halitosis and oral ulcers in association with renal failure, but this is poorly documented. treatment protocols are based on those for traditional pet species, and include fluid diuresis for renal insufficiency and failure, antibiotic therapy for bacterial nephritis, and basic surgical approaches to the kidney. treatment of urinary tract disease follows guidelines established for traditional pet species, such as treatment of bacterial disease based on results of culture and sensitivity. general principles for treatment of renal insufficiency and renal failure are described later in this article. treatment of adrenocortical disease is well described in any current literature about ferret endocrine diseases. large associated prostatic cysts have been managed with marsupialization or omentalization of the cyst. successful nephrotomy for removal of unilateral renoliths has been reported in the ferret. the most common causes of renal disease reported in the literature are encephalitozoon cuniculi, chronic renal failure in older rabbits (oryctolagus cuniculus), and urolithiasis. other renal conditions seen in rabbits include bacterial nephritis, hydronephrosis, and papillary necrosis with renal mineralization. the most common renal disease of rabbits is infection caused by e cuniculi. [ ] [ ] [ ] although disease is often occult, rabbits with severe e cuniculi-associated renal disease may present with pu/pd and a host of other nonspecific signs, such as decreased appetite and weight loss, dehydration, and lethargy. in some rabbits, only renal changes are seen at necropsy. grossly, there may be no lesions or the kidneys may be pitted. on histology, there is a chronic active lesion, which may be granulomatous or pyogranulomatous. aggregates of organisms are found rarely in or around tubules as small, basophilic structures (fig. ) . rare cases of polypoid cystitis and urethritis have been described in rabbits. the masses are white to transparent, spheroidal masses protruding into the lumen. on histology, the polyps are lined by variably hyperplastic to attenuated transitional epithelium over cores of edematous inflamed fibrovascular stroma. there may be transitional epithelium lining cysts within the stroma. these polyps are suspected to arise from a combination of inflammation and a hyperplastic reaction to chronic irritation of the urinary mucosa. bacterial isolates have included proteus and enterococcus spp. the clinical signs associated with polypoid cystitis include perineal scalding, urinary sludge, and possibly hematuria. hematuria is frequently described as a clinical sign with obstructive urolithiasis, cystitis, bladder polyps, and pyelonephritis. red urine in rabbits may not necessarily be hematuria but may be the more common, and normal, porphyrin pigments. urine dipstick is an easy method to differentiate porphyrinuria from hematuria. chronic renal disease or end-stage kidney disease seem to be common in older rabbits. rabbits with renal failure can have lesions similar to those seen in old rats. the kidneys are grossly scarred and histologically there is marked interstitial fibrosis and glomerulosclerosis. this condition may be a sequela of encephalitozoon infection, but the cause is usually not determined. renal cysts are uncommon in rabbits. in a few reports, some cases were speculated to be inherited and to resemble human polycystic kidney disease. , in experimental studies, methylprednisolone acetate has been injected into newborn rabbits to induce polycystic kidneys. the few cases of renal cysts ( out of rabbit renal submissions) in author's database (drr) have been in older rabbits (> years) and associated with lesions of chronic renal disease. tumors of the urinary tract are rare in rabbits, with most involving the kidney. these tumors include benign embryonal nephroma, renal carcinoma, renal adenocarcinoma, nephroblastoma (fig. ) , and kidney hamartoma. a case of renal and ureteral transitional cell carcinoma produced hydronephrosis. a urinary bladder transitional cell carcinoma in a female rabbit presented with acute hematuria, partial vaginal prolapse, and inappropriate urination. the gross appearance was a nodular cystic mass at the cranial central apex of the urinary bladder. urolithiasis and urinary sludge uroliths are described in the kidney and urinary bladder of rabbits. renoliths can be mild with few stones, or severe and produce renomegaly and complete destruction the kidney (fig. ) . uroliths and urinary sludge in rabbits are usually composed of various calcium salts, predominately calcium carbonate. from a study evaluating more than rabbit uroliths, the most common were calcium carbonate ( . %), compound ( %), and mixed ( . %). radiographs are usually confirmatory because calcium carbonate is radiopaque (fig. ) . the predominance of calcium-based uroliths is most likely caused by the unique calcium metabolism of rabbits. excess dietary calcium is absorbed unregulated, resulting in increased plasma calcium concentrations and excretion of excess calcium in the urine. therefore, serum and urinary calcium levels are related to dietary intake. urinary calcium can bind with other constituents when conditions are met for crystallization, such as dehydration, changes in ph, and urine retention, to form both the sludge (which can appear as thick white to yellow material) and uroliths. abnormal accumulations of calcium (sludge) are more common in older rabbits, possibly because of decreased mobility or water consumption. there are rare reports of uroliths composed of silica, struvite, and calcium sulfate dehydrate or gypsum. the gypsum urolith was associated with the rabbit eating gypsumbased plaster that was covered by a white paint containing barium sulfate. hernia hernias can be classified by their anatomic location (body cavities or external, such as inguinal or femoral), cause (true hernia [through an anatomic structure and surrounded by peritoneum] or false hernia [traumatic through induced defects]), and contents. inguinal or scrotal hernias develop when abdominal contents, such as the urinary bladder, enter the inguinal canal or scrotum through a wide or defective inguinal canal. in male rabbits, the inguinal rings are open throughout life, increasing the risk of herniation. the typical appearance of bladder herniation is a fluid-filled mass in the scrotum and no bladder palpable in the abdomen. some cases have concurrent lesions of urinary sediment and multiple uroliths; case featured a testicular tumor on the opposite side. inguinal herniation of the bladder seems uncommon in female rabbits (fig. ) . trauma was suspected to be the cause in case of a female rabbit presenting with hematuria. female rabbits may also evert the urinary bladder through the urethra. recent kindling or a history of multiple litters is common. rabbits present with a vaginal mass, which is the mucosal surface of the everted bladder. rarely, males may evert and prolapse the urinary bladder. this finding seems more likely in castrated males because penis length is reduced after castration. another unusual presentation is diaphragmatic herniation of retroperitoneal fat and a kidney. a case of kidney herniation occurred in conjunction with a diaphragmatic lipoma. treatment general management of renal insufficiency and renal failure is described later. encephalitozoonosis treatment of e cuniculi is problematic because there is a lack of well-controlled scientific studies linking various treatment protocols with documented clearing of organisms. the problem relates to difficulties in establishing a diagnosis, and the observation that many affected rabbits recover spontaneously without treatment. rabbits with severe renal disease secondary to e cuniculi may not harbor renal organisms, and treatment is focused on managing acute or chronic renal failure. treatment of e cuniculi has relied on benzimidazoles, such as fenbendazole, but recently severe disease and deaths have been reported in rabbits treated with this class of drugs. benzimidazoles are radiomimetic and toxicity has been observed in many species; graham and colleagues recently reported deaths in rabbits treated with various dosages of several drugs, including fenbendazole, albendazole, and oxibendazole. clinical signs in rabbits were nonspecific and histopathology showed depletion of all bone marrow cell lines. for this reason, treatment should not be approached lightly, and serial monitoring of the complete blood count may be beneficial. surgical management of renal disease various surgical approaches to the kidney have been described. martorell and colleagues described a lateral flank approach for access to the kidney for removal of uroliths; author (aml) also found this approach simple and straightforward compared with a traditional midline abdominal approach (fig. ) . nephrectomy, performed through a ventral abdominal approach, was reported to manage hydronephrosis in a pet rabbit and to remove a renal carcinoma. cases of diaphragmatic herniation were successfully treated by replacing the herniated kidney and fat followed by diaphragmatic herniorrhaphy, and use of polypropylene mesh. management of bladder sludge strategies for managing urinary sludge include encouraging water consumption and exercise paired with dietary modification as needed. accumulation of sludge can be temporarily managed by bladder catheterization and flushing, or sometimes by gently agitating the bladder to manually resuspend the mineral, followed by repeated expression of urine. urolithiasis is commonly reported in the guinea pig. this condition usually involves the lower urinary tract. inflammatory, degenerative, and other conditions are also reported. chronic nephritis, interstitial nephritis, and pyelonephritis have been reported. , in these conditions the kidneys may be irregular grossly and the ureters may be dilated in pyelonephritis. on histology, the inflammation depends on the cause and the duration of the disease. cystitis in guinea pigs is common. guinea pigs, like chinchillas and degus, have adapted to a semiarid habitat with varying, species-specific adaptations to water deprivation. however, several diseases have been linked to insufficient water intake, such as cystitis, urolithiasis, or obstipation. a decrease in urination frequency may favor bacterial infection and formation of uroliths. the clinical signs of bacterial cystitis include dysuria, polyuria, hematuria, lethargy, or urethral discharge. the urinary bladder may be painful on palpation, small, firm, with a thickened wall. the most common isolate in a review series of adult female guinea pigs was escherichia coli. relapses were common. two guinea pigs were diagnosed with encrusted cystitis (further description in relation to hedgehogs later). renal cysts are a rarely reported and usually incidental finding. given the few reports of renal cysts in guinea pigs, it seems most cases are acquired cysts associated with chronic renal disease. one author (drr) has seen cases in adult guinea pigs ( - years of age), all with chronic renal lesions (fibrosing interstitial nephritis) out of case submissions with kidneys ( . %). chronic (segmental) nephrosclerosis is renal scarring of undetermined cause, which may be the result of vascular disease, infection, or immune-mediated disease. grossly, the renal cortex is irregularly pitted and histologically there is interstitial fibrosis, variable glomerulosclerosis, tubular dilatation, and variable mononuclear inflammation (fig. ) . renal mineralization can be incidental or associated with renal failure, resulting in uremia and soft tissue mineralization. the mechanism is complex but involves tissue death and secondary mineralization. in guinea pigs, exposure to excessive vitamin d is a common mechanism if renal failure is not present. such exposure occurs when feeding guinea pigs a commercial diet with errors in formulation or commercial rodent diets. commercial rodent diets are formulated with more vitamin d than is safe for guinea pigs. , neoplasia primary tumors are rare. lymphoma occasionally involves the kidneys. grossly, there is a diffuse white-gray infiltrate that replaces normal parenchyma. on histology, this is composed of immature lymphoid cells. uroliths may be present from the renal pelvis to the urinary bladder. if in the ureter or bladder, there may be ureteral dilatation and renal swelling or shrinkage. the most common urolith is calcium carbonate, which is radiopaque. similar to rabbits and other hindgut-fermenting herbivores, guinea pigs excrete excess calcium and they can eliminate up to % of their absorbed calcium via urine. urine specific gravity and ph highly influence crystallization. herbivore urine is commonly alkaline, which complicates dietary modification efforts to change urine ph and reduce urolith formation. treatment strategies for treatment of acute and chronic renal failure are described later. surgery of the kidney has not been described in pet guinea pigs. the urethra of the female guinea pig is short and wide. uroliths in the urethra and in the vaginal vestibulum can be visualized and removed manually with the aid of the lone star retractor. primary renal disease is uncommonly reported in pet hamsters, and mostly includes infectious and degenerative causes. sporadic cases of bacterial cystitis and pyelonephritis are seen. gross changes include thickening and exudation in the bladder and yellow-white foci of necrosis and inflammation in the kidney. culture is usually necessary to establish a definitive cause. the most common problem seen in the kidneys is amyloidosis, which has been reported in more than % of hamster necropsies in some colonies. , amyloid is an insoluble pathologic proteinaceous substance, deposited between cells in various tissues and organs of the body. systemic amyloidosis can be classified as primary (amyloid light chain), secondary (amyloid associated), or familial. secondary amyloidosis is most commonly described in animals and has been described as a reaction to diverse inflammatory stimuli. amyloid deposits are more common in female hamsters more than months of age. deposits can also be seen in almost any other organ. amyloidosis can be associated with nephritic syndrome. grossly, the kidneys are pale and irregular. microscopically, amyloid is eosinophilic to amphophilic and can be seen in glomeruli, interstitium, or both. glomerulonephropathy occurs in hamsters as well as other rodents. progressive nephropathy can result in renal failure. grossly, kidneys are pitted and variably shrunken, and histologically there may be variable stages of mesangial and glomerular capillary thickening, periglomerular sclerosis, and variable interstitial nephritis. this condition has been described in both chinese (cricetulus griseus) and siberian hamsters (phodopus sungorus). neoplasia primary tumors are rare in hamsters. two cases of renal adenoma have been reported in aged siberian hamsters. , one author (drr) has seen cases of lymphoma involving the kidney as well as case of a renal adenoma out of submissions that included the kidney. uroliths are also uncommon in hamsters. from study evaluating only samples, the most common uroliths were calcium phosphate ( . %), compound ( . %), and calcium oxalate ( . %). treatment strategies for treatment of acute and chronic renal failure are described later. surgery of the kidney has not been described in pet hamsters. renal disease is common in mice. some conditions are strain related, and determining the genetic heritage of a pet mouse (if possible) may be of value in diagnosis. amyloidosis is seen primarily in older animals. chronic progressive nephropathy or chronic nephritis in mice is similar to the condition in rats and other rodents. , pathogenesis is unknown. interstitial nephritis and pyelonephritis are also reported. , klossiella muris infection is seen primarily in wild mice. grossly, kidneys affected by these conditions have an irregular pitted cortex. microscopically, there is glomerular sclerosis, tubular dilatation and degeneration, interstitial inflammation, and fibrosis. lesions vary in severity according to their duration. in cases of klossiellosis, organisms can usually be seen on histology and may not be associated with significant inflammation. this organism is a parasitic protozoa of the phylum apicomplexa. species in this genus infect the renal tract of mammals and intestinal tract of snakes. this genus of the phylum apicomplexa is unusual in having only a single host in its life cycle. polycystic kidneys occur in balb/c mice, which are used as a model for human disease. cysts vary in size and number, but the condition is progressive and results in loss of renal function and early mortality. the renal surface is irregular and fluid-filled cysts are visible. cysts are also obvious in cut sections. dilated tubules are seen histologically. renal neoplasia occurs sporadically. malignant lymphoma can efface the kidney and carcinomas are occasionally seen. gross lesions are not specific. on histology, lymphoma is typical and carcinomas are usually composed of poorly differentiated cells with a loss of organized structure. hydronephrosis can be unilateral or bilateral. unilateral hydronephrosis is often an incidental necropsy finding and may be strain related or secondary to obstruction or inflammation of the renal pelvis. it is usually progressive and grossly there is variable dilatation of the renal pelvis with loss of renal parenchyma. one cause of obstructive hydronephrosis in male mice, as well as in rats and guinea pigs, is the retrograde movement of seminal plugs in the urethra (fig. ) . these seminal vesicle secretions may coagulate and become impacted in the urethra and urinary bladder. common renal conditions reported include hydronephrosis, polycystic kidneys, and pyelonephritis, as well as neoplasms. [ ] [ ] [ ] disease overview of the urinary tract trichosomoides crassicauda is a nematode that affects the urinary tract of rats. adult worms can be seen in the renal pelvis and urinary bladder, but the condition is usually asymptomatic. eggs are passed in the urine. nematodes are present in the epithelium and there is variable inflammation (fig. ) . the most common renal disease in rats is chronic progressive nephropathy or old rat nephropathy. [ ] [ ] [ ] the incidence can be up to % in affected strains. this disease affects older animals and is more common and severe in males, with a much higher incidence in s-d and fischer rats. high-protein diets are a factor in pathogenesis, as are immunologic factors, such as mesangial deposition of immunoglobulin m, and high levels of prolactin. lesions of progressive nephropathy are characterized by irregular and pitted renal cortices. the cut surface is also irregular. microscopic changes vary with duration. glomeruli are thickened, tubules are dilated and may contain proteinaceous fluid, and there is interstitial fibrosis and inflammation. nephrocalcinosis is seen, usually as an incidental finding but also with diets low in magnesium and/or high in calcium and phosphorous. mineral is deposited within the interstitium and tubules. grossly, there are pale streaks and, histologically, basophilic mineral deposits are seen. spontaneous tumors of the urinary tract are uncommon in rats. tumors reported in the kidney include renal cell adenoma, lipoma/liposarcoma, renal cell carcinoma (with metastasis to the lung and liver), and a nephroblastoma with metastasis to the lung and local lymph nodes. transitional cell carcinoma of the urinary bladder also occurs spontaneously and in case there was metastasis to the lungs. , , obstructive disorders uroliths in rats are generally struvite ( . %) with fewer calcium phosphate ( . %). primary renal diseases are poorly described in chinchillas. urolithiasis has been described, and, although rarely reported, suppurative nephritis is common in younger animals (fig. ) . e coli has frequently been isolated based in author's experience (drr). unlike rabbits and guinea pigs, chinchillas eliminate excessive calcium approximately % via feces and only % to % via urine. nevertheless, their urine calcium concentration may show great individual variation. urolithiasis is reported in chinchillas and most are calcium carbonate ( . %) and miscellaneous ( . %). the clinical signs are as expected, with irritation around the preputial orifice, as well as hematuria, pollakiuria, and stranguria. most uroliths are found in the urinary bladder, with fewer within the urethra. in males, most are in the proximal urethra near the pelvis and cranial to the os penis (fig. ) . prognosis is guarded with urethral stones and recurrence is common. within the urinary bladder there may be thickening of the bladder wall with an inflamed mucosa. perineal hernias are described in sexually intact males. the hernia contents were urinary bladder in animal and lobules of fat in the other. in both cases, the testicles could be moved freely in and out of the inguinal canal. hedgehogs (atelerix albiventris) have been reported to have cystitis and uroliths, but the most common renal problem is chronic nephritis. as in other species, chronic renal disease is likely to have a large number of causes, such as infections, immune-mediated disorders, and nephrotoxins. renal disease may also be a part of a systemic condition. grossly and histologically, the lesion is similar to others that have been previously described in other animals. cystitis is uncommon in hedgehogs, with scarce descriptions in the literature. in females, uterine tumors are a much more common cause of hematuria. however, author (drr) has diagnosed cases of hemorrhagic acute to subacute cystitis. lesions of the urinary system collected from the database of author (drr) are summarized in table . one case was of encrusted cystitis. there was extensive mineralization of the urinary bladder with an ulcerative and/or necrotic cystitis. this type of lesion is uncommon and has been associated with alkaline urine and several bacteria, including staphylococcus pseudintermedius and corynebacterium urealyticum (fig. ). there are very few reports of urinary disease in sugar gliders. renal klossiellosis has been described in pet sugar gliders. in affected sugar gliders there is tubular dilatation and interstitial nephritis. the various life cycle stages can be identified within the renal tubular epithelium. based on morphologic evaluation, the name klossiella dulcis n. sp was proposed. no treatment has been described. bilateral hydronephrosis in a male sugar glider developed secondarily to a functional obstruction suspected to be from hyperplasia and inflammation of the urinary bladder and ureteral epithelium. the animal presented with acute caudal abdominal swelling. one author (aml) has noted several cases of disease of the cloacal and paracloacal glands that resulted in apparent urinary obstruction; obstruction resolved with treatment of the primary cause. both sexes have paracloacal glands (anterior, dorsal, disease overview of the urinary tract and ventral), which are more developed in the male. under the influence of testosterone, the dorsal paracloacal glands and testes increase in size. inflammation and neoplastic disease of these glands can result in functional obstruction of the digestive and urinary tract. there are reports of a paracloacal cyst, sebaceous nodular hyperplasia, and carcinomas in male sugar gliders. self-mutilation, stranguria, and pericloacal swelling are the typical clinical signs. one case had a dorsal paracloacal gland carcinoma and the other a transitional cell carcinoma with squamous differentiation. toxic exposure copper toxicosis has been described in a sugar glider voiding red-brown urine and showing possible seizurelike activity. the kidneys were enlarged and dark red with marked, acute, multifocal renal tubular degeneration and necrosis with hemoglobin casts. icterus was noted and the liver had marked, acute, centrilobular hepatocellular degeneration and necrosis with hepatocellular copper accumulation confirmed with rubeanic stain and a hepatic copper concentration of parts per million (wet-weight basis). the provided mineral and vitamin supplement had . and mg/ml respectively. it was unknown whether the vitamin supplement could have led to the toxicosis. in a review of sugar glider case submissions (drr) from to , out of there were urinary tract lesions. interstitial nephritis was common, as was the nonspecific finding of renal tubular necrosis (nephritis). other lesions included membranoglomerulopathy, renal cysts, and transitional cell carcinoma of the urinary bladder (fig. ) . lesions are summarized in table . without benefit of evidence-based therapies for exotic mammals, treatment of renal insufficiency and failure is based on therapies considered most advantageous in traditional pet species. the international society of feline medicine guidelines for management of chronic kidney disease (ckd) may be useful. even in cats, simple and accurate markers for renal function are currently unavailable; biomarkers such as blood urea nitrogen (bun), creatinine, and urine specific gravity are useful, but interpretation can be difficult. the principles of treatment are supportive, and the overall goal is maintenance of quality of life. even the goal of maintaining hydration has not been formally associated with increased longevity and improved quality of life in cats with ckd; however, the benefits of normohydration are considered obvious enough to continue recommendation of fluid therapy. unstable or decompensated patients benefit from hospitalization and intravenous (iv) fluid therapy. fluid rates are based on calculation of hydration deficit: body weight (kg) times estimated dehydration (%) times . hydration deficit is added to maintenance fluids ( ml/kg/ h in cats) and provided over to hours. once normohydration is achieved, fluid therapy continues at maintenance rate, monitoring the patient carefully for evidence of fluid overload. fluid therapy is tapered over several days once condition and appropriate biomarkers have improved. for long-term maintenance of hydration, many recommendations for cats are appropriate in exotic mammals, and include free access to high-quality water using multiple water sources. in exotic pets, this means providing water in bowls along with traditional water bottles. although moist canned food is not available for rabbits and rodents, moistening greens and hay may aid water intake, as well as supplementing with liquid foods designed for convalescing exotic pets; for example, oxbow critical care or carnivore care depending on the nutritional needs of the species. in cats, placement of feeding tubes for oral hydration may be used long term; this approach is not useful for exotic species. however, subcutaneous fluid administration may have utility, because some owners can be taught to administer fluids to small patients at home. commercial indwelling subcutaneous catheters have been used in cats, and author (aml) has used one in a large rabbit with some success. acute renal failure (arf) may be reversible if identified and treated early and aggressively. as in other species, arf may be prerenal (shock, hypovolemia), renal (renal disease), or postrenal (obstruction). urine specific gravity plus changes in bun and creatinine levels can be helpful. arf is treated by correcting perfusion deficits (discussed later) and dehydration, and diuresis, along with correction of an identified underlying cause. once the patient is rehydrated and normotensive, continue fluid administration as maintenance ( - ml/kg/h) plus estimated losses (diarrhea/vomiting, if present, and estimated urine volume). based on information in domestic species, patients with arf may become % to % dehydrated each day because of ongoing losses. fluid administration is decreased when the patient maintains hydration, begins to eat and drink, and bun/creatinine levels normalize. iv catheterization is a standard procedure in rabbit medicine; iv access is also possible in large guinea pigs and some chinchillas. iv access may not be possible in any patient that is hypovolemic. for these cases, intraosseous (io) catheterization is indicated. placement sites in mammals include the proximal humerus, femur, and tibia. placement is best performed in a sedated patient with parenteral analgesia, and a local block ( - mg/kg lidocaine into the skin, subcutaneous tissue, and periosteum). although short spinal needles are useful in rabbits and large rodents, most io catheters are simple hypodermic needles, to gauge depending on the size of the bone cavity. seat the needle into the proximal bone, and then orient the needle into the bone cavity. cover with an iv catheter cap and secure to the limb with tape (fig. ) . treatment of shock in rabbits and rodents has been described. management of fluid deficits is maintenance fluids estimated at to ml/kg/h in exotic mammals, or via allometric calculations of resting energy requirements (weight [kg]  ) plus hydration deficit, adjusting fluid rates for ongoing losses, such as polyuria in cases of renal insufficiency. other therapies include erythropoietin for treatment of 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(apicomplexa: klossiellidae) in the kidneys of petaurus breviceps (marsupialia: petauridae) bilateral hydronephrosis in a sugar glider (petaurus breviceps) paracloacal gland carcinoma in a sugar glider (petaurus breviceps) paracloacal cyst in a sugar glider (petaurus breviceps) a transitional cell carcinoma with squamous differentiation in a pericloacal mass in a sugar glider (petaurus breviceps) pathology in practice. copper toxicosis isfm consensus guidelines on the diagnosis and management of feline chronic kidney disease emergency and critical care of small mammals key: cord- -x amm g authors: rupali, priscilla title: introduction to tropical medicine date: - - journal: infectious disease clinics of north america doi: . /j.idc. . . sha: doc_id: cord_uid: x amm g tropical medicine deals with infectious and noninfectious diseases geographically located between the tropics of cancer and capricorn. it encompasses diseases that result from poverty, poor sanitation, infrastructure, and inadequate health resources. lack of availability of clean water and food made with unhygienic practices add to the morbidity of these diseases. the tropics are reeling under the onslaught of climate change, deforestation, and air, water, and soil pollution, which worsens an already fragile health system. this article provides an overview of the definition, classification, geophysical problems, syndromic approach to common tropical infections, diagnostic challenges in the tropics, and access to medicines. introduction to tropical spanned many decades in china, hong kong, and taiwan (then called formosa). he eventually came back to london where he lectured on tropical diseases at the st. george's hospital. he then became the chief medical officer to the colonial office. at this point he used his considerable influence to found a school for tropical medicine at the albert dock seamen's hospital. the london school of hygiene and tropical medicine was formally inaugurated on october , . he eventually went on to become the first president of the royal society of tropical medicine and hygiene in . he also made numerous contributions to parasitology, such as ( ) the establishment of mosquito as the intermediate host of wuchereria bancrofti, the causative agent of filariasis; ( ) proposed the mosquito-malaria theory, which eventually spurred sir ronald ross to discover that a mosquito was the definitive host for malaria; and ( ) the discovery of a new species of schistosoma (schistosoma mansoni) and a new parasite, spirometra or sparganum mansoni and spirometra mansonoides. a species of mosquito mansonia spp and a filarial worm mansonella spp were so named to honor him. he is hence known as the "father of tropical medicine." the tropics are regions of the earth that lie on either side of the equator between the tropics of cancer and capricorn (fig. ) . the tropics include parts of central and south america, australia, africa, india, and oceania. the tropics account for % of the earth's landmass, and are home to a third of the world's people. the tropics are warm throughout the year with temperatures ranging from c to c ( f- f) because they are close to the equator with direct sunlight most of the year. however, rainfall does vary remarkably from one area to another with the amazon basin recording a high rainfall and north africa being an arid area recording little to no rainfall most of the year. these climatic conditions often influence the flora, fauna, and insect wildlife in these areas. tropical regions are dominated by equatorial climates with a mean temperature greater than c, and arid zones are characterized by general lack of water, which rupali harms plant and animal life. they host % of the world's biodiversity. environmental factors include the following: climate: climate change has a wide-ranging impact on habitats, species distribution, human health, agriculture, sea levels, and the frequency/intensity of extreme weather events. changing rainfall patterns and increased temperatures have also led to increase in vector borne diseases, such as dengue and malaria, because of increased vector distributions and decreased vector and parasite incubation periods. air pollution: among the tropical regions, southeast asia reported the greatest increase in co emissions leading to a decreased air quality followed by south asia and south america, which has significantly impacted the climate and led to higher weather-related mortality and morbidity, infectious disease rates, and respiratory illnesses. land degradation: although land productivity has gone up because of increased livestock and cereal production, land degradation caused by poor agricultural practices and deforestation has led to altered ecosystems and has not changed the fact that more people in the tropics experience undernourishment compared with the rest of the world. water scarcity: the tropics have more than % of the world's renewable water sources, yet more than half of these areas are considered vulnerable to water stress because of the inequality of water distribution, with southeast asia having the highest pollution discharge in the world. overexploitation of wild marine food resources has led to the coral reef systems to be at high or medium risk of damage. biodiversity: tropical biodiversity is being threatened across all taxonomic groups with a rapid loss of primary forests. protection and maintenance of these fragile ecosystems will have a long-term impact on human health and requires cooperation at global, regional, and international levels. health is defined by the world health organization (who) as a "state of complete physical, mental and social well-being not merely the absence of disease or infirmity." however, there are many social determinants of health that affect a person's disease burden or longevity and these are often underemphasized or overlooked because the impact is often unmeasurable. climate and other environmental factors and such social factors as poverty, overcrowding, undernutrition, and limited access to appropriate health care coupled with the lack of education and poor governance contribute to a higher prevalence of communicable and noncommunicable diseases. according to the state of tropics report , globally extreme poverty has declined by almost % since the s but almost two-thirds of the poorest people in the world continue to live in the tropics. poverty reduction has taken place mostly in southeast asia and central america coupled with a rapid increase in urbanization in these areas. the urbanization rate in the tropics has increased disproportionately to almost % by , in turn giving rise to large populations of slum dwellers as compared with the temperate regions, which in turn brings with it diseases of hygiene and sanitation. about % of the world's population and % of children younger than age also live in the tropics. by , this is expected to increase to % and %, respectively. between and , the life expectancy in the tropics increased by . years to . years and infant mortality reduced by %. despite these enormous strides in the improvement of mortality, . million children younger than age died in , of which % were in low-and low-middle-income countries. infectious agents and toxins are transmitted through water, soil, food, vectors, plants, and animals. freshwater constitutes only . % of the total water resources on the planet. of this only . % is available as surface water, whereas the rest is in polar ice sheets, snow cover, and underground aquifers. water scarcity is defined as less than cubic meters of water available per person per year. in the tropics the number of nations with water scarcity has tripled since . this scarcity is most acute in south asia, where % of the population is considered vulnerable, followed by north africa and the middle east at %. statistics gathered by united nations show that million people lack access to safe drinking water and million live without appropriate sanitation. hence access to safe water and sanitation were recognized as human rights in by the united nations general assembly. waterborne or water-related diseases encompass illnesses resulting from indirect and direct exposure to water. the four main routes of transmission include ( ) waterborne, ( ) water washed, ( ) water based/insect vector, and ( ) water related. these diseases are transmitted through the direct drinking of water contaminated with pathogenic microorganisms. contamination of drinking water often occurs through fecal contamination, caused by poor sewage disposal and improper sanitation. if contamination levels are high, the young, the old, and the immunocompromised are at significant risk of diarrheal diseases and some others. some of the pathogenic organisms and diseases they cause are as follows: these are also known as water-scarce diseases, which thrive in water scarcity and poor sanitation. this depends more on water quantity than quality. a. soil-transmitted helminths: diseases of poor sanitation transmitted through contaminated soil. most prevalent helminths include ascaris spp, trichuris trichura, ancylostoma duodenale, or necator americanus. b. acute respiratory infections: they are responsible for % of the total child deaths every year and good hygienic practices including hand washing with soap can significantly reduce the transmission of acute respiratory infections. c. skin and eye diseases: scabies, impetigo, trachoma, yaws, conjunctivitis, and skin ulcers. d. fleas, ticks, and lice: typhus, scabies, relapsing fever. these are infections caused by parasitic pathogens found in aquatic host organisms. humans become infected either through skin penetration (schistosomiasis) or by ingesting the infective forms (dracunculiasis [ingestion of larvae in crustacean], paragonimiasis [metacercariae ingested in crab or crayfish], clonorchiasis [metacercariae ingested in fish]). these are diseases caused by the insect vectors breeding in and around water bodies. malaria is one of the water-related diseases endemic in countries with . billion people at risk. about % cases occur in africa, % in asia, and % in americas. mosquito-borne diseases include malaria, yellow fever, dengue fever, and filariasis. fly-borne diseases include onchocerciasis and loiasis. foodborne illnesses are defined by the who as diseases of infectious or toxic nature caused by the consumption of contaminated food or water. they are classified into two broad groups: intoxication and infection. intoxication is caused by ingestion of toxin produced by pathogens, whereas infection is caused by ingestion of food containing viable pathogens. intoxication is also possible by eating animals that have consumed toxin-producing organisms. foodborne diseases result in considerable morbidity and mortality, and contribute to significant costs in tropical countries. many of these are caused by bacteria, viruses, parasites, chemicals, and prions through contaminated food. the who estimates from to data that million cases were caused by contaminated food resulting in . million dalys (disease adjusted life years). , norovirus is a leading cause of foodborne illness ( million) followed by campylobacter spp with million cases. in addition, nontyphoidal salmonella diarrheal and invasive infections result in a high burden of . million dalys in the african region followed by the southeast asian region in children less than years. the main pathogens of foodborne illnesses are bacteria ( %), viruses ( %), chemicals ( %), and parasites ( %). bacteria cause % of the foodborne illnesses and botulism, clostridium perfringens gastroenteritis, e coli infection, salmonellosis, and staphylococcal food poisoning. the most common clinical symptoms are diarrhea, vomiting, abdominal cramps, headache, and nausea. foodborne parasitic diseases excluding enteric protozoa cause an estimated . million cases and , deaths annually resulting in an estimated . million dalys. among these foodborne ascariasis and toxoplasmosis were common tropical health contributing to . and . million cases respectively. human cysticercosis with . million, foodborne trematodiasis with . million, and toxoplasmosis with , dalys resulted in a high burden of disease. foodborne enteric protozoa resulted in an additional . million illnesses. clinically foodborne pathogens can cause diarrhea, intoxications, and invasive enteric diseases. bacteria producing acute diarrheas include campylobacter, salmonella spp, shigella spp, staphylococcus aureus, nontyphoidal salmonella, enteropathogenic e coli, and v cholerae. bacteria causing intoxications include clostridium botulinum, c perfringens, bacillus cereus, and enterotoxigenic e coli. bacteria causing chronic diarrheas include brucella spp, listeria spp, and mycobacterium tuberculosis in immunocompetent hosts and nontuberculous mycobacteria in immunocompromised hosts. most of the burden of foodborne illness caused by viruses is transmitted by poor hygienic practices either during food handling or food production. they are transmitted by the fecal-oral route infecting their host after ingestion, followed by invasion of cells in the epithelial lining of the gut and replication at the same site or elsewhere in the body. norovirus and hepatitis a are considered priority pathogens by the who and fao (food and agriculture organization of united nations). increasingly hepatitis e is a pathogen that is assuming increasing importance. zoonotic food borne viruses severe acute respiratory syndrome, monkey pox, and nipah virus have been transmitted through various food-related incidents. numerous parasites are transmitted by food including protozoa and helminths. however, some of these can also be transmitted by water, soil, or person to person contact. a wide variety of helminthic roundworms, tapeworms, and flukes are transmitted in foods, such as undercooked fish; crabs and mollusks; meat; raw aquatic plants, such as watercress; and raw vegetables contaminated by human feces. cryptosporidium spp, giardia intestinalis, cyclospora cayetenensis, and toxoplasma gondii trichinella spp and anisakis spp tapeworms diphyllobothrium spp and taenia spp insecticides used in crops, alcoholic beverages containing methanol, poisonous plants (eg, mushrooms, raw cassava roots), oysters, mussels, and clams (which ingest dinoflagellates producing saxitoxin), large reef fish (which ingest marine algae producing ciguatera toxin), finfish spoiled by bacteria leading to scombroid poisoning, and ingestion of puffer fish containing tetrodotoxin all can contribute to significant morbidity. vector-borne diseases impose heavy economic and health burdens leaving many people who survive the infection permanently debilitated, disfigured, maimed, or blind. vectors thrive in conditions where housing is poor, water is unsafe, and environment is contaminated with filth exacting their toll on the poor in developing countries ( table ) . malaria is the vector-borne disease that causes the largest amount of morbidity and mortality. dengue, yellow fever, and zika are diseases that cause large outbreaks paralyzing health systems and contributing to considerable social and economic disruption. onchocerciasis causes blindness, chikungunya severe arthritis, japanese encephalitis permanent neurologic damage, chagas heart failure and early death, and schistosomiasis poor nutritional status and school performance. insect bites can cause problems and are venomous or nonvenomous. venomous insects attack as a defense mechanism injecting painful toxic venom through their stings. nonvenomous insects bite to feed on the blood of mammals (http://www. traveldoctor.co.uk/stings.htm). snakes are common in rural areas of tropical countries. snakebites are a serious occupational hazard for agricultural laborers and fishermen. generally, the two main families with maximum morbidity and mortality include viperidae and elapidae. snakebites have now been included as a neglected tropical disease with one of the highest rates of mortality as demonstrated by the million deaths study. about % of cancers worldwide are caused by infectious agents in the developing world, and the most important cause after tobacco. if infections are controlled up to in cancers in the developing world can be prevented. human papilloma virus is the most common infectious agent, followed by hepatitis b and epstein-barr virus contributing to cancers worldwide. protein energy malnutrition and micronutrient deficiencies of vitamins and minerals are common in tropical countries and these often contribute to delayed childhood growth and poor child health indices. although universal immunization and nutrient supplementation at child care centers through mid-day meal schemes have played a major role in mitigating this problem, these are often encountered in many poverty-stricken or famine-ridden countries. tropical diseases are classified as communicable and noncommunicable diseases. communicable diseases by definition include diseases that are transmitted to humans and in turn are further classified as those caused by bacteria, viruses, protozoa, parasites, and fungi. noncommunicable diseases are diseases caused by genetic and lifestyle factors, such as cancers, cardiovascular diseases, diabetes, and chronic respiratory diseases. in addition, snakebites, scorpion stings, and marine and terrestrial envenomations are also management dilemmas. it is important to note that because of constraints of poverty in the tropics, often unsafe food practices are condoned leading to high morbidity among individuals. air and water pollution can lead to a myriad of respiratory, gastrointestinal, skin, and neurologic disorders along with serving as carriers of infectious pathogens. although it is impossible to list all the causative pathogens of disease in the tropics, a syndromic approach and a region-wise probability of common tropical diseases is most helpful when dealing with a patient in the tropics or even a returned traveler. each area of the world is unique and endemic for specific tropical diseases. there is now through the geosentinel network a large amount of data about the causes of fever in travelers in contrast with data from the tropical regions. studies that are available use inconsistent definitions about "acute undifferentiated febrile illnesses" and diagnoses are not often confirmed. fever in the tropics is often syndromic and in the absence of appropriate and accurate diagnostics, common diseases are vastly overdiagnosed, such as malaria in africa and typhoid fever in south asia. the advent of rapid diagnostic testing has led to the recognition of other important causes of acute febrile illnesses in the tropics ( table ) . neurologic diseases in the tropics, although rare, are a cause of considerable morbidity and mortality. a history of travel including geographic locale and activities indulged in, possible exposures encountered, vaccines, prophylaxis and protective measures taken, along with the immune status of the host could help determine the etiologic organism of a particular neurologic syndrome. the neurologic syndromes are divided into global and focal syndromes ( table ) . skin lesions are a common problem in the tropics and they could be a primary problem or secondary to an underlying systemic condition. it is important to focus on infections that are treatable, transmissible, and have a high morbidity or mortality. the history must include details of previous travel, previous skin lesions, activities indulged in, immune status of the host, vaccinations, and prophylaxis. exposures to fresh or sea water, animals, arthropods, plants, breaks in skin including tattoos, sexual activities, and although tropics are often considered exotic locations skin problems can often be from cosmopolitan causes. sunburn, scabies, and prickly heat are common and chronic skin problems, such as atopic dermatitis, may exacerbate in a tropical environment. tropical biodiversity also results in a wide variety of plants and hence hypersensitivity to plants, plant products, and drugs may also occur. it is important not to forget mundane causes, such as pyoderma and folliculitis. because skin manifestations are myriad a syndromic approach does help to narrow down a definite diagnosis ( table ). diarrhea as a syndrome in the tropics has been described as "montezuma's revenge" and "delhi belly" because of the associated morbidity. it also is the second most important cause of child deaths younger than the age of years contributing to almost , . in the global burden of disease study diarrhea was a leading cause of death among all ages contributing to . million deaths. most of the deaths in children and adults were attributable to rotavirus, shigella spp, and salmonella spp. however, deaths on the whole have been reduced by . % from to . in the global enteric multicenter study done in children younger than age , interventions to reduce deaths should be directed against five pathogens: stable enterotoxigenic e coli, enteropathogenic e coli, cryptosporidium spp, rotavirus, and shigella spp. different clinical syndromes of diarrhea have been defined, each reflecting different etiology and pathogenesis. these are briefly described in tables and . various challenges exist in the diagnosis of tropical infections. the challenges are multiple and are divided as discussed next. the burden of disease and the kind of setting where the disease is being diagnosed often determines the need of an appropriate diagnostic test. in a high-prevalence, low-resource setting a test that is low cost, point of care, requiring little technical expertise, with a high positive predictive value is required. in contrast in a lowprevalence, low-resource setting, additional tests may need to be performed to confirm the diagnosis. subclinical and asymptomatic manifestations of a tropical disease may also make it difficult for a diagnostic test to distinguish between clinical disease and the former. in addition, if a test is serology-based in an endemic setting it may be difficult to establish causality for the clinical manifestations of the disease if baseline antibody titers is high. although there are obvious infrastructural and financial challenges in low-resource settings there are also impediments with the lack of trained laboratory staff and quality assurance of available laboratory diagnostics. in addition, an ideal laboratory test needs to be rapid, point of care, requiring minimal technical expertise, following norms the who constitution dictates, "all people share the right to the highest attainable standard of health." huge advances are being made toward internationally agreed global health targets, some of which include a reduction in child mortality by % between and and a % decline in aids related deaths since the peak of the hiv/aids epidemic in with at least half the infected people being able to access antiretroviral therapy. many international agencies, such as the who and unaid (joint united nations programme on hiv/aids), pharmaceutical companies and combating antibiotic resistant bacteria biopharmaceutical accelerator and the global antibiotic research and development partnership have incessantly campaigned for increased access to medicines leading to an achievement of many of the health-related millennium development goals. controlling, eliminating, and eradicating neglected tropical diseases has been a major focus for the who since as it moved away from specific diseases to the health needs of poor communities. over the years many disease conditions that were believed to require a concerted effort by the who were included. these were identified as follows ( table ) this has led the who to adopt five major strategies to combat these diseases: veterinary public health at the human animal interface: an integrated human and animal health approach is required for such diseases as cysticercosis, echinococcosis, and rabies, which involve vertebrate hosts. . provision of safe water, sanitation and hygiene: better sanitation and improved safe water supply coupled with vector control is being looked at for long-term economic growth and food production. tropical medicine is an amalgamation of infectious and noninfectious diseases and deals with many important issues, such as water, hygiene, and sanitation, which is out of reach for many low-and middle-income tropical countries. as a result, the health indices for these countries often suffer necessitating global and local public health interventions. research, development, global support, and funding along with access to major health interventions has empowered many of these countries to overcome the challenges faced by them while combating tropical diseases. water and waterborne diseases: a review world health organization estimates of the global and regional disease burden of foodborne bacterial, protozoal, and viral diseases, : a data synthesis world health organization estimates of the global and regional disease burden of foodborne parasitic diseases, : a data synthesis a review on major food borne bacterial illnesses improving food safety through a one health approach estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the global burden of disease study burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study access to medicine index -methodology report accelerating work to overcome the global impact of neglected tropical diseases. a roadmap for implementation. available at: www.who.int/neglected_ diseases/en a review of the global epidemiology of scrub typhus world malaria map global morbidity and mortality of leptospirosis: a systematic review burden of typhoid fever in low-income and middle-income countries: a systematic, literature-based update with riskfactor adjustment key: cord- -m so il authors: riley, david title: looking back, looking forward date: - - journal: glob adv health med doi: . /gahmj. . sha: doc_id: cord_uid: m so il nan i n the mid- s, the medical establishment believed that scurvy was a disease of putrefaction that was treatable with an elixir of vitriol and wort. the benefit of citrus was dismissed as unproven despite the fact that its regular consumption to prevent scurvy had been used by some cultures since antiquity and the english military had recommended citrus for scurvy for more than years. in , scottish physician james lind chose sailors suffering from scurvy for a clinical trial. those treated with citrus fruit quickly recovered. in lind's day, it was not known that vitamin c was necessary for the maintenance of healthy connective tissue. today, people around the world are spending billions of dollars annually on vitamins and supplements despite limited scientific evidence that regular supplementation is useful for the prevention of disease. a recent article concluded that "β-carotene, vitamin e, and possibly high doses of vitamin a sup ple ments are harmful" and that "other antioxidants . . . are ineffective for preventing mortality or morbidity." how and when are vitamins and supplements useful? in , the annual mortality rate from infectious diseases in the united states was approximately per , falling to deaths per in . in , the us food and drug administration issued voluntary "guidance rules" to reverse the steady increased usage of antibiotics to promote animal growth that accounts for % of the antibiotic usage in the united states. according to the us centers for disease control and prevention, more that people die annually from an antibioticresistant infection. have we entered an era in which one of the foundations of modern medicine is marginalized? the emergence of h n influenza virus, a virus with a high case-fatality rate ( deaths) in the populationdense areas of asia, is a concern, as is the reappearance of the h n influenza strain that caused the worldwide pandemic in and the middle east respiratory syndrome, which has a primary case-fatality ratio of almost %. can we develop vaccines that are safe and effective in a timely fashion? statins are routinely prescribed for the prevention of cardiovascular disease. the statin industry, driven by industry-sponsored trials, has been a spectacular commercial success, overshadowing the need for attention to lifestyle interventions, exercise, and nutrition. a recent randomized trial showed that a mediterranean diet reduced the risk of diabetes among persons at high risk for cardiovascular disease. do the new risk prediction algorithms derived from industry-sponsored trials encourage overtreatment at the expense of a healthy diet and lifestyle in the prevention of cardiovascular disease? will there be a shortage of more than physicians in the united states by ? legal battles continue to test the practice boundaries of clinicians who are not medical doctors. can clinicians such as advanced practice registered nurses, chiropractors, naturopaths, and traditional asian medicine practitioners deliver primary care within the scope of their training and licensure and collaborate in team care? the chinese ministry of education recently an nounced programs to train chinese medical researchers in ethics and methodology. these efforts encourage increased collaborations among the global science community as convergences in health and medicine support the development of a worldwide research infrastructure. in , an international group of experts met at the university of michigan, ann arbor, to create reporting guidelines for case reports: the care guidelines. these guidelines were presented at the international congress for peer review and biomedical publication in september and published in multiple medical journals. when case reports are systematically collected and aggregated into larger datasets, that information can be analyzed using techniques matched to big data, offering real-time, datadriven insights into what works for which patients. will this transform how we think about the creation of "evidence" and support the development of communities around data liquidity, transparency, and meaningful use? will we ask why a patient has a diagnosis and integrate the promotion of health with the treatment of disease as our attitude toward "observation" shifts? can systematically collected data from patient care be integrated with the best available research evidence to provide comparative effectiveness information that can measurably improve healthcare delivery and outcomes? stop wasting money on vitamin and mineral supplements trends in infectious disease mortality in the united states during the th century prevention of diabetes with mediterranean diets acc-aha guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults statins: new american guidelines for prevention of cardiovascular disease association of american medical colleges. gme funding: how to fix the doctor shortage the care guidelines: consensus-based clinical case reporting guideline development key: cord- -sdumq z authors: nesse, randolph m; stearns, stephen c title: the great opportunity: evolutionary applications to medicine and public health date: - - journal: evol appl doi: . /j. - . . .x sha: doc_id: cord_uid: sdumq z evolutionary biology is an essential basic science for medicine, but few doctors and medical researchers are familiar with its most relevant principles. most medical schools have geneticists who understand evolution, but few have even one evolutionary biologist to suggest other possible applications. the canyon between evolutionary biology and medicine is wide. the question is whether they offer each other enough to make bridge building worthwhile. what benefits could be expected if evolution were brought fully to bear on the problems of medicine? how would studying medical problems advance evolutionary research? do doctors need to learn evolution, or is it valuable mainly for researchers? what practical steps will promote the application of evolutionary biology in the areas of medicine where it offers the most? to address these questions, we review current and potential applications of evolutionary biology to medicine and public health. some evolutionary technologies, such as population genetics, serial transfer production of live vaccines, and phylogenetic analysis, have been widely applied. other areas, such as infectious disease and aging research, illustrate the dramatic recent progress made possible by evolutionary insights. in still other areas, such as epidemiology, psychiatry, and understanding the regulation of bodily defenses, applying evolutionary principles remains an open opportunity. in addition to the utility of specific applications, an evolutionary perspective fundamentally challenges the prevalent but fundamentally incorrect metaphor of the body as a machine designed by an engineer. bodies are vulnerable to disease – and remarkably resilient – precisely because they are not machines built from a plan. they are, instead, bundles of compromises shaped by natural selection in small increments to maximize reproduction, not health. understanding the body as a product of natural selection, not design, offers new research questions and a framework for making medical education more coherent. we conclude with recommendations for actions that would better connect evolutionary biology and medicine in ways that will benefit public health. it is our hope that faculty and students will send this article to their undergraduate and medical school deans, and that this will initiate discussions about the gap, the great opportunity, and action plans to bring the full power of evolutionary biology to bear on human health problems. ; harris and malyango ) . many medical students do not even accept the theory of evolution (downie ) . most medical students get two or more years of basic science education, including embryology, biochemistry, anatomy, histology, and physiology, and many get a genetics course from a professor who knows evolutionary biology. however, we know of no medical school that teaches a course in evolutionary biology as a basic medical science, and none that requires evolution as a prerequisite. our teaching experience confirms that few doctors have a chance to learn the principles of evolutionary biology most useful for medicine. are medical research and evolutionary biology better connected? new quantitative evidence comes from an innovative strategy for mapping citation patterns. instead of measuring co-citations between traditionally defined fields, rosvall and bergstrom ( ) define the boundaries of disciplines empirically from citation patterns. they then analyze the directed flow of citations between disciplines. the results for evolution and medicine are striking (see fig. ). ecology and evolution journals cite work in medical journals occasionally, but medical journals cite work in ecology/evolution journals too rarely to even show up on the diagram. almost all of the connections have other fields as intermediaries. there are good historical reasons for the gulf between evolution and medicine (zampieri ) . they stem partly from the timing of flexner's ( ) report that recommended bringing basic sciences into the medical cur-riculum. at that time, evolutionary biology was in eclipse. many scientists thought that lord kelvin's arguments about the rate of the earth's cooling proved darwin wrong (kelvin ) . others recognized that darwin's theory of transmission by gemmules was inconsistent with his theory of natural selection (richards ) . natural selection was not re-incorporated into biology until its underpinnings in population genetics were developed in the early to middle years of the th century (fisher ) . even then, those foundations emphasized mutations and genetic variations, not the shaping of complex adaptations by selection, a field that was only developed by evolutionary and behavioral ecologists in the s and later. those insights into trait evolution are just now being incorporated into medical science. mastering medicine is increasingly difficult. it includes far too much knowledge for any one person to learn: on this educators -and medical students! -agree. the challenge is to instill as much useful knowledge as possible in just a few years. the criterion of 'useful' is prioritized because medicine is a practical profession. patients want help and doctors need to know what to do. if a deeper understanding of a disease is useful, fine. otherwise, there is no time. every discipline makes recommendations, even demands, for curriculum content. in addition to the usual or so departments in a medical school, there are demands from groups representing geriatrics, statistics, gender issues, bio-informatics, nutrition, musculoskeletal systems, cancer, law, breast feeding and child abuse experts, among scores of others. each advocates for including more content in one area. put them all together and medical school would take decades. interestingly, proposals for curriculum reform tend not to emphasize ways to include more and more specific content. instead, a review of proposals for medical curriculum reform found that they consistently focused on values, especially the social nature and self-regulation of the medical profession (christakis ) . somehow, from all of these jostling interest groups, priorities and multiple regulations and examination requirements, a dean and faculty must come up with a curriculum. this makes it difficult to warmly welcome visitors who drop by to point out that a whole huge area of basic science has been omitted from the curriculum. however, evolutionary biology is not just another narrow topic, but a fundamental basic science. furthermore, it can help make medical education more coherent by giving students a framework for organizing the required facts. understanding the gap also requires consideration of how well-prepared evolutionary biologists are to apply their knowledge to the problems of medicine. most evolutionary biologists know as little about medicine as physicians know about evolution. if you cannot tell a myocardial infarction from cardiac failure, doctors will not pay attention. if they feel you are just adding to the thousands of facts they need to memorize, they will flee. not many evolutionary biologists are eager to teach medical students; the best are working hard on their own research. the gulf will be understood only by looking from both ends of this two-way street. before we consider opportunities and solutions, it is worth noting that medicine's isolation from evolutionary biology is just one example of the fragmentation that isolates many disciplines. some of the isolation results from academic structures that allow hiring and promotion to be controlled by narrow disciplines. universities talk a lot about promoting interdisciplinary work precisely because their structures so efficiently prevent it. however, disciplines exist for good reasons. there is too much to know. trying to synthesize work from diverse areas is frustrating, especially if the goal is general understanding, not some fine point. also, going beyond your specialty means you will inevitably get some things wrong. it is easier to maintain quality by keeping to a narrow focus. perhaps this list of problems will lead some readers to throw up their hands. we highlight the problems because we want decision makers to recognize that we are fully aware of them. acknowledging problems allows realistic solutions. we want also to emphasize that even large challenges are worth confronting because of the great benefits of bringing more evolution to medicine. most of this article is devoted to examples of rapid progress in applying evolutionary principles to medicine. overviews of evolutionary approaches to health and disease are available in several articles and books (williams and nesse ; nesse and williams ; stearns ; trevathan et al. ; stearns and koella ; trevathan ; o'higgins and elton in press) , and a critical review assesses progress and directions (stearns and ebert ) . the goal here is not to summarize recent work, but to step back to describe the structure of the developing field, the challenges it faces, and its potential. the examples are organized into three categories. some use well-established applications, some are new, and some remain mostly opportunities. they suggest actions that will allow evolutionary biology to provide maximum benefits to human health. at the core of evolutionary medicine is recognition that diseases need both proximate explanations of bodily mechanisms and evolutionary explanations of why natural selection has left the body vulnerable to disease. why do we have an appendix and wisdom teeth, a narrow birth canal, arteries prone to atherosclerotic blockage, and cells that can divide out of control? these are good evolutionary questions; they are fundamentally different from proximate questions. the distinction between evolutionary and proximate questions was emphasized by mayr ( ) , but it was tinbergen's ( ) article that outlined the four questions that must be answered to provide a full explanation for any biological trait. proximate questions . how does the mechanism work? . what is the ontogeny of the mechanism? evolutionary questions . how has this mechanism given a selective advantage? . what is the phylogeny of this mechanism? the first two questions are about the body's proximate mechanisms, from dna transcription and physiological regulation to bones, muscles and behavior. the third and fourth are evolutionary questions about how the body got to be the way it is. the four questions are complementary not competing. all four need to be answered for each trait. medical textbooks address question in detail, question sometimes; questions and only rarely. from this perspective, medicine has been using only one half of biology. understanding jaundice illustrates why both proximate and evolutionary explanations are essential. the yellow color in the skin and eyes is caused by excess bilirubin that accumulates most often because of liver failure. textbooks describe bilirubin as a potentially toxic metabolite of hemoglobin that can be excreted in bile only after it is made water soluble by conjugation with glucuronic acid in the liver. this is a proximate explanation that says nothing about why bilirubin exists in the first place. one might think it is simply a waste product. however, the intermediate step between heme and bilirubin is biliverdin, a chemical that is more soluble than bilirubin. so, why does the body go to the trouble to make a difficultto-excrete toxin? this is the evolutionary question. bilirubin is an effective antioxidant that can protect against the oxidative damage that contributes to aging (stocker et al. ; nesse and williams ) . oxidative damage is partially responsible for atherosclerosis, so many studies have looked to see if higher levels of bilirubin protect against heart attacks. they do, dramatically. levels of bilirubin higher than normal are characteristic in gilbert's disease; middle-aged people with this genetic condition have rates of heart disease sixfold lower than those with normal bilirubin levels (vitek et al. ) . however, there is no mention of evolution or natural selection in the article that reviews the studies of bilirubin protection against atherosclerosis (novotny and vitek ). an evolutionary perspective suggested an experimentlooking to see what happens if you knock out the enzyme that converts biliverdin to bilirubin. when the enzyme is working, potentially damaging oxygen radicals react with bilirubin, turning it into biliverdin, thus reducing the concentration of dangerous peroxide radicals up to -fold. without protection by bilirubin cells die quickly (snyder and baranano ; sedlak and snyder ) . this is a fine example of using the details of a proximate mechanism to test an evolutionary hypothesis. however, many relevant studies remain to be performed. no comparative study has investigated bilirubin levels in other primates to see if they are correlated with life span. more practically, researchers are now considering whether there could be possible disadvantages of using light exposure to reduce mildly elevated bilirubin levels in newborn infants (hammerman et al. ) . high bilirubin levels in the first days of life could be merely a result of the changeover to adult hemoglobin, but they could also help protect against oxidative damage from higher levels of oxygen and free iron exposure. the decision is delicate because too much bilirubin in the early days of life causes irreversible damage. when they first hear about evolutionary medicine, most doctors ask immediately, 'how can i apply it in the clinic today?' this surprises many basic scientists, who expect doctors to share the depth of their curiosity about why the body is the way it is. however, medicine is not a science, it is a practical profession. patients bring their problems; doctors try to help. the question, 'why has natural selection left the body so vulnerable to this disease?' seems very abstract to doctors who need to know right now, 'what is the problem? what treatment is best?' in response to doctors' demands for practical applications, it is tempting to offer quick examples of how evolution can inform everyday medical practice. this article reviews many: preventing antibiotic resistance, the benefits of inflammation, the costs of blocking normal defenses, the phylogeny of hiv, etc. however, offering examples too quickly creates two problems. first, even in these practical examples, evolutionary knowledge does not often change what a physician does in his or her day-to-day practice; instead, it guides research, as in the example of jaundice. treatment decisions are, and should be, based on controlled studies on humans, not on theory or on experiments performed on model organisms alone. darwinian medicine does not often give direct practice guidelines. second, merely listing quick applications sells evolutionary biology short. medical professionals learn other basic sciences not because they are useful everyday in the clinic, but because they provide a crucial depth of understanding and a framework for organizing the myriad facts in which the mind otherwise drowns. knowing the mechanisms and laws of acid-base balance gives a physician the perspective needed to apply formulas in the clinic. evolutionary biology offers the same sort of help, but on a much larger scale. instead of phenomena as specific as acid-base balance, evolution helps doctors make sense of why a disease exists at all, what environments increase the risk, and how treatments work. it has direct applications to medical research, but it also provides an otherwise missing paradigm for understanding why our bodies are vulnerable to disease. while no framework can capture all of the applications of evolution to medicine, recognizing two major distinctions is helpful. table shows the categories created by intersecting the two different evolutionary questions with a selection of things that need explanation. it is important to distinguish the two different kinds of evolutionary questions. answers to questions about phylogeny trace the evolutionary history of the trait in question. answers to questions about the adaptive significance of a trait try to understand why a trait is in the state we find it. historical and adaptive approaches use different methods to test hypotheses; both can deliver usefully different insights, often on the same issues. the other distinction is among the different things we want to explain. often the question is about why our bodies are the way they are, especially why selection has left us vulnerable to a disease. the object of explanation can be a universal trait, such as bilirubin, or it can be traits that differ, for instance, versions of certain genes. for instance, some people have versions of genes that increase depression rates (caspi et al. ; sen et al. ) ; why has selection not eliminated these alleles? many questions are about why all humans are all the same; many are about why we differ. the second major target of explanation is the evolution of pathogens -bacteria, viruses, worms and others. the large issues are the same, but they evolve much faster, so we can often observe their evolution, even in the laboratory where this allows experimental tests of hypotheses. another target for explanation is the evolution of cells within the body, particularly cancer cells and certain classes of cells in the immune system. cancer originates through mutation. cells that divide faster and better evade the body's surveillance systems become more common and spread: a standard evolutionary process. cells in the immune system also undergo a kind of evolution so that those that most effectively fight an infection multiply the most quickly. in both cases selection is not acting on organisms, but on cell lines within individuals; here too evolutionary principles can be useful. the applications of evolution to medicine divide naturally according to the types of questions asked. because answers to questions about phylogeny and adaptive significance require different methods and different skills, it is not surprising that these areas of research remain somewhat separate in evolutionary medicine. some of the most useful applications of evolution often do not use evolutionary theory directly; instead they use technologies developed by evolutionary biologists. in particular, methods for reconstructing phylogenies are being applied to genetic data with very practical results. hiv is especially susceptible to such methods because its fastaccumulating mutations create finely detailed phylogenies. for instance, certain cases of hiv could be traced back to a specific florida dentist (ciesielski et al. ) . phylogenetic analysis also was used to falsify the hypothesis that hiv was introduced into africa via polio vaccine (weiss ) . the sars epidemic was traced quickly to a corona virus similar to one endemic in bats (li et al. ; skowronski et al. ) . tracing pathogen phylogenies can be very useful. influenza phylogenies suggest which strains are likely to spread in future epidemics (bush et al. ; ghedin et al. ; smith ), information vital to decisions about vaccine design. the current h n avian influenza pandemic appears to have originated via reassortment between avian influenza strains circulating in eastern asia (li et al. ) . public health now uses such methods routinely to trace the source of contaminated foods. these phylogenetic methods have a remarkable reach, back even into prehistory. for instance, the complete genome sequence of the severely pathogenic shigella flexneri reveals that it is phylogenetically indistinguishable from the escherichia coli that lives normally in the human gut (wei et al. ) . the difference seems to be in a few virulence factors that result in substantially different ecological niches for the two organisms. technologies for tracing phylogenies have ready application to antibiotic resistance and to pathogen evolution in general. they are particularly powerful in revealing the origins of emerging diseases. for example, hiv originated in chimpanzees in central africa, and hiv originated in sooty mangabeys in west africa (heeney et al. ). importantly, these species do not develop aids. phylogenetic methods have also found recent applications in cancer research and treatment. cell lines differentiate as mutations accumulate, and the genetic differences make it possible to trace the sequence. two tumors that are histologically identical can have very different proteonomic signatures that make it possible to assess the level of cellular differentiation (abu-asab et al. ) . whether a tumor is all derived from the one line of cells, or from different origins arising during the tumor's growth may also be an important indicator (merlo et al. ; frank ) . researchers in every area of medicine use phylogenetic methods to analyze genetic data. sometimes they are used in conjunction with evolutionary theory, but they are also used independently to construct phylogenies with new applications in an era of genetic medicine. doctors who understand these phylogenetic methods and the evolutionary biology behind them will be better prepared to judge the significance of research findings such as those summarized above. as most readers know, evolutionary biology took off only after it was synthesized with population genetics in the s and 's (fisher ) . mathematical treatments of allele frequencies that incorporated selection, drift, mutation, and migration made it possible to begin to understand the forces that shaped the genome. as lewontin ( ) has noted, however, this theory developed separately from breeder's theories about selection for phenotypes; the task of mapping changes in allele frequency to changes in phenotype remains a challenge. while this gap remains substantial in much of medicine, it is being reduced by the explosion of work on quantitative trait loci (qtls) and single nucleotide polymorphisms (snps), e.g., the wellcome trust case control consortium ( ) . the explosion of genetic information tends to focus attention on genetic differences between individuals. much research is trying to explain these differences and their significance. an increasing proportion of this work looks for evolutionary explanations. relatively overlooked, however, are questions about why all members of a species are the same. this is especially important with regards to traits that leave a species vulnerable to disease, such as wisdom teeth, the appendix, or a narrow birth canal. such traits are also in need of evolutionary explanations, and information on genetic variations is not always helpful. many physicians think of genes that cause disease as abnormalities in an otherwise 'normal' genome. this is a nonevolutionary view on two counts. first, it tacitly views the genome as a product of design with a blueprint that defines 'normal.' the genome is, instead, a collection of those genes that have tended to increase reproductive success (or hitchhiked on the success of other genes) while interacting with each other and the environment to construct a functional organism. second, while some dna sequences can be accurately described as 'damaged', it is increasingly clear that many medically relevant genetic variations are helpful or harmful only in interaction with particular aspects of environments. such genes have been called 'quirks' to distinguish them from defective genes that cause problems in all environments (nesse and williams ) . for instance, if you have the genes for nearsightedness, you will almost certainly become nearsighted. unless, that is, you live in a culture where children are not taught to read (norn ) . the problem comes only when certain genes and certain environments interact. similarly, some genes that interact with high fat diets to cause atherosclerosis are quirks that would be harmless if we lived and ate the way people did thousands of years ago. like phylogenetics, population genetics is a mature technology already applied widely and effectively throughout medicine. there are new applications, and one could quibble about whether users of these methods are thinking in evolutionary terms or just using technologies that work. nonetheless, population genetics cannot be separated from evolutionary theory and, as such, is a wellestablished area evolutionary medicine. new applications of evolutionary principles have brought spectacular progress in several areas of medicine during the past years (stearns and ebert ) . studies of infectious disease and aging have been especially transformed. for the sake of continuity, however, we begin with progress coming from increasingly sophisticated evolutionary genetics (maynard smith ; jobling et al. ). established principles of population genetics are being augmented by new ideas and techniques. especially interesting are new strategies for using 'signals of selection' to determine which genes have been strongly selected in the past few thousand generations. just a few years ago, this approach offered a few methods and a few examples (olson ) . now, many new methods are applied to genome scan data to identify loci subject to directional and balancing selection as revealed by the homogeneity of the dna sequences surrounding the loci in question (vallender and lahn ; sabeti et al. ; voight et al. ) , and early overestimates of the number of loci of interest are now being corrected (thornton and jensen ) . these methods provide answers to long-standing questions, such as the origins of genes for lactase persistence (bersaglieri et al. ). most adult humans cannot digest milk because the enzyme that breaks down milk sugar is not made in adulthood. recent studies showed that genes that allow adults to digest milk have evolved separately several times, almost always in dairying cultures (holden and mace ; ingram et al. ; tishkoff et al. ) . similarly, there has been speculation for decades about whether the genetic tendency to feel sick immediately after drinking alcohol could be common in people from asia because it protects against alcoholism in a culture where alcohol has long been available. evidence has been sparse until now. the case has been bolstered by finding a strong signal of selection in asians at the site of the gene (voight et al. ) . the evolutionary backgrounds of alleles that predispose to disease can now be examined. of particular interest is a gene that makes apolipoproteins, substances that bind and transport lipids. individuals with the apoe subtype have a much higher risk of developing atherosclerosis and alzheimer's disease. this allele is universal in other primates. in humans, especially those living in cold climates, selection has increased the rates of the apoe allele (sapolsky and finch ) . this may be a case of selection caught in action, perhaps for genes that prevent health problems for meat eaters (finch and stanford ) . selection has also been proposed as an explanation for cystic fibrosis, given the scores of mutations that can cause it and its systematic variation with latitude. mice heterozygous for the cf allele have less fluid loss from cholera toxin (gabriel et al. ), but the chloride channel is not the rate limiting step for fluid loss in humans (hogenauer et al. ) . the cf gene also prevents entrance of salmonella typhus into gastrointestinal mucosal cells (pier et al. ). however, cystic fibrosis is more common in climates where diarrheal diseases are less common, and although remarkably prevalent, it remains a rare allele. cystic fibrosis offers a fine example of creative tests of interesting hypotheses, and an example of how hard it can be to reach a firm conclusion about the adaptive significance of a genetic variation. until recently, agreement on how to assess the role of selection on vulnerability genes was elusive (chadwick and cardew ) . that is changing fast. we now have systematic reviews of the role of selection in maintaining the prevalence of genes that increase risk for infectious disease (dean et al. ) , and progress in related areas is on the way. naïve thinking that genes exist always for the good of the individual and the species remain common in medicine even though biologists abandoned them in the s. the importance of gene-level selection is highlighted in the work of trivers and others on selfish genetic elements that facilitate their own transmission at the expense of the individual (burt and trivers ) . these are dawkins' selfish genes with a vengeance (dawkins ). the best known examples are the t-allele in mice and segregation distorter in fruit flies. the role of selfish genetic elements in human disease, including cancer (crespi and summers ) , is an especially exciting area that is starting to be elucidated. these advances also suggested looking for conflicts that arise between genes transmitted through males versus those transmitted through females. following the lead of trivers ( ) , haig ( ) pointed out that in pregnancy, the interests of genes from the male differ from those from the female. genes derived from male benefit if they somehow induce the female to make more or larger offspring. energy reserves that a female mouse does not invest in the current litter will not benefit the male unless he happens to mate with her again. conversely, genes from the female benefit by reserving fat stores for future reproduction. the size of offspring that maximally benefits the male is only slightly different from the size optimal for the female, but this small difference may have shaped a complex system. this evolutionary hypothesis is supported by the details of a remarkable proximate mechanism. studies of genetically engineered mice show that the unopposed expression of a gene called insulin-like growth factor (igf ) results in a large placenta and large but otherwise normal offspring; this outcome benefits paternal genes. when transmitted through the mother, this gene is inactivated by a process called imprinting, making the offspring smaller. igf r is a gene with opposite effects; it degrades igf. its effect is decreased by imprinting from passage through the father (haig ) . loss of igf imprinting causes beckwith-wiedemann syndrome, characterized by large babies with very large internal organs. it may be more likely in offspring conceived using artificial reproductive technologies (maher et al. ) . this area of research vividly illustrates the clinical implications of studies that would never be considered without sophisticated applications of evolutionary theory (wilkins and haig ) . addressing a much broader issue, it is worth noting that 'knock-out' studies are about the evolutionary functions of a gene. they are modern equivalents of the old physiological method of extirpation. taking out an organ or a gene and looking to see what goes wrong can generate hypotheses about how an organ or gene is useful. often, no abnormality is observed. of course, this does not mean that the gene is useless, only that its effects are covered by redundant systems, that its benefits are manifest only in special situations, or that the benefit is just too small to be observed in a laboratory setting. for instance, genes involved the capacity for shivering might well appear to be harmful, unless one happened to look at their effects in extreme cold. similarly, some genetic variations associated with faster aging are likely to have compensating advantages, otherwise they would have been eliminated. as we are gaining technologies to manipulate genes, evolutionary thinking about their origins and functions becomes more crucial than ever. aging research shows how evolutionary thinking can transform a field. many doctors still view aging as an inevitable result of body parts wearing out. this knowledge gap is unfortunate for a trait so important to medicine. half a century ago, medawar ( ) saw that selection weakens with age because the surviving number of individuals declines, even in the absence of senescence. then williams ( ) had the insight that pleiotropic genes that cause aging and death can nonetheless be selected for if they also give benefits early in life when selection is stronger. he gave a vivid hypothetical example of a gene that makes bones heal faster in childhood, but that also slowly deposits calcium in the coronary arteries. hamilton ( ) provided mathematical models for the process. these evolutionary insights transformed aging research (finch (finch , . instead of looking only for proximate explanations for aging, the field now also seeks evolutionary explanations for why aging mechanisms exist at all. laboratory (rose ; stearns et al. ) and field evidence (austad ) soon showed that aging was a life history trait shaped by natural selection (stearns (stearns , . for many species, senescence in the wild is a deleterious trait with heritable variation, but life spans do not increase, presumably because the reproductive benefits of longer lives would be balanced by costs that decrease reproduction earlier in the life span (nesse ; austad ; williams et al. ) . the big new news in aging research is the discovery of remarkably strong effects of single genes that influence oxidative metabolism (guarente and kenyon ; austad ). these surprising findings are now being interpreted in evolutionary terms (partridge and gems ; ackermann and pletcher ; mcelwee et al. ) . they suggest that mechanisms that protect against oxidative damage are limited by their reproductive costs or just lack of selection. they also show how selection can shape special states of reduced metabolism that allow some species to survive periods of privation. these states slow aging dramatically, but they are special states precisely because they also so dramatically reduce reproduction. the ancient dream of extending lifespan no longer seems like just a dream, but do not buy beach property on hudson bay just yet. when it comes to aging, males are the weaker sex. it has long been known that men die younger than women, but this has rarely been interpreted in a life-history framework. a recent report about higher mortality rates for male than female mammals attributes it to both external causes and faster aging. the faster rates of aging for males are found mainly in polygynous species because a shortened reproductive span decreases the force of selection for older males (clutton-brock and isvaran ). an evolutionary view of humans suggested looking at the ratio of male to female mortality across the lifespan in different cultures. this found surprising results (kruger and nesse ) . in every culture at every age through late adulthood, mortality rates are higher for men. in modern societies, for every woman who dies at reproductive maturity, three men die. the pattern is consistent in cultures studied. further work looking at the proximate causes of sex differences in mortality rates finds that they result not only from accidents and violence, but also from the full range of causes of mortality. applications of evolutionary biology to infectious disease are also very direct. pathogens evolve fast, right under (and in!) our noses. antibiotic resistance is the classic example. individual bacteria and viruses vary in their susceptibility to antimicrobial agents; those with even slight resistance replicate faster and their genotypes become more common. just a few years after alexander fleming discovered penicillin, he also discovered antibiotic resistance. the basic phenomenon is very simple. antibiotics are selction agents that quickly increase the proportion of organisms that can resist them. (bergstrom and feldgarden ) . shortly after the us surgeon general declared in the mid- s that the war on infectious disease was over, antibiotic resistance became a serious problem. staphylococcus quickly became resistant to penicillin, nearly all other bacteria followed. antibiotic resistance is an arms race; we invent new defenses, the enemy quickly finds ways around them, and we try to find new defenses. we are now faced with many organisms that resist every available antibiotic; some wonder if the war on infectious disease may be lost (normark and normark ; levy and marshall ) . nearly % of staphylococcus aureus are now resistant even to methicillin; infections caused by this resistant organism now cause deaths per year, more than the caused by aids (klevens et al. ). the economic burden of antibiotic resistance is estimated at about $ billion annually in the usa. recognition of antibiotic resistance as an example of natural selection is often missing in medical articles on the topic. in biology journals the phrase 'natural selection' or another direct reference to evolution is used . % of the time to describe antibiotic resistance, but in biomedical journals they were used only . % of the time. instead, medical journals use 'emergence' or some other circumlocution to avoid the 'e-word' (antonovics et al. ) . many doctors view antibiotics as human discoveries, but most are results of selection acting over millions of years in the deadly interactions of bacteria and fungi with each other. the average bacteria isolated from soil demonstrates resistance to seven antibiotics (d'costa et al. ). this is not because of exposure to human-produced chemicals, but because the long co-evolution of bacteria and fungi has shaped toxins, defenses and new toxins (ewald ) . bacteria and fungi have been developing and testing the effectiveness of antibiotics for millions of years! another important aspect of resistance is whether it has costs to the resistant bacteria that will select against the resistance if antibiotics are withdrawn. the answer is sometimes yes, but often the costs seem to be so low that resistance persists, an ecological insight of huge importance for controlling antibiotic resistance (andersson and levin ) . continuous application of antibiotics also produces selection to reduce their costs, yielding resistant strains that persist after the antibiotics are withdrawn (schrag and perrot ) . however, restriction of antibiotic use in danish farm animals resulted in decreased resistance (aarestrup et al. ). more work on these evolutionary responses is of great importance. selection on pathogens is, of course, not a one-way street. hosts evolve too, creating co-evolutionary cycles of deception and ability to detect deception of vast complexity (ewald ; knodler et al. ; frank ) . the genes of vulnerable individuals become less common, and host resistance evolve, but very slowly compared with the rate of pathogen evolution. some of the resulting genetic change is in mechanisms close to the sites of infectivity. for instance, malaria uses the duffy antigen to enter red blood cells. individuals without the duffy antigen are less susceptible to malaria and have a selective advantage where malaria is common (hamblin and di rienzo ) . this is why the duffy antigen is absent in most africans. the ccr- receptor on white blood cells allows hiv to enter. the receptor is absent in about % of europeans; they do not get aids even when infected with hiv (samson et al. ) . some geographical evidence suggested that this genetic difference could result from selection by the plague epidemic in the th century, but in a nice example of hypothesis testing, more careful examination shows the patterns do not match (cohn and weaver ) . would we all be better off without the ccr- receptor? with the advent of hiv the answer may be yes, but this receptor is not useless; at the very least it appears to protect against west-nile infection (lim et al. ) . when a parasite such as malaria deals with both a mosquito and a mammal host, the complexity of its evolution is magnified (mackinnon and read ; grech et al. ) . here host-parasite manipulations can be studied in detail, and their complexity is more than intriguing. doctors learn about the complexity of parasite life cycles, but rarely do they have an opportunity to consider their evolutionary origins. nor do they have the evolutionary principles that would allow them to evaluate proposals to drive genetically engineered strains of mosquitoes into wild populations. such proposals rarely take into account how the introduced strains will evolve in interaction with the wild ones. changes in the phenotype also exert selection forces on pathogens. vaccination of large populations fundamentally changes the environment for a pathogen. for instance, steady pertussis vaccination for years may have selected for more virulent strains of the whooping cough bacteria (diavatopoulos et al. ) , although decreased vaccination may be responsible for the increased incidence. imperfect vaccines can create selection pressures for increased virulence (gandon et al. ). this disturbing possibility has been documented for marek's disease in chickens (davison and nair ) . however, when a vaccine targets a toxin, selection can decrease virulence. this has happened for diphtheria, where lines that do not produce toxin have largely displaced the dangerous forms (soubeyrand and plotkin ) . these findings have obvious major public health implications, but the complex realities of host pathogen interactions make confident prediction difficult (ebert and bull ) . intuitive models for antibiotic resistance are often incorrect (normark and normark ) . for instance, some hospitals have tried rotating the antibiotic of choice over a period of a few months with the idea that by exposing bacteria to changing selective regime, this will prevent antibiotic resistance. but when the process is modeled, this turns out that antibiotic rotation is ineffective at creating a more heterogeneous suite of selective conditions. at least in principle, hospitals would do better to use a mix of different drugs on different patients simultaneously, rather than to cycle through these different drugs over time (bergstrom et al. ) . perhaps equally important are more general but lessrecognized selection forces from infectious agents. we have a wide variety of protective bodily responses, such as fever, cough and vomiting, that are held in reserve until released by a mechanism that detects the presence of pathogens (ewald ) mechanisms that regulate expression of these defenses are under constant selection (nesse c) . individuals vary in how high fever rises during infection, how quickly immune cells are activated, and how much diarrhea is produced for a given level of infection. most symptoms of infectious disease are not caused directly by the pathogens: they result from these useful defenses. some are aspects of the inflammation and immune systems that attack pathogens. others, such as cough, diarrhea and vomiting, extrude pathogens. for all such defenses, one might think that selection would shape regulation mechanisms to be close to the optimal. but what is optimal? the answer is surprising. when the cost of a false alarm is low relative to the possible costs of not expressing a sufficient defense when it is needed, selection shapes regulation mechanisms that express the defense more readily or more intensely than seems sensible. we put up with smoke detectors that sometimes wail when we make toast because we want to be sure they warn us about any real fire. the 'smoke detector principle,' applies signal detection theory to yield quantitative predictions about how selection shaped defense regulation mechanisms (nesse c ). it has clinical relevance because so much everyday medicine involves prescribing medications that block defenses such as fever, pain and cough. this tends to be safe because the body has redundant defense mechanisms and because the thresholds for defense expression are set by the smoke detection principle. sometimes, however, it is fatal. far from suggesting that doctors should let nature take its course, an evolutionary perspective suggests that many defensive reactions are excessive or entirely unnecessary. it also suggests that we have only begun to study a crucial set of principles at the core of general medicine. general practice could have a stronger foundation in science if practitioners had tools for thinking about how selection shaped defense regulation. most already know that using codeine to block cough after surgery is likely to result in pneumonia, and an increasing number recognize the utility of fever. however, only a few are thinking about how natural selection shaped the mechanisms that regulate defenses. such thinking will lead to new studies that provide the evidence we need to make better clinical decisions. in one particularly important example, a debate is now underway about whether influenza kills people directly or via the effects of released inflammatory agents (salomon et al. ). if the former is true, anti-inflammatory drugs will increase death rates, if the latter is true it will decrease them. the central defense against pathogens is, of course, the immune system. the costs as well as the benefits of immune responses need to be analyzed in evolutionary perspective as a life-history trait (zuk et al. ; lochmiller and deerenberg ; zuk and stoehr ; schmid-hempel and ebert ) . in addition to energetic costs, there is tissue damage from immune surveillance, reproductive costs, mate display costs, and others. of particular interest is variation in immune response, either because of limited resources or facultative systems that adapt the response to the current inner and outer situation (schmid-hempel ) . the study of pathogen virulence offers another example of how an evolutionary perspective can transform a field. just a decade ago, many physicians were taught that natural selection tended to shape pathogens and hosts to a benign mutual co-existence. after all, why kill the host that feeds you? rigorous evolutionary analysis revealed that this view is fundamentally incorrect may and anderson ; ewald ; frank ; ebert ) . the most important factor shaping virulence is its influence on the probability of transmission to a new host; virulence is shaped to whatever level maximizes transmission. for instance, prior to modern sanitation, bedridden patients with cholera could infect others and the organisms causing the most diarrhea were transmitted the most. the result is often fatal, but such traits are nonetheless selected for if they maximize transmission. this could have major implications for public health. good water purification systems prevent infection from bedridden patients, thus shifting the advantage to less virulent organisms whose victims can be up and around to spread them. virulence levels can also be influenced when several genetically different pathogen strains compete within a host. this should select for increased virulence. studies of trypanosomiasis (sleeping sickness) suggest multiple infections may be much more common than previously suspected (balmer and tostado ) . much of the recent work in evolutionary medicine asks questions about why natural selection has left the body vulnerable to disease (williams and nesse ; ewald ; nesse and williams ; stearns ; trevathan et al. ) . six categories summarize the main possible explanations: mismatch with the modern environment, pathogens coevolving with hosts, constraints on what selection can do, unavoidable trade-offs, reproduction at the expense of health, and defenses such as pain and fever that are useful despite causing suffering and complications (nesse b) . these are potential evolutionary explanations for why selection has not made the body more resistant to disease. they are fundamentally differ-ent from proximate explanations about how the body works. the last two are not exactly explanations for disease vulnerability, but they need to be on the list because they are so often the source of misunderstandings. some hypotheses can be tested with a definitive experiment, others with comparative data, and some must be assessed by comparing observed features to those expected given the hypothesis (nesse ) . like much in science, this can be challenging. the 'thrifty phenotype' refers most generally to the benefits of weight gain and other mechanisms that conserve calories in environments characterized by erratic nutrition (neel et al. b ). the extraordinary vulnerability to obesity in certain groups, such as pima indians and inhabitants of the south pacific island of palau, has been suggested to result from generations of experience with erratic food supplies. anthropological data on cultural variations in nutritional stability do not well support this interpretation (benyshek and watson ) . however, the more general idea that selection maximizes calorie conservation remains useful. natural selection may also have shaped mechanisms that adjust metabolic systems to cope with different nutritional environments. many studies demonstrate that low birth weight is a significant risk factor for obesity and diabetes in diverse populations (barker et al. ) . the evolutionary question is whether this 'fetal programming' is a 'predictive adaptive response' resulting from a mechanism shaped by selection to monitor fetal nutrition and adjust development in ways that facilitate coping with deprivation (gluckman et al. ) , or whether the association arises for other reasons (wells ) . low birth weight is also correlated with differences in stress reactivity (clark ) and rates of depression (costello et al. ). the adaptive significance of these reactions is as hard to figure out as the reactions are important. whatever the answer turns out to be, these evolutionary applications to medicine and public health nesse and stearns ª the authors studies have called our attention to the importance of the physiological state of mother and infant for the prevalence of lifestyle diseases later in life, with some well-documented effects delayed by several decades. has natural selection shaped a mechanism to detect and reject a fetus that is likely to succumb early to infection? a surprising amount of evidence is consistent with this hypothesis. the early term miscarriage rate is over % (boklage ) , and siblings tend to be more different in their hla immunological types than expected by chance. this suggests that other conceptions who received similar hla genes from both parents may have been selectively lost (ober et al. ) . while giving up a conception is inefficient, continuing to support a fetus who will likely succumb to infection is even more so, thus creating a selection force that could shape such a system. related evidence shows that spouses in small local communities tend to have hla types more different than expected (ober et al. ) . pheromone cues may guide individuals towards mates who differ sufficiently from themselves (jacob et al. ) . that the human female reproductive tract has been shaped to screen defective gametes and concepti is now well supported. that humans detect and choose mates based on immune complementarity is suggested by several studies but not yet definitively confirmed (loisel et al. ). the huge decreases in human mortality in the past century come not mainly from medical treatments, but from public health interventions, vaccination and sanitation in particular (armstrong et al. ) . they have, together, done more than all of the rest of medicine to improve human health. they also have created an environment vastly different from the one we evolved in. one result is a decreased burden of parasites such as worms in the gut. during most of human evolution we lived with helminth parasites. their absence in modern societies may help explain the vastly increased rates of autoimmune diseases, not just allergies, but diabetes and the childhood leukemias (elliott et al. ). regulation systems, including those that screen for antigens that react with self, were shaped with significant helminth loads on board (weinstock et al. ) . new evidence suggests that helminths evolved a capacity to make a protein (es- ) that down-regulates type-ii immunity that would otherwise attack them (melendez et al. ) . where helminth treatment has been initiated, asthma and crohn's disease rates have gone up (hurtado in press) . the cross reactivity between antibodies on schistosomes and dust mites, and different genetic levels of protection against helminths, may help to explain higher rates of asthma in people of african origins (barnes ) . in a bold clinical application, patients with an immune bowel disease, crohn's disease, were treated with the live ova of pig whipworm. about % entered remission . we can expect fast progress in autoimmune disease thanks to improved evolutionary understanding of the rule of modern hygiene. evolutionary approaches to cancer in general have progressed so quickly that their scope can only be suggested here (greaves (greaves , . the very existence of cancer results from an evolutionary process: differential replication of mutated cells (merlo et al. ) . the constant tendency for faster replicating cells to displace others is rigidly controlled by systems that regulate cell division and by surveillance systems that kill cells that are not where they belong. the length of telomeres, the bits of dna that hang from the end of chromosomes, may protect against cancer. each time a cell divides, the telomere get shorter; when it is gone, the cell dies. however, there is a side effect. short telomeres also shorten life-span (blasco ) . mathematical treatments of genes that predispose to cancer (crespi and summers ) and cancer cell evolution (frank ) offer promise of bringing coherence to this difficult field. the greatest opportunities for evolutionary applications relevant to health may be in public health and epidemiology. many have already been mentioned above, from diet to genetic epidemiology. every project needs an individualized application, but a few generalizations may help. for instance, when looking for risk factors for common diseases, the first question is whether the condition is equally common in hunter-gatherer populations. if not, then novel factors in the modern environment should top our list of suspects. some already do, such as too much fat and too little exercise. other factors, like the hygiene hypothesis mentioned above, are increasingly well supported. other apparently innocuous aspects of the modern environment deserve special attention. for instance, ubiquitous lighting has transformed our lives. instead of settling down to slow pursuits when darkness falls, we read, study, dance and watch television until long after we would have otherwise gone to sleep. the light itself may be risky. melatonin levels increase in the dark. a study of visually impaired women -who tend to have higher than normal melatonin levels -found risks of breast cancer about half of the rates for other women (kliukiene et al. ) . a subsequent study of nurses found those doing shift work and others exposed to light at night had increased cancer rates (stevens ) . in a fine demonstration of the value of research connecting proximate mechanisms with evolutionary hypotheses, melatonin-depleted blood from postmenopausal women has been shown to speed the growth of human breast cancer xenografts on nude mice (blask et al. ). more work is needed on this, but even now it suggests a new set of risk factors we should measure, and some simple public health advice -sleep with the lights off. obesity has doubled in the past years in the usa, so that two-thirds of adults are now overweight or obese (wang and beydoun ) . diabetes and obesity are strongly correlated (neel et al. a) . about million adults worldwide have diabetes, and type diabetes (late onset) is exploding. most diabetic patients are in india and china, and diabetes rates are expected to double, from million in to million in (wild et al. ) . much of the individual difference in vulnerability is accounted for by genetic differences (echwald ) , but this does not mean the obesity epidemic results from genetic abnormalities. instead, it means that novel aspects of our modern environment interact with genetic quirks to cause the problem, as it the case for nearly every polygenic disease. while not all ancestral environments were alike (elton in press), it does seem clear that modern diets are vastly different from almost everything that came before. we know what we should do to stay thin. we should eat less and exercise more. so, why don't we? one answer is that in the past individuals who were thin or who wasted calories in nonproductive exercise tended to have fewer children. selection favored those who took advantage of opportunities to eat fat, salt and sugar and who stored some extra calories in good times. selection has shaped mechanisms that limit weight gain, but they are feeble compared with those that prevent weight loss. these arguments have been made many times (eaton and konner ; eaton et al. ; chakravarthy and booth ) , but nutrition researchers sometimes still see these evolutionary hypotheses as alternatives rather than complements to new insights about molecular and physiological mechanisms that regulate caloric intake. an evolutionary view suggests two conclusions about diet, both unwelcome. first, most of us have built in tendencies to overeat and under-exercise when good food is available without much effort. second, there is no such thing as a completely natural diet that is perfectly safe. eating less fat is certainly wise, but it has costs. a diet of all wild vegetables will include poten-tially toxic substances. nonetheless, evolution does offer a way to ground the otherwise faddish area of nutrition research in a solid general understanding of the diets of our ancestors (eaton et al. ; leonard ; ungar ) . evolutionary principles are just beginning to be applied to mental disorders (nesse (nesse , a wenegrat ; baron-cohen ; mcguire and troisi ; badcock and crespi ) , and they promise to bring them into the fold with other medical disorders (nesse ) . perhaps paradoxically, this may finally happen by recognizing the utility of negative emotions such as anxiety and depression (gilbert ; nesse ) . about half of mental disorders are emotional disorders characterized by excesses of negative emotions. while there is no doubt that much anxiety and depression is pathological, the capacities for anxiety and depression were shaped by natural selection along with the mechanisms that regulate them. these disorders are not like diabetes or parkinson's disease where a specific pathological lesion causes the disease. they are, instead, more like chronic pain or chronic cough, where the problem is dysregulation of a response that can be normal and useful. recognition that such evolutionary explanations are needed in addition to proximate explanations of mechanisms is just now dawning, along with recognition that categorical diagnoses that take no cognizance of environmental factors are fundamentally mistaken (nesse and jackson ; wakefield and horwitz ) . genes interact with environmental factors to create mental disorders. for instance, a study of a serotoninrelated polymorphism found that its strong effects on depression vulnerability were almost all mediated via an interaction with the number of severe life events (caspi et al. ) . this has become an exemplar for studies of gene · environment interactions. however, the measure of environmental effects, the number of severe life events, is crude compared with the sophistication of genetic analyses, especially in light of growing knowledge that low mood can be useful in certain special life circumstances (brown et al. ; nesse ; heckhausen et al. ). there are good theoretical reasons for thinking that low mood escalates to depression when an unreachable major life goal cannot be given up, and some supporting laboratory data (carver and scheier ) , but the case has not yet been proved. disorders such as schizophrenia require fundamentally different explanations. older ideas about the adaptive value of schizophrenia are now mostly discredited, although a haplotype associated with higher iq is also associated with a higher risk of schizophrenia (meyer-lindenberg et al. ). also, a haplotype associated with a gaba-a receptor shows clear signs of positive selection, which are weaker in lineages with schizophrenia (lo et al. ) . of particular interest is the hypothesis that autism and schizophrenia may be the flip-sides of extremes of the competition between imprinted genes coming from the father and the mother, the haig idea applied to psychiatry (badcock and crespi ; crespi et al. ). substance abuse is both more straightforward and more difficult. the straightforward aspect is that most drugs that affect the central nervous system evolved in plants to protect them from insects. in modern environments we create increasingly clever ways of purifying and administering them making addiction more common and more devastating. they hijack brain mechanisms that evolved to regulate behaviors such as foraging for ripe nuts (nesse and berridge ) . the problem becomes quickly complex, however, because of profound individual genetic variations in vulnerability to substance abuse that interact in complex ways with social environments that vary even from month to month for individuals (zucker ) . the field in general is gradually moving from an exclusive focus on proximate mechanisms and individual differences, to a broader consideration of the origins of vulnerabilities humans share and how they interact with certain environments to create disease. this review supports a global conclusion: much interesting and important research is taking place at the intersection of evolution and medicine. this research ranges from well-established applications of population genetics and phylogeny to new applications of evolution to specific medical problems such as infectious disease and aging. work in the area is growing rapidly. the fastest growth is in two disparate areas. first are those where evolution helps to make sense of new genetic data. why, for example, do genes that predispose to asthma persist? who would have thought they protect against schistosomes? growth is also fast in research asking new questions about why selection has left the body vulnerable to specific diseases. why, for instance, does bilirubin exist? who would have thought it was to slow aging? another question is whether evolution and medicine is one field, or is it just a collection of applications of different aspects of evolutionary theory? the increasing number of books and conferences that cover the full range of applications has found a large and eager audience. these initiatives have brought together diverse scientists and clinicians who are often delighted to learn about each other's work. in their shared evolutionary foundations, anthropologists, geneticists, physiologists, mathematical modelers and parasitologists turn out to have much in common. this is not artificial interdisciplinarity; advances in understanding evolution illuminate all of these fields and research in each of these fields offers opportunities for advancing evolutionary biology. the structure of evolutionary medicine is still defined mainly by the different contributing disciplines. genetics, paleontology, microbiology and immunology, ecology, reproductive medicine, cancer research, physiology, anatomy, behavioral biology, epidemiology, anthropology, and clinical medicine -all pursue evolutionary questions using somewhat different traditions and methods. a new framework, perhaps one based on questions such as those in the table in the introduction, may emerge. for now, the important observation is that workers in different field are increasingly finding commonalities in their shared foundation: evolutionary biology. awkward tensions always lurk when scientists from diverse disciplines come together. those working at more reductionist levels, sometimes pull away from those working on higher level questions. some doctors are uncomfortable welcoming in another discipline they do not know well. some evolutionary biologists grow quickly impatient with doctors who do not already know all about evolutionary biology. whether this new field can avoid such fragmentation remains to be seen, but many research opportunities provide a unifying force. for instance, research on lactase persistence benefits markedly from close collaboration between geneticists and anthropologists, and their conclusions have clinical relevance. a related challenge is dealing with hypotheses about adaptation (rose and lauder ) . experimental methods are not often available, many scientists are unfamiliar with comparative and other methods, firm conclusions can be elusive, and standards of evidence are still evolving (nesse ) . editors do not always have access to evolutionary expertise, so some good work does not get published where it will be widely seen, and some iffy ideas get presented as stronger than they are. while these problems are not universal, they are real, and they will be solved only by doing science -proposing hypotheses, testing them, discarding those that fail, coming up with new ideas, and finding better ways to test them. the boundary between basic science and applied medicine offers another challenge. preparing this review has impressed us with the number of clinically relevant findings. however, does understanding evolution change dramatically what a physician does in her day-to-day work? in general it does not, and should not. clinical decisions based on theory alone are notoriously suspect. treatment should, whenever possible, be based on controlled studies of treatment outcomes. however, lack of evolutionary understanding among physicians fosters misunderstanding about issues as important as aging, diet, and when it is wise to use medications to block defensive responses. while there is a trend for doctors to just carry out protocols, we want doctors to have a deep knowledge base so their decisions are informed by understanding the body and disease. better decisions come from doctors who understand the ecology of immune responses, the evolutionary reasons for polygenic diseases, the phylogeny of cancer cells, and the origins of antibiotics. we began by describing the magnitude of the gulf between evolutionary biology and medicine. we were struck by how much work is now transcending the gap, but we also were surprised to discover that the gap is even larger than we had thought. the research results described in this article are but a small flock of birds flying over the grand canyon. does it matter? would efforts to bridge the gap pay off in improved human health? many of the findings reviewed here suggest the answer is yes. they serve at the very least to suggest new studies, ranging from whether we need to be concerned about vaccines causing increased virulence to what kinds of disorders are likely to arise from advanced reproductive technologies. however, even aside from suggesting studies with direct clinical and public health relevance, evolutionary approaches increase our fundamental understanding of the body and disease. this is basic science at its most basic. the huge investments in understanding the mechanisms of the cell and gene replication dwarf all investments to date in understanding the evolutionary origins and functions of traits that leave us vulnerable to disease. we predict that increased focus on evolutionary questions will not only offer useful new understanding, it will also synergize with new understanding of mechanisms. in the abstract, we suggested that an evolutionary perspective will be especially helpful in doing away with the incorrect metaphor of the body as a machine designed by an engineer. some readers certainly wondered what we could possibly mean. of course, the body is a machine in the sense that it is composed of chemicals, levers, pulleys and systems that maintain homeostasis. furthermore, machines and bodies are alike in one important way; both are bundles of trade-offs. improving any one trait will likely harm something else. however, because they are products of evolution, bodies are very different from machines (childs ) . a deep understanding of the body as a body is perhaps evolution's greatest single contribution to medicine. machines are designed by an engineer to serve a human purpose. blueprints define the ideal type, and manufac-turing attempts to turn out identical units. when a defect is discovered, engineers change the design. bodies are not designed; they are shaped by natural selection. there is no blueprint, no ideal type. variation is intrinsic. there is no normal genome. there is no normal body. there is no separate manufacturing facility; there is just the process of development -genes interacting with environments to create adult forms. the process involves some chance factors, and also adaptations that monitor the early environment and shift development in ways that adjust the adult form to a particular environment. some traits, such as a birth canal that passes through a narrow circle of bone, cause problems for the species. but there is no engineer with a drawing board to go back to. rerouting birth via the abdominal wall would work better, but the intermediate stages between that and the current route would not work, so the system stays suboptimal. bodies are bodies shaped by selection, not machines designed by intelligence. giving up the machine metaphor gives medicine a stronger foundation in biology. finally, there is the challenge of how best to advance work at the interface of evolutionary biology and medicine. we offer the following brief observations and suggestions in hopes that they will stir discussion and action. at present there is no way to find out what is going on in the field of evolution and medicine. no keywords adequately capture the literature; there is no journal and no society. the resources at the evolution and medicine network (http://evolutionandmedicine.org) offer access to a variety of teaching and information resources and a nucleus for the growing community. the network is being expanded to include the evolution and medicine review: news, recent publications of interest, commentaries on the most notable papers, and questions posed and answered by members of the community. it is intended to meet the need for a central source of information about new research and opportunities in this diverse field. evolutionary applications is a natural outlet for new research in the area of evolution and medicine, and a special issue devoted to the topic is planned for . discussions are underway to organize a society for evolution and medicine. the international alliance of research universities (iaru) evolutionary medicine initiative has budgeted funds for a founding meeting in , likely in conjunction with the darwin festival celebrations honoring the th year of darwin's birth and the th anniversary of the publication of the origin of species. the evolution and medicine network provides updated information on this and other new developments. many young scientists and physicians want to apply evolutionary principles in their areas of medical research, but training programs are not yet available. some are being organized. a proposal to be funded by the iaru will provide research training in evolutionary medicine at six universities including the university of copenhagen, which is recruiting a professor of evolutionary medicine. several other universities, including durham in the uk, are also developing programs. a major research institute is not yet in the works. many have suggested pursuing the strategy that worked so well in the early days of human genetics, summer workshops that bring together established and junior researchers for an intense period of study and discussion with leaders in the basic science. the evolution and medicine network will announce such programs as they become available. funding for projects in evolutionary medicine is available for aging, genetics, and infectious disease. the nih genetic variation and evolution study section has been especially valuable not only in providing funding, but also in providing guidance that increases the quality of work in this subfield. support for work on broader questions is harder to find. private foundations can take the lead in supporting important projects that do not fit the portfolio of government funding agencies, and in supporting efforts to develop the field as a whole. we say reforms, because physicians not now taught even the most basic principles of evolutionary biology as they apply to medicine. it is as if the engineering curriculum included no physics. with no evolutionary biologists on their faculties, no resources to hire them, and an overly-full curriculum, only a very occasional far-sighted dean and faculty will be able to bring evolutionary biology into the curriculum. the modernization of medical education will be helped by curriculum recommendations from advisory bodies such as the institute of medicine that are backed up by new questions on tests administered by the national board of medical examiners and other similar bodies. while some aspects of evolutionary biology must be a part of medical education, it is unrealistic to provide all of the necessary foundations in medical school. like other basic knowledge, much needs to be in courses prior to medical training. undergraduate courses on evolutionary medicine are an excellent solution. most such courses do not provide enough basic evolutionary knowledge, so a combination with a basic evolution course is essential. the logical outcome will be evolutionary questions on the examinations like the mcats in the usa that are used to screen applicants to medical schools. implementation of the above recommendations will require close collaborations among physicians, medical researchers, and basic scientists. creating such connections tends to be difficult because rigid administrative barriers separate the units at most universities, leaving them ill-suited to take advantage of major opportunities such as those at the interface of evolution and medicine. fortunately, however, the barriers are products of human institutions not natural selection. human intelligence and foresight can change those institutions and make action plans that will bring the full power of 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estimates for the year and projections for what good is genomic imprinting: the function of parent-specific gene expression pleiotropy, natural selection, and the evolution of senescence the dawn of darwinian medicine the shaping of senescence in the wild dal darwinismo medico ottocentesco alla medicina darwiniana contemporanea. selezione naturale, disadattamento e predisposizione nell'origine e nella causalità delle malattie. history phd thesis the developmental behavior genetics of drug involvement: overview and comments immune defense and host life history trade-offs in parasitology, evolution and behavior warm thanks to carl bergstrom, pascal gagneux, catriona maccallum, peter jones, and mark thomas for very helpful comments and suggestions; to bernard crespi, robin bush, and an anonymous reviewer for extremely valuable critiques; and to the wissenschaftskolleg zu berlin for a fellowship for rn that made completion of this article possible. key: cord- -yi yjjne authors: desai, angel n.; madoff, lawrence c. title: bending the epidemic curve: advancements and opportunities to reduce the threat of emerging pathogens date: - - journal: epidemiol infect doi: . /s x sha: doc_id: cord_uid: yi yjjne this invited editorial introduces a special issue of epidemiology & infection while also discussing advances in emerging infectious diseases. the world health organization (who) recently enumerated threats to global health for , notably emphasising ebola and other high-threat emerging pathogens as growing priorities [ ] . although many of these diseases have the potential to cause public health emergencies, a lack of timely surveillance and effective interventions continue to hamper preparedness efforts [ ] . moreover, the annualised financial impact of a global pandemic has been estimated to be as high as us$ billion, severely burdening already constrained national budgets and healthcare systems [ ] . outbreaks in resource-limited settings are often further complicated by conflict, fragile health systems, disruptions in healthcare delivery and socio-economic disparities [ ] . these factors can make implementing appropriate outbreak prevention and control strategies difficult. to address these shortcomings, investments in surveillance to inform disease forecasting and ultimately effective prevention strategies are paramount to tackling the challenges posed by emerging pathogens. this edition of epidemiology & infection highlights new insights into many of the emerging infectious diseases mentioned in the who report, as well as re-emerging diseases that are gaining global prominence. they address a variety of diagnostic, therapeutic and epidemiological advances in outbreak preparedness. several papers review data on priority pathogens with a focus on resource-limited settings. for example, sikkema et al. present a systematic review on middle east respiratory syndrome coronavirus with the aim of characterising the distribution and spread of infection in dromedary camels [ ] . aditi et al. review the recent nipah virus outbreaks in bangladesh and india, shedding light on transmission patterns of this emerging pathogen while also highlighting the importance of ongoing surveillance [ ] . a review analysis of h n and h n by parvin et al. discusses genetic variations among avian influenza viruses circulating in bangladesh and the impact of accumulating mutations noted in poultry. lessons learned from the who response to the recent pneumonic plague outbreak in madagascar are presented by heitzinger et al., who highlight specifically the challenges of implementing rapid infection prevention and control measures in epidemic settings [ ] . these studies also underscore the critical importance of the one health approach [ ] . these, and indeed the great majority of emerging disease threats, are zoonotic and require us to consider other hosts and the environment in addressing them. outbreaks of re-emerging infectious diseases in high-income countries are also discussed, often implicating products imported across borders as well as trans-national spread due to as yet unknown causes. other papers discuss zoonotic and vector-borne disease epidemiology and present opportunities for predictive modelling. several diagnostic advances are also mentioned, elucidating changing epidemiological trends in recently recognised re-emerging pathogens. this issue of epidemiology & infection represents a diverse overview of current concerns surrounding emerging infectious diseases globally. all highlight the importance of supporting ongoing surveillance efforts as the cornerstone of disease prevention. early recognition of an outbreak allows control measures to be initiated in a timely way that can shift the epidemic curve, reducing its impact and possibly its geographic spread. enhanced surveillance measures with an emphasis on innovation, transparency and incorporation of the one health model are critical to epidemic preparedness measures in the future. it is also crucial to encourage research during outbreaks through rapid data sharing to facilitate rapid response efforts, as is promoted through organisations such as the international severe acute respiratory and emerging infection consortium (isaric) [ ] . vaccination, if it can be implemented in time, can also bend the epidemic curve. promoting platforms for rapid vaccine development and deployment could provide a significant boost to outbreak control. the coalition for epidemic preparedness innovations (cepi), a public-private coalition that has been working to halt epidemics through the development of appropriate vaccines, is promoting both pathogenspecific and agnostic platform approaches [ ] . these efforts deserve wide support and encouragement. in order for vaccines to be effectively employed, the growing threat of vaccine hesitancy worldwide must also be countered using methods grounded in the social sciences. governments, through their public health agencies and in coordination with efforts like the global health security agenda, must adopt preparedness plans and exercise them before outbreaks become major threats [ ] . emerging and re-emerging infectious diseases will continue to present significant challenges in the coming years, and investing in novel methods for detection, prevention as well as therapeutics should remain priorities for the global public health community. angel n. desai, - - - ; lawrence madoff, - - - ten threats to global health in productive disruption: opportunities and challenges for innovation in infectious disease surveillance the inclusive cost of pandemic influenza risk refugees of the syrian civil war: impact on remerging infections, health services, and biosecurity in turkey global status of middle east respiratory syndrome coronavirus in dromedary camels: a systematic review nipah virus infection: a review using evidence to inform response to the plague outbreak in madagascar: a view from the who african regional office review analysis and impact of co-circulating h n and h n avian influenza viruses in bangladesh coalition for epidemic preparedness innovations international severe acute respiratory and emerging infection consortium acknowledgments. angel desai is supported in part by grant number t ai from the national institute of allergy and infectious disease. the contents of this editorial are solely the responsibility of the authors and do not necessarily represent the official views of the nih. key: cord- - wam aa authors: bevova, m. r.; netesov, s. v.; aulchenko, yu. s. title: the new coronavirus covid- infection date: - - journal: mol doi: . /s sha: doc_id: cord_uid: wam aa in december , the first cases of pneumonia of unknown etiology were found in wuhan (china). later, the pneumonia was associated with a new coronavirus; in february , the world health organization (who) gave the name covid- to the new disease, while the international committee on taxonomy of viruses (ictv) gave the name sars-cov- to the virus causing it. by march , , when the virus had spread to countries, the number of diagnosed patients had reached thousand and the number of deaths was , the who declared the outbreak of the disease a pandemic. in this review, we summarize the relevant information about the origin and spread of sars-cov- , its epidemiology and diagnostics, and the clinical course and treatment of covid- . in december , cases of pneumonia of unknown etiology appeared in china in hubei province. almost all the first cases of the disease were found in individuals working in the market or visiting the seafood market in wuhan. in january , a new coronavirus was detected in patients with pneumonia [ ] . in february , the world health organization (who) gave the name covid- to the new disease, while the international committee on taxonomy of viruses (ictv) gave the name sars-cov- to the virus. by february , , the number of individuals who had been diagnosed with covid- in china exceeded thousand, while a few (less than , except for singapore and the cruise ship diamond princess) cases of the disease were observed in other countries [ ] . despite the quarantine measures that were taken, the disease spread rapidly. on , the who declared the outbreak of the disease to be a pandemic [ ] ; by this time, the virus had spread to countries, with the number of diagnosed patients having reached thousand and the number of deaths thousand [ ] . when this article was written (april , ), the pandemic spanned countries; in the world, there are more than million cases and more than thousand deaths from covid- [ ] . in many countries, the number of cases and deaths are growing exponentially. the current statistics is available on the who website (https://www.who.int/ emergencies/diseases/novel-coronavirus- /situation-reports/), on a russian site (https://www.coro-navirus-monitor.ru), and on a web aggregator supported by johns hopkins university, united states (https:// gisanddata.maps.arcgis.com/apps/opsdashboard/index. html#/bda fd b e ecf ). by mid-april, after china, the largest number of cases occurred in the united states, italy, spain, germany, france, and the united kingdom. while the epidemic in china had been brought under control by this time, with less than new patients per day being detected, the center of the pandemic had moved to the united states and europe, where tens of thousands of new cases were diagnosed in total every day. in the russian federation, there had been more than thousand cases and more than deaths as of april , [ ] . the reproduction number (the number of secondary cases of infection caused by one infected individual, r ) is estimated for covid- as . ( % confidence interval from . to . ) [ ] . for comparison, seasonal influenza usually has a reproduction number of about . [ ] . covid- is transmitted by airborne droplet, airborne dust, and contact ways. it has been demonstrated that infection can be transmitted, including from asymptomatic carriers and infected individuals, already in the incubation period of their disease or even within a few days after clinical recovery [ ] [ ] [ ] . outside the human organism, sars-cov- virus can maintain an ability to infect for up to days on surfaces made of plastic and stainless steel, up to h on cardboard, and up to h on copper surfaces [ ] . the virus genome was for the first time sequenced in china and uploaded to genbank on january , [ ] . the first ten sequenced genomes were identical by . %, which indicates a single initial source of the epidemic. the sequence of sars-cov- was % identical to those for the bat cov ratg virus, % identical to those for the javan pangolin pangolin-cov virus [ ] , % to those for the sars-cov virus, and % to those for the mers-cov virus. it is now believed that bats are a natural reservoir of the virus, but, apparently, there was an intermediate host, which is not currently identified; pangolins, cats, and dogs are under suspicion. phylogenetic analysis of the genomes of virus isolates as compared with genomic sequences of more than natural strains and constructed laboratory strains confirms that sars-cov- virus has a natural origin [ ] [ ] [ ] . sars-cov- virus belongs to the riboviria realm, nidovirales order, cornidovirineae suborder, coronaviridae family, orthocoronavirinae subfamily, betacoronavirus genus, sarbecovirus subgenus, sars-coronavirus species [ ] . sars-cov and mers-cov viruses causing severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) belong to the same genus, subgenus, and species. coronaviruses have a single-stranded plus rna genome ranging in size from to kb [ , ] and are divided into four genera: α, β, γ, and δ [ ] . of them, αand β-coronaviruses are mainly pathogens of mammals, while γand δ-viruses infect mammals and birds. sars-cov- belongs to the groups of β-coronaviruses. sars-cov- virus is the seventh known coronavirus that can cause disease in human beings. of them, α-covs hcov- e, hcov-nl , β-covs hcov-hku , and hcov-oc viruses usually have a low pathogenicity and mainly cause mild or moderate respiratory disease similar to influenza, although sometimes can cause severe pneumonia. sars-cov- and mers-cov viruses also belong to the genus of β-coronaviruses. sars-cov- virus caused the outbreak of atypical pneumonia in - [ ] , while mers-cov virus caused the outbreak of middle east respiratory syndrome in and subsequent years [ ] . to penetrate into human cells, sars-cov- uses the same receptor as sars-cov- -namely, angiotensin-converting enzyme (ace ). the fusion of cell and virus membranes takes place with the involvement of cellular serine protease tmprss [ ] . in vitro, sars-cov- viral particles can penetrate into the same cell cultures as sars-cov- , for example, cultures of hek- t human cells, vero e and vero ccl green monkey cells, mdckii dog cells, human adenocarcinoma cells (a ), and liver cells (huh . ) [ ] . in studies of viral replication and cytotoxicity, a number of cell lines were inoculated with the virus with a high multiplicity and was studied h thereafter. no cytopathic effect was observed, except for in vero cells, in which virus titers reached > pfu h after infection. in contrast to this, huh . and hek- t cells demonstrated only a low level of replication, while a cells were not infected at all. upon observation, the plaques were more distinguishable and visible on vero e culture. two days after infection with a stronger dilution, veroe cells produced separately distinguishable plaques clearly visible when stained with neutral red. in contrast to this, less transparent plaques were obtained on vero ccl cells more distinguishable when stained with neutral red days after infection [ ] . for in vivo studies of sars-cov- , such animals as rhesus macaques and transgenic (humanized) hace laboratory mouse line were used. however, neither macaques nor mice develop a severe respiratory form of infection [ ] . it was demonstrated that sars-cov- can replicate in the upper respiratory tract of ferrets, minks and domestic cats, while cats can infect other cats. at the same time, dogs, pigs, chickens, and ducks are little susceptible to infection [ ] . according to the results of studies of chinese scientists, most patients ( %) were - years of age; %, less than years; %, - years; and %, over years [ , ] . the estimation of the case-fatality rate (portion of deaths divided by the total number of cases) for the disease varies from to % [ , ] depending on the sex and age composition of the population; strategies of testing, diagnostics, and treatment; bureaucratic peculiarities of healthcare in a particular country; and congestion of healthcare systems. on march , , the who estimated the mortality rate to be . % [ ] . however, the results of observations of the crew and passengers of the cruise ship diamond princess, on which out of patients died [ ] , lead to an estimation of the case-fatality rate of . % (a % confidence interval from . to . %). many studies have already demonstrated that the course of the disease and mortality are highly dependent on the patient's age and the presence of other diseases. in children, isolated cases of death have been registered. in the group up to years, the mortality is about . %; after years, the risk increases by three to four times with each additional decade, reaching approximately % in patients aged - years and - % after years [ ] [ ] [ ] . in addition to the elderly, individual with chronic diseases are in the high risk group. it has been demonstrated that arterial hypertension, heart diseases, diabe-tes, and obesity with a body mass index above worsen the prognosis [ , ] . at the same time, a severe course with a fatal outcome is also observed in individuals of middle age ( years or older) without concomitant diseases. it is likely that these individuals have unknown risk factors-for example, an unfavorable genotype. at present, there are insufficient data on the course of covid- in pregnant women and on the effect of the disease on fetal and newborn development. preliminary data from small samples have not shown the course of the disease to be more severe during pregnancy. the course of the disease was mild or moderate in most pregnant women [ ] and newborns [ ] [ ] [ ] [ ] . there are no data on the effect of covid- on fetal development in the first and second trimesters of pregnancy. the incubation period of covid- is from to days, with an average time of the onset of symptoms about days [ ] . cases of the disease with an incubation period of more than days have been described [ ] , but they are isolated and it is likely that they involved repeated and unrecorded contacts with virus carriers. the clinical picture of the disease varies from asymptomatic cases to severe cases requiring hospital and resuscitation treatment. the typical clinical symptoms of covid- include fever, dry cough, shortness of breath, myalgia, and fatigue. other symptoms include headache, confusion, sternal pain, and diarrhea [ ] . in more severe cases, bilateral pneumonia, acute respiratory distress syndrome (ards), multiple organ failure, and sepsis develop [ ] . according to severity, the forms of the disease are classified into mild, moderate, and extremely severe [ ] . most clinical cases ( %) of covid- are classified as mild and moderate. in % of patients, the disease is severe with the presence of shortness of breath, a respiratory rate of ≥ /min, blood oxygen saturation of ≤ %, and a presence of infiltrates in the lungs of > % for - h. about % of patients have the disease in an extremely severe form. the development of respiratory failure, septic shock, and failure of other organs are observed in them [ , ] . after discharge, some patients are still virus carriers, this being confirmed by positive results of laboratory tests. moreover, a relapse of the disease is observed in some patients [ ] . thus, at least in some patients, the immune response does not develop sufficiently to eliminate the virus. this may mean that vaccination can be inefficient for a certain group of individuals. on the basis of clinical symptoms, it is impossible to distinguish covid- from other respiratory infections-in particular, from a cold or other acute respiratory viral infection. the collection of epidemiological data (travel history, patient contacts, etc.) is a key factor in differential diagnostics. the final diagnosis is made according to the results of laboratory tests, such as polymerase chain reaction (pcr), serological tests, and instrumental diagnostics (computed tomography). the diagnostics is based on the amplification (reproduction) of specific viral genome regions by the reverse transcription pcr (rt-pcr) method [ , ] . a nasopharyngeal and/or oropharyngeal swab is the main biomaterial for this study. at present, pcr diagnostics is used for many infectious diseases. its advantages include the possibility of rapid development and production of the test as soon as the genomic sequence of the virus becomes known, as well as a very high sensitivity (up to just ten rna molecules in the sample) and specificity of tests. the testing of sars-cov- is performed on the n, s [ ] , orf ab, and e genes and their combinations. for example, the chinese center for disease control and prevention recommends the use of primers to the orf ab and n genes. the test is considered positive if both genome regions are determined [ ] . a number of russian manufacturers registered their test systems at the end of march and already established production. using serological tests, it is possible to detect the presence of specific antibodies to the virus or the presence of an antigen in an organism. at the same time, combined testing using igm and igg antibodies to the virus is most frequently used. testing for immunoglobulins m allows the fact of recent infection with a virus to be determined, while testing for immunoglobulins g detects either a late stage of infection or immune response after recovery. an antibody test is very useful in the assessment of the immune status of the population and can allow an individual exit from the quarantine, as well as is required to estimate the immune status after vaccination. the minimum testing time is min, while final sensitivity and specificity during testing have been found to be . and . %, respectively [ ] . an enzyme immunoassay test for a viral antigen, especially its express version, helps to identify a coronavirus etiology in the acute phase of infection. however, exact information about these tests or their sensitivity and specificity was unavailable at the time of writing this article. there are no data in the published literature yet concerning the possibility of differentiat- ing different human coronaviruses using serological tests. at present, tests based on the crispr method (for example, sherlock and detectr) are being developed. tests are based on the ability of the crispr system to recognize specific genome regions and cut them. the specific high sensitivity enzymatic reporter unlocking (sherlock) method is based on the use of cas for detecting viral rna at a concentration from to rna molecules per microliter. the results are visualized using an indicator test strip for h. the detectr method uses cas to detect cdna and is faster in execution (about min), but less sensitive, determining the presence of a viral genome in the sample with an initial concentration of - viral rna molecules per microliter. at the time of writing this article, both tests were undergoing clinical trials [ ] . quick tests based on both simple pcr and isothermal pcr (including cartridge, for a field use) have also been developed. however, their sensitivity may be lower than in standard pcr tests. the analysis time for these tests is about min, which is acceptable for testing passengers at airports. a computed tomography is also used for the diagnosis of pneumonia caused by covid- [ ] . bilateral infiltrates in the form of ground glass opacities or consolidation with the primary distribution in lower and middle zones of the lungs are detected with pneumonia caused by coronavirus [ ] . treatment at the moment, there is no specific antiviral therapy for the treatment of covid- . mild cases do not require special treatment. with the development of respiratory failure and pneumonia, the patient is hospitalized and a symptomatic therapy is provided. in severe cases, the treatment is aimed at maintaining the functions of vital organs. when attaching secondary bacterial infections, antibiotics can be prescribed [ ] . oxygen therapy is the main symptomatic treatment in severe cases. ards can develop against the background of a viral infection in severe patients. such patients are transferred to mechanical ventilation (on a mechanical ventilator), and extracorporeal membrane oxygenation is used in more severe cases. with the severe form of the disease, the median time from the first symptoms to the development of shortness of breath is days, to hospitalization days, and to the development of ards days [ ] . at present, a large number of clinical trials for a number of drugs are being carried out. testing drugs commonly used to treat other diseases is of special interest. these drugs were selected from among drugs with a potential antiviral activity or based on the hypotheses about the mechanism of virus action. in particular, the effect of such drugs as favipiravir (inhibitor of viral rna polymerases produced in japan), remdesivir (or gs- , nucleotide analogue, inhibitor of viral rna polymerase developed by gilead sciences co. (united states), but still at an earlier stage of clinical trials compared to other drugs) [ ] , combination of lopinavir and ritonavir (protease inhibitors used in the treatment of hepatitis c and hiv infection), and antimalarial drugs chloroquine and hydroxychloroquine (increase endosomal ph and violate terminal glycosylation of ace ) are being widely investigated. preliminary studies did not confirm the efficiency of treatment with lopinavir and ritonavir [ ] . the efficiency of remdesivir and chloroquine was proven in experiments in vitro [ ] . the efficiency of hydroxychloroquine was demonstrated in preliminary, yet very limited, clinical trials [ ] that are currently criticized for unreliability. it should be noted that hydroxychloroquine can have severe side effects and interact with other drugs. at present, the who has initiated a global clinical study of these drugs, named solidarity [ ] . scientists from different countries are engaged in the development of vaccines against the disease caused by sars-cov- . there are several approaches to the development of antiviral vaccines. in particular, vaccines based on attenuated or inactivated virus, viral vectors, recombinant mrna and dna, and recombinant proteins are undergoing preclinical trials [ ] . phase i clinical trials started on volunteers in united states for a vaccine based on recombinant mrna (modetna co.) and in china for a vaccine based on recombinant live adenovirus (fifth serotype) (can-sino biologics co.). in the event of success in the phase i trial, the phases ii and iii will be held in outbreaks in united states and china in summer. full cycles of testing for the efficiency and safety of vaccines will take at least a year. there are currently no answers to many questions about the covid- disease and the sars-cov- virus itself. there are no accurate estimations of the portion of virus-infected individuals who are asymptomatic carriers or carry the disease in a mild form without getting into the official statistics. if this portion is high and such individuals transmit the disease, then, on the one hand, it will be very difficult to stop infection by currently taking quarantine measures, which, in many countries, are based on isolation of symptomatic cases and their contacts. on the other hand, this means that the estimations of the number of patients requiring hospital treatment and mortality rate are overstated. according to one such estimate, the portion of asymptomatic carriers may be as high as % of the total number of infected individuals [ ] , and at least some asymptomatic infected individuals can infect other individuals [ ] . although the existing estimations of the mortality rate and the amount of patients requiring hospitalization and intensive therapy may be overstated, it is clear that the number of covid- patients who simultaneously need hospitalization and resuscitation measures is many times higher than the rates of habitual infections such as seasonal influenza. this is clear from the fact that the healthcare systems of the regions in hubei and northern italy, in which sars-cov- spread uncontrollably for some time, were close to collapse. the number of patients simultaneously requiring medical assistance is affected by the virulence and the rate of infection in the population. an exact estimation of epidemiological parameters of covid- is important for the development of mathematical models that are used by experts to predict the development of the situation. defining a set of quarantine measures is one of the main tasks solved by these methods. quarantine measures are aimed at suppressing or slowing down the spread of the virus, thus optimizing healthcare opportunities and minimizing the mortality and economic effect from the pandemic [ ] . in particular, the number of patients requiring hospitalization is an important target parameter. their number should not exceed the number of available hospital beds in the healthcare system. a reliable identification of the dynamics of the number of infected individuals is extremely important for making government decisions and for understanding how efficient the measures taken are. at present, the who is calling for as many tests for sars-cov- as possible to control the spread of the disease [ ] , since, apparently, it is impossible to cope with the pandemic without it. establishing the exact number of infected individuals is extremely difficult due to the difficulties of total and repeated screening of the population. the experience of the netherlands and denmark, where monitoring of the dynamics of the number of infected individuals is carried out by determining the content of viral rna in the sewer, is interesting [ ] . however, the efficiency and accuracy of this approach to fighting the epidemic is still unclear. there is also no clear answer to the question of how stable immunity to covid- is. news sites have disseminated information on several cases in which a patient repeatedly contracted covid- [ ] . it is unclear whether these are cases of reinfected individuals, a relapse of the disease, or a result of the first test result being a false positive. however, a recent study on rhesus monkeys showed that reinfection is unlikely [ ] (it should be noted that, by the moment of writing this review, this article had appeared in the form of a reprint and had not yet been reviewed). the issue of the seasonality of covid- remains unclear. there is an hypothesis that the reproductive number will decrease with the onset of summer in the northern hemisphere, which will lead to a slowing down of the spread of infection. there is also a hypothesis that, due to isolation of seriously ill patients, the most pathogenic variants of the virus will not be distributed and will be displaced by weakly pathogenic variants from asymptomatic carriers. the current virus will then become just another coronavirus causing ordinary acute respiratory viral infection. these hypotheses will be tested by nature and life itself in the next - months. it is interesting to note that, although children are infected with the virus, they get sick in a milder form as compared with adults [ ] ; there are currently no data on death of children under the age of years [ ] . it is hypothesized that children may have a fresher immune response to antigenically similar infection, as well as a different level of ace receptors on lung cells [ ] . experiments on model animals indeed demonstrated that specific number of ace receptors plays an important role in the prevention of the development of acute respiratory failure [ ] , while the level of ace decreases with age [ , ] . however, no changes in the activity of ace with age were observed in human beings in studies of ards [ ] . the use of recombinant ace was not efficient in the treatment of human ards [ ] . in the future, it will be important to study the association of the symptoms and severity of the disease with different epidemiological and clinical risk factors, variations in genomic sequences of virus variants, and peculiarities of the genomes and immune systems of patients. the authors are grateful to g.a. bazykin and s.a. moshkovskii for discussion and to n.v. aulchenko for assistance with the preparation of the article. a novel coronavirus from patients with pneumonia in china covid- ) situation report - , world health organization world health organization covid- ) situation report - , world health organization covid- ) situation report - , world health organization early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature transmission of covid- in the terminal stage of incubation period: a familial cluster substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) transmission of -ncov 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macaques, biorxiv clinical characteristics of children with coronavirus disease possible biological explanations for kids' escape from covid- angiotensin-converting enzyme protects from severe acute lung failure agedependent changes in the pulmonary renin-angiotensin system are associated with severity of lung injury in a model of acute lung injury in rats age-and gender-related difference of ace expression in rat lung age-dependent differences in pulmonary host responses in ards: a prospective observational cohort study a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. key: cord- - ghtmhu authors: chua, kaw bing; gubler, duane j title: perspectives of public health laboratories in emerging infectious diseases date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: ghtmhu the world has experienced an increased incidence and transboundary spread of emerging infectious diseases over the last four decades. we divided emerging infectious diseases into four categories, with subcategories in categories and . the categorization was based on the nature and characteristics of pathogens or infectious agents causing the emerging infections, which are directly related to the mechanisms and patterns of infectious disease emergence. the factors or combinations of factors contributing to the emergence of these pathogens vary within each category. we also classified public health laboratories into three types based on function, namely, research, reference and analytical diagnostic laboratories, with the last category being subclassified into primary (community-based) public health and clinical (medical) analytical diagnostic laboratories. the frontline/leading and/or supportive roles to be adopted by each type of public health laboratory for optimal performance to establish the correct etiological agents causing the diseases or outbreaks vary with respect to each category of emerging infectious diseases. we emphasize the need, especially for an outbreak investigation, to establish a harmonized and coordinated national public health laboratory system that integrates different categories of public health laboratories within a country and that is closely linked to the national public health delivery system and regional and international high-end laboratories. infectious diseases have affected humans since the first recorded history of man. infectious diseases remain the second leading cause of death worldwide despite the recent rapid developments and advancements in modern medicine, science and biotechnology. greater than million (. %) of an estimated million deaths that occur throughout the world annually are directly caused by infectious diseases. millions more deaths are due to the secondary effects of infections. moreover, infectious diseases cause increased morbidity and a loss of work productivity as a result of compromised health and disability, accounting for approximately % of all disability-adjusted life years globally. , compounding the existing infectious disease burden, the world has experienced an increased incidence and transboundary spread of emerging infectious diseases due to population growth, urbanization and globalization over the past four decades. [ ] [ ] [ ] [ ] [ ] [ ] most of these newly emerging and re-emerging pathogens are viruses, although fewer than of the approximately pathogen species recognized to infect humans are viruses. on average, however, more than two new species of viruses infecting humans are reported worldwide every year, most of which are likely to be rna viruses. emerging novel viruses are a major public health concern with the potential of causing high health and socioeconomic impacts, as has occurred with progressive pandemic infectious diseases such as human immunodeficiency viruses (hiv), the recent pandemic caused by the novel quadruple re-assortment strain of influenza a virus (h n ), and more transient events such as the outbreaks of nipah virus in / and severe acute respiratory syndrome (sars) coronavirus in . [ ] [ ] [ ] [ ] [ ] in addition, other emerging infections of regional or global interest include highly pathogenic avian influenza h n , henipavirus, ebola virus, expanded multidrug-resistant mycobacterium tuberculosis and antimicrobial resistant microorganisms, as well as acute hemorrhagic diseases caused by hantaviruses, arenaviruses and dengue viruses. to minimize the health and socioeconomic impacts of emerging epidemic infectious diseases, major challenges must be overcome in the national and international capacity for early detection, rapid and accurate etiological identification (especially those caused by novel pathogens), rapid response and effective control (figure ). the diagnostic laboratory plays a central role in identifying the etiological agent causing an outbreak and provides timely, accurate information required to guide control measures. this is exemplified by the epidemic of nipah virus in malaysia in / , which took more than six months to effectively control as a consequence of the misdiagnosis of the etiologic agent and the resulting implementation of incorrect control measures. , however, there are occasions when control measures must be based on the epidemiological features of the outbreak and pattern of disease transmission, as not all pathogens are easily identifiable in the early stage of the outbreak (figure ). establishing laboratory and epidemiological capacity at the country and regional levels, therefore, is critical to minimize the impact of future emerging infectious disease epidemics. developing such public health capacity requires commitment on the part of all countries in the region. however, to develop and establish such an effective national public health capacity, especially the laboratory component to support infectious disease surveillance, outbreak investigation and early response, a good understanding of the concepts of emerging infectious diseases and an integrated country and regional public health laboratory system in accordance with the nature and type of emerging pathogens, especially novel ones, are highly recommended. traditionally, emerging infectious diseases are broadly defined as infections that: (i) have newly appeared in a population; (ii) are increasing in incidence or geographic range; or (iii) whose incidence threatens to increase in the near future. , six major factors, and combinations of these factors, have been reported to contribute to disease emergence and re-emergence: (i) changes in human demographics and behavior; (ii) advances in technology and changes in industry practices; (iii) economic development and changes in land use patterns; (iv) dramatic increases in volume and speed of international travel and commerce; (v) microbial mutation and adaptation; and (vi) inadequate public health capacity. , from the perspective of public health planning and preparedness for effective emerging infectious disease surveillance, outbreak investigation and early response, the above working definition of emerging infectious disease and its associated factors that contribute to infectious disease emergence are too broad and generic for more specific application and for the development of a national public health system, especially in the context of a public health laboratory system in a country. thus, in this article, emerging infectious diseases are divided into four categories based on the nature and characteristics of pathogens or infectious agents causing the emerging infections; these categories are summarized in table . the categorization is based on the patterns of infectious disease emergence and modes leading to the discovery of the causative novel pathogens. the factors or combinations of factors contributing to the emergence of these pathogens also vary within each category. likewise, the strategic approaches and types of public health preparedness that need to be adopted, in particular with respect to the types of public health laboratories that need to be developed for optimal system performance, will also vary greatly with respect to each category of emerging infectious diseases. these four categories of emerging infectious diseases and the factors that contribute to the emergence of infectious diseases in each category are briefly described below. [ ] [ ] [ ] [ ] [ ] [ ] factors that contributed to the occurrence of emerging infectious diseases in this subcategory include population growth; urbanization; environmental and anthropogenic driven ecological changes; increased volume and speed of international travel and commerce with rapid, massive movement of people, animals and commodities; and deterioration of public health infrastructure. subcategory b includes known and unknown infectious agents that occur in new host 'niches'. infectious microbes/ agents placed under this subcategory are better known as 'opportunistic' pathogens that normally do not cause disease in immunocompetent human hosts but that can lead to serious diseases in immunocompromised individuals. the increased susceptibility of human hosts to infectious agents is largely due to the hiv/acquired immune deficiency syndrome pandemic, and to a lesser extent, due to immunosuppression resulting from cancer chemotherapy, antirejection treatments in transplant recipients, and drugs and monoclonal antibodies that are used to treat autoimmune and immune-mediated disorders. a notable example is the increased incidence of progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system that is caused by the polyomavirus 'jc' following the public health laboratories in emerging infectious diseases kb chua and dj gubler increased use of immunomodulatory therapies for anti-rejection regimens and for the treatment of autoimmune diseases. examples of past emerging infectious diseases under this category are antimicrobial resistant microorganisms (e.g., mycobacterium tuberculosis, plasmodium falciparum, staphylococcus aureus) and pandemic influenza due to a new subtype or strain of influenza a virus (e.g., influenza virus a/california/ / (h n )). , - factors that contribute to the emergence of these novel phenotype pathogens are the abuse of antimicrobial drugs, ecological and host-driven microbial mixing, microbial mutations, genetic drift or re-assortment and environmental selection. accidental or potentially intentional release of laboratory manipulated strains resulting in epidemics is included in this category. factors that lead to the spillovers and emergence of these novel pathogens are human population expansion, economic development, changes in land use patterns, modifications to natural habitats, and changes in agricultural practices and animal husbandry. human behavior, such as wildlife trade and translocations, live animal and bush meat markets, consumption of exotic foods, development of ecotourism, access to petting zoos and ownership of exotic pets, also plays a significant role in the transfer of pathogens between species. [ ] [ ] [ ] [ ] [ ] [ ] examples of infectious diseases under category b are gastritis and peptic ulcers due to helicobacter pylori, kaposi sarcoma due to human herpesvirus and chronic hepatitis due to hepatitis virus c and g. [ ] [ ] [ ] [ ] advances in scientific knowledge and technology have contributed substantially to the discovery of these infectious etiological agents. regardless of the category, with some exception for category b, effective early detection, identification, characterization, containment, control and ultimately prevention of the emerging infectious diseases will require a good, functional national public health surveillance system. the system needs to be well supported by a network of primary public health and clinical/medical diagnostic laboratories that are coordinated by a national public health reference laboratory with real-time and harmonious communication between the laboratories and epidemiological surveillance units. confronted with the great diversity of these emerging pathogens and the equally diverse mechanisms and factors that are responsible for their emergence, there is an urgent need to develop a network of diagnostic laboratories, especially in countries where epidemic infectious diseases are likely to emerge. this network should include local laboratories with basic clinical laboratory capabilities, provincial and national public health diagnostic laboratories with greater capability to diagnose known pathogens and support effective laboratory-based surveillance, and a centralized national reference laboratory that can provide laboratory training and quality control for diagnostic assays for the network of diagnostic laboratories in the country. ideally, the national reference laboratory should have state-of-the-art laboratory technology and be able to identify and characterize novel pathogens with specialized university laboratories and foreign institutes that can provide backup capability, but more importantly, the national reference laboratory should be able to conduct research for the development of new diagnostic technologies to detect and identify novel pathogens, especially those classified as category . the us system, which includes local and state public health laboratories that conduct diagnoses of known pathogens, the centers for disease control and prevention and university laboratories that provide research and reference activities, is a good model. disease or pathogen-specific public health diagnostic laboratories established to support world health organization (who)-specific disease surveillance programs and vaccine-preventable diseases, e.g., national influenza, poliovirus and measles laboratories, led to the 'compartmentalization' of laboratory diagnostic services, segregation of functions, and duplication of facilities and equipment. the situation is further complicated by the siting of various pathogen-specific diagnostic laboratories in different buildings or institutes or different locations within a country, thus preventing more cost-effective measures of sharing common equipment and reagents, clinical samples, information and human resources. finally, the problem is compounded by policy makers and laboratory managers lacking flexibility and not allowing these disease or pathogen-specific laboratories to adopt a more generic approach in the investigation of infectious disease outbreaks. past incidents have shown that misdiagnosis or delay in the diagnosis of epidemics can cause substantial economic losses and social disruption and prevent containment or control as a result of the implementation of inappropriate control measures or a delay in implementing the appropriate control measures. the proposed integrated system of public health laboratories is not entirely new; public health laboratories are already in existence in most countries, but most are poorly equipped and are not adequately funded or staffed with trained professional staff. moreover, a lack of knowledge and coordination has led to ineffective operation in the support of infectious disease surveillance. the basic concept of realigning and harmonizing public health laboratories to optimize their roles and functions can be drawn from the system of medical practices. due to rapid and vast expansion of medical knowledge, technology and demand of specialized skills and therapy, medical practices have evolved into a number of specialties and subspecialties, such as infectious disease, cardiology, gastroenterology, neurology, radiology, anesthesiology and oncology. an excellent aspect of the medical system is the continual retention of the general physician (family physician) or general pediatrician as the initial or first entry point for patients seeking consultation for any medical problem before being subsequently referred to the appropriate specialist, if deemed necessary. it is not uncommon for patients, especially older individuals, to have more than one disease or pathology at the time of presentation to the doctor. in a similar manner, in outbreaks of infectious diseases, 'background' endemic pathogens are often present that are capable of similar disease manifestations. thus, public health analytical diagnostic laboratories (both primary and clinical) should adopt a generic approach and serve as the initial or first entry point for the investigation of the causative pathogens in the event of an infectious disease outbreak or the occurrence of any fatal illness with clinical suspicion of infectious etiology. in addition, public health laboratories must have the capability to support the expanded scope and sophistication of public health activities brought about by a rapid increase in population and social, demographic and ecological changes, in addition to the factors mentioned above. despite the presence of several types of health laboratories, they can be classified into three main categories: (i) public health research laboratories; (ii) public health reference laboratories; and (iii) public health analytical diagnostic laboratories. public health analytical diagnostic laboratories can be further subcategorized into primary public health (community-based) and clinical/medical (hospital and clinic-based) analytical diagnostic laboratories. a proposed organizational model to establish an integrated system of public health laboratories within a country to coordinate and link health laboratories under different ministries and in both public and private institutions based on their functional roles is shown in figure . the broken lines indicate the diagnostic laboratories that are not directly regulated by the ministry of health. a schematic flow chart illustrating the functional relationships and linkages between various types of public health laboratories in a country was described previously. a defined and harmonious linkage and collaboration will not only avoid duplication and redundancy, but also enhance and complement the function and output quality of each laboratory. bearing in mind that not all countries in the world have similar resources (financial, man-power and expertise), demography, geopolitical structure, needs and commitment, the proposed model can be appropriately modified to tailor each country's immediate needs with a provision for future upgrading and expansion. ultimately, it is recommended that all countries establish an integrated system covering all three categories of public health laboratories, with a cohesive centralized national public health reference laboratory. in countries with limited resources, an interim centralized national public health diagnostic laboratory can take on some of the roles and functions of a national reference laboratory, especially in supporting laboratory training and quality assurance. for countries without such an idealistic centralized public health reference laboratory, an in-place system of networking should be developed to link to regional and international high-end laboratories or who collaborative centers to rapidly identify and characterize novel pathogens and provide other specialized laboratory diagnostic reagents, assays or validation. in addition, each region should have a regional center for reference public health laboratories in emerging infectious diseases kb chua and dj gubler and research to help the national reference and/or diagnostic laboratories train and maintain laboratory quality control. the us centers for disease control and prevention is a major who collaborative partner and provides laboratory service not only for the american region, but also for many other countries in the world. investigations into the diagnosis of nipah virus, sars coronavirus, pandemic influenza a virus and hemorrhagic fever viruses are just a few examples illustrating its worldwide function. the placement of the centralized reference laboratory under the national center for disease control strengthens close communication and coordination among public health specialists, epidemiologist and laboratory personnel and serves as an important coordinating center to support the functions and activities pertaining to biorisk issues, centralized pathogen characterization and storage, laboratory-based surveillance and laboratory quality assurance, as shown in figure . a national reference laboratory will also be able to play an important role as part of a regional laboratory network to strengthen regional public health laboratory capacity in providing specific referral functions for public health diagnostic laboratories in other countries that do not have a reference laboratory. the public health research laboratories within the research institutes of ministries of health and universities or even private research institutions are best suited and can play a crucial role in collaborating with the national public health reference and diagnostic laboratories to discover novel pathogens of many human diseases under category , especially in subcategory b. the proposed network scheme will provide more cost-efficient laboratory services and ensure a regular flow of laboratory work to maintain the competency of technical staff to produce quality output. because of the increased likelihood of epidemic diseases caused by novel pathogens, diagnostic laboratories serving as the primary entry point of investigation should be able to take a more generic approach in pathogen detection, isolation and identification. the traditional existing system of 'compartmentalization' of national disease/pathogenspecific diagnostic laboratories should thus be reviewed and integrated into the national public health infectious disease diagnostic laboratory system. this proposed model would improve cost-efficiency and allow a more appropriate approach to infectious disease outbreak investigation and control. we thank tikki pang, lee kuan yew school of public policy, national university of singapore, for useful comments and suggestions in the preparation of this manuscript. emerging infections: getting ahead of the curve emerging infectious diseases: a -year perspective from the national institute of allergy and infectious diseases emerging viruses: 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viruses for old disease? a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome molecular cloning and disease association of hepatitis g virus: a transfusion-transmissible agent this work is licensed under a creative commons attribution-noncommercial-noderivative works to view a copy of this license key: cord- -iu e jo authors: taboe, hémaho b.; salako, kolawolé v.; tison, james m.; ngonghala, calistus n.; kakaï, romain glèlè title: predicting covid- spread in the face of control measures in west-africa date: - - journal: math biosci doi: . /j.mbs. . sha: doc_id: cord_uid: iu e jo the novel coronavirus (covid- ) pandemic is causing devastating demographic, social, and economic damage globally. understanding current patterns of the pandemic spread and forecasting its long-term trajectory is essential in guiding policies aimed at curtailing the pandemic. this is particularly important in regions with weak economies and fragile health care systems such as west-africa. we formulate and use a deterministic compartmental model to (i) assess the current patterns of covid- spread in west-africa, (ii) evaluate the impact of currently implemented control measures, and (iii) predict the future course of the pandemic with and without currently implemented and additional control measures in west-africa. an analytical expression for the threshold level of control measures (involving a reduction in the effective contact rate) required to curtail the pandemic is computed. considering currently applied health control measures, numerical simulations of the model using baseline parameter values estimated from west-african covid- data project a % reduction in the daily number of cases when the epidemic attains its peak. more reduction in the number of cases will be achieved if additional public health control measures that result in a reduction in the effective contact rate are implemented. we found out that disease elimination is difficult when more asymptomatic individuals contribute in transmission or are not identified and isolated in a timely manner. however, maintaining a baseline level of asymptomatic isolation and a low transmission rate will lead to a significant reduction in the number of daily cases when the pandemic peaks. for example, at the baseline level of asymptomatic isolation, at least a % reduction in the transmission rate is required for disease elimination. additionally, disease elimination is possible if asymptomatic individuals are identified and isolated within days (after the incubation period). combining two or more measures is better for disease control, e.g., if asymptomatic cases are contact traced or identified and isolated in less than days, only about % reduction in the disease transmission rate is required for disease elimination. furthermore, we showed that the currently implemented measures triggered a % reduction in the time-dependent effective reproduction number between february and june , . we conclude that curtailing the covid- pandemic burden significantly in west-africa requires more control measures than those that have already been implemented, as well as more mass testing and contact tracing in order to identify and isolate asymptomatic individuals early. a new strain of coronavirus (sars-cov- ), that emerged from wuhan, china is the cause of the covid- pandemic that is currently ravaging the world [ - ] . as of june , , about , , confirmed cases of covid- infections and , deaths were reported worldwide [ ] [ ] [ ] [ ] . most of these reported covid- cases (approximately , , ) and deaths (approximately , deaths) were from the united states of america. the epicenter of the pandemic is expected to shift to sub-saharan africa, which as of june , had reported about , confirmed cases and , deaths. of these, about , confirmed cases and , deaths were from west-africa. the first confirmed west-african case was in nigeria on february , , i.e., approximately two months after the first case was officially announced in china [ ] . the highest burden of the disease in west-africa by june , was in nigeria (about , cases and deaths) [ ] . this is in line with a multilayered-risk assessment (based on nine risk factors) in [ ] that identified nigeria as the west-african country with the highest covid- risk. another study by martinez-alvarez et al. [ ] projected that some west-african countries, e.g., burkina faso and senegal might experience sharp increases in the number of covid- cases that are similar to those observed in european countries in march and april, . humans can acquire the novel coronavirus when they come into contact with contaminated surfaces or from droplets released by infectious symptomatic and asymptomatic individuals [ ] . mild to moderate infection symptoms of the disease include fever, cough, sore throat, nasal congestion, malaise, headache, muscle pain, and shortness of breath (or tachypnea in children). in severe cases, fever is associated with severe dyspnea, respiratory distress, and tachypnea [ ] . currently, there is no vaccine or widely accepted drug for covid- . therefore, governments and individuals are forced to rely on public health preventive measures such as basic hygiene, travel or movement restrictions, social-distancing, wearing masks, etc. current control measures being implemented in west-africa include regular hand washing with hydroalkolic solutions, quarantine of suspected cases, isolation of confirmed cases, social distancing (e.g., travel restrictions, school closures, and banning of gatherings involving more than people), contact tracing, and testing and treating identified cases. additionally, wearing of masks in public was recently recommended as another control measure in many countries in this region. unfortunately, it is difficult to implement these basic public health measures effectively in some west-african countries due to wide-spread poverty and poor investment in health care (staff, equipment, and infrastructure) [ ] . for example, in thirteen of the sixteen west-african countries, less than two medical doctors are allocated to every group of , inhabitants [ , ] . in particular, only nigeria, cote d'ivoire, and cape verde have or more medical doctors per , inhabitants [ ] . another possible explanation for this low doctor to population ratio is alternative medicine (especially traditional medicine) that is widely practiced in the west african region. the world health organization estimates that about % of the population of west-times below the average per capita health spending in countries like italy and spain-two european countries that experienced high covid- burden [ ] . as a result of poor investment in health care and health care systems, the region has been an epidemic hotspot for emerging and neglected tropical diseases including the ebola virus (ebov), lassa virus (lasv), and buruli ulcers in recent years [ , ] . with such inadequate healthcare systems and personnel in the region, the impact of covid- might be catastrophic. to compound matters, some countries in west-africa are still not well prepared to tackle this pandemic despite the observed devastating impacts and trend of the pandemic in the us, europe, and asia [ ] . there is, therefore, the need to exploit every existing tool and/or develop new tools that can be useful in guiding public health decision-making in the fight against the pandemic. this includes developing and using new qualitative and quantitative methods such as mathematical models. there has been an influx of mathematical models to assess the impact of covid- and to guide public health response in different cities, countries and regions of the world (see, for example, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). few of these mathematical model frameworks have focused on the covid- pandemic situation in west-africa as a whole. among these few models, some have focused only on one or two countries, e.g. nigeria and ghana, while others have used basic regression or exponential growth models to analyze and predict disease trends [ , ] . martinez-alvarez et al. [ ] used a simple graph-plot based comparative analysis of observed covid- data for the first days of the pandemic to examine the future trajectory of the epidemic in six west-african countries (burkina faso, senegal, nigeria, côte d'ivoire, ghana, and the gambia). this far, only one study has considered the entire region of west-africa [ ] . in this study, a co-variate-based instrumental variable regression framework was used to predict the number of disease cases ( . million by june , ) and to assess the epidemiological, socio-economic and health system readiness of the region to the pandemic. in this study, we develop a new susceptible-exposed-infectious-recovered (seir)-type mathematical model for covid- in west-africa and use it to (i) study the current transmission pattern of covid- , (ii) evaluate the impact of currently implemented public health control measures such contact tracing, social distancing and use of face masks on covid- transmission, and (iii) predict the future course of the pandemic with and without currently implemented and additional control measures in west-africa. in addition to incorporating various basic public health control measures, our model accounts for asymptomatic infectious individuals -a class of individuals that render disease control more difficult since they do not exhibit clinical disease symptoms, although they contribute to disease transmission [ ] . to our knowledge, this is the first study that has used an explicit seir-type compartmental model together with data for the west-african pandemic period (from february , to june , ) to study the spread of the covid- pandemic in the region, assess the impact of current control measures, and discuss further control measures that might be required to better control the pandemic in the region. that lead to a reduction in the force of infection λ, such as social distancing and face mask use. following the approach in ngonghala et al. [ ] , we model the force of infection by the functional form ( − Ψ)(β a i a + β s i s )/(s + e + i a + i s + r), where β k (k ∈ {a, s}) is the disease transmission rate by individuals in the i k class and ≤ Ψ ≤ is the percentage reduction in disease transmission due to public health control measures such as social distancing and face masks use. note that Ψ = implies that no public health control measure leading to a reduction in disease transmission is applied and that Ψ = implies that implemented public health control measures are efficient enough to stop disease transmission. after the average incubation period /σ, a proportion π of the exposed population does not develop symptoms, i.e., joins the i a compartment, while the remaining proportion ( − π) join the i s class. identification and isolation of infectious asymptomatic individuals, e.g., through contact tracing occurs at rate ρ a , i.e., /ρ a is the average time it takes for an infectious asymptomatic individual to be identified and isolated. isolation or hospitalization of symptomatic cases occurs at rate ρ s , i.e., /ρ s is the average time it takes for an infectious symptomatic individual to be isolated. humans from the i a , i s , and i c classes recover at rates γ a , γ s , and γ c , respectively, while individuals in the i s and i c classes die due to covid- at respective rates δ s and δ c . the flow diagram for the model is given in fig. . fig. : flow-chart of covid- model showing the flow of humans between different compartments. the susceptible population is denoted by s, the exposed population is denoted by e, the infectious asymptomatic population is denoted by ia, the infectious symptomatic population is denoted by is, the isolated infectious population is denoted by ic, and the recovered population is denoted by r. the parameters of the model are described in the text. using the flow-chart and the preceding description, we obtain the following model system: where p is the actively mixing population, i.e., p = n − i c = s + e + i a + i s + r. since individuals in the i c class are in some form of confinement or isolation, we assume that they are not part of the actively mixing population and so they do not contribute in disease transmission. we denote the initial conditions of the model ( . ) by s( ) = s > , e( ) = e , i a ( ) = i a , i s ( ) = i s , i c ( ) = i c , r( ) = r , where for the pandemic to take off, at least one of the infected populations must be greater zero. west-africa comprises sixteen countries: benin, burkina-faso, cape verde, ghana, guinea, ivory coast, mali, mauritania, niger, nigeria, senegal, togo, sierra leone, liberia, guinea-bissau and gambia. based on the latest united nations estimates, the current population of west-africa is , , , i.e., about . % of the world population [ ] . the region includes nine of the poorest countries in the world [ ] . application of basic public health control measures against covid- spread in most west-african countries started on march , , i.e., about days after the outbreak in the region. the data considered for this analysis spans the period from february , , i.e., one day after the first case until june , . in west-africa, the reported confirmed cases are infectious symptomatic individuals who have been tested at a treatment center or infectious asymptomatic individuals who have been detected through contact-tracing or systematic testing on target groups and then tested at a treatment centre. hence, we assume that confirmed disease cases correspond to individuals isolated infectious individuals in isolation or at treatment centers (i c ). we use the cumulative number of cases in west-africa, downloaded from the global rise of education website [ ] to fit our model and estimate some of the key parameters presented in table . the fitting that was carried out using a nonlinear least squares method in matlab version (r a) involved finding the best set of parameter values that minimizes the root mean square differences between the observed cumulative covid- confirmed cases in west-africa and the predicted cumulative covid- cases from our model . . the minimization was achieved through the built-in constraint minimization algorithm "fmincon" in matlab. we repeated this procedure , times for each of the parameters and used the normal distribution to compute the mean values of the parameters together with a % confidence interval (ci). the goodness of fit of our model assessed using the root mean squared error (rmse) was . . the fitted and known parameter values are presented in table and the model fit is illustrated in fig. (a). it is worth noting that the choice of parameters to estimate from the data or to extract from the literature was motivated by its importance to the transmission dynamics of the disease. a plot of the observed confirmed daily cases and the predicted daily cases from system ( . ) using the fitted and known parameters is presented in fig. table . . in this section, we derive the basic reproduction number of the model, which is defined as the average number of new infections generated by a typical infectious individual introduced in a population where everybody is susceptible [ ] . it is denoted by r and provides a condition under which a disease can invade a population (when r > ), or can be contained (when r < ). using the next generation matrix approach in [ , ] , the basic reproduction number is: observe from eq. . that the reproduction number r , is the sum of two components, which represent the contribution of the infectious asymptomatic class r a and the infectious symptomatic class r s . for system ( . ) that already include some control efforts, the control reproduction number r c , is given by r c = ( − Ψ)r . the approach in [ , ] also assures us that the continuum of disease-free equilibrium (s , e , i a , i s , i c , r ) = (s( ), , , , , ) of system ( . ) is locally and asymptotically stable whenever the reproduction number r c is less than one. another useful quantity tht is associated with the reproduction number is the time-dependent effective reproduction number, which for model ( . ) is given by r e = r s/(s + e + i a + i s + r). it is worth mentioning that the epidemic grows when the effective reproduction number is greater than unity, peaks when the effective reproduction number equals unity and declines when the effective j o u r n a l p r e -p r o o f journal pre-proof reproduction number is below unity. to derive the threshold reduction in the disease transmission Ψ c , required to reduce the control reproduction number to one, we set r c = , i.e., ( − Ψ)r = in eq. . and solve for Ψ. this yields: note that the fraction in the right hand side of the threshold value Ψ c of Ψ is greater than zero and less than or equal to one since ≤ Ψ ≤ . note also that Ψ = Ψ c , which corresponds to the case in which r c = now serves as a threshold for an outbreak or the disease dying out. in particular, disease elimination is the epidemiological implication of this is that massive adherence to control measures that reduce covid- transmission, e.g., social distancing can lead to disease elimination, while low adherence to such measures will reduce disease burden, but might not lead to elimination. to compute the final size relation, we follow the method in [ ] . in addition to the final size relation calculation, this approach provides an alternative method for calculating the basic reproduction number of the model ( . ). to apply the method, we first identify the vector x of covid- infections, i.e., x = (e, i a , i s , i c ) ∈ r + . then, we identify the vectors y and z of the susceptible and recovered classes, respectively, i.e., y = s ∈ r + and z = r ∈ r + . with this notation, the system ( . ) can now be expressed in the compact form: by theorem . of [ ] , the basic reproduction number is [ ] , the basic reproduction number is given by this final size relation can be used to approximate the number of individuals who remain uninfected, and consequently the number of individuals who were infected during the pandemic. in this section, we use the parameters presented in table to simulate system ( . ) and to investigate the impact of control measures on key model outputs such as the control and effective reproduction numbers r c and r e , respectively. the basic reproduction number r , and the control reproduction number r c computed using this baseline parameter regime in table are to an approximately % reduction in the basic reproduction number due to implemented control measures. this calls for more effective control measures to limit the spread of the disease. all simulations were carried out using the computational software matlab (version r a). when it was necessary solve the system of ordinary differential equations ( . ) we used the inbuilt matlab function "ode ". the first analysis involves evaluating the impact of the percentage reduction in the effective contact rates due to the implementation of public health control measures such as lockdowns, social distancing, the use of masks in public, washing of hands, etc., denoted by Ψ on the control and effective reproduction numbers, as well as the pandemic peak and the time to peak. here, we fixed all the parameters at the baseline values provided in table and varied only the parameter Ψ. changes in the control reproduction number as a function of Ψ, depicted in fig. show that about % reduction in the disease transmission is required to reduce r c to one and possibly bring the pandemic under control. this value ( %) corresponds to the numerical value of Ψ c from eq. ( . ) computed using the baseline parameter values from table and a % increase from the baseline value of Ψ. furthermore, our results show that there will be a % reduction in the effective reproduction number by july , if no control measures are applied and a % reduction by the same date if there is a % reduction in disease transmission, i.e., an additional reduction of % in Ψ (fig. (b) ). we also simulated the system ( . ) to explore the impact of public health control measures on the timing and size of the pandemic peak in west-africa (fig. (c) . the results of our simulations show that in the absence of any control measure that results in a reduction in the effective transmission rates, i.e., if Ψ = , the covid- pandemic in west-africa will peak by july , with about , people infected on the day of the peak (red curve in fig. (c) ). it is worth noting that this is the worst case scenario. applying control measures at the baseline level, i.e., Ψ = % will bring about a % reduction in the number of cases (from , in the worst case scenario to , at the baseline control measures) when the pandemic j o u r n a l p r e -p r o o f journal pre-proof peaks and also delay the peak to october , (blue curve in fig. (c) ). improved control measures resulting in % and % reduction in disease transmission, will lead to reductions of % and %, respectively, in the number of daily confirmed cases when the epidemic attains its peak (black and magenta curves in fig. (c) ). thus, improved control measures that are related to reducing disease transmission have a significant effect in reducing the number of cases and flattening the epidemic curve-an outcome that will prevent the already weak and fragile health care systems in west-africa from being overwhelmed by a high covid- burden. table . other basic public health control measures include isolating symptomatic infectious humans and contact tracing and isolating asymptomatic humans. in our model we assume that isolating symptomatic infectious humans is at rate ρ s and contact tracing and systematic testing and isolating asymptomatic infectious humans is at rate ρ a . heat maps were plotted to investigate the individual and combined effects of pairs of control measures such as contact tracing, isolation, and using control measures that lead to a reduction in disease transmission, e.g., lockdowns, social and physical distancing, mask use, etc., on covid- in west-africa (fig. ) . our analyses show that the spread of the disease decreases with a higher reduction in the disease transmission rate and a decreasing proportion of exposed humans who do not develop clinical disease symptoms (i.e., become asymptomatic) after the incubation period, π ( fig. (a) ). thus, if a higher proportion of exposed humans do not develop clinical disease symptoms after the incubation period, a higher reduction in the disease transmission rate will be required to eliminate the disease. in particular, if half of the exposed individuals develop symptoms at the end of the incubation period, i.e., π = %, then only a % reduction in the disease transmission rate is required to eliminate the disease. but, if % of the exposed become asymptomatic after the incubation period, then a % reduction in the disease transmission rate is required to eliminate the disease (fig. (a) ). the combined impact of detection and isolation of asymptomatic humans (by contact tracing or mass testing) together with a reduction in the disease transmission rate is explored in fig. (b) . if journal pre-proof asymptomatic individuals are detected and isolated fast enough, e.g., within days, i.e., ρ a = . , then a % reduction in the disease transmission rate would suffice for elimination, while if it takes a long time to detect and isolate asymptomatic individuals, e.g., within days, a % reduction on the disease transmission rate is required to contain the pandemic in west-africa ( fig. (b) ). in particular, we found out that disease elimination is possible even if detection and isolation of asymptomatic humans is not complemented with any additional measure (maintaining Ψ = %). but such identification and isolation must occur in a timely manner, e.g., within days, i.e., ρ a = . (fig. (b) ). if only % of humans fail to develop symptoms at the end of the incubation period, then asymptomatic humans must be identified and isolated within days for disease elimination to be possible (fig. (e) ). thus, disease control is more difficult if it takes long to contact trace and isolate asymptomatic humans. our results also show that timely isolation of symptomatic cases is important in reducing the disease burden in west-africa but not enough as asymptomatic isolation do, although disease elimination is only possible if isolation of infectious symptomatic cases is complemented with another control measure (fig. (c) , (d)). in particular, if symptomatic humans are identified and isolated within days, i.e., ρ s = . , then a % reduction in the disease transmission rate is required for disease elimination, while if it takes a long time to isolate symptomatic infectious individuals, e.g., within days (i.e.,ρ s = . ), a % reduction on the disease transmission rate is required to contain the pandemic in west-africa (fig. (c) ). when isolation of symptomatic infectious disease cases is complemented with identification and isolation of asymptomatic infectious cases, disease elimination is only possible through timely identification and isolation of asymptomatic cases (fig. (d) ). thus, identifying and isolating asymptomatic individuals in a timely manner contributes more in curtailing the pandemic in west-africa than isolating symptomatic infectious individuals. furthermore, if more exposed humans develop disease symptoms at the end of the incubation period and more symptomatic infectious individuals are identified and isolated in a timely manner, disease elimination is possible (fig. (f) ). in particular, if % of exposed humans develop clinical disease symptoms at the end of the incubation period (i.e. π = %, then symptomatic infectious humans must be isolated within days in order to eliminate the disease. table . discussion mathematical models have been useful in understanding disease-outbreaks and in informing policy aimed at curtailing such diseases in a timely manner [ - , , , ] . in the context of the covid- pandemic that is currently spreading around the world, mathematical models have been very useful in predicting the course of the disease and in assessing the impacts of basic public health control measures [ - , , ] . in this study, we developed a mathematical model to inform the covid- trend and possible course of control measures in west-africa. the model is trained with covid- data from west-african countries for the period from february , to june , , and used to compute the reproduction number, as well as to assess the impact of basic public health control measures on the disease in the region. we obtained a basic reproduction number of r = [ ] . hence, although the disease is still spreading (r c > ) in west-africa, the spread is slow compared to other parts of world. this low transmission in the region can be attributed to the fact that there is limited movement of individuals within the region and between the region and the rest j o u r n a l p r e -p r o o f journal pre-proof of the world [ ] . furthermore, the low number of confirmed cases in west-africa might result from under-reporting of cases as was the case in peru [ ] . this is supported by the growing number of asymptomatic infectious individuals observed in our study. de leon et al. [ ] observed a similar trend in mexico, where over % of the infectious population was asymptomatic [ ] . in the absence of sufficient test kits in many west-african countries, and with the limitations in health care facilities and personnel, identifying cases is difficult [ ] . irrespective of the current trend, the covid- pandemic is still a more serious health problem in west-africa compared to previous outbreaks like the ebola outbreak ( . ≤ r c ≤ . ) [ , ] or the lassa fever outbreak in nigeria ( . ≤ r c ≤ . ) [ ] . although most countries in west-africa started implementing basic control measures since march , , our simulation results indicate that improvements are necessary to control the pandemic effectively in the region. because there is currently no safe and effective vaccine or drugs against the novel coronavirus, basic public health control measures have been used to curtail the pandemic in many parts of the world including west-africa. these measures include contact tracing, isolation, and measures that lead to a reduction in disease transmission, e.g., lockdowns, social or physical distancing, mask use, etc [ ] . in the absence of such measures, the worst case scenario prediction for west-africa from our model would have recorded around , confirmed covid- cases by july , when the epidemic peaks. this projection is lower than the , - , , confirmed cases predicted by achoki et al. [ ] for the same west-african region. the higher value in [ ] might be linked to the fact that the authors used linear models and considered the rate of infection between the first and second weeks of the epidemic in countries in which the number of cases might have been overestimated. however such prediction was not true since the control measures implemented in many west african countries back in march reduced the disease transmission. in fact, we found out that basic public health control measures, especially those associated with a reduction in the disease transmission rate such as lockdowns, social distancing, and mask use have a significant effect on reducing the burden of the disease, e.g, at baseline values, control measures reduce the number of cases when the epidemic peaks in october , by about %. also, improving control measures so that an estimated % reduction in the disease transmission is attained, will reduce the number of confirmed cases when the epidemic attains its peak by approximately % and shift the peak date to november , . such interventions are useful in reducing the disease spread and ensuring that the already limited and fragile health care systems are not overwhelmed by the covid- burden [ ] . studies have shown that asymptomatic, i.e., pre-symptomatic and asymptomatic infectious individuals and symptomatic individuals contribute in covid- transmission. asymptomatic cases might contribute more in disease spread since they are not even aware of the fact that they are infected with covid- . for example, on the diamond princess, % of all infections were due to asymptomatic individuals [ ] . similarly, the proportion of pre-symptomatic transmission varied between % and % in a study in singapore and in tianjin (china) [ ] . although, we do not have any documented information on covid- spread by pre-symptomatic and asymptomatic infectious individuals in west africa, we believe these groups contribute more in covid- transmission in west-africa. our parameter estimation confirms the fact that asymptomatic transmission in the region is higher than symptomatic transmission-a result that is consistent with recent findings in [ ] . it is also worth mentioning that many symptomatic cases might not be identified in the region, especially if they do not seek medical j o u r n a l p r e -p r o o f journal pre-proof attention [ ] or if they prefer alternative traditional medicine-a common practice in the region [ ] . our results suggest that early identification and isolation of both symptomatic and asymptomatic infectious individuals is an important step towards curtailing the burden of the pandemic. in particular, identifying and isolating asymptomatic individuals early enough, e.g., within - days after the end of the incubation period can result in a significant reduction in disease burden, even without any other control measure. the same is not true with identifying and isolating symptomatic individuals or when it takes a longer time to identify and isolate infectious asymptomatic individuals. in this case, the measure must be complemented with another measure that involves a reduction in the disease transmission rate in order to achieve a significant reduction in disease burden. asymptomatic infectious individuals can be identified through contact-tracing and diagnostic rt-pcr testing. however, since mass testing in most west-african countries only started during the last week of april, the primary way in which asymptomatic individuals were identified in west-africa before the start of mass testing was through contact tracing and then testing the contacts suspected to have the virus. one way in which contact tracing has been used successfully to identify and isolate both pre-symptomatic and asymptomatic infectious individuals is through mobile phone applications, see for example, ferretti et al. [ ] . we showed that a higher reduction in the disease transmission rate, e.g., through social distancing or using masks will be required to eliminate the disease if a higher proportion of exposed humans do not develop clinical disease symptoms after the incubation period. these results highlight the importance of early identification of disease tracing is already being implemented in west-africa, the measure is not very effective due to the lack of resources and high community transmission in the region. for example, as of june , , almost all the reported cases in the region resulted from community transmission [ ] . therefore increased compliance with measures like social distancing and mask use is very important for the region. since the disease can be transmitted from one human to the other through droplets from infected individuals that can travel though a few meters in air [ , ] , using masks and social distancing are very important in the fight against the pandemic. masks are useful in reducing the risk of both uninfected individuals wearing them from contracting the virus and infected individuals wearing masks from spreading the virus to others [ , ] . it is worth mentioning that the efficacy of n masks is about % ( − %), while the efficacy of surgical and cloth are % ( − %) and % ( − %), respectively [ ] . a study in us [ ] suggested that the higher the number of infected asymptomatic people in a population, the more beneficial the use of masks by the general public. both mask-use and social distancing are ongoing in many west-african countries. however, the effectiveness of these measures in reducing the spread of the virus is linked to the extent to which they are applied, the compliance level by the public, and how long they will last [ , ] . our analyses show that covid- can be reduced significantly in west-africa through social distancing and using masks if about % of the population complies with j o u r n a l p r e -p r o o f journal pre-proof these measures. this is consistent with recent results in [ , , ] , where strict compliance with social distancing and mask use can lead to a significant reduction disease burden. some west-african countries, e.g., benin have implemented measures involving isolating some densely populated portions of the country, especially overcrowded urban areas with dilapidated sanitary facilities and limited basic amenities like clean water [ ] . this has a positive effect on reducing the spread of the virus. finally, our study suggests that mass application of control measures coupled with appropriate testing and timely isolation of asymptomatic humans will go a long way in keeping disease numbers low, which is consistent with results in [ ] . however, with the fragile economic conditions and health care systems in the region, interventions like complete lock-down as was the in some european countries, most us states, and china for a couple of months is not feasible [ ] and can be more catastrophic than the disease itself. therefore, the choice of interventions by public policy makers in the region should aim at balancing the prevention of the epidemic with the need for maintaining livelihoods and social cohesion. our study confirms the fact that the novel coronavirus (covid- ) is highly contagious and that infectious humans who do not show clinical disease symptoms (asymptomatic or unreported cases) contribute significantly in disease transmission. this study also indicates that early identification of these unreported cases through contact tracing or systematic testing on target groups, and then isolating individuals who test positive plays a significant role in diminishing the burden of covid- in west-africa. furthermore, this study suggests that social distancing measures such as stay-at-home orders, closing educational institutions, limiting mass gatherings, etc., and using masks can reduce the spread of the disease significantly, with the possibility of disease-elimination if about one in every two people in west-africa respect these control measures. to enhance, the study shows that implementing more than one measure at the same time is better for disease control, and that under current control measures, the disease might not be disappearing from the region any time soon, unless there are improvements in these control measures. we conclude that systematic testing on target group, contact tracing and isolation of confirmed disease cases, as well as improvements to the other existing basic public health measures (e.g., social distancing and mask use) in the region, are required to better manage the pandemic. also, due to uncertainties and various disparities between the economies and health care systems of countries within the region, we conclude that country-level studies are necessary and will provide more insights into disease dynamics and control in the region. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia an 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transmission-a modelbased analysis of the diamond princess outbreak. medrxiv estimating the generation interval for covid- based on symptom onset data quantifying sars-cov- transmission suggests epidemic control with digital contact tracing mathematical model for lassa fever transmission dynamics with variable human and reservoir population feasibility of controlling covid- outbreaks by isolation of cases and contacts airborne transmission of sars-cov- : the world should face the reality modelling transmission and control of the covid- pandemic in australia a final size relation for epidemic models hbt acknowledges the support of imu-cdc through graid program. kvs acknowledges the support of the wallonie-bruxelles international post-doctoral fellowship for excellence, belgium (fellowship # sub/ / ). cnn acknowl- the author declares no conflict of interest. key: cord- -zmizyx e authors: cheng, yuan-yang; chen, chin-ming; huang, wei-chun; chiang, shang-lin; hsieh, pei-chun; lin, ko-long; chen, yi-jen; fu, tieh-cheng; huang, shu-chun; chen, ssu-yuan; chen, chia-hsin; chen, shyh-ming; chen, hsin-shui; chou, li-wei; chou, chen-liang; li, min-hui; tsai, sen-wei; wang, lin-yi; wang, yu-lin; chou, willy title: rehabilitation programs for patients with coronavirus disease : consensus statements of taiwan academy of cardiovascular and pulmonary rehabilitation date: - - journal: j formos med assoc doi: . /j.jfma. . . sha: doc_id: cord_uid: zmizyx e abstract the coronavirus disease (covid- ), which developed into a pandemic in , has become a major healthcare challenge for governments and healthcare workers worldwide. despite several medical treatment protocols having been established, a comprehensive rehabilitation program that can promote functional recovery is still frequently ignored. an online consensus meeting of an expert panel comprising members of the taiwan academy of cardiovascular and pulmonary rehabilitation was held to provide recommendations for rehabilitation protocols in each of the five covid- stages, namely ( ) outpatients with mild disease and no risk factors, ( ) outpatients with mild disease and epidemiological risk factors, ( ) hospitalized patients with moderate to severe disease, ( ) ventilator-supported patients with clear cognitive function, and ( ) ventilator-supported patients with impaired cognitive function. apart from medications and life support care, a proper rehabilitation protocol that facilitates recovery from covid- needs to be established and emphasized in clinical practice. since the end of , coronavirus disease (covid- ), a novel infectious disease emerging from wuhan, china, has continued to spread rapidly, causing an ongoing global outbreak. patients may exhibit dyspnea, hypoxia, remarkable pneumonia, acute respiratory distress syndrome (ards), or even multiple organ failure. , in addition to the possible sequelae of pulmonary fibrosis, which could impair the survivors' ventilation and oxygenation, many other organs could be affected, especially the cardiovascular system. common complications of the cardiovascular system may include arrhythmia, myocarditis, acute coronary syndrome, venous thromboembolism, cardiogenic shock, and heart failure. furthermore, prolonged inactivity can also affect exercise capacity significantly. apart from medical treatment and supportive therapy, rehabilitation plays a vital role throughout the entire disease course. an appropriate rehabilitation program could help patients with mild disease maintain exercise capacity and activities of daily living. for patients with advanced disease severity, a well-designed rehabilitation program is even more crucial to improve pulmonary secretion clearance, ameliorates side-effects related to a prolonged bedridden state, and even prevents intensive care unit-acquired weakness. this expert consensus provides recommendations for rehabilitating patients with varying degrees of covid- severity. although rehabilitation has multiple benefits for patients, the disease remains highly contagious and poses a substantial threat to medical providers. hence, proper protective personal equipment (ppe) should be used when caring for patients with covid- , j o u r n a l p r e -p r o o f considering that covid- can cause significant morbidity and mortality based on certain risk factors, such risk factors need to be identified in order to establish individualized rehabilitation programs. data from , laboratory-confirmed cases of covid- in china have shown that disease severity can be categorized as mild, severe, and critical. approximately % of infected patients were categorized as mild and presented without or with mild pneumonia, while approximately % were categorized as severe and presented with dyspnea, blood oxygen saturation ≤ %, partial pressure of arterial oxygen to fraction of inspired oxygen (p/f) ratio < , or lung infiltrates > % within to hours. the remaining % were classified as critical and presented with respiratory failure, septic shock, or multiple organ dysfunction, which resulted in a high mortality rate of %. those with preexisting comorbid conditions, including cardiovascular disease (cvd) ( . %), diabetes ( . %), chronic respiratory disease ( . %), hypertension ( . %), and cancer ( . %), displayed a higher case-fatality rate (cfr). patients without underlying medical conditions had an overall cfr of . %. a recent meta-analysis that assessed the risk for severe infection in , patients found that those with cvd [odds ratio (or) = . ] had the highest risk for severe infection, followed by respiratory system disease (or = . ) and hypertension (or = . ). moreover, another study outside china showed that older age (or = . ), male gender (or = . ), and hypertension (or = . ) were independently associated with severe disease at admission. altogether, potentially significant risk factors for severe covid- that should be identified and considered when designing rehabilitation programs include old age, male gender, hypertension, diabetes, respiratory disease, and cvd. the world health organization (who) had categorized clinical syndromes associated with covid- as mild illness, pneumonia, severe pneumonia, ards, sepsis and septic j o u r n a l p r e -p r o o f shock. however, this expert consensus categorized patients with covid- differently such that outpatients with various risk factors or inpatients with disparate cognitive status should receive distinct rehabilitation programs. therefore, the current expert consensus categorized patients with covid- into the following five groups: ( ) outpatients with mild disease and no risk factors, ( ) outpatients with mild disease and epidemiological risk factors, j o u r n a l p r e -p r o o f majority of the patients with covid- had mild disease. those with mild disease and no risk factors were allocated to home care, with rehabilitation recommendations similar to those of the american college of sports medicine (acsm) general principles of exercise prescription, though infection control is essential. , while home-based rehabilitation is recommended for this group of patients, hospital-based rehabilitation may be started only when patients have been ( ) at least days since symptom onset ( ) at least hours since resolution of fever without taking antipyretic drugs and ( ) without other covid- related symptoms, according to the recommendation for discontinuation of home isolation from center for disease control and prevention of united states. the objective of rehabilitation is primarily preventing complications of inactivity through conditioning exercises. conditioning exercises should include at least three components, namely warm-up, exercise, cool-down and stretching. a proper warm-up generally includes light intensity endurance activities for at least - minutes. exercises include both aerobic exercise and resistance training. aerobic training involves the activation of large muscle groups in a rhythmic movement (e.g., treadmill walking, bicycling, or upper limb ergometer trainings). training frequency, intensity, and duration should be as follows: -frequency: days or more per week -intensity: maintaining a heart rate reserve of %- % during exercise, while the heart rate reserve refers to the difference between the predicted maximum heart rate and the resting heart rate ( ) hypertension: post-exercise blood pressure (bp) reduction should be considered especially among older patients receiving anti-hypertensive medicine such as α-blockers and vasodilators. therefore, exercise termination should be gradual, while the cool-down phase should be extended and monitored carefully until bp and heart rate return to resting levels. avoid the valsalva maneuver or breath holding during resistance training and static stretching to avoid excessive bp elevation. ( ) cvd: exercise training is safe and effective for most patients with cvd. patient whose angina threshold heart rate had been previously determined by a formal cardiopulmonary exercise test should adjust their exercise intensity to the upper limit of heart rate beats/min below the angina threshold. patients who had not undergone formal exercise testing can exercise at a rated perceived exertion of - on a - scale. however, a formal exercise test should still be considered in patients with advanced cvd with proper clinical disinfection following cdc ( ) pulmonary disease: patients with previous chronic pulmonary diseases could develop much more airway secretions than those without. airway clearance techniques, which will be described in detail in the next section, should include flutter breathing, autogenic drainage, and cough techniques, such as huff coughing and controlled coughing. specific exercises, such as yoga, tai-chi, pilates, and trunk core muscle training, can help stabilize the trunk and promote efficient breathing. inspiratory muscle training, with a threshold loading starting at % of the maximal inspiratory pressure (mip), is recommended given evidence suggesting its benefits in reducing dyspnea and improving, exercise capacity and quality of life for patients with copd. during conditioning exercises, intensity should be set at a rated perceived exertion of - on a - scale. exercise in cold environments or those with allergens or pollutants should be limited to avoid triggering bronchoconstriction among susceptible individuals. approximately % of patients with covid- developed moderate to severe disease that required hospitalization and aggressive treatment. these individuals usually present with fever, cough, dyspnea, tachycardia, tachypnea, and various degrees of oxygen desaturation necessitating support. studies have shown that early rehabilitation interventions for community-acquired pneumonia and interstitial pneumonia within days of admission reduced in-hospital mortality. , the two primary objectives of rehabilitation in this stage are promoting airway clearance and preventing complications of acute illness-related immobilization. about . % of covid- patients could have copious sputum production. incorporating chest physiotherapy into the medical treatment of patients with lung consolidation plays an important role in helping patients with airway secretions. proper chest physiotherapy could promote effective expectoration, enhance mucociliary clearance of secretions to the upper airways, and improve cough effectiveness. chest physiotherapy strategies that promote airway clearance include the following: another important issue in the rehabilitation of patients with moderate to severe disease is preventing deconditioning due to acute illness. immobilization has been shown to speed functional decline with reduced muscle strength and cardiorespiratory fitness, particularly among elderly individuals and those with comorbidities. , therefore, once medical condition stabilized, early mobilization should be encouraged. rehabilitation interventions for such patients should include the following: ( ) active or active-assisted range of motion (rom) exercise: patients with moderate to severe disease who are able to actively move their extremities are encouraged to engage in active or active-assisted rom exercises to maintain or improve joint integrity and prevent joint contracture and soft-tissue shortening. ( ) mobilization and progressive off-bed activities: early mobilization with progressive mobility training, including off-bed transferring, unsupported sitting, standing, level-surface ambulation, and stair climbing, tailored to the patient's general condition should be carried out once medically stabilized. proper walking aid may be used to assist in energy conservation to reduce distress during the activity. the following are the key points and strategies for the rehabilitation of ventilated patients with clear cognitive status: ( ) activities: in-bed cycling in patients with critical illness has been shown to be a safe and feasible procedure for preserving muscle function and muscle fiber cross-sectional area, as well as fostering positive mental effect, including the feeling of control, safety, and hope, during the critical stages of the illness. low resistance levels (i.e., approximately . nm), are recommended with patient self-selected pedaling rate for minutes daily. upper-limb resistance training using elastic bands and pulleys can also be utilized. patients who have stable clinical status can commence out-of-bed mobility depending on their tolerance, which may include sitting on the edge of the bed, moving from bed to chair, standing next to the bed, stepping on the spot, and walking with ambulatory assistive devices. ( ) breathing exercises: inspiratory muscle training, which strengthens the inspiratory muscles through the application of resistance during inspiration, can also be used for intubated patients or those with a tracheostomy. using a threshold training device, with the threshold set at % of the mip, five sets of six breaths performed once per day are recommended. patients can also be educated on diaphragmatic breathing, which involves the negative pressure generated by the diaphragm instead of the accessory respiratory muscles. finally, chest expansion and mobilization are important for increasing chest wall mobility and improving thoracic compliance during mechanical ventilation. j o u r n a l p r e -p r o o f characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china chinese center for disease control and prevention clinical features of patients infected with novel coronavirus in wuhan, china viral infection increases the risk of idiopathic pulmonary fibrosis: a meta-analysis covid- and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options cardiovascular considerations for patients, health care workers, and health systems during the covid- pandemic physiotherapy management for covid- in the acute hospital setting: clinical practice recommendations prevalence of comorbidities in the novel wuhan coronavirus (covid- ) infection: a systematic review and meta-analysis host susceptibility to severe covid- and establishment of a host risk score: findings of cases outside wuhan clinical management of covid- : interim guidance acsm's guidelines for exercise testing and prescription covid- ) for healthcare professionals world health organization. country & technical guidance -coronavirus disease (covid- ) discontinuation of isolation for persons with covid- not in healthcare settings (interim guidance) percentages of maximal heart rate, heart rate reserve and vo max for determining endurance training intensity in male runners test-retest reliability of the one-repetition maximum ( rm) strength assessment: a systematic review interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease (covid- ) in healthcare settings real-time telerehabilitation for the treatment of musculoskeletal conditions is effective and comparable to standard practice: a systematic review and meta-analysis coronavirus disease (covid- ) and pregnancy: what obstetricians need to know epidemiology of covid- among children in china exercise standards for testing and training: a scientific statement from the american heart association cardiac safety of off-label covid- drug therapy: a review and proposed monitoring protocol united states food and drug administration. fda cautions against use of hydroxychloroquine or chloroquine for covid- outside of the hospital setting or a clinical trial due to risk of heart rhythm problems effects of different core exercises on respiratory parameters and abdominal strength effects of inspiratory muscle training in copd patients: a systematic review and meta-analysis effectiveness of early versus delayed physical rehabilitation on in-hospital mortality in interstitial pneumonia: a retrospective cohort study early rehabilitation and in-hospital mortality in intensive care patients with community-acquired pneumonia clinical characteristics of coronavirus disease in china aarc clinical practice guideline: effectiveness of nonpharmacologic airway clearance therapies in hospitalized patients the efficacy of flutter((r)) and active cycle of breathing techniques in patients with bronchiectasis: a prospective, randomized, comparative study airway clearance techniques for bronchiectasis effect of airway clearance techniques in patients experiencing an acute exacerbation of chronic obstructive pulmonary disease: a systematic review physiotherapy for airway clearance in adults reduced j o u r n a l p r e -p r o o f physical activity in young and older adults: metabolic and musculoskeletal implications determining best outcomes from community-acquired pneumonia and how to achieve them the mechanisms and treatments of muscular pathological changes in immobilization-induced joint contracture: a literature review visuo-acoustic stimulation that helps you to relax: a virtual reality setup for patients in the intensive care unit care for critically ill patients with covid- should early mobilization be routine in mechanically ventilated patients? a prospective study of the safety and feasibility of early in-bed cycling in mechanically ventilated patients impact of very early physical therapy during septic shock on skeletal muscle: a randomized controlled trial in-bed cycling in the icu; patient safety and recollections with motivational effects early mobilization of critically ill patients in the intensive care unit: a systematic review and meta-analysis inspiratory muscle training for intensive care patients: a multidisciplinary practical guide for clinicians safety and effectiveness of the high-frequency chest wall oscillation vs intrapulmonary percussive ventilation in patients with severe copd treatment of ards with prone positioning early physical and occupational therapy in mechanically ventilated, critically ill patients: a randomised controlled trial of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome prone position for acute respiratory distress syndrome airway clearance therapy: finding the evidence intrapulmonary percussive ventilation improves lung function in cystic fibrosis patients chronically colonized with pseudomonas aeruginosa: a pilot cross-over study mechanical ventilation, diaphragm weakness and weaning: a rehabilitation perspective a randomized trial of an intensive physical therapy program for patients with acute respiratory failure standardized rehabilitation and hospital length of stay among patients with acute respiratory failure: a the authors would like to thank dr. kai-hua chen, the secretary-general of taiwan academy of cardiovascular and pulmonary rehabilitation, for holding discussion meetings, recording the details of the meetings, and miscellaneous tasks related to the publishing of this expert consensus.j o u r n a l p r e -p r o o f key: cord- - ddlh authors: peeling, rosanna w. title: diagnostics in a digital age: an opportunity to strengthen health systems and improve health outcomes date: - - journal: int health doi: . /inthealth/ihv sha: doc_id: cord_uid: ddlh diagnostics play a critical role in clinical decision making, and in disease control and prevention. rapid point-of-care (poc) tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests vary, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. a new generation of poc molecular tests that are highly sensitive and specific, robust and easy to use are now available for deployment in low resource settings. decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. a feature of many of these poc molecular devices is that they are equipped with data transmission capacities. in a digital age, it is possible to link data from diagnostic laboratories and poc test readers and devices to provide data on testing coverage, disease trends and timely information for early warning of infectious disease outbreaks to inform design or optimisation of disease control and elimination programmes. data connectivity also allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. 'without diagnostics, medicine is blind.' alain merieux. diagnostics and laboratory services have been labelled as the achilles heel of global health. in the past, low quality diagnostics and ineffective laboratory services resulted in a mistrust of laboratory results, which in turn led to a decreasing demand for laboratory services and a low priority for funding. the maputo declaration has called on governments, multilateral agencies, development partners, professional associations and academic institutions to address laboratory challenges that limit the scale-up of services for tb, malaria and hiv diagnosis and care. together with the us centers for disease control and prevention's call to end the neglect of laboratories, this has provided a major milestone on a pathway to place diagnostics and laboratory services as important pillars of a healthcare system. , recent outbreaks of influenza, middle east respiratory syndrome coronavirus (mers-cov) and ebola virus disease illustrate the importance of diagnostics in outbreak investigations. [ ] [ ] [ ] with rapid technological innovations in the last years and donor investments in the development of improved diagnostics for infectious diseases of public health importance, it is time to re-examine the achilles heel and explore the promises and challenges of diagnostics in a digital age. accurate diagnostic tests play a key role in clinician trust and patient management. diagnostic testing is traditionally considered as a tool to rule in or rule out a condition or infection when clinical presentation in a patient is non-specific. for diseases such as hiv and other sexually transmitted infections, most infections are asymptomatic but can cause serious long-term consequences. diagnostics are used to screen for infection so that treatment can be given to prevent the development of long-term complications and to interrupt the chain of transmission to sexual partners or to the fetus in the case of pregnant women. a test for the human papillomavirus helps to determine risk of disease progression to cervical cancer. for pathogens that have developed antibiotic resistance, such as neisseria gonorrhoeae, diagnostic testing is used to determine antibiotic susceptibility to ensure treatment efficacy. for hiv patients, a cd test result is used to determine if the patient is eligible for antiretroviral treatment and, for those on treatment, a viral load assay is used to determine treatment compliance and drug efficacy. a test of cure is used to determine when treatment can be stopped ( figure ). beyond patient management, diagnostics play a critical role in various aspects of public health through disease prevention and control. diagnostics are critical in surveillance, to monitor trends in disease and antimicrobial resistance and assess the impact of interventions. diagnostic tools 'of sufficient sensitivity and specificity to detect levels of infection that can lead to transmission' were identified as one of the three essential requirements for disease elimination or eradication. who has set eradication or elimination targets for a number of neglected tropical diseases (ntds), including guinea worm, polio, yaws, trachoma, schistosomiasis, lymphatic filariasis, onchocerciasis, human african trypanosomiasis, chagas' disease and visceral leishmaniasis (vl) and the elimination of mother to child transmission (emtct) of hiv and syphilis. mass drug administration (mda) is the main control strategy for many of these diseases, resulting in the need for highly sensitive tests to detect the few remaining cases in areas of low transmission intensity after multiple rounds of mda. highly specific tests are then needed to certify elimination and maintain post-elimination surveillance. there is often little commercial interest in the development of diagnostics that are used mainly for surveillance because of the low return on investment. for a ntd such as schistosomiasis, the chinese national control programme has defined stages for schistosomiasis control as morbidity control (prevalence over % as defined by stool examination), infection control (prevalence of - %), transmission control (prevalence lower than %), transmission interruption (no case detected in years successively) and elimination (no case detected in the e years after transmission was interrupted). diagnostics results are used to inform national and regional control policy, redirect resources and to transition to the next phase of the control and treatment strategy. the who targets for emtct and who/unaids - - targets for hiv by also include diagnostic test coverage as indicators for efficiency of service delivery that help countries with setting a course to have an aids free generation and to end the hiv epidemic by . diagnostic testing is usually performed in laboratories. in countries where the laboratory infrastructure is limited, who advocates for the use of a syndromic approach for patient management whereby patients are treated for all the major causes of that syndrome. this often leads to overtreatment and increases risk for the development of antimicrobial resistance. in the last decade, rapid point-of-care (poc) diagnostic tests fulfilling the assured criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable) have become commercially available and are widely used for infectious diseases such as malaria, hiv and syphilis. [ ] [ ] [ ] [ ] [ ] these tests have allowed control programmes to increase access to testing, identify those who need to be put on treatment, optimise disease control and save lives. however, the quality of these tests varies, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. a new generation of immunoassays, molecular and nanotechnology platforms has been developed in recent years that can improve patient management and disease surveillance. these platforms have superior performance and make use of optical readers, mobile phones and data transmission capabilities to improve reading of results and data transmission. such technologies provide real-time results to inform patient management decisions. data, linked to precise geographical locations using gps, can be transmitted to a central database to inform disease control programmes or to monitor progress towards elimination. many of these assays can also yield quantitative results to give estimates of pathogen copy number or, in the case of hiv, to monitor treatment compliance or efficacy. these technology platforms can also be used to detect multiple targets from a single specimen. for geographic areas with multiple diseases targeted for elimination, if surveillance is conducted in the same sentinel population, such as children under the age of years, using the same specimen, such as a blood sample, exposure to multiple ntds can be detected on a multiplex platform. this may be a more cost-effective means of conducting post-mda surveillance than collecting specimens and testing for a single ntd at a time, , although cost-effectiveness of using these platforms for multiple ntds remains to be demonstrated. increased sensitivity of detection using these advances has made it possible to have tests that detect antibodies from oral fluids. the use of these non-invasive specimens has made it possible to design hiv tests for self-testing or for home use. the availability of self-testing at home has allowed testing to be de-stigmatised and should help countries with achieving the first of the unaids/who - - targets. bead-based immunoassays bead-based technologies are more versatile and potentially offer higher sensitivity compared to traditional solid phase immunoassays. instead of using enzymatic amplification of a colorimetric substrate, bead-based assays can use a range of labels, including laser or fluorescence to detect the binding of the secondary antibody to an antigen or antibody target. the beads are in solution, offering more sites for ligand binding compared to a solid phase support. fluorescent intensities can yield quantitative results. protein arrays offer the potential of quantification of the antigen target as a surrogate measure of bacterial, parasite or viral load to inform treatment strategies. detection of multiple targets from the same pathogen can lead to increased efficiency over single-target assays. detection of targets from multiple pathogens using a single specimen can offer substantial cost and sample savings over traditional elisa measurements. the detection platforms are often equipped with data transmission capacities and allows near real-time surveillance of disease trends and monitoring the effect of special interventions. microfluidic devices offer many advantages, such as high throughput, short analysis time, small volume and high sensitivity, which make them an ideal immunoassay format for clinical diagnoses. the current microfluidic immunoassays have limited multiplexing capability compared to flow cytometric assays but are improving. immunoassays developed on a microfluidic platform that reproduce all the steps of a traditional elisa in a miniaturised format are now available. the microfluidic disc can fit into the palm of a hand and is rapid and inexpensive to manufacture. these platforms allow multiplexing, but have only been validated for antibody detection in blood samples. antigen detection from urine or stool samples will require multistep specimen processing and/or purification or concentration before being put into the antigen-antibody reaction within the microfluidic channels. a recent publication showed that it is possible to use the power of smart phones to power a microfluidic immunoassay reaction by plugging the reaction chamber into a smart phone using a dongle. over assays can be run before recharging is necessary. the results are interpreted and displayed on the phone and the data transmitted to a central database if required. a dual test to detect antibodies to hiv and syphilis has been designed on this platform and will have utility not only to improve access to prenatal screening but also to allow surveillance data for the dual elimination of mtct of hiv and syphilis to be available in real time. nanosensor assays a growing number of promising diagnostic tools are based on nanotechnology. the application of nanomaterials to detect host or pathogen biomarkers has the potential to yield ultrasensitive assays. quantum dots are fluorescent semiconductor nanoparticles typically between and atoms in diameter and the technology has been applied to the development of ultrasensitive tests for hiv and other viral infections. it is also possible to use these technologies to combine pathogen detection and speciation with genetic analysis, such as detection of single nucleotide polymorphisms. hence, they can be used for high throughput screening of drug mutation targets. quantum wires are being used in the development of a fully automated diagnostic test for malaria infection, speciation and drug resistance status in less than minutes. these nanosensors can ultimately be configured to a handheld pda-type device or a thin plate about the size of a small business card containing a tiny nanowire sensor. operationally, these tests are simple to use and give answers in less than minutes. they can be used during a doctor's visit or at home by a patient, or early detection of bioterrorism in a community setting. linking mobile connectivity and gps will enable anonymised disease data to be sent to a data cloud for real-time surveillance. advocacy to apply these novel technologies to ntds and antimicrobial resistance monitoring is an urgent priority. this requires precise engineering of nanomaterial surfaces as the interface between the nanomaterial and the specimen is where the reaction occurs. molecular assays offer superior diagnostic performance compared to the limit of detection of immunoassays. in the last two decades, nucleic acid amplification tests (naats) have become the 'gold' or reference standard to which the performance of other diagnostic tests is compared. until recently, they have remained largely laboratory based and are expensive and inaccessible. a number of poc molecular assays, with or without amplification, are now on the horizon that may transform the delivery of health services. , the first sample-in/answer-out real-time pcr platform that can be used wherever there is a source of electricity has been introduced for the diagnosis of tb and detection of rifampicin resistance. [ ] [ ] [ ] the genexpert real-time pcr platform has a large menu of test targets; thus, allowing polyvalency to test for different pathogens. it requires minimal onsite expertise and provides a result in less than hours. it has additional advantages of random access and remote quality control. however, the cartridges are expensive and not affordable or sustainable unless subsidised in most high burden countries. to maximise the impact of these novel technologies, it is critical that patient pathways be modified to take advantage of the speed to result and to find cost-efficient means of implementation compared to traditional laboratory testing. isothermal amplification platforms such as helicase dependent amplification (hda), cross priming amplification (cpa), recombinase polymerase amplification (rpa) and loop-mediated amplification (lamp) assays can be engineered into poc naats as there is no requirement for sophisticated equipment to perform thermal cycling. the details of these technologies have been described elsewhere. , poc naats based on these technologies have largely been applied to high burden diseases such as hiv and tb, but other technologies, such as lamp, have been developed and evaluated for the diagnosis of vl and hat. in the case of vl, the lamp assay for leishmania donovani is highly sensitive and specific for the diagnosis of vl (using a blood sample), and for the diagnosis of post-kala azar dermal leishmaniasis (pkdl) (using a skin biopsy). diagnosis of pkdl will be important for the elimination agenda since patients with this condition are an important source of infection. in the case of hat, the lamp assay was shown to have a sensitivity of . % ( % ci . to . %) and a specificity of . % ( % ci . to . %) compared to a pcr reference standard. advocacy and investments are needed to apply these technologies to the control and elimination of ntds. the promise of diagnostics in a digital age in a digital age, data from social media and poc diagnostics in communities can be used to provide early warning for infectious disease outbreaks, and timely information to inform disease control and elimination programmes. the isense project, an epsrc irc funded project in early warning sensing systems in infectious diseases, aims to create low-cost latent sensing systems to analyse self-reported symptoms on the web, including social networks and micro-blogging sites (twitter) and searches (bing, google). isense will develop a new generation of early warning sensing systems to identify outbreaks of deadly infectious diseases, such as flu, methicillin-resistant staphylococcus aureus (mrsa) and hiv, by linking self-reported symptoms on the web to a new sensor-enabled disease surveillance infrastructure for an early warning sensing system for infectious diseases (http://www.i-sense.org.uk/research). the simplest poc diagnostic tests that are widely used today are rapid diagnostics tests (rdts) in a lateral flow format. these are read with the naked eye, which is subjective and prone to human error and may be further exacerbated by poor lighting in health posts. in addition, rdts lack on-board quality control and are often used in remote areas where health workers receive minimal training. as a result, accuracy of rdts performed in the field can be quite variable. this may adversely affect patient care and the accuracy of the data gathered for surveillance. digital imaging technology in electronic readers and smart phones can be used to capture and interpret rdt results and transmit data. given the large number of brands of rdts for hiv, malaria and syphilis, it would be ideal to have diagnostic test readers that could accommodate a variety of test technologies (e.g., lateral flow, immunofiltration) and formats (e.g., strips, cassettes of various sizes and shapes). a number of rdt readers are already on the market and others are in the pipeline. given the widespread adoption of smart phones in resource-limited settings, rdt readers using this technology have the potential to combine high-resolution test images with the computing capability required to run image analysis software and transmit data. the readers range in price depending on the technical complexity of the instrument and their compatibility with the type of rdts. data collection for the emtct is difficult when testing is highly decentralised and data quality is difficult to verify. to track global progress towards elimination of mtct of hiv and syphilis many challenges need to be addressed. currently, data from antenatal screening need to be manually accessed from individual testing sites. electronic readers for rdts are able to capture and automatically transmit data and may, therefore, serve a critical purpose for strengthening data collection and surveillance in the context of monitoring and evaluation of dual elimination. , improving supply chain management real-time data monitoring via electronic readers could help to improve coordination of supply chain management by multiple partners. operational data on stocks, device usage and condition can be uploaded via wi-fi or cellular networks and transmitted to central databases. by linking the data to supply chain management software, stock-outs can potentially be avoided and health system efficiency improved. in recent years, rdts have been introduced to increase hiv and syphilis screening in antenatal clinics, enabling same day testing and treatment of infected mothers to prevent adverse outcomes of pregnancy and fetal loss. the emtct targets to be achieved are % of pregnant women access antenatal care, % of pregnant women at antenatal care tested for hiv and syphilis, and % of those testing positive receive treatment. to achieve these targets, countries will need to have a system to track progress the capacity for ongoing surveillance once the elimination target is achieved. studies have shown that the introduction of rapid rdts for syphilis for prenatal syphilis screening has strengthened health systems by improving access to diagnostics, health worker job satisfaction and reducing stillbirths and neonatal mortality. in peru, the introduction of rdts for prenatal screening has resulted in infected pregnant women being screened and treated in a single visit instead of - visits over days. , challenges of new technologies assuring quality of poc lateral flow tests and devices decentralisation of testing outside of the laboratory can put tremendous stress on the healthcare system and presents challenges for training and quality assurance. when testing is decentralised, programme managers are often unable to monitor testing coverage and quality, making it difficult to identify problems of sub-standard testing and stock-outs in a timely manner. international health for poc molecular devices, requirements for instrument calibration, ongoing maintenance and frequency of failure, power usage and environmental sustainability should also be considered. creation of a central database to collect surveillance data from village antenatal clinics all the way through to national and global databases is needed. this involves obtaining consensus on where to host the data, what data to collect, who has access to the data and finding affordable software. middleware solution that provides 'open access' will allow for rapid coordinated transmission of data to governments. technology is needed to ensure data is collected, transmitted and stored in a way that conforms to ethical and legal standards, maintaining patient privacy and confidentiality. these governance issues may be resolved by ensuring separate levels of access to operational data versus patient data. in addition, data storage should be in-line with country specific regulations. further discussion needs to take place around data ownership. an excellent example of a national database that informs critical programmatic decisions in real time is the kenyan national early infant diagnosis (eid) portal (www.nascop.org/eid), which has now been extended to cover hiv viral load and other diagnostics. this database is the result of a successful public-private partnership that involved the government of kenya ministry of health, usaid/president's emergency plan for aids relief (pepfar), the us cdc, the clinton health access initiative (chai), hewlett packard, safaricom and strathmore university. hewlett packard built a basic data centre at the national aids/std control programme (nascop) and at testing laboratories for early infant diagnosis. safaricom, the largest provider of mobile services in kenya, set up a short code service for sms. strathmore university students built the application. pepfar, cdc and chai rolled out the eid programme countrywide. the result is a national eid portal running on a government data centre-hosting all the testing and program data entered from computers in laboratories equipped with laboratory information systems (web-based applications). over health facilities that provide eid services have access to all the data and test results over sms, mobile web, web and email in near real time. data analytics include sample transport tracking, volume of testing at each facility, test results, including the number of rejected samples and indeterminate results. results are delivered via electronic and paper means (sms printer, email, courier, web) and real time eid/prevention of mtct programme analytics are available online by facility, and at regional and national levels. the new generation of diagnostics equipped with digital technologies are transforming the field of clinical decision-making and disease control and prevention. rapid poc tests for infectious diseases can improve access to diagnosis and patient management, but the quality of these tests varies, quality of testing is often not assured and there are few mechanisms to capture test results for surveillance when the testing is so decentralised. electronic readers have the potential to provide fast, accurate, standardised rdt interpretation and real-time data reporting with a huge range of positive functions, including improving quality assurance, supply chain management and providing accurate timely data for surveillance. although countries need to consider data governance and confidentiality issues, data connectivity allows control programmes to monitor the quality of tests and testing, and optimise supply chain management; thus, increasing the efficiency of healthcare systems and improving patient outcomes. author's contribution: rp has undertaken all the duties of authorship and is guarantor of the paper. achilles heel" of global efforts to combat infectious diseases laboratory medicine in africa: a barrier to effective health care the maputo declaration on strengthening of laboratory systems. geneva: world health organization laboratory systems and services are critical in global health: time to end the 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diseases surveillance hiv self-testing in resource-limited settings: regulatory and policy considerations unaids. - - -an ambitious treatment target to help end the aids epidemic bead-based microfluidic immunoassays: the next generation microfluidics-based diagnostics of infectious diseases in the developing world smartphone dongle for simultaneous measurement of hemoglobin concentration and detection of hiv antibodies multisite laboratory evaluation of a dual human immunodeficiency virus (hiv)/syphilis point-of-care rapid test for simultaneous detection of hiv and syphilis infection integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth emerging technologies in point-of-care molecular diagnostics for resource-limited settings point-of-care nucleic acid testing for infectious diseases feasibility, diagnostic accuracy, and effectiveness of decentralised use of the xpert mtb/rif test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study point-of-care system for detection of mycobacterium tuberculosis and rifampin resistance in sputum samples geneva: unitaid; geneva opportunities and challenges for cost-efficient implementation of new point-of-care diagnostics for hiv and tuberculosis application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis diagnostic accuracy of loopamp trypanosoma brucei detection kit for diagnosis of human african trypanosomiasis in clinical samples multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an openarray platform using electronic readers to monitor progress toward elimination of mother-to-child transmission of hiv and syphilis: an opinion piece elimination of elimination of mother-to-child transmission (emtct) of hiv and syphilis. global guidance on criteria and processes for validation. geneva: world health organization point-of-care tests to strengthen health systems and save newborn lives: the case of syphilis rapid syphilis tests as catalysts for health systems strengthening: a case study from peru the costs of accessible quality assured syphilis diagnostics: informing quality systems for rapid syphilis tests in a tanzanian setting the author would like to acknowledge david mabey and debi boeras in providing helpful comments in the preparation of this manuscript.funding: none. ethical approval: not required. key: cord- -cq uzziv authors: ma, hui; dong, ji-ping; zhou, na; pu, wei title: military-civilian cooperative emergency response to infectious disease prevention and control in china date: - - journal: mil med res doi: . /s - - -y sha: doc_id: cord_uid: cq uzziv in recent years, the incidence of severe infectious diseases has increased, and the number of emerging infectious diseases continues to increase. the chinese government and military forces have paid a great deal of attention to infectious disease prevention and control, and using military-civilian cooperation, they have successfully prevented numerous severe epidemic situations, such as severe acute respiratory syndrome (sars), influenza a (h n ), avian influenza h n and h n , and ebola hemorrhagic fever, while actively maintained public health, economic development, and national construction. this paper focuses on the mechanisms of the military-cooperative emergency response to infectious diseases--the joint working mechanism, the information-sharing mechanism, the research collaboration mechanism, and the joint disposal mechanism--and presents a sorted summary of the practices and experiences of cooperative emergency responses to infectious diseases. in the future, the chinese military and the civilian sector will further strengthen the cooperative joint command system and emergency rescue force and will reinforce their collaborative information-sharing platform and technical equipment system to further improve military-civilian collaborative emergency infectious diseases disposal, advance the level of infectious disease prevention and control, and maintain public health. in recent years, the prevalence of severe infectious disease has increased, and the number of emerging infectious diseases continues to increase [ ] . the chinese government and military forces have been highly focused on infectious disease prevention and control, and through militarycivilian cooperation, they have successfully prevented numerous severe epidemic situations, such as sudden acute respiratory syndrome (sars), influenza a (h n ), avian influenza h n and h n , and ebola hemorrhagic fever, while actively maintaining public health, economic development, and national construction [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this article introduces military-civilian joint prevention and control as well as prevention and control experiences to provide a basis for reference for future infectious disease prevention and control. the military-civilian collaborative emergency response to infectious diseases refers to the integration of military and government strengths and resources during a certain time period and range, according to the principles of unified command, resource sharing, and close cooperation, in order to solve and jointly respond to epidemic outbreaks of infectious diseases, minimize harm to human health, and maintain social stability [ ] . in , the chinese government disseminated "the law of the prc on the prevention and treatment of infectious diseases," which clearly defined many aspects of infectious diseases, such as epidemic reports, epidemic situation control, medical rescue, supervision and management, guarantee measures, and legal liability, among others; this law provides a legal basis for effectively preventing and eliminating infectious disease occurrence and prevalence and guarantees the health and hygienic safety of the public. the general principle of this law clearly stipulates that military prevention and control of infectious diseases should be consistent with the law and relevant national provisions [ ] . moreover, "regulations on preparedness for and response to emergent public health hazards" was also formulated, which clearly stipulates that the involvement of the people's liberation army's (pla's) medical and health institutions should be consistent with these regulations and relevant military provisions during emergency treatment and health incidents [ ] . the military formulated "the regulations of the pla's prevention and control of infectious diseases" in and the "emergency plan for military disposal of emergency public health events" in , as well as the "military-civilian cooperation mechanism of prevention and control for infectious diseases" in and the "military-civilian cooperation mechanism of emergency disposal for emergency public health events" in . all of these laws and regulations have laid a solid institutional foundation for the militarycivilian collaborative emergency response to infectious diseases [ ] . the operational mechanism of the military-civilian collaborative emergency response under the unified leadership and overall deployment of the nation and the military, the collaborative emergency response mechanism of military-civilian integration and efficient linkage has been established, and it develops a pattern of complementary advantages and efficient collaboration as well as rapidly improves national and military infectious disease prevention and control [ ] [ ] [ ] . for example, five cases of zika virus infection imported from venezuela emerged in guangdong province in february of . the academy of military medical sciences immediately initiated the emergency joint mechanism and acquired samples from guangzhou no. people's hospital. the two zika virus strains were isolated from blood and urine samples acquired from the two patients. the urine test was the first successful isolation of zika from a sample. the gene sequence of the first imported zika virus was also determined through the military-civilian collaboration. the nation has established a joint working mechanism among different departments, and this mechanism indicates that each department is responsibility for itself, coordinates with the other departments, and jointly implements the prevention measures under central unified leadership. the military joint prevention and control came into effect by the unified leading, joint supervision, persevering management, and workforce formation of medical treatment, health, publicity, and rear service among multiple departments. the military and the civilian sector cooperated often and closely, establishing the joint working mechanism; for example, military experts were appointed to participate in leading expert groups for joint prevention and controlled response to emergency events and in the formulation of relevant policies, regulations, and professional documents. meanwhile, the military's working progress was reflected over time, and the military-civilian coordination and communication mechanism was constructed. nationwide epidemic data-sharing mechanism an epidemic information report and sharing mechanism has been established. multiple tasks, such as information report development, joint notification and report release, and joint research on case diagnoses and epidemic announcements, are undertaken daily. the epidemic data from local disease control and prevention institutions are shared nationwide in a timely manner using similar procedures and the epidemic report channel, which establishes the collaborative surveillance and forewarning mechanism that discovers early epidemic outbreaks and provides strong technical support so that prompt and effective military-civilian control measures can be taken. once an epidemic situation has occurred, the military immediately initiates the emergency research mechanism, sets up rapid research channels, and deploys emergency research tasks to scientific research institutions. in the meantime, the national and military disease control and prevention systems immediately integrate scientific research, organize scientific problem-solving measures, collaboratively establish research and development engineering projects, and jointly develop detection reagents and drug-resistant vaccines in response to outbreaks and the prevalence of infectious diseases. these actions can prevent and control epidemic situations. according to the national statement on epidemic information and the national strategy for prevention and control, epidemic prevention and control are jointly deployed at all military-civilian levels, the infection source and close contacts are traced over time, medical quarantine is carefully carried out, and personnel screening and medical observation are put into practice. military experts and military disease control and prevention institutions at all levels, who are obligated to execute these plans due to national requirements, should actively participate, following the laboratory test index, outbreak investigation program, and clinical treatment standards. in addition, the military is also required to participate in the emergent disposal of local epidemic outbreaks; successful past experiences that resulted in preferable public effects can be attributed to the army. the government and the military have always attached great importance to infectious disease prevention and control. after the sars epidemic, the country rapidly intensified construction of its public health system, effectively controlled the spread of several severe epidemic situations, and showed the strong capability of the military-civilian collaborative emergency response, relying heavily on the national military system. this was especially evident when the ebola epidemic outbreaks occurred in west africa in ; china was the first to lend a helping hand, sending approximately , military-civilian medical and health personnel to carry out laboratory tests, make diagnoses, provide treatment and public health training, and build a biological safety laboratory for the affected countries. this is the largest healthcare foreign-aid action of a military-civilian collaborative emergency response since the founding of new china [ ] . the main successful experiences and practices are as follows [ ] [ ] [ ] [ ] . the primary principles of the military-civilian collaborative emergency response are unified command, graded responsibilities, and resident management. by establishing a unified national and military command organization, the comprehensive coordinative command of the military, police, and people was implemented; scientific and effective emergency decisions were made; institutional and functional orientations at all levels were defined; emergency power from all fields was motivated and coordinated for joint participation; unblocked government decree was ensured; and an advantageous configuration of resource integration was achieved. to effectively address severe epidemics, the government and military formulated several laws, regulations, policies, strategic development plans, joint action programs, emergency safety measures, and various types of operable emergency predetermined plans; clearly defined the division of duties and transactions; established an assessment mechanism for military-civilian joint epidemic situations and a docking mechanism for military-civilian relevant departments; and jointly improved the ability to respond to infectious disease emergencies. by using network and communication technologies, an information-sharing mechanism was established and characterized by directional coverage and frequent communication, and a military-civilian collaborative management platform for emergency rescue that can acquire real-time epidemic information, quick responses, and integrated linkage of emergency epidemic situations was built. after the wenchuan earthquake in , an epidemic reporting system was established based on the mobile network, which greatly improved the capability of the military-civilian collaborative emergency response. military emergency prevention and control provides important support for not only the military itself but also national missions, such as emergency disposal during public health emergency events, major activity security, and major domestic and overseas disaster rescue. since , the military emergency medical rescue team, the emergency epidemic prevention team, the anti-nuclear, chemical, and biological (anti-nbc) medical field rescue team; and the veterinary health, prevention and control team have all been included in the national emergency system, which can greatly enhance the capacities of military-civilian collaborative emergency rescue, prevention, and control. in recent years, the national health department has taken the lead and constructed multi-department cooperation, including a health emergency work mode for joint prevention and control that involves the military and coordinates the linkage and efficient decisionmaking across departments and regions [ ] . a series of laws, regulations, and pre-arranged plans were formulated; an effective coordinative command mechanism for military-civilian epidemic situations was perfected; an accurate and quick information reporting system was optimized; and military-civilian collaborative professional emergency disposal teams were cultivated, all of which led to outstanding achievement in preventing and controlling sars, h n , and several other severe infectious diseases [ ] (see also table ). the responses to h n in and to avian influenza h n in highlighted the importance of military-civilian joint prevention and control. test reagents were co-developed in record time; efficient medicine and a specific vaccine for h n were developed and prepared, effectively preventing the prevalence of h n in china. a peramivir and sodium chloride injection was successfully developed and prepared, which played an important role in treating severe h n cases. during the prevention and control of ebola hemorrhagic fever in western africa, military joint national medical rescue contributed significantly to fighting the outbreak in sierra leone and liberia [ , ] . although the chinese military-civilian emergency collaboration has accomplished certain achievements regarding prevention and control of infectious diseases, there are still many problems with the collaborative organization and command, professional power construction, equipment performance level, and emergency theory research, which can be solved by establishing a deep civil-military integration mechanism [ ] . chinese president jin-ping xi, who is also the general secretary of the communist party of china (cpc) central committee and the chairman of the central military commission, has called for further integration of military and civilian undertakings that could cover multiple areas and generate high returns, and he has urged military and local authorities to advance their work while considering the overall development of the country. civilian-military integration has made the synchronized development of economic and national defense capabilities possible. the pla should play an active role in local economic and social development and contribute to wellbeing of the public through actual deeds, including the military-civilian cooperative emergency response to infectious disease prevention and control. this paper focuses on the mechanisms of the militarycooperative emergency response to infectious diseases-the joint working mechanism, the information-sharing mechanism, the research collaboration mechanism, and the joint disposal mechanism-and presents a sorted summary of the practice and experience of the cooperative emergency response to infectious diseases. the chinese military and the civilian sector will further strengthen the military-civilian collaborative joint command system and emergency rescue, reinforce the military-civilian collaborative informationsharing platform and technical equipment system, develop theoretical research on the military-civilian collaborative emergency response, enhance the training and modular exercises of the emergency teams, and strengthen the measures for epidemic information sharing, technical cooperation, disposal linkage, and integral prevention and control, which may further improve military-civilian collaborative emergency disposal, promote infectious disease prevention and control, and maintain public health. emerging and re-emerging infectious threats in the st century surveillance for emerging respiratory viruses coronaviruses: important emerging human pathogens novel reassortant influenza viruses between pandemic (h n ) and other influenza viruses pose a risk to public health populations at risk for severe or complicated avian influenza h n : a systematic review and meta-analysis emergence and development of h n influenza viruses in china providing nursing care to ebola virus disease patients: china ebola treatment unit experience the role of international cooperation in the prevention and control of ebola virus disease epidemic in sierra leone comparison of international ebola virus testing laboratories in ebola virus disease outbreak in sierra leone collaborative emergency management: better community organising, better public preparedness and response disease control and prevention in the military. beijing: people's military medical press army prevention and control of infectious diseases and public health emergency in accordance with the present situation and the countermeasures (author's transl) new progress of military preventive medicine science in china preliminary study on the medical relief mechanism of integrated civil-military resources in non-war states civil-military joint disposal of public health emergencies non war military operations tasks of china's military service historical retrospection of the emergency medical relief of chinese army improve the ability of military hospital emergency medical rescue the think of armed forces deal with major infectious diseases the development of china's emergency medical rescue system present situation and the countermeasure analysis the application and expectation of mobile bsl- laboratory during outbreak of ebola virus disease in serra leone analysis of mechanism for international joint prevention and control of ebola virus disease in sierra leone management mode of military disease control and prevention agencies not applicable. this work was supported by the national social science foundation of china ( - ).availability of data and material not applicable. authors' contributions hm and wp conceived and wrote the manuscript. jpd and nz critically reviewed and revised the manuscript. all authors have read and approved the final manuscript. the authors have no competing interests to declare. submit your next manuscript to biomed central and we will help you at every step: key: cord- -i v u authors: wang, zhen; andrews, michael a.; wu, zhi-xi; wang, lin; bauch, chris t. title: coupled disease–behavior dynamics on complex networks: a review date: - - journal: phys life rev doi: . /j.plrev. . . sha: doc_id: cord_uid: i v u it is increasingly recognized that a key component of successful infection control efforts is understanding the complex, two-way interaction between disease dynamics and human behavioral and social dynamics. human behavior such as contact precautions and social distancing clearly influence disease prevalence, but disease prevalence can in turn alter human behavior, forming a coupled, nonlinear system. moreover, in many cases, the spatial structure of the population cannot be ignored, such that social and behavioral processes and/or transmission of infection must be represented with complex networks. research on studying coupled disease–behavior dynamics in complex networks in particular is growing rapidly, and frequently makes use of analysis methods and concepts from statistical physics. here, we review some of the growing literature in this area. we contrast network-based approaches to homogeneous-mixing approaches, point out how their predictions differ, and describe the rich and often surprising behavior of disease–behavior dynamics on complex networks, and compare them to processes in statistical physics. we discuss how these models can capture the dynamics that characterize many real-world scenarios, thereby suggesting ways that policy makers can better design effective prevention strategies. we also describe the growing sources of digital data that are facilitating research in this area. finally, we suggest pitfalls which might be faced by researchers in the field, and we suggest several ways in which the field could move forward in the coming years. infectious diseases have long caused enormous morbidity and mortality in human populations. one of the most devastating examples is the black death, which killed to million people in the medieval period [ ] . currently, the rapid spread of infectious diseases still imposes a considerable burden [ ] . to elucidate transmission processes of infectious diseases, mathematical modeling has become a fruitful framework [ ] . in the classical modeling framework, a homogeneously mixed population can be classified into several compartments according to disease status. in particular, the most common compartments are those that contain susceptible individuals (s), infectious (or infected) individuals (i), and recovered (and immune) individuals (r). using these states, systems of ordinary differential equations (odes) can be created to capture the evolution of diseases with different natural histories. for example, a disease with no immunity where susceptible individuals who become infected return to the susceptible class after recovering (sis natural history, see fig. where [s] ([i ]) represents the number of susceptible (infectious) individuals in the population, β is the transmission rate of the disease, and μ is the recovery rate of infected individuals. some diseases, however, may give immunity to individuals who have recovered from infection (sir natural history, see fig. where [r] is the number of recovered (and immune) individuals. in these ode models, a general measure of disease severity is the basic reproductive number r = βn/μ, where n is the population size. in simple terms, r is the mean number of secondary infections caused by a single infectious individual, during its entire infectious period, in an otherwise susceptible population [ ] . if r < , the disease will not survive in the population. however, if r > , the disease may be able to persist. typically, parameters like the transmission rate and recovery rate are treated as fixed. however, new approaches to modeling have been developed in past few decades to address some of the limitations of the classic differential equation framework that stem from its simplifying assumptions. for instance, the impact of behavioral changes in response to an epidemic is usually ignored in these formulations (e.g., the transmission rate is fixed), but in reality, individuals usually change their behavior during an outbreak according to the change of perceived infection risk, and their behavioral decisions can in turn impact the transmission of infection. another limitation of the classical compartmental models is the assumption of well-mixed populations (namely, individuals interact with all others at the same contact rate), which thus neglects heterogeneous spatial contact patterns that can arise in realistic populations. in this review we will describe how models of the past few decades have begun to address these limitations of the classic framework. traditionally, infectious disease models have treated human behavior as a fixed phenomenon that does not respond to disease dynamics or any other natural dynamics. for many research questions, this is a useful and acceptable simplification. however, in other cases, human behavior responds to disease dynamics, and in turn disease dynamics responds to human behavior. for example, the initiation of an epidemic may cause a flood of awareness in the population such that protective measures are adopted. this in turn, reduces the transmission of the disease. in such cases, it becomes possible to speak of a single, coupled "disease-behavior" system where a human subsystem and a disease schematic illustration of disease-behavior interactions as a negative feedback loop. in this example, the loop from disease dynamics to behavioral dynamics is positive (+) since an increase in disease prevalence will cause an increase in perceived risk and thus an increase in protective behaviors. the loop from behavioral dynamics back to disease dynamics is negative (−) since an increase in protective behaviors such as contact precautions and social distancing will generally suppress disease prevalence. transmission subsystem are coupled to one another (see fig. ). moreover, because the human and natural subsystems are themselves typically nonlinear, the coupled system is therefore also typically nonlinear. this means that phenomena can emerge that cannot be predicted by considering each subsystem in isolation. for example, protective behavior on the part of humans may ebb and flow according to disease incidence and according to a characteristic timescale (as opposed to being constant over time, as would occur in the uncoupled subsystems). to explore strategic interactions between individual behaviors, game theory has become a key tool across many disciplines. it provides a unified framework for decision-making, where the participating players in a conflict must make strategy choices that potentially affect the interest of other players. game theory and its corresponding equilibrium concepts, such as the nash equilibrium, emerged in seminal works from the s and s [ , ] . a nash equilibrium is a set of strategies such that no player has an incentive to unilaterally deviate from the present strategy. that is, the nash equilibrium makes strategies form best responses to one other, since every player, who has a consistent goal to maximize his own benefit or utility, is perfectly rational. game theory has been applied to fields such as economics, biology, mathematics, public health, ecology, traffic engineering, and computer science [ ] [ ] [ ] [ ] [ ] [ ] . for example, in voluntary vaccination programs, the formal theory of games can be employed as a framework to analyze the vaccination equilibrium level in populations [ , , ] . in the context of vaccination, the feedback between individual decisions of vaccination (or other prevention behaviors) and disease spreading is captured, hence these systems exemplify coupled disease-behavior systems. in spite of the great progress of game theory, the classical paradigm still shows its limitations in many scenarios. it thus becomes instructive to relax some key assumptions, such as the introduction of bounded rationality. game theory has been extended into evolutionary biology, which has generated great insight into the evolution of strategies [ ] [ ] [ ] [ ] [ ] under both biological and cultural evolution. for instance, the replicator equation, which consists of sets of differential equations describing how the strategies of a population evolve over time under selective pressures, has also been used to study learning in various scenarios [ ] . except for temporal concepts, spatial interaction topology has also proved to be crucial in determining system equilibria (also see refs. [ , ] for a comprehensive overview). evolutionary game theory has been extensively applied to behavioral epidemiology, whose details will be surveyed in the following sections. several methods from statistical physics have become useful in the study of disease-behavior interactions on complex networks. most populations are spatially structured in the sense that individuals preferentially interact with those who share close geographic proximity. perhaps, the most simple population structure is a regular lattice: all the agents are assigned specific locations on it, normally a two-dimensional square lattice, just like atoms in crystal lattice sites, which interact with only nearest neighbors. in a regular lattice population, each individual meets the same people they interact with regularly, rather than being randomly reshuffled into a homogeneous mixture, as in well-mixed population models. in addition, another type of homogeneous network attracting great research interest is the erdös-rényi (er) graph [ ] , which is a graph where nodes are linked up randomly and which is often used in the rigorous analysis of graphs and networks. however, in reality, there is ubiquitous heterogeneity in the number of contacts per individual, and recent studies have shown that the distribution of contact numbers of some social networks is not homogeneous but appears to follow a power-law [ ] . moreover, social contact networks also display small-world properties (i.e., short average path length between any two individuals and strong local clustering tendency), which cannot be well described by regular lattices or random graphs [ ] . with both motivations, two significant milestones were born in the late s: the theoretical models of small-world (sw) networks and scale-free (sf) networks [ , ] . subsequently, more properties of social networks have been extensively investigated, such as community structure (a kind of assortative structure where individuals are divided into groups such that the members within each group are mostly connected with each other) [ ] , clusters [ ] , and the recent proposal of multilayer as well as time-varying frameworks [ ] [ ] [ ] [ ] [ ] . due to the broad applicability of complex networks, network models have been widely employed in epidemiology to study the spread of infectious diseases [ ] . in networks, a vertex represents an individual and an edge between two vertices represents a contact over which disease transmission may occur. an epidemic spreads through the network from infected to susceptible vertices. with the advent of various network algorithms, it becomes instructive incorporating disease dynamics into such infrastructures to explore the impact of spatial contact patterns [ ] [ ] [ ] [ ] [ ] [ ] . replacing the homogeneous mixing hypothesis that any individual can come into contact with any other agents, networked epidemic research assumes that each individual has comparable number of contacts, denoted by its degree k. under this treatment, the most significant physics finding is that network topology will directly determine the threshold of epidemic outbreak and phase transition. for example, compared with the finite epidemic threshold of random network, romualdo et al. found that disease with sis dynamics and even a very small transmission rate can spread and persist in the sf networks (i.e., there is absence of a disease threshold) [ ] . this point helps to explain why it is extremely difficult to eradicate viruses on internet and world wide web, and why those viruses have an unusual long lifetime. but the absence of epidemic threshold is only suitable for sf networks with a power-law degree distribution p (k) ∼ k −γ with γ ∈ ( , ]. if γ is extended to the range ( , ) , an anomalous critical behavior takes place [ , ] . to show the condition of disease spread, it is meaningful to define the relative spreading rate λ ≡ β/μ. the larger is λ, the more likely the disease will spread. generally, for an sf network with arbitrary degree distribution, the epidemic threshold is in particular, for an sf network k diverges in the n → ∞ limit, and so the epidemic threshold is expected to die out. similarly, it is easy to derive the threshold of sir model which is related with average degree k and the second moment k of networks as well. along these findings, more endeavors are devoted to the epidemic threshold of spatial networks with various properties, such as degree correlation [ , ] , sw topology [ ] , community structure [ ] , and k-core [ ] . on the other hand, more analysis and prediction methods (such as mean-field method, generation function) are also proposed to explain the transition of disease on realistic networks [ , ] and immunization strategies of spatial networks are largely identified [ ] . to illustrate the meaning of studying disease-behavior dynamics on complex networks, it is instructive to firstly describe a simple example of such a system. consider a population of individuals who are aware of a spreading epidemic. the information each individual receives regarding the disease status of others is derived from the underlying social network of the population. these networks have been shown to display heterogeneous contact patterns, where the node degree distribution often follows a power-law fashion [ , ] . it is possible to use these complex network patterns to model a realistic population that exhibits adaptive self-protective behavior in the presence of a disease. a common way to incorporate this self-protective behavior is to allow individuals to lower their susceptibility according to the proportion of their contacts that are infectious, as demonstrated by bagnoli et al. [ ] . in this model, the authors reduce the susceptibility of an individual to a disease which has a simple sis natural history by multiplying the transmission rate by a negative exponential function of the proportion of their neighbors who are infectious. specifically, this is given by βi (ψ, k) where β is the per contact transmission probability and models the effect an individual's risk perception has on its susceptibility, where j and τ are constants that govern the level of precaution individuals take, ψ is the number of infectious contacts an individual has, and k is the total number of contacts an individual has. the authors show that the introduction of adaptive behavior has the potential to not only reduce the probabilities of new infections occurring in highly disease-concentrated areas, but can also cause epidemics to go extinct. specifically, when τ = , there is a value of j for which an epidemic can be stopped in regular lattices and sw networks [ ] . however, for certain sf networks, there is no value of j that is able to stop the disease from spreading. in order to achieve disease extinction in these networks, hub nodes must adopt additional self-protective measure, which is accomplished by decreasing τ for these individuals. the conclusions derived from this model highlight the significant impact different types of complex networks can have on health outcomes in a population, and how behavioral changes can dictate the course of an epidemic. the remainder of this review is organized as follows. in section , we will focus on the disease-behavior dynamics of homogeneously mixed populations, and discuss when the homogeneous mixing approximation is or is not valid. this provides a comprehensive prologue to the overview of the coupled systems on networks in section . within the latter, we separately review dynamics in different types of networked populations, which are frequently viewed through the lens of physical phenomena (such as phase transitions and pattern formation) and analyzed with physicsbased methods (like monte carlo simulation, mean-field prediction). based on all these achievements, we can capture how coupled disease-behavior dynamics affects disease transmission and spatial contact patterns. section will be devoted to empirical concerns, such as types of data that can be used for these study systems, and how questionnaires and digital equipment can be used to collect data on relevant social and contact networks. in addition, it is meaningful to examine whether some social behaviors predicted by models really exist in vaccination experiments and surveys. finally, we will conclude with a summary and an outlook in section , describing the implications of statistical physics of spatial disease-behavior dynamics and outlining viable directions for future research. throughout, we will generally focus on preventive measures other than vaccination (such as social distancing and hand washing), although we will also touch upon vaccination in a few places. a large body of literature addresses disease-behavior dynamics in populations that are assumed to be mixing homogeneously, and thus spatial structure can be neglected. incorporating adaptive behavior into a model of disease spread can provide important insight into population health outcomes, as the activation of social distancing and other nonpharmaceutical interventions (npis) have been observed to have the ability to alter the course of an epidemic [ ] [ ] [ ] . table disease-behavior models applied to well-mixed populations, classified by infection type and whether economic-based or rule-based. when making decisions regarding self-protection from an infection, individuals must gather information relevant to the disease status of others in the population. prophylactic behavior can be driven by disease prevalence, imitation of others around them, or personal beliefs of probable health outcomes. in this section, we will survey the features and results of mathematical models that incorporate prophylactic decision making behavior in homogeneously mixed populations. the approaches we consider can be classified into two separate categories: economic-based and rulebased. economic based models (such as game theoretical models) assume individuals seek a maximization of their social utility, whereas rule-based models prescribe prevalence-based rules (not explicitly based on utility) according to which individuals and populations behave. both of these methods can also be used to study the dynamics of similar diseases (see table ), and are discussed in detail below. the discovery of human immunodeficiency virus (hiv)/acquired immune deficiency syndrome (aids) and its large economic impacts stimulated research into behaviorally based mathematical models of sexually transmitted diseases (stds). in disease-behavior models, a population often initiates a behavior change in response to an increasing prevalence of a disease. in the context of stds, this change in behavior may include safer sex practices, or a reduction in the number of partnerships individuals seek out. following this prevalence-based decision making principle, researchers have used the concept of utility maximization to study the behavior dynamics of a population [ ] [ ] [ ] [ ] [ ] . in these models, individuals seek to maximize their utility by solving dynamic optimization problems. utility is derived by members of the population when engaging in increased levels of social contact. however, this increased contact or partner change rate also increases the chance of becoming infected. one consequence of this dynamic is that higher levels of prevalence can result in increased prophylactic behavior, which in turn decreases the prevalence over time. as this occurs, self-protective measures used by the population will also fall, which may cause disease cycles [ , ] . nonetheless, in the case of stds which share similar transmission pathways, a population protecting themselves from one disease by reducing contact rates can also indirectly protect themselves from another disease simultaneously [ ] . in general, the lowering of contact rates in response to an epidemic can reduce its size, and also delay new infections [ ] . however, this observed reduction of contact rates may not be uniform across the whole population. for example, an increase in prevalence may cause the activity rates of those with already low social interaction to fall even further, but this effect may not hold true for those with high activity rates [ ] . in fact, the high-risk members of the population will gain a larger fraction of high-risk partners in this scenario, resulting from the low-risk members reducing their social interaction rates. this dynamic serves to increase the risk of infection of high activity individuals even further. these utility-based economic models show us that when considering health outcomes, one must be acutely aware of the welfare costs associated with self-protective behavior or implementing disease mitigation policies [ ] . a health policy, such as encouraging infectious individuals to self-quarantine, may actually cause a rise in disease prevalence due to susceptible individuals feeling less threatened by infection and subsequently abandoning their own self-protective behavior [ ] . also, a population who is given a pessimistic outlook of an epidemic may in fact cause the disease to spread more rapidly [ ] . recently, approaches using game theory have been applied to self-protective behavior and social distancing [ ] [ ] [ ] . when an individual's risk of becoming infected only depends on their personal investment into social distancing, prophylactic behavior is not initiated until after an epidemic begins, and ceases before an epidemic ends. also, the basic reproductive number of a disease must exceed a certain threshold for individuals to feel self-protective behavior is worth the effort [ ] . in scenarios where the contact rate of the population increases with the number of people out in public, a nash equilibrium exists, but the level of self-protective behavior in it is not socially optimal [ ] . nonetheless, these models also show that the activation of social distancing can weaken an epidemic. some models of disease-behavior dynamics, rather than assuming humans are attempting to optimize a utility function, represent human behavior by specifying rules that humans follow under certain conditions. these could include both phenomenological rules describing phenomenological responses to changes in prevalence, or more complex psychological mechanisms. rule-based compartmental models using systems of differential equations have also used to study heterogeneous behavior and the use of npis by a population during an epidemic. a wide range of diseases are modeled using this approach, such as hiv [ ] [ ] [ ] , severe acute respiratory syndrome (sars) [ , ] , or influenza [ , ] . these models often utilize additional compartments, which are populated according to specific rules. examples of such rules are to construct the compartments to hold a constant amount of individuals associated with certain contact rates [ , , ] , or to add and remove individuals at a constant rate [ , , , ] , a rate depending on prevalence [ ] [ ] [ ] [ ] [ ] [ ] , or according to a framework where behavior that is more successful is imitated by others [ , , ] . extra compartments signify behavioral heterogeneities amongst members of a population, and the disease transmission rates associated with them also vary. reduction in transmission due to adaptive behavior is either modeled as a quarantine of cases [ , , ] , or prophylactic behavior of susceptible individuals due to increased awareness of the disease [ , , [ ] [ ] [ ] [ ] [ ] [ ] ] . these models agree that early activation of isolation measures and selfprotective behavior can weaken an epidemic. however, due to an early decrease in new infections, populations may see a subsequent decrease in npi use causing multiple waves of infection [ , , , ] . contrasting opinions on the impact behavioral changes have on the epidemic threshold also result from these models. for example, perra et al. [ ] show that although infection size is reduced, prophylactic behavior does not alter the epidemic threshold. however, the models studied by poletti et al. [ ] and sahneh et al. [ ] show that the epidemic threshold can be altered by behavioral changes in a population. the classes of models presented in this section use homogeneous mixing patterns (i.e., well-mixed populations) to study the effects of adaptive behavior in response to epidemics and disease spread (see table for a summary). often, populations will be modeled to alter their behavior based on reactions to changes in disease prevalence, or by optimizing their choices with respect to personal health outcomes. if possible, early activation of prophylactic behavior and npis by a population will be the most effective course of action to curb an epidemic. homogeneous mixing can be an appropriate approximation for the spread of an epidemic when the disease to be modeled is easily transmitted, such as measles and other infection that can be spread by fine aerosol particles that remain suspended for a long period. however, this mixing assumption does not always reflect real disease dynamics. for example, human sexual contact patterns are believed to be heterogeneous [ ] and can be represented as networks (or graphs), while other infections, such as sars, can only be spread by large droplets, making the homogeneous mixing assumption less valid. the literature surrounding epidemic models that address this limitation by incorporating heterogeneous contact patterns through networks is very rich, and is discussed in the following section. in section , we reviewed disease-behavior dynamics in well-mixed populations. however, in real populations, various types of complex networks are ubiquitous and their dynamics have been well studied. the transmission of many infectious diseases requires direct or close contact between individuals, suggesting that complex networks play a vital role in diffusion of disease. it thus becomes of particular significance to review the development of behavioral epidemiology in networked populations. many of the dynamics exhibited by such systems have direct analogues to processes in statistical physics, such as how disease or behavior percolate through the network, or how a population can undergo a phase transition from one social state to another social state. perhaps the easiest way to begin studying disease-behavior dynamics in spatially distributed populations is by using lattices and static networks, which are relatively easy to analyze and which have attracted much attention in theoretical and empirical research. we organize research by several themes under which they have been conducted, such as the role of spreading awareness, social distancing as protection, and the role of imitation, although we emphasize that the distinctions are not always "hard and fast". the role of individual awareness. the awareness of disease outbreaks may stimulate humans to change their behavior, such as washing hands and wearing masks. such behavioral responses can reduce susceptibility to infection, which itself in turn can influence the epidemic course. in the seminal work, funk and coworkers [ ] formulated and analyzed a mathematical model for the spread of awareness in well-mixed and spatially structured populations to understand how the awareness of disease and also its propagation impact the spatial spread of a disease. in their model, both disease and the information about the disease spread spontaneously by, respectively, contact and word of mouth in the population. the classical epidemiological sir model is used for epidemic spreading, and the information dynamics is governed by both information transmission and information fading. the immediate outcome of the awareness of the disease information is the decrease in the possibility of acquiring the infectious disease when a susceptible individual (who was aware of the epidemic) contacts with an infected one. in a well-mixed population, the authors found that, the coupling spreading dynamics of both the epidemic and the awareness of it can result in a lower size of the outbreak, yet it does not affect the epidemic threshold. however, in a population located on the triangular lattice, the behavioral response can completely stop a disease from spreading, provided the infection rate is below a threshold. specifically, the authors showed that the impact of locally spreading awareness is amplified if the social network of potential infection events and the communication network over which individuals communicate overlap, especially so if the networks have a high level of clustering. the finding that spatial structure can prevent an epidemic is echoed in an earlier model where the effects of awareness are limited to the immediate neighbors of infected nodes on a network [ ] . in the model, individuals choose whether to accept ring vaccination depending on perceived disease risk due to infected neighbors. by exploring a range of network structures from the limit of homogeneous mixing to the limit of a static, random network with small neighborhood size, the authors show that it is easier to eradicate infections in spatially structured populations than in homogeneously mixing populations [ ] . hence, free-riding on vaccine-generated herd immunity may be less of a problem for infectious diseases spreading spatially structured populations, such as would more closely describe the situation for close contact infections. along similar lines of research, wu et al. explored the impact of three forms of awareness on the epidemic spreading in a finite sf networked population [ ] : contact awareness that increases with individual contact number; local awareness that increases with the fraction of infected contacts; and global awareness that increases with the overall disease prevalence. they found that the global awareness cannot decrease the likelihood of an epidemic outbreak while both the local awareness and the contact awareness do it. generally, individual awareness of an epidemic contributes toward the inhibition of its transmission. the universality of such conclusions (i.e., individual behavioral responses suppress epidemic spreading) is also supported by a recent model [ ] , in which the authors focused on an epidemic response model where the individuals respond to the epidemic according to, rather than the density of infected nodes, the number of infected neighbors in the local neighborhood. mathematically, the local behavioral response is cast into the reduction factor ( − θ) ψ in the contact rate of a susceptible node, where ψ is the number of infected neighbors and θ < is a parameter characterizing the response strength of the individuals to the epidemic. by studying both sis and sir epidemiological models with the behavioral response rule in sf networks, they found that individual behavioral response can in general suppress epidemic spreading, due to crucial role played by the hub nodes who are more likely to adopt protective response to block the disease spreading path. in a somewhat different framework, how the diffusion of individual's crisis awareness affects the epidemic spreading is investigated in ref. [ ] . in this work, the epidemiological sir model is linked with an information transmission process, whose diffusion dynamics is characterized by two parameters, say, the information creation rate ζ and the information sensitivity η. in particular, at each time step, ζ n packets will be generated and transferred in the network according to the shortest-path routing algorithm (n hither denotes the size of networks). when a packet is routed by an infected individual, its state is marked by infection. each individual determines whether or not to accept vaccine based on how many infected packets are received from immediate neighbors, and on how sensitive the individual response is to the information as well, weighed by the parameter η. the authors considered their "sir with information-driven vaccination" model on homogeneous er networks and heterogeneous sf networks, and found that the epidemic spreading can be significantly suppressed in both the homogeneous and heterogeneous networks provided that both ζ and η are relatively large. social distancing as a protection mechanism. infectious disease outbreaks may trigger various behavioral responses of individuals to take preventive measures, one of which is social distancing. valdez and coworkers have investigated the efficiency of social distancing in altering the epidemic dynamics and affecting the disease transmission process on er network, sf networks, as well as realistic social networks [ ] . in their model, rather than the normally used link-rewiring process, an intermittent social distancing strategy is adopted to disturb the epidemic spreading process. particularly, based on local information, a susceptible individual is allowed to interrupt the contact with an infected individual with a probability σ and restore it after a fixed time t b , such that the underlying interaction network of the individuals remains unchanged. using the framework of percolation theory, the authors found that there exists a cutoff threshold σ c , whose value depends on the network topology (i.e., the extent of heterogeneity of the degree distribution), beyond which the epidemic phase disappears. the efficiency of the intermittent social distancing strategy in stopping the spread of diseases is owing to the emergent "susceptible herd behavior" among the population that protects a large fraction of susceptible individuals. impact of behavior imitation on vaccination coverage. vaccination is widely employed as an infection control measure. to explore the role of individual imitation behavior and population structure in vaccination, recent seminal work integrated an epidemiological process into a simple agent-based model of adaptive learning, where individuals use anecdotal evidence to estimate costs and benefits of vaccination [ ] . under such a model, the disease-behavior dynamics is modeled as a two-stage process. the first stage is a public vaccination campaign, which occurs before any epidemic spreading. at this stage, each individual decides whether or not to vaccinate, and taking vaccine incurs a cost c v to the vaccinated individuals. the vaccine is risk-free and offers perfect protection against infection. the second stage is the disease transmission process, where the classic sir compartmental model is adopted. during the whole epidemic spreading process, those susceptible individuals who caught the disease incur an infection cost c i , which is usually assumed to be larger than the cost c v for vaccination. those unvaccinated individuals who remain healthy are free-riding off the vaccination efforts of others (i.e., no any cost), and they are indirectly protected by herd immunity. for simplicity, the authors rescale these costs by defining the relative cost of vaccination c = c v /c i ( < c < ) and c i = . as such, after each epidemic season, all the individuals will get some payoffs (equal to the negative value of corresponding costs) dependent on their vaccination strategies and also on whether they are infected or not, then they are allowed to change or keep their old strategies for the next season, depending on their current payoffs. the rule of thumb is that the strategy of a role model with higher payoff is more likely to be imitated. by doing so, each individual i randomly chooses another individual j from the neighborhood as role model, and imitates the behavior of j with the probability where p i and p j are, respectively, the payoffs of two involved individuals, and β ( < β < ∞) denotes the strength of selection. this form of decision alternative is also known as the fermi law [ , ] in physics. a finite value of β accounts for the fact that better performing individuals are readily imitated, although it is not impossible to imitate one agent performing worse, for example due to imperfect information or errors in decision making. the authors studied their coupled "disease-behavior" model in well-mixed populations, in square lattice populations, in random network populations, and in sf network populations, and found that population structure acts as a "double-edged sword" for public health: it can promote high levels of voluntary vaccination and herd immunity given that the cost for vaccination is not too large, but small increases in the cost beyond a certain threshold would cause vaccination to plummet, and infections to rise, more dramatically than in well-mixed populations. this research provides an example of how spatial structure does not always improve the chances of infection control, in disease-behavior systems. the symbols and lines correspond, respectively, to the simulation results and mean-field predictions (whose analytical framework is shown in appendix a). the parameter α determines just how seriously the peer pressure is considered in the decision making process of the individuals to taking vaccine. the figure is reproduced from [ ] . in the similar vein, peer pressure among the populations is considered to clarify its impact on the decision-making process of vaccination, and then on the disease spreading [ ] . in reality, whether or not to change behavior depends not only on the personal success of each individual, but also on the success and/or behavior of others. using this as motivation, the authors incorporated the impact of peer pressure into a susceptible-vaccinated-infected-recovered (svir) epidemiological model, where the propensity to adopt a particular vaccination strategy depends both on individual success as well as on the strategy-configuration of their neighbors. to be specific, the behavior imitation probability of individual i towards its immediate neighbor j (namely, eq. ( )) becomes where n i is the number of neighbors that have a different vaccination strategy than the individual i, and k i is the interaction degree of i, and the parameter α determines just how seriously the peer pressure is considered. under such a scenario, fig. displays how vaccination and infection vary as a function of vaccine cost in er random graph. it is clear that plugging into the peer pressure also works as a "double-edged sword", which, on the one hand, strongly promotes vaccine uptake in the population when its cost is below a critical value, but, on the other hand, it may also strongly impede it if the critical value is exceeded. the reason is due to the fact that the presence of peer-pressure can facilitate cluster formation among the individuals, whose behaviors are inclined to conform to the majority of their neighbors, similar to the early report of cooperation behavior [ ] . such behavioral conformity is found to expedite the spread of disease when the relative cost for vaccination is high enough, while promote the vaccine coverage in the opposite case. self-motivated strategies related with vaccination. generally, it is not so much the actual risk of being infected, as the perceived risk of infection, that will prompt humans to change their vaccination behavior. previous game-theoretic studies of vaccination behavior typically have often assumed that individuals react to the disease incidence with same responsive dynamics, i.e., the same formulas of calculating the perceived probability of infection. but that may not actually be the case. liu et al. proposed that a few will be "committed" to vaccination, perhaps because they have a low threshold for feeling at risk (or strongly held convictions), and they will want to be immunized as soon as they hear that someone is infected [ ] . they studied how the presence of committed vaccinators, a small fraction of individuals who consistently hold the vaccinating strategy and are immune to influence, impacts the vaccination dynamics in well-mixed and spatially structured populations. the researchers showed that even a relatively small proportion of these agents (such as %) can significantly reduce the scale of an epidemic, as shown in fig. . the effect is much stronger when all the individuals are uniformly distributed on a square lattice, as compared to the case of well-mixed population. their results suggested that those committed individuals can have a remarkable effect, acting as "steadfast role models" in the population to seed vaccine uptake in others while also disrupting the appearance of clusters of free-riders, which might otherwise seed the emergence of a global epidemic. one important message taken away from ref. [ ] is that we might never guess what would happen by just looking at the decision-making rules alone, in particular when our choices will influence, and be influenced by, the choice of other people. another good example can be found in a recent work [ ] , in which zhang et al. proposed an evolutionary epidemic game where individuals can choose their strategies as vaccination, self-protection or laissez faire, towards infectious diseases and adjust their strategies according to their neighbors' strategies and payoffs. the "disease-behavior" coupling dynamical process is similar to the one implemented by ref. [ ] , where the sir epidemic spreading process and the strategy updating process succeed alternatively. by both stochastic simulations and theoretical analysis, the authors found a counter-intuitive phenomenon that a better condition (i.e., larger successful rate of self-protection) may unfortunately result in less system payoff. the trick is that, when the successful rate of self-protection increases, people become more speculative and less interested in vaccination. since a vaccinated individual brings the "externality" effect to the system: the individual's decision to vaccinate diminishes not only its own risk of infection, but also the risk for those people with whom the individual interacts, the reduction of vaccination can remarkably enhance the risk of infection. the observed counter-intuitive phenomenon is reminiscent of the well-known braess's paradox in traffic, where more roads may lead to more severe traffic congestion [ ] . this work provides another interesting example analogous to braess's paradox, namely, a higher successful rate of self-protection may eventually enlarge the epidemic size and thus diminish positive health outcomes. this work raises a challenge to public health agencies regarding how to protect the population during an epidemic. the government should carefully consider how to distribute their resources and money between messages supporting vaccination, hospitalization, self-protection, and so on, since the outcome of policy largely depends on the complex interplay among the type of incentive, individual behavioral responses, and the intrinsic epidemic dynamics. in their further work [ ] , the authors investigated the effects of two types of incentives strategies, partial-subsidy policy in which certain fraction of the cost of vaccination is offset, and free-subsidy policy in which donees are randomly selected and vaccinated at no cost on the epidemic control. through mean-field analysis and computations, they found that, under the partial-subsidy policy, the vaccination coverage depends monotonically on the sensitivity of individuals to payoff difference, but the dependence is non-monotonous for the free-subsidy policy. due to the role models of the donees for relatively irrational individuals and the unchanged strategies of the donees for rational individuals, the free-subsidy policy can in general lead to higher vaccination coverage. these findings substantiate, once again, that any disease-control policy should be exercised with extreme care: its success depends on the complex interplay among the intrinsic mathematical rules of epidemic spreading, governmental policies, and behavioral responses of individuals. as the above subsection shows, research on disease-behavior dynamics on networks has become one of the most fruitful realms of statistical physics and non-linear science, as well as shedding novel light on how to predict the impact of individual behavior on disease spread and prevention [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] ] . however, in some scenarios, the simple hypothesis that individuals are connected to each other in the same infrastructure (namely, the so-called single-layer network in section . ) may generate overestimation or underestimation for the diffusion and prevention of disease, since agents can simultaneously be the elements of more than one network in most, yet not all, empirical systems [ , , ] . in this sense, it seems constructive to go beyond the traditional single-layer network theory and propose a new architecture, which can incorporate the multiple roles or connections of individuals into an integrated framework. the multilayer networks, defined as the combination class of networks interrelated in a nontrivial way (usually by sharing nodes), have recently become a fundamental tool to quantitatively describe the interaction among network layers as well as between these constituents. an example of multilayer networks is visualized in fig. [ ] . a social network layer supports the social dynamics related to individual behavior and main prevention strategies (like vaccination); while the biological layer provides a platform for the spreading of biological disease. each individual is a node in both network layers. the coupled structure can generate more diverse outcomes than either isolated network, and could produce multiple (positive or negative) effects on the eradication of infection. because of the connection between layers, the dynamics of control measures in turn affects the trajectory of disease on biological network, and vice versa. under such a framework, which is composed of at least different topology networks, nodes not only exchange information with their counterparts in other network(s) via inter-layer connections, but also diffuse infection with their neighbors through the intra-layer connections. subsequently, more theoretical algorithms and models, such as interdependent networks, multiplex networks and interconnected networks, have been proposed [ ] [ ] [ ] . the broad applicability of multilayer networks and their success in providing insight into the structure and dynamics of realistic systems have thus generated considerable excitement [ ] [ ] [ ] [ ] [ ] . of course, the study of disease-behavior dynamics in this framework is a young and rapidly evolving research area, which will be systematically surveyed in what follows. interplay between awareness and disease. as fig. illustrates, different dynamical processes for the same set of nodes with different connection topologies for each process can be encapsulated in a multilayer structure (technically, these are referred to as multiplex networks [ , ] ). aiming to explore the interrelation between social awareness and disease spreading, granell et al. recently incorporated information awareness into a disease model embedded in a multiplex network [ ] , where the physical contact layer supports epidemic process and the virtual contact layer supports awareness diffusion. similar to sis model (where the s node can be infected with a transmission probability β, and the i node recovers with a certain rate μ), the awareness dynamics, composed of aware (a) and unaware (u) states, assumes that a node of state a may lose its awareness with probability δ, and re-obtains awareness in the probability ν. then, both processes can be coupled via the combinations of individual states: unaware-susceptible fig. . transition probability trees of the combined states for coupled awareness-disease dynamics each time step in the multilayer networks. here aware (a) state can become unaware (u) with transition probability δ and of course re-obtains awareness with other probability. for disease, μ represents the transition probability from infected (i) to susceptible (s). there are thus four state combinations: aware-infected, (ai) aware-susceptible, (as) unaware-infected, (ui) and unaware-susceptible (us), and the transition of these combinations is controlled by probability r i , q a i and q u i . they respectively denote the transition probability from unaware to aware given by neighbors; transition probability from susceptible to infected, if node is aware, given by neighbors; and transition probability from susceptible to infected, if node is unaware, given by neighbors. we refer to [ ] , from where this figure has been adapted, for further details. (us), aware-susceptible (as), and aware-infected (ai), which are also revealed by the transition probability trees in fig. . using monte carlo simulations, the authors showed that the coupled dynamical processes change the onset of the epidemics and allow them to further capture the evolution of the epidemic threshold (depending on the structure and the interrelation with the awareness process), which can be accurately validated by the markov-chain approximation approach. more interestingly, they unveiled that the increase in transmission rate can lower the long-term disease incidence while raising the outbreak threshold of epidemic. in spite of great progress, the above-mentioned findings are based on two hypotheses: infected nodes become immediately aware, and aware individuals are completely immune to the infection. to capture more realistic scenarios, the authors relaxed both assumptions and introduced mass media that disseminates information to the entire system [ ] . they found that the vaccine coverage of aware individuals and the mass media affect the critical relation between two competing processes. more importantly, the existence of mass media makes the metacritical point (where the critical onset of the epidemics starts) of ref. [ ] disappear. furthermore, the social dynamics are further extended to an awareness cascade model [ ] , during which agents exhibit herd-like behavior because they make decisions referring to the actions of other individuals. interestingly, it is found that a local awareness ratio (of unaware individuals becoming aware ones) approximating . has a two-stage effect on the epidemic threshold (i.e., an abrupt transition of epidemic threshold) and can cause different epidemic sizes, irrespective of the network structure. that is to say, when the local awareness ratio is in the range of [ , . ), the epidemic threshold is a fixed and larger value; however, in the range of [ . , ], threshold value becomes a fixed yet smaller value. as for the final epidemic size, its increasing speed for the interval [ , . ) is much slower than the speed when local awareness ratio lies in [ . , ]. these findings suggest a new way of understanding realistic contagions and their prevention. except for obtaining awareness from aware neighbors, self-awareness induced by infected neighbors is another scenario that currently attracts research attention [ ] , where it is found that coupling such a dynamical process with disease spreading can lower the density of infection, but does not increase the epidemic threshold regardless of the information source. coupling between disease and preventive behaviors. thus far, many achievements have shown that considering simultaneous diffusion of disease and prevention measures on the same single-layer network is an effective method to evaluate the incidence and onset of disease [ , , [ ] [ ] [ ] [ ] , ] . however, if both processes are coupled on the multilayer infrastructure, how does it affect the spreading and prevention of disease? inspired by this interesting question, ref. [ ] suggested a conceptual framework, where two fully or partially coupled networks are employed, to transmit disease (an infection layer) and to channel individual decision of prevention behaviors (a communication layer). protection strategies considered include wearing facemasks, washing hands frequently, taking pharmaceutical drugs, and avoiding contact with sick people, which are the only means of control in situations where vaccines are not yet available. it is found that the structure of the infection network, rather than the communication network, has a dramatic influence on the transmission of disease and uptake of protective measures. in particular, during an influenza epidemic, the coupled model can lead to a lower infection rates, which indicates that single-layer models may overestimate disease transmission. in line with this finding, the author further extended the above setup into a triple coupled diffusion model (adding the information flow of disease on a new layer) through metropolitan social networks [ ] . during an epidemic, these three diffusion dynamics interact with each other and form negative and positive feedback loop. compared with the empirical data, it is exhibited that this proposed model reasonably replicates the realistic trends of influenza spread and information propagation. the author pointed out that this model possesses the potential of developing into a virtual platform for health decision makers to test the efficiency of disease control measures in real populations. much previous work shows that behavior and spatial structure can suppress epidemic spreading. in contrast, other recent research using a multiplex network consisting of a disease transmission (dt) network and information propagation (ip) network through which vaccination strategy and individual health condition information can be communicated, finds that compared with the case of traditional single-layer network (namely, symmetric interaction), the multiplex architecture suppresses vaccination coverage and leads to more infection [ ] . this phenomenon is caused by the sharp decline of small-degree vaccination nodes, whose number is usually more numerous in heterogeneous networks. similarly, wang et al. considered asymmetrical interplay between disease spreading and information diffusion in multilayer networks [ ] . it is assumed that there exists different disease dynamics on communication layer and physical-contact layer, only where vaccination takes place. more specifically, the vaccination decision of the node in contact networks is not only related to the states of its intra-layer neighbors, but also depends on the counterpart node from communication layer. by means of numerous simulations and mean-field analysis, they found that, for uncorrelated coupling architecture, a disease outbreak in the contact layer induces an outbreak of disease in the communication layer, and information diffusion can effectively raise the epidemic threshold. however, the consideration of inter-layer correlation dramatically changes the onset of disease, but not the information threshold. dynamical networks play an important role in the incidence and onset of epidemics as well. along this line of research, the most commonly used approach is adaptive networks [ ] [ ] [ ] [ ] , where nodes frequently adjust their connections according to the environment or states of neighboring nodes. time-varying networks (also named temporal networks) provide another framework for the activity-driven changing of connection topology [ , , ] . here, we briefly review the progress of disease-behavior dynamics on adaptive and time-varying networks. contact switching as potential protection strategy. in the adaptive viewpoint, the most straightforward method of avoiding contact with infective acquaintances amounts to breaking the links between susceptible and infective agents and constructing novel connections. along such lines, thilo et al. first proposed an adaptive scenario: a susceptible node is able to prune the infected link and rewire with a healthy agent with a certain probability [ ] . the probability of switching can be regarded as a measurement of strength of the protection strategy. it is shown that different values of this probability give rise to various degree mixing patterns and degree distributions. based on the low-dimensional approximations, the authors also showed that their adaptive framework is able to predict novel dynamical features, such as bistability, hysteresis, and first order transitions, which are sufficiently robust against disease dynamics [ , ] . in spite of great advances, the existing analytical methods cannot generally allow for accurate predictions about the simultaneous time evolution of disease and network topology. to overcome this limitation, vincent et al. further introduced an improved compartmental formalism, which proves that the initial conditions play a crucial role in disease spreading [ ] . in the above examples, switching contact as a strategy has proven its effectiveness in controlling epidemic outbreak. however, in some realistic cases, the population information may be asymmetric, especially during the process of rewiring links. to relax this constraint, a new adaptive algorithm was recently suggested: an infection link can be pruned by either individual, who reconnects to a randomly selected member rather than susceptible agent (namely, the individual has no previous information on state of every other agent) [ , ] . for example, ref. [ ] showed that such a reconnection behavior can completely suppress the spreading of disease via continuous and discontinuous transitions, which is universally effective in more complex situations. besides the phenomena of oscillation and bistability, another dynamical feature, epidemic reemergence, also attracts great interest in a current study [ ] , where susceptible individuals adaptively break connections with infected neighbors yet avoid being isolated in a growing network. under such operations, the authors observed that the number fig. . panel (a) denotes the time course for the number of infected nodes when the network growth, the link-removal process, and isolation avoidance are simultaneously involved into the adaptive framework. it is clear that this mechanism creates the reemergence of epidemic, which dies out after several such repetitions. while for this interesting phenomenon, it is closely related with the formation of giant component of susceptible nodes. panel (b) shows the snapshot of the network topology of th time step (before the next abrupt outbreak), when there is a giant component of susceptible nodes (yellow). however, the invasion of the infection individuals (red) makes the whole network split into many fragments, as shown by the snapshot of th time step (after the explosion) in panel (c). we refer to [ ] , from where this figure has been adapted, for further details. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) of infected agents stays at a low level for a long time, and then suddenly erupts to high level before declining to a low level again. this process repeats several times until the final eradication of infection, as illustrated in fig. (a) . with regard to potential mechanism, it is actually related with the invasion of infected individuals to susceptible giant components. link-removal process can suppress disease spreading, which makes susceptible (infected) agents form giant components (small yet non-isolated clusters), as shown in fig. (b) . but, the entrance of new nodes may bring new infection risk to such giant components, which accelerates next outbreak of infection and network crashing again (see fig. (c) ). interestingly, this finding may help to explain the phenomenon of repeated epidemic explosions in real populations. now, if we carefully look back upon the above-cited bibliography, we will find a common feature: except for disease processes, the adaptive adjustment of individual connections finally changes the degree distribution of networks. an interesting question naturally poses itself: is there an adaptive scenario that preserves the degree distribution of networks? that is, each individual has a characteristic behavior: keeping total number of its neighbors constant. to fill up this gap, neighbor exchange model becomes a very useful tool [ ] , where individual number of current neighbors remains fixed while the compositions or entities of those contacts change in time. similar to famous algorithm of watts-strogatz sw network [ ] , such a model allows an exchange mechanism in which the destination nodes of two edges are swapped with a given rate. incorporating the diffusion of epidemic, this model constructs a bridge between static network model and mass-action model. based on the empirical data, the authors further displayed that the application of this model is very effective to forecast and control sexually transmitted disease outbreak. along this way, the potential influence of other topology properties (such as growing networks [ ] and rewiring sf networks [ ] ) has recently been identified in the adaptive viewpoint, which dramatically changes the outbreak threshold of disease. vaccination, immunization and quarantine as avoidance behaviors. as in static networks, vaccination can also be introduced into adaptive architectures, where connection adjustment is an individual response to the presence of infection risk among neighborhoods. motivated by realistic immunization situations, disease prevention is implemented by adding poisson-distributed vaccination to susceptible individuals [ ] . because of the interplay between network rewiring and vaccination application, the authors showed that vaccination is far more effective in an adaptive network than a static one, irrespective of disease dynamics. similarly, some other control measures are further encapsulated into adaptive community networks [ ] . except for various transition of community structure, both immunization and quarantine strategies show a counter-intuitive result that it is not "the earlier, the better" for prevention of disease. moreover, it is unveiled that the prevention efficiency of both measures is greatly different, and the optimal effect is obtained when a strong community structure exists. vaccination on time-varying networks. in contrast to the mutual feedback between dynamics and structure in adaptive frameworks, time-varying networks provide a novel angle for network research, where network connection and fig. . vaccination coverage as a function of the relative cost of vaccination and the fraction of imitators in different networks. it is obvious that for small cost of vaccination, imitation behavior increases vaccination coverage but impedes vaccination at high cost, irrespective of potential interaction topology. the figure is reproduced from [ ] . dynamics process evolve according to their respective rules [ ] [ ] [ ] . for example, summin et al. recently explored how to lower the number of vaccinated people to protect the whole system on time-varying networks [ ] . based on the past information, they could accurately administer vaccination and estimate disease outbreaks in future, which proves that time-varying structure can make protection protocols more efficient. in [ ] , the authors displayed that limited information on the contact patterns is sufficient to design efficient immunization strategies once again. but in these two works, the vaccination strategy is somewhat independent of human behavior and decision-making process, which leaves an open issue: if realistic disease-behavior dynamics is introduced into time-varying topology (especially combining with the diffusion process of opinion cluster [ ] ), how does it affect the eradication of disease? we continue to discuss some of these and similar issues in section on empirically-derived networks. some research uses networks derived from empirical data in order to examine disease-behavior dynamics. we discuss these models in this subsection. dynamics on different topologies. heterogeneous contact topology is ubiquitous in reality. to test its potential impact on disease spreading, martial et al. recently integrated a behavior epidemiology model with decision-making process into three archetypical realistic networks: poisson network, urban network and power law network [ ] . under these contact networks, an agent can make decision either based purely on payoff maximization or via imitating the vaccination behavior of its neighbor (as suggest by eq. ( )), which is controlled by the fraction of imitators . by means of numerous simulations, they displayed the diploid effect of imitation behavior: it enhances vaccination coverage for low vaccination cost, but impedes vaccination campaign at relatively high cost, which is depicted by fig. . surprisingly, in spite of high vaccination coverage, imitation can generate the clusters of non-vaccinating, susceptible agents, which in turn accelerate the large-scale outbreak of infectious disease (namely, imitation behavior, to some extent, impedes the eradication of infectious diseases). this point helps to explain why outbreaks of measles have recently occurred in many countries with high overall vaccination coverage [ , , ] . with the same social networks, ref. [ ] explored the impact of heterogeneous contact patterns on disease outbreak in the compartmental model of sars. it is interesting that, compared with the prediction of well-mixed population, the same set of basic reproductive number may lead to completely epidemiological outcomes in any two processes, which sheds light to the heterogeneity of sars around the world. impact of network mixing patterns. as ref. [ ] discovered, high vaccination coverage can guarantee herd immunity, which, however, is dramatically affected and even destroyed by clusters of unvaccinated individuals. to evaluate how much influence such clusters possess, a recent work explored the distribution of vaccinated agents during seasonal influenza vaccination through a united states high school contact network [ ] . the authors found that contact table classification of disease-behavior research outcomes according to dynamic characteristics in networked populations reviewed by section . it is clear that the same type of networks can be frequently used to different problems. table observed physical phenomena and frequently used methods in the study of diseasebehavior dynamics on networks. epidemic threshold mean-field prediction phase transition generation function self-organization percolation theory pattern formation stochastic processes bifurcation and stability analysis monte carlo simulation vaccination/immunization threshold markov-chain approximation networks are positively assortative with vaccination behavior. that is to say, large-degree unvaccinated (vaccinated) agents are more likely to contact with other large-degree unvaccinated (vaccinated) ones, which certainly results in a larger outbreak than common networks since these (positively assortative) unvaccinated agents breed larger susceptible clusters. this finding highlights the importance of heterogeneity during vaccine uptake for the prevention of infectious disease once again. in fact, the currently growing available human generated data and computing power have driven the fast emergence of various social, technological and biological networks [ ] [ ] [ ] [ ] . upon these empirically networks, mass diseasebehavior models can be considered to analyze the efficiency of existing or novel proposed prevention measures and provide constructive viewpoint for policy makers of public health [ ] [ ] [ ] [ ] [ ] [ ] . based on the above achievements, it is now clear that incorporating behavior epidemiology into networked populations has opened a new window for the study of epidemic transmission and prevention. to capture an overall image, table provides a summary for the reviewed characteristics of disease-behavior dynamics in networked populations. here it is worth mentioning that some works (e.g., [ , ] ) may appear in two categories because they simultaneously consider the influence of individual behavior and special network structure. fig. . age-specific contact matrices for each of eight examined european countries. high contact rates are represented by white color, intermediate contact rates are green and low contact rates are blue. we refer to [ ] , from where this figure has been adapted, for further details. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) many of these achievements are closely related with physics phenomena (see table ), via which we can estimate the effect of the proposed strategies and measures. on the other hand, these achievements are also inseparable from classical physics methods (table ). in particular, monte carlo simulation and mean-field prediction have attracted the greatest attention due to simplicity and high efficiency. for a comprehensive understanding, we provide a general example of mean-field theory about behavioral epidemiology in appendix a. the first mathematical models studied the adaptive dynamics of disease-behavior responses in the homogeneously mixed population, assuming that individuals interact with each other at the same contact rate, without restrictions on selecting potential partners. networked dynamics models shift the focus on the effects of interpersonal connectivity patterns, since everyone has their own set of contacts through which the interpersonal transmission can occur. the contacts between members of a population constitute a network, which is usually described by some well-known proxy model of synthetic networks, as shown in section . this physics treatment of using evidence-based parsimonious models is valuable in illustrating fascinating ideas and revealing unexpected phenomena. however, it is not always a magic bullet for understanding, explaining, or predicting realistic cases. in recent years, the studies of social experiments become more and more popular. they contribute new insight in parameterizing and designing more appropriate dynamics models. this section briefly introduces the progress in this field. a large-scale population-based survey of human contact patterns in eight european countries was conducted in ref. [ ] , which collects the empirical data of self-reported face-to-face conversations and skin-to-skin physical contacts. the data analysis splits the population into subgroups on the basis of properties such as ages and locations, and scrutinizes the contact rate between subgroups (see fig. ). it reveals that across these countries, people are more probable to contact others of the same age group. combining self-reporting contact data with serological testing data, . we refer to [ ] , from where this figure has been adapted, for further details. recent case studies are able to reveal location-or age-specific patterns of exposure to pandemic pathogens [ , ] , which provide rich information for complex network modeling. sf networks have been widely used to model the connectivity heterogeneity in human contact networks. in sf networks, each hub member can have numerous connections, including all its potential contacts relevant to the transmission. however, such common consideration might not fully agree with human physiological limitations regarding the capacity in preserving a large number of interactions. generally, the size of individual social network is restricted to around people [ , ] . to better characterize the features of human contact behavior, social experiments studying the active contacts in realistic circumstances will be valuable. thanks to the development of information and communication technologies, the usage of digital equipments becomes increasingly popular in collecting empirical data relevant to human contacts in realistic social circumstances. it is instructive to first review a few brief examples. refs. [ , ] referred to the bluetooth technique embedded in mobile phones, which collects the proxy data of person-to-person interactions of mit media laboratory in the reality mining program; with the help of wireless sensors, the social experiment was conducted to trace the close proximity contacts among the members of an american high school [ ] ; refs. [ ] [ ] [ ] [ ] considered the active radio frequency identification devices (rfid) to establish a flexible platform recording the face-to-face proximity contacts among volunteers, which had been deployed in various social contexts such as conference, museum, hospital, and primary school; and the wifi accessing data among all students and staffs were also analyzed as the indirect proxy records of their concurrent communications in one chinese university [ , ] . compared with the abovementioned data of questionnaires, the electronic data generated by digital experiments is more accurate and objective. moreover, some new interesting findings are also listed as follows. the data analysis reveals an unexpected feature that the distribution of the number of distinct persons each individual encounters every day only has a small squared coefficient of variance [ , , [ ] [ ] [ ] [ ] , irrespective of the specific social contexts (see fig. ). this homogeneity in the so-called node-degree distribution indicates the absence of connectivity hubs, which is, to some extent, subject to our physiological limitations. the dynamics of human interactions is not evolving at an equilibrium state, but is highly fluctuating and timevarying in realistic situations. this can be characterized by measuring the statistical distribution of the duration per contact and the time intervals between successive contacts [ ] . as shown in fig. , these two statistics both have a broad distribution spanning several orders of magnitude. most contact durations and inter-contact intervals are very short, but long durations and intervals also emerge, which corresponds to a burst process without characteristic time scales [ ] . the coexistence of homogeneity in degree of nodes and heterogeneity in contact durations lead to unexpected phenomena. for example, the low-degree nodes which are insignificant in conventional network models can act as hubs in time-varying networks [ ] . the usage of electronic devices provides an easy and nonintrusive approach for contact tracing, which can help understand health-related behaviors in realistic settings. to measure close proximity interactions between health-care fig. (a) . similar to fig. , each symbol denotes one venue (sfhh denotes sfhh, nice, fr; eswc (eswc ) is eswc ( ), crete, gr; and ps corresponds to primary school, lyon, fr). we refer to [ ] , from where this figure has been adapted, for further details. workers (hcws) and their hand hygiene adherence, polgreen et al. performed experiments by deploying wireless sensor networks in a medical intensive care unit of the university of iowa hospital. they confirmed the effects of peer pressure on improving the hand hygiene participation [ ] , i.e., the proximity of other hcws, can promote the hand hygiene adherence. they also analyzed the role of "superspreader", who has a high frequency in encountering others [ ] . the disease severity increases with hand hygiene noncompliance of such people. except for empirical data of contact networks, social behavior experiments (or surveys) also play an important role in the vaccination campaign and disease spreading, especially combined with the decision-making process. here we will review the recent progress within this realm. role of altruistic behavior. game theory has been extensively used in the study of behavior epidemiology, where individuals are usually assumed to decide vaccination or not based on the principle of maximizing self-interest [ , ] . however, in reality, when people make vaccination decision, do they only consider their own benefit? to test this fundamental assumption, ref. [ ] recently conducted a survey about individual vaccination decisions during the influenza season. the questionnaires, from direct campus survey and internet-based survey, are mainly composed of two items: self-interest ones (the concern about becoming infected) and altruistic ones (the concern about infecting others), as schematically illustrated in fig. . if agents are driven by self-interest, they attempt to minimize their cost associated with vaccination and infection, which gives rise to selfish equilibrium (or the so-called nash equilibrium). by contrary, if individual decision is guided by altruistic motivation, the vaccination probability reaches community optimum (or the so-called utilitarian equilibrium), at which overall cost of the community is minimal. the authors unveiled that altruism plays an important role in vaccination decision, which can be quantitatively measured by "degree of altruism". to further evaluate its impact, they incorporated the empirical data and altruistic motivation into svir compartmental model. interestingly, they found that altruism can shift vaccination decisions from individual self-interest to a community optimum via greatly enhancing total vaccination coverage and reducing the total cost, morbidity and mortality of the whole community, irrespective of parameter setup. along this line, the role of altruistic behavior in age-structure populations was further explored [ ] . according to general experience, elderly people, who are most likely to be infected in the case of influenza, should be most protected by young vaccinators, who are responsible for most disease transmission. to examine under which condition young agents vaccinate to better protect old ones, the authors organized the corresponding social behavior experiment: participants are randomly assigned to "young" and "elderly" roles (with young players contributing more to herd immunity yet elderly players facing higher costs of infection). if players were paid based on individual point totals, more elderly than young players would get vaccinated, which is consistent with the theoretical prediction of fig. . schematic illustration of questionnaire used in the voluntary vaccination survey. the survey items can be divided into self-interest ones (i.e., outcomes-for-self) and altruism ones (i.e., outcomes-for-others), which have corresponding scores. based on both, it becomes possible to indirectly estimate the degree of altruism, which plays a significant role in vaccination uptake and epidemic elimination. we refer to [ ] , from where this figure has been adapted, for further details. self-interested behavior (namely, nash equilibrium). on the contrary, players paid according to the group point totals make decisions in a manner consistent with the utilitarian equilibrium, which predicts community-optimal behavior: more young than elderly players get vaccinated yet less cost. in this sense, payoff structure plays a vital role in the emergence of altruistic behavior, which in turn affects the disease spreading. from both empirically studies, we can observe that altruism significantly impacts vaccination coverage as well as consequent disease burden. it can drive system to reach community optimum, where smallest overall cost guarantees herd immunity. it is thus suggested that in realistic policies altruism should be regarded as one potential strategy to improve public health outcomes. existence of free-riding behavior. accompanying altruistic behavior, another type of behavior addressed within decision-making frameworks is free-riding behavior, which means that people can benefit from the action of others while avoiding any cost [ , , ] . in a voluntary vaccination campaign, free riders are unvaccinated individuals who avoid infection because of herd immunity, as illustrated by the gray nodes in fig. . to explore the impact of free-riding behavior, john et al. even organized a questionnaire containing six different hypothetical scenarios twenty years ago [ ] . under such a survey, altruism and free-riding were simultaneously considered as the potential decision motivations for vaccination. they found that, for vaccine conferring herd immunity, the free-riding frame causes less sensitivity to increase vaccination coverage than does the altruism frame, which means that free-riding lowers preference of vaccination when the proportion of others vaccinating increases. in addition to homogeneous groups of individuals, yoko et al. recently conducted a computerized influenza experiment, where the groups of agents may face completely different conditions, such as infection risk, vaccine cost, severity of influenza and age structure [ ] . they found that high vaccination rate of previous rounds certainly decreases the likelihood of individuals' vaccination acceptance in the following round, indicating the existence of free-riding behavior. both empirical surveys thus showed that individuals' decision-making may be driven by the free-riding motive, which depresses vaccination coverage. besides the above examples, there exist more factors, such as individual cognition [ ] and confidence [ ] , affecting the decision of vaccination in reality. if possible, these factors should be taken into consideration by public policy makers in order to reach the necessary level of vaccination coverage. the growth in online social networks such as twitter in recent years provides a new opportunity to obtain the data on health behaviors in near real-time. using the short text messages (tweets) data collected from twitter between august and january , during which pandemic influenza a (h n ) spread globally, salathé et al. analyzed the spatiotemporal individuals' sentiments towards the novel influenza a (h n ) vaccine [ ] . they found that projected vaccination rates on the basis of sentiments of twitter users can be in good accord with those estimated by the centers for disease control and prevention of united states. they also revealed a critical problem that both negative and positive opinions can be clustered to form network communities. if this can generate clusters of unvaccinated individuals, the risk of disease outbreaks will be largely increased. we have reviewed some of the recent, rapidly expanding research literature concerning nonlinear coupling between disease dynamics and human behavioral dynamics in spatially distributed settings, especially complex networks. generally speaking, these models show that emergent self-protective behavior can dampen an epidemic. this is also what most mean-field models predict. however, in many cases, that is where the commonality in model predictions ends. for populations distributed on a network, the structure of the disease contact network and/or social influence network can fundamentally alter the outcomes, such that different models make very different predictions depending on the assumptions about human population and diseases being studied, including findings that disease-behavior interactions can actually worsen health outcomes by increasing long-term prevalence. also, because network models are individual-based, they can represent processes that are difficult to represent with mean-field (homogeneous mixing) models. for example, the concept of the neighbor of an individual has a natural meaning in a network model, but the meaning is less clear in mean-field models (or partial differential equation models) where populations are described in terms of densities at a point in space. we speculate that the surge of research interest in this area has been fuelled by a combination of ( ) the individual-based description that characterizes network models, ( ) the explosion of available data at the individual level from digital sources, and ( ) the realization from recent experiences with phenomena such as vaccine scares and quarantine failures that human behavior is becoming an increasingly important determinant of disease control efforts. we also discussed how many of the salient dynamics exhibited by disease-behavior systems are directly analogous to processes in statistical physics, such as phase transitions and self-organization. the growth in research has created both opportunities as well as pitfalls. a first potential pitfall is that coupled disease-behavior models are significantly more complicated than simple disease dynamic or behavior dynamic models on their own. for a coupled disease-behavior model, it is necessary not only to have a set of parameters describing the human behavioral dynamics and the disease dynamics separately, it is also possible to have a set of parameters to describe the impact of human behavior on disease dynamics, and another set to describe the effect of disease dynamics on human behavior. thus, approximately speaking, these models have four times as many parameters as a disease dynamic model on its own, or a human behavioral model on its own: they are subject to the "curse of dimensionality". a second pitfall is that relevant research from other fields may not be understood or incorporated in the best possible way. for example, the concept of 'social contagion' appears repeatedly in the literature on coupled disease-behavior models. this is a seductive concept, and it appears to be a natural concept for discussing systems where a disease contagion is also present. however, the metaphor may be too facile. for example, how can the social contagion metaphor capture the subtle but important distinction between descriptive social norms (where individuals follow a morally neutral perception of what others are doing) and injunctive social norms (where individuals follow a morally-laden perception of what others are doing) [ ] ? social contagion may be a useful concept, but we should remember that it ultimately is only a metaphor. a third pitfall is lack of integration between theoretical models and empirical data: this pitfall is common to all mathematical modeling exercises. the second and third pitfalls are an unsurprising consequence of combining natural and human system dynamics in the same framework. there are other potential pitfalls as well. these pitfalls also suggest ways in which we can move the field forward. for example, the complexity of models calls for new methods of analysis. in some cases, methods of rigorous analysis (including physics-based methods such as percolation theory and pair approximations (appendix b))-for sufficiently simple systems that permit such analysis-may provide clearer and more rigorous insights than the output of simulation models, which are often harder to fully understand. for systems that are too complicated for pen-and-paper methods, then methods for visualization of large and multidimensional datasets may prove useful. the second and third pitfalls, where physicists and other modelers, behavioral scientists and epidemiologists do not properly understand one another's fields can be mitigated through more opportunities for interaction between the fields through workshops, seminars and colloquia. interactions between scholars in these fields if often stymied by institutional barriers that emphasize a 'silo' approach to academic, thus, a change in institutional modes of operation could be instrumental in improving collaborations between modelers, behavioral scientists and epidemiologists. scientists have already shown that these pitfalls can be overcome in the growing research in this area, and this is evidence in much of the research we have described in this review. the field of coupled disease-behavior modeling has the elements to suggest that it will continue expanding for the foreseeable future: growing availability of data needed to test empirical models, a rich set of potential dynamics created opportunities to apply various analysis methods from physics, and relevance to pressing problems facing humanity. physicists can play an important role in developing this field due to their long experience in applying modeling methods to physical systems. where [ss] is the number of susceptible-susceptible pairs in the population, q(i | ss) is the expected number of infected neighbors of a susceptible in a susceptible-susceptible pair, and similarly q(i | si ) is the expected number of infected neighbors of the susceptible person in a susceptible-infected pair. the first term corresponding to creation of new si pairs from ss pairs, through infection, while the second term corresponds to destruction of existing si pairs through infection, thereby creating ii pairs. an assumption must be made in order to close the equations at the pair level, thereby preventing writing down equations of motion for triples. for instance, on a random graph, the approximation might be applied, where q(i | s) is the expected number of infected persons neighboring a susceptible person in the population. equations and pair approximations for the other pair 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gratefully yao yao, yang liu, ke-ke huang, yan zhang, eriko fukuda, dr. wen-bo du, dr. ming tang, dr. hai-feng zhang and prof. zhen jin for their constructive helps and discussions, also appreciate all the other friends with whom we maintained (and are currently maintaining) interactions and discussions on the topic covered in our report. this work was partly supported by the national natural science foundation of china (grant no. , , ) and the natural sciences and engineering council of canada (nserc; ma and ctb). whenever the classical epidemic spreading processes (sis, sir) are taking place in homogeneous populations, e.g., individuals are located on the vertices of regular random graph, er random graph, completed connected graph, the qualitative properties of the dynamics can be well catched by the mean-field analysis, assuming unbiased random matching among the individuals. for simplicity yet without loss of generality, we here derive the mean-field solution for the peer-pressure effect in vaccination dynamics on the er random graph as an example [ ] . let x be the fraction of vaccinated individuals and w(x) be the probability that a susceptible individual finally gets infected in a population with vaccine coverage x. after each sir epidemic season, individuals get payoffs: vaccinated (i, p i = −c), unvaccinated and healthy (j , p j = ), and unvaccinated and infected (ς ,p ς = − ). individuals are allowed to modify their vaccination strategies in terms of eq. ( ). whenever an individual from compartment i goes to compartment j or ς , the variable x drops, which can be formulated aswhere above we have approximated, in the spirit of mean-field treatment, the fraction of neighbors holding opposite strategy of the vaccinated individuals as − x. the quantity i→j is the probability that individuals from the compartment i change to the compartment j , whose value is determined by eq. ( ) . accordingly, the gain of x can be written astake the two cases into consideration, the derivative of x with respect to time is written as dx/dt = x + + x − . solving for x, we get the equilibrium vaccination level f v . note that the equilibrium epidemic size is expected to be f i = w(x), satisfying the self-consistent equationwhere r is the basic reproductive number of the epidemic. pair approximation is a method by which space can be implicitly captured in an ordinary differential equation framework [ , ] . to illustrate the method, consider the variable [s], defined as the number of susceptible individuals in a population distributed across a network or a lattice. for an sir natural history, the equation of motion for s is: key: cord- -qwk jkqm authors: delmage, d. a.; kelly, d. f. title: auricular chondritis in a cat date: - - journal: j small anim pract doi: . /j. - . .tb .x sha: doc_id: cord_uid: qwk jkqm a four‐year‐old male neutered domestic shorthaired cat developed bilateral thickening of the pinnae, with slight curling, intense erythema and pain. no ear canal disease was present. the cat was negative for feline immunodeficiency virus, feline leukaemia virus and feline coronavirus. biopsy of the ear lesion revealed auricular chondritis. in humans, histologically similar lesions may involve the pinnae, nose, trachea, joints, eyes and heart, and the disease is termed relapsing polychondritis. the cat reported had a history of corneal damage, resulting in corneal vascularisation and opacity, eyelid distortion, necessitating an entropion operation, and radiological evidence of mild cardiac enlargement. the ear disease responded rapidly to treatment with prednisolone and, apart from slight thickening and curling of the pinnae, the cat remained normal and pain‐free. after two years, the prednisolone was withdrawn, and there was no recurrence of the condition in a follow‐up period of months. relapsing polychondritis is very rare in the cat, and is diagnosed by characteristic pinna! lesions (scott , bunge and others , gauguere and others , involving thickening, distortion, pain and intense erythema. to the authors' knowledge, only three cases have been reported in the literature. the present report describes a cat with a lesion histologically similar to the previously reported cases. ocular disease was also present, and mild cardiomegaly was found on routine radiography. the latter signs were also described in the case by bunge and others ( ) , but not in the other two cases. a four-year-old male neutered domestic shorthaired cat was presented with thickening and intense erythema of the inner surfaces of both pinnae; the ear flap edges were thickened, slightly curled, distorted and painful (fig ) . the condition had developed on both pinnae simultaneously, and the cat twitched and flicked the ear flaps frequently. the outer (haired) surfaces were unaffected, and no external ear canal disease was evident. the cat had exhibited an altered demeanour for about three months before presentation, manifested as hiding away, cowering, resentment of handling by the owner, and aggression towards the other cat in the household. increased skin scaling was evident on the lumbodorsal area, and was non-pruritic, with no parasites found on coat brushings and scrapings. this was presumed to be due to reduced self-groo ming. no other skin disease was found, and there was no previous history of skin or ear disease. n ine months previo~s l y, the cat had been seen by another veterinary surgeo n for corneal vascularisation of the right eye. the condition was fluorescein-negative, and was diag nosed as an o ld wound; probably a resolvi ng corneal ulcer. one month late r, the cat had conjunctivitis in both eyes, in addition to blepharos pasm and slight entropion of the lower lid of the right eye. steroid/antibiotic drops we re administered for these conditions. two months later, the entropion had worsened, and a surgical correction was performed. both eyes continued to show intermittent conjunctivitis, at times fairly severe, for several months. corneal opacity, extensive vascularisation and a weakly positive uptake of fluorescein stain were noted during this time, but no further inves tigations we re performed. at the time of prese ntation for rhe ear problem, rh e ocular lesions had largel y resolved, leaving a slight corneal opacity in the right eye. both eyes were now fluorescein-negative, and a schirmer tear rest showed a slight reduction in tear producti o n in rhe ri ght eye ( mm; normal range to mm), wirh normal rear production in the left eye ( mm). the cat was anaes rherised ro obtain biopsy sam ples; rh e left pinna was biopsied, wirh a v-sec ri on taken from the edge, and a circular punch biopsy from the centre (including ca rtilage). blood samples were taken for hae matological and serum biochemical analyses. res ults we re unremarkable, except for hypoalbuminaemia ( g/lirre; normal range to g!l itre) of undetermin ed cause. the blood sa mple was negati ve for feline leukaemia virus (felv) (elisa), feline immunodeficiency virus (f iv) (elisa) and felin e coronav irus (immunofluorescence test). an antinuclear antibody tesr was also negative. a direct coombs' res r was negative at °c, but positive at a dilution of : at °c. t horacic radiograp hs showed mild to moderate globular cardiomegaly on borh lateral and dorsoventral views. the ve rtebral heart score was approximately - (no rmal ran ge - to - ). t he hea rt rate was beats/m inute and there was no radi ological or clinical evidence of co ngestive heart failure. it was fel t that the heart changes were sugges ti ve o f ca rdi o myopat hy, either dilared , hypertro phic o r intermediate. h owever, since no echocardiography o r ecg was performed, a diagnosis of ca rdi omyo pathy co uld no r be confirmed. the cat was euthyroi d ar this time (rota! thyroxine nmolllitre; normal range ro nmol/litre). biopsy of the pinn a showed a curling distortion of the rip of the auricular ca rtilage, with ex tensive intense fibropl as ia , ca pillary proliferati o n and infiltration by neutrophilic leucocytes (fig ) . inflam matory cells exte nded into eroded superfi cial ca rtil age, where there was extensive linea r loss of matrix metac hrom as ia (fig ) . t here was intense mastocytosis o f auri cular dermis, but no histo logical evidence of its cause. focal areas of dermal mi xed lymphocyte, plas ma cell and mast cell acc umulati on were noted . t he ca t was give n i mg of o ral predniso lo ne daily ( mg/ kg bodywe ight dail y; prednicare; animalcare) and within i days showed a marked improve ment. t he ea r fl aps were much less painful and eryt hemarous, and the ca t was playi ng with rh e o ther ca t in the ho use ho ld and showed improved demeanour. the dorsal sca ling had reso lved . there was no alrerari o n ro the righr co rn ea l opac ity, and no pulmo nary or ca rdi ac abn o rm alities were detected o n ausc ultati on. after rhree weeks, th e predniso lone was reduced ro mg daily ( i · mg/kg/day), and afte r six mo nths co - mg o n alternate days. eighteen months later, the prednisolone was withdrawn completely. there was no subsequent recurrence in the ear condition during the months follow-up period. relapsing polychondritis in humans is a rare condition in which auricular distortion and discomfort are major signs. cartilage in other sites may also be affected, and in a proportion of cases this results in saddle nose deformity (collapse of the nasal septa! cartilage), joint disease, laryngeal/tracheal disease (leading to collapse of the tracheal rings), ocular disease, cardiovascular disease and skin disease. fever, hearing loss, vertigo and anaemia may also occur. associated ocular diseases can include conjunctivitis, scleritis, keratitis and keratoconjunctivitis sicca. cardiovascular changes include aortic insufficiency, pericarditis, arterial aneurysms and arteritis (white , michet and others ) . the disease in humans is believed to be a true autoimmune disorder in which autoantibodies (predominantly immunoglobulin [ig]g) to native type ii collagen are found in titres of up to : (mckee ) . this contrasts with the situation in rheumatoid arthritis, in which autoantibodies to unfurled rype i, ii and iii collagen are found, suggesting that antibody production in this condition is an epiphenomenon rather than the cause of disease (foidart and others , ebringer and others , meyer and others . in some strains of rats, idiopathic auricular chondritis is common, and is characterised by histological changes similar to those described in the present case (that is, auricular inflammation, invasion of conchal cartilage by inflammatory cells and chondrolysis) (mcewen and barsoum ) . in the cat, relapsing polychondritis is very rare. to the authors' knowledge, only three confirmed cases have been reported in the literature. the case described here is interesting because it is similar to that described by bunge and others ( ) ; both cats had evidence of ocular and cardiac changes in addition to the pinna! disease, as can occur in human cases. the other changes seen in humans have not yet been reported in cats. gauguere and declercq ( ) , calling the condition plasma cell chondritis, suggested a possible viral aetiology, since one reported case was fiv positive (gauguere and others ) and one felv positive (bunge and others ) ; the present case was negative for fiv, felv and feline coronavirus. the significance of the positive cold coombs' test result in the present case is uncertain. a titre of : is a low positive result; the cat was not anaemic, and did not have signs of cold agglutinin disease, such as punched-out necrosis of the earflap edges, or a vascular pattern of ulceration and necrosis. also, the histopathological findings were not compatible with cold agglutinin disease. it is of interest that a weak positive coombs' test result of : was found in the case reported by bunge and others ( ) . although the disease in cats has been designated relapsing polychondritis, the authors prefer the description of auricular chondritis in their patient, since they have no evidence of involvement of other cartilaginous tissues, nor does the benign clinical course in this case warrant the use of the qualifier 'relapsing'. clinical differentiation of auricular chondritis from aural haematoma and aural trauma is straightforward; haematoma and trauma are unlikely to be bilateral, nor to be as painful or intensely erythematous. aural haematomas may exhibit cartilage degeneration and irregular erosion adjacent to the haematoma (joyce and day ) but do not show the severe, aggressive and extensive changes seen in the present case. other differential diagnoses include scarring from irritation associated with ear mite infestation, actinic scarring and distortion in cats with unpigmented eartips, discoid and systemic lupus erythe-journal of small animal practice• vol •october matosus, and vascular diseases, such as frost-bite damage, and immune-mediated vasculitis. cases of floppy pinnae in cats (pearson , rest ) may have the same aetiology as reported here. it is clear that such cases justify as full an investigation as possible, including cardiac and ocular assessment, and detailed histopathological examination, in order to assist with diagnosis and understanding of this condition. relapsing polychondritis in a cat autoantibodies to cartilage and type ii collagen in relapsing polychondritis and other rheumatic diseases antibodies to type ii collagen in relapsing polychondritis guaguere and p. prelaud. veterinary times/ veterinary business development polychondrite auriculaire atrophiante: a propos d'un cas chez un chat lmmunopathogenesis of canine aural haematoma relapsing polychondritis auricular chondritis in wistar rats relapsing polychondritis -pathogenic role of anti-native collagen type ii antibodies: a case report relapsing polychondritis: survival and predictive role of early disease manifestations floppy pinnae in siamese cats floppy pinnae in siamese cats feline dermatology - -'the secret sits' relapsing polychondritis the authors are very grateful to dr s. p. dunham for examining and commenting on the radiographs. key: cord- - i twr authors: esposito, susanna; polinori, ilaria; rigante, donato title: the gut microbiota-host partnership as a potential driver of kawasaki syndrome date: - - journal: front pediatr doi: . /fped. . sha: doc_id: cord_uid: i twr kawasaki syndrome (ks) is a necrotizing vasculitis of small- and medium-sized vessels mostly affecting children under years of age; a host of clinical and epidemiological data supports the notion that ks might result from an infectious disease. however, many efforts have failed to identify a potentially universal trigger of ks. the contribution of the intestinal microbial community—called the “microbiota”—to ks has been evaluated by an increasing number of studies, though limited to small cohorts of patients. differences in the microbiota composition were found in children with ks, both its acute and non-acute phase, with abnormal colonization by streptococcus species in the intestinal tract and a wider presence of gram-positive cocci in jejunal biopsies. in particular, a higher number of gram-positive cocci (of the genera streptococcus and staphylococcus), eubacterium, peptostreptococcus, and hsp -producing gram-negative microbes have been found in the stools of ks children, and their effects on the antigenic repertoire of specific t cells and vβ t cell expansion have been assessed. conversely, lactobacilli were lacking in most children with ks compared with other febrile illnesses and healthy controls. all studies available to date have confirmed that an imbalance in the gut microbiota might indirectly interfere with the normal function of innate and adaptive immunity, and that variable microbiota interactions with environmental factors, mainly infectious agents, might selectively drive the development of ks in genetically susceptible children. further investigations of the intestinal microflora in larger cohorts of ks patients will provide clues to disentangle the pathogenesis of this disease and probably indicate disease-modifying agents or more rational ks-specific therapies. the most insidious primary vasculitis in childhood is kawasaki syndrome (ks), an acute multi-systemic illness which predominantly affects children under years of age ( ) . currently, this disorder of unknown etiology remains the main cause of acquired heart disease among children living in developed countries, where rheumatic fever has been surpassed ( , ) . this condition was originally called "mucocutaneous lymph node syndrome" by dr. tomisaku kawasaki, who was its discoverer, and was thought to be a benign children's disease; nowadays, the illness has been described worldwide in children of every ethnicity following the presence of fever persisting (at least) days together with (at least) of the following signs: bilateral conjunctival injection, oropharyngeal inflammation, abnormalities of hands and feet, polymorphous exanthema, and non-purulent cervical lymphadenopathy, usually unilateral ( ) . several years after the first description, fatalities occurred among children with ks younger than years living in japan, prompting clinicians to reconsider ks's long-term risks related to systemic and necrotizing effects on the vascular endothelium of small-and medium-sized arteries, which have been acknowledged in all guidelines related to the management of ks ( , ) . the most relevant sequelae of ks include variable degrees of damage within coronary arteries in combination with angina, myocardial infarction, ischemic cardiomyopathy, and sudden death; these complications should be preventable with a timely treatment of high-dose intravenous immunoglobulin (ivig), which is the recommended therapeutic strategy in ks ( ) . higher acute phase reactants and younger age at onset of ks are nodal points in determining, respectively, a failure in the response to ivig and an increased occurrence of coronary artery abnormalities ( ) . the prediction of ivig resistance is also crucial in ks patients, as recognizing these high-risk children should consent to start an intensified treatment protocol combined with ivig to prevent coronary injuries ( ) . ks incidence varies widely among different ethnic groups; for instance, in the united kingdom it has stabilized and remains low at . per , population under the age of years. however, general practitioners should be aware that the condition occurs throughout childhood and across the seasons, and-given the potential cardiovascular sequelae-ks should be considered in all children with persistent fever, even in older children and adolescents ( ) . ks is most prominently recognized in japan, korea, and taiwan, reflecting increased genetic susceptibility among asian populations. a recent study reported an incidence of ∼ per , children under years of age in japan ( ) . there is still much controversy about the etiology of ks, though epidemiologic and clinical data suggest that ks might originate from an abnormal response to undisclosed infectious diseases in genetically susceptible children ( ) . there is no agreement whether ks-related infectious agents are of viral, bacterial or fungal origin ( ) , and the underlying immune mechanisms behind ks have not been completely highlighted, remaining only partially known. the absence of a proven unambiguous cause of ks has induced the scientific community to pay attention to other environmental hypothetical triggers, and in particular the composition of the resident intestinal flora as a potential contributor to ks has been evaluated by different research groups. the main aim of this review was to analyze the relationship between the microbial community, or the "microbiota, " and the overall impact of bacterial or viral infections in the potential development of ks. scientific papers have been searched from the electronic databases of pubmed until january ; the retrieving words were "kawasaki disease, " "kawasaki syndrome, " "microbiota, " and "microbiome"; additional reports were identified and analyzed through the specific references cited in the retrieved papers. only papers published in english and those showing evidence-based data were included in our evaluation. the microbiota, a microbial community of trillions of microorganisms and at least , different bacterial species, some eukaryotic fungi and viruses, and which covers every surface of the human body, plays a contributory role in many infections, immune-mediated disorders, rheumatologic diseases, and disorders of the nervous system. the microbiome, on the other hand, is the collection of the whole genome sequences of those microorganisms, consisting of more than , , genes ( , ) . in particular, the gut microbiota is strictly linked to the chronological age of each individual and modulates host physiology and metabolism through different mechanisms. each stage of human life is characterized by a specific intestinal microbial composition: the microbiota that initially colonizes the fetus' intestinal tube consists of aerobic organisms such as enterococcus and streptococcus, is then gradually replaced by anaerobes such as bifidobacterium and lactobacillus and finally reaches the adult composition dominated by bacteroides and firmicutes ( , ) . the fetal microbiota is prone to be conditioned by the type of delivery; the mother's vaginal flora is a relevant source of lactobacillus, prevotella, and bifidobacterium. conversely, a cesarean delivery delays contact with these species, producing a similar-to-skin flora, dominated by staphylococci ( ) . the feeding regimens and food supplements also play a role in modifying the resident flora; a greater complexity is normally seen in infants fed with formula, rather than in breastfed babies who have an "adult-like" structured microbiota with a population rich with bifidobacteria, lactobacilli, and bacteroides ( ). the infant gut microbiota is variable in composition over time and highly changeable during the first year of life, being influenced by specific bacteria to which a baby happens to be exposed, as shown by the resemblance of infants' stool microbial community with mothers' milk and vaginal samples ( ) . thereafter, the infant's intestinal tract progresses toward an extremely dense colonization, ending with a mixture of microbes that is broadly very similar to an adult's intestine. during adulthood, the gut microbiota becomes stable and this intestinal homeostasis remains in equilibrium with the host. food habits influence the composition of the whole intestinal microbiota, as testified by the lower prevalence of bacteroides in those suffering from malnutrition and by different microbiota changes occurring in children with diet-related diseases, such as allergies and obesity ( ) . arumugam et al. identified three distinct enterotypes, namely bacteroides, prevotella, and ruminococcus, which reflect many individual alimentary profiles: bacteroides correlates to a highfat or high-protein regimen, whereas prevotella is associated with higher consumption of fibers and simple sugars ( ) . more recent data have consented to unify bacteroides and ruminococcus enterotypes due to the large similarity between the two ( ) . it is well-established that early events of birth, environmental factors during infancy, sex hormones, diet, body weight, and use of antibiotics can undoubtedly differentiate the composition of the microbiota ( , ) . there are no ideal culture methods to give us a real overview of the complete intestinal flora, though molecular methods, e.g., dna microarrays with comprehensive coverage of most bacterial taxa represented in the available database of small subunit ribosomal rna gene sequences, should allow the characterization of most taxonomic groups of the intestinal bacteria ( , ) . the ancient symbiosis between the human gastrointestinal tract and its resident microbiota involves diverse reciprocal interactions between the microbiota itself and the host, with relevant consequences for human health and physiology. the quality of the microbial flora has an impact on the maintenance of health and also on prevention of diseases. indeed, there are numerous roles carried out by the intestinal ecosystem, as the stimulation of angiogenesis, control of host fat storage and protection against other pathogens. in particular, the microbiota influences the formation and progress of regulation of both innate and adaptive immunity in close interaction with the intestinal mucosal immune system. the intestinal mucosa may be considered as an immunological niche as it hosts a complex immune-functional organ comprised by t cell subpopulations, neutrophils, macrophage-dendritic cells, enterocytes (that possess tight intercellular junctions) and their related anti-and pro-inflammatory cytokines as well as several other mediators of inflammation or antimicrobial peptides, defensins, and secretory immunoglobulin a (iga) ( ) . the intestinal microbiota may also have direct or indirect effects on the natural course of viral infections, interacting with viral particles and leading to differences in either pathogenicity or anti-viral immune response through recruitment and activation of several t cell subpopulations ( ) . a large amount of data has also depicted the relevance of gut microbiota-immune system cross-talk in several diseases, and indeed an "imbalance" of the intestinal flora has been shown in patients with atopic diseases and various noninfectious diseases, including metabolic disorders, chronic inflammatory bowel disease (ibd), irritable bowel syndrome, pancreatic diseases, atherosclerosis, and rheumatoid arthritis ( , ) . furthermore, the gut microbiota, interacting with pattern recognition receptors (prrs), signaling receptors that can recognize molecular structures of pathogens and activate the cascade of innate immunity, plays a crucial role in maintaining the homeostasis of the innate immunity responses in the gut, and leaks in the intestinal mucosal barrier lead to the translocation of bacterial products into portal circulation which promotes systemic inflammation ( ) . in addition, the microbiota can maintain a segregation between intestinal mucosa and bacteria via prrs, though pathogens might usurp innate signals to their advantage ( ) . several immunologic, metabolic and nutritional processes are normally controlled by the intestinal microbiota, and changes in the local microbial communities have been linked to chronic low-grade inflammation ( ) . alexander et al. demonstrated that the microbiota has specific effects on adaptive immunity, such as the induction of regulatory effector cd + cells and production of cytokines and antimicrobial factors, influencing the individual response to various environmental stimuli, as seen in ibd, crohn's disease and ulcerative colitis ( ) . in a recent work, schwiertz et al. have shown microbiota changes characterized by decreased numbers of faecalibacterium praunsitzii and increased numbers of escherichia coli in ibd ( ) . additionally, in celiac disease de palma et al. have described a difference in the microbiota composition with a reduction of bifidobacterium, clostridium histolyticum, clostridium lituseburense, faecalibacterium prausnitzii, and an abundance of bacteroides and prevotella strains ( ) . a significantly higher biodiversity in coeliac children's duodenal mucosa was demonstrated by schippa et al. who also highlighted that the possible pathophysiological role of such microbial differences needs further characterization ( ) . in addition, many immune phenomena were shown to deteriorate under the effect of changes in the microbiota, as revealed by the correlation among intestinal bacterial overgrowth, increased permeability, and development of non-alcoholic steatohepatitis ( ) . much interest has also been paid to the role of the microbiome in the development of "sterile" inflammation, and recent studies have proved that either depletion of the microbiota or changes in the diet and in the gut microbiome might lead to the improvement of inflammasome-mediated manifestations of autoinflammatory disorders, which are caused by dysregulation of specific components of innate immunity ( ) . these diseases can be subdivided into monogenic and multifactorial disorders, with the former being caused by mutations of genes involved in the regulation of the innate immune system and the latter by a combination of genetic background and environmental factors ( ) ( ) ( ) . given the evidence for the role of the intestinal microbiota in the inflammatory state of ibd, atopic diseases and numerous non-infectious diseases, it has been speculated that intestinal microbial agents might also play a trigger role in the development of other inflammatory disorders which do not have a clearly defined etiology, such as ks. the etiology of ks remains obscure, although clinical and epidemiological features suggest a primary infectious cause. indeed, a self-limited and generally nonrecurring illness that manifests itself by fever, rash, mucositis, conjunctival injection, and cervical adenopathy fits well with an infection. more precisely, clinical features of ks resemble some peculiar infectious diseases, such as streptococcal infections, staphylococcal toxic shock syndrome, and atypical measles ( , ) . additionally, age distribution, winter-spring seasonality, high rates of ks in siblings, and occurrence of community outbreaks are suggestive of a transmissible childhood disease, though many efforts with conventional bacterial and viral cultures and serological methods as well as animal inoculation studies have failed to identify a final unique infectious agent of ks ( ) . reported non-infectious factors associated with ks include carpet shampoo, preexisting eczema, environmental pollution, and house dust mites ( ) . the higher incidence of ks in asian populations, presence of familial clustering, and elevated risk of recurrence vs. the risk of a first episode in ks-naïve children are all strong evidence of a genetic contribution to ks susceptibility, probably in association with an infectious trigger ( , ) . however, ks does not appear easily transmittable and does not respond to any antibiotics: these characteristics should contradict a primitively infectious pathogenesis of the illness. the most current theory about the pathogenesis of ks is that the disease results from an exaggerated immune response toward environmental stimuli occurring in a genetically susceptible child ( ) . the prominent role played by the immune system in ks is confirmed by the high number of studies revealing the activation of neutrophils and multiple immune cells with overproduction of pro-inflammatory cytokines, including tumor necrosis factor (tnf)-α ( , ) . manlhiot et al. have very recently proposed a new pathogenetic model of ks in which the disease risk is determined by concurrent interacting processes: genetic susceptibility, habitual exposure to allergens, atmospheric biological particles, and infectious agents ( ) . a study exploring the immune responses during the acute phase of ks showed increased levels of lipopolysaccharide (lps) bound to the surfaces of circulating neutrophils via cd receptor and found markedly increased levels of the soluble cd in the plasma ( ) . furthermore, chen et al. ( ) demonstrated that nlrp inflammasome activation is associated with the development of coronary arteritis in a mouse model of ks, and that infusion of visfatin, a major injurious adipokine, can activate nlrp inflammasome and increase interleukin (il)- production, leading to enhanced endothelial dysfunction. these novel molecular mechanisms of vasculitis mediated by inflammasome activation open more specifically the road to innate immunity pathways in the interpretation of ks ( , ) . unchallengeable proof that an infection is the starting point of ks is not available. different epidemiologic studies have supported this hypothesis, based on documented infections by various microorganisms in many cases of ks. further clues are the occurrence of ks in epidemics, higher incidence during spring and winter and early age at which the disease might be acquired, that is months to years ( ). striking perturbations of many immune pathways occur during the acute phase of ks, which determines a multi-cytokine cascade in the vascular endothelium with focal disruption of small-and mediumsized vessels. however, the exact key steps leading to coronary arteritis are still far from being clarified, though endothelial cell activation; prolonged start-up of neutrophils, cd + monocyte/macrophages and cd + lymphocytes; production of oxygen intermediates and lysosomal enzymes; and oligoclonal iga response by plasma cells all appear to be involved ( , , ( ) ( ) ( ) . the contribution of viruses to ks has been suggested by ultrastructural studies which found cytoplasmic inclusion bodies containing rna of viral origin in the bronchial epithelia ( ) as well as by studies revealing the detection of intracytoplasmic inclusion bodies containing viral proteins and nucleic acid aggregates ( , ) . recent investigations have supported the hypothesis that immune responses in ks are oligoclonal rather than polyclonal (as found typically in superantigen-driven responses), and that iga plasma cells play a crucial role. indeed, higher levels of iga have been found in the vasculature of patients with previous ks, indicating an antigen-driven response against an etiologic agent which might have a respiratory or gastrointestinal portal of entry ( ) . about half of all ks patients might have one or more respiratory viruses detected by polymerase chain reaction, without any particular predominance, but a positive respiratory viral test or presence of respiratory symptoms at the time of presentation should not be used to exclude the diagnosis of ks ( ) . other studies have investigated a potential relationship of ks with coronaviruses, though without finding definite proof of cause and effect ( , ) . many viruses have been suggested to be implicated in the pathogenesis of ks, such as adenovirus, parvovirus b , rotavirus, h n influenza virus, epstein-barr virus, herpesvirus , coxsackie b virus, parainfluenza virus type , measles virus, dengue virus, human immunodeficiency virus, and varicella-zoster virus, but no significant differences emerged from case-controlled studies ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . one of the most difficult infections that are harder to distinguish from ks is caused by adenovirus, due to its frequent incidental detection in ks patients and frequent laboratory finding of increased inflammatory markers ( ) . in particular, adenovirus has been detected in . and % of cases with complete and incomplete ks, respectively, by jaggi et al. ( ) . there are reports that have emphasized the possible relationship of cytomegalovirus (in one child from turkey) and human bocavirus (in a french cohort of patients) with ks through serologic tests and molecular techniques, but this link might be only casual and misleading ( , ) . regarding the bacterial origin of ks, it is debated if the infectious agents might be conventional bacteria or bacteria with superantigen activity. superantigens (sags) are the most powerful t cell mitogens ever discovered, with potent immunostimulatory actions that simultaneously activate the major histocompatibility complex class ii molecules and t cell receptors, leading to massive activation of various immune cells. matsubara et al. conducted a case-control study with a serological approach based on enzyme-linked immunosorbent assay and measured serum antibodies against staphylococcal enterotoxins, including tsst- , and streptococcal pyrogenic exotoxin (spe), such as spe-a. they showed that ks patients had significant elevation of igm antibodies against one or more of five sags throughout the first to the fourth disease week ( ) . in a further paper the same authors analyzed the studies regarding the role of sags produced by staphylococcus aureus and streptococcus pyogenes in the pathogenesis of ks, finding numerous sags implicated, which brought the total number of the known staphylococcal sags to over and streptococcal sags to ( ) . the most recent work that analyzed the relationship between sags and ks examined different sag derived from streptococcus pyogenes in the stools of patients with ks. stool specimens were obtained from patients with ks during the acute phase and age-matched healthy children. the authors examined genes related to five sags-spe-a, spe-c, spe-g, spe-j, and tsst- using polymerase chain reaction; throat and stool cultures were assessed to evaluate the presence of streptococcus pyogenes and staphylococcus aureus in ks patients. they reported that at least one of the sag-related genes was detected more frequently in the stools of children with ks ( ). furthermore, among patients with ks, developed concurrent pneumonia and of these ( . %) had high titers of anti-mycoplasma pneumoniae antibody (> : ), suggesting a potential role of mycoplasma pneumoniae in ks and the importance of anti-mycoplasma treatment ( ) . another study has reported a possible role of fungal infections in the development of ks in japan, san diego, and the island of hawaii through an active role of tropospheric wind patterns leaving from central asia, in which toxins of candida species are aerosolized ( ) . in addition, due to the observation that candida albicans-derived substances, such as candida albicans watersoluble fraction (caws), induce a coronary arteritis in mice similar to that observed in ks, sato et al. evaluated the role of a free-β-glucan diet on caws-induced vasculitis and found that quality of diet might affect the progression of systemic vasculitis ( ) . caws should act as a pathogen-associated molecular pattern in mice and activate lectin pathway of complement by binding to mannose-binding lectin and inducing an acute inflammatory reaction in the vascular system ( , ) . there are three main reasons that led us to postulate a role of the microbiota in ks. the first is the unsatisfactory microbiological data that limited the association between infections and ks and a lack of evidence of any clear relationship between one or more pathogens with this illness. second, the most frequent bacteria or viruses associated with ks have a higher prevalence in the overall pediatric population, but only a limited number of children will develop the disease. third, the association of genetic predisposition and environmental factors in the pathogenetic process of ks makes ks itself similar to other multifactorial diseases. currently, the majority of data has found that the composition of the gut microbiota in ks patients differs from healthy subjects. lee et al. have postulated that the immune system should lose tolerance toward a part of the resident intestinal flora and that environmental factors, i.e., a western lifestyle or improved public hygiene systems, could transform the commensal flora into a pathogen one, as observed in different gastrointestinal disorders ( ) . a recent study about the resident flora and ks was focused on throat flora. horita et al. ( ) assumed that throat flora could be a reservoir of microorganisms triggering ks and, following this hypothesis, they investigated throat microorganisms of ks patients for their content and individual sag activity. in particular, they collected throat swabs at the start of ivig infusion in ks patients and compared them with non-ks controls (displaying other febrile illnesses). the results showed no difference in the throat flora between ks and febrile controls even in the mean mitogenic activity of bacteria isolated ( ) . the gastrointestinal tract could be one of the primary sites of entry of bacterial toxins in children with ks, and a perturbation in the intestinal microbiota has been linked to the disease's pathophysiology in another study by yamashiro et al. ( ) who investigated the microflora of the small intestine in japanese patients with ks. the range of bacterial species isolated from jejunal biopsies was characterized by a wider variety of gram-positive cocci in the acute phase of ks. notably, kinds of streptococci (streptococcus salivarius, streptococcus mitis, streptococcus oralis, streptococcus sangius, and gemella haemolysans) and kinds of staphylococci (staphylococcus capitis and staphylococcus hyicus) were isolated only from ks patients, suggesting that some antigens inducing a delayed-type hypersensitivity reaction in the mucosa might inundate the body by breaching the barrier of the small intestinal mucosa of ks patients ( ) . in fact, nagata et al. ( ) investigated cell surface phenotypes of mononuclear cells and enterocytes in the jejunal mucosa of japanese patients with ks and in patients with diarrhea due to cow's milk protein intolerance. both hla-dr+cd + and dr+cd + cells were significantly increased, and cd + cells significantly reduced in the lamina propria of ks patients during the acute phase compared with patients with cow's milk protein intolerance. these cell patterns returned to normal in the convalescent phase of ks. the authors concluded that a delayed-type hypersensitivity reaction was indeed present in the small intestinal mucosa of ks patients ( ) . due to the fact that the gastrointestinal tract is the largest interface between microbial factors and their host, containing the largest proportion of bacteria and the largest amount of lymphoid tissue in the body, it was hypothesized by eladawy et al. that the intestinal milieu could be altered in children with ks, and indeed ks patients have a higher incidence of gastrointestinal symptoms and complications ( ) . specifically, takeshita et al. ( ) evaluated patients with ks, patients with acute febrile diseases and healthy children, finding that the incidence of lactobacilli isolated from ks patients ( / , %) was significantly lower (p < . ) than in the other cohorts. moreover, no significant differences in the presence of staphylococcus, streptococcus, enterococcus, enterobacteriaceae, bifidobacterium, clostridium, veillonella, or bacteroides were found among the three groups. conversely, the presence of eubacterium and peptostreptococcus was significantly higher in ks patients than in patients with other febrile diseases (p < . and p < . , respectively), though no significant differences were observed between ks patients and healthy children ( ) . this observation confirmed that the majority of lactobacillus species are anti-inflammatory and beneficial for health, particularly during the first years of life, due to their action in protecting against colitis, reducing pro-inflammatory cytokines such as tnf-α, il- , or il- , and increasing the subsets of regulatory t cells ( ) . in fact, several studies have largely described the specific action of lactobacilli in maintaining the epithelial homeostasis of the gut and their striking anti-inflammatory potential ( , ) . nagata et al. ( ) also studied the role of the gut microbiota in ks pathogenesis via sags and heat shock proteins (hsps) produced by gut bacteria: the authors evaluated sags and hsps released by microorganisms isolated from the jejunal mucosa of children with ks compared with age-matched healthy controls, identifying strains of gram-negative microbes from patients with ks, which produced a large amount of hsp , having the power to induce an over-secretion of proinflammatory cytokines. they also identified strains of grampositive cocci with sag properties which induced the expansion of vβ t cells in vitro ( ) . this microbiological analysis disclosed different causative bacteria with a final common pathway of immune activation, which might contribute to the final development of ks. in order to evaluate the differential microbiota composition of ks patients, kinumaki et al. ( ) performed a metagenomic analysis using non-culture-based methods on feces. their study included ks patients ( males and females, aged - months, with a median age of months); the time of admission to the study was defined as the acute phase, while - months after the onset of ks was considered as the nonacute phase. the authors collected a total of samples− samples each for both acute and non-acute phases. it was demonstrated that the genera ruminococcus, roseburia, and faecalibacterium were mostly predominant during the nonacute phase, while a higher presence of streptococcus spp., including streptococcus pneumoniae, pseudopneumoniae, mitis, oralis, gordonii, and sanguinis, was detected in the fecal samples during the acute phase ( ) . this novel interpretation of a disease can be shared by other conditions, as liver cirrhosis and sjögren syndrome, in which major changes of gut microbiota with higher proportions of streptococcus spp. have been demonstrated ( , ) . these findings taken as a whole suggest that many other immune-mediated disorders are likely to be connected to an abnormal bacterial colonization of the intestinal tract, with a main role for streptococcus spp., and that changes in the gut microflora composition might promote systemic and extra-intestinal inflammation. therefore, all presented studies confirm the concept that an imbalance in the gut microbiota might directly or indirectly interfere with the normal functions of the immune system, and that the interaction with other environmental factors, mainly infectious agents, might lead to the final development of ks. table shows the characteristics of the microbiota in children with ks, as emerging by the most relevant studies dedicated to this issue. as no etiologic agent has ever been accused of being directly involved in the etiology of ks, basic research evaluating the pathogenic mechanisms of this disease will probably provide better therapies and probably consent to identify the most vulnerable hosts and protect them. a potential relationship between eubiosis and the protean functions of immunity has been contemplated by different studies, and conversely a relationship should exist between dysbiosis and immunity dysfunction shown by various diseases ( ) . it is strengthened that heterogeneity and abnormalities in the intestinal microflora composition may trigger or contribute to the development of specific diseases, and an increasing amount of research and microbiologic observations have led to a role of the intestinal microbiota for ks to be postulated. while we are becoming convinced that a role of the microbiota is likely, we do not know exactly the basic mechanism of how the microbiota should act. the principal obstacle of today's medical literature is to understand if the microbiota modification during ks can cause the disease or if it is a mark and a consequence of the disease itself, and if modulating the microbiota/microbiome might represent a future target of therapy in ks. this raises the hypothesis of targeting intestinal microflora in order to restore eubiosis through the rational use of antibiotics, xenobiotics, probiotics and nutrients. while multicenter trials and registries may allow us to improve the general outcome of ks, further in-depth analysis in larger cohorts of affected children will probably unravel the tangled pathogenesis of this mysterious disorder and find new targets for identifying disease-modifying agents or more specific therapies. se, ip, and dr contributed to all stages of the preparation of this manuscript, including conception and writing. all authors approved the final submitted version of the manuscript. viral infections associated with kawasaki disease epidemiology, clinical presentation, and outcomes of kawasaki disease among hospitalized children in an inner city hospital before and after publication of the american academy of pediatrics/american heart association guidelines for treatment of kawasaki 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heat shock proteins and superantigenic properties of bacteria from the gastrointestinal tract of patients with kawasaki disease characterization of the gut microbiota of kawasaki disease patients by metagenomic analysis altered mucosal microbiome diversity and disease severity in sjögren syndrome alterations of the human gut microbiome in liver cirrhosis eubiosis and dysbiosis: the two sides of the microbiota key: cord- -a qr nl authors: pires, sara m.; fischer-walker, christa l.; lanata, claudio f.; devleesschauwer, brecht; hall, aron j.; kirk, martyn d.; duarte, ana s. r.; black, robert e.; angulo, frederick j. title: aetiology-specific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: a qr nl background: diarrhoeal diseases are major contributors to the global burden of disease, particularly in children. however, comprehensive estimates of the incidence and mortality due to specific aetiologies of diarrhoeal diseases are not available. the objective of this study is to provide estimates of the global and regional incidence and mortality of diarrhoeal diseases caused by nine pathogens that are commonly transmitted through foods. methods and findings: we abstracted data from systematic reviews and, depending on the overall mortality rates of the country, applied either a national incidence estimate approach or a modified child health epidemiology reference group (cherg) approach to estimate the aetiology-specific incidence and mortality of diarrhoeal diseases, by age and region. the nine diarrhoeal diseases assessed caused an estimated . billion ( % uncertainty interval [ui] . – . billion) cases and , ( % ui , – , ) deaths worldwide in . the largest number of cases were caused by norovirus ( million; % ui – , million), enterotoxigenic escherichia coli (etec) ( million; % ui – million), shigella spp. ( million; % ui – million) and giardia lamblia ( million; % ui – ); the largest number of deaths were caused by norovirus ( , ; % ui , – , ), enteropathogenic e. coli ( , ; % ui , – , ), etec ( , ; % ui , – , ) and shigella ( , ; % ui , – , ). there were marked regional differences in incidence and mortality for these nine diseases. nearly % of cases and % of deaths caused by these nine diarrhoeal diseases occurred in children under five years of age. conclusions: diarrhoeal diseases caused by these nine pathogens are responsible for a large disease burden, particularly in children. these aetiology-specific burden estimates can inform efforts to reduce diarrhoeal diseases caused by these nine pathogens commonly transmitted through foods. diarrhoeal diseases are a major cause of disease burden worldwide [ ] . the global burden of disease study (gbd ) ranked diarrhoeal diseases as the fourth largest disease burden, accounting for . % of the total disease burden globally. diarrhoeal diseases accounted for an even higher proportion ( %) of the total disease burden in children < years of age [ ] . diarrhoeal diseases have a substantially higher impact in low-income countries and regions with poor water quality, sanitation and food safety. diarrhoeal diseases are caused by a variety of bacteria, viruses, and parasites, many of which are commonly transmitted through food [ ] . despite the large disease burden caused by these pathogens, the global contribution of specific aetiological agents of diarrhoeal diseases is largely unknown. for example, recent studies have estimated the worldwide incidence of diarrhoeal diseases in children < years of age [ ] , and in older children and adults [ ] , but did not provide aetiology-specific estimates. lanata et al. [ ] and fisher walker et al. [ ] provided aetiology-specific estimates, but only for diarrhoeal deaths among children < years of age, and diarrhoeal cases in persons years of age, respectively. another large-scale study estimated diarrhoeal aetiologies in children < years of age in specific study sites, particularly in sub-saharan africa and south asia [ ] . other studies have estimated the incidence of specific foodborne diseases, many of which cause diarrhoea, but each focused on a single developed country [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to identify and prioritize targeted interventions to reduce the public health impact of foodborne diseases, public health policy makers and other stakeholders need aetiology-specific regional and global estimates of the incidence and mortality of diarrhoeal diseases caused by pathogens that are commonly transmitted through foods. these estimates, combined with knowledge on the proportion of this burden that is derived from foods, will form the basis for the estimation of the global and regional burden of foodborne diseases. as part of the effort by the world health organization (who) foodborne disease burden epidemiology reference group (ferg; http://www.who.int/foodsafety/areas_work/ foodborne-diseases/ferg/en/) to estimate the disease burden of foodborne diseases, we estimated the global and regional incidence and mortality of diarrhoeal diseases which are commonly transmitted through foods. after reviewing the epidemiology of diseases caused by bacteria, viruses, and protozoa that are commonly transmitted to humans through food, and which are included in ferg, we selected nine pathogens which commonly cause diarrhoea for inclusion in our study: campylobacter global spp., cryptospordium spp., entamoeba histolytica, enterotoxigenic escherichia coli (etec), enteropathogenic e. coli (epec), giardia lamblia, norovirus, salmonella spp., and shigella spp. we did not include shiga-toxin producing e. coli (stec), and vibrio cholerae, diarrhoeal pathogens included in ferg, because the incidence and mortality of diseases caused by these agents have been estimated using different approaches and published elsewhere [ , ] . although some countries have published national incidence and mortality estimates for diseases caused by most of these nine pathogens, such estimates of foodborne diseases, which are considered to be the highest quality burden estimates for these diseases [ ] , are only available from a limited number of countries. we therefore used two approaches to estimate the incidence and mortality of the diseases caused by the nine selected diarrhoeal pathogens, and applied one or the other approach in each country in the six regions. approach was applied to all countries in the european region (euro) and all other countries with overall low mortality as defined by who (http://www.who.int/choice/demography/mortality_strata/en/); approach was applied to all remaining countries. using these two approaches allowed us to utilize the highest quality available data in countries with overall low mortality which have similar sanitary and public health infrastructure and presumed incidence of foodborne diseases. the final results of the two approaches were merged in a last step to determine global estimates and estimates for the regions. the first approach utilized available national incidence and mortality estimates of diseases caused by the nine selected pathogens and was applied to countries in euro (sub-regions a, b, and c), and low-mortality countries in american region (amro; sub-region a) and western pacific region (wpro; sub-region a). we conducted a literature review and consulted with the international collaboration of foodborne disease burden of illness studies (http://www.cdc.gov/ncezid/dfwed/international/enteric-burden-collaboration.html) to identify all published national estimates (with associated uncertainty intervals) of foodborne diseases. such estimates were derived through studies that corrected national surveillance data to account for under-diagnosis and underreporting [ ] . in cases where national estimates were expressed as number of cases and deaths, and not as incidence and mortality rates (per , population), we used united nations population figures for the national study year to create such rates. if a national estimate only included domestically-acquired infections, we used the proportion of infections acquired during international travel in a neighbouring country to derive revised estimates that included infections acquired abroad. we applied the national incidence and mortality estimates, with the associated uncertainty intervals, to each country with such national estimates, and the median incidence and mortality of all studies, with uncertainty intervals associated with those median estimates, to each country in euro, amro sub-region a, and wpro sub-region a that did not have national incidence or mortality estimates. this approach resulted in incidence and mortality estimates (per , population), with uncertainty intervals, for each of the countries for each of the nine diseases. overall estimates were partitioned to the age groups < years of age and years of age on the basis of the age distribution of the population in each region. further details of this approach are available in s appendix. regional incidence and mortality of diarrhoea. first, we identified the total number of diarrhoeal cases in the countries for by combining estimates based on systematic reviews for children < years of age and persons years of age [ , ] . for the estimate of the total number of diarrhoeal deaths, we obtained data on the total number of deaths in the countries in attributed to diarrhoeal diseases from who (http://www.who.int/gho/en/; accessed june ); we used the range of diarrhoeal deaths estimated in the global burden of disease study (gbd ) to derive an uncertainty interval for the death envelope. we derived the final number of diarrhoeal cases ("diarrhoeal envelope") and diarrhoeal deaths ("diarrhoeal death envelope") by subtracting published estimates of the number of diarrhoeal cases and deaths caused by stec [ ] and v. cholerae [ ] . aetiology proportions. our next step was to estimate the proportion of diarrhoeal illnesses and deaths due to the nine pathogens by extracting the aetiological proportions of diarrhoeal cases and deaths for each pathogen by region from systematic reviews of studies reporting stool sample isolation and detection from inpatient, outpatient, and communitybased studies of persons with diarrhoea. we assumed that the distribution of pathogens observed among outpatient and community studies represented the pathogen prevalence among diarrhoeal cases, and that the distribution of pathogens among inpatients hospitalized for severe diarrhoea represented the pathogen prevalence among diarrhoeal deaths for all age groups. due to data scarcity, we also included inpatient studies to estimate the aetiological proportions of diarrhoeal cases in persons years of age (i.e. all available studies). we assumed that g. lamblia infection was unlikely to result in death based upon the data available from the national foodborne disease mortality estimates, and therefore excluded it from mortality estimates. to determine aetiological proportions, we used systematic reviews to identify studies that reported isolation or detection of pathogens from stool specimens or rectal swabs collected from persons with diarrhoea. for norovirus, we used a previously published systematic review including studies published between and and then updated through [ ] . the norovirus-specific review was conducted because of the increased recent use of molecular diagnostics globally and as part of a parallel effort to estimate the global burden of norovirus disease [ ] . for all other pathogens we included two previously published systematic reviews: ) the aetiology of diarrhoeal disease studies for children years of age published between and [ ] and ) the aetiology among children < years of age published between and [ ] . we updated each of these reviews to include studies published through . s appendix details the systematic review methodologies and results of the updated reviews, including search terms and inclusion/exclusion criteria. studies focusing only on norovirus collected in the general reviews were excluded to avoid duplicates with the norovirus-specific review. the general systematic reviews collected data on isolation or detection of the nine pathogens included in our study and on pathogens not commonly transmitted by food (e.g. rotavirus, sapovirus, astrovirus, and coronavirus); the latter were grouped together as "other pathogens". we initially calculated study-specific aetiology-proportions by dividing the number of samples positive for the pathogen by the total number of samples tested in that study (studyproportions); we then estimated regional proportions by calculating the median aetiology-proportion of all study-estimates within each region (regional-proportions). for example, if studies conducted in the western pacific region (wpro) provided data on the number of salmonella isolates among diarrhoeal cases, this region's salmonella-proportion was estimated as the median of the estimated study-specific aetiology proportions. since several of the studies among children < years of age used narrower age ranges, in analyses a and b we calculated an age-adjusted proportion for this age group by calculating a conversion factor as the ratio of the median proportion in the age group - months to the median proportion in age group x ((median (prev - )/median (prevx)). we applied this approach when three or more studies for each pathogen contributed to each of the two medians. if this condition was not met, we borrowed the conversion factor for the age group from a similar age group within the same pathogen (for example, used the conversion factor calculated for studies including infants - months of age for studies that included infants - months of age). for persons years of age, we have assumed that differences between narrower age categories would be diluted in this very broad population group and have chosen not to age-adjust medians. to estimate the proportion of diarrhoeal stools due to unknown aetiology, we included studies that sought pathogens and reported patients with an unknown cause of disease. if only one study tested for a given pathogen in one region, we applied criteria to identify outliers and prevent potentially non-representative studies from misrepresenting the final regional aetiology-proportions. an outlier was defined as a regional estimate ± times the global median. if a regional estimate derived from studies was identified as an outlier, the individual studies' prevalence and sample size were evaluated, and a particular study was excluded if it represented an outlier when compared to remaining studies within the region. outliers were excluded from the results and taken as a missing value. if a regional median aetiological proportion estimate was missing (due to either missing data or exclusion of an outlier), the missing regional estimate was replaced by the global median of the aetiological proportions for that pathogen. all global medians were estimated after the exclusion of potential outliers. statistical analysis. to account for uncertainty in these calculations, we used monte carlo simulation in all steps of the analysis. we applied a bootstrapping analysis to derive % uncertainty intervals (ui) around aetiology-proportions. 'pseudo-data sets' were created by sampling each study with replacement from the real dataset. in each of these , pseudo-datasets, a different random number of positive samples for each study was generated from a binomial distribution defined on the basis of the number of samples and the expected proportion in that study. all pseudo-datasets were used in the estimation procedure described above to generate corresponding , study-specific aetiology proportions, and regional aetiology proportions. the . th and . th percentile of these proportions gave the % ui. data management and analyses were conducted in sas enterprise guide . . we then constrained the aetiological proportions for the nine diarrhoeal pathogens, for other pathogens not commonly transmitted by food, and unknown aetiology to ensure that they did not sum to more than % in each region. for this purpose, we first fitted univariate beta(α, β) distributions to the median proportions and simulated quantiles (i.e., the . th and . th percentiles). the distributions' optimized parameters were estimated via one dimensional optimization, minimizing the squared distance between the estimated and fitted quantiles, and ensuring the median of the fitted distribution to be similar to the estimated median. next, , random deviates were sampled from the fitted beta distributions. if needed, an "unknown aetiology" category was created as minus the sum of simulated proportions across aetiologies, per iteration. finally, the random deviates were normalized iteration-wise by dividing them by the sum of simulated aetiological fractions. once estimates of aetiological proportions for cases and deaths for the nine pathogens were derived, the regional aetiological proportions for each disease were multiplied by the respective estimates for total diarrhoeal illnesses and deaths in those regions, accounting for uncertainty using a stochastic model with , iterations. we then derived age-stratified incidence and mortality estimates, with associated uncertainty, for each disease for each region using country-level population data for from the revision of the united nations world population prospects (esa.un.org/wpp, accessed june , ); among the countries, all countries in the same region were assumed to have the same incidence and mortality, and associated uncertainty, for each pathogen. in order to aggregate results with estimates derived for low-mortality countries (approach ), age-specific estimates were then grouped into overall estimates. the stochastic model was implemented in r version . . (r core team, ). global estimates (combining approach and approach ). the incidence and mortality estimates, with associated uncertainty, for each disease for each region obtained through approach and approach were combined in a final step to produce final global estimates and estimates for each of the six regions. we identified national incidence and mortality estimates of the nine diarrhoeal diseases for seven countries: australia, canada, france, netherlands, new zealand, united states of america, and the united kingdom [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the national estimates from australia and canada included only domestically-acquired infections; revised national estimates were derived using data on international travel-acquired infections from the new zealand and the united states, respectively. estimates of incidence and mortality for each of countries are available in s appendix. the systematic reviews conducted to collect data to estimate the proportion of diarrhoeal illnesses and deaths in the other countries yielded outpatient and community studies (fig ) and inpatient studies (fig ) among children < years of age, and inpatient, outpatient, and community studies (fig ) among persons years of age. table lists the number of studies for each of the nine pathogens by region. the final number of studies providing data for individual pathogens in different regions varied substantially. the data available for persons years of age were scarcer and the number of studies for each region was low, particularly for the afro and amro regions. table a in s file presents the estimates of the regional envelopes of diarrhoeal cases and deaths, and tables b to b in s file present the contribution of each pathogen for these envelopes. we estimated that the nine pathogens included in our study resulted in a total of . billion cases ( % ui . - . ) and . million deaths ( % ui . - . ) in worldwide (table ). in the countries in euro and amro and wpro a regions, we estimated that these nine pathogens resulted in . million cases ( % ui . - , . ) and , deaths ( % ui , - , . in the remaining countries, we estimated these pathogens resulted in a total of . billion cases ( % ui . - . billion) and , , deaths in ( % ui , , - , , ). global and regional estimates of incidence and mortality rates caused by these pathogens are presented in s there were marked regional differences in incidence and mortality caused by the nine diseases (table ; figs and ). among the nine pathogens, norovirus was the most common cause of cases in all regions, particularly among persons years of age (fig ) . among these nine pathogens, norovirus and g. lamblia were the cause of the highest incidence in children < years of age; in afro, amro, emro and wpro campylobacter caused the third highest incidence in afro and wpro, and cryptosporidium spp. made a substantial contribution in emro. the second highest incidence of cases among persons years of age was caused by salmonella spp. in emro, searo and wpro. for deaths caused by the nine pathogens, epec and norovirus were the most frequent causes in children < years of age in afro, amro, searo and wpro (fig ) . etec was also estimated to be an important cause of mortality, particularly in afro and amro, and shigella spp. was the leading cause in emro and third leading in afro. salmonella spp. caused the largest proportion of diarrhoeal deaths in this age group in euro. in persons years of age, norovirus was the leading cause of mortality in all regions. these are the first global and regional estimates of incidence and mortality caused by nine pathogens that cause diarrhoea and are commonly transmitted through foods. of these agents, we showed that norovirus was responsible for the largest number of cases followed by etec, g. lamblia and shigella spp. norovirus caused the most deaths among these nine pathogens, followed by epec and shigella spp. nearly % of all cases and % of deaths caused by these nine pathogens occurred in children < years of age, who represent only % of the global population. we also demonstrated regional differences in both the incidence and mortality of diseases caused by these nine pathogens. these differences are important to consider when defining interventions to reduce the burden of diseases commonly transmitted through foods. as an example, campylobacter and salmonella, which are pathogens that have mainly domestic food-producing animals as reservoirs and usually infect humans due to contamination in the food production chain, play a major role in the incidence of foodborne enteric disease in the euro region but are less frequently observed in who-defined high mortality countries. in high mortality countries where poor sanitary conditions and food and water contamination are more important, pathogens such as g. lamblia and etec play more important roles. the predominance and relative ubiquity of norovirus across all regions suggests that targeted interventions, such as vaccines, may be necessary to reduce the burden of this human reservoir pathogen. our study focused only on diarrhoeal pathogens that are commonly transmitted through foods. it is important to recognize that the estimates we present include disease from all modes of transmission, including contaminated food, water, and environments, along with infected persons and animals. our inclusion of only pathogens that are commonly transmitted through food also meant that we did not present estimates for important diarrhoeal agents, such as rotavirus. by including them into other pathogens, we have assured that we did not overestimate the incidence and mortality of remaining pathogens when distributing the diarrhoeal total cases and deaths to the nine diseases of interest. we used two approaches to estimate the relative contribution of different aetiologies for disease because the quality and availability of data varied substantially between countries and regions. national incidence and mortality estimates were available only from seven low mortality countries, but given the high quality of these estimates, we gave priority to these data by using them as the basis for estimating the incidence and mortality of the diseases caused by the nine pathogens for all euro and other low mortality countries (sub-region a in amro and wpro). for all other countries, we adapted the cherg approach for estimating the burden of diarrhoeal diseases in these countries [ ] . this approach was facilitated by the availability of estimates of the envelope of diarrhoeal deaths, along with recent advances in diarrhoeal disease diagnosis, such as widespread application of polymerase chain reaction (pcr) for norovirus detection. we have divided countries for the two approaches on the basis of their overall mortality status, as defined by who, which we assume can be used as a proxy for differences in the public health status of the countries. the two approaches differed in terms of methodology, assumptions and type of data used. while approach analysed national incidence and mortality of disease by pathogens commonly transmitted through foods estimated primarily by correcting surveillance data to account for underreporting and under-diagnosis, approach relied on systematic reviews of studies identifying causative agents in patients with diarrhoea. applying approach for all regions was not possible due to the scarcity of community, inpatient and outpatient studies from low mortality countries for most pathogens. because of these differences, we acknowledge that comparisons between regions should be made with care. we relied on studies that estimated the incidence of potentially foodborne diseases in a specific country by correcting public health surveillance data for underreporting and under-diagnosis for approach . such studies have their own limitations, such as relying on a variety of data with variable quality and representativeness, multiple modelling approaches and a wide range of assumptions. as an example, most use population surveys on care-seeking behaviours of patients with diarrhoea, which are subject to recall bias that can influence the results [ ] . furthermore, in approach we have assumed that the median aetiology-proportion of the seven collected studies (along with its uncertainty interval) represented the relative *not all studies provide data for pathogens included in our analysis. remaining studies were used to estimate the aetiology-proportion of "others". †studies conducted in countries in the euro region, amro sub-region a, or wpro sub-region a were not included in the analyses. doi: . /journal.pone. .t contribution of each agent for the diarrhoeal morbidity and mortality estimates, and have taken a simplified approach to estimate uncertainty. approach to estimate the contribution of aetiologies for diarrhoeal cases and deaths used studies that tested for varying numbers of pathogens. studies that focused on a single pathogen may tend to overestimate the importance of that pathogen because they are potentially more likely to be conducted in a study site with a high prevalence of that pathogen [ ] or have selective inclusion criteria e.g. acute watery diarrhoea. however, we needed to include them due to estimates exclude the proportion of the incidence envelope attributable to "other pathogens", because these were not considered in approach (applied to countries within euro, amro sub-region a, and wpro sub-region a). doi: . /journal.pone. .g the low number of studies focusing on multiple pathogens, particularly in the older age group. due to scarcity of data, some regional aetiological proportion estimates were informed by a single study. to avoid that studies with potentially over or underestimated aetiology-proportion estimates misrepresented the estimate for that region, we have defined criteria to identify outliers, and have excluded these estimates from the results. these overestimations or underestimations can have various causes, such as study locations with a particularly high or low prevalence of that pathogen, a small sample size, or a country not representative of the whole region. excluded outliers were treated as missing values, and the aetiology-global median used to represent the regional estimate. in this approach, we also assumed that the distribution of pathogens among inpatients hospitalised with severe diarrhoea represented the relative importance of these pathogens for diarrhoeal deaths. this meant that we used severity of disease as a proxy for mortality, which may lead to an over-or under-estimation of the number of deaths caused by some pathogens. in the analyses for persons over five years of age, due to sparseness of data we decided to use data from inpatient studies to estimate aetiology-proportion of cases, which may also have led to an erroneous estimation of the contribution of pathogens to diarrhoeal incidence. additionally, this approach relied on stool samples, but some patients that tested positive for a pathogen may have had asymptomatic infections. this is more likely to happen for pathogens that have long excretion periods after illness (e.g. norovirus). carefully conducted longitudinal studies would be needed to distinguish clinical cases from asymptomatic infections [ ] . in approach , we estimated the proportion of the diarrhoeal cases and deaths that was caused by unknown aetiology. the defined strategy was to base our estimates on studies that had collected data for eight or more pathogens and had reported "aetiology unknown". undiagnosed cases could be caused by truly unrecognized agents, but also could be due to e.g. the use of incorrect or insensitive testing methods, to antimicrobial-therapy prior to stool sampling, or to non-infectious diarrhoeal causes. this strategy could only be adopted for the analyses for children < years of age, and still there were few studies available for the calculations. in remaining analyses, unknown was calculated as the difference between % and the sum of all remaining aetiologies (including others). in both approaches, we assumed that studies conducted in a given country would be representative of the region or sub-region of this country. when several studies providing data for a given pathogen were available for that region, calculating a median estimate ensured that the influence of extreme values was restricted. on the contrary, when only one or few studies were available, this study's/studies' estimate was assumed to represent all countries within a region. in addition, the six regions' classification was based on the geographic distribution of countries, and countries within each are very diverse. we tried to avoid inappropriate extrapolations by applying distinct approaches to low mortality and medium and high mortality countries, assuming that a country's mortality status may also reflect the nation's public health and food safety situation. in the euro region, we made an extra extrapolation and assumed that all countries were similar to sub-region a countries in terms of foodborne diseases' incidence and mortality. this region includes countries with other mortality status (euro b and euro c), and some of these countries may have a different distribution of aetiologies. due to lack of data, we were not able to explore the extent of these differences. a recent large prospective matched case-control study in children < years of age conducted over three years at selected sites in africa and asia (the gems study) estimated the burden of aetiology-specific diarrhoea in these sites, and concluded that rotavirus, cryptosporidium spp., etec and shigella spp. were the most important causes [ ] . this study was fundamentally different from ours: among other differences, it calculated attributable cases of diarrhoea to each aetiology, comparing patients (moderate to severe diarrhoeal cases seen in health facilities) and controls (un-matched healthy children in a community sample). the gems study excluded mild diarrhoeal cases, which along with their moderate diarrhoeal cases would constitute our non-fatal incident cases. by including moderate and severe cases instead of only severe hospitalized cases, the gems study does not have a good proxy for fatal cases as we defined it. still, the study's findings are generally consistent with ours; epec, etec, shigella spp. and cryptosporidium spp. were among the most important causes of diarrhoea. one important difference was on the relative contribution of norovirus for diarrhoea incidence and mortality: our results suggest that norovirus is amongst the most important causes of diarrhoeal cases and deaths, whereas the gems study estimated a lower contribution of this pathogen. this discrepancy may indicate that our approach may lead to an overestimation of the proportion of diarrhoea attributable to norovirus or other pathogens that are likely to be carried asymptomatically after the first exposure (like certain types of etec, e.g. lt-etec) because it does not account for the potential detection of norovirus in the stool of healthy individuals [ ] . however, the gems study required that control individuals did not have a diarrhoeal episode in the days prior to sampling; because norovirus patients can excrete the virus for to weeks after infection, it is likely that some controls included in this study corresponded to asymptomatic carriers of norovirus [ ] . these findings may suggest limitations in ascribing pathogenicity based on case-control studies, particularly in developing country settings in which frequent exposures and a high degree of transmission results in common reinfections [ ] . a recent community based prospective cohort study (the "etiology, risk factors, and interactions of enteric infections and malnutrition and the consequences for child health and development project", the mal-ed study), supported our results and estimated that norovirus was the most important cause of diarrhoea in young children [ ] . our study provides information on the incidence and mortality of nine pathogens that cause diarrhoeal and are commonly transmitted through food. most of these agents can also infect humans through other sources (e.g. environmental, contact with animals and person-toperson transmission), and the relative contribution of these sources for disease varies between aetiologies and regions [ ] . to estimate the number of foodborne aetiology-specific diarrhoeal cases and deaths, our results need to be combined with regional source attribution-proportions and other aetiological agents [ ] . these results, as well as the foodborne disease burden of each disease in terms of disability adjusted life years (dalys) are presented elsewhere [ ] . our results provide public health policy makers, including risk managers, and other stakeholders with information for advocacy for improved regulation and control of diseases commonly transmitted through foods. we highlight the most important diarrhoeal diseases in different regions and age groups, which will allow policy makers to define and improve control strategies targeted at different pathogens, settings and countries. supporting information s appendix. estimating the aetiology-specific incidence and mortality of diarrhoea in countries in the region of the americas (amro) sub-region a, western pacific region (wpro) sub-region a, and european region (euro; sub-regions a, b and c). (docx) s appendix. detailed description of the methods of the systematic reviews used to identify studies that provided data to derive aetiology-proportion estimates for all included pathogens except norovirus. (docx) table b : regional and global etiology-proportions of diarrhea cases in children < , (median % and % ci). table b : regional and global etiology-proportions of diarrhea cases in persons > , (median % and % ci). table b : regional and global etiology-proportions of diarrhea deaths in children < , (median % and % ci). table b : regional and global etiology-proportions of diarrhea deaths in persons > total global incidence and mortality rate (per , ) of foodborne diarrhoeal pathogens (median and % uncertainty intervals) disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study evolving public health approaches to the global challenge of foodborne infections diarrhoea morbidity and mortality in older children, adolescents, and adults. epidemiology and infection global burden of childhood pneumonia and diarrhoea global causes of diarrhoeal disease mortality in children < years of age: a systematic review etiology of diarrhea among older children, adolescents, and adults: a systematic review burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study foodborne illness acquired in the united states-unspecified agents disease burden of foodborne pathogens in the netherlands longitudinal study of infectious intestinal disease in the uk (iid study): incidence in the community and presenting to general practice estimates of the burden of foodborne illness in canada for specified pathogens and unspecified agents, circa foodborne illness, australia, circa and circa foodborne infections in france risk ranking for foodborne microbial hazards in new zealand: burden of disease estimates global incidence of human shiga toxin-producing escherichia coli infections and deaths: a systematic review and knowledge synthesis. foodborne pathogens and disease the global burden of cholera. bull world health organ the global prevalence of norovirus among cases of gastroenteritis estimating the burden of acute gastroenteritis, foodborne disease, and pathogens commonly transmitted by food: an international review epidemiologic implications of asymptomatic reinfection: a mathematical modeling study of norovirus pathogen-specific c burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed) assessing the applicability of currently available methods for attributing foodborne disease to sources, including food and food commodities who foodborne disease burden epidemiology reference group's (ferg) expert elicitation for estimating the relative contribution of food to the global burden of diseases commonly but not only acquired through the consumption of food who foodborne disease burden epidemiology reference group (ferg) estimates of the global and regional disease burden of foodborne bacterial, protozoal and viral diseases the world health organization funded this study under the foodborne disease burden epidemiology reference group through contributions from member states and international agencies. we would also like to acknowledge the support from the bill and melinda gates analyzed the data: smp. wrote the paper: smp clfw cfl bd ajh asrd reb mdk fja. key: cord- - dn at k authors: portillo, aránzazu; ruiz-arrondo, ignacio; oteo, josé a. title: arthropods as vectors of transmissible diseases in spain() date: - - journal: med clin (engl ed) doi: . /j.medcle. . . sha: doc_id: cord_uid: dn at k different aspects related to globalization together with the great capacity of the arthropod vectors to adapt to a changing world favour the emergence and reemergence of numerous infectious diseases transmitted by them. diptera (mosquitoes and sandflies), ticks, fleas and lice, among others, cause a wide spectrum of diseases with relevance in public health. herein, arthropod-borne disease are reviewed, with special emphasis on the existing risk to contract them in spain according to different parameters, such as the presence of arthropod and the circulation or the possible circulation of the causative agents. infectious agents. most avs belong to insecta and arachnida classes (table ) . to review the broad subject of arthropod vector-borne diseases (avbd) in spain is a complex task. just listing the arthropods that transmit diseases in our environment, such as dipterans (culicids and phlebotominae sand flies), fleas, lice, bed bugs and ticks, among others, and the diseases they transmit, or that they can transmit at any given time, would be a reason for one or several treatises. in any case, before discussing the subject, it should be remembered that the infections that they transmit are usually included in the socalled zoonoses. in this regard, the world organization for animal health estimates that at least % of infections that affect humans have a zoonotic origin and, according to the pandemic emerging threats program of the american agency for international development, almost % of current threats also have this origin. one of the many known routes for the acquisition of zoonoses, apart from direct contact with animals or their products, faecal-oral or respiratory routes, bites or scratches, the consumption of undercooked - /© elsevier españa, s.l.u. all rights reserved. products or the intake of milk, is av transmission. it is difficult to limit this issue to our country, since the setting is global and very dynamic, and diseases do not understand political borders. only a few months ago new threats appeared, such as the re-emergence of yellow fever in brazil or the epidemic of plague in madagascar. however, in this review we will put the focus on avbd with greater risk to humans in our environment, without losing the "one health" perspective. avbd are subject to complex interactions (demographic, social and cultural changes, climate change, wars and famine or evolution of microorganisms) among which, undoubtedly, global transport systems and the consequent invasion of exotic species stand out. generally speaking, travel, migration and globalization contribute to the emergence of infectious diseases. its importance has been debated for many years and, possibly, dates back to ancient times. humans carry their usual microbiota, pathogens, ectoparasites and other possible vectors, the immunological history of past infections and vaccines, the genetic load (greater or lesser susceptibility), cultural preferences, behaviours, habits and customs, as well as luggage (pets, goods and others). in the case of avbd, the equation for the appearance of a certain disease would be the following: the presence of competent vector arthropods plus susceptible population, together with the presence of reservoirs and/or intermediate hosts (sick people), could give result an epidemic. the introduction of the tiger mosquito (aedes albopictus) and the threat of its expansion could be a good example. it is thought that a. albopictus was introduced in europe in through albania, by the trade of used tyres, although the first publication on its introduction in the european continent dates back to in italy. since then, a. albopictus has expanded throughout the mediterranean area. in spain, this aggressive mosquito is well established in catalonia, levante, in the coastal area of murcia and andalusia. it has also been detected in guipúzcoa, and little by little it is introduced in other non-coastal areas such as aragon, extending through travel routes (for example, motorways). the last detection was carried out in the community of madrid (fig. ) . a few months ago, the first specimens of this species were also identified in the north of portugal. this mosquito has been incriminated as a vector of numerous arboviruses in different parts of the world, including europe. a. albopictus, is the chikungunya virus vector. in this regard, most of the cases in europe are imported, although in the last two decades there have been different epidemic outbreaks in italy and, recently, in france. in spain, only imported cases have been reported. this mosquito (next to aedes aegypti which, as will be detailed later, has been detected in a timely manner in europe) can act as a vector of the dengue virus and with less effectiveness of the zika virus. in france, a. albopictus was responsible for the first autochthonous cases of dengue and of those reported in the same country during the following years, one of them with a history of travel to madeira, where a large outbreak had been reported (more than a thousand cases) between and . in , the occurrence of autochthonous cases of dengue in croatia were also attributed to a. albopictus, establishing the transmission by this mosquito in europe. in spain we are only suffering from their annoying bites, for now. table shows arbovirus infection and table shows other infections transmitted by dipterans worldwide, with the risk of transmission in spain. we have evaluated the risk of emergence and/or re-emergence of these diseases in spain according to the existing studies for each pathogen, the experience according to other diseases, previous immunity and other criteria, some of them subjective, that do not appear in the text. a. aegypti is the main zika vector and also a transmitter of yellow fever, dengue and chikungunya. in principle, this species is not a problem because it is not settled in europe, although it has been detected in some areas, such as in an airport in the netherlands and, more recently, in fuerteventura (canary islands) (fig. ) . in view of the zika epidemic in the americas, the ministry of health, social services and equality, in collaboration with the carlos iii health institute and the autonomous regions, has established a zika virus disease surveillance in spain. to date (last update in july ), confirmed cases have been reported, all imported, except congenital cases whose mothers were infected in the risk zone and autochthonous cases of sexual transmission. it should be noted that it is not necessary to resort to invasive or exotic species to refer to avbd. thus, anopheles atroparvus is present in spain, a good malaria vector. the official map of the distribution of this mosquito is not updated; however, although the risk of a malarial outbreak is low in our country, recent events have involved this species in the two cases of autochthonous malaria registered in the north of the peninsula. spain was an endemic country of malaria until , when who declared it a the risk of emergence/re-emergence is calculated based on three factors: (a) presence of cases of the disease in humans in the last years in europe, mediterranean, central and south american countries with a significant relationship with spain; (b) presence of the vector in spain; (c) pathogenicity of the virus for humans. each factor is scored with one point (presence in the first two factors and low pathogenicity in the third factor) or with zero points (absence in the first two factors and high pathogenicity in the third factor). the first two factors are added and the third is subtracted. the total score can range between and , with being: low risk; : moderate risk; : high risk. free zone. in europe, after the eradication of malaria, most cases are imported, although sporadic cases have recently been reported in many mediterranean countries such as france, italy, greece. the emergence of autochthonous cases in greece between and has raised doubts about the real situation in europe as a malaria-free zone. other diptera present in spain which we cannot ignore are phlebotominae sand flies. the species phlebotomus perniciosus and phlebotomus ariasi, are the competent vectors of the leishmaniasis agent (leishmania infantum). there are also other potential vectors of l. infantum in spain, as they are phlebotomus papatasi and phlebotomus sergenti. leishmaniasis is endemic throughout the mediterranean basin in europe and its geographic scope is spreading. usually, the epidemiology of leishmaniasis was linked to the rural habitat with the presence of dogs. the great epidemic outbreak of fuenlabrada with a high number of cases in an urban area, in which leishmaniasis was not common, showed the dangers of changing the urban model in spain. many houses were built in rural agricultural areas with gardens and peri-urban green spaces where wildlife was present. in these places the concentration of phlebotominae sand flies was high, and, in their environment, there were not only dogs but also other l. infantum reservoirs, such as hares and rabbits. this fact together with mild temperatures in recent years, decreasing the mortality of the vector, seems to be what caused the great outbreak. [ ] [ ] [ ] undoubtedly, the presence of phlebotominae sand flies throughout the iberian peninsula is a great threat, since not only do they transmit leishmania, but they are also toscana virus vectors, which is causing numerous cases of meningoencephalitis in some areas of spain and other phleboviruses such as the granada virus (without proven pathogenic power), the naples virus or the sicily virus. another species of mosquito to which special attention is to be paid is culex pipiens. this species, which is distributed and well represented throughout the iberian peninsula, is capable of transmitting the west nile virus (wnv). west nile fever is becoming a serious problem in some areas of europe, as in greece, where there has been an outbreak with numerous neuroinvasive forms, and in cases have been reported in france, italy, romania, hungary, croatia, serbia and austria. in spain, according to the data from the situation report and wnv risk assessment, published in , there are several species of mosquitoes capable of virus transmission. for example, culex modestus, culex perexiguus and culex theileri show a high vector competence, while c. pipiens and a. albopictus have a moderate vector competence. the first human case of neuroinvasive disease due to wnv in our country was identified, retrospectively, in a patient diagnosed with meningitis in september who, in the days before the onset of symptoms, visited a village in badajoz, extremadura (spain). in , the ministry of the environment reported the detection of wnv in equine farms in the provinces of cádiz, sevilla and málaga. through this active surveillance, suspected cases were investigated and two human cases of wnv meningoencephalitis were confirmed. between and , virus activity was detected in equines, suggesting that the virus is endemic in our country. in addition, there was previous evidence about its circulation in birds that were considered for the development of a predictive virus circulation model in our country. another factor that clearly influences infections transmitted by arthropod vectors is climate change. in the international conference on climate change and health was held in atlanta, which revolved around the idea that: "health is the human face of climate change". following this meeting, a special article was published in the new england journal of medicine which stated that the distribution of infectious diseases such as lyme borreliosis, rickettsiosis or west nile fever are expanding at the same rate as their avs. we know that climate variations and extreme weather events have a profound impact on avbd. mosquitoes and ticks are devoid of temperature regulation mechanisms and, for this reason, fluctuations in temperature greatly affect their reproduction and survival. in our country, it is more than possible that the great increase in the number of ticks in recent years is due to the fact that winters, in general, are much milder than years ago. just to mention an example, ixodes ricinus, the tick that most frequently bites people in the north of spain, is very sensitive to climate warming. this, among other factors, is increasing its survival. this species of tick transmits very prevalent diseases in europe, such as lyme disease or tickborne encephalitis, or others such as rickettsia monacensis infection, human anaplasmosis, and babesiosis. , in spain, human cases of all of them have been described, except for tick-borne encephalitis. although there is a high suspicion of the circulation of the virus in spain, the molecular screening of hundreds of ticks has been carried out in our laboratory, with negative results. in addition, other pathogens have been detected in i. ricinus specimens collected in spain, such as rickettsia helvetica, candidatus neoehrlichia mikurensis or borrelia miyamotoi, which leads us to be alert to the possible occurrence of human cases. [ ] [ ] [ ] as a consequence of climate change, the hypothesis about the probable changes in the distribution of another species of tick, hyalomma marginatum, which is the crimean-congo hemorrhagic fever (cchf) vector in europe, has also been established. under warmer climate conditions, according to prediction models, it is expected that the distribution of this tick species will extend to new areas which were previously free of the vector. in relation to this issue, the epidemiology of mediterranean spotted fever seems clearly associated with climate change, especially with low rainfall values and it has been shown that warming causes greater aggressiveness in its avs. table shows the tick-borne diseases throughout the world, with the prediction of risk for spain (subjective assessments). to be able to show a perspective on the avbd, it is essential to monitor and identify microorganisms in vertebrates and arthropods, designing strategies before their transmission to humans. early detection and implementation of control strategies allow minimizing the impact on the population. between and , % of emerging infections spread from a wild-type focal point. occasionally, the infection is spread directly from reservoirs such as bats, rats or chimpanzees to domestic animals, which amplify the infection, or to people; other times, infection dissemination (spill-over) occurs through av such as ticks, fleas or mosquitoes. in any case, it is essential to carry out a surveillance and to know the microorganisms carried by av. a recent example in spain is the detection of cchfv in ticks of the species hyalomma lusitanicum collected in deer a few years ago in the province of caceres and the explanation of one of the possible ways of the arrival of the virus to our country. detection of the disease that appeared in as, to some extent, it was predictable. on september , , the ministry of health issued a press release reporting the death of a man by cchf and the contagion of the nurse who had taken care of him in the icu of the vallecas hospital where he had been treated. the first two autochthonous cases of cchf in spain were confirmed (fig. ) . in the last cchfv situation report and transmission risk assessment in spain, the diagnosis of tick-borne diseases is not always easy. we must bear in mind that a tick bite history is usually absent in at least half of the cases and that incubation periods can be very long. depending on the size of the tick, they can be very difficult to see (they can simulate a small mole), and their bite is painless. unless there is awareness or a high index of suspicion when faced with certain signs and/or clinical symptoms, who is going to think of a tickborne disease? it is clear, "what is not sought, is not found". in , who revised the list of emerging pathogens that could cause serious epidemics in the future and those that need to be investigated, including cchfv, ebola/marburg virus, zika virus, middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), lassa virus, nipah virus and rift valley fever virus. in addition, this year the list is completed with disease x, referring to an international epidemic that could be caused by a pathogen whose pathogenic potential and route of transmission is unknown at the moment. this list takes into account the transmissibility between humans, the severity of cases and the percentage of mortality, the difficulty of control and diagnosis and the context of public health and global expansion. in addition, there are other diseases that need more attention as soon as possible: hemorrhagic fevers by other arenaviruses, chikungunya virus, diseases by other highly pathogenic coronaviruses or by emerging enteroviruses and febrile syndrome with severe thrombocytopenia. many of these diseases are avborne. high high debonel/tibola: dermacentor-borne necrosis, erythema and lymphadenopathy/tick-borne lymphadenopathy; lar: rickettsiosis associated with lymphangitis. a in spain, borrelia hispanica relapsing fever. the risk of emergence/re-emergence is calculated based on two factors: (a) presence of cases of the disease in humans in the last years in europe, mediterranean countries and central and south american countries with a significant relationship with spain; (b) presence of the vector in spain. each factor is scored with one point (presence in the two factors) or zero points (absence in the two factors) and both are added. the total score can range between and , with being: low risk, : moderate risk and : high risk. another av that is said to have killed more people than all wars together is the body louse (human pediculus), transmitting exanthematic or epidemic typhus (rickettsia prowazekii), endemic recurrent fever (borrelia recurrentis) and the trench fever (bartonella quintana). body lice have been a serious public health problem until recently. they live in the seams of clothes and multiply in cold weather, lack of hygiene and war conditions. a person can be infested with thousands of lice, and each specimen is capable of biting an average of five times a day. it is said that body lice were one of the main problems during the russian revolution, where three million people affected by exanthematic typhus died. thus, vladimir ilyich lenin ( - ) stated: either socialism defeats the louse, or the louse will defeat socialism". here in spain, it was also a problem during the post-war period and was used as propaganda by franco's regime. we can ask ourselves: is there a risk of an epidemic or an epidemic outbreak of exanthematic typhus? it could happen, as it occurred in burundi in , when a large epidemic affected more than one hundred thousand patients. all the alarms went off when a red cross nurse was diagnosed after returning from work in the affected country. body lice are not seen on the body surface but live in the seams of clothing, in ± • c temperatures. parasitism by body lice should be suspected in persons with signs of scratching and lack of hygiene, more frequently in cold times of the year. at present, body lice have reappeared in refugee camps in europe, as in the second world war. in november , the european center for disease prevention and control reported the emergence of cases of recurrent fever due to body lice at different points along the route followed by refugees arriving in italy from the syrian war. in western europe, although we have not experienced any epidemic since the post-war period, and the fact that the condition had been eradicated, occasionally, there are r. prowazekii and b. quintana infection reports in homeless people parasitized by lice and cases of brill-zinsser disease have been described in people who have suffered from exanthematic typhus which could lead to an epidemic outbreak in certain conditions. fleas are other bloodsucking insects of worldwide distribution with an impact in public health. rickettsia can be transmitted to humans by at least two species: the rat flea (xenopsilla cheopis), which is the endemic or murine typhus vector (caused by rickettsia typhi), and the cat flea (ctenocephalides felis), which is the key vector of rickettsia felis and, occasionally, of r. typhi. as far as we know, rickettsias are not transmitted by the human flea (pulex irritans). in europe, murine typhus is a common avbd in mediterranean countries such as greece, cyprus, croatia and spain, including the canary islands. it develops as a non-specific febrile disease, with or without rash, which is often underdiagnosed. in clinical practice, murine typhus should be included in the differential diagnosis of any patient with fever of intermediate duration, that is, in a patient with fever (more than • c) of more than and less than days of progression, without focality to guide the diagnosis, which remains without a diagnosis after an initial evaluation that includes a complete clinical history, physical examination, complete blood count, chest x-ray and biochemical blood and urine tests. r. felis infection is another rickettsiosis with characteristics similar to murine typhus, of which cases have also been published in spain and that should be considered in patients with fever and/or rash, with a history of contact with cats or flea bites. despite the fact that there have been no reports of autochthonous yersinia pestis infection transmitted by the rat flea (x. cheopis) in spain, there is an alert for travellers at the time of writing this manuscript due to an epidemic outbreak in madagascar that has affected several thousand people. finally, we should remember the phrase written by hans zinsser in in his book entitled: rats, lice and history: "nothing in the world of living creatures remains constant. infectious diseases are constantly changing, new ones are in the process of development and the oldest ones are changing or disappearing". currently, ticks are considered the most dangerous avs in the world, because of their ease in moving from animals to people, because of their ubiquitous nature (they are present on all continents, including antarctica) and because of their capacity to agglutinate inside them a host of pathogenic microorganisms potentially transmissible through their hematophagous habits. the human tampering of ecosystems (deforestation, erosion of geographical limits to facilitate travel, etc.) or climate change are some of the factors that are favouring a greater contact between wild animals (with their ticks and the diseases they transmit) and people, facilitating the expansion of ticks to new previously unoccupied areas. diptera, ticks or other av may play the leading role in the next pandemic. therefore, anticipating the next public health crisis is in our hands. the authors declare no conflict of interest. biology of disease vectors ecology of zoonoses: natural and unnatural histories agency for international 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de alertas y emergencias sanitarias (ccaes), ministerio de sanidad west nile virus in spain: report of the first diagnosed case (in spain) in a human with aseptic meningitis west nile fever outbreak in horses and humans epidemiology and spatio-temporal analysis of west nile virus in horses in spain between risk mapping of west nile virus circulation in spain preventive medicine for the planet and its peoples vector-borne diseases and climate change: a european perspective impact of regional climate change on human health driving forces for changes in geographical distribution of ixodes ricinus ticks in tick-borne rickettsioses in europe the risk of introducing tick-borne encephalitis and crimean-congo hemorrhagic fever into southwestern europe (iberian peninsula). in: virology ii -advanced issues role of birds in dispersal of etiologic agents of tick-borne zoonoses detection of tickborne 'candidatus neoehrlichia mikurensis' and anaplasma phagocytophilum in spain in borrelia miyamotoi: should this pathogen be considered for the diagnosis of tickborne infectious diseases in spain? boutonneuse fever and climate variability crimean-congo hemorrhagic fever virus in ticks, southwestern europe crimean-congo hemorrhagic fever virus in ticks from migratory birds autochthonous crimean-congo hemorrhagic fever in spain informe de situación y evaluación del riesgo de transmisión de fiebre hemorrágica de crimea-congo (fhcc) en españa. ministerio de sanidad fiebre hemorrágica de crimea-congo: «lo que no se busca no se encuentra list of blueprint priority diseases outbreak of epidemic typhus associated with trench fever in burundi rapid risk assessment: communicable disease risks associated with the movement of refugees in europe during the winter season ectoparasitism and vector-borne diseases in homeless people from marseilles zinsser disease in moroccan man fever of intermediate duration: new times, new tools and change of spectrum cluster of cases of human rickettsia felis infection from southern europe (spain) diagnosed by pcr we wish to thank jorge garcía labeaga, of urbe ingeniería, for his collaboration in the preparation of fig. . key: cord- -tt eq e authors: wang, jann-tay; sheng, wang-huei; fang, chi-tai; chen, yee-chun; wang, jiun-ling; yu, chong-jen; chang, shan-chwen; yang, pan-chyr title: clinical manifestations, laboratory findings, and treatment outcomes of sars patients date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: tt eq e clinical and laboratory data on severe acute respiratory syndrome (sars), particularly on the temporal progression of abnormal laboratory findings, are limited. we conducted a prospective study on the clinical, radiologic, and hematologic findings of sars patients with pneumonia, who were admitted to national taiwan university hospital from march to june , . fever was the most frequent initial symptom, followed by cough, myalgia, dyspnea, and diarrhea. twenty-four patients had various underlying diseases. most patients had elevated c-reactive protein (crp) levels and lymphopenia. other common abnormal laboratory findings included leukopenia, thrombocytopenia, and elevated levels of aminotransferase, lactate dehydrogenase, and creatine kinase. these clinical and laboratory findings were exacerbated in most patients during the second week of disease. the overall case-fatality rate was . %. by multivariate analysis, underlying disease and initial crp level were predictive of death. s evere acute respiratory syndrome (sars) is a new infectious disease, first recognized in november ( ) . sars has spread rapidly around the world: > , cases have been reported from countries on five continents ( , ) . previous reports have described some major clinical findings of sars, including the temporal progression of clinical symptoms and chest radiography, the outcomes, suggested treatment protocol, and risk factors for death ( , ) . however, data are still limited on the temporal progression of abnormal laboratory findings, such as leukopenia, lymphopenia, thrombocytopenia, elevated lactate dehydrogenase (ldh), elevated aspartate aminotransferase (ast), elevated alanine aminotransferase (alt), elevated creatine kinase (ck), and elevated c-reactive protein (crp) and the roles each of these plays in predicting outcomes and complications. although so et al. have suggested a treatment protocol with the emphasis on early use of high-dose steroids ( ) , whether this treatment is better than others is unclear. we conducted a prospective study on the clinical, radiologic, and hematologic findings of sars patients with pneumonia, who were admitted to national taiwan university hospital (ntuh) from march to june , . most of these patients were treated following a standard treatment protocol, different from that suggested by the study group in hong kong ( ) . we report on the clinical features of our sars patients with pneumonia, with emphasis on temporal progression of laboratory findings, treatment outcome, and risk factors for poor prognosis. ntuh is a -bed, university-affiliated medical center located in northern taiwan. the center provides both tertiary and primary care for patients. it was also the primary hospital caring for sars patients during the sars outbreak in taiwan. all patients who fulfilled the revised world health organization (who) definition of probable sars ( ) in whom pneumonia developed and who received treatment at ntuh during march to june , , were enrolled in this study. except for the first patient, who did not receive any of the following treatments, and the second, third, and fourth patients, who received steroid in the first week of their disease, all patients received treatments that conformed to the guideline described here. oral ribavirin was prescribed soon after the diagnosis of sars was made; the loading dose was , mg followed by , mg per day if the body weight was > kg, or , mg per day if the body weight was < kg. this treatment lasted days unless adverse effects developed. antimicrobial agents for community-acquired pneumonia, either moxifloxacin alone or ceftriaxone plus azithromycin, were administered at the same time. methylprednisolone was usually administered in the second week of the disease if any of the following occurred: a flare of fever, progression of clinical symptoms (such as dyspnea or diarrhea), a surge or resurge of crp level, or rapid deterioration of chest radiographic findings (development of new infiltration). methylprednisolone was indicated in the first week of disease only if clinical symptoms or laboratory abnormalities (such as elevated ck, ldh, crp) worsened rapidly, and rapidly progressed abnormalities were found on chest radiograph. the dosage was mg/kg/day for days, and then it was tapered off. pulse therapy with methylprednisolone, mg/day for days, was used if there was major disease progress under the standard regimen. intravenous immunoglobulin (ivig) was administered if severe leukopenia (< x /l), thrombocytopenia (< x /l), or both occurred, or if lesions on chest radiography progressed rapidly in the first week of disease. the dosage of ivig was g/kg/day for days. once patients were intubated and supported by a mechanical ventilator, respiratory care followed the principles suggested for managing acute respiratory distress syndrome ( ) . the etiologic workup included the sputum gram stain and acid-fast stain, sputum culture for bacteria, sputum chlamydial antigen, throat swab for virus isolation, urine pneumococcal antigen, and urine legionella antigen. we tested antibody reactions of both acute-and convalescentphase serum specimens, weeks apart, for mycoplasma, chlamydia influenza virus, parainfluenza virus, adenovirus, coxsackievirus, respiratory syncytial virus, and sars-related coronavirus (sars-cov). we also took throat swabs for reverse transcription-polymerase chain reaction (rt-pcr) for sars-cov. the other routine laboratory tests, such as the hemogram, serum ast, alt, ck, ldh, and crp level, were examined every other day during hospitalization. a chest radiography was also performed every other day during hospitalization. once a patient was diagnosed as having sars, he or she was sent to a negative-pressure ventilated room immediately. no visitor or family member was allowed to enter this room. all healthcare workers caring for sars patients were asked to adhere strictly to contact and airborne precautions. before entering isolation rooms to care for sars patients, all healthcare workers washed their hands and put on personal protective equipment, including gowns, gloves, n respirators, goggles, and face shields. after caring for sars patients, such workers were to take off the personal protective equipment in the anteroom and wash their hands before leaving the isolation room. the health of healthcare workers who had any contact with sars patients or their environments was monitored daily for days after the last exposure. once fever developed in a worker, he or she was immediately hospitalized and placed in isolation in a specially designated ward. a standard case report form modified from one designed by the centers for disease control and prevention for sars was used to collect demographic and clinical data ( ) . severity of underlying disease was classified by using the modified risk stratification proposed by mccabe: rapidly fatal (death expected within year), ultimately fatal (death expected within years), or nonfatal (death expected > years or no underlying disease) ( ). all statistical analysis was performed with spss version . (spss, chicago, il). logistic regression was used for univariate and multivariate analysis. continuous variables were compared with the t test. categorical variables were compared by using the fisher exact test. a p value < . was considered significant. during the study period, patients were enrolled. their demographic and clinical data are detailed in table . the male-to-female ratio was : . their age was - years (median . years). twenty-four patients had various underlying diseases, including cardiovascular disorders in patients, diabetes mellitus in , hepatobiliary disorders in , history of cerebrovascular accidents in , chronic renal diseases in , pulmonary fibrosis in , history of intravenous drug abuse in , and adrenal insufficiency in . fourteen of these patients had underlying diseases classified as rapidly fatal (diabetes mellitus, ischemic heart disease, plus congestive heart failure in four patients; diabetes mellitus, ischemic heart disease, plus cerebrovascular accident with being bedridden in three; diabetes mellitus, ischemic heart disease, plus end-stage renal disease in two; diabetes mellitus plus decompensated liver cirrhosis in one; and ischemic heart disease plus massive ischemic bowel in one) or ultimately fatal (severe pulmonary fibrosis in one, ischemic heart disease in two). most frequent initial symptoms were fever, cough, myalgia, dyspnea, diarrhea, and rigor. three of the patients who had diarrhea had previously received various antimicrobial agents. the duration from symptom onset to a patient's visiting ntuh was - days (median days). the initial laboratory data are detailed in table . abnormalities on chest radiography suggesting pneumonia were found in of the patients. lesions were found in one lobe in patients, two lobes in patients, three lobes in patients, four lobes in patients, and five lobes in patients. abnormalities visible on chest radiography developed in the other patients after admission. the duration from disease onset to the time when abnormalities on the chest radiography were first noted was - days (median days). during hospitalization, patients ( . %) had respiratory distress and needed oxygen supplements. the duration from disease onset to severe respiratory distress was a mean of . ± . days (standard deviation [sd]). endotracheal intubation with ventilator support was indicated for patients, but patients refused intubation. among the intubated patients, the duration from disease onset to intubation was . ± . (sd) days. eight of these patients were successfully extubated . ± . (sd) days later. twelve of the patients died, and patients remained intubated at the end of the study because of marked lung fibrosis. thirty-one patients ( . %) experienced exacerbation of diarrhea after admission. all had received various antimicrobial agents since hospitalization. the duration from disease onset to severe diarrhea was . ± . (sd) days. during the disease course, leukopenia, lymphopenia, and thrombocytopenia were found in , , and patients, respectively. elevation of ast and alt was noted in and patients, respectively. elevation of serum ldh, ck, and crp levels was found in , , and patients, respectively. the laboratory values of the various parameters listed above typically peaked in severity in the second week of illness ( table ). the figure demonstrates the relationships between the time points when several clinical and laboratory parameters became most severe. new lesions visible on chest radiography developed in patients during hospitalization, including new lesions limited to one lung lobe in patients, two lobes in , three lobes in , four lobes in , and five lobes in . the duration from disease onset to the most severe chest radiography findings was . ± . (sd) days. sputum gram stain and acid-fast stain, sputum culture for bacteria, sputum chlamydial antigen, throat swab for virus isolation, and urine legionella and pneumococcal antigen tests were available for all patients; all results were negative. paired serum specimens were available for patients. tests for antibody reaction to mycoplasma, chlamydia, influenza virus, parainfluenza virus, adenovirus, coxsackievirus, and respiratory syncytial virus were negative. of these patients, immunoglobulin (ig) g antibody to sars-cov was detected by immunofluorescent assay in patients ( . %). twenty-six ( . %) of the total patients had positive results on throat swab rt-pcr for sars-cov. among the patients whose throat swab results were negative for sars-cov by rt-pcr, seroconverted to sars-cov. the other patients, who had no direct microbiology or serologic evidence for sars-cov infection at the time the study ended, had clinical courses compatible with those of probable sars and clear relationships as well as exposures to the initial immigrant clusters and the later intrahospital outbreaks in taiwan ( ) . their diagnoses of sars had also been confirmed by a committee of the center for disease control (taiwan). the first patient did not receive any treatment specific for sars. five patients did not receive ribavirin treatment because its use was contraindicated for them because of conditions such as cardiac arrhythmia and cardiomyopathy. seven patients did not receive steroids because their cases were rapidly fatal after diagnosis. these seven patients died mainly because of their underlying diseases, especially cardiac events. their sars cases did not progress to the point that steroids were indicated according to our treatment protocol. eight patients received pulse steroid therapy for progressive clinical conditions, with the usual dosage of steroids. forty patients received ivig infusion for severe cytopenia (thrombocytopenia, leukopenia, or both) ( patients in total) or marked local progression of pulmonary lesions on chest radiography in the first week of disease ( patients). those patients who received ivig for severe cytopenia did not receive steroids while on ivig; their leukocyte counts were . ± . x /l and . ± . x /l, before and after the use of ivig, respectively (p = . , by paired t test). their platelet counts were ± x /l and ± x /l, before and after ivig, respectively (p = . , by paired t test). various complications developed in patients during hospitalizations, including rhabdomyolysis, peripheral neuropathy, acute renal failure, and fungal or bacterial superinfection (table ). among the nosocomial infections, were bloodstream infections; of these were caused by enterococci, by methicillin-resistant staphylococcus epidermidis, and by methicillin-resistant s. aureus. eleven episodes were lower respiratory infections, diagnosed by the existence of new infiltration on chest radiography, purulent sputum, phagocytosis of bacteria by neutrophils in sputum (indicated by sputum gram stain), positive sputum culture for bacteria, and response to effective antimicrobial agents. the last episode was a catheter-related infection caused by candida parapsilosis. the overall death rate was . % ( / ). among the patients whose underlying diseases were classified as ultimately fatal or rapidly fatal, the rate of death was . % ( / ) . for the other patients with mild underlying diseases or without underlying disease, the death rate was . % ( / ). the time from disease onset to death in these patients was - days (median days). for the patients whose clinical conditions indicated endotracheal intubation with ventilator support, the rate of death was . % ( / ). when we used the logistic regression model for univariate analysis, age, underlying disease (nonfatal versus ultimately or rapidly fatal), initial crp level, initial absolute neutrophil count (anc), peak ck level, and peak crp level were predictors of death. age, sex, underlying disease, initial chest radiographic findings, initial crp level, initial anc, peak ck level, lowest lymphocyte count, worst chest radiographic findings, peak ldh level, and peak crp level were the predictive factors for respiratory failure (table ) . however, when we used logistic regression for multivariate analysis, underlying disease and initial crp level were the only two factors significantly predictive for death (odds ratio [or], . and . every mg/dl increase, respectively; p < . and p = . , respectively), and age, initial crp level, and worst chest radiographic findings were predictive for respiratory failure (or . , . every mg/dl, and . every one-lobe involvement, respectively; p = . , p = . , and p = . , respectively). for the patients who received steroids as the treatment protocol described above, remained febrile and needed further pulse steroid therapy after the use of methylprednisolone with the dosage of mg/kg/day. for the other patients who became afebrile after steroid was administered, had rebound of fever - days after the temporary defervescence. seven of the patients became afebrile again and had no fever - days after the transient rebound of fever without specific intervention. the other five patients received further pulse steroid therapy to control the fever and exacerbated clinical symptoms. for those patients alive at the end of this study, the time to defervescence after disease onset was . ± . days. major lung fibrosis directly caused by sars developed in of these patients; this condition resulted in exertional dyspnea in patients, oxygen-supplement dependence in patient, and respiratory failure in patients. by the end of the study, of the patients had been successfully discharged from ntuh. the other four patients, including three patients who had respiratory failure and one who was dependent on oxygen supplement, remained hospitalized. the duration of follow-up for these four patients was > weeks. our study of patients with probable sars with pneumonia demonstrated a high case-fatality rate ( . %), especially in patients with major underlying diseases and high initial crp levels. those patients who needed endotracheal intubation with ventilator support during their hospitalization also had a high rate of death ( . %). various complications developed in a high proportion of patients ( . %) during their disease. the yield rate of rt-pcr assay for sars-cov was lower ( . %) than in a previous report ( ) . this finding might be because only throat swabs, not nasopharyngeal aspirations or stools, were obtained for rt-pcr in the present study. as in previous reports from other areas ( , ) , fever was the most frequent initial symptom in our cases. compared to those previous reports, more patients in our case series initially had diarrhea ( . % vs. %- . %). therefore, according to our observations, diarrhea may be also considered as an early symptom and clue for sars. in addition, patients had initial symptoms of diarrhea when fever occurred. gastrointestinal tract should be considered as another important primary infection site of sars-cov. a previous study reported the temporal progression of clinical and radiologic findings in sars patients and indicated that several parameters would become more severe in the second and third week of disease ( ). our study had similar findings. although the exacerbation of diarrhea might be due to the use of antimicrobial agents, the diarrhea improved subsequently without their change or discontinuation. therefore, exacerbation of diarrhea is more likely due to sars itself. our study also demonstrates that most patients' abnormal laboratory findings may become more severe in the second week of disease (table and figure) . our treatment protocol was somewhat different from that suggested by so et al. ( ) . the timing of steroids was modified according to our experiences in treating the second, third, and fourth patients, whose exacerbation of oxygen demand and chest radiography lesions were not prevented by early steroid use. moreover, steroids are immunosuppressive. a previous study pointed out that the viral load of sars co-v in sars patients arrived at peak levels at approximately day of disease ( ). steroids were used as an adjunctive therapy for infectious diseases to reduce the severity of inflammatory damage that could occur in the later stage of disease ( ) . using steroids was also a risk factor for subsequent nosocomial infection ( ) . for all these reasons, we delayed the use of steroids. among the patients who received steroids as the treatment protocol, ( . %) had rebound or persistence of fever after initial steroid use. this fever rebound is less frequent than in prior reports ( . %- . %) ( , ) . however, the overall death rate in our study was similar to that reported from hong kong ( %- . %) ( , ) . comparing the treatment results of our study and previous ones is difficult because of different case definitions, patient backgrounds, and disease severity, as well as obscure descriptions about complications in sars patients in previous reports. all our patients had severe cases. therefore, the best timing of starting steroid usage and the total duration of steroid usage in sars patients to improve treatment outcome remain unclear and need further study. hemophagocytotic syndrome was documented in our second patient by bone marrow biopsy ( ) . her initial clinical signs and symptoms included fever, severe leukopenia, and thrombocytopenia. her hemophagocytotic syndrome was relieved after using ivig. this treatment was suggested because of its immune modulating effect ( ) . the other patients in whom severe leukopenia or thrombocytopenia developed in the first week of disease also received ivig therapy empirically. ivig appeared effective for controlling leukopenia and thrombocytopenia: after infusion, the patients' leukocyte and platelet counts increased to a significantly higher level (p = . ). the increase of leukocyte and platelet counts might have prevented some further complications directly resulting from severe leukopenia and thrombocytopenia, such as infection and tendency to bleed. although we had no control group, we believe that ivig may play a role in treating selected sars patients. advanced age, co-existing conditions, high peak ldh level, and high initial anc count had been reported as factors that predict poor prognosis for sars patients ( , , ) . by univariate analysis, many parameters predicted death or respiratory failure (table ) . however, by using the logistic regression model for multivariate analysis, severe underlying disease and high initial crp level were the only two factors that predicted death; age, initial crp level, and worst chest radiographic findings predicted respiratory failure. the role of crp in predicting the outcome of sars patients has not been discussed in previous studies ( , , ) . the discovery of crp was reported in by tillet and francis ( ) . crp parallels the severity of inflammation or tissue injury and is a useful marker for disease, response to therapy, and ultimate recovery ( , ) . although initial crp level was not available in eight patients in this cohort, our findings suggest that crp also parallels well with the severity and outcome of sars patients. age and underlying disease were strongly correlated: all our patients with severe underlying disease were older (age > years). in a statistical model, these two factors might interfere with each other and lead to the conclusion that age, not underlying disease, was an independent risk for respiratory failure; however, the opposite was true. the worst chest radiographic finding outlined the most severe extent of impaired lung function. this finding might explain why it was an independent factor for respiratory failure. however, other conditions unrelated to pulmonary condition, such as underlying disease or complications during hospitalization, contributed to death. these findings might explain why worst chest radiographic finding was not an independent factor for death. forty-two patients in this cohort were admitted to ntuh through the emergency department, which has no facility to check serum ldh level. also, during intrahospital sars outbreaks ( ) , heavy clinical loads and frequent bed transfers made it difficult for the primary care physician to collect laboratory data as the schedule described above. therefore, initial serum ldh level and crp level were available for only and patients, respectively; thus, initial ldh level could not be factored into our analyses. since both crp and ldh are markers of inflammation, whether the initial ldh level is also an independent risk factor for death or respiratory failure needs further study. complications during the disease courses of sars patients have been seldom or obscurely discussed previously ( , , ) . acute renal failure, which might be more likely caused by methicillin-resistant s. aureus infection and rhabdomyolysis, was found in three patients. acute myocardial infarction occurred in a patient who had been diagnosed with long-standing coronary artery disease. gastronintestinal bleeding, which might be due to critical illness, occurred in two patients. rhabdomyolysis has been reported to be associated with viral infection ( , ) . our observation suggests that sars co-v infection might also be associated with this complication ( ) . although peripheral neuropathy had also been reported with viral infection ( ) , neuropathy caused by steroids or acute illness should also be considered contributing causes in our four patients with neuropathy ( , ) . eleven patients had bacterial or fungal superinfection during hospitalization. all nosocomial infections occurred while patients were intubated and supported with a mechanical ventilator (p < . by fisher exact test). the nosocomial infection rate among sars patients was per , discharges, which was much higher than that of all patients at ntuh ( per , discharges) ( ) . steroid use and more severe clinical conditions than usual patients, such as higher rate of respiratory failure, might be the reasons. our study shows that sars has an overall complication rate of . % and case-fatality rate of . %. clinical symptoms and abnormal radiographic and laboratory findings might become most severe in the second week of disease. in addition to ribavirin and steroids, ivig may play a role in treating selected patients. underlying disease and initial crp level were the two independent predictors of death; age, initial crp level, and worst chest radiographic finding were the three independent factors predicting respiratory failure for adult sars patients. world health organization. sars epidemiology to date world health organization multicentre collaborative network for severe acute respiratory syndrome (sars) diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) a major outbreak of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community-outbreak of coronavirus-associated sars pneumonia: a prospective study development of a standard treatment protocol for severe acute respiratory syndrome severe acute respiratory syndrome (sars): multi-country outbreak updated harrison's principles of internal medicine gram-negative bacteremia severe acute respiratory syndrome-taiwan induction of postinflammatory cytokines in human macrophages by influenza a (h n ) virus: a mechanism for the unusual severity of human disease? the joint associations of multiple risk factors with the occurrence of nosocomial infection patient data, early sars epidemic intravenously administered immune globulin for the treatment of infection-associated hemophagocytotic syndrome clinical features and short-term outcomes of patients with sars in the greater toronto area serological reactions in pneumonia with a nonprotein somatic fraction of pneumococcus applications of acute phase reactants in infectious diseases c-reactive protein in the prediction of cardiovascular events infectious etiologies of rhabdomyolysis: three case reports and review the spectrum of rhabdomyolysis rhabdomyolysis associated with probable sars sabet a. infectious myelopathies ahfs drug information critical illness polyneuropathy: clinical findings and outcomes of a frequent cause of neuromuscular weaning failure nosocomial infection surveillance we thank jou-wei lin for his contribution on statistics.dr. jann-tay wang is an attending staff member at national taiwan university hospital. his specialty is infectious diseases key: cord- - oldaa i authors: murray, kris a.; olivero, jesús; roche, benjamin; tiedt, sonia; guégan, jean‐francois title: pathogeography: leveraging the biogeography of human infectious diseases for global health management date: - - journal: ecography (cop.) doi: . /ecog. sha: doc_id: cord_uid: oldaa i biogeography is an implicit and fundamental component of almost every dimension of modern biology, from natural selection and speciation to invasive species and biodiversity management. however, biogeography has rarely been integrated into human or veterinary medicine nor routinely leveraged for global health management. here we review the theory and application of biogeography to the research and management of human infectious diseases, an integration we refer to as ‘pathogeography’. pathogeography represents a promising framework for understanding and decomposing the spatial distributions, diversity patterns and emergence risks of human infectious diseases into interpretable components of dynamic socio‐ecological systems. analytical tools from biogeography are already helping to improve our understanding of individual infectious disease distributions and the processes that shape them in space and time. at higher levels of organization, biogeographical studies of diseases are rarer but increasing, improving our ability to describe and explain patterns that emerge at the level of disease communities (e.g. co‐occurrence, diversity patterns, biogeographic regionalisation). even in a highly globalized world most human infectious diseases remain constrained in their geographic distributions by ecological barriers to the dispersal or establishment of their causal pathogens, reservoir hosts and/or vectors. these same processes underpin the spatial arrangement of other taxa, such as mammalian biodiversity, providing a strong empirical ‘prior’ with which to assess the potential distributions of infectious diseases when data on their occurrence is unavailable or limited. in the absence of quality data, generalized biogeographic patterns could provide the earliest (and in some cases the only) insights into the potential distributions of many poorly known or emerging, or as‐yet‐unknown, infectious disease risks. encouraging more community ecologists and biogeographers to collaborate with health professionals (and vice versa) has the potential to improve our understanding of infectious disease systems and identify novel management strategies to improve local, global and planetary health. in a globalized world where the spread of infectious diseases appears to ignore all boundaries and the risk of emerging pathogens is on the rise (jones et al. , fisher et al. , there has been a resurgence in interest by academics, the general public and both national and international government authorities in the geography of human infectious diseases at all spatial scales. since most endemic and emerging human pathogens utilise non-human animal species at some stage in their transmission cycles (e.g. reservoir and intermediate hosts and vector species) (taylor et al. , woolhouse and gowtage-sequeria ) , biogeographers and community ecologists are increasingly involved in this pursuit. their contributions have yielded a range of novel and complementary insights on the spatial and temporal patterns of infectious disease occurrence, emergence and burden, their underlying ecological processes, and their surveillance and management (guernier et al. , smith et al. , peterson , reperant , johnson et al. , murray et al. , stephens et al. b . medical geography has a long and rich history (barrett , cliff et al. , rogers et al. , cliff and haggett and its methods and objectives have numerous parallels to those of modern biogeography, with its broad aim of determining how multiple processes (e.g. speciation, adaptation, extinction, ecology, geology, climate) interact with one another to produce distributional patterns in the world's biota (myers and giller ) . for infectious diseases, this history stretches as far back as to the time of the debate between contagionists and anticontagionists, when experts disagreed about the very existence of infectious disease-causing agents; for example, several 'spot maps' in the context of yellow fever in the us were developed in the late th and early th centuries to identify patterns and attempt to infer environmental causes for the disease, well before pasteur and koch's eventual and definitive development of the 'germ theory of disease' in the late s (howe , jones , lederberg . however, the largely correlative methods and findings of medical geography seemed to lose ground as modern medicine developed in favour of a relatively narrow focus on molecules, individuals, individual diseases or sub-components thereof, and small and homogenous populations and areas (e.g. cohort studies, randomized-and case-control trials, small area health statistics), in which causality is presumed easier to stalk (schwartz , mclaren and hawe ) . as a consequence, and despite the availability of theory and methods in other disciplines to overcome, negate or manage key issues related to correlation and scale complexity (chesson ), modern epidemiology has been arguably caught off-guard in a rapidly changing world. so called 'prisoners of the proximate', in reference to a limiting preoccupation with direct individual-level disease risk factors, mcmichael ( ) suggested that modern epidemiologists and public health managers have been sluggish and ill-equipped to recognise, prepare for and proactively respond to some of the most pressing and emerging health challenges of the st century, such as climate change, habitat alteration and degradation, biodiversity loss, invasive species including vectors, demographic shifts, migration and epidemiological transitions. although many global health metrics, such as life expectancy and childhood mortality, have nevertheless continued to improve, researchers from a range of disciplines are increasingly forecasting a collision between ongoing improvements in human health and a range of large-scale and accelerating global change processes, particularly those relating to environmental factors and declining environmental quality (foley et al. , mea , raudsepp-hearne et al. , suk and semenza , costanza et al. , watts et al. , whitmee et al. . health funders are also beginning to recognise these complex risks to human health (e.g. wellcome trust  https:// wellcome.ac.uk/what-we-do/our-work/our-planet-ourhealth , rockefeller foundation  www.rockefellerfoundation.org/our-work/initiatives/planetary-health/ ). given that this type of multi-scale, multi-disciplinary complexity is commonplace in biogeography, and much precedence exists from the study of parasitism, plant and animal diseases and global change ecology, there has never been a better time for biogeographers and ecologists to contribute their knowledge, theory and methods to public and global health. critically, where strong links between human health and the environment are identified and quantified, such collaborations could stimulate novel streams of funding and yield cost-effective co-benefits for health and the environment (myers et al. , waldron et al. . this is particularly salient for research on human infectious diseases, most of which involve animal hosts and vectors in shaping their distributions and are therefore influenced by many of the same ecological processes that govern biodiversity patterns more generally (guernier et al. , murray et al. . analogous to its utility for understanding wildlife distributions, biodiversity patterns and improving conservation management, biogeography has the potential to improve our understanding of infectious disease distributions, describe and explain patterns and processes underlying pathogen diversity ('pathodiversity') and contribute to infectious disease forecasting, risk management and threat abatement. through the analysis of historical disease, host and/or vector occurrence and co-occurrence patterns, biogeographic approaches could even yield some of the earliest, and in some cases the only, insights into poorly known, burgeoning and future infectious disease risks (murray et al. ) . here, we review the building blocks of biogeography and illustrate how it has and could continue to provide novel insights for the study and management of human infectious diseases, an integration we refer to as 'pathogeography' (resurrecting a term coined by plant pathologist israel reichert; reichert and palti ) . better understanding of pathogeography among ecologists and improved biogeographic knowledge among veterinary and medical scientists and public health managers should help improve disease surveillance, combat the global burden of human infectious diseases, and improve environmental management. it may even help rebridge a divide that has opened between the medical and ecological sciences, two powerful, explanatory and potentially predictive disciplines that ultimately share common roots in basic scientific inquiry. the overarching objectives of pathogeography, following biogeography (myers and giller ) , are to determine how the interactions among varying biotic (e.g. speciation, adaptation, extinction) and abiotic (e.g. topography, geology, climate) factors and processes combine to produce distributional patterns in infectious diseases through time. johnson et al. ( ) and stephens et al. ( b) recently reviewed the community ecology and macroecology of infectious diseases, respectively, and a natural intersection between these disciplines and the biogeography of infectious diseases occurs where these fields become spatially explicit. macroecology deals with ecological questions that demand large-scale analysis (brown , brown and lomolino , cox et al. . the limits between biogeography and macroecology are fuzzy. they converge when biogeography is focused on the study of how population-or community-level parameters vary along geographic dimensions (lomolino et al. ) , or when macroecology deals with spatial patterns (e.g. the geographic distribution of a certain pathogen species or diversity patterns). the divergence occurs when spatially explicit components are not crucial to answering questions being addressed by macroecology (blackburn and gaston ) , and when biogeography does not invoke structural and functional patterns of ecological systems (e.g. areography and some evolutionary biogeography approaches) (morrone ). by contrast, community ecology offers further insights on the mechanisms and processes that bridge fine scale processes of individuals and populations with the ecology and evolutionary processes of species and disease distributions at larger spatial scales (johnson et al. ) . whereas biogeography is concerned with the analysis of spatial patterns of biological diversity, pathogeography (i.e. the biogeography of pathogens) is concerned with the analysis of spatial patterns of pathogen (or disease) diversity ('pathodiversity' being the obvious analogue, but also referred to as the 'pathogen pool', 'pathogen community', 'pathosphere' or 'pathobiome'). taylor et al. ( ) constructed the first list of distinct species at a global scale known to be infectious to and capable of causing disease in humans under natural conditions, tallying species ( viruses and prions, bacteria and rickettsia, fungi, protozoa and helminths). discovery and recognition of new human pathogens occurs regularly (woolhouse et al. ) , with the most recent comprehensive inventory that we are aware of listing pathogens in humans ( viruses, bacteria, fungi, protozoa and helminths) (wardeh et al. ) . a comprehensive global database of clinically relevant human infectious diseases, gideon ( www.gideononline. com/ ), tracks more than of these pathogens (berger , victor and edberg ) . surprisingly, the distributions of most of these are very poorly known. as recently as , only infectious diseases were considered 'comprehensively mapped' (including old world coltiviruses, plasmodium falciparum and p. vivax, monkey pox, dengue, lassa fever and mayaro) (hay et al. ) , fundamentally limiting the ability of public and global health resources to be systematically and efficiently directed towards precise geographic areas and populations at highest risk from other impactful diseases. indeed, distributional patterns of human infectious diseases are generally far more poorly compiled and characterized (e.g. often at only country or regional level and as coarse presence vs absence data) than many plant and animal species, for which numerous global stock takes, status assessments, occurrence databases and detailed distribution maps exist following a long tradition of biogeographic study (wallace , murray et al. ) (see also supplementary material appendix table a ). however, with the development of big data approaches and curated databases, resources are slowly improving for human infectious diseases (wardeh et al. , stensgaard et al. . data may even on occasion be far richer or more geographically precise than for many plant and animal species (especially invasive species) owing, for example, to notifiable disease reporting requirements, such as those in place in eu member states for reporting human cases of certain diseases to ecdc and zoonotic and food-borne diseases to other eu registries (e.g. haemorrhagic cases of dengue and rift valley fever, crimean congo haemorrhagic fever, west nile fever, cholera, campylobacteriosis) (lindgren et al. ). in addition, databases for specific groups of diseases (e.g. helminths, neglected tropical diseases) and host-pathogen (including human) associations are increasing (e.g.  www.thiswormyworld.org/ ). in global studies that have classified pathogens according to their epidemiological traits, the majority of known human pathogens ( - %) are classed as zoonotic, defined as diseases and/or pathogens that are naturally transmissible from vertebrate animals to humans, and % are arthropod vector-borne (who , palmer et al. , taylor et al. , woolhouse and gowtage-sequeria . the close association between animals and human pathogens means that the diversity of potentially pathogenic microorganisms that occur in animals including wildlife is also of major interest for human health (morse , murray and daszak ) . however, at present, the full dimensions of this broader 'pathogen pool' are almost entirely unknown. for example, estimates of the total number of viruses within just nine target viral families from the first intensively sampled wildlife species (the natural host of nipah virus, fruit bat pteropus giganteus) suggest that the number of known human pathogens is just a tiny fraction of the total viral diversity that occurs in wildlife (anthony et al. ). the 'operational taxonomic unit' (otu) of pathogeography may differ somewhat from conventional otus in biogeography (e.g. genes, species). while infectious diseases are all caused by specific pathogenic microbial species (which could, or perhaps should, themselves be the relevant otu), it is their infection/presence in human and in some cases livestock or wild animal hosts or vectors that is of primary interest to health stakeholders and the usual target of surveillance programs. 'occurrence' is the presence of a disease or its causative agent in a particular place at a particular time. this can in turn be represented as a presence (i.e. in a human host or a specific geographic unit) or some measure of relative abundance either within single hosts (e.g. the infection load, particularly for macroparasites, such as helminths), within a defined geographic area (e.g. density), time period (incidence) or within the host population (e.g. prevalence). it might also be represented to reflect the process of transmission itself (e.g. 'force of infection'). with some information on the average impact of infections in humans, public health practitioners often also describe spatial and temporal patterns of disease in terms of 'burden', with various available measures that broadly seek to capture the loss of healthy life (e.g. death and disability) attributable to the presence of certain diseases within a population (e.g. disability adjusted life years, dalys) . conversely, the absence of disease is of equal interest for pattern and process analysis, but harder to obtain given the sampling difficulties of asserting freedom from disease (cameron ). furthermore, occurrence records (and its derivatives) will generally be a subset of actual occurrences, because in most cases they will be heavily influenced by observation effort. given the diversity of health studies, all of these epidemiological metrics could be potentially relevant for pathogeographic analysis. the same units of measure (occurrence, abundance, density) for known or potential hosts and vector species are also relevant and will already be familiar to ecologists. some estimates of disease 'risk' (or, perhaps more accurately, 'hazards') are based on these (e.g. tick nymphal abundance as a measure of lyme disease risk) and their ecologies may contribute directly or indirectly to disease patterns (civitello et al. ) , such that data on their potential occurrence can improve biogeographically-informed risk mapping of disease outcomes in humans (messina et al. , pigott et al. , olivero et al. a . one major challenge for biogeographic analyses of human infectious diseases, however, is the availability and utility of appropriate data, which we discuss further in box . supplementary material appendix table a provides some example data types, and some useful databases and sources relevant to biogeographic analyses of human infectious diseases. the study of infectious disease spatial distributions is not new (barrett ) , nor are integrated approaches to studying infectious disease distributions, whether they are labeled biogeographic (peterson ) or not (lambin et al. ) . indeed, the emergence of informatics, geographic information systems (gis) and satellite technology has increased the availability of tools and high quality spatial datasets relevant to both ecologists and medical geographers, driving a recent convergence in the data and in some cases the methods used to examine the distributions of wild species and infectious diseases alike and for developing or evaluating causal hypotheses underpinning them (hay et al. , kraemer et al. . such developments have facilitated the study of the spatial structure of some infectious diseases in unprecedented detail ( fig. ) . in some cases, there has been innovation in the integration of these approaches with mechanistic models traditionally used to explore the population dynamics of infectious diseases (redding et al. ) , and some analyses have been developed and updated in close to real time (e.g. during the west african ebola outbreak) (pigott et al. (pigott et al. , . these advances complement an already strong suite of tools already used in epidemiological studies, which might equally flow the other way into the toolboxes of ecologists (magalhães et al. , caprarelli and fletcher , bhatt et al. . peterson ( ) defined the first explicit 'biogeographic framework' for human infectious diseases. drawing on the work of soberón ( ) on the grinnellian niche and geographic distributions of species, the framework is characterized by considerations of a pathogen's dispersal ability combined with the abiotic and biotic factors that interact to determine whether a disease is able to fulfill its full geographic potential. although not explicitly demonstrated, peterson ( ) emphasizes that a key difference between pathogens and diseases in comparison to free ranging wild species (with the exception of invasive species) is the relative unpredictability of dispersal events (e.g. rapid, long distance introductions), and the relatively lower importance of abiotic and relatively higher importance of biotic factors. this applies particularly to the high degree of inter-specific interactions, from the infection of a pathogen in a host, to the numerous other species that may be involved in pathogen transmission cycles. the distribution of an infectious disease is thus defined by the joint distributions of all species involved in its transmission cycle as dictated by the suitable ecological conditions and dispersal limitations for each. in contrast, lambin et al. ( ) present an alternative framework for understanding generalized disease risks across landscapes, drawing on principals from 'spatial epidemiology' (ostfeld et al. ) and 'landscape epidemiology' (pavlovsky ) . pavlovsky ( ) proposed that infectious disease occurrence is determined by 'a continuous interaction' of five landscape factors: ) animal 'donors' (e.g. reservoir hosts), ) vectors, ) animal 'recipients' (including humans), ) an infective pathogen, and ) environmental factors that are conducive to transmission. lambin et al. ( ) emphasise the use of biogeographical methods for examining the distributions of human infectious diseases will be dependent on the degree of existing knowledge (e.g. about the epidemiology of a particular pathogen or pathogen assemblage in a particular geographic context), data availability (e.g. from publicly accessible databases, surveillance data) and the potential for new data collection (e.g. targeted field collections). these elements could range from no knowledge, no data and large barriers to the collection of new data (e.g. for many emerging infections in developing country contexts, such as at the beginning of the ebola outbreak in west africa), to high degree of knowledge from existing scientific studies, well developed and accessible databases on relevant geographic/environmental, reservoir host, vector, pathogen, human and disease data, and existing structures to streamline the collection of new data (e.g. high impact diseases in developed country contexts, such as malaria in the eu). data on human infectious diseases are normally collected for the needs of a particular discipline or research focus, which may typically limit the extent to which it can be used laterally or opportunistically for answering non-target questions. this may be particularly the case for macroecological and biogeographical studies, which are often data intensive and conducted at scales that may be difficult or impractical to undertake new data collection. there is thus a clear need to expand the scope of research programmes on infectious diseases to encompass the geographic, biological and temporal scales relevant to biogeographical analysis. this involves informing monitoring and surveillance activities on what types of data would be most useful and urgent. improving data capture and quality with standardized sampling methodologies could help lead to analyses and discoveries that transcend the specific epidemiological details of a single site, geographical context or disease. to this end, the following information would be helpful to allow coherent data collection and analysis of infectious disease occurrence and transmission in space and time: • operational taxonomic units: potential complications for biogeographic pattern and process analysis may arise when the 'presence' of a specific disease type is in fact confounded. this could occur, for example, if multiple causal agents result in disease complexes (e.g. leishmaniasis) or if the causal agent is unknown and diseases are instead classified by their symptoms (e.g. syndromes). distributional data and databases on infectious diseases should thus strive for the highest 'taxonomic resolution' possible (murray et al. , stensgaard et al. ). • observation effort: a persistent issue in robustly quantifying occurrence, in any form, is its relationship to observation effort (allen et al. ) . observation effort could vary spatially, temporally or taxonomically. presence, prevalence, burden and diversity patterns of infectious diseases, hosts and vectors may all increase proportionally to observation effort, and confidence surrounding reported absences also increases with observation effort. failing to account for this has the potential to introduce biases in biogeographic studies. numerous studies have taken steps to incorporate measures of observation effort to limit the potential effects of observation bias in biogeographic studies of infectious diseases, typically by including factors hypothesised to be related to observation effort, such as sampling intensity, gdp, health expenditure or publication output, in statistical models (jones et al. , hopkins and nunn , yang et al. , murray et al. ) (see also o in box ). • sampling protocols: a lack of communication between biostatisticians and field workers in both ecological and health fields before collecting samples can lead to a breakdown in robustness of subsequent analyses. for example, sampling too many host individuals of one species could be as problematic as not sampling enough from a target host (e.g. humans), particularly when research resources are constrained. best practices involving probability sampling should be pursued where possible (nusser et al. ). • geographic coverage and resolution: sampling should cover a sufficiently large area(s) to reproduce in space what really exists in the field; for example, metapopulation or metacommunity geographic distributions with sources and sinks of pathogen transmission. the effects of uneven sampling across space and one-shot sampling should be avoided (peterson ) . at the other extreme, improving precision on available data points is a high priority for developing robust geo-referenced databases of disease (or pathogen, host, vector) occurrence. all data should be collected and stored for subsequent access at the highest spatial resolution possible (i.e. gps coordinate locations). • temporal coverage: infected hosts including humans may travel long distances during disease incubation periods, introducing uncertainty in the attribution of the geographic location of infection (peterson ) . surveillance and sampling strategies should thus allow the capture of appropriate temporal scales (i.e. matching scale of disease-relevant processes) so that data can be better aligned with covariate information such as environmental variables and human activities. • phenology: animal and plant phenology should be monitored; the behavior and biology of most species, including humans, are influenced by often relatively predictable annual changes in climate that determine when they start or finish natural events, such as flowering and fruiting, breeding and mass gatherings. many of these activities and departures from norms due to, for example, unusual weather events have implications for disease risks and spatio-temporal distributions, and could have large implications when considering the influences of large scale change (e.g. climate change). • humans as hosts and dispersers: humans will often serve not only as hosts but also as effective dispersal mechanisms for infectious agents. although this can lead to unpredictability and extreme long distance invasion events (peterson ) , data on human populations and their movement are being increasingly well resolved at both fine and coarse spatial scales and this is proving invaluable in decomposing biogeographical components of many infectious disease systems including emergence risks (e.g. use of flight or road traffic data, mobile phone use data, social media) (colizza et al. , balcan et al. , wesolowski et al. , jurdak et al. ). • concurrent covariate sampling and time-lagging: ideally, assessment of environmental and social variables should be conducted at the same time as human or wildlife disease sampling (if it is not available at the appropriate scale retrospectively i.e. from remote sensing data). peterson ( ) discusses a range of issues relevant to modeling the distribution of infectious diseases, including quality control of input occurrence data, sampling design with the reduction of oversampling in certain areas, and design of analysis (see also hosseini et al. ). the dynamic nature of the spatial and temporal interactions between these 'prerequisites' at multiple scales when assessing the impact of landscape changes on vector-borne and zoonotic diseases. although they did not identify their study explicitly as 'biogeographic', there are clear parallels with peterson's ( ) framework (as well as many others, such as plowright et al.'s ( ) recent treatment of spillover ecology). we develop these ideas further in box , focussing on the interactions between a number of inter-dependent elements, including: physical geography (g), environment (e), reservoir host(s) (r), vector(s) (v), pathogen(s) (p), human factors (h), and the management (m) and observation (o) landscapes, the latter serving as the lens through which all other elements and disease distributions (d) are ultimately observed. from this framework, we can envisage how these elements may act and interact to influence both the real and observed distributions of specific diseases in space and through time, as well as multidisease patterns that may emerge at higher levels of organization (see 'emergent patterns and multiple diseases' below). depending on epidemiological characteristics, not all of the elements illustrated in box will be relevant for all human infectious diseases, resulting in a range of 'biogeographic complexity' among human infectious diseases. whereas g and e will almost always have some kind of modifying influences, single-element transmission source systems (i.e. diseases only involving human-human, environmental, single reservoir species or single vector species transmission) represent the least biogeographically complex examples in this framework. examples include tetanus (e), measles (h), and lassa fever (r). the more biogeographically complex diseases involve multiple elements; for example, multiple reservoir host or vector species (r n and v n , respectively) in addition to human-human or environmental transmission (e.g. dengue (hv n r n ); infection with mycobacterium ulcerans (ev n r n )). we describe the potential utility and demonstrate an application of this general framework further in box , by undertaking a 'disease trait profiling' exercise whereby we classify a large number of clinically relevant human infectious diseases according to their transmission sources (i.e. the e, h, v and r elements described in box ). the trends that emerge illustrate how certain disease characteristics are far more common than others (e.g. transmission from animal reservoirs vs arthropod vectors) and the extent to which mapping efforts are already underway. however, the analysis also highlights some important gaps. for instance, . % of diseases with a strong rationale for mapping (as rated by hay et al. ) have not been mapped in any study (box , fig. panel f) , and declining overall quality of mapping efforts correlates with increasing biogeographic complexity (box , fig. panel g) , highlighting a need to expand the breadth of ecological interactions considered within disease systems to improve mapping quality in future studies. an additional consideration not explicitly included in box is that changes in interactions through time can influence the occurrence, transmission and emergence of many diseases. for example, short or longer term changes in land-use and climate (both components of e) can influence d directly or through their influences on v, r and/or h (epstein , patz et al. , nakazawa et al. , jones et al. , hoberg and brooks , mackenstedt et al. . ebola virus disease (evd) outbreaks, for example, have been closely associated with inter-annual anomalies in meteorological seasonality, whereby sharply drier conditions at the end of the rainy season seem to favour the occurrence of outbreaks (pinzon et al. ). more recently, ebola outbreaks have been linked to fragmentation (rulli et al. ) and recent ( yr) deforestation (olivero et al. b ). a promising approach emerging from ecology warranting additional attention here is the increasingly widespread use of species distribution modelling (sdm) (also called ecological niche modelling (enm)). when applied to pathogens, hosts and/or reservoirs and vectors, sdm is useful for understanding risk factors conditioning the distributions, emergence or the accumulation of new outbreaks of disease. in sdms, the available information on the presence or the incidence of disease is linked to a diverse set of environmental variables and allows the evaluation of the degree to which certain environments are favourable for the occurrence of disease, even in areas where it has not been detected before. in the absence of, or in combination with, local-scale data suitable for (pigott et al. ) . illustrating the detail of modern cartographic projections of disease risk based on models of environmental suitability for the zoonotic transmission of ebola virus (shaded colours) and the spatial variation in disease risk that would be masked from country-level chloropleths (thick black lines). dotted lines indicate regions that have reported no index cases to date but are predicted to be partially at risk based on environmental suitability models that utilize a thresholding approach on the model output that captures % of the occurrence records used for model fitting (see also fig. ). boosted regression trees were used to develop the model based on georeferenced records of ebola virus disease outbreaks in humans and infection records in bat reservoirs and a range of environmental covariates. the scale reflects the relative probability that zoonotic transmission of ebola virus could occur at these locations; areas closer to (dark red) are more environmentally similar to locations reporting ebola virus occurrences; areas closer to (light yellow) are least similar. mechanistic models, sdms can take advantage of the large geographic scale to explore macroecological processes that are able to explain and predict the occurrence of disease. this can reveal emergent patterns and processes not perceptible at the local scale (brown ) , and can help lay a foundation for hypothesis testing or provide a geographic context for further studies on the ground (allen et al. ) . the outputs provided by sdms can be summarized in three main categories: probability (e.g. logistic regression, generalized additive models, random forests, boosted regression trees), suitability (e.g. maxent, garp) and favourability (e.g. favourability function). suitability is an idiosyncratic way of ranking local sites according to their capacity to hold the species or pathogen that is not directly related to the probability of occurrence (guisan and zimmermann , royle et al. ) . in contrast, favourability values can be obtained from probability and prevalence (here defined as the proportion of presences in the set of observations) (real et al. ). probability and suitability are biased in their outputs when working with samples differing in prevalence, which is not the case with favourability (acevedo and real ). this quality of favourability enables direct comparison and combination we may represent the challenge of simultaneously understanding patterns and processes of infectious disease systems with respect to a series of interacting elements; including g, the physical geography context (e.g. topography) and e, the abiotic (e.g. climate) and biotic (e.g. habitat) environment; r n and v n , the single or multiple (denoted by superscript n) species of reservoir hosts or vectors; p, the pathogen being transmitted; h, the human population itself; o, the observation effort that may apply to each of the other elements (e.g. surveillance and data collation from existing sources); and m, the management landscape (e.g. interventions). the combinations of these elements ultimately give rise to d, the observed disease distributions. where these elements have consistent effects across multiple diseases, 'higher order' biogeographic patterns emerge; for example, we can observe biogeographic regionalization as a consequence of the more pervasive effects of components of g (e.g. ocean or mountain barriers to dispersal) or e (e.g. unsuitable climates), while the effects of components of h (e.g. population growth, global movement, socio-economic status, immunity heterogeneity) and m (e.g. health infrastructure, vaccination) have fundamentally reshaped the global diversity and burden patterns of infectious diseases. in addition, each layer potentially has additional modifying elements that could further shape disease distributions and diversity patterns, such as temporal fluxes (see main text), or lifehistory and epidemiological traits of hosts, vectors and pathogens (smith et al. , woolhouse et al. . for pathogeographic analyses and as a starting point for risk assessments, a series of 'profiling' steps could help integrate existing information at a scale relevant to a particular research question. this could include geographic and/or environmental profiling (e.g. detailed assessments of g and e for diseases/hosts/vectors in regions of interest relevant to single diseases or disease assemblages) (eisenberg et al. ), disease trait profiling (classifying epidemiological features of diseases, such as the presence or absence of the e, v, r and h elements as sources of pathogen transmission (see box ), and the species known to be implicated in each), and human profiling (assessments of the human population distribution, density and movement patterns and the existing management and observation landscapes). intersection of these geographic, disease trait or human profiles will ultimately yield yet deeper understanding or management relevant insights (semenza et al. ). when several species are involved in the analytical design; for example, when using models for deriving indices based on multiple species (estrada et al. ) , and for the study of biogeographical relationships between species (real et al. , acevedo et al. , including relationships between pathogen and host complexes (olivero et al. a) . sdm approaches represent one of the major recent advances in infectious disease cartography (hay et al. , peterson , kraemer et al. , producing a diverse range of predictions on the presence or risk of human infectious diseases or their causative organisms (peterson et al. , peterson , neerinckx et al. , reed et al. , samy et al. , zhu and peterson , and their animal hosts and vectors (sweeney et al. , de oliveira et al. , giles et al. . modelling the distribution of biotic interactions is a relatively recent advance in macroecology and biogeography (kissling et al. , wisz et al. , ovaskainen et al. ) and could similarly provide a further way forward for the analysis of pathogens with zoonotic cycles based on joint distributions and multispecies assemblages. other methods of representing potential interactions at a community level (e.g. network modeling) or inferring potential hosts/vectors and host/ vector ranges from geographic co-occurrence are similarly being developed alongside (or in some cases integrated with) niche modeling approaches to yield novel insights disease trait profiling could help synthesize and summarize the range of potential ecological interactions of diseases and highlight important priorities or gaps for biogeographic analyses. to illustrate, we classified all clinically relevant diseases within the gideon database with single causative pathogens (n  diseases) into each of the combinations possible considering whether disease transmission sources included the e, h, v and r elements outlined in box (note, although h is by definition always involved for human infectious diseases, here it is treated more specifically as a transmission source i.e. human-human transmission). we then examined how variations in the complexity of diseases, as indicated by the number of elements involved in their transmission (complexity score, cs), was related to geographic range size (as broadly indicated by the number of countries in which the disease is present), the rationale for mapping, whether mapping efforts had already taken place, and the quality of existing mapping as rated by hay et al. ( ) . consistent with other studies, . % of diseases in our dataset involved (but do not necessarily require) transmission to humans from animal reservoirs (zoonotic) (fig. a) . human-human transmission was also common ( . %), while diseases including potential transmission from vectors ( . %) or environmental sources ( . %) were less common. for diseases involving vectors or reservoirs, the great majority involved multiple vector ( %) or reservoir ( %) species rather than single species (fig. a) . diseases ranged considerably in their degree of 'biogeographic complexity', but only of possible combinations were represented in our dataset (fig. b) . the least complex examples involved transmission from the environment (e) only, a single reservoir (r) only, or humanhuman (h) only (cs  ), while the most biogeographically complex diseases involved multiple vectors, reservoirs and could include either environmental (evnrn) or human-human (hvnrn) transmission as well (cs  ). on average, diseases including humanhuman transmission were the least biogeographically complex ('h' mean cs  . ), followed by diseases including transmission from reservoirs ('r' cs  . ), while diseases involving transmission from the environment ('e' mean cs  . ) and vectors ('v' mean cs  . ) were more complex. on average, complexity was not obviously related to commonness (fig. c) , but more complex diseases tended to be more geographically restricted (fig. d) , and had both a stronger rationale for mapping (fig. e) as well as a higher proportion of diseases that had already been mapped (fig. f) , particularly when human-human or environmental transmission were never involved (traits that can radically increase their distributions to the point of making them essentially ubiquitous). however, the quality ratings of existing mapping efforts for more complex diseases were considerably lower on average than for simpler diseases (fig. g) . these trends illustrate the extent to which mapping efforts are already underway for clinically relevant infectious diseases but also highlight some important gaps. for instance, as of hay et al.'s ( ) study, . % of diseases with a strong rationale for mapping had not been mapped in any study (fig. f) , and declining overall quality of mapping efforts for more restricted and biogeographically complex diseases highlights a clear need to address the breadth of ecological interactions within disease systems in future studies. on the spatial distribution of some infectious diseases (stephens et al. (stephens et al. , a . returning to the framework in box , g, e together with time (t) could each have more pervasive effects through their simultaneous influences on the other elements, giving rise to higher-order biogeographic patterns that are defined by multiple diseases, such as co-occurrences (including co-infection patterns), chorotypes, diversity gradients, or biogeographic regionalisation. while biogeography has already made significant contributions to providing a generalized framework for disease mapping (peterson , escobar and craft ) , we emphasise that it is the comparative power of biogeography that could help take pathogeography a step further, diverging more radically from medical geography, to address the factors that govern the structure, assembly, dynamics and spatial patterns of multiple or entire assemblages of human infectious diseases over a more diverse range of spatial and temporal scales. below we provide some illustrative examples relevant to what has or potentially could be applied to human infectious diseases. a chorotype is a type of distribution pattern that is followed by one or several species (baroni urbani et al. , real et al. , passalacqua . as chorotypes represent the shared geographical, ecological and evolutionary context of several species (real et al. , fattorini , these patterns could be useful for generating hypotheses about the causes and origins of host, reservoir and pathogen distributions. although not yet widely explored for infectious diseases, chorotype analysis could significantly contribute to the study of the complex interactions characteristic of human infectious disease systems (peterson , roche et al. . in the analysis of disease distributions, the relative importance of these interactions, compared to the relevance of other environmental factors, is variable. some studies have raised this issue through the comparative analysis of pathogen models and host models based on their respective responses to environmental conditions (maher et al. , costa and peterson ) . however, with the exception of using host distributions as a simple proxy for the potential distributions of pathogens (daszak et al. ) , the distribution of reservoir species has only recently been used as an explanatory variable, together with other environmental descriptors, to define a pathogen distribution model (e.g. compare peterson et al. walsh and haseeb, and pigott et al.' s models for ebola virus) (peterson et al. , walsh and haseeb , pigott et al. . although we are aware of no studies examining chorotypes of human infectious diseases, mammalian chorotypes have been recently incorporated into an infectious disease distribution model (olivero et al. a) . when the ecology of a pathogen is complex and unresolved (e.g. ebola virus, leroy et al. , groseth et al. , olival and hayman , imposing restrictions to the selection of host or vector species considered in a model might under-represent the zoological substrate conditioning a pathogen's transmission and distribution (roche et al. ) . olivero et al. ( a) thus addressed the mapping of favourable areas for the ebola virus in the wild by combining two biogeographical approaches: sdm and chorotype analysis. mammalian chorotypes in africa were employed as surrogates of the types of distributions shown by reservoirs and any wildlife species implicated in the virus spillover cycle. olivero et al. ( a) found that a model based on a number of diversity patterns, each one associated with a different mammalian chorotype, defined favourable areas for the presence of ebola virus with higher accuracy than did a model based on environmental variables alone (i.e. climate, forest type), concluding that mammalian biogeography contributes significantly to explaining the distribution of ebola virus in africa. in addition, vegetation was identified as a factor placing clear limits to the presence of the virus. favourable areas for ebola virus were thus determined from information provided by both models (fig. ) . it is now widely recognized that multiple pathogens may act independently or interact through a variety of different mechanisms to influence disease outcomes in human populations (pederson and fenton , jolles et al. , scholthof , and yet studies of human infectious disease, host and vector diversity or community assembly patterns are rare in comparison to distributional studies of single infectious diseases (see 'single diseases' above). characterizing diversity patterns can thus provide a range of insights on the distributions and processes underlying multiple species of pathogens or diseases. based on components first proposed by whittaker ( ) , inventory diversity quantifies diversity within an environment, where alpha (α) diversity is used to refer to diversity at the local scale (i.e. smallest scale being measured). α-diversity of human infectious diseases has been analysed in a number of studies, typically by comparing the number of diseases occurring in different countries at a global or continental scale due to limited comparative data availability at higher spatial resolutions. although strongly heterogeneous (fig. ) (see also stensgaard et al. ) , some striking patterns suggest that human infectious disease communities are shaped by the same ecological processes that shape the diversity of life more generally (guernier et al. ) . this is evident, for example, from observations of a clear latitudinal gradient in disease diversity and disease range sizes, whereby disease richness decreases and range size increases towards the poles (fig. ) (guernier et al. , guernier and guégan ) . other patterns are consistent with island biogeography theory, such as a positive relationship between land surface area and disease richness (smith and guégan ) , and reduced richness on smaller islands and with distance to the nearest mainland (jean et al. ). together, these findings likely explain why the strongest predictor of human infectious disease richness known to date is wildlife richness (dunn et al. ) , while some vector groups show similar patterns (foley et al. ). these patterns also illustrate that a correlation between human disease diversity and wildlife/ vector diversity may not necessarily imply direct causation. nevertheless, other evidence points to the importance of animals, particularly mammalian and bird wildlife, as a key source of endemic and emerging human pathogens (taylor et al. , woolhouse and gowtage-sequeria , jones et al. , allen et al. . in addition to the causal links, such parallels in patterns of human disease and other taxa reinforce the importance and potential utility of considering wildlife and vector biogeography alongside or as a central component of studies of infectious diseases, including distributional and diversity studies of known or potential zoonotic hosts and vectors (foley et al. , cooper and nunn , han et al. , olivero et al. a . in contrast to inventory diversity, proportional diversity measures the difference in diversity between environments or across gradients of habitats, commonly expressed as beta (β) diversity (whittaker , jost . β-diversity patterns can be expressed in a number of ways, such as biogeographic regionalisations, which define biotic boundaries according to between-area gaps in species composition, and biotic regions based on biotic similarities (olivero et al. ) . such approaches, however, are yet to be widely applied to human infectious diseases. in one study, β-diversity patterns of human infectious diseases appear to parallel patterns in other taxa, consistent with patterns identified to date for α-diversity (richness); murray et al. ( ) show that human infectious disease assemblages exhibit biogeographic regionalisation reminiscent of zoogeographic patterns, particularly for zoonotic (fig. a, b) , vector-borne and parasitic diseases, and that mammalian assemblage similarity is consistently among the strongest predictors of human infectious disease assemblage similarity among countries (fig. c ). such an effect is very likely predictive of as-yet undescribed patterns of microbial diversity, such as the geographic structure recently demonstrated among novel coronaviruses detected in wild bat hosts (anthony et al. ) . in addition to this dominant explanatory effect of biodiversity, other factors, including environmental (climate, land area, population size), social (human connectivity, health expenditure, observation effort) and epidemiological characteristics (e.g. pathogen type, transmission mode) also affect infectious disease αand β-diversity patterns (fig. c ). the strongest β-diversity patterns, for example, can be observed in zoonotic, vector-borne and parasitic infectious diseases, likely due to a more dominant role of environmental factors and persistence of historical dispersal barriers limiting their geographic distributions, while patterns of humanspecific diseases are far more homogenous at the global scale (smith et al. , dunn et al. , just et al. , murray et al. , jean et al. ) (see also box fig. panel d) . guernier et al. ( ) ); (c) nestedness: a hierarchical pattern of human pathogen composition with the pathogen species found at higher latitudes (darker bars) constituting nested subsets of those in progressively richer communities at lower latitudes (lighter bars) (adapted from guernier et al. ( ) ); (d) disease range size: narrower distributional ranges occur in the tropics (darker circles) for human pathogens compared to higher latitudes (lighter bars) (adapted from guernier and guégan ( ) ). β-diversity can be further decomposed into two separable components, nestedness and turnover, which may further help characterize the processes driving differences in the composition of assemblages between sites (harrison et al. , baselga . nestedness occurs where species assemblages are subsets of the biotas at more diverse sites (wright and reeves , ulrich and gotelli ) , and indicates a non-random process arising from any factor that promotes the orderly disaggregation of assemblages (gaston et al. ) . the latitudinal gradient of human infectious diseases exhibits such a pattern, with disease assemblages occurring at higher latitudes being subsets of those occurring closer to the equator (fig. c ) (guernier et al. ). in contrast, turnover indicates the replacement of some species by others as a consequence of environmental sorting or spatial and historical constraints (qian et al. , baselga ). in the only assessment of nestedness vs turnover undertaken so far for human infectious diseases that we are aware of, both nestedness and turnover appear to contribute to overall differences in infectious disease assemblages among countries at a global scale, with the relative contribution varying between major epidemiological classes (murray et al. ) . for example, nestedness dominates differences in human-specific diseases, whereas turnover dominates differences in zoonotic and vector-borne diseases. biogeographic methods and outputs have already contributed and continue to show great promise for a number of health management or research applications on infectious diseases, which could help direct the allocation of scarce public and global health resources more efficiently and effectively. as our abilities to assemble ecological datasets and conduct infectious disease surveillance and analyses are steadily improving, multidimensional ecological data can be mapped and relationships can be identified as data accumulate in close to real-time, providing decision-relevant information for health managers and researchers to respond to. this has already lead to rapid advances in improving disease mapping for single infectious diseases and a closing of the gap between the data types and methods used to characterise disease and species distributions by medical geographers and ecologists, respectively. many other applications are conceivable albeit so far poorly explored. for example, taking inspiration from conservation and ecological applications, diversity analyses and biogeographic regionalisation could be used to test and propose hypotheses about ecological factors and historical events that could underlie the current organization of disease assemblages or . colours represent statistically supported groups (n  groups) of countries that share similar diseases, as derived by evaluating results from the silhouette, elbow, ch index and gap statistic tests; (b) global pathogeographic realms for zoonotic diseases derived from (a) (colours for mapping match country clusters identified in (a)). legend labels indicate the statistically supported regions (first column) and how these align with 'classic' zoogeographic realms (second column) (note although the realm label for nearctic includes greenland for illustration purposes, the 'islands' group (dark blue) actually includes a large number of small islands plus a few other countries scattered globally (a) that may cluster on the basis of being a depauperate or data deficient group); (c) the relative explanatory value of a range of social and environmental covariates for explaining these global patterns in disease beta diversity, illustrating that mammalian biodiversity is the best predictor of zoonotic disease diversity at a global scale (as derived from a relative importance analysis following multiple regression on distance matrices controlling for the effects of spatial autocorrelation (following murray et al. ( ) ). combined disease risks (e.g. identifying processes of disease dispersal, establishment, and extinction and the 'upstream' risk factors or drivers of novel health threats) (following carmona et al. , báez et al. ; to define contexts for representativeness (e.g. improved disease surveillance design) (following austin and margules , carey et al. , mackey ; to provide consistent units for environmental management and for sampling stratification (e.g. for the optimal discovery of novel pathogens) (following bunce et al. , wright et al. ; and as geographic contexts for imputation/extrapolation or forecasting when data from a unit within a region is missing or unavailable (e.g. where disease surveillance coverage is low or patchy) (cooper and nunn ) . biogeographic approaches may also be useful for examining the risks associated with emerging infectious diseases (eids), since data are often extremely limited on eids and yet the priorities for management revolve around anticipating (through forecasting) when, where and why emergence of pathogens in human populations occurs (peterson , morse et al. . large-scale demographic and environmental factors and changes in these factors are increasingly being recognized as key drivers underlying disease emergence, with shifts in the distributions of disease hosts and vectors being central to this process (jones et al. , semenza et al. . given that most pathogen distributions are very poorly characterized or completely unknown (hay et al. ) , and that most of the microbial diversity from which novel and potentially pathogenic agents could originate are as yet undiscovered (anthony et al. ) , biogeographic pattern definition and process identification based on historical patterns of disease occurrence, recent emergence events, or proxy taxa (e.g. mammalian or arthropod vector biodiversity) may provide some of the earliest and in some cases the only insights into such burgeoning or future disease risks (fig. a , b) (murray et al. ) , which may give way to more refined models as data quality or availability increases (fig. c, fig. ). increasing pathogeographic awareness, participation and collaboration among ecologists, biogeographers and veterinary and medical practitioners could thus contribute to closing the gap between environmental and health management, increased inter-disciplinary research and management efficiency, and reductions in the global burden of disease. data available from the dryad digital repository:  http:// dx.doi.org/ . /dryad.p n dv  (murray et al. ) . fig. for raw model output and description). yellow indicates countries that contain some environmentally suitable areas for ebola. darker colour indicates countries that do not contain areas predicted to be suitable for ebola. the overlap in top priority countries between (b) and (c) is ~ %. the approach taken in (a/b) requires no specific information about the target disease and could provide a relevant biogeographic 'prior' for 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and reemerging pathogens. -emerg on the meaning and measurement of nestedness of species assemblages ecoregions as a level of ecological analysis global distribution of outbreaks of water-associated infectious diseases potential geographic distribution of the novel avian-origin influenza a (h n ) virus supplementary material (appendix ecog- at  www. ecography.org/appendix/ecog- ) key: cord- -yl emjef authors: moro, loredana title: mitochondria at the crossroads of physiology and pathology date: - - journal: j clin med doi: . /jcm sha: doc_id: cord_uid: yl emjef mitochondria play a crucial role in cell life and death by regulating bioenergetic and biosynthetic pathways. they are able to adapt rapidly to different microenvironmental stressors by accommodating the metabolic and biosynthetic needs of the cell. mounting evidence places mitochondrial dysfunction at the core of several diseases, notably in the context of pathologies of the cardiovascular and central nervous system. in addition, mutations in some mitochondrial proteins are bona fide cancer drivers. better understanding of the functions of these multifaceted organelles and their components may finetune our knowledge on the molecular bases of certain diseases and suggest new therapeutic avenues. mitochondria are semi-autonomous organelles with a double membrane system, namely the inner and the outer mitochondrial membrane that delimit the intermembrane space. the inner mitochondrial membrane demarcates the matrix, a viscous microenvironment that contains several enzymes catalyzing a plethora of anabolic and catabolic reactions. mitochondria contain their own genome, the mitochondrial dna (mtdna), a circular double-stranded dna molecule of , bp in humans, which encodes only mitochondrial proteins belonging to the electron transport chain (etc), transfer rnas and ribosomal rnas needed to carry out the mitochondrial protein synthesis. all the other mitochondrial components are encoded by the nuclear genome. mitochondria are the energy powerhouses of the cell, being responsible for % of energy production in the form of atp by coupling the flux of electrons throughout the mitochondrial respiratory complexes i-iv with oxidative phosphorylation (oxphos). in brief, complete oxidation of nutrients through the tricarboxylic acid cycle (tca) within mitochondria produces reduced coenzymes (nadh, fadh ) that act as electron donors. the flux of electrons through the mitochondrial respiratory chain complexes produces an electrochemical gradient used by the mitochondrial respiratory complex v to generate atp. notably, the function of mitochondria in cell physiology goes beyond their role as energy producers and metabolic regulators. indeed, these multifaceted organelles play a pivotal role in the modulation of cell death pathways and intracellular signaling [ ] . the etc is also the main cellular source of reactive oxygen species (ros), owing to an incomplete reduction of oxygen by complex i and complex iii. mitochondrial ros production can lead to oxidative damage to proteins, membranes and dna, thus impairing the ability of mitochondria to carry out their biosynthetic and catabolic reactions, including the tca cycle, heme synthesis, fatty acid oxidation, the urea cycle and amino acid metabolism [ ] . mitochondrial oxidative damage can also promote permeabilization of the mitochondrial outer membrane (momp), resulting in release of intermembrane space proteins, such as cytochrome c, and activation of the mitochondrial apoptotic pathway. furthermore, mitochondrial ros production promotes the opening of the mitochondrial permeability transition pore (mptp), leading to permeabilization of the inner mitochondrial membrane to small molecules in pathological conditions, such as during ischaemia (loss of blood flow) and subsequent reperfusion [ ] . two mitochondria quality control mechanisms are in place to meet the functional needs of any given cell under different physiological and pathological conditions: (a) mitochondrial biogenesis, fusion and fission [ ] [ ] [ ] ; (b) mitophagy [ , ] . the first mechanism is a balanced process that allows maintenance of the physiological mitochondrial homeostasis when cells face metabolic or microenvironmental stresses [ ] . mitochondrial fission guarantees an adequate distribution of mitochondria in dividing cells. mitochondrial fusion allows complementation between dysfunctional mitochondria within the cell to maximize mitochondrial performance in response to stress. three gtpases, mitofusin (mfn ), mfn , and optic atrophy (opa ), are primarily involved in the regulation of mitochondrial fusion. instead, mitochondrial fission is mainly controlled by the gtpase dynamin-related protein (drp ) [ ] . disruption of the balance between fusion and fission is associated with neurodegenerative diseases, such as parkinson's, and cancer [ , ] . the second mechanism, mitophagy, is a specific form of autophagy that removes damaged mitochondria and reduces the mitochondrial mass upon microenvironmental stresses, such as hypoxia and nutrient starvation, promoting cell survival [ ] . mitophagy dysregulation has been implicated in cancer development and progression [ ] , neurodegeneration [ ] and cardiovascular diseases [ ] . mitochondrial dysfunction can lead to an array of diseases. depending on the nature of the defect leading to mitochondrial dysfunction, primary and secondary mitochondrial diseases can be distinguished. primary mitochondrial diseases develop as a consequence of germline mutations in mtdna and/or nuclear dna genes that encode proteins affecting mitochondrial functionality and energy production, including etc proteins and proteins involved in mtdna replication, such as polg. the first primary mitochondrial disease was described in [ ] and involved a -year-old woman displaying excessive perspiration, polyphagia, polydipsia without polyuria, asthenia and decreased body weight, symptoms that started when she was seven years old. in addition, her basal metabolic rate was + %, and she presented with creatinuria, myopathy and pathological cardiomyogram. she was diagnosed with a disorder of the enzymatic organization of the mitochondria. studies with mitochondria isolated from the skeletal muscle of this hypermetabolic patient revealed oxphos uncoupling [ ] . since then, a range of primary mitochondrial diseases has been described (reviewed in [ ] ). secondary mitochondrial defects can be caused by germline mutations in genes not involved in respiration/oxidative phosphorylation or can be acquired during the lifetime upon environmental insults. notably, environmental stress can induce mtdna alterations leading to mitochondrial dysfunction during aging, inflammatory response, etc. [ , ] . from a pathological point of view, primary and secondary mitochondrial diseases can cause very similar symptoms, sometimes making diagnosis difficult. at the molecular level, mitochondrial dysfunction can affect the levels of key intracellular signaling regulators, such as ros and ca + , that can be transmitted to the nucleus (mitochondria-to-nucleus signaling or retrograde signaling) resulting in changes in gene expression and modulation of a range of cellular functions [ , [ ] [ ] [ ] . in addition, the release of mtdna and peptides from the mitochondrial matrix can activate an immune response that promotes a pro-inflammatory cascade [ ] . mitochondrial metabolites can also act as signaling molecules and epigenetic modulators. in this context, citrate, an intermediate of the tca cycle, represents the major source of acetyl-coa for protein acetylation, a co-and post-translational modification that regulates protein levels and intracellular signaling in physiological and pathological conditions [ ] . emerging data have also provided new evidences of connections between mitochondrial dynamics and physical contacts among mitochondria and the endoplasmic reticulum (er), known as mitochondrial-associated er membranes (mams), which can finetune the mechanisms of regulation of energy production, ca + homeostasis, survival and apoptosis [ ] . here, a synthetic overview of the role of mitochondria in specific physiopathological conditions is provided ( figure ). cardiovascular diseases are a leading cause of death worldwide. this class of diseases comprises several pathologies, including ischemic heart disease, peripheral vascular disease, cardiac arrest, heart failure, cardiomyopathies, hypertension, atherosclerosis, and arrhythmia. mitochondria have been involved at various degrees in the pathological aspects of these diseases. notably, mitochondrial dysfunction of muscle cells represents a key event in the prognosis of peripheral arterial disease. reduced oxphos activity due to etc impairment increases ros levels and ca + release from mitochondria, causing apoptosis [ ] . however, if ros levels remain below a threshold, the cells activate a defense program involving production of antioxidants and increased mitochondrial biogenesis. these mechanisms, known as mitohormesis, can limit the damage caused by repeated cycles of ischemia-reperfusion in peripheral arterial disease [ ] . pharmacological treatments that can improve mitohormesis might be a promising therapeutic approach for peripheral arterial disease and other cardiovascular diseases. disruption of mitophagy also exacerbates the development of cardiovascular diseases [ ] . growing evidence indicates that the pharmacological targeting of the mitochondria with drugs/natural compounds able to modulate mitophagy can ameliorate cardiovascular disorders in patients and be cardioprotective [ , ] . future studies that aim at a better understanding the pathogenesis of some cardiovascular diseases are crucial to develop mitochondria-targeting drugs in the clinic. inflammation is a complex, protective body response to infections and tissue damage. the inflammatory response signals the immune system to repair damaged tissue and defend against pathogens (viruses, bacteria, etc.) or other harmful stimuli through secretion of specific mediators. however, when inflammation persists, it may drive various diseases and tissue damage. mitochondrial-derived ros play a key role in the inflammatory response. notably, mitochondria are considered the main drivers of the nlrp (nod-, lrr-and pyrin domain-containing ) inflammasome [ ] [ ] [ ] [ ] , representing a central hub that controls innate immunity and response to inflammation. among various inflammatory conditions, mitochondria are involved in the hyper-inflammatory response, also reported as cytokine storm, caused by the sars-cov- (covid- ) respiratory cardiovascular diseases are a leading cause of death worldwide. this class of diseases comprises several pathologies, including ischemic heart disease, peripheral vascular disease, cardiac arrest, heart failure, cardiomyopathies, hypertension, atherosclerosis, and arrhythmia. mitochondria have been involved at various degrees in the pathological aspects of these diseases. notably, mitochondrial dysfunction of muscle cells represents a key event in the prognosis of peripheral arterial disease. reduced oxphos activity due to etc impairment increases ros levels and ca + release from mitochondria, causing apoptosis [ ] . however, if ros levels remain below a threshold, the cells activate a defense program involving production of antioxidants and increased mitochondrial biogenesis. these mechanisms, known as mitohormesis, can limit the damage caused by repeated cycles of ischemia-reperfusion in peripheral arterial disease [ ] . pharmacological treatments that can improve mitohormesis might be a promising therapeutic approach for peripheral arterial disease and other cardiovascular diseases. disruption of mitophagy also exacerbates the development of cardiovascular diseases [ ] . growing evidence indicates that the pharmacological targeting of the mitochondria with drugs/natural compounds able to modulate mitophagy can ameliorate cardiovascular disorders in patients and be cardioprotective [ , ] . future studies that aim at a better understanding the pathogenesis of some cardiovascular diseases are crucial to develop mitochondria-targeting drugs in the clinic. inflammation is a complex, protective body response to infections and tissue damage. the inflammatory response signals the immune system to repair damaged tissue and defend against pathogens (viruses, bacteria, etc.) or other harmful stimuli through secretion of specific mediators. however, when inflammation persists, it may drive various diseases and tissue damage. mitochondrial-derived ros play a key role in the inflammatory response. notably, mitochondria are considered the main drivers of the nlrp (nod-, lrr-and pyrin domain-containing ) inflammasome [ ] [ ] [ ] [ ] , representing a central hub that controls innate immunity and response to inflammation. among various inflammatory conditions, mitochondria are involved in the hyper-inflammatory response, also reported as cytokine storm, caused by the sars-cov- (covid- ) respiratory infection ( [ ] and references therein). when macrophages and other immune cells detect viruses, they start secreting cytokines and chemokines to communicate with other immune cells [ ] . strikingly, wuhan's covid- patients with severe clinical symptoms requiring icu admission displayed higher levels of the cytokines/chemokines ccl , tnf-α and cxcl compared to individuals with less severe symptoms [ ] . the release of large quantities of pro-inflammatory cytokines and chemokines by overdriven immune effector cells sustains an aberrant systemic inflammatory response that results in the immune system attacking the body, which in turn causes the acute respiratory distress syndrome [ ] . immune cells under a hyper-inflammatory state metabolically adapt to this stress condition by favoring aerobic glycolysis over oxphos for energy production. this metabolic rewiring allows macrophages to become more phagocytic and favors anabolic reactions for the synthesis and secretion of cytokines and chemokines in a vicious cycle ( [ ] and references therein). side by side, many biosynthetic reactions occurring in mitochondria of hyper-activated macrophages are inhibited as a consequence of oxphos and tca cycle inhibition. melatonin's synthesis is among these reactions: acetyl-coa, a cofactor in the rate-limiting reaction for melatonin synthesis, lacks due to the tca cycle inhibition [ ] . thus, melatonin cannot be synthetized. notably, melatonin is a potent anti-inflammatory and anti-oxidant and its administration to covid- patients has been recently proposed as potential adjuvant treatment strategy to reduce the severity of the covid- pandemic [ ] [ ] [ ] . though clinical evidences are not yet available, several scientific data supports the potential utility of melatonin to attenuate the worst symptoms of covid- infection [ , ] . mitochondrial dysfunction has long been recognized as a driver of the aging process. early studies have linked accumulation of mitochondrial dna mutations and the concomitant decline in etc and oxphos activity to aging [ , ] . furthermore, genetic studies in mice support a causal relation between mtdna depletion and aging [ ] . recent evidences have confirmed that healthy centenarians retain more "intact" mtdna copies than old people and frail centenarians [ ] , suggesting that "healthy" mtdna is a hallmark of healthy aging. besides the mtdna status, activation of mitochondria-to-nucleus signaling pathways, particularly the mitochondrial unfolded protein response (upr mt ), has been implicated in aging. upr mt activation promotes transcription of several nuclear genes, such as those encoding antioxidant proteins and enzymes, which support survival, gain of the mitochondrial functionality and, thus, longevity and lifespan [ ] . it should be noted that if a heteroplasmic mtdna pool is present, upr mt activation could exacerbate mitochondrial dysfunction as it may lead to accumulation of mutant mtdna [ ] . alterations in the removal of damaged mitochondria through mitophagy have also been implicated in aging. mitophagy markedly decreases during aging in mammalian tissues and organs [ , ] and this may be responsible for the known accumulation of damaged mitochondria in aging tissues. notably, genetic manipulations in c. elegans that increase mitophagy also extend the organismal lifespan [ ] , strengthening the connection between altered mitophagy and aging. neurodegenerative diseases are characterized by changes in mitochondrial morphology and biochemical activity. alzheimer's (ad) and parkinson's (pd) disease are the most diffuse neurodegenerative illnesses among older adults. brain cells from ad and pd patients show reduced respiratory activity and mitochondrial biogenesis [ , ] . a prominent pathological feature of ad is the impaired cerebral glucose metabolism, which is reduced by % in the early stages, preceding neurological impairment and atrophy, and further declines in the late stages of the disease [ ] . the decrease in glucose metabolism is associated with reduced expression and activity of mitochondrial enzymes, including pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, three enzymes of the tca cycle [ ] . in addition, reduced activity of the mitochondrial respiratory complexes i, ii, iii and iv has also been documented [ ] . somatic mutations in the mitochondrial genome have been detected in postmortem brain tissue from ad patients, at levels higher than in healthy brains [ ] . these mutations may not only affect the etc but also trigger other neuropathological consequences, such as increased ros production and oxidative stress in neurons and promotion of amyloidogenic processing of the amyloid precursor protein. mitophagy is also diminished in ad's neurons, and this may contribute to the etiopathogenesis of ad. indeed, mitophagy was able to prevent or reverse the cognitive impairment in several ad models [ ] , confirming the critical involvement of mitochondria in ad. mutations in nuclear genes encoding mitochondrial proteins important for the proper function of mitochondria have been directly linked to pd. notably, mutations in proteins involved in mitochondrial quality control, such as pink , parkin and lrrk , are a frequent cause of monogenic pd [ ] . loss or impaired functionality of these proteins results in mitochondrial fragmentation, dysregulation of calcium homeostasis and changes in mitochondria-endoplasmic reticulum contact sites (mercs). recently, mutations in miro , a protein important for the regulation of the structure and function of mercs, have been causally linked to pd establishing that variants in the gene encoding for miro represent rare genetic risk factors for neurodegenerative diseases like pd ( [ ] and references therein). although there is no doubt about the involvement of mitochondrial dysfunction in ad and pd, still more research is required to identify therapeutic targets that could improve mitochondrial activity and reduce oxidative stress in neurons in the early stages of these neurodegenerative diseases. future studies should be aimed at investigating the chronological sequence of molecular events involved in the pathogenesis of these diseases. further investigations are also needed to assess whether mitochondrial dysfunction represents a primary cause of ad or a consequence of other molecular/genetic events. mitochondrial dysfunction has been involved in different aspects of the pathogenesis of cancer, from the early steps of cancer development to cancer progression to a metastatic phenotype, and resistance to anti-cancer drugs [ , , ] . in this context, mutations in three tca cycle enzymes, namely succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase, have been shown to play a causal role in carcinogenesis [ , ] , thus providing compelling evidence for the involvement of mitochondrial metabolic alterations as cancer drivers. indeed, mutations in succinate dehydrogenase predispose to hereditary paragangliomas, pheochromocytomas, neuroblastomas, gastrointestinal tumors, renal cell cancers and thyroid tumors [ ] . sporadic and hereditary mutations of fumarate hydratase trigger accumulation of an oncogenic metabolite, i.e., fumarate, that favors development of hereditary leiomyomatosis and renal cell carcinoma, ewing sarcoma and osteosarcoma, adrenocortical carcinoma, pheochromocytoma, glioma, neuroblastoma, paraganglioma, and ependymoma [ ] . mutations in isocitrate dehydrogenase are only somatic and have been detected in about % of patients with acute myeloid leukemia or angioimmunoblastic t-cell lymphoma, and at lower frequencies in patients with thyroid, prostate, colorectal cancer and b-cell acute lymphoblastic leukemia [ , ] . besides mutations in nuclear-encoded mitochondrial proteins, mutations in mtdna-encoded proteins have also been implicated in the pathogenesis of cancer. the spectrum of somatic mtdna mutations varies among different tissues, and increasing evidence shows that the load of mtdna mutations could have prognostic value. the majority of cancer-related mtdna mutations have been found in prostate cancer, with a total of more than unique somatic mtdna mutations associated with this cancer [ ] . there is increasing evidence that mtdna mutations/depletion may favor cancer progression to a metastatic and drug-resistant phenotype through increased production of ros and/or activation of a mitochondria-to-nucleus signaling that leads to expression of pro-metastatic and pro-survival nuclear genes [ , , [ ] [ ] [ ] . although mtdna damage may not be the first driver of cancer progression, it is likely that it represents a "supporter" event that facilitates and accelerates different steps of the metastatic cascade, probably within a precise time window that remains to be identified. mitochondrial dysfunction is implicated in several pathological conditions, ranging from neurodegenerative and cardiovascular diseases, to aging, cancer and inflammation. each of these conditions shows a peculiar involvement of mitochondria. for example, up to % of pd patients show a defect in miro function, because this protein, located on the mitochondrial surface, fails to detach from depolarized mitochondria resulting in defective mitochondrial locomotion and clearance by mitophagy [ ] . these new results suggest that miro -based therapeutic strategies may provide new avenues to a personalized medicine for pd. the role of mitochondrial dysfunction in other diseases is still somehow controversial. in some cases, it may represent a driver event, like for mutations in the tca cycle enzymes succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase that predispose to certain types of tumors. in other 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ebola virus disease (evd) is a severe hemorrhagic disease caused by ebola virus. several outbreaks have been reported in africa and often originated from remote agrarian communities where there are enormous misconceptions of the disease, refusal of early isolation and quarantine, and unsafe burial rites practices which aggravates the epidemics. it is on this basis that this study was conducted to (assess) the knowledge, perceptions, beliefs and preventive practices against evd in a predominantly agrarian rural community in southwest nigeria. methods: this was a cross-sectional study conducted in igbogila town, yewa north local government area of ogun state, southwest nigeria in the latter part of during the evd outbreak. mixed methods were used for data collection. quantitative data collection was done using a pre-tested interviewer administered questionnaire. four hundred and seven respondents selected by multi-stage sampling technique were interviewed. descriptive and inferential statistics were done, and the level of significance was set at . . qualitative data collection involved four focus group discussions a year after the epidemic was declared over in the country. the discussions were recorded, transcribed and analyzed along major themes. results: all respondents were aware of evd with radio and television being the major sources of information. knowledge of the disease was however very poor with many misconceptions and it was significantly influenced by educational level of respondent. evd survivors will be welcomed back into the community by few residents ( . %) and a much fewer proportion ( . %) will freely entertain a survivor in their house. most would prefer local herbalists over orthodox medical practitioners to care for their loved one in case they contract evd. although respondents knew that burying a victim is dangerous, they opposed cremation. conclusion: there was poor knowledge of evd with a lot of misconceptions. community members were not pro-active about prevention with dire consequences in the event of an outbreak. continuous public education should be done via mass media, traditional institutions and other community-based channels as part of emergency preparedness. ebola virus disease (evd) is a severe hemorrhagic disease caused by ebola virus: a non-segmented, enveloped, negative-strand rna virus [ ] . the first case of evd was identified in , since then, several outbreaks have been reported in africa [ ] . in the last years, an outbreak of evd has been reported at least every years [ , ] . in ; the deadliest, most widespread (affected ten countries), evd outbreak that lasted approximately years occurred making it a global emergency [ ] . current corona virus disease (covid- ) pandemic has again brought to the fore, the need for countries to maintain a high standard of preventive measures and preparation for emergency response for any emerging or reemerging infectious disease. populace needs to be enlightened on evd preventive measures such as maintenance of careful hygiene (washing hands with soap and water or an alcohol-based hand rub), avoiding contact with non-human primates and bats, avoiding contact with infected person's body fluids or infected items, and avoiding funeral or burial rituals that require handling of the body of someone who has died from evd (confirmed or suspected) [ ] . public health response to evd outbreak include: case finding (suspected, probable and confirmed), contact tracing, isolation and early quarantine, treatment of symptomatic cases, and ensuring appropriate burial for the deceased [ ] . however, a closer look at past evd outbreaks revealed that they often originated from rural agrarian communities where there are many misconceptions about the disease, refusal of early isolation and quarantine, and unsafe burial rites practices which aggravate epidemics [ , ] . it is on this basis that this study was conducted to assess the knowledge, perception, beliefs and preventive practices among residents of an agrarian community in ogun state, southwest nigeria. findings will provide useful information to aid future outbreak prevention and control as well as emergency preparedness efforts. this was a descriptive cross-sectional study which employed mixed-method (quantitative and qualitative) approach in data collection. the study setting was igbogila town, ibeshe ward, yewa north local government area of ogun state, southwest nigeria. yewa north is located at the west end of ogun state sharing border with benin republic (a neighboring country). igbogila is predominantly rural and agrarian with many of the residents engaged in agro-forestry related occupations. at the time of the study, the town had one primary healthcare centre, two public secondary schools, five public primary schools, one major market, few churches and mosques. only residents between and years that had been living in the study area for at least months prior to the study participated in this study. quantitative data were collected during the ebola epidemic in nigeria (july -september ). sample size was determined using cochran's formula (n = z p( -p)/ e ) [ ] . the calculation was based on: prevalence of good knowledge (p) of % obtained from a similar study [ ] , standard normal deviate (z) at % confidence being . and % margin of error (e) resulting in a minimum sample size of . this was increased by % ( ) to make up for non-responses and incomplete questionnaires giving a total sample size of . multi-stage sampling was used to select the respondents. in the first stage, one ward (ibeshe) was selected from the eleven wards in yewa north using simple random sampling technique (balloting). in the second stage, one town (igbogila) was selected from the seven towns in ibeshe ward. igbogila comprises nine smaller communities which were all included in the study. respondents were equally allocated to the communities i.e. about respondents were required from each community. the third stage involved the selection of houses following enumeration and systematic sampling of houses. the houses in the communities largely had no numbering system, so, the research team carried out house numbering. in the fourth stage, households were selected from the houses. only one household was selected per house (simple random sampling (balloting) was used to select one when there were more than one household). in the final (fifth) stage, respondents were selected from households. only one respondent that met the inclusion criteria was interviewed per selected household (simple random sampling (balloting) was used to select only one respondent when there were more than one eligible respondent). respondents were interviewed face-to-face using a pre-tested interviewer administered questionnaire adapted from similar studies [ , ] . eight research assistants who were fluent in yoruba, english and 'pidgin' english were trained for data collection. the questionnaire sought information on socio-demographic characteristics, awareness, knowledge, attitude and perception of evd. knowledge was assessed using questions on cause, transmission, symptoms, prevention and cure of evd. perception and attitude to evd were assessed using respondents' agreement or disagreement to a set of likert statements. data were coded, entered and analyzed using epi info™ . statistical package [ ] . descriptive statistics (frequency, mean and standard deviation) and inferential statistics (chi-square test) was used to test association between categorical variables. level of significance was set at %. in the knowledge section, each correct response given by respondents was allotted one point. overall knowledge was assessed using five domains: cause ( point), transmission ( points), symptoms ( points), prevention ( points) and cure ( point). this gives a total maximum score of points converted to percentage. using % cut-off point; respondents with total score < % were graded as 'poor knowledge' while those with > % were graded as 'good knowledge'. attitude was scored using three points likert scale; the maximum obtainable score was and the least was . using the mid-point ( ) as cut-off point, respondents with score < were graded as having "poor attitude" while those with scores > were graded as having "good attitude". for the qualitative aspect, focus group discussions (fgds) were conducted in november , about a year after the epidemic was declared over by who [ ] . the main purpose for the fgds was to explore explanatory models for the disease in rural communities and their preventive practices against an outbreak. according to the who, this is important in any epidemic preparedness and response [ ] . discussants were approached face-toface and selected into one of four groups: higher secondary education students ( discussants), females of reproductive age ( discussants), adult male ( discussants), elderly female ( discussants). fgd participants were selected by purposive sampling as discussants in each group were selected to be of the same gender and about same age as suggested by ritchie and lewis qualitative research framework [ ] . in each group, one of the discussants volunteered his/her home for the discussion. fgds were moderated by the principal researcher with the assistance of one note taker and a time-keeper. each session lasted for about h. discussions were held mainly in local (yoruba) language understood by all the participants and taperecorded in addition to notes. each discussant was assigned a number. at the end of each session, discussants were given light refreshments. the recordings were later translated and transcribed in english. data was saturated in domains of cause, and spread of evd, but, unsaturated in domains of treatment. thematic analysis was done manuallyrecurring themes from the data were identified, emerging patterns noted, and report written based on these identified patterns. for the purpose of presentation, the groups were coded as follows: higher secondary school students (hs), adult males (am), older females (of), and women of reproductive age (rf). participation was voluntary and formal consent was obtained from each participant. respondents were informed of their right to withdraw at any point of the study without prejudice in line with helsinki declaration [ ] . a total of respondents completed their interviews while respondents withdrew their participation, making the response rate to be . %. mean age of respondents was . + . years with slightly more males (n = , . %). almost one-quarter, ( . %) had no formal education and half (n = ) earning less than , naira monthly (less than us dollars) ( table ) . all the respondents were aware of evd. majority, ( . %) heard of it through radio and television, ( . %) got to know from their friends or family members. print media such as newspapers and flyers were not reported as a source of information by the respondents. 'dirty environment' was the most commonly mentioned cause of evd ( . %), only ( . %) knew that a virus is implicated. few ( . , . and . % respectively) knew that eating poorly cooked bush meat or contact with non-human primates or contact with body fluids of infected persons pose risk of evd transmission. a minority knew fever ( . %), vomiting ( %) and headache ( %) as symptoms of evd. (table ) . a third ( . %) knew hand washing and avoidance of contact with non-human primates as preventive measures while only ( . %) knew that avoiding funeral or burial rituals involving contact with victims' corpses is a preventive measure (table ) . neither age nor sex of the respondents significantly influenced their knowledge of evd. however, those respondents with at least secondary education were more likely to have good knowledge of evd (p = . ) ( table ). a majority ( . %) believed that evd really exists and ( . %) perceived it to be very fatal. while ( . %) agreed that it is not curable, ( . %) believed that victims could survive if given prompt medical intervention. almost a quarter, ( . %) saw it as a political ploy that government officials wanted to use to embezzle funds and only about half ( . %) thought that the country was truly ebola free (table ). in respect to their attitude; ( %) reported that they would accept to be quarantined if they were found to have had close contact with a case, ( . %) would support and empathize with a friend or relative who is infected, however, only two-fifths ( %) would buy from a shopkeeper who has recovered from evd and even a lesser proportion ( . %) would welcome a survivor back to the neighbor-hood. only ( . %) would entertain a survivor in their homes. overall, respondents ( %) had a good attitude towards evd ( table ). sociodemographic characteristics of discussants the mean age of the fgd participants was + . years, ( %) were females, ( %) had no formal education while ( %) had tertiary education. the participants were largely farmers ( %), and petty traders ( %). one discussant was a herbalist (table ) . the recurring themes on how ebola disease can be contacted were: eating infected bush meat, unprotected contact with infected persons, and intercourse with multiple sexual partners. "it is gotten by coming in contact with infected animals, animals such as bats and bush meats" -hs ( years old male student). "the disease is catching whoever has sex with prostitutes …" -am ( year old bricklayer). more respondents believed that local herbalists know the cure for ailment. "i will call a herbalist to come and treat the person (a case) at home" -rf ( years old female farmer). "i believe that they are lying by saying there is no cure for the disease … … , if the victim is taken to good traditional healers, the person will be cured" -hs ( year old male student). when asked how best to handle the corpse of a close relative that died of evd, it was evident that the people knew that burying someone with evd is not without any risk. "i will not move close to the corpse. the people who died of the disease are usually burnt but i cannot allow my own dead family member to be burnt. i will just call them at the centre (primary health facility in the area) to come and help me bury the corpse" -hs ( year old male student). while some ( out of the ) of the respondents did not stop eating bush-meat (bats inclusive), many ( out of ) of the respondents stopped eating bush meat. the precautionary measure was however for a while as it was found that these respondents that initially restrained their intake of bush meat had resumed its consumption. "i stopped eating bush meat and bat, but when i later saw that people who ate bush meat did not die, i started eating them back" -am ( year old male farmer). the important theme that emerged on preventive measures for evd was the use of salt water. some respondents bathed with salt water, drank and mandated its use in their family till they experienced related adverse effects. "i bathed with salt water as instructed by my father" -hs ( years old male student). "my six children and myself used salt water to bath for some days but stopped when we started having skin rashes" -of ( year old female trader). their current preventive practices were explored (without prompting). majority of the respondents confessed that they were eating bush meat as before. they were mostly not taking any pro-active preventive measures to prevent evd such as limiting close physical contact or direct contact with bush animals. "i am not doing anything. i am eating bush meat …." -rf ( year old female farmer). "i am not doing anything … … i did not need to bother myself" -of ( year old female). at the outset of the evd outbreak, the nigerian government embarked on widespread health campaign with major attention on mass media. the mass-media platforms successfully raised evd awareness as all the rural dwellers in this study were aware of evd and they indicated that radio and television were their main sources of information. mass-media played similar pivotal role in purveying awareness for residents of urban communities in lagos, nigeria [ ] and for locals at epicenters in sierra leone [ ] . however, the high level of awareness did not translate to better knowledge of the disease. most respondents had poor knowledge riddled with many misconceptions. for instance, most of them either did not know the cause of evd or misconceived the cause to be dirty environment. there are evidences that have implicated bush-meats especially non-human primates e.g. bats in the spread of evd, yet only few ( %) knew that evd is spread by contact with infected non-primate animals [ , ] . the prominent misconception of the cause of evd as revealed in the fgd was the belief that ebola disease is acquired by leading a promiscuous lifestyle. this apparent disparity between biomedical and traditionally perceived etiology could stymie prevention in the event of another outbreak because based on etiological variances, local perception of prevention will conflict with orthodox suggestions [ ] . apart from the misconception of cause of evd, the knowledge of community-based modes of transmission (from infected individual to others, and from infected fomites/objects to man) of evd were also less known among the residents of the agrarian community. this is worrisome because during outbreaks, community-based transmissions are responsible for most secondary cases and thus responsible for perpetuating the spread of infection [ ] . the knowledge of prevention of evd was also found to be inadequate. more than % did not know that; avoiding direct contact with people, frequent hand washing, avoiding contact with non-human primates' body fluids and blood, and avoiding contact with infected items are precautionary measures. when the respondents were asked how they will handle the corpse of a relative that died of evd; it was evident that the people knew that burying someone with evd is not without risk but they opposed cremation -"… i cannot allow my own deceased family member to be burnt". cremation is rejected because it is not culturally acceptable in most parts of west-africa where autochthonous residents strongly believe that deceased soul will haunt living relatives if not given a traditionally acceptable burial [ ] . this has potential to impede effective burial of dead cases and it can aggravate epidemics as evidenced by catastrophic events that followed unsafe burial of cases at the early stages of the outbreak (in sierra leone and liberia) [ , ] . it may be beneficial to gradually institute interventions involving anthropologists and traditional institutions to discuss and relay such messages at the grass root level. exploring the respondent's knowledge of cure of evd, it was found that although some ( . %) knew that there is no cure for the disease, yet, most preferred local herbalists over orthodox medical practitioners to care for their loved one in case he/she contacts evd. being a rural setting, this is not surprising. the rationale behind this preference is the fear of having their relative isolated from them: "… ..once they carry the person (victims) away from you, you will not be allowed to see them again …" the discussants' preference of local herbalist over medical practitioners is another cause for concern as such misconceptions had made people in gulu district, uganda to resort to traditional practices such as 'ryemo gemo' rituals (wild shouting, jumping and running into nile river), 'chani labolo' rituals (slaughtering and littering intestines of several goats on ground) in kotido district of uganda, all in an attempt to 'cure' the disease. such practices only enhanced the spread of the disease and complicated the economic cost of the outbreak [ , ] . this also has implications for other highly infectious diseases such as lassa fever and covid- that require isolation of confirmed positive cases as part of containment. in such situations, similar preference for alternative treatment options may negatively impact control efforts. the factor that was found to significantly influence participants knowledge about evd was their educational status. the agrarian community dwellers with at least secondary education in this study were more likely to have good knowledge of evd compared to those with only primary or no formal education. this highlights the need to increase education coverage in local communities as the level of education of the populace could play an important role in determining the magnitude of spread as modelled by outcomes in two separate outbreaks in sudan [ ] . most respondents indicated stigmatizing attitudes towards evd survivors. a total of % stated that they will not buy any goods from a survivor, many expressed that they will not welcome a survivor back into the community nor allow survivor into their house. these discriminatory statements were similar to the initial problems local residents at ebola epicenters posed during early phases of the outbreak in liberia [ ] . the danger in this is that persons that suspect that they may have evd, and indeed any infectious disease hide it because of fear of stigmatization. this could drive disease outbreaks further. during the outbreak, the preventive method most respondents in this study observed was avoiding bush meat and use of salt water which are largely misconceptions. the use of salt water may have negative health consequences. though the exposure is there with consumption of bush meat, the key thing is close contact and method of handling during preparation of the animals. this was not really a big issue in evd outbreak in nigeria as the cases recorded were invariably linked to the imported case. the natives already exhibited poor knowledge and bush meat is commonly consumed due to their agro-forestry background hence the need for proper education. one year later, majority of the discussants stated that they had resumed bush meat consumption and were no longer taking any recommended precautions to prevent contracting evd. the main reason for this in-action could be linked to their religious belief, that 'god' protects them from 'evil diseases' like evd (table ) . unfortunately, this behavior may have serious consequences in the re-occurrence of evd outbreak in the country. the study was conducted in a setting that can be described as 'high risk' for evd outbreak. data was collected prospectively, and the mixed-method approach yielded more information necessary for understanding community explanatory models of the disease in the context of outbreak preparedness and control. the study did not emphasize on how local beliefs and practices could aid control efforts in such epidemics. more content could have been covered by adapting dunn's framework [ ] and this could be addressed in larger scale studies. the grading system adopted for measuring 'attitude' could have affected the result of the overall attitude (majority had good attitude) as their 'neutrality' was not factored into the grading system. no case of evd was recorded in the study area during the outbreak, nevertheless the limited data provides relevant information useful to researchers and other public health stakeholders in infectious disease prevention and control. the study has shown very poor knowledge of evd with misconceptions. though majority perceived the disease to be severe, some believed it was a ploy of whites against african countries and avenue for government officials to embezzle money. respondents exhibited stigmatizing attitude which may hinder control efforts in disease outbreaks. they were also not pro-active about prevention of possible future outbreak as most had gone back to harmful practices initially abandoned because the outbreak was declared to be over in the country. immunopathology of highly virulent pathogens: insights from ebola virus world health organization. ebola hemorrhagic fever in zaire, -report of an international commission outbreaks chronology: ebola virus disease emergencies preparedness response: ebola virus disease outbreak news overview, control strategies, and lessons learned in cdc response to the - ebola epidemic ebola response: package and approaches in areas of intense transmission of ebola virus. geneva: world health organization ebola viral hemorrhagic disease outbreak in west africa − lessons from uganda the ebola epidemic: a global health emergency sample size determination study on ebola virus disease knowledge, attitudes and practices of nigerians in lagos state public knowledge, perception and source of information on evd in epi infotm -a database and statistics program for public health professionals world health organization. who declares end of ebola outbreak in nigeria world health organization. recommended guidelines for epidemic preparedness and response: ebola hemorrhagic disease. geneva: world health organization qualitative research practice: a guide for social science students and researchers world medical association declaration of helsinki. ethical principles for medical research involving human subjects study on public knowledge, attitudes and practices relating to ebola virus disease prevention and medical care in sierra leone epelboin a mv. human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo information note: ebola and food safety dilemma with the local perception of causes of illnesses in central africa: muted concept but prevalent in everyday life factors that contributed to undetected spread of ebola virus and impeded rapid containment; one year into the ebola epidemic the impact of traditional and religious practices on the spread of ebola in west africa: time for a strategic shift community perspectives about ebola in bong, lofa and montserrado counties of liberia. results of a qualitative study a time for fear: local, national and international responses to a large evd outbreak in uganda cultural contexts of ebola in northern uganda modelling the role of public health education in ebola virus disease outbreaks in sudan the liberia ministry of health. national knowledge, attitudes and practices study on ebola virus disease social determinants in tropical disease springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors wish to thank the research assistants and participants for their commitment throughout the period of study. special thanks to adebolu olayinka, ogundan olayinka and taiwo toyosi for their assistance with data entry and transcription. authors' contributions abi-was involved in the conception, design, acquisition, analysis, interpretation of data and drafting and revision of the manuscript. ipo-was involved in the conception, design, analysis, interpretation of data, drafting of the manuscript and substantively revised it. eso-was involved in the interpretation of data and substantive revision of the manuscript. tjo-was involved in interpretation of data and substantive revision of the manuscript. all authors read and approved the final manuscript. there were no external funding for this study. the datasets used and/or analyzed during the study are available from the corresponding author on reasonable request. ethical approval was obtained from health research ethics committee of the lagos university teaching hospital (approval number: adm/dcst/hrec/ app/ ). informed consent (in writing) was duly obtained from participants. not applicable. the authors declare that they have no competing interests. key: cord- - oj xtp authors: khan, ali s.; amara, philip s.; morse, stephen a. title: forensic public health: epidemiological and microbiological investigations for biosecurity date: - - journal: microbial forensics doi: . /b - - - - . - sha: doc_id: cord_uid: oj xtp deliberate dissemination of a biological agent via several different routes presents the latest challenge to global public health security. novel pathogens and transmission methods can easily be exploited to cause disease outbreaks. advancements in molecular biology that make it possible to genetically modify, edit, or disrupt the genome of pathogens increase the disease risk of an accidental or intentional release of pathogens with pandemic potential. the occurrence of a disease at more than an endemic level may stimulate an investigation to determine the source of the disease, who has the disease, when it occurred, and how it spreads. when intentional release of pathogens is suspected, investigators have the additional task of attributing the outbreak not only to a pathogen but also to a human source. the deliberate nature of such dissemination may be obvious. however, some forms of bioterrorism may be more covert, requiring molecular methods to uncover. the field of microbial forensics emerged following the anthrax attack in the united states in to extend epidemiologic principles to aid in the investigation of bioterrorism incidents. microbial forensics combines epidemiology with genomic and microbiologic methods, to identify, characterize, and ascribe the cause of an incident resulting from the intentional or unintentional release of a harmful pathogen. unlike routine epidemiologic investigations, microbial forensic investigations are undertaken when there is a potential crime due to the release of a pathogen with disease-causing potential. the investigation is conducted to attribute cause to a source based on indisputable evidence and is used to support criminal charges against the perpetrator(s). however, because bioterrorism may be unannounced, the initial investigation will start the same as to any public health incident of concern. this chapter discusses how epidemiology integrated with laboratory science can be used to identify the source of diseases caused by microorganisms or toxins—especially for attribution purposes. epidemiology is the study of how disease and injury is distributed in populations and of the factors that influence this distribution (gordis, ) . more broadly, it is the study of the distribution and determinants of health-related states or events in specified populations and the application of the results of this study to control health problems (last, ) . epidemiology is based on the premise that disease, illness, and ill health are not distributed randomly in a population, and that individuals have certain characteristics (e.g., genetic, behavioral, social) that interact with the environment and predispose to, or protect against, a variety of different diseases. the specific objectives of epidemiology (gordis, ) are to (i) determine the extent of disease present in the community; (ii) identify the etiology or cause of a disease and the factors that increase a person's risk for disease; (iii) study the natural history and prognosis of disease; (iv) evaluate new preventive and therapeutic measures and new modes of healthcare delivery; and (v) provide a foundation for developing public policy and regulations. the field of microbial forensics emerged following the anthrax attack in the united states in to extend these epidemiologic principles to aid in the investigation of this and other bioterrorism incidents. microbial forensics combines epidemiology with genomic and microbiologic methods, to identify, characterize, and ascribe the cause of an incident resulting from the intentional or unintentional release of a harmful pathogen (rasko et al., ) . unlike routine epidemiologic investigations, microbial forensic investigations are undertaken when there is a potential crime due to the aforementioned release of a pathogen with disease-causing potential. the investigation is conducted to attribute cause to a source based on indisputable evidence and is used to support criminal charges against the perpetrator(s) (sj€ odin et al., ) . however, because bioterrorism may be unannounced, the initial investigation will start the same as to any public health incident of concern. this chapter discusses how epidemiology integrated with laboratory science can be used to identify the source of diseases caused by microorganisms or toxinsdespecially for attribution purposes. disease has been classically described as the result of an epidemiological triad, where disease results from the interaction between a human host, an infectious agent or toxin, and the environment that promotes the exposure (gordis, ) . in some instances, an animal or an arthropod vector such as a mosquito or tick is involved in the maintenance or transmission of the pathogen. among the assumptions necessary for this interaction to take place is that there is a susceptible host. the susceptibility of the host is influenced by a variety of factors, including genetic, nutritional, and immunological factors. bacteria, viruses, prions, fungi, and parasites responsible for disease can be transmitted either directly or indirectly (table . ). different organisms spread in different ways, and the potential of a given organism to spread and produce outbreaks depends on the characteristics of the organism and the route by which it is transmitted from person to person. diseases can be defined as endemic, epidemic, and pandemic. the usual or expected level of a disease is determined through ongoing surveillance. endemic can be defined as either the habitual presence of a disease within a given geographical area or as the usual occurrence of a given disease within such an area. epidemic can be defined as the occurrence of a disease in a community or region, clearly in excess of what is normally expected, and generally derived from a common source or from a propagated source. epidemic and outbreak are interchangeable linguistic choices used differentially to imply degrees of severity or concern. a cluster also implies an apparent excess of cases that may or may not be normal pending an epidemiological investigation or a circumscribed excess of cases when the expected number is near zero. pandemic refers to a worldwide epidemicdoften involving two or more continentsdand usually infecting numerous people. the excess incidence of cases or their widespread distribution is not synonymous with severity. many factors contribute to the emergence of infectious diseases, including human susceptibility to infection, international travel and trade, microbial adaption and change, changing ecosystems, and intent to harm (smolinski et al., ) . the ability to exploit newly created biological conditions is both the hallmark and the challenge of emerging infections (institute of medicine, ). using several . forensic public health: epidemiological and microbiological investigations for biosecurity different strategies and mechanisms, microorganisms are very efficient at infecting humans. these are exemplified both by the various strategies employed by the pathogen to survive before infecting a host, such as spore formation or survival in drought-resistant mosquito eggs, and by the various modes of transmission, such as direct contact (including large droplets) or indirect contact with fomites, or by insect vectors, and airborne via small particle droplets (heymann, ) . natural outbreaks, however, have highlighted the true diversity in the abilities of microorganisms to infect humans and animals: multistate outbreaks of salmonella linked to contaminated spring pasta salad (centers for disease control, ) and to ice cream made from milk contaminated in a tanker that had previously contained raw eggs (hennessy et al., ) , legionellosis associated with grocery store misters (mahoney et al., ) , and pneumonic tularemia on martha's vineyard from mowing over a rabbit (feldman et al., ) . these few examples are a semblance of the seemingly endless list of novel ways that pathogens are spread. changes in technology can influence pathogen dynamics through the creation of new environments in which microbes thrive (e.g., legionellosis, a disease that emerged from the water distribution systems of large buildings including cooling towers). climate change and human alteration of the ecosystem may contribute to the redistribution of pathogens or disease-causing vectors by impacting their life cycles, distribution, transmission, and survival (wu et al., ) . pathogens have the ability to rapidly alter their genetic make-up, evolve, and develop new strains including antibiotic resistant strains. the influenza virus can vary the spikes or proteins on its outer envelope resulting in the emergence of new strains that can cause disease even in vaccinated individuals (mostafa et al., ) . the outbreak of ebola virus disease (evd) in west africa in demonstrated the impact of global travel on the spread of infectious disease and the need to adopt a coordinated approach to the threat they pose. (rathore et al., ) . many of the biological threat agents are also considered to be reemerging or emerging infectious pathogens. viral hemorrhagic fever viruses are considered high-priority threat agents and are a concern as an emerging disease, as illustrated in the west african ebola outbreak in . studies have shown that most human viruses are zoonotic pathogens. of mammalian viruses, have been detected in humans of which . % are zoonotic, i.e., detected at least once in humans and at least once in another mammalian species (olival et al., ) . for early detection and recognition of emerging infections, it is critical that proper epidemiologic investigations are integrated with laboratory surveillance (feldman et al., ) . the occurrence of a disease at more than an endemic level may stimulate an investigation during which investigators may ask three questions (gordis, ) : who has the disease? the answer to this question will help identify those characteristics of the human host that are closely related to disease risk (last, ) . when did the disease occur? some diseases occur with a certain periodicity. this question is also addressed by examining trends of disease incidence over time (rasko et al., ) . where did the cases arise? answers to the previous questions lead to determining the how and why of an outbreak. as stated previously, disease is not distributed randomly in persons, time, and place. these questions are central to virtually all outbreak investigations. investigation of an outbreak may be primarily deductive (i.e., reasoning from premises or propositions proved antecedently), inductive (i.e., reasoning from facts to a general conclusion), or a combination of both. important considerations in the investigation of acute outbreaks of infectious disease include determining that an outbreak has in fact occurred; defining the population at risk; determining the method of spread and reservoir; and characterizing the agent. steps used commonly for investigating an outbreak are shown in table . . deliberate dissemination of a biological agent via several different routes, including air, water, food, and infected vectors, presents the latest challenge to global public health security. the deliberate nature of such dissemination may be obvious, as in the case of multiple mailed letters containing spores of bacillus anthracis. however, some forms of bioterrorism may be more covert, for example, the deliberate contamination of salad bars with salmonella typhimurium in the dalles, oregon, in , by a religious cult to test their ability to incapacitate the local population before an upcoming election (torok et al., ) . this outbreak, which sickened more than persons, was specifically excluded as bioterrorism during the initial investigation and was only recognized as such following a tip from an informant (torok et al., ; carus et al., ) . given the natural ability of infectious agents to emerge, the oregon outbreak serves to highlight difficulties in determining a characteristic signature for an infectious disease outbreak resulting from covert but intentional introduction. these difficulties in identifying a covert dissemination of a biological agent serve as a caution for public health practitioners, because in the aforementioned investigation of a foodborne outbreak, there was a very unusual pattern with a rare strain of s. typhimurium (torok et al., ) . although the possibility of intentional contamination was considered early in the investigation, it was specifically excluded for the following reasons: (i) such an event had never been reported previously; (ii) no one claimed responsibility; (iii) no disgruntled employee was identified; (iv) no motive was apparent; (v) the epidemic curve suggested multiple exposures, which was presumed to be unlikely behavior for a saboteur; (vi) law enforcement officials failed to establish a recognizable pattern of unusual behavior; (vii) a few employees had onset of illness before the patrons, suggesting a possible inside source of infection; (viii) the outbreak was biologically plausibledeven if highly unlikely; and (ix) it is not unusual to be unable to find a source in even highly investigated outbreaks. although one of the initial reasons to exclude terrorism (i.e., no prior incidents) is no longer applicable, based on similar actions since , determining if an unusual outbreak is biologically plausible will remain a challenge. in this context, it is . forensic public health: epidemiological and microbiological investigations for biosecurity important to remember that the first case of inhalation anthrax identified in florida in was initially thought to be natural exposure. it is clear from the two documented cases of bioterrorism in the united statesdthe oregon salmonella outbreak and the anthrax attackdthat a terrorist will not necessarily announce his/her intentions or take credit for such an attack (torok et al., ; jergnigan et al., ) . research with highly transmissible and virulent pathogens has come under increasing scrutiny due to concerns about biosafety and biosecurity. discomposure about the potential for accidental or deliberate escape of pathogens with lethal or pandemic potential from at least one of the several laboratories engaged in research with such agents is not unwarranted. for example, in , cdc reported two incidents at its main campus in atlanta, georgia: (i) the unintentional release of potentially viable anthrax spores and (ii) the potential exposure of one of its lab staff to noninactivated ebola virus (centers for disease control and prevention, ). another incident was reported in when a private company that received regular shipments of specimens from the department of defense (dod) notified the cdc that supposedly "inactivated" b. anthracis spores in its possession were still viable. cdc investigation revealed that the samples came from a dod facility in utah. furthermore, investigators found that over the past decade, facilities in the united states and other countries had received samples of "inactivated" b. anthracis table . commonly used steps in investigation of infectious disease outbreak. step . verify the diagnosis step . establish a case definition (person, place, and time) step . identify cases step . verify you have an epidemic (descriptive epidemiology) time: look for temporal clustering and timeeplace interactions place: look for geographic clustering person: examine the risk in subgroups of affected population according to personal characteristics: sex, age, residence, occupation, social groups, etc. step . develop hypotheses based on the following: existing knowledge (if any) of the disease analogy to diseases of known etiology step . test hypotheses further analyze existing data (e.g., caseecontrol studies) collect additional data, environmental samples, animal/vectors step . recommend and implement control and prevention measures prevention of future similar outbreaks step . communicate findings deliberate introduction of a biological agent spores that also contained low numbers of viable spores from the same facility (department of defense, ) . although none of these incidents were a threat to public health, it made sense to worry that some accidental releases could pose a significant threat especially since there was precedent in the accidental release of variola virus, sars coronavirus, and the influenza a/h n . based on an assessment of historical data on lab accidents, klotz and sylvester ( ) estimated that the probability of an accidental laboratory release of a pathogen with pandemic potential was . % per laboratory per year. with approximately laboratories worldwide working with pathogens such as sars and h n bird flu, they calculated that there was an % likelihood of at least one accidental release occurring in one of these labs over a -year period (klotz and sylvester, ) . advancements in molecular biology make it possible to genetically modify, edit, or disrupt the genome of pathogens. gene editing may result in a loss of function through knock-out, a change of function through gene replacement, or a gain of function through knock-in techniques (zhang et al., ) . genome editing has important therapeutic benefits and holds enormous potential for improving public health (naldini, ) . for example, gain-of-function research (i.e., research intended to increase the transmissibility and/or virulence of pathogens) can actually improve our understanding of how pathogens interact with their hosts, help us assess the potential of pathogens to cause pandemics, and aid in the development of medical countermeasures and public health preparedness (selgelid, ). the cpispr cas gene editing tool has been successfully used to create a gene driverda genetic system use to hijack a population through the propagation of a gene through multiple generationsdto control the spread of malaria that, in time, could be used to edit the dna of any living organism (selgelid, ) . nevertheless, the publication of the results of a successful attempt by researchers to genetically modify influenza a/h n virus so that it was transmissible by the airborne route in ferrets raised serious biosecurity concerns. by demonstrating that avian a/h n influenza virus could be transmitted by the airborne route between mammals, the researchers highlighted its pandemic potential for humans (herfst et al., ) . critics questioned the potential benefits of the results when compared with the biosafety and potential dual-use risks. concerns have been raised not only over the potential misuse of the results of gain-of-function research but also on research in human germline editing and gene drives and the potential for misuse of emerging gene editing technologies. the risk of intentional or unintentional release of a gene driveemodified organism during research and development, transfer between labs, or due to inadequate containment procedures is small but not zero. newly developed gene editing tools such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (crispr-cas) systems are publicly available (dieuliis and giordano, ; maeder and gersbach, ) . the most popular and widely used of these tools are the cpispr systems (crispr-cas , crispr-cpf , and crispr-c c ) (zhang et al., ) . crispr tools for editing prokaryotic and eukaryotic genomes are readily available online at an affordable cost, including user friendly instructions (sneed, ) . in the united states, biohacker boot camps teach basic gene editing skills, and interest in gene editing is growing. just recently, it was demonstrated that mail-order dna could be used to create horse pox virus de novo (kupferschmidt, ; noyce et al., ) . mass casualties could occur if modified strains of a/h n influenza virus with increased virulence and humanto-human transmissibility are produced, aerosolized, and intentionally released. without ignoring the current limitations of gene editing technology for nefarious purposes, a us intelligence community assessment . forensic public health: epidemiological and microbiological investigations for biosecurity stated that genome editing research on pathogens with pandemic potential may pose a national security risk if not regulated. the director of national intelligence in testimony to the us congress in february warned that "given the broad distribution, low cost, and accelerated pace of development of this dualuse technology, its deliberate or unintentional misuse might lead to far-reaching economic and national security implications" (clapper, ) . the european academies science advisory council working group on gene editing acknowledged the potential for misuse but recommended regulating specific applications rather than gene editing itself as a new technology (easac, ) . fortunately, a number of epidemiological clues, alone or in combination, may suggest that an outbreak is deliberate. divining motives behind an attack should be abandoned as a public health tool to assess whether an outbreak is natural or deliberate in nature. it is essential to make this determination not only from a law enforcement standpoint to prevent future such actions but to protect the public health. there is a very short "window of opportunity" in which to implement postexposure prophylaxis for many of the agents likely to be used for bioterrorism (khan et al., ) . therefore, it is critical that all outbreaks be rapidly investigated and assessed for whether they are of deliberate origin. a set of epidemiological clues (table . ) has been proposed by the cdc in collaboration with the federal bureau of investigation (treadwell et al., ) . they are based on distinctive epidemiological and laboratory clues of varying specificity to evaluate whether an outbreak may be of deliberate origin. the clues focus on aberrations in the typical characterization of an outbreak by person, place, and time in addition to consideration of the causative agent. some of the clues, such as a community-acquired case of smallpox, are quite specific for bioterrorism, whereas others, such as a similar genetic type of an organism, may simply denote a natural outbreak. a combination of clues, especially those that suggest suspicious point source outbreaks, will increase the probability that the event is likely due to bioterrorism. although these clues are an important set of criteria to help evaluate outbreaks, no list will replace sound epidemiology to assess an outbreak. it is important to note that forensic investigations are conducted in the context of a rapid and thorough epidemiological investigation. not surprisingly, ongoing surveillance to identify increases in disease incidence is both the first step and the cornerstone of bioterrorism epidemiology. most of the clues described in table . simply suggest an unusual cluster of cases. they have been reorganized by specificity to trigger increasingly broader investigations by state and federal public health officials and to alert law enforcement authorities (tables . and . ). however, even the most specific of clues may signal a new natural disease outbreak. an epidemiological investigation should consider all potential sources and routes of both natural and potential deliberate exposure. for example, the community outbreak of individuals with smallpox-like lesions in the midwest in may, on first blush, have indicated the deliberate release of smallpox virus. however, a thorough integrated epidemiological and laboratory investigation identified the disease as monkeypox, an exotic disease in the united states, which in itself could suggest bioterrorism (centers for disease control, ) . instead, affected individuals were sickened by infected prairie dogs purchased as pets, which had acquired their infection while cohoused with infected giant gambian rats that had been imported from ghana, and not from deliberate dissemination. in , four us soldiers acquired hemorrhagic fever with renal syndrome in the republic of south korea near the demilitarized zone (pasteur et al., ) . despite initial suspicions of deliberate infection, epidemiological and laboratory analysis ultimately linked exposure to rodent hosts at training sites visited by the soldiers (pasteur et al., ) . . single case of disease caused by an uncommon agent (e.g., glanders, smallpox, viral hemorrhagic fever, inhalation, or cutaneous anthrax) without adequate epidemiologic explanation . unusual, atypical, genetically engineered, or antiquated strain of agent (or antibiotic resistance pattern) . higher morbidity and mortality in association with a common disease or syndrome or failure of such patients to respond to usual therapy . unusual disease presentation (e.g., inhalation anthrax or pneumonic plague) . disease with an unusual geographic or seasonal distribution (e.g., plague in a nonendemic area, influenza in the summer) . stable endemic disease with an unexplained increase in incidence (e.g., tularemia, plague) . atypical disease transmission through aerosols, food, or water in a mode suggesting sabotage (i.e., no other possible physical explanation) . no illness in persons who are not exposed to common ventilation systems (have separate closed ventilation systems) when illness is seen in persons in close proximity who have a common ventilation system . several unusual or unexplained diseases coexisting in the same patient without any other explanation . unusual illness that affects a large, disparate population (e.g., respiratory disease in a large heterogeneous population may suggest exposure to an inhaled pathogen or chemical agent) . illness that is unusual (or atypical) for a given population or age group (e.g., outbreak of measles-like rash in adults) . unusual pattern of death or illness among animals (which may be unexplained or attributed to an agent of bioterrorism) that precedes or accompanies illness or death in humans . unusual pattern of death or illness in humans that precedes or accompanies illness or death in animals (which may be unexplained or attributed to an agent of bioterrorism) . ill persons who seek treatment at about the same time (point source with compressed epidemic curve) . similar genetic type among agents isolated from temporally or spatially distinct sources . simultaneous clusters of similar illness in noncontiguous areas, domestic or foreign . large numbers of cases or unexplained diseases or deaths initial investigation at local level a. higher morbidity and mortality than expected, associated with a common disease or syndrome b. disease with an unusual geographic or seasonal distribution c. multiple unusual or unexplained disease entities coexisting in the same patient d. unusual illness in a population (e.g., renal disease in a large population, which may be suggestive of toxic exposure to an agent such as mercury) e. ill persons seeking treatment at about the same time f. illness in persons suggesting a common exposure (e.g., same office building, meal, sporting event, or social event) continued investigation with involvement of state health department and/or centers for disease control and prevention a. at least a single, definitively diagnosed case(s) with one of the following: -uncommon agent or disease -illness due to genetically altered organism b. unusual, atypical, or antiquated strain of agent c. disease with unusual geographic, seasonal, or "typical patient" distribution d. endemic disease with unexplained increase in incidence e. no illness in persons not exposed to common ventilation systems f. simultaneous clusters of similar illness in noncontiguous areas, domestic or foreign g. cluster of patients with similar genetic type among agents isolated from temporally or spatially distinct sources modified from treadwell, t.a., koo, d., kuker, k., khan, a.s., . epidemiologic clues to bioterrorism. public health rep. , e . similarly, the death of a wildlife biologist working for the national park service from yersinia pestis required a thorough epidemiological investigation. the wildlife biologist was found deceased at his home by colleagues and a subsequent postmortem determined cause of death as primary pneumonic plague (wong et al., ). epidemiological, ecological, and laboratory investigations concluded the biologist's source of exposure to y. pestis was most likely during a necropsy that he performed on a mountain lion before his death (wong et al., ) . concerns regarding the potential deliberate use of biological agents such as y. pestis and the presence of emerging infections will continue to complicate efforts to distinguish between naturally occurring disease and disease resulting from deliberate release of a biological agent. the microbiology laboratory has made significant contributions to the epidemiology of infectious diseases. repeated isolation of a specific microorganism from patients with a given disease or syndrome has helped prove infectious etiologies. in addition, isolation and identification of microorganisms from animals, vectors, and environmental sources have been invaluable in identifying reservoirs and verifying modes of transmission. in dealing with an infection, it is often necessary to identify the infecting microorganism and determine its antimicrobial susceptibilities to prescribe effective therapy. many of the techniques that have evolved for such purposes are both rapid and accurate but, in general, do not provide the kind of genetic discrimination necessary for addressing epidemiological questions. historically, the typing methods that have been used in epidemiological investigations fall into two broad categories: phenotypic and genotypic. phenotypic methods are those methods that characterize the products of expressed genes to differentiate strains. for example, the use of biochemical profiles to discriminate between genera and species of bacteria is used as a diagnostic method but can also be used for biotyping. other methods, such as phage typing, can be used to discriminate among groups within a bacterial species. biotyping emerged table . considerations for notifying law enforcement of possible biologic or chemical terrorism initial investigation at local level. a. notification is received from individual or group that a terrorist attack has occurred or will occur b. a potential dispersal/delivery device such as munition or sprayer or questionable material is found notification of the fbi as soon as possible after investigation confirms the following: a. illness due to unexplained aerosol, vector, food, or water transmission b. at least a single, definitively diagnosed case(s) with one of the following: -uncommon agent or disease occurring in a person with no other explanation -illness due to a genetically altered organism notification of fbi after investigation confirms the following (with no plausible natural explanation): a. disease with an unusual geographic, seasonal, or "typical patient" distribution b. unusual, atypical, or antiquated strain of agent c. simultaneous clusters of similar illness in noncontiguous areas, domestic or foreign d. clusters of patients presenting with similar genetic type among agents isolated from temporally or spatially distinct sources e. infection due to novel vehicle or mode of transmission molecular strain typing as a useful tool for epidemiological investigations in the s and early s, while phage typing of bacteria and serological typing of bacteria and viruses have been used for decades. today, the majority of these tests are considered inadequate for epidemiological purposes. first, they do not provide enough unrelated parameters to obtain a good reflection of genotype. for example, serotyping of streptococcus pneumoniae discriminates among only a limited number of groups. in addition, some viruses, such as human cytomegalovirus and measles virus, cannot be divided into different types or subtypes by serology because significant antigenic differences do not exist. second, the expression of many genes is affected by spontaneous mutations, by environmental conditions, and by developmental programs or reversible phenotypic changes, such as high-frequency phenotypic switching. because of this, many of the properties measured by phenotypic methods tend to vary and, for the most part, have been replaced by genotypic methods. the one major exception is multilocus enzyme electrophoresis (mlee) (pasteur et al., ; richardson et al., ) , which is a robust phenotypic method that performs comparably with many of the most effective dna-based methods (pujol et al., ; tibayrenc et al., ) . characteristics of selected phenotypic methods are presented in table . . these methods have been characterized by typeability, the ability of the technique to assign an unambiguous result (i.e., type) to each isolate; reproducibility, the ability of a method to yield the same results on repeat testing of a bacterial strain; discriminatory power, the ability of the method to differentiate among epidemiologically unrelated isolates; ease of interpretation, the effort and experience required to obtain useful, reliable typing information using a particular method; and ease of performance, which reflects the cost of specialized reagents and equipment, technical complexity of the method, and the effort required to learn and implement the method. extremely sensitive and specific molecular techniques have recently been developed to facilitate epidemiological studies. our ability to use these molecular techniques (genotypic methods or proteomic methods for prions) to detect and characterize the genetic variability of infectious agents (bacteria, fungi, protozoa, viruses) is the foundation for most molecular epidemiological studies. the application of appropriate molecular techniques has been an aid in the surveillance of infectious agents and in determining sources of infection. the ability to link isolates to sources has direct implications for investigating both (coleman et al., ) . these molecular techniques can be used to study health and disease determinants in animal (including human) and in plant populations. molecular techniques may also be applied to clinical and environmental samples. it requires choosing a molecular method(s) that can discriminate genetic variants at different hierarchical levels, coupled with the selection of a region of nucleic acid, which is appropriate to the questions being asked (table . ). genotypic methods are those based on an analysis of the genetic structure of an organism. over the past decade, several genotypic methods have been used to fingerprint pathogenic microorganisms (table . ). the methods have been described in detail elsewhere (tenover et al., ; thompson et al., ; soll et al., ; pennington, ; arens, ; foley et al., ) . in general, molecular typing methods can be divided into three general categories: restriction endonucleaseebased methods, amplification-based methods, and sequencebased methods (foley et al., ). among these methods, restriction fragmentelength polymorphism/pulsed-field gel electrophoresis (rflp-pfge) and rflp þ probe and ribotyping have been the most commonly used methods for fingerprinting bacteria (soll et al., ; swaminathan et al., ) . random amplification of polymorphic dna (rapd) and karyotyping have been used for fingerprinting fungi (soll et al., ; soll, ) . mlee, rapd, and polymerase chain reaction (pcr)-rflp have been used for fingerprinting parasitic protozoa (soll et al., ) . multilocus variable number tandem repeat analysis (mlva) has been used to subtype b. anthracis, y. pestis, and francisella tularensis. mlva schemes are now available for most bioterrorism agents (van belkum, ) . single-nucleotide polymorphisms (snps) have been used to analyze strains of b. anthracis and several gram-negative foodborne pathogens (foley et al., ; keim et al., ). an assay used for scoring snps of b. anthracis has been shown to have highthroughput capability and can be performed with small amounts of dna (keim et al., ) . select gene or complete genome characterization, as well as other molecular methods, has been used for viruses (arens, ) . when should fingerprinting be used? strain typing data are most effective when they are collected, analyzed, and integrated into the results of an epidemiological investigation. the epidemiologist must collaborate with the laboratory scientist when investigating a potential outbreak of an infectious disease. microbial fingerprinting should supplement, and not replace, a carefully conducted epidemiological investigation. in some cases, typing data can effectively rule out an outbreak and thus avoid the need for an extensive epidemiological investigation. in other cases, these data may reveal the presence of outbreaks caused by more than one strain. data interpretation is facilitated greatly by an appreciation of the molecular basis of genetic variability of the organism being typed and the technical factors that can affect results. except for whole-genome sequencing (wgs), molecular methods analyze only a small portion of the organisms' genetic complement. thus, isolates that give identical results are classified as "indistinguishable," not "identical." theoretically, a more detailed analysis should uncover differences in the isolates that appeared to give identical patterns but that were unrelated epidemiologically. this is unlikely to occur when a set of epidemiologically linked isolates are analyzed (tenover et al., ) . for this reason, only wgs would provide unequivocal data required for attribution. the power of molecular techniques in epidemiological investigations can be exemplified by a few examples. pulsenet, the national molecular subtyping network for food-borne disease surveillance, was established by the cdc and several state health departments in to facilitate subtyping bacterial food-borne pathogens for epidemiological purposes. twenty-five years ago, most food-borne outbreaks were local problems that typically resulted from improper food-handling practices. outbreaks were often associated with individual restaurants or social events and often came to the attention of local public health officials through calls from affected persons. today, food-borne disease outbreaks commonly involve widely distributed food products that are contaminated before distribution, resulting in cases that are spread over several states or countries. the pulsenet restriction endonucleaseebased methods a. restriction fragmentelength polymorphism without hybridization -frequent cutter ( -to -bp recognition site) coupled with conventional electrophoresis to separate restriction fragments -infrequent cutter (generally -to -bp recognition site) coupled with pulsed-field gel electrophoresis to separate restriction fragments b. rflp with hybridization -frequent cutter ( -to -bp recognition site) coupled with conventional electrophoresis to separate restriction fragments followed by southern transfer to nylon membrane. power and efficacy of typing method depend on the probe. network, which began with laboratories typing a single pathogen (escherichia coli o : h ), achieved full national participation in and includes food safety laboratories of the us food and drug administration (fda) and the us department of agriculture. sister networks have also been established internationally (swaminathan et al., ; gerner-smidt et al., ) . currently, pulsenet usa utilizes standardized pfge protocols for six organisms with mvla as a complementary technique: e. coli o :h , salmonella enterica, shigella spp., thermotolerant campylobacter spp., clostridium perfringens, and vibrio cholerae (gerner-smidt et al., ) . the laboratories follow a standardized protocol using similar equipment so that results are highly reproducible and dna patterns generated at different laboratories can be compared. isolates are subtyped on a routine basis, and data are analyzed promptly at the local level. clusters can often be detected locally that would not have been identified by traditional epidemiological methods alone. pfge patterns are shared between participating laboratories electronically, which increases the ability to link apparently unrelated outbreaks and to identify a common vehicle (centers for disease control, ) . for example, in , pulsenet was critical to facilitating the identification of an s. mbandaka outbreak affecting persons in states in the united states (https://www.cdc.gov/ salmonella/mbandaka- - /index.html). starting in march , cdc and other public health and regulatory officials linked geographically dispersed s. mbandaka isolates from stool samples of symptomatic patients, which had the same pfge pattern. the initial epidemiological investigation revealed that many of the patients reported eating cold cereal, and the vehicle was subsequently identified as contaminated kellogg's honey smacks sweetened puff wheat cereal. additionally, environmental and product samples obtained by the fda at the contract production facility were positive for the outbreak strain of s. mbandaka. without molecular typing, epidemiologists would have found it difficult to identify cases associated with each state cluster and assign attribution to the source. however, the use of pfge subtyping as part of routine surveillance has benefits beyond outbreak detection. for example, the temporal clustering of unrelated cases is not uncommon, and without molecular typing, valuable public health resources would be wasted investigating pseudo or unrelated outbreaks. molecular genotyping of food-borne pathogens continues to evolve. pulsenet has transitioned to the use of wgs for listeria monocytogenes and is expanding wgs to other pathogens to improve the level of resolution. in the future, pulsenet will be evaluating metagenomic approaches and other strategies using nextgeneration sequencing (ngs) technology for direct characterization of patient samples as clinical practice embraces culture-free diagnostic methods. pulsenet remains a powerful tool that can be applied for the early detection of cluster(s) of illness that result from deliberate contamination of food (gerner-smidt et al., ) . another example of the power of molecular techniques is the invaluable information provided during the anthrax attacks. mlva was initially used to subtype isolates obtained from patients, environmental samples, and powders. information from mlva identified the subtype of b. anthracis and was able to link clinical cases to environmental samples and powders, thereby providing information on possible sources of exposure (hoffmaster et al., ) . molecular subtyping also confirmed that clinical cases were caused by the same strain and that suspected cases outside the united states were not linked (hoffmaster et al., ) . both forensic and epidemiological investigations can result in the collection of hundreds of clinical and environmental samples for testing. during this event, mlva assisted with the identification of potential laboratory contamination of samples because of the large number of samples requested to be tested (hoffmaster et al., ) . mlva can be used to reliably and rapidly genotype an isolate within h of receipt by the laboratory. molecular subtyping identified the b. anthracis used in the attack as the ames strain, a strain rarely found in nature (keim et al., ) . this information was a critical epidemiological factor in determining that these cases were most likely the result of a deliberate release (keim et al., ) . additionally, wgs of isolates obtained from spores indicated that the genome and plasmid sequences were identical to those of an ames strain stored at a us army research facility (fricke et al., ) . the utility of molecular typing methodologies was clearly demonstrated in this forensic investigation involving the deliberate release of a biological agent in the united states. in , the cdc was notified of two cases of brucellosis in microbiologists who worked in clinical laboratories in indiana and minnesota (centers for disease control, ). because brucella spp. are considered category b agents (khan et al., ) , infections with brucella spp. should have a thorough epidemiological investigation to determine potential sources of exposure. mlva was utilized to help identify the source of the brucella infections. the cdc compared blood culture isolates from the two microbiologists with the isolates they handled in the laboratory. the epidemiological investigation revealed that the clinical isolate from the infected microbiologist in indiana had been forwarded to the clinical laboratory in minnesota; however, investigation also revealed that the second microbiologist did not handle this clinical isolate (centers for disease control, control, ) . molecular genotyping provided critical confirmation of the source of exposure for these microbiologists and confirmed that these cases resulted from a laboratory exposure. a radical shift in molecular strain typing occurred with the development of technology that allowed for millions of sequencing reactions to be conducted simultaneously on multiple mixed biological specimens. this advancement in sequencing has been termed ngs (behjati and tarpey, ) . the ability to sequence the whole genome, screen mixed dna samples at the same time, detect minor alleles very accurately, and identify causes of disease of unknown etiology has improved the value of dna as evidence in forensic investigations. the throughput diagnostic capacity of ngs technology has the potential to increase the reach and the number of forensic investigations that can be conducted at low cost. ngs is already routinely applied in outbreak investigations to determine the potential source of outbreaks. for example, using dna sequencing, it was determined that the haitian cholera epidemic was associated with the introduction of a strain that was closely related to variant v. cholerae el tor o strains that had been previously isolated in bangladesh in and (chin et al., ) . the e. coli o :h outbreak in europe was epidemiologically linked to seed shipments from egypt that were sent to germany in (grad et al., ) . sequencing capacity was established in liberia during the ebola outbreak to monitor the evolution of the virus during this outbreak (kugelman et al., ) . the technological trend to make portable devices is fueling innovation toward portable ngs devices that are field deployable without the limitations on size, weight, supportive infrastructure, complex sample processing procedures, or need for calibration of sequencing . forensic public health: epidemiological and microbiological investigations for biosecurity machines by field engineers. for example, a pocket-sized, usb-powered sequencer (minion) developed by oxford nanopore was successfully used to rapidly sequence ebola virus at the field diagnostic laboratory in liberia during the ebola virus outbreak in west africa (hoenen et al., ) . advancements in the field of microfluidics also hold promise for the development of lab-on-a-chip systems with capacity to collect and analyze biological specimens on a miniature device. additional advances in molecular laboratory techniques have been used for the rapid detection of antimicrobial resistance. in one prospective study on methicillin-resistant staphylococcus aureus, automated clonal alerts based on real-time subtyping were faster than traditional methods (sintchenko and gallego, ). at present, however, the direct identification of resistance genes by pcr or similar methods is of limited use because only a few resistance genes are strongly associated with phenotypic resistance (jorgensen and ferraro, ) . pcr followed by electrospray ionization mass spectrometry has been used to detect quinolone resistance in acinetobacter spp. (hujer et al., ) . however, this technique must be further evaluated, and limitations must be acknowledged, such as whether detection of a resistance gene indicates that a resistant phenotype is always present (hujer et al., ) . the ability to establish antimicrobial susceptibility patterns rapidly is particularly critical for providing the appropriate antimicrobial agents for treatment or postexposure prophylaxis in a situation where the deliberate dissemination of a potentially engineered drug-resistant organism is being considered. because there are numerous mechanisms for antimicrobial resistance in bacteria, current phenotypic methods will likely continue to be the basis for laboratory determination of antimicrobial susceptibility patterns for the foreseeable future (jorgensen and ferraro, ). unfortunately, molecular genotyping information exists in multiple databases and in a variety of formats. although pulsenet and other systems have web-based access, integration and sharing of data among multiple databases remains a challenge. as information and databases expand, data will also become more challenging to analyze. therefore, there is a need to refine analytic methods including the use of artificial intelligence to improve pattern recognition and integration of multiple streams of epidemiologic and laboratory data so that outbreaks and bioterrorism events can be detected quickly. informatics capacity at local, state, and federal level requires continued investment to maximize the integration of epidemiology and laboratory information. finally, the threat of bioterrorism has initiated the development of mechanisms to quickly identify the presence of biological agents in the environment to rapidly initiate public health and medical response efforts. molecular technologies allow for the rapid identification of genetic material of biological agents from collection devices such as those used for outdoor and indoor air monitoring. public health, forensic, and laboratory assessments must be made based on material collected in a distinct area covered by the monitor or sensor. because these detectors or devices do not preserve the viability of the agent, the assessment cannot indicate that a live organism was released, that individuals were exposed, or that a deliberate release occurred. as a result, it is critical that information from public health and epidemiological investigations be considered when interpreting information from environmental monitors. public health must consider the limits of these new technologies, previous history of environmental detection of a biological agent in each area, and environmental sampling methods. as the recent institute of medicine report on "effectiveness of national bio-surveillance systems: biowatch and the public health system" indicated, the challenge is "understanding the clinical context in which disease detection and reporting occurs and the factors that shape the decision-making process for the state and local public health officials who must interpret the data" from these systems as well as that from traditional public health surveillance systems (institute of medicine and national research council of the national academies, ). with few exceptions, a careful epidemiological investigation will be required to determine whether an outbreak of infectious disease is due to the intentional (or unintentional) release of an agent or is naturally occurring. a number of molecular methods have been developed for subtyping microbes that complement the epidemiological investigation as well as identify related cases. for example, since the establishment of pulsenet, the routine use of molecular subtyping by pfge has improved both the sensitivity and the specificity of epidemiological investigation of food-borne outbreaks at the state and local level (hedberg and besser, ) and mvla was critical in identifying 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evolution through establishment of advanced genomic capacity in liberia bacillus anthracis comparative genome analysis in support of the amerithrax investigation emerging infectious diseases methods for subtyping and molecular comparison of human viral genomes alloenzyme electrophoresis gain eof-function research: ethical analysis laboratory-guided detection of disease outbreaks: three generations of surveillance systems microbial threats to health: emergence, detection and response mail-order crispr kits allow absolutely anyone to hack dna the ins and outs of dna fingerprinting of infectious fungi laboratory procedures for the epidemiological analysis of microorganisms pulsenet: the molecular subtyping network for foodborne bacterial disease surveillance, united states how to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists overview and significance of molecular methods: what role for molecular epidemiology? genetic characterization of six parasitic protozoa: parity between random primer dna typing and multilocus enzyme electrophoresis a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (mlva) primary pneumonic plague contracted from a mountain lion carcass impact of climate change on human infectious diseases: empirical evidence, evidence and human adaptation progress in genome editing technology and its application in plants forensic public health: epidemiological and microbiological investigations for biosecurity we thank dr. nicki pesik for drafting this chapter for the second edition. key: cord- - dannjjm authors: nan title: research abstract program of the acvim forum denver, colorado, june – , date: - - journal: j vet intern med doi: . /j. - . . .x sha: doc_id: cord_uid: dannjjm nan clinics Ãà also see infectious disease abstracts id- -id- (thursday, june , : pm - : pm) Ãà also see pharmacology abstracts p- -p- (thursday, june , : pm - : pm) Ãà also see gasteroenterology abstracts gi- - june , hypertrophic cardiomyopathy (hcm) is the most commonly observed myocardial disease in cats. beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical hcm despite the fact that neither drug category has been proven to slow disease progression or improve survival. ivabradine (procorolan s , servier, france) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. little is known about its efficacy and safety in cats. the purpose of this study was to determine the short-term effects of ivabradine on heart rate (hr), blood pressure, left ventricular (lv) systolic and diastolic function, left atrial (la) performance, and clinical tolerance in healthy cats after repeated oral doses. ten healthy laboratory cats were involved in the present study. physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration ( weeks each) of ivabradine ( . mg/kg, q h) and atenolol ( . mg/cat, q h; . - . mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. a-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at % (f . ) based on predicted clinical relevance, observer measurement variability, and in agreement with fda guidelines. variables were compared by use of -way repeated measures anova. ivabradine was clinically well tolerated with no adverse events observed. hr (ivabradine, po . ; atenolol, po . ; ivabradine vs. atenolol, p . ) and rate-pressure product (ivabradine, p o . ; atenolol, p . ; ivabradine vs. atenolol, p . ) were not different between treatments. at the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (lv) systolic and diastolic function and left atrial performance. ivabradine is non-inferior to atenolol with regard to effects on hr, rate-pressure product, lv function, la performance, and clinical tolerance. clinical studies in cats with hcm are needed to validate these findings and further assess safety. the aim of this study was to compare outcome from cpa in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (cpr). dogs having cpa in the er or icu of a university hospital were randomized to receive either iv epinephrine ( . - . mg/kg) or vasopressin ( . - . u/kg) in a blinded fashion immediately following establishment of iv access and again three minutes later. a standardized cpr protocol was followed. other vasopressors were not permitted during the six minute study period, at the end of the study period additional cpr interventions were at the discretion of the managing clinician. the primary end point was return of spontaneous circulation (rosc) within the study period; secondary end points included rosc at any point, survival to minutes, and survival to one hour. sixty dogs completed the study, received epinephrine and received vasopressin. rosc within six minutes was % ( vasopressin, epinephrine p . ), rosc at any time was % ( vasopressin, epinephrine p . ). survival to minutes was % ( vasopressin, epinephrine p . ), survival to one hour was % ( vasopressin, epinephrine p . ). five dogs survived to hours, one survived to hospital discharge. of animals dying after rosc, / were euthanized and / rearrested. no advantage of routine substitution of vasopressin for epinephrine was seen for rosc, a small survival advantage at one hour was seen in the group receiving epinephrine. the study also demon-strated that prospective clinical cpr research in animals is both possible and practical. three dogs were evaluated in phases. phase- : single-dose diltxr at approximately mg/kg po. phase- : same dose q h for . days. phase- : after a day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. blood pressure (bp), -lead ecg, echocardiogram, and h-ambulatory ecg were performed at baseline, and conclusion of phase- . blood samples and bp was obtained , , , , , , and h after final dosing. peak median plasma diltiazem concentrations (mcg/ml) measured by hplc for each phase were . , . , and . respectively. diltiazem concentrations were below the limit of detection in the majority of samples in phase- . median diltiazem concentration reached purported therapeutic concentrations ( . - . mcg/ml) by h post-pill in phase- and h post-pill in phase- . therapeutic concentrations were maintained for h in phase- , but only h in phase- . median bp (mmhg) was . at baseline and . at peak concentration in phase- . median heart rate (bpm) was . at baseline. h-ambulatory ecg analysis revealed the median hourly heart rate was . at baseline and during phase- . median heart rate at peak concentration in phase- was . . lack of detectable plasma diltiazem during phase- may be due to up-regulation of drug metabolism via p-glycoprotein (abcb - ) mutations. ongoing data collection and analysis will include mutation testing. adiponectin (adpn) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. in the heart, adiponectin activates several pro-survival reactions, including the ampk pathway and cox receptors, which protect the heart following ischemic injury. recent studies have shown that higher levels of adpn influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. in humans, adpn plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit adpn at the level of gene expression. in contrast c-reactive protein (crp), a marker of systemic inflammation is elevated in humans with congestive heart failure (chf) and correlates to the severity of disease. first, we hypothesized that dogs with chf would have reduced adpn and elevated crp compared to normal dogs and that cytokine concentrations would predict severity of chf. second, we hypothesized that adpn receptor- (r ) and adpn protein would be elevated in the myocardium of chf dogs reflecting a compensatory process. we collected serum from dogs ( healthy and chf). circulating adiponectin and crp levels were quantified using a mouse/rat adiponectin elisa and a canine crp elisa. we found lower mean crp concentrations in normal dogs ( . ae . mg/ ml) than dogs with chf ( . ae . mg/ml), however, the results were not statistically significant due to the large variability seen among the chf dogs (p . ). we found greater mean adpn concentrations in normal dogs ( . ae . mg/ml) than chf dogs ( . ae . mg/ml) (p . ). in general, the greater the severity of the heart failure, the lower the level of serum adpn. when the tests the purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (pda) in dogs in our institution. initial and follow up records from all dogs (n ) that underwent attempted transvascular pda occlusion from january -december were examined. dogs were placed into groups depending on the device and route of vascular access (transvenous or transarterial). group : amplatz s canine ductal occluder (acdo) (transarterial) - dogs; group : gianturco s or mreye flipper s detachable embolization (flipper) coil (transarterial) - dogs; group : amplatzer s vascular plug (avp) (transarterial) - dogs; group : flipper coil (transvenous) - dogs. statistical comparisons were made using the kruskal-wallis test with mann-whitney tests to compare pairs of groups when significance was detected. p o . was considered significant. there was no significant difference in ages between the groups. there was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups and ) being significantly smaller than dogs receiving an acdo or avp. this was by design since the acdo and avp cannot be used in small dogs. overall, the success rate of the total procedure (including vascular access and satisfactory pda occlusion) was high ( %) with success rates being comparable between groups ( - %). there was a significant difference in complication rate between groups (p o . ) with the acdo group having a markedly lower complication rate than the remaining groups ( % for acdo versus - % for the other groups). total fluoroscopy time ranged from - minutes (median minutes). fluoroscopy time for the transvenous method was significantly longer (median minutes; range - minutes) than in the remaining groups (median minutes; range - minutes) (p o . ). number of dogs with residual flow immediately following the procedure and hrs later was significantly less in the acdo group than in the remaining groups ( dogs from group and from group had moderate persistent flow while dog from group and from group had severe persistent flow hours after the procedure). the acdo appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. the remaining limiting factor with this device is patient size. until a smaller acdo device is marketed, coils remain the only choice for interventional closure in very small dogs ( o . kg). previously presented at the university of california davis, house officers seminar day. subvalvular aortic stenosis (sas) is one of the most commonly reported canine congenital heart defects and is inherited in newfoundland dogs and human beings. the golden retriever and rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. we performed genome wide association analysis in both normal and sas affected rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (snp) array. ( unaffected/ affected) adult golden retrievers and ( unaffected/ affected) adult rottweilers were included in this study. criteria for affected included a subcostal continuous-wave doppler aortic velocity ! . m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a doppler aortic velocity . m/s. dna samples were obtained from anticoagulated blood. genotypes were obtained using high density ( , ) snp arrays, and genome wide association with sas was evaluated for each breed. significance cut-off was set at p  À , and all snps meeting this criterion were plotted within each breed and compared across breeds using plink. affected golden retriever data implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . affected rottweiler data also implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . other regions of statistical significance were on chromosomes and in the golden retriever and and in the rottweiler. genome wide association with subvalvular aortic stenosis in the golden retriever and rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome . the different secondary chromosomal regions of interest (chr , in golden retrievers and , in rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. these data highlight the need for candidate gene evaluation on chromosome in golden retrievers and rottweilers with sas. heart valves share developmental signaling pathways with bone and cartilage. degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. we tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis. to test this hypothesis, expression of bone morphogenic protein (bmp ), a chondrogenic growth factor; sox , a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type ii collagen were evaluated utilizing immunohistochemistry. normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied. canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets. in conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. this finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. the triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. the fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes. arrhythmogenic right ventricular cardiomyopathy (arvc) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (vpc) . a deletion in the striatin gene has been associated with arvc in at least some boxer families. syncope and sudden death (sd) occur in some affected dogs, although many affected dogs survive for years. the objective of this study was to define clinical characteristics of arvc in boxers that experienced sd, and compare them to those of a contemporaneous group of arvc boxers that had not died suddenly (nsd). data for both groups were collected from adult boxers enrolled in a long term prospective study of arvc in which echocardiograms and hour ambulatory ecg (aecg) are evaluated annually. aecg are quantitated for vpc numbers and arrhythmia grade ( - ). arvc diagnosis requires at least vpcs/ hours in the absence of other disease. forty three adult boxers that entered the study had died suddenly at the time of analysis (sd defined as the absence of observed clinical signs within hours prior to an unexpected and unexplained death). striatin genotype was available for of the sd dogs ( heterozygotes, homozygotes); were female ( intact) and were male ( intact). sd occurred at a mean age of years (range, - ); sd dogs ( %) had no prior history of syncope. twelve sd dogs ( %) were on antiarrhythmics at the time of death (metop-p oooooooooooprolol ( ), sotalol ( ), amiodarone ( ), procainamide ( ), mexiletine & atenolol ( ), atenolol ( )). eleven sd dogs ( %) had decreased myocardial systolic function defined by a shortening fraction (%fs) o % (range - , mean ) on the most recent echocardiogram prior to sd. median vpcs/ hours on annual aecg was , (range - , ) with a median arrhythmia grade of (range - ). twenty one contemporaneously entered arvc boxers that had survived to at least the median age of the sd group with nsd were available for comparison; / were genotyped ( heterozygous, homozygous, negative), were female ( intact) and male ( intact). twelve nsd dogs ( %) had no prior history of syncope. median nsd group age was years (range, - ); / ( %) were on an antiarrhythmics (sotalol ( ), mexiletine & sotalol ( ), mexiletine & atenolol ( )). one nsd dog had decreased %fs (nsd group %fs range - , mean ). the nsd median number of vpcs was , (range - , ), median arrhythmia grade was (range - ). striatin genotype was significantly associated with sd. no significant differences were found between groups with respect to vpc numbers or arrhythmia grade. shortening fraction was significantly lower in the sd group (p o . ). sd in arvc appears to be associated with the presence of the striatin mutation and reduced % fs, it does not appear to be associated with number of vpcs or arrhythmia grade. coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (mmvd) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. the purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with mmvd and chronic cough. we hypothesized that airway collapse was independent of degree of left atrial enlargement. twelve dogs with mmvd and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (bal). group dogs (n ) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [la:ao(a)] . group dogs (n ) had no to mild left atrial enlargement [la:ao(a) ]. the site and severity of airway collapse was graded on bronchoscopy and bal cytology was assessed for evidence of inflammation or infection. the occurrence of bronchoscopic abnormalities was compared between groups using fisher's exact test. p o . was considered significant. age and body weight did not differ between groups. left atrial size was interpreted radiographically as moderately to severely enlarged in of dogs in group and as moderately enlarged in of dogs in group . fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. cervical tracheal collapse was identified during bronchoscopy in both group ( of ) and group ( of ) dogs but was subjectively less severe in group dogs. bronchial collapse % was evident at multiple sites in both groups of dogs with no difference between groups. all dogs had suppurative and/or lymphocytic inflammation on airway cytology. infection was not present in either group of dogs, although non-specific light bacterial growth was detected in of group dogs and of group dogs (p . ). preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with mmvd but did suggest that airway inflammation is common in affected dogs. further studies are needed to identify factors contributing to airway collapse in dogs with and without mmvd. atenolol is often used empirically in cats with asymptomatic hcm, even though clinical and experimental evidence of efficacy is lacking. cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. we hypothesized that serum concentrations of the biomarkers, nterminal pro-brain natriuretic peptide (nt-probnp) and cardiac troponin i (ctni), would improve following the chronic administration of atenolol po to asymptomatic cats with hcm. six maine coon or maine coon cross cats with severe hcm from the research colony at ucdavis were administered atenolol ( . mg po twice a day) for days. no cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. the concentrations of nt-probnp and ctni were assayed prior to drug administration and on the last day of drug administration. there was no statistically significant difference identified in nt-probnp [median before: pmol/l (range: - pmol/l), median after: pmol/l (range: - pmol/l); p . ] or ctni [median before: . ng/ml (range: . - . ng/ml), median after: . ng/ml (range: . - . ng/ml); p . ] concentrations before and after drug administration using the wilcoxon matched pairs test. the ctni finding suggests that atenolol does not reduce chronic myocyte death in cats with hcm. the lack of improvement in nt-probnp suggests that atenolol does not improve myocardial wall stress in cats with hcm. a clinical trial is warranted to confirm or refute the findings from this study. therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (chf; n ) in comparison to dogs presented for cardiac screening (n ) without abnormalities. additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative rt-pcr. leptin blood levels were significantly higher in dogs with chf in comparison to dogs without diseases (p . ). there was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p . ). differences between cardiac regions were present (p o . ) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p . ). interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p . ), whereas acquired cardiac diseases resulted in an increase in leptin (p . ) in comparison to controls. these results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner. predicting risk of chf in asymptomatic dogs with mitral valve disease (mvd) is challenging. we examined ability of nt-probnp to identify asymptomatic dogs at high risk for chf. dogs with isachc- b (la:ao . ) were prospectively recruited; dogs with current or previous chf or diuretic therapy were excluded. physical examination, radiography, echocardiography, and nt-probnp were performed at - mo intervals for dogs (median follow-up d, range, - d). thirty-one patients reached a study endpoint of radiographic pulmonary edema; remained asymptomatic. parameters from the visit immediately previous to onset of chf (future-chf) or prior to the most recent visit without chf (remain-asympt) were analyzed. median nt-probnp of future-chf ( pmol/lpmol/l, iqr - ) was significantly different from remain-asympt ( pmol/l, - ; p . ). median time to chf of future-chf was d (iqr, . groups also differed in median la:ao (future-chf . [ . - . ]; remain-asympt . [ . - . ], p . ); vhs (future- ]; remain-asympt . [ . - . ], p . ); and lvidd:ao ]; remain-asympt . [ . - . ], p . ). roc analysis to predict if chf would occur prior to the next visit yielded auc . ( %ci, . - . ). sensitivity was . % or . % and specificity . % or . % for nt-probnp pmol/l or pmol/l, respectively. mean increase in nt-probnp between penultimate visit and two visits prior to endpoint: future-chf . pmol/l vs. remain-asympt . pmol/l. within mo, . %, . %, . %, and . % of dogs with nt-probnp o , , and pmol/l developed chf. nt-probnp and heart size helped assess risk of chf in asymptomatic mvd. increasing the assay's upper limit of detection would likely improve utility of nt-probnp. piiinp is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. we hypothesised that piiinp concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (mmvd). serum piiinp concentrations (mg/ml) were measured in dogs with mmvd and healthy controls using a validated commerciallyavailable radioimmunoassay. results are reported as (mean ae sd). non-normally distributed variables were logarithmically transformed. comparisons of continuous variables were made between groups using t-tests and one-way anovas with tukey's post-hoc comparisons. univariable analyses were used to evaluate associations between piiinp and clinical characteristics (age, breed [cavalier king charles spaniel (ckcs) yes/ no], sex, weight, heart rate [measured from ecg], treatment with acei [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [la/ao, lvedd/ lvfwd, lveddn, lvesdn]). multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. dogs with mmvd were divided into those with no cardiomegaly (nc) (la/ao o . and lveddn o . ), those with cardiomegaly (la/ao ! . and/ or lveddn ! . ) but no clinical signs (c) and those dogs with cardiomegaly requiring treatment for congestive heart failure (chf). one hundred and fifty-four dogs with mmvd and control dogs were studied. there was no difference in age (p . ) or weight (p . ) between the mmvd and control groups. there was a significant difference in serum piiinp (p . ) between normal ( . ae . ), nc ( . ae . ), c ( . ae . ) and chf ( . ae . ) groups. post-hoc comparisons demonstrated a difference between nc and chf groups (p . ). there was no difference in serum piiinp between genders (p . ). in the univariable analysis ckcs (yes/ no) (p . ) was positively associated with serum piiinp. age (p o . ), log (la/ao) (p . ) and lveddn (p . ) were negatively associated with serum piiinp. in the multivariable model including all dogs, lveddn (p o . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in the multivariable model including only dogs not receiving therapy (n ), lveddn (p . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in conclusion, serum piiinp decreases with age and with increasing lveddn. ckcs have higher serum piiinp measurements independent of age and lveddn, which may reflect a difference in collagen turnover in this breed. left atrial (la) chamber dilation and congestive heart failure (chf) are common consequences of cardiac conditions in cats. in some cases chf is manifest as right-sided chf (r-chf) or pleural effusion, in other cases chf manifests as left-sided chf (l-chf) or pulmonary edema. it is not always readily apparent as to which cats will develop what form of chf. a general impression has been that la enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the la chamber itself. since chf is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict chf in the cat. we hypothesized that cats manifesting r-chf have la failure with the la acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas l-chf cats maintain some booster pump and reservoir function. we measured la maximum and minimum areas from right parasternal long axis four-chamber views on d echo, and la m-mode dimensions at maximum, minimum, and beginning of atrial contraction. la area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. right ventricular internal diastolic diameter was also measured on m-mode views. preliminary data revealed that maximum left atrial size is not significantly different between r-chf and l-chf cats on d or m-mode measurements due to high variability. however, total left atrial fractional shortening is significantly reduced in r-chf cats ( . % ae . ) compared to l-chf cats ( . % ae . )(p . ), and r-chf cats have reduced left atrial active emptying fraction ( . % ae . ) as compared to l-chf cats ( . % ae )(p o . ). left atrial expansion ability is poorer in r-chf cats ( . % ae . ) than in l-chf cats ( . % ae )(p . ). these findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (r-chf). right ventricular diameter on m-mode was increased in r-chf cats ( . mm ae . ) when compared to l-chf cats ( . mm ae . )(p . ) and normal cats ( . mm ae . )(p . ), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. episodic weakness and syncope are common in boxer dogs. reported causes include rapid ventricular tachycardia (vt) and exertion-excitement triggered neurally-mediated bradycardia (nmb) .the purpose of this retrospective study is to describe the features of presumed nmb in boxers. to be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (ec); had absence of vt and fewer than ventricular premature complexes (vpc) on an initial and subsequent hour holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation. a total of boxers were identified. sixteen were male and were female. most ( %) dogs were either less than or more than years of age. most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only ( %) and only once (bradycardia) on the first holter recording. owners were instructed to attempt to precipitate episodes. bradycardia related episodes were subsequently recorded in : during the nd ( ), rd ( ) or th ( ) day of hour holter recordings and during the th day of an event recording ( ). collapse and bradycardia were documented during auscultation in additional dogs. the heart rate during syncope was never documented in ( %) dogs. a presumptive diagnosis of nmb was based on the absence of initial and follow-up of ec abnormalities and the presence of no or few vpc during extended ecg monitoring. multiple holter recordings ( - hours) were performed in of ( %) dogs and event monitoring of days ( ) and days ( ) was performed in additional dogs. in conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only % and was unlikely during the first holter recording. in boxers with suspected nmb, extended ecg monitoring and implantable loop recorders may be best for hr documentation. arrhythmogenic right ventricular cardiomyopathy (arvc) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. though several genes have been linked to the disease both in humans and in boxers, the etiology of arvc is still unclear. several mechanisms for the development of arvc have been suggested, including dysfunction of the canonical wnt pathway, which results in an arvc phenotype in the mouse. the canonical wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. with the recent discovery that the gene encoding striatin, a protein involved in wnt signaling, may be involved in the development of boxer arvc, we hypothesized that changes in the wnt pathway may also play a role in the etiology. here, we show changes in the localization and decreased amount of proteins affiliated with the canonical wnt pathway. afflicted boxers were identified by -hour holter monitoring and histopathological examination of the heart. samples from the right ventricle rv) of arvc afflicted boxers, and unafflicted dogs ( beagle, mongrels, and german shepherds) were collected, fixed in % formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. western blots were performed on unafflicted rv samples, and arvc afflicted rv samples. frozen tissue samples were homogenized in laemmli buffer, and mg of protein was loaded into each well of a - % gradient gel. -catenin, an integral modulator of the wnt pathway, and striatin were colocalized with the endoplasmic reticulum (er) marker, calnexin. in the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the er. in contrast, in the arvc boxers, bothcatenin and striatin were colocalized with calnexin in an er pattern. in the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. western blots of striatin and -catenin revealed no changes in the amount of protein. interestingly, a western blot for the wnt protein revealed a decrease in the amount of protein in arvc samples, compared to unafflicted samples. our preliminary data suggest that disturbances of the canonical wnt pathway may play an etiological role in the development of arvc in the boxer dog. there are numerous benefits of omega- fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. antithrombotic effects of omega- fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. benefits also have been found in canine heart disease, and reduced serum concentrations of the omega- fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have been found in dogs with congestive heart failure (chf) secondary to dcm. to the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. the purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy. serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. cats with cardiomyopathy and at least mild left atrial (la) enlargement (la to aortic ratio . on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or mann whitney u tests, respectively. statistical significance was set at p o . . thirty cats with cardiomyopathy ( neutered males and neutered females) and healthy controls ( neutered males and neutered females) were enrolled. median age was yr (range, - yr) in the cardiomyopathy group and yr (range, - yr) in the control group (p . ). cats in the cardiomyopathy group were classified in the international small animal cardiac health council stage b (n ), (n ), a (n ) and b (n ). compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (p . ) and dha (p o . ), and lower concentrations of linoleic acid (p . ). among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. these findings warrant further investigation into the role of fatty acids in cats with cardiac disease. platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: ma thrombin (maximum platelet activity),ma fibrin (fibrin activity only), an-dma aa or adp (platelet activity not inhibited by arachidonic acid or adp receptor antagonists, respectively). using these three tracings, the % inhibition of platelets can be calculated. the purpose of this study was to evaluate the platelet mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel. employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. clopidogrel ( . mg po q h) was administered for days with platelet mapping performed on days and . platelet mapping values were compared using a paired t test, with significance set at p o . . seven cats ( fs, cm, aged - years) were enrolled. compared to day , ma adp (p o . ) and ma fibrin (p o . ) were lower on day . the latter unexpected result prompted measurement of fibrinogen concentrations at day and in the last of these cats. fibrinogen was not different from day to in these cats. these results suggest that platelet mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. this clopidogrel dose resulted in significant platelet inhibition as measured by ma adp in all cats. further studies correlating antiplatelet effects measured by platelet mapping with clinical outcomes in cats with cardiomyopathy are warranted. this study investigated the hemodynamic effects of application of an itd in a canine model of cardiopulmonary arrest. laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. following euthanasia, cpr was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of - /minute, ventilation with % oxygen was delivered at eight breaths/min and ml/kg tidal volume. cpr was performed with the itd in place (itd-cpr) and without the itd (s-cpr) for one period each in a randomized fashion with the rescuer blinded to its application. baseline, s-cpr and itd-cpr data were assessed for normality, a kruskal-wallis one-way anova on ranks was used (baseline v cpr). when appropriate a pairwise multiple comparison procedure (dunn's method) was used. percentage of baseline s-cpr v itd-cpr was assessed using the student t-test. the right atrial diastolic pressure was significantly more negative with the itd attached than without (p . ), the coronary perfusion pressure and carotid blood flow were significantly higher during cpr with the itd than standard cpr (p . , p . ). no significant differences in diastolic, mean or systolic arterial pressure or end tidal co were seen. application of the itd resulted in significantly improved hemodynamics during cpr in dogs. clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. left ventricular (lv) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. one potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (ticm). in humans, ticm carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. the goal of our study was to describe a population of dogs with ticm and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters. medical records of dogs referred to the cardiology service of ksu vmth from to were reviewed. ticm was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. transthoracic echocardiography, thoracic radiographs and electrocardiography (ecg) were performed in all dogs. ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. arrhythmias were confirmed with a holter monitor in dogs. a total of dogs were included in the study. mean age was years (range - years) with males ( intact, castrated) and females ( spayed, intact). four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. at initial presentation, the meanaesd values were as follows: heart rate ae bpm, m-mode fractional shortening (fs) . ae . %, ejection fraction (ef) using the area-length method . ae . %, and left atrial to aortic root ratio (la/ao) . ae . . initial meanaesd m-mode derived lv internal dimensions corrected for body weight were as follows: diastolic . ae . and systolic . ae . . at least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation ( ), supraventricular tachycardia ( ), junctional tachycardia ( ), and ventricular arrhythmias ( ). ten dogs were available for follow up. seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia ( ), improved systolic function ( ) antidiuretic hormone (adh) has been shown to be elevated in humans with congestive heart failure (chf). recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. adh levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring adh levels via radioimmunoassay. based on the homologous structure of canine and human adh, we aimed first to determine the feasibility of measuring adh in dog plasma using a human elisa kit, and secondly to investigate the level of adh in dogs with congestive heart failure due to acquired cardiac disease. elisa assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. pooled canine plasma was spiked with synthetic adh and intra-assay precision, dilutional parallelism, and linearity were assessed. to address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (cdvd) or dilated cardiomyopathy (dcm). patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. whole blood was collected into edta tubes containing protease inhibitors, cold centrifuged, and plasma was stored at À until analysis. following ether extraction, plasma adh in each sample was measured in duplicate using a human elisa kit. statistical analysis included a d-agostino and pearson test for normality; group results were compared using a nonparametric mann-whitney test. adh was measured in canine plasma using the human elisa kit with acceptable intra-assay precision, linearity, and dilutional parallelism. intra-assay coefficient of variation was %. twenty-four dogs were recruited for the second phase of the study. six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either cdvd or dcm were selected for participation. the remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. median adh values were . ae . pg/ml for the normal group (n ) and . ae . pg/ml for the heart failure group (n ). median adh values for the two groups were statistically different (p . ). our preliminary results indicate that measuring canine adh using a human elisa kit is feasible and provides results with an acceptable coefficient of variation. we also showed that dogs with congestive heart failure due to cdvd and dcm have elevated adh levels in comparison to normal dogs. our findings motivate further investigation to assess the degree of plasma adh level elevation and the possible use of adh antagonists as an adjunct treatment for refractory congestive heart failure in dogs. aortic thromboembolism (ate) occurs in both cats and dogs. whereas ate in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ate in dogs is less well known or understood. further, an effective antithrombotic strategy for ate in dogs has not been reported. medical records of dogs diagnosed with ate between and were examined retrospectively. diagnosis of ate was based on ultrasonography, doppler flow studies, and diminished or absent femoral pulses. dogs were treated with various acute and chronic antithrombotic therapies. the severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. a cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. target international normalized ratio for warfarin therapy was to . twenty-six dogs were diagnosed with ate. all had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. none had structural heart disease at diagnosis. twenty dogs ( %) were still ambulatory at diagnosis. the median duration of ambulatory dysfunction prior to presentation was . weeks (range day - weeks). a majority of dogs ( %) had no concurrent conditions at diagnosis. nine dogs ( %) had protein-losing nephropathy. four dogs ( %) were hypothyroid. fourteen dogs were treated with a standard warfarin protocol for a median period of . months (range . - months). eight dogs were treated concurrently with aspirin, dogs were treated concurrently with clopidogrel, and dogs were treated with warfarin only. ambulatory function improved between and grades in dogs treated with chronic warfarin. the median period until clinical improvement was . days (range - days). two dogs treated with chronic warfarin therapy had documented resolution of ate, and dogs had complete resolution of ambulatory dysfunction. none of the dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ate. the median period of freedom from an adverse event was . months. no serious hemorrhagic events were reported. four dogs were treated with tpa. three of these had an unfavorable outcome. two dogs were ambulatory before tpa and become non-ambulatory after treatment. two dogs underwent surgical thrombectomy. one had a favorable outcome until ate recurred months after surgery. in conclusion, the pathogenesis of ate in dogs is not associated with structural heart disease or left atrial thrombus formation. the presentation tends to be for chronic ambulatory dysfunction. most dogs are still ambulatory at presentation. warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ate. information is known. through previous work, investigators have encountered norfolk terriers (nt) with echocardiographically apparent dmvd in the absence of a heart murmur. in order to more fully understand dmvd in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy nt. overtly healthy nt ! yrs old were recruited by different veterinary hospitals and underwent historical, physical, ecg, and d/color-flow doppler echocardiographic exam. anterior mitral valve length, maximal thickness, area, and prolapse were measured from d images. presence of dmvd was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow doppler evidence of mitral regurgitation. blood samples were obtained for serum biochemistry and serotonin, plasma nt-probnp, amino acid profile, c-reactive protein, and cardiac troponin-i. forty-eight dogs were entered into the study (median age, yrs iqr [ - ]; gender, f, m; median bcs, ). of the dogs, ( %) had murmurs, ( %) had mid-systolic clicks, ( %) had ecg p-pulmonale, and ( %) were deemed to have echocardiographic evidence of dmvd, including nt without a murmur. seven ( %), ( %), and ( %) dogs were classified as isachc , a, and b, respectively. mean indexed echocardiographic mitral leaflet length (p o . ), thickness (p . ), prolapse (p . ), and la:aod (p . ) were significantly different between isachc a/b vs . between isachc a/ b and , there were no differences in serum amino acids, c-reactive protein, troponin, diet, or environmental factors; however different amino acids (ala, gly, phe, pro, trp, tyr) were significantly higher in isachc b vs. a. median serum serotonin was increased in dogs with a/b vs. (p . ). dogs whose diets contained some canned food (p . ) and dogs residing in suburban environments (p . ) had higher serotonin concentrations. nt-probnp tended (p . ) to be higher in isachc a/ b vs. . dmvd appears to be relatively common in nt and echocardiographic changes consistent with mild dmvd can be seen in dogs without a heart murmur. the results of this study establish a foundation of useful information upon which additional prospective studies can be developed. left ventricle (lv) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. however, lv analysis can be made from images obtained by different modes and views of the heart. the aim of this study was to compare lv measurements, shortening fraction (sf) and ejection fraction (ef) obtained from four methods: m mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart. forty normal adult german shepherds were selected. echocardiographic study of lv of each animal was performed by the four methods described above. ancova test was used to examine the effects of axis, mode, weight and gender over lv measurements. isolated effect of the axis was observed for lv end-diastolic diameter (lvedd), with greater values obtained from short-axis views. there was isolated effect of mode over ef and sf, with greater measurements derived from bidimensional mode methods. weight correlated with all linear lv measures at least in one method, but not with ef and sf. weight had positive effect over lv endsystolic diameter and lv end-diastole posterior wall thickness in all methods, except from m mode in short axis in the last one. gender had isolated effect over lvedd, males showing greater values than females in bidimensional mode in short and long axis. the combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness. we concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably. abstract c- assessment of left ventricular diastolic func-tion by color tissue doppler imaging echo-cardiography in maine coon cats tested for mypbc-ap mutation hypertrophic cardiomyopathy (hcm), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. myocardial analysis by color tissue doppler imaging (tdi) is more sensitive than conventional echocardiography. this study evaluated diastolic dysfunction in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation and examined with both echocardiography and tdi. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw) and in radial segment of left ventricular wall, was compared among different groups. a significant decreased (p , ) longitudinal em/am at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. a significant increased (p , ) longitudinal e/em at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. and a significant decreased (p , ) longitudinal sm at the basal segment of the lvw was observed in heterozygous cats compared with negative cats, both without hypertrophy. there was a significant positive correlation between summated early and late diastolic velocities (emam) and heart rate (p o , ); and a positive correlation between sm and em velocities and heart rate (p o , ). the mybpc-a p mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop hcm. the tdi alone is not able to identify cats with mutation before myocardial hypertrophy. diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (hcm) but less is known about systolic function in various stages of hcm. myocardial strain analysis by tissue doppler imaging is a noninvasive echocardiographic method to assess systolic function. this study evaluated systolic function in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation an examined with both echocardiography and strain. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw), left ventricular anterior wall (lvaw), left ventricular posterior wall (lvpw) and radial segment of left ventricular wall, was compared among different groups. whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of ivs) in hcm cats was significantly decreased compared with normal group (p , ). myocardial strain (at basal segment of lvaw) also was significantly decreased in heterozygous cats compared with negative group (p , ); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (p , ). there was a significant negative correlation between strain values and wall thickness (p o , ). this method allows detection of abnormal systolic deformation in maine coons cats with hcm mutation despite apparently normal systolic function. the abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. recently, multiple advanced resting electrocardiographic (ecg) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. this has improved the diagnostic and/or prognostic value of short-time ecg in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ecg. therefore, this study investigates, if advanced ecg can predict the severity of mitral regurgitation (mr) in dogs with myxomatous mitral valve disease (mmvd) and thereby improve the diagnostic value of ecg. the study included privately owned cavalier king charles spaniels (ckcss) (age . ae . years; males and females). all dogs were examined by echocardiography and a short-time ( - min) high-fidelity -lead ecg, with the dog in a resting position and in sinus rhythm. dogs were divided into groups according to the degree of mr estimated as the percentage of the left atrium area using color doppler mapping ( %; % o jet %; % o jet %; jet %; jet % and with clinical signs of congestive heart failure). ecg recordings were evaluated via custom software programs to calculate different parameters, including heart rate variability (hrv), qt variability (qtv), t-wave complexity, wave morphology and -d ecg. one-way anova determined ecg parameters, which were significantly different (p o . ) between the dog groups. principal component factor analysis identified a factor model with . % explained variability. qrs dipolar voltage and two repolarization indices of qtv increased significantly with mr severity, whereas total power of the frequency spectrum of rr interval and the standard deviation of qtv decreased significantly with mr severity. for the selected parameters the prediction of mr jet value was tested by multiple linear regression. a correlation coefficient (r) of . indicated that the prediction value was significant (p o . ). if age was included in the multiple linear regression the prediction value was further increased (r . ). our results indicate that for a cut-off criteria of mr ! % jet the five selected ecg parameters could predict the severity of mr caused by mmvd in ckcss with sinus rhythm with sensitivity % ( % with age inclusion) and specificity % ( % with age inclusion) (p o . ). nt-probnp concentration is increased in canine patients with heart disease. relatively little is known about the stimuli for release of nt-probnp in dogs. physical activity independent of cardiac disease and the stress of being in the hospital could influence nt-probnp release and affect diagnosis and management of patients. we hypothesized that nt-probnp concentration in healthy dogs would not exceed the normal reference value ( pmol/l) following a period of exercise. the goal of this study was to examine whether physical activity could elevate plasma nt-probnp and cause false positive results in healthy dogs. the study population included healthy dogs yr of age without heart murmur or known systemic disease, and normal d/color flow echocardiographic exam. plasma samples for nt-probnp were obtained before, immediately after, and hour after a standardized -minute submaximal exercise regimen. the study included dogs with a median age of . yrs and included females and males. there was no statistical difference in median plasma nt-probnp concentration across the three time points (baseline median, [iqr, immediately post, ; p . ). the average coefficient of variation of nt-probnp concentration across the exercise regimen was . ae . %. in of dogs ( . %), nt-probnp increased from to pmol/l immediately after exercise. the results of this study demonstrate that submaximal exercise does not significantly change median nt-probnp concentration and the incidence of false positive results is low. further studies should investigate effects of exercise on nt-probnp concentrations in dogs with heart disease. obesity is an increasing problem in veterinary medicine. obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of nt-proanp concentration in obese dogs. nt-proanp concentration was determined prospectively in obese dogs ( females; males; - months) and in non-obese dogs (controls; females; males; - months) from a veterinary hospital population. obesity was determined by body condition score [ ( / ); ( / ); ( / ); ( / ); ( / ); ( / )]. dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. commercial kits were used (vetsign s canine cardio screen nt-pro-anp vc -guildhay/biomedica). mann whitney test was used for group comparison. results are presented as median; interval; p and p ). nt-proanp was significantly lower in obese dogs [ . fmol/ml ( . - . ); p . ; p . ] than in controls [ . fmol/ml ( . - . ); p . ; p . ]; (p . ). results were similar to what has been found in obese humans. lower levels of natriuretic peptides are also seen in obese heart failure patients. this study provides important information regarding nt-proanp concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. left-to-right shunting patent ductus arteriosus (pda) is one of the most common canine congenital cardiovascular defects. human studies have shown that bnp and nt-probnp concentrations are elevated in patients with pda, and can be used to detect hemodynamically significant pda. the purpose of this study was to measure nt-probnp concentrations in dogs with pda, and to assess whether additional indicators of hemodynamics correlate with ntprobnp. we hypothesized that nt-probnp will serve as a simple non-invasive marker of hemodynamic significance in dogs with pda prior to and following transcatheter ductal occlusion. nt-probnp was measured in client-owned dogs with echocardiographically normal hearts. ten dogs with pda were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (pcwp) and nt-probnp. nt-probnp and echocardiography were repeated at day and months following ductal occlusion. pcwp was repeated at months. baseline nt-probnp was significantly higher in pda dogs compared to control ( ae pmol/l (mean ae sd), ae ; p o . ). at day and months following ductal occlusion, nt-probnp was ae and ae , respectively. the following decreased significantly from baseline: pcwp ( . ae . to . ae . mmhg; p . ), and indexed left ventricular internal dimensions in diastole ( . ae . to . ae . ; p . ) but not significantly in systole ( . ae . to . ae . ; p . ). nt-probnp is elevated in dogs with pda and transductal closure is associated with a reduction in nt-probnp, pcwp and left ventricular size. cardiac biomarkers, particularly nt-probnp, are becoming more commonly used in dogs and cats as part of a diagnostic work up. multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. in a portion of these reports the manner in which samples were handled was placement of whole blood into an edta tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at À c or À c prior to shipment, either with or without protease inhibition. our objective was to compare the nt-probnp concentrations in feline plasma collected using the previously reported methods to the california animal hospital (cah) collection method using tubes containing a protease inhibitor. this study compared nt-probnp concentrations using the protease inhibitor tubes vs. edta tubes from privately owned feline patients, with confirmed cardiac disease, and control feline patients. for all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ecg, and echocardiogram. in each study participant, at least ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic edta tube. the sample was centrifuged within hour after collection. ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at c until being shipped within hours of collection. the remaining plasma was placed into separate microtubes, which did not contain a protease inhibitor. one microtube was then stored and shipped as previous studies have reported (À c, styrofoam container, shipment within hours), and the second microtube was frozen at À c. all samples were shipped, received and analyzed within hours of collection. results of this study showed that no difference was found between the frozen sample methods ( pmol/l and pmol/l p . ). it was determined that both frozen methods had lower nt-probnp levels ( and pmol/l) when compared to plasma samples shipped in protease inhibitor tubes ( and pmol/l). the findings of this trial demonstrate that the nt-probnp levels are significantly different between samples placed in edta tubes vs. contain protease inhibitor (p . and p . ). utilizing protease inhibitor tubes allows more accurate measurement of plasma nt-probnp. as for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples. degenerative mitral valve disease (dmvd) is one of the most common heart disease and is present approximately % of the canine heart disease. although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. dmvd is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring dmvd. serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous dmvd in dogs. increased circulating ht concentration as a potential source of heightened ht signaling is demonstrated in small dogs with dmvd. the aim of this study was to investigate whether serum ht concentrations were associated with severity of naturally occurring dmvd in small dogs and to investigate potential associations of dog characteristics on serum ht concentrations in our study population. forty-eight dogs were included in this study and were classified into control and dmvd groups according to the results of physical and echocardiographic examinations. based on the la:ao ratio, dogs with dmvd were classified as follow: control (la:ao ratio . and no mr), mild (la:ao ratio . and mr), moderate ( . o la:ao ratio . and mr), severe (la:ao ratio . and mr). serum serotonin concentrations were measured by elisa. an overall significant difference (p o . ) was found among groups and ht concentrations (control, . ng/ml [ . - . dmvd, ). significantly higher ht concentrations were observed in dogs with moderate (p o . ) and severe (p o . ) dmvd, compared with concentration in control group. additionally, ht concentration in dogs with moderate dmvd were significantly higher (p o . ) than concentration in dogs with mild dmvd. also, dogs with severe dmvd had significantly higher ht concentration than dogs with mild (p o . ) and moderate (p o . ) dmvd. there was no significant association of age, platelet, and lvidd, on serum ht concentration, however, weak correlation between serum ht increased significantly and la:ao ratio (r . , p o . ) was observed. the results of this study indicate that serum ht concentrations were higher with increasing severity of spontaneous dmvd, which may be the potential cause to advance the progression of dmvd. further studies should be performed to reveal the role of ht in inducing and accelerating spontaneous dmvd and to investigate if lowering serum ht concentration could alter the progression of dmvd. the objective of this prospective study was to evaluate the utility of cardiac troponin i (ctni) in differentiating between underlying etiologies of pericardial effusion in the canine patient. patients were prospectively recruited at time of diagnosis of novel pericardial effusion. serum samples were collected prior to pericardiocentesis. patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (hsa), heart base tumor (hbt), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. idiopathic pericardial effusion (ipe) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion months, or symptom free months from time of enrollment. patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (creatinine . mg/dl) was present at time of sample collection. serum samples were frozen and analyzed in batches within days of collection by a ctni assay with a . ng/ml lower limit sensitivity. sixty-three patients were recruited over a one year period with patients excluded due to lack of diagnosis ( ) or azotemia ( ). median ctni levels of dogs with hsa (n ), hbt (n ), and ipe (n ) were . ng/dl (interquartile range (iqr) o . - . ), . ng/dl (iqr o . - . ), and o . ng/dl (iqr o . -o . ) respectively. concentrations of ctni differed significantly between dogs with hsa and hbt (p . ) and ipe (p . ). there was no difference between ctni concentrations between hbt and ipe dogs (p . ). receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with hsa and both hbt and ipe revealed a significant (p o . ) area under the curve ( . ). a cut-off point of ctni of . yielded a sensitivity of % ( % ci, - %) and specificity of % ( % ci, - %). utilizing a higher cut-off point of . yielded a lower sensitivity of % ( % ci, - %), but a higher specificity of % ( % ci, - %) which may have more clinical utility given the disparity in prognoses of the etiologies compared. in conclusion, this study supports the diagnostic utility of ctni concentrations to delineate between patients with hsa and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with ipe and other neoplastic etiologies. the pathogenesis of degenerative mitral valve disease (dmvd) in dogs remains to be fully elucidated. the high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule- (icam- ) and e-selectin in affected mitral leaflet tissue. we hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. the goal of this study was to compare plasma levels of icam- and e-selectin in healthy dogs and those with dmvd. the study population included dogs with echocardiographic evidence of dmvd and healthy control dogs year old with no heart murmur or known systemic diseases. dmvd dogs underwent d/color-flow doppler echocardiographic exam. blood samples were obtained for plasma icam- and e-selectin analysis using commercially available elisa kits. the study included dogs, of which had dmvd and were normal. the dmvd group had a median age of . yrs ) and included females and males. two ( %), ( %), ( %) and ( %) dogs were classified as isachc a, b, and a, respectively. of the control dogs, median age was . yrs [ - . ], with females and males. there was no statistical difference in plasma e-selectin between control dogs (median . [ . - . ]) and those with dmvd ( . [ . - . ]); p . . plasma icam- concentrations were higher in dmvd dogs ( . [ . - . ]) than controls (median . [ . - . ], but this difference did not reach statistical significance (p . ). linear regression analysis showed no significant correlation between icam- or e-selectin and serum serotonin level, nt-probnp or echocardiographic measures of dmvd severity (la:ao, lvidd:ao, lvids:ao). the results of this study demonstrate no significant difference in circulating adhesion molecules icam- and e-selectin in dogs with dmvd as compared with healthy controls. further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if icam- and e-selectin play a role in the pathophysiology of dmvd. the rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (hf). glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (gluts), which includes glut- , the major cardiac isoform, and glut- , a recently discovered isoform, the role of which is unknown in the heart. in addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. thus, we hypothesized that glut- and - protein and gene expression would be chamber specific in healthy dogs and during chronic hf. using a canine model of tachypacing induced chronic hf, glut protein and messenger rna in both ventricles and atria were investigated by immunoblotting and real time pcr. in control dogs, glut- , but not glut- , protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (ra, p o . ). glut- and - mrna were homogeneously expressed in all the cardiac chambers. during chronic hf, glut - and - expression was highest in the left ventricle (lv, by . and . fold, respectively, p o . ), with a concomitant increase in glut- and - mrna (p o . ). glut - , but not glut- , was decreased in ra during chronic hf (p . ). our data suggest that glut- protein was differentially expressed across the cardiac chambers in the healthy heart. during chronic hf, lv was the primary site dependent on both glut and glut -mediated glucose transport, which was transcriptionally regulated. in addition, the paradoxical decrease in glut content in ra may induce perturbations in atrial energy production during chronic hf. some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. it has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. the purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs. twenty three obese dogs and controls were included based on a - body condition scoring (bcs). computerized radiography was obtained and vhs measurement was performed as previously described. echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. results for echo and vhs measurements were classified in scores as normal, mild, moderate or severe increase. student's t test was used for comparison of vhs between groups. mann-whitney rank sum test was used to assess echocardiographic scores between groups. spearman rank order correlation was used to assess relationships between any pairs of variables between echo and vhs scores, echo vs bcs and vhs vs bcs. groups were similar regarding age [obese ( ae ); control ( ae ); p . ], breeds and gender distribution. obese dogs had significantly higher vhs and echo scores compared with controls [vhs: ( . ae . ) vs ( . ae . ); p o . ; echo score: range ( - ) vs ( - ); p . ]. there were no relationships between any pair of variables analyzed. these results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but vhs overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. heart rate variability (hrv) is an indirect measurement of the autonomic modulation of heart rate (hr). reduced hrv measured from short-time electrocardiography is seen in dogs with heart failure (hf) secondary to myxomatous mitral valve disease (mmvd). however, hrv is suggested to increase with disease severity at early stages of mmvd. the aims of this study were ) to associate hr and hrv with severity of mmvd in cavalier king charles spaniels (ckcs) and ) to compare hr and hrv between ckcs and other dog breeds in a group of dogs in hf secondary to mmvd. one-hundred dogs were examined by echocardiography and hour electrocardiography. the dogs were divided into five groups: ) ckcs with no/minimal mitral regurgitation (mr) (mr jet % of the left atrial area using color doppler mapping) and no murmur, ) ckcs with mild mr ( % o jet %), ) ckcs with moderate/ severe mr (jet %) and no clinical signs of hf, ) ckcs in hf (hf defined as left atrium to aortic root ratio (la/ao) . , clinical signs of hf and furosemide responsiveness) and ) non-ckcs in hf. dogs in hf were allowed hf therapy. both hr and hrv were analyzed over a -hour period, while hrv were also analysed over a -hour nightly period. analyses of variance were performed with hr or hrv as response variables and the explanatory variables dog group and echocardiographic indices of mmvd were included separately. all p-values were bonferroni corrected. minimum-and mean hr were significantly higher in ckcs with moderate/severe mr and in hf compared to ckcs with no/ minimal and mild mr (all p o . ). seven out of hrv variables were significantly decreased in ckcs with moderate/ severe mr and in hf compared to ckcs with no/minimal and mild mr (all p o . ). another hrv variables showed the same groupwise differences (all p o . ), except that the difference between ckcs with mild mr and ckcs with moderate/severe mr did not reach statistical significance. mminimum hr, mean hr and the hrv variables ( and ) differing between dog groups, also consistently decreased with increasing mr, la/ao and the proximal isovelocity surface area in ckcs. non-ckcs in hf had a lower minimum hr compared to ckcs in hf (p . ) and a higher triangular index measured in both periods (all p o . ). in conclusion, hr increased and most hrv variables decreased with increasing severity of mmvd in ckcs, even prior to the development of hf. other breeds in hf secondary to mmvd had lower minimum hr, but higher triangular index compared to ckcs in hf. although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. different types of respiratory patterns can stimulate autonomic nervous system in different manners. thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate. twenty male, one-year-old healthy new zealand rabbits were enrolled in the study. animals were set in right lateral recumbency and maintained that way by physical contention. chemical sedation was not used. partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. heart rate was obtained by measurement of two consecutives rr intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver. heart rate before the intervention was ae bpm (mean ae standard deviation). all rabbits submitted to the maneuver showed a dramatic reduction in this parameter. after nasal partial obstruction, heart rate was ae bpm. data was submitted to statistical analysis by paired student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o . ). although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. the purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy. the study groups comprised fifteen obese cats with mean body score index (bsi) of , , seven overweight cats (bsi , ) and seven cats with ideal bsi ( , ). the blood pressure was measured by doppler method and the doppler echocardiography was performed in conscious animals. the statistical analysis was performed by analysis of variance followed by tukey's test and pearson's correlation. the blood pressure values of the obese cats were superior ( , ae , mmhg, p o , ) than in overweight ( , ae , mmhg) and normal cats ( , ae , mmhg) and % of the obese cats had blood pressure higher than mmhg. there were observed differences on the ratio of early (e) and late (a) left ventricular filling velocity (p , ) of obese animals (e/a , ae , ) compared to overweight ( , ae , ) and normal cats ( , ae , ). seven obese cats ( %) had inversion of e/a compatible with diastolic dysfunction and there were negative correlation (r À , , p , ) between the e/a ratio and blood pressure values. other differences observed were increases in left ventricular septum in diastole (p , ) and in free wall in diastole (p , ) and systole (p , ) of the obese animals compared to overweight and normal cats. these results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. we hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. a double-blinded, randomized, crossover clinical trial was performed in dogs with stable heart failure receiving bid furosemide and adjunctive medications. dogs were randomized to their current furosemide dose or torsemide (calculated as / of the daily furosemide dose divided into bid dosing). crossover occurred at day and the study ended on day . clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days , and . no dog developed recurrent heart failure during the study. average furosemide dose on day was . mg/kg/day (range, . - . ). following torsemide treatment, blood urea nitrogen (p . ), albumin (p . ), and albumin:globulin ratio (p . ) were significantly increased, and urine specific gravity (p . ) and chloride (p . ) were significantly decreased as compared to baseline and/or furosemide dosing (one-way anova with bonferroni correction). no differences in qol were found. results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. the purpose of this study was to investigate the feasibility of speckle tracking echocardiography (ste) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart. radial and circumferential strain and strain rate were measured by ste using left ventricle short-axis view in clinically healthy cats. eighteen cats with hcm whose lv thickness at end-diastole with mm or more were evaluated with ste analysis, and compared with healthy cats. index of left ventricular synchrony (trs-sd) was also assessed in cats with hcm, and compared to healthy subjects. ste resulted in technically adequate images in % of the cats. fusions of early and late diastolic (e and a) wave in strain rate were seen in of cats. percent errors in analysis with or without simultaneous ecg monitoring were . - . % in all parameters. inter-and intraobserver variability of ste parameters in healthy cats was minimal ( . - . %) except for the systolic circumferential strain rate. sedation using buprenorphine and acepromazine did not affect any ste parameter. e wave in radial and circumferential strain rate of hcm cats was significantly decreased compared with healthy cats. no significant difference was seen in trs-sd. ste analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with hcm. further study is warranted to investigate to assess whether or not ste can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. carvedilol, a rd generation non-selective beta-blocker with ancillary alpha -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. hplc for carvedilol measurement in feline plasma was validated and standardization curves created. the pharmacokinetics (pk) of carvedilol was evaluated in apparently healthy male neutered adult cats (average weight of kg) following single dose intravenous (iv) of . mg/kg and single dose oral administration of . to . mg/kg. concentrations of the active parent compound, carvedilol, were detected in plasma using hplc analysis. lower limit of quantification was ng/ml. the mean peak concentration after iv administration of carvedilol was ng/ml (range, to ), elimination half-life was . hours (range, . to . ), and clearance was . l/hr/kg. the volume of distribution was . l/hr. after a single oral administration of carvedilol, the time to peak plasma concentration was minutes (range, to minutes) and the mean residual time was . hours. the half life was . hours. maximal concentration ng/ ml and the mean bioavailability was . % with a median of . % (range, . % to %). these data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. all cats tolerated the oral dose of carvedilol with no major adverse effects. also, a mean residual time of . hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol. abstract c- effects of sildenafil citrate on dogs with ei-senmenger's syndrome. k nakamura, m yamasaki, h ohta, m takiguchi. department of veterinary clinical sciences, graduate school of veterinary medicine, hokkaido university, sapporo, hokkaido, japan. sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with eisenmenger's syndrome (es) and secondary erythrocytosis has not yet been determined. the objective of this study is to determine the effect of sildenafil for dogs with eisenmeger's syndrome and secondary erythrocytosis. this was a prospective, single arm, open-label study. five clinical dogs with es and secondary erythrocytosis were included. new york heart association (nyha) functional class, pcv, and pulmonary artery acceleration time to ejection time ratio (pa at : et) were evaluated before and after sildenafil therapy ( . mg/kg, bid). nyha functional class was significantly improved after (median ; range - , p . ) and months (median ; range - , p . ) of sildenafil therapy, compared with the baseline (median , range - ). pcv was significantly decreased after month ( . ae . %, p . ) and months ( . ae . %, p . ) of therapy, compared with the baseline ( . ae . %). at : et was significantly increased after month of therapy ( . ae . , p . ) from the baseline ( . ae . ). sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with es. sildenafil therapy could be the treatment of choice for dogs with es. sepsis is the number one cause of mortality in neonatal foals. the role of the raas and hpaa in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. we hypothesized that in septic foals the raas and hpaa will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. blood samples were collected on admission from septic (sepsis score ), sick non-septic (sns), and healthy foals of o days of age. blood concentrations of corticotropin-releasing hormone (crh), adrenocorticotropin (acth), cortisol, aldosterone, angiotensin-ii (ang-ii), arginine vasopressin (avp) and plasma renin activity were determined by radioimmunoassays. acth, cortisol, aldosterone, ang-ii and avp concentrations were higher while crh was lower in septic and sns foals compared to healthy foals (p o . ). septic non-survivor foals had higher concentrations of aldosterone, cortisol, acth and avp and lower concentrations of ang-ii and crh than survivors. avp was associated with ang-ii in septic, and with acth in septic and sns foals (p o . ). there was no difference in renin activity and ang-ii concentrations among foal groups. septic foals had a higher acth:aldosterone ratio than healthy foals (p o . ). this study shows that in response to sepsis there is raas and hpaa activation in critically ill foals. we propose that in sick foals avp is more important than crh in regulating acth secretion. the increased acth:aldosterone ratio further supports relative adrenal insufficiency in septic foals. this prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. physical and hematological parameters were evaluated at times (onset of diarrhea), , , and h, then every h until resolution of diarrhea or death. each hematological analysis included a complete blood count (cbc), thromboelastography (teg), partial-thromboplastin-time (ptt), prothrombin-time (pt), plasma concentrations of lactate, tumor necrosis factor alpha (tnf-a), interleukin (il)- , il- , il- and nt-proc-type-natriuretic peptide (pcnp). horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. group : diarrhea o h without systemic inflammation (si); group -diarrhea ! h without si; group -diarrhea with si. assessment of vital parameters and cbc established a diagnosis of si as previously described (levy, ) . descriptive and univariate outcome analyses were based on data normality. horses were enrolled, of which ( . %) survived to discharge. the mean age was . /- . years. eight horses ( . %) were categorized as group- , ( . %) as group- and ( . %) as group- . two horses developed thrombophlebitis. based on the results of teg, / ( . %) were normocoagulable, / ( . %) were hypocoagulable and / ( . %) were hypercoagulable, at one or more time points. of these, / ( . %) group- horses were coagulopathic. additionally, group- horses had a significantly lower ma than group- horses at baseline ( . ae . vs. . ae . ) and h ( . ae . vs. . ae . ). biomarker analyses are pending. in conclusion, si was associated with coagulation disorders in horses with hospital acquired diarrhea. clostridium difficile and clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. reported carrier rates of toxigenic c. difficile and c. perfringens strains in feces range between - % and - % respectively. toxigenic c. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. the objectives of this study were to investigate the presence of c. difficile and c. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates. intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of euthanized horses free of apparent gastrointestinal disease. enrichment culture was performed for c. difficile and c. perfringens and c. difficile isolates were further characterized via toxin gene detection and ribotyping. c. difficile was isolated from / ( %) samples from / ( %) horses. between zero and three sites were positive per horse, and multiple sites were positive in three horses. isolates were recovered from duodenum (n ), right dorsal colon (n ), small colon (n ) and rectum (n ). in one horse, the rectal sample was negative but c. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. in three horses multiple compartments were positive however different strains were always present within the same horse (n ). all isolates possessed genes encoding toxins a and b. five isolates were ribotype and also possessed genes encoding the binary toxin. the other isolates were ribotype and were negative for the genes encoding the binary toxin. despite using a method with a detection level as low as cfu/g of feces, no c. perfringens was recovered. rectal samples were a good predictor of overall c. difficile carrier status ( / horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. the presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. the predominance of ribotype is consistent with recent emergence of this strain in this region, as earlier studies found other strains ( , ) to be more prevalent and a variety of ribotypes were typically recovered from horses. interestingly ribotype has recently emerged as a hypervirulent strain in humans in our area. clostridium difficile, clostridium perfringens and salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. additionally, antimicrobial resistance is a pressing concern. commensal e.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. the objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for e. coli. fecal samples were collected from adult horses from five farms on a monthly basis over the course of one year. selective cultures were performed for c. difficile, c. perfringens, salmonella and e. coli. c. difficile isolates were characterized via toxin gene pcr and ribotyping. broth microdilution was performed to assess antimicrobial susceptibility profiles of e. coli. clostridium difficile was isolated from / ( . %) samples from / ( %) horses. four horses were positive on more than one occasion, three were positive in two consecutive months. different ribotypes were found in two of the latter horses. most isolates were ribotype (n ) with ribotype (n ) and ribotype c (n ) also identified. ribotypes and c possessed genes encoding toxins a, b and binary toxin, while ribotype only possessed toxin a and b genes. despite a detection threshold of cfu/g feces, c. perfringens was not detected in any samples, nor was salmonella. e coli was isolated from / ( %) samples. resistance to ! antimicrobial was present in only / ( . %) isolates. multidrug resistance (! antibiotics) was present in / ( %). most commonly, isolates were resistant to sulfisoxazole ( / ) and trimethoprim sulfamethoxazole ( / ). the overall detection rate for toxigenic c. difficile in fecal samples of healthy horses was . % which is consistent with previous studies. the cumulative prevalence of % was striking but only one horse shed the same strain for more than one month, indicating c. difficile shedding is a transient and dynamic state. the predominant isolation of ribotype is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. the low prevalence of c. perfringens and salmonella is in agreement with some other studies. the low prevalence of antibiotic resistance in commensal e. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. type polysaccharide storage myopathy (pssm) in horses is associated with a dominant missense mutation (r h) in the skeletal muscle glycogen synthase gene (gys ). since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. belgian / percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (hh), heterozygotes(hr) or normal (rr). subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. sections were stained using haematoxylin and eosin, periodic acid schiff /diastase. anti-dystrophin, nnos and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, there were significant differences in resting ck activity (p . ) (median hh u/l interquartile range(ir) - ; hr u/l ir - ; rr u/l ir - ) and ast activity (p o . ) (ast mean hh u/l sd ; hr u/l sd ; rr u/l sd ) and muscle pathology between the groups, with severity increasing rr o hr o hh. there were significantly more type a (p . ) and fewer type x fibres (p . ) in homozygotes ( a % sd . ; x % sd ) compared with the other groups (hr a % sd . , x % sd . ; rr: a . % sd . x % sd . ). more type a fibres contained polysaccharide inclusions in homozygotes ( % sd . ) than in heterozygotes ( . % sd . ) (p o . ). both dystrophin and nnos expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. in conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma ck and ast activities than heterozygotes, and pssm is associated with a fibre type shift towards type a. although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. the recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. there is currently very little data available on the prognosis and outcome of horses that are recumbent. therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival. records of horses admitted to the school of veterinary medicine, university of california davis from january of to december of with a history of recumbency or horses that became recumbent while hospitalized were evaluated. a horse was defined as recumbent if it was unable to stand on its own. the medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rdvm, breed, age, weight, date of presentation, physical and neurological examination findings, cbc and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. statistical analysis correlating factors associated with survival was performed using logistic regression. overall there were non survivors and survivors. factors that favored survival included early initiation of treatment in the field by the rdvm, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased bun, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease. in conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival. abstract e- plasma peak and trough gentamicin concentra-tions in hospitalized horses receiving once daily gentamicin. jr read , pa wilkins , rd nolen-walston . university of pennsylvania, new bolton center, kennett square, pa. university of illinois, champaign-urbana, il. gentamicin is often used to provide gram negative antimicrobial coverage in horses at . mg/kg iv every hours. therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration ( -  minimum inhibitory concentration) for common bacterial isolates (peak target range - mg/ml). the aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. review of records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) identified horses ! months old receiving once-daily gentamicin with peak and trough assays performed (n sets). spearman rank correlation coefficient analysis revealed a weak (r . ) but statistically significant correlation (p . ) between gentamicin dose and peak plasma concentration. horses receiving . - . and . mg/kg gentamicin (groups and ) had higher median peaks ( mg/ml) than horses receiving . - . mg/kg (group ; . mg/ml). higher doses were more likely to result in peaks mg/ml ( and %, groups and respectively) than horses receiving . - . mg/kg (group ; %). all hour post-gentamicin administration trough values were o mg/ml. no correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. these data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. additionally, these data support an initial dose of . - . mg/kg iv every hours in order to achieve desired peak concentration and an appropriately low trough concentration. heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. this disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. however, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. the objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses. peripheral neutrophils were isolated from heaves-affected (n ) and control (n ) horses using a density gradient technique. the killing capacity was assessed by incubating neutrophils with streptococcus equi spp equi and spp zooepidemicus. after h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. the phagocytosis was evaluated by flow cytometry using fluorescent beads and gfp-transformed streptococcus suis suilysin-negative mutant strain. circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward s. zooepidemicus (p . ). a reduced, although not significant (p . ), killing capacity of s. equi by these neutrophils was also observed. the phagocytosis activity was not different between groups. this impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. obesity is a common disorder of the horse, with current prevalence estimated at %. in people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. in the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. we hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses. sixteen light breed horses, - years of age were included. body condition score (n ) and neck circumference (n ) were recorded. post-mortem biopsy samples of the semi-membranosus muscle were obtained. dna and rna were isolated. relative expression of the metabolic genes, peroxisome proliferator activated receptor g (pparg), pparg coactivator- a (pgc- a), fatty acid translocase (fat) and estrogen related receptor a (erra) was determined by quantitative polymerase chain reaction (qpcr). mitochondrial content was assessed by determining mitochondrial dna/nuclear dna ratio by qpcr, using nadh-dehydrogenase subunit and cytochrome oxidase subunit as mitochondrial genes and beta actin as the nuclear reference gene. non-normal data was log transformed for analysis and a pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. a value of p o . was considered significant. body condition score was strongly correlated with neck circumference (n , r . , p . ). relative expression of erra and glut- increased with body condition score (erra: n , r . , p . ; glut- : n , r . , p . ). copy number of the mitochondrial genes (nadh-dh and cox- ) was not related to body condition score or metabolic gene expression. expression of glut- , erra, pparg, and fat were strongly correlated to each other, but not pgc- a. there was a strong trend towards correlation between pparg and pgc- a in horses with body condition score (n , r . , p . ). in this study, there was no change in mitochondrial content in obese horses. assessment of mitochondrial function in obese horses and horses with ems is under way. the strong correlation between pparg and pgc- a observed only in horses with high body condition scores suggests this pathway is activated with obesity. the role of pparg and pgc- a in equine obesity should be further investigated to determine their potential as therapeutic targets. upregulation of erra and glut- in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. further studies to better define the role of metabolic regulatory gene expression in obese horses and those with ems are ongoing. previously presented at the th annual harold hamm diabetes center research retreat, oklahoma city, ok. inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. this condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. the purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures. eight horses received mg/kg dextrose or d-xylose powder mixed as a % solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks ( tests/horse). a crossover design was employed and the order of treatments was randomized. blood samples were collected at time , , , , , , and min. data were analyzed by repeated measures anova and t-tests. results showed that the xylose response over time differed significantly from the glucose response over time (test  time; p o . ). mean time to maximum concentration differed (p o . ) between tests (glucose min; xylose min). within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. we conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. the purpose of this study was to examine the records of a population of thoroughbreds with cervical vertebral malformation (cvm) and to determine which factors have an effect on these horses achieving athletic function. this was a retrospective case study of thoroughbreds with cvm treated medically from to . forty-one were euthanized after diagnosis, while the remaining were discharged for treatment. racing records were reviewed to determine which horses raced after treatment. horses were separated into groups based on whether or not they raced. medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups. twenty-one of horses treated medically ( %) improved enough to race. median neurologic grade between groups was significantly different (p o . ), with a hind limb grade of . (range - ) for the raced group and . (range . - ) for the unraced. intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. horses with kyphosis (p . ), degenerative joint disease (p . ), or cranial stenosis (p . ) at any site were less likely to return to racing. racing prognosis for horses with cvm treated conservatively is equivalent to that of those treated surgically as reported by rush moore et al (javma, ) . radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. the project consisted of four stages. firstly, behaviors to include in a scale were empirically identified. thirty equine clinicians noted behaviors in each of random film clips of horses with colic using a checklist. nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: . - . ). secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. six expert clinicians independently expressed opinions as to which of behaviors to include and the severity of pain they indicate. two contending scales (equine acute abdominal pain scales (eaaps) & ) were constructed based on both the empirical and the judgmental approaches. each included identical behaviors with a - point score range; eaaps- with gradations to some of the behaviors and eaaps- without. in the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (vas) in horses with colic and controls (spearman rho; lactate . ; cortisol . ; heart rate . ). finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. thirty of films of horses with colic were randomly presented to expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both vas and numerical rating scale (nrs)), and two groups, each by one of the two eaaps scales. inter-observer reliability of both eaaps scales was excellent (intraclass correlation . ). intra-observer reliability based on scores given for identical films demonstrated; % and % agreement, kappa . and . , and spearman's rho . and . for eaaps- and , respectively. both scales varied by score between observations. face validity; each group reported their scale to be valid ( % & %). convergent validity; the scales compared favorably with vas/nrs scores (spearman's rho: . - . ). discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho . - . ). extreme group validity; colic horses scored significantly higher than control horses; scores of . - . in controls versus . - . in cases. in conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. further refinement of the eaaps scale is advised prior to introduction into clinical practice. aortic valve prolapse (avp) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. previously published data has shown that echocardiographic identification of avp in horses is reliable, diagnostic criteria have been established and that development occurs with training. the aims of this study were to evaluate the different rna and protein expressions of smooth muscle actin (sma), transforming growth factor-b (tgf), nitric oxide synthase (nos) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. valve cusps were harvested and processed from a group of horses at a commercial abattoir following disease classification using echocardiography. horses were aged . ae . years, weighing ae kg and with a median body condition score of / . cusps were collected in rnalater s and stored at À c prior to processing. cdna was produced from half a valve using a standard protocoland qrt-pcr performed to assess relative rna expression of sma, tgfb , endothelial (enos) and inducible nos (inos) and compared with the housekeeping gene s. a quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of sma, tgf b , enos and compared to vimentin. specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. the remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays . statistical analyses included one way anova with post-hoc bonferoni, paired student's t-test, linear and logistic regression. there was no effect of gender or age on any of the measurements. valves from animals with avp had lower expression of sma and elastin compared to normal and diseased valves, increased expression of tgfb and enos, whereas inos expression was greater than normal valves (table ) . collagen content of valves from horses with avp was increased compared to normal but lower than horses with valve disease. prolapsing cusps appear to be a different phenotype from diseased cusps. further studies will help to elucidate the significance of these findings in vivo. a clear association between heart rate (hr) and body mass has been observed across a wide range of mammalian species. furthermore, it is well known that electrocardiographic (ecg) time intervals vary with heart rate and body mass. within the equine species, small breeds are generally thought to have higher heart rates than large breeds. however, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ecg time intervals in horses and ponies of different body size. similarly, the relationship between hr and body mass in dogs of various breeds and sizes is still under debate. the goal of this study was to investigate the relationship between heart rate and ecg time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups. adult horses and ponies at an age of . ( - ) y [median (range)] and a body weight of ( - ) kg were included in the study. all animals were considered clinically healthy based on history and physical examination. a standard base-apex ecg was recorded at a speed of (n ) or mm/s (n ) using a multiparameter monitor (datascope passport). during the procedure, the horses were unsedated, standing quiet in a box stall. mean hr over sec was determined for each recording. the following ecg time intervals were measured in triplicate and averaged for further analyses: pq interval, qrs duration, qt interval, and difference between qt and qrs (qt-qrs duration). the relationship between hr, ecg time intervals, and body mass was assessed using linear regression analyses. normal ranges ( . % to . % percentile) were calculated for different weight groups. the level of significance was p . . heart rate was inversely related to body mass (p o . , r . ). the pq interval (p o . , r . ), qrs duration (p o . , r . ), qt interval (p o . , r . ), and qt-qrs duration (p o . , r . ) were directly related to body mass. normal ranges for hr, pq, qrs, and qt within the different weight groups were - bpm, - ms, - ms, - ms (o kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); and - bpm, - ms, - ms, - ms ( kg). we conclude that in healthy horses there is a significant but weak relationship between body mass and hr and between body mass and ecg time intervals, respectively. this study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ecg time intervals than large breeds. therefore, body mass has to be considered when comparing hr and ecg time intervals to normal ranges in horses. horses with pituitary pars intermedia dysfunction (ppid) often have elevated plasma acth concentrations. however, ppidaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. we hypothesized that this apparent dissociation between plasma acth levels and adrenal response in horses with ppid is due to the secretion of acth that is less biologically active than that from normal horses. to test our hypothesis, a bioassay to evaluate acth activity was developed. adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n ) and ppid-affected horses (n ). the assay was performed three times with explants obtained from different horses. cortisol secreted by the explants and plasma acth levels were measured with commercially available radioimmunoassays. cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. cortisol data was normalized for acth concentration in each plasma sample and expressed as a cortisol:protein:acth ratio. ratios from horses with ppid and normal horses were compared by unpaired t-test. horses with ppid had significantly lower cortisol:protein:acth ratios compared to normal horses. (assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ). these results suggest that plasma acth from ppid horses is less biologically active than plasma acth from normal horses. our findings give further insight into the pathophysiology of ppid and may aid in the development of novel diagnostic testing protocols. an online survey was conducted to determine perceived needs of potential employers of new acvim-laim diplomates. the survey was designed as the first step in determining what is needed for success in the various sectors of practice employing acvim-laim diplomates. demographic and background data were collected using questions and drop-down menus on the first page. the survey evaluated skills or concepts in areas of veterinary practice. participants answered questions about each skill or concept using drop-down ranked lists. those participants that had completed an acvim-laim training program were asked additional questions about whether they were taught the skill or concept during their own residency. data were collated and descriptive statistics calculated. the mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate. eighty-eight individuals participated in the survey with respondents being acvim diplomates, respondent was not board-certified and respondent was an act diplomate. nineteen respondents were diplomates of acvim and an additional specialty. eighty-three respondents had completed an acvim residency. the majority of respondents were in academia ( %) with % being in private practice. equine specialists prevailed ( %) followed by mixed large animal ( %) and then food animal only specialists ( %). the distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. most respondents stated that they did not make hiring decisions in their practice. competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. surgical skills, both equine abdominal ( %) and food animal general surgery ( %) were considered important by some respondents. thirty-six per cent of respondents thought that a new diplomate should expect to make o $ , per annum, while only % of respondents thought that a new diplomate should expect to make ! $ , per annum. not all respondents answered questions on all skills or concepts. the mean number of skills or concepts evaluated was (sd ) with only respondents answering all . all skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. at least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. these data will provide a framework for designing future laim residency programs. abstract this study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available cox -sparing nsaid in clinically healthy pony foals in a randomized controlled clinical trial. values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen shetland pony foals treated with firocoxib ( . mg/kg, po, q h) or placebo for days. foals were divided into treatment groups. group and foals received firocoxib while a rd group was administered an oral placebo. gastric endoscopy was performed on group and foals prior to treatment and on days and to monitor for the presence of gastric ulcers. group and foals had blood and urine samples taken sequentially for pharmacokinetic analysis, cbc, serum chemistry evaluation, and urinalysis. physical examinations were performed prior to treatment and daily for days. data were analyzed using anova and paired t-tests (p o . ). none of the foals presented adverse clinical effects. there were no significant changes in cbc, biochemical profiles within groups, or differences between groups. pretreatment gastric endoscopy scores were not significantly different from evaluations at and days. firocoxib was quickly absorbed with an observed maximum concentration at hr, the first sampling interval, for the majority of animals. firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the th dose. based on the sampling times after the final and th dose, an average half life of . days was estimated. administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in - week old foals. equine herpesvirus (ehv- ) is highly prevalent in most horse populations. horses are routinely vaccinated against ehv- , and neutralizing antibodies have helped to prevent disease. however, the usda has recently classified ehv- myeloencephalopathy (ehm) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of ehm that suggests a change in virulence of the virus. it has been reported that cellular immune mechanisms, in particular cytotoxic t-cells (ctls), are important in controlling ehv- viremia. interferon-alpha (ifn-a) has a key function in innate immune regulation by inducing the differentiation and maturation of ctls. here, we investigated the influence of abortogenic (racl , ny ) and neuropathogenic (ab ) ehv- virus strains on ifn-a, il- and il- secretion in equine pbmc. equine pbmc were infected with racl , ny or ab ehv- strains or kept in medium for hours. ifn-a, il- and il- secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. the production of ifn-a increased with increasing viral doses and similarly for all three ehv- strains. the production of the antiinflammatory cytokine il- was significantly decreased after ab infection compared to racl and ny strains at viral infection doses of moi . - . at high doses (moi ), il- production was suppressed by all three ehv- strains. the results suggested that abortogenic and neuropathogenic ehv- strains equally induce antiviral ifn-a production in equine pbmc. they also illustrated the differences in the ability of ehv- strains to modulate anti-inflammatory il- . neuropathogenic ab strain had an increased potential to down-regulate il- production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. the variations in innate il- secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic ab induces more severe disease, including myeloencephalopathy, than abortogenic ehv- strains. previously presented at a conference of research workers in animal disease. rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. treatment protocols are long, expensive and do not always produce good results. rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. recent studies are based on the use of the virulence associated protein a (vapa) which has been described as an important inducer of immunity against r. equi. the present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of s. enterica typhimurium expressing vapa antigen (test group) or s. enterica typhimurium without the vapa gene (control group), previous to and following experimental challenge. two experimental phases were established according to the immunization route: intranasal or oral vaccination up to hrs following birth and at days of age. the experimental and control groups were challenged on day with a virulent stain of r. equi. clinical examination, cbc and image complementary exams were used to evaluate the development of clinical signs. immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of r. equi and cytological profiles of tbw. clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group ( / ) and was due to r. equi pneumonia. the test group produced a more intense iggb response when compared to controls however no statistical difference was observed. lymphoproliferation and th cytokine expression were higher in the test group. in contrast, controls produced an il- response. local iga was significantly higher in animals immunized with salmonella carrying vapa. immunization protocols produced no severe toxic effects. the vaccination of neonatal foals with s. enterica typhimurium expressing vapa was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental r.equi infection. this study was conducted to test the hypothesis that the kd protein, myristolated alanine-rich c-kinase substrate (marcks), is involved in equine neutrophil migration and adhesion. in other species, marcks phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. to investigate marcks phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (paf), leukotriene b (ltb ) or phorbol myristate acetate (pma). western blot was performed using specific phospho-marcks and total marcks primary antibodies. these results determined marcks phosphorylation is maximal seconds following stimulation and that dephosphorylation occurs within minutes. to investigate the requirement for marcks in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with mans (a cell permeant peptide identical to the n-terminal amino acids of marcks), rns (a control peptide) or vehicle control (vc) prior to a migration assay toward known neutrophil chemoattractants (ltb or paf). pre-treatment of equine neutrophils with mans significantly inhibited migration while rns pre-treatment had no effect. to investigate marcks requirement in equine neutrophil adhesion, mans, rns or vc treated cells were stimulated to adhere to immulon plates coated with % fbs. pre-treatment of equine neutrophils with mans significantly inhibited adhesion while rns pre-treatment had no effect. inhibition of marcks using a cell permeant peptide identical to the protein's n-terminus significantly inhibited equine neutrophil adhesion and migration. these results indicate that marcks is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. we hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses. four thoroughbreds without any history or clinical signs suggestive of neurological disease (age range to years) were included in the study. retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. a minimum of trials each with - walk strides for each task were analysed as horses walked across an -force-plate runway i surrounded by a -camera kinematic system. ii force-plate data were processed with semi-automated custom written matlab iii scripts. data were analysed with a mixed model using the statistical software r. there was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o . ) and of the left and right diagonal coupling intervals (p o . ). there was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. the random effect of horse had no influence on the coupling intervals. the decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. we conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin b that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. it affects young quarter horses, appaloosa, and paints. herda shows similarities to the human hereditary connective tissue syndrome ehlers danlos (eds). many eds patients suffer from joint pain and osteoarthritis (oa) as adults. the similarity between eds and herda raises the question whether horses suffering from herda develop oa. in oa, excess production of inflammatory mediators such as prostaglandin e (pge ) activate enzymes that degrade cartilage as well as impede wound healing. the present study examined articular cartilage from yearling horses afflicted with herda. we hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. to test this hypothesis, articular cartilage from carpal and tarsal joints of herda horses were evaluated using histology. pge production by chondrocyte cultures was measured by elisa and analyzed by one-way anova, tukey post-hoc test, p o . significance. we also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (asu), glucosamine (glu), and chondroitin sulfate (cs) mixture (ingredients found in cosequin s asu) and phenylbutazone (pbz) on chondrocytes. cosequin s asu and pbz are used alone or in combination for the management of oa. chondrocyte cultures were incubated for hrs with control media alone, a clinically relevant concentration of pbz ( mg/ml), or the combination of asu (nmx s , . mg/ml) glu (fchg s , mg/ml) cs (trh s , mg/ml). articular cartilage from joints of five herda-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. chondrocyte cultures from cartilage of horses suffering from herda spontaneously produced greater pge than chondrocytes from normal horses ( -fold). pbz significantly decreased pge production by $ % (p o . ). the combination of asu -glu cs also significantly reduced pge production by $ % (p o . ). the present study supports anecdotal findings that horses suffering from herda are likely to develop oa. the inhibition of pge synthesis by asu glu cs suggests that this combination may be beneficial for the management of oa in herda. research supported by nutramax laboratories, inc. equine inflammatory airway disease (iad) is a common condition often treated empirically with corticosteroids. gene expression analysis in the bronchoalveolar lavage fluid (balf) may help understand the effects of corticosteroids in iad. the first part of the study aimed at identifying reference genes in the balf of iad horses treated with corticosteroids. the second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mrna expression of il- b, il- , il- and il- . the expression stability of seven candidate reference genes was determined in balf taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with iad. primers' efficiencies were calculated using linregpcr. normfinder, genorm and qbaseplus softwares were used to rank the genes according to their stability. gapdh was the most stably expressed gene whereas b m was the least stable gene. in addition, genorm analysis revealed that the number of genes required for optimal normalization was four (gapdh, sdha, hprt, rpl ). in the second part of the study the mrna expression of il- b, il- , il- and il- was measured in balf samples from seven iad horses treated in a randomized cross-over design with dexamethasone ( . mg/kg sid, days) or inhaled fluticasone propionate ( mcg bid with aerohippus, days). the balf samples were taken at baseline and after each treatment period. there was no significant effect of the corticosteroids treatment on the mrna expression of il- b, il- and il- in the balf. the mrna expression of il- was suppressed by dexamethasone and fluticasone propionate treatments. pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (tmct). we hypothesize that clenbuterol, a beta- agonist shown to increase tmct in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. six adult horses were enrolled in a cross-over design prospective study. horses were housed with their heads in a fixed position for hours to simulate long distance transport, and treated with clenbuterol ( . ug/kg po q h) or a placebo starting hours before simulated shipping. tmct was measured using a charcoal clearance technique. data were collected at baseline and hours, and included tmct, tracheal wash cytology and quantitative culture, rectal temperature, cbc, fibrinogen, and serum tnfa, il- and il- levels. there was a -week washout between study arms, and each horse served as its own control. the data was analyzed using regression analysis and wilcoxon rank-sum tests. no statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. tmct did not differ after treatment ( . ae . cm/min) versus placebo ( . ae . cm/ min; p . ), and intratracheal bacterial counts were similar for treatment (  ae  cfu; p . ) and placebo (  ae  cfu) groups. a reduction of tracheal b hemolytic streptococcus. spp. after clenbuterol versus placebo was also nonsignificant ( % versus %; p . ). in conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. breathing cold air during strenuous exercise is associated with airway inflammation. under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. the purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation. horses (n ) were examined in a randomized crossover design after exposure to hypertonic aerosols ( minute nebulization with solutions of either isotonic or hypertonic mannitol). airway leukocytes were harvested and hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mrna. hypertonic aerosol challenge resulted in an increase in total number of cells hr after challenge, characterized by increased macrophage (p . ) and neutrophil (p . ) concentrations, but there was no effect on airway leukocyte concentrations hours after nebulization. no significant changes in the relative quantity of mrna for airway cytokines were noted at either time point. these data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. however, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. it is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. we studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. we hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (pi) as a reflection of a necrotizing component of the disease. records of foals days of age hospitalized between and with signs of gastrointestinal disease were evaluated (n ). the association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined. pneumatosis intestinalis was imaged in foals. twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and pi detected sonographically ( ), surgical ( ) or pathological ( ) evidence of gastrointestinal necrosis. there was a difference between overall survival rate ( %) and survival rate in foals with necrotizing disease ( %, p . ) or foals with pi detected sonographically ( %, p . ). pneumatosis intestinalis was the only sonographic finding associated with outcome. sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (p o . ) with surrogates of severity of disease (hospitalization cost or days of hospitalization). hypoproteinemia was associated with pi (p . ). the presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (p o . ). abdominal sonograms have prognostic value in neonatal gastrointestinal disease. pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. the therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. the interaction of insulin and the microvascular endothelial insulin receptor (irc) plays an important role in the normal and insulin resistant (ir) individual. while endothelial irc signaling is normally vasodilatory, this effect is well-documented to reverse in the ir individual, resulting in vasoconstriction. although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. the purpose of this study was to characterize the pattern of irc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (nsc). mixed-breed ponies (body weight . /- . kg) received a diet of hay chop (nsc $ % on a dm basis) for weeks prior to initiation of the experimental feeding protocol. following conditioning, ponies either remained on the control diet (n ) or received the same diet supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for a period of days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. laminar sections were stained immunohistochemically for irc using a commercially-available antibody (abcam); the number of irc ( ) cells was quantified in x light microscopy fields (n ) for each section. the total number of irc ( ) cells was greater in the laminae of challenged ponies than control ponies (p . ), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar irc ( ) endothelial cells (r . ; p o . ). while the number of irc ( ) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p . ), there was no difference in the number of interstitial irc ( ) cells (p . ). no epithelial irc ( ) cells were observed in any laminar section, and irc ( ) cells were conspicuously absent from the deep dermal tissue (including vessels). up-regulation of irc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. the dramatic increase in endothelial irc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial irc present, suggests that hyperinsulinemia associated with exposure to increased dietary nsc may induce laminar injury by causing a similar vasoconstriction in ir equids as described in the microvasculature of ir humans. glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (glut) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. we previously demonstrated that glut , the major isoform, is a key factor in the pathogenesis of equine insulin resistance (ir). while it has been recently demonstrated that glut (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of ir, is not well characterized in any species. in addition, as has recently emerged as a key downstream signaling molecule regulating translocation of glut to the cell surface, the rate-limiting step in glucose uptake. we hypothesized that glut content would be differentially expressed across tissues and that ir would induce alteration in glucose transport by affecting active cell surface glut . biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated ir, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n /group). we specifically quantified active cell-surface glut in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to glut . total glut protein expression was measured by western blots, as well as total and phosphorylated (indicating activation of) as . glut was expressed in all the depots with a significant regional effect. total glut protein content was increased (p o . ) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. ir did not induce alterations in active cell-surface and total glut content nor in total and phosphorylated as in any of the tissues evaluated. our data suggests that glut is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. however, neither glut translocation nor as activation are impaired in insulin-sensitive tissues of ir horses. it is concluded that, in contrast with glut , glut does not appear to contribute to glucose transport alterations during naturally-occurring equine ir. insulin resistance (ir), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. in addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. we hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, ss), and grass hay plus a fat and fiber (ff) feed, were fed to mares, adult ( - yr) and aged ( yr), for a -week adaptation period in a randomized design. glycemic and insulinemic responses to a standardized meal of sweet feed ( g/kg bw offered for hour) were determined for hours from the onset of feeding. peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (auc-g, mg/ dl/ min, and auc-i, mu/ml/ min) were determined and data were analyzed by one-and two-factor repeated measures anova. there were no differences between age groups in glycemic responses to any of the diets. however, in aged horses peak glucose concentration (p o . ) and auc-g (p o . ) were greater after adaptation to the forage-only diet, as compared to the other two diets. in contrast, aged horses had a greater peak insulin concentration (p o . ) and auc-i (p o . ) than adult horses on all diets but no differences in peak insulin concentration or auc-i was found between diets within age groups. as hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. in obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type ii diabetes mellitus. in stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human ir, characterized by hyperinsulinemic euglycemia. few data exist describing the biology of the equine endocrine pancreas in the chronically ir animal that may both: ) explain this unique equine endocrine physiology and ) characterize the animal at-risk for hyperinsulinemia-associated laminitis. the purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. twenty-two mixedbreed ponies (body weight . ae . kg) were conditioned to a diet of chopped hay (nsc $ % on dm basis) for weeks; following conditioning, ponies either remained on the control diet (n ), or received the same hay supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (abcam), and measurements of islet surface area and b-cell surface area were performed (n islets per tissue section) using a commercially-available computer software program (image j). there was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high nsc diet when compared to the ponies on the hay diet (p . ); however, no difference was noted in b-cell surface area between diet treatments (p . ). the change in serum insulin concentration was significantly greater in the high nsc-fed ponies than in controls ( . /- . miu/l vs. . /À . miu/l; p . ); however, this variable was not correlated with total islet surface area (r . ; p . ) or b-cell surface area (r . ; p . ). due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high nsc diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with equine metabolic syndrome (ems). this study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by ems. six healthy adult mares and horses with ems received an intravenous infusion of lipopolysaccharide (lps; ng/kg in ml sterile saline) or saline alone. a crossover design was employed with a -day washout period. physical examinations were performed hourly for h and whole blood was collected at , , , , , and min for assessment of inflammatory cytokine gene expression. a liver biopsy was performed between and min postinfusion. data were analyzed using mixed model anova. mean rectal temperature, heart rate, and respiratory rate increased following lps infusion (treatment  time; p o . ), with higher heart (group  treatment; p . ) and respiratory rates (group; p . ) detected in ems horses. lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (tnfa), interleukin (il)- b (p o . ), il- (p o . ), il- (p o . ), and il- (p . ), and hepatic gene expression of il- (p o . ), il- (p o . ), and il- (p . ). inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. heart rates tended to be higher when lps was administered to horses with ems. elevated serum concentration of cardiac troponin i (ctni) is a biomarker for myocardial damage in horses. preferred times to test blood for ctni levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of ctni in the horse. this information would be necessary to more accurately and reliably test horses for myocardial injury. the aim of this study was to determine the elimination half-life (t / ) of equine ctni. to establish the t / of equine ctni in horses, ctni was recombinantly expressed in e.coli. two healthy ponies received intravenous injections of recombinant equine ctni and plasma ctni concentrations were measured with a point-of-care ctni analyzer at multiple time points after injection. standard pharmacokinetic analysis was performed to establish the elimination half-life of ctni. for comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. elimination of recombinant equine ctni following intravenous administration exhibits a short half-life. establishing the t / of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. this study describes a true biological ctni t / , which has not been documented in any species thus far. stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. the objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (fmd) measurement in healthy ponies, to investigate the hypothesis that fmd occurs in healthy ponies. six healthy, native breed, unrelated pony mares of varying weight ( - kg), body condition score ( / - / ) and age ( - years) were used. the median artery was occluded for minutes. twodimensional ( d) ultrasonographic images of the artery were recorded for seconds prior to and for minutes after occlusion. the peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (fmd). images were obtained from six ponies on one occasion and from one pony on six occasions. analysis of images was performed by two independent analysers and by one analyser twice. the mean (sd) fmd in ponies was . % ( . %) and in pony ( occasions) was . % ( . %). coefficients of variation were . % and . % respectively. agreement between analysers was fair (icc . ) and within analyser was poor (icc . ). fmd is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. fmd occurs and measurement is feasible in ponies. fmd could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. current state-of-the-art technique for measuring blood pressure (bp) in the horse is invasive and involves cannulation of the facial artery. indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. monitoring bp can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, systemic inflammatory response syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). the objective of this study was therefore to a) describe the methodology for application of oscillometric bp using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse. the oscillometric method is simple to apply in a clinical setting. a pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. the tail circumference must match the manufacturers description of the cuffs diameter range. the oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial bp (sap, dap and map). at least consecutive measurements must be obtained. a correction of . mmhg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference. for determination of accuracy and precision of indirect sap, dap and map, eight healthy horses (age to years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i measurements were recorded every minutes for minutes. the data were analysed with the statistical software r using a mixed model with repeated measurements and a bland-altman analysis corrected for repeated measurements. oscillometric bp was accurate and precise for map (mean bias, lower confidence level, upper confidence level, variation in difference, all mmhg) (À . , À . , . , . , respectively) in the conscious horse but not for sap (À . , À . , . , . , respectively) and dap ( . , . , . , , respectively) . there was no significant contribution to the statistical model of either horse or measurement number. all horses tolerated the tail-cuff well and the method was simple to apply. only map could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. reference intervals for thyroid hormone (th) concentrations have not been established for donkeys. therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. we hypothesized that th concentrations are different between donkeys and horses. the purposes of this study were: a) to compare th concentrations between donkeys and horses and, b) to determine whether the age may influence th concentrations. thirty-eight healthy donkeys ( . ae . years), mixed breeds, and healthy andalusian horses ( . ae . years) were used. donkeys were divided into three groups: o years (n ), - years (n ), and years (n ). serum concentrations of total triiodothyronine (tt ), free triiodothyronine (ft ), total thyroxine (tt ), free thyroxine (ft ), reverse triiodothyronine (rt ) and thyroid-stimulating hormone (tsh) were quantified by radioimmunoassay. all blood samples were collected the same day. neither horses nor donkeys had received any treatment for days before sampling and both farms had similar production conditions. total t , ft , ft and tt concentrations were higher (p o . ) in donkeys than horses. in contrast, no statistical differences were found for rt and tsh concentrations. young donkeys ( o years) had higher ft , tt and rt concentrations compared to other donkey groups (p o . ). old donkeys ( years) had lower tt and ft concentrations than both younger donkeys groups (p o . ). this study shows that there are differences in th concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. in humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. the effects of ovariectomy on energy metabolism have not been reported in horses. ovariectomized mares have been shown to respond normally to an acth stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. the aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy. six healthy mares underwent an intravenous glucose tolerance test (ivgtt), an insulin sensitivity test (ist) and a dexamethasone suppression test (dst) before and weeks after bilateral ovariectomy. body weight, cortisol values at baseline, and hours after dexamethasone injection and acth values at baseline, and hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a % decrease in blood glucose in the ist, time to reach baseline glucose concentration in the ivgtt and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an anova for repeated measures. significance level was p o . . average body weight was decreased after surgery ( kg ). the injection of dexamethasone resulted in a serum cortisol concentration of less than mg/dl in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than mg/dl in out of mares. in all cases, acth concentration was within the reference range ( - pg/ml) before and after ovariectomy. however, acth concentrations at t and at t were significantly higher after ovariectomy. each mare had a normal ivgtt, both before and after ovariectomy. additionally, no significant differences were observed in basal blood glucose ( ae mg/dl before and ae mg/dl after) or in the time to reach glucose baseline ( ae min before and ae min after). serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy ( . ae . miu/ml and . ae . miu/ml and . ae . and . ae . , respectively), nor was the average time to reach a % decrease in blood glucose after insulin injection ( ae min and ae min, respectively). these findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. equine gastric ulcer syndrome (egus) is a common medical problem in horses. the high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. berries and pulp from the seabuckthorn plant (hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. the purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (seabuck tm sbt gastro-plus) for treatment and prevention of gastric ulcers in horses. eight thoroughbred and thoroughbred-cross horses ( - years of age, geldings & mares, - kg) were used in a blinded two-period cross-over study. treatments consisted of control (untreated) and treatment (seabuck tm sbt gastro-plus) twice daily mixed with the grain meal. horses were treated for weeks followed by a week alternating feed-deprivation period to induce or worsen existing ulcers. gastroscopies were performed on all horses on day , week , and week (at the end of the alternating feed-deprivation period). gastric juice was aspirated and ph was measured. during gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. horses acted as their own controls, and between each treatment period the horses had a -week washout period. data was analyzed by anova for repeated measures via the glm procedure (sas inst. inc., cary, nc). when significant differences (p o . ) were observed, a post-hoc tukey's test was used to determine differences. non-glandular gastric ulcer scores significantly increased in all control and sbt-treated horses from week to week , after the feed-deprivation phase of the study. there was no significant difference in the non-glandular gastric number (p . ) and nonglandular gastric severity (p . ) scores in sbt-treated horses compared to non-treated controls. glandular ulcer number (p . ) and glandular ulcer severity (p . ) was significantly lower in the sbt-treated horses compared to the control horses. there was no significant difference in the ph (p . ) in sbt-treated horses compared to non-treated controls. thus, seabuck tm sbt gastro-plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach ph. the availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. the abaxis vspro analyzer was evaluated for precision using two levels of human fibrinogen controls ( mg/dl and mg/dl), four different vspro machines, and two different lots of cartridges, assessed over subsequent days. the coefficients of variation of the assay ranged from % ( mg/dl) to % ( mg/ dl). we subsequently evaluated the abaxis vspro fibrinogen assay compared to fibrinogen concentration measured using the beckman coulter acl- in equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. all samples were measured in citrated plasma. fibrinogen samples measured on the acl- ranged from to mg/dl (median mg/dl). vspro samples were run in duplicate, and the mean compared to the acl values. pearson correlation coefficient analysis generated an r value of . (p o . ). duplicate measurements on the vspro were strongly correlated to each other with an r value of . (p o . ). bland-altman analysis of these samples for the vspro compared to the acl- noted a bias of À ae mg/dl the results of this study indicate that the vspro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the acl- reference analyzer. the immune response of foals to r. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. in a recent study foals receiving a large inoculum exhibited th skewing with pneumonia and a small inoculum exhibited th skewing without clinical disease. we hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. alveolar macrophages were obtained by bronchoalevolar lavage from healthy mares and their -week-old foals. macrophage cultures were infected with r. equi ( or -) at a multiplicity of infection (moi) of or . total rna was harvested and hours post-infection, reverse transcribed and used as template for quantita-tive pcr. the ddct method was used to calculate relative gene transcripts for il- , il- p , tnfa and cxcl . cellular infections at moi resulted in significantly higher expression of il- , il- p and tnfa mrna transcripts compared to moi . however, the dose-effect was reversed for cxcl with significantly lower expression at the higher moi. there was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals. dose-dependent responses of alveolar macrophages may represent a novel mechanism by which r. equi could modulate immune responses and therefore disease. significant down-regulation of cxcl mrna transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective th responses. the intent of this study was to develop likelihood ratios (lrs) for infection attributable to corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (shi) test titers. medical records for horses presented to the uc davis veterinary teaching hospital with serum submitted for shi titer determination were evaluated and cases met study inclusion criteria. these cases were grouped based on evidence of internal and/or external infection attributable to c. pseudotuberculosis and likelihood ratios with % confidence intervals determined. results showed increasing lrs indicating increasing odds for any form of active disease as titer increased with all cases considered. lrs for internal infection were for titers ! overall and for titers with external abscess cases excluded. no difference from (and therefore no significant change in pre-test to post-test odds) was seen in any lrs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). overall, the shi test results showed usefulness in determining internal c. pseudotuberculosis infection in horses with no evidence of external abscessation. overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. the shi test was unable to distinguish internal infection when external abscesses were present. salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. rapid detection of s. enterica in fecal samples may facilitate effective infection control practices. current detection methods require - hours (polymerase chain reaction or pcr) or - hours (enriched aerobic culture) to obtain results. alternatives have been developed, lateral flow antigen detection systems (lfads), which are currently marketed for salmonella detection related to food safety microbiology. the objective of this study was to evaluate two commercially available rapid salmonella detection systems in equine feces. fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of salmonella enterica serotype typhimurium (five uninoculated control samples, and samples of each -fold dilution [ .  - .  cfu/gram of feces]). all samples were aerobically cultured using a standard enrichment technique. in a blinded fashion, samples were tested using two different lfads as well as plated on agar media for confirmatory testing. at hours of incubation, using bacterial culture as the reference method, test was correctly identify % of samples ( bacterial contamination of stalls with salmonella sp. is a serious problem in equine hospitals. salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. hospital closure can result in loss of revenue, damage to reputation and interference with patient care. the purpose of this study was to evaluate three stall cleaning methods on eradication of salmonella sp. at an equine veterinary teaching hospital (vth). horses admitted to the vth were assigned to salmonella sp.negative stalls within areas of the vth during the study period (september -january . when the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [pw] , pressure washing and hand scrubbing [pws] , or hand scrubbing only [s]) in a single period, non cross-over design. all stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (super hdq neutral, spartan chemical co., inc, maumee, oh). the pressure-washing system (psc cleaning systems, inc., toronto, canada) used, provided a pressure of psi and a temperature range of - f. following cleaning, each stall was allowed to air dry and within hours, stall surfaces were sampled using three  sponges moistened with sterile saline. the person collecting the samples was masked to the method of cleaning. sponges were submitted to the louisiana animal disease diagnostic laboratory (laddl) for culture of salmonella sp. a chi-squared analysis was used to determine significant differences (limit p o . ) between cleaning methods and salmonella sp. isolation. during the study period, stalls (pw [n ]; pws [n ]; s [n ] were included. all stalls had negative environmental salmonella sp. cultures prior to beginning the study. for pw cleaned stalls, / ( . %) were salmonella sp.-positive, for pws cleaned stalls, / ( %) were salmonella sp.-positive, and for s cleaned stalls, / ( . %) were salmonella sp.-positive. although, there were fewer salmonella sp.-positive stalls ( . %) in the handscrubbed stalls, cleaning method did not significantly (p . ) affect the isolation of salmonella sp. from the stall environment. in conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of salmonella sp. from stalls surfaces in the vth during this study. the objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against streptococcus equi subsp. zooepidemicus (s. zooepidemicus) and rhodococcus equi isolates. a single dose of gamithromycin ( mg/kg of body weight) was administered intramuscularly. concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (pelf), bronchoalveolar lavage (bal) cells, and blood neutrophils were determined using hplc with tandem mass spectrometry detection. the minimum inhibitory concentration of gamithromycin required to inhibit growth of % of r. equi and s. zooepidemicus isolates (mic ) was determined. additionally, the activity of gamithromycin against intracellular r. equi was measured. mean peak gamithromycin concentrations were significantly higher in blood neutrophils ( . ae . g/ml) and bal cells ( . ae . g/ml) compared to pelf ( . ae . g/ml) and plasma ( . ae . g/ml). mean terminal half-lives in neutrophils ( . h), bal cells ( . h), and pelf ( . h) were significantly longer than that of plasma ( . h). the mic of s. zooepidemicus isolates was . g/ml. the mic of gamithromycin for macrolide-resistant r. equi isolates ( g/ml) was significantly higher than that of macrolide-susceptible isolates ( . g/ ml). the activity of gamithromycin against intracellular r. equi was similar to that of azithromycin and erythromycin. intramuscular administration of gamithromycin at a dosage of mg/kg would maintain pelf concentrations above the mic for s. zooepidemicus and phagocytic cell concentrations above the mic for r. equi for approximately days. eight western stock yearling horses were infected with ehv- (ab ) by nasopharyngeal instillation. venous blood samples for collection of plasma were collected in na-citrate tubes on the day prior to infection (d - ) and on d through d . in addition, clinical data, nasal swabs and peripheral blood mononuclear cells (pbmc) for detection of viremia were collected on the day before infection (d - ) and on d through d post-infection. d-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (minutex d-dimer, biopool, ireland). viral load in pbmc was determined using quantitative pcr. all horses showed bi-phasic fevers typical for ehv- infections. one horse developed acute ehm on d and was euthanized after samples were collected. in all horses d-dimers were undetectable on d - and on d , and . in contrast, all horses had increased ddimer concentrations for to consecutive days starting on day post-infection. d-dimer concentrations in horses increased to ug/ml and one of these horses was the horse with acute ehm. interestingly, mean increased d-dimer concentrations showed timely overlap with the mean fever curve and, delayed by day, with the mean viremia curve. because plasma samples for d-dimer measurements were not collected during the first days post-infection, which are typically associated with a primary fever, conclusion on the association of d-dimers with fever of viremia await analysis of a second study currently conducted in our laboratory. in conclusion our data indicates that during ehv- infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of ehm, and can be expected between days and post-infection. lawsonia intracellularis is an emerging pathogen in horses and the causative agent in equine proliferative enteropathy (epe). the goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to lawsonia intracelluaris on several farms in louisiana with a history of epe and compare the results to several farms with no known clinical cases of epe in foals. an additional goal of the study was to identify whether a relationship exists between lawsonia intracelluaris and other gastrointestinal pathogens in foals. whole blood and fecal samples were collected from mares and foals from four breeding farms in louisiana. farms a and b had no known clinical cases of epe, while farms c and d had previous know cases of epe in . serum samples were examined for the presence of antibodies against lawsonia intracellularis using an immunoperoxidase monolayer assay (ipma). dna was extracted from fecal samples using a commercial dna kit and molecular detection of lawsonia intracelluaris was assayed using real-time pcr. fecal ova were determined using quantitative sucrose floatation. the presence of fecal clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (elisa). three of the farms examined had foals and mares with exposure to l. intracellularis as evidenced by serum antibodies against the organism. of the total population sampled, foals ( . %) and mares ( . %) had evidence of antibodies to l. intracellularis based on serology. three foals ( . %) tested positive for l. intracellularis organism by fecal pcr, and all of these foals were located on farm c. of these, one of the foals was seronegative, while the other two were seropositive. farm c also had the highest percentage of mares ( . %) serologically positive for l. intracellularis, while farm a had the highest percentage of foals ( . %) with antibody titers against l intracellaris. farm c also had the only pairs (n ) of serologically positive mares with seropositive foals. while farm a and b had seropositive mares and/or foals, none of the foals were positive for l. intracellularis fecal shedding by pcr. all serum and fecal samples were negative for evidence of l. intracellaris on farm d. ten foals ( %) had fecal egg counts greater than egg per gram and foals ( %) were positive for c. difficile toxin. this study demonstrated evidence of natural exposure to l intracellularis on farms both with and without a history of epe in louisiana. further, this study failed to establish a relationship between l intracellularis and other gastrointestinal pathogens. the objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with anaplasma phagocytophilum presented to a primary care field setting in southeastern pennsylvania. computerized medical records from febrile equids with confirmed anaplasma phagocytophilum infection were reviewed. confirmation of anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (pcr) for anaplasma phagocytophilum. horses and donkey presented with a mean fever of . f and mean fever duration of hours. the mean age at presentation was . years and the mean pack cell volume was . %. / cases were diagnosed in the months of may to december. equids ages to years had significantly lower platelet counts. / cases were positive on blood smear for inclusion bodies and / cases were positive for anaplasma phagocytophilum on pcr. treatments included intravenous oxytetracycline, oral doxycycline, or both. mean treatment duration was . days and mean treatment cost was $ . / cases were normothermic within hours. the treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful. this is the first case series of equine granulocytic anaplasmosis in the mid-atlantic states. all cases were examined and treated in the field. in order to make a definitive diagnosis, some cases required pcr. treatment failures were documented with the use of oral doxycycline alone. % of the cases survived. a high incidence of clinical and possibly genetic abnormalities has been reported amongst friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction. the purpose of the current study was to develop a new electromyography (emg) method to assess neurophysiological function of the esophagus especially for friesian horses. five friesian horses with esophageal dysfunction were included (ranging in age from . - years and comprising mares and a stallion) and two friesian control horses (a -and -year-old gelding). all five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. an endoscopically-guided percutaneous needle emg procedure (viking quest r ; software version . ) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility. esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. mean (ae sd) values were . ae . ms and . ae . mv (n readings) and . ae . ms and . ae . mv (n readings), respectively. in diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. preliminary findings suggest that endoscopically-guided percutaneous needle emg might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in friesian horses. lymphoma affects horses of all ages. unlike in humans, no etiologic agent has been discovered. a year old thoroughbred/warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (empf) and was positive for equine herpes virus (ehv- ) in both the pulmonary tissue and the lymph nodes. retrospective polymerase chain reaction (pcr) testing of six lymphoma cases found that of of the cases were positive on pcr for ehv- ( . %, p . , rr . ). electron microscopy was performed on one sample and herpes virus particles were identified. of the samples in which immunohistochemistry was performed ( of ), only t-cell rich b-cell lymphoma was identified. samples of mesenteric or submandibular lymph nodes from clinically healthy horses were submitted for ehv- pcr analysis; % were positive. gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as kaposi's sarcoma and burkitt's lymphoma. equine herpesvirus , also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. while these events have been documented early in the disease process at both a developmental stage and at the onset of obel grade (og ) lameness in the carbohydrate overload (cho) model of laminitis, the later events occurring at the onset of obel grade lameness(og , time point at which structural failure of the laminae usually occurs) have not been determined. we hypothesized that the inflammatory events described above are sustained through og lameness, likely playing an injurious role culminating in laminar failure. our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at og induced using the cho model. archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous cho study at louisiana state university (control group [n , water], cho group [n , corn starch]. calprotectin (cp) immunohistochemistry (ihc) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; rt-qpcr was used to assess inflammatory gene expression. minimal inflammatory changes were present at og compared to published values at og stage in the cho lameness model including decreased mrna concentrations of cytokines (i.e. -fold increase in il- at og vs. -fold increase at og , no increase in il- b at og vs. -fold increase at og ), chemokines (no change in mcp- at og vs. fold increase at og , -fold increase in il- at og vs. fold increase at og ) and adhesion molecules (no change in e-selectin at og vs. -fold increase at og ). laminar leukocyte emigration was also decreased at the onset of og lameness compared to previously reported leukocyte infiltration at og . interestingly cox- , underwent a greater increase at og (approx. -fold) compared to that reported at og lameness ( -fold). finally, epithelial stress at og evidenced by cp ihc did not follow the uniform widespread distribution reported at og lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased cp signal. overall, laminar inflammation appears to be subsiding at og lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. the sustained increase in cox- , central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. this study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases. retrospective analysis of medical records from our veterinary medical teaching hospital from january , to july , was completed. records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival. thirty equids met inclusion criteria of the study. three of subjects were dead on arrival or died immediately upon arrival ( %). of the remaining equids, % presented with gastrointestinal abnormalities, % were azotemic and % had cardiac arrhythmias. mortality was % for all subjects and % for those treated. the predominant cause for non-survival was cardiac dysfunction. factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization. equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. the quantitative physicochemical approach emphasizes the importance of strong ions (na, k, cl, lactate), pco , and the plasma protein concentrations in determining plasma ph. serum concentrations of strong ions, proteins, and total co are reported on modern biochemical profiles. we hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. the objective was to determine whether blood ph, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis. horses ( adults and foals) presented to the isolation unit of our veterinary teaching hospital for suspected infectious diseases were prospectively enrolled. a venous serum sample was analyzed using a hitachi or copas c automated machine. measured parameters included strong ion difference (sid {na k}-{cl lac-tate}), total protein concentration (tp), and total co (tco ), with lactate being measured by blood gas analyzer. a second venous blood sample was collected into a na-heparin blood gas syringe and analyzed for ph (ph m ), pco and concentrations of na, k, cl, and lactate using a radiometer flex blood gas analyzer; sid was calculated from the measured values, and total solids (ts) were estimated using refractometry. serum/ plasma ph (ph calc ) was calculated using stewart's factor equation from the results of serum or plasma biochemical analysis, assuming pco mmhg for serum and pco accurate for plasma. anion gap (ag) was calculated as: ag (na k)-(cl tco ). strong ion gap (sig) was calculated as: sig . x[total protein, g/l]/ ( {pka-ph} )-ag. linear regression analysis was used to compare ph calc to ph m, as well as ag and sig to blood lactate concentrations. measured ph ranged from . to . ( . ae . ). measured sid from serum biochemistry (sid sb ) ranged from . to . meq/l ( . ae . meq/l) and sid from blood gas analyzer (sid bg ) from . to . meq/l ( . ae . meq/l; r . ; sid bg .  sid sb ). sid sb and sid bg showed small variability in measurements. tp ranged from to g/l ( . ae . g/l) and ts from - ( . ae . g/l; r . ; ts .  tp). using sid sb and tco values with constant pco , ph calc was poorly associated with ph m (r . ; ph calc . . ). in contrast, using sid bg with accurate pco , ph calc was closely associated with ph m (r . ; phcalc . . ) and the equation was not different from the line of identity. anion gap and sig (meq/l) calculated were significantly linearly correlated with lactate concentrations (mmol/l); ag .  [lactate] . (r . ), and sig À .  [lactate] . (r . ). we conclude that ph calc using sid sb , tco and constant pco values is not accurate. however, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. these results reemphasize the importance of strong electrolytes and proteins in acid-base balance. metalloproteinases (mmps) are critically important in remodeling processes and in wound healing. however, excessive activation of mmps by pro-inflammatory mediators including cytokines, prostaglandin e , and nitric oxide lead to tissue breakdown. this is observed in osteoarthritis (oa) which is characterized by erosive lesions in articular cartilage. in hereditary equine regional dermal asthenia (herda), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. herda affects horses with quarter horse bloodlines and is similar to the human hereditary connective tissue syndrome ehlers danlos (eds). many adult eds patients suffer from joint pain and oa. we hypothesized that chondrocytes from articular cartilage of herda horses have increased activity of mmps. to test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous herda carpal and hock joints. chondrocytes from normal horses were also obtained for comparison. chondrocytes were seeded at x /ml into -well plates and incubated at c, % co for up to seven days. activity of secreted mmps was determined by zymography using equal amounts of proteins for loading. secreted mmps were analyzed by western blot. zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at and kda suggestive of the latent form of mmp- and mmp- , respectively. less intense bands of gelatinolytic activity were observed at about and kda suggestive of the active form of mmp- and mmp- . another band of activity was also seen at kda which is suggestive of a dimer of mmp- that has been reported when mmps are in excess of tissue inhibitors of metalloproteinases (timps). chondrocyte cultures from homozygous herda cartilage showed a similar profile but with decreased activity by % at kda and - % increased activity at kda compared to normal chondrocytes. western blot analysis detected mmp- and mmp- immunoreactivity in chondrocyte culture media of herda-afflicted and normal horses. the present study demonstrates for the first time that horses suffering from herda have increased mmp activity which may predispose them to the development of lesions in articular cartilage. research supported by nutramax laboratories, inc. equine polysaccharide storage myopathy (pssm) type is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type (gys- ). the disease has been reported to affect the haflinger breed but so far its prevalence is unknown. aim of this preliminary study was to estimate the occurrence of the gys- mutation in austrian haflingers and establish which of the seven haflinger sire lines appear mostly affected. gys- genotyping of randomly chosen haflingers was performed with a validated restriction fragment length polymorphism assay. resting and post-exercise muscle enzyme activities (creatine kinase (ck), aspartate aminotransferase (ast), lacate dehydrogenase (ldh)) and blood lactate concentrations were compared between horses with and without the mutation. among the horses were heterozygous (hr) carrier of the mutation. no homozygotes (hh) were identified. all horses with the gys- mutation were descendents of the a-or w-sire lines. the estimated hr prevalence was % ( % ci: . - . %). ck activity after exercise (p . ) was significantly higher in hr horses compared with horses not carrying the mutation (rr). ast activity was significantly higher in the hr group at rest and after exercise (p o . ). there was no statistically significant difference in resting ck, resting and post exercise ldh activity or blood lactate between hr and rr. results suggest that the prevalence of hr in the austrian haflinger population is higher than in the overall quarter horse population and might be as high as %, similar to some draft horse breeds. further research is needed to establish the prevalence within the different breeding lines. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder affecting quarter horse lineages. although a mutation in the gene encoding cyclophilin b has been genetically linked to herda, its causal association with the disease is not yet documented. previously, we demonstrated reductions in ultimate tensile strength (uts), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of herda animals. given the presumed relationship between her-da and abnormal collagen structure, and the predominance of type i collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in type i collagen. to evaluate this hypothesis we compared the uts, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (dft) from six herda horses to six age-matched controls. isolated dft was secured and pulled to failure on an instron s universal testing instrument using purpose-built cryogenic clamps. analysis of variance was executed using sas . proc glimmix program (sas institute, ). p-values . were identified as significant. uts and modulus of elasticity were significantly lower in herda dft when compared with controls (p o . ); energy to failure did not differ between groups. these findings document abnormal biomechanics in herda tendon, leading us to postulate that lower uts and modulus of elasticity associated with the herda defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (references on request). a proprietary herbal biocontamination product (bios) approved for cosmetic use in france, inhibits proliferation of medically relevant bacteria, mold, and viruses. these properties make bios potentially useful as a topical wound medication, prompting us to compare bios to silver sulfadiazine (ssd) in a distal extremity wound healing model in horses. using general anesthesia, two . cm wounds were aseptically created on the dorsomedial aspect of all limbs. for the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with % bios and the other with ssd. for each limb the most proximal wound served as an untreated control. every hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (imagej, nih). analysis of variance did not identify significant differences between ssd or bios treatment for wound perimeter (p . ) or area (p . ). at individual time points the effect of bandaging was significant when area was evaluated (p . ) and trended toward significance for perimeter (p . ) comparisons, substantiating published reports that bandaging modifies wound healing. difference in perimeter and area between control and treatment were highly significant (p o . ), substantiating the importance of topical treatment. over the study duration, effects of bandaging (p o . ) and topical treatment (perimeter p o . ; area p . ) continued to be highly significant. bios performance in the equine distal extremity wound model was equivalent to ssd. both bandaging and topical treatment significantly impacted wound healing. this effect was compounded when both variables were evaluated over time. radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. this pilot study compares the accumulation of m tc-labeled igg, peg-liposomes and leukocytes in an equine muscle abscess model. three mixed breed adult horses had  cfu s. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. peg-liposomes were prepared via the film hydration method and labeled using mci m tc-hexamethyl-propylene-amine-oxime ( m tc-hmpao). autologous leukocytes were obtained from ml whole blood and labelled using mci m tc-hmpao. commercial equine polyclonal igg was conjugated with the chelator hydrazinonicotinamide (hynic) and labelled with mci m tc. radiopharmaceutical administration was initiated hours after inoculation. horses and received mg m tc-igg, . mmol/kg m tc-liposomes and m tc-leukocytes, with a hour interval between each radiopharmaceutical. horse received only m tc-leukocytes. scintigraphic examinations were performed at and hours post injection (p.i.) with each radiopharmaceutical. after the final study, horses were euthanized and tissue samples collected. the percentage of injected dose per kilogram of tissue (%id/kg) was calculated for the region of the abscess, normal muscle and multiple organs. scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. at hours p.i. abscess-to-background ratio was highest using m tc-igg ( . ae . ). at hours p.i. abscess to background ratio was highest using m tc-liposomes ( . ae ). tissue biodistribution data revealed abscess to muscle ratios of ( m tc-igg), ( m tc-liposomes), and . ( m tc-leukocytes). this preliminary data demonstrates that m tc-liposomes, m tc-igg and m tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. m tc-igg and m tc-liposomes appear to be superior to m tc-labelled leukocytes in this model. due to its low cost and ease of preparation, m tc-igg has great potential for clinical use where identification of infectious or inflammatory foci is necessary. digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and cox- in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. the p mapk pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. we thus hypothesized that ) p mapk is upregulated and activated in affected laminae in equine laminitis and ) digital hypothermia inhibits inflammatory mediator expression by blocking p mapk phosphorylation (indicator of p mapk activation). western hybridizations using both a total p mapk and a phospho-p mapk antibody were performed on archived pooled laminar samples from black walnut extract (bwe) model ( control, developmental (dev) groups [ . h & h post bwe administration] and the onset of obel grade lameness (og ) [n each]) and carbohydrate overload (cho) models (con [n ], dev [n ], og [n ]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (dh) study. in the dh study, one forelimb of each horse was kept at approximately c in ice water and the other at ambient temperature following administration of g/kg oligofructose (of). dorsal laminae were harvested for snap freezing at either hours after of administration (dev, n ) or at the onset of lameness (og , n ) using protein extracted from treated and untreated digital laminae of each horse. increased laminar concentrations of phospho-p mapk were present in the developmental periods ( . h and h) in the bwe model, and in both the dev and og periods in the cho laminitis models. however, digital hypothermia had no effect on laminar phospho-p mapk concentrations. thus, p mapk is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. therefore, p mapk is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia. abstract e- does cefquinome penetrate the blood brain barrier in the normal horse? hollis ar duggan ve and corley ktt . scott dunn's equine clinic, berkshire, uk; university college dublin, dublin, ireland; anglesey lodge equine hospital, the curragh, ireland. meningitis is a rare but serious condition that occurs in both foals and adult horses. there is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. cefquinome is a fourth generation cephalosporin that has activity against streptococcus, the most commonly reported causative organism in adult horse meningitis. therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. mature, healthy horses were used on separate occasions, seven days apart, in a crossover design. each horse was administered either cefquinome ( mg/kg) or saline (equivalent volume). cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia and hours after administration of cefquinome or saline placebo. blood samples were collected prior to, and and hours after administration of cefquinome or placebo. all samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered. cefquinome was detectable in the cerebrospinal fluid in all horses hours after intravenous administration, and in horses hour after administration. cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. the purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (nsaid) approved for use in horses to control pain and inflammation associated with osteoarthritis. dosages of firocoxib are species dependent, with the recommended dose for horses being . mg/kg as an oral paste every h. the main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated. enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. it is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. a well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. co-administration of multiple drugs can result in unpredictable therapeutic outcomes. often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. various pharmacokinetic interactions between antimicrobials and nsaids have been described. six healthy, adult mares were administered . mg/kg of firocoxib orally. samples were collected by direct venipuncture of the jugular vein at (control), , , and min, , , , , , , , , and h after administration. after a day washout period the six horses were pretreated days with enrofloxacin mg/kg intravenously every h then on the fourth day given . mg/kg of firocoxib orally. samples were collected at (control), , , and min, , , , , , , , , and h after administration. all samples were stored at À c until analysis using a validated hplc method. the t / , c max , t max , auc - and auc -f after firocoxib administration were . angiotensin converting enzyme (ace) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. there is limited information regarding their effects in horses. the purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ace inhibition in horses. six healthy horses were administered quinapril at mg iv, mg po or mg po in a -way crossover design. blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ace concentrations using a radioenzymatic assay. normally distributed data were analyzed with one way repeated measures analysis of variance (rm-anova) and non-normally distributed data were analyzed using friedman rm_anova on ranks. significance was set at p o . . no adverse effects were observed during the study period. plasma quinapril concentrations were low and rapidly declined after iv administration. quinaprilat concentrations were below the limit of quantification ( . mg/ml). ace activity was significantly decreased from baseline at . and hour after iv dosing and at all timepoints after oral dosing. maximum % ace inhibition was , and % with the iv, high and low oral doses, respectively. these results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ace in healthy horses. controlled studies in clinically affected horses are indicated. this study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. foals are more sensitive to the side effects of nsaid, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. firocoxib, a novel, second generation nsaid, is reported to have reduced side effects due to cox- selectivity. the pharmacokinetic profile of firocoxib in neonates has not been established. we hypothesized that firocoxib given po to neonatal foals would achieve therapeutic concentrations in plasma. seven healthy foals of mixed gender were administered . mg/kg firocoxib po q h for nine consecutive days, commencing at h old. blood was collected for firocoxib analysis at (dose # only), . , . , , , , , and h after doses # , and . for all other doses ( , , , , and ) blood was collected immediately prior to the next dose ( h trough). elimination samples were collected after dose # . plasma was stored at À c until analysis. physical examinations were performed on foals daily and body weight obtained every two days during the sampling period. analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. after the initial dose, a maximum plasma concentration was reached in min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. after the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t / of h. seventy-two hours after the final dose, firocoxib was not detectable (o ng/ml). erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. the purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease. ninety-seven horses aged ! year with clinicopathologic or clinical signs of liver disease, a complete blood count (cbc), and hepatic histopathology were included. information on cbc, biochemical variables, and hepatic histopathology was collected from records. data from horses with erythrocytosis (packed cell volume %) were compared to those without using the mann-whitney rank sum test with significance set at p o . . there were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. fibrosis ( %), biliary hyperplasia ( %), inflammatory infiltrate ( %), megalocytosis ( %), degeneration ( %), necrosis ( %), cholestasis ( %), anisocytosis and anisokaryosis ( %), and lipidosis ( %) were observed in livers of horses with erythrocytosis. neoplasia ( %) was observed rarely. this study reports a high frequency of erythrocytosis in horses with liver disease. erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. hepatic neoplasia was rare. this study was performed to determine if horses diagnosed with equine proliferative enteropathy (epe) from lawsonia intracellularis (li) infection had long term effects from disease based on their sale price as yearlings and race earnings. a retrospective review of medical records of thoroughbred horses that were treated for lawsonia intracellularis infection between january , and january , at hagyard equine medical institute in lexington, kentucky was performed. three criteria were used for inclusion in this study. first, each horse had presumptively been diagnosed with li based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. second, horses had hypoalbuminemia of less than . mg/dl (normal reference range: . - . mg/dl). third, each horse had a positive fecal polymerase chain reaction (pcr) for li, a positive serum immunoperoxidase monolayer assay (ipma), or both. an ipma titer greater than or equal to was considered positive for disease. horses met the initial criteria. of the horses sold at public auction as yearlings. the sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). of the horses raced in the united states. their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). earnings of horses that were between and years of age ( / horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. earnings from horses that were two years of age ( / ) at the end of the study were compared to the two year old average earnings of the stallion's progeny. monetary earnings from all races prior to december , were included in the study. horses both sold at public auction and raced. as well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group. this retrospective study indicated that yearling horses previously infected with li do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. these results should assist practitioners in guiding owners in determing if treatment of horses with epe is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential. this abstract was presented at the aaep in december . bronchopneumonia caused by streptococccus equi subsp. zooepidemicus (s. zooepidemicus) is one of the most important causes of morbidity in weanling foals. ceftiofur crystalline free acid (ccfa) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with s. zooepidemicus in adult horses. the objective of the present study was to determine the disposition of ccfa in plasma and pulmonary epithelial lining fluid (pelf) of weanling foals. six healthy -to month-old weanling foals were administered a single intramuscular injection of ccfa at a dose of . mg/kg of body weight. concentrations of desfuroylceftiofur acetamide (dca) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry. following im administration, median time to maximum plasma and pelf concentrations was h ( - h) . mean (ae sd) peak dca concentration in plasma ( . ae . mg/ml) was significantly higher than that in pelf ( . ae . mg/ml). terminal half-life of dca in plasma ( . ae . h) was not significantly different from that of pelf ( . ae . h). time above the therapeutic target of . mg/ml was significantly longer in plasma ( ae h) than in pelf ( ae h). based on the results of the present study, intramuscular administration of ccfa at a dose of . mg/kg would be appropriate for the treatment of bronchopneumonia caused by s. zooepidemicus and other susceptible pathogens in weanling foals. fgf- is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. fgf- enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (ckd). the aim of this study was to determine if plasma fgf- concentrations were associated with the presence of underlying ckd in hyperthyroid cats, and to investigate the changes in plasma fgf- concentrations that occur following treatment of hth. hyperthyroid cats were recruited from two london-based first opinion practices between and . cats that were azotemic at diagnosis were excluded. hth was treated with anti-thyroid medication alone or in combination with thyroidectomy. cats were included in the study if they had a plasma total thyroxine concentration o nmol/l documented for a six month period following commencement of treatment. cats were classified as having azotemic ckd if they developed renal azotemia within six months of establishment of euthyroidism. otherwise cats were deemed to have normal renal function. stored edta plasma samples were assayed for fgf- using a recently validated elisa. the mann-whitney u test and the wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. results are reported as median [ th , th percentiles]. correlations were made using spearman's correlation coefficient. thirty one cats with hth ( azotemic and non-azotemic) were included in the study. plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia ( . [ . , . ] mg/dl vs. . [ . , . ] mg/dl; n , p . ) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia ( . [ . , . ] mg/ dl vs. . [ . , . ] mg/dl; n , p . ). plasma fgf- concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) and post treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) timepoints. plasma fgf- concentrations increased following treatment in both azotemic (p . ) and non-azotemic groups (p . ). plasma fgf- concentrations and plasma phosphate concentrations were not correlated at baseline (r s . , p . ) or following treatment (r s . , p . ). plasma fgf- concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of hth. the reason that fgf- concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. hyperthyroidism is a disorder resulting from the excessive production and secretion of t and t by the thyroid gland. although the disorder and its pathological lesions have been well studied and described the cause remains illusive. whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on t levels and thyroid histology were used in this study. whole blood samples from non-disease cats, severe disease cats and mild disease cats as well as solid thyroid tissue samples from non-disease cats, severe disease cats and mild disease cats were collected and processed. the resulting total rna samples were used for genechip analysis using our custom feline gene chip designed by affymetrix. data analysis was performed using the partek s gs software for gene expression data. the robust multichip average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. anova analysis was performed to find significant differentially expressed genes with a minimal false discovery rate control of . and a fold change of . in each direction. during the mild disease state, pathways associated with dna damage and apoptosis are most prominent. at later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with tgf-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence. the analysis of this unique data set generated from the use of our proprietary genechip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. these mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. although it is well established that concurrent chronic kidney disease (ckd) develops in about % of hyperthyroid cats, no one has reported the use of the iris staging system for ckd before and after treatment of these hyperthyroid cats. the purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage and ckd in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the iris stage in these cats. we evaluated hyperthyroid cats (median age, years) in this study. one day prior to treatment, serum t concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (upc) were measured. all cats were again evaluated with the same parameters again months after treatment with i. prior to treatment, ( %) of the cats had no evidence of azotemia (serum creatinine o . mg/dl), whereas cats ( %) had stage ckd (serum creatinine, . - . mg/dl). in the cats, iris staging revealed proteinuria in cats ( %), with borderline proteinuria (upc, . - . ) and with overt proteinuria (upc . ). hyperthyroidism was cured in all cats (median post-t , . mg/dl). all cats had a good response to treatment; there were no signs of ckd except for polyuria and polydipsia in some cats. a significant (p o . ) increase in median values for both serum urea nitrogen ( mg/dl to mg/dl)) and creatinine ( . to . mg/dl) occurred after treatment. nine of the cats ( . %) classified as nonazotemic or iris stage prior to i progressed to stage ckd after i. all cats with stage ckd before treatment remained azotemic after i, with cats remaining in stage ckd, and cats progressing to stage ckd (serum creatinine, . - . mg/dl). there was a significant inverse relationship (p . ) between pretreatment urine specific gravity (usg) and post-treatment serum creatinine in the cats. of the cats with post-treatment serum creatinine values . mg/dl (stage to ckd), ( %) had pretreatment usg of o . . in contrast, in the cats with post-treatment serum creatinine values o . mg/dl, only ( %) had pretreatment usg of o . . a significant (p o . ) decrease in median upc from . to . occurred after treatment, but there was no relationship between degree of proteinuria and iris stage in these cats. two cats developed iatrogenic hypothyroidism after i, diagnosed by finding low serum t and high ctsh concentrations. both hypothyroid cats had progressed from stage before treatment to stage and ckd, respectively, after i; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. conclusions: ) iris stage ckd is common in untreated hyperthyroid cats. ) progression to next higher iris stage is common after treatment, but most cats with remain relatively asymptomatic for ckd. ) usg may be helpful in predicting which cat's iris stage will progress after i. ) iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. home blood glucose monitoring (hbgm) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. nevertheless, it has proven difficult to objectively establish a clear benefit of hbgm using biological parameters (clinical signs, blood glucose, fructosamine). the current study aimed to assess the impact of hbgm on owner perceived quality of life (qol) aspects of diabetes mellitus (dm) treatment, using the recently validated psychometric tool diaqol-pet. owners of insulin treated diabetic cats were recruited to complete the -item tool, evaluating areas affecting the cat's and owner's qol, including: worry about pet's dm, hypoglycaemia, costs, owner's desire for autonomous control over the pet's dm, etc. item-weighted-impact-scores (iwis), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (awis; average iwis of all items), as an overall measure of diabetes dependent qol. frequencies, iwis and awis were compared between owners practising hbgm and those who did not using mann whitney u test (significance p o . ). two hundred and eleven owners of insulin treated diabetic cats completed the diaqol-pet; owners practised hbgm, whereas the remaining did not practise any form of home monitoring (including urine glucose). iwis for 'excessive drinking' and 'owner wanting more control' were significantly different between the hbgm-group (mean /-standard deviation: À . /À . and À . /À . ) and the non-hbgm-group (À . /À . and À . /À . ). there was no significant difference between the groups with regards to the iwis for other items, including 'worry about hypoglycaemia' or 'worry about pet's dm'. polydipsia was reported significantly more frequently in the non-hbgm-group and this was the reason for the difference between groups in this item's iwis as it was considered of equal importance. frequency and iwis of reported occurrence of hypoglycaemia signs were not significantly different. awis for both groups was not significantly different (hbgm: À . /À . ; non-hbgm: À . /À . ). the current study suggests that hbgm is predominantly practised by owners who desire more autonomous control over their cat's dm. the frequency of polydipsia was lower in the hbgm-group perhaps suggesting superior control. however, hbgm did not detectably affect the impact of the majority of qol-items, nor the frequency of hypoglycaemic episodes. overall diabetes dependent qol of diabetic cat and owner, as measured per diaqol-pet, was unaffected by hbgm. these data argue for the use of hbgm in selected pet-owner combinations rather than as part of a practice's standard dm management protocol, although further studies are indicated. insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (sc) adipose tissue. high plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type diabetes mellitus. however, the mechanisms by which these factors influence insulin action are still unclear. therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate (irs ), the serine/threonine protein kinase b (pkb/akt) and of the principal insulin-dependent glucose transporter protein (glut ) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia. healthy cats were infused through the jugular vein with glucose (n ), lipids (n ) or lipopolysaccharide (lps; n ) for days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: - mmol/l; triglycerides: - mmol/l) or to induce a systemic low-grade inflammation (lps; rectal temperature: . - . c), respectively. healthy control cats were infused with saline (n ). on day , specimens were collected from skeletal muscles, visceral and sc fat and processed for irs mrna expression, total and phosphorylated pkb/akt and glut protein expression. gene transcripts of irs were not different between the groups. compared to controls, skeletal muscle pkb/akt phosphorylation was % lower in cats infused with glucose (p o . ); lipid-infused cats showed a trend for a decrease in pkb/akt phosphorylation ( % lower than saline) and had decreased glut expression (p o . ) in muscle. total (p o . ) and phosphorylated (p o . ) pkb/akt protein expression were decreased in the sc adipose tissue of lps-infused cats compared to controls. in these cats, phosphorylation of pkb/akt protein was also decreased in visceral fat (p o . ). sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. separately used, single photon emission computed tomography (spect) and computed tomography (ct) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. these drawbacks suggest an interest in the fusion of images obtained by the techniques. the aim of this study is to test spect/ct fusion performance in dogs with insulinoma. inclusion criteria were: / a biological diagnosis of insulinoma; / an examination by high resolution ct scan and in-pentetreotide spect followed by spect/ct fusion; / a surgical or post mortem examination completed by histopathological analysis. spect examination showing abnormal foci and ct scan showing pancreas, lymph nodes (ln) or liver abnormalities were considered positive. in case of double positivity, presence (imp ) or absence (imp-) of superimposition of abnormal images was noted. ten dogs were included. in / dogs, superimposition of abnormalities couldn't be tested. ct scan detected abnormal images [ pancreatic nodules (pn), enlarged ln (eln)] while spect failed to show any abnormal uptake. both dogs became euglycemic after removal of pn and ln designed by ct scan. in / , all abnormal images were classified as imp [ pn, eln and diffuse hepatic infiltration (dhi)]. surgery performed on / resulted in euglycemia in ; dog remained hypoglycemic after partial removal of pn. pn localization and dhi were confirmed after necropsy in the th dog. in / dogs imp and imp-images were both recorded. in dog, a dhi was classified as imp but pn localization was imp-: localized in the left lobe by ct scan and in the corpus by spect, the latest localization being confirmed after necropsy. in the other dog pn localization was imp but a diffuse spect signal superimposing to the liver considered as normal on ct scan was noted. hepatic biopsy confirmed spect results. this study confirms an imperfect sensitivity of both ct scan and spect. it confirms that ct scan can be associated with unspecific abnormal images. subject to a confirmation on a larger cohort of dogs, it indicates that imp images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. the majority of dogs with primary hypoadrenocorticism (ph) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. in some of them sodium and potassium levels are normal, a phenomenon currently called atypical addison's. it has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. however, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-acth cortisol levels. the aim of the study was to evaluate aldosterone (aldo) levels in dogs with ph with and without electrolyte abnormalities. seventy dogs with newly diagnosed ph were included. aldo concentrations (ria, coat-a-count s , siemens) were measured before and min after administration of mg synthetic acth (synacthen s , novartis) iv. results were compared to those of healthy dogs and dogs with diseases mimicking ph. to confirm that peak concentrations were not missed aldo was additionally measured , and min after acth in dogs ( with ph, with ph mimicking diseases). results were analysed by means of non-parametric statistical methods (p o . ). post-acth aldo was significantly lower in dogs with ph ( - pg/ml, median pg/ml) than in healthy dogs ( - pg/ml, median pg/ml) and in dogs with ph mimicking diseases ( - pg/ml, median pg/ml). low post-acth aldo was found in / dogs with ph, in / of them levels were below the detection limit of the assay. normal sodium and potassium levels were found in / dogs ( %), / dogs ( %) had hyponatremia and normal potassium, / dogs ( %) had hyponatremia and hyperkalemia. electrolyte abnormalities ranged from mild to severe. there was no correlation between post-acth aldo and sodium and a weak correlation between post-acth aldo and potassium (r À . ). aldo concentrations were not different , and min after acth. the results demonstrate that aldo levels are low in most dogs with ph independent of the degree of electrolyte abnormalities. this implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without aldo. definitive diagnosis of canine hypoadrenocorticism (ha) is based on inadequate cortisol secretion following adrenocorticotropic hormone (acth) administration. an abnormal serum sodium to potassium (na:k) ratio can be used to determine whether an acth stimulation test is warranted. the aim of this study was to examine the utility of combining the na:k ratio with white blood cell counts to determine whether an acth stimulation test is warranted. a retrospective review of medical records of dogs examined between and was performed. dogs diagnosed with ha and control dogs, in which a diagnosis of ha was excluded during the study period, were included. inclusion criteria for all dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum na and k measurements at the time of initial examination. dogs were included in the ha group if they also had pre and post acth stimulation serum cortisol concentrations . mg/dl. dogs were included in the control group if they had resting or post acth stimulation serum cortisol concentration . mg/dl. exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration . mg/dl but . mg/dl. continuous variables were compared between groups using the mann-whitney u test. receiver operating characteristic (roc) curves were produced to assess the sensitivity and specificity of detecting ha with various cutoffs for each variable. data is presented with % confidence intervals (ci) and statistical significance was defined as p o . . the na:k ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with ha than in dogs without ha (p o . for each). lymphocyte and eosinophil counts were significantly higher in dogs with ha compared to dogs without ha (p o . for each). the areas under the curve by roc analysis were largest for na:k ratio ( . , ci: . - . ) and lymphocyte count ( . , ci: . - . ). a na:k ratio . was % sensitive (ci: - %) but only % specific (ci: - %) for detecting ha. a lymphocyte count ! . x cells/ml was % sensitive (ci: - %) and % specific (ci: - %). conversely a na:k ratio . was % sensitive (ci: - %) but % specific (ci: - %) and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) but % specific (ci: - %). a na:k ratio . was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. a combination of this na:k ratio ( . ) and lymphocyte count (! .  cells/ml) was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. these results indicate that the combination of lymphocyte count and na:k ratio results in a better screening test for ha than the use of the na:k ratio alone. pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. elevated plasma concentrations of the catecholamine breakdown products metanephrine (mn) and normetanephrine (nmn) are used to identify pheochromocytoma in humans. this study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness. edta plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between april and october . samples were stored at À c before measurement of free mn and nmn concentrations using high pressure liquid chromatography at the central laboratory for clinical chemistry at the university of groningen ( samples) or the mayo clinic, rochester, minnesota ( samples). kruskal-wallis tests followed by dunn's multiple comparison analysis were used to compare results between groups. significance was set at p o . . results are reported as median [range] . eight dogs with pheochromocytoma, healthy dogs, dogs with non-adrenal illness and dogs with cortical tumors were sampled. pheochromocytoma was diagnosed histologically ( dogs) or cytologically ( dog). cortical tumors were diagnosed histologically ( dogs) or by response to trilostane treatment after obtaining consistent endocrine test results ( dogs occult hyperadrenocorticism (hac) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic hac. however, the ability of sex hormones to cause such alterations has never been closely evaluated. if sex hormones can cause a syndrome similar to classic hac, they should be able to induce expression of classic glucocorticoid-induced genes. the purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced alp (cialp) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult hac. canine hepatocytes were purchased from a commercial source (cellzdirect or invitro) in -well plates. upon arrival ( - plates per shipment), the cells were allowed to recover in general media per supplier recommendations. after hrs, media was changed to william's e media (-l-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with hac or with purported occult hac. each plate was treated with a different hormone (cortisol, -hydroxyprogesterone [ ohp], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. media was changed daily. after days of hormone exposure, rna was extracted. reverse transcription was performed and the product used for quantitative pcr for cialp and beta-actin (roche lightcycler) using a gene-specific fluorescent probe for detection. standard curves were created for each gene. all samples and standards were run in duplicate. using the lightcycler software (vers . ), cialp expression was normalized to that of beta-actin. fold change in expression was determined relative to the negative control. each sex hormone was used to treat plates; one plate in each shipment was treated with cortisol as a positive control. for cortisol, a dose response was seen in expression of the cialp gene. compared to no cortisol, , , , , and nmol cortisol increased expression . , . , . . . , . and . fold, respectively. a -fold increase is considered significant (j.vandesompele et al genome biol ). expression of cialp was not significantly induced in response to any concentration of ohp ( nm maximum), progesterone ( nm maximum), estradiol (max pm maximum) or androstenedione ( nm maximum). we conclude that in vitro these sex hormones do not induce expression of the cialp gene which is classically induced by cortisol in vivo; indeed, elevated serum cialp activity is a hallmark of classic hac. thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (hac), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical hac, i.e. disease due to excess adrenal sex hormone secretion. however, measurements in either setting have not been widely studied. therefore, our objectives were: . to determine the sensitivity of -hydroxy-progesterone ( ohp) and estradiol concentrations pre-and post-acth for diagnosis of typical hac. . to determine the specificity of ohp and estradiol concentrations preand post-acth for diagnosis of occult hac. dogs that had pdh (n ), dogs that were suspected to have hac but proven not to (had non-adrenal illness [nai, n ]) or dogs that were healthy (n , used to establish reference ranges [rr]) were enrolled. acth stimulation tests were performed ( mcg/kg cosyntropin iv); blood samples were drawn pre and min post; ohp and estradiol were measured by previously validated radioimmunoassays. a kruskal-wallis rank sum test was used to compare values between the groups. significance was set at p o . . for basal and acth-stimulated ohp concentrations, the rr were determined to be . - . ng/ml (mean ae s.d.; range . - . ) and . - . ng/ml (range . - . ), respectively. in pdh dogs, and had basal and post-acth ohp concentrations above the rr, respectively; in the nai group, and dogs had concentrations above the rr, respectively. thus, the sensitivity of basal and post-acth ohp measurement for diagnosis of hac is % and %, respectively. specificity of diagnosis is % and %, respectively. post-acth ohp concentration was significantly different between groups. for basal and stimulated estradiol concentrations, the rr were determined to be - pg/ml (range - ) and - pg/ml (range - ), respectively. for both basal and stimulated estradiol, pdh dogs (n ) had concentrations above the rr; for those with nai (n ), and had concentrations above the rr, respectively. thus, the sensitivity of estradiol measurement for diagnosis of hac is % for both pre-and post-acth. specificity of estradiol for diagnosis for hac is % and % for pre-and post-acth, respectively. overall, dogs with nai had at least one elevated estradiol concentration (total specificity %). post-acth estradiol concentration was not significantly different between groups. we conclude that use of ohp and estradiol concentrations for diagnosis of hac can be problematic. sensitivity and specificity are relatively low, potentially leading to misdiagnoses. diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type diabetes. several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. however, to date, no specific genetic risk factors have been identified. genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor (mc r) gene. the aim of the current study was to identify polymorphisms (snps) in the feline mc r gene and to use these to perform a case:control study to determine whether these candidate gene snps were associated with diabetes mellitus in cats. genomic dna from cats ( domestic short hair [dsh], burmese) was initially analysed by pcr and direct sequencing using felmc r-specific primers, which identified a missense mutation (mc r:c. c t) in the region encoding the extracellular domain of the receptor protein in dsh cats only. one hundred and nineteen dsh cats were subsequently recruited into the case:control study. fifty nine cats were obese diabetic ( male, female), mean age . years (range - y); mean weight . kg (range . - kg). sixty lean cats were used as controls ( male, female), mean age . years (range - y), mean weight . kg (range . - . kg). the t to c base change alters a restriction site in the sequence recognized by the enzyme bstoi, such that dna from cats with the mutant (c) allele can be cut, whereas that from the wild-type (t) allele cannot. primers were designed that flanked the mutation to allow pcr amplification of this region of mc r from genomic dna obtained from edta blood. the pcr products were purified and subject to restriction fragment length polymorphism (rflp) analysis. bstoi digestion products were then analysed by agarose gel electrophoresis. of the diabetic cats, ( %) were homozygous for the mutation (cc), compared to ( %) of control cats. statistical analysis (two tailed fisher's square test) revealed that this difference between groups was statistically significant (p . ). in conclusion, this pilot study has identified a missense mutation in the coding sequence of mc r. this could be an important predisposing factor for development of diabetes and/or obesity in dsh cats. polymorphisms in a similar region of human mc r predispose to obesity, which in turn is a major risk factor for type diabetes. hyperadrenocorticism (hac) is one of the most common endocrine disorders of dogs. the two most effective medical treatments are trilostane (vetoryl s ) and mitotane (lysodren s ). previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at min post-acth administration. however, the optimal sampling time would be at the time of maximal secretion, which occurs minutes after the mg/kg dose commonly used for the test (carlson et al, jvim, ). thus, the true effect of either medication on aldosterone secretory capacity is unknown. our objectives were: ) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent hac (pdh) on aldosterone secretory reserve at min post-acth stimulation and ) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations. forty-six dogs being treated for pdh with either mitotane (n ) or trilostane (n ) have been enrolled. the dogs could be treated for any length of time. all had acth stimulation tests performed ( mcg/kg cosyntropin iv); blood samples were drawn before and at and min post-acth for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. ten historical normal controls were also included. serum sodium and potassium concentrations were measured in the basal samples. a kruskal-wallis rank sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. significance was set at the p o . level. acth-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the and min time points. no difference was detected between aldosterone concentrations at and min after acth injection in either treatment group. a positive correlation existed between the -min cortisol and -min aldosterone concentrations in the trilostane-treated group (r . ), i.e. the peak post-acth concentration for each hormone, but not in dogs treated with mitotane. basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group. in conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. measurement of aldosterone concentrations is not predictive of electrolyte concentrations. previously presented at the auburn university phi zeta research emphasis day, november , . antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. in addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. the primary aim of this study was to determine whether deficiencies in glutathione (gsh), ascorbate (aa), or vitamin e, along with increases in urinary -isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. a secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity. ongoing prospective, controlled, observational study. otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n to date) and healthy age-matched controls (n to date) were recruited. all cats were screened with cbc, biochemical panel, urinalysis, and t , as well as red blood cell (rbc) gsh, plasma aa, plasma vitamin e, and urinary -isoprostanes. hyperthyroid cats were re-evaluated months after radioiodine treatment. unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (gsh . mm; aa . mm, and vitamin e, g/ml) compared to controls (gsh . mm; aa . mm, and vitamin e, g/ml). results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. rbc gsh concentrations did increase significantly (to . mm; p . ) after radioiodine treatment. however, this modest change is unlikely to be clinically significant. preliminary data do not indicate clinically significant blood gsh, ascorbate, or vitamin e deficiencies in hyperthyroid cats. with appropriate insulin therapy and a low carbohydrate diet, up to % of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. there are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. the aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission. the study was a prospective study in a feline-only clinic. for inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks. five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.glucose concentration was measured using a meter calibrated for feline blood (abbott alphatrak). a simplified glucose tolerance test was performed after food was withheld for hours. a g catheter was placed in a cephalic vein three hours before the gtt was commenced, to minimize the effects of stress on blood glucose concentration. blood glucose concentration was measured at time and then a g/kg dose of glucose was administered slowly via the intravenous catheter. further blood glucose measurements were made at hours and then hourly until glucose had returned to o mg/dl (o . mmol/l). in the control group, all cats had a fasting blood glucose below mg/dl, and following glucose administration, glucose had returned to o mg/dl by hours. fasting blood glucose in the remission group was o mg/dl ( mmol/l) in all cats except one, which had fasting blood glucose of mg/dl ( . mmol/l). following glucose administration, all five cats in remission had blood glucose above mg/dl ( . mmol/l) at three hours, four were o mg/dl at four hours, and one returned to o mg/dl at five hours. the cat with impaired fasting glucose subsequently became diabetic after steroid administration. the results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. the objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. five domestic shorthair cats ranging in age from - years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (tt ), palpable thyroid nodule and weight loss. serum tt concentrations ranged from - nmol/l (reference range - nmol/l). the cats were then fed a low iodine containing food ( . ppm iodine dmb, as measured by epiboron neutron atomic activation). serum tt concentrations were measured every weeks. biochemistry parameters were also evaluated at weeks , and . at weeks, serum tt concentrations had decreased in all cats with of cats ( %) being euthyroid (mean nmol/l; range - nmol/l). the remaining hyperthyroid cat had an initial serum tt of nmol/l, which decreased to nmol/l after being fed the iodine-restricted food. mean decrease in tt for all cats was nmol/l (range - nmol/l). renal parameters remained stable in all cats. these cats along with additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine ( . ppm dmb). baseline serum tt concentrations in the new cats ranged from - nmol/l. serum tt and other biochemical parameters were monitored every weeks for weeks, and then every weeks for an additional weeks. with the . ppm iodine food the four new cats became euthyroid with a mean tt concentration of nmol/ (range - nmol/l). the euthyroid cats from the earlier feeding study had a further decrease in tt concentration (mean tt nmol/l, range - nmol/l). the single non-euthyroid cat from the first study had a serum tt concentration of nmol/l, a decrease from the baseline concentration of nmol/l. the average decrease in serum tt for all cats was nmol/l (range - nmol/l). finally, of the cats were fed a third iodine-restricted food ( . ppm dmb) along with one other newly diagnosed hyperthyroid cat ( nmol/l serum tt ) and evaluated every weeks. all cats in this evaluation were euthyroid (mean tt nmol/l; range - nmol/ l). this result included the cat whose serum tt remained in the hyperthyroid range in the first two evaluations. the average decrease in tt was nmol/l (range - nmol/l). biochemical features of renal function remained stable and no other biochemical abnormalities were observed. in summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. we have shown previously that restriction of dietary iodine (i) is a safe and effective method for decreasing serum thyroxine concentrations (tt ) in cats with hyperthyroidism. the objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum tt concentrations in hyperthyroid cats currently being controlled on a food containing . ppm i (dmb) as measured by epiboron neutron atomic activation. all cats were previously diagnosed at least months prior to the start of the study and their tt concentrations were maintained in the normal range by dietary iodine restriction for a minimum of months (range months- years). serum tt concentrations ranged from - nmol/l (reference range - nmol/l) at the beginning of the study. the cats were divided into two groups each containing cats. groups were similar in age and gender distribution (mean age . years, range - years). one group (group a) was placed on a food that was formulated for mature adult cats containing . ppm i (dmb). the other group (group b) was placed on a similar food that differed only in that it contained . ppm i (dmb). blood was collected from all cats every three weeks and analyzed for serum tt concentration. biochemistry parameters were also evaluated at weeks , and . all group a cats exhibited increases in serum tt concentration (mean increase of nmol/l above baseline, range - nmol/l). seven of the cats remained in the euthyroid range (mean serum tt nmol/l, range- - nmol/l). two cats exceeded the upper limit of the reference range ( and nmol/l respectively). the cats in group b also exhibited increases in serum tt concentration but to a greater degree than the cats in group a (mean increase nmol/l, range - nmol/l). four cats remained in the euthyroid range (mean serum tt , range - nmol/l). the five remaining cats all exceeded the upper limit of the reference range (mean serum tt nmol/l, range- - nmol/l). all cats returned to a euthyroid state within month of being returned to a diet containing . ppm i (dmb). it was determined that serum tt concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! . ppm i (dmb). hyperthyroidism is a common disease in old cats. excessive production of thyroid hormones is the hallmark of the disease. three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. previously we have shown that limiting dietary iodine to or below . ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing . ppm iodine. the objective of this study was to test whether dietary iodine at . ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism. fourteen cats with hyperthyroidism confirmed by serum tt and ft measurements were stratified into two groups based on gender and age. one group (control: males and females, age ranged from to years) was given a positive control dry cat food ( . ppm iodine) while the other group (test: males and females, age ranged from to years) was fed a commercial dry cat food ( . ppm iodine) for at least weeks before the study. afterwards (week ), the control cats continued to receive the same food while cats in the test group were given a test food ( . ppm iodine) for additional weeks. all cats had free access to their food and deionized water during the study. blood samples were collected during weeks , , , and of the study. the control cats maintained euthyroidism during the study. the test food significantly reduced serum tt ( ae , ae à , ae à , ae à nmol/l in weeks , , and , respectively; à : p o . compared with week , dunnett's t test). it also significantly reduced ft at the end of the study ( ae vs. ae pmol, week vs. week ; dunnett's t test, p o . ). serum ft was within the reference range ( - pmol/l) in cats in both groups. serum tt , ft , and tsh were not affected by the test food and were within the reference ranges (tt : . - . nmol/l, ft : . - pmol/l, and tsh: - mu/l) in cats of both groups during the study. this study demonstrates that dietary iodine at or below . ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. radioactive iodine ( i) is a widely used treatment for feline hyperthyroidism. prior to i administration, many cats receive methimazole therapy. it has been suggested that recent withdrawal of methimazole prior to i may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. to further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received i therapy after methimazole treatment. inclusion criteria included documentation of the time interval between discontinuation of methimazole and i administration, and measurement of thyroxine (t ) at - days after i. cats were divided into groups: those receiving i within day of stopping methimazole, and those receiving i treatment or more days after stopping methimazole. sixty cats met the inclusion criteria. forty received i within day of stopping methimazole. of those, ( %) had a low t (o . mcg/dl), ( . %) had a normal t ( . - . mcg/dl), and ( . %) had an elevated t ( . mcg/dl) at - days after i therapy. fourteen cats received i or more days after stopping methimazole: ( %) had a low t , ( %) had a normal t , and ( %) had an elevated t at - days after i therapy. the results were compared with a fisher's exact test and there was no difference between the groups (p . ). these findings indicate that stopping methimazole therapy within day of i therapy does not inhibit the response to therapy. pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. the purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin elisa with known cross reactivity to several synthetic analogs. pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately minutes following a meal, for a total of samples. dextrose was added to the meals given to two of the cats. blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at À c until analysis. serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin elisas. the elisas were run in duplicate and according to the manufacturer's instructions. concentrations of serum insulin measured by the feline insulin elisa ranged from . ng/l to ng/l. despite the wide range of concentrations of feline insulin, all samples evaluated with the human insulin elisa yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. the aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (pdh). among the client-owned dogs diagnosed with pdh, only the dogs weighing o kg were selected (n ). group a (n ; low-dose treatment group) and group b (n ; high-dose treatment group) received . ae . mg of trilostane/kg orally every hours and mg of trilostane/ body orally every hours, respectively. all of the dogs were reassessed at , , , and weeks after the initiation of treatment. the improvement in post-acth stimulation serum cortisol concentration, as well as clinical signs in group a, required more time than group b; however, of dogs in group b had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for weeks. twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. the present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine pdh and may be safe without the potential adverse effects of once daily, high-dose treatment. however, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. an immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. in pigs, the role of cobalamin has not yet been extensively investigated. thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs. for the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available immulite s cobalamin immunoassay (siemens healthcare diagnostics ltd., deerfield, il, usa) in surplus porcine serum samples from a variety of studies. validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. additional surplus serum samples from piglets from four litters at a texas a&m university farm were obtained. each piglet had been bled twice, the first at weaning ( days of age) and the second one days later. to investigate results in comparison between age groups, serum cobalamin concentrations were compared using a wilcoxon matched pairs test. significance was set at p o . . observed to expected ratios (o/e) for serial dilutions ranged from . to . % (mean ae sd: . ae . %) for four different serum samples at dilutions of : , : , and : , and from . to . % (mean ae sd: . ae . %) for one serum sample at dilutions of : , : , and : . o/e for spiking recovery ranged from . to . % (mean ae sd: . ae . %) for five different porcine serum samples that had been spiked with each other in a : dilution. intraassay coefficients of variation (%cv) for five different serum samples were . , . , . , . , and . %. inter-assay %cvs for five different serum samples were . , . , . , . , and . %. serum cobalamin concentration was significantly lower in piglets post weaning (median: ng/l) compared to those at the time of weaning (median: ng/l; p . ). the immulite s cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. this study also showed that piglets that differ in age by only days have significantly different serum cobalamin concentrations. further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. the prevalence of intramammary infection (imi) ranges from - % of quarters pre-partum and - % at parturition. some pre-partum infections self-cure before parturition, however a number of these imis persist into early lactation. these imis may impact milk production and quality and may serve as a reservoir for contagious pathogens. no study has specifically investigated the risk of an imi persisting from the prepartum period into early lactation. the objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (rr) and attributable fraction of population (afp) for the association between a post-partum and pre-partum imi. two-hundred-ninety-four heifers were systematically assigned to of groups: g ) pre-partum secretions from all mammary quarters (n ), g ) no pre-partum secretions collected (n ) and g ) pre-partum secretions from two diagonal quarters (n ). group assignments were designed to assess whether pre-partum sampling increased the likelihood of imi at calving. mammary quarter secretions were collected for bacterial culture approximately weeks prior to expected calving date. quarter milk samples were collected for bacterial culture once weekly during the st -weeks of lactation. bacterial isolates were classified as staphylococci, non-agalactiae streptococci and gram-negatives. mammary quarter samples yielding different bacteria were classified as mixed infections and those yielding ! bacterial types were classified as contaminated. bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. relative risk and afp were calculated using  tables. forty-five percent of mammary quarters had a pre-partum imi. during the st weeks of lactation the mean prevalence of imi was . % of quarters. staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with s. chromogenes and s. aureus being the most common species. using data from mammary quarters, the rr and afp for the association between a post-partum and pre-partum imi were and %, and %, and and % for all staphylococci, s. aureus only and cns only imis, respectively. mammary quarters sampled pre-partum were no more likely to have a post-partum imi than those not sampled (chisquare, p ! . ). these data demonstrate that pre-partum imis persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting imi at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., s. aureus, into the lactating herd. despite concerns about antimicrobial resistance and clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. this study evaluated the impact of tetracycline treatment on c. difficile shedding in veal calves and the impact on resistance. calves arriving on veal farm received oral oxytetracycline for days as per farm protocols. calves were sampled at arrival and days later. selective culture for c. difficile was performed. isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes. clostridium difficile was isolated from % ( / ) and % ( / ) calves, at the first and second samples, respectively. the percentage of tetracycline resistant isolates increased from % to %. isolates from the second sample were times more likely to be tetracycline resistant (p . ) and times more likely to possess tet(m) (p . ). tet(m) was detected in % ( / ) and % ( / ), tet(o) in % ( / ) and % ( / ) and tet(w) in % ( / ) and % ( / ) of isolates from first and second samples, respectively. tet(l), tet(k) and tet(s) were not detected. resistant isolates were not carrying any of the genes investigated. routine tetracycline use may have had an impact on both the prevalence of c. difficile, as well as the strain distribution and resistance patterns. this is the first report of presence of tet ( the objectives of this study were to ) estimate the prevalence of antimicrobial resistance in the study population and ) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific e. coli (ntsec) recovered from individual feedlot cattle. two-stage random sampling was used to identify cattle for enrollment at western canadian feedlots. a fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. from samples collected at this second time point, a total of , ntsec isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. the least square means estimates and % confidence intervals for the prevalence of resistance at each time point were modeled using poisson regression. multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. regression models were adjusted for clustering of observations among individuals and pens. the most common resistances identified in arrival samples were sulfisoxazole ( . %; %ci: . - . ), streptomycin ( . %; %ci: . - . ) and tetracycline ( . %; %ci: . - . ). at the second sampling point, resistance prevalence was . % ( %ci: . - . ) for sulfisoxazole, . % ( %ci: . - . ) for streptomycin, and . % ( %ci: . - . ) for tetracycline. logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point. abstract fa- premature/dysmature syndrome in cria: a ret-rospective study of cases ( ) ( ) ( ) ( ) ( ) ( ) ( ) . c. gerspach, d. anderson. the ohio state university, columbus oh. prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. a review of medical records for premature cria alive at the time of admission to the veterinary teaching hospital between and was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. medical records for premature or dysmature cria were included in this study. of these cria, were alpaca and llama, were female and were male. reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. cria were presented at a mean age of . days and were premature by a mean estimated time of . days. overall survival rate was . %, with all llama cria surviving. a multivariate logistic regression model was used to identify risk factors associated with not surviving. cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. dyspnea and tachypnea was associated with a poor outcome. all cria that were able to nurse, without assistance prior to referral, survived. clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs ( . mg/kg subcutaneously). furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points. six healthy pigs were used in this study. pigs were recently weaned and weighed an average . kg. on the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. each pig received an injection of enrofloxacin (baytril , bayer animal health) at a dose of . mg/ kg subcutaneously behind the left ear. plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using hplc with fluorescence detection. protein binding was determined with a microcentrifugation system. pharmacokinetic data was analyzed using a one compartment model. the analysis of plasma and isf showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. the plasma half-life (t / ), volume of distribution, clearance, and peak concentration (c max ) for enrofloxacin was . hr (ae . ), . l/kg (ae . ), . l/kg/hr (ae . ), and . mg/ml (ae . ), respectively. the concentrations from each of three tissues were not different in each pig. when pharmacokinetic values from all tissues were combined for the isf, the t / was . hr (ae . ) and the c max was . mg/ml (ae . ). the enrofloxacin plasma protein binding was . % (ae . ) and . % (ae . ) at a high and low concentration, respectively. this study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. the half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. the high tissue concentrations and long half-life produce an auc/mic ratio sufficient for the pathogens that cause respiratory infections in pigs. ceftiofur crystalline free acid (ccfa), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. pharmacokinetic data, however, do not exist for ccfa administered subcutaneously in sheep. the present pharmacokinetic study evaluated the single dose subcutaneous administration of ccfa in sheep (n ) at . mg/kg body weight. concentrations of ceftiofur free acid equivalents (cfae) in plasma were measured by high performance liquid chromatography for days following drug administration. pharmacokinetics of subcutaneous ccfa in sheep were best described using a single compartment model with the following average (ae sd) parameters: area under the concentration time curve ! ( . hÃug/ml ae . ), observed maximum plasma concentration ( . ug/ ml ae . ), and observed time of maximum plasma concentration ( . h ae . ). no significant adverse drug reactions were observed. adequate cfae plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. the purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease. a longitudinal study was performed using female dairy calves from commercial dairy farms in new york state. calves were enrolled based on age. thoracic ultrasound and clinical respiratory scoring were performed on each calf at time points. a standard mhz linear ultrasound probe was utilized to evaluate intercostal spaces through of each hemi-thorax with the calf in lateral recumbency (us ) or standing (us ). lesion appearance, size, and location were recorded. respiratory score (rs) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. abnormal lung on ultrasound was defined as one or more areas of ! cm width or depth of non-aerated lung. farm records were evaluated to identify treated calves. calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or rs. non-parametric methods were used to evaluate the data. ninety-one calves were enrolled into the study, with lost to follow-up. an average of minutes was spent performing the rs and ultrasound on each calf. the median ages at first (us ) and second (us ) examination were (interquartile range - ) and (interquartile range - ) days, respectively. the majority of calves had a low rs (o ) and only . % of calves had a rs high enough to warrant treatment based on previous recommendations (rs! ). the prevalence of calves that had abnormal lungs on ultrasound but a low rs (o ) was . % (us ) and . % (us ). the prevalence of calves that had abnormal lungs on ultrasound and a high rs (! ) was % (us ) and . % (us ). of the calves that had abnormal lungs on ultrasound but a low rs, % were treated with antimicrobials within days of examination. none of the calves with high rs and abnormal lungs on ultrasound were treated with antibiotics within days of examination. this study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. the relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. literature regarding diseases causing lameness in beef cattle is limited. this retrospective study was undertaken to examine beef cattle presented for lameness. medical records of beef cattle having a lameness examination done during the period to were reviewed and descriptive statistics generated. lameness was classified based on clinical diagnosis. the medical records of beef cattle were reviewed of which . % were male and . % were female. beef cattle presented for lameness most often during the summer months ( %) and least during autumn ( %). causes of lameness were categorized as infectious ( . %) or non-infectious ( . %) and infectious lameness subcategorized as either a primary disorder or a secondary infection. all cases of a primary infectious disorder were interdigital phlegmon. secondary infections diseases included sole abscess ( . %), septic arthritis ( . %), tenosynovitis ( . %), and pedal osteitis ( . %). non-infectious lameness included proximal limb lameness ( . %), foot trauma ( . %), hoof horn cracks ( . %), hoof defects ( . %), interdigital fibromas ( . %), overgrown hooves ( . %), sole bruise ( . %), subclinical laminitis ( . %), white line disease ( . %), osteoarthritis ( . %), heel erosion ( . %), sole ulcers ( . %), and sole hemorrhage ( . %). the most frequently affected claw was the lateral digit of the hind limb ( . %), followed by the medial digit of the front limb ( . %), lateral digit of the front limb ( . %), and the medial digit of the hind limb ( . %). the findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. in people, endoscopic ultrasound (eus) has become the technique of choice for assessing pancreatic disease and eus-guided fineneedle aspiration (eus fna) has proven a useful and safe modality for characterizing pancreatic lesions. reported complications include infections, bleeding and acute pancreatitis. in dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however eus and eus fna have not been evaluated in dogs so far. thus the aim of the present study was to assess the practicability and safety of eus examination of the abdominal cavity as well as pancreatic eus fna in healthy dogs. this study was approved by the cantonal committee for the authorization of animal experimentation, zurich, switzerland. the study population consisted of healthy beagle dogs with a median bodyweight of . kg ( . - . ). eus was performed with an olympus gf-uc p-echoendoscope and fna were performed using g needles (cook echotipultra). after completion of the eus-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and eus fna of the pancreas was performed. fna tissue acquisition was made applying negative pressure and to needle passes were made. all dogs received mg/kg metimazole im after eus fna and were re-checked ultrasonographically minutes post eus fna. postoperative activity was assessed using a standardized scoring system. a cbc, serum biochemistry, urinalysis and spec cpl s were measured before, as well as and h after eus fna. the eus examination was complete in / dogs, the pancreas could not be visualized in dog. the pancreas was hypo-( / ) to isoechoic ( / ) to the surrounding mesenterium in all cases. in / dogs parts of the pancreas presented hyperechoic. the mean measured thickness was . cm. the pancreas was aspirated in dogs using a transgastric approach ( ) or transduodenal approach ( ). duodenal transmural puncture was not accomplished in dog where a re-sterilized needle was used. a minimal amount of peripancreatic fluid was observed in / dogs after eus fna. all dogs recovered uneventfully and required no further analgesia. all laboratory results including the spec cpl s measurements were within reference ranges on all three time points. cytologically, conglomerates of exocrine pancreatic cells were seen in / cases, duodenal villous epithelial cells were seen in / cases. in dog the aspirated pancreatic material was sufficient for a histological assessment. the aspirates with exocrine pancreatic cells on cytology were obtained by transgastric ( ) and transduodenal ( ) aspirations. in conclusion, ( ) eus examination of the abdomen is feasible in medium-sized dogs, ( ) the healthy canine pancreas can be difficult to visualize completely, and ( ) eus-guided pancreatic fna using a g needle is a safe procedure in healthy dogs. studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. miniature schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (tg) concentration, with or without an increased serum cholesterol (chol) concentration. a common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. however, the efficacy of this approach has not been evaluated for either condition. the aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of tg, chol, and canine pancreatic lipase immunoreactivity (cpli; measured as spec cpl s ) in apparently healthy miniature schnauzers with hypertriglyceridemia. blood samples were collected from apparently healthy miniature schnauzers with hypertriglyceridemia (serum triglyceride concentrations mg/dl). common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total t , and free t (by ed) concentrations. the owners of the dogs were asked to switch their dog to the study diet (royal canin gastrointestinal low fat s ; fat content: . g/ , kcal) and have a second blood sample collected weeks after their dog had been on the new diet. all blood samples were collected after food had been withheld for hours. serum tg, chol, and spec cpl concentrations were measured both before and after the diet change. results were compared between the two time-points using the wilcoxon signed rank and fisher's exact tests. serum tg concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypertriglyceridemia was significantly higher before ( / ) than after the diet change ( / ; p . ). also, the proportion of dogs with serum tg mg/dl was significantly higher before ( / ) than after the diet change ( / ; p . ). serum chol concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypercholesterolemia was significantly higher before ( / ) than after the diet change ( / ; p . ). finally, the difference in serum spec cpl concentrations before (median: mg/l) and after the diet change (median: mg/l) approached but did not reach significance (p . ). also, the proportion of dogs with high serum spec cpl concentrations before ( / ) and after the diet change ( / ) was different, but this difference was not significant (p . ). in summary, a commercially available low-fat diet was effective in reducing serum tg and chol concentrations in miniature schnauzers with hypertriglyceridemia. toll-like receptor (tlr ) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. we have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). recently, we have confirmed that two of these tlr snps (c t and t c) are significantly associated with ibd in other canine breeds. to further substantiate the role of tlr in canine ibd functional analysis of these polymorphisms would be needed. therefore the aim of this study was to determine the functional significance of the tlr snps by transfecting wild-type and mutant receptors in to human embryonic kidney cells (hek) and carrying out nuclear factorkappa b (nf-kb) luciferase assay and il- elisa. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) as determined by the case-control analysis in gsds with ibd were cloned into plasmids expressing yellow-fluorescent protein (yfp). these were then stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were then stimulated with various ligands ( . mg/ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk) according to the manufacturer's recommendations. the supernatants were harvested and used in an il- elisa (r&d systems). human tlr transfected hek cells (invivogen) served as positive controls in all experiments. independent t-test was used to determine the significance of relative luciferase activity and il- concentration between wild-type and mutated tlr cells. although there was no significant difference between the wild-type and mutated receptor when they were stimulated with . mg/ml of flagellin (p . ), there was a significant increase when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ). similarly, there was a significant increase in il- concentration in the supernatants in the cells with the mutated tlr receptor when stimulated with . mg/ml flagellin compared to the wild-type (p . -one-tailed, . -two-tailed) but not with . mg/ml flagellin (p . ). we show for the first time that polymorphisms associated with ibd are functionally hyper-responsive to flagellin compared to the wild-type receptor. this suggests that tlr may play a role in canine ibd and that blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. however, further in-vivo functional analysis of tlr , especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. tlr has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (ibd). similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the canine tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). therefore the aim of this study was to determine the functional significance of the tlr snps in the breed of gsds. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) were stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were stimulated with various tlr ligands ( . mg/ ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk). the supernatants were harvested and used in an il- elisa (r&d systems). peripheral whole blood from dogs carrying the wild type and mutant tlr genes was cultured and stimulated with tlr ligands as above. canine tnf-alpha was measured in the supernatant by commercially available elisa (r&d systems). t-test was used to determine differences of relative luciferase activity, il- concentration and tnf-alpha concentration between wild-type and mutated tlr cells. there was a significant increase in nf-kb activity when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ), which correlated with il- expression in the supernatant (p . ). similarly, in the whole blood assay the tlr risk haplotype for ibd in gsds (acc) was significantly hyperresponsive to flagellin at a concentration of . mg/ml compared to the tlr wild-type haplotype (gtt) (p . ). we show for the first time that polymorphisms associated with canine ibd in gsds are functionally hyper-responsive to flagellin compared to the wild-type receptor. blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. proton pump inhibitors (ppi) are widely used in human and also veterinary medicine. side-effects of ppi treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. moreover, long-term ppi treatment has been associated with an increased risk for osteoporosis in people. little is known about the effect of ppi treatment on serum gastrin concentration or calcium metabolism in dogs. eight healthy adult research dogs ( males and females) were enrolled into the study. the dogs received an average dose of . mg/ kg of omeprazole orally twice daily for days. blood samples were collected prior to initiating the treatment and every days during the days of treatment and during the days after discontinuation of treatment for determination of serum gastrin, ionized calcium, pth, and oh vitamin d . gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric ph prior to, during, and after the omeprazole treatment period. normally distributed data were compared with a repeated measures anova and post hoc dunnett's test. data that were not normally distributed were compared with a friedman's test and a post-hoc dunn's test. gastric fluid ph was significantly higher (p o . ) at the end of the treatment period (median: . ; range: . - . ) when compared to pretreatment values (median: . ; range: . - . ). serum gastrin concentrations increased significantly from a median baseline of . ng/l (range: . - . ) to a maximum median of . ng/l (range: . - . ) at day of treatment (p o . ). serum gastrin remained significantly increased above baseline values from day to day of the treatment, but was not different from pre-treatment values days after the end of the treatment. omeprazole treatment had no effect on ionized calcium or pth for the duration of the study. marginal, but significant changes of oh vitamin d were observed at day (end of the treatment period -increased by . %) and day ( days after the end of the treatment -decreased by . %). this study shows that treatment with omeprazole for weeks results in a profound and sustained increase in serum gastrin concentration in dogs. this effect is rapidly reversible after cessation of the treatment. no effect on calcium metabolism was observed. however, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (acvim ). however, a sequencing approach can only estimate relative proportions of genomic bacterial targets. therefore, significant changes in the total number of bacteria could not be evaluated. the aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment. eight month-old healthy research dogs ( males and females) were enrolled. the dogs received an average dose of . mg/kg of omeprazole orally twice a day for days. endoscopic gastric and duodenal biopsies were harvested and days before starting omeprazole treatment, on the last day of treatment (day ), and days after the end of treatment (day ). all biopsies were fixed in % formalin for hours, processed, and embedded in paraffin blocks. fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the s ribosomal rna. statistical analysis aimed to compare changes in helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the glimmix and npar way procedures in sas s . . bacteria were counted in , and microscopic fields ( Â) obtained from and gastric and duodenal biopsies, respectively. in the stomach, omeprazole treatment led to a decrease in helicobacter spp. (log of average counts ae standard error: . ae . at day ) when compared to the counts ( . ae . , p . ) and ( . ae . , p . ) days before treatment. after completion of omeprazole treatment, helicobacter spp. increased and returned to baseline counts ( . ae . at day , p . vs day ). also, in the stomach, non-helicobacter spp. bacteria were observed more often during omeprazole treatment (median: , range: - ) than on days (median: , range: - ) and (median: , range: - ) before and days after (median: , range: - ) omeprazole treatment; however, statistical comparison across time points did not reach significance. in the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p . ). our results suggest that omeprazole treatment for weeks leads to a lower abundance of helicobacter spp. organisms in the stomach of healthy dogs. also, this transient decrease in helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. the smartpill ph.p s capsule (the smartpill corporation) is a wireless motility capsule that measures ph, pressure, and temperature as it passes through the gastrointestinal (gi) tract. analysis of this data allows the calculation of gastric emptying time (get), small and large bowel transit time (slbtt), and total gi transit time (tgtt). this study evaluated the variability associated with repeated measurement of gi transit times and the effect of oral administration of ranitidine (zantac s ) on gi transit times in dogs using this system. it was hypothesized that ranitidine would reduce gi transit times. six privately owned healthy adult dogs weighing between . kg and . kg were used. on occasions each dog was fed a standard meal followed by oral administration of a capsule. data were recorded until the capsule had passed in the dog's feces. on a th occasion each dog was given mg of ranitidine po q hrs starting hrs prior to testing. the dogs were then fed the test meal and the capsule was administered as above. ranitidine was given until the capsule had passed in the dog's feces. proprietary smartpill software was used to calculate get, slbtt, tgtt, and the median gastric ph (mgph). mean intra-individual and inter-individual coefficients of variation (cv%) were calculated for get, slbtt, and ttt for the first time points. transit times and gastric ph recorded at all time points were compared using a repeated measures anova. where significant differences were identified, post-hoc testing was performed using a bonferroni's multiple comparisons test. significance was set at p o . . a sharp rise in ph indicating exit of the capsule from the stomach was identified in each experiment. mean (ae sd) get, slbtt, and tgtt without ranitidine were ae , ae , and ae min, respectively. mean get, slbtt, and tgtt during treatment with ranitidine were ae , ae , and ae min, respectively. mean intra-individual cv% before ranitidine for get, slbtt, and tgtt were . , . , and . %, respectively. mean inter-individual cv% before treatment with ranitidine for get, slbtt, and ttt were . , . , and . %, respectively. no significant differences in get, slbtt, or tgtt were found at any of the time points. the mean mgph during treatment with ranitidine (ph . ) was significantly higher than at all other time points (overall mean ph for the time points: . ; p o . ). the smartpill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing gi transit times in medium to large breed dogs. measurements of gi transit times, especially slbtt, were subject to considerable intra-individual and interindividual variation. no significant effect of oral ranitidine on gi motility was identified in this group of dogs. however, as expected, oral ranitidine caused a significant increase in gastric ph. the intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. recent metagenomic data suggest that specific bacterial groups, including bacteria within the clostridium clusters iv and xiva (i.e., faecalibacterium spp., ruminococcaceae, and lachnospiraceae) and bifidobacterium spp. are decreased, while proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. the objective of this study was to establish quantitative polymerase chain reaction (qpcr) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease. fecal samples were collected from healthy dogs ( females and males) and dogs with clinical signs of gastrointestinal disease ( females and males). novel quantitative pcr assays were established for faecalibacterium spp., ruminococcaceae, and lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from s rrna gene clone libraries and sequences available from the ribosomal database project. primers for bifidobacterium spp. and proteobacteria were selected from previously published studies. the specificity of the qpcr assays was confirmed by sequencing of obtained qpcr amplicons. the bacterial dna abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a mann-whitney u test. significance was set at p o . . a significantly lower abundance of faecalibacterium spp. (p o . ) and ruminococcaceae (p . ) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p . ). there was no significant difference in the abundance of lachnospiraceae (p . ) and bifidobacterium spp. (p . ) between both groups. in conclusion, we established novel qpcr assays for faecalibacterium spp., ruminococcaceae, and lachnospiraceae. we observed significant decreases in the abundance of faecalibacterium spp. and ruminococcaceae in dogs with clinical signs of gastrointestinal disease. these bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. the pathogenesis of chronic enteropathies (ce) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. while the application of bacterial s rdna sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. the present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with ce. eleven client-owned dogs with ce involving both the small and large intestines were prospectively enrolled. ce was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (fish) using s rdna-targeted probes directed against all bacteria, enterobacteriaceae, e. coli, eubacterium rectale-clostridium coccoides group, bacteroides/prevotella, and helicobacter spp. sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten x fields of each section. microbial composition in ce dogs was compared to the ileal/colonic microbiota of healthy control (hc) dogs using a mixed effect anova model. p values o . were considered significant. the final diagnoses for dogs with ce included ibd (n ) and lymphosarcoma (n ). when compared to hc dogs, dogs with ce showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. evaluation of colonic biopsies in dogs with ce showed that the total number of bacteria (p o . ), clostridium (p o . ), enterobacteriaceae (p o . ) and e. coli (p o . ) were increased in the adherent mucus regions of dogs with ibd as compared to hc dogs. total bacteria (p o . ) and e. coli (p o . ) were also more numerous in dogs with lsa versus hc and ibd dogs (p o . for e. coli). ileal biopsies from ce dogs similarly showed variable dysbiosis with increased total bacteria (p o . ) but decreased helicobacter spp (p o . ) and bacteroides (p o . ) observed within inflamed intestines as compared to hc tissues. the spatial distribution of these bacteria was also appreciably different from hc dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions. our data demonstrate that dogs with ce affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. although pentatrichomonas hominis (ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent gi infections in dogs with trichomonosis. the aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections. fecal samples from dogs were submitted to ncsu from - for trichomonas spp. pcr testing. dna was extracted using a zr fecal dna mini-prep kit and absence of pcr inhibitors verified by amplification of bacterial s rdna. pcr for ph and tritrichomonas foetus (tf) was performed as well as real-time pcr assays for possible concurrent enteric infectious agents. obtainable medical records were reviewed. all submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery. mean age of the dogs was . years (median . ; range: . - months) and represented a total of breeds. ph, tf, or concurrent ph and tf were diagnosed in , , and dogs respectively (group a). the remaining dogs were negative for ph and tf by pcr no dogs were identified as infected with canine distemper virus or parvovirus. five samples from each group had insufficient quantity or quality of dna for concurrent infectious disease testing. in this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without ph were identified. thus, these findings do not appear to support a primary pathogenic role for ph as a causative agent of diarrhea in dogs. gastrointestinal motility disorders are a common clinical problem in domestic animals. many of the g.i. motility disorders have been treated previously with -ht agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. the dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine d receptors, and putative gastrointestinal prokinetic effects at peripheral d receptors. domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction. portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from - months of age. longitudinal and circular smooth muscle strips from each site were suspended in physiologic (hepes) buffer solution, attached to isometric force transducers, and set to optimal muscle length (l o ) using acetylcholine (ach; À m). muscle strips were treated with domperidone (d; À to À m) in the presence or absence of ach ( À to À m), and maximal force output (p max ) was normalized for cross-sectional area (n  newtons/m ). domperidone (d) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. direct effects were similar whether in the longitudinal or circular muscle orientation. the direct effect of domperidone was dose-dependent and maximal (feline colon p max . - . n; rabbit colon p max . - . n) at a dose of À m. domperidone had a much greater indirect effect in augmenting cholinergic (ach; À m) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. domperidone-augmented cholinergic contractions were - % (feline colon p max . ae . n ach only; feline colon p max . ae . n ach d) of baseline cholinergic contractions. domperidone contractions were of a similar magnitude to those induced by cisapride. domperidone effects were similar in mucosaintact and mucosa-dissected preparations. domperidone contractions were unaffected by prazosin (a receptor antagonist), yohimbine (a receptor antagonist), or terbutaline (b receptor agonist), but were somewhat attenuated by dopamine (d receptor agonist) and a non-specific cholinergic antagonist (atropine). in vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. as recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (nsaids) often in conjunction with tramadol. in people and rats, co-administration increases the risk of perforation and gastric injury over nsaids alone. using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin e (pge ). mucosa from the gastric antrum was harvested from shelter dogs immediately after euthanasia, and mounted on ussing chambers. the tissues were equilibrated for -minutes prior to addition of acidic ringer's solution (ph, . ). after -minutes of injury, the acid was replaced with neutral ringer's and the tissues were treated with indomethacin, tramadol or both. tissues were maintained for minutes total, during which time permeability was assessed electrically. prostanoid concentrations were quantified using a commercially available elisa. western blots were performed for cox- and À . recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( figure ) but not by tramadol or indomethacin alone (data not shown). prostaglandin e increased with acid injury. the increase in pge was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury . ae . , indo tramadol . ae . ). there was no significant effect of treatment on cox- or À expression. co-administration of tramadol with a non-selective nsaid inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. these drugs may exert this effect by decreasing levels of gastroprotective prostanoids. further study is needed to understand the mechanism of this drug interaction. an increased intestinal permeability (ip) has been suggested to be both cause and consequence of gastrointestinal (gi) disease, such as inflammatory bowel and celiac disease, in people. a novel tight junction regulator, larazotide acetate (alba therapeutics, baltimore, md) has been shown to significantly decrease ip in rats and in humans with celiac disease. the purpose of this study was to determine if larazotide acetate reduces ip in soft coated wheaten terriers (scwt) and norwegian lundehunds (nl) with chronic gi disease and ameliorates clinical signs. four nl ( females, males; median age: . yrs, range: . - . yrs) and scwt ( females, males; median age: . yrs, range: . - . yrs) were enrolled based on presence of clinical signs of gi disease and hypoalbuminemia, increased fecal alpha proteinase inhibitor (a -pi) concentrations, and/or hypocobalaminemia. scwt with protein-losing nephropathy were excluded. dogs were fed q hrs and received . mg ( nl and scwt) or . mg ( scwt) of larazotide acetate po before each meal for days. prior to start of treatment (day ) and at the end (day ), ip was evaluated by calculating the lactulose/rhamnose (l/r)-ratio in serum samples obtained at , , , and min after oral dosing. also, consecutive fecal samples each were collected prior to day and day for n-methylhistamine (nmh) measurement. pre-and post-treatment data were compared using a wilcoxon signed rank test. the . mg vs. . mg dose groups were compared using a mann-whitney u test. statistical significance was set at p o . . l/r-ratios (medians) for the min sampling time point were significantly lower on day ( . ) than on day ( . ; p . ). dogs treated with . mg q hrs had significantly lower min l/r ratios on day than dogs treated with . mg ( . vs. . ; p . ). no difference was found between breeds. fecal nmh concentrations were not different between time points, treatment groups, or breeds. fecal a -pi concentrations were available for of the dogs and were significantly higher on day compared to day (p . ). no differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. weight gain was seen in all nl. resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in scwt. another scwt had resolution of diarrhea and a decrease in pruritus. no changes in clinical signs were reported in the remaining scwt. this study indicates that larazotide acetate might be able to reduce ip in dogs. this effect may be dose-dependent. however, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased ip might have been responsible for the clinical signs in these dogs. breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with gi disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with ple and compare them with those in dogs with food-responsive diarrhea (frd) without protein loss. dogs with ple and dogs with frd, referred to the university of bern, ch, were enrolled. selection criteria included a history of chronic diarrhea ( weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. underlying disorders were excluded based on cbc, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. also, canine pancreatic lipase immunoreactivity (spec cpl s ), c-reactive protein (crp), calprotectin and alpha -proteinase inhibitor (a -pi) were measured in serum from dogs and compared with dogs with frd without ple. all dogs were scored using the canine ibd (cibdai) and the canine chronic enteropathy (cce) clinical activity index (ccecai). total protein, albumin ( - . g/l), and total calcium ( . - . mmol/l) were decreased in all dogs. cobalamin was decreased in all but dogs ( o - ng/l). spec cpl was mildly increased in / dogs with ple and normal in / ple and all frd dogs. crp was normal in / dogs with ple ( / frd), mildly increased in / ( / frd), and moderately increased in / ple dogs ( / frd). calprotectin was slightly higher in dogs with ple, but all ple and frd dogs yielded values in the normal range. serum a -pi was significantly lower in dogs with ple than in those with frd (p o . ), with / ple dogs below the reference range ( / frd). cibdai ranged from to and ccecai from to . at the end of the study, / dogs were still alive with survival times between and days. / dogs died with a median survival of days (range - days). dogs with mildly increased crp died earlier than dogs with a normal or moderately increased crp (p . ), whereas albumin, calcium, spec cpl, calprotectin, cibdai, and ccecai had no significant impact on outcome and survival. in conclusion, dogs with ple have a significantly lower a -pi in the serum than dogs with frd. furthermore, most dogs with ple have an increased crp and a decreased cobalamin. a mild increase in crp appears to be a poor prognostic factor. while hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. the aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. we hypothesized that dogs with a protein losing enteropathy (ple) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls). information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time. one hundred seventeen cases fit the inclusion criteria; in the ple group and controls. there was no statistical significance between groups for age (p . ), weight (p . ), weight loss (p . ) and body condition score (p . ). compared to control dogs, ple dogs had decreased serum concentrations of cobalamin (p . ), total calcium (p o . ), globulin (p o . ), cholesterol (p o . ) and ionized calcium (p o . ). survival analysis revealed a significantly decreased survival time for ple dogs (p . ); median survival was days for ple dogs and , days for controls. while the ple group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o . g/dl survived longer than those with mild hypoalbuminemia ( . - . g/dl). this study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. cobalamin (vitamin b ) deficiency in the chinese shar pei (shar pei) is suspected to be hereditary. inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. based on human and veterinary studies, an increased serum methylmalonic acid (mma) concentration has been suggested to reflect cobalamin deficiency at the cellular level. in this context, it has been shown in humans that mma concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. therefore, the aim of this study was to evaluate serum mma concentrations in shar peis and dogs of six other breeds with cobalamin deficiency. from in conclusion, serum cobalamin deficient shar peis had a times higher median serum mma concentration compared to cobalamin deficient dogs of six other dog breeds. further studies are needed to investigate the intracellular processing of cobalamin in shar peis with cobalamin deficiency. chinese shar peis (shar peis) have a high prevalence of cobalamin deficiency. two other conditions reported frequently in this breed are shar pei fever and cutaneous mucinosis. shar pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of c-reactive protein (crp), a nonspecific marker of inflammation. cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. also, hyaluronic acid (ha), the main component of mucin, was shown to be significantly higher in serum from shar peis with cutaneous mucinosis than in healthy controls. to date, a possible association between shar pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in shar peis. thus, the aim of this study was to compare serum concentrations of ha (an indicator of cutaneous mucinosis) and inflammatory markers (crp, calprotectin, and s a ), assumed to be increased in episodes of shar pei fever, in shar peis with and without cobalamin deficiency. serum samples from shar peis, collected from to , were analyzed. serum ha and crp (reference interval (ri): . - . mg/l) were quantified by using commercial elisa kits (echelon biosciences, salt lake city, ut, usa and tridelta, maynooth, ireland; respectively). serum calgranulin concentrations were measured using an in-house elisa (calprotectin; ri: . - . mg/l) and ria (s a ; ri: . - . mg/l), respectively. mann-whitney u tests were used to compare serum ha, crp, calprotectin, and s a concentrations between shar peis with and without cobalamin deficiency. significance was set at p o . . fourteen shar peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o ng/l). in the remaining dogs, serum cobalamin concentrations were within the reference interval ( - ng/l). serum concentrations of ha, crp, calprotectin, and s a were not significantly different between cobalamin deficient shar peis (medians: . ng/ml, . . fifty percent of cobalamin deficient shar peis had serum calprotectin concentrations above the upper limit of the reference interval, and % had serum s a concentrations above the suggested upper reference limit. in this study, serum concentrations of ha, crp, and the calgranulins did not differ between cobalamin deficient shar peis and shar peis with a normal serum cobalamin concentration. this finding leads us to speculate that increased ha and/or inflammatory markers are not associated with cobalamin deficiency in shar peis. further studies are needed to investigate serum cobalamin concentrations in patients with shar pei fever or cutaneous mucinosis. cobalamin deficiency (cd) has been associated with gastrointestinal and pancreatic disease in dogs. hereditary cd has been demonstrated in giant schnauzers and single case reports have suggested congenital cd in the border collie (bc) breed. clinicopathologic findings of cd vary and can be unspecific as cobalamin acts as a co-factor for a multitude of enzymatic reactions. the two most important reactions concern the conversion of methylmalonyl-coa to succinyl-coa and the re-methylation of homocysteine (hcy). these two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. preliminary data from dogs suggested that measurement of methylmalonic acid (mma) may be a better diagnostic test for cd than serum cobalamin concentration. therefore the goals of the study were ( ) to establish reference values for serum cobalamin, urine mma and plasma hcy in healthy pet dogs, ( ) to screen a larger bc population from switzerland for cd, and ( ) to perform genomic analyses on bc with cd. for determination of reference values healthy pet dogs were used. serum cobalamin was measured using an automated chemiluminescence assay (immulite ), urine mma was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma hcy was measured using high pressure liquid chromatography and fluorimetric detection. to calculate reference ranges the th and th percentile were used. data were analyzed using non-parametric tests. reference ranges for cobalamin, hcy, and mma were: cobalamin . - . ng/l; urine mma - . mmol/mol creatinine; and plasma hcy . - . mmol/l. the screened bc population comprised purebred dogs and bc (median . months; range - ) suffering from congenital cd could be identified. clinical signs differed and consisted of tiredness ( ), stunted growth ( ), anemia ( ), dysphagia ( ) and persistent fever ( ). median (ranges) results for healthy bc and bc with cd were: for cobalamin ( - ) and . ( - ) ng/l; for urine mma ( - ) and ( - ) mmol/mol creatinine; for hcy . ( . - . ) and . ( - . ) mmol/l. strikingly, healthy bc with cobalamin concentrations well within the reference range had significantly higher urine mma concentrations compared to control dogs. under the assumption that the four affected bc are inbred to a single founder animal, first results of genotyping on the k illumina canine_hd snp chip suggest that mutations in the cubn and amn gene can be excluded to cause the observed cd in these dogs. we conclude that cd is a rare familial disease in bc with variable clinical signs. to define the genomic region responsible for cd further genetic analysis is in progress. it remains to be determined why some bc have high urine mma concentrations despite a serum cobalamin concentration within the reference range. calprotectin is a protein complex that plays an important role in the innate immune response. preliminary data suggest that canine calprotectin (ccp) is a useful marker for the detection of inflammation in dogs. recently, a radioimmunoassay for the measurement of ccp has been developed and analytically validated, but this test requires the use of a radioactive tracer. therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (elisa) for the quantification of ccp in serum and fecal specimens from dogs. canine calprotectin (ccp) was purified, antiserum against purified ccp was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-elisa was developed. purified antibodies were used for capturing and, after coupling with horseradish peroxidase (hrp), for reporting. a hrp substrate was used for color development. the assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. control intervals for serum and fecal ccp were established from and healthy pet dogs, respectively, using the central th percentile. sensitivity of the assay for serum samples assayed in a : dilution and for fecal extracts assayed in a : , dilution was . mg/l and . mg/g, respectively. over a wide range of the assay, there was no cross-reactivity with cs a , the closest structural analogue of ccp available. observed to expected ratios (o/e) for serial dilutions ranged from . - . % (mean ae standard deviation [sd]: . ae . %) for four different serum samples, and from . - . % (mean ae sd: . ae . %) for five different fecal extracts. o/e for spiking recovery ranged from . - . % (mean ae sd: . ae . %) for four different serum samples and different spiking concentrations, and from . - . % (mean ae sd: . ae . %) for different fecal extracts and different spiking concentrations. intra-assay coefficients of variation (cv) for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. inter-assay cv for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. the control intervals for serum and fecal ccp were established as . - . mg/l and . - . mg/g, respectively. we conclude that this new elisa for the measurement of ccp is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine s a . further studies evaluating the clinical usefulness of measuring serum and/or fecal ccp are currently under way. the syndrome of hemorrhagic gastroenteritis (hge) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. the etiology of hge is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. hge is characterized by an increased vascular/mucosal permeability, thought to represent a type i-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. however, markers of gastrointestinal (gi) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with hge. therefore, the aim of this study was to evaluate fecal canine calprotectin (cp) and s a (a ), a -proteinase inhibitor (a -pi, a marker of gi protein loss), and bacterial groups that have previously been shown to be decreased (i.e., faecalibacterium spp., ruminococcaceae, bifidobacterium spp.) or increased (i.e., proteobacteria) in fecal samples from dogs with hge. fecal samples from consecutive days were collected from dogs with hge. fecal cp, a , and a -pi concentrations were measured by in-house immunoassays. bacterial dna was extracted from each fecal sample and was analyzed for faecalibacterium spp., proteobacteria, rumino-coccaceae, and bifidobacterium spp. using quantitative pcr assays. concentrations of fecal cp, a , and a -pi, and the abundance of bacterial dna were compared using a friedman test with dunn's post-hoc tests. significance was set at p o . . at the time of diagnosis (day ), fecal cp, a , and a -pi were above the suggested reference intervals in , , and of the dogs, respectively. until day , this number decreased to , , and , respectively. decreases in concentrations were significant between days and for a (p . ), and between days and for a -pi (p . ), but not for cp despite a trend (p . ). no differences in the abundance of faecalibacterium spp. (p . ), bifidobacterium spp. (p . ), or proteobacteria (p . ) were observed. however, the abundance of rumino-coccaceae was significantly lower on day when compared to day (p . ). in this study, fecal markers of inflammation and gi protein loss were increased in dogs with hge. although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. these results suggest a loss of protein into the gi tract at the onset of hge. the lack of significant increases of faecalibacterium spp., bifidobacterium spp., and ruminococcaceae, and decreases in proteobacteria may suggest gi dysbiosis. further longitudinal studies are needed and are currently under way to evaluate gi dysbiosis in canine hge patients. the most recent antiemetic approved for use in dogs is maropitant citrate (cerenia s , pfizer animal health). maropitant is a selective nk receptor antagonist that acts by blocking the binding of substance-p within the emetic center and chemoreceptor trigger zone. label dosage recommendations for maropitant citrate are mg/ kg sc or mg/kg orally once daily for up to consecutive days (acute emesis) and mg/kg orally once daily for up to consecutive days (motion sickness). the study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for consecutive days. two groups of eight healthy beagles were administered maropitant citrate at or mg/kg orally once daily for days. concentrations of maropitant and its metabolite were measured in plasma using a lc-ms/ms assay. pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state. the accumulation ratio for maropitant was . (auc - ) and . (cmax) for the mg/kg dose; and . (auc - ) and . (cmax) for the mg/kg dose after days. the model estimate for the number of doses required to reach % of steady-state was . for mg/kg and . for mg/kg. three dogs experienced a single episode of vomiting. dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. steady-state was reached after approximately doses for daily mg/kg and doses for daily mg/kg oral dosing. previously presented at the veterinary cancer society, november . cobalamin (vitamin b ) is involved in a variety of metabolic processes. altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (epi) or severe and longstanding ileal disease. this study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the gastrointestinal laboratory. the study was also aimed at investigating serum trypsin-like immunoreactivity (tli) concentrations that were diagnostic for epi in the dogs with a decreased serum cobalamin concentration. except for csp, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. also, a possible association between an undetectable serum cobalamin and a decreased serum tli in ai needs to be further investigated. calprotectin (cp) is a widely used marker for the diagnosis and monitoring of gastrointestinal (gi) inflammation in humans. studies in humans usually report fecal cp concentrations based on a single stool sample although considerable day-to-day variability of fecal cp was found in patients with gi disease and in healthy controls. intra-individual variation of canine cp (ccp) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic gi disease. thus, the aim of this study was to compare the day-to-day variation of fecal ccp in dogs with chronic gi disease before and during treatment to that in healthy dogs. we hypothesized that fecal ccp would be less variable in patients with chronic gi disease than in healthy controls, and thus collection of a single fecal sample would be sufficient. fecal samples from consecutive days were prospectively collected from dogs (group a; median age: . years) referred for diagnostic work-up of chronic signs of gi disease, from dogs (group b; median age: . years) with stable gi disease while being treated, and from healthy adult dogs (group c; mean age: . years). fecal samples were extracted and ccp was measured by an in-house immunoassay. mean ccp, standard deviation, coefficient of variation (cv), and difference between maximum and minimum ccp for the -day sample collection period were calculated for each dog and were compared among groups using a kruskal-wallis test. fecal ccp ranged from . - . mg/g (median: . mg/g) in dogs with gi disease (group a), from . - . mg/g (median: . mg/g) in dogs of group b, and from . - . mg/g (median: . mg/g) in healthy controls (group c). cvs were - . % in group a (median: . %), . - . % in group b (median: . %), and - . % in group c (median: . %), respectively. patients in group a appeared to have less variable fecal ccp than dogs in group b and c, but this difference was not significant (p . ). the difference between maximum and minimum ccp for the -day sample collection ranged from - . mg/g in group a (median: . mg/g), from . - . mg/g in group b (median: . mg/g), and from - . mg/g in group c (median: . mg/g), and were not significantly different between any of the groups (p . ). in this study, considerable day-to-day variation of fecal ccp was found in dogs with chronic gi disease (regardless of treatment) and was comparable to that in healthy dogs. results of this study suggest that for evaluating fecal ccp in dogs with clinical signs of gi disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. because we did not intend to evaluate the clinical usefulness of fecal ccp as a marker of gi disease in dogs, disease severity, quality, and location differed among dogs in groups a and b. the diagnostic utility of fecal ccp in dogs with gi disease is currently being investigated. it has been suggested that diagnosis of clostridium perfringens related enteropathy should be based on the detection of the c. perfringens enterotoxin gene (cpe-gene) by pcr and/or c. perfringens enterotoxin (cpe) by elisa in feces. however, the prevalence of the cpe-gene and cpe in dogs and especially cats with gastrointestinal disease has not yet been reported. also, there is limited information about the stability of cpe in fecal samples at various storage conditions. the aim of this study was to evaluate the prevalence of the cpe-gene and cpe and the stability of cpe in fecal samples from dogs and cats. to evaluate the prevalence of the cpe-gene, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and fecal samples from those without such signs ( dogs and cats) were examined using pcr. to evaluate the prevalence of cpe, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and dogs without such signs were evaluated using a commercially available elisa kit (techlab, blacksburg, va). the results were analyzed using a fisher's exact test. significance was set at p o . . to evaluate the stability of cpe, fecal samples from dogs and from cats with clinical signs of gastrointestinal disease that were positive for cpe were examined. also, cpe negative samples from dogs were evaluated as negative controls. each sample was subdivided into aliquots and evaluated on day ; on days , , and after being stored at room temperature (rt) or c; and on day after being stored at À c. the prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease ( / ; . %) and dogs without ( / ; . %; p . ). also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease ( / ; . %) was not significantly different compared to cats without ( / ; . %; p . ). pcr and elisa results were available for samples. of the pcr positive samples, only ( . %) were elisa positive. of the pcr negative samples, only ( . %) was elisa positive. the prevalence of cpe was not significantly different between dogs with clinical signs of gastrointestinal disease ( / ; . %) and those without ( / ; . %; p . ). the prevalence of cpe in cats with signs of gastrointestinal disease was / ( . %), but no samples from cats without such signs were available. when evaluating the stability of cpe, results for all aliquots were consistent with the initial result, except for one sample (on day , stored at rt, which was initially cpe positive). these results indicate that only a small proportion of samples that are pcr positive for the cpe-gene are also positive for cpe. studies are warranted to further compare the prevalence of cpe among animals with gastrointestinal disease and those without. furthermore, the results indicate that cpe is relatively stable in fecal samples at various storage temperatures. clostridium perfringens has been implicated as a cause of diarrhea in dogs. the main study objective was to compare two culture methods for the identification of c. perfringens. a secondary objective was to evaluate c. perfringens toxin genes a, b, b , e, ı and cpe from canine isolates using a multiplex pcr and determine their prevalence in a group of normal and diarrheic dogs. fecal samples were collected from clinically normal (nd, n ) and diarrheic dogs (dd, n ) at a primary care veterinary facility. isolation of c. perfringens was performed using direct inoculation of feces onto % sheep blood agar (sba) as well as enrichment of stool in bhi broth followed by inoculation onto sba. isolates were tested by multiplex pcr for the presence of a, b, b , e, ı and cpe genes. c. perfringens was isolated from % ( / ) of nd fecal samples using direct culture and . % ( / ) with bhi enrichment (p . ). in the dd, corresponding isolation rates were . % and . % (p . ). all isolates possessed a toxin gene. b, b , e, ı and cpe toxin genes were identified in . %, . %, . %, . % and . % of nd isolates, respectively. in the dd group, b and b were identified in %, e and ı were not identified and the cpe gene in . % of isolates. bhi enrichment did not significantly increase the yield of c. perfringens compared to sba but increased time and cost involved. c. perfringens (p . ) and c. perfringens toxin genes were present in equal proportions in nd and dd groups (p ! . ). culture of c. perfringens and pcr for toxin genes are of limited diagnostic utility due to the high prevalence of c.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. the purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats. endoscopic biopsies from cats which had duodenal and ileal tissue specimens were evaluated retrospectively. all slides were randomized and reviewed by a single pathologist (jm) for quality, number of biopsies, and diagnosis according to wsava standards. no information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. statistical comparison of the diagnosis of sc-lsa and ibd by intestinal location was conducted using fisher's exact test (p o . significant). of cats ( . %) were diagnosed with sc-lsa in the duodenum and/or ileum. of these cats, ( . %) were diagnosed with only duodenal sc-lsa, ( . %) were diagnosed with only ileal sc-lsa, and ( . %) had sc-lsa in both duodenum and ileum. in cats with only ileal sc-lsa, had severe ibd in duodenal biopsies, possibly consistent with early sc-lsa. of these had duodenal biopsies without evidence of sc-lsa. our results suggest there is a population of cats in which diagnosis of sc-lsa may only be found by evaluating ileal biopsies. clinicians should consider performing both upper and lower gi endoscopic biopsies in cats with suspected infiltrative small bowel disease. periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. in this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats. twelve cats (median age: years; range: - years; dsh and persians; females and males) were enrolled. dental tartar was obtained during scaling for a dental prophylactic procedure. all cats were negative for felv and fiv infection as assessed by a commercial elisa test (snap s fiv/felv combo test). severity of gingivitis (scores: - ; normal, mild, moderate, and severe) and dental tartar (scores: - ) were scored in each cat. endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. the relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. a student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. the association between severity of gingivitis and bacterial type was tested by chi square test. the spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was . (p o . ). interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o . ). the highest scores were found for the molar teeth in all cats. bacterial culture revealed . % anaerobic bacteria species (i.e., bacteroides spp., peptostreptococcus anaerobius, and eubacterium aerofaciens) and . % aerobic bacteria species (i.e., pasteurella multocida, streptococcus spp., enterococcus spp., staphylococcus spp., bacillus cereus, escherichia coli, and pseudomonas aeruginosa). anaerobic bacteria were found mostly in cats with higher gingivitis scores ( - ; chi square: p o . ), while pasteurella multocida was found mostly in cats with lower gingivitis scores ( - ; chi square: p o . ). antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, % were sensitive to erythromycin, and % were sensitive to penicillin. the most abundant aerobic bacterial species, pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured. these results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. these data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. although it can occur in any canine breed, certain breeds are more susceptible. we have previously shown that polymorphisms in the tlr and tlr gene are significantly associated with inflammatory bowel disease (ibd) in the german shepherd dog (gsd), a breed at risk of developing this disease. it would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. therefore the aim of this study was to investigate whether polymorphisms in canine tlr and tlr genes are associated with ibd in other non-gsd canine breeds. four non-synonymous snps in the tlr gene; t c, g a, a t and g a and three non-synonymous snps in the tlr gene; g a, c t and t c previously identified in a mutational analysis in gsds with ibd were evaluated in a case-control study using a snapshot multiplex reaction. sequencing information from unrelated dogs with ibd consisting of different non-gsd breeds from the uk were compared to a breed-matched control group consisting of unrelated dogs from patients treated for noninflammatory disease at the royal veterinary college, london, uk. as in the gsd ibd population the two tlr snps; c t and t c were found to be significantly protective for ibd in other breeds included in this study (p . and p . respectively). this study confirms the protective effects of the two tlr snps (c t and t c) in other canine breeds with ibd. this highlights the importance of tlr in the pathogenesis of canine ibd and may represent common pathological pathways of ibd in different canine breeds due to the high degree of haplotype sharing seen among breeds. this may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. further functional studies looking at the role of tlr in the pathogenesis of canine ibd are needed to confirm these findings. toll-like receptor (tlr ) is an extracellular pattern recognition receptor belonging to the innate immune system. we have recently shown that three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) are significantly associated with inflammatory bowel disease (ibd) in german shepherd dogs. in addition, we confirmed that two of these tlr snps (c t and t c) were significantly associated with ibd in a population consisting of different dog breeds. in order to determine if other novel snps exist in the tlr gene in addition to the ones identified in the gsd population, mutational analysis was carried out in seven boxer dogs with ibd. polymerase chain reaction was carried out to amplify the tlr coding region in the seven dogs with ibd. sequencing was carried out using sequence based typing with the abi prism sequencing kit (applied biosystems, uk) and analyzed using an abi automated sequencer (pe applied biosystems). sequencing information from seven boxer dogs with ibd from the uk were compared to the reference sequence published on the ensemble webserver (www. ensembl.org/canis_familiaris). in addition to the three snps identified previously in the tlr gene, a novel non-synonymous snp; t c was identified in the boxer dog population with ibd. this snp has never been reported before and was present as the homozygote genotype in three dogs with ibd and in one dog as the heterozygote genotype. using the simple modular architecture research tool (smart) web server (http:// smart.embl.de/) we were able to map the t c snp to the leucine rich repeat domain of the tlr protein. the leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the t c snp results in a change in the amino acid from non-polar to polar. our study further confirms the role of tlr in the pathogenesis of canine ibd. our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique snps arising after breed formation which may further affect their susceptibility to this disease. however, a case-control study would be needed in the boxer dog to confirm the significance of the tlr t c snp and further functional data would be needed to elucidate the exact role of this polymorphism in canine ibd. an automated power driver device (oncontrol, vidacare) has recently become available for bone marrow aspiration (bma) and bone marrow biopsy (bmb) in humans. the purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats. twelve healthy research cats were anesthetized using a standardized protocol on different occasions, days apart, to have bmas and bmbs performed by the same operator. on day , half of the cats were randomized to have a bma performed at both the proximal humerus and the iliac crest, and a bmb performed at the iliac wing, using the oncontrol device ( -gauge needle for bma; -gauge needle for bmb). the other half of the cats had the same procedures performed using a manual technique ( -gauge illinois needle for bma; -gauge jamshidi needle for bmb). on day , each cat had bma performed at the opposite humerus and iliac crest, and a bmb performed at the proximal humerus using the opposite technique from day . for each procedure, the operator was given a maximum of attempts to successfully collect a sample. the rate of success, as well as the number of attempts were recorded. the ''ease of use'' of the device was rated by the operator on a -point scale after each procedure. using previously determined criteria, the macroscopic and microscopic qualities of the bma and bmb samples were assessed by a board-certified pathologist, blinded to the technique used. the level of pain experienced by each cat was evaluated , , , , and hours following each set of procedures, using a previously validated pain scoring system. two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for bmas and the humerus to the iliac wing site for bmbs. for all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (p o . ). the duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for bma at the proximal humerus (p o . ). there was no significant difference in the level of pain at any time point following each set of procedures with either technique. performing bma at the proximal humerus was associated with a higher rate of success (p o . ), a lower number of attempts (p o . ), a shorter duration of the procedure (p o . ), a higher-rated ''ease of use'' of the technique (p o . ), and a better quality sample (p o . ) when compared to sampling from the iliac crest, in conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. this technique was easier to use than the manual technique for both bma and bmb, and reduced the duration of the procedure and the number of attempts for successful bma at the proximal humerus. performing bma at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. the fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. it has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint. this study evaluated the effects of a standardizecd anesthetic protocol using ketamine ( mg/kg, iv), midazolam ( . mg/kg, iv) and buprenorphine ( mg/kg, im) on the hematologic parameters of healthy adult research cats. each cat had blood samples collected before and after induction of anesthesia on different occasions, days apart. in total, pairs of complete blood counts were obtained. analyses were performed at a certified veterinary laboratory. paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on different occasions. compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. there was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. on average, there was a . % decrease in the red blood cell count ( .  /l to .  /l) (p o . ), a % decrease in hemoglobin concentration ( . g/l to . g/ l) (p o . ), a . % decrease in the hematocrit ( . l/l to . l/l) (p o . ), a . % decrease in the lymphocyte count ( .  /l to .  /l) (p . ), and a . % decrease in the plasma total protein concentration ( . g/l to . g/l) (p o . ) when samples taken before and after induction of anesthesia were compared. if only the hematocrit was considered as a marker of anemia, % of the samples from these healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o . l/l), using the reference interval established in our laboratory. none of the cats would have been considered anemic before induction of general anesthesia. in practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except had a lymphocyte count that was within the reference interval for cats established by our laboratory. this study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. the mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. rivaroxaban is an oral inhibitor of activated coagulation factor x (xa). it is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats. citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. the rivaroxaban was extracted from commercially available tablets (xarelto s ) and dissolved in dmso prior to addition to the blood. tests performed included kaolin-activated thrombelastography (teg), prothrombin time (pt), dilute pt (dpt), activated partial thromboplastin time (aptt), and anti-factor xa (axa) activity. dose-dependent prolongations were seen in all coagulation parameters. similar to human data, therapeutic axa levels (between . - . axa units) were achieved at in vitro concentrations between and mg/l. at mg/l, dpt measurements were clinically prolonged in all cats ( . ae sec vs. . ae . sec, p . ), while aptt values were only mildly prolonged from baseline ( . ae sec vs. . ae sec, p . ). significant prolongations were seen in dpt at ( . ae ec, p . ). teg r time did not prolong from baseline values until concentrations of mg/l were reached ( . ae min compared to . ae . min, p . ). rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. kaolinactivated teg does not appear to be sensitive to low concentrations of rivaroxaban in the cat. anticoagulated blood is required for platelet function studies. sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to adp-induced platelet aggregation using citrated whole blood. hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the multiplate s platelet function analyzer. ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. venous blood was collected atraumatically from the external jugular vein directly into tubes containing . % trisodium citrate or hirudin. whole blood platelet aggregation was performed (whole-blood lumi-aggregometer, chrono-log corporation, havertown, pa, usa) with collagen ( mg/ml) and adp ( mm) as agonists. maximal platelet aggregation (ohms) was recorded. there was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p o . ). there was also a significant increase in adp-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p . ). the results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the chrono-log impedance aggregometer. while both collagen and adp-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for adpinduced aggregation where almost all samples had no objective measurable platelet aggregation. it is suggested from this data that future whole blood platelet aggregation studies performed on the chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. low-molecular-weight heparin (lmwh) is now used to prevent thrombotic complications in dogs. a functional assay such as the calibrated automated thrombogram (cat) may provide a new approach for monitoring lmwh therapy. we hypothesized that cat would detect decreased endogenous thrombin potential (etp) in healthy dogs receiving lmwh (fragmin s ). twenty-four healthy adult beagles were included in this study and divided equally in four groups. one dose of u/kg, u/kg or u/kg of lmwh were given subcutaneously to healthy dogs and compared to a control group. platelet poor plasma (ppp) was collected over a hour period. using a repeated-measure linear model, effect of lmwh on etp was time and dose dependent with a significant interaction (p o . ). compared to control dogs, significant differences were obtained for group u/kg at t (p . ), for group u/kg at t (p . ) and between t -t minutes (p o . ) respectively, and for group u/ at t (p . ), between t -t minutes (p o . ) respectively and at t (p . the cat assay can be employed to measure the effects of lmwh at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in etp and warrants further investigation as a tool for monitoring lmwh therapy in dogs. the purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (teg). this was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. dogs were evenly divided into either prednisone plus aspirin (pa) or prednisone plus placebo (pp) groups. baseline values for teg parameters (r, k, angle, ma, ly , ly , g, ci) were measured twice two days apart, and thrombin-antithrombin complexes (tat), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (at), fibrinogen) were measured once. each dog received mg/kg/ day of prednisone, and either . mg/kg/day of aspirin (pa group) or placebo (pp group) for days. a complete blood count, biochemistry profile, teg, tat, and traditional coagulation parameters were then repeated on each dog. day to day variation was calculated for the teg parameters using the two baseline measurements. the change from baseline between and within each group were compared using t-tests, or wilcoxon sample test where appropriate, for teg, tat, traditional coagulation parameters, and hematocrit. day to day variation in teg was acceptable ( %) for ma, g, and angle, unacceptable ( %) for r, k, ly and ly , and not meaningful for ci. within both groups, ma, g, ci and fibrinogen significantly increased from baseline (p o . ). within both groups, ly and at significantly decreased from baseline (p o . ). for the pp group, ly significantly decreased from baseline (p . ), and approached significance for the pa group (p . ). all other within group changes from baseline were not statistically significant (p-values . ). for all parameters, there was no difference between groups for change from baseline (p values . ). day to day variation in some teg parameters is high and may preclude their clinical utility. prednisone causes hypercoagulability in healthy dogs based on increased g, ma, and ci. the addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. variability in platelet cyclooxygenase (cox) isoform expression is one proposed mechanism for aspirin resistance in people. two isoforms (cox- and cox- ) have been identified in canine platelets. high (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both cox isoforms in most dogs. this study evaluated the effects of low dose aspirin on platelet function and cox expression in normal dogs. twenty-five healthy client-owned dogs were evaluated before and at two time points (days and ) during aspirin therapy ( mg/kg po sid). platelet response to aspirin (siemens pfa- s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders ( dogs), non-responders ( dogs), or inconsistent responders ( dogs)]. flow cytometry identified platelet cox- and cox- expression. an elisa was used to measure urine -dehydro-thromboxane b ( -dtxb ). there were no significant differences between groups for cox- , cox- or -dtxb at any time point. when all dogs were considered as a single group, there was a significant increase (p o . Ã) in cox- and cox- mean fluorescent intensity (mfi) from baseline to day , . % ae . (mean ae sd) and . % ae . , respectively. there was a significant decrease in mean urine -dtxb :creatinine on day and by . % (p . à ) and % (p o . à ). as with our previous high dose studies, cox- expression was increased with aspirin exposure. however, there was a significant increase in cox- expression with low dose aspirin in contrast to the decrease seen at higher doses. our study suggests that levels of platelet cox- and cox- expression do not influence aspirin response in dogs. although thromboxane levels decreased in most ( of ) dogs on low dose aspirin, platelet function was consistently affected in only % of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. delayed postoperative bleeding is common in retired racing greyhounds (rrgs), despite normal results of routine hemostasis assays. the excessive postoperative bleeding in the rrgs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. providing a method to prevent or minimize the severity of postoperative bleeding in rrgs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. epsilon aminocaproic acid (eaca) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. the objective of this double-blinded, prospective, randomized study was to evaluate the effects of eaca versus placebo on the prevalence of bleeding in rrgs, and to investigate its mechanism of action by using thromboelastography (teg). we compared the effects of eaca and placebo in rrgs that underwent elective ovariohysterectomy or orchiectomy at the veterinary medical center, the ohio state university during years. the main endpoint was bleeding (prevalence and severity); minor endpoints included most teg parameters. thirty percent ( / ) of the rrgs in the placebo group had delayed postoperative bleeding starting to hours after surgery, compared to % ( / ) in the eaca group (p . ). on the teg parameters, the r time (clot formation time) was significantly different between treatment groups (p . ). the postoperative administration of eaca significantly decreased the prevalence of postoperative bleeding in rrgs. thromboembolism associated with protein losing nephropathy (pln) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. in humans, surrogate indicators are frequently used to assess thromboembolic risk. this study aimed to investigate the prevalence of hypercoagulability in pln dogs based on thromboelastography (teg), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure mmhg), hypoalbuminemia (serum albumin o . mg/dl), antithrombin activity (o %), and degree of proteinuria (urine protein:creatinine ratio [upc] ! ). between march -september , twenty-seven dogs were identified with pln at the animal medical center. the prevalence of hypercoagulability based on a teg g-value . was . %. there was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. univariate logistic regression (odds ratio; lower and upper confidence limit; p value) for hypertension was À . ; . , . ; . ; for albumin - . ; . , . ; . ; and for antithrombin activity - . ; . , . ; . . thus, in this patient population, in the absence of teg, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. however, given the documented high prevalence of hypercoagulability in patients with pln, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. thromboelastography (teg) is a test of global hemostasis. due to the effects of extrinsic factors on whole blood coagulation, sample collection method (scm) may influence results. the purpose was to determine if scm influenced teg using kaolin-activated citrated whole blood (wb). healthy dogs with normal platelet counts were prospectively enrolled. three wb samples were obtained from each dog at least hours apart from alternating jugular veins in a randomized order of three methods: ) vacutainer s into citrated tube (vac), ) citrated syringe with transfer into plain tube (cit), or ) plain syringe with transfer into citrated tube (plain). draw time was recorded in seconds. kaolin-activated teg was performed, with measurement of reaction time (r), clot formation time (k), maximum amplitude (ma), and alpha angle. eleven dogs were enrolled. there were no significant differences in teg indices between vac samples and either cit or plain samples. cit samples had a significantly higher k value (p . ) and a lower alpha angle (p . ) compared to plain samples. draw times ranged from - seconds. a longer draw time was significantly correlated (p . ; r À . ) with a shorter r time. higher platelet count was significantly correlated (p . ; r . ) with a higher ma. scm did not have a significant effect on teg parameters when comparing vac samples to either cit or plain samples. minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in teg indices. liquid plasma (lp) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (ffp) that is thawed and stored refrigerated until use. stability studies in people have shown that adequate clotting factor activity is preserved for at least days. lp is transfused in human level i trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost ffp is considered prohibitive. the use of lp has not been described in veterinary critical care. the purposes of this study were to ) determine the length of time required in a water bath for a unit of canine ffp to thaw and ) describe the use of lp in a busy university emergency room (er). for part : six units ( ml) of canine ffp were individually thawed in a c water bath. the duration of time (in minutes) to thaw was recorded. for part : the transfusion log was reviewed for dogs receiving lp in the last months. the indications and outcome were recorded. the mean time ae sd thaw time was . ae . minutes. ten units of lp were transfused to critically ill or injured dogs during the study time. indications for lp transfusion included hypovolemic shock due to intra-abdominal hemorrhage in dogs ( traumatic, non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in dog. lp volume transfused ranged from . to . ml/kg. no transfusion reactions were identified. effect on coagulation was not consistently evaluated. time required to thaw a unit of ffp is greater than minutes which could be detrimental in a bleeding, coagulopathic dog. lp was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary er setting. further investigation of canine lp is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. immune mediated thrombocytopenia (imt) is associated with increased morbidity and mortality. large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. potential explanations include limited availability and low specificity due to nonspecific binding. the focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (psaig) and staining with anti-cd antibodies (cd ab) for the utilization in classifying thrombocytopenic dogs. one hundred clinically ill and apparently healthy dogs were prospectively evaluated. data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of psaig and cd ab. the psaig assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. thrombocytopenia was defined as o , /ml and all dogs were clinically classified into of groups (g): g imt, n , g thrombocytopenia from non-immune mediated diseases, n , g ill with normal platelet counts, n , g healthy dogs, n . median platelet counts, by groups, were g , , ; g , , ; g , , ; and g , , /ml. for the direct and indirect psaigs in dogs with itp (g ), more dogs (n and n ) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n and n ). considering only direct psaig the sensitivity and specificity was % and %, respectively for the diagnosis of imt. for indirect psaig the sensitivity and specificity was % and %, respectively, for the diagnosis of imt. when considering both direct and indirect psaig together, the sensitivity was % with a specificity of %. in g interference from high control antiserum background staining was noted in . % of dogs and resulted in a negative direct psaig classification. minimal background interference was noted in g , g , or g . the percentage of platelets stained with cd ab was significantly less in g (median , p . ) vs. g (median , p . ) vs. g (median . , p . ) and g (median . ). these findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of imt. the decreased cd staining in g (imt group) may reflect decreased surface gpiiia expression, blocking by anti-gpiiia antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of imt. greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (hp) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). the purpose of the present study was to characterize the hp gene in greyhounds. we isolated dna and rna from blood samples of akc-registered and retired racing greyhounds (akcg, rrg), and a german shepherd dog (gsd). we sequenced the hp exons and splice sites, and conducted array comparative genomic hybridization to identify associated dna structural variation (custom m agilent oligonucleotide array). additionally, we tested for the presence of one or multiple haplotypes spanning hp in greyhounds using a high density snp array ( k illumina hd). sequencing results of hp in both dna and cdna revealed three synonymous snps in the racing greyhound. we did not identify structural variation overlapping or near the hp gene. notably, we detected that the rrg and akcg do not appear to share a specific haplotype spanning hp. despite having low or undetectable serum concentrations of hp, we did find that rrg hp mrna is expressed and lacks amino acid variation. this suggests that the clinical absence of the hp is attributable to post-transcriptional hp effects or to an unknown physiological interaction. finally, given the existence of distinct rrg and akcg haplotypes spanning hp, it is important to characterize serum levels of hp in akcg in follow on studies. we reported that hemoglobin in retired racing greyhounds (rrg) has higher oxygen carrying properties and affinity than other breeds. surprisingly, very little is known about canine hemoglobin genetics. the purpose of this study was to characterize genetics of canine beta globins. using computational blast analysis of the dog genome, we identified five beta globin genes in a single locus: two human hbelike followed by three hbb-like genes. we isolated dna and rna from blood of rrgs, akc registered greyhounds (akcg), and german shepherd dog (gsd). all beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom m agilent array). additionally, we determined the number of common haplotypes that span this locus in rrgs and akcgs using high density snp array ( k illumina hd). expression and sequence analysis of cdna showed all five beta globin genes are actively expressed in adults. canhbb and were created by relatively recent segmental duplication and have identical protein sequence. canhbb / are abundantly expressed in adults; canhbb is expressed at greatly reduced levels. sequencing results revealed one rare non-synonymous single nucleotide polymorphism (snp) in hbe of rrgs, but no variation that could explain their abnormal hemoglobin. we did not detect structural variation overlapping or near the beta globin locus. notably, rrg and akcg do not share haplotypes spanning the beta globin locus. this is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. sampling of the bone marrow in the dog from the costochondral (cc) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the united states. the aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the cc junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners. samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. paired samples from separate dogs were obtained by each practitioner using either a gauge needle and cc syringe at the cc junction or an gauge rosenthal needle and cc syringe from either the proximal humerus or femur (clinician preference). three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. slides were prepared by a single investigator and read by a blinded clinical pathologist. data were compared using the paired t-test for normally distributed data and wilcoxen signed rank test for non-gaussian distributions. five pairs of samples from three dogs were evaluated. two dogs had two pairs drawn from opposite limbs and ribs. mean number of attempts to success for traditional sampling sites ( . /À . ) and time to success ( . minutes /À . ) did not differ significantly from attempts ( . /- . , p . ) or time ( . /- . , . ) needed when aspirating from the cc junction. subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (m:e ratio p . , lymphocyte % p . ) and were within normal limits. while there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the cc site and had an increased number of attempts compared to the traditional sites. if the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. based on this data, bone marrow aspiration from the cc junction appears to be equivalent to more traditional sampling sites in healthy dogs. larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase- , a transmembrane serine protease (tmprss ) produced by the liver. while iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (irida) caused by a matriptase- deficiency homologous to a recently described autosomal recessive disorder in humans. the proband, a spayed female cocker spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. there was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. mean values of complete blood cell counts, performed from . - years of age, were: hematocrit % (normal reference range - ); rbc count .  /ml ( . - . x ); mcv fl ( - ); mchc g/dl ( - ). serum iron parameters revealed severe iron deficiency with serum iron mg/dl ( - ); total iron binding capacity mg/dl ( - ); serum iron saturation o % ( - %), and ferritin ng/dl ( - ). prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. however, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. no family members were available for further studies. genomic dna was extracted from the proband's edta blood and the exons of the tmprss gene were amplified with flanking primers and then sequenced. in comparison to the normal canine tmprss sequence and that of a sequenced control dog we found a homozygous missense muation, r h, toward the c-terminal end of the protein in the proband's gene. in conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of irida. the missense mutation in the matriptase- at position from an arginine, which is conserved across all species currently deposited in the genbank, to a histidine is likely the disease-causing mutation. this is the first report of an irida in the dog with features very similar to those observed in humans. dogs with naturally-occurring irida may be helpful in developing and assessing novel therapies. accidental ingestion of copper-coated zinc pennies minted after is the most common causes of zinc toxicity anemia in the dog. zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. the purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. records of dogs presented to the internal medicine service at the veterinary medical center of long island for metallic foreign body ingestion were reviewed for signalment, weight, presenting pcv, and type of metallic foreign body ingested. eighteen dogs met the inclusion criteria and were compared. of the dogs, there were cases of coin ingestion ( %), with ( %) involving ingestion of or more pennies. the other cases involved ingestion of a metallic object ( ), decorative garland ( ), and bb pellets ( ).of the dogs exposed to zinc, ( %) were less than pounds ( . kgs). of those cases ( %) had ingested one or more pennies. eleven out of the ( %) zinc exposure dogs were anemic at presentation. the average weight of the dogs was . pounds ( kg). this study showed that dogs less than pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after . zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. we suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (pc) is used infrequently. data suggest in vitro efficacy of pc and when administered to research dogs, but efficacy is unknown in clinical patients. study objectives were to determine clinical characteristics of dogs receiving pc as well as safety and efficacy of pc in thrombocytopenic dogs. medical records were evaluated retrospectively to identify dogs that received pc. information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. twenty six dogs met study criteria. dogs receiving pc ranged in age from - years (mean . years) and / ( . %) were spayed or intact females. hemorrhage was reported in / dogs ( . %) prior to pc transfusion. platelet counts prior to transfusion ranged from to  /ul (mean . /À .  /ul). change in platelet count was measured in dogs and the mean change was . /À .  /ul. dose of pc administered ranged from . to ml/kg with a mean of . /À . ml/kg. no acute adverse reactions were reported. there was no correlation between transfusion dosage and platelet count change post transfusion. survival to discharge occurred in / ( . %) of dogs. the only variable correlated with survival was age with survivors being younger than non-survivors ( . years-old ae . vs. . years-old ae . .; p . ). efficacy of cryopreserved pc transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, pc is well tolerated in clinical patients. fresh frozen plasma (ffp) is used to treat coagulopathies in dogs. current transfusion guidelines recommend that ffp be administered within hours of thawing to avoid decreasing clotting factor function and bacterial contamination. the purpose of this study was to assess clotting factor activity and bacterial contamination of ffp that had been thawed and refrigerated for days. blood was collected from client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. plasma was separated from whole blood and frozen (À c) within minutes of collection. thawed plasma was maintained at c ( /À c). aerobic and anaerobic bacterial culture, prothrombin time (pt), activated partial thromboplastin time (ptt), and factor ii, vii, ix, and x analyses were tested at time of whole blood collection, ffp thaw, hours post-thaw, hours post-thaw, and hours post-thaw. there were no statistically significant differences in pt and ptt at any of the measured time points. statistically significant differences occurred between initial measurements of factors ii, vii, ix, and x and subsequent time points, but there was no difference in activity levels of the factors once ffp was thawed. one bacterial colony was grown from each of two samples from post-thaw plasma. thawed plasma protocols do not significantly decrease the function of factors ii, vii, ix, and x or prolong pt and ptt. bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. erythrocyte pyruvate kinase (pk) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. the pk enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. the disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. in dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning m -pk expression, but breed-specific r-pk mutation tests have been reported for basenjis, west highland white terriers (whwt), and beagles. we report here on a survey of canine pk deficiency studied at the penngen laboratory. a biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. edta blood samples and/or cheek swabs as well as medical record information were received and genomic dna and/ or enzyme activity testing were performed. among the whwts % and % were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of . . the average age at the time of diagnosis was . years ranging from months to years of age; some samples came from europe and south america. of the beagles studied, % were affected and % were carriers (mutant allele frequency . ). the average age at the time of diagnosis was years ranging from months to years. surprisingly, very few samples from basenjis were received for screening, and none showed the mutant allele. while pk-deficient basenjis lived o years, whwt and beagles often show milder signs and can reach years of age. several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. however, based upon pk enzyme activity studies, chihuahua, dachshund, miniature poodle, spitz, eskimo toy, and labrador retriever dogs were found to be affected; they also had osteosclerosis and at least one labrador retriever developed severe hemochromatosis (hepatic iron , ppm; normal o , ppm, analyzed on a dry weight basis). moreover, sequencing of the r-pk cdna from a pk-deficient labrador retriever revealed a new nonsense mutation in exon . in conclusion, pk deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. pk deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. studies to identify new mutations will confirm and simplify the diagnosis. supported in part by nih grant rr . immune-mediated hemolytic anemia (imha) is a common hematological condition observed in dogs. the diagnosis is based on clinical history, presenting signs and hematological evidence of imha such as regenerative anemia, leucocytosis and presence of spherocytes. the definitive diagnostic procedure is the coomb's test (direct antiglobulin test, dat) which is known to be highly specific but lacks diagnostic sensitivity. direct flow cytometric assay (fca) for igg, igm or c coated red blood cells (rbcs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool. to investigate the usefulness of fca for imha diagnosis, evaluation of igg, igm or c coated rbcs was performed from dogs presented at the veterinary hospital at usp that fulfilled clinical and hematological criteria for imha. thirty eight healthy dogs were included as controls. dat was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with % rbcs suspension at c and c. for fca, % rbcs from anemic and healthy dogs were incubated with fitc anti-dog igg, anti-dog igm and anti-dog c and submitted to flow cytometry evaluation. specific software and mann whitney u test were used for data analysis. five dogs showed positive results for dat with polyvalent coombs reagent at c (titer to ) and c (titer to ) but only three of them had agglutination titer for anti-igg at c ( to ) and c ( to ). no positive results were observed for anti-igm and anti-c dat. by fc, percentage of igg, igm and c coated rbcs in normal and anemic dogs were, respectively, , % and , % (p o , ); , % and , % (p o , ); , % and , % (p o , ). igg coated rbcs percentage were higher in dogs showing dat positive results (min. , %; max. , %; median , %). direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than dat for detection of antibodies coated rbc in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of imzha. bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. in this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of healthy cats weighing . - . kg, with body condition scores of - (on a scale of - ). for sternal aspirates, cats were positioned in sternal recumbency and a -inch, - ga hypodermic needle attached to a cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. aspirates were also obtained from the right iliac crest using an ga illinois needle attached to a cc syringe. difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from (easiest) to (hardest). bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. sample quality was scored from (no marrow particles) to (excellent) based on the number of wellsmeared marrow particles on the slide. particle cellularity was scored from ( % fat) to (o % fat). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for post-biopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and site swelling (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. pneumothorax was not identified. it was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. for samples that could be compared, cellularity was identical for sternal and iliac samples from cat but underestimated in the sternal sample from another cat. myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in and cats, respectively. megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from cats, and overestimated in the sternal sample from cat. bone marrow cell morphology was normal in all acceptable samples. it was concluded that sternal aspiration of bone marrow using a - ga hypodermic needle is ) easier to perform than iliac aspiration; ) safe; but ) provides samples of lower quality than iliac aspiration in cats. the diameter of - ga jamshidi-type needles makes bone marrow core biopsy difficult in cats. in this study, biopsies of the left humeral head were taken under anesthesia using a -inch, ga needle (ez-io s intraosseous infusion system, vidacare) from healthy cats weighing . - . kg with body condition scores of - (on a scale of - ). humeral biopsies were compared to biopsies taken from the left iliac crest using a -inch, ga jamshidi needle. biopsies were performed in randomized order by one investigator. biopsy was repeated to a maximum of attempts until a specimen ! mm long was obtained. difficulty of site localization, needle insertion and needle advancement were scored from (easiest) to (hardest). specimens were wrapped in tissue paper and placed in davidson's fixative for min and then transferred to formalin. biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. biopsy length on the slide was measured, and biopsy quality was scored from (no hematopoietic tissue) to (! intertrabecular spaces free of artifact). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for postbiopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and swelling of biopsy sites (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. there were no significant differences between ga and ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition. six ( %) of ga and ( %) of ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. comparison of acceptable ga to ga biopsy specimens from cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in of the ga biopsies, was assessed as medium in corresponding ga biopsies; and megakaryocytes, assessed as - /low-power field in one ga biopsy, were assessed as /low-power field in the ga biopsy. myeloid:erythroid ratios were greater in ga biopsies compared to ga biopsies in cats, and less in the ga biopsy in one cat. discordant results between biopsies were not related to differences in quality. in conclusion, ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was ga biopsy of the ilium, and resulted in less post-biopsy swelling. reports on canine acute liver failure (alf) include individual or small case series of animals with a specific diagnosis. the aim of this study was to describe the clinical course, outcome and etiology of alf in dogs presenting to a referral hospital. medical records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were reviewed for a diagnosis of alf (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (he)). fifty cases were identified representing breeds: labradors retrievers, golden retrievers, german shepherds, and cocker spaniels. median age was years ( m to humerus, ga . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ilium, ga . ae . . ae . . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ( - ) ( - ) yrs). presenting signs included anorexia ( / ), vomiting ( / ), polydipsia ( / ) and neurologic signs ( / granulomatous hepatitis (gh) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (wsava). published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with gh has detailed clinically relevant findings. the aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of gh, and to identify infectious agents using differential staining techniques, pcr, and fluorescence in-situ hybridization (fish) in archival paraffin-embedded tissues from dogs with gh. medical records of dogs with a histopathological diagnosis of gh (n ) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. two of these samples had sufficient tissue for only pcr. the mean age of dogs with gh was years (median . years; range to years) and included males and females representing different breeds. common presenting complaints included inappetance or anorexia (n ), weight loss (n ), lethargy (n ), fever (n ), and vomiting (n ). high mixed liver enzyme activity ( / ) was the most common clinicopathologic abnormality. leukemia was diagnosed in one dog and copper-associated hepatopathy in dogs. no infectious agents were identified using differential staining techniques. bartonella species dna was not pcr amplified from the extracted archival tissue. furthermore, no bacteria were identified by means of fish using a universal eubacterial probe. these data suggest a possible role for copper accumulation in the genesis of gh in dogs and support further evaluation of dogs with gh for evidence of copper-associated hepatopathy. future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. the purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes. liver biopsies (n ) submitted between july , and june , were evaluated using wsava guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. copper (by rubeanic acid) and iron (by prussian blue) accumulation were graded by increasing amounts ( - ) and location (centrilobular cl, midzonal mz, periportal pp, random r). associations between metal scores and diagnosis category were assessed using the kruskal-wallis test. histologic diagnoses were non-hepatic/normal (n ), congenital (n ), neoplastic (n ), infectious/inflammatory (n ), and other (n ). ninety-two samples were negative for copper; remaining samples were graded (n ), (n ), and (n ). histologic diagnoses (pattern) for positive samples were congenital ( cl), infectious/inflammatory ( : cl, mz, pp, r), neoplastic ( pp), and other ( cl). iron staining was negative in samples; remaining samples were graded (n ), (n ), and (n ). distribution was primarily cl (n ) or r (n ), though mz (n ) and pp (n ) distribution occurred. there were no significant differences by kruskal-wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories. in this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. in contrast, iron accumulation was common and did not correlate with disease category. further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. chronic hepatitis (ch) in dogs is a progressive condition without clearly defined treatment. glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine ch. patient records at the csu veterinary teaching hospital were searched for histologically confirmed cases of ch treated with cyclosporine. data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters. patients over a -month period were identified. serum alanine aminotransferase (alt) decreased by an average of % in dogs. the alt normalized completely in of dogs treated for days. in of dogs on mg/kg/day, the alt also normalized. five of the patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in . post-treatment histopathology, available in one patient, revealed decreased severity of ch. five patients exhibited adverse effects including gastrointestinal signs ( ), gingival hyperplasia ( ), and papillomatosis ( ). cyclosporine was discontinued in dogs with gastrointestinal signs. cyclosporine was an effective therapy for many cases of ch and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in ch. insertion of the veress needle and establishment of pneumoperitoneum is associated with to % of all laparoscopic complications in humans. the purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous dogs euthanized for reasons unrelated to the study. placement order was randomized. a third individual evaluated impedance measurements using a handheld device (sensormed, knoxville tn) to determine correct versus incorrect needle placement. veress needle locations were marked using contrasting colors of india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements, respectively. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. agreement was moderate (kappa . , p . ) for placements by operator and very high (kappa . , p o . ) for placements by operator . results for tissue impedance measurement interpretation are superior to published data for currently available tests. impedance measurements accurately detected all incorrect needle placements. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate veress needle placement and decreases installment phase complication rates. delayed detection of intestinal perforation during veress needle insertion is associated with high mortality. the purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous cats. placement order was randomized. a third individual evaluated impedance measurements (sensormed, knoxville, tn) to determine placement location. needle locations were marked using india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements. all undetected incorrect placements were located within the retroperitoneal fat pad. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. correlation was absent (kappa À . , p . ) for placements by operator and substantial (kappa . , p o . ) for operator . there was no association between correct or incorrect placement and operator on chi-squared analysis. failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant kappa statistics. small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate veress needle placements or decrease installment phase complication rates. best clinicopathologic tests detecting portosystemic shunting (pss) in dogs remains controversial. this retrospective study examined performance of single random "fasting" and paired serum bile acids (sba; pre-and -hr post-feeding) in a large population of non-icteric dogs with confirmed pss (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-ct portography, laparotomy, or necropsy). sba were measured by enzymatic colorimetric method with normal o mmol/l. dogs meeting inclusion criteria (n ) included portosystemic vascular anomalies (psva; extrahepatic [e-psva], intrahepatic [i-psva]), and acquired pss (apss). signalment and laboratory parameters were recorded. non-parametric statistical analyses were used, two-tailed p o . applied with bonferroni corrections. median age and weight of breeds were . ( . - ) yrs and . ( . - ) kg, with equal gender distribution. random "fasting" sba detected % psva and % apss, whereas post-feeding sba detected % psva and % apss. low protein-c (o % activity) occurred in % psva and % apss. low mcv and creatinine occurred in % and % of psva dogs, respectively; other tests were less helpful. in apss, post-feeding sba was superior. compared to apss, psva had significantly (p . ) lower mcv, cholesterol, bun, creatinine, glucose, and protein-c. compared to e-psva, i-psva had significantly (p . ) lower post-feeding sba, mcv, albumin, urine specific gravity, and protein-c but higher cholesterol and glucose. post-feeding sba reflect physiologically provoked bile acid challenge and should be the preferred sba test in non-icteric dogs for pss detection. protein-c assists in identifying psva but its utility in apss may be complicated by concurrent coagulopathies and inflammation. this study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (nsaids) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. medical records were reviewed for dogs diagnosed with blastomycosis at the university of illinois veterinary teaching hospital between and . dogs with a presenting pao of mmhg, and clinical or radiographic signs of respiratory blastomycosis were included. all dogs were treated with either itraconazole, fluconazole, amphotericin b, or a combination of these. group (g ) dogs were treated with nsaids and group (g ) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. the following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. mann-whitney u tests and chi-squared tests were performed on continuous and categorical data, respectively. p o . was considered significant. sixty-eight dogs fit the inclusion criteria. g consisted of dogs and g consisted of dogs. the two groups were found to be similar in weight, age, and sex distribution. there was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival. there does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with nsaids or anti-inflammatory glucocorticoids. further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ards and additional respiratory complications. diagnosis of feline histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. in recent years, an antigen assay (aa) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. the goal of this study was to determine the sensitivity and specificity of h. capsulatum aa in cats with clinical disorders suggestive of histoplasmosis. urine and serum h. capsulatum aa results for feline patients from veterinary hospitals were evaluated. medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis. aa results were available for cats; initial testing was performed on urine samples, serum samples, and unspecified sample. of these cats, / had a definitive diagnosis of histoplasmosis based on organism identification, and had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n ) or other clinical data (n ). an additional cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). the remaining cats had unverified histoplasmosis (n ) or an open diagnosis (n ). of the cats with confirmed histoplasmosis, were positive on initial urine aa. one cat (with rectal involvement) was negative, indicating a test sensitivity of %. one cat was positive on urine aa but negative on serum aa. all of the cats with definitive or clinical alternate diagnoses had negative results on the aa, suggesting an excellent specificity ( %). however, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the patients who underwent necropsy examination. these findings suggest that the aa for h. capsulatum is a reliable diagnostic tool in this species. a positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on aa. in addition, tests performed on urine may be more sensitive that those performed on serum. the purpose of this study was to evaluate the sensitivity and specificity of an aspergillus galactomannan antigen enzyme immunoassay (ga-eia) for the diagnosis of canine systemic aspergillosis. serum and urine samples were collected from sick dogs at hospitals (ucd and tamu). group dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). group dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. group dogs were not suspected to have aspergillosis. samples were tested using the ga-eia and results expressed as a galactomannan index (gmi). gmis . were considered positive. comparisons were performed using the mann-whitney test. there were dogs in group , in group , and in group . serum was collected from all dogs, and urine from , , and dogs, respectively. serum gmis did not differ from urine gmis across groups. serum gmis of group dogs were higher than those of group and group dogs (p o . ). results from dogs in group did not differ from those in group (p . ). two dogs in group tested negative, but had localized pulmonary infections. one dog in group , which had paecilomycosis, tested positive. two dogs in group tested positive. one was being treated with plasmalyte. the other had a cutaneous opportunistic mycosis. these data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. anaplasma phagocytophilum, an ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise. the purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for a. phagocytophilum on a commercially available pcr of peripheral blood (fastpanel tm ). this study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of a. phagocytophilum. the a. phagocytophilum real-time pcr (rt pcr) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. amplicons were confirmed as a. phagocytophilum by dna sequencing. clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. complete blood counts were available from / cats and / blood smears were reviewed. the cats included in this study were all positive for a. phagocytophilum by real-time pcr. the cats ranged from months to years of age with an average age of . years. fifteen of cats had a history of tick exposure and lived in the northeastern region of the us, an ixodes endemic area. all cats presented with lethargy, / were anorexic and / had a fever (temperature o f). other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. hematologic findings included leukopenia ( / ), neutropenia ( / ) and lymphopenia ( / ). thrombocytopenia was not noted in any case. morulae were seen within neutrophils in / cases. all cases in this report responded to treatment with doxycycline. this is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by rt pcr to be infected with anaplasma phagocytophilum in north america. infection with anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. the spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. the objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for bartonella sp. dna (controls) as were the dogs diagnosed with bartonellosis by pcr amplification, dna sequencing and the bapgm (bartonella alpha proteobacteria growth medium) enrichment culture approach. both groups were tested under the same laboratory conditions and in the same time frame. medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment. the study population consisted of bartonella-infected dogs and non-infected dogs. healthy dogs with no historical illnesses, such as blood donors, were excluded. the following species were amplified: b. henselae (n , . %), b. vinsonii subsp. berkhoffii (n , . %), b. koehlerae (n , . %), b. volans-like (n , . %), b. bovis (n , . %). nineteen ( . %) bartonella-infected dogs were febrile and lethargic and ten ( . %) had neurological signs. laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and bartonella sp. infection. there were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. compared to the control population, infection with the genus bartonella was associated with a diagnosis of endocarditis (p . , or . , %ci . - . ) and hypoglobulinemia (p . , or . , % ci . - . ). controls were more likely to have joint effusion (p . , or . , % ci . - . ) and azotemia (p . , or . , %ci . - . ) than were the bartonella sp. infected dogs. bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. when endocarditis or hypoglobulinemia are detected, testing for bartonella should be prioritized. likewise, the detection of bartonella should prompt further testing for endocarditis, if not already investigated. surveillance studies in other species depend on detection of antibodies to the highly conserved influenza a nucleoprotein (np); however, no such antibody detection assay is approved for canine use in the u.s. the purpose of this study was to determine the diagnostic accuracy of a commercial blocking elisa used for avian species in detecting influenza a np antibody in dogs. since the blocking elisa is not a species-specific or viral subtype-specific format, we hypothesized that it would detect np antibodies in dogs infected by influenza a virus. serum samples from uninfected dogs (n ) and dogs naturally infected with canine influenza h n (n ) were tested using the idexx flockchek blocking elisa for influenza a np antibody according to manufacturer instructions. the sample/negative control (s/n) absorbance ratios for infected dogs ranged from . to . compared to . to . for uninfected dogs. a receiver operating characteristic (roc) curve analysis determined optimum diagnostic sensitivity ( . %) and specificity ( . %) at a s/n cutoff ratio of . . using this cutoff ratio, the overall diagnostic accuracy was . %. coefficients of variation for intra-assay ( . %) and inter-assay ( . %) testing demonstrated good repeatability with canine sera. the excellent diagnostic accuracy of the commercial blocking elisa makes it a suitable tool for large-scale surveillance of influenza a virus exposure in dogs. upper respiratory disease (urd) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. the purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (shel), long-term sanctuaries (sanc), home-based foster care (fost), and trap-neuter-return (tnr) programs. conjunctival and oropharyngeal swabs were collected from cats, half of which had clinical signs of urd, and tested for feline herpesvirus (fhv), feline calicivirus (fcv), chlamydiophila felis, bordetella bronchiseptica (bb) and mycoplasma felis by real-time pcr. management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection. a majority of cats in all management models carried one or more organisms capable of causing urd. in many cases, prevalence was similar in cats with or without clinical signs. unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline urd control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. depending on geographical location, sex, age and environment, - % of cats worldwide are infected with the feline immunodeficiency virus (fiv). knowledge of the fiv status of cats is important to limit the spread of disease and to institute appropriate health management. however, like all lentiviruses, fiv is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. detection of antibodies is the most widely employed diagnostic approach, but does not distinguish fiv-infected from fiv-vaccinated cats. in this study, samples from fiv-seronegative cats, fivseropositive cats, and fiv-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative pcr (qpcr) assay and virus isolation. replicate blood samples were coded, and then submitted for ) qpcr (idexx); and ) mononuclear cell isolation with -day culture and viral p antigen detection by elisa. for the p antigen elisa, cutoff absorbance values were established from analysis of fiv-negative samples. fiv infection status was pre-determined based on antibody-elisa results and vaccination history. results indicated that qpcr had a sensitivity of % for samples from fiv-seropositive cats, and a specificity of % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. at a cutoff value of standard deviations above the mean absorbance for p antigen elisa, results from fiv-negative samples yielded a sensitivity of . % for samples from fiv-seropositive cats, and a specificity of . % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. conclusions from this study are ) the commercial fiv qpcr assay has high specificity but limited sensitivity for diagnosis of fiv infection; ) -day virus culture has limited sensitivity and specificity. hence, detection of antibodies remains the most reliable test for diagnosis of fiv infection, but qpcr may be suitable to rule out infection. oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. a number of infectious agents have been associated with private veterinary clinics in the u.s. were recruited to test feline patients presenting with oral disease. presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. all cats were tested using a commercially available point-of-care elisa test (idexx snap combo). confirmatory tests were not performed as part of the study. seroprevalence was calculated as the percentage of positive tests in the study population for each virus. a total of , cats were tested. seroprevalence for felv was . % and for fiv was . %. of these, cats ( . %) were infected with both viruses. seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. of , cats with gingivitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. of , cats with stomatitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. the seroprevalence for felv and fiv reported in this population of cats with oral disease was higher than in a recent large study where samples from u.s. cats not specifically selected for oral disease were tested (felv . %, fiv . %). results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. reliable tests and preventive vaccines and medications for feline retroviral and heartworm (hw) infections are available, but compliance with protocols to reduce transmission is unknown. no largescale longitudinal studies evaluating prevalence over time have been reported. the purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time years previously. veterinary clinics and animal shelters in the us and canada submitted results of testing using a point-of-care elisa for felv antigen, fiv antibody, and hw antigen (idexx snap triple) and risk factor information for cats tested during march-september . bivariable and multivariable analyses were used to evaluate risk factors for infections. a total of , cats were tested. only % of owned cats were prescribed hw preventive. risk of retroviral infections was increased by outdoor access, adulthood, and male gender. the most important risk factor associated with all infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. multivariate analysis revealed differences among geodivisions and across infection types. feline retroviral and heartworm infections are easily prevented, but difficult to treat. despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. during a canine distemper virus (cdv) and parvovirus (cpv) outbreak in sheltered dogs, we used a cdv/cpv point-of-care antibody titer elisa, a cdv quantitative rt-pcr test, and a cpv fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs. serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for cdv pcr) were collected from asymptomatic dogs starting on day of the outbreak. dogs with positive cdv pcr tests were retested every weeks until euthanized for progressive disease or released following recovery from infection. dogs with clinical signs of parvoviral infection were tested using a cpv fecal antigen test. for dogs ! months old, protective antibody titers correlated with resistance to clinical disease, but % of dogs shed cdv. lack of protective cdv antibody titers correlated with susceptibility to clinical infection, but most dogs recovered. risk assessment and outcome in dogs ! months of age feline herpesvirus (fhv- ) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. cyclosporine (csa) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. a small number of cats administered csa to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated fhv- . in this study, young adult cats experimentally inoculated with fhv- several months previously were divided into three groups and administered methylprednisolone acetate ( cats, mg/kg, im, day and day ), csa ( cats, . mg/kg, po, daily for days), or a placebo ( cats, corn syrup; . ml/kg, po, daily for days). each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (day - to day ) and throughout the day treatment period. each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using sas proc glimmix with 'treatment', 'time', and the two-way interaction 'treatment by time' all as fixed effects. statistical significance was defined as p o . . on day of the study, all of the csa treated cats had detectable concentrations of csa in serum (mean . ng/ml; standard deviation . ng/ml; median . ng/ml). when group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups. while clinical signs of activated fhv- occurred in some cats administered methylprednisolone or csa, disease was mild and self-limited in most cats and there were no significant csa sideeffects. these results suggest that the csa protocol described here is unlikely to reactivate latent fhv- infection and cause significant clinical illness. the purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (tnr) programs. fecal samples were collected from cats, half with diarrhea (d) and half with normal feces (n), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. risk factors for infection evaluated include management practices, fecal consistency, and signalment. a majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. for most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being t. foetus in sanctuary cats and fcov in foster cats. prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. toxoplasma gondii infection occurs in approximately % of cats and the effect cyclosporine therapy has on the t. gondii oocyst shedding period is unknown. the objective of this study was to determine whether administration of cyclosporine before or after t. gondii infection influences the oocyst shedding period. the young adult cats were t. gondii seronegative when administered , t. gondii tissue cysts orally on day . group cats (n ) were never administered cyclosporine; group cats (n ) were administered cyclosporine ( . mg/kg, po) daily on days - ; and group cats (n ) were administered cyclosporine ( . mg/kg, po) daily from days - . available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for days after t. gondii inoculation. group shed oocysts for a significantly shorter period than groups or and had a significantly lower oocyst shedding scores than groups and on days - after t. gondii inoculation. group cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug. administration of cyclosporine prior to t. gondii infection lessened oocyst shedding which is likely from the anti-t. gondii effects of the drug. administration of cyclosporine using this protocol is unlikely to induce repeat t. gondii oocyst shedding in client-owned cats. à group with diarrhea significantly different than group with normal feces p o . known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. the aim of this study was to completely sequence the genome of m. haemofelis to further characterise this important pathogen. mycoplasma haemofelis genomic dna was purified and subjected to whole shotgun roche sequencing. gaps were closed using targeted pcr and amplicon sequencing. ribosomal genes and potential open reading frames (orfs) were predicted in silico. putative orfs were annotated and orthologous groups identified. analysis showed a circular genome of . mbp with a gc content of . %. thirty-one transfer rnas (trnas) were identified, accounting for all amino acids, including a tryptophan trna for the opal codon (uga). of the , putative proteins identified, ( . %) matched to proteins from other bacterial species. in common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for m. haemofelis. the majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in m. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. a gene encoding a glyceraldehyde- -phosphate dehydrogenase homolog of the immunogenic msg protein of mycoplasma suis was present. of the uncharacterized hypothetical proteins, , were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately amino acids. the predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an n' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail. these data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. we have also identified a homolog of a known m. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. canine leptospirosis has been recognized as a re-emerging disease in the u.s. over the past years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. the role of cats in the epidemiology of leptospirosis has received little attention. serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the u.s. in the past four decades. the new england states have been found to have a high incidence of canine leptospirosis. the purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central massachusetts. blood was collected from sexually intact feral cats presented to a spay and neuter program. microagglutination titers to leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. three of cats ( . %) had a positive titer to one or more serovars, with autumnalis being the most common. these results are consistent with previously published prevalence rates in feral cats. further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. since years the rivalta's test is routinely used in several european countries as a tool to diagnose feline infectious peritonitis (fip) in cats with effusion. it is inexpensive and easy to perform in private practice. there is, however, only little information about mode of action or its diagnostic value. the objectives of this study were to evaluate sensitivity, specificity, positive (ppv) and negative predic-tive values of the rivalta's test to diagnosis of fip and to examine if there is a correlation with any effusion or blood parameters. medical records of cats with effusion in which the rivalta's test was performed between and were reviewed concerning diagnosis, blood and effusion parameters, and survival time. effusion and blood parameters were compared between rivalta-positive and -negative effusions using the mann whitney u test. prevalence of fip in cats with effusion was . %. the rivalta's test showed a sensitivity of . %, a specificity of . %, a ppv of . %, and a npv of . % for the diagnosis of fip. the ppv improved, when cats with lymphoma or bacterial infection were excluded (ppv . %) and also, when only cats younger than years (ppv . %) or year (ppv . %) of age were included. the most important significantly different parameters between rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion. the rivalta's test in general is a useful tool to diagnose fip, but its sensitivity and specificity are not as high as previously assumed. if the rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. effusion total protein is not highly correlated with test outcome. therefore, it is still unclear, which components in the effusion of cats with fip lead to a positive rivalta's test. canine parvovirus (cpv) and canine distemper virus (cdv) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following weeks after vaccination. serum samples were obtained from incoming shelter dogs aged months and older with no known history of vaccination. immediately following serum collection, the dogs were vaccinated against cpv and cdv using a modified live vaccine (mlv). cpv and cdv antibody protection status was determined using synbiotics titerchek. dogs with unprotective serum antibody levels against cpv and/or cdv were retested at - days post-vaccination and again at - days post-vaccination, if antibody levels were still unprotective against cpv and /or cdv. at the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers. based on the synbiotics titerchek results, / dogs ( . %) were protected against cpv and / ( . %) were protected against cdv at intake. older incoming dogs were more likely to be protected against cpv (p o . ) and cdv (p . ). dogs that were spayed/neutered were more likely to be protected against cpv on intake than intact animals, although this result was not statistically significant (p . ). the number of dogs with protective titers against cpv/cdv was increased at - days post-mlv (cpv - / , . %; cdv - / , . %) and further increased at - days post-mlv (cpv - / , . %; cdv - / , . %). we conclude that incoming shelter dogs often do not have protective antibody titers against cpv and cdv, but older shelter dogs are more likely to be protected against cpv. based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to cpv and cdv within to weeks when vaccinated with a mlv. mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. the objectives of this study was to determine whether mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease. privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. participants were given a free aerobic and anaerobic culture as well as mycoplasma spp. culture and polymerase chain reaction using mycoplasma genus specific primers. mycoplasma spp. amplicons were sequenced to determine the species. all cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone days after beginning treatment. of the cats entered into the study to date, mycoplasma spp. were amplified from cats ( . %). of the positive samples with adequate dna for sequencing, one was consistent with m. felis and the other was consistent with m. equigenitalium. of the cats, responded by day to the initial treatment, including of the mycoplasma spp. positive cats. the cat that failed initial treatment was positive for m. equigenitalium on both day and day and ultimately responded to administration of a fluoroquinolone. the results suggest that while mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. however, as mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. however, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus , parainfluenza, or bordetella bronchiseptica. however, whether this is true for puppies is unknown. puppies (n ) at a breeding facility were moved into area without other dogs at weeks of age. swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days , , , , , , , , , , , , , and after vaccination with an intranasal adenovirus , parainfluenza, and b. bronchiseptica vaccine (intratrac , schering plough). the swabs were shipped on cold packs by overnight express for dna/rna extraction and assay in the fastpanel tm pcr canine respiratory disease profile at antech diagnostics. all puppies were negative for the infectious agents prior to vaccination. after vaccination, positive assay results for parainfluenza and b. bronchiseptica were first detected on day and on day for adenovirus . by day , dna or rna of the agents were amplified from all puppies from both sample sites and most samples were positive for all agents through day . by day , only one dog was still positive for b. bronchiseptica. the results indicate that intranasal administration of adenovirus , parainfluenza, or bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. these findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. antimicrobial resistance in escherichia coli is an increasing concern in both human and veterinary hospitals' patients. the choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (fq) whose activity reflects, in part, ciprofloxacin. among the difficulties in effective e. coli treatment is rapid detection of fq resistance. the purpose of this study was to determine the specificity and sensitivity of a fret based assay for the rapid detection of urinary tract infections caused by fq associated multi-drug resistant e.coli. clinical e. coli isolated from canine urine and clinical veterinary urine samples being examined for e. coli were subjected to susceptibility testing for drugs representing drug classes. pure isolates were designated ndr (no drug resistance), sdr (single drug resistance) and mdr (multi-drug resistant) (n mdr, sdr and ndr). minimum inhibitory concentration (mic) for enrofloxacin ranged from . mg/ml to mg/ml, with high mic generally associated with mdr. extracted dna from culture and from urine were subjected to fret-pcr targeting single nucleotide polymorphisms in gyra. the resulting product was sequenced to detect other polymorphisms. further, to determine the level of detection, microbial free canine urine was inoculated with to cfu/ml of isolates characterized by variable susceptibility to enrofloxacin (mic enro . , . , . , , , , mg/ml). of pure isolates, were confirmed positive for enrofloxacin resistance (mic enro mg/ml), of which were positively identified by the fret-pcr assay giving a sensitivity of . %. only isolate that was resistant was not detected (specificity of . %). however, of the isolates expressing high level resistance (mic x breakpoint [ mcg/ml]), and mdr (n ), sensitivity . %. of the urine samples contained e. coli of which determined to be fq-resistant by the assay. colony dilutions of e. coli confirmed the assay able to detect enrofloxacin resistance at as low as cfu/ml. the relationship between cfus and the peak of the -(d/dt) fluorescence of the melting curve was r . . these results conclude that the assay is capable of detecting not only the presence of escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. the fluoroquinolones (fqs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (utis) caused by e. coli, and fluoroquinolones are common therapy. among the commonly used fqs in dogs and cats are the nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no rd and th generation fq is routinely used. the purpose of this study was to assess the in vitro activity of different generation fqs toward e.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. a total number of canine uropathogenic canine or feline e.coli isolates had been subjected to susceptibility testing to drugs classes ( drugs) and phenotyped as to resistance: none (ndr, n ), single (sdr, n ), or multiple, mdr (resistance to - drug classes; n ). mdr included isolates susceptible (enr s -mdr, n ) or resistant (enr r -mdr) to enrofloxacin. the minimum inhibition concentrations (mics) for quinolones ( - st generation, - nd generation, - rd generation and - th generation) were determined for these isolates using broth microdilution methods according to clsi guidelines (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the results showed that companion animal e. coli expressing ndr or sdr are largely susceptible to nd to th generation fqs. however, isolates expressing resistance to st or nd generation quinolone also express high level resistance based on the mic to rd and th generation fqs. the overall potency (mic) for the drugs for isolates not expressing enr resistance (that is, ndr, sdr and enr s -mdr) is gat canine leproid granuloma (clg) was first reported in brazil in . over the past years, cases of clg were diagnosed in sa˜o paulo, brazil, and clinical and epidemiological findings were similar to those reported in australia. all dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. in general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. short-coated breeds show a marked predisposition for this disease. the definitive diagnosis of clg was obtained by histological examination of skin biopsies that were stained with acid fast (ziehl-neelsen) and diffquik s . thirty one ( . %) of the dogs were purebred; in this study the breed pattern comprised ( . %) boxers, ( . %) german shepherd and labrador retriever, ( . %) dobermann, ( . %) brazilian terrier, ( . %) golden retriever, ( . %) bulldog, ( . %) american pitbull, ( . %) mastiff, ( . %) fila brasileiro and ( . %) cocker spaniel, ( . %) were of unknown breed. nineteen ( . %) of the thirty seven dogs were males. twenty ( . %) dogs were - years old. in most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. the animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. currently, most suspected cases are evaluated for a. phagocytophilum antibodies by immunofluorescence assay (ifa) or elisa. since a. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. it is possible that a. phagocytophilum dna can be amplified from blood or synovial fluid prior to seroconversion. wild caught ixodes scapularis adult ticks from rhode island were allowed to feed on young adult ( - years), mixed sex beagles for up to days. blood (weekly for weeks), serum (weekly for weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for weeks) were collected prior to tick attachment and then weekly after tick attachment. joint fluid cytology was performed and total dna was extracted from blood and synovial fluid and assayed in a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii. serum was assayed for a. phagocytophilum antibodies by ifa. time to first positive results for serology and pcr were compared by paired student's t test. none of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. of the beagles, were positive for a. phagocytophilum dna in blood or synovial fluid or ifa antibodies in at least one sample after tick attachment. antibody titers appeared in of dogs from weeks to (median to st positive weeks ae ). titer magnitude ranged from : to : , . anaplasma phagocytophilum dna was amplified from the blood of of dogs with positive test results ranging from to weeks (median to st positive weeks ae . ). anaplasma phagocytophilum dna was amplified from synovial fluid from of dogs between weeks to (median to st positive weeks ae ). of the dogs, were pcr positive for only one week and dog was pcr positive for two consecutive weeks. of the dogs, were positive for a. phagocytophilum in both blood and joints by dna analysis. anaplasma phagocytophilum dna was amplified from blood more quickly than seroconversion was detected by ifa antibody titer (t À . , p o . ) or dna was amplified from synovial fluid (t . , p o . ). anaplasma phagocytophilum dna can be amplified from the blood prior to development of detectable antibody titers by ifa. amplification of a. phagocytophilum dna from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of a. phagocytophilum infection. canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by anaplasma phagocytophilum (aph) and ehrlichia ewingii (eew), respectively. both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. however, aph and eew have different tick vectors, i.e. ixodes scapularis and ambylomma americanum, respectively, with different, but overlapping geographic distributions. in addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as borrelia burgdorferi (mn) or ehrlichia chaffeensis (ar). therefore, we compared serology and pcr results derived from dogs examined at two private practices located in highly endemic areas for either aph or eew. serum collected between april-december, from minnesota dogs (n ) was tested by snap s dx s and whole blood was tested by aph pcr. serum collected from arkansas dogs (n ) for year beginning in august was tested using microtiter plate elisas for antibodies to eew, e. canis, and e. chaffeensis (ech) while whole blood was tested by ehrlichia pcr. comparisons were evaluated using chi square (Ã) and binomial (w) tests with an alpha of %. the above results indicated that dogs are frequently exposed to both aph and bb in mn, whereas ar dogs are often exposed to eew, but less frequently to ech. antibodies to e. canis peptides were found infrequently in both mn and ar with only seroreactive dogs detected in both locations. active eew infection, as determined by pcr, was four times more frequent in ar pet dog seroreactors as compared to active aph infections among aph seroreactors. although both organisms induce acute disease, the number of aph and eew pcr positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. anaplasma phagocytophilum and ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. while pcr assays that amplify the dna of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. the purpose of this study was to compare the e. canis and a. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals. veterinarians in states (az, md, ct) were recruited to participate in the study based on high prevalence rates for e. canis or a. phagocytophilum infection. blood in edta was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to colorado state and to antech diagnostics. standard operating procedures at each laboratory were followed for total dna extraction and amplification of gapdh as the dna control. at colorado state university, a previously published pcr assay that amplifies the dna of ehrlichia spp., anaplasma spp., neorickettsia spp., and wolbachia was performed on each sample with positive amplicons sequenced to determine the species. at antech diagnostics, a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii was performed. in the study to date, samples from animals ( dogs and cats) have been assayed at both laboratories. dna of a. phagocytophilum ( cats and dogs) and e. canis ( dog) were amplified at both laboratories with a percentage agreement between laboratories of %. the results to date suggest that the assay results of the two laboratories for a. phagocytophilum and e. canis are comparable. ehrlichiosis and bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. human ehrlichiosis is present in peru, with a seroprevalence as high as % in the highlands. bartonella species in humans were also identified in peru since (b. bacilliformis). recently, a new species (b. rochalimae) was isolated from an american woman who became febrile after travelling to peru. dogs can become infected with the same ehrlichia species, and the majority of bartonella species that affect human beings. the role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (tbd), since they share the same environment. the objective of this study was to determine the serological and molecular prevalence of anaplasmosis, ehrlichiosis and bartonellosis in rural dogs in the highlands of peru. a total of healthy adult dogs were enrolled in this study from four communities in the central highlands of peru: ondores, pachacayo, san juan de pachayo, and canchayllo. edta-blood samples were collected from dogs, whereas serum samples were available from dogs. serum samples were tested for ehrlichia canis, anaplasma, borrelia burgdorferi and dirofilaria immitis infections using a qualitative dot-elisa (snap s dx). the edta-blood samples were screened by conventional pcr for the groel gene of the genus anaplasma and ehrlichia, and for the intergenic transcribed spacer of the genus bartonella. speciation was conducted by nucleotide sequencing. bartonella genus dna was detected from seven of the dogs ( . %) and ehrlichia canis dna was detected and sequenced from one dog ( . %). four of the bartonella positive samples were identified by dna sequencing as b. rochalimae (genbank accession numbers hq and hq ). the other three bartonella positive samples were identified as b. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. no dog was infected with anaplasma species by dna amplification, but one dog was seroreactive for this genus ( . %). no specific antibodies against ehrlichia canis and borrelia burgdorferi and no antigens of dirofilaria immitis were detected. this study expands the current knowledge about tbd in peru and describes for the first time the infection of b. rochalimae in dogs in peru. the results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. bartonella spp. dna is commonly amplified from the blood of cats exposed to ctenocephalides felis. in previous work, it was shown that cats administered imidacloprid and experimentally exposed to b. henselae infected cats and c. felis did not become pcr positive for b. henselae whereas untreated cats all developed infection. the purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to bartonella spp. and c. felis in the field lessens prevalence of bartonella spp. infection. veterinary students in tennessee and florida that owned cats that spent at least days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. blood for bartonella spp. pcr assay was collected from the cats seven months after starting imidacloprid administration and assayed at colorado state university. to serve as a control group that was unlikely to have been administered flea control products in the previous months, blood was collected from feral cats during tnr programs in each of the two cities and assayed for bartonella spp. dna. the bartonella spp. dna prevalence rates between the groups were compared by chi square analysis with significance defined as p o . . the overall prevalence rates for bartonella spp. dna in the blood of veterinary student cats ( . %) and the feral cats ( . %) were significantly different (p o . ). the distribution of results is shown in table . the results suggest that florida feral cats were more commonly exposed to c. felis than tennessee feral cats. while the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately days per month and lived in the same cities as the feral cats, so c. felis exposure rates were likely similar. as previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of bartonella spp. amongst naturally-exposed cats. in an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by wolbachia might influence the clinical signs and progression of both diseases. the aims of the present study were ) to determine the prevalence of wolbachia in dogs infected with dirofilaria immitis (di) and other filarial nematodes, ) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and ) to evaluate any associations between leishmania infantum (li) infection, filariosis with or without wolbachia and clinical presentation and outcome. statistical differences between groups were tested for significance by the fisher exact test using spss v. . software (significance: p-value o . ). one-hundred and eighteen owned dogs from southeastern spain presenting for clinical evaluation were included in the study. criteria the results of this study highlight the increased sensitivity of pcr for diagnosis of filariosis, confirm the presence of wolbachia in dogs from the mediterranean basin, show the increased severity of hwd when li-filaria coinfection is present and suggest that wolbachia could play a protective role for leishmaniosis. wolbachia antigens can stimulate a th -type immune response, as has been previously described. however other factors (as treatment with doxycicline) might be responsible for the lower prevalence of wolbachia among filaremic dogs infected with li and further studies must be done to clarify this interaction. the purpose of the present study was investigate the occurrence of leishmaniasis in cats in the municipality of arac¸atuba, sa˜o paulo, brazil, an endemic area for canine visceral leishmaniasis. animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). thirteen ( . %) out of cats studied were diagnosed with visceral leishmaniasis; eight ( %) by parasitological diagnosis through cytological examination of lymphoid organs, six ( %) were considered positive by elisa and one ( . %) by ifat. only two ( . %) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. regarding age five cats ( . %) had between six months and two years, being the others older than years ( . %). only one cat ( . %) was positive for the three employed methods. pcr confirmed leishmania sp infection in nine ( . %) cats, of which six were diagnosed previously by cytological examination, two by elisa and one by the three techniques employed. since its first description in feline leishmaniosis has been reported in several countries. the purpose of this study was to assess the prevalence of leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in brazil. animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). twenty seven ( . %) out of the cats studied were diagnosed with visceral leishmaniosis. twelve ( . %) were positive by parasitological diagnosis; amastigote forms of leishmania sp were identified in lymphoid organs from / ( . %) infected cats, and immunohistochemical technique allowed the identification of nine ( . %) positive animals. the seroprevalence of leishmaniosis was . % ( / ) by elisa and . % ( / ) by ifat. fiv specific antibodies were found in / cats ( . %), of which / ( . %) had leishmaniosis. real time pcr confirmed leishmania chagasi infection in three cats. based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. a recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. with the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. therefore, the purpose of this study was to determine if monitoring the blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs. to this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. the inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. ideally, intervals between testing dates would be between two and five months. radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). urine antigen testing was considered negative if concentrations were less than . enzyme immunoassay units, a reference interval set by the testing laboratory. preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. ceasing treatment month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases. monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. giardia spp. and cryptosporidium spp. are both known to cause infections in dogs and humans in the united states. nevertheless, prevalence rates for dual infection in dogs had not been widely reported. in this study, fecal samples from dogs housed in a northern colorado animal shelter (n ), dogs owned by veterinary students in northern colorado (n ), and dogs from the pine ridge reservation in south dakota (n ) were collected. each sample was assayed with a commercially available fluorescent antibody assay that detects giardia spp. cysts and cryptosporidium spp. oocysts. those samples that were positive for giardia spp. or cryptosporidium spp. with adequate dna available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein- [hsp- ] genes, respectively. overall, ( . %) of the dogs had current evidence of a protozoal infection ( table ). the dogs from pine ridge reservation had the highest prevalence rates for giardia infection and also for dual infections. from the student dogs, sequencing was successful for the three giardia isolates (assemblage d from dogs; assemblage c from one dog) and one cryptosporidium isolate (c. canis). from the reservation dogs, sequencing was successful for nine giardia isolates (assemblage d from dogs; assemblage c from dogs) and one cryptosporidium isolate (c. canis). cryptosporidium and giardia co-infections are commonly detected in dogs; in this study dual infections were more common than cryptosporidium infections alone. further studies will be required to determine the clinical importance of this finding. although the giardia and cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. the current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the veterinary teaching hospital, chiang mai university, northern thailand. fecal samples (n ) were collected and submitted by owners between august to february . demographic and geographic data were recorded. intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available ifa for giardia spp. and cryptosporidium spp. polymerase chain reaction and dna sequencing were performed on all giardia and cryptosporidium positive samples to provide genotyic information. overall prevalence of intestinal parasitic infection in dogs in chiang mai was . %. the most prevalent parasite was giardia spp. ( . %) followed by ancylostoma spp. ( . %), cryptosporidium spp. ( . %), cystoisospora spp. ( . %), toxocara canis ( . %), trichuris vulpis ( . %), coccidian-like ( . %), toxascaris leonina ( . %), and strongyloides spp. ( . %). the prevalence of having at least one parasite in dogs o year, - years, and years were . %, . %, and . %, respectively. of these infected dogs, . %, . %, . %, and . % were infected with one, two, three, and four organisms, respectively. available dna sequences from giardia spp. positive samples were shown to be dog specific. only one adequate dna sequence was available for cryptosporidium spp., which was shown to be c. canis. the findings suggested that intestinal parasitic infection was common in dogs in chiang mai, thailand. dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. regular deworming program is indicated to prevent not only transmission among dogs but also to human. a retrospective study was conducted on parasite positive fecal specimens consisting of canine, feline, equine and from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using standardized methods of parasite concentration: the formalin/ethyl acetate (f/ea) sedimentation concentration and the commercial fecalyzer (flotation) kit procedures. specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. formalin/ethyl acetate concentrations used at a ratio of ml normal saline to ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (saf) solution. flotations with the fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. . ) . the range of parasites recovered from these specimens included flagellate cysts ( total), coccidian oocysts ( total), ova and larvae of nematodes ( total), and ova of trematodes ( total) , and cestodes ( total). recovery rates by fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed fecalyzer in recovery rates. recoveries by f/ea concentrations were higher by . % for giardia, by . % for coccidia and by . % for nematode eggs and larvae. with the exception of coccidian oocysts, based on z-test analyses, recovery rates were significantly higher, at a confidence level of at least %, for all parasites, using formalin/ethyl acetate sedimentation concentration. although capc recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. these results provide good evidence for the use of f/ea concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. cyclosporine inhibits calcineurin-dependent pathways of t-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. little work has been done comparing the effects of these agents on cytokine production in dogs. our study assessed these effects by measuring t-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in activated canine t-cells treated with cyclosporine and dexamethasone. for flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for hours in the presence of cyclosporine ( , , or ng/ml), dexamethasone ( À , À , À m), or cyclosporine plus dexamethasone. for qrt-pcr, whole blood was cultured for hours with the same drugs at the same concentrations, and rna was then extracted from leukocytes. expression of cytokines il- and ifn-g was analyzed in pma/ionomycinactivated t-cells by flow cytometry, and gene expression for il- and ifn-g in activated t-cell populations was assessed via qrt-pcr. flow cytometry and qrt-pcr both demonstrated inhibition of il- and ifn-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. flow cytometry results from the average of samples collected from different dogs are shown in figure a . similar results were achieved using qrt-pcr ( figure b ). suppression of il- and ifn-g in activated t-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine t-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. idiopathic eosinophilic diseases are described in several breeds, but are over represented in rottweilers. the immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. studies in people highlight the importance of cytokines, particularly interleukin- (il- ), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. eotaxin- and eotaxin- also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. the aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy rottweilers, non-rottweilers and rottweilers with non-parasitic eosinophilia. secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options. quantitative real-time reverse transcriptase polymerase chain reaction (qrt-pcr) assays were used to quantify messenger rna (mrna) encoding cytokines il- , il- , il- , il- p , il- p , il- p , il- , interferon gamma (ifn-g) and chemokines eotaxin- and eotaxin- from peripheral blood mononuclear cell (pmbc) samples obtained from healthy non-rottweiler dogs with normal eosinophil counts (n ) and rottweilers with normal (n ), mildly increased (n ) and high (n ) eosinophil counts. quantification of serum ifn-g was also performed using a commercially available canine-specific elisa. all samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin- and - . results were normalised using three stably expressed housekeeper genes (rpl a, sdha and ywaz) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of . no significant differences were found between groups but there was a tendency for ifn-g mrna expression to be lower in the rottweilers with moderate to severe eosinophilia versus control dogs (p . ). this trend was not seen in the concentration of serum ifn-g quantified by elisa as there were no significant differences between normal and diseased animals. in conclusion, there were no significant differences in cytokine mrna profiles between normal dogs and rottweilers with varying degrees of eosinophilia. additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. the presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (rbc) removal process by the mononuclear phagocyte system. an antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate rbc destruction. the study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs. healthy adult dogs (n ) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). blood samples from each animal were collected into edta tubes in two moments: pre (immediately before vaccination) and pos ( to days after vaccination). mononuclear cells were separated by gradient, marked with cfse-fitc and cultured. the stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (aem) and concanavalin a (cona). aem was obtained by hypotonic lysis and tested in two concentrations (m : . ug/ ul; m : . ug/ ul). the proliferation assay was evaluated by flow cytometry and analyzed with specific software. the proliferation index (pi) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. statistical analysis was performed using paired t-test for parametric samples and wilcoxon test for non-parametric samples (a . ). the for the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. e. coli is a common cause of canine urinary tract infection. current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. proanthocyanidins found in cranberry juice inhibit e. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. we hypothesized that purified cranberry extract (ce) inhibits bacterial adhesion to canine uroepithelial cells. five healthy female dogs received an oral ce supplement (vetoquinol; mg ce/tablet) according to body weight for days. voided urine collected from each dog before (pre) and after ( -day) completion of the protocol was membrane filtered ( mm) and stored frozen (- c). bacterial adhesion was determined using an in vitro assay. briefly, urine samples were incubated with an uropathogenic e. coli strain that had been subcultured to promote fimbriae expression. urine samples containing e. coli were next incubated in -well plates containing methanol-fixed madin-darby canine kidney (mdck) cells for -hr ( c) to permit bacterial attachment. after incubation, plates were washed to remove nonadherent bacteria and fresh media added. plates were incubated ( c) for -hr to grow attached bacteria to detection level. bacterial concentration in each well was determined using a spectrophotometer ( nm). results were analyzed using the chi-square test. ce significantly reduced bacterial adhesion by % (n ; p . ) in -day urine samples compared with pre samples. the results show that ce supplementation can reduce adhesion of uropathogenic e. coli to canine uroepithelium and suggests one mechanism by which ce might improve urinary tract health. the purpose of this study was to determine prevalence of urovirulence factors (uvfs) and antimicrobial resistance in canine uropathogenic e. coli (upec) and to evaluate associations between uvfs and antimicrobial resistance. two hundred and twenty-one upec isolates from samples collected from different canine patients submitted to the university of tennessee microbiology laboratory in were evaluated. a multiplex pcr assay was used to detect cnf, hlyd, sfa/foc, and papgiii in dna lysate. in vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class. of the samples, the number of uvf expressed per isolate was: / ( %), / ( %), / ( %), - / ( %), and / ( %). expression of uvf was sfa ( %), hly ( %), cnf ( %), and pap ( %). presence of uvfs was associated with less resistance (p o . ). the combination of hly, cnf, and sfa was associated with less resistance (p o . ). when sfa was present alone, resistance was less (p o . ). average resistance to antimicrobial class by number of uvfexpressed was: uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, and uvf . ae . classes. urovirulence factors were present in a moderate number of upec and correlated negatively with resistance. neither individual nor combinations of uvfs were associated with increased resistance. obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (abuti) than overweight and moderately obese dogs. therefore, a pilot study was conducted to screen for abuti in obese dogs. urinalysis with urine culture and dual energy x-ray absorptiometry (dxa) were performed on fortythree dogs with body fat (bf) percentages ranging from to %. following dxa, subjects were categorized as obese (o)(bf - %, n ) and morbidly obese (mo)(bf %, n ). no dogs had owner-reported symptoms indicative of uti. the prevalence of abuti in o dogs was % (n ) and % (n ) in mo dogs. the dog in the o group with abuti was close to being mo with a bf equaling . %. of the nine dogs with positive cultures, were neutered males and were spayed females. the prevalence ratio of abuti in mo dogs was . , indicating dogs with % or greater bf are . times more likely to have the condition then dogs o % bf. the results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. in conclusion, dogs with body fat percentages greater than % are at risk for abuti, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. calcium carbonate (cac) is recommended to decrease phosphate intake in chronic kidney disease. however, its effect is poorly documented in dogs. our objectives were to assess within-day, postprandial and cac effects on phosphatemia variations in healthy dogs. phosphatemia was measured every hours for hours in eight adult healthy beagle dogs in i) fasted condition and ii) a  crossover design. one group received cac mixed with maintenance diet ( . % phosphorus), while the second group received the diet alone. after a -week wash-out period, groups were switched. a general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (auc - ). a significant (p o . ) circadian variation existed in fasted dogs. the maximum difference (mean: À . mg/dl; % c.i.: À . mg/dl; À . mg/dl) was observed between a.m. and midnight. the auc - with cac ( ae mg.min/dl) was mildly but significantly lower (p . ) than without cac ( ae mg.min/dl). however, it was similar to the auc - in fasted conditions. feeding, with and without cac, has minor effect on phosphatemia. however, circadian variation of fasted phosphatemia might affect its interpretation. gfr measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. accurate and rapid (o min) gfr measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. a recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid gfr determination in larger species. the purpose of this study was to determine correlation and agreement between fluorescent ratiometry (fr) and iohexol plasma clearance (ipc) in dogs over a range of gfrs. acute kidney injury (aki) was induced in female hound-type dogs ( mg/kg gentamicin iv q h), and fr and ipc gfr were simulta-neously determined on days , , and . a -sample, -hr protocol was used for ipc; fr was determined following bolus injection of a dextran conjugate mixture ( -sulfohexamine rhodamine-carboxymethyl kd dextran, -aminofluorescein-carboxymethyl kd dextran) with fluorescence measured over min. gfr was calculated using -compartment model concentration-vs.-time curves for both techniques. correlation was determined via spearman's rho; agreement was analyzed via bland-altman plots. ipc gfr and serum creatinine confirmed progressive aki in all dogs. correlation between fr and ipc was . (p o . ). bland-altman plots confirmed good agreement between techniques with slight underestimation of gfr by fr across most observed values. these results suggest fr is suitable for gfr determination in dogs with aki. importantly, the portable analyzer allowed for point-of-care gfr determination in o min using a peripheral vein. previously presented at the american society of nephrology renal week (related but not identical abstract). dogs with protein-losing nephropathy (pln) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. the purpose of this study was to characterize thromboelastography (teg) in dogs with pln. twenty-eight client-owned dogs with pln (urine protein:creatinine ratio (upc) . ) and control dogs were enrolled. teg parameters, antithrombin activity, serum biochemical profiles, and upc were measured. teg analyses were run in duplicate with kaolin activation; reaction time (r), clot formation time (k), maximal amplitude (ma), and g (global clot strength) were analyzed. a wilcoxon sum rank test was used to evaluate differences between groups. twelve pln dogs ( . %) were azotemic. nineteen pln dogs ( . %) were hypoalbuminemic [serum albumin (salb) o . g/dl]; had salb o . g/dl. dogs with pln had higher k (p o . ), ma (p o . ) and g (p o . ) than controls. r was similar between the two groups. pln dogs with salb o . g/dl had higher g (p o . ) values than dogs with salb . g/dl; however, even pln dogs with normal salb ( . g/dl) had significantly higher g values than controls (p o . ). no significant relationship between upc and g, salb and g, antithrombin and g, or salb and antithrombin was noted using linear regression analysis. these results indicate that antithrombin, salb, and upc cannot be used alone to predict hypercoagulability as assessed by teg in dogs with pln. a comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, cola). the low solubility of cystine in acidic urine predisposes to the formation of uroliths. type i cystinuria in newfoundland and labrador retriever dogs is an autosomal recessive trait caused by mutations in the slc a gene, whereas in other breeds, the cause of cystinuria has not yet been determined. we report here on the clinical, biochemical and molecular features of cystinuria in irish terriers. urine and edta blood were collected from irish terriers from europe and australia. a nitroprusside screening test was used to identify increased cystine in urine. urinary amino acid concentrations were determined by high-pressure liquid chromatography. cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine ( mmol/g creatinine) and/ or a cola concentration of mmol/g creatinine. all females tested nitroprusside negative and had normal urinary cystine (o mmol/g creatinine) and cola (o mmol/g creatinine) concentrations. the intact males that formed calculi as adults exhibited cystine concentrations ranging from - and cola from - mmol/g creatinine. an additional males had similarly high cola values with cystine levels from - mmol/g creatinine. among the affecteds tested, % were nitroprusside positive. the negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. sequencing the coding regions of the slc a and slc a genes from edta blood identified no mutations. the mode of inheritance remains undetermined. however, castration appears to lower the urinary cystine and cola concentrations and to prevent cystine calculi formation, while diet changes have lesser effects. in conclusion, non-type i cystinuria in irish terriers (and several other breeds like mastiffs and scottish deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and cola excretion and fewer and later urolith formation compared to type i cystinuria. castrating cystinuric irish terriers lowers their cystine and cola excretion and thus their risk for calculi formation. cats and dogs that are diagnosed with acute kidney injury (aki) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (ihd). the purpose of this study was to evaluate the long-term outcome of patients with aki treated with ihd, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. medical records of cats and dogs that were diagnosed with aki, treated with ihd, and survived longer than days following the last ihd treatment were retrospectively analyzed. standard methods of survival analysis using kaplan-meier product limit curves and the log-rank test were performed. for all-cause mortality, the median survival time was days ( % confidence interval: , ) for cats and days ( % confidence interval: , ) for dogs. when only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the to day period following the last ihd treatment (p o . for cats, p o . for dogs). this study demonstrates that veterinary patients that are diagnosed with aki, treated with ihd, and survive greater than days after the last ihd treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. renal fine-needle aspiration (r-fna) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. diagnostic utility of fna is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of fna with final diagnosis. objectives of this study were to evaluate the diagnostic utility of r-fna and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. we hypothesized that r-fna is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). dogs and cats that had undergone r-fna from jan , to dec , were identified by database search. patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, r-fna result, and final diagnosis were recorded. patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. a single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-l ratio were evaluated. sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. concordance of r-fna and final diagnosis was determined, and the chi-squared or fisher's exact test were used to determine association of concordance with the above variables; p o . was considered significant. dogs and cats ( animals) met all inclusion criteria. r-fna results were concordant with the final diagnosis in ( . %) patients, discordant in ( . %) patients, and inadequate for cytologic interpretation in ( . %) patients. neoplasia or fip were the final diagnoses in of ( . %) and of ( . %) patients with concordant results, respectively. renal lymphoma (p . ), renal carcinoma (p . ), and renal neoplasia in general (p . ) were not associated with a higher likelihood of r-fna and final diagnosis concordance. there was no association noted between likelihood of r-fna and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables. this study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of r-fna. future studies should use standardized criteria to prospectively identify patients in which r-fna will be performed, evaluate additional variables that may be associated with increased r-fna diagnostic utility, and directly compare the utility of r-fna with that of other diagnostic techniques. feline lower urinary tract disease (flutd) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o years. the goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients. as a study group, cats hospitalized with obstructive flutd at the veterinary university of vienna were examined during a year period ( ) ( ) ( ) . as a control group cats presented for other reasons were randomly chosen during the same time period. the data were examined concerning the signalment and history. furthermore, the long-term outcome was evaluated with a questionnaire. based on assumptions a student's t-test or a chi-square test was used. there were no significant differences in age and breed. the body weight was significant higher in the flutd group than in the control group (p o . ). we could observe a significant risk for the disease of a weight of kg (p o . ). there were significant less cat toilets in the flutd group compared to the control group (p o . ). furthermore we could observe that in the households of flutd cats there was significant less than one toilet per cat (p o . ) and more cats diseased on flutd lived strictly indoor than outdoor (p . ).there were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups. in summary, we could observe that cats over kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive flutd. further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence ( % in months). in dogs, mutation of the urate transporter (slc a ) and portovascular anomalies are common risk factors. however the underlying cause(s) for purine urolith formation in cats is unknown. the purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths. urine concentrations of purine metabolites were measured by high-performance liquid chromatography in cats with ammonium uroliths (cases), clinically healthy, breed and gender matched cats (negative controls), and cats with naturally occurring xanthine uroliths (positive controls). prior to urine collection, all cats were fed a standard maintenance food (protein g/ kcal) for weeks. urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. also, serum pre-and post-prandial bile acid concentrations were measured. when compared to control cats, urinary uric acid concentration was significantly higher in case cats (p . ). xanthine was not detected in the urine of cases or negative controls. a significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (p . , . ).hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. an association between portovascular shunts and purine urolithiasis was not observed in this population of cats. studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. seldi-tof-ms is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. individual low molecular weight (lmw) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; seldi-tof-ms may provide a more global assessment. normotensive, non-azotemic geriatric cats ( years) were recruited prospectively from two first-opinion clinics for routine health screening. at entry cats received a full physical examination, plasma biochemistry, evaluation of total t concentration and urinalysis including urine protein to creatinine ratio. re-examination was offered at and months. cats were divided into two groups based on clinical status at the month re-examination (azotemic; creatinine concentration ! . mg/dl and non-azotemic). optimisation studies were performed to facilitate the automated preparation (biomek ) of cm (weak cation exchange) arrays for seldi-tof-ms analysis (ciphergen enterprise ) of urine samples from cats at entry to the study. results are reported as median [ th , th percentile]. mann whitney u-test and wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. ciphergen express ( . ) software was used to analyse spectral data and a mann whitney u-test was used to identify clusters which differed significantly between groups (p o . ) at entry to the study. twenty non-azotemic cats were recruited, of which cats developed azotemia by months. no significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. as might be expected creatinine increased significantly ( . mg/dl [ . , . ], . [ . , . ], p . ) between study entry and months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration ( . mg/dl [ . , . ], . [ . , . ], p . ). creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. seven clusters with m/z values of , , , , , were found to differ significantly between groups at entry to the study. the low protein concentration of feline urine makes the use of proteomic techniques challenging. however, this pilot study indicates that seldi-tof-ms can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. further work is necessary to identify these proteins/peptides. fibroblastic growth factor (fgf- ) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin d metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. previous studies in cats have shown parathyroid hormone (pth) to be elevated prior to the development of azotemia. the study objectives were to explore the hypothesis that fgf- is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline ckd. healthy, non-azotemic (plasma creatinine concentrations (cr) o . mg/dl) geriatric cats were recruited into the study prospectively and followed for months. at the study end point cats were categorised into the following groups: group (n )-cr . mg/dl, group (n )-cr ! . mg/dl but did not meet the criteria for group and group (n )-cr . mg/dl in association with reduced urine concentrating ability (usg o . ) or demonstration of persistent azotemia (cr . mg/dl). plasma samples were subjected to routine biochemical analysis, intact pth, calcitriol and intact fgf- assay. variables were compared between the groups at the baseline time point. gfr was measured in an additional group of cats ( non-azotemic, iris stage ii, iris stage iii) using a corrected slope-intercept iohexol clearance method. relationships were explored using linear regression analysis and determining the coefficient of determination (r ). results are presented as median [range] . at the baseline time point fgf- concentrations were significantly higher in group ( . [ . - . ], p . ) and group ( . [ . - . ], p . ) compared to group ( . [ . - . ] ). weak positive relationships were identified between fgf- and pth (r . , p . , n ) and fgf- and cr (r . , p . , n ). however, the positive relationships between fgf- and phosphate (r . , p . , n ) and fgf- and calcitriol (r . , p . , n ) were not significant. the additional group of cats in which gfr measurement was performed there was an inverse relationship between fgf- and gfr (r . , p . ). in conclusion, fgf- was elevated in cats prior to the development of azotemia. the role of fgf- in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. however, considering the relationship between fgf- and gfr, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to the progression of this disease. the aim of this study was to evaluate urinary vascular endothelial growth factor (vegf) as a marker of renal hypoxia. cats were recruited through geriatric clinics held at two first opinion london practices. vegf was measured in stored samples using a canine elisa kit validated for use on feline urine and indexed to creatinine concentration to yield a vegf to creatinine ratio (vcr). two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with vcr in cats with ckd. diagnosis of ckd was based on concurrent findings of plasma creatinine ! mg/dl and usg . , with persistence of azotemia for ! weeks. only patients receiving no medical therapies were included. normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure - mmhg on the date of sampling) were not. hyperthyroid cats were also excluded from this cross-sectional study. associations between vcr and clinical data were initially assessed using the spearman's coefficient and mann whitney test. linear regression was then used for multivariate analysis. the second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least months were entered into a randomised cross-over study where they received placebo or benazepril ( . to mg/kg daily) for weeks in turn. vcr on placebo was compared with that on benazepril using the wilcoxon signed ranks test. cats with well controlled hyperthyroidism were included in this intervention study. results are reported as median [ th, th percentile]. vcr was higher ( . [ . , . ] vs. . [ . , . ] fg/g, p . ) in untreated hypertensives (n ) than normotensives (n ). vcr was correlated with pcv (r À . , p . , n ), upc (r . , p o . , n ), plasma phosphate (r . , p . , n ), and usg (r À . , p . , n ), but not plasma creatinine concentration. in the best multivariate model, pcv was associated with vcr independently of upc (r . , n ). vcr was significantly reduced by benazepril therapy ( . [ . , . ] fg/g) compared with placebo ( . [ . , . ] fg/g; p . , n ) with a reduction seen in % of cases. these results suggest urinary vegf excretion is associated with proteinuria in cats with ckd and might be a marker of renal hypoxia induced by low pcv. ace inhibitor therapy might reduce urinary vegf excretion because angiotensin ii causes constriction on efferent arterioles resulting in tubular hypoxia. fgf- is a phosphaturic hormone. fgf- concentrations increase with declining renal function in humans. the objectives of this study were to validate a method for fgf- quantification in feline plasma and to assess the association between fgf- concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (ckd). non-azotemic and azotemic (plasma creatinine concentration (cr) . mg/dl) geriatric ( yrs) cats were recruited into the cross-sectional study from two london first opinion practices. cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. the cats were categorized, using a modified iris staging system, into the following four groups: group (cr . mg/dl), group (cr . - . mg/dl), group (cr . - . mg/dl), group (cr . mg/dl). groups and were further subdivided based on the iris targets for plasma phosphate concentration (po ): group a (po . mg/dl), group b (po . mg/dl), group a (po mg/dl), group b (po mg/dl). fgf- concentrations were measured in feline edta plasma using a human intact fgf- elisa, validated by intraand inter-assay variability and assessment of dilutional parallelism. comparisons between groups were made using the kruskal-wallis test and mann-whitney u test, with statistical significance defined as p o . . bonferroni correction was applied where appropriate (statistical significance then determined as p o . ). results are reported as median [ th, th percentiles]. fgf- concentrations ! pg/ml (upper limit of quantification) were assigned the value of pg/ml. intra-and inter-assay variability of fgf- measurements were o . % and dilutional parallelism between feline samples and the calibration curve were demonstrated. plasma fgf- concentrations increased with increasing creatinine concentrations (group : [ , ] , n , group : [ , ] , n , group : [ , ], n , group : [ , ], n ). fgf- measurements were significantly different between all groups (p . to o . ) except between groups and (p . ). fgf- concentrations were significantly higher in cats with higher plasma phosphate concentrations (group a: [ , ] , n vs. group b: [ , ], n ; p . ) and (group a: [ , ] , n vs. group b: [ , ], n ; p . ). in conclusion, fgf- concentrations were higher in cats with more severe ckd or higher plasma phosphate concentrations as would be predicted from its known biological actions. further work is warranted to explore the role of fgf- in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (pth) and calcitriol in cats at different stages of ckd. progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (aki) in people. pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with aki and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (up), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. we therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with aki than dogs with chronic renal failure (crf). our study objectives were ) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, ) to compare the occurrence of these findings in dogs with aki, crf, or non-systemic illness, and ) to determine if these abnormalities are associated with shorter survival times. records of dogs with renal azotemia evaluated from / / to / / were reviewed; dogs which could be classified as having aki or crf and which had complete thoracic radiograph studies available for review were included. dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. alveolar, bronchial, interstitial, or nodular lesions were described using a -point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. significance was p o . for all analyses. the final study population included aki, crf, and control dogs. crf dogs were older (p o . ) than aki and control dogs. pulmonary-related clinical signs were more commonly diagnosed at first evaluation in aki dogs ( / dogs, . %) than in crf ( / , . %; p . ) or control dogs ( / , . %; p o . ). presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in aki [n , . %, p . ] and crf [n , %, p . ] dogs than in control dogs [n , . %]). there was no association between presence of an alveolar pattern and any other variable. alveolar mineralization was the most common lesion in aki dogs ( / dogs; . %), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in dog each. necropsies had not been performed in any of the crf dogs, but mineralization was not seen in lung tissues from any control dogs (n ). neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with aki (p . and . , respectively) or crf (p . and . , respectively). in this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. the association between and clinical relevance of alveolar mineralization in aki dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from up in people. chronic kidney disease (ckd) is a common cause of morbidity and mortality in cats. the purpose of this study was to investigate the effects of chinese rhubarb (rheum officinale) supplementation on the progression of feline ckd. cats with stable iris stage ii or iii ckd and without comorbidity were included in the study. cats were divided into treatment groups and administered rhubarb extract (group , rubenal s , vetoquinol, mg tablet po q h), benazepril as a positive control (group , . mg/kg po q h), or both (group ). cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. body weight, laboratory data, and blood pressure were recorded every months for up to months. variables between groups at enrollment and within groups over visits were compared with anova and repeated measures ano-va, respectively. a treatment by visit interaction term was included in all repeated measures models. significance was set at p . . except for body weight there was no significant differences between treatment groups at enrollment. there was no significant change in body weight, hematocrit (hct), upc, or creatinine over time as compared to baseline within any group. there was no significant difference between groups over time in regards to change in weight, hct, upc, or creatinine. the treatment by time interaction was non-significant in all models. although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (bun & creatinine) in dogs and cats. cat owners often sprinkle contents onto cat food to facilitate administration. however, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. therefore, we examined the ability of foodsprinkled azodyl to reduce azotemia in cats with ckd. cats with ckd were enrolled in the study and randomized receive azodyl or placebo. owners were provided with - capsules of azodyl prior to enrollment to ensure compliance with administration. baseline blood samples were obtained month apart, and then & months after beginning therapy. clinicians and owners were masked as to medication assignment. we hypothesized that a % decrease in bun and/or creat in the azodyl group would be significant, and set a . . in order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. we compared the % change between groups by mann-whitney u test. bun and creatinine did not differ between groups. based on these results, azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with ckd. whether intact capsule administration reduces azotemia in cats with ckd remains unknown. lower urinary tract disease (lutd) occurs commonly in cats, and idiopathic cystitis (fic) and urolithiasis account for over % of cases in cats less than years of age. although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. in addition to conventional therapy using modified diets, traditional chinese and western herbs have been recommended, although only one, chorieto, has published data. we evaluated commonly used herbal treatments recommended for use in cats with lutd including ( ) san ren tang, ( ) wei ling tang, and ( ) alisma. we hypothesized that these chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite. six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. cats were randomized to of treatments including placebo (p), san ren tang (srt), wei ling tang (wlt), or alisma (a). treatment was for weeks each with a week washout period between treatments. at end of each treatment period, a -hour urine sample was collected using modified litter boxes. urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using equil . b. analysis of variance (anova) was used to analyze data statistically if distributed normally and kruskal-wallis was used to analyze data statistically if data were not distributed normally. a p o . was considered significant. body weights were not different between treatments. no differences were found in -hour urinary analyte excretions, -hour urine volume, urine ph, or -hour urinary saturation for calcium oxalate or struvite between treatments (table) . urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine ph and some geographical and hereditary features in the populations studied have been implicated as risk factors. the effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths. the aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in mexico. this study was developed with dogs with urolithiasis from of the states of the country. chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and x-ray microanalysis. urolithiasis affected nearly the same number of males and females; with ages ranging from two months to years with a median age of years. adult animals were the most affected. breeds more affected were schnauzer miniature, poodle, dalmatian, yorkshire terrier, scottish terrier, chihuahua and bichon frisee´. uroliths were found in the lower urinary tract in . % of the cases. mineral composition of the uroliths was: struvite . %, followed by calcium oxalate . %, purines . %, silicate . %, others . %, mixed . % and compound uroliths . %. struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males. our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths ( . %); in mexico city the frequency reached %. this high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers. acknowledgments: this work has been partially supported by a project of waltham foundation in mexico and the consejo nacional de ciencia y tecnologı´a (conacyt) of mexico. voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. this procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs. the aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. an intravenous catheter was placed, and a combination of medetomidine ( to mg/kg iv) and hydromorphone ( . to . mg/kg iv) was administered, with the addition of ketamine ( mg/ kg iv) in fractious animals; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided. two dogs received the proposed sedation protocol in order to perform urohydropropulsion. dog one was a year old female spayed shih tzu cross, and dog was a year old female spayed standard poodle. ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to mm in dog and . mm in dog . urohydropropulsion was performed and resulted in retrieval of urocystoliths in dog , and approximately urocystoliths in dog . repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. the time from administration of sedation to administration of reversal agent was minutes for dog , and . minutes for dog . records were obtained from dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last years, and the average anesthetic time was minutes. subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a year old neutered male golden retriever with prostatomegaly. medetomidine ( ug/kg iv) and butorphanol ( . mg/kg iv) were administered; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. the time from administration of sedation to administration of reversal agent was minutes for this dog. in conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. this sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. therefore, it is important to know how storage of the sample may affect enzyme activity. we hypothesized that urinary nag and ggt activity would be affected differently in samples stored by refrigeration vs. freezing. thirty-four canine urine samples submitted to the clinical pathology laboratory at kansas state university were included. samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of ml. a complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. nag and ggt activity in the urine supernatant was assessed by colorimetric assay. aliquots of each supernatant were refrigerated for days and frozen at À c for and days at which time enzyme activity was re-assessed. compared to baseline values, enzyme activity for both nag and ggt were stable after days of refrigeration, however there were significant (p o . ) declines in ggt and nag activity when urine supernatants were frozen for and days. treatment for canine urinary tract infections (uti) typically consists of - days of antimicrobial drugs in primary care veterinary practice. compliance with this drug regimen can be difficult for some clients. enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine uti. fluoroquinolones are often used in human medicine to treat uncomplicated utis in women and can be prescribed for as little as days. the primary objective of this study was to determine if dogs with naturally occurring uncomplicated uti have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol. client-owned adult dogs with naturally occurring, uncomplicated uti were prospectively enrolled in a multi-center clinical trial and assigned to of groups in a randomized blinded manner. group received treatment with - mg/kg oral enrofloxacin once daily for consecutive days. group dogs were treated with . - mg/kg oral amoxicillin-clavulante twice daily for days. both groups had urinalyses and urine cultures submitted on day , , and . at the time of this interim analysis, thirty-six dogs have completed the trial. bacteriological cure was achieved in dogs ( %) treated with enrofloxacin and dogs ( %) treated with amoxicillinclavulante, respectively. these data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine uti in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. azotemia is frequent in dogs with dmvd (nicolle et al; jvim ; : - ) and could result from renal hemodynamic alterations. renal resistive index (ri) allows assessment of renal vascular resistance. the aim of this prospective study was to assess ri in dogs with different dmvd stages. fifty-five dogs with dvmd were used (isachc class (n ), (n ), and (n )). physical examination, renal ultrasonography and echo-doppler examinations were performed in awake dogs by trained observers. plasma creatinine, urea and nt-probnp were measured. statistical analyses were performed using a general linear model. whereas ri of renal and arcuate arteries were unaffected by isachc class, left interlobar ri increased (p o . ) from . ae . (mean ae sd) in class to . ae . in class . left interlobar ri was also higher (p o . ) in azotemic ( . ae . ) than in non azotemic ( . ae . ) dogs. similar findings were observed for right interlobar ri. a positive effect of nt-probnp (p . ), urea (p o . ), creatinine (p . ), urea-to-creatinine ratio (p o . ), left atrium-to-aorta ratio (p o . ), regurgitation fraction (p . ), systolic pulmonary arterial pressure (p o . ) and shortening fraction (p . ) on ri was also observed. in conclusion, interlobar ri increases with the severity of dmvd and azotemia. a cause-effect relationship remains however to be established. antibodies against alpha-enolase are associated with immunemediated nephritis in people. it was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. the purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats. clinically stable privately owned cats ! years of age, with and without azotemia (creatinine mg/dl), and with an available vaccine history for ! years were recruited for the study. sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. results from cats with and without azotemia were compared by student's -tailed t test or fisher's exact test with significance defined as p o . . median ages were years (range: - ) and years (range: - ) for cats with (n ) and without azotemia (n ), respectively. there was no significant difference in vaccine events (number, type, or route of administration) between groups. azotemic cats ( . %) were more likely than normal cats ( . %) to be positive for antibodies against alpha-enolase (p . ). in addition, alpha-enolase antibody concentrations were greater (p . ) in azotemic cats (mean % elisa . %) than cats with normal creatinine concentrations (mean %elisa . %). results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. additional prospective evaluation in a larger number of cats is indicated. aki is used in human medicine as a predictor of mortality based on the akin (acute kidney injury network) scoring system which utilizes relative increases in creatinine to determine stage. with this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by akin score) increases. accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. we retrospectively evaluated dogs and cats ( ) ( ) ) that had ! creatinine measurements within days, and whose first creatinine was o . mg/dl. patients were categorized as: level (no aki); level (second creatinine value o . mg/dl, but creatinine increased ! . mg/dl); or level (second creatinine . mg/dl with a creatinine increase ! . mg/dl). thirty and day survival for each level was compared to level . adjusted odds ratio (or) in dogs for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level ; or for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level . for cats, or at days was . (ci %, . - . ) for level and . (ci %, . - . ) for level ; or for day survival was . (ci %, . - . ) for level and . (ci %, . - . ) for level . thus, detecting increasing stage of aki helps predict mortality in dogs and cats. abstract n/u- feline urate urolithiasis: cases ( - . j dear , r shiraki , a ruby , j westropp . william r pritchard veterinary medical teaching hospital, university of california, davis, ca, gerald v. ling urinary stone analysis laboratory, university of california, davis, ca and the department of veterinary medicine and epidemiology, university of california, davis, ca. feline urate urolithiasis accounts for % of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies. the objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present. a retrospective analysis of feline urate uroliths submitted to the stone lab between january -december were included. from these data, primary veterinarians were solicited to submit records. furthermore, all records from cats with urate uroliths from the vmth were analyzed separately. records were received from the primary care veterinarians. sixteen cases were identified from the vmth. median values for the cbc and chemistry panels available were within the reference ranges provided, with only a few outliers present. of the cats with radiographic reports, ( %) had visible evidence of uroliths. two external cases had confirmed pss; five cases from the vmth had a pss. cats with urate uroliths and pss were younger than cats without a documented hepatopathy ( years vs. years). the siamese breed was overrepresented. all stones were ammonium hydrogen urate. the pathogensis of urate uroliths in cats is poorly understood. most cats were not completely evaluated for pss, however, there were few clinicopathologic parameters which indicated hepatopathies were present. further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. -indoxyl and p-cresyl sulfate (is, and cs, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (ckd). in the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in ckd appear to be driven by the free fraction. the objectives of the present study were to assess the normal, physiological levels of is and cs in healthy cats and to evaluate the correlation of the respective free and protein-bound levels. blood samples were taken from clinically healthy adult cats enrolled at five participating veterinary practices in germany. after centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. serum creatinine and urea levels were quantified by vettest s (idexx laboratories, inc.). total and free is and cs, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the % confidence interval for reference values of is and cs, ii) a calculation of various pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) wilcoxon-mann-whitney utest for an estimation of the effect of hemolysis on serum is or cs levels. six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values. median levels of is in cat serum were . mg/l with upper and lower bound % confidence intervals at . and . mg/l, respectively. the corresponding median levels of cs were . mg/l (median) and . vs . mg/l (upper vs lower bound levels, respectively). these values showed a low, non-significant correlation with serum creatinine or urea levels. however, is and cs serum levels were moderately correlated (total levels r . , p o ). their respective free levels constituted about % of the total serum levels (r ! . , p o . ). non-hemolytic samples tended to yield lower values than hemolytic samples. due to the low number of hemolytic samples (n ) , the group difference could, however, not be statistically confirmed. the results indicate that it is sufficient to determine total levels of either is or cs in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline ckd. reference values are provided for orientation towards clinically relevant changes. disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (fic). studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent -hydroxy-prostaglandin dehydrogenase (pgdh) activity and subsequent metabolism of cytoprotective prostaglandins. the goal of this study was to confirm persistent pgdh expression in fic bladders compared to desmoplakin i ii expression, a marker of urothelial differentiation. urinary bladder biopsy specimens were obtained by cystotomy from symptomatic cats with chronic fic. cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, nsaids, antihistamines, antidepressants, or glycosaminoglycans were excluded. urinary bladder tissue specimens were also obtained from untreated clinically normal specific-pathogen-free cats. tissue specimens were fixed in buffered % formalin and embedded in paraffin. tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. tissues were stained for pgdh using a rabbit anti-pgdh antibody, an isotype negative control or goat anti-desmoplakin i ii and developed using the avidin-biotin peroxidase complex method. all fic ( / ) and normal ( / ) cat bladder samples showed similar staining of urothelial cytoplasm for pgdh. however, desmoplakin i ii staining, found on the luminal cell surface in / normal tissues, was disrupted in / fic bladder samples. desmoplakin i ii staining confirmed altered urothelial differentiation in fic cats. however, pgdh expression remained intact in fic samples. we hypothesize that pgdh expression in fic may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. additional studies will explore this hypothesis. the university of tennessee college of veterinary medicine's picture archiving and communication system was searched over a month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. one hundred and three cats were identified (age range o to yrs; median yrs). kidney size was determined based on radiographic and ultrasound findings. of the included cats, . % had two normal sized kidneys, . % had one small and one normal, . % had one large and one normal, . % had two small, . % had two large, and . % had one small and one large kidney. the presence of mineralization, uroliths and hydronephrosis was also noted. medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. no significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. the presence of uroliths was significantly associated with hydronephrosis. of the cats with at least one large kidney, ( %) had hydronephrosis. of the cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, ( . %) had at least one small kidney. small kidneys were commonly found in older cats, however, this correlation was not statistically significant. based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. this study aimed to evaluate ife, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis. one australian shepherd and two miniature australian shepherds were included. all three dogs were homozygous for the mdr- gene mutation. two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. ivermectin was administered to the third dog ( mg/kg po). all three dogs exhibited tremors, ptyalism, and cns depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. a % formulation of ife (liposyn ii, hospira) was administered as a bolus ( . ml/kg) followed by a slow iv infusion ( . - ml/kg over minutes). no change was observed in the neurologic status of any patient. lipemia visible upon blood sampling persisted for hours in one dog. no other adverse effects were noted. serum ivermectin levels confirmed ivermectin exposure in each case. in this study, ife administration did not result in clinical benefit in cases of ivermectin toxicosis. brain ivermectin concentrations in mdr mutant/mutant genotype dogs may be too high to be overcome by ife. additionally, these dogs may lack p-glycoprotein-mediated biliary clearance mechanisms needed for optimal ife function. further investigation is needed to determine the utility and optimal dosing of ife in canine toxicoses, to characterize its safety, and to determine how mdr- status may alter the efficacy of ife in treatment of canine ivermectin intoxication. rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. rufinamide is administered at a dose of mg/kg divided twice daily to achieve therapeutic concentrations of mg/ml. the objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy. six healthy adult dogs were included. the pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of . mg/kg (range . - . mg/kg), extrapolated from the dose used in human patients. dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. plasma rufinamide concentrations were determined using high-performance liquid chromatography. pharmacokinetic data were analyzed using winnonlin version . . no adverse effects were observed. the mean terminal half-life was . /À . hours. the mean maximum plasma concentration was . /À . mg/ml and the mean time to maximum plasma concentration was . /À . hours. mean clearance was . /À . l/hr. auc inf was . /À . mgÃh/ml. results of this study suggest that rufinamide given orally at mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. the aims of this study were to investigate the abg for (i) the prevalence of skull abnormalities; (ii) the prevalence of sm; (iii) an association between lateral ventricular size, cerebellar size and sm; and (iv) associations between sm, skull abnormalities, csf pleocytosis and clinical signs. seventy-six abgs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal mri evaluation ( . t general electric signa hdx, milwaukee, wi). all dogs were evaluated neurologically, recording deficits and the presence of spinal pain. sequences acquired included t w, t w pre-and postcontrast, and t w flair, sagittal and transverse. cervical spinal cord central canal (cc) and or syrinx size and its percent area of spinal cord was measured using osirix s . the presence of chari-like malformation (cm) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume qa respectively. forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of mri and the white blood cell (wbc) count was recorded. student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs. the mean age of the males ( intact) and the females ( intact) was . months (range - ; median months). neurological deficits and neck pain were noted in ( %) and ( . %) of dogs respectively; dogs ( . %) exhibited both. cerebellar deviation and vermal herniation were present in ( . %) and ( . %) dogs respectively; twenty-three dogs ( . %) had both. mean height of the cc was . mm ( - . mm). forty ( . %) ccs were greater than mm in height; the mean length of these lesions was . vertebrae ( . - ). mean csf wbc count was . /ml ( - ). syrinx height and extent were significantly higher in dogs with neurological signs (size p . ; extent p . ). there were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. there were no associations of syrinx height or extent with csf wbc count or age of dog. intact females had a significantly lower syrinx extent than intact males (p . ). there were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. there was a significant negative association of ventricular percentage and cerebellar percentage (p o . ). there was a significant association of ventricular percentage with syrinx percentage (p . ) and height (p . ). this study suggests that sm and cm are prevalent in abgs. syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. however, sm may not be associated with cm as defined by cerebellar herniation and deviation and is not associated with csf inflammation. the power tissue resection device (ptrd) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. this device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. the purpose of this study is to describe the use of the ptrd for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the ptrd. fifteen cadaveric lumbar spines were randomly placed into three study groups: group was the control group on which no fenestrations were performed, group was the manual fenestration group and group was the ptrd fenestration group. the effectiveness of fenestration via both manual and ptrd was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. discs with lower ratios were more effectively fenestrated. results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the ptrd ( . ae . ) as compared to manual fenestration ( . ae . ). these results did not show statistical significance. when fenestrated samples were compared to control samples ( . ae . ), there was a statistically significant reduction in ratios. in conclusion, the ptrd is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration. according to the human who classification gliomatosis cerebri (gc) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. gc has not been reported to occur as a hereditary disease, neither in man nor in animals. here, we report the temporally clustered occurrence of gc in a family of bearded collies. a years old female bearded collie with forebrain signs was presented. differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. within a time period of months, offspring of this bitch were presented with similar clinical signs. two dogs were full siblings ( males). the remaining female dog originated from a match with a different male dog. mri was performed in all dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. the ill defined lesion showed mainly a white matter distribution with hyperintense signal in t -w and flair images and iso-to hypointense signal in t -w images without contrast enhancement. the lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface. neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all dogs. based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of gc was made. this is the first report of familial occurrence of gc, which is likely the result of a germ-line mutation. several human hereditary cancer syndromes are associated with cns tumors including amongst others the li-fraumeni cancer family syndrome (p mutation), neurofibromatosis (type and ) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. in the dog, hereditary cns tumors are not known. the exact mode of inheritance and putative gene mutations of gc in this bearded collie family are currently under investigation. preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. mutations in the tp gene were not found following amplification and sequencing of exons - in affected dogs. previously presented at the ecvn annual meeting in cambridge, uk. the gm gangliosidoses are characterized by a deficiency of bhexosaminidase. there are two isoforms: hex a composed of an a and b subunit encoded by hexa and hexb genes respectively and hex b with two b subunits. hex a requires an activator encoded by gm a. two japanese chin dogs with confirmed gm gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase a activity, a pattern associated with the ab variant in humans and consistent with prior reports in the breed. this study was performed to identify the mutation responsible using resequencing with an applied biosystems xl dna analyzer as previously described (awano ). mutations in gm a cause the ab variant in humans, but resequencing gm a revealed no mutation that could account for the disease. resequencing hexa and hexb revealed a c. g a mutation in hexa which was homozygous in both affected dogs. sixty-five normal japanese chin dogs were screened for the mutant allele; were homozygous for the ancestral allele and heterozygous. this mutation predicts a p. e k substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of gm ganglioside. substitution of a lysine residue at this site is likely to eliminate subunit a enzymatic activity. the apparently normal levels of hexosaminidase a activity in affected dog samples may be a result of b subunit overexpression. human hex b possesses low levels activity against the artificial substrate used to assess hex a activity, but specificity of activity of the canine enzyme is not known. previously presented at the american society for neurochemistry: additional data in this abstract. phenytoin (pht) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. iv fosphenytoin (fos) is a pht pro-drug which causes less administration related adverse events. while the short half-life of pht is not suitable for chronic oral therapy in dogs, iv fos has not been studied. two dogs received mg/kg phenytoin equivalent (pe) and two dogs received mg/kg pe of fosphenytoin intravenously at a rate of mg pe/min. blood for plasma levels were drawn at time-points over hours; total and unbound drug levels were measured by hplc. vital signs including ekg, blood pressure, and neurological examination were monitored. the half-life of metabolism of fos to pht was $ min, with % of fos metabolized to pht by minutes. eighty to % of pht was protein-bound during the first minutes after dosing, compared to - % in humans. the elimination half-life for total pht ranged from . - . hours and for unbound pht ranged from . - . hours. dogs receiving mg/kg pe intravenously achieved unbound pht plasma maximum concentrations of . - . ug/ml at minutes, consistent with human loading dose levels. adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. all dogs were clinically normal within minutes of all doses. a mg/kg pe dose of iv fos appears adequate for production of pht levels predicted to be effective for the treatment of canine seizure emergencies. further studies in clinical canine patients are warranted. acquired myasthenia gravis (mg) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia. therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. therapeutic plasma exchange (tpe) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. tpe is used routinely to treat mg in human patients with severe disease or disease unresponsive to conventional therapy. we report the successful use of tpe to treat large breed dogs with confirmed mg (aceytlcholine receptor antibody concentration: . and . nm/l, respectively; normal concentration: o . nm/ l) that was severe and not adequately responsive to traditional therapies. both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia. three tpe treatments ( plasma exchange each) were performed over and days, respectively, in each dog without complication. both dogs became ambulatory within days of starting tpe treatment with subsequent resolution of regurgitation and megaesophagus. pyridostigmine was continued during tpe sessions and discontinued in both dogs within - months. both dogs remain asymptomatic and have had no recurrence of mg during and months of follow-up, respectively. tpe is a viable treatment option for dogs with mg that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. tpe may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. the aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst. medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the veterinary teaching hospital between and were reviewed. signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. all meningiomas were histologically classified and graded following the international who human classification for cns tumors. seven dogs were included, males and females. median age at presentation was . years (range, . - . years), and median weight was kg (range, - kg) . median time between onset of clinical signs and diagnosis was days (range, days - year). cerebrospinal fluid (csf) analysis was performed in dogs, showing increased protein concentration in cases, and being normal in the other . spinal radiographs revealed vertebral canal widening in one case. myelography ( / ) showed intradural/extramedullary lesions in three cases, one of them consistent with a csf-filled subarachnoid cavity, and an extradural lesion in one case. magnetic resonance imaging (mri) was performed in all cases and revealed mild to marked hyperintensity on t w and precontrast t w images and homogeneous contrast enhancing (ce) intradural/extramedullary masses ( cervical and thoracic) in six cases, with one of these showing an additional intramedullary ce pattern. a dural tail was identified in two dogs. one dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. this lesion was hyperintense on t w, hypointense on t w and flair images, and did not enhance. it was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade i cystic meningioma. five other cases underwent cytoreductive surgery, two transitional meningiomas (grade i) that survived (alive at the time of writing) and months; and three anaplastic meningiomas (grade iii) that survived - . months before neurological deterioration and euthanasia. another anaplastic meningioma was euthanized right after diagnosis. there are few reports grading canine spinal meningiomas, with most being grade i or ii. of the few grade iii tumors reported, only one had been treated surgically and was euthanized days later because of neurological deterioration. we report four grade iii (anaplastic) meningiomas, three of which surgically treated and with longer survival times. finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. a recent meta-analysis showed that higher plasma glucose, lower plasma ph and lower hemoglobin at admission were associated with increased risk of death in human head trauma. the goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma. fifty one dogs presenting to the cornell university hospital for animals with head trauma from to that had a blood gas analysis done within hour of presentation were eligible for inclusion. parameters assessed included glucose, base excess (be), anion gap (ag), ph, hemoglobin, and sodium. biochemical data were found to be normally distributed using the kolmogorov-smirnov test. t-tests or welch tests were used to compare parameters between survivors (s,n ) and non-survivors (ns, n ). of glucose, be, ag, ph, hemoglobin, and sodium, only mean glucose (s mg/dl, ns . mg/dl, p . ) was significantly different between groups, although there was a trend for a difference in mean be (s À . , ns À , , p . ). logistic regression analysis showed that of the parameters, only be was independently associated with outcome (odds ratio . , % ci . - . , p . ). these results suggest that two easily measured biochemical parameters (glucose and be) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. type i intervertebral disc disease (ivdd) commonly affects chondrodystrophic dogs. neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. the purpose of the study was ) to determine the relationship between serum and csf lactate levels and ) to compare lactate levels with neurological outcome following decompressive surgery in dogs with ivdd. healthy, chondrodystrophic dogs diagnosed with ivdd localized to the t -l spinal cord were included. serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. in patients with hyperlacatemia at extubation, additional samples were obtained. csf was analyzed for lactate concentration. neurological status was recorded at presentation and multiple times during the recovery period. dogs were included in the study ( - years old). / dogs had normal lactate levels throughout the study. / dogs had serum hyperlactatemia prior to anaesthetic induction; / dogs returned to normal during anaesthesia and / dogs had continued hyperlactatemia until the end of the observation period. neurological status of the dogs varied similarly between all groups. in / dogs where csf lactate levels were measured, initial serum levels were lower than csf lactate levels; in / dogs where csf and serum were collected simultaneously, serum lactate concentration was consistently lower than csf lactate. no association between presenting neurological status or neurological outcome and serum or csf lactate concentration was made. neither serum nor csf lactate concentration is useful for predicting neurological outcome in dogs with ivdd. chiari-like malformation (cm) has been associated with syringomyelia (sm) in cavalier king charles spaniel (ckcs) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. the objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia. three dimensional models were created using t -weighted transverse magnetic resonance images in the commercial software package mimics s . volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups. forty-five small breed dogs (sb), ckcs and labradors (ld) were compared. as sm is thought to be a late onset disease process, two subgroups were formed for comparison: ckcs younger than years with sm (group ) and ckcs older than years without sm (group ). ckcs had a larger cerebellar caudal cranial fossa percentage than the other groups . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). the cerebellar brain percentage was also larger in ckcs compared to the other groups (ckcs . % [ . - . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). group had a significantly larger cerebellar caudal cranial fossa percentage than group ( . % ae . vs. . % ae . , p . ) and a significantly larger cerebellar brain percentage ( . % ae . vs. . % ae . , p . ). our findings show that the ckcs has a relatively larger cerebellum than small breed dogs and labradors and there is an association between increased cerebellar volume and sm in ckcs. chiari-like malformation (cm) is nearly omnipresent in the cavalier king charles spaniel (ckcs) breed. the mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (sm). there is currently a lack of information if the morphological changes seen in ckcs with cm are progressive or non-progressive. in this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (mr) imaging of the brain of the same dog. electronic patient records were reviewed for ckcs with cm which had two separate mri scans, which were a minimum of months apart. ckcs with diseases affecting measurements were excluded. for the volumetric measurements three-dimensional models were created using t -weighted transverse mr images in the medical imaging software (mimics v . , materialise n.v, ) . volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. the volume of the ventricular system was recorded as a percentage of total parenchymal volume. data was assessed for normality and the appropriate statistical test was used to compare means/medians. twelve ckcs were included with a median scan interval of . months ( - months). the size of the foramen magnum increased significantly between the first and second scan ( . ae . cm vs. . ae . cm; p . ), as did the length of cerebellar herniation ( . ae . cm vs. . ae . cm; p . ) and the caudal cranial fossa percentage ( . % [ . - . %] vs. . % [ . - . %]; p . ). there was no significant difference noted between the two time points in any of the other volumetric measurements ( this work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. this leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. the cord dorsum potential (cdp) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. cdps have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. this study was undertaken to determine whether cdps could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality. ten adult dogs weighing from . to . kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. recording needles were placed onto the dorsal lamina at intervertebral sites from l /s to l / . stimulations were made on the lateral aspect of the caudal vertebrae approximately - cm from the tail base. recordings from stimulations were averaged. cdps were recorded successfully in all dogs. onset latency varied from . to . ms. the cdp was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in dogs. administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the cdp. these data show that cdps can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. canine intracranial glial tumors and many human brain tumors express heat shock proteins (hsps) associated with their degree of malignancy. the up-regulation of hsps during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. ki expression and ec have been strong indicators of cell proliferation and dedifferentiation, respectively.the aims of this study were to determine (i) if canine meningiomas express hsp and/or hsp ; (ii) whether the expression of the hsps was associated with ki and/or e-cadherin (ec) expression; and (iii) whether peritumoral edema was associated with hsp, ki and/or ec expression. forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-hsp , or antibodies. these tumor samples were also immunohistochemically stained for ki and ec expression. canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express hsps. skin was used as control for ki and ec. four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for hsps and ki ; a qualitative assessment was used for ec. all analyses were performed using sas v . (cary, nc). descriptive statistics of staining percentages were calculated for all tumors tested. simple pearson's correlation was used to test for correlations of ec area with hsp areas and ki- percent positive cells and of ec intensity with hsp intensities and ki- percent positive cells. all hypothesis tests were sided and the significance level was a . . thirteen meningiomas had mr images quantitatively evaluated for peritumoral edema using t flair sequences. the edema index (ei) was evaluated for an association with hsp , hsp , ec and ki expression. hsp was expressed in % (mean . % of cells; range - %), hsp in % (mean . % of cells; range - %) and ec in % of meningiomas. there was no association demonstrated between either hsp expression variable and ec or ki- expression. there was also no association between the ec expression variables and ki- . however, there was a significant negative association between hsp extent (p . ) and area (p . ) with ei. in conclusion, hsp and expression was demonstrated in canine intracranial meningiomas but was not associated with ki- or ec expression. this study suggests that hsps may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. however, hsp may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. an extended release (xr) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. the potential benefits of the xr formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. the aim of this study was to compare the pharmacokinetics of levetiracetam xr tablets with immediate release (ir) tablets following single dosing in dogs. five clinically and neurologically normal mixed breed dogs were used in a cross-over design. all dogs (mean body weight . kg; range . - . ) had normal hematology, serum chemistry and urinalyses. following a hour fast, each dog was administered oral ir levetiracetam ( mg; mean dose . mg/kg; range . - . ). heparinized blood for drug analysis was taken from each dog prior to administration and . , . , . , , , and hours after. blood was immediately centrifuged and supernatant plasma was stored at À c until analysis. after a day wash-out period, each dog was administered mg oral xr levetiracetam and blood samples were taken at identical timings. plasma samples were thawed at room temperature before preparation by solid phase extraction for hplc analysis. reverse phase chromatographic separation was performed. levetiracetam and an internal standard were detected using ultraviolet spectroscopy at nm. concentrations of levetiracetam were determined by peak area comparison to the internal standard. mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for xr formulation. no adverse clinical effects were noted in any of the dogs. the auc associated with xr was hr ug/ml, a . fold increase over that with ir ( . hr ug/ml). the absorption half-life was . hr with xr and . hr with ir, a . fold difference. the elimination halflife was . hr with xr and . hr with ir, a . fold difference. the tmax associated with xr . hr and . hr with ir, a . fold difference. the cmax associated with xr was . mg/ml and . mg/ml with ir, a . fold difference. the plasma concentration of ir levetiracetam was not detectable at hr after administration whereas it was greater than mg/ml at hr after xr administration. based on the auc data, there is an approximately fold increase in bioavailability of the xr compared to the ir formulation. the cmax was approximately times greater following xr administration and a high plasma level in excess of the suggested canine therapeutic concentration ( mg/ml) for at least hours. although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of xr over ir suggests that single, daily administration could be efficacious. thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. the aims of this study were to retrospectively evaluate the effect of different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (tl) and lumbosacral (ls) spinal trauma. medical records were searched for dogs with radiographic evidence of a tl or ls vertebral fracture and or luxation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; signalment, body weight and duration of disease were recorded. dogs were retrospectively scored neurologically ( - ; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. lesion location was classed as t -l and l -s ; dogs were evaluated as one group and as two separate groups with respect to outcome. a subset of lesions were classed as cord compression or not based on advanced imaging. three repair techniques were evaluated (i) pins and polymethylmethacrylate (pmma); (ii) screws and pmma; and (iii) spinal stapling. regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). simple bivariate analyses were performed to investigate for variables predictive of a successful outcome. fifty-nine dogs were included. twenty-eight dogs were classed as t -l and were l -s . there were dogs with fractures and with luxations; dogs had both. thirty-one of dogs evaluated had spinal cord compression. ten dogs were repaired with im pins and pmma, dogs with screws and pmma and dogs with spinal stapling. overall, there was a . % success rate; there was no significant difference in outcome between the anatomic sites (p . ). all dogs initially graded as - pre-operation were classed as a successful outcome after at least one week following surgery; % of dogs initially graded as (plegic with pain perception) were classed as successful recovery. one dog ( . %) initially as graded as (plegic with no pain perception) had a successful outcome. a low admission score was statistically predictive of a successful outcome (p o . ). surgery type was not associated with a successful recovery (p . ). signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome. from this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. the choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. cranial thoracic intervertebral disc disease (ivdd) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest german shepherd dogs (gsd) can share some predisposition for this disorder. the objective of the study was to retrospectively evaluate through mri if cranial thoracic ivdd is significantly more common in gsd compare to other large breed dogs. a search was done through database of the ontario veterinary college. any gsd were a spinal mri including t -t spine was performed was recruited. a group of large-breed non-gsd was used as a control. in the midsaggital t wi plane, three variables were assessed and graded for each intervertebral disc space t -t : spinal cord compression (scc), disc degeneration (dd), and herniation. wilcoxon sign rank test was used to assess if scores were different between groups. exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. gsd and large breed non-gsds were recruited. the gsd group had significantly higher scores than the non-gsd for scc, and herniation. regarding the individual intervertebral discs, in the gsd group t - , t - , t - discs had significantly an increased risk for scc, and t - for herniation. the results of this study show that gsd have a higher risk of cranial thoracic disc ivdd than other large breed dogs. that risk was higher in discs t -t , t - , and t - , particularly in t - . genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose gsd to this lesion. diskospondylitis is a common disease of the canine spine; however, few reports of mr imaging findings in dogs are available. the purpose of this study was to describe the signalment, clinical and mr imaging features in affected dogs. twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, mr imaging, and urine, blood, csf and/or intervertebral disk cultures were included. large breed dogs ( kg) accounted for of the cases. the mean age was . years with males and females equally represented. most dogs ( / ) were ambulatory with varying degrees of pain and paresis. mr imaging characteristics of sites were reviewed. on t w images, vertebral endplates were of mixed signal intensity ( / ) while the vertebral body was hypointense ( / ). the intervertebral disk space was hyperintense on t w ( / ) and stir ( / ) images and mixed signal intensity ( / ) on t w images. paravertebral soft tissue hyperintensities were noted on / t w and / stir images. contrast enhancement occurred at / endplates and / intervertebral disk spaces. only / vertebral bodies and / parvertebral soft tissues contrast enhanced. intramedullary spinal cord t w hyperintensity was noted at / sites. spinal cord or cauda equina compression occurred at / sites. based on the spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (p . ). the incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. these procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. a proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. the purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning. ten canine cadavers had both procedures performed on opposite sides to access the t - , t -l , and l - spinal canal. measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. the surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. dorsal and ventral remnant lamina heights were also compared. the height of the defect relative to the spinal canal was - % with hemilaminectomy and - % with pediculectomy. the observed difference in defect height of - % (p o . ) and varied with spinal canal height. dorsal remnant lamina height was . - . % of spinal canal height with hemilaminectomy and - % with pediculectomy. ventral remnant lamina height ranged from - % and . - . %, respectively, though the difference was not statistically significant. while a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. the difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. no effect of vertebral site was detected. of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of % of the articular facet height. this result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. low level laser therapy (lllt) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, tmj disorders, chronic joint disorders, and wound healing. research is currently on-going to determine the adequate wavelengths to promote effective treatment results with lllt in these conditions. it is purported that lllt acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. in this study, we hypothesized that dogs treated with lllt in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation. seventeen dogs ( dachshunds, chihuahuas, french bulldogs, lhasa ahpsos, and each of a pembroke welsch corgi, and a miniature poodle) were selected and divided into two groups. the dogs ranged in age from to years old, weighed between and pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). one group received laser treatments on days through of hospitalization. the second group did not receive lllt, but followed the same peri-operative medication protocol. the laser used in this study was an erchonia laser model pl ( nm). the hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (ivdd) with pulse rate ranging from hz to hz. all dogs received advanced imaging pre-operatively with myelogram or mri. results of the study revealed that treatment with lllt of nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to ivdd after hemilaminectomy procedures. dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. a lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. lllt did not appear to have an effect on this result. however, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. thus the purpose of this study was to assess the normal distribution of fdg uptake of canine brain structures using a high-resolution research tomography-pet and t-magnetic resonance imaging (mri) fusion system. fdg-pet and t -weighted mr imaging of the brain were performed on healthy laboratory beagle dogs. acquired pet and mr images were automatically co-registered by the image analysis software. on mr images, regions of interest (roi) were manually drawn over intracranial structures, including gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). a standard uptake value (suv) and relative suv ratio (rsuv suv of roi/suv of whole brain) were calculated for each roi. t-mr images compensated the low anatomical resolution of pet qj;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. among gross structures, mesencephalon and ventral metencephalon had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) fdg uptake respectively. when suvs were calculated on detailed regions, rostral colliculus and corpus callosum had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) value respectively. these data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. degenerative lumbosacral stenosis (dlss) is a multifactorial condition affecting predominantly large breed dogs. the combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. previous reports describe urinary and fecal incontinence in severely affected dogs. the objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of dlss in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment. medical records from the university of georgia and the university of missouri between and of dogs were reviewed. inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of dlss. dysuria was defined as inability to initiate or sustain a urine stream. urine residual volume was evaluated postvoiding. dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry ( ) and cystometry ( )), ultrasonography ( ), and urine culture ( ). presumptive diagnosis of dlss was based on imaging using plain radiography and epidurography ( ), computed tomography ( ) or magnetic resonance imaging ( ). breeds represented included the german shepherd dog (n ), golden retriever (n ), burnese mountain dog (n ), and each labrador retriever, weimaraner, rottweiler and mixed-breed. all dogs were male. were intact at onset of clinical signs. median body weight was . kg (range . - ) and median age was years (range - ). median duration of clinical signs prior to admission was months (range . - ). other pertinent presenting clinical signs included dyschezia ( ), fecal incontinence ( ), general proprioceptive ataxia ( ), weakness ( ), and difficulty rising ( ). physical examination findings included pelvic limb muscle atrophy ( ) and prostatomegaly ( ). abnormal neurologic examination findings included postural reaction deficits ( ), hyporeflexia ( ), decreased tail tone ( ) and lumbosacral hyperesthesia ( ). neurologic examination was normal in dogs. dorsal laminectomy was performed and diagnosis confirmed in dogs; recovery was monitored for a median of . months (range . - ). three of the dogs ( %) regained normal micturition within . - . months of surgery. though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median . months, range . - ) versus dogs that remained dysuric (median months, range - ). two of the dogs that regained micturition were neutered at the onset of clinical signs, but only of dogs that remained dysuric was neutered. signs improved in all dogs with postural reaction deficits and decreased tail tone. hyperesthesia resolved in of dogs ( %) and fecal continence returned in of dogs ( %). these findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in dlss affected dogs presenting with signs of dysuria. glycogen storage disease type ia (gsdia; von gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose -phosphatase-a (g pase). previous reports indicate that clinical manifestations of gsdia occur only in individuals with homozygous expression of a p.i l mutation. heterozygote dogs (het) have been previously reported to exhibit an overall normal outward phenotype. the purpose of this report is to briefly describe some differences that have been observed between het and homozygous wild type (wt) dogs. a colony of dogs at the university of florida contains a mix of affected, wt, and het individuals. in the course of studies designed to determine the effectiveness of gene therapy for correction of gsdia in dogs, both wt and het dogs have been utilized as controls. available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both wt and het dogs and compared. although birth weights are similar, het dogs have a slower average rate of weight gain than wt dogs and this difference is especially prominent during the first few months of life (figure ) . in contrast to affected dogs, both wt and het dogs are able to maintain normal blood glucose concentrations for up to - hours of fasting, however, after longer fasts of - hours, het dogs have lower glucose and higher lactate concentrations (table ). in addition, liver biopsy samples from het dogs had greater apparent levels of glycogen suggested by pas staining than did samples from wt dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated . times greater glycogen content in a liver biopsy sample from a het dog compared to a sample from a wt dog. together, these findings suggest that the level of g pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. the glucokinase gene (gck) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. gck mrna is present in feline pancreas but the gene is not expressed in feline liver. hepatic gck expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. we hypothesized speciesspecific features in the gck hepatic promoter may underlie the gene expression pattern observed in cats. the putative feline gck (fgck) promoter region was located using bioinformatic software to identify homology with human gck (hgck). genomic dna from a dsh cat was subjected to direct sequencing using a series of pcr reactions with speciesspecific primers. dna clones thus obtained were aligned to generate the feline sequence. direct sequencing yielded . kb of genomic dna sequence with high homology with sequences (acbe , acbe ) archived in the feline genome project. the feline sequence had six regions homologous with non-coding regions of hgck; four of these conserved regions are upstream of the putative fgck start. a . kb segment immediately upstream of feline hepatic exon is not present in hgck. the . kb insert is the reverse complement of a conserved sequence located downstream of exon in feline and human sequences. in conclusion, the putative hepatic promoter of fgck shares extensive homology with the hgck promoter but contains a . kb insert not found in hgck. functional studies are needed to confirm the role of the unique insert in regulation of fgck gene expression. deuterium oxide (d o) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. the objective of this study was to assess if the volume of distribution (vd) of creatinine could be proposed as an alternative in dogs for such a measurement. creatinine and d o vd were measured before (c) and after induction (o) of obesity (by giving an hypercaloric diet ( kcal/ kg) for months) in six healthy adult beagle dogs. creatinine ( mg/kg) and d o ( mg/kg) were simultaneously injected by bolus iv. blood was collected before administration and then at , , , , , , , , and min post-injection (creatinine), and , , , , , , and min (d o) . plasma concentrations of both markers were determined. vd was calculated using pharmacokinetic equations. the body weight increased from . ae . (c) (mean ae sd) to . ae . kg (o). d o vd decreased from ae (c) to ae (o) ml/kg. similarly, creatinine vd decreased from ae (c) to ae (o) ml/kg. the individual difference between creatinine and d o vd (expressed in % of d o vd) ranged from À . to . % (c) and from À . to À . % (o). in conclusion, creatinine vd provides a good estimate of d o vd in both normal and obese conditions. a wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. all work was approved by an iacuc. healthy beagle dogs ( - years old, mixed gender) were used. affected (a) and non-affected (na) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. a and na dogs were evenly distributed between two locations. foods had nutrient profiles adequate for maintenance according to the aafco official publication. the test food contained greater amounts of methionine, manganese, carnitine, vit. e,, vit. c, alpha linolenic acid (ala), and eicosapentaenoic (epa) acid: the food provided mg n fatty acids and mg n fatty acids per kcal. all dogs were fed the control food for wks followed by a wk feeding period where a and na dogs consumed the test food and a and na dogs the control. blood and urine were collected at weeks , , and and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined. evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. orthopedic exams were performed by two veterinary surgeons at each site on weeks , , and . the same two evaluators examined the same dogs throughout. the data was evaluated for the difference between a and na dogs and between foods with age, gender and location as covariates. body weight, disease status, age and gender were blocked. analysis included anova repeated measures mixed procedure (sas version . ) to determine treatment effects over time.serum epa was greater and arachidonic acid lower at weeks , and in the test food fed dogs (p o . ). urine thromboxane:creatinine ratios were decreased in the a dogs fed the test food compared to the a dogs fed the control food at wks (p o . ). lameness score was significantly improved (p o . ) within and between groups of dogs fed the test food. a significantly greater proportion of a dogs fed the test food had improvement in total het (n ) blood glucose (mg/dl) (ae ) (ae ) blood lactate (mmol/l) . (ae . ) . (ae . ) joint score, lameness, functional disability and overall assessment score at wks compared to a dogs fed the control food. % of a dogs had an improved overall assessment score on the test food after wks and at wks compared to % at wks and % at wks of a dogs consuming the control food. this study shows that a food with moderate amounts of added linolenic acid and epa can have a positive impact on systemic inflammation and mobility in - weeks. a similar abstract will be presented at the orthopedic research society meeting in january to an audience largely of orthopedic researchers interested in human orthopedics. fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (fa). medium-chain triglycerides (mct) contain intermediate length fa that do not rely on l-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in fa oxidation. longchain (n- ) polyunsaturated fatty acids (pufa) from fish oil (fo), and in particular eicosanoids derived from eicosapentaenoic acid (epa), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n- ) arachidonic acid (aa). this study investigated the effects of adding mct:fo and l-carnitine to a control diet (prescription diet s k/d s ) on lean body mass, and serum fa and metabolites. forty healthy beagles ( . to . y) were fed one of three foods (n to dogs each) for mo. the study protocol was reviewed and approved by iacuc, hill's pet nutrition, inc. all foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. . %, . %, . %, respectively). composition of serum fa was determined by gas chromatography of fa methyl esters. metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on gc/ms and lc/ ms/ms platforms, for identification and relative quantification of small metabolites. body composition was determined by dual energy x-ray absorptiometry. serum concentrations of lauric and myristic fa increased; epa and dha increased in a dose-dependent manner; and aa decreased in dogs fed treatment food (proc-mixed procedure in sas; all p . ) when compared to dogs fed treatment foods or . serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating lcarnitine incorporation, in dogs fed treatment foods and . thus, a diet enriched with mct:fo significantly altered serum fa composition, enriching (n- ) pufa and lowering aa concentrations. there was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. ten owned dogs, obese for more than months (body condition score [bcs] of ; fat mass [fm] . ae . %) were studied. these dogs had their weight reduced by % (bcs ; fm . ae . %; p o . ) being designated weight reduced (wr) group and then were fed to maintain constant body weight during days (bcs . ; p . ), designated maintenance (main) group. a control (ct) group of beagles was also included (bcs . ; fm . ae . %; p o . ). in all groups the glucose postprandial response test was performed after hours of fasting. blood samples were taken prefeeding and after , , , , , , , , , and minutes of the consumption of cooked rice enough to the ingestion of g of starch/kg body weight. tnf-a and il- were dosed in milliplex tm map panel, insulin and leptin by radioimmunoassay. statistical analysis included paired or non-paired t-tests and wilcoxon (p o . ). the regimen normalized meal glucose response, the area under the curve (auc) of glucose for wr was lower than for obese (p o . ) and similar to main and ct (p . ). insulin secretion did not normalize immediately, as obese and wr exhibited similar auc of insulin and higher values than for ct (p o . ). main, however, presented similar auc of insulin than ct, with lower values than obese and wr (p o . ), suggesting that dogs require some time to adapt their metabolism. leptin, tnf-a, and il- presented significant reductions after weight loss (p o . ), without differences between wr, main and ct (p . ), suggesting an improvement of the pro-inflammatory state consequent to obesity. studying food base excess (be) modification, methionine intoxication was described. in a basal kibble dog diet (be meq/kg; . g/kg of s) two dosages of ammonium sulphate and methionine was added, resulting in diets with be of meq/kg ( . g/kg of s) and À meq/kg ( . g/kg of s), or be of meq/kg ( . g/kg of s) and meq/kg ( . g/kg of s), respectively. a  factorial plus a control diet design, resulting in five treatments, and adult health beagle dogs were used, in a completed randomized design with six dogs per diet. a -d adaptation phase followed -d of total urine collection (in bottles with mg of thymol). urine were pooled by dog and analyzed for density, volume and ph. food macroelements were determined by standards methods (aoac, ) and used for be calculation. dog's acid-basic status was studied by blood gas analysis of venous blood, at : h (pre feeding) and hours after meal. a dose-dependent reduction of urinary ph was verified for both compounds (p o . ). blood bicarbonate (r . ; p o . ), and blood base excess (r . ; p o . ) were highly correlated with food be. acidemia and reduced blood be were verified in diets with be close to zero (higher dose of both compounds, or . g/kg of s), resulting in daily or each other day vomiting episodes in the dogs. ataxia, seizures, and vomiting were previously describe in dogs fed g/kg of methionine, but our results suggest that a much lower value ( . g/kg) was toxic and that the safe upper limit should be between this value and . g/kg (the lower evaluated dose). in people with chronic kidney disease and heart failure, obesity is associated with longer survival times. this association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. the purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (ckd). dogs diagnosed with ! iris stage ii ckd between and at iowa state university and tufts cummings school of veterinary medicine were eligible for the study. dogs o year of age and those with acute renal failure or suspected congenital renal diseases were excluded. medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (bcs, - scale), clinicopathologic values, changes in body weight and bcs, comorbidities, and treatments. dogs were classified as underweight (bcs - ), moderate weight (bcs - ), or overweight (bcs - ). a change in body weight was defined as . kg. survival times were determined for all dogs that were discharged from the hospital and lived day. associations between survival and bcs or body weight changes were analyzed using cox proportional hazards models. one hundred two dogs were enrolled in the study. at the time of diagnosis, dogs were classified as iris stage ii, dogs were stage iii and dogs were stage iv. median body weight at baseline was . kg (range, . - . kg). for dogs with body condition scores recorded (n ), were underweight ( %), were moderate ( %), and were overweight ( %). for dogs that had at least two body weights recorded over the course of their disease, gained weight, lost weight, and had no change in weight. changes in body weight were not associated with survival; however, bcs at the time of diagnosis was significantly associated with survival. dogs classified as underweight had a significantly shorter survival time compared to both moderate (p o . ) and overweight dogs (p o . ). these results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with ckd. protein restriction is the cornerstone of dietary management of kidney disease. the national research council recommends % crude protein and the american association of feed control officials (aafco) recommends a minimum of % crude protein for maintenance for healthy adult cats. protein requirement is unknown for adult cats with kidney disease. most commercially produced cat foods for adult maintenance contains % or more crude protein on a dry matter basis. a typical therapeutic food for cats with kidney disease contains about % crude protein. the objective of the present study was to investigate whether dietary crude protein at . % would be adequate for the maintenance for adult cats with impaired kidney function. seven adult cats, female and male, with age ranging from to . years old (mean: . years) were used in the study. all cats had elevated serum creatinine concentration ( . mg/dl, range: . - . mg/dl) and reduced glomerula filtration rate (mean: % reduction; range: - % reduction) during the study. they did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. the food contained . % crude protein (chemical analysis) and kcal/kg (calculated) on a dry matter basis, or . g protein/ kcal. each essential amino acid in the food was at least % of that recommended by aafco. other nutrients in the food also exceeded aafco's recommendations for maintenance for adult cats. cats were fed the food for weeks. lean body mass (dual x-ray absorptionmetry; hologic, hologic, inc, ma) and serum albumin concentration were measured periodically to monitor protein status of cats. the average lean body mass (mean ae sd) was . ae . kg, . ae . kg, . ae . kg, and . ae . kg in weeks , , , and of the study, respectively. paired t-test did not detect statistical difference (p . ) when comparing the lean body mass in weeks versus weeks , , and , respectively. serum albumin concentration were within the normal reference range during the study (mean ae sd: . ae . %, . ae . %, . ae . %, and . ae . % in weeks , , , and , respectively) . these data show that . % dietary crude protein in a dry food with kcal/kg on a dry matter basis, or . g protein/ kcal, is adequate for maintenance for cats with impaired kidney function. in humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. indeed there have been reports of decreased plasma arachidonic acid and reduced delta- desaturase activities in dogs with atopy and other skin disorders. the present study investigated serum fatty acid profiles in dogs and cats presented to the texas a&m university veterinary teaching hospital, clinical pathology laboratory over the past one year period. results were compared with normative data generated among dogs and cats from earlier feeding studies. sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than months after collection. the samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. total lipids were extracted using chloroform:methanol ( : v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n ; cats, n ) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. normalcy ratios were then plotted on a logarithmic scale with normal at . . the ratio was then compared to changes greater than , and standard deviations of the normal mean values. in this way a graphical presentation of resultant values was obtained. although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at p o . ; oleic acid was increased at p o . . remarkably, these findings were similar for all canine disease categories evaluated (n , heart; n , kidney; n , liver, and n digestive disorders). in cats, a slight decrease in arachidonic acid and large decrease in : was observed but only in heart disorders. by contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o . . sample sizes of the feline sera were considerably smaller (range of - per group). a limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable however, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. additional studies to verify these findings are warranted. the objective of this study was to determine whether or not lalanyl-l-glutamine (ala-gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. this randomized, double-blinded, placebo-controlled clinical trial included client-owned dogs. the dogs were randomly assigned into two groups and administered ala-gln solution (dipeptiven; . g/kg) or an equivalent volume of placebo orally twice a day. all of the dogs (ala-gln group [n ] and placebo group [n ]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for days. among the dogs, (ala-gln-treated group [n ] and placebo group [n ]) were vaccinated and (ala-gln-treated group [n ] and placebo group [n ]) were not vaccinated. the population consisted of purebreds and mixed breed dogs, with a mean age of . ae . weeks. the survival data were compared statistically by means of a log-rank test for the kaplan-meier survival curves. the clinical scores of ala-gln-treated dogs improved significantly relative to the placebo group. there was a significant difference between the two groups in the survival distribution (p . ); specifically, of the ala-gln-treated dogs ( . %) died, whereas of the dogs in the placebo group ( . %) died. no side effects were associated with the administration of ala-gln. these results suggest that the oral administration of ala-gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. the objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (teg) in normal dogs with varying concentrations of a % soybean oil emulsion (intralipid s ). twelve clinically healthy dogs were used for this study. whole blood platelet aggregation, using adp and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of , , , and mg/ml. the teg parameters r, k, a-angle, and maximum amplitude (ma) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation. there was no significant difference between groups with collageninduced platelet aggregation. there was a significant increase in the area under the curve (auc) with adp-induced aggregation at a lipid concentration of mg/ml (p . ). the ma was significantly reduced at both the mg/ml (p o . ) and mg/ml (p o . ) lipid concentration. there was no statistical difference between groups evaluating the other teg parameters. while platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. the reduction in ma seems discordant but both fibrinogen and platelets contribute to the ma. therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. in vivo studies are indicated to determine if any of these changes are clinically significant. rosiglitazone is a peroxisome proliferator-activated receptor gamma (pparg) agonist and an fda-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control. the purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. all dogs received carboplatin intravenously concurrently with oral rosiglitazone. ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs. peak plasma concentrations varied with dose and ranged from . - . ng/ml and occurred between minutes and hours post administration and rapidly declined after the peak. the dose limiting toxicity was hepatic at a dose of mg/m . there was one grade iii, two grade i alt, and one grade iii ast elevations noted. no changes in total bilirubin, alkaline phosphatase, or ggt values were noted. blood glucose values remained within normal limits. mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin. based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is mg/m orally once daily. side effects of the combination appear similar to side effects noted with carboplatin alone. further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (tcc). however, gemcitabine has shown synergism with carboplatin in human cell lines. the purpose of this study was to evaluate the activity of gemcitabine against canine tcc cell lines alone or in combination with carboplatin. we hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. the results of this study could provide a rationale for treatment of canine tcc with the combination of these drugs. tcc cell lines tcc-kiss, tcc-knapp-js, tcc-axa, tcc-hxc, and tcc-sh were treated with gemcitabine, carboplatin, or the combination. cell proliferation was assessed using cyquant assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase- / activation. synergy was quantified by combination index analysis using compusyn software. treatment of canine tcc cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. when tcc cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o um, carboplatin o um), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in of cell lines, and additive in the remaining lines. gemcitabine exhibits biologic activity against canine tcc cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. our results support further evaluation of these drugs in dogs with tcc to determine the clinical efficacy of this combination. metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory t cells (treg). treg are a subset of t lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. the purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma. client owned dogs with histologically confirmed grade i or ii soft tissue sarcoma were administered cyclophosphamide at . mg/m or mg/m orally once daily for days. whole blood and tumor biopsies were obtained on days , , and . flow cytometric analysis of blood was performed to assess changes in t lymphocyte subsets, including cd and cd cells as well as cd foxp treg. tumor microvessel density (mvd) was assessed by performing immunohistochemistry for cd . five dogs were enrolled in the . mg/m /day dose cohort and six dogs were enrolled in the . mg/m /day dose cohort. in patients that received cyclophosphamide at . mg/m /day, the mean number of treg decreased from day to but there was no change in the mean percentage of treg or mvd. for patients that received . mg/m /day, both the mean number and percent of treg as well as mvd decreased over the day time period. cyclophosphamide at . mg/m /day or greater selectively depletes treg and inhibits angiogenesis in dogs with soft tissue sarcoma. arsenic trioxide (ato) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. we hypothesized that ato will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death. the osw canine lymphoma cell line was cultured with um dexamethasone. remaining viable dividing cells were considered resistant. resistant cells were exposed to . um and . um of ato without dexamethasone, after which cells were washed and re-exposed to um dexamethasone. after , and hours of dexamethasone exposure, cells were counted using trypan blue stain. apoptosis was assessed by tunel assays on cytospin preparations collected at , , and hours from ato-exposed and control groups. statistical analysis was performed using way anova and tukey's test. the proportion of dead cells increased over time in both . um and . um ato exposed groups. the proportion of dead cells was greater for . um ato (p o . ) and . um (p o . ) groups compared to control. apoptosis increased with increasing ato concentration and duration of dexamethasone exposure compared to control. these results support the effectiveness of ato at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. ongoing gene expression studies aim to elucidate this mechanism. additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. lymphoma is the most common hematopoietic tumor of dogs. protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. serum protein electrophoresis (spe) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. the purpose of this study was to compare spe changes in lymphoma patients without elevated total proteins with a population of healthy dogs. agarose gel electrophoresis was performed on residual serum from healthy control dogs and untreated dogs with multicentric lymphoma (stage iii -v) after measuring total protein (tp) using the biuret method. densitometric traces of the protein bands were obtained using computer software (totallab ) and the albumin, alpha- , alpha- , beta- and gamma globulin subfractions were identified by visual inspection. the total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations. in lymphoma dogs, tp, absolute albumin, beta- and gamma globulin concentrations and both relative and absolute concentrations of the alpha- globulins were significantly lower however relative and absolute alpha- globulin concentrations were significantly elevated. no monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram. this study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in tp. further investigation is required to identify the proteins responsible for these changes. it is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with t-cell lymphomas having a worse prognosis than b-cell lymphomas. the recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between t and b cells, but also estimate possible aberration on immunophenotype. in human oncology, although some controversy persists, it seems that non-hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis. the aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry. eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. the most common aberrations found were: positivity to cd , biphenotypes, double expression of tantigens (cd , cd ), diminished expression of cd . all dogs were treated with a chop-based protocol. there was a significant difference for the time to achieve response to chemotherapy (partial or complete). / non aberrant lymphomas went into cr or pr after the first treatment (l-asparaginase), while aberrant lympho-mas needed more than treatment to reach cr or pr. there was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant. aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. class ii major histocompatibility expression is a significant and independent predictor of prognosis in human b cell lymphoma. low class ii mhc is consistently associated with poorer outcome. the mechanism underlying this relationship is not clear, but one hypothesis is that high class ii mhc allows for better antigen presentation and tumor-specific immune responses. in the this study, we investigated whether that class ii mhc expression in canine b cell lymphoma was associated with remission and survival times. a total of patients were categorized by level of class ii mhc,expression of cd and cell size for on neoplastic b cells. multivariable cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining / of patient data. results suggested that low class ii mhc expression was associated with decreased times to relapse and death as is seen in human b cell lymphoma, and that large neoplastic cells were associated with decreased survival time. cd expression was not associated with patient outcomes. these findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with b cell lymphoma with a high level of specificity. one of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. the major form of this resistance is provide by multidrug resistance abc transporters. abc transporters proteins comprise a large superfamily of transmembrane proteins, atp-dependent, that extrude a large variety of drugs from the cells. multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. abcg , also known as bcrp, is a residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. the aim of this study was to investigate the expression of bcrp (abcg ) in canine multicentric lymphoma. samples were collected by fine needle aspiration of an enlarged lymph nodes, from dogs with multicentric lymphoma (stage iii to v) at diagnosis, and normal lymph nodes (control). dogs that were previously treated with prednisone or chemotherapy were excluded from the study. quantitative rt-pcr was used to measure the mrna expression level of bcrp and flnb expression as a endogenous reference canine gene. a widely range expression value for abcg expression was found for canine multicentric lymphoma. high gene expression was observed in % ( / ) canine lymphoma, but % of dogs had a lower expression when compared with normal lymph node. gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. in conclusion, abcg was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. osteosarcoma (osa), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. prognostic factors for canine osa include tumor location, distant metastatic disease, and serum alkaline phosphatase (alp) concentration. an increased serum alp concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. during normal bone development alp may be used as a marker for osteoblasts. additionally, alp is a downstream target of activated canonical wnt/b-catenin signaling. therefore, we hypothesized that increased serum alp would be associated with increased expression of b-catenin in canine osa. the goals of this study were: ( ) characterize and compare cellular alp expression in osa tissue from patients with normal and high serum alp; and ( ) assess b-catenin expression in those same patient populations. we used frozen osa samples collected from patients with either high alp (n ) or normal alp (n ). total rna was isolated from the frozen tissue, converted to cdna, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) with either target gene alp (aim ), or target gene b-catenin (aim ). additionally, b-catenin expression was analyzed by western blot. qpcr data for bcatenin and alp expression were normalized to s, and relative expression was calculated by the ddct method. the relative expression of cellular alp was higher in high serum alp samples compared to normal serum alp samples: . ae . (mean relative expression ae standard deviation; p o . ). further, the relative expression of b-catenin was also increased; b-catenin expression of high serum alp samples relative to low serum alp samples was . ae . (p o . ), which is also seen by western blot. this study begins to clarify the mechanism behind high serum alp in canine osa, and suggests the wnt signaling pathway may be active in this population of patients. further work will focus on elucidating the role active wnt signaling plays in the biology of osa. in the future, serum alp status of osa patients may help identify patients that would benefit from therapies targeting this pathway. accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (agaas). pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. in addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with agaas. imaging studies in people indicate that magnetic resonance imaging ( to determine and compare the plasma concentration of cyclophosphamide and its metabolite -ohcp, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide. in this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of mg/m . based on a priori power calculation eight patients per treatment group were enrolled. plasma was obtained at times , , , minutes, and then at , , , , hours post administration for evaluation of -ohcp concentrations by liquid chromatography-dual mass spectrometry (lc/ms/ms). average values were obtained for both cyclophosphamide and -ohcp concentrations within the plasma of both groups. the following values were obtained, half life (hl), time to maximum concentration (tmax), maximum concentration (cmax), and area under the curve (auc). the mann-whitney statistical test was used to compare the groups. the auc for cyclophosphamide was statistically significant (p o . ) when compared between the two groups. the auc for -ohcp was not statistically significant between the groups. the difference between cmax for cyclophosphamide and -ohcp was statistically significantly (p o . ) between the groups. although the auc for cyclophosphamide was statistically significant between the two groups, the auc for the active metabolite -ohcp was not different when administered intravenously or orally. thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally. previously the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver by a wide spectrum of novice participants in feline cadavers were evaluated. novice users' mean io catheter insertion time using the ez-io g driver was also compared to the mean iv catheter insertion time in normovolemic feline and canine patients presented to the western college of veterinary medicine (wcvm) small animal hospital. novice users included wcvm personnel ( technicians, veterinary students, interns, residents, clinicians). after watching a -minute ez-io g training video, each participant inserted io catheters using the ez-io g driver. site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. participants then graded the ''ease of use'' of the ez-io g device on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by wcvm small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the mean io catheter insertion time in feline cadavers by study participants using the ez-io g device. the io catheter was inserted correctly on every attempt by % ( / ) of participants. no difference was found between participant groups for mean io catheter placement confirmation percentage (p . ). percentage of io catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p . ). mean io catheter insertion times were all less than seconds and did not differ significantly as a function of attempt number (p . ) or as a function of participant group (p . ). participants rated the ez-io g 's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p . ). compared to the mean insertion time for iv catheterization ( sec), mean io catheter insertion by participants using the ez-io g ( sec) was significantly faster (p . ). regardless of their level of clinical experience, participants rated the ez-io g device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. regardless of their level of clinical experience, study participants successfully placed io catheters using the ez-io g device and did so significantly faster than the reported iv catheter insertion time in normovolemic feline and canine patients in the wcvm small animal hospital. intraosseous catheterization using the ez-io g has the potential to provide very rapid vascular access and is a skill that can be easily learned. previously presented at the western college of veterinary medicine undergraduate poster competition. multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. the goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. a retrospective analysis of cases of multicavitary effusion admitted to the icu of a tertiary referral center (ontario veterinary college) from to was performed. twenty-three different breeds, with golden and labrador retrievers ( . % and %, respectively) being most commonly seen, were included in the study. ages ranged from to years with a median age of years and a mean of . years. . % of cases were males ( / cases). most common presenting signs included lethargy ( . %), anorexia ( . %), vomiting ( %) and dyspnea ( %). cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). bicavitary effusion was present in cases ( . %) whereas cases ( . %) had tricavitary effusion. neoplasia was found to be the most common underlying cause overall ( . %), with hemangiosarcoma being the leading type ( . % of neoplasia cases), followed by congestive heart failure ( . %), gastrointestinal lymphangectasia ( . %), peritonitis/pancreatitis ( . %), cirrhotic liver disease ( . %) and acute renal failure ( . %). in cases ( . %), no underlying cause could be found. of these, ( . % of all cases) were diagnosed as having idiopathic pericardial effusion. taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. while guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. the objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement. observation of preparation practices was performed in each of ten clinics over - days using - small wireless surveillance cameras. data was coded for surgical patients, and surgeons performing a total of hand scrubs. patient hair removal was most commonly performed after induction of the animal ( / , %) and using clippers ( / , %) . steps in surgical site aseptic preparation ranged from - . contact time with soap ranged from - s (mean s, median s), and with alcohol from - s (mean s, median . s). application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent ( / ( %) and / ( %), respectively). potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation ( / , %). preoperative alcohol hand rub was used in / facilities, but soap and water hand scrub was still more commonly used even at these clinics. proximal-to-distal scrubbing was noted in / ( %) of soap and water scrubs. contact time during surgeon hand preparation ranged from - s (mean s, median s) for soap and water and from - s (mean s, median s) for alcohol-based hand rub. approximately % of the variation in contact time was due to inter-surgeon variation. no significant changes in practices were identified over the course of the observation period. some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. the camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. this prospective randomized study compared the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver to manual io catheterization in feline cadavers. the io catheter insertion time in cadavers using the ez-io g device was also compared to iv catheter insertion time in normovolemic feline and canine patients. after a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed io catheter placements in feline cadavers ( io insertions by placing an illinois needle manually and io insertions using the ez-io g ). order of technique (manual or ez-io g ), site of io placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. when using the ez-io g , a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. after each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by western college of veterinary medicine (wcvm) small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the student's mean io catheter insertion time using the ez-io g .median io catheter insertion times for the techniques were significantly different (manual io technique sec; ez-io g sec) (p o . ); the manual method took seconds longer ( % confidence interval of to seconds) than the ez-io g method. insertion time was more variable for the manual technique than for the ez-io g . percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual io catheter compared with placement of the ez-io g catheter (p o . ). student's subjective ratings were more favorable and more consistent for the ez-io g technique compared to the manual technique for io catheter insertion. compared to the mean insertion time for iv catheterization in the wcvm small animal hospital, io catheter insertion by the student using the ez-io g was significantly faster (iv catheter sec; ez-io g io catheter sec) (p o . ). intraosseous catheter insertion using the ez-io g can be said to be significantly faster, less traumatic, more user-friendly, and as effective as io catheter placement using the manual technique. vascular access via io catheter insertion using the ez-io g device may be suggested to be faster than iv catheter insertion. previously presented at the western college of veterinary medicine undergraduate poster competition. computed tomography (ct) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. the aim of this study was to assess age-related changes and anatomic variation in bone mineral density (bmd) using quantitative ct in normal cats. seventeen normal cats were included in this study and divided into the following age groups: o year (n ); - years (n ); and years (n ). a computed tomographic scan of each vertebra from the th thoracic to the th lumbar spine, and the pelvis, was performed with a bone-density phantom ( , , and mg/cm , calcium hydroxyapatite, cirs phantom s ). on the central transverse section, the elliptical region of interest (roi) was drawn to measure the mean hounsfield unit value. those values were converted to equivalent bmd by use of the bone-density phantom and linear regression analysis (r . ). the mean bmd value of the thoracic vertebrae ( . ae . mg/cm ) was significantly higher than of the lumbar vertebrae ( ae . mg/cm ). the maximum bmd occurred at the t , t , and l levels in all age groups. there was a statistically significant difference in the mean bmd value among the age groups at the t (p o . ), t (p o . ), and l levels (p . ), respectively. in addition, there was no significant difference between the mean bmd value of the left and right iliac bodies ( . ae . mg/cm and . ae . mg/cm , respectively). the present study suggests that age-related changes and anatomic variation in bmd values should be considered when assessing bmd using quantitative ct in cats with bone disorders. dynamic contrast-enhanced computed tomography (dce-ct) is a rapid and widely available method of cerebral perfusion imaging. however, there is no established reference value of cerebral blood flow (cbf) measured by dce-ct according to a dog's age. the purpose of this study was to identify the correlation between regional cbf and aging in clinically normal dogs using dce-ct. fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. dogs were assigned to the following age groups: o year (group ); - years (group ); and o years (group ). dce-ct scans were performed at the level of the third ventricle and mesencephalic aqueduct. cbf in the gray and white matter was calculated using stroketool-ct s software. the overall mean ae standard deviation quantitative estimate for regional cbf in clinically normal dogs was . ae . ml/min/ g, . ae . ml/min/ g, and . ae . ml/min/ g in groups , , and , respectively. there was no significant regional cbf difference between the right and left sides of the brain in each group. also, a statistically significant difference in the regional cbf was observed between groups and (p o . ). thus, aging affects the regional cbf in normal dogs and the values should be considered assessing the results of dce-ct. according to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). by contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. however, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). this might be associated with an apprehensive state or simply a co-morbid state. as part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of ''spraying'' and ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. the case-control dyads were brought by the owners to the veterinary hospital of the university of sa˜o paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound). no significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. both groups had a similar proportion of cats affected by medical illnesses (sprayers: . %, toileters: . %; chi , p . ), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). in both groups, control cats also had a relatively high occurrence of medical concerns ( . % and . %, respectively for each control group). these results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. the relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. the way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding. this study aimed at testing the following hypotheses: (a) owners' perceived quality of life affects cats' global levels of stress; (b) cats' global levels of stress are influenced by cats' personality; (c) cats' living style (single housing versus large group housing) does affect stress levels in cats. to our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality. in this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer. significant differences in mean glucocorticoids metabolites concentrations (mgcm) between the two populations (i.e. single versus in-group cats) were not detected (random effect model, p . ). however, when mgcm were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (random effect model, p . ) and bossy (random effect model, p . ) cats. as to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mgcm was found for single cats only (i.e. the better the owner felt itself social wise the higher the mgcm of the cat; random effect model, p . ). social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. in dogs, raas activation is a major feature of congestive heart failure (chf). benazepril (fortekor s ) is a potent ace inhibitor with well-documented effectiveness in canine chf. although ace activity (ace a ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ace a and changes in angiotensin ii (aii) or aldosterone (al). the purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (pra/prc), angiotensin i (ai), aii, al, and fractional excretion of potassium (ufek), sodium (ufena) and aldosterone (ufeal). sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets ( mg po, q h) for days. blood and urine samples were collected on day (d ) and day (d ) over -hour periods. data were analyzed by repeated measures anova of baseline corrected values, and anova of auc hours . compared with placebo, benazepril induced a significant increase in pra and ai at d (p-value [pra] : . , p-value [ai] : . ) and d (p-value [pra] : . , p-value [ai] o . ). no differences in prc were noticed. based on auc hours, aii levels were % lower in the benazepril group at d (p-value [aii] : . ). ufeal and al decreased by up to % and % at d and d , respectively, though differences did not reach statistical significance. benazepril markedly influences raas dynamics in dogs. decreased exposure to aii and al are likely to be the key events required to counteract pathological remodeling of the heart in chf. this study compared two intravenous anesthetic agents, alfaxalone (alf) (alfaxan s , jurox pty. ltd.) and propofol (ppf) (rapinovet s , schering plough animal health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses. this randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs ( m- f). for each drug, each dog was dosed incrementally at , , , and times the labeled anesthetic induction dose rate (alf mg/kg, ppf . mg/kg) or until a dose was reached that rendered the dog apneic. a minimum of three days was allowed between doses. for each dose administration, the entire calculated dose was delivered constantly over min. the primary variable was apnea, defined as an absence of spontaneous ventilation for minute. apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. for each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. pulse rate, respiratory rate, spo and etco were each measured every min. within-dog comparisons were made using the paired student's t-test. for both alf and ppf all dogs respired voluntarily at the labeled ( x) dose. for ppf at and x doses, and dogs respired voluntarily respectively. for alf at , and x doses, all , and dog respired voluntarily respectively. for all six dogs to become apneic required x dose of ppf and x dose of alf. the mean no observable adverse effect dose (noael) expressed as a multiple of the labeled dose was higher for alf ( . x) than for ppf ( . x) (p . ). there were no significant differences between times to extubation, head lift or attaining sternal recumbency after alf and ppf at , and x doses. at the x dose, dogs took longer to stand after alf ( . ae . min) than ppf ( . ae . min). we concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of mg/kg for alf and . mg/kg for ppf were equivalent. however, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with ppf having a narrower safety margin, i.e., ppf caused more respiratory depression than alf. parenteral levetiracetam (lev) has been shown to rapidly attain therapeutic levels in dogs when given iv or im, and has been used offlabel for the treatment of seizure emergencies. the purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. potential application of these results would be use of sq lev instead of or in addition to rectal diazepam for the treatment of cluster seizures at home. lev was administered sq between the shoulder blades to healthy, purpose-bred hound dogs, at a dose of mg/kg (undiluted). blood samples were collected at , and minutes after lev administration via jugular venipuncture. plasma lev concentrations were measured by high pressure liquid chromatography. none of the dogs became sedated, nor was there pain evident on palpation of the injection site. mean (standard deviation) lev concentration was . ( . ), . ( . ) and . ( . ) mg/ml at , and minutes, respectively. administration of sq lev was well tolerated, and exceeded the suggested therapeutic range ( - mg/ml) within minutes of administration, and remained above the range for at least hours. these data indicate that sq lev administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. the purpose of this study was to assess the effects of cyp inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy greyhound dogs. the iacuc approved this study. cyp inhibitors were administered po for hours prior to methadone administration. methadone hydrochloride was administered po at a targeted dose of mg/kg. blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. the area under the curve (auc) of methadone for each treatment group was compared statistically to the auc of methadone administered without inhibitors using the mann-whitney rank sum test. significant increases (p o . ) in the methadone auc occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. the magnitude of increase was at least fold. mean concentrations of methadone exceeded ng/ml for at least hours in all groups administered concurrent chloramphenicol. no significant increases in the auc occurred in any of the groups which did not include chloramphenicol. in conclusion, chloramphenicol significantly inhibits the metabolism of methadone in greyhound dogs. as a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for - hours after a single oral administration. doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. the commercially available doxycycline calcium mg/ml oral suspension, vibramycin s (pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. a high pressure liquid chromatography (hplc) assay with uv absorption at nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the united states pharmacopeia (usp). doxycycline hyclate mg tablets were first tested for potency. the tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: . mg/ml and . mg/ml. the vehicle used was a : mixture of a vehicle for oral solution (ora-sweet, usp-nf) and vehicle for oral suspension (ora-plus, usp-nf). the suspensions were prepared in replicates of . each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. doxycycline was extracted from the formulations and measured by hplc at day , , , , , , and . each replicate was tested and the potency reported as the percent doxycycline relative to the usp reference standard. on day , , , and , the potency of each formulation was within - % of the reference standard (range . - %). this value is within the accepted range cited in usp o on pharmaceutical compounding-non-sterile preparations. however, starting at day , the potency declined dramatically and remained low for the tests performed on day and . the potency on day , , and was below % of the reference standard (range - %). there was also a noticeable change in the quality of the formulation starting on day , and a change in the color of the formulation to a dark brown. these results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at . and . mg/ml under the storage conditions used in this study, potency of the formulation cannot be assured beyond days. we recommend a beyond-use-day (bud) of days for formulations prepared and stored at room temperature in light or dark conditions. therapeutic options for multidrug resistance (mdr) escherichia coli urinary tract infections (uti) are limited. fosfomycin (fos) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated uti in humans. the purpose of this study was to determine time dependency of fos and the disposition of fos tromethamine in dogs. using a randomized, double crossover design, client-owned dogs received fos sodium iv ( mg/kg) and fos tromethamine (po, mg/kg) either with (n ) or without food (n ). serum and urine were collected for hr; fos was quantitated with a bioassay (atcc e. coli , serum or atcc proteus vulgaris , urine). in-vitro killing curves (cell counts through hours) were performed at (control), . , , , , and x mic for mdr e. coli canine fos susceptible (e-test s ) uropathogens. killing curves indicated fos to be time dependent. after iv administration, clearance (mlÃkg/hr), volume of distribution (l/kg), elimination half-life (hl; hr) and mean residence time (mrt, hr) were (mean ae sd): ae , . ae . , . ae . , . ae . and . ae . , respectively. for po, c max , hl and mrt were ae , . ae . and . ae . , respectively. serum fos exceeded the mic reported for multidrug resistant (mdr) e. coli ( . mg/ml) for hr (iv; . mg/ml) and hr (po, mg/ml diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. in a noncontrolled case series, of cats with clinical cytauxzoonosis given mg/ kg of diminazene aceturate survived infection. dosage frequency was two intramuscular injections given one week apart. commercial formulations contain the diminazene diaceturate salt. the active base is diminazene with the salt consisting of two aceturate molecules. currently there is no data available on the pharmacokinetics of either diminazene compound in cats. the objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats. four purpose bred cats with normal physical examination, cbc, chemistry and urinalysis were used. a powdered commercial drug formulation (veriben s , ceva sanet animale) was freshly reconstituted with sterile water to a concentration of mg/ml prior to administration and sterile filtered solution. heparinized blood samples were collected just before (hour ) or . , , , , , , , , , , , , and hours after intramuscular administration of mg/kg ( . mg/kg of diminazene base) diminazene diaceturate. the plasma was separated by centrifugation within minutes of collection and frozen (À c) until analysis. concentrations of diminazene were measured by hplc analysis using uv absorption and ion-pairing conditions. the pharmacokinetic profile was analyzed using a simple one-compartment model. in these cats, diminazene had a mean terminal half life (t / ) of . ( /- . ) hrs and mean peak plasma concentration (c max ) . ( /À . ) mg/ml. the mean residence time (mrt) of diminazene was . hrs ( /À . ). systemic clearance (cl/f) was . ( /À . ) l/kg/hr. the volume of distribution per fraction absorbed (vd/f) was . ( /À . ) l/kg. a single intramuscular dose of diminazene diaceturate was well tolerated by all cats. without knowing the concentration required to inhibit or kill cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. the objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs. eight male dogs were treated with . mg/kg of meloxicam on day one and . mg/kg of meloxicam on day two. all treatments were administered orally. on day three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of ml sodium urate crystal suspension ( mg/ml). in four dogs synovitis was induced in the l stifle and in four dogs the same procedure was used in the r stifle. in each dog the stifle without induction of synovitis served as the ''normal'' joint sample. a synovial fluid sample was collected from both the r and l stifle of each dog. sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam. synovial meloxicam concentration was analysed using high performance liquid chromatography-mass spectrometry (hplc/ ms-ms). the concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. the results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. no national surveillance system exists for monitoring emergent resistance in companion animals. however, e. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. the purpose of this study was to describe current resistance patterns of canine and feline pathogenic e. coli throughout the united states and identify risk factors of antimicrobial resistance. isolates (n ) of clinical e. coli collected from dogs or cats from may through may located in different regions. susceptibility was determined to drugs ( drug classes) by broth microdilution methods. pharmacodyamaic statistics were described regionally. phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (ndr), single (sdr) and multi (mdr). the majority of isolates were from urinary tract ( . %) and dogs ( . %). the proportion of resistance type for each drug was: ndr ( . %), sdr ( . %) and mdr ( . %). the proportion of mdr was greatest in the southwest ( . %) and least in the northwest ( . %) (p o . ). for all regions, the proportion of resistance was: cephalothin (cph, . %) amoxicillin-clavulanic acid (amx, . %), ampicillin (amp, . %), tricarcillin-clavulanic acid (tcx, . %), doxycyline (dxy, . %) cefoxitin (cfx, . %), cefpodoxime (cpx, . %), chloramphenicol (chp, . %), enrofloxacin (enr, . %) , ciprofloxacin (cif, . %), trimethoprim-sulfamethoxazole (tmx, . %), ceftazidime(cfz, . %), gentamicin (gtm, . %), cefotaxime (cft, . %) meropenem ( . %) (p o . ). the mic exceeded the resistant breakpoint for amp, amx, cpx, cph, cif, cfx, dxy and enr whereas mic did not surpass the susceptible breakpoint. beta-lactams ( . %) was the most and aminoglycosides the least ( . %) sdr. the drug class most frequently involved in mdr was beta-lactams ( . %) and least, gen ( . %). resistance differs regionally, being greatest in the southwest. cph is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. the fluoroquinolones (fqs) are common choices for treatment of e. coli urinary tract infections (utis) in animals and humans. nd generation drugs approved in animals include enrofloxacin (enr), marbofloxacin (mar), orbifloxacin (orb); human drugs include ciprofloxacin (cip). rd and th generation fq for humans include moxifloxacin (mox), gatifloxacin (gat) and ofloxacin, (ofl]), its lisoform levofloxacin (lev). for animals, pradofloxacin (pra) is approved for use in europe. the purpose of this study was to assess the in vitro activity of st (naladixic acid [nal] through th generation fqs (n ) toward dog or cat e.coli uropathogens (n ). isolates were subjected to susceptibility testing to drugs classes ( drugs). isolate phenotypes included no (ndr; n ), single (sdr; n ) or multidrug (to more than drug classes; mdr; n ) resistance (including enr resistant [enr r -mdr; n ] or enr susceptible (enr s -mdr, n ). the minimum inhibition concentrations (mics) were determined for each isolates using broth microdilution (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the overall potency (mic ) for all enr susceptible isolates (ndr, sdr and enr s -mdr) was gat pra, mox, mar, lev, cip sar, orb, ofl enr nal. each e. coli isolate expressing ndr or sdr was susceptible to all fq. however, isolates expressing resistance to st or nd generation fq were also resistance to later generation drugs. glucocorticoids (gc) are standard therapy for allergic asthma but do not reverse the underlying type i hypersensitivity. allergenspecific immunotherapy (asit), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. the purpose of this study was to determine if oral or inhaled gc administered at routinely used dosages would interfere with allergen identification. we hypothesized that oral but not inhaled gc would interfere with accurate identification of allergen-specific ige using skin and serum testing in experimentally asthmatic cats. asthma was induced in eighteen cats using bermuda grass allergen (bga). cats (n /group) were randomized to receive oral gc ( mg prednisolone q hr po), inhaled gc ( ug budesonide q hr) or placebo (gelatin capsule q hr po) for one month. intradermal skin testing (idst) and bga-specific ige amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. a paired t test was used to compare serum ige among groups pre-and post-treatment (p o . significant). idst reactivity was eliminated in / cats on oral gc, / on inhaled gc, and / placebo-treated cats. within two weeks after stopping treatment, idst was again positive in all cats. contrary to our hypothesis, serum ige reactivity to bga was not significantly diminished by any treatment. in conclusion, a two week withdrawal from gcs is adequate for idst identification of allergen but no withdrawal is required prior to serum ige testing to identify the sensitizing allergens. previously in people, increasing severity of asthma is associated with low serum concentrations of -hydroxyvitamin d ( -oh-d). -oh-d is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine il- . in people, serum -oh-d concentration is associated with sunlight exposure as well as dietary intake. cats do not rely on sunlight for vitamin d synthesis; all vitamin d comes from dietary intake. cats have a naturally occurring lower airway disease syndrome (lad) that shares many features with human asthma. the goal of this study was to evaluate serum -oh-d concentrations in cats with lad. cats with naturally developing lad were enrolled. criteria for a diagnosis of lad included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. dietary history was obtained. -oh-d concentrations were determined on serum samples by a commercial laboratory. twelve cats with lad were enrolled. all cats ate commercial cat food. the median -oh-d concentration was nmol/l with a range of - nmol/l which is within the reported reference range of - nmol/l. in contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of -oh-d. interstitial lung diseases (ild) are uncommon in dogs, with the most commonly recognized ild idiopathic pulmonary fibrosis (''westie fibrosis''). in human medicine, ild represent a large umbrella of pulmonary diseases, with ipf only a subset. other, more treatable, ilds are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. the goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ild. the computed tomography (ct) log was reviewed for dogs that underwent thoracic ct scanning for evaluation of respiratory signs, and had changes consistent with ild as the primary abnormality, including the presence of diffuse disease in all lobes, and at least of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. survival time from ct date was calculated. the presence of moderate pulmonary hypertension [phtn] ( mmhg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a mann-whitney rank sum with p o . considered significant. thirteen dogs were identified. terriers and chihuahuas were the most commonly affected breeds. two dogs were adolescents, the remaining dogs ranged from - years, with a median of years. histopathology results (n ), including moderate to severe interstitial fibrosis ( ) alveolar proteinosis with fibrosis ( ), and interstitial eosinophilic pneumonia ( ). one had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. eight dogs died of respiratory failure, with a median post ct survival time of days (range - ), two dogs died of non-pulmonary disease, dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at years since diagnosis, terrier is alive at months and was lost to follow up. dogs had phtn, with a median survival of days ( - ), while the dogs without had a survival of days (range - ), [p . ]. interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. following respiratory infection, young dogs may develop an ild with a relatively indolent course and rare ild is steroid responsive. ct is useful to identify ild but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. idiopathic pulmonary fibrosis (ipf) is an interstitial pulmonary disease, mainly described in west highland white terriers (whwt). identification of molecular pathways important in the pathogenesis of ipf would improve our understanding of this disease and may help identify therapeutic targets. the aim of the present study was to investigate gene expression in lungs of whwt with ipf using oligonucleotide microarray. total rna was extracted from post-mortem pulmonary samples from five whwt with ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. the most significant biological functions for these genes were identified using ingenuity pathways analysis. more than genes were identified as having greater than twofold difference in expression. the significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. most notable among these were genes encoding the leukocyte chemotactic proteins: ccl (fold change . ), ccl ( . ) and il ( . ); the proteins involved in fibroblast migration; and the matrix metalloproteinases (mmps) involved in matrix degradation: mmp (À . ), mmp (- . ), mmp (À . ). this study has identified genes which may be important in pathogenesis of ipf, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. however, real-time quantitative rt-pcr studies are needed to confirm these results before any definitive conclusions can be drawn. idiopathic pulmonary fibrosis (ipf) is an interstitial disease, mainly described in west highland white terriers (whwt). defini-tive diagnosis ultimately relies on lung histopathology. identification of specific biomarkers would be very helpful. expression microarray is a powerful screening tool to study local gene expression in a disease state. the aim of the present study was to measure gene expression profiles in lungs of whwt with ipf to identify potential blood or bronchoalveolar lavage fluid (balf) biomarkers. total rna was extracted from post-mortem pulmonary samples from five whwt with histopathologically confirmed ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. ipa-biomarkers analysis (ingenuity system) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between ipf and ctrl; expression of the gene in lung tissue; possible detection of the protein in blood or in balf. fifty-four molecules met all the criteria. based on difference in expression, promising proteins included ccl (fold change . ), a -actinin ( . ), ccl ( . ), serum amyloid a ( . ), il ( . ), plunc (À . ), mmp (À . ). some are well-known biomarkers of ipf in humans either for diagnosis (mmp , il ) or prognosis (ccl ). these results provide novel potential biomarkers of canine ipf. measurement of these proteins in blood and balf of healthy dogs, dogs with ipf and with other respiratory diseases is needed to assess their use as biomarkers of canine ipf. heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. the purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation. eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. flow-volume loops were collected using commercial software (buxcor) while breathing : helium: oxygen (heliox) and : nitrogen:oxygen (nitrox) in a randomized order via a low dead-space face mask. due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. respiratory rate, tidal volume (ml), minute ventilation (l), inspiratory time (ti), expiratory time (te), peak inspiratory flow (pif) and peak expiratory flow (pef) were recorded while breathing heliox or nitrox. values were compared using a paired sample t-test, with p o . considered significant. all dogs cooperated with testing. there was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. peak expiratory flow was significantly higher (p . ) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (p . ). heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (c-reactive protein [crp] , haptoglobin [hp]) and cardiac troponin i (ctni). thirty three brachycephalic dogs with boas were included and evaluated before and, approximately two months, after surgical correction. the most common components of boas found were elongated soft palate ( / ; %), stenotic nares ( / ; %) and everted laryngeal saccules ( / ; . %). staphylectomy was performed by means of two different surgical techniques: laser (n ) or electrical scalpel (n ). there were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o . ) and a better postsurgical improvement (p o . ) with the use of laser. the levels of crp, hp and ctni were categorized into normal or elevated. before surgical treatment three ( . %), six ( . %) and thirteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. two months after surgical correction, five ( . %), eleven ( . %) and fourteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. there were no statistical differences between values of crp and ctni before and after surgical correction but the levels of hp increased significantly after surgical treatment (p o . ), probably due to postsurgical treatment with corticosteroids. as previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with boas submitted to surgical correction (p o , ). according to the results obtained in the present study, the determination of crp, hp and ctni before and two months after surgical treatment do not have a prognostic value in dogs with boas. even though, near half of the dogs studied had elevated levels of ctni ( . %) that persisted after surgical treatment ( . %), suggesting some degree of myocardial damage is present. further studies are needed considering the influence of breed and age. to the authors' knowledge, this is the first description of crp, hp and ctni determination in dogs with boas. overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. the objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia. twenty nine adult cats were arranged in three groups: obese (n ), overweight (n ) and with ideal body score index (bsi) (n ). mean of bsi in the groups were: , (obese), , (overweight) and , (ideal bsi). cats did not had respiratory, cardiac or others systemic diseases. the respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. the anesthesia was performed with propofol ( ae , ml/kg) and the cats were maintained in the same anesthetic plan. the three groups were compared by analysis of variance followed by tukey's test and conscious and anesthetized cats were compared by student's t test, with a % significance level. there were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal co (petco )) and on arterial gas parameters (pao e paco ) in the three groups. the pao of cats with ideal bsi was , ae , mmhg, although was not significantly different (p , ) from overweight ( , ae , mmhg) and obese cats ( , ae , mmhg). comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in petco in respiratory rate in the anesthetized cats. only petco , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats. these results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. osteosarcoma (osa) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. breeds such as rottweilers and greyhounds are at higher risk for developing osa, suggesting that heritable factors play a role in this disease. mirnas have tumor/tissue specific roles in regulating gene expression and dysregulated mirna expression is found frequently in cancer. we hypothesize that canine osa is characterized by a unique mirna expression profile(s) with dysregulation of some mirnas being associated with specific breeds. mirna expression profiling of primary osa tumors from greyhounds and rottweilers was performed using the nanostring technologies ncounter mirna expression assay kit, interrogating the mirna expression profile of human mirnas, of whose mature sequences are % conserved between human and dog. mirnas were differentially expressed in greyhound versus rottweiler tumors (p o . ), suggesting that breed-specific dysregulation of mirnas may contribute to the development and progression of spontaneous osa. hierarchical clustering revealed distinct mirna expression signatures in greyhound osa tumors as compared to rottweilers. based on these preliminary results, we are evaluating a larger cohort of osa tumor samples including greyhounds, rottweilers, golden retrievers, and a mixed population of other breeds. statistical analysis will be performed to determine the association of mirna transcript levels with specific breeds and overall outcome. characterization of mirna expression in canine osa will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention. originally combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. the hypothesis intended to be tested is that palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (vbl), at dosages known to have activity against mast cell tumors. dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase i dose-finding trial ( cohort), at a starting dose of . mg/m iv vbl (weekly for a total of treatments) and . mg/kg po palladia s eod, concurrently. dose escalation of palladia s was scheduled in . mg/kg increments until mtd was established or fda label dose completed ( . mg/kg). safety evaluation was performed weekly throughout the week study period. dose-limiting toxicities were described following established vcog-ctcae(v . ) criteria. while antitumor response is not a primary endpoint of phase i trials, activity was documented prior to vbl treatments - , and monthly thereafter, based on recist criteria. nine dogs have been enrolled; cohort is filled and approaching completion of the evaluation period. hematologic dose limiting toxicity led to de-escalations of vbl. the current safe combination appears to include vbl at . mg/m every other week and palladia s at . mg/kg eod. response was seen in all but one dog. without head to head trials comparing efficacy of bi-weekly vbl combined with palladia s and vbl alone, choice of therapy should remain at the clinician's discretion. originally prostate specific membrane antigen (psma) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. based upon its selective expression in endothelial cells associated with cancer, psma may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies. to investigate the feasibility of targeting a homogenous population of psma-expressing endothelial cells as a novel anticancer strategy, we have investigated psma expression in several canine hemangiosarcoma (chsa) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to psma by means of the a aptamer, a commercially-available oligonucleotide. the expression of psma by chsa cells was confirmed transcriptionally and translationally by real time pcr and immunohistochemistry, respectively. selective binding and endocytosis of a decorated nanoparticles was studied by fluorescent microscopy. the ability of a decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in chsa cells was assessed by colony forming assays. using a chsa xenograft murine tumor model, clinically-relevant anticancer effects of a decorated nanoparticles encapsulating doxorubicin were tested. all chsa cell lines expressed psma mrna and protein. a decorated nanoparticles were selectively endocytosed by psma-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. finally, a decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic chsa tumor burdens in transplanted mice. diagnostic and therapeutic nanoparticles can be targeted to psma-expressing endothelial cells, and chsa provides a comparative model for the future study of nanoparticle therapeutics. canine transitional cell carcinoma (tcc) is the most common tumor of the urinary tract, and is similar to human invasive tcc in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species. personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. a genomic algorithm has been developed termed ''coexpression extrapolation'', or coxen, that aims to use expression microarray data to predict drug activity in patient tcc samples. the utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined. validation studies of coxen in canine tcc cell lines were conducted. the goal was to determine the value of coxen in predicting baseline sensitivity of canine tcc to chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. additionally, expression data from canine treatment-naı¨ve primary tumor samples were generated on an affymetrix array platform (canine genome v . ). both the expression data and tcc cell line data (antiproliferative effects, % growth inhibition or gc ) were used to establish a canine specific predictive coxen algorithm. coxen scores for canine tcc cell-line drug activity were then analyzed. scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all cell lines, and of carboplatin in cell lines. because all of the cell lines were sensitive to vinblastine (gi o . mm), the coxen score was not predictive of its potency. interestingly, coxen fails to predict vinblastine response in human tcc cell line data as well. in concurrent work, comparative genomic studies to define and compare the gene expression signatures of tcc in dogs and humans provides further evidence that canine tcc is a valuable genomic model of the human disease. current studies involve testing the chemo-predictivity of this derived canine coxen algorithm in additional canine tcc cell lines. canine tcc offers an excellent model for in vitro and in vivo studies of the coxen approach. this preclinical work will be used to guide the feasibility of future coxen clinical trials in dogs and humans with tcc. a small molecule complex (aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (egfr) and insulin-like growth factor receptor- (igfr- ). ingestion of aminoact in people with cancer results in lower serum tnf-alpha, an increase in antioxidant superoxide dismutase (sod) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. this study was designed to first assess safety and secondly the efficacy of three dosage levels of ax- in sustaining progression free survival (pfs) for dogs with refractory advanced and/or metastatic cancer. the prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. the first dogs received aminoact at g/m ; the second group of dogs subsequently received the same dosage mg of aminoact; and the third group of dogs subsequently received g/ m . each dog was treated orally daily for six weeks along with mg betaine hcl, that aids in peptide absorption. all patients were evaluated for toxicity using the vcog-ctcae and efficacy using the recist criteria via assessment of clinical parameters, blood work and client questionnaires. no toxicity other than mild, transient (grade i) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. dogs with tumors that were confirmed as responders ( % reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. it appears in limited studies that the response rate may be more durable at higher dosages. the response to aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. further clinical studies will be valuable in determining the effective dosage and response duration. treating cancer in dogs with aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. stereotactic radiation therapy (srt) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. the purpose of this study was to evaluate the efficacy of srt as a means of achieving local tumor control for canine nasal tumors. retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of srt at csu.srt was administered via the varian trilogy linear accelerator once daily for three consecutive days. the varian eclipse treatment planwas reviewed to determine the planned target volume (ptv) and dose to % of the ptv. kaplan-meir survival analysis was performed for disease free interval (dfi) and overall survival (os). sixteen patients with nasal tumors ( adenocarcinoma/carcinomas, squamous cell carcinomas, chondrosarcomas, osteosarcomas, and undifferentiated sarcoma) were treated with srt. a median dose of . gy was administered to % ptv with a median ptv of . cc. srt was well tolerated by the normal tissues with minimal, manageable side effects. to date, the median dfi is days, while the median os is days. based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. canine leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. the objective of this study was to evaluate the ability of a canine leptospira bacterin to prevent infection and disease following challenge with virulent leptospira canicola, l. pomona, l. grippotyphosa, or l. icterohaemorrhagiae. groups of week-old beagles were vaccinated (day ) and boosted (day ) with placebo (n ) or the -way bacterin (n ! ) and subsequently challenged with each serovar. the results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. in addition, placebo-recipients developed kidney and liver dysfunction. in contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! . ). a few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o . for each serovar). administration of the bacterin also prevented thrombocytopenia ( ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not fda-approved for veterinary use. however, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. this is surprising since mean urine ciprofloxacin concentration ( . mg/ml) in dogs following a modest iv dose ( mg/kg) was times higher than the solubility of ciprofloxacin in water ( . mg/ml). to identify the occurrence of ciprofloxacin uroliths in dogs, records from the minnesota urolith center were reviewed. between january and december , ciprofloxacin was identified in uroliths from dogs; uroliths were composed of % ciprofloxacin in , mixed uroliths containing ciprofloxacin were identified in , a shell of ciprofloxacin was observed in , and ciprofloxacin surface crystals were identified in . based on an experimental study in which % of human volunteers consuming mg of ciprofloxacin with nahco exhibited ciprofloxacin crystalluria (urine ph . ), while no volunteers consuming mg of ciprofloxacin and nh cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. to test this hypothesis, canine uroliths composed of % ciprofloxacin from a single source ( -yr-old male, english bulldog receiving mg/kg of ciprofloxacin po, q hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected mg of ciprofloxacin/g of urolith) were incubated in urine at selected ph's and monitored for dissolution. urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into aliquots. aliquots were adjusted with hcl or naoh to a ph of , , , , or . aliquots were capped and preserved by refrigeration; ph was monitored and readjusted weekly. ten uroliths of approximately equal weight were randomly assigned to individual flasks containing mls of urine. flasks were constantly agitated and maintained at c. every hours, urine was discarded and replaced with mls of urine of identical ph until stone dissolution was complete. ciprofloxacin urolith dissolution times at each urine ph are reported below. ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. in vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. urolith dissolution times were shortest at lower and higher ph's, which is consistent with the pka ( . and . ) of this amphiprotic antimicrobic (more soluble at ph below the acidic pka and above the alkaline pka). foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine ph to allow for dissolution and then life-long maintenance feeding minimizing recurrence. the purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food. sixteen client-owned adult cats ( fs, mc) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (test) or a dry food known to dissolve struvite uroliths (control). the clinical care team and owner were blinded to treatment assignment. the test food was formulated to provide . % mg (dm), . % p, % protein, and a calculated target urine ph value (uph) of . - . . the control food was formulated to provide . % mg (dm), . % p, % protein, and a targeted urine ph of . - . . owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. after diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. a physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. statistical analysis was by anova. all uroliths dissolved in all cats and both foods were palatable. radiographs of cats fed the control food indicated the uroliths dissolved in a significantly shorter time (mean ae std dev of . ae . weeks) compared to cats consuming the test food (mean . ae weeks; po . ).). cats in the control group finished the study at (n ), (n ) and weeks. cats in the test group finished the study , , (n ), , , , and weeks. all the minnesota urolith center occasionally receives uroliths for analysis that are immersed in formalin. results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. the purpose of this study was to determine if formalin alters the mineral composition of uroliths. urolith submissions containing stones of either % struvite (n dogs and cats), % calcium oxalate (n dogs and cats), % calcium phosphate apatite (n dogs and cats), % cystine (n dogs and cats), % ammonium urate (n dogs and cats), and % silica (n dogs) preserved by only air drying were tested. one urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. a subsequent urolith from the same submission was immersed in ml of % buffered formalin for hours at room temperature. uroliths were then air dried for minutes and the analysis was repeated. after exposure to formalin, portions of all struvite uroliths were transformed into newberyite. three ( dog and cats) of ammonium urate uroliths were completely dissolved. newberyite was not detected in any of the remaining uroliths. likewise quantitative mineral analysis of non-struvite uroliths remained unchanged. to avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. we previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (prbcs) that had been stored for days causes a profound inflammatory response ( x increase in leucocyte count and fibrinogen, x increase in c-reactive protein). we speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (f) prbcs would elicit less inflammation than would stored (s) prbcs. a whole blood unit was collected from healthy dogs (n ) for prbcs on day , then again on day . on day dogs received an autologous transfusion of prbcs stored for either days (s, n ) or days (f, n ). cbcs and in-tem thromboelastometry (ct:coagulation time, cft:clot formation time, a:alpha, mcf:maximum clot firmness) were evaluated on blood samples collected at (pre) and , , , , and hours after transfusion. fresh prbcs did not elicit any change in leucocytes, platelets, or thromboelastometry. stored prbcs elicited a degenerative left shift ( hr) followed by a regenerative left shift ( - hr), thrombocytopenia ( % decrease at hr), and marked hypocoagulability characterized by prolonged ct ( , , hr) and cft ( , hr), and decreased a ( , hr) and mcf ( , , hr). data are mean(sd). a: p o . between groups f and s by t test. b: p o . compared to '' '' by rm anova. transfusion of autologous stored prbcs elicits a greater inflammatory response than fresh prbcs, and results in hypocoagulability on thromboelastometry. clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. the parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. the majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. there are multiple hepatic isozymes responsible for the formation of the active metabolite. a non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. we have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. the goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes. six healthy, purpose-bred dogs were used for this study. the pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through lc/ms/ms. the pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. blood samples were collected prior to clopidogrel administration (baseline), after days of mg/kg clopidogrel po q hrs, and after days of mg/kg clopidogrel po q hrs mg/ kg po q hrs rifampin. given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. there was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. however, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of . ae . . the development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. this is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. this increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. this data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. critical illness-related corticosteroid insufficiency (circi) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to acth stimulation. our purpose was to determine if circi exists in critically ill horses. endogenous plasma acth and serum cortisol concentrations, and cortisol at t and t min after . mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days , and of hospitilization. horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. inappropriately low cortisol was defined as endogenous cortisol o mean- sd achieved after administration of . mg/kg cosyntropin to normal horses ( o nmol/l). inadequate delta cortisol was defined as o mean delta cortisol in normal horses after . mg/kg cosyntropin ( o nmol/l). cortisol, acth and delta cortisol were compared using anova between groups, with p o . considered significant. fifty-eight horses classified as having mild ( ), moderate ( ) and severe ( ) disease at admission had survival rates of %, % and % respectively. admission acth and cortisol concentrations were highest in severely ill horses ( ae pg/ml, ae nmol/l) compared to moderate ( ae , ae ) and mildly ill horses ( . ae . , ae ). admission cortisol concentrations were higher overall in severely ill horses (p . ), but were low in % ( / ). admission delta cortisol was low in % ( / ) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors. severely ill horses have high cortisol and acth, but low cortisol and delta cortisol may indicate circi secondary to adrenal hemorrhage. equine pituitary pars intermedia dysfunction (ppid) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. while have previously demonstrated that horses with ppid have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. we hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with ppid compared to controls. neurointermediate lobe pituitary tissue was from collected from horses with ppid (n ) and healthy horses (n , - years). realtime pcr was used to determine the relative expression of autophagy genes (mtor, beclin , atg , atg , atg , pink, lamp ) and a-synuclein relative gene expression from horses with ppid were compared to healthy horses by t-test following log transformation. a pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression. the expression of a-synuclein, autophagy-related genes (atg , beclin, lamp ), and mtor was greater in horses with ppid than in healthy horses. age was not correlated to a-synuclein or autophagy gene expression. there was a significant positive correlation between expression of a-synuclein and beclin , atg , atg , atg , and pink, but not mtor expression. accumulation of a-synuclein protein in horses with ppid may result from increased a-synuclein expression. autophagy genes are upregulated in horses with ppid, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mtor, suggesting a-synuclein may stimulate autophagy in an mtor independent manner. acvim forum session a efficacy of delayed antiviral therapy against ehv- challenge. lk maxwell , ll gilliam , n pusterla , r carmichael , rw eberle , jw ritchey , tc holbrook , t gull , gb rezabek , d mcfarlane , cg macallister . oklahoma state university, stillwater, ok. university of california, davis, ca. equine herpes virus type- (ehv- ) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (ehm). the objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for ehm. eighteen aged ( years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. drug administration was initiated at the onset of the second febrile phase, between days - after ehv- inoculation (pi), and continued for one week. neurological examinations were performed prior to the study and for three weeks pi. one horse was excluded from the study for failure to become febrile. body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days - pi (p o . ), whereas valacyclovir-therapy horses did not differ from control horses. viremia in whole blood, as determined by pcr, was also lower in the ganciclovir-therapy horses on days - pi and on day pi in the valacyclovir-therapy horses (p o . ). although antiviral drug administration did not reduce the risk of ataxia (p . ) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (p o . ) as compared to valacyclovir-therapy and control horses, where / , / , and / horses, respectively, developed at least a two grade change in ataxia. in summary, ganciclovir administration provided better protection against ehm than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (eiv), a pathogen in which continuous antigenic drift can lead to vaccine failure. a month duration of immunity (doi) challenge infection study was conducted using commercial inactivated vaccines containing different strains of a/equine/ /influenza virus's, including innovator tm , containing kentucky/ (pfizer animal health, new york, ny), and calvenza, containing a combination of ohio/ , kentucky/ , and newmarket (boehringer ingelheim vetmedica, st. joseph, ms) . the challenge virus strain was colorado/ , the most contemporary challenge strain currently in use. the study design was a blinded, randomized challenge trial. three groups of yearling ponies, with no history or serological evidence of eiv infection were established. each group received one of three treatments: vaccination with innovator tm ; vaccination with calvenza tm ; or injection with a saline placebo. each treatment was administered times, at intervals of month between the first two treatments, and months between the second and third treatments. all ponies were challenged by nasal nebulization of x eid influenza virus a/eq/ /colorado/ months after the third treatment. clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for days prior to challenge infection, and days post-challenge. nasal shedding of eiv was measured on the same days, using a realtime pcr test procedure. eiv-specific antibody responses were measured by elisa. differences between groups were analyzed by non-parametric repeated measures anova, and differences were declared significant when p o . . all control group ponies demonstrated clinical signs of disease consistent with eiv infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. these signs were significantly lower in both vaccine groups; mean body temperature was elevated ( . f) for days in controls, but only days in vaccine groups. nasal shedding of eiv was detected in all ponies in all groups: over the duration of the study the calvenza group shed significantly less virus than innovator and control. over time antibody titers were significantly higher in the calvenza than the innovator group, and both were significantly greater than controls. this study demonstrated that both current commercial inactivated eiv vaccines have a duration of clinical protection of at least months after a highly pathogenic challenge with a recent eiv isolate. both antibody responses and virological protection differed between the vaccines. formulation difference between the vaccines, including the eiv antigens employed, may have contributed to this performance difference. degenerative myelopathy (dm) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. the aims of this study were to determine (i) presence or absence of abnormalities in concentrations of csf amino acid (aa) neurotransmitters (glutamate, glycine and gÀaminobutyric acid (gaba)) and cytokines in dogs with dm and if present (ii) investigate associations with disease severity. twenty-two dogs histopathologically confirmed for dm and dogs with suspected dm based on thorough diagnostic investigations and clinically normal age-matched control dogs were included in the study. the neurological severity of the dm dogs was graded ( - ) using an established scale. csf was evaluated for presence of glutamate, glycine and gaba by high performance liquid chromatography and for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc (keratinocyte chemoattractant), mcp- (monocyte chemotactic protein- ) and tnf-a using a commercially available, canine multiplex immunoassay (millipore, billerica, ma). all data analyses were performed using sas v . (cary, nc). analyte levels were compared between dm confirmed, dm suspected and control dogs by an analysis of variance (anova). spearman correlation was used to test for correlations of analyte levels and neurological grades. all hypothesis tests were -sided with a . . there were no significant differences between individual csf analytes in dm confirmed and dm suspected dogs. glutamate levels were not significantly different between dm affected (mean . mg/ ml; range . - . ; sd . ) and control dogs (mean . mg/ ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) had significantly higher levels of gaba (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) also had significantly higher glycine concentrations (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). dm-affected dogs also had significantly higher levels of il- (p . ), kc (p o . ) and mcp- (p . ) than control dogs. neurotransmitter levels were not significantly associated with neurological grade. kc levels were significantly higher in the least affected dogs (p . ). there were no associations with disease severity and analyte concentrations. dm affected dogs have an imbalance of csf aa concentrations creating a relatively excitotoxic environment. reports in human als confirm an imbalance between csf excitatory and inhibitory aas suggesting a pathogenic role for excitotoxicity in als. it also appears that dm affected dogs have increases in csf cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to als. further prospective analysis of dm is warranted to evaluate the role of treatment on csf variables. the pathogenesis of neuropathic pain (np) and syringomyelia (sm) in association with chiari-like malformation (clm) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (csf) flow abnormalities. neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. the aim of this study was to investigate the csf neurotransmitter and cytokine levels in brussels griffon dogs (bgs) with clm, sm and np. as part of an mri study investigating the prevalence of sm in bgs, atlanto-occipital csf was acquired from dogs and stored at - c until analysis. all dogs underwent a neurologic exam prior to mri; osirix s software was used to measure sm and the presence of cerebellar herniation and deviation were recorded. deproteinized csf samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and gaba (mg/ml) by high performance liquid chromatography. all csf samples were evaluated simultaneously for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc, mcp- and tnf-a. a commercially available, canine multiplex immunoassay (millipore, billerica, ma) was used for the cytokine analysis (pg/ml). student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. simple pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. all hypothesis tests were -sided and the significance level was a . . np was detected in dogs ( %); sm was present dogs ( %); and cm was detected in dogs ( %). ifn-g levels were significantly lower in dogs with np than without (p . ). there were significant positive correlations between syrinx size and il- (p . ), kc (p . ) and mcp- (p . ). there were significant negative correlations between ifn-g and syrinx height (p . ) and extent (p . ). there was a significant negative correlation between il- and syrinx height (p . ). neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with il- (p . ) and mcp- with glutamate (p . ) and serotonin (p . ). the size of the syrinx in bgs with sm is associated with several cytokine elevations but only a decrease of ifn-g was associated with np. based on this study it does not appear that excitotoxicity plays a role in either sm development or np. further work is justified on the role of the immune system in cm, sm and np. current knowledge about the conservative management of disk associated cervical spondylomyelopathy (da-csm) is rather limited and mainly based on retrospectively retrieved data. the goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for da-csm. additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (mri) and transcranial magnetic stimulation (tms) were investigated. twenty-one dogs were included. after neurological evaluation, neurological status was graded from ( normal) to ( tetraplegia). all animals underwent low-field mri and tms with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. from the mr images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (cbr); canal height to body length ratio (cblr); and the canal compromise ratio. intraparenchymal intensity (isi) changes were graded from to . all dogs were reevaluated by the same person after , , , , and months. eight of dogs ( %) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. all dogs with a negative clinical evolution month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. the opposite was true for all dogs with a positive clinical evolution after month. outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. prognosis was not significantly affected by clinical presentation or tms. progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. there were no significant correlations between clinical presentation, mri and tms. two dogs underwent necropsy and histopathological examination. this revealed in both cases chronic wallerian degeneration and segmental myelomalacia. the results of this study suggest that conservative treatment of da-csm is associated with a rather guarded prognosis. clinical evolution month after diagnosis and selected mri parameters can be considered as prognostic indicators. the lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. a mri-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. the purpose of this study was to determine the ability of the brainsight tm system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for hours after the biopsy. five dogs with mri definable lesions in the brain have been enrolled. breeds included a pitbull mix, pembroke welsh corgi, french bulldog, border terrier and west highland white terrier. age ranged from - years. weight ranged from . - . kg.dogs presented with seizures (n ), ambulatory paresis(n ), unilateral blindness(n ) and head tilt(n ). one dog had a normal neurologic exam. lesions chosen for biopsy were in the olfactory and/or frontal lobes (n ), parietal lobe(n ), and pyriform lobe(n ). lesions were between - mm in diameter. all lesions were well-circumscribed and contrast enhancing except for one. histologic diagnosis of meningioma(n ) and granulomatous meningoencephalitis(n ) were made. the poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. following biopsy, three dogs returned to pre-biopsy neurologic status within hours. the french bulldog took hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. one dog had acute respiratory arrest hours post-biopsy. necropsy is pending. these results suggest that this mri-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. concurrent malformations of the craniocervical junction are commonly identified in humans with chiari type i malformation. recent evidence suggests such craniocervical junction abnormalities (cjas) also occur in dogs suspected of having chiari-like malformation (clm). the purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected clm and to investigate for associations between these features and the occurrence of other malformations in this region. magnetic resonance (mr) and computed tomographic (ct) images from dogs with clm were evaluated. three regions of neural tissue compression were assessed: cerebellar compression (cc); ventral compression at the c /c articulation, termed ''medullary kinking'' (mk); and dorsal compression (dc) at the c /c articulation. a compression index (ci) was calculated for all abnormal regions for each dog. multiple logistic regression analysis was performed (p o . ) to ascertain whether ci values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities. % of dogs had mk and % of dogs had dc. % of dogs also had evidence of atlanto-occipital overlapping (aoo medical infrared imaging (mii) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, mii provides information about the function of the autonomic nervous system. because of recent advances in technology and lack of sedation needed to image patients, mii has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like chiari-like malformation in dogs (clm). the purposes of this study were to establish a mii protocol for dogs suspected of having clm, to identify thermal imaging patterns for various regions of interest (roi), to evaluate changes in thermal patterns and compare the results to those of mri findings, considered the standard for diagnosing clm in dogs. one hundred and five cavalier king charles spaniel dogs with clinical signs attributable to clm and confirmed clm with mri were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector ct scan of the craniocervical junction, whole body mri and mii. the protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. thermal patterns were assessed with custom image recognition software. after each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. mri findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with mii results. the top of head and front of head roi were . % and . % successful in identifying dogs with clm. based on these preliminary findings, mii may be a viable screening tool to detect clm in dogs. medical infrared imaging (mii) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. mii has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. acute thoracolumbar intervertebral disk disease (tlivdd) is common in dogs. the purpose of this study was to: ) determine the success of mii in identifying dogs with tlivdd, ) compare the mii localization with mri results and surgical findings ) determine if the mii pattern returns to that of normal dogs following decompression surgery. small breed chondodystrophic dogs with tlivdd confirmed with mri and dogs with no tlivdd were evaluated with mri and mii. regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. thermal patterns were assessed with computer recognition pattern analysis (crpa) software. dogs were re-evaluated weeks after surgery using the same protocol. when analyzing temperature averages over a region, no significant difference was found between control and affected dogs. crpa was % successful in differentiating normal from affected dogs. crpa was % successful in identifying the intervertebral disk space when compared with mri and surgical findings. based on these findings, mii may be a viable screening tool to detect tlivdd in dogs. microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. spinal cord injuries (sci) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. it has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells. the purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following sci. microglia from dogs with sci was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. the results were compared to region-specific findings obtained from healthy control dogs (n ). the histopathological exam confirmed the diagnosis of sci in the cervical (n ) and thoracolumbar (n ) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with sci days or longer prior to euthanasia. microglial ex vivo examination showed significantly increased expressions of b - , b - , mhc ii, cd c, icam- , cd , cd , and cd , and significantly enhanced phagocytosis and generation of reactive oxygen species (ros) in sci compared to healthy controls. microglial cells seem to be highly activated following sci with an immunophenotype indicating their active role in co-stimulation of t cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. however, as ros can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. modulating the microglial response in sci might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. thymidine kinase (tk) is a soluble biomarker present in s-phase of a salvage pathway for dna synthesis, and can be measured in serum. tk activity correlates with stage, prognosis, and relapse in canine and human lymphoma. we previously reported the results of a pilot study evaluating tk activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (hsa) sera, and found elevated tk activity in % of canine hsa sera evaluated. the purpose of this study was to prospectively evaluate serum tk activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if tk activity could be used as a noninvasive means to distinguish hsa versus benign conditions in this population. dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. tissue from all patients was evaluated histologically by a single pathologist. sera from age-matched normal dogs comprised a control population. an elisa using azidothymidine as a tk substrate was used. comparisons between groups were made using -tailed student t-tests, and receiver-operator characteristic (roc) curves were generated. sixty-two patients and normal controls were studied. there were dogs with hsa, dogs with other splenic neoplasia, and dogs with benign diseases. using a training set of normal dogs, a cutoff of . u/l was established from the roc curve. tk activity was significantly higher (p o . ) in dogs with hsa than in the validation set of normal dogs (mean /Àsd . /À . and . /À . respectively), but not between dogs with hsa and benign splenic disease (mean /Àsd . /À . , p . ). using a cutoff of . u/l, tk activity demonstrated a sensitivity of . , specificity of . , positive predictive value of . and negative predictive value of . for distinguishing hsa versus benign splenic disease. when interval thresholds of o . and . u/l were used together, diagnostic utility was markedly increased for distinguishing both hsa versus normal and hsa versus benign disease. in conclusion, serum tk evaluation may assist in detection of canine hsa, and may also discriminate between benign disease and hsa in dogs with hemoabdomen and a splenic mass. t cell chronic lymphocytic leukemia (cll) is a heterogeneous disease that affects a number of dog breeds. cll patients have variable disease outcomes. the objectives of this study were to use gene expression profiling of cd t cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy. gene expression profiling of cd t cell leukemias ( good, poor prognosis) was conducted. samples from normal dogs were also profiled. several differentially expressed genes were found including cd , cd , and cd . these were selected for further study using flow cytometry to determine expression of protein on the cell surface. seventy nine cases of cd t cell leukemia were screened for cd expression. forty seven had associated outcome information. based on analysis to date, cd expression as assessed by flow cytometry does not appear to provide prognostic information. a monoclonal antibody to cd was recently made available. to date patients with cd t cell leukemia have been profiled. cd is variably expressed on t cell leukemias compared to normal cd t cells. cd is the receptor for interleukin . cyclosporin, a commonly used immunosuppressive drug, inhibits il- production, and has been used to treat a subset of t cell leukemias in people. thus, the finding that cd is up regulated on t cell leukemias compared with normal t cells suggests a possible new therapeutic avenue. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. there is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (ibd). similarly, compositional changes of the intestinal bacterial ecosystem have been associated with ibd in humans. the aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique. fecal samples were obtained from healthy dogs (n ), dogs with acute uncomplicated diarrhea (n ), dogs with acute hemorrhagic diarrhea (ahd; n ), and dogs with active (n ) and therapeutically controlled ibd (n ). the bacterial composition was analyzed by massive parallel s rrna gene -pyrosequencing. differences between groups were analyzed using mann-whitney u tests and kruskal-wallis tests followed by dunn's multiple comparison tests. statistical significance was set at p o . . significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. dogs with gastrointestinal disease had significantly higher proportions of proteobacteria (p o . ). proportions of firmicutes were lower in diseased dogs, but this difference did not reach significance (p . ). within the firmicutes the most notable findings were decreases in bacterial groups belonging to clostridium clusters iv and xiva (i.e., ruminococcus, dorea, and faecalibacterium spp.; p o . for all). dogs with ahd had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active ibd. faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active ibd, but was not significantly different between healthy dogs and dogs with therapeutically controlled ibd (p . ). results of this study revealed bacterial dysbiosis in fecal samples of dogs with various gi disorders. bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled ibd, suggesting that the microbiota is stable in non-active disease. the bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of gi disease. future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. subsequently, published doses for dogs in veterinary formularies have varied from to mg/kg. this study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. six healthy adult beagle dogs were used for the study ( . kg mean weight). after placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin ( mg tablet; mean dose mg/kg), or an intravenous (iv) dose ( mg/kg; mg/ml solution). a randomized crossover design was used with a washout time between treatments. blood was collected for plasma drug analysis for hours. ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (hplc) and pharmacokinetics analyzed using a computer program. oral absorption was also evaluated via deconvolution analysis. the oral dose was well-tolerated, but the iv dose produced transient vomiting and depression in some dogs. after the oral dose, the peak plasma concentration (c max ) was . mg/ ml (cv . %), terminal half-life (t / ) . hr (cv . %), auc . mg Á hr/ml (cv . %), and systemic absorption (f) . % (cv . %). after the iv dose, the t / was . hr (cv . %), systemic clearance . l/kg/hr (cv . %), and volume of distribution . l/kg (cv . %). after examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately %), but poorly in others (approximately %). to explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a mg/ml solution ( mg total dose) via gastric tube. after administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. absorption of the oral solution of ciprofloxacin was . % (cv %) with a t / of . (cv . %) hr and c max of . mg/ml (cv . %). therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. doses were calculated using the data for oral tablets in these dogs. the pharmacokinetic-pharmacodynamic (pk-pd) target was an auc/ mic ratio of . because of the wide range in oral absorption of tablets, a dose to reach the pk-pd target ranged from canine distemper (cd) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (mlv) vaccines. however, mlv strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians. the purpose of this study was to use quantitative real-time pcr viral load information to differentiate between vaccine virus used in mlv vaccines and wildtype infections in dogs. a real-time pcr test for cd virus (cdv) based on the p gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. a total of respiratory mucosal swab samples from mlv vaccinated and asymptomatic dogs were obtained within the first weeks after mlv vaccination. based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of cdv. two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value. the cdv real-time pcr proved to be of high analytical and diagnostic sensitivity: a standard curve was established using known numbers of cdv molecules to allow absolute quantitative cdv viral load data. the limit of detection was in the single molecule range while the limit of quantitation was established at around molecules per pcr reaction. a comparison to ifa showed real-time pcr to be % more sensitive. the cdv viral load in vaccinated animals averaged , viral particles per swab. a cutoff value of , viral particles was calculated by adding standard deviations to the average value. this cutoff value correctly detected . % of the vaccinated samples. acutely infected dogs with cdv compatible clinical signs have high viral loads normally several logs higher than the cutoff value. in dogs with clinical distress, recent cdv mlv vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time pcr tests. testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that cdv is not the cause of clinical signs. in conclusion, quantitative real-time pcr is a sensitive, rapid and reliable test regardless of recent vaccination. the use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. feline nephrostomy tubes are reported as being effective when pelvic drainage is required. the biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. the development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. the objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (sub) in cats with ureteral obstructions. fifteen cats ( kidneys) had a sub placed for: ureterolithiasis ( ), ureteral stricture ( /À stones) ( ), and ureteral stent rejection ( ). the median pre-and post-procedure creatinine was mg/dl (range: . - ) and . mg/dl (range: . - ), respectively. the median pelvis diameter pre and post-procedure were (range: - ) and mm (range . - ), respectively. six french tubes were placed in , and fr. in . the bypass remained indwelling for a median of days (range - ). there were major complications resulting in nephrostomy tube dislodgement ( ) and port leakage ( ) days after surgery. one patient with severe coagulopathy developed a clot which resolved with tpa infusion through the port. no sub got occluded/obstructed long-term. overall, the use of a sub for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. oxidative stress is considered central to the pathogenesis of many systemic diseases. in humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. therapeutic antioxidant supplementation with nac in glutathione (gsh)-deficient patients has shown clinical benefits, including repletion of intracellular gsh levels. we have shown that clinically ill dogs are gsh-deficient, and that gsh deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. the purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of nac to normalize blood antioxidants (rbc reduced gsh (rbc gsh), plasma cysteine (cys), serum vitamin e (vit e), and whole blood selenium (se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (u i/ c)), and improve illness scores (spi ) and outcome (survival to discharge) in clinically ill dogs. clinically ill client-owned dogs, admitted to the uw veterinary medical teaching hospital that did not receive blood transfusions, tpn, vitamins, or antioxidants were eligible for the study. dogs enrolled in the study were randomized to receive iv infusions q. h. of either nac (  mg/kg and  mg/kg) or equal volumes of % dextrose (placebo) over hours. at the time of enrollment, and hours following the final hour infusion, blood and urine were collected to quantify rbc gsh, cys, vit e, and se concentrations; u i/c ratios; and calculate spi scores. rbc gsh and cys concentrations were quantified by hplc. commercially available hplc, atomic absorption spectroscopy, and eia were used to quantify vit e, se, and u i/c ratios, respectively. nonparametric statistical analyses were used, with results reported as medians and p o . considered significant. sixty-one ill dogs were randomized to either nac (n ) or placebo (n ). overall this group of ill dogs had significantly decreased rbc gsh ( . vs. . mm; p . ), vit e ( vs. mg/ml; p . ), and se ( . vs. . mg/ml; p . ) levels and elevated u i/c ratios ( vs. pg/mg; p . ) in comparison to healthy control dogs. dogs in the placebo group showed a significant further decrease in rbc gsh over the next hours ( . to . ; p . ). nac supplementation significantly increased plasma cys levels ( . to . mm; p o . ), and prevented a further decline in rbc gsh ( . to . mm; p . ). however, serum vit e ( vs. mg/ml), se ( . vs. . mg/ ml), u i/c ratios ( vs. pg/mg), spi scores ( . vs. . ), and outcome ( % vs. %) were not significantly different between the nac and placebo groups after treatment. the results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with nac within the first hours of hospitalization prevents further rbc gsh depletion. further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. diabetes mellitus in cats is very similar to type ii diabetes in humans, preceded by a period of insulin resistance. evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. there is a need for a simple biomarker based test predictive of insulin resistance. there is a biomarker based assay predicative of insulin resistance in humans. the purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance. the insulin resistance assay is based on the quantitative analysis of metabolites ( -hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-lpc). a proprietary algorithm (metabolon, inc, durham nc) was used to generate a predictive rd (rate of disposal) value (normal range in cats . - . ). individuals with an rd value less than will have a greater than % chance of being insulin resistant and an rd value less than will have a greater than % chance of being insulin resistant. initial studies demonstrated that the rd values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. in a feeding study of cats ( % vs. o % body fat) rd values improved from . ae . to . . (p . ). during weight maintenance, % body fat for months, further improvement was observed (rd, . . (p . e- )). these results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. the use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. however, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. to overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. these libraries consist of single chain variable fragments (scfv) comprised of canine variable heavy (vh) and variable light (vl) immunoglobulin chains displayed on the surface of bacteriophage (fig. ) . the antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. we can now use simple panning techniques to isolate scfv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques. canine hsa is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. although there are no confirmed immunological targets for hsa, serum levels of vascular endothelial growth factor (vegf) are elevated in these patients and, as in many human cancers, vegf may represent an important therapeutic target for neutralization. we used simple panning techniques to screen canine scfv libraries generated from the spleens of dogs with hsa against canine vegf and successfully isolated scfv clones that bind and neutralize canine vegf in vitro. these scfvs are now being taken into a murine model of canine hsa to determine whether they can inhibit tumor growth and metastases. in addition, we have panned the same antigen-experienced scfv phage display libraries against allogeneic primary canine hsa cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. early results demonstrate enrichment of scfv phage libraries for malignant endothelial cell binders. these scfv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. this novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. calcitriol, (vitamin d ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. ccnu, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (mct). vitamin d receptor is expressed in the majority of canine mcts, suggesting a role for calcitriol in the management of dogs with these tumors. the purpose of our study was to examine the in vitro effects of calcitriol in combination with ccnu, vinblastine, imatinib, or toceranib on canine mastocytoma c cells. also, we evaluated the antitumor activity of dn , a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring mcts. c cells were incubated with serial dilutions of calcitriol ( . - nm). twenty-four hours later, cells were then treated with vehicle control or serial dilutions of ccnu ( . - um), vinblastine ( . - nm), imatinib ( . - . nm), or toceranib ( . - nm). cell viability was assessed with an mtt assay after hours and data was used to derive a combination index (ci: values o , , indicate synergism, additivity, antagonism, respectively). in the phase ii clinical trial, dogs were eligible if they had at least measurable, histologically confirmed, mct. calcitriol was administered orally. recist criteria were used to assess tumor response. calcitriol, ccnu, vinblastine, imatinib, and toceranib each suppressed c cell viability in a dose-dependent manner. ci values o were obtained for calcitriol ( . - . nm) combined with ccnu ( and um), vinblastine ( . and nm), imatinib ( . - . nm) and toceranib ( . - . nm). due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase ii trial was terminated early; only of planned patients were treated. one dog with a metastatic muzzle mct had a complete response that lasted days. three dogs achieved partial response lasting from - days. in summary, our in vitro data demonstrate that calcitriol combined with ccnu, vinblastine, imatinib or toceranib has synergistic effects on c mastocytoma cells. antitumor responses were observed in dogs with spontaneously occurring mcts treated orally with single-agent calcitriol, but the frequency of adverse effects was high. together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine mcts but refinement of the calcitriol-dosing regimen must be done. cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on t-cells, the drug's main cellular target in the body. our acvim foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect t-cell function. we have completed several studies using flow cytometry to evaluate activated t-cell expression of surface molecules (cd & cd ) and cytokines (il- , ifn-g & il- ) as potential biomarkers. our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine t-cells exposed to varying concentrations of cyclosporine. this study established consistent drug-associated suppression of the cytokines il- , ifn-g and il- . our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least ng/ml) and a lower dose used to treat atopy ( mg/kg sid), on t-cell expression of these three cytokines. significant suppression of il- and ifn-g expression was seen at the high cyclosporine dose, while at the lower dose only ifn-g expression was suppressed. because tcell expression of il- was not significantly suppressed at the high cyclosporine dose, il- was not evaluated at the lower drug dose. because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as pcr) for routine use in pharmacodynamic testing. we have therefore conducted an in vitro study comparing the effects of cyclosporine on activated t-cell expression of il- and ifn-g using flow cytometry and qrt-pcr, and demonstrated dose dependent and comparable suppression of il- and ifn-g using either methodology. we are currently evaluating, using qrt-pcr, the effects of oral cyclosporine on t-cell expression of il- and ifn-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients. effect of hypothyroidism on reproduction in bitches. dl panciera , bj purswell , ka kolster , sr werre . departments of small animal clinical sciences, large animal clinical sciences, and laboratory for study design and data analysis, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va. numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. we previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of weeks. the purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine. twelve multiparous bitches were studied. hypothyroidism was induced in dogs by administration of mci/kg i. hypothyroidism was confirmed by finding serum t concentrations before and hours after iv administration of human recombinant tsh that were o nmol/l. levothyroxine ( . mg/kd q h) was administered to all hypothyroid bitches. six bitches served as euthyroid, untreated controls. dogs were evaluated daily for signs of estrus and were bred by of males when serum progesterone was ! ng/ml. interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. the student's t-test and anova were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. the wilcoxon rank sum test was used to analyze data between groups that was not normally distributed. the mean duration of hypothyroidism prior to levothyroxine administration was ae . weeks. breeding took place after levothyroxine treatment for ae . weeks in the hypothyroid group. all dogs in the hypothyroid group and / control dogs were pregnant, while / hypothyroid and all control bitches became pregnant prior to levothyroxine administration. no difference in interestrus interval or gestation length was noted between groups. during whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. litter size, birth weight and peirparturient mortality were similar between groups. levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from - % of dogs running at least miles in a day or less. omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. the purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis. experiment # was a randomized placebo-controlled study using sled dogs ( - years) competing in a mile race over - h. treatment groups were famotidine (approx mg/kg qd) or no treatment, beginning days prior to the start of the race and proceeding until gastroscopy was performed h after the race. experiment # was a randomized positive-control study using sled dogs ( - years) running a mock race of miles in h. dogs were divided into omeprazole (approx mg/kg qd, administered min prior to a meal) or famotidine (approx mg/kg bid) groups beginning days prior to the exercise challenge and continuing for h after completion. gastroscopy was performed immediately prior to the start of dosing and h after completion of the exercise. in all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system. famotidine ( mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment ( / vs / , p . ). compared to famotidine at mg/kg bid, omeprazole significantly decreased the severity ( . vs . , p . ) and prevalence ( / vs / , p . ) of gastric lesions. although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between - % of the dogs receiving famotidine had clinically significant lesions. a previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a % prevalence of clinically significant gastric lesions. however, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over %. the conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing alaskan sled dogs. mares may be an important source of environmental contamination with rhodococcus equi on breeding farms. attempts to reduce fecal shedding of r equi by the mare and the effects of the mare's fecal r equi concentration on airborne concentrations in the foaling stall have not been previously reported. twenty-one arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. fecal samples were collected at day of gestation (time ), the week before foaling (time ), and the week after foaling (time ). airborne concentration of r equi were measured in the stall within hours post foaling using a microbial air sampling system into which standard ( -mm) culture plates with a media selective for r. equi have been loaded. concentration of total r equi were determined by morphological characteristics. the concentration of virulent r equi was determined using a modified colony immunoblot method. concentrations of total and virulent r equi were compared among mares to examine effects of treatment, time, and treatment by time interaction. there were significant (p o . ) effects of treatment that depended on time of sample collection. at sample times and there were no significant differences between groups in the fecal concentration of virulent r equi. at time concentrations of virulent r equi were significantly lower among mares in the treatment group (p o . ) compared to control. effects of time depended significantly on groups: for the control group, there were no significant effects of time. for the treatment group, concentrations tended to decrease over time, and concentrations at time were significantly (p o . ) lower than those at time . no other differences among times for concentrations in the treatment group were statistically significant. there were no significant effects of treatment, sample time, or their interaction on the concentration of total r equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. no significant differences were determined between treatment groups for airborne concentrations of virulent or total r equi. treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent r equi over time, but had no impact on the airborne concentration of r equi shortly after foaling. the purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (balf) in horses affected with recurrent airway obstruction (rao) and in control horses using proteomics and western blot techniques. rao-affected (n ) and control horses (n ) were subjected to an experimental exposure trial; when the rao-affected horses showed clinical signs of disease, balf was collected from all horses. balf was also collected from client-owned rao-affected horses (n ) with naturally-occurring clinical signs of disease and client-owned control horses (n ) from the same environments. the balf from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (ms). peaks detected with ms were identified using tandem ms analysis and database searches. western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the balf of all horses. data from ms experiments were analyzed with the student's t-test to compare peak intensity between rao-affected and control horses. western blot band density data was analyzed with the kruskal-wallis anova for comparison between groups of horses. significance level was set at p o . . with ms proteomic analysis of the balf from the experimental exposure trial horses, total peaks (peptides) were identified. of these peaks, were differentially expressed between the rao-affected ( over-expressed) and control horses ( over-expressed). identifications were made for balf proteins. transferrin and secretoglobin were chosen for validation with western blot. proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with western blot analysis. western blot also showed that clientowned rao-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. according to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to rao-affected horses. while the western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned rao-affected horses. in addition, both secretoglobin and transferrin band densities on western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation. this study demonstrates that proteomics techniques can be used in the investigation of equine balf proteins. the proteins identified as differentially expressed between rao-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of rao, and as potential targets of new therapeutic agents for rao. cardiotoxic effects of rattlesnake venom in the horse are not well defined. the first aim of this study was to document cardiac damage in naturally envenomated horses. twenty horses with clinical diagnosis of snake bite were included. a snake venom elisa was utilized to confirm envenomation when possible. serum and plasma were collected at selected intervals. plasma was assayed for cardiac troponin i (ctni) using a flurometric assay (stratus cs s , dade behring). holter monitors (zymed s , philips) were placed at admission, week and month post presentation. echocardiography was performed on available horses - months after envenomation. the second aim of this study was to investigate potential mechanisms of the cardiac damage. serum samples were assayed for tnfalpha using a commercial assay (endogen). antibody titers to crotalus atrox venom were measured at admission, week and month after natural envenomation and compared to titers in vaccinated horses (crotalus atrox toxoid, red rock biologics). a significant number of horses showed elevations in ctni (p o . ) at one or more time point indicating myocardial damage. holter readings revealed the presence of arrhythmias or persistent tachycardia in horses. five of twenty horses were available for echocardiography; no abnormalities were noted. horses with increased ctni tended to have greater tnfalpha concentrations compared with horses without increased ctni. peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o . ). rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. our laboratory previously reported that a diverse microbial flora with a predominance of streptococcus spp. and lactobacillus spp. exists in healthy horses using fluorescence in situ hybridization (fish). the present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel srrna bacterial tag encoded flx-titanium amplicon pyrosequencing (btefap). biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from healthy horses via gastroscopy after a -hour fast and horses immediately post-mortem. dna was extracted from the mucosal biopsies and btefap and data processing was performed. hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses. pyrosequencing yielded between , and , reads per horse with , , reads in the antrum, squamous and glandular regions, respectively. the microbiome segregated into two distinct clusters: cluster comprised of horses that were stabled, fed hay and sampled at post-mortem and cluster consisted of horses that were pastured on grass, fed hay and biopsied gastroscopically after a -hour fast. samples from different antomic regions clustered by horse rather than region. despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. the dominant bacteria in cluster horses were firmicutes ( % reads/sample) consisting of mainly streptococcus spp., lactobacillus jensenii, l. fornicalis and sarcina maxima. cluster had more diversity with a predominance of proteobacteria, bacteroidetes and firmicutes and genera identified such as streptococcus spp., moraxella spp., actinobacillus spp., and others. though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by shanon weaver, ace and choa i diversity indices). helicobacter spp. sequences were not identified in any sample (out of , reads). the ulcerated mucosa from horse (group ) had lower diversity and higher numbers of bacteria predominated by lactobacillus equigenerosi. this data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. seasonal pasture myopathy (spm; atypical myopathy [am] in europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. clinical signs rapidly progress from muscular weakness to recumbency and frequently death. extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. recently, a defect of lipid metabolism called madd has been identified in european horses with am. this report documents the first cases of equine madd in the united states. six midwestern us horses suspected of having spm in the spring or fall of were evaluated for madd by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. one horse was found dead on pasture after days of rear limb stiffness and inappetance. urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine madd. plasma acylcarnitine profiles from horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters. spm appears to be a highly fatal emerging disease of pastured horses in the us characterized by weakness, colic-like signs and myoglobinuria. the disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. candidatus mycoplasma haemolamae (cmhl) is a common red blood cell parasite of new world camelids. the high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic dna. this dna can then be used in the development of genetically-derived tools such as pcr and in-situ hybridization. thus, one splenectomized animal can replace many immunologically intact animals within a research setting. the purpose of this study was to track the natural progression of cmhl parasitemia and associated clinical signs in a splenectomized alpaca. an intact, -month-old, . kg male alpaca was used in this study. he had tested positive via pcr for cmhl on three different occasions, although no organisms were seen on peripheral blood smears. the alpaca was placed under general anesthesia and a ventral midline incision was made. the spleen was located, the vessels ligated, and the organ removed. buprenorphine and flunixin meglumine were given for and days after surgery respectively. body weight, attitude, rectal temperature, blood glucose, and pcv were recorded daily. in addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with mycoplasma organisms was determined. the alpaca was not parasitemic prior to surgery. one percent of the rbc's contained mycoplasma on days and after splenectomy. parasitic bloom developed on day with % of the red blood cells infected, and over % containing or more organisms. the alpaca was treated with mg/kg oxytetracycline i.v. on day . on postoperative day no parasites were seen in the peripheral blood. the peripheral blood remained free of parasites for days. on the morning of the th day, % of the peripheral red blood cells contained mycoplasma. by late that afternoon, % of the observed rbc's contained - organisms. the alpaca again received oxytetracycline. there were no more parasites observed from that time until the alpaca was euthanized days later. the alpaca lost . kg between days À and after surgery. his weight fluctuated between . and . kg for the remainder of the study period. blood glucose ranged between and mg/dl there was no major change in pcv (range - %), a finding that was expected as the spleen was not available to remove infected red blood cells. body temperature ranged between . and degrees celsius except for days and when more than % of red blood cells contained parasites. on those days rectal temperature reached . and . degrees respectively. this study confirmed that a non-parasitemic, yet pcr positive alpaca did indeed harbor cmhl. the time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. gastro-intestinal (gi) disease frequently results in increased wall thickness in many species. identification of changes in gi wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. although gi motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. the aims of this study were to report normal values for wall thickness of various gi structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. eight healthy adult holstein friesian (hf) cattle ( ae kg), eight jersey (j) cattle ( ae kg), thirteen adult sheep ( ae kg) and eleven adult goats ( . ae kg) were recruited and examined on three consecutive days. ultrasonographic images were optimised for the structure of interest. structures were identified based upon appearance and anatomical position. a minimum of three cineloops were obtained of the abdominal organs per intercostal space (ics) and three along the ventral midline in each ics; images were analysed offline. data were analysed using anova and post-hoc bonferoni, student's ttest and intra-class correlation coefficients. each structure was measured per ics per species; if no differences were noted for structures in different ics, then measurements were pooled. no differences were noted between hf and j cattle so data were pooled. data are displayed in table . good repeatability (icc . ) was obtained for all measurements and no differences were noted between animals of the same species or between days. these measurements for assessment of normal gi thickness are repeatable and may allow valuable additional information to be gained from ruminants with gi disease. ocular infections with the infectious bovine keratoconjunctivitis (ibk) agent moraxella bovis (m. bovis) are associated with significant economic loss in the cattle industry.although antibiotic therapy is the treatment of choice for ibk, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. as a result, our laboratory has been actively investigating the therapeutic potential of bdellovibrio bacteriovorus j (b. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of gram-negative bacteria, as a new treatment for ibk. we have previously shown that b. bacteriovorus can reduce the number of m. bovis attached to bovine epithelial cells in an in vitro model of ibk and that b. bacteriovorus can be trained to kill m. bovis as effectively as e. coli using serial passages. in this study, we hypothesized that b. bacteriovorus can remain viable in bovine tears without its prey for up to hours. this hypothesis was addressed by incubating inocula of active b. bacteriovorus in its preferred media peptone yeast extract (pye) and comparing b. bacteriovorus viability in bovine tears or phosphate buffered saline (pbs) at time , , , , and hours. using a plaque assay to quantify the mean amount of plaque forming units (pfus) of b. bacteriovorus exposed to each treatment, it was determined that viability of b. bacteriovorus over time was comparable between treatment groups. overall, the results supported that b. bacteriovorus can remain viable in tears for up to hours in the absence of prey bacteria. further studies are needed to determine the therapeutic potential of b. bacteriovorus in an in vivo model of ibk. correction of the measured ionized calcium concentration (cca ) to a ph . is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. the equation most commonly used for ph correction in bovine plasma is: cca ph . cca  (- . Â{ . -ph}) . the validity of this equation for bovine plasma is unknown. accordingly, our first objective was to characterize the in vitro relationship between cca and ph for bovine plasma. feeding rations with a low dietary cation-anion difference (dcad) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. the mechanism for this beneficial effect remains unclear. our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cca . the in vitro relationship between plasma log(cca ) and ph in was investigated using lithium heparin anticoagulated blood from healthy holstein-friesian calves. plasma was harvested and tonometered with co at c over a ph range of . - . . plasma chloride concentration (ccl -) was altered by equivolume dilution of plasma with electrolyte solutions of varying ccl -( ae , ae , and ae meq/l; mean ae sd). the slope of the linear regression equation relating log(cca ) to ph for tonometered plasma samples from the calves was - . ae . at normal values for cca ( . ae . meq/l), albumin concentration ( . ae . g/l), and ccl -( . ae . meq/l). the experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. the formula for correcting cca in bovine plasma for change in ph from . is therefore: cca ph . cca  (À .  { . -ph}) . this equation is only valid at normal concentrations of albumin and chloride in plasma. equivolume dilution of plasma by electrolyte solutions of varying cclindicated that cca ph . increased by . meq for every meq/l increase in ccl -. in other words, plasma cca at a given ph increases directly in response to an increase in plasma ccl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. furthermore, the increase in cca is independent to the change in plasma ph induced by an increase in ccland decrease in plasma strong ion difference. our finding that hyperchloremia directly increases plasma cca provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, nh cl, cacl ) are more effective in increasing cca than equimolar quantities of acidogenic salts such as mgso . coagulase negative staphylococci (cns) are among the most common bacteria isolated from the bovine mammary gland. historically, these bacteria were lumped together as minor mastitis pathogens. modern molecular techniques have allowed accurate speciation and fingerprinting of the cns species. these methodologies have recently been applied to the study of cns in bovine mastitis. the aim of the studies presented here was to evaluate the role of individual cns species on milk somatic cell count (scc) and duration of intramammary infection (imi). in the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($ head) at the university of missouri dairy research center once monthly for months for bacterial culture and milk scc. staphylococcal isolates were speciated by sequencing the rpob gene and strain-typed using pulsed-field gel electrophoresis (pfge). using species and fingerprint data along with published definitions for staphylococcal imi, cns imis were identified. overall, species of cns were identified with staphylococcus chromogenes, s. cohnii, s. epidermidis, and s. simulans being most prevalent. duration of imi and scc data were analyzed using regression models accounting for repeated measures. mean milk scc and duration of imi were found to differ between cns species (p o . ). although most imis were of short duration ( month), staphylococcus capitis and s. chromogenes imis had longer mean durations of infection than or more of the other species isolated. mean sccs were under , cells/ml in most cases. however, staphylococcus simulans and s. xylosus imis were more inflammatory (mean , cells/ml) and had a higher mean scc than s. cohnii, s. epidermidis, and s. haemolyticus. to examine the relationship between cns imi and milk scc in a larger population of cattle, cns isolates from the canadian bovine mastitis research network (cbmrn) culture collection were obtained for speciation. speciation and fingerprinting were performed as above. isolates were from subclinical imi from before and after the dry period and from subclinical imi during lactation. data associated with each isolate were obtained from the cbmrn database. nine-hundred-thirty-eight isolates from mammary quarters in herds were successfully speciated. twenty-two different species of cns were identified. staphylococcus chromogenes was the most frequent species identified accounting for % of the infections. three species, s. chromogenes, s. xylosus, and s. simulans accounted for % of all infections. data were analyzed using a linear hierarchical repeated measures mixed model. differences in mean scc were found between some cns species and culture negative control quarters and also between different species of cns (p o . ). overall, our data demonstrate potential differences in pathogenicity between strains of cns that cause bovine mastitis. passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first hours of life is crucial for the health and future productivity of dairy calves. however, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. while pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral igg concentrations. the purpose of this study was to investigate the effect of high pressure processing (hpp) on the viscosity, igg concentration, and bacterial contamination of bovine colostrum. first milking colostrum samples were collected from cows from different farms, and ml aliquots of each sample were pooled for analysis. pooled colostrum was processed in triplicate using an isostatic press at mpa ( , psig) for , , , , and minutes. samples were tested for the effects of hpp on the viscosity, bacterial load (cfu/ml), and igg concentration. there was a significant decrease (p o . ) in bacterial load at each time point when compared to time . no significant difference in igg concentration was found between any time points. subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time. hpp appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate igg concentrations. minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of hpp on specific bovine pathogens. the heme-associated iron-binding apoprotein lactoferrin (lf) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: saliva; tears; mammary secretions, as well as at mucosal surfaces. it is also released from immune cells under pathogenic stimulation. the purpose of this study which has been approved by western university's institutional animal care and use committee is to further characterize the mechanisms through which lf modulates inflammation in the face of bacterial endotoxin. it was hypothesized that lf would inhibit p phosphorylation. numerous studies speak to the ability of lf to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (lps); mechanisms through which it is believed to achieve its anti-inflammatory effects. recently, investigators demonstrated its ability to interact with host dna while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling. in earlier studies, dawes me, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase- and the gelatinase, matrix metalloproteinase - by lps-induced macrophages. the generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin- b (il- b) and tumor necrosis factor-alpha (tnf-a), the production of both being dependent on signaling through the p mitogenactivated protein kinase (mapk) pathway. peripheral mononuclear cells (  )isolated from buffy coat cells of healthy neonatal to -month old holstein calves were cultured in the presence and absence of lf ( ng/ml), lps ( mg/ml), anisomycin ( mg/ml), a known p activator -the positive control, and mm of sb , a known p inhibitor -the negative control. sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. reactions were terminated under reducing conditions and evaluated using western immunoblotting. phosphorylation of activated transcription factor- (atf- ) by phosphorylated p served as the marker of investigation. immunologically reactive atf- expression by lps and anisomycin-treated cells was compatible with a prominent band at kd. evidence of lf-induced inhibition of lps-induced p- activation was observed in lanes representative of co-cultures of lf lps; lf anisomycin; and anisomycin sb , which was demonstrated by decreased immunological reactivity at kd. the findings here, suggest that lf interferes with lps-induced p- activation of transcription factor atf- , in vitro. this serves as additional proof of its potential use in attenuating the systemic effects of lps. six ( ) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ f] fluorodeoxyglucose ([ f]fdg) and [ f]ftha by using dynamic cardiac-gated fused pet/ct for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. pet data were acquired over a hour period with the heart in the center of the scanner field of view. regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. serial blood samples were also taken during pet imaging for comparison with image data. the equilibrium biodistribution of both tracers was documented hour post-injection in a whole body pet/ct image. standard echocardiographic examination of cardiac structures was also performed. both radiotracers remained in the plasma fraction; however, [ f]ftha was cleared from the more rapidly than [ f]fdg (t / $ and $ min, respectively). the tracers were readily visualized within the feline myocardium in dynamic pet images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range - bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. physiologic [ f]fdg was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ f]ftha seen in the myocardium, liver and renal cortex. all cats were normotensive with normal echocardiographic parameters. this study demonstrates the utility of kinetic imaging using the left ventricle (lv) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. real-time three-dimensional echocardiography (rt de) offers new modalities for lv assessment. the aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (mmvd) in dogs using rt de. privately owned dogs were classified by standard echocardiography into: healthy ( ), mild ( ), moderate ( ) and severe mmvd ( ). a lv cast was obtained using semi-automated endocardial border tracking from rt de dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on lv long-axis dimension) end-diastolic (edv) and endsystolic volumes (esv), lv long-axis dimension and rt de sphericity index, were derived. global and regional edv and esv increased significantly with increasing mmvd severity, assessed by mmvd group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and lv end-diastolic and end-systolic dimensions. all three segments contributed to the overall increased global volumes, but the mid edv segment was strongest associated with increasing lv end diastolic dimension (p . ). furthermore, lv long axis distance and lv sphericity index increased with increasing mmvd severity. the basal and apical edv segments were strongest associated with sphericity index (p o . ). in conclusion, this rt de study showed that increased lv edv, primarily in the mid segment, leads to rounding of lv apical and basal segments in response to increasing mmvd severity in dogs. dogs from shelters in florida with naturally acquired di infection were euthanized and necropsied. all adult di in each dog were sexed using morphological features. total worm burdens and numbers of males and females were recorded. no other information was available for any dog. all data, raw and transformed, were examined visually and descriptively. raw numerical data were further examined by a paired t-test; log-odds transformed data were examined by logistic regression. we also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a m:f . . worm intensities ranged from to di per dog. eight dogs had unisex infections: / had all-female infections. dogs with lowintensity dual-sex infections were more likely to have greater numbers of female di. overall, sex ratios were equal (paired t-test, p . ). however, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms. our data show that di sex ratios in naturally-infected dogs equal when examining the entire dog population, but deviate to favor female worms at low worm intensities. these data could impact adulticide treatment strategies. the reasons for sex ratio distortion in di are unknown. we evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (cpb) for mitral regurgitation in small breed dogs. fifty dogs (body weight . - . kg, age - years) with mitral regurgitation were treated between august and november . the cardiac murmur was grade / - / . the preoperative chest x-rays showed cardiac enlargement (vertebral heart scale (vhs) . - . ). echocardiography showed severe mitral regurgitation and left atrium enlargement (la/ao . - . ). after inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. cpb was started by using a cpb circuit connected to carotid artery and jugular vein catheters. after inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. the chordae tendineae were replaced with expanded polytetrafluoroethylene. a mitral annulus plasty was also done, and the left atrium was closed. after de-clamping for restarting the heart, the chest was closed. heart rate decreased from - bpm to - bpm. the grade of cardiac murmur was reduced to / - / three months postoperatively, and the heart shadow was reduced (vhs . - . ) in the chest x-rays. echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (la/ao . - . ). mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. pulmonary arterial hypertension (pah) is a well recognized condition in dogs leading to considerable morbidity and mortality. the majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin- . more recently, it has been shown that the mitochondria play an important role in the development of pah as oxygen sensors and regulators of cellular proliferation. in pah, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. dichloroacetate (dca) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the krebs cycle, thus increasing mitochondrial respiration. in three different rat models of pah, dca has been shown prevent and reverse pah by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. dca has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring pah. the objective of this open label pilot study is to evaluate the therapeutic and toxic effects of dca in naturally occurring canine pah. three dogs with pah diagnosed by doppler echocardiography and no correctable underlying cause are enrolled in the study. dogs are orally administered mg/kg of dca divided daily for weeks, and then . mg/kg of dca divided daily for the remainder of the study. at baseline, , , , and weeks, an echocardiogram, cbc, serum chemistry profile, urinalysis, nt-probnp, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough dca level ( hr post-dose) are obtained. the measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time. the basis for the role of the mitochondria in pah and the results of this pilot study will be presented to determine if dca warrants further study as a therapy for dogs with pah. study produced the strongest associations between the ncl phenotype and cfa markers. all ncl-affected tibetan terriers were homozygous for the same haplotype which extended for consecutive snps spanning . mb. none of the annotated genes within this target region had previously been associated with human or rodent ncl. we used dna from ncl-affected tibetan terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c. delg, in exon of positional candidate atp a . this deletion produces a frame shift and a predicted premature termination codon. we genotyped all tibetan terrier dna samples in our collection and found all ncl-affected tibetan terriers to be homozygous for the c. delg allele. eleven additional c. delg homozygotes were either less than years old, or lost to follow up. there were no known cases of ncl in the remaining tibetan terriers which were either heterozygous (n ) or homozygous for the ancestral allele (n ). atp a is a member of group of ion transport genes and has been associated with lysosomes. mutations in human atp a cause kufor-rakeb syndrome (krs), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. post-mortem findings in krs have not been reported. we conclude that ncl in tibetan terriers is caused by a mutation in atp a . our results suggest that krs may be a form of adult onset ncl in humans. niemann-pick type c (npc) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat npc disease have been ineffective to date. the recent use of intravenously administered -hydroxypropylbeta-cyclodextrin (hpbcd), an fda-designated orphan drug (may ), in a small number of children with npc disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. to rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with hpbcd therapy in npc disease, we have utilized the spontaneous feline npc model harboring a missense mutation in npc (pc s), orthologous to the most common mutation in juvenile-onset patients. the feline npc model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. we identified that intrathecal administration of hpbcd ameliorated all clinical aspects of neurological disease at least up to weeks of age (an age when untreated cats die) but had no effect on hepatic disease. we identified that while subcutaneous therapy with hpbcd at all doses ameliorated liver disease, only mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. finally, we identified a dose-related toxic effect of hpbcd on hearing function that had not been described in any other species. leukodystrophies are disorders of myelin synthesis and maintenance that affect cns myelin. they are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. although infrequently seen, several forms have been described in various dog breeds. we present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in border terrier puppies. three border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. the youngest dog was a -week old male. it was the only dog affected in the litter. the other two dogs were -week old female littermates. there were two unaffected males in the same litter. physical examination revealed no abnormalities. on neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). the tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. severe cerebellar ataxia was observed when the dogs ambulated. proprioceptive positioning was delayed in the pelvic limbs of all dogs. spinal reflexes and nociception appeared normal. necropsy was performed in all puppies. no macroscopic changes were observed. histologic evaluation of the cns revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. white matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. the combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. the number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process. the clinical and pathological features of this disease have not been described in any other canine leukodystrophy. the primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. genetic studies are underway to determine if the disease is inherited and the inheritance mode. a syndrome of border collie collapse (bcc) appears to be common in dogs used for working stock. this syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. a presumptive diagnosis of bcc can only be made by eliminating other causes of exercise intolerance and weakness. the purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis. seven adult border collies with a history of collapse during sheep herding (affected) and adult border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. exercise was halted at minutes or earlier if there were signs of gait or mentation abnormalities. pre-exercise evaluation included physical examination, orthopedic and neurological exam. pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ecg, cbc, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to minutes after exercise. additional testing in affected dogs included measurement of acetylcholine receptor antibodies (achrab) and dna testing for dynamin-associated exercise induced collapse (deic) and the ryanodine receptor mutation associated with canine malignant hyperthermia(mh). one week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for emg and muscle biopsies. there were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. no arrhythmias were detected. affected dogs were negative for the dna mutations tested and for achr ab. thoracic radiographs, echocardiograms, emgs and muscle biopsies were normal. the normal dogs had no alterations in mentation or gait during or after exercise. three of the affected dogs had exercise halted early ( min- min) because of altered gait or mentation. all of the affected dogs were abnormal in the minutes following exercise. abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. all dogs returned to normal by minutes. bcc appears to be an episodic nervous system disorder that can be triggered by exercise. genetic testing excluded deic and the described canine mh mutation. common causes of exercise intolerance were eliminated, but the cause of collapse in bcc was not determined and no clinical or biochemical marker to aid diagnosis was established. equine cushing's disease (ecd) is common in older horses. the purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. the study was a retrospective cohort design evaluating equine accessions reported to the veterinary medical data base (vmdb) and the ohio state university from - . proportional accessions, annual incidence and demographic characteristics of horses with ecd were compared with all accessions in the vmdb. medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information. two hundred seventeen new cases of ecd were reported to the vmdb. incidence increased from . / , in to . / , in . eighty-one percent of horses were ! years of age. average delay from onset of signs to diagnosis was days (range to , days). hirsutism ( %) and laminitis ( %) were the most common clinical signs. improvement in one or more signs months after diagnosis was reported by / ( %) of horse owners. none of the clinical or laboratory data were associated with survival and, % of horses were alive, . years after diagnosis. / ( %) of horses were euthanatized and / ( %) were euthanatized due to conditions associated with ecd. twenty-eight of ( %) of horse owners said they would treat a second horse for ecd. ecd is becoming a more frequent diagnosis. fifty percent of horses survived . years after diagnosis and owners were satisfied with the horse's quality of life. supported by centers of excellence in livestock diseases and human health, college of veterinary medicine, university of tennessee. the role of the hypothalamic-pituitary-adrenal (hpa) axis in sepsis has been a subject of a great deal of research. the role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. the purpose of this study was to assess inter-relationships of adrenocorticotropin (acth), cortisol, and insulin-like growth factor-i (igf-i), in septic and non-septic term foals. blood samples were obtained from term septic foals less than days of age (n ) admitted to texas a&m university veterinary medical teaching hospital or mid-atlantic equine hospital. the foals were classified as septic by a sepsis score ! and/ or a positive blood culture. non-septic term foals less than days of age (n ) and having a sepsis score o and a negative blood culture, were obtained from texas a&m university veterinary medical teaching hospital and mid-atlantic equine hospital. plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at hours post admission and at days post admission or at the time of discharge. plasma concentrations of acth, and serum concentrations of cortisol and igf-i were determined by specific rias. data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of sidak. significance was set at p o . , and analyses were performed using s-plus software (tibco, inc., seattle, wa). plasma concentrations of acth were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol ( ae ng/ml vs ae ng/ml) but lower serum igf-i ( ae ng/ml vs ae ng/ml) relative to non-septic foals pooled overall sampling times. the positive association of the peripheral blood concentrations of acth and cortisol depended on disease status of the foals. specifically, cortisol and acth were positively correlated for the septic foals (p . ) but not significantly correlated in the non-septic foals. peripheral concentrations of acth and igf-i were not significantly correlated whether data were pooled overall or stratified by sepsis status. however, peripheral concentrations of cortisol and igf-i were negatively associated (p . ); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. the negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide igf-i may reflect interactions of these homeorhetic hormones. further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. the purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals. four healthy foals were used for its comparison with foals with suspected botulism. controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. the common peroneal nerve was chosen for its superficial location and easy access. stimulating electrodes were placed along the common peroneal nerve. for recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies ( to hz). amplitude, area under the curve and percentages of decrement or increment for each m wave over subsequent potentials for each set of stimuli were analyzed. baseline m waves were decreased in affected foals compared to controls. a decremental response was seen at all frequencies in control foals. decremental responses were also observed in affected foals at low frequencies. however, an incremental response in amplitude and area under the curve was seen in all affected foals at hz. reduced baseline m waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals. this study examined the frequency with which dogs are exposed to e. chaffeensis and e. ewingii relative to e. canis, which is transmitted by the more ubiquitously distributed brown dog tick (rhipicephalus sanguineus). a total of , canine serum samples, ranging from to from each of the participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. all serum samples were tested by three microtiter plate elisas using species-specific peptides for antibodies to e. canis, e. chaffeensis and e. ewingii. zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. comparisons were evaluated using the chi-square test. seroreactivity for at least of ehrlichia spp was found in samples from every institution both mississippi and oklahoma had greater than a % samples from ohio had the lowest aggregate seroprevalence ( . %) with only dogs e. canis seropositive, one e. ewingii seropositive and no e. chaffeensis seroreactors. the geospatial pattern of e. chaffeensis and e. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to e. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (hme) cases reported by the cdc. this study provides the first large scale regional documentation of canine exposure to these three ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector urinary incontinence (ui) occurs in approximately % of spayed female dogs. the most common cause is urethral sphincter mechanism incompetency (usmi). pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. new synthetic compounds have been used in human medicine; the most promising is polydimethylsiloxane (pdms), which has been shown to be more effective than glutaraldehyde cross-linked collagen. the purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of pdms urethral bulking agent (pdms uba) in client-owned, spayed female dogs with naturally-occurring ui due to usmi.twenty-two, spayed female dogs were included. dogs had a median age of years ( to years). eighteen dog breeds were represented, and dogs weighed a median of . kg ( . to . kg). average length of time of ui was . ae . years; / dogs had been treated medically, of which / were continent, / were improved, and / had no improvement. dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a . mm, -or -degree, cm rigid cystoscope. urethral bulking was performed with pdms uba. on average, . ae . ml were injected in to locations approximately to . cm distal to the trigone submucosally in the proximal urethra. good coaptation was achieved in all dogs. the procedure took on average . ae . minutes. one dog experienced urethral obstruction after the procedure; a foley catheter was inserted for approximately hours and removed at which time she urinated normally and was continent. three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. all dogs were treated with meloxicam ( . mg/kg po q h for days).owners were contacted on day after discharge and / dogs were continent; / dogs was improved. dogs were re-evaluated week after discharge and / dogs were continent and / dogs polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. the first cases of polyneuropathy in the leonberger breed (leonberger polyneuropathy or lpn) were identified in by one of the authors (gds) and a report published in (musclenerve : - ) . in this report a spontaneous, distal and symmetrical polyneuropathy with onset between to years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. there were striking similarities between lpn and the charcot-marie-tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.a genome-wide case-control association study for lpn was performed with cases and controls on high-density k canine snp arrays and revealed a significantly associated region on cfa (p raw .  À p genome .  À ). a clear association of an approximately mb cfa haplotype with cases (p .  À ) was observed, particularly with those cases that were affected more severely and at a younger age (p .  À ). a positional candidate gene, arhgef , which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. of all leonbergers with young onset lpn (before years), . % ( of ) have two copies of this deletion, and, of all young onset leonbergers that are nerve biopsy positive for lpn, . % ( of ) have two copies of this deletion. importantly, nearly all dogs carrying two copies of the deletion ( of or . %) are affected with lpn by the age of years.the leonberger breed was generated from crossing several breeds, including the st. bernard, and a polyneuropathy clinically and histologically similar to lpn occurs in this breed. to determine if the arhgef mutation was associated with polyneuropathy in the st. bernard, dna was extracted from archived frozen muscle biopsy specimens from clinical cases (n ). the identical arhgef startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. in humans and animals, startle disease is typically caused by mutations in one of three genes (glra , glrb, and slc a ) encoding postsynaptic glycine receptor subunits (a and b) or a presynaptic glycine transporter (glyt ). a litter of seven irish wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at - days of age post-partum. signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.based on the clinical signs, exons from the three candidate genes were amplified from genomic dna isolated using pcr and directly sequenced. no deleterious polymorphisms were identified in either glra or glrb. however, difficulties were experienced in amplifying slc a exons and from affected animals, although control samples were positive, suggesting that the pcr primer designs and conditions were not at fault. further pcrs revealed that the reason for this anomaly was the presence of a homozygous . kb deletion encompassing exons and of the glyt gene in both affected animals. this deletion is predicted to result in the loss of part of the large cytoplasmic n-terminus that is vital for trafficking of glyt to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. this genetic lesion was confirmed by defining the deletion breakpoint, southern blotting and multiplex ligationdependent probe amplification (mlpa). this analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. wider testing of related animals has identified a total of carriers of the slc a deletion and enabled the identification of non-carrier animals to guide future breeding strategies. insulin resistance (ir), obesity, and type diabetes affect glucagon-like peptide (glp- ) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. we therefore hypothesized that glp- concentrations would change in horses as obesity and ir were induced or exacerbated by overfeeding. six horses previously diagnosed with equine metabolic syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for weeks. intravenous and oral glucose tolerance tests (ogtts) were performed at and weeks. effects of time and period ( and weeks) were assessed by repeated measures anova.mean body weight increased from ae kg (range, to kg) to ae kg (range, to kg) over weeks, with individual horse weight gain varying from to %. mean body condition score increased (p . ) from ae (range, to . ) to ae (range, to ). three horses developed mild laminitis. glucagon-like peptide concentrations increased over time during ogtts (p . ), but the period  time effect was not significant (p . ). area under the glp- curve remained unaffected by weight gain, whereas area under the insulin curve increased (p . ) over time, indicating a reduction in insulin sensitivity. obesity and ir were induced or exacerbated when horses previously diagnosed with ems were overfed, but glp- concentrations did not change as a result. hypertonic saline solution ( . %) (hss) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. the use of this fluid in horses with severe dehydration is controversial. the purpose of this study was to compare the use of hss and isotonic saline solution ( . %) (iss) for the emergency treatment of endurance horses.endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. twenty-two horses were randomly assigned to receive ml/kg of either hss or iss along with l lactate ringer's solution (lrs). following this bolus, all horses were treated with an additional l of lrs. blood and urine samples were collected before, during and after treatment. data was compared using two-way anova with repeated measures.as compared to iss, hss horses showed a greater decrease in pcv (p . ), total protein (p . ), albumin (p . ), and globulin (p . ). hss horses showed a greater increase in sodium and chloride (p o . ) as compared to iss horses. horses receiving hss had a shorter time to urination (p . ) and lower specific gravity (p o . ) than those receiving iss.results of this study indicate that hss may provide faster restoration of intravascular volume deficits than iss in endurance horses receiving emergency medical treatment. more profound electrolyte changes should be expected with hss however. b -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the jacobs-stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. increase in pulmonary transvascular fluid fluxes (j v-a ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar o exchange during the fast erythrocyte transition time across the pulmonary capillaries. therefore, we hypothesised that treatment with inhaled b -adrenergic receptor agonist will increase j v-a and the alveolar-arterial po difference (aado ) during exercise.six stb horses were exercised on a high-speed treadmill at % vo peak until fatigue. horses were randomly assigned to treatment with salbutamol (sal: mcg) or placebo (control: con) inhalation via aeromask à min prior to exercise, with cross over treatment used at the repeated exercise test ( days later). arterial and mixedvenous blood, as well as co elimination and o uptake, were sampled simultaneously at rest, during exercise at sec intervals until fatigue, and into recovery. blood gases were analyzed. aado was calculated using the inspired po ( mmhg), and blood partial pressure of o and co . blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. cardiac output (q) was calculated using the fick equation. j v-a was calculated using q and blood volume changes across the lung. variables were analyzed using two-way repeated-measures anova (p o . ).the duration of exercise to fatigue was . ae . min and . ae . min in both con and sal, respectively. at rest sal had no effect on j v-a , oxygen consumption (vo ), blood oxygen saturation (so ) or aado (p . ). at the onset of exercise j v-a increased in con and sal (p o . ) and at fatigue reached . ae . l/min and . ae . l/min, respectively. treatment with sal had no effect on j v-a during exercise (p . ). at the onset of exercise so and vo increased in con and sal (p o . ). treatment with sal had no effect on so or vo during exercise (p . ). aado increased during exercise in con and sal (p o . ) and at fatigue reached . ae . mmhg and . ae . mmhg, respectively. treatment with sal had no effect on aado during exercise (p . ).inhaled b -adrenergic receptor agonist salbutamol at the dose of mcg given min before exercise did not affect the duration of exercise to fatigue, j v-a , vo , so or aado . therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. inflammatory airway disease (iad) and recurrent airway obstruction (rao) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. there is evidence that th cytokines and il- play some role in rao. iad is a common condition in horses, but its pathophysiology is still not understood. the aim of the present study was therefore to determine the mrna expression of th , th and th inflammatory cytokines, to understand the immunological mechanisms of iad.the mrna expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (balf) of seventeen horses with iad and compared with ten control horses. the horses were selected based on -their clinical signs, -the inflammatory cells count in the balf, -their physical examination and -their medical history. the mrna expression of il- , il- b, il- , il- and il- was significantly up-regulated in balf from horses with iad.furthermore, the balf samples were subdivided in two groups based on the differential cells count -balf with increased mast cells (iad-mast) and -balf with increased neutrophils (iad-neutro). il- was significantly down-regulated in the iad-neutro group compared to the iad-mast group. il- , il- and il- were significantly up-regulated in the iad-neutro group compared to the iad-mast group.the present study shows that iad in horses is characterized by a th and a th mrna inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the balf of horses with iad. b -adrenoreceptor (b -ar) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. activated human peripheral blood lymphocytes (pbls) also respond to b -ar agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and recurrent airway obstruction (rao). the aim of this study was to develop an in vitro technique for measuring the response of equine pbls to stimulation with salbutamol, a b -ar agonist. this method was then used to compare the response of pbls from rao-affected and non-affected horses to b -ar agonist stimulation. pbls from rao and nonaffected horses were cultured ( x /ml) in rpmi complete media with concanavalin a (cona, ug/ cells) for , , or days then stimulated with salbutamol ( minutes). using flow cytometric techniques, response was measured by detecting protein kinase a phosphorylation of vasodilator stimulated phosphoprotein (vasp). results were verified by western blot analysis. activated pbls were then incubated with cona for one day were pre-incubated with b or b-adrenoreceptor antagonist (ici , , sigma s ; atenolol, sigma s ) for minutes, followed by minutes salbutamol ( nm) stimulation. results were analyzed by anova or ancova and differences were considered significant when p o . .response to b-antagonist was only observed in activated pbls (pre-cultured with con a) and was greater in cells from rao horses as compared to cells from non-affected horses. the addition of b-antagonist attenuated the response of pbls to salbutamol while the addition of a b -antagonist had no effect. these findings indicate that activated pbls from rao-affected horses have a greater response to salbutamol as compared to pbls from non-affected horses, and this response is mediated mainly through the b -ar.human b -ar are known be polymorphic and this polymorphism results in a variable response to b -agonist binding that affects long term outcome in human asthmatics. further studies are required to determine if the difference in response of pbls from rao affected as compared to non-affected horses is due to genetic polymorphism in the equine b -ar, and whether this difference is associated with a propensity for horses to develop equine rao. key: cord- -k coui authors: mckeown, alex; turner, andrew; angehrn, zuzanna; gove, dianne; ly, amanda; nordon, clementine; nelson, mia; tochel, claire; mittelstadt, brent; keenan, alex; smith, michael; singh, ilina title: health outcome prioritization in alzheimer’s disease: understanding the ethical landscape date: - - journal: journal of alzheimer's disease : jad doi: . /jad- sha: doc_id: cord_uid: k coui background: dementia has been described as the greatest global health challenge in the st century on account of longevity gains increasing its incidence, escalating health and social care pressures. these pressures highlight ethical, social, and political challenges about healthcare resource allocation, what health improvements matter to patients, and how they are measured. this study highlights the complexity of the ethical landscape, relating particularly to the balances that need to be struck when allocating resources; when measuring and prioritizing outcomes; and when individual preferences are sought. objective: health outcome prioritization is the ranking in order of desirability or importance of a set of disease-related objectives and their associated cost or risk. we analyze the complex ethical landscape in which this takes place in the most common dementia, alzheimer’s disease. methods: narrative review of literature published since , incorporating snowball sampling where necessary. we identified, thematized, and discussed key issues of ethical salience. results: eight areas of ethical salience for outcome prioritization emerged: ) public health and distributive justice, ) scarcity of resources, ) heterogeneity and changing circumstances, ) knowledge of treatment, ) values and circumstances, ) conflicting priorities, ) communication, autonomy and caregiver issues, and ) disclosure of risk. conclusion: these areas highlight the difficult balance to be struck when allocating resources, when measuring and prioritizing outcomes, and when individual preferences are sought. we conclude by reflecting on how tools in social sciences and ethics can help address challenges posed by resource allocation, measuring and prioritizing outcomes, and eliciting stakeholder preferences. health outcome prioritization is the ranking in order of desirability or importance of a set of diseaserelated objectives and their associated cost or risk, obtained collaboratively with or from patients and their carers. it can occur at several levels and by different stakeholders, including: between a doctor [ ] , nurse [ ] , pharmacist [ ] , or other healthcare professional and an individual patient; within families of an individual or individuals with ad [ , ] ; between cohorts of patients and researchers in a randomized controlled trial (rct) [ ] ; by national healthcare systems such as the national health service (nhs) and associated resource allocation bodies such as the national institute for clinical and care excellence (nice) [ ] ; and at the international level between governments when collaborating on meeting global health priorities [ ] . situations in which outcome prioritization is useful include: ) regulatory scenarios for assessing benefit to risk ratio by, for example, establishing which endpoints would be the most relevant to measure in an rct; ) economic scenarios considering 'value for money', as the finitude of resources precludes being able to fund every intervention; ) questions of individual benefit-torisk ratios to establish what kind of care a particular patient wishes to receive. alzheimer's disease (ad) is the most common type of dementia and a 'global public health priority'. [ , ] some evidence suggests the prevalence of all-cause dementia rates may be declining [ ] ; however, with an aging population and older people living longer, the incidence of specific dementias such as ad may continue to rise [ ] . the prospect of a global prevalence of . million [ ] imposes a significant and growing costs, necessitating innovation in the management of dementia [ ] . in this paper we describe and analyze the evidence base for the complex ethical landscape of health outcome prioritization as it takes place in the most common form of dementia, ad. the paper was produced as an ethical, legal, and social implications (elsi) output for the european union and innova-tive medicines initiative (imi)-funded consortium project, roadmap: real world outcomes across the ad spectrum for better care [ ] . the authorial team is comprised of researchers working on the roadmap project, including clinicians, scientists, and patient representatives with expertise in ad, led by three bioethicists with expertise in analyzing ethical issues in healthcare and medicine. although there are several types of dementia, roadmap focuses exclusively on ad dementia; as such, the paper analyses ethical issues in health outcome prioritization for ad alone. the research questions we seek to answer are: • what are the ethical issues that drive outcome prioritization? • what balances need to be struck when prioritizing outcomes? our analysis highlights eight key areas of ethical salience to fair outcome prioritization in ad. there is currently no curative treatment for ad; as such, while we assume that if it were possible to cure ad, most individuals would be likely to prioritize this over other outcomes, since no cure exists, the review focuses on how outcomes other than this might be weighed and prioritized. the themes that emerged from our analysis illustrate the challenges of realizing outcomes and making prioritization choices. given the growing global prevalence of ad analysis of these ethical issues is timely and necessary. a narrative review based on a mixture of structured and ad-hoc searching of academic literature was conducted. this is a narrative, rather than systematic, review, capturing the landscape of the debate rather than surveying the prevalence of different views in the literature. while establishing the relative prevalence of different themes or theories would be independently interesting, our aim was the identification of these in the literature rather than their relative prominence. this choice reflects our presumption that the normative legitimacy of ethical challenges is not a function of the frequency of their discussion. relevant manuscripts were identified in several stages. first, a structured search was carried out in january and updated in september across five academic indexes: embase, pubmed (incl. medline), scopus, web of science, and google scholar. the search identified articles with relevant titles using the search strings listed in table . the keyword search was limited to the 'article title', for three reasons. first, a search of titles was likely to yield those articles most directly pertinent to our analysis. second, additional snowball sampling via further references supplied by co-authors, and via screening of the references of already included studies identified further relevant research representing a broad range of stakeholders in ad. third, a preliminary search of abstracts as well as titles yielded an extremely large sample that, on the basis of screening a sample of the papers returned, added noise but little value in relation to capturing the principles of the ethical debate, when compared to the results of a title only search. this is to say that numerous papers are available which happen to use some of our keywords, but on reviewing are not pieces of research which are about ethics in any way that is relevant to our purpose. for example, a title and abstract search on scopus for articles published in english for the specified date range using key terms ethic* priorit* and health* returned , documents. to ensure a manageable and contemporary sample, we restricted our search to literature in peer-reviewed journal articles and conference proceedings published from , ten years before work on the study began. additionally, we only reviewed articles written in english. article abstracts and, if needed, full texts were then assessed for relevance by a single reviewer. a subset of articles was screened by a second reviewer, and the results of this screening by the two reviewers were compared for consistency in assessing the relevance of the study to the purposes of the review. this comparison yielded an agreement of %, which indicated a reliable degree of consistency, and discussion of the reasons for the discrepancies in the remaining helped to refine shared understanding of the criteria when further screening was carried out. finally, we screened the references of included articles to identify further relevant articles and allowed all authors to suggest further relevant articles not identified by the initial search ("snowball sampling"). articles were retrieved by the initial search. of these were rejected, either due to being judged insufficiently relevant after review; because they were irretrievable; or having been published in a language other than english, leaving included and reviewed here. snowball sampling and the introduction of additional previously collected articles of contextual relevance yielded a further references, giving a total of documents pertaining to ethical issues in outcome prioritization in ad reviewed and discussed. three references pertaining to research methodology were also provided for support in study design and data analysis. analysis of the papers aimed to map the landscape of prevalent ethical themes in the academic and clinical discussion around outcome prioritization in healthcare generally and ad specifically. to identify themes for discussion, a pre-defined thematic framework was not used; rather, themes emerging from the literature were identified [ ] . each paper was read in full, key passages were highlighted that addressed ethical issues or concepts, understood here as areas of 'right' and 'wrong' or competing values and normative interests. highlighted segments were labelled with codes reflecting the lead author's interpretation of the text [ ] . our analytic approach was informed by established 'realist' and 'meta-narrative' methods of qualitative synthesis, drawing on previous ethics-focused review studies for guidance [ ] [ ] [ ] . discussion of the themes occurred between co-authors, who come from disciplines including philosophy, epidemiology, psychology and psychiatry, public health, as well as the pharmaceutical industry. themes were reviewed and discussed at each draft iteration of the study until consensus was reached and there was agreement among all of the authors that the themes accurately reflected the findings of the analysis. this required five such iterations in total, at which point the thematic categorization was finalized. below, we outline eight areas of ethical salience in outcome prioritization that emerged from the analysis. these eight areas are not themselves ranked in order of priority or importance; rather they reflect the landscape of matters of key ethical importance that arose from the review: ) public health and distributive justice, ) scarcity of resources, ) heterogeneity and changing circumstances, ) evidence, ) values and circumstances, ) conflicting priorities, ) communication, autonomy and caregiver issues, and ) disclosure of risk. a key ethical aspect of contemporary ad management is the shift toward public health prevention [ ] away from clinical medicine [ ] , to contain the costs of supporting an expanding and aging population in which ad prevalence will increase [ ] [ ] [ ] . however, the benefits of a preventive approach should not be overstated [ , ] ; rather, the benefits of promoting general health advice may extend to dementia [ ] . this shift is important for determining an ethical prioritization of outcomes in two respects. first, public information about how to reduce the risk of dementia through lifestyle modifications from early life onwards affects the relationship between the state and its citizens [ ] . pressure to make certain choices may be viewed as an infringement of liberty [ , ] , particularly where the state establishes policies that expect individuals to modify their behavior; or prioritize treatment based on following preventative recommendations. moreover, recommending lifestyle modifications that provide no guarantee to prevent ad may be viewed as unacceptably paternalistic and corrode trust. second, the application of quality adjusted life years (qalys) to individual priorities may not adequately capture the subjectively bound nature of 'quality of life' or 'well-being', despite the centrality of both to ensuring a successful outcome for people with ad, given that personal evaluations of concepts such as these differ [ , ] . furthermore, 'benefit' and 'value' may be interpreted differently. from a health economic perspective it is essential to attach a particular price to a particular level of benefit [ ] , however, the priorities of individuals with ad are likely to differ from other stakeholders, such as patients, their carers, health care professionals, providers, and payers of medical treatments [ ] . outcome prioritization requires distributing limited resources to achieve desired outcomes. how these distribution decisions are made differs geographically. for example, in the uk, nice prioritizes cost-effectiveness, whereas cost-effectiveness does not inform decision-making in germany, and in the netherlands it informs decision-making but only above a certain price threshold (very cheap drugs do not attract scrutiny) [ ] . irrespective of particular metrics used, the finitude of resources means that some people will be denied access to interventions that would benefit them [ ] [ ] [ ] . this is ethically significant since the needs of some will be de-prioritized in favor of the needs of others, and the resulting gap may conflict with the clinical duty of care (this also holds for prioritization between people with the same condition and people with different conditions). as hermeren et al. [ ] summarize baldly, to prioritize is inevitably to say 'no' to somebody. this is especially acute in the ad context, in which many people are unable to derive significant value, given that no curative treatments are yet available and evidence for the effectiveness of preventative interventions is weak. in light of this, some have concluded that the greatest need is for high-quality long term care, and similarly high-quality support for family carers, such that the lives of people with ad and their families are made as satisfying as possible in the absence of any treatment that would decisively reverse the disease [ , ] . indeed, research carried out by kelly et al. ( , p. ) [ ] with people with dementia, their carers, clinicians, and other health professional finds preferences of this kind in the top ten overall prioritized outcomes across all groups. encouragingly, for the findings of our own analysis, there is also significant parity with the kelly et al. [ibid.] study insofar as ethical dilemmas over finite resources report shared concerns, for example: negotiating the balance of benefits between pharmacological and lifestyle or social interventions; evaluating the value of early diagnosis in light of its psychological impact in the absence of a cure; how to optimize outcomes for people with advanced dementia at the end of life; and how to also support carers of people with ad as well as people with ad themselves. prioritization decisions are ultimately concerned with both outcomes and endpoints, which are related but distinct parameters. outcomes denote measured effects of treatment, whereas endpoints are preidentified targets of a study built into its design [ ] . from an international perspective, the outcomes and endpoints considered important may differ, depending on characteristics of particular markets [ ] . moreover, the definition, diagnostic cut-off and mechanism of measuring the numerous potential outcomes and endpoints continue to be debated; and the difficulty of determining preferences for these-for example living longer, declining more slowly, or dying sooner before more serious incapacity-is compounded by the plethora of available assessment tools. a further challenge in ad, and in the context of other conditions, is that assessment tools measuring a wide range of domains including memory, spatial orientation, semantic processing, speech, emotion, mood, apathy, aggression, and mobility [ ] [ ] [ ] are not ideally sensitive or specific and so do not yield perfect validity either in earlier and preclinical stages of the disease [ ] or in more severe stages [ , ] . in addition to this heterogeneity in assessment tools, changing background circumstances also affect prioritization decisions. prioritization is also made complex by the heterogeneity of ad itself, in that the disease affects different individuals in different ways, in different cultural contexts, and at different stages of the disease; indeed, priorities are likely to differ in line with the progression of the disease, since the impact of mci, for example, is less severe than very advanced ad later in life [ , ] . as such the ad community itself cannot be assumed to be homogeneous with respect to how members of it are affected by the disease [ , ] , which underlines the risk of assuming that the priorities of one ad population subgroup is necessarily representative of other subgroups or the ad population at large. moreover, not only are resources for people with ad limited (as above), but their availability is not necessarily constant. for example, external or short-term political factors and election cycles can affect both health policy and budgets, which shape prioritization decisions [ ] . equally, prioritization is shaped by new advances in medical science that expand what is therapeutically possible. furthermore, global trends towards population growth and increasing life expectancy may mean a future in which more people are competing for fewer resources [ ] . outcome prioritization should therefore be viewed as an ongoing process, requiring reflection and revision, according to changing circumstances, as needs and options change [ ] [ ] [ ] . determining what counts as fair prioritization of outcomes depends partly on what needs to be known to make the relevant decision [ , ] . data from randomized trials is considered the 'gold standard' for establishing efficacy; however, there are a number of challenges that make it expensive and time consuming to test the efficacy (or effectiveness) of a drug, and make it difficult to determine optimal clinical practice for long term treatment management in ad. first, there is the challenge of attrition compromising the validity and/or generalizability of results from trials [ ] . there is evidence that older, sicker individuals in particular are more likely to drop out of studies [ , ] , die before the trial is concluded or to discontinue the study treatment due to adverse effects. second, people in symptomatic stages of ad are typically older and more likely to have comorbidities, making them complex [ ] [ ] [ ] . rcts of new drug products may investigate drug-drug interactions to some degree, but this is not always exhaustive. data from rcts about drug-drug combinations may be underpowered or simply unavailable. since interventions may have trade-offs between desirable effects and undesirable side-effects, weighing these is less straightforward where comorbidities requiring different types of treatment are present [ ] . polypharmacy is common, without clinicians fully knowing the risk of harm due to lack of evidence. indeed, separate studies are designed to address these issues [ ] , and knowledge of the mechanism of action of certain drugs contributes usefully to decision-making. notwithstanding the challenges outlined, therefore, clinicians are not uninformed when prescribing certain drugs. rather, the biggest challenge arises when patients have multiple comorbidities which all require treatment [ ] since it may become necessary to prioritize the treatment of some rather than other in order to realize an all-thingsconsidered, rather than ideally, desirable outcome. as such, ensuring an optimal balance of outcomes in the context of chronic disease in old age is particularly challenging [ ] . this uncertainty poses a further challenge for realizing priority outcomes which depend on the effectiveness of a particular drug. the importance of patients being able to make their own decisions is reflected in the contemporary centrality of 'patient-centered care' [ , ] . properly respecting the values and circumstances of patients does not mean swapping a paternalistic approach for an equally polar norm in which patients have absolute autonomy over all courses of action [ , ] . nevertheless there is an inevitable imbalance between the clinical expertise of doctors and patients who are experts in their own experience and the subject of the disease and its treatment [ ] [ ] [ ] . as such, patient-centered care and prioritization of outcomes in ad should be conceived as a collaboration known as 'shared decision-making' [ , ] . knowledge-related power imbalances underline the ethical importance of remembering that it is the patient who must have the final say. indeed, this is especially important in the case of ad, where treatment options are limited. for example, if faced with the prospect of cognitive decline a patient may, for example, decide not to accept a pacemaker to prevent heart failure. it is not obvious in such circumstances that the decision against prolonging life as long as possible is either irrational or to be avoided. as van summeren et al. [ ] report, a dilemma often faced by patients, carers, and clinicians is how to balance factors such as the benefit of palliation of symptoms and maintenance of life or independence against the discomfort caused by the side effects of a potentially complicated drug regime. examples such as this thus remind us of the primacy of the ad patient's right to choose, even within a shared decision-making model. complications arise when determining and combining preferences. for example, the difference between qualitative and quantitative findings present a challenge when pooling and attempting to weigh different forms of evidence [ , ] . to mitigate this, preference elicitation techniques [ ] [ ] [ ] exist to help individuals to arrive at a decision or ranking of the various outcomes and permutations. ordering outcomes according to their desirability may not be straightforward, given the different ways in which a disease and its symptoms may affect daily living and wellbeing. for example, in patients with multiple conditions or comorbidities, countervailing factors will need to be taken into account [ ] to arrive at what is, overall, the best balance between benefits and risks of one approach to treatment rather than another [ , ] . ordering outcomes is also complex because the 'best' balance of risks and benefits may be plural, that is, there may be more than one 'right' decision in any given scenario. this is especially pertinent in the ad context, given that no cure is available at present. were there curative treatments it may be easier to calibrate the relative desirability of different options since, presumably, reversing the disease would be always be prioritized over other outcomes. in the absence of curative treatment, deciding on the 'right' course of action must be made by the careful weighing of other, suboptimal, options. ethical challenges in outcome prioritization also emerge when priorities conflict, for example between short-and long-term treatment goals, considering that the development of drugs and tests is slow and resource intensive [ ] , even when research funding prioritizes a particular condition [ ] . moreover, there may be challenges in involving all relevant stakeholders in the prioritization process [ ] . for example, factors such as linguistic barriers and insensitivity to different cultural values mean that minority ethnic groups are under-represented in research [ , [ ] [ ] [ ] . there may also be intractable conflicts of priorities in relation to decisions concerning the end of life. qualitative research by goodman et al. [ ] reminds us that for many people with dementia, death may be preferable to a severely impaired life with dementia. since family members and carers cannot (legally) hasten death to bring an end to unwanted suffering, it is not obvious how such wishes can be meaningfully accommodated. as hunter [ ] writes, questions about whose responsibility it is to engage in the process of prioritizing outcomes are also ethically contentious. this creates a further set of practical ethical complexities around the balance of duties and responsibilities between patients, carers, society at large, clinicians, policy makers, health economists, and other bodies with a stake in optimizing care. for instance, prioritization can be complex when making choices regarding prevention or disease progression, such as whether to provide treatment to those who are most in need, or those who would benefit the most. there is no straightforward way to resolve dilemmas such as this, which reminds us we cannot assume that priorities will be uniform and agreed between patients, their carers, clinicians, and the wider general public [ , , ] . indeed, such dilemmas may be fundamentally unresolvable, given that, ultimately, one group's interests will be relatively de-prioritized. we can go further still than this to reinforce the point if we keep in mind that allocation dilemmas in a specific context such as ad are not unique in their complexity, since in the vast majority of instances, notwithstanding certain paradigmatically immoral acts such as torture or rape, ethical judgements are always contestable. since there is no objectively agreed standard of measurement or value as is more readily available in the sciences, it is the nature of moral deliberation that grounds for rational disagreement can never be completely extinguished. returning to the extant context of health outcome prioritization in ad, therefore, this general characteristic of ethical discourse underwrites the claim that what counts as a successful decision and legitimate prioritization of outcomes will consist in the fastidiousness of the process by which the decision was reached in terms of considering the views of as wide a range of stakeholders as possible [ ] , rather than the decision itself. ad impedes communication [ ] , cognition [ ] , autonomous decision-making [ ] , social participation [ ] , and independence [ ] and has a psychological and emotional impact affecting relationships and perceptions of self and personhood [ , ] as the disease progresses from mild cognitive impairment (mci) onwards [ , ] . this becomes increasingly significant as the disease advances and compromises more aspects of life, as family carers may often need to spend an increasing amount of time supporting their relative with ad and making decisions on their behalf on the basis of what they take to be the relative's wishes [ , ] . questions of self-determination are immediately relevant in this context. seeking out the preferences of people with ad and other dementias is crucial as far as it is possible to do so [ ] , and it is important not to assume that people with dementia are necessarily unable to speak for or represent themselves, albeit that sensitivity and flexibility must be shown to the varying lengths of time that someone with ad may need to make choices or communicate their wishes [ ] . for example, at the end of life it is of the utmost importance that the dying person's wishes are understood as explicitly as possible in advance [ ] [ ] [ ] , moreover, it should not be assumed that people will be unable to express their preferences in some form, even at later stages of the disease or at the end of life [ , ] . this is vital when considering who has a say in determining which outcomes matter: firstly to ensure that those who can contribute do so; and secondly to ensure that first-hand experiences of different disease stages are represented. this is an issue of both fairness-in that there should be no systematic exclusion based on assumptions about capacity at particular stages; and validity-in that the actual priorities of patients are heard above the hypothetical priorities of those imagining being in such circumstances. the completion of advance directives by individuals before their dementia becomes too advanced to communicate their preferences towards the end of life are one option for negotiating likely future communication issues [ ] . however, these are not without potential ethical problems; for example that they are often left incomplete such that the individual's wishes are uncertain or unknown to carers and others [ ] ; and the validity of advance directives can be disputed or undermined given that it is possible an individual's preferences change after they are no longer able to communicate this to others [ ] . as such, when attempting to make prioritization decisions in the later stages of ad, it is important to keep in mind that advance directives, while undoubtedly a vital tool for eliciting preferences, are nevertheless fallible. given the progressive, degenerative nature of ad, it can become increasingly difficult to know the priorities and preferences of the person affected. often caregivers, whether professional or unpaid, family members, or partners, are able to understand the affected person's wishes as their ability to clearly communicate their preferences diminishes [ , [ ] [ ] [ ] . however, this presents a challenge for ensuring that carers properly represent the affected person's interests and, crucially, that they respect and protect the dignity of the person with dementia [ ] . this is a considerable ethical challenge, as knowing what we ought to do for people with ad if their preferences are only indirectly discernible or require interpretation raises the risk of erroneous decisions. again, the limitations of advance directives should also be taken into account in relation to this point. people with ad are vulnerable if they cannot communicate effectively and may be more reliant on their carers to represent their interests. while most carers will do this, it cannot be assumed that all risks of misrepresentation or mistreatment are eliminated [ ] . for example, carers' evaluations of the quality of life of people with ad are typically negative [ ] , but we cannot verify this precisely because of the impaired communication that the disease brings about. similarly, there is evidence to suggest that carers wish patients to remain in the milder stages for as long as possible and decline rapidly toward the end to minimize the impact that it will have on them, but we cannot assume that this is what the person being cared for would also wish [ ] . one reason may be that the burden and risk of isolation experienced by carers increases as the patient's disease progresses, and their judgement of the patient's quality of life can be negatively influenced by the deterioration in their own quality of life [ , ] . nevertheless, it is important here to consider carers as stakeholders with a legitimate voice of their own regarding outcome prioritization, separate from the extent to which they can reliably interpret the wishes of those for whom they are caring [ , ] . the risk of progressing from mci to ad may be an important outcome for people in early stages of cognitive decline; however, the uncertainty of such predictions and the limited ability to act on it, make knowledge of one's risk ethically complex. for instance, although preclinical testing for ad is advancing in accuracy and scope [ ] , understanding the wishes of all those to whom such testing may be relevant is important for harm reduction [ ] and ensuring individuals have the greatest scope for decision-making that they wish to have, whatever their decisions and preferences following risk disclosure might be. people identified at high risk of developing ad, such as some mci patients, will already feature in such considerations, since, as rose [ ] points out, the best indicator of major disease in future is often the existing presence of minor disease. indeed, the current diagnostic guidelines state that ad dementia is preceded by mci, and mci is preceded by an asymptomatic preclinical ad phase [ ] . however, as algorithms become better at predicting risk in asymptomatic individuals, namely, those who have biomarkers of the disease but no symptoms, those to whom this will become relevant will increase in line with the increasing accuracy of predicting ad at the asymptomatic stage. in each group deemed at risk, the ethical ramifications of testing center on how individuals, and those close to them, should respond to risk information as well as what responsibilities fall on those disclosing the risk status. for example, this will extend to family members, as knowledge that a relative will or is likely to develop ad will affect their lives, not least because some of those family members or partners may have to become carers [ , ] . moreover, in the case of genetic risk factors for ad which can be identified at any age, such as apoe, genetically related relatives may be faced with a decision about whether they too wish to undergo testing for presence of such markers [ ] . these scenarios are ethically challenging as long as ad remains incurable. for example, while cognitively normal, asymptomatic, individuals may be able to reduce their risk of developing mci or ad by making lifestyle changes in mid-life, there is an absence of high-quality, reliable evidence for the effectiveness of these changes, and as such they do not guarantee it [ , ] . as such, it cannot be assumed that individuals will necessarily prioritize receiving risk information in the absence of a guarantee that successful prevention is possible. nevertheless, there may be benefits to early risk assessment and diagnosis, since it enables patients to more capably make legal and care arrangements and change their lifestyle so that they can maximize the time they have with significant others [ , ] or to make plans to end their lives before dementia makes that impossible. to the extent that a foundational duty of healthcare professionals is avoiding and preventing harm, it is necessary to keep in mind a broad conception of harm that encompasses not only physical and cognitive impairment caused by the progression of ad, but psychological and emotional damage that may come to people by knowing their risk. it is straightforward to state that individuals are entitled to be informed of all the findings of their scan, however distressing these might be, if they wish to and understand the implications of what they might be told. however, it should not be assumed that, for example, cognitively normal but amyloid positive individuals [ ] should automatically be informed of the results of their scan without prior discussion as to the individual's preferences for disclosure [ ] , as it may cause significant distress even if the information is sought. indeed, amyloid positive individuals may live to old age [ ] or die before developing notable symptoms, either because of another condition or an accident, even though there is a consensus among clinicians that these individuals would have developed dementia had death not intervened first. in these cases, it is important to elicit what an individual's preferences are with respect to ad, relative to other health risks that they face, also taking into account how legal rights to the disclosure of information to individuals are framed in different jurisdictions. aside from the harms of the disease, therefore, disclosure may pose a risk of psychological and emotional harm to affected individuals [ ] , their carers and families, and the interpersonal relationships between them [ ] . finally, for individuals with private health coverage rather than the majority in the european context who depend on state health provision, known information relating to the risk of disease may affect individuals' insurance premiums and coverage and cover if they are obliged to disclose this to insurers [ , , , , ] , and a the risk of harm that may come from the exploitation of this information by insurers should be taken into account when balancing the priority of potential outcomes. the risks outlined are some of those adduced in arguments against dementia screening [ , ] and help to demonstrate that although an assumption that diagnosis and knowledge of one's condition would and should always be one's priority may look prima facie reasonable, when subjected to scrutiny the situation reveals itself too complex for the assumption to be applicable or necessarily beneficial. our analysis indicates that the themes identified can be grouped into three larger arrangements of overlapping ethical issues in outcome prioritization, which we now discuss. we will also make some remarks about what normative approaches might be appropriate for negotiating them; for example, philosophical tools such as differing theories of justice can inform debate and decision-making in arriving at conclusions about how resources should be distributed; and (social) scientific tools such as quantitative and qualitative research methodologies can help to elicit personal perspectives that are required for understanding what outcomes are important to whom, and why. it is important to note here that there are research consortia which draw together and integrate these expertise toward developing fair and equitable methods of outcome prioritization in ad, such as imi eu efpia roadmap [ , ] the first group of issues relates to the importance of ensuring the adequacy of the procedures according to which finite resources are allocated. for example, if the needs of a certain group of people cannot be met because it would not be costeffective to do so, then it is important: first, that relevant professionals are trained to give individuals difficult and potentially distressing news; and second, that the commissioning process is thorough and comprehensively justified, with efforts to prevent geographical disparities, or 'postcode lotteries' in provision (themes , , , ). similarly, procedures for prioritizing outcomes must be able to respond effectively to advances in therapy and the consideration of these under whatever protocol for allocation is applied. if it is important to maximize the benefit from available treatments, then it commensurately important to ensure that potentially beneficial new developments can be incorporated into resource allocation decision procedures. for these kinds of considerations, we adopt the most general, macrolevel perspective. given that due consideration for the correct balance of rights and responsibilities between the state and the individual falls within the purview of ethics, it is here that the philosophical tools derived from understanding competing theories of justice are instructive for negotiating resource allocation and prioritization dilemmas. the second set of issues relates to the specific outcomes to be measured and prioritized and how to ensure that those measured are most important and meaningful to relevant stakeholders (themes , , , , , ) . prioritization can only meet the needs and wishes of the affected parties the outcomes valued by the people to whom allocation decisions pertain are known. this may change at different stages of the disease and hence it is important also to have valid and reliable outcome assessment tools to measure the priority outcomes that are appropriate to these different stages. this obviates the need to elicit the preferences of people with ad and their carers, rather than assuming what outcomes are desired and in what order of preference. moreover, as we pointed out, understanding what outcomes are prioritized and in what order is also complicated in view of the complexity of ad. medications may have side-effects and/or drug-drug interactions that may affect the overall outcome for a patient, and since ad is largely a disease of old age it is frequently important to take comorbidities into account when attempting to attach a value to a particular outcome and course of action [ ] . to the extent that a duty of prioritization decisions is to optimize outcomes for people with ad, understanding the preferences of these people is a necessary step in discharging that duty. these demands can be met by qualitative and quantitative research methods in the form of interviews, focus groups, surveys, questionnaires, and digital data collection. as such, social science research tools can be usefully employed for seeking out and understanding individual preferences and the reasons behind them. the careful application of these tools is also valuable for negotiating the third group of ethical challenges, which, like the issue of age-related comorbidities, is highly relevant to ad, and concerns the way in which individual preferences are sought (themes , , ) . ad can impair the ability of affected people to clearly make and convey their preferences and wishes, and so there is a potential ethical risk in not prioritizing outcomes in a way that meets the needs and wishes of those people. furthermore, given that ad can impair communication, carers may need to make decisions on a patient's behalf, and there may be conflicting accounts of the patient's best interests. adequate and ethically robust outcome prioritization processes depend partly on first having identified what is needed by and important to people with ad, and as such the procedures used to elicit this information must be capable of doing so. since cost-effectiveness decisions are necessarily utility-driven, unless preferences for outcomes are successfully elicited and appropriately quantified, there is a risk that these important qualitative aspects of ad are lost against the background of the aggregative method by which prioritization is directed. this is a non-systematic narrative review. one potential limitation may come from the type of conducted search, where only the titles were searched for presence of keywords. this limitation was mitigated by subsequent snowball sampling of further relevant manuscripts, which significantly increased the number of papers eligible for review. another potential limitation may come from the single-reviewer process applied to this study at the abstract selection and initial full-text review stages. this limitation was to some extent mitigated by the iterative analysis process, where all contributors reviewed, discussed, and contextualized the results, reaching back to the original sources as needed; and by the reviewing of a subset of titles by a co-author and comparison of decisions for inclusion and exclusion. a further limitation is that since we have only used english language studies in this paper, we are unsure of the reliability of our findings for non-english speaking ad communities. however, this was unavoidable given the resources available to the authors. a final limitation is that since the later stages of editing post-peer review were carried out during the initial lockdown phase of the covid- pandemic, the authors are unable to review literature about the ethical implications of outcome prioritization in ad in the context of the current pandemic and potential future ones. given that the paper is based on a review of the relevant literature, while adrelevant studies produced in light of the pandemic will undoubtedly emerge, the paper has not been produced sufficiently long after the initial global infection for such material to be published made available for analysis. nevertheless, we can make a tentative remark that it would be worthwhile to investigate in future work. it is likely that situations such as the covid- pandemic may change what are considered acceptable trade-offs in outcome prioritization within the eight themes that we identify in this study. a possible concrete example of this might be accepting the additional safety risks associated with remote monitoring of people with ad living on their own, in order to shield them from the more immediate infectious disease risk that would come with usual levels of face-to-face contact. our analysis demonstrates the essential role that ethical deliberation plays in ensuring the just prioritization of outcomes. given that discussions about how we 'should' allocate resources is an irreducibly normative question, expertise in ethical and philosophical reasoning are indispensable for a task such as this, irrespective of the fact that the task is an applied one and these kinds of expertise are putatively theoretical. of course, the evidence to which we refer is similarly indispensable, since without empirical information about how and why different stakeholders prioritize outcomes in the way that they do, no rational negotiation of these towards a just outcome can be carried out. nevertheless, what ought to be done cannot simply be read off these descriptive data. for this reason, allied with the compelling need to find new strategies for managing ad in view of the growing societal pressure that it is exerting, we conclude by reiterating the central role that ethical reflection contributes in decision-making processes regarding the prioritization of patient outcomes. a descriptive review on methods to prioritize outcomes in a health care context personcentred care of people with severe alzheimer's disease: current status and ways forward outcome prioritisation tool for medication review in older patients with multimorbidity: a pilot study in general practice family decision-making in advanced dementia: narrative and ethics preferences and priorities for ongoing and end-oflife care: a qualitative study of older people with dementia resident in care homes randomised trials in context: practical problems and social aspects of evidencebased medicine and policy saying no isn't 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disease appendix i: patient and public involvement consultations quality of life for caregivers of people with alzheimer's disease quality of life and depression in carers of patients with early onset dementia genetic research into alzheimer's disease: a european focus group study on ethical issues ethical aspects of research into alzheimer disease. a european delphi study focused on genetic and nongenetic research alzheimer's disease: definition, natural history, and diagnostic criteria introduction to the recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease sick individuals and sick populations preclinical alzheimer disease-the challenges ahead family caregivers of people with dementia apoe and alzheimer disease: a major gene with semi-dominant inheritance lifestyle change and the prevention of cognitive decline and dementia modifiable lifestyle risk factors for alzheimer's disease early identification and treatment of alzheimer's disease: social and fiscal outcomes progress on dementia-leaving no one behind disclosure of amyloid imaging results to research participants: has the time come? frequent amyloid deposition without significant cognitive impairment among the elderly early dementia diagnosis and the risk of suicide and euthanasia the biomarkerbased diagnosis of alzheimer's disease. -ethical and societal issues ethical issues in alzheimer's disease research involving human subjects dementia screening in primary care political drive to screen for pre-dementia: not evidence based and ignores the harms of diagnosis challenges for optimizing real-world evidence in alzheimer's disease: the roadmap project authors' disclosures available online (https:// www.j-alz.com/manuscript-disclosures/ - r ). key: cord- -jrvl ykn authors: nelson, martha i.; lloyd-smith, james o.; simonsen, lone; rambaut, andrew; holmes, edward c.; chowell, gerardo; miller, mark a.; spiro, david j.; grenfell, bryan; viboud, cécile title: fogarty international center collaborative networks in infectious disease modeling: lessons learnt in research and capacity building date: - - journal: epidemics doi: . /j.epidem. . . sha: doc_id: cord_uid: jrvl ykn due to a combination of ecological, political, and demographic factors, the emergence of novel pathogens has been increasingly observed in animals and humans in recent decades. enhancing global capacity to study and interpret infectious disease surveillance data, and to develop data-driven computational models to guide policy, represents one of the most cost-effective, and yet overlooked, ways to prepare for the next pandemic. epidemiological and behavioral data from recent pandemics and historic scourges have provided rich opportunities for validation of computational models, while new sequencing technologies and the ‘big data’ revolution present new tools for studying the epidemiology of outbreaks in real time. for the past two decades, the division of international epidemiology and population studies (dieps) of the nih fogarty international center has spearheaded two synergistic programs to better understand and devise control strategies for global infectious disease threats. the multinational influenza seasonal mortality study (misms) has strengthened global capacity to study the epidemiology and evolutionary dynamics of influenza viruses in countries by organizing international research activities and training workshops. the research and policy in infectious disease dynamics (rapidd) program and its precursor activities has established a network of global experts in infectious disease modeling operating at the research-policy interface, with collaborators in countries. these activities have provided evidence-based recommendations for disease control, including during large-scale outbreaks of pandemic influenza, ebola and zika virus. together, these programs have coordinated international collaborative networks to advance the study of emerging disease threats and the field of computational epidemic modeling. a global community of researchers and policy-makers have used the tools and trainings developed by these programs to interpret infectious disease patterns in their countries, understand modeling concepts, and inform control policies. here we reflect on the scientific achievements and lessons learnt from these programs (h-index = for rapidd and for misms), including the identification of outstanding researchers and fellows; funding flexibility for timely research workshops and working groups (particularly relative to more traditional investigator-based grant programs); emphasis on group activities such as large-scale modeling reviews, model comparisons, forecasting challenges and special journal issues; strong quality control with a light touch on outputs; and prominence of training, data-sharing, and joint publications. in the fogarty international center celebrates its th year as the only part of the us national institutes of health focused singularly on global health. the fogarty international center (fic) uses high return-on-investment approaches to maximally impact global health, including (a) establishing strong international collaborative research networks, (b) building capacity for research in lowand middle-income countries (lmics) through training, and (c) liaising with a wide range of other us government agencies and private partners with a shared interest in advancing global health. here, we describe two flagship fic-led programs that identified critical gaps in infectious disease research and formed global networks of researchers to address the scientific questions most needed to inform policy and outbreak response. the foot-and-mouth disease outbreak in the uk, the influenza a/h n pandemic, and the - epidemic of ebola in west africa underscore the continual threat of emerging and re-emerging acute infections. knowledge of the mode of spread of a pathogen and the subpopulations at highest risk of transmitting the pathogen and experiencing severe disease can inform key decisions about social distancing and prioritization of therapeutics and vaccines. designing disease models rooted in empirical data, enhancing existing tools for data analysis, and teaching collaborators around the world how to apply these tools represent highly cost-effective ways to prepare for the next infectious disease outbreak. past disease events have highlighted the capabilities of infectious disease modeling and molecular epidemiology to effectively guide outbreak responses, but also revealed persistent challenges in making predictions in data-scarce and rapidly changing field settings (woolhouse, chase-topping et al. ; keeling, woolhouse et al. ; heesterbeek, anderson et al. ; woolhouse, rambaut et al. ; dudas, carvalho et al. ) . the increasing availability of large, electronic datasets, or "big data," presents new opportunities for scientists to understand drivers of disease. electronic health records, social media, satellite imagery, and cell phone records provide highly granular information on human behavior, the environment, transmission patterns, and disease burden (lazer, kennedy et al. ; bansal, chowell et al. ; simonsen et al., ) . recent advances in next-generation sequencing techniques have markedly increased the amount of pathogen genetic data available for study, providing almost real-time insights into how pathogens from different locations are genetically related and infer transmission patterns (dudas, carvalho et al. ) . increasingly, researchers are devising methods to collect data even in resource-limited settings. the highly portable minion sequencer demonstrated how real-time wholegenome sequence data could be generated in remote locales during the zika epidemic (quick, loman et al. ; faria, quick et al. ) . mobile phone records and remotely-sensed nightlights present additional strategies to track human movements and contacts in resourcelimited settings wesolowski, eagle et al. ; wesolowski et al., b) . since its inception in , the division of international epidemiology and population studies (dieps) has played a central role in promoting the growth of a vibrant and multi-disciplinary infectious disease modeling community through its misms and rapidd programs. together, these programs have strengthened infectious disease modeling on a global scale by developing research and training networks, facilitating collaboration and data sharing, developing and disseminating new methodologies, and building a stronger collective voice for evidence-based policymaking. most critically, these programs have been catalysts for other larger us agencies and governments worldwide to expand support for infectious disease modeling research and to incorporate models into policy decision-making. the misms project was initiated in to build global research capacity for the study of influenza viruses using computational methods (http://misms.net, box ). the acronym originally referred to the multinational influenza seasonal mortality study, although the program quickly expanded beyond mortality studies. misms has a mission to promote research, training and inform policy, more specifically (i) to characterize the global epidemiological and evolutionary dynamics of influenza in different host species, and (ii) to empower influenza researchers globally to study influenza and inform policymaking in their own countries. towards this end, technical training workshops were conducted on six continents during - . these workshops focus on reviewing the state of influenza research and epidemiology in different global regions, and hands-on teaching of time series analysis of contemporary and historical outbreaks, control measures, mathematical transmission models, and phylogenetic approaches (fig. ) . misms supports interdisciplinary approaches by fostering collaborations between historians, demographers, ecologists, evolutionary biologists, vaccine manufacturers, microbiologists, epidemiologists, and policymakers, as evidenced by the rich diversity of publications (fig. ). none of the last three pandemics -influenza a/h n in , a/ h n in , and a/h n in -originated in a high-income country. building capacity for influenza research in low-and middleincome countries is an essential but underfunded part of global pandemic preparedness and a key goal of the misms program. a central feature of misms workshops has been partnership with local organizations to help identify talented scientists in under-resourced settings. workshops are held either in low-or middle-income countries, or the program provides funding for researchers from these countries to attend workshops in high-income countries (box ). the misms workshop in dakar, senegal, was co-organized with institut pasteur and was the first influenza workshop ever held in africa. in addition to periodic workshops that provide intense short-term training to - participants, misms staff also host junior and mid-career researchers from central america, europe, africa, and asia for further training at the nih for periods ranging from a few months to several years. these scientists gain deep expertise in epidemiological and phylogenetic approaches and establish research programs to inform policy in their home countries. organizing workshops in under-studied tropical settings brought critical attention to the burden of influenza and the variability of seasonal patterns in less connected locales and warm, humid climates bloom-feshbach, alonso et al. ) . increased recognition of the global burden of influenza in tropical and semi-tropical regions also has encouraged uptake of routine annual vaccination in many middle-income countries. however, several misms studies rooted in epidemiological and virological data have pointed out that the semi-annual schedule is designed for wealthier countries in the temperate regions in the northern and southern hemispheres, and are suboptimal for many tropical countries that experience influenza at different times of the year, or have multiple peaks (alonso et al., ; de mello, de paiva et al. ; tamerius et al., ; bloom-feshbach, alonso et al. ; green, andrews et al. ; alonso et al., a,b; ayora-talavera, flores et al. ) . misms research has informed whether a tropical country should opt for the northern or southern hemisphere vaccine formulation -or in some cases both. large countries with heterogenous influenza patterns and climate, such as china, mexico and brazil, may require different vaccine recommendations for their northern and southern regions (alonso et al., ; de mello, de paiva et al. ; ayora-talavera, flores et al. ) . additionally, misms research demonstrating the low impact of senior vaccination programs in the us and italy paved the way for expanded vaccination why have rapidd and misms workshops been so successful? since , rapidd has sponsored workshops on topics ranging from gain-of-function experiments to the latest developments in particle filtering methods. these workshops have had a remarkable track record of enabling constructive syntheses of current frontiers in the science of disease modeling, and in spurring innovation and analysis of case studies to advance those frontiers. they also have earned a strong reputation in the field as important venues for scientific discourse. here we summarize distinctive elements of those workshops that we believe have contributed to their success. • lightweight and nimble proposal process, steered by two generous and visionary scientists that had deep expertise in the field. the program 'let a thousand flowers bloom' by accepting workshop proposals on a rolling basis with a two-page initial proposal. for promising proposals, program leadership then iterates with proposers to refine the focus on rapidd goals, ensure solid deliverables, and optimize the participant list. • small size and balanced composition. research workshops are generally kept to - participants, to ensure free-flowing and natural discussion. age structure is key, with emphasis on including junior scientists (especially postdocs) so there are people with the time and incentive to do follow-up work. • flexible structure. adapt meeting structure to meet scientific needs. some workshops focus on conceptual or methodological issues, others on particular disease systems. some of the best workshops had a central theme combined with data in hand and time to analyze it. • keep workshops short (typically . - days) and maximize the value of in-person meetings. pre-workshop teleconferences (among leaders or the whole group) help to reach consensus on goals and scope, so talks are on target and preliminary work can get done. avoid overloading the schedule with formal talks, to ensure ample time for discussion and breakout groups; some participants can act as synthesizers or reactors rather than giving talks. relaxed interactions on a hike or at a pub usually pay off. • designed for follow-through. before the meeting, specify deliverable and timelines, and identify who will lead these efforts (often rapidd postdocs or workshop organizers). get key pieces such as data sets and foundational analyses in place beforehand. require workshop reports within a few months, summarizing the scientific content of the meeting, progress toward deliverables, and new opportunities that have emerged. support follow-up when warranted, including 'working meetings' of project leaders, but also be prepared to stop threads that are not productive or not aligned with program priorities. since , misms has convened training workshops on all continents and trained several hundreds of scientists on computational methods for infectious diseases. the unique characteristics of these workshops include: • the primary output of these workshops is technology transfer; i.e. capacity building in infectious disease analytics. participants are not expected to become modeling experts after a short workshop, but instead they should understand principles of data analysis and modeling (to the point that they are able to understand a disease modeling article). • misms workshops are typically regionally-focused and organized in tandem with a local university, research institute, or ministry of health. regional participants are identified based on an exhaustive pubmed search (especially in early years of the misms program), word-ofmouth, and existing misms contacts (particularly as the program matured). public health experts from who, cdc, and local institutions, are always invited. • workshops are sometimes organized in conjunction with a larger influenza scientific meeting to decrease travel costs and optimize participation. • weeklong workshops include days of general scientific session and days of hands-on training. the general session is meant to expose the state of the art of influenza epidemiology in the region and highlight success stories in influenza modeling and misms collaborations. the hands-on training session demonstrate methods in time series analysis, transmission models, and evolutionary analyses. • workshops are open to scientists and public health experts who do not have prior training in infectious disease modeling, e.g. veterinarians, clinicians, virologists, lab technicians, policy makers, etc. • flexible travel support for participants based on abstract selection process; focus on participants bringing data that may be amenable to modeling, those who have a clear training plan, and those from underserved areas. • training portion of the workshop is limited to participants, for - faculty, to facilitate one-on-one interactions. • participants are encouraged to bring their own data; at least a day is devoted to small-group analyses of these data. sample datasets are always available for demonstration purposes and for those unable to bring data (or with sparser datasets). • while the workshops are focused on influenza, participation is open to scientists working on related infectious diseases, recognizing that relevant (or specific) influenza data may be scarce in some countries, and that computational skills are cross-cutting. • workshop deliverables include joint publications and supplementary issues that spur collaborative work (for instance, supplementary issues on big data for infectious disease surveillance or historical pandemics bansal, chowell et al. ; simonsen et al., )) • similarly to rapidd, there is opportunity for follow-up beyond the workshop, primarily via longer visits to nih for more extensive training with misms staff, and occasionally, through remote collaborations. workshops can also be organized in the same region a few years later to gauge progress. misms staff provides support for completion of analytical work and manuscripts, where workshop participants are recognized as first authors. programs targeted at children who are important foci of transmission (simonsen et al., ; simonsen, reichert et al. ; rizzo, viboud et al. ; simonsen et al., ) . global vaccine strategies also require an understanding of how viruses migrate long distances between countries, and how viruses evolve antigenically to require vaccine strain updates (ferro, budke et al. ; adler, eames et al. ) . active areas of misms research include a deeper understanding of influenza migration pathways at different spatial scales, climatic and demographic drivers of influenza seasonality, and the mapping between vaccine match and population-level protection, all of which can affect vaccine policy (box ). the global network of collaborators that grew out of the misms program is a unique resource to leverage in the event of the next pandemic influenza or another infectious disease threat. as a case in point, in , the presence of misms-trained researchers in mexico facilitated early reporting of severity and striking age-shifts in mortality patterns that are signatures of new pandemic viruses (chowell et al., a , c ). during the critical early stages of the pandemic, rapid reporting of these epidemiological patterns informed use of limited resources, including vaccine and m.i. nelson et al. epidemics ( ) - therapeutics, as well as interventions such as school closures (chowell et al., a , c , chowell, echevarria-zuno et al. . misms collaborations also focus on "archeo-epidemiology" studies, or studies of past influenza pandemics and other historic scourges . such studies have provided estimates of influenza transmission intensity, age-structured mortality patterns, spatial dynamics, and the distribution of mortality across multiple pandemic waves as far back as the s (andreasen, viboud et al. ; bloom-feshbach et al., ; simonsen et al., ) . quantitative studies of past patterns can help predict the course of future pandemics and inform the range of plausible scenarios for preparedness, including elevated mortality occurring several years after the initial policy impact of rapidd and misms: select success stories. • vaccination: misms studies in large and climatologically-diverse countries have identified geographic differences that affect annual influenza vaccination programs. because of the timing of influenza epidemics and composition of circulating strains in southern china and southern brazil, southern provinces should use the southern hemisphere influenza vaccine, while the northern part of these countries should use the northern hemisphere vaccine (miller, viboud et al. ; chowell, viboud et al. ; yu, alonso et al. ; alonso et al., a,b ). • surveillance: misms work on the phylodynamics of swine influenza viruses highlight the importance of undersampled "source" locations with large pig populations and intense swine flows to other regions, such as russia. surveillance efforts should target these regions that are expected to generate important global diversity of swine viruses ). • pandemic control measures: mexico implemented nationwide social distancing measures to control the influenza pandemic (combination of mandatory school closure, restaurant closure, and cancellation of large gatherings) during an -day period in late april and early may . misms researchers were among the first to work with the ministry of health to assess the dynamics of the outbreak and the impact of interventions. by fitting transmission models to surveillance data, they found that social distancing reduced influenza transmission by one-third, lending support to these costly measures. beyond the pandemic, this works suggests that social distancing interventions could be implemented to mitigate severe outbreaks and alleviate the pressure on healthcare, for limited periods of time and until other interventions (such as vaccination) can kick in (chowell, bertozzi et al. ; chowell, viboud et al. ; chowell, echevarria-zuno et al. ). • addressing data gaps: on the rapidd front, modelers thought creatively to find ways to fill data gaps that were essential obstacles to resolving policy-relevant health problems. for example, a common problem in livestock diseases is the lack of information on farm locations and animal movements, especially in the us. these are key ingredients to design transmission models, evaluate interventions, and help prepare for potential outbreaks such as foot-and-mouth disease. the rapidd network designed a method to leverage veterinary records and mandatory licenses filed by farmers. further, much simulation work was done based on the richer uk dataset informing farm locations, animal movements, and observed foot-and-mouth disease outbreaks, to strengthen us models and remedy data gaps. • gain-of-function studies and pandemic risk prediction: as the debate on gain-of-function studies intensified, rapidd organized a twoworkshop series on "assessing the outbreak potential of nonhuman influenza viruses using sequence-based risk approaches'" and "modeling and predicting influenza phenotypes" to explore modeling of pandemic potential. gain-of-function studies indicated that an avian influenza virus undergies a series of genetic changes for human adaptation, which are required for efficient transmission among mammals. the workshops assembled global experts to synthesize current knowledge about the three key steps of the adaptation process: switch of the hemagglutinin surface protein to mammalian alpha- , sialic acid binding, enhancement of ph-and temperature-dependent stability of the hemagglutinin, and adaptation of the viral polymerase to function in mammalian cells. these workshops spurred modeling work to infer pandemic potential based on ferret transmission experiments, and further work is underway to explore whether mapping of genotype-tophenotype traits is possible, despite rampant epistatic interactions across the influenza genome (buhnerkempe et al., a) . other avenues for policy-oriented research include the optimization of global influenza surveillance strategies to identifies early precursors of pandemic strains. • vaccination decline in the aftermath of ebola: another example of the link between rapidd research and policy is a study on the precipitous decline in childhood immunization during the - ebola outbreak (takahashi et al., ) . rapidd modelers estimated that around one million children in liberia, sierra leone and guinea were vulnerable to measles following the suspension of vaccination campaigns during the ebola outbreak. the study concluded on the urgent need to mount an aggressive vaccination campaign as soon as the ebola outbreak subsided. this advice was heeded by public health authorities, as measles vaccination campaigns targeting several million children were launched in sierra leone and guinea in october and in liberia a few months earlier. emergence of a pandemic virus (box ). misms also has worked actively to bridge human and veterinary research communities and understand the emergence of pandemic viruses at the animal-human interface. two workshops held in ames, iowa, usa, in collaboration with us department of agriculture, focused on swine influenza, while another held in padua, italy, in collaboration with istituto zooprofilattico sperimentale delle venezie, focused on avian influenza, providing training opportunities in an active area of influenza research. improvements to genetic sequencing technologies have reduced costs worldwide and advanced the field of 'phylodynamics', in which epidemiological dynamics are inferred from pathogen sequence data. in the early s, new computational packages, including bayesian evolutionary analysis sampling trees (beast), provided the capacity to infer detailed evolutionary, demographic and spatial patterns from sequence data (lemey et al., a,b; lemey, rambaut et al. ) . however, in-depth studies of influenza virus evolution require large numbers of viral sequences carefully sampled over time and space, which are unavailable in many low-and middle-income countries. to address this gap, fogarty's misms staff partnered with the national institute of allergy and infectious diseases (niaid) and national library of medicine (nlm), to provide whole-genome sequencing of influenza virus collections free of cost via the influenza genome sequencing program (box ). when this sequencing project began in , there were fewer than complete influenza virus genomes available on genbank. as of , almost , influenza virus genomes have been sequenced through this project and made publicly available. a strong linkage between the influenza genome sequencing program and fogarty's misms network of global researchers, and their viral collections, has helped address important gaps in influenza sequence availability and in turn transmission dynamics. the program targeted understudied aspects of influenza virus evolution, including large regions of the viral genome not typically sequenced, under-sampled populations in tropical settings, influenza b virus, and non-human mammalian hosts (box ). the program fostered a global culture of data-sharing that facilitates large-scale comparative studies across subtypes, regions, and species, advancing our fundamental understanding of influenza virus evolution and ecology. despite increased availability of influenza genetic and epidemiological data, there are still important gaps, particularly regarding surveillance in africa and south america and at the animal-human interface (viboud, nelson et al. ) , and poor integration of different data streams (e.g., epidemiological, antigenic and genetic information), representing target areas of future misms efforts. because influenza is unique in combining long-term pandemic risk with a continued need for annual vaccine updates to reduce the burden of seasonal influenza, the field has traditionally enjoyed far better attention than other respiratory infections. as genomic and epidemiological data for other respiratory pathogens increases, a natural extension for misms would be to broaden its scope beyond that of influenza. recognizing this potential, misms collaborators have embarked on modeling studies of pneumococcus and respiratory syncytial virus, and their interactions with influenza (weinberger, simonsen et al. ; shrestha, foxman et al. ; weinberger, harboe et al. ; shrestha, foxman et al. ; weinberger, klugman et al. ) . with additional resources, a more systematic broadening of the scope of the misms study to other important human respiratory pathogens would provide fresh opportunities to enhance understanding of multiple disease systems while drawing on an established global research network and a wealth of computational tools. development of infectious disease models deeply rooted in empirical data can improve planning for, and response to, infectious disease threats; however, the link between modeling groups and policy makers remains tenuous in many countries (heesterbeek, anderson the influenza genome sequencing project, a large-scale collaboration between nih institutes and j. craig venter institute, transforms our understanding of influenza virus evolution. et al. ; metcalf, edmunds et al. ) . fogarty played an important role in promoting the field of modeling population-based impact of biological threats within the u.s. government. immediately following the events of september th , and before the anthrax-laced letters that began just one week later, dieps helped establish intergovernmental networks to model and address biological threats. these efforts contributed to launch an extramural modeling funding program on models of infectious disease agents (midas) managed by the nih national institute of general medical studies and nascent funding to nih fogarty to study bioterror threats. in , the rapidd program was established with funds from the department of homeland security to build a hub-and-spokes network of infectious disease modelers working at the interface of policy and academic research, in close collaboration with government. the program's mandate was to advance us capacity in infectious disease modeling by pioneering new methodologies, studying key emerging disease threats, and training the next generation of scientists (box ). rapidd departed from traditional grant-based programs in its flexibility and concentration on working group and workshop activities, and on developing research capacity. a key success of rapidd since has been to promote collaborative research and synergies between modeling groups beyond core participants of the program (box ). rapidd research has been at the forefront of responding to the greatest infectious disease crises of recent years, particularly the - ebola epidemic in west africa and the zika epidemic in the americas. modeling work helped to identify key routes of ebola transmission and characterize effectiveness of intervention policies (team, aylward et al. , camacho, kucharski et al., a , b , kucharski, camacho et al., team, agua-agum et al. ) . further work, in collaboration with who's consortium for ebola modeling, identified risk factors for transmission including severe symptoms, death, non-hospitalization, older age, and travel history (international ebola response team et al., ) . modeling work further demonstrated the unintended consequences of the ebola-associated healthcare disruption on childhood immunization, and stressed the need to promote supplemental vaccination campaigns, particularly for measles (takahashi et al., ) . further, rapidd collaborators have developed predictive maps of the spread of the zika virus in the americas, driven by environmental conditions and population mobility (bogoch, brady et al. ) . another rapidd modeling study analyzed factors shaping the efficacy of screening air travelers for emerging pathogens, including ebola and mers-coronavirus . taken together, careful literature surveys of prior modeling work, in addition to primary research done by rapidd, form a substantial body of "case law" for infectious disease modeling, which can be used as reference to understand and model outbreaks caused by new pathogens with similar properties. as an important demonstration of this principle, prior modeling work on dengue provided the groundwork for rapid development of regional and global models for the spread of zika virus (bogoch, brady et al. ). during - , rapidd functioned primarily via parallel working groups focused on four areas identified as having important research gaps: (i) methodological and data issues, (ii) zoonoses and pathogen emergence, (iii) vector-borne infections, and (iv) disease dynamics in small mammals. the working groups conducted primary research and led multi-disciplinary workshops on emerging research frontiers (box ). since , rapidd organized workshops across a wide range of topics, with participation from thousands of scientists and government employees globally. workshops produced large-scale reviews of the state-of-the-art of disease modeling in particular areas, and opinion pieces discussing new frontiers of model-data synthesis (katz, plowden et al. ; buhnerkempe et al., b; frost, pybus et al. ; gog, pellis et al. ; heesterbeek, anderson et al. ; lloyd-smith et al., a,b; metcalf et al., a, b) . working group members then pursued follow-up research to address the identified gaps by developing new modeling approaches or new applications of models to understudied but important diseases. the direct link between ra-pidd research and policy was ensured by periodic presentations to the us white house scientific offices. overall, since , rapidd has made substantive contributions across a remarkable range of public health challenges (box ), from strategies to deploy current and future vaccines to the unique role of bats as zoonotic reservoirs. while a comprehensive review of the more than rapidd publications is beyond the scope of this review (fig. , table ), below we single out a few achievements of the program. many of the most important emerging disease threats involve zoonotic pathogens originating from animal reservoirs. by conducting a seminal review of published modeling studies of zoonotic pathogens, rapidd researchers identified critical areas in need of further attention ). the review highlighted the marked predominance of studies of directly transmitted infections with simple life cycle (e.g., influenza, sars), over infections involving multiple hosts with complex life cycles (vector-borne diseases, protozoans, food-borne infections). it identified a dearth of modeling research on looming threats including ebola, chikungunya and yellow fever viruses, which was regrettably prescient in light of subsequent epidemics around the world (who, ). crucially, the review noted that very few studies linked dynamics across the animal-human interface, where the defining process of zoonotic spillover occurs, and few box : training the next generation of scientists: rapidd post-doctoral fellows. • outstanding fellows handpicked and hired for years minimum, and up to . • comfortable prestigious fellowship, with independent and flexible funding for travel. • strong impetus to work with the larger rapidd network of outstanding scientists, particularly by organizing workshops and working groups, monthly webex presentations, periodic policy-oriented seminars at the white house, and annual rapidd network meeting. • post-doctoral fellows helped drive major collaborative reviews of modeling gaps (eg, zoonoses and emerging infections, vector-borne diseases) -a superb bonding exercise leading to high-profile publications in their chosen fields. • community of rapidd fellows learned from each other on topics ranging from communication skills to methodological issues. • of the rapidd fellows, are in tenured or tenure-track academic positions, works for the us government and for a private company (as of july ). m.i. nelson et al. epidemics ( ) - models incorporated evolutionary processes. to address these gaps, the zoonosis working group launched a series of methodological developments to link models across the animal-human interface, analyze the stuttering chains of transmission that foreshadow emergence events, and describe the evolutionary processes linked to pathogen emergence, with key applications in monkeypox, mers-coronavirus and influenza h n and h n viruses rimoin, mulembakani et al. ; blumberg and lloyd-smith, a, b; park, loverdo et al. ; blumberg, enanoria et al. ; chowell, blumberg et al., ; kucharski and althaus, ; kucharski, mills et al. ; lloyd-smith et al., a,b; plowright, parrish et al. ) . similarly, the rapidd vector-borne disease working group's review of studies of mosquito-borne pathogen transmission determined that most models were still primarily derived from mid- th century methods pioneered by ross and macdonald smith et al., ) . in response, the group developed more refined models to capture important features of mosquito-borne infections, including mosquito life history, heterogeneous biting and fine-scale spatio-temporal variation in transmission. applying these methods to malaria and dengue highlighted the impact of incorporating realistic mosquito biology and biting heterogeneities on control efforts perkins, garcia et al. ; reiner, le menach et al. ) . in recent years, a major focus of rapidd research has been infectious disease forecasting, responding to a rising need in the public health community, particularly for influenza and emerging infections yang, cowling et al. ; yang et al., a; yang, olson et al. ). in addition to rapidd's work on evaluating the impact of interventions in the midst of the west african ebola epidemic, rapidd launched an after-the-fact forecasting challenge using synthetic epidemiological data sets to assess model performances in a controlled environment (merler, ajelli et al. ; viboud et al., a, b) . this unprecedented group effort revealed that the most accurate disease forecasts stemmed from ensemble predictions combining outputs from different models, since even the best models have weaknesses, and that prediction accuracy does not scale with model complexity. the forecasting challenge also strengthened communication and collaboration between modeling groups, laying the foundation for more effective response for the next public health crisis (viboud et al., a (viboud et al., , b . influenza forecasting represents an area of rich synergy between the rapidd and misms programs. a major aim of ongoing influenza research is to predict the emergence and trajectory of new seasonal strains, and hence improve the imperfect semi-annual vaccine strain selection process. forecasting work in this area was greatly enhanced by a workshop, organized in collaboration with who, that highlighted the need for better flow of genetic and antigenic information from public health agencies to the institutions and mathematical modelers to improve prediction accuracy and horizon (morris, gostic et al. ) . further, rapidd and misms collaborators were the first to analyze the potential benefits of broadly cross-protective influenza vaccination programs (arinaminpathy, ratmann et al. ; subramanian, graham et al. ). this is a particularly fruitful area of overlap between rapidd and misms, as universal influenza vaccine candidates enter pre-clinical trials, and an ideal opportunity to integrate network members' expertise on relevant evolutionary and immunological concepts with the global source-sink dynamics so unique to influenza russell, jones et al. ). although the four rapidd working groups had separate areas of focus, several synergistic themes rapidly emerged (box ). one dominant theme was the spatiotemporal dynamics of pathogens, encompassing major strides in spatial dynamics and mapping of vector-borne infections, a broad effort to advance data-driven models for foot-andmouth disease in the us and abroad, and cutting-edge syntheses of novel and traditional data streams informing human demographics and mobility to produce a new generation of spatio-temporal epidemic models. another major rapidd theme on pathogen evolution -entailing phylodynamics, virulence evolution, host jumps and drug resistance -has generated methodological breakthroughs and important applications to influenza, dengue, malaria, and more. focused workshops re-examined current thinking on optimal drug treatment to minimize resistance evolution (read, day et al. ; kouyos, metcalf et al. ) , and the potential to predict influenza pandemic risk from viral sequence data lipsitch, barclay et al. ) . bibliometric analysis of the rapidd and misms programs. rapidd publications are based on any mention of the rapidd program in acknowledgments; this includes fellows and faculty funded by rapidd and/or participation to rapidd workshops that led to publications. misms publications are based on influenza research articles published by staff of the fogarty international center, as listed in affiliations. data collection performed on july , . since rapidd has advanced the methods and applications of data-driven modeling of infectious diseases, with a clear focus on policy-relevant research, and has fostered a new generation of talented researchers in this field (box ). the program's remarkable success arises from strong collaborations developed among the many participants over the years, its flexibility and agility in convening workshops on emerging research frontiers, and from providing freedom and unparalleled networking opportunities for talented post-doctoral scientists. looking forward, pending availability of funds, rapidd will continue its research emphases on emerging infections and vectorborne diseases, while growing new areas of focus on topical challenges such as on modeling the dynamics of anti-microbial resistance, and predicting the impact of next-generation vaccines such as for respiratory syncytial virus, malaria, typhoid, dengue, and the universal influenza vaccine. the fogarty-led misms and rapidd programs have been catalysts in advancing state-of-the-art global infectious disease modeling and training with tangible impacts in building capacity globally and guiding policies, such as defining age priority groups for epidemic and pandemic influenza vaccines, addressing optimal delivery of influenza vaccines in tropical and low-income countries, and highlighting gaps in measles vaccine coverage following the ebola outbreak in west africa. in addition to influencing policy, a major success of the misms and rapidd programs is reflected by the outstanding professional trajectories of their alumni, domestically and internationally, and the excellence and international scope of their publications (table , box ). of the more than publications of the misms and rapidd programs, which have been cited over , times, % include authors from lmics. many of the rapidd and misms trainees and collaborators are now established as senior researchers. both programs rely on global multi-disciplinary networks to identify and address research questions at the forefront of infectious disease transmission and evolution. both programs build off the "big data" revolution that provides increasingly detailed and abundant information on disease patterns and host behavior, leveraging a surge of pathogen genetic sequence data, exquisitely detailed epidemiological information derived from digital and social media, and human mobility and demographic proxies derived from mobile phones or remote sensing. new methodological approaches are needed to handle such a vast and diverse amount of information. further, both programs have long histories of promoting data sharing and development of publicly available methodological tools, with key contributions to the influenza genome sequencing program and database, the beast package for phylogenetic and associated analyses, and development of new transmission modeling packages (pomp, tsir) and spatial analysis software (epipoi) (alonso and mccormick, ; becker and morris, ; king, ) the misms and rapidd programs are also well connected with parallel efforts at nih and cdc and in the broader scientific community. for instance, within nih, there are strong synergies between rapidd and midas, a funding program in infectious disease modeling managed by the national institute for general medical sciences, and between misms and the centers of excellence in influenza research and surveillance (ceirs), funded by the national institute of allergy and infectious diseases. in parallel, there are strong ties between rapidd and misms and the cdc's influenza division, involving joint workshops, forecasting challenges (e.g., (merler, ajelli et al. ; viboud et al., a viboud et al., , b ) and post-doctoral exchanges. further, there is a tight link between misms and rapidd and national and global public health agencies, with regular participation of collaborators in the white house pandemic preparedness working group, us government modeling coordination groups, and who modeling groups. several of the misms workshops have been co-organized with, and in support of, foreign government agencies (fig. ) , while many international government representatives have taken part in misms and ra-pidd workshops. despite these successes, some challenges are worth noting. the most key aspects of diseases dynamics investigated by rapidd. problematic is the lack of sustainable long-term funding for these programs more than years after they were created, even though their combined annual costs are under m$, which is negligible relative to the costs of public health crises (see (office of science, ; world bank and ecohealth alliance, ) for a discussion of the value of infectious disease preparedness and modeling). it is worth nothing however that the low cost of computational modeling does not include funding for generation of biomedical, epidemiological and surveillance data, which are essential to support modeling work. and with their extended duration and distributed network structure, the rapidd and misms programs fall outside standard funding streams, despite their extraordinary efficiency in propelling leading-edge science and addressing problems of national interest (box ). as the ebola and zika outbreaks clearly demonstrate, infectious disease threats are not in retreat advances in multi-pathogen diagnostics and sequencing will facilitate real-time molecular epidemiology during future outbreaks, but will require people on the ground and international partners who can rapidly interpret large amounts of data. development of new vaccines (e.g., respiratory syncytial virus, universal influenza) and the growing challenge of anti-microbial resistance will require improved models to optimize public health policies and understand pathogen evolutionary responses. in conclusion, the misms and rapidd programs illustrate the power of scientific diplomacy and collaborative research networks, with demonstrable successes in improving research and control of infectious diseases. we see no shortage of policy-relevant research themes for these networks to explore, including the emergence of new threats at the animal-human interface, microbial interactions within and between hosts, integration of traditional and novel data streams into ever more sophisticated models, short-and long-term disease forecasts, projections of the impact of novel vaccines, and anti-microbial resistance. fogarty-led disease modeling programs will continue to strengthen capacity in lmics for outbreak analysis, and in turn help control emerging infectious disease threats domestically and internationally. this article is dedicated to our fogarty colleague, dr. ellis mckenzie, who was instrumental in launching, growing and shepherding the rapidd program from until his death in . ellis played a key role in promoting use of infectious disease models in government and mentored countless infectious disease modelers. the establishment and success of the program would have been impossible without ellis's gentle and inspired guidance. the rapidd program is indebted to the leadership, vision, and guidance of two outstanding and incredibly generous scientists who shepherded this program since , bryan grenfell and ellis mckenzie. we acknowledge support from the department of homeland security and fic for the rapidd program, and the hhs pandemic threat unit, office of global affairs and fic for the misms program. we are grateful to felix wu, amherst college, for performing a thorough bibliometric analysis of the rapidd and misms publications and generating maps of co-authors. we are thankful to kate skoczdopole for continued administrative support of the rapidd and misms programs. the funders did not play a role in study design; collection, analysis, and interpretation of data; or in the writing the 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of mobile phone data quantifying the impact of human mobility on malaria yellow fever in brazil: update epidemiology. foot-and-mouth disease under control in the uk lessons from ebola: improving infectious disease surveillance to inform outbreak management a synchronized global sweep of the internal genes of modern avian influenza virus forecasting influenza epidemics in hong kong forecasting influenza outbreaks in boroughs and neighborhoods of new york city transmission network of the - ebola epidemic in sierra leone characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data key: cord- - rblhwb authors: mahy, b.w.j title: emerging and reemerging virus diseases of vertebrates date: - - journal: reference module in biomedical sciences doi: . /b - - - - . - sha: doc_id: cord_uid: rblhwb in the last two decades, a large number of new viruses have been discovered, many of which are pathogenic in humans or other vertebrates. among the more important causes of virus emergence have been changes in human behavior, population, and increases in travel to distant countries. in addition, the application of new molecular technologies has led to the recognition of many viruses that hitherto went undetected. many of the new, emerging viruses have an rna genome, and many are zoonoses. the spread of human immunodeficiency virus, causing acquired immune deficiency syndrome, and the use of immunosuppressive drugs following transplant surgery, have increased the numbers of people in the population that are highly susceptible to emerging virus infections. the threat of a new pandemic of influenza virus in the human population stresses the need for development of better methods for detection, surveillance, and control of emerging virus diseases. it became apparent during the last two decades of the twentieth century that new infectious diseases were increasingly being recognized in the human and animal populations. this led to the establishment of a formal committee of the institute of medicine of the national academy of sciences, usa, who reported on their deliberations in , in a report edited by joshua lederberg and richard shope. this was followed years later by a second report, edited by mark smolinski, margaret hamburg, and joshua lederberg, which appeared in . among the factors they cited as contributing to emergence were microbial adaptation and change, human susceptibility to infection, climate and weather, changing ecosystems, economic development and land use, human demographics and behavior, technology and industry, international travel and commerce, breakdown of public health measures, poverty and social inequality, war and famine, lack of political will, and finally intent to harm. the advent of highly specific molecular techniques such as the polymerase chain reaction (pcr) in the early s permitted the detection and grouping of viruses on the basis of genome nucleotide sequence analysis, and in several respects these techniques have replaced serological analyses for the characterization of viruses. although it is still important to isolate viruses in cell culture for their complete characterization, it is now possible directly to detect viruses in diseased tissues by pcr, then, by sequencing the amplicon, to determine whether a new virus has emerged to cause the disease. in fact, many viruses which do not readily grow in cell culture can only be differentiated by sequence analysis. the papillomaviruses are an example. their study was very difficult until the advent of sequence analysis, which now has revealed more than types in humans, and many more in animals and birds. for differentiation, three virus genes (e , e , and l ) are sequenced, and if the combined sequence of these three genes differs by more than % from known papillomaviruses, the virus is considered to be a new type. other viruses which have not been grown in cell culture include many caliciviruses, and the ubiquitous anelloviruses, such as torque-teno (tt) virus, which can be detected and sequenced in the blood of most humans and many other vertebrate species. hepatitis c virus was originally described as non-a, non-b hepatitis virus because of the severe disease it caused but the virus would not grow in cell culture, and eventually was detected in blood known to be infected with the virus by reverse transcription of the rna present using random primers then expressing the resultant dna in the bacteriophage lambda gt . thousands of clones were screened using patient blood as a source of antibody before positive clones were detected, which then allowed the ☆ change history: july . bwj mahy added new section on mers coronavirus. added several new references development of enzyme immunoassays that could detect the virus in blood and so were used to screen blood destined for transfusion, saving millions of lives worldwide. once the complete genome of hepatitis c virus was sequenced, it became apparent that there are many different genotypes circulating in the world, with different pathogenic properties. nucleotide sequence analysis has also been extremely useful in tracing the origins of viruses. for example, when hantavirus pulmonary syndrome, caused by a bunyavirus of rodents, sin nombre virus, was initially detected in in the four corners region of western usa, it was found that rodents inside a house where people had been infected carried a virus identical in sequence to virus isolated from human cases. however, rodents caught at various distances from the house had increasingly variable genome rna sequences, providing strong evidence that these rodents, deer mice (peromyscus maniculatus), were the source of the infection. subsequently more than other hantaviruses, some of which also cause hantavirus pulmonary syndrome in humans, were isolated from rodents throughout north and south america. each new virus seems to be associated with a different genetic variant of rodent host, and all rodents that carry the virus belong to the subfamily sigmodontinae, unique to the american continent. a particularly powerful tool for the initial recognition of an emerging virus is the application of immunohistochemistry to diseased tissues. provided a comprehensive collection of antibodies is available, the particular virus or related group of viruses can often be detected. for example, when hendra virus first appeared in in australia, causing the death of a horse trainer and of his horses, antibody against the virus was sent to the centers for disease control and prevention (cdc). then, in , cdc was asked to investigate a newly emerged epidemic that had appeared in malaysia, killing more the people and causing disease in many pigs. initially, it was suspected to be caused by a virus related to japanese encephalitis virus, but a virus was isolated from the pigs that replicated in vero cells, and reacted in an immunofluorescence test against the hendra virus antiserum. this could subsequently be used on patient tissues to study the pathogenesis of the disease, and after comparison of the genome sequences of hendra virus and nipah virus they were found to be closely related and are now classified in the genus henipavirus of the paramyxoviridae. finally, molecular methods can be used to detect new, emerging viruses in the absence of disease in the host. in , allander and colleagues searched for rna viruses in human respiratory secretions using random primer pcr, and discovered a hitherto unknown parvovirus with a sequence related to the bovine and canine parvoviruses, which are grouped together in the genus bocavirus. the new virus was called human bocavirus, and many research groups worldwide have now confirmed the presence of the virus, particularly in pediatric samples, although it is still not certain how important this virus is in causing morbidity and mortality. their method also amplified a human coronavirus from the respiratory samples, and when sequenced this turned out to be hku , a recently emerged coronavirus detected by scientists at hong kong university. it is possible that a systematic search of human samples using such molecular techniques might reveal more hitherto unknown human viruses. in some cases, the emerging viruses themselves have contributed to other viruses emerging and reemerging in the population. this is especially true of human immunodeficiency virus (hiv), the cause of acquired immune deficiency syndrome (aids), which rapidly spread following its emergence in the early s to infect more than million people worldwide by the end of the twentieth century. because of its severe effects on the immune system, the virus leads to numerous other infections in the hiv-infected population. for example, picobirnaviruses, that had been detected in fecal samples from chickens and rabbits, were difficult to detect in human fecal samples until a cohort of men with aids was examined, and in these humans picobirnavirus was detected for the first time. some rare diseases have become common in persons with aids. for example, the human polyomavirus known as jc virus can cause the rare brain disorder known as progressive multifocal leukoencephalopathy (pml). normally, the virus remains dormant in the kidney, but in hiv-infected individuals the hiv-encoded transactivator tat acts as a transactivator of jcv leading to pml which progresses to death within months after infection. other important virus infections which emerge in aids patients are human herpesviruses (cytomegalovirus, herpes simplex viruses and , varicella-zoster virus, and human herspesvirus , which causes kaposi's sarcoma). hiv is mainly spread through sexual activity between an infected and a noninfected person, and is most common in those who indulge in high-risk sexual behavior with multiple partners. it can also be transmitted by direct contact with infected blood, and is common in persons who indulge in intravenous drug use, particularly when needles, syringes, or equipment used to prepare drugs for injection are shared. it is therefore an example of a virus disease which is dependent on risky human behavior for its maintenance in the human population. the ability of such new infections to spread in the population has been greatly enhanced by population growth and ease of movement as a result of rapid air travel. a dramatic recent example of this was the appearance of the coronavirus causing severe acute respiratory syndrome (sars) in late which spread by air travel from a single infected chinese physician who infected persons in a hong kong hotel. these infected persons then traveled by air and spread the infection to more than individuals worldwide, % of whom died. the virus then apparently receded from the human population in july . only recently was it discovered that the sars coronavirus has a natural reservoir in chinese horseshoe bats (rhinolophus sinicus). some other species such as himalayan palm civets and raccoon dogs from which the virus has been isolated may serve as amplification hosts. following the recognition of the human coronavirus sars, research on coronaviruses intensified, and this led to the discovery in of two previously unrecognized human viruses, one found by hong kong university, called hku virus, and another reported almost simultaneously from the netherlands, called nl , and from yale university, called new haven coronavirus. the latter viruses probably represent two isolates of the same virus species. they are clearly associated with lower respiratory tract infection in children, but initially it was claimed that new haven coronavirus was also associated with kawasaki disease in children. this intriguing claim was rapidly investigated and refuted by several different groups in japan, taiwan, and elsewhere, and the cause of kawasaki disease, which has features resembling a virus infection, remains unknown. in , another novel human coronavirus was isolated from sputum from a fatal case of severe respiratory disease in the arabian peninsula. the virus, now called middle east respiratory syndrome coronavirus (mers-cov), has since been detected in more than patients with respiratory disease, of whom died. in november ,the mnistry of health in saudi arabia was notified of case of mers-cov infectionin a year-old male patient who had cared for ill dromedary camels in his herd of animals starting in early october. the patient was confirmed with mers-coronavirus by real-time pcr, and limited sequencing found the mers-coronavirus in three of the dromedary camels. other studies have detected mers-coronavirus-infected dromedary camels in several other regions in saudi arabia.but it remains unclear whether camels are an important source of the virus fror transmission to humans, and whether bats also play a role, as was the case with the sars-coronavirus. a majority of recent emerging virus diseases have been zoonoses (i.e., diseases transmitted from animals to humans under natural conditions). some of the more important of these include hiv- , which was transmitted to humans from chimpanzees in central africa around , and hiv- , transmitted from sooty mangabeys to humans in west africa around . other important recent examples are the viruses of the genus henipavirus. hendra virus was first recognized through a disease outbreak in some horse stables in hendra, queensland, australia, when horses and their trainer died from pulmonary disease with hemorrhagic manifestations in . the reservoir of the virus was found to be in large fruit-eating bats (pteropus spp.), and one year later a horse farmer miles away in mackay, queensland, died of encephalitis from the same virus. then, in , a related virus was discovered in malaysia following a major outbreak of respiratory disease in pigs and neurological disease in humans in their close contact. more than humans died, and in a successful effort to control the disease . million pigs were slaughtered. the causative virus was isolated from a fatal human case that had lived in nipah river village, and so was named nipah virus. hendra and nipah viruses are clearly members of the family paramyxoviridae, but have been placed in a separate genus as their rna genome is about kb in length, larger than that of any other paramyxovirus. nipah virus, like hendra virus, was found to have a reservoir in pteropus bats, and has since been identified in fatal human disease outbreaks in india in and bangladesh in . other new viruses which apparently have a reservoir in fruit bats include menangle virus, a new paramyxovirus which emerged in a commercial piggery near sydney, australia, to cause stillbirths and abortion in pigs. menangle virus also caused disease in two workers in the piggery. a new virus related to menangle virus emerged during an investigation of urine samples from pteropid bats collected on tioman island, off the coast of malaysia, in , and was named tioman virus. during the same investigation, a new orthoreovirus was isolated from pteropus hypomelanus in and called pulau virus, and more recently a related orthoreovirus called melaka virus was isolated from a human case of acute respiratory disease in melaka, malaysia. serological studies of sera collected from human volunteers on tioman island showed that % had antibodies against both pulau and malaka viruses. another important group of zoonotic diseases are rodent-borne, and caused by members of the genus hantavirus of the family bunyaviridae. these viruses first emerged during the korean war of - , when thousands of un troops developed a mysterious disease with fever, headache, hemorrhage, and renal failure with a fatality rate of - %. it was more than a quarter of a century before the causative virus was isolated from field mice in korea, and named hantaan virus, the cause of hemorrhagic fever with renal syndrome (hfrs) in humans. then, in , a new hantavirus emerged in the four corners region of southwestern usa as the cause of a severe acute respiratory disease syndrome, with a fatality rate close to %, and named sin nombre virus. this virus was shown to be transmitted to humans by inhalation of virus present in the urine, feces, or saliva of deer mice (peromyscus maniculatus). it seems likely that this disease had existed for many years, and was only recognized in because of a clustering of human cases as a result of a regional upsurge in the rodent population resulting from climatic conditions causing increased availability of rodent food. fortunately, in most of these infections, humans appear to be a dead-end host, and transmission between humans does not occur except with the andes virus in south america. rodent-borne viruses of the family arenaviridae also cause a number of serious zoonotic diseases in humans. the 'old world' arenaviruses such as lassa fever virus have been known for some time, but still cause thousands of fatal hemorrhagic fever cases every year in west africa. however, 'new world' arenaviruses such as junin virus causing argentinian hemorrhagic fever and machupo virus causing bolivian hemorrhagic fever have long been recognized in south america. recently, new arenaviruses have emerged, probably as a result of deforestation, which results in rodents seeking shelter in human habitation, and brings them into closer contact with people. these viruses include guanarito virus that causes venezuelan hemorrhagic fever with % mortality rate from confirmed cases, and sabia virus isolated in that causes brazilian hemorrhagic fever with a high fatality rate, including two laboratory acquired cases. rabies is a zoonotic disease of great antiquity that has mainly been associated with carnivores, such as dogs. the virus is excreted in the saliva of infected animals, and following infection it moves through the nervous system to attack the brain, causing aggressive behavior which results in the animal biting humans and animals with which it comes into contact and thereby spreading the virus infection. fortunately, due to early work by louis pasteur, a vaccine was developed that protects humans or other animals from infection, and can also be given immediately post exposure, and the domestic dog population in the developed world is vaccinated and does not pose a risk to humans. however, in some developing countries, it is not uncommon for a rabid dog to bite and infect more than people before it can be put down, and worldwide there are still some human rabies deaths per year. using molecular sequencing techniques, it is now possible to distinguish the genotypes of rabies viruses associated with different species of host, as the virus has become adapted through frequent transmission between members of the same host species. in the usa, there are six recognized terrestrial animal genotypes, in raccoons in eastern states, skunks in north-central states, skunks in southcentral states, coyotes in southern texas, red foxes in alaska, gray foxes in arizona, and several genotypes associated with particular species of bat. in fact, most fatal cases of human rabies in the usa can now be traced to bats, which are often not detected when the person is bitten; so rabies is not suspected and vaccination is not undertaken until the disease has taken hold. many important virus diseases are spread by arthropods, and exposure to new arthropods and the viruses they carry is critical to the emergence of new virus diseases. dengue hemorrhagic fever is caused by dengue virus which is transmitted mainly by the asian mosquito (aedes albopictus), and dengue fever is one of the most rapidly emerging diseases in tropical regions of the world. there are four serotypes of dengue virus, and it seems that consecutive infections with two antigenic types can lead to the more serious disease of dengue hemorrhagic fever with shock syndrome, which, if untreated, can result in up to % mortality. unfortunately, through the importation of vehicle tires containing water from korea, the asian mosquito was introduced into the usa, and is now present in several regions of the southern states. it can act as a vector not only for dengue virus, but also for california encephalitis virus. in europe, the emergence of two important animal diseases has occurred through the movement of arthropod vectors into the iberian peninsula. african horse sickness virus causes a disease that can be fatal to horses, mules, and donkeys, and is transmitted by nocturnal biting flies of the genus culicoides. these were introduced inadvertently into spain, and the disease is now endemic around madrid and regions to the south. african swine fever virus is transmitted by ticks of the genus ornithodorus and it causes a fatal disease resembling classical swine fever in domestic pigs. it first emerged in portugal and spain in , france in , italy in , and cuba in . through slaughter of infected animals, the disease was eradicated from europe, except sardinia, by . the most recent dramatic example of the movement of a virus vector is provided by west nile virus, a flavivirus first isolated in uganda in . this virus uses birds as a reservoir host, and is transmitted from birds to humans and other vertebrates by mosquitoes. in , cases of encephalitis in new york were found to have been caused by a strain of west nile virus that was phylogenetically similar to a virus isolated from geese in israel. at the same time, many birds, especially corvids, began dying in new york state. since the introduction in , west nile virus has become well established throughout the usa and moved north into canada and south into the caribbean and into mexico. it is not known how the virus moved from israel to the usa, but the most reasonable explanation is that it was carried in an infected mosquito or possibly an infected bird in the hold of an aircraft. transmission by an infected human seems less likely since the titer of virus in human blood is usually too low for efficient mosquito transmission. it is clear, nevertheless, that once it arrived in north america, west nile virus found an extremely favorable environment with abundant avian and arthropod hosts that facilitated its spread throughout the american continent. the emergence of new viruses is likely to continue as viruses evolve and find new ecological niches in the human and animal population. it is noticeable that most newly recognized viruses have been rna viruses, perhaps since rna evolves at a faster rate than dna, for which host cells have developed efficient proofreading enzymes. it will be important in the future to detect new viruses before they can emerge to cause disease in the population. the sars epidemic provides an excellent example. before the epidemic, only two human coronaviruses were known, human coronaviruses e and oc . despite the fact that serious coronavirus diseases were well known in other vertebrates, such as feline infectious peritonitis and avian infectious bronchitis virus, it was not until the sars epidemic that research on human coronaviruses led to the discovery of three new human coronaviruses -sars, hku , and nl /new haven. there are other genera of viruses that cause serious disease in animals but have not been adequately investigated in humans. an example is the genus arterivirus, which has members causing serious disease in horses and pigs, but has not been reported at all in humans. this could be a worthwhile area for future investigation. another critical factor in the future control of emerging viruses is better vector control. when mosquito control was conducted using ddt, dengue fever virus was virtually eliminated from the americas in the s, but environmental concerns led to the widespread banning of the use of ddt, so that since the s there has been a considerable expansion of dengue fever in south america, with the appearance of dengue hemorrhagic fever there for the first time. there is a real need to improve mosquito control measures to control this disease. although there are prospects for a dengue virus vaccine, this is so far not available. in , a new outbreak of ebola virus occurred in west africa, causing several thousand deaths in liberia and sierra leone. the ebola virus first emerged as the causative agent of viral hemorrhagic fever in two outbreaks occurring almost simultaneously along the ebola river in the democratic republic of congo in . there were some human cases in this episode with a mortality rate of %. since that time, a number of smaller outbreaks of ebola hemorrhagic fever affecting humans and monkeys were reported from west africa, but although most infections have been associated with hunting and handling of great apes, the actual origin of the virus has never been ascertained. it is clear hat transmission of the virus requires close contact with an infected individual, and does not involve airborne transmission. ebola virions are usually long filamentous sometimes branched forms with a uniform diameter of about nm, and varying in length up to nm, from which they are called filoviruses, belonging to the family filoviridae. finally, one of the most important viruses that continue to emerge in different antigenic forms is influenza virus. the main reservoir of influenza viruses is in birds, and over the past century several pandemics of influenza have emerged, the most serious of which was in . pandemic strains usually arise by a process of antigenic shift, where one of the genes encoding the hemagglutinin and/or the neuraminidase of influenza virus is replaced by one from birds. new pandemics occurred in (h n subtype), (h n subtype), and (h n subtype). since , there have been no new pandemics, but it is widely expected that another will occur. at the time of writing, there is worldwide concern that a highly pathogenic avian influenza virus (h n subtype), which has caused some human infections and deaths in persons in close contact with infected birds, might mutate or recombine to generate a virus which would be highly transmissible in the human population. plans are being developed in many countries and by the who to try to prepare for such an event by generating possible vaccines against such a virus and stockpiling antiviral drugs. biomedical research emerging trends in lassa fever:redefining the role of immunoglobulin m and inflammation in diagnosing acute infection identification of the major, parenteral non-a, non-b hepatitis agent (hepatitis c virus) using a recombinant cdna approach nipah virus: a recently emergent deadly paramyxovirus identification of new papillomavirus types critical review of the vector status of aedes albopictus west nile virus: epidemiology and clinical features of an emerging epidemic in the united states nipah virus encephalitis reemergence the widening scope of coronaviruses timing the ancestor of the hiv- pandemic strains lessons to be learned from the ebolavirus outbreak in west africa a novel coronavirus associated with severe acute respiratory syndrome emerging zoonoses: crossing the species barrier the emergence and re-emergence of viral diseases molecular and biophysical characterization of tt virus: evidence for a new virus family infecting humans genetic identification of a hantavirus associated with an outbreak of acute respiratory illness microbial threats to health, emergence, detection and response unraveling the mysteries of middle east repiratory syndrome coronavirus key: cord- -a jewy h authors: bianchini, juana; humblet, marie‐france; cargnel, mickaël; van der stede, yves; koenen, frank; de clercq, kris; saegerman, claude title: prioritization of livestock transboundary diseases in belgium using a multicriteria decision analysis tool based on drivers of emergence date: - - journal: transbound emerg dis doi: . /tbed. sha: doc_id: cord_uid: a jewy h during the past decade, livestock diseases have (re‐)emerged in areas where they had been previously eradicated or never been recorded before. drivers (i.e. factors of (re‐)emergence) have been identified. livestock diseases spread irrespective of borders, and therefore, reliable methods are required to help decision‐makers to identify potential threats and try stopping their (re‐)emergence. ranking methods and multicriteria approaches are cost‐effective tools for such purpose and were applied to prioritize a list of selected diseases (n = including zoonoses) based on the opinion of experts in accordance with drivers‐related criteria. diseases appearing in the upper ranking were porcine epidemic diarrhoea, foot‐and‐mouth disease, low pathogenic avian influenza, african horse sickness and highly pathogenic avian influenza. the tool proposed uses a multicriteria decision analysis approach to prioritize pathogens according to drivers and can be applied to other countries or diseases. the oie from different european member states including belgium (world organisation for animal health, ) . another very important recent emerging livestock disease reported specifically in belgium at the end of was african swine fever, although cases so far have been reported only in wild boars . its emergence is of great concern for the pig industry of the region and being a disease, which until now has been exotic for belgium. it shows how diseases may re-emerge unexpectedly with most likely origin attributable to human activity (saegerman, ) . the (re-)emergence of diseases shifts in relation to several underlying set of factors inherent to modern society, that is the so-called 'drivers'. the joint presence of these drivers can create an environment in which infectious disease can (re-)emerge and be maintained in animal and/or human compartments (king, ) . many drivers have been identified, such as climate change, global travel, immigration patterns, increase in the human population, environmental degradation and others (altizer, ostfeld, johnson, kutz, & harvell, ; daszak, cunningham, & hyatt, ; king, ) . the threat of (re-)emergence is more likely to increase and past experience has shown that no country, however economically welldeveloped it may be, is capable of ensuring % security of its borders, even by imposing measures such as quarantine protocols or import bans on animals and animal products (ben jebara, ) . in belgium, the monitoring and reporting of livestock diseases are subjected mostly on self-reporting of suspected clinical cases by the farmers to the federal agency for the safety of the food chain (fasfc), with an established list of mandatory notifiable diseases for livestock and other species (aquatic, exotic) (federal agency for the safety of the food chain, ). each suspicion is then confirmed by laboratory analysis (federal agency for the safety of the food chain, ). thus, a rational priority setting approach is needed to assist decision-makers in identifying and prioritize diseases that are more likely to (re-)emerge and as such allocating the right resources tailored to a particular disease threat. one such approach used is disease prioritization, which has as main objectives: to optimize financial and human resources for the surveillance, prevention, control and eradication of infectious disease and to target surveillance for early detection of any emerging diseases (humblet et al., ) . some studies identified key characteristics of potential emerging infectious diseases and prioritized infectious diseases according to their risk of (re-)emergence or impact in some countries (cardoen et al., ; cox, sanchez, & revie, ; havelaar et al., ; humblet et al., ) . hence, these focused on human or zoonotic diseases and the impact they would have in certain countries. in this study, the focus is livestock epidemic diseases and the aim was to identify (re-)emergence drivers' criteria and with it use expert elicitation to prioritize livestock epidemic diseases that may emerge in belgium. a multicriteria decision analysis (mcda) method was chosen because it provides a systematic way to integrate information from a range of sources (cox et al., ) and it aims to improve transparency and repeatability (european centre & for disease prevention & control, ) . multicriteria decision analysis requires identifying criteria and scoring criteria according to the pathogen/disease. by weighting each criterion and calculating weighted scores from the criteria, an overall score per pathogen/disease was calculated (european centre & for disease prevention & control, ; humblet et al., ) . this is the first study to prioritize livestock epidemic disease using drivers as criteria. this prioritization list could be an aid to decision-makers to make an informed decision on course of actions to be taken and use the correct resources when there is a threat of a disease (re-)emerging in belgium. we compiled a list of livestock-associated infectious diseases ( figure ) using a systematic approach. this was done by collating in a single database notifiable terrestrial animal diseases from different governmental official lists from belgium (federal agency for the safety of the food chain, ) and neighbouring countries (luxembourg was excluded because of high similarity), that is germany (federal ministry of food & agriculture of germany, ), france (légifrance, a (légifrance, , b , the netherlands (ministerie van landbouw, ) and great britain (scottish government, ) . in order to broaden the spectrum, diseases included in two other lists of official international organizations, that is the world organisation for animal health (oie) (world organisation for animal health, ) and the european union (european commission, ) , were also added to the database. only diseases that affect cattle, sheep, goats, swine and poultry (livestock) were selected from the official lists and included in database. (azkur et al., ; chaintoutis et al., ; lievaart-peterson et al., ; yilmaz et al., ) . thus, the risk of any of these viruses to (re-) emerge may be present, which further prompted the necessity of adding these three viruses to the list of diseases to be prioritized. the main objective was to prioritize the diseases according to drivers of (re-)emergence. a driver was defined as a factor, which has the potential to directly or indirectly precipitate ('drive') or lead to the (re-)emergence of a livestock infectious disease. we identified different criteria considered as drivers through scientific literature and previous disease prioritization exercises, and discussion with experts from academia, government agencies and international bodies. a total of criteria were identified and classified under different domains (table ) each criterion had a definition of the coefficient, which ranged from to accordingly (appendix a). each domain spreadsheet had a number of criteria. for each criterion, coefficients were clearly defined for a good comprehension and standardization. coefficients were from scores of to or from to (a number of criteria could not be scored with a zero, e.g. current f i g u r e systematic process for selecting the livestock diseases. * livestock diseases were those which affected cattle, sheep, goats, swine and poultry ta b l e list of criteria used to prioritise (re)emerging infectious diseases, according to their likelihood of (re)emergence in belgium in response to different categories of drivers (gore, ) . the number of points to be distributed was proportional to the number of criteria per category multiplied by ten. indeed, the criterion with the most points allocated is considered the one that weighs the most in the category. if, on the other hand, all the criteria have the same weight in the category, the distribution is equitable, with points for each criterion. for example, points were to be distributed between the criteria of the 'pathogen characteristics' domain. indeed, the criterion with the most points allocated is considered the one that weighs the most in the pathogen characteristics. such process illustrated the experts' opinion on the relative importance of criteria within one domain. the last spreadsheet was dedicated to the inter-domain weighting. experts were asked to distribute a total of points (n = domains) among the domains to classify the domains according to their opinion. two rounds of expert elicitation were implemented. the first round consisted in the questionnaire assessment; experts were asked to verify whether the questions were in relation with the drivers and whether the scoring systems were correctly defined and identified. the questionnaire and related instructions were sent to experts (appendix b) by e-mail. the experts were asked to complete questionnaire by scoring and additionally to assess and give comments on the criteria and coefficient definitions. the questionnaire was then refined according to experts' comments and suggestions. for the second round, experts were identified (appendix c) via internet searching and recommendations from the project partners and recruited participants. these experts were asked to answer the questionnaire in order to rank the diseases. thus, they had to choose the defined coefficient for each criterion (i.e. criterion scoring), then distribute the points for within each domain (i.e. the intradomain weighting), and lastly distribute the points within the domains (i.e. inter-domain weighting). they were invited to participate via a project summary e-mail and were sent the reviewed questionnaire via e-mail if they agreed to participate. experts were recruited until a minimum of experts per disease was obtained with a maximum of experts. in some cases, one expert could answer several questionnaires (one per disease) if the diseases were within is area of expertise. to obtain the overall score for the ranking, an aggregation method that combined the types of weighting (i.e. the intra-and inter-domain) was used. first, the criterion score (coefficients attributed by experts) had to be standardized. indeed, some criteria were allocated coefficients from to and others from to . this standardized score was then multiplied by the intradomain weight as given by the expert. these results were summed to obtain a domain score. in this formula, dsj = domain score, crit = criterion, scj = standardized score of the criterion and wdwj = intradomain weight for each criterion. each domain score was then multiplied by the inter-domain weight. these results were summed and an overall weighted score calculated, per expert and per disease. in this formula, ows = overall weighting score of each expert for a specific disease, cat = category, dsj = domain score and idwj = inter-domain weight. each disease had or ows (since there were or experts per disease), and thus, for each disease, the final score was the average of all disease experts' ows. the final score was then used to rank the diseases, based on drivers, from the highest score to the lowest. the highest score corresponded to the disease with the highest risk of (re-) emerging according to the drivers. in addition, the median and range among the scores of all the disease experts were also obtained. with the median, a ranking was done to observe whether there was any significant difference with the ranking obtained using the mean. the range was used to note which diseases had the highest and lowest level of variation/uncertainty among the final experts' average score. in order to determine which driver(s) was/were considered as the most influential for the (re-) emergence of diseases, the domains were ranked. domain ranking was performed using the inter-domain scores (weights). the sum of each domain weight (∑idwj) per disease and per domain given by each expert was ranked from the high to the low, that is to . then, for each domain, the frequency of their rank was used to display in graph. a cluster analysis was implemented using regression tree analysis (salford predictive modeler ® , version . , salford systems, san diego, california, usa). the normalized disease score is a continuous variable, and the aim was to obtain groups in qualitative categories of importance (e.g. very high, high, moderate and low) with minimal within-group variance. two sensitivity analyses were assessed, that is on expert elicitation and influence of a domain. this was achieved by repeating the disease ranking with a 'reduced' version of the model and comparing the new ranking to the complete model. the experts' sensitivity analysis consisted in dividing them into groups. scores were then re-calculated by deleting a group of experts. each reduced ranking model was compared to the full complete model by using the spearman's rank test to establish whether the ranking was correlated between the complete and the reduced models. the sensitivity analysis on the domains was done by deleting one domain and re-calculating the mean scores to rank the diseases. this 'reduced' ranking was then compared with the complete model, and the spearman's rank test was applied. if the ranking position changed to less than three places, then the final score was considered as robust. if it changed to more than two places, then it was considered as a domain of drivers influencing greatly disease (re-) emergence. we compiled a list of diseases ( all experts contacted for the first phase (questionnaire assessment) answered positively (appendix b). there was a general agreement on which criteria and coefficients were clear or not. neither criterion nor coefficient were deleted but only amended according to experts' suggestions. for the second phase of expert elicitation, a total of experts agreed to participate and answered the questionnaires (appendix c). the objective of minimum of experts per disease was reached, and the maximum of experts was reached for diseases. the final disease ranking based on the average final scores is shown in figure . the higher the mean score, the higher the ranking, which means the disease is most likely to (re-)emerge in belgium. the top diseases in decreasing order were porcine epidemic diarrhoea (ped), fmd, low pathogenic avian influenza (lpai), african horse sickness (ahs) and hpai (table ) . on the other end, the diseases with the lowest mean scores were haemorrhagic septicaemia, japanese encephalitis, wnf, peste des petits ruminants (ppr) and nipah disease. ( ) the regression tree analysis determined clusters ( figure ). the clusters distinguished five, eleven, nine and four diseases, and were classified, respectively, as of 'low importance', 'moderate importance', 'high importance' and 'very high importance' (i.e. highly influenced by drivers). the diseases belonging to the node 'highest importance' were ped, fmd, lpai and ahs. the node of the lowest importance included haemorrhagic septicaemia, japanese encephalitis, ppr, nipah disease and wnf. the relative importance of the domains varied depending on the disease. however, when considering all domains for all diseases, 'economy and trade activities' obtained the highest number of points, being ranked first times and zero times last ranked ( th). the opposite can be said about 'characteristics of farm/production system', as it was never ranked st nor nd ( figure ). the sensitivity analysis done on the groups of experts showed that the ranking of diseases was not affected in the reduced models. indeed, the spearman's rank-order correlation indicated a strong positive association of ranks when using different groups of experts for different reduced models, showing that there was a consistency among the scoring of the experts. ta b l e (continued) as for the domain sensitivity analysis, table the mcda approach allowed the selection of livestock diseases exotic to belgium and their prioritization based on drivers. whilst such an approach was used in previous disease prioritization exercises, this is one of the first to consider livestock epidemic diseases only and to use criteria related to drivers of (re-)emergence. only diseases exotic to belgium were prioritized. the diseases that fitted the eligibility criteria were all of viral origin, except haemorrhagic septicaemia (pasteurella multocida, serotypes :b, :e), ccpp and cbpp. few zoonoses were included in the list (n = ) as the prioritization exercise focused on livestock epidemic diseases. therefore, several zoonoses included in other prioritization processes were excluded. regarding prioritization, ped ranked top of the list. although currently not reportable neither in the eu (except in the uk) nor to the oie, it ranked high in all models (high mean score), possibly due to its highly transmissible character and the difficulty to control it; furthermore, the disease mainly concerns intensive production. cases have already been reported in eu member states: for example in may , an outbreak of diarrhoea occurred in fattening pigs on german farms. an outbreak of diarrhoea occurred on a belgian fattening pig farm at the end of january ; this was the first confirmed ped case in belgium in decades (theuns et al., ) . when the list of diseases was compiled, the outbreak had not occurred yet, but when the experts answered the questionnaire it had, and therefore, this was most likely the reason why it ranked at the top of the prioritized list. low pathogenic avian influenza ranked slightly higher than hpai in this multicriteria analysis on the risk of (re)-emergence (lpai ranked rd whilst hpai ranked th ( ) ( ) ( ) (monne et al., ) . hence, these characteristics of the virus give in this prioritization lpai a higher score than hpai, but hpai is more likely to be detected and notified. african horse sickness surprisingly ranked th, although its last know incursion in europe (portugal and spain) was in and its eradication dates back to . such high position in the ranking could be related to its vector-borne transmission, that is by culicoides biting midges. these vectors are often highly abundant, across most of africa, the middle east, europe and southern asia (carpenter, mellor, fall, garros, & venter, ) . additionally, the recent changes in the epidemiology of bluetongue and its latest epidemic in europe and the emergence of schmallenberg disease (afonso et al., ; anonimous, ; carpenter et al., ; highlight the uncertainty about the variables controlling the spread and persistence of culicoides-borne arboviruses. these different factors have raised concerns that ahs may also amount similar incursions, hence explaining such high mean final score in the prioritization process. in this prioritization, most of the diseases were in clusters (high importance, n = ) and (moderate importance, n = this score can only be compared with the prioritization work done by humblet and collaborators (humblet et al., ) as other prioritization works using the mcda method, such as those by cardoen et al. ( ), and havelaar et al. ( ) , only included zoonoses. indeed, in regression tree analysis of prioritized diseases of food-producing animals and zoonoses, asf also fell in the th group of importance out of the th group (humblet et al., ) , just like in this prioritization work. another study, which may be used for comparison as it used mcda approach and had swine diseases, done by brookes, hernandez-jover, cowled, holyoake, and ward ( ), asf ranked higher, but in this study only exotic diseases for the pig industry in australia were ranked using criteria related to impact and the experts were pig producers, which changes the importance in the scores, giving asf a higher ranking. the livestock diseases at the bottom of the list were nipah disease, ppr, wnf, japanese encephalitis and haemorrhagic septicaemia. in other prioritization exercises, nipah, japanese encephalitis and wnf were ranked in a higher category (cox et al., ; havelaar et al., ; humblet et al., ) . the prioritiza- drivers are a complex set of factors, and their convergence can cause the (re-)emergence of a disease. several drivers have a stronger impact on diseases compared to others, as shown in the results section. porcine epidemic diarrhoea ranked at the top in all models, except in the reduced models of production system characteristics. porcine epidemic diarrhoea affects mainly intensive production systems; thus, the driver category 'production system characteristics' logically influences a lot. when using the reduced model, the mean score decreases and the disease moved from the st place to the th place. in comparison, fmd ranked high in the prioritization process ( nd), but lowered to the th place in the reduced model, which excluded disease pathogen characteristics. for fmd, the strongest driver was the 'pathogens characteristics'. the virus is highly contagious, spreads via airborne and direct contact and affects different livestock species, giving this driver category a strong weight. all experts considered that 'economy and trade activities' was the most important driver (high weight). it was ranked first more often than others. in the reduced model (without the 'economy and trade activities' domain), all diseases with the exception of moved up or down in the ranking by more than places. this is of no surprise, as economic and trade activity has priority in the age of globalization; increased movement of live animals and animal products crossing oceans and international boundaries increase the risk of spread for animal and zoonotic diseases (domenech, lubroth, eddi, martin, & roger, furthermore, the sensitivity analysis of experts also showed a high correlation among the ranking of models, which confirms that experts were in agreement in regards to the scores. overall, the importance of validating each generated model is the authors declare no conflict of interest. ethical statement is not applicable to this study as the data were gathered through questionnaire survey without any animal experimentation. number of criteria = , hence points to be distributed within this domain for the intra-domain weighing. mono species farms-one single farmed animal (e.g. only bovines) or multi species farms (farms with more than one species, for example goats and bovines in the same farm/land/premises). score score negligible: the type of farm does not influence in any form (re)emergence of the disease among the livestock population. score low: mono or multi species farm has a low effect on the risk of disease to emerge or re-emerge. score moderate: the type or types of farmed animals has a moderate effect on the emergence of the disease in belgium. high: the type of farmed animals has a high influence for the disease to emerge and spread in belgium. farm demography/management: such as type of dairy or beef (cattle) production. for pigs-reproduction, fattening, finishing farm or both. chickens-only laying eggs chickens or solely finishing broilers score score negligible: population demography does not influence in any form the (re)emergence of the disease among the livestock population. score low: the demographic population of the farm is a low influencing factor for disease (re)emergence. for example, disease only clinically affects only one age strata (i.e.) newborns, therefore adults are immune to it. moderate: the demographic of the population has a moderate effect on the (re)emergence of the disease, as it can (re)emerge in more than one type of demography but other conditioning factors have to occur in conjunction. high: the type of demographic of the farm has a high effect on the (re)emergence of the disease as it can (re)emerge in different types of farmed animals and all types of age groups d animal density of farms. extensive (small holders with a few animals) v/s intensive farming score score negligible: animal farm density is not a risk factor for the disease to emerge in belgium score low: farm density (extensive or intensive) of animals has a low effect on the pathogen's/disease (re)emergence score moderate: farm density of animals in the farm (extensive v/s intensive) has a moderate effect on the emergence of pathogen/ disease score high: farm density of animals has a high effect on the (re)emergence of pathogen/disease. feeding practices of farms potential roles of zoo's in the (re)emergence of the pathogen score score negligible: the disease can be present in zoo animals but it is not known to have been transmitted from zoo animals to livestock. score low: the disease can enter a zoo (e.g. with introduction of an infected exotic animal) but only accidental transmissions of the disease from zoo animals to livestock have been reported. hence, zoos have a low effect on the (re)emergence of the disease in belgium's livestock score moderate: the disease can enter a zoo and be present in zoo animals but it needs a vector (biological/mechanical) for its transmission into livestock. therefore, zoos have a moderate effect on the (re)emergence of the disease in belgium. high: disease can be introduced to a zoo via an infected imported animal, zoo animals can carry the disease that can easily jump to livestock animals the rural(farm)-wildlife interface score score negligible: the disease has never (re)emerged from the narrowing of the farm-wild interface score low: the disease has a low probability to (re)emerge via the livestock farm-forest interface. the disease has been known to (re)emerge from the wild bush but very rarely score moderate: the disease has a moderate probability of (re)emergence via the farm/wildlife interface. barriers ( natural or artificial) are needed to keep the disease/pathogen (re)emerging in livestock score high: there is a high probability for the disease to (re)emerge via the farm/forest interface. barriers (natural or artificial) separating farms from natural forests are ineffective score low: there is a low probability of the disease (re)emerging and spreading through increased populations of endemic/migrating wild birds. disease has spread from the endemic/migrating wild birds but only accidentally or under exceptional circumstances score moderate: there is a moderate probability of disease being introduced and spread through increased populations of endemic/migrating wild birds. they are hosts and in close contact with domestic livestock (i.e. poultry farms) may spread the disease score high: there is a high probability for a disease to (re)emerge through increased populations of wild/migrating birds. these are hosts or reservoirs of the disease hunting activities: hunted animals can be brought back to where livestock is present score score negligible: the risk of the disease/pathogen of (re)emerging in livestock due to hunting activities is practically null score low: disease is present in hunted wildlife and birds and only accidental cases have been reported in livestock that have (re)emerged because of hunting. the risk of the disease/pathogen of (re)emerging in livestock due to hunting activities is practically null score moderate: disease is present in hunted wildlife and birds but a certain control is established by the hunter score high: disease is present in hunted wildlife and birds and hunting is one of the main modes of transmission of the disease to livestock f transboundary movements of terrestrial wildlife from other countries score null: disease is not carried by terrestrial wildlife score negligible: (re)emergence of the disease by terrestrial movements of wildlife has only been suspected but never confirmed. low: there is a low probability for the disease to (re)emerge and spread through transboundary movements of terrestrial wildlife score moderate: there is a moderate probability for the disease to (re)emerge and spread through transboundary movements of terrestrial wildlife score high: there is a high probability for the disease to (re)emerge and spread through transboundary movements of terrestrial wildlife. these are host and may spread/carry the disease along. number of criteria = , hence points to be distributed within this domain for the intra-domain weighing. (continued) g in-and out-people movements linked to tourism score score negligible: the movement of tourism is a negligible driver on the emergence or re-emergence of the disease score low: tourism increase has a low driver of the (re)emergence of the disease. score moderate: tourism increase has a moderate driver for the (re)emergence of the disease. biosecurity measures are enough to stop the entering of the pathogen. high: tourist movement is a high driver on the (re)emergence of a disease. tourists are highly likely to bring the disease into belgium in their belongings and biosecurity measures are insufficient to stop the pathogen g human immigration score score negligible: the immigration movements are a negligible driver of the disease (re)emergence in belgium score low: the immigration movements are a low driver of the disease (re)emergence in belgium score moderate: the disease is currently present in countries where more immigrants come from and pathogen highly likely to enter through, clothes, shoes and or possession, but the current biosecurity measures in place are able to prevent the emergence of the disease in belgium score high: the immigration movement has a high effect as a driver on the emergence or re-emergence of disease in belgium. disease is highly likely to emerge using this route as biosecurity measures are not enough to avoid emergence of the disease g transport movements: more specifically commercial flights, commercial transport by ships, cars or military (excluding transport vehicles of live animals). score score negligible: the role of commercial movements as a driver on the (re)emergence of the disease in belgium is negligible. score low: the role of commercial movements as a driver on the (re)emergence of the disease in belgium is low. it is easily preventable by implementing biosecurity measures score moderate: the role of commercial movements as a driver on the (re)emergence of a disease in belgium is moderate. disease can be prevented if biosecurity measures are tightened. high: the role of commercial movements as a driver on the (re)emergence of a disease in belgium is high. disease is hard to control via the current biosecurity measures. transport vehicles of live animals score score negligible: the role of transport vehicles of live animals as a driver for the (re)emergence of the disease in belgium is negligible score low: the role of transport vehicles of live animals as a driver for the (re)emergence of the disease in belgium is low. moderate: the role of transport vehicles of live animals as a driver for (re)emergence of the disease in belgium is moderate. high: the role of transport vehicles of live animals as a driver for (re)emergence of the disease in belgium is high g bioterrorism potential score score negligible: the role of bioterrorism as a driver for a disease to (re)emerge is negligible: agent is available but difficult to handle or has a low potential of spread or generates few economic consequences score low: the role of bioterrorism as a driver for a disease to (re)emerge is low: agent is available and easy to handle by professionals and labs but has a low spread score moderate: the role of bioterrorism as a driver for a disease to (re)emerge is moderate: agent available and easy to handle by professionals and labs and rapidly spreads score high: the role of bioterrorism as a driver for a disease to (re)emerge is high: agent is available and easy to handle by individuals and rapidly spreads g inadvertent release of an exotic infectious agent from a containment facility, for example laboratory score score negligible: the pathogen is not currently present in any laboratory score low: the pathogen is present in a containment facility but its release is very unlikely as it is very easily contained score score score negligible: modification of the disease status due to a reduced national budget has a negligible effect on the (re) emergence of the disease in belgium score low: modification of the disease status due to a reduced national budget has a low effect on the (re) emergence of the disease in belgium score moderate: modification of the disease status due to a reduced national budget has a moderate effect on the (re) emergence of the disease in belgium score high: modification of the disease status due to a reduced national budget has a high effect on the (re) emergence of the disease in belgium decrease of resources allocated to the implementation of biosecurity measures at border controls (e.g. harbours or airports). score score negligible: decreasing the resources allocated to the implementation of biosecurity measures has a negligible effect on the (re)emergence of the disease in belgium. disease has never been detected in the past in a harbour or airport score low: decreasing the resources allocated to the implementation of biosecurity measures has a low effect on the (re)emergence of the disease in belgium. the disease has been suspected to have entered other countries because of deficient biosecurity at border controls. score medium: decreasing the resources allocated to the implementation of biosecurity measures has a moderate effect on the (re) emergence of the disease in belgium. the disease has been introduced in other countries because of deficient biosecurity at border controls score high: decreasing the resources allocated to the implementation of biosecurity measures highly increases the risk of (re)emergence of the disease in belgium. in the past, the disease has been introduced in other countries and in belgium because of deficient biosecurity at border controls most likely influence of (il)legal movements of live animals (livestock, pets, horses, etc.) from neighbouring/european union member states (ms) for the disease to (re)emerge in belgium. (continued) h influence of increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states for the disease/pathogen to (re)emerge in belgium score score negligible: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a negligible influence on the pathogen/disease (re)emergence in belgium. score low: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased (il)legal imports of animal subproducts such as skin, meat and edible products from eu member states have a high influence on the pathogen/disease (re)emergence in belgium. most likely influence of increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states for the disease/pathogen to (re)emerge in belgium. score score negligible: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a negligible influence on the pathogen/disease (re)emergence in belgium. score low: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased (il)legal imports of non-animal products such as tires, wood, furniture from eu member states have a high influence on the pathogen/disease (re)emergence in belgium. most likely influence of (il)legal movements of live animals (livestock, pets, horses, etc.) from third countries for the disease to (re)emerge in belgium. most likely influence of increased imports of animal subproducts such as skin, meat and edible products from third countries, for the disease to (re)emerge in belgium. score score negligible: increased imports of animal subproducts such as skin, meat and edible products from third countries have a negligible influence on the pathogen/disease (re)emergence in belgium. score low: increased imports of animal subproducts such as skin, meat and edible products from third countries have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased imports of animal subproducts such as skin, meat and edible products from third countries have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased imports of animal subproducts such as skin, meat and edible products from third countries have a high influence on the pathogen/disease (re)emergence in belgium. a p p e n d i x a (continued) h most likely influence of increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries, for the disease to (re)emerge in belgium. score score negligible: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a negligible influence on the pathogen/disease (re)emergence in belgium. score low: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a low influence on the pathogen/disease (re)emergence in belgium. moderate: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a moderate influence on the pathogen/disease (re)emergence in belgium. high: increased (il)legal imports of non-animal products such as tires, wood, furniture from third countries have a high influence on the pathogen/disease (re)emergence in belgium. number of criteria = , hence points to be distributed within this domain for the intra-domain weighing. a p p e n d i x b list of experts enrolled (n = ) in the phase i (questionnaire assessment) with their gender, affiliation, country and field of expertise the schmallenberg virus epidemic in europe- - 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( , . ) , . epizootic haemorrhagic disease african swine fever key: cord- - td a authors: lazcano-ponce, eduardo; allen, betania; gonzález, carlos conde title: the contribution of international agencies to the control of communicable diseases date: - - journal: arch med res doi: . /j.arcmed. . . sha: doc_id: cord_uid: td a although inequality is often measured through three critical indicators—education, income and life expectancy—health-related differences are also essential elements for explaining levels of equality or inequality in modern societies. investment and investigation in health also involve inequalities at the global level, and this includes insufficient north-south transfer of funds, technology and expertise in the health field, including the specific area of communicable diseases. globally, epidemics and outbreaks in any geographic region can represent international public health emergencies, and this type of threat requires a global response. therefore, given the need to strengthen the global capacity for dealing with threats of infectious diseases, a framework is needed for collaboration on alerting the world to epidemics and responding to public health emergencies. this is necessary to guarantee a high level of security against the dissemination of communicable diseases in an ever more globalized world. in response to these needs, international health agencies have put a number of strategies into practice in order to contribute to the control of communicable diseases in poor countries. the principle strategies include: ) implementation of mechanisms for international epidemiologic surveillance; ) use of international law to support the control of communicable diseases; ) international cooperation on health matters; ) strategies to strengthen primary care services and health systems in general; ) promotion of the transfer of resources for research and development from the north to the south. many obstacles to the improvement of global health persist in the st century, something that is evident in the large degree of health-related inequality between rich and poor countries. infectious diseases constitute the second cause of death worldwide, are an incalculable source of human suffering and cause immense economic loss at every level. in point of fact, % of all deaths worldwide and % of the global burden of disease can be attributed to infectious diseases ( figure ). unfortunately, not only are the majority of these deaths potentially preventable, but over % occur in developing countries, where poverty is often the common denominator. the three infectious diseases that cause the largest number of deaths are hiv/aids, tuberculosis and malaria. against this background, the need to achieve a greater degree of social justice and uphold the human rights of the populations of poor countries is the principle justification of north-south assistance related to emerging and re-emerging infectious diseases ( ) . in this context, there are a number of strategies that international health agencies have used in order to contribute to the control of communicable diseases in poor countries. this essay reviews these efforts, beginning with the mechanisms created to facilitate international epidemiologic surveillance. the use of international health law as a tool for global collaboration on the control of communicable diseases is also touched upon. next, international cooperation on a range of health matters and efforts to strengthen primary health care and health systems in general in poor countries are referred to. finally, a discussion is included about how international agencies facilitate the north-south transfer of resources for research and development and contribute to the implementation of these investments in the areas of research, surveillance systems and improvements to health. diseases that are rapidly spread, including a number of emerging and re-emerging infectious diseases, require surveillance systems with a high degree of sensitivity, which are also opportune. surveillance systems with these characteristics allow rapid decision making and action to stop an outbreak from growing or to control an epidemic. given the above, a new paradigm for global collaboration has been developed based on the establishment of surveillance networks at the international level. epidemics and outbreaks in local regions can represent an international public health emergency. although such situations require a global response, there is no single institution with the ability to guarantee health safety worldwide. therefore, collaboration by international agencies, national governments and individuals with pertinent expertise is required. collaboration in this area has included the establishment of the networks for surveillance of emerging infectious diseases, three regional structures operating in latin america with the support of the pan american health organization (paho) ( ) . these networks carry out epidemiologic and laboratory-based surveillance of emerg-ing and re-emerging infectious diseases in the amazon, central american and the southern cone regions and provide a forum for information exchange, cooperation on capacity building and collaboration on quality control mechanisms, all aimed at the prevention or control of epidemics ( ). another initiative in this area is the global outbreak alert and response network (goarn), which was established in under the guidance of the world health organization (who). goarn provides a technical, multidisciplinary response to outbreaks and epidemics with a global outlook. this network aims to improve coordination of international responses to situations involving emerging and re-emerging infectious disease by focusing its actions on technical and operational support for national or regional efforts. goarn assists countries in actions targeted at disease control by ensuring rapid technical support; investigation and risk calculation in epidemics; controlling outbreaks of diseases with the potential for spreading rapidly; providing technical advice and guidance; carrying out epidemiologic research; advising on clinical management issues; confirming laboratory diagnoses; handling dangerous pathogens; and giving logistic support and sending supplies (drugs, vaccines, reagents, medical equipment). goarn constitutes a global resource that guarantees rapid access to experts and necessary operational resources for infectious disease control (see box ) ( ). traditionally, international law has been a central tool in the global surveillance of communicable disease. throughout the th century, international law was decisive for coordinating quarantines in different european countries, which were not coherent from one nation to the next. international legislation and norms have contributed to the exchange of epidemiologic information about infectious diseases in diverse geographic areas, justification of the establishment of international health organizations, and support for epidemiologic surveillance systems for communicable diseases. as the world enters the st century, communicable diseases continue to stretch the limits of global health policy, carried out through the use of legally binding instruments and voluntary norms. such legislation is discussed and adopted within the framework of multilateral institutions such as the who, world trade organization (wto), food and agriculture organization of the united nations (fao) and the world organization for animal health (oie). international law has constituted an indispensable tool for the protection and promotion of health in the context of globalization. international legislation has also been useful in the application of global health policy aimed at reducing human vulnerability to mortality and morbidity due to communicable diseases ( ) . the international health regulation, and particularly global regulations related to the control of infectious disease, have not been significantly modified since its proposal in . therefore, the who and paho have been involved in coordinating the review and modification of the international health regulation, which constitutes a series of guidelines for cooperation by countries on the control of disease outbreaks ( ) . the recognition of the need for changes in the international health regulation grew out of the emergence of new infectious diseases such as severe acute respiratory syndrome (sars, see box ), ( ) as well as the resurgence of re-emerging infectious diseases which for the latin american region include cholera ( ) and typhoid fever ( ) , while in africa reemerging diseases that indicate the need for this modernization of health regulations include influenza, ( ) measles ( ) and cholera ( ) as well ( figure ). the efforts to modify the international health regulation proceed from an understanding of the issues raised by increasing globalization; specifically, public health emergencies with international repercussions. important in this respect is the need to take into account the potential for the international spread of an emerging or re-emerging disease while also measuring the repercussions for the free circulation of people and goods ( ) . the who proposal for modernization of the international health regulation includes the following: ) a mission with a stronger focus on control of infectious diseases, ) emphasis on broader health care coverage and better access to treatment schemes, ) global surveillance including data from official and non-official sources, ) strengthening of national public health systems through the establishment of comparable productivity indicators and outcome measurements, ) giving priority to the protection of human rights, ) guidelines for good health governance defined as adoption of the principles of impartiality, objectivity and transparency ( ). above all, who needs to ensure all geographic regions establish health norms and structures that facilitate the transfer to poor countries of economic and technical assistance related to health. the need for increased global capacity to deal with infectious diseases is what drives the creation of a collaborative framework for epidemic alerts and responses to public health emergencies that are of international concern. such a structure should guarantee the highest level of security against the spread of disease with the least possible interference in other globalized processes such as commerce or travel. the best way to prevent the international spread of diseases is through opportune detection of and intervention into public health threats, while the problem is still limited. this requires early detection of unusual events the largest ebola outbreak ever recorded was successfully controlled in uganda in through the efforts of the ugandan government and an international team coordinated by the global outbreak alert and response network (goarn). more than ebola cases were isolated and treated and , contacts were tracked. after initial containment of the outbreak, follow-up was carried out through a community-based early warning surveillance system, establishment a field laboratory and creation of an isolation ward. goarn provided logistic support and coordination in the field, which made quick and effective control of the outbreak possible ( ). through national epidemiologic surveillance and international coordination as part of an effective response to public health emergencies of international importance. when common challenges exist, common strategies are necessary to find solutions, as is sharing high-quality information in order to provide effective, evidence-based responses. globalization has had multiple repercussions on international health, including the dissemination of certain infectious and vector-borne diseases, greater reach for bioterrorism and new health behaviors, among others. against this global backdrop, cooperation among countries would seem the best way to ensure worldwide progress in public health matters ( ) . however, international-and specifically north-southcooperation on actions to promote greater health should not be limited to the control of communicable diseases. particularly in many poor countries, it is important to reduce the burden of illness or ill health related to malnutrition ( ) . at times this may imply the existence of conflicting health priorities. international agencies and national institutions and governments will have to decide how limited resources can best be invested to achieve the greatest gains in the fight against ill health, be they through the control of communicable diseases or in the fight against malnutrition ( ) . of course, international cooperation on health issues cannot be the sole responsibility of poor countries; instead, developed and developing nations must collaborate. developed countries should commit to resolving global problems, making an effort to recognize precisely their global qualities even when originally they are located (in geographic terms) in developing countries. on their part, the less-developed nations should work towards guaranteeing the sustainability of their health policies. a specific proposal for dealing with issues such as these is the establishment of a global research council, which would contribute to making action-research more efficient as well as promoting faster uptake of new applied knowledge in the public health field. in general, in order to reach these goals, north-south collaboration is essential ( ) . the initiative for global eradication of poliomyelitis in has various lessons to teach us about international cooperation. in the first place, each goal should be defined based on strategies that are technically feasible for large geographic areas. secondly, before a strategy is implemented, an informed, collective decision should be negotiated and a consensus reached. in addition, financial risk should be minimal while the possibilities for implementation in a short time period should be maximized. finally, global health interventions should take into consideration the available infrastructure within the local health systems and ensure sufficient resources-financial and in terms of health care systems-as was the case in the eradication of poliomyelitis ( ) . there is a lack of efficacy in existing measures for stopping the spread of communicable diseases among countries. in order to create a foundation upon which to build communicable disease control strategies, to start with, health system infrastructure in developing countries must be strengthened (table ). this will involve the continual development of institutional capabilities for early detection and efficient and opportune intervention in emergencies linked to epidemics. for too long, many international agencies have given priority to other matters, including managerial capacity. although these issues may be important, the end result has been the postponement of support for improving primary care. therefore, international support for the control of communicable diseases should begin to include resources for strengthening local health systems ( ) . although some aspects of health problems, priorities and policy have become global, most of the responsibility for communicable disease control continues to be exercised at the local level. therefore, a network for the ''global public good'' has been proposed to improve communicable disease control in developing countries. this initiative proposes that failures and omissions in collective efforts to control communicable diseases can be overcome through the following actions: a) providing additional or matching funds to those offered at the local level; b) promoting investment by developed countries in the health systems of less developed nations; c) offering joint strategies for the global control of communicable diseases; and d) guiding the political process that will establish mechanisms for financing global communicable disease control programs ( ) . in this context, an alliance of a number of agencies has been formed, including the united nations, the governments of developing countries, governmental donors in developed countries, private foundations and corporations and non-governmental organizations. the goal is to mobilize, manage and distribute additional resources for the control, to begin with, of hiv/aids, tuberculosis and malaria. one high priority in the use of funding is the purchase of vaccines. however, there is still no consensus on implementing a strategy for financing and improving the health services of poor countries ( ) , and this will be something that should receive priority in the near future. research, development and funding priorities in the health field vary greatly in different parts of the world (particularly developed vs. underdeveloped), something which is linked to the insufficient north-south transfer of investment in health. one of the reasons this situation exists is simply that communicable diseases make up a much larger proportion of the burden of disease in underdeveloped countries as compared to developed ones (see box ) ( ) . a related problem is that research priorities are different in richer countries where chronic diseases are a priority than in poorer nations where infectious diseases and malnutrition are of greater concern. even when chronic diseases constitute an important proportion of the burden of disease in developing (often middle-income) countries, research needs may be different from those in developed nations. specifically, interventions to prevent chronic diseases, or to improve adherence to treatment once acquired, which may be successful in developed countries can be either not feasible or inappropriate (in cultural, social or economic terms) in developing nations. in addition, the development of vaccines in developed countries, for the control of communicable diseases, can be of little use in poorer nations, where they may be ineffective given the existence of different viral strains or bacterium. quality of health care services is quite heterogeneous from one country to another, both when comparing developing countries with one another or with their developed country counterparts, which again implies different research and funding needs. finally, the high cost of patented medicines and medical technology limits their transferability from richer to poorer nations. the new health environment is highly complex and therefore the proposals being made to improve it are extremely heterogeneous ( ) . certain international agencies have contributed to successful inter-institutional and international collaboration on scientific capacity building, joint research programs and technology transfer. to establish the basis for north-south discussion and transfer of resources and technology, as well as south-south collaboration, these successful examples will need to be examined and learned from. the identification of the necessary conditions for developing sustainable research, control efforts and health services will also be essential elements in the control of communicable diseases. recently there has been growing interest in the study of how priorities for investment in health research are established in different parts of the world ( ) . there are a number of perspectives from which an analysis of these issues can be carried out, including a focus on the economic, health or human rights aspects of priority setting. in economic terms, there has been an increase in investment in health research, from billion usd in ( ) to . billion usd in ( ) . the health sector generates trillions of dollars at the global level; among the products and services to be invested in are prevention of disease and health promotion, as well as diagnosis and treatment. the world bank is the agency that provides the largest amount of health-related financing worldwide, at close to one billion usd each year. the world bank's principal health-related aims are to contribute to the improvement of the health of the poor and to the reduction of the impoverishing effects of disease, as well as increasing equitable access to health care and promoting sustainable financing for health systems ( ) . the . billion usd provided by the world bank for the fight against hiv/aids in recent years constitutes an example of how the emergence of a public health problem-specifically an emerging disease-can lead to the creation of new investment priorities in terms of health research, prevention activities and treatment. human development can be measured through three critical indicators: education, income and life expectancy, all of which interact in complex ways with health. the large north-south differences can be expressed in terms of these four elements and their reciprocal influences. these indicators would seem to indicate a lack of equity in the way health research priorities are established, which in turn translate into insufficient north-south transfer of applicable after antibiotics began to be used to treat tuberculosis patients in developed countries beginning in the s, the control programs for this disease underwent radical transformations in these nations and tuberculosis incidence and mortality rates declined steadily in the industrialized world. the developed nations began to ignore the disease, and resources available to developing countries for dealing with it dried up. the treatment options used in developed nations were unaffordable for developing countries, where instead strategies such as ambulatory care and passive case detection were preferred in order to lower treatment costs and avoid expensive mass screening. studies carried out in developing contexts produced useful schemes for other resource poor settings. investigation undertaken in india confirmed the effectiveness of treatment of tuberculosis in patients' homes and provided alternatives to costly mass screening. research supported by the ministry of health of tanzania provided the groundwork for the development of dots (directly observed treatment shortcourse), which is now the leading global intervention against tuberculosis. however, these research results were not applied in many poor countries, given the almost total absence of tuberculosis on the international health agenda and especially the lack of funding. it was not until tuberculosis incidence began to rise in developed countries such as the united states and a number of european nations in the s that international concern was again focused on this disease, including resources. at this juncture, the world bank made tuberculosis a priority and provided loans for the implementation of who-dots, after which countries adopted this scheme ( ) . health research results, technology and health investment. among the many reasons offered to explain this situation is that in rich countries most infectious diseases are not endemic, as opposed to the reality of many poor countries, where emerging infections (such as hiv/aids) and reemerging diseases (such as malaria, tuberculosis or cholera) are priority public health issues (see box ). perhaps the most heart-wrenching example of this is the fact that epidemiologic surveillance and especially therapeutic interventions for hiv/aids are not available in poorer countries, where the large majority of people living with the disease are concentrated ( % of people living with hiv/ aids reside in developing countries, and only % of the million people living with the disease in resource poor areas received antiretroviral treatment in ( ) . a communicable disease has been controlled if through public policy the spread of an infectious agent is restricted to its pre-epidemic status, which is to say that the epidemic has been reversed. on the other hand, a communicable disease is eliminated if it is sufficiently controlled to prevent the occurrence of an epidemic in a specific geographic area. control and elimination are achieved locally, but a disease is eradicated only if it has been eliminated in all geographic regions. thus, eradication is clearly the most difficult goal to achieve, although it has large advantages over control. the economic effects of eradication can be extremely favorable in that it not only reduces the infection but also eliminates the need for future vaccination efforts. eradication generally becomes feasible, from an economic point of view, when a disease is first eliminated in one or more of the richer countries. the incentives for participation by the poorer countries in eradication initiatives begin with the existence of an international control program, which allows them to take advantage of financial support for elimination efforts ( ) . to promote and facilitate participation by developing and developed countries in epidemiologic surveillance systems, as well as initiatives for the control, elimination or eradication of communicable diseases, poor countries need to develop their capacity for early detection, dissemination of precise and high quality information and a high degree of transparency ( ) . all countries should adhere to international regulations, including the international health regulation and those of the world trade organization. finally, developed countries should provide financial and technical support for countries undergoing emergencies linked to communicable diseases, because globally this is the only way they can guarantee the safety of their own populations and ensure the usefulness of their internal health-related investments. ultimately, developed countries should also share expertise, technology, and funds as a contribution to social justice, because health is a fundamental human right. infectious diseases, non-zero-sum thinking, and the developing world international law and communicable diseases el boletín de la organización panamericana de la salud, noviembre eid updates: emerging and reemerging infectious diseases eid weekly updates: emerging and reemerging infectious diseases, region of the americas eid weekly updates: emerging and reemerging infectious diseases gostin lo. international infectious disease law: revision of the world health organization's international health regulations globalization and disease: in an unequal world, unequal health a decade of child health research in developing countries the emergence of global disease control priorities global public goods and health: taking the agenda forward global health goals: lessons from the worldwide effort to eradicate poliomyelitis district health systems in a neoliberal world: a review of five key policy areas communicable disease control: a 'global public good' perspective a global health fund: a leap of faith? the burden of disease among the global poor public health. grand challenges in global health commission on health research for development. health research: essential link to equity in development global forum for health research. monitoring financial flows for health research. geneva: global forum for health research economics, health and development: some ethical dilemas facing the world bank and the international community resumen mundial de la epidemia del vih/sida: diciembre de eradication versus control: the economics of global infectious disease policies impediments to global surveillance of infectious diseases: consequences of open reporting in a global economy sars vaccine development after the initial efforts to eradicate malaria through the use of ddt beginning around the mid-twentieth century, although some areas achieved important reductions in malaria incidence and mortality, frequent resurgences of endemicity continued in a number of poor countries. the initial eradication strategies failed for a number of reasons, including the development of resistance to ddt, a lack of high quality epidemiological knowledge and managing capacity as well as the inadequacy of the health care systems that existed in less developed countries. although for at least two decades malaria ceased to be an international priority, research continued, including the development of antimalarial drugs and attempts at vaccine development supported by such international agencies as the who, the world bank, the united nations development programme (undp) and usaid. during the last decade of the twentieth century a different set of strategies was developed, including evaluation of national malaria situations by a number of african countries and joint efforts to strengthen malaria control programs by african governments and the who, which were financed primarily by the world bank but also with resources from g countries, the european commission and development banks. it was this transnational alliance of international agencies and national governments and a more complex and realistic appraisal of the work needed to control malaria (instead of relying on a single strategy, ddt use) that constituted perhaps the most important difference between these two phases in the fight against malaria ( ) . key: cord- -yp ta o authors: nacul, luis; o'boyle, shennae; palla, luigi; nacul, flavio e.; mudie, kathleen; kingdon, caroline c.; cliff, jacqueline m.; clark, taane g.; dockrell, hazel m.; lacerda, eliana m. title: how myalgic encephalomyelitis/chronic fatigue syndrome (me/cfs) progresses: the natural history of me/cfs date: - - journal: front neurol doi: . /fneur. . sha: doc_id: cord_uid: yp ta o we propose a framework for understanding and interpreting the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (me/cfs) that considers wider determinants of health and long-term temporal variation in pathophysiological features and disease phenotype throughout the natural history of the disease. as in other chronic diseases, me/cfs evolves through different stages, from asymptomatic predisposition, progressing to a prodromal stage, and then to symptomatic disease. disease incidence depends on genetic makeup and environment factors, the exposure to singular or repeated insults, and the nature of the host response. in people who develop me/cfs, normal homeostatic processes in response to adverse insults may be replaced by aberrant responses leading to dysfunctional states. thus, the predominantly neuro-immune manifestations, underlined by a hyper-metabolic state, that characterize early disease, may be followed by various processes leading to multi-systemic abnormalities and related symptoms. this abnormal state and the effects of a range of mediators such as products of oxidative and nitrosamine stress, may lead to progressive cell and metabolic dysfunction culminating in a hypometabolic state with low energy production. these processes do not seem to happen uniformly; although a spiraling of progressive inter-related and self-sustaining abnormalities may ensue, reversion to states of milder abnormalities is possible if the host is able to restate responses to improve homeostatic equilibrium. with time variation in disease presentation, no single me/cfs case description, set of diagnostic criteria, or molecular feature is currently representative of all patients at different disease stages. while acknowledging its limitations due to the incomplete research evidence, we suggest the proposed framework may support future research design and health care interventions for people with me/cfs. the lack of progress in myalgic encephalomyelitis/chronic fatigue syndrome (me/cfs) research has been attributed to a range of factors, including the paucity of large, high quality, hypothesis-driven studies, and controversy around diagnosis. without recognized and validated biomarkers or diagnostic tests, there is an over-reliance on patient history for diagnosis, which is based on criteria with limited sensitivity and specificity ( ) and which ignore disease sub-groups. furthermore, the lack of consistency in the choice and application of research case definition has led to problems with reliability and comparability of research findings ( ) . an additional factor complicating diagnosis and case definition for research studies is the timerelated variation in phenotype both in the short- ( , ) and longterm ( ) , which has seldom been considered in research studies. in addition to often marked variability in disease presentation, severity, progression, and duration among different individuals, the way disease manifests in each individual may change with time. inter-and intra-individual phenotypic variations lend toward the categorization of different subtype trajectories of me/cfs that may differ in pathogenesis and prognosis. in some studies, female sex, increased age ( ) ( ) ( ) , and lower socioeconomic status ( ) have been found to predict poor prognosis; however, the variable nature of both population sampling and diagnostic criteria has led to ambiguous results and has reinforced the need for ongoing research in this area ( ) . further subtypes have been defined on the basis of "minor" symptoms i.e., musculoskeletal, infectious, or neurological ( ), through genetic studies ( , ) , metabolomics studies ( ) , and, duration of disease studies ( ), highlighting the multitude of possible ways me/cfs patients can be categorized. other studies have identified variations in symptom profiles as disease progresses; however, such results are often limited by cross-sectional study design ( ) , and/or recall bias ( ) . the breadth of subtype studies available follow a similar model of looking for patterns across patient groups at single time-points; far fewer consider longitudinal subtyping and disease progression of a single patient cohort over time. the concept of the natural history of disease is wellunderstood in public health and medicine: many, if not all, diseases are framed using this construct to formulate how they progress from a pre-illness stage to a final disease outcome, which may vary from full recovery to death. a good understanding of the disease course is vital not only for the design of preventative and intervention studies ( ) , but also to assess the timing and type of intervention that minimizes disease risk or optimizes prognosis. although there is some understanding of the natural history of me/cfs, this has been limited by problems in case definition (as above) as well as by the paucity of longitudinal studies, and in particular those that follow up individuals' preillness. a review of studies on cfs prognosis ( ) suggested recovery rates under % in adults, and an improvement rate over % for people with fatigue lasting < months. the prognosis was worse: when more stringent case definitions were used; in older people; in cases with more severe symptoms; and, in the presence of psychiatric co-morbidity. a subsequent systematic review on prognosis found a median recovery rate of %, and median proportion of people improving of . % ( ) with most reporting symptoms still present at follow-up. this conceptual paper explores the long-term course of me/cfs and how presentation and pathophysiological abnormalities may vary with time. the pathophysiological concepts discussed are based on evidence from clinical observations and research, where available, and, as such, are not claimed to be original or indeed conclusive. instead, they serve to highlight our proposed characterization of me/cfs's distinct stages within the framework of the natural history of the disease. prior to exploring the course of me/cfs, we propose to revisit some concepts related to mechanisms of disease that have been used in the context of life-threatening emergencies and to potential return to homeostasis, such as those occurring in sepsis or poly-trauma. although very different to me/cfs, these acute injuries have been extensively studied, and the high intensity and speed of events result in changes that are easily identified and well-described, from potential homeostatic failure to recovery. we present the following models as a paradigm for the understanding of disease mechanisms, based on well-studied examples. they merely serve as a reference for mechanisms that the host may partially engage with in the presence of insults of different severities. hence, in the following paragraphs, we explore the pathophysiological mechanisms that may be taking place in me/cfs, which have been related to abnormal homeostasis guided by these established disease descriptions. the response to an insult frequently involves multiple bodysystems and has components that are independent of the etiology of the insult and, to some extent, its severity. there are many commonalities between the response to sepsis and to polytrauma: both are acute and severe insults, to which many of the aspects of the host response are indistinguishable. our proposal is based on the idea that there may be some similar mechanisms at play when individuals predisposed to me/cfs are faced with a range of "insults" or "stressors." needless to say, the hyperacute changes and co-factors in both sepsis and poly-trauma occur in very rapid sequence, whereas in me/cfs, physiological changes, even if they resemble those of acute injury in some respects, take place at a much slower pace with less obvious and uniform patterns. in both sepsis ( ) and poly-trauma, ( , ) a state of hyperinflammation is observed initially as the host responds to the infection or traumatic stress with marked production of pro-inflammatory mediators, e.g., cytokines and polypeptides. a failing circulatory system is associated with activation of the hypothalamic-pituitary-adrenal (hpa) axis and increased sympathetic drive, contributing to metabolic changes and to increased energy expenditure ( , ) . in these conditions, the acute pro-inflammatory state is usually followed by a compensatory anti-inflammatory response, with a different profile of biochemical and molecular mediators. the success of the host in balancing pro-with anti-inflammatory responses alongside injury-related factors, are key to improved long-term outcomes. the direct and indirect effects of immune cells and active products derived from immune, neural, and endocrine systems (some of which cause pathology if present in excess) contribute to a number of physiological changes, including those leading to the formation of reactive oxygen species (ros, oxidative stress) and reactive nitrogen species (rns, nitrosative stress). endothelial and parenchymal (organ) cell damage may result because of a combination of factors, such as polymorphonuclear leukocyte infiltration and the action of reactive oxygen and nitrogen species, cytokines, vasoactive amines, and other products. endothelial dysfunction results in capillary leakage, accelerated inflammation, platelet aggregation, coagulation, and loss of vascular tone ( ) . vascular dysfunction is associated to peripheral vasodilation due to increased nitric oxide and prostacyclin synthesis ( ) and to a decrease in the proportion of perfused vessels and an increase in the heterogeneity of blood flow distribution ( ) . this results in relative hypovolemia, decreased capillary flow, haemoconcentration, and micro-thrombi formation, and further contributes to reduced exchanges of oxygen and nutrients at the microcirculatory level. the consequent decreased cellular oxygen delivery eventually leads to cytopathic hypoxia. adenosine triphosphate (atp) increased consumption and ensuing deficits cascade into a range of metabolic disturbances with systemic effects ( ) , and promote changes in membrane permeability that lead to dysfunctional transmembrane ion transport. in acutely and severely ill patients, reperfusion results in further oxidative damage ( , ) . additional failures of biological and cell processes lead to multiple dysfunction, to system and organ failure, and to potentially irreversible disease ( ) . concepts that are relevant here are those of homeostasis and allostasis. while homeostasis refers to the "stability of physiological systems," allostasis has been defined as "the adaptive processes aimed to maintain homeostasis following acute stress, and which contribute to wear and tear on the body and the brain, or allostatic overload" ( ) . a central characteristic of individuals with me/cfs points to a state of homeostatic failure ( ) , aggravated by the incidence of, or increase in, levels of new stressors or by the increase in allostatic load ( ) . typical stressors include infection [( ): [ ] [ ] [ ] [ ] [ ] , physical exertion and cognitive effort (e.g., reading or solving mental puzzles) triggering post-exertional malaise (pem) ( ) , comorbid conditions (e.g., sleep disturbances) ( ) and a range of environmental and individual factors ( ) ( ) ( ) ( ) ( ) ( ) . in those who do not develop me/cfs or prolonged illness following an insult such as an acute infection, external stressors may initially cause physiological changes accompanied by nonspecific symptoms, but the state of homeostatic equilibrium that operated before the insult is quickly restored. failing reestablishment of this equilibrium, there may be a shift to a state of "aberrant homeostasis, " where physiological processes converge to a new or alternative state of functioning; a state that remains homeostatic in nature, but functions at a less optimum level ( ) . while such a state may be adequate for many physiological processes, it will be inadequate or inefficient for a number of other processes and functions and the prolongation of such aberrant functioning will represent another potential source of ongoing stress. there is a growing body of evidence on biological abnormalities in me/cfs that has been reviewed elsewhere ( , , ) , and summarized by komaroff ( ) . of note, many of the abnormalities shown in severe injury have also been identified in me/cfs such as: immune dysfunction, including pro-inflammatory response (especially at early stages of disease) ( , ); autonomic nervous system ( ) ( ) ( ) ; hpa axis dysfunction ( ); hypovolemia ( ); nitrosamine and oxidative stress ( ); endothelial dysfunction ( ); metabolic dysfunction ( - ); dysfunction of membrane transport ( ) ; and, tissue hypoxia ( ). other tools widely used in clinical medicine are staging systems. using sepsis again as an example, such a system was proposed at the international sepsis definitions conference in to introduce the stratification of patients with sepsis ( ). by applying piro (predisposition, infection/insult, response, and organ dysfunction) patients are stratified into appropriate subgroups allowing for more accurate prognostication in emergency medical services ( ) . the idea of classifying people with me or cfs into distinct categories or stages has been explored previously by several theorists. one school of thought proposes categories based on the psychological process of coming to terms with this new and evolving state of health rather than addressing biological differences, and are defined as such by the emotions common to any trauma experience: e.g., denial, fear, frustration, and acceptance ( , ) . alternatively, schweitzer ( ) proposes the different presentations of cfs according to more physical categories (prodrome, relapse and remission, improvement and plateau, and collapse followed by slow worsening with no remission); it is these that we aim to expand on, as follows. we show a tentative representation of the key pathophysiological mechanisms operating in each stage of me/cfs in figure . as in severe injury or sepsis, the range and order of occurrence of biological processes taking place in me/cfs may vary, as may their relative significance and impact on each individual. therefore, it is important to note that although the various abnormalities may occur continuously and often simultaneously, the predominance of furthermore, we propose a characterization of disease stages in me/cfs, based on the natural history of disease framework considering available descriptions from the literature ( ) , and the life-stories reported by our own cohort of research participants with me/cfs (including those with mild/moderate or severe symptoms) ( ) . this characterization is summarized in table , which may be used in support of research designs that consider the disease presentation in distinct phases. individuals with a combination of genetic predispositions and exposures to environmental factors may first manifest symptoms of me/cfs following their encounter with a specific trigger, of which acute infections of various etiologies are the most commonly reported ( , ) ; other patients report a more insidious onset with no obvious initiating factor ( ) . while it remains unclear exactly which individuals are predisposed to develop me/cfs and why, some patterns have emerged. for example, gender-and age-specific factors are thought to contribute to the risk of me/cfs ( ) , with epidemiological studies consistently reporting higher rates of the disease in females ( , ) . although most cases are endemic, there have been reports of epidemic cases, suggesting an infectious or other environmental cause play a role ( , ( ) ( ) ( ) ( ) ; although discrepancies in onset patterns and case definitions make these epidemics difficult to compare ( ). many studies have reported an association between acute viral infection and the development of me/cfs ( ) ( ) ( ) ( ) . cases are predominantly reported in north america, europe, and oceania; however, the occurrence of me/cfs is thought to be global with evidence of cases in other parts of the world ( ) ( ) ( ) . psychiatric morbidity, experiences of stress and trauma, either physical or emotional have been reported to precipitate the disease ( , ( ) ( ) ( ) and to predict disease progression ( ) , under the explanatory biopsychosocial models. however, these models have not been replicated ( , ) . furthermore, chu et al. ( ) found that even when a significant proportion of their research population report stress or a major life event as a precipitating factor for me/cfs, "stressful events were rarely chosen as the only precipitant though, endorsed only by % of our subjects, and appeared mostly in conjunction with infection or other precipitants." we acknowledge that stress may play a role in the development and perpetuation of me/cfs through its role on the immune system and hpa axis dysfunction ( ), or by aiding transmission or reactivation of viral infections ( ), or as a consequence of the loss of normal functioning experienced by the individual. the role of genetic variation has been supported by a number of family-based studies assessing the possibility of a heritable component ( ) ( ) ( ) . genes underpinning immune system function and inflammatory response may contribute to genetic susceptibility for me/cfs; some studies suggest associations with human leucocyte antigen class ii alleles ( , ) and in genes related to the complement cascade, chemokines, cytokine signaling, and toll-like receptor signaling ( ) . small genome-wide association studies (gwas) have had little overlap in results save for two snps in the grik gene: a gene implicated in a number of neurological conditions such as autism and frontiers in neurology | www.frontiersin.org is based on reviews on prognosis ( ) . ¶ the prevention level will be considered further in a subsequent publication which is being prepared by the authors. schizophrenia ( ); in the grik gene: relating to a pattern recognition receptor capable of binding to a broad range of pathogens; and in the non-coding regions of t-cell receptor loci ( ) . a further study reported snp markers in candidate genes involved in hpa axis function and neurotransmitter systems that distinguished individuals with me/cfs ( ). it is important to preface here that, with the current diagnostic methodology of me/cfs stipulating the presence of symptoms for more than months ( , ) and the absence of a positive validated diagnostic test, the following processes (occurring prediagnosis) are difficult to substantiate from existing biomedical research. however, based on the published work on me/cfs and considering the pathophysiological events happening in sepsis and polytrauma may be similar (though in a much slower pace), we hypothesize that the following may occur. in addition to any manifestations specifically related to the acute insult or triggering event, the mechanisms involved in producing the first symptoms of me/cfs may be similar to what has been described in relation to "sickness behavior" ( ) or in those with severe acute disease, i.e., "systemic inflammatory response syndrome" ( ) . these result from the interaction of an infective agent or other insult with the host's immune system, as well as their potential effect on the host's central nervous system (cns). the immune system-nervous system interactions involve bidirectional signals ( - ): while immune system activity may interfere with cns function via various mechanisms, e.g., release and action of pro-inflammatory cytokines and other mediators, various neurotransmitters, neuropeptides, and neurohormones may also affect immune function. additionally, the hpa system and the autonomic nervous system (ans) are affected, with consequences that may be observed well-beyond the cns. these effects may vary according to different factors, such as host susceptibility, the nature and persistence (or return to normality) of systemic and local immune dysfunction, altered cns metabolism, neuro-transmission, brain perfusion changes, and the integrity of the blood-brain barrier ( ) ( ) ( ) ( ) . particular characteristics of the specific infectious agent or stressor may also play a role during this prodromal stage, which would explain the different risks of disease development following acute infection. for example, there has long been an interest in the association between me/cfs and infections such as epstein-barr virus (ebv) and other herpesviruses ( , ( ) ( ) ( ) ( ) ( ) ( ) . herpesviruses tend to be neurotropic and persist following acute infection in a latent state. similar to ebv infection ( ) , the risk of chronic fatigue has been shown to be substantially increased following viral meningitis, a relatively severe infection of the cns ( ). early disease represents a continuation of the processes initiated at the prodromal period, when there is a failure of physiological and homeostatic processes to resume previous levels of equilibrium and normality. fatigue and other symptoms may be largely explained by a combination of the local and systemic effects of pro-inflammatory and other mediators or toxins, cns metabolic dysfunction (with enhanced excitability and other changes), and a systemic hyper-metabolic state. with higher energy demands for essential biological processes, there will be a reduction in the available energy for less essential tasks, including those demanding increased physical or mental exertion. the increased production and action of anti-inflammatory mediators, as well as their ability to counter-balance pro-inflammatory stimuli, modulate physiological responses, and symptoms and affect disease progression or reversibility. as mentioned previously, without a validated biomarker to diagnose me/cfs early it is difficult to substantiate the exact mechanisms occurring in the early disease phase. research into potential diagnostic markers, such as the recent study on impedance signatures ( ) , are crucial not only clinically, but to identify these mechanisms as possible targets for early intervention. the persistence of immune and cns dysfunction with the initial over-production of pro-inflammatory and neurotoxic factors may result in a prolonged state of low-grade neurological and systemic inflammation. in the cns, a status of glial activation with microglial hypersensitivity to peripheral ( ) and regional stimuli is established ( , ( ) ( ) ( ) , akin to what has been described in chronic pain states ( ) . in support of cns dysfunction, neuroimaging studies have shown various abnormalities in me/cfs, often associated with symptoms of fatigue and other indications of severity ( ) . glial activation in several areas of the brain has also been demonstrated in positron emission tomography (pet) scans of patients with fibromyalgia (fm), compared to controls, which was correlated to the severity of fatigue ( , ) . neuro-glial bidirectional signaling is associated with increased production of neuro-excitatory neurotransmitters and immune-inflammatory mediators ( ). nervous system dysfunction affecting parts of the brain, brain stem, and ans, could explain not only the encephalopathic or neuro-cognitive type of symptoms, but also those resulting from disruption of key central regulatory mechanisms, such as those involved in endocrine, circulatory, thermoregulation, and respiratory control ( , , , , ) . examples of these include intolerance to extremes of temperature, chills and temperature variations, intolerance to exertion, hyperventilation or irregular breathing, orthostatic intolerance, with hypotension or postural orthostatic tachycardia, and other symptoms related to autonomic and endocrine control function ( ) . among the various by-products produced as a consequence of ongoing abnormalities, are highly ros and nitric oxide synthase (nos) or free radicals, which affect cell signaling and cell functioning and structure, particularly when present at high levels. it has been hypothesized that free radicals, and increased levels of nitric oxide and peroxynitrite in particular, play a significant role in me/cfs ( , ) ; their links to immune and neuro signaling, cell integrity, mitochondrial function, and energy metabolism may play an important part in the long term abnormalities in me/cfs. the nature of neuro-immune and other dysfunctions may change as disease progresses. while a pro-inflammatory state is typical of the early response to insults, immune abnormalities may become less marked (and less pro-inflammatory) with time ( ) , and patients with longer periods of illness may show fewer inflammatory immunological abnormalities. in support of this, our preliminary results from the analysis of over me/cfs patients participating in the uk me/cfs biobank (ukmeb), showed that the reported time since disease onset was significantly associated with cytokines, namely scd l and il ra (manuscript in preparation). these results were found after aliquots of peripheral blood mononuclear cells (pbmc) from participants were stimulated (i.e., subjected to an infection resembling stimulus) and analyzed with magpix r multiplexing system. the statistical analyses were conducted after transforming each cytokine measurement to the logarithm scale to approximate normality; linear regression of these logtransformed values (adapted for truncated outcome variables to account for the assay's limits of detections) was applied to the variables' time since onset, level of severity (mild to moderate vs. severe) and the interaction between severity and time since onset, while also adjusting for age and sex. the results evidenced a decrease of scd l-a pro-inflammatory cytokine-and an increase of il ra-an anti-inflammatory cytokine-for every additional year since onset of me. as the disease progresses, physiological, and systems abnormalities take their toll and cell dysfunction becomes more pronounced. endothelial dysfunction may arise as a consequence of a range of factors, including, but not limited to, persistent oxidative and nitrosative stress and circulatory dysfunction ( , , , , ) . the associated reduced delivery of oxygen and nutrients to the cell leads to a deterioration of cell function and impaired energy metabolism ( , , ) and a decreased ability of the cell to extract oxygen and produce energy, a condition known as cytopathic hypoxia. as suggested by naviaux et al. ( ) , in cases of me/cfs with mean duration of symptoms over years, there is a shutting down of various metabolic processes leading to a hypometabolic state, i.e., a move to an energy-saving mode. at this stage, symptoms are likely to be severe, with profound fatigue, intolerance to effort, pem and other systemic symptoms, which are largely explained by the slowness of physiological and metabolic processes and decreased energy production. it is unknown how the initial host response to a stressor or insult compares in individuals who do or do not develop typical symptoms of me/cfs. however, the return to good health, which happens to most people following exposure to mild or moderate levels of insult, seems to be impeded in me/cfs when symptoms persist for longer than - months; the time interval that is featured in some of the currently used diagnostic criteria ( , , ) . this suggests that subsequent mechanisms involved in the host response will differ at some point in those who develop me/cfs from those who regain full health. therefore, a key question is what determines full recovery? or alternatively, what determines the perpetuation and transformation of symptoms? while the abnormalities observed in acute disease are general and mostly reversible once the challenge from the stressor ceases, some degree of dysfunction may persist for longer periods. the degree of reversibility of various physiological abnormalities is likely to decrease with time, and some permanent functional, and even structural, damage may occur consequently. this is likely caused by either the persistence or frequent reactivation of the initial stressor ( , ) , an accumulation of insults, a continuing dysfunctional host-response, or the effects of the numerous psychosocial risk factors that influence disease development and progression ( ) , or a combination of all of these. although our framework focuses on the underlying biological mechanisms that may be at play in the development and progression of me/cfs, it is important to acknowledge the impact of psychosocial and behavioral aspects in the progression of chronic diseases. stressors such as stressful life events, low satisfaction with social and medical support, and excessive use of coping mechanisms, have been shown to contribute to the neuroendocrine and immune responses by acting through complex pathways that ultimately affect health and health outcomes ( ) ( ) ( ) . the interplay between these three dimensions (biological, psychosocial, and behavior) has been noted in the development and the progression of a number of chronic diseases and to influence disease outcomes ( ) ( ) ( ) ( ) . the combined effects of stress from work or family life, social deprivation, and depression have been found to contribute to the risk of cardiovascular diseases, including coronary heart disease ( ) and myocardial infarction ( ) , and to a worse prognosis ( ) by enhancing cortisol secretion, increasing sympathetic activation, and elevating plasma catecholamine levels ( ) . a higher cumulative average number of stressful life events, when coping involves denial, and higher levels of serum cortisol have been found to be associated with a faster progression to aids ( ) . correspondingly, low stress levels and low scores of avoidance coping behaviors were shown to be protective against relapse in crohn's disease patients ( ) in contrast to high levels, which act as mediators, overloading the sympathetic nervous system. in the case of me/cfs, the effect of these dimensions is the same. in fact, one framework has been used to propose a model for managing patients with this disease in which it is considered that genes predispose, life events precipitate, and behaviors perpetuate ( ) ( ) ( ) . however, this model may downplay the important role of the biological mechanisms involved in me/cfs and overstate the role of psychosocial and behavioral factors ( ) . the pathophysiological distinction between cases from the milder to the more severe end of the me/cfs spectrum may relate to near-normal homeostatic regulation in milder cases, and established "aberrant homeostasis" or homeostatic dysregulation with multi-systemic consequences in moderate to severe cases. alternatively, homeostatic failure, along with variable multi-system physiological failure and increasing degrees of irreversibility, may happen in the most severe cases. the early stage of me/cfs is of variable duration but is usually considered to be between and months to years after the start of prodromal symptoms. reversibility is possible, but often people will evolve to chronicity or established me/cfs with either: (a) partial reversal of dysfunctional physiological mechanisms (mild cases with slow improvement over time); (b) persistence of dysfunctions and symptoms (mild or moderate cases with stable symptoms or slow changes over time); or (c) worsening dysfunctions and symptoms (moderate and severe cases) ( ) . note that some cases present early with severe symptoms, which not uncommonly evolve to a milder form ( ) . the use of coping mechanisms, such as pacing, can also help improve energy management in people with me/cfs over time and reduce the risk of relapse into a more severe state; however, there is little evidence that these will lead to a reversibility ( ) . there is some indication that rates of resolution are higher in cases of epidemic cfs compared to sporadic cases, although very few of these individuals will recover to their pre-morbid level ( ) . one way of thinking about these phases is as interconnected spirals, each representing a distinct disease phase. individuals may either remain for long periods in a single phase with symptoms fluctuating within the "spiral section" or move between phases either upwards (i.e., toward better health status) or downwards (i.e., toward disease deterioration). figure represents an illustration of the multi-spiraling disease course suggested for me/cfs, and shows how patients may move across spirals, with different molecular and system abnormalities. there are a number of comorbid conditions associated with me/cfs and, as such, these comorbidities can complicate diagnosis, treatment and research of the disease. comorbidities have been found in up to % of people with me/cfs (pwme) ( , ) with some developing before, with, or after me/cfs onset ( ) . the complexity of me/cfs is in part due to the number of different systems affected that contribute to the many and varied symptoms experienced. me/cfs and fm share a number of overlapping core symptoms that mean the two are commonly experienced together; fm has been reported to cooccur in - % of pwme ( , , ) . however, there is evidence to suggest the two conditions differ in their hormone dynamics, genetic/molecular biology, and autonomic function ( , ) . this is reiterated by the absence of post-exertional malaise in fm ( , ) , which is one of the key features of me/cfs ( , , , ) . sleep disturbances can cause some symptoms that are also present in me/cfs including fatigue, joint pain, and impaired cognition ( - ). additionally, as part of a bidirectional relationship, comorbid pain conditions may further impact sleep quality ( ) . sleep disturbances are also present in a number of neurological diseases ( ) , which would explain their presence as an important feature in me/cfs ( , ) ; however, differences in sleep cycle patterns and distinct sleep phenotypes suggest that me/cfs and primary sleep disorders are, in fact, different entities ( , ) with many pwme showing normal sleep study results ( ) . primary sleep disturbances are considered exclusionary for me/cfs by a number of diagnostic criteria ( , , ) , however, with little evidence that treatment of these disorders improves symptoms of me/cfs it is argued they are better considered as comorbid conditions ( , , ) . also highly prevalent in those with me/cfs is orthostatic intolerance (oi), a common multifactorial disorder commonly accompanying neurodegenerative, cardiovascular, metabolic, and renal disorders ( ) . disruptions to ans and reduced blood volume contribute to oi ( ) and the same systemic dysfunctions have been reported in those with me/cfs ( ); however, not all people with oi disorders have me/cfs ( , ) . intestinal dysbiosis thought to be associated with some cnsrelated disorders via the gut-brain-axis ( ) . ibs is another largely overlapping syndrome with both me/cfs and fm but metabolic profiles are distinct in me/cfs and me/cfs with ibs subgroups ( ) . some authors hypothesize ibs could be considered an initial symptom of me/cfs, as they reported that % of ibs patients followed up developed me ( ) . authors of a co-twin control study found significant associations between cfs and fm, ibs, chronic pelvic pain, multiple chemical sensitivities, and temporomandibular disorder. after controlling for psychiatric risk factors, they argued that these associations could not be attributed to uniquely psychiatric illness, thus suggesting a "complex interplay of genes and environmental factors" to help explain the clinical picture ( ) . while healthcare costs likely increase following the diagnosis of additional comorbidities ( ) , treating comorbidities may improve the quality of life of pwme ( ) not only symptomatically but also in what they might be able to contribute to the economy. we argue that by using the proposed natural history framework, how and when common comorbidities develop in relation to me/cfs may be highlighted, allowing researchers, and clinicians to better tailor potential interventions according to each phase, thus resulting in a more efficient management of costs. these distinct hypothetical stages may help explain the apparent inconsistency of findings from me/cfs studies, which likely include cases at distinct stages of disease with potentially diverse systems abnormalities. hence, we consider that the conceptual approach presented in this paper may help to elucidate pathophysiological mechanisms that may be more prominent at different stages of disease; and consequently, could indicate potential target therapeutic approaches in future. we argue that the different stages patients go through during the course of the disease, their severity, and the presence and degree of complications are key parameters for disease stratification. research leading to an understanding of what is occurring during the first three stages of progression to me/cfs is greatly needed but requires the recruitment of individuals for research at pre-illness stage. such research could be invaluable to understanding the biological mechanisms at play before, during and after an insult, and research using proxy disease models for me/cfs ( ) or follow up of patients after an acute viral infection [e.g., mononucleosis ( ) or more presently covid- ] could begin to address this knowledge gap. electronic health records could also be a valuable source of retrospective pre-illness data in people with me/cfs. well-designed longitudinal studies, with strict protocols, would help refine this attempted description of the natural course of the me/cfs, and the interpretation of the findings. the concept of the natural history of disease, common in the field of public health and medicine, serves to frame a disease according to how it progresses from a pre-illness stage to the final disease outcome. due to the lack of knowledge surrounding the etiology of me/cfs, the heterogeneous presentation of symptoms and their severity, and the lack of a recognized and validated biomarker to determine diagnosis, the natural history of this disease has been hard to determine. while current research efforts tend to group me/cfs subtypes according to clusters of symptoms, few studies have considered me/cfs as a continuum. pathophysiological patterns and changes along and across disease stages result in the expression of different, albeit overlapping phenotypes as seen in the preliminary ukmeb findings related to changes in cytokine levels and symptoms scores with time of disease, reported here. ignoring phenotype temporal variation in me/cfs may have an impact on the outputs and the interpretation of research investigating disease mechanisms, pathways, and interventions. this paper sought to provide a simple framework, similar to those of other chronic diseases, in an effort to extend the temporal perception of me/cfs and better incorporate the less defined pre-illness stages of the disease. we believe that by applying this framework to me/cfs research efforts could better elucidate the pathophysiological mechanisms of the disease and identify potential therapeutic targets at distinct stages. the datasets generated for this study are available on request to the corresponding author. . all biobank participants provided written consent for questionnaire, clinical measurement and laboratory test data, and samples to be made available for ethically approved research, after receiving an extensive information sheet and consent form, which includes an option to withdraw from the study at any time and without any restrictions. ln and el conceived the paper. lp and el provided the preliminary findings from data from the ukmeb participants and possible interpretation of them. so'b contributed to drafting, referencing, and formatting. all authors contributed to drafting and to revising the manuscript and approved the final version to be published. the uk me/cfs biobank was established with a joint grant from the charities me association (including continuing support), me research uk and action for me, as well as private donors. research reported in this manuscript was supported by the national institutes of health under award number r ai . the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. how have selection bias and disease misclassification undermined 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orthostatic intolerance dysbiosis of the gut microbiota in disease fecal metagenomic profiles in subgroups of patients with myalgic encephalomyelitis/chronic fatigue syndrome. microbiome from ibs to methe dysbiotic march hypothesis comorbid clinical conditions in chronic fatigue. a cotwin control study the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © nacul, o'boyle, palla, nacul, mudie, kingdon, cliff, clark, dockrell and lacerda. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -w cmljm authors: sharon, donald; chen, rui; snyder, michael title: systems biology approaches to disease marker discovery date: - - journal: dis markers doi: . /dma- - sha: doc_id: cord_uid: w cmljm our understanding of human disease and potential therapeutics is improving rapidly. in order to take advantage of these developments it is important to be able to identify disease markers. many new high-throughput genomics and proteomics technologies are being implemented to identify candidate disease markers. these technologies include protein microarrays, next-generation dna sequencing and mass spectrometry platforms. such methods are particularly important for elucidating the repertoire of molecular markers in the genome, transcriptome, proteome and metabolome of patients with diseases such as cancer, autoimmune diseases, and viral infections, resulting from the disruption of many biological pathways. these new technologies have identified many potential disease markers. these markers are expected to be valuable to achieve the promise of truly personalized medicine. disease markers are of vital importance to clinicians and their patients as early detection, accurate prognosis/diagnosis and monitoring of therapy can lead to increased overall survival and cure rates. as our knowledge of diseases quickly expands, the field of disease marker discovery will play increasingly important roles in the delivery of improved diagnosis and treatment. these markers, such as protein (including autoantibodies, which are antibodies specific to self-antigens [ ] ), hormonal markers (such as lack of insulin in type i diabetic patients [ ] ), and genetic/genomic markers (such as brca mutation in breast cancer patients [ ] ), enable clinicians to diagnose the disease while it is still at early stages, to ensure appropriate surgical intervention, efficient drug treat-ment and monitoring, and to predict an individual's risk of developing specific diseases before they experience symptoms. traditionally, discovery and detection of these disease markers relied on low throughput technologies such as enzyme-linked immunosorbant assay (elisa) or d-gel plus edman degradation for protein markers, reverse transcription-polymerase chain reaction (rt-pcr) for mrna markers, and restriction enzyme digestion, cloning and sanger sequencing for dna markers. before the dawn of high-throughput technologies these methods played important roles in marker identification and yielded significant discoveries in various diseases such as systemic lupus erythematosis, rheumatoid arthritis and breast cancer [ , , ] , which greatly enhanced the diagnostic efficiency in these diseases. during the past two decades, high-throughput technologies emerged and have displayed great potential in large-scale studies for marker discovery. these technologies include protein microarrays [ , ] , mass spectrometry for large-scale shotgun studies [ ] , and, more recently, high-throughput parallel sequencing (including rna-sequencing) [ , , ] . this article will review disease marker discoveries using these systems biology approaches, with a focus on high-density protein microarray technologies. we will also briefly review current progress in disease marker identification using parallel sequencing and mass spectrometry technologies. currently, high-density protein microarrays contain hundreds to thousands of proteins that are arrayed on coated glass microscope slides (e.g. nitrocellulosecoated slides) in an addressable format [ ] . these arrays are usually probed with fluorescently labeled molecules and the signals are then acquired with a confocal laser scanner. a number of surface chemistries have been employed for their ability to bind proteins efficiently although there is often a trade-off between retention and decreased protein function or improper folding. there are several broad categories of protein microarrays: arrays composed of cell or tissue lysates or protein fractions isolated from crude lysates [ , ] , antibody or analytical arrays that contain types of antibodies directed specific analytes [ ] as well as socalled functional protein arrays [ , ] . functional protein arrays contain full length proteins with intact catalytic function and proper epitope folding, which are often generated by arraying purified proteins produced individually prior to printing [ ] or proteins produced in situ by in vitro transcription and translation of dna that is printed directly on the surface of the array [ ] . our group has been developing the last type of protein microarrays by arraying purified proteins on nitrocellulose-coated glass slides. this type of functional protein microarray has clear advantages over the other alternatives: the ability to specifically identify individual proteins compared with cell lysate arrays and the ability to ensure the quality of each arrayed protein compared to the in situ transcription-translation arrays. after development of the first protein microarray that contains full-length proteins of the budding yeast saccharomyces cerevisiae [ ] , our group produced a number of protein arrays including the yeast n-terminal and c-terminal arrays, a protein arabidopsis array, and a coronavirus array [ ] . in conjunction with protometrix corporation (now part of invitrogen corporation) we also collaborated in the development of a human protein array that currently holds more than , proteins expressed individually using a baculovirus/sf expression systems. these various arrays were used for a variety of applications including assaying for protein-protein, protein-lipid and proteinnucleic acid interactions as well as probing for substrates of protein kinases [ , , ] . we also developed algorithms for positive signal calling and large dataset processing [ ] . recently, we have applied this technology in a novel proteomics-based approach to screen for human antibodies that react with foreign and self-antigens [ , , ] . particularly notable in this review is our use of these protein microarrays to analyze the immune response to coronaviruses [ ] as well as our screening projects to analyze ovarian cancer [ ] , myeloma, multiple sclerosis and asthma. in this section we will review disease marker identification in several fields using high-density protein microarrays. currently, tests for the detection of microbial infections are the only clinical tests that rely on measuring antibody responses. elisa-based detection methods are often used to detect a patient's antibody titer to epitopes of the microorganisms for diagnosis of the infection. in late , an outbreak of a novel coronavirus (cov), the severe acute respiratory syndrome (sars) virus, resulted in that killed over deaths. novel diagnostic tests were required to identify and monitor this disease, and elisa, immunofluorescence and nucleic acids tests were employed. it was shown that protein array-based methods proved to be more accurate than any of the existing antibody based methods [ ] . our lab developed a coronavirus proteinmicroarray that contained coronavirus proteins including all sars-cov proteins and proteins from five additional coronaviruses. the microarray was used to probe sera obtained from canadians and chinese during the sars outbreak, including samples from confirmed sars-cov cases, other respiratory disease patients, and healthcare professionals. after detection with cy -labeled anti-human igg antibodies, the bound reactive antibodies to coronavirusencoding proteins from sera were visualized and quantified. the reactivity results of the different proteins were analyzed using a variety of computational methods, and we developed computer algorithms based on the reactivity results to predict which patients were infected with sars [ ] . the protein microarray platform displayed a very high sensitivity, and reliably detected sars-cov reac-tive antibodies even when serum was diluted at , fold. the assay showed good reproducibility with less than % variance in signal intensity between duplicate slides. importantly, the method requires less than one microliter of serum for detection, which is desirable when serum samples are limited. moreover, probing of the coronavirus protein microarrays with sars infected serum samples also shows high specificity to sars-cov-specific proteins, as very little crossreactivity with proteins of other coronaviruses has been observed in sars infected serum samples. to determine the best classifiers to distinguish sars-positive from sars-negative sera, we used one unsupervised clustering method and two supervised methods: k-nearest neighbor (k-nn) and logistic regression (lr). both supervised models showed high sensitivity ( % for k-nn and % for lr) and specificity ( % for k-nn and % for lr) with a panel of (k-nn) or (lr) best classifiers, and these numbers were greater than % when the assay was performed in triplicate. the prediction methods were then tested on sera from chinese fever patients for sars-infection prediction and were determined to have % sensitivity and % specificity, which are superior to two elisa-based detection methods that were used during the sars outbreak. our study in sars-cov infection diagnosis via detection of sars-cov-specific antibodies by protein microarrays demonstrated that this approach is sensitive ( -fold more sensitive than elisas), specific (little crossreactivity with other coronavirus proteins), and rapid (performed in a few hours). nevertheless it should be noted that tests based on immune responses to foreign antigens are more likely to achieve higher accuracy than those based on autoantibody-autoantigen responses since there is no self-tolerance to the foreign antigens and the presence of pathogen associated molecular patterns (pamps) significantly increases the immune response. overall, this study demonstrated for the first time that protein microarrays could be used to diagnose and monitor human antibodies as protein markers that are generated during the course of a disease. moreover, it demonstrated the power of using a panel of multiple classifiers for diagnostics. while protein microarray technology can efficiently and accurately detect antibodies generated against foreign antigens from infectious organisms, perhaps the most intriguing application of this technology is in the discovery of novel protein markers for the early detection of various cancers. the identification of disease markers holds the promise of increasing the effectiveness of clinic therapies and marker-based routine screening programs and can potentially enable diagnosis at the earliest stages of the disease, before the development of clinically recognizable cancers that are usually at advanced stages. for instance, in heavy smokers autoantibodies recognizing mutant forms of the tumor suppressor p have been detected prior to the diagnosis of lung cancer [ ] . early detection and treatment would result in markedly improved survival rates, especially for patients whose cancers do not present symptoms during early stages such as pancreatic and ovarian cancer [ , , ] . oncoproteomics is a rapidly expanding field aimed at applying high-throughput proteomics approaches to understanding the mechanisms involved in cancer. proteomic approaches to discover cancer markers have been an area of strong interest in recent years. in the past, these projects often involved serum screening with phage expression libraries prepared from cancer tissues, or serex (serological analysis of cdna expression libraries), or by immunoblotting cancer cell lysates after two dimensional polyacrylamide gel electrophoresis ( de-page). these approaches have yielded some promising candidate markers but suffer from particular issues, such as the fact that phage expression libraries often contain out of frame and truncated protein targets and protein candidates discovered by de-page are difficult to identify since the proteins are unknown. mass spectrometry is often required in order to identify the candidate autoantigens [ , , , ]. an additional problem with these approaches is that the samples are usually limited in amount and are difficult to reproduce. protein microarrays overcome those difficulties as all of the spotted proteins are derived from known, well-characterized clones. additionally, even a small amount of purified protein is sufficient to print hundreds of arrays for patient screening [ , , , , ] . as most traditional disease markers are proteins that have become over-or under-expressed during the course of disease, there is much interest in the potential use of autoantibodies as a novel class of disease markers. recently, detection in serum of circulating autoantibodies targeting tumor-associated antigens (taas) has emerged as an effective approach for identifying cancer early detection markers (e.g. breast, lung and ductal pancreatic cancer [ , , ] ). this approach is based on the fact that the immune system produces an-tibodies against abnormal/mutated proteins generated from apoptotic/necrotic cancer cells. these autoantibodies can then be detected with immunosorbant assays like elisa. because the levels/stability of autoantibodies are potentially much greater than those of the original autoantigens, they would be more easily detected. by comparing autoantibody profiles between different groups (cancer patients versus controls), it is possible to identify markers that are significantly differentially expressed. this method is expected to be superior to dna array-based methods since changes in rna expression levels do not necessarily correlate with protein expression. the area of research in autoantibody marker discovery using protein microarrays has rapidly expanded over the last several years as the protein array platform continues to mature. the recent availability of high content protein microarrays allows for global profiling of autoantibodies to cancer antigens in both highthroughput (thousands of protein candidates) and high sensitivity ( fg of protein) [ , ] . improvements in printing techniques and increases in protein spot quantity have made these arrays promising vehicles for exploring the repertoire of autoantibodies in human disease. this approach has been applied, by various groups, for the discovery of autoantibody markers in breast cancer [ ] , lung cancer [ ] and ovarian cancer [ ] , as well as a smaller study in pancreatic cancer [ ] . here we will review past and ongoing research in immune response profiling using protein microarrays relating to a number of disease states. while self-tolerance usually abrogates the antibody response to self-proteins it is possible to elicit an autoimmune response under certain conditions present in cases of disease. the antigenicity of self proteins may result from overexpression of normal proteins such as in the case of her- in breast cancer subtypes and prostate specific antigen (psa) in prostate cancer, from aberrant post-translational modification such as different mucin- glycoforms in breast cancer, or from mutations in the proteins as has been found to be the case with the tumor suppressor p in multiple cancer types [ , , , ] . additionally, proteins that are usually restricted to expression in germ line cells or are expressed only in the early stages of development may be aberrantly expressed in cancer. this is the case with the testis antigen ny-eso- and carcinoembryogenic antigen (cea) respectively. because many of the proteins mentioned above are detected only at very low levels in serum, even in late stages of disease, they would be of little utility for screening purposes. however, even slight increases in the expression of those antigens can lead to detectable increases in the corresponding autoantibody. generally, we find the existence of a basal autoantibody level to many self-antigens, however, this response has been shown to be markedly increased in cases of diseases such as those mentioned above [ , , ] . ca- is currently the only clinically approved marker for ovarian cancer screening. unfortunately, although ca- serum levels are significantly elevated in advanced stages of the disease, its positive predictive value for the detection of early stage ovarian cancer is less than % [ ] . for this reason the identification of new markers for this disease is of critical importance. scientists, such as the group led by gil mor at yale university, recruited proteomics-based approaches using antibody-based protein microarrays to identify new serum biomarkers, which, in combination with ca- , may enhance the early detection of ovarian cancer [ , , ] . our group also launched a pilot study to profile ovarian cancer-associated autoantibodies with protein microarrays containing , fulllength human proteins [ ] . we compared the autoantibody profiles in cases of epithelial ovarian cancer patients and healthy controls, and after statistical analysis, identified proteins to have significantly different immune reactivity in the patient group versus the control group. the results were validated by immunohistochemistry (ihc) and demonstrated high sensitivity ( %) and specificity ( . %) when the top two markers (lamin a/c and ssrp ) were combined. however, further validation is required before the candidate markers can be adopted in a clinical setting. therefore we carried the top ranking candidates through to the validation phase in which a much larger set of samples will be tested to evaluate the performance of the potential markers. we have generated focused protein microarrays containing these candidates as well as control proteins such as ca- (fig. ) . these arrays are printed in twelve blocks per slide allowing as many as twelve samples to be screened per array. this approach will allow hundreds to thousands of samples to be screened in order to determine which markers or combination of markers demonstrate the best receiver operator characteristic (roc) performance [ , ] . autoantigens in breast cancer subtypes such as her- /neu positive tumors have been shown to correlate with increased autoantibody responses in patients. her- /neu autoantibodies in those patients demonstrate approximately % sensitivity and % specificity. other groups have adapted similar approaches to multiplex- ing immune responses using high-density protein microarrays in breast cancer [ , ] . among these studies, anderson et al. used nucleic acid programmable protein arrays (nappa) for sera screening in breast cancer [ ] . these arrays were generated by printing individual genes as plasmid dna along with capture antibodies to gst tags on the fusion proteins. the arrays were then incubated with in vitro transcription and translation coupled cell-free lysates to produce the proteins and anchor them to the array surface. each array consisted of cancer associated candidate proteins including p as well as the epstein bar nuclear antigen (ebna) as a control. sera from four breast cancer patients and four healthy controls were used to probe the arrays and they found anti-p autoantibodies in cancer patients. because the proteins are not produced until the arrays are ready to be probed they do not suffer from degradation during periods of storage. however, the protein quantity is more variable between spots compared to directly printed arrays. we have found that spot to spot variation in protein amount may be overcome by evaluating the autoantibody response relative to the protein amount (i.e. the ratio of autoantibody signal to the signal from an epitope tag on the autoantigens). in our protein array immune response profiling studies we have found that this approach results in decreased signal variance between replicate spots (unpublished data). to date, no studies that attempt to identify novel breast cancer markers have been performed using high-density protein microarrays. pre-vious high-throughput serum screens in breast cancer have relied on serex and de-page and involved relatively small sample sets [ , ] . a more interesting immune response profiling study may be the autoantibody responses to self-antigens in cancers of the blood and lymph, such as multiple myeloma. our lab has been involved in a protein microarray based screening project aimed at elucidating the autoantigen repertoire in multiple myeloma. multiple myeloma is a cancer of the bone marrow system resulting from the uncontrolled proliferation of monoclonal plasma cells (precursors of the bcells responsible for the production of antibodies) [ , ] . monoclonal gammopathies of unspecified significance (mgus) is a precursor disease to multiple myeloma characterized by bone marrow plasmacytosis and increased m-protein levels in the blood [ ] . we have probed high-density protein arrays using plasma from dozens of cases of mgus, multiple myeloma, and healthy controls. by comparing igg responses to individual antigens on the arrays between the healthy and diseased groups we have identified multiple autoantigens that are significantly differentially targeted by igg autoantibodies in early stage disease. this study was unique as it employed the highest density protein arrays for multiple myeloma immune response profiling to date and the patient samples were from a prospective collection. because samples were drawn prior to disease onset there are no artifacts resulting from medical treatment of the patients. by querying such early stage samples we have a better chance of identifying markers that will be effective for diagnosing patients at early stages when they are more treatable and will experience better outcomes. the markers identified in this study may yield insight into the biological processes and mutational events that contribute to the development of aggressive forms of multiple myeloma. in addition to early detection, cancer markers may also enable clinicians to offer personalized treatment. recent advances using the anti-cancer drugs herceptin and iressa illustrate this point. these drugs target specific patient populations: herceptin is effective against those tumors expressing the her receptor and iressa is effective for patients with specific mutations in the epidermal growth factor receptor [ , ] . these drugs offer limited benefits to patients with the same cancer types when these markers are not present. thus, determining the marker profile of an individual's disease can enable the identification of distinct patient populations, allowing tailored and more effective treatment. one issue that we would like to note is that no single autoantibody response to an autoantigen has been confirmed to have sufficient sensitivity and specificity for screening purposes in early stage disease. however, by evaluating the antibody responses involving a panel of autoantigens, accuracy has been markedly improved [ , , ] . thus, future protein microarray immune response screening tests will likely combine multiple autoantigens. numerous approaches have been applied to combining disease markers. some of the common methods for combining multiple autoantibody responses are linear regression, split-point analysis, and k-nearest neighbor (k-nn) [ , ] . still there are currently no clinical screening or diagnostic tests that rely on a panel of protein markers,although multi-parameter dna microarray tests are becoming commonplace in diseases such as breast cancer [ ] . clearly, more work is needed before autoantigen based microarray tests can be implemented in a clinical setting. the use of protein microarray technology for biomarker discovery in autoimmune diseases seems a natural extension of the technique as autoantibody responses have already been shown to contribute to disease progression in diseases such as lupus [ ] . while antinuclear antibody tests are sometimes used to confirm diagnosis of certain autoimmune diseases, they are not disease specific [ , ] . multiple sclerosis (ms) is a debilitating disease of the central nervous system characterized by rounds of axonal demyelination and repair. it affects mostly younger people and is more common in women than men. the underlying cause remains unknown, though there is mounting evidence that antibody responses to self proteins play a role in both demyelination and repair [ ] . previous efforts have identified a number of myelin specific proteins that demonstrate increased autoantibody responses in ms. these autoantibodies have been detected in both serum and cerebrospinal fluid (csf). recently, protein microarray screening has been applied to evaluate autoantibody responses to subsets of myelin proteins that are known to be associated with ms and other neurodegenerative diseases [ , , ] . the antigens that were shown to elicit autoantibody responses include classical ms antigens such as myelin-basic protein (mbp), myelin associated glycoprotein (mag) and myelin oligodendrocyte glycoprotein (mog) as well as proteins that have not been demonstrated to play a significant role in ms previously. these studies have suffered from the fact that the arrays were focused on a relatively small number of previously known candidate autoantigens. our group is currently conducting larger-scale screenings based on high-density protein microarrays. with this platform, we have tentatively identified a number of novel candidate markers in multiple sclerosis. we are currently working to validate these markers in a larger sample set. this less biased approach may result and the identification of novel autoantigens leading to a better understanding of the underlying mechanisms in the etiology of ms and result in improved screening and diagnostic tests. asthma, a common disease with a prevalence of % for all ages [ ] , and % in children under years of age in the united states [ ] , is another disease involving an autoimmune mechanism [ , ] . this heterogeneous inflammatory disease of the airways is marked by recurrent episodes of airway obstruction and wheezing [ , ] , and is anatomically characterized by bronchoconstriction, inflammation and thickening of the airway walls [ ] . considering asthma as an aberrant chronic wound healing process [ ] , it would not be surprising that some of the released/leaked cellular contents from the airway epithelium due to damage and remodeling, similar to necrotic cancer cells, may elicit autoimmunity. in fact, aberrant autoantibodies have been detected in asthmatic sera by autologous serum skin tests as compared to normal controls [ ] . autoreactive antibodies have also been de- tected in the asthmatic sera against the high-affinity ige receptor fcεri [ , ] . a few specific autoantigens have been identified in the serum of asthmatic patients, including the autoigg-reactive β-adrenergic receptor [ , ] , cytokeratin [ ] , dfs [ ] , and α-enolase [ ] . moreover, studies on atopic dermatitis, which often occurs with asthma, revealed autoreactive ige antibodies against hom s - (hom s = sart ; hom s = α-nac; hom s = bcl b; hom s = a protein with calcium-binding motif; hom s = a type ii cytokeratin) [ , , ] and dfs [ ] . however, these studies focused on small patient groups with no cross validation, as well as limited number of potential targets investigated. since both autoreactive igg and ige may be involved in the pathogenesis of asthma, we conducted a large-scale screening for asthma-associated auto-igg and ige reactive autoantigens using protein microarrays with more than , protein candidates (unpublished data). this is the first large-scale study to profile asthma-associated autoantigens, and the results will greatly improve our understanding of the role of autoimmunity in the etiology of asthma. one unique feature of this study is that, in order to maintain uniform probing conditions, we multiplexed the detection for both autoreactive igg and autoreactive ige in the serum samples simultaneously on the same array, with a mixture of anti-human igg and ige secondary detection antibodies labeled with distinct fluorescent dyes. our result suggested that the protein array is capable of detecting both igg and ige reactive signals in distinct emission channels with high specificity and no/detectable signal bleeding across the channels (fig. ) . similar applications of protein arrays have been performed in studies of other autoimmune diseases. song et al. discovered novel autoantigens, namely rps , alba-like and dutpase for autoimmune hepatitis (aih) using a protein microarray containing nonredundant proteins [ ] . horn et al. profiled the repertoire of igg autoantibodies in plasma samples from dilated cardiomyopathy (dcm) patients with a redundant protein microarray containing , total proteins and identified autoreactive proteins to igg (with of them reactive specifically to the igg subclass) [ ] . autoantigens were also identified for the chronic disease alopecia areata by protein microarray technology [ ] . these examples demonstrate the great potential of protein microarray technology in the application of autoantigen marker identification in autoimmune diseases. although protein microarray technology provides a high-throughput method with high sensitivity and specificity for protein marker discovery, there are particular limitations that investigators need to be aware of before applying the technology to their research. first of all, as probing protein microarrays for autoantibodies are in vitro studies with all the protein targets arrayed in a -d platform, one has to take into consideration off-target binding. therefore findings from protein microarray screenings should be validated in larger sample sets, and the autoantigens have to be confirmed by direct detection methods such as western blotting, elisa, or ihc in patient samples before an autoantigen can be confidently associated with the disease. secondly, microarrays that contain fulllength, folded proteins may not be recognized by autoantibodies that are directed against misfolded or degraded proteins expressed in disease cases, contributing to false negative detections. patwa et al. developed a method to chemically digest the proteins with cnbr before printing them on the arrays, which may help to overcome this problem [ ] , however, as the digestion rate is hard to control, the final complex mixture of digested proteins at different levels may complicate normalization efforts and experimental control. normalization of the array data is another important consideration. as we have already discussed, normalization of protein spot morphology and quantity can be achieved by probing with a labeled antibody directed against an epitope-tag appearing on all of the arrayed proteins (e.g. gst). various software based methods have been adopted to adjust for regional defects and background that has traditionally been an issue for protein and dna based microarrays [ ] . nevertheless, analysis and interpretation of the acquired large-scale data is still a challenge to both biologists and statisticians, therefore the development of improved algorithms is an ongoing effort. under real probing conditions, uncontrollable events such as scratches on the slides, deposition of salt and non-homogeneous local concentrations can further complicate the analysis of the array data, although internal controls are often included to help overcome these defects and improved array surface chemistries have significantly decreased local and regional background defects. in recent years it has become feasible to sequence entire genomes and transcriptomes using massively parallel sequencing platforms such as the and solexa genome analyzer. these platforms use a highly sensitive light sensor (such as cmos sensors or ccd cameras) to capture fluorescent signals emitted from each deoxynucleotide as they are added to the dna chain simultaneously in up to millions of parallel reactions in a flowcell [ ] , thus performing sequencing in a high-throughput manner to obtain short sequences (vary from to bp depending on the platform) from one or both ends. currently, related platforms and products are available through illumina ig, applied biosystems solid, roche life science, and the helicos biosciences tsms [ ] . another company, pacific biosciences, will manufacture a new sequencer that will perform single molecule sequencing by the end of [ ] . a typical parallel sequencing procedure consists of the following steps: dna/rna isolation, fragmentation and dna/cdna library construction, highthroughput sequencing and read assembly and mapping. this method has many advantages compared to the traditional tiling microarray hybridization-based methods, or the more traditional rt-pcr and sanger sequencing method. these new platforms achieve single-base resolution in a high-throughput manner, have low background, no cross-hybridization noise, low dependence on the availability of existing genomic sequence, high reproducibility and low cost per base, and there is no upper limit for quantification [ ] . in this section we will briefly review recent efforts in genetic marker discovery with next-generation parallel sequencing. this new generation of sequencing technology is shaping a new paradigm in disease marker research, in which massive amounts of sequence information from genomic dna and expression libraries are screened for linkages and associations of genetic and genomic markers to specific diseases by comparing disease patients and healthy individuals [ , , , ] . genomic dna sequencing provides rich information on genetic variations (such as single nucleotide polymorphisms, insertions and deletions) and structural variations (such as copy number variations, transposition and transloca-tion) of the investigated genomes and is a powerful tool to reveal novel disease-associated markers. genome sequencing can also detect integrated viral sequences which may help address studies of virus-associated diseases. whole genome sequencing has already been applied in organisms with small genomes, such as acinetobacter baumannii [ ] , toxoplasma gondii [ ] , and drosophila melanogaster [ ] , however, due to the large size of human genome and the high cost of parallel sequencing, human whole genome sequencing is still in its infancy. ley et al. were the first to sequence the entire genome of one type of cancerous tissue, the acute myeloid leukemia (aml) cells ( . x haploid coverage), as well as corresponding normal tissue, the patient's skin tissue ( . x haploid coverage) [ ] . due to the unbiased nature of the sequencing methods, they were able to use read frequency to establish how rates of mutations vary within the cancer tissue. this concept is important for future works as we seek to understand the progression of mutational events that lead to the development of diseases like cancer. the researchers found that , single nucleotide variations were unique in the cancer tissue sample. these mutations resulted in changes to the coding regions in ten genes, two of which were previously implicated in cancer. nonetheless, as sequencing costs continue to decrease with the maturation of the platforms, whole genome sequencing of larger sample sets is shedding light on new venues of genetic and genomic marker identification in various diseases. both biologists and clinicians are preparing for this coming revolution, and projects have already been conceived such as clinseq, a pilot project led by green et al. which currently enrolls about participants for whole genome sequencing [ ] . whole transcriptome sequencing by rna sequencing (rna-seq) is another promising application of parallel sequencing technology and has already been applied to marker discovery in various cancers. gene expression profiling has been shown to predict the outcome of breast and other types of cancer [ , ] . shah et al. used paired end rna-seq to canvas the transcriptomes of four granulose-cell tumors (gct) from ovarian cancer patients [ ] . they found a common missense mutation in the foxl gene (c g) in those tumors that was not present in other ovarian cancer transcriptomes that they also sequenced. additionally, this mutation was confirmed to be present in % of other gct cancers that they tested. leven et al. performed illumina sequencing on tiling-array-hybridization enriched transcripts representing cancer associated genes from the k- chronic myeloid leukemia cell line and detected a wide range of dna and rna sequence alterations in the targeted transcripts [ ] . these alterations included fusion transcripts such as bcr-abl and nup -xkr , as well as snps within and splice isoforms of these transcripts. while whole genome sequencing is still a relatively expensive proposition, transcriptome sequencing can be performed at much lower cost, facilitating the discovery of any mutations in the transcriptome that may contribute to the development of disease. in addition, rna sequencing also provides information that would not be obtained through whole genome sequencing, such as information on the expression level of each transcript, alternative splicing and rna editing, as well as trans-splicing events. we should expect to see genetic and genomic biomarkers identified as causative or contributing factors in human disease in the near future thanks to the utility of rna sequencing. one other promising application for transcriptome sequencing is to identify viruses in the host sample. in a study carried out by sorber et al., the authors successfully detected hepatitis b virus (hbv) sequences in the serum sample from a patient with hbv infection [ ] . similarly, palacios et al. used rna-seq and identified a novel old world arenavirus in rna samples extracted from the liver and kidney of three deceased patients who had transplantation-related infection [ ] . nakamura et al. obtained - reads of influenza virus sequence in nasopharyngeal aspirates and - , reads of norovirus sequence in fecal specimens from patients suffering from influenza or norovirus infections [ ] . meanwhile, rwahnih et al. sequenced the rna from a grapevine with the roche system and revealed infection of plant viruses as well as one novel virus in the diseased grapevine [ ] . our group has also carried out an rna-seq experiment to detect west nile virus sequences in infected macrophages (unpublished data). we obtained reads ( . % of the total mapped reads) mapped to the west nile virus genome from the infected cells and very few reads ( , ∼ . % of the total mapped reads) mapped to viral sequences in rna isolated from mock control cells. after analysis recheck of the few reads that did map to the virus genome in the control cells, we found that they were redundant with sequences in the human genome. these studies proved that rna-seq can achieve high sensitivity and specificity to identify and profile infecting viruses, or the "virome", us-ing rna samples isolated from the host. moreover, rna-seq will also provide information on virus-host interactions by monitoring expression changes of the host's genes. while mutations in protein-coding sequence are well known to contribute to multiple diseases, rna-seq is limited to only those actively transcribed sequences of the genome. this bias results in overlooking variations in non-transcribed regions of the genome that can be important contributors to disease as mutations in these regions can result in aberrant gene regulation. besides whole genome sequencing, chip-seq, or chromatin immunoprecipitation-sequencing, is another approach to address mutations in functional non-transcribed regions of the genome. this technology sequences the genomic regions bound by transcription factors or other dna-binding proteins (such as histones), and provides information on the position of these binding sites as well as possible mutations in these sites. the binding site profiles of multiple transcription factors, as well as the identified sequence variations within, may act as new markers for diseases such as leukemia [ , ] . sono-seq is a related technology developed in our lab, which parallel sequences sonicated formaldehyde cross-linked chromatin dna via illumina sequencing, and identifies the chromatin regions that are open and accessible (nucleosome-free therefore susceptible to sonication) [ ] . with this technology we identified multiple highly accessible chromatin regions including actively transcribed promoter regions as well as the ctcf insulator protein binding sites. this technology is similar to another open chromatin finding technology, termed faire (formaldehyde-assisted isolation of regulatory elements), which selects open chromatin regions for dna microarray hybridization by phenolchloroform extraction of sonicated cross-linked samples [ ] . when interrogated in the background of a disease compared with healthy controls, the identified profiles of these nucleosome-free regions may provide a new type of disease marker for future studies. while next-generation sequencing holds great promise for the discovery of novel disease markers, there are issues with the current technology, such as artifacts due to sample preparation (both reverse transcription and polymerase chain reaction can generate biases) and data processing (assembly of short reads can result in errors, especially in regions of repetitive sequence). newer technologies from companies like helicos biosciences and pacific biosciences are on the horizon that could overcome these issues by direct rna sequencing and long single molecule sequencing, fulfilling the promise of personalized medicine in the post genome era [ , ] . mass spectrometry technology (ms) has been growing rapidly in the past several decades. since john bennet fenn and koichi tanaka developed new soft desorption methods that made mass spectrometric analyses of biological macromolecules possible, this technology has been widely used in proteomic studies [ , ] . the ability to identify and quantify target molecules (e.g. peptides) makes mass spectrometry methods a popular tool for disease marker discovery. disease marker discovery with mass spectrometry is usually combined with varied sample separation methods such as de ( -dimentional electrophoresis) and d-dige ( -dimentional differential in-gel electrophoresis) [ ] . in a typical procedure, mixed proteins from pooled disease samples and pooled controls are separated with d or d electrophoresis, and individual protein bands or spots are visualized and differential bands or spots are then excised followed by enzyme digestion (e.g. trypsin). the digested peptides are then subjected to mass spectrometry analysis for protein identification. with this method, shen et al. identified potential markers for pancreatic adenocarcinoma, and the spectrum of these markers covered antioxidant proteins, chaperones, calcium-binding proteins, catalytic enzymes, signal transduction proteins and extracellular matrix proteins [ ] . similarly, wang et al. identified differentially expressed proteins (including novel markers) associated with oral squamous cell carcinoma, and validated one of the eight markers named rack using immunostaining and gene silencing studies [ ] . even though d electrophoresis can improve protein separation and assist in further identification by mass spectrometry, the discovery of low-abundance disease markers has been greatly limited by the poor resolution and sensitivity of de or d-dige methods. furthermore, the pooled samples will not only lose important information such as personal variation of the disease markers among different individuals, but also miss protein that are only present in a subset of the sample population. recent improvement of mass spectrometry techniques as well as data analysis algorithms has enabled analysis of complex protein samples [ ] . currently, liquid chromatography (lc)-ms/ms using electrospray ionization (esi) is one of the commonly used methods for large-scale shotgun proteomic studies. gel-free methods, such as mudpit (multidimensional protein identification technology) has been more and more popular and greatly enhanced detection limits [ ] , and the improved resolution and accuracy of mass spectrometers makes this technology more useful in disease marker discovery [ ] . with d lc-ms/ms, ralhan et al. identified nonredundant proteins in head-and-neck squamous cell carcinoma from individual cancer samples compared with one pooled normal control, and the panel of the three best performing markers achieved a sensitivity of % and specificity of % in cancer classification [ ] . a most recent study to identify ovarian cancer biomarkers from patient ascites samples with d lc-ms/ms also yielded a panel of known and novel protein markers [ ] . these studies demonstrated that the improved ms techniques not only enabled researchers to search for biomarkers in unpooled samples, but also could lead to the identification of a larger number of potential markers due to improved sensitivity. in addition to protein marker identification, mass spectrometry is also capable of identifying metabolom-ic markers [ ] . metabolomics is a novel field that studies the global profiles of all metabolites in a given sample. since diseases such as cancer usually have unique metabolomes [ ] , the over-or under-presented metabolites could serve as potential markers of the disease. moreover, certain metabolites in the cells will also influence the activity of larger biomolecules such as kinases (unpublished data), therefore identification of the cancer-specific metabolomes would be of great value. currently, multiple metabolites have been associated with various tumors, such as alanine, saturated lipids, ccms, glycine, lactate, myo-inositol, nucleotides, pufas and taurine [ ] , and it will not be surprising if this list will grows dramatically in the coming years. although mass spectrometry is a powerful tool in molecular marker identification, the clinical application of this technology for diagnostic purposes is still limited. mass spectrometry may one day become the platform of choice for detection of disease-associated markers, however, the high cost of mass spectrometers as well as lack of standardized methods are preventing it from being adopted by clinicians as a diagnostic tool. moreover, the high level of molecular complexity of biological samples is still a large obstacle in both marker identification and application. there remains great space for improvement before mass spectrometry realizes its ultimate potential in the clinic. disease markers are important for the efficient diagnosis, prognosis and treatment of a disease, therefore identifying these markers is crucial especially for diseases with high mortality rates such as cancer. each technology reviewed in this article has a unique niche in disease marker discovery. protein microarray technology excels in finding protein markers, especially antibody markers; next-generation parallel sequencing is designed for rna and genetic/genomic marker discovery; and mass spectrometry specializes in the identification of protein markers as well as metabolomic markers. each technology has its unique advantages and limitations, and the "-omics" information obtained with the help of these technologies may complement each other and leading to a comprehensive view of disease (fig. ) . this information will greatly expand our understanding of the etiology and course of human diseases, resulting in more efficient diagnosis and treatment of disease. moreover, by adopting a comprehensive "-omics" view of human diseases, it will be interesting to discover the extent to which these systems interact with each other. will we find that a disease associated with certain genetic markers also develops specific autoantibodies? or a certain autoantigen response is actually due to the dysregulation of a specific metabolite or trans-spliced mrna? is it possible that diseases such as multiple sclerosis and asthma actually are caused by the coordinating effect of genetic susceptibility and, say, viral infection? while systems biology approaches to disease marker discovery are still in their infancy they have already led to the discovery of many promising genetic and protein markers in various diseases. additionally, new dimensions in the development and application of these approaches may further revolutionize this field by interrogating additional "-omes", such as the methyl-genome, the "kinome" and the "virome". these systems may be as important as the systems reviewed above to establish a complete understanding of, and efficient treatments for, many diseases. comprehensive genomic characterization defines human glioblastoma genes and core pathways immune responses in cancer deep sequencing analysis of rnas from a grapevine showing 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comparative proteomics approach to screening of potential diagnostic and therapeutic targets for oral squamous cell carcinoma asthma genetics: personalizing medicine large-scale analysis of the yeast proteome by multidimensional protein identification technology overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics autoantibodies as potential biomarkers for breast cancer global analysis of protein activities using proteome chips severe acute respiratory syndrome diagnostics using a coronavirus protein microarray procat: a data analysis approach for protein microarrays asthma and autoimmunity: is there a connection? we cordially thank our collaborators, drs. gil mor (ovarian cancer studies), geoffrey chupp (asthma studies) and ruth montgomery (west nile virus studies) at yale university, and dr. jonas bergqvist at uppsala university, sweden (multiple sclerosis study) for their contributions to the projects outlined in this review. key: cord- -ju vuywm authors: rohde, rodney e. title: common myths and legends of rabies date: - - journal: rabies doi: . /b - - - - . - sha: doc_id: cord_uid: ju vuywm humankind has somewhat of a dark, yet almost fascinating, supernatural relationship with rabies. even after pasteur's rabies vaccine discovery, globally people continue to be stricken with it today. history has carried along the myths and legends that surround this diabolical virus. some still believe that rabies treatment requires or more shots to the stomach by some monstrously long needle. however, today's treatment regimen is typically only four vaccinations (five for immunocompromised) in the arm, plus human rabies immune globulin. this chapter explores the misunderstood concepts of rabies prevalence, signs and symptoms, exposures, and treatment protocols. rabies has been a part of human history for as long as it has been recorded in writing and art. from odysseus's story to achilles, the actual word (term) that provoked and transformed hector to a rage and frenzy, "lyssa," is closely linked to the word "lykos" or "wolf" and is used to invoke images and feelings of an animal's anger, madness, and wolfish rage. lyssavirus is a genus of rna viruses in the family rhabdoviridae, order mononegavirales. lyssaviruses are bullet-shaped, single-stranded, negative-sense rna viruses and the causative agents of the ancient zoonosis rabies. africa is the likely home to the ancestors of taxa residing within the genus lyssavirus, family rhabdoviridae. diverse lyssaviruses are envisioned as coevolving with bats, as the ultimate reservoirs, over seemingly millions of years ( fig. . ). through eons of time, a wide range of myths and legends have developed pertaining to rabies. many people still believe today that rabies treatment requires or more shots into the stomach by some monstrously long needle. while in fact, today's treatment regimen is typically only four vaccinations (five for immunocompromised individuals) in the arm, plus a dose of humane rabies immune globulin (hrig). this myth is but one of a long list of questions, untruths, or exaggerations about the disease known as rabies. • is there a way to vaccinate wildlife using a recombinant vaccine dropped from aircraft? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • can scientists actually determine the origin of a rabies virus variant by way of genetic testing and methodologies? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • are there special government laboratories that conduct specialized rabies testing? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • can international public health teams actually halt and remove a moving viral epizootic outbreak in a geographical area? [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] • is there a relationship between rabies and zombies? in this chapter, some of the more common misconceptions (and facts) about rabies will be described. these misconceptions are categorized under concepts of prevalence, signs and symptoms, exposure, treatment, and pop culture influence with respect to rabies. where exactly do most cases of rabies occur in the world? which type of animals are often more "at risk" for contracting rabies virus, otherwise known as highrisk animals? are there places in the world where one might travel without worrying about rabies? do coldblooded animals, insects, or birds have a risk for acquiring this deadly disease? there are many untruths to unpack with respect to the prevalence of rabies. learn more in the following accounts: • many people believe that rabies infections only occur in poor, third-world countries. however, the truth is that rabies or rabies-like viruses are present on nearly every continent, with the exception of antarctica. numerous and diverse variants of lyssaviruses are found in a wide variety of animal species throughout the world, all of which may cause fatal human rabies. rabies virus is by far the most common lyssavirus infection of humans. in the united states, only hawaii is rabies free. interestingly, in hawaii and other rabies-free areas that are often islands, there are very strict laws and restrictions on the transport of animals from rabies-endemic states or countries to rabies-free geographic locations. in the united states, rabies as a disease is most prevalent along the east coast (raccoon and bat rabies virus variants) from florida to maine and in southern arizona (arizona gray fox) along the mexican border. rabies is also prevalent in texas with south central skunk and bat rabies virus variants predominating. common myths and legends of rabies rodney e. rohde, phd, ms, sm(ascp) cm , sv cm , mb cm , facsc as a side note, one should always consider the risk of infectious disease when traveling to other countries by discussing travel plans with an appropriate person such as a travel physician or other health-care professional. there may be a need to plan for preexposure vaccinations (e.g., rabies, yellow fever, etc.) or medications (e.g., malaria, antibiotics) to take on their travels. • a frequent belief is that this disease kills many people in the united states. however, approximately % of human deaths from the disease occur in africa and asia, where access to health-care facilities and treatment protocols is limited. the estimated annual figure of almost , rabies fatalities in humans is probably an underestimate. almost all cases of rabies in humans worldwide result from bites from infected dogs. table ). • all warm-blooded animals can contract rabies, particularly mammals. a viral disease of the central nervous system, rabies transmits between animals, including humans, when saliva containing the virus enters an opening in the skin. usually, the rabies virus enters through the bite of a rabid animal, but transmission can also occur when infected saliva enters through mucous membranes or a break in the skin. viral familial relatives can be found with invertebrates and plants (distant hosts), but warmblooded vertebrates are the hosts for the rabies virus. among warm-blooded vertebrates, birds are susceptible to infection, but rabies predominates naturally among various mammalian populations. only rare accounts occur in nonmammalian hosts, but in the mammalia family, rabies cases have occurred from the armadillo to the zebra. rabies is a significant disease of domestic and wild mammals alike, yet its zoonotic aspect is the cause of major historical infamy. according to the centers for disease control and prevention (cdc), the first clinical signs and symptoms of rabies may be very similar to those of the flu including general weakness or discomfort, fever, or headache. these clinical signs and symptoms may last for days and are directly related to the difficulty of a differential diagnosis of rabies in the absence of an obvious animal bite (or other) exposure. there may be also discomfort or a prickling or itching sensation at the site of bite, progressing within days to signs and symptoms of cerebral dysfunction, anxiety, confusion, and agitation. as the disease progresses, the person may experience delirium, abnormal behavior, hallucinations, and insomnia. the acute period of disease typically ends after - days (see chapter ). once clinical signs of rabies appear, the disease is nearly always fatal, and treatment is typically supportive. disease prevention includes both passive immunity, through an injection of human rabies immune globulin, and active immunity produced via a round of injections with rabies vaccine. if a person has already begun to exhibit signs of the disease, survival is rare. to date, less than documented cases of human survival from clinical rabies have been reported and only two have not had a history of pre-or postexposure prophylaxis. one of the more bizarre, but not uncommon, signs and symptoms of rabies that has been reported is male patients exhibiting hypersexual behavior. the virus sometimes acts on the limbic system of the brain causing men to show this behavior: increased sexual desire, involuntary erections, and in some reports continuous orgasms occurring at a rate of one per hour! in general, most people think of dogs when the topic of animals that can carry or transmit rabies is mentioned. this may be due to the long-standing fascination with the popular media (and ancient writings) that target the canine as the primary vector for rabies. for example, if you have seen the movie old yeller or cujo, then you probably understand how the popular media can influence the public to fear rabies and scrutinize the family dog for any signs of "frothing or foaming" at the mouth. however, the disease affects both domestic and wild animals. early clinical signs of the illness include fever, pain, and/or a tingling sensation around the wound. however, unlike the popular notion of old yeller or cujo, the most typical clinical signs of rabies are unexplained paralysis and a change in behavior (refer to chapter for details). still, there are many reports of other strange clinical behaviors by a rabid animal. . exposure histories are categorized as bite, contact (eg, waking to find bat on exposed skin) but no known bite was acknowledged, or unknown (ie, no known contact with an animal was elicited during case investigation). † variants of the rabies virus associated with terrestrial animals in the united states and puerto rico are identified with the names of the reservoir animal (eg, dog or raccoon), followed by the name of the most definitive geographic entity (usually the country) from which the variant has been identified. variants of the rabies virus associated with bats are identified with the names of the species of bats in which they have been found to be circulating. because information regarding the location of the exposure and the identity of the exposing animal is almost always retrospective and much information is frequently unavailable, the location of the exposure and the identity of the animal responsible for the infection are often limited to deduction. ‡infection was not identified until . when an organ recipient developed rabies. one of the difficult decisions about rabies, if not the most difficult, is "what constitutes an exposure" when trying to discern whether to treat or not to treat an individual. likewise, there are important implications for the animal (domestic). for example, whether to quarantine the animal for observation or to euthanize an animal followed by laboratory testing for confirmation of rabies. the world health organization (who) has recommendations intended as a general guide. it is recognized that, in certain situations, modifications of these procedures are warranted. such situations include exposure of infants or mentally disabled persons and other circumstances where a reliable history cannot be obtained, particularly in areas where rabies is enzootic, even though the animal is considered to be healthy at the time of exposure. there are other myths and legends surrounding the question of a true rabies exposure. the following are a few of the common ones: • on a given warm summer evening in areas of texas just as dark approaches, bats start swooping in their feeding mode of eating insects, which is an ecologic and biologic benefit. this could initiate a warning from some folks about "bats getting tangled in your hair," leading to being exposed to rabies. of course, bats are not attracted to a person's head of hair and do not try to "nest" there, but if a bat did get tangled in someone's hair, they would receive pep if the bat could not be tested as its too close a contact to rule out a bite occurring. however, contributing to this misconception could be media and historical writings with respect to vampire lore surrounding bats. additionally, there were even popular television series like the andy griffith show that had an episode with barney fife discussing bats in caves and their potential for laying "their eggs" in a person's hair (fig. . ) . • can i get rabies if i handle blood, feces, or urine? the simple answer is no in this scenario. rabies is not transmitted through the blood, urine, or feces of an infected animal, nor is it spread airborne through the open environment. saliva provides the primary transmission medium when the animal is in the infectious stage of rabies. for the rabies virus to get to the salivary glands, it has to travel first from the site of entry (usually a bite wound) through the animal's nervous system, then to the brain. this is what causes most rabid animals to exhibit abnormal behaviors, depending on what part of the brain is infected. finally, the virus travels to the salivary glands during the terminal stage of rabies. it is this later stage of rabies when an animal is most infectious because the virus is in the saliva. treatment for rabies, one of the world's most diabolical viruses, has a long history of creative and bizarre origins. rabies is a terrifying disease that dates as far back as the beginnings of humankind. in a sense, we humans fear rabies because it crosses the line between humanity and animal. think about it, the disease is at the intersection of humans and animals. somewhere deep in the psyche, one can become terrified by the thought of a bite from a rabid animal because it symbolizes the very metamorphosis of a human becoming that very rabid animal-a vampire or werewolf if you let your brain take you to that place. it should not be a surprise that humans have a longstanding and deep-seated fear of diseases with animal origins, or as science calls them, zoonotic diseases. a majority of new diseases are zoonotic. for example, swine flu, west nile virus, anthrax, severe acute respiratory syndrome, tuberculosis, influenza, ebola, nipah, powassan, and plague represent diseases from a wide range of eras. it is not a far reach to realize that the collective conscience of the general public has been swayed and biased by diseases with animal origins. so, along with that understanding comes an almost guttural need to find ways to treat or cure those diseases. this is where some of the earliest misconceptions for treating rabies originate in our history. let us examine some of those treatments that seem to perpetuate in time regardless of the modern advances in medicine. • in many of the earliest recordings of the disease, the treatments centered on the wound. this makes sense when you consider that humans from early in history understood that the origin of rabies manifested in humans after a canid bite, often while the animal was drooling copious amounts of saliva (frothing at the mouth). without listing every single early thought on treatment of these wounds, the common thread is to "bleed and cauterize (burn) the wound." for example, in early traditional indian medicine, there is acknowledgment of the fatality of hydrophobia (fear of water in rabies patients) and a prescription of treatment for it-bleeding and cauterization of the wound. the process involved cauterizing the wound with clarified butter, which the patient is then asked to drink. it is accepted even today that after an animal bite, it is wise to let it bleed, followed by rigorous washing and flushing of the wound with soap and water. however, simply closing the wound or cauterizing it will not cure one from rabies. • interestingly, the very pathogenesis of rabies in how it takes the central nervous system hostage to spread its deadly virions is now being studied to treat and possibly cure disease in brain cancer patients. the rabies virus, which kills tens of thousands of people a year, has a rare ability to enter nerve cells and use them as a conduit to infect brain tissue. now, scientists are trying to mimic this strategy to ferry tumorkilling nanoparticles into brain tumors. in laymen's terms, this means using viruses to carry tiny cancer killing agents to the tumor. so far, the approach has been shown to work only in mice. if successful in people, these nanoparticles could one day help doctors send treatment directly to tumors without harming healthy cells. • alarmingly, homeopathic and neuropathic treatments have gained some traction in the world in a variety of applications for mild to deadly diseases. rabies is not immune to these "treatments." while there is truth that some modern day medicines and drugs come from nature (e.g., penicillin is derived from the fungus penicillium), in most instances it does not replace peer-reviewed and sound clinical trial-based medical treatments or procedures. homeopathy principles roughly state that substances that produce similar symptoms of a particular ailment can cure said ailment ("like cures like") and that diluting a substance increases its potency ("law of infinitesimals"), which brings to mind the "hair of the dog" remedy for some hangover sufferers. recently, there was a report in which diluted saliva from a rabid dog was used to "treat" a -year-old boy for aggressive behavior. the first question that comes to mind is "where did the united kingdom homeopathic pharmacy obtain rabid dog saliva?" regardless, the rabid dog treatment, called lyssinum (aka lyssin or hydrophobinum), is one of more than homeopathic products approved by health canada. there is cause for concern, including those at the us food and drug administration, that such homeopathic products can be harmful and/or delay actual medical interventions and treatments. the homeopath who administered the treatment admitted that "there is no common consensus about how the remedies work" and continued to claim that it was effective and safe, plus added that the saliva was diluted to the point that it would not contain any trace of rabies virus. most rabies experts would have concerns about this treatment. • in an earlier section of this chapter, it was mentioned that some individuals (including some in health care or other biological science majors or backgrounds) incorrectly believe that rabies treatment requires or more shots into the stomach by some monstrously long needle. actually, almost all modern treatment regimens for rabies include four key features: • generous cleaning and lavage of the bite wound with soap or topical antiseptic and warm water. • avoidance of wound repair. closed bite wounds from dogs (or other animals) often become bacterially infected with early closure. the recommendation is to use very few sutures early and wait - days to more definitively close the wound. additionally, tetanus immunization boosters are indicated. • human rabies immune globulin (hrig) is essential for bridging immunity until the response to rabies vaccination occurs - days later. the hrig is most effective when infiltrated in the wound and not intramuscularly (im) remote from the bite wound. • rabies vaccine should be a modern cell-based, inactivated vaccine. rabies vaccine is effective when given intradermally or im, although only the latter route is licensed in the united states. the who recommends several schemata for rabies immunization. in the united states, only four doses (from the original five) are now recommended ( , , , and days) because an immune response is invariably present by day . however, a fifth dose is still recommended on day for individuals who are immunocompromised. simply put, rabies usually kills its victims without early intervention. with the recent and increased attention from stories of survivors of rabies, there have been recent reports from india of natural survivors, but most often with poor functional outcomes. , the exciting new milwaukee protocol and its use globally has produced four survivors who have had excellent cognitive recovery and outcomes. however, two had spastic diplegia that has been described in animal survivor models of rabies. it really should not be a surprise that rabies, a disease that goes back to the dawn of human civilization, continues to influence our pop culture (movies, television, art and literature, and tales told over generations). perhaps it is because rabies has always been known to be the very transformation of a disease from animal to man that is easily observed. the cdc estimates that zoonotic diseases are very common, both in america and globally. they estimate that more than out of every known infectious diseases in people are derived from animals and out of every new or emerging infectious diseases in people are transmitted from animals. historically, humans just did not see the connection of infectious disease to animals. scientists and scholars blamed nearly everything but animals. even some of the nastiest culprits of disease like smallpox (albeit this infection is not zoonotic) or black death (bubonic plague), which spreads to humans via respiratory droplets or by way of hitching a ride on a rat or other rodents via a flea vector, were often misunderstood regarding transmission or cause. usually, this meant that things such as demons, bad air (literal meaning of malaria), heavenly bodies/stars, and even human behavior were the root of disease. almost all of them were blamed on nonhuman hosts, except for rabies and anthrax. regardless of how far one goes back in time, one did not need to look further than the consequences of what happened after a human was bitten by a "mad dog" or other animal. in addition, to add insult to injury (literally), it was often the owner's very own best friend-the dog. if one looks to the time of greek myth, we find lycaon, king of arcadia, transforming into a slavering wolf with rabid and foamy jaws. in fifteenth century spain, we read about witch-hunters called saludadores and they were known as healers of rabies. during the interim of the th and th centuries, our european counterparts were building two well-known legendsthe werewolf and vampire-which had the ability to be human and animal and pass on this ability via the bite. even as we approach the th century with viruses becoming more understood by science and the discovery of pasteur's rabies vaccine, people of france were still transfixed and terrified by the fantasy and horror that a rabies infection converted people into maddened animals. ironically, their horror (fantasy) had some foundation. even after pasteur's vaccine, which miraculously could save one from certain death by rabies if given prior to signs and symptoms, humankind's ongoing rabid fantasy about possible monster metamorphoses with rabies continues in the pop culture and is going strong to this day. one needs to look no further than even possibly american's most trusted media icon, walt disney, when he released old yeller or about years later when the novel cujo (and later film) transfixed audiences to fear rabies (figs. . and . ). vampire movie after vampire movie arrived followed by zombie movies (i am legend, world war z, etc.) and the more current television series (the walking dead, fear the walking dead, etc.) (fig. . ). rabies even shows up in our comedy with appearances in shows like king of the hill and beavis and butt-head, as well as in an episode of the office. in the popular seinfeld episode-classic tv-one of the main characters, elaine, gets bitten by a dog, the owner evades her, she has to get pep (the doctor tells her the shot will hurt very much), and she keeps thinking she is showing signs of rabies (spitting back water, frothing at the mouth, etc.). it seems that rabies will always have a place in shaping pop culture, especially in the realm of the horror and science fiction genre. one can assume that because of this ongoing fascination with rabies in the arts and in our passing on of stories, often handing them down from one generation to the next, that we will continue to be fed misconceptions (although often based on science) about this diabolical virus known as rabies. it is incumbent for all of us in the modern era to dispel these myths and legends so that we can move forward in our efforts to assist humankind and downplay the sometimes-misguided fascination with this ongoing threat. realistically, those in the rabies medical, public health, and research community have a long way to go for increased disease survivorship and increased health literacy between health-care professionals and the public. to give just one example about our need to learn and communicate in the world of rabies, one needs to look no further than the variances in virulence and possible better outcomes from rabies virus variant phylogeny differencesthe reasons for the overrepresentation of the silver-haired bat, lasionycteris noctivagans (fig. . ) rabies virus variant in human infections are unclear. the frequency of infection, shedding, and dissemination of rabies virus in l. noctivagans, compared with myotis lucifugus and eptesicus fuscus, suggests the discrepancy of human rabies cases may be due to increased infectivity in heterospecific hosts, human susceptibility, and/or behavioral factors. in the interim, misconceived notions will most likely continue to be generated regarding the prevalence, signs and symptoms, diagnosis, and treatment protocols pertaining to this notorious and ancient killer known as rabies. it is incumbent upon all of us to be better stewards in the art of science communication with respect to decreasing the misunderstanding and sometimes panic surrounding this ancient disease. a silver-haired bat, the type that has been responsible for numerous human rabies cases and deaths. (reprinted with pemission by source, fair use: https://com mons.wikimedia.org/wiki/file:silver-haired_bat.jpg.) a cultural history of the world's most diabolical virus lyssaviruses and rabies: current conundrums, concerns, contradictions and controversies the new rabies plague. college station molecular epidemiology of rabies epizootics in texas typing of rabies virus isolates by dna enzyme immunoassay rabies: methods and guidelines for assessing a clinical rarity bat rabies evaluation of oral rabies vaccination programs for control of rabies epizootics in coyotes and gray foxes bat-associated rabies virus in skunks rabies: an old disease for a new generation controlling rabies at its source: the texas experience -oral rabies vaccination program rabies in skunks in texas epidemiology of rabies in bats in texas molecular diagnosis and epidemiology of rabies invited interview for outbreak news today radio podcast -rabies: history, myths and diagnosis on outbreak news this week centers for diseases control and prevention -explore travel health with the cdc yellow book obscure and little known facts about rabies current status of rabies and prospects for elimination rabies surveillance in the united states during centers for disease control and prevention. what are the signs and symptoms of rabies the many faces of rabies things you may not know about rabies -but should world health organization. rabies -guide for post-exposure prophylaxis misconceptions about rabies how to stop brain cancer-with rabies treated" -yr-old boy's behavior problems with saliva from rabid dog rabies: rare human infection -common questions survival from rabies encephalitis atypical rabies encephalitis in a six-year old boy: clinical, radiological, and laboratory findings rabies: still a uniformly fatal disease? historical occurrence, epidemiological trends, and paradigm shifts overwintering of rabies virus in silver haired bats key: cord- -o zspgrk authors: ippolito, g.; fusco, f.m.; caro, a. di; nisii, c.; pompa, m.g.; thinus, g.; pletschette, m.; capobianchi, m.r. title: facing the threat of highly infectious diseases in europe: the need for a networking approach date: - - journal: clin microbiol infect doi: . /j. - . . .x sha: doc_id: cord_uid: o zspgrk in recent years emerging and re-emerging infections, as well as the risk of bioterrorist events, have attracted increasing attention from health authorities because of the epidemic potential that renders some of them a real public health challenge. these highly infectious diseases (hids) are occurring more and more frequently in europe, and despite the many initiatives in place to face them, many unsolved problems remain, and coordinated efforts for dealing with hids appear mandatory. whereas uncoordinated measures would lead to only partial and poor responses to these emerging threats, networking represents a valuable approach to these diseases, in order to: (i) ensure a rapid and effective response; (ii) stimulate complementarity and prevent duplication; (iii) promote international cooperation, exchange of experience, good practice and protocols; and (iv) support the less prepared countries in the european community. despite hopes to the contrary, infectious diseases appear far from being defeated and continue to claim the attention of public health authorities. particularly in recent years, yet to be fully understood changes in the environment, increased movement of goods and persons, and the local influence of global warming and other phenomena concerning vectors and hosts, seem to have promoted and accelerated changes in the presentation of old infectious diseases and the development of new ones [ ] [ ] [ ] . the relevance of the 'emerging and re-emerging' infectious diseases, usually defined as 'infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range', is further noted by the who in its recent world health report [ ] . the who stressed that infectious diseases are spreading faster and emerging more quickly than ever before. some emerging and re-emerging diseases represent a real challenge because of their epidemic potential. recently, many global alarms involving infectious diseases-such as the anthrax crisis in the usa, the emergence of sars, the pandemic threat posed by the highly pathogenic avian influenza a (h n ), and the cases of imported or autochthonous viral haemorrhagic fever (vhfs) in europe-have highlighted the need to improve preparedness for these highly infectious diseases (hids), also in order to increase certain aspects of what is perceived in many areas as an issue of collective and national security [ ] . emerging hids are of particular concern because they usually hit relatively unprepared public health systems, and appropriate diagnostic tests, vaccines, drugs, containment and mitigation measures are frequently not available or not immediately so. a similar situation could occur if a pandemic strain of influenza virus emerges: several surveys conducted in european countries and in the usa have revealed many gaps in their preparedness plans, in particular in terms of making such plans truly operational, in stepping up prevention measures against seasonal influenza, in ensuring essential services, in enhancing collaboration with adjacent countries, and in extending and better directing influenza research [ ] [ ] [ ] . research on emerging infectious diseases has been funded since the inception of the european union (eu) framework programmes (fp) for research in . in the eu developed recommendations for early diagnosis and management of bioterrorism-related infections, with the aim of providing member states with a common basis for dealing with these diseases [ ] . among activities/projects covered in the fp - , more than half are focused on various aspects of influenza, which makes the commission's fps arguably the single largest funding source for influenza research in europe. the other topics covered include: vhfs, sars, transmissible spongiform encephalopathies, food-and water-borne diseases, other zoonoses, as well as issues such as preparedness and capacity building for different diseases in a more generic fashion. in total, both influenza research and research on other emerging infectious diseases have received more than € million of eu funding each since . a complete searchable list and short descriptions of all projects, grouped into different categories (as well as a downloadable pdf version) is available online [ ] . concurrent with the increasing awareness of the threat of a new influenza pandemic, the current fp - introduces for the first time a specific area dedicated to 'potentially new and re-emerging epidemics', specifying that its 'focus will be on confronting emerging pathogens with pandemic potential including zoonoses'. the term 'potentially new and re-emerging epidemics', which is uncommon in the scientific literature, refers mainly to those emerging viral diseases of current or future relevance for europe. this new mandate to cover research systematically in the area of emerging epidemics establishes a focal point within the fp from which calls for proposals in this area can be strategically planned. the past calls in the area of emerging and re-emerging infectious diseases were frequently published ad hoc, in response to specific threats, and the lack of a dedicated area was responsible for the limited coordination and long-term planning. the new mandate in fp specifically dedicated to diseases should overcome these problems. although research on influenza will continue to receive support in view of the magnitude and likelihood of an influenza pandemic, future calls will increasingly build a strategic european research capacity for other emerging and re-emerging hids. a definition of hids and the agents/diseases included are summarized in table . several cases of these diseases have been reported in europe since : cases of sars were imported in eight countries, and approximately imported confirmed or suspected cases of vhfs have been reported, mainly lassa fever [ ] [ ] [ ] [ ] . very recently, two isolated cases of lassa fever have been diagnosed in london in travellers who returned to the uk from nigeria and mali [ , ] , and several cases of autochthonous crimean-congo haemorrhagic fevers have been reported in the european region (in turkey and in some states in the balkans) and in some countries within the eu (bulgaria and greece) [ , ] . no human cases of highly pathogenic influenza a (h n ) virus have occurred in europe, but two suspected cases were managed in the netherlands and belgium, and public health authorities in greece faced a pseudo-outbreak [ ] [ ] [ ] . moreover, several recent cases of cowpox infections have been reported recently in europe: confirmed cases in germany, one suspected case in the netherlands, five confirmed and seven suspected cases in france. although human cowpoxvirus infections are not classified as hid, these cases are worth mentioning here as an example of how an unexpected agent can disseminate rapidly. some of the cases described above were proven to be caused by the same virus, indicating exposure to a common source of infection related to an international trade in pet rats by a czech rat breeder [ ] . two cases of human infection with an orthopoxvirus, similar to but distinct from cowpox, have been identified in north-eastern italy in two veterinary doctors who had been exposed to infected cats [ ] . this finding, and the fact that the two infections occurred independently of one another, underscore the need to enhance awareness of zoonotic poxvirus transmission (possibly endemic) also in regions where this problem has not been addressed so far, e.g. the southern alps. almost all of the cases requiring isolation were first admitted to a general hospital without adequate isolation capabilities, and later transferred to a high-level isolation unit. despite the fact that no outbreaks occurred in europe, these experiences exposed weaknesses in terms of recognition, public health response, and diagnostic and clinical management. indeed, despite the wide availability of national and international plans and guidelines, their application in 'real-life' scenarios remains poor. not surprisingly, public health policies and diagnostic and clinical approaches to hids differ widely among european countries, and a common platform that would enable scientists to respond in a quick and powerful manner is still lacking. hids require multidisciplinary expertise. experts in microbiology (especially virology), public health, epidemiology, infectious diseases, and communication need to work together to respond to such incidents. for hids in particular, because of the rarity of their occurrence, strong collaboration and exchange of data, and attention to lessons learned from previous episodes, are advisable. for these reasons, creating new networks and enhancing those functioning well should be strongly promoted, in order to: ensure a rapid and effective response to health threats deriving from natural infection by or deliberate release of hid agents; stimulate complementarity and prevent duplication; promote international cooperation, exchange of experience, good practice and protocols; support the less prepared countries in the european community. a continuous effort is necessary for sustaining and promoting research on hids, whether basic or translational, in order to promote the increase of general knowledge concerning on these issues and to support the development of new tools for facing them in an effective manner. the development and refinement of new diagnostic tests, new therapeutics and innovative vaccines is mandatory as never before. the use of networking and international partnership could represent the successful strategy in this context. the traditional boundaries between basic science and clinical medicine should be dropped, and through effective networks the few hid events that occur worldwide should be studied thoroughly. a network of top-quality scientists and clinicians will provide the complementarity required for the development of these new approaches. moreover, improved funding for research on hids could come from the involvement of networks in the private sector, which could be encouraged to invest in this area because of the epidemic potential and the possible large-scale economic consequences. early recognition and prompt reaction to hids rest upon adequate preparedness, which should include the availability of adequate infrastructures and specific training for healthcare workers. several differences exist among european countries, due to government policies, as well as to pre-existing conditions, and these may result in delayed and dissimilar public health interventions and non-standardized training programmes. a better coordination of public health approaches to hids may lead to standardization of interventions and protocols, such as the prompt isolation within structures with adequate technical and logistic features, to the development of a common core-curriculum, and substantial improvement in the application of international health regulations. moreover, from a practical perspective, a network involving the main public health institutes may play a key role in the management of: a returning hid patient travelling through more than one country (e.g. in the case of one or more connecting flights), in order to coordinate public health interventions; an hid patient admitted in a country without adequate healthcare settings for isolation (in this case, a cross-border transport by ground or air may be the most appropriate solution); multi-country outbreaks. due to the current perceived international security threats, several eu member states are considering establishing biosafety level (bsl)- diagnostic facilities. to improve and sustain the existing initiatives and networks aimed at promoting collaboration among the existing bsl- laboratories appears mandatory, as well as to provide assistance, through these networks, to other european countries not equipped with such sophisticated and costly facilities [ ] . moreover, among the critical points identified in the context of the laboratory diagnosis of hid agents are the scarcity of biological samples to validate the diagnostic methods and the fact that few commercial diagnostic tests are available for these pathogens. thus, a well-functioning network is essential for: the sharing of diagnostic and research experience of the currently operating bsl- european laboratories, as well as diagnostic protocols, samples, reagents, and personnel for training; the review of current laboratory diagnostic capability for hid agents; the development of new hazard-free diagnostic tests suitable to be transferred to other non-bsl- laboratories; the standardization of procedures for biosafety and biosecurity. intra-hospital procedures for clinical assistance and infection control for hid cases represent 'the core' of managing these diseases, and represent effective measures for hid containment. on the other hand, hospitals may play an important role in the amplification of an outbreak if infection control measures are inconsistently applied. consequently, common protocols for infection control and biosafety during the clinical and diagnostic management of hids patients, based on the available evidence and on 'reallife' experiences, are strongly advisable [ ] [ ] [ ] [ ] . moreover, in order to offer to these patients the best available standards of care, a set of specific skills is required, and thus, given the scarcity of these events, a functioning network for expert consultation, second opinion, and scientific support is needed. as more and more persons, animals and goods move within europe, the need for improved and coordinated responses to hids continues to grow. furthermore, it is increasingly recognized that hids can pose a significant threat to each country's national security. innovative research and coordinated efforts, through the establishment of well-functioning networks, are the only way to deal with these issues, in order to improve preparedness and to react quickly: in short, to be 'prepared for the unknown'. a key role may be played by the european centre for disease prevention and control, whose mission is, among others, to 'coordinate the european networking of bodies operating in the fields within the centre's mission' [ ] . uncoordinated measures can lead to only partial and poor responses, and different approaches to similar health threats in various eu countries are likely to negatively affect the compliance by health professionals, and the perception of the population. although well-functioning networks are already in place, many gaps still exist, as well as opportunities for future collaboration. fortunately, new scientific developments, and new perceptions of these health threats, make this field one of the most stimulating research areas with a direct impact on the health of millions of people. global trends in emerging infectious diseases climate change and infectious disease: a dangerous liaison? european lab network prepares for high-risk pathogen threat world health organisation: geneva infectious diseases and national security how prepared is europe for pandemic influenza? analysis of national plans challenges remain in preparedness european centre for disease prevention and control gouvras g task force on biological and chemical agent threats, public health directorate, european commission, luxembourg. bichat clinical guidelines for bioterrorist agents european commission quality assurance for the diagnostics of viral diseases to enhance the emergency preparedness in europe no authors listed e-alert july: case of lassa fever imported into germany from sierra leone marburg hemorrhagic fever -the netherlands ex uganda a fatal case of lassa fever in london the first case of lassa fever imported from mali to the united kingdom crimean-congo hemorrhagic fever in greece: a public health perspective probable cases of crimean-congohaemorrhagic fever in bulgaria: a preliminary report management of potential human cases of influenza a/h n : lessons from belgium a dutch case of atypical pneumonia after culling of h n positive ducks in bavaria was found infected with chlamydophila psittaci a pseudo-outbreak of human a/h n infections in greece and its public health implications european centre for disease prevention and control. cowpox in germany and france related to rodent pets cat-to-human orthopoxvirus transmission, northeastern italy networking for infectious-disease emergencies in europe risk management of febrile respiratory illness in emergency departments the initial hospital response to an epidemic framework for the design and operation of highlevel isolation units: consensus of the european network of infectious diseases infection control in the management of highly pathogenic infectious diseases: consensus statement from the european network for infectious diseases the authors wish to thank c. schmaltz (european commission, research directorate general, brussels) for the data about eu-funded research projects, and for his invaluable suggestions and critical reading of the manuscript. all authors declare no dual or conflicting interests. key: cord- - ickafd authors: kapil, sanjay; yeary, teresa; johnson, bill title: diagnostic investigation of emerging viruses of companion animals date: - - journal: vet clin north am small anim pract doi: . /j.cvsm. . . sha: doc_id: cord_uid: ickafd in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable, because viruses are highly mutagenic. even the immune response dictates the nature of virus that evolves in a host. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with the diagnostic laboratory to solve the diagnostic puzzle. c linicians and laboratorians are usually the first to detect most outbreaks of emerging diseases in animals. much attention is rightfully given to emerging diseases of commercial food animals; however, small animal practitioners also have an obligation to be vigilant to the possibility that new and devastating viral diseases might emerge that infect the companion animals in their charge. canine parvovirus (cpv) type , emerged in and spread worldwide within less than years [ ] . in , a new antigenic type, cpv- c, was reported in italy [ ] , which has since caused outbreaks in western europe, asia, south america, and the united states [ ] because current vaccines offer no protection for this type. in this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. the term emerging virus is defined broadly and includes these categories: variants of a known virus that has gained enhanced virulence or that is able to infect completely vaccinated animals a known virus that has reappeared in the population after a decline in incidence novel or previously unidentified viral agents detected for the first time because of improved diagnostic capabilities ''mystery diseases'' with large numbers of naive animals involved that are caused by previously uncharacterized viruses spread of an emerging virus among small companion animals is multifactorial and includes animal health and sanitation practices; migration of a pathogen from a wild reservoir to domestic animals because of changes in populations, trade, climate, land use, and the introduction of invasive species (eg, plant, animal, insect); and, finally, globalization, as was the case with west nile virus (wnv). emerging viral infections may take a heavy toll on the health of cats or dogs whenever they are brought into situations in which groups of animals are housed together, even temporarily, such as at greyhound racetracks, kennels, catteries, animal shelters, animal obedience training classes, dog parks, pet stores, pet day care facilities. this is especially true when pets are allowed by their owners to roam at will, commingling with ownerless feral dogs and cats and wildlife. for example, the rapid spread of cpv- , which is extremely stable in the environment and highly contagious, was caused not only by the movement of dogs by their owners but by the transfer of fecal material on shoes and clothing of travelers and, unintentionally, through national and international mail [ ] . according to the to national pet owners survey conducted by the american pet products manufacturers association, the us pet cat population is estimated to be . million and the pet dog population is estimated to be . million [ ] . municipalities throughout the united states commonly pass animal control ordinances to protect the public health and safety and general welfare of the citizens and animals residing within the city. typically, animal control codes limit the numbers of companion animals that individuals may own or keep on their private property, require that cats and dogs be licensed annually by owners and vaccinated against rabies, prevent animals from running at large, require proper disposal of animal waste, and prevent the feeding of wild or feral cats or dogs. vaccination of dogs and cats by compliant pet owners for rabies prevention has, since , dramatically reduced the occurrence of this disease; currently, most animal cases reported to the centers for disease control and prevention (cdc) now occur in wildlife [ ] . compliance with other animal control ordinances is variable, particularly among pet owners with respect to leash laws for dogs and cats and among well-intentioned individuals who maintain wild or feral colonies of cats and dogs by providing food, water, and shelter. statistics from the humane society of the united states indicate that to million companion animals are admitted to shelters each year and nearly half are adopted or reclaimed by their owners, whereas the remaining animals are euthanized [ ] . no census of ownerless dogs and cats is available. estimates of the feral cat population in the united states range from million to million animals living primarily in or near urban settings with ample opportunity to interact with pets that are allowed to roam and with wildlife [ ] . thus, ownerless, wild, or feral dog and cat populations may transmit infectious and zoonotic diseases between wildlife and companion animals. from a public health standpoint, this is of particular importance because emerging viral infections from wildlife are often transmitted to human beings by means of a pet that is allowed to stray. it is widely believed by virologists and public health epidemiologists that most viruses emerging from wildlife have an rna or single-strand dna genome [ ] because they have a high propensity for mutation. two significant canine viruses have emerged recently and meet this hypothesis: cpv and canine distemper virus (cdv). canine distemper has re-emerged in the past decade [ , ] because of antigenic and genetic drift in the surface protein (h glycoprotein). in a multicontinent study, variant cdv strains, (but not the vaccine strain of cdv virus) were the cause of illness within weeks after vaccination. in and , large outbreaks of cpv variants (cpv- c and cpv- b*) in kennels occurred in oklahoma and other states [ ] . diagnostic and molecular studies detected mutations in the parvovirus isolates that explained the failures of current commercial cpv vaccines from conferring protection and of approved commercial diagnostic kits from detecting these new viral isolates. another recent example is outbreaks of hemorrhagic symptoms associated with virulent feline calicivirus (fcv) in the united states [ ] ; however, molecular basis of gain of virulence in fcv is not yet understood. in addition to virus evolution, in some cases, the virus can be reintroduced back after the population immunity has declined after a period of disease-free status. thus, diseases that have been eradicated from developed countries but are still circulating in developing countries [ ] may re-emerge by reintroduction from trade or movement of animals. there is a major commitment by the us department of agriculture (usda) in this country and in cooperation with foreign governments and international agencies worldwide to monitor the health of food animals and certain wildlife but not of companion animals [ ] . the primary mission of the cdc is to promote and protect human health. to this end, the cdc performs surveillance for noninfectious and infectious diseases, including zoonoses [ ] ; however, the only chosen reportable viral diseases of animals that are collected by the cdc are rabies and avian influenza (h n ), and those that are reported to the cdc arbonet system are avian, animal, or mosquito wnv infections. largely, surveillance of companion animal diseases, many of which have zoonotic potential, has not been considered to be a priority until recently [ , ] . in , the cdc partnered with the purdue university school of veterinary medicine to establish a pilot surveillance system to monitor clinical syndromes and diseases of small animals [ ] to determine whether animals can serve as sentinels of health hazards to human beings. the national companion animal surveillance program (ncasp) initially drew exclusively on the database of the privately owned organization, banfield, the pet hospital, which provides medical care to approximately . million pet dogs and cats in states, and it now integrates data from antech diagnostics to detect potential emerging and zoonotic infections. a long-term goal of the ncasp is to become a national resource in veterinary public health. in the meantime, the front line of companion animal surveillance for emerging diseases is at the home front, with astute small animal clinicians playing a major role. it can be a challenge for busy and isolated veterinary practices to receive the information on emerging viruses. linking to a health-related network for companion animals might fill the gap. recently, a space-time permutation scan statistic, which was applied in the anthrax terrorist attacks in [ ] , wnv outbreaks [ ] , and enzootic raccoon rabies [ ] , has been applied to veterinary diagnostic data in the unite states and europe [ ] . this analysis provides important information about potential clusters of medical conditions and issues medical alerts about the developing situations based on mortality and confirmed diagnosis of important disease conditions. earlier and more timely notifications should lead to more thorough investigations and reduce losses, especially from emerging viral diseases. it is important to keep in mind that clinical syndromes tend to be multifactorial, and it is essential to review the entire history, including environmental factors, with the specialist in a small animal specialty practice and also with a small animal teaching hospital before arriving at a conclusion about the case. the purpose of this article is to encourage companion animal veterinarians to think outside the routine diagnostic plan when atypical cases of infectious disease are presented at their practices. detecting emerging viral diseases of companion animals requires interaction and discussion among clinicians, pathologists, and virologists, and practicing small animal veterinarians must stay engaged in communication with these specialists through their state diagnostic laboratories or nearby colleges of veterinary medicine. veterinary diagnostic medicine is rapidly progressing, and it is critical for the successful practitioner to stay abreast of new developments in small animal infectious diseases and their diagnosis through continuing education [ ] [ ] [ ] . the development of monoclonal antibody technology in the s and the advent of the polymerase chain reaction (pcr) assay in the s have reshaped veterinary diagnostic strategies, especially in the subspecialty of virology. now, these molecular techniques, which are becoming mainstream applications in routine viral diagnoses, are proving their merit in facilitating the diagnosis of emerging animal viruses. the authors offer practical information on the applications of diagnostic techniques for investigating viral disease outbreaks in companion animals. the authors provide this brief overview of diagnostic techniques in the modern virology laboratory that are used for routine diagnosis and in identifying novel and emerging viruses. every step of diagnostic investigation-history, specimen collection, transportation, and laboratory examination-has to be carefully aligned for optimal outcome. small animal clinicians are familiar with symptoms of common infectious diseases and are often the first to recognize the emergence of new disease problems. in some cases, there may be a history of vaccination compliance, yet some animals develop disease [ , ] . it is important to record the complete history, including the body system involved (eg, respiratory, gastrointestinal, reproductive tract, nervous system), clinical symptoms and their duration, the presence of lesions, and vaccination history. particularly when the case is confounding, the client must be carefully and thoroughly interviewed as to how he or she manages the pet (ie, is the pet free to roam; has the pet traveled recently and where; if this is a new pet, where and how was it obtained; are there other pets in the household). consulting a book on differential diagnoses can be useful to list the potential causes [ , ] . when a history of unusual symptoms is presented, clinicians, recognizing that these cases may be important to individual and universal animal health, should refer these cases to an accredited veterinary diagnostic laboratory. it is convenient to attach copies of all relevant hospital records to the laboratory submission form to aid the diagnostician. correct diagnosis depends on a thorough case history of the affected animal and submission of appropriate specimens that are collected and transported in a manner to preserve the integrity of the viral agent. submitting a comprehensive collection of specimens in a timely manner to the diagnostic laboratory from affected animals when the disease does not fit a familiar clinical picture, as is the case with emerging viral diseases, is of paramount importance. all the system(s) that are potentially involved and all the tissues with gross lesions should be sent to the diagnostic laboratory. it is important to check for concurrent infections. viral diagnosis depends on the quality and type of specimen collected [ ] . the best time for collection of specimens is immediately after symptoms of disease are first noticed. samples from all body systems involved in the acute stage of the disease of affected animals should be submitted to the diagnostic laboratory in a timely manner by overnight delivery. at least to g or ml of each sample should be collected. recovery of virus in cell culture depends on the condition of the specimen received by the diagnostic laboratory. freezing specimens can be detrimental to virus isolation efforts (and also to electron microscopic identification) and should only be done (À c) if it is not possible to deliver the specimen to the laboratory within hours. use wet ice for shipping virology samples, because dry ice (solid carbon dioxide gas) can inactivate many viruses, preventing isolation in cell culture. tissues intended for virus isolation should always be shipped in separate packages from specimens that are immersed in formalin to prevent fumes of formaldehyde from reaching the fresh tissues. it is imperative that tissues and organs from animals that have died be harvested as soon as possible after death. postmortem tissues should be placed in sterile containers with a small amount of transport medium ( - ml), if possible. when the clinician is unsure as to what specific organs and fluids should be retrieved, the entire carcass of the dog or cat may be delivered to the laboratory for examination. to obtain more specific details regarding specimen collection, packaging, and submission, contact the diagnostic laboratory of your choice by telephone or consult its specimen submission and fee schedule guidelines, which are often available on an internet web site. individuals who ship biologic substances for diagnostic testing are required by federal law to be in compliance with all regulations governing packaging and labeling of interstate shipments of causative agents. failure to follow the regulations results in heavy fines (fig. ) . complete instructions on appropriate packaging for laboratory specimens to be mailed or shipped by a common carrier may be accessed in several sections of the code of federal regulations (cfr). health and human service regulations define such terms as diagnostic specimen and etiologic agent and describe requirements for packaging and labeling viruses have a simple structure with a protein coat enclosed with only one type of nucleic acid (dna or rna) rather than both. thus, methods for viral diagnosis target one of the components of the virus structure. for a definitive viral disease diagnosis, four basic approaches are used: direct detection by virus isolation or direct identification, viral serology for detection of a specific antibody, viral antigen detection, and molecular-based detection of genetic material. a brief discussion of the principles of diagnostic assays representative of each approach follows. gross pathologic and histopathologic findings histologic (fig. ) and cytologic examination (fig. ) of tissues and fluids by a board-certified veterinary pathologist contributes valuable information about the pathologic signs, gross and microscopic, that distinguish infections caused by viral or bacterial pathogens and other possible etiologies. tissue tropism, mononuclear infiltrates, development of inclusion bodies (intranuclear, cytoplasmic, or both), and the formation of syncytia are some of the characteristics that differ among viruses and can sometimes distinguish different viral infections. for example, most dna viruses replicate in the nucleus, and thus tend to produce intranuclear inclusions, whereas most rna viruses form cytoplasmic inclusions, although there are exceptions. as part of the pathologist's examination, immunohistochemistry testing (figs. and ), fluorescent antibody testing, and possibly in situ hybridization (ish) studies on tissues may be ordered; these methods are considered elsewhere in this article. a complete histopathology report should include possible differentials for the lesions. the pathologist might note that some findings do not exactly fit the routine lesions he or she has observed in previously. in cases in which there are deviations in lesion type or distribution or when gross lesions and histopathologic findings suggest the involvement of a viral disease but routine virology tests do not detect the expected conventional viral agents, variant or ''emerging'' viruses or even iatrogenic infections may be suspected. in early , blue tongue virus serotype was introduced in canine populations from a commercial modified-live multivalent canine vaccine that was associated with high mortality in dogs [ , ] . in some situations, second or even third opinions from pathologists at other laboratories who have special expertise should be solicited [ ] . with the application of telepathology to veterinary case materials, networks of specialists, including veterinary pathologists, small animal clinicians, infectious disease specialists, and laboratory diagnosticians, are able to exchange patient histories, clinical data, and images (gross and microscopic) through the internet for consultation, diagnosis, and education. this allows timely access to expert opinions at other locations throughout the world [ , ] . the use of telepathology can facilitate rapid intervention through the synergy of computer technology and special pathology expertise (eg, system-and speciesspecific pathologic findings) to understand the lesions in difficult cases better. conventional virus isolation techniques are often the backbone of investigation of novel viral diseases, provided that the virus is cultivable in available cell lines or primary cell cultures. virus isolation may be relatively slow depending on the growth characteristics of the virus; however, roller culturing or centrifugation of samples onto cell monolayer(s) can enhance viral replication and recovery. in many of the recent emerging viruses from wildlife (eg, bats), the virus was first cultivated, allowing further characterization of the virus. it is important to keep in mind that virus isolation, even if the effort is successful, may have a slow turn-around time, approximately to weeks. definitive identification of virus in cell culture can only be accomplished with specific antibody nucleic acid testing, and in the case of an ''emerging'' virus, existing reagents may not be reactive with the ''new'' virus. if culture is successful, however, the viral material may be studied by electron microscopy (em) and by molecular techniques, as described in this article, to characterize the new isolate. virus isolation requires fresh tissues and cannot be done on formalin-fixed tissues. em is often used in veterinary diagnostic laboratories to detect enteric viruses in fecal samples retrieved during the course of viral diarrheal disease. additionally, em is indispensable for identification of emerging and previously unidentified viruses in clinical samples [ ] , and this method has helped in the identification of many new viruses, including, most recently, bat lyssavirus [ ] . viruses can be classified up to the virus family based on size, shape, and distinctive structural features, such as envelopes or protein spikes, particularly for parvovirus, rotavirus (fig. ) , coronavirus, astrovirus, herpesvirus, poxvirus, and picornavirus. em allows detection of multiple viruses simultaneously. application of antibodies to supplement the em diagnosis provides higher sensitivity and further confirmation of the viral diagnosis. sensitivity is the major limitation of em, and at least to virus particles per milliliter must be present in the sample being examined. because the electron microscope is an expensive piece of equipment that requires special technical skills and a high level of expertise, it is not available in many laboratories. viral components can also be determined by several basic biochemistry experiments. acridine orange (ao) staining can determine the nature of the nucleic acid of purified viral particles [ ] . differentiation as to whether the nucleic acid is single-or double-stranded in nature is based on the color developed on ao staining; double-stranded dna or rna nucleic acids stain yellow green, whereas single-stranded dna or rna acids stain flame red. nuclease susceptibility of the purified virions differentiates dna from rna. the presence of envelope on viruses can be determined by susceptibility to the virus to heat, ether, or other lipid solvents [ ] . the titrated virus preparation is treated with ether or chloroform. a decrease in virus titer of greater than log is considered to be significant to indicate the presence of envelope on the virus. the presence of envelope indicates that virus is susceptible to common disinfectants. lack of envelope indicates that the virus is resistant to the use of common disinfectants. classic serology tests indirectly determine the viral etiology of disease by detecting the presence of antibody in serum (red-topped tube) to a specific test viral antigen, and thus provide retrospective evidence of an immune response or exposure to a virus. serologic methods still provide powerful tools in the virology laboratory of today for diagnosing viral diseases that are seen routinely and for discovering and characterizing novel viral diseases. serologic tests are now used to detect antibody or antigen in serum and body fluids. typically, methods used in the virology laboratory are serum neutralization (sn), hemagglutination-inhibition (hai) test, indirect fluorescent antibody test (ifat), and elisa. serologic results require interpretation by an expert diagnostician based on critical clinical observations, confirmation by pathology examination, virus isolation, and mass screening of the populations by serology. if animals in populations that have never been exposed to or vaccinated against a given virus have specific antibodies detected in their serum, it is expected that this is most likely attributable to recent exposure to the emerging virus. paired serum samples are important to demonstrate a fourfold significant increase in antibody titers, which indicates that the diagnosis of recent exposure may be attributable to infection as opposed to previous exposure or vaccination depending on the vaccination history. serology is also useful to study the antigenic distance of the emerging virus and provides clues as to whether the newly emerged agent is or is not likely to be protected by an available vaccine(s), such as heterologous virus in another species of animal. viral hemagglutination (ha) occurs between the viral protein; hemagglutinin (hn), which is present on the viral capsid or envelope of only certain families of viruses; and specific receptors on red blood cells (rbcs) that bind to hn, causing their agglutination and precipitation from solution. this phenomenon is the basis for a powerful and sensitive assay, the hai test. when a hemagglutinating virus is mixed with serum containing antibodies specific to that virus, rbcs that are added to the mixture do not agglutinate and precipitate from solution. feline panleukopenia, cpv, influenza a, and parainfluenza antibodies may be detected by hai testing. the hai method may also be used to identify unknown virus utilizing antibodies of known specificity; however, most often, this test is applied to detect the presence of antibodies in a serum sample against specific hemagglutinating viruses. variants of cpv and feline parvovirus can differ in the hemagglutinating activity of swine erythrocytes [ , ] . sn measures the inhibitory activity of a hyperimmune serum against viral isolates in cell culture. commonly performed in a cell culture microwell format, this is a long-standing method for quantifying virus-specific antibodies, and it is usually performed to test for antibodies to viruses that typically cause cell damage (cytopathic effect [cpe] ) to the host cell culture they infect. when a virus is mixed with hyperimmune serum containing antibodies specific to that virus, the antibodies bind the virus, preventing infection of the cell culture. the sn test can diagnose current infection using acute and convalescent serum samples from individual animals. it may also be used to determine immune status conferred on vaccinated animals. vaccination antibody titers often differ from antibody titers developed in response to natural infection. usually, vaccination titers are lower relative to infection titers, and maximal titers occur approximately to days after vaccination. sn assays are commonly performed to detect antibodies to fcv, herpesvirus, enteric coronavirus, and syncytial viruses and to canine herpesvirus, cdv, coronavirus, parainfluenza virus, and adenovirus. elisa this is useful for screening large numbers of samples for the presence of antibodies against viruses. the elisa format is flexible, and it may be used to detect antibody or antigen in clinical specimens. in either case, the detection system is an antibody conjugated to an enzyme. when the enzyme-linked antibody binds to the analyte being measured, the enzyme reacts with a chromogenic substrate, causing a color change to occur that may be measured spectrophotometrically or evaluated visually. several elisa kits are available to detect antiviral antibodies in companion animals, including cpv and cdv, feline leukemia virus (felv), feline immunodeficiency virus (fiv), and feline coronavirus. the immunoglobulin m (igm) elisa is a method used to distinguish current infection from past infection. during acute disease or immediately after vaccination with modified-live viruses, igm is the first class of immunoglobulin produced in response to infection, appearing to weeks before there are detectable levels of igg in the serum. because it is short-lived, igm levels typically disappear months after infection. a single acute-phase serum test sample is sufficient to diagnose current infection with an igm elisa. testing of igm titers is available for several viral agents, including cdv and cpv among others. elisa is useful for screening naive animal populations for the presence of antibodies against viruses to track the origin and spread of emerging infections. antibodies to wnv have recently been detected in dogs and cats by igm-capture elisa [ ] . a related method known as virus neutralization can be used to identify the serotype of a newly discovered virus. western blot (wb) may be used as a supplementary test to confirm antibody elisa results for fiv testing [ ] . to perform the assay, purified virus is disrupted using detergent; the constituent proteins are then separated on the basis of molecular weight by electrophoresis in a polyacrylamide gel. the proteins are transferred (blotted) from the gel to a nitrocellulose or polytetrafluoroethylene (ptfe) membrane for stabilization. the electrophoretically separated proteins are the antigen substrates for analyzing the test sera for the presence of specific antibodies. as with the elisa format, the western immunoblot uses an enzyme-labeled antispecies antibody that binds to the test serum antibodies that have bound to the separated viral antigens. substrate reacting with the enzyme-labeled antibody in the presence of a colorless soluble benzidine derivative results in conversion to colored insoluble precipitate at the protein bands where test serum antibodies are bound. the molecular weight of the protein detected is characteristic for a particular viral component. immunoblot results of the unknown test antisera are compared with positive control test sera for interpretation. a major advantage of the immunoblot technique is that a full antibody profile of a single serum sample is made simultaneously, identifying each of the individual particulate viral antigens that patient antibodies bind. as an epidemiologic tool, wb analysis may be used to detect currently circulating viral subtypes within a population and to characterize new emerging viral subtypes. immunoblotting is also a valuable research technique for antigen detection that is often used to characterize novel viruses by comparing them with known related viral family members using standard antisera or monoclonal antibodies. immunofluorescence assays on cells from clinical samples can be applied for rapid diagnostic investigations ( - minutes), provided that the fluorescent microscope and expertise are available in a laboratory. with the pooling of primary monoclonal antibodies against potential viral agents, the assay can be used as a screening tool and the sample tested again with individual conjugates to obtain specific virus diagnosis (fig. ) . the elisa is also a means for detecting viral antigens present in clinical specimens, and it offers a relatively quick turn-around time. antigen test elisa kits are available to detect antiviral antigens in companion animals, including cpv, felv, and fiv. additionally, it is a common practice by many veterinary diagnostic laboratories to appropriate the use of some rapid antigen test kits intended for the human diagnostic market, specifically, rotavirus test kits. when monoclonal antibodies are used as capture antibodies in elisa test kits, however, they fail if there is a mutation in the epitope of the viral surface protein present in the specimen that is being tested. lateral flow immunoassay is a special application of the elisa that provides a rapid, economic, portable, sensitive, and specific technique that is convenient for performing testing outside of the laboratory. it is the technique of choice for emerging viral infections [ , ] , and it has gained attention for use in diagnosing foreign animal diseases and zoonotic and emerging viral infections of animals, such as influenza virus and wnv, in the field. the test kits are small in size (size of credit cards), extremely stable at ambient temperature ( c), and take minutes to perform. an advantage of nucleic acid-based testing is that specimens submitted for analysis do not have to have viable viral particles present to be detected by this means. there is a trend toward application of molecular or gene sequence-based techniques to routine virology testing in diagnostic laboratories, which is justified under several circumstances. first, a molecular technique may be the test of choice if conventional methods of diagnosis are technically weak, such as when a viral agent is noncultivable or there are biocontainment concerns with culturing the virus, the virus has amorphous morphology by em, antibodies are unavailable or not specific to the virus, and serologic tests result in a confounding diagnosis. second, molecular techniques may be essential to detect and classify the sequence type or genotype of a virus. third, a viral agent may be characteristically slow to replicate, such as c-herpes virus; thus, a molecular method might provide a better turn-around time for diagnosis. in this instance, a rapid diagnosis might be achieved by pan-herpesvirus pcr. finally, a novel viral isolate that cannot be definitively identified by the routine diagnostic methods described previously may merit investigation and characterization by molecular-based techniques, which are indispensable in the classification of new and emerging viruses. these advanced techniques may confirm a diagnosis of viral etiology when other tests have failed; however, they are, unfortunately, relatively expensive. furthermore, the presence of nucleic acid does not equate to infection, and infections are attributable to subclinical, latency-associated nucleic acids or defective interfering virus particles, such as in paramyxoviruses, produced in nonproductive infections in genetically resistant hosts. clients, who bear the financial burden, should be counseled as to the benefit and shortfalls of this testing before ordering molecular-based tests. an excellent review of molecular-based techniques for diagnostic testing of infectious diseases has appeared in a previous issue in this series [ ] . the most familiar nucleic acid testing technique, pcr, has been used for more than a decade; however, over the past few years, real-time pcr has taken its place, revolutionizing diagnostic virology. in this procedure, the pcr chemistry may be combined with detection using a single-stranded dna probe with a fluorescent label [ ] . moreover, the procedure may be completed within an hour, and it allows for quantitation of results. because the hands-on steps are reduced and the pcr reactions are not opened, it eliminates the chances of cross-contamination in the laboratory. real-time pcr protocols are gaining more acceptance in routine veterinary diagnosis. in situ hybridization ish involves using nucleotide probes with an attached label. non-isotopelabeled probes (digoxigenin or fluochrome) can be applied in veterinary diagnostic laboratories. diagnostic applications of ish involve identification of virus-specific sequences (dna or rna) in the tissues or cells [ ] . although uncommon in veterinary diagnostic laboratories, ish is in routine use in human diagnostic laboratories for detection of the genotype of human papilloma viruses in cervical samples. for ish, smears and tissues (fresh, unfrozen, and fixed tissues) are suitable. in electropherotyping and restriction fragment length polymorphism (rflp), double-stranded dna (rflp) or rna (electropherotypes) is purified and size-separated on agarose or acrylamide gel electrophoresis. because nucleic acids are charged and double-stranded molecules bind more ethidium bromide compared with single-stranded nucleic acids, under the electric field, the nucleic acids migrate and larger sized molecules separate out higher than smaller sized molecules. for dna molecules to be tested, the double-stranded viral dna-or pcr-amplified fragments are digested with restriction enzymes. these techniques allow quick differentiation of viral genomes (dna or rna). both techniques have applications in molecular epidemiology of rotaviruses [ ] . new generation molecular techniques viral genome sequencing technologies viral genome or mrna sequencing is a powerful molecular epidemiologic tool and has been applied for epidemiology of rabies virus [ ] . sequences of novel or emerging viruses may be derived based on known conserved sequences of previously characterized viruses within the same family. although virus sequencing is gaining more routine application in veterinary laboratories, it does add cost, and thus should be used judiciously. when these methods fail to identify a newly discovered virus, which is truly novel, metagenomic analysis, which is largely used in research laboratories, may be applied. pyrosequencing is a recent variation on sequencing short stretches of pcrgenerated dna without the need for labeled primers, labeled nucleotides, and gel electrophoresis [ ] . although this variation on pcr and nucleic acid sequencing is currently used exclusively as a research tool, it is likely to be adapted for clinical diagnostic work in future years because it has been demonstrated to detect many different unrelated viruses simultaneously in a single reaction and to identify viral serotypes and detect viral isolates that could not previously be typed by classic procedures [ , ] . a biochip or microarray is small solid support, such as a nylon membrane, silicon chip, or glass slide, on which nucleic acid fragments, antibodies, or proteins are immobilized in an orderly arrangement. thousands of different molecules, referred to as probes, may be machine-printed as spots on the support, allowing for high throughput of samples using lower volumes of analyte in less time than conventional laboratory techniques take to complete. microarrays are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions that are most commonly detected through the use of fluorophores. the fluorescent signal patterns formed by each analyte are then compared by the computer software using complex algorithms to make an identification of its contents. biochips enable researchers to screen large numbers of biologic analytes quickly for a variety of purposes, ranging from disease diagnosis to detection of bioterrorism agents. biochip technology is still relatively new and has not yet entered the mainstream of clinical diagnostics techniques, although it is widely used in research institutions. as an epidemiologic tool, the use of nucleic acid microarrays was instrumental in the rapid identification of the first severe acute respiratory syndrome (sars) coronavirus outbreak in china [ ] . coronavirus protein microarrays have been used to screen canadian sera [ ] for specific antibodies to sars and to other coronaviruses in a comparative study with the traditional elisa. scientists around the world are assessing the feasibility of using microarrays as tools for surveillance and diagnosis of influenza viruses [ , ] . once issues of sensitivity and assay validation have been addressed satisfactorily and the cost of the technology has become more affordable, microarray technology may find a place in clinical diagnosis. pathogenic virus or ''orphan'' virus or ''vaccine-source'' virus molecular methods for detecting and identifying viral pathogens are powerful. it is possible to detect a virus in a specimen, but it may have no association with the clinical condition. these types of viruses are called ''orphan viruses.'' minute virus of canine is a parvovirus, and it causes no clinical disease [ ] . as a result of the advent of sensitive molecular techniques, it is quite common to detect viral sequences of agents that may be present in a sample but not associated with the disease (orphan viral agents). it is possible to study the association of the viral agent with the pathologic findings observed to support the diagnosis. moreover, the pcr protocols targeting structural genes that are expressed only during active infection are useful and avoid the potential false-positive results attributable to latency or persistent viral infections. moreover, the sense and antisense probes offer the opportunity for resident and replication intermediates of viruses. obviously, the history of recent vaccination should be known, and the vaccine virus from the same lot of vaccine should be simultaneously included in the testing run and sequenced over critical regions to ensure that the virus in the sample is the same or different from the vaccine. when fluorescent antibody testing or immunohistochemistry testing is performed, false-negative findings result even when a related virus is present. because of changes in the sequence of the target protein epitopes, antibodybased detection methods may fail to provide the diagnosis; monoclonal antibodies used may fail to react and polyclonal antibodies may cross-react weakly when a variant strain of virus is present. thus, a sudden trend in lack of correlation between tests may signal an emerging variant of the virus. if a new variant of the virus arises, it may be associated with a change in the clinical profile and we may or may not understand the molecular basis of this shift. it is possible that the polyclonal antibodies may react weakly with the new variant of the virus. in many cases, the pcr primers may fail to amplify the new variant if the mutation occurs in the hypervariable region of the target gene amplified. for example, in the recent emergence of cpv variants, many practitioners noted clinical symptoms compatible with cpv but the commercial field tests were not working. if a new variant of virus emerges, a polyclonal antibody antiserum prepared in a heterologous species (rabbit or goat) can be used as a primary antibody against the whole virus, because it is possible that the monoclonal antibody might fail. the molecular techniques are more likely to fail compared with the antibody-based techniques because of the degeneracy of codons. it is important to keep in mind that factors other than emerging viruses can also affect the performance of usda-approved tests. for example, local anesthetic can also affect the outcome of antibody tests. in one study, the use of lidocaine was recommended over oxybuprocaine to avoid false-positive results [ ] . it should be clear to the readers that veterinarians are bound to encounter emerging viruses in their practice. the problem is unavoidable because viruses are ''perfect'' obligate parasites. even the immune response dictates the nature of virus that evolves in a host. thus, vaccines are to be viewed as preventive tools rather than as a cure for emerging viruses. in some situations, the best vaccine is bound to fail. similarly, the diagnostic methods have to be tailorfitted to keep up with the emerging viruses. if the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with diagnostic laboratory to solve the diagnostic puzzle. your state veterinary diagnostic laboratory may be the first place that issues an alert to veterinary professionals and the public at large to possible emerging viral diseases. newsletters from your state diagnostic laboratory can be a good source of information about emerging viral diseases in your area. additional sources that are dedicated to dog and cat health issues and public health are available on the internet [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an annotated historical account of canine parvovirus evidence for evolution of canine parvovirus type in italy canine parvovirus types c and b circulating in north american dogs in and american pet products manufacturers association. industry statistics and trends: - national pet owners survey rabies surveillance in the united states during feral cat population statistic the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia canine distemper viruses circulating in north american dogs an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus future risks from infectious diseases of animals. presented at the world association of veterinary laboratory diagnosticians linking human and animal health surveillance for emerging diseases in the united states-achievements and challenges united states department of health and human services centers for disease control and prevention animals as sentinels of environmental health hazards world health organization. future trends in veterinary public health. who technical report series purdue university-banfield national companion animal surveillance program for emerging and zoonotic diseases early statistical detection of anthrax outbreaks by tracking over-the-counter medication sales dead bird clustering: a potential early warning system for west nile virus activity spatial and temporal patterns of enzootic raccoon rabies adjusted for multiple covariates a space-time cluster investigation of an outbreak of acute respiratory disease in norwegian cattle herds methodology in diagnostic virology a primer on diagnostic virology: specimen selection and serology. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian a primer on diagnostic virology: direct and molecular-based detection of viral pathogens. a primer of veterinary diagnostic laboratory testing. compendium on continuing education for the practicing veterinarian antemortem diagnosis of cdv infection by rt-pcr in distemper dogs with neurological deficits without the typical clinical presentation clinicopathological findings in dogs with distemper encephalomyelitis presented without characteristic signs of the disease differential diagnosis in small animal medicine small animal medical differential diagnosis diagnostic virology bluetongue disease in dogs associated with contaminated vaccine iatrogenic infection of a pregnant dog with bluetongue virus, serotype interobserver variation among histopathologic evaluations of intestinal tissues from dogs and cats telepathology: its role in disease diagnosis in meat hygiene. presented at the world association of veterinary laboratory diagnosticians th international symposium static image telepathology in perspective overview of electron microscopy and its role in infectious disease diagnosis. presented at the world association of veterinary laboratory diagnosticians th international symposium diagnostic electron microscopy: historical review and future. presented at the world association of veterinary laboratory diagnosticians th international symposium acridine orange staining of purified rat virus strain x recovery and characterization of a minute virus of canines characterization of a nonhemagglutinating mutant of canine parvovirus characterization of a canine parvovirus strain isolated from an adult dog serologic survey of cats and dogs during an epidemic of west nile virus infection in humans use of western blot and radioimmunoprecipitation for diagnosis of feline leukemia and feline immunodeficiency virus infections a comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats, and mink simple rapid, on-site detection for diagnosis of animal disease update on molecular techniques for diagnostic testing of infectious disease real-time pcr in clinical microbiology: applications for routine laboratory testing in situ hybridization and its diagnostic applications in pathology the isolation of rotavirus from calves, foals, dogs and cats in new zealand clinical laboratory advances in the detection of rabies virus pyrosequencing sheds light on dna sequencing type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology identification of enterovirus serotypes by pyrosequencing using multiple sequencing primers micro-array-based detection and genotyping of viral pathogens severe acute respiratory syndrome diagnostics using a coronavirus protein microarray comparison of the mchip to viral culture, reverse transcription-pcr, and the quickvue influenza aþb test for rapid diagnosis of influenza detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray minute virus of canines (mvc, canine parvovirus type- ): pathogenicity for pups and seroprevalence estimate oxybuprocaine induces a false-positive response in immunochromatographic sas adeno test american association of public health veterinarians cdc: healthy pets healthy people available at national association of state public health veterinarians united states animal health association code of federal regulations (search engine) key: cord- -mxdlii authors: arji, goli; ahmadi, hossein; nilashi, mehrbakhsh; a. rashid, tarik; hassan ahmed, omed; aljojo, nahla; zainol, azida title: fuzzy logic approach for infectious disease diagnosis: a methodical evaluation, literature and classification date: - - journal: biocybern biomed eng doi: . /j.bbe. . . sha: doc_id: cord_uid: mxdlii this paper presents a systematic review of the literature and the classification of fuzzy logic application in an infectious disease. although the emergence of infectious diseases and their subsequent spread have a significant impact on global health and economics, a comprehensive literature evaluation of this topic has yet to be carried out. thus, the current study encompasses the first systematic, identifiable and comprehensive academic literature evaluation and classification of the fuzzy logic methods in infectious diseases. papers on this topic, which have been published from to and related to the human infectious diseases were evaluated and analyzed. the findings of this evaluation clearly show that the fuzzy logic methods are vastly used for diagnosis of diseases such as dengue fever, hepatitis and tuberculosis. the key fuzzy logic methods used for the infectious disease are the fuzzy inference system; the rule-based fuzzy logic, adaptive neuro-fuzzy inference system (anfis) and fuzzy cognitive map. furthermore, the accuracy, sensitivity, specificity and the receiver operating characteristic (roc) curve were universally applied for a performance evaluation of the fuzzy logic techniques. this thesis will also address the various needs between the different industries, practitioners and researchers to encourage more research regarding the more overlooked areas, and it will conclude with several suggestions for the future infectious disease researches. fuzzy logic approach for infectious disease diagnosis: a methodical evaluation, literature and classification show that the fuzzy logic methods are vastly used for diagnosis of diseases such as dengue fever, hepatitis and tuberculosis. the key fuzzy logic methods used for the infectious disease are the fuzzy inference system; the rule-based fuzzy logic, adaptive neuro-fuzzy inference system (anfis) and fuzzy cognitive map. furthermore, the accuracy, sensitivity, specificity and the receiver operating characteristic (roc) curve were universally applied for a performance evaluation of the fuzzy logic techniques. this thesis will also address the various needs between the different industries, practitioners and researchers to encourage . an infectious disease can be defined as a situation that is created by the incursion of a human body by harmful agents which will in turn hurt the body and may spread to other people as well [ ] . infectious diseases are the major cause of illness and death for any given population [ , ] . human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids), severe acute respiratory syndrome (sars), h n influenza and poliomyelitis are examples of an infectious disease [ , ] . while globally the incidence of the infectious disease varies greatly between countries, in the st century, the main incidences are hand-foot-mouth disease, hepatitis b, and tuberculosis [ ] . these diseases have a significant impact on the global health and economies [ ] . despite numerous valuable achievements regarding the procedures for preventing and controlling of various diseases, infectious diseases remain a massive threat to a population's well-being [ ] . aspects such as the increasing in antimicrobial resistance, increase in population and environmental changes are important in an infectious disease transmission [ ] . of million mortality reported per year throughout the world, . million were related to infectious diseases; which represent more than % of the overall deaths [ ] . additionally, clinical diagnosis and detection of the contaminated population are critical elements in controlling and supervising an infectious disease [ ] . for the diagnosis of different diseases, there are different essential criteria that should be interpreted by the caregivers [ ] . in a clinical situation, an analysis physician combines a patient's medical history, clinical symptoms, physical examination and laboratory findings [ ] . due to the elusive and complex nature of clinical decisionmaking, they may be accomplished with unwanted errors [ ] . in other word, medical diagnosis is an error-prone process, which occurs as the result of logical thinking [ ] . mathematical models are essential means for demonstrating the cause and effect relationship and evaluating the evidence for decisiveness regarding infectious diseases [ ] . computer-aided methods have the potential for examining the complexity of an infectious disease dynamics [ , ] . computational tools are essential for understanding epidemiological patterns in a disease outbreak [ ] . for handling the uncertainty of decision-making, studies have tried to explain the decision-making process in a medical setting by using the boolean or binary structure [ ] . fuzzy logic is considered as a vigorous technique for modelling ambiguity in medical practice [ ] . in medical field, most medical concepts are fuzzy [ , ] . these concepts usually are difficult to formalize and measure [ ] . fuzzy logic is making a decision in an inaccuracy, uncertainty and incompleteness environment [ ] . fuzzy methods deal with the classes whose boundaries are unclear and elusive [ ] . fuzzy logic concepts were introduced in [ ] . the medical field was one of the first fields in which fuzzy theory was implemented [ ] . fuzzy classes are given a degree of membership that is intermediary between and [ ] . some of the cases for a medical use of the fuzzy set theory are the myci, internist and doctormoon applications [ , ] . therefore, for these application methods, different types of research have been conducted in the disease diagnosis field. so, the major objective of the current study is to examine the researches in which fuzzy logic techniques have been applied in infectious diseases so to determining its trends and methods, through the processes of conducting a systematic literature review (slr). the current thesis is structured in the fallowing order. section exemplifies the procedures or the methodology of the current slr, section will present a complete report of the current systematic review. section will be dedicated to the discussion of this research. section will be dedicated to the implications of this study, its future research prospects and to the limitations of this evaluation and the last section will be the conclusion. we carried out an slr to define the influence of using the fuzzy logic methods in infectious diseases. the three main questions of this slr are as follow: (rq ) which domain of an infectious disease was more interesting in past researches? (rq ) which fuzzy methods were more dominant in infectious disease data analysis? (rq ) which performance evaluation methods were more frequently applied in previous reviews? for the gathering of the relevant information from all the eligible articles, a data extraction method was used for getting the detailed answer of the research questions. (rq ) which domain of an infectious disease was more interesting in past researches? (rq ) which fuzzy techniques were more dominant in an infectious disease data analysis? (rq ) which performance evaluation procedures were more frequently applied in the previous reviews? to answer the research questions and to produce the extracted data, numerous methods were used. generally, a narrative combining approach to answering different research questions was applied. furthermore, various visualization techniques such as tables and charts were used according to the research questions. current part is dedicated to the outcome of the current slr. first, we will demonstrate an overall description of the outcome of selecting the appropriate studies; and then, all the obtained outcomes will be categorized for each study questions independently. by searching for the four aforementioned databases, the candidate paper was removed as shown in fig. . then, based on using the exclusion conditions, studies were excluded. the other articles were investigated meticulously to choose relevant studies. by reviewing the title, abstract and the keywords, merely papers that have at least one of the inclusion measures were used. eventually, papers were used to get an answer to the research questions and data extraction for this methodical evaluation. fig. is showing the distribution of eligible papers per year. according to the diagram below, the frequency of papers relevant to the use of fuzzy logic in an infectious disease was roughly stable during the first years. however, as shown in the next chart, there is a persistent trend in the number of finally, the spread of the relevant articles based on a journal or conference, has been explained in table . based on the obtained results expert systems with applications journal was used by researchers for publishing their studies in the . percent of instances. rq : which domain of an infectious disease was more interesting in past researches? articles related to the precise domain of fuzzy methods application in an infectious disease, which have been considered from the past section, are summarized in the following section. fig. illustrates the distribution of numerous fuzzy techniques that have been used in an infectious disease data analysis. based on this figure, the results revealed that the critical application domain of the fuzzy methods in an infectious disease were relevant to dengue fever ( % of papers), hepatitis and tuberculosis ( % of papers). in addition, the other papers were related to diseases such as pulmonary infection ( . % of papers), urinary tract infections (utis), human immune deficiency viruses (hiv) and meningitis ( % of papers). for all the other diseases, there is one paper per each disease ( . % of papers). appendix a, table a lists eligible articles according to the specific type of disease, their objective, their inward and outward variables and findings. as shown in fig. , the distribution of the different fuzzy methods in various disease data analysis was as fallows. in articles published for dengue fever, . % of the papers applied fuzzy set theory and the adaptive neuro-fuzzy inference system (anfis) method in data analysis while . % of the papers used association rule mining. % of selected papers that related to hepatitis, employed neuro-fuzzy classifier as a fuzzy technique, while the other % were devoted to fuzzy inference system and fuzzy decision support system (fdss). furthermore, in tuberculosis studies, % of the papers used rule-based fuzzy logic and % of the papers applied the gaussian-fuzzy neural network method. rq : which fuzzy techniques were more dominant in an infectious disease data analysis? fuzzy inference systems are increasingly becoming more predominant in the area of fuzzy logic methods where % of the selected papers in the current slr are related to this particular method. fuzzy inference systems are used for the processes of mapping the inward variables to appropriate outward [ , ] . fuzzy inference process incorporates three key concepts: the membership functions, the fuzzy set operations, and the inference procedures [ ] . a fuzzy inference system can be divided mainly to four portions as follows: fuzzification, weighting, assessment of inference procedures, and the de-fuzzification [ ] . dragovit et al. utilized the fuzzy inference system to decide the probability of having peritonitis in patients. by using this system, the fuzzy rules enable the automation of the clinical decision making process in an imprecise and complicated conditions [ ] . urinary tract infections (utis) are considered amongst one of the utmost predominant bacterial infections. ibrisimovic et al. has suggested a fis fuzzy model, to provide the necessary support that the caregivers need for explaining the aftermath of a microscopic urine analysis. to create the model's various variables such as the colony forming units (cfu), white blood cells (wbc) and the red blood cells (rbc) as well as the turbidity of urine specimen used for inward variables, and the risk of a uti as an outward variable. the end result has revealed that the use of the fuzzy methods simplifies and secures the clarification of urine analysis [ ] . putra and munir proposed a method for diagnosis of measles, german measles and varicella. the data for those diseases were used because of the similarity of their infection mechanism and symptoms. a built fuzzy inference system b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - takes in the inward variables that represent the possible symptoms that may appear in each disease. cough, runny nose, sore throat, conjunctivitis, koplik's spot, diarrhea, headache, swollen neck or ear, loss of appetite, malaise, pimples/crust skin, joint pain used as inward variables. the application effectively identified out of accurate diseases throughout its testing stage [ ] . the anfis method was proposed by jang and its notions then were used in other fields [ ] . this method works by setting a list of features by using an amalgam of learning rules which will incorporate the back-propagation incline in error digestion and a tiniest squares method. this method can be implemented as the basis for creating a set of if-then guidelines with suitable association of functions to brand the inward and outward variables [ ] . anfis method has been notably successful in disease diagnosis in the past few years. as an example, the major groups of hepatitis in human beings are hepatitis a, hepatitis b and hepatitis c with the main symptoms being malaise (a common sick feeling), fever, and muscle pain, loss of appetite, nausea, vomiting, diarrhea, and jaundice. viral hepatitis disease can be diagnosed by blood test analysis and interpretations. dogantekin et al. developed an anfis system to be used for hepatitis diagnosis. the precision achieved in this automated system of diagnosis was at about . % [ ] . faisal et al. conducted a research using an adaptive neurofuzzy inference system to diagnose a dengue fever. the general precision of the developed method is . % with its sensitivity being at . % and its specificity being at . % [ ] . this method was used in the campisi research as well to determine the amount of the risk factors of an infection disease in relationship with oral candidiasis (oc) [ ] . additionally, shariati has recommended a method of diagnosis for both hepatitis and thyroid diseases. the researchers in this study compared the outcome of the anfis technique with the support vector machine (svm) and the artificial neural networks techniques. then, they demonstrate that this technique had an improved outcome in being a precise diagnosis in comparison with the previous techniques [ ] . the information in a fuzzy rule-based system is typically represented by using an if-then statement. rule-based fuzzy logic includes two portions: the precursor portion are the relevant conditions which are known as the inward variable(s), and the subsequent portion which expresses the outward variable(s). mamdani and sugeno are two different types of fuzzy rulebased systems. in the mamdani technique, the precursor, as well as the subsequent section, comprises fuzzy statements that reveal the value of the variables, while in sugeno method, the subsequent portion displays a nonlinear affiliation among both the inward and outward variables [ , ] . because of the complex nature of the numerous diseases, this technique can improve the infectious disease diagnosis and the treatment of such diseases as hiv and tuberculosis. based on this, sloot et al. efficiently used rule-based fuzzy logic to uncover the drug-resistance in hiv patients [ ] . furthermore, semogan et al. has created a clinical decision supporting system based on the fuzzy logic and the rule-based model that determine different classes of tuberculosis to assess respiratory diseases. in this system variables such as cough, cough duration, body temperature, fever duration, sputum discoloration, nose sputum, afternoon chills, night sweats, weight loss, and loss of appetite have been used in the diagnosis [ ] . b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - the fuzzy cognitive map (fcm) is a modelling technique that defines the connections between concepts such as variables, the inwards and the outwards by the methods of previous knowledge and experience. kosko has introduced fcm in . fcm is usually used to demonstrate the cause and effect relationship between the different concepts in a given system. fcms are used in numerous fields such as in engineering, error detection and medicine [ , ] . in this regard, mei et al. has applied fcm to describe how factors such as people's emotions and cognition functions influence each other to create epidemic infection [ ] . fcm was also used by the mago et al. research to develop the required knowledge-based structure used for recognizing the specific causes and the symptoms of meningitis disease in children. this system can be implemented as a dependable instrument in supporting the physician's to better their decision making processes [ ] . rq : which performance evaluation techniques were more frequently applied in the previous reviews? performance evaluation procedures are among the furthermost significant indicators for determining the quality of the artificial intelligence techniques [ ] . overall, performance evaluation procedures are classified into two main types; the first being the single scaler techniques and second being the graphical techniques. the sensitivity, specificity and the accuracy indicators are grouped into the single scaler techniques. receiver operating characteristic (roc) curve, cost-line, and lift graph are clustered together in graphical methods. anyhow, graphical methods cannot simply be clarified and analyzed as single scalar method [ ] [ ] [ ] . fig. indicates the most noticeable performance evaluation indicators used in eligible papers. for the current slr, most qualified studies did not use any kind of indicators for analyzing the performance of the fuzzy techniques. among the fuzzy techniques, anfis and fis techniques were evaluated by single scaler techniques and graphical evaluation techniques. the major objective of this slr was to select and examine the various studies relevant to the employment of the fuzzy logic techniques in an infectious disease. in this regard, studies were selected and analyzed from an original number of candidate studies. this section is dedicated to the discussion of the major findings of this study. rq : which domain of an infectious disease was more interesting in past researches? in last few years, it has been obvious from the results of analytic studies conducted in our research that there is a growing interest in studying the relevance of the fuzzy logic techniques for an infectious diseases diagnosis applications. the studies have shown that there is an increase in the number of published articles from % in up to . % of the evaluated papers in . this growing trend may prove the popularity of the fuzzy techniques between different academic papers and its valuable effects in identifying the infectious disease trends. additionally, different fuzzy logic techniques have been used in the evaluated papers. from a medical point of view, in fig. we found that the main application domain of fuzzy logic in infectious disease was related to dengue fever, hepatitis and tuberculosis respectively. the other noticeable outcome that we have found was the b i o c y b e r n e t i c s a n d b i o m e d i c a l e n g i n e e r i n g ( ) - fact that the evaluated papers were very diverse in terms of disease types. because of the big impact on global health by infectious diseases, determining the different features of these diseases are a valuable source for improving our knowledge and ability to predict how a disease will spread in a population [ , ] . the ability to comprehend and control an infectious disease can be obtained by the usage of mathematical models [ ] . the newly arising infectious agents such as hiv, the severe acute respiratory syndrome (sars), the mid-eastern respiratory syndrome (mers) coronaviruses; the west nile virus; the nipah virus; the drug-resistant pathogens; novel influenza a strains and the ebola virus outbreak were considered as big challenge to health in the recent century [ ] . using computeraided diagnosis techniques like the fuzzy logic technique can be useful in determining the main factors associated with the infectious diseases occurrence and epidemics. regarding this, in another field of research in relationship to the vaccination strategies for infectious diseases it has been shown that infectious diseases such as influenza have a seasonal pattern in which case mathematical techniques such as the fuzzy logic techniques is considered a vital instrument in the process of forecasting the viral development from one year to another. this technique can also deliver the required scientific confirmation which will help us decide the amount of vaccine treatment, its efficiency, its financial costs and its pattern of contact in any given population [ , , ] . it has been shown that the fuzzy based techniques have an essential part in the determination of infectious disease outbreaks. as an example for that, we have the hiv outbreaks, these techniques can identify at what time a viral transmission has occurred, its outbreak stage and the sexual behavior pattern of the specific population in which a disease is spreading. moreover, the infectious diseases annihilation and elimination techniques dictate that the transmission process itself has to be the target not the disease. regard this, the data sources are typically scattered and the collective effect of the numerous control techniques are complex and are dependent on the rate of transmission [ ] . rq : which fuzzy techniques were more dominant in an infectious disease data analysis? as shown in fig. , there is a various number of fuzzy techniques employed in the chosen papers. based on this studied section we can mostly classify the fuzzy techniques into four main classes; the fuzzy inference system, the rulebased fuzzy logic, anfis and the fuzzy cognitive map. the fuzzy inference system and the anfis technique were used as shown in . % and . % of candidate papers, respectively. fis is based on the 'if-then' rules and it can be used to predict the behavior of a various uncertain situations [ ] . as seen in the distribution of the studied articles, we can say that fuzzy techniques are efficient means for modelling unclear disease conditions such as in the case of transmissible disease diagnosis. additionally, in the last few years, there has been substantial interest amongst the researchers to apply the anfis technique in the case of an infectious disease. anfis combines the positive effects of both ann and fis in an influential tool for disease diagnosis. this technique does not require excessive knowledge in the modelling and training system. these techniques are usually valuable for the situation, which are in many cases complicated, with a nonlinear behavior pattern. this work approach has created a relationship between the inward and the outward features by the means of the neurons [ ] . rq : which performance evaluation procedures were more frequently applied in the previous reviews? performance evaluation procedures are usually employed as a valued method in determining the quality of the numerous fuzzy techniques [ ] . as shown in fig. in the current slr, amongst the single scaler techniques, the sensitivity, specificity and the accuracy are frequently utilized in the evaluation of a developed technique. additionally, in graphical evaluation techniques, the roc curve was the other measure of performance evaluation that was used in the adequate papers. these indicators are to be considered significant when reporting and assessing a diagnostic technique. scientists have to provide suitable information about the sensitivity, specificity, and the projected values when describing a computer based diagnostic technique end results and this information must contain how those metrics are concluded and also what are its appropriate interpretations [ , ] . although those indicators are promising in qualified studies, nonetheless, they typically generate an inadequate image of the indicators performance and thus it is possible to lose a certain amount of valuable information. moreover, it is possible that the employment of a single-scaler technique will not identify the full scope of a performance assessment. therefore, a comprehensive and dependable assessment must reflect all the numerous parts of performance distinctive quality. in this methodical review, the studies related to the employment of the fuzzy logic techniques in an infectious disease were assessed, and depending on the acquired outcomes, we can notice an interest amongst the researchers regarding this specific field of research. in last few years, a large number of the transmissible diseases that were supposed to be eliminated have made a comeback. certain factors such as manufacturing, agricultural practices, wars, changes in lifestyles, development and urbanization, and environmental change are all effective in the appearance and reappearance of an infectious disease [ ] . this slr's result demonstrates that even though these are the most of the infectious diseases that were investigated, but there is still more area that needs to be covered. nevertheless, more work should be carried out on the appearance and reappearance diseases domains such as h n , sars, zoonosis and the rift valley fever. internationally, there is a lack of an integrated framework for reporting infectious disease [ ] . additionally, an infectious diseases information system has an inadequate support for data analysis and generating predictive techniques centered on artificial intelligence. an integrated analytical framework that offers functions as in a progressive data analysis capability and a visualization support is of a critical importance [ , ] . there is serious need for creating an atmosphere for collecting, distributing, reporting, assessing, and picturing the infectious disease data and to provide support for decisionmaking tools regarding disease prevention, recognition, and controlling [ , ] . infectious disease observation and controlling has demanded an interdisciplinary work. to have the ability to achieve those objectives, the employment of geographic information systems (gis), three-dimensional information analysis, machine learning and visualization applications and techniques became a must. because of the significance of the infectious diseases at the global level, it is essential to simultaneously develop an incorporated infectious disease dataset and to make the specialized analytical and diagnostic methods all at the same time. additionally, there was slight conversation in the incorporated literature about three-dimensional information analysis for an infectious disease occurrence. three-dimensional information evaluation techniques may be useful in determining the concentration pattern of a disease occurrence and to make the required association from the determined patterns to the measureable procedures [ , ] . furthermore, the social networks information study has facilitated the evaluation of the association amongst the population in a particular social setting. methods that correlate spatial and social network data analysis are unusual but have the capacity to promote the determination of a spreading progress of an infectious disease [ ] . or we can say, the deployment of a unified spatial and social network structure to define the spreading of an infectious pathogens in a given populace will allow insights into both the understanding to the disease method of distribution and the possible process associated with the observed patterns. since there are various studies regarding social media information analysis, we recommend the use of this dataset for the prediction of an infectious disease epidemic. this research has its limitations. even with the use of a broad search approach, some of the publications regarding the fuzzy logic deployment in an infectious disease could not be recovered, as in the case of grey literature and reports that were not published in surveyed digital databases, which we have reviewed. thus, it is recommended that additional slr papers should be carried out to go through the other noticeable databases. additionally, some studies did not report clearly on the performance assessment technique. in conclusion, only the english language publications were included, therefore future studies could be expanded to incorporate relevant papers which are published in other languages. in this study we have identified, classified, and defined the use of the fuzzy logic techniques in infectious diseases. studies were scrutinized and the main conclusions can be briefed as follows: ( ) the key application field of the fuzzy logic in an infectious disease was related to dengue fever, hepatitis and tuberculosis, ( ) amongst the fuzzy logic techniques fuzzy inference system, rule-based fuzzy logic, anfis and fuzzy cognitive map are commonly used in many studies, and ( ) the major performance evaluation indicators such as the sensitivity, specificity, and the accuracy the roc curve is employed. in addition, this study highlights the absence of an integration of infectious disease information systems in order to provide a valuable datasets in this domain. additionally, using machine learning and visualization applications for information analysis is essential. even though in the current slr there is a diversity of infectious diseases that are investigated, there is only one article per each disease. there is additional need to use the fuzzy logic methods for infectious disease detection and prediction. it appears that one of the causes for a limited number of relevant articles to infectious diseases is in the difficulty in obtaining adequate research data. finally, we expect that a mounting number of infectious diseases datasets will be mostly obtainable in the forthcoming years because of raising collaboration amongst medical practitioners and researchers and that this would lead to additional studies of the machine learning techniques that can be useful in this regard. the generated results are more reliable for the prediction of tb in patients. langarizadeh [ ] meningitis rule-based fuzzy logic this system is used to distinguish between bacterial and aseptic meningitis, by using fuzzy logic. gram stain, white blood cell (wbc) count in cerebrospinal fluid (csf), percentage of polymorphonucleocytes in csf, csf protein, csf/ serum glucose ratio, wbc count in blood, percentage of blood neutrophils, blood c-reactive protein (crp), and platelet (plt) count. bacterial and aseptic meningitis 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intelligent curation system for blood infections using fuzzy inference system in android mathematical modelling and a systems science approach to describe the role of cytokines in the evolution of severe dengue mathematical modelling of immune parameters in the evolution of severe dengue optimal neuro-fuzzy control of hepatitis c virus integrated by genetic algorithm evaluation of pulmonary function tests by using fuzzy logic theory ebinformatics: ebola fuzzy informatics systems on the diagnosis, prediction and recommendation of appropriate treatments for ebola virus disease (evd) an experimental comparison of fuzzy logic and analytic hierarchy process for medical decision support systems a grid-based hiv expert system gfnn: gaussian-fuzzy-neural network for diagnosis of tuberculosis using sputum smear microscopic images a belief rule based expert system to assess tuberculosis under uncertainty a fuzzy expert system for distinguishing between bacterial and aseptic meningitis a genetic-neuro-fuzzy inferential model for diagnosis of tuberculosis application of evolutionary fuzzy cognitive maps for prediction of pulmonary infections supporting meningitis diagnosis amongst infants and children through the use of fuzzy cognitive mapping key: cord- -hbd euq authors: søborg, christian; mølbak, kåre; doherty, t. mark; ulleryd, peter; brooks, tim; coenen, claudine; van der zeijst, ben title: vaccines in a hurry date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: hbd euq preparing populations for health threats, including threats from new or re-emerging infectious diseases is recognised as an important public health priority. the development, production and application of emergency vaccinations are the important measures against such threats. vaccines are cost-effective tools to prevent disease, and emergency vaccines may be the only means to prevent a true disaster for global society in the event of a new pandemic with potential to cause morbidity and mortality comparable to the spanish flu, the polio epidemics in the s, or the sars outbreak in if its spread had not been contained in time. given the early recognition of a new threat, and given the advances of biotechnology, vaccinology and information systems, it is not an unrealistic goal to have promising prototype vaccine candidates available in a short time span following the identification of a new infectious agent; this is based on the assumption that the emerging infection is followed by natural immunity. however, major bottlenecks for the deployment of emergency vaccine are lack of established systems for fast-track regulatory approval of such candidates and limited international vaccine production capacity. in the present discussion paper, we propose mechanisms to facilitate development of emergency vaccines in europe by focusing on public–private scientific partnerships, fast-track approval of emergency vaccine by regulatory agencies and proposing incentives for emergency vaccine production in private vaccine companies. although progress in medical science has eradicated one infectious disease (smallpox) and threats from other infections such as polio have been reduced by widespread vaccination, new infectious diseases emerge at historically surprisingly high rates-more than one disease per year. there are several explanations. globalisation with its correspondingly increased transport of persons, products and animals can rapidly spread new infectious diseases around the world. furthermore, the condensing of populations with worldwide urbanization and encroachment of humans into new habitats, facilitating close contact with wild animals creates new hazards for transmission of zoonotic infectious agents from animals to man and possibly in the reverse direction, transmitting human pathogens to animals [ , ] . it has been suggested that more than % ( / ) of all new human pathogens in the last century originated from an animal reservoir [ ] . there is an international recognition of the importance of emerging infectious disease in an age of changes, as recently under-lined by the world health organisation: "it would be extremely naïve and complacent to assume that there will not be another disease like aids, another ebola or sars, sooner or later" [ ] . the recent experience with emergence of chikungunya virus in italy underlines these issues. the spread of chikungunya exemplifies how easily a well-known virus from a subtropical region in africa is able to shift vector from one mosquito vector (aedes aegypti) to another (aedes albopictus), disseminate to other climatic zones -including europe -and cause disease in a susceptible population [ ] . the adaptation to a new vector can probably be ascribed to a point mutation in the virus, whereas international travel served as the means of introduction of the virus to the competent vector. a. albopictus have recently become prominent around the mediterranean basin from greece to spain and other arboviral diseases including dengue and west nile virus may use the same vector-possibly causing the next outbreak in europe. climate change may boost this development further by expanding the range of vectors and their capacity to spread disease, together with other activities that transfer potential vectors to new areas. a. albopictus has extended its distribution to europe and the americas as its larvae can be transported in used automobile tyres [ ] . emerging infections have impact not only on the health but also on the economics of the afflicted region. the sars epidemic was estimated to have a direct cost of billion us$ in asia, and the international community was on the verge of a true disaster of even larger dimensions. this blow could have disrupted health care services, and affected societies and economies for years. it was a fortunate coincidence that sars was not transmissible before the onset of the patient's signs and symptoms of disease. hence, the epidemic was contained by traditional measures of disease control such as early case finding, isolation and quarantine. if this had not been the case, the rapid development and use of an emergency vaccine might have been the only feasible measure to prevent further spread. over the last century, vaccines have been shown to be one of the most cost-effective ways to prevent and control diseases. in some situations, such as the re-emergence of smallpox or a new influenza pandemic with a severity comparable to the spanish flu, emergency vaccinations may be the only way to prevent a true disaster for europe and the global society. following the early recognition of a new threat, the current advances in biotechnology, vaccinology (including reverse genetics) and information systems offer us the possibility of developing promising vaccine candidate shortly after the identification of a new infectious agent, under the assumption that this emerging infection is followed by natural immunity. however, additional major bottlenecks for the deployment of emergency vaccines include the lack of established systems for fast-track regulatory approval of such candidates and limited international vaccine production capacity. in the present discussion paper, we address mechanisms to facilitate development of emergency vaccines in europe by focusing on regulatory aspects and proposing incentives for emergency vaccine production in private vaccine companies. the control of poliomyelitis by rapid development of a vaccine shows that it can be done. it exemplifies what is possible with a strong governmental commitment, public demand and dedication. it also underlines the importance of government institutions taking the lead and responsibility in vaccine development. within only one year -from to -double blinded placebo controlled studies were conducted involving , vaccines and , controls receiving placebo. these trials proved safety and efficacy, leading to licensing of the vaccine shortly after [ ] . the fruit of basic immunology and vaccinology research led to the success of the vaccine and can be condensed into four major discoveries. firstly, characterization of the poliovirus and the definition of three serotypes leading to a trivalent vaccine [ ] . secondly, pathogenicity: the discovery that poliovirus causes paralysis [ ] . thirdly, proof of principle: confirmation that neutralizing antibodies protect against disease [ ] and finally, that virus could be grown in cell cultures for mass vaccine production [ ] . the vaccine campaign was a huge success, and was accepted very well by the population, leading to a steep decrease in polio cases in the years immediately following the vaccine's deployment in europe and usa. today, polio is eradicated in most parts of the world and remains present only in a few of the poorest countries. on the other hand, diseases selected for mass vaccination have to be chosen carefully. the attempt to prevent the spread of swine influenza by vaccination, in usa , is a good example of why to think twice before initiating mass vaccination. influenza specialists were worried that an influenza strain isolated from swine might cross the species barrier and cause a repeat of the spanish flu pandemic from . although no human cases were detected, the decision to start mass vaccination was made and more than million people were vaccinated within a few months. however, suspicion that vaccination was increasing the risk of guillain-barré syndrome as a side effect soon stopped the vaccination campaign [ ] . thus, with a strong public commitment and vital basic knowledge about the pathogen, a successful vaccine can be developed within a short timeframe. however, the decision whether to implement a new vaccine needs to be based on solid risk assessment. potential and unknown hazards associated with the early mass deployment of a new vaccine must be weighed against the risk and nature of the disease. vaccine development -in particular for emergency vaccinesneeds a different business plan than the market-driven approach that underlies the pharmaceutical industry [ ] . private vaccine companies cannot be expected to use resources on improving existing vaccines or developing new vaccine candidates for emerging infectious diseases when there is no current market or if the market is too small or diffuse to be economically feasible. hence, it is important that governments find mechanisms and funding to ensure the fundamentals for the success of a vaccine, namely basic and applied research in public health institutions and academia. furthermore, clear communication is necessary between governments and the vaccine industry on which vaccines need to be developed from a public health perspective. the challenge is to find incentives for the vaccine industry to take part in the development of products that currently do not have a clear market. one solution might be public support to public-private research and development of vaccine candidates in their early preclinical/clinical phases or advance assurances of confirmed purchases of certain volumes of vaccines if they make it to licensure. the european union has promoted the concept of public-private partnerships, but this concept has not resulted in important changes so far [ ] . it is important to find new ways to achieve these aims: it will be too late by the time we suddenly need new vaccines against an emerging epidemic. public-private partnerships in particular are necessary to secure vaccine production from laboratory bench through pilot plant to mass scale industrial production. specific contracts between governments and vaccine companies must be in place to secure that private production capacity is available for emergency vaccination production if needed. as mentioned, modern biotechnology has opened novel approaches for the development of new vaccines allowing production to be carried out in only a few months under the best circumstances (see table ). but obtaining data on clinical efficacy for licensure and regulatory approval will be a major bottleneck for making use of the current technology. without some indication of the immune response required for protection, basic efficacy studies will be difficult. without an animal model, they will be all but impossible. that leaves only the possibility of going to human studies without an indication that the vaccine is effective-a step that is highly unlikely, even in a dire situation. this leads us back to the responsibility of governments and international agencies -including the european union -for laying the groundwork. the first vaccines for sars were developed this way, leveraging preclinical work that had already been done on other coronaviruses [ ] . seasonal influenza vaccines are also made this way; working on the assumption that what has proved efficacious in the past against a related virus will prove efficacious in the future table a toolbox for the rapid development, production and deployment of an emergency vaccine. early recognition of an emerging microbial threat identification and characterization of the causative agent rapid understanding of natural history, pathogenesis, molecular biology and epidemiology; building on work in related pathogens as well as ongoing clinical, laboratory and epidemiological studies identification of potential vaccine candidates identification of potential delivery systems and suitable adjuvant to improve immunogenicity and sparing of antigen and dosages production at pilot plant level development and acceptance of correlates of immunity development and acceptance of correlates of safety limited trials in animals and humans based on these correlates as outcome measures fast-track approval of the vaccines enhancing production capacity by public-private partnerships based on risk assessment and defined objectives: implementation of emergency vaccination post-licensure follow-up of emergency vaccination with data accessible in real-time to medicine-and public health agencies as a surrogate for phase iii trials and ensuring development with advance purchase agreements to establish a market. testing novel vaccine candidates also becomes a bottleneck: just any combination of antigen and delivery system may not be effective. toxicity testing -which is designed to catch only acute problems with a vaccine -can be generally applied to most vaccines. in theory, it may be possible to produce basic safety data even before a complete product is available by testing "mock-up vaccines" in which a placeholder antigen or antigens are used [ ] . this would provide information on the safety of the delivery system including adjuvant, which is generally the most reactogenic part of a vaccine. safety testing of a specific product would still be required, but the reactogenicity of the generic components would already be defined and be one less variable to consider in designing the vaccine. however, efficacy studies require animal models of the infection or the disease and this is both time-and money-intensive. in the absence of a defined animal model, the obvious choice is nonhuman primates, as the closest match to humans, but even if this is possible (i.e. the pathogen will infect primates and produce disease) it may not be practical. both primate and non-primate animal research facilities are in short supply. support of animal facilities is likely to pay dividends when a need arises for rapid assessment of new vaccines. this should be relatively easily put into place, by making it an explicit goal, since it is really only an expansion of existing activities by research supporting agencies. certainly, in the united states, the biodefense initiative has led to a significant expansion in capacity. but it cannot be put into place on an ad hoc basis, nor is it likely that the private sector will become involved-the return on investment in possible emerging diseases is highly uncertain. such facilities take years to establish and their benefits are primarily in research and public health: therefore bodies involved in research and public health will need to take the initiative. once a potential vaccine has been produced and some evidence for efficacy and safety produced in animals through accepted correlates, the same data needs to be reproduced in humans. the current procedures were designed with safety as the foremost consideration and rapidity is not a characteristic of the process: it can take - years (occasionally longer) for a new vaccine to pass through clinical trials. under normal conditions, this is a sensible application of risk/benefit analysis with the emphasis on "first, do no harm". by definition, emerging diseases are not a major health risk-until a significant outbreak occurs. thus, clinical assessment is built around gradual steps-first screening for major risks (phase i, trials generally conducted in very small groups) then subsequently screening for less frequent risks (phase ii, in larger, but still small groups). it is not until phase iii studies, which are often large (thousands to tens of thousands), that efficacy data are expected to be produced. and while phase iii studies are large, they still occasionally fail to uncover rare risks, which only emerge after hundreds of thousands of people have been vaccinated, for example, intussusception of the bowel after administration of rotashield, a vaccine to prevent diarrhoea caused by rotavirus infection [ ] . such events are only uncovered during post-licensure pharmacovigilance. in an epidemic situation, however, the risk/benefit balance changes: if morbidity and mortality due to the pathogen is high, then even a vaccine with significant side effects becomes much more acceptable. it is therefore important to develop procedures for alternative pathways of approval. this should be done in close collaboration with regulatory agencies, and be based on accepted correlates of immunity and safety plus [probably] limited human data. while it will ultimately be up to governments with who guidance under international health regulations to decide what constitutes an "emergency" in which it could be invoked, the bare bones of a "fast-track approval" system for new treatments already exists (influenza mock-up vaccines emea) [ ] , and this procedure could be expanded to accelerate vaccine-testing in clinical trials during a public health emergency. in most cases, the greatest amount of time in a clinical trial is devoted to paperwork, to ensure that the trial complies with regulations designed to minimize risk to participants, ensure transparency and provide a paper trail as a shield against future litigation, should things go awry. in diseases with a poor survival chance -aggressive cancers, for example, or anti-retroviral therapy in the early days of the hiv epidemicregulatory agencies tended to be more forgiving. in such a situation, while safety remains a major issue (particularly for preventive vaccines administered to healthy individuals), testing for efficacy assumes greater importance.the demands for faster processing of vaccines can be addressed by the following steps: . an already-defined regulatory framework within which fasttrack clinical trials can be conducted. this should contain rules for priority review and approval, some (limited) protection against liability to open the process to commercial entities (as they are best equipped for large scale production and distribution) and rules for invoking such a process. they may not (perhaps should not) lead to open-ended approval of a product, being instead intended to allow limited release. . rapid access for vaccine developers to the appropriate regulatory authorities within the emea and authorization for regulators to draw on necessary expertise (perhaps in the form of expert panels) to enable assessments to be made quickly. regulations on relaxing approval (perhaps in the form of approval for a limited time) for import of vaccines not currently approved for use in europe. . a process whereby approval can be granted under the understanding that the complete necessary paperwork can be submitted retrospectively-enabling a rapid progress of efficacy trials, as soon as initial data suggests a vaccine is safe, without waiting for collation, submission and approval before progressing to the paperwork for the next step. . a broader acceptance of surrogate data, e.g., correlates of protection and safety, in the early steps. if (for example) animal toxicity studies raise no concerns, it may be possible to proceed directly to combine phase i/ii studies. this would provide human safety data, but at the cost of placing slightly more participants at risk. the payoff would be earlier access to data indicating whether the vaccine is immunogenic and stimulates the desired type of immune response, plus a more rapid assessment of vaccine safety. acceptance of immunogenicity data as a surrogate for efficacy, based on animal models (and it is here that expert panels will be crucial) might allow rapid release of a product from phase iii trials, subject to the study continuing to collect efficacy data. such an approach could shorten vaccine-testing time by months to years. . finally, while it is possible to enter phase i trials with an experimental product, phase ii has stricter requirements and the product tested needs to be closer to, if not identical with, that which will be taken into phase iii trials-which itself will be the final product. some flexibility on vaccine composition would allow a more rapid progress to phase iii. phase iii trials are intended to demonstrate that a product is efficacious. this is never an easy task and for emerging diseases is complicated by the fact that such diseases are, by definition, not endemic. that means that without reliable surrogate markers, efficacy studies can only be done in endemic countries or selected high-risk populations. it can be expected that even in countries where the disease is endemic, people will be reluctant to accept testing of a vaccine for which safety data and approval has been fast-tracked: some form of compensation mechanism is almost certain to be required to encourage manufacturers and the public to participate in a clinical trial. this passes out of the remit of organizations such as emea and into that of international cooperation, which needs to be arranged at the governmental level. the risks of rapidly proceeding into phase iii can be ameliorated by compromising (to some extent) impartiality. to avoid bias, such large studies are normally blinded and results assessed at the end of the study. however, in a situation where large numbers of people are being vaccinated with a "fast-tracked" vaccine safety concerns will be higher than normal. by putting enhanced surveillance into place and assessing data from cohorts within the main phase iii study, data on adverse events possibly associated with vaccination and efficacy of the vaccine could be collected much faster. objectivity could be maintained by maintaining blinding with regard to the study monitors. this approach essentially expands the role of the data safety management board, whose role is normally to oversee the safety of the study and who do review results on an ongoing basis, to cover decision-making on vaccine efficacy. in such a case, they would need to involve the study designers, which may raise issues of conflict of interest. this can be addressed by again involving expert review panels, but that will almost certainly face resistance from commercial developers who would face exposure of their operating procedures. but where there is an overwhelming public interest in rapid assessment of vaccine efficacy, the conventional rules may need to be relaxed and increased transparency is the safest counter to decreased regulatory oversight. in conclusion, new infectious diseases are emerging at a historically high rate. to secure both public health and economic stability in the future effective countermeasures have to be instituted in advance at governmental levels. implementing fast-track approval systems for emergency vaccines by the regulatory agencies, and underpinning public private partnerships to enable production in the absence of a market would be an important step in order to be prepared for a new pandemic. finally, innovative research towards the understanding of vaccine safety and efficacy and leading to shorter development times should be promoted. american association of physical anthropologists meeting perceived vaccination status in ecotourists and risks of anthropozoonoses surveillance and response to disease emergence a safer future, global health in the century. the annual health report who infectious diseases chikungunya: no longer a third world disease the used tyre trade: a mechanism for the worldwide dispersal of container breeding mosquitoes making history: thomas francis m.d. jr. and the salk poliomyelitis vaccine field trial differentiation of types of poliomyelitis viruses; the grouping of strains into three basic immunological types viremia in human poliomyelitis evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis iv. final report of results based on clinical diagnoses cultivation of the lansing strain of poliomyelitis virus in cultures of various human embryonic tissues guillain-barre syndrome following vaccination in the national influenza immunization program, united states - why certain vaccines have been delayed or not developed at all european union dg interternal policies of the union a dna vaccine induces sars coronavirus neutralization and protective immunity in mice pandemic vaccines: promises and pitfalls rotavirus vaccines: an overview key: cord- -z h sc authors: semenza, jan c; ebi, kristie l title: climate change impact on migration, travel, travel destinations and the tourism industry date: - - journal: j travel med doi: . /jtm/taz sha: doc_id: cord_uid: z h sc background: climate change is not only increasing ambient temperature but also accelerating the frequency, duration and intensity of extreme weather and climate events, such as heavy precipitation and droughts, and causing sea level rise, which can lead to population displacement. climate change-related reductions in land productivity and habitability and in food and water security can also interact with demographic, economic and social factors to increase migration. in addition to migration, climate change has also implications for travel and the risk of disease. this article discusses the impact of climate change on migration and travel with implications for public health practice. methods: literature review. results: migrants may be at increased risk of communicable and non-communicable diseases, due to factors in their country of origin and their country of destination or conditions that they experience during migration. although migration has not been a significant driver of communicable disease outbreaks to date, public health authorities need to ensure that effective screening and vaccination programmes for priority communicable diseases are in place. population growth coupled with socio-economic development is increasing travel and tourism, and advances in technology have increased global connectivity and reduced the time required to cover long distances. at the same time, as a result of climate change, many temperate regions, including high-income countries, are now suitable for vector-borne disease transmission. this is providing opportunities for importation of vectors and pathogens from endemic areas that can lead to cases or outbreaks of communicable diseases with which health professionals may be unfamiliar. conclusion: health systems need to be prepared for the potential population health consequences of migration, travel and tourism and the impact of climate change on these. integrated surveillance, early detection of cases and other public health interventions are critical to protect population health and prevent and control communicabledisease outbreaks. travel medicine will increasingly see the health consequences of significant changes associated with global change, particularly climate change and socioeconomic development. together these changes mean more opportunities for people to travel to pathogen-endemic countries and for pathogens to be imported to new locations, with the potential for unexpected communicable disease cases and outbreaks. moreover, people who migrate or are displaced can also be at increased risk for chronic disease and mental health issues. thus, travel medicine needs to take a broader perspective than just asking patients where they are going or where they have been, considering how the ongoing and projected shifts in the magnitude and pattern of disease could affect the health and well-being of individuals and populations. modifications to the approaches used by health systems to manage adverse health outcomes are needed to ensure health care providers have the most up-to-date information. migration describes the movement of a person away from their usual residence whereas travel describes a person who passes from place to place, for any reason. migration and travel have shaped the history of humanity and enriched societies economically, socially and culturally. multiple and interacting factors drive migration in particular. climate change, including increasing climate variability, can be one of these factors. we discuss the potential impacts of climate change on migration and travel and the implications for travel medicine and public health practice. following a brief review of climate change, we discuss the potential health implications of migration and travel and the way in which they are affected by climate change and provide suggestions for health systems to better manage the potential impact of climate change, migration and travel on population health. the intergovernmental panel on climate change special report on warming of . • c concluded that human activities have caused ∼ . • c of global warming since pre-industrial times and that, if it continues to increase at the current rate, warming is likely to reach . • c between and . the special report concluded that climate change is increasing, and will continue to increase, land and ocean temperatures and the frequency, intensity and duration of heat waves in most land regions (high confidence). it also concluded that climate change will continue to increase the frequency and intensity of heavy precipitation events globally and the risk of drought in the mediterranean region specifically (medium confidence). climate change is a long-term process, but the extent to which individual weather events are influenced by climate change can increasingly be estimated. , migration the greatest human migration started or years ago when homo sapiens left africa for other parts of the world - ; a series of 'mega-droughts' may have contributed to this. [ ] [ ] [ ] migration has continued since and has become a defining characteristic of our times, affecting every inhabited continent. migrants may move internally, settle in neighbouring countries or move to other continents. currently, europe, north america and oceania are the main destinations for migrants although, overall, europe and asia have received the greatest number of migrants in recent decades. migration has political, social, economic, cultural, environmental and public health implications. migrants generally contribute more in taxes and social contributions than they receive in benefits. [ ] [ ] [ ] [ ] but migration can also pose chal-lenges, particularly when there is a rapid influx of large numbers of people. the international importance of migration was recognized in by the united nations general assembly when the agenda for sustainable development was adopted; of the sustainable development goals (sdgs) directly or indirectly relate to migration (table ). in , the united nations general assembly adopted the new york declaration for refugees and migrants to support a comprehensive approach to migration. these international accords were also thematically linked to other major international agreements, including the sendai framework for disaster risk reduction and the paris agreement under the united nations framework convention on climate change. , the drivers of migration are complex and include political, economic, social, cultural and environmental factors (figure ). these same factors can determine whether migration is permanent or temporary. the drivers of migration can be grouped into five categories - : ) push factors that force people to migrate including war, unemployment, food scarcity, over-population or prosecution. ) pull factors that attract people to a new destination including work and educational opportunities, political or religious freedom. ) network drivers that facilitate the migration process such family ties, social support, kinship, safety and feasibility of transport. ) national policies that allow or prohibit migration. ) personal aspirations and motivations. these drivers are often interconnected and situation specific ( figure ). they can also interact with climate change, which can itself be a trigger for migration ( figure ). , although environmental drivers do not appear to have been the most significant contributor to international migration to date, , migration in response to the consequences of climate change can be a pragmatic adaption strategy that balances the risks of staying and the risks of moving. , [ ] [ ] [ ] [ ] for example, for the tonga-speaking people in southern zambia, migration is an adaptation strategy to climate variability when the availability of water affects both agricultural production and livestock survival. additional warming of . • c is projected to further exacerbate climate-related drivers of migration; studies suggest that an increase in extreme weather and climate events and disasters associated with climate change could trigger an increase in migration. [ ] [ ] [ ] [ ] climate change impacts on migration and health. the complex and multiple drivers of migration mean that it is difficult to attribute specific migrations to climate change (figures and ) . as a result, projections of migration associated with climate change vary widely, ranging from million to million, with these numbers based on extrapolations. , in , a world bank report on the impact of climate change on migration estimated that by -in just three regions-climate change could force more than million people to move within their countries. table summarizes the world bank findings from three case studies in sub-saharan africa (ethiopia), south asia (bangladesh) and latin america (mexico). evidence for the possible role of climate change on migration comes from different sources (figure and ) . these include studies of the extent to which 'temperature and precipitation' are associated with out-migration. for example, in recent decades, temperature had a statistically significant effect on out-migration from agriculture-dependent countries. there is a nexus between climate change and migration in that temperature and precipitation are positively associated with migration: a • c increase in average temperature in the sending country is associated with a . % increase in bilateral migration flows; an additional millimeter of average annual precipitation is associated with an increase in migration of . %. another study suggests that an increase in precipitation anomalies from the long-term mean may also be associated with an increase in out-migration. under a moderate climate change scenario, if global mean temperature rises by • c, tropical populations would have to move considerable distances in order find places where temperatures are similar to those in places they currently live in and to preserve their annual mean temperatures. 'extreme weather events' linked to climate change, such as heat waves, droughts, storms, floods and wildfires, cause shortterm population displacement ( figure ). in , disastersmost of which were related to extreme weather-displaced . million people in countries ( figure ). in , california only low-income countries show marked increases in population under spp . b the higher number of climate migrants under the more climate-friendly scenario in ethiopia is in part driven by the regional climate models, which project lower water availability by in general compared with the other two scenarios (pessimistic reference and more inclusive development scenarios), which are coupled with higher emissions. experienced the most destructive wildfire season on record with more than , people being evacuated from their homes. a number of factors led to these fires, including accumulated natural fuel and compounding atmospheric conditions that were linked to climate change. based on the experience of other events in the usa, some people will eventually return to their communities, but others will permanently relocate. for example, following hurricane katrina, it was estimated that between % and % of the population returned, with young adults being more likely to have moved further away. , in , after hurricane andrew in florida, % of displaced residents relocated within the same county, % moved within the state and % moved out of state. in some cases, extreme weather events can cause longerterm migration. for example, an increase in the frequency and intensity of droughts in tanzania has killed livestock and forced the maasai people to migrate from rural to urban settings, with documented implications for their mental health. similarly, prior to the syrian migrant crisis in , the fertile crescent was affected by a severe and sustained drought associated with anthropogenic increases in greenhouse gas emissions and this, combined with unsustainable agricultural and environmental practices, contributed to internal migration, political instability and conflict and eventually to an exodus of one million migrants to europe. 'river flooding and erosion' has adversely affected agriculture in bangladesh and resulted in population migration to india ( figure ). climate change models project an increased frequency and likelihood of severe storms, with implications for sea level and flooding, in the ganges-brahmaputra-meghna delta, and this is likely to displace a large number of people. in pakistan, the adverse economic impact of heat waves on agricultural and non-agricultural activity has increased outmigration. in contrast, flooding in pakistan was not associated with significantly increased migration, possibly due to the larger scale of the response by relief agencies to floods compared with heat waves. in other regions, food scarcity due to flooding has contributed to internal migration. in in mali, flooding destroyed fields, damaged grain stores and impeded livestock production and, as a result, an estimated . million people are food insecure and internally displaced. 'gradual changes' due to climate change, such as changing rainfall patterns, rise in sea level, increased salinization and decreased soil fertility, can also be a driver for migration ( figure ). practice. migration has implications for health and public health practice. , migrants can be exposed to health risks in their country of origin and country of destination (figure ) ; they may be particularly at risk during their journey, due to lack of shelter, exposure to harsh climatic conditions, heat and overcrowded, inadequate transit and detention centres. public health interventions need to target migrants in all of these settings, and especially during migration, in order to minimize associated health risks (figure ) . sudden displacement because of a disaster or other event can be very stressful and manifest in post-traumatic stress disorders, depression or anxiety that can continue for months or even years. other factors that can affect the mental health of migrants include family separation, acculturation, job insecurity, restricted mobility, dangerous border crossings, stigmatization and marginalization. stress is associated with higher rates of depression and anxiety disorders in the short term and with cardiovascular disease in the longer term. [ ] [ ] [ ] epidemic intelligence data from the european centre for disease prevention and control (ecdc) suggests that migration is a relatively infrequent driver of communicable disease threat events in europe, compared with travel and tourism. , there was no evidence during the migrant crisis in europe that migrants posed a risk to european union citizens from communicable diseases, and despite the barriers to accessing immunization that migrants experience, the contribution of migrants to the current measles outbreaks in europe is minimal. however, migrants may be at increased risk of communicable disease due to exposure or conditions during migration and in the country of destination (table ) . , , , [ ] [ ] [ ] [ ] for example, there is evidence that tuberculosis transmission occurs on migration routes to europe, including during long-term stays in refugee camps. overcrowded and inadequate refugee camps, detention centres and housing can spread vectors (e.g. lice and fleas) that transmit diseases such as relapsing fever, trench fever and epidemic or murine typhus. poor and overcrowded living conditions can also increase the risk of respiratory diseases, such as seasonal influenza. migrants living in overcrowded conditions with poor sanitation may also be at risk of cholera outbreaks. overcrowding, poor sanitation and limited access to health services can also facilitate the emergence of antimicrobial resistance (amr) among migrants, particularly refugees and asylum seekers. migrants may be at increased risk of communicable disease in their country of destination due to factors including lack of vaccination, low socioeconomic status and poor living conditions and limited access to health care (table ) . recent evidence from the european union (eu) and the european economic area (eea), based on cd cell counts and hiv-rna trajectories from seroconversion also suggests that migrants may be at risk of hiv transmission in their country of destination; two-thirds of a sample of hiv-positive migrants from sub-saharan africa, latin america and the caribbean acquired their hiv infection in the country of destination. essential public health measures include ensuring adequate living conditions, access to health care in refugee camps, detention centres, screening for communicable diseases and assessment offer serological screening and treatment (for those found to be positive) to all migrants from countries of high endemicity in sub-saharan africa and focal areas of transmission in asia, south america and north africa. offer serological screening and treatment (for those found to be positive) for strongyloidiasis to all migrants from countries of high endemicity in asia, africa, the middle east, oceania and latin america. offer vaccination against mmr to all migrant children and adolescents without immunization records as a priority. offer vaccination to all migrant adults without immunization records with either one dose of mmr or in accordance with the mmr immunization schedule of the host country. offer vaccination against diphtheria, tetanus, pertussis, polio and hib a (dtap-ipv-hib) to all migrant children and adolescents without immunization records as a priority. offer vaccination to all adult migrants without immunization records in accordance with the immunization schedule of the host country. if this is not possible, adult migrants should be given a primary series of diphtheria, tetanus and polio vaccines. see table for details. cxr means chest x-ray; ltbi, latent tb infection; tst, tuberculin skin test; igra, interferon-gamma release assay; mmr, measles/mumps/rubella. source: ecdc. public health guidance on screening and vaccination for infectious diseases in newly arrived migrants within the eu/eea. https://ecdc.europa.eu/sites/portal/files/documents/public% health% guidance % on% screening% and% vaccination% of% migrants% in% the% eu% eea.pdf. vaccination against hib is only recommended to children up to five years of age. of vaccination status on arrival in countries of destination (table ). available evidence shows that it is cost-effective to screen child, adolescent and adult migrants for active and latent tuberculosis, hiv, hepatitis c, hepatitis b, strongyloidiasis and schistosomiasis (table ) . targeted hiv prevention, testing and treatment programmes are also required in countries where migrants are at risk of hiv acquisition after arrival. there is also a clear benefit from ensuring that migrants are included in vaccination programmes and providing catch-up vaccinations where needed (table ) . particular attention should be given to vulnerable children, women and the elderly, who might also suffer from non-communicable diseases, such as cardiovascular diseases, diabetes, cancer, chronic lung diseases, mental health and trauma. sudden migration can result in complications from noncommunicable diseases if treatment and care are discontinued or if access to medications is interrupted. these conditions can cause a life-threatening deterioration and acutely exacerbate preexisting conditions in the event of sudden migration (table ). travel, including for business and tourism, has increased dramatically in recent decades. globally, in , commercial airlines carried more than billion passengers; this is projected to have risen to . billion in and to continue to increase in the coming years. air travel contributes to greenhouse gas emissions and, hence, to climate change. at the same time, the effects of climate change on tourism assets, such as biodiversity, coral reefs, glaciers and cultural heritage, are leading to an increase in 'last chance' travel to destinations before they are further degraded. [ ] [ ] [ ] for example, in the southern hemisphere, sea level is projected to rise by - cm by the end of this century, submerging much of the maldives. in northern latitudes, climate change is already having an adverse impact on the winter tourism industry in lapland and may make it impossible to maintain its snowcovered winter wonderland in future. in many ski resorts, artificial snowmaking, which has high energy costs, is increasingly being used to supplement natural snow, to improve reliability and to extend the season. climate change can be a factor in creating conditions suitable for vector-borne disease transmission. importation of pathogens from endemic areas can then result in the subsequent spread of these diseases under suitable climatic conditions. extended seasonal transmission seasons due to climate change can also increase the window of opportunity of pathogen importation. climatic suitability, including lower and upper temperature thresholds, affects the growth, abundance and survival of mosquito vectors such as aedes aegypti and aedes albopictus that can transmit dengue, zika and chikungunya. in europe, a warmer climate appears to be associated with more frequent outbreaks of dengue and chikungunya are projected to become more suitable for onward transmission of the chikungunya pathogen. climate change may have contributed to the explosive spread of zika in brazil. the epidemic was preceded by 'record warmest' climatic conditions, accompanied by a severe drought. the utilization of home water storage containers during the drought might have provided breeding sites for aedes mosquitoes and increased exposure to mosquitoes. virus importation into a susceptible population could then have triggered the epidemic. precipitation, another important environmental factor affected by climate change, also influences the availability of conditions suitable for mosquito vectors. for example, heavy rainfall increases the abundance of a. albopictus and, hence, the risk of transmission of dengue and chikungunya. climate change can increase the risk of other vector-borne diseases, including tick-borne and rodent-borne infections, and can also influence the range, seasonality and incidence of water-borne diseases such as cholera. , global air travel may facilitate the spread of 'resistant pathogens'. for example, it is thought to have facilitated the rapid dissemination of methicillin-resistant staphylococcus aureus from the uk and north america across europe and then to asia. vancomycin-resistant enterococci and klebsiella pneumoniae carbapenemase-producing k. pneumoniae followed a similar trajectory. the burden of antibiotic resistance in destination countries can also be exacerbated by climate change, because an increase in ambient temperature is associated with a significant increase in antibiotic resistance for common pathogens, including k. pneumoniae and s. aureus. it is hypothesized that temperature accelerates horizontal transfer of resistance genes or the uptake of genetic material. ambient temperature may also facilitate environmental growth of resistant strains and enhance transmission from food, agriculture and environmental sources. climate change impacts on travel: implications for public health practice. air travel can facilitate the spread of communicable diseases around the globe. passenger volume has been identified as a significant driver of the importation of viremic passengers. , flights directly connect millions of passengers between europe and asia, africa, north america and south america, with many of these locations 'hot spots' for the emergence of communicable diseases. a disease outbreak in one part of the world can quickly spread to another part via air travel. , for example, in-flight transmission of severe acute respiratory syndrome (sars) in resulted in the rapid spread of sars-associated coronavirus (sars-cov) around the world. international air travellers departing from mexico in unknowingly carried and disseminated the novel influenza virus a(h n )v around the world. similarly, air transport networks contributed to the spread of middle east respiratory syndrome from saudi arabia in , chikungunya dispersion in the americas in , ebola from west africa in and zika spread in the americas in . [ ] [ ] [ ] [ ] more specifically, air travel can increase the risk of importation of pathogens from endemic areas into regions with competent mosquito vectors and suitable climatic and environmental conditions for vector-borne diseases. dengue, a mosquito-borne viral disease, is a significant public health concern, threatening almost half of the world's population. table . vaccinations to be offered to migrants in the absence of documented evidence of prior vaccination within the eu/eea priority vaccinations mmr administer to individuals ≥ months of age. two doses of mmr a should be administered at least month apart but preferably longer according to national guidelines. measles vaccine provided before months of age does not induce protection in all and should be repeated after months of age. loss of access to medication or devices, loss of prescriptions, lack of access to health care services leading to prolongation of disruption of treatment degradation of living conditions loss of shelter, shortages of water and regular food supplies and lack of income add to physical and psychological strain interruption of care due to destruction of health infrastructure, disruption of medical supplies and the absence of health care providers who have been killed, injured or are unable to return to work interruption of power supplies or safe water life-threatening consequences, especially for people with end-stage renal failure who require dialysis source: http://www.euro.who.int/en/health-topics/health-determinants/migration-and-health/migrant-health-in-the-european-region/migration-and-health-key-issues# transmission occurs largely in tropical and sub-tropical regions, but outbreaks have occurred in europe around the mediterranean where a. albopictus is present. infected air passengers from endemic areas can arrive in europe during their viremic period, be bitten by local mosquito vectors and transmit the dengue virus locally and trigger an outbreak due to a warming climate. other means of transport are also implicated in the spread of disease-causing pathogens. for example, ballast from cargo ships played a role in moving pathogenic vibrios to new locations that were environmentally and climatically suitable, even in northern latitudes. travel and tourism can also facilitate the importation of pathogens from developed countries with low vaccination coverage, to developing countries with high rates of susceptible individuals due to lack of health care access. , responding to the public health challenges associated with travel and climate change requires robust national surveillance systems, including effective tracking of vector location and disease importation. integrated surveillance of invasive and endemic mosquito species is crucial for effective prevention and control of vector-borne diseases. early detection of outbreaks can be aided by seasonal surveillance in areas where competent vectors are active and in close proximity to airports with a large influx of passengers from endemic areas. for example, the risk of dengue importation can be described by a model that relates air travellers from dengue-affected areas to dengue importation. this approach can delineate in space and time where the risk is the highest, and target seasonal surveillance to high-risk areas, for early case detection and intervention. an important consideration when projecting risk of disease transmission associated with climate change and travel is that the timing of risks may be under-estimated, i.e. transmission may occur sooner than models suggest. for example, the modelled future environmental suitability of a. aegypti in the contiguous usa in the period - projects potential suitability in southern areas of mid-western states, such as illinois, indiana and ohio. however, breeding colonies of a. aegypti were found in ontario in southern canada in , indicating that the risk of transmission of diseases such as dengue fever and zika may occur earlier than the projections suggest. the importation and spread of tropical pathogens in temperate regions due to climate change is another aspect to consider for public health practice. it is a potential threat to the safety of the blood supply and, hence, of blood transfusions, particularly with respect to pathogens for which there are no diagnostic tests. [ ] [ ] [ ] [ ] [ ] expansion of areas with an increased risk of communicable diseases due to climate change also increases the risk of blood bank contamination with communicable pathogens as a higher number of prospective blood donors are exposed to potential infections for a longer period, e.g. if climate change alters the length of the annual mosquito activity season. moreover, the asymptomatic phase of many infections, even if it is relatively short, increases the potential for transmission by transfusion. climate change projections of the probability of infection should therefore be used for preparedness activities. in addition, certain pathogens, such as the west nile virus, can persist in stored blood components and subsequently cause infection through intravenous application. pathogen reduction technology that inactivates pathogens in donated blood is a strategy that could be used for pre-emptive risk reduction. the world is warmer today than in pre-industrial times, and warming is projected to continue. the frequency, duration and intensity of extreme weather events have increased, with implications for migration in particular. global warming is associated with migration from farming communities and from small islands and coastal areas. evidence of climate change impacts on migration in other settings is less clear. the combined impact of travel and climate change can favour the importation, establishment and spread of tropical diseases in temperate regions. these developments require public health action, including targeted and culturally appropriate interventions and novel technologies, to prevent and control emerging health threats associated with the interaction between climate change, migration and travel. it calls for training of health care professionals to provide appropriate health care (prevention, screening, and treatment) for migrants and for risk assessment of diseases among travellers/ tourists. adequate living conditions, screening and vaccination programmes, and medical interventions for migrants can prevent outbreaks of communicable diseases and the spread of resistant pathogens. assessment and interventions also need to encompass chronic conditions, mental health and trauma due to migration. targeted hiv prevention, testing and treatment programmes are also required in countries where migrants are at risk of hiv acquisition after arrival. effective national surveillance systems that include tracking of vector location and disease importation can inform targeted prevention and control interventions. novel approaches including modelling the arrival of air passengers into environmentally and climatically receptive areas, to improve assessment of the risk of importation of pathogens from endemic areas, and assessment of climatic suitability, can inform seasonal surveillance or active case finding. specific measures may be required to ensure blood safety, including pathogen reduction technology, where there is a risk of the importation and spread of tropical pathogens in temperate regions. the authors report no financial interests or connections, direct or indirect or other situations that might raise the question of bias in the work reported or the conclusions, implications or opinions stated-including pertinent commercial or other sources of funding for the individual author(s) or for the associated department(s) or organization(s), personal relationships or direct academic competition. j.c.s. wrote the first draft of the paper. both authors approved the final paper. herein are the authors' own and do not necessarily state or reflect those of ecdc. ecdc is not responsible for the data and information collation and analysis and cannot be held liable for conclusions or opinions drawn. we should like to thank wei-yee leong from the lee kong chian school of medicine, nanyang technological university, singapore for her help with the figures. none declared. an ipcc special report on the impacts of 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population changes on human exposure to the virus vector mosquito aedes aegypti county health unit. aedes aegypti mosquito climate change projections of west nile virus infections in europe: implications for blood safety practices blood supply under threat emerging infectious threats to the blood supply emerging infectious diseases that threaten the blood supply current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections emerging infectious disease agents and their potential threat to transfusion safety we would like to thank the editor and the three anonymous reviewers for detailed and constructive feedback on the manuscript. we also appreciated the insightful comments and suggestions from teymur noori, pontus naucler and senia rosales-klintz at ecdc. the views and opinions expressed key: cord- -vsswxwdi authors: hammou, rahma ait; benhassou, mustapha; bessi, hlima; ennaji, moulay mustapha title: chapter scientific advances in the diagnosis of emerging and reemerging viral human pathogens date: - - journal: emerging and reemerging viral pathogens doi: . /b - - - - . -x sha: doc_id: cord_uid: vsswxwdi abstract despite scientific advances, the diagnosis of infectious diseases is primarily possible through vaccination and later by antibiotics. emerging and reemerging pathologies are still considered to be dangerous to humanity because of the unique nature of these diseases: it is the encounter between two living organisms that have coexisted for millions of years within the people on the same planet without being previously recognized. these infectious agents, such as bacteria, viruses, fungi, or parasites, pose no threat to humans. in fact, only a few hundred are able to inflict damage to the human host. in addition, the spectrum of human disease caused by a particular pathogen varies considerably depending on the factors related to the ecological agent, the host, and the infectious agents. several emerging or reemerging infectious agents are organisms that could be used in biological control. the differentiation of a natural epidemic from a bioterrorian event is based on several epidemiological indices as well as on the molecular characterization of the pathogen(s) involved. the role of pathologists is indeed very important. it is in this context that this chapter aims to discuss the various scientific advances, particularly molecular, in terms of diagnosis of these diseases; the new discoveries in the role of nanotechnologies and nanobiosensors; and also the implication of biomarkers, especially micrornas (mirnas), since it was reported that a single mirna has the ultimate capacity to target multiple genes simultaneously. in a viral infection context, mirnas have been connected with the interplay between host and pathogen and occupy a major role in the host–parasite interaction and pathogenesis. it is in this context that various molecular and nanomethods for the detection of emerging viruses and experimental validation of mirnas during quelling viruses target transcripts will be discussed in this chapter. the diagnosis of any infectious disease requires active communication between clinicians and clinical laboratory personnel, usually in the form of "consultations" from clinicians to the laboratory director "on call" or directly with the laboratory technologists. arriving at a correct diagnosis in a timely fashion begins with the acquisition of an adequate clinical specimen (blood, cerebrospinal fluid, urine, respiratory secretions, bronchoalveolar lavage, feces, etc.) and its transportation to the laboratory in an appropriate container. specimens for molecular diagnostic tests sometimes require different methods of transportation/ preservation from those of regular specimens so as to ensure that the techniques, used efficiently, will detect the suspected infectious agent. the classic diagnostic principles in clinical medicine still apply when using molecular methods of diagnosis. patients and populations are evaluated clinically and epidemiologically, and case definitions are created so that test parameters, such as sensitivity, specificity, and predictive values, can be defined. likewise, understanding the concept of screening versus confirmatory testing is also important. the three major steps performed in molecular assays are specimen processing, nucleic acid amplification or hybridization, and product detection (nolte and caliendo, ; persing et al., ) . processing of the specimen is one of the most important steps for the successful detection of nucleic acids. protocols vary, depending on the specimen received, and the specimen "matrix" plays an important role in nucleic acid extraction, owing to the presence of extraneous material in some matrices, that could potentially interfere with the molecular assays being performed. the most widely known example of such interference is the presence of taq polymerase inhibitors in nucleic acid extracts, leading to false-negative results. nucleic acid extraction protocols are usually cumbersome, and automation has been relatively challenging. however, several instruments have been designed for the clinical laboratory in which automated extraction protocols have been incorporated. automation has also been incorporated for nucleic acid amplification methods, including conventional and real-time polymerase chain reaction (pcr), branched dna, and isothermal technologies that obviate the usual thermal cycling and potentially speed up the time required for amplification. automated platforms, however, have decreased the cycling time by changing more quickly the temperatures required for different pcr cycles. real-time pcr systems are usually coupled with automated detection systems that also vary in complexity. a few instruments are also capable of complete automation, including extraction, nucleic acid amplification, and detection systems. tables . and . present a summary of the techniques available for the detection of nucleic acids from infectious agents. fortunately, the biotechnology boom of the late s and early s fueled the development of highly automated nucleic acidÀbased testing methods, which had important implications for the identification of infectious pathogens in human specimens (monecke and ehricht, ) . one of these technologies, commonly referred to as real-time pcr, has gained considerable popularity. this method combines nucleic acid amplification and fluorescent detection of the amplified product in the same closed system (morse, ; relman et al., ; hjelle et al., ; aragon et al., ) . the promulgation of real-time pcr as an important testing platform in clinical microbiology was catapulted by us homeland security efforts to produce rapid, reliable testing methods for identifying potential agents of bioterrorism. the laboratory response network (lrn), an integrated group of public health, armed forces, and private referral laboratories, was created by the centers for disease control and prevention (cdc) to serve as a reference laboratory network for identifying and confirming agents of bioterrorism. in a very short period, scientists at cdc successfully developed a number of realtime pcr assays for the detection of agents of bioterrorism, and these assays are now available at many of the lrn laboratories. therefore molecular tools have improved the initial diagnosis of emerging and reemerging infectious diseases especially for pathogens that are uncultivated or difficult to isolate in clinical laboratories (nolte and caliendo, ; persing et al., ) . in addition, conventional diagnostic tests for infectious agents are slow, sensitive, expensive, or unavailable. therefore since the identification of several pathogens has been obtained with molecular techniques that have contributed primarily to the diagnosis of these infectious agents relman, ) . nucleic acidÀbased assays are now gold standards for several infectious agents, including hepatitis c virus (hcv), enterovirus, bordetella pertussis, and herpes simplex virus (hsv) (in the context of herpes encephalitis, chlamydia trachomatis, and others). cost-effectiveness is undoubtedly an extremely important factor when introducing molecular diagnostics into the clinical laboratory to replace conventional techniques. the best example is that of common bacterial pathogens, some of which require, in addition to identification, sensitivity tests to guide clinicians in choosing the most effective antibiotic. one of the most promising platforms in clinical microbiology laboratories is that of molecular diagnostics using multiplexing technology (loeffelholz, ; smithn et al., ) . the ability of real-time pcr to amplify and detect the product to be amplified, using specific probes at the same time, allows for multiple amplifications with the same clinical sample. the design of primers is the most critical step in multiplex pcr so that efficiency and specific amplification are not compromised. real-time multiplex coupling pcr with liquid-stained microarrays provides the multiplex detection of pathogens from clinical syndromes. fda-approved diagnostic products are already available for viral respiratory pathogens. we could imagine the use of multiplex systems to diagnose enteric pathogens, nosocomial infections, including identification of genes responsible for antibiotic resistance (according to the hospital's epidemiological data), pathogens of the central nervous system, hemorrhagic fever, and sepsis. we could also consider the inclusion of emerging and reemerging infectious agents in some of the syndrome-based diagnostic products to establish true incidence and prevalence of these highly neglected infectious diseases. furthermore, numerous reports have described the utility of this user-friendly technology for the rapid (same-day) and accurate detection of many emerging (new) and reemerging pathogens as well as pathogens commonly encountered in medical practice. a search for all articles published in the journal of clinical microbiology from through , which evaluated real-time pcr as a test method for pathogen detection and/or identification of genes or mutations associated with antimicrobial resistance in pathogens, revealed in articles. among these articles, described assays with the lightcycler instrument (roche diagnostics corporation, indianapolis, in); described assays with the abi prism , , or h instrument (applied biosystems, foster city, ca); described assays with the smartcycler instrument (cepheid, sunnyvale, ca); and described assays with the icycler instrument (bio-rad laboratories, hercules, ca). the availability of nucleic acidÀbased technology, such as real-time pcr, along with conventional staining and culture methods and immunoassays, can provide laboratories of many sizes with a comprehensive and responsible approach for the detection of both commonly encountered and emerging or reemerging pathogens. the clinical presentations of concerned patients infected with common viruses that cause cutaneous vesicular lesions [hsv, varicellazoster virus (vzv), enterovirus, or disseminated vaccinia virus following smallpox vaccination] might mimic those of patients with smallpox. another complicating feature is that some recipients of the smallpox vaccine may develop erythema multiforme, which can also present as vesicular lesions. it has been our experience that by using real-time pcr assays, one can rapidly discriminate among these possibilities. analyte specific reagents (asrs) or kits for the detection of hsv or vzv with the lightcycler instrument are available from at least two vendors [realart hsv / kit and realart vzv pcr kit (artus); lightcycler herpes simplex virus / and lightcycler vzv orf (roche diagnostics corporation)]. kits are also available for testing for vzv (realart vzv pcr kit; artus) with the abi prism , , and h instruments. researchers have used the assays with the roche lightcycler instrument to routinely detect hsv and vzv and have developed an in-house realtime pcr assay for poxviruses, including variola virus that uses the lightcycler instrument. these assays have been invaluable for providing a rapid result, especially for military personnel who have developed cutaneous vesicular lesions as a complication of receiving the smallpox vaccine and who have been on assignment in areas of the world at significant risk for bioterrorism events. importantly, the home-brewed real-time assay that they have developed can discriminate among several poxviruses and was useful in the identification of viremia in a recent case of monkey poxvirus disease in a patient from the upper midwest. an asr for the detection of variola virus with the lightcycler instrument is also available from artus (realart orthopox pcr kit). west nile virus, a rna virus of the family flaviviridae, has a predilection for the central nervous system and can be associated with significant morbidity and mortality. the first human cases of west nile virus infection occurred in the northeastern united states in the summer of ; since then the disease has progressed relentlessly from east to west across the continental united states. yet, no effective therapy has been defined. traditionally, during the summer and early fall in the united states, viral central nervous system disease is most frequently caused by enterovirus. in most regions of the united states, west nile virus infection must now also be considered during this time of the year. hsv can cause encephalitis at any time of the year, and antiviral therapy is available and effective. therefore ruling out hsv infection should be a priority, especially when encephalitis is encountered. real-time pcr has replaced viral culture as the gold standard for the rapid and accurate detection of hsv in cerebrospinal fluid. as mentioned previously, asrs or kits for the detection of hsv are available from artus and roche. artus also has a kit that can be used to test for enterovirus (realart enterovirus rt-pcr kit) with the lightcycler instrument. limited studies have shown that the pcr detection of west nile virus in cerebrospinal fluid is less sensitive than immunoassay for immunoglobulin m antibodies. currently, only a few referral and public health laboratories have the capability to perform immunoassays. at least two companies offer asrs or kits for real-time pcr [realart wnv rt-pcr kit (artus); lightcycler wnv detection kit (roche applied science)] with the lightcycler platform. if effective antiviral therapy becomes available, the rapid on-site diagnosis of west nile virus disease in areas of endemicity may be desirable. one important lesson learned from the À winter outbreaks of severe acute respiratory syndrome (sars) was that the early identification and quarantine of individuals with suspected cases of sars were essential for controlling the disease, especially in institutional settings. this effective approach toward the control of a communicable infectious disease adds credence to the concept that similar measures can be effective for controlling and preventing nosocomial vre and methicillin-resistant staphylococcus aureus (mrsa) outbreaks. no laboratory tests were available for the detection of sars coronavirus (sars cov) during much of the outbreak, as the etiological agent was not confirmed until early march . eventually, real-time pcr tests were developed and were available commercially from at least two manufacturers for use with several real-time pcr testing platforms [realart hpa-coronavirus rt-pcr kits (artus) for use with the lightcyler instrument, the abi prism , , and h instruments, and the rotor-gene instrument; and lightcycler sars cov (roche diagnostics corporation) for use with the lightcycler instrument]. during the outbreak, it was important to rule out treatable influenza virus types a or b infections, whose clinical presentations can mimic those of sars cov. rapid antigen tests for the detection of influenza virus (both types a and b) are relatively easy to perform and may be useful in the local setting for the detection of cases of influenza; however, these tests lack sensitivity. as infections due to both influenza virus types a and b are now treatable, rapid on-site diagnostic capabilities are important. recently, a real-time pcr assay that uses the lightcycler platform was demonstrated to have much greater sensitivity than antigen detection ( % and %, respectively) for the detection of influenza virus type a infections. following the À sars outbreak, many lrn member laboratories developed the capability to detect sars cov. should another outbreak occur, this public health laboratory network should facilitate the laboratory diagnosis of cases, especially when testing at the local level is not available. the prognosis of infectious diseases has also been significantly affected by the availability of molecular techniques. viral load in several chronic infections, such as human immunodeficiency virus (hiv), human papillomavirus, hepatitis c, bk virus, and cytomegalovirus, plays an important role in predicting better or worse outcomes and the need to start specific antiviral therapy to improve survival (humar et al., ; mellors et al., ; reidn et al., ; walsh et al., ) . viral and bacterial quantification by molecular methods could also be used in the future to differentiate a mild subclinical infection from a disease caused by the agent under certain conditions. a microarray is a "collection of microscopic features," usually dna, which can be probed with target molecules to produce quantitative or qualitative data (miller and tang, ). indeed, the diagnosis of infectious diseases is most often qualitative (presence or absence of the pathogen), although the quantitative data become more numerous under certain conditions, such as the hiv/immunodeficiency syndrome acquired and hepatitis c. the microarray platforms can be classified as follows: printed, synthesized in situ, high-density bead networks, electronic suspension, and liquid bead (loeffelholz, ; miller and tang, ). all microarray platforms have the ability to multiplex, a characteristic that becomes indispensable in the diagnosis of infectious diseases. for all approaches the probe is the dna sequence bound to the solid surface of the array, and the target is the nucleic acid to be detected. the probes are synthesized and immobilized on microscopic spots. it has been reported that of all platforms, suspension beads probably offer the most practical technology to clinical microbiology labs (loeffelholz, ; miller and tang, ) . universal heel sets are available, and user-defined applications are easily implemented because of their flexibility. therefore the use of wide-range or multiplex pcr followed by microarrays provides an excellent platform for the rapid and efficient identification of bacterial, viral, or fungal pathogens (mikhailovich et al., ) . for the diagnosis of bacterial infections, microarrays are also incorporated in clinical laboratories for the rapid detection and characterization of mrsa, determination of antimicrobial drug resistance in several pathogens, such as enterococcus and mycobacterium tuberculosis, and the diagnosis of sepsis (mancini et al., ; aragon et al., ; monecke and ehricht, ) . several difficult problems remain before the microarrays can be widely used in the clinical setting, including pre-and postanalytical variables, such as the type of clinical specimens (matrices), extraction techniques, labeling, and quality controls (miller and tang, ). the introduction into microbiology laboratories of additional diagnostic tests based on molecular technology will depend heavily on automation. many molecular tests remain difficult because of complex and lengthy problems of processing and interpretation of samples. biosurveillance has become an important health priority because of the increased risk of emerging and reemerging pathologies and their effects on human populations (sintchenko and gallego, ). the potential consequences of large-scale epidemics or epidemics can be economically, socially, and environmentally profound, as evidenced by the global spread of coronavirus associated with severe acute respiratory syndrome. traditional biosurveillance relies heavily on disease reporting by clinical laboratories to local, state, and federal health-care organizations (sintchenko and gallego, ) . most clinically diagnosed cases are confirmed using standard microbiological methods, such as serologic markers and cultures, followed by biochemical identification, if necessary, or dna-based typing methods, once a pure isolate is obtained. as a result, these methods cannot be described as slow and insensitive. traditional biosurveillance has improved through electronic reporting to some extent, with an increase in the proportion of diagnosed cases reported in all cases occurring in the community (panackal et al., ; effler et al., ) . on the other hand, other factors include variability in case definitions (clinical and laboratory criteria), screening practices, contact tracking methods, and the quality of performance of the diagnostic tests used. the second surveillance system is said to be syndromic and is based on the collection of vast amount of data from the various services, including emergency departments, intensive care units (icus), admission and hospitalization systems, and clinical laboratories (sintchenko and gallego, ; bravata et al., ; lewis et al., ) . indeed, the purpose of this system is to play the role of an alarm signal when the models obtained over time move away from the "normal" state. the normal state is determined by the collected data and by creating "baselines" for a given area or community (wang et al., ; berger et al., ) . as expected, the specificity of these systems is rather low but can be improved by combining syndromic surveillance systems and laboratory surveillance (sintchenko and gallego, ; buehler et al., ; weber and pitrak, ) . the latest generation of surveillance is based on the genomics of surveillance systems and is essentially based on the development of rapid molecular tests applied to the diagnosis of infectious diseases. a technique based on molecular subtyping has increased sensitivity and specificity in the investigation of epidemics at all levels (hospital, local, state, and national) (hedberg and besser, ; monecke et al., ; mellmann et al., ) . similarly, modern typing methods, based on direct data, such as sequencing and powerful software, to analyze gene clusters and genetic evolution, have also made it possible to detect possible epidemics by new pathogens or reemerging agents (sintchenko and gallego, ) . typical examples include surveillance systems for influenza a viruses and severe acute respiratory syndrome. powerful molecular techniques cannot be used in isolation. modern biomonitoring systems are important elements, including traditional epidemiological tools and newly developed "computer-based" systems in which complete integration of microbial profiling is merged with epidemiological surveillance and spatial surveillance (depending on the use of systems geographical information). such a "network" or "global laboratory" would provide an exquisitely comprehensive view of potential biotherapies (urwin and maiden, ; casadevall and relman, ; reis et al., ; o'connor et al., ; layne and beugelsdijk, ; heymann and rodier, ) . nanotechnology is a broad term that encompasses several disciplines and techniques, some of which currently have or will have a major impact on health care. for the purpose of this review, nanobiotechnology and its applications in molecular diagnostics are discussed. nanotechnology is the study of the control and manipulation of matter at the atomic and molecular scales (johnson et al., ) , the materials used are nm or less in at least one of the dimensions of the material. on the other hand, molecular diagnosis is an essential element in the development of personalized medicine, thus presenting punctual performance of diagnostic procedures. this report focuses on the application of several technologies in the clinical laboratory setting. the different interrelationships between nanotechnology and molecular diagnostics and their role in nanomedicine and personalized medicine are shown in fig. . . these are nanofluidic networks for the isolation and analysis of biomolecules, such as nucleic acids or proteins. the networks use nanotubes for the isolation of molecules and the detection of molecules trapped with electrode-based systems. the volumes required for such tests are very small, compared to those required with conventional instruments, and turnaround times are significantly reduced (jain, ) . whenever a single molecule of dna moves in the nanotube, the electric current changes abruptly. the current returns to its reference value when the dna molecule exits the nanotube. nanofluidic technology is expected to have wide applications in systems biology, personalized medicine, pathogen detection, drug development, and clinical research. gold nanoparticles, nanocrystals (also called quantum dots) and magnetic nanoparticles are the main examples in this category. gold nanoparticles range from . to nm and can be functionalized with a variety of biomolecules including antibodies, nucleic acids, peptides, proteins, and carbohydrates. thus gold nanoparticles have better optical absorption and scattering properties because of a unique property known as plasma resonance. like nanocrystals (see the next section), their optical properties can be modified by changing size, shape, and composition. on the other hand the surface of nanocrystals can also be functionalized with nucleic acids, antibodies, proteins, and peptides, allowing adaptation to several diagnostic platforms (bruchez et al., ; hotz, ; michalet et al., ) . the magnetic nanoparticles are made of iron and incorporated into copolymer beads. the surface charge of the beads can be manipulated with the polymer coating to enhance nonspecific protein adsorption to the surface of the beads, increasing the specificity of the assay (johnson et al., ; jain, ) . nanobiosensors are nanosensors that are allowed for the detection of chemical or biological materials. these materials are extremely sensitive (jain, ) . prototype sensors have demonstrated the detection of nucleic acids, proteins, and ions. these sensors can operate in liquid or gaseous phase, which allows them to be used for different downstream applications. these sensors are inexpensive in their manufacture and are portable. thus they can serve even as bases for the implementation of implantable devices for detection and monitoring. this technology provides an alternative approach to pcr and complements current dna and protein microarray methods. using this method, it is not necessary to label or copy the target molecules. the advantages of cantilevers are that they provide fast, unlabeled recognition of specific dna sequences for single-nucleotide polymorphisms, oncogenes, and genotyping. nanocantilevers could be crucial in the design of a new class of ultrasonic sensors to detect viruses, bacteria, and other pathogens (gupta et al., ) . finally, a real-time cantilever biosensor can provide continuous monitoring of clinical parameters in personalized medicine. the virus particles are biological nanoparticles. giving examples of hsv and adenovirus that can be used to trigger the assembly of nanomagnetic beads as nanobiosensors for clinically relevant viruses (perez et al., ) . thus this technique offers the possibility of detecting up to five virus particles in a ml serum sample. this system has the advantage of having an increased sensitivity compared to the enzymelinked immunosorbent assay (elisa)-based methods as well as an improvement over the pcr-based detection because it is less expensive and faster and has fewer artifacts. micrornas (mirnas) represent a recently uncovered class of small noncoding rnas from to nucleotides that function as posttranscriptional regulators of gene expression. these mirnas are coded by separate genes that are localized in the nonprotein coding part of the genome. micrornas predict to regulate about %À % of human genes (griffiths-jones et al., ; malumbres, ) . these small noncoding rnas bind their target messenger rnas (mrnas) in the untranslated region (utr) and coding sequence (cds) regions and act as negative regulators. this action affects various biological processes including cell growth, differentiation, signal transduction, metabolism, and development (inui et al., ) . dysregulation of mirna expression has been described in several diseases, and their role appears to be pivotal in driving tumorigenesis (iorio and croce, ) . mirna dysregulation is considered to be an early event in tumorigenesis (cortez et al., ) . it was discovered in in the caenorhabditis elegans nematode of a dna fragment containing a small sequence that encodes a nucleotide rna, lin that regulates the transition from larval stage l to stage l . this regulation is achieved by repressing the expression of the proteins lin and lin by this mirna by binding to the noncoding regions ( -utr) of the corresponding mrnas. in another mirna let was identified, which regulates the transition from larval stage l to adult stage by the same mechanism of action. since then, several discoveries have been made and have demonstrated the conservation of these mirnas in several species. studies have revealed that more than mirnas can potentially target nearly % of mrnas in humans (friedman et al., ) . in the nucleus the gene of mirna is transcribed into a pri-mirna by the action of a rna polymerase type ii or iii (borchert et al., ; yoontae et al., ) . pri-mirna will then be cleaved by the nuclear microprocessor complex formed by the association of the enzyme drosha and the dgcr (digeorge critical region ) protein into an intermediate precursor called a pre-mirna (han et al., ) . once the complex is attached to primer-mirna, the dgcr protein will allow to define the cleavage distance that generally measures base pairs from this junction, while the two drosha domains will cleave the and ends of the pri-mirna. the resulting pre-mirna is a loop stem having two unmatched nucleotides at the end and a phosphate at the end, this asymmetry is specific for type iii rnases. the pre-mirna will migrate to the cytoplasm by the exportin (huang et al., ; garza, ; sotillo and thomas-tikhonenko, ) . in the cytoplasm the pre-mirna undergoes another stage of maturation where the rlc (risc loading complex) is composed by rnasedicer acytoplasmic endonuclease that performed the second stage of processing, which includes a leading strand or mir and a passenger strand or mir*. for mirnas with a high degree of complementarity throughout the loop stem, an additional cleavage at the middle of the strand is effected by the triteness activity of the ago protein before that cleaved by the enzyme dicer in order to generate an ac-pre-mirna (ago -cleaved precursor mirna) (eulalio et al., ) . then, the resulting mirna will form a risc complex, one of the two strands called the "passenger strand" is degraded, whereas the strand having the least stable end called the "guide strand" is retained-it is the mature mirna. the evidences have shown that the strand with more unstable (weaker base pairing) has lower chance of degradation (sotillo and thomas-tikhonenko, ) (fig. . ). although new established methods such as reverse transcription quantitative pcr (rt-qpcr) have been used to detect viral infections, there is still a lack of robust biomarkers for early diagnosis and prognosis of the infectious disease. increasing evidence indicates that cell free mirnas are present in body fluids including blood and saliva. they are produced endogenously in response to the molecular change in cells, and therefore they can be used as diagnostic reporters for various diseases such as cancer and viral infections (zhu et al., ) . mirna is a class of small ( À nucleotides in length) noncoding rnas, which involves in gene regulation and plays important roles in cell proliferation, differentiation, apoptosis, and tumorigenesis (kaladhar, ) . from the previous reports, mirnas have distinct expression profiles in virus-infected cells in comparison to their healthy counterparts (tambyah et al., ; zhu et al., ) . moreover, the expression levels of many mirnas in virus-infected samples and normal controls exhibit fold change difference (song et al., ) . this studies show that in addition to regulating multiple processes, mirnas themselves may be independent effectors of innate immunity by directly targeting viral transcripts. in vitro studies show mirna target influenza; vesicular stomatitis virus, human t-cell leukemia virus ; human papillomavirus; and enterovirus , and they inhibit viral replication (fig. . ) , which facilitates the clearance or potentiating viral latency of the pathogen (bai and nicot, ; hen et al., ; zheng et al., ) . mirnas were also essential to show malaria transcripts translocation into the parasite ( ). indeed, due to their targeting of viral transcripts, mirnas have the potential to partially dictate the cell tropism of a virus, the resistance of resting t-cells to human t-cell leukemia virus appears to be due to their expression of mir- - p rna polymerase ii produces a À nucleotide transcript, called pri-mirna, that is mature to pre-mirna hairpin by a drosha (_ À nucleotides). this double-stranded hairpin structure is exported from the nucleus into cytoplasm by exportin (ran gtpase). lastly, the pre-mirna is processed by dicer and produces sense and antisense strands, approximately nucleotides in length, the effective strand called antisense and known as mature mirna and short-lived complementary sequence called passenger strand (mir*). the antisense-stranded mirna is combined into risc, which then targets it to the target untranslated region mrna sequence to facilitate repression and cleavage. aa, poly a tail; m g, -methylguanosine cap; mirnas, microrna; orf, open reading frame; rna, ribonucleic acid. (bai and nicot, ) . although a few scientists agree on the direct interaction between mirnas and viral transcripts, bogerd et al. ( ) argue that cellular mirnas do not target viruses as global downregulation of host cell mirnas (via dicer knockout) does not lead to the enhancement of viruses in human embryonic kidney cell line t. however, bogerd et al.'s model is problematic as viruses may be dependent on cell mechanisms that are controlled by mirnas, and the usual host cell of the viruses in their study is not human embryonic kidney cells. contrary to bogerd et al.'s study, there is evidence that direct targeting of viral genome/transcripts occurs in vivo as several groups have successfully attenuated viral vaccines by incorporating human mirna seed sites in viral genome (see the clinical applications of mirnas: improving vaccines) (barnes et al., ) . the relative importance of mirna direct targeting of viruses in innate immunity remains to be seen however as in vivo and in vitro studies show viral mutation of mirna seed sites in viral genomes means viruses can quickly evolve to avoid being targeted by mirnas (zheng et al., ; heiss et al., ) . there is another category of mirnas, which are secreted from cells and called extracellular mirnas (ex-mirnas, circulating mirnas). this ex-mirnas can be isolated from most biological fluids (de candia et al., ; irmak et al., ; weber et al., ) . often, we find these ex-mirnas in extracellular vesicles (exosomes, microvesicles, and apoptotic bodies), and through their association with argonaute protein (a component of the risc complex-see fig. . ) and high-density lipoprotein (de candia et al., ; arroyo et al., ; vickers et al., ; zernecke et al., ) . their biological function is debated since their secretion may be activated as intercellular communicators of gene regulation, or as cellular waste disposal method, or passively secreted as a by-product of cell death (turchinovich and cho, ) . although these three theories can be valid, there is increasing evidence that ex-mirnas are functional, can be passed between leukocytes in vitro and in vivo, and play a role in disease (alexander et al., ; bell and taylor, ; lehmann et al., ; mittelbrunn et al., ) . regardless of their functions, one clinical application of ex-mirna is the use of these as biomarkers of infectious disease. furthermore, these mirnas have shown the potential to be used as a biomarker for the prognosis and therapy of infectious diseases. mirnas have also shown a significantly altered expression during infection. the altered expression of mirna level in an infected human can be identified by the use of advanced diagnostic tools. in addition to their availability in numerous body fluids, mirnas are highly stable in these fluids. these features make mirnas, in single or in a combination (peng et al., ) , ideally suited as biomarkers for disease diagnosis. presently, only few standardized procedures are available for the isolation and characterization of specific mirna. experimental research and its observation have shown that small interfering rna, premature mirnas, and transfer rna may interfere with specific mirna during the process of isolation and characterization. therefore this interference leads to the false-positive result, which should be taken care of during diagnosis. the necessity of the large amount of rna input for the northern blot technique can generate difficulties in the quantification of the mirna. rt-qpcr, microarray profiling, and next-generation sequencing have been found to be useful for the identification of novel mirna. an experienced researcher having a good knowledge of molecular biology as well as bioinformatics should do the characterization of mirna. the immunomodulatory functions of mirnas represent a promising application of mirnas in the target of promoting antimicrobial pathways during infection and controlling dysregulated inflammatory responses during sepsis. in the lungs of mice infected with nontypeable haemophilus influenza, hock et al. noted that the physiological downregulation of mir- - p promotes phagocytosis by neutrophils and macrophages and bacterial killing and found that it boosts this downregulation by intratracheally administering an antagomir of mir- - p enhanced bacterial killing when they made challenge with nontypeable h. influenza. in another case, alexander et al. observed that mice inflammatory response to endotoxin in vivo was ameliorated and enhanced with administration of exosomes containing mir- a and mir- , respectively, which prompts the authors to conclude that such treatments could be useful adjuncts in managing sepsis (in the case of mir- a) or vaccination (in the case of mir- ). moreover, another work by wang et al. ( ) supports the idea that mirnas can be used in diseases therapy, especially sepsis, when they administrate mesenchymal stem cell exosomes containing mir- , which confer cardiac protection in septic mice. overall, using mirna-based therapies to leverage immune response may prove useful adjuncts to standard antimicrobial therapies, for example, in multidrug-resistant gram-negative infections, or chronic viral infections such as hepatitis c. nevertheless, there are significant challenges in implementing mirna-based antimicrobial therapeutics, which include devising methods of administration, and drug design that will protect mirna mimics/antagomirs from circulating rnase enzymes. delivery systems have to ensure targeted efficient delivery of mirnas to the site of infection, because, as noted earlier, a mirna may appear in many cell types, serving very different functions making offtarget effects a real possibility, limiting efficacy, and safety (chen et al., ) . a detailed analysis of the outcomes of phase trials of two mirnabased cancer treatments will provide more important data on the feasibility of mirna-based therapeutics generally (beg et al., ; reid et al., ) . as noted in the earlier rg- trial, viral mutation and resistance is an issue that will need tackling ( van der ree et al., ) . the class of ex-mirnas is ideal biomarker candidates due to their possible isolation from biological fluids (boon and vickers, ) . molecular methods such as rt-pcr are already used routinely in the clinical setting to quickly identify infections (e.g., respiratory infections in babies with bronchiolitis) and could be used to quantify ex-mirnas in patient samples. a search of the literature identified studies assessing ex-mirnas in infectious diseases through whole micronome profiling and candidate mirna approaches. thus the huge majority of these works are based on serum and plasma, but ex-mirnas in cerebrospinal fluid (csf), saliva, and sputum have also been implicated. until now, most of the studies have focused on hcv, hepatitis b virus, hiv, tb, and sepsis with the aim to improve diagnosis and prognosticate infection outcome (e.g., death in sepsis, liver cirrhosis in hepatitis) or treatment response. these infection studies are based on comparison of ex-mirna profile of patients and healthy controls. then, many studies identify that expressions of ex-mirna are highly predictive of infection. in the study by zhang et al. ( ) , they found a differentiated expression of mir- , mir- - p, mir- , mir- c, mir- , and mir- b between pulmonary tb and healthy controls with a high sensitivity and specificity going up to % and . %, respectively. the limitation of healthy controls used for comparison is that differentially expressed ex-mirnas may represent a nonspecific marker of infection, and this leads to limited clinical translatability of these studies given most people undergoing tests are symptomatic of some disease process. although, to solve this problem, a handful of studies have chosen more pragmatic comparator groups and promisingly suggest ex-mirna signatures can differentiate particular infectious disease from other differential diagnoses. furthermore, a promising application of ex-mirna biomarker work may be to differentiate viral from bacterial infection, identify or prognosticate sepsis, and in monitoring of response to antimicrobial treatment. there are substantial interstudy discrepancies in mirnas identified as potential biomarkers. this may be due to heterogeneity in study design (e.g., data normalization methods) and confounders such as hospital differences in defining sepsis and icu admission criteria. differences in the lengths of illness between patients create noise in the data; longitudinal studies that measure serial mirna levels would provide temporal information on mirna expression in sepsis and may resolve some conflicting findings. however, there are challenges in using mirnas as biomarkers of infectious disease, and this is underlined by a lack of interstudy cross validation of many results. conflicting study results may arise from heterogeneity in study design including differences in populations and control groups, methods of mirna extraction and the circulating fraction under investigation (serum, plasma, microvesicles, or exosomes), micronome expression profiling platforms (next-generation sequencing, probe-based hybridization microarrays, or rt-pcr arrays) and the dearth of mirnas assessed, the limited statistical power of many studies at the profiling stage, data normalization methods, whether p-values were adjusted to take account of multiple testing issues (usually not done), and whether confirmatory cohorts were used to validate results. given there is good evidence that mirna contained in exosomes is functionally secreted as intercellular mediators of gene regulation, it is tempting to speculate that biomarker studies which profile mirnas in exosomes rather than ex-mirna in total plasma/serum (which will include a background of mirna present from dead cells) could be more sensitive or specific biomarkers; comparisons of different extraction methods within the same biomarker study could help resolve this possibility. in the previous studies, it was reported that mirnas play an important role in influenza virusÀhost interaction. in a study by fang peng et al., the mirna expression profiles in the sera of h n -infected patients and healthy controls were analyzed using mirna microarray. among the mirnas that were significantly differentially expressed in h n serum samples when compared with that of healthy controls, mirnas were upregulated and downregulated. five serum mirna candidates (hsa-mir- - p, hsa-mir- a, hsa-mir- d, hsa-mir- e, and hsa-mir- ) were further verified by rt-qpcr. receiver operating characteristic curve analysis was performed to evaluate the potential use of these mirnas for the h n infection diagnosis from the serum samples. in this study, mirna microarray assays revealed differential expression of mirnas in h n patients' serum samples when compared to that in healthy controls. they identified five mirnas that can be used for the diagnostic biomarkers for the early detection of the h n infection, and this mirna signature will advance our understanding of the molecular mechanisms involved in the influenza h n infectious disease. rapid and accurate diagnosis of highly transmissible, lethal illnesses such as ebola virus (ebov) disease (evd) is critical for restricting pathogen spread and for applying appropriate therapeutic strategies. as demonstrated by the recent evd outbreak in western africa, early detection and confirmation of suspected cases are essential to halting disease spread (blackley et al., ) . current diagnostics rely on identifying ebov in blood samples by targeting viral antigens using enzyme immunoassays or by amplifying specific viral sequences through quantitative reverse transcriptase pcr (rt-pcr) (trombley et al., ) . in the study by janice duy et al., the authors analyzed the expression of circulating mirna sequences in archived plasma from rhesus macaques exposed to ebov infected either through intramuscular injection or through aerosol inhalation. we identified mirnas that showed significant changes in abundance during lethal ebov infection for each group. they found mirnas correlated with viral titer in both rhesus macaque as well as human samples. as a proof of concept for a host mirnaÀdriven diagnostic, they identified eight mirna classifiers predictive of acute infection with high accuracy in both nonhuman primates and humans, and this classifier identified half of the presymptomatic macaque hosts. keeping these caveats aside, this work shows that mirnas are potential diagnostic candidates via a proof of concept acute evd classifier while also establishing the potential basis for presymptomatic or asymptomatic diagnosis of the disease. clinical microbiology laboratories at the local level have an increasing responsibility to provide rapid and accurate diagnostic services for emerging (new) and reemerging infectious diseases, especially those diseases for which significant mortality or morbidity may occur as the result of a delay in diagnosis. rapid, accurate diagnosis of emerging and reemerging infectious diseases may also be critical at the local level to ensure optimal infection control. detection of these pathogens has often required esoteric procedures such as conventional pcr, which could be performed only at referral laboratories or, recently, at public health laboratories. recent technical advances in molecular diagnostics have resulted in the development of user-friendly automated testing platforms, such as real-time pcr. these novel-testing methods can be used to detect emerging and reemerging pathogens as well as common pathogens and have the potential for broadscale use in smaller laboratories in close proximity to the delivery of care. while writing this review, a large outbreak of influenza virus type a (h n ) was peaking in the united states, and new influenza virus type a strains (h n , h n ) have been associated with both avian and human influenza in regions of the far east. the apparent significant morbidity and mortality associated with these new influenza virus strains emphasize the need for rapid, accurate laboratory diagnostic capabilities at the local level. as is the case for sars, agents of bioterrorism, and the other pathogens, rapid diagnostic methods, such as real-time pcr, and microarray will likely play a major role in the early and sensitive detection of emerging and reemerging infectious diseases encountered in the future. otherwise, a class of small rnas implicated in the diagnosis of these diseases is mirnas and is considered an essential mediator of host response to pathogens. since several microbes have evolved to exploit their pleiotropic characteristics, identification of key genes and pathways in terms of activation, enhancement, repression, or silent, which are essential to facilitate the immune response, is based on the elucidation of the roles of mirnas in host response to infectious disease. the complex regulatory network within which mirnas are embedded makes unpicking the roles of mirnas tough but not impossible. integrating large mirna and mrna datasets using advanced statistical techniques (in a "systems biology" approach) will facilitate the unpicking of these complex networks. overall, mirnas have multiple targets, and therefore any vaccines or treatments that harness mirnas may produce off-target effects compromising safety; however, there are challenges that must be overcome. with the objective to improve the cross-study reproducibility of the findings, especially in the context of ex-mirna biomarkers identification, universal endogenous controls are needed, and a more standardized approach to biomarker studies may also help. initiatives devoted to harnessing the diagnostic and therapeutic potential of extracellular rnas such as the national institute for health extracellular communication consortium can facilitate this. as the literature and experimental studies on mirnas are developing, the potential for new mirna therapeutics, diagnostics/prognostics, and vaccines becomes tangibly closer. translating the insights of mirna studies into improving the lives of patients is the critical next step. 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combination biomarkers for pulmonary tuberculosis diagnosis human microrna hsa-mir- - p suppresses enterovirus replication by targeting the viral genome comprehensive characterization of serum microrna profile in response to the emerging avian influenza a (h n ) virus infection in humans all the authors are thankful to the contributors of the team of virology, oncology, and medical biotechnologies of laboratory of virology, microbiology, quality, and biotechnologies/etb and also to the fondation lalla salma de lutte contre le cancer. key: cord- -g oes authors: nemzek, jean a.; lester, patrick a.; wolfe, a. marissa; dysko, robert c.; myers, daniel d. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: g oes historically, the dog played an important role as a laboratory animal in biomedical research. although numbers are declining, the use of dogs continues to be common in pharmacokinetics and cardiovascular studies. the normal biology of the dog as both a laboratory and a companion animal has been well studied and reference values are presented here as a clinical and experimental resource. this provides the necessary background to discuss the spontaneous diseases, including infectious and neoplastic conditions, prevalent in purpose bred as well as random source dogs used in biomedical research. in addition, diseases and conditions that arise secondary to the housing and experimental manipulation of dogs is discussed with emphasis on treatment and prevention. laboratory animal medicine mellitus. a comprehensive but concise review of the use of the dog as a research subject is available in gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger dog breeds for use in surgical research studies. some specific breeds with congenital or spontaneous disorders have also been maintained by research institutions (see examples below). random-source dogs used in research are most frequently mongrels or larger dog breeds (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for "beagle" for the years - , a significant portion of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. other common areas of research using beagles were dental and periodontal disease and surgery, orthopedic surgery, skeletal physiology, and imaging studies. other research areas that utilized beagles included canine infectious disease, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established and the organs of larger dog breeds are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies had been maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of the blood, most notably the neutrophil population. these dogs can be used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by the absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . other genetic disorders studied in dog colonies include hereditary canine spinal muscle atrophy (cork, ) and narcoplepsy in doberman pinschers (ripley et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease) and the development of spontaneous diabetes mellitus and hypothyroidism has been studied in several breeds of dogs for comparisons with the human conditions. although historically the dog has been a common laboratory animal, their use in research has waned over the past years. according to the u.s. department of agriculture (usda), animal and plant health inspection service ( , ) , the number of dogs used in research has declined from , in to , in (prior to the previous edition of this text) and , in . this decrease was caused by a variety of factors, including (but not limited to) decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), increased cost, and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purposebred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against rabies virus, canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola, icterohaemorrhagiae, grippotyphosa, and pomona, and bordetella bronchiseptica (jasmin, personal communication). purpose-bred dogs are also usually treated prophylactically for intestinal helminths and ectoparasites, and possibly given a heartworm preventative. random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting and racing) or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. random-source dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations and tissue/organ harvest. options for procurement of dogs for biomedical research typically include purchase from a usdadesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . definitions, and . requirements and application (office of the federal register, ) . briefly, class a licensees are breeders who raise all animals on their premises from a closed colony. class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and record-keeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. in december , the national institutes of health (nih) issued notice not-od- - entitled notice regarding nih plan to transition from use of usda class b dogs to other legal sources (national institutes of health, ) . this nih policy begins in the fiscal year and prohibits the procurement of dogs from class b dealers using nih grant funds. from that point forward, dogs on nihfunded studies will have to be obtained from class a vendors, privately owned colonies (such as institutional breeding colonies), or client-owned animals (e.g., animals participating in veterinary clinical trials). the best resource for identification of possible vendors are online 'buyer's guide' sites or 'buyer's guide' issues of trade periodicals. online sites include the buyer's guide of the american association of laboratory animal science (http://laboratoryanimalbuyersguide.com), and the trade journals lab animal (http://guide.labanimal. com) and animal lab news (http://www.alnmag.com/ content/buyers-guide). a 'buyer's guide' typically lists sources for both purpose-bred and random-source dogs, and denotes such features as pathogen-free status, health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within issues of the journals. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare (office of the federal register, ) . regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures-additional requirements for dogs) because the space required for housing dogs is calculated using body length rather than weight (a parameter used for other species and also for dogs in the national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute for laboratory animal research (ilar) has written the guide for the care and use of laboratory animals (national research council, ) . the 'guide' is the primary document used by institutional animal research units to develop their programs and by animal care evaluation groups, such as the association for assessment and accreditation of laboratory animal care international (aaalac international), to facilitate site visits and inspections. the primary difference between the th and th editions of the 'guide' (national research council, regarding the care of dogs is the notation that "enclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs, or kennels) are preferable." the ilar committee on dogs authored dogs: laboratory animal management (national research council, ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. the information presented in the tables represents a range of normal values that can vary depending on the analytical method, as well as the age, breed, and sex of the animal. cohen, covance laboratories, inc., cumberland, va ( ) . physiological data for a mixed population of dogs of both sexes. fig. . demonstrates the normal weights and corresponding ages for both male and female beagle and hound dogs. tables . and . feature hematology data from beagles of both sexes from two commercial facilities. tables . and . list serum chemical data for beagles of both sexes from two commercial facilities. representative blood gas, coagulation data, and normal urinalysis parameters can be found in tables . - . , respectively. finally, the reviews in arterial and venous blood gas anaylses (rieser, ) and the manual of canine and feline cardiology (tilley et al., ) are excellent resources. good nutrition and a balanced diet are essential to the health, performance, and well-being of the animal. the nrc of the united states national academy of sciences is the leading provider of nutrient recommendations for dogs and provides average requirements needed to maintain growth and prevent deficiencies (subcommittee on dog and cat nutrition, ) . the nrc publications form the basis for the association of american feed control officials (aafco) nutrient profiles, which are updated periodically (baldwin et al., ) . the aafco is an advisory body comprising state representatives from across the united states. it provides a mechanism for developing and implementing uniform and equitable laws, regulations, standards, and enforcement policies, and establishes nutrient profiles for cat and dog foods (dzanis, ; thatcher et al., ) . additional resources should be consulted for details on the nutritional requirements for dogs of all ages (dzanis, ; subcommittee on dog and cat nutrition, ; baldwin et al., ; thatcher et al., ; hand et al., ) . recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment. the mer for most breeds may be calculated using the following equation: mer (metabolizable kcal/day) = bw × . × kj de, where bw = body weight (kg), kj = kilojoules, de = digestable energy (kienzle and rainbird, ) . in-depth overviews of diets used in biomedical research are available in diet-specific literature. open-formula diets have defined concentrations of all ingredients and the information is publicly available. this allows researchers to control for this important environmental variable and enables retrospective analysis of possible diet composition effects on research results (barnard et al., ) . open-formula diets occasionally may require changes in formulation to maintain nutrient composition or meet changing nutrient requirements. these changes in quantitative ingredient formulation are made public when open-formula diets are modified. in contrast, closed-formula diets are commercially available, balanced diets that meet and label the minimum requirements for protein and fat and the maximum values for ash and fiber; however, the exact composition of ingredients may vary from batch to batch. ingredient composition varies as the manufacturer applies a leastcost strategy, referring to formulating diets to maximize profit by using the least-expensive ingredients. although the ingredients are listed, the quantitative ingredient formulation is not publicly available and can vary without public disclosure, due to proprietary nature of commercial diets produced and marketed under vendor trade names. closed-formula diets have also been referred to as as 'fixed formula' or 'constant nutrition' (labdiets, pmi nutrition international, st. louis, missouri) by manufacturers (barnard et al., ) . in fixed-formula diets, the quantitative ingredient formulation does not change; however, this information is proprietary and therefore laboratory animal medicine not disclosed publically (barnard et al., ) . semipurified and purified diets provide the strictest control of ingredients and are formulated from purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. although purified and semipurified diets do differ in the types of ingredients used, the terms are generally used to mean the same thing. purified-ingredient diets are generally 'open' formulas, meaning that they are published and available to the scientific community. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf-life, but the best strategy is to feed only fresh diets and use each lot based on the date of manufacture. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. from dr. asheley wathen, covance laboratories, inc., madison, wi, and dr. kimberley cohen, covance laboratories, inc., cumberland, va ( ) . overall health, body condition, nutrition, and age greatly influence reproductive efficiency (gavrilovic et al., ; johnson, ) . therefore, only normal, healthy animals in excellent body condition should be used in breeding programs. beagles between and . years of age have the best conception rates and litter size with the lowest neonatal mortality. after years of age, conception rates and litter size decline and neonatal mortality increases (johnson, ) . the vagina is a long, musculomembranous canal that extends from the uterus to the vulva. during physical examination, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva, avoiding the deep ventral clitoral fossa. examination should proceed at an angle of approximately ° until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. female dogs are monoestrous, typically nonseasonal, spontaneous ovulators that have a spontaneous luteal phase approximately days longer than the ± days of pregnancy followed by obligate anestrus. puberty (beginning of the first estrus) occurs between and months in most breeds. the time of onset positively correlates with the body size (concannon, ) . the canine cycle is divided into four phases: proestrus, estrus, diestrus, and anestrus. the duration of laboratory animal medicine proestrus is - days with an average of days and reflects the follicular phase rise in estrogen. during this stage, the vulva is enlarged and turgid, and a serosanguinous vaginal discharge is present (concannon, ) . estrus may be from to days in duration but generally lasts days. the endocrine feature of estrus is the first abrupt increase in progesterone (> ng/ml), which occurs concomitantly with the luteinizing hormone (lh) surge % of the time, followed by ovulation within - h. the vulva is softer and smaller than in proestrus. the vaginal discharge persists and may remain serosanquinous or become straw colored. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. defined behaviorally as starting when estrous behavior ceases (concannon, ) , diestrus represents the peak of serum progesterone. anestrous may last from to days and is the stage of reproductive quiescence. it is characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. the onset of puberty in the male ranges from to months of age and is affected by breed, season, nutrition, and disease status. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial or leydig's cells. at this time, the testicular growth is rapid, the seminiferous tubules begin to differentiate, and sertoli cells form the blood-testis barrier. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including, inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, whereas inhibin and estrogen provide negative feedback to the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from the initiation of spermatogonial mitosis to the delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality semen production. supression of sexual behavior and problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities. animals with poor hind limb conformation or trauma to the back may be unable to properly mount the female. there is a positive correlation between scrotal circumference and the number of sperm produced. finally, the quality of sperm is assessed by motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities is a good indicator of normal male fertility. complete anatomy of the bitch and dog can be found in miller's anatomy of the dog (evans and de lahunta, ) . cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear/ cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear 'anuclear' and are classified as 'cornified' or 'anuclear squames.' the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear/cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, observation of behavioral estrus is the best criterion to use in breeding management. during proestrus, the male is attracted to the bitch and will investigate her hindquarters, but she laboratory animal medicine will not accept breeding. estrus is characterized by proactive receptivity to mounting by males and increased male-seeking behavior (concannon, ) . during this stage, the bitch will exhibit 'flagging,' or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate and litter size, it is recommended to breed the bitch on days , , and of the standing heat. due to the long life span of canine sperm, fertilization occurs in the oviduct up to days after coitus. the ovulated oocyte is a primary oocyte that must undergo two meiotic divisions before fertilization can occur. this overall maturation process takes approximately days. after maturation, the oocyte remains viable for - days. optimal conception rates tend to occur when the bitch is bred from days before to days after ovulation; best litter size is achieved when the bitch is bred days after ovulation. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary, indicating the placental villi are arranged in a belt, and deciduate, reflecting that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, and then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility (johnson, ; verstegen-onclin and verstegen, ) . pregnancy detection can be performed by several methods. abdominal palpation of the uterus may be most informative at approximately days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings and are approximately inches in length at - days. by day , the uterus begins to enlarge diffusely and the vesicles become difficult to identify by palpation. radiology can be used to confirm pregnancy and facilitate determination of gestational age, beginning days after the lh surge (lopate, ) . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. ultrasonography can be used to confirm pregnancy beginning on days - , at which point the gestational sacs will be approximately cm in diameter, and until parturition (shille and gontarek, ; lopate, ) . ultrasonography can assess fetal viability by visualizing fetal heartbeats and fetal movement beginning on gestational days - and , respectively (lopate, ) . it can also predict gestational age using the inner diameter of the chorionic cavity in early pregnancy and the biparietal diameter in late pregnancy luvoni and beccaglia, ) . however, ultrasonography for determination of gestational age is most accurate at day of pregnancy when using correction factors for small (< kg) and large (> kg) body weight dogs (kutzler et al., ) . thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged because they may adhere to the umbilical cord and predispose to ascending infections. heat lamps may be placed h prior to parturition and remain until all neonates demonstrate vigorous suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. monitoring of parturition is important, but human intervention should be minimal in order to prevent stressinduced cannibalism. an abrupt drop in body temperature to less than °f indicates impending parturition within - h. the process of parturition has been divided into three stages. stage of labor lasts - h and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include panting and increased pulse rate (johnson, ) . fetal expulsion occurs during stage , which lasts approximately - h. as the fetus engages the cervix, there is release of oxytocin, referred to as the ferguson reflex, which strengthens the uterine contractions and may elicit abdominal contractions as well. the bitch is able to inhibit this stage of labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between deliveries of each pup is irregular, but the average is less than h between pups. veterinary assistance laboratory animal medicine is necessary if the bitch remains in stage for more than h without delivering the first pup, or for more than h before delivering subsequent pups. during stage of labor, the placentas are expelled either immediately or within min of delivery of each pup. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. the peripartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. oxytocin should not be used in the event of systemic illness or abnormalities precluding vaginal delivery. indications for its use include lack of delivery h after onset of stage labor, greater than h of unproductive stage labor, inadequate contractions, or abnormal vaginal discharge. in these cases, radiographs are recommended to assess fetal size in relation to the birth canal and any possible obstructions, followed by . - . iu of oxytocin intramuscularly or subcutaneously. the oxytocin can be repeated - min after the first dose for a total of two doses (plunkett, ) . in some cases, treatment with . - . ml/kg of % calcium gluconate, delivered slowly iv while monitoring closely for bradycardia, and % dextrose iv may be indicated. uterine involution occurs during anestrus within - weeks of parturition. during this time, a greenish to red-brown vaginal discharge, or lochia, is considered normal. the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the th postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies, the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as a vaginal-vestibular stricture, narrow vagina, vaginal septum, and vaginal hyperplasia, or if there is a behavioral incompatibility between the male and female dogs (kutzler, ) . semen is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the scent of an estrous bitch and manually stimulated. the first two fractions are collected followed by a sufficient amount of the third fraction (predominantly of prostatic fluid) to bring the total semen volume to - ml. the semen can be introduced into the cranial vagina or directly into the uterus either through trans-cervical catheterization with a norwegian ai catheter or utilizing fiberoptic endoscopy. use of the norwegian ai catheter for intrauterine insemination of frozen-thawed, fresh, and chilled-extended semen results in significantly higher whelping rates than intravaginal insemination (linde-forsberg et al., ; thomassen and farstad, ) . for trans-cervical insemination, the bitch is either standing on all four legs or standing with hindquarters raised. the ai catheter and guiding tube are inserted into the vestibulum as far as the pseudocervix. firm abdominal palpation is then used to locate and fix the cervix in the other hand, at which point the catheter is further inserted along the dorsal vaginal fold until the cervical opening is located and semen is deposited into the uterus lumen (thomassen and farstad, ) . surgical and laparoscopic ai has been used successfully for intrauterine and intratubal insemination; however, these techniques are invasive and require anesthesia. therefore, the nonsurgical techniques mentioned above are recommended, as these approaches are less invasive and can be completed without anesthesia in nonsedated or sedated dogs depending on the experience of the personnel and personality of the dogs. ai with freshly collected sperm can be done on days , , and of standing heat or on days of maximal vaginal cornification. the viability of frozen-thawed sperm is significantly reduced compared to fresh or chilled sperm that may live up to or days in the reproductive tract of the bitch; frozen-thawed sperm live only a few hours. therefore, the ova must be mature and insemination with frozen-thawed semen must be done - days after ovulation in the bitch as determined by serum progesterone concentrations (thomassen et al., ) . false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. a comprehensive review of canine pseudocyesis exploring its cause, clinical features, and treatments is covered by c. gobello (gobello et al., ) . reproductive performance in the bitch is optimal prior to years of age. cycling does not completely cease; however, after - years of age, bitches demonstrate significant decreases in conception rate and the number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia, are extremely common. dogs prefer living in a social environment. dogs have well developed olfactory glands, vision, and auditory and tactile senses that allow them to gain environmental cues and information from other dogs and humans (field and jackson, ; joint working group on refinement, ) . much of their instinctive behavior is dependent on learning to interact with other members of their species. beagles have been a popular animal model because of their docile nature. they are easily handled and, for the most part, respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiograms (ecgs), oral gavage, and venipuncture. although sexually mature by - months of age, dogs are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from to weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age, dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the canine behavior section of the manual of clinical behavioral medicine for dogs and cats (overall, ) . by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of its disorders. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting, especially infectious diseases associated with the use of random-source dogs and conditions seen frequently in the beagle. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks. etiology canine infectious respiratory disease (cird) is a highly contagious illness and several organisms have been incriminated including bordetella bronchiseptica; streptococcus equi subsp. zooepidemicus; canine parainfluenza virus (cpiv); canine influenza virus (civ); canine respiratory coronavirus; canine adenovirus type (cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasma and ureaplasma. naturally occuring infection can result in coinfection by two or more organisms (garnett et al., ; ford, ) . clinical signs cird can be subdivided into mild or severe forms. the mild form is more common and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough may be elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise, dogs are typically asymptomatic. mild tracheobronchitis usually lasts - days, even when untreated. the severe form results from poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and can be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. epizootiology and transmission the natural reservoir for b. bronchiseptica is the respiratory tract (bemis, ) , and it is very easily spread by aerosol and direct contact. transmission is heightened by confined housing of multiple animals. bordetella bronchiseptica is highly infectious with an incubation period of - days. pathogenesis the most common clinical isolates are cpiv and b. bronchiseptica (mochizuki et al., ) . however, b. bronchiseptica is often recovered from clinically healthy animals (chalker et al., ) . during clinical infection, b. bronchiseptica attaches to the cilia of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv laboratory animal medicine or cav- alone are usually subclinical but can cause necrotizing tracheobronchiolitis (dungworth, ) . diagnosis and differential diagnosis diagnosis is often based on clinical signs and known history; however, cough elicited by tracheal palpation may be inconsistent and should not be used for definitive diagnosis. presumptive diagnosis can be made by isolation of b. bronchiseptica or mycoplasma by nasal swabs. viral isolation or paired serology is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. differential diagnoses include civ, canine distemper virus, pneumonia, heartworm disease, tracheal collapse, mycotic infections, and diseases resulting in tracheal compression (johnson, ) . prevention prevention is best achieved by avoiding exposure to infected animals. dogs should be vaccinated prior to or upon admission to the animal facility. intranasal vaccines protect against infection and disease and can be given to dogs as young as weeks of age (greene and levy, ) . combination vaccines for b. bronchiseptica, cav- , and cpiv are preferred. vaccinations should be boostered every months when multiple animals are housed in a confined area. control staff must practice proper hygiene to prevent transmission by fomites. sanitation, proper ventilation, and proper humidity are critical for control. symptomatic animals should be isolated and kennels should be disinfected with agents such as bleach, chlorhexidine, or quaternary ammonium chloride. treatment bordetella bronchiseptica is sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur and should be continued for days. for severe or unresponsive infection, treatment should be based on bacterial culture/sensitivity patterns. nebulized gentamicin or kanamycin may be helpful in severe cases. antitussives should be avoided if the cough is productive; however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction. research complications due to the altered respiratory tract physiology, infected animals should not be used for pulmonary studies. etiology β-hemolytic lancefield's group c streptococcus (s. equi ssp. zooepidemicus) is a gram-positive, non-spore-forming coccus that causes pneumonia and sepsis in dogs. clinical signs clinical signs vary based on the organ system affected. pneumonic disease is typically associated with sudden onset of clinical signs including coughing, weakness, fever, dyspnea, and hematemesis. the rapid progression of disease is similar to that seen in humans with toxic shock syndrome (tss) caused by streptococcus pyogenes. peracute death has been reported in research and shelter dogs (bergdall et al., ; pesavento et al., ) . epizootiology and transmission streptococcus equi ssp. zooepidemicus is not considered a commensal of healthy dogs as most of the β-hemolytic commensal organisms belong to group g, specifically streptococcus canis. asymptomatic carriers are suspected to be the route by which infection enters populations. streptococcus equi ssp. zooepidemicus is considered an opportunistic pathogen and stressful factors such as transport can predispose to disease (priestnall et al., ) . pathologic findings in peracute cases, hemorrhage from the mouth and nose and within the pleural cavity can be the most striking lesion. ecchymotic and petechial hemorrhages can be noted on several organs ( fig. . ). 'bull's-eye' lesions may be observed on the pleural surface of affected lung lobes. histologic lesions can include fibrino-suppurative, necrotizing, and hemorrhagic pneumonia. gram-positive cocci can be found in intracellular clusters throughout the lung ( fig. . ), tonsils, and spleen of affected animals (bergdall et al., ; priestnall and erles, ) . pathogenesis predisposing factors such as transport stress and viral coinfection have been shown to contribute to the virulence of s. zooepidemicus (priestnall and erles, ) . due to the similarities with the clinical signs seen in human cases of tss, superantigens are thought to contribute to the virulence of s. zooepidemicus in cases of acute hemorrhagic pneumonia. these superantigens work by bypassing the conventional mechanisms of antigen presentation and binding to major histocompatibilitity complex class ii receptors. as a result, there is a hyperactive proinflammatory response and an 'avalanche' of cytokines including interleukin β (il- β), interleukin (il- ), and tumor necrosis factor alpha (tnf-α). three novel superantigen-encoding genes have been identified from a case of acute fatal hemorrhagic pneumonia, szef, szen, and szep. however, it is currently unclear what effect these superantigens have in vivo priestnall et al., ) . while superantigens have been detected in some isolates, there is not enough data to determine if superantigens play a role in the pathogenesis (byun et al., ; kim et al., ) . diagnosis and differential diagnosis definitive diagnosis is based on bacterial culture of nasal swabs or transtracheal lavage. polymerase chain reaction (pcr) can be done on post-mortem lung tissue. bacterial pneumonias or bacteremias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and b. bronchiseptica. nonbacterial causes of respiratory disease include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control there is no vaccine for prevention of s. zooepidemicus. the organism has been isolated from the environment during active outbreaks (pesavento et al., ) , so dogs diagnosed with s. zooepidemicus should be quarantined and any potential fomites (e.g. food bowls, enrichment) should be properly disinfected. treatment antibiotic therapy should be based on culture and sensitivity. resistance to doxycycline and tetracycline has been demonstrated (garnett et al., ; pesavento et al., ) . research complications dogs with severe hemorrhagic pneumonia or systemic disease are not appropriate for research study. the association between epizootics of this disease and transportation supports operational policies that require adequate acclimation periods for animals upon arrival. etiology serovars canicola, bratislava, and grippotyphosa result in renal or hepatic disease, whereas serovars icterohaemorrhagiae and pomona predominantly result in hepatic disease . clinical signs canine leptospirosis can present as subclinical, acute, or chronic disease. clinical signs in acute infection can be nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, vomiting, muscle tenderness, and pyrexia. clinical signs can be related to renal failure including polyuria and polydipsia, with or without azotemia, oliguria, or anuria. leptospirosis can also lead to hepatic failure with signs such as icterus or bleeding abnormalities. peracute leptospirosis is characterized by shock, vascular collapse, and rapid death. uveitis, abortions, stillbirths, and pulmonary hemorrhage have also been associated with leptospirosis (klopfleisch et al., ; van de maele et al., ) . bivalent vaccines against the most common canine serovars, icterohaemorrhagiae and canicola, have resulted in the increased prevalence of other serovars including grippotyphosa, pomona, bratislava, and autumnalis. increased movement of wild animal reservoirs (rats, raccoons, skunks, opossums) into urban/suburban areas have also contributed to the greater prevalence of previously uncommon serovars (sykes et al., ) . transmission occurs primarily through environmental contact, although direct transmisson between hosts may also occur. leptospires passing from urine into water is the most common route of contamination (goldstein, ) . leptospirosis is a zoonotic disease. pathologic findings the kidneys consistently have gross and microscopic lesions. in the acute phase, the kidneys are swollen with subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and necrotic foci (searcy, ) . in chronic stages of leptospirosis, the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis the severity and course of leptospirosis depend on the causative serovar as well as the age and immune status of the dog. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly, reaching the renal tubular epithelium several days postinfection. acute or progressive renal failure leading to oliguria or anuria may occur. nephritis may or may not be accompanied by hepatitis, uveitis, pulmonary hemorrhage, and meningitis. disseminated intravascular coagulation is often a secondary complication. diagnosis and differential diagnosis paired serology for the microscopic agglutination test is the most reliable means of definitive diagnosis, and successive serum sampling should be done - days after the first sample. pcr can be used to identify active infection early in the disease when serologic testing is negative or in previously vaccinated animals (sykes et al., ) . differential diagnoses include other causes of acute renal failure and hepatitis. prevention and control according to the american animal hospital association's vaccination guidelines, vaccination for leptospirosis is recommended based on geographic location and exposure risk (welborn et al., ) . both quadrivalent and bivalent inactivated bacterins are available. quadrivalent bacterins protect against canicola, icterohaemorrhagiae, grippotyphosa, and pomona serovars, whereas bivalent bacterins cover only canicola and icterohaemorrhagiae. immunization does not prevent the development of the carrier state or protect against other serovars. control requires preventing contact with wildlife reservoirs as well as identification of carrier animals. treatment doxycycline is the drug of choice as it can eliminate renal colonization. if vomiting or allergic reactions prohibit treatment with doxycycline, ampicillin or other penicillins should be utilized. aggressive fluid therapy and supportive care may also be needed. research complications due to the zoonotic potential, dogs with clinical leptospirosis should not be used in research studies. etiology campylobacter spp. are thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rods. many species of campylobacter have been isolated from normal and diarrheic animals; however, the most common pathogenic species include campylobacter jejuni ssp. jejuni and c. coli (marks et al., ) . clinical signs most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, mild and intermittent mucoid or watery diarrhea, with or without frank blood, is most commonly noted. signs typically last - days but can persist for several months. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea (marks et al., ) . bacteremia and cholecystitis secondary to c. jejuni have also been documented in dogs (fox, ) . epizootiology and transmission the role of campylobacter spp. as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . stress or immunosuppression may make animals more susceptible. transmission is via the fecal-oral route, mostly through contaminated food or water. campylobacter jejuni can be zoonotic with immunocompromised individuals at greatest risk. pathologic findings lesions depend on the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated, fluidfilled bowel loops, with little or no histopathologic alteration. cytotoxin-mediated disease results in a friable, hemorrhagic mucosal surface. histologically, the mucosa is ulcerated with lymphoplasmacytic infiltration. translocation can result in edema and congestion of the lamina propria with focal accumulation of granulocytes. epithelial hyperplasia and decreased goblet cell numbers are also noted. campylobacter jejuni may be visualized between enterocytes with warthin-starry silver-stained sections. pathogenesis clinical disease may be produced by several different mechanisms as campylobacter spp. have a variety of virulence factors including enterotoxins, cytotoxins, and adherence or invasion properties. campylobacter jejuni can cause an erosive enterocolitis by invasion of epithelium and production of the cytolethal distending toxin (cdt) (fox, ; van kruiningen, ) . in addition, c. jejuni can produce illness via translocation to regional lymph nodes causing a mesenteric lymphadenitis. diagnosis and differential diagnosis fresh feces (per rectum) can be used for presumptive diagnosis by demonstration of highly motile, curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and johnson, ) . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of °c. a pcr multiplex assay for differentiation of c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus ssp. fetus has been developed (wang et al., ) . any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis. prevention and control proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. in enzootic situations, group housing should be avoided. outbreaks are controlled by isolation and treatment of affected individuals. treatment antibacterial treatment should be considered in severely ill dogs. erythromycin, neomycin, enrofloxacin, clindamycin, and doxycycline are all effective. resistance to quinolones and ciprofloxacin has been documented (acke et al., ) . treatment should be a minimum of - days with bacterial cultures repeating and weeks after treatment. research complications dogs with clinical campylobacteriosis have temporary derangements to digestive and absorptive functions. etiology helicobacters are gram-negative, microaerophilic, spiral bacteria that infect the gastrointestinal tract. helicobacter spp. can be separated into gastric and enterohepatic groups. the gastric helicobacters commonly identified in dogs are referred to as non- (haesebrouck et al., ; joosten et al., ) . the most common enterohepatic species found in dogs include h. bilis, h. canis, and h. cinaedi (castiglioni et al., ; dewhirst et al., ; fox, (haesebrouck et al., ; fox, ) . clinical signs most infections are subclinical in the dog. gastric infections may present with vomiting, diarrhea, and fever, accompanied by anorexia, pica, or polyphagia. enterohepatic helicobacters have been linked with inflammatory bowel disease in experimental animal models. heavy infections in dogs have been associated with inflammatory lesions of the large intestine (castiglioni et al., ; nguyen et al., ) . epizootiology and transmission the epizootiology and transmission of helicobacter spp. in the dog remain unknown. both oral-oral and fecal-oral routes for transmission have been suggested in humans, but transmission via canine saliva is a less reliable source of infection (craven et al., ) . enterohepatic infections of pet dogs are as high as % (castiglioni et al., ) . prevalence of gastric helicobacter infections in colony or shelter dogs can be as high as - % (fox, ; hermanns et al., ) . pathologic findings gastritis is usually mild and characterized by reduced mucus content of the surface epithelium with vacuolation, swelling, karyolysis, and karyorrhexis of parietal cells. multifocal infiltrates of plasma cells and neutrophils occur around blood vessels and between gastric pits (hermanns et al., ) . intestinal lesions include mild to moderate lymphoplasmacytic infiltration as well as crypt dilation and crypt hyperplasia (castiglioni et al., ) . pathogenesis gastric helicobacters are urease positive, which assists with survival in the acidic environment of the stomach (kusters et al., ; uberti et al., ) . enterohepatic helicobacters are urease negative and typically reside in the lower intestine. the mechanism by which enterohepatic helicobacters colonize the liver is thought to be through portal circulation after uptake by enterocytes or through retrograde movement from the intestine into the bile duct (fox, ) . diagnosis and differential diagnosis organisms may be demonstrated with histopathology on endoscopic or surgical biopsy tissue samples. warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. culture may be difficult depending on the helicobacter spp. for species that produce urease, a positive urease test on a gastric biopsy specimen may give a presumptive diagnosis. the urea breath test has been successfully used to diagnose helicobacter spp. in laboratory beagles with a sensitivity and specificity of % (kubota et al., ) . western blot has been used to detect serum antibodies to enterohepatic species and pcr can be used to detect helicobacter spp. in fecal samples (oyama et al., ; wadström et al., ) . any causes of acute or chronic vomiting and diarrhea in the dog are differential diagnoses. prevention and control until more is known about the epizootiology and transmission of helicobacter spp., specific recommendations cannot be made for prevention and control. treatment for gastric species, combination therapy of amoxicillin ( mg/kg q h), metronidazole ( mg/kg q h), and sucralfate ( . - . mg/kg q h) has proven to be most effective (hall and simpson, ) . replacing sucralfate with famotidine, omeprazole, or bismuth subsalicylate may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . recurrence rates within days of treatment can be as high as % (anacleto et al., ) . treatment of enterohepatic helicobacters may depend on species susceptibility. aminoglycosides have been successful in treating h. cinaedi, but resistance to fluoroquinolones has been documented (tomida et al., ) . combination therapy of amoxicillin, clarithromycin, metronidazole, and omeprazole in medicated chow has been successful in eliminating various enterohepatic helicobacters from mice (del carmen martino-cardona et al., ) . long-term antibiotic treatment at a minimum of days is suggested for enterohepatic and gastric helicobacters. research complication dogs used in gastrointestinal physiology or oral pharmacology studies should be free from helicobacteriosis. etiology parvoviral enteritis in dogs is caused by canine parvovirus strain (cpv- ) of the family parvoviridae, genus protoparvovirus, species carnivore protoparvovirus . currently, there are three antigenic variants, a, b, and c. parvoviruses are nonenveloped, single-stranded dna viruses. clinical signs while parvoviral infection can affect the gastrointestinal tract, bone marrow, myocardium, and nervous tissues, the most common manifestation of disease is acute enteritis. clinical signs usually appear days after fecal-oral inoculation and include anorexia, fever, depression, vomiting, and profuse intractable diarrhea which may become hemorrhagic. excessive fluid and protein losses through the gastrointestinal tract result in rapid and severe dehydration. dogs can develop severe leukopenia with a total leukocyte count of cells/μl or less. repeated hemograms may provide prognostic value, as rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. epizootiology and transmission parvovirus can infect dogs of any age, but puppies between and weeks of age are particularly susceptible. puppies less than weeks of age are protected by passive maternal antibody. strain cpv- c has been associated with severe disease in adult vaccinated dogs (calderon et al., ) . pathogenesis canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes acute enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus, leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis parvovirus can be detected with a commercially available fecal enzyme-linked immunosorbent assay (elisa). due to intermittent and brief shedding of the virus, fecal elisas can have false-negative results. pcr can be used to confirm an elisa result and to differentiate the viral strain. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. prevention and control parvoviral-positive animals should be quarantined for at least days as the infectious virus is shed for several days after onset of clinical signs. although pcr has been used to detect viral dna in feces for up to weeks (decaro et al., ) , it is currently unknown if the material being shed at this time is still infectious. disinfection of exposed areas with dilute bleach ( : ) or a commercial disinfectant is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a modified live vaccine until at least weeks of age. treatment treatment is largely supportive and aimed at restoring fluid and electrolyte balance. antimicrobial therapy is recommended due to intestinal compromise and risk of sepsis. early nutritional support continued throughout the disease has been shown to decrease recovery times (mohr et al., ) . research complications infection with parvovirus precludes the use of a particular dog in an experimental protocol. due to the significant discomfort of the animal, as well as the intensive therapy required, humane euthanasia is usually chosen in a research setting. etiology rabies virus is a lyssavirus belonging to the family rhabdoviridae. clinical signs clinical progression of neurologic disease occurs in three stages. the first, prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, furious, stage, animals are easily excited or hyperreactive to external stimuli and will readily bite at inanimate objects. the third, paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death due to respiratory failure usually occurs after onset of the third stage. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact with infected saliva, usually via bite wounds. pathogenesis the incubation period for rabies is - weeks to the onset of clinical signs but can range from week to year. bites to the head and neck result in shorter incubation periods due to the close proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to neurons within the brain, resulting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis definitive diagnosis is based on immunofluorescence of the virus in negri bodies of hippocampal cells. submission of the whole, unfixed brain, including the cerebellum and proximal brain stem, should be done within h of collection. the tissue should be kept refrigerated as freezing can cause delays in testing. differential diagnoses laboratory animal medicine include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. prevention and treatment puppies should be vaccinated by weeks of age, again at year, and then annually or triennially, depending on state and local laws. research complications immuno-prophylaxis is recommended for animal care and research personnel who may have work-related risks of exposure. due to risk of human exposure, animals with suspected infection should be humanely euthanized and brain tissue should be submitted for confirmation. giardiasis giardia lamblia, also known as g. duodenalis and g. intestinalis, is a binucleate flagellate protozoan that usually causes subclinical infestation of the small intestine. clinical disease is usually seen in young dogs and the characteristic sign is voluminous, light-colored, foul-smelling, soft to watery diarrhea, which is the result of malabsorption and hypersecretion. giardia has a direct life cycle with infection resulting after consuming cyst-contaminated food or water. the change in ph between the stomach and duodenum activates excystation and trophozoites then attach to the enterocytes. for diagnosis, direct fecal smears are considered best for observing trophozoites and zinc sulfate centrifugation is preferred for detection of cysts. a commercial elisa kit is licensed for use in dogs, but the positive predictive value is poor and zinc sulfate centrifugation techniques should be used in conjunction with elisa (rishniw et al., ) . pcr assays are also available for diagnosing giardiasis. differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. metronidazole at - mg/kg po q h for - days is effective at treating giardiasis as well as other enteric protozoans, which may be potential differential diagnoses or coinfections. albendazole, fenbendazole, pyrantel, and praziquantel are also effective. coccidiosis intestinal coccidia associated with enteropathy in dogs include isospora canis, i. ohioensis, i. neorivolta, i. burrowsi, and hammondia heydorni (dubey and greene, ) . coccidian oocysts can be found in feces of clinically healthy dogs, as well as animals with diarrhea. clinically affected animals are young or immunosuppressed and develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. coccidia oocysts are typically spread by fecal-oral transmission, but dogs can ingest monozoic cysts in intermediate host tissues. the coccidian life cycle is both sexual and asexual, and results in the release of unsporulated eggs, which sporulate under appropriate environmental conditions. other causes for diarrhea should be excluded before a coccidial etiology is implicated. treatment may not be necessary, as infections are typically self-limiting and clinically insignificant. treatment may help to limit the number of oocysts shed in a kennel-housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/kg po q h for - days) or trimethoprim sulfa ( mg/kg po q h for days). ascarids roundworms of dogs are most often toxocara canis; however, toxascaris leonina can also affect dogs. clinical illness is usually only seen in young animals with large worm burdens. diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization can be seen. puppies may have a classical 'potbellied' appearance. heavy infestations can cause intussusception and/or intestinal obstruction. puppies that experience lung migrations of larval worms can develop fatal pneumonia. toxascaris canis can infect dogs by transplacental migration, transmammary migration, or ingestion of infective eggs. the infective stage of t. canis is the third-stage larva (l ). in transplacental infections, puppies may be born with l larvae in their lungs (sherding, ) . for diagnosis, large ( - μm in diameter) and relatively round ascarid eggs can be seen by standard fecal flotation methods. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention (hall and simpson, ) . most anthelmintics are effective for treatment. puppies should be treated early and often (every other week until weeks of age) because of the possibility of prenatal or neonatal infection. pregnant bitches can be treated with extended fenbendazole therapy ( mg/kg po once a day from day of gestation through day of lactation). hookworms the most common and most pathogenic hookworm of dogs is ancylostoma caninum. ancylostoma braziliense can also be found in dogs, but only a. caninum infestation typically results in clinical illness. puppies with hookworm infections can present as anemic with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. these signs are a direct result of the worms' consumption of blood and body fluids. infective larvae (l ) are ingested from the environment and develop directly in the intestinal tract. infestation can also be transmammary, from ingestion of a paratenic host, and, less often, by transplacental migration. on histological sections, embedded worms with mouthparts may be identified. diagnosis is made by identification of eggs or larvae by either fecal flotation or direct smear. a differential diagnosis of parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in young dogs with anemia. pyrantel pamoate is the anthelmintic of choice because it is safest in young ill animals. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention and control (hall and simpson, ) . due to transplacental or milkborne infection, puppies should be treated q weeks from to weeks of age. whipworms trichuris vulpis, the canine whipworm, can cause acute or chronic large intestinal diarrhea. the adult worm resides in the cecum or ascending colon. most infections are subclinical, but in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss may also be seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. trichuris vulpis has a direct life cycle with eggs passed in the feces. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to diarrhea. factors that influence development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. whipworm infestation is diagnosed by the presence of barrel-shaped, thickwalled eggs with bipolar plugs on fecal flotation. adult worms intermittently release eggs; therefore, negative results do not exclude infection. differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . several species of cestodes parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. most cestode infestations are subclinical, but severe infestations with dipylidium can cause diarrhea, weight loss, and poor growth. the cestode requires an intermediate host, which for d. caninum are fleas and lice. ingestion of these arthropods results in transmission of the tapeworm. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. the most significant means to limit cestode infestation is to control flea and/or louse exposure. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against d. caninum (hall and simpson, ) . demodicosis canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles and is passed from dams to nursing pups. localized demodicosis is typically asymptomatic, but disease can present with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and face, and around the ears (demanuelle, a). generalized demodicosis can develop in juvenile or adult populations and is indicative of an underlying immunosuppressive disorder. demodex has a characteristic 'cigar shape' and can be identified from deep skin scrapings mounted on mineral oil (campbell, ; noli, ) . differential diagnoses include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma, which is also a common secondary complication of generalized demodicosis. ivermectin at - μg/kg and oral milbemycin at - mg/kg/day are effective treatments. treatment duration can be extensive and must be accompanied by repeated skin scrapings. sarcoptic mange canine sarcoptic mange is caused by sarcoptes scabiei var. canis, which is zoonotic. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas of the ear pinnae, elbows, ventral thorax, and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. adult mites, mite eggs, or mite feces may be observed on superficial skin scrapings, but diagnosis may be difficult because multiple skin scrapings may yield negative results. even if scrapings are negative, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige either in the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ) . histologic examination is nondiagnostic; however, suggestive lesions include small foci of edema, exocytosis, degeneration, and necrosis . an important differential diagnosis is flea allergy dermatitis (fad). unless antiparasitic therapy would interfere with research objectives, all dogs with sarcoptic mange should be treated. in addition, their kennel mates should also be treated due to the contagious nature of the disease and its zoonotic potential. the usual means of treatment is either ivermectin at - μg/kg q days or milbemycin at mg/kg q days for three oral doses . ticks ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, amblyomma, and ixodes. the primary significance of tick infestation is vector-borne infectious diseases, including rocky mountain spotted fever (rickettsia rickettsii), lyme disease (borrelia burgdorferi sensu stricto), thrombocytic anaplasmosis (anaplasma platys), and canine monocytic ehrlichiosis (ehrlichia canis). ticks alone cause minimal signs unless the dog develops a hypersensitivity reaction leading to a more granulomatous response at the bite location (merchant and taboada, ) . some species (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that causes an ascending flaccid paralysis (malik and farrow, ) . uncomplicated tick bites and tick-bite paralysis are diagnosed by identification of the tick and clinical signs of paralysis. dogs with tick-bite paralysis usually show improvement within h of tick removal, with complete recovery within h (malik and farrow, ) . formamidines (amitraz), pyrethroids, and phenylpyrazoles (fipronil) are available as spot-ons, collars, sprays, and foggers to treat tick infestations in both the animal and the environment (halos et al., ; beugnet and franc, ) . differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . fleas the most common flea to infest dogs is ctenocephalides felis felis, the cat flea (sousa, ) . flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop severe fad, which features papules, crusting, and excoriations over the lumbosacral region, flanks, thighs and abdomen. these animals may require oral corticosteroids to relieve clinical signs (muller et al., ) . secondary bacterial and fungal infections can also develop. fleas can also transmit other parasitic diseases, such as dipylidium tapeworms. flea infestations and fad are definitively diagnosed by observing the fleas on the host's skin; however, the presence of flea excrement can support a presumptive diagnosis (demanuelle, b) . treatment of flea infestations should use an integrated pest management (ipm) approach that targets adult fleas, immature stages, and environmental contamination in order to limit the risk of chemoresistance. combining ovicidal treatments, such as lufenuron and selamectin, with adulticidal treatments, such as fipronil, spinosad, selamectin, and imidacloprid, is recommended (halos et al., ; beugnet and franc, ; dryden et al., ) . certain chemicals (i.e., imidacloprid and selamectin) have both adulticidal and larvacidal abilities, but the principles of ipm preclude the use of one product solely for both adulticidal and larvacidal properties (schwassman and logas, ). differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs. canine dermatophytoses are commonly caused by microsporum spp., trichophyton spp., and epidermophyton spp. (moriello and deboer, ) . uncomplicated infections are characterized by circular areas of alopecia and crusting with or without follicular papules, usually around the face, neck, and forelimbs. dermatophytes infect the hair shaft and follicle, as well as the surrounding skin. infected hairs become brittle and broken shafts remain infective in the environment for months. dermatophytoses are zoonotic and easily transmitted to other animals through the environment or by direct contact. definitive diagnosis is made using dermatophyte test medium for culture. hair and crust material from infected sites can be plucked and placed on culture; however, the 'toothbrush' method is more effective for sampling multiple sites. the brush is used to comb hairs and scales from several infected sites and then pressed into the culture media. media plates should be visually inspected daily for days. positive cultures will become red at the same time as growth of a fluffy white colony. microscopic examination of hairs and scales to visualize fungal elements can be done using skin scrapings in % koh or mineral oil; however, this method is not very sensitive. topical and systemic therapy should be initiated together after all suspected areas are clipped to reduce spreading of contaminated fragile hairs. wholebody topical therapies with antifungal shampoos, rinses, and creams are recommended rather than spot treatment. systemic therapy can be achieved with griseofulvin, ketoconazole, itraconazole, or fluconazole. due to the highly infective nature of this disease, animals should be isolated and the environment thoroughly disinfected. chlorhexidine and virkon ® s are ineffective at clearing environmental spores, but lime sulfur ( : ), enilconazole ( . %), and bleach ( : ) are effective across many strains of microsporum canis (moriello and deboer, ) . although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly laboratory animal medicine affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology primary hypothyroidism affects the thyroid gland directly, whereas secondary hypothyroidism has indirect effects through dysfunction of the pituitary gland (seguin and brownlee ) . both of these causes result in a gradual loss of functional thyroid tissue (avgeris et al., ; kemppainen and clark, ) . the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including nonpruritic, bilaterally symmetrical alopecia, hyperpigmentation, seborrhea, and pyoderma (avgeris et al., ; peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic, normochromic, nonregenerative anemia may be seen in approximately one-half of the cases (avgeris et al., ) . increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bichsel et al., ; panciera, ) , and a true causal relationship with hypothyroidism has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause abnormalities of the cardiovascular system including bradycardia, hypocontractility, increased vascular volume, and atherosclerosis (seguin and brownlee ) . abnormalities that may be detected by ecg include a decrease in p-and r-wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these ecg abnormalities are caused by lowered activity of atpases and calcium channel function. an association between hypothyroidism and von willebrand disease has been suggested. however, the relationship is probably one of shared breed predilection and not a true correlation. contradictory studies have shown either deficient (avgeris et al., ) or normal (panciera and johnson , ; avgeris et al., ) von willebrand factor antigen and bleeding times in hypothyroid dogs. most importantly, hypothyroidism does not appear to cause overt, clinical von willebrand disease. however, it may exacerbate existing subclinical von willebrand disease (seguin and brownlee, ) . epizootiology the prevalence of hypothyroidism in the general canine population is reportedly less than % (panciera, ) . the disorder occurs most often in middle-aged, larger breed dogs (avgeris et al., ) , and reports suggest a higher incidence of hypothyroidism in spayed, female dogs (panciera, ; peterson and ferguson, ) . doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . diagnosis and differential diagnosis because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t and is heavily protein bound. free t represents the laboratory animal medicine unbound fraction that is available to the tissues (peterson and ferguson, ) . the measurement of total t carries a sensitivity of around % and can be used as a good screening tool. with the measurement of both serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial may be in order (peterson and ferguson, ) . however, nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present and animals should not be treated solely on the basis of serum hormone levels if clinical signs are not present. if the clinical signs are equivocal or only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone, the measurement of serum t levels is an unreliable indicator of hypothyroidism (peterson and ferguson, ; ferguson, ) . serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released and conversion of t to t may be enhanced by the failing thyroid (peterson and ferguson, ; ferguson, ) , particularly early in the disease. in one study, t was within normal limits in % of the hypothyroid dogs (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous thyroid-stimulating hormone (tsh) levels provide reliable information on thyroid status, and an assay is available for dogs. however, endogenous tsh levels can be normal in some dogs with hypothyroidism and high tsh levels have been noted in normal dogs and sick animals that are actually euthyroid. it is therefore recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and h after. a normal response to the administration of tsh should create an increase of t levels at least μg/dl above the baseline levels or an absolute level that exceeds μg/dl (peterson and ferguson, ; wheeler et al., ) . treatment the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . - . mg/kg once a day (avgeris et al. ). if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - h after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must address obesity and its potential effects on animal welfare and research results. etiology obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy, the result of overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household because access to food is more restricted, and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and limitation to exercise reduces energy expenditure. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy laboratory animal medicine expenditure are followed (butterwick and hawthorne, ) . as in humans, genetics plays an important role in the development of obesity in dogs, and certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . several metabolic or hormonal changes are also associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ). in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . differential diagnosis the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict the total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see above), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to have rebound weight gain after restrictions are relaxed. there has been a great deal of attention in humans as to the correct diet to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorie-restricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick et al., ; butterwick and markwell, ) . research complications it is important to control weight gain in research animals because of the association of obesity with metabolism. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity and obesity-induced hyperinsulinism in several experimental models (macewen, ) . a recent study demonstrated metabolic disease, typified by hyperinsulinemia and hypoadiponectinemia, in approximately % of obese dogs (tvarijonaviciute et al., ) . research that requires anesthesia may be complicated by a greater risk of cardiovascular diseases (edney and smith, ) including hypertension and compromise to the respiratory tract. etiology in the laboratory setting, the majority of traumatic wounds will be small in size and quickly observed. occasionally, dogs may sustain minor trauma during transport or have a small, previously undetected, chronic wound upon arrival at the facility. when dogs are group housed, they may sustain bite wounds during early socialization periods. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or when the basic principles of wound management are not followed. clinical signs the signs and appearance of a traumatic wound will vary with the cause and the duration of time since wounding. abrasions, sustained by shear forces, are partial thickness skin wounds characterized laboratory animal medicine by minimal bleeding or tissue disruption. puncture wounds have a small surface opening but penetrate into deep tissues with the potential for contamination. lacerations are wounds caused by sharp separation of skin that may extend to deeper tissues. acute wounds are characterized by bleeding tissue, sharp edges and no obvious devitalization. they have variable degrees of contamination. chronic wounds generally do not exhibit active bleeding and will have curled or rounded edges. these wounds often have necrotic tissue and are considered contaminated. treatment to aid decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a 'golden period.' it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the competence of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as clean, clean-contaminated, contaminated, or dirty (see table . ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds require more aggressive therapy. postsurgical infections or complications of initial therapy would be considered dirty wounds. when in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of its classification. when first recognized, the wound should be covered with a sterile dressing until definitive treatment can be rendered. bleeding should be controlled with direct pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . anesthesia or analgesia may be necessary and the choice of agent will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed. then a water-soluble lubricant gel may be applied directly to the wound to prevent it from further contamination during the hair removal process. a wide margin of hair should be clipped and a surgical scrub performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a, b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. several irrigation solutions have been recommended (lozier et al., ; waldron and trevor, ; sanchez et al., ) , but type may not be as important as the volume and pressure of delivery. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a -ml syringe with an -or -gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of waldron and trevor ( ) . extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be placed. subcutaneous closure should be performed with absorbable suture such as polydioxanone, polyglactin , or polyglycolic acid. it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. the wound may be covered by a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . second-intention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than with surgical repair, and, in the case of large wounds, it will be more expensive because of the cost of bandaging materials. several factors must be weighed when considering the use of antibiotics in traumatic wound care, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. topical application of bacitracin, neomycin sulfate, and polymixin b combinations may be used in wounds with minor contamination. in skin wounds with more extensive contamination, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. when systemic antibiotics are necessary, cephalosporins, amoxicillinclavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . prevention in facilities with good husbandry practices and a diligent staff, potentially injurious equipment or surfaces are identified quickly. appropriate attention to surgical technique and to initial wound care will generally reduce the occurrence of postprocedure wound infection. etiology pressure sores (decubital ulcers) can be a problem in long-term studies and housing situations that require chronic skin contact with hard surfaces. decubital ulcers often develop over a bony prominence such as the elbow, tuber ischii, tarsus, or carpus. the compression of soft tissues between hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores, including poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting coaptation devices (swaim and angarano, ) . clinical signs initially, the skin will appear red and irritated. over time, constant trauma can result in full-thickness skin defects and can progress to necrosis of underlying tissues. the severity of the sores may be graded from i to iv according to the depth of the wound and the tissues involved, from superficial skin irritation to involvement of underlying bone (waldron and trevor, ) . epizootiology the problem usually occurs in large dog breeds, but any type of dog can be affected. prevention and control minimizing or eliminating predisposing factors is important to both the prevention and treatment of this condition. if a dog will experience long periods of recumbency, adequate bedding or padding must be provided. recumbent animals should be moved frequently, ideally every h, to prevent continuous compression on a specific laboratory animal medicine area (waldron and trevor, ) . skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain body weight will minimize compressive forces experienced over areas susceptible to ulceration (swaim and angarano, ) . treatment the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive diagnostics and therapy must be performed. the affected area should be radiographed to assess bone involvement and the wound should be cultured. all of the damaged tissue should be debrided and basic wound management guidelines should be followed (see above). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary (waldron and trevor ) . bandaging should be performed on all full-thickness wounds; however, it is important to remember that illfitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound, in order to displace pressure over a larger area and onto healthier tissue. the doughnut is then incorporated into a padded bandage. if a cast has been applied to the area for treatment or research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ) . bandages should be removed at least once or twice a day to allow wound care. etiology an acral lick granuloma is a skin lesion caused by self-trauma. in a few cases, the self-trauma is due to initial irritation caused by an identifiable neurologic or orthopedic condition (tarvin and prata, ) . allergy may also be a source of irritation that leads to self-trauma. however, the majority of cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory environment could promote the abnormal behavior and lead to acral lick granuloma. epizootiology the lesions associated with acral lick granuloma are seen most often in large dog breeds, particularly dobermans. however, any type of dog can be affected (walton, ) . clinical signs early lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ) . the predilection for the limbs may be due to accessibility or possibly a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated and the wound develops a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of the aforementioned problems can be ruled out by the history of the animal. however, a complete history may be unavailable in the laboratory setting. fungal cultures and allergy testing may aid in diagnosis. biopsy of the affected area would rule out neoplasia. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. environmental enrichment including exercise, co-housing and various toys is already a basic requirement and may be increased to combat self injurious behaviors. treatment several treatments have been reported for acral lick granuloma and the selection of a treatment should be based on the underlying cause. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been applied as treatments for acral lick granulomas and self-injurious behaviors with the theory that this will block the effects of endogenous opioids. naltrexone and nalmefene have been used successfully to reduce excessive licking behaviors and resolve associated lesions. however, lesions did recur after the drugs were discontinued (dodman et al., ; white, ) . the topical administration of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide has also been shown to be effective (walton, ) . in addition, psychoactive drugs have been suggested to relief of laboratory animal medicine boredom or anxiety. these have included phenobarbital, megestrol acetate, and progestins. however, side effects have been reported (swaim and angarano, ) . other behavior-modifying medications such as clomipramine may be effective in the treatment of compulsive anxiety disorder. the potential for effects that could interfere with experimental results must be determined prior to initiation of treatment. it is important to note that none of the above-mentioned treatments have been successful in all cases. the overall prognosis for acral lick granuloma should be considered guarded since the lesions often recur when treatment is discontinued. etiology hygromas are fluid-filled sacs that develop as a result of repeated trauma or pressure over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology elbow hygromas are most frequently reported in large and giant breeds of dogs, less than years of age (johnston, ; white, ; cannap et al., ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment of research dogs, especially cage bottoms and cement runs, may predispose them to hygromas. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs the clinical presentation will depend upon the chronicity of the problem. a dog with an elbow hygroma usually presents with a painless, fluctuant swelling over the point of the elbow without signs of lameness. the condition may be unilateral or bilateral. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma becomes secondarily infected, the animal may exhibit pain and fever (johnston, ; white, ) . pathology the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is a yellow or red serous transudate. this fluid is less viscous than joint fluid and elbow hygromas do not communicate with the joint (johnston, ) . treatment the treatment of elbow hygromas should be conservative whenever possible. conservative management of the elbow hygroma is aimed at relieving the source of pressure at the point of the elbow. in early and mild cases, simply providing padding to cover hard surfaces will result in resolution of the hygroma. a soft padded bandage or doughnut bandage around the affected site may also be of benefit. neoprene/polyester sleeves that cover the elbows and fit over the shoulders are also available as an option for either prevention or treatment of hygromas (cannap et al., ) . more aggressive therapies, including needle drainage and injection of corticosteroids into the hygroma, have been described but are not recommended due to the risk of infection (johnston, ) . surgical options should be reserved for complicated or refractory cases. even simple excision can be associated with complications such as wound dehiscence and ulceration (johnston, ) due to the location of the bony prominence at the surgical site. this issue may be avoided by using a skin advancement flap (white, ) that allows intact, healthy skin to cover the boney prominence. a muscle advancement flap has also been described (green et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology in the research environment, corneal ulcers are most often associated with direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these would be rare in the laboratory setting. clinical signs the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may appear normal however, in cases of deeper ulceration, the cornea may appear roughened or have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. diagnosis a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers is made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies, abnormal eyelids, or abberant cilia. treatment the treatment of corneal ulcers will depend on the depth and size of the affected area, as well as the underlying cause. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given three times a day for - days usually provides adequate treatment. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. deep ulcers may require further debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. ulcers caused by entropion, ectropion, or dystichiasis will not resolve until the condition is repaired, and descriptions for this can be found elsewhere. prevention the proper application of lubricant eye ointment at the time of anesthesia will prevent drying due to exposure and may also protect the eye from scrub solutions applied near the eye. early treatment of superficial ulcers should prevent self-trauma and progression of the wound. etiology research protocols often require the placement of chronic implants. implants such as cardiac or other biomedical devices may be the primary focus of the research study. implants may also be used as chronic monitoring devices, for delivery of compounds, or to collect serial samples. infection may occur at the time of implant. alternatively, the implant may serve as a nidus after hematogenous spread from other sources. one of the most common sources of infection is from colonization of the device from an external component, which is a frequent complication with indwelling catheters. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters, which included traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. these lesions were primarily associated with catheter-induced trauma or secondary to embolization of fibrin. in a veterinary clinical setting, infections in peripheral catheters were more likely when the catheters were used for blood collection immediately after placement and when a 't' connector rather than a 'y' connector was used. . intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., ; kwei et al., ) . clinical signs dogs with implant infections may not exhibit signs initially . localized swelling around the implant may occur. in the case of indwelling catheters, signs may include redness and swelling of the skin around the external port or discharge from the skin wound. vascular access ports may develop fluctuant subcutaneous abscesses. in more severe cases, systemic signs may be noted (bach et al., ; hysell and abrams, ) . the systemic signs of infection are covered elsewhere in this chapter. treatment the treatment of catheter infections almost invariably requires removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ). superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes, and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. localized abscesses or sinus tracts may be managed by establishing drainage and copious flushing. aerobic and anaerobic cultures of blood and locally infected sites should be performed prior to initial treatment (ringler and peter, ) . systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. if retention of a catheter is important, the catheter lumen may be safely disinfected with chlorine dioxide solution (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used with an infected catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. prevention it is highly desirable to prevent complications that may result in loss of an implanted device. catheters and other implants should be made of nonthrombogenic material and be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. intravenous catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. ideally, catheters are secured to reduce movement and irritation of the skin, which may predispose to infection around external ports. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that long extension tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . for intestinal access ports, catheter security may be improved with a synthetic cuff added to the end of the catheter allowing better attachment to the intestine (meunier et al., ) . after any catheter placement, animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide. in addition, a solution of the antibiotic ceftazidime used on alternate days with the heparin locking solution has been shown to effectively reduce infections in indwelling vascular catheters (bach et al., ) . throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. periodically, the placement of an indwelling catheter may be verified by radiography. when placed and managed correctly, catheters and ports of any kind may remain in place for months without complications. etiology sepsis is defined as the systemic response to infection caused by bacteria (gram negative and/or gram positive), fungi, or viruses. in laboratory animals, sepsis is most often seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course, dogs may present with signs of a hyperdynamic sepsis, including increased heart rate, increased respiratory rate, red mucous membranes, and a normal-to-increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals may show classic signs of septic shock including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell (wbc) count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of band neutrophils than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several areas should be evaluated for infection, including the urinary, reproductive, respiratory, alimentary, and cardiovascular systems, as well as the abdominal cavity (kirby, ) . treatment the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is infected, it should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or thirdgeneration cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. this category of sepsis treatment is the focus of much research. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no 'magic bullet' for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology in research animals, aspiration may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. pathogenesis aspirated compounds can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response, probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - h. clinical signs the severity and clinical manifestation of aspiration lung injury are dependent upon the ph, osmolality, and volume of the aspirate. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on witness of aspiration, history consistent with aspiration, and/or the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. treatment the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. if the aspiration is witnessed, the mouth and, idealy, the upper airway should be cleared of residual material. when small amounts of a relatively innocuous substance (e.g., barium) have been aspirated, treatment may not be necessary. when severe inflammation is present, systemic as well as localized therapy may be necessary. oxygen therapy may be instituted; however, the concentration and time frame are controversial, because lung injury may be exacerbated by long-term administration of oxygen at high concentrations (nader-djahal et al., ) . fluid therapy may also be necessary in severe cases; however, cardiovascular support should be performed judiciously as fluid overload could lead to an increase in pulmonary edema. the use of colloids is also controversial because of the increase in vascular permeability that occurs in the lungs. several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. corticosteroids are contraindicated (raghavendran et al., ) . in humans, antibiotics are reserved for cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be immediately treated with antibiotics when the aspirated material is not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. prevention aspiration of drugs and other compounds may be avoided through careful administration of oral medications by experienced individuals. likewise, gavage or orogastric administration of liquids should be performed by experienced individuals, and the procedure should be aborted if coughing or other respiratory signs occur. the aspiration of stomach contents can largely be avoided by appropriate fasting prior to anesthesia for at least h for food and h for water. if appropriate fasting times are not observed, anesthesia should be postponed whenever possible, particularly if intended procedures require manipulation of the viscera or head-down positioning of the dog. if anesthesia cannot be avoided, it should be rapidly induced and the dog should be intubated. during recovery from anesthesia, the endotracheal tube should be removed with the cuff partially inflated and with the dog in a head-up position (haskins, ) . based on the source of energy, burn wounds may be categorized into four groups: thermal, chemical, radiation, and electrical. in laboratory animals, accidental burns are usually the result of thermal injury (heating pads, water bottles), chemicals (strong alkalis, acids, disinfectants, drugs), or experimental irradiation protocols. etiology inappropriate use of external heating devices is the most common cause of burns in laboratory animal medicine. the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs the clinical signs vary with the depth, location, and surface area of burn injury. classification systems for thermal burns are generally based on the depth of the injury, varying from superficial involvement of only epidermis to complete destruction of skin and subcutaneous tissues (bohling, ) . superficial burns appear erythematous and inflamed. in some cases, matting of the overlaying hair with exudate may be the first sign of a previously undetected skin lesion. progressive hair and skin loss may be evident over the first few days after injury (johnston, ) . although blistering is a characteristic of partial thickness burns in humans, this is rarely seen in dogs (bohling, ) . uncomplicated, superficial burn wounds heal by reepithelialization within - days. deeper burn wounds are characterized by a central area of nonviable tissue surrounded by edematous, inflamed tissues. a thick eschar, composed of the coagulated proteins and desiccated tissue fluid, develops over deep burn wounds. these wounds heal by granulation under the eschar, which will eventually slough. the amount of pain associated with burns depends upon several factors including the depth and area of the wound, procedural manipulations, and movement at the affected site (bohling, ) . pain associated with superficial burn wounds usually subsides in - days. theoretically, deep burns destroy nerve endings and result in less pain than superficial burns. however, inflammatory pain may still be present due to the tissue reaction around the necrotic site. in addition, sharp procedural pain and breakthrough pain have been described in humans during the healing phases of burn injuries and should be considered as potential complications in dogs as well (bohling, ) . severe and widespread accidental burn injury can result in clinical signs associated with multiple organs including the pulmonary, gastrointestinal, hematopoietic, and immune systems. in addition, extensive burn injury can predispose to infection and even sepsis. this type of injury with the associated complications would be extremely rare in the laboratory setting. treatment appropriate and timely treatment of a burn wound will reduce the extent of tissue damage and associated pain. thermal injuries should be immediately exposed to cool water ( °c) to reduce edema and pain. exposure to very cold water and ice does not improve outcomes (bohling, ) . topical wound dressings are recommended in the early stages of treatment for both partial-and full-thickness burns that are of small size. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, a thin film of a water-soluble, broad-spectrum antibiotic ointment should be applied to the wound surface. silver sulfadiazine has a broad spectrum, penetrates eschar, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it has been associated with pain upon application (demling and lalonde, ) . once a topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds (demling and lalonde, ; bohling, ) . after the initial treatment, burn wounds should be gently cleansed two to three times a day, followed by reapplication of the topical antibiotic and rebandaging (demling and lalonde, ) . systemic antibiotics are indicated in cases where local or systemic infection is present and their ultimate selection should be based on culture results. burn wounds can be extremely painful, and analgesia should be instituted immediately and adjusted accordingly throughout the treatment period. surgical intervention may be necessary in some cases. with small or moderately sized wounds, the eschar over the burn wound may actually impede wound contraction and reepithelialization. in such cases, once the eschar has become fully defined, a complete resection may improve wound healing. with large and severe burn wounds, repeated debridement by surgery or other means might be necessary. in the laboratory setting, a decision to pursue extensive surgical intervention would be dependent upon full consideration of the effects on animal welfare and research results. prevention thermal burns can be prevented in the research setting. electric heating pads and heat lamps should be avoided if possible. only heated water blankets or circulating warm air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore, these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. basic fire prevention precautions should be taken particularly around oxygen sources and flammable agents etiology chemical injury may be due to skin contact with concentrated solutions such as disinfectants or inadvertent exposure to laboratory chemicals. in addition, perivascular injection of certain drugs (pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin) have been associated with extensive tissue damage (swaim and angarano, ; waldron and trevor, ) . the mechanism of action will vary depending upon the ph, osmolality, and chemical composition of the agent and may include oxidation, reduction, disruption of lipid membranes, or other reactions (bohling, ; swaim, ; waldron and trevor, ) . clinical signs surface contact with chemicals may result in mild irritation and redness of superficial layers of the skin. however, many agents may cause progressive injury until the chemical reaction has been neutralized. this may result in tissue necrosis and secondary infection. the immediate signs of perivascular injection are withdrawal of the limb or other signs of discomfort and swelling at the injection site. the area may appear red, swollen, and painful as inflammation progresses. there may eventually be necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a -to -month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . treatment in cases of skin contact with chemical agents, the affected area should be thoroughly and repeatedly lavaged with warm water to dilute or remove the substance. the material safety data sheet for the substance should be consulted for any possible neutralization protocol. additional treatment will depend upon the severity of the tissue damage and will follow the same guidelines as for the thermal injury described earlier. for the treatment of perivascular injections, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions. the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) . the local infiltration of hyaluronidase accompanied by warm compresses has been suggested for perivascular vinblastine (waldron and trevor, ) and for doxirubicin. the use of dmso or another free radical scavenger, dexrazoxane, infused at the site has also been suggested for doxorubicin toxicities. despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful and analgesia should be addressed. prevention prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. the material safety data sheets should be available for all compounds and storage recommendations followed closely. for intravenous administration of toxic compounds, insertion of an indwelling catheter is extremely important. prior to the injection, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. etiology radiation burns are generally a complication of therapeutic administration and are a result of free oxygen radical formation (waldron, ) . the severity of radiation burns and their treatment will depend upon the dose, frequency, total surface area, and location of the radiation. damage to epithelial layers of the skin can lead to desquamation. direct injury to fibroblasts results in decreased collagen production and poor wound healing. in addition, there may be fibrosis of blood vessels (pavletic, ) and subsequent hypoxia causing necrosis of deeper tissues. clinical signs the tissues most often affected are the skin and mucus membranes. with superficial injury, affected skin may exhibit hair loss and erythema, and produce a clear exudate. the intensity of the inflammation may increase for - weeks after the completion of radiation treatment. deeper and more serious injury manifests with subcutaneous fibrosis and can lead to disfigurement . the skin and underlying deep structures including the bone may become necrotic over several weeks (pavletic, ) . these deeper injuries are prone to infection due to their lack of blood supply. systemic signs such as vomiting are rare in dogs unless there has been direct radiation treatment to organs . treatment with superficial skin burns, the wound should be kept clean and should be covered if possible. in cases of oral mucous membrane damage, there may be special feeding requirements. when wounds are ulcerated, avascular tissues should be excised. treatments with silver sulfadiazine, mafenide acetate, or other topical agents are recommended to control infection. in addition, infection is avoided by closure of the wound as soon as possible. the goal of surgery is to cover the wound with healthy tissue to promote vascularization of the area. in some cases, this may require muscle and/ or skin grafts. prevention radiation burns can be limited by selection of appropriate, fractionated therapy and application of shielding to reduce exposure. prompt treatment of the injuries can reduce the occurrence of infection. since radiation is associated with poor wound healing, complications may arise when additional procedures are required. it is recommended to wait at least week (pavletic, ) or even longer (laing, ) prior to administering radiation to a surgical site. after radiation, routine surgeries should be avoided for - months (pavletic, ). the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. a complete review of clinical oncology in the dog is beyond the scope of this chapter but can be found elsewhere (withrow et al., ) . fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients with local anesthesia. an instrument such as a tru-cut ® needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a -mm × - . -cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies aid in histopathological examination and are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large softtissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. canine lymphoma represents - % of canine tumors and a majority ( %) of canine hematopoetic disease (ettinger, ; vail and young, ) . whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . in addition, tobacco smoke, environmental chemicals, and waste emissions are considered possible risk factors (marconato et al., ; gavazza et al., ) clinical signs multicentric high-grade lymphoma (mhgl) accounts for the majority of reported cases of canine lymphoma. depending upon grade, immunophenotype, and location involved, dogs with mhgl usually present with painless, enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, fever, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. less commonly, dogs develop alimentary, mediastinal, cutaneous, and extranodal lymphomas. alimentary lymphoma is associated with vomiting and diarrhea, in addition to clinical signs associated with mhgl. dogs with mediastinal lymphoma often present with respiratory signs (dyspnea and exercise intolerance) secondary to pleural effusion or cranial vena caval syndrome. hypercalcemia is most frequently associated with this form of lymphoma and may result in polyuria, polydypsia, and weakness. cutaneous lymphoma is an uncommon epitheliotrophic form of lymphoma. it is often referred to as mycosis fungoides and is typically of a cd + t-cell immunophenotype. it varies in presentation from solitary to generalized and may mimic any of a number of other inflammatory skin disorders including oral mucosal lesions. the lesions may occur as erythema, plaques, erosions, scales, nodules, crusts, hypopigmentation, and alopecia (fontaine et al., ) . approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement (e.g., nervous system: seizures, paresis, paralysis). epizootiology the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . no gender predilection has been reported. diagnosis and pathologic findings a fine-needle aspirate is initially performed on accessible lymph nodes. thoracic radiographs and abdominal ultrasound ± fine needle aspiration of the liver or spleen can be used if mediastinal or abdominal involvement is suspected. additional staging can be determined through complete blood counts, serum biochemistry, flow cytometry for immunotyping, bone marrow aspiration, or surgical lymphadenectomy and histology. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. flow cytometry and lymphoblastic markers (cd ) can aid in diagnosis and subtyping of tumors. in addition, positron emission tomography is being explored for detection of extranodal and metastatic lymphoma (leblanc et al., ; marconato, ; elstrom et al., ) . classification of lymphoma types is based upon cytological, morphological, and immunological characteristics using the kiel classification criteria (vail and young, ) . histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. lymphoma subtypes can be further characterized based upon genetic, molecular, and immunological criteria (ponce et al., ) . pathogenesis all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . median survival time with aggressive therapy is generally less than months. hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. differential diagnosis differential diagnoses for multicentric lymphoma include systemic mycosis; salmon-poisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections for histopathologic examination may be needed. treatment therapy for lymphoma primarily consists of one or a combination of several chemotherapeutic agents. in addition, radiation therapy and bone marrow transplantation have been utilized. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. although treatment may induce clinical remission and prolong short-term survival, most treatment is palliative and aimed at improving quality of life. a thorough discussion of therapeutic options for the treatment of lymphomas in the dog can be found elsewhere (chun, ; marconato, ) . future directions include development of molecular and cellular targeted therapies to enhance traditional chemotherapy treatment, prolong remission, and treat immunologic subtypes of lymphoma (e.g., t-cell lymphoma). research complications given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with significant clinical illness. etiology mast cells are derived from cd + bone marrow progenitor cells. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog and may account for up to % of canine skin laboratory animal medicine tumors (bostock, ; welle et al., ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin, lung, liver, and gastrointestinal tract. current research has linked mast cell tumor development to multifactorial causes including breed predisposition and a genetic component, chronic inflammation, and mutations in the surface growth factor, c-kit (ma et al., ; reguera et al., ; webster et al., ) . clinical signs well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, -to -cm nodules in the dermis and subcutaneous tissue. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema to surrounding tissues. mast cell tumors can be found on any portion of the dog's skin but frequently affect the trunk and hind limb extremeties along with perineal and preputial areas. the tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. abdominal organs are rarely involved but may be associated with anorexia, vomiting, melena, abdominal pain, and gastrointestinal ulceration. mast cell tumors have also been reported in extracutaneous areas such as the salivary glands, larynx, nasopharynx (london and thamm, ) and conjunctiva (fife et al., ) . mast cell tumors within the perineal, preputial, or inguinal areas are associated with a greater predilection for recurrence or metastasis (misdorp, ) . epizootiology these tumors tend to affect middleaged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ) . pathologic findings because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis and are well differentiated, with round to ovoid, uniform cells with distinct cell borders. the nuclei are round and regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have the worst prognosis. the cells contain large, irregular nuclei with multiple prominent nucleoli and few cytoplasmic granules. mitotic figures are much more frequent. cells are pleomorphic with indistinct borders. in addition to associated skin lesions (e.g., ulceration, collagenolysis, necrosis, and infection), mast cell tumors have been associated with gastric ulcers in the fundus, pylorus, and/or proximal duodenum, most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . pathogenesis although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver, and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis using fineneedle aspiration, mast cell tumors can be distinguished cytologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue to metachromatically stain the cytoplasmic granules red or purple. mast cell granules can also be stained with wright's, giemsa, and romanowsky stains. in addition, mast cells may contain tryptase, chymase, or both (fernandez et al., ) . histological evaluation is generally required for grading. examination of regional lymph nodes may be warranted if metastatic or systemic disease is suspected. in addition, radiographs and ultrasound with guided aspirates of the liver, spleen, or sublumbar lymph nodes can be used to determine metastatic disease. treatment depending upon the grade, initial treatment for mast cell tumors is generally wide surgical excision ( -cm margins), which may be followed by radiation, chemotherapy, or glucocorticoid therapy. aspiration or surgical removal of regional lymph nodes is recommended if lymphatic tumor drainage is suspected. if the tumor is not completely resectable or is grade ii or iii (moderately to undifferentiated), then debulking surgery and adjunct therapy may be used. treatment algorithms are outlined elsewhere (withrow et al., ) research complications because of the potential for systemic release of substances such as histamine, vasoactive substances, heparin, eosiniphilic chemotactic factor, and proteolytic enzymes, along with the possibility of delayed wound healing and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed monthly. grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the canine transmissible venereal tumor (ctvt) is transmitted horizontally to the genitals by coitus (nielsen and kennedy, ) . ctvt is a 'parasitic-like' tumor that appears to have originated from dogs or wolves thousands of years ago and despite immense mutation, ctvt adapted, survived, and spread across multiple continents making it the oldest known continuously passaged somatic cell line (rebbeck et al., ; murchison et al., ; murgia et al., ) . it has been described as a round cell tumor of histiocytic origin. although this tumor has been reported in most parts of the world, it is most prevalent in tropical or temperate climates (macewen, ) . clinical signs the tumors are usually cauliflowerlike masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis. less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission ctvts are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for exfoliation and transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions may be the result of oral contact with previously traumatized areas. pathologic findings histologically, cells are arranged in compact masses or sheets. the cells are round, ovoid, or polyhedral, and have large, round nuclei with coarse chromatin. the cytoplasm is eosinophilic with small vacuoles arranged in a 'string of pearls' pattern. pathogenesis tumor growth occurs within - months after mating or implantation, and then growth generally slows. metastasis is rare (< - % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. spontaneous regression may occur within - months of tumor development. diagnosis and differential diagnosis transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. however, cytological examination of impression smears, swabs, and fine-needle aspirates generally provide a definitive diagnosis. although not usually required, histopathology of a biopsy from the mass can aid in diagnosis. prevention thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control removing affected individuals from a breeding program should stop further spread through the colony. treatment surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for four to six treatments will induce remission and cure in greater than % of the cases (macewen, ) . research complications experimental implantation of ctvts has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger, invasive, and coalescent with adjacent tissues. inflammatory mammary carcinomas may mimic mastitis or severe dermatitis and must be ruled out to prevent misdiagnosis. epizootiology mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. the median age at diagnosis is - years. a longitudinal study of a large beagle colony showed that significant risk for development of mammary tumors begins at approximately years of age (taylor et al., ) . mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings the t (tumor size), n (lymph node involvement), and m (metastasis) system is commonly used to stage mammary tumors. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . histopathologic grades are scored based upon tubule formation, nuclear pleomorphism, and mitosis (elston and ellis, ) . extensive discussions of classification, staging, and histopathologic correlations can be found elsewhere (moulton, ; sorenmo et al., ) . pathogenesis mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs and extraskeleton. diagnosis and differential diagnosis both benign and malignant mammary tumors must be distinguished from mammary hyperplasia, mastitis, and severe dermatitis. cytological evaluation from fine-needle aspirates correlates well with histological examination of benign and malignant tumors (simon et al., ) . radiographs and possibly ultrasound should be performed to rule out metastatic disease prior to surgery. prevention the lifetime risk of developing mammary tumors can effectively be reduced to . % by spaying bitches prior to the first estrus (schneider et al., ) . this is commonly done in the general pet population at months of age. the protective effects of early spay rapidly decrease after several estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. treatment surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. single mammary tumors should be surgically removed with -cm lateral margins or margins wide enough for complete resection. deep margins may include removing sections of abdominal fascia or musculature en bloc with mammary tumor. multiple mammary tumors should be removed via regional or unilateral chain mastectomies. bilateral, staged mastectomies are reserved for more aggressive tumors. there is insufficient evidence at this time to recommend routine complete unilateral or bilateral chain mastectomies. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. sorenmo et al. ( ) provide a thorough review of canine mammary gland neoplasia. research complications treatment of early-stage or low-grade mammary tumors may be rewarding, allowing dogs to continue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. beagles are subject to many of the inherited and/ or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, ) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). other defects observed include cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology benign prostatic hyperplasia (bph) is an age-related condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs. epizootiology and transmission bph typically affects older dogs (> years), although glandular hyperplasia begins as early as years of age. approximately % of inact male dogs will develop bph by years of age (smith, ) . pathologic findings in the early stages of bph, there is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular with a honeycomb appearance (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to α-dihydrotestosterone (kustritz and klausner, ) mediating bph. diagnosis and differential diagnosis bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis that may be characterized by hemorrhage. a prostatic biopsy can be performed to confirm diagnosis. differential diagnoses include squamous metaplasia of the prostate, para-prostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve with castration. prevention castration is the primary means for prevention of benign prostatic hyperplasia. treatment the first and foremost treatment for bph is castration. in pure cases of bph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies, this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases where semen collection is necessary from a valuable breeding male (e.g., genetic diseases). if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. finasteride, a synthetic α-reductase inhibitor, has been used in dogs to limit the metabolism of testosterone to α-dihydrotestosterone. treatment at daily doses of . - . mg/kg orally for weeks was shown to reduce prostatic diameter and volume without affecting testicular spermatogenesis (sirinarumitr et al., ) . upon discontinuation of finasteride, the prostate generally returns to its pretreatment size within several months (smith, ) . gonadotropin-releasing hormone analogs such as desorelin inhibit production of testosterone and estrogen via negative feedback on the hypothalamus-pituitary axis. this is available in a sustained release subcutaneous implant, which has demonstrated efficacy in reducing prostatic size in dogs (junaidi et al., ) . however, medical therapy has not shown to be as advantageous as castration. research complications bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. research model older dogs with benign protastic hypertrophy are used in research to evaluate the use of ultrasonic histotripsy as a precise nonsurgical urethralsparing alternative to prostate surgery (lake et al., ; hall et al., ; schade et al., ) . etiology juvenile polyarteritis syndrome (jps), also known as steroid-responsive meningitis-arteritis, is a painful disorder seen in young beagles (occasionally reported in other breeds) caused by a systemic necrotizing vasculitis. the cause of the vasculitis has not been established but appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to have a hunched posture and/ or an extended head and neck. most dogs seem to be in pain when touched, especially in the neck region. neurological examination may reveal proprioceptive deficits, paresis, or paralysis. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission jps typically affects young beagles ( - months), with no sex predilection. jps has been reported in other breeds including sibling welsh springer spaniels (caswell and nykamp, ) . pathologic findings on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to mediumsized arteries are seen. these lesions are most noticeable where gross lesions are observed, but they may be seen in other visceral locations. arterial fibrinoid necrosis leading to thyroid gland hemorrhage and inflammation was also reported (peace et al., ) . the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries. the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen. a subclinical vasculitis has also been diagnosed in beagles post mortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclic nature and respond to treatment with corticosteroids, and the affected dogs have elevated α -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resulted in one in seven affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). prevention and control no prevention and control measures are known at this time. treatment clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q h, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. research complications because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes. the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym 'sterile pyogranuloma complex'). clinical signs dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . a retrospective study of beagles housed in a research industry setting, linked development of interdigital cysts to body codition score, age, location of cyst, and type of caging, and may result from chronic interdigital dermatitis (kovacs et al., ) . pathologic findings histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg q h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require surgical removal. excision includes the removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weight-bearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. etiology 'cherry eye' is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating membrane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit. excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. pathologic findings typically, the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . prevention hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland or its removal might compromise 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with chronic indwelling catheters clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound burn trauma chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis discordant s and s rrna gene phylogenies for the genus helicobacter: implications for phylogenetic inference and systematics use of narcotic antagonists to modify stereotypic selflicking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma evaluation of the ovicidal cctivity of lufenuron and spinosad on fleas' eggs from treated dogs enteric coccidiosis the respiratory system the association of american feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the united states study of obesity in dogs visiting veterinary practices in the united kingdom pathological prognostic factors in breast cancer. i. the value of histological grade in breast cancer: experience from a large study with long-term follow-up utility of fdg-pet scanning in lymphoma by who classification principles of treatment for canine lymphoma miller's anatomy of the dog update on diagnosis of canine hypothyroidism immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors the laboratory canine canine conjunctival mast cell tumors: a retrospective study canine cutaneous epitheliotropic t-cell lymphoma: a review canine infectious respiratory disease helicobacter-associated gastric disease in ferrets, dogs, and cats 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canine testis and prostate gland five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism maintenance energy requirement of dogs: what is the correct value for the calculation of metabolic body weight in dogs? outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs kirk's current veterinary therapy xii: small animal practice an emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome? tarsal joint contracture in dogs with golden retriever muscular dystrophy an epidemiological study of interdigital cysts in a research beagle colony value of the ( )c-urea breath test for detection of gastric helicobacter spp. infection in dogs undergoing endoscopic examination pathogenesis of helicobacter pylori infection prostatic diseases semen collection in the dog accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs problems in wound healing associated with chemotherapy and radiation therapy histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model fdg-pet imaging in canine lymphoma and cutaneous mast cell tumor comparison of fertility data from vaginal vs intrauterine insemination of frozenthawed dog semen: a retrospective study withrow & macewen's small animal clinical oncology estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs the prediction of parturition date in canine pregnancy clustering of activating mutations in c-kit's juxtamembrane coding region in canine mast cell neoplasms transmissible venereal tumors kirk's current veterinary therapy : small animal practice canine lymphoma and lymphoid leukemias the staging and treatment of multicentric highgrade lymphoma in dogs: a review of recent developments and future prospects association between waste management and cancer in companion animals tick paralysis in north america and australia thyroid gland and arterial lesions of beagles with familial hypothyroidism and hyperlipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs mast cells and canine mast cell tumours: a review etiologic study of upper respiratory infections of 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dog. part : clinical trial in dogs manual of clinical behavioral medicine for dogs and cats. mosby-year book identification of and screening for human helicobacter cinaedi infections and carriers via nested pcr identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and buccal bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs atlas of small animal wound management and reconstructive surgery what's your diagnosis? fever and leukocytosis in a young beagle. canine juvenile polyarteritis syndrome (beagle pain syndrome) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus thyroid diseases 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development and postsurgical survival how to treat common parasites safely muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology, second ed. mosby-year book thyroid and parathyroid glands diseases of the small bowel canine infectious tracheobronchitis (kennel cough complex) diseases of the intestines the use of ultrasonography for pregnancy diagnosis in the bitch cytologic examination of fine-needle aspirates from mammary gland tumors in the dog: diagnostic accuracy with comparison to histopathology and association with postoperative outcome effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment pathologic features of naturally occurring juvenile polyarteritis in beagle dogs development, anatomy, histology, lymphatic drainage, clinical features, and cell 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serum concentrations of thyroxine and , , ′-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs surgical treatment of specific skin disorders naltrexone for treatment of acral lick dermatitits in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs mammal species of the world: a taxonomic and geographic reference withrow & macewen's small animal clinical oncology the authors thank dr. bambi jasmin of marshall farms for contributions on preventive health practices for class a dogs. key: cord- - imc f authors: schneider, susanne a.; hizli, baccara; alcalay, roy n. title: emerging targeted therapeutics for genetic subtypes of parkinsonism date: - - journal: neurotherapeutics doi: . /s - - - sha: doc_id: cord_uid: imc f in recent years, a precision medicine approach, which customizes medical treatments based on patients’ individual profiles and incorporates variability in genes, the environment, and lifestyle, has transformed medical care in numerous medical fields, most notably oncology. applying a similar approach to parkinson’s disease (pd) may promote the development of disease-modifying agents that could help slow progression or possibly even avert disease development in a subset of at-risk individuals. the urgent need for such trials partially stems from the negative results of clinical trials where interventions treat all pd patients as a single homogenous group. here, we review the current obstacles towards the development of precision interventions in pd. we also review and discuss the clinical trials that target genetic forms of pd, i.e., gba-associated and lrrk -associated pd. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. parkinson's disease (pd) is the second most common neurodegenerative disorder, affecting more than million people worldwide [ ] . numerous drugs for the treatment of motor and non-motor symptoms of pd are available on the market. while these drugs improve motor symptoms and quality of life of people with pd, there is no evidence that any of these interventions modify the progression of pd pathology. there are no fda-approved interventions to slow down pd progression even though there have been multiple clinical trials for treatments that would modify the pd pathophysiological process. one potential explanation of this challenge is that pd is not a single patho-biological process. consequentially, a tailored approach, where interventions are selected by the patients' genotype or other biomarkers (as a measure of a biological process associated with a disease state), is required. in parallel, our knowledge of genetic risks has expanded significantly in the past decade and makes a precision medicine approach in pd very timely. here, we review the obstacles towards the completion of clinical trials in pd along with the efforts to overcome these challenges. we outline the currently available data on precision medicine clinical trials, foc u s i n g p r i m a r i l y o n a l p h a s y n u c l e i n ( s n c a ) , glucocerebrosidase (gba), and leucine-rich repeat kinase (lrrk ). precision medicine aims to tailor treatment in a personalized manner, for the right person at the right time. to achieve this, it uses diagnostic tools to identify specific biomarkers, often genetic, to help assess which medical treatments will be best for each patient [ ] . thus, in addition to the mission of providing personalized medicine, the term precision medicine also conveys the concept that genomics and other emerging biodata sciences will improve medicine's clinically defined nosology [ ] . the hypothesis underlying precision medicine is that diseases previously treated as a single condition are actually biologically different, and that treatment should be tailored based on the biological signature of the individual (stratified medicine [ ] ). a successful example is oncology, where precision medicine led to the identification of effective therapies based on tumor profiling and a better understanding of how tumors become resistant to cancer therapy (https://www. cancer.gov/research/areas/treatment/pmi-oncology). in pd, precision trials are ongoing for genetic forms of pd. in recent years, our understanding of the genetics of pd has expanded significantly. in addition to over a dozen mendelian loci (fig. ) , multiple pd genetic risk factors have been identified. these risk factors are mostly associated with a small increase in risk of pd (e.g. up to~ . -fold) but occur as commonly as % in the general population [ ] . it is thought that the overall heritable component of disease is about % ( - % due to monogenic causes and around % driven by common variants identified by genome-wide association studies) [ ] [ ] [ ] . a prevailing hypothesis is that many of the identified genes and risk factors map into two closely related and overlapping cellular pathways: mitochondrial metabolism and autophagy. in addition, other pathways such as endosomal trafficking, synaptic transmission and immune response pathways have been identified to play a role in the pathogenesis of pd. [ , ] while these biological pathways have been linked to pd via genetic studies, it is very likely that dysfunction in these same pathways due to environmental exposures could also lead to pd. figure describes the genetic architecture of pd. the genetic risks for pd can be roughly divided into three groups, based on their frequency in the general population and the pd risk they convey. accordingly, clinical trial designs for each one of these groups may also differ. group a includes most of the mendelian genes linked to pd (shown in the left upper part of fig. ). these mutations convey a major risk factor for pd, but are extremely rare. in most cases, mutations are present in too few patients for a standard clinical trial design (e.g., pink or park ). intervention on such mutations would require a fig. genetic architecture of parkinson's disease, modified from [ ] and [ ] , showing the continuum of variants of different effect strengths and allele frequencies. the size of the bubbles roughly corresponds to population allele frequencies. colors symbolize modes of inheritance: dominant (red), recessive (yellow), risk loci (green). the genetic risks for pd can be roughly divided into three groups (dotted areas; see main text for more details). the group of common low-risk genes (lower right corner) includes more than putative independent genome-wide significant signals identified in a gwas meta-analysis based on more than , patients and , controls [ ] . similar to lrrk and snca, some mutations in vps c and gch are likely causal or confer high risk for pd [ ] different study design such as patient-customized therapy. a recent report of patient-customized intervention in a child with neuronal ceroid lipofuscinosis (cln , a form of batten's disease) demonstrates the potential of this type of study [ ] . among all of these rare pd mutations, mutations in snca and parkin may be more common. indeed, if a sufficient amount of snca mutation carriers are identified, there may be promising interventions in this patient population group including silencers of the snca gene (e.g., antisense oligonucleotides (asos), viralmediated delivery of sirna, and mirna. of these, asos have recently shown favorable results in patients with spinal muscular atrophy [ ] and huntington's disease [ ] . as described below, these treatments may also be useful for a broader range of pd patients. group c includes variants which convey a very low increased risk for pd (shown in green on the right in fig. ), but may be present in a large percentage of people with pd. these variants may be common enough that intervention on the biological pathway of one of these variants may not require pre-screening clinical trial candidates, but instead result in stratifying the participants based on the presence of the variant. for example, a major pathway highlighted by gwas analysis in large pd cohorts is the adaptive immune system. this was first identified through a human leukocyte antigen association but is now increasingly recognized as a major determinant of risk across the genome [ ] . we are not aware of any current clinical trials that focus on a group c gene for pd. however, asos (developed by ionis) targeting tau (mapt) are being studied in a phase i clinical trial for alzheimer's disease. in addition, retroactive analyses of published clinical trials may identify responders versus non-responders based on variant status. it is possible that unsuccessful clinical trials for disease-modifying agents would have been successful in a select group of people with pd. group b includes variants that are shown in the middle of fig. . they are deleterious enough (associated with a high enough risk for pd) to warrant an intervention, but also common enough to make clinical trials targeting mutation carriers with a reasonably high number of participants feasible. currently, this group includes variants in gba and lrrk . given tremendous efforts to identify research cohorts, (e.g., by the michael j. fox foundation-funded ppmi studies) our knowledge of the pd phenotype and the risk for pd among these mutation carriers has improved [ , ] . the differences among the groups (a/b/c) are derived from the frequency of the variant in the population (x-axis), and the risk for pd associated with the variant (y-axis). the relationship between frequency and pd risk can guide the study design and who will benefit from particular interventions. specifically, mutations in group a are rare, and interventions on these targets may not benefit all pd cases. one example of this is homozygous carriers of prkn mutations. we are not aware of such studies in group a in pd but an example from the dementia field would be for intracisternal pr administration of the progranulin protein (pgrn) in carriers of the progranulin gene mutation (https:// clinicaltrials.ucsf.edu/trial/nct ). this type of intervention would not be for all patients with dementia but would only include mutation carriers. the study design would have to include genotype as an inclusion criterion and a large number of pd cases would have to be genotyped to identify a small number of participants. variants in group c are common and intervention on them may be beneficial for all pd cases. an example would be interventions on alpha-synuclein (αsyn) in pd. these studies do not require prescreening for genetic variants prior to study participation. discovered in , snca, which encodes the protein product α-syn, was the first cloned gene for pd. soon after, it was discovered that α-syn is a key component of lewy bodies which are considered to be a pathological hallmark of pd. therefore, the link between α-syn and pd seems clear and extends beyond the genetic cases caused by snca mutations. the physiological state of α-syn is an intrinsically disordered monomer or helically folded tetramere that has toxic effects in its oligomeric form. the protein is degraded mainly by the autophagic system. pathogenic missense mutations (a t [ ] , a p [ ] , e k [ ] and h q [ ] and g d [ ] [park ] which leads to misfolding and promotes apoptosis) and changes in gene dosage (duplications, triplications [park ] which leads to an excess and aggregation of αsyn) are associated with pd (fig. ) . the prevailing hypothesis is that excess α-syn is pathogenic and that mutations in the snca gene lead to gain-of-function. indeed, the clinical phenotype of mutation carriers has been associated with earlyonset rapid motor progression and frequent dementia; the phenotype may also overlap with multiple system atrophy (msa) or dementia with lewy bodies (dlb). following the dosage effect, the disease may be more rapidly progressive in triplication carriers versus duplication carriers. multiple clinical trials are targeting alpha-synuclein. while snca mutation carriers would naturally benefit from such interventions, given the difficulty recruiting large enough cohorts of these carriers, none of the trials focuses solely on this population. since α-syn deposits are universally present in pd and msa, the trials include both pd and msa patients. while a very attractive drug target, one of the challenges of the field is that there are no α-syn tracers available. developing α-syn tracers would help advance the field as they may serve as biomarkers, specifically for target engagement. multiple mechanisms of actions are currently being studied for α-syn, including immunotherapies, gene therapies, and small molecules (table ) . immunotherapies include active immunization (i.e., a vaccination which triggers the immune system to generate an immune reaction, including antibodies, against α-syn), and passive immunization (i.e., administration of antibodies directed against different domains of α-syn). since it is estimated that antibodies would be most effective against extracellular proteins, these immunotherapies are designed to reduce the load of extracellular α-syn, stop the spread of α-syn pathology in the brain, and/or neutralize its toxic effects. while active vaccinations are generally associated with a higher potential risk of side effects, so far no relevant safety and tolerability issues have occurred in the current α-syn studies. however, active vaccination strategies (such as affitope) have only been tested in phase human trials. of the passive vaccinations, two compounds have reached the clinical phase study level and are currently tested in patients with early pd: prx (pasadena study, sponsored by prothena) and biib (spark study, sponsored by biogen). the first antibody, prasinezumab (prx / ro ), binds to the c-terminus of α-syn. a study in healthy volunteers [ ] and in mild-to-moderate pd patients [ ] demonstrated a favorable safety and tolerability profile which led to the international pasadena study. a total of centers in the usa and europe (i.e., austria france, germany, and spain) participate in this -week trial. completion is estimated for early . the second α-syn antibody, biib , binds to the nterminus of α-syn, to residues - [ ] . biib is highly selective for aggregated forms of α-syn with at least an fold higher apparent affinity for fibrillar versus monomeric recombinant α-syn and it has a strong preference for human pd brain tissue. efficacy studies in three different mouse models with intracerebrally inoculated preformed α-syn fibrils showed that biib treatment attenuated the spreading of α-syn pathology, rescued motor impairment, and reduced the loss of dopamine transporter density in dopaminergic terminals in the striatum. this led biogen to initiate the international spark study which consists of monthly iv infusions (at mg, mg, or mg) over weeks. another [ ] compound in the pipeline for passive immunization is medi [ ] (phase ). another approach that takes a different route of action is small molecules which penetrate the blood-brain barrier and modify α-syn biology. these include inhibition of α-syn misfolding and aggregation and reduction of cortical pathology (npt - [ ] , currently in phase ). another small molecule mechanism utilized is targeting misfolded or aggregated proteins (npt , a second-generation immunoglobulin-general amyloid interaction motif (ig-gaim) molecule, which is being developed by the company neurophage). in the case of an α-syn overexpression-based pd rat model, vh *pest (a nanobody which targets the non-amyloid component region) showed maintenance of striatal dopaminergic tone and modest behavioral improvement [ ] . nanobodies are genetically engineered fragments of antibodies which can modulate monomeric concentrations of target proteins. another potential strategy is gene therapy, namely the crispr-dcas epigenome-editing approach which manipulates endogenous snca levels. researchers at duke u n i v e r s i t y ( n o w f u r t h e r d e v e l o p e d b y s e e l o s therapeutics) designed an all-in-one lentiviral which harbors grna, dcas nuclease, and the catalytic domain dnmt a to target specific hypomethylated cpg islands in the snca intron region. this led to the downregulation of snca mrna and protein levels and phenotypic perturbations in pluripotent stem cells of a snca triplication carrier [ ] . given pd's shared pathophysiological basis with msa (i.e., both are synucleinopathies), it will be interesting to follow current developments in msa as treatments may also be effective in pd. α-syn aggregation inhibitors (e.g., anle b, clr , pbt- , and vx- ), passive immunization (rec ), and α-syn degradation enhancers (e.g., rapamycin/ sirolimus) have been investigated for msa; for a review, see heras-garvin and stefanova [ ] . more than lrrk cases have been reported in the literature. the international lrrk consortium study estimated that the most common mutation in lrrk , g s, accounts for % of sporadic and % of familial pd patients [ ] . mutations in lrrk are more common in certain ethnicities. north african arabs ( % in familial, % in sporadic) and ashkenazi jews ( % in familial, % in sporadic) have the highest frequencies [ ] . the g s mutation is common in whites, while g r and r p are the most frequent variants in asian patients (detected in around - % of asian pd patients) [ ] . penetrance of lrrk is age dependent and estimations in the general population are variable, ranging between % and % [ ] . there are multiple reasons for this wide range of penetrance estimates, including differences in study design, the investigation of different populations, and the heterogeneity of lrrk presentation. regardless, the wide range of penetrance estimation makes trial design for preventative interventions extremely difficult. while the mechanisms by which lrrk mutations cause pd have not been completely disentangled, there is increasing evidence of increased lrrk kinase function in mutation carriers with pd. the g s mutation, for example, results in a direct two-to-threefold increase in kinase activity [ , ] .the potential gain-of-function effect is an attractive target for treatment because inhibition is easier to achieve than improvement of reduced protein activity (as in gba). furthermore, kinase inhibitors are widely used in oncology and the pd field can benefit from advancements in other fields. since the first generation of lrrk inhibitors, newer compounds have progressively improved in potency, selectivity, and brain penetrance. however, efficacy and safety remain a concern. this is mainly due to the fact that other tissues, particularly the kidney, lung, and a subtype of peripheral immune cells, robustly express lrrk . for example, the kidney has a~ . -fold higher expression of lrrk compared to the brain [ ] . the expression of lrrk in other tissues is a potential source of peripheral side effects, which can include the development of lamelar bodies and impaired autophagylysosomal function induced by lrrk inhibitors [ ] [ ] [ ] . more recent data, however, suggest compounds that only partially downregulate lrrk levels or kinase activity, i.e., by % or less, are unlikely to produce major side effects related to on-target safety [ ] and lipid droplets in lamelar bodies are absorbed after the drug is stopped. one alternative to avoid systemic toxicity is to find a way to specifically modify lrrk activity in the brain without modifying activity peripherally. new drug modalities that keep peripheral side effects to a minimum include protacs (proeolysis targeting chimera) which selectively induce intracellular proteolysis via the ubiquitinproteasome system of the mutant without affecting wt-lrrk [ ] . however, due to their molecular size, protacs show poor permeability across cell membranes which might affect brain penetration [ ] . another alternative to avoid systemic side effects is currently tested by biogen, who launched a phase antisense oligonucleotide (aso) trial. participants will receive a single intrathecal injection of the compound biib , an aso, on multiple days. recently, asos have been approved by the fda for spinal muscular atrophy and are being tested for huntington's disease and non-neurological disorders such as cancer [ , ] . asos reduce the expression of a mutated gene by binding to target mrnas and either block the translation of the abnormal protein or induce its degradation [ ] . asos can also promote splicing around mutations. for disorders caused by toxic gain-of-function mutations such as lrrk , further investigation regarding asos is warranted. in a preclinical study, administration of lrrk asos to the brains of mice reduced lrrk protein levels and fibril-induced α-syn inclusions [ ] ; data from humans are not yet available. several structurally different lrrk inhibitors from genentech, gsk, merck, and pfizer are in the pipeline ( table ) [ ] . the compounds developed by denali are already in clinical trials and a phase b trial in healthy individuals has been completed which included pulmonary and renal safety parameters. the company is advancing dnl (gne- ) to a phase b safety and biomarker study in lrrk -linked pd and ipd (idiopathic pd). twenty-nine mild to moderately affected pd patients with or without lrrk mutation were randomized to either low dose dnl , high dose dnl , or placebo in this day randomized placebo-controlled trial. in january , denali announced that both doses achieved more than % inhibition of ps lrrk and prab phosphorylation in blood, and improved the lysosomal biomarker bmp in urine (by % or % at the low and high dose) (https:// denalitherapeutics.gcs-web.com/node/ /pdf [online]). the same company also is testing another compound, dnl . in a trial in healthy volunteers, safety and biomarker goals were met (https://denalitherapeutics.gcsweb.com/node/ /pdf [online]). the compound is now being tested in pd patients with recruitment in belgium, the netherlands, and the uk; however, the trial is delayed because of the covid- pandemic. according to the press release, denali intends to select either dnl or dnl in mid- to advance into phase / clinical trials in patients with parkinson's disease (https:// denalitherapeutics.gcs-web.com/node/ /pdf [online]). to facilitate recruitment, a "direct-to-consumer" approach for testing and counseling will be available [ ] -a strategy that proved successful in genetic testing with the ppmi initiative. most recently, denali has announced a strategic collaboration with centogene, a gene diagnostic lab, to enhance recruitment [ ] . pharmaceutical companies and academic centers are also conducting observational studies to identify best endpoints related to the lrrk mutation, e.g. typical neurocognitive abnormalities (https://clinicaltrials.gov/ct /show/ nct ?term=lrrk &draw= &rank= ). interestingly, a different level of benefit was noted after deep brain stimulation surgery in lrrk -associated pd. the outcome was better in patients with the most common lrrk mutation, p.g s, compared to the poor response seen in patients with the rarer p.r g mutation. however, overall numbers remain small. the overall benefit was compromised by the rapid progression of cognitive and neuropsychiatric symptoms [ ] . clinical trials that target the glucocerebrosidase (gba) pathway are probably in the most advanced stages towards precision medicine in pd. this is a result of the relatively high frequency of gba mutations in pd and the availability of basic science data collected about gba mutations from gaucher disease research. gba mutations are a common risk factor for pd and are present in - % of pd patients worldwide. among ashkenazi jews, around % of pd patients carry a gba mutation [ ] . a high prevalence has also been reported in the netherlands, where . % of pd patients carry a gba mutation [ ] . homozygous gba mutations cause gaucher disease (gd), the most common autosomal recessive lysosomal storage disease, with an estimated annual incidence of / , and an estimated carrier frequency [ ] of . to . % in the general population. the clinical presentation of gd can be divided into systemic and neurological manifestations. the former, present in all forms of gd, includes hepatosplenomegaly, painful skeletal disorders, and pancytopenia. the latter is present in the more severe types of gd, gd-type , and gd-type [ ] . more than mutations in gba have been reported [ ] , and they are traditionally divided based on the gd phenotype they are associated with. mild mutations (e.g., the n s mutation) are associated with type gd, while severe mutations (e.g., the l p mutation) are associated with type or type gd. some ethnicities show higher mutation rates; phase b phase b n/a n/a n/a phase design multicenter, randomized, placebo-controlled multicenter n/a n/a n/a international, multicenter, placebo-controlled total n of patients n/a n/a n/a lrrk -pd √ √ n/a n/a n/a √ √ √ n/a n/a n/a √ age - n/a n/a n/a - duration days days n/a n/a n/a n.d. three doses n/a n/a n/a single-and multiple-ascending-dose specifically, in the ashkenazi jewish (aj) population, the annual incidence is / and carrier frequencies are as high as % in all aj. both gd patients and healthy heterozygous carriers are at increased risk for pd [ ] . about % of gba mutation carriers will develop pd and studies suggest that penetrance is age dependent [ ] . as in lrrk , there is incomplete penetrance of pd among carriers and a wide range of penetrance estimates. parkinsonism in gba heterozygotes may be indistinguishable from ipd. however, they may have an earlier age at onset, more prevalent cognitive impairment, and may not respond to levodopa as well as ipd patients [ , ] . gba mutations are also associated with other alpha-synucleinopathies, including dementia with lewy bodies [ ] (pathologically confirmed) and, in some studies, with msa [ ] [ ] [ ] [ ] [ ] . in contrast, there is no association with essential tremor or alzheimer's disease. pathologically, the brains of patients with heterozygous gba mutations strongly resemble those from patients with ipd. however, there is also widespread cortical lewy body involvement in gba mutation carriers [ , ] . a few studies showed a reciprocal relationship between levels of glucocerebrosidase (gcase; the enzyme encoded by gba) and levels of the aggregate-prone protein α-syn [ ] . notably, ipd patients also have reduced gcase activity (about % decrease) in the substantia nigra and cerebellum, making treatments that target gba relevant for ipd and patients with pd dementia as well [ ] . the gba-encoded protein, glucocerebrosidase, is a lysosomal enzyme which plays a role in the breakdown of glucocerebroside and glucosylsphingosine into glucose and ceramide or sphingosine respectively. in gd, there is a lysosomal buildup of the substrate glucocerebroside in the reticulo-endothelial system with reduced clearance. gaucher disease manifestations are a result of this diminished glucocerebrosidase (gcase) activity. fda-approved interventions for gd include enzyme replacement therapy (ert) and substrate reduction therapy (srt), which are inhibitors of the glucosylceramide synthase enzyme. none of the currently approved drugs penetrate the blood-brain barrier, but theoretically similar interventions that penetrate the blood-brain barrier may modify the pd phenotype. while the pd field can benefit from decades of gd research, the underlying mechanisms of how gba leads to the development of pd are not fully understood. one hypothesis is that there is a self-propagating bidirectional feedback loop between gcase and α-syn. loss of gcase activity causes αsyn accumulation and oligomerization, resulting in neurotoxicity through aggregate-dependent mechanisms [ ] . furthermore, elevated α-syn inhibits lysosomal maturation and normal gcase activity while hindering gcase transport from the endoplasmic reticulum to the lysosome. all of these mechanisms continue over time until the threshold for neurodegeneration is reached [ ] . based on this, targeted treatments can take a variety of approaches including modulation of glycosphingolipid turnover and restoration of enzyme function (fig. , table ). substrate reduction therapy inhibits the biosynthesis of lipid substrates and thereby prevents their accumulation. while this approach does not target the mutant gene or dysfunctional enzyme itself, it is an effective fda-approved treatment of the systemic symptoms of gaucher disease. however, the approved inhibitors show no effective cns concentration and do not affect the neurological symptoms of gaucher disease (i.e., gaucher type and ). new glucosylceramide synthase inhibitors show good brain penetration, improved α-syn processing, and behavioral outcomes in mouse models [ , ] . based on these findings, sanofi launched the moves-pd study, a randomized, double-blind, placebo-controlled trial, in order to assess the efficacy and safety of the glucosylceramide synthase inhibitor venglustat (gz/ sar ). initial results from a phase i study were recently published [ ] . briefly, gba-pd were enrolled ( on venglustat, on placebo; mean age years, mean disease duration years) into this -week randomized, placebo-controlled, double-blind, sequential cohort study of once-daily venglustat at three escalating doses. no serious adverse events occurred. side effects included psychological, neurological, and gastrointestinal-related symptoms. plasma and cerebrospinal fluid (csf) glucosylceramide levels decreased in a dose-dependent manner (up to %), confirming target engagement. this favorable safety and tolerability profile of venglustat at all doses led the company to advance to a phase ii study (a -week trial which is currently ongoing) [ ] . other therapies focus on the restoration of enzyme function, thus increasing glucocerebrosidase activity, especially in the brain. this can be achieved by [ ) ] ert (enzyme replacement therapy), [ ) ] gene therapy, or [ ) ] small molecules. ert is currently available for patients with gaucher disease. however, ert is based on large molecules and we are not familiar with any clinical trials of ert that may penetrate the blood-brain barrier. furthermore, there are no data on the use of ert in pd. gene therapy uses adeno-associated virus vectors to deliver engineered dna to human cells [ , ] . for gba, preclinical studies in mice demonstrated that adeno-associated virus-mediated expression of glucocerebrosidase corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau, and α-syn aggregates [ ] . prevail therapeutics, a new company launched in , started a gene therapy clinical trial of pr in gba-pd patients in late (https://ir.prevailtherapeutics.com/newsreleases/news-release-details/prevail-therapeutics-providesprogram-update-pr -parkinsons- ). the company will also test the compound in children with neuropathic gaucher disease starting in the first half of . furthermore, small molecules have also attracted attention [ ] . early glucocerebrosidase chaperones that underwent clinical trials for gaucher disease included isofagomine (afegostat-tartrate, at ). this treatment did not lead to significant clinical improvement and further clinical development for this indication was discontinued [ ] . ambroxol, which is a promising small molecule chaperone widely used in europe as a mucolytic agent, may potentially facilitate the transit of the misfolded gcase protein to the lysosome [ ] . its stabilizing and enhancing effects on the levels of gcase were identified in a high-throughput screen in the context of gaucher disease [ ] . it is ph dependent, binds to the active site of the gcase protein, and reduces gcase activity. ambroxol has also been shown to improve lysosomal function and increase enzyme activity in in vitro studies utilizing dermal fibroblasts with gba mutations (i.e. [ ] as well as in studies performed on non-human primates (i.e. cynomolgus monkeys) with gba mutations [ ] ). the effects of ambroxol at high doses were recently tested in a single-center, openlabel, non-controlled, clinical trial (escalating oral dose to . g per day [ mg times per day] which is considerably higher compared to the - mg/day used for the treatment of respiratory disease) [ ] . eighteen patients with moderately severe pd (mean age years) completed the study, eight with gba mutations and ten mutation-negative. ambroxol penetrated csf, but led to a % reduction from the mean baseline csf gcase activity at day . there was an increase of csf α-synuclein which the authors interpreted as an increase of extracellular export of the protein from the brain parenchyma. the drug was well tolerated, and no serious adverse events were reported [ ] . for other indications, ambroxol has been studied in > , patients in more than trials with an excellent safety record [ ] . as mentioned above, gcase activity is also reduced in ipd patients' brains (sn) [ ] , making the therapy potentially relevant for ipd. the effect of ambroxol in non-gba pd [ ] and non-gba pd dementia [ ] will be better understood once results from the two ongoing studies become available. these studies include ten non-gba pd and pd dementia patients [ , ] (treated at daily doses of mg/day or - mg/day, respectively). the effects of lti- , an activator of the gcase enzyme and another small molecule therapy, were studied in a month phase b trial conducted in the netherlands, where the frequency of gba mutations was reported to be around % [ ] . around gba-pd patients participated. there were no safety events and data showed a good dosedependent brain penetration (personal communication). the company, lysosomal therapeutics, inc. (lti), is a massachusetts-based biotech venture which plans to develop therapies for gaucher disease and other lysosome-based neurodegenerative diseases. small molecules can also modify gba-pd by modifying gba-independent pathways. one such example is rtb , which is an inhibitor of the target of rapamycin complex (torc ) [ ] . rapamycin reached public attention when bloomberg magazine publicized it as a potential "forever pill" in . known for its immunosuppressant properties, rapamycin prolongs lifespan by - % in various nonmammalian organisms even when given late in life; it has also been found to increase healthspan. a -year study in dogs is planning on testing the geroprotective effects of rtb in mammals [ ] . torc plays a role in cell growth and aging and is the switch between fasting and feeding states [ ] . mutations in torc cause focal cortical dysplasia, an established cause of epilepsy. the role of mtorc in regulating autophagy may also have implications for neurodegenerative diseases. in preclinical models, torc inhibition induces autophagy and prevents neuronal loss [ ] . it improves motor function in multiple pd models including α-syn transgenic mice and mptp models [ ] . in oncology cell cultures, treatment with rtb reduced the levels of glucosylceramide, the main substrate of gcase. there is a current phase b/ a trial of rtb in combination with sirolimus that involves pd patients with or without a gba mutation. the study was initiated in early ; data is expected in . outcome data after dbs are available for around gba-pd patients, % of whom showed a favorable response, whereas % showed a poor response [ ] . however, sample sizes remain small and genetic testing does not yet provide added prognostic value over what can already be obtained through careful clinical assessment when the patient is evaluated for dbs surgery [ ] . the second major pathway implicated in pd pathogenesis is mitochondrial metabolism, which is susceptible to damage by oxidative stress. a prevailing hypothesis is that mutations in prkn and pink cause pd due to mitochondrial dysfunction. clinically, people with prkn pd and pink pd display an earlier-onset, are sensitive to levodopa, and are prone to motor fluctuations. and, these patients have minimal cognitive decline, even after many years of disease [ ] . hence, antioxidative strategies targeting parkin (encoded by prkn) and pink -mediated mitophagy are another emerging therapeutic approach for new treatments of pd. several small molecule drugs that enhance mitophagy are in preclinical development and exploratory biomarkers for mitochondrial dysfunction and mitophagy activation are being tested. however, there are many aspects that need to be considered such as selecting the most effective and feasible targets in the mitophagy pathway, potential side effects, and stratification of the ideal population (for recent review, see miller and muquit [ ] ). it is important to note that several compounds targeting mitochondria have been systematically studied in pd, including coenzyme q and creatine. however, these trials failed to demonstrate a disease-modifying benefit in patients with pd. of these, coenzyme q is currently revisited in a "smaller, smarter trial" where patients are recruited based on their genetic risk profile [ ] . hence, patients will be stratified according to their risk for mitochondrial dysfunction (i.e., biallelic or monoallelic mutation in prkn or pink vs. patients in whom omics suggest little or minimal mitochondrial dysfunction). surprisingly, the major challenge towards precision medicine clinical trials for pd is not in the basic understanding of the genes and their function, but rather in identifying mutation carriers for clinical trials (table ). there are many ways studies are trying to overcome this difficulty. in the case of interventions targeting α-syn, the challenge in identifying carriers of point mutations or copy number variants has led to clinical trials targeting patients with sporadic pd. in the case of gba and lrrk , it is less clear whether interventions on these pathways would help patients with sporadic pd. given the relative rarity of mutation carriers, gba and lrrk trials may include patients in different stages of pd, as opposed to other clinical trials of disease modification which recruit patients in early pd who are often medication naïve. the inclusion of patients at different stages of pd requires the development of more sophisticated outcome measures that are less sensitive to symptomatic, dopaminergic therapy than the standard motor examination, the unified parkinson's disease rating scale (updrs). the feasibility of genetically-targeted clinical trials would require a significant change in the way genotyping is currently practiced in pd. only a small fraction of pd patients are clinically genotyped in clia-approved laboratories and know their genetic status [ ] . a few pharmaceutical companies launched major genotyping efforts, e.g., denali therapeutics in collaboration with centogene (https://www.centogene. com/company/article/centogene-and-denali-therapeutics- announce-strategic-collaboration-to-recruit-lrrk -patients-forcli.html [online]). in this context, the parkinson's foundation launched the pd generation initiative (currently conducted at sites in the usa), which offers free genetic testing for clinically relevant pd-related genes and free genetic counseling to help participants better understand their results. more about current efforts in "overcoming barriers to pd trial participation" are discussed in chapter of this special issue. another major challenge is the identification of reliable and validated disease markers and outcome measurements that are suitable for targeted intervention. past trials largely relied on clinical motor impressions (e.g. updrs in the "off" state during clinic visits) which may not robustly detect diseasemodifying effects. better biomarkers are needed to evaluate the effectiveness of treatments by reflecting drug target engagement, disease severity, and progression of disease. having a reliable marker would enhance drug development. for example, in the case of huntington's disease, trials now include femtomolar-sensitive quantification (single molecule counting immunoassay) of the mutant protein in csf [ ] . for pd, real-time quaking-induced conversion (rt-quic) techniques are being tested to determine their sensitivity and specificity [ ] . there is also still a lack of reliable αsyn imaging methods which is a barrier to testing promising compounds; however, targeting a-syn is a major pursuit on the research front [ , ] . using artificial intelligence may advance the field further by identifying new biomarkers or facilitating biomarker validation and application. furthermore, better understanding of disease mechanisms linking these genes to pd is necessary. in lrrk and snca, while detailed mechanisms are not fully understood, treatments are focused on reducing activity or expression. in the case of gba, as outlined above, enhancing or restoring enzyme function are currently favored approaches. drug development for other genetic forms lags behind (e.g., prkn), because suitable druggable targets have not been identified. concerted efforts are being made to identify a variety of disease progression markers including biospecimen-based (i.e., blood, urine, csf or biopsy; molecular, genomic, cellular), clinical, imaging, or others (e.g., electrophyiological, behavioral, and environmental). among these, the ppmi initiative is a valuable source that brings together longitudinal data and specimen collection from more than volunteers with pd [ ] . recent failures in large phase iii clinical trials for pd suggest that disease modification will not succeed as long as pd is treated as a single disease with one pathophysiology. therefore, we believe that precision medicine in pd may be a promising alternative and a timely approach. as summarized in this review, several gene-based therapies are being tested in clinical trials and numerous more are in the pipeline. these are exciting times. however, the process of bringing a drug into the clinic is cumbersome [ ] . pharmaceutical research and manufacturers of america (phrma) estimate that for every to , compounds screened, only five enter human clinical trials, only one is approved by the food and drug administration, and only two in ten drugs generate enough revenue to recoup their research and development costs [ ] . thus, setbacks are expected. one unanswered question is who will benefit from new "precision" drugs. will these be useful only for mutation carriers (and therefore require an orphan drug assignment) or will they be beneficial for the larger group of idiopathic pd or atypical parkinsonian disorders? it seems unlikely that these broad groups will all respond to the same drug unless there are shared downstream disease mechanisms. one may even have to differentiate among mutations within gba due to the effect a specific mutation has on the protein. for example, the affinity of chaperones to a mutated enzyme may be different depending on the mutation. furthermore, a new drug that facilitates protein function may fail in patients with null mutations who do not express any protein. the term "super precision medicine" has been used to capture this phenomenon. to be able to make the most out of clinical trial data, future clinical trials will benefit from pretrial genetic adjustment or, at the minimum, post-trial adjustment, and analysis for failed trials [ ] . however, in addition to addressing the purely scientific aspects, success in drug development also depends on funding opportunities for late preclinical development phases which are significantly more expensive than early-stage discovery. experience shows that promising ideas have often failed in this "valley of death" (a term coined by former nih director, elias zerhouni) [ ] . feasible regulatory frameworks and efficient data-sharing ecosystems [ ] that also ensure ethical leadership and governance [ ] as well as new funding instruments (for example, similar to the recently initated publicprivate partnership set up to boost research and drug development in infectious diseases and thereby to address market failures [ ] ) will facilitate drug development. raising awareness and educating the community, including physicians, patients, and caregivers, will be an important step towards successful future studies [ ] . advancing precision medicine will not only shift current reactive approaches to proactive detection and prevention, but will also help the next generation of scientists develop creative new approaches for detecting, measuring, and treating parkinson's disease. required author forms disclosure forms provided by the authors are available with the online version of this artic funding s. schneider was supported by lmu clinician scientist programme, the ara parseghian medical research fund, and the verum stiftung. r. alcalay research is funded by the nih, dod, parkinson's foundation, and the micheal j. fox foundation. he received consultation fees from sanofi, roche, janssen, and restorbio. conflict of interest dr. alcalay reports receiving consultation fees from genzyme/sanofi, roche, janssen, and restorbio. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not 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her careful work editing the manuscript. key: cord- -gdplbrsf authors: arget, michael; kosar, justin; suen, brandon; peermohamed, shaqil title: successful treatment of legionnaires’ disease with tigecycline in an immunocompromised man with a legion of antibiotic allergies date: - - journal: cureus doi: . /cureus. sha: doc_id: cord_uid: gdplbrsf legionella species are gram-negative bacilli that are relatively rare causes of community-acquired pneumonia but can be associated with significant morbidity and mortality if unrecognized or improperly treated. limited data exist regarding the use of tigecycline, a third generation glycylcycline, in the treatment of legionnaires' disease. we present an immunocompromised patient with legionnaires' disease and allergies to both fluoroquinolones and macrolides, which are first-line treatment options for legionnaires' disease. he was successfully treated using tigecycline, a third generation glycylcycline, indicating that tigecycline may serve as a safe and effective alternative therapeuticl option for treatment of legionnaires’ disease. legionnaires' disease is a relatively rare cause of community-acquired pneumonia caused by legionella species; however, %- % of patients who are hospitalized with legionnaires' disease require invasive mechanical ventilation and average mortality rates for sporadic disease range from % to % [ ] . legionnaires' disease is caused by inhalation of legionella species, which are intracellular, gram-negative bacilli ubiquitously found in the environment [ ] [ ] . host risk factors for legionnaires' disease include male gender, age older than years, cigarette smoking, diabetes, end-stage renal failure, organ transplantation and immunosuppression, such as glucocorticoids or anti-rejection drugs following organ transplantation [ ] . travel is an important and underappreciated risk factor associated with legionellosis in a community setting [ ] . treatment options for legionnaires' disease include macrolides, fluoroquinolones, or tetracycline; however, preferred therapies for immunocompromised patients with legionnaires' disease include levofloxacin and azithromycin [ ] [ ] [ ] . we describe an immunocompromised and severely ill patient with legionnaires' disease and who also has allergies to both fluoroquinolones and macrolides; he was successfully treated using tigecycline, a third generation glycylcycline, indicating that tigecycline may serve as a safe and effective alternative therapeutic option for treatment of legionnaires' disease in select cases. a -year-old caucasian man presented to the emergency department in autumn with one week of dyspnea, productive cough, myalgia, and fever. he denied any chest pain or hemoptysis. his past medical history was significant for hypertension, diabetes mellitus, chronic kidney disease, and non-hodgkin's lymphoma with receipt of an allogeneic stem cell transplant years prior. given prior complications due to graft versus host disease, he was receiving prednisone at a maintenance dose of mg daily for several years. he had multiple documented allergies to penicillin, sulfa drugs, macrolides and fluoroquinolones, with reported reactions including rash, hives, and anaphylaxis. approximately one week prior to the onset of symptoms, he was traveling in the midwest united states with his partner and staying in various hotels. upon arrival to the hospital, he was noted to have a heart rate of beats per minute, a blood pressure of / mmhg, a respiratory rate of breaths per minute with an oxygen saturation of % requiring eight liters of supplementary oxygen, and an oral temperature of . °c ( . °f). he was in acute respiratory distress and had evident decreased breath sounds and crackles bilaterally. he was noted to have normal heart sounds without any murmurs, rubs, or gallops. he did not have any rash on examination. two sets of blood cultures were collected and sputum samples were sent for culture as well as stains and polymerase chain reaction (pcr) testing for pneumocystis jirovecii (p. jirovecii). a nasopharyngeal sample was collected for respiratory virus pcr testing for influenza a and b, respiratory syncytial virus a and b, coronavirus, parainfluenza virus, rhinovirus, enterovirus, adenovirus, bocavirus, and metapneumovirus. in addition, a serum cytomegalovirus (cmv) pcr and legionella urine antigen were sent. he was empirically started on meropenem, vancomycin, oseltamivir, and intravenous pentamidine. despite initiation of broad spectrum antimicrobials, he continued to deteriorate with increasing oxygen demands, persistent fever, hemodynamic instability, and worsening radiographic infiltrates ( figure ). showing worsening bilateral pulmonary opacification (arrows). sputum and blood cultures were negative for any bacterial growth; sputum stains and pcr were negative for p. jirovecii. his serum cmv pcr was negative. his nasopharyngeal swab for respiratory virus testing was negative for influenza a and b, respiratory syncytial virus a and b, coronavirus, parainfluenza virus, rhinovirus, enterovirus, adenovirus, bocavirus, and metapneumovirus; however, his legionella urine antigen was positive. according to his pharmacy records, he had previously received and tolerated a five-day course of moxifloxacin approximately one year prior to this hospital admission. given his diagnosis of legionnaires' disease, intravenous moxifloxacin therapy was initiated. however, shortly following receipt of his first dose of moxifloxacin, he developed an allergic reaction with a generalized, erythematous, maculopapular rash and angioedema, necessitating administration of epinephrine. given his documented allergies to both fluoroquinolones and macrolides, hemodynamic instability, and concern for poor gastrointestinal absorption of oral antimicrobials, he was subsequently treated with intravenous tigecycline with an initial, loading dose of mg, followed by mg twice daily for a total of days of therapy. intravenous doxycycline is not readily available in our institution. his oxygen requirements decreased and fever resolved following hours of treatment with tigecycline. all other antimicrobials were discontinued once the diagnosis of legionnaires' disease was made. there was no recurrence of infection after three months of follow-up; his repeat chest radiograph showed resolution of his bilateral air space opacities. later in discussion with the north dakota department of health and centers for disease control and prevention, it was determined that there was an ongoing outbreak of legionnaires' disease associated with five cases over a -month period; all cases including our patient had stayed at the same hotel. subsequent environmental testing of the hotel was negative, but this may have been impacted by a recent deep clean of the hotel's ventilation system. legionnaires' disease and its causative pathogen were first recognized in , following a common-source outbreak of severe pneumonia involving people at an american legion convention in philadelphia, pennsylvania in [ ] . outbreaks and clusters of cases of legionnaires' disease have been associated with contaminated cooling towers, whirlpools, hospital decorative water fountains, hot spring spas, and water births [ ] [ ] . legionnaires' disease can be associated with a prodromal illness with symptoms including fever, headache, myalgia, and anorexia; however, the clinical presentation of legionnaires' disease is often nonspecific and difficult to distinguish from other causes of communityacquired pneumonia [ ] [ ] . blood and sputum cultures are relatively insensitive in diagnosing legionnaires' disease; in contrast, urine antigen testing has a sensitivity of %- % and specificity greater than % [ ] . urine antigen testing for legionella only detects legionella pneumophila serogroup (lp ) and is most sensitive for the detection of the pontiac subtype of lp , which causes the majority of cases of community-acquired legionnaires' disease [ ] . legionella urine antigen testing will often be positive on the first day of illness and remain positive for several weeks [ ] . molecular testing of lower respiratory tract specimens can also be used to identify both legionella pneumophila and legionella species by pcr [ ] . preferred therapies for immunocompromised patients with legionnaires' disease include levofloxacin and azithromycin [ ] [ ] [ ] . tigecycline is a third generation, intravenous glycylcycline and minocycline derivative that inhibits bacterial protein synthesis by binding to bacterial s ribosomal subunits [ ] . prior in vitro and animal model studies have shown that tigecycline achieves high intracellular concentrations [ ] . however, demonstrated clinical effectiveness of tigecycline in the treatment of community-acquired pneumonia in humans with legionnaires' disease remains limited. two prior case reports describe successful use of tigecycline in the treatment of immunocompromised patients with legionellosis; however, fluoroquinolones were used as initial therapy in both of these cases and tigecycline was later added to their antimicrobial regimen [ ] [ ] . a recently published case series describes eight patients with legionnaires' disease who were switched to tigecycline, often due to worsening sepsis and/or respiratory status, following initial exposure to macrolide and/or fluoroquinolone therapy (median of three days) [ ] . all but one of these eight patients received combination therapy with tigecycline plus either levofloxacin or azithromycin as part of their treatment regimen once tigecycline was added. furthermore, the one patient in this case series who received days of tigecycline monotherapy had received eight days of azithromycin prior to switching therapy. thus, it is difficult to ascertain whether clinical improvement in these cases was due to the addition of tigecycline or post-antibiotic effect and delayed response from fluoroquinolone/macrolide therapy. integrated results from two randomized controlled trials showed comparable cure rates between tigecycline and levofloxacin in the treatment of hospitalized patients with community-acquired pneumonia, of which a small proportion were diagnosed with legionnaires' disease in each treatment arm [ ] . while the integrated results of these two randomized controlled trials support the early use of tigecycline as empiric treatment of community-acquired pneumonia, one of these trials permitted switching to oral levofloxacin following at least three days of intravenous therapy if evidence of clinical improvement. current evidence, albeit limited, suggests that tigecycline may be added as combination therapy in severe cases of legionnaires' disease. this case, however, demonstrates that tigecycline can be effective as a second-line treatment option for legionnaires' disease in the setting of allergies to traditional mainstays of therapy. in , the food and drug administration (fda) approved a new boxed warning about the higher risk of death among patients receiving tigecycline compared with other antibiotics, particularly apparent for hospital-acquired pneumonia and ventilator-associated pneumonia [ ] . while both the fda and health canada have approved tigecycline for treatment of community-acquired bacterial pneumonia, complicated skin and soft tissue infections, and complicated intra-abdominal infections, its use should be reserved for situations when alternative treatments are not suitable [ ] [ ] . legionnaires' disease is a rare cause of community-acquired pneumonia but can be associated with significant morbidity and mortality, especially amongst immunocompromised individuals. although the evidence regarding the use of tigecycline in treating legionnaires' disease is limited, this case report provides evidence supporting the use of tigecycline as a second-line therapeutic option in select cases where fluoroquinolone or macrolide therapy may be contraindicated. human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. legionnaires' disease and pontiac fever . mandell, douglas, and bennett's principles and practice of infectious diseases legionella and legionnaires' disease: years of investigation treatment strategies for legionella infection principles and practice of infectious diseases activities of tigecycline (gar- ) against legionella pneumophila in vitro and in guinea pigs with legionella pneumophila pneumonia a case of legionellesis pneumonia verified by isolation of legionella pneumophila serogroup from bronchoalveolar lavage fluid treated with levofloxacine and tigecycline disseminated legionella pneumophila infection in an immunocompromised patient with tigecycline tigecycline as a second-line agent for legionnaires' disease in severely ill patients. open forum infect dis integrated results of phase studies comparing tigecycline and levofloxacin in community-acquired pneumonia the role of tigecycline in the treatment of infections in light of the new black box warning epidemiologist with the north dakota department of health in providing details pertaining to this outbreak of legionellosis. key: cord- -r c authors: greco, s.; madè, a.; gaetano, c.; devaux, y.; emanueli, c.; martelli, f. title: noncoding rnas implication in cardiovascular diseases in the covid- era date: - - journal: j transl med doi: . /s - - - sha: doc_id: cord_uid: r c coronavirus disease (covid- ) is caused by the infection of the severe acute respiratory syndrome coronavirus (sars-cov- ). although the main clinical manifestations of covid- are respiratory, many patients also display acute myocardial injury and chronic damage to the cardiovascular system. understanding both direct and indirect damage caused to the heart and the vascular system by sars-cov- infection is necessary to identify optimal clinical care strategies. the homeostasis of the cardiovascular system requires a tight regulation of the gene expression, which is controlled by multiple types of rna molecules, including rna encoding proteins (messenger rnas) (mrnas) and those lacking protein-coding potential, the noncoding-rnas. in the last few years, dysregulation of noncoding-rnas has emerged as a crucial component in the pathophysiology of virtually all cardiovascular diseases. here we will discuss the potential role of noncoding rnas in covid- disease mechanisms and their possible use as biomarkers of clinical use. the novel human severe acute respiratory syndrome coronavirus (sars-cov- ), isolated on th january , has been identified as the cause of acute respiratory distress syndrome (ards) cases of unknown etiology detected in wuhan city, hubei province of china and then indicated as coronavirus disease (covid- ) [ ] . because of the rapid global spread of the covid- , on th march , the director-general of the world health organization (who) defined the disease as a pandemic [ ] and up to october th , there have been more than million confirmed cases and more than million deaths worldwide [ ] . the main clinical manifestations of covid- are respiratory. however, many patients also display a severe involvement of the cardiovascular system [ ] [ ] [ ] [ ] [ ] [ ] . thus, it is of paramount importance to understand the direct and indirect damage caused to the cardiovascular system by sars-cov- infection, as well as the underpinning pathogenetic mechanisms. here we will review the importance of transcriptomics techniques in our understanding of human coronavirus disease mechanisms in the cardiovascular system and for the identification of biomarkers of potential clinical use. specifically, we will focus on noncoding rnas (ncrnas), an emerging class of regulatory rnas [ ] . given their fundamental role in gene expression regulation, we propose ncrnas as promising candidates for understanding the consequences of sars-cov- infection on the cardiovascular system. threshold: small noncoding rnas (small ncrnas) and long noncoding rnas (lncrnas). the most studied small ncrnas are micrornas (mirnas) and long ncr-nas, which include also circular rnas (circrnas) [ , ] . micrornas (mirnas) are short noncoding rnas ( - nucleotides) that modulate gene expression by sequence-specific recognition of their target transcripts. most mirna genes are transcribed by rna polymerase ii from intergenic, intronic or polycistronic loci forming an intermediate hairpin of - nucleotide, named ''precursor mirna'' (pre-mirna) [ ] . the pre-mirna is then transported out of the nucleus and cleaved by the cytoplasmic rnase iii dicer into a short mirna duplex [ , ] . one strand of this duplex binds to the argonaute (ago) protein forming the rna-induced silencing complex (risc), which can recognize the target mrna by sequence complementarity [ , ] . this binding leads to the degradation and/or translational inhibition of the target mrnas. each mirna can have hundreds of rna targets and also a single mrna may have several mirna recognition sequences, generating complex regulatory networks [ ] . lncrnas are ncrnas regulating the transcription and translation of protein-coding gene expression [ ] [ ] [ ] [ ] . they have some similarities with coding genes, such as the presence of epigenetic marks, introns and the existence of splice variants, and the transcription driven by promoter elements. moreover, they can be either polyadenylated or non-polyadenylated [ ] . they may originate from either the sense or antisense dna strand, can overlap coding genes entirely or partly. they can be divided in sense, antisense, intronic, intergenic, and bidirectional lncrnas, enhancer-associated rnas (ernas), and promoter associated long rnas (palrs) [ ] . finally, there are circrnas, that are circularized rnas generated by back-splicing events of pre-mrnas [ ] . lncrnas can function as epigenetic regulators, can regulate the transcription rate by assembling transcriptional activators and repressors [ ] . some nuclear lncrnas have been implicated in maintaining nuclear structures, including interchromatin granules, nuclear speckles and paraspeckles [ ] . lncrnas can also regulate the gene expression by binding to mrnas and modulating their translation and/or stability [ , ] . despite their generally low-abundance, some lncrnas can accumulate because they high stability and, by sequestering mirnas, can function as competing endogenous rnas (cernas) [ , ] . sars-cov- belongs to the beta-coronavirus genus of the coronaviridae family; like the other members of the family, sars-cov- is enveloped and its genome is a single-stranded positive-sense rna of around kb [ ] . coronaviruses genome encodes for nonstructural proteins and for four structural proteins: spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins ( fig. ) [ ] . the s protein can recognize the receptor of the host cell and is responsible for cell membrane fusion [ ] , the n protein interacts with the viral rna to assemble the ribonucleoprotein [ ] , the e protein is necessary for virion assembly [ ] and the m protein has a pivotal role in virus assembly [ ] . all the members of the coronaviridae family seem to have the same replication mechanism: the genomic rna represents the template used to translate two partly overlapping open reading frames in two polyproteins, which encode the nonstructural proteins necessary to the replication-transcription complexes assembly in association with cytoplasmic membranes [ , ] . coronaviruses can infect many animal species, causing different symptoms, such as respiratory and intestinal diseases [ , ] . among human coronaviruses, severe acute respiratory syndrome-coronavirus (sars-cov) and middle east respiratory syndrome-coronavirus (mers-cov) are the best studied, due to the epidemics that originated in and , respectively [ ] . virology and genetic studies indicate that human coronaviruses have a reservoir in nature not corresponding with the intermediary host species, which is responsible for dissemination to humans [ ] [ ] [ ] . since sars-cov- genome is more than % identical to a bat coronavirus, it is very likely that bats may have been the initial zoonotic host [ ] . however, the intermediary species is unknown. the most common human-human transmission is through respiratory droplets generated by sneezing and coughing [ ] . genetic sequence analysis revealed that sars-cov- has . % nucleotide identity to sars-cov and . % identity to mers-cov [ , ] . comparing sars-cov- and sars-cov genomes, six regions of difference have been recognized (fig. ). based on proteomic comparison, sars-cov- proteins are highly homologous (about %- %) to the sars-cov virus proteins [ ] . the similarity of sars-cov and sars-cov- viruses is also confirmed by the fact that they both use angiotensinconverting enzyme (ace ) as cellular receptor, via the receptor-binding domain of surface spike glycoprotein s [ ] [ ] [ ] . for their similar structural and pathogenicity features, the better studied sars-cov and mers-cov constitute important models when developing hypothesis for sars-cov- disease mechanisms. patients with pre-existing cardiovascular diseases (cvds) have a higher risk of severe disease and death upon sars-cov- infection [ ] [ ] [ ] ] . it is plausible that covid- can affect the cardiovascular system at various levels. indeed, ace is expressed by a multitude of cell types, including cardiac and vascular cells [ ] , which may represent direct targets. accordingly, sars-cov- infection has been associated with multiple direct and indirect cardiovascular complications including arrhythmias as well as myocardial injury due to hypoxia, microvascular thrombosis and systemic cytokine release syndrome ("cytokine storm") [ , ] . indeed, the immune response is crucial for infection resolution, but this response can also result in immunopathogenesis. during the disease course, sars-cov viral loads were observed to decrease while disease severity increased, suggesting that immunopathogenesis may contribute to ards [ ] and may be an important cause of cardiovascular damage (fig. ) . moreover, antiviral therapies under investigation for covid- may damage the heart and have other cardiovascular side effects, such as arrhythmias and repolarization abnormalities [ ] . ace is an integral membrane carboxymonopeptidase that has been identified as a functional receptor for coronaviruses, including sars-cov and sars-cov- [ , [ ] [ ] [ ] . in this process, it is crucial the s-protein priming by the transmembrane serine protease tmprss [ ] . ace is expressed in alveolar epithelial cells, in line with the respiratory symptoms of covid- , but also in other epithelial and non-epithelial cells, in the kidneys and the gut. with relevance to the potential cardiac impact of covid- , ace is also expressed by cardiomyocytes, fibroblasts, pericytes, macrophages and the epicardial fat [ , [ ] [ ] [ ] . ace is an important member of the renin angiotensin system [ ] . as shown in fig. , ace activity is necessary to generate angiotensin - , which, via the activation of the g protein-coupled mas receptor, leads to vasodilatory, anti-hypertrophic, and anti-fibrotic effects. this represents an important mechanism to compensate the negative cardiovascular action of an excessive angiotensin ii stimulation on the angiotensin ii type receptor (at r) (fig. ) . in keeping with an important role of ace in the heart, ace knockout mice display impaired cardiac contractility and the upregulation of the hypoxia-associated gene program [ ] . in both the mouse model of acute myocardial infarction (ami) and in idiopathic and ischemic heart failure (hf) patients, increased expression of ace protein and mrna has been observed [ , ] . moreover, ace is a transmembrane protein, whose catalytic domain is located at the extracellular side of the cell and it can be released into the blood after cleavage by adam (also named tace). previous studies have reported increases in circulating ace , possibly due to augmented ace shedding, to be associated with cardiovascular risk factors, such as advanced age and diabetes mellitus [ ] , which are also risk factors for covid- [ , ] . given ace expression in the heart, it is conceivable that the sars-covs virus can have a direct effect the renin-angiotensin system. ace can cleave both angiotensin i and angiotensin ii to generate ang - and ang - , respectively. ang - and ang - , in turn, bind angiotensin ii type receptor (at r) and mas and have vasodilatory, pro-apoptotic, anti-fibrotic and anti-inflammatory effects. ace converts angiotensin i to angiotensin ii, which through the binding to at r has opposite effects, i.e. vasoconstriction, anti-apoptotic, pro-fibrotic and pro-inflammatory effects on cardiac function. accordingly, in several sars-cov patients, viral rna was detected not only in the lungs, but also in the myocardium [ , ] . increased ace expression in cardiovascular patients [ , ] may increase their susceptibility to sars-covs infection. however, in the heart of both sars-cov infected mice and of sars patients, decreased ace -levels were observed [ ] , suggesting a complex virus/receptor dynamic that needs to be elucidated. further complicating the scenario, ace levels can also be increased by inhibitors of the renin angiotensin system [ ] . however, while the impact of these drugs on covid- is still largely unknown, a retrospective study did not identify inhibitors of the renin angiotensin system as independent predictors of poor outcome [ , ] . different studies found that the values of cardiac troponins were increased in covid- patients with more severe disease [ , , [ ] [ ] [ ] , indicating an association of sars-cov- with myocardial damage. indeed, in a study surveying covid- hospitalized patients [ ] , myocardial injury identified by increased troponin t and n-terminal pro brain natriuretic peptide (nt-pro-bnp) levels was significantly associated with death, while the prognosis of patients with underlying cvds, but without myocardial injury, was less severe. moreover, cardiac injury was associated with cardiac dysfunction and arrhythmias. accordingly, there are several studies reporting a direct effect on heart function of coronaviruses with a pathogenicity similar to that of sars-cov- . in a rabbit model of coronavirus infection, viral-cardiomyopathy and progression into dilated cardiomyopathy (dcm) have been described [ ] . moreover, mers-cov infection has been associated with acute myocarditis and hf [ ] . interestingly, in the hearts of both sars-cov infected mice and sars patients, macrophage infiltration with evidence of myocardial damage was observed [ ] . a similar pattern with low-grade myocardial inflammation and viral particles in the heart has been reported in one covid- case [ ] . cardiomyocytes showed non-specific features such as focal myofibrillar lysis, and lipid droplets, but no viral particles were observed in myocytes and endothelia, suggesting infected macrophage migration from the lung or during a viraemic phase. other myocarditis single cases in which endomyocardial biopsy was performed scored negative for sars-cov- genome presence [ , ] . thus, further studies with higher numerosity are definitely needed to ascertain the nature of covid- myocarditis. li et al. [ ] reported in sars patients an impairment in left ventricular performance during the acute phase, but reversible on clinical recovery. this impairment was more severe in critically ill patients and elevated lactate dehydrogenase levels, reflecting the severity of tissue damage, were associated with decreased ejection fraction [ ] . while the expression of ace is well known in heart, it is controversial the expression of tmprss [ ] . these data draw the attention on the mechanism by which the virus could enter in the cardiomyocytes, and on the existence of secondary effects related to hypoxia and systemic inflammation during complicated covid- courses. indeed, the finding of a more prominent immune reaction in patients with more critical illness, and the association of cytokines as pro-inflammatory mediators in hf, support the hypothesis that the heart function impairment may be due to the cytokine storm in response to sars infection [ , ] . likewise, also in covid- patients, a cytokine storm triggered by the sars-cov infection may result in damage to myocardial cells. this strong inflammatory response may also confer risk for atherosclerotic plaque rupture in coronary artery disease patients, increasing the risk of acute coronary syndrome in more severely affected covid- patients, akin to what has been observed in influenza viral illness [ ] . also important in elevating the risk of cardiac injury is hypoxemia caused by respiratory dysfunction, higher risk of capillary embolism [ ] and the increased metabolic demands. moreover, many antiviral drugs can cause cardiac damage [ ] , further complicating the clinical situation. in hospitalized covid- patients, cardiac arrhythmia was present in more than % of the patients and was far more common in those presenting serious symptoms and requiring intensive care [ , ] . while myocarditis should be considered, high prevalence of arrhythmia might be also attributable to hypoxia, cytokine storm syndrome and metabolic disorder in the setting of viral infection, possibly in the presence of prior cvds. indeed, there are several possible pathophysiological causes of arrhythmias such as: ) the direct injury to cardiomyocytes that alters the membrane electrical conduction, ) massive edema, ) ischemia originating from microvascular disease following the infection, ) re-entrant arrhythmias caused by myocardial fibrosis or scars and ) proinflammatory cytokines storm. the first three events could occur in the acute setting, while the fourth and fifth are associated with chronic or healed myocarditis. moreover, the proinflammatory cytokines storm (e.g., il- ) might cause alteration in the desmosomal proteins of the cardiomyocyte membrane that could be arrhythmogenic [ , ] . there is increasing evidence that patients with cvds and/or diabetes mellitus are more likely to develop severe symptoms when affected by covid- [ - , , ] . among covid- patients with severe symptoms, many had hypertension, heart disease and arrhythmia. acute coronary syndrome seems to have a particularly poor prognosis in covid- patients. indeed, upon sars-cov- infection, cardiac insufficiency is more likely to occur in these patients where cardiac function is already compromised by myocardial ischemia [ - , , ] . moreover, sars-cov infection leads to long term disorders of lipid and glucose metabolism [ ] . considering the similarities between sars-cov and sars-cov- , this novel virus might also inflict a similar chronic damage. transcriptomics has been extensively used to understand cvd pathogenesis, to identify coding and ncrnas with a potential as therapeutic targets and clinically useful biomarkers. these studies have been extensively reviewed elsewhere [ , [ ] [ ] [ ] [ ] . here we will only give few examples that may be paradigmatic also in the investigation of the cardiovascular implications of covid- . transcriptomics first focused on mrnas expressed in the heart, identifying transcripts encoding mostly contractile sarcomeric proteins, cytoskeletal proteins, ion channels, intracellular signal transducers, including apoptosis and cell proliferation genes, and proteins maintaining the redox state of the myocardium [ , ] . transcriptomic profiling identified several mirnas deregulated in left ventricles (lv) of both dilated (dcm) and ischemic cardiomyopathy (icm) patients [ ] . additional studies allowed identification also of signatures specific to particular cardiomyopathies, such as dcm in patients with reduced catecholamine sensitivity [ ] , dcm in patients with a reverse-remodeling response following β-blocker treatment [ ] and icm in patients affected by type diabetes mellitus [ ] . mirna expression is also dysregulated in viral myocarditis [ ] [ ] [ ] . in particular, mir- - p is one of the mir-nas consistently modulated in both human and mouse viral myocarditis, contributing to myocardial damage through the modulation of monocyte-macrophages cardiac infiltration and t lymphocyte activation [ ] . genome-wide analyses of lv samples of hf patients identified a subset of lncrnas significantly deregulated compared with healthy controls; despite the limited evolutionary conservation of lncrnas, many of these findings were also confirmed in relevant mouse models of disease [ , [ ] [ ] [ ] [ ] [ ] . particularly relevant are circrnas originating from the multi-exon gene titin, that are dysregulated in both dilated and hypertrophic cardiomyopathies, and are regulated by the splicing factor rna binding motif protein (rbm ) [ ] . of great translational relevance is the fact that ncrnas are released into the blood where they are sufficiently stable to be readily measured with common laboratory techniques, such as qpcr, and that their concentration levels can differentiate diseased patients from healthy subjects. in addition, their measurement requires a minimally invasive procedure, thus representing an enormous reservoir for biomarkers discovery, for both diagnostic and prognostic applications. many genome-wide profiling studies of circulating ncrnas have been performed. among mirnas, heart and muscle-enriched mirnas, also named myomirs (mir- - p, mir- a- p, mir- b- p, mir- a/b- p, and mir- - p) seem to be particularly relevant [ ] [ ] [ ] [ ] [ ] [ ] . indeed, in patients with ami, myomirs have been reported to be elevated in plasma, likely due to cardiomyocyte necrosis and thus released into the circulating system [ , ] . accordingly, mir- a- p and mir- - p were also elevated in the plasma of viral-cardiomyopathy patients and their levels correlated positively with myocardial damage assessed by measuring troponin t levels [ ] . among myomirs, mir- is of particular interest, since its expression is highly cardiacspecific. moreover, voellenkle et al., analyzing the pattern of expression of peripheral blood mononuclear cells (pbmcs) identified a mirna signature characterizing dcm patients [ ] . also lncrnas can be found in the peripheral blood and hold a promising biomarker potential. the expression of lipcar (long intergenic noncoding rna predicting cardiac remodeling) in plasma has been found to be correlated to cardiac remodeling progression after ami [ , ] . other potential biomarkers are miat (myocardial infarction associated transcript), discriminating st-elevation from non st-elevation ami [ ] , sencr (smooth muscle and endothelial cell-enriched migration/ differentiation-associated long noncoding rna), associated to lv remodeling [ ] , as well as nron (noncoding repressor of nfat) and mhrt (myosin heavy chain associated rna transcripts), lncrnas elevated in hf patients and independent predictors of hf events [ , ] . greco et al. [ ] , analyzing icm patients found that anril, hotair, and loc /tusc had similar modulation in pbmc and heart tissue, suggesting a potential role as functional biomarkers. among circrnas, micra (myocardial infarctionassociated circular rna) levels in the blood are associated with ischemic hf [ ] , while hsa_circ_ and hsa-circ- are potential diagnostic biomarkers of coronary artery disease [ ] and hypertension [ ] respectively (fig. b) . the sars-cov- host-response transcriptomics changes have just started to be investigated, but reports on them are increasing over time. the gene expression profile of blood samples derived from covid- patients not only allows the definition of the host responses to the infection and, thus, a better understanding of the disease pathogenesis, but also the identification of potential biomarkers that could help monitoring patient responses to the disease. specifically, a distinct pattern of inflammatory cytokines was identified in bronchoalveolar lavage fluid and in the peripheral blood mononuclear cells, including ccl- , - and - , and cxcl- , as well as the activation of the p signaling-pathway in lymphocytes that may be related to covid- lymphopenia [ ] . in addition, several differential expressed transcripts were identified in pbmcs of covid- patients with severe or mild symptoms [ ] compared to controls. cytokinemediated signaling, the natural killer cell mediated toxicity and t cell activation pathways were enriched terms in common between the two disease stages, while interleukins-and tnf-signaling pathways were enriched terms the release of ncrnas by diseased cardiac tissues in the peripheral blood and the immunomodulation associated to cvd allowed the identification of dysregulated ncrnas in whole blood, plasma/serum or in pbmcs, to be used as potential biomarkers [ ] . moreover, the single-cell rna-sequencing (scrnaseq) approach has been used to profile the sars-cov- host-response in the pbmcs of covid- patients, and to comprehensively characterize the immunological changes [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in eight covid- patients, scrna-seq of pbmc indicated the depletion of the innate immune subsets compared to healthy pbmc [ ] . in addition, a new population, which was annotated as 'developing neutrophils' , has been found increased only in patients with ards; this population expressed several genes in common with the neutrophil progenitors, but not canonical neutrophil markers, indicating that they could be neutrophils at various developmental stages [ ] . the monocyte subset was enriched in cd , but without a substantial expression of pro-inflammatory cytokine genes (tnf, il , il b, ccl , ccl or cxcl ), which is in contrast with other reports [ , ] . additionally, cd + monocytes showed the upregulation of a signature of interferon (ifn)-stimulated genes that correlated with clinically relevant parameters [ ] . this strong activation of interferon-stimulated genes was also confirmed by arunachalam et al. [ ] , primarily in patients with mild or moderate disease, that is associated to reduced expression of genes encoding proinflammatory cytokines [ ] . similarly, a more pronounced expression of type i ifn in mild covid- patients and lack of il b mrna levels increase were also confirmed in a recent study that analyzed by scrna-seq two independent large cohorts of covid- patients [ ] . conversely, an increased tnf/il- β-driven inflammatory response was observed in severe covid- patients as compared to severe influenza, and to mild covid- patients [ ] . the scrna-seq approach has also been used to study the immune cell landscape of two severe-stage covid- patients prior to and following tocilizumab-induced remission [ ] . in this study, a monocyte subpopulation that contributes to the inflammatory cytokine storms was identified. bioinformatics analysis predicted a severe stage-specific interaction-networks formed by monocyte receptors and cytokines [ ] . in addition, tocilizumab treatment in severe-stage covid- patients led to an increased number of antibody-secreting plasma b cells, while cd + t cell-related cytotoxicity and cytokine production did not change [ ] . these data provide insights into the understanding of the inflammatory storms observed in covid- patients and identify also potential drug targets. zhang et al. analyzed by scrna-seq the pbmcs of thirteen covid- patients with clinical conditions ranging from moderate to severe and to convalescent [ ] . differentially expressed genes of cd + and cd + covid- monocytes, especially in severe covid- state, were associated with ifn responses, myeloid leukocyte activation, cytokine production and nuclear factor (nf)-κb signaling pathway [ ] . interestingly, in patients at the early phase of convalescence, despite of the recovery of most of the clinical parameters to a normal range, the immune system was still fully activated, as demonstrated by a still high naive t and t reg subsets ratios [ ] . early and late stages of recovery have been analyzed in detail by wen et al., confirming that covid- patients are still vulnerable after hospital discharge [ ] . covid- early recovery stage was characterized by cd ++ monocytes with high inflammatory gene expression as well as by the abundance of cd ++il β + cells, while t cells decreased remarkably, compared with both late recovery stage and healthy control subjects. due to the limited cardiac tissue availability, data on sars-cov- -mediated heart transcriptome changes have not been reported yet. however, sars-cov- infection of human induced pluripotent stem cell-derived cardiomyocytes (ipsc-cms) induced cytotoxic effects and rna-seq findings highlighted significant transcriptional changes in gene pathways related to cellular metabolism and immune response [ ] [ ] [ ] . indeed, sharma et al. [ ] observed in infected ipsc-cms a downregulation of transcriptional pathways related to mitochondrial function, oxidative phosphorylation, and cardiac function, whereas upregulated pathways included immune cytokines, immunomodulators, antiviral response, and apoptosis [ ] . accordingly, bojkova and collaborators showed that sars-cov- infection of ipsc-cms induced cytotoxic and proapoptotic effects and abolished cardiomyocyte beating [ ] . virus infection produced a transcriptional response including the up-regulation of genes associated to viral response and interferon signaling, apoptosis and reactive oxygen stress [ ] . on the same line, pérez-bermejo et al. [ ] , found that human ipsc-cms exposed to sars-cov- demonstrated a productive infection and morphological signatures of damage, which included a distinct pattern of myofibrillar fragmentation and numerous ipsc-cms lacking nuclear dna. these morphological changes were also confirmed in human autopsy specimens from covid- patients [ ] . rnaseq transcriptomic data obtained in infected ipsc-cms suggested a compensatory overexpression of myosin heavy chain genes in response to targeted degradation and also a significant depression of the ubiquitin-proteasome system upon infection [ ] . as for the heart, a limited number of reports on lung transcriptomic changes mediated by sars-cov- infections has been published so far. one of the first studies on lung cells was the analysis of the transcriptional footprint in post-mortem lung samples of covid- patients [ ] . in this study a reduced ifn-i and -iii response and a consistent chemokine signature compared to other respiratory viruses were observed [ ] . the reanalysis of a previously reported dataset identified, as expected, upregulated expression of chemokines and neutrophils in the lung tissue and bronchoalveolar lavage fluid of covid- patients and, in addition, the upregulation of genes coordinating heme biosynthesis [ ] . this effect, which has been shown in sepsis secondary to pneumonia to have a protective role against oxidative stress [ ] , could be responsible of pro-inflammatory cytokine production amplification [ ] or cause intravascular coagulation [ ] . the possible involvement of altered coagulation following sars-cov- infection has been also proposed by a study analyzing three publicly available rna sequencing datasets obtained from clinically isolated samples of bronchoalveolar lavage fluid, pbmcs and from in vitro sars-cov- infected primary normal human bronchial epithelial cells compared to the respective controls [ ] . gene expression analysis of both bronchoalveolar lavage and bronchial epithelial cells, but not of pbmcs, highlighted the activation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system [ ] . moreover, a study performed in autopsy lung specimens from patients who succumbed to sars-cov- infection supported two phases of disease evolution in patients with severe covid- pneumonia [ ] . in the first phase, high expression of ifn pathway genes and of endothelial genes related to tissue damage and fibrosis were observed [ ] . this first phase was morphologically characterized by high viral rna, a histological pattern of exudative diffuse alveolar damage, a shorter disease duration, while the second phase showed a low (or undetectable) viral rna, and an organizing form of diffuse alveolar damage [ ] . of note, the aforementioned pro-inflammatory cytokines, such as tnf-α, ifn-γ, il- β, il- , il- , and il- , are also elevated in hf and in viral myocarditis, and their sustained elevation correlates to hf progression. they are responsible for both compensatory cardiac hypertrophy and fibrosis in the setting of cardiac injury and induce further inflammation [ ] . there are contrasting effects attributed to ifn-γ in the heart. upon adverse stimuli, such as myocarditis or hypertension, the release of ifn-γ by recruited inflammatory cells to the heart results in cardiac fibrosis and hypertrophy. however, other studies have found that ifn-γ has also protective effects, limiting cardiac hypertrophy [ ] . the signaling of the pro-inflammatory cytokines is counterbalanced by the release of anti-inflammatory cytokines and tgf-β, which mitigate hypertrophic cardiac remodeling [ ] . these data indicate that, along with the direct effect mediated by the virus, such as in myocarditis, the cardiac sequelae may be due to the cytokines storm following the infection, which reinforces the cytokines release, observed in dilated cardiomyopathies. noncoding rnas regulating inflammation and the cardiovascular system: are they playing a role in covid- ? an over-activation of the inflammatory response and the ensuing cytokine storm seem to be a crucial pathogenetic mechanism in covid- patients and, as illustrated in sect. "transcriptomics studies of sars-covs infection: focus on inflammation", the innate immune response pathway has emerged as profoundly dysregulated in sars-covs infections. the immune system contributes to heart development, composition and function and, in specific circumstances, immune cells can also cause damage, participating to cardiac disease [ ] . for instance, in patients with hf, a chronic activation of the innate immune system is often observed, with t-cells and macrophages myocardial infiltration and increased pro-inflammatory cytokine levels (i.e. tnf-a, il- b, and il- ) [ ] . ncrnas are important regulators of these processes and we propose that ncrnas may play a fundamental role also in the cardiovascular dysfunctions of covid- patients. as mentioned above, the expression of ace is well known in adult cardiomyocytes [ , [ ] [ ] [ ] ] , as well as in neonatal rat cardiomyocytes and in human ipsc-cms [ ] . bioinformatics analysis predicted mir- b, mir- c and mir- among the mirnas targeting ace , and in vitro experiments demonstrated that mir- c modulation regulated the expression of ace [ ] . mir- c is upregulated by oxidative stress [ ] and is also involved in cvds [ ] , suggesting that mir- c-mediated regulation of ace may be important for sars-cov- entry. computational analyses have predicted that sars-cov- can divert the cellular mirnas from their transcriptional regulating activity, contributing to the abnormal immunity activation in patients with covid- [ ] , or synthesizes its own viral mirnas to reduce the host cell apoptosis preventing host defense [ ] [ ] [ ] . on the other side, host mirnas were predicted to target sars-cov- spike proteins, suggesting a potential role as therapeutic molecules [ , ] . direct or indirect effects of sars-cov- infection can alter host response and produce noncoding rna differential expression. in particular, in bronchial epithelial cells infected with sars-cov- , a complex bioinformatics and computational pipeline, revealed several activated networks, including those involved in immunoglobulin g and interferon lambda [ ] . in addition, acute inflammatory response and activation of tnf were also observed. this analysis also revealed several host-derived lncrnas differentially expressed in covid- patient-derived lung tissue, and in sars-cov- infected epithelial cells, including malat (metastasis-associated lung adenocarcinoma transcript ) and neat (nuclear-enriched autosomal transcript ) [ ] (fig. ) . the computational reanalysis of rna-seq dataset of sars-cov- infected bronchial epithelial cells [ ] identified several protein-coding rnas and lncrnas differentially modulated [ ] . the interaction of lncrnas with the differentially expressed protein-coding genes was analyzed by network enrichment analysis indicating significant interactions involved in cellular signaling, metabolism, immune response and rna homeostasis [ ] . the rna-seq of rna extracted from peripheral blood samples from covid- patients compared to controls demonstrated that mirnas were upregulated and mirnas were downregulated in the human patients with covid- [ ] . enrichment analysis of differentially expressed mirna target genes revealed that peptidases, protein kinases, and the ubiquitin system were shown to be the highest enrichment categories [ ] (fig. ) . there are also some hints provided by transcriptomics studies on sars-cov. when this infection had a multicountry outbreak in to , coding rnas were still the focus of attention for the scientific community and microarrays were the preferential profiling tool. nevertheless, some studies performed in the following years on lung samples or on bronchoalveolar stem cells collected from mouse infected with adapted sars-cov are available [ ] [ ] [ ] [ ] . these studies identified lncrnas and mirnas involved in innate immune response in cvds. in particular, the lncrnas neat (nuclear paraspeckle assembly transcript ) and malat (metastasis-associated lung adenocarcinoma transcript ) [ ] were up-and down-regulated after infection, respectively, and mir- - p [ ] , mir- - p and mir- - p were among the most deregulated mirnas upon sars-cov infection (fig. ) . mir- - p, mir- - p and mir- - p are expressed in immune cells and involved in both innate immune response [ , ] and in myocardial infarction or hf [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these mirnas control the production and secretion of pro-inflammatory cytokines in the heart by toll-like (trl) receptors and their downstream signaling pathway, which involves the transcription nuclear factor-kappa b (nf-κb). indeed, mir- - p, along with mir- a- p, represents a unique regulatory network for the fine-tuning of the macrophage inflammatory response via regulation of nf-κb activity [ ] . in particular, the activation of nf-κb by a stressor stimulates mir- - p expression which, amplifying nf-κb activity, enables a strong macrophage activation. as the inflammatory response develops, mir- a- p transcription increases, inhibiting its targets irak and traf , whereby dampening nf-κb activation (fig. ) . in addition, also mir- - p targets a component of the nf-κb pathway, pdcd (programmed cell death ), thus stimulating the release of pro-inflammatory cytokines and inhibiting the release of anti-inflammatory cytokines [ , ] . finally, mir- - p seems to have cardio-protective and anti-inflammatory roles by enhancing glucose metabolism and inhibiting granulocyte activation. accordingly, a decrease of mir- - p levels in tissue and plasma has been observed in diabetes and cvds [ , [ ] [ ] [ ] . another ncrna of relevance in cardio-immunology is the lncrna neat , which was reported to induce intima thickening in vascular smooth muscle cells [ ] . neat is also part of the innate immune response by promoting the activation of the inflammasome in macrophages and the release of il- β [ ] . together, these data indicate that some of the ncrnas reported to be dysregulated by transcriptomic profiling in cvds, are also involved in viral innate immune responses, and that they may be identified as candidate transcripts for our query in understanding the pathogenesis of covid- . investigating the interplay between sars-cov- , the host antiviral defenses and the cardiovascular system, is fundamental to understand the viral pathogenesis and the infection outcome. indications from sars-cov and other coronaviruses are very helpful; however, sars-cov- is a novel human pathogen and many aspects of its interaction with the host could be unique. it is now clear that pre-existing cvds increase both the severity of the primary respiratory syndrome and the risk of adverse outcomes. sars-cov- infection consequences on the cardiovascular system should be investigated both for their acute and prolonged sequelae. in this view, transcriptomics may be a powerful approach to study the ncrna involvement in the disease mechanisms and for the identification of biomarkers. significant hurdles are represented by difficulties in measuring and studying ncrnas due to low abundance of many of them or to specific structural features (e.g. circrnas). identification of rna-based biomarkers and targets requires heavy reliance on relatively expensive sequencing approaches that still lack universally adopted standard operative procedures [ , , ] . insufficient structural and functional annotation of the noncoding genome is also a significant problem. these limitations should be overcome in order to make significant scientific progresses in our understanding of covid- pathogenesis, facilitating prognosis and hopefully, paving the way to potential therapeutic approaches. in this respect, international cooperation to share knowledge, patient samples and data collection across many institutes and countries seems as an almost obligatory strategy [ ] . a novel coronavirus outbreak of global health concern covid- : a fast evolving pandemic who coronavirus disease clinical 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pectoris assessment of micrornas in patients with unstable angina pectoris microrna signatures in peripheral blood mononuclear cells of chronic heart failure patients prospective study on circulating micro-rnas and risk of myocardial infarction circulating micrornas are new and sensitive biomarkers of myocardial infarction plasma microrna- a is a new marker for both acute myocardial infarction and underlying coronary artery stenosis circulating microrna- b and microrna- reflect myocardial damage in cardiovascular disease increased plasma levels of lncrna h and lipcar are associated with increased risk of coronary artery disease in a chinese population circulating long noncoding rna, lipcar, predicts survival in patients with heart failure long noncoding rnas in patients with acute myocardial infarction circulating long-non coding rnas as biomarkers of left ventricular diastolic function and remodelling in patients with well-controlled type circulating long non-coding rnas nron and mhrt 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the role of type i interferons in development of severe covid- single-cell landscape of immunological responses in patients with covid- human ipsc-derived cardiomyocytes are susceptible to sars-cov- infection sars-cov- infects and induces cytotoxic effects in human cardiomyocytes sars-cov- infection of human ipsc-derived cardiac cells predicts novel cytopathic features in hearts of covid- patients imbalanced host response to sars-cov- drives development of covid- immune and metabolic signatures of covid- revealed by transcriptomics data reuse genetic signature related to heme-hemoglobin metabolism pathway in sepsis secondary to pneumonia characterization of heme as activator of toll-like receptor excess of heme induces tissue factor-dependent activation of coagulation in mice unique transcriptional changes in coagulation cascade genes in sars-cov- -infected lung epithelial cells: a potential factor in covid- coagulopathies. medrxiv: the preprint server for health sciences temporal and spatial heterogeneity of host response to sars-cov- pulmonary infection role of inflammation in heart failure could interferon-gamma be a therapeutic target for treating heart failure? cardioimmunology: the immune system in cardiac homeostasis and disease upregulation of cell adhesion molecules and the presence of low grade inflammation in human chronic heart failure micrornas targeting the sars-cov- entry receptor ace in cardiomyocytes mir- c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via zeb inhibition implications of the virus-encoded mirna and host mirna in the pathogenicity of sars-cov- the prediction of mirnas in sars-cov- genomes: hsa-mir databases identify key mirs linked to host responses and virus pathogenicity-related kegg pathways significant for comorbidities epigenetic regulator mirna pattern differences among sars-cov, sars-cov- , and sars-cov- world-wide isolates delineated the mystery behind the epic pathogenicity and 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altered innate immune signaling. mbio microrna- promotes autoimmune inflammation by enhancing inflammatory t cell development mir- and mir- : two key modulators of immune response and tumor development conditional dicer gene deletion in the postnatal myocardium provokes spontaneous cardiac remodeling microrna- contributes to myocardial disease by stimulating map kinase signalling in fibroblasts author correction: an nf-kap-pab-microrna regulatory network tunes macrophage inflammatory responses engulfment of apoptotic cells by macrophages: a role of microrna- in the resolution of wound inflammation micrornas in nf-kappab signaling low circulating microrna levels in heart failure patients are associated with atherosclerotic disease and cardiovascular-related rehospitalizations microrna- regulates glut expression and cardiomyocyte glucose metabolism noncoding rnas in cardiovascular disease: pathological relevance and emerging role as biomarkers and therapeutics : • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from long noncoding rna neat (nuclear paraspeckle assembly transcript ) is critical for phenotypic switching of vascular smooth muscle cells the lncrna neat promotes activation of inflammasomes in macrophages circular rnas: methodological challenges and perspectives in cardiovascular diseases call to action for the cardiovascular side of covid- publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are thankful to jeremy c. hill (imperial college london) for editorial assistance. figures were partly generated by using biorender app and https :// www.somer sault .com/scien ce-illus trati ons/. all authors contributed to the conception, design and revision of the article. sg, am and fm wrote most of the text and prepared the figures. cg, yd and key: cord- - ksn fc authors: rodriguez, j. m. title: detection of animal pathogens by using the polymerasechain reaction (pcr) date: - - journal: the veterinary journal doi: . /s - ( ) - sha: doc_id: cord_uid: ksn fc summary the polymerase chain reaction (pcr) is a nucleic acid-based technique that enables the rapid and sensitive detection of specific micro-organisms. although this technique is widely used in veterinary research, it has not yet found applications in routine microbiological analysis of veterinary clinical samples. however, advances in sample preparation together with the increasing availability of specific gene sequences will probably lead to the more widespread diagnostic use of pcr in the future. pcr is likely to have a strong impact in the epidemiology, treatment and prevention of animal infectious diseases. the development of polymerase chain reaction (pcr) has revolutionized the field of molecular biology. the technique consists basically of the enzymatic synthesis of millions of copies of a target dna sequence (saiki et al., ) . using a thermostable dna polymerase (saiki et al., ) , and a succession of cycles that includes denatnration of the template dna, hybridization of specific dna primers to the template and extension of the primers, it is possible to generate multiple copies of the target region enzymatically. thus, pcr provides a method [or obtaining large quantities of specific dna sequences from small amounts of dna, including degraded dna samples. the technolo~, has been extensively reviewed (see for example, ehrlich, ; innis el al, ; griffin & gritt]n, ) . although pcr is widely used in an increasing number of applications, those in the area of microbiolo~, and diagnosis of infectious diseases have undergone outstanding advances in recent ),ears. traditionally, strategies for identifying - / / x - /$ . / most microbial pathogens invoh'e isolation on selective agar media or cell cultures, and the use of phenotypical tests but these techniques are usually slow and laborious. the important cost that animal infectious diseases can have on national economies has therefore stimulated the search for faster, mo,'e sensitive and more specific methods to identid microbial pathogens. many useful nucleic acid probes and immunological assays have been developed for diagnostic purposes, but these techniques also have some deficiencies (jones & bej, ) . the emergence of pcr, however, offers the potential to improve the laboratory-based diagnosis of pathogens (mahbubani & bej, ) . althougla pcr has some shortcomings, such as the problems caused by contaminants and inhibitors or the lack of suitable sequences for designing specific primers, the outstanding research effort focused on tiffs technique, together with the remarkable development of molecular biology have minimized the deficiencies and allowed its increased general use as a diagnostic tool. foot-and-mouth disectse vires (fmdv) . fmdv is one of the most dangerous viruses of ruminants. its speed of spread and ability to change its antigenic identip,,' makes fmdv ve d, threatening to the beef and dai d, industries of many countries. fast and accurate detection of fmd outbreaks is needed to limit spread of the disease. the virus consists of copies of each of the four proteins vp to , of which vp is the main protein determining antigenic identig,. pcr systems for detecting fmdv have been developed by different laboratories (meyer et al., ; laor et al., laor et al., , . there are also repo,'ts on the use of pcr to determine fmdv serotype (rodrfguez el al., : strata el al., . rinderpest (r-p . animal diseases greatly influence animal production and trade. diagnosis should enable fast implenlentation of control measures to minimize losses. this is particularly important in the case of highly-contagious pathogens such as rpv and peste des petit ruminants (pprv) viruses. they are, at present, confined to developing countries where they remain a constant threat to livestock. rpv may infect all artio-dac~,ls, with cattle and buffaloes being the most susceptihle species, while pprv specifically causes disease in small ruminants. field diagnosis of classical rinderpest, with its many indicative clinical signs, is easy but those signs are not ahvays clearly seen, particularly in countries where the disease is endemic (diallo et al, ) . moreover, some mild strains can fail to produce clinical signs unless the infected animals are stressed. the situation is more complicated in small ruminants becanse they can be infected with rpv and/or pprv, and the disease produced by both viruses is similar. the laboratory tests commolaly used are either expensive (animal inoculation), slow (virus isolation or neutralization) or insensitive (agar gel immunodiffusion). pcr can drastically improve the diagnosis. chamberlain el al. ( ) grouped different isolates of rpv combining pcr with sequencing studies. using the same procedure, barrett el al. ( ) identified two different rpv strains in the same clinical sample and warmwayi el al. ( ) showed that there was co-circulation of two different lineages of rpv in nigeria during the epizootics of the s. bvdv is another important pathogen of cattle, causing considerable economic losses throughout the world. three syndromes caused by bvdv have been described: an acute gastroenteritis with severe diarrhoea, mucosal disease, and chronic infections of several weeks' dnration in calves up to l-year-old. persistently infected animals are the main source of infection to herdmates because they continually shed large quantities of virus in body secretions and excretions. due to the obvious impact of b\q)v infections, screening of animals must be carefully made and current methods of detection, such as virus isolation or immunoassays, either lack optimal sensitivity or rapidity for consistent and large scale testing in animal specimens (radwan et al., ) . pcr, however, can readily detect b\q)v (belak & ballagi-pordany, ; brock, ; hertig el al, ; ward & misra, ; hoft van iddekinge et al., ; gruber el al, ) , and pcr analysis of hulk tank milk samples has provided a rapid and sensitive method to screen herds for the presence of the virus. sensitive studies using reference strains of bvdv fi-om persistently infected carriers have shown that reverse transo-iption (rt)-pcr has greater sensitivity than other tests, including enzyme-linked immunosorbent assay (elisa) (horner el al, ) ; unfortunately, cost currently makes this technique unsuitahle for large-scale testing but it should be valuahle as a coniirmatm t test in cases where elisa resuhs are in the 'suspicious range' or where the viral titre is low, such as in batches of foetal bovine serum. additionally, it is possible to discriminate among different bvdv strains using pcr (tajima el al., ) and pcr-restriction fi'agment length polymorphism (rflp) tests have demonstrated that b\q)v isolates fi'om ruminants on four different farms in sweden were herd-specific rather than speciesspecific, and that the virus is readily transmitted between cattle and sheep (paton et al., ) . bt is an arthropod-borne viral infection of rmninants caused by bluetongue virus (btv). clinically, bt varies depending on factors such as population density and competence of the cullicoides sp. vector, the distribution of susceptible hosts and the virulence of the different serotypes of btv. among ruminants, only sheep are clinically affected while cattle are usually asymptomatic reservoirs. pcr-based procedures have been developed for the diagnosis of btv (gould el al., ; dangler el al., ; wade-evans et al., ; mccoll & gould, ; akita el al., ; parsonson el al., ) . whole hlood seems to he the most convenient clinical sample, but fractions of blood have also been successfully used for pcr detection of btv infection in sheep (mccoll & gould, ) . in a comparison of methods tbr isolation of btv in infected cah,es, virus was detected in elnbrionating chicken eggs for - weeks, whereas pcr detected btv nucleic acid tot - weeks (maclachlan et al, ) . the sensitivity of the technique means that it can be adapted to detected btv in cuuicoides sp. samples (wilson & chase, ) . ehdv is an orbivirus related to btv that causes tatal haenaorrlaagic disease in domestic and wild runainants. clinical signs and pathological changes caused by ehdv and btv are indistinguishable. aradaib el al. ( ) compared the value of pcr with virus isolation for the detection of ehdv in clinical samples taken fi'om naturallyinfected deer, and concluded that pcr assays for edhv can provide a diagnostic ahernative superior to the current cumbersome and timeconsunfing x rus isolation procedures. bovine immunodeficien o, vimts (bi]' . bw is structurally and genetically related to human immunodeficiency virus (hiv). biv causes lymphoproliferative changes and enlargement of subcutaneous lymphatic nodules in cattle. although infection by bw is widely prevalent in beef and dai d, cattle, there is no accurate diagnostic test for the virus. using pcr, nash et al. ( ) detected biv-infected leucocytes in the blood and milk of biv-seropositive cows. these data confirmed the presence of bw in milk and laighlighted the potential for lactogenic transmission of the virus. suarez el al. ( ) examined blood samples from biv-experimentally infected calves by virus isolation, protein immunoblot and nested pcr and showed that the nested pcr test is more sensitive than any other method o " the detection of biv infection in cattle. bovine he~pesvirus . bhv- causes infectious bovine rhinotracheitis (ibr), an economically important disease of cattle characterized by acute respiratory infection and reproductive problems such as abortion, infertility, vulvovaginitis and balanoposthitis. latently infected animals can be reservoirs of bhv- in the herd. virus detection is often requested for the laboratot t diagnosis of most cases of respiratm t and reproductive problems in cattle. several reports have described the pcr of different bhv- genes from tissue cultures (vilcek, ; kibenge et al., ; yason et al., ) and bovine semen (wiedman et al., ; xia et al., ) . spanish sheep encephalitis (sse). all three viruses belong to the tick-borne encephalitis virus group, within the genus flavivirus. these viruses produce a similar clinical syndrome, and the histological changes that they produce in the brains of affected sheep are indistinguishable. moreover, animals fi'om affected flocks have antibodies that cross-react with the other viruses (gonzalez et al, ) . sequencing of pcr products obtained from cdna of sse have permitted the location of specitic genetic markers for this flavm~rus (marin el al., ) . pcr has also enabled the construction of recombinant vaccinia virus expressing prm and e glycoproteins of louping-ill virus (venugopal et al, ) . cap~ine arth~qtis encephalitis (cae). cae is a worldwide multisystemic disease of domestic goats, characterized by progressive arthritis, leucoencephalomyelitis and mastitis. although the virus persists for life, infection of goats with cae is often subclinical. isolation of cae is not attempted routinely as a diagnostic tool but pcr has recently been adapted for the detection of proviral dna in caev-infected cells from clinical specimens (clavijo & thorsen, ) . the technique has a sensitivity which is several orders of magnitude higher than direct hybridization, and may represent an important alternative procedure for identification of persistently infected animals. other rmninalats viruses for which pcr protocols have been successfully developed include bovine leukaemia virus (naif et al., (naif et al., , murtaugh et al., ; ballagi-pordany et al, ; sherman et al., ; agresti et al., ; kelly et al., ) , bovine coronaxdrus (verbeek & tijssen, ) , rotavirus (xu et al, ) , and maedi-visna virus (staskus et al., ; zanoni et al., ) . porcine pa~vo virus (ppio. the role of ppv in inducing swine reproductive failure characterized by embryo and fetal deaths has been extensively described, often when other clinical signs of disease are lacking. sources of ppv include contaminated semen, the female reproductive tract or exposure during gamete/embryo manipulation. molitor et al. ( ) developed a pcr amplification test for the detection of ppv thereby minimizing the risk of transmission of ppv to seronegatives recipients through embryo transfer (gradil et al., ) . swine influenza. swine influenza induces high morbidity and low mortality in pig populations throughout the world. although the disease usually resolves, infected pigs represent a substantial source of economical loss to the producer because of their weight loss and poor weight gain. the results obtained by schorr et al. ( ) proved that rt-pcr from nasal swabs specimens of pigs is significantly more sensitive than the techniques currently used such as the infectivity assay in embrionating chicken eggs. porcine reproductive and respirato~ syndrome (prrs) . the disease complex known as prrs has become an economically important health problem throughout europe and north america. pcr has been used to confirm the presence of prrs genes in infected monolayers (katz et al., ) , thus providing the first steps for the development of a pcr test to analyze prrs virus in clinical samples. . prv is the aetiological agent of a major disease that has substantial economic impact in swine industry. the disease is fatal to young pigs but in adults the infection is less severe, and sometimes clinical signs are not apparent. pigs surviving prv infection remain latently infected for life. pcr has become the recommended method for evaluating prv latency; reports from several laboratories have indicated that neuronal tissues, and especially the trigeminal ganglia, are the most reliable sources for detection of latent prv genome (belak et al., ; wheeler & osorio, ; volz et al., ; brockmeier et al, ) but trigeminal ganglion assay can be performed only after death of the affected animal. tonsil biopsy specimens can be obtained from live animals and used to amplify prv sequences by pcr (chung, ) . prv has also been detected in the semen of boars (guerin et al., ) . the method is simple and allows the detection of around viral dna sequences per microlitre of sample. prv infects cells of the lymphatic tissue and white blood cells of a variety of mammals. these cells are also present in sausages, and schunk & rziha ( ) established a pcr method specifically to detect prv in artificially contaminated sausages and showed that pcr was less affected by extreme ph values than tissue culture techniques usually employed to recover the virus. other important swine virus that have been detected by pcr include hog cholera virus (boye et al, ; liu et al, ; wirz et al, ) and african swine fever virus (steiger et al, ) . intensive breeding of poultry means that high populations often live in confined spaces. under such conditions, the entry of a virulent virus can cause high mortality and big economical losses. rapid diagnostic tests are needed to minimize the consequences of viral outbreaks in these environmerits. when compared with virus isolation and other classic techniques, pcr is the method of choice for diagnosis of many poultry viruses including marek's disease virus (becket et al., (becket et al., , silva, ; zhu et al., ; davidson et al, ; zerbes et al., ) , reticuloendotheliosis virus (aly el al., ; davidson et al., ) , avian leucosis virus (van woensel et al., ) , infectious bronchitis virus (andreasen et al., ; linet al., ; jackwood et al., ; zwaagstra et al., ; kwon et al., a, b) , newcastle disease virus (jestin & jestin, ) , lymphoproliferative disease virus (sarid et al., ) and infectious bursal disease virus (lee et al., ; wu et al, a, b) . equine viral arteritis (eva). eva is a ubiquitous disease present throughout mainland europe. the variety and sevelity of clinical signs vary widely from inapparence to abortion and death. a proportion of seropositive stallions shed the causal organism, equine arteritis virus (eav), in their semen, and play a primary role in its dissemination and perpetuation in the equine population. therefore, when a stallion is identified as eav positive, the first priority is to ascertain whether virus is being shed before the animal is allowed to cover mares. pcr is included among the three methods that may be used to establish the presence of virus in the semen (chirnside & spaan, ; horserace betting levy board, ) . equine herpesvirus. pcr has been successfully applied to detect ehv and in aborted equine fetuses (ballagi-pordany el al., ) and in nasopharyngeal swab specimens from horses with respiratory or neurological disease (sharma el al., ; wagner et al., ) . other equine viral diseases which have been diagnosed by pcr include equine infectious anaemia (o'rourke el al, ) and african horse sickness (zientara el al., ; stone-marschat et al., ) . rabies. rabies is still one of the most lifethreatening zoonosis in some regions of tile world. obviously, fast and accurate detection of infected animals is of vital importance. research resuhs have shown that pcr can play a remarkable role in the rapid, sensitive and specific detection of the rabies virus (ermine et al, ; sacramento et al., ; shankar el al., ; kamolvarin et al., ; mccoll et al., ) and the technique should spread among the reference laboratories located in regions at risk. canine pa)vovirus (cpi . cpv is the causative agent of haemorrhagic enteritis and myocarditis, and at present is one of the most common pathogenic vi,'uses causing diarrhoea in dogs. cpv is not easily inactivated with the usual disinfectants, and can survive more than months once a hospital or kennel is contaminated, often leading to secondary infections. as a result, it is important to have a rapid, specific and sensitive method to distinguish infected fl'om uninfected dogs. pcr assays based on vp and vp genes have been used to detect cpv in paraffin-embedded tissues (truyen el al., ; uwatoko el al., ) and in faeces of diarrhoeic dogs (hirasawa et al, ) . additionally, pcr-rflp analysis is a practical and reliable method for differentiating wild-and vaccine-type cpvs (hirasawa el al., ; senda et al., canine distemper virus (cdi/). cdv induces a multifocal demyelinating disease in the central nervous system of dogs, in which virus persistence plays a key role. pcr has been an essential research tool to study the virus's nucleocapsid protein, and to provide a molecular basis for the observed differences in virus release and spread between attenuated and virulent cdv (stettler & zurbriggen, ) . feline infectious peritonitis vinls (fipv) . fipv causes a severe, often fatal disease in domestic and wild cats. despite considerable research, no routine diagnostic method is available. detection of fipv by nested pcr has been attempted (egberink el al, ) but the authors concluded that the value of pcr for the identification of sick animals and asymptomatic carriers needed to be further studied. in their work a positive pcr in healthy animals failed to provide an absolutely definitive diagnosis of fip; equally, a negative pcr result from a sick animal did not completely exclude fip. better results have been achieved in the pcr detection of active and latent feline herpesvirus (nunberg et al., ; reubel et al., ) and feline immunodeficiency virus (rimstad & ueland, ) . morbillivi)'us infections in marine mammals were first reported in , and are known to be distributed among a wide spectrum of seals and cetaceans in the atlantic ocean and the mediterranean sea. rt-pcr has revealed that there were no obvious links between the morbillivirus outbreak ill marine seals in northern europe in and that which occurred in freshwater seals in lake baikal in (visser et al., barrett et al., ) . direct sequencing of pcr products that included the haemagluttinin protein gene of the lake baikal seals isolate (pdv- ) revealed that it was closely related to two isolates of cdv from germany but different from cdv vaccines currently used in the lake baikal region (mamaev et al., ) . staphylococcal mastitis is an important problem in dairy farms. several staphylococci, mainly staphylococcus aureus strains, cause acute and chronic mastiffs, and can lead to gangrenous mastitis. human handling of the udder or the milking machine is a potential source of staphylococci, and contaminated milk can be the cause of foodborne intoxication in man. rapid detection of staphylococci, including those killed by heat treatment, in suspected food could prevent foodborne staphylococcal gastroenteritis, and differentiation of s. aureus strains has been achieved by dna amplification fingerprinting (saurnier et al., ; van belkum et al, ) . although listelia monocytogenes infection may produce clinical syndromes of abortion and neonatal septicaemia, encephalitis is most common in adult animals. the clinical diagnosis of listeric encephalitis in ruminants is difficult because of the existence of a broad spectrum of central nervous system diseases with similar clinical symptoms. in addition, listeria can only rarely be cultured from the cerebrospinal fluid (csf) of affected animals. because pcr is able to detect low numbers of bacteria, it may be a tool for increasing the sensitivity of listeria detection in csf of ruminants (peters et al., ) . it is also important to detect asymptomatic carriers because of the zoonotic nature of the infection. during the last decade several outbreaks and single cases of human listeriosis have demonstrated that the disease is often transmitted by contaminated food. jaton et al. ( ) developed a sensitive nested pcr assay for the detection of l. monoc),togenes in human csf. additionally, pcr has confirmed its usefulness to detect specific strains in the epidemiological investigations of listeriosis (ericsson et al, ) . anthrax is a fatal infection of humans and livestock that is caused by the gram-positive, endospore-forming bacterium bacillus anthracis. humans are infected primarily through contact with products derived fi'om contaminated animals. there is a growing need for methods to detect b. anthracis spores and vegetative cells, not only to prevent large-scale livestock destruction, but also to protect humans that may come into contact with them. pcr amplification of some b. anthracis genes has already been reported (carl el al., ; turnbull el al, ; hutson et al., ; johns et al., ; reif et al., ) , allowing the detection of even a single spore of b. anthracis (reif et al, ) . henderson et al. ( ) examined the variation among isolates of b. anthracis using restriction patterns and pcr and found that the b. anthracis profiles were unique when compared with those of closely related species, including b. cereus, b. thuringiensis and b. mycoides. their results showed that isolates of b. anthrads are ahnost completely homogeneous and distinct from other members of the b. cereus group. botulism is a severe foodborne disease caused by clostridium botulinum and is characterized by generalized flaccid paralysis. botulinal neurotoxins, produced by seven distinct serological t)qoes of c. botulinum are among the most potent biological substances known and neurotoxins a, b, c, d, e and f have all been implicated as causes of human and/or animal disease. the mouse bioassay is the established method for the detection of neurotoxin but alternatives to the use of animals for diagnostic purpose are ethically desirable and should be encouraged. some immunological methods have been proposed but the use of dna-based techniques has not been extensively explored. however, some authors have confirmed that pcr has a great potential for the identification of botulism neurotoxin-producing strains (szabo et al, (szabo et al, , fach et al., ) , and clearly demonstrated that pcr methods should be used for the development of highly sensitive and specific assays for organisms harbouring botulismneurotoxin genes. closhfdium p~fifngens enterotoxin genes have also been detected in stools without isolation of tile organism (saitoet al., ; fach et al., ) . although the isolates were fiom human foodpoisoning outbreaks or sporadic diarrhoeal cases, c. perfringens is also a well-known animal pathogen, being the aetiological agent of haemorrhagic and necrotic enteritis. thus, the application of pcr should be desirable and appropriate in veterinal t laboratories. a pcr assay has in fact been developed recently for the rapid detection of genes encoding c. pe~fringens enterotoxins (buogo et al., ) , and successfully applied in samples of small and large intestine from infected piglets. enterotoxigenic eschenlchia coli (etec) is a major cause of diarrhoea in neonatal and postweaned calves, lambs and piglets. several fimbrial adhesins and enterotoxins are recognized as the virulence factors of etec. the sequencing of the enterotoxins and fimbrial genes have made possible the application of nucleic acid-based methods for their detection (harel et al., ; woodward et al., ) . these methods have the advantage that they are readily applicable to a large number of isolates, in contrast to classic methods such as agglutination, infant mouse, ligated swine intestine and cell culture assays. pcr resuhs obtained in sweden by kennan et al. ( ) showed that the gene for the major subtmit of f fimbria was present on approxiinately half of the strains not expressing k , k , p and f fimbria isolated from piglets older than week with diarrhoea. this suggested that f fimbria are of major importance among etec strains causing post-weaning diarrhoea. ojeniyi et al. ( ) applied two different genotyping methods, colony laybridization and pcr, to detect enterotoxin, verotoxin and fimbrial genes in e. coli strains from piglets with diarrhoea, and the results were compared with those obtained by phenotypic methods. the correlation between the genotypic and phenotypic resuhs was . - %. detection of fimbrial and enterotoxin genes detected more pathogenic strains than the serotyping using a set of rabbit ok antisera. using such techniques, the verotoxin and the fimbrial f genes were found to be more frequent in post-weaning than in neonatal e. coli strains and genotypic tests are becoming valuable tools in the identification of pathogenic e. coli. together with staphylococcal mastitis, coliform mastitis is a major problem in dairy farms. identification of e. coli strains from cows with clinical mastitis can be accomplished by pcr amplification using repetitive extragenic palindromic (rep) and enterobacterial repetitive intergenic consensus (eric) sequences. such procedure has revealed that e. coli strains isolated from repeated episodes of clinical mastitis in the same cow have similar genotypes (lipman et al., ) . in western countries, enterohaemorrhagic e. coli (ehec), especially serotype o :h , have become a major concern for human health. ehec strains produce verocytotoxins, and have been identified as causative agents of human diarrhoea, haemorrhagic colitis (hc), haemolytic-uraemic syndrome (hus) and thrombotic thrombocytopaenic purpura (ttp). cattle seem to be the most important reservoir of ehec, and although ehec can produce haemorrhagic colitis in calves, many healthy animals are carriers. the high levels of ehec carriage among young animals is of concern as meat may be a significant source of transmission from bovines to humans. because verocytotoxin genes can be detected by pcr (smith et al., ; tyler et al., ) , this technique has become useful to determine the prevalence and clinical significance of ehec isolated from cattle herds with and without calf diarrhoea. burnens et al. ( ) found a % level of ehec carriage among cows, but it was reassuring that no ehec were detected in milk samples. enteric disease caused by infection with salmoneua is an important cause of morbidity in animals. s. enteritidis in particular is associated with human food-borne illness resulting from the consumption of contaminated poultry eggs or meat. salmonellas are generally identified by microbiological culture of faeces, tissue or body fluids. although elisas may be used to identify salmonellas, full identification still requires culture. amplification of salmonella genes offers a specific and direct means of detection (rahn et al., ; widjojoatmodjo et al., ; aabo et al., ; cohen et al., ; way et al., ; nguyen et al., ; wood et al., ) . booster pcr methods for the genus-specific detection of salmonellas in equine and chicken faeces have been developed (cohen et al., a, b) with detection possible within - h from the time of submission of samples. although booster pcr is highly sensitive, its cost is about twice that of a simple pcr reaction. cohen et al. ( ) described an alternative method using enrichment followed by a simple pcr reaction that enabled salmoneua to be detected in faeces within h of submission of samples. a quantitative method using a known quantity of competitor dna to quantify the numbers of sahnonellas in chicken faeces has also been developed (mahon & lax, ) , but some problems with inhibitor), substances have been reported. comparison of pcr and microbiological cultures for the detection of salmonellas in drag-swabs from poultry houses have revealed that pcr is significantly more sensitive than culture for environmental monitoring (cohen et al., c) . y. enterocolitica also causes food-borne human gastroenteritis, with pigs implicated as the major reservoir for the pathogenic serotypes : , : and : . detection of y. enterocolitica often includes enrichment and biochemical confirmation but the whole process can take up to weeks. pcr can be successfully used for recognition of pathogenic y. enterocolitica (kapperud et a/., ; koeppel et al., ; rasmussen et al., ) , and the best results are achieved if the bacteria are concentrated by immunomagnetic separation (ims) before pcr. this approach has been used to detect y. enterocolitica : in faecal samples and tonsil swabs from pigs (rasmussen et al, ) and the authors concluded that ims-pcr was a reliable method when used on pre-enriched medium, enabling the detection of positive samples which are not recognized by traditional methods. h. pyrlori is a microaerophilic, gram-negative spiral organism that has received great attention for its association with human gastritis, peptic ulcers and even gastric cancer. other species of the genus have been isolated from the gastric mucosa of animals and mostly associated with gastritis of the host. because it has been suggested that some strains of helicobacter canis are capable of zoonotic transmission, sensitive methods for their detection are needed, and pcr has already been shown to be useful (stanley et al., ) . clinical samples (cousins et al., ; buck et al., ; yule et al., ; wards et al., ) . mycobacterium paratuberculosis causes johne's disease, a commonly diagnosed disease of sheep, goats and other ruminants. the organisms can be detected by pcr from intestinal and lymph node tissue of infected animals (ridge et al., ) . ovine loot rot is a highly contagious, economically serious disease of sheep with worldwide distribution, especially in temperate farming areas. although [hot rot results fi'om a mixed bacterial infection, dichelobacter nodosus has been shown to be the essential pathogen for the initiation and estahlishment of the disease. clinical diagnostic methods currently available for foot rot are subjective and lack precision. consequently, there is a demand for rapid and precise tests to differentiate virulent strains. the use of pcr based on specific regions of s rrna constitutes a competent assay for foot rot (la fontaine et al., ) . pcr assays employing virulent-and benign-specific primers are capable of specific and sensitive differentiation ot" strains causing virulent, intermediate or benign foot rot (liu & webber, ) . bacteria of the genus bruceua are well-known as intracelhflar pathogens that cause animal and human infections. rapid and sensitive pcr detection of brucellas with or without extraction of dna has been accomplished (fekete et al., a (fekete et al., , b ouahrani et al., ) . mycobacterium bovis, the causative agent of tuberculosis in cattle, is a member of the tuberculosis complex, a group of related species that includes mycobaclerium tuberculosis, the major cause of human tuberculosis. histological examinations enable rapid decisions to be made on suspect carcasses during meat inspection. however, agents other than m. bovis can induce similar lesions, and additionally, the microscopic detection of acid-fast organisms can only detect bacteria in great concentrations. laboratory culture of m. bovis is sensitive but requires viable bacteria, and the growth of this organism may take - weeks. species identification procedures extend the reporting time even further. tests based on pcr have been shown to be very promising for mycobacterial detection in leptospirosis is probably one of the world's most widespread zoonoses. rapid diagnosis of leptospirosis is important in view of the need for adequate early treatment. clinically, it is sufficient to know whether or not a patient is infected with pathogenic leptospires but, epidemiologically, it would be of considerable value if tile causative leptospira can be identified at the strain level. serology does not contribute to early diagnosis as antibodies become detectable on approximately the seventh day of infection. conventional methods to detect leptospires in blood are either unreliable or too slow to give early results. pcr is a promising tool for early detection of leptospires in blood, urine or csf in the period between the first appearance of clinical symptoms and the time when antibodies become detectable (van eys et al., ; gerritsen et al., ; hookey, ; merien et al., ; gravekamp et al., ) . the genus borrelia contains several human and animal pathogens. the aetiological agent of lyme disease is bo~'elia burgdorfe~, which is primarily transmitted by ixodes ticks. several authors have successfully employed pcr for diagnosis of lyme disease (rosa & schwan, ; marconi & garon, ; kawabata et al, ) . it is well-known that ticks feed on deer species, and using pcr, kimura et al. ( ) demonstrated the presence of b. imrgdmfefi in the skin of naturally infected wild sika deer, thus confirming the potential of deer as a source of transmission. pcr data also support the notion that birds are partly responsible for the heterogeneous distribution of lyme disease borrelia spirochetes in europe (ols n et al., ) . zingg and lefebvre ( ) have developed a high-sensitive pcr assay for bon'elia cm aceae that does not cross-react with any other closely related spirochetes. disease in chickens which results in reduced egg production and significant downgrading of carcasses at slaughter. chlamydia psittaci includes a heterogeneous group of mammalian and avian isolates but, at present, there is no generally accepted and accessible method for typing these. the major outermembrane protein (momp) is the most important antigen at the cell surface of chlamydia. recently, pcr-rflp analysis of the momp encoding gene has been used for t q~ing of c. psittaci strains (denamur et al., ; kaltenboeck et al., ; sayada et al., ) . mycoplasmas are known to produce a wide spectrum of animal diseases. cattle infected with ~,coplasma mycoides subsp, mycoides infection can either remain apparently healthy or develop contagious bovine pleuropneumonia (cbpp), a disease characterized by respiratory problems. post mortem findings should be followed by bacteriological culture of the organism from affected tissue which can take up to weeks to complete. the serological detection of antibodies is highly specific but asymptomatic animals in the early stages of infection and chronically-infected animals may not have detectable levels of antibodies. bashiruddin et al. ( ) described the use of pcr to detect specific dna in clinical material and isolates from outbreaks of cbpp in cattle and buffaloes in italy. these data showed that pcr can identify the aetiological agent within days of extraction of clinical material, and the specificity of the pcr test to distinguish lvl. subsp, nqcoides from other subspecies was confirmed. m~,coplasma hyopneumoniae has been identified as the causative agent of mycoplasmal pneumonia in pigs. because an effective vaccine is not currently available, efforts to control the disease have focused on the elimination of sick animals. unfortunately, efforts have been hampered by difficulties in differentiating m. hyopneumoniae fi'om crossreacting mycoplasma flocculare and mycoplasma hyorhinis. stemke et al. ( ) developed a method for differentiation of those three species on the basis of amplification of a s rrna gene sequence. pcr methodolog , for detection of mycoplasma gallisepticum have also been reported (nascimento et al., ; kempf et al., kempf et al., , . the organism is the cause of chronic respiratory coxiella lntrnetii, a zoonotic organism, is the aetiological agent of q fever. in humans, q fever occurs as a influenza-like illness, pneumonia, granulomatous hepatitis or chronic endocarditis. in animals, coxiella can reach high concentrations in the female reproductive system and infection can be followed by abortion or infertility. although the infection of cattle is usually latent, c. imrnetii may be shed via milk by infected cows for one o " several lactation periods. the organism can survive, in low numbers, for a long time in dairy, products made from non-pasteurized milk of infected cows and detection in milk requires a high-sensitive method. a pcr approach with primers based on repetitive transposon-like sequences have been established for the highlysensitive and specific detection of c. lncrnetii in cow's milk (willems et al., ) . leishmaniasis is a group of infestations of the viscera, skin and mucous membranes caused by protozoa of the genus leishmania. multicopy s rrna has been the basis of some pcr assays that specifically detects leishmania sp. (guevara et al., ; van eys et al., ) . kinetoplast dna (kdna) is a target of interest because both maxiand minicircles are present in each cell in multiple copies. however, it has proved to be difficult to select species-specific kdna sequences for diagnosis by pcr (smyth et al., ; l pez et al., ) , and it is important to investigate only small regions of minicircles to find species-specific sequences consmwed among strains of the same species. pcr has been used to detect leishmanias in conjunctival biopsies (roze, ) , showing that a number of cases of ocular inflammation can be attributed to this parasite. in some tropical countries, the protozoan parasites of the genus to,panosoma are responsible for life-threatening diseases in animals and humans, and pcr is now being used to evaluate the vectorial ability of glossina longipalpis in western africa (solano et al., ; weiss, ) . the cyst-forming apicomplexan parasite toxoplasma gondii infects a broad spectrum of vertebrates. domestic and feral cats are the definitive hosts but humans and other animal species can be infested by ingestion of oocysts or tissue cysts. overwhelming infestations, especially in innnunosuppressed individuals, may be fatal. application of pcr can quickly and accurately detect ". gondii in a varieg, of clinical specimens including formalin-fixed and paraffin embedded tissues (macpherson & gajadhar, ; wastling et al., ; hyman et al., ) . cryptosporidiosis is now recognized as an important cause of human and animal diarrhoea. pcr amplification combined with chemiluminescence can specifically detect cuptospofidium pa~vum dna present in fixed paraffin-embedded tissues (laxer et al., (laxer et al., , . species and strain differentiation of domestic fowl coccidia of the genus eimeria has also been achieved by pcr (procunier et al., ) . echinococcosis is a disease caused , larval stages of different cestode species of the genus echinococeus, especially echinococcus granulosus and echinococcus multiloeulafis. these species are widely prevalent and may cause severe disease in animals and humans. a pcr study including several independent e. multilocula, s isolates and various other cestodes revealed that the pcr product was obtained from genomic dna of all e. multilocula~ s isolates but not from dna of other cestode species (gottstein & mowatt, ) . the sensitivity of the e. granulosus pcr was evaluated experimentally and approached . pg of template dna, which con'esponds to the dna content of a single ecbinococcus egg (rishi & mcmanus, ) . a random amplified polynaorphic dna (rapd) method has permitted a detailed genetic analysis of swiss and spanish isolates ofe. granulosus (siles-lucas et al., ) . the application of pcr to detect echinococci can allow the identification of biopsy material ol)tained from liver lesions of unknown aetiology and the demonstration of adult-stage parasite tissue or eggs in samples derived from faeces, small intestines or anal swabs of definitive carnivore hosts (gottstein, ) . tapeworms of the genus taenia can cause human and animal taeniasis and cysticercosis. although the eggs fi'om tnenia solium and taenia saginata cannot be differentiated morphologically, a bp sequence that h ,bridize specifically to a single-copy gene sequence of t. sofium and not to t. saginata dna may be available in the future for rapid pcr diffe,'entiation (rishi & mcmanus, ) . lungworms are common parasites of ruminants, and to a lesser extent, horses. in cattle, they cause considerable economic losses due to weight loss and deaths. rapd-pcr has proved to be a valuable tool to examine genome differences among dict~,ocaulus species fi'om cattle, sheep and fallow deer (epe et al, ) . the nematode )ichinella spimlis can infect nearly all meat-eating animals. trichinellosis is transmitted within two cycles that can interact; a sylvatic cycle in wild animals and a domestic cycle in pigs which is the major source of human infestation. two different sets of primers have been developed specifically to discriminate domestic from sylvatic isolates (dupouy-camet et al, ; dick et al., ) . pcr has been able to detect, in situ, a single excysted lmwa, as well as a single encysted larva, in infected mouse muscle following boiling (dick et al., ) . rapd-pcr has also been useful for the identification of trichinella species (bandi et al., ; dupouy-camet et al., ) . pcr has ah'eady played an important role in studies of the epidemiology, taxonomy and patho-genesis of micro-organism infections in animals but is not yet used routinely for the diagnosis of any animal infectigus disease. in fact, pcr has become a routine tool only in research laboratories. however, infectious diseases will remain among the major areas for application of pcr detection and genotyping, offering the potential to analyse most micro-organisnas of veterinary importance by a single technique. although many systems have been developed, few have proceeded towards field trials or large-scale clinical evaluation, and pcr application to the routine analysis of biological salnples is still a major diagnostic challenge. most of the assays to detect microorganisms have high sensiti~t t with purified dna samples, but advances in sample preparation and detection of amplified products under field or clinical laboratol , conditions are needed in order to achieve high sensitivity with animal specimens. diagnosis of viral diseases should be a major target for pcr application because laboratou, tests tbr identification of viruses are either slow, expensive or insensitive. the technique has found large-scale application for the routine detection of human patlaogens such as hiv and hepatitis viruses. among animal viral diseases, pseudorabies, equine viral arteritis, bovine leukaemia and bovine viral diarrhoea are good candidates for early development. the approach should also be focused on viral diseases that have a deep socioeconomic impact in endemic regions, such as african swine fever or rinderpest. eradication programmes must include the diagnosis of sick animals, asymptomatic carriers and vectors, and often involve the rapid screening of a large number of samples for which pcr would be vel t useful. in relation to bacterial diseases, pcr can be used for the rapid detection of those pathogens whose in vitro cultivation is difficult, time-consuming or unavailable. rflp patterns using pcramplified dna is an excellent method for bacterial typing and has already been used for the identification of the bacterial strains involved in human foodborne outbreaks (hill, ) . parasitic infestations will probably be the last field of veterinal-y clinical diagnosis to incorporate pcr techniques, partly because of the relative scarcity of important parasitic diseases in the main countries where pcr research is being developed (weiss, ) . in conclusion, pcr will most likely become the standard diagnostic test in situations where either the micro-organism level is low, differentiation between, morphologically identical organisms is required, or whether the immune response to the infection is uninformative. as happened with the progressive introduction of enzyme-linked immunosorbent assays (elisa) as routine diagnostic tools, the existence of a strong demand for improved diagnosis methods will surely lead, in the next decades, to the development of pcrbased test kits suitable for field application. salnlonella identification by the polymerase chain reaction use of polymerase chain reaction to diagnose bovine leukemia-virus infection in calves at birth detection of bluetongue xfrus in clinical samples by pol)~nerase chain reaction detection of reticuloendotheliosis virus infection using the polymerase chain reaction polymerase chain reaction amplification of the genome of infectious bronchitis virus comparison of polymerase chain reaction and virus isolation for detection of epizootic hemorrhagic disease in clinical samples from naturally infected deer equine herpesvirus type : detection of viral dna sequences in aborted fetuses with the polymerase chain reaction direct detection of bovine leukemia x rus infection: practical applicability of the polymerase chain reaction random amplified polymorphic dna technique for the identification of tfichinella species & bostock, tiation of wild-and vaccine-type canine parvoviruses by pcr and restriction-enzyme analysis application of polymerase chain reaction to the detection of bovine ~firal diarrhea virus detection of leptospiraceae by amplification of s ribosomal dna comparison of an antigen capture enzyme-linked assay with reverse transcription-polymerase chain reaction and cell ctdture immunoperoxidase tests for the diagnosis of ruminant pesti~ rus infections. i/etefinmy microbiolo~ the horserace betting levy board's code of practice for equine viral arteritis for the breeding season. veterinar) the development and assessment of dna and oligonucleotides for the specific detection of baduus anthracis specificity of pol)anerase chain reaction identification of toxoplasma gondii in paraffin-embedded animal tissues pcr protocols: a gume to methods and applications infections bronchitis virus detection in allantoic fluid using the polyrnerase chain reaction and dna probes developlnent of polymerase chain reaction assays for detection of lis-te~vta monoo, togenes in clinical cerebrospinal fluid samples detection of newcastle disease virus rna in infected allantoic fluids by in vitro enzymatic amplification (pcr). archives of vimlo~ improved methods for the detection of bacillus anthracis spores by the polymerase chain reaction detection of foodborne microbial pathogens using polymerase chain reaction methods two-step polymerase chain reaction and restriction endonuclease analyses detection and differentiation of ompa dna of chlamydia spp diagnosis of rabies by polymerase chain reaction with nested primers detection of pathogenic yersinia entercolitica in foods and water by immtmomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified dna antigenic differences between european and american isolates of porcine reproductive and respirator), syndrome virus (prrsv) are encoded by the carboxyterminal portion of viral open reading frame polymerase chain reaction analysis of borrelia species isolated in japan early detection of hovine leukemia virus in cattle by use of the polymerase chain reaction the polymerase chain reaction for the detection of l~'l~coplasma gallis'epticum mycoplasma gallisepticum infection in drugtreated chickens: contparison of diagnosis methods including polymerase chain reaction detection of i}ovine irnmunodelicie,+~cv virus in bh}{}d arm milk-<lerived leukocvtes i}v use of polymerase chain reaction. american .+]ourna'l of "elerina~ identil]cati{m of the thvmidine kinase gene {} leline herpesvirus: use {} degenerate {}ligonucleotides in the l}olymerase chain ,'eaction t{} isolate he,-l}esvirus gene homoh}gies detection {}t thnbrial and toxin genes in l'scherir'hia col~ an{i their prevalence in piglets with diarrhea. the application of cohmv h\'l}ridizati<}n assay, i}{}lymcrasc chain ,cat +-tion and pl{en{}tyl}ic assass prevalence of born'lia im~ gdo(fi'ri sensu htt{}-inli'{ted ticks on mig pr()viral scqtte,tces detected }v the p()lymerasc chain rcacti{}n in peripheral i} <}{}{ cells {}l horses with equine infectious ,uremia lentivirus. .-i)chive.s +!/ roh)g identilicalion and sequell{t' anal\'sis ()f is . an insertion sequence in b)ucelkt spp ts()xs( experimentally induced int+cction with i)luetonguc virus ser()type in cows. amrr/can tim;,-hal o/ i "elevina+ ' re ( `¢) ). l{lentilicalion (}t her{i-specific b()\'ine viral dia nini"i, b. & bii.i.i", j. (i`¢).{-) ). studies of the detection ol + l.isleria mom,o'lo(-em,s i}v cuhure and pcr in cerel}r()spinal lluid santpl{:s from runfinants with listeric encephalitis species and strain differentiation {)t eimeria spp. <}f the d{)mestic owl using dna polynaorplaisn~s amplified i}v arl}itrarv primers h{){;.-\x developmer~t {}fa pcr amplification assay as a screening test using bulk milk samples ti}r identity'trig dairy herds infected with bovine viral diarrhea virus. "ele/'ina u a.licrobiolo~' amplification of an inva gene sequence of sahnonella lyphimurium by i}olymerase chain reacti(m as a specific method of detection of salmonella specific detection of pathogenic }+ersinia enterocoli/ica by two-step pcr using hot-start de tection of }'et:sinia entemcolitica : in [aecal slunples and tonsil swabs from pigs using ims and p(:r identit]clttion o capsule-forming bacillu.s anlhraci,~ spo,es with the p(:r and a novel dual probe hybridization lin i.)electit}n of ,(clive ,ind latent i+cline herl}esvirus i infi:ctions ttsing the polynwrase chain reaction+ air'hive+ detection ot + t('lint" inmnulodcficiencv virus by a nested +<}lvmet'asc chaiu reaction (;cu{m/ic chining {}l" httlllall lic/fin,u'm'cu.s ffranul, su.+ dna is{}htti{m <}f rec<mtl}inanl pl,lsmids and their use as genetic nlarkers in strain characterizatitm, ljara.+ilolo.&q molecular chining {}f l'aenia .wdium gen{>mic dna aud charactcriz-ati<m {}f taeniid cest{}{ics by dna anah'sis. i}ara.+iloh~q ' primer design., for specific diagnosis by. p(:r {} highly vatial}h: rna vnuses: typing of ib<}t-and-mouth disease virus. i'iroh~& a specific a,td sensitive assay fi}r the ixmc disease spir{}chete borrelia blt~:&~dorfi,ji itsing th{: i}{}lymerase clmi,t reaction p{}h'nlerase chain reaction: a rev{}l-uti{}n ill diagn{}sis {'}f ocular lcishmaniosis? pcr technique as an alternative meth<}d li} enzymatic amplitication of [b-gl{}bin genomic sequences and restricti{m site analysis li}r diagnosis of sickle cell armmia primer-directed enzymatic aml}lification {}f dna with a tlaerntostal}le dna polymerase pcr of wim-type c:mine parvm'i,us which colll~llllillales dog vaccines rabies virus into the cenlral nela.'()tls svsleln willlout prior local replication diagn<~sis ofequid herl)esvirus and l)y polymerase chain rcaclion. equi,e l'eleri amplithation and anah'sis of specific dna and rna sequences of bm'iile leukemia \'irus from infected cows by polynterase chain reaction compa,ative genetic anah'sis of swiss and spanish isolates of kchi,ocmcu.s ,~,rrt',ulo.s,.~ by soulhern hvl)l"idization and randomh differenliation o pallmgenic and n<m-pathogenic serotvl)e marek's disease \'iruses (mdvs) by the l)olymerase chain reaction amplification of the tandem di,ect repeats within tile mdv ge verocvtotoxin productio,l and presence of vt genes in l rapid and sensitive detection of ldshmania kenetoplast dna from spleen and blood samples of kala-aza utilisation de l'hybridisation mol culaire et de i'amplification en chaine par polym rase pour la cha,'act risation des trypanosomes et l'epidemiologie des tltpanosomes en af,ique de i'quest. veterinmy re hdicobacter cants, sp. nov, a new species fi'om dogs: an integrated study of phenotype and genolype in sire amplification of visna virus dna in tissue sections ,eveals a reservoir of latenth rapid and biologically sail: diagnosis of afi-ican swine fever by using polwnerase chain reaction diffe,entiation of mycoplasma hyolmeumoniae, mycoplasma /loccuhtre, and mwvpla.~ma hvorhi,i.~ on tile basis of ampliticalion of a s rrna gene sequence nucleotide and deduced amino acid sequence of the nucleocal)sid protein of the virulent a / -cdv strain of canine distemper virus, i "eterinm)' microbiolo detection of .mrican horse sickness vi,us by reverse transcription-pcr, flmr, al o~ cli,ical microbir identification of foot and mouth disease virus (fmdv) ser()t.vpes using the i)olymerase chain reaction. l~rael.flmrnal r!/ i ?leri detection and subtypi,]g of foot-and-mouth disease virus in infccted cattle by polymerase chain reaction and amplified vp sequencing../our, al oj i >leri,a u diaff hnproved earh' and long-term detection o bovine lentivirus by a (lested polymerase chain ,eaction test in experimentally infected cah'es. america (.t ). specific detection of clostridium botulinum type g by using the polymerase chain ,eaction. applied a,rl ' n vimn mental ~ licrobiolo detection of the genes encoding botulinum neurotoxin types a to e by the polymerase chain reaction. applied and envirimmental microbiolg~ attempt to discriminate between bovine viral diarrhea virus strains using polp tt:aym' identification of verotoxin type variant b subunit genes in ewhe~qchia coli by the polymerase chain reaction and restriction fiagment length polymorphisna analysis rapid method utilizing pol)qnerase chain reaction tor detection of canine parvovirus in feces of diarrheic clogs. i:eterina o' microbiolo~ comparison of phage typing and dna fingerprinting by pcr lor discrimination of methicil]in-resistant staphyh)coccus aureus strains detection of leptospires in urine by polymerase chain reaction sequence analysis of small subunit ribosolnal rna genes and its use for detection and identification of leishmania parasites detection of proviral dna and viral rna in various tissues early after avian leukosis infection \:l-:xt'~ reco,nbinant vaccinia virus expressing prm and e glycoproteins of louping-ill virus: induction of partial homologous and heterologous protection in mice polymerase chain reaction for probe synthesis and for direct amplification in detection of bovine coronavirus detection of bovine herpesvirus- (bhv-i) genome by pcr comparison of two morbilliviruses isolated from seals during outbreaks of distemper in latency of a thymidine kinase-negative pseudorabies vaccine virus detected by the polymerase chain reaction. archives of ~'rology development of tile polymerase chain reaction for the detection of hluetongue virus in tissue samples detection of eqnine herpesvirns and differentiation of equine herpesvirus type i characterisation of african isolates of rindepest virus. l'elelqnmy microbiolo~ detection of bovine viral diarrhea virus using degenerate oligonucleotides primers and the polymerase chain reaction. american ./ou rnal of i "eterina o' reseaith detection of mycobacterium boris in tissues by polymerase chain reaction. vete~ nan., micmbiolo© comparation of two gene amplification methods for the detection of )moplasma gondii in experimentally infected sheep..fimrnal of medical microbiolo© specific detection of sahnonella spp. by multiplex polylnerase chain reaction. ap/died and environmental mioobiolo© dna probes and pcr for diagnosis of parasitic infections investigation of sites of pseudorahies virus latency, using polymerase chain reaction. ame~ can .fimrnal of l:t wlt~l(~loa'rm<)t) the magnetic im,ntmo-polymerase chain reaction assay for direct detection of sahnonellae in fecal samples detection of bovine herpesvirus- in bovine semen by a nested pcr detection of coxiella burnetti in cow's milk using the polymerase chain reaction nested multiplex polylnerase chain reactions lor the identification of bluetongtte virus infection in the biting midge cul-licoide~" variipennis detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction development of a probe and pcr primers specific to the virulence plasmid of salmoneua entet~tidis detection ot" entero-and verocyto-toxin genes in esch ion of pathogens elqchia coil fi'om diarrhoeal disease in animals using the pol)~nerase chain reaction. vete~qnmy microbiolod detection of infectious bursal disease virus in digested formalin-fixed paraffin embedded tissue sections by polymerase chain reaction iviolecular detection of infectious bursal disease virus by polymerase chain reaction comparison of dot-blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus- (bhv- ) in at'tificiallv-infected bovine semen the application of polymerase chain reaction to the detection of rotaviruses in lheces establishment of conditions lbr the detection of bovine herpesvirus- by polymerase chain reaction using primers in the thymidine kinase region amplification-based diagnostics target tb genomic heterogeneity of small ruminant lentiviruses detected by pcr some characteristics of a recent virus isolate of marek's disease virus differentiation of oncogenic and nononcogenic strains of marek's disease virus type by using polymerase chain reaction dna amplification diagnosis of the african horse sickness virus serotype by a one-tube, one manipulation rt-pcr reaction fi'om infected organs polymerase chain reaction for detection of bm~elia coriaceae, putative agent of epizootic bovine abortion rapid detection and identification of avian infectious bronchitis virus key: cord- -s ezxi r authors: principi, nicola; rigante, donato; esposito, susanna title: the role of infection in kawasaki syndrome date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: s ezxi r objectives: to analyse the evidence suggesting a possible infectious origin of kawasaki syndrome (ks). methods: pubmed was searched for all of the studies published over the last years using the key words “kawasaki syndrome” or “mucocutaneous lymph node syndrome” and “infectious disease” or “genetics” or “vasculitis” or “pathogenesis”. results: various levels of evidence support the hypothesis that ks is a complex disease triggered by an infection due to one or more pathogens. viruses or bacteria may be the primum movens, although no specific infectious agent can be considered definitely etiological. a number of genetic polymorphisms have been identified in subjects with ks, but none of them can currently be considered a real marker of susceptibility. conclusions: various data suggest that ks is intimately related to infectious diseases and that its clinical expression is influenced by predisposing genetic backgrounds, but our knowledge of the infectious agent(s) involved and the genetic characteristics of susceptible children remains only partial. further studies are needed to address the many still open questions concerning the disease. kawasaki syndrome (ks), originally called "mucocutaneous lymph node syndrome" in , is an acute multisystem vasculitis that causes generalised inflammatory cell tissue injury starting in the vascular endothelium and is mainly encountered in children aged less than five years regardless of their ethnicity. the injuries are particularly severe in the coronary arteries, which are frequently affected by dilatations, aneurysms or fistulae, especially in patients who do not receive prompt treatment with intravenous immunoglobulins and anti-inflammatory doses of acetylsalicylic acid. now that rheumatic fever is largely controlled, ks has become the leading cause of acquired heart disease among children in industrialised countries. as no specific diagnostic test is available, ks is identified on the basis of a constellation of non-specific clinical signs. according to the american heart association guidelines, which are shared by most scientific communities throughout the world, a diagnosis requires prolonged fever lasting more than five days and at least four of the following signs: ) diffuse oral cavity inflammation (including pharyngeal infection), dry fissured lips, and a strawberry tongue); ) bilateral non-purulent conjunctivitis; ) heterogeneous skin rashes; ) angioedema of the extremities (including peripheral erythema, oedema, or induration of the hands or feet); and ) non-purulent cervical lymphadenopathy exceeding ! . cm in diameter. in what are known as "incomplete" cases, one or more of these clinical signs may be absent but a diagnosis can still be made if there is echocardiographic evidence of coronary artery abnormalities. furthermore, a broad range of unusual clinical finding have been reported as defining the "atypical" variant of ks, including aseptic meningitis, peripheral facial nerve palsy, uveitis, gastrointestinal complaints, acalculous gallbladder hydrops, urethritis, testicular swelling, pulmonary nodules, liver impairment with jaundice, and even hemophagocytic syndrome. laboratory investigations (listed in table ) can only support the diagnosis of ks, but they still need to be validated before they can be used in everyday clinical practice. however, although the clinical features of ks are usually recognisable, its underlying immune mechanisms are still being investigated. most experts consider it to be the consequence of an abnormal immunological response evoked by one or more widely distributed infectious agents in genetically susceptible individuals, but it still remains a medical enigma. the main aim of this review is to analyse the available evidence suggesting that ks may have an infectious origin. pubmed was used to search for all of the studies published over the last years using the key words: "kawasaki syndrome" or "mucocutaneous lymph node syndrome" and "infectious disease" or "genetics" or "vasculitis" or "pathogenesis". more than articles were found, but only those published in english or providing evidence-based data were included in the evaluation. various levels of evidence support the hypothesis that ks is a complex disease initiated by an infection due to one or more pathogens (table ) . however, no strict and unmistakable correlation between specific infectious agents and the development of the disease has ever been identified. a number of bacterial and viral infectious agents have been sporadically isolated from ks patients. the most frequently implicated bacteria are staphylococcus aureus, streptococcus pyogenes, and atypical pathogens, e and the viruses associated with ks over recent years are epsteinebarr virus, adenovirus, parvovirus b , herpesvirus , parainfluenza type , measles, rotavirus, dengue virus, and human immunodeficiency virus. varicella, h n pandemic influenza and coxsackie b virus have also been described in patients with ks, but they were equally found in the blood or body fluids of both patients and healthy subjects. moreover, no relationship was reported between ks and the circulation of the commonest respiratory viruses. the most recent and numerous studies of ks-related viruses have postulated the etiological role of human coronavirus (hcov) nl and bocavirus, e but this has not been confirmed by subsequent studies. in order to evaluate the importance of hcov-nl in ks, shimizu et al. established a multi-institutional collaborative research project to test respiratory samples using realtime polymerase chain reaction (rt-pcr), and found that only one out of ks patients ( %) was positive ; dominguez et al. found exactly the same prevalence in nasopharyngeal wash samples from ks patients and healthy controls over a period of seven months ; and lehmann et al. measured the concentrations of igg, igm and iga antibodies against hcov nl and oc in the blood of children showing the signs and symptoms of ks for e days and healthy controls, but did not find any difference between the two groups. the data regarding bocavirus (a virus that has recently emerged as a possible cause of respiratory infection) are also unconvincing : although it was identified in the serum, stool and cerebrospinal fluid of some children with ks, no definitive conclusion could be drawn concerning its etiological role. the limited etiological importance of the pathogens so far identified seems to be supported by the studies of benseler et al. and jordan-villegas et al., who found that that concomitant infections in children with ks did not alter the response to treatment with intravenous immunoglobulins, and did not influence the risk of coronary artery involvement or affect overall cardiovascular outcomes. however, the lack of any clear relationship between one or more pathogens and the development of ks does not exclude the possibility that a real infectious disease may be involved, and other factors support this hypothesis. on the other hand, the unsuccessful identification of a specific pathogen to which ks could be ascribed has led some authors to postulate that variants of normal flora in the gut, oral cavity or skin of young children with a genetic defect of proper immune maturation do not induce immune tolerance as self commensals, but rather induce an imbalance of the immune system, leading to a hyperimmune reaction and the manifesting ks. table laboratory findings supporting a diagnosis of kawasaki syndrome. . c-reactive protein ! . mg/dl . erythrocyte sedimentation rate ! mm/h . albumin . g/dl . age-relative anaemia . high alanine aminotransferase levels . platelet count ! , /mm in the subacute phase of the disease . white blood cell count ! , /mm . white blood cells/high-power field ! in urinalysis histopathological data some histological findings strongly suggest that ks has an infectious origin, although they do not identify the responsible pathogen. the most important is that persistent intracytoplasmic inclusion bodies (iibs) showing amphophilic staining by haematoxylin-eosin and stain for rna have been found in most of the tissues of patients who have died because of ks. as transmission electron microscopy (tem) of bronchial epithelia has revealed virus-like particles associated with the iibs, it was thought that these may be a "footprint" of a viral infection that may persist indefinitely: rowley et al. speculated that the infection associated with the development of ks could be due to a new and ubiquitous rna virus that caused only asymptomatic infection or very mild disease in the general population, but ks in genetically selected subjects. it was thought that the first site of infection was the respiratory tract, with further dissemination through macrophages to all body sites, including the medium-sized arteries (mainly the coronary arteries) that are the most crucial targets in ks. further findings that strongly support an infectious origin of ks are those of orenstein et al., who used light microscopy and tem to study tissue specimens from autopsies, eight heart transplants and an excised coronary aneurysm of patients with ks and identified three different vasculopathic processes: acute self-limited necrotising arteritis (na), subacute/chronic vasculitis, and luminal myofibroplastic proliferation. on the basis of the changes in coronary and non-coronary arteries during the different phases of ks, they considered na the only self-limiting process of the three, and that it was consistent with an acute viral infection. many epidemiological findings regarding ks are consistent with an infectious origin, as they are quite similar to those of various infectious diseases. first of all, there is the occurrence of epidemics because, although cases of ks are diagnosed, there are also sometimes widespread epidemics. in japan, where nationwide epidemiological surveys of ks have been carried out almost every two years since , three large-scale epidemics were recorded in , , and , with incidence rates that were several times higher than those reported in the previous and following year. secondly, as in the case of a number of infectious diseases, , ks is more frequent in boys, and the male to female ratio is about . . thirdly (once again as in the case of many infectious diseases), the incidence of ks seems to be closely related to weather conditions, although the predominant season varies from country to country: winter and spring in the united kingdom, australia and the usa, and spring and summer in china. e pitzer et al., examined seasonal changes in the age and incidence of ks hospitalisations in the usa, and found that periods of high incidence corresponded to a low average age, and vice versa. comparison of the observed pattern with those predicted by a suite of models based on different etiological hypotheses, the ageincidence pattern of ks suggested the involvement of an imperfectly immunising infection and/or a persistently infectious agent. the possible relationship between seasonal variations in the incidence of ks and an infectious aetiology is also supported by data showing that its incidence in the usa inversely correlates with average monthly temperature (r z À . ; p < . ) and positively correlates with the role of infection in ks average monthly precipitation (r z À . ; p < . ). moreover, analyses of the three major ks epidemics in japan, major non-epidemic inter-annual fluctuations of ks cases in japan and san diego, and seasonal variations in the incidence of ks in japan, hawaii and san diego have revealed a consistent pattern linking ks cases to large-scale wind currents originating in central asia and crossing the north pacific. this seems to indicate that the environmental trigger of ks may be wind-borne, and this has led some experts to suggest that efforts to isolate the causative agent should concentrate on the microbiology of aerosols. finally, children in the first months of life only exceptionally develop ks, which supports the hypothesis that most infants are protected by passively acquired specific maternal antibodies against the possible causative agent(s). on the other hand, the very low incidence of ks beyond the fifth year suggests that most children are infected uneventfully by one or more of the infectious agents associated with ks in early life and can then mount a long-lasting, strong and protective immune response. recurrences of ks have been reported in only e % of children, and are best documented in japan. the clinical findings of fever, an erythematous pharynx, conjunctival injection, rash, oedema of the extremities and cervical adenitis in patients with ks, and the clear tendency of these signs to resolve spontaneously even without treatment also support an infectious aetiology. a number of viral diseases have a similar clinical picture. moreover, some of the clinical features of ks, such as mucous membrane erythema and desquamation of the fingers and toes (usually beginning within e weeks of the onset of fever), overlap those associated with some bacterial diseases that are considered to be a consequence of the superantigen (sa)-mediated activation of t cells leading to the overproduction of cytokines, systemic inflammation and shock. e the best examples in this regard are toxic shock syndrome (tss) and scarlet fever due to s. aureus, and streptococcal toxic shock syndrome (stss) due to streptococcus pneumoniae. the similarity between these conditions and ks has led to the conclusion that, at least in some cases, ks may follow an infection due to an saproducing pathogen. a number of sas have been identified: the most widespread bacterial pathogens are s. aureus and s. pneumoniae, but yersinia pseudotuberculosis, mycoplasma pneumoniae and mycobacterium tuberculosis have also been associated with sa formation ; among viruses, sas have been found in epstein barr virus, rabies virus and mouse leukaemia virus. sas are a family of potent immunostimulatory proteins whose particular structures and sequences lead to a shared ability to by-pass the mechanism of conventional major histocompatibility complex (mhc)-restricted antigen processing. when an sa is involved, t cell responses are quantitatively and qualitatively different from conventional t cell activation by normal antigens. in particular, sas activate t cells in a manner that depends on the t cell receptor variable domain (vb), and so a large number of t cells can be simultaneously activated. the activation is extremely potent and a number of studies have found that ks is characterised by the marked activation of t cells and monocytes/macrophages, and increased production of il- b, tnf-a and il- , which are the same immunological findings as those of tss. although some attempts to demonstrate the presence of sa-producing pathogens in children with ks have led to negative results, e others have provided data suggesting the direct involvement of sas. leung et al. blindly studied bacterial sas potentially involved in the pathogenesis of ks in cultures of patients in the acute phase and controls, and found sa-producing bacteria in of the patients but in only one of the controls (p < . ). eleven of the toxin-positive cultures from the patients with ks contained toxic shock syndrome toxin (tsst)- secreting s. aureus, and the remaining two contained streptococci producing streptococcal pyrogenic exotoxin b (speb) and streptococcal pyrogenic exotoxin c (spec). twelve of the culture-positive patients had toxin-producing s. aureus isolated from pharyngeal or rectal cultures, thus suggesting the gastrointestinal tract as the primary entry site. similar findings of tsst- -producing s. aureus and spec-producing streptococci in children with acute ks have been recently published. the sa theory may be supported by anecdotal reports of ks patients with guttate psoriasis because it has been suggested that this form of psoriasis is due to toxinmediated t cell activation. furthermore, a number of studies analysing the t lymphocyte receptor repertoire and the titres of antibodies against selected sas have indirectly demonstrated that these proteins may be related to the development of ks. , e in addition, suenaga et al. examined five sa genes in the stools of ks patients, febrile controls and healthy children, and found at least one of the genes in specimens from the patients with ks ( %), in from the febrile group ( . %), and in seven from the healthy group ( . %). the detection rate between subjects with and without ks was of at least one of the sa genes (p < . ), and more than two sa genes were significantly different (p z . ), thus suggesting the direct involvement of sas in the development of ks. despite the direct and indirect evidence supporting the hypothesis that ks is an infectious disease, only "susceptible" subjects seem to develop it and genetics seem to play a role in selecting the patients. ks occurs throughout the world, but is significantly more common in some asian countries, such as japan, korea and taiwan. it has been reported that the annual incidence of ks in japan in and was respectively . and . per , children aged e years ; on the contrary, a recent analysis found that the ks-related hospitalisation rate in the usa was per , children aged < years, and even lower incidence rates have been calculated for a number of european countries. e theoretically, various factors could explain this large difference. the incidence of ks is rapidly increasing throughout the world, and so surveys carried out at different times may lead to different results. moreover, although ks is significantly more frequent in younger children than in older children, adolescents or adults, its frequency in younger patients also varies. a comparison of the incidence of ks in northern european and japan found that . % of the japanese patients were aged < years, but . % of the cases diagnosed in norway, finland, sweden and denmark (p < . ). the incidence of ks in different regions may be affected by differences in surveillance methods, clinical diagnostic and treatment practices, physician awareness of ks, and the occurrences of ks clusters or outbreaks. however, a global evaluation of all these factors suggests that they are unlikely to be the only reason for such a striking difference. furthermore, american studies have clearly shown ethnicity-related variations in the incidence of ks: in comparison with white subjects, the incidence is twice as high among asians and pacific islanders, and about . times higher among black subjects. similarly, its incidence in hawaii (the state with the highest proportion of asians and pacific islanders) is . times higher than that reported for the continental usa. it has also been reported that the incidence of ks is e times higher among the siblings of ks patients than in the general population, and that children with ks are more likely to have parents who have had the disease. it has been calculated that the inheritability of ks (i.e. the ratio of the incidence of ks between siblings and the general population) is only slightly less than that of type diabetes and about four times more than that of allergic asthma. all of the above findings strongly suggest that genetic factors play a substantial role in the occurrence of ks, but studies of the genetic characteristics of ks patients have not definitively identified which genetic marker(s) may favour or protect humans from developing of ks, or regulate its clinical severity. several candidate genes, mainly chosen among those related to innate and acquired immunity, cardiovascular function, and vascular remodelling, have been tested in patients with ks (table ). e results were frequently negative or conflicting, particularly when the studies enrolled a limited number of patients and were carried out in populations with significant racial differences that impact the allele frequency of some of the single nucleotide polymorphisms (snps) analysed in the studies. good example at this regard are the data collected on the role of matrix metalloproteinases (mmps), fc gamma receptors (fcgr) and of cd ligand snps in conditioning susceptibility, evolution and outcome of ks. mmps play an important role in processes that degrade extracellular matrices. their activity is controlled by specific inhibitors (timps) and imbalances between mmps and timps are associated with several pathological conditions, including vascular aneurysm. association of increased mmp /timp and mmp / timp ratios with risk of coronary artery lesions was found in japanese children. on the contrary, no association was found between snp of mmp- in the korean population. debated is also the role of cd l, a transmembrane protein that engages with cd and transduces signals related to cell activation and development because a strict association between snps and development and severity of ks was demonstrated in the japanese patients but not in the taiwanese population. however, in some studies more convincing results were found particularly when they could evidence that the same polymorphisms were present in populations with different racial characteristics. onouchi et al. reported that multiple variants in the caspase- gene (casp ) that were in linkage disequilibrium conferred susceptibility to ks in both japanese and us subjects of european ancestry. these authors found that a g to a substitution of one commonly associated snp located in the untranslated region of casp (rs ; p z . Â À in the japanese and p z . Â À in the european americans) abolished binding of nuclear factor of activated t cells to the dna sequence surrounding the snp suggesting that altered casp expression in immune effecter cells influences susceptibility to ks. interesting results were also reported by shimizu et al. that investigated genetic variation in genes belonging to the tgf-b pathway in a total of kd subjects of mainly european descent from the united states, the united kingdom, australia, and the netherlands. genetic variants in tgfb , tgfbr , and smad and their haplotypes were consistently and reproducibly associated with ks susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. a smad haplotype associated with ks susceptibility replicated in independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (a/g, rs ; p z . ; or, . ; % ci, . e . ). pathway analysis using all genes further confirmed the importance of the tgf-b pathway in ks pathogenesis. because similar data regarding susceptibility were reported by kuo et al. it was concluded that genetic polymorphisms in tgfb signalling pathway are strictly associated with the risk of development of ks. however, more reliable results have been obtained using genome-wide association studies (gwas) because genome scanning without a defined hypothesis has the advantage of identifying disease genes even if the functions of these genes are not associated with previous knowledge about the disease's pathophysiology. even in this case, the most important results were those repeatedly reported in different populations. by linkage disequilibrium mapping performed on the region of chromosome q . , it was found that an snp within the inositol , , -trisphosphonate -kinase c (itpkc) gene, a gene that regulates the signal transduction in t lymphocytes and the degree of inflammatory response, was associated with increased susceptibility to ks and with the development of coronary artery in both a japanese and a usa population. further data confirming the relationships between genetics and ks were collected by burgner et al.. these authors conducted a gwas with dutch ks cases and controls and followed up associations signals with a family-based association study of ks families from australia, usa, and uk. they reported that snps in intron of n-acetylated a-acidic dipeptidase-like (naaladl ), a protein possibly involved in immune homeostasis, were associated with ks (p z . Â À ) and that mrna expression of the same protein in erythrocytes was significantly lower in the acute phase of ks than in the convalescence period. the presence of a predisposing genetic system favouring the development of ks and regulating its severity was confirmed by the study of kim et al., korean ks patients and healthy controls, and found that genomic regions with one or more sequence variants were associated with ks, and were associated with coronary artery lesions (cals) (p < Â À ). an snp within the disabled homologue (dab ) gene locus in chromosome (rs ) was replicated in children with ks and the most strongly associated snps detected in the joint analysis corresponded to three novel loci. among kd-associated snps, three were close to the copb (coatomer protein complex beta- subunit) gene: rs (p z . Â À ), rs (p z . Â À ), and rs (p z . Â À ). moreover, an snp in the intronic region of the erap (endoplasmic reticulum amino peptidase ) gene (rs , p z . Â À ) and six snps (rs , rs , rs , rs , rs , and rs ) clustered in an area containing immunoglobulin heavy chain variable regions genes, with p-values between . Â À and . Â À , were also identified. because these kd candidates have been implicated in t cell receptor signalling, regulation of proinflammatory cytokines, as well as antibody-mediated immune responses, these findings strongly supported the relationships among genetics, alterations in immune response and development of ks. association of snps within the fcgr gene cluster on chromosome with ks was identified by khor et al. in european and asian populations. in a large study sample ( individuals with ks and controls) including european and asian populations, these authors found that two loci exceeded the formal threshold for genome-wide significance. the first was a functional polymorphism in the igg receptor gene fcgr a encoding an h r substitution (rs ; p z . Â À , or z . ), with the a allele (coding for histidine) leading to a high risk of disease. the second was at q (p z . Â À , or z . for the rs snp near mia and rab b; p z . Â À , or z . for rs in itpkc ), thus confirming the data previously with previous studies. the involvement of the fcgr a locus may have implications for understanding immune activation in ks pathogenesis and the mechanism of response to intravenous immunoglobulin, the only proven therapy for this disease. more recently, japanese and taiwanese groups independently reported a significant association between ks and polymorphisms in the intergenic region on chromosome p -p between b lymphoid kinase (blk ), a tyrosine kinase involved in b-cell receptor signal transduction and fam a, a functionally uncharacterized gene. , onouchi et al. undertook a gwas involving japanese individuals with ks and japanese controls genotyped at , snps. they validated the results in two independent replication panels of cases and controls, and observed significant associations in the fam a-blk region (rs , p z . Â À ). similar results were obtained by lee et al. in individuals with ks and controls in a han chinese population residing in taiwan, with replication in an independent han chinese sample of cases and controls. they found that polymorphisms at blk gene together with genetic abnormalities at cd , were associated with ks at genomewide significance (p < . Â À ) confirming the role of immune activation and inflammation in the pathogenesis of the syndrome. however, despite these findings, the correlations between genetic markers the risk of developing and severity of ks are far from clear. at the moment the most convincing evidences of a strict correlation between genetic abnormalities and ks regards polymorphisms of itpkc, fcgr, casp and tgfb genes. although various data suggest that ks is an infectionrelated clinical syndrome that can only develop in children with predisposing genetic backgrounds, our knowledge of the infectious agent(s) involved and the genetic characteristics of susceptible children is still unsatisfactory. either viruses or bacteria could act as disease, but no specific infectious agent can be considered the definite cause of ks, and so no specific anti-infective therapy can be developed. moreover, although potential genetic determinants have been hypothesised in subjects with ks, none of them can yet be considered real markers of disease susceptibility. consequently, the pathogenesis of ks is only partially known and measures to prevent it remain elusive. further studies are needed to address the many still open questions concerning this still enigmatic disease. a new infantile acute febrile mucocutaneous lymph node syndrome (mlns) prevailing in japan current recommendations for the pharmacological therapy in kawasaki syndrome and management of its cardiovascular complications kawasaki syndrome: an intriguing disease with numerous unsolved dilemmas diagnosis, treatment, and long-term management of kawasaki disease: a statement for health professionals from the committee on rheumatic fever, endocarditis, and kawasaki disease, council on cardiovascular disease in the young discrimination between incomplete and atypical kawasaki syndrome versus other febrile diseases in childhood: results from an international registry-based study kawasaki syndrome-like illness associated with infection caused by 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explore possible infectious aetiologies relationship of climate, ethnicity and socioeconomic status to kawasaki disease in san diego county association of kawasaki disease with tropospheric wind patterns infectious disease: blowing in the wind cardiac sequelae of kawasaki disease among recurrent cases superantigen: structureefunction relationships superantigen-induced regulatory t cells in vivo the role of superantigens of group a streptococcus and staphylococcus aureus in kawasaki disease tcr vb family repertoire and t cell activation markers in kawasaki disease characterization of cd þ t helper cells in patients with kawasaki disease (kd): preferential production of tumor necrosis factoralpha (tnf-a) by vb _ or vb _ cd þ t helper cells the absence of evidence of staphylococcal toxin involvement in the pathogenesis of kawasaki disease serologic evidence that streptococcal superantigens are not involved in the pathogenesis of kawasaki disease prevalence of superantigen-secreting bacteria in patients with kawasaki disease association of psoriasis-like eruption and kawasaki disease analysis of t-cell receptor v-beta in peripheral blood lymphocytes as a diagnostic marker for kawasaki disease selective increase of v beta þ t cells in the small intestinal mucosa in kawasaki disease t cell activation profiles in kawasaki syndrome detection of multiple superantigen genes in stools of patients with kawasaki disease epidemiologic features of kawasaki disease in japan: results of the e nationwide survey epidemiology of kawasaki disease in asia, europe, and the united states rising incidence of kawasaki disease in england: analysis of hospital admission data kawasaki syndrome hospitalizations in ireland increased detection rate of kawasaki disease using new diagnostic algorithm, including early use of echocardiography incidence of kawasaki disease in northern european countries hospitalizations for kawasaki syndrome among children in the united states racial/ethnic differences in the incidence of kawasaki syndrome among children in hawaii kawasaki disease in families kawasaki disease in parents and children the value of isolated populations in genetic studies of allergic disease high incidence of angiotensin i converting enzyme genotype ii in kawasaki disease patients with coronary aneurysm polymorphism of angiotensin- converting enzyme gene and kawasaki disease insertion/deletion polymorphism of angiotensin converting enzyme gene in kawasaki disease possible synergic effect of angiotensin-i converting enzyme gene insertion/deletion polymorphism and angiotensin-ii type- receptor a/c gene polymorphism on ischemic heart disease in patients with kawasaki disease common variants in casp confer susceptibility to kawasaki disease genetic variations in the receptor-ligand pair ccr and ccl l are important determinants of susceptibility to kawasaki disease the ccr (- c/t) polymorphism may be associated with the development of kawasaki disease in korean children polymorphisms in chemokine receptor genes and susceptibility to kawasaki disease genetic polymorphisms in the cd ligand gene and kawasaki disease inflammatory gene polymorphisms and susceptibility to kawasaki disease and its arterial sequelae polymorphism of fc gamma riia may affect the efficacy of gamma-globulin therapy in kawasaki disease the involvement of fc gamma receptor gene polymorphisms in kawasaki disease association of il- ra gene polymorphism, but no association of il- beta and il- gene polymorphisms, with kawasaki disease the - c/t and a/g interleukin- polymorphisms are not associated with kawasaki disease in taiwanese children interleukin gene promoter polymorphism is not associated with kawasaki disease the il- (- a/c) promoter polymorphism may be associated with coronary aneurysms and low serum albumin in korean children with kawasaki disease influence of interleukin promoter polymorphisms in susceptibility to kawasaki disease in taiwan inducible and endothelial constitutive nitric oxide synthase gene polymorphisms in kawasaki disease increased frequency of alleles associated with elevated tumor necrosis factor-alpha levels in children with kawasaki disease association of mannose-binding lectin genotype with cardiovascular abnormalities in kawasaki disease polymorphisms in the mannose-binding lectin gene as determinants of age-defined risk of coronary artery lesions in kawasaki disease modulating effects of mannose binding lectin genotype on arterial stiffness in children after kawasaki disease monocyte chemoattractant protein gene regulatory region polymorphism and serum levels of monocyte chemoattractant protein in japanese patients with kawasaki disease polymorphism of transmembrane region of mica gene and kawasaki disease circulating matrix metalloproteinases and their inhibitors in patients with kawasaki disease polymorphism of matrix metalloproteinase- promoter gene as a risk factor for coronary artery lesions in kawasaki disease genetic analysis of mmp gene polymorphisms in patients with kawasaki disease association of the matrix metalloproteinase- (- c/g) promoter polymorphism with kawasaki disease in korean children methylenetetrahydrofolate reductase polymorphism in kawasaki disease a polymorphism in plasma platelet-activating factor acetylhydrolase is involved in resistance to immunoglobulin treatment in kawasaki disease polymorphism of slc a (formerly nramp ) gene confers susceptibility to kawasaki disease transforming growth factor-beta signaling pathway in patients with kawasaki disease polymorphisms of transforming growth factor-b signaling pathway and kawasaki disease in the taiwanese population tissue inhibitor of metalloproteinase and coronary artery lesions in kawasaki disease analysis of tumor necrosis factor-alpha production and polymorphisms of the tumor necrosis factor-alpha gene in individuals with a history of kawasaki disease association between levels of tnf-alpha and tnf-alpha promoter - a/a polymorphism in children with kawasaki disease tumor necrosis factor-alpha levels and promoter polymorphism in patients with kawasaki disease in korea association of vascular endothelial growth factor (vegf) and vegf receptor gene polymorphisms with coronary artery lesions of kawasaki disease vascular endothelial growth factor gene haplotypes in kawasaki disease association of vascular endothelial growth factor c- g polymorphism in taiwanese children with kawasaki disease lack of association of the vascular endothelial growth factor gene polymorphisms with kawasaki disease in taiwanese children itpkc functional polymorphism associated with kawasaki disease susceptibility and formation of coronary artery aneurysms a genome-wide association study identifies novel and functionally related susceptibility loci for kawasaki disease a genome-wide association analysis reveals p and p . as susceptibility loci for kawasaki disease identification of novel susceptibility loci for kawasaki disease in a han chinese population by a genome-wide association study genome-wide association study identifies fcgr a as a susceptibility locus for kawasaki disease a genome-wide association study identifies three new risk loci for kawasaki disease two new susceptibility loci for kawasaki disease identified through genome-wide association analysis this review was supported by a grant from the italian ministry of health (bando giovani ricercatori ). the authors have no potential conflict of interest to declare. key: cord- - fctxk authors: proudfoot, chris; lillico, simon; tait-burkard, christine title: genome editing for disease resistance in pigs and chickens date: - - journal: anim front doi: . /af/vfz sha: doc_id: cord_uid: fctxk nan for thousands of years, humans have used selective breeding to improve desirable traits in both livestock and companion animals. in livestock, targeted breeding has been common practice since the british agricultural revolution of the th century, with measurable production traits such as feed conversion in cattle or wool production in sheep actively selected for. in the late th century, genomic selection was added to the livestock breeding tool box; by reading specific locations in the genome and assigning them to measurable production traits, faster improvement in livestock production efficiency has been achieved. one of the inherently difficult production traits to measure is resistance to a specific disease, as animals with less severe symptoms or pathology may simply have been exposed to less pathogen. experimental infections guaranteeing equal pathogen exposures are expensive and require large numbers of animals for genetic association studies, making them ethically questionable. genome editing offers new opportunities to livestock breeding for disease resistance, allowing the direct translation of laboratory research into disease-resistant or resilient animals. made? genome editors are custom enzymes that allow scientists to cut the dna strands in the nucleus of a cell at a specific position. the researcher can then influence how the dna is repaired, introducing very precise genetic changes at a target locus in their species of interest. this technology has been revolutionary and provides exciting possibilities for the production of livestock resistant to viral diseases. such opportunities are particularly pertinent given state efforts to improve global food security and reduce food waste throughout the production chain. the most prominent editor technology today, crispr/cas, uses a nucleotide rna guide to target its enzyme component to a designated locus in the genome. the probability of off-target cutting with a high fidelity cas enzyme is very low, because with four potential base combinations at each of the nucleotides there are over one trillion unique guide combinations. once the enzyme has cut the dna strands, the predominant repair pathway in most cells is nonhomologous end joining, an error-prone process which often introduces small insertions or deletions into the genetic code at the break site. if the target is within a gene, such perturbations can result in a disruption to the function of that gene, potentially leading to a loss of protein function. this can be very useful to basic science as it allows researchers to discover functions associated with novel genes. for many applications, a more precise change to the genome is required. to that end, scientists regularly make an alternative dna repair process called homology-directed repair. to do this, researchers provide a novel dna sequence alongside the crispr/cas reagents, whereby the cellular repair machinery uses the new dna as a template when fixing the break. this approach facilitates the introduction of defined implications • genome editing technology enlarges the tool box of trait-selective breeding. • methods for genome editing have developed over the past decades, making the technology more efficient and specific. • technology to generate edited pigs and chickens is developing alongside genome editors to generate animals faster and more affordable. • for two major pig diseases, it has been shown that resistant animals can be generated that are refractory to infection. in chickens there are promising laboratory results but no genome-edited, resistant chickens yet. • genome editing allows us to overcome bottlenecks in trait-selective breeding and allows the incorporation of genetic traits from other breeds, related species, or laboratory results. • two major hurdles still to be faced prior to the implementation of this promising technology are consumer acceptance and the regulatory framework. changes at the genomic target locus and has sufficient refinement to alter a single nucleotide, allowing precise modification of gene function. finally, by introducing a pair of editors, it is possible to generate two concurrent dna breaks on the same chromosome. the cellular repair machinery then joins the ends of the cut sites, promoting the deletion of the intervening sequence. all the editor reagents introduced to the cell are rapidly degraded, with only the alteration to the genomic sequence remaining to be propagated following cell division. genome editing has been applied to a wide variety of agricultural species including salmonids, poultry, and ruminants. however, due to its global economic value, relatively short generation time, and multiparous nature, the most edited livestock species to date is the pig. there are two main methods widely used for the generation of edited pigs: cloning of edited fibroblasts or direct injection of the zygotes with editor reagents. both work well, and each has specific advantages. in cloning, fibroblast cells can be maintained in the lab for prolonged periods. this allows researchers to introduce editor reagents into the cultured cells typically by lipofection, electroporation, or microinjection. editing events in each cell of a population can be characterized and individual cells with the desired alteration to their genome selected for the cloning process, whereby the fibroblast cell is fused with an enucleated oocyte shell in a process called somatic cell nuclear transfer ( figure a ). the reconstituted "zygote" is then transferred to a recipient gilt or sow (carlson et al., ) . despite the benefit of being able to prescreen the donor cells, cloning is generally inefficient with hundreds of reconstituted zygotes being transferred to a single recipient. cloning also yields reduced litter sizes when compared with standard breeding and offspring often demonstrate reduced viability. as an alternative to cloning, newly fertilized zygotes can be directly microinjected with genome-editing reagents and transferred immediately back to the oviduct of a recipient animal ( figure b ). in contrast to cloning, this approach (lillico et al., ) results in the efficient establishment of pregnancies and robust litters. however, without the prescreening of cells that is routine in cloning, offspring from direct zygote manipulation inevitably encompasses a range of editing outcomes, since selection of a specific edit is not possible. porcine zygotes can also be generated by maturation of oocytes extracted from slaughterhouse-derived ovaries and in vitro fertilization. unfortunately, in vitro fertilization in pigs often results in polyspermy, rendering the resulting embryo inviable. however, in this controlled environment, editing rates can be increased and costs and animal use reduced. an emerging alternative to these proven methods could be the use of surrogate sires. as a first step towards this goal, pigs have been edited to remove a gene required for male fertility, generating an empty spermatagonial stem cell niche in the testis . spermatogonial stem cells can be isolated and cultured in vitro, opening the possibility to edit and characterize these cells before transfer to a recipient (park et al., ) ( figure c ). genetic modification of poultry poses unique challenges due to the very different physiology of the avian egg compared with a mammalian oocyte. as a result, isolation and transfer of a chicken yolk is not practical. one approach that has been taken is in ovo electroporation of editing reagents, which allowed the analysis of gene function in the neural crest (gandhi et al., ) . however, others reported that electroporation resulted in mosaicism with editing limited to a subset of cells as the chicken embryo is already much further developed when an egg is laid compared with a zygote (veron et al., ) ( figure d ). as a result, it is unlikely that this approach could be efficiently utilized to generate edited birds. an alternative approach involves sperm transfection-assisted gene editing, whereby sperm are lipofected with editing reagents before use in artificial insemination (cooper et al., ) ( figure e ). however, advances in chicken stem cell technology show the greatest promise for genome editing in chicken. primordial germ cells (stem cells that eventually develop into germ cells) can be isolated from the blood of developing chicks in ovo and cultured in vitro. as with mammalian fibroblasts, these cells can be edited and selected in vitro before transfer into the bloodstream of a stage-matched recipient where they migrate to and populate the developing gonad. the chicken embryo is accessed through an opening in the egg shell, which is sealed again until the chicken hatches. genome editing in primordial germ cells has been successfully demonstrated by a number of groups (park et al., ; taylor et al., ; idoko-akoh et al., ) and one group has generated modified birds (park et al., ) . the founder birds generated from this editing method are chimeric due to the presence of preexisting germ cells. the resulting offspring generated from breeding with the founders will be a mixture of edited or nonedited. recipient chicken embryos devoid of germ cells are currently being developed that will significantly increase the efficiency of this process (m. mcgrew, unpublished results) ( figure f ). genome editors will undoubtedly have a significant role on the generation of disease-resistant animals as exemplified below. it is important to note that currently the technology is limited to modifying a single gene or a snp with large effects; however, disease resistance in many cases is likely to be a polygenic trait. multiplexing technology is under development such that in the future polygenic traits could be altered in a single step. progress so far? porcine reproductive and respiratory syndrome virus. porcine reproductive and respiratory syndrome (prrs) is arguably the most economically important pig disease worldwide. the causative agent of prrs is an arterivirus, named prrs virus (prrsv), that affects pigs of all ages but most importantly causes late-term abortions and stillbirth in sows and severe respiratory disease in piglets with severe morbidity and high mortality. prrsv also incapacitates the pig's immune response, providing an ideal breeding ground for severe secondary infections, mostly by bacteria, which leads to increased use of antibiotics. prrsv exclusively infects cells of the monocyte/macrophage lineage and two macrophagespecific proteins, cd and cd , were identified as receptors for the virus: cd acting on the surface of the cells and cd inside the internalizing transport vesicles (calvert et al., ; van gorp et al., ) . the virus was thought to attach to cd to be taken up into the cells; however, genome-edited pigs lacking cd were not resistant to prrsv infection (prather et al., ) . cd on the other hand is thought to act through a key-lock interaction with the virus to allow it to escape from the internalizing transport vehicles into the cytosol where it replicates. cd consists of nine globular domains, organized like beads on a string, with domain determined to mediate the key-lock interaction allowing viral entry into pig cells (van gorp et al., ) . using genome editing to generate pigs lacking cd whitworth et al. showed for the first time that this approach could be used to produce livestock resistant to important viral diseases, in this case prrs (whitworth et al., ) . cd is known to have a range of important biological functions in homeostasis, inflammation, and immune responses. as a refinement on functional knock out of the entire cd protein, editing reagents were designed to remove only domain leaving the remainder of the protein intact. the resulting animals were completely resistant to prrsv infection and maintained the biological functions associated with the remaining domains of cd (burkard et al., ; burkard et al., ) . porcine epidemic diarrhea virus/transmissible gastroenteritis virus. the two coronaviruses porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) both cause severe diarrhea in preweaned piglets and are associated with high morbidity and mortality. in vitro host-pathogen studies identified aminopeptidase n as the receptor for tgev and a potential receptor for pedv (delmas et al., ; li et al., ) . the use of genome editing to generate pigs lacking aminopeptidase n successfully showed that pigs resistant to tgev infection could be generated. however, the edited animals remained susceptible to pedv infection (whitworth et al., ) . aminopeptidase n is important for peptide digestion in the small intestine and knockout mice were shown to have delayed mammary gland development. in humans, aminopeptidase n defects are associated with different types of leukemia and lymphoma. therefore, further investigation into the potential consequences of the absence aminopeptidase n in pigs is warranted as it may affect the overall health and/or productivity of the animals. african swine fever virus. african swine fever virus (asfv) causes a severe hemorrhagic disease in domestic pigs (sus scrofa domesticus) and wild boars (sus scrofa ferus) with high mortality in pigs of all ages. asfv is highly contagious and can be transmitted by soft ticks of the ornithodoros genus. it was identified in and contained to africa with occasional transmission around the strait of gibraltar into portugal and spain. in an introduction of the virus into the caucasus region showed that the virus does not solely rely on ticks for transmission in the wild, as transport of contaminated material and direct contact between animals have been shown to be major routes of disease dissemination. since then, the virus has spread across eastern europe and russia and was recently found in western europe and china. asfv poses a huge risk to the pig industry worldwide and is a limiting factor to a sustainable pig industry in many parts of africa. interestingly, asfv also infects wild suids, such as warthogs (phacocherus africanus) and bushpigs (potamocherus porcus), without causing overt disease. such infected wild suids are thought to act as a reservoir of the virus in africa. in the late stages of asfv infection, a cytokine storm, i.e., an overreaction of the immune system, is observed, which is thought to strongly contribute to the lethal outcome of disease. a comparison of the warthog and domestic pig genomes identified differences in the rel-like domain-containing protein a (rela, also known as p ) protein, which is involved in nf-κb cytokine signaling, was thought to underlie the different responses of the related species to asfv infection (palgrave et al., ) . researchers used genome editing to convert a key region of the encoded domestic pig protein sequence to the warthog equivalent (lillico et al., ) . data on susceptibility of the edited animals to asfv infection have yet to be reported. in this instance, it is important to differentiate between disease resistance, the ability of an animal to suppress the establishment and/or development of an infection, and disease resilience, where an infected host manages to maintain an acceptable level of productivity despite challenge pressure. should these pigs prove to be resilient to asfv infection it is likely that their use may not be permitted in many jurisdictions, since they could act as reservoirs of infection. however, in environments where the disease is endemic use of such animals could be beneficial. avian leucosis virus. avian leukosis virus infection results in inappetence, diarrhea, weight loss, a reduction in eggs laid, and often causes tumor formation in the chicken. the virus is divided into six subgroups, with the avian leucosis virus subgroup j (alv-j) shown to be responsible for major disease outbreaks in china. the cellular receptor of alv-j was identified to be the chicken sodium/hydrogen exchanger protein on the cell surface. chicken somatic cell lines have been edited to introduce changes to this gene-conferring resistance to avian leucosis virus in vitro (lee et al., ) . despite cells showing resistance to alv-j infection, no edited chickens have been produced to date. in both mice and humans, a lack of the sodium/hydrogen exchanger protein is associated with severe neurological disease; however, targeted changes to single amino acids may retain biological functions of the protein in chicken while resulting in disease resistance. avian influenza virus. in chickens, disease resistance to avian influenza is at the top of the wish list due to the serious impact on chicken health but also the risk of transmission to humans. similarly, influenza a is also one of the diseases on the resistance wish list for pigs, as they can act as an intermediate host-aiding virus adaptation to humans. the acidic leucine-rich nuclear phosphoprotein- a (anp a) was found to play a key role in avian influenza virus replication in both chicken and water fowl. although the virus polymerase protein readily interacts with the avian anp a, the human version of the same protein supports only limited replication of the viral genome. it has been demonstrated in vitro that deletion of a small region of chicken anp a can prevent replication of avian influenza virus (long et al., ; long et al., ) . although the functional consequence of edited anp a has yet to be demonstrated in vivo, such approaches offer exciting opportunities that have the potential to benefit both industry and animal welfare. as exemplified above, currently many gene editing approaches focus on targeting host genes involved in mediating entry of the virus, with a special focus on receptors. however, as the example for avian influenza shows, host genes play an important role in other steps of the pathogen replication cycle and also provide editing targets for disease resilience or resistance. more in-depth host-pathogen interaction studies, including genome-wide editing studies in vitro, will no doubt produce a variety of further candidate genes for genetic disease resistance. an alternative antipathogen approach pursued for decades is the generation of transgenic livestock, expressing antiviral or antibacterial agents, such as enzymes or small interfering rnas. genome editing can be used to improve the integration efficiency of these transgenes at specific locations in the genome; however, the discussion of transgenic disease-resistant animals is beyond the scope of this review. how does genome editing fit within existing selective breeding structures and how will it be regulated? selective breeding has generated highly productive, robust animals that are adapted to a modern production environment. livestock production is dynamic, with evolving challenges such as climate change and disease outbreaks coupled with societal pressure to reduce antimicrobial use. selective breeding for disease resistance has proven difficult, as outbreaks are often sporadic and resistant/resilient animals often difficult to identify. in circumstances where a genetic trait for disease resistance can be identified in the breeding population, then selection through the selective breeding can be achieved. a good example of this is pigs with resistance to f type enterotoxigenic e. coli. association studies revealed that a polymorphism in the fucosyl transferase gene conferred resistance to these bacteria. there was initial concern that selection for the locus figure . genetic resistance to disease and how genome editing can help integrate traits into highly productive lines. (a) genetic resistance to disease may be present in a small percentage of production animals and genetic selection for these animals may be associated with the risk of inbreeding, productivity loss, or the risk of losing other desirable traits. genome editing allows integration of the disease-resistance trait into a wider selection of pigs, ensuring genetic variability and maintenance of desirable traits. (b) genetic resistance to disease may be present in an indigenous or less productive breed. crossbreeding would result in productivity loss and the risk of losing other desirable traits, such as fur color. genome editing allows for incorporation of genetic disease resistance into highly bred lines without losing productivity. (c) genetic resistance may be observed in a closely related species, e.g., wild boar or wild suids in the case of the domestic pig. integration into highly bred domestic pig lines would only be possible by genome editing. (d) resistance genes may be identified in laboratory research but not in highly bred lines, making integration into those productive animals only possible using genome editing. harboring this gene may counterselect for another gene associated with stress resistance. however, this proved not to be the case and genetic selection for the favorable fucosyl transferase allele has been integrated into many pig-breeding programs (coddens et al., ) . this was possible, in part, because the favorable allele was present at sufficient prevalence (in most studies between % and %) in the breeding population to allow for selection while avoiding inbreeding. in circumstances where an allelic variant associated with a resistant phenotype is present at a much lower frequency, it may prove difficult to incorporate effective selection into a standard breeding regime without the risk of inbreeding and related longer-term productivity loss (figure a) . genome editing has the potential to contribute in such circumstances, allowing the direct introgression of a beneficial allele into the offspring of diverse, highly productive animals. similarly, disease-resistance traits associated with less productive indigenous breeds are unlikely to be introduced to highly productive populations by standard crossbreeding as this would result in a significant set-back in productivity, abrogating decades or even centuries of advances made through genetic selection ( figure b ). in circumstances where resistance or resilience is observed in a related species, crossbreeding is simply not possible. genome editing could bridge these gaps. one example of this is resilience of wild suids to african swine fever virus while domestic pigs can suffer from severe disease. it is not possible to crossbreed these species, so introduction of the genetics underlying resilience is not possible by this route. genetic comparison can be used to identify the functional differences underlying such traits, and genome editing employed to introduce appropriate variants into domestic pigs ( figure c ). finally, with a good understanding of host-pathogen interactions, novel genetics that has not been observed in live animals can be created and tested for efficacy in a laboratory environment. this was the case for both the cd /prrsv and apn/tgev examples in pigs and would be the case for the anp a/ influenza and the alv-j resistance in chicken, described above. in such circumstances, integration through genome editing presents a practical route to benefit from the findings ( figure d ). it is imperative that in such circumstances thorough phenotypic characterization of the edited animals be carried out as deletion of all or part of a functional protein could result in a loss of (systemic) biological function. a second measure worthy of consideration before embarking on an editing project is whether the gene is located within a locus that has been actively selected in breeding programs. this could indicate whether a potential target is associated with known production traits. this approach has been taken for prrsv-resistant pigs, with evaluation as to whether the cd gene locus has been selected for in pig breeding programs (johnsson et al., ) . overall, genome editing holds vast promise for the future production of animals resistant or resilient to disease, improving productivity and animal welfare while reducing food waste throughout the production chain. through reduction of primary and secondary infections, it should also be possible to reduce antimicrobial use in livestock production. technical improvements in the generation of genome editing animals will undoubtedly reduce the cost implications of this technology. the two major hurdles still to be faced prior to implementation of this promising technology are consumer acceptance and the regulatory framework. approval of edited animals for human consumption relies on national and multinational legislation, which is currently at early stages. encouragingly, some international jurisdictions such as argentina and brazil have about the authors dr. chris proudfoot is research fellow at the roslin institute/university of edinburgh since . his work centers on generation of genome-modified livestock, with particular emphasis on genome editors, to improve disease resistance or to accurately model human disease. dr. proudfoot has worked extensively with zfns, talens, and crispr/cas to produce a variety of edited animals. he was a member of the team that produced the first edited livestock using this method. dr. simon lillico is a research associate at the roslin institute/university of edinburgh. he joined the institute on an industrial collaboration to produce highvalue therapeutic proteins in hens eggs and then applied his expertise in lentiviral transgenesis to generate livestock models of human diseases. the rapid expansion of the field of genome editors over the last yr has made practicable genome modifications which had previously been unattainable. dr. lillico has been at the forefront of application of these editors to livestock, creating either disease-resistant/resilient strains, or accurate models of human disease. dr. christine tait-burkard is an assistant professor at the roslin institute/university of edinburgh since in the departments of genetics and genomics and infection and immunity. her research focuses on understanding host-pathogen interactions on a cellular and genetic level, developing new in vitro tools for virus research, improving and developing easy-to-use diagnostics, and devising strategies to combat viral disease in livestock in general and pigs in particular. she employs genome editing and genetic selection to generate animals genetically resistant to viral disease. corresponding author: christine.burkard@roslin.ed.ac.uk already ruled that modifications, such as the prrsv-resistant pig, that do not have any new genetic information integrated into the animal, will be exempt from regulation. precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd srcr domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain of cd are resistant to porcine reproductive and respiratory syndrome virus infection cd expression confers susceptibility to porcine reproductive and respiratory syndrome viruses efficient talen-mediated gene knockout in livestock the possibility of positive selection for both f (+)escherichia coli and stress resistant pigs opens new perspectives for pig breeding generation of gene edited birds in one generation using sperm transfection assisted gene editing (stage) aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev optimization of crispr/cas genome editing for loss-of-function in the early chick embryo high fidelity crispr/cas increases precise monoallelic and biallelic editing events in primordial germ cells precise gene editing of chicken na+/h+ exchange type (chnhe ) confers resistance to avian leukosis virus subgroup j (alv-j) porcine aminopeptidase n is a functional receptor for the pedv coronavirus live pigs produced from genome edited zygotes mammalian interspecies substitution of immune modulatory alleles by genome editing species difference in anp a underlies influenza a virus polymerase host restriction avian anp b does not support influenza a virus polymerase and influenza a virus relies exclusively on anp a in chicken cells species-specific variation in rela underlies differences in nf-κb activity: a potential role in african swine fever pathogenesis successful genetic modification of porcine spermatogonial stem cells via an electrically responsive au nanowire injector generation of germline ablated male pigs by crispr/cas editing of the nanos gene targeted gene knockout in chickens mediated by talens an intact sialoadhesin (sn/siglec /cd ) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus efficient talen-mediated gene targeting of chicken primordial germ cells sialoadhesin and cd join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd protein domains involved in infection of the porcine reproductive and respiratory syndrome virus crispr mediated somatic cell genome engineering in the chicken gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs we acknowledge financial support from the biotechnology and biological science research council (bbsrc) (bb/ r / , bb/n / ) and the bbsrc institute strategic programme grant funding to the roslin institute (bbs/ e/d/ and bbs/e/d/ ). key: cord- -yfh d io authors: su, yu-ching; jalalvand, farshid; thegerström, john; riesbeck, kristian title: the interplay between immune response and bacterial infection in copd: focus upon non-typeable haemophilus influenzae date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: yfh d io chronic obstructive pulmonary disease (copd) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. it is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. acute exacerbations of copd (aecopd) are characterized by increased cough, purulent sputum production, and dyspnea. the aecopd is mostly associated with infection caused by common cold viruses or bacteria, or co-infections. chronic and persistent infection by non-typeable haemophilus influenzae (nthi), a gram-negative coccobacillus, contributes to almost half of the infective exacerbations caused by bacteria. this is supported by reports that nthi is commonly isolated in the sputum from copd patients during exacerbations. persistent colonization of nthi in the lower airway requires a plethora of phenotypic adaptation and virulent mechanisms that are developed over time to cope with changing environmental pressures in the airway such as host immuno-inflammatory response. chronic inhalation of noxious irritants in copd causes a changed balance in the lung microbiome, abnormal inflammatory response, and an impaired airway immune system. these conditions significantly provide an opportunistic platform for nthi colonization and infection resulting in a “vicious circle.” episodes of large inflammation as the consequences of multiple interactions between airway immune cells and nthi, accumulatively contribute to copd exacerbations and may result in worsening of the clinical status. in this review, we discuss in detail the interplay and crosstalk between airway immune residents and nthi, and their effect in aecopd for better understanding of nthi pathogenesis in copd patients. the lungs are vital organs involved in gas exchange between the vascular system and the external environment, thus they are greatly exposed to the environment-derived microorganisms, including fungi, viruses, and bacteria. the bronchial tree and parenchymal tissues of the lungs, that until recently were considered as sterile, are colonized by phylogenetically-diverse microbes. the genera of firmicutes, bacteroidetes, and proteobacteria are the most common phyla identified and represent % of the total bacterial microbiome in the healthy airway ( , ) . the majority of the lung microbiota belongs to the normal flora that play an important role in the pulmonary epithelial integrity, colonization resistance, and homeostasis of the immune system in the respiratory tract ( ) . a small fraction of them are, however, potentially pathogenic microorganisms that are involved in a variety of lung diseases, as exemplified by the genus haemophilus. non-typeable haemophilus influenzae (nthi) is a gram-negative coccobacillus that are commonly residing in the human airways. uniquely and yet unexplained, nthi is a commensal when colonizing the nasopharynx or throat, but pathogenic in the lower airways triggering a robust inflammatory response [for reviews see ( , ) ]. nthi is considered a potential opportunistic pathogen as it frequently infects the lower respiratory tract of lungs with structural damage as a consequence of non-infectious lung diseases or mechanical injuries. moreover, nthi occasionally causes bronchitis and pneumonia ( ) . in addition, lower airway colonization by nthi has been associated with disease progression of several more or less non-infectious lung diseases such as bronchiectasis ( ), cystic fibrosis ( ) , interstitial lung diseases ( , ) , but mostly in chronic obstructive pulmonary disease (copd) ( , ) . copd is a severe inflammatory lung disease characterized by airflow limitation with a range of pathological changes. both genetics and environmental factors trigger the onset of copd, however, microbes including nthi play an important role in the acute exacerbations. this review describes the disease progression of copd in the context of host immuneinteractions linked to nthi, and the overall impact in disease exacerbation. copd is the third leading cause of morbidity and mortality worldwide expected to affect more than million people by ( , ) . according to the global initiative for chronic obstructive lung disease (gold), copd is a pulmonary disease that is manageable, but significant exacerbations and co-morbidities may, however, contribute to the overall severity in individual patients ( ) . copd is characterized by chronic airflow limitation of the peripheral airways with a range of pathological changes in the lung that are not fully reversible, and usually become progressively worse over time. the progression of copd is associated with an abnormal inflammatory response of the lung to noxious particles or gases. from a pathological point of view, copd comprises a group of pulmonary abnormalities related to the inflammatory reaction of the airways, alveoli, and pulmonary vessels ( ) ( ) ( ) ( ) . these include (i) pulmonary emphysema, (ii) chronic bronchitis, and (iii) disease in the small airways. the pulmonary abnormalities progressively affect all parts of the lung, resulting in increased resistance of the conducting airways and thus chronic airflow obstruction that eventually will lead to a declined lung function. emphysema is a permanent loss of elastic lung recoil caused by elastolytic destruction and enlargement of the alveolar wall distal to the terminal bronchioles. this consequently results in the loss of alveolar attachments to the small airways and thus limitation of airflow and gaseous exchanges. chronic bronchitis is characterized by consecutive and chronic cough with expectorations that last for more than months within years. it is associated with inflammation of the bronchial walls with increased inflammatory infiltrates, hyperplasia of goblet cells, hypertrophy of tracheobronchial submucosa, increased mucous secretion and, finally, dilatation of the airway ducts (airways of about - mm in internal diameter). the majority of the ciliated epithelium lining the airways are also either compromised or dysfunctionnal, and may be replaced by nonciliated squamous epithelial cells. small airway diseases, on the other hand, involve hyperplasia and metaplasia of mucosal glands and goblet cells, hypersecretion of intraluminal mucus, macrophage bronchiolitis, and accumulation of lymphocytes in the small bronchioles (airways of ∼ mm or less in diameter and terminal bronchioles). in addition, distortion, fibrosis, stenosis, tortuosities, hyperplasia, and hypertrophia of the small airway smooth muscles also contribute to the loss of elasticity in the lung parenchyma. although copd mainly affects the lungs, it also produces significant extrapulmonary consequences as a results of an escalated inflammatory response orchestrated by airway cells and immune mediators ( , ) . the comorbidities are commonly seen in copd patients despite the actual mechanism responsible for the systemic inflammation remains to be elucidated. the development of copd is multifactorial, with cigarette or tobacco smoking being the primary cause of copd ( , ) . other risk factors that may promote the onset and progression of copd includes prolonged occupational exposure to particles/gases in mining and textile industries, air pollution resulting from biomass combustion, and bronchial hyperresponsiveness ( , , ) . the variability of copd incidences among smokers is also explained by a genetic predisposition, such as α -antitrypsin deficiency and cutis laxa [mutation of the elastin gene (eln)] ( , ) . the α -antitrypsin deficiency is caused by deleterious homozygous mutations in serpina which contributes to - % of copd cases. the deficiency results in increased neutrophil elastase activity that ultimately leads to the degradation and collapse of the alveoli. importantly, meta-analyses of genome-wide association studies (gwas) and other genotyping studies have revealed that multiple single nucleotide polymorphism (snps) in at least genes from different pulmonary genomic loci are associated with copd susceptibility ( ) ( ) ( ) ( ) . airway epithelium exposed to cigarette or tobacco smoke has compromised tight junctions and delayed epithelial wound repair ( ) ( ) ( ) ( ) . moreover, cigarette smoke alters basal cell differentiation and subepithelial extracellular matrix (ecm) composition, and thus causes airway remodeling (i.e., goblet cell hyperplasia and small airway squamous metaplasia) ( ) ( ) ( ) . this results in mucus hypersecretion, impaired mucocilliary clearance, and airway obstruction. tobacco or cigarette smoke also enhances proliferation and ecm deposition by activating the extracellular signal related kinase (erk) and the p signaling pathway ( ). the alteration of major ecm components are widespread in all lung compartments in copd patients with a total increase of type i and iii collagens, fibronectin, and laminin in parallel with reduced concentrations of proteoglycans, perlecan decorin, versican, biglycan, tenascin and elastin ( , ). cigarette induced-overexpression of matrix metalloproteases (mmps- , , , , , and ) and elastase has also been reported, and may contribute to the airway tissue destruction and fibrosis ( - ). in addition, harmful volatile chemicals derived from cigarette smoke (i.e., acetaldehyde, acrolein, and crotonaldehyde) are prone to form carcinogen adducts with dna and various proteins (i.e., apoliprotein e and surfactant protein a). they also dysregulate airway epithelial ion transport, disrupt the phagocytic activity of airway phagocytes, and diminish the airway surface liquid volume ( - ). numerous proteomics and transcriptomic analyses have unveiled the crucial impact of cigarette or tobacco smoke and copd disease progression on airway gene expression ( , ). the differential gene expression studies were done using copd experimental models or clinical samples [i.e., bronchial epithelial cells, sputum, plasma, blood, and bronchoalveolar lavage (bal) fluid]. collectively, most of the altered genes are involved in oxidative stress, xenobiotic metabolism, antioxidant responses, dna repair, ecm remodeling, inflammatory responses, and immune defenses, which the latter two are our major interest of discussion in this review. the omics data aid in the increased knowledge of molecular mechanisms in copd. they may reflect the dynamic response and attempts by the airway epithelial cells to repair the cytotoxic injury primarily triggered by inhaled irritants. deleterious and irreversible alterations occurring and interfering with the airway epithelial homeostasis and immune defense may promote copd development and progression. notably, gene alterations in phagosomal-and leukocyte transendothelial migration pathways (ltm) are significantly correlated with the level of t cells and airway obstruction in smokers ( ). the ltm, however, were found to be further dysregulated in copd patients. hence, in addition to clinical/physiology variables, a number of gene products with significant differential gene expression may be targeted as specific proteomic signatures or biomarkers for early copd detection, patient monitoring, disease subgrouping, and finally treatment selection ( , ). tobacco or cigarette smoke regulates airway gene expression via two main mechanisms, by altering the status of (i) chromatin remodeling, and (ii) dna methylation of the target genes (figure ) ( - ). chromatin remodeling is a result of a disrupted balance in histone acetylation/deacetylation ( ). excessive activation of more than transcription factors including nf-κb, and lipoprotein peroxidation products (peroxinitrite, acrolein, and hne from tobacco smoking) contributes to such anomaly. nf-κb is a key inflammatory and redox-sensitive transcription factor that plays a direct role in cigarette smoke-induced airway inflammation. nf-κb has been described as a "smokesensor" due to its sensitive activation by tobacco residues ( ). stimulation of multiple signaling cascades [p mitogenactivated protein (mapk) kinases, mitogen and stress-activated kinase (msk ), protein kinase c zeta (pkcζ), and iκb kinase (ikk) complex (ikkα, ikkβ, and nemo)] by tobacco residues promotes the activation and nuclear translocation of transcription factor nf-κb rela/p ( , - ). this is followed by a complex formation of nf-κb/cbp-p [coactivator, crebbinding protein (cbp) or cbp/p ] at target dna sequences. it should be noted that cbp/p also has intrinsic histone acetyltransferase (hat) activity. subsequent acetylation and phosphorylation of the subunit p in the nf-κb/cbp-p complex by the activated msk /pkcζ-signaling pathways (and other different kinases), and cbp/p , respectively, are required for the full activation of nf-κb ( , , ). this enhances the dna binding affinity of the complex. histones h and h in the chromatin complex of target sequences are then being acetylated (histone h at lys ; h at lys and lys ) and phosphorylated (histone h at ser ) by the subunit cbp of the nf-κb/cbp-p complex, and the activated msk and pkcζ, respectively. the hyperacetylated core histones, however, fail to be neutralized or deacetylated by a dysfunctional histone deacetylase (hdac ). peroxinitrite nitrates the tyrosine residues of the hdac and causes inhibition of activation and reduced expression of the protein. of note, peroxinitrite is a by-product generated from the immune cell-derived nitrite oxide (no) and reactive oxygen species (ros) of cigarette smoke ( , ). cigarette or tobacco smoke disturbs the dna methylation status of target genes through several mechanisms. firstly, dna damage caused by cigarette smoke stimulates the dna methyltransferase (dnmt) to actively induce cpgs methylation at the damaged site ( ). the hypermethylation is prone to introduce error of methylation in some target genes, resulting in reduced gene expression. secondly, activation of nicotine signaling pathway by tobacco smoke causes camkii/iv and erk/mapk pathway activation that subsequently induces the activity of cbp to suppress the expression of dnmt . this may result in reduced dna methylation and thus altered level of gene repression by dnmt ( - ). finally, enhanced activities of transcription factors such as hypoxia inducible factor due to the high level of carbon monoxide and hypoxia have also been reported to influence airway gene expression ( ) . consequently, the combinatorial effect from both aberrant acetylation of histone and dna methylation promotes the transformation of chromatin from a condensed structure to an activated open conformation. this facilitates irregular accessibility of dna for transcription machineries, hence figure | cigarette and tobacco smoke has several effects on gene regulation. nicotine and other compounds in the smoke alter gene expression by two pathways, firstly, chromatin remodeling (left) and secondly, dna methylation (right). chromatin remodeling involves activation of kinases signaling pathways, activation and nuclear translocation of transcription factor nf-κb (rela/p ), and complex formation with cbp/p on specific dna sites. cbp/p is intrinsically a histone acetyltransferase (hat). subunit p is further phosphorylated at ser and ser , respectively, by msk and pkcζ, whereas cbp acetylates p at lys . the phosphorylation and acetylation enhance the interaction within the nf-κb/cbp/p complex while stabilizing the dna binding of nf-κb. the complex of nf-κb/cbp/p then modifies the histones through cbp-mediated acetylations of histone h (at lys ) and h at lys and lys , and phosphorylation of h at ser by msk and pkcζ. this results in the structure change of chromatin, from a condensed structure (repressed) to an activated open conformation. the transcription of target genes is therefore increased. in the second mechanism, several side effects resulting from cigarette smoking such as dna damage and nicotine signaling could trigger the hypermethylation or decreased methylation of target dna. this may lead to dna methylation anomalies and thus altered dna expression. resulting hypoxia due to high concentrations of carbon monoxide also contributes to altered gene expression. the aberrant gene expression by cigarette smoke mostly occurs in pro-inflammatory genes with resulting increased production of inflammatory mediators, and amplified inflammation in the copd lung upon exposure. irregular gene expression by various cell types in the airway. the mechanisms reported are responsible for increased expression of nf-κb-dependent proinflammatory gene products [i.e., il- β, il- , il- , ccl- cyclooxygenase (cox)- , and mip- /cxcl ] in both pulmonary structural cells (bronchial, small airway, and alveolar epithelial cells) and immune cells (alveolar macrophages), increased vegf and inos in nasal fibroblasts and lymphocytes (jurkat t cells), respectively, and decreased activity of antioxidant transcription factor nrf and α -antitrypsin in bronchial epithelial cells ( , , , , - , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these may contribute to the anatomical anomalies in the airway and excessive inflammatory responses among smokers during the course of copd. copd is associated with chronic inflammation in the peripheral airways orchestrated by both innate and adaptive immune responses that are interconnected via dendritic cells ( ) . increasing numbers of inflammatory cells (neutrophils, macrophages, t and b lymphocytes, mast cell, eosinophils, and dendritic cells) and inflammatory mediators are accumulated in the airway lumen/wall in the lung parenchyma ( , ) . these immune cells and inflammatory mediators can hence be detected in the sputum and bal fluid of copd patients. the level of accumulation is positively correlated with disease severity. an increasing number of studies using animal models and clinical tissues have reported the nature of excessive airway inflammatory responses in copd. despite this, the heterogeneity in symptoms progression among copd patients remain unexplained. the overall mechanism of copd inflammatory immune response is depicted in figure . the first line of defense in the lung-the innate immunity and inflammasome lung structural cells (epithelial and endothelial cells, fibroblasts, and airway smooth muscle cells) are activated by inhaled irritants through the stimulation of several pattern recognitions receptors (prrs), with toll-like receptor (tlr)- being reported as the key player in most of the inflammatory responses ( ) ( ) ( ) ( ) . this causes an increased expression and release of an array of pro-inflammatory mediators and chemokines through the oxidative pathway by the activated bronchial epithelial cells and immune cells (alveolar macrophages). the inflammatory mediators [(interleukin (il)- β, il- , il- , il- , c-x-c motif chemokine ligand (cxcl) , granulocyte-macrophage colonystimulating factor (gm-csf), granulocyte-colony stimulating factor (g-csf), tumor necrosis factor (tnf)-α, fibroblast growth factor and (fgf / ), transforming growth factor (tgf)-β , c-c motif chemokine ligand (ccl) , ccl , and thymic stromal lymphopoietin (tslp)] act on recruited immune cells and resident cells to initiate a series of innate immune responses ( , ( ) ( ) ( ) ( ) ( ) . meanwhile, activated alveolar macrophages, which are usually patrolling the lung parenchyma, further release more pro-inflammatory mediators and chemokines [il- β, il- , il- , il- , tnf-α, ccl , cxcl , cxcl (ena- ), cxcl , cxcl , cxcl , ccl , leukotriene b (ltb )], ros, elastolytic enzyme [matrix metalloprotease protein (mmp)- ,− , and− ; and cathepsin-k,-l, and -s], gm-csf, and g-csf ( , , ) . the enhanced levels of ccl and cxcl result in recruitement of blood monocytes expressing ccr and cxcr (receptors for ccl and cxcl , respectively), to the lung and differentiate locally into macrophages. interestingly, there are higher expression levels of the ccr and cxcr found on blood monocytes in copd subjects ( ) . this may explain the rapid recruitment and excessive accumulation of monocyte-derived interstitial macrophages in the lung tissue of copd patients ( , ) . upregulation of neutrophil chemoattractors (ltb , cxcl , cxcl , il- , and tnf-α) induces a massive migration of circulating neutrophils into the lung parenchyma ( ) . the transmigration of blood neutrophils occurs through adherence of the granulocytes to e-selectin of endothelial cells that is found to be upregulated in copd ( ) . this results in airway neutrophilia in several copd patients ( , , ) . the recruited neutrophils (to the lung) are then activated to secrete granule proteins [myeloperoxidase (mpo) and neutrophil lipocalin] while releasing its own il- for further neutrophilic recruitment and amplification of the inflammation ( ) . in addition to the macrophage-derived proteases, neutrophils also secrete serine proteases [neutrophil elastase (ne), cathepsin g, proteinase- , mmp- , and mmp- ] that are associated with serious alveolar destruction in emphysema ( ) . the protease activity may be further enhanced in conditions with genetic deficiencies or suppressed expression of α -antitrypsin by tobacco smoke. in addition, ne, cathepsin g, and proteinase- are involved in the stimulation of mucus secretion from submucosal glands and goblet cells, resulting in airway mucus hypersecretion and airway obstruction in copd ( ) . the nlrp (nlrp : nucleotide-binding domain, leucinerich-containing family, pyrin domain-containing- or nod-like receptor protein ) inflammasome is a cytosolic multi-protein complex (consisting of the inflammation sensor protein nlrp , adapter protein asc, and the effector protein caspase- ) ( ). the nlrp inflammasomes are involved in the copd airway inflammation by regulating the production of pro-inflammatory cytokines il- α, il- β, and il- . these cytokines are important for neutrophil survival and activation of t helper (th) cells ( ) . interestingly, local airway nlrp inflammasome activation is positively correlated with acute exacerbations and lower airway microbial colonization in copd patients ( , ) . moreover, in an elastase-induced emphysema model, the nlrp inflammasome is activated in addition to hyperproduction of mucin muc ac by diesel extract particles, extracellular atp, and inflammatory protein s ( , ) . the adaptive immunity is initiated at a later stage, and is recognized by the increased number of t and b lymphocytes and pulmonary dendritic cells. dendritic cells are the major antigen-presenting cells (apc) in the airways, and link the innate and adaptive immunity. circulating dendritic cells (expressing receptors ccr and ccr ) are recruited to the airway via dendritic chemoattractants ccl and ccl released by activated airway epithelial cells in response to cigarette smoke ( , ) . dendritic cells act by endocytosis of inhaled irritants that subsequently are processed into antigen peptides during maturation and further migration to lymph nodes. uncommitted t lymphocytes are thereafter primed by the presented antigen. these important cells are activated by il- released from dendritic cells for subsequent commitment into antigen-specific t cell lineages, i.e., t helper (th ; cd + cd + ) cells, whereas immature dendritic cells in the airway promote th differentiation ( , ) . interestingly, in copd patients, pulmonary th and cytotoxic t cells (tc; cd + cd + ) express more cxcr receptors compared to healthy individuals ( , ) . this enhances their migration toward chemoattractants cxcl , cxcl , and cxcl that are actively released by alveolar macrophages in copd subjects. activated cd + t cell subset type (tc ) releases perforins, granzyme b, and tnf-α to induce alveolar cells apoptosis, contributing to the emphysema ( ) . in parallel, pulmonary th t cells are activated by alveolar macrophage-derived il- and il- to secrete il- a and il- causing neutrophilic inflammation ( , ) . inflammatory cytokines are also released by type figure | non-typeable h. influenzae-dependent immune responses in the lower airway of copd patients result in inflammation. airway epithelium exposed to cigarette or tobacco smoke display an increased permeability with compromised tight junctions, and airway remodeling (goblet cell hyperplasia and small airway squamous metaplasia). cigarette smoke causes the activation of airway epithelium and alveolar macrophages. the activated airway structural and resident immune cells release an array of chemotactic factors responsible for recruitment of inflammatory and immune cells to the lung. activated epithelium produces tgf-β and fgf that triggers the production of ecm molecules by fibroblasts. increased deposition of ecm causes progression of fibrosis and air flow limitation. the chemokines cxcl and il- , and ltb attract the circulating neutrophils through the receptors cxcr and blt , respectively. meanwhile, cxcl and ccl targeting the receptors cxcr and ccr on blood monocytes are also released. recruited blood monocytes differentiate into macrophages in the airway tissue. activated alveolar macrophage and epithelium cell also release inflammasome ( l- β and il- ) for neutrophils survival and activation of helper t cells th . the chemokine il- are released by macrophages to attract t helper cell subset th , and ilc . both th and ilc will release il- and il- that will act on the alveolar epithelium to release cxcl and il- for enhanced recruitment of neutrophils, resulting in neutrophilic inflammation. activated neutrophils are thereafter degranulated and release myeloperoxidase (mpo), lipocalin, neutrophil elastase (ne), cathepsin-g (cg), proteinase- (prot- ), and matrix metalloprotease (mmp) and . the granulated products are proteolytic and elastilolytic to aveolar, causing alveolar destruction and emphysema. in addition, ne, cg, and prot- are also targeting goblet cells and submucosal glands to induce hypersecretion of mucus. dendritic cells carrying the receptors ccr and ccr are recruited to airway tissue via chemottractants ccl and ccl . the dendritic cells uptake the antigen (smoke residues), and present the antigens to the naïve t cells at lymph nodes. uncommitted t lymphocytes are thereafter primed to the presented antigen and activated by il- derived from dendritic cells (professional antigen presenting cells; apc). mature/activated t cells expressing receptor cxcr are chemotactic toward cxcl , cxcl , and cxcl and are recruited to the lung tissue. cytotoxic cd + t cell subtype tc releases perforin and granzyme b resulting in epithelial apoptosis contributing to emphysema progression. for the humoral immune response, b cells are activated by th , enter the circulation via high-endothelial venule (hev)-like vessel and transported to lung tissue, and organized into lymphoid follicles at peripheral airway. b cell-derived plasma cells from lymphoid follicles release iga, and secreted into airway lumen as secretory iga (siga) via the polymeric immunoglobulin receptor. mucosal antibodies play an important role to eradicate pathogens and noxious antigens via immune exclusion. however, the airway defense by siga is diminished by nthi iga protease that degrade the antibodies. tlr and tlr of the airway phagocytes and epithelium following exposure to cigarette smoke are not responding to p and los of nthi. this results in defective phagocytosis and delayed bacterial clearance from the airway. the suppressed tlr induction in t cells has also lead to th predominant immune response, with low production of ifn-γ and reduced t cell-mediated immune killing of nthi. moreover, nthi downregulates foxp of tregs and thus impairs the anti-inflammatory/pro-inflammatory balance of tregs. the extensive immunosuppressive activity by tregs diminishes the response of effector t to (continued) figure | nthi stimulation. lastly, plasma cells from copd patients fail to produce nthi-specific antibodies and compromised immunoglobulin class switching. the impairment of the host immune response in copd toward nthi infection are labeled in blue. in total, nthi infection in copd lung adversely reduces the production of il- β, il- , il- , cxcl- , il- , tnf-α, antimicrobial peptide (amp), and ifn-γ. this may explain the inefficient eradication of airway pathogens in copd patients whereby persistent nthi infection concomitantly escalates the inflammation and thus exacerbation in copd. innate lymphoid cells (ilc ) ( ) . the ilcs are involved in the homeostasis of lung immunity and are regulated by epithelially produced il- and tslp ( , ) , and are further stimulated in response to cell damage. the accumulation of b lymphocytes in the peripheral airway and within lymphoid follicles is associated with airway autoimmunity in the progression of copd ( ). airway tissue damage in conjunction with impaired t-regulatory cells (tregs), both related by cigarette smoke, contributes to the formation of autoantibodies against airway components. autoantibodies against elastin, epithelial, endothelial, carbonylated, and citrullinated proteins are found in the circulation of copd patients ( ) ( ) ( ) ( ) ( ) ( ) . the generation of autoantibodies might activate plasma exudate-derived complement components resulting in a chronic inflammation, and consequently damage of the airways with emphysema progression ( ) ( ) ( ) ( ) . from a physiological point of view, a modulated inflammatory process is important for a protective and optimal immune response. however, the prolonged airway inflammation in copd as a results of impaired homeostasis leads to serious side effects since it amplifies the tissue damage and impairs the local immune defenses. the abrogated local immune system may make the airways of copd patients susceptible for opportunistic or recurrent infections by viruses and bacteria that in turn might exacerbate the disease. acute exacerbations of copd (aecopd) are episodes of acute symptom worsening that usually are associated with both respiratory (increased airway inflammation) and non-respiratory (system inflammation/co-morbidities) effects ( ) ( ) ( ) . the typical symptoms of an aecopd include increased production of purulent sputum, dyspnea, cough, wheezing, and symptoms of a cold that may last from days up to weeks ( , , ) . it commonly occurs in patients with advanced copd and results in additional therapy based on the level of exacerbations. exacerbations are classified in three levels according to gold. there is the mild disease that can be treated with short acting bronchodilators (sab); moderate disease with sab combined with antibiotics and/ or oral corticosteroids; and finally severe exacerbations with acute respiratory failure which requires emergency room visit and eventually hospitalization ( , ) . aecopd is a complex yet multifactorial consequence of copd. most of the exacerbations could be triggered by infectious (up to %) or non-infectious agents (∼ %) (aecopd with known etiology), whereas up to % of cases are of unknown etiology ( , ) . respiratory tract infections are the major causes for aecopd with known etiology and are mainly attributed to infections by viruses, bacteria, and atypical bacteria (not detected with conventional gram-staining) ( , , ) . non-infectious causes of aecopd include air pollution, environmental factors, meteorological effects, and comorbidities of the patients, all of which are partially contributing to copd exacerbations ( , , ) . respiratory viral infections are often the primary cause in the infection-dependent aecopd, and virus was identified as single or multiple infecting strains from up to % of copd patients with exacerbations recorded between years - ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the most common infecting viruses are, by far, human rhinovirus, influenza virus a, and respiratory syncytial virus, whereas parainfluenza virus, coronavirus, echovirus, human metapneumovirus, and adenovirus are considerably rare. bacterial infections contribute to an average of % of infective acute exacerbations with a prevalence being reported ranging from to % ( , , ( ) ( ) ( ) . the most commonly pathogenic bacterial species isolated from the lower airway of copd patients during aecopd are nthi, moraxella catarrhalis, streptococcus pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, and klebsiella pneumoniae ( , , , , ( ) ( ) ( ) ( ) . it has been suggested that infection with new strains of the infecting species, rather than a new species, is highly associated with an increased risk of exacerbation ( , , ) . atypical bacteria that cause exacerbations are chlamydia spp., legionella pneumophilia, and mycoplasma spp. in contrast to viral infections that are diagnosed in - % of copd patients with stable disease and increase to . - % during copd exacerbations, bacterial colonization in the airways are more common with the same species during both stable disease ( - %) and exacerbations ( . - %) ( , , , , , ( ) ( ) ( ) ( ) . hence the precise or direct role of bacterial infection as the primary cause in triggering aecopd remains controversial although a significantly increased bacterial load is observed during exacerbation in several patients. this further suggests that bacteria might be more involved as secondary invaders after an initial viral infection. viral infections have been reported to cause several physiological changes in the lung that in turn facilitates secondary bacterial invasion. the mechanism of bacterial superinfection has been described for h. influenzae, s. pneumoniae, s. aureus, and many other airway pathogens ( ) ( ) ( ) . firstly, viral infections destroy the tight junctions of the airway epithelial barrier while inducing epithelium apoptosis. this results in the onset of airway epithelium lining repair whereby the sloughed off dead cells would become a rich nutrient source for growth of infecting bacteria. the damaged epithelium lining also enables bacterial adherence to the exposed basement membrane and ecm. secondly, the demolished ciliated clearance as a result of the virus-damaged airway epithelium lining further promotes bacterial colonization and subsequent epithelial transmigration into deeper tissues ( ) ( ) ( ) . lastly, viral infections are also detrimental to the airway immune defense by causing degradation of antimicrobial peptides (amp), and by triggering ifn-γ secretion by immune cells. this results in suppressed macrophage and neutrophil responses to infecting bacteria, and thus enables bacterial evasion of the airway immune defense ( ) ( ) ( ) ( ) . nevertheless, viral and bacterial coinfection have greater impact in the aecopd airway inflammatory responses than bacteria or virus infection alone ( , ) . this is in parallel with the co-isolation of both respiratory viruses and bacteria from to % of aecopd patients ( , , , ( ) ( ) ( ) ( ) ( ) . infective aecopd is also attributed to impaired functions of amp, macrophages, and neutrophils triggered by inhaled irritants such as tobacco smoke. expression of microbialinduced amp (human β-defensin ) is suppressed in airway epithelial cells when exposed to cigarette smoke ( , ) . both the alveolar and monocyte-derived macrophages in patients with copd are defective in phagocytosis of bacteria such as h. influenzae and s. pneumoniae ( , ) , and in efferocytosis of apoptotic neutrophils and epithelial cells. in addition, neutrophils from copd patients are aberrant in chemotactic response with defective accuracy ( ) . all these factors contribute to the failure to resolve inflammation in copd leading to facilitated chronic microbial colonization, also during exacerbations. the low number of cultivable bacteria found in healthy individuals previously led to the conclusion that healthy and normal lungs are virtually sterile. this hypothesis is currently being revised, since the introduction of s rdna based molecular diagnostics has shown that even healthy lungs have a distinct microbial community, different from that seen in the upper respiratory tract ( , ) . this has led to the concept of a core human lung microbiome which can be altered in copd stable disease and during exacerbations ( ) . the role of the lung microbiome in the pathogenesis of copd by influencing host immune response has also been suggested ( , ( ) ( ) ( ) ( ) ( ) ( ) . the stability of the lung microbiome has profound impact on maintaining local immune homeostasis ( ) . according to the "vicious circle" hypothesis, airway inflammation and impaired immune defenses caused by either viral infections or irritant inhalation have ecological influence on the airway microenvironment and growth conditions that would eventually lead to dysbiosis of the lung microbiota ( , ) . the changed lung microbiome would then cause a maladaptive immunological response resulting in further inflammation and damage of the lung immune defenses, and additional alteration of the lung microbiome. the chain of events thus generates a vicious circle that contributes to copd progression and exacerbation. several studies have documented that copd progression from stable state to an exacerbation could induce microbiota shift in the lower airway (bronchioles), sputum, and throat ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . alteration in the microbiome complexity or richness is associated with the inflammatory process and changes in ecm protein expression in the lung, as observed in copd ( , ) . declined diversity in the lung microbiome has been reported to be related to disease severity, inflammation and decreased lung functions in copd. this includes the increased emphysematous destruction, bronchial tissue remodeling, lymphoid follicle formation, elevated autoantibodies, and il- a production, and finally increased neutrophil extracellular traps (net) formation in the airway of animal models or aecopd patients ( ) ( ) ( ) ( ) . it has recently been reported that lung microbiome diversity is also associated with genetic factors. mannose-binding lectin (mbl) deficiency has also been associated with disease severity and exacerbations in patients with cystic fibrosis and bronchiectasis ( ) . however, copd patients with a genetic deficiency in mbl are less susceptible to haemophilus spp. colonization, lowering the risk of exacerbations while their lung microbiota is more diverse than normal copd patients ( ) . in this review we will focus on nthi, one of the dominant genera that is relatively abundant in the total copd-dependent lung microbiome, due to its role of infection in copd immunological responses ( , , - , , - ) . the microbiology of h. influenzae has recently been reviewed in detail by our group and others ( , , ) . it is a gramnegative coccobacillus that commonly colonizes the human nasopharynx, and is typed as capsulated (type a-f) or nonencapsulated strains (nthi). h. influenzae may cause both invasive and mucosal disease ( ) . since the introduction of capsule polysaccharide conjugate vaccines against type b (hib), nthi dominate, followed by capsule type f (hif) ( , ) . mucosal infections, including acute otitis media, sinusitis, and exacerbations in copd, are nowadays mainly associated with nthi. there has also been a significant shift in the epidemiology of severe invasive disease, from hib infections in small children to nthi in adults ( , ) . the most common principal infection focus by h. influenzae is now community acquired pneumonia (cap), whereas the incidence of historically common diagnoses such as meningitis and epiglottitis have significantly decreased ( , ) . patients with underlying conditions, notably copd, seem to be at higher risk for invasive infections ( ) . there is consensus that h. influenzae is one of the key bacterial pathogens involved in pathogenesis of both stable copd disease and acute exacerbations ( ) . however, the relative abundance and significance of nthi in copd varies between different studies. several factors, such as sampling methodology, choice of microbiological analysis and, if the patient has a stable disease or an exacerbation, or has been subject to previous antibiotic therapies, tend to affect the outcome of the studies ( ) . common sampling methods from the lower respiratory tract include both bronchoscopy techniques such as protected specimen brush (psb) and collection of bal fluid as well as non-invasive methods like sputum sampling ( ) . all of these methods, particularly sputum, are to some extent subject to the risk of contamination from the normal microbial flora of the oro-and nasopharynx, which might reduce their specificity ( ) . however, several studies still show a distinct association between lower respiratory tract samples and clinical parameters in copd patients, making the information valuable ( ) . cultivable bacteria are seldom found in the lower airways of healthy individuals ( ) , whereas copd patients show bacterial growth in - % of cases even during stable disease ( table ) . on top of that, several studies have shown a significant increase in the proteobacteria phylum, which includes haemophilus spp., in individuals with both stable disease and aecopd ( table ) . nthi is consistently one of the predominating bacterial species isolated in those cultures; other important pathogens include s. pneumoniae, m. catarrhalis, and p. aeruginosa ( ) . during aecopd, the bacterial load is increased even further, and nthi continues to be the predominating species ( ) . furthermore, acquisition of a new nthi strain has, in one study, been linked to the onset of aecopd ( ) . moreover, the growth and dominance of h. influenzae following rhinovirus infection was observed in the sputum microbiome of patients with copd ( ) . the chronic inflammation that characterizes copd pathogenesis causes significant changes to the pulmonary tissue. the lower respiratory tract of patients suffering from this disease is marked by epithelial denuding, hypersecretion of mucus, disproportionate phagocyte presence and imbalances in antioxidant/oxidants ( ) . this altered milieu selects for specific bacterial species that are genetically equipped to competently address these environmental stressors ( , , ) . nthi is the most common pathogen isolated from the sputum of copd patients, and the primary cause of exacerbations ( ) , indicating a unique ability to colonize and persist in the chronically inflamed lower respiratory tract. in recent years, great efforts have been made in understanding how nthi colonizes the pulmonary tissue. in addition to the regular arsenal of virulence factors associated with nthi ( ), the bacterial pathogen undergoes specific adaptations to increase its fitness in the copd setting. specific genetic islands that include ureabcefgh, lic b, hgba, iga, hmw , and hmw have been reported to be enriched in nthi strains isolated from copd patients compared to commensal nthi ( ) . these genes are involved in raising the ph of the environment, lipooligosaccharide (los) synthesis, iron uptake, immune evasion, and attachment to host tissue. the validity of these findings is strengthened by previous work identifying upregulation of many of the same bacterial gene products during growth in copd sputum ( ) . moreover, peroxiredoxinthioredoxin, an antioxidant enzyme, was found to be one of the most enriched proteins in nthi during growth in copd sputum, suggesting that the bacteria upregulate oxidative stress-countermeasures when facing oxidative imbalances in the diseased lung ( ) . oxidative stress resistance has previously been shown to be vital for nthi survival in infection models ( ) . in a seminal investigation by pettigrew et al., wholegenome sequencing (wgs) was conducted to follow the in vivo adaptation of nthi to the copd environment over time ( ) . several interesting findings were reported in this work. firstly, the median duration of persistence by the pathogen was found to be days, but it could persist in patients for up to as many as , days. secondly, slipped-strand mispairing-mediated phase variation was identified as the primary genetic adaptation to the niche. poignantly, the genes affected by the regulation mechanism encoded for (among others) the hmw adhesins, los biosynthesis, and iron uptake, that is, the same processes identified in the previous studies as important for copd adaptation ( , ) . thirdly, and somewhat surprising, it was observed that a very limited number of genes were gained/lost during persistent colonization, meaning that selection for strains that thrive in the inflamed lower airways occurs at the very onset of colonization. finally, the authors reported that genetic changes occurred in of the investigated vaccine antigens during persistent infections, a fact that might be taken into consideration for potential vaccine development against nthi. another virulence factor that has been reported by murphy and co-workers to play a pivotal role for nthi survival in copd settings is iga-protease, a hydrolytic enzyme that cleaves secretory iga (siga) antibodies in the mucosal epithelium ( ) ( ) ( ) . four genes encode for the same number of different variants of the endopeptidase with various cleavage site specificities: two igaa (igaa and igaa ) and two igab (igab and igab ). the igaa is present in all nthi whereas igab is present in ∼ % of the strains ( ) . the igab gene has been reported to be more prevalent in copd exacerbation-causing strains, although the in vivo expression levels did not differ from asymptomatic colonization strains that also carried the gene ( ) . however, iga-protease b and b have been found to promote the intracellular survival of nthi in human epithelial cells, providing a secondary function (in addition to hydrolysis of iga antibodies) that could facilitate nthi growth in inflamed environments ( ) . while a majority of the persistent nthi strains that dwell in copd patients continuously express one or more variants of the enzyme, it has recently been found that a phase variation to an off-state can occur via slipped-strand mispairing over time ( ) . this suggests that during certain conditions, there is a fitness benefit in not expressing iga in the airways of copd patients, albeit the specifics of this process are currently unknown. another interesting aspect of nthi colonization of copd patients is with regard to biofilm formation ( ) . nthi strains that colonize the eustachian tube causing otitis media are known to build up biofilms in situ ( ) . however, strains isolated from copd patients tend to have significantly diminished ability to form biofilm compared to invasive strains or those isolated from otitis media patients ( ) , suggesting that this mechanism is not important for survival in the copd niche. as biofilms tend to protect the bacterial community from external assaults, these findings could indicate that the hypermucoid milieu in the copd airways is severely impaired in its ability to deliver an apt immune response for optimal clearance of residing microorganisms. in light of this impairment, biofilm formation might not be necessary for nthi to persist in this particular environment. infections with nthi have also been shown to reduce cellular levels of e-cadherin, a protein required for tight junction formation and epithelial cell integrity in human cells ( ) . considering that perturbations in the epithelial cell barrier caused by the loss of e-cadherin is a common symptom of copd, nthi-mediated exacerbations likely contribute to this step of copd pathogenesis. the subsequent denuding of the epithelium could facilitate microbial colonization of the basal lamina, a well-established virulence mechanism employed by nthi and other pathogens ( ) . it is currently unknown which bacterial virulence factor(s) that induce the reduction of e-cadherin levels in the host. in summary, investigations from recent years show that the environment of the lower respiratory tract of copd patients selects for nthi strains that can upregulate adhesins, modify los biosynthesis pathways, increase antioxidant stress responses and cellular invasion strategies, and, finally, trigger tolerance against acidic ph. these important colonization mechanisms thus provide researchers with viable targets for developing novel therapies. nthi is a commensal in the nasopharyngeal site but is often associated with strong inflammatory responses in the lower respiratory airways, especially in patients with copd, bronchiectasis, cystic fibrosis, pneumonia, or idiopathic pulmonary fibrosis ( , ) . colonization and subsequent infection of nthi in the lower airways of copd patients elicits episodes of immune responses orchestrated by both the innate and adaptive immunity. nthi infection is thus commonly associated with inflammation that is mainly mediated by transcription factor nf-κb-dependent production of proinflammatory mediators. the activation of nf-κb requires induction of cross-signaling networks and cascades via activation of prrs (pattern recognition receptors) of host innate immune cells ( ) . unresolved or prolonged (chronic) inflammation or failure to restore the homeostatic inflammatory status potentially contributes to exacerbations. this is clearly shown in murine copd simulation models with nthi-triggered inflammation ( - ). mice exposed to nthi lysates display inflamed airways loaded with increased levels of inflammatory mediators and phagocyte infiltrates. moreover, multiple exposures to bacterial lysates which may represent a chronic nthi infection caused extremely high infiltration of phagocytes and lymphocytes in the airways of this particular mouse model. in addition, the airway walls of the infected animals were also thickened due to increased collagen deposition (fibrosis) that reflects the typical copd features. the host immune response and specific interactions during nthi infection in copd is summarized in figure . the epithelium and alveolar macrophages are predominant cell types in the airway compartment. they comprise the first line of defense in the cellular immune response against potential inhaled pathogens and antigens. the sensing of bacteria, and particularly nthi in the lower airways is initiated via prrs expressed on innate immune cells and endothelium in addition to epithelial cells ( ) ( ) ( ) . tlrs are prrs that sense stimulation by nthiderived pathogen-associated molecular patterns (pamps), and play a primary role in initiating effector cellular responses and intracellular signaling for nf-κb activation ( ) . among the different tlrs, most of the studies on nthi infection have by far been focused on tlr and . lipoproteins including nthi p , and los are potent immunomodulators for activation of tlr and tlr , respectively, and has been described in several studies on airway epithelial cells and alveolar macrophages ( ) ( ) ( ) ( ) . interaction of nthi lipoprotein p with tlr on human epithelial cells [type ii alveolar a and human middle ear epithelial cells (hmee)] causes nf-κb-dependent activation via two distinct tlr-signaling pathways, that is, the nf-κb translocation-dependent, and -independent pathways ( ) . the nf-κb nuclear translocation-dependent pathway requires activation of nf-κb-inducing kinase ikk complex. in the second pathway, the mkk / -p mapk signaling cascade is recruited for direct nuclear phosphorylation, and thus activation of nf-κb. the branching of both pathways may occur at the tgf-β activated kinase (tak ) signaling junction. nthi stimulation via tlr and downstream activation of p mapk/nf-κb-dependent pathways result in expression of cox- and prostaglandin (e ) (pge ) that promote inflammatory responses ( ) . tlr stimulation by nthi los also contributes to the activation of nf-κb via two signaling pathways, the primary activating pathway of myd cascade and the alternative pathway of toll/il- r domain-containing adapter-inducing interferon-β (trif). both pathways activate nf-κb through phosphorylation and degradation of inhibitor iκbα ( , ) . nthi-tlr signaling mediates an effective innate immune response that leads to upregulation of tnf-α, il- β, il- , macrophage-inflammatory protein (mip)- α, mip- , and neutrophil infiltration in the airways of mice. the tlr response promotes efficient pulmonary clearance of bacteria in tlr expressing animals compared to cd /tlr knockout mice ( , ) . a recent study by jungnickel et al. revealed that, in parallel with the infection-induced pulmonary neutrophilic inflammation, nthi-dependent stimulation of both tlr and tlr in a transgenic mouse [(kras la ) with oncogenic kras allele in the lung epithelium] additionally promotes the proliferation of kras-induced early adenomatous lesion in the lung in an tlr-dependent manner ( ) . the association or role of nthi-induced airway inflammation in lung cancer progression, however, is not supported by another recent cohort study showing the lack of differences in nthi specific-antibodies between cancer-and non-cancer copd patients ( ) . lastly, dectin- and the epidermal growth factor receptor (efgr) pathway also have proinflammtory effects upon interaction with nthi ( , ) . activation of the dectindependent proinflammatory response requires nthi-induced phosphorylation of the dectin- hem-immunoreceptor tyrosinebased activation motif (hemitam) ( ) . direct activation of efgr in alveolar cells and hmee by nthi-derived egflike factor has been shown to contribute to nf-κb activation. the efgr-dependent nf-κb activation is mediated via an nf-κb nuclear translocation-independent pathway, which involves both mkk / -p and pi k/akt signaling pathways ( ) . surprisingly, the interaction of efgr and nthi also results in negative regulation and suppression of the induction of tlr via the src-mkk / -p α/β map kinase-dependent signaling cascade, and this in turn may facilitate nthi infection ( ) . the actual components of nthi that exhibit the egf-like factor activity have, however, yet to be defined. the efgrdependent negative regulation of tlr may thus suggest a novel mechanism targeted by nthi for immune evasion by attenuating the responses of host prr, despite the contradicted role of efgr in proinflammatory and innate immune responses of the airway epithelium ( ) . nthi infection also upregulates the nrlp -inflammasome during nthi-induced inflammation in the airway epithelium and alveolar macrophages, leading to increased secretion of il- β and il- , and thus neutrophilic influx to the lung ( ) . some of the endogenous inflammatory mediators that are produced in response to nthi infection, including tnf-α, il- α, and tgf-β , may act synergetically with nthi on the airway epithelial and immune cells. the synergetic interaction drives a positive feedback loop to amplify the nf-κb transcriptional activity on proinflammatory genes and further augments airway inflammation. the synergetic activation of nf-κb by nthi and tnf-α in hmee and normal human bronchial epithelial (nhbe) cells occurs via nf-κb nuclear translocation-dependent and independent pathways. the latter pathway involves mapk/extracellular signal regulated kinase kinase kinase (mekk )-dependent activation of mapk kinase / -p mapk pathway ( ) . however, the synergetic action of nthi with tgf-β is mediated by another mechanism which involves smad / -protein kinase a (pka)-p -dependent signaling cascade. the pathway components, pka and p , phosphorylates residue ser and acetylates lys of the nf-κb subunit p , respectively. this results in enhanced dna-binding activity of nf-κb ( ) . the synergetic action of nthi with both tnf-α and tgf-β enhances the production of tnf-α, il- β, and il- from airway epithelial cells and interstitial polymorphonuclear infiltrates. recently, it has been reported that co-infection of human rhinovirus and nthi on the airway epithelial cells (nhbe cells and the beas- b cell line) also results in synergetic induction of ccl and il- , albeit the exact mechanism remains to be elucidated ( ) . of note, activated macrophages also release increased concentrations of tnf-α and il- α ( ) , further enhancing the inflammatory synergetic effect of surrounding immune cells. finally, il- α acts synergetically with nthi to upregulate the expression of amp β-defensin (defb- ) via the p /mapk pathway ( ) . of note, il- α could also act individually to upregulate the expression of defb- via the src-dependent mek / -erk / signaling pathway ( ) . taken together, the synergetic action may aid in the expansion of the inflammatory response and in some cases worsen the clinical outcome. alveolar macrophages located in the air-parenchyma interface are the primary professional phagocytes in the lung ( , ) . these cells are responsible for infection eradication through its phagolysosomal machinery while releasing a plethora of inflammatory cytokines and chemokines for promoting a local inflammatory response and recruitment of neutrophils. neutrophils are the first responder cells recruited from circulation to the airway for efficient killing of pathogens through an array of microbicidal strategies ( , ) . during nthi lung infection, both alveolar macrophages and neutrophils are the main innate immune cells involved in the pulmonary bacterial clearance through phagocytosis. they are also an important source of cytokine secretion required for induction of other immune cells and enhanced bacterial killing. eradication of nthi by alveolar macrophages involves adhesion or contact, phagocytosis and phagolysosomal processing of bacteria, in addition to secretion of tnf-α. phagocytic clearance of nthi by alveolar macrophages is orchestrated through actin polymerization, plasma membrane lipid rafts, and phosphatidylinositol -kinase (pi k) signaling cascade upon induction of macrophage prrs by nthi ( ) . interestingly, in response to nthi infection, human alveolar macrophages, and blood neutrophils produce extensive amount of intracellular and extracellular ros as a component of the antimicrobial defense. this leads to the formation of macrophage and neutrophil extracellular traps (mets and nets, respectively), with co-expression of mmp- for enhanced bacterial killing ( , ) . nevertheless, the overexpression of mmps may adversely result in a protease imbalance and contribute to alveolar emphysematous destruction and bronchiectasis in copd ( ) . moreover, excessive endogenous ros production could also introduce airway oxidative stress that is detrimental by causing chronic inflammation and tissue damage in the lung, and thus contributing to the copd exacerbation ( , ) . the net formation is elicited mainly by nthi los in addition to other haemophilus pamps ( ) . several studies by king et al. have revealed that t cellmediated adaptive immune responses against nthi airway infection in patients with idiopathic bronchiectasis and copd has been predominated by a th /tc response ( ) ( ) ( ) . the activated t cells produce reduced level of the cd ligand and ifn-γ, and increased levels of tnf-α, il- , and il- , as well as altered igg subclass production by plasma cells. it is to be noted that the th /tc -mediated immune response is less effective in suppressing nthi infection. redirecting the th /tc -mediated immune response to th /tc dominant (which is more protective) by adding the th /tc mediators (cd ligand and ifn-γ) has helped to restore the t cellmediated immune killing of nthi ( ) . however, a separate study in a copd mouse model by lu et al. reported that nthi infection causes increased production of airway type interferon ( -ifn) ( ) . it was further reported that dna of nthi acts as a pamp in stimulating the sting/tbk /irf pathway, and thus the production of -ifn. the impact of the bacterial dnainduced -ifn in host immune/inflammatory response, which may potentially induce a th /tc response requires further investigations. copd patients also have abnormally higher number of treg cells, myeloid-derived suppressor cells (mdsc), and exhausted effector t cells (pd- + ) than healthy individuals ( , ) . cigarette smoke-induced anti-inflammatory activity of tregs in a copd model is further suppressed by nthi infection. the pathogen causes downregulation of foxp (biomarker of tregs), and thus impairs the anti-inflammatory/pro-inflammatory balance of tregs ( , ) . this may lead to the extensive immunosuppressive activity by tregs on the proliferation of nthi p -specific effector t cells, causing a diminished response of effector t cells to sputum il- and il- induction, and increased levels of il- and tgf-β ( , ) . recently, it has been reported that mucosal-associated invariant t cells (mait) from copd patients are more effective in response to nthi stimulation and thus produce increased levels of ifn-γ, -, to -fold more than the copd th (cd + ) and tc (cd + ) cells ( ) . however, the pulmonary mait cell immune responses are compromised in the presence of corticosteroids that are commonly used for the treatment of copd. this may potentially prone the t cell-mediated immunity to a th /tc response in copd patients treated with corticosteroids ( ) . interestingly, antigen-specific th cells from nthi-immunized non-copd mice model recognize both homologous and heterologous strains of nthi, and are able to confer protection upon adoptive transfer ( ) . however, it is unclear whether the th cell which is prone to the inflammatory response could be "trained" to counteract the nthi infection in copd patients, particularly during exacerbations. during the systemic humoral immune response in nthiinfected copd patients, greater concentrations of nthi-specific igg, iga, igm, and ige serum antibodies are produced compared to non-infected controls ( , ( ) ( ) ( ) . some of the nthispecific serum immunoglobulins are specific to p , p , and p ( , , ) . however, decreased mucosal antibodies associated with siga deficiency, or decreased total igg in the small airways have been reported in copd patients, and might be associated with disease severity ( , ) . importantly, nthi-specific mucosal siga has been found to be lower in the airways of nthi-infected copd patients than the non-colonized patients ( , ) . the epithelial polymeric immunoglobulin receptor (pigr) is essential for the generation of mucosal siga. it is, however, downregulated in copd patients with a positive correlation to disease severity and increased level of tgf-β ( ) . the combinatorial effects of downregulated plgr and elevated tgf-β contribute to an impaired mucosal iga immunity in copd patients. a mouse model lacking the pigr ( −/− ) is therefore devoid of siga and are susceptible to airway stimulation by an nthi lysate resulting in increased inflammation and airway neutrophilia. interestingly, introduction of exogenously added siga mitigated the airway inflammation ( ) . nthiinfected copd patients with greater airway inflammation have also decreased nthi-specific mucosal igg in the bal fluid compared to the non-colonized patients ( ) . interestingly, the phenomenon with decreased nthi-specific antibodies seems to be restricted to the airways, since the specific serum antibodies are not affected. therefore, the reduced mucosal igg is unlikely to be associated with hypogammaglobulinemia (igg deficiency), despite the latter was reported as a contributing factor in nthi infection ( ) . decreased airway iga might be attributed to the expression of iga proteases by nthi. the bacterial iga protease degrades the local airway iga during airway colonization to avoid immune exclusion by siga ( , ) . reduced mucosal antibodies might promote host immune evasion and resistance to complement-mediated killing of nthi, thus enable persistent colonization of nthi in the airways of copd patients, in addition to a plethora of various other virulence mechanisms ( , , ) . in a cohort study of stable copd patients, augmented airway inflammation and plasma fibrinogen, but not systemic inflammation, were found to be constantly correlated with the increased bacterial load ( ) . higher numbers of nthi has a greater impact than s. pneumoniae and m. catarrhalis in triggering inflammatory responses as measured by the augmented levels of inflammatory cytokines in sputum including il- , mpo, and l- β. the increased inflammatory response in affected patients is potentially attributed to the persistent colonization of nthi in the lower airway ( , ) . the compromised innate immune response in copd, particularly the decreased microbicidal activity, has been regarded as one of the culprits for persistent airway colonization by nthi, and is highly associated with copd exacerbations (figure ). whilst the role of macrophage extracellular traps (met) for killing of nthi remains unknown, it has been reported that blood neutrophils and net from copd patients are defective in the killing of planktonic or biofilm/net-entrapped nthi, respectively ( , , ) . a series of studies by berenson et al. revealed that alveolar macrophages derived from copd patients are basically dysfunctional in eradication of nthi ( , ( ) ( ) ( ) . intriguingly, tlr and tlr expressed on alveolar macrophages from copd patients are intrinsically unresponsive to the potent immunomodulatory lipoprotein p and los, respectively. this causes decreased los/p -induced expression of tlrs, reduced nf-κb nuclear activation and consequently diminished il- , tnf-α, and il- β responses by alveolar macrophages from copd patients. the compromised tlr expression and signaling potentially contribute to the defective complement-dependent and independent phagocytosis of nthi. the defective phagocytosis is greater for nthi than for m. catarrhalis, and correlates with disease severity. interestingly, the phagocytosis disability was not detected in monocyte-derived macrophages in copd. in contrast, however, taylor et al. reported that monocyte-derived macrophages from copd patients are also defective in phagocytosis of nthi and s. pneumoniae. the author also suggested that the defective monocytederived macrophages are not attributed to the alteration in cell surface tlr or tlr expression, macrophage receptor with collagenous structure (marco), cd , cd or the mannose receptor ( ) . the unresponsive tlr and tlr in copd alveolar macrophages to nthi lipoprotein and los might be explained by the recently reported phenomenon of tlr tolerance ( ) . repetitive stimulation of copd alveolar macrophages with the same tlr ligands, pam csk and lps desensitizes the tlr and tlr , respectively, and generates tlr tolerance. moreover, the repetitive tlr stimulation further reduced the production of tnf-α, ccl , and il- without affecting the constantly augmented level of il- and il- in alveolar macrophages. this may provide alternative explanations for diminished immune responses against the recurrent/repetitive infection by nthi. the intrinsically reduced expression of tlrs in copd patients may also contribute to the impaired pulmonary immune response thus facilitating nthi persistent colonization. expression of tlr or tlr are found to be lower on sputum neutrophils, alveolar macrophages, nasal epithelium, and t cells in copd patients despite high concentrations of il- and mmp- ( ) ( ) ( ) ( ) . the lack of the more protective th /tc immune response in copd patients against nthi infection might be attributed to upregulated antagonists (a , irak-m, and myd s) of the myd /irak/mapk signaling pathway in copd t cells ( ) . it should be noted that the myd /irak/mapk pathway is required for expression of tlr in th , whereas production of ifn-γ in th /tc is tlr -dependent via the tlr /trif/ikke/tbk signaling pathway. the antagonists prevent the nthi los-induced tlr expression in th and tc and thus a reduced secretion of ifn-γ. in addition, unusual high numbers of tregs in copd patients have also contributed to effector t cell dysfunction or a th /tc predominant immune response ( ) . however, freeman et al. reported that tc (cd + ) cells from copd patients have increased expression of tlr , tlr , tlr , tlr , and tlr / as well as tc cytokines (ifnγ and tnf-α) compared to healthy individuals that may imply the auto-aggressive response of lung tc cells in copd lung inflammation ( ) . however, the copd tc cells can only be stimulated by ligands for tlr / (pam csk ) yet tolerant to other agonists, indicating the dysfunctional tlrs or tlr tolerance on t cells despite their high level of receptor expression. inversely, peripheral blood neutrophils isolated from copd patients have increased expression of tlr , tlr , and nlrp ( , ) . nevertheless, the increased tlrs expression might not improve the microbicidal ability of copd peripheral neutrophils probably due to the inaccurate responses to cytokines ( ) . in addition, certain types of snps (snps) in tlr and tlr have also been associated with decreased lung function, enhanced inflammatory responses and increased immune cell infiltration in copd ( ) . interestingly, the diminished il- responsiveness of copd alveolar macrophage to nthi infection has a strong association with the carriage of tlr (t c) polymorphism instead of tlr (arg gln), tlr (thr ile; asp gly), and tlr (t c) ( ) . the carriage of tlr (t c) is also positively correlated with diminished lung function. of note, the activation of tlr -signaling cascade in pro-inflammatory cytokine response requires stimulation from microbial dna ( ) . the microbicidal malfunction in both innate and adaptive immune cells is also potentially linked to the deleterious effect of tobacco smoke, the major risk factor for copd. it has been reported that, exposure of tobacco or cigarette smoke can impair phagocytosis/engulfment of nthi by alveolar macrophages isolated from copd patients ( , ) . moreover, the chemical exposure also suppressed the tlr-induced tnfα, il- , and il- production in copd alveolar macrophages that have been pre-stimulated with tlr , , or ligands (pam csk , lps, or phase i flagellin, respectively), or whole nthi bacteria ( ) . this may potentially delay the macrophagedependent bacterial clearance. the suppressive effect of cigarette smoke in macrophage-dependent phagocytosis is due to the suppression of the pi k signaling cascade which is required for optimal phagocytic activity and movement ( ) . meanwhile, the cigarette smoke also inhibits the activation of the p -erk signaling pathway and p /nf-κb, thus dampens the nthi losinduced cytokine production of copd alveolar macrophages ( ) . the diminished alveolar macrophage responsiveness could also be related to anticholinergic agents used by copd patients that results in lower concentrations of nthi-induced tnfα ( ). nevertheless, the impaired phagocytosis of nthi by copd alveolar macrophages could be improved in the presence of nuclear erythroid related factor and microrna mir- ( , ) . interestingly, in addition to the constant exacerbated inflammatory effect observed in different murine model studies, gaschler et al. observed a rapid pulmonary clearance of nthi in mice upon exposure to cigarette smoke, and this was positively correlated with an increased neutrophilia in the animal bal fluid ( , ( ) ( ) ( ) . however, in other copd animal studies, cigarette smoke also impaired the il- production that has a potential anti-bacterial activity while delaying the airway clearance of nthi ( ) ( ) ( ) . interestingly, il- might play a protective role in copd exacerbation as supplementation of il- manages to restore the homeostasis of airway immune response and improve nthi clearance ( ) . the increased airway neutrophilia might be due to the enhanced production of pulmonary il- triggered by cigarette smoke ( , , , ) . this may imply the important microbicidal role of neutrophils (neutrophilia) in compensating the copd-or cigarette smoke-associated dysfunctional alveolar macrophages ( , ) . however, such compensation may not be adequate to provide optimal immune defense to eradicate persistent nthi lower airway colonization, since the cigarette smoke also has profound suppressive effect on the host adaptive immunity, thus constantly risking the copd patients to episodes of exacerbation and relapsed infection. in adaptive immunity, cigarette smoke impairs the antigen-specific b and t cells responses to nthi infection. it suppresses the secretion of ifnγ and il- by nthi-specific t cells. antibody production by b cells has also been attenuated, with lower levels of specific anti-p antibodies and compromised igg , igg a, and iga class switching ( , ) . a recent and some previous cohort studies revealed that the level of airway antimicrobial cathelicidin (hcap /ll- ) in copd patients increase gradually from the stable disease to exacerbation states ( , ) . moreover, higher levels of cathelicidin are positively associated with nthi airway colonization, sputum neutrophilia, and higher concentrations of il- , particularly in the nthi-infected copd patients. of note, cathelicidin and other amps play important roles in the innate immune defense against different pathogens and persist immunomodulatory properties ( ) ( ) ( ) . ironically, it is plausible that the increased level of cathelicidin could diminish or alter the balance in lung microbiota, and the immune/inflammatory response. this might contribute to the "vicious circle, " thus considerably increasing the risk for nthi infection during copd exacerbations ( , ) . moreover, the microbicidal property of cathelicidin could be compromised by the inflammatory conditions in the airway, such as low ph, or the effect of cigarettes that causes peptide citrulination and modification ( , ) . finally, expression of amps (human beta defensin and s a ) by copd airway epithelium in response to nthi infection, is also disturbed by cigarette smoke. the insulted airway cells have also a reduced expression of tlr and il- , and impaired nthi-induced nf-κb activation ( , ) . thus, a large body of evidence exists on the deleterious effects of tobacco smoke. antibiotic treatment of aecopd has been shown to significantly reduce the risk of treatment failure, especially for in-patients with severe exacerbations and patients requiring intensive care ( ) . the efficacity of antibiotic treatment for out-patients with exacerbations is less clear ( , ) . recommendations on which empirical treatment to use for aecopd varies between different countries, but common antimicrobial agents that are frequently used as definitive therapy against nthi include aminopenicillins (with or without a beta-lactamase inhibitor), tetracyclines, trimethoprimsulfamethoxazole, and fluoroquinolones. in addition, the clinical and laboratory standards institute (clsi) has developed clinical breakpoints for the macrolides azithromycin and clarithromycin ( ) , whereas the european committee on antimicrobial susceptibility testing (eucast) have not set any clinical breakpoints against this class of antibiotics due to lack of clinical data ( ) . one study shows that nthi frequently develops resistance to macrolides during prolonged treatment and that treatment failure may occur, making fluoroquinolones more reliable for eradication in copd-patients ( ) . as for aminopenicillins, resistance is also common, with up to - % of nthi isolates expressing beta-lactamases and an additional - % of the isolates having amino acid substitutions in penicillin-binding protein (pbp ), which reduces their susceptibility to these agents ( , ) . the fraction of isolates expressing beta-lactamases has been stable during the last years, whereas an increase has been seen in isolates displaying altered pbp ( , ) . this is worrisome, since some of these amino acid substitutions also confer resistance to third generation cephalosporins ( ) . moreover, there seems to be a correlation between isolates expressing altered pbp and increased invasiveness. studies have shown that strains that express a mutated pbp with certain key amino acid substitution have a significantly higher rate of invasion of bronchial epithelial cells compared to strains with a wild type pbp ( ) . however, when such mutated pbp was cloned into a susceptible wild type strain, invasion efficacy did not increase, suggesting that pbp is only indirectly linked to invasion ( ) . besides using antibiotics for acute management of copd exacerbations, some studies have considered the use of continuous prophylactic antibiotics in the management of patients with copd ( ) . there is some evidence that continuous administration of macrolide antibiotics would prevent future exacerbations in a selected population of the most severely ill patients, but a cochrane review revealed no support for a reduced all-cause mortality or less hospital readmissions ( ) . however, more recent studies have shown a significant decrease in both the frequency of exacerbations and hospitalizations when long-term azithromycin treatment was chosen ( ) . the fact that macrolide antibiotics display not only antimicrobial effects, but also have anti-inflammatory and immunomodulatory properties, has made them interesting to use as prophylactic therapy ( ) . it has been shown that azithromycin inhibits mucus hypersecretion in the respiratory tract by significantly inhibiting tnf-α induction of the muc ac mucin secretion from human nasal epithelial cells ( ) . more specifically, it has been shown that azithromycin can reduce the nthi-dependent induction of muc ac expression by suppressing the transcription factor activator protein- ( ) . apart from affecting mucus secretion, it also seems that low-dose azithromycin has the ability to improve phagocytosis of bacteria by airway macrophages ( ) . one study showed that azithromycin concentrations that were unable to kill nthi still increased the uptake rate of the bacteria into alveolar macrophages by enhancing their phagocytic function ( ) . however, the risk of development of antimicrobial resistance limits the use of low-dose azithromycin solely for its immunological properties. this has triggered an interest in finding new macrolide substances that lack antibiotic effect and solely interact with the airway immune system ( ) . the considerable clinical problems caused by nthi with regard to copd exacerbations and otitis media has prompted the scientific community to investigate whether a vaccine can be developed against the pathogen ( , , ) . the search has been intensified due to a steady increase in antibiotic resistance and a trend of more invasive infections caused by nthi over the last decade ( ) . whereas, a highly efficient glycoconjugate vaccine has previously been developed against hib, an identical strategy cannot be employed against nthi due to the lack of a polysaccharide capsule. vaccine developments efforts have thus been concentrated on identifying nthi surface structures that are immunogenic, have low antigenic variability, and are conserved across this genetically highly heterogeneous species. several promising vaccine candidates have been identified in the last years, as excellently reviewed elsewhere ( , ). two of these antigens, fused into one protein, protein e-pila, are together with protein d currently being tested by glaxosmithkline in a phase iib proof-of-concept clinical trial (randomized, observer-blind, placebo-controlled, and multicentric) for infection prophylaxis in copd patients ( - years old) ( ) . notably, the m. catarrhalis ubiquitous surface protein a (uspa ) is also included in the vaccine so that an immune response against both exacerbation-causing pathogens could be elicited by the same preparation. this clinical study (nct ) is the only one currently being conducted on nthi (and m. catarrhalis) according to clinicaltrials.gov, and as the investigations are on-going, the results are currently unknown. due to an increase in the difficulty to treat nthi infections, an efficient and protective nthi vaccine likely considerably raises the quality of life of copd patients. since nthi-mediated exacerbations contribute to the progression of the disease and a steady deterioration of the pulmonary capacity of those patients, prevention against nthi infections potentially slows down the debilitating effect of the disease. it is therefore critical to continue this line of research until such a vaccine has been obtained. it could also be worth targeting non-conventional structures with a vaccine, such as the secreted enzymes urease and iga protease that have proven important for nthi infections in copd patients in several studies ( ) . copd is a multifaceted airway disease. several factors influence the clinical outcome of copd. importantly, the crosstalk between intrinsic factors (the stability and integrity of the airway immune response and structure in addition to hereditary factors), and the extrinsic factors (lung microbiome, viral and bacterial infections, meteorological factors, and noxious inhalation) determines the fate of lower airway opportunistic infection by h. influenzae. intriguingly, nthi has been one of the most isolated pathogens at both stable and exacerbation states of copd. such persistent airway colonization of nthi costs virulence fitness to counteract with the bactericidal effect of the host immune response. adversely, the impaired defense mechanisms in copd are not only unable to protect the lung structure from inhaled physical assaults, but they also fail to suppress nthi infection. the disoriented immune response in copd instead allows the pathogen to cause more harm and inflammation in the airways. the currently used bronchodilator and inhaled corticosteroid therapies have limited efficacy in preventing disease progression in copd. moreover, the inhaled corticosteroid therapies might have side effects that may weaken the immune response. hence, more investigations are needed to garner a more adequate knowledge regarding the variabilities in immune networking of copd. this knowledge will be an important platform for a more efficient drug design. in addition, a vaccine targeting nthi is another important approach in controlling the infective exacerbations in copd as the antibiotic treatment is also getting dampened by the emergence of nthi antibiotic resistance. y-cs coordinated and drafted the major part of the manuscript, and prepared the figures; fj participated in the literature study of virulence and vaccine research of nthi; jt prepared the review section for nthi epidemiology in copd and antibiotic studies; y-cs and kr edited and critically revised the manuscript. all authors read and approved the final manuscript. topographical continuity of 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in chronic obstructive pulmonary disease subjects relationship between azithromycin susceptibility and administration efficacy for nontypeable haemophilus influenzae respiratory infection nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases vaccines for nontypeable haemophilus influenzae: the future is now we thank the following funding agencies for their financial support during the preparation of the manuscript. they are the alfred Österlund, the anna and edwin berger, the swedish medical research council (grant number k - x- - - , www.vr.se), the physiographical society (forssman's foundation and, endowments for the natural sciences, medicine and technology), skåne county council's research and development foundation, and heart lung foundation (kr: grant number , www.hjart-lungfonden.se). the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © su, jalalvand, thegerström and riesbeck. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -kwu otpi authors: judson, gregory l.; kelemen, benjamin w.; njoroge, joyce n.; mahadevan, vaikom s. title: cardiovascular implications and therapeutic considerations in covid- infection date: - - journal: cardiol ther doi: . /s - - - sha: doc_id: cord_uid: kwu otpi the ongoing severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic has profoundly impacted all fields of medicine. infection with sars-cov- and the resulting coronavirus of (covid- ) syndrome has multiorgan effects. the pandemic has united researchers from bench to bedside in attempts to understand the pathophysiology of the disease and define optimal treatment strategies. cardiovascular disease is highly prevalent and a leading cause of death across gender, race, and ethnic groups. as the pandemic spreads, there is increasing concern about the cardiovascular effects of the viral infection and the interaction of infection with existing cardiovascular disease. additionally, there are concerns about the cardiac effects of the numerous treatment agents under study. it will be essential for cardiologists to understand the interplay between underlying cardiac comorbidities, acute cardiovascular effects of covid- disease, and adverse effects of new treatments. here we describe emerging evidence of the epidemiology of sars-cov- infection and underlying cardiovascular disease, the evidence for direct myocardial injury in sars-cov- infection, the specific presentations of cardiovascular involvement by sars-cov- , and the cardiac effects of emerging treatments. coronavirus disease of is caused by the severe acute respiratory syndrome coronavirus (sars-cov ) and was first reported in wuhan, china, in december of [ ] . initial presentations from wuhan were consistent with viral pneumonia and subsequent deep sequencing confirmed a novel rna-based virus of the coronaviridae family. as of june , , there are . million confirmed worldwide cases of covid- with over , deaths [ ] . covid- has become a worldwide pandemic and has mobilized healthcare workers in all disciplines. since the initial presentation of covid- , much has been learned about the pathophysiology and specific cardiovascular manifestations of the disease. early in the pandemic, there was evidence that those with preexisting cardiovascular conditions were over-represented in cases of severe infection [ ] . since that time, it has become clear that certain racial and ethnic groups are disproportionately affected by covid- [ ] . additionally, covid- appears to have several unique cardiovascular manifestations that cardiologists must understand, including worsening of heart failure, myocarditis, a proclivity for thrombosis, and arrythmia. finally, the speed at which clinical trials for covid- are being conducted, using both repurposed and new therapies, means that cardiologists must be aware of potential cardiovascular side effects for agents that are not commonly used in cardiovascular practice. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. pathophysiology sars-cov- is a novel rna coronavirus that has emerged as the causative infection responsible for the current covid- pandemic. other viruses from the coronavirus family include the mers-cov virus, responsible for the middle east respiratory syndrome (mers) outbreak, and the sars-cov virus, responsible for the severe acute respiratory syndrome (sars) outbreak [ ] [ ] [ ] [ ] . initial infection with sars-cov- is a process that is still being understood but is known to involve viral s proteins and host angiotensin-converting enzyme (ace ) receptor and the transmembrane protease, serine (tmprss). sars-cov- expresses numerous spike (s) proteins on the surface of the viral envelope, which facilitates viral entry into the host cell. tmprss, a serine protease expressed on the host cell, primes the viral s proteins to enable interaction with the host ace receptor [ ] . the primed s protein then binds the s subunit of the host ace receptor allowing for viral entry into the cell [ ] . the role of the ace receptor in human physiology remains under investigation but it is believed to provide counter regulation to the renin-angiotensin system [ , ] . the ace receptor is commonly found in the lungs in type alveolar cells but is also expressed in vascular endothelium, gut epithelium, and in the proximal tubule in the kidneys [ ] [ ] [ ] . the location of the ace receptor is implicated in a multitude of disease presentations associated with sars-cov- infection. after binding the ace receptor, the virus is internalized, and the viral rna is released. immediately, the rna-dependent rna polymerase is translated, which allows for viral replication and spread of the infection. the role of the ace receptor expression and blockade within both sars-cov- infection and disease severity remains controversial, with both protective and deleterious effects hypothesized. the united states has surpassed other countries worldwide in case numbers with over . million cases as of june , [ ] . the epicenter has spread from china to many european countries and now the usa, with significant clusters around the country. the number of covid- -related deaths worldwide has now climbed past , . due to the heterogeneity of testing strategies and case ascertainment, mortality rates remain controversial, with estimates ranging from . to . %. covid- has a broad spectrum of severity and phenotypic presentation. the most typical presentation involves consequences of direct respiratory infection ranging from cough and dyspnea to hypoxia to acute respiratory distress syndrome (ards). the initial cohort study of covid- patients included data from hospitals in china with patients with covid- disease [ ] . the study reported that the most common symptoms at presentation were cough ( . %), fatigue ( . %), and temperature c . °c ( . %). in the largest published cases series of sequential covid- cases from the united states in new york city, of the patients admitted to different hospitals, % of patients presented with an initial fever and % presented with hypoxia [ ] . tachypnea was present in . % and tachycardia in . %. mechanical ventilation was required in . % of patients and early mortality was seen in . %, with . % of patients surviving to discharge and . % remaining in the hospital. thus, sars-cov- infection most commonly causes respiratory illness with a wide range of severity and outcomes that is relatively consistent in presentation (cough, dyspnea, fever, and hypoxia) across the globe. the preexisting burden of comorbid chronic disease is well recognized to contribute to the severity of sars-cov- infection and there are concerns that underlying comorbid illnesses play a role in the diverse spectrum of phenotypic presentation. the initial study of patients in wuhan, china, demonstrated a predominance of male patients ( %) with pre-existing comorbidities including diabetes ( %), hypertension ( %), and cardiovascular disease ( %) [ ] . the median age of patients in this group was years old. only % of these initial patients had a history of tobacco use. however, since these initial studies, a number of prospective and retrospective studies out of china have demonstrated higher rates of underlying comorbid conditions. common underlying comorbidities include hypertension ( - %), diabetes ( - %), and cardiovascular and coronary artery diseases ( - %) [ ] [ ] [ ] . smoking history or obstructive lung disease diagnoses were low, when documented ( - %). in these studies, these comorbidities were associated with worse severity of illness and mortality. a review of , confirmed covid- cases from wuhan, china, demonstrated increased mortality in patients with cardiovascular disease ( . %), diabetes ( . %), and hypertension ( %), which was significantly higher than the overall case-fatality rate of . % [ ] . in the united states, there are similarities and notable differences from the european and chinese experience. among patients admitted with covid- , . % were men and . % were over the age of years, with the majority ( . %) being over years old [ ] . comorbidities included hypertension ( . %), obesity ( . %), diabetes mellitus ( . %), chronic lung disease ( . %), cardiovascular disease; including coronary disease and heart failure ( . %), and, less frequently, renal disease ( . %) and immunocompromised conditions ( . %). these initial cases series have shown a similar relationship between underlying cardiac comorbidities with a higher prevalence of hypertension, diabetes, coronary artery disease, and obesity in patients requiring mechanical ventilation [ ] . a concerning trend is the alarming rate at which black and hispanic/latino americans are being infected and dying from covid- [ ] . there were early data from cities and states across the country indicating higher mortality from covid in black and hispanic/latino populations. these included milwaukee, wi, experiencing a % mortality rate in black patients who make up % of the population; chicago, il ( % of the mortality with % of the population); and the states of louisiana ( % of the mortality with % of the population) and michigan ( % of the mortality with % of the population). in the covid-net catchment population of the usa, a higher percentage of black patients were infected ( %) compared to the percentage of overall population being black ( %) [ ] . this trend has been repeated across the country and has brought into stark reality the devastating impact covid- has had on communities of color [ ] . while it is well known that black and hispanic/latino patients have a higher incidence of diabetes and hypertension, this alone does not explain the increased infected and mortality rates. studies during prior epidemics have continued to demonstrate similar healthcare disparities on smaller scales likely related to multiple factors including access to care, racial and ethnical stigmas among medical professionals, crowded living conditions, income instability, and continued rates of exposure and protection [ ] [ ] [ ] [ ] . it is crucial to acknowledge the institutionalized barriers to health as a first step to incite change and narrow the outcome gap and improve healthcare for marginalized individuals. de novo cardiac injury has also been described in patients presenting with covid- disease. initial studies provide insight into the prevalence and incidence of acute cardiac injury patients presenting with covid- . early studies reported a prevalence of acute cardiac injury of % in the entire cohort as defined by either high sensitivity troponin (hs tn) or the mb fraction of creatinine kinase (ck-mb) [ th percentile or new echocardiographic or electrocardiographic abnormalities with greater elevations in cardiac biomarkers among patients requiring icu care [ , ] . a metaanalysis further clarified the prevalence of acute cardiac injury among chinese patients presenting with covid- disease reporting a similar prevalence of acute cardiac injury defined as ck-mb or hs tn exceeding the th percentile [ ] . case cohort studies included data in patients for whom the outcome and illness course helped further elucidate the role of cardiac injury in covid- disease. zhou et al. showed that the initial hs tn was not dramatically elevated at illness onset, but that in non-survivors there was a rapid rise starting after day that was not seen in survivors [ ] . ruan et al. reported the cause of death in the non-survivors with the majority ( %) due to respiratory failure but a clinically significant number related to combined respiratory/cardiac failure ( %) and cardiac failure ( %) [ ] . based on these studies, the authors hypothesize that myocardial injury occurs frequently in patients with severe covid disease and that myocardial dysfunction is involved in the pathophysiology of severe disease. as the pandemic progressed and patient outcomes were defined, investigators sought to evaluate the association of myocardial injury with mortality. in a multivariable cox regression, acute cardiac injury at any point during hospital stay was associated with an increased risk of mortality (hr . , % confidence interval . - . ). guo et al. examined the association of cardiac comorbidities and acute cardiac injury as defined by hs tn above the th percentile with mortality in a retrospective cohort of patients [ ] . in this cohort, % of patients had acute cardiac injury. the proportion of non-survivors was increased in the groups with underlying cardiac comorbidities (hypertension, coronary artery disease, and cardiomyopathy) or myocardial injury and highest in groups with both. the authors did not assess measures of association beyond descriptive statistics. thus, myocardial injury occurs not infrequently with covid- disease and that the presence of myocardial injury portends both more severe disease and higher mortality. the mechanism of this acute cardiac injury remains elusive. data from zhou et al. and guo et al. have shown that the rise in troponin typically occurs late in the disease process, typically occurring - days after the onset of symptoms and coincides with progressive respiratory decline and multiorgan failure [ , ] . this would suggest that myocardial injury may be a marker for more severe infection and not causally associated. however, there are few reports of systematic cardiac testing of patients with covid- disease, and cardiac injury limited our ability to understand causal associations. a number of mechanisms have been proposed including direct myocardial injury and myocarditis, systemic hyperinflammatory response leading to cytokine storm and myocardial injury, microvascular thromboses, acute plaque rupture physiology, stress cardiomyopathy, and extreme oxygen supply-demand mismatch due to critical illness [ , ] . the relative mystery surrounding the pathophysiology of acute cardiac injury has resulted in conflicting recommendations regarding the use of troponin to screen and evaluate cardiac dysfunction in patients with covid- disease. a communication by the american college of cardiology (acc) recommended against routine cardiac biomarker testing in patients presenting with covid- disease [ ] . subsequent review papers have advocated for systematic use of high-sensitivity troponin as a biomarker of severe illness with proven prognostic value in covid- disease [ , ] . however, this risk prediction strategy has not yet been evaluated prospectively. sars-cov has been implicated in cases of myocarditis and acute decompensated heart failure. initial reports of myocarditis from china described cases of cardiogenic shock and reduced left ventricular ejection fraction among covid- patients [ , ] . these patients had extremely elevated levels of cardiac biomarkers (namely troponin, ck-mb, and bnp) and required inotropy or extracorporeal membrane oxygenation (ecmo) to maintain adequate cardiac output. based on the clinical presentation and on these elevations in biomarkers, these cases were deemed ''fulminant myocarditis'' and were treated with a combination of steroids, ivig, antivirals, antibiotics, anti-inflammatory agents, renal replacement, and mechanical ventilation. during other outbreaks of similar coronaviruses in the last years, namely sars-cov and mers, there was evidence to support direct myocardial involvement. among patients who died from sars-cov in toronto, canada, viral rna was detected in % of samples and the presence of viral rna was associated with increased levels of interstitial fibrosis and macrophage activity within the myocardium, thus potentially leading to a more rapid clinical course compared to patients without viral rna in the myocardium [ ] . however, hemodynamically significant myocarditis appears to have been a relatively rare complication of sars, with one case series noting only one instance of potential myocarditis requiring inotropy among cases [ ] . myocarditis also appears to have been a potential complication during the mers outbreak, with mri findings consistent with myocarditis reported [ ] . there is now additional evidence that sars-cov has a similar effect on the myocardium as sars-cov and mers. myocardial involvement has been confirmed in sars-cov- -positive patients via mri and endomyocardial biopsy [ ] [ ] [ ] . cardiac biopsy findings in covid- patients have shown inflammatory infiltration of the myocardium with t-lymphocyte and macrophages, interstitial edema, and in some cases, evidence of cytoplasmic vacuoles indicating direct viral involvement of myocardial cells [ , ] . in a post-mortem case series of patients from china, % of covid- patients died of circulatory failure with some degree of myocardial involvement, as marked by elevations in troponin [ ] . however, it is unclear if these cases can truly be classified as myocarditis given the lack of ejection fraction assessment via tte, mri, or biopsy. from a different case series in seattle, wa, troponin elevation was seen in % of patients, but none of the patients who underwent tte had evidence of a reduced ejection fraction [ ] . there is currently no standardized approach to treatment of covid- cardiomyopathy. in cases of more severe ''fulminant'' myocarditis resulting in cardiogenic shock, treatments with immunomodulating drugs, namely steroids and ivig, but also more specific agents like tocilizumab to inhibit il- , are being used. clinical trial data is as of yet lacking on the efficacy of such therapies in covid- -related myocarditis and are being extrapolated from other causes of fulminant myocarditis, namely giant cell subtype. decompensation of pre-existing heart failure appears to be a more common cardiovascular manifestation of sars-cov- infection than clinically significant myocarditis. prior to the covid- pandemic, influenza-like respiratory infections significantly increased the risk of hospitalization for decompensated heart failure, and in high influenza activity months, may contribute to % of heart failure hospitalizations across the united states [ ] . the relationship between respiratory infection and decompensated heart failure appears to also be true for covid- -like seasonal influenza. in two retrospective case series from china of and patients, heart failure was observed in % and % of deaths, respectively [ , ] . there appears to be a broad spectrum of disease severity, ranging from stable to fulminant heart failure requiring inotropes, and potentially mechanical circulatory support for refractory shock and or hypoxia. the use of ecmo in covid- cases is being studied in a prospective manner as part of the extracorporeal life support organization (elso) registry. as of june , , ecmo has been used in covid- -confirmed cases worldwide, with % discharged alive, and the vast majority of patients receiving veno-venous ecmo for respiratory support [ ] . there is interest in the role of acei/arb in both the overall risk of covid- and the severity of lung disease with studies suggesting both the possibility of deleterious and beneficial effects [ , ] . major guideline committees have continued to advocate for the use of ace/ arb medications in hemodynamically stable heart failure patients, hypertension, and those with ischemic heart disease [ , ] . covid- has been hypothesized to increase the risk of acute coronary syndromes (acs) and systemic thrombosis. this premise is based on prior experience with viral infections such as influenza, which predisposes patients to developing acs, and may increase the risk of myocardial infarction (mi) by at least sixfold in the week following infection [ ] . this increase in mi incidence is also seen following infection with other respiratory infections such as respiratory syncytial virus, and for non-viral respiratory illnesses such as bacterial pneumonia. many mis due to covid- disease are likely related to supply-demand mismatch from hypoxia resulting in myocardial injury and troponin release. however, mi from acute atherothrombotic events were first reported in wuhan, china, soon after the onset of the pandemic. as such, chinese hospitals soon developed treatment algorithms aimed at providing urgent reperfusion to covid- -suspected patients while maintaining the safety of catheterization lab staff. in the setting of st-elevation mi (stemi) and suspected sars-cov- infection, thrombolytics were often the first choice for acute reperfusion therapy in china [ ] . following this early experience in china, acc/scai released a joint recommendation outlining care for covid- -suspected patients [ ] . this document highlights the need for adequate personal protective equipment for catheterization lab staff, the need to differentiate true acs from supply-demand mismatch, consideration of deferring invasive angiography in low-risk non-st elevation myocardial infarction (nstemi) patients until hospital resources improve, and the potential use of thrombolytics for st segment elevation myocardial infarction (stemi) cases where the risks of exposing staff to sars-cov outweigh the benefits of a primary percutaneous coronary intervention (pci)-based approach. however, the authors still expect primary pci to remain the standard of care for patients with stemi and possible covid- and thrombolytics reserved for non-pci-capable hospitals [ ] . further adding to the complexity of treating these patients, st-segment elevations are seen in covid- patients who are then found to have non-obstructive coronary disease. in a case series of patients in new york, ny, who developed st-segment elevations, nine patients underwent invasive angiography. of these nine patients, three had no obstructive coronary artery disease and / with obstructive disease underwent pci ( after receiving thrombolytics) [ ] . these cases of st-segment elevation with no obstructive coronary disease on angiography may be related to peri-myocarditis, although the pathophysiology remains under investigation. importantly, the mortality among patients without no obstructive coronary lesion was higher ( %) than among those with obstructive coronary lesions ( %), although the absolute numbers were limited. a similar incidence of obstructive to non-obstructive coronary lesions was reported in a population of italian patients with stemi [ ] . the high prevalence of stemi mimics in this population further emphasizes the need for angiography (either invasive or non-invasive) as opposed to empiric fibrinolytic therapy given the potential for harm when administering fibrinolytics for non-acs presentations. while covid- may potentially increase the risk of acs, activations for stemi in the united states have decreased significantly during the pandemic. among nine high-volume centers in the us, there was a % reduction in stemi activations compared to the -month period before the pandemic [ ] . this finding is similar to the % reduction in stemi activations seen in spain [ ] . it is unclear what is responsible for the significant reduction in stemi activations, but it may be related to patients' fear of exposure to sars-cov when presenting to the hospital. in hong kong, china, patients who presented with stemi during the height of the outbreak presented to the hospital significantly longer after onset of symptoms compared to a year prior ( vs. min) [ ] . it is currently unknown how many people worldwide may not be seeking medical care for possible acs due to fear of covid- . it is possible that due to delays in seeking appropriate medical care, patients may eventually present to the hospital with heart failure, cardiogenic shock, or mechanical complications from acs. studies must be performed to assess the impact covid- could have on cardiovascular mortality through such indirect mechanisms. sars-cov infection is theorized to predispose to a hypercoagulable state through inflammation, immune dysregulation, and activation of cytokines. this process has been implicated in acs, but also in cases of thrombosis outside the coronary arteries. among icu patients in the netherlands, % were diagnosed with pulmonary embolism, deep-vein thrombosis, ischemic stroke, myocardial infarction, or systemic embolism-all while receiving standard prophylactic doses of anticoagulation as recommended by the who [ ] . remarkably, none of the patients in this study developed disseminated intravascular coagulopathy (dic), a known risk factor for the development of arterial and venous thromboembolism in critically ill patients. ddimer levels above ng/ml had a sensitivity of % and specificity of % for detecting venous thromboembolism in a cohort from china [ ] . some centers have advocated for the use of treatment dose anticoagulation for select patients with covid- and elevated d-dimer levels, however this practice has yet to be well validated. among patients with confirmed sars-cov- in wuhan, china, arrhythmia occurred in . % of all patients admitted [ ] . elevated troponin t was a statistically significant prognostic marker with % having either ventricular tachycardia or fibrillation. it is unclear whether elevated troponin preceded or succeeded the arrhythmic events. furthermore, among patients admitted to a single hospital in wuhan, china, arrhythmia (type not specified) was reported in % with the majority ( %) occurring in critically ill patients in the intensive care unit [ ] . among patients who suffered a cardiac arrest, the predominant rhythm was asystole/pulseless electrical activity ( %), followed by shockable ventricular tachycardia/fibrillation ( %) [ ] . similar rates in the new york city population were reported by goyal et al. arrhythmia was seen in . % of the entire cohort, with higher rates in the patients receiving icu care ( . %) as compared to non-icu care ( . %) [ ] . arrhythmias may be induced by the presence of acidosis and metabolic disturbances, as seen in critical illness with multiorgan dysfunction or catecholaminergic pressor infusion for hypotension and shock. finally, qt-prolonging agents given to some covid- patients may increase the susceptibility to arrhythmia as discussed below [ , ] . unfortunately, with the limited data available, no trends have been apparent as of yet [ , ] . there is suspicion that the aggressive nature of the coronavirus pneumonia is related to an exaggerated immune response mediated by interferon and interleukins, as has been seen in prior coronavirus infections [ , ] . targets for covid- therapies are therefore not only focused on the intrinsic viral make up (including proteins such as ace- cell surface receptor, -chymotrypsin-like protease, spike, rna-dependent rna polymerase, and papain-like protease) but also the human immune system [ ] . the ongoing covid- pandemic poses an enormous threat globally. given the high prevalence of existing cardiovascular disease worldwide, it is not surprising that cardiovascular comorbidities significantly impact disease severity. the unique properties of the sars-cov- infection and pathophysiology further raise concern for de novo cardiac injury with covid- . the cardiovascular presentations of covid- are still being elucidated, but appear to at least include decompensated heart failure, myocarditis, acs, arrhythmia, and thrombosis. the effect of the covid- pandemic on cardiovascular systems of care and whether patients are delaying necessary treatment will be of great importance to understand as the pandemic progresses in the months and years to come. lastly, emerging therapeutic approaches have expected and unexpected cardiac effects that must be monitored as treatment algorithms expand. cardiologists will play a vital role in the care of covid- patients worldwide and in the ongoing research into the pathophysiology and population-based effects of the sars-cov virus. funding. no funding or sponsorship was received for this study or publication of this article. authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. disclosures. gregory l. judson, benjamin w. kelemen, joyce n. njoroge, and vaikom s. mahadevan have nothing to disclose in relation to this article. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. open access. this article is licensed under a creative commons attribution-noncommercial . international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the 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of severe covid- patients with tocilizumab treatment of critically ill patients with covid- with convalescent plasma key: cord- -egd gxby authors: barh, debmalya; chaitankar, vijender; yiannakopoulou, eugenia ch; salawu, emmanuel o.; chowbina, sudhir; ghosh, preetam; azevedo, vasco title: in silico models: from simple networks to complex diseases date: - - journal: animal biotechnology doi: . /b - - - - . - sha: doc_id: cord_uid: egd gxby in this chapter, we consider in silico modeling of diseases starting from some simple to some complex (and mathematical) concepts. examples and applications of in silico modeling for some important categories of diseases (such as for cancers, infectious diseases, and neuronal diseases) are also given. mathematically and computed models are established. these in silico models encode and test hypotheses about mechanisms underlying the function of cells, the pathogenesis and pathophysiology of disease, and contribute to identification of new drug targets and drug design. the development of in silico models is facilitated by rapidly advancing experimental and analytical tools that generate information-rich, high-throughput biological data. bioinformatics provides tools for pattern recognition, machine learning, statistical modeling, and data extraction from databases that contribute to in silico modeling. dynamical systems theory is the natural language for investigating complex biological systems that demonstrate nonlinear spatio-temporal behavior. most in silico models aim to complement (and not replace) experimental research. experimental data are needed for parameterization, calibration, and validation of in silico models. typical examples in biology are models for molecular networks, where the behavior of cells is expressed in terms of quantitative changes in the levels of transcripts and gene products, as well as models of cell cycle. in medicine, in silico models of cancer, immunological disease, lung disease, and infectious diseases complement conventional research with in vitro models, animal models, and clinical trials. this chapter presents basic concepts of bioinformatics, systems biology, their applications in in silico modeling, and also reviews applications in biology and disease. biotechnology will be the most promising life science frontier for the next decade. together with informatics, biotechnology is leading revolutionary changes in our society and economy. this genomic revolution is global, and is creating new prospects in all biological sciences, including medicine, human health, disease, and nutrition, agronomy, and animal biotechnology. animal biotechnology is a source of innovation in production and processing, profoundly impacting the animal husbandry sector, which seeks to improve animal product quality, health, and well-being. biotechnological research products, such as vaccines, diagnostics, in vitro fertilization, transgenic animals, stem cells, and a number of other therapeutic recombinant products, are now commercially available. in view of the immense potential of biotechnology in the livestock and poultry sectors, interest in animal biotechnology has increased over the years. the fundamental requirement for modern biotechnology projects is the ability to gather, store, classify, analyze, and distribute biological information derived from genomics projects. bioinformatics deals with methods for storing, retrieving, and analyzing biological data and protein sequences, structures, functions, pathways, and networks, and recently, in silico disease modeling and simulation using systems biology. bioinformatics encompasses both conceptual and practical tools for the propagation, generation, processing, and understanding of scientific ideas and biological information. genomics is the scientific study of structure, function, and interrelationships of both individual genes and the genome. lately, genomics research has played an important role in uncovering the building blocks of biology and complete genome mapping of various living organisms. this has enabled researchers to decipher fundamental cellular functions at the dna level, such as gene regulation or protein-protein interactions, and thus to discover molecular signatures (clusters of genes, proteins, metabolites, etc.), which are characteristic of a biological process or of a specific phenotype. bioinformatics methods and databases can be developed to provide solutions to challenges of handling massive amounts of data. the history of animal biotechnology with bioinformatics is to make a strong research community that will build the resources and support veterinary and agricultural research. there are some technologies that were used dating back to , b.c. many of these techniques are still being used today. for example, hybridizing animals by crossing specific strains of animals to create greater genetic varieties is still in practice. the offspring of some of these crosses are selectively bred afterward to produce the most desirable traits in those specific animals. there has been significant interest in the complete analysis of the genome sequence of farm animals such as chickens, pigs, cattle, sheep, fish, and rabbits. the genomes of farm animals have been altered to search for preferred phenotypic traits, and then selected for better-quality animals to continue into the next generation. access to these sequences has given rise to genome array chips and a number of web-based mapping tools and bioinformatics tools required to make sense of the data. in addition, organization of gigabytes of sequence data requires efficient bioinformatics databases. fadiel et al. ( ) provides a nice overview of resources related to farm animal bioinformatics and genome projects. with farm animals consuming large amounts of genetically modified crops, such as modified corn and soybean crops, it is good to question the effect this will have on their meat. some of the benefits of this technology are that what once took many years of trial and error is now completed in just months. the meats that are being produced are coming from animals that are better nourished by the use of biotechnology. biotechnology and conventional approaches are benefiting both poultry and livestock producers. this will give a more wholesome affordable product that will meet growing population demands. moreover, bioinformatics methods devoted to investigating the genomes of farm animals can bring eventual economic benefits, such as ensuring food safety and better food quality in the case of beef. recent advances in highthroughput dna sequencing techniques, microarray technology, and proteomics have led to effective research in bovine muscle physiology to improve beef quality, either by breeding or rearing factors. bioinformatics is a key tool to analyze the huge datasets obtained from these techniques. the computational analysis of global gene expression profiling at the mrna or protein level has shown that previously unsuspected genes may be associated either with muscle development or growth, and may lead to the development of new molecular indicators of tenderness. gene expression profiling has been used to document changes in gene expression; for example, following infection by pathological organisms, during the metabolic changes imposed by lactation in dairy cows, in cloned bovine embryos, and in various other models. bioinformatics enrichment tools are playing an important role in facilitating the functional analysis of large gene lists from various high-throughput biological studies. huang et al. discusses bioinformatics enrichment tools, which helps us understand their algorithms and the details of a particular tool. however, in biology genes do not act independently, but in a highly coordinated and interdependent manner. in order to understand the biological meaning, one needs to map these genes into gene-ontology (go) categories or metabolic and regulatory pathways. different bioinformatics approaches and tools are employed for this task, starting form go-ranking methods, pathway mappings, and biological network analysis (werner, ) . the awareness of these resources and methods is essential to make the best choices for particular research interests. knowledge of bioinformatics tools will facilitate their wide application in the field of animal biotechnology. bioinformatics is the computational data management discipline that helps us gather, analyze, and represent this information in order to educate ourselves, understand biological processes in healthy and diseased states, and to facilitate discovery of better animal products. continued efforts are required to develop cost-effective and efficient computational platforms that can retrieve, integrate, and interpret the knowledge behind the genome sequences. the application of bioinformatics tools for biotechnology research will have significant implications in the life sciences and for the betterment of human lives. bioinformatics is being adopted worldwide by academic groups, companies, and national and international research groups, and it should be thought of as an important pillar of current and future biotechnology, without which rapid progress in the field would not be possible. systems approaches in combination with genomics, proteomics, metabolomics, and kinomics data have tremendous potential for providing insights into various biological mechanisms, including the most important human diseases. we are witnessing the birth of a new era in biology. the ability to uncover the genetic code of living organisms has dramatically changed the biological and biomedical sciences approach towards research. these new approaches have also brought in newer challenges. one such challenge is that recent and novel technologies produce biological datasets of ever-increasing size, including genomic sequences, rna and protein abundances, their interactions with each other, and the identity and abundance of other biological molecules. the storage and compilation of such quantities of biological data is a challenge: the human genome, for example, contains billion chemical units of dna, whereas a protozoan genome has billion units of dna. data management and interpretation requires development of newly sophisticated computational methods based on research in biology, medicine, pharmacology, and agricultural studies and using methods from computer science and mathematics -in other words, the multi-disciplinary subject of bioinformatics. bioinformatics enables researchers to store large datasets in a standard computer database format and provides tools and algorithms scientists use to extract integrated information from the databases and use it to create hypotheses and models. bioinformatics is a growth area because almost every experiment now involves multiple sources of data, requiring the ability to handle those data and to draw out inferences and knowledge. after years of rapid evolution, the subject is now quite ubiquitous. another challenge lies in deciphering the complex interactions in biological systems, known as systems biology. systems biology can be described as a biology-based interdisciplinary field of study that focuses on complex interactions of biological systems. those in the field claim that it represents a shift in perspective towards holism instead of reductionism. systems biology has great potential to facilitate development of drugs to treat specific diseases. the drugs currently on the market can target only those proteins that are known to cause disease. however, with the human genome now completely mapped, we can target the interaction of genes and proteins at a systems biology level. this will enable the pharmaceutical industry to design drugs that will only target those genes that are diseased, improving healthcare in the united states. like two organs in one body, systems analysis and bioinformatics are separate but interdependent. computational methods take an interdisciplinary approach, involving mathematicians, chemists, biologists, biochemists, and biomedical engineers. the robustness of datasets related to gene interaction and co-operation at a systems level requires multifaceted approaches to create a hypothesis that can be tested. two approaches are used to understand the network interactions in systems biology, namely experimental and theoretical and modeling techniques (choi, ) . in the following sections is a detailed overview of the different computational or bioinformatics methods in modern systems biology. experimental methods utilize real situations to test the hypothesis of mined data sets. as such, living organisms are used whereby various aspects of genome-wide measurements and interactions are monitored. specific examples on this point include: protein-protein interaction predictions are methods used to predict the outcome of pairs or groups of protein interactions. these predictions are done in vivo, and various methods can be used to carry out the predictions. interaction prediction is important as it helps researchers make inferences of the outcomes of ppi. ppi can be studied by phylogenetic profiling, identifying structural patterns and homologous pairs, intracellular localization, and post-translational modifications, among others. a survey of available tools and web servers for analysis of protein-protein interactions is provided by tuncbag et al., . within biological systems, several activities involving the basic units of a gene take place. such processes as dna replication, and rna translation and transcription into proteins must be controlled; otherwise, the systems could yield numerous destructive or useless gene products. transcriptional control networks, also called gene regulatory networks, are segments within the dna that govern the rate and product of each gene. bioinformatics have devised methods to look for destroyed, dormant, or unresponsive control networks. the discovery of such networks helps in corrective therapy, hence the ability to control some diseases resulting from such control network breakdowns. there has also been rapid progress in the development of computational methods for the genome-wide "reverse engineering" of such networks. aracne is an algorithm to identify direct transcriptional interactions in mammalian cellular networks, and promises to enhance the ability to use microarray data to elucidate cellular processes and to identify molecular targets of pharmacological drugs in mammalian cellular networks. in addition to methods like aracne, systems biology approaches are needed that incorporate heterogeneous data sources, such as genome sequence and protein-dna interaction data. the development of such computational modeling techniques to include diverse types of molecular biological information clearly supports the gene regulatory network inference process and enables the modeling of the dynamics of gene regulatory systems. one such technique is the template-based method to construct networks. an overview of the method is shown in flow chart . . the template-based transcriptional control network reconstruction method exploits the principle that orthologous proteins regulate orthologous target genes. given a genome of interest (goi), the first step is to select the template genome (tg) and known regulatory interactions (i.e. template network, tn) in this genome. in step , for every protein (p) in tn, a blast search is performed against goi to obtain the best hit sequences (px). in step three these px are then used as a query to perform a blast search against tg. if the best hit using px as a query happens to be p, then both p and px are selected as orthologous proteins in step four. if orthologs were detected for an interacting p and target gene then the interaction is transferred in goi in the final step. note that this automated way of detecting orthologs can infer false positives. signal transduction is how cells communicate with each other. signal transduction pathways involve interactions between proteins, micro-and macro-molecules, and dna. a breakdown in signal transduction pathways could lead flow chart . template-based method for regulatory network reconstruction. to detrimental consequences within the system due to lack of integrated communication. correction of broken signal transduction pathways is a therapeutic approach researched for use in many areas of medicine. high-throughput and multiplex techniques for quantifying signaling and cellular responses are becoming increasingly available and affordable. a high-throughput quantitative multiplex kinase assay, mass spectrometrybased proteomics, and single-cell proteomics are a few of the experimental methods used to elucidate signal transduction mechanisms of cells. these large-scale experiments are generating large data sets on protein abundance and signaling activity. data-driven modeling approaches such as clustering, principal components analysis, and partial least squares need to be developed to derive biological hypotheses. the potential of data-driven models to study large-scale data sets quantitatively and comprehensively will make sure that these methods will emerge as standard tools for understanding signal-transduction networks. the systems biology and mathematical biology fields focus on modeling biological systems. computational systems biology aims to develop computational models of biological systems. specifically, it focuses on developing and using efficient algorithms, data structures, visualization tools, and communication tools. a mathematical model can provide new insights into a biological model of interest and help in generating testable predictions. modeling or simulation can be viewed as a way of creating an artificial biological system in vitro whose properties can be changed or made dynamic. by externally controlling the model, new datasets can be created and implemented at a systems level to create novel insights into treating generelated problems. in modeling and simulation, sets of differential equations and logic clauses are used to create a dynamic systems environment that can be tested. mathematical models of biochemical networks (signal transduction cascades, metabolic pathways, gene regulatory networks) are a central component of modern systems biology. the development of formal methods adopted from theoretical computing science is essential for the modeling and simulation of these complex networks. the computational methods that are being employed in mathematical biology and bioinformatics are the following: (a) directed graphs, (b) bayesian networks, (c) boolean networks and their generalizations, (d) ordinary and partial differential equations, (e) qualitative differential equations, (f) stochastic equations, and (g) rule-based formalisms. below are a few specific examples of the applications of these methods. mathematical models can be used to investigate the effects of drugs under a given set of perturbations based on specific tumor properties. this integration can help in the development of tools that aid in diagnosis and prognosis, and thus improve treatment outcome in patients with cancer. for example, breast cancer, being a well-studied disease over the last decade, serves as a model disease. one can thus apply the principles of molecular biology and pathology in designing new predictive mathematical frameworks that can unravel the dynamic nature of the disease. genetic mutations of brca , brca , tp , and pten significantly affect disease prognosis and increase the likelihood of adverse reactions to certain therapies. these mutations enable normal cells to become self-sufficient in survival in a stepwise process. enderling et al. ( ) modeled this mutation and expansion process by assuming that mutations in two tumor-suppressor genes are sufficient to give rise to a cancer. they modified enderling's earlier model, which was based on an established partial differential equation model of solid tumor growth and invasion. the stepwise mutations from a normal breast stem cell to a tumor cell have been described using a model consisting of four differential equations. recently, woolf et al. ( ) applied a novel graphical modeling methodology known as bayesian network analysis to model discovery and model selection for signaling events that direct mouse embryonic stem cells (an important preliminary step in hypothesis testing) in protein signaling networks. the model predicts bidirectional dependence between the two molecules erk and fak. it is interesting to appreciate that the apparent complexity of these dynamic erk-fak interactions is quite likely responsible for the difficulty in determining clear "upstream" versus "downstream" influence relationships by means of standard molecular cell biology methods. bayesian networks determine the relative probability of statistical dependence models of arbitrary complexity for a given set of data. this method offers further clues to apply bayesian approaches to cancer biology problems. cell cycle is a process in which cells proliferate while collectively performing a series of coordinated actions. cell-cycle models also have an impact on drug discovery. chassagnole et al. ( ) used a mathematical model to simulate and unravel the effect of multi-target kinase inhibitors of cyclin-dependent kinases (cdks). they quantitatively predict the cytotoxicity of a set of kinase inhibitors based on the in vitro ic measurement values. finally, they assess the pharmaceutical value of these inhibitors as anticancer therapeutics. in cancer, avascular tumor growth is characterized by localized, benign tumor growth where the nearby tissues consume most of the nutrients. mathematical modeling of avascular tumor growth is important to understanding the advanced stages of cancer. kiran et al. ( ) have developed a spatial-temporal mathematical model classified as a different zone model (dzm) for avascular tumor growth based on diffusion of nutrients and their consumption, and it includes key mechanisms in the tumor. the diffusion and nutrient consumption are represented using partial differential equations. this model predicts that onset of necrosis occurs when the concentrations of vital nutrients are below critical values, and also the overall tumor growth based on the size effects of the proliferation zone, quiescent zone, and necrotic zone. the mathematical approaches towards modeling the three natural scales of interest (subcellular, cellular, and tissue) are discussed above. developing models that can predict the effects across biological scales is a challenge. the long-term goal is to build a "virtual human made up of mathematical models with connections at the different biological scales (from genes to tissue to organ)." a model is an optimal mix of hypotheses, evidence, and abstraction to explain a phenomenon. hypothesis is a tentative explanation for an observation, phenomenon, or scientific problem that can be tested by further investigation. evidence describes information (i.e. experimental data) that helps in forming a conclusion or judgment. abstraction is an act of filtering out the required information to focus on a specific property only. for example, archiving books based on the year of publication, irrespective of the author name, would be an example of abstraction. in this process, some detail is lost and some gained. predictions are made through modeling that can be tested by experiment. a model may be simple (e.g. the logistic equation describing how a population of bacteria grows) or complicated. models may be mathematical or statistical. mathematical models make predictions, whereas statistical models enable us to draw statistical inferences about the probable properties of a system. in other words, models can be deductive or inductive. if the prediction is necessarily true given that the model is also true, then the model is a deductive model. on the other hand, if the prediction is statistically inferred from observations, then the model is inductive. deductive models contain a mathematical description; for example, the reaction-diffusion equation that makes predictions about reality. if these predictions do not agree with experiment, then the validity of the entire model may be questioned. mathematical models are commonly applied in physical sciences. on the other hand, inductive models are mostly applied in the biological sciences. in biology, models are used to describe, simulate, analyze, and predict the behavior of biological systems. modeling in biology provides a framework that enables description and understanding of biological systems through building equations that express biological knowledge. modeling enables the simulation of the behavior of a biological system by performing in silico experiments (i.e. numerically solving the equations or rules that describe the model). the results of these in silico experiments become the input for further analysis; for example, identification of key parameters or mechanisms, interpretation of data, or comparison of the ability of different mechanisms to generate observed data. in particular, systems biology employs an integrative approach to characterizing biological systems in which interactions among all components in a system are described mathematically to establish a computable model. these in silico models complement traditional in vivo animal models and can be applied to quantitatively study the behavior of a system of interacting components. the term "in silico" is poorly defined, with several researchers claiming their role in its origination (ekins et al., ) . sieburg ( ) and (danchin et al. ) were two of the earliest published works that used this term. specifically, in silico models gained much interest in the early stages by various imaging studies (chakroborty et al., ) . as an example, microarray analysis that enabled measurement of genome-scale expression levels of genes provided a method to investigate regulatory networks. years of regulatory network studies (that included microarray-based investigations) led to the development of some well-characterized regulatory networks such as e. coli and yeast regulatory networks. these networks are available in the genenetweaver (gnw) tool. gnw is an open-source tool for in silico benchmark generation and performance profiling of network inference methods. thus, the advent of high-throughput experimental tools has allowed for the simultaneous measurement of thousands of biomolecules, opening the way for in silico model construction of increasingly large and diverse biological systems. integrating heterogeneous dynamic data into quantitative predictive models holds great promise for significantly increasing our ability to understand and rationally intervene in disease-perturbed biological systems. this promise -particularly with regards to personalized medicine and medical intervention -has motivated the development of new methods for systems analysis of human biology and disease. such approaches offer the possibility of gaining new insights into the behavior of biological systems, of providing new frameworks for organizing and storing data and performing statistical analyses, suggesting new hypotheses and new experiments, and even of offering a "virtual laboratory" to supplement in vivo and in vitro work. however, in silico modeling in the life sciences is far from straightforward, and suffers from a number of potential pitfalls. thus, mathematically sophisticated but biologically useless models often arise because of a lack of biological input, leading to models that are biologically unrealistic, or that address a question of little biological importance. on the other hand, models may be biologically realistic but mathematically intractable. this problem usually arises because biologists unfamiliar with the limitations of mathematical analysis want to include every known biological effect in the model. even if it were possible to produce such models, they would be of little use since their behavior would be as complex to investigate as the experimental situation. these problems can be avoided by formulating clear explicit biological goals before attempting to construct a model. this will ensure that the resulting model is biologically sound, can be experimentally verified, and will generate biological insight or new biological hypotheses. the aim of a model should not simply be to reproduce biological data. indeed, often the most useful models are those that exhibit discrepancies from experiment. such deviations will typically stimulate new experiments or hypotheses. an iterative approach has been proposed, starting with a biological problem, developing a mathematical model, and then feeding back into the biology. once established, this collaborative loop can be traversed many times, leading to ever increasing understanding. the ultimate goal of in silico modeling in biology is the detailed understanding of the function of molecular networks as they appear in metabolism, gene regulation, or signal transduction. this is achieved by using a level of mathematical abstraction that needs a minimum of biological information to capture all physiologically relevant features of a cellular network. for example, ideally, for in silico modeling of a molecular network, knowledge of the network structure, all reaction rates, concentrations, and spatial distributions of molecules at any time point is needed. unfortunately, such information is unavailable even for the best-studied systems. in silico simulations thus always have to use a level of mathematical abstraction, which is dictated by the extent of our biological knowledge, by molecular details of the network, and by the specific questions that are addressed. understanding the complexity of the disease and its biological significance in health can be achieved by integrating data from the different functional genomics experiments with medical, physiological, and environmental factor information, and computing mathematically. the advantage of mathematical modeling of disease lies in the fact that such models not only shed light on how a complex process works, which could be very difficult for inferring an understanding of each component of this process, but also predict what may follow as time evolves or as the characteristics of particular system components are modified. mathematical models have generally been utilized in association with an increased understanding of what models can offer in terms of prediction and insight. the two distinct roles of models are prediction and understanding the accuracy, transparency, and flexibility of model properties. prediction of the models should be accurate, including all the complexities and population-level heterogeneity that have an additional use as a statistical tool. it also provides the understanding of how the disease spreads in the real world and how the complexity affects the dynamics. model understanding aids in developing sophisticated predictive models, along with gathering more relevant epidemiological data. a model should be as simple as possible and should have balance in accuracy, transparency, and flexibility; in other words, a model should be well suited for its purpose. the model should be helpful in understanding the behavior of the disease and able to simplify the other disease condition. several projects are proceeding along these lines, such as e-cell (tomita, ) and simulations of biochemical pathways. whole cell modeling integrates information from metabolic pathways, gene regulation, and gene expression. three elements are needed for constructing of a good cell model: precise knowledge of the phenomenon, an accurate mathematical representation, and a good simulation tool. a cell represents a dynamic environment of interaction among nucleic acids, proteins, carbohydrates, ions, ph, temperature, pressure, and electrical signals. many cells with similar functionality form tissue. in addition, each type of tissue uses a subset of this cellular inventory to accomplish a particular function. for example, in neurons, electro-chemical phenomena take precedence over cell division, whereas, cell division is a fundamental function of skin, lymphocytes, and bone marrow cells. thus, an ideal virtual cell not only represents all the information, but also exhibits the potential to differentiate into neuronal or epithelial cells. the first step in creating a whole cell model is to divide the entire network into pathways, and pathways into individual reactions. any two reactions belong to a pathway if they share a common intermediate. in silico modeling consists not only of decomposing events into manageable units, but also of assembling these units into a unified framework. in other words, mathematical modeling is the art of converting biology into numbers. for whole cell modeling, a checklist of biological phenomena that call for mathematical representation is needed. biological phenomena taken into account for in silico modeling of whole cells are the following: . dna replication and repair . translation . transcription and regulation of transcription . energy metabolism . cell division . chromatin modeling . signaling pathways . membrane transport (ion channels, pump, nutrients) . intracellular molecular trafficking . cell membrane dynamics . metabolic pathways the whole cell metabolism includes enzymatic and nonenzymatic processes. enzymatic processes cover most of the metabolic events, while non-enzymatic processes include gene expression and regulation, signal transduction, and diffusion. in silico modeling of whole cells not only requires precise qualitative and quantitative data, but also an appropriate mathematical representation of each event. for metabolic modeling, the data input consists of kinetics of individual reactions and also effects of cofactors, ph, and ions on the model. the key step in modeling is to choose an appropriate assumption. for example, a metabolic pathway may be a mix of forward and reverse reactions. furthermore, inhibitors that are part of the pathway may influence some reactions. at every step, enzymatic equations are needed that best describe the process. in silico models are built because they are easy to understand, controllable, and can store and analyze large amounts of information. a well-built model has diagnostic and predictive abilities. a cell by itself is a complete biochemical reactor that contains all the information one needs to understand life. whole cell modeling enables investigation of the cell cycle, physiology, spatial organization, and cell-cell communication. sequential actions in whole cell modeling are the following: . catalog all the substances that make up a cell. substances (for qualitative modeling). . add rate constants, concentration of substances, and strength of inhibition. . assume appropriate mathematical representations for individual reactions. . simulate reactions with suitable simulation software. . diagnose the system with system analysis software. . perturb the system and correlate its behavior to an underlying genetic and/or biochemical factor. . predict phenomenon using a hypothesis generator. in silico modeling of disease combines the advantages of both in vivo and in vitro experimentation. unlike in vitro experiments, which exist in isolation, in silico models provide the ability to include a virtually unlimited array of parameters, which render the results more applicable to the organism as a whole. in silico modeling allows us to examine the workings of biological processes such as homeostasis, reproduction, evolution, etc. for example, one can explore the processes of darwinian evolution through in silico modeling, which are not practical to study in real time. in silico modeling of disease is quite challenging. attempting to incorporate every single known interaction rapidly leads to an unmanageable model. furthermore, parameter determination in such models can be a frightening experience. estimates come from diverse experiments, which may be elegantly designed and well executed but can still give rise to widely differing values for parameters. data can come from both in vivo and in vitro experiments, and results that hold in one medium may not always hold in the other. furthermore, despite the many similarities between mammalian systems, significant differences do exist, and so results obtained from experiments using animal and human tissue may not always be consistent. also there are many considerations that cannot be applied. for example, one cannot investigate the role of stochastic fluctuations by removing them from the system, or one cannot directly explore the process that gave rise to current organisms. in silico modeling has been applied in cancer, systemic inflammatory response syndrome, immune diseases, neuronal diseases, and infectious diseases (among others). in silico models of disease can contribute to a better understanding of the pathophysiology of the disease, suggest new treatment strategies, and provide insight into the design of experimental and clinical trials for the investigation of new treatment modalities. in silico modeling of cancer has become an interesting alternative approach to traditional cancer research. in silico models of cancer are expected to predict the complexity of cancer at multiple temporal and spatial resolutions, with the aim of supplementing diagnosis and treatment by helping plan more focused and effective therapy via surgical resection, standard and targeted chemotherapy, and novel treatments. in silico models of cancer include: (a) statistical models of cancer, such as molecular signatures of perturbed genes and molecular pathways, and statistically-inferred reaction networks; (b) models that represent biochemical, metabolic, and signaling reaction networks important in oncogenesis, including constraint-based and dynamic approaches for the reconstruction of such networks; and (c) models of the tumor microenvironment and tissue-level interactions (edelman et al., ) . statistical models of cancer can be broadly divided into those that employ unbiased statistical inference, and those that also incorporate a priori constraints of specific biological interactions from data. statistical models of cancer biology at the genetic, chromosomal, transcriptomic, and pathway levels provide insight about molecular etiology and consequences of malignant transformation despite incomplete knowledge of underlying biological interactions. these models are able to identify molecular signatures that can inform diagnosis and treatment selection, for example with molecular targeted therapies such as imatinib (gleevec) (edelman et al., ) . however, in order to characterize specific biomolecular mechanisms that drive oncogenesis, genetic and transcriptional activity must be considered in the context of cellular networks that ultimately drive cellular behavior. in microbial cells, network inference tools have been developed and applied for the modeling of diverse biochemical, signaling, and gene expression networks. however, due to the much larger size of the human genome compared to microbes, and the substantially increased complexity of eukaryotic genetic regulation, inference of transcriptional regulatory networks in cancer presents increased practical and theoretical challenges. biochemical reaction networks are constructed to represent explicitly the mechanistic relationships between genes, proteins, and the chemical inter-conversion of metabolites within a biological system. in these models, network links are based on pre-established biomolecular interactions rather than statistical associations; significant experimental characterization is thus needed to reconstruct biochemical reaction networks in human cells. these biochemical reaction networks require, at a minimum, knowledge of the stoichiometry of the participating reactions. additional information such as thermodynamics, enzyme capacity constraints, time-series concentration profiles, and kinetic rate constants can be incorporated to compose more detailed dynamic models (edelman et al., ) . microenvironment-tissue level models of cancer apply an "engineering" approach that views tumor lesions as complex micro-structured materials, where three-dimensional tissue architecture ("morphology") and dynamics are coupled in complex ways to cell phenotype, which in turn is influenced by factors in the microenvironment. computational approaches of in silico cancer research include continuum models, discrete models, and hybrid models. in continuum models, extracellular parameters can be represented as continuously distributed variables to mathematically model cell-cell or cell-environment interactions in the context of cancers and the tumor microenvironment. systems of partial differential equations have been used to simulate the magnitude of interaction between these factors. continuum models are suitable for describing the individual cell migration, change of cancer cell density, diffusion of chemo-attractants, heat transfer in hyperthermia treatment for skin cancer, cell adhesion, and the molecular network of a cancer cell as an entire entity. however, these types of in silico models have limited ability for investigating singlecell behavior and cell-cell interaction. on the other hand, "discrete" models (i.e. cellular automata models) represent cancer cells as discrete entities of defined location and scale, interacting with one another and external factors in discrete time intervals according to predefined rules. agent-based models expand the cellular automata paradigm to include entities of divergent functionalities interacting together in a single spatial representation, including different cell types, genetic elements, and environmental factors. agent-based models have been used for modeling three-dimensional tumor cell patterning, immune system surveillance, angiogenesis, and the kinetics of cell motility. hybrid models have been created which incorporate both continuum and agent-based variables in a modular approach. hybrid models are ideal for examining direct interactions between individual cells and between the cells and their microenvironment, but they also allow us to analyze the emergent properties of complex multi-cellular systems (such as cancer). hybrid models are often multi-scale by definition, integrating processes on different temporal and spatial scales, such as gene expression, intracellular pathways, intercellular signaling, and cell growth or migration. there are two general classes of hybrid models, those that are defined upon a lattice and those that are off-lattice. the classification of hybrid models on these two classes depends on the number of cells these models can handle and the included details of each individual cell structure, i.e. models dealing with large cell populations but with simplified cell geometry, and those that model small colonies of fully deformable cells. for example, a hybrid model investigated the invasion of healthy tissue by a solid tumor. the model focused on four key parameters implicated in the invasion process; tumor cells, host tissue (extracellular matrix), matrix-degradative enzymes, and oxygen. the model is actually hybrid, wherein the tumor cells were considered to be discrete (in terms of concentrations), and the remaining variables were in the continuous domain in terms of concentrations. this hybrid model can make predictions on the effects of individual-based cell interactions (both between individuals and the matrix) on tumor shape. the model of zhang et al. ( ) incorporated a continuous model of a receptor signaling pathway, an intracellular transcriptional regulatory network, cell-cycle kinetics, and three-dimensional cell migration in an integrated, agent-based simulation of solid brain tumor development. the interactions between cellular and microenvironment states have also been considered in a multi-scale model that predicts tumor morphology and phenotypic evolution in response to such extracellular pressures. the biological context in which cancers develop is taken into consideration in in silico models of the tumor microenvironment. such complex tumor microenvironments may integrate multiple factors including extracellular biomolecules, vasculature, and the immune system. however, rarely have these methods been integrated with a large cell-cell communication network in a complex tumor microenvironment. recently, an interesting effort of in silico modeling was described in which the investigators integrated all the intercellular signaling pathways known to date for human glioma and generated a dynamic cell-cell communication network associated with the glioma microenvironment. then they applied evolutionary population dynamics and the hill functions to interrogate this intercellular signaling network and execute an in silico tumor microenvironment development. the observed results revealed a profound influence of the micro-environmental factors on tumor initiation and growth, and suggested new options for glioma treatment by targeting cells or soluble mediators in the tumor microenvironment (wu et al., ) . trauma and infection can cause acute inflammatory responses, the degree of which may have several pathological manifestations like systemic inflammatory response syndrome (sirs), sepsis, and multiple organ failure (mof). however, an appropriate management of these states requires further investigation. translating the results of basic science research to effective therapeutic regimes has been a longstanding issue due in part to the failure to account for the complex nonlinear nature of the inflammatory process wherein sirs/mof represent a disordered state. hence, the in silico modeling approach can be a promising research direction in this area. indeed, in silico modeling of inflammation has been applied in an effort to bridge the gap between basic science and clinical trials. specifically, both agent-based modeling and equation-based modeling have been utilized . equation-based modeling encompasses primarily ordinary differential equations (ode) and partial differential equations (pde). initial modeling studies were focused on the pathophysiology of the acute inflammatory response to stress, and these studies suggested common underlying processes generated in response to infection, injury, and shock. later, mathematical models included the recovery phase of injury and gave insight into the link between the initial inflammatory response and subsequent healing process. the first mathematical models of wound healing dates back to the s and early s. these models and others developed in the s investigated epidermal healing, repair of the dermal extracellular matrix, wound contraction, and wound angiogenesis. most of these models were deterministic and formulated using differential equations. in addition, recent models have been formulated using differential equations to analyze different strategies for improved healing, including wound vacs, commercially engineered skin substitutes, and hyperbaric oxygen. in addition, agent-based models have been used in wound healing research. for example, mi et al. ( ) developed an agent-based model to analyze different treatment strategies with wound debridement and topical administration of growth factors. their model produced the expected results of healing when analyzing for different treatment strategies including debridement, release of pdgf, reduction in tumor necrosis factor-α, and increase of tgf-β . the investigators suggested that a drug company should use a mathematical model to test a new drug before going through the expensive process of basic science testing, toxicology and clinical trials. indeed, clinical trial design can be improved by prior in silico modeling. for example, in silico modeling has led to the knowledge that patients who suffered from the immune-suppressed phenotype of late-stage multiple organ failure, and were susceptible to usually trivial nosocomial infections, demonstrated sustained elevated markers of tissue damage and inflammation through two weeks of simulated time. however, anti-cytokine drug trials with treatment protocols of only one dose or one day had not incorporated this knowledge into their design, with subsequent failure of candidate treatments. by now the reader is expected to be familiar with the meaning and the basics of in silico modeling. in this section we discuss the application of in silico modeling in the understanding of infectious diseases and in the proposition/ development of better treatments for infectious diseases. in fact, the applications of in silico modeling can help far beyond just the understanding of the dynamics (and sometimes, statistics) of infectious diseases, and far beyond the proposition/development of better treatments for infectious diseases. the modeling can be helpful even in the understanding of better prevention of infectious diseases. the level of pathogen within the host defines the process of infection; such pathogen levels are determined by the growth rate of the pathogen and its interaction with the host's immune response system. initially, no pathogen is present, but just a low-level, nonspecific immunity within the host. on infection, the parasite grows abundantly over time with the potential to transmit the infection to other susceptible individuals. to comprehensively understand in silico modeling in the domains of infectious diseases, one should first understand the "triad of infectious diseases," and the characteristics of "infectious agent," "host," and "environment" on which the models are always based. in fact, modeling of infectious diseases is just impossible without this triad; after all, the model would be built on some parameters (also called variables in more general language), and those parameters always have their origin from the so-called "triad of infectious diseases." at this point, a good question would be: what is a "triad of infectious diseases?" "triad of infectious diseases" means the interactions between ( ) agent, which is the disease causing organism (the pathogen); ( ) host, which is the infected organism, or in the case of pre-infection, the organism to be infected is the host (thus in this case the host is the animal the agent infects); and ( ) environment, which is a kind of link between the agent and the host, and is essentially an umbrella word for the entirety of the possible media through which the agent reaches the host. now that we have an idea on what in silico modeling of infectious diseases are generally based on, we will outline a better understanding of the parameters that are considered in most in silico disease models. to discuss the parameters in an orderly manner, we just categorize them under each of the three components of the "triad of infectious diseases," and summarize them in the next subsection. it must be emphasized at this point that ( ) even though all the possible parameters for in silico modeling of infectious diseases can be successfully categorized under the characteristics of one of any of the three components of the "triad of infectious diseases" (agent, host, and environment), ( ) the parameters discussed in the next sub-section are by no means the entirety of all the possible parameters that can be included in in silico modeling of infectious diseases. in fact, several parameters exist, and this section cannot possibly enumerate them all. that is why we have discussed the parameters using a categorical approach. some of the parameters for in silico modeling of infectious diseases are essentially a measure of infectivity (ability to enter the host), pathogenicity (ability to cause divergence from homeostasis/disease), virulence (degree of divergence from homeostasis caused/ability to cause death), antigenicity (ability to bind to mediators of the host's adaptive immune system), and immunogenicity (ability to trigger adaptive immune response) of the concerned infectious agent. the exact measure (and thus the units) used can vary markedly depending on the intentions for which the in silico infectious diseases model is built, as well as the assumptions on which the in silico disease model is based. from the knowledge of the agent's characteristics, one should know that unlike parameters related to the other characteristics of the agent, the parameters related to infectivity find their most important use only in the modeling of the preinfection stage in infectious disease modeling. finally, some of the agent-related parameters of great importance in in silico modeling of infectious diseases are concentration of the agent's antigen-host antibody complex, case fatality rate, strain of the agent, other genetic information of the agent, etc. the parameters originating from characteristics of the host can also be diversified and based on the intentions for which the in silico infectious diseases model are built and the assumptions on which the in silico disease model are based; however, the parameters could then be grouped and explained under the host's genotype (the allele at the host's specified genetic locus), immunity/health status (biological defenses to avoid infection), nutritional status (feeding habits/food intake characteristics), gender (often categorized as male or female), age, and behavior (the host's behaviors that affect its resistance to homeostasis disruptors). typical examples of host-related parameters are the alleles at some specifically targeted genetic loci; the total white blood cell counts; differential white blood cell counts, and/or much more sophisticated counts of specific blood cell types; blood levels of some specific cytokines, hormones, and/or neurotransmitters; daily calories, protein, and/or fat intake; daily amount of energy expended and/or duration of exercise; etc. at first parameters originating from the environment might seem irrelevant to the in silico modeling of infectious diseases, but they are relevant. even after the pre-infection stage, the environment still modulates the host-agent intersections. for example, the ability (and thus the related parameters) of the agent to multiply and/or harm the host are continually influenced by the host's environmental conditions, and in a similar way the hosts defense against the adverse effects of the agents are modulated by the host's environmental conditions. but somehow, not so many of these parameters have been included in in silico infectious disease models in the recent past. a few examples of these parameters are the host's ambient temperature, the host's ambient atmospheric humidity, altitude, the host's light-dark cycle, etc. now that we know the parameters for in silico infectious disease modeling, the next reasonable question would be "what form does a typical in silico infectious disease model take?" so, this sub-section attempts to answer this very important question. let us view the in silico model as a system of wellintegrated functional equations or formulae. such well-integrated functional equations can be viewed or approximated as a single, albeit more complex, functional equation/formula. it is hence possible to vary any (or a combination) of the variables contained in this equation by running numerical simulations on a computer depending on the kind of prediction one wants to make. such in silico models can hence investigate several (maybe close to infinite) possible data points within reasonable limits that one sets depending on the nature of the variables considered. so the equations behind a typical infectious disease in silico model could take the form (equation . ): where h is the output from a smaller equation that is based on host parameters; β is a constant; f and g are link functions which may be the same as or different from each other and other link functions in this system of equations; a is the output from a smaller equation that is based on agent parameters; g is a link function which may be the same or different from other link functions in this system of equations; e is the output from a smaller equation that is based on environment parameters; and Ɛ is a random error parameter. readers should know that we use the term "link function" to refer to any of the various possible forms of mathematical operations or functions. this means that based on the complexity of the model, a particular "link function" might be as simple as a mere addition or as complex as several combinations of operators with high degree polynomials. where β a is a constant; f a , f a , … f ax are link functions that may be same or different (individually) from (every) other link function in this system of equations; a , a , … a x are a set of the agent's parameters (e.g. case fatality rate, agent's genotype, etc); and Ɛ is a random error parameter. where β e is a constant; f e , f e , …f ex are link functions which may be the same or different (individually) from (every) other link function in this system of equations; e , e , … e x are a set of environmental parameters (e.g. host's ambient temperature, host's ambient atmospheric humidity, etc.); and Ɛ is a random error parameter. muñoz-elías et al. ( ) documented (through their paper "replication dynamics of mycobacterium tuberculosis in chronically infected mice") a successful in silico modeling of infectious diseases (specifically, tuberculosis). in their in silico modeling of tuberculosis in mice, the researchers investigated both the static and dynamic host-pathogen/agent equilibrium (i.e. mice-mycobacterium tuberculosis static and dynamic equilibrium). the rationale behind their study was that a better understanding of host-pathogen/agent interactions would make possible the development of better anti-microbial drugs for the treatment of tuberculosis (as well as provide similar understanding for the cases of other chronic infectious diseases). they modeled different types of host-pathogen/ agent equilibriums (ranging from completely static equilibrium, all the way through semi-dynamic, down to completely dynamic scenarios) by varying the rate of multiplication/growth and the rate of death of the pathogen/ agent (mycobacterium tuberculosis) during the infection's chronic phase. through their in silico study (which was also verified experimentally), they documented a number of remarkable findings. for example, they established that "viable bacterial counts and total bacterial counts in the lungs of chronically infected mice do not diverge over time," and they explained that "rapid degradation of dead bacteria is unlikely to account for the stability of total counts in the lungs over time because treatment of mice with isoniazid for weeks led to a marked reduction in viable counts without reducing the total count. readers who are interested in further details on the generation of this in silico model for the dynamics of mycobacterium tuberculosis infection, as well as the complete details of the parameters/variables considered, and the comprehensive findings of the study, should refer to the article of ernesto et al. published in infection and immunity. another one of the many notable works in the domain of infectious disease in silico modeling is the study by navratil et al. ( ) . using protein-protein interaction data that the authors obtained from available literature and public databases, they (after first curating and validating the data) computationally (in silico) re-examined the virus-human protein interactome. interestingly, the authors were able to show that the onset and pathogenesis of some disease conditions (especially chronic disease conditions) often believed to be of genetic, lifestyle, or environmental origin, are, in fact, modulated by infectious agents. models have been constructed to simulate bacterial dynamics, such as growth under various nutritional and chemical conditions, chemotactic response, and interaction with host immunity. clinically important models of bacterial dynamics relating to peritoneal dialysis, pulmonary infections, and particularly of antibiotic treatment and bacterial resisitance, have also been developed. baccam et al. ( ) utilized a series of mathematical models of increasing complexity that incorporated target cell limitation and the innate interferon response. the models were applied to examine influenza a virus kinetics in the upper respiratory tracts of experimentally infected adults. they showed the models to be applicable for improving the understanding of influenza in a virus infection, and estimated that during an upper respiratory tract infection, the influenza virus initially spreads rapidly with one cell, infecting (on average) about others (daun and clermont, ) . model parameter and spread of disease: model parameters are one of the main challenges in mathematical modeling since all models do not have a physiological meaning. sensitivity analysis and bifurcation analysis give us the opportunity to understand how model outcome and model parameters are correlated, how the sensitivity of the system is with respect to certain parameters, and the uncertainty in the model outcome yielded by the uncertainties in the parameter values. uncertainty and sensitivity analysis was used to evaluate the input parameters play in the basic productive rate (ro) of severe acute respiratory syndrome (sars) and tuberculosis. control of the outbreak depends on identifying the disease parameters that are likely to lead to a reduction in r. difficulty in finding the most appropriate set of parameters for in silico modeling of infectious diseases is often a challenge. it is hoped this challenge will subside with the advancement in infectonomics and high-throughput technology. however, another important challenge lies in the understanding (and the provision of reasonable interpretations for) the results from all the complex interactions of parameters considered. in this sub-section we focus on the application of in silico modeling to improve knowledge of neuronal diseases, and thus improve the applications of neurological knowledge for solving neuronal health problems. it is not an overstatement to say that one of the many aspects of life sciences where in silico disease modeling would have the biggest applications is in the better understanding of the pathophysiology of nervous system (neuronal) diseases. this is basically because of the inherent delicate nature of the nervous system and the usual extra need to be sure of how to proceed prior to attempting to treat neuronal disease conditions. by this we mean that the need to first model neuronal disease conditions in silico prior to deciding on or suggesting (for example) a treatment plan is, in fact, rising. this is not unexpected; after all, it is better to be sure of what would work (say, through in silico modeling) than to try what would not work. obtaining appropriate parameters for the in silico modeling of a nervous system (neuronal) disease is rooted in a good understanding of the pathophysiology of such neuronal disease. since comprehensive details of pathophysiology of neuronal diseases is beyond the scope of this book, we only present the basic idea that would allow the reader to understand how in silico modeling of a nervous system (neuronal) disease can be done. to give a generalized explanation and still concisely present the basic ideas underlying the pathophysiology of neuronal diseases, we proceed by systematically categorizing the mediators of neuronal disease pathophysiology: ( ) nerve cell characteristics, ( ) signaling chemicals and body electrolytes, ( ) host/organism factors, and ( ) environmental factors. readers need to see all these categories as being highly integrated pathophysiologically rather than as separate entities, and also that we have only grouped them this way to make simpler the explanation of how the parameters for in silico modeling of neuronal diseases are generated. when something goes wrong with (or there is a marked deviation from equilibrium in) a component of any of the four categories above, the other components (within and/ or outside the same category) try hard to make adjustments so as to annul/compensate for the undesired change. for example, if the secretion of a chemical signal suddenly becomes abnormally low, the target cells for the chemical signal may develop mechanisms to use the signaling chemical more efficiently, and the degradation rate of the signaling chemical may be reduced considerably. through these, the potentially detrimental effects of reduced secretion of the chemical signal are annulled via compensation from the other components. this is just a simple example; much more complex regulatory and homeostatic mechanisms exist in the neuronal system. despite the robustness of those mechanisms, things still get out of hand sometimes, and disease conditions result. the exploration of what happens in (and to) each and all of the components of this giant system of disease conditions is called the pathophysiology of neuronal disease, and it this pathophysiology that "provides" parameters for the in silico modeling of neuronal diseases. some of the important parameters (that are of nerve cell origin) for a typical in silico modeling of a neuronal disease (say, alzheimer's disease) are the population (or relative population) of specific neuronal cells (such as glial cells: microglia, astrocytes, etc.), motion of specific neuronal cells (e.g. microglia), amyloid production, aggregation and removal of amyloid, morphology of specific neuronal cells, status of neuronal cell receptors, generation/regeneration/ degeneration rate of neuronal cells, status of ion neuronal cell channels, etc. based on their relevance to the pathophysiology of the neuronal disease being studied, many of these parameters are often considered in the in silico modeling of the neuronal disease. more importantly, their spatiotemporal dynamics are often seriously considered. the importance of signaling chemicals and electrolytes in the nervous system makes parameters related to them very important. the secretion, uptake, degradation, and diffusion rates of various neurotransmitters and cytokines are often important parameters in the in silico modeling of neurodiseases. other important parameters are the concentration gradients of the various neurotransmitters and cytokines, the availability and concentration of second messengers, and the electrolyte status/balance of the cells/systems. the spatiotemporal dynamics of all of these are also often seriously considered. the parameters under host/organism factors can be highly varied depending on the intentions and the assumptions governing the in silico disease modeling. nonetheless, one could basically group and list the parameters collectively under genotype (based on the allele at a specified genetic locus), nutritional status (feeding habits/food intake characteristics; e.g. daily calories, protein intake, etc.), gender (male or female), age, and behavior (host's behaviors/lifestyle that influences homeostasis and/or responses to stimuli). a few examples of these parameters are ambient temperature, altitude, light-dark cycle, social network, type of influences from people in the network, etc. just like other in silico models, a neuronal disease in silico model is also based on what could be viewed as a single giant functional equation, which is composed of highly integrated simpler functional equations. so the equations behind a typical neuronal disease in silico model could take the form (equation . ): where n could be a parameter that is a direct measure of the disease manifestation; β is a constant; f, g, j, and k are link functions which may be the same or different from other link functions in this system of equations; c, s, h, and e are the outputs from smaller equations that are based on parameters from neuronal cell characteristics, signaling molecule and electrolyte parameters, host parameters, and environment parameters, respectively; and Ɛ is a random error parameter. the reader should know that each of n, c, s, h and e could have resulted from smaller equations that could take forms similar to those (equations . to . ) described under in silico modeling of infectious diseases (previous sub-section). in silico models edelstein-keshet and spiros ( ) used in silico modeling to study the mechanism and/formation of alzheimer's disease. the target of their in silico modeling was to explore and demystify how various parts implicated in the etiology and pathophysiology of alzheimer's disease work together as a whole. employing the strength of in silico modeling, the researchers were able to transcend the difficulty of identifying detailed disease progression scenarios, and they were able to test a wide variety of hypothetical mechanisms at various levels of detail. readers interested in the complete details of the assumptions that govern in silico modeling of alzheimer's disease, the various other aspects of the model, and more detailed accounts of the findings should look at the article by edelstein-keshet and spiros. several other interesting studies have applied in silico modeling techniques to investigate various neuronal diseases. a few examples include the work of altmann and boyton ( ) , who investigated multiple sclerosis (a very common disease resulting from demyelination in the central nervous system) using in silico modeling techniques; lewis et al. ( ) , who used in silico modeling to study the metabolic interactions between multiple cell types in alzheimer's disease; and raichura et al. ( ) , who applied in silico modeling techniques to dynamically model alpha-synuclein processing in normal and parkinson's disease states. a more specific example of a molecular level in silico alzheimer's disease model can be found in ghosh et al. ( ) . among the amyloid proteins, amyloid-β (aβ) peptides (aβ and aβ ) are known to form aggregates that deposit as senile plaques in the brains of alzheimer's disease patients. the process of aβ-aggregation is strongly nucleation-dependent, and is inferred by the occurrence of a "lag-phase" prior to fibril growth that shows a sigmoidal pattern. ghosh et al. ( ) dissected the growth curve into three biophysically distinct sections to simplify modeling and to allow the data to be experimentally verifiable. stage i is where the pre-nucleation events occur whose mechanism is largely unknown. the pre-nucleation stage is extremely important in dictating the overall aggregation process where critical events such as conformation change and concomitant aggregation take place, and it is also the most experimentally challenging to decipher. in addition to mechanistic reasons, this stage is also physiologically important as lowmolecular-weight (lmw) species are implicated in ad pathology. the rate-limiting step of nucleation is followed by growth. the overall growth kinetics and structure and shape of the fibrils are mainly determined by the structure of the nucleating species. an important intermediate along the aggregation pathway, called "protofibrils," have been isolated and characterized that have propensities to both elongate (by monomer addition) as well as to laterally associate (protofibril-protofibril association) to grow into mature fibrils (stage iii in the growth curve). aggregation ghosh et al. ( ) generated an ode-based molecular simulation (using mass-kinetics methodology) of this fibril growth process to estimate the rate constants involved in the entire pathway. the dynamics involved in the protofibril elongation stage of the aggregation (stage iii of the process) were estimated and validated by in vitro biophysical analysis. ghosh et al. ( ) next used the rate constants identified from stage iii to create a complete aggregation pathway simulation (combining stages i, ii, and iii) to approximately identify the nucleation mass involved in aβ-aggregation. in order to model the aβ-system, one needs to estimate the rate constants involved in the complete pathway and the nucleation mass itself. it is difficult to iterate through different values for each of these variables to get close to the experimental plots (fibril growth curves measured via fluorescence measurements with time) due to the large solution space; also, finding the nucleation phase cannot be done independently without estimating the rate constants alongside. however, having separately estimated the post-nucleation stage rate constants (as mentioned above) reduces the overall parameter estimation complexity. the complete pathway simulation was used to study the lag times associated with the aggregation pathway, and hence predict possible estimates of the nucleation mass. the following strategy was used: estimate the pre-nucleation rate constants that give the maximum lag times for each possible estimate of the nucleation mass. this led to four distinctly different regimes of possible nucleation masses corresponding to four different pairs of rate constants for the pre-nucleation phase (regime , where n = , , , , ; regime , where n = , , ; regime , where n = , , ; and regime , where n = , , , ) . however, it was experimentally observed that the semi-log plot of the lag times against initial concentration of aβ is linear, and this characteristic was used to figure out what values of nucleation mass are most feasible for the aβ -aggregation pathway. the simulated plots show a more stable relationship between the lag times and the initial concentrations, and the best predictions for the nucleation mass were reported to be in the range - . such molecular pathway level studies are extremely useful in understanding the pathogenesis of ad in general, and can motivate drug development exercises in the future. for example, characterization of the nucleation mass is important as it has been observed that various fatty acid interfaces can arrest the fibril growth process (by stopping the reactions beyond the pre-nucleation stage). such in depth modeling of the aggregation pathway can suggest what concentrations of fatty acid interfaces should be used (under a given aβ concentration in the brain) to arrest the fibril formation process leading to direct drug dosage and interval prediction for ad patients. despite the fact that we have mentioned several possible parameters for in silico modeling of neuro-diseases, it is noteworthy that finding a set of the most reasonable parameters for the modeling is in fact a big challenge. on the other hand, understanding (and thus finding reasonable biological interpretations for) the results from the complex interaction of all parameters considered is also a big challenge. in addition, a number of assumptions that models are sometimes based on still have controversial issues. accurately modeling spatio-temporal dynamics of neurons and neurotransmitters (and other chemicals/ligands) also constitutes a huge challenge. understanding the complex systems involved in a disease will make it possible to develop smarter therapeutic strategies. treatments for existing tumors will use multiple drugs to target the pathways or perturbed networks that show an altered state of activity. in addition, models can effectively form the basis for translational research and personalized medicine. biological function arises as the result of processes interacting across a range of spatiotemporal scales. the ultimate goal of the applications of bioinformatics in systems biology is to aid in the development of individualized therapy protocols to minimize patient suffering while maximizing treatment effectiveness. it is now being increasingly recognized that multi-scale mathematical and computational tools are necessary if we are going to be able to fully understand these complex interactions (e.g. in cancer and heart diseases). with the bioinformatics tools, computational theories, and mathematical models introduced in this article, readers should be able to dive into the exhilarating area of formal computational systems biology. investigating these models and confirming their findings by experimental and clinical observations is a way to bring together molecular reductionism with quantitative holistic approaches to create an integrated mathematical view of disease progression. we hope to have shown that there are many interesting challenges yet to be solved, and that a structured and principled approach is essential for tackling them. systems biology is an emerging field that aims to understand biological systems at the systems level with a high degree of mathematical and statistical modeling. in silico modeling of infectious diseases is a rich and growing field focused on modeling the spread and containment of infections with model designs being flexible and enabling adaptation to new data types. the advantages of avoiding animal testing have often been seen as one of the advantages offered by in silico modeling; the biggest advantage is that there are no ethical issues in performing in silico experiments as they don't require any animals or live cells. furthermore, as the entire modeling and analysis are based on computational approaches, we can obtain the results of such analysis even within an hour. this saves huge amounts of time and reduces costs, two major factors associated with in vitro studies. however, a key issue that needs to be considered is whether in silico testing will ever be as accurate as in vitro or in vivo testing, or whether in silico results will always require non-simulated experimental confirmation. tracqui et al. ( ) successfully developed a glioma model to show how chemo-resistant tumor sub-populations cause treatment failure. similarly, a computational model of tumor invasion by frieboes et al. ( ) is able to demonstrate that the growth of a tumor depends on the microenvironmental nutrient status, the pressure of the tissue, and the applied chemotherapeutic drugs. the d spatio-temporal simulation model of a tumor by dionysiou et al. ( ) was able to repopulate, expand, and shrink tumor cells, thus providing a computational approach for assessment of radiotherapy outcomes. the glioblastoma model of kirby et al. ( ) is able to predict survival outcome post-radiotherapy. wu et al. ( ) has also developed an in silico glioma microenvironment that demonstrates that targeting the microenvironmental components could be a potential anti-tumor therapeutic approach. the in silico model-based systems biology approach to skin sensitization (tnf-alpha production in the epidermis) and risk of skin allergy assessment has been successfully carried out; it can replace well known in vitro assays, such as the mouse local lymph node assay (llna) used for the same purpose (by maxwell and mackay ( ) at the unilever safety and environmental assurance centre). similarly, davies et al. ( ) effectively demonstrated an in silico skin permeation assay based on time course data for application in skin sensitization risk assessment. kovatchev et al. ( ) showed how the in silico model of alcohol dependence can provide virtual clues for classifying the physiology and behavior of patients so that personalized therapy can be developed. pharmacokinetics and pharmacodynamics are used to study absorption, distribution, metabolism, and excretion (adme) of administered drugs. in silico models have tremendous efficacy in early estimation of various adme properties. quantitative structure-activity relationship (qsar) and quantitative structure-property relationship (qspr) models have been commonly used for several decades to predict adme properties of a drug at early phases of development. there are several in silico models applied in adme analysis, and readers are encouraged to read the review by van de waterbeemd and gifford ( ) . gastroplus™, developed at simulations plus (www.simulations-plus.com), is highly advanced, physiologically based rapid pharmacokinetic (pbpk) simulation software that can generate results within seconds, thus saving huge amounts of time and cost in clinical studies. the software is an essential tool to formulation scientists for in vitro dose disintegration and dissolution studies. towards next-generation treatment of spinal cord injuries, novartis (www.novartis. com) is working to model the human spinal cord and its surrounding tissues in silico to check the feasibility of monoclonal antibody-based drug administration and their pharmacokinetics and pharmacodynamics study results. the in silico "drug re-purposing" approach by bisson et al. ( ) demonstrated how phenothiazine derivative antipsychotic drugs such as acetophenazine can cause endocrine side effects. recently aguda et al. ( ) reported a computational model for sarcoidosis dynamics that is useful for pre-clinical therapeutic studies for assessment of dose optimization of targeted drugs used to treat sarcoidosis. towards designing personalized therapy of larynx injury leading to acute vocal fold damage, li et al. ( ) developed agent-based computational models. in a further advancement, entelos ® (www.entelos.com) has developed "virtual patients," in silico mechanistic models of type- diabetes, rheumatoid arthritis, hypertension, and atherosclerosis for identification of biomarkers, drug targets, development of therapeutics, and clinical trial design, and patient stratification. entelos' virtual idd mouse (nod mouse) can replace diabetes resistance type- diabetes live mice for various in vivo experiments. apart from diseases, systems level modeling of basic biological phenomena and their applications in disease have also been reported. an in silico model to mimic the in vitro rolling, activation, and adhesion of individual leukocytes has been developed by tang et al. ( ) . developing virtual mitochondria, cree et al. ( ) vi p r ( h t t p : / / w w w. v i p r b r c . o rg / b r c / h o m e . d o ? decorator=vipr) is one of the five bioinformatics resource centers (brcs) funded by the national institute of allergy and infectious diseases (niaid). this website provides a publicly available database and a number of computational analysis tools to search and analyze data for virus pathogens. some of the tools available at vipr are the following: . gatu (genome annotation transfer utility), a tool to transfer annotations from a previously annotated reference to a new, closely related target genome. . pcr premier design, a tool for designing pcr primers. . a sequence format conversion tool. . a tool to identify short peptides in proteins. a meta-driven comparative analysis tool. as there are many different kinds of tools available the tools on the website are organized by the virus family. the rat genome database (rgd) the rat genome database (http://rgd.mcw.edu/wg/ home) is funded by the national heart, lung, and blood institute (nhlbi) of the national institutes of health (nih). the goal of this project is to consolidate research work from various institutes to generate and maintain a rat genomic database (and make it available to the scientific community). the website provides a variety of tools to analyze data. influenza resource centers (brcs) funded by the national institute of allergy and infectious diseases (niaid). this website provides a publicly available database and a number of computational analysis tools to search and analyze data for influenza virus. this website provides many of the same tools that are provided at vibr. there are numerous other tools such as models of infectious disease agent study (midas), which is an in silico model for assessing infectious disease dynamics. midas assists in preparing, detecting, and responding to infectious disease threats. the wellcome trust sanger institute the sanger institute (http://www.sanger.ac.uk/) investigates genomes in the study of diseases that have an impact on global health. the sanger institute has made a significant contribution to genomic research and developing a new understanding of genomes and their role in biology. the website provides sequence genomes for various bacterial, viral, and model organisms such as zebrafish, mouse, gorilla, etc. a number of open source software tools for visualizing and analyzing data sets are available at the sanger institute website. an in silico modeling approach to understanding the dynamics of sarcoidosis models of multiple sclerosis. autoimmune diseases kinetics of influenza a virus infection in humans discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs using a mammalian cell cycle simulation to interpret differential kinase inhibition in antitumour pharmaceutical development in silico models for cellular and molecular immunology: successes, promises and challenges introduction to systems biology a reduction of mitochondrial dna molecules during embryogenesis explains the rapid segregation of genotypes from data banks to data bases in silico modeling in infectious disease determining epidermal disposition kinetics for use in an integrated nonanimal approach to skin sensitization risk assessment a four-dimensional simulation model of tumour response to radiotherapy in vivo: parametric validation considering radiosensitivity, genetic profile and fractionation in silico models of cancer mathematical modeling of radiotherapy strategies for early breast cancer exploring the formation of alzheimer's disease senile plaques in silico in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling farm animal genomics and informatics: an update an integrated computational/experimental model of tumor invasion dynamics of protofibril elongation and association involved in abeta peptide aggregation in alzheimer's disease bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists mathematical modeling of avascular tumour growth based on diffusion of nutrients and its validation in silico models of alcohol dependence and treatment large-scale in silico modeling of metabolic interactions between cell types in the human brain a patient-specific in silico model of inflammation and healing tested in acute vocal fold injury application of a systems biology approach to skin allergy risk assessment agent-based model of inflammation and wound healing: insights into diabetic foot ulcer pathology and the role of transforming growth factor-β . wound repair regeneration replication dynamics of mycobacterium tuberculosis in chronically infected mice when the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases dynamic modeling of alphasynuclein aggregation for the sporadic and genetic forms of parkinson's disease physiological studies in silico computational and experimental models of ca +-dependent arrhythmias dynamics of in silico leukocyte rolling, activation, and adhesion whole-cell simulation: a grand challenge of the st century a mathematical model of glioma growth: the effect of chemotherapy on spatio-temporal growth computational cardiology: the heart of the matter a survey of available tools and web servers for analysis of protein-protein interactions and interfaces admet in silico modelling: towards prediction paradise? translational systems biology of inflammation bioinformatics applications for pathway analysis of microarray data bayesian analysis of signaling networks governing embryonic stem cell fate decisions in silico experimentation of glioma microenvironment development and anti-tumor therapy development of a three-dimensional multiscale agent-based tumor model: simulating gene-protein interaction profiles, cell phenotypes and multicellular patterns in brain cancer further reading silico' simulation of biological processes silico toxicology: principles and applications multiscale cancer modeling silico immunology silico: d animation and simulation of cell biology with maya and mel algorithm any well-defined computational procedure that takes some values, or set of values, as input, and produces some value, or set of values, as output. bioinformatics bioinformatics is the application of statistics and computer science to the field of molecular biology. biotechnology the exploitation of biological processes for industrial and other purposes. data structures a way to store and organize data on a computer in order to facilitate access and modifications. genome the complete set of genetic material of an organism. genomics the branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes. gene ontology a major bioinformatics initiative to unify the representation of gene and gene product attributes across all species. informatics the science of processing data for storage and retrieval; information science. in silico in silico is an expression used to mean "performed on a computer or via computer simulation." in vivo in microbiology in vivo is often used to refer to experimentation done in live isolated cells rather than in a whole organism. in vitro in vitro studies in experimental biology are those that are conducted using components of an organism that have been isolated from their usual biological surroundings in order to permit a more detailed or more convenient analysis than can be done with whole organisms. kinomics kinomics is the study of kinase signaling within cellular or tissue lysates. oncogenesis the progression of cytological, genetic, and cellular changes that culminate in a malignant tumor. pathophysiology the disordered physiological processes associated with disease or injury. proteomics the branch of genetics that studies the full set of proteins encoded by a genome. sequencing the process of determining the precise order of nucleotides within a dna molecule. systems biology an inter-disciplinary field of study that focuses on complex interactions within biological systems by using a more holistic perspective. . the template-based transcriptional control network reconstruction method exploits the principle that orthologous proteins regulate orthologous target genes. in this approach, regulatory interactions are transferred from a genome (such as a genome of a model organism or well studied organism) to the new genome. . the ultimate goal of in silico modeling in biology is the detailed understanding of the function of molecular networks as they appear in metabolism, gene regulation, or signal transduction. there are two major challenges in modeling infectious diseases: a. difficulty in finding the most appropriate set of parameters for the in silico modeling of infectious diseases is often a challenge. b. understanding the results from all the complex interactions of parameters considered. . there are three types of cancer models. continuum models: in these models extracellular parameters can be represented as continuously distributed variables to mathematically model cell-cell or cell-environment interactions in the context of cancers and the tumor microenvironment. discrete models: these models represent cancer cells as discrete entities of defined location and scale, interacting with one another and external factors in discrete time intervals according to predefined rules. hybrid models: these models incorporate both continuum and discrete variables in a modular approach. there are three types of parameters considered for in silico modeling of infectious diseases: a. parameters derived from characteristics of agent: examples: concentration of the agent's antigen-host antibody complex; case fatality rate; strain of the agent; other genetic information of the agent; etc. examples: the total white blood cell counts; differential white blood cell counts, and/or much more sophisticated counts of specific blood cell types; blood levels of some specific cytokines, hormones, and/or neurotransmitters; daily calories, protein, and/or fat intake; daily amount of energy expended and/or duration of exercise; etc. c. parameters derived from characteristics of environment: examples: host's ambient temperature; host's ambient atmospheric humidity; altitude; host's lightdark cycle; etc. key: cord- - u idlon authors: tong, joo chuan; ranganathan, shoba title: infectious disease informatics date: - - journal: computer-aided vaccine design doi: . / . sha: doc_id: cord_uid: u idlon abstract: throughout history, infectious diseases have posed a serious burden to mankind. more recently, there has been an alarming increase in drug-resistant microbes. furthermore, new pathogens are emerging due to microbial evolution and adaptation. the spread of these diseases is a result of pathogen mutations and changes in human behavior patterns. then, there are diseases that are lurking in the background, waiting for the right conditions before they strike again. in the war against these diseases, we have come to understand the behaviors of microbes in a heterogeneous world and the mechanisms governing disease transmission. these works have profoundly shaped modern knowledge of emerging and re-emerging infections. more recently, computational techniques have led the way into this new era by allowing rapid high-throughput analysis of pathogens which was previously not possible using traditional laboratory techniques. this chapter introduces methods in mathematical modeling, computational biology, and bioinformatics that have been used to study infectious diseases. abstract: throughout history, infectious diseases have posed a serious burden to mankind. more recently, there has been an alarming increase in drug-resistant microbes. furthermore, new pathogens are emerging due to microbial evolution and adaptation. the spread of these diseases is a result of pathogen mutations and changes in human behavior patterns. then, there are diseases that are lurking in the background, waiting for the right conditions before they strike again. in the war against these diseases, we have come to understand the behaviors of microbes in a heterogeneous world and the mechanisms governing disease transmission. these works have profoundly shaped modern knowledge of emerging and re-emerging infections. more recently, computational techniques have led the way into this new era by allowing rapid highthroughput analysis of pathogens which was previously not possible using traditional laboratory techniques. this chapter introduces methods in mathematical modeling, computational biology, and bioinformatics that have been used to study infectious diseases. epidemics, pandemics, and outbreaks of infectious diseases are regular features of life on earth. in bc , thucydides described the very fi rst pandemic in recorded history -the athenian plague that reportedly killed up to one-half of the citizens of athens. in ad - , an outbreak occurred in the byzantine empire, causing deaths every day. the outbreak, named the justinian plague after the reigning emperor justinian i, resulted in over million deaths and wiped out nearly half the inhabitants of the city. in - , the plague returned to europe under the name of the black death, killing up to % of the continent's population. in march , an infl uenza outbreak was fi rst reported in a us military camp in kansas. the outbreak, later known as the "spanish fl u," subsequently spread and infected up to a billion people, or half the world's population at the time, causing some million deaths within six months. all over the world, changes in socio-economic, demographic and environmental factors brought about by urbanization and industrialization have led to the resurgence of old and new infectious diseases. over the past years, there has been an alarming increase in drug-resistant microbes in diseases such as malaria and tuberculosis. furthermore, the world is also witnessing the emergence of more new pathogens due to microbial evolution and adaptation. then, there are diseases that are lurking in the background, waiting for the right conditions before they strike again. in , west nile virus re-emerged in new york and spread across the united states to long island, connecticut, maryland, florida, california, arizona, and colorado, with over reported cases and associated deaths within a span of fi ve years. previously known to be a mild disease, the re-emergence of epidemic chikungunya virus (chikv) in africa, indian ocean, south-east asia, pacifi c, north america, and europe in the past decade has caused severe morbidity with some cases of fatality. in april , a new strain of human infl uenza a (h n ) virus containing genes from human, swine and avian infl uenza a viruses emerged in mexico. over the course of one year, the virus had spread to more than countries and overseas territories or communities, causing more than deaths. more recently, in january , a human case of avian infl uenza a (h n ) virus infection was reported in china. if history is our guide, we can assume that the threat of these diseases will continue to grow and pose a serious problem to the security of countries worldwide. similarity between related sequences can give clues to the structure, function, or homology to the common ancestor. computational methods that can compare sequence features are, therefore, particularly useful. sequence alignment is the determination of residue-residue correspondences between two or more character strings, usually preserving the relative order. this method allows us to measure similarity and infer evolutionary relationships between two or more sequences. pairwise sequence alignment is useful for analyzing the degree of similarity between two biological sequences. where more than two sequences are involved, multiple sequence alignment can be used to identify regions of similarity that may help explain functional and/or phenotypic variability. the h n fl u was not the fi rst human pandemic caused by infl uenza a viruses. it is related to the russian fl u that killed ∼ million people, the spanish fl u that infected ∼ % of the global population and killed at least million people worldwide, the asian fl u that resulted in ∼ million deaths and the hong kong fl u that caused ∼ million deaths. in cases where the ancestry is unclear, sequence alignment methods can be used to infer their phylogenetic relationships. this includes: ■ identifying globally optimal alignment solutions for studying highly conserved sequences; ■ identifying maximally homologous subsequences among sets of long sequences for detecting distantly related proteins. in information theory and computer science, four types of metrics are commonly used to measure the edit distance between two strings of characters. they include: ■ the hamming distance, which is the number of positions with mismatched characters between two strings of the same length. ■ the levenshtein distance, which is the minimum number of operations that is needed to transform one string into the other, which may be of different length. an operation can be a deletion, insertion, or substitution of a single character in the strings. ■ the damerau-levenshtein distance, which is the minimum number of operations that is needed to transform one string into the other, which may be of different length. an operation can be a deletion, insertion, or substitution of a single character, or a transposition of two adjacent characters in the strings. ■ the jaro-winkler distance, which is a measure of similarity between two strings using the jaro distance metric. this method fi rst identifi es the common characters between two strings of characters. two characters are common if there is an exact match and if the difference in positions between the two strings is less than half the length of the shorter string. once all the common characters are determined, the number of transpositions of common characters are determined and used to compute the jaro similarity. strings that are more similar will have a higher jaro distance. in biological systems, certain amino acid changes are more likely to occur than others. for example, a hydrophobic residue is more likely to be replaced by another hydrophobic residue than a hydrophilic residue. to account for such transformations, a weight can be assigned to the different edit operations. this can take the form of a matrix that shows the substitution frequencies of observed pairs of amino acid residues. two popular substitution matrices are: ■ the percent accepted mutation (pam) matrices by dayhoff, which measure sequence similarity in closely related species. two sequences pam apart have an average of one accepted point mutation event per amino acids. they need not be % identical, as two point accepted mutations can occur at the same position. to analyze sequences that are more divergent, we can use the pam matrix as a base for calculating other matrices. this is based on the assumption that repeated mutations would follow the same pattern as those in the pam matrix. ■ the block substitution matrix (blosum) matrices by henikoff and henikoff, which measure sequence similarity in divergent sequences. the matrices are constructed from the blocks database of aligned conserved regions in divergent protein families. these regions are assumed to be of functional importance. once the substitution matrix is selected, the optimal alignment can be found using dynamic programming algorithms. a related concept is the use of theoretical statistics, such as information entropy, to quantify the rate of information transfer in biological sequences. the shannon entropy is a measure of uncertainty that is associated with a random variable. it is commonly used to assess the variability of microbial proteomes and epitope sequences. for a given alignment, the information content (i.e. entropy) of an amino acid position h ( x ) is defi ned by: where x is one of amino acid residue types. p ( x ), the probability of occurrence of x , is estimated by f ( x ), the frequency of the appearance of residue type within the alignment column: where n ( x ) is the number of appearances of amino acid residue x , and l is the length of the column. this method has been used to analyze the genetic diversity and antigenic relationships of chikungunya virus (chikv) proteomes from its introduction in to . antigenic switches refer to changes in gene expression at a specifi c site which may abrogate binding to hla molecules or antagonize/ interfere with t cell response, leading to cellular immune evasion. the study suggested that chikv is undergoing mild positive selection, with signifi cant amounts of "antigenic switches" clustered over the entire genome. an effective way to identify amino acid residues that are involved in virus adaptation is to fi nd interdependencies between mutations in multiple proteins. a simple way to do this is to calculate mutual information (mi) between variable pairs. mi is an information theoretical statistic that measures the strength of association between a pair of variables. the mutual information between two variables a and b is defi ned by: the evolutionary inertia of a pathogen can be qualitatively examined by studying the nucleotide usage patterns at single amino acid sites. the neutral theory of molecular evolution by kimura in states that most evolutionary changes at the molecular level are caused by random genetic drift of selectively neutral nucleotide substitutions. due to the degeneracy of the genetic code, some point mutations are silent with no amino acid replacements. silent or synonymous substitutions are primarily transparent to natural selection, whereas replacement or non-synonymous substitutions may be a result of strong selective pressure. a simple method to calculate the extent of adaptive evolution at highly variable genetic loci is to compare the fi xation rates between nonsynonymous (d n ) and synonymous (d s ) substitutions. the d n /d s ratio ( ω ), otherwise known as the "acceptance rate," provides a sensitive measure of selection pressure at the amino acid level. ω = indicates neutral expectation, ω < suggests negative (purifying) selection, while ω > suggests positive (diversifying) selection. a group of genes that often show the ω > relationship are antigenic genes in human immunodefi ciency virus- , plasmodia, and other parasites. the hemagglutinin gene from infl uenza a virus is probably one of the fastest evolving genes in terms of the rate of nucleotide substitution, which was estimated at . × − per site per year. this high genetic variation confers a fi tness advantage to the pathogen in its attempt to evade host defenses. the simple counting method of nei and gojobori is commonly used for estimating d n and d s . however, the reliability of this technique is low when the rate of transitional nucleotide change is higher than that of transversional change. the model-based maximum likelihood (ml) methods such as those proposed by muse and gaut and goldman and yang represent a viable and widely used alternative for this purpose. the original ml model of goldman and yang assumes a single ω for all lineages and sites, and has been extended to account for variation by allowing ω to vary either across lineages, among substitution sites, or both among sites and among lineages. lineagespecifi c models assume that ω do not vary among sites, and can detect positive selection for a lineage only if the averaged d n over all sites is greater than the average d s . site-specifi c models, on the other hand, allow ω to vary among sites but not among lineages. as such, these models can detect positive selection at individual sites only if the averaged d n over all lineages is greater than the average d s . by allowing ω to vary both among sites and among lineages, the method can be applied to detect positive selection that occurred at a few time points and affects a few sites. upcoming challenges for multiple sequence alignment methods in the high-throughput era founder effects in the assessment of hiv polymorphisms and hla allele associations prediction and entropy of printed english hla class i restriction as a possible driving force for chikungunya evolution complete-proteome mapping of human infl uenza a adaptive mutations: implications for human transmissibility of zoonotic strains mining mutation chains in biological sequences unifying the epidemiological and evolutionary dynamics of pathogens selection-driven evolution of emergent dengue virus a method for detecting positive selection at single amino acid sites adaptsite: detecting natural selection at single amino acid sites evolutionary rate at the molecular level selectionism and neutralism in molecular evolution molecular evolution of mrna: a method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its applications sequence relationships among the hemagglutinin genes of subtypes of infl uenza a virus simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions a likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome a codon-based model of nucleotide substitution for protein-coding dna sequences a maximum likelihood method for detecting directional evolution in protein sequences and its application to infl uenza a virus codon-substitution models for detecting molecular adaptation at individual sites along specifi c lineages key: cord- - xkaa authors: sahu, govind prasad; dhar, joydip title: dynamics of an seqihrs epidemic model with media coverage, quarantine and isolation in a community with pre-existing immunity date: - - journal: journal of mathematical analysis and applications doi: . /j.jmaa. . . sha: doc_id: cord_uid: xkaa abstract an autonomous deterministic non-linear epidemic model seqihrs is proposed for the transmission dynamics of an infectious disease with quarantine and isolation control strategies in a community with pre-existing immunity. the model exhibits two equilibria, namely, the disease-free and a unique endemic equilibrium. the existence and local stability of the disease free and endemic equilibria are explored in terms of the effective reproduction number r c . it is observed that media coverage does not affect the effective reproduction number, but it helps to mitigate disease burden by lowering the number of infectious individuals at the endemic steady state and also lowering the infection peak. a new approach is proposed to estimate the coefficient of media coverage. using the results of central manifold theory, it is established that as r c passes through unity, transcritical bifurcation occurs in the system and the unique endemic equilibrium is asymptotically stable. it is observed that the population level impact of quarantine and isolation depend on the level of transmission by the isolated individuals. moreover, the higher level of pre-existing immunity in the population decreases the infection peak and causes its early arrival. theoretical findings are supported by numerical simulation. sensitivity analysis is performed for r c and state variables at endemic steady state with respect to model parameters. mathematical modeling has become an important tool in analyzing the spread and control of infectious diseases taking into account the main factors governing development of a disease, such as transmission and recovery rates. mathematical models are being used to predict how the disease will spread over a period of time. in recent years, many attempts have been made to develop realistic mathematical models for investigating the transmission dynamics of infectious diseases, and the asymptotic behaviors of these epidemic models are studied [ , , , ] . vaccination and antiviral drugs are the two most effective pharmaceutical interventions used for the control of an infectious disease. but due to strain's novelty most people may lack innate immunity to the disease, available vaccine may not provide protection against the pathogen and effective antiviral drug may not be available in sufficient amount. for example, bird flu viruses h n and h n that have sporadically infected humans, could, with a few mutations to a key protein on their surface, become capable of infecting cells along the human upper airway and thereby take a step towards turning into pandemic-causing strains [ , ] . in that scenario, the role of isolation, quarantine and other non-pharmaceutical interventions stimulated by media coverage becomes more significant as disease control strategies. despite initial concern that little protective immunity existed in the general population for pandemic h n ( ), subsequent epidemiological data showed that morbidity in the elderly was lower than that in younger individuals, suggesting the existence of pre-existing immunity [ , , , ] . the centers for disease control and prevention (atlanta, ga, usa) reported that among persons > years old, % have pre-existing, cross-reactive neutralizing antibodies against the new virus of pandemic (h n ) [ ] . phylogenetic analyses on the ha of the pandemic h n virus demonstrated its close relationship with the - spanish h n virus [ , ] . low virulence of the virus and pre-existing immune status are among the main factors that account for lower death rates in influenza outbreaks [ ] . pre-existing immunity is an important factor countering the pandemic potential of an emerging infectious disease. thus, studying of pre-existing immunity will advance our understanding of the pathogenesis and disease dynamics of the emerging pathogen. when an infectious disease breaks out in a population, people's response to the threat of the disease is dependent on their perception of risk, which is affected by public and private information disseminated widely by the media [ , , ] . media coverage about an epidemic gives a sense about the risk level and the relative need for precautions in risk areas and encourage the public to take precautionary measures against the disease such as wearing masks, avoiding public places, avoiding travel when sick, frequent hand washing, etc. massive news coverage and fast information flow can generate a profound psychological impact on public health [ ] . this is extremely important in the early stages of an epidemic, when pharmaceutical interventions are not often possible because treatment or vaccination options have not yet been developed [ ] . media coverage and education may reduce the contact rate of human beings and the use of npis may reduce the transmission probability. many researchers investigated the impact of media awareness using mathematical modeling [ , , , , , , , , [ ] [ ] [ ] , , , [ ] [ ] [ ] , , , , ] . one method is to form an information set summarized by a new state variable, mostly based on the publicly available information on both present and on the recent past spreading of the disease [ , [ ] [ ] [ ] ] . the population is broadly classified as educated and non-educated to reflect the awareness through media coverage [ ] [ ] [ ] ] possible multiple outbreaks and sustained periodic oscillations of the infection were observed. cui et al. used similar transmission coefficient and established that multiple positive equilibria are possible when the media effect is sufficiently strong [ ] . reduction function of the negative exponential form to describe the reduction factor either by large number of infectious cases or by significant change in the number of infectious cases is used to model the media coverage [ , ] . cui et al. [ ] proposed a general contact rate, β(i) = c − c f (i), to reflect some intrinsic characters of media coverage. using the same contact rate proposed by liu and cui [ ] , tchuenche et al. [ ] studied the impact of media coverage on the spread and control of an influenza strain. sun et al. [ ] used similar non-linear contact rate as in [ ] to study media-induced social distancing in a two patch setting. li and cui [ ] studied the effect of constant and pulse vaccination on sis epidemic models incorporating media-induced incidence function similar as in [ , ] . emerging infectious diseases have devastating impacts on public health and impose great financial burden on the community, which attracts a major concern to public health agency. so it is of great importance to evaluate optimal methods for controlling these diseases [ ] . historically, quarantine (of individuals feared exposed to a communicable disease) is one of the oldest public health control measures for the spread of communicable diseases. these measures have been successfully applied dating back to the plague epidemic of the th century, to the influenza epidemics of the th century. more recently, this measure was successfully used to combat the spread of some emerging and re-emerging human and animal diseases, such as the severe acute respiratory syndrome (sars) [ , , , , , ] , foot-and-mouth disease [ ] and the swine influenza pandemic [ ] . the sars outbreaks of provided an important example of a novel disease that was effectively controlled using quarantine and isolation [ , , ] . implementing these measures, however, can inflict significant socio-economic and psychological costs. public health officials need to be able to present comprehensive, understandable assessments of the options to other government officials in a timely manner [ ] . for the purpose of this study, quarantine means the removal of individuals suspected of being infected (yet exhibiting no clinical symptoms) from the general population. thus, these individuals could be asymptomatically-infected or susceptible. on the other hand, isolation refers to the removal of infected individuals exhibiting clinical symptoms of the disease. although isolation is probably always a desirable public health measure, quarantine is more controversial. mass quarantine can inflict significant social, psychological, and economic costs without resulting in the detection of many infected individuals [ ] . isolation is primarily used for controlling the disease when it suddenly emerges or reemerges [ ] . a successful example is the isolation of those infected with sars during - . however, the disadvantages of this strategy are the difficulty of detecting infected individuals and the cost of isolation. in general, to achieve perfect isolation is difficult at large scale resulting in a leaky isolation causing nosocomial infections. numerous mathematical modeling works have been carried out to assess the impact of quarantine and isolation in controlling the spread of communicable diseases in human and animal populations [ , , , , , , , [ ] [ ] [ ] [ ] , , ] . further, the emergence of sars in led to the formulation of numerous quarantine and isolation models for curtailing its spread [ , , , , ] . most of the disease modeling studies, published in the literature, provided quantitative evaluation of the control measures (quarantine and isolation) by simulating the models with available epidemiological and demographic data [ , , , , ] . the primary goal of this article is to theoretically study the impact of use of npis stimulated by media coverage, quarantine and isolation for an infectious disease in a community with pre-existing immunity. the rest of the paper is organized as follows: in section , the proposed model in formulated. in section , existence and local behavior of disease free (dfe) and endemic equilibria are explored. global stability of dfe and uniform persistence is established. in section , important thresholds are calculated. numerical simulation is performed in section . sensitivity analysis is performed for effective reproduction number and steady states at endemic level with respect to model parameters in section . finally, results are discussed in section . in this section, we will formulate an epidemic model incorporating quarantine, isolation, use of nonpharmaceutical interventions stimulated by media coverage in presence of pre-existing cross-protective immunity. the total population at time t , denoted by Ñ (t), is sub-divided into six mutually exclusive compartments of susceptible (s(t)), exposed (Ẽ(t)), quarantined (q(t)), infectious (Ĩ(t)), hospitalized (h(t)) and recovered (r(t)) individuals, so that Ñ (t) =s(t) +Ẽ(t) +q(t) +Ĩ(t) +h(t) +r(t). in the modeling of infectious diseases, the incidence function plays a very important role, it can determine the rise and fall of epidemics [ ] . in many epidemic models, the bilinear incidence rate βsĨ and the standard incidence rate βsĨ /Ñ are frequently used, where β measures the effect of both the infectiousness of the disease and the contact transmission rates. however, these incidence functions do not consider the impact of media coverage to the spread and control of infectious diseases. the media induced transmission rate β (Ĩ) =βe −mĨ , used in [ , ] , has two limitations. first, β e −mĨ → as Ĩ → ∞, independent of the value of m. it is not reasonable since the media coverage is not the intrinsic deterministic factor responsible for the transmission and hence the transmission rate cannot be reduced below a certain level merely through media awareness and alert. second, even for a fixed m, the minimum transmission rate differs for different population sizes, regardless the similarity in social structure (i.e., education and awareness level) and climatic condition. we propose media induced transmission rate as β (Ĩ) =βe −mĨ n , which is more reasonable than that used in [ , ] overcoming the aforementioned limitations (see fig. ). keeping in view the above, in our proposed model we choose the media induced transmission rate of the form β e −m Ĩ n −m h n . the parameters m and m represent the coefficients of media coverage using nonpharmaceutical interventions corresponding to infectious (Ĩ) and isolated (h) individuals, respectively. now, the next important question is how to measure the media coefficient m. one innovative way of estimation of the media awareness coefficient m will be discussed in numerical simulation section . other modeling assumptions are as follows: the population grows at constant rate Λ. population in all the compartments decreases at rate μ due to natural death. susceptible individuals acquire infection through effective contact with infectious individuals at rateβ the parameter β is the effective contact rate (that is, contact capable of leading to infection), while the modification parameter, < η < , accounts for the assumed reduction in disease transmission by isolated individuals in comparison to non-hospitalized infectious individuals in the Ĩ class. thus, η measures the efficacy of isolation or treatment given to hospitalized individuals (isolation is perfect if η = ; leaky if < η < and completely ineffective if η = ). the schematic flow diagram of the proposed model is shown in fig. . based on the aforementioned modeling assumptions, the proposed model is govern by the following system of ordinary differential equations: table description of parameters for the system ( )- ( ). contact rate (in absence of npis through media coverage) days − η modification parameter for reduction in infectiousness of hospitalized individuals m , m coefficients of media coverage corresponding toĨ andh σ progression rate from exposed to infectious class days − ξ recovery rate due to pre-existing cross-protective immunity days − k quarantine rate for exposed individuals days − α hospitalization rate for quarantined individuals days − φ hospitalization rate for infectious individuals days − γ recovery rate for non-hospitalized infectious individuals days − γ recovery rate for hospitalized infectious individuals days − /θ average waning period of disease-induced immunity days δ disease-induced death rate for non-hospitalized infectious individuals days − δ disease-induced death rate for hospitalized individuals with initial conditions: descriptions of all the parameters are summarized in table . note that we consider only solutions with initial conditions inside the biologically feasible region Γ = (s,Ẽ,q,Ĩ,h,r) ∈ r + : ≤s,Ẽ,q,Ĩ,h,r,s +Ẽ +Ĩ +q +h +r ≤ Λ μ in which the usual existence, uniqueness of solutions and continuation results hold. we study the system ( )- ( ) and claim that the region Γ is bounded and positively invariant with respect to the proposed system ( )- ( ). all the solution trajectories of system ( )- ( ) initiating inside Γ approach enter or stay within the interior of Γ . denote the non-negative cone in six-dimensional euclidean space. from the system ( )-( ), we observe that , r (t) are continuous functions oft. thus the vector field on each bounding hyperplane of r + is pointing inward direction of r + . hence all the solution trajectories initiating in r + will remain inside r + for all the time. this establishes the fact that r + is positively invariant for the system ( )- ( ) . also, the total population Ñ (t) satisfies dÑ dt = Λ −μÑ −δ Ĩ −δ h . then, dÑ dt < Λ −μÑ , applying birkhoff's and rota's theorems on differential inequality [ , ] , as t → ∞, we have ≤Ñ (t) ≤ Λ μ =Ñ . therefore the solution of system ( )-( ) is bounded and hence any solution of the system originated from Γ remains in Γ . we reduce the above system into non-dimensional form using the equivalent non-dimensional system is given by: where β =β μ , and the initial conditions: in the following sections, we will study the dynamical behavior of the system ( )- ( ) with initial condition ( ) . in this section, we calculate all feasible steady states and the basic reproduction number for the system. observe that the biologically feasible region for the non-dimensional system is which is positively invariant for the system ( )-( ). we consider only solutions with initial conditions inside the region Ω. the system ( )-( ) always has the disease-free equilibrium (dfe) (e = ( , , , , , )). the local stability of dfe e will be explored using the effective reproduction number r c . the non-negative matrix f, of the new infection terms, and the matrix v, of the remaining terms are given, respectively, by the corresponding linearized matrices evaluated at the dfe e are respectively. it follows that the effective reproduction number r c = ρ(fv − ), where ρ is the spectral radius, is given by it is worth mentioning that in the absence of a combined quarantine and isolation program (k = , α = , φ = , δ = , γ = ), the effective reproduction number reduces to the basic reproduction number . using theorem in [ ] , one can establish the following result. theorem . . the disease-free equilibrium of the system ( )- ( ) is locally-asymptotically stable if r c < , and unstable if r c > . biologically speaking, r c represents the average number of secondary infections produced by a typical infected individual in a community that adopts isolation and quarantine programs. the epidemiological implication of this result is that if r c < , then the influx of a few infected individuals will not generate large outbreaks (and the disease will die out). disease outbreak will occur if r c > . the possible endemic equilibria of proposed model are derived by solving the system of non-linear equations obtained from the system ( )-( ) equating the derivatives to zeroes. the endemic equilibrium Ē = (e * , q * , i * , h * , r * , n * ) of the model ( )-( ) is given by the value of i * is given by the solution of the equation in case, there is no media effect, i.e., m = , we get . it follows that i * exists at positive level (and so as the unique endemic equilibrium Ē ) if and only if r c > . otherwise, the value of i * is given from eq. ( ) . now we establish the existence of i * for r c > though graphical approach. in figs. and , we plot the curve e mi * r c and straight line − b i * against i * in the range [ - ]. note that since b > , we have /b < . from fig. , it follows that there is no point of intersection when r c ≤ , resulting the non-existence of the endemic equilibrium, but as r c > , i * exists uniquely at positive level and hence the unique endemic exists in this case (see fig. ). from the above discussion we conclude that theorem . . the system ( )- ( ) has no endemic equilibrium for r c ≤ , but has a unique endemic equilibrium Ē if r c > . now we will state and prove the local stability of the endemic equilibrium in the following theorem: the endemic equilibrium Ē is locally asymptotically stable for r c > , but close to . proof. the jacobian matrix j at dfe is given by here, we use the method based on the central manifold theory to establish the local stability of endemic equilibrium taking β as bifurcation parameter [ ] . a critical value of bifurcation parameter β at r c = is given as it can be easily verified that the jacobian j at β = β c has a right eigenvector (corresponding to the zero eigenvalue) given by w = (w , w , w , w , w , w ) t , where × γ ( + γ + δ )σ + γ σφ + ( + γ + δ )ξ( + γ + δ + φ) and furthermore, the components of the left eigenvector (corresponding to the zero eigenvalue), v = (v , v , v , v , v , v ), must satisfy the equalities v.j = and v.w = , so that we obtain use the notations substituting the values of all the second order derivatives evaluated at dfe and β = β c , we get and finally, substituting the values of v and w, we obtain where since a < and b > at β = β c , therefore using theorem . and remark stated in [ ] , a transcritical bifurcation occurs at r c = and the unique endemic equilibrium is locally asymptotically stable for r c > . in this section, we analyze the global stability of the disease-free steady states for a special case. we state the following theorem: theorem . . suppose r c < and δ = δ = , then the disease-free equilibrium e is globally asymptotically stable. proof. here, we prove global stability of dfe applying the method used in [ ] . when δ = δ = , we have dn dt = − n . then n → as t → ∞. take the n in the limiting case, i.e., n = , then the system ( )- ( ) reduces to let x = (r) and z = (e, q, i, h), here u = (x , z ), where x = ( ) and z = ( , , , ). we have clearly, b is an m-matrix. for i ≥ , h ≥ , we have < e −m i−m h ≤ , therefore g(x, z) ≥ since ≤ s ≤ . thus both the conditions (h ) and (h ) are satisfied. hence, the dfe e is globally asymptotically stable if r c < . now, we explore the uniform persistence for the system ( )- ( ) . again, the system ( )-( ) is said to be uniformly-persistent if there exists a constant c such that any solution (e(t), q(t), i(t), h(t), r(t)) satisfies . similar as in [ ] , we can state the following theorem for persistent: theorem . proof. from theorem . , the dfe of the model ( )-( ) is unstable whenever r c > . apply the uniform persistence result stated in [ ] , finally it can be proved in a similar manner as proposition . of [ ] . the consequence of this result is that in limiting case, all the infected state variables e, q, i and h of the model will remain above a certain positive threshold and the disease will persist in the population. in this section, the effect of quarantine and isolation on the transmission dynamics of the disease is measured qualitatively. a threshold analysis on the parameters associated with the quarantine of exposed individuals (k) and the isolation of the infected individuals (φ) is performed by computing the partial derivatives of the effective reproduction number r c with respect to these parameters. we observe that so that, from the above analysis it is clear that if the relative infectiousness of the hospitalized individuals (η) does not exceed the threshold value η k , then quarantining of exposed individuals results in reduction of the effective reproduction number r c and therefore, reduction in disease burden (new infections, hospitalization etc.). on the other hand, if η < η k , then due to increase in the rate of quarantine, the effective reproduction number r c will increase and consequently, the disease burden also increases. thus, the use of quarantine is detrimental in this case. the result is summarized as follows: theorem . . for the model ( )- ( ) , the use of quarantine of the exposed individuals will have positive similarly, the impact of isolation of infectious individuals is assessed by calculating the partial derivatives of r c with respect to the isolation parameter φ. thus, we obtain ∂r c ∂φ = βσ(η(γ + δ + ) − ( + γ + δ )) (γ + δ + )(γ + δ + φ + ) (k + ξ + σ + ) . table parameter values used in the simulation for the system ( )- ( ) . ∂r c ∂φ < (> ) iff η < η φ (η > η φ ), nominal values (per day) therefore, the use of isolation of infected individuals will be helpful to control the disease in the community if the relative infectiousness of the hospitalized individuals (η) does not exceed the threshold η φ . the result is summarized below: hence, we conclude that the combine use of quarantine of exposed individuals and isolation of individuals with symptoms will have positive population-level impact if and only if the quarantine and isolation strategies will have negative population-level impact if the effective reproduction number r c is a decreasing (non-decreasing) function of the quarantine and isolation parameters k and φ if condition ( ) [ ( )] is satisfied (see figs. and obtained from simulation of the model in which the results are consistent with the analytical findings discussed earlier). in this section, we provide numerical simulations to illustrate previously established results with the biological feasible parametric values as shown in table case the disease-free equilibrium is las (see fig. ). when β = . , the effective reproduction number r c = . > , the unique endemic equilibrium exists and is locally asymptotically stable as shown in fig. . whenever r c < , the system ( )- ( ) has no endemic equilibrium and the dfe of the model with δ =δ = is gas in Ω, from theorem . . fig. depicts the numerical experimentation of the model ( )-( ) taking δ =δ = , with β = . and r c = . < , for different initial infected population. from the figure it is clear that all solutions converge to the dfe (e ). similarly, for β = . , we get r c = . > , and all the solutions converge to the ee (Ē) as shown for infectious individuals i in fig. . the coefficients of media coverage m and m should depend on the disease under consideration, the social structure (education, awareness, responsiveness, economy, etc.) of the population and the npis used in a particular region. here, we use the formula m j = − log e (p + q j − pq j ), to quantify the coefficients m and m of media coverage, where q j quantifies the response of the population aware to media recommended npis with respect to the number of infective and the hospitalized individuals. if people are not responding to media alert, then q j = and if all the people are adopting the recommended npis, then q j = . it is assumed that the disease transmission rate can be reduced by p fraction when all individuals follow the it is observed from the analysis that the coefficients of media coverage m and m do not affect r c and the qualitative features of the model remain unaltered. from ( ) , we observe that a is always negative, which precludes the existence of backward bifurcation in the system and hence ensures transcritical (i.e., forward) bifurcation about r c = . hence in this case, the classical requirement of r c < is necessary and sufficient for disease control. moreover, from ( ) we observe that one can easily observe that the use of npis stimulated by media coverage helps to mitigate the disease burden from the environment by lowering the level of infectious individuals at steady state. the effect of m and m on the fraction of infectious individuals (i) is shown in figs. and taking β = . , and β = . , respectively and the rest of the parametric values are as in table . it is observed that the level of endemic equilibrium is significantly affected by media coefficients m and m . the pre-existing immunity in the population has significant role in the disease outbreak as it lowers the basic as well as effective reproduction numbers. the effect of pre-existing immunity parameter ξ on the fig. . it is clear form the figure that higher level of pre-existing immunity helps to reduce disease burden. the effectiveness of quarantine and isolation depends on the size of the modification parameter (η) for the reduction in infectiousness of hospitalized individuals. for β = . , the threshold value of η with respect to quarantine parameter k is η k = . and with respect to isolation parameter φ is η φ = . . from fig. , it is clear that quarantine k has positive population-level impact (r c decreases with increase in k) for η < . and have negative population level impact for η > . . similarly for η < . , isolation has positive level impact, whereas isolation has negative impact if η > . (see fig. ). in this section, we perform sensitivity analysis of effective reproduction number r c and endemic equilibrium taking parametric values given in table . sensitivity indices allow us to measure the relative change in a state variable/derived parameter when a model parameter changes. table the sensitivity indices, Υ r c y j = ∂r c ∂y j × y j r c , of the effective reproduction number r c to the parameters, y j , for parameter values given in table . definition. (see [ , ] .) the normalized forward sensitivity index of a variable, u, that depends on a parameter, p, is defined as: estimation of highly sensitive parameter should be done very carefully, because a small variation in the parameter will lead to relatively large quantitative change. on the other hand, a less sensitive parameter does not require as much effort to estimate, since a small variation in that parameter will not produce large change to the quantity of interest. the normalized sensitive indices of effective reproduction number r c with respect to parameters are shown in table . from table , we observe that β, η, σ, and α have positive impact on r c and the rest of the parameters have negative impact. for example, % increase (decrease) in σ, resulting in . % increase (decrease) in r c , on the other hand % increase (decrease) in γ , will decrease (increase) r c by . %. moreover, parameters β, δ and γ are most sensitive to r c , hence we observe significant change in r c by small changes in these parameters. again, we perform sensitivity analysis of state variables at endemic steady state with respect to model parameters. sensitivity indices of state variables at endemic equilibrium are shown in table using parametric values shown in table . from table , we observe that parameters β, η, δ , α, θ and σ have positive impact on i * and the rest of the parameters have negative impact. moreover, parameters φ, σ, β, γ , θ and δ are most sensitive parameters to i * , hence we observe significant change in i * by small changes in these parameters. an seqihrs epidemic model for the transmission dynamics of an infectious disease is proposed and rigorous mathematical analysis is carried out to get insight into the qualitative dynamics in presence of pre-existing immunity and the use of npis stimulated by media coverage. the main mathematical and epidemiological findings of the proposed model presented in this article are as follows: (i) the model ( )-( ) has a locally-asymptotically stable disease-free equilibrium whenever the associated effective reproduction number is less than unity (theorem . ). moreover, if the disease-induced death rates are neglected then the dfe is globally-asymptotically stable (theorem . ). (ii) the model has a unique endemic equilibrium whenever the effective reproduction number exceeds unity and then ee is locally asymptotically stable (theorems . and . ). (iii) the presence of pre-existing immunity (ξ) in the population has significant impact on the transmission dynamics of the disease. higher level of pre-existing immunity in the population decreases the infection peak and causes its early arrival. (iv) the coefficients of media awareness m and m do not affect the effective reproduction number r c . hence, it does not change the qualitative behavior of the model, but it helps to mitigate disease burden by lowering the level of infection over time. (v) the use of quarantine and/or isolation could have positive, no or negative population level impact depending on the relative infectiousness of isolated individuals (η). (vi) since disease transmission is directly related to the effective reproduction number, and the disease prevalence is directly related to the endemic equilibrium point Ē , specifically to the magnitude of i * , therefore normalized forward sensitivity indices of the effective reproduction number and i * will be helpful to determine decisive parameter(s) for containing the infectious disease. sensitivity indices of the effective reproduction number and i * are calculated and highly sensitive parameters are discovered. ordinary differential equations global stability of an sir epidemic model with information dependent vaccination on the computation of r and its role on global stability dynamical models of tuberculosis and their applications on the dynamics of a two-strain influenza model with isolation determining important parameters in the spread of malaria through the sensitivity analysis of a mathematical model the impact of media on the control of infectious diseases an sis 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(h n ) structural basis of preexisting immunity to the h n pandemic influenza virus optimal and sub-optimal quarantine and isolation control in sars epidemics analysis of an epidemic model with awareness programs by media on complex networks analysis of stability and bifurcation for an seiv epidemic model with vaccination and nonlinear incidence rate the authors are grateful to the anonymous reviewers for their helpful suggestions and comments that have improved the quality and presentation of the manuscript. key: cord- -tbdsr iw authors: carvalho, c.l.; lopes de carvalho, i.; zé-zé, l.; núncio, m.s.; duarte, e.l. title: tularaemia: a challenging zoonosis date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: tbdsr iw in recent years, several emerging zoonotic vector-borne infections with potential impact on human health have been identified in europe, including tularaemia, caused by francisella tularensis. this remarkable pathogen, one of the most virulent microorganisms currently known, has been detected in increasingly new settings and in a wide range of wild species, including lagomorphs, rodents, carnivores, fish and invertebrate arthropods. also, a renewed concern has arisen with regard to f. tularensis: its potential use by bioterrorists. based on the information published concerning the latest outbreaks, the aim of this paper is to review the main features of the agent, its biology, immunology and epidemiology. moreover, special focus will be given to zoonotic aspects of the disease, as tularaemia outbreaks in human populations have been frequently associated with disease in animals. seventy-five per cent of emerging infectious diseases are zoonotic [ ] . some wildlife species have been recognised as being major reservoirs for infectious diseases and the proximity of wildlife habitats and the existence of arthropod vectors with a wide geographical spread have rendered epidemiological cycles more complex [ ] . tularaemia is a zoonosis caused by the francisella tularensis bacterium, which was first isolated in in tulare county, california, by george mccoy and charles chapin [ ] [ ] [ ] . initially termed bacterium tularense, it was allocated to a new genus and named f. tularensis in honour of the pioneer of research on the organism, edward francis [ , ] . arthropod-borne transmission of tularaemia was first demonstrated by francis in when he isolated the etiologic agent in a patient with "deer fly fever" [ , , ] . tularaemia was recognised as an important disease in the last century and since then there has been a growth in enthusiasm for research on this pathogen [ , ] . interest has arisen with regard to f. tularensis as it has emerged in new locations, populations and settings, and increasingly figured in scientific research gauging its potential use in bioterrorism [ , ] . the european centre for disease control and prevention (ecdc) surveillance report refers a total of confirmed cases of tularaemia in a number of european countries in , with sweden reporting the highest confirmed case rate, followed by finland and hungary [ ] . tularaemia is considered an unusual disease and the confirmed case rate in europe has remained stable from to . recent outbreaks of tularaemia have occurred in several european countries, presented in table , including the czech republic, kosovo, bulgaria, germany, sweden, finland, spain, turkey, france and norway [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . besides these outbreaks, sporadic case notifications have occurred in austria, estonia, italy, lithuania, poland, romania, slovakia and the united kingdom [ ] . although there are no reports of tularaemia for denmark during this period, a confirmed case of the disease in a human was recorded there in [ ] . in portugal, the bacterium has been detected in the blood of an asymptomatic man and in a dermacentor reticulatus tick by molecular methods [ ] . f. tularensis is one of the most virulent microorganisms currently known, while as few as ten microorganisms can cause potentially fatal disease in man and animals [ , ] . this high rate of infectivity has led the centre for disease control and prevention (cdc) to classify f. tularensis as a category a biowarfare agent [ ] . f. tularensis is a gram-negative, catalase-positive, pleomorphic and non-motile cocobacillus, characterised as a facultative intracellular pathogen that can grow within different types of cells including macrophages, hepatocytes and epithelial cells [ , , , ] . the cell wall of f. tularensis has an unusually high level of fatty acids with a unique profile for the genus, and wild strains have a lipid-rich capsule, with neither toxic nor immunogenic properties [ , , ] . capsule loss has been related to a decrease in virulence, although the viability or survival of the bacterium within neutrophils may remain unaltered. f. tularensis is a gamma ( )-proteobacteria of the francisellaceae family [ , , ] . f. tularensis is the most common and pathogenic species and is formally divided into three subspecies with different pathogenicities and geographic distributions: tularensis, holarctica and mediasiatica. the species francisella novicida is currently widely accepted as a fourth subspecies of f. tularensis [ , , [ ] [ ] [ ] [ ] [ ] [ ] , as it shares with f. tularensis an average of . % nucleotide identity over a . mbp of genome sequence [ , , , ] . however, some objections to the transfer of f. novicida to the subspecies rank of f. tularensis have been recorded, based on recent multiple genome sequencing results, which show divergent evolutions for f. tularensis and f. novicida populations. therefore, separate species may be retained [ ] . the f. tularensis subspecies tularensis, regarded as the most virulent subspecies and classified as type a, occurs predominantly in north america [ , , , , ] . two distinct genetic sub-populations have been identified, ai and aii, which have different geographic distributions, hosts and vectors [ , , , , , ] . sub-population ai has been additionally sub-divided into groups aia and aib [ , , , ] . the subspecies holarctica, related to milder forms of the disease and classified as type b, occurs throughout the northern hemisphere [ , , , ] . human infection with aib strains usually have a fulminant clinical progression and are associated with high mortality rates, in contrast with infections by aia and aii strains or type b tularaemia [ , , ] . recently, this subspecies has also been detected in tasmania, australia [ ] . subspecies mediasiatica presents a similar virulence to subspecies holarctica, but its geographic distribution is restricted so far to central asia [ , ] . f. novicida is less virulent and has been isolated in north america, australia and thailand [ , , [ ] [ ] [ ] [ ] [ ] ] . based on a high degree of similarity between s rrna gene sequences, other microorganisms have been classified as probable members of the francisellaceae family; these include the francisella-like endosymbionts or fles [ , , ] . fles belong to a distinct phylogenetic clade from f. tularensis species [ ] . the effect of fles, if any, on vector competency and in the transmission of f. tularensis by ticks is still unknown [ ] . fles have a worldwide distribution and are vertically transmitted by hard and soft ticks of the genera amblyomma, dermacentor, ixodes and ornithodoros [ ] [ ] [ ] [ ] . fles have been detected in ticks in north america [ , , ] a information unavailable. (texas, california, minnesota), canada (alberta) and european countries such as spain, portugal, hungary, serbia and bulgaria [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . their pathogenicity to humans is undetermined. they have recently been detected in freeliving small mammals in europe, suggesting the possible transmission of some fle types from ticks to small mammals, although, to date, attempts to demonstrate it have failed [ , , , , ] . the phylogeographic distribution of f. tularensis is given in fig. ; the geographic locations where fles have been detected in ticks are also indicated. in nature, f. tularensis has been detected in a high number of wild species including lagomorphs, rodents, insectivores, carnivores, ungulates, marsupials, birds, amphibians, fish, and invertebrates [ , , , , [ ] [ ] [ ] . lagomorphs and rodents are considered as the main reservoirs of f. tularensis [ , , ] . wild lagomorphs, such as the european brown hare (lepus europaeus), are thought to be suitable sentinels for f. tularensis and disease surveillance [ , ] . recently, there have been serological evidences that foxes and raccoon dogs could also act as biological indicators for tularaemia [ ] . natural infections with f. tularensis have also been documented in different arthropods, although only a subset of these have been identified as important in f. tularensis transmission to humans. still, few pathogens show the adaptability of f. tularensis to such a wide range of arthropod vectors capable of infection dissemination [ ] . arthropod found infected in nature include ticks of the genera amblyomma, dermacentor, ixodes and ornithodoros, mosquitoes of the genera aedes, culex, anopheles and ochlerotatus excrucians, and flies from the tabanidae family (tabanus spp., chrisozona spp. and chrisops spp.) [ , , , ] . nevertheless, vector competence has only been demonstrated in ticks of the genera dermacentor [ ] . tick-borne transmission of f. tularensis usually results in sporadic cases, although occasional outbreaks have also been reported [ ] . although regarded as merely mechanical vectors, mosquitoes have been associated with widespread epidemics of tularaemia and are capable of transient disease transmission [ , ] . both ticks and mosquitoes may be infected in the larval phase. transtadial transmission has been demonstrated in ticks although in mosquitoes evidences for transtadial transmission are only based in molecular methods [ , ] . although transovarial transmission of f. tularensis in ticks was reported [ , , ] , a recent study in dermacentor variabilis has proved otherwise [ ] . despite dissemination to ovaries and then to the oocytes, the pathogen was not recovered from the subsequently hatched larvae. tabanid flies are regarded as mechanical vectors for f. tularensis and the long-term survival of this bacterium does not occur in these arthropods [ ] . the epidemiologic characteristics of vector-borne tularaemia vary throughout the northern hemisphere and also within a given geographic location. this is thought to be related to the abundance of different vectors and host species. this could explain why, in the usa, sweden, finland and russia, the arthropod bite is a common mode of transmission to humans, whilst in western and central europe, contact with infected animals and the ingestion of contaminated food or water have been reported as more common transmission modes. differences in transmission patterns have also been recorded within the usa: in western states, both ticks and deer flies are considered to be important vectors of tularémia, while in the east only ticks are considered relevant. in sweden and finland, mosquitoes have been identified as the primary vectors [ ] . in portugal, the role of ticks and small mammals in the transmission of tularaemia is still the subject of research. a collection of mosquitoes belonging to the genus culex ( . %), ochlerotatus ( . %), anopheles ( . %), culiseta ( . %) and a small number of aedes aegypti females from the island of madeira ( . %) have been analysed, although all the results were found to be negative [ ] . so far, this is in accordance with previous findings regarding the epidemiology characteristics of vector-borne tularaemia, suggesting that, in portugal, mosquitoes have no role in the transmission of this disease. ticks are thought to be the most important vectors of tularaemia in the majority of countries where tularaemia is endemic [ ] . nevertheless, major on-going research on tularaemia, aiming at gauging the overall impact of the disease in portugal, is expected to throw further light on the main f. tularensis sources. in endemic areas, tularaemia is a seasonal disease, with higher incidence in late spring, summer and autumn, occurring annually over a -year period or unreported for more than a decade. often, the number of cases varies widely from year to another, which is thought to be due to temperature or precipitation variability. however, the association between climactic conditions and tularaemia outbreaks has yet to be demonstrated [ ] . f. tularensis has been found to be extremely resistant to environmental stress, surviving for weeks in soil, water and animal carcasses, at low temperatures [ ] . human tularaemia outbreaks are often preceded by animal outbreaks, particularly in wild lagomorphs and rodents. this is usually related to an increase in the numbers of these species, increasing the probability of exposure to infected animals [ , , , ] . the transmission of tularaemia to humans can occur either by direct contact with infected animals or indirectly due to arthropod vector bites, the ingestion of contaminated water, food or aerosols inhalation. aerosols can be dispersed by ventilators, farming, and the deposition of contaminated hay, either intentionally or unintentionally [ ] . domestic dogs and cats can also transmit tularaemia to humans after contact with an infected animal, environment or infected ticks [ ] [ ] [ ] . personto-person transmission has not been described so far [ , , , ] . tularaemia has been reported to occur in any age group. men tend to present a higher prevalence than women [ , ] . professions that are prone to contact with reservoirs or arthropod vectors have been associated with a higher infection risk: these include laboratory technicians, hunters, farmers, veterinary surgeons, and anyone handling the flesh of infected animals [ , ] . few pathogens show the adaptability of f. tularensis to varying vector, host and environmental conditions. variations occur in local transmission cycles in association with differing ecologies. both f. tularensis type a and type b are associated with different life cycles in which different animal hosts and arthropod vectors intervene [ ] . type a tularaemia is more commonly associated with the terrestrial cycle of the disease, with wild lagomorphs such as rabbits and hares acting as vertebrate hosts in which amplification of the agent occurs and where arthropods are disease-disseminating vectors [ , , , ] . type b tularaemia is more frequently associated with the aquatic cycle, although outbreaks of tick-borne tularaemia involving subspecies holarctica have been reported [ , , ] . in this life cycle, f. tularensis circulates in rodents such as beavers, muskrats and voles, and can be introduced in water courses from animal carcasses [ , , , ] . there is also evidence that f. tularensis can persist in water courses in association with amoebas [ , , ] . contaminated water can be the source of infection to humans, flies and mosquitoes [ ] . an unusual waterborne outbreak of human tularaemia has been described in spain associated with crayfish (procambarus clarkii) caught in a contaminated freshwater stream. the crayfish acted as mechanical vectors, through mud-or water-contaminated carapaces, although the presence of f. tularensis in crayfish stomach and hepatopancreas could indicate their eventual role as hosts [ ] . a diagrammatic representation of the terrestrial and aquatic cycles of tularaemia is shown in fig. . f. tularensis is a remarkable bacterial pathogen that can invade and multiply in a wide range of cell types [ , , , , ] . antigen-presenting cells (apc) such as macrophages or dendritic cells, appear to be the primary cell types targeted by the bacterium at the outset of infection [ ] . the virulence of the bacterium is directly related to its capacity to replicate within the cytosol of infected cells [ ] . f. tularensis clearly possesses several mechanisms by which it manipulates immunity. the bacterium evades detection at the point of entry in the host in three ways: (a) it has modified cell-surface structures that enable it to avoid interaction with host receptors that are associated with the induction of inflammation; (b) it targets cells that lack co-receptors which facilitate binding to receptors that might alert the host cell to invasion; (c) it utilises receptors that fail to initiate the production of proinflammatory cytokines [ ] . the entry of f. tularensis in macrophages occurs by means of a specific mechanism inherent to francisella spp. [ ] . the bacterium induces the macrophage to produce asymmetric spacious pseudopod loops in a "looping phagocytosis" process [ , ] . uptake of f. tularensis is markedly enhanced by serum opsonisation, which depends on serum intact complement factor c and host cell receptors (cr ), involving bacterial surface polysaccharides [ , ] . utilisation of cr (and of mannose receptors of dendritic cells (mr) under non-opsonising conditions) is considered to be a fairly innocuous route for entry of f. tularensis, since it is not associated with the induction of signalling cascades that result in pro-inflammatory cytokines production. when opsonised by serum, f. tularensis binds ic b and gains entry to host cells via the cr receptor [ ] . the lipopolysaccharide (lps) of subspecies tularensis is only moderately inflammatory and acts as an extremely weak toll-like receptor (tlr) agonist stimulating a reduced production of pro-inflammatory cytokines [ , ] . these is attributed to the presence of only four acyl groups on the lps that do not bind to the "lps-binding proteins", subverting tlr recognition [ , , ] . in addition to lps, f. tularensis possesses two other tlr agonists [ ] : tul and ftt lipoproteins. these interact with tlr and may alert the host cell for the presence of the bacterium prior to phagocytosis [ , , ] . tlr /myeloid differentiation primary response gene ( ) (myd ) signalling is essential for the production of pro-inflammatory cytokines and is critical for host defence against francisella infection [ , , , ] . f. novicida has been used as a model organism to study immunity to f. tularensis. nevertheless, f. novicida expresses a structurally distinct chemotype of lps that is more pro-inflammatory in mice than the dominant lps chemotype, and is expected to result in different inflammasome activations [ ] . f. novicida escapes the phagossome and replicate in the cell cytosol where it is recognised by the inflammasome signalling system [ , , , ] . inflammasome stimuli activate the protease cysteine aspartate-specific caspase- , promoting the release of potent pro-inflammatory cytokines responsible for cell apoptosis [ , ] . this results in f. novicida release from infected cells and enables the infection of new ones [ , ] . f. tularensis survival and replication within macrophages is enabled by a large set of virulence genes that include the "macrophage growth locus" (mgl) a and b and the "francisella pathogenicity island", fpi [ ] . fpi encodes for a putative type vi secretion system [ , ] and contains genes that have been demonstrated as essential for intra-cellular growth and virulence [ ] . less virulent f. novicida presents only one copy of fpi in contrast with f. tularensis subspecies tularensis and holarctica that present two copies [ , ] . genes within the fpi are regulated by mgla [ ] . although current knowledge of the gene's functions is far from complete, this is one of the most active areas of francisella research [ ] . following phagocytosis of opsonised f. tularensis by polymorphonuclear cells (pmn), the bacterium actively inhibits superoxide anion generation (ros) via nadph oxidase. this allows f. tularensis to evade the phagosome and persist in the cell cytosol. the contribution of polymorphonuclear cells seems to be related to the secretion of cytokines and chemokines that recruit effector cells to the infection site [ ] . however, an excessive recruitment of neutrophils, modulated by an increase in metaloprotease- from the matrix, plays an important role in modulating leucocyte recruitment and seems to be directly related to f. tularensis pathogenesis [ , ] . natural killer (nk) cells from the liver, spleen and lung also play an important role in the innate immune response, in particular by producing inf-␣ following primary infection by f. tularensis [ ] . as f. tularensis is an intracellular pathogen, cellular immune response is believed to be the main defence mechanism. memory effector t cells cd + and cd + are clearly important for the primary control of infection. these cells produce type th cytokines like inf-␥, tnf-␣ and il- that are critical for the initial response to f. tularensis infection [ ] . although the role of humoral immunity in f. tularensis infection is believed to be less important, some studies have demonstrated the enhanced recovery of infected humans that have received hyper-immune serum [ ] . also, infection-specific igm, iga and igg antibodies produced are good exposition indicators and may interfere with the ability of bacteria to infect host cells [ , , ] . the contribution of b cells in defence is thought to be dependent on strain virulence [ , ] . research on anti-francisella antibodies targets is expected to allow for the identification of new diagnostic or reactive antigens and the development of vaccines [ ] . furthermore, f. tularensis is capable of influencing multiple pathways, and continued research into the specific mechanisms by which f. tularensis evades, modulates and suppresses the host immune response will improve our understanding of tularaemia pathogenesis and the regulation of host immunity [ ] . relevant clinical disease has been reported with f. tularensis subsp. tularensis and holarctica. clinical manifestations of tularaemia depend on strain virulence, infective dose and infection route, the extent of systemic involvement and host immune status [ , , ] . the incubation period averages - days but ranges from to days. the disease has an acute onset, with the occurrence of fever ( - • c), chills, fatigue, generalised myalgia and headaches, resembling a flu-like syndrome [ , , ] . the subspecies tularensis (type a) causes severe disease, potentially fatal if untreated. the subspecies holarctica (type b) causes less severe disease and fatalities are rare [ ] . depending on the route of infection, the following forms of the disease are described: ulceroglandular, glandular, oculoglandular, oropharyngeal, pneumonic, typhoidal and septic [ , ] . ulceroglandular and glandular forms of the disease are the most common and frequently result from an arthropod bite or animal contact [ , , ] . in ulceroglandular tularémia, a soft, painless ulcer develops at the inoculation site and evolves to a scar [ , ] . this presentation is associated with fever, lymphadenopathy and, in type a tularaemia, pneumonia and pleural effusion can occur [ ] . in glandular tularaemia, the primary ulcer is unrecognisable [ , , , ] . direct contamination of the eye through contaminated fingers, splashes or aerosols, may be followed by oculoglandular tularaemia. unilateral conjunctivitis, with ulcers or papules in some patients, photophobia and epiphora are the main signs of this form of the disease [ , ] . oropharyngeal tularaemia is acquired by means of contaminated food or water intake and aerosol inhalation [ ] . it develops with ulcerative and exudative stomatitis and pharyngitis [ ] . pneumonic tularaemia occurs by means of contaminated aerosol inhalation but can also arise as a complication of any of the other disease forms by haematogenous generalisation [ , , ] . initial disease development is characterised by fever, cough, pleuritic chest pain and dyspnoea, along with other unspecific symptoms. type a tularaemia is associated with significantly severer and more fulminant forms of pneumonia [ , ] . typhoidal tularaemia refers to a systemic and febrile form of the disease in which no route of infection acquisition can be established [ , ] . septic tularaemia is a severe and often fatal form of the disease that can occur as a complication of the ulceroglandular form in type a tularaemia [ , ] . patients can present unspecific and neurologic symptoms, and septic shock, sirs (systemic inflammatory response syndrome), dic (disseminated intravascular coagulation), haemorrhages, sars (severe acute respiratory syndrome) and multiple organ failure [ , ] . in type b tularaemia, complications of meningitis and septicaemia have only occasionally been described [ ] . clinical manifestations largely depend on the susceptibility of animal species to f. tularensis [ ] . in wild animals, clinical signs of tularaemia are not well documented, and post-mortem findings are highly unspecific and include splenomegaly and punctual necrotic lesions in the liver and spleen [ , ] . in one experimental study in european brown hares (lepus europaeus), clinical signs developed -day postinoculation with a f. tularensis subspecies holarctica strain. these included fever, lethargy and anorexia. two of the five hares in the study succumbed to the infection on days and following inoculation. pathological findings included splenomegaly, diffuse spleen necrosis and focal liver necrosis with hepatocytes vacuolisation. the remaining three hares were euthanised and revealed no pathological lesions. both bacterial culture and mouse inoculation test failed to produce f. tularensis isolation [ ] . in a natural outbreak of tularaemia in brown hares in france, all eight hares involved presented splenomegaly, congestion and haemorrhagic lesions of several organs, tracheitis and bronchitis [ ] . a similar study carried out in hungary on european brown hares naturally infected with f. tularensis subspecies holarctica also showed very similar results [ ] . in another study, female new zealand white rabbits (oryctolagus cuniculus) were exposed to type a tularaemia aerosols, with three different doses. seven of them died while the others developed fever, anorexia and weight loss, with all infecting doses. haematological findings in six rabbits included lymphopenia, monocytopenia and thrombocytopenia. a bibasilar pneumonia and gastrointestinal tract gas distension were the only radiological findings. necropsy findings demonstrated hepatosplenomegaly with extensive spleen necrosis and small white nodules. some of the rabbits presented nodular lesions in the lungs while others showed haemorrhagic lesions [ ] . a situation of particular public health significance, given the risk of pet-to-human transmission, is associated with infected prairie dogs (cynomys ludovicianus) sold as pets in the usa and exported internationally [ , ] . a ban was put in place in the european union and other countries regarding the import of prairie dogs and other rodent species after the usa monkeypox outbreak in [ , ] . wild-caught prairie dogs are particularly susceptible to environmental stress, such as capture, transit and crowding, which can enhance disease manifestations. clinical signs include lethargy, dehydration and grossly enlarged cervical lymph nodes. prairie dogs can produce specific antibodies against f. tularensis and survive tularaemia infection, suggesting their potential role as f. tularensis reservoirs in nature. moreover, one study found that all seropositive animals harboured live infectious bacteria, suggesting persistent infection [ ] . tularaemia has also been described in domestic dogs and cats [ , ] , which may be infected by means of arthropod bites, direct contact with infected animals, their ingestion, or contaminated aerosols [ , ] . cats usually develop severe illness with unspecific clinical signs like fever, lethargy, prostration, vomiting and anorexia, dehydration, regional or generalised lymphadenopathy, splenomegaly, tongue and oropharyngeal ulceration and jaundice [ , , ] . pathological findings include multiple necrotic foci on the lymph nodes, spleen, liver and lungs. frequently, panleukopenia with toxic degeneration of the neutrophils and hyperbilirubinaemia with bilirubinuria are present [ ] . dogs are less susceptible and rarely manifest signs of the disease [ , ] . nevertheless, they can act as carrier hosts [ ] and transmit the bacterium by means their fur after contact with contaminated dead animals or soil [ ] . in most cases, infection is self-limiting and recovery is spontaneous. however, only few cases of natural infection in dogs have been reported [ , ] . in humans, samples should preferably be collected before the onset of antibiotherapy and depend on the clinical form of the disease. samples may include non-heparinised whole blood, serum, respiratory tract secretions and washes, swabs from visible lesions, lymph node aspirates or biopsies, urine, and autopsy materials [ ] . in animals, serum is the preferential sample for all disease forms, but plasma and dry blood on paper filters can also be used. blood samples should be collected at least days after the onset of the symptoms. lymph nodes or bone marrow aspirates, organs (lung, liver, spleen) and cerebrospinal fluid can also be used [ ] . in the context of an outbreak or epidemiologic studies, samples should include arthropod vectors as well as environmental samples like water, soil and rodent faeces [ , ] . culture is the gold standard for f. tularensis and must be carried out in biosecurity level facilities (bsl- ) [ , , ] . f. tularensis is a fastidious microorganism. optimal growth conditions occur at • c and ph . [ , ] . cysteine-enriched media, such as enriched chocolate agar (ca) or % cysteine heart agar with blood medium (chab) must be used for this purpose [ , , ] . growth in a chab medium enables the presumptive identification of f. tularensis by characteristic growth at - h of round and smooth green opalescent shiny colonies, - mm in diameter [ , , , , ] . antibiotic supplementation of chab is possible in order to optimise growth and inhibit contaminants [ , , ] . for cultures made from blood, the use of the bactec tm (bd) system or equivalent, bact/alert tm (biomérieux) is recommended [ , ] . liquid media is not suitable for f. tularensis growth, even when supplemented with cysteine [ , , ] . basic biochemical tests provide a presumptive identification of isolates and may be further complemented by immunological and molecular methods. some additional biochemical tests, such as the ability to ferment glucose or glycerol, or the presence of the citrulline ureidase pathway are useful for subtyping purposes [ ] . the commercial microlog microstation tm system (biolog inc., hayward, ca) based on the ability to ferment glucose has been successfully used for differentiating between subspecies tularensis and holarctica [ , ] . also, the commercially available microbial identification system (mis) and library generation system (lgs) (midi, inc, newark, nj) enables cell-wall fatty-acid analysis and can be used for the identification of francisella at the genus level. it has also enabled the identification of atypical f. tularensis strains lacking cysteine requirements [ , ] . immune based techniques have also been employed for identification: immunoblot analysis and immunofluorescence microscopy, either from grown cultures or clinical samples [ ] . antibodies against f. tularensis reach detectable levels - days post-infection [ ] . a fourfold increase in the titre between acute and convalescent sera or a titre of : or greater of agglutinating antibodies is considered for diagnostic purposes [ , , , ] . titres peak at a level of - and decline slowly [ ] . serologic methods include the whole-cell agglutination test (widal's reaction), the tube agglutination test, microagglutination assays, haemagglutination, elisa (enzyme-linked immunosorbent assay) and immunoblot [ , ] . elisa has repeatedly been more sensitive than agglutination assays, with the additional advantage of determining separately different antibody classes (igm, igg and iga) [ ] . a combination of a first elisa screening test complemented by an immunoblot confirmatory test, with higher specificity, is the current recommended two-step approach for the serological diagnosis of tularaemia [ ] . the same approach can be used for animals. serology has a limited use in highly susceptible species since death usually precedes the development of specific antibodies [ ] . however, in endemic areas, antibodies for f. tularensis are frequently detected in wild animals that have developed immunity, including foxes and coyotes. this seroconversion is suspected as being related to subspecies holarctica infection since infection by the subspecies tularensis is expected to be fatal [ , ] . molecular methods are valuable diagnostic tools whenever culture is either not possible or is negative [ , , ] . moreover, they reduce the high risk of laboratory-acquired infections over conventional biochemical typing [ , , ] . during recent years, polymerase chain reaction (pcr)based methods have been successfully used for the rapid identification and classification of francisella isolates, with increased sensitivity and specificity [ , ] . however, false positive results related to non-pathogenic closely related francisella subspecies, occurring naturally in the environment, may hamper species and subspecies identification [ ] . conventional pcr targets are tul and fopa genes, which encode for f. tularensis superficial membrane lipoproteins. both protocols show a good level of sensitivity and reasonable specificity in f. tularensis detection and may be used in blood, tissue or aerosol samples [ , , ] . pcr product specificity is confirmed by sequencing, reverse-line blotting (rlb) or restriction fragment-length polymorphism (rflp) [ ] . real time pcr for f. tularensis detection has been developed, in particular, taqman tm (applied biosystems) real time pcr multiple assay shows high specificity and sensitivity using four target genes: isftu , kda, tul and fopa [ , ] . real-time pcr for the differentiation between the subspecies tularensis and holarctica is also now available [ ] . further discrimination has been achieved using highresolution genotyping methods including pulse-field gel electrophoresis (pfge), amplified fragment-length polymorphism (aflp), ribotyping, s rdna gene sequencing, canonic insertion deletions and paired-end sequence mapping [ , , , ] . still, as f. tularensis exhibits highly conserved genomic sequences among strains of diverse origin, genetic polymorphisms allowing for individual strain typing have been difficult to find [ ] . as for other bacteria, more recent pcr-based techniques such as variable-number tandem repeats (vntr), multiple-locus vntr analysis (mlva) and short-tandem repeats (str) typing have been successfully used for identification at the subspecies level and for molecular epidemiology purposes [ , , ] . one of the most discriminatory methods for the molecular subtyping of f. tularensis is mlva, which consists of a series of vntr loci that are pcr amplified via flanking primer sites and examined for size variation [ ] . one mlva system designed for f. tularensis is based on polymorphisms of vntr loci, ft-m to ft-m . this mlva typing system has a greater discriminatory power when applied to a worldwide set of f. tularensis isolates and provides accurate classification at the subspecies level [ ] . this mlva system has recently been improved by redesigning the subset of the previously identified vntrs to produce a new optimised, multiplexed mlva system with a similar level of discrimination but with fewer time and cost requirements [ ] . ten of the previously described vntr loci were selected based on their discrimination ability within the subspecies: ft-m , ft-m , ft-m , ft-m , ft-m , ft-m , ft-m , ft-m , ft-m and ft-m . locus ft-m was split into two loci, ft-m a (which contains the originally described bp repeat and is polymorphic across subspecies) and ft-m b (which contains the insertion with its bp repeat and varies only among type a.ii and f. novicida isolates) [ ] . while providing discrimination among strains, vntrs are unsuited for determining deeper phylogenetic relationships due to mutational saturation. in this case, more accurate and alternative markers should be used, such as whole-genome sequence single nucleotide polymorphism (snps) [ ] . additional studies have shown a remarkable degree of discrimination of the f. tularensis phylogenetic structure, using a combined analysis with canonical whole-genome snps for major clade typing, and mlva for high-resolution typing [ , ] . in a different study, the combined analysis of insertion-deletion markers, for subspecies and major clade typing, along with mlva, was used [ ] . microarrays have also allowed for the differentiation of the four f. tularensis subspecies and have been proven useful for pathogenicity and virulence marker identification [ ] . tularaemia usually responds to antibiotic therapy. historically, aminoglycosides have been the drugs of choice for humans. although clinically effective, they are rarely used now due their ototoxicity and nephrotoxicity. nevertheless, gentamicin has been used for treatment of pneumonic tularaemia and aminoglycosides are now generally used in the most serious cases. chloramphenicol is effective but seldom the first choice due to its possible irreversible effects on haematopoiesis. tetracyclines have been associated with high relapse rates on withdrawal. fluoroquinolones, such as ciprofloxacin, have been shown to be highly effective in per os and are the best choice for uncomplicated tularaemia. also, ciprofloxacin has proved suitable and effective in the treatment of tularaemia in children and pregnant women [ , ] . in domestic animals, gentamicin, enrofloxacin, doxycycline and chloramphenicol are referred to as therapeutic options for dogs [ , ] . in cats, there are reports of the use of doxycycline or enrofloxacin and amoxicilin-clavulanic acid as being beneficial in the early stages of the disease [ ] . currently, there is no available licensed vaccine against f. tularensis although an attenuated type b strain, known as the live vaccine strain (lvs) was developed in the united states during the s and used to vaccinate military personnel and laboratory workers [ , , [ ] [ ] [ ] . lvs failed to uniformly protect against pneumonic tularaemia and when delivered in high titres caused mild tularaemia as an undesirable side-effect [ ] . one focus of current research work in the usa and in europe is to develop a vaccine for protection against f. tularensis intentional release [ ] . the restricted efficacy of the lvs has fostered extensive research with a view to providing alternative vaccine formulations, including the exploration of different live and killed attenuated strains and immunogenic components to produce subunit vaccines [ , ] . in view of its immunogenic antigens, an effort has been made to develop attenuated strains of schus , a representative strain of type a tularaemia, for vaccine production. in fact, between lvs and schus strains there are about genes that encode for different protein sequences, whose functions are not well defined, and may represent important immunogenic antigens. still, given the increased virulence of the schus strain, only a small number of bacteria should be required to generate effective protection against wild type f. tularensis [ ] . a recently published study demonstrated that inoculation with low doses of specific attenuated mutants of the f. tularensis strain schus provided protection against parenteral and intranasal challenge with a fully virulent wild type schus strain [ ] . this favours the role of t-cell memory response as a critical determinant of f. tularensis immunity, additionally to the humoral response. this feature is the basis of the challenges foreseen for vaccine development, aiming at identifying antigen determinants that elicit an effective cellular-mediated immune response [ , , , ] . cell-mediated immunity was found to persist three decades after tularaemia vaccination. a recent study sought to identify the t-cell responses present in immune individuals in order to characterise f. tularensis-specific immune response [ , ] . the findings showed that the production of inf-␥, macrophage inflammatory protein (mip)- ␤ and cd a (lysosomeassociated membrane protein or lamp- ) by peripheral blood mononuclear cells appeared to be a characteristic of protective immune responses and that a correlation exists between these parameters and immunity [ ] . several factors such as human demographics and behaviour, international travel and commerce, including the animal trade, climactic changes and microorganism adaptation, have a potential impact on disease ecology and the emergence of zoonosis. the same factors are thought to be related to the emergence of tularaemia. special concerns regarding this bacterium exist in relation to its high infectivity, and easy dispersion through aerosols and contaminated water, which make it a potential bioterrorism weapon. also, tularaemia presents a wide geographic distribution and has recently emerged in new settings, particularly in europe. in portugal, an on-going research project on tularaemia aims to increase our knowledge about the disease, particularly its impact in this country, which is still poorly understood, in view of the fact that there is little information available to risk population and health professionals, with the result that there is a possible underestimation of prevalence in man and animals. to this regard, efforts have been made by the national institute of health to increase awareness of the disease among risk populations, particularly hunters and health professionals. in accordance with the preliminary results, on-going research will further identify and characterise f. tularensis circulating strains and develop molecular and typing methods with increased sensitivity, specificity and discriminatory power. the role of autochthon wild lagomorphs in the f. tularensis life cycle, their involvement in animal-to-human transmission and their suitability as tularaemia sentinels will be accessed. moreover, considering the economic and social relevance of hunting-related activities in this country, with very few studies having acknowledged its relation to zoonotic disease transmission risks, research into infection in game species is of major importance. f. tularensis is also associated with a considerably wider range of hosts and vectors than most zoonotic pathogens, although there is little information on bacterium mechanisms for adaptation to such a wide diversity of arthropod vectors. despite our increasing knowledge of tularaemia and its etiological agent, many aspects of f. tularensis biology and epidemiology need to be further examined, particularly its pathogenicity and virulence, vaccine development, and the specific mechanisms by which f. tularensis evades, modulates and suppresses the host immune response. as with any zoonotic emergent disease, the role of wild and domestic animals in f. tularensis epidemiology needs to be further evaluated, in particular, those which may act as reservoirs. other epidemiologic data such as the population dynamics of susceptible animals, particularly lagomorphs and rodents in europe, should be part of surveillance programmes, as they are thought to be directly associated with disease transmission patterns. from a public health perspective, disease surveillance in animals is crucial in order to prevent and monitor human outbreaks, particularly in endemic areas, where contact between humans and wildlife reservoirs or vectors is likely. although tularaemia is not regarded as a common disease, and there is little awareness of the disease among health authorities and practitioners, its eventual future impact as an emergent zoonosis should not be neglected. the authors declare that they have no conflict of interest. emerging zoonosis and vector-borne infections affecting humans in europe douglas, and bennet's principles and practice of infectious diseases francisella tularensis: unraveling the secrets of an intracellular pathogen topley and wilson's microbiology and microbial infections francisella tularensis: an arthropod-borne pathogen tularemia: emergence/re-emergence special topic on francisella tularensis and tularemia francisella tularensis reporting on 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hugo osório, maria joão alves and rita de sousa from the doctor ricardo jorge national health institute, i.p., centre for vectors and infectious diseases research key: cord- -la vi j authors: brower, jennifer l. title: the threat and response to infectious diseases (revised) date: - - journal: microb ecol doi: . /s - - - sha: doc_id: cord_uid: la vi j the threat from microorganisms is complex, and the approaches for reducing the challenges the world is facing are also multifaceted, but a combination approach including several simple steps can make a difference and reduce morbidity and mortality and the economic cost of fighting infectious diseases. this paper discusses the continually evolving infectious disease landscape, contributing factors in the rise of the threat, reasons for optimism, and the policies, technologies, actions, and institutions that might be harnessed to further reduce the dangers introduced by pathogens. it builds upon and updates the work of other authors that have recognized the dangers of emerging and re-emerging pathogens and have explored and documented potential solutions. in just the past year, the united states has been bombarded with headlines on the dangers of infectious diseases: "hiv 'epidemic' triggered by needle-sharing hits scott county, indiana [ ] ;" "american with ebola now in critical condition [ ] ;" "seasonal flu vaccine even less effective than thought: cdc [ ] ;" "'superbug' outbreak at california hospital, more than exposed [ ] ;" "deadly cre bugs linked to hard to clean medical scopes [ ] ;" "painful virus [chikungunya] sweeps central america, gains a toehold in u.s. [ ] . " the ebola outbreak that began in and the measles outbreak initiated at disney world in particular brought the threat of "exotic" infectious diseases back to the american and global consciousness. this coupled with the fact that the most commonly circulating strains of the influenza a virus h n drifted [ ] from that used in the - influenza vaccines serve as reminders that the threat from microorganisms is continuously evolving and is persistent. the threat of emerging and re-emerging pathogens has been discussed in the scientific literature, the medical community, by policy makers, and the general public over the past years, but much of the discussion was among directly affected populations and their caregivers. general interest flourished after a series of events in the s and early s. in , a report by russian general kuntsevich followed by boris yelstin's decree in april of that year to end all offensive biological weapons programs revealed that the former soviet union had an extensive biowarfare program and that facilities and expertise still existed which would enable russia to unleash deadly pathogens on the world [ ] . in when shoko asahara, the spiritual leader of a japanese religious cult, was arraigned, the magnitude of the organization's attempts to deploy anthrax in was exposed [ ] . in october , the united states was transfixed by the first bioterrorism attacks on its own soil: envelopes containing bacillus anthracis spores were sent through the mail to targets ranging from media companies to government officials [ ] . five people died and thousands were treated with prophylactic antibiotics. the attacks and other attempted and planned attacks, along with widely publicized outbreaks such as west nile virus in [ ] and severe acute respiratory syndrome (sars) in [ ] , brought the topic of infectious disease to the forefront. in addition, more incessant threats such as influenza and lower respiratory infections continue to kill and cause economic harm through lost productivity and hospitalizations. furthermore, zoonotic diseases such as salmonella and listeria, which represent more than two-thirds of emerging and re-emerging diseases [ ] , raise the visibility of the economic and human and animal health issues caused by pathogens. in april , the sabra dipping company voluntarily recalled about , cases of hummus potentially contaminated with listeria monocytogenes. at the same time, blue bell recalled nearly products also similarly contaminated. while there were no known casualties as a result of the sabra contamination, authorities in kansas and texas reported that three deaths in each state might be attributed to the blue bell incident [ ] . the threat from microorganisms is complex, and the approaches for lowering the challenges the world is facing are also multifaceted, but several simple steps can make a difference. this paper will discuss the emerging infectious disease landscape, contributing factors in rise of the threat, reasons for optimism, and the actions, policies, technology, and institutions that might be harnessed to further reduce the dangers introduced by pathogens. it builds upon the work of other authors who have recognized the dangers of emerging and re-emerging pathogens and have explored and documented potential solutions [ ] [ ] [ ] . microorganisms pose health and economic threats and may pose a strategic threat if a large percentage of the population is overcome or if the potential transmission of infectious diseases across borders causes an increase in tension among state allies or enemies. one organism alone, clostridium difficile, is estimated to cost the united states between $ and $ billion per year [ ] , with its primary impact on american children [ ] . initially identified in the early s as a commensal organism in the digestive tract, c. difficile infection (cdi) has only been recognized as a significant threat to pediatric health over the last decade [ ] . the threat to both children and adults is global. infections since have become more common, more acute, less treatable by standard therapy, and more likely to reoccur [ ] . initially, the c. difficile infections were associated with the use of the antibiotic clindamycin, but fluoroquinolones and cephalosporins are currently the more likely cause of disturbed gut microbiota, which increasingly lead to colonization with ribotype , a severe variant of c. difficile [ ] . according to the centers for disease control and prevention (cdc), emerging infectious diseases are those "whose incidence in humans has increased in the past two decades or threatens to increase in the near future [ ] ." while there may be debate about the specifics, for the purposes of this article, re-emerging and emerging diseases are distinguished as follows: re-emerging diseases are those that were known to impact humans or animals in the past and were thought to be brought under control with zero or few infections in the past several decades. these include infections resulting from changes or evolution of existing organisms and changes in the geographic distribution of an organism or populations affected by the organisms. previously unrecognized (in the past several decades) infections are considered emerging. according to this definition, c. difficile would be considered an emerging pathogen as its dangers were not recognized when it was first identified. other outbreaks and trends of concern include the following: tuberculosis (tb), while no longer among the leading causes of death in , was still among the leading causes, killing over , people in [ ] . in the united states, while overall tb incidence is decreasing, it is still a large problem for foreign-born residents and for the homeless population at a cost of nearly $ million per year [ ] . lyme disease caused by the spirochete borrelia burgdorferi was recently recognized as an epidemic. the disease is difficult to diagnose, causes long-term disability if untreated, and may impact as many as , people in the united states [ ] . more than % of lyme disease patients continued to exhibit symptoms after six months, and for % of infected people, symptoms continued for more than three years [ ] . the spread of diseases such as multidrug resistance acinetobacter in at-risk populations is also of increasing concern. "within the last years, members of the bacterial genus acinetobacter have risen from relative obscurity to be among the most important sources of hospital-acquired infections. the driving force for this has been the remarkable ability of these organisms to acquire antibiotic resistance determinants, with some strains now showing resistance to every antibiotic in clinical use [ ] ." acinetobacter resistance to drugs such as imipenem and ampicillin/sulbactam increased % from to [ ] . leptospirosis, one of the most widely distributed zoonotic diseases worldwide, is an emerging public health concern particularly in large urban centers of developing countries [ ] . it is also important in the united states in humans, pets, and wildlife. experts believe incidence in humans is underreported, but the cdc estimates that - leptospirosis cases occur annually with approximately half of those in hawaii [ ] . in , triathletes in illinois were exposed to leptospirosis of which became symptomatic [ ] , representing the largest human outbreak in the united states. recently, cases in pets have caused concern in california [ ], michigan [ ], and florida [ ] . more than a quarter of the tested deer population in michigan was infected with the disease [ ] . west nile virus (wnv) is another zoonotic disease of concern, and the us population and health practitioners have become more aware of this disease over the past decade. birds carry the virus, which is then transmitted by mosquitoes to humans, horses, and other mammals. disease symptoms range from fever to neurological complications, such as encephalitis or meningitis. mortality is observed mostly in older and immunocompromised individuals. in , wnv was introduced to the united states, and its range soon extended across north america [ ] . not only is the number of wnv outbreaks increasing but also novel strains are emerging, which display higher virulence. wnv has also developed sophisticated avoidance mechanisms to avoid its elimination [ ] . noroviruses are the leading cause of foodborne disease outbreaks worldwide and may soon eclipse rotaviruses as the most common cause of severe childhood gastroenteritis, because rotavirus vaccine use is becoming more prevalent [ ] . norovirus rapidly undergoes genetic mutations and recombinations so that new epidemic strains are constantly evolving. although norovirus infection is generally not fatal, infections in children, the elderly, and the immunocompromised can cause morbidity and even death. research into a vaccine or treatment has been impeded by the lack of a cell culture or small animal model. however, vaccines based on norovirus capsid protein virus-like particles show potential and may become broadly available through transgenic expression in plants [ ] . vibrio vulnificus, a common gram-negative bacterium in warm coastal waters globally, is an emerging pathogen [ , ] . up to million vulnerable americans are at risk when consuming raw or improperly prepared seafood tainted with v. vulnificus which can cause primary septicemia [ ] . additionally, all individuals are at risk of serious wound infection that may lead to secondary septicemia [ , ] . even with antibiotic treatment, half of patients may die from primary septicemia and a quarter from secondary [ , ] . other environmental organisms of concern include the waterborne pathogen that causes legionnaires' disease, legionella bacterium; naegleria fowleri, which causes amebic meningoencephalitis; other mycobacterium (hospital environment) such as mycobacterium abscessus and m.massiliense in lung disease; the mosquitoborne chikungunya virus and the tickborne bourbon virus. in addition to causing acute illness, research has uncovered links between infectious diseases and cancer. in one study by wu et al. [ ] , researchers found measurable differences in fecal microbiota between healthy individuals and those with colorectal cancer as determined by pyrosequencing of the s rrna gene v region. as early as , researchers found that hepatitis b surface antigen (hbsag) carriers had a greater incidence of primary hepatocellular carcinoma (phc) than among non-carriers [ ] . the list of emerging and re-emerging pathogens could fill up a tome. these organisms vary in virulence and distribution, but all of them share common characteristics in that the incidence or virulence or both are increasing and humans must find methods of preventing, detecting, and treating them. to combat infectious disease, it is important to understand the factors that are working to increase the occurrence and severity of infections. human behavior has a large impact on the creation of environments where microorganism can evolve and mutate. these changes can sometimes make organisms more infectious and/ or virulent. examples include the following: antibiotics in the environment through overuse and misuse; changes in sexual norms; patterns of drug use and incarceration; global climate change; human incursion into new environments; and changing patterns of human interactions with wild and domesticated animals; expanding travel patterns; vaccination avoidance; and population concentrations in large cities. recent cases are used to illustrate how differences in human behavior have modified the threat from bacteria and viruses. the problem of antibiotic resistance is threefold: there has been a rise in the number or identification of resistant bacterial strains; the pipeline for the development of new medicines to treat infection dried up significantly over the past years; and the most significant problem is the lack of stewardship of existing antimicrobials. these issues have led to a reduction in the efficacy and number of responses available to physicians and their patients. the biological processes that lead to resistance are extremely complicated and not fully understood, resulting in sometimes limited progress in the control and treatment of resistant microorganisms and the diseases they cause [ ] despite recognition of the problem nearly a century ago. davies and davies [ ] compiled a list of "suberbugs," which have increased pathogenicity and are more impervious to treatment. their list includes the following: multidrug-resistant (mdr) m. tuberculosis; nosocomial (hospital-linked) infections with acinetobacter baumannii, burkholderia cepacia, campylobacter jejuni, citrobacter freundii, clostridium difficile, enterobacter spp., enterococcus faecium, enterococcus faecalis, escherichia coli, haemophilus influenzae, klebsiella pneumoniae, proteus mirabilis, pseudomonas aeruginosa, salmonella spp., serratia spp., staphylococcus aureus, staphylococcus epidermidis, stenotrophomonas maltophilia, and streptococcus pneumoniae. their list does not include the new delhi metallo-beta-lactamase- (ndm- ) resistant strains discussed below. as the authors point out, in addition to the direct human toll, treatment is often more costly [ ] when resistant organisms are involved. in fact, the issue has become so acute that new terms have developed over the past decades: microorganisms that are pan-drug resistant (pdr) or extremely drug resistant (xdr). one of the most widely dispersed antibiotic resistant organisms is m. tuberculosis. worldwide, this organism is often resistant to multiple drugs, and in , completely drug-resistant forms of tuberculosis were reported in citizens of four countries: afghanistan, azerbaijan, iraq, and iran [ ] . in many organisms, such as enteric bacteria which are acquired both in community and hospital settings, resistance (often to β-lactam antibiotics in this case) spreads through horizontal gene transfer on plasmids; however, there have been no documented cases of this in tuberculosis, where all resistance occurs by spontaneous mutation [ ] . multidrug resistant pseudomnas aeruginosa is also of concern as it is deadly and widespread [ , ] . m. tuberculosis is one example of the multitudes of resistant organisms. other widespread and dangerous bugs include staphylococcus aureus ( . per inpatient prevalence rate in [ ] ) and c. difficile (in us hospitals in , c. difficile was the most commonly reported pathogen causing . % of health careassociated infections and staphylococcus aureus caused the second highest percentage, . %. klebsiella pneumoniae and klebsiella oxytoca . % and escherichia coli . % followed closely behind [ ] ). at a single hospital in and , resistant acinetobacter baumannii infected . % of patients who were not previously infected [ ] . infections with resistant organisms are harder to control; standard treatments are less effective; illness and hospital stays are longer; and mortality is higher. gram-positive organisms resistant to antibiotics were the first concern, but resistance in gramnegative organisms emerged: gram-negative bacteria resistance increases faster than in gram-positive bacteria [ ] , and there are fewer antibiotics in the pipeline that work against gram-negative bacteria [ ] . cosgrove et al. [ ] performed a meta-analysis of studies published between and on the impact of methicillin resistance on mortality. these studies included nearly patients, a third of whom were infected with methicillin resistant staphylococcus aureus (mrsa). mortality was significantly lower in the group infected with susceptible bacteria. in another study, cosgrove's group found that mrsa bacteremia also increased median length of hospital stay by almost % and not surprisingly (given the longer stay), increased hospital charges from an average of $ , to $ , [ ] . a prospective study found similar results in hemodialysis patients at the duke university hospital [ ] as did a study on orthopedic patients [ ] . vancomycin-resistant enterococci (vre) [ ] and enterobacter species resistant to third generation cephalosporins [ ] showed a similar trend; however, penicillin-and cephalosporin-resistant streptococcus pneumonia results were dissimilar, and the authors surmised that this might be due to the specific use of vancomycin [ ] . chemicals in daily use may also change microorganism susceptibility to antimicrobial agents. for instance, it has been regularly demonstrated in the laboratory that resistance to triclosan, an antimicrobial agent used in many household products including hand sanitizer, and crossresistance to antimicrobials increases with use of triclosan containing products; however these results have not yet been observed in the community. based on the available evidence, the risk of potential antimicrobial resistance outweighs the benefit of widespread triclosan use in antimicrobial soaps [ ] . resistance is not something that can be conquered: bacteria with their relatively short lifespans can mutate quickly; however, with knowledge of the , resistance genes of types [ ] , it may be possible to stay one step ahead of resistance and find new ways to treat bacterial infections. other changes that have impacted infectious disease distribution and prevalence are changes in sexual norms, drug use, and incarceration. needle sharing itself can spread infections, and the use of drugs can affect sexual and risk taking behavior which can put people in jeopardy [ ] . while homophobia is decreasing in the united states and worldwide, homophobia has been one of the major social determinants of infection particularly with hiv/ aids and other sexually transmitted diseases. for example, men sleeping with men accounted for % of new hiv infections in [ ] . historical legal restrictions, which are now being relaxed in this decade, had ostracized gay people, limiting their self-identification and therefore efforts to target gay communities for education and prevention as well as diagnosis and treatment efforts. injecting drug users account for % of new hiv infections often due to inadequate access to sterile needles and syringes and addiction treatment programs [ ] . as noted below, drug use also changes behavior which also leads to increased transmission. drug use and incarceration patterns go largely hand-in-hand. in part because of the united states hard line on drug use, united states incarceration rates are the highest in the world with minorities accounting for a disproportionate percent of the prison population. incarceration rates disrupt community and sexual relationships and compound poverty issues, amplifying the exposure of communities and individuals to hiv infection and other infections [ ] . in a second example, methamphetamine use has been shown to affect a person's judgment and may lead to unsafe behaviors such as reduced condom use, multiple partners, and increased drug injection. methamphetamines also increase physical susceptibility because their use dries mucosa intensifying chafing and abrasions, which, in turn, allow microorganisms to enter the body during sexual and other activity [ , ] . aquaculture contributes to the pollution of rivers, bays, and even our oceans with antibiotics and antibiotic resistance genes (args). from china to the united states, antibiotics and args have been found in surface water of all types. for example, in the coastal water of the bohai bay, china, fluoroquinolones, macrolides, sulfonamides, tetracyclines and chloramphenicoles, and polypeptides were found at concentrations up to several micrograms per liter with higher concentrations where human activity was concentrated [ ] . in a review, comparing aquaculture and land animal production with the respect to type, mechanism, and quantity of antibiotic resistance, done, venkatesan, and halden [ ] found that aquaculture was similar to terrestrial agriculture in terms of the resistance mechanisms, that antibiotics used in aquaculture are important in human health, and that pathogens isolated from the farmed fish were resistant to multiple antibiotics. due to improper use and disposal of antibiotics, the presence of antibiotic-resistant organisms and genes in natural waterbodies, wastewater, and treated municipal water has been widely demonstrated and reviewed [ ] [ ] [ ] [ ] . without additional treatment, this water is commonly used on crops; humans and animals then consume the products, and serious outbreaks have occurred that are difficult to treat because the microorganisms do not respond to commonly used antibiotics [ ] [ ] [ ] [ ] . pruden et al. [ ] found concerning levels of args in colorado (united states) dairy lagoon water, irrigation ditch water, river sediments, treated drinking water, and recycled wastewater. ramsden et al. [ ] similarly found antibacterial resistance in municipal wastewater treatment plants. zuccato et al. [ ] discovered that the concentrations of atenolol, bezafibrate, clofibric acid, cyclophosphamide, diazepam, erythromycin, furosemide, lincomycin, oleandomycin, ranitidine, salbutamol, spiramycin, and tylosin were in the nanogram per liter range in river or drinking water or river sediments in several sites in italy. munir et al. [ ] examined the presence of antibiotic-resistant genes and bacteria in several types of wastewater effluents in michigan and found that advanced water treatment systems such as membrane bioreactors were significantly more effective than conventional wastewater treatment at removing the tetracycline-resistant gene teto and sulfonamide-resistant gene (sul-i) as well as tetracycline and sulfonamide-resistant bacteria. anaerobic digestion and lime stabilization treatment of wastewater was more effective than the conventional dewatering and gravity thickening methods for removing antibiotic-resistant genes and bacteria [ ] . burch et al. [ ] were able to significantly reduce the concentrations of the args tet(a), tet(w), and erm(b) using conventional wastewater treatment (aerobic); however, removal of inti required batch treatment, while the others required relatively long-term semi-continuous treatment. tet(x) increased in concentration. according to the world bank, nearly million travelers visited the united states and approximately one billion people traveled globally in [ ] . the incidence of tuberculosis in the united states is largely due to foreign visitors and citizens and residents born in other countries [ ] . another, travel related resistance threat emerged in the united states in when three patients were reported to have the gene for new delhi metallo-beta-lactamase (ndm- ), an enzyme that destroys beta-lactam antibiotics including commonly used penicillins, cephalosporins, and carbapenems. the first case was reported in india in [ ] , and to date, india and pakistan have reported the most instances of ndm- , but the gene is spreading globally, and cases have now been detected in many countries, including great britain, canada, sweden, australia, japan, and the united states. antibiotics are widely used in india and some researchers [ ] have demonstrated that overuse of carbapenems led to the development of ndm- [ ] . research also points to medical tourism as a cause [ ] [ ] [ ] [ ] . ndm- is a newly identified problem, only recognized since about december in the medical literature, but it is only one example of diseases transmitted through medical tourism which is defined as travel to a country to get medical care that is not available or is more expensive in one's own country. precise data on the economic value and the number of patients seeking medical procedures are not easily available. in , smith et al. [ ] estimated that approximately four million patients crossed borders seeking treatment. in , guidelines to unify definitions of medical tourism and methodologies for reporting its extent were published and accuracy of the types and amounts of medical tourism may improve in the near future [ ] . a greater potential threat is related to the increasing travel of immunocompromised patients. lortholary et al. [ ] illuminated the fact that as more and more people are living with hiv, having organ transplants, using immunodilators, or suffering from diabetes, more individuals are infected when traveling. the authors suggested preparations and responses to prevent severe illnesses when traveling. infections spread within the united states from travel as well. for example, during the period from through , cryptococcus gattii infections were reported to the cdc. c. gattii, an environmental fungus typically prevalent in tropical and sub-tropical regions, can cause an uncommon infection of the lungs and/or the central nervous system in those who inhale the fungus. more than % of the cryptococcosis cases occurred in people who had traveled to the pacific northwest. the infection was fatal for % of the patients [ ] . many factors have reduced the number of new antibiotics approved in the united states each year as well as reduced domestic production including demanding food and drug administration (fda) regulations, the cost and time to market of development, the consolidation in the pharmaceutical industry, and the lack of financial impetus to produce and distribute antibiotics, which are generally used on a one-off basis versus drugs used to treat chronic conditions such as statins, viagra, and allergy medications. in a may speech, janet woodcock, the director of the center for drug evaluation and research (cder), acknowledged that new antibiotics were not sufficient to address growing antibiotic resistance and that fda's approach to approval was a significant factor [ ] . the fda introduced new regulations for clinical trials at the beginning of the twenty-first century, which led to a cooling of antimicrobial development in the pharmaceutical industry [ ] . first, the newly required approach doubled the cost of phase iii clinical trials, already a substantial barrier for development. in phase iii, it is expected that testing will include pairs of relatively large (usually > total subjects per study) groups of people conducted for the selected pathogen at the relevant body location(s). this has become challenging as new antibiotics focus on particular pathogens including resistant pathogens, making it difficult to enroll large numbers of patients [ ] . in part because of the cost of the new regulations, eli lilly, bristol-myer squib, glaxo smithkline, proctor and gamble, roche, and wyeth left the development business [ , ] . in addition, while the amount of antibiotics prescribed has continued to grow, the market value has not changed and was estimated at $ billion in [ ] as compared to a $ . billion market in for statins alone [ ] . companies are getting out of the market because the regulatory burden is high, antimicrobials are typically used for short periods of time, public pressure is building to lower use, and the medicines are often subject to price controls outside of the united states [ , ] . while development has slowed, in the past years, new antibiotics have been brought to market. two approved more recently, fidaxomicin and bedaquiline, have new modes of action. fidaxomicin was shown to effectively treat c. difficile [ ] . because the financial incentives are few, much antibiotic production has been outsourced from the united states to india, china, and other countries where labor, raw material, and energy costs are lower [ ] . in fact, it has been more than years since the active ingredient for penicillin was last manufactured in the united states. this presents a significant strategic problem for the united states in the case of an outbreak, particularly during times of conflict or worldwide scarcity. global climate change is increasingly accepted as causing extreme, unusual weather patterns [ ] . changing weather patterns can impact the presence of infectious agents in many ways. for instance, in may and june , an initially unidentified disease killed ten people in the four corners region of arizona and new mexico. at the outset, % of the patients died of the infection, and after the medical staff developed enhanced protocols, the death rate was only reduced to %. scientists isolated a hantavirus [ ] , and later, researchers determined that an unusually wet spring led to increased rodent carrier density which in turn impacted human infection rates; however, these factors alone are not enough to explain persistent hantavirus infection in the southwestern united states [ ] . ecosystem changes and human interactions with the environment may increase the transmission of infectious disease [ ] . for instance, three studies found robust correlations between the threat to humans from west nile virus and low bird diversity in the united states [ ] [ ] [ ] . the spread of emerging infectious diseases among animals has significant human health and economic costs. zoonotic diseases kill more than two million people per year and transmission occurs from both wild and domesticated animals [ ] . halsby et al. [ ] reviewed the english literature with respect to infectious diseases caused by pet store animals and found discussions of infections related to pet shops. the most commonly observed diseases were salmonellosis and psittacosis: other diseases such as tularemia were also identified. the human animal interaction has impacted civilization throughout history. according to daszak et al. [ ] , "parallels between human and wildlife emerging infectious diseases (eids) extend to early human colonization of the globe and the dissemination of exotic pathogens. in the same way that spanish conquistadors introduced smallpox and measles to the americas, the movement of domestic and other animals during colonization introduced their own suite of pathogens. the african rinderpest panzootic of the late s and s is a paradigm for the introduction, spread, and impact of virulent exotic pathogens on wildlife populations. this highly pathogenic morbillivirus disease, enzootic to asia, was introduced into africa in . the panzootic front traveled km in years, reaching the cape of good hope by , extirpating more than % of kenya's buffalo population and causing secondary effects on predator populations and local extinctions of the tsetse fly." more recently, bovine tuberculosis, while responsible for only cases of human tuberculosis in the uk, prompted the slaughter of tens of thousands of cattle in the first decade of the twenty-first century [ ] . in , throughout the united states, domestic poultry and wild birds have been suffering from a highly pathogenic strain of avian influenza (hpai) h [ ] . through june , , more than million birds were put to death. the cost of the government response is tagged at $ million primarily to fund the work of staffers and contractors [ ] . on the commercial side, analysts used economic models and found that for a million dollars in direct losses there are $ . million in overall economic losses. in mid-may direct losses in poultry production were estimated at $ million leading to overall losses of more than $ million [ ] . transmission to humans in the united states has not been detected, although related viruses have caused serious illness and death around the world [ ] . typically, people have focused on wildlife diseases that affect human health and agriculture. recently, researchers, policy makers, and others have begun to pay attention to wildlife infectious diseases, because a number of endangered species including birds, amphibians, and invertebrates [ ] are impacted [ ] . human's changing relationship with the environment "deforestation and ensuing changes in land use, human settlement, commercial development, road construction, water control systems (dams, canals, irrigation systems, reservoirs), and climate, singly, and in combination have been accompanied by global increases in morbidity and mortality from emergent parasitic disease [ ] ." lyme disease is a prime example of how human destruction of the environment (forests) can lead directly to increased risk for disease exposure. allan, keesing, and ostfeld [ ] found that as forest patch size decreased ioxdes nymphal infection prevalence and nymphal density with increased, resulting in a noticeable rise in the density of infected nymphs and concluded that habitat fragmentation affects human health. as humans change or destroy the local environment, they tend to interact with or disturb wildlife populations, creating further instances for exposure to infectious diseases. goldberg et al. [ ] found increased rates of interspecific gastrointestinal bacterial exchange between people and nonhuman primates when humans visited chimpanzee and ape habitats. chimpanzees carried antibiotic-resistant bacteria although there had never been treatment with antibiotics. many of the factors discussed above coexist to increase the threat from microorganisms. the antivaccine lobby, especially in the united states, has led to a significant decline in the vaccination rates of infants and children, particularly among specific demographics despite the overwhelming success of vaccines in the fight against vaccine-preventable diseases. for instance, in (pre-vaccine), , cases of measles were reported with mortalities. in , there were cases of measles but no deaths. similarly in , cases of diphtheria were reported resulting in deaths. in , there were no reported cases of diphtheria [ ] . up to two % of parents in the united states refuse vaccination completely for their children with up to % more who are cautious or elect to delay vaccination [ ] . the reduction in vaccination coverage is typically attributed to the lack of perceived threat due to the success of vaccination, combined with false medical research and media reporting [ ] . the reduction in vaccination rates has resulted in the highest number of cases of measles in the united states since it was declared eliminated in [ ] . while native measles has been eliminated in canada, several measles cases are imported each year by international travelers and due to inadequate vaccination, these cases often lead to secondary spread. in the first five months of , cases in five provinces from known importations occurred through infected travelers arrived from the philippines, india, the united states, thailand, pakistan, italy, and the netherlands [ ] . travel patterns in canada are exemplary of much of the world. in years, international travel (excluding travel to the united states) more than doubled from . million to million trips [ , ] . if the antivaccine trend does not abate, and in conjunction with widespread global travel, the threat from diseases once thought under control may pose a significant threat to the population. influenza outbreaks kill and hospitalize more than , americans each year. the predominant strategy in the united states is to encourage all eligible populations to get vaccinated; however, for the - flu season, more than half of influenza a (h n ) viruses had drifted from the h n vaccine virus. this mismatch leads to decreased vaccine effectiveness [ ] . it may also discourage individuals from getting the flu vaccine in the future. population growth, urbanization, and travel along with deterioration in public health infrastructure have contributed to the resurgence of infectious diseases. dengue fever provides a prime example of the intersection of the triad. while dengue viruses were dispersed throughout the tropics in the first half of the twentieth century, epidemics were infrequent because urban populations were comparatively small, and the viruses and mosquito vectors were transported on ships versus the air transport of today. the travel of both goods and people during world war ii set the stage for the spread of dengue fever. in the post war era with unparalleled urban growth and travel, serious epidemics occurred more frequently. scarcely years later, dengue hemorrhagic fever became a principal cause of hospitalization and mortality in the pediatric population throughout southeast asia [ ] . with respect to the intentional use of microorganisms as a weapon, the united states and the world have an outmoded threat-view focused on soviet era biological weapons, but travel, medicine abuse, and the lack of a us capability to approve and manufacture new antimicrobial and antiviral agents have changed many dimensions of the threat as discussed above. with the dissolution of the soviet union, the fact that the us biological weapons program ended decades ago, and the intellectual, medical, manufacturing, and weaponization knowledge needed to start a bioweapons program, the threat from naturally occurring organisms is far greater than the threat of bioterrorism or biowarfare in . the threats of infectious diseases dwarf that of terrorism and other asymmetric threats to human life. approximately three million people died in due to lower respiratory infections [ ] , and infectious diseases are the major cause of death of children under five. "the most important pathogens are rotavirus for diarrhea and pneumococcus for lower respiratory infections [ ] ." however, there is hope that new antibiotics will be identified and developed. recent research such as that performed by ling et al. [ ] found new ways to identify antibiotics [ ] in the environment and companies are beginning to invest again. under the direction of dr. kim lewis, ling and colleagues identified teixobactin. to do this, the team used the novel screening method to examine , strains. in both in vitro and in vivo tests, teixobactin was demonstrated to be operative, without major side effects, against the organisms that cause common illnesses such as pneumonia, tuberculosis, and staph infection, diseases which sicken more one million americans yearly. while teixobactin was effective against diseases of public health concern, it was ineffective against gram-negative bacteria. teixobactin binds on several targets triggering cell wall break down. the ability to bind on multiple sites lessens the chance of early teixobactin resistance. in addition to developing the new antibiotic, the researchers commercialized the screening technology, which can examine organisms that cannot typically be cultured in the lab [ ] . researchers are also developing techniques to enhance the impact of probiotics in fighting infections and other diseases such as cancer [ ] . while recent events bring the threat of microorganisms to the forefront of the public mind, the work of doctors, researchers, public health professionals, and other experts have continued unabated for decades. these attempts include scientific, technological, policy, and commercial attempts to reduce or eliminate the deaths and other losses caused by pathogens. to a large extent, these efforts have succeeded. in , the average lifespan in the united states was . for men and . for women, and one of the predominant causes of death was infectious disease. by the end of the century, lifespan had increased to . for men and . for women [ ] . in , infectious diseases accounted for more than half of all deaths: in , this percentage was reduced tenfold [ ] . the increases in life expectancy have been distributed across the world, although some areas have benefitted more than others from breakthroughs in sanitation, nutrition, and medical advances. one of the primary contributors to the reduction in the death rate was the reduction of infant deaths due to infectious diseases. prior to the mid- s, infectious disease played the predominant role in infant mortality with half of the (out of ) infant deaths due to pathogens [ , ] . by , the united states infant mortality rate had decreased to . per live births [ ] . also in the united states, in the midnineteenth century, foodborne and waterborne diseases such as typhoid, cholera, and dysentery resulted in deaths per , . these diseases were eliminated in the united states by the early s [ ] . one noteworthy exception to the steady progress in increased life expectancy is due to an infectious disease: hiv/aids decreased life expectancy dramatically in parts of africa over the past years [ ] . the leading causes of death and illness have shifted from infectious and parasitic diseases to noncommunicable diseases and chronic conditions. with the introduction of widespread antibiotics [ , ] in the s and antivirals in the late s [ ] , a new era of public health was ushered in, and the death rate due to infectious diseases accounted for less than % of mortality worldwide [ ] ; however, the optimism was short lived. even before there was prevalent proof that bacteria could quickly evolve to thwart antibiotics, evidence indicates that bacteria exhibit resistance in nature even without human pressure [ ] ; however, mechanisms of resistance impacting disease treatment were first noticed in the late s with regards to the use of sulfonamides [ ] . due to overuse, underuse, and incorrect disposal, antibiotic resistance has become a worldwide threat to public health [ ] . in addition, the cost and difficulty in developing new antibiotics has stunted the pipeline. finally, environmental [ ] , behavioral, and other physical and cultural changes have fostered situations where new pathogens can emerge and old enemies reemerge or spread to new locations. global climate change is altering where species thrive, and more localized or temporary changes modify infectious disease risk to humans as well. while ndm- strains are difficult to treat, many of them remain sensitive to an older, seldom used antibiotic, colistin, or aztreonam [ , ] years, clinical trial number n c t ; a n d s a f e t y, to l e r a b i l i t y, a n d immunogenicity study of a clostridium difficile toxoid vaccine in healthy adult volunteers, clinical trial number nct (a total of studies were found on www. clinicaltrials.gov when searching for 'c. difficile vaccine [ ] .' improvements are needed in dosage and timing to achieve high level immunity, however the investment required is large with estimates ranging from $ , , to $ , , [ ] to take a vaccine or antibody, respectively, through clinical trials. until a vaccine is developed, antibiotics will be used to treat infections. fidaxomicin, the first new antibiotic approved by the fda to treat cdi was approved in may . it was shown to be as effective as oral vancomycin, previously the only fdaapproved therapy for mild-to-moderately severe cdi. vancomycin is expensive and resistance in enterococci is a concern. oral metronidazole has been used by the medical community off label (it was approved for the treatment of certain anaerobic bacteria and parasites); however, relapse was observed in a quarter of patients within a month following treatment. fidaxomicin, in addition to being as effective as standard treatment, is a narrow spectrum antibiotic, allowing patients to maintain healthy native gut microbiota [ , ] . on a larger scale, according to the world health organization (who), hiv mortality was reduced from . million in to . million in , and diarrhea fell from one of the top five causes of death to number seven, with a similar number of deaths to hiv/aid in [ ] . tuberculosis distribution has declined since the turn of the century, in part because of the reach of the who's directly observed therapy short-course strategy and the implementation of the stop tb partnership plan [ ] . malaria cases and mortality has been meaningfully reduced by over cases and four million people respectively over the years between and through the use of artemisinin-based drugs, distribution of insecticide-treated bed nets, and indoor residual spraying of insecticide [ ] . this demonstrates that research, infrastructure, and other health-based investments have improved prevention and response to infectious diseases. all of this comes at a cost: between and governments including the united states, the uk, australia, canada, france, and germany and large non-profits and international institutions such as the gates foundation and the global fund contributed more than $ billion to the fight against hiv/ aids and nearly $ billion for international maternal and child health, which is in large part funding for vaccination [ ] . in addition, president obama has recognized that infectious diseases pose a national security threat. on september , , in his weekly address [ ] , the president stated, "so this is an epidemic that is not just a threat to regional security-it's a potential threat to global security if these countries break down, if their economies break down, if people panic. that has profound effects on all of us, even if we are not directly contracting the disease. and that's why, two months ago, i directed my team to make this a national security priority." because the challenges of new and re-emerging infections are complicated, a combination of science and technological advances, policy initiatives, and cooperative institutions are required. to make a significant difference, the united states and other countries must invest in technology and have systems capable of making these advancements available to those who need them, build technology development, and public health infrastructure; put in place policies and institutions that encourage these investments both in the public and private sectors. the success of programs such as the malaria initiative that combine these approaches is self-evident, but more needs to be done. an illustrative, but not complete, discussion of recent and additional proposals/initiatives is below. the united states, other countries, states, and international institutions have taken many steps to combat the threat. below are many of the important efforts and characteristics needed for resilience to the microbial threat. most importantly, it is critical to have a well-defined leader who is responsible for directing and monitoring progress as well as communicating risks. in president obama's september executive order [ ] , he directed the "national security council staff, in collaboration with the office of science and technology policy, the domestic policy council, and the office of management and budget to coordinate the development and implementation of federal government policies to combat antibiotic-resistant bacteria [ ] ." the president also created both a task force and an advisory council; however, he did not put a single individual in charge. identifying and developing a central, qualified, trusted person in charge of coordinating the investments in research, infrastructure and outreach; policies to incentivize behaviors to improve medicine development, infection control in medical and community settings; and communicate risks and responses in a directed and trusted manner at the federal government level, will enhance accountability and the likelihood of success. during times of low or chronic threat (e.g., flu season), the named person can develop a trusted relationship with the public, the medical and public health communities, the pharmaceutical industry, the defense department, international peers, and others involved in infectious disease response and defense. this is particularly difficult in diverse countries with divided political parties. a history of purposeful and innocent ethical lapses and scientific mistakes have contributed to a lack of trust such as the inaccurate flu vaccine in the - season and the confusing messages from the texas hospital and the cdc on ebola in . when a man traveled from africa and came down with a high fever and other symptoms, he was sent home by the hospital with antibiotics for two days [ ]: ebola was not well diagnosed in texas. one of the last trusted public health officials was the surgeon general under ronald reagan, dr. c. edward koop. by the time he stepped down in , he had become a household name, a rare distinction for a public health administrator. "dr. koop issued emphatic warnings about the dangers of smoking, and he almost single-handedly pushed the government into taking a more aggressive stand against aids [ ] ." dr. anthony fauci, director of the united states national institutes for allergy and infectious disease, has been a source of trusted and accurate infectious disease related information recently with regards to the ebola outbreak of . fauci is a natural leader for the us infectious disease/public health message, "he is someone who is really trusted by all the different organizations and people surrounding the aids challenge, ranging from the scientific community, the academic community and the activist community," according to louis sullivan, m.d., secretary of health and human services during the first bush administration and president emeritus of morehouse school of medicine in atlanta. "i don't know of anyone as broadly accepted by all those disparate groups [ ] ." the head of the cdc can also be a valuable spokesperson, but the cdc may have lost some of the public's trust during the ebola crisis [ ] . to centralize response, president obama appointed rob klain as the ebola coordinator. he was neither a doctor nor a scientist, and he left the job after six months, while ebola was still spreading in africa. while additional capability was developed at medical centers in the united states under klain's tenure, there were few noticeable signs of progress; he was not open to the media [ ] ; and likely as a result, was not embraced by the public. if the president chose a well-respected individual with healthcare and pharmaceutical industry expertise to serve in the white house to coordinate policies, funding and messages from nih, the cdc, the department of defense, the state department, state public health agencies, and other national and international institutions involved in the chain of prevention, detection, and treatment of infectious disease, it would be optimal. critical manufacturing capabilities have moved overseas, particularly to india and china. the us government could provide tax and other incentives and clear policies for approval for drugs, biologics, and manufacturing facilities to get manufacturing of key ingredients back to the united states. this would allow a faster and more certain response in times of emergency and the allow the government to initiate emergency medicine production under president obama's march executive order [ ] -national defense resources preparedness for manufacturing and distribution of medicines during times of crisis and the defense production act of as amended [ ] . international institutions are making significant efforts in preventing, detecting and responding to infectious diseases, and the continued work and support through the who, un, nato, the pan american health organization, the g , the cdc global health initiative, and other domestic and international bodies will improve international surveillance, reporting, prevention, and response. mechanisms for early reporting would avoid punishment such as travel bans for acknowledgement of dangerous infectious diseases within countries' borders. in addition, leaders in the united states would work to develop trusted relationships with peers in other countries. with more us foreign aid directed towards building public health infrastructure, the funds would have the primary impact of bolstering response and reducing transmission and casualties from infectious diseases within a country and secondary impacts of stabilizing societies (studies have shown that countries with healthy populations are more stable [ ] ). these outcomes would result in a safer and more secure world as there would be reduced disease transmission across borders. there are many existing global and domestic health initiatives such as the following: [ ] . lessons learned from this work can be utilized to further the goals of improving prevention and response to infectious disease. a research and response focus on diseases we encounter in the modern era as opposed to an emphasis on old soviet threats (unless the intelligence community identifies specific threats in the areas of bioterrorism and biowarfare) would enhance prevention and response capacities and funnel limited resources to current health and disease issues. preparations for naturally occurring outbreaks will not only prevent deaths year to year, but will also help exercise countries to fight intentionally introduced diseases by developing policies, procedures, infrastructure, and new technologies that foster quick innovation and therefore response to any microorganism, natural or manmade. each day, there are technological advances for preventing and combatting infectious diseases in addition to the progress specifically in medical research. for instance, adoption of advanced wastewater treatment systems can reduce exposure to antibiotics and args. this can be accomplished by tax incentives and partial payment by the federal government when wastewater treatment systems are replaced and advanced systems are used. in , $ billion federal dollars were spent on water utilities (water supply or treatment) accounting for approximately one quarter of public infrastructure spending [ ] . state and local governments spent $ billion for the operation and maintenance of infrastructure double the spending on capital improvements ($ billion). "although state and local governments rely primarily on their own revenues to purchase capital, federal grants also are an important source of funds. since , federal grants have accounted for one-third or more of the capital spending on infrastructure by states and localities. that share was considerably larger from the mid- s through the mid- s as a result of federal support for water utilities after passage of the clean water act in [ ] ." a renewal of this investment, with a focus on improving water treatment to remove antibiotics, args and other pollutants and destroying resistant organisms, would expand the positive results. regulations limiting the concentrations of antibiotics and args in treated municipal water, if enacted, in concert with meaningful financial penalties for those violating these standards, may significantly reduce the risk of population exposure. this can be difficult because the source of the contamination is often hard to identify. current antiviral drugs have several disadvantages including their specificity, toxicity and expense. researchers at the charles draper stark laboratories developed draco (double-stranded rna activated caspase oligomerizer). in lab-grown cells, draco killed different viruses, including ones that cause the common cold, influenza, polio and dengue fever with minimal effects on healthy cells [ ] ; however, there is still much work to be done before this drug can be fda approved and used by the general public. vectored vaccines use a live-vaccine made with a partial pathogen. they have been developed against sars-cov and demonstrated in mice, but the safety of vesicular stomatitis virus vaccine (vsv) in humans requires further research. newcastle disease virus, a host range-restricted virus, has been developed as a vaccine vector for intranasal immunization against emerging pathogens [ ] . science informs advances in drug development. for instance, authors reviewed a variety of genome sequence and gene knockout data for acinetobacter spp., with a focus on the critical systems to find the most appropriate sequences to target for therapies [ ] . this is just one early example in the explosive field of bioinformatics. in , in recognition of the importance of bioinformatics as a tool to diagnose and develop therapeutics for infectious diseases, the national institute of allergy and infectious diseases established four bioinformatics resource centers (brcs) to collect, store, and share bioinformatics information on bacteria, viruses, eukaryotic pathogens, and invertebrate vectors of human pathogens. as with the factors involved in the rise of the threat the responses are interrelated. the fda is, and must continue to, evolve its policies and regulations in the approval process so that research can proceed to the stage where drugs and biologics are ready for human use. this is discussed in more detail in the policies section below. because infectious diseases do not respect borders, it is in the strategic interest of the united states, the european union, and other countries with developed public health systems to invest in global public health infrastructure. this requires both a long-term investment as well as an acute response capability. president obama recognized both of these in the fall of . first on september , at the global health summit, president obama discussed long-term capacity building: "we, collectively, have not invested adequately in the public health capacity of developing countries." "this speaks to a central question of our global age-whether we will solve our problems together, in a spirit of mutual interest and mutual respect, or whether we descend into the destructive rivalries of the past. when nations find common ground, not simply based on power, but on principle, then we can make enormous progress. [ ] " a few weeks later, president obama discussed the acute strategic needs, "as i have said from the start of this [ebola] outbreak, i consider this a top national security priority. this is not a matter of charity-although obviously the humanitarian toll in countries that are affected in west africa is extraordinarily significant. this is an issue about our safety [ ] ." the president also signed the executive order on combating antibiotic-resistant bacteria in september of [ ] . recent outbreaks of diseases thought banished from the united states demonstrate the need for full vaccination. several communities resist vaccination, and incentives to vaccinate will increase population safety and prevent those who cannot be vaccinated from coming down with vaccinepreventable diseases. one common incentive is the requirement to be vaccinated to enter public school. waivers can be sought, but to boost the vaccination rates, state and local governments can reduce the numbers of exemptions provided. mississippi has already followed this course, and it has the highest vaccination rates in the united states. other potential policies include requiring exemption forms to be filed yearly; requiring parents to complete an education component; and requiring private as well as public school children to be vaccinated [ ] . several states are implementing one or more related measures. while only four states do not recognize a religious exemption from vaccinations, states do not allow exemptions for personal reasons (all states allow exemptions for medical reasons). in part due to the measles outbreak, on july of california will eliminate all non-medical vaccine exemptions. pennsylvania is also pondering eliminating personal exemptions. colorado has made the exemption process more burdensome [ ] . dina fine maron of scientific american [ ] suggested the following common sense approach: improved education and communication, sustain and enhance immunization outreach, maintain vigilance and rapidly contain imported infections. anthony fauci proposed partnerships, among government, industry, and academia to develop additional timely solutions to the threat of new and resurgent infectious diseases [ ] . one example of a successful academia-industry partnership is the response to the hiv/aids epidemic. aids was first recognized in the early s and the death rate steadily increased through the mid- s when it was recognized as a worldwide epidemic. research at and collaboration among academic institutions (including wayne state university) and investment by the public and private sectors (burroughs wellcome which later became glaxosmithkline) led to the development of the antiretroviral treatments used today. the partnerships transformed a deadly infection into a principally chronic disease within two decades [ ] [ ] [ ] . partnerships now work to ensure prevention, testing, distribution of anti-hiv/aids drugs and treatment worldwide. over the years fda has introduced innovations for the development and approval of pharmaceuticals including fast track, parallel track, orphan drugs, surrogate endpoints, noninferiority [ ] . according to the fda guidance [ ] , a non-inferiority (ni) study is used to demonstrate that the degree of inferiority of the drug being tested as compared to the control (an already approved drug) is less than the noninferiority margin. recently, to facilitate the development of biopharmaceuticals, a cross-industry group, including members from astra zeneca, university of texas medical school houston and smaller pharmaceutical companies, proposed a tiered evidence-based regulatory approach. in this approach tier a is the typical large phase iil approach and tier d is equivalent to the animal rule, which states that "for drugs developed to ameliorate or prevent serious or life threatening conditions caused by exposure to lethal or permanently disabling toxic substances, when human efficacy studies are not ethical and field trials are not feasible, fda may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is reasonably likely to produce clinical benefit in humans [ ] ." tiers b and c rely heavily on preclinical data and combined animal and human pharmacokinetic and pharmacodynamic (pk-pd) data fully integrated into a limited clinical program [ ] . in the c. difficile study discussed above, suggestions for prevention include: limit contact, limit inappropriate antibiotic usage, and increase surface cleaning. handwashing with soap from dispensers with sealed refills instead of open refillable dispensers can lower the risk of infection [ ] and is just one example of a common sense technique to prevent the spread of many bacterial infections. another common sense response is increased monitoring. cryptosporidium parvum did not appear to pose a risk until , people became ill, and approximately people died of cryptosporidiosis in milwaukee's water service area in . today, regulators and public health scientists are trying to identify microbes that pose a similar risk in the future. if these microbial contaminants occur in raw water supplies, they may need monitoring and treatment prior to these waters entering the potable water distribution system. the contaminant candidate list (ccl) developed by the united states environmental protection agency outlines a series of biological contaminants of concern that are not currently regulated but may pose a threat. should these contaminants move from the ccl to a regulatory framework, water supply utilities will incur added monitoring and testing of their water supply sources, and potentially added monitoring and treatment costs in their operations, but safety will likely increase as a result of these expenditures. the article discusses many of the problems and solutions due to emerging pathogens with a focus on the impact and response in the united states. these challenges are exacerbated in less well-off countries with poor sanitation, lack of access to preventative health care, unstable governments, or weak public health infrastructure. awareness is key, and this and other articles are working to spread the message. the threat from emerging diseases is continuously evolving as evidenced by the recent appearance of the zika virus. while the virus itself was isolated from the zika forest in uganda in the first half of the twentieth century, it did not begin to take a serious human toll until when it traveled from the pacific islands to brazil [ ] : it is now considered a global threat, with its vector, the aedes species mosquito living on all continents [ ] . there have been more than one million cases in brazil, and researchers noticed a surge in fetal microcephaly, a small head size for gestational age and sex indicating issues with brain growth, in zika-prone locations [ ] . it is now widely accepted that maternal infection with zika can lead to serious consequences for a fetus. for most infected, the effects will be minimal, but in addition to the fetal effects guillain-barre increases have been associated with zika infections. reliable diagnosis is not yet widely available, but reverse-transcriptase polymerase chain reaction (rt-pcr) testing of serum in the first seven days after symptom onset or igm-capture enzyme-linked immunosorbent assay (mac-elisa) analysis of samples are the most promising methods [ ] . animal models for further research, therapeutics and vaccines are required [ ] to stop the negative impacts of the disease since the vector is widespread and difficult if not impossible to eradicate. the general growing awareness of the threat posed by infectious disease because of travel, urbanization and all of the other factors described above combined with the serious consequences, primarily for pregnant mothers and their fetuses led to one of the fastest global responses to an infectious disease in the history of humankind. on april , , president obama announced that he would direct $ million in federal dollars remaining from the fund to fight ebola to fight the zika virus. the money will primarily be used for cdc and nih research on the virus, its role in birth defects, and vaccines for prevention. funds will also go to the formation of cdc response teams. this funding falls short of the $ . billion in emergency money president obama initially requested, and the shortfall is likely to delay a complete, effective response. internationally, the who designed and disseminated a global strategic response framework and joint operations plan, which can be accessed at http://www.who. int/emergencies/zika-virus/response/en/ . compare this to the response to polio, an enterovirus that causes few symptoms in the vast majority of cases, but can cause paralysis and even death in - % of cases. though poliovirus circulated in the population for hundreds of years, it did not reach 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bulk-soap-refillable dispensers zika virus the global distribution of the arbovirus vectors aedes aegypti and ae albopictus acknowledgments dr. ralph mitchell inspired me to look at the world in a new way, from the perspective of the tiny organisms that make the world what it is, but also threaten that world. key: cord- -tbtlnv b authors: massó sagüés, elena; fernández-carrión, eduardo; sánchez-vizcaíno, jose manuel title: risk of introduction of infectious animal diseases for europe based on the health situation of north africa and the arabian peninsula date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: tbtlnv b the current growth of the human population, the intensification of animal production, climate change or globalization favors an increase in the transmission of infectious diseases. risk analysis is the tool that allows the identification of the factors involved in the introduction and the spread of infectious diseases. the main objective of this work is to evaluate the risk of entry of animal infectious zoonotic and non-zoonotic diseases from north africa and the arabian peninsula to countries of the european union. a probabilistic formulation has been developed to obtain the probabilities of introduction of diseases associated with each possible route of entry in the european union. the results show that, among the infectious diseases analyzed in this study, avian influenza and newcastle disease are the ones with a higher risk of entry in the european union and the wild bird's migration is the route with greater impact. it is confirmed a moderate probability of entry of some vector-borne diseases, bluetongue and epizootic haemorrhagic disease, through wind flow from morocco, algeria and tunisia. due to the absence of live dromedary movement to europe, the more likely way of entry of the middle east respiratory syndrome is through the infected people movement from saudi arabia, kuwait, qatar and oman. this study includes different methodologies. a model of vectors dispersion in wind currents has been established to assess the risk of introduction of vector borne diseases. it is applicable both in animal health and public health. a periodical update would be useful to obtain a periodically updated risk analysis and to allow early detection of potential hazard with an increased risk over the previous years. the current growth of human population, the intensification of animal production, climate change or globalization favors an increase in the transmission of infectious diseases between distant geographical areas ( , ) . these diseases represent a threat for both human and animal health, resulting in higher mortality rates and serious economic losses with the decline in production. the risk analysis is an important tool in epidemiology and it is essential to assess the risk of introduction and possible spread of diseases in areas with little impact (emerging and re-emerging diseases). it enables the design and implementation of control measures for the disease eradication, in order to avoid, prevent and minimize the losses or consequences arising from the transmission of diseases ( ) . the development of a conceptual model is necessary to analyze the risk of introduction of diseases. it has to integrate all the identified elements that are going to intervene in the assessment of the risk. the final risk will be estimated from the evaluation of each one of the elements considered in the conceptual model, called "parameters, " and each parameter will be defined by "variables" ( , ) . the parameters considered in this study are the different routes of entry of diseases coming from north africa and the arabian peninsula into the european union (figure ) . the movement of animals, both domestic and wild, is one of the main routes of entry of diseases. animal products such as meat, milk or eggs, along with the transport of semen for artificial insemination, are possible ways of entry of pathogens ( , ) . wild birds are an important agent of transmission of diseases, hosting diseases such as newcastle disease and avian influenza ( , ) , or carrying vectors like ticks that could transmit pathogens (coxiella, anaplasma, babesia, borrelia, rickettsia, and tickborne encephalitis virus) ( , ) . the wind can be a vehicle of pathogens. many of the epidemiological suspicions are based on the fact that certain vector-borne diseases are introduced to distant regions by the dispersion of vectors in the wind flow. it has been suggested that with a wind speed of between and km/h, with a temperature of between and • c, the vector can be transported up to about km ( ) . rift valley fever, epizootic haemorrhagic diseases, lumpy skin disease and west nile fever are examples of vector-borne diseases, transmitted by insects as culicoides and mosquitoes like aedes. scientific studies prove that culicoides imicola, mainly afro-asiatic dispersion, is spreading through europe and it is expected a rise favored by global warming ( , ) . there are other diseases, such as foot and mouth disease, which can also be transmitted by the wind flow (especially in warm regions) up to km by land and km by sea, without being a vector-borne disease ( ) . the tourism and the high frequency of travels may favor an increase in the risk of transmission of diseases. spain is the route in and out europe. immigrants coming from africa and european citizens of african descent are driving frequently from morocco to europe across the iberian peninsula. we consider this entry route for diseases such as the middle east respiratory syndrome (mers), a zoonotic emerging disease with possible transmission within the human species. the majority of the cases have been related to healthcare centers, but transmission is possible between relatives. in an outbreak on south korea in , only one patient infected more than people during his stay in a hospital emergency room for days ( ). according to the data provided at the who database ( ) another route of entry is the movement of contaminated vehicles, potential source of transmission of diseases and can also vehicle vectors. vehicles transporting animals that have not been correctly disinfected could be an important source of pathogens; therefore the animals that are loaded after have a high probability of infection during transport ( , ) . glanders is a zoonotic infectious disease, endemic in africa. it is important to maintain the vigilance with this disease due to the importance of the horses' social network and the high number of movements for international competitions, and to control the disease to avoid its use as a biological weapon ( ) . there are other possible routes of entry such as bioterrorism, biological leaks, illegal trade; but not referred to this work for not having access to detailed and concrete data for a quantitative analysis of the risk of introduction of diseases by these pathways. the main objective of this work is to evaluate the risk of entry of animal infectious zoonotic and non-zoonotic diseases from north africa and the arabian peninsula to countries of the european union by different pathways. sixteen countries of north africa and the arabian peninsula are included in this study (table ) the possible routes of entry of each selected disease, based on an extensive literature review, are provided ( table ). the identification of the routes of entry has been conceptually based on the possibility of occurrence, regardless of its likelihood and frequency. the necessary data for the analysis is collected from different official databases of free access. the information relating to the movements of live animals and animal product origin between countries, are extracted from the faostat database ( ) and completed with cites trade database ( ) . it provides animal movement and animal product data for imports and exports between countries. the data is obtained in quantities. to obtain information relating to the movement of vehicles and people between countries, eurostat ( ) database is queried. it provides flights data (origin, destiny ( ) it is extracted the data referring to animal population in each selected country, the health status for each disease of the different countries and the annual immediate notifications of each disease. demographic information is obtained from the world health organization database ( ) . after carrying out a literature review of the migratory areas in the world and the more frequented areas by migratory birds, "critical site network tool-species search" ( ) was used to obtain data on the species of birds present in each country and the census, and to visualize its distribution on the global map. for the study of the possible introduction of vector borne diseases through the drag of culicoides and/or mosquitoes by wind currents, simulations (figure ) are made for the estimation of wind and particle's dispersion trajectories that reached the european territory during the year . the program "hysplit-hybrid single particle lagrangian integrated trajectory model" ( ) was used to locate areas and periods of maximum diffusion of particles. this program, facilitated by u.s. national oceanic and atmospheric administration's air resources laboratory (noaa-arl), allows creating simulations in any geographical coordinate of the globe, to different heights and dates, by using a file of gdas (global data assimilation system) climate data. this model has been used in other studies of dispersion of arthropods, especially of culicoides as carriers of bluetongue ( , ) . the program allows the creation of dispersion models for different densities (ρ) and particles size. densities of . g/cc for culicoides and . g/cc for aedes aegypti has been obtained using the formula ρ = m/v, m mass and v volume. the following data has been used for this calculation: culicoides with . mm size and . mg mass ( ) , and aedes aegypti with mm size and mg mass ( ) . our interest is to calculate the probability of occurrence of specific events: the probability of entry of j diseases by different routes from country i; where, henceforth, i∈{ ,. . ., } and j ∈{ ,. . ., }. the probability of occurrence of an event a, p (a), is defined as the quotient between the number of favorable cases to the event a and the number of possible cases ( , ) : each probability is calculated as the probability of a set of independent events (x , x . . . ), being the first event the probability of infection in the country of origin and the last one the likelihood of introduction into the country of destination. the first event is the probability of the country i being affected by the disease j, named as ppa j i , calculated as an average based on the number of outbreaks notified in the last years (from to ). we used a weighted moving average formula that gives a high probability when the country of origin had notified outbreaks in the last few years ( , , . . . ) and a lower probability to those countries that had notified outbreaks in the first years included in the study ( , , . . . ) . therefore, in the extreme cases, when a disease has never been notified or, conversely, it has been endemic in a country during the period of study, the probability will be zero or one, respectively. the formula that is needed for the estimation of the likelihood of a country i being affected by the diseases j is: the b j i, xx represents the binary value / -the value is associated to the occurrence of an outbreak of the disease j on the country i in the year xx. the results are shown in table . the probability of introduction of the j disease into the european union through live animal's trade from the country i (pia i j ) is calculated as the proportion of animals that are annually the formula chosen to estimate the probability that a j disease reaches the european union coming from the country i through transport-vehicles (pit i j ) is a ratio between the vehicles or load transported arriving to the european union from the country i and the total vehicles or load transported to the european union, multiplied by the probability of the country i being affected by the disease j (ppa j i ) and the probability of the vehicles being contaminated by the pathogen responsible for the disease j. the probability of a vehicle being contaminated is obtained from the average survival of the pathogen in surfaces (psa j ) and the average disinfection of vehicles ( . ). it is estimated that one out of every ten vehicles cannot be effectively disinfected allowing the survival of pathogens ( ) . the average survival of pathogens in surfaces (psa j ) is obtained based in the literature review ( ) and available data. glanders, newcastle disease and heartwater infective pathogens have a long average survival, more than days (psa j = ). foot and mouth disease and highly pathogenic avian influenza infective pathogens have an intermediate average survival there is no available information about some diseases in some countries; therefore, no probability has been assigned for those countries. in addition, for the countries that had never notified a disease, it has been assigned a zero probability. the probability of introduction of the j disease into the european union through the importation of animal products from the country i (pis i j ) is the ratio between the proportion of animal products that are annually transported to the european union coming from the country i and the total animal products transported to the european union, multiplied by the probability of the country i being affected by the disease j (ppa the probability of the vector or infective particle remaining viable during the route (psv j i ) is established with a review of the conditions of rainfall and humidity in the countries of study (weather history|weather underground). according to the sources consulted, the foot and mouth disease virus needs specific conditions for the dispersal over long distances by air and its survival is determined by the relative humidity, below per cent the virus is inactivated ( ) . for mosquitos and culicoides, above • c temperature and below • c temperature, their survival is limited ( ). a zero probability is assigned when the weather conditions do not allow the survival of the vector or infective particle, and a probability one is assigned when the conditions are optimal for their survival and dispersion. there is no available information about some diseases in some countries; therefore, no probability has been assigned for those countries. in addition, for the countries that had never notified a disease, it has been assigned a zero probability. there is no available information about some diseases in some countries; therefore, no probability has been assigned for those countries. in addition, for the countries that had never notified a disease, it has been assigned a zero probability. with all the probabilities calculated already, we can calculate the total probability of entry of the disease j from the country i to the european union, taking into account all the routes of entry already evaluated(pi j i ). to do this, we calculate the probability of occurrence of the opposite case, the probability of no introduction of the j disease by any of the routes of entry, using the following formula: there is no available information about some diseases in some countries; therefore, no probability has been assigned for those countries. in addition, for the countries that had never notified a disease, it has been assigned a zero probability. for the validation of the model, historic data has been used to relate the obtained results with the historic events. the information available in the oie database wahis interface ( ) has been used to the extraction of the data referring to the exceptional epidemiological events in the european union countries during the last years ( and ). following the methodology described in the previous section quantitative probabilities were obtained. the probabilities of entry for each route, country and disease are detailed in the following tables: tables - . there are some countries without information available regarding certain diseases, therefore, no probability has been assigned. with all the probabilities already calculated, the total probability of entry of the disease j from the country i into the european union by any combination of routes of entry (pi j i ) is obtained and detailed in table . furthermore, the likelihood of entry of a disease j in the european union is represented in figure . the results show that contagious bovine pleuropneumonia has no risk of entry. this disease is not only absent in europe at the moment, but it is only transmissible by direct contact between cattle, and according to the databases consulted, the movement of cattle among the affected countries and the european union is not made. there is no available information about some diseases in some countries; therefore, no probability has been assigned for those countries. in addition, for the countries that had never notified a disease, it has been assigned a zero probability. on the other hand, highly pathogenic avian influenza and newcastle disease have a major risk of entry into the european union. these disease have a high risk of entry into the european union through wild birds migration, due to the existence of different migratory routes that link the european breeding areas with african wintering areas ( , ) , resulting in a constant flow of wild birds among the affected countries of africa and the arabian peninsula, and the european union. bluetongue, epizootic haemorrhagic disease, foot and mouth disease and rift valle fever resulted to have a moderate risk of entry into the european union. it has been demonstrated the possibility of entry of this diseases into the european union through wind dispersion of vectors for bluetongue, epizootic haemorrhagic disease and rift valley fever; or through wind dispersion of virus for foot and mouth disease. there is no available information about some diseases in some countries; therefore, no probability has been assigned for those countries. in addition, for the countries that had never notified a disease, it has been assigned a zero probability. a low risk of entry has been obtained for the following diseases: middle east respiratory syndrome, peste des petits ruminants, glanders and heartwater. to complete the study, the results obtained are compared with the historic events occurred during the last years, shown in table . in and , european countries had notified outbreaks of bluetongue, highly pathogenic avian influenza, newcastle disease and rift valley fever (among all the diseases included in this study). this work demonstrates the possibility of assessing the risk of entry of different infectious diseases at the same time, and by different routes of entry into a large geographical area. different methods has been used; some of them already deployed in other studies about one single disease, african swine disease ( ), avian influenza ( ), bluetongue ( ), etc. in particular, the dispersion of particles in wind currents model has proved to be a very useful tool for the analysis of risk of spreading of certain vector borne diseases. it is applicable both in animal health and in public health, in the interest to improve the health and well-being through the prevention of risk and mitigation of the effects of emerging diseases that originate from the interface between humans, animals and the natural environment ( ). once the model is complete, an annual review may be useful to update the variables and parameters that are being used and to obtain a periodically updated risk analysis which allows detecting potential hazard with an increased risk over the previous years. in this study we worked with a few parameters and variables that can be subject to improvement. it is possible to increase table | total probability of entry of the j disease from the country i to the european union. disease "j" country "i" the number of diseases studied as well as the routes of entry included in this model. it would be interesting, with the necessary data, to include the illegal trade as another possible way of entry. we have evaluated the risk of introduction, not the risk of spread of disease, it may also be interesting to add the probability of the infectious agent that has been introduced, to come into contact with sensitive populations and disseminate. with the obtained results, the following diseases are not currently presenting a risk of entry through animal trade: heartwater, bovine contagious peripneumonia, middle east respiratory syndrome, and peste des petits ruminants. glanders has no risk of introduction through equine meat, although it has risk of entry through horse trade from bahrain and through people and transport vehicles movement (as fomites). rift valley fever has no risk of entry in the european union through wind dispersion of viruses, although it has a certain risk of entry through animal and dairy products trade, and transport vehicles movement containing vectors of the disease. the probability of entry of infectious diseases through animal and animal products trade is relatively low, due to the shortage of commercial movements between these countries and the european union (see supplementary material) . in the movement of live animals, tunisia represents a greater risk of entry of bluetongue and epizootic haemorrhagic disease for the european union, bahrain of glanders and united arab emirates of foot and mouth disease. there is a lower risk of entry of highly pathogenic avian influenza through the movement of live birds from egypt and saudi arabia. in the movement of animal products, the countries representing a higher risk of entry of diseases are egypt, mauritania, niger and saudi arabia, the three of them with a higher risk of introduction of foot and mouth disease into the european union. the probability of entry of infectious diseases through people movement and transport-vehicles is low, but they are the only likely routes of entry of certain diseases such as heartwater, peste des petits ruminants, and middle east respiratory syndrome (mers). cowdriosis is a vector-borne disease transmitted by ticks (amblyoma genus in the majority of cases). the only likely route of entry of this disease into the european union is through the movement of infected animals or carriers of the tick (with a cero probability obtained) of through the transport of the infected tick in transport-vehicles. the most likely entrance of the middle east respiratory syndrome in the european union is through infected people movement (travelers of immigrants) from countries of the arabian peninsula. outbreaks of this disease had occurred in europe in , the origin was a person coming from saudi arabia ( ) . the peste des petits ruminants, transmissible by direct contact and fomites, represents a risk for europe only by the movement of vehicles contaminated by previous trips in which carrying this type of cattle. the probability of introduction of highly pathogenic avian influenza and newcastle disease through wild bird migration is high due to the existence of different migratory routes that link the european breeding areas with african wintering areas ( , ) , resulting in a constant flow of wild birds among the affected countries of africa and the arabian peninsula, and the european union. this pathway may be responsible for the recent entry of newcastle disease in the european union, in the last years ( and ) outbreaks of newcastle disease has been notified in portugal, france, rumania, bulgaria, sweden, switzerland, check republic and cyprus ( table ). the origin of the outbreaks is not defined but following the results obtained, it may be possible that a percentage of these outbreaks may be caused by wild bird's migration from african and middle east wintering areas. there is a high risk of introduction of bluetongue and epizootic haemorrhagic disease in the european union through dispersion of particles in wind currents from morocco, algeria, tunisia and libya, and a moderate risk of introduction of foot and mouth disease through this pathway. the result of bluetongue is validated by the recent notifications; in the last years ( and ) outbreaks of bluetongue have been notified in greece, italy, and switzerland (table ) . in the other hand, there is no risk of entry of rift valley fever through this pathway. if the north african coast countries, the countries of the study that show in the simulations a certain probability of particles dispersion arriving to the european union, remain free of rift valley fever, there will be no risk of introduction of this disease. to confirm the absence of risk of introduction of rift valley fever through this pathway, the situation of the disease in the countries of the study, which so far are not affected, should be checked periodically. the main limitation of the model is the information available. the majority of information managed in this study is coming from official databases (pubmed, oie , gdas , eurostat , faostat ), although they do not have information from all countries of interest. there is only complete information of five countries: morocco, algeria, tunisia, egypt, and saudi arabia. there is not enough information for certain emerging diseases such as middle east respiratory syndrome, so this study can be updated as the research on the different diseases transmission routes progresses. in the same way, there are countries without information available, for lack of declaration on the current health situation or underreporting of diseases outbreaks. once the model is complete, could conduct an annual or biannual review to update the variables and parameters in use, to obtain an updated risk analysis that allows detecting potential dangerous routes of entry by an increase in the risk from the previous period. this work has made possible to assess the risk of entry of different infectious diseases at the same time, and through different routes of entry into a large geographical area. the use of spread sheets for the development of probabilistic formulation has been of vital importance for the collection and analysis of data, although its validity depends on the confidence and quality of the available information. in this case, there is only complete information of five countries: morocco, algeria, tunisia, egypt, and saudi arabia. it has been established a model for vectors introduction in wind flow that confirms the potential entry by this pathway of some vector-borne diseases, bluetongue and epizootic haemorrhagic disease, from morocco, algeria and tunisia. of all the diseases analyzed in this study, newcastle disease and avian influenza are the ones with a higher risk of entry in the european union. the pathway with more relevance in the risk of entry of these diseases is the wild bird's migration. the diseases with a moderate risk of entry are bluetongue, epizootic haemorrhagic disease and foot and mouth disease. these diseases have in common the possible entry through wind dispersion. in the case of vector-borne diseases it is possible by vectors dispersion in wind currents, and in the case of foot and mouth disease it is possible by virus spreading through wind currents. due to the absence of live dromedary movement to europe, the more likely way of entry of the middle east respiratory syndrome is through infected people movement, from saudi arabia, kuwait, qatar and oman. the contagious bovine pleuropneumonia is the only disease with no risk of introduction in the european union, due to the absence of cattle movement from the countries affected by this disease, chad, niger, mali, and mauritania. publicly available datasets were analyzed in this study. this data can be found here: http://www.fao.org/faostat/en/#data/ ta, http://www.fao.org/faostat/en/#data/tp, https://ec.europa. eu/eurostat/data/database, http://www.oie.int/wahis_ /public/ wahid.php/wahidhome/home, http://apps.who.int/gho/data/ node.country, http://csntool.wingsoverwetlands.org/csn/default. html#state=speciessearch, not available any more and changed into http://critical-sites.wetlands.org/en, http://ready.arl.noaa. gov/hysplit.php. js-v conceived the idea for the work. em and ef-c designed the work. em carried out the data extraction for the risk analysis. em and ef-c developed the probabilistic formulation. em designed the tables with the results for interpretation. ef-c contributed in the interpretation of the program hysplit. em obtained the wind and particle dispersion simulations and estimated trajectories. em obtained data from critical site network tool-species search and studied species distribution in the global map. js-v reviewed the design of the work and the results obtained. em wrote the manuscript. ef-c and js-v provided critical feedback and helped shape the research, analysis, and manuscript. cambio climático en españa y riesgo de enfermedades infecciosas y parasitarias transmitidas por artrópodos y roedores foot-and-mouth disease veterinary epidemiology análisis probabilístico del riesgo potencial de entrada y difusión de la análisis probabilístico del riesgo de introducción de la peste porcina clásica en españa risk assessment applied to spain's prevention strategy against highly pathogenic avian influenza virus h n transport of ixodid ticks and tick-borne pathogens by migratory birds transport of ixodes ricinus infected with borrelia species to norway by northwardmigrating passerine birds bluetongue virus serotype outbreak in the basque country (northern spain) - . data support a primary vector windborne transport using climate data to map the potential distribution of culicoides imicola (diptera: ceratopogonidae range expansion of the bluetongue vector, culicoides imicola middle east respiratory syndrome coronavirus: a comprehensive review middle east respiratory syndrome coronavirus (mers-cov)-emergencies glanders: off to the races with burkholderia mallei statistical databases of the food and agriculture organization of the united nations available online at: https://trade.cites. org/ . eurostat. statistical databases of the european commission available online at air resources laboratory-hybrid single particle lagrangian integrated trajectory model long-distance aerial dispersal modelling of culicoides biting midges: case studies of incursions into australia aedes aegypti (l.), the yellow fever mosquito: its life history, bionomics and structure significance of fomites in the spread of respiratory and enteric viral disease sánchez-vizcaíno f, martínez-lópez b, sánchez-vizcaíno jm. identification of suitable areas for the occurrence of rift valley fever outbreaks in spain using a multiple criteria decision framework manual of procedures for wildlife disease risk analysis. paris: world organisation for animal health the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fvets. conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © massó sagüés, fernández-carrión and sánchez-vizcaíno. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -eeh a t authors: lambert, paul-henri; ambrosino, donna m.; andersen, svein r.; baric, ralph s.; black, steven b.; chen, robert t.; dekker, cornelia l.; didierlaurent, arnaud m.; graham, barney s.; martin, samantha d.; molrine, deborah c.; perlman, stanley; picard-fraser, philip a.; pollard, andrew j.; qin, chuan; subbarao, kanta; cramer, jakob p. title: consensus summary report for cepi/bc march - , meeting: assessment of risk of disease enhancement with covid- vaccines date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eeh a t a novel coronavirus (cov), severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in late in wuhan, china and has since spread as a global pandemic. safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of coronavirus disease (covid- ) disease and ease the major economic impact. there has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. however, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. a syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. animal models allowed scientists to determine the underlying mechanism for the former in the case of respiratory syncytial virus (rsv) vaccine and have been utilized to design and screen new rsv vaccine candidates. because some middle east respiratory syndrome (mers) and sars-cov- vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for sars-cov- vaccines. to address this challenge, the coalition for epidemic preparedness innovations (cepi) and the brighton collaboration (bc) safety platform for emergency vaccines (speac) convened a scientific working meeting on march and , of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. this report summarizes the evidence presented and provides considerations for safety assessment of covid- vaccine candidates in accelerated vaccine development. since the identification of a novel coronavirus, sars-cov- , as the cause of pneumonia in patients from wuhan china, a pandemic has erupted, resulting in enormous health care, social and economic disruption to our global society [ ] . as of may , there have been , , cases and , deaths worldwide [ ] . in rapid response to the pandemic, academic and industry scientists from around the world have initiated efforts to develop vaccines and therapeutics for disease prevention and patient management. the coalition for epidemic preparedness innovations (cepi), a global partnership between public, private, philanthropic, and civil organizations, is funding work to develop sars-cov- vaccines using a variety of technology platforms. several vaccine candidates are already in phase studies with others likely to enter the clinic in the next few months [ ] . one of the challenges facing rapid vaccine development for sars-cov- is the need to adequately assure the safety of these vaccines. one such safety concern is disease enhancement syndrome that occurred in the s with inactivated rsv and measles vaccines. vaccinemediated disease enhancement is characterized by a vaccine that results in increased disease severity if the subject is later infected by the natural virus. during early trials with inactivated rsv vaccine, the vaccine did not prevent infection, % of those infected required hospitalization and two children died [ ] . lung pathology in patients showed an unexpected inflammatory response with both neutrophils and eosinophils, evidence of immune complex formation and complement activation in small airways [ ] . scientists later learned that the vaccine caused a similar disease enhancement in animals characterized by immunopathology and a t helper cell type (th ) biased response and antibody responses with poor neutralizing activity [ ] [ ] [ ] . since that time, the animal models have been relied upon to predict safety for new rsv vaccines that are developed. of note, the pathogenesis of rsv disease enhancement is distinct from antibody disease enhancement (ade) which occurs for macrophage tropic viruses, demonstrated most notably for dengue in humans and the coronavirus feline infectious peritonitis virus in cats, and is directly caused by non-neutralizing or sub-neutralizing antibodies leading to more efficient viral uptake via fcγ receptor binding [ ] . since pathology consistent with the rsv vaccine enhanced disease (and perhaps ade) has been demonstrated for some sars-cov- vaccine candidates in animal models, there is also a concern that a similar syndrome could occur in humans immunized with sars-cov- candidate vaccines. therefore, cepi and the brighton collaboration safety platform for emergency vaccines (speac) convened a scientific working meeting https://brightoncollaboration.us/brighton-collaboration-cepi-covid- -web-conference/) on march and , of experts in the field of vaccine immunology and coronaviruses to discuss current knowledge that could form the basis for the assessment of the risk of enhanced disease during sars-cov- vaccine development. this consensus report presents considerations for vaccine developers and can serve as a guide for the development and testing of vaccine candidates to avoid these safety concerns. ultimately, the door to clinical trials is controlled by regulators in the context of the risk/benefit for the entire dataset provided by developers and within the local trial context. microbiology and immunology and pediatrics at the university of iowa, both reviewed their work and that of others in animal models developed for sars-cov- and mers-cov. the lessons from these models can inform the development priorities for useful sars-cov- animal models to address both efficacy and safety. in inbred mouse strains, sars-cov- replicates efficiently in the respiratory tract and can cause pneumonitis, but clinical signs and pneumonia were only observed in old balb/c mice [ ] . subsequent passage of sars-cov- through mouse lungs resulted in the isolation of virus that caused severe disease in both young and old mice [ , ] . this virus was used in many subsequent studies. ferret models of sars-cov- also demonstrate virus replication in respiratory tracts with induction of a neutralizing antibody response but also demonstrated little evidence of clinical disease [ ] . hamsters, in contrast to mice and ferrets, demonstrate high levels of viral replication, develop pneumonitis, and can be shown to have clinical signs of disease [ ] . following the identification of human ace as the receptor for sars-cov- , transgenic murine models expressing human ace receptor (hace ) were developed and shown to develop mild pulmonary disease. of note, these mice also developed lethal viral encephalitis, attributed to viral spread through the olfactory nerve, despite the relative scarcity of hace expression in the brain which may have relevance to sars-cov- disease [ ] . efficacy of several sars-cov- vaccines was evaluated in these models with spike (s) protein based vaccines demonstrating neutralizing antibody and protection against pulmonary replication of the challenge virus in mice and hamsters [ ] . for dna vaccine studies, it was shown that candidate vaccines encoding the s protein conferred antibody mediated protection from challenge in mice and that vaccines encoding the n protein induced humoral and cellular immunity [ , ] . for vectored vaccines expressing sars-cov- proteins, it was shown that viral proteins were expressed in mice, ferrets, and hamsters. in these studies, neutralizing antibodies were elicited by b/hpiv , vsv, rabies, mva and adeno viruses expressing s protein, that protected against sars-cov- replication in lungs of challenged animals. however, one mva vaccine expressing the s-protein did not protect against infection [ ] . in contrast to sars-cov- , inbred mice were found to be resistant to mers-cov, thus infection was studied by creating models that expressed the mers receptor, human dpp (hdpp ). ad -hdpp transduced mice could be infected with mers virus but infection was associated with minimal clinical disease except in immunocompromised mice that developed weight loss after infection. of note, hdpp -transgenic mice developed lethal viral encephalitis with concurrent inflammatory changes on histopathological examination of the lung, similar to hace -tg mice with sars-cov- . subsequently, investigators developed mice "knocked-in" for expression of hdpp and after virus passage in these mice, identified mouse-adapted mers strains that caused more severe disease and increased histopathology with more pulmonary edema than those infected with the original mers strain [ ] . importantly, mice without functional t cells, such as rag -/-and tcr alpha-/-, had delayed viral clearance whereas mice that could not produce antibodies, mumt mice, did not show delay in clearance. similar models were developed by crispr/cas mutagenesis of two residues in the mouse ace molecule, followed by mouse adaptation with serial passage, leading to an ards model of lethal infection [ , ] . taken together this evidence supports the notion that t cells are important in viral clearance for mers [ ] . non-human primate (nhp) models have also been established for both sars-cov- and mers-cov. there was evidence of upper respiratory and lower respiratory tract sars-cov- replication in african green monkeys to a greater extent than in cynomolgus macaques, and least in rhesus macaques, with little evidence of clinical disease in all three species [ ] . of note, consistent with findings in older humans and mice, increased pathology has been documented in aged cynomolgus macaques with sars-cov- wild type infection [ ] . there is some controversy on the disease severity in the mers models with different groups seeing different levels of pathology. this has not been resolved [ , ] . both vaccine efficacy and safety have been studied in animal models with many sars-cov- candidate vaccines. the group of experts discussed how the vaccine models were utilized to characterize the response of specific vaccines and to examine both disease enhancement and antibody dependent enhancement (ade) signals. there is evidence for disease enhancement in vaccinated animals after challenge with live virus in multiple studies with sars-cov- vaccine candidates as summarized in table. we are limiting our comments in this report to data in animal models and not discussing in vitro data except to mention that there is some evidence of ade in human primary monocytes [ , ] . different animal models exhibit different pulmonary pathology but generally characterized by cellular infiltrates including eosinophils. in this summary, we provide an overview of the consensus opinion on vaccine related outcomes in animal models that were of concern for risk of disease enhancement and could guide assessments of sars-cov- vaccine candidates. in murine models, evidence for vaccine related disease enhancement has been demonstrated for inactivated whole vaccine (with and without alum), vectored vaccine expressing n protein (but not seen with vectored vaccine expressing s protein in same report), a replicon particle platform expressing s protein, and a vectored vaccine expressing s proteins. in general, the pathology described included pulmonary infiltrates often with eosinophils observed. th dominant responses were documented in some reports by expression of th driven cytokines [ ] [ ] [ ] [ ] [ ] . in a ferret model, hepatitis was demonstrated in animals vaccinated with a recombinant modified vaccinia virus ankara vaccine expressing s protein and then challenged with virus [ ] although questions have been raised about this study [ ] . [ , ] . non-human primate models have also produced evidence of enhanced disease after sars-cov- vaccine immunization. chinese macaques immunized with a modified vaccinia virus expressing s protein then challenged with sars-cov- did not develop clinical disease, but histopathology showed lung injury. this injury was characterized by decreased wound healing, and increased pro-inflammatory macrophages expressing il- , il- , and ccl [ ] . this report also demonstrated that passively administered anti-s antibody was associated with lung pathology after challenge with the live virus although the mechanism may not be through fc receptor and thus not classic "ade". of note, a second report similarly demonstrates the effect with certain anti-s antibody preparations and without fc involvement [ , ] . the relevance of these reports remains unclear as there are multiple studies with administration of neutralizing monoclonal antibodies to different models that did not induce disease enhancement. other investigators have reported absence of disease enhancement in both hamsters and monkeys immunized with a whole inactivated vaccine although these models differed in a number of ways, most notably by the use of bpl (β-propiolactone) instead of formalin for inactivation of the virus [ , ] . finally, we note that there has not be an agreed upon positive control applied in these animal studies and thus interpretations are hampered.ba animal models with sars-cov- are being rapidly developed by multiple research human ace transgenic mice (hace tg) aged - weeks and - months of age were studied and hace expression was observed in lung, heart, kidney and intestinal tissues. following intranasal inoculation with sars-cov- , weight loss was observed, and viral rna was detected in the lungs as well as in the intestine [ ] . gross pathology demonstrated swollen and enlarged lungs with moderate interstitial pneumonia. histological studies documented an accumulation of inflammatory cells including monocytes and lymphocytes in alveolar interstitium, with thickening of alveolar walls. sars-cov- s protein was detected by ihc in alveolar macrophages and epithelia [ ] . nhp were also infected with sars-cov- with rhesus macaques aged - years inoculated intratracheally and although no fever was observed, weight loss and asthenia were seen on multiple days. viral rna was detected from nasal and throat swabs and to a lesser degree in anal specimens, peaking on days to and lasting until day post infection. one animal was euthanized on day for necropsy and viral rna was detected in multiple organs including cns, skeletal muscle and heart. for the two surviving rhesus macaques, positive neutralization titers were documented by day post infection. there was radiographic evidence of multiple ground glass opacities in the lungs on days , and post infection. microscopically the lung lesions represented an acute interstitial pneumonia characterized by mild to moderate thickening of alveolar septum, increased number of macrophages, degeneration of pneumocytes and an inflammatory cell infiltration. presence of viral antigen was confirmed, predominately in alveolar monocytes and macrophages [ ] . analysis of blood samples showed a decline in counts of total white blood cells, lymphocytes and monocytes with no observed changes in percentages. a decrease in both cd +cd + and cd + cd + t-cell counts was observed. importantly, these hematological findings are similar to those seen in sars-cov- infected patients. this model could serve as a critical tool for detailed studies of pathogenesis and the evaluation of intervention strategies including vaccines. of note, following the meeting another group has confirmed sars-cov- infection in rhesus macaques with viral antigen detected in type i and type ii pneumocytes and diffuse pulmonary alveolar damage noted [ ] . experts agreed that these models and others under development should be utilized to evaluate vaccine candidates for any evidence of disease enhancement as specified in later sections. design barney the sars-cov- s protein structure was solved shortly after its emergence and shows similar structure and mobility as the sars-cov- s [ ] . the timing from first knowledge of sars-cov- to the beginning of the phase study was a remarkable sixty-five days. the advantages of mrna vaccines include ability to create a highly precise type of protein to elicit the correct antibodies, to elicit t cell responses that are th predominant, and the rapidity of manufacturing. of course, disadvantages include the novel nature of both mrna and dna vaccines without any licensed vaccine with either technology to date and lack of experience for mass production. therefore, multiple platforms for sars-cov- are under development that mitigate against some of the potential disadvantages of nucleic acid vaccines. although mrna and dna vaccines elicit t cell responses without adjuvants, adjuvants may be important for subunit and whole cell inactivated vaccines to increase their immunogenicity and drive an immune response that could limit the risk of disease enhancement. multiple sars-cov- vaccines are in development including vectored vaccines, whole cell inactivated vaccines, and recombinant protein vaccines. the experts discussed how the choice of adjuvants will be important for both efficacy and safety with these platforms. dr. arnaud didierlaurent from the centre of vaccinology at the university of geneva presented background on the effects of different adjuvants on animal and human immune responses. several adjuvants are now being used in commercial vaccines or are in clinical development [ ] . oil-in-water emulsions such as mf or as have been shown to increase the breadth of the antibody repertoire, binding affinity and affinity maturation when compared to unadjuvanted vaccines [ , ] in human studies with influenza vaccines, h n vaccine adjuvanted with mf (squalene-based emulsion) increased the levels of h -specific antibody for subclasses igg and igg and the binding to fcγr but not to fcγr when compared to alum adjuvanted vaccines. this demonstrates that the use of an adjuvant can skew the functionality profile of antigen-specific antibodies, with the potential to activate different innate effectors based on their fcγr expression [ ] . use of squalene-based emulsion vaccines for influenza have also been shown to increase cd + t cell response frequencies and crossreactivity. even if pre-existing cross-reactive antibodies are present prior to immunization, such adjuvants could activate naïve b cells and promote the adaptability of memory b cells [ ] [ ] [ ] [ ] . in addition to antibodies, adjuvants can promote cellular responses. human malaria challenge studies provided early evidence that the choice of adjuvants(combined with the malaria antigen rts,s) was critical in achieving optimal protection and highlighted the importance of cellular response [ ] . more recently, studies with hepatitis b surface antigen (hbs) vaccine adjuvanted with as , as , as or alum showed that vaccines formulated with as and as induced the highest antibody levels while as promoted best hbs-specific cd t cell response [ ] . these differences were associated with the magnitude of the initial inflammatory response triggered by the different adjuvanted formulations [ , ] . interestingly, although the level of cd t cell response was lower in the alum group compared to the as group, both adjuvants led to similar memory subset profiles and cytokine production profiles given the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for subunit vaccines in providing antigen-dose sparing, increased immunogenicity, breadth and duration of response, potentially eliciting cross-protection against new cov strains and minimizing the risk of enhanced disease. following the presentations, attendees participated in discussion of the suggested consensus statements and all attendees were asked to comment on the draft statements available online. these comments were reviewed and discussed again on the second day of the meeting and resulted in the summary consensus statement that follows. • sars-cov- has a low affinity for murine ace receptor and murine models will require the use of hace transgenic mice, preferably with a 'knock-in' approach. preliminary data indicate the possibility of infecting hace transgenic mice with demonstration of viral replication and mild lung lesions. mouse adaptation of sars-cov- , as done with sars-cov- , will likely be required to obtain a virus that causes more severe disease in mice. models that develop acute lung injury with some lethality and that mimic the human condition will be important for evaluating vaccine safety. • previous studies from sars-cov- and mers-cov indicated that some vaccines, especially those using whole inactivated virus, could enhance the disease induced in mice challenged with sars-cov- or mers-cov. the lung lesions were highly inflammatory, with a dominance of eosinophil infiltration and occurred in animals despite presence of a neutralizing antibody response and reduced challenge virus replication in the lungs. such studies have not yet been completed for sars-cov . • in mice, this immunopathology was considered a consequence of a dominant th type response to the vaccine antigens. it was not seen after including adjuvants (e.g. cpg) in the vaccine or other vaccine formulations known to drive immune responses towards th . the timing of challenge after vaccination may be critical. it would be of major interest to explore the outcome following challenge at later timepoints when antibodies are significantly decaying. • one should be aware of the potential confounding effect of cell-culture excipients in the vaccine and challenge strain material. it is known that impurities such as fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx in any mouse model if the challenge strain also contains the same excipients. • in these models, characterization of the immune response to the candidate vaccine (e.g., igg isotypes, th markers) may have some predictive value. • other small animal models which can be infected by sars-cov- can be considered (e.g. ferret, hamster). development of small animal models of severe disease will also inform studies of vaccine-enhanced disease. • passive transfer in nhps of human antibodies generated during phase trials, followed by viral challenge could be considered to assess the risk of disease enhancement. • challenge of immunized animals with a closely related heterologous cov strains may assess the risk of enhancement during future outbreaks. • in case of disease enhancement, in-depth studies in animal models may give some indications on the mechanism of immunopathology. they can inform human trial designers on the critical immunological risk markers to be monitored in phase trials. • given what will be the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for sub-unit vaccines in providing increased immunogenicity, breadth of response, dose sparing, duration of response, potentially cross-protection against new cov strains, and possibly minimize the risk of enhanced disease. preference should be given to th -driving adjuvants with an established safety profile in humans. • understanding of the role of cross-reacting antibodies from prior coronavirus infections may have on natural disease caused by sars-cov- or influence the risk of enhanced disease following vaccination may inform vaccine design. • data are needed on whether antibody waning could increase the risk of enhanced disease on exposure to virus in the long term. it was the opinion of the experts that animal data to support clinical development could address: • post-vaccination (neutralizing) antibody responses, and t cell analysis to demonstrate a th response. • post-vaccination challenge data from nhps with careful evaluation for immunopathology and quantitative virology in the animals. • small animal data may also provide important supporting evidence of safety, and hamster, ferret and mouse models are likely to be available for developers. • where possible, immunopathology experiments with a positive control (e.g., formalin inactivated alum-adjuvanted sars-cov- or sars-cov- vaccine) and a mockimmunized negative control will provide best guidance. it was felt that it will be important to establish broadly accepted endpoints and scoring systems to allow comparison of various vaccine candidates. who is working on this issue. • for vaccine constructs likely to induce a predominant th response, the group felt that animal studies should be considered before entering human phase trials in more than one animal species including nhps where possible. it was noted that the absence of a th response does not eliminate the risk of enhanced disease. • since not all studies that have begun or are about to begin will prescreen to determine preimmunization serostatus of participants, although this shall be determined retrospectively, appropriate baseline blood specimens should be obtained and stored. because the virus is spreading rapidly, such specimens will allow assessment of the immune response in both seronegative and seropositive persons as both are likely to be vaccinated. • level of neutralizing antibodies and determination of the relative ratio of binding to neutralizing antibodies will be important to assess the potential risk of enhanced disease. also, detection of initial priming that includes cd t cells and/or a cd th biased response is likely to mitigate the risk of disease enhancement. determination of memory responses will be useful, particularly if sars-cov- continues to circulate. • consideration should be given to the use of post-vaccination sera from vaccinees which could be used for antibody transfer studies in animals to look for enhanced disease and for evidence of cross-protection against other coronaviruses. • monitoring for enhanced disease in immunized participants may require longer follow-up than is usual in phase trials but need not delay phase trials. • investigators on the call requested frequent updating with both preclinical and evolving clinical data that are being developed by the different academic and industrial developers to help in decision-making about the various vaccine clinical trials. creation of a central information hub was encouraged for this purpose. • participants on the call expressed the need for standardization of protocols, data collection forms, critical assays (including reagents) and biobanking of samples from initial clinical trials to allow future re-assay once standards are agreed to and enable comparison of results across trials • the group of experts considers that the demonstration of some disease enhancement with any candidate vaccine after viral challenge in animal models should not necessarily represent a no-go signal for deciding whether to progress into early trials in clinical development of a covid- vaccine. • continuous monitoring of this risk during clinical trials in an epidemic context will be needed. • each observed effect should be discussed by the developers with their regulators who will ultimately define the actual requirements for clinical studies. the considerations in this document should be interpreted as general scientific remarks based on current knowledge to inform a research agenda that could be beneficial for vaccines in development. these considerations are not of a regulatory nature and cannot in any sense replace the need for proper regulatory consultations on the requirements for vaccines clinical trials. vaccine developers are therefore encouraged to seek individual scientific advice from regulatory authorities. a novel coronavirus from patients with pneumonia in china covid- data center developing 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induced with malaria vaccine, rts,s, is linked to plasmodium falciparum circumsporozoite protein-specific cd + and cd + t cells producing ifn-gamma impact of adjuvants on cd (+) t cell and b cell responses to a protein antigen vaccine: results from a phase ii, randomized, multicenter trial different adjuvants induce common innate pathways that are associated with enhanced adaptive responses against a model antigen in humans. front immunol systems analysis of human vaccine adjuvants innate transcriptional effects by adjuvants on the magnitude, quality, and durability of hiv envelope responses in nhps all authors attest they meet the icmje criteria for authorship key: cord- - pw y authors: koch, lionel; lopes, anne-aurelie; maiguy, avelina; guillier, sophie; guillier, laurent; tournier, jean-nicolas; biot, fabrice title: natural outbreaks and bioterrorism: how to deal with the two sides of the same coin? date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: pw y nan f or the last three decades, the outbreak events have constantly increased and became more complex to prevent, predict and contain. nowadays, health authorities concern is to identify which ones are bioterrorist outbreaks. however, natural outbreaks and biological attacks have a too intertwined nature to be considered separately and hence, in the absence of any attack evidence, differentiate them is a delicate task requiring complex, long and rigorous scientific investigations. furthermore, and as demonstrated by the covid- outbreak, the effectiveness of the response to an outbreak is closely dependent on the timeliness of the response: the effort should thus rather focus on the development of early detection and preparation measures such as the development of global contingency plans organising the action of all entities (civilians, militaries, governmental and non-governmental) in a common effort. innovative artificial intelligence is becoming unavoidable to detect the crisis and to manage it, especially in the phases of preparedness and response effectiveness. this technology largest impact will be to complement and enhance human capabilities but cannot substitute them. the human experts monitoring new threats and able to work with these systems will stay at the centre of the stage. in the last thirty years, the pace of emerging infectious disease outbreaks has significantly increased [ ] . the globalisation of international exchanges contributes to the inefficiency of common quarantine measures to contain the disease [ ] . the last ebola outbreak in in west africa was regarded as a paradigm of the issues caused by emerging infectious diseases nowadays: this extremely deadly pathogen has naturally emerged in a large new area, and its overwhelming spread has subsequently impacted europe and the united states [ ] . this observation was confirmed and emphasised by the coronavirus disease pandemic caused by the new human coronavirus sars-cov- . the effectiveness of the ongoing lockdown of billions of people during the covid- will have to be evaluated and compared to other strategies. thus, outbreaks can no longer be considered as a local and distant issue but should be regarded as a global concern [ ] . natural outbreaks and bioterrorism: how to deal with the two sides of the same coin? of course, in history, some outbreaks have been the starting point of biological attacks, even long before the discovery of microbiology. in , mongols exploited the second plague pandemic by catapulting the bodies of soldiers died from plague over the city walls of caffa [ ] . in the same lines, in the th century, the distribution of infected blankets from a smallpox hospital of english settlers probably caused the deadly smallpox outbreaks in the native americans population [ ] . in the th century, after the discovery of microbiology, a period of extensive industrial biological weapon programs started with scenarios of massive biological attacks against military troops. since , the threat is considered more focused on actions against the population or vital interest points of the nations. these biological attacks could be perpetrated by state or non-state groups in the context of low intensity or hybrid wars and bioterrorist attacks [ ] . nowadays, when an outbreak occurs, one of the first concern of the authorities is to separate a natural outbreak [ ] from an intentional act involving a biothreat agent [ ] in order to adapt their management. even the sars-cov- did not escape the suspicion to have been laboratory-engineered [ , ] . thus, this review will show that there are no easy ways to distinguish one from each other but that they share the same consequences and hence should have a shared management. accordingly, group together preparation measures and response tools against both the emergence of an unknown pathogen and an unpredictable attack will optimise the effectiveness of the response. the biological weapons convention signed in outlaws the use of biological weapons [ ] . since then, the identification of a biological attack is a major international political and judiciary issue [ ] . however, multiple nested events such as global warming [ ] , natural catastrophes [ ] , human actions [ ] and conflicts [ ] affect natural outbreaks in an unpredictable way [ ] . several authors proposed algorithms to determine, during crisis or shortly after, if the biological event had natural or induced causes [ ] [ ] [ ] . except for some criteria, like evidence of a release explicitly referring to attacks, the great part of the arguments should be carefully analysed before being attributed to a biological attack. the use of some spontaneously rare agents could denote a criminal origin, as it has been the case with the use of bacillus anthracis during the amerithrax crisis in [ ] , and, to a lesser extent, with the aum shinrikyo sect in japan in [ ] . however, the agent is not a sufficient criterion to identify natural or induced biohazard. for example, the rajneesh sect used a quite common salmonella enteric [ ] to perpetrate attacks, and some bacterial toxins are considered as a potential warfare agent precisely because of their high prevalence [ ] . in sharp contrast, recent natural outbreaks involved top select agents like ebola virus in west africa in - [ ] or yersinia pestis causing pulmonary plague in madagascar in [ ] . even the emergence of a peculiar new strain cannot be a stand-alone criterion to differentiate both events. indeed, even if there is no evidence of using such agents through history, natural agents can be modified by humans to increase their transmission, lethality or drug resistance capabilities [ ] . at the same time, some natural outbreaks were caused by naturally altered pathogens like the escherichia coli o :h in europe in , a strain that acquired and combined unusual virulence factor and drug resistance genes [ ] or in the new human coronavirus (sars-cov) identified with surprise in front of severe acute respiratory syndrome cases [ ] . if a pathogen is detected in a location where it has never been detected before, it can constitute a hint for a biological attack suspicion. it was the case with the amerithrax crisis in when a texan b. anthracis strain was found on the east coast of the usa [ ] . however, the biggest outbreak of the ebola virus occurred in a part of the continent recognised as free of the disease until then [ ] . one other clue for biological attacks could be the seasonality, arguing that if an outbreak appears during a season not compatible with the pathogen time-life, human activity could be the cause [ ] . here too, some natural outbreaks disrupted both experts and ai will have to learn how to work together and assist each other by developing collaborative intelligence. all rules like the influenza virus h n pandemic in , which appeared in april in north america with two epidemiological spikes [ ] . it unusually emerged from infected pig populations and was followed by a unique global spread [ ] . multiple starting points are commonly considered a sign of a biological attack like the five letters containing b. anthracis spores [ ] as well as the several restaurants targeted by the rajneesh cult [ ] . an attack can also occur in a single place, like the "shigella dysenteriae poisoning" in a laboratory in in the us, where one unique set of pastries has been deliberately contaminated by a laboratory strain [ ] . in contrast, the natural tularaemia outbreak in kosovo in - reached several districts simultaneously in a tensed geopolitical context [ ] and, in , the plague outbreak in madagascar had multiple index cases [ ] . even the assumption that an unusual swift spread or a large share of the population rapidly affected could be evidence for a biological attack is disputable: recent terrorist actions used non-contagious pathogen and hence reliable epidemiological data for the intentional use of a contagious disease do not exist [ ] . by contrast, the influenza virus [ ] , the sars-cov [ ] and the sars-cov- [ ] propagated very fast all around the world with more than countries affected in one year for the first one and countries in months for the second. for the current covid- pandemic, the centre for systems science and engineering (csse) of johns hopkins university (baltimore, md, usa) created a website to visualise and track the reported cases in real-time [ ] . in april , less than five months after the first alert, countries declared at least one case of infection (https://coronavirus.jhu.edu/map. html). in the same vein, the last zika virus natural outbreak showed an unusual spread, as it emerged in africa, travelled across the pacific ocean to finally trigger a pandemic in south america [ ] . is there any interest to identify one from the other? thus, to characterise an infectious phenomenon, we should merge the most advanced technics with thorough epidemiological investigations. results have to be interpreted very carefully by taking into account contextual elements and technical biases to avoid any misunderstanding [ ] . the list of common-sense items is beneficial to process data and improve awareness but should not be solely used to distinguish the origin of an ongoing event because of a lack of reliability (). it should be noted that all criteria and weightings have been determined retrospectively based on past outbreaks and bioterrorist attacks. one of these algorithms has been reviewed in the light of new infectious events but have not yet proven its effectiveness on a prospective basis [ ] . the confusion surrounding these criteria confirms that both phenomena have intertwined nature. moreover, during a natural outbreak, depending on the knowledge about its hazardousness and transmission, the infectious agent can be secondarily regarded as a biothreat agent, like it is now the case with the us department of justice currently considering people who intentionally spread the sars-cov- as terrorists [ ] . however, these political considerations are far away from health workers and do not consider the public health issues of quick detection and response. indeed, even if the substantial remaining risk in the case of an attack is the possibility of secondary actions aiming to maximise damages to the emergency infrastructure [ ] , the real challenge for global safety remains the early detection, the accurate characterisation and the establishment of specific measures, whatever the outbreak origin [ , ] . during the covid- crisis, it had been estimated that the early detection and isolation of cases would have been more efficient to prevent infections than travel restrictions and contact reductions [ ] . the challenge of an early detection some diseases like influenza are internationally monitored [ ] while some others are subject to active surveillance in an outbreak context, like the ebola virus during the last outbreak in west africa [ ] . for such well-known diseases, the case definition is clear and an outbreak is declared when the number of cases exceeds what has been expected [ ] . this classic and passive way of detection is efficient but has numerous drawbacks as it requires an expensive and complex public health network and is often activated with a certain delay. however, when it comes to a new disease or pathologies with polymorphic or nonspecific symptoms, the case definition and the outbreak declaration threshold are subject to debate [ ] . the source of the infection can be as unpredictable as the local outbreak of anthrax in reindeers triggered by a permafrost melting [ ] or the detection of the variola virus in ancient mummies [ ] . most of the time, the high volatility lying in the infectious process complicates the record of the cases. for the same exposition, symptoms can differ according to individual variables like health status or genetic factors [ ] or to collective variables involved, for example, in the chain of transmission [ ] but also cultural or socio-economic factors: the most-disadvantaged individuals will develop more severe and hence more specific forms of the disease but will have a belated use of health care [ ] . on the other hand, systematic environment monitoring for all diseases is, for now, impossible due to technological barriers and cost challenges. experts in biodefense suggested that more targeted measurements in delimited spaces or during a large gathering of people should be the priority to improve the sensitivity and specificity of the early detection of a biological attack but, also for a natural outbreak, while reducing the cost [ ] . for example, the analysis of wastewater could be a good way to monitor the spread of sars-cov- in the community [ ] . however, these measurements should always be paired with epidemiological investigations to avoid any misinterpretation of the results [ ] . thus, for the moment, health workers would first notice an unusual event (disease or an unusual number of cases) and should be able to alert public health officials [ ] as protecting themselves from contagiousness. given the importance of early detection, training has to be a building block in infectious diseases programs in order to promote unusual event awareness [ ] . the implementation of information technologies leaves room for improvement in the outbreak detection process [ ] as more and more stakeholders of the health care system use informatics tools in their daily practice. yet, considerable efforts have been made on information technologies and electronic query of a data set to improve the efficiency of surveillance [ ] . it's an imperative prerequisite for the implementation of an electronically assisted surveillance. currently, data management tools can aggregate several inputs and are already used for epidemiological studies or trigger an alert [ ] . internet-based surveillance systems offer a logistically and economically appealing extension to this traditional surveillance approach. the results are immediate and allow access to a paucisymptomatic population or people who are not using the health care system [ ] . this methodology has been used in in china to reconstruct the progression of the sars-cov- outbreak [ ] . despite ethical concerns and regulatory barriers, social networks appear to be a powerful data collection tool and can also be used as a medium to communicate sanitary messages or alerts [ ] . however, here again, these data are subject to many biases and should be carefully interpreted. indeed, the simple act of online documentation is just an indi-rect marker of disease, and such detection system could trigger an alert just because a worldwide released blockbuster movie increases the anxiety of population or a massive hacking produces millions of requests. taken to its logical extreme, the next step of this epidemiologic watch would probably allow the contribution of the internet of things (iot) already used to follow chronic illness [ ] and for biomedical research [ ] . a smartphone or a smartwatch is now able to detect modifications of vital parameters like temperature or heart rate. the capability of crossing these types of information with, for example, geotracking solutions, could alert competent authorities on an ongoing infection and help them to implement more rapidly appropriate measures and focus on a possible source of contamination. this seems to be only the beginning of iot possibilities as the future could be even more connected with the development of projects like smart cities. nowadays, china is already using video surveillance systems to follow its citizens and detect incivilities [ ] . likewise, criminality hot spots prediction by artificial intelligence (ai) is no more fictional in los angeles [ ] . these new technologies already have some applications in epidemiology as the detection in real-time of restaurants with a higher risk to be sources of foodborne diseases [ ] . in the medical field, computers start to help clinicians in the diagnostic of mental illness through the facial expressions and head gesture in a video [ ] but could also be used to detect an infectious disease at the prodromal phase with potential highest efficiency than thermic portals. the crossing data obtained from surveillance systems combined with machine learning capabilities in prediction and diagnostic could be used to help early detection of an infectious phenomenon in a population. this early detection could guide further specific actions of screening to identify potential patients and even the source of the infection. in korea, during the covid- crisis, gps from cellular phone, credit card transaction log and video footage had been used to monitor the patient's contact and avoid further transmissions [ ] . however, the implementation of such surveillance systems is not without legal and ethical issues and should be carefully considered. the privacy policy has to be carefully examined as well as the securing of the transmission and storage of sensitive medical data, not to mention the possible human rights abuses in non-democratic countries [ ] . these concerns have already been raised during the current covid- pandemic [ ] but there is still no international consensus on the use of personal data. pending the advent of ai tools, many initiatives have been recently proposed to facilitate the investigation of epidemics in the genomic era. the whole-genome sequencing already can help to determine the origin of an outbreak and also to explain the dispersion of the pathogen through its local evolution [ ] . new tools may include online data processing [ ] up to the development of original algorithms to aggregate spatial, temporal, epidemiological and genomic data [ ] . the interactions of this technological surveillance system with the previously described classic one are also possible at the condition to continue to improve the data-sharing practices [ ] . the use of the nextstrain tool [ ] in the context of sars-cov- (https://nextstrain.org/ncov) perfectly illustrates the potential of such approaches in the context of spreading epidemics [ ] . in the years to come, the epidemiological monitoring system of our societies will probably rely on economic capacities, technical development capabilities and societal choices, with the common objective to early detect outbreaks, regardless of their causes (). even if the epidemiological monitoring is the crucial step to respond to an outbreak, detection is useless if the resources to deal with the crisis are unavailable. being pre- pared includes but is not limited to health workers being trained to detect, react and alert the health authorities: quick and reliable equipment has to be available and health workers have to be used to work with them. dedicated infrastructures have to be prepared and ready for activation and personal protective equipment (ppe), intensive care devices and treatments have to be stockpiled. the covid- crisis revealed that the lack of simple ppe put the all health system at high risk [ ] . several authorities (civilians or militaries, governmental or non-governmental entities) already have some contingency plans but the compartmentalisation between different governmental branches and the nebulous labelling of the means between natural outbreak or bioterrorist attack dedicated sometimes prevent an accurate global appreciation [ ] . as it is, and as unfortunately still demonstrated during the covid- pandemic, if an outbreak would occur, there is a risk, even for the highly trained first aid service in the most developed countries, to get in each other's way. by learning how to work together, synergies could be developed to improve health response [ ] . after the failure in the control of the last ebola virus outbreak by the who, international agencies called for better international preparation to respond to future outbreaks [ ] . thus, international and european research networks managed to improve the speed and effectiveness of the present deployment on a validated diagnostic workflow for sars-cov- [ ] . this demonstrating the response capacity that can be released through the coordinated action of academic and public laboratories like pre-pare [ ] . in , in china, coordination by the central authority allowed to deploy medical staff and built new hospitals in wuhan in a tight schedule. in europe, crisis management strategies were different among countries, but cooperation helped relieve overloaded intensive care units in some regions and saved lots of patients. in the meantime, other consortiums like grace may also help us to prepare the possible future sanitary crisis [ ] . developments of ai do not only help for early detection, but make available a full set of possibilities in crisis management to the authorities. by using classic risk analysis documentation with ai tools, it is now possible to generate predictions to improve the resilience of a system and to mitigate the risk [ ] . the preparation phase of the crisis can also benefit from ai tools by ordering the reuse of information from previous crises [ ] , improving the stockholders' training with a serious game approach [ ] , helping to design realistic plans [ ] or even boosting the discovery of new drugs [ ] . resources management can also be improved by the use of ai in terms of network structuration [ ] as well as for the mean's allocation [ ] . during the crisis, ai can also sort information from many sensors, merge it and make it relevant for the user responsible of the situation assessment [ ] , which will be helped by a decision-support system [ ] to design the best crisis response. for example, during the covid- crisis, social contact matrices had been used to project the benefit to maintain social distancing measures [ ] . over the past ten years, epidemiological and mathematical modelling data were essential for risk characterisation and management during infectious disease outbreaks [ ] but ironically, the rising power of ai systems will not erase the role of human experts [ ] . indeed, intuition and emotions are known for a long time to be part of the decision-making process [ ] . during crisis management, expert intuition developed through years of practice is described as more realistic than pure analytical thinking [ ] . moreover, both intuition and creativity are part of the problem-solving process [ ] . both experts and ai will have to learn how to work together and assist each other by developing collaborative intelligence, which will be the best way to solve complex problems (). this was experienced during the covid- crisis in which experts, assisted by simulations, had to make decisions to control the spread of a virus still little known. inevitably, to develop an anticipation-centred view required investment. the economic justification of such an investment was underlined for a long time (even before the amerithrax crisis) [ ] , and recently, a panel of usa experts recommended reinforcing the biological threat characterisation research at a federal level with clear safety, ethical and practical guidelines [ ] . splitting outbreaks into two causes is not cost-efficient and seems absurd as dangerous pathogens to human can be used for biological attacks but are first and foremost causing natural outbreaks [ ] . however, studies about the burden-adjusted research intensity showed that diseases with a greater impact are still underfunded [ ] in an economical context where citizens are more and more concerned by public expenses. indeed, if the vaccine policy implemented were economically profitable in the usa during the influenza pandemic [ ] , a sim-ilar strategy caused substantial wastage in australia [ ] . thus, authorities have to be very careful to dimension their actions appropriately, even though a delayed response is severely judged by public opinion as during the ebola outbreak [ ] . hence, authorities and experts should improve the global contingency plans, especially on catastrophic biological risks, which represent a small portion of the biological threats but with substantial potential consequence for humanity [ ] . for a health care system, the preparation for a biological attack [ , ] or a natural outbreak [ , ] is globally the same challenge. moreover, preparedness for biological attacks has a significant added value that helps to strengthen preparedness for natural outbreaks, and vice versa [ ] . it is therefore economically interesting to consider the natural biological risk and the possibility of an attack as a single threat in the preparation of the response to an infectious event with epidemic potential. the crisis generated by the numerous deaths of covid- and the lockdown of billions of people will probably trigger a new evaluation of public policies to control outbreaks with the opportunity that the public opinion will look at it through fresh eyes. indeed, the uncertainty associated with scientific knowledge often challenges decision making and opens the way to the contestation of expertise [ ] . sometimes, the best intentions can result in a health disaster, such as the deployment of a peacekeeping force and the cholera outbreak in haiti in [ ] or the project of spreading some modified mosquitoes to fight against malaria [ ] . technology allows us to modify organisms specifically leading to the reconstitution of the spanish influenza virus [ ] or to unexpected results as a test for a new poxvirus vaccine resulted with an ultra-virulent strain able to neutralise the immune system [ ] or, during research experiments mimicking natural phenomenon, the generation of highly-resistant b. anthracis spores [ ] and viruses acquiring airborne transmission [ ] . nowadays, these widely used technics appear to be almost obsolete in comparison with the new capacities of gene synthesis: a horsepox virus has been reconstructed using only internet-bought sequences [ ] , and a new bacterium has been created de novo in a laboratory [ ] . currently, the possibilities of genome editing technologies like crispr-cas seems to be limitless [ ] . some malicious scenarios have already been imagined with a genetically modified virus infecting mosquitoes able to perform gene modification of crops in a field [ ] . the south african « coast » project [ ] that aimed at developing a bacterial agent able to selectively kill a part of the population could now be a terrifying technical possibility. thus, even if applications of some of these modified organisms may be highly beneficial, as the recycling complex wastes [ ] , they are swamped in the middle of the wanderings reported by the media [ ] . due to all these miscalculations and misguidances, society lost confidence in the authorities and national programs. it leads to society-born threats with notably the growing emergence of highly antibiotic-resistant bacteria due to the improper antibiotic use [ ] or the re-emergence of nearly forgotten pathogens linked to the mistrust in public health programs like vaccination programs [ ] . this lack of confidence extends to crisis management programs and can compromise outbreak management measures the same way it happened with the ebola outbreak in [ ] or currently, with the beginning of the management of the covid- pandemic and the lockdown decision [ ] . however, during the co-vid- pandemic, the transparency about its progress reported in real-time, for the first time in the outbreaks' history, lead to better comprehension and cooperation of people [ ] . thus, every decision can have a dual nature and should be considered carefully before being implemented (). that is why, while encouraging research, technologies and their application must be controlled to avoid any misuse and major communication actions are needed to overcome the public reluctance. ethics in research and data publication must also be placed at the centre of researchers' concerns. the intricate nature of natural outbreaks and biological attacks is too important to consider them separately. to create an efficient way to detect and contain them, the first step is to anticipate them by performing continuous scientific and epidemiological monitoring. still, the most serious and unpredictable events are referred as "black swan events" and despite our inability to foresee their occurrence, knowledge keeps being the key concept to anticipate them [ ] . thus, we need to continue and intensify networking at local, regional and global levels. stakeholders from a broader range of backgrounds must be involved to monitor the evolution of threats and update existing procedures by developing concrete and immediately applicable solutions in crisis. the biological crisis is becoming a field of expertise by itself, and it is no longer enough to be a specialist in crisis management, 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the covid- pandemic covid- : fighting panic with information emerging risk -conceptual definition and a relation to black swan type of events -sciencedirect phd institut de recherche biomedicale des armees departement de microbiologie et maladies infectieuses unite de bacteriologie bretigny sur orge france lionel the authors ensure the quality and integrity of their research and declare that their research the authors completed the icmje unified competing interest form (available upon request from the corresponding author), and declare no conflicts of interest. key: cord- - fty yi authors: ni lochlainn, m.; lee, k. a.; sudre, c. h.; varsavsky, t.; cardoso, m. j.; menni, c.; bowyer, r. c. e.; nguyen, l. h.; drew, d. a.; ganesh, s.; lavigne du cadet, j.; visconti, a.; freydin, m. b.; modat, m.; graham, m. s.; capdevila pujol, j.; murray, b.; el-sayed moustafa, j. s.; zhang, x.; davies, r.; falchi, m.; spector, t. d.; chan, a. t.; ourselin, s.; steves, c. j. title: key predictors of attending hospital with covid : an association study from the covid symptom tracker app in , , individuals date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: fty yi objectives: we aimed to identify key demographic risk factors for hospital attendance with covid- infection. design: community survey setting: the covid symptom tracker mobile application co-developed by physicians and scientists at kings college london, massachusetts general hospital, boston and zoe global limited was launched in the uk and us on th and th march respectively. it captured self-reported information related to covid- symptoms and testing. participants: , , users of the covid symptom tracker app. uk ( . %) and us ( . %) population. data cut-off for this analysis was st april . main outcome measures: visit to hospital and for those who attended hospital, the need for respiratory support in three subgroups (i) self-reported covid- infection with classical symptoms (sr-covid- ), (ii) self-reported positive covid- test results (t-covid- ), and (iii) imputed/predicted covid- infection based on symptomatology (i-covid- ). multivariate logistic regressions for each outcome and each subgroup were adjusted for age and gender, with sensitivity analyses adjusted for comorbidities. classical symptoms were defined as high fever and persistent cough for several days. results: older age and all comorbidities tested were found to be associated with increased odds of requiring hospital care for covid- . obesity (bmi > ) predicted hospital care in all models, with odds ratios (or) varying from . [ . ; . ] to . [ . ; . ] across population groups. pre-existing lung disease and diabetes were consistently found to be associated with hospital visit with a maximum or of . [ . , . ] and . [ . ; . ]) respectively. findings were similar when assessing the need for respiratory support, for which age and male gender played an additional role. conclusions: being older, obese, diabetic or suffering from pre-existing lung, heart or renal disease placed participants at increased risk of visiting hospital with covid- . it is of utmost importance for governments and the scientific and medical communities to work together to find evidence-based means of protecting those deemed most vulnerable from covid- . trial registration: the app ethics have been approved by kcl ethics committee remas id , review reference lrs- / - as the coronavirus disease (covid- ) pandemic escalates and countries struggle to contain the virus, healthcare systems are under increasing pressure as unprecedented numbers require hospitalisation and respiratory support. emergency departments and intensive care units worldwide are under strain, and medical resources are being diverted to tackle the crisis. there is a pressing need to identify risk factors for severe disease, and particularly to identify key predictors of hospitalisation amongst patients with covid- . in order to address this, we used self-reported data collected on the covid symptom tracker app ( ) to identify key demographic risk factors for hospitalisation and the need for respiratory support in the context of covid- . as part of the coronovirus pandemic epidemiology (cope) consortium ( ), the covid symptom tracker smartphone application ("app") co-developed by king's college london, massachusetts general hospital, and zoe global limited was launched in the uk on th march and was available in the us beginning th march ( , ). individuals without symptoms are encouraged to use the app. it captured self-reported information related to covid- symptoms. on first use, the app records the user's self-reported location, age, and core health risk factors (table ) . at this point height and weight are self-reported, allowing calculation of body mass index (bmi). with continued use, participants provide daily updates on symptoms, information on health care visits, covid- testing results, and whether they are seeking healthcare, including the level of intervention and related outcomes. what sex were you assigned at birth? what is your height? what is your weight? do you have lung disease or asthma? do you smoke? do you have heart disease? do you have diabetes? do you have kidney disease? what treatment did you receive while in the hospital / what treatment are you receiving right now? a) none b) oxygen and fluids (breathing support administered through an oxygen mask, no pressure applied c) non-invasive ventilation breathing support administered through an oxygen mask, which pushes oxygen into your lungs d) invasive ventilation; breathing support administered through an inserted tube. people are usually asleep for this procedure table . relevant questions asked in the covid symptom tracker app exposure, outcome and covariates were all ascertained via the app. a subset of individuals reported being tested for covid- . bmi was calculated as kg/m and considered as a categorical variable ( ). classical symptoms of covid- are defined as "high fever and persistent cough for several days". this was either directly selfreported as an outcome of an enrolment question or if participants reported jointly fever and persistent cough for more than two days. visit to hospital was recorded if the location was ever self-recorded as hospital or "back from hospital". data from the app were downloaded and only records where the self-reported characteristics fell within the following ranges were utilised for further analyses: age between ( in the us) and ; bmi between and kg/m . separate logistic regression models were fit to predict two different outcomes: . visit to hospital as outcome were fit to test for association between i) self-reported obesity and ii) chronic lung disease and asthma, heart disease, diabetes and kidney disease in the following groups: ) self-reported covid- infection with classical symptoms (sr-covid ); ) self-reported positive covid- test results (t-covid ); ) imputed/predicted covid- infection based on symptomatology (i-covid ) imputation for testing positive for covid was performed using the data at day of maximum sum of symptoms and applying a logistic regression using coefficients defined previously ( ) . samples from the coefficient distribution were sampled to create the predictions. a participant was considered as positive % of the time. please see supplementary table of the coefficients for the logistic regression. . the need for respiratory support (oxygen or ventilation) as the outcome to test for similar association with comorbidities in the specified groups was carried out, for participants who had provided information about the treatment received in hospital. sign of recovery was defined as a decrease of more than two points in the sum of reported symptoms compared to the maximum sum of symptoms. the total studied sample comprised , , individuals who supplied their self-assessment on the covid symptom tracker app after quality control, including . % males. . % of the sample reported at least one of the following comorbidities: diabetes, lung, heart or kidney disease. table summarises the demographic characteristics in the different population groups following the removal of those for whom the outcome (recovery or hospital visit) was still unknown. sr-covid- figure illustrates the differrence in age and bmi distribution across the different groups. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . requirement for respiratory support (rs) amongst those who reported a visit to hospital and then subsequently updated the app with the medical support they received, respiratory support (oxygen and/or ventilation) was reported in . , . and . % of the cases, for the t-covid- , sr-covid- and i-covid- groups respectively. plotted ors for needing respiratory support, according to the three defined groups are shown in figure . pre-existing lung disease and diabetes were consistently associated with a higher risk of requiring respiratory support with or ranging from . for lung disease in t-covid to . for diabetes in sr-covid . individuals with obesity were notably more likely to require medical support for the t-covid and i-covid groups with or of . , t-covid- and i-covid- respectively. age was also more predictive of treatment course than of hospital visit with or around . in all three groups. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . in this study we found that age, obesity, diabetes and pre-existing lung, renal and cardiac disease, were risk factors for a hospital visit with covid- amongst a large but relatively young, community-based population of app users. while a number of studies have now reported the increased risk of sars-cov- in those with diabetes, hypertension and cardiovascular disease (cvd) ( - ), all of which are associated with obesity, the reporting of body mass index (bmi) or obesity has been left out of most initial studies. only studies have reported bmi in the context of covid- (table ). the omission of obesity from initial cohort studies is particularly lamentable when one considers that during the h n outbreak ('swine flu'), obesity was found to be an independent risk factor for ventilation, as well as increased morbidity and mortality ( , ) . since then, kwong et al. have published a study that explored the relationship between bmi and seasonal influenza infection using a series of canada's cross-sectional population-based health surveys covering twelve influenza seasons ( ) . analysis of the retrospective cohort demonstrated that obese people are at greater risk for respiratory hospitalisations during the seasonal flu periods. the world health organisation reports that . % of adults in the uk are obese, the third highest rate in europe. worldwide, more than million adults are obese ( ). obesity is associated with an increased risk of a wide range of diseases ( ) , along with being associated with greater risk of infection, and of developing serious complications of common infections ( ) . importantly, obese individuals are at higher risk of developing acute respiratory distress syndrome (ards) ( ), a common complication of sars-cov- ( ). in the developed world age is now the leading risk factor for many of our most common diseases, including cvd, cancer and neurodegenerative disease ( ) . older adults are most affected by annual influenza outbreaks with % of influenza-associated deaths in the us in the - season being adults aged > years ( ) . initial data emerging from the countries first affected by the covid- pandemic reveals that older adults are again disproportionately affected, with multiple studies reporting higher mortality, a more severe disease course and longer length of hospital stay amongst the older population ( , ( ) ( ) ( ) , ) . the increased vulnerability of older adults to infection is often attributed to 'immunosenescence', a term which refers to decline in immunity with advancing age ( ). immunosenescence is associated with increased susceptibility to infection ( ), re-. cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . emergence of latent infections ( ) , reduced immune surveillance, contributing to diseases such as cancer ( ) and the reduced efficacy of vaccines ( , ) . related to this is the concept of 'inflammageing', referring to the increase in systemic inflammation noted in older adults ( ) . thus, the ageing immune system of an average older adult has higher numbers of circulating inflammatory cells but each with reduced ability to fight infection. however, ageing is a heterogeneous process, and some older adults, especially the physically active have immune systems more comparable to younger adults ( ) . we show older age as a risk factor for hospitalisation with covid- but also urge more definition and careful consideration by policymakers and healthcare researchers alike. advice from governments recommending them to stay indoors and "shield" or "cocoon" ( , ) , due to their increased risk of morbidity and mortality from covid- may be appropriate on average, but more sophisticated approaches (including immunity testing) should be conducted to avoid blanket decision making on the basis of age alone. the number of people over has doubled since , now exceeding million. careful consideration must be given to older people in the development of diagnostic, therapeutic, rehabilitation and vaccination programs, in order for such programs to maximise autonomy and minimise the burden on older people both from covid- and the measures introduced to manage it. research in other respiratory viruses clearly suggests that patients with chronic respiratory diseases, particularly chronic obstructive pulmonary disease (copd) and asthma, would be at increased risk of sars-cov- infection, and of more severe presentations of covid- ( , ) . however, chronic lung disease appears to be under-represented in the literature to date for covid- ( ); a similar pattern was seen with sars. with the exception of one single-centre retrospective sars study ( ) , clinical studies to date that report on comorbidities in either covid- or sars, reveal little evidence that chronic lung disease is a key predictor for hospitalisation in these groups (table ). to place this table in context, the world health organisation (who) estimates copd prevalence to range between - %, depending on the country, and rising considerably with age ( ) .the lower reported prevalence of chronic lung disease in the literature amongst patients diagnosed with covid- is unexplained. it is possible that, in contrast to the diagnosis of diabetes, there was substantial underdiagnosis or poor recognition of pulmonary disease in patients with covid- patients described in early chinese cohort studies. notably, the covid symptom tracker app asked participants: "do you have lung disease or asthma?", and we believe that the group answering yes to this question likely includes a large proportion of individuals who self-reported mild asthma (or a previous history of mild asthma). such diagnoses would possibly not be deemed sufficiently significant for hospital doctors to document as a relevant comorbidity when reporting patients in cohort studies, and so we caution against overinterpretation of the lung disease data herein. as previously mentioned, many of the initial studies on covid- reported an increased risk of sars-cov- in those with diabetes, hypertension and cvd ( - ), in the case of diabetes, covid- has also been associated with developing diabetic ketoacidosis, even amongst those who usually have good glycaemic control ( , ) . less well-reported in the context of the pandemic, however, is renal disease. a number of the initial studies from china found that pre-existing renal disease was a risk factor for more severe covid- infection, as summarised in a meta-analysis by henry and lippi ( ) ; an overall or of . ( % ci . - . ) was found for the association of chronic kidney disease to severe covid- across early studies. in addition, it is becoming apparent that covid- can cause acute kidney injury (aki) in covid- , and this confers a worse prognosis: a chinese prospective cohort study (n= ) reported that % and % of covid- patients had proteinuria and haematuria on admission, respectively, with . % of this cohort developed aki during their hospital stay ( ) . importantly, this study also reported higher mortality from covid- for those who had preexisting kidney disease. as we learn more about aki in covid- , it is becoming paramount to disentangle whether pre-existing renal disease predisposes patients to significant aki in covid- , as one might expect. while we did find a significantly increased risk of attending hospital for those with renal disease, relatively few reported this condition in our cohort (table ) , which may represent a problem with self-reporting bias. more research is needed to examine the effect of pre-existing renal disease on covid- infection. despite the literature consistently showing males to be at higher risk for more severe covid- ( , , ) , we did not find gender assigned at birth to be a predictor of attending a hospital in this study. however, male gender was associated with a substantially greater risk of requiring respiratory support, in all groups. men have been shown to be less likely to utilise medical services than women ( ) and large studies have consistently shown women to report greater numbers of physical symptoms than men ( , ) . such health behaviours may lead to men having a higher threshold for visiting the hospital with covid- than women, along perhaps with less awareness of early symptoms. this may result in males being more unwell when they do seek medical help and contribute somewhat to the stark difference in mortality between genders ( ) ( ) ( ) . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . our sample size is the largest reported to date, adding significant strength to many predictors of hospitalisation or severe covid- that have been identified in smaller cohort studies to date. we hope that our data will guide clinicians to protect their at-risk patient cohorts and policymakers to consider these vulnerable groups when planning and allocating resource, both during this pandemic and for future similar eventualities. clearly many questions remain, and the mechanisms by which a number of these predictors of hospitalisation are unclear; much research is needed to define these questions, and quickly. our study has a number of limitations. first, all the data collected is self-reported, and questions on comorbidities were somewhat simplified to ease reporting at large scale on an app. both symptoms and test results may be subject to reporting bias. secondly, the sampling using an app will under-represent individuals without smartphone devices, including older participants, and is likely to under-represent those severely affected by the disease. additionally, we are reporting visits, rather than admissions, to hospital; we do not know how many visits resulted in an inpatient stay. while we believe that our sampling provides useful information about the risk of most symptomatic infection, it will not provide insight into very severe disease as the most unwell patients may not record hospitalisation due to incapacitation or even death. additionally, covid- diagnoses, where confirmed by testing, were likely to be based on rt-pcr which is thought to be between - % sensitive for a single test ( , ) . another important caveat of note is that the individuals on which the model was trained are highly selected because covid- tests are not performed at random ( ) . the participants were tested because they either displayed severe symptoms, were in contact with covid- positive individuals, were healthcare workers or had travelled to an area of particular risk. additionally, the app captured whether participants had been diagnosed with covid- but did not specifically ask when. given that symptoms are recorded at the time of data entry, it is possible that some individuals may no longer have been symptomatic from the virus. the key predictors of hospitalisation in the context of covid- affect many in our society. lung disease, diabetes, heart disease, advanced age and increased bmi were associated with risk of hospitalisation with covid. careful planning of the use of immune testing and contact tracing could be particularly relevant for people in these higher-risk groups. this may help minimise the risk that patients living with comorbidities and older people are disproportionately isolated in the months to come. finally, the presence of these factors should be regarded as an important factor in future risk stratification models for covid- . it is of utmost importance for governments and the scientific and medical communities to work together to find evidence-based means of protecting those most vulnerable from covid- in a way which minimises their risk of economic, mental and social implications of isolation. in the uk, app ethics has been approved by kcl ethics committee remas id , review reference lrs- / - . in the us, the informed consent process was approved by the partners human research committee (protocol p ). all subscribers provided informed consent before submitting responses. the study was registered with clinicaltrials.gov as nct . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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of severe acute respiratory syndrome in hong kong key: cord- - syce n authors: domínguez-andrés, jorge; netea, mihai g. title: impact of historic migrations and evolutionary processes on human immunity date: - - journal: trends immunol doi: . /j.it. . . sha: doc_id: cord_uid: syce n the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host–pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. the evolution of mankind has constantly been influenced by the pathogens encountered. the various populations of modern humans that ventured out of africa adapted to different environments and faced a large variety of infectious agents, resulting in local adaptations of the immune system for these populations. the functional variation of immune genes as a result of evolution is relevant in the responses against infection, as well as in the emergence of autoimmune and inflammatory diseases observed in modern populations. understanding how host-pathogen interactions have influenced the human immune system from an evolutionary perspective might contribute to unveiling the causes behind different immune-mediated disorders and promote the development of new strategies to detect and control such diseases. infectious diseases are arguably the main source of evolutionary pressure that humanity has ever confronted. the dispersion of different human communities around the globe has exposed each population to different infectious agents, exerting a selective pressure (see glossary) on them; thus, adaptation to the new environment has favored the selection of the most beneficial genetic variants for the host. as a result, infectious agents have caused the expansion of alleles behind the induction of either protection or tolerance to these diseases; heritable variations, that increased the survival to deadly infectious agents, may have been naturally selected before the hosts had the opportunity to reproduce [ ] . natural selection driven by pathogens is probably more remarkable for those infectious agents that have been among us for a longer time, namely the causative agents of well-known diseases such as leprosy, smallpox, malaria, or tuberculosis. the genetic imprint of pathogen-driven selection depends on the length and the virulence of the infections and also the geographical distribution. the human genome presents more than genetic loci with traces of selective pressure [ ] . this group includes more than immune-related genes with functional variations between populations, which are probably behind the variability of responses to immune-related diseases reported nowadays [ , ] . besides natural selection, other evolutionary mechanisms, such as genetic drift, greatly influence the frequencies of the genetic variants found within diverse populations throughout the world [ ] (box and figure ). with the burst of next-generation sequencing and the development of cutting-edge technologies such as transcriptomics, proteomics, and systems biology, we are starting to witness the great impact of evolutionary processes on human immunity and how the interactions between microorganisms and humans that took place millennia ago might play a fundamental role not only in the response against modern pathogenic threats, but also in the emergence of autoimmune and inflammatory diseases observed in modern populations worldwide. in this review we offer a novel perspective on the role of infectious diseases as agents of natural selection and as forces behind the evolutionary pressure encountered by human ancestors and modern humans in their migrations around the globe. specific genetic variants selected throughout different periods of human history may have influenced immune responses of present-day populations against pathogenic microorganisms and may have played a role in the development of certain inflammatory and autoimmune diseases. the majority of experts agree that africa is where our species originated. genetic studies conducted in diverse contemporary populations suggest connections with ancestors that lived on the continent up to years ago [ ] . human evolution has been constantly influenced by pathogens; therefore, a great number of human genes linked to immune functions and immunity-related disorders have evolved along with humans. the heterogeneity in the immune response to infectious diseases across different populations is under genetic control and is the result of evolutionary processes. genetic variants that have been under evolutionary pressure can contribute to explaining the differences in the susceptibility to diseases observed across different populations. the ancestry of individuals from different populations across the globe greatly influences their possibility of developing certain autoimmune diseases and inflammatory disorders. the lifestyle of western societies affects the symbiotic relationships between humans, viruses, and other organisms and might contribute to the rise of certain autoimmune and inflammatory diseases. pathogens have played a central role as agents of natural selection from those very early days. among various infectious diseases, malaria has exerted the highest evolutionary pressure on the communities across the african continent ( figure ) [ ] . populations remaining in sub-saharan africa have been exposed to malaria for such long periods of time that their genetic structures have been shaped by the severity of malaria (plasmodium sp.) infections. in , allison described that sickle cell disease distribution was confined to africa and was associated with the geographical presence of malaria [ ] . this finding led to the more recent description of the existence of mutations in the hemoglobin-b (hbb) gene as a result of natural selection driven by evolutionary pressure for protection against malaria [ ] (table ) . similarly to hbb, some areas of west africa with a high incidence of malaria the gene variations that pass from one generation to the next are often transmitted as a random process known as genetic drift, while selection of advantageous variants tends to be preferentially transmitted. mutations, genetic drift, migration, and environmental selective pressure are among the fundamental processes behind the evolution of humans. the influence of these mechanisms in the diverse communities that were mobilized and then became isolated, as well as severe external factors such as epidemics, caused successive genetic bottlenecks in populations (see figure in main text) [ ] . human evolutionary studies are currently considered under 'modern synthesis', which merges darwin's ideas of natural selection with theoretical population genetics and mendelian principles, stating that evolution occurs via small genetic changes that are regulated by natural selection [ ] . these beneficial adaptations subsequently expand within the members of a population and become evident in the ancestral specificity and the geographical distribution of the advantageous alleles in the genomes of contemporary humans. genetic bottlenecks occur when the number of individuals in a population is reduced drastically due to a catastrophic event such as an earthquake, a flood, a famine, or the outbreak of an infectious disease. these events limit the genetic variation of a population and can lead to genetic drift. as a result, a smaller population, with a correspondingly reduced genetic diversity, remains to transmit genes to future generations through sexual reproduction. even if this reduction in the genetic diversity is temporary, it can lead to long-lasting effects on the genetic variation of the offspring populations. genetic drift: changes in the allele frequencies of a population over generations due to chance. genetic locus: fixed position on a chromosome (e.g., the position of a gene or a genetic marker). histocompatibility complex: region of approximately kb, located on human chromosome , that contains a large number of genes whose products are expressed as proteins on immune cells. of these genes, the best known are hla genes. introgressive hybridization: incorporation of genes from one species into the genetic reserves of another by interspecific hybridization and backcrossing with the parent species. present a high frequency of hemoglobin-c (hbc) in their populations, which is associated with a - % decrease in the possibility of developing the disease [ ] . this is also the case for the duffy antigen receptor gene (darc) in erythrocytes and single nucleotide polymorphisms (snps) in human leukocyte antigen (hla), which have been associated with protection against plasmodium vivax malaria in certain areas of africa where this disease is endemic [ , ] . another example of natural selection driven by evolutionary pressure for protection against malaria are thalassemia (a and b) pathologies, a group of hemoglobin disorders that presents an incidence of up to % among communities of west africa [ ] . the human casp t c snp, expression of which is restricted to the african subcontinent, south america, and certain areas of asia, can modulate immune and inflammatory responses to malaria by antagonizing interleukin (il)- b and nf-kb signaling in innate immune cells; moreover, caspase -deficient mice (casp -/-) exhibit decreased interferon (ifn)-g production and clearance of the parasite, relative to wild type (wt) infected mice [ ] . however, others have questioned these findings, given that caspase -deficient mice also lack caspase expression, so the effects observed might not be specific to caspase [ ] . mycobacterium tuberculosis (mtb) has caused infections in our species and ancestors for at least years [ ] . this long-standing relationship between humans and mtb probably underlies the large variety of immune-related factors that modulate susceptibility to mtb infection, including vitamin d receptor (vdr), natural resistance-associated macrophage protein (slc a ), tir domain containing adaptor protein (tirap), hla, monocyte chemoattractant protein (mcp- ), and cytokines such as il- and ifn-g [ ] ( table ) . patients with african ancestry present a higher frequency of mtb-related genetic variants than individuals from other populations, including variants in the gene encoding for toll-like receptor (tlr ), mediating cellular responses to bacterial malaria is one of the greatest causes of morbidity and mortality in the history of humanity. most human populations with a long history of endemic malaria have evolved genetic adaptations to malaria parasites due to the strong selective pressure that this infection has exerted. since the parasite infects erythrocytes, the evolutionary pressure has selected genetic variants that affect red blood cells and, therefore, the survival of the parasite as well. genetic variants conferring resistance to the disease have spread through human populations over time, including several abnormal hemoglobins that protect against malaria but usually cause erythrocyte-associated diseases in the populations where these adaptations are prevalent. these factors include the t c polymorphism in the caspase gene (casp ); the hemoglobin b (hbb) and hemoglobin c (hbc) variants; mutations in the duffy antigen receptor gene (darc); thalassemias (a and b); sickle cell disease; and polymorphisms in the human leukocyte antigen (hla) loci. dna nucleotide. for example, an snp may replace cytosine (c) with thymine (t) in a certain segment of dna. selective pressure: phenomenon that alters the behavior and fitness of living organisms within a given environment. it is the driving force of evolution and natural selection. thalassemias (a and b): inherited hemoglobinopathies characterized by a failure in the synthesis of the globin alpha or beta chains. toll-like receptors: family of transmembrane pattern recognition receptors expressed by immune and nonimmune cells that recognize conserved pathogenassociated molecular patterns. they play a pivotal role in innate immunity. transgenerational inheritance: transmission of traits from generation to generation. trends in immunology, december , vol. , no. lipoproteins [ , ] . selective pressure has also shaped the mechanisms that modulate the expression of genes implicated in immune responses against lassa virus, such as il- (il ) and the glycosyltransferase-like protein large (large), suggesting that the natural selection exerted by the virus drove the expansion of genetic variants that enhance immunity against lassa fever [ ] . these examples indicate that infectious diseases have contributed to shaping the genetic landscape of african populations and their descendants, and highlight the great impact of pathogens as an evolutionary force in humans. our homo sapiens ancestors were not the only species to venture out of africa, with other homo species performing a similar migration much earlier, such as homo ergaster, homo erectus, or homo heidelbergensis [ ] . from these early migrations, local populations such as the denisovans and neanderthals evolved [ ] . these lineages were not geographically isolated, but lived side by side with modern humans and interbred with them, leaving a genetic footprint in their common progeny. accordingly, - % of the genome of european and asian populations is thought to derive from these now-extinct hominid lineages [ ] . neanderthals spent close to years adapting to their environment and their immune systems were shaped by the infections they faced. by interbreeding with archaic humans, modern humans incorporated these advantageous adaptations in the genome of their descendants. this was highlighted by different studies that showed that the introgression of diverse genes related to immune functions, such as the oas cluster, tlr , or the histocompatibility complex from denisovans and neanderthals shaped the genetic landscape of present-day eurasian, but not african, communities. genomic sequences and expression data from lymphoblastoid cell lines from individuals of european and african ancestry confirmed that the tlr -tlr -tlr genetic loci, presenting signs of local positive selection and repeated introgression from both neanderthal and denisovan genomes [ ] [ ] [ ] , showed a significantly higher expression in individuals carrying archaic-like alleles than in individuals carrying the nonintrogressed modern human alleles [ , , ] . the expression of these genes has shaped human immune responses against different types of pathogens. for example, the gp protein of the hiv- virus has been recently recognized as a tlr ligand [ ] . in this regard, increased tlr expression has been correlated with higher il- production by the macrophage cell line thp- and higher titers of hiv- in the breast milk of hiv- -infected nigerian women relative to controls [ ] . tlr and tlr form dimers with tlr , triggering immune responses against different types of bacteria, fungi, virus, and parasites [ ] . variation in tlr -tlr -tlr is the major genetic determinant of human interindividual differences in tlr / -mediated responses, including cytokine production to a number of clinically relevant pathogens such as staphylococcus aureus and listeria monocytogenes [ ] (table ). this inheritance from archaic humans may have also left some human individuals more prone than others to developing asthma, hay fever, and other allergies (of snps associated with susceptibility to allergic disease, had a neanderthal or denisovan origin) [ ] , although these associations remain to be fully demonstrated [ ] . these reports demonstrate that by interbreeding with archaic humans, modern humans incorporated a group of advantageous adaptations to the genome of their descendants and contributed to shaping immune responses in modern human populations. the migration of our human ancestors out of africa implied the exposure to different types of infectious diseases (box and figure ). one study tested the responsiveness of human macrophages to pathogenic bacteria in vitro, finding that almost % of the genes present in human macrophages infected with the bacteria salmonella typhimurium or l. monocytogenes present different regulatory responses directly linked to the lineage of the donors and, also, that macrophages obtained from individuals of african origin display enhanced bactericidal activity compared with those from individuals of european lineage [ ] . the trend towards lower inflammatory responses in european populations is strengthened by the fixation of a tlr gene variant that results in lower proinflammatory gene expression in populations with a european ancestry compared with those with an african one [ ] . the largest population differences in gene expression between africans and europeans have been found in the macrophage receptor with collagenous structure (marco), a protein implicated in responses against viral infections and tlr-induced dendritic cell activation [ ] , the chemokine receptor cx cr , which mediates effector lymphocyte functions, and also several ifn-stimulated genes [ ] . west eurasian populations present a high frequency of tirap ser leu snps [ ] . tirap is an adaptor protein in tlr and tlr signaling pathways, involved in inflammatory responses and cytokine production. the heterozygous expression of the ser leu snp is protective against invasive pneumococcal disease, bacteremia, malaria, and tuberculosis, as shown in a case-control study of individuals from the uk, vietnam, and several african countries, and it is associated with lower tlr signaling in humans [ ] . this variant is considered to be a consequence of the natural selection that may have taken place in an early period following the migration of modern humans out of africa [ ] . european populations present a selective adaptation of the ifn gene that allows a high production of ifn-g in infectious scenarios due to the positive selection of ifng variants + g and + t; this suggests the existence of strong environmental pressures linked to higher ifn-g concentrations in plasma during mtb infection in european individuals relative to other populations [ , ] . in line with this, a database meta-analysis showed that individuals expressing the + t/a variant of ifng presented higher susceptibility to tuberculosis mtb infection than individuals without it, which might be considered a putative prognostic factor for the development of tuberculosis [ ] , although this remains to be robustly demonstrated. one of the most interesting aspects of humans is their ability to adapt to almost every ecosystem of the planet. the history of mankind is also the history of millions of individuals wandering around the world, looking for a better place to live. a glimpse to the migratory legacy of humanity around the globe reveals the great impact that the massive population movements defined the world as we know it today (see figure in main text). the distances ancient humans travelled are impressive, from the first hominids colonizing africa to the conquest of the americas in a time when the bering strait was not yet under water. the historical exodus of mankind started almost million years ago with the migration of homo erectus from africa through eurasia. from this event on, relatively isolated human populations evolved separately on different continents, leading to the emergence of different human species, such as neanderthals in europe, the denisovans in asia, and, later, modern homo sapiens in africa [ , ] . h. sapiens first colonized large areas of the continent around years ago [ ] , spread towards the middle east at some point between and years ago, and migrated through eurasia, reaching australia within years [ ] . asian human ancestors went through the frozen waters of the bering strait in two distinct waves to colonize the american continent approximately years before the present time [ ] . when humans ventured out of africa, they faced different types of pathogens than the communities that stayed in the african continent. with time, the series of events faced by diverse populations has generated differences in the immune responses to pathogens in the populations with an african or eurasian origin and which have spread throughout the world. the indigenous populations of south america are descendants of migrating populations of north-east asians that crossed the bering strait around years ago [ ] . five centuries ago, european settlers disembarked on the american continent, bringing a large collection of pathogens such as those causing measles, pneumonic plague, and influenza infections, which the indigenous populations had never faced before. these diseases rapidly spread and caused mortality rates above % [ ] . the consequences of these pandemics are still visible in current populations; one report studied dna from the bones of ancient humans from the tsimshian community, living in the british columbia region in canada until the th century, identifying marks of positive selection in a number of immune-related variants [ ] . specifically, the hla-dqa variant was present in almost the totality of tsimshian individuals, but only in one third of present-day humans studied; this suggested that ancient american genomes were evolutionarily selected to respond to local diseases but not to fight against pathogens brought by the europeans [ ] . another study compiled information on infectious diseases that have killed more than individuals among indigenous communities of the amazonia in the past two centuries, showing that the mortality rates and the incidence of infectious diseases rapidly decayed within the time following the first encounter with the pathogen, compatible with genetic adaptation [ ] . european colonizers underwent purifying selection in situations of intense pressure. such scenarios were documented when dutch colonists migrated to surinam and encountered epidemics of yellow and typhoid fever that caused a % mortality rate among settlers [ ] . variations in the frequencies of c , glo, and hla-b genes among the descendants were not likely caused by genetic drift, but rather, it has been proposed that these populations were probably selected through genetic control of survival to the epidemics [ ] (box ). africans and americans with an african ancestry present a much higher number of genetic variants related to robust inflammatory reactions, increased cytokine secretion, and bactericidal activities compared with the other populations [ ] , including more than genes with traces of recent selection, such as il a and il b gene variants [ ] . the degree of african ancestry, analyzed by fine-mapping analysis refined to the duffy-null allele of rs , was correlated with an increased amount of the proinflammatory chemokines ccl and ccl in plasma relative to controls [ ] . a study involving african american and hispanic american women found that the higher values of c-reactive protein (crp) in blood found in these populations compared with european americans were related to a crp-associated variant of triggering receptors expressed by myeloid cells (trem ) [ ] . moreover, comparison of health record data from individuals with connective tissue diseases, including rheumatoid arthritis and systemic lupus erythematosus (sle), as well as atherosclerotic cardiovascular disease from almost african american and european american adults was conducted; the study reported for the latter, a prevalence of atherosclerotic cardiovascular disease in . % african americans (particularly high in young individuals), relative to . % in european americans [ ] . these studies highlight certain genetic links to inflammatory predisposition/manifestation. however, increased proinflammatory activity is a double-edged sword. in the absence of regular pathogen challenges that require maintained modulation of the balance between inflammation and suppression of the immune response, the organism can overreact to inflammatory stimuli and trigger exacerbated responses. for instance, descendants of african populations generally present higher susceptibility to a variety of autoimmune syndromes such as inflammation-associated carcinomas, lupus, asthma, and multiple sclerosis (ms), the overall prevalence of which is up to three times higher in individuals with african ancestry relative to individuals with european ancestry [ , , ] . there are extensive differences in immune cell gene expression between americans with african and european ancestry. the increased proinflammatory responses observed in american individuals relative to other populations might be beneficial to combatting infections, but might also increase the chances of developing inflammatory and autoimmune disorders, which warrants further investigation. our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms, harboring ten million different microbial genes in the human fecal microbiome [ ] . the microbiome has its own evolutionary scenario across different populations with divergent lifestyles, nutrition, and exposure to environmental agents, generating extraordinary heterogeneity. the ongoing process of 'lifestyle westernization' of different societies has an important impact on the mutualistic relationships between humans and commensal organisms worldwide. african tribes are adopting western subsistence patterns, leading to remarkable changes in the composition of their microbiota [ ] . the comparison of the intestinal flora of the baaka hunter-gatherers and the bantu agriculturalists (both from the central africa republic), with a group of us-born african americans showed a great example of the evolution of the human microbiome [ ] . specifically, the bantu, still engaged in hunting, have a greater bacterial gut diversity than their baaka neighbors, who left the jungle for agriculture, and even more than urbanized westerners (us african-americans) [ ] . this reduced microbiota diversity in western societies has been associated with a higher incidence of the so-called 'diseases of civilization' such as cardiovascular diseases, diabetes, obesity, and autoimmune disorders, which are very unusual in hunter-gatherer societies compared with communities living a western-type lifestyle [ , ] . although viruses are mainly seen as pathogenic agents, they also play a fundamental role in the evolution and maturation of the human immune system [ , ] . approximately % of the human genome is composed of endogenous retroviruses (ervs), sequences derived from past retroviral infections and permanently inserted into different regions of the human genome [ ] . one study showed that ervs played a central role in the induction of ifn-dependent immune responses and that the removal of one or more of these viral dna elements in the hela human cell line severely impaired the recruitment of transcription factors necessary to trigger the expression of ifng against vaccinia virus infection relative to controls [ ] . viruses can also influence the severity of infections caused by other viruses. for example, cytomegalovirus infection in hiv- seropositive humans can potentiate the effects of hiv- infection by expanding the pool of circulating regulatory t cells (immunosuppressive); these were shown to inhibit the proliferation of autologous peripheral blood mononuclear cell the origins of the hiv virus are still a matter of scientific discussion. the most accepted scenario argues that hiv originated in simians and was transmitted to humans in west africa in the s, likely due to local ingestion of ape meat infected with the simian immunodeficiency virus. around , the virus reached wide parts of the continent and finally spread overseas thanks to a group of haitian professors coming back from africa. in the following decades, the virus spread worldwide and generated the pandemic we now know. today, there are approximately million people worldwide living with hiv- /aids [ ] . ccr is a receptor of chemokines that plays a fundamental role in hiv- pathogenesis and it is also one of the most promising targets to restrict the infection, since mutations in this receptor turn individuals resistant [ ] . the ccr -d mutation results in a deletion that eliminates the hiv- co-receptor on lymphocytes, providing robust protection against hiv- and, therefore, aids [ ] . ccr -d allele frequencies reach % in northern europe populations, whereas it is not present in populations with different ancestry, such as east asian, native american, or african groups [ ] . this regional distribution of ccr -d variants is most likely related to a naturally selective episode that struck european populations around years ago and involved a strong infectious agent that also employed ccr [ ]. a mathematical model studying the changes in the european populations in the middle ages suggested yersinia pestis (bubonic plague) as the most probable infectious agent behind the pressure that selected this particular genetic variant [ ] . this is in agreement with the finding that european rroma populations, but not northwestern indian populations that inhabit the area where the rroma originally lived, present signatures of positive selection in tlr -tlr -tlr , which influence cytokine responses in y. pestis infections [ ] . (pbmc) in response to cytomegalovirus infection in vitro [ ] . in one study, patients with chronic hepatitis c virus (hcv) infection and hepatitis a virus (hav) superinfection presented lower titers of hcv rna than patients harboring only hcv, suggesting that hcv replication might be potentially suppressed during hav infection [ ] , although this will still require further investigation. the relationships between humans and pathogenic or nonpathogenic organisms are extraordinarily complex and include tripartite evolutionary interactions between humans and microbes competing with each other. this is the case of parasites that infect other parasites, such as bacteriophage viruses, that can influence the outcome of bacterial infections. for example, in a cohort of individuals with chronic wounds, a report showed that the phage pf, which coexists with pseudomonas aeruginosa in infected wounds, triggered the production of type i ifn, the inhibition of tumor necrosis factor (tnf) production, and the suppression of phagocytosis in human primary monocytes and mouse bone marrow-derived macrophages, dampening the antibacterial response and promoting the bacterial infection [ ] . however, bacteriophages can also provide protection to the human host by directly attacking pathogenic bacteria and by upregulating in human pbmcs the expression of proinflammatory genes such as il a, il b, il , tnfa, cxcl , and cxcl , as shown for several s. aureus and p. aeruginosa phages, including pnm, luz , - , and ge-vb_pae-kakheti [ ] . cooperative relationships between organisms are evolutionary processes themselves. the way microorganisms and their hosts associate can lead to interactions of mutualism, in which the interplay may be so intimate as to provide benefit for each party and influence immune responses against different types of pathogens. a great number of humans live far away from the original settlements of their ancestors and are subject to radically different environmental conditions. between two and three million people with european genealogy suffer from autoimmune diseases, the prevalence of which is also increasing in other populations across the globe [ ]. there is rising evidence that the emergence of autoimmune diseases is associated with the presence of a number of immune-related alleles that have been selected via evolutionary processes; and, furthermore, that the contrasting differences in the prevalence of autoimmune diseases between populations may be a result of different selective pressures [ ] . alleles associated with inflammatory diseases that present marks of modern positive selection include the risk allele fut at rs for crohn's disease (cd) or the risk variant sh b at rs for celiac disease [ ] ; such variants have been linked to the development of several human autoimmune diseases, such as type diabetes, ms, and celiac disease [ , ] . the analysis of the integrated haplotype score [ ] of loci associated with sle that might provide protection against infections, such as tnip , itgam, ptpn , tnfsf , uhrf bp , tet -dguok, and blk, has suggested that these loci exhibit robust signs of positive selection [ ] (figure , key figure and table ). african and asian human populations exposed to trypanosoma brucei or plasmodium sp. have presented positive selection of snps in the apol and fcgr b genes; indeed, by enhancing human macrophage-mediated phagocytosis of infected erythrocytes, despite their association with an sle predisposition, these snps have been associated with protective roles against sleeping sickness and malaria, respectively [ , ] . an analysis of human loci associated with inflammatory bowel disease (ibd) concluded that the majority of the loci associated with cd are also linked with a higher risk of developing ulcerative colitis [ ] ( table ). many of these loci were also associated with the development of other autoimmune diseases, namely psoriasis and ankylosing spondylitis [ ] . pbmcs from individuals carrying the sh b variant rs *a (associated with a risk for developing cd) [ ] presented higher production of il- and il- b after stimulation with lipopolysaccharide or muramyl dipeptide due to enhanced activity of the nod inflammasome pathway relative to controls [ ] ; this has suggested an immune-related role for sh b in the context of bacterial infections, which might help explain the positive selection of sh b approximately years ago [ ] . others found an association between genetic variants in nod , cd , and increased susceptibility to cd [ ] , in line with previous results showing that mutations in nod and tlr /cd are related to an increased risk of developing ibd [ ] . from another angle, changes in hygiene patterns seen in the past two centuries brought vast improvements in sanitation, drinking water, and garbage collection, which greatly reduced the exposure to many infectious diseases. however, these conditions may have reduced the exposure to viral and microbial agents that help the immune system to develop tolerance during childhood. the hygiene hypothesis proposes that the lack of exposure to microbial agents in the early stages of life is related to a higher risk of developing hypersensitivity reactions, based on the fact that children that are exposed to higher amounts of microbial stimuli (e.g., by growing on farms) are less prone to develop allergies and asthma [ , ] . moreover, reduced exposure to infectious agents can have a much wider effect than initially believed. lack of exposure to microbes in childhood can cause aberrant responses to infection and potentiate the effects of etv -runx mutations in the pathogenesis of acute lymphoblastic leukemia [ ] . by contrast, a meta-analysis of six observational studies, including participants, showed a correlation between low helicobacter pylori infection and ms, suggesting that low h. pylori prevalence might be a putative protective factor in ms, although this remains to be experimentally validated [ ] . one study also reported that antibodies against toxoplasma gondii were detected less often in patients with ms compared with healthy controls [ ] . however, these findings warrant further and robust investigation. overall, it is clear that evolutionary processes can drive the fixation of genetic variations that increase (or decrease) our defense against infections upon sensing microbial ligands, but can also lead to a greater risk of developing certain autoimmune diseases in which endogenous ligands can cause tissue damage and inflammation. a growing number of reports suggest that inheritance is not always governed by classical darwinian evolutionary processes. exposure to certain environmental stimuli can cause effects in the progeny of an exposed individual, even though the stimuli are no longer present. this type of transgenerational inheritance might be explained through the effects of epigenetic processes, which are hypothesized to be transmitted through the germline and passed on to the offspring [ ] . for example, the worm caenorhabditis elegans can transmit improved resistance to infections to pathogenic bacteria to their offspring through alterations of the histone landscape [ ] . indian meal moths exposed to low doses of virus are subsequently less susceptible to viral challenge, a protection offered to their offspring as well [ ] . transgenerational inheritance of diverse traits has also been observed in mice, in which the variation of the color of the coat is passed on the next generation [ , ] . offspring of male rats subjected to a high-fat diet present glucose intolerance and reduced insulin secretion, linked to reduced methylation at the il ra gene relative to controls [ ] ; and mice fed scorpions are more resistant to a challenge with scorpion venom than mice on a normal diet [ ] . since infections are one of the strongest factors impacting survival, it is conceivable that transgenerational transmission of traits in mammals, including humans, evolved to improve host defense. the number of studies of the potential role of epigenetic inheritance in shaping the human immune system is still scarce. however, different experiences undergone by certain communities indicate that these mechanisms might be important. for example, the babies of pregnant women who suffered during the early stages of pregnancy (the effects of the dutch hunger winter in ), years later showed reduced dna methylation marks in several genes that control metabolism and cell differentiation during development, such as igf , pim , txnip, abcg , pfkfb , and mettl , compared with their siblings [ , ] . this was related with higher rates of obesity, heart disease, cancer, and depression in individuals whose pregnant mothers suffered the effects of the famine [ , ] . some of these effects seemed to be present in the progeny of this group, that is, in the grandchildren of those who had passed the famine during pregnancy [ ] . the rapid growth in the number of reports covering the impact of epigenetic mechanisms in different human processes warrants further and robust studies on the impact of epigenetic inheritance in shaping the evolution of the human immune system. human immune responses have been shaped by the evolutionary pressure exerted by microorganisms and viruses throughout history. generating a broad range of genetic variations and immune functions in different populations favors the adaptation to new environments and increases the chance of survival of the human species against potential pandemics. much remains to be learned in this exciting field over the coming years in order to identify the main regulatory forces and the time window necessary for the fixation of an advantageous genetic trait in a population (see outstanding questions). the combination of the selective pressure caused by infectious diseases with other evolutionarily relevant processes, such as genetic drift, migratory events, bottlenecks, and introgressive hybridization, contribute to driving the expansion, fixation, or elimination of characteristic immune response-related traits in different populations around the world. these specific genetic variants are able to boost the host response against pathogens by improving the sensing of microbial ligands but can also lead to the development of autoimmune diseases, in which the immune system responds to endogenous ligands and induces abnormal responses targeted against the host's own tissues. of note, it is very difficult to assign certain variants, a specific role in the protection or induction of autoimmunity. to assign changes in the genetic landscape of human populations to certain diseases is an extraordinary challenge. moreover, our species is in constant evolutionary interaction with various microorganisms and viruses. populations of bacteria and their viruses (phages) undergo, under natural conditions, reciprocal evolution in terms of resistance and infection; this, in turn, also affects the evolutionary traits of our immune system. thus, an extraordinarily complex scenario exists in which organisms of different phyla interact, compete, and coevolve, to ensure their own survival. as novel tools, the development and refinement of methods that study large sections of the human genome, epigenome, and microbiota, will help to obtain genome-wide data in diverse human populations, allowing us to follow the evolutionary trails left from the encounters with different organisms, further unveiling the roots of human immunity. high-throughput biotechnology and an expanding computational capacity can enable the study of global population genomics and might contribute to decoding the origin and consequences of functional changes in adaptive alleles down to the single cell level. however, these methods also have limitations associated with the difficulty in linking gene variations to clinical phenotypes and disease, the generation of false positives, or the high number of samples necessary to reach reliable conclusions. expanding the heterogeneity of populations studied for immune gene association studies relevant to disease will be key, as generally, a large focus is placed on certain european or american communities, thus generating results that are difficult to extrapolate to other populations. the knowledge of the evolutionary and genetic basis of human immune traits and their impact on diverse pathologies (e.g., autoimmunity, infections, inflammatory diseases, cancer) increasingly suggests that the genetic basis of disease may be derived from a large number of rare variants of modest effect. the mechanisms described here acquire special importance in the current scenario of world globalization, in which the migration fluxes and the admixture of different populations are reaching unprecedented levels, allowing faster expansion of advantageous alleles. however, these processes may also accelerate the spread of new epidemics, as seen in the cases of hiv infection, or more recently, sars-cov, ebola, and chikungunya viruses; as well as the emergence of multiresistant bacteria and fungi, such as methicillin-resistant s. aureus or candida auris. this is just the starting point to unveil the evolutionary history of the relationships between pathogens, the immune system, and humans. further investigation of the functional adaptations of human populations is warranted to provide a broad picture of the functional consequences of evolution in human immunity. acknowledgments m.g.n. is supported by a spinoza grant of the netherlands organization for scientific research and an erc advanced grant (# ). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. we thank laszlo a. groh for which are the strongest evolutionary forces that drive the evolution of the human innate immune system? how long does the genome of a given population take to adapt to a new infectious threat? are the mechanisms of resistance to infection transmitted only via genetic modifications, or can epigenetic adaptations to resistance also be transmitted to the progeny, and under what circumstances? the development of single-cell sequencing technologies has opened new fields of study. how does genetic variation of the expression of a specific variant vary between different cell subsets and how does it affect the overall phenotype of an individual within a population? are there specific immune processes that are preferentially impacted by evolutionary pressures? in western societies we enjoy a life expectancy vastly superior to that of our predecessors, but at the same time, we suffer diseases that they did not suffer. can some of the reasons for these changes lie among some of those bacteria that we have somehow lost in our microbiome? identifying these bacteria and understanding their effects on the human body might be the first step to developing putative therapies based on bacterial restoration. since many of the variants causing autoimmune diseases are linked to an enhanced responsiveness to pathogens that is no longer needed in developed countries, could these genes and their related pathways be employed as targets for new putative therapeutic approaches against inflammatory/ autoimmune syndromes? are other newly described genetic regulatory pathways, such as interfering rna or long noncoding rnas, also influenced by pathogen-driven evolutionary processes in different populations globally? proofreading and correction of the manuscript. we additionally thank srinivas agra for providing the figure icon 'bacteria' as deposited on https://thenounproject.com. natural selection and infectious disease in human populations genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes genome-wide identification of regulatory sequences undergoing accelerated evolution in the human genome divergent selection of 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severe malaria full-exon resequencing reveals toll-like receptor variants contribute to human susceptibility to tuberculosis disease multiple common variants for celiac disease influencing immune gene expression epitope selection for hla-dq presentation: implications for celiac disease and viral defense card /nod mutational analysis and genotype-phenotype correlation in patients with inflammatory bowel disease association of nod leucine-rich repeat variants with susceptibility to crohn's disease nod and nod : beyond peptidoglycan sensing global, regional and countrylevel - - estimates for : assessing progress towards the targe key: cord- -z l vdsr authors: río, francisco garcía; clau, luis borderías; macario, ciro casanova; celli, bartolomé r.; sanglás, joan escarrabill; mangado, nicolás gonzález; torrent, josep roca; romero, fernando uresandi title: air travel and respiratory disease date: - - journal: archivos de bronconeumología ((english edition)) doi: . /s - ( ) - sha: doc_id: cord_uid: z l vdsr nan in recent years there has been a progressive rise in the number of people who travel by air. according to data from the international civil aviation organization, million people traveled by air in and, despite problems related to security restrictions and severe acute respiratory syndrome (sars), it is anticipated that the number of passengers will increase annually by . % until . more than million air traffic operations were handled during in airports managed by the spanish aviation authority (aeropuertos españoles y navegación aérea, aena), representing travel by million passengers. those figures correspond to a % increase in the number of passengers since , with an annual increase of %. in addition, advances in the monitoring and treatment of many chronic respiratory diseases have allowed changes in the lifestyle of patients. thus, patients are now able to consider leisure and professional activities that were not possible some years ago. although adverse respiratory events as a result of air travel are not common, this form of transport does present potential risks. data from airline companies forming part of the international air transport association (iata) show that between and there were deaths in flight, corresponding to . deaths per million passengers or . deaths per million takeoffs. respiratory complications represented the third highest known cause of death ( %) after cardiac causes ( %) and deaths due to cancer ( %). in addition, it was noteworthy that while there was prior knowledge of the presence of heart disease in only % of deaths due to cardiac events, there was prior knowledge in % of those due to respiratory disease, suggesting that there are problems in the assessment of patients prior to the flight or in their in-flight care. aside from fatal events, respiratory symptoms are responsible for a good proportion of the emergencies that occur on board aircraft. analysis of all cases in which the first-aid kit was used on commercial aircraft belonging to the iata between august and july showed that chest pain and dyspnea were of the most common causes, along with loss of consciousness. , likewise, % of passengers who required medical assistance had a known medical condition associated with the episode that occurred on board the aircraft, further indicating the importance of careful assessment prior to flight. along similar lines, a service offering the assistance of experts by radio during in-flight emergencies received calls in , of which % corresponded to respiratory problems. , thus, respiratory problems may represent up to % of in-flight emergencies. in response to this situation, various guidelines and recommendations have been prepared by scientific societies or the airline companies themselves. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, little scientific information supported by a high level of evidence is available in this field, meaning that the majority of the recommendations are based solely upon expert consensus. in fact, in recent years, conflicting results have been reported using the regimens recommended in previous guidelines. furthermore, there is a local problem generated by differences in the legislation and the wide range of criteria, resources, and attitudes of the different airline companies. the aim of these guidelines is to define assessment protocols for patients with chronic respiratory disease intending to travel by plane that are adapted to the situation in spain and the most recent available data. in addition, the guidelines aim to establish specific recommendations for the most common respiratory diseases. extensive information is available on respiratory physiology during air travel in both healthy individuals and patients. , - , - some of those detailed reviews of francisco garcía río a (coordinator), luis borderías clau, b ciro casanova macario, c bartolomé r. celli environmental conditions and their control are published by the airlines themselves and are available on the internet. , it is worth remembering that the atmosphere surrounding the earth's crust is made up of different layers or strata: the troposphere, the stratosphere, the mesosphere, the thermosphere, and the exosphere. the layer closest to the earth is the troposphere, which extends from sea level to m ( feet) at the poles and to m ( feet) at the equator (appendix ). today's commercial aircraft fly within this zone. atmospheric pressure depends on the column of air above the measurement point; consequently, the higher the altitude, the lower the pressure. since the reduction in atmospheric pressure is logarithmic (figure ), at lower levels small changes in altitude produce substantial changes in pressure. thus, at m ( feet) the atmospheric pressure is less than half that at sea level. the composition of the troposphere is constant and contains approximately % nitrogen and % oxygen. since the partial pressure of a gas is a function of its concentration and the total pressure, oxygen tension is directly dependent upon altitude and drops exponentially as altitude increases ( figure ). this hypoxia is the cause of the limitations and risks faced by mountaineers and also of acclimatization problems in high-altitude populations. in addition, adaptation to this type of environment is affected by the amount of exercise that is performed. in terms of the physiologic response of the human body, the atmosphere can be divided into zones: the physiologic zone, the physiologically deficient zone, and the zone equivalent to space. the physiologic zone is where the human body is well adapted and where the oxygen level is sufficient to maintain normal processes. this zone extends from sea level to an altitude of m. nevertheless, rapid changes in altitude within this zone can cause minor problems due to the expansion of gases trapped within the body. the physiologically deficient zone extends from to m. in that zone, the reduction in barometric pressure causes a critical environmental hypoxia, necessitating the use of supplementary oxygen at higher altitudes. from a physiologic point of view, space begins at an altitude of m. in this zone, the low ambient pressure means that humans are unable to survive even with supplementary oxygen and they require pressurized suits. above m the barometric pressure is lower than the vapor pressure of water at ºc and body fluids evaporate. commercial aircraft generally fly at an altitude of around to m ( - feet). , , if the internal pressure of the aircraft were to be directly dependent upon the external atmospheric pressure the environment would be incompatible with life. consequently, aircraft must be pressurized, that is, have elevated pressure compared with that of the external environment. to achieve this, they take ambient air and compress it. since the gas heats up in this process, it must subsequently be cooled. the pressure is controlled according to the quantity of air injected and through the use of escape valves set to the desired pressure. to support the pressure difference, the structure of the aircraft must be reinforced and that increases its weight. as a result of both the increased weight and the additional energy required to compress the air, cabin pressurization increases aircraft fuel consumption and thereby decreases their independence. the pressurization system used by commercial aircraft is known as isobaric. initially, as the aircraft climbs in altitude, it maintains the same ambient pressure as its environment, and then, from a certain altitude, it maintains a constant (isobaric) pressure, irrespective of changes in altitude. many military aircraft employ a different system known as differential-isobaric pressurization, which imposes fewer structural requirements and thereby saves weight. due to the technical limitations mentioned and the cost, aircraft pressure is not maintained at that of sea level but rather at an intermediate pressure; that pressure depends on the type of aircraft but is usually approximately equivalent to that of an altitude of m. altitude, the atmospheric oxygen tension is equivalent to breathing . % oxygen at sea level. although international legislation establishes that minimum cabin pressure should correspond to an altitude of m ( feet), the pressure does not remain constant throughout a flight. in a large series of measurements performed during commercial flights, it was determined that the conditions within aircraft cabins usually correspond to an altitude of to m ( - feet) above sea level. , , survival in the event of a sudden reduction in cabin pressure necessitates the use of oxygen masks (obligatory equipment on commercial flights). it is also important to note that at an altitude of m a person will lose consciousness in to seconds. the degree of pressurization also depends on the type of plane. the old concorde was pressurized at a comfortable level corresponding to an altitude of m ( feet). the current tendency for new models of aircraft, whether manufactured by boeing or airbus, is to pressurize at this more comfortable, safer pressure. however, the new airbus is expected to carry around passengers with a cabin pressure equivalent to an altitude of more then m ( feet) for up to hours. in addition to the difficulties caused by changes in barometric pressure, the external environment presents additional problems for commercial flights. the concentration of ozone, which is very low at sea level, increases with altitude and peaks in the stratosphere. ozone, which is important to filter ultraviolet radiation, is toxic to the respiratory system, even at concentrations below part per million (ppm), which can be reached at some common flight altitudes. to manage this problem, planes have catalytic ozone converters installed to reduce the concentration of the gas. the regulations of the federal aviation administration establish a maximum mean concentration of . ppm and a maximum peak concentration of . ppm. the temperature falls by approximately ºc for every m increase in altitude, necessitating warming of the air inside the cabin. this air normally has a low humidity ( %), which can cause problems for some individuals. most commercial aircraft recirculate approximately % of the air to improve humidity and energy efficiency. the air must be filtered to retain particles smaller than . µm in diameter using highefficiency particulate air (hepa) filters similar to those used in hospital operating theaters. in addition to particles in suspension, this system is considered effective for the retention of bacteria, fungi, and even viruses released during speech, coughing, or sneezing ( figure ). the air is renewed to times per hour, although this may vary according to the model and the zone of the plane. the cabin ventilation system generates transverse airflow and is able to renew the air more effectively than in buildings with air conditioning. complex electronic systems with sensors located throughout the cabin control the temperature and regulate valves in order to maintain a temperature that is as homogeneous as possible. finally, it is worth mentioning that the carbon dioxide content of this filtered and conditioned air is usually very low ( ppm). the partial pressure of inspired oxygen (pio ) is a function of the atmospheric pressure and the vapor pressure of water. as the vapor pressure of water at the same body temperature remains stable with altitude, pio will decrease with altitude (hypobaric hypoxia). breathing ambient air at m ( feet) is equivalent to breathing . % oxygen at sea level, meaning pio falls from mm hg at sea level to mm hg at m. , in healthy subjects, this can represent a reduction in pao from to mm hg, , , which is usually well tolerated and does not produce symptoms. however, in patients with chronic respiratory diseases and some degree of baseline hypoxemia, the reduction in pio during the flight can cause more marked reductions in oxyhemoglobin saturation. [ ] [ ] [ ] acute exposure to a hypobaric environment triggers hyperventilation, which is essentially induced by stimulation of peripheral chemoreceptors and is usually mediated by an increase in tidal volume. it also generates an increase in cardiac output to compensate for the residual systemic hypoxia. this increase is mainly mediated by tachycardia and is usually proportional to the drop in oxygen saturation. the increased pulmonary perfusion caused by the rise in cardiac output is associated with hypoxic vasoconstriction of the pulmonary artery and increased systolic pulmonary pressure. as a consequence of the increase in pulmonary vascular resistance, there is a redistribution of pulmonary blood flow and an increase in perfusion of certain areas of the lungs compared with the situation at sea level. altitude is also associated with limitation of oxygen diffusion from the atmosphere into the pulmonary capillaries as a consequence of the interaction of various factors. both the reduced pio and the reduction in affinity of hemoglobin for oxygen in conditions of low pao lead to a more marked drop in the oxygen content of the pulmonary capillaries than at sea level. finally, the transit time of blood through the pulmonary capillaries is shortened due to the tachycardia caused by the altitude and this limits the time available to establish an adequate oxygen equilibrium. the net result is an increase in the alveolar-arterial oxygen difference. , , in addition, the oxyhemoglobin saturation is significantly reduced during physical exercise in a hypobaric environment. exercise at high altitudes also increases the alveolar-arterial oxygen difference in subjects who normally reside at sea level, while it does not affect those native to high altitudes. studies performed using the multiple inert gas elimination technique have shown that hypobaric hypoxia is associated with a greater heterogeneity in the ventilation-perfusion ratio and a limitation of diffusion that together worsen hypoxemia as exercise intensity increases. limited diffusion secondary to reduced pio appears to exert the greatest influence on blood gas alterations during exercise in a hypobaric environment. additionally, the interstitial edema caused by extravasation of fluids into the extravascular space appears to potentiate the ventilation-perfusion imbalance. the changes described have few consequences in healthy subjects, who might only note a slight increase in tidal volume and heart rate. however, hypobaric hypoxia represents a risk for some patients with chronic respiratory disease, in whom it can aggravate preexisting hypoxemia and favor the development of cardiovascular complications. in fact, it is recognized that hypoxia reduces the ischemic threshold in men with exerciseinduced ischemic heart disease as well as favoring some atrial arrhythmias and being associated with ectopic ventricular beats as a result of increased sympathetic activity. with increasing altitude, barometric pressure is reduced and gases expand if they are trapped in the body, unable to escape. this phenomenon is explained by boyle's law, which establishes that the volume of a gas is inversely proportional to the pressure: although the expansion of the trapped gases is limited, it occurs rapidly, and in healthy subjects can cause discomfort in organs such as the ear, paranasal sinuses, teeth, and gastrointestinal system. in patients with respiratory diseases, and even in young, apparently healthy individuals with small apical bullae, the phenomenon can generate more serious problems. [ ] [ ] [ ] [ ] ears. air trapping can occur in the ears due to partial or complete obstruction of the eustachian tube, which normally equalizes air in the middle ear with the outside. this can occur both during ascent and descent and is also one of the main problems associated with underwater diving. it can be the result of a chronic intrinsic or acquired obstruction or an acute process caused by an infection or allergic reaction. with increasing altitude, the air expands and exerts a pressure on the tympanic membrane, which expands outward. when a pressure increase of to mm hg is reached, a small bubble of air is expelled into the nostrils and is sometimes accompanied by a small noise. upon descent, the reverse situation occurs. the external pressure increases and the tympanic membrane is pressed inwards. it is much more likely for obstruction to occur in this situation since the eustachian tube functions less effectively in this direction. this air block can produce sounds, nausea, and pain in the ears that is sometimes very intense, particularly if the finally phase of the descent occurs very rapidly. a useful maneuver to prevent this obstruction involves repeated swallowing of saliva. consumption of liquids or food can also help. if the condition persists, gentle valsalva maneuvers are recommended. paranasal sinuses. the paranasal sinuses can present similar problems to those experienced in the ear. in this case, the obstruction may be due to chronic lesions such as polyps or to acute problems such as mucus generated in response to infections or allergies. in general, the problem appears during descent and in % of cases affects the frontal sinuses. the pain can become very intense. [ ] [ ] [ ] [ ] barodontalgia. some subjects may experience dental pain, mainly during ascent to between and m. it was initially thought that small pockets of air trapped during dental restoration or other manipulations were the cause of the problem. however, it has not been possible to confirm that hypothesis, despite the association of symptoms with different types of dental complaints. gastrointestinal tract. the gastrointestinal tract usually contains some quantity of gas, and consequently, gastrointestinal discomfort is common during air travel. nevertheless, such problems are of minor significance at the cabin pressures reached during commercial air travel. lungs. in healthy subjects without structural abnormalities there are usually no problems of this type associated with the lungs since pulmonary gas pressure is rapidly equalized with the ambient pressure. nevertheless, some young, apparently healthy subjects may have apical bullae, which can burst during ascent and cause a pneumothorax. in some cases this may be a tension pneumothorax and become serious. given that the gas in the body cavities is saturated with water vapor, the expansion caused by increased altitude is greater than that calculated according to boyle's law. given that body temperature remains constant, in the case of bullae or closed pneumothorax the increase in volume can be calculated with the following formula: ∆volume= pressure of gas at sea level-water vapor pressure if it is assumed that the gas pressure is mm hg at sea level and mm hg at an altitude of m, and that water vapor pressure remains constant at mm hg, it can be estimated that the volume of trapped gas will increase by . % during ascent. the problem is much more severe in patients with chronic obstructive pulmonary disease (copd), since those patients usually have regions of emphysema that are poorly connected with the exterior or separated from it and can cause rupture and pneumothorax, in addition to the problems generated by hypoxia. airline companies usually recommend that individuals do not fly within weeks of the resolution of a spontaneous pneumothorax, although the scientific evidence supporting this recommendation is very limited. if the pneumothorax has been treated surgically or by pleurodesis with talc it is highly unlikely that there will be a relapse during flight. diving and flight. a particular problem may occur following scuba diving activities. dissolved nitrogen can accumulate in the tissues (residual nitrogen) during scuba diving, particularly when diving is deep and repeated. during ascent, that nitrogen may be released and give rise to symptoms of decompression, which in some cases can be severe. in general, it is recommended that individuals do not fly within hours following scuba diving, and that they abstain longer periods if diving required decompression breaks. tables and computer programs are available that can help determine the amount of residual nitrogen and the recommended delay before flying. [ ] [ ] [ ] [ ] as mentioned, cabin humidity is usually less than % to %. this can cause skin dryness and discomfort in the eyes, mouth, and nostrils. the dehydration caused by a long flight can also be significant in patients with bronchiectasis. if nasal irritation is particularly acute, use of a hypertonic saline spray is recommended. prolonged immobility, particularly in a sitting position, contributes to the accumulation of blood in the legs, and this can cause swelling, tightness, and discomfort in the lower limbs. in turn, immobility can favor the development of deep vein thrombosis (dvt). for some subjects, the aircraft environment and the flight itself can trigger increased anxiety, which can lead to an exaggerated perception of some respiratory symptoms or contribute to the deterioration of an existing respiratory condition. it is difficult to establish definitive guidelines based on currently available information. in fact, a wide variety of procedures are used for the assessment of patients with respiratory disease. in a review of in-flight requests for oxygen, information on oximetry or spirometry results were only available in % of cases. furthermore, a survey of specialists in respiratory medicine in england and wales revealed that they followed highly diverse criteria in prescribing use of oxygen in flight. in any case, to establish a medical opinion on risk in air travel, the type, reversibility, and degree of functional impairment caused by the disease must be assessed along with the tolerance of the patient for the predicted flight altitude and the length of exposure. although all patients with chronic respiratory disease may benefit from a clinical assessment prior to undertaking air travel, such assessment should be considered obligatory in those situations shown in table . the following procedures should be considered in this preliminary examination: -medical history, in which special attention should be paid to recognizing all cardiorespiratory disease, with particular interest in comorbidity that could be worsened with hypoxemia (cerebrovascular disease, ischemic heart disease, heart failure). it is also important to assess dyspnea and other respiratory symptoms and compile previous experiences of the patient on other flights. -measurement of oxyhemoglobin saturation by pulse oximetry (spo ) or arterial blood gas analysis, following a period of rest sufficient to guarantee stability of the recordings. in the case of clinical suspicion of hypercapnia, blood gas analysis should obviously be performed. -forced spirometry , and single-breath determination of the diffusing capacity of the lung for carbon monoxide (dlco). -walk test. the medical departments of some airlines propose walking for m as a way to assess tolerance of flight conditions. in such a test, the aim is to verify that the patient is capable of walking m without limitation due to dyspnea. although it is a crude procedure that has not been sufficiently validated, it allows an estimate to be made of the cardiorespiratory reserve by assessing the increase in ventilation and cardiac output in response to exercise. in principle, there is no reason to use a m walk test in place of the minute walk test, which is commonly used in many patients with respiratory disease and is well standardized. criteria for concern should be the inability of the patient to continue walking for minutes, a distance covered of less than m, or the development of severe dyspnea (score of more than on the borg scale). garcÍa pressure of gas at m-water vapor pressure -incremental cardiorespiratory exercise test. the incremental exercise test is not recommended for the systematic assessment of all patients, although it could be useful if the results of simulated altitude-induced hypoxia were unclear. it has been confirmed that a peak oxygen consumption (vo max) greater than . ml/min/kg in patients with moderate or severe copd is associated with a pao of greater than mm hg during the flight. that relationship between vo max and pao was confirmed in the first and fourth hour of flight in a study involving patients with severe copd. in fact, in a multivariate analysis, pao at sea level and vo max were selected as independent predictors of pao during the first hour of flight. however, in the fourth hour the only independent variable associated with vo max was pao . identification of at-risk patients. the information collected in the aforementioned procedures should allow identification of patients who should not fly (table ) along with those in whom the hypoxemia in flight could prove dangerous. in general, it is accepted that patients with acute respiratory failure should not fly. this should also apply to patients with sputum-positive tuberculosis. in the case of patients who are negative for the human immunodeficiency virus (hiv), it would be necessary to have taken antituberculosis treatment for at least weeks. in hiv-positive patients, negative sputum stains or a negative sputum culture are required during the course of the treatment. passengers with respiratory symptoms who come from areas of local transmission of sars should also be prohibited from flying, as should contacts of probable or confirmed cases of sars who have been exposed within the last days. patients with undrained pneumothorax, subcutaneous or mediastinal emphysema, or a pulmonary contusion, or who have undergone a major thoracic surgical procedure in the last weeks are also considered to have a respiratory contraindication for air travel. most current guidelines only consider the results of pulse oximetry or baseline arterial blood gas analysis in screening for patients at risk of developing severe hypoxemia. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in fact, pao greater than mm hg or spo above % are usually considered acceptable for air travel in the majority of cases. , however, in recent years it has been shown that screening based on pao or spo alone are insufficient. for instance, a study was performed in which in-flight hypoxemia was assessed in a group of patients with copd who had a resting pao of more than mmhg, without hypercapnia, and a forced expiratory volume in second (fev ) less than % of reference. in % of the patients, pao was less than mm hg at an altitude of m and % had a pao of less than mm hg. what was even more noteworthy in that study was that % of the patients had a pao less than mm hg when they undertook lowintensity exercise similar to that necessary to walk along the aisle of the cabin or to go to the bathroom. similar findings have been obtained in patients with interstitial disease. figure shows a proposed algorithm for patient assessment. in those patients who receive home oxygen therapy, it is recommended that the oxygen flow be increased during the flight, usually by to l/min. in other patients, in-flight hypoxemia should be estimated if they have a pao less than mm hg or an spo less than %, if the forced vital capacity (fvc) or dlco is less than % of reference, or if other risk factors are present ( table ) . based on the level of hypoxemia during air travel in healthy subjects, a pao of more than to mm hg has been arbitrarily considered acceptable. [ ] [ ] [ ] [ ] [ ] consequently, it is important to estimate the pao during the flight, since below mm hg provision of supplementary oxygen during the flight is recommended. pao at altitude can be estimated in ways: through the use of prediction equations or with a hypoxia-altitude simulation (hypoxic challenge) test. absolute acute respiratory failure sputum-positive tuberculosis passengers from areas with recent local outbreaks of severe acute respiratory syndrome (sars) with respiratory symptoms contacts of probable or confirmed cases of sars who have been exposed in the last days undrained pneumothorax thoracic surgery within the last weeks lung contusion subcutaneous or mediastinal emphysema relative resolution of a spontaneous pneumothorax in the last weeks major thoracic surgery within the last weeks scuba diving in the last hours (table ) . , [ ] [ ] [ ] [ ] [ ] [ ] [ ] some of them allow pao to be determined for any given altitude based on values obtained at sea level ( figure ). , in most cases, the equations were established for patients with copd and the measurements of pao at altitude were performed in hypobaric chambers or following altitude simulation via respiration with a fraction of inspired oxygen (fio ) of %. the accuracy improves when measurements of fev , or fev /fvc are included. in addition, greater accuracy is obtained when they are applied to copd patients with an fev less than % of reference. despite the simplicity of equations to estimate in-flight hypoxia and their widespread availability, they also have drawbacks. the most important is the consequence of their very large % confidence interval, which is ± . mm hg, mainly due to the use of very small samples in their calculation. it is notable that in patients with severe copd differences have been detected between the actual pao during the flight and that estimated in the equation of gong et al of - ± mm hg (range, - to mm hg). in almost all cases, patient series used to develop the equations have involved healthy men or men with copd, meaning that accurate information on women is lacking. nor have flight duration and cabin conditions been considered. in addition, the equations have not been validated with another hypoxia test repeated after the test used to generate them. it is possible that equations that include fev underestimate the severity of hypoxemia triggered by altitude in hypercapnic patients, since some authors have demonstrated that pao at altitude is inversely proportional to paco at sea level. in the same way, equations that use fev or fev /fvc in healthy subjects probably overestimate pao at altitude. it is also likely that the cause of the hypoxemia should be taken into account. for instance, hypoxemia as a result of shunt is affected very little by altitude, while that caused by ventilation-perfusion imbalance is highly dependent upon pio . , recently, a specific prediction equation that includes dlco was developed for patients with restrictive disease. another equation relevant to patients with copd or interstitial disease has also been proposed. in addition, in recent years models have incorporated paco , both for healthy subjects and patients with copd. in the light of available data, the equation published by muhm would be the most recommendable in healthy subjects and patients with copd, while that of christensen et al would be advisable for patients with restrictive disease. hypoxia-altitude simulation test. although hypobaric hypoxia is the ideal method to estimate the degree of hypoxemia during a commercial flight, it can not be used in ordinary clinical practice due to the limited availability of hypobaric chambers (appendix ). as an alternative, it is recommended to resort to the isobaric hypoxia-altitude simulation (hypoxic challenge) test, initially described by gong et al. this test assumes that respiration of a hypoxic gas mixture at sea level (normobaric hypoxia) simulates the hypobaric hypoxia characteristic of higher altitude. the maximum altitude corresponding to cabin pressure ( m) can be simulated by respiration of a mixture of % oxygen in nitrogen. no specific preparation is required for the test. it is recommended that the test be performed without interruption of the patient's usual medication, attempting to avoid changes in the dose or intervals of the medication. once patients are seated, they can be made to breathe a hypoxic gas mixture using a douglas bag, a plethysmography chamber, or a venturi mask. the most traditional and simple method is to ask the subject to breathe the gas mixture contained in a to l douglas bag, which is filled with % oxygen and nitrogen as a carrier using pressurized cylinders. in this case, the patients can breath through a mouthpiece with a nose clip, or through a face mask with a valve to prevent rebreathing. , the second option involves filling a sealed plethysmography chamber with a gas mixture ( % oxygen in nitrogen) that can be kept constant by introducing oxygen or nitrogen through a port. this procedure has the advantage of not requiring a mask or mouthpiece and also allowing titration of the oxygen flow required to correct the hypoxemia by administration of oxygen through nasal prongs within the hypoxic environment of the chamber. however, while the patient remains in the chamber it is not possible to obtain samples of arterial blood and monitoring is therefore limited to spo . as a third possibility, a venturi mask can be used in which oxygen is replaced with nitrogen as the carrier gas. it has been confirmed with various devices that a venturi system at % generates an fio of %, while % produces an fio of %, both in healthy subjects and patients with copd. however, it must be remembered that not all commercial models based on the venturi principle are able to administer oxygen with an error of less than %, as claimed in their specifications. in addition, the fio can be reduced if the inspiratory flow of the patient exceeds the total flow generated by the apparatus. although its role is more limited, the dead space inside the mask also affects the concentration of oxygen provided. it is also necessary to consider that nitrogen is % less dense than oxygen, meaning that the carryover capacity for air through the venturi system is lower than that of oxygen, thereby making the fio achieved less accurate. thus, it appears reasonable to suggest that if this system is used to administer the hypoxic gas mixture then fio should be monitored simultaneously. during the test, the patient will be asked to breathe at tidal volume and the test will be ended after minutes , or when a stable situation is achieved, defined as the absence of variability in spo (± %) or heart rate (± beats per minute) for at least minutes. it is recommended that spo be monitored continuously and that arterial blood gas analysis be performed at the beginning and end of the test. in terms of pulse oximetry, it should not be forgotten that true oxygenation can be slightly overestimated in smokers, given that the technique does not discriminate between oxyhemoglobin and carboxyhemoglobin. furthermore, most pulse oximeters display a certain degree of inaccuracy and variability in the saturation range between % and %. therefore, spo should only be used to monitor the test, while interpretation of the test results should be based on pao . in both healthy subjects and patients with copd, the hypoxic challenge test provides a measure comparable to that obtained by simulating the same altitude in a hypobaric chamber. the relationship between isobaric hypoxia and hypobaric hypoxia appears not to be affected by the age or the sex of the subjects. in turn, it has also been demonstrated that there is a good correlation between the pao obtained during simulation of altitude-induced hypoxia and that determined during flight, although this correlation is weakened when the interval between the measurements is longer than months. in terms of safety, the tolerance of hypoxic challenge is good and only mild side effects such as tachycardia, dyspnea, vertigo or nausea, headache, and sleepiness have been described. hypoxic challenge offers certain advantages over prediction equations. it provides a more accurate assessment of the individual's response to hypoxia. in addition, it allows assessment of the possible effects of hypoxia, such as symptoms or electrocardiographic (ecg) abnormalities. although initial studies involved continuous ecg monitoring, , few arrhythmias related to hypoxia were identified and all of them were benign; consequently, systematic ecg monitoring is not recommended. however, it may be considered on an individual basis in patients with cardiovascular comorbidity. despite these considerations, hypoxic challenge is a procedure that also presents limitations. it does not reproduce cabin conditions of pressure or air density. however, in order for reduced air density or flow turbulence to generate an increase in fev or a reduction in work of breathing, altitudes of more than m are required, suggesting that these factors will have little influence. in addition, the potential beneficial effect of the reduced air density will never be greater than the negative effect caused by the reduction in pio , the increase in lung elasticity and air trapping, and the poor distribution of ventilation. , the length of the flight is also not taken into account during hypoxic challenge. however, changes in arterial blood gases during a flight lasting hours have recently been analyzed in patients with copd. it has been demonstrated that when patients remain seated pao falls until cruising altitude is reached and then remains stable for the rest of the flight. there is less consensus regarding the application of these recommendations in children with respiratory diseases. little information is available on physiologic changes at altitude in children. in addition, the spectrum of disease can be very broad. in premature babies with acute viral respiratory infection there is a greater risk of apnea due to immaturity of the breathing pattern. in that case, environmental hypoxia can increase the risk of apnea and it is therefore recommended that infants do not fly until months after the date for full-term birth. on the other hand, some children with cystic fibrosis are better adapted to a hypoxic environment, probably through changes in the dissociation characteristics of hemoglobin. as a result, the current recommendation considers that children with an fev less than % of reference for cystic fibrosis or other chronic lung disease should undergo a hypoxic challenge test and that if spo is less than % during the test then provision of oxygen during the flight should be prescribed. , the most recommendable route for administration of the hypoxic gas mixture in children is breathing in a plethysmography chamber. supplementary oxygen is recommended during air travel for patients who have an estimated in-flight pao of less then mm hg obtained with prediction equations or, preferably, a hypoxic challenge test ( figure ). , the criteria on which this cutoff is based are arbitrary. since healthy individuals can reach a pao of to mm hg at cabin altitude, mm hg was considered to represent the lower limit for a clinically acceptable pao . therefore, that cutoff is based on expert consensus and does not have scientific support. patients with an estimated pao greater than mm hg could fly without a requirement for supplementary oxygen. finally, the group of patients with an estimated pao between and mm hg should be assessed on an individual basis. in this case, if there is serious deterioration of resting lung function, marked exercise limitation in either the walk test or the incremental cardiorespiratory exercise test, or comorbidity, provision of oxygen during the flight could also be recommended ( figure ). oxygen is usually provided during the flight through nasal prongs. in patients with severe copd subjected to conditions of hypobaric hypoxia similar to those in the cabin of a commercial aircraft, it has been shown that provision of oxygen through nasal prongs at a rate of l/min produces a greater increase in pao than when administered using a venturi mask at % or %. in fact, ventimask systems may favor dilution of ambient air at relatively low flow rates. an oxygen flow of l/min appears sufficient to correct the hypoxemia in most cases. it has been confirmed that provision of oxygen through nasal prongs at l/min in healthy subjects and patients with obstructive or restrictive disease who breathe an ambient fio of % achieves an spo similar to that recorded when they breathe at an fio of %. in restrictive diseases, a flow rate of l/min also appears to be sufficient to maintain adequate oxygenation during the flight, although when the patient moves about the aircraft it may be advisable to increase the flow to l/min, so long as an extension is available. finally, provision of supplementary oxygen should be considered a safe and effective procedure for the management of many patients with chronic respiratory diseases who undertake a journey by air. , , for example, it has recently been described that provision of oxygen during flights of up to km allowed a group of patients with severe lung disease to reach their destinations satisfactorily. in that study, only a few episodes of near fainting were observed due to insufficient oxygenation when going to the bathroom without supplementary oxygen. consultation prior to air travel. however, the response of physicians cannot currently be clear and robust, since there is insufficient scientific evidence and many elements remain to be clarified. in general, the first recommendation for a patient with copd and hypoxemia would be to avoid air travel and look for other means of transport. this indication might have been valid some years ago but it is currently insufficient for many patients, since it may affect their quality of life and in some cases their work. in fact, a small survey performed in the united states of america on patients with severe copd showed that each year approximately in traveled by air. however, those results cannot be extrapolated to spain, where the proportion is likely to be lower. as for other diseases, it is accepted that patients with copd should maintain a pao of more than mm hg during a flight. , , with this threshold, no problems have been observed in studies involving hypoxic challenge and it seems reasonable given the clinical experience accumulated in patients with copd treated by continuous home oxygen therapy. however, this level is arbitrary and no studies have analyzed its possible consequences in periods of time closer to those of flights, although flight duration appears to have less effect than the altitude reached. despite the potential impact of copd, few studies have addressed the problem of hypoxemia at high altitude during air travel in this setting. furthermore, the studies performed have involved small samples of patients without severe hypoxemia, the majority eucapnic, and without significant cardiovascular comorbidity. , , , , the results of those studies indicate that patients can have reductions in pao of up to mm hg when they reach an in-flight altitude of m ( feet). this situation is not uncommon in normal flights, and although the incidence of medical problems appears minimal in the general population, , the same is not true of copd patients, in whom symptoms and the requirement for in-flight medical assistance are more common. nevertheless, these events do not normally appear to be particularly serious, and when they are, they are usually cardiovascular in origin. , although the interpretation of these data may be erroneous due to the limitations of their collection, it is also possible that the tolerance of hypoxemia in patients with copd (without other factors that could alter oxygen transport such as heart disease or anemia) is greater than might be expected. according to current knowledge, it could be recommended that all patients with moderate or severe copd who wish to travel by air should be clinically assessed, with attention to the following elements: ) ruling out the presence of exacerbation or that the patient is in an early phase of recovery from an exacerbation, ) identifying the treatment being taken, and ) reducing comorbidity. once clinical stability has been confirmed and treatment optimized, arterial blood gas analysis and spirometry should be performed in the days prior to flying. the values obtained for pao must be adjusted to sea level; in some regions of spain that may imply an increase of up to mm hg. in order to simplify the assessment, the following algorithm could be recommended in response to the presence of hypoxemia ( figure ): . pao > mm hg. in general, patients with this pao will not present severe hypobaric hypoxemia, making systematic estimation of in-flight pao unnecessary. nevertheless, the presence of symptoms (dyspnea or chest pain) during previous flights should be assessed, and if they are present, oxygen support at low flow rates ( - l/min) should be recommended. it also seems wise to extend that treatment option to those cases and in which the in-flight cabin pressure corresponds to an altitude of greater than m ( feet) and the patient has very severe copd (fev ≤ %), where limitations may be present in the mechanisms of compensation for hypoxemia, or diseases that alter oxygen transport. . pao = - mm hg. an estimate of in-flight pao should be made using a prediction equation or, preferably, hypoxic challenge. prescription of oxygen at low flow rates is recommended in the following situations: -estimated in-flight pao less than mm hg -flights in which the cabin pressure corresponds to an altitude greater than m ( feet) -presence of cardiovascular comorbidity and/or anemia . pao < mm hg. patients in this situation usually already receive continuous home oxygen therapy. the goal would be maintenance of the same oxygen levels during the flight, necessitating an increase of to . l/min over the patient's usual oxygen support. such treatment should not normally create problems in eucapnic copd patients, in whom a tendency toward hypocapnia due to hyperventilation has been observed. however, in the presence of hypercapnia, prior assessment of variations in gas exchange following increased oxygen support should be undertaken. it is important to mention that patients who are not receiving continuous home oxygen therapy have a lower sense of the severity of the disease and a substantial proportion may not consult their doctor prior to undertaking air travel. thus, improved treatment education should be developed for this patient population. alongside preflight planning based on pao , other general measures to prevent deterioration of hypoxemia include the following: -avoid excessive physical effort: do not carry weight and reserve a seat close to the bathroom. however, this should not be a contraindication for the necessary movement of the lower limbs to prevent dvt -avoid sleep -do not eat large meals it is advisable for airline companies to have trained staff available who are able to monitor spo in patients who require oxygen during the flight (spo between % and % could be acceptable). in addition, they might be able help to detect abnormalities in heart rhythm, which although rare, show a high between-individual variability. this monitoring is essential if the patient has to travel urgently whilst clinically unstable. while awaiting new studies that improve upon the substantial limitations in our understanding, the overall message is that all patients with copd should be assessed by their pneumologist prior to air travel. supplementary oxygen should be provided for those patients whose estimated in-flight pao is less than mm hg, taking particular care with those who have cardiovascular comorbidity. commercial flights represent a favorable environment for the spread of pathogens transported by passengers or flight personnel, as was shown during the recent outbreak of sars. few studies or data are available on this topic and it is difficult to quantify the global repercussions, which may be underestimated, since almost all of the diseases involved have incubation periods that are shorter than the length of the trip, some of the diseases are treated as trivial processes, and the studies that have been performed have included a significant proportion of passengers who could not be located. the international health regulations adopted worldwide in to limit the spread of disease are in the process of revision. , recently, the world health organization (who) published guidelines on infectious diseases and air travel. risk factors. the respiratory infections that have been the object of the greatest interest are pulmonary tuberculosis, sars, and infections caused by the influenza virus. since the microorganisms responsible for those infections are mainly transmitted through the air, the risk of transmission during flights is affected by duration, the proximity of the index case, and the cabin ventilation, in addition to the pathogenic characteristics, the epidemiology of the infection in each region, and the immune status of the subject. the use of appropriate filters and correct recirculation of air in the plane reduces the risk of infection. although the safety of hepa filters in protection against viruses has been questioned, a more serious concern is the absence of legislation obliging their use in most countries. hepa filters were found not to be used on % of flights carrying more than passengers in the usa, and that figure is considerably higher in small planes that undertake local flights. based on the cases analyzed and studies involving mathematical models, individuals seated in either of the rows of seats closest to the affected passenger are at the highest risk for transmission of mycobacterium tuberculosis and if ventilation is doubled, the risk is reduced by half. the probability of transmission is also reduced to almost zero in passengers seated rows from the zone of infection. , however, this "safe distance" does not apply in the case of a patient with sars, who could infect any other healthy passenger seated in the next rows. studies performed by the who have failed to demonstrate that air recirculation by itself facilitates transmission of infectious disease on board aircraft. however, it should be confirmed that the cabin ventilation system functions correctly and continuously while passengers are on board, independently of whether or not the plane is in flight or held on the runway, as inadequate functioning clinical calls have recently been made in scientific journals and in the general media for serious consideration to be given to regulations on the use of hepa filters and for an increase in the number of checks made on aircraft by the authorities. , tuberculosis. a third of the world's population is infected by m tuberculosis, and consequently, it is the most extensively studied model of transmission during air travel. evidence is available that transmission from smear-positive individuals is more common during long flights (longer than hours) and can affect both the passengers and crew members. seven episodes of possible tuberculosis transmission during airplane journeys have been studied, of the episodes corresponding to strains resistant to isoniazid and rifampicin. possible transmission of the infections (mantoux conversion) to other passengers or crew members could only be established in of the episodes, although it was not possible to demonstrate development of the disease as a result of exposure during a commercial flight in any of the cases. , in the remainder, the studies found no evidence of transmission, were inconclusive, , or the likelihood of transmission was considered very low. in all of the cases, the index patient had substantial radiographic involvement and sputum stains revealed acid-fast bacilli with positive sputum cultures. despite the fact that acquisition of the disease and possibly transmission of the infection is less likely than in other modes of transport, a great deal of anxiety has been generated among the public, health authorities, and airline companies, and consequently, the who has published guidelines with a protocol that ends with a series of recommendations for passengers, physicians, health authorities, and airlines (appendix ). severe acute respiratory syndrome. the epidemic outbreak of sars, for which the causative agent is a coronavirus, is the most recent and representative example of a disease transmitted by a very small number of travelers to other countries and continents within a few weeks. studies showed that in of the flights investigated for carrying patients infected with the sars virus transmission of the virus to other passengers was likely to have occurred. . - the majority of the patients who were infected had been seated in the rows closest to the index case, although at least in flight lasting hours (hong kong-beijing) an outbreak occurred that affected a high percentage of passengers seated up to rows from the index case and subsequently in more than secondary cases. possible explanations for that outbreak have been sought, and although no conclusive results have been obtained, it has been suggested to have occurred mainly through aerial transmission from a direct or indirect contact, that some of the passengers were infected prior to the flight, or that it occurred as a result of defective cabin ventilation. the cabin crew may have an increased risk of acquiring the disease due to their movement through the aircraft. the who developed a series of recommendations and guidelines, which included a series of measures that should be followed by all countries (appendix ) . , once those measures were put into practice, no new cases of long-distance propagation of the disease were identified. influenza. epidemic infection with the influenza a virus appears between the months of october and april in the northern hemisphere and between may and september in the southern hemisphere. in a recent study undertaken in switzerland, almost % of passengers who suffered fever during a journey to subtropical or tropical regions had a significant antibody titer against influenza viruses when they returned and in more than % it was possible to demonstrate a seroconversion of more than times the initial titer. the most common pathogens in fever episodes outside the periods of local epidemic were influenza viruses. that source may be the cause of some of the limited outbreaks that occur during the nonepidemic period. , other viruses such as influenza b and parainfluenza also have demonstrated pathogenic capacity. , as in conventional epidemic outbreaks, a series of risk factors affect acquisition of infection, such as age over years, presentation of comorbid conditions, and close contact with the index case, meaning that tourism in groups can facilitate infection. nevertheless, only studies have reported infection during air travel. , , the passengers seated in the rows closest to the index case were the most often affected, although given the high infectiousness of the virus, between % and % of the passengers was possible in flights lasting longer than hours and up to % of secondary familial contacts developed the disease. suspension or failure of the ventilation system favors disease transmission, as demonstrated in a flight in which an individual with flu infected % of the passengers. some countries recommend flu vaccination for those passengers undertaking journeys to the southern hemisphere during the summer and who were not vaccinated during the previous year. some microorganisms that do not produce respiratory symptoms, or at least are not associated with respiratory conditions as the principal symptoms, are nevertheless transmitted through the airways. among them, meningococcus and measles virus are the most noteworthy as a result of their infectiousness, morbidity and mortality. between and , cases were studied of patients with meningococcal disease who had traveled by plane during the infectious period without evidence of a single secondary case. nevertheless, given the severity of the disease, it is advised that individuals seated near the index case begin prophylactic treatment in the hours following the case being reported, so long as less than days have elapsed since the contact. , the measles virus is highly contagious, with up to % of exposed individuals developing the disease, and cases have been described of transmission during air travel. [ ] [ ] [ ] [ ] currently, the vaccination schedule in the different autonomous communities of spain includes vaccination against meningococcus from the age of years and measles from months, making the risk of transmission of those diseases presumably minimal, although individuals without antibodies or those from other countries who have not been vaccinated could be affected. no epidemic outbreaks have been reported for the virus that causes the common cold, but this absence is presumably due to the high frequency of the disease and the difficulties associated with investigating it. one study found no evidence that the air recirculation system in the cabin aided appearance of symptoms of infection in the upper airways. there is currently a great deal of concern regarding spread of the avian flu virus (h n ). this virus has a shorter incubation period and is more contagious than the sars virus. the usa has prepared a national plan to prevent the spread of outbreaks through the establishment of a series of specific health measures in airports. in addition to an increase in the number of health care workers, medical consulting rooms have been built that allow the health of passengers to be assessed and isolation rooms created to establish a quarantine area in international airports. those facilities are in permanent contact with the centers for disease control and prevention (cdc) and have access to passenger information for all flights in order to identify contacts of a possible index case. to date, the benefits of such a strategy have not been demonstrated and it is quite unlikely that it would prevent or slow an epidemic caused by introduction of the influenza or sars virus. detection of individuals with the disease exclusively in the destination airport would only have consequences for the detection of individuals who developed the clinical features during the flight and of contacts, thereby making the sensitivity low. most experts are in favor of strategies similar to those followed in the sars outbreak, including monitoring to detect individuals with symptoms in the departure airport, in an effort to prevent individuals with the disease from boarding the flight. [ ] [ ] [ ] [ ] if a case of infection with the avian influenza virus is confirmed, isolation measures similar to those followed for patients and contacts with sars must be established, treatment with neuraminidase inhibitors should be initiated immediately, and in contacts, prophylactic measures with those drugs should be started during the first hours. if a specific vaccine is available it should be immediately administered to contacts. the who has established a global plan in which these elements are considered. , recently, a series of recommendations and considerations were prepared on the management of exposure to an infectious disease during commercial air travel : -although passenger transport companies can refuse to transport individuals with a disease, they cannot undertake systematic examination in an effort to identify ill passengers. -early diagnosis is necessary to establish measures for the other passengers. -governments have the legal authority, in accordance with international law, to establish controls on passengers with transmissible diseases for which declaration is obligatory. -the authorities may establish measures to quarantine passengers who arrive at their airports. -physicians must identify those subjects who are not in a good enough state of health to travel by air and inform them of how a flight might affect their health. -prevention is the best course of action and postponement of the journey should be advised. -hand washing reduces the risk of transmission of contagious diseases and should be performed as a matter of course during travel and always prior to eating. -the mouth and nose should be covered in the event of sneezing or coughing and hands should be washed afterwards to protect others. -in the case of a passenger with suspected sars during the flight, a us national institute for occupational safety and health n mask should be provided and an isolation zone established in the aircraft. survival and quality of life have improved in patients with cystic fibrosis, making it not uncommon for them to want to go on holidays and even undertake work that may involve air travel. few studies have assessed the effects of commercial flights on patients with cystic fibrosis. there is some disagreement regarding estimation of the level of hypoxemia in those patients. although in a study performed in a small group of patients aged between and years, hypoxic challenge predicted with a high level of sensitivity and specificity the development of desaturation during the flight, later studies have not confirmed those findings. a study undertaken by the same group that contained a larger number of subjects and involved longer flights ( - hours) contradicted the earlier findings and showed that an fev less than % of reference better identified patients who desaturated than did the results of hypoxic challenge. only a small percentage of the patients who displayed reductions in spo to below % presented symptoms and required oxygen supplementation. however, it should be noted that the patients included in those studies were stable, had disease that was not very advanced, and were younger than other groups of patients with cardiac or respiratory diseases for whom reduction of pao to below mm hg necessitates the implementation of oxygen therapy during the flight. this would explain the greater tolerance of hypoxia seen in patients with cystic fibrosis, confirmed both in acute exposure in hypobaric chambers and during time at altitude. in addition, in patients with cystic fibrosis, the results of hypoxic challenge are particularly variable over time and can change within a few weeks. , consequently, the decision to have a cystic fibrosis patient use oxygen therapy during a flight should not be based exclusively on hypoxic challenge tests but also on clinical parameters and the degree of bronchial obstruction. other recommendations to consider in patients with cystic fibrosis who intend to travel by air are summarized in table . some authors have described an increase in exacerbations following a holiday, , related to poorer management of the disease. correct compliance with treatment and, in particular, physiotherapy improves the conditions in which the return flight is undertaken and reduces the likelihood of complications. the estimated incidence of venous thromboembolic disease (vtd) in the general population is per person-years. the pathogenesis of dvt was first described by virchow in , and the description remains valid today. it is based on a triad formed by stasis of venous blood flow, damage to the vascular endothelium, and hypercoagulability. these circumstances coincide in acquired-transient or persistent-or congenital conditions defined as risk factors, present in approximately % of patients with vtd. extended journeys have been associated with an increased incidence of vtd and have been included in the list of risk factors. , in , the term "economy class syndrome" was coined following the description of cases of vtd after flights in economy class. the aim was to highlight that the limited space within which to stretch the legs during an extended period of time reduces venous return and favors stasis of venous blood flow. this situation is not exclusive to air travel in economy class. it has also been described in business class and in other forms of travel, such as cars and buses, involving long periods of time with the lower limbs flexed and at rest. apart from venous stasis, there is a lack of agreement regarding other factors associated with air travel that could contribute to dvt such as dehydration, favored by the low humidity of the cabin and in some cases increased by the diuretic effect of coffee or alcoholic drinks, and the hypobaric hypoxia associated with pressurized cabins. dehydration could predispose to dvt as a result of hemoconcentration and blood hyperviscosity, although this hypothesis has not been confirmed. it has been observed in experimental studies that hypobaric hypoxia favors activation of clotting , and reduces physiologic fibrinolytic activity of endothelial cells ; however, those results have not been reproduced in subsequent studies. incidence and risk of vtd. studies addressing the incidence and risk of thrombosis associated with long-distance flights have employed a variety of different methods and yielded disparate results. for passengers with a high risk of thrombosis due to the presence of additional risk factors the incidence of vtd appears to be high, from % to %. , in patients at low or moderate risk the incidence drops to between % and %. , most of the vtd events that were identified were asymptomatic dvt that exclusively affected the venous territory of the calf, although the screening method used in almost all of the studies involved venous compression ultrasound with or without doppler, raising questions over the results due to the limited sensitivity of the technique for distal clots. the influence of other individual risk factors appears to be decisive in generating dvt. the incidence of pulmonary embolism has been assessed in cohort studies. [ ] [ ] [ ] according to data collected in paris airports between and , the incidence of this entity has increased. significant differences have been described in incidence rates according to distance traveled, ranging from . per passengers for distances of less than km to . cases per passengers in flights of more than km. differences were also seen according to distance traveled in a study performed at madrid barajas airport. in flights lasting more than hours the incidence of pulmonary embolism was . per passengers and in flights lasting to hours it was . per passengers, while no cases were observed in flights lasting less than hours. consequently, hours has been considered the cutoff for recommending general measures for the periodic movement of the limbs. the relative risk of vtd is difficult to establish due to the heterogeneity of the studies. , , considering only air travel, the risk is not clear (odds ratio, . ), and consequently, it could not be considered as an independent risk factor. however, in passengers with additional risk factors for thrombosis, the odds ratio increased in all studies to represent a -fold to -fold higher risk of vtd. recently, it has been demonstrated that the immobility during a flight lasting more than hours increases the levels of certain markers of clotting in subjects without risk factors for thrombosis, but it remains to be established whether this represents an increased risk of vtd. prophylactic measures. patients must be assessed individually and the presence of other risk factors for venous thrombosis identified (table ) in order to adopt prophylactic interventions. classification of the risk as moderate or high in these circumstances is not well established. it seems reasonable to extrapolate the impact of each of these factors on vtd. general measures. adequate hydration, regular movement of the lower limbs, and avoiding keeping the legs bent for long periods of time are the measures recommended by most experts. these measure are recommended for general application in flights lasting more than hours. drink lots of liquids to avoid noxious effects of the dry cabin air on the secretions and mucosa of the airway if the patient uses a nebulizer, some airlines allow the patient's own nebulizer to be used or provide one for long-haul journeys if possible, physiotherapy exercises should be performed during stopovers on long journeys compression stockings. in passengers at high risk of thrombosis, compression stockings, generally knee length and with a pressure of to mm hg have proven to be effective in reducing the incidence of vtd; [ ] [ ] [ ] ; no adverse effects are associated with their use and they are well tolerated. prophylactic drug treatment. the use of acetylsalicylic acid and low molecular weight heparins has been tested in passengers at high risk of thrombosis. a dose of mg acetylsalicylic acid for days proved to be ineffective and caused gastrointestinal discomfort in % of subjects. in contrast, a single dose of enoxaparin, both at a therapeutic weight-adjusted dose and as a high-risk prophylactic dose, administered to hours prior to the flight reduced the incidence of dvt without side effects. the general conclusions on vtd and air travel are summarized in table . few studies have addressed the effects of air travel on patients with respiratory diseases who present respiratory failure or severe abnormalities in control of ventilation. issues that must be taken into account in relation to air travel in such patients, in addition to the characteristics and length of the flight, are the following: ) the total length of the journey (flight time plus predicted waiting time and risk of unexpected delays), ) travel from the airport to the final destination, ) logistic aspects such as provision of oxygen or the feasibility of charging the batteries of the apparatus or a wheelchair during the flight and at the destination, and ) the altitude of the destination point and the length of time the individual will remain there. most patients can travel despite limitations, so long as the journey is sufficiently prepared and no elements are left to chance. in general, an increase in oxygen flow of to l is recommended in patients who receive home oxygen therapy. it is also essential to know the conditions of each airline company prior to embarking upon a journey, both in terms of the transport and provision of oxygen and in relation to the accessories required by the patient (wheelchair, ventilator) and the requirement to travel with an escort. some companies allow the passenger to carry small oxygen bottles (a maximum of bottles less than . m long and mm in diameter), but other companies do not accept transport of oxygen, although they allow the use of some oxygen concentrators, according to very strict regulations, so long as the user has sufficient batteries available to last the entire duration of the flight. cases have been described of patients with kyphoscoliosis or neuromuscular diseases in whom long air journeys generated right heart failure, presumably linked to the hypoxia maintained during the flight. from a theoretical point of view, in patients with nonhypercapnic restrictive disease (caused by involvement of the parenchyma), who present a risk of hypoxia during the flight, oxygen would be indicated to reduce the impact of hypoxemia on pulmonary hypertension. in patients with restrictive diseases who use mechanical ventilation (for extrapulmonary involvement), it is recommendable that they carry the apparatus with them during the flight, even if they only use it at night. clearly, patients with continuous ventilation should carefully assess the journey since they will need to use the ventilator throughout the travel period, including during airport transfers. from a logistic perspective, it is very important to confirm the hand luggage that the patient can carry, especially in relation to wheelchairs, the ventilator, and the spare battery. in the case of patients with severe disability, most airlines require the presence of an escort and consider that person can take responsibility for there are few reports in the literature on the impact of air travel in patients with sleep apnea-hypopnea syndrome (sahs). some complications have been associated with long journeys followed by a period at altitude. all patients with sahs should avoid consumption of alcohol immediately before and during the flight. patients in a severe condition should employ continuous positive airway pressure (cpap) during long flights. to this end, they should have a dry cell battery available for use as an energy source for the equipment. although the low humidity of the air in aircraft cabins may favor the development of bronchospasm due to loss of water from the bronchial mucosa, asthma attacks during air travel are thought to be rare. in addition, it is sometimes difficult to differentiate them from dyspnea due to hyperventilation or panic. more recently, a higher incidence of episodes of bronchospasm requiring treatment during flight has been described. patients with controlled asthma and no respiratory failure do not present problems for air travel, although they should ensure that they have their medication to hand. patients with severe asthma with frequent exacerbations and serious attacks should ensure that the disease is well controlled prior to the day of the flight. since , the emergency medication in most aircraft includes bronchodilators, both in pressurized cartridges and for injection. however, in case of an attack, patients are recommended to take their normal rescue medication. patients with primary tumors or metastases can generally fly safely. nevertheless, it may be necessary to consider measures to alleviate hypoxemia or pain. pneumothorax is a contraindication for air travel. a patient will only be allowed to fly when the lung has been completely reinflated. the patient should not be allowed to fly until hours after pleural drainage has been withdrawn and with a radiograph performed hours after completion of drainage to confirm resolution of the pneumothorax. optionally, some airline companies may accept transport of a passenger with a pleural drain. in that case, since it is difficult to guarantee continuous aspiration during the flight, it is recommended that a heimlich valve be used. in exceptional cases it may be necessary to evacuate a pneumothorax during the flight. this should only be done by trained staff and when the cabin pressure corresponds to sea level. simple rib fractures do not usually present problems during the flight, particularly when there is no lung damage or prior pulmonary disease. the main problem associated with such fractures is pain, which can reduce ventilation. therefore, it is important that adequate analgesia is guaranteed during the flight. multiple fractures may cause thoracic instability and, in that case, the requirement for specialized transport should be considered. flights should be postponed in all patients with acute respiratory failure due to lung contusion until lung function returns to normal. , likewise, mediastinal or subcutaneous emphysema constitutes a contraindication for travel on commercial flights. in any of those situations, if air travel is essential an air ambulance is required. although individual assessment is necessary, as a general rule patients are advised not to fly until at least weeks after the operation. patients with respiratory diseases who require oxygen on board or some form of health care during the flight are the association between venous thromboembolic disease (vtd) and air travel is weak. the clearest risk is for presentation of asymptomatic deep vein thrombosis (dvt) restricted to the calf area. symptomatic episodes of vtd, including fatal pulmonary thromboembolism, are rare. the risk is increased in journeys lasting more than hours in patients with additional risk factors. regular movement of the legs and hydration should be a general recommendation. in passengers with other risk factors for venous thromboembolism, the decision to implement other prophylactic measures should be made on an individual basis. knee-length compression stockings are effective and reduce the incidence of dvt. low molecular weight heparins are effective in patients at high risk of thrombosis. in general, a single high-risk prophylactic dose of low molecular weight heparin appears to be sufficient but should be assessed on an individual basis. aspirin is ineffective and should not be recommended. considered as ill patients who require medical authorization (medical fitness for air travel [meda] case). all patients who report such a condition must be informed when making the reservation of the process that needs to be followed in order to obtain medical authorization, of the limitation and requirements that exist, of the number of escorts required, and of the cost of the service requested. in turn, they must complete the incad/medif form provided by the company (appendix ), based on iata recommendations, and send it by fax to the medical department of the airline company to receive authorization and initiate the corresponding procedures. oxygen is normally supplied through a mask, although patients may use their own nasal prongs. three sources of oxygen can be used in aircraft. if cabin pressure is lost, passengers may receive oxygen through masks located above their seats. however, this oxygen source, which has a limited duration, cannot be used for provision of supplementary oxygen to sick patients during the flight. the most common practice is to use cylinders with a capacity of cubic feet. at a flow rate of l/min, those cylinders can provide oxygen for hours, , making it important to estimate the number of cylinders that the patient will need based on the flow prescribed and the length of the journey. recently, the american department of transportation approved the use of portable oxygen concentrators, which can be used during takeoff and landing and while moving inside the cabin. this equipment can also help the patient while moving inside the plane and in the terminal. to date, the only approved models are manufactured by inogen (www.inogen.net) and airsep (www.airsep.com). it should be noted that most companies do not allow the use of liquid oxygen on board. if the patient wishes to transport a portable liquid oxygen system it must be checked in empty and filled on arrival at the destination. in general, in-flight oxygen is administered at flow rates of or l/min, and exceptionally, at l/min. the medical department of the airline company may require that the patient be accompanied by an escort trained in the used of the oxygen therapy system. in most cases, provision of oxygen during the flight is a service paid for by the passenger. as a guide, from january the spanish airline iberia charges € per flight and requires at least hours notice prior to departure of the flight or hours in the case of emergencies. in more exceptional cases, some companies may insist that a second seat is purchased for the oxygen source. previous experiences of travel with patients requiring oxygen therapy or mechanical ventilation show that the main problems arise during transfer of the patients. in general, most companies only provide oxygen during the period of time inside the plane or during transfer between planes of the same company. if oxygen is required during boarding or while waiting in the airport, the passenger should inform the medical services of the company to organize specialized transport, such as ambulance transfer to the plane. transport with oxygen during the flight does not represent an exceptional situation. data from the airline iberia indicate that persons require supplementary oxygen in flight each year. it is also possible to use cpap equipment or ventilators during flights. in that case, patients should carry their own equipment, since it is not provided by airlines. it is important to mention that, since the great majority of commercial aircraft do not have plug sockets in the cabin, the patient should carry a dry cell battery to independently power the equipment. permission to use cpap or a ventilator on board must also be requested when making the reservation and requires authorization by the medical department of the company. in general, an escort is not required for the use of cpap, obtain a report of the clinical condition of the patient that includes the most recent functional assessment and treatment. this is essential if the stay is for a number of weeks and the destination does not have the usual health care resources. in countries in which smoking is still allowed inside the aircraft, the patient must be seated in a nonsmoking area. avoid excessive alcohol consumption prior to and during the flight, especially in cases of apnea-hypopnea syndrome and risk of venous thromboembolic disease. move around during long flights, unless oxygen is required. if oxygen is required, it should be used if possible while moving inside the plane (with an extension to allow movement). prophylactic measures should be taken to reduce the risk of thromboembolism. carry required medication, especially rescue inhalers, in hand luggage. if medication is checked with baggage, ensure that it is not affected by the extreme conditions in the hold. use spacer chambers rather than nebulizers. if continuous positive airway pressure is required on a long-haul flight, carry a dry cell battery, which must be switched off prior to landing. patients who require a ventilator must be able to tolerate temporary disconnection of the apparatus during takeoff and landing. the requirement for oxygen or any other form of medical assistance must be indicated when the reservation is made, at least hours. prior to departure. if necessary, assistance must be organized with the medical department of the company to transfer the patient within the airport. whereas mechanical ventilation usually demands the presence of an assistant trained in its use. patients who are completely dependent on a ventilator and cannot tolerate temporary disconnection of the equipment during takeoff and landing, or in the event of other occurrences, cannot fly in commercial aircraft. in such cases, the use of air ambulances is necessary. nevertheless, there is a marked diversity in the regulations, availability, cost, and ease of oxygen provision during air travel, making it advisable for patients or their representatives to determine the criteria established by the company with which they intend to fly. this information can be obtained directly from travel agencies, when making a reservation, or via the webpage of the british lung foundation. finally, all patients with respiratory diseases who intend to fly are advised to consider certain general recommendations ( table ) and even to access specific information sources for patients. [ ] [ ] [ ] . individuals with tuberculosis (tb) with the possibility of between-individual transfer, such as sputum-positive patients, must postpone their journey until they are no longer a potential source of transmission. . if the history of a patient with tb who could transmit the disease shows that he or she has recently undertaken a journey by air (eg, within the last months), the physician should immediately inform the health authorities in the declaration of the tb case. . the health authorities should immediately contact the airline company if the person has undertaken a journey lasting at least hours in a commercial aircraft during the last months. for the airline companies . airline companies should work closely with health authorities in the provision of information to passengers and flight crew who may have been exposed to mycobacterium tuberculosis as well as in the identification of those passengers who should be informed. airline companies should cooperate closely with health authorities in the provision of information to passengers and flight crew who may have been exposed to m tuberculosis as well as in the identification of those passengers who should be informed. airline companies should require the home and work addresses and telephone numbers of passengers so that they can be informed in the event of potential health risks (exposure to m tuberculosis or other infectious diseases, exposure to toxins, etc). airline companies should ensure that all crew receive appropriate training in first aid and the use of universal precautions regarding exposure to biologic fluids. all aircraft must be equipped with emergency medical supplies (including gloves, masks containing high efficiency particulate air [hepa] filters, and biohazard bags). . airline companies must have prearranged access to physicians with experience in transmissible disease who are available for subsequent consultation by health authorities. records of all diseases and medical emergencies must be kept for at least years. . long delays should be reduced to a minimum and hepa filters should be installed and maintained at maximum efficiency ( . % at . µm). centro travel by air: health considerations flying with respiratory disease inflight deaths during commercial air travel. how big a problem? utilization of emergency kits by air carriers. oklahoma city: faa civil aeromedical institute response capability during civil air carrier inflight medical emergencies. oklahoma city: faa civil aeromedical institute british thoracic society standards for care committee. managing passengers with respiratory disease planning air travel: british thoracic society recommendations expert care, everywhere managing passengers with respiratory disease planning air travel: british thoracic society recommendations standards for the diagnosis and care of patients with chronic obstructive pulmonary disease the canadian thoracic society standards committee. recommendations for 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flight: the need for more rational guidelines recomendación separ sobre gasometría arterial recomendaciones separ ats/ers task force: standardisation of lung function testing. standardization of spirometry ats/ers task force: standardisation of lung function testing. standardisation of the single-breath determination of carbon monoxide uptake in the lung ats statement: guidelines for the sixminute walk test development of severe hypoxaemia in copd patients at m ( ft) altitude reevaluation of birth weights at high altitude hypoxia-altitude simulation test. evaluation of patients with chronic airway obstruction hypoxaemia during altitude exposure. a meta-analysis of chronic obstructive pulmonary disease hypoxia-altitude simulation test hypoxaemia during aeromedical evacuation the preflight evaluation. a comparison of the hypoxia inhalation test with hypobaric exposure predicted arterial oxygenation at commercial aircraft cabin altitudes effect of hypobaric hypoxia on blood gases in patients with restrictive lung disease effect of simulated commercial flight on oxygenation in patients with interstitial lung disease and chronic obstructive pulmonary disease detection and correction of hypoxemia associated with air travel air transportation of patients with acute respiratory failure: theory ventilationperfusion inequality in chronic obstructive pulmonary disease assessment of oxygen supplementation during air travel an air-entrainment device for preparing precision gas mixtures the effect of carbon monoxide inhalation on pulse oximetry and transcutaneous po accuracy of oxyhemoglobin saturation monitors during simulated altitude exposure of men with chronic obstructive pulmonary disease is normobaric simulation of hypobaric hypoxia accurate in chronic airflow limitation? pulmonary mechanics at altitude in normal and obstructive lung disease patients changes in lung volume, lung density, and distribution of ventilation during hypobaric decompression predicting hypoxaemia during flights in children with cystic fibrosis oxygen supplementation during air travel in patients with chronic obstructive pulmonary disease the accuracy of venturi masks at altitude the safety of air transportation of patients with advanced lung disease. experience with patients requiring lung transplantation or pulmonary tromboendarterectomy preflight medical screenings of patients. analysis of health and flight characteristics air travel in patients with chronic obstructive pulmonary disease altituderelated hypoxia: risk assessment and management for passengers on commercial aircraft short-term adaptation to moderate altitude kardiopulmonale belastung von flagpassagierien mit obstruktiven ventilationsstorungen prevalence of in-flight medical emergencies on commercial airlines hemodynamic effects of altitude exposure and oxygen administration in chronic obstructive pulmonary disease transmission of multidrug resistant mycobacterium tuberculosis during a long airplane flight revision of the international health regulations international infectious disease law. revision of the world health organization's international health regulations geneve: who using a mathematical model to evaluate the efficacy of tb control measures aviation safety: more research needed on the effects of air quality on airliner cabin occupants estimation of tuberculosis risk on a commercial airliner transmission of infectious diseases during commercial air travel transmission of severe acute respiratory syndrome on aircraft an outbreak of influenza aboard a commercial airline ticket to ride: spreading germs a mile high transmission of mycobacterium tuberculosis associated with air travel exposure to mycobacterium tuberculosis during air travel exposure of passengers and flight crew to mycobacterium tuberculosis on commercial aircraft, - tuberculosis risk after exposure on airplanes a passenger with pulmonary/laryngeal tuberculosis: no evidence of transmission on two short flight tuberculosis and air travel. guidelines for prevention and control. who/tb . . geneve: world health organization update . -more than cases reported globally, situation in taiwan, date on in-flight transmission, report on henan province, china consensus document on the epidemiology of severe acute respiratory syndrome (sars). who/cds/csr/gar/ who recommended measures for persons undertaking international travel from areas affected by severe acute respiratory syndrome (sars) world health organization. summary of sars and air travel in flight transmission of severe acute respiratory syndrome virus (sars): a case report influenza virus infection in travelers to tropical and subtropical countries an outbreak of influenza a/taiwan / (h n ) infections at a naval base and its association with airplane travel an outbreak of influenza a caused by imported virus en united states mixed outbreak of parainfluenza type and influenza b associated with tourism and air travel influenza outbreak related to air travel specific recommendations for vaccination and disease prevention: influenza exposure to patients with meningococcal disease on aircraft -united states imported measles in the united states low risk of measles transmission after exposure on an international airline light epidemiological notes and reports. interstate importation of measles following transmission in an airport-california multistate investigation of measles among adoptees from china aircraft cabin air recirculation and symptoms of the common cold triple airport quarantine stations. health program aims to prevent infectious diseases from entering country.washington post staff writer entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation are we ready for pandemic influenza? h n influenza pandemic: contingency plans global task force for influenza will it be the next pandemic influenza. are we really? aviar influenza. a new pandemic threat? who global influenza preparedness plan. the role of who and recommendations for national measures before and during pandemics prediction of hipoxaemia at high altitude in children with cystic fibrosis respiratory function and blood gas variables in cystic fibrosis patients during reduced environmental pressure lung function in adults with cystic fibrosis at altitude: impact on air travel flying cystic fibrosis: getting there and back safely exacerbations of cystic fibrosis after holidays at high altitude-a cautionary tale intensified physiotherapy improves fitness to fly in cystic fibrosis patients the epidemiology of venous thromboembolism relative impact of risk factors for deep vein thrombosis and pulmonary embolism: a population-based study an epidemiologic study of risk factors for deep vein thrombosis in medical outpatients: the sirius study guía para el diagnóstico, tratamiento y seguimiento de la tromboembolia pulmonar thrombosis of the deep leg veins due to prolonged sitting pulmonary thromboembolism after travel síndrome del pasajero de clase económica the best study -a prospective study to compare business class versus economy class air travel as a cause of thrombosis travel as a risk factor for venous thromboembolic disease: a case-control study association between acute hypobaric hypoxia and activation of coagulation in human beings changes of biochemical markers and functional tests for clot formation during long-haul flights is mild normobaric hypoxia a risk factor for venous thromboembolism? venous thromboembolism from air travel: the lonflit study venous thrombosis after long-haul flights frequency of venous thromboembolism in low to moderate risk long distance air travellers: the new zealand air traveller's thrombosis (nzatt) study venous thromboembolism in passengers following a -h flight: a case-control study thromboembolic syndrome from prolonged sitting and flights of long duration experience of the emergency medical service of the paris airports severe pulmonary embolism associated with air travel incidence of air travel-related pulmonary embolism at the madrid-barajas airport prevention of venous thromboembolism long-haul flights and deep vein thrombosis: a significant risk only when additional factors are also present activation of coagulation system during air travel: a crossover study prevention of edema, flight microangiopathy and venous thrombosis in long flights with elastic stockings. a randomised trial: the lonflit concorde edema-ssl study prevention of venous thrombosis with elastic stockings during long-haul flights: the lonflit jap study venous thrombosis from air travel: the lonflit study -prevention with aspirin vs low-molecularweight heparin (lmwh) in high-risk subjects: a randomised trial prevention of flight venous trombosis in high risk subjects with stockings or one-dose enoxaparin travel techonology-dependent patients with respiratory disease oxygen and air travel cor pulmonale presenting in a patient with congenital kyphoscoliosis following intercontinental air travel assisted ventilation at home. . oxford: medical publications emergencies in the air responding to medical events during commercial airline flights viajar con oxígeno. reflexiones a propósito de la primera reunión internacional de pacientes con déficit de alfa- antitripsina a comparative analysis of arranging in-flight oxygen aboard commercial air carriers air travel with a lung condition safe flying for people with lung disease medical guidelines for airline passengers the authors would like to thank dr fernando merelo de barberá, head of aerospace medicine at iberia, and dr francisco ríos tejada, head of the department of aerospace medicine at the aerospace medicine training center, for advice on preparation of the manuscript. . establish a screening system organized by the authorities in the affected regions in which all passengers are assessed by health workers at the point of departure. in case of suspicion during the flight, isolation measures should be taken for subjects who are suspected to carry the disease (provision of an exclusive bathroom, covering the mouth and nostrils of the patient with an appropriately protective mask) and the health authorities at the destination point should be informed about the suspicion. . management of contacts. contacts are considered as all individuals seated in the rows closest to the index case and all those who have had close contact with the index case prior to or during the journey. if the affected individual is a member of the cabin crew, all passengers are considered contacts. it is obligatory for the health authorities to identify and locate the whereabouts of those individuals for the following days and to contact the health authorities immediately if they develop any symptoms. . the aircraft should be disinfected according to world health organization guidelines. key: cord- -v spbo u authors: peterson, a. townsend title: biogeography of diseases: a framework for analysis date: - - journal: naturwissenschaften doi: . /s - - - sha: doc_id: cord_uid: v spbo u a growing body of literature offers a framework for understanding geographic and ecological distributions of species; a few applications of this framework have treated disease transmission systems and their geography. the general framework focuses on interactions among abiotic requirements, biotic constraints, and dispersal abilities of species as determinants of distributional areas. disease transmission systems have key differences from other sorts of biological phenomena: interactions among species are particularly important, interactions may be stable or unstable, abiotic conditions may be relatively less important in shaping disease distributions, and dispersal abilities may be quite variable. the ways in which these differences may influence disease transmission geography are complex; i illustrate their effects by means of worked examples regarding west nile virus, plague, filoviruses, and yellow fever. the past decade or so has seen considerable progress toward a general framework for understanding species' geographic distributions, essentially addressing the question, why is a species where it is and why is it not where it is not? key steps toward this understanding have included ( ) mathematical formulation and clarification of ecological niches of species and how they affect species' geographic distributions (pulliam ; hirzel et al. ; soberón ) , ( ) exploration of the role of historical events in shaping species' geographic distributions (wiens and graham ; martínez-meyer and peterson ; peterson and nyári ; waltari et al. ), and ( ) analysis of the time scale on which change in ecological niche characteristics is likely to occur (holt ; holt and gomulkiewicz ; peterson et al. ) . altogether, this body of work provides a basis for understanding the complexities of distributions of species in both geographic space and ecological dimensions. diseases are caused by pathogens-generally viruses, bacteria, fungi, and protozoa-that invade an individual's body and cause ill effects (for the purposes of this review, i distinguish between pathogens and parasites simply on the basis of size-pathogens being microorganisms and parasites being of larger size-and focus on pathogens). because disease processes are dynamic, taking place on extremely diverse scales of space (microscopic to continental) and time (minutes to centuries), and are the products of interactions among species (pathogens, reservoirs, vectors, etc.) , their ecological and distributional dynamics may differ from those of more "normal" species. these differences are the focus of this review, which i will illustrate via a series of examples. a recent concept paper presented a simple heuristic for understanding questions regarding species' distributions (soberón and peterson ) , which aims to provide a framework for thinking about species' distributional patterns, even if not a comprehensive summary of the phenomenon. this framework (fig. ) centered on a venn diagram showing interactions among three factors: abiotic conditions, biotic conditions, and accessibility considerations. the former two factors correspond roughly to the fundamental and realized niches as outlined by hutchinson ( ) ; although hutchinson focused more specifically on the role of competition among abiotic conditions, a more modern view would include other biotic interactions, such as predation, parasitism, and mutualism. accessibility considerations, including both current dispersal ability and opportunity for dispersal and colonization in the past (waltari et al. ) , were not integrated into hutchinson's ( ) framework. the heuristic diagram centers on requirements of particular abiotic conditions (i refer to this circle as "a" for abiotic conditions) and how they relate to biotic interactions (referred to as "b" for biotic conditions) and how they modify the abiotic requirements. for example, a species may have fairly broad tolerances of abiotic conditions such as temperature, solar radiation, rainfall, and soil chemistry. the interactions of this species with other species, however, may restrict it to only a subsector of the abiotically suitable areas-that is, some areas may not be suitable owing to the presence of a particular predator or pathogen or owing to the absence of a key mutualist or symbiont. as such, the potential distribution of the species can be seen as a∩b-or to put it into words, in these areas, both abiotic and biotic conditions are appropriate for the species to maintain populations. finally, the species may be limited from occupying the entirety of its distributional potential by accessibility considerations (referred to as "m" for mobility). that is, a hypothesis of the actual distribution of the species would be a∩b∩m, which are those areas that are simultaneously fig. diagrammatic representation of three important factors in determining species' geographic distributions. circle a summarizes abiotic variables that circumscribe species' geographic potential, circle b adds biotic considerations, and circle m indicates limitations on dispersal or movement ability. a∩ b is the potential geographic distribution of the species, and a∩b∩m is a hypothesis of the actual distribution of the species. three example disease systems are illustrated: west nile virus, the filoviruses (ebola and marburg viruses), and plague: changes are illustrated as the difference between broken (original) and entire (present) outlines of circles; particular geographic occurrences of the disease are labeled to illustrate points discussed in the text appropriate from both abiotic and biotic perspectives and that are accessible to the species in terms of dispersal. although this area cannot simply be assumed to hold populations of the species (consider, e.g., metapopulation dynamics, which may lead to absences in suitable, accessible areas), this area does provide an index to the likely distribution of the species. considerable exploration and testing has demonstrated both that a∩b∩m is an appropriate prediction of the actual distributions of species (lópez-cárdenas et al. ; elith et al. ) and that a∩ b offers highly accurate predictions of species' distributional potential when dispersal constraints are relaxed (peterson ; benedict et al. ). this framework (the "bam" diagram), although simple, offers considerable inferential power regarding distributions of species (soberón and peterson ) . i emphasize that this framework is only heuristic and cannot cover the full diversity and complexity of disease phenomena-a more complete and mathematical treatment has been developed elsewhere (soberón ) . a wide variety of biodiversity phenomena (including diseases) can be placed in this framework and understood with greater clarity-for example, species' invasions are simply the broadening of the m circle to include more of the potential distribution of the species (peterson ) . particular factors (e.g., abiotic conditions) can be visualized as more or less constraining by increasing or decreasing the size and overlap of the circle relative to the other circles. the bam framework encounters some intriguing challenges in applications to disease biogeography. for example, in coarse-scale biodiversity applications, the focus is generally on the role of abiotic conditions in constraining species' distributions (soberón )-in contrast, disease applications can emphasize biotic interactions as dominating the process. the following paragraphs highlight these differences. interactions rule a basic characteristic of a disease is its transmission cycle. generally, these cycles involve several species-one or more reservoirs, vector(s), incidental hosts, and the pathogen itself. in an autecological world, then, a disease transmission system could be seen as a suite of species, each distributed according to its own ecological needs (i.e., its own particular a). in this sense, we could consider each element in a disease transmission system (species , , ... i ... up to n) to have its own particular version of a (which we can denote a i )-disease transmission would then occur only where a ∩a ∩...∩ a i ∩...∩a n . from the perspective of any single species in the system, the combined intersections of the a's for all other species in the system compact into that species' b (peterson ) . put more simply, however, in disease transmission systems, interactions rule. much of the dynamics of these systems will be determined not simply by abiotic considerations but by the interactions among many species. examples are more than common across the world of diseases: malaria transmission occurs only when appropriate mosquito vector species are present (gu et al. ); plague transmission is most efficient when certain flea species are present (krasnov et al. ) ; much more complex examples can be found in any introductory parasitology text. this interactions-dominated landscape appears to stand in sharp contrast with the abiotic-dominated landscape in the biodiversity world (peterson ) . stable and unstable interactions beyond simply being dominant, interactions in the disease world can be stable or unstable (i.e., enzootic or epizootic). specifically, some disease systems show dramatic associations of pathogens and hosts (dragoo et al. ; field et al. )-in these cases, pathogens and hosts coevolve over evolutionary time and may establish stable relationships that may even involve evolution of resistance on the part of the host and/or avirulence on the part of the pathogen (lenski and may ) . these stable relationships can also lead to the evolution of parallel phylogenetic patterns, indicating a long period of shared evolutionary change (charleston and page ) . on the other hand, species interactions in disease transmission systems can also be extraordinarily unstable, particularly when interactions are relatively new. some disease systems have epizootic transmission phases in which hosts are 'burned through' at surprising rates: excellent examples include plague (yersinia pestis) transmission among prairie dogs (cynomys spp.; ubico et al. ; cully et al. ) , rabies (rabies virus, rhabdoviridae) transmission among mammals other than vampire bats (desmodus spp.; wandeler ; davis et al. ) , and ebola (ebolavirus spp., filoviridae) transmission among great apes (leroy et al. ) . in these (and other) cases, the host-pathogen interaction is so unstable that host mortality almost inevitably results, producing either unstable epizootic transmission systems or brief outbreaks that burn out after a few generations of transmission, although some instances of unstable transmission maintenance of diseases are known (e.g., bingham ) . a can be of minor importance another characteristic of disease systems that differs markedly from much of the remainder of biodiversity is the potential for a to be of relatively minor importance. that is, particular for microbial pathogens (viruses, bacteria) that may not have free-living stages, abiotic considerations may place few constraints on the distributional potential of species. some examples are obvious-influenza transmission on the international space station or at an antarctic research base-but more subtle examples should be considered more carefully, such as outbreaks of afrotropical diseases such as ebola virus in virginia and marburg virus (marburgvirus spp., filoviridae) in germany (murphy et al. ). more generally, some pathogens may not have free-living life stages or any other interface with the outside environmentas such, their "ecological niche" requirements may constitute simply that of having a live host. in other words, in many disease transmission systems, a may place only broad constraints on the potential geography of the system, and b and m may determine much more of the spatial dynamics of the system. m can vary dramatically pathogens and parasites are generally of small body size and often are not particularly well equipped for movement. as such, one might expect their dispersal abilities to be quite limited. however, because of the tight associations between pathogens and their much larger hosts, dispersal events of surprising magnitude become possible. that is, because pathogens can ride around with their much bigger and much more mobile hosts, dispersal events can yield surprising results, often termed disease "emergence" events, but really just constituting extreme dispersal events. disease dispersal ability can thus be surprising in its effects. for instance, bat-hosted coronaviruses related to that which causes severe acute respiratory syndrome (sars) had certainly been present in asia for what is effectively the entirety of human history in the region (guan et al. ; li et al. ) . although such viruses may, from time to time, have infected humans and caused disease events, the major recent sars outbreak not only had serious public health consequences in asia but also jumped rather dramatically to north america (mcdonald et al. ) . similarly, monkeypox (monkeypox virus, orthopoxviridae) is well known as a central african disease (arita and henderson ), but a seemingly innocent importation of african rodents for pets in the usa resulted in a small-scale monkeypox outbreak in north america (hutson et al. ). these dramatic variations in dispersal ability must be borne in mind in considering disease biogeography. in this section, i offer three brief case studies intended to illustrate the oddities of disease biogeography, as contrasted with the biogeography of most customary elements of biodiversity. the three studies are designed to illustrate different aspects of distributional biology and offer some intriguing insights into disease biogeography. these case studies also offer an illustration of the basics of tools for modeling species' ecological niches-i have used simple niche-modeling tools to develop some interesting coarsescale results about disease biogeography. in niche modeling, known occurrences of species (or diseases, in some cases) are related to raster geographic information system (gis) coverages summarizing relevant environmental parameters in an evolutionary computing environment; the result is a picture of the species' ecological distribution, which can be projected onto geography to identify a potential distribution for the species (peterson ; soberón ) . west nile virus west nile virus (wnv; west nile virus, flaviviridae) was first described and characterized from a patient in uganda in and came to be known as a cause of mildly serious encephalitis cases across eastern and southern africa (taylor et al. ; nir et al. ; mcintosh et al. ); eventually, its sporadic occurrence, occasionally in the form of major outbreaks, in the southern tier of europe was also documented (hubálek and halouzka ) . in , however, wnv appeared in new york, in the usa, and quickly spread west and south across essentially all of the americas (komar and clark ) . the disease is clearly now "endemic" to (i.e., established in) much of the americas as a permanent component of the pathogen landscape of the western hemisphere. figure , however, paints a picture that contrasts actual and potential distributions of wnv. the area shaded brown in the figure in africa, southern europe, and southwest asia represents the virus' known distribution as of ; the blue-shaded area represents areas globally that fit the same precipitation-temperature profile as the brown-shaded area, showing that the same climate regime was present across a much broader portion of the world. in , wnv managed to jump across the atlantic ocean and become established in new york city-in the ensuing - years, thanks probably to dispersal via migratory birds, the virus has spread west to the pacific ocean and south to argentina (komar and clark ) , fulfilling much of the spatial extent of its potential distribution in the americas. ebola and marburg viruses discovered only in (marburg) and (ebola), the two virus genera that make up the filoviridae family are exclusively african insofar as their known geographic distributions (peters et al. ) . what is more, however, ebola virus is restricted to humid lowland evergreen tropical forests in africa (congo basin and a small area along the liberia-ivory coast border), and marburg virus is restricted to less humid tropical forests in eastern and southern africa. as such, these two related virus genera occupy a predictable ecological niche space across african landscapes (peterson et al. a; peterson et al. ) . as an illustration of the volatility and unpredictability of disease geography given the association of pathogens with larger hosts, it is worthy of note the several times that ebola or marburg viruses have appeared outside the usual suite of conditions for filoviruses (feldmann et al. ) . several filovirus-caused disease outbreaks have appeared under conditions distinct from the norm for these viruses and in areas well outside the usual distributional area of these viruses: ( ) marburg virus in marburg, germany, in ; ( ) marburg virus in johannesburg, south africa, in ; and ( ) ebola (reston species) in virginia, texas, and the philippines at various points in the s (peters et al. ; miranda et al. ) . as can be seen in both geographic and ecological dimensions (fig. ) , these filovirus occurrences are outliers and clearly illustrate how pathogens can be carried around and into odd situations by their larger and more mobile vertebrate hosts. clearly, this example illustrates the point that the "normal," endemic transmission mode may lend itself much better to predictive modeling than the irruptive, epizootic phases. plague finally and the most complex, plague is a zoonotic disease that is held in a small mammal reservoir and vectored by several flea species (gage ) ; the particular species involved depend on the region in question. plague was originally-apparently-restricted to central asia and interior china (pavlovsky ) ; with the advent of largescale movements (i.e., silk route trade, intercontinental shipping), however, a series of pandemics began, in which plague became established in urban rattus populations (gage ) . particularly in the early twentieth century, however, these plague outbreaks could be extinguished by reducing the contact between humans and rattus; however, in a number of cases around the world (e.g., western north america, andean and northeastern south america, eastern and southern africa, madagascar), the plague "jumped" into native rodents and has established itself as an endemic zoonosis (levy and gage ; enscore et al. ) . viewed ecologically, the endemic (i.e., not epizootic) plague appears to occur under a consistent suite of environmental conditions (enscore et al. ) . as can be appreciated in fig. , the environmental conditions across its original range in asia are fairly restricted with respect to the extent to which it spread via global shipping (echenberg ) . then, however, plague retreated somewhat as it moved from epizootic to enzootic, for example, not being maintained on hawaii or in australia (fig. ) . overall, then, in the history of the plague, two shifts have been observed: ( ) broadened distributional potential thanks to improved dispersal abilities and ( ) reduced distributional potential thanks to reduction in urban rat populations. the above framework can be applied to understanding the geography of many biological phenomena, including diseases of various types (soberón and peterson ) . the distribution of disease occurrences can be seen as the joint spatial distribution of suitable ecological conditions for all of the biological species involved in the transmission fig. actual and potential distributions of wnv as of : brown shading indicates an approximate boundary around areas of known wnv occurrence prior to ; blue shading indicates areas matching the annual mean temperature×annual precipitation profile of the brown areas using the bioclim algorithm (nix ) . note that the dispersal event of allowed the colonization of essentially the entire american portion shaded blue in the course of just years cycle: pathogen, reservoir, vector, etc.-in essence, the geographic projection of the ecological distribution of the pathogen as limited by the ecological and geographic potential of each of its interacting species. this interplay among ecological and geographic spaces, among the ecological niche characteristics of various species and as constrained by the spatial configuration of suitable habitats and dispersal abilities of the species involved, provides a framework for understanding disease distributions (peterson ) . the discussions above emphasize the role of humans in increasing the geographic scope of disease transmission, yet many diseases have yet to expand distributions broadly beyond their original distributional areas. for example, chagas disease remains uniquely american, although it may be expanding northward . the filoviruses and monkeypox are endemic only to tropical africa. scrub typhus (orientia tsutsugamushi) and nipah virus (nipah virus, paramyxoviridae) are uniquely asian. these diseases either have yet to disperse successfully to other regions, or perhaps they have dispersed but have failed to find appropriate interacting species (reservoirs, vectors, etc.) upon arrival. the distributions of such endemic diseases will be governed by the joint ecological requirements outlined above. some enigmas remain, however, in understanding disease geography. for example, yellow fever (yellow fever virus, flaviviridae) was originally distributed across humid tropical africa; early shipping (probably via slave ships) transported it to south america, and it spread broadly across the humid tropical portions of that continent. curiously, however, yellow fever has never colonized humid tropical portions of asia, in spite of ample areas presenting appropriate climatic conditions (fig. ) . similarly, particular strains of malaria (e.g., falciparum malaria) have patchy and odd distributional patterns that would seem to challenge purely ecology-based explanations of distributions. this review may be seen as a nontraditional way of presenting disease geography. instead of a broad review of dark gray areas correspond to countries with current yellow fever transmission (who ) ; red areas indicate portions of asia that match yellow fever-endemic regions in terms of annual mean temperature and annual precipitation distributional patterns disease by disease (e.g., ackerknecht ) , i have instead attempted to present a general framework for understanding geographic distributions of species and to point out the ways in which disease transmission systems differ from other biological systems. my hope is that the generalities that emerge will assist in a new, more synthetic view of disease geography. many challenges remain to be addressed in this field. in particular, the way in which interactions among species act (and how these interactions can vary across geography) remains almost completely unexplored. that is, the a∩b framework described above is certainly an oversimplification-interactions among species may easily vary spatially and may even vary as a function of interactions with still other species. these considerations could produce complexity well beyond what has been appreciated in disease geography studies to date and indeed might explain some of the complexities and nonlinearities that confound the understanding of disease geography. another issue is the many diseases that have poorly known or unknown components to their transmission cycles. in such cases, niche modeling tools can only be applied to the overall cycle as a "black box"-human cases would be used as an integration over the entire a ∩a ∩...∩a i ∩...∩a n , even though the species involved are not identified-several such studies have already been developed (e.g., peterson et al. ) . indeed, under some circumstances, niche-modeling tools can be used to identify the particular species participating in the transmission cycle or at least to identify likely candidate species (peterson et al. b . when participating species can be identified clearly, however, the power of these approaches increases considerably. not only can the ecology and geography of the transmission cycle be reassembled in detail in a gis environment, but exceptions to these predictions offer insights into the additional complexities in these systems. that is, where + + does not equal may indicate that some additional factors are acting. such next-step insights will become available as more detailed, range-wide views of the ecology and biogeography of disease transmission systems are developed. history and geography of the most important diseases chagas disease in a domestic transmission cycle in southern texas spread of the tiger: global risk of invasion by the mosquito aedes albopictus canine rabies ecology in southern africa world distribution of plague treemap . β: a macintosh program for the analysis of how dependent phylogenies are related, by cophylogeny mapping dynamics of plague in a gunnison's prairie dog colony complex from new mexico the evolutionary history and dynamics of bat rabies virus phylogeography of the deer mouse (peromyscus maniculatus) provides a predictive framework for research on hantaviruses novel methods improve prediction of species' distributions from occurrence data modeling relationships between climate and the frequency of human plague cases in the southwestern united states ebola virus ecology: a continuing mystery henipaviruses: emerging paramyxoviruses associated with fruit bats source reduction of mosquito larval habitats has unexpected consequences on malaria transmission isolation and 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reservoirs of sars-like coronaviruses fine-scale predictions of distributions of chagas disease vectors in the state of guanajuato conservatism of ecological niche characteristics in north american plant species over the pleistocene-to-recent transition sars in healthcare facilities ecological studies on sindbis and west nile viruses in south africa. v. the response of birds to inoculation of virus epidemiology of ebola (subtype reston) virus in the philippines filoviridae: marburg and ebola viruses isolation of arboviruses from wild birds in israel a biogeographic analysis of australian elapid snakes natural nidality of transmissible diseases filoviruses. in: morse ss (ed) emerging viruses predicting the geography of species' invasions via ecological niche modeling ecological niche modelling and understanding the geography of disease transmission ecological niche conservatism and pleistocene refugia in the thrush-like mourner, schiffornis sp conservatism of ecological niches in evolutionary time ecologic niche modeling and potential reservoirs for chagas disease ecological and geographic distribution of filovirus disease potential mammalian filovirus reservoirs geographic potential for outbreaks of marburg hemorrhagic fever mammal taxa constituting potential coevolved reservoirs of filoviruses on the relationship between niche and distribution ebola (subtype reston) virus among quarantined nonhuman primates recently imported from the philippines to the united states grinnellian and eltonian niches and geographic distributions of species biodiversity informatics: managing and applying primary biodiversity data interpretation of models of fundamental ecological niches and species' distributional areas a study of the ecology of west nile virus in egypt a plague epizootic in the white-tailed prairie dogs (cynomys leucurus consilience testing to determine location of pleistocene refugia: comparing phylogeographic, fossil and ecological niche model predictions wildlife rabies in perspective who report on global surveillance of epidemic-prone infectious diseases-yellow fever. world health organization niche conservatism: integrating evolution, ecology, and conservation biology acknowledgments i thank my many "disease" colleagues for their many kind hours spent educating me about their areas of expertise. i thank in particular d. carroll for his insightful comments on an earlier version of this manuscript. this work was supported in part by a grant from microsoft research. key: cord- -fr tme authors: kearns, shawn title: infectious hepatopathies in dogs and cats date: - - journal: top companion anim med doi: . /j.tcam. . . sha: doc_id: cord_uid: fr tme this article serves to review the various infectious diseases that affect the liver primarily or as a part of systemic infection. although bacterial infections are probably the most common cause of infectious hepatitis, the clinician should be aware of other potential organisms and other commonly involved systems. therefore, this article includes a description of common bacterial, mycobacterial, viral, fungal, protozoal, parasitic, and rickettsial diseases in dogs and cats. alimentary flora circulates to the liver under various clinical conditions. these bacteria are extracted by kupffer cells, killed by neutrophils, or excreted in bile in healthy clinical states. a low-flow, low-pressure perfusion of hepatic sinusoids may allow superior removal of bacteria by phagocytes, and pressure differentials in the biliary system may limit retrograde access of enteric organisms. , , changes in this sinusoidal flow may decrease the effectiveness of phagocytosis when portal flow is compromised. bowel disease, cholestasis, immunosuppression, and altered gut motility result in altered portal circulation, and the subsequently unchecked bacterial access to the liver may result in bacterial hepatitis or cholangiohepatitis. common isolates implicated in bacterial hepatitis and cholecystitis include escherichia coli, enterococcus spp, bacteroides spp, streptococcus spp, and clostridium spp. cultures can be obtained by liver aspirate, liver biopsy, and cholecystocentesis. a combination of liver and gall bladder samples (fig ) may increase the likelihood of identification of the offending organism(s). surgical or laparoscopic biopsies may be more rewarding for culture growth compared with aspirates. in suspected cases, broad-spectrum antibiotics for common enteric isolates should be initiated pending specific culture results. focal micro-and macro-abscesses have also been documented in dogs and cats. , predisposing causes include alterations in blood flow, trauma, ascending biliary infections, liver lobe torsions, immunocompromised clinical states, and neoplasia. , microabscesses are often identified in association with extrahepatic infection and sepsis. , ultrasound has greatly enhanced the early diagnosis of hepatic abscesses. greater than % of solitary abscesses are polymicrobial. antimicrobial treatment should be directed at both anaerobes and aerobes regardless of whether anaerobic cultures are negative if a polymicrobial hepatic infection is documented. bacterial isolates in hepatic abscesses are similar to those identified in diffuse bacterial hepatic disease. however, clinically rare isolates including klebsiella, listeria, salmonella, brucella, yersinia pseudotuberculosis, actinomyces, nocardia, and pasturella have also been documented. focal abscesses may require surgical drainage and antibiotic therapy. treatment in all cases must be implemented for a minimum of to weeks. leptopirosis is an extremely common nonenteric bacterial infection in the canine liver. leptospires are thin, filamentous, spiral-shaped motile bacteria with a lipopolysaccharide outer envelope. direct transmission occurs via contact with infected urine, venereal and placental tissues, or fluids. indirect transmission can occur through contaminated water sources, soil, food, or bedding. the organism can stay stable for several months with the right environmental conditions. the organism initially penetrates the mucous membranes and rapidly multiplies after entry into the vascular space. dissemination and replication occur in many tissues, including the liver. however, the organism tends to persist in the kidney and can be shed for weeks to months after infection. certain serovars are more frequently associated with hepatic involvement and include leptospira icterohaemorrhagiae and l. pomona. young dogs (Ͻ months of age) seem to develop signs of hepatic dysfunction more frequently in disease outbreaks. profound hepatic dysfunction may occur without significant histologic changes because of subcellular damage produced by bacterial toxins. the endothelial damage, subsequent thrombosis, and possible disseminated intravascular coagulation seen in acute disease may contribute to hepatic damage. chronic hepatitis has been reported as a sequelae to leptospiral infection. , diagnosis is usually made based on clinical signs and serologic titers. however, leptospirosis polymerase chain reaction (pcr) performed be-fore treatment may increase testing sensitivity given vaccinal interference and delayed seroconversion in the acute phase. penicillins are the treatment of choice in the acute phase and must be followed by appropriate antibiotics to eliminate the carrier state. alternatively, doxycycline may be used for both the acute and carrier states. bartonella spp are gram-negative fastidious bacteria and are well adapted for the intracellular environment. a recent case report documented b. henselae and b. clarridgeiae dna in the liver of dogs with granulomatous inflammation. both had a positive clinical response to azithromycin and demonstrated biochemical reduction in hepatocellular enzymes. another dog with peliosis hepatitis (a rare vascular condition characterized by multiple, randomly distributed blood-filled cavities throughout the liver) had b. henselae dna amplified from multiple hepatic specimens by pcr. helicobacter canis has been isolated from the liver of a single dog with hepatitis. helicobacter spp have also been amplified from hepatic tissue in cats with cholangiohepatitis. further studies are required to determine whether these organisms are associated with inflammatory liver disease. these organisms are difficult to culture, and this failure may reflect the fastidious nature of these bacteria. pcr positivity may reflect the presence of intestinal helicobacter from the enterohepatic circulation or transient colonization rather than a true disease association. francisella tularensis (tularemia) is a pleomorphic, gramnegative, nonspore-forming bacillus. this disease frequently occurs as a result of exposure to ticks or wildlife. macrophages are the primary host cells, and bacteremia with multiorgan involvement is common. lungs, spleen, liver, and skin are common sites for embolic spread, resulting in microabscesses and granulomatous disease. puppies and young cats appear more susceptible to infection, and dogs are generally more resistant to infection. clinical findings include depression, oral/lingual ulceration, regional or generalized lymphadenomegaly, hepatosplenomegaly, panleukopenia with severe toxic neutrophil changes, hyperbilirubinemia, and bilirubinuria. [ ] [ ] [ ] examination for evidence of microscopic agglutinating antibody is most frequently used for diagnosis, although indirect fluorescent antibody testing may be useful as well. aminoglycosides are the primary treatment in humans. however, tetracyclines (doxycycline), chloramphenicol, and quinolones are commonly used in dogs and cats. unfortunately, clinical relapse is common with these antibiotics. tyzzer's disease (clostridium piliforme) is caused by a flagellated, spore-forming, gram-negative intracellular parasite. although spores have been identified in rodent species, interspecies transmission via ingested feces has not been documented. however, spontaneous disease has been documented in dogs and cats. [ ] [ ] [ ] [ ] colonization of the liver results in multifocal, periportal hepatic necrosis and may result from a currently unidentified toxin. minimal inflammation may be present despite extensive necrosis. death usually occurs within to hours once the organism is in the liver. [ ] [ ] [ ] [ ] [ ] [ ] [ ] rhodococcus is a soil-borne pleomorphic, gram-positive bacteria normally associated with suppurative infections in figure . fine-needle aspirate and cytology from the gallbladder of a cat with cholangiohepatitis. the aspirate consists predominantly of bacteria of mixed type. the bacteria are frequently present in chains (black arrow). also, note dark brown-staining amorphous material (bile pigment: yellow arrow). the finding of bacteria in cytologic specimens of bile is considered abnormal. the following organisms were cultured from the bile: escherichia coli, streptococcus pneumoniae, an anaerobic bacterial rod, prevotella oralis, and a gram-positive rod that could not be classified. courtesy of the pathology department, angell animal medical center, boston, massachusetts. domestic livestock. inhalation from soil or wound inoculation are the suspected routes of transmission. disseminated infection and death have been reported in a single dog. clinical reports are rare in cats. mycobacterium spp are aerobic, nonspore-forming, nonmotile bacteria with a wide host affinity and pathogenic potential. they are typically classified based on growth in culture and by the pathologic production of tubercles or granulomatous disease. mycobacterium tuberculosis and m. bovis are the most pathogenic, and humans are reservoirs for these species. aerosolized organisms in sputum are considered the primary mode of transmission. however, m. bovis can be acquired via uncooked meats and wildlife reservoirs. mycobacterial disease is often subclinical in dogs and cats, but signs may be associated with granuloma formation in various organs. , nontuberculous mycobacterium, including those in the mycobacterium avium complex, are saprophytic opportunistic organisms primarily implicated in disseminated disease in cats [ ] [ ] [ ] [ ] [ ] and occasionally in dogs. - no clear associations have been identified with retroviral diseases. canine and feline breeds with potentially increased susceptibility include basset hounds, miniature schnauzers, siamese, and abyssinians. dogs with m. avium complex-induced disease will often demonstrate extensive granulomatous disease of the intestine, spleen, liver, and mesenteric lymph nodes. animals undergoing immunosuppressive drug therapy with inhibition of cell-mediated immunity may be at risk for disseminated disease, including renal transplant patients. acidfast cytology can demonstrate bacilli, although false negatives can occur. negative bacterial images may be identified on routine stains (fig ) . pcr may provide greater sensitivity and safety than culture. combination therapies are often required, because organisms build resistance quickly, particularly with disseminated disease. although not a risk for immunocompetent individuals, dogs and cats infected with saprophytic mycobacterium pose a risk for immunodeficient people. mycobacterium lepraemurium was considered the main causative agent for feline leprosy until recently. however, m. visibilis has been associated with feline multisystemic granulomatous mycobacteriosis, resulting in diffuse cutaneous disease and widespread dissemination to multiple internal organs. organisms responsible for disseminated fungal infections include histoplasma capsulatum, coccidioides immitis, blastomyces dermatitides, aspergillosis sp, cryptococcussp, and sporothrix schenckii. most are dimorphic, saprophytic, opportunistic fungi that exist in the mycelial stage in the environment. spores are produced in the mycelial stage and be-come pathogenic on inhalation, ingestion, or inoculation. dissemination occurs via the hemolymphatic system. specific environmental conditions are required for the individual organisms, and this dictates their geographic range. histoplasma capsulatum is located primarily in the temperate and subtropical areas of the world. organisms are phagocytized by mononuclear cells and replicate intracellularly once they are inhaled and converted to the yeast phase. the primary clinical signs in dogs are associated with the gastrointestinal system (diarrhea, tenesmus, mucous, fresh bloods in stools). clinical signs in cats are vague. dissemination to other visceral organs (including the liver) has been documented in both species. [ ] [ ] [ ] clinically affected animals are usually young ( - years of age). diagnosis is usually achieved with fine-needle aspirate or exfoliative cytology of affected organs. aspergillosis is primarily associated with rhinitis. however, several reports have documented systemic infections in german shepherds and in non-shepherd breeds. aspergillus terreus - and a. deflectus , have been most frequently implicated in systemic infection. predisposing factors include optimal climatic conditions, access to a partial strain, or subtle defects in mucosal immunity. disseminated aspergillosis has also been documented in cats. , neurologic deficits, spinal column pain, urinary system disorders, and respiratory pathology are the primary presenting clinical signs. prototheca is a saprophytic, achlorophyllous alga found in the southeastern united states. three species of prototheca have been identified, but p. zopfii is the only one associated with disseminated disease. the organism is associated with sewage, slime flux of trees, and animal waste. transmission generally occurs through ingestion or penetration of injured skin or mucosa. disease can develop with diminished host resistance or concurrent diseases. concomitant large intestinal diarrhea and ocular signs should prompt clinical consideration of prototheca infection. dissemination via blood or lymph to other organs including the liver is common. various stages of development of the organism may be identified on cytology or histopathology. urine culture and sediment are also useful in organism identification. this disease is invariably fatal, although disease progression may be delayed with various antifungal and antibacterial agents. - coccidioides immitus is a dimorphic fungus with preference for the alkaline sandy soil environment found in the lower sonoran life zone in the southwestern united states, western mexico, and central and south america. mycelia are produced during rainfall, but arthroconidia develop with soil drying and become airborne under dry and windy conditions. inhalation is the primary mode of infection in dogs and cats. the spherule (tissue parasitic form) undergoes division with eventual rupture. the severity and extent of clinical disease depend on immunocompetence and range from a mild, asymptomatic, pulmonic form to severe, life-threatening disseminated disease. dissemination most commonly involves the axial and appendicular skeleton and overlying skin. tissues from abdominal viscera, the central nervous system (cns), pericardium, myocardium, and prostate can also be involved. , cytology or histology may reveal spherules, although diagnosis is often made based on history, clinical signs, and positive serology. antigens for sero-testing commonly use tube precipitin and complement fixation with agar gel immunodiffusion. sporothrix schenckii causes a chronic granulomatous disease of worldwide distribution. infection is usually the result of trauma and inoculation with infective conidiophores. the skin is the primary target organ. however, disseminated disease has been reported, particularly in the cat. no clear dissemination pattern has been identified because of low case numbers, but affected organs include the internal lymph nodes, liver, lungs, eyes, bone, muscles, and cns. , diagnosis is frequently made by cytology. blastomyces dermatitidis is found primarily in mississippi, missouri, the ohio river valley, the mid-atlantic states, and some canadian provinces. growth of the organism requires sandy, acidic soil with some proximity to water. preferred sites for dissemination include the skin, eyes, bones, and lymph nodes, although dissemination to the liver has been reported. , cryptococcus neoformans has a worldwide distribution. inhalation may be the primary mode of infection, and sites of infection tend to be areas of the body with cooler temperatures, including the respiratory passages and subcutaneous tissues. the fungus is occasionally disseminated to the kidneys and rarely to the liver. treatment of most disseminated fungal infections involves the use of triazoles, including itraconazole and fluconazole, as well as amphoterocin b. , - clinical signs may resolve in many cases, but relapses occur and patients with severe clinical illness generally have a poor prognosis. leishmania, transmitted by the sandfly (lutzomyia in the new world, phlebotymus in the old world), frequently causes cutaneous and visceral lesions in the dog. promastigotes transmitted by the female sandflies become amastigotes in the vertebrate and are phagocytized by mononuclear cells. the organism travels through hemolymph organs to remote dermal sites and other organs. clinical signs will not develop in all exposed animals, and the immune response at the time of infection appears important in determining development of disease. leishmania infection should be considered in dogs from endemic areas with marked hyperglobulinemia or in those with a travel history to endemic areas. mild increases in liver enzymes are often noted. however, unlike the kidneys, the liver is not a primary target organ. infection can be associated with chronic hepatitis. definitive diagnosis is made by demonstration of organisms on cytology or histopathology, or by serology, culture, or pcr. amphotericin b in a soybean oil lipid emulsion has been intravenously administered for higher clinical cure success rates and greater numbers of negative posttreatment parasitologic tests compared with other treatments. other less successful treatment options include allopurinol and the pentavalent antimonials. hepatozoon canis is a worldwide protozoal disease reported in domestic dogs and is most prevalent in subtropical and temperate climates. the primary vector is the rhipicephalus sanguineous tick, which is primarily located in warm and temperate regions. transmission occurs through ingestion of ticks containing mature protozoal oocyts. sporozoites are released in the intestine on tick ingestion and penetrate the gut wall, invade mononuclear cells, and disseminate. target organs include the bone marrow, spleen, and lymph nodes but can involve other internal organs such as the liver, kidney, and lungs. , the most striking clinicopathologic abnormality is leukocytosis with evidence of parasitemia of the white cells on peripheral blood smears. clinical findings can range from incidental hematologic findings to severe life-threatening illness. hepatitis, glomerulonephritis, and pneumonitis have all resulted from h. canis infection. coinfections with other protozoal diseases (toxoplasma, leishmania, and babesia spp) or other tick-borne diseases (ehrlichia spp) and immunosuppressive states can predispose animals to clinical illness. the hepatitis is associated with developing meronts within the liver and their associated neutrophilic and mononuclear inflammation. hepatozoon has also been documented in felines. [ ] [ ] [ ] microscopic detection of gamonts in peripheral blood smears is the most frequently used diagnostic test. imidocarb is the treatment of choice in dogs. subcutaneous or intramuscular injections are administered every days until gamonts are no longer visualized in the leukocytes. a new species, hepatozoon americanum, was identified in , with the amblyomma maculatum tick as its definitive host. this emerging disease has spread to the north and the east since its initial identification in the gulf coast region. clinical signs are often severe, even in the absence of other diseases or in the presence of immunosuppression. waxing and waning clinical signs are attributed to repeated cycles of asexual reproduction and pyogranulomatous inflammation. the primary site of infection for the merozoites is the cardiac and skeletal muscle. however, single zoites can enter circulation and reproduce asexually at distant locations. diagnosis is most often made with muscle biopsy, although a recent study has identified promise in the use of pcr testing. an enzyme-linked immunosorbent assay has been developed with sporozoites as the antigen. no treatment effectively eliminates the tissue stages of h. americanum. however, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and an excellent quality of life. the microsporidial parasite encephalitozoon cuniculi is an obligate intracellular protozoan. infection likely occurs by inhalation or ingestion of spores from contaminated urine or feces shed by infected hosts. the organism undergoes asexual reproduction or binary fission after infecting host cells and ruptures, leading to infection of new cells or shedding of resistant spores into the environment. typical organs of localized infection include the kidney, liver, lungs, and brain with resultant granulomatous inflammation. , cats and older dogs are not commonly affected, and renal disease predominates in young dogs. cytological examination of fluids (particularly urine) is important in making a diagnosis in animals with disseminated disease as other tests are commercially unavailable. cytauxzoon felis is a tick-borne protozoal disease of domestic and wild cats. the bobcat is the natural reservoir in north america and is usually asymptomatic despite persistent erythroparasitemia. the tissue phase of infection consists of the development of large schizonts in mononuclear phagocytes. the schizonts line the lumens of vessels in most organs, eventually leading to vessel occlusion. merozoites are released into blood or tissue fluid once the host cells rupture and infect red blood cells. late-stage parasitemia can often be detected on blood films at about to days before death. most clinical signs, including those associated with liver abnormalities, are due to schizont-associated mechanical obstruction. however, parasite by-products may also be toxic, pyogenic, and vasoactive. the anemia is regenerative but mild in comparison with clinical icterus. this may be useful in differentiating this infection from hemotropic mycoplasmas. demonstration of piroplasms in wright's-stained or giemsa-stained blood films most frequently provides a definitive diagnosis. histopathology reveals schizont-laden mononuclear phagocytes in the veins of the lungs, liver, and spleen. the prognosis is generally considered poor, but different geographic strains may have varying virulence. treatment with diminazene or imidocarb has been somewhat successful. toxoplasma gondii is an obligate intracellular coccidian parasite that infects almost all warm-blooded animals. do-mestic cats are the definitive hosts and excrete the infective oocyts. three stages of the life cycle are considered infectious, including oocyst sporozoites, tissue cyst tachyzoites, and tissue cyst bradyzoites. transmission can occur through ingestion of oocysts or tissue cysts and via congenital transmission. other reported modes include lactation, transfusions, and transplantation. a higher frequency of disease is reported in dogs and cats fed raw meat or those in a rural/ feral environment. the extra-intestinal life cycle is the same in all hosts, and sporozoites encyst in the intestinal lumen, penetrate cells, and divide into tachyzoites. the tachyzoites can form cysts in the cns, muscle, and visceral organs, and may persist for the life of the host. clinical signs were diverse in cats with histologically confirmed toxoplasmosis, and more than % had pulmonary, cns, and liver manifestations. , in dogs, disseminated infection is most often associated with canine distemper, other infections including ehrlichiosis and immunosuppression, or vaccination with live attenuated vaccines. clinical cases in cats have been seen with steroids, cyclosporine use, hemotropic mycoplasms, and viral disease. [ ] [ ] [ ] liver and lung involvement is associated with quicker mortality than other organ involvement. tachyzoites may be detected on cytology of various organs and body fluids. however, diagnosis is most frequently based on clinical signs, serology (immunoglobulin g, immunoglobulin m), and response to treatment. clindamycin is the treatment of choice. neospora caninum is a protozoan similar to toxoplasma. dogs and coyotes are considered definitive hosts, and deer and cattle are intermediate hosts. the predominant mode of transmission is transplacental in the dog, and clinical signs are usually secondary to exacerbation of a congenital infection. acute phases of infection include widespread dissemination to many organs, including the liver, whereas chronically infected animals are restricted to muscular and neuronal sites. serology and muscle biopsy often provide a diagnosis, although tachyzoites may be detected in other parasitized tissue or body fluid. sarcocystis canis is an apicomplexan protozoan with no particular geographic distribution. infection results in disseminated disease, including protozoal hepatitis. , many reports involve puppies, suggesting the presence of congenital infection. however, the life cycle is still unknown. sarcocystis canis is the only sarcocystis species known to form schizonts in canine tissue. infectious canine hepatitis (ich) is caused by adenovirus type . this is the only virus with primary tropism for the liver. infection leads to severe hepatic necrosis and can also cause ocular and renal changes. the virus localizes in the tonsils after oronasal exposure, spreads to regional lymph nodes, and disseminates via the thoracic duct. hepatic parenchymal cells and vascular endothelial cells are the prime targets of viral localization, and injury leading to centrilobular to panlobular hepatic necrosis ranges from self-limiting to fatal. most affected dogs are less than year of age and unvaccinated. severely affected dogs can become moribund and die within hours of disease onset and with few predictive clinical signs. if patients survive the acute phase, they may develop clinical signs including vomiting, diarrhea, and abdominal pain. , those that survive may go on to develop chronic hepatitis and fibrosis, likely secondary to self-perpetuating liver inflammation rather than chronic infection. diagnosis is frequently made based on clinical signs and serology, although the virus can be isolated in cell cultures. this disease is rarely encountered because of the high efficacy of vaccination. canine acidophil hepatitis is believed to be caused by a viral agent. however, the specific agent is not yet identified. disease has been reproduced via injections of sterile liver homongenates from spontaneously affected animals. acute infections can lead to acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. diagnosis is made on histology because acidophils are scattered throughout lesions. this disease has only been reported in great britain. , canine herpesvirus causes tissue necrosis and localized mucosal or generalized systemic infections in young or immunocompromised animals. the virus only infects dogs because of specific cell-surface receptors. replication occurs via viral dna synthesis within the host nucleus. transmission occurs through direct contact with mucosal secretions from the respiratory or genital tract of animals. factors predisposing to infection in puppies include hypothermia and a poorly developed immune system. newborns can acquire disease in utero, during passage through the birth canal, during contact with infected littermates, from oronasal secretions of the dam, and from fomites. puppies less than week of age are more susceptible to generalized fatal infections. dissemination leads to hemorrhagic necrosis in several organs including the adrenal glands, kidney, liver, lungs, and spleen. clinical signs include loss of interest in nursing, loss of body weight, soft yellow-green feces, abdominal discomfort, and dullness. a marked increase in alanine aminotransferase is often noted on biochemistry profile. definitive diagnosis is by viral isolation. feline leukemia virus is a single-stranded retrovirus that replicates in many tissues. clinical illness is generally related to the hematopoietic system and the immune system. feline leukemia virus has also been associated with icterus and various inflammatory and degenerative liver diseases including focal liver necrosis. feline infectious peritonitis (fip) is a feline coronavirus that has undergone frequent rna mutations, resulting in an ability to enter and replicate in macrophages. an immunemediated vasculitis occurs if the virus is not eliminated. affected cats develop signs related to target organ lesions (kidney, liver, cns, intestine) or due to fluid redistribution. abnormal liver enzymes can occur because of hepatitis, hepatic lipidosis, or prehepatic sequalae of vasculitis, erythrocyte destruction, and hypoxia. hyperbilirubinemia is common and usually secondary to vasculitis in the liver. histopathology is required for definitive diagnosis but is sup-ported by history, physical examination, and laboratory findings. a new pcr test may also prove useful in the diagnosis of fip. treatment is generally unrewarding. conflicting information exists on the usefulness of feline recombinant interferon, although it may be beneficial for a subpopulation of fip-infected cats. , rickettsial diseases the most common agents encountered in dogs with clinical evidence of liver involvement include the ehrlichia sp, rickettsia rickettsii, and borrelia burgdorferi. these organisms can infect either hepatocytes or endothelial cells. hepatic involvement in erhlichia infections occurs in more than % of human patients, leading to mild transient increases in transaminases. liver injury may be related to organism proliferation in hepatocytes and stimulation of immunologic and nonspecific inflammatory mechanisms. rocky mountain spotted fever is vasculotropic in nature and can cause moderate increases in transaminases. experimental evidence with borrelia suggests direct hepatic invasion by the spirochetes in conjunction with cellular and humoral immunologic mechanisms. an association with borrelia was observed and confirmed with liver biopsy in dogs. lesions were consistent with lobular dissecting hepatitis and mixed multifocal inflammation leading to focal pyogranulomas in the other. chronic cholangitis associated with liver fluke infestation in endemic areas is primarily observed in cats and less frequently in dogs. most infections are due to opistorchus and metorchis, which require intermediate hosts. the first hosts are water snails, and the second hosts include a wide variety of fish with encysted metacercariae. the final host acquires infection by ingestion of fish, and the young liver flukes migrate to the liver through the bile ducts. this results in bile duct thickening and dilation. rarely, cysts may be formed as well. a slight to moderate inflammation may be seen both within the ducts and in the portal areas. although eosinophils may be present, they are usually limited in numbers. the number of liver flukes and eggs within the dilated bile ducts varies markedly, and often only limited evidence of liver flukes or eggs is identified. platynosomum concinnum is a trematode of the feline biliary system. terrestrial snails, lizards, toads, and terrestrial isopods act as intermediate hosts based on geographic location. disease is most prevalent in the tropical and subtropical climates. clinical cases involve adult indoor or indoor-outdoor cats. the severity of clinical signs is proportional to the number of adult flukes as well as to the duration of parasitemia. early diagnosis can be difficult. however, diagnosis is easier when eggs have been identified in the bile. treatment of p. concinnum and liver fluke infections is best accomplished with praziquantel. there are many infectious diseases that ultimately affect the liver. few, however, have primary tropism for hepatic tissue. testing should be directed based on signalment, geographic locale, and primary presenting complaint. cytology and/or histopathology of the liver will most frequently provide a definitive diagnosis in clinical situations with liver involvement. the prognosis is guarded with many disseminated infections. toxic, metabolic, infectious, and neoplastic liver diseases infectious diseases of the dog and cat detection of portal and systemic bacteremia in dogs with severe induced hepatic disease and multiple portosystemic shunts diseases of the gallbladder and biliary tree cholangitis/cholangiohepatitis complex in the cat bacterial culture results from liver, gallbladder, or bile in dogs and cats evaluated for hepatobiliary disease: - hepatic abscesses in cats: cases hepatic abscesses in dogs: a review of the ultrasonographic findings, clinical data and therapeutic options liver lobe torsion and liver abscess in a dog hepatic abscesses associated with diabetes mellitus in two dogs hepatocellular carcinoma with secondary abscessation in a cat hepatic abscesses in dogs hepatic abscesses in dogs: cases ( - ) infectious diseases of the dog and cat chronic active hepatitis in dogs associated with leptospires chronic hepatitis associated with leptospiral infection in vaccinated beagles clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease peliosis hepatis in a dog infected with bartonella henselae helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis association of helicobacter with cholangiohepatitis in cats feline tularemia on tularemia in a cat acute tularemia in three domestic cats bacillus piliformis infection in an adult dog tyzzer's disease in kittens with familial primary hyperlipoproteinaemia tyzzer's disease in a puppy tyzzer's disease in puppies infectious diseases of the dog and cat the liver in systemic disease. an innocent bystander naturally occurring tyzzer's disease as a complication of distemper and mycotic pneumonia in a dog tyzzer's disease in a dog tyzzer's disease in an adult cat naturally occurring tyzzer's disease in a cat naturally occurring tyzzer's disease in a puppy tyzzer's disease complicated with distemper in a puppy tyzzer's disease in puppies vapa-negative rhodococcus equi in a dog with necrotizing pyogranulomatous hepatitis, osteomyelitis, and myositis mycobacterium tuberculosis complex infection in a dog putative transmission of mycobacterium tuberculosis infection from a human to a dog disseminated mycobacterium avium infection in a cat disseminated mycobacterium avium infection in young cats: overrepresentation of abyssinian cats disseminated mycobacterium avium complex infection following renal transplantation in a cat tuberculosis in cats disseminated mycobacterium avium complex infection in three siamese cats fatal mycobacteriosis with hepatosplenomegaly in a young dog due to mycobacterium avium a case of disseminated tuberculosis in a dog caused by mycobacterium avium-intracellulare systemic mycobacterium smegmatis infection in a dog disseminated mycobacterium avium infection in a dog disseminated mycobacterium avium complex infection in a dog tuberculosis in five basset hounds systemic mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat disseminated mycobacterium avium infection in three miniature schnauzer litter mates histologic and genotypic characterization of a novel mycobacterium species found in three cats atypical histoplasma capsulatum infection in a dog disseminated histoplasmosis in dogs: cases ( - ) disseminated histoplasmosis in cats: cases ( - ) disseminated aspergillosis in two dogs in israel long-term survival of four dogs with disseminated aspergillus terreus infection treated with itraconazole disseminated aspergillus terreus infection in a dog disseminated aspergillosis in a dog with diskospondylitis and neurologic deficits disseminated aspergillosis in a dog systemic mycosis due to aspergillus deflectus in a dog disseminated aspergillosis attributable to aspergillus deflectus in a springer spaniel canine disseminated aspergillosis systemic aspergillosis and mucormycosis in cats feline disseminated aspergillosis altered immune function in a dog with disseminated protothecosis urinary tract manifestations of protothecosis in dogs more than meets the eye: subretinal aspirate from an acutely blind dog disseminated protothecosis causing acute blindness and deafness in a dog disseminated protothecosis in a dog infectious diseases of the dog and cat deep mycotic infections in cats disseminated coccidioidomycosis in a dog pathology of sporotrichosis in cats in rio de janeiro disseminated sporotrichosis in a cat ocular changes in a cat with disseminated blastomycosis legendre am: blastomycosis, in greene ce fatal disseminated cryptococcosis and concurrent ehrlichiosis in a dog disseminated opportunistic fungal disease in dogs: cases infectious diseases of the dog and cat cryptococcosis coccidioidomycosis and paracoccidioidomycosis infectious diseases of the dog and cat chronic hepatitis associated with canine leishmaniosis (leishmania infantum): a clinicopathological study of cases initial and long term efficacy of a lipid emulsion of amphotericin b desoxycholate in the management of canine leishmaniasis hepatozoon canis infection in two dogs canine hepatozoonosis in oklahoma hepatozoon canis infection feline hepatozoonosis granulomatous cholangiohepatitis in a cat due to a protozoan parasite resembling hepatozoon canis hepatozoon species infection in domestic cats: a retrospective study a new hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in north america characterization of stages of hepatozoon americanum and of parasitized canine host cells diagnosis of canine hepatozoon spp. infection by quantitative pcr an indirect enzymelinked immunosorbent assay for diagnosis of american canine hepatozoonosis treatment of dogs infected with hepatozoon americanum: cases ( - ) mammalian microsporidiosis experimental encephalitozoonosis in neonatal dogs cats surviving natural infection with cytauxzoon felis: cases ( - ) administration of diminazene aceturate or imidocarb dipropionate for treatment of cytauxzoonosis in cats toxoplasmosis and neosporosis infectious diseases of the dog and cat acute primary toxoplasmic hepatitis in an adult cat shedding toxoplasma gondii oocysts fatal toxoplasmosis in five cats acute toxoplasmosis following renal transplantation in three cats and a dog feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis neonatal toxoplasmosis in littermate cats histologically confirmed clinical toxoplasmosis in cats: cases ( - ) neospora caninum associated with septic peritonitis in an adult dog fatal cutaneous and visceral infection in a rottweiler dog associated with a sarcocystis-like protozoan fatal hepatic sarcocystosis in a puppy with eosinophilia and eosinophilic peritoneal effusion canine viral diseases viral hepatitis of dogs (rubarth's disease) infectious canine hepatitis (hepatitis contagiosa canis use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis a new transmissible agent causing acute hepatitis, chronic hepatitis, and cirrhosis in dogs persistent hepatitis and chronic fibrosis induced by canine acidophil cell hepatitis virus canine herpesvirus frequency and significance of feline leukemia virus infection in necropsied cats feline infectious peritonitis and feline enteric coronavirus feline infectious peritonitis: typical findings and a new pcr test use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis effect of feline interferonomega on the survival time and quality of life of cats with feline infectious peritonitis ehrlichial diseases of humans: emerging tickborne infections host-pathogen interactions in the immunopathogenesis of lyme disease severe cholestatic liver disease secondary to liver fluke key: cord- -tuq z gm authors: weiss, robin a; mcmichael, anthony j title: social and environmental risk factors in the emergence of infectious diseases date: journal: nat med doi: . /nm sha: doc_id: cord_uid: tuq z gm fifty years ago, the age-old scourge of infectious disease was receding in the developed world in response to improved public health measures, while the advent of antibiotics, better vaccines, insecticides and improved surveillance held the promise of eradicating residual problems. by the late twentieth century, however, an increase in the emergence and re-emergence of infectious diseases was evident in many parts of the world. this upturn looms as the fourth major transition in human–microbe relationships since the advent of agriculture around , years ago. about new diseases have been identified, including legionnaires' disease, human immunodeficiency virus (hiv)/acquired immune deficiency syndrome (aids), hepatitis c, bovine spongiform encephalopathy (bse)/variant creutzfeldt-jakob disease (vcjd), nipah virus, several viral hemorrhagic fevers and, most recently, severe acute respiratory syndrome (sars) and avian influenza. the emergence of these diseases, and resurgence of old ones like tuberculosis and cholera, reflects various changes in human ecology: rural-to-urban migration resulting in high-density peri-urban slums; increasing long-distance mobility and trade; the social disruption of war and conflict; changes in personal behavior; and, increasingly, human-induced global changes, including widespread forest clearance and climate change. political ignorance, denial and obduracy (as with hiv/aids) further compound the risks. the use and misuse of medical technology also pose risks, such as drug-resistant microbes and contaminated equipment or biological medicines. a better understanding of the evolving social dynamics of emerging infectious diseases ought to help us to anticipate and hopefully ameliorate current and future risks. popular writing on emerging infectious diseases resounds with dire warnings about the threat of modern 'plagues' and losing the 'war against microbes.' this adversarial language obscures the fact that most of the microbial world is either neutral toward, or supportive of, human well-being and survival. indeed, we would not survive long without commensal microbes such as the beneficial strains of escherichia coli in our gut. that aside, the study of emerging infections is more than a passing fad. the recent rate of identification of such infections, the impact of the sars outbreak, the devastation caused by aids, and the ever-present threat of a new influenza pandemic indicate that we cannot control our disease destiny. nor are emerging infections unique to humans; the irish potato famine in and the english foot-and-mouth disease epidemic in underscore the consequences for human societies of disease emergence in crops and livestock. emerging infectious diseases in humans comprise the following: first, established diseases undergoing increased incidence or geographic spread, for example, tuberculosis and dengue fever; second, newly discovered infections causing known diseases, for example, hepatitis c and helicobacter pylori; and third, newly emerged diseases, for example, hiv/aids and sars. this perspective will discuss the human ecology of both the (apparently) new and re-emerging diseases. interest in infectious disease has itself recently re-emerged. in , burnet and white commented, "the most likely forecast about the future of infectious disease is that it will be very dull. there may be some wholly unexpected emergence of a new and dangerous infectious disease, but nothing of the sort has marked the past fifty years" . today, we may criticize the short-sightedness of our mentors' generation, yet in demographic terms they were essentially correct because the proportion of deaths from infectious disease has fallen throughout the twentieth century , (fig. ) . humankind currently faces neither apocalyptic extinction nor even a population reduction such as occurred in europe during the black death of the fourteenth century. rather, overpopulation in relation to environmental resources remains a more pressing problem in many developing countries, where poor economic and social conditions go hand-in-hand with infectious disease. in industrializing countries during the nineteenth century, a major reduction in enteric infections was achieved by separating drinking water from sewage-an environmental change that probably saved more lives than all the twentieth century vaccines and antibiotics together. today, however, the growth of shanty towns without sanitation around the megalopolis cities of asia, africa and south america is recreating similar conditions, and in the past years cholera has made a remarkable re-emergence through its longest ever (seventh) pandemic . in most countries, life expectancy has risen over the past years (fig. ) . the most important exception is those regions where hiv infection is rife. moreover, during the past years, falling living standards in some african countries and the breakdown of public health infrastructure in ex-soviet nations has aided the re-emergence of transmissible diseases like tuberculosis , . further, severe outbreaks such as the - influenza a pandemic temporarily reversed the decline of deaths caused by infectious disease. the million estimated deaths from that pandemic represented about % of the global population at that time, and is twice as many as the cumulative aids mortality of the past years. the next influenza pandemic may be just around the corner, and may spread even faster , if access to appropriate vaccines and drug treatment is not available , . for other newly emerging infections that make headlines, such as sars, ebola or vcjd, it is important to keep a sense of demographic proportion. placing these emerging infections on a 'richter' scale of human mortality (box ) shows that they elicit scarcely detectable minor tremors in numbers of fatalities -despite the fear they invoke. we do not know, however, which one might leap to the top of the scale like hiv has done; indeed, it may be a completely unknown agent, as the sars coronavirus was two years ago. a major challenge is to predict which infection presages the next big quake, hopefully allowing preventive action. like any other animal or plant species, humans have been prone to infection by pathogens throughout their evolutionary history. such ancient infections by helminth and protozoan parasites, bacteria, fungi and viruses are endemic, eliciting a range of effects from a heavy burden of disease (e.g., malaria) to being essentially commensal in immunocompetent hosts (e.g., most types of herpesvirus and papilloma virus). other infections depend on an animal reservoir for their maintenance; their infection of humans may be pathogenic, but it has little part in the evolving ecology of the microbe or parasite. an estimated % of the , species of infectious organisms known to be pathogenic in humans are transmitted by animals , for which the human represents a dead-end host. occasionally, however, a zoonotic infection adapts to human-to-human transmission and diversifies away from its animal origin. epidemic diseases are generally caused by infections that are directly transmissible between humans. hiv is a recent example of a long line of human infections initiated by a switch of host species, stretching back to the origins of measles and smallpox. free-living microbes may also find a human niche that suits their lifestyle, such as the lung for legionella pneumophila and the gut for vibrio cholerae. legionnaires' disease, first recognized in philadelphia in , is the environmental equivalent of a zoonosis. it is seldom passed directly from person to person but it was human ingenuity in designing warm, aerated, humid 'artificial lungs' called air-conditioning systems that allowed the microbe to proliferate and become an opportunistic colonizer of the human lung. cholera, which was unknown beyond the ganges delta before it spread widely in asia and the middle east during the period - , at around that time horizontally acquired a toxin gene and other factors in a genetic package that helped it to colonize the gut; the resultant diarrhea aids dispersal of the microbe , . human society has undergone a series of major transitions that has affected our pattern of infectious disease acquisition and dissemination . these transitions illustrate the interrelationship between environmental, social and behavioral influences on the emergence and subsequent spread of infectious disease. some infections were acquired when our australopithecine ancestors left their arboreal habitat to live in the savannah. this ecological change included exposure to new species of mosquito and tick as vectors for infection. after the emergence of homo sapiens, the eventual migration of neolithic hunter-gatherers out of africa , to , years ago exposed them to new infections in distant regions. the first major transition of prehistoric/early historic times gave rise to the epidemic, or 'crowd,' infections. this change must have started in the millennia following the advent of agriculture-from around , years ago-as agriculturally based society developed larger, denser populations. the domestication of livestock and the rich dividends available in human settlements to other animals (e.g., rodents, dogs and various insects) provided further opportunities for pathogens to move between species. sometimes such a pathogen (or a mutant strain thereof) would have been well suited to humans as a new host species, and, if human numbers were adequate, could therefore persist indefinitely as a human infection. thus, measles emerged about , years ago, probably from rindepest of cattle, and diverged to become an exclusively human infection when population size and density became sufficient to maintain the virus without an animal reservoir. similarly, smallpox became epidemic about , years ago, possibly evolving from camelpox, its closest phylogenetic relative. the next two transitions were primarily to do with great extensions in the spread of infectious diseases, entering distant populations as 'new infections.' thus, the second historical transition occurred in classical times as large eurasian civilizations came into commercial and military contact. they inadvertently exchanged their pools of infections, and vectors such as rats and fleas, across the mediterranean basin, the middle east, india and china. the plague of athens in bce during the peloponnesian war vividly described by thucidides may represent the first report of typhus. this rickettsial infection is transmitted from rats to humans and thence among louse-ridden humans. typhus frequently accompanies human conflict and deprivation, as seen in a recent outbreak among rwandan refugees in burundi . the justinian plague of ce devastated the eastern mediterranean region and probably extended as far as china like the black death years later (and both are attributable to yersinia pestis ). the third historical transition accompanied the era of worldwide exploration and colonization by europeans from circa ce onward. a contemporary account by one of hernan cortes' fellow conquistadors, bernal diaz, recalls that they might well have failed to overthrow the mighty aztec empire had they not been aided by a raging epidemic. this was possibly a combination of smallpox and measles, both wholly unknown to the new world population. curiously, the columbian exchange was unidirectional regarding infectious diseases; the one contentious possible exception being syphilis. the new world is believed to have had substantially fewer human zoonotic infections , , and vector-borne infections like chagas' disease did not travel in the absence of an appropriate vector. two centuries later, captain cook unwittingly repeated the decimation of indigenous peoples through syphilis, measles and tuberculosis in many of the pacific islands, whereas lord jeffrey amherst deliberately attempted to spread smallpox among 'hostile' native americans, one of the better documented cases of germ warfare . the transmission dynamics of infections in naive populations is markedly different from those in which the majority of adults are immune . onboard the beagle, charles darwin observed with his customary acuity, "wherever the values are approximate global death rates for the year , taken from the world health organization (who) and other sources. hbv and hcv, hepatitis b and c viruses; rsv, respiratory syncytial virus; hpv, human papilloma viruses; vcjd, variant creutzfeldt-jakob disease. two major, novel causes of mortality top the list: cigarette smoking and hiv infection; they emerged in the twentieth century and continue to increase in many developing countries. among the chronic and re-emerging infections, malaria and tuberculosis are near the top, so it becomes apparent why there is a need for the global fund for malaria, tuberculosis and aids. accidental injuries, particularly road deaths, continue to rise, with % occurring in developing countries . although was the year of the sars outbreak , , less than , people actually died as a result of sars coronavirus infection despite the collateral damage to daily life, psychological wellbeing and economic activity in the affected cities. this richter scale represents a snapshot in time. twenty years ago, hiv was three logs further down the scale, whereas polio was three logs higher. fifty years ago, malaria was finally eradicated from europe, where it had formerly been widespread, including in england (shakespeare's 'ague'). bacterial respiratory diseases used to have a more important role in human mortality and, despite concern over multi-resistance to antibiotics , , the situation is considerably better than in the era before the advent of antibiotics. common bacterial infections of childhood, such as diphtheria and whooping cough, have become rarities in the developed world, largely through vaccination. viral diseases have similarly been reduced. thanks to effective immunization policies of the who, smallpox was eradicated in ; polio and measles viruses, which have no animal reservoir, may soon be eliminated in the same way. the european has trod, death seems to pursue the aboriginal …most of the diseases have been introduced by ships and what renders this fact remarkable is that there might be no appearance of the disease among the crew which conveyed this destructive importation." today we are living through the fourth historical transition of globalization. urbanization, dense and usually impoverished peri-urban settlements, social upheaval, air travel, long-distance trade, technological developments, land clearance and climate change all influence the risks of infectious disease emergence and spread. although some of the apparent increase in infectious disease may be attributable to better diagnostic methods and surveillance, there seems little doubt that more incidents are occurring, and have the potential to spread more widely than years ago, as outbreaks and spread of infections like nipah virus and sars would not have passed unnoticed. as humans encroach further into previously uncultivated environments, new contacts between wild fauna and humans and their livestock increases the risk of cross-species infection . this process will only diminish as wild species become rarer and eventually endangered, like the great apes today. an example of such contact followed the establishment of piggeries close to the tropical forest in northern malaysia, where, in , the nipah virus first crossed over from fruit bats (flying foxes, pteropus spp.) to pigs and thence to pig farmers . destruction of natural forest has also encouraged fruit bats to relocate nearer human habitation, like the large colony in the botanic gardens in the heart of sydney. indeed, in , hendra, a related paramyxovirus of australian fruit bats , fatally infected a veterinarian examining a sick horse. rodents continue to be sources of re-emerging infections, as witnessed in the s with hantaviruses in the united states. rodentborne hantavirus is prevalent in agricultural systems in south america and east asia, in arid grasslands in north america and elsewhere. in mid- , an unexpected outbreak of acute, sometimes fatal, respiratory disease occurred in humans in the southwestern united states . this 'hantavirus pulmonary syndrome' was caused by a previously unrecognized virus, maintained primarily within the native deermouse, and transmitted through excreta. the - el niño event, with unseasonal heavy summer rains and a proliferation of piñon nuts, hugely amplified local rodent populations which led to the outbreak , . in south america, there have been several outbreaks of hantavirus and arenavirus infections linked to forest clearance and the growth of rodent populations in the new grasslands . habitat destruction is not the only cause of increased human infection, however. dengue virus is extending its range and prevalence because its mosquito vector breeds rapidly in the urban environment . in the united states, nature conservation and increased woodland in the eastern states has led to the emergence of lyme disease. this disease is caused by a tick-borne spirochete and the presence of tick-infested deer near suburban homes leads to ticks residing on bushes adjacent to baseball diamonds and gardens. intensification of production of meat and meat products has led to new infections . most notorious is vcjd in the uk arising from consumption of contaminated food products of cattle affected by bse . bse, or 'mad cow disease,' emerged in british cattle in because of industrialized cannibalism, whereby rendered neural tissue and bone meal from slaughtered cattle were recycled into cattle feed, as well as into pies and hamburgers for human consumption . originally, infectious prions from scrapie in sheep were the suspected source, but it now seems more likely that it arose from a bovine with sporadic prion disease. the extent of the human epidemic remains unclear. although natural transmission is unsustainable (r < in both cattle and humans), there are concerns that vcjd might be transmissible through blood transfusions . without effective diagnostic tests for presymptomatic vcjd infection, this situation is extremely unfortunate. other recently emergent food-borne infections include e. coli o :h , which is harmless to cattle but toxic to humans, and salmonella enteriditis in chickens. better hygiene in abattoirs, butchers and domestic kitchens can greatly reduce the incidence of infection. in theory, closed and intensive farming of a single species should reduce the risk of cross-species infection (fig. ) . but it also allows large-scale epidemics to emerge, as seen recently for avian influenza strains in southeast asia and the netherlands , . ancient dietary taboos, such as those of hindus, muslims and jews regarding pork as unclean, doubtless had their roots in protection from infectious disease. today, an increasing demand for consumption of exotic and wild animals raises new risks of infectious diseases such as sars (box ). changing patterns of human behavior and ecology affect two distinct steps in the emergence of new infectious disease. the first is an increased opportunity for animal-to-human infection to occur owing to greater exposure, which may be necessary but not sufficient to lead to the emergence of a new human infection. the second step is the opportunity for onward transmission once a person has become infected. for each novel epidemic, such as the - influenza pandemic or aids, there are probably thousands of failed transfers. some infections simply do not take in the new host. innate hostspecific restrictions on viral replication have recently become evident for primate lentiviruses , which may explain why certain species that harbor simian immunodeficiency virus, but not others more commonly in contact with humans, gave rise to hiv- and hiv- . even in the case of hiv- , only one pedigree of three independent chimpanzee-tohuman crossover events has given rise to the aids pandemic, whereas the other two smolder as poorly transmissible infections. fatal pathogenesis is not necessarily coupled with infectiousness , which is evident for h n avian influenza in humans . but genetic reassortment between avian and human influenza viruses could easily give rise to a new, rapidly spreading strain . a poorly infectious pathogen may not spread at all from the index case, as is usual with rabies, or may only infect close contacts and soon peter out, as seen with lassa fever and ebola virus. sars nearly became self-sustaining but was brought under control. some of the most insidious infections are those with long, silent incubation periods during which the person is infectious. these emerge surreptitiously so that when the new disease is eventually recognized, as aids was in , the infection has already spread far beyond control. like the ships of centuries past, the speed of modern air travel works wonders for the dispersal of infectious diseases. sars was eventually constrained by quarantine and strict adherence to infection control guidelines in hospitals, but not before it quickly traveled from guangzhong to hong kong and on to toronto. if ebola broke out in a city with a busy international airport, it might also travel across continents in a similar manner. brockmann has modeled how rapidly such infections can move once they reach a major airport hub; closing the hubs becomes an immediate imperative. we cannot be sure what the initial vector was for the arrival of west nile virus into north america in : a migratory bird blown off course, an infected human with a valid air ticket or a stowaway mosquito on a similar flight. whatever the means of entry and early colonization of crows in new york, it has taken less than four years to reach the pacific coast . thus, west nile virus has found a new reservoir in american birds, just as yellow fever virus reached new world primates years earlier. microbes frequently capitalize on situations of ecological, biological and social disturbance. biologically weakened and vulnerable populations-especially if also socially disordered and living in circumstances of privation, unhygienic conditions and close contact-are susceptible to microbial colonization. the severity of the bubonic plague (black death) in mid-fourteenth-century europe seems to have reflected the nutritional and impoverishment consequences of several preceding decades of unusually cold and wet weather with crop failures compounding the incipient destabilization of the hierarchical feudal system. many of the rapid and marked changes in human social ecology in recent decades have altered the probabilities of infectious disease emergence and transmission. these changes include increases in population size and density, urbanization, persistent poverty (especially in the expanding peri-urban slums), the increased number and movement of political, economic and environmental refugees, conflict and warfare. political ignorance, denial and obduracy often compound the risk of infectious disease transmission-as has been tragically observed with hiv/aids in parts of africa, where widespread poverty, a culture of female disempowerment and political instability further "if there is any conceivable way a germ can travel from one species to another, some microbe will find it," wrote william mcneill in his classic text plagues and peoples . for millennia, small farmsteads accommodated mixed species living closely with humans-goats, pigs, cattle, ducks, geese, chickens and perhaps a water buffalo or a donkey-and exchanged infections. when species are raised separately but are sold together, the opportunity for cross-infection moves from the farm to the marketplace. the outbreak of avian influenza in hong kong occurred in mixed markets, where live chickens, quail and ducks were stacked together in close quarters with humans. the h n virus that emerged may have been derived by recombination between those of different avian hosts . after , mixed species were separated into different areas of the markets. but this year's h n virus is spreading among intensively reared chickens across southeast asia. the increasing predilection for meat of exotic species has exacerbated the risk of exposure to infections not previously encountered, and this situation probably triggered the sars epidemic . although we are still not sure of the natural reservoir species of sars coronavirus, the live markets and restaurants in guangzhong sold small carnivores, and several species of civet cat, racoon dog and ferret badger captured in china, laos, vietnam and thailand, were brought into close proximity . clearly, some of the palm civet cats were infected with sars-related viruses, but it is less clear whether they represent the original source species. there is a danger in incriminating the wrong species; if the true reservoir resides in the rodent prey of these carnivores, then culling the predators may be counterproductive. stopping the exotic meat trade altogether would seem to be a simple solution to prevent the reappearance of sars, but once the taste for it has been established, that may prove no more practical than attempting to prohibit the tobacco trade. in africa, bushmeat also poses a serious problem for emerging infectious diseases, as well as for nature conservation. sick animals may be more easily captured. for example, human deaths owing to ebola virus infection ensued from the butchering of a single chimpanzee . hiv has crossed from chimpanzees to humans on at least three occasions, and a higher number of zoonotic events from sooty mangabeys are indicated for hiv- (ref. ). whether these cross-species infections arose from butchering the animals or from keeping them as pets is unknown, but a recent survey of primate hunters in africa showed that they are susceptible, like handlers of primates in captivity, to infection (though not disease) from foamy retroviruses . the escalating intercontinental trade in exotic pets can lead to unexpected infectious disease outbreaks. the united states has only recently imposed more stringent regulations and quarantine following cases of monkeypox in humans and in prairie dogs introduced by rodents imported from africa as pets . exacerbate the problem , . but we have little understanding of why the prevalence of hiv infection varies so greatly between cities in sub-saharan africa . the urban environment has only recently become the dominant human habitat. urbanism typically leads to a breakdown in traditional family and social structures, and entails greater personal mobility and extended and changeable social networks. these features, along with access to modern contraception, have facilitated a diversity of sexual contacts and, hence, the spread of sexually transmitted diseases . this risk is further amplified by the growth in sex tourism in today's internationally mobile world, which capitalizes on the desperation and ignorance of poverty, combined with exploitative behaviors, in developing countries. more generally, cities often function as highways for 'microbial traffic' . rapid urbanization boosts certain well established infectious diseases, such as childhood pneumonia, diarrhea, tuberculosis and dengue, and facilitates dissemination of various 'emerging' diseases-as occurred for sars in the high-rise housing of hong kong. crowded and dilapidated public housing can potentiate infectious disease transmission through drug abuse and sexually transmitted infections . technological advances in medicine and public health can also inadvertently promote the emergence and spread of infectious disease. it has become commonplace to quip that you go to the hospital at the peril of acquiring an intractable nosocomial infection such as methicillin-resistant staphylococcus aureus , and such infections killed around times as many people as sars did in (box ). multidrug-resistant tuberculosis has also become a major problem, and, paradoxically, regions with health programs that reduced wildtype tuberculosis strains can develop into 'hot zones' for multidrugresistant tuberculosis . by far the most effective medical vector of infectious disease has been the syringe and needle. drucker et al. have charted the massive increase in the use of injecting equipment over the past years. individuals with hemophilia treated with pooled clotting factors became almost universally infected with hepatitis b and c viruses before diagnostic screening tests were developed. over % of such affected individuals also became infected with hiv , and more recently, transmission of west nile virus by blood transfusion and by organ transplantation has been reported , . the use of contaminated needles among intravenous drug users has had similar consequences. infectious diseases have also been amplified by the use of nonsterile medical injections in developing countries . egypt has the highest prevalence of hepatitis c infection in the world because of the use and reuse of syringes and needles in an earlier public health campaign to reduce bilharzia by medication given by injection. the transmission of cjd through contaminated surgical instruments is another example of iatrogenic spread of infection . biological medicines produced from animal-cell substrates present an inherent potential hazard for introducing new infections. great care must be taken to ensure that live attenuated vaccines grown in animal cells or eggs are devoid of pathogens ; for example, several early batches of live and inactivated polio vaccine unwittingly contained live sv virus, a polyoma virus of macaques. after sv was discovered in , polio vaccine production shifted to virus propagation in primary kidney cells of african green monkeys. these cultures were free of sv but possibly contained sivagm, a relative of hiv that fortunately does not infect humans . the irony of the sv story is that the united states food and drug administration prohibited the use of well known, permanent cell lines demonstrably free of adventitious infectious agents, for fear that such immortalized cells might exert oncogenic properties on the vaccine. there is no epidemiological evidence of increased tumor incidence in those populations who are known to have received sv -contaminated polio vaccine. but there have been a number of recent claims of an association of sv dna sequences in a variety of human malignancies , although these findings remain controversial . the ultimate medical means of introducing animal viruses into humans is xenotransplantation. the implantation of animal cells or tissues into immunosuppressed individuals seems to be a perfectly designed way to encourage cross-species infection. it is astonishing that trials were started without much thought about the consequences for potentially emerging pathogens, for example, porcine retroviruses . the generation of genetically modified knockout or transgenic animals to prevent hyperacute rejection of donor tissues may exacerbate the infection hazard , . happily, there is no evidence so far of retrovirus infection in individuals who were exposed to living pig cells , and clinical xenotransplantation is now stringently regulated; so it seems all the more extraordinary that cellular therapies with fetal lamb cells and extracts continue to be practiced with impunity in alternative medicine clinics in europe and the far east. novel infectious diseases can emerge in any part of the world at any time. hiv and ebola came out of africa, avian influenza and sars from china, nipah virus from malaysia, bse/vcjd from the uk and hantavirus pulmonary syndrome from the americas. it is difficult to predict what new disease will come next or where it will appear, but changing ecological conditions and novel human-animal contacts will be useful clues as to which horizons require scanning with most scrutiny. we must expect the unexpected. as a codicil, another factor that needs to be taken into account is the potential impact of the hiv pandemic on the emergence of other infectious diseases . we already know that persons with aids act as 'superspreaders' of tuberculosis, and we can only speculate what course the sars outbreak might have taken had someone incubating the disease flown to durban rather than toronto . people with aids may persistently harbor infections that would otherwise be transient, and this could hamper the eradication of measles and polio. multivalent pneumococcus vaccines are ineffective in hiv-infected people with cd + lymphocyte levels below /µl, whereas live 'attenuated' vaccines such as vaccinia can cause virulent disease in the immunocompromised host. immunodeficient persons living at high density could also be the seed-bed for microorganisms that are initially ill adapted to human infection to evolve into transmissible human pathogens. thus, an infection from a zoonotic or environmental source-for example, the mycobacterium avium intracellulare complex-could conceivably emerge as the tuberculosis of the twenty-first century, although direct transmission between individuals with aids of such opportunistic infections have not been documented so far. we shall give girolamo frascatoro the last word on emerging and re-emerging infectious diseases by quoting from his treatise de contagione, published almost years ago, "there will come yet other new and unusual ailments in the course of time. and this disease [syphilis] will pass way, but it later will be born again and be seen by our descendents." we are grateful to m. e. chamberland, h. w. jaffe and s. leff for critically reading the manuscript. natural history of infectious disease human frontiers, environments and disease. past patterns, uncertain futures the challenge of emerging and re-emerging infectious diseases environmental and social influences on emerging infectious diseases: past, present and future mortality trends and setbacks: global convergence or divergence? betrayal of trust: the collapse of global public health (hyperion updating the accounts: global 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travels in hivland what have we learnt from sars? mission now possible for aids fund research on preventing road traffic injuries in developing countries is needed severe acute respiratory syndrome antimicrobial resistance worldwide: causes, challenges and responses animal origins of sars coronavirus: possible links with the international trade in small carnivores zoonoses and haemorrhagic fever. in safety of biological products prepared from mammalian cell culture naturally acquired simian retrovirus infections in central african hunters human monkeypox: an emerging zoonosis the authors declare that they have no competing financial interests. key: cord- - abupl authors: vokó, zoltán; pitter, jános györgy title: the effect of social distance measures on covid- epidemics in europe: an interrupted time series analysis date: - - journal: geroscience doi: . /s - - - sha: doc_id: cord_uid: abupl following the introduction of unprecedented “stay-at-home” national policies, the covid- pandemic recently started declining in europe. our research aims were to characterize the changepoint in the flow of the covid- epidemic in each european country and to evaluate the association of the level of social distancing with the observed decline in the national epidemics. interrupted time series analyses were conducted in european countries. social distance index was calculated based on google community mobility reports. changepoints were estimated by threshold regression, national findings were analyzed by poisson regression, and the effect of social distancing in mixed effects poisson regression model. our findings identified the most probable changepoints in european countries. before changepoint, incidence of new covid- cases grew by % per day on average. from the changepoint, this growth rate was reduced to . %, . % increase, and to . % and . % decrease by increasing social distancing quartiles. the beneficial effect of higher social distance quartiles (i.e., turning the increase into decline) was statistically significant for the fourth quartile. notably, many countries in lower quartiles also achieved a flat epidemic curve. in these countries, other plausible covid- containment measures could contribute to controlling the first wave of the disease. the association of social distance quartiles with viral spread could also be hindered by local bottlenecks in infection control. our results allow for moderate optimism related to the gradual lifting of social distance measures in the general population, and call for specific attention to the protection of focal micro-societies enriching high-risk elderly subjects, including nursing homes and chronic care facilities. after a million confirmed and , fatal european cases, the covid- pandemic started declining in europe in april (european centre for disease prevention and control ). this much awaited decline was headed by the introduction of unprecedented "stay-at-home" national policies in most countries, including border closure, public gathering bans, school and workplace closure, and temporary restrictions on free internal movements of the citizens (european centre for disease prevention and control ). these economically and socially disruptive control measures are not sustainable on the longer term (petersen et al. ) , and gradual restart of economy and social life is now on the political agenda throughout europe (european centre for disease prevention and control ; european commission ). a european roadmap to lifting the coronavirus containment measures has been framed, proposing a gradual, slow approach backed with adequate monitoring and healthcare capacity to ensure sufficient control of potential flareups (european commission ). planning the consecutive steps is supported by general provisions and considerations of the roadmap; however, ultimately, it remains a trial-and-error-based process due to the high uncertainty in possible consequences of any change in containment measures. importantly, all national epidemic containment measures were introduced within a narrow time period in most countries (european centre for disease prevention and control ; hale et al. a) . hence, the contribution of unique interventions to the overall impact on covid- spread is hard to estimate retrospectively (imai et al. ; imperial college covid- response team report ). as an overall measure of policy response intensity, the blavatnik school of government proposed a composite stringency index, integrating rigor and scope of multiple containment and closure policies (school and workplace closure, restrictions on gathering, international and internal movements, public transport, cancelation of public events, and information campaigns) into a single numeric parameter in the - range (hale et al. a) . the same team organizes global data collection on all included indicators, as well as on economic responses and adaptations of public health systems, providing a freely available but very precious tool to overview and visualize global policy efforts. however, this stringency index has important limitations when tested as an explanatory factor of sars-cov- infection spread. first, the adopted categories of policy rigor and scope may aggregate heterogeneous policies (as illustrated by an amendment on april -see the details at (hale et al. b) ). more importantly, the stringency index is based on sterile policy decisions, while the compliance of the population with the corresponding restrictions may vary across countries and over time. the google covid- community mobility reports provide an alternative option to derive a composite stringency measure of multiple containment and closure policies (google ) . these community mobility reports provide daily, country-level (and subregional) aggregated anonymized data on time spent at different categories of places such as retail and recreation, groceries and pharmacies, parks, transit stations, workplaces, and residential areas, compared with a baseline period before the epidemic. telemonitored mobility trends are dynamic in time and reflect real-world changes in social behavior, making them promising explanatory factors in sars-cov- infection spread control analyses. recently published covid- microsimulation models based on social network data in the uk and usa revealed that epidemic suppression would require a complex intervention package including social distancing of the entire population, home isolation of cases, and household quarantine of their family members, supplemented with school closure, in intermittent periods adjusted to epidemic intensity and unoccupied critical care capacity (imperial college covid- response team report ; kucharski et al. ) . however, adaptation of these microsimulation models to other countries would require rich and solid input data on local social networks. a semi-mechanistic bayesian hierarchical model of social distancing interventions across european countries was also reported, calculating daily infections from observed death rates (imperial college covid- response team report ). the authors inferred that the combined application of five intervention types (lockdown, public events ban, school closure, self-isolation, and social distancing) could prevent about , covid- deaths in the investigated countries until the end of march . nonetheless, individual contributions of the five intervention types to the overall effect showed high uncertainty, probably because many interventions occurred on the same day or within days of each other. important limitations of this study were the assumptions on identical effect of interventions across countries and over time, and the possible over-representation of countries with more advanced epidemics (imperial college covid- response team report ). additional reassurance whether the covid- transmission was truly slowing has been warranted. to estimate the effect of social distancing on the time trend data of the epidemic, interrupted time series analysis is an alternative approach (aminikhanghahi and diane ) . this analysis can be extended to a broader range of european countries without need for sophisticated local input data collection and assumptions on between-country similarities; hence, it can broaden our current understanding of the epidemic and its association with changes in population social distance patterns. our research aims were to identify the date when the covid- pandemic started declining in each european country and to evaluate the association of the level of community mobility restrictions (social distancing) with the observed extent of decline in the national epidemics. establishing an association of telemonitored population mobility patterns with a decline in covid- spread may support policymakers in assessing the benefits of previously implemented stay-at-home policies, and in planning the gradual lifting of current restrictions. daily incidence of new covid- cases by countries was obtained from the open-access database of the european centre for disease prevention and control (european centre for disease prevention and control data ). data from european union member states and the european free trade association countries were included to focus our analyses on countries with similar sociocultural characteristics and reliable estimates of changes in daily covid- incidence. data from cyprus, iceland, and liechtenstein were dropped, due to the lack or scarcity of related google community mobility reports. data from latvia have also been dropped, because it covered only days in the observation period (see below). accordingly, the analyses included data from countries: austria, belgium, bulgaria, croatia, czech republic, denmark, estonia, finland, france, germany, greece, hungary, ireland, italy, lithuania, luxembourg, malta, netherlands, norway, poland, portugal, romania, slovakia, slovenia, spain, sweden, switzerland, and the uk. within the study period of february to april, the first day of observation was defined in each country as the last day when the number of the new cases was at least following days with less than new cases. due to missing data for some calendar days, the start of observation period was postponed to march in finland and to march in luxembourg (table ) . the most likely changepoint date was determined in each country by linear threshold regression models of the logarithm of daily cases over time, replacing zero daily cases in these analyses by one, and looking for threshold in the - percentile range of the country time series, using the threshold application of the statistical package stata . (statacorp ). the threshold regression using the logarithm of cases in linear regression with the usual gaussian, homoscedastic and independent errors is a correct method to identify the threshold, but when the extent of change is estimated the count nature of the data needs to be taken into account as the non-gaussian errors might give incorrect standard errors of the regression coefficients. therefore the extent of change at the most probable changepoint in the reported daily incidence was estimated by country via poisson regression models using poisson application of stata . . independent variables in the models were time from start of observation and time from the estimated changepoint. as the observations by country were not independent, huber/white/sandwich variance estimator was used to estimate confidence intervals. results of changepoint identification and poisson regression are summarized in table and fig. . the google covid- community mobility reports provide daily, country-level (and sub-regional) aggregated anonymized data on time spent at six different categories of places, compared with a baseline period before the epidemic and controlled for the weekday effect (google ) . in the investigated countries, largest reported decline in staying in retail and recreation, grocery and pharmacy, parks, transit stations, and workplace areas were − %, − %, − %, − %, and − %, respectively, while highest reported change in staying in residential areas was + %. these dimensions of community mobility were integrated into a social distance index. first, data on staying in parks was omitted, since its implications on social distance were considered ambiguous: staying in parks may reflect either individual or social activity. as a next step, daily change from baseline in each mobility report dimension was normalized between baseline and international maximum (see above). finally, the normalized values were averaged, yielding a country-specific daily social distance index with a baseline of and a theoretical maximum of . country-specific social distance index data over time are shown in fig. . for multivariate regression analysis, the average social distance index was estimated for a -day incubation period ending at the changepoint for each country, separately, and countries were grouped by four quartiles of this parameter ( table ) . effect of social distancing on the spread of the epidemic daily new cases were modeled via mixed effects poisson regression with gamma random effect (sutradhar and jowaheer ) in the xtpoisson application of stata . , using countries as random effect. fixed effects in the model were time from the start of observation period, time from changepoint, and the interaction between the latter and the quartiles of the average social distance index in days ending at changepoint, reflecting an incubation period of - days before diagnosis of new cases (european centre for disease prevention and control ). all analyses were conducted in stata . (statacorp ), and double-checked in r (r core team r ) using packages chngpt (fong et al. ) and hglm (rönnegård et al. ; alam et al. ) . most likely changepoints and the estimated extent of change are summarized in table and depicted in fig. . the identified changepoints were associated with statistically significant alteration in daily covid- incidence in of the investigated countries, and all significant findings exhibited a decline in epidemic spread. findings of the multinational regression analysis are summarized in table . translating the model coefficients into incidence rate ratios shows that before changepoint, incidence of new covid- cases grew by % per day (irr . ) on average. from the changepoint, this growth rate reduced to . %, . % increase, and to . % and . % decrease by increasing sdi quartiles. the beneficial effect of higher social distance quartiles (i.e., turning the increase into decline) was statistically significant for the fourth quartile. our analysis identified the most probable changepoint in the flow of the covid- epidemic in european countries and found a clear dose-response association of the observed flattening of the epidemic curve with increasing social distance index derived from google community mobility reports. countries in the highest sdi quartile achieved a statistically significant decline of the epidemic, with less and less new cases every day, while countries with the least stringent sdi increase also greatly reduced the initially high growth rate of incident covid- cases. accordingly, it can be inferred that the unprecedented "stay-at-home" national policies meaningfully contributed to the suppression of the covid- pandemic in europe. countries which achieved on average only % of the maximum observed level of the decrease in social contacts showed already a large reduction in the spread of the epidemic. on the other hand, restrictions on internal movements of the citizens are obviously not the only contributors to this decline: contact tracing and isolation, widescale use of individual protective equipment, keeping safe interpersonal distance in public places, and proper hand hygiene are all plausible contributors to stopping the first wave of the pandemic in europe (european commission ; imperial college covid- response team report ). notably, the importance of local micro-epidemic chains in the overall covid- epidemic is better and better recognized. nursing homes are known to be predisposed to having high transmission rates for infectious diseases for many reasons including crowding, sharing bathroom facilities, social contacts, and low preparedness for infection control. unfortunately, covid- does not seem to be an exception in this respect (davidson and szanton ; trabucchi and de leo ) . according to a who report on april, up to half of those who have died from covid- in the european region were resident in long-term care facilities (world health organization statement ). to sdi social distance index, sd standard deviation prevent covid- transmission in nursing homes and other chronic care facilities enriching high-risk elderly patient groups, effective local infection control measures are clearly more relevant than general interventions targeting the country population as a whole, without specific focus on critical hot spots of the epidemic. such a discrepancy between global and local containment measures may also explain the relatively small difference in the slowing of the epidemic by different level of social distancing. therefore, in parallel with the gradual lifting of country-level covid- spread control measures, special attention must be paid to ensure adequate local infection control in nursing homes and chronic inpatient care facilities, in compliance with the european roadmap to lifting coronavirus containment measures (european commission ) and the corresponding recommendations of the centers for disease control and prevention (centers for disease control and prevention key strategies ). the unprecedented "stay-at-home" national policies meaningfully contributed to the suppression of the covid- pandemic in europe, which can be detected in macro level time trend analysis. however, the importance of several other interventions introduced in parallel must be noted as well, and our findings could be shaped also by the important distinction between country-level and institution-level preparedness. our findings allow for moderate optimism related to the gradual lifting of social distance measures in the general population, and call for specific attention to the protection of focal micro-societies enriching high-risk elderly subjects, including nursing homes and chronic care facilities. code availability (software application or custom code) threshold, poisson, and xtpoisson applications of the statistical software of stata . were used for the analysis as described in the methods. author contributions jgp initiated the project and prepared the data for the analysis. zv developed the analysis plan and performed the analysis in stata . . jgp checked the analysis by repeating it in r . . . the first version of the manuscript was drafted by jgp. both authors contributed to the interpretation of the results and to the writing of the successive versions of the manuscript. funding information open access funding provided by semmelweis university (se).data availabilitypublicly available data was used for the analysis (see in the acknowledgments). conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. fitting conditional and simultaneous autoregressive spatial models in hglm a survey of methods for time series change point detection centers for disease control and prevention key strategies to prepare for covid- in long-term care facilities (ltcfs). nursing homes and covid- : we can and should do better rapid risk assessment: coronavirus disease (covid- ) in the eu/eea and the uk-ninth update european centre for disease prevention and control data on the geographic distribution of covid- cases worldwide a european roadmap to lifting coronavirus containment measures chngpt: threshold regression model estimation and inference google covid- community mobility reports covid- government response tracker. blavatnik school of government oxford covid- government response tracker. what's changed? adoption and impact of nonpharmaceutical interventions for covid- estimating the number of infections and the impact of nonpharmaceutical interventions on covid- in european countries impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand effectiveness of isolation, testing, contact tracing and physical distancing on reducing transmission of sars-cov- in different settings covid- -we urgently need to start developing an exit strategy a language and environment for statistical computing. vienna: r foundation for statistical computing hglm: a package for fitting hierarchical generalized linear models statacorp. stata statistical software: release . college station: statacorp llc on familial longitudinal poisson mixed models with gamma random effects nursing homes or besieged castles: covid- in northern italy world health organization statement -invest in the overlooked and unsung: build sustainable people-centred long-term care in the wake of covid- key: cord- -uy r lt authors: greenspan, hayit; san josé estépar, raúl; j. niessen, wiro; siegel, eliot; nielsen, mads title: position paper on covid- imaging and ai: from the clinical needs and technological challenges to initial ai solutions at the lab and national level towards a new era for ai in healthcare date: - - journal: med image anal doi: . /j.media. . sha: doc_id: cord_uid: uy r lt in this position paper, we provide a collection of views on the role of ai in the covid- pandemic, from clinical requirements to the design of ai-based systems, to the translation of the developed tools to the clinic. we highlight key factors in designing system solutions - per specific task; as well as design issues in managing the disease at the national level. we focus on three specific use-cases for which ai systems can be built: early disease detection, management in a hospital setting, and building patient-specific predictive models that require the combination of imaging with additional clinical data. infrastructure considerations and population modeling in two european countries will be described. this pandemic has made the practical and scientific challenges of making ai solutions very explicit. a discussion concludes this paper, with a list of challenges facing the community in the ai road ahead. the covid- pandemic surprised the world with its rapid spread and has had a major impact on the lives of billions of people. imaging is playing a role in the fight against the disease, in some countries as a key tool, from screening and diagnosis through the entire treatment process, but in other countries, as a relatively minor support tool. guidelines and diagnostic protocols are still being defined and updated in countries around the world. where enabled, computed tomography (ct) of the thorax has been shown to provide an important adjunctive role in diagnosing and tracking progress of covid- in comparison to other methods such as monitoring of temperature/respiratory symptoms and the current-gold standard, molecular testing, us- * corresponding author: hayit@eng.tau.ac.il;hayitg@gmail.com ing sputum or nasopharyngeal swabs. several countries (including china, netherlands, russia and more) have elected to use ct as a primary imaging modality, from the initial diagnosis through the entire treatment process. other countries, such as the us and denmark as well as developing countries (southeast asia, africa) are using mostly conventional radiographic (x-ray) imaging of the chest (cxr). in addition to establishing the role of imaging, this is the first time ai, or more specifically, deep learning approaches have the opportunity to join in as tools on the frontlines of fighting an emerging pandemic. these algorithms can be used in support of emergency teams, real-time decision support, and more. in this position paper , a group of researchers provide their views on the role of ai, from clinical requirements to the design of ai-based systems, to the infrastructure necessary to facilitate national-level population modeling. many studies have emerged in the last several months from the medical imaging community with many research groups as well as companies introducing deep learning based solutions to tackle the various tasks: mostly in detection of the disease (vs normal), and more recently also for staging disease severity. for a review of emerging works in this space we refer the reader to a recent review article shi et al. ( a) that covers the first papers published, up to and including march -in the entire pipeline of medical imaging and analysis techniques involved with covid- , including image acquisition, segmentation, diagnosis, and follow-up. we also want to point out several special issues in this space-including ieee special issue of tmi, april ; ieee special issue of jhbi, ; as well as the current special issue of media. in the current position paper, it is not our goal to provide an overview of the publications in the field, rather we present our own experiences in the space and a joint overview of challenges ahead. we start with the radiologist perspective. what are the clinical needs for which ai may provide some benefits? we follow that with an introduction to ai based solutions -the challenges and roadmap for developing ai-based systems, in general and for the covid- pandemic. in section of this paper we focus on three specific use-cases for which ai systems can be built: detection, patient management, and predictive models in which the imaging is combined with additional clinical features. system examples will be briefly introduced. in section we present a different perspective of ai in its role in the upstream and downstream management of the pandemic. specific infrastructure considerations and population modeling in two european countries will be described in section . a discussion concludes this paper, with a list of challenges facing the community in our road ahead. as of this writing, according to the johns hopkins resource center (https://coronavirus.jhu.edu/), there are, approximately, . million confirmed cases with , deaths throughout the world, with , deaths in new york state alone. the rate of increase in cases has continued to rise as demonstrated by the log scale plot in figure . the most common symptoms of the disease, fever, fatigue, dry cough, runny nose, diarrhea and shortness of breath are non-specific and are common to many people with a variety of conditions. the mean incubation period is approximately days and the virus is probably most often transmitted by asymptomatic patients. knowing who is positive for the disease has critical implications for keeping patients away from others. unfortunately, the gold standard lab test, real time reverse transcription polymerase chain reaction (rt-pcr) which detects viral nucleic acid, has not been universally available in many areas and its sensitivity varies considerably depending on how early patients are tested in the course of their disease. recent studies have suggested that rt-rpr has a sensitivity of only - %. consequently, repeat testing is often required to ensure a patient is actually free of the disease. fang et al (fang et al. ( ) ) found that for the patients they studied with thoracic ct and rt-pcr assay performed within days of each other, the sensitivity of ct for covid- was % compared to rt-pcr sensitivity of % (p < . ). on cxr and ct exams of the thorax, findings are usually bilateral ( %) early in the progression of disease and even more likely bilateral ( %) in later stages , zhao et al. ( ) ). the typical presentation in icu patients is bilateral subsegmental areas of air-space consolidation. in non-icu patients, classic findings are transient subsegmental consolidation early and then bilateral ground glass opacities that are typically peripheral in the lungs. pneumothorax (collapsed lung) and pleural fluid or cavitation (due to necrosis) are usually not seen. distinctive patterns of covid- such as crazy paving in which ground glass opacity is combined with superimposed interlobular and intralobular septal thickening and the reverse halo sign where a ground glass region of the lung is surrounded by an irregular thick wall have been previously described in other diseases but are atypical of most pneumonias. the use of thoracic ct for both diagnosis of disease and tracking has varied tremendously from country to country. while countries such as china and iran utilize it for its very high sensitivity to disease in the diagnosis and tracking of progression of disease, the prevailing recommendation in the us and other countries is to only use lab studies for diagnosis, use chest radiography to assess severity of disease, and to hold off on performing thoracic ct except for patients with relatively severe and complicated manifestations of disease (simpson et al. ( ) ). this is due to concerns in the us about exposure of radiology staff and other patients to covid- patients and the thought that ct has limited incremental value over portable chest radiographs which can be performed outside the imaging department. additionally, during a surge period, the presump-tion is made that the vast majority of patients with pulmonary symptoms have the disease, rendering ct as a relatively low value addition to the clinical work-up. as a diagnostic tool, ct offers the potential to differentiate patients with covid- not only from normal patients, but from those with other causes of shortness of breath and cough such as tb or other bacterial or alternatively, other viral pneumonias, bronchitis, heart failure, and pulmonary embolism. as a quantitative tool, it offers the ability to determine what percentage of the lung is involved with the disease and to break this down into areas of ground glass density, consolidation, collapse, etc. this can be evaluated on serial studies which may be predictive of a patient's clinical course and may help to determine optimal clinical treatment. complications of covid- are not limited to acute lung parenchymal disease. these patients have coagulopathies and are at increased risk for pulmonary embolism. diffuse vascular inflammation can result in pericarditis and pericardial effusions. renal and brain manifestations have been described by many authors and are increasingly recognized clinically in covid- patients. long term lung manifestations will not be apparent for many months or years, but there is the potential that these patients will develop higher rates of chronic obstructive pulmonary disease (copd) such as emphysema, chronic bronchitis and asthma than the general population. objective metrics for assessment and follow-up of these complications of the disease would be very valuable from a clinical perspective. the extraordinarily rapid spread of the covid- pandemic has demonstrated that a new disease entity with a subset of relatively unique characteristics can pose a major new clinical challenge that requires new diagnostic tools in imaging. the typical developmental cycle and large number of studies required to develop ai algorithms for various disease entities is much too long to respond effectively to produce these software tools on demand. this is complicated by the fact that the disease can have different manifestations (perhaps due to different strains) in different regions of the world. this suggests the strong need to develop software more rapidly, perhaps using transfer learning from existing algorithms, to train on a relatively limited number of cases, and to train on multiple datasets in various locations that may not be able to be easily combined due to privacy and security issues. it also suggests that we determine how to balance regulatory requirements for adequate testing of the safety and efficacy of these algorithms against the need to have them available in a timely manner to impact clinical care. ai technology, in particular deep learning image analysis tools, can potentially be developed to support radiologists in the triage, quantification, and trend analysis of the data. ai solutions have the potential to analyze multiple cases in parallel to detect whether chest ct or chest radiography reveals any abnormalities in the lung. if the software suggests a significantly increased likelihood of disease, the case can be flagged for further review by a radiologist or clinician for possible treatment/quarantine. such systems, or variations thereof, once verified and tested can become key contributors in the detection and control of patients with the virus. another major use of ai is in predictive analytics: foreseeing events for timely intervention. predictive ai can be potentially applied at three scales: the individual scale, the hospital scale, and the societal scale. an individual may go through various transitions from healthy to potentially contaminated, symptomatic, etc. as depicted in figure . at the individual level, we may use ai for computing risk of contamination based on location, risk of severe covid- based on co-morbidities and health records, risk of acute respiratory distress syndrome (ards) and risk of mortality to help guide testing, intervention, hospitalization and treatment. quantitative ct or chest radiographic imaging may play an important role in risk modeling for the individual, and especially in the risk of ards. at the hospital level, ai for imaging may for example be used for workflow improvement by (semi-) automating radiologist's interpretations, and by forecasting the future need for icu and ventilator capacity. at the societal level ai may be used in forecasting hospital capacity needs and may be an important measure to aid in assessing the need for lock downs and reopenings. so far, we have here concentrated on disease diagnosis and management, but imaging with ai may also have a role to play in relation to late effects like neurological, cardiovascular, and respiratory damage. before entering the discussion on specific usages of ai to ease the burden of the pandemic, we briefly describe the standard procedure of creating an ai solution in order to clarify the nomenclature. the standard way of developing deep learning algorithms and systems entails several phases (greenspan et al. ( ) , litjens et al. ( ) ) : i. data-collection, in which a large amount of data samples need to be collected from predefined categories; expert annotations are needed for groundtruthing the data; ii. training phase in which the collected data are used to train network models. each category needs to be represented well enough so that the training can generalize to new cases that will be seen by the network in the testing phase. in this learning phase, the large number of network parameters (typically on the order of millions) are automatically defined; iii. testing phase in which an additional set of cases not used in training is presented to the network and the output of the network is tested statistically to determine its success of categorization. finally, iv, the software must be validated on independent cohorts to ensure that performance characteristics generalize to unseen data from other imaging sources, demographics, and ethnicity. in the case of a new disease, such as the coronavirus, datasets are just now being identified and annotated. there are very limited data sources as well as limited expertise in labeling the data specific to this new strain of the virus in humans. accordingly, it is not clear that there are enough examples to achieve clinically meaningful learning at this early stage of data collection despite the increasingly critical importance of this software. solutions to this challenge, that may enable rapid development, include the combination of several technologies: transfer learning will utilize pretraining on other but somehow statistically similar data. in the general domain of computer vision, ima-genet has been used for this purpose (donahue et al. ( ) ). in the case of covid- this may be provided by existing databases of annotated images of patients with other lung infections. data augmentation is a trick used from the beginning of applying convolution neural networks (cnns) to imaging data (lecun et al. ( ) ), in which data are transformed to provide extra training data. normally rotations, reflections, scaling or even group actions beyond the affine group can be explored. other technologies include semi-supervised learning and weak learning when labels are noisy and/or missing (cheplygina et al. ( )). thus, the underlying approach to enable rapid development of new ai-based capabilities, is to leverage the ability to modify and adapt existing ai models and combine them with initial clinical understanding to address the new challenges and new disease entities, such as the covid- . in this section we briefly review three possible system developments: ai systems for detection and characterization of disease, ai systems for measuring disease severity and patient monitoring, and ai systems for predictive modeling. each category will be reviewed briefly and a specific system will be described with a focus on the ai based challenges and solutions. the vast majority of efforts for the diagnosis of covid- have been focused on detecting unique injury patterns related to the infection. automated recognition of those patterns became an ideal challenge for the use of cnns trained on the appearance of those patterns. one example of a system for covid- detection and analysis is shown in figure , which presents an overview of the analysis conducted in gozes et al. ( a) . in general, as is shown here, automated solutions are comprised of several components. each one is based on a network model that focuses on a specific task to solve. in the presented example, both d and d analysis are conducted, in parallel. d analysis of the imaging studies is utilized for detection of nodules and focal opacities using nodule-detection algorithms, with modifications to detect ground-glass (gg) opacities. a d analysis of each slice of the case is used to detect and localize covid- diffuse opacities. if we focus on the d analysis -we again see that multiple steps are usually defined. the first step is the extraction of the lung area as a region of interest (roi) using a lung segmentation module. the segmentation step removes image portions that are not relevant for the detection of withinlung disease. within the extracted lung region, a covid- detection procedure is conducted, utilizing one of a variety of possible schemes and corresponding networks. for example, this step can be a procedure for (undefined) abnormality detection, or a specific pattern learning task. in general, a classification neural network (covid- vs. not covid- ) is a key component of the solution. such networks, which are mostly cnn based, enable the localization of covid- manifestations in each d slice that is selected in what have become known asheat maps per d slice. to provide a complete review of the case, both the d and d analysis results can be merged. several quantitative measurements and output visualizations can be used, including per slice localization of opacities, as in figure (a), and a d volumetric presentation of the opacities throughout the lungs, as shown in figure (b), which presents a d visualization of all gg opacities. several studies have shown the ability to segment and classify the extracted lesions using neural networks to provide a diagnostic performance that matches a radiologist rating ; bai et al. ( ) ). in zhang et al. ( ) , , manually annotated ct slices were used for seven classes, including background, lung field, consolidation (cl), groundglass opacity (ggo), pulmonary fibrosis, interstitial thickening, and pleural effusion. after a comparison between different semantic segmentation approaches, they selected deeplabv as their segmentation detection backbone (chen et al. ( ) ). the diagnostic system was based on a neural network fed by the lung-lesion maps. the system was designed to classify normals from common pneumonia and covid- specific pneumonia. their results show a covid- diagnostic accuracy of . % tested in subjects. in bai et al. ( ) , a direct classification of covid- specific pneumonia versus other etiologies was performed using an efficientnet b network (tan and le ( )) followed by a two-layer fully connected network to pool the information from multiple slices and provide a patientlevel diagnosis. this system yielded a % accuracy in a testing set of subjects compared to an % average accuracy for six radiologists. these two examples exemplify the power of ai to perform at a very high level that may augment the radiologist, when designed and tested for a very narrow and specific task within a de-novo diagnostic situation. time delay in covid- testing using rt-pcr can be overcome with integrative solutions. augmented testing using ct, clinical symptoms, and standard white blood cell (wbc) panels has been proposed in mei et al. ( ) . the authors show their ai system that integrates both sources of information is superior to an imaging-alone cnn model as well as a machine learning model based on non-imaging information for the diagnosis of covid- . integrative approaches can overcome the lack of diagnostic specificity of ct imaging for covid- (rubin et al. ( )) it is well understood that chest radiographs (cxr) have lower resolution and contain much less information than their ct counterparts. for example, for covid- patients, the lungs may be so severely infected that they become fully opacified, obscuring details on an x-ray and making it difficult to distinguish between pneumonia, pulmonary edema, pleural effusions, alveolar hemorrhage, or lung collapse ( figure ). still, many countries are using cxr information for initial decision support as well as throughout the patient hospitalization and treatment process ). deep learning pipelines for cxr opacities and infiltration scoring exist. in most publications seen to-date, researchers utilize existing public pneumonia datasets, which were available prior to the spread of coronavirus, to develop network solutions that learn to detect pneumonia on a cxr. in (selvan et al. ( ) ), an attempt to solve the issue of the compact lungs is presented using variational imputation. a deep learning pipeline based on variational autoencoders (vae) has shown in pilot studies > % accuracy in separating covid- patients from other patients with lung infections, both bacterial and viral. a systematic evaluation of one of those system has demonstrated comparable performance to a chest radiologist (murphy et al. ( ) ). this demonstrates the capability of recognizing covid- associated patterns, using the cxr data. we view these results as preliminary, and to be confirmed with more rigorous experimental setup which includes access to covid- and other infections from the same sources with identical acquisition technology, time-window, ethnicity, demographics, etc. such rigorous experiments are critical in order to assess the clinical relevance of the developed technology. in this section we focus on the use of ai for hospitalized patients. image analysis tools can support measurement of the disease extent within the lungs, thus generating quantification for the disease that can serve as an image-based biomarker. such a biomarker may be used to assess relative severity of patients in the hospital wards, enable tracking of disease severity over time, and thus assist in the decision-making process of the physicians handling the case. one such biomarker, termed the corona score, was recently introduced in gozes et al. ( a,b) . the corona score is a measure of the extent of opacities in the lungs. it can be extracted in ct and in cxr cases. figure presents a plot of corona-score measurements per patient over time, in ct cases. using the measure, we can assess relative severity of the patients (left) as well as extract a model for disease burden over the course of treatment (right). additional very valuable information on characterization of disease manifestation can be extracted as well, such as locations of opacities within the lungs, opacities burden within specific lobs of the lungs (using a lungs lobe segmentation module) and analysis of the texture of the opacities using classification of patches extracted from detected covid- areas (using a patch-based classification module). these characteristics are important biomarkers with added value for patient monitoring over time. the clinical covid- lung infections are diagnosed and monitored with ct or cxr imaging where opacities, their type and extent, may be quantified. the picture of radiological findings in covid- patients is complex (wong et al. ( ) ) with mixed patterns: ground-glass opacities, opacities with a rounded morphology, peripheral distribution of disease, consolidation with groundglass opacities, and the so called crazy-paving pattern. first reporting of longitudinal developments monitored by cxr (shi et al. ( b) ) indicate that cxr findings occur before the need for clinical intervention with oxygen and/or ventilation. this fosters the hypothesis that cxr imaging and quantification of findings are valuable in the risk assessment of the individual patient developing severe covid- . in the capital region of denmark, it is standard practice to acquire a cxr for covid- patients. the clinical workflow during the covid- pandemic does not in general allow for manual quantitative scoring of radiographs for productivity reasons. making use of the cxr already recorded during real time risk assessment therefore requires automated methods for quantification of image findings. several scoring systems for the severity of covid- lung infection adapted from general lung infection schemes have been proposed (wong et al. ( ) , shi et al. ( b) , cohen et al. ( ) ). above, in figure , it is shown how opacities may be located in ct images. similar schemes may be used for regional opacity scoring in cxr, as shown in figure . for the administration and risk profiling of the individual patient, imaging does not tell the full story. important risk factors include age, bmi, co-morbidities (especially diabetes, hypertension, asthma, chronic respiratory or heart diseases) (jordan et al. ( ) ). combining imaging with this type of information from the ehr and with data representing the trajectory of change over time enhances the ability to determine and predict the stage of disease. an early indication is that cxr's contribute significantly to the prediction of the probability for a patient to be on a ventilator. here we briefly summarise the patient trajectory prognosis setup: we have in preliminary studies from the cohort from the capital and zealand regions of denmark, combined clinical information from electronic health records (ehr) defining variables relating to vital parameters, comorbidities, and other health parameters with imaging information. modeling was performed using a simple random forest implementation in a -fold cross-validation fashion. in figure are as illustration auc for prediction of outcome in terms of hospitalisation, requirement for ventilator, admission to intensive care unit, and death. these have been illustrated on , covid- positive subjects from the zealand and capital region of denmark. these are preliminary unconsolidated results for illustrative purposes. however, these support the feasibility of an algorithm to predict severity of covid- manifestations early in the course of the disease. the combination of cxr into these prognostic tools have been performed by including a number of quantitative features per lung region as a feature vector in the random forest described above. imaging has played a unique role in the clinical management of the covid- pandemic. public health authorities of many affected countries have been forced to implement severe mitigation strategies to avoid the wide community spread of the virus (parodi and liu ( ) ). mitigation strategies put forth have focused on acute disease management and the plethora of automated imaging solutions that have emerged in the wake of this crisis have been tailored toward this emergent need. until effective therapy is proven to prevent the widespread dissemination of the disease, mitigation strategies will be followed by more focused efforts and containment approaches aimed at avoiding the high societal cost of new confinement policies. in that regard, imaging augmented by ai can also play a crucial role in providing public health officials with pandemic control tools. opportunities in both upstream infection management and downstream solutions related to disease resolution, monitoring of recurrence and health security will be emerging in the months to come as economies reopen to normal life. pandemic control measurements in the pre-clinical phase of the infection may seek to identify those subjects that are more susceptible to the disease due to their underlying risk factors that lead to the acute phase of covid- infections. several epidemiological factors, including age, obesity, smoking, chronic lung disease hypertension, and diabetes, have been identified as risk factors (petrilli et al. ( ) ). however, there is a need to understand further risk factors that can be revealed by image-based studies. imaging has shown to be a powerful source of information to reveal latent traits that can help identify homogeneous subgroups with specific determinants of disease (young et al. ( ) ). this kind of approach could be deployed in retrospective databases of covid- patients with pre-infection imaging to understand why some subjects seem to be much more prone to progression of the viral infection to acute pulmonary inflammation. the identification of high-risk populations by imaging could enable targeted preventive measurements and precision medicine approaches that could catalyze the development of curative and palliative therapies. identification of molecular pathways in those patients at a higher risk may be crucial to catalyze the development of much needed host-targeting therapies. the resolution of the infection has been shown to involve recurrent pulmonary inflammation with vascular injury that has led to post-intensive care complications (ackermann et al. ( ) ). detection of micro embolisms is a crucial task that can be addressed by early diagnostic methods that monitor vascular changes related to vascular pruning or remodeling. methods developed within the context of pulmonary embolism detection, and clot burden quantification could be repurposed for this task ). another critical aspect of controlling the pandemic is the need to monitor infection recurrence as the immunity profile for sars-cov- is still unknown (kirkcaldy et al. ( ) ). identifying early pulmonary signs that are compatible with covid- infection could be an essential tool to monitor subjects that may relapse in the acute episode. ai methods have shown to be able to recognize covid- specific pneumonia identified on radiographic images (murphy et al. ( ) ). the accessibility and potential portability of the imaging equipment in comparison to ct images could enable early pulmonary injury screening if enough specificity can be achieved in the early phases of the disease. eventually, some of those tools might facilitate the implementation of health security screening solutions that revolve around the monitoring of individuals that might present compatible symptoms. although medical imaging solutions might have a limited role in this space, other kinds of non-clinical imaging solutions such as thermal imaging may benefit from solutions that were originally designed in the context of x-ray or ct screening. one of the fascinating aspects that has emerged around the utilization of ai-based imaging approaches to manage the covid- pandemic has been the speed of prototyping imaging solutions and their integration in end-to-end applications that could be easily deployed in a healthcare setting and even ad-hoc makeshift caring facilities. this pandemic has shown the ability of deep neural networks to enable the development of end-to-end products based on a model representation that can be executed in a wide range of devices. another important aspect has been the need for large-scale deployments due to the high incidence of the covid- infection. these deployments have been empowered by the use of cloud-based computing architectures and multi-platform web-based technologies. multiple private and open-source systems have been rapidly designed, tested, and deployed in the last few months. the requirements around the utilization of these systems in the general population for pandemic control are: • high-throughput: the system needs to have the ability to perform scanning and automated analysis within several seconds if screening is intended. • portable: the system might need to reach the community without bringing them to hospital care settings to avoid nosocomial infections. • reusable: imaging augmented with ai has emerged as a highly reusable technique with scalable utilization that can adapt to variable demand. • sensitive: the system needs to be designed with high sensitivity and specificity to detect early signs of disease. • private: systems have to protect patient privacy by minimizing the exchange of information outside of the care setting. web-based technologies that provide embedded solutions to deploy neural network systems have emerged as one of the most promising implementations that fulfill those requirements. multiple public solutions in the context of chest xray detection of early pneumonia and covid- compatible pneumonia have been prototyped, as shown in figure . the covictory app, part of the slow-down covid project (www. slowdowncovid .org) implements a classification neural network for the detection of mild pneumonia as an early risk detection of radiographic changes compatible with covid- . the developers of this system based their system in a network architecture recently proposed for tuberculosis detection that has a very compact and efficient design well-suited for deployment in mobile platforms (pasa et al. ( ) ). the database that trained the network was based on imaging from three major chest x-ray databases: nih chest x-ray, chexpert, and padchest. the developers sub-classified x-ray studies labeled as pneumonia in mild versus moderate/severe pneumonia fig. . illustration of a public ai systems for covid- compatible pneumonias on chest x-rays from two covid- subjects using covictory app (www.covictoryapp.org) with mild pneumonia signs (left) and more severe disease (right). by consensus of multiple readers using spark crowd, an open source system for consensus building (rodrigo et al. ( ) ). another example is the coronavirus xray app that included public-domain images from covid- patients to classify images into three categories: healthy, pneumonia and covid- . both systems were implemented as a static web application in javascript using tensorflow-js. although the training was carried out using customized gpu hardware, the deployment of trained models is intrinsically multi-platform and multi-device thanks to the advancement of web-based technologies. other commercial efforts like cad covid-xray (https://www. delft.care/cad covid/) has leveraged prior infrastructure used for the assessment of tuberculosis on x-ray to provide a readily deployable solution. the covid- crisis has seen the emergence of multiple observational studies to support research into understanding disease risk, monitoring disease trajectory, and for the development of diagnostic and prognostic tools, based on a variety of data sources including clinical data, samples and imaging data. all these studies share the theme that access to high quality data is of the essence, and this access has proven to be a challenge. the causes for this challenge to observational covid- research are actually the same ones that have hampered large scale data-driven research in the health domain over the last years. owing to the data collection that takes place in different places and different institutes, there is fragmentation of data, images and samples. moreover there is a lack of standardization in data collection, which hampers reuse of data. consequently, the reliability, quality and re-usability of data for datadriven research, including the development and validation of ai applications, is problematic. finally, depending on the sys-tem researchers and innovators are working in, ethical and legal frameworks are often unclear and may sometimes be (interpreted as being) obstructive. a coordinated effort is required to improve the accessibility to observational data for covid- research. if implemented for covid- , it can actually serve as a blueprint for large, multi-center observational studies in many domains. as such, addressing the covid- challenges also presents us with an opportunity, and in many places we are already observing that hurdles towards multicenter data accessibility are being addressed with more urgency. an example is the call by the european union for an action to create a pan-european cohort covid- including imaging data. in the netherlands, the health-ri c initiative aims to build a national health data infrastructure, to enable the re-use of data for personalized medicine, and similar initiatives exist in other countries. in light of the current pandemic, these initiatives have focused efforts on supporting observational covid- research, with the aim to facilitate data access to multi-center data. the underlying principle of these infrastructures is that by definition they will have to deal with the heterogeneous and distributed nature of data collection in the healthcare system. in order for such data to be re-usable, harmonisation at the source is required. this calls for local data stewardship, in which the different data types, including e.g. clinical, imaging and lab data, need to be collected in a harmonized way, adhering to international standards. here, the fair principle needs to be adopted, i.e. data needs to be stored such that they are findable, accessible, interoperable and reusable wilkinson et al. ( ) . for clinical data, it is not only important that the same data are collected (e.g. adhering to the world health organisation case report form (crf), often complemented with additional relevant data), but also that their values are unambiguously defined and are machine-readable. the use of electronic crfs (ecrfs) and accompanying software greatly supports this, and large international efforts exist to map observational data to a common data model, including e.g. the observational health data sciences and informatics (ohdsi) model. similarly the imaging and lab data should be processed following agreed standards. in the health-ri c implementation, imaging data are pseudonimized using a computational pipeline that is shared between centers. for lab data, standard ontologies such as loinc can be employed. the covid- observational project will not only collect fair metadata describing the content and type of the data, but also data access policies for the data that are available. this will support the data search, request, and access functionalities provided by the platform. an illustration of the data infrastructure in health-ri c is provided in fig . next to providing data for the development of ai algorithms, it is important to facilitate their objective validation. in the medical imaging domain, challenges have become very popular to objectively compare performance of different algorithms. in the design of challenges, part of the data needs to be kept apart. it is therefore important that, while conducting efforts to provide access to observational covid- data, we already plan for using part of the data for designing challenges around relevant clinical use cases. fig. . design of covid- observational data platform. in order for hospitals to link to the data platform, they need to make their clinical, imaging and lab data fair. tools for data harmonization (fair-ification) are being shared between institutes. fair metadata (and in some cases fair data) and access policies are shared with the observational platform. this enables a search tool for researchers to determine what data resources are available at the participating hospitals. these data can subsequently be requested, and if the request is approved by a data access committee, the data will be provided, or information how the data can be accessed will be shared. in subsequent versions of the data platform, also distributed learning will be supported, so that data can stay at its location. during the pandemic, setting up such an infrastructure from scratch will not lead to timely implementation. health-ri c was already in place prior to the pandemic, and some of its infrastructures could be adjusted to start building a covid- observational data platform. in denmark, a similar initiative was not in place. however, in eastern denmark, the capital and zealand regions share a common data platform in all hospitals with a common ehr and a pacs at each region covering in total hospitals and . million citizens making data collection and curation relatively simple. at continental scale, solutions are being created, but will likely not be in place during the first wave of the pandemic. the burdens to overcome are legal, political, and technical. access to un-consented data from patients follows different legal paths in different countries. in uk the department of social and mental care issued on march , a notice simplifying the legal approval of covid- data processing. in denmark, usual regulation and standards were maintained, but authorities made an effort to grant permission by the usual bodies in fast track. as access to patient information must be restricted, not every researcher with any research goal can be granted access. without governance in place prior to an epidemic, access will be granted on an ad hoc and first-come-first-served basis, not necessarily leading to the most efficient data analysis. finally, data are hosted in many different it systems and the two major technical challenges lie in bringing data to a common platform, and having a (in eu gdpr) compliant technical setup for collaboration. building such infrastructure with proper security and data handling agreements in place is complex and will lead to substantial delays if not in place prior to the epidemic. in the netherlands, the health-ri c platform was in place. in denmark, the efforts have been constrained to the eastern part of the country sharing common ehr and pacs and having infrastructure in place for compliant data sharing at computerome. at a european scale, the commission launched the european covid- data platform on april building on existing hardware infrastructure. this was followed up by a call for establishing a pan-european covid- cohort. funding decision will be in august . even though a tremendous effort has been put in place and usual approvals of access and funding have been fast-tracked, proper infrastructures have not been created in time for the first wave in europe. the current covid- pandemic offers us historic challenges but also opportunities. it is widely believed that a substantial percentage of the (as of this writing) . million confirmed cases and , deaths and trillions of dollars of economic losses would have been avoided with adequate identification of those with active disease and subsequent tracking of location of cases and prediction of emerging hotspots. imaging has already played a major role in diagnosis and tracking and prediction of outcomes and has the potential to play an even greater role in the future. automated computer based identification of probability of disease on chest radiographs and thoracic ct combined with tracking of disease could have been utilized early on in the development of cases, first in wuhan, then other areas of china and asia, and subsequently europe and the united states and elsewhere. this could have been utilized to inform epidemiologic policy decisions as well as hospital resource utilization and ultimately, patient care. this pandemic also represented, perhaps for the first time in history, that a disease with relatively unique imaging and clinical characteristics emerged and spread globally faster than the knowledge to recognize, diagnose and treat the disease. it also created a unique set of challenges and opportunities for the machine learning/ai community to work side by side and in parallel with clinical experts to rapidly train and deploy computer algorithms to treat an emerging disease entity. this required a combination of advanced techniques such as the use of weakly annotated schemes to train models with relatively tiny amounts of training data which has only become widely available recently, many months after the initial outbreak of disease. the imaging community as a whole has demonstrated that extremely rapidly developed ai software using existing algorithms can achieve high accuracy in detection of a novel disease process such as covid- as well as provide rapid quantification and tracking. the majority of research and development has focused on pulmonary disease with developers using standard chest-ct dicom imaging data as input for algorithms designed to automatically detect and measure lung abnormalities associated with covid- . the analysis includes automatic detection of involved lung volumes, automatic measurement of disease as compared to overall lung volume and enhanced visualization techniques that rapidly depict which areas of the lungs are involved and how they change over time in an intuitive manner that can be clinically useful. a variety of manuscripts describing automated detection of covid- cases have been recently published. when reviewing these manuscripts one can see the following interesting trend: all are focusing on one of the several key tasks, as defined herein. each publication has a unique system design that contains a set of network models, or a comparison across models; and the results are all very strong. the compelling results, such as the ones presented herein may lead us to conclude that the task is solved; but is this the correct conclusion? it seems that the detection and quantification tasks are in fact solvable with our existing imaging analysis tools. still, there are several data-related issues which we need to be aware of. experimental evidence is presented on datasets of hundreds and we need to go to real world settings, in which we will start exploring thousands and even more cases, with large variability. our systems to date are focusing on detection of abnormal lungs in a biased scenario of the pandemic in which there is a very high prevalence of patients presenting with the disease. once the pandemic declines substantially, the shift will be immediate to the need to detect covid among a wide variety of diseases including other lung inflammatory processes, occupational exposures, drug reactions, and neoplasms. in that future in which the prevalence of disease is lower,will our solutions that work currently be sensitive enough, without introducing too many false positives? that is the crux of many of the current studies that have tested the different ai solutions within a very narrow diagnostic scope. there are many possibilities and promising directions, yet the unknown looms larger than the known. just as the current pandemic has changed the way many are thinking about distance learning, the practice of telemedicine, and overall safety in a non-socially distanced society, it seems that we are similarly setting the stage with our current on the fly efforts in algorithm development for the future development and deployment of ai. we need to update infrastructure including methods of communication and sharing cases and findings as well as reference databases and algorithms for research, locally, country-based and globally. we need to prove the strengths, build the models and make sure that the steps forward are such that we can continue and expand the use of ai, particularly just in time ai. we believe that imaging is an absolutely vital component of the medical space. for predictive modeling we need to not limit ourselves to just the pixel data but also include additional clinical, patient level information. for this, combined effort among many groups, as well as state and federal level support will result in optimal development, validation, and deployment. many argue that we were caught unaware from a communication, testing, treatment and resource perspective with the current pandemic. but deep learning-augmented imaging has emerged as a unique approach that can deliver innovative solutions from conception to deployment in extreme circumstances to address a global health crisis. the imaging community can take lessons learned from the current pandemic and use them to not only be far better prepared for recurrence of covid- and future pandemics and other unexpected diseases, but also use these lessons to advance the art and science of ai as applied to medical imaging in general. pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- ai augmentation of radiologist performance in distinguishing covid- from pneumonia of chest ct findings in coronavirus disease- (covid- ): relationship to duration of infection rethinking atrous convolution for semantic image segmentation not-so-supervised : a survey of semi-supervised, multi-instance, and transfer learning in medical image analysis covid- image data collection decaf: a deep convolutional activation feature for generic visual recognition rapid ai development cycle for the coronavirus (covid- ) pandemic: initial results for automated detection and patient monitoring using deep learning ct image analysis coronavirus detection and analysis on chest ct with deep learning guest editorial deep learning in medical imaging: overview and future promise of an exciting new technique peneta scalable deeplearning model for automated diagnosis of pulmonary embolism using volumetric ct imaging covid- : risk factors for severe disease and death covid- and postinfection immunity: limited evidence, many remaining questions backpropagation applied to handwritten zip code recognition a survey on deep learning in medical image analysis artificial intelligence-enabled rapid diagnosis of patients with covid- covid- on the chest radiograph: a multi-reader evaluation of an ai system from containment to mitigation of covid- in the us efficient deep network architectures for fast chest x-ray tuberculosis screening and visualization factors associated with hospital admission and critical illness among people with coronavirus disease spark-crowd: a spark package for learning from crowdsourced big data the role of chest imaging in patient management during the covid- pandemic: a multinational consensus statement from the fleischner society lung segmentation from chest x-rays using variational data imputation review of artificial intelligence techniques in imaging data acquisition, segmentation and diagnosis for covid- radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study. the lancet infectious diseases radiological society of north america expert consensus statement on reporting chest ct findings related to covid- efficientnet: rethinking model scaling for convolutional neural networks. arxiv.org the fair guiding principles for scientific data management and stewardship frequency and distribution of chest radiographic findings in covid- positive patients the role of imaging in novel coronavirus pneumonia (covid- ) uncovering the heterogeneity and temporal complexity of neurodegenerative diseases with subtype and stage inference relation between chest ct findings and clinical conditions of coronavirus disease (covid- ) pneumonia: a multicenter study the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -a zcggf authors: antolin, michael f.; jenkins, kristin p.; bergstrom, carl t.; crespi, bernard j.; de, subhajyoti; hancock, angela; hanley, kathryn a.; meagher, thomas r.; moreno‐estrada, andres; nesse, randolph m.; omenn, gilbert s.; stearns, stephen c. title: evolution and medicine in undergraduate education: a prescription for all biology students date: - - journal: evolution doi: . /j. - . . .x sha: doc_id: cord_uid: a zcggf the interface between evolutionary biology and the biomedical sciences promises to advance understanding of the origins of genetic and infectious diseases in humans, potentially leading to improved medical diagnostics, therapies, and public health practices. the biomedical sciences also provide unparalleled examples for evolutionary biologists to explore. however, gaps persist between evolution and medicine, for historical reasons and because they are often perceived as having disparate goals. evolutionary biologists have a role in building a bridge between the disciplines by presenting evolutionary biology in the context of human health and medical practice to undergraduates, including premedical and preprofessional students. we suggest that students will find medical examples of evolution engaging. by making the connections between evolution and medicine clear at the undergraduate level, the stage is set for future health providers and biomedical scientists to work productively in this synthetic area. here, we frame key evolutionary concepts in terms of human health, so that biomedical examples may be more easily incorporated into evolution courses or more specialized courses on evolutionary medicine. our goal is to aid in building the scientific foundation in evolutionary biology for all students, and to encourage evolutionary biologists to join in the integration of evolution and medicine. the scientific and societal benefits each has delivered, and we acknowledge that potential conflicts can arise between the evolutionary and medical viewpoints. as evolutionary biologists, we think in terms of traits and genes in populations or lineages over time. we view traits as continuously evolving in a population context, driven by processes such as adaptation by natural selection, mutation, and genetic drift, but constrained by genetic and physiological trade-offs. we know that interactions between evolutionary forces are mediated by the effective size of populations over long time periods, and we are accustomed to seeing traits as the products of genotype-by-environment interactions. medical doctors, on the other hand, are trained to view patients as individuals in need of care, with injuries and symptoms at a particular time. population-level and public health concerns must be balanced with alleviating an individual patient's suffering. these apparently contradictory viewpoints need not be at odds (e.g., childs et al. ) . the integration of evolutionary biology into medicine, often referred to as "evolutionary medicine," provides a dynamic view of genetic, environmental, and infectious diseases. an individual patient's ailments represent a particular point in time at the convergence of ancestries, environment, and exposures, like the tips of growing and changing trees whose branches intermingle through time but stay mostly out of sight. evolutionary biologists who train undergraduate premedical students are in a position to teach them the fundamental evolutionary principles that underlie biology. by explicitly using biomedical examples, we can form a truly interdisciplinary science that actively engages evolutionary biologists as well as medical researchers and practitioners. this requires collaboration between the communities to develop methodologies and curricula that integrate evolutionary biology into training future generations of doctors, public health workers, and biomedical researchers. here, we give a brief overview of the historical relationships between evolutionary biology and medicine (box ), and discuss how evolutionary biologists can use medically relevant examples to teach basic evolutionary biology concepts to all undergraduates, but particularly to students preparing to enter medical professions. we frame these examples in terms familiar to evolutionary biologists, and point to resources currently available for the classroom. our message is that evolutionary medicine is an exciting and useful field that many of our students will need to understand and will find engaging, and that evolutionary biologists can actively connect evolutionary biology and the biomedical sciences. we, the authors, contributed to a symposium on evolutionary medicine during the meeting of the society for the study of evolution in norman, oklahoma, to honor seminal contributions made by george williams, who passed away on sept . williams' writings on the evolution of senescence and life histories provided fundamental conceptual developments in evolutionary biology (williams ) , as did his thoughts on the role of natural selection in adaptive evolution on multiple levels from genes to individuals to groups of organisms (williams ) . further, his work in collaboration with randolph nesse spurred interest in applying evolutionary biology to medicine and public health. the essay on "the dawn of darwinian medicine" (williams and nesse ) and the book "why we get sick" (nesse and williams ) are cornerstones of the synthesis between evolutionary biology and the biomedical sciences. williams' work inspired research projects, articles, and books that have further addressed the many ways that evolutionary biology influences biomedical research and how an evolutionary approach can improve medical diagnostics and therapies (e.g., a special issue of the proceedings of the national academy of science featuring presentations from a sackler symposium, held in april (stearns et al. ); a special issue of evolution: education and outreach originating in part from the sse evolutionary medicine symposium (jenkins and antolin ) . awareness of the potential impact of evolutionary biology on medicine is also growing beyond the biology research community, as illustrated by a joint report from the american association of medical colleges and the howard hughes medical institute (aamc-hhmi ), entitled scientific foundations for future physicians. it describes core scientific competencies that should be taught during premedical training and in medical school, and explicitly covers evolutionary biology, genetics, and genomics (download the full report from http://www.hhmi.org/grants/pdf/ - _aamc-hhmi_report.pdf). nesse et al. ( ) substantially expand on these core competencies and provide further recommendations for integrating evolutionary biology into medical education. the "future physicians" report makes the case that because scientific knowledge and medical practice continually grow in response to new discoveries, one goal of medical school must be to train doctors to integrate and apply new clinical and scientific knowledge into their daily work, and to absorb the "fundamental scientific principles that are key to lifelong learning and biomedical scientific literacy" (aamc-hhmi , p. ) . as an integrative scientific discipline at the core of the life sciences, evolutionary biology has a role in helping physicians and biomedical researchers understand how the human body works and why it sometimes fails. human diseases are in part a reflection of the evolutionary history that shaped our genetic make-up and responses to our current environment, including the parasites and pathogenic microbes that plague us. but understanding evolutionary history provides only one perspective on disease. as pointed out by ernst mayr ( ) and niko tinbergen ( ) , traits have both proximate and ultimate causes (see also nesse and williams ) . proximately, diseases arise from combinations of biochemical, physiological, and immunological responses of individuals to the environment and to pathogens. in this sense, disease can be defined mechanistically in terms of malfunctions, disorders, anatomical flaws, and infections that cause morbidity and/or death. ultimately, however, the causes of diseases can be explained in three parts-by ancestry and common descent of organisms, by variation in the environment over time, and by ongoing genomic and evolutionary forces (williams and nesse ; nesse and williams ; nesse and stearns ) . a key to understanding vulnerability to disease is that adaptive evolution increases fitness in terms of reproductive success, and not health, over many generations. natural selection will compromise health and survival if that increases overall reproductive success, and natural selection may not keep pace with the rapid or recent environmental changes in industrial and postindustrial societies. the viewpoint that diseases are shaped by both proximate and ultimate causes represents one of the central concepts underlying evolutionary medicine. it is important to recognize that evolution may not act to shape disease per se, but rather that evolution acts on traits that mediate vulnerability to disease. for example, the potential to form cancers is an inherent risk of multicellularity. moreover, symptoms such as vomiting and fever may actually be defenses against disease by reducing the effects of pathogens. legacies of evolutionary history, including interactions between an individual's genetic susceptibilities, immune system responses, and environmental exposure to poor nutrition, toxins, and pathogens, are important ultimate explanations for disease. ongoing natural selection may also influence disease, for example, through the genomic mechanisms that underlie many cancers or parent-parent and parent-offspring conflicts for resources. these conflicts begin with different genomic contributions by the parents, and continue through prenatal development and into childhood. further, human bodies are not optimally designed, but represent a series of developmental, biophysical, and physiological compromises driven by trade-offs that contribute to risks of injury and disease. on another level, natural selection acts rapidly on microbes via their short generation times and high mutation rates, and microbes rapidly evolve resistance to immune defenses, drugs, and vaccines, often with tragic consequences. the evolutionary approach to the health sciences has clear implications for research on the prevalence of disease within human populations, improved public health practice, and ultimately more effective care for the sick (nesse and stearns ; stearns and koella ; wolfe et al. ; omenn ). the goal of biology courses, from introductory to advanced, is twofold: for students to learn how to think scientifically, and for students to master content in biology. understanding the nature and process of science informs personal and policy decisions, with local, regional, and global impacts, and some knowledge of biology can be helpful for everyone in making life decisions. education research suggests that one way to support those goals is to teach with key concepts such as biological evolution (bransford et al. ; ambrose et al. ). this "big picture" approach helps students build a reference framework to categorize information, which facilitates building connections between new and old content, and improves student retention and ability to transfer information. the ability to categorize and connect information is one of the traits that enable experts to navigate easily through disciplinary content. the ap biology revisions (college board ) and the vision and change report (nsf/aaas ) emphasize teaching key concepts including evolution. evolution is an excellent theme for teaching about the nature of science (national academy of sciences ), and a poor understanding of the nature of science is correlated with a low acceptance and understanding of evolution (johnson and peeples ) . undergraduate biology educators can serve all their students well by introducing an evolutionary view into their courses, beginning with introductory biology. using examples from evolutionary medicine to engage all students-premedical, majors, and nonmajors-in learning about biological evolution may be a particularly effective approach to support students' development both as scientific thinkers and biologists (e.g., see hillis ) . as we show below, applying basic evolutionary principles to human health leads to interesting, novel, and useful conclusions. students who are not typically excited by basic evolutionary concepts may be drawn in by focusing on human health. exploring the research behind theories such as the "stone age diet" and the hygiene hypothesis gives students an accessible entry to discovering the nature and process of science. in particular, this approach demonstrates that premedical students should learn evolutionary biology for the same reasons they study biochemistry, physics, and mathematics-because broad scientific knowledge gives future medical practitioners the tools to understand how a patient may have developed sets of symptoms, and how to best to treat the patient. evolutionary medicine is easily incorporated into undergraduate curricula, through specific examples illustrating basic evolutionary concepts as well as more complex ideas. here, we highlight key aspects of evolutionary medicine in the form of familiar evolutionary concepts. additional examples are provided in the evolution-focused science competencies in the aamc-hhmi report ( ), in an expanded analysis by nesse et al. ( ) , and in boxes - . a growing number of resources are available to support advanced courses or seminars on evolutionary medicine, courses that should complement those in evolution, phylogenetics, and population genetics. these include nesse and williams' ( ) classic as well as more recent collections (stearns and koella ; trevathan et al. ) and a textbook (gluckman et al. ). the website evolution and medicine review (http://evmedreview) provides a resource for sharing news about events and relevant articles, as well as a list of syllabi and other resources useful for educators (box ). the december issue of evolution: education and outreach includes by stearns ( b), omenn ( ) , and alcock and schwartz ( ) that provide specific suggestions for courses linking evolution to medicine and public health. other articles in the same issue provide excellent examples of the synthesis of evolution and medicine in cancer (casas-selves and degregori ), vaccine development (hanley ) , and scurvy (buklijas et al. ) and highlight a new module for teaching evolution and medicine (beardsley et al. ). however, we will know that evolution has truly become an integral part of premedical scientific training when testing competency in evolution becomes part of gaining admission to medical school. the aamc-hhmi report recommends that "assessment of the newly defined scientific competencies must be credibly and reliably accomplished by the medical college admission test (mcat ® ) exam" (aamc-hhmi , p. ). the just-released (november ) preview of the revised mcat ® for specifically includes evolution in section c on transmission of heritable information from generation to generation and the processes that increase genetic diversity and in section a on understanding social structure (https://www.aamc.org/students/applying/mcat/mcat /). although this is a positive step forward, indicating support from the medical community for increasing evolutionary thinking in medical education, evolution biology is still not listed as one of the "foundations of living systems" or integrated throughout the lists of life sciences topics to be tested. our undergraduate premedical students should be willing to learn evolutionary medicine, but greater synthesis is possible. with the goal of continuing development of an interdisciplinary and integrated framework for evolutionary medicine in the undergraduate curriculum, we provide some principles of evolutionary biology aligned with medical examples that students may find particularly enticing. evolutionary medicine can provide engaging illustrations of the genotype to phenotype connection, and students' experience with variation in disease presentation is a familiar way to demonstrate genetic variation. in-depth genetic studies generally show that even commonly taught diseases that at first appear to have a relatively simple genetic basis are found to be more complex. multiple functional and mutant alleles combine with environmental effects to cause variable symptoms and disease severity (templeton ) . this is clearly demonstrated by phenylketonuria (pku), a condition caused by mutations in the single-copy gene encoding phenylalanine hydroxolase and tested for in neonatal health screening. pku arises from the inability to metabolize the amino acid phenylalanine, the buildup of the amino acid and phenylketones, and eventually microcephaly, incomplete brain development, seizures, and severe learning disabilities. although in most cases, disease can be avoided by excluding foods rich in phenylalanine from the diet, the treatment is not equally effective for all variants, and the frequencies of both functional and mutant alleles differ among human populations. this is a familiar problem to evolutionary biologists: although genetic variation is the stuff of evolution, it is seldom straightforward to map genotypic (now genomic) variability onto phenotypic differences between individuals and populations (see boxes and ). students often miss (or reject) this basic concept in their first introduction to evolution, but examples from evolutionary medicine can help drive this idea home. common ancestry explains why biomedical research in animals is applicable to humans (antolin ). but some caution is in order, as in the case of differences between humans and mouse strains in the p family of enzymes important for drug metabolism, which limits the mouse as a model for drug testing for humans (nelson et al. ) . further, it is important for medical practitioners to be aware that each patient has a different ancestry (evolutionary history), and therefore a different genetic makeup, different reactions to drugs, and often different disease symptoms (meyer ; omenn ) . in managing health care, such differences can result in life, death, or long-term disability and morbidity. individual patients' presenta-tion of disease will depend on complex interactions at the convergence of patients' genetic legacies, the evolutionary history of pathogens to which they are exposed, and the environmental context where the patients and pathogens meet. combining concepts of ancestry with individual-level genetic variation culminates in the newly emerging approach of "personalized medicine" (knight ; costa et al. ; jiang and wang ; see box ). a common misconception among students is that "fittest" is the biggest, strongest, or fastest. examples from evolutionary medicine demonstrate that fitness depends on how rates of reproduction and environmental circumstances interact (see boxes , , and ). for example, "diseases of civilization" imply a mismatch hypothesis: alleles that cause disease in modern times may have been adaptive in the past, but produce traits that are maladaptive (mismatched) under current diet and living conditions. a corollary is that modern humans may be changing their environment more rapidly than potential responses to selection can occur. the mismatch hypothesis provides some of the best ultimate explanations for modern rates of obesity, heart disease, type- diabetes, breast and prostate cancer, goiter, iodine deficiency, birth defects, and aging (harper ; eaton et al. ; williams and nesse ; greaves ; diamond ; swynghedauw ; gluckman et al. ). the understanding that contemporary genetic diseases and conditions may be consequences of adaptive mismatches can help explain the persistence of maladies in the face of improved sanitation and living standards. genetic disease varies among human populations because both long-term population sizes and histories of natural selection determine how many disease-causing mutations they carry (antolin ). as an example, the prevalence of specific allelic variants of the cftr gene that cause cystic fibrosis, a debilitating and in the long-term fatal genetic disorder, is potentially linked to selection imposed by past outbreaks of tuberculosis (poolman and galvani ) . this is similar to the long-established link between sickle cell anemia in individuals who inherit at least one copy of the s allele of the hbb globin gene, but where heterozygotes have some degree of resistance to malaria (allison ). in addition to leading to better diagnoses and treatments, seeing diseases as unfortunate genetic legacies inherited from the past may also help ameliorate some of the social stigma associated with genetic diseases. a related concept is that we are specifically adapted to the premodern environment in which humans were primarily huntergathers, before the domestication of plants and animals. central to this concept is that humans are adapted to a stone-age "environment of evolutionary adaptation" (eea). we caution against uncritical use of the eea, as the view is speculative, struggles to account for differences among ancestral human populations that probably experienced different environments, and ignores ongoing human evolution (also see strassmann and dunbar ; méthot ) . nonetheless, the concepts of a mismatch and an eea provide opportunities to introduce students to the scientific process, with discussion, gathering of information, and forming testable hypotheses. evolutionary biologists view phenotypic plasticity-the ability for organisms to display a range of phenotypes in different environments-as a way to maximize fitness under variable environmental conditions (also see box ). in health and medicine, phenotypic plasticity may explain why early developmental problems may lead to disease in adults (bateson et al. ). an example of developmental trade-offs leading to disease in later life is increased susceptibility to heart disease in adults related to periods of nutritional deficiencies and slowed growth in fetal development and early childhood. the damage is potentially higher if subsequent weight gain (catch-up growth) is too rapid when better nutrition becomes available. in the case of growth rates and body size, adult heart disease is a cost of rapid growth following nutritional stress, and should be considered during care of low birth weight or premature babies (bateson et al. ; gluckman et al. ). weak selection on the diseases of aging helps explain their persistence in modern times, especially as postreproductive longevity increases (also see box ). in evolutionary biology, fitness accrues via reproductive success summed across all stages of an individual's life history, and reproductive events early in life contribute more to fitness than do those late in life. the evolutionary theory of aging suggests that early-life fitness components including developmental rate, age at first birth, and fecundity early in life are linked by trade-offs to fecundity and mortality later in life. if these trade-offs result from the same genes acting at both life stages, selection on early-life traits will increase their frequency even if they negatively impact fitness-related traits later in life, a genetic effect called antagonistic pleiotropy (cf. williams ) . the role of testosterone in males provides an example. although increased testosterone has clear benefits for maximizing male reproductive success, this may come at the expense of disease re-sistance because increased testosterone also reallocates immune function away from immune reactions that protect against infections (braude et al. ; bribiescas and ellison ) . higher reproductive success early in life may come at the expense of disease and morbidity, especially if infections are chronic. whether the correlation between testosterone and altered immune function represents a genetic trade-off, or whether reallocation represents adaptive phenotypic plasticity, may be determined using modern genomic analyses and association studies. another striking trade-off is embodied in stem cells. stem cells are essential for the differentiation and renewal of all tissues, but their genetic characteristics place them only a few mutational steps away from cancer cells, and almost all cancers originate through a short series of somatic mutation in stem cells, which are increasingly being revealed as a double-edged sword (e.g., janzen et al. ; krizhanovsky and lowe ) . exploring evolution on a microbial time scale can help students understand the roles of genetic variation and reproductive success in evolution (box ). short generation time, huge populations, high mutation rate, genetic reassortment, and horizontal gene transfer in microbes and viruses ensures that infectious diseases will evade therapies and vaccines in the future, and that new diseases will continue to emerge from reservoirs in wild animals: an "arms race against an adaptable opponent" (lederberg ) . this dynamic view of disease accounts for the variability in human-adapted pathogens such as influenza viruses and malaria, where evolutionary escape hinders development of vaccines with long-lasting protection and results in multidrug resistance. medical practice is directly impacted by rapid evolution of resistance to antimicrobial drugs as well as avoidance of natural and vaccine-induced immunity (bergstrom and feldgarden ; nesse and stearns ) . understanding how pathogen variability arises leads to public health interventions that may reduce exposure and limit chains of transmission (galvani ) . in our germophobic society, students need to realize that we have a long evolutionary history with microbes and parasites, and that these organisms are not always debilitating even if they cause mild disease (also see box ). in some cases, microbes are helpful, and removing all commensals can be unhealthy: consider the occurrence of vaginal yeast infections in conjunction with systemic antibiotic use (eckert et al. ; reid ) . as we learn more about the complex interactions between humans and the diverse microbial species that colonize our bodies, we discover just how important commensal organisms are to our well-being (e.g., grice and segre ). many gut bacteria play important roles in our nutrition, such as the bacterium escherichia coli in the large intestine providing us with the essential vitamin k, which we cannot produce ourselves. some pathogens and parasites may, paradoxically, also be necessary to our well-being. obsession with hygiene in some industrialized societies has for the first time in our evolutionary history relieved us of our helminth parasites. strachan ( ) first proposed the "hygiene hypothesis" that attributed the recent epidemic in asthma, allergy, and other autoimmune diseases to this loss of our old companions (bach ; osada and kanazawa ) . this hypothesis predicts that restoration of helminth infections, or some facsimile thereof, should relieve symptoms of at least some of these diseases. indeed, in a clinical trial, the majority of crohn's disease patients who ingested pig whipworm (trichuris suis) experienced disease remission (summers et al. ) , and more studies indicating the utility of helminth infections to treat autoimmune disease have followed (reddy and fried ) . particularly, striking is the observation that patients with both multiple sclerosis and worm infections experience a much slower development of disease than do those lacking worm infections (correale and farez ) . finally, our long evolutionary history with pathogens has led to many defensive adaptations, including fever, coughing, and vomiting. medical practitioners need to be aware of symptoms that are actually defenses, because it is possible that in some cases, treating symptoms may prolong the course of infections and opportunities for pathogen transmission (williams and nesse ; carey ) . in a memoir (the edge blog [http://www.edge.org/documents/ williams_index.html], downloaded june ), george williams sums up why evolutionary medicine should resonate in the community of evolutionary biologists, and how evolutionary thinking can help physicians to become better practitioners: in twenty or thirty years, medical students will be learning about natural selection, about things like balance between unfavorable mutations and selection. they will be learning about the evolution of virulence, of resistance to antibiotics by microorganisms, they will be learning about human archaeology, about stone age life, and the conditions in the stone age that essentially put the finishing touches on human nature as we now have it. these same ideas then will be informing the work of practitioners of medicine, and the interactions between doctor and patient. they'll be guiding the medical research establishment in a fundamental way, which isn't true today. at the rate things are going, this is inevitable. these ideas ought to reach the people who are in charge-the doctors and the medical researchers-but it's even more important that they reach college students, especially future medical students, and patients who go to the doctor. they'll have questions to ask that doctor, who will have to have answers. i hope this set of ideas produces a certain amount of bottom-up influence on the medical community, via students and patients. but i hope also that there's some top-down influence-that it will be influencing the faculties in medical schools and the researchers on human disease. george williams' work spurred us to ask "what is the role of evolutionary biologists in promoting evolutionary thinking in medical education and practice?" we encourage evolutionary biologists working in schools of medicine and public health, as well as colleges and universities where evolutionary research and instruction occurs, to explore relationships between evolution and health. we encourage evolutionary biologists to reach out to premedical students and others preparing for the medical professions, and to directly connect evolutionary science to the intellectual development of physicians and public health practitioners at every phase of their education and training. financial support for this symposium was provided by the education and outreach committee of the society for the study of evolution, by the national evolutionary synthesis center, and by the co-authors home institutions. we thank the local organizers of the evolution meetings, particularly r. broughton, i. schlupp, and l. weider (special thanks for the use of the bicycle!). the historic relationship between medicine and evolutionary biology has been mixed, and traditionally evolutionary biology has not played a prominent role in medical training. even so, general natural history was part of the medical school curriculum when charles darwin attended edinburgh in - , but evolution certainly was not (antolin a,b). darwin had familiarity with links between evolutionary biology and medicine through his grandfather erasmus darwin's volume "zoonomia," which included both a linnaean classification of disease and a clear statement about transmutation and common descent in nature (darwin ). darwin also consulted with his father, the physician robert waring darwin, on human heredity and disease (bynum ). the question of heredity was briefly covered issues in the "descent of man" (darwin ) and in "variation of plants and animals under domestication" (darwin ) , along with observations on the similarity of diseases (and responses to medication) in humans and apes, differing disease prevalence among human populations, effects of disease on aboriginal populations coming into contact with europeans, and sex-limited maladies such as hemophilia and gout. as evolutionary biology developed as a field, various scientists and physicians also advocated for inclusion of evolutionary thinking in medicine (e.g., huxley ; aitken ; morton ; see zampieri ). in the late s, most applications of evolution in medicine were holistic, fitting in with the traditional medical ideas of "constitutions" and "diatheses." these were defined as tendencies to display groups of maladies in general ways, for instance "tubercular" individuals (bynum ; zampieri ). at the same time, the medical sciences were moving discovering specific causes for specific ailments, resulting in improved diagnosis and treatment (porter ) . as an example, the use of anesthesia and aseptic methods in surgery was developed during this period. the work of pasteur, koch, and others in identifying microbes and the birth of germ theory made it possible to identify the pathogens underlying common diseases such as whooping cough, syphilis, and cholera. further, industrialization and concentration of human populations in cities created new public health challenges that needed attention (porter ) . as the mechanisms underlying evolution and genetics were explored during the late s and early s, so were the physiological, developmental, genetic, and microbial bases of disease. the flexner report of marked a major turn toward placing medicine on a firm scientific footing. this classic of medical education explicitly called for bringing medical education onto a uniform foundation of human biology (flexner ) , and for experimental analysis of physiology, development, endocrinology, biochemistry, and anatomy of specific tissue and organ systems. flexner also called for broad coverage of the general sciences as part of medical training and practice. this period saw a step toward a reductionist view of the causes of disease, diverging from previous holistic medical approaches. although medical knowledge grew rapidly, medical applications from the emerging field of evolutionary biology were not as clear and, as a result, the evolutionary viewpoint was not broadly incorporated into medical school curricula. evolution was not mentioned in the flexner report, but one response to the report was for medical schools to establish departments of anthropology to bring human natural history, via classes in comparative anatomy and geographical medicine, explicitly into medical training (morton ) . this period spanning through the s also saw the modern synthesis in evolutionary biology and breakthroughs in evolutionary research. but evolutionary biology still was not incorporated into medical training. in part, this was because the successful clinical application of antimicrobial drugs during this same period led to the idea that infectious diseases would soon to be completely manageable (burnet and white ; anderson ) . ironically, it is evolutionary biology that explains why pathogenic microbes remain an ongoing challenge (e.g., see box ). but three serious misunderstandings and abuses of evolutionary theory resulted in evolution being expunged from medical training by the late s (porter ; zampieri ). the first was related to the previously mentioned "constitutions," "diatheses," and "degeneracy" used to explain disease vulnerabilities in certain racial groups and in women. the misapplication of this typologically racist and sexist thinking to disease, which was wrongly tied to evolutionary principles, was largely dealt with by implementation of flexner's ( ) recommendations. the mechanistic understanding of disease attenuated societal biases of race and gender, although social disparities in the delivery of medical treatment still persist. the second reason was the horrific application of eugenics in the first half of the th century culminating in the genocidal nazi regime in the s and s in europe (zampieri ). to be sure, eugenics was a malignant social policy that arose from misapplication of the science of evolution, and was discarded by evolutionary biologists after the population genetics of mutation and natural selection were better understood (j. b. s. haldane [ ] provides an amusing essay defending mathematical theories of population genetics, including the balance between mutation and selection that was overlooked by eugenicists, and some lovely verse about his own life-ending fight with colorectal cancer). but the backlash was severe enough that even today many scientists within biomedical fields do not fully acknowledge how much evolutionary science informs medicine (antonovics et al. ; nesse and stearns ) . the third reason was that the dominant reductionist scientific paradigm advocated by flexner ( ) trained scientists to reject teleological thinking, which was mistakenly conflated with the process of natural selection, a misunderstanding that persists among some to this day. currently, few medical schools teach evolutionary topics beyond human genetic variation, drug resistance, pathogen virulence, and adaptation by natural selection (nesse and schiffman ; downie ; childs et al. ; harris and malyango ) . much of the new interest in evolutionary approaches to medicine has come from addressing questions about why natural selection has left the body vulnerable to disease. why does the human eye have a blind spot? why does the appendix persist despite causing appendicitis? why are autoimmune diseases becoming more prevalent in recent decades? why has not natural selection shaped childbirth to be less painful and risky? why are humans so vulnerable to drug and alcohol abuse? what is the evolutionary explanation for aging? is menopause a life-history trait shaped by selection, or an epiphenomenon? students are quickly engaged in discussions about such interesting questions. the challenge for educators is to help them think critically about the questions, without getting prematurely discouraged by the substantial challenges of forming and testing evolutionary hypotheses about disease vulnerability. for many questions, such as the adaptive significance of menopause, the unknowns are so substantial that consensus remains elusive despite extensive research. some may see this as a reason to teach simpler topics. however, students already get plenty of experience memorizing the answers to scientific questions. topics in evolutionary medicine give them an opportunity to grapple with current questions. students will tend to latch onto a favorite hypothesis and try to defend it. this gives educators an opportunity to emphasize the importance of considering all possible hypotheses, and strategies for determining which are correct and which are false. in contrast to proximate studies, evolutionary questions often have more than one answer, in the sense that multiple factors may be involved in accounting for a trait. emphasizing this can help students recognize the need to consider multiple possibilities. in short, topics in evolutionary medicine are well suited to advancing a major goal of modern education-helping students to learn how to formulate hypotheses, how to go about the scientific process of testing them, and how to cope with the inevitable complexity and confusion that attend almost any scientific endeavor. some of the more common errors are summarized in an article that lists questions worth asking for any evolutionary study of disease (nesse ) . human populations span a tremendous diversity of environments, and our ability to inhabit such a broad range of habitats results in part from genetic adaptations. the availability of genome-wide data on genetic variation from diverse populations offers unprecedented opportunities to identify the loci responsible for these adaptations and elucidate the genetic architecture of human adaptive traits. some of the earliest and most convincing evidence of adaptation to the environment comes from correlations between phenotypes and environmental variables. classic examples of these patterns include correlations between sickle cell anemia, β-thalassemia and malaria endemicity (haldane ; allison ) , body mass and temperature (roberts ; katzmarzyk and leonard ) , skin pigmentation and solar radiation (jablonski and chaplin ) , and lactase persistence and dairying (simoons ) . for several of these examples, the genetic variants underlying these patterns are now known (enattah et al. ; lamason et al. ; sulem et al. ; tishkoff et al. ; piel et al. ). using genome-wide data, it is now possible to turn this approach around to scan the human genome to identify genetic variants that correlate with predicted environmental selective pressures, even without knowing the phenotypic adaptations that may drive the associations between specific alleles and the environment. association studies of this kind have been carried out for variation related to climate, subsistence, diet, and pathogen diversity (fumagalli et al. ; hancock et al. ; hancock et al. ) . integrating results from these environmental genome scans with studies of genotype-phenotype association and other sources reveals the relationships between disease risk and adaptation to the environment. for example, scans show that alleles correlated with diet and subsistence influence folate and energy metabolism. in addition, among genes with strong correlation with climate, many have alleles or genotypes implicated in traits such as pigmentation phenotypes and other aspects of ultraviolet radiation response, cardiovascular disease, immune response, and cancer. together with ongoing efforts to identify variants associated with disease phenotypes in diverse populations (rosenberg et al. ) , environmental genome scans and scans for adaptive genetic loci can identify the genetic variants that underlie differences in disease susceptibility among populations (e.g., nielsen et al. ; pickrell et al. ; grossman et al. ). it is repeatedly stated in medical practice that each case is unique and should be treated individually, but in most cases the underlying factors determining such variation remain poorly understood. part of the answer however may lie in our genes and thus in our evolutionary history. for example, broad patterns of population structure among humans are largely the result of ancient demographic events. the bottleneck during the out of africa exodus of modern humans about , years ago contributed substantially to reduced heterozygosity in individuals living outside of africa and remains the most remarkable demographic imprint reflected in their genomes (cavalli-sforza and feldman ) . in contrast, fine-scale population structure stems from recent demographic processes, reflecting local mating within popu-lations living in geographically circumscribed locations. the impact of inbreeding on the frequency of rare diseases has been demonstrated in historically isolated groups, such as the ashkenazi jewish population (reich and lander ) . if geographic regions throughout the world are finely structured, as suggested by global genomic surveys (auton et al. ), it may be predicted that populations in some regions harbor disease-risk alleles that are population specific and otherwise rare in humans. for example, acuña-alonzo et al. ( ) recently reported that a novel cholesterol transporter abca gene variant (r c, rs ) is exclusive to native americans and has been associated with low levels of high-density lipoprotein (hdl), obesity and type diabetes in admixed populations in mexico (villarreal-molina et al. ) . the authors also identified signatures of positive natural selection around the abca variant, suggesting that r c carriers could have had a selective advantage during the initial peopling of the americas. because the c abca protein shows decreased cholesterol efflux, the presence of this variant could favor intracellular cholesterol and energy storage, conferring an advantage during severe famine periods. however, under current westernized lifestyle, this allele may have become a major susceptibility allele for low hdl-c levels and other metabolic traits. it becomes clear that genetic variation observed today, including medically relevant variation, is the consequence of the interplay between different evolutionary forces acting over time during human population history. therefore, medical students would benefit from learning evolution theory, as it will allow them to better understand genomic variation and its implications in health and disease. cancers account for almost % of all human deaths worldwide (jemal et al. ) , representing a major medical problem for the modern world. it is not a single disease, but an ensemble of diseases with common principles. all cancers harbor groups of cells that show uncontrolled growth, invasion of neighboring tissues, and metastasis to distant organs (hanahan and weinberg ) . ultimately, most cancers cases result in death. cancer arises via accumulation of mutations and selection-a case of aberrant evolution of somatic cells during the lifetime of the host, although instances of transmissible cancer are also known (murgia et al. ) . although somatic cells in healthy individuals often harbor many mutations without any apparent consequences (de ) , cancer cells carry specific mutations that result in immortality, uncontrolled growth, and the ability to metastasize (hanahan and weinberg ) . many of the key mutagenic processes that are active in cancer cells also operate in germ-line evolution, which together with selection, gives rise to population-and species-level divergence in genomes over thousands or millions of years. moreover, many of the genes and pathways associated with cancer are also present in other organisms such as yeast or fruit flies. for instance, the first discovery of a class of cancer genes, the rat sarcoma (ras) family, was in rat (harvey ; kirsten et al. ) . therefore, much can learned about the mechanistic bases of cancer, and we may exploit weakness of cancer cells to develop better diagnostic and prognostic strategies by borrowing knowledge from germ line evolution. genetically modified animals provide insights into cancer-related biological processes; and even today we test new cancer drugs on mice for efficacy and side effects before prescribing them to human patients. we are now moving into an era of personalized diagnostics and treatment, which will require low cost, rapid analysis using more minimal biological samples more than ever before. evolutionary medicine can help to achieve these goals by translating knowledge from evolution into clinical strategies. box . public health and evolution. gilbert s. omenn. public health courses are emerging as popular undergraduate offerings, especially at universities with schools of public health. evolution has shaped burden of disease in the modern world in which we practice public health (omenn ) . human cultures and technologies have modified life on planet earth and have coevolved with myriad other species, including microorganisms, plant and animal sources of food, invertebrate vectors of disease, and intermediate bird, mammal, and primate hosts. molecular mechanisms of evolution have produced differential resistance or susceptibility to infectious agents, including malaria, plague, smallpox, tb, measles, and diarrheal and respiratory diseases. the domestication of sheep and cattle has selected for humans able to digest milk throughout life through persistence into adulthood of lactase enzyme expression in the intestine, a major story of anthropology (itan et al. ). the emergence of a "western diet" of dairy, refined cereal grains, refined sugars, vegetable oils, alcoholic beverages, salt, and omega- -rich meats has dramatically altered glycemic load, fatty acid composition, macronutrients, acid-base balance, sodium/potassium ratio, and fiber content. the results include epidemics of atherosclerotic cardiovascular disease, obesity, diabetes, high blood pressure, osteoporosis, certain cancers, and bowel, inflammatory, and autoimmune disorders (cordain et al. ) . a newly recognized phenomenon is the selection of excessive hemostatic activity from platelets and the plasma-clotting proteins; what was protective against death from bleeding after injuries among hunter-gatherer, warring humans, or against pregnancy-related hemorrhage now contributes to thrombosis underlying heart attacks and strokes. conversely, there is little pressure against hemostasis and thrombosis because deaths resulting from blood clots and coronary disease associated with hemostasis and thrombosis occur mostly after the reproductive years of life (coller ) . learning about evolution over millennia for humans and over hours or days for microbes enlivens the experience of understanding evolution in public health context. synthesis of data from multiple disciplines has led to hypotheses for understanding the evolutionary-genomic underpinnings of autism spectrum conditions and psychotic-affective spectrum conditions, mainly schizophrenia, bipolar disorder, major depression, and borderline personality disorder (crespi and badcock ) . the synthesis integrates inclusive fitness theory, comparative primatology, and developmental psychology with information from mouse gene knockouts, human genomic disorders, psychiatric genetics, and human and mouse gene expression. the primary hypotheses focus on autism and psychoticaffective conditions as diametric disorders mediated by opposite alterations to the development of human-elaborated social-brain and sexual-brain phenotypes. by this hypothesis, social-brain architecture develops predominantly in the context of mother-child interactions, whereby early, basic mother-child attachment, underlain in part by paternally expressed imprinted genes, serves as a scaffold for development of the highly social, neocortical brain. in turn, development of the highly social neocortical brain is mediated in part by effects of maternally expressed imprinted genes. alterations to the expression of imprinted genes (genes that are silenced under inheritance from one or the other parent, and that evolve under genomic conflict, (see haig [ ] ) play important roles in early social-brain development, with deviations toward relative paternal-gene expression increasing risk of autism. deviations toward relative maternal-gene expression increase risk of psychotic-affective spectrum conditions (crespi and badcock ; crespi ) . autism and psychotic-affective conditions are thus hypothesized to arise in part through epigenetic variation that influences maternalpaternal genomic conflicts. more generally, the human social brain may underdevelop in childhood, manifesting as autism spectrum conditions, or selectively and relatively over-develop, usually in adolescence or early adulthood, leading to psychotic-affective spectrum phenotypes and conditions. cognitive and affective variation between females and males strongly modulates liability to these conditions. thus, major epidemiological and phenotypic features of autism and psychotic-affective conditions can be predicted from interactions between a malefemale brain continuum and a maternal-paternal imprinting bias continuum (crespi and badcock ) this conceptual framework spans two levels. first, the diametric nature of autism versus psychotic-affective conditions is modulated by multiple genetic, genomic, epigenetic, and social-environmental causes based in human brain evolution. second, this diametric nature may arise from imprinted genes, which can be dysregulated in two opposite directions. the framework makes novel, testable predictions with direct implications for cognitive-behavioral and pharmacological therapy, human evolution, the molecular evolution of psychiatric risk genes, and human brain development. one important prediction, that imprinting is relatively common among genes that underlie risk of autism and schizophrenia, is supported by analyses of data from a recent genome-wide study of imprinted genes in mice (gregg et al. ). box . can any organisms be potentially immortal? staphen c. stearns. weismann ( ) suggested that only organisms with a soma and germ line would age, and that organisms that divided symmetrically would not age. recent experiments have shown just how precisely symmetric that division would have to be. their results suggest that all known organisms must age, for even a bacterium like e. coli divides into two nonidentical cells, one of which contains older parts than the other and ages faster (stewart et al. ) . the lineages with the old parts suffer a fitness disadvantage and disappear, and the lineages with the young parts persist. the goal of potential immortality (barring accidents) may also be unattainable if another basic claim of the evolutionary theory of aging remains unrefuted: aging and death result not from a single mechanism that can be repaired but from the diffuse degradation of multiple processes across the entire genome and body (williams ) . repair of one problem will simply reveal the next in line, and that process will repeat hundreds or thousands of times. the byproducts of repair are also expected to connect through trade-offs to costs paid in other traits. recently, some genes with major effects on aging have been discovered in other organisms, but they are involved in trade-offs with reproductive performance early in life, and whether those trade-offs can be compensated remains to be seen (stearns a) . the evolutionary view of aging is not optimistic about life-extending genetic therapies, but it may provide some needed wisdom about the human condition. box . why does antibiotic resistance persist after antibiotic use stops? carl t. bergstrom. the evolution and spread of antibiotic resistance powerfully illustrates the basic process of natural selection. bacterial populations manifest variability in antibiotic susceptibility; some of this variation is heritable; in the presence of antibiotics, less-susceptible strains survive and reproduce more rapidly. the inevitable result-as we have seen with every antibiotic yet introduced-is the evolution of resistance in bacterial populations (genereux and bergstrom ) . a related phenomenon offers an opportunity to teach more subtle aspects of evolutionary biology and population genetics (bergstrom and dugatkin ) . when antibiotic use is terminated in a community, what happens to antibiotic resistance in the bacterial populations? given that antibiotic resistance commonly imposes some cost to bacteria that express it, we might predict that when a particular antibiotic is discontinued, resistance to that antibiotic should decline. although this sometimes occurs (e.g., seppälä et al. ) , it is by no means guaranteed. sometimes resistance to the drug persists long afterward. one example is sulfonamide resistance in great britain, which has not declined even though sulfonamides were essentially discontinued in the mid-nineties (bean et al. ) . why? a number of multilocus evolutionary processes may contribute, including the phenomenon of associated linkage selection (levin et al. ) . because bacteria have haploid genomes and limited opportunities for recombination, most bacteria exhibit strong linkage along the entire circular chromosome. as a result, hitchhiking and related processes are very important. in the process of associated linkage selection, resistance to a new drug arises on a chromosome or plasmid carrying resistance alleles for older drugs. this generates positive linkage disequilibrium between the new and old resistance alleles. even if an older drug is discontinued, the resistance alleles to this drug may be indirectly favored by selection for resistance to the newer drug. associated linkage selection can thus explain why older resistant strains are not outcompeted by unrelated sensitive strains: those sensitive strains are less likely to carry resistance to whatever new drugs are currently in use. but associated linkage selection does not explain how resistant strains are able to avoid being replaced by mutants that retain resistance to the new antibiotic but revert to sensitivity to the old one. to make sense of this, we turn to another phenomenon: compensatory mutation. although resistance mechanisms impose fitness costs, compensatory mutations can ameliorate these costs and restore fitness of a resistant strain-in the absence of the antibiotic-to near that of the sensitive strain (schrag and perrot ; andersson and hughes ) . an obvious consequence is that compensatory mutations reduce the selective difference between resistant and sensitive strains in the absence of antibiotic use, and thus slow the rate at which sensitive strains are able to replace resistant ones. many compensatory mutations have a more subtle effect as well. they make it difficult for bacteria to revert to full sensitivity, by creating a fitness valley between the resistant, compensated strain and the sensitive uncompensated strain (levin et al. ) . this occurs because the compensatory mutation, while beneficial in the presence of the resistance mutation, is deleterious in its absence. because it is easy to "get in" to the resistant compensated state in the presence of the antibiotic but difficult to get out in its absence, this phenomenon has been described as an evolutionary lobster trap (tanaka and valckenborgh although the negative impacts of rapid microbial evolution are well understood, humanity has turned the evolutionary lability of viruses into a means of fighting disease. "applied evolu-tion" has been used to generate many live-attenuated virus vaccines, including those against measles, mumps, rubella, varicella (chickenpox), influenza, and poliovirus (fda ). in this classical approach, pioneered by pasteur in his effort to create a rabies vaccine, wild-type virulent viruses are serially passaged in a novel host or in a novel temperature regime. the resulting adaptations to the new environments results in loss of fitness in the original host (plotkin ) . a successfully attenuated virus shows decreased virulence in humans while retaining the ability to stimulate an immune response. this process can require as few as passages, as for the varicella vaccine (takahashi et al. ), or hundreds, as was needed for the yellow fever vaccine (monath et al. ) . however, one of the drawbacks of classical vaccine design is that the phenotype of the virus, that is, its attenuation, determines genotype. thus, although it is clearly desirable to secure attenuation by multiple or large attenuating mutations (burch et al. ) , the mutations responsible for attenuation of many liveattenuated virus vaccines are either unknown or, in the case of the three strains of the oral poliovirus vaccine (opv) (kew et al. ) , few in number. predictably, ∼ in every , children receiving their first dose of opv experience vaccine-associated paralytic polio due to reversion of virulence in the vaccine (kew et al. ) . revertant strains may be transmissible between humans and thus threaten the success of the global effort to eradicate polio. in contrast, the nasal influenza vaccine carries attenuating mutations on four of its eight genomic segments, and to date has never reverted to virulence within a vaccinee (tosh et al. ) . fortunately, reverse genetics (rational vaccine design) informed by evolutionary thinking suggest novel approaches to safeguard the attenuation of live vaccine viruses. for example, vignuzzi et al. ( ) have attenuated poliovirus by enhancing the fidelity of its rnadependent rna polymerase and thereby slowing its mutation rate. because this virus is less likely to mutate, it is less likely to revert to a less-faithful, and more fit, polymerase. finally, live vaccines may not only evolve themselves but may also shape the evolution of their wild-type counterparts. there is evidence that some veterinary vaccines are driving the evolution of antigens in their wild-type targets (park et al. ), but the impacts of such evolution on virulence and transmission are not yet known. at the extreme, vaccines have the potential to eradicate their wild-type targets, potentially creating an empty niche into which new viruses may emerge from human or zoonotic reservoirs (rieder et al. ; rimoin et al. ; vasilakis et al. ) . evolution, medicine, and the darwin family evolution by any other name: antibiotic resistance and avoidance of the e-word global distribution of genomic diversity underscores 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evolutionary medicine and health: new perspectives fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health engineering attenuated virus vaccines by controlling replication fidelity association of the atp-binding cassette transporter a r c variant with early-onset type diabetes in a mexican population a mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine ueber die dauer des lebens pleiotropy, natural selection and the evolution of senescence the dawn of darwinian medicine key: cord- -fb rtmx authors: joseph, maxwell b.; mihaljevic, joseph r.; arellano, ana lisette; kueneman, jordan g.; preston, daniel l.; cross, paul c.; johnson, pieter t. j. title: taming wildlife disease: bridging the gap between science and management date: - - journal: j appl ecol doi: . / - . sha: doc_id: cord_uid: fb rtmx . parasites and pathogens of wildlife can threaten biodiversity, infect humans and domestic animals, and cause significant economic losses, providing incentives to manage wildlife diseases. recent insights from disease ecology have helped transform our understanding of infectious disease dynamics and yielded new strategies to better manage wildlife diseases. simultaneously, wildlife disease management (wdm) presents opportunities for large‐scale empirical tests of disease ecology theory in diverse natural systems. . to assess whether the potential complementarity between wdm and disease ecology theory has been realized, we evaluate the extent to which specific concepts in disease ecology theory have been explicitly applied in peer‐reviewed wdm literature. . while only half of wdm articles published in the past decade incorporated disease ecology theory, theory has been incorporated with increasing frequency over the past years. contrary to expectations, articles authored by academics were no more likely to apply disease ecology theory, but articles that explain unsuccessful management often do so in terms of theory. . some theoretical concepts such as density‐dependent transmission have been commonly applied, whereas emerging concepts such as pathogen evolutionary responses to management, biodiversity–disease relationships and within‐host parasite interactions have not yet been fully integrated as management considerations. . synthesis and applications. theory‐based disease management can meet the needs of both academics and managers by testing disease ecology theory and improving disease interventions. theoretical concepts that have received limited attention to date in wildlife disease management could provide a basis for improving management and advancing disease ecology in the future. population density decreased due to disease, contact rates would become too low for transmission to continue; thus, pathogens would be extirpated before host populations (anderson & may ) . however, disease-induced declines and extinctions of wildlife resulting from small population sizes, reservoir hosts, host switching and heterogeneity in contact rates, susceptibility and transmission within and among populations have forced a re-evaluation of this perspective (de castro & bolker ) . for example, when contact rates among individuals do not depend on host density, pathogens are more likely to drive populations to extinction because transmission continues as host populations are reduced, as seen with tasmanian devil sarcophilus harrisii (boitard ) facial tumour disease where transmission appears to be related to mating behaviours (mccallum ) . white nose syndrome in bats also seems to be more likely to cause extinctions owing to social behaviours in which hosts cluster in hibernacula, reducing the correlation between contact rates and population densities (langwig et al. ) . in recent decades, wildlife disease management (wdm) has been increasingly used to conserve threatened wildlife populations (deem, karesh & weisman ) . for example, wdm has controlled outbreaks of feline leukaemia in critically endangered iberian lynxes lynx pardinus (temminck ) and rabies in endangered ethiopian wolves canis simensis (r€ uppell ), both of which are associated with domestic animal disease reservoirs (haydon et al. ; l opez et al. ). despite the wealth of empirical wdm research, management outcomes can be difficult to predict because system-specific information is lacking for novel pathogens and many theoretical concepts in disease ecology (see table for a subset) have not been widely tested in the field, leading to uncertainty in their generality. this is unlike other environmental management disciplines such as fisheries ecology, which has effectively used theoretical models to predict yields, manage harvest timing and limits and design reserves (e.g. gerber et al. ) . indeed, theoretical applications in fisheries ecology have also produced insights into density-dependent population dynamics, metapopulation theory and the evolution of life-history table . selected theoretical concepts in disease ecology: theoretical concepts in disease ecology theory that apply to wildlife disease management, some direct management implications and a theoretical reference for each concept theoretical concepts management applications selected references host population regulation by disease disease reductions may increase host abundance and/or survival anderson & may ( ) trade-offs between transmission and virulence artificial stocking may increase virulence, and culling may reduce or increase virulence depending on pathogen life-history, culling selectivity and transmission dynamics frank ( ) seasonal drivers of disease emergence and dynamics intervention timing and frequency matters; control efforts can target transmission peaks altizer et al. ( ) pathogen interactions within hosts managing one pathogen alters the transmission and virulence of other pathogens fenton ( ) multi-host species disease dynamics reservoir hosts can drive the extinction of alternate hosts; rates of interspecific transmission may be inferred by managing one host species; management may need to target multiple host species dobson & foufopoulos ( ) spread of disease in spatially structured hosts corridor vaccination can reduce disease in metapopulations; movement controls are unlikely to work for chronic infections keeling & eames ( ) transmission increases with host density host density reductions may reduce disease transmission, and density thresholds for disease persistence may exist anderson & may ( ) transmission increases with disease prevalence independent of host density transmission associated with sexual interactions is more likely to cause host extinction, and non-selective culling may not reduce transmission getz & pickering ( ) predation as a regulator of host population and disease predator conservation may reduce disease in prey populations packer et al. ( ) community composition, diversity and disease risk biodiversity loss and community disassembly may increase disease as predators and less-competent hosts are extirpated, depending on community composition and transmission dynamics keesing, holt & ostfeld ( ) environmental reservoirs and indirect transmission duration of disease control must scale with the environmental persistence; host extinction is more likely joh et al. ( ) individual-level variation and superspreading heterogeneity in individual resistance and infectiousness within a host population can lead to 'superspreaders' that account for a large portion of transmission; management can target superspreaders lloyd-smith et al. ( ) strategies (frank & leggett ) . in this review, we assess the extent to which a similar union between theory and practice has been achieved in wdm. we use a quantitative, case-based approach to provide a critical retrospective of wdm over the last four decades to: (i) quantify how frequently specific theoretical concepts from disease ecology have been applied in the literature, (ii) identify prevailing management objectives, groups and reported outcomes and (iii) assess taxonomic biases in wdm literature. we then present methodological and conceptual opportunities to facilitate the newly emerging synthesis of disease ecology and management, drawing from environmental management and biomedicine to outline steps towards more cost-effective, efficacious and informative wdm. this synthesis aims to facilitate the development of a more predictive framework for disease interventions while simultaneously building empirical support for understanding of disease processes across systems. we compiled wdm case studies using a systematic, twostep search process with specific criteria for inclusion in our review. in the first stage, we searched titles and abstracts of records included in isi web of science using specific terms [(wild*) and (disease* or infect* or pathogen* or parasit*) and (manage* or conserv*)] to capture breadth in published wdm records. additionally, we searched for case studies in grey literature using the following online resources: national wildlife health center, wildpro, national biological information infrastructure wildlife disease information node, u.s. government printing office and the u. s. fish and wildlife service. no case studies identified in the grey literature met our criteria that were independent of cases identified in the scientific literature. case studies were also identified using previous review papers and books (lafferty & gerber ; wobeser ; hudson et al. ; wobeser ; ostfeld, keesing & eviner ) . we conducted a follow-up search with isi web of knowledge to capture subject depth for each managed disease or pathogen identified in the first step, using a search string that included all pathogen and disease names along with terms related to management interventions: (e.g. (rabi* or lyssavir*) and (vaccin* or treat* or manag* or control* or preval* or incidence or cull*) and (wild* or free-ranging or free ranging). the initial web of science search returned articles dating back to , but our disease-specific search strings often returned results dating back to the s or earlier. historical accounts of wdm are probably under-represented in the literature available online, and those returned by our search strings were often less readily accessible than recently published articles. as a result, the cases reviewed here primarily represent recently published cases of wdm. the publication dates of included cases range from to , and % of the cases included in our review were published after . for each article that met our criteria, we recorded (i) pathogen and host characteristics, (ii) management motivations, strategies and outcomes and (iii) whether and how disease ecology theory was incorporated in each article that satisfied our criteria. we only included cases that provided quantitative data on disease in a population or area (number of cases, seroprevalence, prevalence, incidence, etc.). when multiple records were encountered for a single management event, we used the most recent record (as of spring ). cases that only described disease management in humans, livestock or plants were excluded. finally, we only included studies that described management of diseases in populations (operationally defined as groups of > individual) of free-ranging wildlife. incorporation of disease ecology theory was defined broadly as the explicit use or discussion of theoretical concepts relating to transmission dynamics, host population regulation by disease, pathogen evolution, host or pathogen community effects on transmission, spatial heterogeneity in disease dynamics, life stage-or age-specific disease dynamics, endemic vs. epidemic disease states and herd immunity (see table for a list of specific concepts used to define theory in the literature search). four broad management objectives were identified, including conservation of a host species, prevention of disease transmission to humans, prevention of disease transmission to livestock and basic research. studies falling into our basic research category were usually an attempt to better understand the system, determine the extent of the disease problem or provide insight into future management opportunities. to investigate differences in theory application and objectives among managing groups, we also classified author affiliations for each paper as academic, governmental, private or some combination thereof. university or university laboratory affiliations were considered academic, and we used the same criteria for governmental and private affiliation. mixed author affiliations (e.g. academic and governmental) were recorded for individual authors and for papers with multiple authors with different affiliations. we characterized management outcomes by recording whether the disease was eradicated, and if not, whether there were changes in the prevalence, incidence or intensity of disease. ideally, these changes could be quantified and compared across disease systems, but in many cases, inconsistent reporting of results and a lack of pre-management or control data complicate meaningful quantitative comparisons of effect sizes across studies. finally, we considered whether the original management objective was attained using the following categories: 'apparent success', meaning that there was no unmanaged control population or area to compare to the treated area; 'partial success', meaning that at least some of the management objectives were reported as fulfilled; and 'success,' for cases that had controls and reported fulfilment of all management objectives. while management outcomes are rarely clear-cut in this practice, this simplified classification system facilitated coarse comparisons across disease systems and among management studies with variable monitoring time-scales. in total, scientific articles among the identified from the search strings satisfied our criteria (see appendix s and table s in supporting information). many ( %) cases consisted of collaborations between government agencies and academic researchers (fig. ) . conservation motivated % of management that involved private groups, whereas basic research was only conducted when academics were involved. overall, host conservation was the most common objective ( % of cases), while reducing disease risk to humans and domestic animals were the next most common objectives ( % and % of cases, respectively; fig. ). disease ecology theory as defined above has only recently been incorporated consistently into wdm literature (fig. a) . some theoretical concepts such as density dependence in transmission were frequently applied, while others such as pathogen evolution and the role of predators and biodiversity in regulating disease were not (fig. , table ). unexpectedly, papers authored by academics were not more likely to incorporate theory (fisher's exact test, p = Á ). management outcomes were related to theory incorporation (fisher's exact test, p = Á ). the three papers that reported disease increases following intervention explained their results in terms of disease ecology theory, providing insights into transmission and optimal control strategies (e.g. cross et al. ; fig b) . however, there was no relationship between management objective attainment and theory incorporation (fisher's exact test, p = Á ). nevertheless, some counter-intuitive but successful management distribution of management outcomes according to whether disease ecology theory was incorporated. reductions and increases refer to changes in prevalence, incidence, infection intensity or diseaseinduced mortality; eradication refers to local rather than global eradication. programmes clearly benefited from theory. for example, control of classical swine fever in wild boar sus scrofa (linnaeus ), is often hampered by stage-dependent transmission dynamics. susceptible piglets are hard to target with baited vaccines and act as disease reservoirs. by allowing an epidemic to peak such that most adults are immune, then culling only piglets, swiss academics and governmental groups successfully eradicated the disease from a -km region near the italian border (schnyder et al. ) . reductions in prevalence, incidence or infection intensity were reported in % of cases, with vaccination and host treatment as the most commonly applied intervention strategies (fig. b) . ninety-four percentage of cases reported management in terrestrial systems, with % and % of cases reporting management in freshwater and marine systems, respectively. the majority ( %) of reported management efforts were directed towards mammals, with birds and fish representing % and % of cases, respectively. however, mammals are less speciose and less threatened by disease than amphibians (vi e, hilton-taylor & stuart ), for which we found no published wdm records. taxonomic bias could arise because vaccines and drugs are developed primarily to protect human, livestock or poultry health. relatively few cases ( %) reported a failure to meet management objectives, possibly due to negative publication bias. collectively, our analyses indicate that while academics and government agencies collaborate to manage wildlife diseases, collaborations do not necessarily lead to an integration of disease ecology theory with management. density-dependent transmission was often assumed to justify control efforts, but other theoretical concepts were rarely applied (fig. ). data quality issues and potential publication biases currently hinder the application of metaanalytical techniques for wdm, and there is a paucity of published records on non-mammalian management. overcoming challenges to theory-based management while collaboration alone may not necessarily lead to an integration of disease ecology theory and wdm, it should provide a starting point for such integration. academics and managers have unique needs, constraints and knowledge-seeking behaviour that challenge such collaborations. for instance, untreated control areas or pre-treatment data can be unavailable or even unethical in wdm, but are critical for experiments in disease ecology. while academics may design field experiments to test and refine theoretical models, managers need practical, effective and uncontroversial management strategies that succeed in particular systems. such strategies may not be easily identified in the literature from model systems, which managers may be unable to access. modelling wildlife disease systems requires decisions about model complexity. in our experience, theoreticians prefer simpler, more general models that may be applicable to many systems. these models are easier to parameterize and analyse, and the resulting papers are likely to have a wider academic audience. on the other hand, simple models are easily discarded by managers because they lack system-specific detail. this tension is likely to continue, but we recommend additional flexibility on both sides. in particular, managers should appreciate that the addition of modelling details that are only weakly supported by data may not lead to better predictions. meanwhile, theoreticians may develop general models that bear little resemblance to any biological system. furthermore, individuals may be most interested in a particular suite of theoretical concepts, but a narrow approach can impede management by ignoring the full range of phenomena relevant to producing desired management outcomes (driscoll & lindenmayer ) . thus, academics and managers are challenged to take a broad view that incorporates relevant theoretical concepts and an appropriate amount of biological realism, which may require collaboration among researchers with different areas of expertise (driscoll & lindenmayer ) . unfortunately, such large collaborative efforts may bring a loss of autonomy at odds with academic or governmental bureaucracy. a diverse body of literature addresses the gap between academics and environmental managers and provides examples of successful strategies for integration. for instance, international symposia have improved information transfer in invasion biology (shaw, wilson & richardson ) . social networking, joint appointments, interinstitutional sabbaticals, fellowships, concise reporting table , and their application in the literature was included in this review, showing that some concepts such as densitydependent transmission are well represented, while others were less frequently (or not at all) applied. of relevant science to managers and targeted calls for research proposals by managers can all help to foster cooperation (gibbons et al. ) . interdisciplinary working groups for particular management issues can ensure that the needs of multiple stakeholders are considered together when organizing such activities (gibbons et al. ) . groups such as the wildlife disease association and applied journals including the journal of wildlife diseases have encouraged interdisciplinary collaboration, and a broader recognition of the complementarity between disease ecology theory and wdm can provide the impetus for expanding interdisciplinary work in this important field. theory can help address unprecedented management challenges and can be refined in the process. disease outbreaks are often caused by novel pathogens or the appearance of known pathogens in new hosts. often, details of host-pathogen interactions are unknown. by combining limited information with general principles of disease ecology (table ) theory is often refined by evaluating competing hypotheses. therefore, adaptive management is one way to integrate theory and management, especially if multiple management hypotheses can be tested (holling ) . differentiation among competing hypotheses is synonymous with identifying optimal management in this framework. thus, monitoring the effects of disease interventions on prevalence, virulence and host vital rates can help to estimate model parameters including transmission and recovery rates and help in evaluating management outcomes. when agencies have limited flexibility in decisionmaking, thus precluding adaptive management, the best available theoretical and system-specific knowledge can at least produce a 'best guess' management strategy (gregory, ohlson & arvai ) . failed management is still valuable in this framework because outcomes can be compared to predictions from competing models of disease dynamics that can be selectively eliminated, as with tasmanian devil facial tumour disease (mcdonald-madden et al. ). this approach produces mechanistic insights that might be missed if management strategies are characterized simply as effective or ineffective based on management outcomes. if many groups apply adaptive management separately in similar systems without communicating, generalities that benefit management and disease ecology may remain elusive. systematic reviews, invaluable to biomedicine, can help establish which interventions are effective and explain heterogeneity in effectiveness with a standardized meta-analytic approach. guidelines for systematic reviews in environmental management exist, but have not been applied in wdm (sensu pullin & stewart ) . our metadata indicate that this may be due to a lack of data quality and quantity. simple recommendations to facilitate the production of data suitable in quality for systematic review include: (i) establishing unmanaged control areas and/or baseline data, (ii) achieving replication, (iii) reporting precision for estimates of model parameters, prevalence and effect size, (iv) publishing and mechanistically explaining failed management and (v) reporting the spatiotemporal extent of management. data quantity may be lacking because of publication biases and a lack of incentives for managers to publish when working independently. this latter issue is minor if collaborations involve academics, but even motivated scientists may have difficulty publishing if management has no effect. however, management failures are as important to report in the literature as successes for systematic reviews and meta-analyses. an evaluation of the applicability of a theoretical concept in a particular case will rely on comparisons of observed data with explicit predictions from theoretical models, which can often be derived through mathematical modelling. theoretical concepts can be explicitly built into systemspecific mathematical models to identify and evaluate management strategies, as exemplified in a modern wdm challenge: chronic wasting disease (cwd). in , the state of wisconsin began culling white-tailed deer odocoileus virginianus (zimmermann ) and lengthened the hunting season in an attempt to eradicate cwd. these efforts were mandated despite uncertainty over transmission dynamics, the environmental persistence of prions that cause cwd and the time of cwd arrival to the state (bartelt, pardee & thiede ) . five years later, prevalence was still slowly increasing (heisey et al. ) . as this epidemic has unfolded, several models have been used to describe the dynamics of cwd (gross & miller ; wasserberg et al. ; wild et al. ) . simple models of cwd do not tend to produce plausible results. purely density-dependent transmission models predict increases in prevalence that are too rapid, while frequencydependent transmission models predict rapid host extinction or epidemics that are very slow to develop (on the order of centuries). modelling indirect transmission via environmental contamination results in a wider range of outcomes and produces several patterns observed in the field including a slow disease progression with prevalences of over % and significant host population reduction without rapid extinction (almberg et al. ) . recent analyses did not provide much support for models with variable increases in transmission over models with variable starting prevalence, suggesting that host density effects may be relatively weak in this system (heisey et al. ) . taken together, these results suggest that managers would have to reduce deer densities to extremely low levels, probably for decades, at which point other stakeholders such as hunters may wonder whether it is worse to have a lower deer density due to disease impacts or disease control efforts. disease ecology theory is not a 'silver bullet' for solving management problems. indeed, some have pointed out that application of theory under certain circumstances can lead to poor management (driscoll & lindenmayer ) . misapplication of theory at an inappropriate scale, or in a system that does not meet necessary assumptions, could cause undesired consequences. for instance, an assumption of broad-scale culling as a disease management intervention is that pathogen transmissions scale positively with host population density. however, density-dependent changes in social behaviour can alter dispersal patterns that violate this assumption, increasing transmission, as seen with bovine tuberculosis (tb) in cattle and european badgers meles meles (linnaeus ) (woodroffe et al. ) . work in the badger-tb system has refined our understanding of the effects of culling on social animals. however, one could argue that if culling-induced dispersal had been discovered in another disease system, the unintended increase in tb transmission to cattle following badger culling might have been avoided. unfortunately, had this been the case, the applicability of the social perturbation-transmission increase phenomenon to the badger-tb system would have remained uncertain. this underscores the value of moving beyond a case studydominated paradigm, towards a rigorous and empirically verified contingency-based understanding of theory applicability to disease management. such a framework could test and refine theoretical concepts that have shown promise in model systems, but have been infrequently applied in the wdm literature (fig. ) . what are the future directions for wdm? recent advances in disease ecology based on co-infection provide new ways to reduce disease susceptibility and transmission. for example, in african buffalo syncerus caffer (sparrman ), gastrointestinal nematodes reduce individual resistance to mycobacterium tuberculosis, which causes bovine tb, because of cross-regulatory immune responses to micro-and macroparasites (ezenwa et al. ) . hence, deworming drugs may increase resistance to tb and improve tb vaccination efficacy, raising the possibility that tb could be managed indirectly through nematode control (elias, akuffo & britton ; ezenwa et al. ) . management involving immunological trade-offs could improve general understanding of immune-mediated parasite interactions. for instance, interventions aimed at helminth parasites, which accounted for % of cases in our review, are predicted to differentially affect microparasite transmission depending on recovery rates and virulence (ezenwa & jolles ) . these predictions could be evaluated opportunistically by monitoring non-target pathogens. similarly, management in systems with co-infecting parasites could be used to understand virulence evolution in response to changing co-infection dynamics (alizon & van baalen ) . there is increasing recognition that microbial symbiosis can play a role in host health. using mutualistic microbes to control disease, a technique known as probiotics therapy, has benefitted aquaculture, agriculture and human medicine. for example, addition of bacillus and pseudomonas bacteria controls pathogenic vibrio that infect prawns, salmon and crabs in aquaculture (irianto & austin ; panigrahi & azad ) . bifidobacterium and lactobacillus can ameliorate escherichia coli infection in pig farms (zani et al. ; shu, qu & gill ) . in humans, probiotics can treat diarrhoea caused by clostridium difficile infection and antibiotic therapy (mcfarland ; rohde, bartolini & jones ). probiotics may prove useful for wdm. frogs with certain skin bacteria may be less susceptible to population extirpation caused by chytridiomycosis, a fungal disease that implicated global amphibian declines (lam et al. ) . experimental augmentation of skin bacteria reduces mortality of susceptible amphibians in captivity, and field experiments are underway to determine whether probiotics can prevent extirpations in nature (harris et al. ; rex ) . probiotics can also reduce vector populations. for instance, laboratory-reared mosquitoes with a maternally heritable probiotic that disrupts dengue fever virus transmission can locally replace wild mosquito populations and reduce dengue fever risk (hoffmann et al. ) . the successful use of probiotics depends on an understanding of microbial ecology, especially with respect to long-term probiotic maintenance in a host or environment. risks associated with introducing non-native microbes may be ameliorated by isolating probiotic agents from native hosts. as data accumulate, it will be important to evaluate whether the risks of probiotics outweigh those associated with antibiotic treatment in terms of antibiotic or probiotic resistance and pathogen virulence evolution. finally, linking these within-host processes to among-host processes (e.g. microbial community structure and transmission) is an important frontier for wdm and disease ecology. optimal management strategies depend on the degree to which transmission is driven by host population density and the amount of individual and population heterogeneity in contact or transmission rates. host population size, aggregation patterns and contact rates can be altered through hunting, artificial feeding, predator and scavenger conservation, fertility control, culling, translocation of individuals, artificial stocking, movement barriers, etc. understanding the functional form of the relationships among host contacts, density and transmission in real systems is critical to predicting the impacts of such interventions. therefore, field manipulations will play a crucial role in refining our mechanistic understanding of disease transmission. optimal management strategies are not static; contact rates, host abundance and demography can change naturally over time, in response to disease and due to management. for example, group sizes and contact rates may remain constant for highly social species despite management-induced population reduction. reservoir hosts may increase disease risk for other species if infected individuals maintain high fitness via increased reproductive output (fecundity compensation, for example, schwanz ) . fertility control of such reservoir hosts may protect other species that are less tolerant to infection. lastly, if transmission peaks in a short time period, perhaps due to breeding or a pulsed influx of juveniles (altizer et al. ) , management may be applied optimally in a narrow time interval. brucellosis in the elk (cervus elaphus linnaeus ) populations of the greater yellowstone ecosystem of wyoming illustrates how management can capitalize on temporal transmission dynamics. every year, wildlife managers provide supplemental feed to elk at sites in the region. contrary to theoretical predictions, elk abundance at each feeding site is uncorrelated with brucellosis seroprevalence (cross et al. ), but locally, host contact rates correlate positively with elk density (creech et al. ) . these seemingly contradictory findings are explained by variation and interaction between transmission and host density over time, which suggests that brucellosis seroprevalence may be reduced by shortening the length of the feeding season in early spring when transmission is highest (maichak et al. ). this option is appealing because vaccination has had limited, if any, effect (cross et al. ) , and a testand-remove programme, although effective, is financially prohibitive to implement across a broad region. establishing contact networks for a variety of disease systems across a range of densities and over time will help to identify life-history traits, social structures and other species characteristics that predictably influence transmission. taken together, these population-level tools can advance general understanding of transmission dynamics and optimize the application of disease control strategies. community-level interactions including predation and competition can influence disease management outcomes. predation on hosts can increase or decrease disease prevalence and the likelihood of epidemics depending upon predator selectivity, as well as behavioural and demographic effects on host populations that influence transmission and disease susceptibility (packer et al. ; holt & roy ) . interspecific competition can also influence host background mortality and thus the net effect of disease in a population (bowers & turner ) . unintended consequences when managing predators or competitors may be of less concern if coupled with ongoing management such as predator restoration and invasive species control. interspecific transmission of generalist parasites is hard to quantify, but attempts to control generalist parasites in one host species can reveal the extent to which other hosts contribute to transmission. for example, tsao et al. ( ) vaccinated white-footed mice peromyscus leucopus (rafinesque ) in southern connecticut to reduce the prevalence of borrelia burgdorferi, the bacterium that causes lyme disease. based on the strains of b. burgdorferi found in ticks in vaccinated plots, and the relationships between mouse density and tick infection prevalence, the authors concluded that other host species contributed more to tick b. burgdorferi infections than previously thought. thus, vaccination would have to target multiple host species to be effective. contact prevention between wildlife and livestock also provides an opportunity to prevent disease spillover, and when linked with monitoring of both wildlife and domestic populations, can be used to estimate relative rates of within-and among-species transmission. novel management strategies may target ultimate causes of disease emergence once they have been identified. for instance, lyme disease risk in the north-eastern united states increases with habitat fragmentation, which leads to extirpations of (i) predators and competitors that limit white-footed mouse abundance and (ii) less-competent hosts for b. burgdorferi and ticks (ostfeld & logiudice ) . in this system, biodiversity conservation might be an option for proactive wdm. management interventions that recognize and target community-or ecosystem-level processes are rare, but in some cases may more directly address disease threats than focusing solely on individuals or populations. a black box? common wdm interventions have evolutionary consequences that remain largely unexplored. in contrast, a vast literature in the biomedical sciences describes the effects of vaccination on the evolution of human pathogens. generally, (i) some pathogens tend to evolve vaccine resistance, (ii) imperfect vaccines that confer partial immunity select for increased virulence, and (iii) live attenuated vaccines can revert to virulence if inadequately distributed (anderson, crombie & grenfell ; gandon & day ; mackinnon, gandon & read ) . together, these observations provide strong incentives for an 'all or nothing' approach to vaccine-laden bait distribution programmes, which may jeopardize long-term success if low-coverage field trials using vaccines of limited or unknown efficacy precede full distribution of an effective vaccine. selective culling (analogous to selective predation) whereby managers remove infected individuals from the population to prevent disease spread may also have unintended consequences. it can select for increased virulence, because there are relatively more susceptible hosts available for the pathogen, and pathogens must transmit to susceptible hosts faster to avoid being culled along with their hosts (choo, williams & day ) . in many cases, selective culls are based on serological tests that do not discriminate between recovered and infectious individuals. removal of recovered individuals may actually result in more explosive epidemics later on due to a reduction in herd immunity (ebinger et al. ) . experiments and genetic analyses of wildlife pathogens that are often treated by vaccination or culling could reveal the extent to which these concerns are realized. aside from developing new vaccines, these risks may be mitigated if management capitalizes on immune-mediated parasite interactions, employ probiotic approaches and consider population-and community-level management interventions. the use of multiple strategies (seen in % of our case studies) may provide one means with which to avoid problems such as antibiotic or vaccine resistance resulting from the overuse of any one strategy. a more complete integration of disease ecology with wdm can benefit both disciplines. management provides unique opportunities to test disease ecology theory while building system-specific understanding. by evaluating management outcomes in terms of theory, managers can better identify effective strategies even in the face of management failures. we have presented specific recommendations, methodological tools and conceptual approaches to achieve a stronger integration of theory and practice, which we hope will facilitate the development of a strong predictive framework for wdm. the generality of this framework is currently limited by the lack of theoretical and taxonomic breadth of coverage. however, these biases are beginning to be addressed, and disease ecology theory is being integrated with wdm with increasing frequency. by continuing to incorporate ecological and evolutionary ideas in the development and evaluation of management actions, disease ecology and wdm are likely to continue to advance towards a more unified body of theory and evidence. multiple infections, immune dynamics, and the evolution of virulence modeling routes of chronic wasting disease transmission: environmental prion persistence promotes deer population decline and extinction seasonality and the dynamics of infectious diseases the epidemiology of mumps 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mule deer: disease dynamics and control skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus low-coverage vaccination strategies for the conservation of endangered species linking process to pattern: estimating spatiotemporal dynamics of a wildlife epidemic from cross-sectional data successful establishment of wolbachia in aedes populations to suppress dengue transmission adaptive environmental assessment and management, st edn predation can increase the prevalence of infectious disease the ecology of wildlife diseases, st edn probiotics in aquaculture dynamics of indirectly transmitted infectious diseases with immunological threshold global trends in emerging infectious diseases networks and epidemic models dynamics of the uk foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape effects of species diversity on disease risk good medicine for conservation biology: the intersection of epidemiology and conservation theory proportion of individuals with anti-batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog rana muscosa sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome should we expect population thresholds for wildlife disease? management measures to control a feline leukemia virus outbreak in the endangered iberian lynx virulence evolution in response to vaccination: the case of malaria effects of management, behavior, and scavenging on risk of brucellosis transmission in elk of western wyoming disease and the dynamics of extinction active adaptive conservation of threatened species in the face of uncertainty meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of clostridium difficile disease infectious disease ecology: effects of ecosystems on disease and of disease on ecosystems community disassembly, biodiversity loss, and the erosion of an ecosystem service keeping the herds healthy and alert: implications of predator control for infectious disease microbial intervention for better fish health in aquaculture: the indian scenario guidelines for systematic review in conservation and environmental management scientist at work: toiling to save a threatened frog the use of probiotics in the prevention and treatment of antibiotic-associated diarrhea with special interest in clostridium difficile-associated diarrhea epidemiology and control of an outbreak of classical swine fever in wild boar in switzerland. the veterinary record persistent effects of maternal parasitic infection on offspring fitness: implications for adaptive reproductive strategies when parasitized initiating dialogue between scientists and managers of biological invasions probiotic treatment using bifidobacterium lactis hn reduces weanling diarrhea associated with rotavirus and escherichia coli infection in a piglet model an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle wildlife in a changing world. an analysis of the iucn red list of threatened species host culling as an adaptive management tool for chronic wasting disease in white-tailed deer: a modelling study the role of predation in disease control: a comparison of selective and nonselective removal on prion disease dynamics in deer disease management strategies for wildlife. revue scientifique et technique de l'office international des epizooties disease in wild animals: investigation and management culling and cattle controls influence tuberculosis risk for badgers effect of probiotic cenbiot on the control of diarrhea and feed efficiency in pigs we thank y.p. springer, v.j. mckenzie, s.h. paull, s.a. orlofske, s. ellis, t.j. zelikova, the cu disease discussion group and the johnson lab for insightful comments. any use of trade, product or firm names is for descriptive purposes only and does not imply endorsement by the u.s. government. a.l.a., d.l.p., j.g.k., j.r.m. and m.b.j. were supported by the nsf graduate research fellowship program. p.c.c.'s work was supported by u.s.g.s. and the nsf/nih ecology of infectious disease program deb- , and some ideas stem from working groups sponsored by the nih/dhs-funded rapidd program. p.t.j.j. was supported by a fellowship from the david and lucile packard foundation and grants from the national science foundation (deb- , ) and the morris animal foundation. the authors have no conflict of interests with regard to this research or its funding. additional supporting information may be found in the online version of this article.appendix s . references for reviewed articles. table s . metadata from reviewed articles. key: cord- - amiljnm authors: clements, bruce w.; casani, julie ann p. title: emerging and reemerging infectious disease threats date: - - journal: disasters and public health doi: . /b - - - - . - sha: doc_id: cord_uid: amiljnm this chapter describes the potential public health impact of emerging and reemerging disease. factors contributing to the emergence of diseases include increasing international travel and commerce, changes in human demographics and behavior, advances in technology and industry, microbial adaptation and the breakdown of public health systems. of emerging diseases, % are zoonotic, making the human–animal biome interaction critical. preparedness for an emerging disease relies on strong biosurveillance systems for early detection. control measures to prevent transmission must be implemented early. these include: rapid epidemiologic surveillance and investigations to characterize the disease; transmission prevention through containment and control measures; development and deployment of medical countermeasures; and emergency public information and warning. recovery after the outbreak of an emerging disease can result in a “new normal” with persistent endemic infection in the community. emerging and reemerging infectious disease threats objectives • describe why diseases "emerge" or "reemerge." • discuss the impact of emerging infectious diseases on public health preparedness. • list the likely sources of emerging infectious diseases in the future. • describe how international travel and commerce contribute to emerging infectious disease threats. • discuss how microbial adaptation contributes to emerging infectious disease threats. • list human demographic factors and behaviors contributing to emerging infectious disease threats. • identify the epidemiological clues indicating a possible emerging disease. • describe various types of surveillance approaches. • discuss the breakdown of public health measures and systems. • recognize the actions needed for responding to an emerging disease. on a friday afternoon before the labor day holiday weekend in , the phone at the new york city department of health was answered by the on-call epidemiologist. an infectious disease specialist was calling to report an unusually large number of encephalitis or meningitis cases (inflammation of the brain or covering of the brain and spinal cord) at several hospitals in the borough of queens. blood and spinal fluid were tested at the new york state and cdc laboratories and reported positive for saint louis encephalitis virus (slev), a virus known to occur in the united states but never in new york city. new york city officials immediately began mosquito control programs in an attempt to stop transmission from mosquitoes, the vector for slev. over the next week, test results appeared to be conflicting. unknown to many of the investigators of the human outbreak, a veterinarian at the bronx zoo was investigating an outbreak of central nervous system disease in birds. wildlife veterinarians were also observing large bird die-offs but could not find a clear cause. by mid-september, both sets of investigators believed the causative agent was not slev. by september , after expanding testing services to several federal and academic laboratories as well as to the wider family of flaviviridae, it was confirmed that the causative agent for the human outbreak, the avian outbreak, and sentinel mosquito sampling was west nile virus (see fig. - ). by , the new york city outbreak included confirmed cases and deaths (johnston and conly, ) . by that time, the west nile virus was documented as having spread halfway across the united states to the rocky mountains (roehrig, ) . on april , , a sample was routinely tested from a young girl with influenza-like illness as part of the us sentinel influenza network. the results indicated this was a uniquely novel strain of influenza a (see fig. - ). on april , , a similar result was confirmed miles from the originally identified case. testing also confirmed that this virus was resistant to both amantadine and rimantadine but sensitive to both oseltamivir and zanamivir, two readily available antiviral medications. there had been sporadic cases of influenza with infection of viruses in the swine lineage, and it was suspected and later confirmed that this novel influenza a, h n , was a unique combination of genes most closely related to north american swine-lineage h n and eurasian swine-lineage h n . in the two initial cases, no contact with pigs was discovered through extensive investigation, and it was determined that this novel virus had been spread to these cases by human-to-human transmission, a new behavior for h n . by april , additional cases were identified in texas, and samples collected from an outbreak in mexico were also positive for this new strain. by april , a public health emergency, the first in the history of the united states, was declared to allow for the rapid development of a vaccine, mobilization of antiviral medications through the federally resourced strategic national stockpile, and enhanced surveillance through reporting and testing. travel advisories were put into place, and upon discovery of additional cases internationally, the world health organization (who) raised the pandemic level from phase to the pandemic level phase . the largest number of cases occurred in people between the ages of and years old. major figure - a culex quinquefasciatus mosquito is known as one of the many arthropodal vectors responsible for spreading arboviral encephalitis or west nile virus to human beings through their bite. photo by james gathany courtesy of the centers for disease control and prevention, public health image library. morbidity (illness) and mortality (death) occurred in pregnant women, and middle-aged people with chronic diseases and obesity, but overall the fatality rate was low. few cases were reported in people over the age of , indicating that this group may have had immunity. levels of influenza illness remained high in the summer of , unusual for the north american continent. rapid molecular analysis of the viral genome led to rapid production of a vaccine. in the late summer, the food and drug administration announced the monovalent vaccine would be licensed via a "strain change" pathway, which is similar to how seasonal influenza vaccines are licensed. this meant production would be expedited, since the same methods would be used as those used to produce seasonal flu vaccine and additional safety and validation studies would not need to be completed. by september, , months after the first case was identified, prototype vaccine was delivered to us states for use. h n is now included in seasonal vaccines (gatherer, ). ebola virus disease (evd) was first described in in two simultaneous outbreaks in sub-saharan africa. in december , a small outbreak of evd was reported in a forested area in southeastern guinea (see fig. - ). it has been postulated that the index case, a boy, was in direct contact with bats. this was the th reported ebola outbreak in history but the first to be reported in west africa. evd then spread to liberia and sierra leone, all through direct contact with the outbreak in guinea (who, ) . in guinea, liberia, and sierra leone as of august , , there have been over , cases and , deaths. a small outbreak of cases occurred in nigeria, and one case occurred in senegal. several cases were reported outside of the area, mostly in healthcare or humanitarian workers returning to their home countries. there were also imported cases in the united states and spain which led to secondary infections of medical workers but did not spread further. in both countries, the management of companion animals and environmental cleanup were challenging issues. several factors contributed to the devastation of this epidemic: poverty, population density, infrastructure decline after years of armed conflict, serious gaps in health and medical infrastructure with little to no surge capacity, and a delay in coordinated response. in spite of the significant risk to underresourced healthcare workers (comprising % of the dead), a major humanitarian medical response was launched internationally. in october , controls on people traveling out of the area went into effect with exit screening for symptoms, entry screening in most countries, and active monitoring of travelers in some countries after arrival. this screening program controlled movement and by actively monitoring attempts to identify people who may become ill early in the course of the disease, getting them to healthcare in a controlled manner and with appropriate infection control practices in place. healthcare facilities and providers in many countries stockpiled personal protective equipment, implemented infection control training, and implemented screening processes in order to be prepared. on january , , the who reported for the first time since the week ending june , , that there had been fewer than new confirmed cases reported. the focus of the response shifted from slowing transmission to ending the epidemic. in july , results of early testing of a vaccine appeared very promising. by the end of , the ebola outbreak in west africa reached over , cases and , deaths making it the largest ebola outbreak in history (cdc, ) . prior to this, the largest was a - uganda outbreak with cases and deaths (cdc, ) . at the time this chapter was written, public health professionals around the world continued to watch closely the progression toward stopping, and continued success in recovering from, this devastating ebola epidemic. bacteria plural of bacterium. a single-celled microorganism that can exist independently as a free-living organism or as parasite dependent upon a host organism. emerging infectious diseases illnesses caused by pathogenic organisms with an increasing incidence in humans. infectious diseases with increased incidence over the past two decades or those which threaten to increase in the near future are considered "emerging." emerging infectious diseases include pathogens which are newly evolving, spreading to new geographic areas, are previously unrecognized, or are old infections reemerging due to lapses in public health measures. fungi single-celled or multicellular organisms which cause infections in healthy persons or serve as opportunistic pathogens in persons who are immune compromised. examples include histoplasmosis and aspergillosis. helminths parasitic worms which live in humans or other animals and derive nourishment from their host. examples include the tapeworm, fluke, or nematode. healthcare-associated infection (hai) an adverse localized or systemic infectious condition occurring in a healthcare setting with no evidence that the infection was present at the time of admission. pathogen a biological organism capable of causing disease or illness to its host. prion the smallest infectious particle. it is an infectious strand of protein which replicates and leads to disease, and is similar to a virus. prions are the causative agents of diseases such as mad cow disease and creutzfeldt-jakob disease. protozoa a single-celled parasitic organism that can only multiply inside a host organism. r nought (r ) a metric widely used in assessing disease transmissibility or the basic reproductive rate. it represents the average number of subsequent cases which one case generates during its infectious period. rickettsia a group of microorganisms requiring other living cells for growth like viruses, but having cell walls, using oxygen, and having metabolic enzymes like bacteria. rickettsia are typically transmitted by ticks, mites, or lice. typhus is one example of a disease caused by rickettsia. virus an infectious organism consisting of a nucleic acid molecule in a protein coat. it is only able to multiply inside the living cells of a host. emerging infectious diseases (eids) are some of the most challenging public health issues facing the global community. the hypothesis of "disease emergence" may have helped shaped the growth of global health initiatives, particularly at the world health organization (brown et al., ; lakoff, ) . eids are caused by pathogens that: ( ) have increased in incidence, geographic, or host range; ( ) have changed pathogenesis; ( ) have newly evolved; or ( ) have been discovered or newly recognized (lederberg et al., ) . in most developed countries, routine and seasonal outbreaks challenge health departments and healthcare systems, but reporting, investigation, and treatment protocols are typically in place, trained on, and easily implemented. vaccination programs and antiviral and antimicrobial medications mitigate many recurring infectious disease risks. those who have been exposed to or infected with recurring illnesses have developed some immunity. however, novel infectious diseases pose challenges that often exceed the immune function of populations and the capabilities of public health systems around the world. there are several factors that permit infections previously not seen globally or in specific locations to emerge or reemerge after periods of quiescence. those factors include: international travel and commerce and the movement of goods and people permits the movement of sick people and disease vectors into areas previously not visited, thereby exposing others to pathogens previously not encountered. international tourism has expanded consistently for over years. from to it grew more than ten-fold from million in to million in . it nearly doubled again between to reaching million and again by reaching . billion. annual growth in tourism is expected to grow by over % each year reaching . billion international annual tourist arrivals in (un, ). modern transportation systems allow for rapid movement of people and with them diffusion of illness at a greater speed than during the preavia tion travel era. when travel was slower, often by caravan or ship, those who were ill could recover, layover, or succumb without transporting disease as easily. increased international travel is believed to have played a major role in the spread of hiv/aids. some virologists suspect that hiv was present at very low levels in remote areas of west africa for perhaps as long as years in animals before the disease reached epidemic proportions and was officially isolated by scientists in (krause). hunting animals as a source of protein created greater exposure of humans to the disease, and development of the transcontinental highway from point-noire, zaire (now the democratic republic of congo) to mombasa, kenya, may have allowed truck drivers and traders along this route to carry the virus into the general population. airline travel has its own unique risks, with recirculated air in the confined space of an aircraft which could expose travelers to airborne diseases such as tuberculosis (see fig. - ) . passenger compartment conditions have some similarities to the holds of ships in sea-crossings known historically for harboring eids. with the ease of global travel, people with increased luxury income can visit developing countries, exposing those populations to novel diseases, or return home with novel infections after several hours of airline travel and before signs or symptoms of illness become apparent. economic development may result in changes of land use from agriculture to industry, disrupting established ecosystems. altering natural habitats by building dams and creating deforestation-reforestation programs alters the balance of ecosystems, allowing some species to overflourish or die out. additionally, there may be movement of people into land previously occupied by vegetation and animals, exposing those who resettle to novel or previously contained pathogens and/or vectors. zoonotic diseases, those which infect animals, comprise - % of eids (taylor et al., ) . in the coming plague: newly emerging diseases in a world out of balance, medical journalist laurie garret writes "in this fluid complexity, human beings stomp about with swagger, elbowing their way without concern into one ecosphere after another (garrett, ) ." in addition, the effect of climate change on emerging diseases is unknown; however, lindgren et al. ( ) point out that climate change interacts with "a complex web" of all drivers of emerging diseases and therefore cannot be ignored. alterations of the geographic ranges of birds in europe and north america have also been demonstrated. many migrating birds carry pathogens, and changes to migratory bird habitats may result in human exposures to new pathogens (fuller et al., ) . climate change may also have effects on vector development, vector physiology, and vector habitat, ultimately affecting human vector-borne disease risks (parham et al., ) . human demographic factors and behavior including population density, population growth, and population distribution not only may affect the spread of people into geographic regions not previously inhabited, but it also can expose them to new pathogens. in addition, it may affect how disease transmission occurs from human to human. in many parts of the world, an increase in urban population is not matched by an increase in urban infrastructure and is accompanied by poverty, poor sanitation, and inadequate housing. conflict can result in population shifts to new geographic areas, disruption of critical infrastructure (including public health and health systems), and economic stress. poverty from any cause not only affects sanitation and vector control, but may force people into risk behaviors such as entering the sex trade. once ill, people living in poverty may not have access to healthcare or even have basic hygiene resources. they also may be unable to comply with isolation measures. as populations continue to age and advanced medical interventions such as chemotherapy and immunosuppressive biologicals alter immune function, diseases previously not known to infect humans can emerge. behaviors such as sexual activity and illicit drug use may also impact novel disease transmission. understanding human relationships with animals provides additional insights into eid risks (see fig. - ). sixty percent of recent emerging diseases are zoonotic. colocation of open poultry markets and cohabitation with poultry has been identified as an important risk factor for human cases of avian influenza (dinh et al., ; thorson et al., ; choi et al., ) . human cases of high pathogenic avian influenza have occurred in workers depopulating flocks in the netherlands and canada (koopmans et al., ; tweed et al., ) . fortunately, few of these outbreaks to date have sustained human-to-human transmission. in , an investigation of a large multistate outbreak of monkeypox was traced back to prairie dogs sold as exotic pets. the prairie dogs had been in close contact with giant gambian rats from ghana (cdc, a) . this outbreak resulted in increased controls of imported exotic animals and markets. the source of the severe acute respiratory syndrome (sars) outbreak has never clearly been identified. the index cases were initially linked to catlike exotic pets related to raccoons called "civet cats" (cdc, b) . this resulted in extensive bans on civet cat transportation and commerce. however, detailed investigation links the causative coronavirus to several wild animals used as food, and there are suggestions that the trade of many types of animals used in food was the source (cdc, a,b; he, a,b; normille and enserink, ; guan et al., ; ng, ) . bats play an important role in ecosystems in vector control, seed dispersal, and pollination. however, it is increasingly recognized that bats also play a significant role in the reservoir and transmission of zoonotic infections. contact with bats has long been recognized as a potential source for rabies, but several findings indicate that bats have a wider spectrum of diseases and may be a reservoir for paramyxoviruses such as measles, mumps, distemper, parainfluenza (drexler et al., ; messenger et al., ) , and ebola (leroy et al., ) . freidl et al. ( ) recently discovered antibodies for influenza a h in bats. influenza a h is a zoonotic disease and is a possible candidate for a novel pandemic strain. human interactions with bats and their habitats can occur in the workplace, home or recreational venues. in an observational study in western ghana, nearly half of the residents had contact with bats and bat habitats (anti et al., ) . reengineering and reopening closed facilities where bats have populated must be made with prevention methods in place. entering caves where bats and other animals live must also be approached with infectious disease precautions in mind. technology and industry certainly have tremendous human benefits but may also expose populations to conditions which foster eids. some of the most significant issues are changes in food production. mass agricultural compounds and facilities create environments where changes occur in microbial ecology. globalization of the food supply allows transportation of organisms on and in food and through accompanying vectors. food transportation over large distances may also introduce breaches in food security. advances in medical technology introduce techniques that allow bacteria to infect people and spaces not formerly at risk. increases in invasive procedures can result in the introduction of novel organisms. n n n an epidemiological investigation in traced the source of a mysterious meningitis outbreak to contamination of methylprednisolone acetate, a steroid injected to relieve back pain. several lots of the drug were contaminated with a rare fungus called exserohilum rostratum. over , patients were potentially exposed and had confirmed infections, resulting in deaths across states (smith et al., ) . the cdc healthcare-associated infection (hai) survey of a large sampling of us acute care hospitals found on any given day, about in hospital patients has at least one hai (see table - ). there were an estimated , hais in us acute care hospitals in , and about , patients died during their hospitalizations. more than half of all hais occurred outside of the intensive care unit. contaminated gastrointestinal and bronchoscopy endoscopic devices are among the many advanced technologies associated with increasing outbreaks in healthcare settings, including those of organisms such as pseudomonas aeruginosa and salmonella spp. (kovalevaa et al., ) . the us food and drug administration, whose responsibilities include medical devices, issued a warning in march of raising awareness that several devices with complex designs may be difficult to adequately clean. many causative agents of healthcare associated infections are especially dangerous but their transmission is preventable. while some causative agents are more common bacteria and viruses such as escherichia coli or norovirus, many are less common, including acinetobacter, burkholderia, clostridium, and klebsiella. in addition, antibiotic-resistant strains such as carbapenem-resistant enterobacteriaceae, methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci and s. aureus are more difficult to control and treat. after the establishment of the european union in the s and increased economic growth, patient transfer within and between countries expanded. after political changes and the opening of borders, medical care and technology improved. for example, the number of bone-marrow transplant units in central europe increased from to by , and the number of dialysis units increased from approximately to more than (krcmery, ) . however, vincent et al. ( ) reported in a one-day prevalence study that % of icu patients in western europe had nosocomial or healthcare-associated infections by microorganisms including p. aeruginosa and methicillin-resistant staphylococcus. microbial adaptation contributes to the development of "super-bugs." this includes both bacteria and viruses (see figs. - and - ). as discussed in the previous section, antimicrobial resistance is pervasive and is increasing in the face of widespread overuse and misuse of antibiotics. in addition, viruses rapidly adapt to immunologic responses. influenza viruses provide an example of genetic drift and shift which make them a persistent health threat. once formed, novel influenza viruses sometimes pose health risks for which humans and/or animals have little to no immunologic protection. these viruses can also develop an increased capacity for animal-to-animal or human-to-human sustained transmission. breakdown in public health systems and measures as a result of conflict, economicbased program cuts, or poor infrastructure can produce environments with increased risk and decreased prevention. this situation may result in disease emergence, but may also to read the full report, please visit the cdc hai prevalence survey (magill et al., ) . result in reemergence of diseases. diseases which had previously been controlled through sanitation, vector control, or vaccination programs can reemerge if any of the prevention programs fail or stop. in developed countries with successful vaccination campaigns, many diseases have been declared "controlled" or "eradicated." because of this, political and public interest in continued vaccination programs is waning, or personal risk is considered low relative to the risk (real or perceived) of vaccinations. in the united states there are also many states allowing "opt-out" programs so people may choose not to get vaccinated or have their children vaccinated. in numerous outbreaks this has proved problematic. n n n the us antivaccination movement has contributed to increased preventable illnesses and deaths. it is led by misguided conspiracy theorists, minor celebrities, and a small number of "healthcare professionals" who are either unfamiliar with or in denial of the science supporting vaccinations. the above headline appeared in january in the new york times (nagourney and goodnough, ) . notable quotes from this report include: • "we can expect to see many more cases of this preventable disease unless people take measures to prevent it," dr. gilberto f. chavez, the deputy director of the california center for infectious diseases, said. "i am asking unvaccinated californians to consider getting vaccinated against measles." • dr. james cherry, a specialist in pediatric infectious diseases at ucla, said the outbreak was " percent connected" to the anti-immunization campaign. "it would not have happened otherwise-it would not have gone anywhere," he said. "there are some pretty dumb people out there." • "the problem is that there are these pockets with low vaccination rates," said dr. jane seward, deputy director of the viral diseases division at the cdc. "if a case comes into a population where a lot of people are unvaccinated, that's where you get the outbreak and where you get the spread." n n n in ghent, belgium, a measles outbreak was associated with an anthroposophic school which promotes "complementary medicine" (braeye et al., ) . over cases of measles were identified in a school that had low vaccination rates because of the philosophical beliefs of the parents. leaders of the school were not against vaccination. striking the balance between individual rights and beliefs, relative risk perception and acceptance, and public health priorities is always challenging. while the next emerging disease can be of any species of infectious organism, trends in recent cases display patterns suggesting which threats may be imminent (see table - ). coronaviruses such as the causative agents of sars and middle east respiratory syndrome co-v (mers co-v); filoviruses such as ebola; and novel influenza viruses, are considered likely candidates. in a study by taylor et al., an analysis of emerging species revealed that of those known, there are currently emerging species. viruses and prions are less than half of the total ( %), and bacteria or rickettsia comprise just under one-third. these potential pathogens may be transmitted by several routes, and the most common route is direct contact ( %), followed by indirect contact ( %), vectors ( %), and for % the route of transmission is unknown (taylor et al., ) . while it may not be possible to predict which pathogens may emerge or reemerge, it is possible to build infrastructure and take general steps to make populations and public health systems better prepared for the next novel infectious disease outbreak. at the heart of these measures is epidemiological surveillance. identification of a new illness or disease requires surveillance systems which not only continually monitor "routine" illness, but also have the ability to recognize anomalies when something is not "routine." as described in chapter "bioterrorism," there have been extensive efforts toward developing early warning epidemiological and environmental surveillance systems for unusual diseases and pathogens. the vital link in identifying a novel disease in a community is the astute clinician. clinicians must be able to recognize a novel disease, report it appropriately, and feel confident that the information will be quickly analyzed and acted upon. laboratory support is vital to quickly identifying and characterizing an emerging threat. this includes testing for drug resistance to inform decision-making concerning appropriate medical countermeasures. continued epidemiological surveillance throughout an outbreak can produce data which may be useful in evaluating and improving the public health and medical response. n n n • surveillance • robust outbreak investigation practices • transmission prevention through containment and control measures • delivery of medical countermeasures, if any • public messaging • recovery to a "new normal" n n n along with surveillance is the ability of public health responders to perform detailed outbreak investigations to determine the characteristics of the disease. characterization of the outbreak, identifying the natural course of the illness, and recognizing key risks for infection are necessary in order to learn how the new illness behaves in a population (see fig. - ) . this information will also inform control measure decisions. a priority item in the initial characterization of an outbreak is the identification of transmissibility. unfortunately, as surveillance methods are enhanced and cases are increasingly recognized and reported, transmissibility may be a very difficult thing to quantify. for example, the current analyses of transmissibility for mers co-v suggest that it is not a likely pandemic threat because of its low reproduction number or "r nought" (r ). however, different cluster sizes, demographics, geographies, and public health and healthcare infrastructures may alter the outbreak characteristics and the reproduction number (r ) (fisman et al., ) . capturing critical data will allow for accurate characterization within the context of the outbreak and will direct response activities such as control measures and medical surge management. the primary control strategy in all infectious disease outbreaks is preventing transmission. even minimal biosecurity actions can prevent animal-to-human transmission. separation of potential source animals, such as exotic imported animals and poultry, from routines such as cooking can reduce risk. effective cleaning of animal waste and habitats should be performed with at least basic respiratory protection. to prevent human-to-human transmission, good hygiene practices such as hand washing, general surface cleaning, and careful waste disposal should be reinforced. during the - h n pandemic, people were advised to "cough hygienically" into their sleeve to prevent contamination of the hands and then others. of note, this technique was not without some controversy and was not recommended in the united kingdom and spain (anderson, ) . supplying hand wipes and hand sanitizer may be implemented in some locations of businesses, critical industries, and government agencies where other measures may not be reasonable. social distancing by school closures, limiting mass gatherings, and canceling large events may be necessary (see figs. - and - ). quarantine may be considered for individuals who have been exposed but are not yet sick, typically for two incubation periods of the disease. isolation separates those who are already ill from those who are not ill. in healthcare settings, isolation may begin from early screening of patients at points of entry. this may limit disease transmission to other patients who often have underlying conditions which increase their risks for severe illnesses and complications. risk management for communicable diseases in emergency departments includes signage to encourage patients to self-identify if they are sick or have an associated travel history. this allows for rapid triage to isolation areas and early institution of personal protective equipment for staff (puro et al., ) . in a study conducted of facilities in european countries, % had isolation rooms, but not all had anterooms, negative pressure, or hepa filtration. only . % had all components. personnel trained in the recognition of highly infectious diseases were available in of the , and management protocols were available in of the (fusco et al., ) . it would be interesting to see how attention paid to diseases such as ebola has impacted these numbers. in addition to human-to-human spread, animal-to-human spread is a common transmission route for eids. zoonotic disease professionals can develop recommendations for vector control when zoonotic diseases threaten human populations. mosquitoes are the most pervasive disease-carrying vectors. control measures include reducing standing water breeding sources or using larvicides and adulticides. the recent introduction of engineered aedes aegypti mosquitoes (ox a), which can reproduce and produce offspring which die rapidly, shows promise in areas with emerging dengue fever (specter, ) . once control measures are determined, public health agencies must have the legal and social authority to implement recommended measures. the majority of the population typically complies with basic containment measures such as improved hygiene, avoiding ill people, etc. however, some individuals may be reluctant or unable to comply. for example, minimally ill people without paid sick leave may not be able to stay at home without significant economic loss. people may, out of fear, choose to flee quarantine zones. in these cases, agencies need to have flexible, scalable authorities to limit travel outside areas known to have disease. the challenges with quarantine measures were apparent throughout the international response to the - west african ebola epidemic. in the united states as in many countries, the government has the authority to restrict stateto-state and international travel. state and local health agencies have authority to control intrastate activities. however, political pressure may make it difficult to enact some control measures. school closure is one of the more difficult decisions for government agencies during a public health emergency. for many communicable diseases, children can act as superspreaders because of their behaviors and hygiene lapses. however, many schools function not only as places of education but also deliver significant social programs: safe havens, meals, and day care for working parents with marginal income. to close these schools disrupts very necessary social welfare programs. quarantine laws must also respect civil liberties. in the united states for example, quarantine orders must be the least restrictive form of control, have very clear reasons, be timelimited and allow for appeal. enforcement of quarantine orders requires law enforcement officers to interact, sometimes in close physical proximity, with potentially contagious individuals. additionally, any enforcement of quarantine of an individual must be heard in the judicial system. the judiciary has its own constitutional constraints of process which are frequently in opposition to the goals of quarantine. if the eid is susceptible to antibiotics or a vaccine is quickly developed, rapid distribution and dispensing of these medical countermeasures must be conducted. if the illness is highly contagious, programs limiting public interaction during mass dispensing and mass vaccinations will need to be implemented. as discussed throughout this text, it is unlikely that there will be large quantities of medical countermeasures available early on in an emerging or reemerging infectious disease outbreak. therefore, plans must also be in place for the utilization of scarce resources while complying with ethical and legal frameworks. throughout the entire outbreak, appropriate public messaging must be delivered. messages will contain information about the illness, personal protective actions, and where to go for health care. these messages must communicate risk and how individuals can limit morbidity and mortality. general prescripted public health messages can often be adapted early in an outbreak when detailed information on the threat is not yet specific. trusted leaders in the community should be used to deliver messages in order to improve compliance and alleviate fear. a "new normal" may follow as a community recovers from a novel outbreak. some eids become endemic diseases in affected communities and must be included in recurring public health activities. for example, west nile virus was first introduced to the western hemisphere in the late s and is now an annual threat, causing , cases from to and deaths (cdc, ) . however, public health responses help to establish new practices in order to limit reemergence. these activities also prepare the community for similar eid threats. because of ongoing west nile virus threats in north america, the region is better prepared for emerging threats such as chikungunya and dengue. public health has been challenged with eids throughout human history. with globalization of populations, commerce, and travel comes globalization of infectious diseases. this exposes people and animals to novel diseases for which they have little to no natural immunity. rapid transportation and free movement allows sick individuals to spread illness and disease faster than ever before. preparedness efforts cannot predict the next emerging infection, but public health and healthcare capabilities developed, lessons learned from prior outbreaks, and institutionalization of routine infection control practices may serve to lessen the impact. european centre for disease prevention and control 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and bronchoscopy trends in nosocomial infections in europe: comparison between western and central europe two regimes of global health emerging infections: microbial threats to health in the united states. institute of medicine human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo monitoring eu emerging infectious disease risk due to climate change multistate point-prevalence survey of health careassociated infections bats, emerging virus infections and the rabies paradigm measles cases linked to disneyland rise, and debate over vaccinations intensifies tracking the roots of a killer possible role of an animal vector in the sars outbreak at amoy gardens climate, environmental and socio-economic change: weighing up the balance in vector-borne disease transmission risk management of febrile respiratory illness in emergency departments west nile virus in the united states-a historical perspective fungal infections associated with contaminated methylprednisolone injections the mosquito solution risk factors for human disease emergence. philos is exposure to sick or dead poultry associated with flulike illness? a population-based study from a rural area in vietnam with outbreaks of highly pathogenic avian influenza human illness from avian influenza h n , british columbia world tourism organization. unwto tourism highlights the prevalence of nosocomial infection in intensive care units in europe: results of the european prevalence of infection in intensive care (epic) study ebola response team . ebola virus disease in west africathe first months of the epidemic and forward projections west nile virus fact sheet key: cord- - us smz authors: mcfee, robin b. title: travel-related illness date: - - journal: disease-a-month doi: . /j.disamonth. . . sha: doc_id: cord_uid: us smz nan international travel, the expansion of transnational business enterprises, growing reliance on imported goods, including food, all continue to raise the likelihood that infectious diseases, as well as the vectors that spread them, will enter the united states, remain, and pose significant public health challenges. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diseases that have historically been confined to regions, even continents can now spread with relative ease. given the globalization of commerce, agriculture, tourism, and the job market, raising awareness, improved surveillance, and greater collaboration must go beyond traditional public health to include nonmedical resources and partnerships. although many of us were taught in medical school and residency training that when you hear hoof beats, think horses, not zebras, such advice must now be called into question, considering global travel, immigration into the united states from countries with endemic illnesses not commonly found in north america, and societal factors (domestic and international) that include poverty, homelessness, institutionalization, overcrowding-in health care facilities as well as domiciles-and lack of access to timely health care, all of which set the stage to create conditions that facilitate the spread of diseases that are from previously unknown, little recognized, or emerging pathogens that are endemic to or novel strains in foreign lands, and which can become very quickly clinical realities and community-wide problems in the u.s. this is one of the reasons obtaining a thorough travel and occupational history from patients who present with an atypical or severe, progressive illness, is critically important, as it may give valuable insights into the origin of the infection, how to access timely information from sources experienced in treating the cascade of symptoms (biodrome) (fig.) and the etiological pathogen, and allow you to rapidly initiate appropriate, potentially life-saving interventions. as who notes in its international travel and health updates, , , , , international travelers are at risk depending upon the area visited, the purpose of the trip, itinerary, resources, accommodations, hygiene, sanitation, and individual behaviors, as well as innate immunity and vaccinations, but unfortunately such pretravel guidance is lacking. , , general precautions can reduce the risk of exposure to infection risks. the most common infectious illness to affect travelers is "traveler's diarrhea"-of which many food and waterborne pathogens can be implicated. knowing the modes of infectious disease transmissions provides opportunities for prevention. these include the following: food-borne (e.g., avoid raw or undercooked foods) waterborne (e.g., ice cubes, local water at restaurants, as well as standing water, and rivers) zoonoses (e.g., animal sources) sexually transmitted diseases (stds) (e.g., local brothels) blood-borne [including poorly cleaned medical instruments as well as iv drug use (ivdu)] airborne (respiratory) (crowds or persons exhibiting respiratory signs/symptoms) soil [farms and farm lands can spread disease-footwear can carry soil/feces/animal products, and proximity to animals (e.g., avian influenza), plants (carry insects, for example) contributes to disease spread] respiratory diseases are common community-acquired diseases, as well as tia; viruses creating epidemics of more severe viral pneumonitis, as was seen when a new coronavirus-sars-continued to emerge. , typically coronaviruses cause low-grade respiratory infections. that changed in . by , the world health organization (who) emphasized the importance of travel history in identifying possible and probable cases-a critically important step in characterizing the epidemiology and ultimately containing the infection. soon thereafter, sars coronavirus seemed to disappear, only to have another novel one appear in ; mers cov. unlike sars that emerged from asia, mers seems to have originated on the saudi peninsula in the middle east. it is important to note that several illnesses endemic abroad have a variety of incubation periods. health care professionals should encourage their patients to discuss travel plans and/or work out of the country, be knowledgeable about travel specialists in their community as important pretravel resources and referral, as well as some of the more common tai and emerging global threats. this includes infectious mononucleosis (im) from epstein-barr virus and im-like syndromes (cytomegalovirus-cmv) that have been reported in febrile travelers returning from the tropics. it is important not to forget our servicemen and servicewomen deployed in developing nations-a unique source of tai to be sure, who will return from the middle east and other countries that may harbor endemic illnesses that u.s. physicians and health care professionals may be unaware of or unaccustomed to recognizing, let alone treating. as described elsewhere, but worth repeating, malaria is one of the most important infectious diseases of concern for returning military as well as travelers, especially in the middle east, where our troops are deployed. in anticipation of our troops returning from the persian gulf, afghanistan, and elsewhere, it is important to take some time for training and familiarize ourselves with pathogens and symptom cascades-biodromes-that are likely to infect them. at a minimum, patients presenting with respiratory or gastrointestinal illnesses, especially an unusual or unexpected cascade of symptoms, require a thorough history of travel and contact information to be obtained. an index of suspicion as well as travel, contact, and occupational history are all critically important pieces of information to aid in developing management strategies, especially if the infectious agent is not known or clearly identified. world tourism organization. unwto tourism highlights travel-associated illness trends and clusters health problems after travel to developing countries spectrum of disease and relation to place of exposure among ill returned travelers the role of the traveler in emerging infections and magnitude of travel knowledge, attitudes and practices in travel-related infectious diseases: the european airport survey epidemiology of travel-related hospitalization severe respiratory syndromes: travel history matters infectious diseases of potential risk for travelers. international travel and health avian influenza: the next pandemic? dis mon health disparities among travelers visiting friends and relatives abroad illness in travelers visiting friends and relatives: a review of the geosentinel surveillance network human coronavirus emc is not the same as severe acute respiratory syndrome coronavirus mbio gulf war servicemen and servicewomen: the long road home and the role of health care professionals to enhance the troop's health and healing key: cord- -dc oyftd authors: koehlmoos, tracey pérez; anwar, shahela; cravioto, alejandro title: global health: chronic diseases and other emergent issues in global health date: - - journal: infectious disease clinics of north america doi: . /j.idc. . . sha: doc_id: cord_uid: dc oyftd infectious diseases have had a decisive and rapid impact on shaping and changing health policy. noncommunicable diseases, while not garnering as much interest or importance over the past years, have been affecting public health around the world in a steady and critical way, becoming the leading cause of death in developed and developing countries. this article discusses emergent issues in global health related to noncommunicable diseases and conditions, with focus on defining the unique epidemiologic features and relevant programmatic, health systems, and policy responses concerning noncommunicable chronic diseases, mental health, accidents and injuries, urbanization, climate change, and disaster preparedness. prevailing concerns and expected future trends, as seen clearly in the reemergence of tuberculosis and malaria as key health problems that have become global and individual country health priorities. infectious diseases have always had a decisive and rapid impact on shaping and changing health policy with global pandemics such as severe acute respiratory syndrome (sars) and h n , emerging without warning and challenging approved priorities within days if not hours. however, it is important not to lose sight of other areas of health and to maintain a close and watchful eye on trends and developments in those diseases that do not generate the immediate impact that some infectious diseases have been able to do. noncommunicable diseases fall into this group; they may not have garnered as much interest or importance over the past or years, but in fact have been affecting public health around the world in a very steady and critical way, becoming the leading cause of death in both developed and developing countries. this article discusses emergent issues in global health related to noncommunicable diseases and conditions. trying to offer an in-depth discussion on such a wide range of issues in just one article is clearly not possible, and therefore focus and emphasis is given to defining the unique epidemiologic features and relevant programmatic, health systems, and policy responses concerning noncommunicable chronic diseases (ncds), mental health, accidents and injuries, urbanization, climate change, and disaster preparedness. in the shadow of global efforts to achieve the millennium development goals (mdgs), by far the largest killer on the planet has continued to advance in low-income and middle-income countries. ncds cause % of all global deaths but receive just . % of international development assistance for health. approximately % of deaths caused by ncds occur in developing countries, generally in a younger population than those in high-income countries. , over the next years, the world health organization (who) predicts that ncd deaths will increase by % globally with the greatest increases in the african ( %) and the eastern mediterranean ( %) regions. in terms of the highest absolute number of deaths, the western pacific and south-east asia are projected to lead the field. noncommunicable diseases are a group of illnesses and include those conditions that have been identified as the leading causes of death around the world: heart disease, stroke, cancer, chronic respiratory diseases, and diabetes. these diseases are characterized by their long latency period often influenced by exposure to risk factors for extended periods over a patient's lifetime. the situation becomes more acute with the addition of the word "chronic," indicating that these diseases are mostly incurable and the duration of treatment may cover decades of a person's life. cardiovascular disease (mainly heart disease and stroke) is the biggest killer worldwide, contributing to % of global deaths each year. the importance of such a high figure can be seen in the countries that make up latin america and the caribbean, where cardiovascular disease alone accounts for % of the total mortality burden while aids, tuberculosis, malaria, and all other infectious diseases combined are responsible for only % of that burden. globally, chronic disease deaths have been predicted to increase by % between and . although research on multimorbidity has been based primarily on high-income countries, experts estimate that around % of the population living with chronic disease may actually be living with multiple chronic conditions. sometimes erroneously referred to as "lifestyle diseases," ncds are affected by a variety of risk factors that are often outside the control of the individual. there is very little that can be done about some risk factors, such as age and genetic inheritance, and increasing evidence suggests that what happens before a person is born and during early childhood plays a key role in the onset of adult chronic disease, demonstrated by the proven association between low birth weight and increased rates of high blood pressure, heart disease, stroke, and diabetes. however, the most common chronic diseases share some of the same highly preventable or avoidable risk factors including physical inactivity, tobacco use, and obesity, leading researchers to study mortality for ncds by risk factor. the who estimates that each year approximately . million people die from tobacco use, . million from being overweight or obese, . million as a result of raised cholesterol levels, and . million as a result of raised blood pressure. raised cholesterol and raised blood pressure (hypertension) are particularly dangerous risk factors because they can exist in an individual for a long time without presenting any obvious symptoms. in its seminal book preventing chronic disease: a vital investment, the who presents what it defines as effective and feasible interventions to reduce the threat of ncds, with low-income and middle-income countries being specifically targeted. the who seeks ideally to reduce the burden of ncd mortality by % per year through the implementation of the who framework convention on tobacco control (fctc), which was the first global treaty negotiated by the who in . as of it had been signed by nations, although stages of ratification vary. the fctc contains guidelines for implementing demand-reducing policies toward tobacco including health policies aimed at protecting the public with respect to commercial and other vested interests of the tobacco industry, protection from exposure to tobacco smoke, packaging and labeling of tobacco products; and limits or bans on tobacco advertising, promotion, and sponsorship. tax increases for tobacco control are considered to be clinically effective and very cost-effective relative to other health interventions, while the implementation of smoking bans in public areas appears to reduce the risk of heart attacks significantly, particularly among younger individuals and nonsmokers, according to a study published in the journal of the american college of cardiology (september , issue). researchers reported that smoking bans can reduce the number of heart attacks by as much as % per year. , policy level programs are also being discussed for reducing salt and sugared beverages , in the diet, consumer products, and food outlets. the who report also encourages screening for which there are clear public health benefits and cost benefit, and in situations in which the ability to treat the condition (such as raised blood pressure and cervical cancer) exists. however, at present the quality and quantity of research investigating the actual benefits of different intervention programs to prevent noncommunicable diseases in developing countries is sparse and exists primarily as case studies. , low-income and middle-income countries have developed their health provision and policies according to a primary care or alma ata model, focused on meeting the needs of pregnant women and children younger than years, and developing services for a variety of high-impact communicable diseases such as human immunodeficiency virus (hiv)/aids, tuberculosis, and malaria. the health systems in these countries are unprepared to deal with risk-factor education and behavior modification for the prevention, diagnosis, and treatment of ncds, or the long-term management of these conditions. despite growing interest among the population and health system leadership, one high-ranking health official pointed out that currently, donor countries are operating a policy ban on funding ncds, thereby starving low-income governments of the financial and technical assistance needed to turn around the ncd epidemic. this policy has to change, with overseas development assistance aligned to the priorities of recipient countries. this situation continues to be an issue for developing countries despite numerous calls for action in the area of ncds and funding. , , [ ] [ ] [ ] furthermore, there is a clear inequity inherent in noncommunicable diseases, as the poor and less educated are more likely to be exposed to several preventable risk factors including tobacco use, high-fat and energy-dense food consumption, physical inactivity, and obesity. there is no denying that noncommunicable diseases are linked to economic loss, and the who highlighted this in , predicting that national income loss due to heart disease, stroke, and diabetes for china, india, and the united kingdom are expected to be $ billion, $ billion, and $ billion, respectively, with part of the losses being the result of reduced economic productivity. the global burden of disease (gbd) project began in and since then chronic diseases have exceeded the burden of infectious diseases. despite this, the international community has yet to display a sense of urgency toward reducing ncds or supporting ncd-focused interventions in developing countries, even though they are threatening development and economic progress. perhaps the situation will change in the near future with the participation of united nations (un) member states in a highlevel summit on noncommunicable diseases scheduled to take place in new york in september . although nothing can be guaranteed, similar un summits have provided the catalyst for change, as seen following the summit on hiv/aids in that resulted in significant funding and political commitment to a coordinated action plan. since , the who has defined health as "a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity." however, mental illness and related conditions have never received the same importance or consideration as other areas of health despite their enormous burden on the population. this fact is exemplified by the routine exclusion of mental health services from primary health care (phc) and the absence of any mental health-related objectives in the mdgs. , mental illnesses, including behavioral, neurologic, and substance use disorders, affect a significant number of the world's population. in , the who estimated that globally million people suffered from depression, million from schizophrenia, and million from substance use disorders, with around , people committing suicide every year. in the same year, unipolar depressive disorders were ranked as fourth in terms of burden of disease, well on the way to prove the prediction of the gbd analysis that estimated mental illness, specifically unipolar major depression, would become the second leading cause of burden of disease by , second only to ischemic heart disease. studies in phc settings in turkey, the united arab emirates, france, vietnam, and zimbabwe revealed that the prevalence of mental illness ranges between % and % among adults, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] with depression being the most common ranging from % to %, followed by generalized anxiety disorders ( %- %) and dependency on addictive substances ( %- %). [ ] [ ] [ ] children are not immune to mental health problems, with those aged between and years exhibiting a prevalence of mental illness of between % and %, , the most common diagnoses being anxiety disorders, major depression, behavioral disorders, and attention-deficit/hyperactivity disorder. , mental illness has an effect on other family members, which is seen clearly in a study looking at growth rates of children with mothers suffering from mental illness. the study showed that % of these children suffered from stunted growth, which could have been averted if interventions to treat the maternal depression had been performed. , individuals suffering from severe form of depression are at increased risk of attempting suicide, as are women who experience abuse. meanwhile, the prevalence of mental health problems among elderly people is %, the majority of whom suffer from depression. , cost-effective treatment for most mental illnesses exists and, if correctly applied, most patients become functioning members of society, leading normal lives even in low-resource areas, and suicide risk is reduced. of interest, poverty indicators are related to mental disorders - with low education level being the most influential determinant. extrapolating these data, it is feasible to suggest that developing countries with low education levels will tend to have a higher proportion of the population suffering from mental health problems. despite this, however, most low-income and middle-income countries spend less than % of their health expenditure on mental health. explicit mental health policy, legislation, mental health treatment facilities, and community care are all lacking. injuries as a global health issue include many types that are routinely reported to and published by the who, such as poisoning, falls, drowning, burns, and intentional injuries including interpersonal violence such as elderly, partner, or child abuse, and collective violence such as war. however, two of the most important injuries that contribute to high global death rates are road traffic accidents and occupational injuries. in , an estimated % of all global deaths were the result of an injury. injuries not only affect morbidity and mortality rates but also have a tremendous effect on the individual, the family, and the community. box presents the scope of injuries and their importance as a national health issue. it is predicted that by , road traffic injuries will be the fifth leading cause of death. already, approximately . million people die due to road traffic accidents each year, and an additional to million are injured or disabled. despite being home to fewer than % of the world's motor vehicles, low-income and middleincome countries have % of the mortality burden for road traffic accidents. one injuries prove to be the largest killer of children between and years of age, accounting for % of all classifiable deaths. this means that children per day die of injuries or children per hour. the leading cause of injury-related deaths among children is drowning ( . %) followed by road traffic accidents ( . %), animal bites ( . %), and suicide ( . %). it is estimated that injuries permanently disable around , children per year in bangladesh. nonfatal injuries occur in approximately million children per year or per minute (institute of child and mother health, ). when injury-related deaths are broken down by type and by age group, children aged - and - years are most likely to die from drowning with a mortality rate of per , and per , child deaths, respectively. in the - year age group, road traffic accidents account for per , child deaths, and in the - year age group, suicide accounts for per , child deaths. of the most important reasons for this apparent discrepancy is the high number of vulnerable road users in developing countries. vulnerable road users include pedestrians, cyclists, and both the rider and passenger of motorcycles and scooters. vulnerable road users account for % of deaths, and in low-income countries pedestrians account for nearly half of all road accident-related deaths. there are proven interventions that can lead to a reduction in the amount of road traffic deaths and injuries. such measures include controlling or reducing the speed of traffic with speed bumps or low-speed zones in urban areas, establishing and enforcing blood alcohol concentration limits, enforcing the use of helmets for both riders and passengers on motorcycles, and enforcing the use of seat belts, infant seats, and child booster seats. the wearing of seatbelts in automobiles can reduce front-seat passenger deaths by % to % and rear-seat passenger deaths by % to %; however, only % of countries require the wearing of seat belts by all passengers. the problem is that because of the high numbers of both people and different types of vehicles in developing countries and the lack of resources to police traffic effectively, traffic laws are not easily enforced, despite evidence showing the benefit of specific interventions in the reduction of traffic-related morbidity and mortality. occupational injuries are a significant problem in global public health, contributing to between , and , deaths worldwide each year. with great shifts in industrialization from the developed to the developing countries, it is logical that the highest number of occupational injuries is shifting in the same way toward the developing world. however, it is very likely that published figures are underestimated, with numbers probably being % below the actual figure for the united states and as much as % for some locations such as rural africa. , although several factors come into play when analyzing the causes of underreporting in developing countries, one of the main reasons is the lack of adequate data. determining the actual prevalence of occupational injury is critical for several reasons: ( ) to provide accurate data to health providers, policy makers, nongovernmental organizations (ngos), and the public; ( ) to provide baseline data against which to measure interventions; ( ) to aid priority setting and targeting for policy change and interventions; and ( ) to estimate societal costs of rising occupational injuries. tools to capture occupational injury have been designed and widely circulated by the un's specialized agency, the international labour organization. however, field testing of the tools has been limited to small-scale surveys in diverse settings such as vietnam, ghana, and bangladesh, - and larger, nationally representative studies are needed. in many developing countries, there is a lack of policy for or enforcement of safe working environments, which naturally means that wood cutting, mining, agriculture, construction, and manufacturing are more hazardous than in developed countries. the developed world has accepted that poor working conditions and practices are unacceptable and has legislated against them, leading to a reduction in occupational injuries over the past century. however, it seems that globally the same care has not been forthcoming, and developing countries have taken on the burden of heavy industry and poor working conditions that generate increases in occupational injuries. this trend is perfectly exemplified by the phrase "export of hazard" to describe when an outdated and dangerous technology is relocated from a high-income country to a developing country, despite the knowledge that the risk of injury with this technology is high. cost of production plays a key role in maintaining poor working conditions, and many industries in developing countries manage cost control through the use of manual labor, which is cheaper than the infrastructure and equipment needed to upgrade a process that produces the same amount of product at a much safer level. manual labor is particularly exploited in the construction industry in developing countries, which have a disproportionate number of deaths from workers falling and injuries from falling objects. working conditions at all levels of commerce are also full of risk factors to health, from the lack of ergonomically designed offices to avoid back injuries and repetitive stress disorders, to building materials used in construction, which may offer a long-term risk of health problems. the latter is of particular concern in many low-income and middle-income countries, with construction still making use of asbestos despite the documented links to lung cancer. , urbanization urbanization is a major public health challenge for the twenty-first century, with significant changes in our living standards, lifestyles, social behavior, and health. previously more of a phenomenon in developed countries; it is now taking hold and being seen at a greater level in developing countries. the united nations population fund (unfpa) predicts that over the next to decades, almost all the world's population growth will be in urban areas in developing countries. who figures for the period to already show an alarming increase in urban population growth, with developing countries' urban areas growing at an average of . million people per week or around , people every day. while urban settings offer many opportunities including access to better health care, they can affect existing health risks and introduce new health hazards. the living and working conditions of those living in rapidly expanding and poorly planned urban areas often experience risks to health in some of the most basic areas such as unsafe drinking water, unsanitary conditions, poor housing, overcrowding, hazardous locations, and exposure to extremes of temperature. these increases in health risks are particularly critical for those most vulnerable: children younger than years, infants, and the elderly. , the rapid growth of urban settlements is often due to poor economic performance of the area in question and lack of urban planning and regulation, which has resulted in an increase in the number and size of informal settlements or slums in many cities. it is estimated that in the developing regions, more than % of urban residents live in slums. the urban health situation the current pattern of urban growth is expected to have a multiplier effect on many dimensions of illness and disease. child mortality is already high in the urban areas of developing regions. in nairobi, where % of the city's population lives in slums, child mortality in these slums is . times greater than in other areas of the city. evidence from various surveys and studies points to a heavier burden of diseases such as diarrheal diseases, acute respiratory diseases, malnutrition among children, hiv/aids, tuberculosis, malaria, diabetes, and obesity on the urban poor. , , migration, increased mobility, changes in the ecology of urban environment, high population density, poor housing, and poor provision of basic services all act as pathways for emerging and reemerging communicable diseases. , the consequence of these changes is evident in the spread of multidrug-resistant strains of tuberculosis that is placing the urban poor of india, indonesia, myanmar, and nepal at a higher global health risk. vector-borne diseases such as dengue and malaria are also increasing in many urban areas, due to migration, climate change, stagnant water, insufficient drainage, flooding, and improper disposal of solid waste. , unhealthy lifestyles characterized by unhealthy nutrition, reduced physical activity, and tobacco consumption due to rapid and unplanned urbanization are associated with common modifiable risk factors for chronic diseases such as hypertension, diabetes mellitus, and obesity. urban environments tend to discourage physical activity and promote unhealthy food consumption. overcrowding, heavy use of motorized transport, poor air quality, and lack of safe public spaces are some urban factors that restrict participation in physical activities. in the larger populated cities of asia obesity is becoming a significant problem, and the rapid transition of diets in developing countries is typified by the coexistence of child malnutrition and maternal obesity in the same household. one of the main factors identified as causing an increase in diabetes worldwide is the change in traditional diets caused by urbanization. urbanization is exacerbating the health risks in terms of traffic accidents, injuries on the street or in the home, and mental health problems. the changes in climate and rising sea levels work toward increasing urbanization, with million people living in the low-elevation coastal zones being at heightened risk of flooding, which will lead to migration to higher elevations and larger cities. adopting preventive measures to control communicable diseases, upgrading the infrastructure of existing health facilities, increasing human resource capacity, and taking appropriate measures for providing equitable health services to all, especially the most vulnerable groups, are vital for improving urban health. recently, the who identified key areas of action for improving urban health: . promote urban planning for healthy behaviors and safety . improve urban living conditions, including access to adequate shelter and sanitation for all . involve communities in local decision making . ensure cities are accessible and age-friendly . make urban areas resilient to emergencies and disasters. however, these actions will only be effective if there is strong collaboration between health authorities, urban planning agencies, environmental agencies, energy providers, and the transportation systems. climate change is an emerging threat to global public health. it is now widely accepted that climate change is occurring as a result of emission of greenhouse gases, especially from fossil-fuel combustion. climate change is predicted to affect many natural systems and habitats, for example, increasing the frequency and intensity of heat waves, increasing the number of floods and droughts, altering the geographic range and seasonality of certain infectious diseases, and disturbing food-producing ecosystems, which in turn will affect human health both directly and indirectly. direct health effects include changes in mortality and morbidity, and changes in respiratory diseases from heat waves. in terms of indirect health effects, these are much more extensive and include changes in the distribution of vector-borne diseases, the nutritional and health consequences of regional changes in agricultural productivity, and the various consequences of rising sea levels, flooding, and droughts. [ ] [ ] [ ] climate change is highly inequitable, and the paradox is that those at greatest risk are the poorest populations in developing countries who have contributed least to koehlmoos et al greenhouse gas emissions. however, the rapid economic development and concurrent pollution means that developing countries are now vulnerable to adverse health effects from climate change and, simultaneously, are becoming an increasing contributor to the problem. , although the effects of climate change affect all levels and ages of any single population, the elderly and those with preexisting medical conditions are seen as being the most vulnerable. conversely, major diseases that are most sensitive to climate change such as diarrhea, malaria, and infection associated with malnutrition are most serious in children living in poverty, making them highly vulnerable to the resulting disease burden. heat waves are expected to increase the occurrence of heat-related illnesses such as heat exhaustion and heat stroke, and aggravate existing conditions related to circulatory, respiratory, and nervous system problems, especially among the elderly. , in , a major heat wave affected most of western europe and caused additional deaths in england and wales. another consequence of high temperatures is that they raise the levels of ozone and other air pollutants, which in turn aggravate respiratory diseases such as asthma. meanwhile, health impacts due to natural disasters, such as floods, droughts, and storms, range from immediate effects that include physical injury, mortality and morbidity, and communicable diseases, to possible long-term effects such as malnutrition and mental health disorders. from to , flooding was the most frequent natural disaster ( %), killing almost , people and affecting over . billion people worldwide. droughts increase the risk of food shortages and malnutrition, and increase the risk of diseases spread by contaminated food and water, because viral load increases in water sources when levels drop dramatically. rising temperatures, irregular rainfall patterns, and increasing humidity affect the transmission of many vector-borne and water-borne diseases such as malaria, dengue, cholera, and other diarrheal diseases. vector-borne diseases currently kill approximately . million people each year while . million die from diarrheal diseases. studies suggest that by , climate change may put million people in africa at risk of malaria, , and by the s the global population at risk of dengue is likely to increase to billion. , recent published data provides evidence of an association between the el niñ o and la niñ a phenomena, which are major determinants of global weather patterns, and some infectious diseases. evidence shows that there is an association between el niñ o and malaria epidemics in parts of south asia and south america, and with cholera in coastal areas of bangladesh. , studies of malaria have already revealed the health impacts of climate variability associated with el niñ o, including large epidemics on the indian subcontinent, colombia, venezuela, and uganda. one of the most immediate problems related to changes in climate and climate patterns is that on food production and availability. each year approximately . million people, mostly children from developing countries, die from malnutrition and related diseases. it is projected that climate change will decrease agricultural production in many tropical developing regions, thus putting tens of millions more people at risk of food insecurity and adverse health consequences of malnutrition. disasters in certain areas of high food production will also affect global prices, thereby affecting not only those people living in the affected region but others around the world who depend on food produced from that region. the who gbd study in indicated that the climatic changes that have occurred since the mid- s would be having an effect by the year , with , deaths ( . % deaths globally each year) and . million lost disability-adjusted life years (dalys) per year ( . % global dalys lost per year). the estimated effects are predicted to be most severe in those regions that already have the greatest disease burden of climate-sensitive health outcomes, such as malnutrition, diarrhea, and malaria. , , many of the projected impacts on health are avoidable, and public health policy makers need to act to reduce or negate the impact caused by climate change through a combination of short-term public health interventions that aim to adapt measures in health-related sectors, such as agriculture and water management, and long-term strategy. the most effective responses are likely to be strengthening of the key functions of environmental management, surveillance and response to protect health from natural disasters and changes in infectious disease patterns, and strengthening of the existing public health systems. , however, countries need to assess their main health vulnerabilities and prioritize adoptive action accordingly, keeping in mind the costs involved. natural disasters know no boundaries, and any nation or population can be subject to a catastrophic disaster at any time. however, some nations and populations are more at risk of disasters than others due to geographic location, poverty, and several sociopolitical factors. this issue of disaster risk reduction (drr) rose to global prominence in the aftermath of the tsunami in the indian ocean in december . following a disaster, some populations suffer more acutely than others. it is worth considering the complex issues of how societies organize themselves in terms of risk and actual prevention and care, for access to clean water and sanitation, and how they communicate and initiate behavioral change among the displaced or fragile populations. at the forefront of most discussions when planning post-disaster management and action is the priority placed on certain elements of disaster relief, such as the building of embankments, the distance to clean water, or the time from incident to response. recent examples of varying responses and outcomes were seen following the two cyclones in south asia. there was a relative success in bangladesh in terms of lives saved and response coordination after cyclone sidr in november , compared with the devastating loss of more than , lives after cyclone nargis in myanmar in may , not to mention the loss of draft animals and dykes, and the flooding of fields during planting season. bangladesh reverted to its welldeveloped program for drr that includes national-level coordination, whereas in myanmar there was no national platform for disaster preparedness, and delays occurred in the coordination of international response to the disaster. in addition to the immediate and obvious impact of natural disasters, conditions often worsen in poorly coordinated settings, as evidenced in when vibrio cholerae emerged in post-flood pakistan, and for the first time since the s in post-earthquake haiti. in general, there are factors that can turn a natural disaster into a complex disaster regardless of the severity or magnitude of the initiating event such as a hurricane, earthquake, or tsunami. according to the un department of humanitarian affairs, the factors are: poverty, ungoverned population growth, rapid urbanization and migration, transitional cultural practices, environmental degradation, lack of awareness and information, and war and civil strife. poverty is by far the single greatest factor that contributes to the vulnerability of a population to complex disasters. in addition to lacking financial resources to prepare for or recover from a disaster, impoverished people are also more likely to have low levels of education and low amounts of political influence to properly deal with a disaster situation. in addition to increases in birth rates, rapid population growth can be the consequence of urbanization or migration. population growth without limits produces a population that is more likely to settle in areas that are unsuitable or at risk for natural disasters, meaning that more people are at risk of disease and, most importantly, are more likely to undergo civil strife while competing for scarce resources. as mentioned previously, rapid urbanization and migration lead to impoverishment. former rural populations make themselves more vulnerable to disaster by settling in less developed or high-risk city environs, often leading to homelessness or living in urban slums that have circumvented any planning controls or regulations. such populations therefore are made more vulnerable to floods, landslides, and the destruction of their dwelling during a hurricane or earthquake. transition of cultural, economic, or government practices such as the increase in migration from rural to urban areas, economic advancements, families moving away from traditional support networks and to unfamiliar surroundings, and the shift from an agrarian to an industrialized society leave certain societies vulnerable to natural disasters. environmental degradation can play a role by either causing or exacerbating a disaster. for example, deforestation can work in two ways: firstly enabling runoff or secondly, making landscapes vulnerable to storms, due to lack of natural wind breaks. everyone is aware of the natural conditions that provoke droughts, but through the construction of dams, unchecked urbanization, implementation of poor cropping patterns, and the depletion of water supplies, man-made droughts are becoming more widespread. it is clearly of upmost importance to ensure that populations are informed about what to do to prepare in advance of a natural disaster such as a hurricane, and also are able to fend for themselves following the event. a lack of awareness and the dissemination of accurate information is a major factor that can turn one disaster into a multiple or complex disaster involving, for example, subsequent outbreaks of cholera, malnutrition, and physical injury. war and civil strife are extreme events that can both produce disasters or be caused by disasters, normally as a result of the preceding factors. the phrase for disasters that specifically strike war-torn populations is complex humanitarian emergencies. global efforts to address and capture the importance of disaster risk and poverty have been hampered by a lack of data, especially from asia, latin america, and the caribbean. empirical evidence linking disaster risk to poverty tends to come from microstudies within one community, making it impossible to generate generalized findings across regions or entire countries. prompted by the devastation that followed the tsunami on december, , there was widespread acceptance that an early-warning system should be installed and other actions taken to prevent loss of life where possible. the world conference on disaster reduction was held in japan in january , and resulted in the creation of the hyogo framework for action - (hfa), which was endorsed by un member states and urges all countries to make major efforts to reduce their disaster risk by . the hfa outlines the need to increase awareness and understanding about drr, the importance of knowing the real and potential risks, and taking action against them. specific recommendations included the need to create or enhance early-warning systems, build drr into education, and reduce risk factors such as deforestation, unstable housing, and the location of communities in risk-prone areas. although different areas of the planet experience different risks, the one common factor is that drr "concerns everyone, from villagers to heads of state, from bankers and lawyers to farmers and foresters, from meteorologists to media chiefs." to support common needs within regions, associations and networks have been established to support drr, such as the south asian saarc disaster management center and the caribbean disaster emergency response agency. types of activities that can feature in a national or regional drr program can include: establishing early-warning systems; using local knowledge of events; building an awareness of risk and risk preparedness through community activities; building flood-resistant buildings and safe homes; developing contingency plans; helping communities and individuals develop alternative sources of income; and establishing insurance or microfinance programs to help transfer the risk of loss and provide additional resources to the community. in addition to chronic diseases, mental health problems, injuries, and complex disasters, communities should consider increasing risks from more than new or reemerging diseases that have appeared since the s: liver disease due to the hepatitis c virus; lyme disease; food-borne illnesses caused by escherichia coli o :h ; cyclospora, a water-borne disease caused by cryptosporidium; hantavirus pulmonary syndrome; and human disease caused by the avian h n influenza virus. the increasing number of new and reemerging diseases is not the only risk factor that should be added to the planning processes for developing a drr program. drug resistance in treating many diseases and illnesses is a major concern, as witnessed in malaria and tuberculosis, and with a highly mobile world population, global pandemics such as sars, h n , and h n , for which treatments either are not available or levels of suitable drug are clearly not sufficient for a worldwide epidemic, are proving to be very challenging. this clear inability to predict and maintain sufficient levels of treatment for potential threats makes health risk reduction extremely difficult, and in developing countries where resources are already stretched to cope with existing health issues, creating effective programs will require intervention from social partners, global support organizations, and aid from the developed world. an ever quickening pace of globalization means that public health-related problems in one area of the world will have an impact on those living in another area and therefore, it is in everyone's interest to ensure that all countries, irrespective of their economic development and available resources, are sufficiently supported to maintain and review strategies that will effectively reduce morbidity and mortality rates in all spheres of public health. preventing chronic diseases: a vital investment a race against time: the challenge of cardiovascular disease in developing economies non-communicable diseases: time to pay attention to the silent killer. press release missing in action: international aid agencies in poor countries to fight chronic disease when people live with multiple chronic diseases: a collaborative approach to an emerging global challenge the developmental origins of chronic adult disease world health organization framework convention on tobacco control [who fctc]. guidelines for implementation 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evolution of complex disasters united nations-international strategy for disaster reduction united nations-international strategy for disaster reduction [un-isdr] living with risk. a global review of disaster reduction initiatives disaster risk management and climate change adaptation in south asia. dhaka: portfolion new and reemerging diseases: the importance of biomedical research key: cord- -gozt aur authors: tambo, ernest; xiao-nong, zhou title: acquired immunity and asymptomatic reservoir impact on frontline and airport ebola outbreak syndromic surveillance and response date: - - journal: infect dis poverty doi: . / - - - sha: doc_id: cord_uid: gozt aur the number of surveillance networks for infectious disease diagnosis and response has been growing. in , the world health organization (who) established the global outbreak alert and response network, which has been endorsed by each of the who african members since then. yet, taming the dynamics and plague of the vicious ebola virus disease (evd) in african countries has been patchy and erratic due to inadequate surveillance and contact tracing, community defiance and resistance, a lack of detection and response systems, meager/weak knowledge and information on the disease, inadequacies in protective materials protocols, contact tracing nightmare and differing priorities at various levels of the public health system. despite the widespread acceptance of syndromic surveillance (ss) systems, their ability to provide early warning alerts and notifications of outbreaks is still unverified. information is often too limited for any outbreak, or emerging or otherwise unexpected disease, to be recognized at either the community or the national level. indeed, little is known about the role and the interactions between the ebola infection and exposure to other syndemics and the development of acquired immunity, asymptomatic reservoir, and ebola seroconversion. can lessons be learnt from smallpox, polio, and influenza immunity, and can immunization against these serve as a guide? in most endemic countries, community health centers and disease control and prevention at airports solely relies on passive routine immunization control and reactive syndromic response. the frontline and airport ebola ss systems in west africa have shown deficiencies in terms of responding with an alarming number of case fatalities, and suggest that more detailed insights into ebola, and proactive actions, are needed. the quest for effective early indicators (eee) in shifting the public and global health paradigm requires the development and implementation of a comprehensive and effective community or regional integrated pandemic preparedness and surveillance response systems tailored to local contexts. these systems must have mechanisms for early identification, rapid contact tracing and tracking, confirmation, and communication with the local population and the global community, and must endeavor to respond in a timely manner. electronic supplementary material: the online version of this article (doi: . / - - - ) contains supplementary material, which is available to authorized users. please see additional file for translations of the abstract into the six official working languages of the united nations. the current widespread ebola epidemic is estimated to infect , people before it is contained by early . the total number of probable and confirmed cases of the ebola virus disease (evd) in the five affected countries as reported by the ministries of health of guinea, liberia, nigeria, senegal and sierra leone is cases and has claimed more than deaths so far. more than % of the total number of cases have occurred and are concentrated within a few localities. the average case fatality rate is %; this ranges from % in sierra leone to % in guinea. a separate outbreak of the evd, which is not related to the outbreak in west africa, was laboratory confirmed on august by the democratic republic of congo (drc). there have been cases and deaths so far [ ] . priority responses and actions are needed to tackle the ongoing ebola crisis in west africa and this requires improvements in access to diagnostic technologies and healthcare resources, as well as improved surveillance and communication. as it stands, there is little incentive for vulnerable communities to seek professional diagnosis of suspected ebola. most people with the flu and febrile illnesses self-medicate and are treated at home or by traditional healers/practitioners, making difficult to define the true extent and nature of the outbreak [ ] . syndromic surveillance (ss) has been advocated and used to monitor illness syndromes and events, and to detect epidemics and bioterrorist attacks early, thus increasing and ensuring that the response from public health departments is timelier. however, its effectiveness and usefulness in ebola outbreak surveillance remains unclear [ , ] . up until now, no unified definition for ss coupled with limited predictive abilities of emerging diseases and ebola seroconversion with no associated clinical signs has been determined. the source of high rate of health workers and volunteers infections and death is worrisome and urgently required further investigations, probably resulting from poor adherence and poor compliance to protective measures protocols and standard operating procedures, hard to implement standard clinical and laboratory operating procedures in such challenging environments, stress and anxiety poor incentives and lack of health insurance of those high risk health and humanitarian workers and quality assurance of the local and humanitarian protective products, the protocols. a number of remarkable similarities exist between the humoral responses to filoviruses, in particular the ebola virus, and the response to hiv- infections, and these have been invaluable in demonstrating that antibodies can indeed provide protection against a virus [ ] . therefore, the efficiency of frontline and airport ss is compromised by a number of confounders/factors, such as acquired immunity, human-animal host asymptomatic reservoirs, inadequacies in diagnostic tools, infrastructure of health and social support systems, and various biosocial, environmental and climatic factors. these are still poorly understood in ebola bottlenecks and thus hinder the establishment of adequate and reliable responses to prevent new cases, control further infections, and contain the ongoing geo-distribution trend and pattern in west and central africa. it is common in ss that what is to be detected is unknown, and with asymptomatic and acquired immunity ebola appears to present long incubation periods and prodromes. in order to test detection methods, a simple disease simulation model examination of a spectrum of ultraviolet biosensors of temperature (fever) sensitivity and specificity can provide advantages and limitations in frontline as well as airport syndromic surveillance and could be applied at all times in monitoring any early symptoms in the absence of disease outbreak. it is then sufficient to run the detection method on a subset of simulated disease scenarios or on the challenges present in predicting unbiased diseases that are sufficiently different in order to determine the strengths and weaknesses of different methods at the point of care [ , ] . specific definitions for ss are lacking, and the name itself is imprecise. certain programs monitor just surrogate data sources (e.g., over-the-counter prescription sales or school absenteeism) rather than specific disease syndromes. meanwhile, certain well-defined disease or clinical syndromes (e.g., hemolytic uremic syndrome or kawasaki's syndrome) are not included in syndrome definitions, often leading to confusion about what "syndromic" surveillance actually monitors. the different types of public health surveillance systems for early detection of outbreaks include: ( )early warning systems in the region, disease control policies to restrict border crossings, as well as sales and accustomed consumption of bush meats, which have been ineffective implemented and sustained in diseases or outbreak surveillance and response. in addition, the handling of information by politicians, who have a history of partisan gain and constantly manipulate the media for political agendas, has been worsened over the years, underscored by corruption and nepotism. these coupled with weak health programs-continues to jeopardize genuine efforts to convey timely, trustworthy, and reliable information in a language accessible to the most vulnerable and remote communities [ , ] . symptom-based surveillance relies on self-reporting, health-based or routine admission procedures with clinical examinations, specific diagnosis, and recognition and reporting by clinicians to public health authorities/departments. smart phones should play a greater role in these systems as well. laboratories and other bodies in the region have shown that routine ss systems can be designed to rely on mobile phones which have become ubiquitous in west africa. some researchers forecast that mobile internet use in africa will increase -fold in the next five years, and will consequently double the growth rate in rest of the world, and could be of potential use for individual or community-based surveillance. ( )prodromal surveillance of flu or febrile like diseases has low specificity and may have insidious uncertainty based on differential diagnosis and overlapping early clinical multiple syndrome syndemics that trigger false alarms compared to post exposure, which can cause severe or life threatening disability. the type of prodrome varied from one individual to the next based on previous illness trends, pre-immunity, early indicators for response, over counter or self-medications, and various genetic, ecological and environmental factors [ , , ] . in west africa, the poor evidence-oriented approaches and tools, principles, and guidelines for communication before or during an outbreak require pivotal redesigning based on local contexts and taking into account social media and web-based health information and communication. the usefulness of who tools, guidelines, and recommended practices in ebola outbreak prodrome surveillance in this context is underscored by local limitations, gaps in knowledge, and other challenges. prodromal surveillance tools could be very timely and vital in the realm of emergency communication, advice, behavior change, hygiene and sanitation, and provide an integrated grassroots-based participatory approach. this could lead to more effective prevention, thus curbing the spread and containing the outbreak at the different levels of the disease, and at different places in time and space [ , ] . ( )outbreak detection systems urgently require reliable, effective, and cost-effective tools and interventions which allow for constant access to diagnostic tools and personal protective equipment in healthcare centers across the region [ ] . ( )information system-based sentinel surveillance. ( )biosurveillance systems are used in practice to augment classical outbreak investigations. the major advantages of syndromic systems include sensitivity, timeliness, and flexibility, and being able to provide data for situational awareness. however, biosurveillance precincts lack in specificity, rely on chief complaint data, and lack formal training for users. linking syndromic data to triage notes and medical chart data would substantially increase the value of biosurveillance in outbreak investigations and thus reduce the health burden [ , ] . ( )laboratory-guided detection of disease outbreak surveillance systems relies on detection and monitoring of biothreats enabled by laboratory methods of diagnosis and identifies trends in biosurveillance research. it is based on three approaches, namely: ( ) laboratory-initiated infectious disease notifications, ( ) ss based on health indicators, and ( ) genotyping-based surveillance of biothreats. the insufficient and delayed support for biosurveillance alerts for public health users and the inadequate integration of surveillance signals into action plans remain the major barriers, and require coordination between syndromic and laboratorybased surveillance for efficient public health outbreak monitoring and response [ ] . ( )health indicator surveillance provides authorities with vital health indicators such birth rates, motility rates, and life expectancy. however, trustful and reliable information and communication, health education on preventable fatalities and cautious behavior are also required in order to prevent fear, panic and community resistance to stem out the spread of the disease outbreak. ( )digital or electronic bio-epidemiology surveillance systems, including social media networking and web-based systems, provide valuable channels for timely collection of public health data; give information on the early detection of, and response to, disease outbreaks; and enhance situational awareness to communities. the creation of blogs and user-generated content has turned social networking into a conversation space in which everyone can participate. however in west africa, the low level of literacy and high inequality indices compromised the usefulness of such tools to trace and map the ebola outbreak, as compared to their usefulness and effectiveness in a community with high literacy, for example during the sars and h n influenza outbreak in hong kong and mainland china. social media (e.g., twitter, facebook) and web-based communication provide epidemiological knowledge dissemination, and creates virtual communities based on shared values about critical outbreak perceptions, seriousness of the crisis, and population evidence-based guidance. however, it is not subject to experts or authorities' advice and assessment, and doesn't receive guidance from associated communication or information risk management and security. this requires further development and attention. the implications of social media and web-based information and communication in creating fear, anxiety and stigmatization about ebola in some communities in west africa are noteworthy. during this outbreak, web-based activities were also responsible for fuelling rumors that led to counter-productive behaviors. improved communication between reliable health officials and the media, community leaders, health professionals, and the general public is necessary to reduce misinformation and improve compliance with ebola prevention and control measures that have proven effective. these include population dynamics of emerging infections and the optimal design of monitoring and management strategies in prevention, control, and containment of ebola in other countries in africa [ ] . nonetheless, the term "syndromic surveillance" (ss) has persisted to describe this kind of surveillance as its fundamental goal is to identify signs and symptoms of illness clusters early before diagnoses are confirmed, report to public health authorities or agencies, and mobilize responses rapidly. syndromic surveillance targets the threshold number of early symptomatic cases allowing outbreaks to be detected earlier than conventional reporting of confirmed cases would allow [ , , ] . response protocols for investigating ss alerts present some limitations in most endemic countries with syndemics acquired/partial immunity, diagnosis and identification of co-infections clusters, and sources of exact human-animal reservoirs [ ] . contact tracing and epidemiological case investigation of the nature and severity of the outbreak could provide timely and scientifically reliable information to curb the risk of propagation. hence, more effective surveillance and response systems such as point-of-care ebola molecular typing and immune-detection assays and rapid diagnostic kits in frontline and airport detection are urgently needed. for ebola outbreaks, ss is able to provide the early symptom (prodrome) period before clinical or laboratory confirmation of the disease, as is explained below, although difficult in endemic areas in africa where many tropical diseases with similar and/or differential signs and symptoms co-exist. ebola will continue to be a global threat if prompt and effective commitment is not directed towards control and containment. surveillance and response systems are of interest in public health and veterinary epidemiology for the early detection of the emergence or re-emergence of infectious diseases. in relation to several confounders of ebola outbreaks such as flu-like signs and symptoms of unknown sources, ss, which consists of the routine monitoring of indicators to detect adverse health events, may allow for early detection depending on continuous indicator measurements and sensitivity and specificity (timeliness tools for the detection or diagnosis of diseases outbreak emergence). the limitations in detection and spot diagnosis of asymptomatic reservoirs and preexisting immune confounders have been the challenges since ebola broke out guinea in december , was detected in march , and finally spread to liberia, sierra leone, and nigeria. it is the most severe outbreak of ebola since the discovery of the virus in , with the number of cases from the current outbreak outnumbering the combined cases from all known previous outbreaks. the who has declared the ebola outbreak in west africa to be a public health emergency of international concern and called for action [ , ] . below, we outline the characteristics of the ss approach. ( )the concept and application of ss is doubly attractive because in addition to its potential to increase the speed and effectiveness of the public health response to natural or deliberate disease outbreaks with a certain degree of assurance, it costs far less to implement than traditional, labor-intensive approaches to disease surveillance (both should complement each other) [ ] . however, the ability of ss to reduce disease-related morbidity and mortality remains to be demonstrated, as does its cost-effectiveness and warning devices. it will be critical to assess its utility, sensitivity, and accuracy in outbreak or bioterrorism within the context of health systems that respond to both "true" and "false" alarms in infectious disease syndemic settings. this involves the collection of information and clinical data that might indicate if an infectious disease outbreak might be happening in the community and whether it warrants further public health response. before an outbreak occurs, little is documented in health centers and by airport active ss, except for passive checks of yellow card immunization for bcg, polio, and hepatitis vaccines programmes, in addition to medical referrals for passengers requiring medical or surgical interventions abroad. during the ongoing outbreak of ebola in west africa, ss, along with collaborative efforts between local health departments, has been used on patients, ground staff, health workers, passengers across community/national borders, and airports across africa and in some other major airport hubs worldwide. ( )this approach is confronted by a lack of effective and accurate spot invasive frontline and airport rapid diagnostics tools, district and provincial health laboratories being equipped with little or no advanced molecular technologies, lack of drugs and vaccines to treat ebola, inadequacy in coordinated ebola frontline planning efforts in the community, as well inefficient or nonexistent community and national active infectious disease surveillance systems. syndromic surveillance systems monitor existing descriptive data of these behaviors (e.g., school and work absenteeism, sales of over-the-counter medications, illness-related information, emergency room admissions for symptoms indicative of infectious diseases) for patterns or clusters of behaviors suggestive of an illness outbreak [ ] . hence, ss is not sufficiently equipped to control and contain ebola in africa due to its complex web of interactions and challenges. the usefulness of laser thermal detection of febrile state or other characteristic symptoms of individuals in frontline and airport surveillance systems could be very challenging in hyper-, holo-, and meso-endemic settings, and present several limitations with the rampantly increasing confounders of poverty-related diseases in africa and elsewhere. in addition, it should be noted that several factors such as travel syndrome, menopausal or post-menopausal syndrome in women and other lifestyle stressors associated with an increase in temperature-although normal-can trigger false alarms. moreover, syndemics and partial acquired immunity in the region poses concerns about the spread and the burden of the disease due to asymptomatic reservoirs and the long latency period of infection [ , ] . efforts should be devoted to enhancing research and developing innovative, more sensitive detection and diagnostic tools for early-stage epidemic warning and preparedness in frontline and airport spot surveillance mechanisms and response, rather than increasing the use of empirical broad-spectrum detectors. the bottlenecks to ebola outbreak frontline and airport syndromic surveillance and response systems diagnosis is "the cornerstone of effective outbreak and disease control and prevention efforts, including surveillance" [ ] . current challenges in diagnosis of ebola by frontline and airport surveillance systems underscored the existing detection and diagnostic tools, and highlighted the importance of combining diagnostic needs with appropriate technologies. the need for rapid, accurate, inexpensive, and robust diagnostic low-detection thresholds can be met by recent advances in genomics, proteomics, and material science; profitable public-private partnerships; and sustainable profits in low-resource settings. the continued development and deployment of efficient, low-cost diagnostic platforms is essential for containment. detection methods suitable to local/international standard laboratories or sentinel for imported cases epidemiology must be validated prior to transition from malaria sustained prevention and control programs and interventions. the importance of developing and implementing sensitive diagnostic approaches to accurately quantify and monitor ebola reservoirs is imperative in curbing the persistent transmission dynamics, and preventing, controlling, and containing the disease given africa's engagement in achieving the millennium development goals (mdgs), and in the london declaration international health regulations ( ), the universal human rights declaration, and the new partnership for african's development (nepad) in africa. however, a number of challenges remain to be overcome before deployment of rapid, low-cost, sensitive, and specific point-of-care disease diagnostics become a reality. spot frontline and airport surveillance using laser imaging of febrile conditions with a latency period of - days for clinical manifestations require rethinking, more research, and funding for the development of simple, rapid, field adaptable, and effective detection tools in asymptomatic, presymptomatic, and symptomatic cases, to be used in addition to spot airport passengers' diagnostic kit(s). immune variability across african countries with syndemics is poorly understood, although it is believed that populations develop varied degrees of acquired/partial to complete immunity resulting from repeated exposure to infectious diseases, and can carry a certain load of virus for months or years before it becomes a clinical manifestation of the ebola disease [ , ] . the concept of immunization or vaccination as described by edward jenner ( - ) observed that people with cowpox infection developed immunity to smallpox, with several lifelong survivors. hence, smallpox was declared eradicated in the wild in after a worldwide immunization campaign took place similar to the ongoing polio eradication with effective immunity response to outbreak depending on the age and level of individual antibodies (cell-mediated immunity) and protective threshold. consequently, large asymptomatic population-animal reservoirs in the case of the ebola infection in africa may not be surprising, and further screening of ebola exposed and non-exposed populations is required. what are the lessons and challenges learned in shaping future research priorities? this makes the need for in-depth knowledge on the exposure of viral infectious (e.g., measles, yellow fever, chickenpox, or hiv/aids) even more urgent as this can contribute to ebola resurgence and the quest for an ebola vaccine [ ] . genetic and clinical variability have shown that genetic make-up or traits vary from one ethnic group to another, and within and between populations with different clinical manifestations, but the relationship between population genetic changes and ebola seroconversion and progression over time and space is still poorly understood. the precise role and efficacy of biosurveillance in public health has yet to be determined, as well as the limitations of ss systems to detect ebola infection or other outbreaks. health professionals should continuously aspire to accurately diagnose and treat patients, as well as to identify public health outbreaks or emergencies, combined with adequate local integration of infrastructure, facilities, and capacity building. environment, ecological, and animal interface, and encroachment factors due to landscape use and misuse, mining, deforestation, forest degradation, wildfire, conflicts/wars, and man and animal interactions also require further research. . infrastructure and capacity of health and social systems challenges in ebola control and containment in west africa are obvious due to a lack of humanitarian response models for fragile and under resourced health systems, and the local government and affected community's inability to contain the wide spread of the disease. these challenges include: insufficient regional and international political commitment, insufficient resources and funding, lack of an ebola vaccine or drug, detection and diagnostic limitations, and a lack of resources or infrastructure to support such activities. additional challenges include inadequacies in programs and approaches, weak or nonexistent primary healthcare infrastructure, poor access to health facilities, and a lack of effective mental, traumatic or neurological assessment tools, as well as national and regional functioning early-warning alert and surveillance response systems. other associated factors include social media and web-based information and communication; an artificial country colonial landscape demarcation and barriers amongst african countries with cross-border families; marriage; employment and commercial/trading activities; sociocultural realities and practices; attitudes to care seeking and utilization; environmental and ecological risk factors; human-animal migration and movement dynamics; conflicts/wars and violence in the region; intense mining activities in the region with an associated impact on the political sphere; and the socioeconomic, ecological, and epidemiologic impact of ebola and others infectious and chronic diseases. . acquired or partial immunity of local populations due to the scarcity of data on immune parameters and exposure doses, the exact impact of the disease on humans is hard to quantify. evaluating 'acquired immunity' may improve outbreak estimates when evaluating the risk of microbial illness from food or environmental exposures. this suggests that some current approaches may significantly overestimate their role in causing such illnesses. immune status is a major factor in susceptibility to disease outbreak, and the impact of acquired immunity to a pathogen needs careful insight when assessing the potential health risks of outbreaks and other infectious diseases of different sources of exposure, including ( ) low-frequency, low-dose exposure (recreational water); ( ) low-frequency, high-dose exposure (consumption of raw chicken liver); ( ) high-frequency, low-dose exposure (direct contact with sheep and goats, i.e., farmers); and ( ) high-frequency, high-dose exposure (visiting petting zoos, wildlife hunters or bush meat sellers/consumers). the public health community should also take acquired immunity into account in order to improve estimates of the potential impacts of infectious diseases and to assist in preventing and managing outbreaks. further studies to better characterize and quantify the effects of acquired immunity on ebola outbreaks are also needed. in humans, there may be apparent ecological, ethnic susceptibility and geographical landscape variations, but it is always important to disentangle such factors-as well as climate, nutrition, environmental, and economics drivers-from those that might be genetically determined in both animal and human transmission dynamics. ongoing efforts to control and contain the ebola outbreak have been limited by the estimated mass asymptomatic population and animal-human reservoirs, which enhance the tenacious transmission dynamics between and within some communities, provinces, and across african countries and elsewhere. viral infectious diseases such as hiv/aids, hepatitis coupled with malaria, tuberculosis, and other neglected and emerging infectious diseases are rampant in africa [ ] . assessment of transmissibility requires tools that can accurately identify the various developmental stages of the animal-human and/or human-human interphases. moreover, in most epidemic areas, asymptomatic carriers are not uncommon and, as potential carriers, represent a significant reservoir for ebola transmission regardless of successful local interventions. these are the challenges to the current humanitarian and national prevention, control, and containment programs [ ] . many of these asymptomatic infections may be present at densities below the limit for microscopic and rapid diagnostic tests threshold detection and thus lead to underestimation persistence of the epidemic burden and probably resurgence. there is very limited, if no, accurate information or data available on submicromolar asymptomatic carriers or presymptomatic surveillance, the detection and diagnosis responsible for ebola virus survival, and persistent transmission on susceptible populations. paucity information pertaining to the current status of the effectiveness of microscopic and rapid diagnostic test tools necessary for ebola control and containment interventions, except for molecular confirmation of cases done in very few selected research centers in africa and across the country since the first ebola outbreak in is also lacking. the development and deployment of active surveillance at all levels coupled with monitoring and evaluation (m&e) of outbreak risk factors and transmission dynamics in early active detection asymptomatic and presymptomatic cases, as well as prompt management of either local or imported cases, is paramount to understanding the viral seroconversion dynamics in suspected communities and travellers in africa and worldwide. sensitive and effective serological, immunological, and biochemical ebola biomarkers that can be used in these remote communities with uncertain or low animal and population reservoirs alongside spot airport testing, mass deployment in mapping geographical distribution, evidence informed policy decision, and prompt interventions-are also essential. understanding the ebola epidemiological trend and patterns including reservoirs and transmission dynamics can provide valuable information for the success of ebola control and containment strategies. although ss response systems are able to detect ebola outbreaks earlier than traditional surveillance, it will be more efficient for these systems to prepare for the standard operational protocol to avoid unexpected occurrences of events. therefore, we recommended the strengthening of the following activities in order to improve the frontline and airport ss responses to ebola outbreaks: . improving case investigation, tracking of susceptible populations, and quarantine period most african countries are challenged by insufficient or nonexistent facilities, a lack of qualified personnel, and the vicious cycle of poverty. cultural practices and myths, challenges in african traditional and alternative medicine implementation in healthcare systems, and attitudes towards health seeking should also be noted. the poor landscape mapping, rural and urban town planning, and especially the poor or nonexistent accessible roads to these communities are other contributing factors. tracking can be very difficult in areas with poor documentation habits, lack of appropriate reporting or a contact tracing system, uncontrolled migration and population movement across borders, unlimited cross-border marriages and trade, as well as animal in-and out-flow of foreigners at the entrance or departure terminuses (airport) in africa and elsewhere. the porous nature of west african country borders stresses the need for automated robust, high-sense human and animal health and movement detectors, in partnership with communities and governments so appropriate data can be collected to answer essential ebola questions. public health surveillance and powerful analytical tools are needed to accurately interpret the findings. the strengthening of the epidemiological capacity through surveillance response systems at the local level needs to be advocated in order to inform the interpretation of syndromic findings in light of "local epidemiological peculiarities," as well as to ensure a rapid response to syndromic alerts. under enabling conditions, community-based mobilization and empowerment in recognizing, informing and active case investigation and contact tracing could build strong relationships between public health and healthcare providers in effective early alert, prevention, control of current and future outbreaks. these relationships are critical for reliable and effective emergency outbreak response and follow up epidemiological investigation, and for evidence policy-building regardless of the type of intervention [ , ] . . nurturing "one health and one world" surveillance and response systems infectious diseases primarily affecting animals can have direct and indirect impacts on humans including significant economic consequences. two important factors can contribute to the proliferation of zoonotic diseases: the explosive growth of human and domestic animal populations, and the increasingly close physical proximity within which humans and domestic and wild animals live [ , ] . timely identification of current and future emerging microbial threats (on the order of sars, the west nile virus, and h n avian influenza) will require an integrated international approach to disease surveillance. however, progress has been hampered by a variety of mining, environmental, climatic, socioeconomic, and political factors, in addition to a weak and fragile or nonexistent surveillance infrastructure and technology, and inadequate expertise in africa. success in ebola control and containment requires a comprehensive and integrated strategy in human disease surveillance among the underserved populations that live in close contact with bat fruits, gorillas, and other wildlife animals. this strategy should incorporate capacity building, training, and empowerment of the local community by integrating simple data collection with basic laboratory diagnosis to identify the link between human outbreaks of the ebola virus, and poaching, the consumption of bush meats, basic hygiene measures such as hand washing and cooking meat thoroughly, overall food safety in communities, and early warning of outbreaks in animals [ ] . in view of the recognized potential of ss systems, there are many practical concerns about sensitivity and false-positive rate trade-offs and the time required to accumulate enough evidence of an outbreak to trigger a detection algorithm, as well as on the available control strategies of the local, national, and regional public health practice and utilization of these systems. the broad and multifaceted practices of surveillance approaches are used to monitor the progress and outcome of interventions to mitigate or stop the progression of an outbreak, including of economically and ecologically important animal or plant species and the transmission of zoonotic diseases among animal and human populations over space and time, as well as to predict future transmission patterns [ , ] . currently, disease outbreak surveillance and detection relies heavily on the astute individual: the clinician, the veterinarian, the grower, and the livestock manager noticing both routine and suspicious symptoms and bringing them to the attention of the public health or veterinary community including academics and zoological parks. most developed countries have a surveillance system in place and the ability to detect and diagnose human and animal diseases. innovations and strategies for the surveillance and detection of human and animal diseases, and assessing the resource needs and opportunities for improving and coordinating infectious disease surveillance, early detection, tracing, case investigation, prompt reporting, and management are needed in upholding health systems strengthening and future sustainable development in most under resourced countries [ , ] . technological advances in disease surveillance and detection that have benefited public health surveillance such as rapid, automated, and sensitive biosensors; portable sampling and assay systems; and dna-based diagnostic tools remain to be adapted to track animal diseases. models and interventions incorporating m&e systems and true coordination and collaboration would enable optimal surveillance response, thereby driving policy and action, with a feedback process to facilitate continuous evolution and adaptation [ , ] . information would be drawn from a broad range of disciplines relevant to physical and mental health, as well as domestic and wild animal health and plant health, through the complementary processes of agent or disease surveillance and host and environmental monitoring with potential economic benefits of surveillance systems for all. nevertheless, the release of surveillance information should be evaluated on a case-by-case basis, as trust is not built by merely sharing data, but by helping people understand information that is context-specific. active engagement to discuss the perception of risk of outbreak and identifying priorities for action is also essential. community or regional active surveillance systems, new effective rapid diagnostic methods, and prompt reporting have the potential to advance infectious disease control and prevention efforts in africa and elsewhere. although, the electronic surveillance system for the early notification of community-based epidemics (essence), operated by the department of defense, allows epidemiologists to track-in real-timesyndromes reported in daily data feeds from regional hospitals and clinics, it is yet to be actively implemented in most africa countries. due to the persistent outbreaks of infectious diseases across africa, it is crucial to analyze the applicability of surveillance response systems in order to improve the ability of hospital/health center triage systems to identify and appropriately treat patients who show symptoms associated with an emerging infectious disease, a threat of an infectious disease (e.g., influenza, sars, and ebola, as well as potential bioterrorism agents such as anthrax and smallpox), or an emerging infectious disease. laboratory diagnosis may be possible by building a network of information about early warning alert and response systems that travels up or down the public health hierarchy, from the local to the international level and vice versa [ ] [ ] [ ] . . shifting towards an effective public and global health paradigm due to globalization, health for all under the "one health" initiate calls for immediate ebola and other outbreak actions plans. a future, in which outbreaks and bioterrorism agents are continually reengineered to evade standard detection and diagnostic methods, as well as therapeutics, is imagined. hence, africa and the global community has no choice but to move from postsymptomatic to presymptomatic detection and diagnosis, and to prompt effective surveillance response systems that seek to benefit the global community. ultimately, to reach the best-case scenario stage in which microbes are ubiquitous, constantly evolving, and adapting requires community and national surveillance policies to inform and guide action on the basis of importance, not for reaction and emergency to dictate priority [ , ] . there is no magic bullet for changing paradigms; steady progress, albeit being slow, can be made through small successes. this needs to be properly recognized as an effective engine for change to educate the next generation of leaders early in their careers and encourage greater global, inter-, and trans-disciplinary awareness in future public health professionals. the quest for eee in shifting the public and global health paradigm to achieve the mdgs post the - agenda, the "one health, one world" and other global health initiates requires: community outreach and advocacy, and local and international mobilization to combat outbreaks in africa and globally. multidisciplinary approach studies to understand the drivers, determinant dynamics, and risk factors of persistent ebola outbreaks. strengthening south-south and public-private partnerships to build local capacity, health education, and empowerment in health and environmental community health for sustainable development. more research in host-based early-warning alert models and understanding of the contribution of context, culture, and ecosystems on asymptomatic/ presymptomatic factors in ebola pre-exposure diagnosis prior to the appearance of symptoms. monitoring a person's blood serum chemistry for changes that suggest a compromised health status or non-invasive sampling of breath and saliva is attractive in theory. rapid molecular markers for mass population screening and diagnosis-based triage and increasing the effectiveness of quarantine or other social distancing measures including the development of synthetic antibody techniques for monitoring infection-related changes in protein levels. monitoring the biological signatures of infectious disease devices: easily accessible (e.g., in the home), robust, inexpensive, and capable of quickly measuring thousands of ebola outbreak spatiotemporal minimum effective data for mining variables in understanding the progression from asymptomatic to clinical ebola cases and forecasting future ebola trends and geo-distribution. more infrastructure and facilities in rural and remote areas, especially in mining african countries, as well as research and development (r&d) funding for ebola drug and vaccine development. development and implementation of country and cross/regional active and integrated communitybased surveillance response systems and m&e initiatives to formulate alternative and innovate community/national recovery and rehabilitation programs, measures, and interventions post-ebola outbreak surveillance and response systems. given the considerable interdependence of surveillance, detection, and diagnostic activities and infectious diseases, it is not surprising that the key challenges identified in this paper can be overcome by innovative surveillance strategies and future prospects as described above. early detection is essential to control and contain the spread of the ebola outbreak. a disease such as this-in a profoundly interconnected world-requires active vigilance for rapid recognition, and prompt diagnosis, case investigation, and tracking of its causes and sources, as well as the mitigation of reliable and robust strategies and resources for an appropriate and efficient response. this paper illuminates the major gaps in frontline and airport ebola control and containment, and provides structured opportunities for leaders, governments, academia, industry, and stakeholders to more robustly mobilize and combine resources. we examine issues of shared concern regarding research, prevention, detection, and management of the ebola outbreak and other emerging and re-emerging infectious diseases. additional file : multilingual abstracts in the six official working languages of the united nations. effective early indicators; evd: ebola virus disease; hiv/aids: human immunodeficiency virus infection and acquired immune deficiency syndrome; mdgs: millennium development goals; m&e: monitoring and evaluations; r&d: research and development; sars: severe acute respiratory syndrome; ss: syndromic surveillance; who: world health organization ebola virus disease in west africa -no early end to the outbreak world health organization: no early end to the ebola outbreak syndromic surveillance and bioterrorism-related epidemics pre-and postexposure prophylaxis of ebola virus infection in an animal model by passive transfer of a neutralizing human antibody veterinary syndromic surveillance: current initiatives and potential for development evaluation of animal and public health surveillance systems: a systematic review use of syndromic surveillance at local health departments: movement toward more effective systems biosurveillance in outbreak investigations syndromic surveillance: review and prospect of a promising concept laboratory-guided detection of disease outbreaks: three generations of surveillance systems developing open source, self-contained disease surveillance software applications for use in resource-limited settings ebola in sierra leone: a call for action disease outbreak news syndromic surveillance: is it a useful tool for local outbreak detection? scaling up impact of malaria control programmes: a tale of events in africa and people's republic of china from prioritizing research for "one health -one world global infectious disease surveillance and detection: assessing the challenges-finding solutions, workshop summary. institute of medicine persistent infection with ebola virus under conditions of partial immunity spatiotemporal environmental triggers of ebola and marburg virus transmission need of surveillance response systems to combat ebola outbreaks and other emerging infectious diseases in african countries elimination of tropical disease through surveillance and response surveillance-response systems: the key to elimination of tropical diseases submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank all of our researchers, field workers, and stakeholders, including humanitarian organizations and volunteers on the frontline of ebola and other outbreaks prevention, control, and containment worldwide, for all their hard work. this paper is especially dedicated to all the health workers and volunteers that contracted ebola and lost their battle with the disease. no funding organization supported this work. the authors declare that they have no competing interests. this study was conceived and designed by et. zx provided guidance on the technical aspects of the study. et and zx provided additional detailed scientific and technical information and commentary on the manuscript. et assembled the final version and performed extensive revisions. all authors read and approved the final manuscript. key: cord- -z n ys authors: murray, jillian; cohen, adam l. title: infectious disease surveillance date: - - journal: international encyclopedia of public health doi: . /b - - - - . - sha: doc_id: cord_uid: z n ys world health organization retains copyright in the manuscript and provides elsevier the permission to publish the manuscript as a chapter in this book. infectious disease surveillance is an important epidemiological tool to monitor the health of a population. the goals of infectious disease surveillance are threefold: ( ) to describe the current burden and epidemiology of disease, ( ) to monitor trends, and ( ) to identify outbreaks and new pathogens. first, describing the burden and epidemiology (including seasonality, age distribution, age groups, etc.) of disease is critical for demonstrating the need and advocating for interventions, such as vaccination and mass drug administration. surveillance is also used to detect antimicrobial resistance in certain pathogens (for example, fluoroquinolone resistance in gonorrhea) and the circulating strains of disease, which helps target vaccine interventions (for example, annual influenza vaccine composition). second, infectious disease surveillance is used to monitor disease trends, such as the impact of interventions like vaccination. disease trends do not only mean the number of cases, but also the etiology of cases. for example, after pneumococcal conjugate vaccine introduction, the distribution of serotypes causing disease should be surveyed for serotype replacement, when the incidence of disease caused by serotypes not covered in the vaccine may increase following the decline in disease due to vaccine serotypes from vaccination. information garnered from vaccine effectiveness studies can be coupled with burden and cost information to describe the cost-effectiveness of interventions. surveillance also monitors the control, elimination, and eradication of diseases. disease control refers to reducing the incidence of disease to a desired level (which will vary depending on the disease) and includes diseases such as malaria (dowdle, ) . disease elimination is defined as zero disease in a defined geographic area as a result of control measures. progress toward disease elimination requires control measures to stay in effect. disease eradication is defined as zero disease globally as a result of control measures, which are no longer required. smallpox is the only human disease and rinderpest is the only animal disease that have been eradicated from the world, but efforts are underway to eradicate polio and dracunculiasis. finally, a key aspect of infectious disease surveillance is the cycle of detecting, responding to, and preventing outbreaks. ongoing surveillance for an outbreak-and epidemic-prone disease can facilitate early detection of an outbreak, allowing a more rapid response and therefore mitigation of the outbreak. epidemic meningococcal disease in the meningitis belt in africa requires ongoing surveillance to identify outbreaks in the region. cholera surveillance is maintained globally to detect outbreaks and requires mandatory reporting to the world health organization (who). emerging and reemerging diseases also pose a big risk to public health. these diseases include both unknown pathogens that appear for the first time in a population as well as known pathogens that increase in geographic spread or severity or are reintroduced into the population. the zika outbreak in south america in - demonstrates how rapidly a known pathogen in a naïve population can spread. infectious disease surveillance can have different approaches based on the epidemiology and clinical presentation of the disease and the goals of surveillance. we will discuss some distinctions between infectious disease surveillance methods and give examples below. in passive surveillance systems, medical professionals in the community and at health facilities report cases to the public health agency, which conducts data management and analysis once the data are received. public health staff do not engage in identifying cases but rather assess data completeness and reliability of the reported cases. in contrast, active surveillance requires public health staff to engage actively in the system and take action in order to receive reports of disease cases. this may involve calling or visiting health facilities to encourage follow-up or having staff review medical records to identify cases meeting prescribed case definitions. active surveillance aims to detect every case, and passive surveillance likely misses cases due to the reporting structure. although active surveillance is more comprehensive, it requires significant human and financial resources, so passive surveillance is often implemented. notifiable disease surveillance is an example of passive surveillance. notifiable diseases are classified as such because they are of public health importance: they can be a severe risk to human health, outbreak prone, considered to be an emerging or reemerging disease, or have a timely intervention available for control of the disease. countries mandate which diseases are notifiable, many of which are infectious diseases. globally, the who, as described in the international health regulations, defines what is notifiable by every country to who, such as mers-cov (middle east respiratory syndrome coronavirus) and ebola. nationally notifiable diseases depend on the country. in the united states, the centers for disease control and prevention (cdc) and the council of state and territorial epidemiologists compile a list of diseases that have mandated reporting to the cdc. these include foodborne and sexually transmitted infections, other infectious diseases such as dengue, malaria, and hiv, and noncommunicable diseases such as cancer. the list is updated every year. on january , the list was amended to include zika virus disease after an outbreak in south america resulted in cases being imported into the united states. although not commonly used for surveillance purposes, administrative data or vital statistics are another example of routinely gathered data that can be used as passive surveillance. the international classification of diseases (icd- ) is used globally for the standard naming of diseases in hospitalized patients. administrative data such as hospital billing data using icd codes can be used for syndromic surveillance, such as for pneumonia, if it is available for ongoing monitoring of disease. these data may provide information on the clinical characteristics of patients across different regions and hospitals. active surveillance can have many approaches, including country-wide (e.g., for polio, measles, and rubella) or restricted to sentinel sites that capture cases within a demined catchment population. for example, as of , the whocoordinated global invasive bacterial vaccine-preventable disease (ibvpd) sentinel site surveillance network is a system of more than hospitals in more than countries that conducts active surveillance for meningitis, pneumonia, and sepsis ( figure ). within this network, staff are engaged to work specifically on finding all cases meeting the case definition at the sites. cases are enrolled into surveillance after they have been admitted to the medical facility. a case report form, filled out by the dedicated staff member, details their clinical symptoms. laboratory testing is done at the hospital for initial diagnosis or at a national or regional reference laboratory to monitor for trends in culture positivity and serotype/serogroup incidence. these data are reported to the ministry of health and who. data are analyzed to look at trends of disease, including pre-and postintroduction of vaccine. other diseases that have globally coordinated sentinel surveillance networks include rotavirus, influenza, and congenital rubella syndrome. surveillance for some diseases can be a mixture of passive and active surveillance wherein passive surveillance is complemented by active surveillance to investigate outbreak signals detected through passive surveillance. for example, surveillance for ebola virus disease is ongoing throughout the year as it is a notifiable disease for many countries and globally. during an outbreak, active case finding in the community is enacted to find symptomatic patients as well as contact tracing to find those at risk for developing the disease. choosing where to conduct surveillance is based on a number of considerations: how severe is the disease and how does it present? how important is it to find every single case? how outbreak prone is the disease? cases of infectious disease can be identified at medical facilities (hospitals and outpatient clinics) or in the community. the location of individuals enrolled into surveillance can vary based on clinical presentation of disease and access to health care. more severe cases of disease can often be identified at hospitals. hospitalized cases are those that are severe enough to be admitted to the hospital for treatment and have the resources to seek care. hospitalized cases can be enrolled prospectively or retrospectively when a case report form is filled out based on their medical chart. identifying cases in hospitals can be easier than identifying cases in the community, but the cases may only represent a small proportion of cases and miss cases that do not seek health care. an example of hospital-based surveillance is severe acute respiratory illness (sari) surveillance for influenza. for diseases like ebola, where fear in the community might prevent cases from going to seek health care, hospital surveillance would be insufficient alone. individuals with milder cases of disease may also seek medical care, such as at outpatient clinics, so surveillance may be conducted at medical facilities outside of hospitals, such as with influenza-like illness (ili) surveillance. some cases of disease are so mild or the patient's situation is such that they will not be able to seek care at medical facilities. in that case, community-based surveillance can monitor disease outside of health facilities, such as at schools, homes, traditional medicine practitioners, and other community facilities. this type of surveillance aims to capture cases beyond those that are admitted to a health facility, therefore enrolling a wide range of disease severity and access to medical care. community-based surveillance is useful for surveying diseases targeted for eradication because all cases must be traced and is not limited to those severe enough to be admitted to a hospital or those that have access to a health-care facility. acute flaccid paralysis (afp) surveillance is an active surveillance network that aims to identify every case of polio, which is currently targeted for eradication. suspect cases are sought in the community and at health facilities to identify any unreported cases. this type of surveillance was also a method used in the ebola virus disease epidemic of - . community members and volunteers would report individuals with symptoms meeting the case definition for ebola, who would then be visited by health personnel for testing. community-based surveillance was supplemented by contact tracing, where individuals who had been in contact with confirmed ebola patients were sought out in the community and monitored for symptoms. a sentinel surveillance site is a single or small number of health facilities that are responsible for collecting data on cases enrolled with the case definition under surveillance including global networks surveying for diarrhea or pneumonia. most sentinel sites do not have a predefined catchment population (or denominator to calculate incidence), and therefore data at these sites are simply numbers of cases (numerators). sentinel site surveillance provides useful epidemiological information on proportions caused by different pathogens, age distribution, and risk factors and could also be used for monitoring trends of hospitalized cases within a health facility if health-care patterns and population have been stable. furthermore, these data may be used in case-control studies to assess effectiveness of a vaccine or other preventive measures. surveillance focused on one or a small number of surveillance sites often allows for gathering more data of higher quality. in contrast, with population-based surveillance, every appropriate health facility reports on the predefined diseases with the goal of identifying all cases in a specific geographic area. population-based surveillance can either represent the whole country (national) or a defined subnational population area. since the population is defined, these surveillance sites can produce rates of disease (for example, incidence and mortality rates), which allows for comparison of rates of disease between other population-based surveillance sites. population-based surveillance is more costly than sentinel site surveillance, but produces more generalizable data on incidence of disease. aggregate surveillance data can exist in a variety of forms, but the main feature is that it lacks detailed information on specific cases. aggregate data typically include the number of cases (for example, number of suspect and confirmed neonatal tetanus cases, or by age group) for a specific region and time period. this information can monitor the number of cases but lacks the individual-level data required for specific analyses. an example of this is the integrated disease surveillance and response (idsr) system which asks clinicians to report the number of cases of specific diseases. case-based surveillance refers to surveillance systems that collect information about each case at the individual level. this type of surveillance system has a case investigation form where information can be gathered from the patient or their family members, their medical records, and their laboratory records. at a minimum, more detailed information on person (who is infected), place (where they live, where they might have been infected), and time (when they became ill) is collected. a line list from this investigation form is created and reported up their normal reporting channels. in some scenarios, a case-based surveillance system might transition to aggregate as the number of cases becomes large as it overwhelms the system, like what happened during the h n outbreak. in contrast, an aggregate surveillance system might become case-based temporarily in an outbreak to understand more of the epidemiology of the disease. certain diseases, such as polio and measles, are recommended to be case-based. measles surveillance has seen a movement away from aggregate and toward case-based surveillance (who, ) . initially, when the united nations (un) development goals were established in , measles was endemic in many countries, and mortality reduction was the primary goal. given this, aggregate data were the most feasible approach and were conducted in most countries. by , all six who regions have measles elimination goals. as measles has moved away from control and toward elimination, case-based surveillance is needed to ensure every case is reported and investigated. when disease was endemic, case-based surveillance would quickly be overwhelmed given the time and resources, but as countries have fewer and fewer cases, it is relatively easier to conduct an investigation on every single case. who recommends the type of data to be collected in an investigation. one key advantage of case-based surveillance is that it allows one to analyze which age cohorts are being infected and their individual vaccination status to help to target vaccination efforts and close existing immunity gaps. surveillance networks identify and enroll cases that meet a specific case definition. case definitions have three essential components: person, place, and time. case definitions vary in sensitivity and specificity. sensitive case definitions are more inclusive, are less likely to miss cases, but will include patients that do not have the disease. specific case definitions have stricter criteria and exclude more patients that do not have the disease but can also miss patients with milder or atypical disease presentations. both sensitive and specific case definitions can be used in infectious disease surveillance depending on the goals of surveillance. for example, sensitive case definitions may be preferred if it is important not to miss cases. in general, case definitions should be as sensitive and specific as possible. however, since a highly sensitive and specific case definition is not always possible, it is important that the case definition is at least applied systematically and consistently over the surveillance period. syndromic surveillance involves monitoring cases that meet a clinical case definition for the disease under surveillance, typically without laboratory confirmation (henning, ) . this allows for rapid identification of a cluster of cases that might warrant further investigation. an example of syndromic surveillance includes acute fever/rash surveillance in many countries, which is used to monitor measles and rubella. the fever and rash could be due to a multitude of causes, and if there is an increase in the number of fever/rash cases reported, this could indicate an outbreak. as field investigations are ongoing, laboratory testing can be performed on some or all of the cases identified by syndromic surveillance to determine the etiology. in the acute fever/rash surveillance system, laboratory specimens might be collected to undergo testing for measles and rubella. a wellestablished global who-coordinated measles laboratory network provides support to monitoring measles cases and provide genotype information globally. syndromic surveillance case definitions can be used in emergency or outbreak situations as an alert system to identify suspect cases that meet a broad case definition to then be further investigated. during the ebola outbreak in - , airport security was increased to identify people with a fever and a history of travel to an ebola-affected country in order to stop the disease from traveling between countries. in contrast, some surveillance case definitions are based on confirmed cases in a laboratory where the etiologic agent can be identified through a variety of laboratory tests (e.g., serology testing, bacterial culture, or molecular diagnostics) or at the bedside with well-validated commercial rapid diagnostic tests (e.g., malaria and streptococcus pneumoniae). as an example, virologic influenza surveillance networks use laboratoryconfirmed influenza to determine the circulating strains to provide information for vaccine composition. a critical objective of laboratory-based surveillance is to monitor for emerging drug resistance in pathogens or shifts in serotype distribution. cases meeting a suspect case definition (a sensitive case definition) may undergo laboratory testing leading to a more specific case definition. for example, the case definition for suspect meningitis as part of the who invasive bacterial vaccine-preventable disease network is very sensitive: a hospitalized patient at a surveillance hospital with sudden onset of fever and at least one meningeal sign during the surveillance period. after being enrolled into surveillance, additional clinical and laboratory information can reclassify a patient as having probable bacterial meningitis (namely having abnormal white cell count, protein or glucose levels in cerebrospinal fluid). this definition has a greater specificity but lower sensitivity. the most specific meningitis definition is confirmed meningitis by polymerase chain reaction assay or other laboratory test. this definition may lose some sensitivity because confirmatory tests can have false negatives, especially in areas with high antibiotic usage. zoonotic diseases cause disease in humans and can be challenging to control since both animals and humans can be hosts. many zoonotic diseases of public health importance are covered in other articles of this encyclopedia, including west nile virus, avian influenza, ebola (and other hemorrhagic fevers), lyme disease, sars, nipah virus, and rabies. historically, zoonotic and human disease surveillance existed separately, but there is a push to harmonize these systems to improve surveillance for diseases affecting both populations. illness in one species might be a harbinger of illness in humans, and an integrated comprehensive surveillance system can help identify potential disease transmission that might be ongoing. for example, surveillance for borrelia burgdorferi, the causative agent of lyme disease, in the tick population can help public health authorities determine proper interventions to decrease the transmission from ticks to humans. one health emphasizes the link of human health to the surrounding environment and animals. one of the mission statements of one health is to improve the lives of all species by harmonizing both animal and human disease surveillance and control efforts. international organizations participating in one health include who, the un food and agricultural organization, and the world organization for animal health. serosurveillance involves the use of blood specimens to determine the burden of disease or immunity gaps in a population. serosurveillance is frequently done as a periodic survey for multiple diseases of interest simultaneously. however, serosurveillance cannot provide information in a timely manner; thus an outbreak might have occurred that is discovered by serosurveillance, but it might be potentially too late for an intervention to decrease disease transmission. serosurveillance is sometimes the only type of surveillance conducted for an infectious disease. for example, hepatitis b is frequently asymptomatic in children, making evaluating the impact of vaccination efforts extremely challenging (who, ) . the standard has become to perform serosurveillance among cohorts of vaccinated children to identify the burden of disease and determine the impact of vaccination efforts. in some countries, national health surveys, such as the national health and nutrition examination survey (nhanes) and malaria indicator surveys, are conducted periodically and include a serologic component, allowing one to monitor trends in diseases and immunity over time. for example, nhanes includes data on hepatitis b, c, and d antibodies. adverse events following immunization (aefi) surveillance is a critical component of ensuring vaccine safety in the populations where the vaccines are being used. surveillance often begins at the health facility level, where health workers are trained to recognize adverse events from immunizations, and is reported to national regulatory agencies and who. this surveillance is critical for investigating problems that could occur with bad lots of vaccines and mishandling of vaccines in the cold chain (improper storage) which can contribute to the public perception of the vaccine program. technology is increasing the availability of data on health that can be used for infectious disease surveillance, including sources that go beyond that of traditional passive or active surveillance systems. new sources of data include mobile data, electronic health records, and social media. these aggregate sources and the speed at which they can be compiled are referred to as 'big data' (wyber et al., ) . these sources of data can provide more real-time information to help mitigate outbreaks or improve the health of a population. in , google started a venture called 'google flu' which was an algorithm tracking global search habits (such as search engine queries for 'influenza') with the hope that it could act as a real-time syndromic surveillance system. it was one of the first 'nowcasting' surveillance technologies and was able to predict influenza disease with some accuracy, close to the us cdc influenza reports based on laboratory-confirmed influenza surveillance. however, after a couple of years, it was found to overpredict the number of influenza cases given the generic case definition used. the system is no longer active, but is used to help groups develop newer public health analytics. the use of mobile technology to improve systems is an important area for public health (also referred to as mhealth) and has a growing use for surveillance. mobile data can monitor the movement of people during an outbreak, and this information can allow health officials to better predict where a given disease will spread. the un pulse project supports infectious disease mapping in kampala, uganda, using m-health (un global pulse, ) . in , there was a typhoid outbreak in uganda. the pulse lab in kampala provided mobile data to complement data which the ministry of health collected on cases. these data sources combined allowed better visualization of the outbreak and where clusters of infections were happening and therefore permitted improved mobilization of resources to respond to the outbreak. flowminder is another organization developing the use of mobile technology in outbreak situations. it currently has projects supporting monitoring the spatial patterns of individuals during outbreaks using data from mobile phones. during a cholera outbreak in haiti in , researchers from flowminder mapped the movement of people using anonymous data from mobile usage from the affected areas (bengtsson et al., ) . following the outbreak, the data were analyzed, and it was shown that this was an effective way of mapping the spread of the outbreak. many partnerships between academic, programmatic, and global organizations exist to facilitate ongoing infectious disease surveillance and promote global health security. partnerships can take different forms and often include providing technical and operational support and resources to facilitate ongoing surveillance. some examples are as follows. in , the idsr strategy was first drafted by the who regional office for africa in order to harmonize existing surveillance networks (including afp and neonatal tetanus) in the african region (who, ) . the strategy aims to integrate surveillance being done at the community, health facility, district, and national level to improve the data collected and to conserve resources. idsr includes standard case definitions and protocols and involves collecting only data necessary for disease control, often aggregated data. this helps to decrease the work burden at all levels on health staff, is more efficient, and costs less than nonintegrated surveillance. however, the challenge of integrated disease surveillance is that sometimes more information is needed than is readily available to target intervention activities. the global outbreak and response network (goarn) is a who-coordinated network comprised of over partners worldwide, including government, technical, and academic institutions involved in epidemic surveillance. the purpose of goarn is to coordinate a rapid response to international disease emergencies through deployment of resources to the affected countries. goarn coordinates a multidisciplinary team comprising clinicians, epidemiologists, social mobilization, and communications experts. the increase in international travel is an important risk factor in the spread of infectious diseases. travelers can contract many infectious diseases, from common travelers' diarrhea to more serious conditions such as ebola. this can pose a serious public health risk if conditions are right for an outbreak or when novel pathogens are introduced into a naïve (not vaccinated or without protective antibodies) community. geosentinel is a global network of clinics assessing travelers' and migrants' health for illnesses acquired while abroad (leder et al., ) . this network of clinics confirms and registers cases of infectious diseases acquired while traveling. this surveillance information is critical for tracking the movement of diseases and informing guidelines for travel medicine. surveillance is an action-oriented public health tool. time lags in surveillance can affect outcomes if there is not a rapid response with interventions. surveillance information can be used at the global, regional, national, local, and individual levels. new technologies are being developed to assist with more real-time data dissemination. surveillance bulletins and reports are a frequently used method for disseminating surveillance information. many surveillance networks use them to send information to stakeholders and partners involved with the surveillance. these can be frequent (weekly or monthly reports) or more infrequent such as annual or biannual surveillance bulletins. these normally include case counts for the disease under surveillance or detection of new outbreaks. the scientific literature (peer-and non-peer-reviewed publications) and scientific conferences are also important venues for disseminating surveillance data. the audience for publications can be much wider than bulletins since they are accessible by a wide range of individuals. there can be a long lag-time between data generation and publication. these modes of communication are critical for improving the wealth of available knowledge and advancing research, but are not timely enough to mobilize a response to an outbreak. the morbidity and mortality weekly report (mmwr) from cdc and the weekly epidemiological record (wer) from who are two examples of periodic, non-peer-reviewed dissemination tools. the mmwr publishes an annual list of notifiable diseases using weekly data on the cdc surveillance systems. the mmwr publishes weekly reports for outbreaks and case reports for diseases under the international health regulations. with the advance of social media and the internet, there have been innovative strategies for more quickly disseminating surveillance information for rapid public health intervention. for example, the program for monitoring emerging diseases (promed) is a popular tool run by the international society for infectious diseases. it consolidates and verifies reports from media, observers, and news and disseminates via email and their website. they have a large audience since their information is free and easily available on the internet. they act as an important early warning of outbreaks to facilitate public health preparedness. online platforms are creating innovative ways of displaying the surveillance data that are collected. an online platform called healthmap run by boston children's hospital is one example (figure ) . reports of disease cases come from a series of vetted online sources (including promed, who, and online news outlets) and are mapped on an interactive interface allowing users to view the geographic distribution of multiple diseases. physicians and individuals can use healthmap to identify diseases occurring close to them or their travel destination. other tools include the health alert network (han) at cdc, which is used for quickly disseminating confirmed disease reports and information to medical and public health professionals at a national level. han disseminates four types of information: ( ) health alerts (where an action required); ( ) health advisories (information on health events where no action required), ( ) health updates (information on given events); and ( ) general public health information. in february , han released an official cdc health advisory on preventing sexual transmission of zika virus after a confirmed sexually transmitted case in texas. event-based surveillance entails monitoring cases and outbreaks of disease through formal and informal news and online reporting platforms. traditional surveillance can miss many outbreaks or delay the opportunity to intervene. eventbased surveillance includes reports from the community, health facilities, universities as well as media and online sources in order to develop alerts of health situations that are developing. the data and reporting methods are much less structured than other surveillance, but allow for quick detection of events that need to be investigated. surveillance is in and of itself a critical tool for public health. using an existing surveillance network as a platform for surveillance of additional diseases allows streamlining resources and can be a cost-effective measure to improve public health. for example, influenza surveillance is being leveraged to conduct surveillance for other respiratory viral diseases, such as respiratory syncytial virus. additionally, the laboratory, clinical, and epidemiological capacity built to run a surveillance network can be utilized for other public health studies. surveillance sites can be used as platforms for research and special studies. since infectious disease surveillance sites often conduct surveillance for vaccine-preventable diseases, studies on vaccine effectiveness and vaccine impact can be built on the platform of surveillance. vaccine impact studies can use surveillance to demonstrate reduction of disease after introducing an intervention such as a vaccine. these impact studies require baseline data before the vaccine was introduced in order to compare the postvaccine era to the prevaccine introduction disease incidence. special studies that gather additional information may complement surveillance disease trends. vaccine effectiveness studies evaluate the ability of a vaccine to control the disease in a real-world setting, which differs from vaccine efficacy studies where the vaccine impact is estimated in a controlled clinical situation. a good example of this is rotavirus diarrheal sentinel site surveillance, which has been used both to show the decline in rotavirus disease among age groups vaccinated as part of routine immunization and has also been used as a platform to conduct vaccine effectiveness studies. estimating the burden of disease at the country or global level with epidemiological models can be a critical part of using surveillance data and advocacy for disease interventions. in many countries, surveillance data alone may not be sufficient to provide informative data for a specific disease for a number of reasons: surveillance data may not be available, there might not be laboratory confirmation, or the data necessary to answer a certain question may not have been collected. in these situations, models using local and nonlocal data can be very useful. in addition to data from one region being extrapolated to inform on the disease within that region, data from similar regions can also be used to fill in gaps where surveillance is missing. there are many global burden estimation projects updated regularly to give global prevalence and mortality estimates by different government, research, and academic groups for a number of diseases (for example, influenza, s. pneumoniae, and rotavirus). burden estimation modeling can also be done on the national level using surveillance data collected locally. surveillance data have been used in a model to extrapolate the burden of influenza-associated hospitalizations in south africa, guatemala, and kenya using local surveillance data from the country (murray et al., ) . using mobile phone data to predict spatial spread of cholera the principles of disease elimination and eradication overview of syndromic surveillance: what is syndromic surveillance? geosentinel surveillance of illness in returned travelers figure healthmap displaying reported measles cases for the past month determining the provincial and national burden of influenza-associated severe acute respiratory illness in south africa using a rapid assessment methodology data visualisation and interactive mapping to support response to disease outbreak who-recommended standards for surveillance of selected vaccine-preventable diseases [online]. who document production services technical guidelines for integrated disease surveillance and response in the african region documenting the impact of hepatitis b immunization: best practices for conducting a serosurvey big data in global health: improving health in low-and middle-income countries united nations global pulse the views expressed in this article are those of the authors and do not necessarily reflect the views of who.see also: childhood infectious diseases: overview; ebola and other viral hemorrhagic fevers; geographic information systems (gis) in public health; health-care delivery systems; influenza; measles; poliomyelitis; surveillance of disease: overview. key: cord- -rrwy osd authors: neiderud, carl-johan title: how urbanization affects the epidemiology of emerging infectious diseases date: - - journal: infect ecol epidemiol doi: . /iee.v . sha: doc_id: cord_uid: rrwy osd the world is becoming more urban every day, and the process has been ongoing since the industrial revolution in the th century. the united nations now estimates that . billion people live in urban centres. the rapid influx of residents is however not universal and the developed countries are already urban, but the big rise in urban population in the next years is expected to be in asia and africa. urbanization leads to many challenges for global health and the epidemiology of infectious diseases. new megacities can be incubators for new epidemics, and zoonotic diseases can spread in a more rapid manner and become worldwide threats. adequate city planning and surveillance can be powerful tools to improve the global health and decrease the burden of communicable diseases. t he industrial revolution in the th century led to larger cities with greater potential for growth and development both for the individual and the community. living in a city can provide you with several advantages, such as the possibility for higher education, a new job with higher income, the security of better health care, and the safety of social services. in , the united nations estimated that % of the world's population, . billion, lived in urban centres ( ) . economic growth for countries has been linked to urbanization and countries with high per capita income are among the most urbanized, whereas countries with low per capita income are the least urbanized ( ) . the financial and political power is often concentrated in the cities, which leads to unique possibilities for action and quick response if needed. the process of urbanization refers to increased movement and settling of people in urban surroundings ( ) . however, the meaning of the word 'urban' does not have a universal definition. a wide variety of different interpretations can be found in various countries, and often they do not share the same understanding. different versions could be: living in the capital, economic activities in the region, population size, or even density. the lack of a universal definition makes it hard to compare different countries and cities in regard to public health and the burden and impact of infectious diseases ( ) . many of the studies conducted address the differences between urban and rural areas, and do not compare different urban settings. it can thus be difficult to get a global overview and get a better understanding of the burden of infectious diseases in these specific environments. cities from around the world can also be very heterogeneous and the local diseases and health challenges can greatly differ. the challenges for one city can be completely different for another location ( ) . about a century ago, only % of the world's population lived in cities, and in the least developed countries the percentage was only % ( ) . approximately half of the world's population now live in these urban centres. the two inhabited continents, which currently are the least urbanized, are asia and africa, with respectively and % of the population living in cities. these percentages are expected to rise dramatically by the year to and % respectively ( ) . in the last decade, the growth in the urban population has been the highest in asia, adding . million urban migrants per week. africa was the second highest contributor with . million. the total figure of new urban residents per week during the last decade was on average . million. it is in africa and asia where the current rapid growth is taking place. years are that almost all of the population growth will be in urban areas, but the growth in developed countries is expected to remain largely unchanged ( ) . chronic illnesses have been increasing in importance for the developing world. worldwide the leading causes of death in were ischaemic heart disease followed by stroke, lower respiratory infections, chronic obstructive lung disease, and diarrhoeal diseases. however, if you look at the list for low-income countries, infectious diseases still have a profound impact. the top three causes of death in these settings are all infectious diseases: lower respiratory infections, hiv/aids, and diarrhoeal diseases ( ) . many of the lower income countries are expected to have a major growth among the urban population, which leads to considerable challenges for the governments and health care to keep up to pace and develop their social services and health care as these regions grow. the rise of the new modern cities also creates potential risks and challenges in the aspect of emerging infectious diseases. different risk factors in the urban environment can, for example, be poor housing which can lead to proliferation of insect and rodent vector diseases and geohelminthiases. this is connected to inadequate water supplies as well as sanitation and waste management. all contribute to a favourable setting for both different rodents and insects which carry pathogens and soil-transmitted helminth infections. if buildings lack effective fuel and ventilation systems, respiratory tract infections can also be acquired. contaminated water can spread disease, as can poor food storage and preparation, due to microbial toxins and zoonoses ( ) . the density of inhabitants and the close contact between people in urban areas are potential hot spots for rapid spread of merging infectious diseases such as severe acute respiratory syndrome (sars) and the avian flu. criteria for a worldwide pandemic could be met in urban centres, which could develop into a worldwide health crisis ( ) . adequate city planning can be a key factor for better overall health, and such considerations must be in the mind of the governing bodies. today's megacities are very heterogeneous with large slum settlements, which lead to challenges for overall health and health care in the community. within one large urban setting, there can be huge differences in health conditions depending on where you live. in general, the urban health is better, but in some areas, it can actually be worse compared to certain rural environments ( ) . of the estimated billion people living in urban centres, about one-third live in slum areas ( ) . the ever-changing environment of cities has made certain infectious diseases both emerge and re-emerge. pathogens which adapt to urban environments from rural settings can spread in a more rapid manner, and be a greater burden to the health care services ( ) . this review article examines the urban world and how the current rapid urbanization around the world is affecting the epidemiology of emerging infectious diseases. currently the most rapid growth in urban population is taking place in the developing countries, and poses many different challenges compared to traditional highincome countries. this review focuses on these growing regions and their implications and how emerging infectious diseases affect the community. urban population Á a heterogeneous group with different living conditions cities around the world can look very different if you compare the living conditions for the residents. however, it is not only different cities that can have completely diverse standards of infrastructure and social security. the same city can provide very varying conditions for their residents. living in the slums compared to more wealthy neighbourhoods, will expose the inhabitants to different risks. traditionally cities can offer many advantages compared to rural settings, but under certain circumstances they can rather be a health hazard. the rapid migrations of people to cities can lead to overcrowding, which can generate slums or shanty towns. these slums are characterized by poor housing, lack of fresh water, and bad sanitation facilities ( ) . all of these shortages can be a threat to the residents' health and be a possible breeding ground for infectious diseases. the location of slums are often outside of the city centres, in more hazardous locations and the population feels a lack of social and economic opportunities compared with other residents. in sub-saharan africa, % of the urban population in lived in shanty towns ( ) . for example, in , % of the urban population in central african republic lived in these slums ( ) . in kenya's capital nairobi, % of the population lives in slums, and child mortality there is . times greater than other parts of the city ( ). the community and health care services have great challenges to provide the entire population with equal and adequate service. the collected parties need to be aware of the differences in threats with respect to infectious diseases, both at the local and governmental levels. certain infectious diseases have been shown to be more widespread in the slums. an example of this is the diarrhoeal disease cholera. infections have been linked to slums in dar es salaam, tanzania, with high population density and low income ( ) . in several other countries, cholera incidence is the highest in urban regions with high population density ( , ) . differences in prevalence of asymptomatic carriers of antimicrobial drug-resistant diarrhoeagenic escherichia coli have also been found in brazil between slum settlements and more wealthy parts of the community ). the poor infrastructure in the slum can be a barrier for improvement, but at the same time targeted interventions for safer water and better sanitation carl-johan neiderud facilities could potentially have a profound effect of the overall health. overcrowded housing in high-density populations in the slums can be a breeding ground for infectious diseases such as tuberculosis. the rate of tuberculosis has traditionally been higher in urban centres compared to rural ( , ) . studies in slum settlements in dhaka city, bangladesh, indicate a high prevalence of tuberculosis, which was almost twice as high compared to the overall national average and four times higher than the overall urban levels ( ) . however, different patterns can be seen in different countries; for example, in poland the rates of tuberculosis have shown only slightly lower incidence in rural population compared to urban, . per , versus . per , respectively ( ) . tuberculosis in the united states has declined in the twentieth century, and several factors such as improved nutrition status, socioeconomic status, overall public health, and new drug regimens have been thought to play a major role. however, in the mid- s a resurgence occurred which reached its peak in , especially in urban areas among the homeless and incarcerated population ( ) . the knowledge regarding symptoms, transmission, and prevention has been shown to be greater among the urban population in pakistan's punjab province compared to the rural population. health-seeking behaviour was also better among the urban population, in the aspect of when to seek medical advice for early diagnosis and potential treatment ( ) . information about infectious diseases and how they spread in the community can help the individuals to protect themselves, but knowledge about the slums and the infectious diseases panorama is also crucial for local physicians. they need to know how to look for the correct diagnosis, even if their diagnostic tools might be limited. the right hypothesis from the start in these cases is even more important. the rapid urbanization around the world leads to great challenges in city planning. the rapid influx of migrants can lead to overcrowding and local governments might not be able to provide safe housing, drinking water, and adequate sewage facilities, all of which are potential health hazards and must be taken into account for safe city planning. today more than half of the world's population, almost billion people, have access to piped water connected to their homes. since , well over billion people have gained improved drinking water facilities, and almost billion people have access to improved sanitation. however, more than million people still lack access to improved sources of safe drinking water, and in sub-saharan africa half of the population lack such facilities. globally the decline of open defecation between and went from to %. however, billion people in the world still practice open defecation. in this group, % live in rural areas, but the actual amount of residents from urban settings is gradually increasing. between and , the group in urban settings which lacked sanitation actually significantly increased from million to million, which could be explained by population growth ( ). much of the hard work to improve sanitation facilities has benefited large population groups, but the rapid influx of new urban residents shows that there is still much hard work to be done. residents who are subject to overcrowding and who lack access to safe drinking water or proper sanitation can be more susceptible to soil-transmitted helminths ( ) . these infections are among the most important causes of physical and intellectual growth retardation in the world and have a major impact on public health ( ) . good hygiene practices and good sanitary conditions have lowered the prevalent levels of contamination. in the brazilian city of salvador, with a population of . million, an improvement of sewerage coverage from to % of the households led to an estimated overall reduction of diarrhoeal diseases of % ( ) . neglected tropical diseases can cause substantial health problems in developing countries, and some of these diseases have a faecal-oral transmission pathway. examples of such diseases could be schistosomiasis, trachoma, and soiltransmitted helminthiases. improved sanitation could contribute to a significant improvement for the public health. in many countries, however, the focus is on treatment by medication and not improved sanitation. the reason could be that it would be much more expensive to carry out the necessary infrastructural improvements ( ) . safe drinking water and proper sanitary facilities must be taken into account in city planning. factors like this can potentially have a profound positive effect in lowering infectious diseases with a faecalÁoral route. however, the real challenge lies in the uncontrolled growth of slum settlements. poor housing and overcrowding can also contribute to vector proliferation. one example of this is for chagas disease, which is a parasitic infection caused by the protozoan trypanosoma cruzi. an important mode of transmission is vectorial infected bites of triatomine bugs. living in close contact to domestic animals and poor hygienic habits have also been identified as risk factors ( ) . chagas disease affects an estimated million people every year, and is an important health challenge in latin america. in recent decades, progress has been made to reduce the burden of disease, by vector control, screening blood donors, improved housing, and epidemiological surveillance. chagas disease is a growing health problem in non-endemic areas because of population movements ( ) . it is estimated that , individuals in the united states are infected ( ) and the most affected country in europe, spain, is thought to have , Á , cases ( ) . the example of chagas disease shows that physicians who practice in countries where the disease is not present must be aware of the travel history of the patient to connect the potential symptoms to the correct diagnosis. the environment in urban cities has proven to be favourable for the rat population (rattus spp.) and close encounters between rats and humans can lead to transmission of zoonotic infectious diseases. they can carry pathogens such as yersinia pestis, leptospira spp., rickettsia typhi, streptobacillus moniliformis, bartonella spp., seoul hantavirus, and angiostrogylus cantonensis ( ) . new york city has one of the largest populations of rats in the united states. it has been shown that encounters between rats and humans have been linked to proximity to open public spaces and subway lines, the presence of vacant housing units, and low education of the population ( ) . information like this can be useful for health officials when they launch specific control initiatives. the changes in human population with increased urbanization and urban poverty has also altered our perception of some zoonoses linked to the rat population. leptospirosis has traditionally been perceived as a primarily rural disease, but the incidence in urban centres is increasing ( , ) . in chinese cities, the incidence of seoul hantavirus haemorrhagic fever with renal syndrome has been linked to urban growth, growing rat population, and increase ratÁhuman contact ( ) . large megacities all over the world have large rat populations, but the surveillance and local knowledge seem to be inadequate. a better understanding of how to prevent uncontrolled growth in rat population can potentially lead to a decline of these zoonotic diseases. the growing trend of urbanization around the world has shifted some infectious diseases, which have traditionally been perceived as rural, to urban settings. the world health organization (who) has published a list of neglected tropical diseases. several of them have now become a reality in the urban environment, these diseases are something the practicing physicians in these areas have to be aware of ( ) . many of the diseases on the list are present in the developing world, which sometimes lack the opportunity to solve these problems by themselves. these countries need help from the global community. one of the neglected infectious diseases is lymphatic filariasis (lf) with billion people at risk, and . million in urban areas. one of the main reasons is the lack of proper sanitation facilities ( ) . lf still has its major impact in rural settings, but the increasing urbanization in the developing world has made lf an infectious disease that also has to be considered elsewhere. one of the parasite species wuchereria bancrofti has been located in many urban areas and has the potential for transmission in this environment. moreover, one of the vectors for the parasite is the mosquito culex quinquefasciatus, which thrives in these surroundings, especially in overcrowded areas with poor sanitary and draining facilities. however, within one city the transmission can vary substantially depending on the standard of the sanitary conditions. the mosquito vector culex spp. can be found in large parts of central and south america, east africa, and asia ( ) . another vector which has adapted to urban surroundings is the mosquito aedes aegypti, which is a key component for dengue transmission. dengue is on who's list of neglected tropical diseases, and is on the rise worldwide. the number of infections has drastically increased in the tropical regions of the world in the last years. recent studies have estimated million cases each year, and the burden is the highest in india with onethird of all the new infections ( ) . several factors have played a big role in the escalation, such as urbanization, globalization, and lack of mosquito control. aedes aegypti lay their eggs in artificial water containers made by humans, which is a key component in the urban transmission cycle. the adaption of dengue through its vector has made dengue an infectious disease on the clear rise ( ) . thailand is a country with all four serotypes of dengue virus, and the epidemics of dengue haemorrhagic fever have shown a possible correlation to originate from the urban capital of bangkok and then spread geographically in an outward manner to more rural settlements and provinces. a model to understand this mechanism could lead to more effective use of the health systems in the affected areas ( ) . dengue has become a global problem and is no longer restricted to the developing world. despite better knowledge, it seems tough to control the vector, which has adapted to the urban environment and living close to people. an efficient vaccine is not yet commercially available, but could be a powerful factor in the fight against the global dengue epidemic. often several different factors need to be favourable for a vector-borne disease to adapt to the conditions in an urban environment. for example, west nile virus (wnv) infection is an infectious disease which has become a reality in the urban environment. the primary vector is the mosquito culex pipens, which lay their eggs in water resources which are often man-made. however, for a successful transmission cycle wnv also need the american robin (turdus migratorius), which has several broods per season and hatchlings are more susceptible to wnv infection than adult birds ( ) . the county of dallas, texas, experienced an epidemic of wnv infections in . surveillance reports revealed % of the cases in the united states were found in dallas county ( ) . it shows for a vector-borne disease to have a successful transmission cycle several different factors need to be in place to affect the human population. leishmaniasis is a disease caused by the protozoa leishmania, which affects million and threatens million people in different countries. there can be different clinical presentations such as cutaneous and visceral ( ) . leishmaniasis is transmitted by the vector phebotomine sandflies. when rural migrants bring their domesticated animals to urban settings, often slums, they create favourable conditions for an urban transmission ( ) . it has been shown that it is a growing health problem and the ongoing urbanization has contributed to the increase ( ) . if the different vectors can adapt to the urban environment and man-made resources, the potential health implications can be of major concern. control programs and adequate surveillance is of importance, but in rapidly growing cities and slums it can be tough to implement such measures. emerging infectious diseases can also make the jump to stable transmission in the urban surroundings and surveillance of these can potentially prevent major health concerns and high cost for the health care services. who can play a major role in the fight for better control and knowledge. many of the countries in the developing world do not have the proper resources and the problem is not concentrated to one region, but is a global concern. numerous of the neglected tropical diseases play a major role in the developing world, which is currently experiencing a much faster pace of urbanization compared to the developed world. the who's call for help is important and, for example, dengue is now turning into a global crisis. safe and targeted assistance can be a huge factor for overall health; such assistance could be an effective vaccine or safe and easy vector control programs. urban centres can be catalysts for rapid spread of infectious diseases. the basis of large population groups in a restricted area can provide the perfect conditions for different epidemics. international travel has connected the world in the last century, and this mobility creates a potential threat of many emerging diseases. international tourist arrivals have shown an exceptional growth from million in to , million in . according to the latest forecast from the world tourism organization, international tourism arrivals will continue to increase, and in the figure is expected to be . billion ( ) . with the pace of modern travel, highly contagious infectious diseases can be a potential threat in a completely different setting compared to the original outbreak. urban population and the density of residents can meet the criteria for a new epidemic and create a public health disaster, if not taken seriously. international trade and travel can potentially also contribute to the occurrence of a worldwide pandemic. sars emerged as a global threat in . sars is thought to originate from the sars-like coronavirus (scov) of bats and reached the human host in china due to hunting and trading of bats for food ( ) . the disease was first recognized in wildlife markets in guandong, china. investigations have found this scov from the himalayan palm civets in live-animal markets in the region. the first cases of sars reportedly occurred in individuals who handled these animals to prepare exotic food, and the virus is thought to have crossed over to their human host ( ) . sars could then spread throughout the world by, for example, international travel. it spread in urban dwellings in large cities and in wellequipped city hospitals. public fear of travelling led to considerable economic losses that affected entire countries ( ) . the example of sars shows that food markets in southern china can be the origin of a worldwide health crisis. travel routes around the world have connected the urban world and large megacities like never before. accordingly it is important to take necessary preventive measures before the epidemic gets out of control, and here big organizations like who, but also governments, play an important role. early action is of utmost importance, and functional surveillance programs needs to be in place. the zoonotic disease dengue is endemic in most tropical and subtropical regions, which often are also popular tourist destinations. travellers to endemic countries can contribute to the spread of the disease. the burden of disease is on the rise, and estimations are that in returning travellers from southeast asia, dengue is now a more frequent cause of febrile illness compared to malaria ( ) . dengue is now an urban health problem, which is one of the major reasons why the rise is exceptional. the global rising problem of antibiotic resistance has also been linked to international travel. the worldwide spread of certain antibiotic resistant staphylococcus aureus has been linked to tourism, which shows the potential impact on international health ( , ) . faecal colonization with esbl-producing enterobacteriaceae has also been linked to international travellers in several studies ( Á ). the physician needs to take into account the recent travel activities of the patient to better evaluate the current condition and need for potential treatment and care. global travel shows no signs of decline and the interconnected megacities around the world make global surveillance even more important when it comes to contagious infectious diseases. measurements to stop the spread need to be taken at the original location, but knowledge about the specific disease needs to be passed on to the global community and local health workers in other parts of the world. this global surveillance and alert system needs to be fast and efficient to, if possible, reduce the impact. the expected rise of travel makes it critical for the future global health and the possibility to react in time for possible threats. zoonotic disease a challenge for the future rapid and sometimes uncontrolled urbanization can, in certain circumstances, lead to closer encounters with wildlife. human influence on the ecosystems creates meeting points for new and potential zoonotic diseases, which could have a profound impact for both local and global health. the global trends of urbanization push people to previously untouched ecosystems. new housing in the outskirts of big cities can potentially be meeting points for new and already known zoonotic diseases. of emerging infectious diseases, which have been recognized between and , more than % have been zoonotic diseases ( ) . living in close contact to domesticated animals and hunt for 'bush-meat' can also be risk factors for an infectious disease to make the jump from the animal host to humans. major deforestation creates closer contact between humans and bats and even primates, who can potentially be host for 'new' viruses. a better understanding, surveillance, and prevention of zoonotic diseases would be of great value, to both prevent and manage this upcoming threat for global health. hot spots for this transmission have been found and they often correlate where the process of urbanization is on the clear rise ( ) . even if it is not always the urban population who is at the front of new encounters with wildlife, it can still have an effect on urban health. the trend of people moving to cities are at the highest, where many of these new encounters with ecosystems take place, and infectious diseases can be introduced to these growing urban environments. the sometimes uncontrolled growth of cities pushes residents to untouched ecosystems when new housing expands. ebola virus disease (evd) has had a profound impact on the world in . since the spring of , the world has witnessed an unprecedented epidemic of this zoonotic disease. the hub of the epidemic has been the three countries in western africa: sierra leone, liberia, and guinea. it all began in december in guinea, in the providence of guéckédou, in the eastern rainforest region. the disease transmission in the capital of conakry is thought to be the first major urban setting for evd ( ) . who was first notified of the evd outbreak in march , and on august , the who declared the current situation as 'public health emergency of international concern' ( ). before, evd outbreaks in central africa had been limited in size and geographical spread to a few hundred persons, mostly in remote areas and not large urban settings ( ) . the centre of the epidemic (guinea, liberia, and sierra leone) has, as many of their neighbouring countries, a large population living in rural settings; only , , and % of their population live in urban centres ( Á ). the population is, however, highly interconnected in these countries with travel and crossborder traffic, with good road access between rural and urban settings. these communications have made the magnitude of the evd epidemic possible. despite cases of evd in nigeria and lagos, a megacity with million inhabitants, the transmission has been limited, which proves that implementation of control measures can limit the transmission ( ) . the mortality rate has been high in previous outbreaks, up to % ( ) . the fatality rate in the west africa epidemic has been estimated to around % for guinea, liberia, and sierra leone when data for patients with recorded definitive clinical outcomes ( ) . this unprecedented epidemic points out the importance of better surveillance, understanding, and preventions measures for this potentially deadly virus. ebola virus (ebov) is thought to be a zoonotic disease, and fruit bats are under investigation to be the natural reservoir. ebov sequences have been found in these animals near the human outbreaks which implies where the virus might originate from ( , ) . closer contact with humans and fruit bats are thus risks for a new global health crisis and the severity of an ebola epidemic has already been witnessed. the high costs, both from an economic and overall health perspective, have affected entire countries and have even cost lives on the other side of the earth. urban centres offer their residents greater possibility for health and social services. different factors, such as education, direct primary care services, and the governments' capacity for rapid response to upcoming health threats, can contribute to the opportunities in a city. however, in many cities the poor can find it difficult to access proper health care, due to the cost of such services. in more rural areas, the problem can instead be the distance to the nearest clinic, which in reality makes it impossible for prompt and efficient treatment ( ) . malaria has historically been and is still a major health concern in large parts of the world. who estimates million cases ( Á million) of malaria and , deaths ( , Á , ) in . the highest mortality rates have been shown to be closely linked to poor countries with a low gross national income (gni) per capita ( ) . estimations have been made that nearly % of the total african population, million, currently live in urban settings where malaria transmission is a reality. the annual incidence is estimated at . Á . million cases of clinical malaria among the urban population in africa ( ) . the relationship between the malaria mosquito vector and the human host determines the burden of morbidity and mortality. this interface is dependent on many different factors and the degree of urbanization is an important one. a significant reduction in malaria transmission has been observed over the last century. increased urbanization and decreased transmission have correlated in several different studies ( ) . however, whether it was the increased urbanization that led to a reduction in transmission or the malaria reduction that led to development that promoted urbanization of societies is a challenge to determine ( ) . a clear connection has been shown between reduced transmission of plasmodium falciparum and urbanization; however, for plasmodium vivax it is less obvious. for p. vivax, a connection has been found globally and in asia and africa; inconsistent results, however, were found in the americas. several possibilities could explain these incoherent results, such as more widespread transmission of p. vivax, lower transmission intensity, the wide distribution in asia, and high prevalence of duffy negativity in africa, which protects against p. vivax ( ). the overall decrease of the burden of malaria has been a positive effect of urbanization, but the exact mechanisms are not yet known. however, it seems that urbanization can have a favourable influence. immunization status between residents in urban centres and rural areas can differ. coverage of measles vaccination in indonesia have shown to be . % in rural areas, compared with . % in urban regions ( ) . studies in nigeria have shown that sometimes the coverage can actually be better in more rural areas, and it might be explained by better mobilization and participation in the delivery of immunization services ( ) . in a study in uganda, % of the urban group compared to % in the rural areas were fully immunized, but polio vaccine was given to % in the urban group and % in the rural group ( ) . immunization coverage can also vary considerably among different settings, not only between rural and urban surroundings, but also between urban, rural, and slum settlements. in changdigarh, a union territory of india, full immunization of children at the age of was % in slums, % in urban, and . % in rural settings ( ) . it shows that there can be a wide variety of reasons for immunization status among the population in different regions and countries of the world. effective immunization can be a cost-effective measure in poorer countries. high coverage can prevent epidemics in large cities and save many lives; however, immunization needs to be available both for the rural and urban population to achieve the greatest benefit. a study in tanzania has compared the knowledge about certain zoonotic diseases among general practitioners in urban and rural areas. the rural practitioners had poor knowledge of how sleeping sickness is transmitted and clinical features of anthrax and rabies. laboratories in rural areas are often poorly equipped and cannot always diagnose certain zoonotic diseases, which could limit the doctors' capability for correct diagnosis and treatment ( ) . public knowledge about certain infectious diseases can also vary depending on many different factors. the knowledge about sexually transmitted diseases (stis) among bangladeshi adolescents was higher among people in urban areas compared to rural, both in general and hiv and aids ( ) . the same results about hiv and aids have been found among a canadian population ( ) . studies in chengdu and shanghai, china, have shown risk perception about stis and hiv and aids is profoundly changed in rural-to-urban migrants ( , ) . the same result has been shown in a study among rural-to-urban migrants in ethiopia ). the rapid influx of migrants moving to cities makes it hard to get adequate information to all the different groups in the society. to educate the public is one of the many challenges for local governments and health officials. campaigns to improve the public knowledge are useful to fight the threat of infectious diseases. residents need to be aware of symptoms of infectious diseases to gain knowledge about when to seek health care and when it is safe to treat yourself. knowledge about food storage, waste management, vector control, and sanitary facilities are all aspects that can lower the burden of communicable diseases. these campaigns can sometimes be easier in the urban environment because of the density of the population. urbanization is an ongoing process in the world at the moment, but the pace of the process is not universal. the developed countries, which have traditionally been thought of as high-income countries, are already urbanized, and it is in the developing world that the rapid rise is taking place. infectious diseases still have a big impact on the global health, and urbanization is now altering the characteristics of these diseases. living conditions in cities are overall better in urban environments compared to rural settings; better housing, sanitation, ventilation, and social services all play an important role in this improvement. certain pathogens can, however, adapt to the different conditions and thus create a new challenge for both local governments and the global community. the capacity for surveillance, control programs, prevention, and public knowledge programs is far better in cities. it is here where the resources and political and financial power are gathered. but some countries do not have the resources and because these diseases can be of global concern, it is also the international community's responsibility to help and support with knowledge and resources. the rapid urbanization has also interfered in previously untouched ecosystems. these new settlements create new and closer encounters with wildlife, which can be a potential source of zoonotic diseases. these can be both previously known or new pathogens, which make the shift from their animal host to generate infections in humans. surveillance is of primary importance to monitor the burden of disease and will give both local authorities and the global community a chance for a quick response to public health threats. world urbanization prospects: revision highlights hidden cities: unmasking and overcoming health inequities in urban settings urbanization and human health urbanisation and infectious diseases in a globalised world the transition to a predominantly urban world and its underpinnings united nations human settlements programme (un-habitet) the top causes of death our cities, our health, our future Á acting on social determinants for health equity in urban settings facts: urban settings as a social determinant of health informal urban settlements and cholera risk in dar es salaam spatial and demographic patterns of cholera in ashanti region-ghana outbreak of cholera 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west africa Á the first months of the epidemic and forward projections ebola haemorrhagic fever fruit bats as reservoirs of ebola virus recent common ancestry of ebola zaire virus found in a bat reservoir world malaria report urbanization in sub-saharan africa and implication for malaria control urbanization, malaria transmission and disease burden in africa urbanization and the global malaria recession the effects of urbanization on global plasmodium vivax malaria transmission determinants of apparent rural-urban differentials in measles vaccination uptake in indonesia community participation and childhood immunization coverage: a comparative study of rural and urban communities of bayelsa state, south-south nigeria factors influencing childhood immunization in uganda reproductive and child health inequities in chandigarh union territory of india knowledge of causes, clinical features and diagnosis of common zoonoses among medical practitioners in tanzania nyströ m l. urban-rural and socioeconomic variations in the knowledge of stis and aids among bangladeshi adolescents talking about, knowing about hiv/aids in canada: a rural-urban comparison vulnerable but feeling safe: hiv risk among male rural-to-urban migrant workers in chengdu sexual behavior among employed male rural migrants in hiv-related sexual behaviors among migrants and non-migrants in rural ethiopia: role of rural to urban migration in hiv transmission i would like to thank the two anonymous reviewers for their insightful opinions. the author have not received any funding or benefits from industry or elsewhere to conduct this study. key: cord- -bbbq ylr authors: tong, michael xiaoliang; hansen, alana; hanson-easey, scott; xiang, jianjun; cameron, scott; liu, qiyong; liu, xiaobo; sun, yehuan; weinstein, philip; han, gil-soo; bi, peng title: china's capacity of hospitals to deal with infectious diseases in the context of climate change date: - - journal: soc sci med doi: . /j.socscimed. . . sha: doc_id: cord_uid: bbbq ylr objectives: infectious diseases are a major cause of morbidity and mortality in china. the capacity of hospitals to deal with the challenge from emerging and re-emerging infectious diseases due to climate change is of great importance to population health. this study aimed to explore the capacity of hospitals in china to deal with such challenges. methods: a cross-sectional questionnaire survey was utilized to gauge information regarding capacity of hospitals to deal with infectious diseases in the context of climate change among clinical professionals whose roles pertained to infectious disease diagnosis, treatment and management in anhui province of china. descriptive analysis and logistic regression analysis were performed on the data. results: more than % of participants believed climate change would have an adverse influence on population health and infectious disease control in china. most indicated that their hospitals were well prepared for emerging infectious diseases at present, and they considered that logistical support in hospitals (e.g. administrative and maintenance services) should be strengthened for future capacity building. the majority of participants suggested that effective prevention and control measures, more interdisciplinary collaborations, more funding in rural areas for health care, and improved access to facilities enabling online reporting of infectious diseases, were extremely important strategies in building capacity to curb the population health impact of emerging and re-emerging infectious diseases due to climate change in china. conclusions: clinical professionals recognized that climate change will likely increase the transmission of infectious diseases. although rural health care and hospitals’ logistical support need to be improved, most professionals believed their hospitals to be capable of dealing with emerging diseases. they thought that interdisciplinary and cross-regional collaborations, together with necessary resource support (e.g. improved facilities for rural health care) would be important control strategies. currently there are roughly million cases of reported infectious diseases in china, which result in approximately , deaths annually (national health and family planning commission of the prc, ) . previous studies have found the incidence of infectious diseases has been sharply reduced due to great improvements in health care services (hipgrave, ) . however, the emergence and re-emergence of some climate-sensitive diseases, such as malaria, dengue and hemorrhagic fever with renal syndrome (hfrs) have occurred in recent decades in china (huang et al., ; xiang et al., ; zhang et al., b) . malaria was a serious public health problem during the s with an incidence of approximately per , population . in , the incidence was only per , with about , cases reported . however, malaria cases in china increased to , in (zhou et al., ) dengue cases were reported prior to , but frequent outbreaks have occurred during the last few decades in china (lai et al., ) . hfrs, a serious zoonotic disease caused by hantaviruses, can be transmitted to humans by contact with infected rodents or their excreta . the disease is frequently reported in china where roughly % of the world's cases occur (xiao et al., ) . although the incidence of hfrs significantly declined during the s, there has been an increasing trend since (xiao et al., ; zhang et al., ) . the possible reasons for the emergence and re-emergence of these infectious diseases may be linked to climate change, population movement, rapid urbanization and increased surveillance efforts (tong et al., ; wu et al., ) . it is widely known that the global average combined land and ocean surface temperatures have increased by . °c over the period from to (intergovernmental panel on climate change, ). the intergovernmental panel on climate change (ipcc) indicates that the global average surface temperature will increase by . - . °c by - compared to - , and other climatic variations, such as changes in rainfall patterns and relative humidity will also occur (intergovernmental panel on climate change, ). climate change has, and will continue to, impact the transmission of vector and rodent-borne diseases by affecting the growth and development of the vectors or hosts, shortening the incubation period of the pathogens within the vectors, and impacting human behaviour (e.g. more time spent outdoors) (world health organization, ). the projected temperature increase and change in rainfall patterns may bring about an increase in cases of infectious diseases such as malaria, dengue and hfrs zhang et al., a; zhou et al., ) . in china, annual average land surface air temperature has increased by . - . °c over the past years, and is projected to increase by . - . °c by as compared to (china national development and reform commission, ) . extreme climate events, such as extremes in temperature, drought in north china, and flooding in south china, may become more frequent and intensive (china national development and reform commission, ) . the variations in temperature, rainfall, humidity and extreme events posed by climate change could facilitate infectious disease transmission and result in the possible increase in cases of infectious diseases in china (tong et al., ) . in the chinese health care system, both the clinical health sector and preventive medicine (public health system) play important roles in the protection of population health. in the clinical health system, hospitals provide patient diagnosis, treatment and management. in the public health system, the centers for disease control and prevention (cdc) work to protect and improve public health and safety, and focus on disease prevention, control and surveillance; and cases of notifiable diseases are reported by clinical professionals to the local cdc (he, ) . therefore, hospital clinical staff and cdc public health professionals perform different roles, and may have different views on infectious disease control and prevention. given that the ability of the public health system (i.e. cdcs) to deal with emerging and re-emerging infectious diseases in the context of climate change has been extensively studied (tong et al., a; tong et al., ) , a similar investigation of the clinical health system where diseases are diagnosed and treated, is warranted. this study will contribute to a better understanding of infectious disease diagnosis, treatment, prevention and control in the face of climate change in china, and may also benefit disease control in other countries . the study employs a cross-sectional questionnaire survey among clinical professionals to explore china's capacity of hospitals to deal with infectious diseases in the context of climate change. further, the study explores participants' views on capacity building in the hospital sector to curb potential emerging and re-emerging infectious diseases due to climate change in china. a questionnaire was administered to clinical professionals in november . the questionnaire instrument design was informed by previous studies and relevant literature on climate-sensitive diseases (national development and reform commission of the prc, ; semenza et al., ; tong et al., ; wei et al., ) . the questions asked about clinical professionals' thoughts on climate change, disease occurrence, the capacity of hospitals to deal with infectious diseases, and strategies to build the capacity of the hospital sector and curb the health impacts of climate change related to infectious diseases. an open-ended question was included to explore in greater depth, participants' understandings of disease control, diagnosis, treatment and management in the context of climate change. the questionnaire is included in appendix a. the study site of anhui province was selected because it has a high incidence of infectious diseases, especially malaria and hfrs (bi et al., ; jiao et al., ; the people's government of anhui province, ). anhui province is located in east china (see fig. ), and has a warm-temperate, semi-humid monsoonal climate with an average annual temperature between and °c, and annual precipitation between and mm (the people's government of anhui province, ). participants were clinical professionals from three major hospitals in anhui. a total of clinical professionals were surveyed in two general hospitals in the capital city of anhui. a further were surveyed in an infectious diseases hospital in a prefectural-level city. the clinical professionals included both doctors and nurses whose roles pertained to infectious disease diagnosis, treatment, and management. investigators administered the questionnaire in the three hospitals. to maximize the response rate, the principal researchers selected six key senior contacts from hospitals to assist with the distribution of questionnaires to potential participants. the process of participation was voluntary, and no incentives were offered. there are clinical professionals relating to infectious diseases in the three major hospitals, and for ease of administration, the entire population was used as sample. in total, questionnaires were distributed, and after omitting incomplete questionnaires, were analyzed, with a response rate of %. all returned questionnaires were entered using epidata . software (lauritsen, ) to create a database. statistical analyses were performed with stata . (statacorp, ). participants' demographic characteristics were descriptively analyzed. binary logistic regression was used to explore the association between binary responses and demographic variables. ordinal logistic regression was used to explore the association between ordinal responses and demographic variables. the demographic variables were age, gender, professional level, length of employment, education, and occupation. the binary responses were "yes", and "no/unsure". the ordinal responses were "very concerned", "concerned", "slightly concerned", and "not concerned"; or "extremely important", "very important", "important", "less important" and "not important". data were analyzed with a two-sided test and p-values less than . were considered statistically significant. table shows the demographics of the participants. in this study, % of the clinical professionals who participated were doctors, and . % were nurses. while ages ranged from to years, . % of professionals were aged below years, with the mean age being . years. of the participants, . % were female. junior level professionals accounted for . %, intermediate level professionals . % and senior level . % of the total participants. over % of the participants had worked in hospitals more than five years. more than % held a university degree or higher qualifications, especially the doctors who were mostly highly educated with a bachelor of medicine degree or above (see supplementary table s in appendix b). as shown in table , more than % of the professionals were either very concerned or concerned about climate change. there was a statistically significant association between age group, professional level and the concern about climate change (see supplementary table s in appendix b). those who were over years (or = . , % ci: . - . , p = . ) and the senior staff (or = . , % ci: . - . , p = . ) were more likely to be concerned about climate change. furthermore, % of the professionals agreed with the statement that the weather was becoming warmer, especially nurses and the professionals who had been employed more than ten years (see supplementary table s in appendix b) . nearly all ( . %) believed climate change would have an adverse influence on population health. furthermore, . % and . % of professionals agreed that predicted increasing temperatures and changes in precipitation patterns would affect infectious disease transmission. specifically, . %, . %, and . % of professionals were either extremely or very likely to believe that there was an association between climate change and malaria, dengue, and hfrs, respectively. table shows that about % of participants thought that there had been an increasing number of patients with malaria, dengue, and hfrs, and roughly % and % of professionals thought that climate change and population migration, respectively, were contributing factors. more than % of participants believed that malaria and hfrs hospital-reporting protocols were in place, and . % believed so for dengue. in terms of the diagnostic capability of hospital laboratories, . % of professionals responded that their hospitals were 'always' or 'mostly' able to rapidly provide diagnostic tests for malaria, . % for dengue, and . % for hfrs, respectively. some % of participants rated diagnostic and treatment capacity as excellent/good for malaria and hfrs, while % thought this was the case for dengue. moreover, if an unusual cluster of cases was noticed, most professionals would take actions such as discussing with colleagues and laboratory technicians, informing the public health officer, and consulting with the cdc. in addition, there were no significant differences in these perceptions of infectious diseases between doctors and nurses. clinical professionals' perceptions of the current capacity of their hospitals to deal with infectious diseases are shown in table . specifically, . % of participants agreed that they had sufficient staff to deal with disease outbreaks. also, more than % believed that their staff were well informed about current infectious disease trends, and that the quality of reported data from their hospitals to the cdc was excellent. however, . % of professionals thought that logistical support in hospitals (for example administrative and maintenance services) needed to be strengthened. overall, . % either agreed strongly or somewhat that hospitals were well prepared for the threat of a serious emerging disease. there were no significant differences between perceptions of doctors and nurses regarding the current capacity of the hospital. additionally, . % agreed that more research on the health impacts of climate change was needed. to build capacity to meet the challenge of emerging and re-emerging infectious diseases due to climate change, . % of participants thought that prevention and control measures were extremely important strategies, and . % believed more collaboration with their local cdc was extremely important (table ) . furthermore, multivariate ordinal logistic regression analysis showed that compared to those under years, professionals aged over years were more likely to indicate that prevention and control measures are extremely important (or = . , % ci: . - . , p = . ). senior level staff were more likely to believe that more collaboration with the cdc was extremely important (or = . , % ci: . - . , p = . ); while doctors were less likely to believe so (see supplementary table s in appendix b). about % believed more funding was required for rural health care and that improving the accessibility of the online infectious disease reporting system for rural hospitals was also extremely important. more than % of those surveyed believed that the health impacts of emerging and re-emerging infectious diseases due to climate change could be addressed with strategies such as: better response mechanisms; strengthening the climate change poses a significant threat to global population health (intergovernmental panel on climate change, ). clinical professionals are at the frontline of health care provision to the population and are likely to witness firsthand the health impacts of a changing climate (blashki et al., ) . they, therefore, provide a unique perspective on the health impacts of climate change and an indepth understanding of local community health (blashki et al., ) . to the best of our knowledge, this study is the first of its kind to gauge clinical professionals' perceptions of the capacity of china's hospitals to manage infectious diseases under climate change and to provide insight for policymakers, practitioners and medical educators regarding climate change adaptation in the health sector. concern about climate change was acknowledged by most professionals, especially among senior staff and the group aged over years. the majority of professionals, particularly nurses and those with longer terms of employment indicated that the weather was getting warmer. nearly all respondents indicated climate change would adversely affect population health, and most indicated it would facilitate infectious disease transmission. less than % of participants thought climate change was unlikely to be associated with malaria incidence compared to % for dengue and hfrs. the results were consistent with our previous studies among cdc staff who indicated climate change was more likely to have an influence on malaria than hfrs, whilst the perception of an association between climate change and dengue was not as strong as in a previous study conducted in guangdong (tong et al., ) . this is likely due to guangdong having the highest incidence of dengue in china, whilst in anhui there are fewer dengue cases reported (lai et al., ) . moreover, compared with previous studies among cdc public health professionals in other provinces (tong et al., a) , the hospital clinical professionals in this study were more likely to indicate that climate change would have an influence on hfrs. this could be due to the relatively high number of hfrs cases in anhui province, and hence firsthand knowledge of the disease and its determinants. additionally, another study conducted among health experts in europe also indicated their perceptions of the negative impact of climate change on vector-borne, food-borne, water-borne and rodent-borne diseases (semenza et al., ) . despite . % of participants agreeing strongly or somewhat that hospitals were currently well prepared for the threat of a serious emerging disease, climate change may present new challenges for health sectors such as increased incidence in certain climate-sensitive diseases, or diseases either not previously seen in the area, or not seen for some time. such an influx in cases could stretch the coping capabilities of the present system. this may explain why the public health and clinical health sectors share concerns about potential emerging and re-emerging climate-sensitive diseases. the majority of professionals indicated that climate change and population migration were considered as the most significant factors associated with the increase of these infectious diseases. this is in line with other studies that indicated the impact of climate change and migration would affect infectious disease transmission (gao et al., ; mcmichael, ; sang et al., ) . higher temperatures and changing precipitation patterns can contribute to the increasing m.x. tong et al. social science & medicine ( ) - population of vectors/rodents and more frequent contact with humans. simultaneously, population movement can facilitate disease transmission from one region to another. in china, currently it is estimated that approximately million people have migrated from poor rural areas to gain work in the cities (national health and family planning commission of the prc, ). this "floating population" (highly mobile population) of internal migrants chiefly work in insecure lowwage jobs. moreover, they often lack health insurance and defer seeking timely medical treatment, which could have a negative influence on infectious disease control (qin et al., ) . future infectious disease control and prevention should be cognizant of climate change impacts and population movements on the transmission of these diseases. in addition, building a more comprehensive national health insurance system covering the internal migrant population's health expenses in any part of china may provide benefits for population health (qin et al., ) . most professionals claimed that there was a hospital protocol in place for the reporting of notifiable diseases and believed that the capacity for disease diagnosis and treatment was either excellent or good, especially for malaria and hfrs. as anhui historically has a high incidence of malaria and hfrs (chen and qiu, ; gao et al., ) , professionals are more likely to have experience and confidence in dealing with these diseases, whereas historically there has been a low incidence of dengue in the province. the majority of professionals believed that their hospital laboratories were able to provide rapid diagnostic test results for malaria, dengue and hfrs. additionally, most professionals purported they would take comprehensive actions if they detected an unusual cluster of cases. this indicates there is a strong likelihood that outbreaks of emerging or re-emerging diseases would be detected early by the clinical professionals or the cdc. this detection of unusual trends could aid in curbing transmission if preventive measures are activated early. regarding the capacity of hospitals to deal with disease risks, most participants believed they were well prepared for the threat of a serious emerging disease. of particular note, participants indicated there was a need to strengthen the hospital's logistical support. this is in line with another study conducted by xu and chu in focusing on logistics capacity in hospitals, which advocated that a reliable logistical support system is one of the most important components in modern health care systems and should be given higher priority to sustain and promote better health care services in the long-term (xu and chu, ) . currently, the main obstacles to improving hospitals' logistical support in china are the lack of high-quality staff and regulatory frameworks for logistics management within hospitals (lin, ) . promoting specialized training for logistics staff and implementing management and quality control guidelines would be important. moreover, the problems in rural areas were highlighted, especially regarding the funding for rural health care support and accessibility to an online reporting system. the severe acute respiratory syndrome (sars) outbreak demonstrated that rural health care was the weakest component of china's disease control and prevention system (knobler et al., ) . more resource allocation to rural areas would be a vital step in advancing china's capacity building in response to emerging infectious diseases due to climate change. in this study, older and senior staff were more likely to indicate that primary prevention measures and interdisciplinary collaboration were important, compared with the younger or non-senior staff. such differences could be due to the rich working experience of the groups, who may have a better understanding of the infectious disease control and prevention strategies. most participants also suggested a need for improved environmental health, more health education programs and financial support to improve vector/rodent-borne disease control, diagnosis, treatment and management in the context of climate change. some studies have made future predictions about the effect of climate variation on dengue and malaria (caminade et al., ; ebi and nealon, ) , although there are many confounding factors to consider. although some participants in this study indicated that changes have already been noted, it would be useful in future studies to gain an insight into stakeholders' perceptions of when global warming would reach a point where there is a marked effect on the burden of climate-sensitive diseases. some limitations of this study deserve mention. firstly, this study was conducted in one province. the results may not be generalizable to clinical professionals in other provinces of china. secondly, as dengue is an emerging and uncommon disease in anhui, participants in this region may be less likely to have hands-on experience in dealing with dengue cases, and feel less confident about dengue diagnosis and treatment. lastly, the study was conducted among three major hospitals in cities, and results may not be generalizable to rural areas, townshiplevel hospitals or village-level clinics. nevertheless, these findings may provide information for policymakers and clinical professionals pursuing initiatives to strengthen the capacity for hospitals to cope with a potential increase over time, in cases of climate-sensitive infectious diseases. this study revealed that most clinical professionals thought climate change would have an impact on infectious diseases, and believed climate change and population migration were significant factors associated with infectious disease transmission. health professionals in our study thought that the overall capacity of the hospital healthcare system to deal with infectious diseases was excellent. however, logistical support should be strengthened in hospitals and more climate change related research is needed. issues that could be addressed include prevention and control measures, collaboration with the cdc, inhouse staff training and improved healthcare systems in rural areas. the multiple direct and indirect impacts of climate change will continue to threaten the health of the chinese population. these findings may help health policymakers develop organizational adaptation policies to address the adverse impact of climate change on health. the authors declare no conflicts of interest. el nino-southern oscillation and vector-borne diseases in anhui, china. vector borne zoonotic dis general practitioners' responses to global climate change -lessons from clinical experience and the clinical method impact of climate change on global malaria distribution epidemiologic surveillance on the hemorrhagic fever with renal syndrome in china china's national climate change programme. people's republic of china: national development and reform commission dengue in a changing climate change in rainfall drives malaria re transmission of haemorrhagic fever with renal syndrome in china and the role of climate factors: a review bringing into full play the role of general hospitals in the prevention of contagious diseases communicable disease control in china: from mao to now epidemiologic characteristics of haemorrhagic fever with renal syndrome in mainland china from climate change : the physical science basis climate change : 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and meteorological factors in animal reservoir, natural and socioeconomic variations and the transmission of hemorrhagic fever with renal syndrome in adhere to scientific development to improve the overall logistics capacity in hospital historical patterns of malaria transmission in china climate variability and hemorrhagic fever with renal syndrome transmission in northeastern china spatiotemporal transmission dynamics of hemorrhagic fever with renal syndrome in china meteorological variables and malaria in a chinese temperate city: a twenty-year time-series data analysis malaria situation in the people's republic of china in . chin geographical, meteorological and vectorial factors related to malaria re-emergence in huang-huai river of central china this work has been funded by the department of foreign affairs and trade through the australian development research awards scheme under an award titled 'how best to curb the public health impact of emerging and re-emerging infectious diseases due to climate change in china' [project id: ] and the national basic research program of china ( program) [grant no. cb ]. the views expressed in the publication are those of the authors and not necessarily those of the department of foreign affairs and trade or the australian government. the commonwealth of australia accepts no responsibility for any loss, damage or injury resulting from reliance on any of the information or views contained in this publication. we thank the anhui medical university, anhui provincial hospital, the second hospital of anhui medical university and fuyang no. people's hospital getting involved in this study for their assistance in the distribution and return of questionnaires. all survey participants are greatly appreciated for their valuable contributions. supplementary data related to this article can be found at http://dx. doi.org/ . /j.socscimed. . . .m.x. tong et al. social science & medicine ( ) - key: cord- - ad fw z authors: monath, thomas p. title: vaccines against diseases transmitted from animals to humans: a one health paradigm date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: ad fw z abstract this review focuses on the immunization of animals as a means of preventing human diseases (zoonoses). three frameworks for the use of vaccines in this context are described, and examples are provided of successes and failures. framework i vaccines are used for protection of humans and economically valuable animals, where neither plays a role in the transmission cycle. the benefit of collaborations between animal health and human health industries and regulators in developing such products is discussed, and one example (west nile vaccine) of a single product developed for use in animals and humans is described. framework ii vaccines are indicated for domesticated animals as a means of preventing disease in both animals and humans. the agents of concern are transmitted directly or indirectly (e.g. via arthropod vectors) from animals to humans. a number of examples of the use of framework ii vaccines are provided, e.g. against brucellosis, escherischia coli o , rabies, rift valley fever, venezuelan equine encephalitis, and hendra virus. framework iii vaccines are used to immunize wild animals as a means of preventing transmission of disease agents to humans and domesticated animals. examples are reservoir-targeted, oral bait rabies, mycobacterium bovis and lyme disease vaccines. given the speed and lost cost of veterinary vaccine development, some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. opportunities for vaccine-based approaches to preventing zoonotic and emerging diseases that integrate veterinary and human medicine (the one health paradigm) are emphasized. zoonoses (diseases transmissible from animals to humans) account for approximately % of all infectious pathogens of human beings and % of all emerging infectious diseases [ , ] . the origins of these diseases, and underlying factors (including man-made factors) in their emergence have been the subject of considerable interest [ , ] . certain zoonotic diseases have the potential for pandemic spread by human contagion, such as avian influenza, sars and the middle east respiratory syndrome coronavirus, and others for regional cross-border epizootics, such as yellow fever, venezuelan equine encephalitis and rift valley fever. the cost of a short list of zoonotic disease emergences in the interval between and , including bovine spongiform encephalopathy, sars, highly pathogenic avian influenza, west nile, and pneumonic plague (india), was estimated to exceed $ billion [ ] . of major emergencies that concerned public health (including hurricanes, earthquakes, and terrorist attacks) occurring between and , more than % were zoonotic disease outbreaks [ ] . however, the toll on public health is much greater than that caused by such dramatic outbreaks. it is estimated that different zoonotic diseases are responsible annually for . billion cases of human disease with . million deaths and substantial reductions in livestock production [ ] . animals, including livestock and companion animals, also suffer illness and death following infection with many zoonotic infections, and livestock and poultry are subject to large-scale intentional destruction as a means of preventing human infections, resulting in huge economic losses. wild animals, including endangered species, may also be mortally affected, examples being west nile disease in birds, yellow fever in neotropical monkeys, plague in black-footed ferrets, and ebola in the great apes. vaccines are an important means of prevention and control of zoonotic infectious diseases in humans and domesticated animals. however, the target for vaccination is, in almost all cases, the directly affected species, and there are few practically implemented illustrations of the potential for indirectly preventing human disease by immunizing the domesticated or companion animal sources of infection. the concept of immunizing wild animal reservoirs for the prevention of disease in humans or domestic animals is even more challenging and has received limited attention. moreover, human and animal health divisions of the biopharmaceutical industry are generally separate and segregated, and there is no organized approach to the development of new vaccines indicated for the prevention of spread of diseases from domestic or wild animals to humans. in addition, the major funding sources for research in human and animal diseases tend to be stove-piped into different government agencies, stifling cross-cutting approaches. the purpose of this review is to stimulate the science and policy communities to seek innovative ways to interdict zoonotic diseases by integrating human and veterinary medicine and vaccine development, and by creating new streams of funding aimed at the intersection of human and animal health. these aims are consistent with the one health initiative, which seeks to establish "collaborative efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals and our environment" [ ] . a framework for considering one health vaccine interventions is provided, as well as a brief review of past and current efforts, including a few successes. it will be obvious to the reader that this is a wide-open field with many opportunities that deserve more attention than they have received. the complexity, timeline, and cost of development of animal vaccines and the regulatory hurdles for product approval are far less than for human vaccines. thus some interventions based on the immunization of animals could lead to rapid and relatively inexpensive advances in public health. it is obvious that the benefit to human health deriving from vaccination of animals is far easier to justify when there is also a benefit to animal health, the latter often being closely linked to economic value. causative agents of zoonotic diseases, including viruses, parasites, bacteria, fungi, and prions, have extraordinarily varied life cycles and modes of transmission. some may persist between periods of active transmission in soil or invertebrate species. many have silent transmission cycles involving wild animals that have coevolved with the infectious agent and exhibit no signs of disease. some zoonotic diseases occur when a causative agent harbored by a wild animal reservoir jumps species to domesticated animals and thence to humans. others are primarily diseases of domesticated animal species. humans may be infected by direct contact with wild or domesticated animals, or indirectly by ingestion of contaminated milk or meat, inhalation of aerosolized secretions or excreta, fomites, or hematophagous insect or tick vectors. despite this complexity of epidemiological patterns, the opportunities for intervention often boil down to a few simple bottlenecks in the transmission process. for example, milk-borne diseases can be prevented by pasteurization, certain meat-borne diseases by inspection and animal husbandry improvements (e.g., trichinella, bovine tuberculosis), and other diseases avoided by limiting contact with known high risk species (e.g., tularemia, turtle-borne salmonellosis, exposure to bats carrying henipaviruses). where the risk of infection is high, or the resulting disease severe, vaccines may be the most efficient and cost-effective means of prevention and control. alternative methods for control of zoonotic diseases have generally employed trapping, poisoning, or other means of destroying the offending animal reservoir/vector; these methods have a mixed (often negative) record of success and are in any case becoming socially unacceptable. this review focuses on the use of vaccines for animals as an acceptable means of interdicting zoonotic disease in both animals and humans. three major epidemiological frameworks are identified for the control of zoonotic disease by means of vaccination of animals ( table ). the scope of this review encompasses only those diseases for which a strategy targeting vaccination of animals is actually used or is under development. there are many diseases of table frameworks for vaccine development and utilization as a means of preventing zoonotic diseases, and status of approval of existing vaccines for humans and animals. the first major framework includes zoonotic infectious diseases that affect both humans and economically important animals, where wild animals are the source of infection. livestock and humans are dead-end hosts, and neither contributes to the transmission cycle. in this case, vaccines are required to prevent disease in both humans and economically important animals, but do not interrupt transmission of the disease in nature. for framework i diseases, there is an important opportunity to accelerate the development of new vaccines by concurrent veterinary and human product research ( table ) . moreover the affected animal species represents a natural disease model of infection, pathogenesis and immunity that may be useful in testing efficacy and immunological correlates of protection of a new vaccine intended for human use, and thus provide data supporting regulatory approval under fda's animal rule [ ] . a list of framework i diseases and vaccines is shown in table , and an example is given below. west nile virus is a mosquito-borne, single strand, positivesense, enveloped rna virus (genus flavivirus, family flaviviridae), closely related to japanese encephalitis virus. west nile virus is a recognized cause of human disease ranging from mild feverrash-headache syndromes to lethal encephalomyelitis. horses, domesticated geese, farmed alligators (as well as a number of wild birds and mammals) are also susceptible to severe and fatal disease. although recognized as a cause of disease as early as the s, west nile became an increasing problem in the s, with outbreaks affecting humans and/or horses in northern africa, western europe, the eastern mediterranean, the black sea region and the volgograd oblast of russia [ ] . the most dramatic development was the introduction of west nile virus into north america in [ ] [ ] [ ] , the occurrence of large outbreaks in and , rapid spread across the us, and subsequent introduction to the caribbean and south america. horses and a number of wild bird species, notably crows and jays, were affected in addition to humans. between and , a total of , cases of west nile fever and , cases of neuroinvasive west nile disease were reported in the us, with the highest incidence in the middle of the country from texas north to the dakotas [ ] . the incidence of neuroinvasive disease in the us has varied between . and . per , in this interval [ ] . over , horses have been affected since , and in these animals the disease is more severe ( % case-fatality, % of survivors with neurological sequelae) than in humans ( - % case fatality, % of encephalitis survivors with sequelae) [ ] . moreover, the incidence of west nile in horses (∼ per , ) is substantially higher than in humans [ ] . the animal health industry rapidly responded to this veterinary emergency, and multiple west nile vaccines were rapidly developed, including a live vaccine (discussed below), whole virion inactivated, dna, and poxvirus vectored vaccines. the first vaccine for horses was marketed in , only years after introduction of the virus into the us. multiple human vaccine development programs were also initiated, but many of these efforts were discontinued or decelerated due to the high market risk associated with low incidence, a slackening of public concern with the disease, and the uncertain regulatory pathway for vaccine approval. although efficacy of a vaccine for equids is established [ ] , field trials to prove vaccine efficacy in humans would be large, expensive, and difficult due to the unpredictable occurrence of west nile outbreaks. the application of the animal rule to licensing a west nile vaccine, while plausible, has not been adjudicated by the fda with a sponsor. although approaches to developing veterinary and human vaccines against west nile were technologically similar, in only one case was a concurrent development program undertaken by a single company. this fact illustrates the current status of stove-piped and separate animal and human health biopharmaceutical industries. the exceptional case is of interest as a model of how vaccine development for zoonotic diseases could be improved. in , within months of the identification of west nile as the etiological agent of the initial outbreak affecting humans and horses in new york, acambis, a publically traded human-vaccine biotechnology company, applied its platform technology for yellow fever dvectored vaccines to the development of a west nile vaccine, with the intent to develop both vaccines for both horses and humans. this technology involved replacing the gene encoding the yellow fever d vaccine virus' envelope (e) protein with the corresponding gene of west nile virus. this chimeric vector vaccine platform had been previously used by the company to construct a chimeric vaccine against the closely-related japanese encephalitis virus [ ] ; at the time, that vaccine had proven to be highly effective, protecting non-human primates against intracerebral challenge with virulent japanese encephalitis virus [ ] . two new vectors were engineered, one with the wild-type west nile ny strain e protein gene and one with an e gene containing three attenuating mutations [ ] [ ] [ ] . the former was neurovirulent in mice, while the latter was more attenuated and thus deemed more suitable as a human vaccine. the principal question was whether the yellow fever d vector would infect and immunize horses, since yellow fever is a host-restricted primate virus. to find out, studies were sponsored by acambis in early at the colorado state university school of veterinary medicine. horses were vaccinated with the west nile/yellow fever chimeric virus or with yellow fever d vaccine, and viremia and antibody responses were determined [ ] . in addition, vaccinated and control horses were challenged by the intrathecal injection of a high dose of virulent west nile virus, following the same model referred to above for protection studies of the chimeric japanese encephalitis vaccine wherein monkeys were challenged by the intracerebral route. these studies showed that horses inoculated with chimeric vaccine developed neutralizing antibodies against west nile and were protected against a severe intrathecal challenge. a similar development plan was successfully followed for the mutated human vaccine version, using non-human primates as the test host. the veterinary and human vaccine candidates were developed side by side, and the data obtained in both programs were designed to support transition to advanced clinical trials in horses and humans. in , acambis licensed the veterinary vaccine technology to a major animal health company (intervet), and in the same year clinical trial materials were made for human trials. intervet completed development of the veterinary vaccine (prevenile ® ) [ ] , which was approved by usda in , and acambis brought the human vaccine (chimerivax-wn ) into clinical trials in the same timeframe [ ] , subsequently outlicensing the technology to sanofi pasteur. as expected, development of the veterinary vaccine and its progression through the regulatory pathway substantially outpaced the human vaccine; importantly, the veterinary application significantly informed the human vaccine table strengths and limitations of vaccination of animals as a means to control of zoonotic diseases. framework (see table development program in providing useful information on safety, durability of immunity and immune correlates or protection. co-development of the veterinary and human west nile vaccines is as a potential model for other vaccines. there are a few other examples where vaccines were co-developed for animals and humans, including vaccines against venezuelan equine encephalitis and rift valley fever (described below). certain issues arise, however, that may need to be considered in the context of such integrated efforts. first, a safety problem arising during use of a veterinary vaccine could provoke regulatory concerns or a liability problem for the human analog. although a safety issue did arise briefly due to anaphylaxis type reactions in horses, resulting in temporary withdrawal of prevenile ® from the market [ ], there were no repercussions for the human analog vaccine (chimerivax-wn ). this raises the interesting question whether the human and veterinary regulatory agencies are integrating information that might be of value, either during development of similar vaccines for different species or after marketing approval. this is an obvious overlooked area for application of one health principles. second, the immune response to veterinary vaccines generally should differentiate naturally infected from vaccinated animals (diva) on the basis of a laboratory test, which allows compliance with trade restrictions. this is, of course not a concern for regulatory approval of human vaccines, although it can be useful in seroepidemiological and vaccine coverage studies. an example of problems associated with diva is the off-label use of commercial horse vaccine to protect emus against eastern equine encephalitis (eee), since some combination vaccines against eee contain equine influenza antigen and such vaccines elicit cross-reactive antibodies to avian influenza resulting in quarantine. the chimeric west nile vaccine described above potentially allows for diva testing (using responses to the yellow fever nonstructural proteins expressed by the vector) [ ] . a number of important diseases are transmitted between domesticated animals and thence to humans. vaccination of domesticated animals has the potential to protect humans against these zoonoses, either indirectly by interrupting transmission where domesticated animals are amplifying hosts in the transmission cycle or directly by preventing spread from infected animals to humans. a list of framework ii diseases and vaccines is shown in table . selected examples are used to illustrate the role of vaccination of domesticated animals in preventing human disease. brucella spp. are facultative, intracellular gram-negative bacteria, pathogenic for domestic animals and humans. brucellosis, caused mainly by brucella melitensis (which infects sheep and goats), brucella abortus (cattle), and brucella suis (swine), occurs worldwide, with the highest prevalence in the middle east, asia, africa, tropical america, and the mediterranean region [ , ] . the annual incidence of human infections is estimated at , cases but the disease is widely acknowledged to be underreported [ ] . brucella canis, an infection of dogs, occurs worldwide with highest prevalence in tropical america. b. canis disease in humans has been reported, especially in persons handling breeding dogs and in immunosuppressed individuals. human brucellosis is acquired by contact or aerosol spread from infected animals, fomites, or ingestion of unpasteurized milk or undercooked meat. not surprisingly, brucellae survive for long periods in dust, animal excreta, soil, meat and dairy products. wild animals are also affected and can be the source of infection of livestock and humans. in animals, brucellosis causes epididymitis in males and abortion, placentitis, infertility and reduced milk production in female animals. the human disease is protean, manifested by chronic fatigue, relapsing fever, endocarditis, spondylitis, osteomyelitis, arthritis, and meningitis [ ] . prevention of human disease by control of brucellosis in livestock has long been a public health priority [ ] . control of brucellosis relies principally on surveillance, testing, removal of infected animals, import/export animal and animal product control provisions, protection from exposure to wild reservoirs (such as elk, deer, and bison), and vaccination. antibiotic treatment of animals is regulated and discouraged due to the large doses and long treatment required and concern about resistance. old, empirically developed live attenuated vaccines, b. melitensis rev vaccine for goats and sheep; b. abortus s and rb vaccines for cattle; and the oral b. suis s vaccine used widely in china for multiple species, elicit cellular immunity against the intracellular pathogen and are more effective than other types of vaccine [ ] . the rb vaccine (a spontaneous rifampin-resistant rough mutant) is approved in the us [ ] . however, the live vaccines have a number of drawbacks. the latter include lack of the ability to differentiate s and rev vaccine immunity from natural immunity (diva) and interference with surveillance and export control procedures; pathogenicity (especially abortion when animals are vaccinated during pregnancy); antibiotic resistance of the vaccine strains; and modest efficacy. live vaccines used in livestock can also cause illness in humans. vaccination as a stand-alone strategy has rarely been carefully evaluated, in large part due to concerns over the quality of the existing vaccines. however, vaccination is largely credited with elimination of brucellosis in the us [ , ] (the us was declared free of brucellosis in cattle in ) and for control of brucellosis in china [ ] . a recent study in greece showed that a mass vaccination program with the b. melitensis rev vaccine resulted in a decrease in human infections [ ] . a number of new vaccine approaches, including diva vaccines, designed to induce th oriented cellular immunity are under investigation, including safer rationally designed, mutated live vaccines [ ] ; recombinant, invasive escherischia coli [ ] ; recombinant subunit microencapsulated vaccines; and dna vaccines [ ] . experimental b. canis vaccines have been investigated in mice. where brucellosis-free status has been achieved, as in the us, wild animal reservoirs (especially bison and elk) threaten to reintroduce the disease. vaccination with existing vaccines is feasible, but delivery is challenging [ ] . this vero cytotoxin secreting gram-negative bacteria is an important cause of sporadic and epidemic food-borne illnesses of humans, including gastroenteritis and hemorrhagic colitis, with potentially lethal complications (hemolytic-uremic syndrome). cattle and sheep are the principal reservoirs of infection and transmission to humans occurs via food (meat, seeds and vegetables) contaminated with animal feces. undercooked ground beef is a source of infection in approximately one-third of human cases and recalls are a significant economic threat to the meat packing and distribution industry. animals concentrated at feed lots and slaughter that shed bacteria can produce lots of meat with high rates of o [ , ] , but there is considerable variability in the occurrence of contaminations [ ] . in developed countries, various sanitary measures and testing have been instituted to reduce the risk to consumers, but these remain imperfect. vaccination of feed lot cattle has been proposed as a measure to reduce the prevalence and duration of shedding and the risk to consumers. o -specific bacterial extract vaccines containing protective outer membrane proteins have been conditionally approved by usda (manufactured by epitopix, willmar, mn) and fully approved by the canadian food inspection agency (bioniche life sciences, belleville, ont.). feedlot cattle receiving or doses of the bioniche vaccine - weeks apart had - % reduction in colorectal colonization or fecal shedding and significant reduction in magnitude and duration of shedding [ ] [ ] [ ] . hurd and malladi [ ] modeled the impact of vaccinating cattle on human health outcomes. assuming % efficacy of the vaccine and % adoption rate, the model indicated a % reduction in the incidence of e. coli o -related human illness. the model also predicted significant reductions in the number of lots of contaminated ground beef and detection by usda, which would have substantial economic benefit to packers and distributors. vaccine effectiveness under conditions of field use will be highly dependent on adoption rate (vaccine coverage), and in part by whether cattle complete the -dose vaccination series on the proscribed schedule [ ] . an interesting question is: who will pay for vaccination of feedlot cattle? is the economic benefit there for the meat industry, and will vaccination reduce the cost of other preventive measures and testing or will vaccine costs simply be on top of other costs? will government agencies concerned with human health subsidize the cost, without empirical demonstration of a human health benefit? since ground beef only contributes about one-third of human infections, how could vaccines be used as a means of reducing other sources of food contamination? another issue for use of the current vaccines is the role of other enterohemorrhagic e. coli (e.g. o , o , and o ) in human disease. this disease is caused primarily by the gram-negative bacterium, bartonella henselae, transmitted between domestic cats by the agency of cat fleas, ctenocephalides felis. human infection occurs by contact spread from cats, including scratches by claws contaminated with blood or flea feces, or possibly by flea bite [ , ] . the prevalence of infection in household cats in the us is approximately %, but in stray animals it is % and high prevalence rates have been found in developing countries [ , ] . disease in humans is manifest by fever, a papule followed by a pustule at the site of infection and lymphadenopathy. rare complications include meningitis, encephalitis, endocarditis, glomerulonephritis, osteomyelitis, neuropsychiatric abnormalities, and relapsing fever and splenomegaly. human infections in immune-compromised individuals are particularly severe. approximately , - , cases and hospitalizations caused by cat scratch disease are estimated to occur annually in the us. in the late s, heska corporation, an animal heath company in colorado, initiated a program to develop a vaccine for household cats, with the goal of limiting the potential for transmission of the bacteria from cats to humans. unfortunately, the program was not completed and no vaccine is available at present. given the fact that cats rarely become ill with b. henselae, this would have been an unusual product providing protection to pet owners, with marginal if any real benefit to the target species. moreover, it would likely have been extremely challenging to demonstrate a health benefit to humans. rabies is a fatal infection of the central nervous system caused by rabies virus, a member of the lyssavirus genus, family rhabdoviridae. it is estimated that up to , - , cases of human rabies occur annually, and dog bite is the cause of over % [ ] . successful vaccination of dogs and humans against rabies was first demonstrated in by louis pasteur, using crude nerve tissue preparations. however, until the first decades of the th century in developed countries, and continuing in many developing countries today, the ancient practice of dog population reduction campaigns have been the main approach to rabies control, a method that has repeatedly proven to be ineffective. dog licensing and vaccination requirements were introduced in the united kingdom in , and gradually at the local and then state levels in the us beginning in the s, with national requirements attained by [ ] . many successful dog vaccination campaigns have been reported in latin american countries and asia [ ] . some countries have declared eradication of canine rabies, notably the united kingdom in , japan in , the us in , as well as malaysia, singapore, taiwan, hong kong. in , the first world rabies day event, the ultimate vision was promulgated of canine rabies elimination through systematic vaccination. however, rabies remains a significant public health problem, due to absent or incomplete dog vaccination in many areas of the world. more than . million post-exposure treatments with rabies vaccines are given annually, at a cost of over $ billion worldwide [ , ] . in china, due to the low prevalence of canine vaccination, the sales of rabies vaccines for human post-exposure prophylaxis outstrips any other human vaccine, accounting for % of all annual vaccine sales, i.e. million doses costing $ million [ ] . the requirement for human post-exposure vaccination at this scale represents an obvious failure of public health, since it plays no role in containing the spread of rabies in the canine vector. a very high rate of vaccine coverage in the dog population (exceeding %) is required for interruption of the rabies transmission cycle [ ] . the development of oral rabies vaccines has allowed vaccination of free-roaming dogs that could not be restrained and vaccinated by injection, as well as the vaccination of wild animal species that are the source of infection in dogs [ ] . oral bait vaccine for dogs has been successfully deployed in trials in many countries, including turkey, thailand, sri lanka, south africa, and the philippines [ ] ; the world health organization has supported this approach as a supplemental program where there are substantial populations of free-ranging or feral dogs [ ] . significant increases in canine vaccination coverage have been achieved when oral rabies vaccine was added to a program of standard parenteral vaccination. oral vaccination of dogs has been accomplished using commercial baits containing live modified rabies vaccines, such as the street-alabama-dufferin (sad) strain, variant b [ ] and the attenuated copenhagen strain of vaccinia expressing the rabies glycoprotein gene (g protein) [ ] . a more detailed review of oral rabies vaccines is provided below (framework iii). hendra virus disease, a severe and fatal infection of horses and humans in australia, has been noted as an example illustrating one health principles in disease prevention and control [ ] , and is especially relevant now that a new vaccine for horses has been introduced. hendra is a member of the henipavirus genus, family paramyxoviridae. the reservoir hosts are fruit bats (pteropus spp.), and the virus is spread from bats to horses by contact (including respiratory droplets), by food or fomites contaminated with bat urine, or by contact with sick horses. all reported human infections have resulted from contact with infected horses [ ] [ ] [ ] . hendra virus disease was first described in . human and equine cases have occurred in coastal queensland and new south wales and positive bats have been detected in the northern territory. a total of deaths in equids have been reported in outbreaks, with a very high case-fatality rate ( %), and cases ( fatal) have occurred in humans, including horse trainers and veterinarians, all of whom had contact with sick horses [ ] . the disease is manifested by severe systemic illness, respiratory symptoms or acute and relapsing encephalitis [ ] . swine appear to be susceptible to experimental infection. nipah virus, a closely related bat-borne agent in se asia has caused outbreaks of severe and fatal disease in swine and humans [ ] [ ] [ ] . in november , pfizer animal health launched equivac ® hev, an adjuvanted subunit protein vaccine for the prevention of hendra virus disease of horses in australia [ ] . since horses are a major source of contact spread of hendra virus to humans, the vaccine promises to make an important contribution to human health as well. fear of acquiring the disease has also constrained equine veterinary practice in australia [ ] , and the vaccine should mitigate this problem. development of equivac ® hev was a collaborative effort between pfizer and csiro's australian animal health laboratory. however, support for the development program was also provided by human medical researchers in the us, at the uniformed services university of the health sciences supported by the henry jackson foundation for the advancement of military medicine. a provisional approval for limited use of the vaccine was obtained in early , with full approval in november. the vaccine is a soluble, recombinant glycoprotein (g) of hendra virus, the ligand for cell attachment and antibodies to the protein neutralize cell receptor binding of the virus [ , ] . the vaccine protects horses and ferrets against experimental infection [ ] , and appears to cross-protect against nipah virus [ ] . the availability of equivac ® hev should lead to rapid uptake by horse owners in australia. the equine and horse racing industry in australia is large, contributing billions of dollars, and over % of total gross domestic product [ ] . hendra virus in horses is a notifiable disease in all australian jurisdictions; the property where the horse cases are located is quarantined and animals that are infected are euthanized. the occurrence of at least one hendra virus outbreak annually since , and the high lethality of the disease have raised considerable awareness in australia. since all human cases of this zoonosis have resulted from contact with infected horses, vaccination of horses against hendra virus promises to be a highly effective strategy for preventing human cases. rift valley fever is an enveloped, single-strand, segmented rna virus belonging to the phlebovirus genus, family bunyaviridae, occurring in africa, with intermittent extensions to the arabian peninsula. rift valley fever virus causes explosive and economically damaging outbreaks of disease in cattle and sheep, with stillbirth, abortion, and very high mortality of young animals; in adult animals, it causes % mortality in sheep, - % in cattle, and - % in goats [ ] . humans typically develop self-limited nonspecific febrile illness, but - % have a complicated course with hemorrhagic fever syndrome, encephalitis, hepatitis, renal failure, or retinitis, and case-fatality rates in severely ill and hospitalized patients is as high as % [ , ] . the virus is transmitted between livestock and from livestock to humans by the agency of mosquito vectors. in addition, humans commonly acquire infection by contact and aerosol routes when handling, treating, or butchering infected livestock, and the virus can persist in meat for weeks. the ecology of rift valley fever and the reasons behind its periodic emergences have been the subject of intensive study. in brief, the reservoir of infection is aedes mosquitoes, especially ae. linneatopennis, which maintain the virus by transovarial transmission [ ] . the dessication-resistant ova containing virus remain in depressions in the earth ('dambos') that are flooded during the rainy season, with the subsequent emergence of infected adult aedes mosquitoes. infected, viremic, livestock in turn served as source for amplified virus transmission by a variety of mosquito vectors [ , ] . there are no approved vaccines for prevention of rift valley fever in humans, although a number of candidates are in development by academic and government laboratories. vaccination of cattle and sheep represents a strategy for preventing disease in these species, and thereby for interrupting virus transmission to humans. rift valley fever epizootics are to some extent predictable based on rainfall patterns and surveillance of disease in livestock [ ] . surveillance provides opportunities for rapid intervention, particularly with a single-dose vaccine (most likely a live, attenuated vaccine) that would protect against viremia in cattle and sheep and prevent mosquito infection. in addition, routine vaccination of livestock in inter-epizootic periods with a product capable of inducing durable protective immunity is a long range goal supported conceptually by modeling [ ] . however, there are many obstacles to livestock immunization in africa, including access, policy, regulatory approval, cold chain, and commercial viability. additionally, diva requirements are driven by regulations prohibiting export of livestock or meat by countries experiencing rift valley fever [ ] . some of the obstacles to vaccination implementation could be overcome by development of improved vaccines. two old veterinary vaccines, the live smithburn vaccine, developed using techniques of serial passage in mouse brain similar to that applied to the early development of the french neurotropic vaccine against yellow fever [ , ] , and a formalin inactivated vaccine [ ] are commercially available from the onderstepoort institute in south africa. however, the smithburn vaccine, now produced in bhk- cells, is reported to cause teratogenicity and abortion when used in pregnant animals [ ] and is used only in countries endemic for rift valley fever due to concerns about reversion. the inactivated vaccine, also grown in bhk- cell culture, requires multiple doses to be effective and probably has relatively short durability [ ] , making it less desirable for the interventions proposed above. nonetheless, the inactivated vaccine was successfully used to interrupt a rift valley fever outbreak in south african sheep [ ] . indeed, following a large epidemic of rift valley fever in egypt in - , the veterinary serum and vaccine research institute (cairo, egypt) produced a formalin-inactivated vaccine in bhk- cells using the epidemic strain (zh ), which matched locally circulating strains compared to the south african strain [ ] . another inactivated vaccine (tsi-gsd ) developed by the us army for human immunization and grown in diploid fetal rhesus lung cells was tested clinically and shown to be well tolerated and immunogenic. ninety % of the subjects developed a neutralizing titer > , which was shown to be higher than the protective level in a passive immunization-challenge study in hamsters [ , ] . in addition to the requirement for multiple doses for primary and booster immunization, inactivated rift valley fever vaccines have the disadvantage of requiring high biocontainment facilities for manufacturing. in recent years, there has been substantial progress in development of newer vaccines, and some of these vaccine development projects have been collaborations between human and veterinary research groups. additionally, there has been a moderate level of support from the us government because rift valley fever is a credible threat of natural or intentional (bioterrorist) introduction. nevertheless, despite very promising technical results, there has been insufficient support from industry and government to propel any of these new vaccine candidates into use. because of the obvious advantages of rapid onset and durable immunity associated with live vaccines, development of an improved live vaccine has been the focus of research. us army investigators attempted to induce attenuating mutations in two rift valley fever virus strains isolated during the egyptian epidemic [ ] . mp- is a live vaccine that was developed from the virulent zh- strain by passages in mrc- cells in the presence of the mutagen, -fluorouracil, resulting in a temperature sensitive virus with amino acid mutations. attenuation was demonstrated in multiple animal models, and reassortment studies showed that attenuating mutations were redundant and resided in all three gene segments [ , ] . development of mp- was undertaken by the us army medical research institute of infectious diseases with the intention to produce a vaccine for both human and animal immunization. the vaccine was clinically tested in human volunteers and shown to be well-tolerated and highly immunogenic [ ] . army investigators, in collaboration with usda, conducted a number of studies of mp- vaccine in sheep and cows, including neonatal, pregnant and lactating animals. these studies showed that mp- caused a low viremia, but with no attendant clinical signs; there was no virus secretion in milk, and no abortions or teratogenicity when vaccine was given in in mid-to late term pregnancy. mp- was highly immunogenic and protected livestock against virulent challenge [ , ] . however, ewes vaccinated with mp- early in pregnancy showed a low incidence of abortion and teratogenicity, indicating some residual virulence of the vaccine [ ] . to improve genetic stability and safety of mp- , reverse genetic techniques were used to introduce deletions in the s and m rna segments in genes encoding, respectively, nss and nsm proteins [ , ] . two deletion mutants were evaluated for safety and immunogenicity in pregnant ewes [ ] and in calves (morrill jc personal communication, ) with positive results for the nsm deletant, making it an attractive candidate as a veterinary and human vaccine. there were no clear safety signals when ewes were inoculated in early-mid pregnancy. further safety studies are required to rule out the low incidence of abortion/teratogenicity seen with parental mp- in early-term ewes. unfortunately, human trials have not yet been performed with the rationally designed mp- derivative. a third live vaccine designated clone , is a plaque-derived clone of a central african strain of rift valley fever isolated from a human subject, and was found to be naturally attenuated for mice and to have an in-frame deletion of most of the nss gene [ ] . this observation was the basis for modifying the mp- vaccine by ns gene deletion, as described above. once again, a collaboration between the human and veterinary researchers led to a study in ewes, showing that clone was highly immunogenic but did not cause abortions [ , ] . another attenuated vaccine designated r , has been developed by reassorting clone and mp- so that it contains the s segment of clone and the l and m segments of mp- . this strain has attenuation domains from both parental vaccine candidates. a number of live, replicating, and non-replicating heterologous viral vectors expressing rift valley fever g and g glycoproteins and nonstructural proteins have been have been investigated in mice, elicited immune responses and protected against challenge. the vectors included lumpy skin disease (capripoxvirus) [ ] , alphavirus (vee and sindbis) replicons [ ] , newcastle disease virus [ ] , adenovirus, and modified vaccinia ankara. several of these constructs were used to immunize sheep and/or cattle (lumpy skin disease virus, newcastle disease virus, and sindbis replicons) with somewhat variable success. for a more comprehensive review see indran and ikegami [ ] and boshra et al. [ ] . live vectors are a promising approach for new rift valley fever vaccines, particularly veterinary vaccines, but may have problems for homologous boosting in light of anti-vector immunity. various prime-boost strategies have been proposed, as for plasmid dna vaccines (see below), but these would be exceptionally difficult to implement for immunization of livestock in the field, and are thus impractical. subunit protein produced in insect cells, virus-like particles [ ] , and dna vaccines [ ] against rift valley fever are also in early stage development. these approaches have potential advantages of safety and thermostability during storage and distribution, but may require multiple dosing and provide less durable immunity than live vaccines, and thus are less desirable products for framework ii implementation. overall, it remains to be seen which of the many rift valley fever vaccines in development progress to regulatory approval and whether an integrated veterinary and human health policy based on the immunization of livestock in africa together with predictive surveillance, can abort impending outbreaks, and lead to long range control of this important disease. vee is a mosquito-borne single strand, positive-sense, enveloped rna virus belonging to the alphavirus genus, family togaviridae. other medically important members of the alphavirus genus include eastern and western equine encephalitis viruses. there are vee virus subtypes identified by antigenic and genomic analyses, and a number of additional varieties. subtype iab and ic cause epizootic disease in equids and associated zoonotic infections of humans [ ] . during epizootics, horses and donkeys infected with these strains develop high viremias, serve as the primary hosts for infection of mosquito vectors and therefore are the indirect source of human infections acquired by mosquito bite. in contrast, the enzootic subtypes ii-vi, are maintained in nature in cycles involving rodent species and mosquitoes, are not amplified by equid viremic hosts, and cause sporadic illness in humans and equid dead-end hosts. epizootics of ic virus are the result of mutation and selection of virulent equine-competent viruses from enzootic strains, particularly the id variant [ ] . in the - s vee iab viruses caused large epizootics in south america, with associated human epidemics of encephalitis. between and , a series of major subtype iab and ic epizootics occurred in northern south america, and between and the virus spread north to central america, mexico, and texas [ ] . the cumulative economic and medical impact of vee outbreaks between and was devastating, with over , equid and , human cases. some of the vee iab epizootics are believed to have been spawned by the injection of horses with inactivated veterinary vee vaccines containing residual live virus [ ] . this likely occurred in trinidad in and again in nicaragua in , but probably was a widespread problem in the past. between and , vee ic re-emerged in venezuela and colombia, with an estimated equid deaths and over , human cases of which had encephalitis [ , ] . since vee causes an acute incapacitating illness in humans and the virus efficiently infects via the aerosol route, it was developed by both the us and soviet union as an offensive biological weapon [ ] . as part of these programs, vaccines for the protection of military personnel were also developed. in the us, a live, attenuated virus (tc- ) was developed by the us army medical research & development command (usamrdc) by empirical passages of the prototype trinidad donkey (subtype iab) virus in fetal guinea pig heart cell culture [ ] . the development of the live vaccine followed poor experiences with chemically inactivated vaccines; in animal models, only the live vaccine protected against aerosol challenge. however, tc- vaccine has a number of drawbacks as a human vaccine, including failure to immunize about % of vaccinees, and moderate-to-severe reactogenicity in about % of subjects. in humans, it remains an investigational product, used solely for the protection of laboratory workers [ ] , with approximately persons vaccinated since . the tc- virus acquired mutations during the empirical passage series in guinea pig heart cells, but attenuation appears linked to only of these, in the -noncoding region and the e envelope glycoprotein, and these substitutions appear to be subject to reversion in the vaccinated host [ ] . in addition, tc- has been isolated from mosquito vectors during field use, illustrating the potential for secondary spread and mutation and recombination events. an investigational formalin-inactivated tc- vaccine (designated c- ) was also developed by usamrdc and used following tc- priming to seroconvert tc- non-responders. since horses and related species are severely affected during epizootics and are the source of mosquito vectors infecting humans, there is an obvious need for a single dose veterinary vaccine that evokes rapid immunity. us army investigators explored the use of tc- live, attenuated human vaccine for immunization of equids beginning in [ ] , and there was limited field use of the vaccine in colombia in . however, when epizootic vee appeared for the first time in central america (guatemala) in may , and then spread southwards to costa rica and northwards to the us, there was considerable urgency to utilize a vaccine strategy for control of the disease in horses, donkeys and mules. in , the us military responded rapidly to requests for tc- vaccine from guatemala and el salvador. the vaccine had been produced and stockpiled at the merrell national laboratories, swiftwater pa under contract to the usamrdc for the purposes of biological defense. by , over million doses of tc- had been given to equidae in the us, mexico and central america [ ] . collaborative studies were also undertaken by agencies concerned with human and animal health (usamrdc, nih and usda) to fully explore the biology of the vaccine in horses [ , ] , ultimately leading to licensure and commercialized by the animal health industry, both as a live vaccine and then an inactivated vaccine combo with eastern and western equine encephalitis vaccines. tc- vaccine was credited with a rapid curtailment of the - outbreak. the history of vee exemplifies many one health principles, including the prevention of human cases through domesticated animal vaccination, use of a single vaccine product for animals and humans, and an agency (the us army) concerned with human health engaged in both veterinary and human vaccine development, and providing a solution for curtailing an emerging zoonosis. after the large epizootic in the s, tc- vaccine was again deployed during the epidemic in in colombia to create an immune barrier to spread of the virus. recent efforts have focused on development of improved vee vaccines for humans that are less reactogenic and more immunogenic than tc- , can be manufactured in a more acceptable substrate, and have a lower risk of reversion to virulence and of mosquito transmission [ ] . in addition, vaccines that crossprotect against the enzootic vee subtypes are needed. vee id is endemic in colombia, peru, venezuela, and ecuador, and the ie subtype circulates in southern mexico. aguilar et al. [ ] postulated that disease caused by vee is confused clinically with dengue, and that, in endemic areas, up to % of dengue cases may actually be due to vee enzootic subtype viruses. subtype id poses the ever-present risk of mutational change to produce high viremia and epizootic transmission in equids, as happened in the s. v vaccine is a rationally designed vaccine from the epizootic subtype iab genome, with insertion of a pe cleavage-signal mutation combined with an e gene resuscitating mutation. v had a good safety profile and was immunogenic and protective in laboratory animals, including nonhuman primates [ ] . while retaining a degree of neurovirulence for suckling mice, v is not virulent when inoculated intracranially in juvenile monkeys [ ] . v has also been evaluated in horses [ ] . the vaccine was safe and highly immunogenic, with subcutaneous doses as low as plaque-forming units shown to protect horses against challenge with virulent subtype iab virus. unfortunately, v proved to be too reactogenic for humans in a phase trial [ ] , and thus development for both human and veterinary use has stopped. the v virus was subsequently formalin inactivated and has been investigated with adjuvants replacement for the c- vaccine. other live and live vector approaches to improved vee vaccines have been investigated only in laboratory animals, including a chimeric virus constructed from nonstructural genes of sindbis and the structural genes from vee [ ] , vee replicon vaccines, and vaccinia recombinants. none of these approaches have reached advanced development. it is only a matter of time before another vee outbreak emerges in tropical america, and there is a substantial risk of cross-border spread. the prospects for vaccine interventions have diminished with dwindling support for new vaccines and increased concerns for vaccine safety. most zoonotic diseases are maintained in transmission cycles involving wild mammals or birds. however, because of the difficulties in vaccinating specific host species, wildlife immunization as a means of preventing spread to domestic animals and humans has been applied in only a few diseases. some of the barriers to implementing wild animal vaccination include (i) involvement of multiple species in natural transmission cycles; (ii) safety concerns for non-target species; (iii) high reproductive rates and population turn-over; (iv) fastidious feeding behaviors and difficulty in designing effective baits; (v) difficult delivery due to very high or, conversely, very low population densities of the target species; (vi) environmental concerns, and release of genetically modified organisms; (vii) difficulty in designing an effective formulation for oral immunization; (viii) instability of a vaccine or vector under prevailing environmental conditions; and (ix) requirement for low unit cost and government funding for vaccine purchase and delivery. nevertheless, targeted immunization of wild animal reservoirs is a subject of considerable interest for future research, not only for control of infectious agents affecting domestic animals and humans but also for control of wildlife diseases. one example of the latter was the effort to develop a means of immunizing great apes affected by ebola virus in central africa with vaccines previously developed for human use. aside from rabies vaccines delivered in oral baits, which is well-established, wildlife vaccination has had limited success. two promising examples of early-stage vaccine applications are described below (lyme disease and mycobacterium bovis). in addition experimental immunization and protection of prairie dogs (cynomys ludovicianus) using a raccoon poxvirus recombinant oral bait vaccine [ ] , and ballistic vaccination of bison against b. abortus [ ] have been described. plague, a global but localized zoonotic disease with rodent wildlife reservoirs, would appear to be a target of particular interest for future research [ ] . there are many other possible targets for new framework iii vaccines, and future research in this field is encouraged. in the united states, lyme disease is the most common vectorborne disease and the th most common infectious disease overall. it is also a major and increasing public health problem in europe. approximately , cases are reported in the us annually, and the number has doubled in the last years [ ] . however, at a meeting in august, , the centers for disease control and prevention (cdc) reported that the annual incidence of infection is believed to be -fold higher, i.e. , cases. although lyme disease occurs across the country, the incidence is highest in the northeast and north central states. in the us, lyme disease is caused by the spirochete borrelia burgdorferi, which is amplified each spring and summer in a cycle principally involving ixodes scapularis ticks and field mice. mice are persistently infected and represent the reservoir of infection in nature [ ] . b. burgdorferi is passed transtadially to nymphal and adult ticks which infect humans and dogs; these species develop clinical disease but are dead-end hosts. the human disease is manifested by a protean syndrome, starting with a localized skin infection (erythema migrans), and progressing to a multisystem disease variably with lassitude, arthritis, carditis, meningitis and other neurological manifestations [ ] . because of the increasing incidence and geographic expansion of lyme disease, the high incidence of tick exposure, and the difficulty in recognizing and removing attached ticks due to their small size, difficult differential diagnosis, troublesome and potentially severe clinical manifestations and medical controversies over treatment and chronicity of the disease, lyme disease has emerged as a high priority for public health interventions [ ] . vaccination of humans would appear to be a logical and cost-effective means to prevent the disease [ ] , and veterinary vaccines for dogs are widely used and have proven to be modestly effective [ ] . however, whereas safe and highly effective vaccines for humans have been developed, none is available for distribution today. glaxo smithkline's lymerix ® vaccine was approved in , but withdrawn in by the company, principally for commercial reasons, a decision that is lamentable given the increasing incidence of the disease [ , , ] . a new lyme disease vaccine for humans active against both b. burgdorferi and species causing lyme disease in europe developed by baxter bioscience is now in phase ii development, but it is uncertain whether it will reach the market. nevertheless, these vaccines established critical immunological principles; the human vaccines are composed of recombinant ospa protein, the dog vaccines of both ospa and ospc, and work via antibody-mediated mechanisms. ospa is expressed by the borrelia spirochete in the midgut of infected ticks. since the tick vector only begins to transmit borrelia - h after initiating blood feeding, ospa specific antibodies imbibed in the blood meal of a vaccinated host kill the bacteria and block transmission [ , ] . if a similar ospa antibody response could be evoked in the natural reservoir hosts of b. burgdoferi (peromyscus spp. field mice), it may be possible to interrupt the transmission cycle and reduce the prevalence of infected nymphal and adult ticks responsible for human and canine infections. proof of concept was obtained in a field study where peromyscus leucopus mice were trapped and vaccinated by subcutaneous injection of ospa; a reduction in the prevalence of b. burgdorferi in nymphal ticks was seen in the following year [ ] . however, practical vaccine delivery and effective immunization of mice in the wild, requires a thermostable oral bait vaccine matched to the high population density and rapid population turnover of the reservoir hosts, the effects of which are not diluted by non-targeted species that play a role in b. burgdorferi transmission [ ] . two promising live oral vaccine approaches have been investigated in the laboratory: a bacterial (e. coli) vector [ , ] and a viral vector (vaccinia) [ , ] expressing ospa. the e. coli vector contained in an oral bait formulation and ingested multiple times elicited anti-ospa antibodies and protected laboratory and wild p. leucopus mice against needle and tick challenge. a -year field study of the oral bait vaccine, sponsored by cdc, has been performed and results are anticipated with interest. a company, us biologics inc., is engaged in bringing this vaccine to market. the vaccinia technology, which rests on the shoulders of the successful oral bait vaccine against wildlife rabies (see below), has been tested in the laboratory. laboratory mice immunized by gavage with vaccinia expressing ospa were successfully immunized after a single dose and were protected against tick challenge. peromyscus consuming oral bait vaccine were also significantly protected against challenge with infected ticks. although the vaccinia vector looks promising, no commercial endeavor has yet emerged to support development. both the e. coli and vaccinia oral vaccines require specialized formulations in baits that incorporate the vaccine in the bait itself, rather than in a liquid sachet embedded in the bait used for delivery of rabies vaccines. many questions surround the application of an oral bait vaccine targeting the reservoir host, including efficacy of this approach in the field, the high density of baits required, cost and sustainability of local and state funded programs aimed at distributing baits, and the role of species not targeted by the vaccine in lyme disease maintenance cycles. if only partially effective, the risk of acquiring lyme disease may be reduced, but the public would still need to take precautions against tick bite. nevertheless, given the lack of a vaccine for humans, the high level of public concern about lyme disease, the high risk to children, the localized nature of b. burgdorferi transmission allowing geospatially focused control efforts, and the possibility that homeowners may be motivated to play an active role in distributing baits, the idea has appeal. m. bovis is the cause of tuberculosis in a wide array of domesticated and wild animals, and it remains a major veterinary health problem worldwide, causing severe economic losses from livestock disease, death and export restrictions. humans become infected by ingesting raw milk or undercooked meat, or by the aerosol route from infected animals or humans. in developed countries where pasteurization and test-and-slaughter programs have controlled the disease, zoonotic infections are relatively rare, accounting for . - . % of tuberculosis cases [ ] [ ] [ ] ; in developing countries which do not practice these measures, it remains more common, although few data on prevalence exist. wild animals are a major source of infection of domestic livestock [ ] . control measures aimed at control of m. bovis by culling wildlife reservoirs is problematic, with inconsistent results and ethical concerns. vaccination of wildlife is an attractive alternative control measure, especially since the traditional tuberculosis vaccine (bacille calmette-guerin, bcg) derived from m. bovis is effective when orally administered [ ] . examples of wildlife that serve as maintenance hosts of m. bovis and sources of infection in livestock, include white-tail deer in the us [ ] ; wild boar, red and fallow deer in europe [ ] ; badgers in the united kingdom [ ] ; african buffalo (syncerus caffer) in south africa [ ] ; and brushtail possums (trichosurus vulpecula) in new zealand [ ] . brushtail possums have been experimentally vaccinated using oral bcg and shown to be resistant to challenge with m. bovis [ ] . proof of concept has been provided by a field study in an endemic area of new zealand. possums were trapped, manually vaccinated using orally delivered bcg in a lipid matrix formulation, and vaccinated and control animals were recaptured at intervals [ ] . vaccinated animals received - vaccinations during the -year study. at the end of study, the ha study area was depopulated, and all animals assessed for clinical and subclinical m. bovis infections. vaccine efficacy against naturally acquired tuberculosis was - %. the authors concluded that oral vaccination of possums could be a practical strategy contributing to elimination of m. bovis in livestock. although the field study did not demonstrate control via freely consumed bait vaccine, captive possums have been shown to consume vaccine in flavored baits [ ] . rabies is transmitted between specific wild carnivore reservoir hosts, which serve as a source of spill-over infections of other wild carnivores, and infection of domesticated animals and humans. oral rabies vaccine was initially deployed in europe for control of rabies in the red fox (vulpes vulpes), using modified live virus vaccine [ , ] . the concept began in the s with the work of george baer at the cdc, which showed that foxes could be orally immunized with modified live virus [ ] . the live, attenuated evelyn-rokitnicki-abelseth (era) vaccine or street-alabama-dufferin (sad) viruses were employed in experimental and field studies. numerous studies in the s confirmed that captive and wild foxes could be orally immunized with a variety of baits containing vaccine [ ] . in , steck and colleagues initiated a wild fox rabies control program in the swiss alps using oral bait vaccine consisting of chicken heads with vaccine and tetracycline biomarker in a container made of polyvinyl chloride and aluminum foil inserted under the scalp [ ] . the trial demonstrated that % of foxes had ingested bait. over the next years, successful fox rabies control programs were carried out in many european countries, after the late s using baits distributed by fixed wing aircraft and helicopters rather than by ground [ ] , and resulting in elimination of terrestrial rabies in several countries [ , ] . for large scale distribution, the laborious chicken head method bait gave way to commercially manufactured molded or extruded baits of various kinds, consisting of fish meal or bone meal, fat, and a pouch or blister containing liquid vaccine virus [ ] . the vaccines currently used in europe are ( ) sag (e.g., rabigen ® , virbac laboratories, france), a modified live attenuated rabies virus derived from the original sad vaccine and having an additional mutation in the codon for amino acid of the rabies g protein, which increases genetic stability of the virus [ ] ; and ( ) recombinant vaccinia virus (copenhagen strain) expressing the era ® strain rabies g protein (raboral ® , merial corp.) [ ] . the rabies g protein gene has been inserted into the thymidine kinase gene of vaccinia, which results in further attenuation compared to the parental virus [ , ] . duration of oral rabies immunity, at least months in adult red foxes, is sufficient to provide herd immunity and reduce the reproductive rate (r ) to less than . the modified live virus vaccines are more thermolabile than vaccinia, require − • c storage, retain some pathogenicity for non-target species, and pose safety risks to humans exposed inadvertently. consequently, recombinant vaccinia is the only oral rabies vaccine approved for wildlife immunization in the us. this vaccine consists of fishmeal and fish oil bound by a polymer (ethylene vinyl acetate) and containing the vaccine in a plastic pouch held in place by a wax mixture. immunization with vaccinia or modified live virus occurs in the buccal mucosa and tonsils, and vaccines are poorly effective after ingestion [ ] . in one study, consumption by red foxes of a single bait containing vaccinia resulted in protection against virulent rabies in only half of the animals [ ] . this observation suggests that high bait densities and repeated vaccinations are important to effective control in the wild. bait densities distributed in europe generally range between and baits/km , resulting in - % of animals potentially immunized (positive for the tetracycline biomarker) [ ] . in addition to distribution density, feeding habits may also be important, since animals have been observed to pick apart baits and consume only the bait portion. while control of terrestrial wildlife rabies has been successful in parts of europe, it has been more challenging in other areas due to the diversity of carnivores involved in transmission of different rabies virus variants. in the arctic regions, a specific rabies variant is maintained in the arctic fox, with spill-over infections of red foxes, skunks, and raccoon dogs. where vaccination is not practiced in domesticated sledge dogs, these animals may be severely impacted by contacts with rabid foxes, and human dog owners placed at considerable risk. while experimental oral vaccination of arctic foxes has been successful, there is limited experience in the field [ ] . in ontario, canada control of arctic rabies variant in red foxes using oral bait vaccine has been successful, but the virus still occurs as a result of spill-over transmission to skunks, which are not efficiently immunized with recombinant vaccinia vaccine [ , ] . oral bait recombinant vaccinia vaccine has been primarily used to control raccoon rabies, which expanded beginning in the mid- s from enzootic areas in florida northwards and westwards to involve many states in the eastern us, as well as new brunswick and quebec, canada [ , ] . the control program relies on distribution of vaccine baits specific zones of rabies activity, particularly along the appalachian ridge, enhanced surveillance and ring vaccination with evidence of spread of the disease. judged from the absence of spread beyond the zones of vaccine distribution, the program has worked well, despite relatively low prevalence of rabies antibody (approximately %) in sampled raccoons [ ] . it is possible that pre-existing immunity to raccoonpox virus may interfere with immunization with vaccinia [ ] . the economics of large-scale oral vaccination programs in the us have been modeled and are generally favorable [ ] . in addition to control of raccoon rabies, successful use of the vaccine has been made in the control of gray fox (urocyon cinereoargenteus) variant rabies in west texas [ ] . in contrast to raccoons, a higher prevalence of rabies antibody ( %) attributed to vaccination is found in gray foxes. rabies in coyotes (canis latrans) was responsible for epizootic canine rabies in the s and s in parts of the us, and was controlled by an oral bait vaccination campaign, contributing to the elimination of canine rabies by [ ] . skunks remain a problematic species for vaccine control of rabies. skunks are an important spill-over host for the arctic fox, raccoon rabies, and big brown bat rabies virus variants [ ] . although the vaccinia vector vaccine is not sufficiently effective in skunks [ ] , promising results were obtained with a replicationcompetent adenovirus type vector expressing the rabies g protein [ ] . aerial distribution of this vaccine (onrab ® , artemis technologies, guelph) showed high rates of immunization of raccoons, and arctic foxes and modest seroconversion ( - % in different plots) in skunks, probably due to lower rates of bait acceptance [ , ] . onrab ® is approved by the canadian regulatory authorities for control of rabies in skunks, raccoons, and foxes, and is under investigation in the us. there are some notable failures of animal vaccination as a means to preventing zoonotic diseases of humans, and these illustrate some of the limitations of the approach shown in table . japanese encephalitis, a mosquito-borne flavivirus closely related to west nile virus, is endemic in asia, with nearly billion people at risk of infection [ ] . horses and humans are dead-end hosts, and framework i immunization of both is widely practiced in many parts of asia, with a long record of success. pigs are an important domesticated animal amplifying host for infection of rice paddy-breeding culex spp. vectors, resulting in spill-over infections of humans. moreover, infection of pregnant sows can lead to abortion and stillbirth, and infected boars may have reduced spermatogenesis and infertility [ ] . framework ii immunization of swine was previously a major initiative in japan, using live, attenuated vaccines. however, it was exceedingly difficult to vaccinate piglets born in spring and early summer during the narrow window between loss of interfering maternal antibody and contribution to virus amplification. the practice of vaccination was abandoned in favor of re-locating piggeries from areas of culex breeding and biting activity. elsewhere in asia, other obstacles precluded consideration of framework ii vaccination against japanese encephalitis, including the prevalence of small piggeries located near vector breeding sites and of feral swine, and the importance of wild ardeid birds and waterfowl in virus transmission. moreover in many areas of asia, less developed than japan, and undergoing rapid urbanization, the locations of pig holdings are not controlled and are often located near rice paddies and urban centers [ ] . consequently, the focus has long been on human vaccination as the primary means of prevention. this case study exemplifies some of the factors that can limit application of framework ii vaccination: ( ) the need to customize vaccination to the breeding and husbandry practices and timing of domesticated animal targets; ( ) the role of wild animals and feral domesticated animals as additional amplifying hosts in the transmission cycle; and ( ) difficult access given the very large scale and geographic complexity of domesticated animal populations. q fever is caused by the intracellular gram-negative bacterium, coxiellla burnetii and an important worldwide infection of ruminants, which serve as the source of infection of humans, especially where large numbers of animals are concentrated [ ] . q fever is a major occupational hazard of abattoir and farm workers, veterinarians, and persons involved in the handling and distribution of animals or animal products. a dramatic outbreak recently occurred in high-intensity goat farms in the netherlands ( ), with human cases and deaths [ ] . transmission of c. burnetii occurs between direct spread between domesticated animals, and from animals to humans. infected animals shed bacteria in urine, feces, vaginal secretions and products of conception, and in milk. ticks are also a reservoir of bacteria in nature and a source of infection of livestock. ruminants, particularly goats and sheep develop pneumonia, abortion, stillbirth, premature delivery, and delivery of weak offspring, and herds can be affected for prolonged periods, causing significant economic losses [ ] . there are two developmental stages of c. burnetii, a small-cell variant (scv, the extracellular form) and the metabolically active intracellular large-cell variant. the scv is highly resistant to degradation and can persist in the environment for long periods of time. infection of humans is acquired principally by the aerosol route via dust containing spore-like scv forms, with oral routes of infection (ingestion of unpasteurized milk) being secondary. the human disease is characterized by an influenza-like illness, and may be complicated by pneumonia, endocarditis, and (pregnant women) abortion and fetal death. person-to-person transmission occurs, but is rare. approximately % of infected persons may develop chronic infections, with various manifestations. prophylactic vaccines have played a role in the control of q fever in livestock, but the practice is not a reliable means of preventing human infections. in russia, a live, attenuated m- vaccine was used for many years in animals and humans, but causes a persistent infection and has not been considered safe for use elsewhere. in europe, coxevac ® , a formalin inactivated strain rsa /nine-mile phase bacterial form (smooth forms, with complete surface lipopolysaccharide) has been used in goats and cattle and reduced the incidence of shedding, but is not effective in pregnant or chronically infected animals [ , ] . the live and phase vaccines are also not diva, which presents substantial issues for export controls. in australia, an inactivated phase vaccine prepared from henzereling strain is marketed for humans by csl ltd (qvax ® ) [ ] . however, severe reactions occur in persons who have previously been naturally infected with c. burnetii, and skin testing to ensure absence of exposure is required [ ] . clearly, improved vaccines are needed for both livestock and humans, but various attempts at recombinant vaccines have been disappointing. the problem for framework ii vaccination against q fever is due to multiple factors, including imperfect vaccines, limited efficacy of vaccines in parous animals, the difficulty in recognizing the disease and intervening expeditiously, and the rapid and widespread contamination of the environment with scv forms. the last problem is the major reason that livestock vaccination is not a reliable means of protecting humans against exposure. a year study of sheep vaccinated with the phase vaccine showed that the proportion of animals shedding bacteria in feces was markedly reduced after year and then eliminated after years, but c. burnetii was still present in environmental samples [ ] . finally, live veterinary vaccines may pose a risk of inadvertent infection of humans handling the vaccine or vaccinated animals. examples include live brucella and orf (contagious ecthyma) vaccines. while this topic is beyond the scope of this review, it is important in several contexts, and indeed little is known about the risk of human pathogens to animals. immunization of swine and poultry workers against influenza as a means of preventing introduction of human influenza viruses to these animals has been emphasized as a means of preventing emergence of reassortant strains [ ] . immunization of humans to prevent spread of viruses to captive nonhuman primates is often practiced, including vaccines for influenza, hepatitis a and b, and measles. prevention of animal diseases and human diseases by use of vaccines is a well-established principle, and there are potential synergies that can be achieved in concurrent delivery of human and animal vaccines in developing country settings [ ] . for some diseases affecting both livestock and humans there is a clear commercial incentive to develop vaccine products (framework i vaccines) ( table ) . however, such development efforts are generally segregated in the animal and human health divisions in industry and academia, and have separate regulatory pathways. this results in a potential waste of resources and duplicated scientific endeavors. interestingly, when one company (akso nobel), an animal health company, decided in on a strategic move into human vaccine development, it drew on its veterinary scientists to staff the program. as pointed out in this review, there have been isolated successful examples, e.g. a west nile vaccine, of co-development of a vaccine for both veterinary and human indications, an obviously efficient strategy that broadened both the commercial and public health opportunity. future efforts along similar lines should be considered on a case by case basis, depending on medical need, but in general there is value in closer connections between human and veterinary vaccines and regulatory science, and in the application of domesticated animals as models for development of infectious disease and cancer vaccines. several issues related to diva requirements and liability concerns have been mentioned. prevention of zoonotic diseases of humans by means of vaccination of domesticated (framework ii) or wild (framework iii) animals is an attractive but under-exploited concept. an obstacle is that there may be limited commercial incentives (table ) . where a market exists, governments may be the principal customers, as is the case for the approved oral bait rabies vaccines and the reservoirtargeted lyme disease vaccine in development. thus, the public health gains for such an intervention need to be compelling and must offset the cost of development and implementation. the goal is far easier to justify when vaccination also prevents disease in an economically valuable animal species, there is a profit incentive for animal vaccination or a clear social gain from improved animal health, and when the public health spin-off is an added benefit. examples of the latter may include vaccines against hendra and nipah virus diseases, brucellosis, chlamydophila felis, rift valley fever, and venezuelan equine encephalitis. the potential for elimination of a disease through vaccination (employed together with other strategies), as has been demonstrated for brucellosis in the us and terrestrial rabies in some european countries is a compelling economic concept, though applicable in only selected cases. the cost of preventative programs is almost always lower than emergency response programs, as illustrated by the significantly lower cost of oral wildlife rabies programs over contingency actions to control epizootic spread [ ] . the recent announcement of a fold higher incidence of lyme disease than previously believed will lead to a reassessment of the economics of preventive strategies for this disease, including wildlife vaccines. economics represent the key determinant for development and utilization of framework ii and iii vaccine strategies. a low unit cost of such vaccines will always be a requirement. the economic barriers are particularly relevant when considering vaccines for developing countries. on the positive side, the cost of developing a new animal vaccine through licensure is a small fraction, approximately %, of the cost of a typical human vaccine (the latter being $ - million by one estimate [ ] , but often far higher) [ ] . the relatively lower cost and shorter timelines for developing animal vaccines reflects the simpler path to regulatory approval, and is driven by the significantly lower market potential for these vaccines. despite the lower cost of developing new veterinary vaccines, high and middle income countries still pay higher prices until the fixed costs of development are paid off, there is an over-supply of vaccine, or competing products enter the market. to redress the pricing barriers in the case of human vaccines, there has been strong advocacy for new approaches to secure vaccine supply and access for developing countries where the burden of infectious diseases is greatest [ ] . as part of this strategy, emerging market manufacturers provide access to low unit cost vaccines, and such manufacturers of veterinary vaccines could play a substantial role in a public health strategy for animal immunization. indeed all of the principles being applied to human vaccines could be extended to vaccines for animals, particularly if there is both a clear rationale for public health and the "pull" of a potentially expanded market or of guaranteed purchase agreements. up to now public-private financing for developing and distributing veterinary vaccines has represented a tiny fraction of support available for human developing-world vaccines, and has focused on vaccines for livestock as a means of improving animal production and protein supply rather than preventing zoonotic diseases [ ] . given the public health impact of zoonotic diseases described in the introduction to this review and the potentially lower cost of interventions targeting animals vs. humans, there should be a new emphasis on the public health improvements that could result from animal immunization. zoonotic diseases that merit consideration because they occur at high incidence or are poorly controlled, include rabies, zoonotic leishmaniasis, brucellosis, rift valley fever, m. bovis, lyme disease, and several enteric bacterial infections. as mentioned above, the regulatory pathway for animal vaccines is considerably simpler than for human vaccines [ ] . this is due to multiple factors, including less onerous requirements for manufacturing and control of veterinary products, the simpler and far less expensive clinical trial requirements for marketing approval, the ability to challenge animals to demonstrate vaccine efficacy, as well as an established regulatory mechanism for conditional approval allowing commercial sales while still gathering more definitive data. there is no requirement for large, statistically powered efficacy field trials to obtain marketing approval, as is the case for human vaccines. the development of veterinary vaccines is consequently far faster than for human vaccines. for example, west nile vaccine for horses was commercialized years after introduction of the virus into the u.s., whereas the first (phase ) human trial of a west nile vaccine was completed years after introduction. the lower costs and accelerated timeframe for development of animal vaccines represent an important rationale for novel investments in public health, particularly in developing countries. to justify investments in a framework ii or iii vaccine where an economic incentive for animal immunization is marginal and a public health benefit is a goal, it is important to plan for vaccine effectiveness studies showing that vaccination of animals actually reduces human disease prevalence, as was postulated in greece following vaccination against b. melitensis [ ] . such demonstrations would really drive the field 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direct doi: . /s - - - sha: doc_id: cord_uid: ybqj z r chron’s disease is a chronic inflammatory intestinal disease, first described at the beginning of the last century. the disease is characterized by the alternation of periods of flares and remissions influenced by a complex pathogenesis in which inflammation plays a key role. crohn’s disease evolution is mediated by a complex alteration of the inflammatory response which is characterized by alterations of the innate immunity of the intestinal mucosa barrier together with a remodeling of the extracellular matrix through the expression of metalloproteins and increased adhesion molecules expression, such as maccam- . this reshaped microenvironment enhances leucocytes migration in the sites of inflammation, promoting a t(h) response, through the production of cytokines such as il- and tnf-α. il- itself and il- have been targeted for the medical treatment of cd. giving the limited success of medical therapies, the treatment of the disease is invariably surgical. this review will highlight the role of inflammation in cd and describe the surgical approaches for the prevention of the almost inevitable recurrence. crohn's disease (cd) is a chronic inflammatory intestinal disease, first described as regional ileitis by crohn, ginzburg and oppenheimer in a case series presented at american medical association annual meeting in [ ] . cd inflammation interests the whole intestine, being the most frequently affected part the distal ileum. patients with cd experience periods of flares and periods of remissions during their disease course. pathogenesis results from the interactions of environmental factors, immune system, susceptibility genes and host's microbiome changes, leading to disruption of the intestinal mucosa. the role of inflammatory cells in maintaining an active disease is well known and most of the therapies aim to stop the cascade of inflammatory and proinflammatory cytokines. treatment of cd is multidisciplinary: medical treatment is focused on mucosal healing and symptoms reduction; surgery maintains a key-role in treating complications such as stenosis, perforations, fistulas and abscesses. surgical recurrence is known to afflict over % of the operated patients [ , ] . multiple surgical strategies have been investigated to improve outcome. introduction of laparoscopic techniques has permitted several improvements but failed in reducing recurrences; other surgical techniques are currently under evaluation in view to retard or prevent the ineluctable recurrence but the surgical cure for cd is yet to be discovered. this review is focused on cd inflammation and will discuss potential strategies to prevent recurrences, such as novel surgical approaches. crohn's disease pathogenesis is based on tissue inflammation, caused by an unrestrainable immune response against luminal bacterial antigens (fig. ) . immune cells like cd t-cells, cd t-cells, b-cells, cd monocytes and natural killers, are involved in this process as they infiltrate the gut of cd patients. part of the immune-mediated susceptivity to cd resides in some innate mechanisms of defense form infectious diseases and the intestinal mucus secretion is part of those. it has been shown that variants of the muc gene reducing mucus production are associated to cd in a mouse model. moreover, molecules that are mediating bacterial adhesion have been correlated to the disease. this is the case of fut , which encodes for the fucosyltransferase enzyme, responsible for the secretion of soluble forms of the abo antigens. people harboring a fut variants decreasing the secretion of the antigens, have an altered interaction with bacteria and are more prone to developing cd [ ] . the pathogenesis is also sustained by the interaction of these cells with integrins, adhesion molecules and multiple chemokines, responsible for the production of elevated levels of inflammatory cytokines, representing the target of immune and non-immune cells and the promotion of mucosal inflammation. as such, among many adhesion molecules, some evidences on the involvement of the leucocyte maccam- , receptor for the α β integrin, seems to play a crucial role. together with leucocyte adhesion, the role of the extracellular matrix on their activation has been explored. proteins like cd and cd where shown to play a role as well as metalloproteins (mmp), being mmp and mmp abundant in the granular tissue close to cd sites of inflammation, therefore responsible for leucocytes activation [ ] [ ] [ ] [ ] . in the mucosa of cd patients, a dysregulation of various components of the immune system is invariably found. the most pronounced alteration is the hyperactivity of t cells with excessive production of cytokines, between which il- and ifn-γ, promoting a t h lymphocytic phenotype, opposed to the t h one, correlating to ulcerative colitis. moreover, tnf-α production has also been demonstrated to increase the number of cd + foxp + t reg cells, especially in the mucosa of children affected by cd [ ] . the inhibition of fig. immuno-mediated pathogenesis of crohn's disease. crohn's disease is a multifactorial pathology in which a major role is played by alterations at the level of immunity and inflammation. innate immunity is involved in terms of defects in the mucous barrier (mut and fut genes) while adaptive immunity relies on a t h lymphocitic response and t reg cells mediated by cytokines like tnf-α, il- , il- and il- . the increased migration to the sites of inflammation is also determined by a reshaping of the extra cellular matrix through the action of metalloproteins (mmp- and mmp- ) and the overexpression of adhesion molecules such as maccam- and integrin α β . finally, also the host pathogen interaction between the intestinal epithelium and the microbiota has been linked to the evolution of the disease. picture created with biorender.com the effector cytokines, like tnf-α, attenuates the detrimental effects in subsets of cd patients. furthermore, the expression of the interleukins, a subgroup of cytokines implicated in the enhanced or inhibition of other cytokines in many different regulatory pathways such as maturation, growth and responsiveness of immune cells population, is to be considered anomalous in cd patients [ ] . further analysis of t cell subsets has revealed the presence of t h and t h cells in cd, whereas the cytokines considered more involved are tnf, il and il . apart from the cited cytokines, il- has also been associated to ibd and cd in particular. il expression is more pronounced in the areas of active inflammation, especially in cd, and seems to induce tnf-α and il expression through a erk-mediated mechanism. moreover, il- has been described as an inducer of ccl through the interaction with its receptor the m-csfr , abundantly expressed in the inflamed colonic epithelium but not in the healthy controls. on the contrary, il- inversely correlates to the inflammatory state of the patients with ibd, being reduced in cd patients as opposing to healthy subject, and being reduced in the affected areas of the colon if compared to the surrounding adjacent normal tissue from the same subject. among all the possible interleukins associated to cd pathogenesis, il- and il- represent the target of still inadequate therapies because of potential side effects, such as increased risk for infection, and the blockade of specific immunological targets, capable of induction of alternative signaling or homing pathways. the latter mechanism may also partially explain the frequent lack of response to therapy with biologics such as infliximab (remicade©), a chimeric monoclonal antibody used to treat autoimmune diseases, that works by binding to tnf-α causing the reduction of il- expression, implicated in monocyte and macrophage differentiation, survival and function [ ] [ ] [ ] [ ] [ ] . although t-cells are the main effector lymphocytes in intestinal tissue inflammation, also humoral immune system plays a crucial role. plasma cells differentiation indeed is promoted by cd t-cells, through a mechanism that is firmly dependent on il- , overproduced in cd patient's gut. il- converts naive b-cells into b-cells expressing granzyme-b: it possesses a cytotoxic activity on the intestinal mucosa and perpetuates the epithelial damage. these proofs indicate that an altered balance between effector and counter regulatory factors is probably involved in the sustainment of the tissuedamaging immune response in cd [ ] . gut microbiota also plays a recognized role in designing the inflammatory response in ibd and especially in cd. there is growing evidences that some microbial among them, anti-inflammatory drugs as mesalazine, antibiotics such as fluorochinolones and metronidazole and immunosuppressants (methotrexate). more targeted treatment options are directed towards tnf-α (infliximab, adalimumab, certolizumab) or against integrins (vedolimumab) and interleukins il- and il- (ustekinumab). picture created with biorender.com gene products can influence gene expressions in the host [ ] [ ] [ ] . the complex network arising from this assumption is referred to as the microbial-associated molecular pattern (mamp) which is sensed by toll-like receptors on immune cells, contributing to their activation in the context of the chronic inflammation [ ] . microbiome moreover represent a source of potential pathogenic inputs that can be approached through the methods used in the omics era, such as metagenomics studies, also impacting on our knowledge on geographical variations on the clinical manifestation of the disease [ ] [ ] [ ] [ ] . the inflammation is generally transmural and, on pathology examination, granulomas may be identified on biopsies, with a discontinuous distribution along the longitudinal axis. this inflammatory process often leads to irreversible tissue damage in the form of intestinal stenosis or fistulas, inflammatory masses or intra-abdominal abscesses. patients can develop one or more of these disease behavior and they often tend to evolve from inflammatory to penetrating or stricturing disease [ ] . medical management (fig. ) should be tailored based on various factors such as disease severity, subtype, behavior and location [ ] . moreover, it is important to consider other factors such as age at diagnosis, extension of the lesions and extra-intestinal manifestations [ ] . as matter of fact, none of the drugs used in the treatment of cd has been demonstrated to be curative or completely safe. mesalazine, which belong to the -asa compounds category, has been evaluated in many studies and it has never shown to definitely induce or maintain remission in cd. its benefits are related to its safety outline [ ] . antibiotics are principally recommended to treat septic complications; like mesalazine, they do not show a real efficacy in the treatment of cd, except in the shortterm treatment of perianal fistula in association with anti-tnf. most frequently used agents are fluoroquinolone and metronidazole [ ] . systemic corticosteroids show a fast onset of action and are indicated to induce remission. unfortunately, steroid dependency or steroid resistance can jeopardize their use that is often accompanied to a wide range of side effects like obesity, hypertension, glaucoma, cataracts and adrenal insufficiency [ ] . another major part is played by immunosuppressant like thiopurines and methotrexate: thiopurines are used to maintain remission in moderate cd, usually in combination with steroids. before starting thiopurines treatment, it is mandatory to assess tpmt (thiopurine s-methyltransferase) activity, crucial for their metabolism [ ] . methotrexate may be considered as an option for steroid-dependent patients. included in the side effects are hepatotoxicity and more rarely myelosuppression; they are prohibited during pregnancy because teratogenic and abortifacient [ ] . anti-tnf drugs are considered the most powerful tools to treat moderate and severe form of cd, alone or in association with immunomodulators to obtain and maintain remission. the most frequently used anti-tnf are: infliximab (remicade©), a chimeric antibody that is administered intravenously; adalimumab (humira©), a fully humanized monoclonal antibody administered subcutaneously; certolizumab (cimzia©), a fab antibody fragment of humanized anti-tnf molecule [ ] . more recently super-selective target monoclonal antibodies have been developed, directed against a specific pattern of inflammation. in this class there are vedolizumab (entyvio©), that targets the adhesion molecular inhibiting leukocyte migration [ ] , and interleukininhibitors like ustekinumab (stelara©), a fully humanized monoclonal antibody targeting the p- subunit of il- and il- [ ] . when cd was described for the first time, therapy was exclusively surgical. since the beginning of cd surgery experience, there was no consensus on the optimal procedure. at the mount sinai hospital in new york, dr. berg was the surgeon who operated fourteen patients presented by crohn. the "berg" operation, also known as "mount sinai" operation, implied exclusion bypass of the ileocecal region, transecting small bowel proximal to the diseased ileum, over sewing distal ileum, and anastomosing the proximal ileal end into the mid-transverse colon [ , ] . in fact, this was a staged management, being the second planned step the resection of the diseased bowel. performing more and more cases, mount sinai surgeons noted that during the second stage of surgery, the bypassed bowel seemed to have "healed" in several cases. starting from this observation and as patients manifested clinical improvements, they decided to omit the planned second procedure. obviously, a great debate about this procedure and all early and late risks linked to it arose: blow out of the blind end, reactivation of cd in the excluded segment with abdominal pain and infections, deprivation of a large portion of the colon for water absorption. eventually, bypass procedure was abandoned due to findings of adenocarcinoma occurring in the excluded segment [ , [ ] [ ] [ ] . surgical resection of the diseased bowel emerged as the procedure of choice for most patients with cd of the terminal ileum or with ileo-colitis, including complicated cases [ ] . in tandem, advances in perioperative care, such as nutritional improvement, anesthesia and fluid and electrolyte management, guaranteed cd surgery improvements and safety. at this point physician focused on the amount of resection. in fact, it was commonly accepted practice for surgeons to resect all macroscopically involved intestine with large resection margins. consciousness of cd as a pan-enteric affection and, above all, the evidence of the inevitable recurrence and possible development of short bowel syndrome due to repetitive surgery, suggested the adoption of a conservative policy, avoiding wide resections [ ] . for this reason, in order to avoid short bowel syndrome -in particular in those scenarios characterized by extensive jejunal-ileitis with fibrotic stenosing segments scattered along the diseased intestine -lee from oxford, in , reported another advancement in surgical management. lee was inspired by the work of indian surgeons on the management of tuberculous strictures: they observed that small bowel preservation could be achieved in patients with multiple tuberculous strictures by strictureplasty [ ] . lee applied strictureplasty to the short intestinal strictures of crohn's disease and from that moment, with a great variability on techniques according to the different situations, strictureplasty assumed a fundamental role for cd surgeons [ , ] . different strictureplasty techniques have been described. heineke-mikulicz strictureplasty consists of a longitudinal enterotomy closed in a transverse direction and it is best applied to stricture up to cm in length [ ] . finney strictureplasty is used for longer stenosis, up to - cm: after an antimesenteric longitudinal incision, the opened bowel segment is bent into a u shape and posterior and anterior layers are close with continuous absorbable suture [ ] . michelassi strictureplasty is indicated for the treatment of multiple strictures, interesting up to cm long bowel segment; in this case, a segment of diseased bowel is anastomosed to a nonaffected segment of intestine [ ] . interestingly this technique has shown to induce remission in the diseased part. the mechanism is still unknown, however there seems to be a process in cd whereby obstruction is responsible for the pathogenesis of many complications [ ] . this technique allows the mitigation of fecal stasis, which may play a central role in postoperative mucosal healing, modifying the microbial-mucosal interaction. possibly, the resolution of chronic obstruction may interrupt the cascade of events causing active disease [ ] [ ] [ ] . another cornerstone in cd surgery was represented by the advent of minimally invasive surgery. feasibility and safety of laparoscopic ileo-cecal resection has been assessed and it was found not inferior in terms of outcomes when compared to open surgery [ ] [ ] [ ] . nowadays, laparoscopy is largely accepted as the first line approach for cd, in the presence of adequate expertise [ ] . laparoscopy demonstrated advantages in terms of cosmetics and postoperative recovery and assured some long-term advantages, including fewer incisional hernias, fewer adhesions and a significant impact on female fertility [ , ] ; unfortunately, no clear differences on time to recurrence was found. from a surgical point of view cd recurrence should be considered as an inevitable consequence. the same factors that underline the pathogenesis of cd at its first stages are thought to be responsible for post-operative recurrence (por) setting, being the result of interplay of microbial, environmental, immunological and genetic variables [ ] . within this contest, microbial flora role seems to be linked to the fecal stream, as demonstrated by rutgeerts et al. [ , ] investigating the rapid recurrence of microscopic inflammation in the mucosa of excluded ileum when newly interested by fecal content. the term post-operative recurrence is used to define the appearance of new lesions after bowel resection. active surveillance for an early diagnosis is considered mandatory. rutgeerts endoscopic index is possibly the most widely used scoring system to detect recurrent lesions [ , ] . previous studies demonstrated that the lesions are located more often in or near the area of anastomosis, usually reproducing the same initial pattern of the disease, though it has been suggested that postresection lesions should be considered new. the presence of microscopic lesion, detected during endoscopic examinations year after surgery, reinforces their role as precursors of por. timing of endoscopic surveillance has been discussed taking into account available evidence; recommendation is to perform endoscopic examination after months from surgery or within the first year [ , ] . other techniques have been investigated to assess por [ ] ; in particular, there is a great interest in non-invasive techniques such as ultrasonography (us). among several emerging us technique, small intestine contrast ultrasonography (sicus) resulted more sensitive in detecting small bowel lesions in cd patients [ ] . sicus has been demonstrated to be comparable to ileocolonscopy, also after surgery, allowing the visualization of extra luminal lesions related to cd (bowel wall thickness, mesenteric and lymph nodes enlargement). hence, in expert hands, sicus could be considered a valid alternative for the follow-up and early diagnosis of por after surgery in cd [ ] [ ] [ ] [ ] . other recent interesting fields of investigation focus on the role of anastomosis configuration and the mesentery function in the pathogenesis of por. for what concern anastomosis, a great debate was raised regard which technique should be considered optimal, in particular between the more frequent choices: side-to-side versus end-to-end configuration and mechanic stapled versus hand sewn. in he [ ] carried out a meta-analysis to compare stapled sideto-side anastomosis (sssa) and hand sewn end-to-end anastomosis (heea) in terms of postoperative early and late complications and por after ileo-colic resection for cd. the conclusion was that sssa should be preferred because of its larger luminal diameter, thus showing lower overall incidence of complications including anastomotic leak, lower recurrence and re-operation for recurrence. in , feng [ ] carried out a similar meta-analysis, looking specifically at the orientation of the anastomosis. feng concluded that sssa isoperistaltic is probably the optimal anastomosis because it can significantly reduce incidence of overall postoperative complications and clinical por. the underlying idea is that, with its wide lumen configuration, sssa isoperistaltic reduces recurrence by preventing early stenosis, colonic reflux and secondary ischemia. in gajendran [ ] published his series, supporting the superiority of heea when compared to anti-peristaltic sssa. he also looked into the impact on quality of life and inflammatory activity. gajendran developed an experimental animal model to provide a mechanistic explanation for his clinical findings, showing that antiperistaltic orientation alters anatomy and physiology, creating an anti-peristaltic reservoir, which causes dysmotility and alteration in contractility. in particular, the animal model showed that this is due to the perpendicular surgical trans-section of the intestinal circular muscle layers: disruption of motility seems to lead to significant structural and functional changes with local stasis of enteric contents and local distension at the anastomotic site. gajendran concluded that, according to his data, the restoration of physiologic intestinal function with surgical reconstruction of the bowel as an intact tube could contribute to a better outcome in cd patients. in the same year, aaltonen's group [ ] published its series regarding risk factors for anastomotic recurrence. aaltonen's et al. proposed a technical variant heea, adding an opening of the small bowel's anti-mesenteric border to ensure enough wide bowel lumen, describing this modified technique as a safe choice for ileo-colonic resection. current guidelines from the american society of colon and rectal surgeons [ ] , states that anastomosis can be constructed as deemed most appropriate by the surgeon. ecco guidelines [ ] more recently, seems to favor sssa, taking into account he and feng's meta-analysis [ , ] and stressing the concept that a wider anastomosis will have a lower rate of clinical and surgical recurrence. within this context, toru kono developed a new anastomotic technique. the first kono-s anastomosis' (ksa) work was published in [ ] . ksa is an antimesenteric functional end-to-end hand sewn anastomosis, configured so that the mesentery side is located in the center of the stump. both stumps are sutured to create a "supporting column" to maintain the diameter and dimension of the anastomosis, preventing distortion and stenosis associated with recurrent disease at the anastomotic side, especially on the mesenteric side, which represents a locus minoris resistentiae and so a typical recurrence location. in fichera [ ] highlighted ksa innovations: the theoretical advantages of the complete exclusion of the mesentery, the initial site of cd por, with a true antimesenteric anastomosis; lower susceptibility to mechanical distortions due to the stability provided by the "supporting column"; better preservation of blood supply and innervation, achieved by dividing the mesentery close to the bowel. moreover, in , katsuno published his series confirming safety, feasibility and good results and highlighted the easier endoscopic access to the ksa [ ] . results from the first international multicenter study [ ] , leaded by kono himself, were available in and the authors suggested resection and ksa for cd patients who are not candidates for anti-tnf therapy due to adverse effects, loss of efficacy or financial reasons. in , seyfried [ ] and shimada [ ] published two more series and in , during the ecco congress in vienna, luglio presented the results of "the supreme-cd study" [ ] , the first randomized clinical trial comparing ksa and sssa in terms of endoscopic and surgical recurrence, confirming a reduction in por when ksa is performed. multicenter trials are still needed to further confirm these preliminary results. indeed, the interest on anastomotic configuration is still open and recently celentano developed a model for a v-modified side-to-side, antimesenteric, iso-peristaltic anastomosis in which a strictureplasty is added to the inlet and the outlet of the anastomosis. celentano's configuration target is the widening of the lumen of the bowel in these two critical areas, with the aim of minimizing the risk of clinical and surgical anastomotic recurrence; this is just a model ex vivo but representative of the wide interest in this field [ ] . the role of the mesentery could be considered the other trending topic of the last decade. the underlying idea is that the mesentery plays a prominent role in cd pathogenesis and in recurrences. mesentery is seen as an independent organ, interposed between the intestine and the body. from its privileged location, mesentery is responsible for the conduction of local intestinal and systemic response: it is the reservoir of various cell types, in particular inflammatory ones, contained in lymph nodes and related mediators [ ] . moreover, cd mesentery shows the pathognomonic phenomenon known as fat wrapping or creeping fat [ ] , consisting of a peculiar form of adipose tissue hypertrophy. creeping fat is characterized by small adipocytes, increased in number with a specific gene expression profile, accompanied to immune cell infiltration, comprising regulatory m macrophages and t-cells [ ] . guedj et al. [ ] , thanks to in vitro experiments on resection specimens of ileum from patients operated for cd, proposed a mechanism in which mesenteric adipocyte, through their production of key chemokines in response to inflammatory/bacterial stimuli, orchestrate an immune response in cd-affected mesentery. between and , li published two different papers regarding this topic. in the first one [ ] he focused on the contribution of the mesenteric adipose tissue, measuring the fat visceral areaassimilated to the creeping fat phenomenonin ct scan performed before surgery; in this retrospective study also subcutaneous fat area and mesenteric fat index, defined as the ratio of visceral and subcutaneous fat were considered. as result, high fat visceral area was found to be and independent predictor of early clinical recurrence of cd por. in the second paper [ ] , li highlighted the contribution of mesenteric nerves, vessels, lymphatics and fat mass, concluding that all these structures play a crucial role in cd pathogenesis and disease progression. he provided the basis for the copernican revolution in cd pathogenesis, querying the current dominant theory in cd, based on the unidirectional, "outside-in" axis of dysbiosis, innate immunity-adaptive immunity-mesenterybody system. emerging clinical evidence strongly suggest that the axis is bidirectional, involving also all the cited mesenteric structures, and not only endoluminal agents as already cited in the pathogenesis section. coffey and rivera [ , ] gave other further hints to remove the veil of maya on the pathogenesis and the mesenteric role. they started from the assumption that topographic distribution of crohn's disease along the intestinal tract may have a bodily mesenteric basis in terms of tissue volume and thickness. as a proof, they noted that the largest mesenteric region is the ileocolic region, which happens to be also the commonest localization of cd. in mao [ ] gave more insights on how creeping fat influences stricture formation in cd; he took into account mechanisms involved in the microenvironment at interfaces of different tissue compartments, such as creeping fat, considered as an extension of mesenteric fat beginning at the intestinal hilum, and the intestine muscularis mucosa itself. as already emphasized, in creeping fat immune and nonimmune cell types are represented and increased in number, producing mediators responsible for intestinal stricture formation. among these cells, fatmesenchymal cells seem to play a pivotal role because their interactions appear to be important in tissue remodeling in multiple organs, including the intestine. hence, creeping fat abandons the old role of innocent bystander and is acknowledged as an active participant in inflammation and immunity. mao hypothesized also that stricture formation incidence has remained unchanged because no target-therapy is available and so cells involved in this process should represent a pharmacological target. therefore, the awareness of the central mesentery's role provides a copernican revolution also in terms of clinical target. in fact, from this point of view, mesentery could be considered as an anatomical sacrarium: there are no valid pharmacotherapeutic modalities designed specifically to manipulate it. at present, the only means of targeting the mesentery are surgical, so there is the need for development of new strategies in this field. even though conservative approach to intestinal resection in cd could be considered an established dogma, in the light of the recent discovered role of mesentery, the attitude is changing. up to the present, mesentery resection has not become standard practice. this is mainly due to technical concerns regarding risk of bleeding when manipulating tissues with significant inflammation, disease-related perforation, fistula formation, adhesions, and thickened mesentery, potentially leading to complications like hemorrhage, hematomas and other injuries. overcoming these issues and starting from the previous considerations, coffey et al. [ ] published a series on the inclusion of mesentery in ileocolic resection for cd. clinical findings were coherent with formulated theories: inclusion of the mesentery as part of intestinal resection is associated with reduced por, that means improved clinical outcomes, and advanced mesenteric disease resulted to be a predictor of increased risk of por. moreover de groof et al. presented a series on proctectomy in cd, demonstrated that perineal complications were more frequent after close rectal dissection than after total mesorectal excision [ ] . these results suggested a pathogenic role for the mesorectal tissue in cd. in the footsteps of these assumptions, an international, multicenter, randomized controlled trial [ ] is ongoing about the mesenteric excision surgery versus conservative limited resection in cd. moreover, our group is running the panacea study (pathophysiological, nodal-based approach for crohn's disease excision), a pilot yet unpublished study, based on the belief that the majority of t-cells -especially memory t-cellslies in lymph nodes [ ] [ ] [ ] [ ] . our hypothesis is that mesenteric resection, including lymph nodes, should free the organism from a great number of cells involved in intestinal inflammation. results from this study altogether with the others, will contribute to understand which of the proposed approach will be valid in reducing por. crohn's disease has been seen, in the last two decades, as a multifactorial inflammatory disease. much is known in terms of its pathogenesis from a molecular point of view from the involvement of the mucosal mucinous barrier and the role played by variants of the mut or the fut genes, which alters the barrier interaction with both pathogens and harmful substances, to the complex mechanisms involving aberrant expression of adhesion molecules. the leucocytic maccam- is a mediator of integrin dependent adhesion, which is part of the mechanism leading to migration of leucocytes in cd inflamed region. on the other front, the migration of inflammatory cells is also mediated by the alteration of the extracellular matrix, often mediated by mmp proteins, being mmp- and mmp- the most abundant in this clinical context. the complex milieu that is created by the interaction of the inflammatory cells with the intestinal epithelium is sustained by a complex network of cytokines and chemokines, which direct t lymphocytes towards a t h response, mediated by the expression of the foxp transcription factor in the t reg population. among the cytokines involved in cd pathogenesis, il- and il- seem to play opposing roles, the first increased in the injured tissue, the second one diminished. other cytokines such as il- and il- have been demonstrated paying a key role in the inflammatory response and are some of the few current medical therapeutic targets for cd. anyhow, considering that a consistent part of cd therapeutic approaches remains surgical. we reviewed emerging surgical approaches 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surgery publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. lorenzo petagna, amedeo antonelli, carlo ganini and giuseppe sica, wrote the manuscript and approved the final version. marzia franceschilli, andrea guida, sara ingallinella, bruno sensi, leandro siragusa, michela campanelli, vittoria bellato, andrea divizia, did the systematic bibliography research and contributed equally in retrieving data and analysing results. they all approved the final manuscript. cesare efrati and fabrizio montagnese, gave their clinical gastroenterology expertise, adding valuable material to the manuscript. they both critically review the whole text and approved the final version of the manuscript. the european society degenerative disease supported the publication of this review.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.author details key: cord- - lgup yj authors: robbins, r.c.; almond, g.; byers, e. title: swine diseases and disorders date: - - journal: encyclopedia of agriculture and food systems doi: . /b - - - - . - sha: doc_id: cord_uid: lgup yj swine diseases and disorders that are significant in modern, commercial swine production systems are organized by body system; the reader will need to know basic anatomy and physiology. the industry significance, etiology, epidemiology, pathogenesis, clinical signs, postmortem and histpathologic lesions, diagnostic testing, and generic treatment, control, and prevention are described. diseases of a particular system are summarized in a differential diagnosis table. r elsevier inc. all rights reserved. autogenous vaccines vaccine made from microorganisms isolated from the animal it will be used on; in the united states these are killed and, by permit, are extended for use in a farm, production system, or region. farrow process of parturition; location where a pig is born and stays till weaning, usually - weeks of age. grow-finish phase of production that follows the nursery period where pigs reach slaughter weight; used to describe pigs from to weeks of age. histopathology; -ic the science and microscopic examination of formalin-fixed and paraffin-embedded sections of diseased tissues. lesion visible (microscopic or macroscopic) deviation from normal. national animal health monitoring service a program administered by united states department of agriculture-animal and plant health inspection service. nursery phase of production that begins after weaning, used to transition pigs from the farrowing house to finishing; used to describe pigs from to weeks of age. oie world organization for animal health created on january . pathognomonic characteristic of a specific disease or disorder that, when present, is sufficient to make a diagnosis. veterinary diagnostic laboratory location where samples are submitted and tests are run to determine the cause of disease. in an approach to investigating any suspected disease or disorder in swine production, a history should be gathered first. important history to understand from caretakers includes: age of pigs affected, duration of clinical signs, morbidity rate, mortality rate, treatments administered, response to treatments, and any other important information regarding previous diagnoses or disease in the affected group of animals. this is also the time to examine any production records that have been kept on the affected group of swine as well as previous groups for comparison. records include but are not limited to: where the animals originated from; number in the herd; age; daily mortality; number treated; name of treatment, route of delivery and dose; feed and water usage; high-low temperatures; and vaccinations received or administered. after examining the production records and obtaining a history, proceed with a visual examination of the herd. typically, it is a biosecurity custom to observe youngest groups first; however, in cases of suspected infectious diseases, it may be best to begin with the healthiest group advancing in order of increasing severity or prevalence. often, a definitive diagnosis is not achieved without an extensive clinical and pathological investigation. a postmortem examination, or necropsy, of affected pigs should occur last. any pigs recently deceased of natural causes should be examined to establish trends, with the understanding that submission of tissues from these animals may not yield valuable diagnostic results. tissues for diagnostic evaluation should be collected from clinically affected pigs that are euthanized immediately before necropsy. sampling of five or more pigs may be required to obtain a valuable diagnosis. when investigating signs referable to the central nervous system (cns), it is important to preserve brain and spinal cord tissue for microscopic evaluation in cases of neurological disease; therefore, blunt force trauma and brain penetration by captive bolt are not preferred methods of euthanasia. at minimum, fresh and formalin-fixed tissue samples should include: brain, tonsil, heart, lung, lymph nodes, spleen, kidney, liver, and intestine. additional samples that may be beneficial for diagnosis include: premortem whole blood and ethylenediaminetetraacetic acid-chelated blood (for serum chemistry and complete blood count), spinal cord, intact stifle and hock joints (remove the leg at the hip), intact eyeball with optic nerve attachment, urine, feed, and water. consult a diagnostic lab regarding any additional samples that may be required in determining an etiologic diagnosis. the etiologic diagnosis should be based on consistent history, signs, and pathology derived from a list of differential diagnoses that are most common or most likely to occur in that herd or production system. a treatment, control, or prevention program should be formulated simultaneously. before using any chemical, pharmaceutical, or biologic in swine intended for food, know the domestic use guidelines, importer requirements or producer-packer agreements regarding withdrawal times, residue and tolerance limits, prescribing guidelines, and prohibited substances. this section will focus on a practical approach to investigating signs of neurological disease in swine summarized in table . it is important to determine if clinical signs are consistent with cns or peripheral nervous system lesions (pns). common cns signs in pigs include behavioral abnormalities (most commonly stupor), ataxia, loss of righting, seizures or seizure-like activity (paddling), nystagmus, and blindness. musculoskeletal disorders may clinically confuse or complicate perceived pns signs and must be differentiated from each other. streptococcus suis is a gram-positive cocci with reported serotypes. observational studies implicate sows as carriers and piglets are colonized as they pass through the birth canal (amass et al., ) . disease occurs most frequently during the suckling and postweaning period. commingling pigs from different herds, concurrent infection with porcine reproductive and respiratory syndrome (prrs), and other stress factors may increase the risk of developing s. suis meningitis (villani, ; thanawongnuwech et al., ) . variable morbidity and mortality: mortality depends on early recognition and treatment. clinical signs of s. suis meningitis include paddling, recumbency, nystagmus, and seizure. isolation of s. suis from the lung, nasal secretions, or tonsil from normal pigs is clinically insignificant. in contrast, s. suis isolation from cerebrospinal fluid (csf), meninges, joints, endocardium, or serosal surfaces with or without lesions is relevant (pijoan, ) . few to no gross lesions may be observed during necropsy. early recognition of clinical signs followed by injection with an antimicrobial that s. suis is susceptible is the most effective means of treatment. administering an antimicrobial that s. suis is susceptible to in the drinking water has been proposed to control morbidity (villani, ) . antimicrobial susceptibility patterns for s. suis isolates from regional diagnostic laboratories can be used to assist in selection of an appropriate antimicrobial while diagnostic tests are pending; ceftiofur is effective . commercial and autogenous vaccines are available but due to s. suis serologic diversity may not be effective . haemophilus parasuis (hps), also called glässer's disease, causes bacterial meningitis, arthritis, and polyserositis similar to s. suis. infections are not clinically or grossly distinguishable from s. suis. definitive diagnosis is by bacterial isolation. however, hps is a fastidious gram-negative rod and culture media must be supplemented with v factor for successful isolation. owing to the difficulty in isolating hps, polymerase chain reaction (pcr) tests are a suitable alternative (oliveira et al., ) . like s. suis, isolation from the airways has little significance unless lesions are present (hoefling, ) . antimicrobial susceptibility testing identifies ceftiofur or florfenicol that are typically effective first choice therapeutics (oliveira, b) . prevention may be achieved with medicated early weaning. edema disease results when a fimbrial (f or f ) and shiga-like toxin (stx- e) positive strain of escherichia coli successfully attaches to brush border receptors releasing toxin that damages blood vessels including those of the blood-brain barrier causing edema and encephalomalacia. edema disease most commonly affects rapidly growing pigs, weeks postweaning. morbidity is moderate to high and mortality is high. acute death of robust pigs, ataxia, eyelid swelling, and diarrhea are typical clinical signs (rademacher, ) . at necropsy, edema may be observed in the mesentery between the loops of the spiral colon and in the cardiac region of the gastric mucosa. stomachs are usually full of feed. bacteriologic isolation of a β-hemolytic strain of e. coli from affected pigs with meningoencephalitis is not sufficient for a diagnosis. genotyping is necessary to confirm that the e. coli isolated was f or f and stx- e positive and thus capable to induce such lesions. there is no effective treatment. vaccination using an avirulent live culture of e. coli postweaning, thorough cleaning and disinfection between groups, and use of genetically resistance breeds that lack the fimbrial receptor are preventatives (fairbrother and gyles, ) . pseudorabies (prv), also known as aujezsky's disease, is caused by a herpesvirus. prv was eradicated from the us commercial swine herd in (usda aphis, ) . feral swine are potential reservoirs. cattle, sheep, dogs, and cats can also be infected with prv. high morbidity is due to large quantities of virus shed in saliva and nasal secretions for several weeks following infection. mortality is inversely related to age approaching % in neonates. clinical signs are also age dependent. neonates may die without signs. suckling and recently weaned pigs are those that commonly exhibit ataxia, tremors, excess salivation, and seizures. at necropsy, the brain appears congested and hemorrhagic. necrotic foci occur in the spleen, liver, lung, lymph node, and specifically tonsils. histopathologic lesions are characterized by nonsuppurative meningitis and intranuclear inclusion bodies. pcr, virus isolation (vi), immunohistochemistry (ihc), or fluorescent antibody can be used to confirm the diagnosis. no specific treatment is available. vaccination and eradication are effective for control (usda aphis, ) . in areas free of prv, suspicion of the disease should be reported to state and federal agencies as required. congenital tremors result when hypomyelination or demyelination of the brain and spinal cord. clinical signs are clonic muscle contractions that cause a general tremor of the entire body. pigs are affected at birth but severity subsides with note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). age (dewey, ) . mortality is variable and is the sequela of malnutrition because piglets are unable to nurse. there is no known treatment or prevention. hypoglycemia occurs when piglets fail to nurse the sow. this condition is observed within h afterbirth. there is a low morbidity but high mortality. pigs may appear disoriented, ataxic, recumbent, or dead. on necropsy, affected piglets will have empty stomachs. assigning an employee to attend farrowing to ensure piglets nurse will reduce incidence. water deprivation, also referred to as salt poisoning, is an idiopathic disease resulting from a period of inadequate water intake (carson, ) . high morbidity with variable mortality occurs. the disease is suspected when there is a history of power outage or poor management (thacker, ) . fighting over water access is the first clinical sign and occurs within hours. dog-sitting, opisthotonous, convulsions, and fighting over water follow and develop after h without water. removal of the brain from an affected animal reveals edema and eosinophilic meningoencephalitis with perivascular cuffing and this is pathognomonic (gudmundson and meagher, ) . serum or csf with a sodium level above meq l À may also be used for supporting evidence (osweiler and hurd, ) . treatment of swine showing signs with an anti-inflammatory is variably effective. when water is restored, limit intake to short, - min intervals until all animals have had a chance to drink and fighting has ceased after which water can be provided ad libitum. prevention is daily observation to ensure each animal can access water, adequate water delivery system, and equipping the facility with a generator or alternative method to deliver water during power outages. gastrointestinal diseases and disorders can occur in all ages of swine as summarized in table . most digestive diseases are referable to the gastrointestinal tract and result in diarrhea and occasional vomiting. diarrhea is the result of an intestinal dysfunction caused by malabsorption, excessive secretion, or effusion. unfortunately, this is not an exclusive characterization of diarrhea and overlap occurs (moeser and blikslager, ) . rather, differentials for diarrhea should be referable to age at onset and site of infection. gastric ulcers are noninfectious and result when glandular mucosa specifically the pars esophagea is traumatized by gastric acid. gastric ulcers have a wide variety of causes but are most commonly associated with small feed particle size (ayles et al., ) and interruption of feed intake whether caused by disease or poor management. it is common to see signs consistent with gastric ulceration increase following an acute prrs or influenza outbreak. morbidity and mortality vary with the scope of the underlying cause. clinical signs include regurgitation, vomiting, pallor or jaundice, and acute death. an acutely dead pig with blood in its stomach is indicative of an active ulcer and is sufficient evidence for a diagnosis. in chronic cases, ulceration causes hyperplasia resulting in stricture of the pars esophagea and regurgitation. feeding a coarse ground diet for weeks significantly decreases severity (ayles et al., ) but is impractical in modern production facilities. rotavirus is a nonenveloped rna virus with a doublelayered capsid allowing it to remain stable and infective in the environment for months and intrinsically resistant to some disinfectants. four serogroups infect swine: a, b, c, and e (the latter only reported from the united kingdom). in addition, infections with particular serogroups vary by age: type c mostly in suckling piglets and type a predominately in nursery pigs (stephenson et al., ) . type a is the most prevalent serogroup. severity of disease decreases with age and is self-limiting. the virus infects and destroys villous enterocytes resulting in villous atrophy. in response, crypt cells fill in the gaps but, because they are incapable of absorption, suckling piglets quickly loose body condition and have a gaunt or wasted appearance. neither clinical signs nor gross lesions are pathognomonic although loops of small intestine appear thinwalled with moderate to large amounts of watery contents. a histopathologic report of blunted villi and crypt hyperplasia is suggestive of rotavirus infection. infection with rotavirus can be confirmed by pcr or electron microscopy (em). enzymelinked immunosorbent assay (elisa) is also available but limited to detection of serogroup a. ihc and em detect rotavirus and confirm its role in pathology, but both tests lack table common gastrointestinal diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). sensitivity. treatment is supportive by administration of oral rehydration solutions. acidifiers and antibiotics are sometimes administered to control secondary bacterial infections. treatment success is variable and depends on the degree of malnourishment. prevention among neonatal piglets is through ingestion of lactogenic virus neutralizing antibody from the sow, which is stimulated by administering feedback of rotavirus positive piglet feces or intestines (arruda et al., ) or a modified-live commercial type a vaccine no less than weeks before farrowing. a modified-live commercial type a vaccine is also available for pigs. it does not induce cross-protection for other serogroups and may be cost-prohibitive. tge is caused by tgev, a coronavirus that is heat labile at temperatures above c, prone to dessication and photosensitization (bay et al., ) . the epidemic form causes acute disease in all age groups within as little as h of infection. morbidity and mortality is high, approaching %, in an epizootic outbreak. the severity of disease is age dependent but all ages will develop diarrhea (moeser and blikslager, ) . postweaning infections result in high morbidity but low mortality; the most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. necropsy reveals that the small intestine and colon are fluid filled, the small intestinal wall is thin almost translucent, and lacteals are empty. necrosis and atrophy are observed throughout the length of the villus. the colon and cecum are spared. the endemic form occurs when susceptible animals are introduced to the herd or after maternal antibody wanes. prior exposure to porcine respiratory coronavirus (prcv) may cause false-positive antibody test results. a tgev/prcv differential elisa is available. in an outbreak, sows are fed tissue of diarrheic pigs to stimulate herd immunity and new introductions of animals are stopped. after the exposure and a subsequent cool-down period of - months or after clinical signs cease, sentinels can be introduced and monitored for seroconversion (saif and sestak, ) . absence of seroconversion indicates successful elimination of tgev. commercial vaccines are available but should be used with caution and only when elimination is not an option. ped is caused by pedv, a coronavirus that causes signs and histolopathologic lesions indistinguishable from tgev. unlike tgev, pedv is more environmentally resistant making elimination more difficult. the disease has been described in europe, asia, and, as of , the united states. prevalence of the enzootic form is approximately % (chae et al., ) . morbidity approaches % and mortality is % or more in a naïve sow herd resulting in - weeks of production losses. clinical signs appear within h; piglets develop a watery, fetid diarrhea leading to dehydration, metabolic acidosis, and death before caretakers are able to humanely euthanize them. vomiting also occurs. the severity of disease is age dependent but all ages will develop diarrhea. postweaning infections result in a high morbidity but low mortality; most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. viral shedding occurs up to days postinfection. reproductive failure and inefficiency is a sequela of an outbreak (olanratmanee et al., ) . tgev/pedv differential pcr is available to confirm a presumptive diagnosis of ped. serum can be submitted for elisa or immunofluorescent antibody but collected no sooner than weeks after diarrhea was o bserved. immunoprophylaxis using egg antibody or hyperimmune serum and supportive care including electrolyte administration have been used for treatment. in an outbreak, sows are fed tissue and feces from diarrheic pigs to stimulate herd immunity (olanratmanee et al., ) . hygiene is the key to reducing environmental contamination. preventing introduction of virus into a herd with biosecurity alone may not be sufficient because the virus has been found in aerosol up to miles from a positive farm (goede et al., ) . porcine coccidiosis is most often caused by isospora suis. farm hygiene, specifically farrowing rooms, and sow infestation influence the persistence of disease; however, age at infection rather than infectious dose has the greatest impact on severity (worliczek et al., ) . the prepatent period is approximately days. morbidity is variable and mortality is low. pasty diarrhea, unthrifty to potbellied appearance of - day old pigs, and below average wean weight is suspicious for coccidiosis. on necropsy, the small intestine often is thickened and the mucosal surface is necrotic and has an adherent pseudomembrane. histopathologic examination of the affected portion of the intestine reveals larvae in the lamina propria. sensitivity of fecal flotation is moderate. there is no effective treatment. prevention is by oral administration of an anticoccidial (maes et al., ) . heat treatment (flaming) of flooring may reduce environmental contamination. concrete, rubber coated and plastic flooring in the farrowing crates are difficult to clean and disinfect so removal may be the only option. swine dysentery (sd) is a spirochete of the genus brachyspira that is an oxygen-tolerant anaerobe giving it the ability to survive for long periods of time in manure, pits, and lagoons (schwartz et al., ) . rodents, particularly mice, are known vectors and can serve as reservoirs. brachyspira hyodysenteriae is the species known for causing sd. other species of brachyspira have been recently described in dysentery-like disease (burrough, ) . incubation is - days but disease occurs in a - week cycle. administration of tiamulin or lincomycin in the feed or water may alter the time to onset of signs after exposure. morbidity is high and mortality is low to moderate characteristically causing disease in only the finisher and mature groups. economic significance is mostly lost performance due to reduced daily gain and feed conversion. the specific mechanism of pathogenesis is not well understood but the spirochete does not invade the lamina propria. clinical signs are the presence of mucohemorrhagic diarrhea containing flakes of frank blood or appearing as a generalized brick red to rust color. lesions are mostly observed in the spiral colon where epithelial sloughing and mucosal invasion cause necrosis resulting in the formation of a pseudomembrane. the colonic walls may be thickened due to vascular congestion and mucosal hyperplasia. bacterial culture produces strong β-hemolysis. pcr test for confirmation and speciation is recommended for any isolate with characteristic growth. introduction of infected pigs and contaminated equipment or facilities are the source of infection. pleuromutilins, like tiamulin, and the lincosamide, lincomycin, are effective for treatment. however, if the environment remains contaminated, clinical signs will recur. depopulation has resulted in successful eradication (harms, ) . medicated elimination that combines thorough pulse medication with tiamulin, cleaning and disinfection, and employment of an aggressive rodent control program is also effective (burrough and sexton, ) . escherichia coli is a gram-negative rod that infects all ages of swine but must express virulence factors to cause diarrhea. escherichia coli colonize the small intestine by fimbria that binds receptors on the villous surface of enterocytes. enterotoxigenic e. coli (etec) then produce toxin(s) that increase osmolality leading to diarrhea (moeser and blikslager, ) . etec is subdivided by fimbria, toxin, and age of pig affected. neonatal diarrhea (nd) is most common in pigs - days of age. the onset of postweaning diarrhea (pwd) caused by f is delayed, occurring - days postweaning, compared to that caused by f and its severity is indirectly related to wean age. clinical signs are profuse diarrhea, rapid dehydration leading to emaciation, or death due to metabolic acidosis. fluid filled and hyperemic sections of jejunum and ileum may be present at necropsy but few consistent gross lesions occur. intestinal contents have a distinctly alkaline ph. isolation of large numbers of e. coli and with dense layers of rod-shaped bacteria covering villi seen on histopathology in samples from pigs with diarrhea is sufficient for diagnosis of e. coli but not etec. genotyping is necessary to determine fimbria and toxin types, which are essential to confirm diagnosis of etec. treatment of affected pigs/litters/groups includes administration of antibiotics and oral rehydration solution or electrolytes to correct hyperkalemia (kiers et al., ) . control and prevention of nd is by passively derived lactogenic immunoglobins from vaccinated females (kohler, ) . prevention of pwd include selection of genetically resistant breeds lacking k and f receptors, administration of an oral avirulent live culture to stimulate active immunity or competitively exclude field strains (genovese et al., ) , feeding ppm zinc oxide postweaning and probiotics. immunity and exclusion is unique to each fimbria; vaccines should include the prevalent genotype(s) causing the diarrhea. clostridium perfringens type a (cpa) is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of neonatal infection. frequency of cpa diarrhea is on the rise in the usa. in uncomplicated cases, mortality is low whereas morbidity is high and below average weaning weights result. cases of cpa diarrhea are associated with the expression of α and β toxin. cpa is cultured from the stomach and upper third of small intestine but does not bind intestinal epithelium causing few to no histologic lesions. because of its ubiquitous nature and prevalence among health pigs, cpa may be an opportunist and its role as a primary cause of neonatal enteritis is not definitive. large numbers ( þ or þ ) of gram-positive bacilli cultured from feces or intestinal contents of diarrheic pigs is suggestive of cpa. genotyping by pcr to confirm presence of cpb gene in cpa isolates and rule out of other causes of nd are supportive to the diagnosis (bueschel et al., ). treatment has variable success rates and is limited to administration of empirically selected antibiotics and oral rehydration solutions to affected piglets. control of cpa enteritis is best accomplished by preventing other causes of nd. following a thorough cleaning, sporicidal disinfectant should be applied to farrowing crates and equipment between litters and be allowed to dry before reloading. feeding of bacitracin to sows has resulted in significant increases in weaning weights (schultz, ) . a commercial cpa toxoid vaccine is available (hammer et al., ) . autogenous whole cell vaccines are also in use. if vaccine is unavailable, feedback might be considered but should be pursued with caution (robbins and byers, a) . cpc is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of infection. pathogenesis of type c is due to expression of β toxin leading to necrosis of intestinal epithelium resulting in hemorrhagic diarrhea or acute death of piglets less than days of age. gross necropsy reveals hemorrhagic and blood-filled loops of small intestine. a pseudomembrane may form on the luminal surface, and intestinal mucosa is edematous. gross and histopathologic lesions in the presence of large numbers ( þ or þ ) of grampositive bacilli cultured from feces or intestinal contents warrant a presumptive diagnosis. genotyping by pcr to confirm presence of the cpb gene is confirmatory (songer and uzal, ) . treatment of affected piglets is unrewarding due to the rapid and debilitating course of this disease. prevention is accomplished by vaccination of gestating females with a commercial toxoid and ensuring piglets consume sufficient colostral antibodies to result in protection. clostridium difficile is a gram-positive bacillus that easily sporulates making it environmentally resistant to many disinfectants. clostridium difficile associated diarrhea leads to a - % reduction in wean weights (songer and uzal, ) . although more than a third of piglet diarrhea involves c. difficile, it is the better known to cause healthcare-associated infections among humans. the pathogenesis of c. difficile infections is in response to the expression of toxins a and b. a watery diarrhea occurs in - day old piglets. mesocolonic edema may be observed at necropsy. clostridium difficile is difficult to culture and can be isolated from healthy piglets. therefore, volcano lesions on histologic exam and confirmation of toxins in fecal contents by antigen elisa are diagnostic. treatment is ill-defined but is likely similar to that for cpa enteritis, because it is likely to be initiated based on clinical signs, which are similar. autogenous vaccines are used to aid in prevention but efficacy is unclear. ppe, commonly referred to as ileitis, is the general categorization of infections caused by lawsonia intracellularis, an obligate intracellular bacterium. because the bacteria cause lesions in the ileum, ppe is also referred to as ileitis. seroprevalence in grow-finish herds can reach %. ppe can further be divided into four clinical forms (kroll et al., ) . porcine intestinal adenomatosis (pia) is most common in - week pigs and causes little mortality. porcine hemorrhagic enteritis (phe) affecting pigs weeks of age and older including breeding swine and can be associated with increased mortality and dark, bloody stools. necrotic enteritis (ne) and subclinical ileitis, the most common form, occur among postweaning pigs. in all forms, transmission is by the fecaloral route. crypt enterocytes infected with l. intracellularis become hyperplastic. the altered ratio of villous and crypt enterocytes leads to malabsorption and subsequent increases in feed conversion and time to reach market weights. pia results in variable degrees of thickened ileum that can be found at necropsy. the ileal lumen may contain a blood clot in phe or pseudomembrane in ne. when diarrhea ranging in color from normal (pia, ne, and subclinical) to dark-red or black (phe) is observed, ppe should be considered as a possible cause. subclinical ileitis usually causes no clinical signs (gebhart, ) . histopathologic lesions containing intracellular s-shaped organisms are suggestive of lawsonia infection but ihc should be used to confirm diagnosis. pcr is helpful to detect infection and is highly specific but moderately sensitive. cross-sectional or longitudinal serologic profiling using a widely available elisa is the best tool for determining timing of exposure. treatment is with effective antibiotics, such as tylosin, administered by injection or in the feed or water. control is by administration of a commercially available modified-live oral vaccine before infection or feeding antibiotics when infection is known to occur. vaccination should take place at least weeks before seroconversion (walter et al., ) . salmonellosis causing gastrointestinal disease in swine is most commonly associated with the species typhimurium. salmonella typhimurium is commonly isolated from swine. isolation of multidrug resistance strains of s. typhimurium from swine at slaughter have garnered attention from public health and food safety professionals and it is this that make this infection significant to the pork industry (foley et al., ) . some european union member states have implemented meat-juice serologic monitoring at slaughter to assess on-farm salmonella control programs. pathogenesis of s. typhimurium is similar to salmonella cholerasuis by invading enterocytes and subsequently macrophages leading to an infectious carrier state. initial infection causes inflammation and cytokine release that result in watery, yellow diarrhea containing feed particles. button ulcers may be visible on the mucosal surfaces of the colon and cecum on gross necropsy examination and, on histopathology, can be found to extend into the lamina propria. bacterial isolation without using enrichment media and the presence of histopathologic lesions is consistent with a diagnosis of salmonella enteritis. treatment is with antibiotics administered symptomatically to diarrheic pigs. antibiotic susceptibility of the isolate should be considered before initiating treatment. rearing pigs on slatted floors, decreasing stocking density, and acidification of digesta are effective in reducing the prevalence of salmonella infections in swine (funk and gebreyes, ; boyen et al., ) . cross-protection with s. cholerasuis vaccine has been reported and reduces carcass colonization (husa et al., ) . whipworm infestations of swine are the result of trichuris suis infection. pigs kept on pasture, in outdoor lots, or facilities with a history of t. suis diagnosis are at greatest risk for disease (pittman et al., a) . the prepatent period is - weeks. the egg is not immediately infective, which requires - weeks in the environment. the infectious larva hatches from the egg and invades enterocytes in the small intestine and cecum. the entire life cycle of t. suis is completed in the intestine. ulcerations in the mucosa and damage to capillary blood supply of intestinal epithelium lead to hemorrhage, anemia, and hypoalbuminemia. clinical signs are depressed weight gain, increased feed conversion, bloody diarrhea, ill thrift, and death. adult worms imbedded in the ileum, cecum, or proximal colon are sufficient for diagnosis of whipworms. eggs are intermittently shed and thus not a reliable method of diagnosis (pittman et al., a) . treatment and control are synonymous and require administration of an effective anthelmintic like fenbendazole. prevention is by steam sanitation and drying; however, eggs are resistant to common disinfectants and remain infective for years. the porcine integument or skin, like that of other domestic species, serves as a protective barrier between fragile internal tissues and harsh external hazards. skin is comprised of layers (from external to internal): epidermis, dermis (superficial and deep), and subcutis. blood vessels, hair follicles, sebaceous glands, and muscles are found in the dermis. notably, the pig's skin does not contain sweat glands; therefore, modern swine facilities are outfitted with evaporative cooling systems for thermal regulation in hot climates. skin diseases and disorders can be the result of viral or bacterial infections, parasite infestations, immunologic reactions, and idiopathic or iatrogenic causes that are summarized in table by their various macroscopic and microscopic lesions. greasy pig is a skin disease of swine caused by a toxin produced by staphylococcus hyicus. a break in the skin is the typical sequela. gilt litters reportedly have a higher incidence of this disease, presumably due to deficient maternal immunity. all ages of pigs may be affected but suckling and nursery pigs are most likely to develop disease. affected pigs develop focal crusts on the face, neck, and axillary region, and the crusts may coalesce as the disease progresses. affected areas are greasy to touch and may appear black due to dirt adhering to it. if pigs are untreated or fail to respond to treatment, the trunk and extremities may become involved. pyrexia and lethargy can be observed in severe cases and are followed by growth reduction. gross appearance of affected skin is rarely confused with other skin conditions of swine. submission of formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis for histopathologic examination is needed for a diagnosis. the pathognomonic histologic lesion is exudative epidermitis. table common integument diseases and disorders of pigs greasy pig x x x erysipelas x x porcine dermatopathy and nephropathy syndrome (pdns) x x x sarcoptic mange x x the first column provides the diseases. the remaining columns represent the type of lesion that occurs. the s. hyicus can be cultured from the surface of clinically normal skin sections. treatment includes topical application of antimicrobials or disinfectants. unaffected pigs with direct contact with affected pigs should also be treated to control spread. in cases where pigs exhibit systemic signs, administration of an injectable antimicrobial and anti-inflammatory is warranted. in the united states, no antimicrobials are labeled for the treatment, control or prevention of s. hyicus so all antimicrobial therapy is extra-label. prevention should focus on facility hygiene and include a soap degreaser and disinfectant regimen to reduce contamination. in addition, scarification of the skin of breeding age females with the farmspecific s. hyicus strain can reduce disease incidence in suckling pigs (murray and rademacher, ) . erysipelas or diamond skin disease is caused by a soilborne gram-positive bacterium, erysipelothrix rhusiopathiae. this zoonotic pathogen is transmitted by migratory fowl, turkeys, and pigs. humans may become sickened when direct contact with blood from affected animals contaminates an open wound (brooke and riley, ) . the finding of lesions at slaughter results in partial or complete carcass condemnation (bender et al., ) . the disease is most common in growing, finishing, and breeding age swine. bacterial emboli lodge in blood vessels causing vasculitis, thrombosis, and ischemia leading to lameness, abortions in gestating females, and raised, red to purple rhomboid skin lesions for which erysipelas is best known. skin biopsies from the affected area should include epidermis and dermis, but histologic lesions are only supportive. bacteriologic isolation or pcr identifying e. rhusiopathiae confirms the diagnosis. treatment with β-lactam antibiotics including penicillin is effective. commercial bacterins and avirulent live cultures are available for prevention (wood, ) or in the face of outbreaks to prevent the chronic form. pdns has been associated with porcine circovirus type (pcv ) infection, but any disease process resulting in ischemia could cause result in pdns. the condition is characterized by red to purple discoloration of skin that begins on the caudal surface of the hind limbs and the ventral surface of the abdomen resulting from ischemia. on necropsy, gross examination of the kidney cortex may be speckled with pinpoint, white foci caused by infarcted blood vessels. pig of any age can be affected with pdns, but it is more commonly observed during growing and finishing stages. submission of fresh and formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis is required. there is no specific treatment or prevention; rather, diagnose the underlying cause to determine appropriate therapy ( figure ) . sarcoptic mange is the result of an allergic reaction to the saliva of ectoparasites, sarcoptes scabiei. mange may also be caused by demodex phylloides. mortality is low and morbidity is moderate. economic losses are the result of reproductive inefficiency, growth reduction, and carcass condemnation. infestation and subsequent clinical signs in the breeding herd, most notably an incessant scratching, develop following the purchase of infested genetic replacements. in addition, growing pigs placed in facilities that previously housed infected swine or facilities that reuse straw bedding or have solid wood partitions may also become infested. the mite is rare in modern, high-health swine operations. the burrowing mite causes red pustules and flaking skin. individual pigs may develop signs in as few as weeks but a herd may not show signs for several months. in the chronic stage, thick crusts develop at the corners of and inside the ears. examination of a scraping from the crusts will reveal the mite (averbeck and stromberg, ). an elisa test is used to determine prior exposure and determine success of eradication programs. treatment can be applied topically using an antiparasitic, such as amitraz, to temporarily alleviate clinical signs. control and eradication programs utilize feeding or injection of ivermectins (mohr, ) . the musculoskeletal system is comprised of tendons, ligaments, muscles, and bones. disorders and disease of this system are typically characterized by lameness. lameness is any deviation in normal locomotion including favoring a limb or failure to bear weight on the limb. neurologic conditions, which also cause changes in locomotion, may be ruled out by postmortem examination of articular surfaces and diagnostic testing. investigation of musculoskeletal diseases and disorders should always start with the claws that are easily traumatized causing pain resulting in lameness. flooring and genetics also influence the incidence of lameness. common musculoskeletal diseases and disorders of swine can be divided into osteopathies and myopathies and summarized in table . mycoplasma hyosynoviae colonizes upper airways and tonsils resulting in a carrier state. transmission is vertical from sow to pigs and lateral between pigs (ross and spear, ) . m. hyosynoviae is most often diagnosed during the grow-finish phase. morbidity is variable but mortality is low. clinical signs are a stiff gait and difficulty in standing, most often the stifle or elbow and less frequently the hock, hip, and shoulder. signs often occur - weeks after a stressful event; lesions begin to resolve weeks postinfection. the affected joint contains yellow or blood-tinged effusion with moderate villous proliferation but is not always observed despite lameness and does not necessarily correlate with presence of histopathologic lesions. aseptic collection of synovial fluid by needle aspiration or sterile swab or submission of the affected joint intact is recommended for diagnosis. pcr is the most sensitive test; culture requires special media and lacks sensitivity (gomes neto et al., ) . histopathologic examination of formalin-fixed synovium reveals nonsuppurative fibrinous polyarthritis and lymphoplasmacytic perivascular synovitis. elisa is also available. lincomycin has historically been an effective therapeutic choice (burch and godwin, ) . treatment should be initiated when lameness is first observed; however, spontaneous resolution is common. no commercial vaccines are available. mycoplasma hyorhinis is a ubiquitous bacterium that is an early colonizer of upper airways. transmission is vertical from sow to pigs and then between pigs postweaning (rovira, ) . infection can progress to polyarthritis, polyserositis, and otitis in the pre-or postweaning phases; arthritis develops postweaning. clinical signs include lameness, arthritis, and fever that develop - days after septicemia occurs and persists for - days (gomes neto et al., ) . disease may become chronic resulting in ill thrift, reduced growth, and death. articular surfaces may be eroded. in cases of lameness, synovial fluid and formalin-fixed synovium can be submitted. alternatively, the entire affected leg can be submitted; disarticulate above the infected joint keeping the affected joint intact. submission of fibrin or fibrin covered tissue(s) should be included for pcr testing to differentiate m. hyorhinis from other bacteria that form fibrin on serosal surfaces like hps and s. suis (rovira, ). histopathology reveals fibrinosuppurative inflammation in affected tissues. treatment is empirical. erysipelas is the result of a chronic e. rhusiopathiae infection causing arthritis and endocarditis that follows the initial septicemia. lameness and joint swelling is mostly noticeable in hock and carpal joints. lameness may also occur in stifle and elbow but swelling cannot be appreciated. synovial fluid appears serosanguinous and can be submitted for testing by bacterial culture or pcr. alternatively, the entire affected leg can be removed to prevent contamination; disarticulate the leg above the infected joint. histopathologic examination of formalin-fixed synovium reveals a proliferative synovitis. other lesions that occur are nonsuppurative fibrinous polyarthritis and erosion of cartilage that can progress to pannus and ankylosis. treatment with β-lactamase antibiotics including penicillin is effective. an anti-inflammatory is added to a treatment program for pain management. commercial bacterins or avirulent live cultures are available for control and prevention. oc is the result of a delay in ossification of articular cartilage, and represents the most common lesion among culled sows. morbidity is most often reported in adult and breeding age pigs (dewey et al., ) . mortality is variable and is the result of humane euthanasia because the animal becomes nonambulatory. oc causes lameness, pain, and joint swelling. a noninfectious lameness most often affects the distal part of the humerus or femur. lesions are typically bilateral and symmetrical. diagnosis is made by ruling out other causes of lameness. ricketts occurs as a result of phosphorus deficiency, vitamin d deficiency, or secondary to iron toxicity but is not caused by dietary calcium deficiency. the condition should be suspected when there is an increase in nonambulatory pigs and broken bones during the finishing stage particularly at and immediately before marketing. occasionally, joint swelling in the nursery stage is observed. rachitic rosary (enlargement of costochondral junctions) and soft bones are observed on necropsy. if rickets are present, a bone ash analysis of the second rib will be below normal. feed analysis can identify low levels of vitamin d or phosphorus. low levels of vitamin d or phosphorous serum chemistry also will occur (madson et al., ) . supplementation of vitamin d is the only reported treatment and response that may be considered diagnostic. prevention includes proper diet formulation for the stage of production. mhd is a noninfectious disease of muscle caused by deficiency of vitamin e or selenium. it can occur if pigs are fed grain grown in selenium deficient soils (dewey, ) . clinical signs are limited to acute death of large, robust pigs. on necropsy, the heart muscle has a mottled appearance. feed analysis, response to vitamin e supplementation, and ruling out other causes support diagnosis of vitamin e/selenium deficiencies like mhd (hooser, ) . supplementation with selenium is impractical in the united states because of environmental regulations, and overzealous supplementation may cause toxicosis. splayleg is a noninfectious, congenital condition resulting from delayed myofibril development with no known cause. splayleg has low morbidity and mortality as long as it is identified and corrected before it leads to starvation or being crushed by the sow. treatment includes the use of nonslip note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). flooring in farrowing crates and application of harness or tape that holds the rear legs under the pig until it is strong enough to walk on its own. reproductive failure occurs when insemination fails to result in pregnancy or pregnancy fails to produce viable pigs due to infectious and noninfectious causes summarized in table . reproductive failure should be considered when a low conception or farrowing rate, irregular returns to estrus, abortions, stillbirths, or mummies persist at an abnormal rate. infertility occurs when fewer than four embryos are present at the time of maternal recognition of pregnancy resulting in a regular return to estrus and reduced conception rate for that breeding group. irregular returns to estrus result from embryonic death or early term abortion after implantation but before calcification of the fetuses. embryonic death of some or all of the embryos will result in low total born or irregular return to estrus, respectively. early term abortion also will reduce farrowing rates. mummies and stillborns can occur any time after calcification of the fetuses. the normal rates for mummies and stillborns are o . and o pig per litter, respectively. late-term abortions are classified as those occurring after days of gestation. total abortion rate should remain o % of a breeding group. these are general guidelines; thus, familiarity with the herd's normal reproductive performance is the most sensitive means to identify a reproductive problem. prrs is, at this time, known only to occur among swine. the estimated cost of prrs to the us pork industry is us$ million annually (holtkamp et al., ) . prrs usually results when susceptible swine are infected with either the leylystad or north american strains of prrs virus (prrsv), a member of the arteriviridae family. viremia lasts up to days, but shedding of infectious virus can last much longer (murtaugh and genzow, ) . prrsv is most commonly transmitted by introduction of infected swine or contaminated fomites, use of contaminated semen, and aerosol. the pathogenesis of the reproductive form is believed to be arteritis of fetal umbilical cords during gestation (lager and halbur, ) . swine may show no signs when reinfected with a homologous strain. conversely, infection with a heterologous strain will reproduce lesions and disease but is usually less severe than that of naïve swine (murtaugh and genzow, ) . clinical signs of prrs in a breeding herd start with an epidemic of abortions followed by an increase in low viable piglets, stillbirths, and mummies. abortions result due to fetal death or pyrexia of the gestating female. sows and gilts may be anorexic, pyrexic, or lethargic. periparturient females may become agalactic. in severe outbreaks of prrs, sow mortality also increases. in utero infection of feti can result in persistently infected piglets (rossow, ) . prewean mortality commonly increases and may remain above the herd average for weeks. diagnosis can be made by submitting lung, spleen, and lymph node from fetuses or low viable piglets. whole fetuses can also be submitted but should be refrigerated to prevent autolysis. lesions are not pathognomonic so confirmatory testing such as pcr, ihc, or vi should be conducted. tissues and thoracic fluid from stillbirths, aborted, or mummified feti can be submitted but may result in false negatives. serum collected from aborted sows or low viable piglets and tested for prrsv by pcr is another option for diagnosis. prrsv elisa indicates previous exposure but is not useful in a previously exposed herd. treatment of prrs is supportive. anti-inflammatories to reduce fever and antibiotics for control and treatment of secondary bacterial pneumonia may be necessary. the most common methods for control include depopulationrepopulation and herd closure and rollover, also called loadclose-homogenize, using commercial vaccine or herd-specific live virus exposure (corzo et al., ) . periods of closure vary based on facility capacity but a minimum of days is recommended. commercial modified-live and killed vaccines are available but do not prevent infection and should be used in accordance with label and domestic guidelines. ppv is sometimes described by the acronym smedi (stillborns, mummies, embryonic death, and infertility). ppv is an enzootic infection of swine breeding herds in the united states. the virus is ubiquitous and is transmitted through ingestion of infected feces, afterbirth, or fetal tissue. the disease most commonly affects gilts and younger parity sows (christianson, ) . the pathogenesis is through damage to table common reproductive diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the clinical signs. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). the placental epithelium resulting in fetal death. clinical signs of ppv range from low total born, mummies of various sizes, irregular returns to estrus, and females diagnosed pregnant but fail to farrow. diagnosis is based on vaccination history, clinical signs, and pcr testing of mummified fetuses. ppv elisa may provide diagnostic value if acute and convalescent serum samples are used. there is no effective treatment for ppv; however, commercial killed vaccines are available and very effective. exposure of unbred females to tissue or cull sows from a seropositive herd has been used for immunization when vaccine is unavailable. leptospirosis is caused by infection by spirochete bacteria. leptospira species may be zoonotic (leptospira canicola, l. icterohemorrhagiae), swine-adapted (l. pomona and l. bratislava), or incidentally infect swine (l. grippotyphosa and l. hardjo). infection has been associated with exposure of swine to contaminated soil or untreated surface water, and exposure to urine from infected vectors, such as rodents. infected swine can become carriers resulting in chronic disease. the pathogenesis is due to bacteremia resulting in transplacental infection followed by fetal death. clinical signs include pyrexia, low conception rate, abortion, stillbirths, and low viability pigs resulting in increased prewean mortality. diagnosis is made using dark field microscopy or ihc performed on tissues, particularly kidney, of aborted feti or stillbirths. paired or matched serology for hemagglutination inhibition (hi) testing may be useful if suspected. treatment with antibiotics, such as chlortetracycline, may be pursued (henry et al., ) . commercial killed bacterins are available to aid in prevention and should be given at least semiannually to breeding stock (christianson, ) but may not be available in all countries. for example, federal regulations prohibit the use of these bacterins in france and the netherlands (figure ) . pcv is a ubiquitous virus in swine facilities. pigs become infected with pcv through ingestion (oral nasal contact). in addition, breeding females can become infected via insemination with contaminated semen (madson et al., a) . gilts and low parity sows are affected most often, whereas boars show no clinical signs. pcv -associated reproductive failure may occur in conjunction with ppv. infection results in variable lengths of viremia. pcv -reproductive failure is due to transplacental infection of fetuses. clinical signs depend on the stage of gestation when the infection occurs. embryonic death, early term abortions, stillbirths, mummies, low total born, or low viable pigs can result from infection. mummies may vary in size, like ppv, and measuring crown to rump length is useful to determine the time when that fetus was infected. pcv -reproductive failure is diagnosed by the presence of viral antigen confirmed by ihc or deoxyribonucleic acid confirmed by pcr along with the presence of lesions in fetal tissue notably myocardial mineralization. pcr testing of fetal thoracic fluid is sufficient to diagnose in utero infection of piglets (madson and opriessnig, ) . commerical killed baculovirus vectored vaccines are available and effective for prevention of disease but not infection or viremia (madson et al., b) . prv or aujezsky's disease virus was eradicated from the us commercial swine herd in ; a comprehensive review is available (usda aphis, ). prv is a member of the herpesviridae family and, like other herpesviruses, infection can result in a carrier state or latency within nervous tissue with the potential for recrudescence. the pathogenesis of prv results from viremia, and then replication and necrosis of epithelial tissue including the placenta (christianson, ) . the period of viremia gives prv time to cross the placenta and cause fetal death. clinical signs following acute infection include embryonic death, abortion, mummies, and stillborns. necrotic foci can be found in fetal spleen, liver, lung, and lymph node. histopathology is not definitive; ihc is required to confirm presence of antigen. diagnosis may also be made through serology; a commercial elisa test is available and can differentiate between exposure to the gene-deleted vaccine and wild-type virus used extensively in the us eradication. commercial prv vaccines are available but only should be used in accordance with federal guidelines. brucellosis is a zoonotic infection caused by the bacteria, brucella suis biovars and . brucella suis is transmitted through direct contact with susceptible swine, ingestion of infected tissue, or fluids including milk and contaminated semen. pathogenesis of b. suis is initiated when the mucosal epithelium is penetrated, thereby resulting in bacteremia that commonly persists for weeks and results in placentitis among other lesions. clinical signs of infection in gilts and sows include abortion with or without vaginal discharge, whereas boars have reduced libido and fertility. bacteriologic isolation of b. suis from vaginal discharge or tissue confirms diagnosis. serology reflects prior exposure (or vaccination) to b. suis but not for diagnosis of acute disease. the us commercial swine herd is brucellosis free. swine erysipelas (se) is a zoonotic, gram-positive bacterium, which is ubiquitous among swine. erysipelothrix rhusiopathiae is the sole causative species. carrier swine shed the bacteria in saliva, nasal discharge, and feces. infection may result from direct contact with carriers, exposure to infected facilities or soil (wood, ) . a bacteremia lasting several days precedes lesions. reproductive failure is most often due to abortion but infertility and low total born following high fevers or endometritis at the time of breeding is also possible. clinical signs including rhomboid skin lesions, high fevers, lethargy, inappetence, withdrawal, and response to treatment with penicillin of affected sows and gilts are suggestive of acute and subacute se. serology is available; availability is by veterinary diagnostic laboratory (vdl) and value is limited when vaccine is in use. bacterial culture and histopathologic examination of fetal tissue is unrewarding for diagnosis of se but is helpful to rule out other causes of abortion. in chronic se, culture of e. rhusiopathiae from vulvar discharges was successful (gertenbach and bilkei, ) . treatment involves injections of antibiotics and anti-inflammatories. commercial vaccines are available and effective. co poisoning induces hypoxia resulting in an increased number of stillborns (hooser, ) . concentrations of ppm are toxic. malfunctioning heating units or poorly ventilated farrowing rooms are the cause. diagnosis is done by ruling out infectious causes of stillbirths. fetal blood or thoracic fluid can also be measured for co concentrations. zearalenone is a luteotropic mycotoxin produced by fusarium rosea. it binds estrogen receptors resulting in irregular returns to estrus, signs of estrus in prepubertal gilts, and reduced litter size (hooser, ) . diagnosis is by detection of elevated levels in feed samples. however, definitive diagnosis is rarely possible because the contaminated feed has long been consumed by the time reproductive failure occurs. aas and seasonal infertility is a noninfectious cause of reproductive failure. the declining photoperiod and temperature fluctuations during the fall months result in declining progesterone levels. high-ambient temperature experienced during lactation and the postweaning period are suspected but not confirmed as a cause. diagnosis is done by ruling out infectious causes and careful assessment of management, facilities, and reproduction records (rueff, ) . modern facilities that utilize gestation crates and evaporative cooling systems may improve but not prevent infertility during the fall months (leman, ) . the respiratory system can be simply divided into upper and lower portions. the upper portion includes the nasal cavity and sinuses, throat, trachea, and bronchi for air conduction. the lower portion is the lung comprised of bronchioles and alveoli responsible for air exchange. the respiratory system is commonly involved in numerous infectious diseases of swine summarized in table . the most notable infectious agents are the viral pathogens, prrs and pcv , which cause primary pathologic lesions to both the respiratory and the immune system. this damage to the immune system often leads to respiratory or systemic disease incited by secondary infectious agents. such mixed respiratory infections can occur at any age, and, when they occur in growing and finishing pigs, are termed porcine respiratory disease complex (prdc). multifactorial respiratory disease can obscure histopathologic lesions complicating the diagnostic process. app is a host-adapted, fastidious, and gram-negative encapsulated rod that is transmitted vertically from sow to piglet. morbidity and mortality are strain-specific; virulence varies with expression of apxi and apxii toxins. inhalation of strains of app expressing apx toxins results in lung lesions within - h. the disease is economically significant because mortality occurs during the later part of the finishing phase, usually just before slaughter. clinical signs of fever, lethargy, dyspnea, and acute death are common. pigs found dead may have a frothy, blood-tinged discharge from the nose and mouth. focal hemorrhage occurs in the diaphragmatic lung lobe, which is firm, and its appearance is likened to that of a bull's eye. fibrinous, necrotizing bronchopneumonia containing streaming leukocytes is a key histopathologic feature. bacterial culture is difficult and requires nicotinamide adenine dinucleotide (nad)-supplemented media so diagnosis is traditionally made on finding characteristic postmortem and histopathologic lesions. a pcr test is also available. in an outbreak, the entire population should receive antimicrobial therapy parentally. unlike many other gram-negative bacteria, app is sensitive to a variety of antimicrobials; at iowa state university vdl % of isolates were sensitive to ceftiofur, enrofloxacin, florfenicol, tiamulin, tilmicosin, and tulathromycin. prevention is aimed at eliminating carrier swine through depopulation or by pulse medication (marsteller and fenwick, ) . actinobacillus suis causes a hemorrhagic, necrotizing pneumonia during nursery and grow-finish phases. actinobacillus suis infection has similar clinical signs and pathologic appearance to app. affected pigs are frequently observed in a dog-sitting position with elbows abducted. unlike app, lung lesions are random in their distribution and petechial table common respiratory diseases and disorders of pigs postweaning nursery postweaning grow-finish adult note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). hemorrhages may be seen in other organs due to the septicemia that follows a. suis infection (yaeger, ) . a pcr test is available to help differentiate disease from app and hps (oliveira, a) . like app, outbreaks should be treated by parental delivery of an antimicrobial; however, treatment of only those individual pigs with clinical signs is usually sufficient. autogenous vaccines can be used for control but response is variable because most pigs are already seropositive at the time of vaccination. ascaris suum, the swine roundworm, is the most common parasitic infection of swine. the reduced growth performance and liver condemnations are responsible for economic losses (stewart and hoyt, ) . the prepatent period is - days. adult roundworms are present in the manure but it is the migration of larvae through the lungs, occurring - days after ingestion of an infective egg that causes respiratory signs. a persistent cough and dyspnea result due to verminous pneumonia. the liver develops whitish spots, called 'milk spots' that are the cause of condemnations but resolve within days (stewart and hoyt, ) . the presence of eosinophils is suggestive of a parasite infestation. treatment and control is accomplished using anthelmintics: dichlorvos, fenbendazole, levamisole eliminate adults and larvae; piperazine kills only adults. proper cleaning and disinfection particularly removing fecal material between groups reduces potential for exposure but it is virtually impossible to get rid of a. suum once a premise is infested (pittman et al., b) . it is necessary to prevent access to contaminated soil. ar is described in two forms: progressive (par) and nonprogressive (npar). par is caused by toxigenic strains of pasteurella multocida, whereas npar is the result of toxigenic strains of bordetella bronchiseptica. in both forms, the bacteria attach to cilia in the nasal passages and the cytotoxin production causes hypoplasia of nasal turbinates. clinical signs include sneezing, deviated snouts, and, in cases of par, bloody nasal discharge occurring in a large number of grow-finish pigs. mortality is low but the reduced growth that results due to ar makes it economically important. beacause the cytotoxins are responsible for ar, isolation of either bacterium from nasal passages is not sufficient for diagnosis. in addition, b. bronchiseptica and p. multocida colonize the lung leading to bronchopneumonia causing cough and dyspnea in pigs postweaning, often part of prdc (hansen et al., ) . therefore, examination of nasal turbinates at slaughter is the recommended method for diagnosis of ar (gatlin et al., ) . transmission is vertical; therefore, prefarrow vaccination of sows can protect piglets up to weeks of age. if vaccination does not prevent ar, depopulation of the herd may be necessary. hps is also called glässer's disease. there are serovars identified and prefer to colonize the nose (macinnes et al., ) . hps may not actually result in pneumonia but does cause signs of respiratory disease including nasal discharge and dyspnea. in addition, fever, lethargy, and acute death are observed. on necropsy, one or all of the pleural, pericardial, epicardial, and peritoneal serosal surfaces become covered in fibrin. effusion commonly occurs. histopathologic lesions are described as fibrinopurulent. definitive diagnosis is by bacterial isolation on culture media supplemented with v factor. owing to the difficulty in isolating hps, pcr testing is now available (oliveira et al., ) . isolation from the airways in the absence of lesions has little significance (hoefling, ) . ceftiofur, enrofloxacin, or tulathromycin delivered parenterally to affected animals are effective therapeutic drugs. use of water-soluble antimicrobials is for control. maternal immunity, medicated early weaning, and controlling infections with prrs, pcv , and influenza postpone or prevent disease onset (rapp-gabrielson et al., ) . commercial and autogenous vaccines are available but may experience limited efficacy due to serologic diversity; controlled exposure to low dose, live virulent culture is another option (oliveira et al., ) (figure ) . mh is known to infect pigs in production systems worldwide causing reduced growth performance and mortality. the disease is classified as enzootic pneumonia or a component of prdc. both manifestations of mh cause paralysis of the mucociliary escalator resulting in a severe cough and dyspnea known as thumping. vertical and lateral transmission can occur; but, owing to its slow rate of transmission between pigs, the disease primarily occurs in grow-finish pigs (meyns et al., ) . in addition, time of colonization with mh and disease severity are directly related (fano et al., ) . on necropsy, well-demarcated (red to purple lobular consolidation occurs in the apical) diaphragmatic, and accessory lung lobes is visible. histopathologic lesions characteristic of mh is bronchopneumonia with lymphocytic perivascular, peribronchial, and peribronchiolar cuffing. because mh is difficult to isolate, pcr is the most sensitive method of detection. elisa is available and is helpful in establishing herd status but must be interpreted in the context of vaccination as tests do not distinguish between antibodies produced subsequent to vaccination or field infection. treatment of affected pigs with parental antimicrobials like enrofloxacin, tulathromycin, or lincomycin or administration of water-soluble lincomycin, tiamulin, or tetracyclines to affected groups is effective in outbreaks. control can be achieved through pulse-medication in feed of chlortetracycline (thacker et al., ) beginning figure epicarditis, heart, nursery pig. fibrin gives surface a granular appearance, caused by hps infection. note the enlarged (draining) mediastinal lymph nodes located cranial to the base of the heart and the excess thoracic fluid (reddish-brown) indicative of septicemia. courtesy dr. glen almond. week before the historical onset of disease . commercial vaccines are whole cell bacterins marketed to reduce lesions but do not prevent disease or slow transmission rate. simultaneous infection with prrsv reduces efficacy of mh vaccination thacker, ) . eradication from the herd is preventative but practically difficult to accomplish. pcvad is any disease process where pcv infection results in lesions and includes pmws (ellis et al., ) and pdns. infection with pcv is widespread. morbidity and mortality is variable, often dependent on the occurrence of secondary infections and their virulence. survivors of pcvad remain stunted, owing to the economic significance of this collection of diseases. clinical signs include wasting, dyspnea, depression, ill thrift, and diarrhea. lungs are wet, heavy, and fail to collapse; pulmonary edema and lymphadenopathy also can be found at necropsy. histopathology results include presence of interstitial pneumonia, lymphoid depletion, enteritis, nephritis, and dermatitis. for a diagnosis of pcvad the following must occur: pcv antigen within characteristic lesions and lymph nodes are depleted (sorden, ) . ihc is used to confirm presence of pcv antigen within the histopathologic lesion. pcr has little value in diagnosing pcvad unless the herd is considered free. commercial vaccines are very effective and available with flex labels for administration to sows and pigs and as or doses (chae, ) . nonvaccinated, subclinically infected pigs have poorer weight gain compared to their vaccinated counterparts (kristensen et al., ) ; therefore, it is part of most vaccination protocols by us pork producers ( figure ) . prrs is the result of infection with the leylystad or north american strain of prrsv. the estimated cost of prrs to the us pork industry is us$ million annually (holtkamp et al., ) . prrsv is the most commonly diagnosed viral respiratory pathogen at vdls (gauger, ) . infection is observed to increase susceptibility to other infections, particularly opportunistic bacteria. this apparent increased susceptibility to secondary and opportunistic infections is the result of the pathologic process in which prrsv recruits and replicates in pulmonary alveolar macrophages, and then disseminates systemically (rossow, ) . clinical signs are nonspecific including fever, lethargy, and dypsnea but not cough. signs also depend on the type of secondary infection(s) present. lungs fail to collapse and appear heavy, wet, and gray on postmortem examinations. lymphadenopathy is caused by hyperplasia of germinal centers. interstitial pneumonia, alveoli are lined with hyperplastic type ii pneumocytes and contain necrotic debris, whereas the lining of bronchi and bronchioles is normal (rossow, ) . vasculitis also occurs. pcr is the most sensitive method for confirming infection. owing to the genetic diversity of prrsv, sequencing of the orf region is a common adjunct to pcr testing. sequences are then used to create dendrograms for use by production systems pursuing prrs control and epidemiologic investigations (murtaugh, ) . prrsv elisa is helpful for establishing herd status; national animal health monitoring service reports that a large percentage of us herds are seropositive. treatment is limited to maintaining pig comfort, minimizing stress, and controlling secondary infections. commercial modified-live vaccines (mlv) are available and administration during the nursery phase significantly reduces mortality and improves growth performance during the grow-finish phase of production (robbins et al., b) . mlv vaccines do replicate and should not be used in negative populations. salmonella cholerasuis is the swine-adapted salmonella from the c serogroup and, unlike s. typhimurium, is not a foodborne pathogen. ingestion or inhalation of the bacteria causes a septicemia resulting in low to moderate morbidity with high mortality within - days of infection that occurs postweaning, predominately during the grow-finish phase (baskerville and dow, ) . signs include high fevers ( c), lethargy, dyspnea, acute death, and cyanotic extremities and abdomen. the latter makes it impossible to differentiate clinically from classical swine fever virus (csfv). pleuropneumonia, interlobular edema, mediastinal, and tracheobronchial lymphoadenopathy, and occasionally white foci in the liver are apparent postmortem (turk et al., ) . acute histopathologic lesions that form in the lung are purulent bronchitis, lobular necrosis, and abscessation, whereas paratyphoid nodules are observed in the liver. isolation is best achieved from the draining lymph nodes, lung, or liver using selective culture media. serogrouping and typing is necessary for speciation and diagnostic confirmation. owing to the rapid onset of disease, parental treatment is recommended. salmonella cholerasuis isolates are commonly susceptible to ceftiofur. increased hygiene particularly eliminating access to waste and vaccination is preventive (husa et al., ) . siv is more accurately described as influenza, to encompass the infections occurring in swine, avian, and human species. influenza virus is classified by its hemagglutinin and neuraminidase proteins; the three predominant strains in pigs are h n , h n , and h n . rapid transmission and onset are characteristic; in the experimental inoculation of one nonvaccinated nursery age pig resulted in . more becoming infected (romagosa et al., ) . virus is shed for - days and uncomplicated lesions resolve days postinfection (gramer, ) . nasal discharge, fever, and lethargy occur but resolve quickly. cough and dyspnea can last up to figure pulmonary edema, lung, grow-finish pig. interlobular edema associated with pcvad, ventral portion of apical and diaphragmatic lung lobes is consolidated (purple) due to secondary bacterial infection. courtesy dr. glen almond. weeks postinfection. pcr and vi detect virus for diagnosis of clinical cases. elisa and hi detect antibodies; elisa is helpful in establishing herd status, whereas hi is best for vaccination timing and measuring postvaccination titers (allerson et al., ) . necrotizing bronchitis, bronchiolitis, and alveolitis as lesion resolves affected areas appear vacuolated. pigs recover quickly so treatment should focus on maintaining pig comfort, minimizing stress, and controlling secondary infections. all licensed vaccines are killed; commercial and autogenous products are in use in the united states. vaccination reduces lung lesions and rate of transmission, but does not prevent infection and is complicated by antigenic shift and drift. in the united states, it is typical to vaccinate the sows rather than pigs to control disease and infection (allerson et al., ) . trade diseases are those listed by the oie. when one of these diseases is suspected or confirmed, it results in closure of international market access, which would be economically devastating to import-export businesses. the primary method for managing diseases that affect trade is to prevent their introduction. foot-and-mouth disease (fmd) is caused by a picornavirus, fmdv, which causes mucosal lesions exclusively in cloven hoofed species. clinical signs are excessive salivation, anorexia, and lameness causing high morbidity but low mortality. gross lesions are vesicles at cutaneous junctions, on the snout, or in the oral cavity. similar lesions can be caused by seneca valley virus, vesicular stomatitis, swine vesicular disease, and vesicular exanthema of swine; therefore, any blister in swine warrants diagnostic investigation. fmdv is highly transmissible within and between species. african swine fever (asf) is caused by asfv, currently classified as an iridovirus. soft ticks can act as reservoirs or vectors. current outbreaks are reported throughout eastern europe and russia that have been associated with improper garbage feeding. the virus damages blood vessels resulting in clinical signs and gross lesions consistent with septicemia; including red to purple skin discoloration and enlarged spleen, liver, and lymph nodes. excess blood and fluid in body cavities may occur. classical swine fever, historically referred to as hog cholera, is caused by csfv, a pestivirus, eradicated from the united states in . transmission is associated with infected feeding, uncooked or undercooked garbage containing pork or pork by-products to swine. the virus remains infectious for months when refrigerated and years when frozen. clinical signs are nonspecific and are easily confused with s. cholerasuis. the virus replicates rapidly in tonsils, which makes it the ideal tissue to collect for diagnosis of csfv. see also: animal health: ectoparasites. animal health: foot-and-mouth disease. animal health: global antibiotic issues. slum livestock agriculture. vaccines and vaccination practices: key to 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epidemiology and clinical manifestations of an occupational pathogen prevalence of cpb , encoding beta toxin, in clostridium perfringens field isolates: correlation of genotype with phenotype use of tiamulin in a herd of pigs seriously affected with mycoplasma hyosynoviae arthritis brachyspira-associated colitis − update and review swine dysentery: diagnostic criteria and elimination strategies toxic minerals, chemicals, plants, and gases commercial porcine circovirus type vaccines: efficacy and clinical application prevalence of porcine epidemic diarrhea virus and transmissible gastroenteritis virus infection in korean pigs stillbirths, mummies, abortions, and early embryonic death control and elimination of porocine reproductive and respiratory syndrome virus diseases of the nervous and locomotor systems clinical and postmortem examination of sows culled for lameness isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome postweaning escherichia 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of test results sodium salt poisoning of swine efficacy of antimicrobial treatments and vaccination regimens for control of porcine reproductive and respiratory syndrome virus and streptococcus suis coinfection of nursery pigs serological evaluation of a clostridium perfringens type a toxoid in a commercial swine herd an investigation of the pathology and pathogens associated with porcine respiratory disease complex in denmark practitioner experiences with swine dystentery leptospira pomona: a case report in growing swine and breeding stock the various forms of haemophilus parasuis assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers swine toxicosis. swine health production a comparison of the safety, cross-protection, and serologic response associated with two commercial oral salmonella vaccines in swine effect of osmolality on net fluid absorption in non-infected and etec-infected piglet small intestinal segments protection of pigs against neonatal enteric colibacillosis with colostrums and milk from orally vaccinated sows a meta-analysis comparing the effect of pcv vaccines on average daily weight gain and mortality rate in pigs from weaning to slaughter proliferative enteropathy: a global enteric disease of pigs caused by lawsonia intracellularis gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus optimizing farrowing rate and litter size and minimizing nonproductive sow days prevalence of actinobacillus pleuropneumoniae, actinobacillus suis, haemophilus parasuis, pasteurella multocida, and streptococcus suis in representative ontario swine herds effect of porcine circovirus type (pcv ) on reproduction: disease, vertical transmission, diagnostics and vaccination rickets: case series and diagnostic review of hypovitaminosis d in swine reproductive failure experimentally induced in sows via artificial insemination with semen spiked with porcine circovirus type effect of porcine circovirus type (pcv ) vaccination of the dam on pcv replication in utero control of mycoplasma hyopneumoniae infections in pigs effects of toltrazuril on the growth of piglets in herds without clinical isosporosis actinobacillus pleuropneumoniae disease and serology quantification of the spread of mycoplasma pneumoniae in nursery pigs using transmission experiments mechanisms of porcine diarrheal disease sarcoptic mange control/eradication and their impact on pig performance: a literature review and comparison of different protocols and monitoring programs successfully implemented worldwide with ivomec s products for swine system wide sick pig management immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) use and interpretation of sequencing in prrsv control programs impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows update on actinobacillus suis diagnosis, epidemiology, and control: on the path from good to great development of a pcr test to diagnose haemophilus parasuis infections evaluation of haemophilus parasuis control in the nursery using vaccination and controlled exposure determination of sodium content in serum and cerebrospinal fluid as an adjunct to diagnosis of water deprivation in swine diagnosis of streptococcus suis infections trichuris suis in finishing pigs: case report and review prevalence of internal parasites in a production system: part ii-finishing pigs diagnostic approaches to swine central nervous system disorders haemophilus parasuis what do we really know about feedback to gestating dams? prrsv control in finisher pigs, a large scale barn study in a high dense area in usa vaccination of influenza a virus decreases transmission rate in pigs role of the sow as a reservoir of infection for mycoplasma hyosynoviae porcine reproductive and respiratory syndrome review of mycoplasma hyorhinis diagnostic approaches to reproductive failure in pigs. swine health production transmissable gastroenteritis and porcine respiratory coronavirus effect of bmd s in sow gestation/lactation diets on clostridial infection, piglet pre-weaning performance, and sow body condition effect of waste environment on survival of brachyspira hyodysenteriae clostridial enteric infections in pigs update on porcine circovirus and postweaning multisystemic wasting syndrome (pmws). swine health production rotavirus and undifferentiated diarrhea in suckling piglets: what's new and diagnostics criteria internal parasites efficacy of a chlortetracycline feed additive in reducing pneumonia and clinical signs induced by experimental mycoplasma hyopneumoniae challenge effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (prrsv)-induced pneumonia by mycoplasma hyopneumoniae water in swine nutrition pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection pleuropneumonia in missouri swine pseudorabies (aujeszky's disease) and its eradication: a review of the u.s. experience a retrospective evaluation of actions taken to control streptococcus suis infection serologic profiling and vaccination timing for lawsonia intracellularis swine erysipelas − a review of prevalence and research age, not infection dose, determines the outcome of isospora suis infections in suckling pigs actinobacillus suis septicemia: an emerging disease in highhealth herds key: cord- -il s lgp authors: tam, lai‐shan; tanaka, yoshiya; handa, rohini; chang, chi‐chen; cheng, yew kuang; isalm, nazrul; li, mengtao; lorenzo, jose paulo; song, yeong‐wook; yamamoto, kazuhiko; zeng, xiaofeng; haq, syed atiqul title: care for patients with rheumatic diseases during covid‐ pandemic: a position statement from aplar date: - - journal: int j rheum dis doi: . / - x. sha: doc_id: cord_uid: il s lgp nan the outbreak of coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) was first reported in china in december . this disease now affects the whole world. patients with rheumatic diseases are at higher risk of respiratory infections including influenza and pneumococcal pneumonia, which is attributed to the underlying disease, comorbidities and immunosuppressive therapy, but to date we lack good information about the virus sars-cov- . nonetheless, immunosuppressive treatments are essential to control disease activity and prevent functional deterioration in these patients. rheumatologists need to be vigilant in preventing rheumatic disease patients from contracting the disease during this pandemic, especially patients with chronic lung problems (eg scleroderma with lung fibrosis) and chronic kidney disease (eg lupus nephritis) and those on high-dose glucocorticoids and immunosuppressants (appendix ). in the desperate search to find effective treatments for covid- , drugs largely used by rheumatologists have entered the spotlight, including the caution against use of non-steroidal anti-inflammatory drugs (nsaids), the potential of antimalarials and biologic disease-modifying anti-rheumatic drugs (bdmards), for example anti-interleukin- (il- ) and targeted synthetic dmards (tsdmards) janus-activated kinase (jak) inhibitors to manage cytokine storm syndrome (css)/cytokine release syndrome associated with covid- . here, we try to provide guidance regarding clinical decision-making both for patients with covid- and those with rheumatic diseases, and strategies to mitigate further harm to these patients. an asia-pacific league against rheumatism (aplar) covid- task force comprising rheumatologists from asia-pacific countries was convened on march, . a set of guidance statements was developed and refined based on best available evidence up to april, and expert opinion. given the overall limited nature of the data, a systematic review was not performed. the final guidance statements integrate both the task force members' assessment of the evidence quality and the ratio of risk and benefit from the treatment or action. we assert that the key guiding principle should be to "first do no harm," especially given the unknown efficacy of proposed dmards and biologics and their established potential harms. this guidance document has been reviewed and endorsed by the aplar executive committee and the aplar scientific committee chairpersons. in the absence of a vaccine or a therapeutic agent, a "mitigation approach", including "social distancing", frequent hand washing and quarantining strategies are the primary interventions to hamper the spread of infection. smoking can cause an increase in the release of il- in bronchial epithelial cells, and upregulate angiotensin-converting enzyme- (ace ) receptors, the known receptor for sars-cov. this is particularly relevant as some of the asia-pacific countries, for example china, has a high male smoking rate. globally the quality of evaluation, monitoring and treatment of comorbidities in rheumatic disease patients is variable with considerable scope for improvement. rheumatologists should be vigilant in assessing and managing comorbidities not only to improve morbidity and mortality, but hopefully to minimize risk of covid- in rheumatic disease patients. in patients with acute respiratory tract infections, short-term use of nsaids are associated with increased risk of cardiovascular events and nephrotoxicity, [ ] [ ] [ ] higher rates of complications, and delays in the prescription of effective antibiotic treatment. despite the lack of evidence relating specifically to people with covid- , regular nsaid use should not be recommended as the first line option for managing the symptoms of covid- . nonetheless, arthritis patients taking nsaids for symptomatic relief should continue their treatment as needed. epidemiologic studies have identified advanced age, male gender and presence of comorbidities (hypertension, obesity, diabetes, coronary heart disease, chronic obstructive lung disease and chronic kidney disease) as poor prognostic factors for covid- . despite the lack of data on the true prevalence and risk of covid- in rheumatic disease patients, immunosuppressed status (the use of chemotherapy or conditions requiring immunosuppressive treatment) was not reported to be a risk factor and risk for adverse outcome. one patient with systemic sclerosis-associated interstitial lung disease (ssc-ild) on tocilizumab and patients on bdmards or ts-dmards who developed covid- recovered uneventfully. [ ] [ ] [ ] nonetheless, at least patients on rituximab developed respiratory failure and of them died despite treatment with tocilizumab. in order to gather real-world data to inform treatment strategies and better characterize individuals at increased risk of infection, the covid- global rheumatology alliance has successfully de- preclinical and limited clinical data suggested that hydroxychloroquine (hcq) and chloroquine (clq) have antiviral activities against sars-cov- . [ ] [ ] [ ] in contrast, a small but randomized study from china in patients with mild to moderate covid- treated with hcq or placebo found no difference in recovery rates, once hospitalized, for some patients with covid- , death can occur within a few days, many with ards, and some with multiorgan dysfunction syndrome. in those critically ill patients, there are both clinical signs and symptoms, as well as laboratory abnormalities, that suggest a css is occurring in response to the viral infection. according to data from the chinese cohorts, patients with severe disease and requiring intensive care often show leucopenia, lymphopenia, significantly higher levels of c-reactive protein (crp), il- , il- , and tumor necrosis factor-α (tnf-α). in this setting, biologic drugs selectively blocking inflammatory cytokines, such as tnf-α inhibitors, anti-il- , anti-il- and jak inhibitors are currently employed in the treatment of severe cases of covid- in an experimental manner or undergoing clinical trials (appendix ). tocilizumab, has been shown effective in treating css, a common complication of chimeric antigen receptor-t cell therapy used for treating refractory acute lymphoblastic leukemia and may be effective in chinese covid- patients with severe and critical disease. anti-il- r antibody is currently included in the treatment recommendation for chinese covid- patients (appendix ). these concepts have led to interests in jak inhibitors, for example baricitinib, as potential treatments for css complicated with severe covid- . ace is a cell-surface protein widely existing on cells in the heart, kidney, blood vessels, especially alveolar epithelial cells. sars-cov- was believed to invade and enter lung cells through ace -mediated endocytosis. one of the known regulators of endocytosis is the ap - the following link s are from national or international or ganiz ations to help rheumatologis t s an d patient s to manage their diseases during covid - incidence and prevalence of vaccine preventable infections in adult patients with autoimmune inflammatory rheumatic diseases (aiird): a systemic literature review informing the update of the eular recommendations for vaccination in adult patients with scientific and ethical basis for social-distancing interventions against covid- prevalence of underlying diseases in hospitalized patients with covid- : a systematic review and meta-analysis the effect of electronic cigarette and tobacco smoke exposure on copd bronchial epithelial cell inflammatory responses smoking upregulates angiotensin-converting enzyme- receptor: a potential adhesion site for novel coronavirus sars trends in cigarette smoking among older male adults in china: an urban-rural comparison prevalence of comorbidities in rheumatoid arthritis and evaluation of their monitoring: results of an international, cross-sectional study (comora) risk of stroke associated with use of nonsteroidal anti-inflammatory drugs during acute respiratory infection episode acute respiratory infection and use of nonsteroidal anti-inflammatory drugs on risk of acute myocardial infarction: a nationwide case-crossover study non-steroidal anti-inflammatory drug induced acute kidney injury in the community dwelling general population and people with chronic kidney disease: systematic review and meta-analysis risks related to the use of non-steroidal anti-inflammatory drugs in community-acquired pneumonia in adult and pediatric patients non-steroidal anti-inflammatory drugs and covid- asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus (sars-cov- ): facts and myths covid- in a patient with systemic sclerosis treated with tocilizumab for ssc-ild clinical course of covid- in a series of patients with chronic arthritis treated with immunosuppressive targeted therapies what is the true incidence of covid- in patients with rheumatic diseases? rituximab for granulomatosis with polyangiitis in the pandemic of covid- : lessons from a case with severe pneumonia incidence and clinical course of covid- in patients with connective tissue diseases: a descriptive observational analysis rheumatic disease and covid- : initial data from the covid- global rheumatology alliance provider registries the role of cytokines including interleukin- in covid- induced pneumonia and macrophage activation syndrome-like disease clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical evidence does not support corticosteroid treatment for -ncov lung injury hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection clinical course of coronavirus disease (covid- ) in a series of patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine clinical and immunologic features in severe and moderate coronavirus disease management of cytokine release syndrome related to car-t cell therapy effective treatment of severe covid- patients with tocilizumab baricitinib as potential treatment for -ncov acute respiratory disease baricitinib for covid- : a suitable treatment? lancet infect dis european league against rheumatism (eular) guidance for patients on covid german society for rheumatology -patient section guida nce-on-shiel ding-and-prote cting-extre mely-vulne rable-perso ns-from-covid- /guida nce-on-shiel ding-and-prote ctingextre mely-vulne rable-perso ns-from-covid- ) national rheumatoid arthritis society: coronavirus: what we know so far telemedicine practice guidelines (ncov)-infec tion-is-suspe cted national institute of health treatment guideline https://covid tre atmen tguid elines.nih.gov/intro ducti on/ us food and drug administration (fda) cautions against the use of antimalarial agents outside hospital setting or clinical trial: https:// www.fda.gov/drugs/ drug-safety-and-avail abili ty/fda-cauti ons-again st-use-hydro xychl oroqu ine-or-chlor oquine-covid- -outsi de-hospi tal-setti ng-or treatment recommendation for chinese covid- patients hydroxychloroquine for the treatment of patients with mild to moderate covid- to prevent progression to severe infection or death the covid- global rheumatology alliance key: cord- -dde nlhh authors: antabe, roger; ziegler, bianca rosa title: diseases, emerging and infectious date: - - journal: international encyclopedia of human geography doi: . /b - - - - . - sha: doc_id: cord_uid: dde nlhh emerging and infectious diseases have persisted as leading causes of global morbidity and mortality. caused by pathogens including bacteria, viruses, parasites, or fungi, they are known to pose serious health threats to the world's population dating back to ancient egypt. in the th century alone, infectious diseases were responsible for decimating – % of the world's population. the discovery of vaccines, coupled with improved sanitation, hygiene, and health care, witnessed the eradication of several infectious diseases, although some have resurfaced or are resurfacing since the latter part of the th century. while geography partly define hotspots for emerging and infectious diseases, low socioeconomic development, poverty, and underfunded health care systems remain driving forces for the reoccurrence of these diseases among vulnerable populations who experience material deprivation. to eradicate infectious diseases, a global response will have to prioritize the allocation of resources by way of expertise and technology to areas that are most affected. furthermore, an effective surveillance system, and a rigorous vaccine deployment regime targeting vulnerable persons and regions is desirable in mitigating the impacts of these diseases. infectious diseases are illnesses, which are spread directly or indirectly, from person to person. infectious diseases can be classified under one of the following origins: zoonotic, vector-borne, or drug-resistant. the majority (over %) of emerging and reemerging infectious diseases are caused by bacteria, viruses, parasites, or fungi, which were passed from animals to humans, known as zoonotic pathogens. zoonotic pathogens, which originated in wild life, are the causative agents for diseases such as severe acute respiratory syndrome (sars) and ebola. as a result, zoonotic infectious diseases have led to epidemics that drew attention to the interactions between humans and wildlife, including the domestication of animals, food production and intensive farming, moving animals and plants to foreign environments, urban sprawl and migration, and climate change and deforestation. vector-borne infectious diseases are spread between humans and animals by vectors such as mosquitoes and ticks. these diseases, including malaria and lyme disease, impact specific geographic regions. tropical and subtropical areas are disproportionately affected, and the poorest and most vulnerable tend to face the highest burden of these diseases. the history of these diseases includes widespread illness and high mortality rates affecting populations on a global scale. the history of infectious diseases goes back as far as smallpox being found on egyptian mummies and the plague infecting and killing - % of the world's population in the th century. however, by the end of the s, most infectious diseases were considered a health issue of the past. with improved public health measures including sanitation processes and sewage treatments, as well as improved healthcare services and the introduction of antibiotics and vaccination protocols, the prevalence of infectious diseases appeared to be declining. as a result, health care professionals and researchers turned their attention and available resources toward the rising issue of chronic disease. yet, during the last quarter of the th century, infectious diseases began to reemerge with new and resurgent diseases cited as causes of morbidity and mortality for large populations. since this time, infectious disease rates have continued to rise, new diseases have emerged, and previously eradicated diseases have reemerged. the emergence and reemergence of infectious diseases has mostly been attributed to the unintentional consequences of human behavior, activities, and development. the threat of emerging and infectious diseases (eids) to human health, society, and the global economy is increasing as infectious diseases continue to evolve and spread. to illustrate, in alone over million people died from hiv, and million people contracted malaria. furthermore, the sars outbreak in resulted in death for out of the infected patients within months, with the survivors experiencing ill health and, in some cases, disability. clearly, infectious diseases such as sars place added stress on healthcare systems in affected countries as these systems must mobilize resources to effectively deal with the epidemics. they also create tensions within communities and decrease economic prosperity. for example, travel, tourism, and retail sales dropped dramatically during the sars epidemic, especially in asia where the disease originated and was most prevalent. globally, the economic impact of sars was estimated to be $ - billion usd. emerging and reemerging infectious diseases are largely preventable, and yet with their profound impact and increasing prevalence, they remain a threat to global health, which must be addressed. humans and pathogens have always been in a dynamic state of interaction; however, current trends reveal that this relationship has become unbalanced as human activities are causing pathogens to appear and spread at an increasingly alarming rate. as aforementioned, the st century has seen a rise in infectious disease, largely due to the societal and global trends that are reinforced by an increasingly technological, globalized, and interconnected world. between and , infectious diseases, such as tuberculosis, sars, and h n , emerged or reemerged in both the developed and developing world. these diseases caused the death of an estimated million people per year, with % of these deaths occurring in the concentrated region of south-east asia and the sars outbreak in china affecting other countries. by the end of april , a measles outbreak in the united states affected people, % of whom were unvaccinated. similarly, in the european region, the who reported a total of , measles cases by march , with related deaths in the preceding year. additionally, the world is still battling with diseases such as hiv/aids, malaria, ebola, h n , and dengue fever. each year, approximately million travelers transport pathogens between places and provide a means for infectious disease to spread across borders. the increased globalization of the world has resulted in travel and trade being facilitated on a global scale, which has caused infectious diseases to spread more easily to countries that were previously unaffected. moreover, there is a higher prevalence of infectious diseases in low-income and developing countries due to increased poverty, social inequality, and conflict, leading to increased susceptibility and a decreased capacity to effectively provide sufficient treatment. additionally, environmental variability due to climate change has lengthened the season in which vectors are present and impacted agricultural practices, allowing for increased transmission. furthermore, vectors have also become resistant to numerous pesticides making it harder to control the spread of the diseases, which they carry. another important issue that is feeding the emergence and reemergence of infectious diseases is the issue of drug resistance to treatment. increasingly microorganisms are evolving and adapting which makes it difficult to treat these diseases, thus increasing their rate of transmission. many factors lead to drug-resistant infectious diseases including antibiotic misuse (such as not finishing an entire dose of antibiotics), over-prescription of antibiotics, and the use of antibiotics for agriculture and animal production that can lead to antimicrobial resistance. moreover, vaccinations not being kept up to date or not being administered to the majority of the population to maintain herd immunity has led to drug-resistant diseases, which have evolved and rendered current vaccines ineffective. many infectious diseases are evolving more quickly than effective treatments can be created and tested, as a result, as drug-resistant diseases continue to increase in prevalence, their threat to human health continues to increase. other health trends have also contributed to the infectious disease epidemic. an aging population with a higher life expectancy has resulted in an increased proportion of the population being elderly, thus susceptible to infectious disease, and less likely to have the capacity to combat sickness. much of the world also suffers from multimorbidities, with chronic diseases such as diabetes and heart disease on the rise, once again increasing susceptibility. similarly, the hiv/aids epidemic has resulted in a large immunocompromized population who are vulnerable to contracting multiple infectious diseases. the prevalence of hiv/aids declined globally by % between and . however, as of , . million people were infected globally with hiv/aids, with africa having the highest proportion of hiv/aids cases, a trend which like the general trend of emerging and reemerging infectious disease prevalence is not declining quickly or equitably enough. eids have been posited by researchers to persist as a global health concern for the next couple of decades particularly in poor and under-resourced settings in the global south. however, efforts at the global level yield the potential to address incidences of eids. in line with these objectives, there have been proposed strategies to address current trends of eids. the sustainable development goals (sdgs), created in , identified infectious diseases as a priority area for health policy; sdg . set out to end numerous infectious disease epidemics, such as hiv/aids and malaria by . the sdgs posit that through increased surveillance and allocating more resources and funding to this health issue, diagnostic and treatment programs will be improved, and the epidemic of emerging and reemerging infectious diseases will once again begin to decline. in the meantime, there is urgency in building a global network consisting of a team of specialists in eids. this network will be hinged on improved communication surrounding risks of outbreaks in global hotspots that are difficult to access. this will be foundational to addressing outbreaks, as the who has observed that areas with more frequent outbreaks and endemic cases of reemerging infectious diseases such as hiv, hbv, and ebola tend to be in low-and middle-income countries. budget constraints in allocations for emergencies and laboratory research may therefore be undermining efforts at total eradication of eids in these areas. the lack of coordinated effort in understanding the nature and extent of outbreaks among specialists may be a major contributory factor where isolated cases of eids easily escalate to full-blown epidemics. therefore, a global network of specialist and experts is key in designing future responses to eids. the introduction of vaccines led to the eradication of major infectious disease such as smallpox and measles that plagued earlier centuries as leading causes of death. currently, immunizations alone are estimated to avert two to three million global annual deaths. targeting populations at increased risk, including high-density areas and those in the geographies of elevated risk of zoonosis, with vaccines will lead to a substantial decline in new outbreaks. the trial of ebola vaccines marks a major milestone in reducing mortalities associated with the current perennial outbreak of the ebola virus in west and central africa. as well, after an initial trial of malaria vaccines ended in , the who has recommended an expanded trial in three countries in sub-saharan africa, based on this trial, deployment of this vaccine could be sanctioned across the globe to eradicate malaria. similarly, discussion on the deployment of preexposure prophylaxis (prep) and post exposure prophylaxis (pep) for hiv in both endemic and nonendemic contexts in the global south and north respectively, and among exposed populations, holds the key to achieving sections of the sdgs and the unaids - - . it is worth noting that, an effective surveillance system of people and areas at risk, will lead to early detection of instances of microbial and drug resistance in vaccines and treatment of eids. particularly for eids that are asymptomatic, providing access to testing and screening services is desirable to obtain an early warning and to devise a response to outbreaks. this is largely dependent on available resources at the locale by way of laboratory or clinical technology that will be most effective in the timely detection of new incidences of eids. for instance, only % of people with viral hepatitis have been diagnosed due to limited access to testing services. given that a large percentage of eids are concentrated in countries where governments are under-resourced in responding to outbreaks, well-equipped testing and screening facilities for the public at risk are needed. currently in low-income settings where health infrastructure is under-developed, point-of-care (poc) diagnostics have emerged with the potential for inexpensive, effective, and timely diagnoses of infectious diseases. this also calls for the development and deployment of diagnostic techniques that are suitable for specific regions or geographies. in view of the disproportionate global burden of infectious diseases where some regions are more prone relative to others, a key consideration in eradicating eids may be the reallocation of resources, including expertise and clinical technology to areas that are most impacted. currently, areas least affected by eids receive a higher allocation and concentration of resources to respond to outbreaks. in this regard, in the context of developing countries, hbv, hiv, and ebola have persisted largely due to the absence of funding to secure vaccines and treatment for populations at heightened risk of infection. this is in sharp contrast to the context of the global north where eids are less prevalent but have control of global resources in eradicating infectious diseases. zoonotic pathogens account for . % of all eids, of which an estimated . % are contracted through contact with wildlife. in this context, understanding human contact with wildlife and how to minimize it in hotspots with highly biodiverse fauna can help contain new incidences. moreover, the limited outbreak of eids in areas where conservation efforts have drastically reduced anthropogenic contacts with wildlife means more conservation efforts will lead to a substantial decline of eids. it is also crucial to institute smart surveillance in areas of high population density to avoid new strains of infection from reemerging. see also: epidemiological transition; medical geography. emerging and re-emerging infectious diseases: the third epidemiologic transition emerging infectious diseases: public health issues for the st century global trends in emerging infectious diseases point-of-care testing for infectious diseases: past, present, and future thomas hepatitis b virus epidemiology, disease burden, treatment, arid current and emerging prevention and control measures geography, ecology and emerging infectious diseases emerging infectious diseases: threats to human health and global stability factors in the emergence of infectious diseases the epidemiology of hiv and other sexually transmitted infections in african, caribbean and black men in toronto developments in the diagnostic techniques of infectious diseases: rural and urban prospective shweta sustainable development knowledge platform. www.who.int/whr/ /media_centre/press_release/en/ world health organization www.who.int/news-room/fact-sheets/detail/immunization-coverage world health organization: immunization coverage key: cord- -vbhilmve authors: santos, c. sieiro; morales, c. moriano; Álvarez, e. díez; castro, c. Álvarez; robles, a. lópez; sandoval, t. perez title: determinants of covid- disease severity in patients with underlying rheumatic disease date: - - journal: clin rheumatol doi: . /s - - - sha: doc_id: cord_uid: vbhilmve background: over the month of april, spain has become the european country with more confirmed cases of covid- infection, after surpassing italy on april nd. the community of castile and león in spain is one of the most affected by covid- infection and the province of león has a total of cases and deaths so far. rheumatic patients should be given special attention regarding covid- infection due to their immunocompromised state resulting from their underlying immune conditions and use of targeted immune-modulating therapies. studying epidemiological and clinical characteristics of patients with rheumatic diseases infected with sars-cov is pivotal to clarify determinants of covid- disease severity in patients with underlying rheumatic disease. objectives: to describe epidemiological characteristics of patients with rheumatic diseases hospitalized with covid- and determine risk factors associated with mortality in a third level hospital setting in león, spain. methods: we performed a prospective observational study, from st march until the st of june including adults with rheumatic diseases hospitalized with covid- and performed a univariate and multivariate logistic regression model to estimate ors and % cis of mortality. age, sex, comorbidities, rheumatic disease diagnosis and treatment, disease activity prior to infection, radiographic and laboratorial results at arrival were analysed. results: during the study period, patients with covid- were admitted to our hospital, of whom ( %) had a rheumatic or musculoskeletal disease. fifty-three percent were women, with a mean age at hospital admission of . (iqr – ) years. the median length of stay was days. a total of patients died ( %) during their hospital admission. patients who died from covid- were older (median age . iqr . – . ) than those who survived covid- (median age . iqr . – . ) and more likely to have arterial hypertension ( [ %] vs [ %] patients; or ( % ci . – . ), p . ), dyslipidaemia ( ( %) vs ( %); or ( % ci . – ), p . ), diabetes (( ( %) vs ( %) patients; or , p . ), interstitial lung disease ( ( %) vs ( %); or . ( % ci . – ), p . ), cardiovascular disease ( ( %) vs ( %); or . ( % ic . – . , p . ) and a moderate/high index of rheumatic disease activity ( ( %) vs ( %); or . ( . – . ), p . ). in univariate analyses, we also found that patients who died from covid- had higher hyperinflammation markers than patients who survived: c-reactive protein ( (iqr – ) vs . (iqr – ; p . ); lactate dehydrogenase ( . (iqr . – . ) vs (iqr – ), p . ); serum ferritin ( (iqr . – . ) vs . (iqr – . ), p . ); d-dimer ( , . (iqr . – , . ) vs . (iqr – ), p . ). no differences in sex, radiological abnormalities, rheumatological disease, background therapy or symptoms before admission between deceased patients and survivors were found. in the multivariate analysis, the following risk factors were associated with mortality: rheumatic disease activity (p = . ), dyslipidaemia (p = . ), cardiovascular disease (p = . ) and interstitial lung disease (p = . ). age, hypertension and diabetes were significant predictors in univariate but not in multivariate analysis. rheumatic disease activity was significantly associated with fever (p = . ), interstitial lung disease (p = . ), cardiovascular disease (p = . ) and dyslipidaemia (p = . ). conclusions: our results suggest that comorbidities, rheumatic disease activity and laboratorial abnormalities such as c-reactive protein (crp), d-dimer, lactate dehydrogenase (ldh), serum ferritin elevation significantly associated with mortality whereas previous use of rheumatic medication did not. inflammation is closely related to severity of covid- . introduction sars-cov- infection has spread rapidly across the planet. it is thought to have originated in china's wuhan province; however, it has spread to more than countries on continents, according to the who. over % of sars-cov- infection cases have flu-like symptoms; however, % require hospitalization and - % intensive care [ ] . patients with rheumatic patients deserve special attention since they are considered at-risk for serious infections due to their immunocompromised state resulting from their underlying immune conditions and use of targeted immune-modulating therapies such as biologics and immunomodulatory therapies [ ] . some drugs that m o d i f y t h e a c t i v i t y o f t h e d i s e a s e , s u c h a s hydroxychloroquine, used as treatment of rheumatic diseases have been used for the treatment of covid- [ ] . the use of biological therapies such as il- (tocilizumab, sarilumab) and il- (anakinra) inhibitors are also being implemented in patients who developed pathological immune responses such as cytokine storm [ ] . it is not clear whether the use of immunosuppressive medication for rheumatic pathology may suppose a risk of developing sars-cov- infection, if rheumatic patients have a higher mortality rate than general population or what factors may be associated with covid- severity [ ] . all patients with rheumatic diseases with positive test for sars-cov admitted to the hospital from the community of león were included in the study. for the purpose of this study, we included patients: (a) aged > years old, (b) with medical diagnosis of inflammatory rheumatic disease, (b) with sars-cov- pcr positive diagnostic test or positive antibody serology and (c) patients that required hospitalization. we excluded patients who did not require hospitalization or had a negative test for sars-cov . registry data elements included the following co-variables that were considered: ( ) sociodemographic baseline characteristics: including sex and age; ( ) type of inflammatory rheumatic disease diagnosis: including systemic autoimmune conditions (polymyalgia rheumatica (pmr), vasculitis, systemic sclerosis (ss), sjogren's syndrome (sjs), systemic lupus erythematosus (sle) and chronic inflammatory arthritis (rheumatoid arthritis (ra) inflammatory polyarthritis, psoriatic arthritis (pa) and ankylosing spondyloarthritis (as). ( ) baseline comorbid conditions including hypertension, dyslipidaemia, hyperuricemia, diabetes mellitus, heart disease (including valvulopathies, arrythmias, myocardiopathies, heart failure or pericarditis) and interstitial lung disease. ( ) treatment for inflammatory rheumatic disease: glucocorticoids, conventional synthetic disease-modifying antirheumatic drugs (csdmards) including antimalarials (chloroquine/ hydroxychloroquine); sulfasalazine; leflunomide; methotrexate; azathioprine or mycophenolate mofetil, bdmards including targeted synthetic or biologic (ts/bdmards), including: anti-tnf agents and other biologics: anti-il (tocilizumab, sarilumab); rituximab; abatacept. ( ) rheumatic disease activity prior to admission. we also compiled information on covid- -related illness including diagnosis date, symptoms, laboratory and radiological finding, treatment, the need for icu care, subsequent complications and death during follow-up. we performed a univariate and multivariate logistic regression model to estimate ors and % cis of mortality. age, sex, comorbidities, rheumatic disease diagnosis, treatment for rheumatic disease and disease activity prior to infection, duration of hospital stay, symptoms before admission, radiographic abnormalities and laboratorial results at arrival were analysed. we defined radiographic abnormalities using the following classification system ( - ): for no lung abnormalities, for unilateral interstitial abnormalities, for bilateral interstitial abnormalities and for interstitial and alveolar infiltrates. the presence of hyperinflammation was defined as lymphocyte counts lower than cells per ml, serum ferritin higher than ng/ml, lactate dehydrogenase higher than u/l, c-reactive protein higher than and d-dimers higher than ng/ml. we revised consultation charts and calculated rheumatic disease activity prior to infection and used the following classification system: for remission, for low/minimal, for moderate, for high/severe. the medical treatment was prescribed according to our hospital's guidelines. hydroxychloroquine was prescribed at mg bid the first day, followed by mg bid for total of days. lopinavir/ritonavir / mg bid was initially indicated in patients with moderate to severe infection. treatment with systemic steroids and/or biologics (tocilizumab or anakinra) was started in case of worsening of respiratory symptoms or acute respiratory distress syndrome. the study was conducted according to the principles of the declaration of helsinki and approved by our hospital's institutional ethics committee. categorical variables were reported as percentages, whereas continuous variables were expressed as median and iqr values. quantitative variables were compared by use of the non-parametric mann-whitney test. categorical variables were compared by use of contingency tables and p values were calculated with χ or fisher's exact tests, when appropriate. p values less than . were considered significant. the effect size for retrospective studies was then evaluated with odds ratios (ors) with % cis. the demographic and clinical characteristics of the cases in our registry are shown in table . during the study period, patients with covid- were admitted to our hospital, of whom ( %) had a rheumatic or musculoskeletal disease. fifty-three percent were women, with a mean age at hospital admission of . (iqr - ) years. the median length of stay was days. a total of patients died ( %) during their hospital admission. most patients had important comorbidities: % patients had hypertension, % had dyslipidaemia, % had diabetes mellitus, % had cardiovascular disease (cd), % had interstitial lung disease (ild). the most frequent symptoms of covid- were dyspnoea ( %), cough ( %), fever ( %), % reported gastrointestinal symptoms (diarrhoea and vomit), % muscle pain, % odynophagia and dysgeusia. forty-two percent had a previous diagnosis of rheumatoid arthritis (ar), % of polymyalgia rheumatica (pmr), % of systemic lupus erythematosus (sle), % of psoriatic arthritis (pa), % of ankylosing spondylitis (as), % of giant cell arteritis and there was one case of limited systemic sclerosis and sjogren's disease. at the time of sars-cov infection, % received oral corticosteroids at a mean dose of . mg/day, % were taking csdmards, % received treatment with bdmards and % were taking hydroxychloroquine. ten patients died. patients who died from covid- were older (median age . iqr . - . ) than those who survived covid- (median age . iqr . - . ) and more likely to have arterial hypertension ( [ %] vs [ %] patients; or ( % ci . - . ), p . ), dyslipidaemia ( ( %) vs ( %); or ( % ci . - ), p . ), diabetes (( ( %) vs ( %) patients; or ( % ic . - . p . ), interstitial lung disease ( ( %) vs ( %); or . ( % ci . - ), p . ), cardiovascular disease ( ( %) vs ( %); or . ( % ic . - . ), p . ) and a moderate/high index of rheumatic disease activity ( ( %) vs ( %); or . ( . - . ), p . ). in univariate analyses, we also found that no differences in sex, radiological abnormalities, rheumatological disease or background therapy or symptoms before admission between deceased patients and survivors were found. glucocorticoids were used for severe respiratory manifestations related to lung involvement in patients ( %) and tocilizumab in ( %). to control for possible confounding variables, sequential multivariate regression analyses were performed. in the multivariate analysis, this work describes a collection of covid- -hospitalized cases among patients with rheumatic diseases in a third level hospital, in león, spain. we identified factors associated with mortality in patients with underlying rheumatic diseases, including age, sex, comorbidities, rheumatic diagnosis, treatment, rheumatic disease activity, laboratorial/radiographic abnormalities and complications during admission. our data showed that, in a real-world setting, there is a % of patients with underlying rheumatic diseases with covid- that required hospital admission, mainly elderly patients, with more comorbidities and systemic autoimmune condition. in accordance with previous studies of covid- in different populations, we found that patients with comorbidities such as hypertension, dyslipidaemia, diabetes, interstitial lung disease and cardiovascular disease and diabetes had higher odds of mortality [ , ] . the covid- global rheumatology alliance has recently published a cohort of patients with covid- and underlying rheumatic disease, with over cases [ ] . compared to that cohort, patients in our study were more likely to be aged older than years and have a higher rate of comorbidities (hypertension, diabetes, and dyslipidaemia) and a more severe disease course. in several studies, the importance of diabetes as comorbidity in covid- has already been seen. it was stated that diabetes is an independent risk factors for morbidity and mortality in covid- [ ] . we did not find a significant association between mortality and antimalarial use before hospitalization. thirty-five percent of the patients were negative for reverse transcriptasepolymerase chain reaction of nasopharyngeal and sputum swabs after receiving treatment; however, almost all of these patients had mild disease and would likely have a favourable clinical outcome and viral clearance regardless of treatment. there has been speculation about the use of hcq in the treatment of covid- and many randomized studies have been conducted, claiming nasopharyngeal virus clearance in cases treated with antimalarial agents; however, the evidence supporting the use of hcq is not compelling [ ] . recently, an observational study found no significant benefit with either hydroxychloroquine alone or combined with azithromycin on clinical outcomes including mortality [ ] . we also did not detect differences in background therapies when comparing deceased patients with survivors, such as csdmards, corticosteroid and biologic agents; however, we found differences in hyperinflammation markers between the two groups and found that patients who died from covid- are more likely to have moderate/high rheumatic disease activity prior to infection. several studies have theorized that sars-cov infection can induce cytokine release, leading to an increase in il- , il- , and tnf. in cytokine release syndrome, common laboratory abnormalities in hospitalized patients involve elevated liver enzymes, serum ferritin value, c-reactive protein, d-dimer, coagulation times (pt/ptt) and lactate dehydrogenase (ldh) [ ] . in fact, we observed that patients with severe prognosis often associate a rate of hyperinflammation or need for respiratory support, which required the use of glucocorticoids or tocilizumab. fadel et al. have recently pointed out the use of [ ] . in our hospital, the use of glucocorticoids or tocilizumab, or both, was based on the severity of respiratory symptoms, in accordance with our hospital's protocol. the number of patients taking biologic drugs was very small in our study which may be insufficient to demonstrate other underlying effects. there has been talk about the effect of biologic treatment in covid- infection. several studies have highlighted the use of biologic agents, as evidenced by those with more severe disease having higher levels of cytokines, including il- and tnf [ ] . treatment with anti-il receptor monoclonal antibody, tocilizumab has been shown effective in treating cytokine release. il- levels can be easily measured in blood in hospitalized covid- patients. a recent study showed that elevated il- concentration was associated with detectable serum sars-cov rna in covid- patients and reflects the severity of the disease. the same study also associates parameters of multiple organ dysfunction, like troponin t levels with higher il- levels [ ] . these data reinforce the idea that the prognosis of covid- is more likely to be related to the presence of other risk factors rather than the rheumatic and musculoskeletal disease itself or the background therapy; and that hyperinflammation is associated with the severity of covid- . the results of our study should be interpreted considering limitations. first, we have included a small sample size with underlying rheumatic diseases who needed hospitalization; second, we should consider the observational nature of the study and that our patients were treated at a single centre. overall, the prognosis of patients with rheumatic diseases hospitalized with covid- is good; however, our findings should be interpreted with caution. studies including inflammatory rheumatic disease patients with covid- are still limited; however, we hope these data can shed some light as to know which of the factors may be associated with an increased mortality. our results suggest that comorbidities, rheumatic disease activity and laboratorial abnormalities such as c-reactive protein (crp), d-dimer, lactate dehydrogenase (ldh), ferritin serum elevation significantly associated with mortality whereas previous use of hydroxychloroquine, corticosteroid, dcdmards and biologic therapy did not. laboratory abnormalities like elevated ldh, crp, serum ferritin, d-dimer were found more frequently in critically ill patients over the course of hospitalization, suggesting inflammatory cytokine storm is closely related to severity of covid- . time course of lung changes on chest ct during recovery from the risk of infections with biologic therapies for rheumatoid arthritis a rush to judgment? rapid reporting and dissemination of results and its consequences regarding the use of hydroxychloroquine for covid- covid- : consider cytokine storm syndromes and immunosuppression covid- infection and rheumatoid arthritis: faraway, so close! autoimmun rev clinical characteristics of covid- in new york city presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area ) characteristics associated with hospitalisation for covid- in people with rheumatic disease: data from the covid- global rheumatology alliance physician-reported registry plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars festina lente: hydroxychloroquine, covid- and the role of the rheumatologist no evidence of clinical efficacy of hydroxychloroquine in patients hospitalized for covid- infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial ford hcovid- management task force, early short course corticosteroids in hospitalized patients with covid- clinical features of patients infected with novel coronavirus in wuhan for the infectious diseases society of america publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments the authors acknowledge the assistance of study participant, radiographers, study nurses and laboratory staff who participated in the study. the study was conducted without any financial support. disclosures none. key: cord- -c xm n authors: bakaletz, lauren o. title: developing animal models for polymicrobial diseases date: journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: c xm n polymicrobial diseases involve two or more microorganisms that act synergistically, or in succession, to mediate complex disease processes. although polymicrobial diseases in animals and humans can be caused by similar organisms, these diseases are often also caused by organisms from different kingdoms, genera, species, strains, substrains and even by phenotypic variants of a single species. animal models are often required to understand the mechanisms of pathogenesis, and to develop therapies and prevention regimes. however, reproducing polymicrobial diseases of humans in animal hosts presents significant challenges. there is now compelling evidence that many infectious diseases of humans (fig. ) and animals (table ) are caused by more than one microorganism. the mixed microbial nature of these diseases has been recognized since the early s but there has been renewed interest in this topic since the s , signalled by the publication of four important reviews from to the present date. polymicrobial diseases (see box for nomenclature) can be caused by the synergistic or sequential action of infectious agents from either the same or different kingdoms, genera, species, strains or substrains, or by different phenotypic variants of a single species . polymicrobial diseases share underlying mechanisms of pathogenesis, such as common predisposing factors (box ) , but each disease has unique aspects. although the molecular mechanisms of some polymicrobial infections are known, other polymicrobial diseases are not well understood. owing to their complexity, the study of polymicrobial infections requires a multidisciplinary approach and specific in vitro methodologies and animal models. the development of assay systems and treatment and prevention regimes is needed. multiple diverse in vitro systems have been used to study polymicrobial diseases (box ) . although in vitro methods are crucial for understanding polymicrobial diseases, rigorous, reproducible and relevant animal models of human diseases are essential for the prevention and treatment of these co-infections [ ] [ ] [ ] [ ] .all animal models of human diseases have inherent limitations but they also have important advantages over in vitro methods, including the presence of organized organ systems, an intact immune system and, in inbred mice, specific genetic backgrounds, and the availability of many reagents for characterizing the immune response to sequential or co-infecting microorganisms. the availability of mice with specific genetic backgrounds can have a pivotal role in understanding the mechanisms of pathogenesis of polymicrobial diseases, as exemplified by studies on septic peritonitis - , periodontal disease and lyme arthritis , . understanding the molecular mechanisms underlying polymicrobial diseases of veterinary importance has also been facilitated by the use of animal models. these veterinary systems are useful examples for those researchers attempting to develop animal models of complex human diseases. so far, most animal models for human polymicrobial diseases are rodents, usually mice, but also rats, gerbils, cotton rats and chinchillas. other animal models include non-human primates, which are useful for modelling diseases that are caused by microorganisms with a restricted host range. for most human viral co-infections of clinical importance, good animal models and culture systems are lacking and are urgently required. this review provides an overview of the pathogenesis of selected polymicrobial diseases, the molecular basis for some of these co-infections and describes animal models that have been developed to mimic these diseases. human co-infections with multiple hepatotropic viruses from the hepatitis virus group are well documented. co-infection with multiple hepatitis viruses is possible owing to their similar routes of transmission and ability to chronically infect the host. hepatitis a virus (hav) co-infection of individuals that are chronically infected with hepatitis b virus (hbv) and/or hepatitis c virus (hcv) results in a disease of increased severity and risk of death. moreover, hbv-hcv co-infection occurs in - % of hbv patients, and hepatitis g virus (hgv)-hcv co-infection occurs in - % of individuals with chronic hcv infection; however, hbv-hepatitis d virus (hdv) coinfection occurs only in the setting of co-infecting hbv. viral interference, in which replication of one virus is suppressed by another virus, is an intriguing aspect of triple hbv-hcv-hdv infection -hdv can suppress both hbv and hcv replication . in a retrospective study of patients with hepatitis virus co-infections, hdv was dominant by rt-pcr detection of hdv rna in triple co-infections, but in dual co-infections there were alternating dominant roles for either hbv or hcv. multiple hepatotropic viral infections are associated with reduced hcv replication but increased pathology. patients with dual or triple co-infections have more severe liver disease pathologies than patients that are in cattle, infections with bovine viral diarrhoea virus (bvdv) can be clinically asymptomatic or can cause severe symptoms. the outcome depends on whether the primary infection occurred in utero or after birth and whether the primary infection was with a cytopathic or non-cytopathic biotype of bvdv. milder, congenital persistent infection follows foetal infection with noncytopathic bvdv. death, or culling from the herd within year of birth after failure to thrive, is common; however some persistently infected calves seem healthy at birth and survive for several years. mucosal disease follows congenital persistent infection and is due to coinfection with cytopathic and non-cytopathic bvdv in utero. conversely, acute bovine diarrhoeal disease is induced by primary post-natal infection with either of in some instances production of a virulence factor by one microorganism can increase the risk of infection or colonization by a second microorganism. this might include sharing virulence factors, such as adhesins; for example, h. influenzae shows enhanced adherence when pretreated with bordetella pertussis adhesins. infection with a microorganism that results in an impaired immune system predisposes the affected individual to infection with other microorganisms, or can allow infection of a niche that is usually protected in the body. [ ] [ ] [ ] [ ] [ ] [ ] . brdc pathology results from the effects of pathogen and host virulence factors. m. haemolytica produces multiple virulence factors, including a leukotoxin of the repeat in toxin (rtx) family that activates pmns, induces production of inflammatory cytokines, results in cytoskeletal changes and causes apoptosis. leukotoxin-activated pmns are crucial to pathogenesis and inflammatory mediators released by neutrophils are thought to be essential because inflammation and most of the pathology in brdc is absent human viruses, in contrast with rodent hosts, the use of greater primates for modelling human viral disease is limited by differences in the clinical presentation of disease -some diseases are asymptomatic in primates -and the expense of using primate models in research . given the difficulties of modelling diseases caused by individual viruses, it is not surprising that models of virus co-infections, such as hiv and hcv, have not been established. a variety of small animal and lower-order nonhuman primate model systems have been developed to model human viral co-infections. mice and ferrets have been used to study interference between influenza a virus (iav) strains, as well as interference between cold-adapted influenza a and b vaccine reassortants and wild-type viruses [ ] [ ] [ ] . murine hosts have been used to study how one retrovirus can block infection by a second retrovirus , and to define the role of the tissue tropisms of helper viruses on the disease specificity of a co-infecting oncogene-containing retrovirus such as the type of tumour that is induced . balb/c and nih swiss mice have been used as models to analyse a putative pathogenic interaction between a murine leukaemia virus and a polyomavirus . rabbits have been used to produce models of mixed htlv- and hiv- co-infection and co-infection with htlv types i and ii . rhesus and pig-tailed macaque monkeys have been used to model co-infection with simian immuno deficiency virus (siv) and simian acquired immunodeficiency syndrome retrovirus type (srv- ) . more recently, macaques have been used to define the susceptibility to co-infection with two human hiv- isolates . in this model, co-infections were established in macaques that were simultaneously exposed to both viruses, whereas in macaques that were sequentially challenged, co-infections were only observed if challenge with the second hiv- isolate occurred early after challenge with the first hiv- isolate and before full seroconversion. chimpanzees have also been used to study hiv- subtype b strain co-infections there are several new in vitro methods, including: • genomic sequencing of individual microorganisms and mixed microbial ecosystems and the use of meta-genomics to study the genomes of uncultured microbial communities. • molecular phylogenetic studies, such as genotyping or s rrna analyses, to determine the genetic relatedness or diversity of microbial community members. • genome-wide transcription profiling using microarrays to assess the rates of transcription during polymicrobial infection. • fluorescence-based imaging and detection methods such as laser confocal microscopy using fluorescent probes, fluorescent in situ hybridization (fish) using species-specific s rrna-directed oligonucleotide probes and the use of transcription and translation reporter gene constructs. • analyses of inter-genera bacterial signalling such as quorum sensing. • use of biofilm chambers and continuous culture flow cell reactors to study polymicrobial diseases. • co-infections of cell lines, tissue and organ cultures and extracted teeth. • laser capture microdissection of colonized infected tissues. the mechanisms of synergy between pathogens in om have been analysed using in vitro methods and animal models (reviewed in ref. ). briefly, viral infection compromises the protective functions of the eustachian tube, alters respiratory-tract secretions, damages the mucosal epithelial lining, interferes with antibiotic efficacy, modulates the immune response and enhances bacterial adherence and colonization to predispose the host to bacterial om. influenza and parainfluenza viruses have neuraminidases that remove sialic acids from host-cell glycoproteins, which results in the exposure of receptors for pneumococci. the activity of neuraminidases allows the adherence of and/or colonization by s. pneumoniae , which is one of the primary aetiological agents of acute om. although all upper respiratory tract viruses can disrupt the host respiratory tract defences, each virus has a specific pathology. not surprisingly, there are specific partnerships between viruses and bacteria in om. in the chinchilla model (fig. ) iav predisposes the host middle ear to s. pneumoniae-induced or pneumococcal om and adenovirus infection predisposes the host middle ear to nthi om. iav does not predispose the chinchilla host to nthi-induced om, nor does adenovirus predispose the host to either m. catarrhalis-induced om or to pneumococcal om , .virus and bacteria synergy seems to be maintained in adults and children. the oropharynges of % of adults with experimental iav infection were heavily colonized with s. pneumoniae six days after viral challenge , whereas isolation rates for other middleear pathogens were unaffected. in children, s. pneumoniae is cultured more often from middle-ear fluids that contain iav than from those that are culture-positive for either rsv or parainfluenza virus . cystic fibrosis polymicrobial diseases. upper respiratory viruses predispose the host to bacterial invasion of the lower respiratory tract and are often detected in patients with copd or cystic fibrosis (cf). in addition to bacterial factors, host determinants also have a role in co-infections of the cf lung. cf patients do not have a higher incidence of viral disease compared with non-cf individuals, but viral disease produces more significant pathology. it has been proposed that cf patients have impaired innate immunity, which allows increased virus replication and upregulated cytokine production. in turn, this results in increased bacterial colonization of the lung. zheng and co-workers showed that increased virus replication in cf patients is due to the absence of the antiviral nitric oxide synthesis pathway. this was attributed to impaired activation of signal transducer and activator of transcription (stat ), which is an important component of the antiviral defences of the host. compromising innate immunity provides a mechanism for the severity of viral disease in cf and the establishment of bacterial co-infections. expression of virulence determinants by pseudomonas aeruginosa, a pathogen of cf patients, can depend on signals produced by other bacteria. transcriptional profiling in vitro coupled with research in an animal model showed that adding exogenous signalling molecules, in neutrophil-depleted animals. in pigs, porcine respiratory disease complex (prdc) is a similar disease complex that is caused by co-infection with one of several porcine respiratory tract viruses and members of the pasteurellaceae family [ ] [ ] [ ] [ ] [ ] . porcine reproductive and respiratory syndrome. prrs is caused by prrsv co-infection with multiple bacterial pathogens including streptococcus suis type ii , bordetella bronchiseptica , mycoplasma hyopneumoniae and actinobacillus pleuropneumoniae . in turkeys, pems is caused by turkey coronavirus, avian pneumovirus or newcastle disease virus co-infection with enteropathogenic escherichia coli , . ) . despite the diverse spectrum of diseases and anatomical niches, there are common underlying mechanisms involved in these co-infections. often, viral disruption of host defences has a role in the development of bacterial co-infections. in otitis media, which is a middle ear infection, a synergistic interaction that results in disease owing to co-infection with an upper respiratory tract virus and three bacterial species -streptococcus pneumoniae, nontypeable haemophilus influenzae (nthi) and moraxella catarrhalis -is well documented. however, certain viruses such as respiratory syncytial virus (rsv) and rhinovirus seem to predispose affected individuals more often to bacterial om. the saying that children ''get a cold and a week later develop om'' is substantiated by epidemiological data that indicate a seasonal influence on the coincidence of 'colds' and om, as well as evidence for a peak incidence of virus isolation that is coincident with, or immediately preceding, peak incidence of om (fig. ) . in the recent finnish om cohort study and finnish om vaccine trial, the relationship between viruses and om was supported by data that showed the presence of a virus in either nasopharyngeal aspirates or middle-ear fluid specimens in % or % of om cases in these studies, respectively . rhinovirus was the most commonly isolated virus, followed by enterovirus and rsv. a specific virus was detected in two-thirds of all cases of acute om in young children, but only those viruses that are tested for can be detected, so this figure is likely to underestimate the proportion of acute om events with viral co-infection. a sequential inoculation model has been developed in mice to probe the mechanisms of the interaction between s. pneumoniae and iav. mice infected simultaneously with s. pneumoniae and iav displayed gradual weight loss and increased mortality, commensurate with an additive effect. conversely, mice infected with s. pneumoniae seven days after iav infection uniformly died within hours and had significant bacteraemia -lethality was due to overwhelming pneumococcal septicaemia . this model is being used to define the molecular mechanisms of the lethal synergy of iav with s. pneumoniae . the activity of viral neuraminidase was found to be crucial to this synergistic relationship and, in common with om, has an important role in predisposing both the upper and lower respiratory tracts to invasion by s. pneumoniae. such as autoinducer- , or the production of this molecule by the oropharyngeal bacterial flora upregulated the expression of genes that encode virulence factors . modulation of gene expression by interspecies communication between normal flora and pathogenic bacteria could therefore have a role in polymicrobial diseases. periodontitis. some herpesviruses, including human cytomegalovirus (hcmv), epstein-barr virus type (ebv- ) and hsv, have been implicated in the pathogenesis of a severe and highly aggressive form of periodontitis through co-infection with porphyromonas gingivalis , . hcmv and hsv were detected at significant levels using pcr in periodontal disease and were shown to be good predictors of the presence of p. gingivalis. ebv- was not linked to isolation of p. gingivalis but was also predictive of active disease . a common theme has emerged from these models that upper respiratory tract viruses of both animal and human hosts can predispose the respiratory tract to infection by pasteurellaceae in brdc and prdc in animals and periodontitis, sinusitis, copd and om in humans. a mouse model has been developed to evaluate the role of respiratory dendritic cells (rdcs) in viral-bacterial co-infections . rdc migration from the lungs to the secondary lymph nodes after infection with pulmonary virus is monitored by the use of a fluorescent dye. after inoculation with influenza virus, the rate of rdc migration to the draining peribronchial lymph nodes increased, but this only occurred during the first hours after virus infection. after hours, rdcs did not migrate, despite virus replication and pulmonary inflammation. moreover, viral infection suppressed additional rdc migration in response to either a second pulmonary virus infection or administration of bacterial cpg dna. in addition to suppressed rdc migration, there was also suppression of an antiviral pulmonary cd + t-cell response. it seems likely that the transient suppression of rdc migration and the delayed development of an effective adaptive immune response to a second infection might be another mechanism by which influenza virus predisposes the host to bacterial co-infection. atrophic rhinitis. infection with more than one bacterial species is common in animals and man. in pigs, atrophic rhinitis (ar), which is characterized by severe atrophy of the nasal turbinates, is caused by co-infection with strains of b. bronchiseptica and heat-labile toxin-producing strains of p. multocida [ ] [ ] [ ] . p. multocida can adhere to respiratory tissues, but co-infection with b. bronchiseptica allows more efficient colonization by p. multocida. p. multocida produces a dermonecrotic toxin called pmt (for p. multocida toxin), which interferes with normal bone modelling in both the nasal turbinates and long bones in swine, and is distinct from the b. bronchiseptica dermonecrotic toxin (dnt). in porcine models, pmt causes a more serious form of ar known as progressive ar, whereas b. bronchiseptica infection alone induces a milder, or non-progressive, form of the disease. bacterial co-infections of humans include orofacial infections , adenotonsillitis , persistent osteomyelitis , peritonitis , chronic sinusitis , abscesses , , necrotizing fasciitis and approximately one-third of urinary tract infections (utis) in the elderly and in renal transplant patients . two important bacterial co-infections are periodontitis and vaginosis. periodontitis. periodontal disease causes tooth loss and is associated with systemic vascular diseases such as atherosclerosis and carotid coronary stenotic artery disease . periodontitis in an expectant mother can contribute to both low birth weight and pre-term labour . periodontal disease is initiated by the formation of a a murine model has been developed to reproduce the pathogenesis of human meningococcaemia, which often results in serious symptoms or death . in this model, adult balb/c mice are infected intranasally with a mouse-adapted iav, then seven or ten days later, are co-infected with neisseria meningitidis. fatal meningococcal pneumonia and bacteraemia occurred in mice challenged at seven, but not ten, days after iav infection. meningococcal pneumonia and bacteraemia did not develop in mice that were not co-infected with iav. susceptibility to lethal infections correlated with peak interferon-γ production in the lungs and decreased iav load and production of il- , which indicates that transient iav-induced modulation of host immunity has a role in susceptibility to n. meningitidis co-infection. the only viral-bacterial co-infection model for om is the chinchilla. before , most om studies in the chinchilla used inoculation of pathogens directly into the middle ear. although this induces disease in almost all of the animals inoculated and is therefore extremely useful for studies of therapeutics and surgical intervention strategies, it bypasses all of the early steps in the development of the pathogenesis of the disease, including colonization of the nasopharynx, ascension of the eustachian tube and initiation of infection in the middle ear. giebink and co-workers , developed a clinically relevant model in which chinchillas were challenged intranasally with both s. pneumoniae and iav. this study showed that % of the chinchillas that were infected with iav alone, and % of those inoculated with s. pneumoniae alone, developed om, but of the animals that were coinfected with both microorganisms,~ % developed om (fig. ) . this model has been useful in defining the molecular mechanisms of iav predisposition to pneumococcal om [ ] [ ] [ ] and to investigate the role of pneumococcal virulence determinants in om . to study the pathogenesis of om mediated by nthi, a chinchilla model that uses a co-challenge method was developed. in this model, adenovirus infection can predispose the chinchilla to nthi invasion of the middle ear (fig. ) ; however, iav infection has no effect. this adenovirus-nthi co-infection model has been used to study the mechanisms of adenovirus predisposition to nthi-induced om , , to identify new nthi virulence determinants and to assay the relative efficacies of different nthi-derived vaccine candidates for om [ ] [ ] [ ] . a cotton rat model of rsv and nthi co-infection has also been developed to study co-infections of the respiratory tract . in the cotton rat, colonization of the respiratory tract with nthi increased to a maximum level four days after infection with rsv and colonization was increased compared with rats that had not been infected with rsv. nthi colonization of the respiratory tract was increased by rsv co-infection and, although the mechanisms underlying this relationship are not understood, this model might be useful to determine the mechanisms of rsv predisposition to bacterial om. bacterial formed, in terms of microbial constituents, is the main predictor for periodontal disease. actinobacillus actinomycetemcomitans, p. gingivalis and bacteroides forsythus are the main periodontal pathogens. periodontal disease covers a range of clinical symptoms and there are multiple forms of periodontitis in children and adults. in individuals under years of age, a. actinomycetemcomitans is the main bacterial pathogen, whereas in adults aged years or older, periodontitis has been linked to p. gingivalis and b. forsythus. spirochaetes, especially treponema denticola, have been implicated in periodontitis despite the fact that most oral spirochaetes have not been successfully cultured. recent studies using molecular phylogenetic techniques have implicated new bacterial biofilm on the tooth surface followed by bacterial invasion of gingival tissues . teeth have a non-shedding surface and are located in a warm, moist environment, so are a particularly suitable niche for biofilm formation by the oral microbial flora. in periodontitis, coaggregation -a process in which genetically distinct bacteria become interconnected by specific adhesins -is central to the formation of complex multispecies biofilms (fig. ) . in dental plaque, primary colonizers such as streptococcus gordonii, and other oral streptococci that express adhesins, provide a film on which other bacterial colonizers assemble the biofilm. it was thought that the abundance of plaque that formed was responsible for the induction of periodontitis but, at present, the favoured hypothesis is that the quality of the plaque periodontal disease. in most individuals, periodontal pathogens trigger an inflammatory response that effectively prevents microbial colonization and invasion of adjacent gingival tissues. however, individuals that have specific il- polymorphisms that result in increased levels of il- expression are predisposed to periodontal disease. using this criterion, a mouse model of polymicrobial-induced osteoclastogenesis, bacterial penetration, leukocyte recruitment and softtissue necrosis has been developed to clarify the role of cytokines in periodontal disease. in this model, the dental pulp of the first mandibular molars is exposed by surgically clipping the mesial cusps and then a mixture of putative oral pathogens is inoculated into the dental pulp (fig. a) . by monitoring the size of osseous lesions, tissue necrosis, osteoclastogenesis, osteoclastic activity, inflammatory cell recruitment and bacterial penetration into tissue periodontal disease, pathogenic mechanisms can be investigated . il- or tnf receptor signalling does not seem to be required for bacteria-induced osteoclastogenesis and bone loss in this model, but does have a crucial role in protecting the host against anaerobic co-infections. a rat model of periodontitis was developed to test adherent (rough) and non-adherent (smooth) variants of a. actinomycetemcomitans for virulence, as well as to assess phenotypic reversion in vivo . in this model, the normal flora of the oral cavity of sprague-dawley rats is reduced by antibiotic treatment, after which rats are inoculated with a. actinomycetemcomitans by either normal ingestion of food layered with bacterial cultures, oral swabbing or gastric lavage (fig. b) . when clinical isolates of a. actinomycetemcomitans were compared with laboratory-adapted variants, fine et al. found that the clinical strains were more efficient at colonization and persisted longer in the rat oral cavity than laboratory strains. rough variants were more efficient colonizers of the rat oral cavity than smooth variants, regardless of the method of inoculation, although feeding was the preferred method owing to the similarity with human disease. importantly, rats that were orally infected with a. actinomycetemcomitans by feeding developed immunoglobulin g (igg) antibodies to the bacteria and had bone loss that was typical of periodontitis. this model has not been used to study the process of bacterial co-infection in periodontitis, but has been used to identify a gene locus that is important in virulence and which mediates tight adherence by a. actinomycetemcomitans . a primate model (macaca fascicularis) of periodontal disease uses silk ligatures tied around the posterior teeth to induce plaque accumulation and the initiation of periodontitis . so far, this model has only been used for single pathogen studies, but is considered to be a relevant animal model of periodontal disease owing to the similarity of clinical and histological features with those of periodontal disease of humans, and because, in this model, periodontal destruction is clearly triggered by bacterial infection . species or phylotypes -including members of the uncultivated bacterial division tm -in periodontitis, dental caries and halitosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in many of these studies, not only were new species and phylotypes identified, but bacteria that are known to be oral pathogens were found to be numerically minor, which was expected because ~ % of oral flora have not been cultivated . in localized juvenile periodontitis (ljp), the leukotoxin of a. actinomycetemcomitans, like that of m. haemolytica, is the best-studied virulence factor. this leukotoxin selectively kills pmns and macrophages in vitro, and pmns and macrophages are important components of the host defence in vivo. expression of this leukotoxin is variable among a. actinomycetemcomitans isolates and the leukotoxin-expression phenotype correlates with differences in the promoter region of the leukotoxin gene operon . a subset of leukotoxin-overproducing strains are more virulent and are associated with ljp in humans. neutrophil abnormalities seem to be an important predisposing condition for periodontal disease. in addition, loss of tooth attachment and bone resorption, which are important events in periodontal disease, occur together with increased il- and tumour-necrosis factor (tnf) activities. the production of il- and tnf (both of which are pro-inflammatory cytokines) has been correlated with the spread of inflammatory cells to connective tissues, the loss of connective tissue attachment, osteoclast formation and the loss of alveolar bone. an overzealous host response to periodontal pathogens, resulting in excessive production of il- and tnf, is hypothesized to be responsible for much of the damage that occurs in periodontal disease . ial co-infection. the mucosal environment of the vagina is influenced by developmental and hormonal changes . the most common bacterial constituents of the vaginal microflora are lactobacilli, including lactobacillus crispatus and lactobacillus jensenii . when these hydrogen peroxide (h o )-producing lactobacilli are outcompeted by anaerobic and facultatively anaerobic members of the vaginal flora, bv develops with a concomitant rise in vaginal ph, which further promotes the growth of the resident lactobacilli. bv is common, occurring in - % of the global female population and the role of lactobacilli in the maintenance of vaginal homeostasis has been well studied. women with stable bacterial colonization have a reduced risk of developing bv . normal vaginal flora has a role in defence against the acquisition of other pathogenic microorganisms, including those that are responsible for sexually transmissible diseases (stds), and bv is a strong predictor of std acquisition . compared with subjects with normal vaginal flora, subjects that have bv are more likely to test positive for neisseria gonorrhoea and chlamydia trachomatis. recently, bv has also been found to be associated with an increased risk of hsv- infection . candida species. co-infection with multiple candida strains and substrains are also found. regardless of the co-pathogens, mycotic co-infections of the oral and vaginal cavities, on indwelling prosthetic devices, or systemic infection of the blood can present significant therapeutic challenges. difficulty in treating some of these infections is partly attributed to the formation of biofilms by candida spp. biofilm formation on devices such as prosthetic heart valves and catheters has been studied in vitro . when cultured on a variety of catheter materials, candida spp. form biofilms comprising a matrix of microcolonies of both the yeast and the filamentous hyphal forms. in studies of mixed microbial populations, candida spp. form biofilms with several bacterial species, including staphylococcus epidermidis and oral streptococcal species. the receptor for candida albicans co-aggregation with s. gordonii is a complex cell surface polysaccharide that is expressed on the surface of the bacterium. the interaction between yeast cells and oral streptococci or other bacteria has important implications for the mechanisms of yeast infections of the oral cavity, in addition to promoting biofilm formation on a variety of surfaces. in the oral cavity, candida-bacterial interactions are responsible for denture stomatitis, angular cheilitis and gingivitis, and also have a role in periodontitis . although the role of pathogenic enterococci and their role in peritonitis is not understood, many putative virulence factors have been identified using animal models. available animal models include systemic infection in mice and compartmentalized infection in rats, and the bacterial virulence factors that have been identified using each model differ . this indicates that both host and pathogen factors contribute to peritonitis and, perhaps, that the animal models are quite different. nevertheless, these models have identified a role for cytokines in septic shock, a protective role for il- against lethal shock , a role for stat in the mortality seen in bacterial co-infection sepsis and helped to define the role of the classical pathway of complement activation in defence against polymicrobial peritonitis . animal models of bacterial co-infection peritonitis and/or sepsis can involve any of the following methods for induction of infection: peritoneal implantation of microbe-filled gelatin capsules , ; intraperitoneal injection of faecal suspensions or caecal ligation and puncture , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . candida and mixed infections. as defined by soll and his colleagues bacterial suspensions that are being tested for the ability to cause periodontal disease are injected into the dental pulp and the mouse is monitored for signs of periodontal disease by methods that include examination of osseous lesions, tissue necrosis, inflammatory cell recruitment, bacterial tissue penetration and osteoclastogenesis. b | in the rat model bacteria are grown using standard laboratory procedures and washed times with phosphate-buffered saline (pbs) supplemented with % sucrose. the rats are pretreated with antibiotics and the mouth is swabbed with chlorhexidine to deplete the oral flora. the bacterial suspension is mixed into the rat's food so that this animal model replicates, as far as possible, the natural route of infection for periodontal disease. after daily inoculation the rats are assessed for bacterial colonization and bone loss. using this model, different strains of bacteria and the contribution of different virulence loci can be tested. cfu, colony-forming units. of these genetic manipulations . a murine host has been used as a model of systemic candidiasis and there is also a murine model for c. glabrata-induced vaginitis . in the c. glabrata model, the increased susceptibility of non-obese diabetic mice to c. glabratainduced vaginitis compared with their non-diabetic counterparts indicates a link between susceptibility to diabetes and infection with c. glabrata. in addition to studies of candida genetics and pathogenicity , this model is useful for the evaluation of the relative efficacy of antimycotic agents and probiotics for the prevention of vaginitis. an animal model of haematogenously disseminated candidiasis has recently been developed that can investigate the role of phenotype switching in candidiasis. in this model (fig. ) mice were injected with engineered c. albicans strains in which the transition between yeast and filamentous forms is under the control of a doxycycline-regulated promoter. mice that were infected with strains that switched to the filamentous form died, whereas those infected with strains that could not switch from the yeast to the filamentous form survived, despite the fact that the fungal burdens in both groups were nearly identical. these data indicate that the filamentous form is important for mortality but that the yeast form of c. albicans is important for dissemination to deeper tissues. parasite-parasite co-infections. several human diseases have mixed parasitic aetiologies, including co-infections with plasmodium spp. and nematodes. in some studies, co-infection with a helminth seemed to confer protection against severe complications of malaria , but this is not always the case. when infected controls with a low helminth burden were compared with those with circulating helminth schizonts, co-infection with ascaris lumbricoides was found to be associated with protection from cerebral malaria. in addition, a later study showed a significant association between ascaris infection and the risk of co-infection with plasmodium falciparum and plasmodium vivax, indicating that pre-existing ascaris infection might increase host tolerance to coexisting plasmodium spp. subsequently, helminthinfected patients were found to be more likely to develop falciparum malaria compared with those that were not co-infected . moreover, the risk of developing falciparum malaria increased with the number of coinfecting helminth species. collectively, these findings indicate that a helminth-mediated helper t cell (t h ) shift (an immune response that is biased towards that which is characteristic of a t h -mediated response) might have a complex impact on malaria co-infection -decreasing antisporozoite immunity but inducing a protective outcome against severe complications of malaria. although the underlying mechanism is less clear, mwatha et al. showed that exposure of schistosoma mansoni-infected children to p. falciparum had a significant influence on the severity of hepatosplenomegaly (enlargement of the liver and spleen) that was observed in co-infected children. a new category of polymicrobial diseases has been proposed for candida spp. in which the infection is due to phenotypic heterogeneity . in addition to the hypha-bud transition, c. albicans has a reversible, high-frequency phenotype switch that can be identified by differences in colony morphology. c. albicans cells of two phenotypic phases have different virulence characteristics. the ability of this human pathogen to rapidly switch between phenotypes could be a higher-order pathogenic trait. support for this hypothesis comes from studies in which strains that cause deep tissue mycoses were shown to switch at higher frequencies than those that cause superficial infections. furthermore, pathogenic c. albicans strains that were isolated from the oral cavity switch at higher rates than commensal strains that were isolated from the same site. a clinically relevant example of the role of both phenotype and mating-type switching in disease was characterized by brockert et al. , who investigated oral cavity and vaginal isolates of c. glabrata in three patients with vaginitis. the results of this study showed that switching occurs at sites of infection, that different switch phenotypes of the same strain can dominate in different anatomical locations in the same host and that mating-type switching occurs in vivo. these co-infections can cause disease in the lower respiratory tract. in cf patients, clinical specimens that also harbour c. albicans contain nine times the amount of p. aeruginosa compared with patients that do not harbour c. albicans . moreover, sputum samples of - % of cf patients contain c. albicans in addition to p. aeruginosa. hogan and kolter showed that p. aeruginosa forms a dense biofilm on c. albicans filaments in vitro and, in doing so, kills the fungus. p. aeruginosa fails to bind to, or kill, the yeast form of c. albicans. it is unclear if a similar relationship between these two pathogens functions in vivo but, as several p. aeruginosa virulence factors that are important in human disease are also involved in killing the fungal filaments, this co-culture system could prove useful for the study of the pathogenesis of p. aeruginosa-induced disease. owing to the ability of candida spp. to switch between bud and hypha (or hypha-like) forms as well as to switch phenotype, all animal models of candida infection are likely to represent one or another of the multiple polymicrobial states that have been proposed for this microorganism. a rat model of oral colonization has been used to compare the relative pathogenicity of different candida strains as well as to determine the effect of chemotherapeutic immunosuppression on the ability of candida spp. to switch from a commensal to an invasive phenotype . a rat model of oral candidiasis has also been developed and used to assay isogenic derivatives of a virulent c. albicans strain for the biological consequences co-infection with schistosoma species and plasmodium species has been modelled in field voles and mice since (ref. ) , with conflicting observations concerning the ability of one parasite to suppress the capacity of the other to infect the host , . results obtained seem to depend on the plasmodium species used as well as the immune status of the host. s. mansoni is, however, a potent inducer of a t h dominant response, not only to itself but also to other bystander antigens that are present in a host, so it does have an influence on the clinical outcome in these co-infections , . synergistic interactions between specific protozoans and helminths are often ascribed to the immunosuppression that is characteristic of protozoan infections and which is observed in the mouse, which is the main model for these infections a mouse model has been used to model the arthritis and carditis that can occur in co-infections with b. microti and b. burgdorferi . co-infection resulted in a significant increase in symptoms of arthritis. this increase was correlated with a reduction in concentrations of the cytokines il- and il- . a mouse model for tick-borne lyme arthritis mediated by co-infection with b. burgdorferi and a causative agent of human granulocytic ehrlichiosis (hge) has been developed , (fig. ) . co-infection results in increased titres of both pathogens and more severe arthritis than does infection with b. burgdorferi alone. co-infection resulted in reduced concentrations of il- , ifn-γ and tnf-α and increased concentrations of il- . ifn-γ expression in macrophages was suppressed, which might indicate a reduction in phagocytic activity in co-infection. these models will allow us to define the modulation of host immune responsiveness that occurs in those individuals that are simultaneously or sequentially infected with multiple tick-borne pathogens . co-infections can arise as a result of the virus-induced immunosuppression that is characteristic of a subset of human viral pathogens, the best characterized of which is hiv. schistosomiasis is a chronic helminth infection that is caused by s.mansoni. in hcv and s. mansoni co-infection, there is a higher incidence of viral persistence and accelerated damage to the liver than when the patient is infected with either infectious agent alone. in a recent study, stimulation of cd + t cells with hcv antigens produced a type cytokine profile in patients infected with hcv, whereas in patients that were co-infected with hcv and s. mansoni, a type cytokine predominance was evident despite the fact that t cells that were recovered from both patient populations responded in the same manner to stimulation with schistosomal antigens . the helminth-induced inability to generate an hcv-specific cd + /t h t-cell response has been shown to have a role in the persistence and severity of hcv infection, which indicates that the induction of a strong cellular immune response through new therapeutic approaches might limit subsequent liver damage in those individuals with chronic hcv infection . parasite-bacteria co-infections. one example of coinfection with a parasite and bacterium in humans is that of borrelia burgdorferi (the causative agent of lyme disease) and the intra-erythrocytic parasite babesia microti. both of these pathogens are transmitted by the tick vector ixodes scapularis. co-infection can occur by a bite from a single tick carrying multiple pathogens, or from multiple tick bites. the first cases of lyme borreliosis and babesiosis co-infection were reported in the mid s, with parasite-bacteria co-infection rates of up to % among those with confirmed tick-borne infection in certain populations. although ticks can also harbour the human pathogen anaplasma phagocytophilum, lyme borreliosis and babesiosis coinfection accounts for ~ % of polymicrobial disease in the eastern united states. consistent with the theme for other co-infections involving a parasite, patients that harbour both of these pathogens had more severe and longer-lasting symptoms than those with lyme borreliosis alone (nug) and is characterized by a surface biofilm of mixed microbial flora overlying a subsurface flora comprising dense aggregates of spirochaetes. in contrast to nug, high levels of yeast and herpes-like viruses were observed using transmission electron microscopy examination of tissues recovered from the former patient group. herpes-like particles were observed in . % of biopsies obtained from hiv-infected patients with nup. these findings correlate well with those of contreras and co-workers , in which co-infection with herpesvirus was associated with high levels of periodontopathic bacteria. the role of viruses in the pathogenesis of nup or periodontitis is not known but, in addition to inducing immunosuppression, it has been suggested that viruses might promote the overgrowth of bacterial pathogens and/or induce the release of tissuedestroying cytokines by host cells . , . in addition to systemic diseases, localized infections with candida spp., such as thrush in the oral cavity, are common co-infections in hiv-infected individuals . the commensal oral flora acquires an invasive phenotype in the hiv-infected host, and c. albicans is indicative of a defect in host t-cell immunity in hiv infection . oropharyngeal candidiasis develops iñ - % of hiv-infected patients and often precedes the development of a more invasive candida infection, oesophageal candidiasis. the progressive immunosuppression that is characteristic of hiv infection provides a mechanism for the development of oesophageal candidiasis, which is a reportable aidsdefining opportunistic illness. another disease of the oral cavity in hiv-seropositive patients is necrotizing ulcerative periodontitis (nup), which is a disease that is characterized by ulcerated gingival papillae figure | animal model for lyme disease and human granulocytic ehrlichiosis (hge) co-infection. these diseases share a tick vector, ixodes scapularis, and to analyse whether ehrlichia sp. and borrelia burgorferi (the causative agents of hge and lyme disease, respectively) co-infection leads to increased severity of spirochaete-induced lyme arthritis a mouse model has been developed. mice are infected intradermally with either spirochaetes (b. burgdorferi cultured in vitro) or hge (blood culture from a scid mouse, see inset panel) . arthritis and presence of the two pathogens can then be determined through histopathology, pcr to detect bacterial dna and by assessing immune responses. ticks were allowed to feed on all groups of mice to assess transmission of the pathogens. after feeding, pcr (hge) and immunofluorescence (b. burgdorferi) were used for pathogen detection. and not due to either increased apoptosis or altered distribution of these cells between the spleen, blood or lymphatics. the ability of measles virus to suppress both innate and adaptive immune responses is thought to be responsible for the increased susceptibility to bacterial co-infection. the future of polymicrobial disease research molecular methods are now being used together with conventional culture techniques to determine the identity of the full complement of microorganisms that are involved in co-infections and to determine the interactions between these microorganisms. as a result, additional diseases of polymicrobial origin will be identified. this will necessitate the development of new animal models and new in vitro methods for the study of polymicrobial diseases. uncovering the molecular mechanisms that are involved in the pathogenesis of complex diseases might show that changes in lifestyle, such as smoking cessation or dietary changes, could prevent co-infections. developing methods to disrupt biofilms are one target for researchers. new antimicrobials and vaccine candidates for both the predisposing and the co-infecting microorganisms will be sought. therapeutic approaches for polymicrobial diseases might include the use of probiotics for the treatment or prevention of vaginal infections, gastroenteritis, inflammatory bowel disease, utis and periodontitis. moreover, advances in nanotechnology and biomedical engineering will allow the development of new ways to deliver these therapeutic or preventative agents in a disease-or site-specific manner such as the design and use of 'intelligent implants' . these indwelling devices might be embedded with sensors to detect the biofilm-forming microorganisms and signal the release of antimicrobial agents stored in an internal reservoir. as the organizers of the first satellite conference on diseases of mixed microbial aetiology (see the online links box) stated -polymicrobial diseases are ''a concept whose time has come'' . virus cannot present antigen to t cells and have a diminished capacity to secrete ig or proliferate. susceptibility to bacterial co-infection is likely owing to these underlying immune defects that result in the hallmark of measles virus infection -inhibition of the proliferation of cd + and cd + t cells [ ] [ ] [ ] [ ] . a primate model of hiv-induced immunosuppression that developes cutaneous leishmaniasis has been developed in rhesus macaques. in this model, macaques are chronically infected with siv, and then co-infected with leishmania major metacyclic promastigotes by intradermal injection. lesion size, parasite load and siv viraemia are measured weekly. this model has been used to assay both the synergistic relationship between these two pathogens and the responsiveness to, and relative protective efficacy of, cpg oligodeoxynucleotides delivered to co-and mono-infected macaques . recently, a rhesus monkey model for siv predisposition to mycobacterium leprae co-infection has been developed, which showed that co-infection increases the susceptibility to leprosy regardless of the timing between the two infections . as mentioned earlier, measles virus-induced immunosuppression often leads to bacterial co-infection. to understand the mechanisms of co-infection, a murine model of combined measles virus and listeria monocytogenes infection was developed . in this model system, transgenic mice expressing the human measles virus receptor cd are co-infected with measles virus and l. monocytogenes, or are challenged with the bacterial pathogen alone. mice co-infected with measles virus were more susceptible to infection with l. monocytogenes and this susceptibility corresponded with a reduction in the macrophage and pmn populations in the spleen, as well as a reduction in ifn-γ production by cd + t cells. a reduction in cd b + macrophages and ifn-γ producing t cells was found to be due to reduced proliferative expansion introduces and provides a concise overview of polymicrobial diseases the role of microbial interactions in infectious disease mechanisms of bacterial superinfections in viral pneumonias respiratory viral infection predisposing for bacterial disease: a concise review molecular pathogenesis of pneumococcal pneumonia polymicrobial diseases polymicrobial diseases regulation of gene expression by cell-to-cell communication: acylhomoserine lactone quorum sensing bacterial biofilms: an emerging link to disease pathogenesis endotoxemia and polymicrobial septic peritonitis resistance to acute septic peritonitis in poly(adp-ribose) polymerase- -deficient mice interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury cd contributes to lethality in acute sepsis: in vivo role for cd in innate immunity role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis mice lacking monocyte chemoattractant protein have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment provided the background for the development of an animal model of parasitic co-infection, as well as demonstrating the potential importance of mouse strain -genetic background -in disease outcome coinfection with borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses il- and ifn-γ production and promotes an il- response in c h/hej mice dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study pathogenesis and clinical aspects of a respiratory porcine reproductive and respiratory syndrome virus infection dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus induction of dual infections in newborn and three-week-old pigs by use of two plaque size variants of porcine reproductive and respiratory syndrome virus experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus pathogenesis of postweaning multisystemic wasting syndrome reproduced by co-infection with korean isolates of porcine circovirus and porcine parvovirus replication status and histological features of patients with triple (b, c, d) and dual (b, c) hepatic infections polymicrobial diseases hepatitis c virus (hcv) and human immunodeficiency virus type (hiv- ) infections in alcoholics hiv/hcv co-infection: putting the pieces of the puzzle together polymicrobial diseases kaposi's sarcoma-associated herpesvirus (kshv/hhv ): key aspects of epidemiology and pathogenesis kaposi's sarcoma-associated herpesvirus (kshv)/human herpesvirus (hhv- ) as a tumour virus hiv and herpes co-infection, an unfortunate partnership epidemiology of herpes and hiv co-infection the interaction between herpes simplex virus and human immunodeficiency virus the role of sexually transmitted diseases in hiv transmission g protein-coupled receptors in hiv and siv entry: new perspectives on lentivirus-host interactions and on the utility of animal models interference between cold-adapted (ca) influenza a and b vaccine reassortants or between ca reassortants and wild-type strains in eggs and mice interference by a non-defective variant of influenza a virus is due to enhanced rna synthesis and assembly interference following dual inoculation with influenza a (h n ) and (h n ) viruses in ferrets and volunteers post-entry restriction of retroviral infections the helper virus envelope glycoprotein affects the disease specificity of a recombinant murine leukemia virus carrying a v-myc oncogene a model for mixed virus disease: coinfection with moloney murine leukemia virus potentiates runting induced by polyomavirus (a strain) in balb/c and nih swiss mice evidence for dual infection of rabbits with the human retroviruses htlv-i and hiv- dual infection of rabbits with human t cell lymphotropic virus types i and ii infection of macaque monkeys with simian immunodeficiency virus from african green monkeys: virulence and activation of latent infection identification of a window period for susceptibility to dual infection with two distinct human immunodeficiency virus type isolates in a macaca nemestrina (pig-tailed macaque) model one of the few examples of an attempt to model a human viral co-infection in which viral host restriction presents significant challenges extensive diversification of human immunodeficiency virus type subtype b strains during dual infection of a chimpanzee that progressed to aids interference between non-a, non-b and hepatitis b virus infection in chimpanzees hepatitis δ-virus cdna sequence from an acutely hbv-infected chimpanzee: sequence conservation in experimental animals molecular genetic analysis of virulence in mannheimia (pasteurella) haemolytica coinfection with bhv- modulates cell adhesion and invasion by p. multocida and mannheimia (pasteurella) haemolytica smoke and viral infection cause cilia loss detectable by bronchoalveolar lavage cytology and dynein elisa ultrastructural features of lesions in bronchiolar epithelium in induced respiratory syncytial virus pneumonia of calves experimental infection of lambs with bovine respiratory syncytial virus and pasteurella haemolytica: immunofluorescent and electron microscopic studies pathological study of experimentally induced bovine respiratory syncytial viral infection in lambs role of α/β interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking ns proteins nonstructural proteins ns and ns of bovine respiratory syncytial virus block activation of interferon regulatory factor bovine viral diarrhea virus isolated from fetal calf serum enhances pathogenicity of attenuated transmissible gastroenteritis virus in neonatal pigs effects of intranasal inoculation with bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with pasteurella multocida in pigs experimental reproduction of severe disease in cd/cd pigs concurrently infected with type porcine circovirus and porcine reproductive and respiratory syndrome virus association of porcine circovirus with porcine respiratory disease complex in utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, bordetella bronchiseptica, or a combination of both organisms in pigs differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract high mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic escherichia coli and turkey coronavirus experimental infection of turkeys with avian pneumovirus and either newcastle disease virus or escherichia coli invasive group a streptococcal disease in children and association with varicella-zoster virus infection. ontario group a streptococcal study group risk factors for invasive group a streptococcal infections in children with varicella: a casecontrol study invasive group a streptococcal infections in children with varicella in southern california presence of specific viruses in the middle ear fluids and respiratory secretions of young children with acute otitis media adenovirus infection enhances in vitro adherence of streptococcus pneumoniae effect of adenovirus type and influenza a virus on streptococcus pneumoniae nasopharyngeal colonization and otitis media in the chinchilla reviews the evidence in support of the crucial role of a viral virulence factor in predisposing both the upper and lower respiratory tract to bacterial secondary infections adenovirus serotype does not act synergistically with moraxella (branhamella) catarrhalis to induce otitis media in the chinchilla comparison of alteration of cell surface carbohydrates of the chinchilla tubotympanum and colonial opacity phenotype of streptococcus pneumoniae during experimental pneumococcal otitis media with or without an antecedent influenza a virus infection effect of experimental influenza a virus infection on isolation of streptococcus pneumoniae and other aerobic bacteria from the oropharynges of allergic and nonallergic adult subjects prevalence of various respiratory viruses in the middle ear during acute otitis media infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable haemophilus influenzae impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis modulation of pseudomonas aeruginosa gene expression by host microflora through interspecies communication the herpesvirus-porphyromonas gingivalis-periodontitis axis herpesviral-bacterial interactions in aggressive periodontitis a mouse model of dual infection with influenza virus and streptococcus pneumoniae lethal synergism between influenza virus and streptococcus pneumoniae: characterization of a mouse model and the role of plateletactivating factor receptor role of neuraminidase in lethal synergism between influenza virus and streptococcus pneumoniae a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice the chinchilla superinfection model developed in this study was the first animal model to demonstrate conclusively the important role of the upper respiratory tract viruses eustachian tube histopathology during experimental influenza a virus infection in the chinchilla different virulence of influenza a virus strains and susceptibility to pneumococcal otitis media in chinchillas polymorphonuclear leukocyte dysfunction during influenza virus infection in chinchillas synergistic effect of adenovirus type and nontypeable haemophilus influenzae in a chinchilla model of experimental otitis media evidence for transudation of specific antibody into the middle ears of parenterally immunized chinchillas after an upper respiratory tract infection with adenovirus kinetics of the ascension of nthi from the nasopharynx to the middle ear coincident with adenovirus-induced compromise in the chinchilla nontypeable haemophilus influenzae gene expression induced in vivo in a chinchilla model of otitis media protection against development of otitis media induced by nontypeable haemophilus influenzae by both active and passive immunization in a chinchilla model of virus-bacterium superinfection passive transfer of antiserum specific for immunogens derived from a nontypeable haemophilus influenzae adhesin and lipoprotein d prevents otitis media after heterologous challenge relative immunogenicity and efficacy of two synthetic chimeric peptides of fimbrin as vaccinogens against nasopharyngeal colonization by nontypeable haemophilus influenzae in the chinchilla effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable haemophilus influenzae: implications for otitis media accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection evaluation of vaccines for atrophic rhinitis -a comparison of three challenge models the pathological effect of the bordetella dermonecrotic toxin in mice immunopathological changes in mice caused by bordetella bronchiseptica and pasteurella multocida the virulence of mixed infection with streptococcus constellatus and fusobacterium nucleatum in a murine orofacial infection model bacteriology of adenoids and tonsils in children with recurrent adenotonsillitis bacterial biofilms: a common cause of persistent infections an excellent review of the role of polymicrobial biofilms in diverse anatomical niches that are involved in persistent and chronic human diseases handbook of animal models of infection bacteriologic findings in patients with chronic sinusitis microbiology of polymicrobial abscesses and implications for therapy synergistic effect of bacteroides, clostridium, fusobacterium, anaerobic cocci, and aerobic bacteria on mortality and induction of subcutaneous abscesses in mice the etiology of urinary tract infection: traditional and emerging pathogens culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients involvement of periodontopathic biofilm in vascular diseases periodontal disease: bacterial virulence factors, host response and impact on systemic health bacterial coaggregation: an integral process in the development of multi-species biofilms an overview of the molecular mechanisms by which bacteria interact with one another to construct complex multispecies biofilms phylogeny of porphyromonas gingivalis by ribosomal intergenic spacer region analysis association of bacteroides forsythus and a novel bacteroides phylotype with periodontitis diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients prevalence of bacteria of division tm in human subgingival plaque and their association with disease single-cell enumeration of an uncultivated tm subgroup in the human subgingival crevice genetic relatedness and phenotypic characteristics of treponema associated with human periodontal tissues and ruminant foot disease new bacterial species associated with chronic periodontitis beyond the specific plaque hypothesis: are highly leukotoxic strains of actinobacillus actinomycetemcomitans a paradigm for periodontal pathogenesis? the contribution of interleukin- and tumor necrosis factor to periodontal tissue destruction the identification of vaginal lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections bacterial vaginosis is a strong predictor of neisseria gonorrhoeae and chlamydia trachomatis infection association between acquisition of herpes simplex virus type in women and bacterial vaginosis interleukin- and tumor necrosis factor receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss but is essential for protecting the host from a mixed anaerobic infection provided the background for the development of an animal model of the relative bacterial colonization and persistence by single phenotypic variants of a bacterium tight-adherence genes of actinobacillus actinomycetemcomitans are required for virulence in a rat model inflammation and tissue loss caused by periodontal pathogens is reduced by interleukin- antagonists non-human primates used in studies of periodontal disease pathogenesis: a review of the literature exemplifies the potential for different animal models to provide disparate data and shows that there can be species and model dependency to our ability to define and characterize microbial virulence determinants in polymicrobial diseases origin microbiological and inflammatory effects of murine recombinant interleukin- in two models of polymicrobial peritonitis in rats stat is required for antibacterial defense but enhances mortality during polymicrobial sepsis comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture early activation of pulmonary nuclear factor κb and nuclear factor interleukin- in polymicrobial sepsis granulocyte colony-stimulating factor and antibiotics in the prophylaxis of a murine model of polymicrobial peritonitis and sepsis antibiotics delay but do not prevent bacteremia and lung injury in murine sepsis mortality in murine peritonitis correlates with increased escherichia coli adherence to the intestinal mucosa polymicrobial sepsis induces organ changes due to granulocyte adhesion in a murine two hit model of trauma modulation of the phosphoinositide -kinase pathway alters innate resistance to polymicrobial sepsis the importance of systemic cytokines in the pathogenesis of polymicrobial sepsis and dehydroepiandrosterone treatment in a rodent model the activity of tissue factor pathway inhibitor in experimental models of superantigen-induced shock and polymicrobial intra-abdominal sepsis candida biofilms and their role in infection adherence of candida albicans to a cell surface polysaccharide receptor on streptococcus gordonii reviews the pathogenic potential and phenotypic variability of c. albicans and illustrates that the coinfecting microorganisms can be phenotypic variants of a single microbial strain phenotypic switching and mating type switching of candida glabrata at sites of colonization asm conference on polymicrobial diseases abstract pseudomonas-candida interactions: an ecological role for virulence factors the relative pathogenicity of candida krusei and c. albicans in the rat oral mucosa avirulence of candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis in vivo pathogenicity of eight medically relevant candida species in an animal model a murine model of candida glabrata vaginitis the protective immune response against vaginal candidiasis: lessons learned from clinical studies and animal models engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of candida albicans during infection provided the background for the development of a murine model of fungal co-infection that allowed the demonstration of the importance of the ability of candida albicans to switch morphology from a yeast to a filamentous form in pathogenesis ascaris lumbricoides infection is associated with protection from cerebral malaria contemporaneous and successive mixed plasmodium falciparum and plasmodium vivax infections are associated with ascaris lumbricoides: an immunomodulating effect? intestinal helminth infections are associated with increased incidence of plasmodium falciparum malaria in thailand specific cellular immune response and cytokine patterns in patients coinfected with hepatitis c virus and schistosoma mansoni kinetics of intrahepatic hepatitis c virus (hcv)-specific cd + t cell responses in hcv and schistosoma mansoni coinfection: relation to progression of liver fibrosis associations between anti-schistosoma mansoni and anti-plasmodium falciparum antibody responses and hepatosplenomegaly, in kenyan schoolchildren epidemiology and impact of coinfections acquired from ixodes ticks. vector borne zoonotic dis coinfection in patients with lyme disease: how big a risk? some aspects of concomitant infections of plasmodia and schistosomes. i. the effect of schistosoma mansoni on the course of infection of plasmodium berghei in the field vole (microtus guentheri) infection of mice concurrently with schistosoma mansoni and rodent malarias: contrasting effects of patent s. mansoni infections on plasmodium chabaudi, p. yoelii and p. berghei suppression of schistosome granuloma formation by malaria in mice altered immune responses in mice with concomitant schistosoma mansoni and plasmodium chabaudi infections schistosoma mansoni infection cancels the susceptibility to plasmodium chabaudi through induction of type immune responses in a/j mice heterologous synergistic interactions in concurrent experimental infection in the mouse with schistosoma mansoni, echinostoma revolutum, plasmodium yoelii, babesia microti, and trypanosoma brucei coinfection with borrelia burgdorferi and the agent of human granulocytic ehrlichiosis alters murine immune responses, pathogen burden, and severity of lyme arthritis coinfection with borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses il- and ifn-γ production and promotes an il- response in c h/hej mice tuberculosis in hiv-infected patients: a comprehensive review a tem/sem study of the microbial plaque overlying the necrotic gingival papillae of hiv-seropositive, necrotizing ulcerative periodontitis molecular epidemiology of candida albicans strains isolated from the oropharynx of hiv-positive patients at successive clinic visits relationship between herpesviruses and adult periodontitis and periodontopathic bacteria herpesviruses in human periodontal disease herpesviruses: a unifying causative factor in periodontitis? modulation of immune system function by measles virus infection: role of soluble factor and direct infection modulation of immune system function by measles virus infection. ii. infection of b cells leads to the production of a soluble factor that arrests uninfected b cells in g /g manganese superoxide dismutase induction during measles virus infection suppression of antigen-specific t cell proliferation by measles virus infection: role of a soluble factor in suppression cpg oligodeoxynucleotides protect normal and siv-infected macaques from leishmania infection interactions between mycobacterium leprae and simian immunodeficiency virus (siv) in rhesus monkeys this paper reviews how virus-induced immunosuppression of both innate and adaptive immune responses can provide an underlying mechanism for bacterial co-infection this paper introduces the concept of one approach to treat or prevent polymicrobial diseases by using bioengineering and nanotechnology to specific anatomic sites and crucial time points in the disease course for intervention and/or prevention communication among oral bacteria. microbiol the author would like to thank j. neelans for help with manuscript preparation. discusses our increasing understanding of the link between biofilms and the pathogenesis of human disease, as well as identifying the need for relevant animal models with which to study these infectious states. . brogden key: cord- -ikepr p authors: tulchinsky, theodore h.; varavikova, elena a. title: expanding the concept of public health date: - - journal: the new public health doi: . /b - - - - . - sha: doc_id: cord_uid: ikepr p ancient societies recognized the needs of sanitation, food safety, workers’ health, and medical care to protect against disease and to promote well-being and civic prosperity. new energies and knowledge since the eighteenth century produced landmark discoveries such as prevention of scurvy and vaccination against smallpox. the biological germ theory and competing miasma theory each proved effective in sanitation, and immunization in control of infectious diseases. non-communicable diseases as the leading causes of mortality have responded to innovative preventive care of health risk factors, smoking, hypertension, obesity, physical inactivity, unhealthful diets, and diabetes mellitus. health promotion proved effective to modern public health in tackling disease origins, individual behavior, and social and economic conditions. the global burden of infectious and non-communicable diseases, aging and chronic illness faces rising costs and still inadequate prevention. the evolution of concepts of public health will have to address these new challenges of population health. the development of public health from its ancient and recent roots, especially in the past several centuries, is a continuing process, with evolutionary and sometimes dramatic leaps forward, and important continuing and new challenges for personal and population health and well-being. everything in the new public health is about preventing avoidable disease, injuries, disabilities, and death while promoting and maximizing a healthy environment and optimal conditions for current and future generations. thus, the new public health addresses overall health policy, resource allocation, as well as the organization, management, and provision of medical care and of health systems in general within a framework of overall social policy and in a community, state, national, transnational, and global context. the study of history (see chapter ) helps us to understand the process of change, to define where we came from and where we are going. it is vital to recognize and understand change in order to deal with radical transformations in direction that occur as a result of changing demography and epidemiology, new science, evolving best practices in public health and clinical medicine, and above all inequalities in health resulting from societal system failures and social and economic factors. health needs will continue to develop in the context of environmental, demographic and societal adjustments, with knowledge gained from social and physical sciences, practice, and economics. for the coming generations, this is about not only the quality of life, but the survival of society itself. over the past century there have been many definitions of public health and health for all. mostly they represent visions and ideals of societal and global aspirations. this chapter examines the very base of the new public health, which encompasses the classic issues of public health with recognition of the advances made in health promotion and the management of health care systems as integral components of societal efforts to improve the health of populations and of individuals. what follows in succeeding chapters will address the major concepts leading to modern and comprehensive elements of public health. inevitably, concepts of public health continue to evolve and to develop both as a philosophy and as a structured discipline. as a professional field, public health requires specialists trained with knowledge and appreciation of its evolution, scientific advances, concepts, and best practices, old and modern. it demands sophisticated professional and managerial skills, the ability to address a problem, reasoning to define the issues, and to advocate, initiate, develop, and implement new and revised programs. it calls for profoundly humanistic values and a sense of responsibility towards protecting and improving the health of communities and every individual. in the twenty-first century, this set of values was well expressed in the human development index agreed to by nations (box . ). public health is a multidimensional field and therefore multidisciplinary in its workforce and organizational needs. it is based on scientific advances and application of best practices as they evolve, and includes many concepts, including holistic health, first established in ancient times. the discussion will return to the diversity of public health throughout this chapter and book many times. in previous centuries, public health was seen primarily as a discipline which studies and implements measures for control of communicable diseases, primarily by sanitation and vaccination. the sanitary revolution, which preceded the development of modern bacteriology, made an enormous contribution to improved health, but many other societal factors including improved nutrition, education, and housing were no less important for population health. maternal and child health, occupational health, and many other aspects of a growing public health network of activities played important roles, as have the physical and social environment and personal habits of living in determining health status. in recent decades recognition of the importance of women's health and health inequalities associated with many high-risk groups in the population have seen both successes and failures in addressing their challenges. male health issues have received less attention, apart from issues associated with specific diseases, or those of healthy military personnel. the scope of public health has changed along with growth of the medical, social, and public health sciences, public expectations, and practical experience. taken together, these have all contributed to changes in the concepts and causes of disease. health systems that fail to adjust to changes in fundamental concepts of public health suffer from immense inequity and burdens of preventable disease, disability, and death. this chapter examines expanding concepts of public health, leading to the development of a new public health. public health has evolved as a multidisciplinary field that includes the use of basic and applied science, education, social sciences, economics, management, and communication skills to promote the welfare of the individual and the community. it is greater than the sum of its component elements and includes the art and politics of the funding and coordination of the wide diversity of community and individual health services. the concept of the interdependence of health in body and in mind has ancient origins. they continue to be fundamental to individuals and societies, and part of the fundamental rights of all humans to have knowledge of healthful lifestyles and to have access to those measures of good health that society alone is able to provide, such as immunization programs, food and drug safety and quality standards, environmental and occupational health, and universal access to high-quality primary and specialty medical and other vital health services. this holistic view of balance and equilibrium may be a renaissance of classical greek and biblical traditions, applied with the broad new knowledge and experience of public health and medical care of the nineteenth, twentieth, and the early years of the twenty-first centuries as change continues to challenge our capacity to adapt. the competing nineteenth-century germ and miasma theories of biological and environmental causation of illness each contributed to the development of sanitation, hygiene, immunization, and understanding of the biological and social determinants of disease and health. they come together in the twenty-first century encompassed in a holistic new public health addressing individual and population health needs. medicine and public health professionals both engage in organization and in direct care services. these all necessitate an understanding of the issues that are included in the new public health, how they evolved, interact, are put together in organizations, and are financed and operated in various parts of the world in order to understand changes going on before our eyes. great success has been achieved in reducing the burden of disease with tools and concepts currently at our disposal. the idea that this is an entitlement for everyone was articulated in the health for all concept of alma-ata in . the health promotion movement emerged in the s and showed dramatically effective results in managing the new human immunodeficiency virus (hiv) pandemic and in tackling smoking and other risk factors for non-communicable diseases (ncds). a health in all policy concept emerged in promoting the concept that health should be a basic component of all public and private policies to achieve the full potential of public health and eliminate inequalities associated with social and economic conditions. profound changes are taking place in the world population, and public health is crucial to respond accordingly: mass migration to the cities, fewer children, extended life expectancy, and the increase in the population of older people who are subject to more chronic diseases and disabilities in a changing physical, social, and economic climate. health systems are challenged with continuing development of new medical technologies and related reforms in clinical practice, while experiencing strong influences of pharmaceuticals and the medicalization of health, with prevention and health promotion less central in priorities and resource allocation. globalization of health has many meanings: international trade, improving global communications, and economic changes with increasing flows of goods, services, and people. ecological and climate change bring droughts, hurricanes, arctic meltdown, and rising sea levels. globalization also has political effects, with water and food shortages, terrorism, and economic distress affecting billions of people. in terms of health, disease can spread from one part of the world to others, as in pandemics or in a quiet spread such as that of west nile fever moving from its original middle eastern natural habitat to the americas and europe, or severe acute respiratory syndrome (sars), which spread with lightning speed from chinese villages to metropolitan cities such as toronto, canada. it can also mean that the ncds characteristic of the industrialized countries are now recognized as the leading causes of death in low-and middle-income countries, associated with diet, activity levels, and smoking, which are themselves pandemic risk factors. the potential for global action in health can also be dramatic. the eradication of smallpox was a stunning victory for public health. the campaign to eradicate poliomyelitis is succeeding even though the end-stage is fraught with setbacks, and measles elimination has turned out to be more of a challenge than was anticipated a decade ago, with resurgence in countries thought to have it under control. global health policies have also made the achievements of public-private partnerships of great importance, particularly in vaccination and acquired immunodeficiency syndrome (aids) control programs. there have been failures as well, with very limited progress in human resources development of the public health workforce in low-income countries. the new public health is necessarily comprehensive in scope and it will continue to evolve as new technologies and scientific discoveries -biological, genetic, and sociological -reveal more methods of disease control and health promotion. it relates to or encompasses all community and individual activities directed towards improving the environment for health, reducing factors that contribute to the burden of disease, and fostering those factors that relate directly to improved health. its programs range broadly from immunization, health promotion, and child care, to food labeling and fortification, as well as to the assurance of well-managed, accessible health care services. a strong public health system should have adequate preparedness for natural and human-made disasters, as seen in the recent tsunamis, hurricanes, biological or other attacks by terrorists, wars, conflicts, and genocidal terrorism (box . ) . the concepts of health promotion and disease prevention are essential and fundamental elements of the new public health. parallel scientific advances in molecular biology, genetics and pharmacogenomics, imaging, information technology, computerization, biotechnology, and nanotechnology hold great promise for improving the productivity of the health care system. advances in technology with more effective and less expensive drug and vaccine development, with improved safety and effectiveness, and fewer adverse reactions, will over time greatly increase efficiency in prevention and treatment modalities. the new public health is important as a conceptual base for training and practice of public health. it links classical topics of public health with adaptation in the organization and financing of personal health services. it involves a changed paradigm of public health to incorporate new advances in political, economic, and social sciences. failure at the political level to appreciate the role of public health in disease control holds back many societies in economic and social development. at the same time, organized public health systems need to work to reduce inequities between and inside countries to ensure equal access to care. it also demands special attention through health promotion activities of all kinds at national and local societal levels to provide access for groups with special risks and needs to medical and community health care with the currently available and newly developing knowledge and technologies. the great gap between available capabilities to prevent and treat disease and actually reaching all in need is still the the mission of the nph is to maximize human health and well-being for individuals and communities, nationally and globally. the methods with which the nph works to achieve this are in keeping with recognized international best practices and scientific advances: . societal commitment and sustained efforts to maximize quality of life and health, economic growth with equity for all (health for all and health in all). collaboration between international, national, state, and local health authorities working with public and private sectors to promote health awareness and activities essential for population health. . health promotion of knowledge, attitudes, and practices, including legislation and regulation to protect, maintain, and advance individual and community health. . universal access to services for prevention and treatment of illness and disability, and promotion of maximum rehabilitation. . environmental, biological, occupational, social, and economic factors that endanger health and human life, addressing: (a) physical and mental illness, diseases and infirmity, trauma and injuries (b) local and global sanitation and environmental ecology (c) healthful nutrition and food security including availability, quality, safety, access, and affordability of food products (d) disasters, natural and human-made, including war, terrorism, and genocide (e) population groups at special risk and with specific health needs. . promoting links between health protection and personal health services through health policies and health systems management, recognizing economic and quality standards of medical, hospital, and other professional care in health of individuals and populations. . training of professional public health workforces and education of all health workers in the principles of ethical best practices of public health and health systems. . research and promotion of current best practices: wide application of current international best practices and standards. . mobilizing the best available evidence from local and international scientific and epidemiological studies and best practices recognized as contributing to the overall goal. . maintaining and promoting equity for individual and community rights to health with high professional and ethical standards. source of great international and internal national inequities. these inequities exist not only between developed and developing countries, but also within transition countries, mid-level developing countries, and those newly emerging with rapid economic development. the historical experience of public health will help to develop the applications of existing and new knowledge and societal commitment to social solidarity in implementation of the new discoveries for every member of the society, despite socioeconomic, ethnic, or other differences. political will and leadership in health, adequate financing, and organization systems in the health setting are crucial to furthering health as an objective with defined targets, supported by well-trained staff for planning, management, and monitoring the population health and functioning of health systems. political leadership and professional support are both indispensable in a world of limited resources, with high public expectations and the growing possibilities of effectiveness of public health programs. well-developed information and knowledge management systems are required to provide the feedback and information needed for good management. it includes responsibilities and coordination at all levels of government. non-governmental organizations (ngos) and participation of a well-informed media and strong professional and consumer organizations also have significant roles in furthering population health. no less important are clear designations of responsibilities of the individual for his or her own health, and of the provider of care for humane, high-quality professional care. the complexities and interacting factors are suggested in figure . , with the classic host-agent-environment triad. many changes have signaled a need for transformation towards the new public health. religion, although still a major political and policy-making force in many countries, is no longer the central organizing power in most societies. organized societies have evolved from large extended families and tribes to rural societies, cities, regions, and national governments. with the growth of industrialized urban communities, rapid transport, and extensive trade and commerce in multinational economic systems, the health of individuals and communities has become more than just a personal, family, and/or local problem. an individual is not only a citizen of the village, city, or country in which he or she lives, but a citizen of a "global village". the agricultural revolutions and international explorations of the fifteenth to seventeenth centuries that increased food supply and diversity were followed only much later by knowledge of nutrition as a public health issue. the scientific revolution of the seventeenth to nineteenth centuries provided the basics to describe and analyze the spread of disease and the poisonous effects of the industrial revolution, including crowded living conditions and pollution of the environment with serious ecological damage. in the latter part of the twentieth century, a new agricultural "green revolution" had a great impact in reducing human deprivation internationally, yet the full benefits of healthier societies are yet to be realized in the large populations living in abject poverty in sub-saharan africa, south-east asia, and other parts of the world. global water shortages can be addressed with new methods of irrigation, water conservation and the application of genetic sciences to food production, and issues of economics and food security are of great importance to a still growing world population with limited supplies. further, food production capacity can and must be enlarged to meet current food insecurity, rising expectations of developing nations, and population growth. the sciences of agriculture-related fields, including genetic sciences and practical technology, will be vital to human progress in the coming decades. these and other societal changes discussed in chapter have enabled public health to expand its potential and horizons, while developing its pragmatic and scientific base. organized public health in the twentieth century proved effective in reducing the burden of infectious diseases and has contributed to improved quality of life and longevity by many years. in the last half-century, chronic diseases have become the primary causes of morbidity and mortality in the developed countries and increasingly in developing countries. growing scientific and epidemiological knowledge increases the capacity to deal with these diseases. many aspects of public health can only be influenced by the behavior of and risks to the health of individuals. these require interventions that are more complex and relate to societal, environmental, and community standards and expectations as much as to personal lifestyle. the dividing line between communicable and non-communicable diseases changes over time. scientific advances have shown the causation of chronic conditions by infectious agents and their prevention by curing the infection, as in helicobacter pylori and peptic ulcers, and in prevention of cancer of the liver and cervix by immunization for hepatitis b and human papillomavirus (hpv), respectively. chronic diseases have come to the center stage in the "epidemiological transition", as infectious diseases came under increasing control. this, in part, has created a need for reform in the funding and management of health systems due to rapidly rising costs, aging of the population, the rise of obesity and diabetes and other chronic conditions, mushrooming therapeutic technology, and expanding capacity to deal with public health emergencies. reform is also needed in international assistance to help less developed nations build the essential infrastructure to sustain public health in the struggle to combat aids, malaria, tuberculosis (tb), and the major causes of preventable infant, childhood, and motherhood-related deaths. the nearly universal recognition of the rights of people to have access to health care of acceptable quality by international standards is a challenge of political will and leadership backed up by adequate staffing with public health-trained staff and organizations. the challenges of the current global economic crisis are impacting social and health systems around the world. the interconnectedness of managing health systems is part of the new public health. setting the priorities and allocating resources to address these challenges requires public health training and orientation of the professionals and institutions participating in the policy, management, and economics of health systems. conversely, those who manage such institutions are recognizing the need for a wide background in public health training in order to fulfill their responsibilities effectively. concepts such as objectives, targets, priorities, cost-effectiveness, and evaluation have become part of the new public health agenda. an understanding of how these concepts evolved will help the future health provider or manager to cope with the complexities of mixing science, humanity, and effective management of resources to achieve higher standards of health, and to cope with new issues as they develop in the broad scope of the new public health for the twenty-first century, in what breslow called the "third public health era" of long and healthy quality of life (box . ). health can be defined from many perspectives, ranging from statistics on mortality, life expectancy, and morbidity rates to idealized versions of human and societal perfection, as in the world health organization's (who's) founding charter. the first public health era -the control of communicable diseases. second public health era -the rise and fall of chronic diseases. third public health era -the development of long and high-quality life. preamble to the constitution of the who, as adopted by the international health conference in new york in and signed by the representatives of states, entered into force on april , with the widely cited definition: "health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity". this definition is still important conceptually as an ideal accepted as fundamental to public policy over the years. a more operational definition of health is a state of equilibrium of the person with the biological, physical, and social environment, with the object of maximum functional capability. health is thus seen as a state characterized by anatomical, physiological, and psychological integrity, and an optimal functional capability in the family, work, and societal roles (including coping with associated stresses), a feeling of well-being, and freedom from risk of disease and premature death. deviances in health are referred to as unhealthy and constitute a disease nomenclature. there are many interrelated factors in disease and in their management through what is now called risk reduction. in , claude bernard described the phenomenon of adaptation and adjustment of the internal milieu of the living organism to physiological processes. this concept is fundamental to medicine. it is also central to public health because understanding the spectrum of events and factors between health and disease is basic to the identification of contributory factors affecting the balance towards health, and to seeking the points of potential intervention to reverse the imbalance. as described in chapter , from the time of hippocrates and galen, diseases were thought to be due to humors and miasma or emanations from the environment. this was termed the miasma theory, and while without a direct scientific explanation, it was acted upon in the early to mid-nineteenth century and promoted by leading public health theorists including florence nightingale, with practical and successful measures to improve sanitation, housing, and social conditions, and having important results in improving health conditions. the competing germ theory developed by pioneering nineteenthcentury epidemiologists (panum, snow, and budd), scientists (pasteur, cohn, and koch), and practitioners (lister and semmelweiss) led to the science of bacteriology and a revolution in practical public health measures. the combined application of the germ (agent-host-environment) and miasma theories (social and sanitary environment) has been the basis of classic public health, with enormous benefits in the control of infectious and other diseases or harmful conditions. the revolutionary changes occurring since the s have brought about a decline in cardiovascular and cancer mortality, and conceptual changes such as health for all and health in all to bring health issues to all policies at both governmental and individual levels. the concepts of public health advanced with the marc lalonde health field concept (new perspectives on the health of canadians, ) , stating that health was the result of the physical and social environment, lifestyle and personal habits, genetics, as well as organization and provision of medical care. the lalonde report was a key concept leading to ideas advanced at the alma-ata conference on primary care held in and more explicitly in the development of the basis for health promotion as articulated in the ottawa charter of on health promotion. this marked the beginning of a whole new aspect of public health, which proved itself in addressing with considerable success the epidemic of hiv and cardiovascular diseases. in the usa, the surgeon general's reports of on smoking and health, and of defining health targets as national policy promoted the incorporation of "management by objectives" from the business world applied to the health sector (see chapter ). this led to healthy people usa and later versions, and the united nations (un) millennium development goals (mdgs), aimed primarily at the middle-and low-income countries (box . ). the identification of infectious causes of cancers of the liver and cervix established a new paradigm in epidemiology, and genetic epidemiology has important potential for public health and clinical medicine. in the basic host-agent-environment paradigm, a harmful agent comes through a sympathetic environment into contact with a susceptible host, causing a specific disease. this idea dominated public health thinking until the midtwentieth century. the host is the person who has or is at risk for a specific disease. the agent is the organism or direct cause of the disease. the environment includes the external factors which influence the host, his or her susceptibility to the agent, and the vector which transmits or carries the agent to the host from the environment. this explains the causation and transmission of many diseases. this paradigm (figure . ), in effect, joins together the contagion and miasma theories of disease causation. a specific agent, a method of transmission, and a susceptible host are involved in an interaction, which are central to the infectivity or severity of the disease. the environment can provide the carrier or vector of an infective (or toxic) agent, and it also contributes factors to host susceptibility; for example, unemployment, poverty, or low education level. the expanded host-agent-environment paradigm widens the definition of each of the three components ( figure . ), in relation to both acute infectious and chronic noninfectious disease epidemiology. in the latter half of the twentieth century, this expanded host-agent-environment paradigm took on added importance in dealing with the complex of factors related to chronic diseases, now the leading causes of disease and premature mortality in the developed world, and increasingly in developing countries. interventions to change host, environmental, or agent factors are the essence of public health. in infectious disease control, the biological agent may be removed by pasteurization of food products or filtration and disinfection (chlorination) of water supplies to prevent transmission of waterborne disease. the host may be altered by immunization to provide immunity to a specific infective organism. the environment may be changed to prevent transmission by destroying the vector or its reservoir of the disease. a combination of these interventions can be used against a specific risk factor, toxic or nutritional deficiency, infectious organism, or disease process. vaccine-preventable diseases may require both routine and special activities to boost herd immunity to protect the individual and the community. for other infectious diseases for which there is no vaccine (e.g., malaria), control involves a broad range of activities including case finding and treatment to improve the individual's health and to reduce the reservoir of the disease in the population, and other measures such as bed nets to reduce exposure of the host to vector mosquitoes, as well as vector control to reduce the mosquito population. tb control requires not only case finding and treatment, but understanding the contributory factors of social conditions, diseases with tb as a secondary condition (substance abuse and aids), agent resistance to treatment, and the inability of patients or carriers to complete treatment without supervision. sexually transmitted infections (stis) which are not controllable by vaccines require a combination of personal behavior change, health education, medical care, and skilled epidemiology. with non-infectious diseases, intervention is even more complex, involving human behavior factors and a wide range of legal, administrative, and educational issues. there may be multiple risk factors, which have a compounding effect in disease causation, and they may be harder to alter than infectious diseases factors. for example, smoking in and of itself is a risk factor for lung cancer, but exposure to asbestos fibers has a compounding effect. preventing exposure to the compounding variables may be easier than smoking cessation. reducing trauma morbidity and mortality is equally problematic. the identification of a single specific cause of a disease is of great scientific and practical value in modern public health, enabling such direct interventions as the use of vaccines or antibiotics to protect or treat individuals from infection by a causative organism, toxin, deficiency condition, or social factor. the cumulative effects of several contributing or risk factors in disease causation are also of great significance in many disease processes, in relation to infectious diseases such as nutritional status as for chronic diseases such as the cardiovascular group. the health of an individual is affected by risk factors intrinsic to that person as well as by external factors. intrinsic factors include the biological ones that the individual inherits and those life habits he or she acquires, such as smoking, overeating, or engaging in other high-risk behaviors. external factors affecting individual health include the environment, the socioeconomic and psychological state of the person, the family, and the society in which he or she lives. education, culture, and religion are also contributory factors to individual and community health. there are factors that relate to health of the individual in which the society or the community can play a direct role. one of these is provision of medical care. another is to ensure that the environment and community services include safety factors that reduce the chance of injury and disease, or include protective measures; for example, fluoridation of a community water supply to improve dental health, and seat-belt or helmet laws to reduce motor vehicle injury and death. these modifying factors may affect the response of the individual or the spread of an epidemic (see chapter ). an epidemic may also include chronic disease, because common risk factors may cause an excess of cases in a susceptible population group, in comparison to the situation before the risk factor appeared, or in comparison to a group not exposed to the risk factor. these include rapid changes or "epidemics" in such conditions as type diabetes, asthma, cardiovascular diseases, trauma, and other non-infectious disorders. disease is a dynamic process, not only of causation, but also of incubation or gradual development, severity, and the effects of interventions intended to modify outcome. knowledge of the natural history of disease is fundamental to understanding where and with what means intervention can have the greatest chance for successful interruption or change in the disease process for the patient, family, or community. the natural history of a disease is the course of that disease from beginning to end. this includes the factors that relate to its initiation; its clinical course leading up to resolution, cure, continuation, or long-term sequelae (further stages or complications of a disease); and environmental or intrinsic (genetic or lifestyle) factors and their effects at all stages of the disease. the effects of intervention at any stage of the disease are part of the disease process (figure . ). as discussed above, disease occurs in an individual when agent, host, and environment interact to create adverse conditions of health. the agent may be an infectious organism, a chemical exposure, a genetic defect, or a deficiency condition. a form of individual or social behavior, such as reckless driving or risky sexual behavior, may lead to injury or disease. the host may be immune or susceptible as a result of many contributing social and environmental factors. the environment includes the vector, which may be a malaria-bearing mosquito, a contaminated needle shared by drug users, lead-contaminated paint, or an abusive family situation. assuming a natural state of "wellness" -i.e., optimal health or a sense of well-being, function, and absence of disease -a disease process may begin with the onset of a disease, infectious or non-infectious, following a somewhat characteristic pattern of "incubation" described by clinicians and epidemiologists. preclinical or predisposing events may be detected by a clinical history, with determination of risk including possible exposure or presence of other risk factors. interventions, before and during the process, are intended to affect the later course of the disease. the clinical course of a disease, or its laboratory or radiological findings, may be altered by medical or public health intervention, leading to the resolution or continuation of the disease with fewer or less severe secondary sequelae. thus, the intervention becomes part of the natural history of the disease. the natural history of an infectious disease in a population will be affected by the extent of prior vaccination or previous exposure in the community. diseases particular to children are often so because the adult population is immune from previous exposure or vaccinations. measles and diphtheria, primarily childhood diseases, now affect adults to a large extent because they are less protected by naturally acquired immunity or are vulnerable when their immunity wanes naturally or as a result of inadequate vaccination in childhood. in chronic disease management, high costs to the patient and the health system accrue where preventive services or management are inadequate, not yet available, or inaccessible or where there is a failure to apply the necessary interventions. the progress of diabetes to severe complications such as cardiovascular, renal, and ocular disease is delayed or reduced by good management of the condition, with a combination of smoking cessation, diet, exercise, and medications with good medical supervision. the patient with advanced chronic obstructive pulmonary disease or congestive heart failure may be managed well and remain stable with smoking avoidance, careful management of medications, immunizations against influenza and pneumonia, and other prevention-oriented care needs. where these are not applied or if they fail, the patient may require long and expensive medical and hospital care. failure to provide adequate supportive care will show up in ways that are more costly to the health system and will prove more life-threatening to the patient. the goal is to avoid where possible the necessity for tertiary care, substituting tertiary prevention, i.e., supportive rehabilitation to maximum personal function and maintaining a stable functional status. as in an individual, the phenomenon of a disease in a population may follow a course in which many factors interplay, and where interventions affect the natural course of the disease. the epidemiological patterns of an infectious disease can be assessed in their occurrence in the population or their mortality rates, just as they can for individual cases. the classic mid-nineteenth-century description of measles in the faroe islands by panum showed the transmission and the epidemic nature of the disease as well as the protective effect of acquired immunity (see chapter ). similar, more recent breakthroughs in medical, epidemiological, biological, and social sciences have produced enormous benefit for humankind as discussed throughout this text, with some examples. these include the eradication of smallpox and in the coming years, poliomyelitis, measles, leprosy, and other dreaded diseases known for millennia; the near-elimination of rheumatic heart disease and peptic ulcers in the industrialized countries; vast reduction in mortality from stroke and coronary heart disease (chd); and vaccines (against hepatitis b and hpv) for the prevention of cancers. these and other great achievements of the twentieth and early part of the twenty-first centuries hold great promise for humankind in the coming decades, but great challenges lie ahead as well. the biggest challenge is to bring the benefits of known public health capacity to the poorest population of each country and the poorest populations globally. in developed countries a major challenge is to renew efforts of public health capacity to bear on prevention of chronic conditions such as diabetes and obesity, considered to be at pandemic proportions; and the individual and societal effects of mental diseases. in public health today, fears of a pandemic of avian influenza are based on transmission of avian or other animal-borne (zoonotic) prions or viruses to humans and then their adaptation permitting human-to-human spread. with large numbers of people living in close contact with many animals (wild and domestic fowl), such as in china and south-east asia, and rapid transportation around the world, the potential for global spread of disease is almost without historical precedent. indeed, many human infectious diseases are zoonotic in origin and transferred from natural wildlife reservoirs to humans either directly or via domestic or other wild animals, such as from birds to chickens to humans in avian influenza. monitoring or immunization of domestic animals requires a combination of multidisciplinary zoonotic disease management strategies, public education and awareness, and veterinary public health monitoring and control. rift valley fever, equine encephalitis, and more recently sars and avian influenza associated with bird-borne viral disease which can affect humans, each show the terrible dangers of pandemic diseases. ebola virus is probably sustained between outbreaks among fruit bats, or as recently suggested wild or domestic pigs, and may become a major threat to public health as human case fatality rates decline, meaning that patients and carriers, or genetic drift of the virus with possible airborne transmission, may spread this deadly disease more widely than in the past (see chapter ). the health of populations, like the health of individuals, depends on societal factors no less than on genetics, personal risk factors, and medical services. social inequalities in health have been understood and documented in public health over the centuries. the chadwick and shattuck reports of - documented the relationship of poverty and bad sanitation, housing, and working conditions with high mortality, and ushered in the idea of social epidemiology. political and social ideologies thought that the welfare state, including universal health care systems of one type or another, would eliminate social and geographic differences in health status and this is in large part true. from the introduction of compulsory health insurance in germany in the s to the failed attempt in the usa at national health insurance in (see chapters , and ) and the more recent achievements of us president obama in - , social reforms to deal with inequalities in health have focused on improving access to medical and hospital care. almost all industrialized countries have developed such systems, and the contribution of these programs to improve health status has been an important part of social progress, especially since world war ii. but even in societies with universal access to health care, people of lower socioeconomic status (ses) suffer higher rates of morbidity and mortality from a wide variety of diseases. the black report (douglas black) in the uk in the early s pointed out that the class v population (unskilled laborers) had twice the total and specific mortality rates of the class i population (professional and business) for virtually all disease categories, ranging from infant mortality to death from cancer. the report was shocking because all britons have had access to the comprehensive national health service (nhs) since its inception in , with access to a complete range of services at no cost at time of service, close relations to their general practitioners, and good access to specialty services. these findings initiated reappraisals of the social factors that had previously been regarded as the academic interests of medical sociologists and anthropologists and marginal to medical care. more recent studies and reviews of regional, ethnic, and socioeconomic differentials in patterns of health care access, morbidity, and mortality indicate that health inequities are present in all societies including the uk, the usa, and others, even with universal health insurance or services. the ottawa charter on health promotion in placed a new paradigm before the world health community that recognized social and political factors as no less important ion health that traditional medical and sanitary public health measures. these concepts helped the world health community to cope with new problems such as hiv/aidsfor which there was neither a medical cure nor a vaccine to prevent the disease. its control came to depend in the initial decades almost entirely on education and change in lifestyles, until the advent of the antiretroviral drugs in the s. there is still no viable vaccine. although the epidemiology of cardiovascular disease shows the direct relationship of the now classic risk factors of stress, smoking, poor diet, and physical inactivity, differences in mortality from cardiovascular disease between different classes among british civil servants are not entirely explainable by these factors. the differences are also affected by social and economic issues that may relate to the psychological needs of the individual, such as the degree of control people have over their own lives. blue-collar workers have less control over their lives, their working life in particular, than their white-collar counterparts, and have higher rates of chd mortality than higher social classes. other work shows the effects of migration, unemployment, drastic social and political change, and binge drinking, along with protective effects of healthy lifestyle, religiosity, and family support systems in cardiovascular diseases. social conditions affect disease distribution in all societies. in the usa and western europe, tb has re-emerged as a significant public health problem in urban areas partly because of high-risk population groups, owing to poverty and alienation from society, as in the cases of homelessness, drug abuse, and hiv infection. in countries of eastern europe and the former soviet union, the recent rise in tb incidence has resulted from various social and economic factors in the early s, including the large-scale release of prisoners. in both cases, diagnosis and prescription of medication are inadequate, and the community at large becomes at risk because of the development of antibioticresistant strains of tubercle bacillus readily spread by inadequately treated carriers, acting as human vectors. studies of ses and health are applicable and valuable in many settings. in alameda county, california, differences in mortality between black and white population groups in terms of survival from cancer became insignificant when controlled for social class. a -year follow-up study of the county population reported that low-income families in california are more likely than those on a higher income to have physical and mental problems that interfere with daily life, contributing to further impoverishment. studies of the association between indicators of ses and recent screening in the usa, australia, finland, and elsewhere showed that lower ses women use less preventive care such as papanicolaou (pap) smears for cervical cancer than women of higher ses, despite having greater risk for cervical cancer. many factors in ses inequalities are involved, including transportation and access to primary care, differences in health insurance coverage, educational levels, poverty, high-risk behaviors, social and emotional distress, feeling a lack of control over one's own life, employment, occupation, and inadequate family or community social support systems. many barriers exist owing to difficulties in access and the lack of availability of free or low-cost medical care, and the absence or limitations of health insurance is a further factor in the socioeconomic gradient. the recognition that health and disease are influenced by many factors, including social inequalities, plays a fundamental role in the new public health paradigm. health care systems need to take into account economic, social, physical, and psychological factors that otherwise will limit the effectiveness of even the best medical care. the health system includes access to competent and responsible primary care as well as by the wider health system, including health promotion, specific prevention and population-based health protection. the paradigm of the host-agent-environment triad (figures . and . ) is profoundly affected by the wider context. the sociopolitical environment and organized efforts at intervention affect the epidemiological and clinical course of disease of the individual. medical care is essential, as is public health, but the persistent health inequities seen in most regions and countries require societal attention. success or failure in improving the conditions of life for the poor, and other vulnerable "risk groups", affect national or regional health status and health system performance. the health system is meant to reduce the occurrence or bad outcome of disease, either directly by primary prevention or treatment as secondary prevention or by maximum rehabilitation as tertiary prevention, or equally important indirectly by reducing community or individual risk factors. the the effects of social conditions on health can be partly offset by interventions intended to promote healthful conditions; for example, improved sanitation, or through good-quality primary and secondary health services, used efficiently and effectively made available to all. the approaches to preventing disease or its complications may require physical changes in the environment, such as removal of the broad street pump handle to stop the cholera epidemic in london, or altering diets as in goldberger's work on pellagra. some of the great successes of public health have been and continue to be low technology. examples, among many others, include insecticide-impregnated bednets and other vector control measures, oral rehydration solutions, treatment and cure of peptic ulcers, exercise and diet to reduce obesity, hand washing in hospitals (and other health facilities), community health workers, and condoms and circumcision for the prevention of stis, including hiv and cancer of the cervix. the societal context in terms of employment, social security, female education, recreation, family income, cost of living, housing, and homelessness is relevant to the health status of a population. income distribution in a wealthy country may leave a wide gap between the upper and lower socioeconomic groups, which affects health status. the media have great power to sway public perception of health issues by choosing what to publish and the context in which to present information to society. modern media may influence an individual's tendency to overestimate the risk of some health issues while underestimating the risk of others, ultimately influencing health choices, such as occurred with public concern regarding false claims of an association between the measles-mumps-rubella (mmr) vaccine and autism in the uk (see the wakefield effect, chapter ). the new public health has an intrinsic responsibility for advocacy of improved societal conditions in its mission to promote optimal community health. an ultimate goal of public health is to improve health and to prevent widespread disease occurrence in the population and in an individual. the methods of achieving this are wide and varied. when an objective has been defined in "social justice is a matter of life and death. it affects the way people live, their consequent chance of illness, and their risk of premature death. we watch in wonder as life expectancy and good health continue to increase in parts of the world and in alarm as they fail to improve in others. a girl born today can expect to live for more than years if she is born in some countries -but less than years if she is born in others. within countries there are dramatic differences in health that are closely linked with degrees of social disadvantage. differences of this magnitude, within and between countries, simply should never happen. these inequities in health, avoidable health inequalities arise because of the circumstances in which people grow, live, work, and age, and the systems put in place to deal with illness. the conditions in which people live and die are, in turn, shaped by political, social, and economic forces. social and economic policies have a determining impact on whether a child can grow and develop to its full potential and live a flourishing life, or whether its life will be blighted. increasingly the nature of the health problems rich and poor countries have to solve are converging. the development of a society, rich or poor, can be judged by the quality of its population's health, how fairly health is distributed across the social spectrum, and the degree of protection provided from disadvantage as a result of ill-health." preventing disease, the next step is to identify suitable and feasible methods of achieving it, or a strategy with tactical objectives. this determines the method of operation, course of action, and resources needed to carry it out. the methods of public health are categorized as health promotion, and primary, secondary, and tertiary prevention (box . ). health promotion is the process of enabling people and communities to increase control over factors that influence their health, and thereby to improve their health (adapted from the ottawa charter of health promotion, ; box . ). health promotion is a guiding concept involving activities intended to enhance individual and community health and well-being (box . ). it seeks to increase involvement and control by the individual and the community in their own health. it acts to improve health and social welfare, and to reduce specific determinants of diseases and risk factors that adversely affect the health, well-being, and productive capacities of an individual or society, setting targets based on the size of the problem but also the feasibility of successful intervention, in a cost-effective way. this can be through direct contact with the patient or risk group, or act indirectly through changes in the environment, legislation, or public policy. control of aids relies on an array of interventions that promote change in sexual behavior and other contributory risks such as sharing of needles among drug users, screening of blood supply, safe hygienic practices in health care settings, and education of groups at risk such as teenagers, sex workers, migrant workers, and many others. control of aids is also a clinical problem in that patients need antiretroviral therapy (art), but this becomes a management and policy issue for making these drugs available and at an affordable price for the poor countries most affected. this is an example of the challenge and effectiveness of health promotion and the new public health. health promotion is a key element of the new public health and is applicable in the community, the clinic or hospital, and in all other service settings. some health promotion activities are government legislative and box . modes of prevention l health promotion -fostering national, community, and individual knowledge, attitudes, practices, policies, and standards conducive to good health; promoting legislative, social, or environmental conditions; promoting knowledge and practices for self-care that reduce individual and community risk; and creating a healthful environment. it is directed toward action on the determinants of health. l health protection -activities of official health departments or other agencies empowered to supervise and regulate food hygiene, community and recreational water safety, environmental sanitation, occupational health, drug safety, road safety, emergency preparedness, and many other activities to eliminate or reduce as much as possible risks of adverse consequences to health. l primary prevention -preventing a disease from occurring, e.g., vaccination to prevent infectious diseases, advice to stop smoking to prevent lung cancer. l secondary prevention -making an early diagnosis and giving prompt and effective treatment to stop progress or shorten the duration and prevent complications from an already existing disease process, e.g., screening for hypertension or cancer of cervix and colorectal cancer for early case finding, early care and better outcomes. l tertiary prevention -stopping progress of an already occurring disease, and preventing complications, e.g., in managing diabetes and hypertension to prevent complications; restoring and maintaining optimal function once the disease process has stabilized, e.g., promoting functional rehabilitation after stroke and myocardial infarction with long-term follow-up care. health promotion (hp) is the process of enabling people to increase control over, and to improve their health. hp represents a comprehensive social and political process, and not only embraces actions directed at strengthening the skills and capabilities of individuals. hp also undertakes action directed towards changing social, environmental, and economic conditions so as to alleviate their impact on public and individual health. health promotion is the process of enabling people to increase control over the determinants of health and thereby improve their health. participation is essential to sustain health promotion action. the ottawa charter identifies three basic strategies for health promotion. these are advocacy for health to create the essential conditions for health indicated above; enabling all people to achieve their full health potential; and mediating between the different interests in society in the pursuit of health. these strategies are supported by five priority action areas as outlined in the ottawa charter for health promotion: regulatory interventions such as mandating the use of seat belts in cars, requiring that children be immunized to attend school, declaring that certain basic foods must have essential minerals and vitamins added to prevent nutritional deficiency disorders in vulnerable population groups, and mandating that all newborns should be given prophylactic vitamin k to prevent hemorrhagic disease of the newborn. setting food and drug standards and raising taxes on cigarettes and alcohol to reduce their consumption are also part of health promotion. promoting a healthy lifestyle is a major known obesity-preventive activity. health promotion is provided by organizations and people with varied professional backgrounds working towards common goals of improvement in the health and quality of individual and community life. initiatives may come from government with dedicated allocation of funds to address specific health issues, from donors, or from advocacy or community groups or individuals to promote a specific or general cause in health. raising awareness to inform and motivate people about their own health and lifestyle factors that might put them at risk requires teaching young people about the dangers of sexually transmitted diseases, smoking, and alcohol abuse to reduce risks associated with their social behavior. it might include disseminating information on healthy nutrition; for example, the need for folic acid supplements for women of childbearing age and multiple vitamins for elderly, as well as the elements of a healthy diet, compliance with immunization recommendations, compliance with screening programs, and many others. community and peer group attitudes and standards affect individual behavior. health promotion endeavors to create a climate of knowledge, attitudes, beliefs, and practices that are associated with better health outcomes. international conferences following on from the ottawa charter were held in adelaide in , sundsvall in , jakarta in , mexico in , bangkok in , and nairobi in . the principles of health promotion have been reiterated and have influenced public policy regarding public health as well as the private sector. health promotion has a track record of proven success in numerous public health issues where a biomedical solution was not available. the hiv/aids pandemic from the s until the late s had no medical treatment and control measures relied on screening, education, lifestyle changes, and supportive care. health promotion brought forward multiple interventions, from condom use and distribution, to needle exchanges for intravenous drug users, to male circumcision in high-prevalence african countries. medical treatment was severely limited until art was developed. the success of art also depends on a strong element of health promotion in widening the access to treatment and the success of medications to reduce transmission, most remarkably in reducing maternal-fetal transmission (see chapter ). similarly, in the battle against cardiovascular diseases, health promotion was an instrumental factor in raising public awareness of the importance of management of hypertension and smoking reduction, dietary restraint, and physical exercise. the success of massive reductions in stroke and chd mortality is as much the result of health promotion as of improved medical care (see chapter ). the character of public health carries with it a "good cop, bad cop" dichotomy. the "good cop" is persuasive and educational trying to convince people to do the right thing in looking after their own health: diet, exercise, smoking cessation, and others. on the other side, the "bad cop" role is regulatory and punitive. public health has a serious responsibility and role in the enforcement of laws and regulation to protect the public health. some of these are restrictive box . elements of health promotion . address the population as a whole in health-related issues, in everyday life as well as people at risk for specific diseases. . direct action to risk factors or causes of illness or death. . undertake activist approach to seek out and remedy risk factors in the community that adversely affect health. . promote factors that contribute to a better condition of health of the population. . initiate actions against health hazards, including communication, education, legislation, fiscal measures, organizational change, community development, and spontaneous local activities. . involve public participation in defining problems and deciding on action. . advocate relevant environmental, health, and social policy. . encourage health professional participation in health education and health advocacy. . advocate for health based on human rights and solidarity. . invest in sustainable policies, actions, and infrastructure to address the determinants of health. . build capacity for policy development, leadership, health promotion practice, knowledge transfer and research, and health literacy. . regulate and legislate to ensure a high level of protection from harm and enable equal opportunity for health and well-being for all people. . partner and build alliances with public, private, nongovernmental, and international organizations and civil society to create sustainable actions. . make the promotion of health central to the global development agenda. of individual rights that may damage other people or are requirements based on strong evidence of benefits to population health. readily accepted are food and drug standards, such as pasteurization of milk, and iodization of salt; requirements to drive on the right-hand side of the road (except in some countries such as the uk), to wear seat belts and for motorcyclists to wear safety helmets; and not smoking in public places. enforcement of these and similar statutory or regulatory requirements is vital in a civil society to protect the public from health hazards and to protect people from harm and exploitation by unscrupulous manufacturers and marketing. cigarette advertising and sponsorship of sports events by tobacco companies are banned in most upper income countries. the use of transfats in food manufacturing and baking is now banned and salt reduction is being promoted and even mandated in many us local authorities to reduce cardiovascular disease. advertising of unhealthy snack foods on children's television programs and during child-watching hours is commonly restricted. banning high-sugar soda drink distribution in schools is a successful intervention to reduce the current child obesity epidemic. melamine use in milk powders and baby formulas, which caused widespread illness and death of infants in china, is now banned and a punishable offence for manufacture or distribution in china and worldwide. examples of this aspect of public health are mentioned throughout this text, especially in chapters and on nutrition, and environmental and occupational health, respectively. the regulatory enforcement function of public health is sometimes controversial and portrayed as interference with individual liberty. fluoridation of community water supplies is an example where aggressive lobby groups opposing this safe and effective public health measure are still common. this is discussed in chapter . equally important is the public health policy issue of resource allocation and taxation for health purposes. taxation is an unpopular measure that governments must employ and enforce in order to do the public's business. the debate over the patient protection and affordable care act (ppaca or "obamacare"), discussed elsewhere in this and other chapters, shows how bitter the arguments can become, yet the goal of equality of access to health care cannot be denied as a public good, demonstrably contributing to the health of the nation. primary prevention refers to those activities that are undertaken to prevent disease or injury from occurring at all. primary prevention works with both the individual and the community. it may be directed at the host to increase resistance to the agent (such as in immunization or cessation of smoking), or at environmental activities to reduce conditions favorable to the vector for a biological agent, such as mosquito vectors of malaria or dengue fever. landmark examples include the treatment and prevention of scurvy among sailors based on james lind's findings in a classic clinical epidemiological study in , and john snow's removal of the handle from the broad street pump to stop a cholera epidemic in london in (see chapter ). primary prevention includes elements of health protection such as ensuring water, food and drug, and workplace safety; chlorination of drinking water to prevent transmission of waterborne enteric diseases; pasteurization of milk to prevent gastrointestinal diseases; mandating wearing seat belts in motor vehicles to prevent serious injury and death in road crashes; and reducing the availability of firearms to reduce injury and death from intentional, accidental, or random violence. it also includes direct measures to prevent diseases, such as immunization to prevent polio, tetanus, pertussis, and diphtheria. health promotion and health protection blend together as a group of activities that reduce risk factors and diseases through many forms of intervention such as changing smoking legislation or preventing birth defects by fortification of flour with folic acid. prevention of hiv transmission by needle exchange for intravenous drug users, promoting condom usage, and promoting male circumcision in africa, and the distribution of condoms and clean needles for hivpositive drug users are recent examples of primary prevention associated with health promotion programs. primary prevention also includes activities within the health system that can lead to better health. this may mean, for example, setting standards and to reduce hospital infections, and ensuring that doctors not only are informed of appropriate immunization practices and modern prenatal care or screening programs for cancer of the cervix, colon, and breast, but also are aware of their vital role in preventing cardiovascular and other non-communicable diseases. in this role, the health care provider serves as a teacher and guide, as well as a diagnostician and therapist. like health promotion, primary prevention does not depend on health care providers alone; health promotion works to increase individual and community consciousness of self-care, mainly by raising awareness and information levels and empowering the individual and the community to improve self-care, to reduce risk factors, and to live healthier lifestyles. secondary prevention is early diagnosis and management to prevent complications from a disease. public health interventions to prevent the spread of disease include the identification of sources of the disease and the implementation of steps to stop it, as shown in snow's closure of the broad street pump. secondary prevention includes steps to isolate cases and treat or immunize contacts so as to prevent further cases of meningitis or measles, for example, in outbreaks. for current epidemics such as hiv/aids, primary prevention is largely based on education, abstinence from any and certainly risky sexual behavior, circumcision, and treatment of patients in order to improve their health and to reduce the risk of spread of hiv. for high-risk groups such as intravenous drug users, needleexchange programs reduce the risk of spread of hiv, and hepatitis b and c. distribution of condoms to teenagers, military personnel, truck drivers, and commercial sex workers helps to prevent the spread of stis and aids in schools and colleges, as well as among the military. the promotion of circumcision is shown to be effective in reducing the transmission of hiv and of hpv (the causative organism for cancer of the cervix). all health care providers have a role in secondary prevention; for example, in preventing strokes by early identification and adequate care of hypertension. the child who has an untreated streptococcal infection of the throat may develop complications which are serious and potentially life-threatening, including rheumatic fever, rheumatic valvular heart disease, and glomerulonephritis. a patient found to have elevated blood pressure should be advised about continuing management by appropriate diet and weight loss if obese, regular physical exercise, and long-term medication with regular follow-up by a health provider in order to reduce the risk of stroke and other complications. in the case of injury, competent emergency care, safe transportation, and good trauma care may reduce the chance of death and/or permanent handicap. screening and high-quality care in the community prevent complications of diabetes, including heart, kidney, eye, and peripheral vascular disease. they can also prevent hospitalizations, amputations, and strokes, thus lengthening and improving the quality of life. health care systems need to be actively engaged in secondary prevention, not only as individual doctors' services, but also as organized systems of care. public health also has a strong interest in promoting highquality care in secondary and tertiary care hospital centers in such areas of treatment as acute myocardial infarction, stroke, and injury in order to prevent irreversible damage. measures include quality of care reviews to promote adequate longterm postmyocardial infarction care with aspirin and betablockers or other medication to prevent or delay recurrence and second or third myocardial infarctions. the role of highquality transportation and care in emergency facilities of hospitals in public health is vital to prevent long-term damage and disability; thus, cardiac care systems including publicly available defibrillators, catheterization, the use of stents, and bypass procedures are important elements of health care policy and resource allocation, which should be accessible not only in capital cities but also to regional populations. tertiary prevention involves activities directed at the host or patient, but also at the social and physical environment in order to promote rehabilitation, restoration, and maintenance of maximum function after the disease and its complications have stabilized. the person who has undergone a cerebrovascular accident or trauma will reach a stage where active rehabilitation can help to restore lost functions and prevent recurrence or further complications. the public health system has a direct role in the promotion of disability-friendly legislation and standards of building, housing, and support services for chronically ill, handicapped, and elderly people. this role also involves working with many governmental social and educational departments, but also with advocacy groups, ngos, and families. it may also include the promotion of disability-friendly workplaces and social service centers. treatment for conditions such as myocardial infarction or a fractured hip now includes early rehabilitation in order to promote early and maximum recovery with restoration to optimal function. the provision of a wheelchair, walkers, modifications to the home such as special toilet facilities, doors, and ramps, along with transportation services for paraplegics are often the most vital factors in rehabilitation. public health agencies work with groups in the community concerned with promoting help for specific categories of risk group, disease, or disability to reduce discrimination. community action is often needed to eliminate financial, physical, or social barriers, promote community awareness, and finance special equipment or other needs of these groups. close follow-up and management of chronic disease, physical and mental, require home care and ensuring an appropriate medical regimen including drugs, diet, exercise, and support services. the follow-up of chronically ill people to supervise the taking of medications, monitor changes, and support them in maximizing their independent capacity in activities of daily living is an essential element of the new public health. public health uses a population approach to achieve many of its objectives. this requires defining the population, including trends of change in the age and gender distribution of the population, fertility and birth rates, spread of disease and disability, mortality, marriage and migration, and socioeconomic factors. the reduction of infectious disease as the major cause of mortality, increased longevity coupled with declining fertility rates, resulted in changes in the age composition, or a demographic transition. demographic changes, such as fertility and mortality patterns, are important factors in changing the age distribution of the population, resulting in a greater proportion of people surviving to older ages. declining infant mortality, increasing educational levels of women, the availability of birth control, and other social and economic factors lead to changes in fertility patterns and the demographic transition -an aging of the population -with important effects on health service needs. the age and gender distribution of a population affects and is affected by patterns of disease. change in epidemiological patterns, or an epidemiological shift, is a change in predominant patterns of morbidity and mortality. the transition of infectious diseases becoming less prominent as causes of morbidity and mortality and being replaced by chronic and non-infectious diseases has occurred in both developed and developing countries. the decline in mortality from chronic diseases, such as cardiovascular disease, represents a new stage of epidemiological transition, creating an aging population with higher standards of health but also long-term community support and care needs. monitoring and responding to these changes are fundamental responsibilities of public health, and a readiness to react to new, local, or generalized changes in epidemiological patterns is vital to the new public health. societies are not totally homogeneous in ethnic composition, levels of affluence, or other social markers. on one hand, a society classified as developing may have substantial numbers of people with incomes that promote overnutrition and obesity, so that disease patterns may include increasing prevalence of diseases of excesses, such as diabetes. on the other hand, affluent societies include population groups with disease patterns of poverty, including poor nutrition and low birth-weight babies. a further stage of epidemiological transition has been occurring in the industrialized countries since the s, with dramatic reductions in mortality from chd, stroke and, to a lesser extent, trauma. the interpretation of this epidemiological transition is still not perfectly clear. how it occurred in the industrialized western countries but not in those of the former soviet union is a question whose answer is vital to the future of health in russia and some countries of eastern europe. developing countries must also prepare to cope with increasing epidemics of non-infectious diseases, and all countries face renewed challenges from infectious diseases with antibiotic resistance or newly appearing infectious agents posing major public health threats. demographic change in a country may reflect social and political decisions and health system priorities from decades before. russia's rapid population decline since the s, china's gender imbalance with a shortage of millions of young women, egypt's rapid population growth outstripping economic capacity, and many other examples indicate the severity and societal importance of capacity to analyze and formulate public health and social policies to address such fundamental sociopolitical issues. aging of the population is now the norm in most developed countries as a result of low birth and declining mortality rates. this change in the age distribution of a population has many associated social and economic issues as to the future of social welfare with a declining age cohort to provide the workforce. the aging population requires pension and health care support which make demands of social security systems that will depend on economic growth with a declining workforce. in times of economic stress, as in europe, this situation is made more difficult by longstanding short working weeks, early pension ages, and high social benefits. however, this results in unemployment among young people in particular and social conflict. the interaction of increasing life expectancy and a declining workforce is a fundamental problem in the high-income countries. this imbalance may be resolved in part through productivity gains and switching of primary production to countries with large still underutilized workforces, while employment in the developed countries will depend on service industries including health and the economic growth generated by higher technology and intellectual property and service industries. the challenge of keeping populations and individuals healthy is reflected in modern health services. each component of a health service may have developed with different historical emphases, operating independently as a separate service under different administrative auspices and funding systems, competing for limited health care resources. in this situation, preventive community care receives less attention and resources than more costly treatment services. figure . suggests a set of health services in an interactive relationship to serve a community or defined population, but the emphasis should be on the interdependence of these services with one other and with the comprehensive network in order to achieve effective use of resources and a balanced set of services for the patient, the client or patient population, and the community. clinical medicine and public health each play major roles in primary, secondary, and tertiary prevention. each may function separately in their roles in the community, but optimal success lies in their integrated efforts. allocation of resources should promote management and planning practices to assist this integration. there is a functional interdependence of all elements of health care serving a definable population. the patient should be the central figure in the continuum or complex of services available. effectiveness in use of resources means that providing the service most appropriate for meeting the individual's or group's needs at a point in time are those that should be applied. this is the central concept in currently developing innovations in health care delivery in the usa with organizations using terms such as patient centered medical home, accountable care organizations (acos), and population health management systems, which are being promoted in the obamacare health reforms now in process (see chapter ) (shortell et al., ) . separate organization and financing of services place barriers to appropriate provision of services for both the community and the individual patient. the interdependence of services is a challenge in health care organizations for the future. where there is competition for limited resources, pressures for tertiary services often receive priority over programs to prevent children from dying of preventable diseases. public health must be seen in the context of all health care and must play an influential role in promoting prevention at all levels. clinical services need public health in order to provide prevention and community health services that reduce the burden of disease, disability, and dependence on the institutional setting. health was traditionally thought of as a state of absence of disease, pain, or disability, but has gradually been expanded to include physical, mental, and societal well-being. in , c. e. a. winslow, professor of public health at yale university, defined public health as follows: "public health is the science and art of ( ) preventing disease, ( ) prolonging life, and ( ) winslow's far-reaching definition remains a valid framework but is unfulfilled when clinical medicine and public health have financing and management barriers between them. in many countries, isolation from the financing and provision of medical and nursing care services left public health with the task of meeting the health needs of the indigent and underserved population groups with inadequate resources and recognition. health insurance organizations for medical and hospital care have in recent years been more open to incorporating evidence-based preventive care, but the organization of public health has lacked the same level of attention. in some countries, the limitations have been conceptual in that public health was defined primarily in terms of control of infectious, environmental, and occupational diseases. a more recent and widely used definition is: "public health is the science and art of preventing disease, prolonging life, and promoting health through the organized efforts of society." this definition, coined in in the public health in england report by sir donald acheson, reflects the broad focus of modern public health. terms such as social hygiene, preventive medicine, community medicine, and social medicine have been used to denote public health practice over the past century. preventive medicine is the application of preventive measures by clinical practitioners combining some elements of public health with clinical practice relating to individual patients. preventive medicine defines medical or clinical personal preventive care, with stress on risk groups in the community and national efforts for health promotion. the focus is on the health of defined populations to promote health and well-being using evidence-based guidelines for cost-effective preventive measures. measures emphasized include screening and follow-up of chronic illnesses, and immunization programs; for example, influenza and pneumococcal pneumonia vaccines are used by people who are vulnerable because of their age, chronic diseases, or risk of exposure, such as medical and nursing personnel and those providing other personal clinical services. clinical medicine also deals in the area of prevention in the management of patients with hypertension or diabetes, and in doing so prevents the serious complications of these diseases. social medicine is also primarily a medical specialty which looks at illness in an individual in the family and social context, but lacks the environmental and regulatory and organized health promotion functions of public health. community health implies a local form of health intervention, whereas public health more clearly implies a global approach, which includes action at the international, national, state, and local levels. some issues in health can be dealt with at the individual, family, or community level; others require global strategies and intervention programs with regional, national, or international collaboration and leadership. the social medicine movement originated to address the harsh conditions of the working population during the industrial revolution in mid-nineteenth-century europe. an eminent pioneer in cellular pathology, rudolph virchow provided leadership in social medicine powered by the revolutionary movements of , and subsequent social democrat political movements. their concern focused on harsh living and health conditions among the urban poor working class and neglectful political norms of the time. social medicine also developed as an academic discipline and advocacy orientation by providing statistical evidence showing, as in various governmental reports in the mid-nineteenth century, that poverty among the working class was associated with short life expectancy and that social conditions were key factors in the health of populations and individuals. this movement provided the basis for departments in medical faculties and public health education throughout the world stressing the close relationship between political priorities and health status. this continued in the twentieth century and in the usa found expression in pioneering work since the s at montefiore hospital in new york and with victor sidel, founding leader of the community health center movement the usa from the s. in the twenty-first century this movement continues to emphasize relationships between politics, society, disease, and medicine, and forms of medical practice derived from it, as enunciated by prominent advocates such as harvardbased paul farmer in haiti, russia and rwanda, and in the uk by martin mckee and others (nolte and mckee, ) . similar concepts are current in the usa under headings such as family medicine, preventive medicine, and social medicine. this movement has also influenced sir michael marmot and others in the world health commission of health inequalities of , with a strong influence on the un initiative to promote mdgs, whose first objective is poverty reduction (commission on inequalities report ). application of the idea of poverty reduction as a method of reducing health inequalities has been successful recently in a large field trial in brazil showing greater reduction in child mortality where cash bonuses were awarded by municipalities for the poor families than that observed in other similar communities (rasella, ). in the usa, this movement is supported by increased health insurance coverage for the working poor, with funding for preventive care and incentives for community health centers in the obamacare plan of for implementation in the coming years to provide care for uninsured and underserved populations, particularly in urban and rural poverty areas. the political aspect of social medicine is the formulation of and support for national initiatives to widen health care coverage to the percent of the us population who are still uninsured, and to protect those who are arbitrarily excluded owing to previous illnesses, caps on coverage allowed, and other exploitative measures taken by private insurance that frequently deny americans access to the high levels of health care available in the country. the ethical base of public health in europe evolved in the context of its successes in the nineteenth and early twentieth centuries along with ideas of social progress. but the twentieth century was also replete with extremism and wide-scale abuse of human rights, with mass executions, deportations, and starvation as official policy in fascist and stalinist regimes. eugenics, a pseudoscience popularized in the early decades of the twentieth century, promoted social policies meant to improve the hereditary qualities of a race by methods such as sterilization of mentally handicapped people. the "social and racial hygiene" of the eugenics movements led to the medicalization of sterilization in the usa and other countries. this was adopted and extended in nazi germany to a policy of murder, first of the mentally and physically handicapped and then of "racial inferiors". these eugenics theories were widely accepted in the medical community in germany, then used by the nazi regime to justify medically supervised killing of hundreds of thousands of helpless, incapacitated individuals. this practice was linked to wider genocide and the holocaust, with the brutalization and industrialized murder of over million jews and million other people, and corrupt medical experimentation on prisoners. following world war ii, the ethics of medical experimentation (and public health) were codified in the nuremberg code and universal declaration of human rights based on lessons learned from these and other atrocities inflicted on civilian populations (see chapter ). threats of genocide, ethnic cleansing, and terrorism are still present on the world stage, often justified by current warped versions of racial hygienic theories. genocidal incitement and actual genocide and terrorism have recurred in the last decades of the twentieth century and into the twenty-first century in the former yugoslav republics, africa (rwanda and darfur), south asia, and elsewhere. terrorism against civilians has become a worldwide phenomenon with threats of biological and chemical agents, and potentially with nuclear capacity. asymmetrical warfare of insurgencies which use innocent civilians for cover, as with other forms of warfare, carries with it grave dangers to public health, human rights, and international stability, as seen in the twenty-first century in south sudan, darfur, dr congo, chechnya, iraq, afghanistan, and pakistan. in , kerr white and colleagues defined medical ecology as population-based research providing the foundation for management of health care quality. this concept stresses a population approach, including those not attending and those using health services. this concept was based on previous work on quality of care, randomized clinical trials, medical audit, and structure-process-outcome research. it also addressed health care quality and management. these themes influenced medical research by stressing the population from which clinical cases emerge as well as public health research with clinical outcome measures, themes that recur in the development of health services research and, later, evidence-based medicine. this led to the development of the agency for health care policy and research and development in the us department of health and human services and evidence-based practice centers to synthesize fundamental knowledge for the development of information for decision-making tools such as clinical guidelines, algorithms, or pathways. clinical guidelines and recommended best practices have become part of the new public health to promote quality of patient care and public health programming. these can include recommended standards; for example, follow-up care of the postmyocardial infarction patient, an internationally recommended immunization schedule, recommended dietary intake or food fortification standards, and mandatory vitamin k and eye care for all newborns and many others (see chapter ). community-oriented primary care (copc) is an approach to primary health care that links community epidemiology and appropriate primary care, using proactive responses to the priority needs identified. copc, originally pioneered in south africa and israel by sidney and emily kark and colleagues in the s and s, stresses medical services in the community which need to be adapted to the needs of the population as defined by epidemiological analysis. copc involves community outreach and education, as well as clinical preventive and treatment services. copc focuses on community epidemiology and an active problem-solving approach. this differs from national or larger scale planning that sometimes loses sight of the local nature of health problems or risk factors. copc combines clinical and epidemiological skills, defines needed interventions, and promotes community involvement and access to health care. it is based on linkages between the different elements of a comprehensive basket of services along with attention to the social and physical environment. a multidisciplinary team and outreach services are important for the program, and community development is part of the process. in the usa, the copc concept has influenced health care planning for poor areas, especially provision of federally funded community health centers in attempts to provide health care for the underserved since the s. in more recent years, copc has gained wider acceptance in the usa, where it is associated with family physician training and community health planning based on the risk approach and "managed care" systems. indeed, the three approaches are mutually complementary (box . ). as the emphasis on health care reform in the late s moved towards managed care, the principles of copc were and will continue to be important in promoting health and primary prevention in all its modalities, as well as tertiary prevention with followup and maintenance of the health of the chronically ill. copc stresses that all aspects of health care have moved towards prevention based on measurable health issues in the community. through either formal or informal linkages between health services, the elements of copc are part of the daily work of health care providers and community services systems. the us institute of medicine issued the report on primary care in , defining primary care as "the provision of integrated, accessible health care services by clinicians who are accountable for addressing the majority of personal health care needs, developing a sustained partnership with patients and practicing in the context of the family and the community". this formulation was criticized by the american public health association (apha) as lacking a public health perspective and failing to take into account both the individual and the community health approaches. copc tries to bridge this gap between the perspectives of primary care and public health. the community, whether local, regional, or national, is the site of action for many public health interventions. moreover, understanding the characteristics of the community is vital to a successful community-oriented approach. by the s, new patterns of public health began to emerge, including all measures used to improve the health of the community, and at the same time working to protect and promote the health of the individual. the range of activities to achieve these general goals is very wide, including individual patient care systems and the community-wide activities that affect the health and well-being of the individual. these include the financing and management of health systems, evaluation of the health status of the population, and measures to improve the quality of health care. they place reliance on health promotion activities to change environmental risk factors for disease and death. they promote integrative and multisectoral approaches and the international health teamwork required for global progress in health. the definition of health in the charter of the who as a complete state of physical, mental, and social well-being had a ring of utopianism and irrelevance to states struggling to provide even minimal care in severely adverse political, economic, social, and environmental conditions (box . ). in , a more modest goal was set for attainment of a level of health compatible with maximum feasible social and economic productivity. one needs to recognize that health and disease are on a dynamic continuum that affects everyone. the mission for public health is to use a wide range of methods to prevent disease and premature death, and improve quality of life for the benefit of individuals and the community. the world health organization defines health as "a state of complete physical, mental and social well-being, not merely the absence of disease or infirmity" (who constitution, ) . in at the alma-ata conference on primary health care, the who related health to "social and economic productivity in setting as a target the attainment by all the people of the world of a level of health that will permit them to lead a socially and economically productive life". three general programs of work for the periods - , - , and - were formulated as the basis of national and international activity to promote health. in , the who, recognizing changing world conditions of demography, epidemiology, environment, and political and economic status, addressed the unmet needs of developing countries and health management needs in the industrialized countries, calling for international commitment to "attain targets that will make significant progress towards improving equity and ensuring sustainable health development". the object of the who is restated as "the attainment by all peoples of the highest possible level of health" as defined in the who constitution, by a wide range of functions in promoting technical cooperation, assisting governments, and providing technical assistance, international cooperation, and standards. in the s, most industrialized countries were concentrating energies and financing in health care on providing access to medical and hospital services through national insurance schemes. developing countries were often spending scarce resources trying to emulate this trend. the who was concentrating on categorical programs, such as eradication of smallpox and malaria, as well as the expanded program of immunization and similar specific efforts. at the same time, there was a growing concern that developing countries were placing too much emphasis and expenditure on curative services and not enough on prevention and primary care. the world health assembly (wha) in endorsed the primary care approach under the banner of "health for all by the year " (hfa ) . this was a landmark decision and has had important practical results. the who and the united nations children's fund (unicef) sponsored a seminal conference held in alma-ata, in the ussr ( kazakhstan), in , which was convened to refocus health policy on primary care. the alma-ata declaration stated that health is a basic human right, and that governments are responsible to assure that right for their citizens and to develop appropriate strategies to fulfill this promise. this proposition has come to be increasingly accepted in the international community. the conference stressed the right and duty of people to participate in the planning and implementation of their health care. it advocated the use of scientifically, socially, and economically sound technology. joint action through intersectoral cooperation was also emphasized. the alma-ata declaration focused on primary health care as the appropriate method of assuming adequate access to health care for all (box . ). many countries have gradually come to accept the notion of placing priority on primary care, resisting the temptation to spend high percentages of health care resources on high-tech and costly medicine. spreading these same resources into highly costeffective primary care, such as immunization and nutrition programs, provides greater benefit to individuals and to society as a whole. alma-ata provided a new sense of direction for health policy, applicable to developing countries and in a different way than the approaches of the developed countries. during the s, the health for all concept influenced national health policies in the developing countries with signs of progress in immunization coverage, for example, but the initiative was diluted as an unintended consequence by more categorical programs such as eradication of poliomyelitis. for example, developing countries have accepted immunization and diarrheal disease control as high-priority issues and achieved remarkable success in raising immunization coverage from some percent to over percent in just a decade. developed countries addressed these principles in different ways. in these countries, the concept of primary health care led directly to important conceptual developments in health. national health targets and guidelines are now common in many countries and are integral parts of box . declaration of alma-ata, : a summary of primary health care (phc) . reaffirms that health is a state of complete physical, mental, and social well-being, and not merely the absence of disease or infirmity, and is a fundamental human right. existing gross inequalities in the health status of the people, particularly between developed and developing countries as well as within countries, are of common concern to all countries. . governments have a responsibility for the health of their people. the people have the right and duty to participate in planning and implementation of their health care. . a main social target is the attainment, by all peoples of the world by the year , of a level of health that will permit them to lead a socially and economically productive life. . phc is essential health care based on practical, scientifically sound, and socially acceptable methods and technology. . it is the first level of contact of individuals, the family, and the national health system bringing health care as close as possible to where people live and work, as the first element of a continuing health care process. . phc evolves from the conditions and characteristics of the country and its communities, based on the application of social, biomedical, and health services research and public health experience. . phc addresses the main health problems in the community, providing promotive, preventive, curative, and rehabilitative services accordingly. . phc includes the following: (a) education concerning prevailing health problems and methods of preventing and controlling them (b) promotion of food supply and proper nutrition (c) adequate supply of safe water and basic sanitation (d) maternal and child health care, including family planning (e) immunization against the major infectious diseases (f) prevention of locally endemic diseases (g) appropriate treatment of common diseases and injuries (h) the provision of essential drugs (i) relies on all health workers … to work as a health team. . all governments should formulate national health policies, strategies and plans, mobilize political will and resources, used rationally, to ensure phc for all people. national health planning. reforms of the nhs -for example, as discussed in chapter , remuneration increases for family physicians and encouraging group practice with public health nursing support -have become widespread in the uk. leading health maintenance organizations, such as kaiser permanente in the usa and district health systems in canada, have emphasized integrated approaches to health care for registered or geographically defined populations (see chapters - ). this approach is becoming common in the usa in acos, which will be fostered by the obamacare legislation (ppaca). this systematic approach to individual and community health is an integral part of the new public health. the interactions among community public health, personal health services, and health-related behavior, including their management, are the essence of the new public health. how the health system is organized and managed affects the health of the individual and the population, as does the quality of providers. health information systems with epidemiological, economic, and sociodemographic analysis are vital to monitor health status and allow for changing priorities and management. well-qualified personnel are essential to provide services, manage the system, and carry out relevant research and health policy analysis. diffusion of data, health information, and responsibility helps to provide a responsive and comprehensive approach to meet the health needs of the individual and community. the physical, social, economic, and political environments are all important determinants of the health status of the population and the individual. joint action (intersectoral cooperation) between public and non-governmental or community-based organizations is needed to achieve the well-being of the individual in a healthy society. in the s and s, these ideas contributed to an evolving new public health, spurred on by epidemiological changes, health economics, the development of managed care linking health systems, and prepayment. knowledge and self-care skills, as well as community action to reduce health risks, are no less important in this than the roles of medical practitioners and institutional care. all are parts of a coherent holistic approach to health. the concept of selective primary care, articulated in by walsh and warren, addresses the needs of developing countries to select those interventions on a broad scale that would have the greatest positive impact on health, taking into account limited resources such as money, facilities, and human resources. the term selective primary care is meant to define national priorities that are based not on the greatest causes of morbidity or mortality, but on common conditions of epidemiological importance for which there are effective and simple preventive measures. throughout health planning, there is an implicit or explicit selection of priorities for allocation of resources. even in primary care, selection of targets is a part of the process of resource allocation. in modern public health, this process is more explicit. a country with limited resources and a high birth rate will emphasize maternal and child health before investing in geriatric care. this concept has become part of the microeconomics of health care and technology assessment, discussed in chapters and , respectively, and is used widely in setting priorities and resource allocation. in developing countries, cost-effective primary care interventions have been articulated by many international organizations, including iodization of salt, use of oral rehydration therapy (ort) for diarrheal diseases, vitamin a supplementation for all children, expanded programs of immunization, and others that have the potential for saving hundreds of thousands of lives yearly at low cost. in developed countries, health promotions targeted to reduce accidents and risk factors such as smoking, high-fat diets, and lack of exercise for cardiovascular diseases are low-cost public health interventions that save lives and reduce the use of hospital care. targeting specific diseases is essential for efforts to control tb or eradicate polio, but at the same time, development of a comprehensive primary care infrastructure is equally or even more important than the single-disease approach. some disease entities such as hiv/aids attract donor funding more readily than basic infrastructure services such as immunization, and this can sometimes be detrimental to addressing the overall health needs of the population and other neglected but also important diseases. the risk approach selects population groups on the basis of risk and helps to determine interventional priorities to reduce morbidity and mortality. the measure of health risk is taken as a proxy for need, so that the risk approach provides something for all, but more for those in need, in proportion to that need. in epidemiological terms, these are people with higher relative risk or attributed risk. some groups in the general population are at higher risk than others for specific conditions. the expanded programme on immunization (epi), control of diarrhoeal diseases (cdd), and acute respiratory disease (ard) programs of the who are risk approaches to tackling fundamental public health problems of children in developing countries. public health places considerable emphasis on maternal and child health because these are vulnerable periods in life for specific health problems. pregnancy care is based on a basic level of care for all, with continuous assessment of risk factors that require a higher intensity of follow-up. prenatal care helps to identify factors that increase the risk for the pregnant woman or her fetus/newborn. efforts directed towards these special risk groups have the potential to reduce morbidity and mortality. high-risk case identification, assessment, and management are vital to a successful maternal care program. similarly, routine infant care is designed not only to promote the health of infants, but also to find the earliest possible indications of deviation and the need for further assessment and intervention to prevent a worsening of the condition. low birth-weight babies are at greater risk for many short-and long-term hazards and should be given special treatment. all babies are routinely screened for birth defects or congenital conditions such as hypothyroidism, phenylketonuria, and other metabolic and hematological diseases. screening must be followed by investigating and treating those found to have a clinical deficiency. this is an important element of infant care because infancy itself is a risk factor. as will be discussed in chapters and and others, epidemiology has come to focus on the risk approach with screening based on known genetic, social, nutritional, environmental, occupational, behavioral, or other factors contributing to the risk for disease. the risk approach has the advantage of specificity and is often used to initiate new programs directed at special categories of need. this approach can lead to narrow and somewhat rigid programs that may be difficult to integrate into a more general or comprehensive approach, but until universal programs can be achieved, selective targeted approaches are justifiable. indeed, even with universal health coverage, it is still important to address the health needs or issues of groups at special risk. working to achieve defined targets means making difficult choices. the supply and utilization of some services will limit availability for other services. there is an interaction, sometimes positive, sometimes negative, between competing needs and the health status of a population. public health identifies needs by measuring and comparing the incidence or prevalence of the condition in a defined population with that in other comparable population groups and defines targets to reduce or eliminate the risk of disease. it determines ways of intervening in the natural epidemiology of the disease, and develops a program to reduce or even eliminate the disease. it also assesses the outcomes in terms of reduced morbidity and mortality, as well as the economic justification in cost-effectiveness analysis to establish its value in health priorities. because of the interdependence of health services, as well as the total financial burden of health care, it is essential to look at the costs of providing health care, and how resources should be allocated to achieve the best results possible. health economics has become a fundamental methodology in policy determination. the costs of health care, the supply of services, the needs for health care or other health-promoting interventions, and effective means of using resources to meet goals are fundamental in the new public health. it is possible to err widely in health planning if one set of factors is overemphasized or underemphasized. excessive supply of one service diminishes the availability of resources for other needed investments in health. if diseases are not prevented or their sequelae not well managed, patients must use costly health care services and are unable to perform their normal social functions such as learning at school or performing at work. lack of investment in health promotion and primary prevention creates a larger reliance on institutional care, driving health costs upwards, and restricting flexibility in meeting patients' needs. the interaction of supply and demand for health services is an important determinant of the political economy of health care. health and its place in national priorities are determined by the social-political philosophy and resource allocation of a government. the case for action, or the justification for a public health intervention, is a complex of epidemiological, economic, and public policy factors (table . ). each disease or group of diseases requires its own case for action. the justification for public health intervention requires sufficient evidence of the incidence and prevalence of the disease (see chapter ). evidence-based public health takes into account the effectiveness and safety of an intervention; risk factors; safe means at hand to intervene; the human, social, and economic cost of the disease; political factors; and a policy decision as to the priority of the problem. this often depends on subjective factors, such as the guiding philosophy of the health system and the way it allocates resources. some interventions are so well established that no new justification is required to make the case, and the only question is how to do it most effectively. for example, infant vaccination is a cost-effective and cost-beneficial program for the protection of the individual child and the population as a whole. whether provided as a public service or as a clinical preventive measure by a private medical practitioner, it is in the interest of public health that all children be immunized. an outbreak of diarrheal disease in a kindergarten presents an obvious case for action, and a public health system must respond on an emergency basis, with selection of the most suitable mode of intervention. the considerations in developing a case for action are outlined above. need is based on clinical and epidemiological evidence, but also on the importance of an intervention in the eyes of the public. the technology available, its effectiveness and safety, and accumulated experience are important in the equation, as are the acceptability and affordability of appropriate interventions. the precedents for use of an intervention are also important. on epidemiological evidence, if the preventive practice has been seen to provide reduction in risk for the individual and for the population, then there is good reason to implement it. the costs, risks and benefits must be examined as part of the justification to help in the selection of health priorities. health systems research examines the efficiency of health care and promotes improved efficiency and effective use of resources. this is a vital function in determining how best to use resources and meet current health needs. past emphasis on hospital care at the expense of less development of primary care and prevention is still a common issue, particularly in former soviet and developing countries, where a high percentage of total health expenditure goes to acute hospital care with long length of stay, with smaller allocation to preventive and community health care. the result of this imbalance is high mortality from preventable diseases. new drugs, vaccines, and medical equipment are continually becoming available, and each new addition needs to be examined among the national health priorities. sometimes, owing to cost, a country cannot afford to add a new vaccine to the routine. however, when there is good evidence for efficacy and safety of new vaccines, drugs, diagnostic methods or other innovations, it could be applied for those at greatest risk. although there are ethical issues involved, it may be necessary to advise parents or family members to purchase the vaccine independently. clearly, recommending individual purchase of a vaccine is counter to the principle of equity and solidarity, benefiting middleclass families, and providing a poor basis of data for evaluation of the vaccine and its target disease. on the other hand, failure to advise parents of potential benefits to their children creates other ethical problems, but may increase public pressure and insurance system acceptance of new methods, e.g., varicella and hpv vaccines. mass screening programs involving complete physical examinations have not been found to be cost-effective or to significantly reduce disease. in the s and s, routine general health examinations were promoted as an effective method of finding disease early. since the late s, a selective and specific approach to screening has become widely accepted. this involves defining risk categories for specific diseases and bearing in mind the potential for remedial action. early case finding of colon cancer by routine fecal blood testing and colonoscopy has been found to be effective, and pap smear testing to discover cancer of the cervix is timed according to risk category. screening for colorectal cancer is essential for modern health programs and has been adopted by most industrialized countries. outreach programs by visits, telephones, emails or other modern methods of communication are important to contact non-attenders to promote utilization, and have been shown to increase compliance with proven effective measures. these programs are important for screening, follow-up, and maintenance of treatment for hypertension, diabetes, and other conditions requiring long-term management. screening technology is changing and often the subject of intense debate as such programs are costly and their cost-effectiveness is an important matter for policy making: screening for lung cancer is becoming a feasible and effective matter for high-risk groups, whereas breast cancer screening frequency is now in dispute; while nanotechnology and bioengineering promises new methods for cancer screening. the factor of contribution to quality of life should be considered. a vaccine for varicella is justified partly for the prevention of deaths or illness from chickenpox. a stronger the right to health public expectation and social norms argument is often based on the fact that this is a disease that causes moderate illness in children for up to weeks and may require parents to stay home with the child, resulting in economic loss to the parent and society. the fact that this vaccination prevents the occurrence of herpes zoster or shingles later in life may also be a justification. widespread adoption of hepatitis b vaccine is justified on the grounds that it prevents cancer of the liver, liver cirrhosis, and hepatic failure in a high percentage of the population affected. how many cases of a disease are enough to justify an intervention? one or several cases of some diseases, such as poliomyelitis, may be considered an epidemic in that each case constitutes or is an indicator of a wider threat. a single case of polio suggests that another persons are infected but have not developed a recognized clinical condition. such a case constitutes a public health emergency, and forceful organization to meet a crisis is needed. current standards are such that even one case of measles imported into a population free of the disease may cause a large outbreak, as occurred in the uk, france, and israel during through , by contacts on an aircraft, at family gatherings, or even in medical settings. a measles epidemic indicates a failure of public health policy and practice. screening for some cancers, such as cervix and colon, is cost effective. screening of all newborns for congenital disorders is important because each case discovered early and treated effectively saves a lifetime of care for serious disability. assessing a public health intervention to prevent the disease or reduce its impact requires measurement of the disease in the population and its economic impact. there is no simple formula to justify a particular intervention, but the cost-benefit approach is now commonly required to make such a case for action. sometimes public opinion and political leadership may oppose the views of the professional community, or may impose limitations of policy or funds that prevent its implementation. conversely, professional groups may press for additional resources that compete for limited resources available to provide other needed health activities. both the professionals of the health system and the general public need full access to health-related information to take part in such debates in a constructive way. to maintain progress, a system must examine new technologies and justify their adoption or rejection (see chapter ). the association between health and political issues was emphasized by european innovators such as rudolf virchow (and in great britain by edwin chadwick; see chapter ) in the mid-nineteenth century, when the conditions of the working population were such that epidemic diseases were rife and mortality was high, especially in the crowded slums of the industrial revolution. the same observations led bismarck in germany to introduce early forms of social insurance for the health of workers and their families in the s, and to britain's national health insurance, also for workers and families. the role of government in providing universal access to health care was a struggle in individual countries during the twentieth century and lasting into the second decade of the twenty-first century (e.g. president obama's affordable health care act of ). as the concept of public health has evolved, and the cost effectiveness of medical care has improved through scientific and technological advances, societies have identified health as a legitimate area of activity for collective bargaining and government. with this process, the need to manage health care resources has become more clearly defined as a public responsibility. in industrialized countries, each with very different political make-up, national responsibility for universal access to health has become part of the social ethos. with that, the financing and managing of health services have developed into part of a broad concept of public health, and economics, planning, and management have come to be part of the new public health (discussed in chapters - ). social, ethical, and political philosophies have profound effects on policy decisions including allocation of public monies and resources. investment in public health is now recognized as an integral part of socioeconomic development. governments are major suppliers of funds and leadership in health infrastructure development, provision of health services, and health payment systems. they also play a central role in the development of health promotion and regulation of the environment, food, and drugs essential for community health. in liberal social democracies, the individual is deemed to have a right to health care. the state accepts responsibility to ensure availability, accessibility, and quality of care. in many developed countries, government has also taken responsibility to arrange funding and services that are equitably accessible and of high quality. health care financing may involve taxation, allocation, or special mandatory requirements on employers to pay for health insurance. services may be provided by a state-financed and -regulated service or through ngos and/or private service mechanisms. these systems allocate between percent and percent of gross national product (gnp) to health services, with some governments funding over percent of health expenditure; for example, canada and the uk. in communist states, the state organizes all aspects of health care with the philosophy that every citizen is entitled to equity in access to health services. the state health system manages research, staff training, and service delivery, even if operational aspects are decentralized to local health authorities. this model applied primarily to the soviet model of health services. these systems, except for cuba, placed financing of health low on the national priority, with funding less than percent of gnp. in the shift to market economies in the s, some former socialist countries, such as russia, are struggling with poor health status and a difficult shift from a strongly centralized health system to a decentralized system with diffusion of powers and responsibilities. promotion of market concepts in former soviet countries has reduced access to care and created a serious dilemma for their governments. former colonial countries, independent since the s and s, largely carried on the governmental health structures established in the colonial times. most developing countries have given health a relatively low place in budgetary allotment, with expenditures under percent of gnp. since the s, there has been a trend in developing countries towards decentralization of health services and greater roles for ngos, and the development of health insurance. some countries, influenced by medical concepts of their former colonial master countries, fostered the development of specialty medicine in the major centers with little emphasis on the rural majority population. soviet influence in many ex-colonial countries promoted state-operated systems. the who promoted primary care, but the allocations favored city-based specialty care. israel, as an ex-colony, adapted british ideas of public health together with central european sick funds and maternal and child health as major streams of development until the mid- s. a growing new conservatism in the s and s in the industrialized countries is a restatement of old values in which market economics and individualistic social values are placed above concepts of the "common good" of liberalism and socialism in its various forms. in the more extreme forms of this concept, the individual is responsible for his or her own health, including payment, and has a choice of health care providers that will respond with high-quality personalized care. market forces, meaning competition in financing and provision of health services with rationing of services, based on fees or private insurance and willingness and ability to pay, have become part of the ideology of the new conservatism. it is assumed that the patient (i.e., the consumer) will select the best service for his or her need, while the provider best able to meet consumer expectations will thrive. in its purest form, the state has no role in providing or financing of health services except those directly related to community protection and promotion of a healthful environment without interfering with individual choices. the state ensures that there are sufficient health care providers and allows market forces to determine the prices and distribution of services with minimal regulation. the usa retains this orientation in a highly modified form, with percent of the population covered by some form of private or public insurance systems (see chapters and ). modified market forces in health care are part of health reforms in many countries as they seek not only to ensure quality health care for all but also to constrain costs. a free market in health care is costly and ultimately inefficient because it encourages inflation of provider incomes or budgets and increasing utilization of highly technical services. further, even in the most free market societies, the economy of health care is highly influenced by many factors outside the control of the consumer and provider. the total national health expenditure in the usa rose rapidly until reaching over . percent of gross domestic product (gdp) in , the highest of any country, despite serious deficiencies for those without any or with very inadequate health insurance (in total more than percent of the population). this figure compares to some . percent of gdp in canada, which has universal health insurance under public administration. following the defeat of president clinton's national health program, the conservative congress and the business community took steps to expand managed care in order to control costs, resulting in a revolution in health care in the usa (see chapters and ). in the - decade health expenditure in the usa is expected to rise to . percent of gdp, partly owing to increased population coverage with implementation of the ppaca (obamacare). reforms are being implemented in many "socialized" health systems. these may be through incentives to promote achievement of performance indicators, such as full immunization coverage. others are using control of supply, such as hospital beds or licensed physicians, as methods of reducing overutilization of services that generates increasing costs. market mechanisms in health are aimed not only at the individual but also at the provider. incentive payment systems must work to protect the patient's legitimate needs, and conversely incentives that might reduce quality of care should be avoided. fee-for-service promotes high rates of services such as surgery. increasing private practice and user fees can adversely affect middle-and low-income groups, as well as employers, by raising the costs of health insurance. managed care systems, with restraints on fee-for-service medical practice, have emerged as a positive response to the market approach. incentive systems in payments for services may be altered by government or insurance agencies in order to promote rational use of services, such as reduction of hospital stays. the free market approach is affecting planning of health insurance systems in previously highly centralized health systems in developing countries as well as the redevelopment of health systems in former soviet countries. despite political differences, reform of health systems has become a common factor in virtually all health systems since the s, as each government searches for costeffectiveness, quality of care, and universality of coverage. the new paradigm of health care reform sees the convergence of different systems to common principles. national responsibility for health goals and health promotion leads to national financing of health care with regional and managed care systems. most developed countries have long since adopted national health insurance or service systems. some governments may, as in the usa, insure only the highest risk groups such as the elderly and the poor, leaving the working and middle classes to seek private insurers. the nature and direction of health care reform affecting coverage of the population are of central importance in the new public health because of its effects on allocation of resources and on the health of the population. the effects of the economic crisis in the usa are being felt worldwide. while the downturn has largely occurred in wealthier nations, the poor in low-income countries will be among those affected. past economic downturns have been followed by substantial drops in foreign aid to developing countries. as public health gained from sanitary and other control measures for infectious diseases, along with mother and child care, nutrition, and environmental and occupational health, it also gained strength and applicability from advances in the social and behavioral sciences. social darwinism, a political philosophy that assumed "survival of the fittest" and no intervention of the sate to alleviate this assumption, was popular in the early nineteenth century but became unacceptable in industrialized countries, which adopted social policies to alleviate the worst conditions of poverty, unemployment, poor education, and other societal ills. the political approach to focusing on health and poverty is associated with jeremy bentham in britain in the late eighteenth century, who promoted social and political reform and "the greatest good for the greatest number", or utilitarianism. rudolf virchow, an eminent pathologist and a leader in recognizing ill-health and poverty as cause and effect, called for political action to create better conditions for the poor and working-class population. the struggle for a social contract was promoted by pioneer reformists such as edwin chadwick (general report on the sanitary condition of the labouring population of great britain, ), who later became the first head of the board of health in britain, and lemuel shattuck (report of a general plan for the promotion of public and personal health, ) . shattuck was the organizer and first president of the american statistical association. the social sciences have become fundamental to public health, with a range of disciplines including vital statistics and demography (seventeenth century), economics and politics (nineteenth century), sociology (twentieth century), history, anthropology, and others, which provide collectively important elements of epidemiology of crucial significance for survey methods and qualitative research (see chapter ). these advances contributed greatly to the development of methods of studying diseases and risk factors in a population and are still highly relevant to addressing inequalities in health. individuals in good health are better able to study and learn, and be more productive in their work. improvements in the standard of living have long been known to contribute to improved public health; however, the converse has not always been recognized. investment in health care was not considered a high priority in many countries where economic considerations directed investment to the "productive" sectors such as manufacturing and large-scale infrastructure projects, such as hydroelectric dams. whether health is a contributor to economic development or a drain on societies' resources has been a fundamental debate between socially and market-oriented advocates. classic economic theory, both free enterprise and communist, has tended to regard health as a drain on economies, distracting investment needed for economic growth. as a result, in many countries health has been given low priority in budgetary allocation, even when the major source of financing is governmental. this belief among economists and banking institutions prevented loans for health development on the grounds that such funds should focus on creating jobs and better incomes, before investing in health infrastructure. consequently, the development of health care has been hampered. a socially oriented approach sees investment in health as necessary for the protection and development of "human capital", just as investment in education is needed for the long-term benefit of the economy of a country. in , the world bank's world development report: investing in health articulated a new approach to economics in which health, along with education and social development, is seen as an essential precondition for and contributor to economic development. while many in the health field have long recognized the importance of health for social and economic improvement, its adoption by leading international development banking may mark a turning point for investment in developing nations, so that health may be a contender for increased development loans. the concept of an essential package of services discussed in that report establishes priorities in low-and middle-income countries for efficient use of resources based on the burden of disease and cost-effectiveness analysis of services. it includes both preventive and curative services targeted to specific health problems. it also recommends support for comprehensive primary care, such as for children, and infrastructure development including maternity and hospital care, medical and nursing outreach services, and community action to improve sanitation and safe water supplies. reorientation of government spending on health is increasingly being adopted, as in the uk, to improve equity in access for the poor and other neglected sectors or regions of society with added funding for relatively deprived areas to improve primary care services. differential capitation funding as a form of affirmative action to provide for highneeds populations is a useful concept in public health terms to address the inequities still prevalent in many countries. as medical care has gradually become more involved in prevention, and as it has moved into the era of managed care, the gap between public health and clinical medicine has narrowed. as noted above, many countries are engaged in reforms in their health care systems. the motivation is largely derived from the need for cost containment, but also to extend health care coverage to underserved parts of the population. countries without universal health care still have serious inequities in distribution of or access to services, and may seek reform to reduce those inequities, perhaps under political pressures to improve the provision of services. incentives for reform are needed to address regional inequities, and preserving or developing universal access and quality of care, but also on inequities in health between the rich and the poor countries and within even the wealthy countries. in some settings, a health system may fail to keep pace with developments in prevention and in clinical medicine. some countries have overdeveloped medical and hospital care, neglecting important initiatives to reduce the risk of disease. the process of reform requires setting standards to measure health status and the balance of services to optimize health. a health service can set a target of immunizing percent of infants with a national immunization schedule, but requires a system to monitor performance and incentives for changes. a health system may also have failed to adapt to changing needs of the population through lack, or misuse, of health information and monitoring systems. as a result, the system may err seriously in its allocation of resources, with excessive emphasis on hospital care and insufficient attention to primary and preventive care. all health services should have mechanisms for correctly gathering and analyzing needed data for monitoring the incidence of disease and other health indicators, such as hospital utilization, ambulatory care, and preventive care patterns. for example, the uk's nhs periodically undertakes a restructuring process of parts of the system to improve the efficiency of service. this involves organizational changes and decentralization with regional allocation of resources (see chapter ). health systems are under pressures of changing demographic and epidemiological patterns as well as public expectations, rising costs of new technology, financing, and organizational change. new problems must be continually addressed with selection of priority issues and the most effective methods chosen. reforms may create unanticipated problems, such as professional or public dissatisfaction, which must be evaluated, monitored, and addressed as part of the evolution of public health. literacy, freedom of the press, and increasing public concern for social and health issues have contributed to the development of public health. the british medical community lobbied for restrictions on the sale of gin in the s in order to reduce the damage that it caused to the working class. in the late eighteenth and the nineteenth centuries, reforms in society and sanitation were largely the result of strongly organized advocacy groups influencing public opinion through the press. such pressure stimulated governments to act in regulating the working conditions of mines and factories. abolition of the slave trade and its suppression by the british navy in the early nineteenth century resulted from successful advocacy groups and their effects on public opinion through the press. vaccination against smallpox was promoted by privately organized citizen groups, until later taken up by local and national government authorities. advocacy consists of activities of individuals or groups publicly pleading for, supporting, espousing, or recommending a cause or course of action. the advocacy role of reform movements in the nineteenth century was the basis of the development of modern organized public health. campaigns ranged from the reform of mental hospitals, nutrition for sailors to prevent scurvy and beriberi, and labor laws to improve working conditions for women and children in particular, to the promotion of universal education and improved living conditions for the working population. reforms on these and other issues resulted from the stirring of the public consciousness by advocacy groups and the public media, all of which generated political decisions in parliaments (box . ). such reforms were in large part motivated by fear of revolution throughout europe in the mid-nineteenth century and the early part of the twentieth century. trade unions, and before them medieval guilds, fought to improve hours, safety, and conditions of work, as well as social and health benefits for their members. in the usa, collective bargaining through trade unions achieved wage increases and widespread coverage of the working population under voluntary health insurance. unions and some industries pioneered prepaid group practice, the predecessor of health maintenance organizations and managed care or the more recent acos (see chapters and ). through raising public consciousness on many issues, advocacy groups pressure governments to enact legislation to restrict smoking in public places, prohibit tobacco advertising, and mandate the use of bicycle helmets. advocacy groups play an important role in advancing health based on disease groups, such as cancer, multiple sclerosis, and thalassemia, or advancing health issues, such as the organizations promoting breastfeeding, environmental improvement, or smoking reduction. some organizations finance services or facilities not usually provided within insured health programs. such organizations, which can number in the hundreds in a country, advocate the importance of their special concern and play an important role in innovation and meeting community health needs. advocacy groups, including trade unions, professional groups, women's groups, self-help groups, and many others, focus on specific issues and have made major contributions to advancing the new public health. the history of public health is replete with pioneers whose discoveries led to strong opposition and sometimes violent rejection by conservative elements and vested interests in medical, public, or political circles. opposition to jennerian vaccination, the rejection of semmelweiss by colleagues in vienna, and the contemporary opposition to the work of great pioneers in public health such as pasteur, florence nightingale, and many others may deter or delay implementation of other innovators and new breakthroughs in preventing disease. although opposition to jenner's vaccination lasted well into the late nineteenth century in some areas, its supporters gradually gained ascendancy, ultimately leading to the global eradication of smallpox. these and other pioneers led the way to improved health, often after bitter controversy on topics later accepted and which, in retrospect, seem to be obvious. advocacy has sometimes had the support of the medical profession but elicited a slow response from public authorities. david marine of the cleveland clinic and david cowie, professor of pediatrics at the university of michigan, proposed the prevention of goiter by iodization of salt. marine carried out a series of studies in fish, and then in a controlled clinical trial among schoolgirls in - , with startlingly positive results in reducing the prevalence of goiter. cowie campaigned for the iodization of salt, with support from the medical profession. in , he convinced a private manufacturer to produce morton's iodized salt, which rapidly became popular throughout north america. similarly, iodized salt came to be used in many parts of europe, mostly without governmental support or legislation. iodine-deficiency disorders (idds) remain a widespread condition, estimated to have affected billion people worldwide in . the target of international eradication of idds by was set at the world summit for children in , and the who called for universal iodization of salt in . by , nearly percent of households in developing countries consumed adequately iodized salt. china and nigeria, have had great success in recent years with mandatory salt fortification in increasing iodization rates, in china from percent to percent in years. but the problem is not yet gone and even in europe there is inadequate standardization of iodine levels and population follow-up despite decades of work on the problem. professional organizations have contributed to promoting causes such as children's and women's health, and environmental and occupational health. the american academy of pediatrics has contributed to establishing and promoting high standards of care for infants and children in the usa, and to child health internationally. hospital accreditation has been used for decades in the usa, canada, and more recently in australia and the uk. it has helped to raise standards of health facilities and care by carrying out systematic peer review of hospitals, nursing homes, primary care facilities, and mental hospitals, as well as ambulatory care centers and public health agencies (see chapter ). public health needs to be aware of negative advocacy, sometimes based on professional conservatism or economic self-interest. professional organizations can also serve as advocates of the status quo in the face of change. opposition by the american medical association (ama) and the health insurance industry to national health insurance in the usa has been strong and successful for many decades. the passage of the ppaca has been achieved despite widespread political and public opposition, yet was sustained in the us supreme court and is gaining widening popular support as the added value to millions of formerly uninsured americans becomes clear. in some cases, the vested interest of one profession may block the legitimate development of others, such as when ophthalmologists lobbied successfully against the development of optometry, now widely accepted as a legitimate profession. political activism for reform in nineteenth-century britain led to banning and suppressing the slave trade, improvements in working conditions for miners and factory workers, and other major political reforms. in keeping with this tradition, samuel plimsoll ( - ), british member of parliament elected for derby in , conducted a solo campaign for the safety of seamen. his book, our seamen, described ships sent to sea so heavily laden with coal and iron that their decks were awash. seriously overloaded ships, deliberately sent to sea by unscrupulous owners, frequently capsized, drowning many crew members, with the owners collecting inflated insurance fees. overloading was the major cause of wrecks and thousands of deaths in the british shipping industry. plimsoll pleaded for mandatory load-line certificate markers to be issued to each ship to prevent any ships putting to sea when the marker was not clearly visible. powerful shipping interests fought him every inch of the way, but he succeeded in having a royal commission established, leading to an act of parliament mandating the "plimsoll line", the safe carrying capacity of cargo ships. this regulation was adopted by the us bureau of shipping as the load line act in and is now standard practice worldwide. jenner's discovery of vaccination with cowpox to prevent smallpox was adopted rapidly and widely. however, intense opposition by organized groups of antivaccinationists, often led by those opposed to government intervention in health issues and supported by doctors with lucrative variolation practices, delayed the implementation of smallpox vaccination for many decades. ultimately, smallpox was eradicated in , owing to a global campaign initiated by the who. opposition to legislated restrictions on private ownership of assault weapons and handguns is intense in the usa, led by well-organized, well-funded, and politically powerful lobby groups, despite the amount of morbidity and mortality due to gun-associated violent acts (see chapter ). fluoridation of drinking water is the most effective public health measure for preventing dental caries, but it is still widely opposed, and in some places the legislation has been rescinded even after implementation, by wellorganized antifluoridation campaigns. opposition to fluoridation of community water supplies is widespread, and effective lobbying internationally has slowed but has not stopped progress (see chapter ). despite the life-saving value of immunization, opposition still exists in and harms public health protection. opposition has slowed progress in poliomyelitis eradication; for example, radical islamists killed polio workers in northern nigeria in , one of the last three countries with endemic poliomyelitis. resistance to immunization in the s has resulted in the recurrence of pertussis and diphtheria and a very large epidemic of measles across western europe, including the uk, with further spread to the western hemisphere in - (see chapter ). progress may be blocked where all decisions are made in closed discussions, not subject to open scrutiny and debate. public health personnel working in the civil service of organized systems of government may not be at liberty to promote public health causes. however, professional organizations may then serve as forums for the essential professional and public debate needed for progress in the field. professional organizations such as the apha provide effective lobbying for the interests of public health programs and can have an important impact on public policy. in mid- , efforts by the secretary of health and human services in the usa brought together leaders of public health with representatives of the ama and academic medical centers to try to find areas of common interest and willingness to promote the health of the population. in europe too, increasing cooperation between public health organizations is stimulating debate on issues of transnational importance across the region, which, for example, has a wide diversity of standards on immunization practices and food policies. public advocacy has played an especially important role in focusing attention on ecological issues (box . ). in , greenpeace, an international environmental activist group, fought to prevent the dumping of an oil rig in the north sea and forced a major oil company to find another solution that would be less damaging to the environment. an explosion on an oil rig in the gulf of mexico in led to enormous ecological and economic damage as well as loss of life. damages levied on the responsible company (british petroleum) amount to some $ . billion dollars and several criminal negligence charges are pending. greenpeace also continued its efforts to stop the renewal of testing of atomic bombs by france in the south pacific. international protests led to the cessation of almost all testing of nuclear weapons. international concern over global warming has led to growing efforts to stem the tide of air pollution from fossil fuels, coal-burning electrical production, and other manifestations of carbon dioxide and toxic contamination of the environment. progress is far from certain as newly enriched countries such as china and india follow the rising consumption patterns of western countries. public advocacy and rejection of wanton destruction of the global ecology may be the only way to prod consumers, governments, and corporate entities such as the energy and transportation industries to change direction. the pace of change from fossil fuels is slow but has captured public attention, and private companies are seeking more fuel efficiency in vehicles and electrical power production, mainly though the use of natural gas instead of fuel oil and coal for electricity production or better still by wind and solar energy. the search for "green solutions" to the global warming crisis has become increasingly dynamic, with governments, the private sector, and the general public keenly aware of the importance of the effort and the dangers of failure. in the latter part of the twentieth century and the early twenty-first century, prominent international personalities and entertainers have taken up causes such as the removal of land mines in war-torn countries, illiteracy in disadvantaged advocacy is a function in public health that has been important in promoting advances in the field, and one that sometimes places the advocate in conflict with established patterns and organizations. one of the classic descriptions of this function is in henrik ibsen's play an enemy of the people, in which the hero, a young doctor, thomas stockmann, discovers that the water in his community is contaminated. this knowledge is suppressed by the town's leadership, led by his brother the mayor, because it would adversely affect plans to develop a tourist industry of baths in their small norwegian town in the late nineteenth century. the young doctor is taunted and abused by the townspeople and driven from the town, having been declared an "enemy of the people" and a potential risk. the allegory is a tribute to the man of principle who stands against the hysteria of the crowd. the term also took on a far more sinister and dangerous meaning in george orwell's novel and in totalitarian regimes of the s to the present time. populations, and funding for antiretroviral drugs for african countries to reduce maternal-fetal transmission of hiv and to provide care for the large numbers of cases of aids devastating many countries of sub-saharan africa. rotary international has played a key role in polio eradication efforts globally. the public-private consortium global alliance for vaccines and immunization (gavi) has been instrumental in promoting immunization in recent years, with participation by the who, unicef, the world bank, the gates foundation, vaccine manufacturers, and others. this has had an important impact on extending immunization to protect and save the lives of millions of children in deprived countries not yet able to provide fundamental prevention programs such as immunization at adequate levels. gavi has brought vaccines to low-income countries around the world, such as rotavirus vaccine, pentavalent vaccine in myanmar, and pneumococcal vaccine for children in countries in sub-saharan africa, including dr congo. the bill & melinda gates foundation pledged us $ million in to establish gavi, with us $ million per year and us $ billion in to promote the decade of vaccines. international conferences help to create a worldwide climate of advocacy for health issues. international sanitary conferences in the nineteenth century were convened in response to the cholera epidemics. international conferences continue in the twenty-first century to serve as venues for advocacy on a global scale, bringing forward issues in public health that are beyond the scope of individual nations. the who, unicef, and other international organizations perform this role on a continuing basis (see chapter ). criticisms of this approach have focused on the lack of similar effort or donors to address ncds, weak public health infrastructure, and that this frees national governments from responsibility to care for their own children. no one can question, however, that this kind of endeavor has saved countless lives and needs the backing of other aid donors and national government participation. consumerism is a movement that promotes the interests of the purchaser of goods or services. in the s, a new form of consumer advocacy emerged from the civil rights and antiwar movement in the usa. concern was focused on the environment, occupational health, and the rights of the consumer. rachel carson stimulated concern by dramatizing the effects of ddt on wildlife and the environment but inadvertently jeopardized anti-malarial efforts in many countries. this period gave rise to environmental advocacy efforts worldwide, and a political movement, the greens, in western europe. ralph nader showed the power of the advocate or "whistle-blower" who publicizes health hazards to stimulate active public debate on a host of issues related to the public well-being. nader, a consumer advocate lawyer, developed a strategy for fighting against business and government activities and products which endangered public health and safety. his book unsafe at any speed took issue with the us automobile industry for emphasizing profit and style over safety, and led to the enactment of the national traffic and motor safety act of , establishing safety standards for new cars. this was followed by a series of enactments including design and emission standards and seat-belt regulations. nader's work continues to promote consumer interests in a wide variety of fields, including the meat and poultry industries, and coal mining, and promotes greater government regulatory powers regarding pesticide usage, food additives, consumer protection laws, rights to knowledge of contents, and safety standards. consumerism has become an integral part of free market economies, and the educated consumer does influence the quality, content, and price of products. greater awareness of nutrition in health has influenced food manufacturers to improve packaging, content labeling, enrichment with vitamins and minerals, and advertisement to promote those values. low-fat dietary products are available because of an increasingly sophisticated public concerned over dietary factors in cardiovascular diseases. the same process occurred in safe toys and clothing for children, automobile safety features such as mandatory use of car seats for infants, and other innovations that quickly became industry standards in the industrialized world. dangerous practices such as the use of lead paint in toys and melamine contamination of milk products from china capture the public attention quickly and remind public health authorities of the importance of continuous alertness to potential hazards. consumerism can also be exploited by pharmaceutical companies with negative impacts on the health system, especially in the advertising of health products which leads to unnecessary visits to health providers and pressure for approval to obtain the product. the internet has provided people with access to a vast array of information and opinion, and to current literature otherwise unavailable because of the often inadequate library resources of medical and other health professionals. the very freedom of information the internet allows, however, also provides a vehicle for extremist and fringe groups to promote disinformation such as "vaccination causes autism, fluoridation causes cancer", which can cause considerable difficulties for basic public health programs or lead to self-diagnosis of conditions, with often disastrous consequences. advocacy and voluntarism go hand in hand. voluntarism takes many forms, including raising funds for the development of services or operating services needed in the community. it may take the form of fund-raising to build clinics or hospitals in the community, or to provide medical equipment for elderly or handicapped people; or retirees and teenagers working as hospital volunteers to provide services that are not available through paid staff, and to provide a sense of community caring for the sick in the best traditions of religious or municipal concerns. this can also be extended to prevention, as in support for immunization programs, assistance for the handicapped and elderly in transportation, meals-on-wheels, and many other services that may not be included in the "basket of services" provided by the state, health insurance, or public health services. community involvement can take many forms, and so can voluntarism. the pioneering role of women's organizations in promoting literacy, health services, and nutrition in north america during the latter part of the nineteenth and the early twentieth centuries profoundly affected the health of the population. the advocacy function is enhanced when an organization mobilizes voluntary activity and funds to promote changes or needed services, sometimes forcing official health agencies or insurance systems to revise their attitudes and programs to meet these needs. by the early s, canada's system of federally supported provincial health insurance plans covered all of the country. the federal minister of health, marc lalonde, initiated a review of the national health situation, in view of concern over the rapidly increasing costs of health care. this led to articulation of the "health field concept" in , which defined health as a result of four major factors: human biology, environment, behavior, and health care organization (box . ). lifestyle and environmental factors were seen as important contributors to the morbidity and mortality in modern societies. this concept gained wide acceptance, promoting new initiatives that emphasized health promotion in response to environmental and lifestyle factors. conversely, reliance primarily on medical care to solve all health problems could be counterproductive. this concept was a fundamental contributor to the idea of health promotion later articulated in the ottawa declaration, discussed below. the health field concept came at a time when many epidemiological studies were identifying risk factors for cardiovascular diseases and cancers that related to personal habits, such as diet, exercise, and smoking. the concept advocated that public policy needed to address individual lifestyle as part of the overall effort to improve health status. as a result, the canadian federal government established health promotion as a new activity. this quickly spread to many other jurisdictions and gained wide acceptance in many industrialized countries. concern was expressed that this concept could become a justification for a "blame the victim" approach, in which those ill with a disease related to personal lifestyles, such as smokers or aids patients, are seen as having chosen to contract the disease. such a patient might then be considered not to be entitled to all benefits of insurance or care that others may receive. the result may be a restrictive approach to care and treatment that would be unethical in the public health tradition and probably illegal in western jurisprudence. this concept was also used to justify withdrawal from federal commitments in cost sharing and escape from facing controversial health reform in the national health insurance program. during the s and s, outspoken critics of health care systems, such as ivan illytch, questioned the value of medical care for the health of the public. this became a widely discussed, somewhat nihilistic, view towards medical care, and was influential in promoting skepticism regarding the value of the biomedical mode of health care, and antagonism towards the medical profession. in , thomas mckeown presented a historicalepidemiological analysis showing that up to the s, medical care had only a limited impact on mortality rates, although improvements in surgery and obstetrics were notable. he showed that crude death rates in england averaged about per population from to , declining steeply to per in , per in , and per in , when medical care became truly effective. mckeown concluded that much of the improvement in health status over the past several centuries was due to reduced mortality from infectious diseases. this he related to limitation of family size, increased food supplies, improved nutrition and sanitation, specific preventive and therapeutic measures, and overall gains in quality of life for growing elements of the population. he cautioned against placing excessive reliance for health on medical care, much of which was of unproved effectiveness. this skepticism of the biomedical model of health care was part of wider antiestablishment feelings of the s and s in north america. in , milton roemer pointed out that the advent of vaccines, antibiotics, antihypertensives, and other medications contributed to great improvements in infant and child care, and in the management of infectious diseases, hypertension, diabetes, and other conditions. therapeutic gains continue to arrive from teaching centers around the world. vaccine, pharmaceutical, and diagnostic equipment manufacturers continue to provide important innovations that have major benefits, but also raise the cost of health care. the latter issue is one which has stimulated the search for reforms, and search for lower cost technologies such as in treatment of hepatitis c patients, a huge international public health issue. the value of medical care to public health and vice versa has not always been clear, either to public health personnel or to clinicians. the achievements of modern public health in controlling infectious diseases, and even more so in reducing the mortality and morbidity associated with chronic diseases such as stroke and chd, were in reality a shared achievement between clinical medicine and public health (see chapter ). preventive medicine has become part of all medical practice, with disease prevention through early diagnosis and health promotion through individual and community-focused activities. risk factor evaluation determines appropriate screening and individual and community-based interventions. medical care is crucial in controlling hypertension and in reducing the complications and mortality from chd. new modalities of treatment are reducing death rates from first time acute myocardial infarctions. better management of diabetes prevents the early onset of complications. at the same time, the contribution of public health to improving outcomes of medical care is equally important. control of the vaccine-preventable diseases, improved nutrition, and preparation for motherhood contribute to improved maternal and infant outcomes. promotions of reduced exposure to risk factors for chronic disease are a task shared by public health and clinical medical services. both clinical medicine and public health contribute to improved health status. they are interdependent and rely on funding systems for recognition as part of the new public health. during the s, many new management concepts emerged in the business community, such as "management by objective", a concept developed by peter drucker at general motors, with variants such as "zero-based budgeting" developed in the us department of defense (see chapter ). they focused the activities of an organization and its budget on targets, rather than on previous allocation of resources. these concepts were applied in other spheres, but they influenced thinking in health, whose professionals were seeking new ways to approach health planning. the logical application was to define health targets and to promote the efficient use of resources to achieve those targets. this occurred in the usa and soon afterwards in the who european region. in both cases, a wide-scale process of discussion and consensus building was used before reaching definitive targets. this process contributed to the adoption of the targets by many countries in europe as well as by states and many professional and consumer organizations. the usa developed national health objectives in for the year and subsequently for the year , with monitoring of progress in their achievement and development of further targets for and now for . beginning in , state health profiles are prepared by the epidemiology program office of the centers for disease control and prevention based on health indicators recommended by a consensus panel representing public health associations and organizations. the eight mdgs adopted by the un in include halving extreme poverty, reducing child mortality by twothirds, improving maternal health, halting the spread of hiv/aids, malaria, and other diseases, and providing universal primary education, all by the target date of . the mdgs form a common blueprint agreed to by all countries and the world's leading development institutions. the process has galvanized unprecedented efforts to meet the needs of the world's poorest, yet reviews of progress indicate that most developing nations will not meet the targets at current rates of progress. the united nations development programme (undp) global partnership for development report on the mdgs states that if the national development strategies and initiatives are supported by international development partners, the goals can be achieved by . the mdgs were adopted by over nations and provided guidance for national policies and for international aid agencies. the focus was on middle-and low-income countries and their achievements have been considerable but variable (see box . and chapter ) . as of july , extreme poverty was falling in every region, the poverty reduction target had been met, the world had met the target of halving the proportion of people without access to improved sources of water, and the world had achieved parity in primary education between girls and boys. further progress will require sustained political commitment to develop the primary care infrastructure: improved reporting and epidemiological monitoring, consultative mechanisms, and consensus by international agencies, national governments, and non-governmental agencies. the achievement of the targets will also require sustained international support and national commitment with all the difficulties of a time of economic recession. nevertheless, defining a target is crucial to the process. there are encouraging signs that national governments are influenced by the general movement to place greater emphasis on resource allocation and planning on primary care to achieve internationally recognized goals and targets. the successful elimination of smallpox, rising immunization coverage in the developing countries, and increasing implementation of salt iodization have shown that such goals are achievable. while the usa has not succeeded in developing universal health care access, it has a strong tradition of public health and health advocacy. federal, state, and local health authorities have worked out cooperative arrangements for financing and supervising public health and other services. with growing recognition in the s that medical services alone would not achieve better health results, health policy leadership in the federal government formulated a new approach, in the form of developing specific health targets for the nation. in , the surgeon general of the usa published the report on health promotion and disease prevention (healthy people). this document set five overall health goals for each of the major age groups for the year , accompanied by specific health objectives. new targets for the year were developed in three broad areas: to increase healthy lifespans, to reduce health disparities, and to achieve access to preventive health care for all americans. these broad goals are supported by specific targets in health priority areas, each one divided into four major categories: health promotion, health protection, preventive services, and surveillance systems. this set the public health agenda on the basis of measurable indicators that can be assessed year by year. reduce child mortality -progress on child mortality is gaining momentum. the target is to reduce by two-thirds, between and , the under- -year-old mortality rate, from children of every dying to of every . child deaths are falling, but much more needs to be done in order to reach the development goal. revitalizing efforts against pneumonia and diarrhea, while bolstering nutrition, could save millions of children. l mdg . improve maternal health -maternal mortality has nearly halved since , but levels are far removed from the target. the targets for improving maternal health include reducing by three-quarters the maternal mortality ratio and achieve universal access to reproductive health. poverty and lack of education perpetuate high adolescent birth rates. inadequate funding for family planning is a major failure in fulfilling commitments to improving women's reproductive health. l mdg . combat hiv/aids, malaria, tuberculosis, and other diseases -more people than ever are living with hiv owing to fewer aids-related deaths and the continued large number of new infections. in , an estimated . million were living with hiv, up percent from . this persistent increase reflects the continued large number of new infections along with a significant expansion of access to lifesaving antiretroviral therapy, especially in more recent years. l mdg . ensure environmental sustainability -the unparalleled success of the montreal protocol shows that action on climate change is within grasp. the th anniversary of the montreal protocol on substances that deplete the ozone layer, in , had many achievements to celebrate. most notably, there has been a reduction of over percent in the consumption of ozone-depleting substances. further, because most of these substances are also potent greenhouse gases, the montreal protocol has contributed significantly to the protection of the global climate system. the reductions achieved to date leave hydrochlorofluorocarbons (hcfcs) as the largest group of substances remaining to be phased out. l mdg . a global partnership for development -core development aid fell in real terms for the first time in more than a decade, as donor countries faced fiscal constraints. in , net aid disbursements amounted to $ . billion, representing . percent of developed countries' combined national income. while constituting an increase in absolute dollars, this was a . percent drop in real terms over . if debt relief and humanitarian aid are excluded, bilateral aid for development programmes and projects fell by . percent in real terms. equitable and sustainable funding of health services. . developing human resources (educational programs for providers and managers based on the principles of the health for all policy). . research and knowledge: health programs based on scientific evidence. . mobilizing partners for health (engaging the media/ television/internet). . policies and strategies for health for all -national, targeted policies based on health for all. a - review has been commissioned by the european office of the who to assess inequalities in the social determinants of health. while health has improved there are still significant inequalities. factors include variance in local, regional, national, and global economic forces. the european union and the european region of who are both working on health targets for the year . there are competing demands in society for expenditure by the government, and therefore making the best use of resources -money and people -is an important objective. the uk has devolved many of the responsibilities to the constituent countries (england, wales, scotland, and northern ireland) within an overall national framework (box . ). of the health consequences of their decisions and to accept responsibility for health. health promotion policy combines diverse but complementary approaches, including legislation, fiscal measures, taxation, and organizational change. it is a coordinated action that leads to health, income, and social policies that foster greater equity. joint action contributes to ensuring safer and healthier goods and services, healthier public services, and cleaner, more enjoyable environments. health promotion policies require the identification of obstacles to the adoption of healthy public policies in non-health sectors, and ways of removing them. built on progress made from the declaration on primary health care at alma-ata, the aim was to make the healthier choice the easier choice for policy makers as well. the logo of the ottawa charter has been maintained by the who as the symbol and logo of health promotion. health promotion represents activities to enhance and embed the concept of building healthy public policy through: l building healthy public policy in all sectors and levels of government and society l enhancing both self help and social support l developing personal skills through information and education for health l enabling, mediating, and advocating healthy public policy in all spheres l creating supportive environments of mutual help and conservation of the natural environment l reorienting health services beyond providing clinical curative services with linkage to broader social, political, economic, and physical environmental components. (adapted from ottawa charter; health and welfare canada and world health organization, ) an effective approach to health promotion was developed in australia where, in the state of victoria, revenue from a cigarette tax has been set aside for health promotion purposes. this has the effect of discouraging smoking, and at the same time finances health promotion activities and provides a focus for health advocacy in terms of promoting cessation of cigarette advertising at sports events or on television. it also allows for assistance to community groups and local authorities to develop health promotion activities at the workplace, in schools, and at places of recreation. health activity in the workplace involves reduction of work hazards as well as promotion of a healthy diet and physical fitness, and avoidance of risk factors such as smoking and alcohol abuse. in the australian model, health promotion is not only the persuasion of people to change their life habits; it also involves legislation and enforcement towards environmental changes that promote health. for example, this involves mandatory filtration, chlorination, and fluoridation for community water supplies, vitamin and mineral enrichment of basic foods. primary care alliances of service providers are organized including hospitals, community health services serving a sub-district population for more efficient and comprehensive care. these are at the level of national or state policy, and are vital to a health promotion program and local community action. community-based programs to reduce chronic disease using the concept of community-wide health promotion have developed in a wide variety of settings. such a program to reduce risk factors for cardiovascular disease was pioneered in the north karelia project in finland. this project was initiated as a result of pressures from the affected population of the province, which was aware of the high incidence of mortality from heart disease. finland had the highest rates of chd in the world and in the rural area of north karelia the rate was even higher than the national average. the project was a regional effort involving all levels of society, including official and voluntary organizations, to try to reduce risk factors for chd. after years of follow-up, there was a substantial decline in mortality with a similar decline in a neighboring province taken for comparison, although the decline began earlier in north karelia. in many areas where health promotion has been attempted as a strategy, community-wide activity has developed with participation of ngos or any valid community group as initiators or participants. healthy heart programs have developed widely with health fairs, sponsored by charitable or fraternal societies, schools, or church groups, to provide a focus for leadership in program development. a wider approach to addressing health problems in the community has developed into an international movement of "healthy cities". following deliberations of the health of towns commission chaired by edwin chadwick, the health of towns association was founded in by southwood smith, a prominent reform leader of the sanitary movement, to advocate change to reduce the terrible living conditions of much of the population of cities in the uk. the association established branches in many cities and promoted sanitary legislation and public awareness of the "sanitary idea" that overcrowding, inadequate sanitation, and absence of safe water and food created the conditions under which epidemic disease could thrive. in the s, iona kickbush, trevor hancock, and others promoted renewal of the idea that local authorities have a responsibility to build health issues into their planning and development processes. this "healthy cities" approach promotes urban community action on a broad front of health promotion issues (table . ). activities include environmental projects (such as recycling of waste products), improved recreational facilities for young people to reduce violence and drug abuse, health fairs to promote health awareness, and screening programs for hypertension, breast cancer, and other diseases. it combines health promotion with consumerism and returns to the tradition of local public health action and advocacy. the municipality, in conjunction with many ngos, develops a consultative process and program development approach to improving the physical and social life of the urban environment and the health of the population. in , the healthy cities movement involved countries with cities in europe, canada, the usa, the uk, south america, israel, and australia, an increase from cities in . the model now extends to small municipalities, often with populations of fewer than , . networks of healthy cities are the backbone of the movement, with more than member towns and cities across europe. the choice of core themes offers the opportunity to work on priority urban health issues that are relevant to all european cities. topics that are of particular concern to individual cities and/or are challenging and cutting edge for innovative public health action are especially emphasized. healthy cities encourages and supports experimentation with new ideas by developing concepts and implementing them in diverse organizational contexts. a healthy city is a city for all its citizens: inclusive, supportive, sensitive and responsive to their diverse needs and expectations. a healthy city provides conditions and opportunities that encourage, enable and support healthy lifestyles for people of all social groups and ages. a healthy city offers a physical and built environment that encourages, enables and supports health, recreation and well-being, safety, social interaction, accessibility and mobility, a sense of pride and cultural identity and is responsive to the needs of all its citizens. the apha's formulation of the public health role in , entitled the future of public health in america, was presented at the annual meeting in . the apha periodically revises standards and guidelines for organized public health services provided by federal, state, and local governments ( table . ). these reflect the profession of public health as envisioned in the usa where access to medical care is limited for large numbers of the population because of a lack of universal health insurance. public health in the usa has been very innovative in determining risk groups in need of special care and finding direct and indirect methods of meeting those needs. european countries such as finland have called for setting public health into all public policy, which reflects the vital role that local and county governments can play in developing health-oriented policies. these include policies in housing, recreation, regulation of industrial pollution, road safety, promotion of smoke-free environments, bicycle paths, health impact assessment, and many other applications of health principles in public policy. public health involves both direct and indirect approaches. direct measures in public health include immunization of children, modern birth control, and chronic disease case finding -hypertension, diabetes, and cancer. indirect methods used in public health protect the individual by community-wide means, such as raising standards of environmental safety, ensuring a safe water supply, sewage disposal, and improved nutrition (box . ). in public health practice, the direct and indirect pproaches are both relevant. to reduce morbidity and mortality from diarrheal diseases requires an adequate supply of safe water and waste disposal, and also education of the individual in hygiene and the mother in use of ort, and rotavirus vaccination of all children. the targets of public health action therefore include the individual, family, community, region, or nation, as well as a functioning and health system adopting current best practices for health care and health protection. the targets for protection in infectious disease control are both the individual and the total group at risk. for vaccine-preventable diseases, immunization protects the individual but also has an indirect effect by reducing the risk even for non-immunized persons. in control of some diseases, individual case finding and management reduce risk of the disease in others and the community. for example, tb requires case finding and adequate care among high-risk groups as a key to community control. in malaria control, case finding and treatment are essential together with environmental action to reduce the vector population, to prevent transmission of the organism by the mosquito to a new host. control of ncds, where there is no vaccine for mass application, depends on the knowledge, attitudes, beliefs, and practices of individuals at risk. in this case, the social context is of importance, as is the quality of care to which the individual has access. control and prevention of noninfectious diseases involve strategies using individual and population-based methods. individual or clinical measures include professional advice on how best to reduce the risk of the disease by early diagnosis and implementation of appropriate therapy. population-based measures involve indirect measures with government action banning cigarette advertising, or direct taxation on cigarettes. mandating food quality standards, such as limiting the fat content of meat, and requiring food labeling laws are part of the control of cardiovascular diseases. the way individuals act is central to the objective of reducing disease, because many non-infectious diseases are dependent on behavioral risk factors of the individual's choosing. changing the behavior of the individual means addressing the way a person sees his or her own needs. this can be influenced by the provision of information, but how someone sees his or her own needs is more complex than that. an individual may define needs differently from the society or the health system. reducing smoking among women may be difficult to achieve if smoking is thought to reduce appetite and food intake, given the social message that "slim is beautiful". reducing smoking among young people is similarly difficult if smoking is seen as fashionable and diseases such as lung cancer seem very remote. recognizing how individuals define needs helps the health system to design programs that influence behavior that is associated with disease. public health has become linked to wider issues as health care systems are reformed to take on both individual and population-based approaches. public health and mainstream medicine have found increasingly common ground in addressing the issues of chronic disease, growing attention to health promotion, and economics-driven health care reform. at the same time, the social ecology approaches have shown success in slowing major causes of disease, including heart disease and aids, and the biomedical sciences have provided major new technology for preventing major health problems, including cancer, heart disease, genetic disorders, and infectious diseases. technological innovations unheard of just a few years ago are now commonplace, in some cases driving up costs of care and in others replacing older and less effective care. at the same time, resistance of important pathogenic microorganisms to antibiotics and pesticides is producing new challenges from diseases once thought to be under control, and newly emerging infectious diseases challenge the entire health community. new generations of antibiotics, antidepressants, antihypertensive medications, and other treatment methods are changing the way many conditions are treated. research and development in the biomedical to improve the quality of public health practice and performance of public health systems sciences are providing means of prevention and treatment that profoundly affect disease patterns where they are effectively applied. the technological and organizational revolutions in health care are accompanied by many ethical, economic, and legal dilemmas. the choices in health care include heart transplantation, an expensive life-saving procedure, which may compete with provision of funds and labor resources for immunizations for poor children or for health promotion to reduce smoking and other risk factors for chronic disease. new means of detecting and treating acute conditions such as myocardial infarction and peptic ulcers are reducing hospital stays, and improving long-term survival and quality of life. imaging technology has been an important development in medicine since the advent of x-rays in the early twentieth century. technology has forged ahead with high-technology instruments and procedures, new medication, genetic engineering, and important low-technology gains such as impregnated bed nets, simplified tests for hiv and tb, and many other "game changers". new technologies that can enable lower cost diagnostic devices, electronic transmission, and distant reading of transmitted imaging all open up possibilities for advanced diagnostic capacities in rural and less developed countries and communities. molecular biology has provided methods of identifying and tracking movement of viruses such as polio and measles from place to place, greatly expanding the potential for appropriate intervention. the choices in resource allocation can be difficult. in part, these add political commitment to improve health, competent professionally trained public health personnel, the public's level of health information, and legal protection, whether through individuals, advocacy, or regulatory approaches for patients' rights. these are factors in a widening methodology of public health. the centers for disease control and prevention (morbidity and mortality weekly report) in summarized great achievements of public health in the usa, with an extension of the lifespan by over years and improvements in many measures of quality of life. they were updated in a similar summary report in , showing continuous progress, and a global version which was also encouraging in its scope of progress (table . ). these achievements were also seen in all developed countries over the past century and are beginning to be seen in developing countries as well. they reflect a successful application of a broad approach to prevention and health promotion along with improved medical care and growing access to its benefits. in the past several decades alone, major new innovations are leading to greater control of cardiovascular disease, cancer prevention, and many other improvements to health affecting hundreds of millions of people. a similar report by the cdc shows global progress in the first decade of the twenty-first century, while mdg reports show progress on all eight target topics, although not at uniformly satisfactory rates. these achievements are discussed throughout this text. this successful track record is very much at the center of a new public health involving a wide range of programs and activities, shown to be feasible and benefiting from continuing advances in science and understanding of social and management issues affecting health care systems worldwide. public health issues have received new recognition in recent years because of a number of factors, including a growing understanding among the populace at different levels in different countries that health behavior is a factor in health status and that public health is vital for protection against natural or human-made disasters. the challenges are also increasingly understood: preparation for bioterrorism, avian influenza, rising rates of diabetes and obesity, high mortality rates from cancer, and a wish for prevention to be effective. health systems offer general population benefits that go beyond preventing and treating illness. appropriately designed and managed, they: l provide a vehicle to improve people's lives, protecting them from the vulnerability of sickness, generating a sense of life security, and building common purpose within society l ensure that all population groups are included in the processes and benefits of socioeconomic development l generate the political support needed to sustain them over time. health systems promote health equity when their design and management specifically consider the circumstances and needs of socially disadvantaged and marginalized populations, including women, the poor, and groups who experience stigma and discrimination, enabling social action by these groups and the civil society organizations supporting them. health systems can, when appropriately designed and managed, contribute to achieving the millennium development goals. the mdgs selected by the un in have eight global targets for the year , including four directly related to public health (discussed above, box . ). these are a recognition and a challenge to the international community and public health as a profession and as organized systems. formal education in newly developing schools of public health is increasing in europe, including many countries of eastern europe, and beginning to develop in india and sub-saharan africa. but there is delay in establishing centers of postgraduate education and research in many developing countries which are concentrating their educational resources on training physicians. many physicians from developing nations are moving to the developed countries, which have become dependent on these countries for a significant part of their supply of medical doctors. progress in implementation of the mdgs is mixed in sub-saharan africa, making some progress in immunization, but falling back on other goals. proposals to renew global health targets following the end-stage of the mdg health goals will need to add a focus on ncds, which account for percent of global deaths, including . million premature deaths below the age of (undp). economic growth has been hampered by the global recession since , which will affect continued progress with many other factors of changing population dynamics, the economics of prevention versus expensive treatment costs, and the high costs of health care. environmental degradation with high levels of carbon dioxide contamination is a growing concern, with disastrous global warming and consequent effects of drought, flooding, hurricane, and elevated particulate matter-induced asthma and effects on cardiovascular disease. the potential for the development of basic and medical sciences in genetics, nanotechnology, and molecular biology shows enormous promise for health benefits as yet unimagined. at the same time, the effectiveness of health promotion has shown dramatic successes in reducing the toll of aids, reducing smoking, and increasing consciousness of nutrition and physical fitness in the population, and of the tragic effects of poverty and poor education on health status. the ethics of public health issues are complex and changing with awareness that failure to act on strong evidence-based policies is itself ethically problematic. the future of public health is not as a solo professional sector; it is at the heart of health systems, without which societies are open to chronic and infectious diseases that are preventable, affecting the society as a whole in economic and development matters. there is an expanding role of private donors in global health efforts, such as the rotary club and the polio eradication program, gavi with immunization and bed-nets in sub-saharan africa, and bilateral donor countries' help in reducing the toll of aids in sub-saharan africa. the new public health has emerged as a concept to meet a whole new set of conditions, associated with increasing longevity and aging of the population, with the post-world war ii baby-boom generation reaching the over- age group facing the growing importance of chronic diseases. inequalities in health exist in and between affluent and developing societies, as well as within countries, even those having advanced health care systems. regional inequalities are seen across the european region in an east-west gradient and globally a north-south divide of extremes of inequality. the global environmental and ecological degradation and pollution of air and water present grave challenges for developed and developing countries worldwide. yet optimism can be derived from proven track records of success in public health measures that have already been implemented. many of the underlying factors are amenable to prevention through social, environmental, or behavioral change and effective use of medical care. the new public health idea has evolved since alma-ata, which articulated the concept of health for all, followed by a trend in the late s to health in all policies and establishing health targets as a basis for health planning. during the late s and early s, the debate on the future of public health in the americas intensified as health professionals looked for new models and approaches to public health research, training, and practice. this debate helped to redefine traditional approaches of social, community, and preventive medicine. the search for the "new" in public health continued with a return to the health for all concept of alma-ata (renewed in ) and a growing realization that the health of both the individual and the society involves the management of personal care services and community prevention, with a comprehensive approach taking advantage of advancing technology and experience of best practices globally. the new public health is an extension of the traditional public health. it describes organized efforts of society to develop healthy public policies: to promote health, to prevent disease, and to foster social equity within a framework of sustainable development. a new, revitalized public health must continue to fulfill the traditional functions of sanitation, protection, and related regulatory activities, but in addition to its expanded functions. it is a widened philosophy and practical application of many different methods of addressing health, and preventing disease and avoidable death. it necessarily addresses inequities so that programs need to meet special needs of different groups in the population according to best standards, limited resources, and population needs. it is proactive and advocates interventions within legal and ethical limits to promote health as a value in and of itself and as an economic gain for society as well for its individual members. the new public health is a comprehensive approach to protecting and promoting the health status of the individual and the society, based on a balance of sanitary, environmental, health promotion, personal, and community-oriented preventive services, coordinated with a wide range of curative, rehabilitative, and long-term care services. it evolves with new science, technology, and knowledge of human and systems behavior to maximize health gains for the individual and the population. the new public health requires an organized context of national, regional, and local governmental and non-governmental programs with the object of creating healthful social, nutritional, and physical environmental conditions. the content, quality, organization, and management of component services and programs are all vital to its successful implementation. whether managed in a diffused or centralized structure, the new public health requires a systems approach acting towards achievement of defined objectives and specified targets. the new public health works through many channels to promote better health. these include all levels of government and parallel ministries; groups promoting advocacy, academic, professional, and consumer interests; private and public enterprises; insurance, pharmaceutical, and medical products industries; the farming and food industries; media, entertainment, and sports industries; legislative and law enforcement agencies; and others. the new public health is based on responsibility and accountability for defined populations in which financial systems promote achievement of these targets through effective and efficient management, and cost-effective use of financial, human, and other resources. it requires continuous monitoring of epidemiological, economic, and social aspects of health status as an integral part of the process of management, evaluation, and planning for improved health. the new public health provides a framework for industrialized and developing countries, as well as countries in political-economic transition such as those of the former soviet system. they are at different stages of economic, epidemiological, and sociopolitical development, each attempting to ensure adequate health for its population with limited resources. the challenges are many, and affect all countries with differing balances, but there is a common need to seek better survival and quality of life for their citizens (table . ). the object of public health, like that of clinical medicine, is better health for the individual and for society. public health works to achieve this through indirect methods, such as by improving the environment, or through direct means such as preventive care for mothers and infants or other atrisk groups. clinical care focuses directly on the individual patient, mostly at the time of illness. but the health of the individual depends on the health promotion and social programs of the society, just as the well-being of a society depends on the health of its citizens. the new public health consists of a wide range of programs and activities that link individual and societal health. the "old" public health was concerned largely with the consequences of unhealthy settlements and with safety of food, air, and water. it also targeted the infectious, toxic, and traumatic causes of death, which predominated among young people and were associated with poverty. a summary of the great achievements of public health in the twentieth and in the early twenty-first century in the industrialized world is included in chapter and throughout this text. these achievements are reflective of public health gains throughout the industrialized world and are encourage and leverage national, state, and local partnerships to build a stronger foundation for public health preparedness and investigate health problems and health hazards in the community . inform, educate, and empower people about health issues . mobilize community partnerships to identify and solve health problems . develop policies and plans that support individual and community health efforts . enforce laws and regulations that protect health and ensure safety evaluate effectiveness, accessibility, and quality of personal and population-based health services vision, mission and goals guidelines on food fortification with micronutrients. who, geneva. alliance for health policy and systems research essential public health services healthy communities, . model standards for community attainment of the year national health objectives determinants of adult mortality in russia: estimates from sibling data commission on social determinants and health. closing the gap in a generation: health equity through action on the social determinants of health compression of morbidity in the elderly institute of medicine. who will keep the public healthy? educating public health professionals for the st century global alliance for vaccine and immunization (gavi) chronic disease prevention and the new public health the evolution, impact and significance of healthy cities/healthy communities world health organization. ottawa charter for health promotion: an international conference on health promotion behavioral and social sciences and public health at cdc. mmwr health in all policies: seizing opportunities, implementing policies. ministry of social affairs and health new perspectives on the health of canadians: a working document new perspective on the health of canadians: years later the us healthy people initiative: its genesis and its sustainability mortality from cardiovascular and cerebrovascular diseases in europe and other areas of the world: an update strategic review of health inequalities in england post. department of health primary care (extended version): ten key actions could globally ensure a basic human right at almost unnoticeable cost public health in europe: power, politics, and where next health: a vital investment for economic development in eastern europe and central asia. european observatory on health systems and policies. who, european regional office it is not just the broad street pump addressing the epidemiologic transition in the former soviet union: strategies for health systems and public health reform in russia what is the "new public health"? millenium development goals: progress chart united nations development programme, millennium development goals. eight goals for healthy people healthy people. the surgeon general's report on health promotion and disease prevention the millennium development goals: a cross-sectoral analysis and principles for goal-setting after selective primary health care: an interim strategy for disease control in developing countries declaration of alma-ata. international conference on primary health care healthy cities networks across the who, european region preamble to the constitution of the world health organization as adopted by the international health conference regional office for europe. health -health for all in the st century. who regional office for europe, copenhagen. world health organization, . regional office for europe. who european healthy cities network. available at:. who regional office for europe leading health indicators selected for incorporate the original objectives in healthy people , which served as a basis for planning public health activities for many state and community health initiatives. for each of the leading health indicators, specific objectives and subobjectives derived from healthy people are used to monitor progress. the specific objectives set for healthy people are listed in box . . thirteen new topic areas are listed for , such as older adults, genomics, dementias, and social determinants of health. these provide guidelines for national, state, and local public health agencies as well as insurance providers, primary care services, and health promotion advocates. a key issue will be in reducing regional, ethnic, and socioeconomic health disparities.the process of working towards health targets in the usa has moved down from the federal level of government to the state and local levels. professional organizations, ngos, as well as community and fraternal organizations are also involved. the states are encouraged to prepare their own targets and implementation plans as a condition for federal grants, and many states require county health departments to prepare local profiles and targets.diffusion of this approach encourages state and local initiatives to meet measurable program targets. it also sets a different agenda for local prestige in competitive terms, with less emphasis on the size of the local hospital or other agencies than on having the lowest infant mortality or the least infectious disease among neighboring local authorities. the who european region document "health -health for all in the st century" addresses health in the twentyfirst century, with principles and objectives for improving the health of europeans, within and between countries of europe. the health targets include: . closing the health gap between countries. . closing the health gap within countries. . a healthy start in life (supportive family policies). . health of young people (policies to reduce child abuse, accidents, drug use, and unwanted pregnancies). . healthy aging (policies to improve health, self-esteem, and independence before dependence emerges). . improving mental health. . reducing communicable diseases. . reducing non-communicable diseases. . reducing injury from violence and accidents. . a healthy and safe physical environment. . healthier living (fiscal, agricultural, and retail policies that increase the availability of and access to and consumption of vegetables and fruits). . reducing harm from alcohol, drugs, and tobacco. . a settings approach to health action (homes should be designed and built in a manner conducive to sustainable health and the environment). . multisectoral responsibility for health. . an integrated health sector and much stronger emphasis on primary care. . managing for quality of care using the european health for all indicators to focus on outcomes and compare the effectiveness of different inputs. the uk national health service (nhs) has semi-autonomous units in england, scotland, wales, and northern ireland. they are funded from the central uk nhs but with autonomy within national guidelines. the nhs has defined national health outcomes for improvements grouped around five domains, each comprised of key indicators aimed at improving health with reducing inequalities. l preventing people from dying prematurely from causes amenable to health care for all ages: l the target diseases include cardiovascular, respiratory, and liver diseases, and cancer (with focus on cancer of breast, lung, and colorectal cancer) l reducing premature death in people with serious mental illnesses l reducing infant mortality, neonatal mortality, still births, and deaths in young children l increasing -year survival for children with cancer. health improvement; help people to live healthy lifestyles, healthy choices, reduce health inequalities, protection from major incidents and other threats, while reducing health inequalities. l health care, public health and preventing premature mortality; reduce the numbers of people living with preventable ill-health and people dying prematurely, while reducing the gap between communities.source: uk department of health. available at: https://www.gov.uk/government/organisations/department-of-health/about#our-priorities, https:// www.gov.uk/government/uploads/system/uploads/attachment_data/ file/ /improving-outcomes-and -supporting-transparency-part- a.pdf. pdf, and https://www.gov.uk/government/uploads/system/uploads/attach-ment_data/file/ / -nhs-outcomes-framework- - .pdf. pdf [accessed june ] . national policy in health ultimately relates to health of the individual. the various concepts outlined in the health field concept, community-oriented primary health care, health targets, and effective management of health systems, can only be effective if the individual and his or her community are knowledgeable participants in seeking solutions. involving the individual in his or her own health status requires raising levels of awareness, knowledge, and action. the methods used to achieve these goals include health counseling, health education, and health promotion (figure . ).health counseling has always been a part of health care between the doctor or nurse and the patient. it raises levels of awareness of health issues of the individual patient. health education has long been part of public health, dealing with promoting consciousness of health issues in selected target population groups. health promotion incorporates the work of health education but takes health issues to the policy level of government and involves all levels of government and ngos in a more comprehensive approach to a healthier environment and personal lifestyles.health counseling, health education, and health promotion are among the most cost-effective interventions for improving the health of the public. while costs of health care are rising rapidly, demands to control cost increases should lead to greater emphasis on prevention, and adoption of health education and promotion as an integral part of modern life. this should be carried out in schools, the workplace, the community, commercial locations (e.g., shopping centers), and recreation centers, and in the political agenda.psychologist abraham maslow described a hierarchy of needs of human beings. every human has basic requirements including physiological needs of safety, water, food, warmth, and shelter. higher levels of needs include recognition, community, and self-fulfillment. these insights supported observations of efficiency studies such as those of elton mayo in the famous hawthorne effect in the s, showing that workers increased productivity when acknowledged by management in the objectives of the organization (see chapter ). in health terms, these translate into factors that motivate people to positive health activities when all barriers to health care are reduced.modern public health faces the problem of motivating people to change behavior; sometimes this requires legislation, enforcement, and penalties for failure to comply, such as in mandating car seat-belt use. in other circumstances it requires sustained performance by the individual, such as the use of condoms to reduce the risk of sti and/or hiv transmission. over time, this has been developed into a concept known as knowledge, attitudes, beliefs, and practices (kabp), a measurable complex that cumulatively affects health behavior (see chapter ). there is often a divergence between knowledge and practice; for example, the knowledge of the importance of safe driving, yet not putting this into practice. this concept is sometimes referred to as the "kabp gap". the health belief model has been a basis for health education programs, whereby a person's readiness to take action for health stems from a perceived threat of disease, a recognition of susceptibility to disease and its potential severity, and the value of health. action by an individual may be triggered by concern and by knowledge. barriers to appropriate action may be psychological, financial, or physical, including fear, time loss, and inconvenience. spurring action to avoid risk to health is one of the fundamental goals in modern health care. the health belief model is important in defining any health intervention in that it addresses the emotional, intellectual, and other barriers to taking steps to prevent or treat disease.health awareness at the community and individual levels depends on basic education levels. mothers in developing countries with primary or secondary school education are more successful in infant and child care than less educated women. agricultural and health extension services reaching out to poor and uneducated farm families in north america in the s were able to raise consciousness of safe self-health practices and good nutrition, and when this was supplemented by basic health education in schools, generational differences could be seen in levels of awareness of the importance of balanced nutrition. secondary prevention with diabetics and patients with chd hinges on education and awareness of nutritional and physical activity patterns needed to prevent or delay a subsequent myocardial infarction. the who sponsored the first international conference on health promotion held in ottawa, canada, in ( figure . ) . the resulting ottawa charter defined health promotion and set out five key areas of action: building healthy public policy, creating supportive environments, strengthening community action, developing personal skills, and reorienting health services. the ottawa charter called on all countries to put health on the agenda of policy makers in all sectors and at all levels, directing them to be aware a typical healthy city has a population in the multiple thousands, often multilingual, with an average middleclass income. a healthy cities project builds a coalition of municipal and voluntary groups working together in a continuing effort to improve quality of service, facilities, and living environment. the city is divided into neighborhoods, engaged in a wide range of activities fostered by the project. municipalities have traditionally had a leading role in sanitation, safe water supply, building and zoning laws and regulation, and many other responsibilities in public health (see chapter ). the healthy cities or communities movement has elevated this to a higher level with policies to promote health in all actions. some examples are listed of municipal, advocacy group, and higher governmental activities for healthier city environments: working with senior levels of government, other departments in the municipalities, religious organizations, private donors, and the ngo sector to innovate and especially to improve conditions in poverty-afflicted areas of cities is a vital role for health-oriented local political leadership. human ecology, a term introduced in the s and revived in the s, attempted to apply theory from plant and animal life to human communities. it evolved as a branch of demography, sociology, and anthropology, addressing the social and cultural contexts of disease, health risks, and human behavior. human ecology addresses the interaction of humans with and adaptation to their social and physical environment.parallel subdisciplines of social, community, and environmental psychology, medical sociology, anthropology, and other social sciences contributed to the development of this academic field with wide applications in health-related issues. this led to the incorporation of qualitative research methods alongside the quantitative research methods traditionally emphasized in public health, providing crucial insights into many public health issues where human behavior is a key risk factor.health education developed as a discipline and function within public health systems in school health, rural nutrition, military medicine, occupational health, and many other aspects of preventive-oriented health care, and is discussed in later chapters of this text. directed at behavior modification through information and raising awareness of consequences of risk behavior, this has become a longstanding and major element of public health practice in recent times, being almost the only effective tool to fight the epidemic of hiv and the rising epidemic of obesity and diabetes.health promotion as an idea evolved, in part, from marc lalonde's health field concepts and from growing realization in the s that access to medical care was necessary but not sufficient to improve the health of a population. the integration of the health behavior model, social ecological approach, environmental enhancement, or social engineering formed the basis of the social ecology approach to defining and addressing health issues (table . ).individual behavior depends on many surrounding factors, while community health also relies on the individual; the two cannot be isolated from one another. the ecological perspective in health promotion works towards changing people's behavior to enhance health. it takes into account factors not related to individual behavior, which are determined by the political, social, and economic environment. it applies broad community, regional, or national approaches that are needed to address severe public health problems, such as controlling hiv infection, tb, malnutrition, stis, cardiovascular disorders, violence and trauma, and cancer. beginning to affect the health situation in countries in transition from the socialist period. countries emerging from developing status are also showing signs of mixed progress in the dual burden of infectious and maternal/child health issues, along with growing exposure to the chronic diseases of developed nations such as cardiovascular diseases, obesity, and diabetes. the new public health synthesizes traditional pub lic health with management of personal services and community action for a holistic approach. evaluation of costeffective public health and medical interventions to reduce the burden of disease also contributes to the need to seek and apply new approaches to health. the new public health will continue to evolve as a framework drawing on new ideas, science, technology, and experiences in public health throughout the world. it must address the growing recognition of social inequality in health, even in developed countries with universal health programs with improved education and social support systems. for a complete bibliography and guidance for student reviews and expected competencies please see companion web site at http://booksite.elsevier.com/ bibliography key: cord- -w pm fs authors: riad, abanoub; boccuzzi, michela; pold, ave; krsek, martin title: the alarming burden of non‐communicable diseases in covid‐ new normal: implications on oral health date: - - journal: oral dis doi: . /odi. sha: doc_id: cord_uid: w pm fs the coronavirus disease (covid‐ ) outbreak has triggered massive debates within dental professional organizations about prioritization of offered services, served groups, and required protective measures.(volgenant et al., ) while navigating through the post‐outbreak era, we aim to demonstrate the importance of continuous global focus on the burden of non‐communicable diseases (ncds) such as oral diseases. oral diseases are the most prevalent ncds worldwide consuming one‐fifth of out‐of‐pocket health expenditure and being recognized as the third most expensive condition to treat in europe.(peres et al., ) the world health organization (who) recommends prioritizing common risk factor approaches in all interventions targeting ncds in order to draw attention to the multifaceted relationship between oral diseases and chronic conditions like diabetes, cardiovascular disease, and cancers.(sheiham & watt, ) the coronavirus disease outbreak has triggered massive debates within dental professional organizations about prioritization of offered services, served groups, and required protective measures. (volgenant et al., ) while navigating through the post-outbreak era, we aim to demonstrate the importance of continuous global focus on the burden of non-communicable diseases (ncds) such as oral diseases. oral diseases are the most prevalent ncds worldwide consuming one-fifth of out-of-pocket health expenditure and being recognized as the third most expensive condition to treat in europe. (peres et al., ) the world health organization (who) recommends prioritizing common risk factor approaches in all interventions targeting ncds in order to draw attention to the multifaceted relationship between oral diseases and chronic conditions like diabetes, cardiovascular disease, and cancers. (sheiham & watt, ) ncds have been recognized for long as the leading cause of mortality and disability worldwide; in addition, they are highly associated with the severity and fatality rates of covid- indicating that prevention and control of ncds are integral parts of the covid- response. unfortunately, the ncds burden is predicted to rise in the next period due to the paradigm in prioritizing covid- over ncds. (kluge et al., ) however being inevitable; non-pharmacologic interventions impose unprecedented risks to people living with ncds. physical distancing can enhance behavioural risk factors like smoking and physical sedentary. reorientation of national health budgets will negatively impact the continuity of palliative care due to decreased supply of essential medicines and technologies and restricted access to healthcare workers. this increase in the levels of ncds will create a vicious circle between the two pandemics-the ncds and covid- -thus exacerbating health inequities. (kluge et al., ) besides the bidirectional relationship between periodontitis and diabetes, periodontal diseases is a risk factor of lung diseases, including chronic obstructive pulmonary disease (copd). periodontitis is strongly linked to hypertension with an array of pathophysiologic mechanisms, including pro-inflammatory cytokines. chronic inflammation is recently suggested as the common factor in both periodontitis and cancers. (cullinan et al., ; yao et al., ) oral diseases and major ncds share common etiological factors, chronicity mechanisms, and control requirements, implying that the burden of oral diseases may unprecedentedly increase. a recent cochrane review revealed that access to elective dental care may be substantially restricted during the covid- "new normal" period. this global ban on elective dental procedures will have a strong impact on public oral health, and patients' oral health-related quality of life.(covid- dental services evidence review working group, this article is protected by copyright. all rights reserved ) the immediate increase in stress and anxiety levels in response to the covid- outbreak, especially in patients with ncds, can deteriorate adherence to health-promoting behaviours, including oral hygiene. (horenstein et al., ) public apprehension of infection may contribute to resistance to dental treatment; which in turn will increase the levels of dental anxiety. (gonzález-olmo et al., ) as pandemics hit the lower socio-economic groups the most, the financial recession will restrain millions of people from seeking dental treatments, including emergency interventions. to conclude, the burden of oral disease should be adequately investigated during the next months in order to avoid a surging demand for dental care that may collapse our limitedly operating facilities. oral health promotion programs and tele-dentistry applications are now needed more than ever to stabilize the curve of oral diseases. recommendations for the re-opening of dental services: a rapid review of international sources periodontal disease and systemic health: current status perceived vulnerability to coronavirus infection: impact on dental practice how does anxiety sensitivity increase risk of chronic medical conditions prevention and control of non-communicable diseases in the covid- response. lancet accepted article this article is protected by copyright oral diseases: a global public health challenge the common risk factor approach: a rational basis for promoting oral health. community dentistry and oral epidemiology infection control in dental health care during and after the sars-cov- outbreak association of periodontal disease with oral cancer: a meta-analysis the authors declare that there is no conflict of interest. key: cord- -m tbdfri authors: khandia, rekha; dadar, maryam; munjal, ashok; dhama, kuldeep; karthik, kumaragurubaran; tiwari, ruchi; yatoo, mohd. iqbal; iqbal, hafiz m.n.; singh, karam pal; joshi, sunil k.; chaicumpa, wanpen title: a comprehensive review of autophagy and its various roles in infectious, non-infectious, and lifestyle diseases: current knowledge and prospects for disease prevention, novel drug design, and therapy date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: m tbdfri autophagy (self-eating) is a conserved cellular degradation process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. autophagy dysfunction can have various pathological consequences, including tumor progression, pathogen hyper-virulence, and neurodegeneration. this review describes the mechanisms of autophagy and its associations with other cell death mechanisms, including apoptosis, necrosis, necroptosis, and autosis. autophagy has both positive and negative roles in infection, cancer, neural development, metabolism, cardiovascular health, immunity, and iron homeostasis. genetic defects in autophagy can have pathological consequences, such as static childhood encephalopathy with neurodegeneration in adulthood, crohn’s disease, hereditary spastic paraparesis, danon disease, x-linked myopathy with excessive autophagy, and sporadic inclusion body myositis. further studies on the process of autophagy in different microbial infections could help to design and develop novel therapeutic strategies against important pathogenic microbes. this review on the progress and prospects of autophagy research describes various activators and suppressors, which could be used to design novel intervention strategies against numerous diseases and develop therapeutic drugs to protect human and animal health. phosphatidylinositol- -kinase (pi k) inhibitors and beclin inhibits the starvation-induced mitochondrial autophagy, but not the neurotoxin ( -methyl- -phenylpyridinium)-mediated autophagy [ ] [ ] [ ] . although autophagy was discovered over years ago [ ] , its molecular mechanisms were only understood in the late s following a genetic screening in yeast, which revealed mutations in autophagy-related genes. at least yeast autophagy genes (atgs) have been identified, many of which have mammalian cell homologs [ ] . many molecular mechanisms have been explored to reveal the basic processes underlying autophagy. multiple signaling pathways focus on two protein complexes to initiate autophagy, the ulk (unc -like autophagy activating kinase ) protein kinase complex and the pi kc -c (class iii phosphatidylinositol -kinase complex i) lipid kinase complex [ ] . novel autophagy regulators with rna-related activities have also been shown to be involved in this process [ ] . furthermore, upstream signaling pathways common to both autophagy and apoptosis are known to be induced by er stress via signaling molecules such as perk/atf , ire α, atf , and ca + [ ] . the details of these mechanisms will shed light on the different forms of autophagy and the numerous intermediates involved. three types of autophagy [macroautophagy, microautophagy, and chaperone-mediated autophagy (cma)] are depicted in figure . autophagy refers to the process of delivering cytoplasmic or extracellular components to the lysosomes of an animal cell or the vacuoles of plant or yeast cells [ ] . the production and maturation of autophagosomes are directly regulated by location, timing, and intensity [ ] . the phosphoinositide-binding protein, hs bp , is a negative regulator of autophagosome biogenesis that regulates the lipid composition and phosphatidic acid (pa) levels of autophagosome precursor membranes [ ] . increased levels of systemic autophagy have been reported in caenorhabditis macroautophagy is initiated when a portion of cytoplasm containing a cellular organelle is sequestered to form the autophagosome [ ] . the autophagosome fuses with the lysosome or late endosomal multivesicular bodies (mvbs) to degrade the materials within it. atg (microtubule-associated protein a/ b-light chain , lc , is an atg homolog in humans) was the first autophagosomal protein to be characterized [ ] . macroautophagy can be classified as cargo-specific or non-selective [ , , ] . mitophagy has been observed in yeasts when a shift occurs between non-fermentable and fermentable carbon sources, such as glucose, following which the surplus mitochondrial population undergoes mitophagy [ , ] . the first protein identified to cause mitophagy in yeast was uth p, a member of the sun family, which is present in the outer mitochondrial membrane and allows excessive mitochondria to be removed during starvation [ ] . the mitochondrial outer membrane protein, atg , is a receptor for selective autophagy [ ] that is not conserved in mammalian species; instead, fundc and bnip , bnip l/nix, and sqstm /p act as mitophagy receptors, and are dependent upon hypoxia, erythrocyte maturation, and damage-induced mitophagy, respectively [ , , ] . pexophagy is also induced in saccharomyces cerevisiae and pichia pastoris via the atg and ppatg receptors, respectively, when the fungal medium is switched from an oleic acid or methanol to a glucose or nitrogen starvation medium [ , ] . starvation has also been shown to induce non-selective macroautophagy [ ] , whereas mitochondrial phospholipids have been demonstrated to be required for autophagy [ ] . the machinery required for selective autophagy has been studied extensively using yeast cells, revealing that the cytoplasm-to-vacuole targeting (cvt) pathway is used to specifically transport vacuolar hydrolases into the vacuole of budding yeast cells [ ] . a high degree of curvature in the initiating membranes (phagophores or isolation membranes) is a prominent feature of cvt vesicles during mammalian autophagy [ ] . after the lysosome has formed vesicles by invaginating and engulfing small sections of the cytoplasm, lysosomal proteases degrade the contents of these vesicles [ ] . microautophagy occurs during the biogenesis of multi-vesicular bodies (mvbs), which deliver soluble proteins to the late endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for transport (escrt) i and iii and the heat-shock cognate protein (hsc ). hence, microautophagy involves both endocytic and autophagic components [ , ] . only proteins with a c-terminal pentapeptide kferq motif undergo cma; the hsc cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome [ , ] . chaperones bound to the substrate are transported to the lysosomal surface, where they interact with the monomeric lamp- a [ , ] . lamp- a must form a multiprotein complex to translocate the substrate [ ] ; lamp- a complex assembly is a dynamic process that occurs when the substrate binds to the receptor. the unfolded substrate protein (chaperon-mediated) is then translocated into the lysosome by lamp- a for degradation, following which lamp- a disassembles and its monomers are degraded in lipid microdomains. the levels of lamp- a tightly regulate the rate of cma at the lysosomal membrane [ , ] . in the mammalian anti-viral defense system, a cell-autonomous autophagy mechanism has been identified wherein cellular p adaptor-mediated autophagic viral protein clearance induces cell survival [ ] . some positive-strand rna viruses, including picornaviruses and influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal fusion) [ ] [ ] [ ] . consequently, studying the interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic virotherapies [ ] . the process of cma is depicted in figure . after the lysosome has formed vesicles by invaginating and engulfing small sections of the cytoplasm, lysosomal proteases degrade the contents of these vesicles [ ] . microautophagy occurs during the biogenesis of multi-vesicular bodies (mvbs), which deliver soluble proteins to the late endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for transport (escrt) i and iii and the heat-shock cognate protein (hsc ). hence, microautophagy involves both endocytic and autophagic components [ , ] . only proteins with a c-terminal pentapeptide kferq motif undergo cma; the hsc cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome [ , ] . chaperones bound to the substrate are transported to the lysosomal surface, where they interact with the monomeric lamp- a [ , ] . lamp- a must form a multiprotein complex to translocate the substrate [ ] ; lamp- a complex assembly is a dynamic process that occurs when the substrate binds to the receptor. the unfolded substrate protein (chaperon-mediated) is then translocated into the lysosome by lamp- a for degradation, following which lamp- a disassembles and its monomers are degraded in lipid microdomains. the levels of lamp- a tightly regulate the rate of cma at the lysosomal membrane [ , ] . in the mammalian anti-viral defense system, a cell-autonomous autophagy mechanism has been identified wherein cellular p adaptormediated autophagic viral protein clearance induces cell survival [ ] . some positive-strand rna viruses, including picornaviruses and influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal fusion) [ ] [ ] [ ] . consequently, studying the interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic virotherapies [ ] . the process of cma is depicted in figure . autophagy is an evolutionarily conserved process induced via multiple signaling pathways by numerous stimuli including nutrient starvation [ , ] , hypoxia [ , ] , oxidative stress [ , ] , pathogen infection [ , ] , and er stress [ ] . in the presence of nutritional substances and cytokines, mechanistic/mammalian target of rapamycin (mtor) can prevent apoptosis and stimulate cell growth [ ] , whereas stress and nutrient starvation inhibit mtor to initiate autophagy via at least four molecular complexes, including the unc- -like kinase (ulk) complex, consisting of ulk- , atg , atg , and fak-family interacting protein (fip ); the pi k complex, consisting of atg , vacuolar protein sorting (vps) , vps , beclin , and beclin -regulated autophagy protein (ambra ) [ ] [ ] [ ] ; transmembrane protein complexes, including atg and wipi; and two ubiquitin-like protein conjugation systems (atg and lc ) [ , ] . autophagy is initiated by the assembly of the ulk complex, which phosphorylates ambra and leads to activation of the pi k complex [ , ] . class iii pi k is known to participate in various membrane trafficking events, whilst pi k and beclin mediate membrane nucleation. the atg -atg -atg complex is recruited to the pre-autophagosomal structure (pas) where it associates with the outer membrane of the phagophore, essentially preventing the premature fusion of vesicles and lysosomes [ ] . the second ubiquitin-like system stimulates the binding of phosphatidylethanolamine (pe) and atg /microtubule-associated protein light chain (lc ). lc has a high affinity for the lysosome when bound to the phagosome (laposome); thus, any engulfed pathogens will be killed and degraded at a higher rate [ ] . atg , atg , and atg process lc into lc -ii, a molecular marker for autophagosomes [ ] that is present on both its inner and outer surfaces and is essential for the expansion and completion of the autophagic membrane. following autophagosomal closure, the atg -atg -atg complex dissociates from the autophagosome. atg is required for the formation of intraluminal vesicles and is localized within the autolysosome for acidification [ ] ; atg is also translocated to the site of autophagosome formation where it provides a membrane to elongate the limiting membrane, known as the phagophore [ ] . the autophagosome then fuses to the lysosome to form the autolysosome, which is regulated by lysosomal membrane proteins and cytoskeletal proteins [ ] . the lamp- / protein controls autophagosomal maturation. genetic mutations in lamp- are known to cause danon disease, a glycogen storage disorder linked to hypertrophic cardiomyopathy, skeletal muscle weakness, and intellectual disability [ ] . within the autolysosome, hydrolytic enzymes digest the internalized cargo and the internal autophagosome membrane, then the digested products such as amino acids are released into the cytosol to be recycled. autophagosomes are also directly related to cell trafficking pathways. recently, holland et al. identified that the phosphoinositide-binding protein hs bp negatively regulates autophagosome production [ ] . hs bp is thought to reduce phospholipase d (pld ) activity and its localization to atg l and transferrin receptor (tfrc)-positive vesicles. it is also known to regulate the levels of pa and the lipid content of autophagosome precursor membranes [ ] . two large families of e ubiquitin ligases, trim and cullin, have been recognized as important autophagy regulators which promote or inhibit the process, respectively [ ] . the gtpase ras-related protein in brain (rab ) also plays a key role in autophagy regulation, particularly in modulating its flux [ ] . knockdown of the small gtpase rab has been shown to inhibit pterostilbene-induced autophagy in vascular endothelial cells (vecs), whilst its upregulation stimulates autophagy in vecs [ ] . under basal autophagy conditions in humans, proteomic analysis of the autophagy interaction network (ain) revealed a network of interactions between candidate proteins [ ] . in order to identify the proteins modulating starvation-induced autophagy, genome-wide screening of sirna in a gfp-lc -expressing human cell line was carried out [ ] , shortlisting nine proteins. one of these, short coiled-coil protein (scoc), forms an essential starvation-sensitive trimeric complex with uv radiation resistance-associated gene (uvrag) and wac (ww domain-containing adapter protein with coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. genome-wide studies in c. elegans identified genes that promote autophagy when inactivated [ ] . long ncrnas (lncrnas), which are longer than nucleotides and do not encode proteins, often possess regulatory functions; for example, mir - has been found to regulate atg expression. rna-linked strategies have revealed several autophagy regulators such as rna-binding proteins (rbps), which are post-transcriptional and co-translational regulators with rna-related functions. surprisingly, various key autophagy proteins, including lc b and lamp- c, have been found to bind rna [ ] . a considerable amount of autophagy research is being carried out worldwide; however, these innovative findings have raised numerous additional questions. to some extent, autophagy research has been protein-centric, and innovative new approaches have been developed to strengthen this focus in recent years. among these, genome-wide screens and proteomics-based strategies have revealed substantial interlinking between autophagy and rnas; however, the precise mechanisms underlying this association require further investigation. future studies must develop and evaluate novel agents that specifically target the autophagy pathway. a pictorial representation of the process of autophagosome formation is presented in figure . starvation-sensitive trimeric complex with uv radiation resistance-associated gene (uvrag) and wac (ww domain-containing adapter protein with coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. genome-wide studies in c. elegans identified genes that promote autophagy when inactivated [ ] . long ncrnas (lncrnas), which are longer than nucleotides and do not encode proteins, often possess regulatory functions; for example, mir - has been found to regulate atg expression. rna-linked strategies have revealed several autophagy regulators such as rna-binding proteins (rbps), which are post-transcriptional and co-translational regulators with rna-related functions. surprisingly, various key autophagy proteins, including lc b and lamp- c, have been found to bind rna [ ] . a considerable amount of autophagy research is being carried out worldwide; however, these innovative findings have raised numerous additional questions. to some extent, autophagy research has been protein-centric, and innovative new approaches have been developed to strengthen this focus in recent years. among these, genomewide screens and proteomics-based strategies have revealed substantial interlinking between autophagy and rnas; however, the precise mechanisms underlying this association require further investigation. future studies must develop and evaluate novel agents that specifically target the autophagy pathway. a pictorial representation of the process of autophagosome formation is presented in figure . liu and levine [ ] described a novel form of non-apoptotic autophagic gene-dependent cell death, termed autosis, which is mediated by the na + /k + -atpase pump. autosis involves enhanced cell-substrate adhesion, focal ballooning of the perinuclear space, and dilation and fragmentation of the endoplasmic reticulum. the tat-beclin peptide complex may initiate autosis, with the fusion of the evolutionarily conserved, -amino acid-long beclin domain with amino acids from the hiv tat protein transduction domain aiding the cellular entry of the fusion peptide [ ] . the tat-beclin fusion peptide has been shown to inhibit the replication of hiv, chikv, sindbis (sinv), and wnv, as well as intracellular bacteria such as listeria monocytogenes [ ] . tat-beclin treatment also reduced mortality in neonatal mice infected with chikv and wnv, demonstrated using a tunel assay, and cleared mutant huntingtin protein aggregates [ ] . autosis can be partially rescued by knocking down atg or atg or using -methyladenine. under serum/amino acid starvation, approximately % of dying cells exhibit a morphology similar to that of cells treated with tat-beclin and autosis is selectively blocked when the na + /k + -atpase pump is inhibited [ ] . during cerebral hypoxia or ischemia, the neonatal brain releases cardiac glycosides (ouabain or endobain), which inhibit the na + /k + -atpase pump and reduce autosis [ ] . autosis has also been observed in patients with severe liver anorexia nervosa who display focal ballooning of the perinuclear space, convoluted nucleus, dilated and fragmented er, empty vacuoles, and several autolysosomes in their hepatocytes [ ] . ischemic injury can also lead to autosis in other organs, including the kidney and heart, which is attenuated in beclin +/− mice [ , ] . autophagy can promote or inhibit cell death depending on the cellular context; many other death mechanisms are intricately involved in the processes, with several mechanistic links elucidated between autophagy and other death mechanisms. autophagy and apoptosis regulation overlap when the bh domain of the beclin autophagy protein interacts with anti-apoptotic proteins of the bcl- family, including bcl- , bcl-xl, and mcl- [ ] [ ] [ ] [ ] . the bh domain has a critical role in the interaction between these proteins and has been shown to interact with the receptor domain of the bcl- family in nutrient-rich cells [ ] . beclin -mediated autophagy is inhibited by er-localized bcl- [ ] ; the transgenic expression of bcl- was shown to inhibit autophagy in mouse heart muscles. beclin mutants, which are unable to bind to bcl- , induce higher levels of autophagy than their wild-type counterparts [ , ] ; hence the physical beclin -bcl-xl/bcl- interaction regulates beclin -mediated autophagy [ ] . abt , a compound which mimics the bh domain and thus inhibits this interaction, increases the aggregation of lc , an autophagy marker which is present on autophagosomes [ ] , in both nutrient-rich and nutrient-deprived media. furthermore, the knockdown of beclin and other essential atg proteins using sirna heteroduplexes was shown to reduce abt -stimulated lc aggregation [ ] . atg is a dual-functioning protein that participates in both autophagy and apoptosis [ ] ; non-conjugated atg can bind and inhibit mcl- and bcl- via a bh -like domain to positively regulate mitochondrial apoptosis. atg knockout inhibits the release of cytochrome c from the mitochondria and apoptosis, whilst abnormal atg expression represses the anti-apoptotic activity of mcl- [ ] . autophagy promotes apoptosis by degrading a negative regulator of the fas ligand [ ] ; however, it can also protect cells against apoptosis induced by tumor necrosis factor (tnf)-related apoptosis-inducing ligand (trail) by altering the concentrations of bcl family members [ ] . similarly, components were found to be degraded by autophagy during developmental apoptosis [ ] , whilst it was recently shown that inhibiting autophagy increased apoptosis and accelerated mortality in murine sepsis models with inadequate autophagy pathways in cd + t cells, indicating that autophagy has a functional role against apoptosis and immunosuppression in t cells in sepsis [ ] . furthermore, trail combined with a novel chalcone derivative, chal- , was found to remarkably increase lung cancer cell cytotoxicity via autophagy-mediated apoptosis [ ] . necroptosis is often associated with inflammation [ ] . the relationship between autophagy and necroptosis is complex, elusive, and slightly controversial since reports have indicated that necroptosis may promote [ ] , inhibit [ , ] , or do not affect autophagy [ ] . in several cell lines, including l cells, lymphocytes, and cancer cells, autophagy is activated in the presence of tnfα and under starvation to suppress necroptosis [ ] . the apoptosis-inhibiting peptide, carbobenzoxy-val-ala-asp (zvad), prevents autophagy and induces necroptosis in response to tnfα by regulating lysosomal cathepsins, highlighting the pro-survival function of autophagy against necroptosis [ , ] . similarly, inhibiting the mtor signaling pathway can prevent apoptosis and even enhance necroptosis, whereas starvation, which induces autophagy, protects cells from zvad-mediated necroptotic death [ ] . sirtuins (sirt) are nad + -dependent protein deacetylases which are actively involved in both autophagy and necroptosis, as well as transcription, stress resistance, and aging. sirt- deacetylates various components of the autophagy pathway, including atg , atg , and atg [ ] , thus promoting autophagy. in cancer cells, dissociation of the foxo transcription factor from sirt- during oxidative stress or starvation results in the acetylation and binding of foxo to atg , which subsequently induces autophagy [ ] . the binding of sirt- to receptor-interacting protein (rip) mediates rip deacetylation in response to tnfα; rip and rip then form a complex, which triggers necroptosis [ ] . the switch between necroptosis and apoptosis is achieved by recruiting necrosome components to autophagy machinery. atg knockdown reduced the association between ripk and mlkl, suggesting that atg is important in trail-induced necrosome activation. furthermore, atg knockout in the atg -/-df- cell line inhibited autophagy but promoted apoptosis [ ] . autophagy machinery also affects the mechanism of cell death by promoting efficient necrosomal activation and mlkl phosphorylation, thus inducing necroptosis [ ] . several anti-cancer agents, including sorafenib, cause deficient autophagosome formation and facilitate the interaction between p and ripk, resulting in cell death by necroptosis [ ] ; however, there is still much to be elucidated about the interplay between these two processes. necrosis refers to the increase in cell volume caused by organelle swelling, which results in plasma membrane rupture and the loss of intracellular contents. when atp is depleted, the cell is unable to undergo apoptosis and undergoes necrosis instead [ ] . poly adp ribose polymerase (parp ) is an enzyme with roles in dna repair, transcriptional regulation, and chromatin modification [ ] . parp over-activation decreases the atp reservoir and induces necrotic cell death by bypassing energy-dependent apoptotic cell death [ ] . atp depletion also activates amp-activated kinases (ampk) [ ] , which induce autophagy by activating the ulk complex or inhibiting mtor signaling [ ] . thus, dna damage-induced parp activation leads to a decline in atp levels, ampk activation, mtor inhibition, and autophagy induction [ ] . parp plays a dual role in autophagy and necrosis since autophagy is a pro-survival mechanism, whilst necrosis is a pro-death mechanism. the fate of the cell depends on the balance between autophagy and necrosis, where autophagy represents the final attempt of the cell to survive before necrosis. autophagy plays a beneficial role against infectious diseases by simultaneously degrading pathogens and activating the host immune system [ ] . this enables infections to be countered directly, by killing infectious agents, and indirectly, by inducing host immunity against pathogens. autophagy provides an excellent intracellular defense system against bacterial pathogens, including salmonella enterica serovar typhimurium [ , ] , listeria monocytogenes [ , ] , and shigella flexneri [ ] . anti-bacterial autophagy is termed xenophagy [ , , ] . numerous cellular, membrane-associated, or cytoplasmic moieties modulate xenophagy; and those cells unable to carry out xenophagy, exhibit higher rates of infection. bcl-xl regulates xenophagy, and bcl-xl knockout cells are more susceptible to streptococcus pyogenes infection [ ] . the infection of non-phagocytic cells by shigella flexneri is dependent upon type-iii secretion system (t ss) effector proteins [ ] which reorganize the host cell cytoskeleton, ruffle the cell membrane, and cause bacterial uptake. following internalization, bacterial peptidoglycans are detected by nucleotide-binding oligomerization domain (nod)-like receptors (nlrs) which trigger a pro-inflammatory immune response [ ] (figure ). the bacteria-sensing nod proteins interact with atg l to initiate anti-bacterial autophagosome biogenesis in response to bacterial invasion [ ] . intracellular bacterial sensing either by nlrs or sequestosome- -like receptors (slrs) recruits autophagy proteins, including unc- -like kinase (ulk) / and lipid kinase complexed with beclin and atg l , to initiate phagophore membrane nucleation and engulf invading bacteria [ ] . mutant c. elegans with defective autophagy genes exhibit increased susceptibility to bacterial infection [ ] . in addition, it has been reported that hlh- /tfeb-mediated autophagy and autophagy pathways can regulate the tolerance of c. elegans to bacillus thuringiensis infection by protecting against its pore-forming toxins [ ] , suggesting a novel association between intrinsic epithelial defenses and hlh- -mediated autophagy against in vivo bacterial attacks. liang et al. [ ] reported that beclin overexpression inhibits sindbis virus replication, indicating that autophagy protects against infectious pathogens. autophagy may activate innate immunity against mycobacteria via pattern recognition receptors (prrs) or non-receptor-mediated processes [ ] . infection with group a streptococcus species (gas; streptococcus pyogenes) induces anti-apoptotic bcl-xl expression which inhibits autophagy directly by suppressing autophagosome-lysosome fusion, and indirectly by interacting with beclin -uvrag to suppress gas internalization [ ] . in addition, mycobacterium tuberculosis is known to induce mir expression in human macrophages and monocytes and adversely affect their antimicrobial activities and innate host immune responses against the bacterial infection by targeting dram (dna damage-regulated autophagy modulator ), which is a critical element of the autophagy response [ ] . the ubiquitin ligase, smurf , has also been shown to control m. tuberculosis replication in human macrophages by associating with bacteria in the lungs of patients with pulmonary tuberculosis. the murine macrophage cell line, raw . , has been used to study bacillus amyloliquefaciens sc -induced autophagy and its anti-bacterial response against escherichia coli; b. amyloliquefaciens stimulated autophagy by increasing the expression of beclin and the atg -atg -atg complex, but not activating the akt/mtor signaling pathway [ ] . several autophagy-inducing drugs have been used to treat microbial infections; for example, ar- [ -amino-n-[ -[ -( phenanthrenyl)- -(trifluoromethyl)- h-pyrazol- -yl] phenyl]-acetamide] inhibits phosphoinositide-dependent kinase- and eliminates salmonella typhimurium in murine macrophages and francisella tularensis in human leukemic thp- macrophages [ , ] . furthermore, α, -dihydroxycholecalciferol, a form of vitamin d, can enhance autophagy and inhibit human immunodeficiency virus (hiv) replication in macrophages [ ] . the bacteria-sensing nod proteins interact with atg l to initiate anti-bacterial autophagosome biogenesis in response to bacterial invasion [ ] . intracellular bacterial sensing either by nlrs or sequestosome- -like receptors (slrs) recruits autophagy proteins, including unc- -like kinase (ulk) / and lipid kinase complexed with beclin and atg l , to initiate phagophore membrane nucleation and engulf invading bacteria [ ] . many bacteria have evolved mechanisms to overcome autophagy and allow them to replicate within infected cells or even within autophagosomes. these bacteria may express receptors to prevent or enhance phagosome formation, capture nutrient containing phagosomes, subvert autophagy machinery, prevent fusion, or resist autophagy. certain bacteria hijack autophagosomes and use the by-products of autophagic degradation for microbial replication [ ] . anaplasma (formerly ehrlichia) phagocytophilum, the causative agent of human anaplasmosis, uses the effector anaplasma translocated substrate (ats- ) to enhance autophagosome formation and acquire nutrients from inside the autophagosome [ ] . after entering the cell, a. phagocytophilum replicates inside a double-lipid bilayer membrane associated with lc (atg ), beclin , and atg but lacking lysosomal markers. inhibiting autophagy with -methyladenine did not prevent bacterial internalization but arrested its growth [ ] , indicating that the autophagic machinery had been subverted to facilitate bacterial proliferation. another bacterium, yersinia pseudotuberculosis, replicates intracellularly inside specific compartments called yersinia-containing vacuoles (ycvs), which contain autophagy markers; however, ycvs are not acidified and sustain bacterial replication [ ] . during y. pestis infection, lc -i is conjugated with pe to recruit lc -ii, a marker of autophagy progression, to the phagosomal membrane [ ] . a similar mechanism is used by coxiella burnetii, the causative organism of q fever. coxiella-replicative vacuoles contain lc , beclin , and rab ; overexpression of these proteins increases the number of coxiella-replicative vacuoles [ ] . brucella abortus replicates within brucella-containing vacuoles (bcvs) which traffic from the endocytic compartment to the endoplasmic reticulum, where the bacteria proliferate. bacterial proliferation requires the autophagy-initiation proteins ulk , beclin , and atg l; however, atg , atg l , atg b, atg , and lc b are not required [ ] . pathogens such as brucella spp. and porphyromonas gingivalis have evolved to survive inside autophagosomes by preventing its fusion with the lysosome, thus escaping host innate immunity mechanisms [ , ] . salmonella typhimurium studies have revealed that autophagy targets invading intracellular bacterial pathogens for degradation [ ] ; s. typhimurium regulates the sirt /lkb /ampk complex of the mtor pathway by targeting sirt , lkb , and ampk to lysosomes for rapid degradation, restricting autophagy and disrupting ampk-mediated mtor regulation [ ] . autophagy is differentially regulated in tuberculoid and lepromatous leprosy [ ] ; in tuberculoid skin lesion cells, autophagy controls mycobacterium leprae, whereas in lepromatous cells, the blocking of bcl- -mediated autophagy promotes bacterial persistence. ifn-γ may counteract the m. leprae-mediated inhibition of autophagy in lepromatous macrophages as autophagy levels were restored in lepromatous patients who developed the reversal reaction, an inflammatory state associated with augmented ifn-γ and rapamycin treatment, indicating that autophagy is an important innate mechanism associated with m. leprae control in skin macrophages [ ] . autophagy has a beneficial role in cellular defense against invasion by viruses; therefore, it has been used for antiviral immunity [ , , ] . autophagy helps to clear viral pathogens during infection via various molecular mechanisms, regulates immune responses, and prevents harmful overactivation and inflammation [ ] . for example, autophagy increases the presentation of endogenous viral antigens in the peptide grooves of major histocompatibility complex (mhc) class i molecules on the cell surface during herpes simplex virus type (hsv- ) infection. studies of viral peptides have suggested a complex interaction between vacuoles and mhc class i presentation pathways in autophagosomes [ ] . in contrast, mhc class ii molecules continuously accept input from autophagosomes, which facilitates antigen presentation by mhc class ii molecules [ , ] . autophagy is a major component of drosophila immunity against vesicular stomatitis virus (vsv) [ ] as it can deliver viral antigens to tlrs for presentation. during anti-viral signaling, pattern recognition receptors (prrs) at the plasma membrane (i.e., toll- ) that are engaged by vsv stimulate an autophagy-dependent innate immune response mediated by pi k-akt-signaling [ , ] . furthermore, toll/tlr signaling has been shown to regulate the rift valley fever virus (rvfv) replication in both flies and mammals [ ] , whilst sirt , an nad(+)-dependent deacetylase, modulates the activation of dendritic cells and autophagy during induced immune responses against respiratory syncytial virus (rsv) [ ] , thereby directing an effective anti-viral immune response. furthermore, autophagy is stimulated by the salicylamide derivatives against cytopathic bovine viral diarrhea virus (cp-bvdv), a flaviviridae pestivirus [ ] . foot-and-mouth disease virus (fmdv) infection suppresses autophagy and nf-κb anti-viral activities by degrading atg -atg using the viral protein, c pro , suggesting that atg -atg positively modulates anti-viral nf-κb and irf pathways during fmdv infection to limit fmdv proliferation [ ] . however, autophagy is often hijacked by viral pathogens and can be modulated to their own benefit. subverting the autophagic pathway can have adverse consequences by giving pathogens access to nutrients for growth and reproduction [ ] . autophagy plays an important role in viral replication and pathogenesis [ ] , with coronaviruses [ ] , coxsackievirus b [ ] , poliovirus [ ] , hepatitis c virus (hcv) [ , [ ] [ ] [ ] , and denv [ ] all known to stimulate and require autophagy for accelerated replication. for instance, autophagy has been demonstrated to be actively involved in the replication of influenza a virus (iav), which induces autophagosome formation during the early phase of infection and later inhibits autophagosomal maturation by preventing autophagosomal-lysosomal fusion and promoting autophagosomes to accumulate in virus-infected cells [ ] . autophagy-deficient cells are more susceptible to apoptosis upon influenza infection [ , ] , while using pharmacological reagents or rna interference to alter cellular autophagy can impair viral protein accumulation [ ] . human single-chain antibody variable fragments (scfvs) which bind to the influenza a virus ion channel protein (m ) and inhibit viral replication [ ] were found to restore autophagosome maturation suppressed by the infecting virus (personal communication). it has also been reported that hcv can trigger autophagy via immunity-related gtpase m, which promotes hcv replication [ ] . paramyxoviruses such as newcastle disease virus (ndv) have been shown to trigger autophagy in u glioma cells to enhance viral replication [ ] . in addition, modulating ndv-induced autophagy using rapamycin, chloroquine, or small interfering rnas which target genes critical for autophagosome formation (atg and beclin ) affects virus production, suggesting that ndv may utilize autophagy to promote its replication [ ] . human immunodeficiency virus (hiv) uses multiple methods to regulate autophagy and enhance its replication [ , ] . hiv induces the early stages of autophagy but inhibits the later stages which would suppress the production of new virions. the hiv- accessory protein, nef, inhibits autophagosomal maturation by interacting with beclin [ ] , whilst the hiv protein vpr can trigger autophagy in transfected thp- macrophages, indicating that autophagy may be involved in maintaining hiv reservoirs in macrophages [ ] . hsv- [ ] , kaposi's sarcoma-associated herpesvirus (kshv) [ ] , and mouse herpesvirus (mhv- ) encode proteins that bind beclin to prevent autophagy initiation [ ] . during poliovirus (pv) infection, vesicle acidification, which can mature autophagosomes, has been shown to induce the maturation of virions into infectious particles [ ] . one of the most important characteristics of high-risk human papillomavirus (hrhpv) etiopathogenesis is that inhibiting host autophagy could cause cervical cancer via hrhpv [ ] . in epithelial cells, flavivirus ns a-induced autophagy protects infected cells and induces viral replication [ ] . autophagy also plays a critical role in the replication of coronaviruses and the generation of their replicative structures [ ] . coronavirus nonstructural proteins (nsp ) induce the formation of omegasomes and autophagosomes from the er via an omegasome intermediate [ ] . in addition, autophagy has been shown to induce the replication of infectious spleen and kidney necrosis virus (isknv) in the chinese perch brain (cpb) cell line, suggesting complex interactions between isknv and host cells during viral pathogenesis and for anti-viral treatment strategies [ ] . treating fmdv-infected cells with rapamycin, an autophagy inducer, was shown to increase viral replication, whilst inhibiting the autophagosomal pathway using -methyladenine or small-interfering rnas decreased viral replication [ ] . furthermore, disrupting autophagy using the knockdown approach in hepatitis c virus (hcv)-infected hepatocytes stimulated the interferon signaling pathway and induced apoptosis, indicating that hcv-induced autophagy can impair the innate immune response [ ] . suppressing hcv-induced autophagy could be a promising approach for inhibiting exosome-mediated viral transmission [ ] , besides autophagy has been shown to reduce hcv clearance following ifn-α/ribavirin (rbv)-based anti-viral therapy [ ] . a denv study revealed that autophagy inhibitors are better candidate targets than conventional anti-viral therapies using interferons (ifns) [ ] ; upregulating cellular autophagy was reported to inhibit rlr-mediated type-i ifn-independent signaling and cause the antibody-dependent enhancement (ade) of denv [ ] . suppressing autophagic vacuoles has been demonstrated to stimulate the maturation of infectious bursal disease virus [ ] . adenoviral infection may be favored by autophagy via an increase in atp, essential to increase anabolism of the infected cells and amino acid pools for the synthesis of viral proteins. in the later stages of adenoviral infection, atg -atg complex is significantly upregulated as an evidence of enhanced autophagy [ ] ; therefore, autophagy may improve the virulence of some viruses. autophagy genes are involved in the regulation and execution of autophagy [ ] . beclin was the first mammalian gene identified to stimulate autophagy [ ] . some viruses, such as α-, βand γ-herpesviruses, encode the neurovirulence protein, icp . , which associates with atg /beclin and inhibits autophagy by preventing the formation of the pi kinase complex [ ] . the autophagy genes fip , beclin , atg , atg l , atg , atg , and atg have been found to promote the reactivation of latent murine gamma-herpesvirus by inhibiting virus-induced systemic inflammation molecules, such as ifn-γ [ ] . in contrast, autophagy inhibition has been reported as a new molecular mechanism by which hsv- escapes innate immunity, resulting in fatal disease [ ] . the autophagic cell death of alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by h n influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic effects against this virus [ ] . activating the pi k /akt/mtor pathway and inhibiting autophagy have been shown to promote the cellular entry of hpv type [ ] , contrarily autophagy has been shown to be essential for the replication of coronavirus and mouse hepatitis virus (mhv) [ ] . hsv- mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [ ] . autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens through the mhc class ii pathway [ ] . the proviral and anti-viral actions of autophagy are illustrated in figure . [ ] . in contrast, autophagy inhibition has been reported as a new molecular mechanism by which hsv- escapes innate immunity, resulting in fatal disease [ ] . the autophagic cell death of alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by h n influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic effects against this virus [ ] . activating the pi k /akt/mtor pathway and inhibiting autophagy have been shown to promote the cellular entry of hpv type [ ] , contrarily autophagy has been shown to be essential for the replication of coronavirus and mouse hepatitis virus (mhv) [ ] . hsv- mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [ ] . autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens through the mhc class ii pathway [ ] . the proviral and anti-viral actions of autophagy are illustrated in figure . initially, autophagy was thought to be involved in tumor suppression by stimulating gene expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products, and stimulating signaling pathways. the essential atg /beclin gene was found to be lost monoallelically in - % of human prostate, breast, and ovarian cancers [ ] , whereas excessive autophagy stimulation by beclin overexpression has been reported to inhibit tumor progression [ , ] . autophagy causes necrosis and chronic inflammation by inhibiting the release of proinflammatory hmgb , which is involved in tumorigenesis [ ] . in cell-based assays, inhibiting autophagy was shown to enhance cancer cell growth [ ] . p (a signaling adaptor/scaffold protein) is involved in the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency. atg -deficient mice exhibit enhanced accumulation of p and ubiquitinated protein aggregates in hepatocytes and neuron [ ] . autophagy has also been implicated in benign hepatomas [ ] , and the inactivation of beclin and atg was shown to increase the incidence of initially, autophagy was thought to be involved in tumor suppression by stimulating gene expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products, and stimulating signaling pathways. the essential atg /beclin gene was found to be lost monoallelically in - % of human prostate, breast, and ovarian cancers [ ] , whereas excessive autophagy stimulation by beclin overexpression has been reported to inhibit tumor progression [ , ] . autophagy causes necrosis and chronic inflammation by inhibiting the release of pro-inflammatory hmgb , which is involved in tumorigenesis [ ] . in cell-based assays, inhibiting autophagy was shown to enhance cancer cell growth [ ] . p (a signaling adaptor/scaffold protein) is involved in the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency. atg -deficient mice exhibit enhanced accumulation of p and ubiquitinated protein aggregates in hepatocytes and neuron [ ] . autophagy has also been implicated in benign hepatomas [ ] , and the inactivation of beclin and atg was shown to increase the incidence of cancer in mice [ ] . atg -and atg -deficient mice exhibited liver tumors, indicating that defective autophagy can affect the suppression of tumorigenesis [ ] . heterozygous beclin (beclin +/-mutant) was shown to have a high incidence of spontaneous tumors [ , ] , whilst beclin inhibited tumor growth in cell lines such as the breast cancer cell line, mcf- , in which beclin expression was lower than in normal epithelial breast cells [ ] . the uvrag protein was found to suppress the tumorigenicity and proliferation of human colonic cancer cells [ ] (figure ). cancer in mice [ ] . atg -and atg -deficient mice exhibited liver tumors, indicating that defective autophagy can affect the suppression of tumorigenesis [ ] . heterozygous beclin (beclin +/mutant) was shown to have a high incidence of spontaneous tumors [ , ] , whilst beclin inhibited tumor growth in cell lines such as the breast cancer cell line, mcf- , in which beclin expression was lower than in normal epithelial breast cells [ ] . the uvrag protein was found to suppress the tumorigenicity and proliferation of human colonic cancer cells [ ] (figure ). ( ) mtor is implicated in cancer and its substrates include the eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and the ribosomal s kinases (s ks) and , which promote cell cycle progression. the mtor, which is inhibited by rapamycin, induces autophagy. ( ) a novel anti-cancer molecule, ha , which targets hspa /bip was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability [ ] [ ] [ ] [ ] . the conserved protein kinase, mtor, has been implicated in cancer since its substrates (eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and ribosomal s kinases (s ks) and ) promote cell cycle progression [ ] . mtor, which is inhibited by rapamycin [ ] , and molecules such as phosphatase and tensin homolog (pten) and tuberous sclerosis (tsc) (products of tumor suppressor genes) can induce autophagy [ , ] . pogostone, a medicinal herb widely used to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating autophagy and apoptosis via the pi k/akt/mtor axis [ ] . in addition, the novel anti-cancer ) mtor is implicated in cancer and its substrates include the eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and the ribosomal s kinases (s ks) and , which promote cell cycle progression. the mtor, which is inhibited by rapamycin, induces autophagy. ( ) a novel anti-cancer molecule, ha , which targets hspa /bip was shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in cancer cell death through autophagy and apoptosis. autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability [ ] [ ] [ ] [ ] . the conserved protein kinase, mtor, has been implicated in cancer since its substrates (eukaryotic initiation factor e (eif e)-binding proteins ( e-bps) and ribosomal s kinases (s ks) and ) promote cell cycle progression [ ] . mtor, which is inhibited by rapamycin [ ] , and molecules such as phosphatase and tensin homolog (pten) and tuberous sclerosis (tsc) (products of tumor suppressor genes) can induce autophagy [ , ] . pogostone, a medicinal herb widely used to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating autophagy and apoptosis via the pi k/akt/mtor axis [ ] . in addition, the novel anti-cancer molecule ha , which targets hspa /bip, was shown to induce er stress and increase the unfolded protein response, resulting in cancer cell death via autophagy and apoptosis [ ] . trichosanthin (tcs), a kda protein from the bioactive component of the root tuber of trichosanthes kirilowii (chinese cucumber plant; gua lou in mandarin), also exhibited anti-cancer properties against different human ovarian cancer cells via a pathway common to both autophagy and apoptosis [ ] . various factors and mechanisms are involved in autophagy-mediated tumor progression. tumor-induced nutrient shortage, cell debris (degraded proteins), inflammation, and oxidative cascades are all molecular mechanisms affecting tumor progression. during nutrient starvation, autophagy induction promotes the survival of normal cells and may also promote tumor cell survival; however, hypoxia, metabolic stress, energy shortage, oxidative stress-damaged mitochondria, and organelles can be caused by cancer-causing genes or cancer treatments [ ] . the undifferentiated colon cancer cell line, ht- , and other transformed cells have shown an increased tendency to degrade autophagic proteins [ , , ] . the elevated expression of the autophagy signature protein, bnip , a pro-apoptotic bcl- member, has been demonstrated in colorectal and gastric epithelial carcinomas, suggesting that bnip expression may be important for the development of these cancers [ ] . the activation of autophagy and peroxisome proliferator-activated receptor gamma (pparγ) was shown to protect colon cancer cells against apoptosis induced by the interaction between butyrate and docosahexaenoic acid (dha) in a cell type-dependent manner [ ] . additionally, an atg /lc family member implicated in autophagy and tumor suppression was associated with alterations to cell death and cytokine secretion in mice lacking gamma-aminobutyric acid receptor-associated protein (gabarap) [ ] . it has been well documented that inhibiting autophagy in cancer cells increases their death, with this strategy proving most useful in tumors that behave like ras-activated tumors [ ] . inhibiting autophagy is expected to cause ubiquitinated proteins to accumulate and p levels to increase; in hepatic tumors, autophagy suppresses spontaneous tumorigenesis via cell-intrinsic pathways whilst p accumulation promotes tumor formation [ ] . the elimination of damaged organelles via autophagy may allow cancer cells to survive despite the stress caused by chemotherapeutic agents [ ] . it has also been reported that upregulating autophagy after chemotherapy causes cancer cells to enter a dormant state, which may then propagate at a later stage [ , ] . the state of cell cycle arrest, termed senescence, has been postulated to underlie autophagy-induced tumor cell dormancy [ ] . ras-induced senescence is mediated by autophagy, with autophagy inhibition delaying senescence [ ] . moreover, it has been reported that psmd /gankyrin stimulates autophagy in hepatocellular carcinoma (hcc) in response to starvation or stress. a physical association occurs between psmd and atg , and is translocated to the nucleus to bind to atg promote and upregulates atg expression [ ] . there is growing evidence that autophagy performs both physiological and pathological functions in the nervous system, and that autophagic stimulation plays critical roles in neuronal survival and activity. autophagy is the only method by which neurons degrade and excrete expired organelles, and is responsible for clearing abnormal intracytoplasmic contents from normal cells which would otherwise cause protein accumulation and damage neuronal activity, inducing severe functional impairment. autophagy also clears protein aggregates from old neurons; thus, inhibiting autophagy can lead to neuronal degeneration and intraneuronal protein accumulation. mutations in atg confined to neural tissues can cause impaired growth, progressive motor and behavioral deficits, prominent neurodegeneration, and axonal swelling in regions of the brain with increased levels of ubiquitinylated proteins, indicating that autophagy has a neuroprotective role [ ] . in mammals, the absence of atg and atg can cause severe neurodegeneration, further supporting the neuroprotective role of autophagy [ , ] . furthermore, neuronal death can be attributed to the loss of basal autophagy or an imbalance in autophagic flux. in some neurodegenerative diseases, such as alzheimer's, parkinson's, and huntington's, as well as in the brain or spinal cord trauma, the damaged neurons exhibit abnormally high numbers of autophagosomes. therefore, understanding the interaction between pathophysiological mechanisms and autophagy could be a promising approach for therapies against neurological disorders [ ] . prenatal alcohol exposure has been shown to increase the number of autophagic vacuoles in the cortical micro-vessels of human fetal and mouse neonatal brains, impairing autophagy [ ] . furthermore, autophagy can modulate notch degradation, stem cell development, and neurogenesis [ ] . several reviews have evaluated the relationship between autophagy and neurodegenerative diseases [ , ] . autophagy is vital for neuronal homeostasis [ ] , and its deregulation is highly associated with numerous neurodegenerative effects, such as the accumulation of damaged and toxic molecules with pathological consequences in neurodegenerative disorders such as alzheimer's, parkinson's, and huntington's diseases [ , ] . lysosomal system inactivation is responsible for the accumulation of autophagosomes observed in alzheimer's disease [ ] , whilst the disease is thought to be due to either excessive or impaired autophagosomal degradation, or the activation of autophagy genes in response to temporary injury/stress in neuronal tissues. alzheimer's [ ] , parkinson's [ , ] , and huntington's diseases [ ] are key examples where autophagosomal accumulation and anomalies in the endosomal-lysosomal pathway have been observed in post-mortem human brain tissues via electron microscopy. autophagy deficiency resulted in neuronal loss in the cerebral and cerebellar cortices in a mouse model [ ] . dysfunction or abnormalities in autophagy, including mutations in autophagy-regulating genes, are accompanied by neurodegenerative diseases across the age spectrum with exceptional frequency. atg -deficient mice exhibited ubiquitin accumulation in their cns, causing nervous symptoms, neurodegeneration, and ultimately death [ , ] , whilst atg -deficient mice developed cytoplasmic inclusions and exhibited motor dysfunctions [ ] , and ambra -deficient mouse embryos displayed neuronal tube defects [ ] . autophagy is involved in the cytoplasmic clearance of α-synuclein (α-syn), which is observed in parkinson's disease [ ] . in a mouse model, beclin overexpression was found to reduce the clearance of α-syn, leading to pathological neuron abnormalities [ ] . pharmacological and genetic pathways are involved in the degradation of α-syn by polo-like kinase via autophagy, suggesting that these two proteins are concomitantly co-degraded [ ] . the pink and parkin genes regulate mitophagy [ ] , indicating that mutations in these genes can cause defects in mitophagy which have been correlated with parkinson's disease [ , ] . beclin expression is lower in the brains of patients with alzheimer's disease and not only affects autophagy but also increases the deposition of β-amyloid proteins causing neurodegeneration [ ] . huntington's disease is caused by the extension of the polyglutamine (polyq) proteins aggregate intracellularly, which causes neuronal death. atg-knockout c. elegans exhibit increased polyq toxicity [ ] , whilst in drosophila the autophagy-enhancing small molecule -( -phenylphenyl)- , -dihydroimidazo[ , -b] [ , ] thiazole, also known as autophagy enhancer- (auten- ), has been shown to prevent the symptoms of neurodegenerative diseases [ ] . the dual role observed for autophagy may be due to our poor understanding of this ubiquitous cellular recycling system. the differences between physiological and pathological autophagy may help design therapeutic strategies specifically targeting pathological autophagy without hindering its physiological roles [ ] . for example, the apoptosis-stimulating protein p - (aspp / bp l) was reported to have different effects on autophagy in neurons stimulated with different levels of gp , a soluble envelope glycoprotein of hiv- that interacts with chemokine receptors such as cxcr and ccr . thus, regulating autophagy in the cns could be a potential therapeutic approach against hiv-associated neurocognitive disorders [ ] . . . autophagy in the immune system and autoimmune diseases . . . autophagy in the immune system the roles of cellular autophagy in immunological processes and autoimmune diseases have been reviewed extensively [ , , ] . autophagy plays important roles in both innate and adaptive immunity [ ] , modulates cellular and humoral immune responses [ ] [ ] [ ] , and has roles in the non-metabolic and metabolic functions of immune cells [ ] . furthermore, autophagy is involved in innate immune cell differentiation, degranulation, phagocytosis and extracellular trap formation involving neutrophils, eosinophils, mast cells, and natural killer cells, and plays an essential role in the renewal, differentiation, and homeostasis of immune cells [ ] . autophagy also regulates the functional responses of immune cells, such as phagocytosis, antigen presentation, cytokine production, control of inflammasome activation, tolerance, and their consequences on overall host defense via monocytes, macrophages, dendritic cells, and antigen presentation [ ] . additionally, autophagy plays important roles in b cell development, activation, and differentiation, which enables b cells to adapt to various events, and determines their fate, survival, and function [ ] . since b cells produce antibodies, autophagy can determine humoral immune responses. in one study, the b cells of atg -deficient mice had defective antibody responses, indicating that autophagy has a role in antibody production [ ] . several studies have documented interplay between autophagy and the nf-κb signaling pathway. members of the nf-κb family of transcription factors regulate the transcription of genes involved in cell proliferation, survival, differentiation, and development, whilst activation of the inhibitor of nf-κb (iκbα) kinase complex is essential for autophagy induction. t-cell receptor-mediated nf-κb activation in b-cell lymphoma/leukemia is linked with the autophagy adaptor p /sqstm [ ] , which modulates the nlrp -inflammasome activation and il- β production in macrophages [ ] . il- α secretion is enhanced in atg -deficient macrophages, whilst inhibiting autophagy results in il- β overexpression [ ] . the anti-inflammatory cytokine, il- , inhibits autophagy by activating mtor complex [ ] and inhibits starvation-and ifn-γ-induced autophagy via bcl- and beclin in various autoimmune and inflammatory disorders [ ] . autophagy has predisposing, pathogenic, and therapeutic roles in autoimmunity. defects in autophagy pathways and/or autophagy-related genes have been implicated in numerous autoimmune and autoinflammatory diseases, including multiple sclerosis, systemic lupus erythematosus (sle), rheumatoid arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease, diabetes mellitus, crohn's disease, and vitiligo [ , [ ] [ ] [ ] . abnormalities in the maintenance of homeostasis via autophagy result in the accumulation of dysfunctional or defective cellular organelles, abnormal proteins, infectious agents, and metabolite accumulation. this predisposes cells to the generation of autoantibodies and proinflammatory mediators and exposes vital and susceptible cellular structures to deleterious agents that can cause disease [ ] [ ] [ ] [ ] . a recent study revealed a correlation between the expression pattern of autophagy-related genes and the type of lupus nephritis (ln); thus, autophagy could indicate of the type of ln when formulating a treatment regimen [ ] . several atgs are known to be involved in autoimmune disorders, including atg , pr domain zinc finger protein (prdm ; also known as blimp- ), and dna-damage regulated autophagy modulator (dram ) in sle patients and atg l and immunity-related gtpase m (irgm) in crohn's disease and ulcerative colitis. autophagy defects have been observed in t cells, b cells, and macrophages [ ] . mhc class ii antigen presentation by macrophages occurs via cma; lysosomal proteins have a central role in antigen processing, which is essential for a correct immune system function. studies in mrl/lpr mice which develop a full panel of lupus autoantibodies revealed that increased lysosomal ph might be an important lysosomal malfunction involved in autoimmunity, and that perturbed lysosomal turnover may lead to hyperactive antigen presentation by antigen presenting cells (apc) in autoimmune disorders [ ] . in innate immunity, reduced atg and mtor expression result in defective autophagy, affecting the clearance of dead cells, increasing levels of nucleic acid remnants and self-antigens, increasing type ifn by dcs, and inducing b cell hyper-differentiation and autoantibody production [ , , ] . it has also been reported that autophagy-related gene knockdown can have therapeutic effects on autoimmune diseases [ ] . modulating autophagy can manage immunity-related and inflammatory diseases [ , , , ] by regulating cytokine and antibody production against immunogenic insults to prevent autoimmune diseases. therefore, regulating autophagy has clinical potential in cancer immunotherapy [ ] , whilst autophagy and adenoviral combinations are proving beneficial in adenoviral-based oncolytic virotherapy [ ] . under normal conditions, the myocardium exhibits low levels of autophagy, whilst stressful conditions can increase the level of autophagy to increase cell survival [ , ] . patients with congestive heart failure, coronary artery disease, hypertension, and aortic valvular disease display increased autophagosomal accumulation in their myocardial biopsies [ ] . autophagy levels vary in normal and affected or stressed hearts, with constitutive autophagy maintaining normal structure and function, and upregulated autophagy occurring during cardiac disease or stress [ ] . in atg -deficient mice, contractile dysfunction and hypertrophy have been observed during cardiomyopathy [ ] , whilst cell culture studies have revealed that autophagic gene deficiency can cause the accumulation of unwanted proteins and contribute to myocardial disease [ ] . similarly, lamp- -deficient mice displayed increased autophagic vacuole accumulation and could not degrade proteins, thereby promoting cardiomyopathy [ , ] . it has been postulated that during early life, between birth and suckling, autophagy provides the energy required for cardiac cells [ ] , whilst mitophagy protects cardiac muscles under ischemic stress [ ] . increased autophagy can cause heart failure [ ] , with autophagy-induced degeneration resulting in the death of cardiomyocytes. this knowledge has helped our understanding of the pathogenic role of autophagy in cardiac failure models and helped devise therapeutic targets [ , ] . autophagy can cause myocardial cell damage via parp , which promotes autophagy in cardiomyocytes by modulating foxo a transcription [ ] . increased autophagy causes pathological remodeling of the heart, whilst decreased autophagy reduces remodeling [ ] . thus, it has both protective and destructive roles in the cardiovascular system. iron homeostasis involves a form of macroautophagy known as ferritinophagy, wherein ferritin, an iron storage protein, is degraded in the lysosome [ ] . iron levels are tightly regulated in cells; nutrient deficiency induces autophagy, during which cellular proteins and organelles are engulfed by the autophagosome, which then fuses with the lysosome. the degradation of these contents provides essential resources that either promote cell survival or lead to cell death. iron (fe), copper (cu), zinc (zn), and aluminum (al) react with molecular oxygen to produce reactive oxygen species (ros) and reactive nitrogen species (rns). besides acting as a cofactor for metalloprotein enzymes involved in redox reactions, iron also plays a major role in mitochondrial atp metabolism and other cellular processes. the fenton and haber-weiss redox reaction is highly involved in ros production and alzheimer's progression [ ] . to maintain iron homeostasis, storage and recycling are critical. when engulfed by macrophages, the iron within erythrocytes is either stored as a ferritin complex or exported from the cell by the ferroportin iron-exporter [ ] . hsp and ferritin bind iron in the cytosol and autophagocytosis of these proteins can sequester redox-active iron in the lysosomes [ ] . cells rich in these proteins exhibit increased resistance to oxidative stress; therefore, autophagy plays a major role in maintaining cellular redox status [ , ] . the autophagy inhibitor ncoa , which is a substrate of pi k, has been shown to physically bind to the ferritin protein complex and direct it to autolysosomes for degradation [ ] . ncoa knockdown prevents the localization of ferritin in the lysosomes and increases the levels of iron-responsive element-binding protein (irp ), which is a free intracellular fe antagonist that prevents cell death via exogenous ros [ ] . ncoa also acts as an autophagy receptor for ferritin and delivers it to the lysosome to maintain iron homeostasis. experimentally simulating low-iron conditions by chelating iron revealed that ferritin is degraded to release the stored iron [ ] . autophagy is involved in obesity [ ] and diabetes mellitus [ ] . improper lipid and glycogen processing can affect the liver activity and thus, insulin synthesis, resulting in diabetes. studies have shown that hepatocytes from mouse models of obesity display reduced autophagy [ ] , with decreased atg expression causing er stress and affecting insulin signaling [ ] . mutant atg mice also exhibit reduced β cell mass, reduced insulin circulation, and glucose intolerance, indicating that autophagy defects can reduce insulin levels and cause hyperglycemia [ , ] . a genetic mosaic screen for mutations that increase lysosomal and/or autophagic activity in d. melanogaster larva revealed that autophagy-lysosome pathways underlie novel cytoprotective features in drosophila [ ] . obesity impairs autophagy in the liver via s-nitrosylation, a process induced by nitric oxide (no). s-nitrosylation of the lysosomal enzymes cathepsin b (ctsb) and hexosaminidase subunit β (hexb) impairs normal lysosomal functioning and is carried out by denitrosylation enzymes, particularly s-nitrosoglutathione reductase (gsnor) and thioredoxin [ ] . obesity inhibits the denitrosylation ability of the liver, impairing hepatic autophagy and insulin resistance [ ] . in obese animals, hepatic insulin signaling is impaired by no-induced hepatic autophagy repression, which ultimately causes the progression of type diabetes [ ] . the potential roles of autophagy in ameliorating diseases while maintaining homeostasis have been enumerated in table . details of applied/granted patents for treating autophagy-related ailments and dysfunctions are presented in table . human nuclear ribonucleoprotein k (hnrnp-k) and ubiquilin (ubqln ) help in viral replication. ndp human autophagy receptor interacts with chikv nsp and acts as proviral factor wong and chu, [ ] increased autophagy-associated protein lc and bnip -linked to colorectal and gastric cancers; elevated expression of nip (a pro-apoptotic member of the bcl- family of cell death factor) in gastric carcinomas lee et al., a [ ] autophagy inhibition leads to cell death in tumors acting like an ras-activated tumor guo et al., [ ] in the absence of autophagy -accumulation of ubiquitinylated protein aggregates and higher p level-responsible for liver tumor takamura et al., [ ] activation of autophagy and peroxisome proliferator-activated receptor gamma (pparγ) protect colon cancer cells against apoptosis tylichová et al., [ ] in ras-activated tumors, inhibition of autophagy leads to increased cancer cell death guo maintaining homeostasis is the most important role of autophagy, which is the final survival mechanism used to escape cell death. mutations or genetic dysfunctions in autophagy-associated genes can have a variety of pathological consequences, as described below: . . . static encephalopathy of childhood with neurodegeneration in adulthood (senda) senda is a recently discovered type of neurodegeneration associated with iron accumulation in the brain [ ] . it begins with early-onset spastic paraplegia and mental retardation during childhood, followed by symptoms of parkinsonism and dystonia during adulthood along with eye movement abnormalities, dysautonomia, and sleep disorders. whole-exome next generation sequencing has revealed that senda is associated with a mutation in the wipi gene (also known as wdr ), located at xp . [ , , ] . the disease is characterized by iron accumulation in the globus pallidus [ ] . wipi , which is the mammalian homolog of yeast atg , is involved in autophagosome formation [ ] by binding phosphatidylinositol -phosphate, which is recruited to the autophagosome formation site [ ] . a severe decline in wipi expression has been reported in lymphoblastoid cell lines derived from patients with senda [ ] . as the wipi gene is found on the x chromosome, one of which undergoes inactivation in females, the loss of wipi function is expressed in a mosaic pattern in females. crohn's disease is a major inflammatory bowel disease whose symptoms include abdominal pain, diarrhea, vomiting, and weight loss. atg l mutations have been found to be linked with crohn's disease [ ] . atg l forms a complex with atg -atg which initiates autophagosome formation by inducing the conjugation of lc with pe [ ] . a study on the t > a atg l mutation revealed that the mutation is differentially involved in crohn's disease and canonical autophagy [ ] . in mice, the mutations that eliminated or reduced atg l expression indicated a relationship between atg l mutations and crohn's disease [ ] . other autophagy-associated proteins, including irgm- , , and and nod - , , and have also been linked to crohn's disease in humans [ ] . autophagy is thought to be involved in the pathogenesis of crohn's disease, but it can also control disease severity by reducing the levels of inflammatory mediators [ ] . hsp is a neurodegenerative disorder that causes axonal degeneration in the corticospinal or pyramidal motor and sensory tracts that control distal organs and is caused by a recessive mutation in the tecpr gene. tecpr interacts with six human atg orthologs to positively regulate autophagy, with tecpr knockdown in hela cells reducing autophagic activity and indicating its role in autophagy [ ] . the zinc-finger protein spastizin interacts with the beclin -uvrag-rubicon complex and is involved in autophagosome maturation. in fibroblasts derived from patients with hsp, knockout of the spastizin gene reduced autophagy and caused autophagosome accumulation by impairing lysosomal fusion [ ] . the zfyve /spastizin and spg /spatacsin mutations were recently associated with hsp [ ] ; however, there are currently no treatments available to reverse nerve degeneration in hsp, with efforts instead directed towards reducing symptoms by physiotherapy and improving spasticity using medication. danon disease is a rare cardiomyopathic disease caused by lamp- deficiency and characterized by the accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscles, cardiomyopathy, and intellectual dysfunction [ ] . the lamp- gene is alternatively spliced into three isoforms (lamp- a, - b, and - c) which have different functions in autophagy. lamp- a acts as a receptor for cma, in which proteins carrying the kferq motif are selectively trafficked into the lysosome and degraded. lamp- b has been suggested to play a prominent role in macroautophagy and is responsible for the danon phenotype. lamp- b-deficient mice exhibit higher mortality and autophagic vacuole accumulation in the liver, kidney, pancreas, and cardiac and skeletal muscles [ ] . whole genome sequencing identified a nonsense mutation (codon c>t in exon ) in lamp in a family with danon disease [ ] . lamp- knockout resulted in failed autophagic progression, as evidenced by inappropriate cathepsin d processing in the autophagic vacuoles and abnormally high numbers of mannose- -phosphate receptors that limited the degradation of long-lived proteins during starvation [ ] . . . . x-linked myopathy with excessive autophagy (xmea) xmea is a rare x-linked recessive skeletal myopathy caused by a single-nucleotide substitution (c. - t > g) in vma , whose protein product assembles proton pumps in the lysosome which generate and maintain the acidity required for the activity of various lysosomal hydrolases [ ] . impaired hydrolase activity can block the final step of autophagy or induce autophagy by inhibiting mtor. if autophagy is impaired, a feed-forward pathogenic loop is activated, which results in the accumulation of autophagolysosomes containing incompletely-digested products [ ] . xmea is characterized by progressive muscle weakness, particularly in the proximal muscles of the legs, and muscle degeneration (atrophy) in adulthood. recently, two vma non-coding microdeletions [ ] , one intronic (c. - _ - del) and the other in the utr (c.* _* del), were reported to result in more severe clinical manifestations with extra-ocular and upper extremity involvement and earlier disease onset. besides long-lived proteins and membranes, xmea also affects membrane repair, interrupts sarcolemmal membrane homeostasis, and increases serum creatine phosphokinase (cpk) levels [ ] . sibm is an age-related progressive muscle disorder which presents in the elderly and is characterized by the presence of autophagic vacuoles and ubiquitinylated misfolded multiprotein aggregates. two major lysosomal proteases, cathepsins d and b, exhibit decreased activation in sibm muscle fibers [ ] whilst lc -ii is increased, indicating that autophagosome formation is increased due to reduced cathepsin d and b activity during autophagosome maturation [ ] . t cells may have a role in sibm [ ] , whilst allelic variants of the hla-dr locus, other genetic factors, and the hla genotype are also thought to play major roles in the progression and severity of the disease [ ] . dysphagia (difficulty in swallowing) may occur due to weakness in the neck muscles of patients with sibm, whilst myalgia (muscle pain) and difficulty in manipulating objects may occur due to weakness in the fingers. an overview of diseases caused by genetic defects in autophagy genes is presented in table . to alleviate autophagy-associated diseases, a variety of drugs, biomolecules, chemicals, and epigenetic strategies have been developed to either promote or inhibit autophagy. autophagy can be suppressed during any stage of autophagic flux. the pro-apoptotic role of autophagy may lead to hyperstimulation-induced excessive activation, which results in detrimental self-cannibalism that can go beyond the limit of cellular recovery. numerous chemical inhibitors have been identified and tested in various cell and animal models. most autophagy inhibitors are poorly selective, limiting their wider applications. -methyladenine ( -ma), a class-iii pi k inhibitor, is commonly used to inhibit autophagy [ ] . class iii pi k and beclin are essential during the first step of autophagy induction [ ] ; thus, inhibiting pi k reduces autophagosome formation. bafilomycin a is a vacuolar h + -atpase inhibitor, which functions at concentrations as low as nm to inhibit both the early and late stages of autophagy by activating the mtor pathway [ ] . in early , bafilomycin a was reported to inhibit autophagic vacuoles in rat hepatoma [ ] by dissociating the beclin -vps complex and preventing autolysosome formation, thus attenuating functional autophagy. bafilomycin a has been tested in a mouse model and found to be safe [ ] . furthermore, disruption of the vacuolar-type h + -atpase complex in mouse liver cells has been shown to induce the rapamycin complex (mtorc )-independent aggravation of autophagic vacuoles and lysosomes [ ] . concanamycin a belongs to the same class of vacuolar h + -atpase inhibitors as bafilomycin a and in the presence of autophagosomes causes autophagic bodies to accumulate in the central vacuole and cytoplasm of tobacco by- cells [ ] . therefore, vacuolar-type h + -atpases have been proposed as potential therapeutic agents. cycloheximide is a protein biosynthesis inhibitor that is frequently used in biomedical research. in mouse pancreatic acinar cells, cadmium chloride-or hyperosmotic sucrose-stimulated autophagy was found to be inhibited by cycloheximide [ ] , which likely inhibits autophagy at the segregation step. cycloheximide is used to prevent the autophagy-lysosome pathway [ ] and its effects can be rapidly reversed by its removal [ ] . chloroquine, hydroxychloroquine, nh cl, and neutral red are chemicals that can rapidly neutralize the acidic environment of the lysosome; therefore, they are used to block autophagosome maturation. chloroquine and hydroxychloroquine are repurposed drugs that have been used to treat malaria, sle, and rheumatoid arthritis [ ] ; however, higher hydroxychloroquine concentrations are required to induce active autophagy as a cancer treatment, which are usually not achievable in cancer patients. chloroquine-mediated lysosomal dysfunction is thought to have increased anti-cancer functions when combined with nutrient deprivation [ ] . lys , a dimeric form of chloroquine in which each molecule is separated by the spacer molecule n-bis( -aminoethyl)-methylamine, has been reported to inhibit autophagy at a level -fold higher than chloroquine lys , a water-soluble salt of lys , effectively accumulates within the lysosome to concurrently deacidify and inhibit autophagy [ ] . lysosomal proteases are active at an acidic ph. the protease inhibitor leupeptin inhibits cysteine, serine, and threonine peptidases to block protein degradation and thus inhibits the final step of autophagy, as evidenced by the accumulation of vacuolar autolysosomes [ ] . cathepsins are aspartic, cysteine, and serine proteases that are transported to lysosomes via mannose- -phosphate receptors [ ] and can be inhibited by the lysosomal protease inhibitors e d and pepstatin a. cathepsins b, h, and l are inhibited by e d, whilst cathepsins d and e are inhibited by pepstatin a. cells treated with e d and pepstatin a exhibit increased lc -ii levels [ ] . bafilomycin and chloroquine, which inhibit autophagy by targeting lysosomes, have been reported to increase levels of the autophagosome marker lc -ii, block key aspects of autophagy, decrease mitochondrial quality, and increase mitochondrial dna damage in primary neurons [ ] . the atg genes are crucial in autophagy; therefore, the inactivation or knockdown of these genes may be a useful way to manipulate the process [ ] . mir- a has been reported to downregulate beclin and atg expression, whilst mir- can inhibit basal and rapamycin-induced autophagy. autophagy is induced by the activation of the ulk complex, which consists of ulk / , atg , fip , and atg . in melanoma cells, mir- - clusters can inhibit ulk and atg expression to suppress autophagic cell death [ ] . mir- - p targets ulk to regulate autophagy [ ] , whereas mirna- a/b, mirna- b, mir- a, mir- a, and mir- - p inhibit beclin expression to suppress vesicle nucleation [ ] [ ] [ ] . recently, the leucine-rich repeat kinase gene (lrrk ), which is associated with crohn's disease, parkinson's disease, and leprosy, was shown to inhibit the non-canonical autophagy cascade, indicating that the negative regulation of this cascade could directly induce disease [ ] . in addition, the atg l t > a polymorphism has been demonstrated to disrupt unconventional tmem . -mediated autophagy in mice [ ] . rapamycin, also known as sirolimus, is a natural mtor inhibitor [ ] that stimulates autophagy both in vitro and in vivo; however, its long-term use is often complicated as it suppresses ribosome biogenesis and protein translation [ ] . the rapamycin ester cci- /temsilorimus, which is also an mtor inhibitor, prevents the development of intracellular tau protein inclusions (abundant protein that stabilizes microtubules in cns neurons) which are known to accumulate in neurons in alzheimer's disease and other tauopathies and can also reduce the density of neurofibrillary tangles by stimulating mtor-dependent autophagy [ ] . rad and ap are rapamycin derivatives with comparatively high safety and low toxicity [ , ] . during the early stages of carotid atherosclerosis, which is an inflammatory step in the primary pathogenesis of cerebrovascular diseases, mirna- plays an important role in the activation of autophagy by rapamycin [ ] . rapamycin has also been shown to increase autophagy, decrease cyclin d expression, and attenuate aggressive iga nephropathy progression in a rat model [ ] . the immunosuppressive activities of rapamycin limit its frequent use; therefore, safer molecules are required. three smers were identified from , compounds that can induce autophagy [ ] ; smers , , and can reduce the pathogenesis associated with polyglutamine aggregation and a t α-synuclein, which are linked to huntington's disease and familial parkinson's disease, respectively [ ] , and induce autophagy in an mtor-independent manner. in addition, the small molecule auten- activates autophagy in cell cultures and animal models and inhibits the progression of neurodegenerative symptoms [ ] . trehalose is a natural disaccharide known to stimulate autophagy via ampk [ ] . in cultured cells from an animal model of huntington's disease, trehalose has been shown to stimulate autophagy and reduce huntingtin protein aggregation during starvation by inhibiting a family of glucose transporters known as the solute carrier or the glucose transporter family [ ] . as well as being an autophagy activator and a non-reducing disaccharide, trehalose can also reduce the levels of aggregate-prone proteins and ameliorate cytotoxicity in neurodegenerative disease models [ ] . trehalose treatment has been shown to increase the conversion of lc -i to lc -ii via an mtor-independent pathway, whilst sucrose and raffinose are also known to induce autophagy [ ] . it has been shown that trehalose can be used to safely induce autophagy in neurodegenerative disorders such as parkinson's disease [ ] , alzheimer's disease [ ] , and prion disease [ ] . the bioavailability of trehalose in the body for autophagy induction is low due to the hydrolytic enzyme, trehalase, which is expressed in the intestine and kidney; therefore, the enzyme-stable trehalose analogs, lentztrehaloses a, b, and c, were synthesized and found to be as effective as trehalose [ ] . lithium chloride (licl) can induce autophagy by inhibiting inositol monophosphatase (impase), which reduces inositol and inositol- , , -triphosphate (ip ) levels [ ] . since lithium has already been approved by the fda for patients with bipolar disorder, it can easily be adapted for other diseases [ ] . long-term oral lithium administration enhanced autophagy in a tauopathy mouse model by inhibiting glycogen synthase kinase- . l- , , a bisphosphonate inhibitor of impase, exhibits a similar function to lithium by clearing mutant synucleins and egfp-hdq [ ] . valproic acid is an angio-suppressive compound which suppresses the akt/mtor signaling pathway by acting as a histone deacetylase inhibitor and promotes autophagy, as evidenced by the increased concentrations of lc -ii and beclin after its administration in prostate cancer cell lines [ ] . carbamazepine and valproic acid can both reduce intracellular inositol- , , trisphosphate levels [ ] . therefore, other impase inhibitors could also be incorporated into therapeutic strategies. anacardic acid, curcumin, garcinol, and spermidine increase autophagy by reducing the level of acetylation in cultured human cells, as evidenced by depleted sequestosome- levels and mtorc inhibition [ ] . spermidine also inhibits ep and the major autophagy proteins, atg , atg , atg , and lc to repress autophagy [ ] . supplementing the diet of mice with coffee beans rich in polyphenols concomitantly increased autophagy and decreased acetylation levels [ ] , whilst natural polyamines can inhibit acetyltransferases, and their dietary intake can improve the life span of short-living mouse strains and the health of long-living ones [ ] . fluoxetine, which selectively improves the secretion of the pro-inflammatory cytokine tnf-α, and gefitinib, which inhibits the epidermal growth factor receptor (egfr), can also enhance autophagy to impart neuroprotection and restrict m. tuberculosis growth [ ] . the antiepileptic compound, carbamazepine, has been found to protect cells against m. tuberculosis infection, likely by inducing autophagy [ ] . elevated intracytosolic ca + levels can inhibit autophagy; penitrem a, which inhibits ca + -dependent k + channels, is known to induce autophagy and has been used to treat neurodegenerative disorders [ , ] . furthermore, bacterial pore-forming toxins can induce the expression of the transcription factor hlh- (tfeb) in c. elegans, which stimulates autophagy genes and thus inhibits bacterial infection [ ] . metformin, an ampk activator, has been shown to induce autophagy and reduce the risk of hepatocellular carcinoma (hcc) in diabetic patients [ ] . a patent studied by gorski and qadir [ ] revealed the potential of a thioxanthone-based autophagy inhibitor for suppressing atg gene expression by using sirna-directed therapy against cancer cells receiving anti-cancer therapies, and for treating cancer in combination with other therapies. the use of dimeric quinacrine derivatives to inhibit lysosomes and autophagy in cancer cells where autophagy allows them to survive metabolic and therapeutic stresses [ ] is currently at the patent filing phase. the herbal product onjisaponin b, from radix polygalae (polygala tenuifolia)-a traditional chinese herb, is known to enhance autophagy. upon administration, it prevents, treats, or delays the onset of neurodegenerative diseases, thus a patent for this product has been granted to law et al. [ ] . imidazo [ - f] [ , ] phenanthroline derivatives ( - ) have been evaluated for their anti-cancer effects, with their use of upregulating lc -ii and beclin expression and thus inducing autophagy. of the different compounds evaluated, the phenanthroimidazole derivative was found to induce autophagy and apoptosis; thus, it could prove to be a novel anti-cancer drug [ ] . in nanosilica stimulated raw . cells, melatonin was found to increase lc expression and decrease bax expression, suggesting that autophagy was promoted, and apoptosis was inhibited [ ] . sasanquasaponin iii obtained from schima crenata korth was found to upregulate lc -ii expression in melanoma cells and induce autophagy; hence, it can be used as an anti-cancer drug to treat melanoma [ ] . epigallocatechin gallate (egcg) was found to promote autophagosome formation and increase lysosome acidification in müller cells, thus increasing autophagy. a gliosis experimental model revealed that egcg reduced reactive gliosis and retinal damage; hence, egcg should be explored as a treatment of diabetic retinopathy [ ] . compounds that inhibit and activate autophagy are depicted in figure . therapies. the use of dimeric quinacrine derivatives to inhibit lysosomes and autophagy in cancer cells where autophagy allows them to survive metabolic and therapeutic stresses [ ] is currently at the patent filing phase. the herbal product onjisaponin b, from radix polygalae (polygala tenuifolia)a traditional chinese herb, is known to enhance autophagy. upon administration, it prevents, treats, or delays the onset of neurodegenerative diseases, thus a patent for this product has been granted to law et al. [ ] . imidazo [ - f] [ , ] phenanthroline derivatives ( - ) have been evaluated for their anti-cancer effects, with their use of upregulating lc -ii and beclin expression and thus inducing autophagy. of the different compounds evaluated, the phenanthroimidazole derivative was found to induce autophagy and apoptosis; thus, it could prove to be a novel anti-cancer drug [ ] . in nanosilica stimulated raw . cells, melatonin was found to increase lc expression and decrease bax expression, suggesting that autophagy was promoted, and apoptosis was inhibited [ ] . sasanquasaponin ΙΙΙ obtained from schima crenata korth was found to upregulate lc -ii expression in melanoma cells and induce autophagy; hence, it can be used as an anti-cancer drug to treat melanoma [ ] . epigallocatechin gallate (egcg) was found to promote autophagosome formation and increase lysosome acidification in müller cells, thus increasing autophagy. a gliosis experimental model revealed that egcg reduced reactive gliosis and retinal damage; hence, egcg should be explored as a treatment of diabetic retinopathy [ ] . compounds that inhibit and activate autophagy are depicted in figure . compounds that inhibit and activate autophagy. autophagy inhibitors: -methyladenine inhibits pi k. bafilomycin a causes dissociation of the beclin -vps complex and prevents the formation of autolysosome. chloroquine/hydroxychloroquine, nh cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. autophagy activators: rapamycin inhibits the mtor. rad and ap are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. trehalose causes lc -i to lc -ii conversion in an mtor-independent pathway. valproic acid increases lc -ii and beclin concentrations. compounds that inhibit and activate autophagy. autophagy inhibitors: -methyladenine inhibits pi k. bafilomycin a causes dissociation of the beclin -vps complex and prevents the formation of autolysosome. chloroquine/hydroxychloroquine, nh cl, and leupeptin rapidly neutralizing the acidic environment of the lysosome and are used to block lysosomal degradation of substrates. leupeptin inhibits cysteine, serine and threonine peptidases, and hence blocking protein degradation at the last step of autophagy. autophagy activators: rapamycin inhibits the mtor. rad and ap are rapamycin derivatives having comparatively higher safely with minimum dose toxicities. trehalose causes lc -i to lc -ii conversion in an mtor-independent pathway. valproic acid increases lc -ii and beclin concentrations. autophagy is a fast-moving area of science which has had an excellent positive impact on animal and human health-related issues and threatening diseases and has considerable future potential. autophagy is a highly complex process, and understanding the complexity of its mechanisms and internal regulations will be highly beneficial for developing novel methods such as simple ameba-based experimental models. emerging studies have established many interesting connections in the field of autophagy research. since autophagy is one of the most conserved evolutionary processes, present in lower organisms and all highly evolved mammals, much work has been carried out to understand its physiological and pathological features. pathogenesis-associated autophagy is involved in degenerative, inflammatory, infectious, and neoplastic diseases, whereas physiological autophagy is associated with maintaining liver iron homeostasis, cns development, endothelial cell alignment, atheroprotection, and preventing infection. numerous mechanisms of action involving various signaling pathways have been linked to autophagy, with novel mechanisms currently being investigated. mtor acts as a nutrient sensor in autophagy; however, the process may also be regulated via mtor-independent pathways. intracellular ca + regulates autophagy, with intracellular ca + mobilization via the ip receptor stimulating calmodulin and erk pathways to inhibit autophagy. notably, ca + also inhibits autophagy by increasing mitochondrial atp levels, which inhibits the camkkβ/ampk pathway, yet promotes autophagy by promoting the ampk-dependent inhibition of mtor. autophagy is linked with other cellular mechanisms such as apoptosis, necrosis, autosis, and necroptosis, and can take the form of microautophagy, macroautophagy, and cma. autophagy can be selective, neutralizing specific substances, or non-selective, degrading different materials irrespective of their nature. it can play physiological and pathological roles by maintaining homeostasis and causing disease, thereby affecting both health and disease. autophagy encounters infectious and non-infectious diseases, either mediating protection or aggravating the disease. autophagy has dual roles in an anti-bacterial, anti-viral, and tumor suppressing capacity and by favoring bacterial infections, viral infections, and tumor progression. among its dual physio-pathological roles, autophagy can promote brain development, immunity, and cardiovascular and endocrine development, or cause neurodegeneration, autoimmune diseases, cardiovascular diseases, obesity, and diabetes. autophagy has a genetic basis and many diseases are caused by genetic defects in autophagy genes, including senda, crohn's disease, hsp, danon disease, xmea, and sibm. efforts are being made to explore the positive aspects of autophagy to overcome various health and disease problems. novel therapeutics and interventions are being investigated to counter autophagy-associated diseases, including apoptosis inhibitors and activators. excessive autophagy stimulation may cause self-destruction which could be controlled with drugs such as vacuolar-type h (+)-atpase inhibitors, cycloheximide, lysosome alkalizers (chloroquine, hydroxychloroquine, nh cl, and neutral red), and acidic protease inhibitors (e d and pepstatin a), whilst knocking down beclin and atg using mir- a could be used to inhibit the excessive cannibalism caused by autophagy. upregulating autophagy could therapeutically benefit a range of diseases caused by reduced neuronal apoptosis, including the neurodegeneration-associated impairment of learning/memory capabilities, motor dysfunction, seizures, adult stroke, neonatal asphyxia, cardioskeletal myopathy, and cancers. autophagy could also prevent various bacterial and viral diseases, inflammatory and autoimmune conditions, and also increase lifespan. rapamycin, small-molecular enhancers of rapamycin (auten- ), trehalose, impase inhibitors (carbamazepine and valproic acid), epigenetic modulators (anacardic acid, curcumin, garcinol, and spermidine), and chemicals (fluoxetine, penitrem a, and metformin) are all autophagy stimulators which help to ameliorate disorders associated with reduced autophagy. the apparent dual role of autophagy may be due to our poor understanding of this ubiquitous cellular recycling system. understanding the differences between physiological and pathological autophagy may help us design therapeutic strategies to target pathological autophagy without hindering its physiological effects. studies on mouse models combined with human genetic studies provide an important insight into the role of autophagy in neurological diseases like parkinson's and inflammatory disorders like crohn's disease. some critical issues have yet to be addressed regarding the role of autophagy in therapeutics and diagnostics. there are few efficient markers for studying autophagy modulation and those that exist have limitations. these markers are important for determining the effect of autophagy on disease initiation and progression. furthermore, autophagy modulating drugs are imprecise and nonspecific; hence, more specific drugs are required. similarly, autophagy upregulation appears to be beneficial for removing misfolded and aggregated proteins, intracellular pathogens, damaged mitochondria, and other organelles; however, it is not yet clear how selective autophagy could be upregulated in other situations. these are just some of the questions that need to be addressed in order to use autophagy as a therapeutic molecular process. nonetheless, autophagy modulation-based therapies will soon become a reality and will help safeguard human health against various pathological conditions. author contributions: all the authors substantially contributed to the conception, design, analysis, and interpretation of data, checking and approving the final version of the manuscript, and agree to be accountable for its contents. funding: this compilation is a review article written, analyzed, and designed by its authors and required no substantial funding to be stated. till death do us part: the marriage of autophagy and apoptosis autophagy, process 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knockout of atg inhibits proliferation and promotes apoptosis of df- cells the autophagy machinery controls cell death switching between apoptosis and necroptosis sorafenib-induced defective autophagy promotes cell death by necroptosis nad+ depletion triggers macrophage necroptosis, a cell death pathway exploited by mycobacterium tuberculosis adp-ribose) polymerase , parp , modifies ezh and inhibits ezh histone methyltransferase activity after dna damage receptor interacting protein kinase- determines cellular necrotic response to tnf-alpha amp-activated protein kinase, an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases role of ampk-mtor-ulk / in the regulation of autophagy: cross talk, shortcuts, and feedbacks atm signals to tsc in the cytoplasm to regulate mtorc in response to ros autophagy recognizes intracellular salmonella enterica serovar typhimurium in damaged vacuoles listeria monocytogenes acta is a key player 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activity of a novel beclin -binding protein uvrag autophagy, mitochondrial quality control, and oncogenesis autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis autophagy suppresses tumor progression by limiting chromosomal instability the expanding role of mtor in cancer cell growth and proliferation inhibition of mtor induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of huntington disease the coordinate regulation of the p and mtor pathways in cells autophagy and cancer, dynamism of the metabolism of tumor cells and tissues pogostone induces autophagy and apoptosis involving pi k/akt/mtor axis in human colorectal carcinoma hct cells new anti-cancer molecules targeting hspa /bip to induce endoplasmic reticulum stress, autophagy and apoptosis trichosanthin inhibits human ovarian cancer cells growth due to apoptosis and autophagy autophagy is activated in colorectal cancer cells and contributes to the 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inhibitors for detecting autophagy in plants cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose lysosomal turnover, but not a cellular level, of endogenous lc is a marker for autophagy inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons the mir- - cluster suppresses autophagic cell death of melanoma cells phospho-∆np α/mir- - p axis in tumor cell life and cell death upon cisplatin exposure mir- a: a link between endothelial dysfunction and autophagy mir- - p downregulation contributes to paclitaxel resistance of lung cancer cells through altering beclin expression interaction of autophagy with micrornas and their potential therapeutic implications in human cancers genetic control of autophagy underlies pathogenesis of inflammatory bowel disease the t a crohn's disease risk polymorphism impairs function of the wd domain of atg l mtor interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery a liaison between mtor signalling, ribosome biogenesis and cancer rapamycin ester analog cci- /temsirolimus alleviates tau pathology and improves motor deficit in mutant tau transgenic mice mtor-targeted therapy of cancer with rapamycin derivatives autophagy inhibition enhances rad -induced cytotoxicity in human bladder cancer cells. drug des expression of mirna- in carotid atherosclerotic plaques of apolipoprotein e knockout (apoe −/− ) mice and the interventional effect of rapamycin rapamycin enhances repressed autophagy and attenuates aggressive progression in a rat model of iga nephropathy small molecule enhancers of rapamycin-induced tor inhibition promote autophagy, reduce toxicity in huntington's disease models and enhance killing of mycobacteria by macrophages polyglutamine aggregation in huntington's disease and spinocerebellar ataxia type : similar mechanisms in aggregate formation mystery solved: trehalose kick starts autophagy by blocking glucose transport focus: drug development: variable effects of autophagy induction by trehalose on herpes viruses depending on conditions of infection trehalose activates autophagy and decreases proteasome inhibitor-induced endoplasmic reticulum stress and oxidative stress-mediated cytotoxicity in hepatocytes trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mtor-independent pathway treatment with trehalose prevents behavioral and neurochemical deficits produced in an aav α-synuclein rat model of parkinson's disease autophagic degradation of tau in primary neurons and its enhancement by trehalose autophagy induction by trehalose counteracts cellular prion infection novel autophagy inducers lentztrehaloses a., b and c lithium and autophagy lithium protects against oxidative stress-mediated cell death in alpha-synuclein over-expressing in vitro and in vivo models of parkinson's disease lithium induces autophagy by inhibiting inositol monophosphatase valproic acid induces autophagy by suppressing the akt/mtor pathway in human prostate cancer cells induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through hdac-independent and ip -mediated prkaa activation spermidine induces autophagy by inhibiting the acetyltransferase ep regulation of autophagy by the p acetyltransferase coffee induces autophagy in vivo essential role for autophagy in life span extension identification of host-targeted small molecules that restrict intracellular mycobacterium tuberculosis growth functional drug screening reveals anticonvulsants as enhancers of mtor-independent autophagic killing of mycobacterium tuberculosis through inositol depletion rapamycin and mtor-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases metformin promotes apoptosis in hepatocellular carcinoma through the cebpd-induced autophagy pathway phenanthroimidazole derivatives act as potentinducer of autophagy by activating dna damage pathway melatonin enhances autophagy and decreases apoptosis induced by nanosilica in raw . cells sasanquasaponin iii from schima crenata korth induces autophagy through akt/mtor/p s k pathway and promotes apoptosis in human melanoma a cells epigallocatechin- -gallate stimulate autophagy and reduces apoptosis levels in retinal müller cells under high-glucose conditions this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: all the authors acknowledge and thank their respective institutes and universities. all authors declare that there exist no commercial or financial relationships that could in any way lead to a potential conflict of interest. key: cord- -e yfo authors: rainwater-lovett, kaitlin; rodriguez-barraquer, isabel; moss, william j. title: viral epidemiology: tracking viruses with smartphones and social media date: - - journal: viral pathogenesis doi: . /b - - - - . - sha: doc_id: cord_uid: e yfo the science of epidemiology has been developed over the last years, using traditional methods to describe the distribution of diseases by person, place, and time. however, in the last several decades, a new set of technologies has become available, based on the methods of computer sciences, systems biology, and the extraordinary powers of the internet. technological and analytical advances can enhance traditional epidemiological methods to study the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. social media are increasingly used to detect the emergence and geographic spread of viral disease outbreaks. large-scale population movement can be estimated using satellite imagery and mobile phone use, and fine-scale population movement can be tracked using global positioning system loggers, allowing estimation of transmission pathways and contact patterns at different spatial scales. advances in genomic sequencing and bioinformatics permit more accurate determination of viral evolution and the construction of transmission networks, also at different spatial and temporal scales. phylodynamics links evolutionary and epidemiological processes to better understand viral transmission patterns. more complex and realistic mathematical models of virus transmission within human and animal populations, including detailed agent-based models, are increasingly used to predict transmission patterns and the impact of control interventions such as vaccination and quarantine. in this chapter, we will briefly review traditional epidemiological methods and then describe the new technologies with some examples of their application. the science of epidemiology has been developed over the last years, using traditional methods to describe the distribution of diseases by person, place, and time. however, in the last several decades, a new set of technologies has become available, based on the methods of computer sciences, systems biology, and the extraordinary powers of the internet. technological and analytical advances can enhance traditional epidemiological methods to study the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. social media are increasingly used to detect the emergence and geographic spread of viral disease outbreaks. large-scale population movement can be estimated using satellite imagery and mobile phone use, and fine-scale population movement can be tracked using global positioning system (gps) loggers, allowing estimation of transmission pathways and contact patterns at different spatial scales. advances in genomic sequencing and bioinformatics permit more accurate determination of viral evolution and the construction of transmission networks, also at different spatial and temporal scales. phylodynamics links evolutionary and epidemiological processes to better understand viral transmission patterns. more complex and realistic mathematical models of virus transmission within human and animal populations, including detailed agent-based models, are increasingly used to predict transmission patterns and the impact of control interventions such as vaccination and quarantine. in this chapter, we will briefly review traditional epidemiological methods and then describe the new technologies with some examples of their application. insight into the epidemiology of viral infections long preceded the recognition and characterization of viruses as communicable agents of disease in humans and animals, extending at least as far back as the treatise of abu becr (rhazes) on measles and smallpox in the tenth century. successful efforts to alter the epidemiology of viral infections can be traced to the practice of variolation, the deliberate inoculation of infectious material from persons with smallpox (see chapter on the history of viral pathogenesis). documented use of variolation dates to the fifteenth century in china. edward jenner greatly improved the practice of variolation in using the less-virulent cowpox virus, establishing the field of vaccinology. an early example of rigorous epidemiological study prior to the discovery of viruses was the work of the danish physician peter panum who investigated an outbreak of measles on the faroe islands in . through careful documentation of clinical cases and contact histories, panum provided evidence of the contagious nature of measles, accurate measurement of the incubation period, and demonstration of the long-term protective immunity conferred by measles. the discovery of viruses as "filterable agents" in the late-nineteenth and early twentieth centuries greatly enhanced the study of viral epidemiology, allowing the characterization of infected individuals, risk factors for infection and disease, and transmission pathways. traditional epidemiological methods measure the distribution of viral infections, diseases, and associated risk factors in populations in terms of person, place, and time using standard measures of disease frequency, study designs, and approaches to causal inference. populations are often defined in terms of target and study populations, and individuals within study populations in terms of exposure and outcome status. the purpose of much traditional epidemiological research is to quantify the strength of association between exposures and outcomes by comparing characteristics of groups of individuals. exposures or risk factors include demographic, social, genetic, and environmental factors, and outcomes include infection or disease. in viral epidemiology, infection status is determined using diagnostic methods to detect viral proteins or nucleic acids, and serologic assays to measure immunologic markers of exposure to viral antigens. infection status can be defined as acute, chronic, or latent. standard measures of disease frequency include incidence, the number of new cases per period of observation (e.g., person-years), and prevalence, the number of all cases in a defined population and time period. prevalence is a function of both incidence and duration of infection and can increase despite declining incidence, as observed with the introduction of antiretroviral therapy for human immunodeficiency virus (hiv) infection in the united states. although the number of new cases of hiv infection declined, the prevalence of hiv infection increased as treated individuals survived longer. commonly used study designs include, l cross-sectional studies in which individuals are sampled or surveyed for exposure and disease status within a narrow time frame, l cohort studies in which exposed and unexposed individuals are observed over time for the onset of specified outcomes, l case-control studies in which those with and without the outcome (infection or disease) are compared on exposure status, and l clinical trials in which individuals are randomized to an exposure such as a vaccine or drug and observed for the onset of specified outcomes appropriate study design, rigorous adherence to study protocols, and statistical methods are used to address threats to causal inference (i.e., whether observed associations between exposure and outcome are causal), such as bias and confounding. much can be learned about the epidemiology of viral infections using such traditional methods and many examples could be cited to establish the importance of these approaches, including demonstration of the mode of transmission of viruses by mosquitoes (e.g., yellow fever and west nile viruses), the causal relationship between maternal viral infection and fetal abnormalities (e.g., rubella virus and cytomegalovirus), and the role of viruses in the etiology of cancer (e.g., epstein-barr and human papilloma viruses). the epidemiology of communicable infectious diseases is distinguishable from the epidemiology of noncommunicable diseases in that the former must account for "dependent happenings." this term was introduced by ronald ross to capture the fact that infectious agents are transmitted between individuals or from a common source. traditional epidemiological and statistical methods often assume disease events in a population are independent of one another. in infectious disease epidemiology, individuals are defined in terms of susceptible, exposed, infectious, and recovered or immune. key characteristics of viral infections that determine the frequency and timing of transmission, and thus the epidemiology, include the mode of transmission (e.g., respiratory, gastrointestinal, sexual, bloodborne, and vector-borne), whether infection is transient or persistent, and whether immunity is short or long lasting. temporal changes in the transmission dynamics of viral infection can be displayed with epidemic curves, by plotting the number or incidence of new infections over time to demonstrate outbreaks, seasonality, and the response to interventions. key metrics in infectious disease epidemiology that capture the dependent nature of communicable diseases include: ( ) the latent period, the average time from infection to the onset of infectiousness; ( ) the infectious period, the average duration of infectiousness; ( ) the generation time, the average period between infection in one individual and transmission to another; and ( ) the basic reproductive number (r ), the average number of new infections initiated by a single infectious individual in a completely susceptible population over the course of that individual's infectious period. if r is larger than one, the number of infected individuals and hence the size of the outbreak will increase. if r is smaller than one, each infectious individual infects on average less than one other individual and the number of infected individuals will decrease and the outbreak ceases. the reproductive number (r) is a function not only of characteristics of the viral pathogen (e.g., mode of transmission), but also the social contact network within which it is transmitted and changes over time in response to a decreasing number of susceptible individuals and control interventions. an important concept related to the interdependence of transmission events is herd immunity, the protection of susceptible individuals against infection in populations with a high proportion of immune individuals because of the low probability of an infectious individual coming in contact with a susceptible individual. the concepts and methods of infectious disease epidemiology provide the tools to understand changes in temporal and spatial patterns of viral infections and the impact of interventions. traditional epidemiological methods provide powerful analytical approaches to measure associations between exposures (risk factors) and outcomes (infection or disease). recent technological advances enhance these methods and permit novel approaches to investigate the emergence, epidemiology, and transmission dynamics of viruses and associated diseases. expanded access to the internet and social media has revolutionized outbreak detection and viral disease surveillance by providing novel sources of data in real time (chunara, ) . traditional epidemiologic surveillance systems rely on standardized case definitions, with individual cases typically classified as suspected, probable, or confirmed based on the level of evidence. confirmed cases require laboratory evidence of viral infection. surveillance systems are either active or passive. active surveillance involves the purposeful search for cases within populations whereas passive surveillance relies on routine reporting of cases, typically by health care workers, health care facilities, and laboratories. data acquired through active surveillance are often of higher quality because of better adherence to standardized case definitions and completeness of case ascertainment but are more expensive and resource intensive. however, both active and passive surveillance are prone to delays in data reporting. the major advantage of using the internet and social media to monitor disease activity is that the signal can be detected without the lag associated with traditional surveillance systems. influenza is the most common viral infection for which the internet and social media have been used for disease surveillance because of its high incidence, wide geographic distribution, discrete seasonality, short symptomatic period, and relatively specific set of signs and symptoms. however, the internet and social media have several limitations compared to traditional active and passive surveillance systems and complement rather than replace these methods. these limitations include lack of specificity in the "diagnosis," and waxing and waning interest and attention in social media independent of disease frequency. in , the internet company google developed a webbased tool called google flu trends, for early detection of influenza outbreaks. google flu trends is based on the fact that millions of people use the google search engine each day to obtain health-related information (ginsberg, ). logs of user key words for pathogens, diseases, symptoms, and treatments, as well as information on user location contained in computer internet protocol (ip) addresses, allow temporal and spatial analyses of trends in search terms ( figure ). early results suggested that google flu trends detected regional outbreaks of influenza - days before conventional surveillance by the centers for disease control and prevention (carneiro, ). however, accurate prediction was not as reliable as initially thought, and google estimates did not closely match measured activity during the - influenza season. google now reevaluates estimates using data from traditional surveillance systems (specifically those of the centers for disease control and prevention) to refine model and parameter estimates. these refinements more accurately capture the start of the influenza season, the time of peak influenza virus transmission, and the severity of the influenza season. a similar approach, called google dengue trends, is used to track dengue virus infections by aggregating historical logs of anonymous online google search queries associated with dengue, using the methods developed for google flu trends. early observations suggest google queries are correlated with national-level dengue surveillance data, and this novel data source may have the potential to provide information faster than traditional surveillance systems ( figure ). other internet sources are being explored to enhance viral surveillance. wikipedia is a free, online encyclopedia written collaboratively by users and is one of the most commonly used internet resources since it was started in . as with google searches, the use of disease-specific queries to wikipedia are expected to correlate with disease activity. the number of times specific influenza-related wikipedia sites were accessed provided accurate estimates of influenza-like illnesses in the united states weeks earlier than standard surveillance systems and performed better than google flu trends (mciver, ) . similarly, social media data are being evaluated for surveillance purposes. twitter is a free social networking service that enables users to exchange text-based messages of up to characters known as tweets. as with google flu trends, the number of tweets related to influenza activity is correlated with the number of symptomatic individuals. several published studies reported correlations between twitter activity and reported influenza-like illnesses (chew, ; signorini, ; figure ). limitations to using social media, such as twitter, to monitor disease activity are illustrated by the ebola virus outbreak in west africa in early . despite the fact that ebola had not yet occurred in the united states, posts to twitter on ebola rose dramatically, likely in response to intense media coverage and fear. clearly, such tweets could not be interpreted to indicate ebola disease activity in the united states. studies reporting misleading associations, or the lack of correlation between social media and disease activity, are rarely published, providing a cautionary note. while initial efforts using data from the internet for viral disease surveillance offer promising results, concerns have been raised regarding the utility and robustness of these approaches (lazer, ) . integration into existing surveillance frameworks will be necessary to maximize the utility of these data streams. the internet allows rapid processing and communication of health-related information, including the aggregation and display of surveillance data for viral infections. traditional surveillance networks can be linked through the internet to allow rapid integration and dissemination of information. information on viral disease outbreaks available through internet postings of health care agencies such as the world health organization (who) and centers for disease control and prevention (cdc), as well as press reports and blogs, can provide data that are more current than traditional surveillance systems. information from these online sources can be made available to a large, global audience. several of the most commonly used surveillance sites report animal as well as human diseases (see sidebar and figure ). mapping spatial patterns of disease and relationships with environmental variables preceded the development of modern epidemiology. the classic example is john snow's hand-drawn map of london cholera cases of . however, routine mapping of health data only became commonplace in the s after desktop geographic information systems became widely available. combined with satellite imagery and remotely sensed environmental and ecological data, spatial mapping of viral infections is a powerful tool for surveillance and epidemiological research. spatial epidemiology is typically used to identify and monitor areas of differential risk. an early example was a large outbreak of st. louis encephalitis virus infection in houston, texas in . spatial analysis showed that the outbreak was concentrated in the city center, with lower incidence at the outskirts. further investigation revealed that the city center was associated with the lowest economic strata, unscreened windows, lack of air-conditioning and pools of standing water, factors facilitating virus transmission. investigation into the spatiotemporal dynamics of viral diseases at smaller spatial scales has become promed, the program for monitoring emerging diseases, is an internet-based reporting system established in that compiles information on outbreaks of infectious diseases affecting humans, animals, and food plants. promed relies on official announcements, media reports, and local observers, including the network of subscribers. a team of experts screen, review, and investigate reports before posting and often provide commentary. reports are distributed by email to direct subscribers and posted on the promed-mail web site. promed-mail currently reaches over , subscribers in at least countries. started in by epidemiologists and software developers at boston children's hospital, healthmap monitors disease outbreaks and provides real-time surveillance of emerging public health threats, including viral infections (figure ) . healthmap organizes and displays data on disease outbreaks and surveillance using an automated process. data sources include online news aggregators, eyewitness reports, expertcurated discussions and validated official reports. google flu trends http://www.google.org/flutrends google dengue trends http://www.google.org/denguetrends healthmap http://healthmap.org promed http://www.promedmail.org possible with increasing availability of global positioning systems devices and geocoding algorithms. such studies have revealed spatial heterogeneity in the local transmission of some directly (e.g., hiv and influenza) and indirectly (e.g., dengue and chikungunya) transmitted viruses. for example, clustering analyses of the residential locations of people with dengue in bangkok over a -year period showed evidence of localized transmission at distances less than km (salje, ; figure ). analyses of data from a large population-based cohort of hiv-infected persons in rakai district, uganda revealed strong within-household clustering of prevalent and incident hiv cases as well clustering of prevalent cases up to m (grabowski, ) . beyond descriptive applications, mapping spatiotemporal patterns of viral infections can provide fundamental insights into transmission dynamics at different spatial scales. traveling waves from large cities to small towns were shown to drive the spatiotemporal dynamics of measles in england and wales (xia, ) . the incidence of dengue hemorrhagic fever across thailand manifested as a traveling wave emanating from bangkok and moving radially at a speed of km/month (cummings, ) . insight into the spatial epidemiology of viral infections and associations with environmental risk factors can be greatly enhanced when information on the spatial location of cases is combined with remotely sensed environmental data (rodgers, ) . the spatial coordinates of cases can be overlaid on satellite imagery to demonstrate relationships with environmental features-such as bodies of water-and formally analyzed using spatial statistical techniques. satellite sensors that detect reflected visible or infrared radiation provide additional information on temperature, rainfall, humidity, and vegetation among other variables, which are particularly important for the transmission dynamics of vector-borne viral infections. satellite data for epidemiologic analyses are provided by a number of sources such as: ( ) earth-observing satellites with high spatial resolution ( - m) but low repeat frequencies such as ikonos and landsat satellites; ( ) oceanographic and atmospheric satellites such as modis and aster with lower spatial resolution ( . - km) that provide images of the earth surface twice a day; and ( ) geostationary weather satellites such as geos with large spatial resolution ( - km). the statistical relationships between cases and environmental risk factors can be used to construct risk maps. risk maps display the similarity of environmental features in unsampled locations to environmental features in locations where the disease is measured to be present or absent. spatial analysis of the initial cases of west nile virus infection in new york city in identified a significant spatial cluster (brownstein, ) . using models incorporating measures of vegetation cover from satellite imagery, the risk of west nile virus could be estimated throughout the city. a more recent risk map for west nile virus in suffolk county, new york, was generated with data on vector habitat, landscape, virus activity, and socioeconomic variables derived from publicly available data sets (rochlin, ; figure ). population movement plays a crucial role in the spread of viral infections. in the past, quantifying the contribution of movement to viral transmission dynamics at different spatial scales was challenging, due to limited data. as an early example, the impact of restrictions of animal movement on transmission of foot-and-mouth disease in was estimated, using detailed contact-tracing data from farms in the united kingdom (shirley, ) . however, such detailed data are rarely available for patterns of human movement. studies have attempted to model the impact of long-range human movement on the spread of viral diseases using measures such as distance between cities, commuting rates, and data on air travel. this approach has been used to explain regional and interregional spread of influenza viruses. data on air traffic volume, distance between areas, and population sizes have been invoked to describe and predict local and regional spread of chikungunya virus in the americas (tatem, ) . new technologies have greatly enhanced the capacity to study the impact of human movement on transmission dynamics of infectious diseases. data from mobile phones and gps loggers can be used to characterize individual movement patterns and the time spent in different locations (figure ) . individual movement patterns can be overlaid on risk maps to quantify movement to and from areas of high (sources) and low risk (sinks) as well as to estimate potential contact patterns. gps data loggers generated . million gps data points to track the fine-scale mobility patterns of residents from two neighborhoods in iquitos, peru, to better understand the epidemiology of viral infections (vazquez-prokopec, ) . most movement occurred within km of an individual's home. however, potential contacts between individuals were irregular and temporally unstructured, with fewer than half of the tracked participants having a regular, predictable routine. the investigators explored the potential impact of these temporally unstructured daily routines and contact patterns on the simulated spread of influenza virus. the projected outbreak size was % larger as a consequence of these unstructured contact patterns, in comparison to scenarios modeling temporally structured contacts. in addition to identifying individual and environmental characteristics associated with temporal and spatial patterns of viral infections, transmission networks are critical drivers of the dynamics of viral infections. analysis of transmission networks defines the host contact structure within which directly transmitted viral infections spread. network theory and analysis are complex subjects with a long history in mathematics and sociology, but have recently been adapted by infectious disease epidemiologists. the epidemiologic study of social networks is facilitated by unique study designs, including snowball sampling or respondent-driven sampling, in which study participants are asked to recruit additional participants among their social contacts. differing sexual contact patterns serve as an example of the importance of contact networks to the understanding of viral epidemiology. concurrent sexual partnerships amplify the spread of hiv compared with serial monogamy. this could partially explain the dramatic differences in the prevalence of hiv in different countries. social networks were shown to affect transmission of the h n influenza virus, and were responsible for cyclical patterns of transmission between schools, communities, and households. technological advances in quantifying contact patterns, with wearable sensors and the use of viral genetic signatures, have greatly enhanced the ability to understand complex transmission networks. self-reported contact histories and contact tracing are the traditional epidemiological methods to define transmission networks. contact tracing has a long history in public health, particularly in the control of sexually transmitted diseases and tuberculosis, and is critical to the control of outbreaks of viral infections such as the middle east respiratory syndrome coronavirus (mers-cov) and ebola virus. to better understand the nature of human contact patterns, sensor nodes or motes have been used to characterize the frequency and duration of contacts between individuals in settings such as schools and health-care facilities. these technologies offer opportunities to validate and complement data collected using questionnaires and contact diaries. as an example, investigators used wireless sensor network technology to obtain data on social contacts within m for high school students in the united states, enabling construction of the social network within which a respiratory pathogen could be transmitted (salathe, ) . the data revealed a high-density network with typical small-world properties, in which a small number of steps link any two individuals. computer simulations of the spread of an influenza-like virus on the weighted contact graph were in good agreement with absentee data collected during the influenza season. analysis of targeted immunization strategies suggested that contact network data can be employed to design targeted vaccination strategies that are significantly more effective than random vaccination. advances in nucleic acid sequencing and bioinformatics have led to major advances in viral epidemiology. population (sanger) sequencing has been the standard method for dna sequencing but is increasingly replaced by deep sequencing in which variants within a viral swarm are distinguished. sequencing allows for the detection of single nucleotide polymorphisms (snps) and nucleotide insertions or deletions ("indels"), analysis of synonymous and nonsynonymous mutations, and phylogenetic analysis (see chapter on virus evolution). sequencing techniques can be applied to both viral and host genomes. snps may be associated with changes in viral pathogenesis, virulence, or drug resistance. molecular techniques applied to pathogens also have been fundamental to the study of the animal origins of many viral infections including hiv and mers. phylogeographic approaches were used to trace the origins of the hiv pandemic to spillover events in central africa (sharp, ) . more recently, sequence data were used to track the animal reservoirs of mers-cov associated with the outbreaks (haagmans, ) , and to compare the ebola virus strain circulating in the west africa outbreak to strains from prior outbreaks (gire, ) . epidemiologic studies that probe host genomes can be either candidate gene studies or genome-wide association studies. the goal of these studies is to link specific changes with an increased risk of infection or disease. as an example, a small subset of individuals who failed to acquire hiv infection despite exposure, prompted studies to determine how these individuals differed from those who acquired infection. a -base-pair deletion in the human ccr gene, now referred to as ccr -delta , accounted for the resistance of these subjects. individuals who are ccr -delta homozygotes are protected against hiv infection by ccr tropic hiv strains, while heterozygotes have decreased disease severity. infectious disease epidemiologists are increasingly linking evolutionary, immunologic, and epidemiological processes, a field referred to as phylodynamics voltz, ) . because of the high mutation rates of viral pathogens, particularly rna viruses, evolutionary and epidemiological processes take place on a similar timescale (see chapter on virus evolution). according to this framework, phylodynamic processes that determine the degree of viral diversity are a function of host immune selective pressures and epidemiological patterns of transmission ( figure ). intrahost phylodynamic processes begin with molecular characteristics of the virus as well as the host's permissiveness and response to infection. for example, a single amino acid substitution in epstein-barr virus was shown to disrupt antigen presentation by specific human leukocyte antigen polymorphisms (liu, ) . this resulted in decreased t-cell receptor recognition and successful viral immune escape. the virus must also induce an "optimal" host immune response to maximize transmission to new hosts. if the virus induces a strong, proinflammatory immune response not balanced by the appropriate anti-inflammatory responses, the host may succumb to the overabundance of inflammation and cannot propagate viral transmission. alternatively, a virus that fails to stimulate an immune response may also replicate uninhibited, overwhelming, and killing the host prior to transmission. selective pressures maximize replication while sustaining transmission between hosts. interhost dynamics are affected by several factors including evolutionary pressures, timescales of infection, viral latent periods, and host population structures. typically, only a small number of virions are transmitted between hosts, creating a genetic bottleneck that limits viral diversity. a virus that mutates to cause highly pathogenic disease but is not transmitted cannot propagate its pathogenicity. cross-immunity between viral strains also precludes the replication of particular viral lineages. influenza vaccine strains require annual changes due to new circulating influenza strains that have escaped immune pressures through high mutation rates and gene re-assortment. the strong selection pressure of cross-immunity is reflected in the short branch lengths in a phylogenetic tree of influenza viruses isolated from infected individuals. thus, the selection of influenza strains for future vaccines is partly determined by cross-immunity to prior circulating strains, because influenza viral strains that circulated in the past may elicit immune protection against currently circulating strains. at the population level, phylodynamic methods have been used to estimate r for hiv and hepatitis c virus, for which reporting and surveillance data are often incomplete (volz, ) . phylodynamic and phylogeographic models also have been useful in reconstructing the spatial spread of viruses to reveal hidden patterns of transmission. for example, epidemiological and molecular studies of influenza virus transmission were compared at different spatial scales to highlight the similarities and differences between these data sources (viboud, ) . the findings were broadly consistent with large-scale studies of interregional or inter-hemispheric spread in temperate regions with multiple viral introductions resulting in epidemics followed by interepidemic periods driven by seasonal bottlenecks. however, at smaller spatial scalessuch as a country or community-epidemiological studies revealed spatially structured diffusion patterns that were not identified in molecular studies. phylogenetic analyses of gag and env genes were used to assess the spatial dynamics of hiv transmission in rural rakai district, uganda, using data from a cohort of , individuals residing in communities (grabowski, ) . of the phylogenetic clusters identified, almost half comprised two individuals sharing a household. among phylodynamics links evolutionary, immunologic, and epidemiologic processes to explain viral diversity, as shown here for equine influenza virus. for viral evolution, these processes take place on a similar timescale. within host mutations ( ) result from an interplay between optimization of viral shedding, immunologic selective pressures and host pathogenicity. transmission bottlenecks and host heterogeneity ( ) further determine the population genetic structure of the virus, which in turn influences and is determined by the epidemic dynamics. larger scale spatial dynamics at local, regional, and global levels ( ) the remaining clusters, almost three-quarters involved individuals living in different communities, suggesting transmission chains frequently extend beyond local communities in rural uganda. the timescale of infection is also important for viral diversity and transmission dynamics. some viruses are capable of initiating an acute infection that is cleared within days, while other viral infections are chronic and persist for a lifetime. the duration of infection impacts how quickly a virus must be transmitted and has implications for the infectious period and the potential to be transmitted to new hosts. viruses with long latent periods create interhost phylogenetic trees with longer branch lengths. the long duration between infection and transmission permits accumulation of viral changes through many rounds of viral replication before transmission to the next host. examples include hepatitis b virus, hepatitis c virus, and human immunodeficiency virus. availability of computational resources allows widespread use and development of classic approaches to the mathematical modeling of viral transmission dynamics, such as compartmental, metapopulation and network models, to address epidemiologic questions (see chapter on mathematical methods). these models have been used extensively in the study of viral dynamics and to explore the potential impact of control interventions. new sources of high-resolution spatial, temporal, and genetic data create opportunities for models that integrate these data with traditional epidemiological data. such analyses improve estimates of key transmission parameters and understanding of the mechanisms driving virus spread. agent-based models (also known as individual-based models) can now be run using desktop computers, and offer advantages over more traditional mathematical models. because each unit in a population is modeled explicitly in space and time and assigned specific attributes, agent-based models can reproduce the heterogeneity and complexity observed in the real world. more traditional compartmental, differential equation models often require simplifying assumptions that limit applicability. agent-based models have been used to study the spread of viruses in populations as well as the evolution of viruses within and across populations. while agent-based models are intuitive and easy to formulate, these models are often difficult to construct due to the large number of parameters necessary to describe the behavior and interaction between individual units. commercial frameworks that offer large computational power and intuitive user interphases have also become increasingly available. the global epidemic and mobility model (gleam) on the gleamviz platform (www.gleamviz.com), for example, contains extensive data on populations and human mobility, and allows stochastic simulation of the global spread of infectious diseases using user-defined transmission models. our understanding of the epidemiology of viral infections is being revolutionized by the integration of traditional epidemiological information with novel sources of data. l data streams from the internet are promising sources to enhance traditional surveillance but have yet to be fully validated. l molecular data on viral genomic sequences provide unprecedented opportunities to characterize viral transmission pathways. l phylodynamic and phylogeographic models have been used to estimate r , and characterize the spatial spread of viruses. l network analysis reveals hidden patterns of transmission between population subgroups that are not easy to capture with traditional epidemiological methods. l novel analytical and computational resources are playing a key role in integrating information from multiple large data banks. these more comprehensive methods improve our ability to estimate the impact of infection control measures. the combination of traditional and evolving methodologies is closing the gap between epidemiological studies and viral pathogenesis. these developments have laid the foundation for exciting future research that will complement other approaches to the pathogenesis of viral diseases. with these evolving technologies in mind, it is timely to ask: is the world able to control viral diseases more effectively? it is a mixed score card. on the one hand, smallpox has been eradicated and we are on the verge of elimination of wild polioviruses. furthermore, deaths of children under the age of years (which are mainly due to viral and other infectious diseases) have decreased by almost % in the last few decades. on the other hand, the aids pandemic continues to rage in low-income countries, with only a slight reduction in the annual incidence of new infections. the united states has not done any better in reducing hiv incidence which has been unchanged for at least years. the - ebola pandemic in west africa reflects the limited capacity for dealing with new and emerging viral diseases on a global basis. in conclusion, epidemiological science continues to advance with evolving new technologies, but their application to public health remains a future challenge and opportunity. new technologies for reporting real-time emergent infections google trends: a web-based tool for real-time surveillance of disease outbreaks unifying the epidemiological and evolutionary dynamics of pathogens studying the global distribution of infectious diseases using gis and rs spatial analysis of west nile virus: rapid risk assessment of an introduced vector-borne zoonosis pandemics in the age of twitter: content analysis of tweets during the h n outbreak travelling waves in the occurrence of dengue haemorrhagic fever in thailand detecting influenza epidemics using search engine query data rakai health sciences program. the role of viral introductions in sustaining community-based hiv epidemics in rural uganda: evidence from spatial clustering, phylogenetics, and egocentric transmission models middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation the parable of google flu: traps in big data analysis wikipedia usage estimates prevalence of influenza-like illness in the united states in near real-time a molecular basis for the interplay between t cells, viral mutants, and human leukocyte antigen micropolymorphism assessing and maximizing the acceptability of global positioning system device use for studying the role of human movement in dengue virus transmission in iquitos predictive mapping of human risk for west nile virus (wnv) based on environmental and socioeconomic factors a high-resolution human contact network for infectious disease transmission revealing the microscale spatial signature of dengue transmission and immunity in an urban population the evolution of hiv- and the origin of where diseases and networks collide: lessons to be learnt from a study of the foot-and-mouth disease epidemic the use of twitter to track levels of disease activity and public concern in the u.s. during the influenza a h n pandemic air travel and vectorborne disease movement usefulness of commercially available gps data-loggers for tracking human movement and exposure to dengue virus using gps technology to quantify human mobility, dynamic contacts and infectious disease dynamics in a resource-poor urban environment contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission viral phylodynamics measles metapopulation dynamics: a gravity model for epidemiological coupling and dynamics key: cord- - jx u js authors: sulmasy, daniel p. title: “diseases and natural kinds” date: journal: theor med bioeth doi: . /s - - -x sha: doc_id: cord_uid: jx u js david thomasma called for the development of a medical ethics based squarely on the philosophy of medicine. he recognized, however, that widespread anti-essentialism presented a significant barrier to such an approach. the aim of this article is to introduce a theory that challenges these anti-essentialist objections. the notion of natural kinds presents a modest form of essentialism that can serve as the basis for a foundationalist philosophy of medicine. the notion of a natural kind is neither static nor reductionistic. disease can be understood as making necessary reference to living natural kinds without invoking the claim that diseases themselves are natural kinds. the idea that natural kinds have a natural disposition to flourish as the kinds of things that they are provides a telos to which to tether the notion of disease – an objective telos that is broader than mere survival and narrower than subjective choice. it is argued that while nosology is descriptive and may have therapeutic implications, disease classification is fundamentally explanatory. sickness and illness, while referring to the same state of affairs, can be distinguished from disease phenomenologically. scientific and diagnostic fallibility in making judgments about diseases do not diminish the objectivity of this notion of disease. diseases are things, not kinds. injury is a concept parallel to disease that also makes necessary reference to living natural kinds. these ideas provide a new possibility for the development of a philosophy of medicine with implications for medical ethics. twenty-five years ago, in the pages of this journal, david c. thomasma and edmund d. pellegrino challenged the field by arguing that medical ethics must begin with the philosophy of medicine. before his untimely death, dave let us know that such a comprehensive philosophy of medicine -one that could serve as a foundation for medical ethics -still eluded us. perhaps, he seemed to suggest, it had only become more difficult to achieve. in , he wrote, practice of medicine today, the movement back towards foundations will be difficult indeed. in this essay, dedicated to david's memory, i would like to set forth a possible new direction for pursuing this challenge. as he noted, such an endeavor amounts to swimming upstream against the currents of contemporary ''bioethics.'' despite the difficulties, however, it would appear that substantial new work in analytic philosophy might (surprisingly) lead to the sort of foundation for the philosophy of medicine and medical ethics that david sought. the notion of natural kinds has become very important in contemporary anglo-american philosophy, but scholars have not yet taken seriously how the notion that human beings are a natural kind might have implications for the philosophy of medicine and medical ethics. i will not attempt to lay out all of the implications of this idea in a single essay. rather, i will attempt to explain the notion of a natural kind, and to outline some of the ways in which this notion might help to illuminate a few fundamental questions about disease and health care. in the long run, a philosophy of medicine based centrally upon the notion of the human as a natural kind might be exactly the sort of foundation for medical ethics that david thomasma sought. essences have not been very popular in recent philosophy. positivists, for instance, complained that they had never seen one. the philosophy of medicine has also largely discarded essentialism as a sort of lingering, outmoded aristotelian habit of speech, or as jensen has written, ''the child of a static (pre-darwinian) view of nature.'' jensen goes on to say that, ''a unit of classification is a human construct,'' and argues that since diseases are evolving entities, they cannot be assigned essences, which are necessarily static. the idea that all classifications are human constructs is not new. it traces its origins to th century nominalism. but mere nominalism is not radical enough for the st century. for the post-modern bioethicist, medical terms are all socially constructed and thus, little more than disguised attempts to assert power and domination over others. thus, one group of postmodern feminists has critiqued the research agenda in women's health, writing that the concept to which the word 'woman' refers is '''crowded with the overdeterminations of male supremacy,' and... contaminated with misogyny and sexism.'' they argue further that 'gender' and 'sex' are also tainted with essentialism. the idea of pure sex difference uninfluenced by social constructions presupposes a mythical time prior to gendering. since it is impossible to be both inside culture and outside it at the same time, sexual differences are always already informed by gender. poststructuralist critiques have demonstrated that women's bodies and their experiences are only knowable through the discourses that constitute them, so both sex and gender are socially constructed. nonetheless, to advance their own political agenda of claiming power for women and righting past injustices, they advocate a cynical appropriation of the essentialist terms 'woman', 'sex', and 'gender' as tools for their movement. we argue that a ''strategic essentialism,'' as advanced by spivak, provides feminists the theoretical ground from which to argue their political agendas. in this formulation, essentialist categories such as woman/women, sex, or gender are used strategically to achieve specific purposes or aims. in spivak's terms, strategic essentialism is the ''strategic use of a positivist essentialism in a scrupulously visible political interest.'' in this sense, the strategy suits the situation, and the essentialized term (i.e., woman) becomes a mobilizing slogan aimed at specific political ends. in an academic climate such as this, small wonder that dave thomasma thought that the movement back to foundationalism would be difficult. and yet, powerful arguments are emerging in recent analytic philosophy that are making the concept of essentialism not only respectable once again, but even changing the antiessentialist received view. consider the work of saul kripke on identity and necessity. kripke has argued that ''identity statements are necessary and not contingent.'' he has used the term ''rigid designator'' to describe what might in an earlier era have been called an essential characteristic of a thing. a rigid designator is ''a term that designates the same object in all possible worlds.'' he does not imply that the thing must exist, of necessity, in all possible worlds, but rather that if it were to exist in any possible world, the rigid designator would designate it in that world. this is not trivial or obscure in its consequences for medical ethics. every time, for instance, that one requests a substituted judgment, the question is framed as a contrary to fact conditional that posits a possible world that is not our own. a clinician asks the daughter of a comatose patient, ''what would your mother, mrs. penelope smith, have wanted in terms of life sustaining treatment if she were able to speak to us today?'' kripke would insist that the mrs. penelope smith to whom we refer in the possible world in which comatose people's thoughts can be understood is not some other person who happens to look like mrs. penelope smith, but is identical with the mrs. penelope smith lying in the bed in the icu. it is this woman's values we are after. it is critically important to note that this routine exercise in clinical ethics, the elicitation of a substituted judgment, hailed by purportedly anti-essentialist bioethicists everywhere, is not even conceptually possible unless there is something akin to the essence of mrs. penelope smith. she is rigidly designated by her name. baruch brody's analysis would also support this interpretation, but on more forthrightly essentialist, aristotelian grounds. brody argues that ''identity across possible worlds is prior to rigid designation,'' and that something must already have been picked out as the same in any possible world before it can be designated as such. kripke's work on naming, identity, and natural necessity has also been complemented and even extended by david wiggins' work on natural kinds. wiggins has developed the notion of ''sortal predicates'' by which entities of a certain kind are picked out, identified, and re-identified over time. he has come to the conclusion that the predicate calculus simply cannot account for much of what is (particularly living things) unless it is enriched by the addition of the concept of a sortal predicate. in other words, one cannot say ''smith is the same man i saw yesterday'' without predicating that smith and the man i saw yesterday belong to the same natural kind (in this case, man). and if this is the case, then wiggins says, we must embrace at least a ''modest'' form of essentialism. these essences are not platonic forms. as wiggins puts it, essences are not ''fancied vacuities parading in the shadow of familiar things as the ultimate explanation of everything that happens in the world. they are the natures whose possession by their owners is the precondition of their owners being divided from rest of reality as anything at all.'' with respect to the philosophy and ethics of medicine, one would think it obvious that at least this sort of modified essentialism would be necessary in order to function in the clinical world. but while this might be obvious to clinicians, it has not been obvious to philosophers. how might the concept of natural kinds bridge the gap between the common sense essentialism of clinicians and the philosophy of medicine? building on the work of putnam, wiggins very clearly, if densely, tells what a natural kind entails: the determination of a natural kind stands or falls with the existence of law-like principles that will collect together the actual extension of the kind around an arbitrary good specimen of it; and these law-like principles will also determine the characteristic development and typical history of members of this extension. diseases and natural kinds i will argue that one cannot understand human disease unless one understands that human beings are a natural kind. this is not to argue that diseases are natural kinds, but that the concept of a disease must make necessary reference to a natural kind. if one is sincerely interested in acquiring knowledge about a subject, rather than beginning with obscure cases proposed as counterarguments in support of all manner of skeptical conclusions, one should begin by studying what is common and paradigmatic. the concept of natural kinds provides a framework for doing so. taking biology, pathology, plant science, human medicine, and veterinary medicine very seriously seems critical to any contemporary discussion of the general notion of disease. in this light, the following points are worth noting: ( ) a disease is always a disease of (at least one) living natural kind. for example, powdery mildew is a disease that afflicts members of at least one living natural kind -rosemary bushes (rosmarinus officinalis). but it is not a disease of the human natural kind. human diseases are diseases of members of the human natural kind. ( ) diseases can afflict more than one natural kind. plants other than rosemary, such as grapes and roses, can become diseased with powdery mildew. dogs and human beings both can be afflicted with myasthenia gravis. ( ) diseases of one natural kind may be caused by another natural kind -e.g. -erysiphe spp. and sphaerotheca spp. are among the natural kinds causing powdery mildew. plasmodium falciparum causes one type of malaria in human beings. but clearly not all diseases are caused by one natural kind invading another. there is no known pathogenic natural kind that causes myasthenia gravis. the important point is that to understand any of the conditions named above as diseases, one must understand how these conditions diseases and natural kinds affect the afflicted individual as the kind of thing that it is. there are law-like principles that determine the characteristic history and typical patterns of development that collect together the actual extension of those individual entities one calls members of the human natural kind. in fact, two arbitrary, good specimens of this natural kind have been picked out, radiologically dissected, and their anatomical features placed on line at the national library of medicine in bethesda, maryland. and amazingly enough, the average man or woman (or even child), is easily able to tell which entities belong to this natural kind (and their sex), in the absence of technical anatomical knowledge and without reference to a political agenda. one can also readily recognize those deviations from the characteristic development and typical history that render some members of this natural kind defective. so, children born with syndactyly (webbed fingers) are defective members of the human natural kind. they are not members of some other natural kind, say dolphins. to say something is a defective x implies that one knows what kind of thing an x is. without at least this much essentialism, medicine would not even be conceptually possible. natural kinds have a natural teleology. to say that they have characteristic patterns of development and typical histories implies a teleology. anthony lisska has called the properties that determine the pattern of development of a natural kind its ''dispositional predicates.'' the successful unfolding of these dispositions over developmental time -a program and a pattern -allows the individual to flourish as the kind of thing that it is. natural kinds have dispositions. this much teleology must be granted. uranium undergoes a characteristic pattern of radioactive decay. various types of stars have dispositional predicates. they develop and change over periods of time that may seem long by human standards, but there is a pattern by which a star's history must unfold temporally if it is to behave as the kind of thing that it is. dispositional predicates seem especially characteristic of living natural kinds. in fact, philippa foot has written that the word 'good' is an attributive adjective, not a predicative adjective, and cannot be understood apart from an understanding of what kind of thing something is. so, for instance, the word 'good', as used in the phrase, ''good roots,'' cannot be understood unless one knows that it is being attributed to a rosemary bush and not to a rhinoceros. having deep roots would not be good for a rhinoceros. natural kinds have natural tendencies. much of the scientific enterprise consists of coming better to understand these natural tendencies. this teleology is natural. it does not imply the anthropomorphizing of things or the ascribing of conscious goal-directed activity to them, nor does it imply that there is a deity that directs their development. that natural kinds have law-like principles that determine how they develop and flourish as the kinds of things that they are is simply a fact about the world as we encounter it. natural kinds are not timeless, changeless platonic entities. the concept of a natural kind is perfectly compatible with biological evolution. that living natural kinds evolve over time is simply one of the law-like generalizations that characterize them. some will go extinct. some may change so dramatically that they become a new natural kind. but for human medicine (as well as veterinary medicine and botany), the slow time frame of evolutionary change is just not significant for clinical pathology and practice. human understanding of the law-like generalizations that determine the characteristic development and typical history of natural kinds may also change over time. but this does not mean that the kind has changed, merely our understanding of it. this understanding has a mind-to-world direction of fit -i.e. -our understanding must be better fitted to the kind; the kind is not shaped to our understanding of it. thus, there is scientific progress in medicine. humoral theories of disease have been abandoned. bloodletting has been replaced by plasmaphoresis. but the gene of mendel and the gene of watson and crick are one and the same gene, now understood in greater depth. genetics is the study of one aspect of the lawlike principles that determine the characteristic development and typical history of living natural kinds. we understand human genetic principles better now than we did years ago. but it is still the same natural kind that is studied and treated by human medicinethe human natural kind. the idea of a natural kind is not reductionistic. the fundamental way that complex systems are understood is through a hierarchical analysis of their structure, recognizing that there is both ''top-down'' and ''bottom-up'' causation along the hierarchical levels. natural kinds can be understood at multiple hierarchical levels of organization, and lower levels of organization are neither individually nor jointly sufficient to explain higher levels of organization, or even sufficient to ask the questions that would be relevant to the understanding of the kind at that higher level of organization. these properties, those that can only be understood in relation to the hierarchical level of organization to which they belong, are said to be ''emergent.'' that is to say, they ''supervene'' only at successively higher levels of organization, but cannot be fully explained by lower levels of organization. thus, physics is insufficient to explain chemistry, chemistry is insufficient to explain biology, biology is insufficient to explain psychology, and psychology is insufficient to explain morality. for example, cell walls contain both quarks and l-mesons, but quarks and l-mesons cannot explain what a cell wall is. this does not preclude the work by which scientists can arrive at partial explanations of various states of affairs of a given natural kind, considered at a given level of organization, by invoking an understanding of one or another state of affairs of lower levels of organization of the kind. thus, at a chemical level, understanding that a base pair mutation results in the substitution of valine for glycine at the th position of the b chain of the hemoglobin molecule provides a substantial causal explanation for sickle cell disease. this information is useful and important. it has diagnostic and therapeutic implications. but as an explanation of what sickle cell disease is, it is radically incomplete. one cannot understand this disease without also understanding the biology of plasmodium falciparum, the ecological concept of inclusive fit, population genetics, patterns of migration, the physiology of blood flow, the existence of capillaries, the biology of pain, and the concept of infarction, none of which can be fully reduced to chemistry. the chemical explanation deepens the biological explanation, but only biology and levels of scientific hierarchy above the biological (so-called ''top-down causation'') fully explain the biological phenomena. a moral, rational, living natural kind can be understood, nonreductionistically, at multiple, hierarchically arranged levels of organization at which level-specific properties emerge, featuring both top-down and bottom-up causation. for the human natural kind, these might be sketched out as: evolutionary ecology sociology, economics, history psychology physiology anatomy molecular biology and genetics physics the domain of medicine is primarily the domain of the biology of individual members of the human natural kind -particularly the levels of anatomy, physiology, and psychology. those law-like principles that determine the characteristic development and typical history of members of this natural kind at these hierarchical levels of scientific understanding are the subject of medicine as an applied science. these are the biological principles of the kind. this is not to say that physics and sociology are irrelevant to medicine -patients can develop cancer because of radon exposure and economic factors may help to explain why a particular patient came to be exposed to radon. there is both top-down and bottom-up causation. but medical science cannot claim to be the complete human science. it must have boundaries if it is to be a field of inquiry distinguished from others. and this is determined by its praxis, with its emphasis on caring for the individual with disturbed biology at the level of experience and function. so, for example, molecular biology matters intensely to medicine because it can explain alterations in anatomy, physiology, and psychology and offer possibilities for corrective intervention. there can be ''allied'' sciences of medical sociology, medical economics, public health, and medical ethics. there is also a field of medical engineering, applying physics to medical care. but these fields merit the adjective, 'medical' precisely because they can help to explain or can help produce effective interventions in matters of anatomy, physiology, and psychology. this much being said, i am now ready to offer a formal definition of disease. a disease is a class of states of affairs of individual members of a living natural kind x, that: ( ) disturbs the internal biological relations (law-like principles) that determine the characteristic development and typical history of members of the kind, x, ( ) in a pattern of disturbance shared with at least one other member of the kind, x. ( ) the aim of this classification must be to provide at least a provisional basis for explaining the causes and/or natural history of a disturbance in the internal biological relations of the affected members of x (and, if x is a self-reflective natural kind, can serve as an explanation of the illness of those so affected), ( ) and at least some individuals of whom (or which) this class of states of affairs can be predicated are, by virtue of that state, inhibited from flourishing as xs. i must further explicate this fairly dense definition. a. a disease is not a natural kind. it is a classification of a certain state of affairs that can occur in members of particular living natural kinds. b. the ''biological internal relations (law-like principles)'' encompass anatomy, physiology, and psychology. not all variations in the law-like principles that govern natural kinds at these levels of organization are diseases, but only those variations that also meet the other criteria. c. there are no diseases that are unique to particular individuals. individuals may be sick or ill, but until the pattern of disturbance is detected in other members of the kind, it is not a disease. disease is a scientific concept and concerns more than the individual. d. the purpose of disease classification (nosology) is, in the first place, explanatory. even if the disease does not provide a causal explanation for the illness, the purpose of bringing a pattern of disturbance under a particular name is to predict an expected natural history and provide the first step towards explanation. even if the explanation is not scientific by contemporary standards (e.g., possession by demons or angry ancestors), the classification of a pattern of disturbance in the law-like principles that govern the biological organization of a living natural kind as a disease serves an explanatory function. e. the hope of medical practice is that better explanatory knowledge of a disease will provide better treatment. the th and th centuries rewarded medicine handsomely for pursuing explanatory knowledge of diseases. the eventual payoff was better therapy. but the aim of nosology is fundamentally explanatory, not therapeutic. f. there can be asymptomatic disease. but if a pattern of disturbance in the law-like biological principles that determine the characteristic development and typical history of a living natural kind is to be called a disease, at least some individuals with the disease must be inhibited from flourishing as the kinds of things that they are. for example, prostate cancer at age may be ''incidental'' and never interfere with a man's flourishing. but unless prostate cancer interfered with at least some men's flourishing, it would not be called a disease. g. the telos in this definition is that the individual should flourish as the kind of thing that it is. this is an important difference from christopher boorse's specification of the telos in his definition of disease as survival and reproduction. survival and reproduction might be sufficient for an amoeba to flourish as the kind of thing that it is. but survival is scarcely enough for a rosemary bush, never mind a human being. a rosemary bush might survive a bout of powdery mildew, live a normal lifespan, and reproduce well. but while infected, it is not flourishing as a rosemary bush. likewise, a human being with a coronavirus infection has a disease (the common cold). it is almost inconceivable that this could affect her survival or reproduction. but while infected, she fails to flourish as the kind of thing she is. h. setting as the telos the flourishing of the individual as the kind of thing that it is also explains why it can be controversial to classify as diseases certain patterns of variation in the law-like biological principles that determine the characteristic development and typical history of a living natural kind. in particular, patterns of human behavior are most susceptible to being controversially called diseases. but this does not undermine the definition of a disease. it is only to say that the task of deciding whether to designate as disease a pattern of variation in the lawlike principles that govern a thing as a member of a kind will have some very clear cases. by virtue of being the kinds of things that they are, human beings are incredibly varied -biologically, psychologically, and socially. human beings make choices, some of which preclude one good in order to pursue another. there is, therefore, a subject-relativity to the flourishing of human beings that ought not be confused with subjectivity. thus, human flourishing includes a certain diversity, and this diversity is objectively good. while the purpose of this essay is not to engage in a discussion of medical ethics, it must be stated that this diversity is not morally unbounded. human choices ought not detract from the common good (as integrally understood) and ought not undermine the basic conditions by which human beings flourish as the kinds of things that they are. with minor variations, i accept the by-now classical distinction between disease and illness, and i also accept the distinction between sickness and illness, but with a significant variation on the use of these terms. in sum, i hold that these three words designate three ways of referring to the same state of affairs -individually, socially, and scientifically -corresponding to the terms sickness, illness, and disease, respectively. sickness and illness are not the same as disease. distinctions between these terms have phenomenological and sociological meaning for human beings. while the first two terms, in particular, are largely synonymous in ordinary language, they can be used as technical terms to stipulate different phenomenological and sociological aspects of the lived experience of diseased human beings. sickness, as i use the term, is a state of disturbed internal biological relations that appears (from a non-professional perspective) to be inhibiting the individual from flourishing as the kind of thing that it is. individual members of any living natural kind, whether a rosemary bush or a fruit fly, can be said to be sick. generally, this disturbed state has observable manifestations, but not always. to know that an individual member of a kind is sick, one can observe the pattern of disturbance, and, with at least some knowledge of what it takes for such a kind of thing to flourish, one can make the judgment that the individual is sick. if a crow is walking around in clockwise circles and is not flying, not flapping its wings, not eating, and not cawing, most adult north american observers who know enough about what a crow is and how it typically behaves can make the judgment that the bird is sick. one can also ask for a report (from an individual capable of so reporting) about abnormal sensations or other subjective indications of disturbed biology associated with diminished flourishing. before one hears a report from the individual, however, one can make one's own judgment and simply ask for confirmation, saying, ''you look as if you are sick.'' illness is a socially mediated (but also unscientific) judgment about the exact same state of affairs to which sickness refers. illness connotes the recognition of a pattern, but is not a judgment that is made with an explanatory intention. an individual manifests a pattern of disturbed internal biological relations and appears not to be flourishing (is sick). another observer further adduces that this pattern of disturbance is not unique, and that this judgment has been made regarding other members of the kind. unscientifically, one judges, ''this illness is something that has been going around.'' in a rational, self-reflective, and socially interdependent natural kind such as the human, the individual, subjectively appreciated state that i have called sickness must generally be validated by other member(s) of the kind, thus allowing the ill individual to assume ''the sick role.'' disease serves as an explanatory designation for the same state of affairs. this individual's sickness, the illness that has been ''going around,'' is explained by judging that the individual suffers from a disease, e.g., influenza. human disease is therefore also social, but its aim is scientific -an explanation for the observed biological disturbance that does, scientifically, cause at least some individuals, so afflicted, to fail to flourish as the kinds of things that they are. symptoms are the sensory signals by which a sensible natural kind is alerted that internal biological relations are askew. when symptoms are sensed by self-reflective individuals, such individuals can use these sense data to judge that they suffer from sickness -feeling feverish, nauseated, pruritic, being in pain, etc. many living natural kinds have such signals. many of these signals have biological purpose -e.g. -pain may signal that a behavioral response to avoid the offending stimulus is in order. some are biologically inappropriate, or appropriate in the short-term but not in the long-term. symptoms are ultimately subjective, even if the cause of the symptoms is not. among the members of self-reflective natural kinds, symptoms assume meaning. one author has recently suggested the primary symptom is der unheimliche (a sense of not being at home in oneself). an individual sensing symptoms is often initially uncertain of the veracity of a symptom complex or its meaning. this leads the individual to seek validation by others. when this occurs, the patient enters the sick role and concludes, ''i am ill.'' disease, by contrast, is a class of patterns of actual disturbance in the internal biological relations of members of a living natural kind that can explain the sickness of individuals of whom the disease can be predicated. disease can also explain the sickness or illness of afflicted individuals if the natural kind in question is self-reflective and social. diagnosis is the art by which specialists determine that an individual's illness can be explained by a particular disease. this art requires recognizing a pattern of disturbance that typifies the state of affairs that defines the particular disease. diagnosis is an epistemic project. the data are the symptoms (if the individual has the linguistic capacity to report on internal sensations), and signs. signs are observable manifestations of disturbances in the internal relations or law-like principles that determine the characteristic development and typical history of sick members of the kind to which the individual belongs. signs are what others (e.g., doctors and nurses) observe as the effects of illness. some symptoms cannot be observed and can only be reported, for example, the symptom of seeing yellow halos around lights. some signs are only observed directly by others, and cannot be experienced subjectively, or even observed of oneself directly, such as an asymptomatic change in the surface of the retina. often, signs and symptoms will be intrinsically linked. the same phenomenon can have two sides -it can be experienced as a symptom and observed as a sign. for example, a patient may experience the symptom of feeling feverish. if a doctor observes this patient, the doctor will note that the patient has, perhaps among other signs, a fever. a diagnosis is a disease name that has been judged to be predicable of a state of affairs of a given member of a given natural kind, serving at least as a provisional explanation for the observed pattern of disturbance in the internal, kind-typical, biological relations of the individual. a diagnosis is a judgment that the pattern of disturbance in an individual can be classified as belonging to a named disease. diseases are thus classes of disturbed states of affairs in natural kinds. a diagnosis is a judgment that a certain state of affairs in a particular individual belongs to a certain class. lonergan's distinction between classical and statistical heuristic structures provides an important insight into the ways that diseases are currently understood. from a scientific point of view, there are two basic ways of understanding phenomena -classically and statistically. in classical science, the data converge upon some formula or understanding, such as pv = nrt, or e= mc , or the notion that stars form clusters called galaxies. in statistical science, one appreciates that the phenomenon of interest is itself stochastic, and that one can only understand this phenomenon by coming to know a value such as a mean, from which this inherently non-systematic process cannot systematically deviate. so, for instance, one comes to know that the mean temperature in paris in july is °c, or that the mean temperature of the background cosmic microwave radiation in space is . °k. this distinction is also helpful in understanding medical science, some of which is understood via a classical heuristic structure, and some of which is understood via a statistical heuristic structure. so, for instance, classical physiological laws obtained in medicine, such as the henderson-hasselbach equation: ph = pk a þ log ½conjugate base ½weak acid . the data about acid-base balance in the human natural kind converge upon this formula. but other processes can only be understood statistically, such as the fact that the ulnar artery is absent in about % of members of the human natural kind. diseases likewise are understood both classically and statistically. pneumococcal pneumonia is an example of a classical disease. the data converge on a particular description of the pattern of disturbance in the internal biological relations afflicting individual members of the human natural kind. this does not mean there will be no variations. in fact, the variation one sees in biology is greater than the variation one sees in physics. there is variation even in physics, and a classical law such as pv = nrt does not describe each measurement of pressure, temperature, molar quantity, and volume, but the for-diseases and natural kinds mula upon which multiple such measurements converge. likewise, the data do converge on a classical description of pneumococcal pneumonia even if it is not true that each and every case of pneumococcal pneumonia is exactly the same. there are also statistical diseases. since human diseases are disturbances in the biological law-like properties that govern the typical history and characteristic development of the human natural kind, it is not the mean value or central tendency but variations from the mean that are of concern to medicine. medicine characterizes some such variations as diseases, to the extent that they also fulfill the other criteria for classifying something as a disease -especially that they inhibit human flourishing. thus, some statistical variations (such as the absence of an ulnar artery) are not classified as diseases, while others (such as hypertension) are classified as diseases. the patterns of reasoning by which one makes the judgment that a particular state of affairs in a particular individual belongs to the class defined by a particular disease are multiple and complexdeductive, analogical, statistical, pragmatic, and more. a full discussion of diagnostic reasoning is beyond the scope of this essay. the philosophical literature on diseases has raged on for decades as a pitched battle between realists and anti-realists. the modest essentialism that accompanies the notion of natural kinds provides the basis for a modified form of realism about disease. the view proposed here may provide a solution to the problems posed by previous defenses of disease realism. diseases are not primary existents. 'systemic lupus erythematosis' does not pick out a primary existent, but a class of states of affairs occurring in members of a natural kind(s). diseases are not natural kinds, but states of affairs. diseases have no essences. saying this does not imply, however, that diseases have no objective basis, or are merely human constructions, or that diseases are merely value judgments. diseases make necessary reference to natural kinds, and natural kinds admit of at least a modest essentialism. it is essentialism about living natural kinds and their natural dispositions that provides the foundation for realism about diseases. the patterns of disturbance that one classifies as diseases are not arbitrary. it is the pattern of disturbed internal biological relation-ships in the natural kind that imposes itself upon the observer; the observer does not impose the pattern upon the affected members of the kind. that is to say, there is a mind-to-world direction of fit -the mind of the observer must conform to the world for the observer's beliefs about disease to be true. the world is the standard by which the observer's beliefs are judged. further, many diseases of one natural kind are caused by other natural kinds, and the pattern of disturbed development of a diseased natural kind may reflect the flourishing of the one causing the disease (e.g., in malaria, the illness of a member of the human natural kind is explained by the characteristic development and typical history of plasmodium falciparum). thus, the essentialism of two natural kinds may necessarily be invoked in defining some diseases. arguments are often proposed in (roughly) the following form: it was once thought that x was a disease. now, we no longer think that x is a disease. therefore diseases are social constructions. such arguments seem superficially to be sound, but they are specious. that the medical community can change its beliefs about disease does not imply that diseases are constructed by physicians according to the mores of their era. an alternative interpretation is that the former belief or the present belief or both are wrong. medical judgments are fallible. but fallibility does not imply subjectivity. for example, it may turn out that edwin hubble was wrong and that the universe is not expanding. but whether hubble was right or wrong depends upon the universe, not upon the mental states of human beings. in thinking about fallibility in medicine, it is important to distinguish two types of fallible judgment about disease -scientific judgments and diagnostic judgments. among scientific judgments about diseases, there are two sub-types, both of which are fallible: ( ) one may judge incorrectly that what appears to be a pattern of disturbance in the law-like principles governing the internal biological relations of a natural kind constitutes a disease (i.e., one may be wrong in one's scientific judgment that apparent pattern x is a disease) and ( ) one may incorrectly formulate a causal explanation for the pattern of disturbance picked out by a disease name (i.e., one may be wrong in one's judgment about the cause of x). the objective basis for disease classification must be that it makes necessary reference to a natural kind and to disturbances in the internal biological relations that inhibit at least some of the affected members of the kind from flourishing as the kinds of things they are. i will argue that, at the end of the day, this is what settles the question of whether a certain observable pattern constitutes disease. those who offer putative counter-examples, such as the th century medical theory that a slave's habitual tendency to try to escape was a disease (''drapetomania''), do not in any way thereby provide the slightest bit of proof that disease is a ''value-judgment.'' the objective basis for deciding this matter is to ask whether a desire no longer to be a slave is a disturbance in the internal biological relations that inhibits afflicted members of the human natural kind from flourishing as the kinds of things they are. if someone once thought this was true of slaves who wanted to escape, this opinion is simply wrong on both counts. it might be understandable that someone would render such an erroneous opinion given the socioeconomic and historical conditions under which physicians who made such judgments lived. but it is an absolute non-sequitor to conclude that this implies that all diseases are value judgments and that therefore appendicitis is simply a human construct. that judgments are fallible does not mean that they have no objective basis. behaviors are particularly susceptible to this sort of error. even today, whether some behaviors (such as homosexuality or substance abuse) are diseases can be hotly contested by various groups. but again, this does not mean that there is no basis for settling the case. the basis for settling this question is the same: whether there is at least a plausible biological explanation and whether the behavior inhibits human beings from flourishing as the kind of things that they are. that we are not % settled on what constitutes human flourishing does not mean that there is no basis for making such judgments or that there are not thousands of other clear cut cases (such as appendicitis and malaria) where it is obvious that the observed pattern of kind-atypical activity has a biological basis and that this pattern inhibits human flourishing. medical science can also be mistaken in its causal explanations about diseases. malaria (as its name implies) was once attributed to ''bad air.'' thus, a pattern of disturbance in the law-like characteristics governing the typical development and history of human beings was noted, cases fitting this pattern were carefully classified according to patterns of fever, and each thought to have a particular biological explanation. that explanation made epidemiological sense, and it ultimately fit with the causes of malaria as presently under-stood -anopheles mosquitoes, carriers of the malarial parasites, breed where the air is ''bad.'' doubtless our future understanding of malaria will deepen further than our understanding today. but the ''mal'aria'' of medieval italy and the disease we know today are not two different diseases. they are the same disease, better understood. the fact that we can incorporate previous observations into deeper explanations of the same disease is inconsistent with the assertion that diseases are merely human constructions. throughout the history of medical science, all we have ever tried to do has been to shape our minds better to fit the world as we encounter it. the fact that we have made mistakes does not mean that disease concepts are merely subjectively or intersubjectively chosen values. the ultimate standard of the truth about malaria and other disease concepts is in the world, not in our heads. that is how we can learn that we have been mistaken in our views about diseases. human medical science will establish the criteria for deciding that a particular pattern of disturbance in the biological internal relations of an individual member of the human natural kind falls within the extension of each disease category. one can be mistaken in the criteria that one establishes, because the pattern one is attempting to capture has an objective unity. one discovers that unity; one does not create it. in lonerganian terminology, a disease is a real thing, even if it is not a natural kind. one can progress in one's understanding of a thing. for example, the medical community once judged that a diagnosis of the condition known as lyme disease required manifestations of an oligoarthritis. as more research was conducted, it was noted that oligoarthritis was one stage in this disease, and that earlier stages had different manifestations such as an erythema migrans rash. the pattern of disturbance in the internal biological relations of the human natural kind to which the disease name 'lyme disease' refers did not change. our scientific understanding of lyme disease changed and the diagnostic criteria were adjusted. mistakes are also possible in diagnostic judgments. whether someone does or does not have a disease is fallible judgment, as all physicians (and medical students) know well. mistakes are possible at multiple levels. first one must correctly interpret the observable signs and the communicated symptoms (history and physical). second, one must be knowledgeable about the patterns of signs and symptoms manifested by the diseases as they are classified by the medical science of one's era (differential diagnosis). third, one must be knowledgeable about the appropriate confirmatory tests (diagnostic testing), and sufficiently experienced to use only those that are necessary (diagnostic elegance). fourth, in the setting of uncertainty, one must avoid the extremes of rashness (making a diagnosis before there are sufficient data) and indecisiveness (deferring judgment when sufficient data are at hand). the art of medicine is the mediation between medical science (concerned with universals) and the individual who must be diagnosed and treated. diagnosis is a judgment about individuals. as aristotle has said, the doctor does not cure 'a man' universally taken, except accidentally, but callias or socrates or someone else to whom also the essence of man happens to belong. if, then, someone without the experience has the theory and knows the universal but is ignorant of the individual included under this universal, he will often fail to cure; for it is rather the individual that is curable. (metaphysics a - ) . the fact that one can be mistaken in applying the science of one's era in making a judgment about whether a particular disease name can be correctly applied to a particular pattern of disturbance in the internal biological relations of an individual member of the human natural kind does not imply that diseases are value judgments. the logic of diagnosis is intensional. the ultimate standard for whether or not one has correctly applied the disease name (as defined by the science of one's day) to a particular patient is the match between that definition and the state of affairs that actually exists in the affected individual member of the human natural kind who is being diagnosed. further, the fact that the standards that are given for defining a disease must, to some extent, be arbitrary and admit of borderline cases, does not mean that diseases are primarily human constructions and value judgments. one must be careful not to confuse the epistemological with the ontological. definition is an epistemic exercise. whatever definition one gives will exclude some cases and include others, because this is what a definition does. giving intensional definitions for ontologically real entities is an inherently fallible enterprise. in a definition of multiple myeloma, for instance, one must arrive at cutoff values, currently set at > % plasma cells in the bone marrow and > . gms/dl of an igg m-protein. as every experienced clinician knows, some individuals who do not meet these criteria actually have multiple myeloma. however, this only means that the reality to which the definition points is prior to the definition. the definition is open to revision, based on its ability to correctly classify cases as belonging to the recurring pattern of disturbance in internal biological relations of the human natural kind that the definition is attempting to capture. there will be mistakes. some cases will only come to be knowable as the pattern of disturbance in the individual progresses along its own natural history. there will also be borderline cases in which the diagnostic judgment will be difficult. but as anscombe once remarked (in a quote she attributes to samuel johnson), ''the fact of twilight does not mean there is no difference between night and day.'' borderline cases do not suffice to refute objectivity. thus, one must conclude that none of these sources of fallibility in reasoning about diseases provides a philosophical justification for radical skepticism about the realism with which clinicians must undertake the tasks of studying and diagnosing diseases. diseases are objective perturbations in the reality of the natural kinds of which they can be predicated. finally, something must be said about the distinction between disease and injury. i am very sympathetic to the spirit of boorse's defense against those who criticize his theory for failing to make this distinction. this distinction is not fundamental to the notion of disease. however, i do think the distinction has some usefulness and should be maintained by the philosophy of medicine. injury and disease are highly related but distinct concepts. the words are used quite distinctly by both medical laypersons and medical professionals. the sub-specialists who treat diseases (e.g., internists, pediatricians, and family physicians) are different from those who treat injuries (e.g., orthopedists, burn surgeons, and trauma physicians). so, it is probably worthwhile to maintain the distinction. nonetheless, disease and injury are sufficiently related that the definition of injury is really parallel to that already given for disease. like disease, injury makes necessary reference to the notion of a living natural kind. an injury is a state of affairs of an individual member of a living natural kind x, that: ( ) disrupts the physical structure or physical integrity that is characteristic of the developmental stage and is typical of the history of members of the kind, x, and ( ) this state of affairs inhibits the individual from flourishing as an x. this definition of injury is parallel to the definitions of sickness and illness. that is to say, it is a ''lay'' definition. unfortunately, there is no ready-made cluster of synonyms that one can use to distinguish the various phenomenological and sociological aspects of injury to parallel the vocabulary of sickness, illness, and disease discussed above. instead one can therefore call this lay definition, injury sense . in this sense, one can say, as a medical layperson, in parallel to the notion of sickness, ''my finger is bleeding,'' or ''i hurt my wrist.'' parallel to the sociology i associated with the word, 'illness,' the injured sense individual may seek intersubjective validation. while the same definition applies, there is therefore a usage we can call injury sense . thus, my - / year old nephew asks, ''do i have a splinter, uncle danny?'' or someone says, ''you broke your wrist,'' or ''you've burned your hand rather badly.' ' however, there are many specific injuries that are more like diseases than they are like sickness or illness in that they have been named and categorized by medical science. in the absence of synonyms to use as technical terms, one can call this injury sense . this sense of injury can be defined in a manner more formally parallel to the definition of disease. a named injury (injury sense ) is a class of states of affairs of individual members of a living natural kind x, that: ( ) disrupts the physical structure or physical integrity that is characteristic of the developmental stage and is typical of the history of members of the kind, x, ( ) in a pattern of disruption shared with at least one other member of the kind, x. ( ) the aim of this classification must be to provide at least a provisional basis for explaining the causes and/or natural history of this disruption in members of x (and, if x is a self-reflective natural kind, can serve as an explanation of the injury sense of those so affected), ( ) and at least some individuals of whom this class of states of affairs can be predicated are, by virtue of that state, inhibited from flourishing as xs. thus the physician says, ''you have a non-displaced colle's fracture,'' or ''this is a second-degree burn injury involving . % of the body surface.'' this is scientific, explanatory vocabulary. the notion of injury sense is parallel to the notion of disease in every way. while it is a different class of states of affairs of the biology of living natural kinds, injuries (sense ), like diseases, can be predicated of any living natural kind. and the concept of injury sense , like the concept of disease, is objective, even if scientific and diagnostic judgments regarding injuries can be just as fallible as those regarding diseases. the notion of natural kinds can be a powerful starting point for the philosophy of medicine. it provides a philosophically credible basis for objectivity about diseases. the modest essentialism that accompanies the notion of a natural kind meets most objections raised in the literature. it provides a basis for discussion of value in disease discourse, while maintaining that such values are objective and inherent in the dispositional predicates of natural kinds in accordance with a theory of natural goodness and the teleology of a thing flourishing as the kind of thing that it is. it provides a basis for disease realism while avoiding the pitfalls of characterizing diseases as natural kinds themselves. it may provide just the sort of foundation for the philosophy of medicine upon which to develop a medical ethics, following the plan david thomasma outlined for us but could not carry out before his untimely death. philosophy of medicine as the source for medical ethics antifoundationalism and the possibility of a moral philosophy of medicine a critique of essentialism in medicine,'' in health, disease, and causal explanations in medicine abstracting women: essentialism in women's health research identity and necessity naming and necessity,'' in semantics of natural language sameness and substance see the debate about the notion of diseases as natural kinds: robert d'amico however, the inference both seem to share, that if disease is not a natural kind then it must be a ''value judgment aquinas' theory of natural law: an analytic reconstruction philippa foot, natural goodness intentionality: an essay in the philosophy of mind on the moral nature of the universe concepts of health,'' in health care ethics: an introduction autonomy, subject-relativity, and subjective and objective theories of well-being in bioethics four basic notions of the common good dependent rational animals: why human beings need the virtues - ; and his later refinements of this distinction in ''a rebuttal on health while i accept more or less the same distinction, i am using the terms 'illness' and 'sickness' in the opposite manner as has become standard in the philosophy of medicine (see bjørn hofman since it is preferable to assign to technical terms the use that is closest to ordinary language, i propose a reversal of the standard use of these terms. i will use 'sickness' to refer to the individual's own subjective experience of disturbed internal biological relations, or to an observer's assessment of the disturbed state of an individual. i will use 'illness' to refer to the intersubjectively supported, socially mediated understanding of the same state of affairs. so, we often say that a dog is ''sick,'' but we do not usually say that a dog is ''ill das unheimliche: towards a phenomenology of illness insight: a study of human understanding i am tempted to argue that ''statistical'' diseases are only epidemiological risk factors for classical ones, but i will not explore this hypothesis further in this essay a variation on this fallacious line of argument is the illicit inference that since there are heated arguments in the present about whether certain states such as homosexuality or menopause or aging or infertility are diseases, one must conclude that diseases are socially constructed. this is the tack taken by arthur l. caplan in ''the 'unnaturalness' of aging -give me a reason to live report on the diseases and physical peculiarities of the negro race,'' in health, disease, and illness: concepts in medicine plasma cell neoplasms,'' in principles and practice of oncology war and murder toward a pragmatic theory of disease.'' in what is disease war and murder.'' in war and morality concepts of health.'' in health care ethics: an introduction on the distinction between disease and illness a rebuttal on health the unnaturalness of aging -give me a reason to live.'' in health, disease, and illness report on the diseases and physical peculiarities of the negro race.'' in health, disease, and illness: concepts in medicine is disease a natural kind the foundations of bioethics natural goodness on the triad disease, illness and sickness a critique of essentialism in medicine.'' in health, disease, and causal explanations in medicine identity and necessity.'' in identity and individuation naming and necessity.'' in semantics of natural language theory of natural law: an analytic reconstruction insight: a study of human understanding dependent rational animals: why human beings need the virtues abstracting women: essentialism in womens health research plasma cell neoplasms.'' in principles and practice of oncology on the moral nature of the universe minneapolis definitions of health and illness in the light of american values and social structure.'' in patients, physicians, and illness dis-ease about kinds: reply to damico intentionality: an essay in the philosophy of mind four basic notions of the common good das unheimliche: towards a phenomenology of illness philosophy of medicine as the source for medical ethics antifoundationalism and the possibility of a moral philosophy of medicine autonomy, subject-relativity, and subjective and objective theories of well-being in bioethics sameness and substance key: cord- -q w fb i authors: ewald, paul w. title: evolution of virulence date: - - journal: infect dis clin north am doi: . /s - ( ) - sha: doc_id: cord_uid: q w fb i at the close of the th century, the germ theory had generated a new understanding of the causes of acute infectious diseases and revealed new directions for study. this understanding contributed to the greatest improvements in health in the history of medicine. at the end of the th century, the second stage of this disciplinary development is occurring. the old germ theory is being expanded into a new germ theory, which, by integrated the full spectrum of biologic disciplines. this new germ theory is emphasizing how environments and human activities influence the characteristics of infectious agents and the broader role of infection as a cause of chronic diseases. human history cannot be understood well without understanding the causes and consequences of human disease. this fact has become amply apparent over the past few decades as the impacts of infectious diseases have been studied in the context of war, colonization, and competition [ ] [ ] [ ] [ ] [ ] . it is much less widely appreciated that the reverse is also true. historical studies of infectious diseases may help guide modern health sciences to recognize options for controlling diseases of the present and future. ecologic and evolutionary perspectives are enmeshed with the historical perspective of infectious diseases, because infectious agents spread and evolve over times scales that accord with historical events. they may influence historical events and may be influenced by such events. the influence of historical events on the evolution of pathogens largely has been neglected until the past quarter century. it has become clear that activities that were undertaken for one purpose can have unforeseen effects on the evolution of important characteristics of pathogens, such as virulence (which is defined broadly here to mean the degree of harm imposed on the host). an understanding of these evolutionary effects helps in understanding why some pathogens cause more harm than others, the environmental circumstances that permit this harm, and, most importantly for the future, the human activities that can ameliorate or prevent this harm. for most of the th century, the prevailing dogma was that disease organisms eventually should evolve toward benign coexistence with their hosts; harmful diseases were interpreted as a transitory state of maladaptation [ ] [ ] . this belief has not been useful for ameliorating the suffering caused by infectious diseases, because it suggests that the occurrence of severe disease is bad luck and that not much can be done to control this evolutionary process. this view arose more from assumptions about the harmony of nature than from rigorous application of evolutionary principles. specifically, it failed to cast the problem in the context of natural selection. rather than asking whether harmful or mild variants would win out in competition with each other over the short run, the focus was on what was stable over the long run. natural selection is powerless to favor long-term stability if the variants that win in the short term destabilize the system. natural selection may favor the evolution of extreme harmfulness if the host exploitation that causes this harm enhances the competitive success of the harmful variants over benign variants in the short run. if predator-like variants of a pathogen population out-produce and out-transmit benign variants, benign coexistence may be precluded. instead of generating longterm stability, the evolutionary conflict of interest between a predator-like pathogen and its host generates an evolutionary arms race in which pathogen and host each evolve characteristics that give them a leg up on the other, shifting the level of host exploitation closer to the optimum for the pathogen or that of the host. before the last decades of the th century, a few authors expressed reservations about the traditional dogma [ ] [ ] [ ] , but they largely were ignored. over the past quarter century, however, the evolution of virulence has been broadly investigated by a theoretical framework that is based on the principles of natural selection [ ] [ ] [ ] [ ] [ ] . this framework offers explanations for the broad range of virulence found among host parasite relationships and offers possibilities for virulence management (ie, the control of diseases by controlling the evolution of virulence) [ ] . the evolutionary framework also provides insight into the true scope of infectious causation of chronic disease and a sense of which diseases can be prevented or cured by developing disease-control strategies, such as vaccines and antibiotics. for acute infectious diseases, theory about the evolution of virulence focuses on the negative effects of virulence on transmission of pathogens between hosts. much of the variation in these negative effects of virulence depends on whether pathogens can be transmitted from hosts that have become immobilized by the infections. pathogens that can be transmitted readily from immobile hosts should be molded by natural selection to exploit hosts severely and to be highly virulent [ ] . the reason is simple. variants that exploit severely gain the competitive advantages of this exploitation while incurring little, if any, competitive cost from the illness that their exploitation generates, because they still can be transmitted from hosts that have the severe, immobilizing illness. specific applications of this idea are presented. parasites transmitted by biting arthropods can be transmitted effectively from immobilized hosts and therefore should evolve to a higher level of virulence than directly transmitted parasites. a comparison of viral, bacterial, and protozoal agents of human diseases showed that vector-borne pathogens are more lethal on a per-infection basis than are directly transmitted pathogens [ ] . the association between vector-borne transmission and virulence explains why diseases such as malaria, yellow fever, dengue, sleeping sickness, and visceral leishmaniasis are so severe, whereas most of the respiratory-tract pathogens of humans are relatively benign. a follow-up comparison of vector-borne pathogens indicates that this greater virulence of vector-borne pathogens is related to adaptation to the conditions of vector-borne transmission rather than to some spurious correlate of vector-borne transmission, such as injection of a pathogen below the surface of the skin. this follow-up comparison used historical data to assess the virulence of particular vector-borne pathogens in humans in relation to the degree to which the pathogen had evolved in response to vector-borne transmission between humans. specifically, it compared the virulence of vector-borne pathogens that had just been transmitted to humans with the virulence of the same kind of vector-borne pathogen that had been cycling extensively in humans and should be better adapted to vector-borne transmission between humans. as expected from evolutionary theory, the pathogens that had been cycling in humans were more severe in humans than those that recently had been introduced to humans from some other vertebrate host [ ] . the yellow fever virus, for example, was less deadly in humans just after it entered the human population than it was in outbreaks that involved extensive cycling of transmission between mosquitoes and humans. evolutionary management of the virulence of vector-borne diseases requires interventions that elevate the immobilization of hosts more costly to the infecting pathogens. logic dictates that this goal can be accomplished by mosquito proofing of dwellings. people who are immobilized by illness are more likely to be at home or in a hospital than people who do not feel ill; transmission from homes and hospitals therefore should tend to involve relatively virulent variants. when such dewllings are mosquito-proof, however, vectors cannot gain access to the severely ill people who are incapacitated inside these dewllings. the vectors will instead transmit pathogens from those infected people who feel healthy enough to get up out of bed and walk outdoors. these pathogens should tend to be relatively benign. the vector proofing of dwellings therefore should favor transmission of the benign strains from people in the outside environment instead of transmission of the more virulent strains that infect the bed-ridden. this favoring of benign strains through the mosquito proofing of dewllings would be manifested as an evolutionary decline in virulence. though this idea has not been tested directly, geographic variations in virulence and the demonstrated effect of vector proofing of houses on disease transmission suggests that it will work [ ] . strains of malaria are mild where the potential for vector-borne transmission is low and sporadic [ ] [ ] , and mosquito proofing of houses has had a strong inhibitory effect on transmission of plasmodia [ ] . as with vector-borne pathogens, evolutionary theory predicts that waterborne pathogens should evolve to relatively high levels of virulence, because they can be transmitted from immobilized people. reliance on the mobility of infected hosts is low for water-borne pathogens, because the wastedisposal activities of attendants and the movement of water can contaminate sources of drinking water. the lethality of diarrheal bacteria is correlated positively with the extent to which they are water borne [ ] . geographic comparisons also support the idea that the virulence of diarrheal diseases is linked to water-borne transmission; among shigella, for example, severe strains have been disproportionately common where the potential for waterborne transmission is high [ ] . changes in such ratios over time support the idea that the virulence of diarrheal pathogens couuld be managed evolutionary by blocking waterborne transmission. diarrheal pathogens, such as shigella and vibrio cholerae, evolved toward lower virulence as the water supplies were cleaned up in north america, south america, europe, and asia. [ , ] . like vector-borne and water-borne pathogens, pathogens acquired while in the hospital can be transmitted from immobile hosts. in this case, the transporting is done on the hands of doctors, nurses, and other attendants and the objects they touch. such attendant-borne transmission is the major route for most serious hospital-acquired pathogens, such as the staphylococci, streptococci, enterococci, pseudomonas, and clostridium difficile [ ] . attendants usually do not get infected, partly because they are less vulnerable than their patients, they wash their hands before leaving the environment, and they may have generated some immunity to the hospital organisms. although the evolution of virulence in hospitals has been studied only superficially, the available information supports the idea that cycling in hospitals makes pathogens more harmful. a review of all hospital outbreaks of escherichia coli-related infection that occurred in the united states and united kingdom before the effective use of antibiotics assessed whether increased attendant-borne transmission was associated with increased lethality [ ] . a statistically significant association was found; strains that had been circulating for a week rarely caused death, but strains that had circulated for many months killed about in infants [ ] . the implications for virulence management of attendant-borne transmission in hospitals mirror the implications for vector-borne and waterborne pathogens. if the attendant-borne transmission is blocked through proper hand washing and glove use, strains circulating in hospitals increasingly are represented by strains that are brought from the outside community; such community strains depend on host mobility for transmission and tend to be less virulent than strains that have been cycling in hospital environments. pathogens that are durable in the external environment also can be transmitted from very ill people, because such pathogens can reach susceptible individuals by relying on the mobility of susceptible, rather than infected, people. the high durability of smallpox, mycobacterium tuberculosis, and corynebacterium diphtheriae in the external environment helps explain why these pathogens have been scourges throughout history. the agent of plague, yersinia pestis, can be transmitted as a durable pathogen by the respiratory route and as a vector-borne pathogen. a re-analysis of plague from evolutionary and historical perspectives suggests that both routes were important in the black death of the th century [ ] . perhaps it was this combination that led this outbreak of y pestis to be so unusually destructive. as is the case with the preceding categories, virulence management of sitand-wait pathogens requires selective blocking of transmission from immobile individuals. in this case, however, the intervention requires selective inhibition of durable pathogens from the chain of transmission; this goal could be accomplished by requiring frequent air exchanges and decontamination of surfaces. vaccination programs generally do not eradicate target pathogens; at the global level, only the smallpox vaccine has eradicated its target from the human population. when eradication does not occur, policymakers must consider effects of vaccination not only on the frequency of infection but also on the virulence of the pathogens that are left in the wake of the vaccination program. vaccination programs that cause evolutionary reductions in the virulence tend to be successful because they leave behind mild variants that may circulate and protect unvaccinated individuals against virulent variants that might remain in the population, arise by mutation, or enter from other areas. the circulating, benign strains may protect unvaccinated individuals and the population as a whole against the spread of harmful strains. this process of virulence management can be accomplished by a virulence antigen strategy. this strategy dictates that vaccines should be based on virulence antigens (ie, antigens that make mild but transmissible organisms harmful) [ , ] . the virulence antigen strategy differs from the traditional approach to vaccine development, which selects antigens on the basis of the protection conferred to study subjects regardless of whether the antigens are virulence antigens. by selectively suppressing the virulent variants, virulence antigen vaccines force the target pathogens to evolve toward benignity. the virulence-antigen strategy is well illustrated by the diphtheria toxoid vaccine, which is based on a modified diphtheria toxin. the intact toxin liberates nutrients to the bacterium by killing nearby human cells. the immunologic response to the toxoid vaccine neutralizes the toxin and causes the toxin to be a net drain on the bacterium's nutrient budget. toxinless c diphtheriae still can infect and be transmitted from people [ ] ; the toxin therefore qualifies as a virulence antigen, because it makes viable benign pathogens harmful. when vaccination prevents the negative effects of the toxin, the toxinless strains should have a competitive advantage over the toxigenic strains, because the toxinless strains do not waste valuable resources by producing an ineffective toxin. toxinless strains therefore should increase in frequency relative to toxigenic-strains wherever toxoid vaccines have been administered extensively. this transition is confirmed by the historical data [ ] [ ] [ ] [ ] . the most detailed data set came from the vaccination program administered in romania from through . as the acquired immunity rose to %, the percentage of isolates that produced toxin dropped from % to %, and diphtheria vanished [ ] . if all of the costs of vaccine development and administration could be tallied and health benefits per dollar spent calculated, the control of diphtheria by the toxoid vaccine surely would be one of the most costeffective vaccine programs in history. only the smallpox vaccination program would rank higher, because it eradicated smallpox, which allowed for the abandonment of continuous vaccination. theory about the evolution of virulence is fundamentally different for chronic infectious diseases than for acute infectious diseases. this difference is well illustrated by the virulence of sexually transmitted diseases, which are intermediate between acute and chronic infectious diseases. syphilis has an acute phase that is characterized by a primary chancre, an early chronic phase that is characterized by a pervasive rash, and a late chronic phase (tertiary syphilis), which may involve mental illness, tumors, paralysis, meningitis, tremors, and cardiovascular disease. in other sexually transmitted diseases, the acute phase is inconspicuous or entirely lacking. hiv type (hiv- ) causes a mild flu-like illness within about a month of the onset of infection but is generally lethal in its chronic phase. the human tlymphotropic virus type causes asymptomatic acute infection soon after the onset of infection but causes paralysis, leukemia, or lymphoma decades later in a minority of infected people. because infected hosts generally must be mobile to engage in sexual activity, natural selection favors benignity of sexually transmitted pathogens over the short run. to be successful in the context of natural selection, however, sexually transmitted parasites must be infectious over relatively long periods of time, because options for sexual transmission of a given infection are generally less frequent than opportunities for transmission of typical agents of acute infectious diseases-a person generally has sex with many fewer people per week than he can sneeze on. natural selection therefore favors long-term persistence and contagiousness of sexually transmitted pathogens within each host. accordingly, most sexually transmitted pathogens are characterized by adaptations that allow the pathogen to evade the immune system to persist in and be transmitted from the body. although sexually transmitted pathogens are molded by natural selection to be benign over the short run, this long-term persistence within hosts raises the possibility of long-term damage, even though there is low probability of severe damage during any small period of time during the first years of infection. according to this framework, the evolution of virulence depends on the potential for sexual transmission in the host population. if the population is characterized by a high potential for sexual transmission (high rates of partner changes and unprotected intercourse), pathogen variants that replicate to relatively high levels soon after infection tend to have a greater chance of being transmitted to new partners. if the host population is characterized by a low potential for sexual transmission, the chance of a partner change occurring soon after infection is low, and the advantages of a high shedding of pathogens soon after infection is also low. if sexual partners remain together for a long period of time, a low probability of infecting the partner per act of sexual intercourse is of little consequence to the probability of pathogen transmission between the partners, because a large number of sexual contacts will occur during the long-term relationship. the low potential for sexual transmission thus favors low levels of exploitation and low virulence. a high potential for sexual transmission should favor elevated exploitation, which should increase the chances that negative side effects eventually will occur in the long run. this theoretical framework leads to two central predictions: the virulence of sexually transmitted pathogens ( ) should be greater in populations in which the potential for sexual transmission is greater and ( ) should increase within a population in response to an increase in the potential for sexual transmission. tests of these predictions uniformly have confirmed them whenever comparisons provide clear differences in the potential for sexual transmission and the virulence of infections. these comparisons involve hiv, human papillomavirus, human herpesvirus , and human t-lymphotropic viruses [ ] . the confirmations suggest that the virulence of sexually transmitted pathogens could be reduced by reducing the potential for sexual transmission through interventions designed to reduce partner changes and increase the use of barrier-type contraception. information on the virulence of sexually transmitted pathogens from restricted regions provides a sense of the potential effect of such interventions. the evidence from senegal is perhaps the most informative in this regard. the population in senegal has a low potential for sexual transmission relative to populations of other countries in sub-saharan africa. the relatively benign hiv type has not been replaced by the more virulent hiv- in senegal, as it has in other areas of west africa [ ] . the hiv- subtype that predominates in senegal is more benign than the hiv- subtypes that predominate in sub-saharan countries with a higher potential for sexual transmission [ ] . a similar difference occurs among the strains of the sexually transmitted bacterium chlamydia trachomatis. the strains of c trachomatis that predominate in senegal are more mild than the strains that predominate in sub-saharan countries with a higher potential for sexual transmission [ ] . these comparisons illustrate how a low potential for sexual transmission can favor benign sexually transmitted pathogens even in relatively small populations that are not isolated from surrounding populations. chronic infectious diseases may seem passe´relative to the acute emerging diseases that have monopolized the headlines, such as ebola virus and severe acute respiratory syndrome. chronic diseases, however, pose a much greater threat over the near term, and something important probably can be done to control them if their causes are examined. these two claims may seem presumptuous at first. the worst plagues of history have been acute infectious diseases that spread swiftly and lethally through human populations. the most damaging examples generally have been well adapted to transmission through human populations, either directly from person to person or indirectly through a biologic vector, such as a mosquito, or a nonbiologic vehicle, such as water. these diseases as a rule were longadapted to humans and caused their harm when they spread through previously unexposed human populations. measles and smallpox decimated native populations in the americas when they were introduced during the early colonial period [ ] [ ] [ ] [ ] . syphilis probably caused large amounts of death in previously unexposed populations in europe as a result of a reciprocal introduction into europe from the new world [ ] [ ] . these outbreaks were devastating largely because they were introduced from human populations with which they had been in evolutionary arms races into populations that had no acquired immunity and little if any evolved resistance. terrible new outbreaks of long-standing human diseases are unlikely to be a great threat in the future because the current high level of worldwide transportation is far greater than the level needed for global transport of well-adapted human pathogens. so far as is known, the only pathogens of humans that have not already been mixed globally by human travel are zoonotic (ie, newly introduced into humans from other species). zoonotic diseases generally have limited potential for spread in human populations and therefore have little potential for causing devastating epidemics. aids is the only exception; however, even aids causes minor damage compared with the decimation that was caused in new world populations on contact with old world pathogens. the diseases that are known to be a threat in the near future are those that currently are killing massive numbers of humans. in rich countries, these diseases are chronic diseases that have been and still widely are presumed to be caused by bad genes and harmful environments rather than by infectious agents. for most of the th century, the accepted wisdom has been that the scope of infectious chronic diseases is narrow, largely limited to the chronic phases of sexually transmitted diseases and a handful of other diseases, such as tuberculosis and shingles, diseases that were thought of as chronic sequelae to acute infectious diseases. evolutionary theory, however, suggests that many if not most of the major chronic diseases of humans are caused by infection [ ] . the logic leading to this conclusion involves a simple application of natural selection. there are three general categories of disease causation: genetic, parasitic, and nonparasitic environmental. (''parasitic'' is broadly defined to include infectious causes.) evolutionary considerations severely limit the feasibility of genetic causation for the most common severe chronic diseases, because such diseases tend to reduce any causal alleles down to a frequency that can be maintained by mutation [ ] . if an allele provides some compensating benefit (as is the case with the allele for sickle cell anemia), it can be maintained, but few of the common and harmful diseases with unknown causes have characteristics that are consistent with such a scenario. although a great amount of research effort has been spent on attempts to discover genetic causes of chronic diseases, this research generally has identified only genetic predispositions to common damaging diseases rather than direct causes. even in the case of cancer, where mutational causes have been identified, these causes are insufficient to explain any more than a minuscule portion of human cancer without invoking other categories of causation. in no case has the research on genetic causation of chronic disease led to a practical breakthrough that decisively controls or cures any common and damaging chronic disease. in contrast to this lack of success, infectious causes of chronic diseases have been documented (table ) , and preventive or curative interventions have been enacted (with vastly less funding). peptic ulcers, stomach cancer, and liver cancer are recognized as being caused by infection. peptic ulcers and some stomach cancer can be cured and prevented by antibiotic treatment [ ] , and many cases of liver cancer have been prevented by screening the blood supply for hepatitis b and c viruses. infectious agents have been associated with a large proportion of the most common severe chronic diseases of unknown cause, such as diabetes, alzheimer's disease, atherosclerosis, and schizophrenia ( table ) . because infectious causation of chronic diseases generally cannot be demonstrated with the same level of certainty as infectious causation of acute diseases (eg, koch's postulates generally cannot be satisfied), acceptance of infectious causation is more protracted for chronic diseases [ ] . the evidence for infectious causation of these diseases is steadily mounting and often is making sense of the evidence for genetic and noninfectious environmental causation. this coalescence of perspectives is well illustrated by the e alleleassociated diseases: atherosclerosis, stroke, alzheimer's disease, rheumatoid arthritis, and multiple sclerosis. the e allele is maintained at frequencies that range from about % to % in different human populations. its frequency is lowest in populations that have been living in high densities for the past few thousand years. the frequency is higher in populations that have been relatively small and isolated during this time, and it is highest in people who have been hunter-gatherers into the th century. even the lowest frequency of the e allele is too great to be maintained simply by mutation. the e allele is the primary allelic form of the apolipoprotein e gene in other primates and cannot be considered a defective allele. one possible explanation is that the e allele increases vulnerability to at least one infectious cause of the e allele-associated diseases. although many pathogens have been associated with these diseases (table ) , one pathogen, chlamydia pneumoniae, has been associated with all of them. this finding raises the possibility that the e allele increases vulnerability to c pneumoniae infection. as a respiratory-tract pathogen, c pneumoniae undoubtedly inflicts a heavier cost on dense human populations than on sparse populations. if so, the longer that a particular ethnic group has lived in high-density populations, the greater the cumulative selective pressure against the e allele. in accordance with this scenario, individuals who are infected with c pneumoniae are about four times as likely to have the e allele as are individuals from the general population [ ] . c pneumoniae apparently has evolved to take advantage of people who have the e allele and has driven down the frequency of e allele over time. the theoretical framework for understanding the evolution of virulence of sexually transmitted pathogens provides clues about which infectious agents are the most likely causes of these illnesses. the primary requirement for infectious causation of chronic disease is persistent infection, and this a question mark indicates transmission route is uncertain. abbreviations: ebv, epstein-barr virus; n, nonsexually transmitted; s-o, sexually transmitted by oral contact; s-g, sexually transmitted by genital contact. theoretical framework proposes that sexual transmission favors persistent infections more than any other mode of transmission. one caveat applies. pathogens transmitted by sexual oral contact should be selected to be persistent for the same reason that pathogens transmitted by sexual genital contact are selected to be persistent (ie, because sexual oral contact occurs rarely relative to contact through coughing or sneezing). if sexual transmission is defined broadly to include transmission through sexual oral and genital contact, sexually transmitted pathogens over the past quarter century have been responsible for a disproportionately large fraction of the chronic diseases that have been accepted as being caused by infection; about % of all human pathogens are sexually transmitted by this definition, but about half of the pathogens that cause these chronic diseases are sexually transmitted (see table ). they are also candidate causes of the chronic diseases for which infectious causation strongly is implicated but not yet accepted (see table ). to identify infectious causes of chronic diseases, one should look closely at the sexually transmitted pathogens. ecological imperialism. the biological expansion of europe plagues and peoples guns, germs and steel epidemics and 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atherosclerosis in non-obese japanese type diabetic patients periodontal disease and cardiovascular disease: epidemiology and possible mechanisms maternal herpesvirus infections and risk of acute lymphoblastic leukemia in the offspring human papillomavirus type infection and squamous cell carcinoma of the head and neck in never-smokers: a matched pair analysis identification of a proviral structure in human breast cancer mouse mammary tumor virus-like gene sequences in breast tumors of australian and vietnamese women jc virus dna is present in the mucosa of the human colon and in colorectal cancers association of human polyomavirus jcv with colon cancer: evidence for interaction of viral t-antigen and beta-catenin hepatitis c virus infection and incident type diabetes is crohn's disease caused by a mycobacterium? comparisons with leprosy, tuberculosis, and johne's disease i thank gregory m. cochran and levi g. ledgerwood for contributing to the development of the ideas presented in this article. key: cord- - eqh t authors: hwang, grace m.; mahoney, paula j.; james, john h.; lin, gene c.; berro, andre d.; keybl, meredith a.; goedecke, d. michael; mathieu, jennifer j.; wilson, todd title: a model-based tool to predict the propagation of infectious disease via airports date: - - journal: travel med infect dis doi: . /j.tmaid. . . sha: doc_id: cord_uid: eqh t epidemics of novel or re-emerging infectious diseases have quickly spread globally via air travel, as highlighted by pandemic h n influenza in (ph n ). federal, state, and local public health responders must be able to plan for and respond to these events at aviation points of entry. the emergence of a novel influenza virus and its spread to the united states were simulated for february from international metropolitan areas using three basic reproduction numbers (r( )): . , . , and . . empirical data from the ph n virus were used to validate our seir model. time to entry to the u.s. during the early stages of a prototypical novel communicable disease was predicted based on the aviation network patterns and the epidemiology of the disease. for example, approximately % of origins (r( ) of . ) propagated a disease into the u.s. in under days, % of these origins propagated a disease in under days. an r( ) of . reproduced the ph ni observations. the ability to anticipate the rate and location of disease introduction into the u.s. provides greater opportunity to plan responses based on the scenario as it is unfolding. this simulation tool can aid public health officials to assess risk and leverage resources efficiently. influenza transmission; susceptible-exposedinfectious-recovered (seir) disease-spread modeling; public health aviation screening; pandemic response; points of entry summary epidemics of novel or re-emerging infectious diseases have quickly spread globally via air travel, as highlighted by pandemic h n influenza in (ph n ). federal, state, and local public health responders must be able to plan for and respond to these events at aviation points of entry. the emergence of a novel influenza virus and its spread to the united states were simulated for february from international metropolitan areas using three basic reproduction numbers (r ): . , . , and . . empirical data from the ph n virus were used to validate our seir model. time to entry to the u.s. during the early stages of a prototypical novel communicable disease was predicted based on the aviation network patterns and the epidemiology of the disease. for example, approximately % of origins (r of . ) propagated a disease into the u.s. in under days, % of these origins propagated a disease in under days. an r of . reproduced the ph ni observations. the ability to anticipate the rate and location of disease introduction into the u.s. provides greater opportunity to plan responses based on the scenario as it is unfolding. this simulation tool can aid public health officials to assess risk and leverage resources efficiently. ª elsevier ltd. all rights reserved. as the world's population becomes ever more closely connected and as the numbers of international flights and air passengers continue to increase, so too has the spread of communicable diseases of public health concern via air travel. novel infectious diseases have emerged and rapidly spread around the globe during the modern jet travel era. examples include the severe acute respiratory syndrome (sars) outbreak that started in southern china in and the pandemic influenza a (ph n ) virus that was first reported in mexico in . recent evidence, including analyses of the spread of ph n , e has demonstrated how quickly transmissible diseases can be spread by air travelers. , due to these health issues, the dramatic increase of international aviation travel and security concerns, the term "border" no longer denotes a static, fixed entity that begins and ends at political boundaries, but instead has been extended virtually to include pre-and post-travel geotemporal space. because of this global interconnectedness, adverse health consequences and economic and travel disruptions can result from the emergence of rapidly spreading novel communicable diseases anywhere in the world. whenever global or regional public health threats emerge, countries predictably implement mitigation measures at their international points of entry. , e anticipating how such events will emerge and unfold is the essence of preparedness planning, and a critical practice for public health authorities who wish to mitigate the impact of such events. previous aviation point of entry modeling studies have evaluated the potential effectiveness of some measures for mitigating global outbreaks of communicable disease, such as improving the timeliness of diagnostic testing for epidemic diseases, , and rapidly developing and distributing sufficient quantities of pharmaceutical countermeasures, such as vaccines and antimicrobial drugs. , e post-ph n airport-based public health interventions including traveler screening have been described. while collectively these reports remain useful in analyzing the potential value of public health interventions at airports, to our knowledge no studies have used modeling to provide operational planning guidance assumptions for policy makers and public health authorities who would implement such measures. for this paper, and as part of pre-pandemic preparedness planning, we estimated the geo-temporal components of disease spread between city pairs via air travel. more specifically, we examined the time-course for infectious air travelers to arrive in the united states (u.s.) from international cities with the highest u.s.-bound flight traffic for the month of february. previous u.s. modeling and planning efforts for point of entry response to pandemic influenza assumed a system-wide "all or nothing" initiation of traveler screening, aligned with world health organization phases. temporal and geographical risk-based response stratification was underrepresented in those analyses, while spatial considerations and disease epidemiological characteristics guided most airport response planning. this paper describes a method that would allow for a more flexible approach, which could be applied to threats other than pandemic influenza. we used a traditional susceptible-exposed-infectious-recovered (seir) model , and an illustrative scheduled-flight dataset, to demonstrate how public health authorities could prioritize the allocation of responseresources in the u.s. at point of entry in response to a novel disease that was spreading rapidly outside of north america. to characterize possible patterns and rates of spread for an emerging infectious disease that could enter north america from various geographic points of origin, a prototypical novel pandemic influenza virus was simulated as an example of a human-to-human transmissible disease that is known to spread rapidly via air travel. the model propagates disease based on population sizes from metropolitan areas using calculations that estimate the dynamics of disease spread within each city and by air travel of individuals from one city to another, as previously described and calibrated by epstein et al. and bobashev et al. , the simulated population is mutually assigned into one of the following disease states: susceptible (s), exposed (e), symptomatically infectious (i_s), asymptomatically infectious (i_a), and recovered (r). these disease states are illustrated in fig. . a person can move from being susceptible, to exposed, then to either asymptomatically or symptomatically infectious prior to recovery. the model is implemented such that people in the infectious state have a . % chance of being symptomatic and a . % chance of being asymptomatic. people who transition to the recovered state would no longer be susceptible and remain in the recovered state for the duration of the simulation. unlike prior implementation of this model, people in the infectious states were disallowed from entering a dead state in part to maximize person-to-person spread in the early stages of a pandemic and to create an upper boundary for this modeling study. as such the case-fatality proportion was intentionally set to zero. the model was propagated so that a susceptible person could become exposed, on average for . days. the exposed person could then become symptomatically infectious or asymptomatically infectious on average for . days, with a maximum infectious duration of days. our seir model does not differentiate symptomatic and asymptomatic infection in the average infectious period calculation, nor does the model account for enhanced level of infectivity which could for example occur during early figure mutually exclusive, allowable disease states of the model: susceptible (s), exposed (e), asymptomatically infectious (i_a), symptomatically infectious (i_s), and recovered (r). stages of influenza while a person is asymptomatic. the simulation evolved in discrete units of day. selected parameters used in this model are given in supplemental table s . unlike prior implementations of the model e which permitted only asymptomatically infectious people to travel, the present implementation permitted both asymptomatically and symptomatically infectious people to travel. in addition, the model includes major metropolitan areas (see supplemental table s ) around the world, including the largest airports, the largest cities worldwide, , and cities in the u.s. (supplemental tables s and s ). this approach led to some parts of the globe being sparsely represented, commensurate with the level of direct aviation traffic those regions send to the u.s. all estimates of population data were from sources released in or later. e international metropolitan population data were taken from united nations estimates and the world gazetteer. u.s. metropolitan population data were based on us census estimates for the metropolitan statistical areas, totaling million people. points of origin were selected on the basis of the numbers of actual flights between international metropolitan areas and the u.s. during february e , . the study used the top points of origin, which accounted for approximately % of all international air passengers traveling to the u.s. america's neighbor countries, mexico and canada, each have numerous ground border crossings with the u.s., many of them heavily trafficked; therefore it has been assumed that infectious diseases that originated in mexico or canada would quickly propagate to the u.s. through these ground channels. in fact, in the recent ph n pandemic, the virus spread rapidly into the u.s. from mexico through multiple points of entry. for this reason in the points of origin analysis presented here, mexico, canada and the u.s. have been treated as a single world region, and we have not calculated times required for an infectious disease to propagate to the u.s. from points of origin in canada or mexico. in contrast, honolulu was treated as an international point of origin because the state of hawaii has limited nonaviation links to the rest of the world, including the u.s. in fact, public health authorities watching for the spread of disease to north america from east asia consider honolulu an important sentinel site for surveillance. thus, honolulu's role in the model closely resembles that of other points of origin outside north america. to populate the disease-spread model, weekly direct flight data were extracted from scheduled flights listed in a market intelligence source for aviation industry data (i.e., diio Ò llc ) for one week of february , because the and h n pandemics emerged in late winter. database extraction included origins and destinations from airports in metropolitan areas (supplemental table s ). weekly seats for flights between different airports in the same city pair were combined. if the summed seats between a city pair differed in directionality, the average seat counts were applied. weekly seat counts were converted to daily seat counts and then multiplied by % to obtain enplanement estimates for all city pairs. the % load factor is consistent with information reported by the bureau of transportation and statistics for the average of scheduled and non-scheduled flights (http://www.bts.gov). the flight matrix used in this study is available upon request. since this study focused on forecasting and predictions, best available scheduled flight data were used, with the understanding that not all flights into the u.s. were recorded in scheduled flight databases (e.g., chartered flights and unscheduled flights were not represented). known assumptions for pandemic influenza are summarized in supplemental table s . model output included how quickly the disease reached the u.s. via the global airline transportation network and also which u.s. metropolitan areas the disease reached first. time to reach the united states, referred to as "early disease arrival time," was defined as the number of days it took for the tenth symptomatically infectious person from anywhere in the simulated worldwide network to appear in u.s. metropolitan areas. it is important to note that the "early disease arrival time" includes the period of time during which the virus is spreading person-to-person but unknown to public health authorities. the -person threshold was selected on the basis of a report by the u.s. department of homeland security's national infrastructure and simulation analysis center, which showed that the arrival of as few as infected people would be sufficient to propagate a disease in the u.s. for simulations seeded with an attack rate of % or greater. in this case, "attack rate" referred to the cumulative number of people infected at the peak of the pandemic, normalized by the population at the start of the pandemic. in a given simulation, the symptomatically infectious people used to determine the early disease arrival time could be infectious travelers arriving in the u.s., or a combination of infectious travelers and people within the u.s. who became infected following contact with an infectious traveler. in addition to modeling an r of . which best approximates the ph n , we selected two additional r 's, . and . , to simulate based on evaluations of r values from all actual th century influenza pandemics. , , , although r values for some of these pandemics have been estimated as being greater than . , , , , research has shown that mitigation measures such as point of entry interventions may be less effective at such high r values. , for the purpose of pandemic response planning, we assumed that well over exposed persons at each point of origin was a realistic scenario. the model accounted for seasonality by assuming that cities within the tropics have the same viral transmission year round, while cities outside the tropics exhibit transmission rates that vary sinusoidally with peak transmission occurring on january in the northern hemisphere and july in the southern hemisphere. although the model can be implemented with flight data files reflecting true traveler patterns across city pairs for each simulated increment in time, we choose to use representative data from the month of feb. for the purpose of this prepandemic baseline study. clearly, the analysis presented in this paper would depend on the granularity of the actual data used by the model. all simulation outputs were interactively accessed via a visualization tool, written in anylogic , xj technologies, st. petersburg, russian federation. we vertically grouped points of origin by world regions, as follows: ( ) central america, caribbean, south america, ( ) africa, ( ) europe including russia, ( ) asia, ( ) southeast asia with india, ( ) near east including north african arab states and middle east mediterranean states, and ( ) oceania (see supplemental table s for world region classification). results are shown in the left-, middle-, and right-hand panels of fig. respectively for the three reproduction numbers modeled: r z . , . , and . . gray-scale raster plots represent the trial-average number of symptomatically infected people over time as a fraction of the aggregate simulated u.s. metropolitan populations. a solid red dot was superimposed onto each aggregate wave to indicate the median early disease arrival time across all trials for a given point of origin. a green bar illustrating the minimum and maximum range of the early disease arrival time was also superimposed on each aggregate wave. to determine which airports have the highest probability of being affected by disease spread via air travel, we computed the number of times a particular u.s. airport received any of the first ten symptomatically infectious passengers for all points of origin and for points of origin segregated by their respective world regions. the time-course of disease entry into the u.s is presented in fig. as aggregate pandemic waves from all points of origin seeded for three r 's. for response planning purposes, we are most interested in the time leading up to the day in which ten infectious people appear in the u.s. (red dot). it is apparent that at least two clusters of median early disease arrival times appeared for each panel of fig. . the separation in clusters was most pronounced from the r z . simulation, followed by a decrease in separation as the r values increased (see fig. a ). more specifically, median early disease arrival times in the th and th percentile were under and over days, respectively (see table ). this observation suggests that response planning could be conducted differently for points of origin depending on their respective quartile and r classifications. when points of origin were grouped by their respective world regions (see table ), median early disease arrival times from central america, the caribbean, south america, europe, the near east, and oceania (honolulu) were shorter than those from asia, africa, southeast asia including india, and oceania (australia). fig. b , in which median early disease arrival times into the u.s were plotted in decreasing magnitude for each point of origin and r , further elucidates this trend. a summary examination of how u.s. airports would be affected by the first ten symptomatic people entering the u.s. revealed that new york, miami, newark, atlanta, los angeles would experience the earliest impact (fig. a) . further, detailed examinations of how these airports would be affected by points of origin from specific world regions are also presented: central america, caribbean, or south america (fig. b) , africa (fig. c) , europe (fig. d) , asia and southeast asia (fig. e and f) , near east (fig. g) and oceania (fig. h ). our analysis indicated that los angeles and san francisco airports would experience the earliest impact for disease originating from asia, southeast asia and oceania, while new york and atlanta airports would experience one of the earliest impacts for diseases originating from all other world regions. unlike our points of origin analysis, when we validated our model based on data from the ph n , we did not treat the u.s. and mexico as one mixing body. rather, we used mexico city as a proxy for the village of la gloria, veracruz, mexico, from where some of the earliest ph n cases were reported. in addition, we replicated the mean u.s. incidence rate of . %, as reported by the cdc for ph n cases in the u.s. between april and july , based on the u.s. cities used in the model (listed in supplemental table ). in order to replicate the mean incidence rate of . %, we evaluated two parameters: ) the basic reproduction number, which represents a measure of the average number of people in a totally susceptible population to whom one infected individual transmits a disease, was allowed to vary between . and . in increments of . . ) the number of days it takes the model to predict the targeted mean incidence rate which best matches july , , i.e., the th day after feb th, . we confirmed that our model generated a u.s. incidence rate of . % (standard deviation of . %) on the th day after disease onset for an r of . ; these parameters are very close to the reported mean incidence rate of . % by july , . an r of . is well within published estimates of r for the ph n which ranged from . to . . , since the h n (hong kong) influenza pandemic, global air travel has nearly increased by a factor of ten, from million passengers worldwide to more than . billion passengers in . , air travel contributes greatly to the rapidity of communicable disease transfer across international borders and thus federal, state, and local public health authorities must understand and plan for aviation point of entry response to these threats. , , the model results presented here resemble those from related modeling studies in that they suggest an important influence of geographic origin of the outbreak on the timing and location of disease introduction into the u.s. during an emerging disease event. in this respect, the range of public health tools available for point of entry intervention (observation, health information distribution, health questionnaires, individual screening, illness response, contact investigations, isolation and quarantine, etc.) could be activated based on anticipated city-to-city spread of the disease in question, rather than being "turned on" systemwide unnecessarily, at great potential cost to taxpayers and need for use of government resources. our model output shows how quickly the disease reached the u.s. via the global airline transportation network and which u.s. metropolitan areas the disease reached first. we found variance in arrival times and locations of infectious passengers from origin points at different global locales. model results indicated that a staggered "turn on" of point of entry responses according to strategic risk assessments, resources available, and time required to mount an effective response, would be feasible and should guide response planning. the model, as well as the actual events during the sars and ph n outbreaks of the past decade, reinforced that a short time window would be available to implement measures at points of entry that could be effective in mitigating disease spread. as occurred during ph n , the initial outbreak and subsequent spread could go undetected for weeks or even months before the first cases were figure predicted disease spread time-course. simulations were based on exposed people from each international metropolitan point of origin across three reproduction numbers: . , . , and . . the y-axis displays origins that are first grouped by continent and then sorted alphabetically. the x-axis denotes time in days relative to the start of the disease-spread simulation. gray-scale raster plots represent the number of infected people across time as a fraction of the aggregate u.s. metropolitan population size employed by the model. each row shows the median disease spread for all suprathreshold trials (out of ) in which at least symptomatically infectious people appeared in the united states. in the r z . simulations, early disease arrival times varied from about days to slightly under days from disease emergence in a population. approximately % of origins resulted in median early disease arrival under days, with % of those origins resulting in median early disease arrival in less than days. in the r z . simulations, early disease arrival time ranged from about days to approximately days. approximately % of origins resulted in median early disease arrival in less than days, with % of those origins resulting in median early disease arrival in less than days. in the r z . simulations, early disease arrival time varied from about days to slightly under days. approximately % of origins resulted in median early disease arrival under days, with % of those origins showing median early disease arrival in less than days. identified and laboratory-confirmed, drastically curtailing the time and options available to respond effectively. since the u.s. essentially serves as a global hub for aviation travel with international arrival and departure points for regularly scheduled commercial flights ; with an additional u.s. airports that received passengers from international charter, private and/or air ambulance flights in , it is not surprising that once a novel infectious disease emerges and begins person-to-person spread near international transportation hubs anywhere in the world, that novel disease will in all likelihood appear in the u.s. within days or, at most, a few weeks' time. spurred by a rise in global traveler numbers and favorable changes in aviation regulations in many countries, the dramatic increase in the sheer number of aviation international arrival and departure points located within geographically and politically distinct entities, has resulted in the creation of new pathways for passengers e and diseases e to enter into local communities worldwide. our model could prove useful for those countries with multiple entry points to consider when planning for a pandemic. although this paper describes new data for policy makers and planners to use for planning of public health interventions at point of entry, the effectiveness of point of entry interventions should be examined closely so that resources are not diverted from community infection control and prevention efforts, where they could have greater impact. point of entry interventions for influenza have been questioned, for example, because of the many travelers who could be infected but not symptomatic at the time of entry (thus avoiding detection), greatly reducing the efficacy of any public health intervention. computer simulations have demonstrated that even drastic travel restrictions (e.g., border closure) would achieve only limited delays in the introduction of a severe novel influenza virus, for example, into the u.s. during the early stages of an outbreak. , , , e indeed, the world health organization (who) discouraged point of entry screening measures both before and during ph n , e and the who director-general margaret chan asserted very early in the pandemic that travel restrictions would be counterproductive and "serve no purpose". , however, prudence dictates planning for such a contingency as one possible component of a comprehensive and coordinated public health response to an emerging disease threat. there are many drivers behind the decision to screen aviation travelers during novel communicable disease outbreaks spreading regionally or globally; despite recommendations to the contrary, and known and potential limitations to aviation traveler screening activities for novel influenza, many countries mounted broad screening efforts at their borders during ph n nonetheless. in calibrating an effective public health response, knowing the "where" is just as important as knowing the "what" and the "when." thus, the unique geospatial characteristics of specific originating locations and regions need to be identified to accurately assess risk and develop effective response plans for point of entry interventions. for example, if a rapidly unfolding outbreak were taking place in central america, it would make sense to anticipate higher need for public health resources focused on direct flights coming to the u.s. from high-volume contributors such as managua and guatemala city. although other passengers from central america may come to the u.s. via connecting flights, these travelers would be so low in number as to present minimal risk, and to expend resources to address them initially may divert from more effective efforts with the bulk of travelers arriving on direct flights at a few u.s. locations. our analysis of flight data revealed that, by volume, the airports most affected by flights from central this approach seems intuitive, but others have observed that this risk-based, targeted approach to bolstering surveillance and response capacities at key airports has been the exception rather than the norm, and that careful planning for public health response at key points of entry can be more effective than the usual first response of implementing travel restrictions. , , in light of current global economic uncertainties and the overall decline of resources for public health in the u.s. and other countries, current planning and future point of entry interventions for global disease outbreaks must optimize the resources that are available, quickly and accurately assess risks and prioritize efforts to maximize impact of intervention efforts. the "node-to-node" approach embodied in our model will allow public health officials to preserve resources for longer-term, community-based mitigation and prevention activities by targeting response to those points of entry likely to be affected earliest. although scientific modeling and simulation have proven to be effective tools for visualizing and planning responses to outbreaks of infectious diseases, in particular for influenza pandemics, , , these analyses should not be the only drivers for planners or policy makers when anticipating future outbreaks. e as with other studies reported in the literature, this model shows that the speed with which a communicable disease spreads to the u.s. will depend on the r of the disease in question. g this finding could represent a limitation of using the model for planning, since r is quite difficult to determine for an emerging disease. further, the model employed in this study is not agentbased, and the attack rates reported by agent-based models cannot be computed identically using this equation-based model. this is because the total number of people who became infectious during a complete model run cannot be tallied without modeling each person as an agent. although we used the well-known and accepted assumptions of a prototypical pandemic influenza virus to examine early disease arrival times, the recent ph n pandemic makes it clear that novel viruses do not always behave as expected. some disease-specific assumptions and outcomes of the model may therefore not be useful in planning for future disease outbreaks. since this study focused on forecasting and predictions, future scheduled nonstop flights for a single month were utilized as the basis for estimating travel for a simulated period of one year. this approach simplifies the complex phenomenon of international travel. a rigorous comparison between scheduled seats and actual enplanements (which would include, among other things, chartered flights and actual load factors), was not conducted in part because such data for non-u.s. cities were not readily available. the spread of disease during travel from infectious passengers to susceptible passengers was not modeled separately from spread in the general population. variation in travel based on seasonality was not included and thus the analysis presented in this study only applies to a disease that spread in the month of february based on travel in that month. finally, this approach neglects travel between cities that does not take place via nonstop flights on the same day e in particular, connecting flights through gateway cities, or non-aviation means such as car or train for nearby cities such as paris, france, and amsterdam, the netherlands. thus, new analyses should be conducted periodically, and especially as an event is unfolding which may require point of entry intervention. policy makers and public health authorities must always consider aviation points of entry as potential foci for interrupting or slowing the global spread of disease. therefore, future modeling should consider how to maximize effectiveness of public health interventions while minimizing passenger delays and travel disruptions, by analyzing the efficacy and cost of specific interventions such as dispensing antivirals at points of entry, health questionnaires with real-time data entry using handheld devices, and fever detection using technology such as thermal imaging. further analysis and reporting of worldwide government responses to the emergence of ph n and other regional disease outbreaks would help to fill in some of the information gaps regarding point of entry interventions. future models should also examine various pathogens (e.g., smallpox, pneumonic plague, a sars-like virus) and their rates of spread. the rate of global spread from specific geographic locations around the world should also be studied using various known assumptions and pathogen characteristics. those results could usefully be compared with the ph n and sars experiences. our findings indicate that time to disease entry to the u.s. during the early stages of an emerging pandemic would vary and can be predicted based on point of origin and point of entry into the u.s. this ability to anticipate the rate and location of disease introduction into the u.s. provides greater opportunity to plan responses based on the scenario as it is unfolding. this simulation tool can aid public health officials to assess risk and leverage resources efficiently via targeted and scalable border mitigation measures, especially at key u.s. airports that would be most expected to bear the initial brunt of an international outbreak. the findings and conclusions in this paper are those of the author(s) and do not necessarily represent the views of the centers for disease control and prevention, the federal aviation administration (faa), or the u.s. department of transportation (dot). the united states government, the faa, and the dot make no any warranty or guarantee, expressed or implied, concerning the content or accuracy of these views. approved for public release; distribution unlimited. case numbers - numbers - , - infectious disease movement in a borderless world: workshop summary the severe acute respiratory syndrome pandemic potential of a strain of influenza a (h n ): early findings clinical features of the initial cases of 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globalized world a framework for public health action: the health impact pyramid modeling targeted layered containment of an influenza pandemic in the united states dynamically modeling sars and other newly emerging respiratory illnesses: past, present, and future population-based simulations of influenza pandemics: validity and significance for public health policy large-scale spatial-transmission models of infectious disease all authors have confirmed that they have no conflict of interest either financially or via personal relationships as defined by travel medicine and infectious disease. supplementary material associated with this article can be found, in the online version, at doi: . /j.tmaid. . . . key: cord- -srq bo v authors: fèvre, eric m.; bronsvoort, barend m. de c.; hamilton, katie a.; cleaveland, sarah title: animal movements and the spread of infectious diseases date: - - journal: trends microbiol doi: . /j.tim. . . sha: doc_id: cord_uid: srq bo v domestic and wild animal population movements are important in the spread of disease. there are many recent examples of disease spread that have occurred as a result of intentional movements of livestock or wildlife. understanding the volume of these movements and the risks associated with them is fundamental in elucidating the epidemiology of these diseases, some of which might entail zoonotic risks. the importance of the worldwide animal trade is reviewed and the role of the unregulated trade in animals is highlighted. a range of key examples are discussed in which animal movements have resulted in the introduction of pathogens to previously disease-free areas. measures based on heightened surveillance are proposed that mitigate the risks of new pathogen introductions. domestic and wild animal population movements are important in the spread of disease. there are many recent examples of disease spread that have occurred as a result of intentional movements of livestock or wildlife. understanding the volume of these movements and the risks associated with them is fundamental in elucidating the epidemiology of these diseases, some of which might entail zoonotic risks. the importance of the worldwide animal trade is reviewed and the role of the unregulated trade in animals is highlighted. a range of key examples are discussed in which animal movements have resulted in the introduction of pathogens to previously disease-free areas. measures based on heightened surveillance are proposed that mitigate the risks of new pathogen introductions. animal movements and disease transmission infectious diseases are transmitted between hosts by a variety of mechanisms, including direct, airborne and vector-borne transmission. control of animal-to-animal transmission of disease agents is a key concept in infectious disease epidemiology; however, a more sensible approach might be to prevent the types of contact that lead to transmission in the first place. in humans, it is often difficult to prevent contacts, particularly with the ease of long-distance travel [ ] . however, in livestock and animals, movements can be the subject of legislation or strict controls and there is a real opportunity to reduce disease transmission. the importance of animal movements is, of course, well understood and international regulations [e.g. from the world organisation for animal health (oie; box )] exist to mitigate the risks involved [ ] . in spite of these regulations, outbreaks occur regularly as a result of both legal and illegal animal movements. in this review, we examine several issues that relate to the movement of domestic and wild animals and discuss the risks that these movements entail -at local, regional and global scales -with regard to the spread of disease. first, we present data on livestock movements at a global level, and highlight the scale of wildlife movements as a result of translocation by humans. second, we provide key case studies of animal and animal-to-human (zoonotic) disease introductions in different parts of the world that have resulted in subsequent disease transmission and new outbreaks, with an emphasis on how introduction of the disease agents could have been prevented either through intervention or regulation. the trade in livestock, wildlife and animal products is enormous and complex, and occurs on many different scales. there are no overriding rules to control these movements and much of the trade is still based on bilateral agreements between countries. however, countries that are members of the world trade organization are bound by the sanitary and phytosanitary agreement (http://www.wto.org/english/tratop_e/sps_e/ spsagr_e.htm), which concerns food safety and animal and plant health regulations. countries are encouraged to base their sanitary and phytosanitary measures on existing international standards. the international standards relating to animal health and zoonoses were developed by the oie and are stated in the aquatic and terrestrial animal health codes (http://www.oie.int/eng/ normes/fcode/en_index.htm and http://www.oie.int/eng/ normes/mcode/a_summry.htm, respectively). the aim of the codes is to assure the sanitary safety of the international trade in terrestrial animals and their products by detailing the health measures that should be used by the veterinary authorities. the important role of a good veterinary infrastructure to minimize the risks of disease spread has been emphasized [ ] . the relevant legislation for international wildlife trade [ ] relates to three main areas: animal health, animal welfare and the international movement of endangered species. the animal health regulations relevant for livestock trade (see earlier) also apply to non-domesticated animals. however, additional regulations exist for wildlife to protect endangered species from overexploitation from trade, in the form of the convention on international trade in endangered species of wild fauna and flora (cites) and the convention on biological diversity (cbd). detailed guidelines have also been developed by the world conservation union (iucn) to minimize disease risks associated with the intentional movement of wildlife for conservation or game management purposes: for example, the supplementation of populations, reintroduction of endangered species, removal of problem animals and the release of confiscated animals [ ] ( table ) . the international trade in livestock is big business; for example, in , china exported us$ million, us$ million and us$ million worth of cattle, swine and poultry, respectively [ ] unregulated (illegal or informal) trade is, by its nature, difficult to quantify, although available data for some regions of the world show that unregulated trade is substantial. although there are no official records, it has been documented that, for example, head of cattle move from somalia to kenya annually, and that up to goats move from somalia to the middle east (which accounts for o % of all goat imports from the eastern africa region) [ ] . indeed, in the case of cattle trading with kenya, political instability resulted in a large increase in the value of unofficial trade. this coincided with a collapse in the animal health infrastructure of somalia and a resulting lack of animal export controls. diseases known to have been circulating in somalia include both zoonotic and non-zoonotic infectious diseases: anthrax, babesiosis, brucellosis, contagious bovine pleuropneumonia, contagious caprine pleuropneumonia, foot and mouth disease (fmd), heartwater, peste des petits ruminants, rabies, rift valley fever, rinderpest and trypanosomiasis, among others [ ] . unofficially traded animals are a much greater risk factor for disease spread because they are not necessarily subject to veterinary controls. it has been reported that much of the human brucellosis problem in saudi arabia, which has an incidence of cases per people nationally, is the result of unscreened and unregulated imports that come mainly from africa [ ] . the picture that emerges from a consideration of these various data sources is one of a highly interconnected world in which animals move locally, regionally and across large international distances. the global wildlife trade is also huge, with an annual turnover estimated at billions of dollars and involving hundreds of millions of individual plants and animals (http://www.traffic.org/ /wild .htm). precise estimates of its scale are difficult because much is conducted through informal or illegal networks but recent figures suggest that w live primates, four million live birds, live reptiles and million live tropical fish are traded globally each year [ ] . in asia, the substantial regional trade in wild animals is estimated to result in several billion direct and indirect contacts among wildlife, humans and domestic animals each year [ ] . despite the widespread recognition of the risks of disease transmission associated with wildlife translocation, and the legislation and regulation in place to minimize disease risks [ , [ ] [ ] [ ] , new diseases continue to emerge as a result of wildlife trade. here, we present key examples from the past five years that illustrate the continuing threat to human, livestock and wildlife health. severe acute respiratory syndrome (sars) and highly pathogenic avian influenza (e.g. h n ) are emerging infections that have the potential for pandemic spread with massive public health and economic consequences. both diseases are maintained in wildlife reservoir hosts: h n in wild fowl and sars in horseshoe bats (rhinolophus species) in china [ , ] . for sars, the trade in bats is likely to have brought infected animals into contact with susceptible amplifying hosts such as the masked palm civet (parguma larvata) at some point in the wildlife supply chain, establishing a market cycle in which susceptible people and animals could subsequently become infected [ , ] . for avian influenza, these 'wet markets' could also function as network hubs for potential cross-species transmission. however, the international trade in birds also poses considerable risks for longdistance transmission of h n , as highlighted by the detection of infected hawk eagles imported illegally from thailand to belgium [ ] . the disease risks of the wild bird trade have been brought into sharp relief by the importation of h n -infected birds destined for the uk pet market [ ] and, at the time of submission of this review, a temporary ban on the importation of wild birds had been implemented by the uk government. the recent detection of pseudamphistomum truncatum (an opisthorchid fluke parasite) in the english otter population has been linked with the introduction of two freshwater fishes: the sunbleak (leucaspius delineatus) and the topmouth gudgeon (pseudorasbora parva), which can function as intermediate hosts for the fluke. these species were newly introduced into the uk by an ornamental fish supplier in hampshire in the mid- s and their escape led to the colonization of many river systems in southern england [ ] . the translocation of amphibians is implicated in the emergence of amphibian diseases such as chytridiomycosis and ranavirus infections, which might be a major contributing factor in the widespread decline and extinction of amphibian species worldwide [ ] . chytridiomycosis, which has been associated with amphibian mortalities and population declines in central america and australia, has also been linked to the introduction of cane toads (bufo marinus) into australia [ ] . also, chytridiomycosis has appeared recently in the uk, with confirmed cases in an established breeding population of north american bullfrogs (rana catesbeiana), which is an introduced species [ ] . recent movements of r. catesbeiana and b. marinus might also have disseminated ranaviral diseases such as tadpole oedema virus [ ] . in australia, where b. marinus was introduced to queensland in , ranaviral antibodies can be identified in this species throughout its geographic range [ ] . in addition to the well-recognized threat that animal translocations and invasions into new geographic areas pose for species extinctions and biodiversity, the large wildlife trade clearly poses great dangers for the emergence of human and animal pathogens. it is doubtful whether the disease risks associated with the international wildlife trade, let alone additional welfare and conservation-related problems, can ever be justified simply to supply a demand for pets or recreational hunting. the dilemma is perhaps more acute in areas where the wildlife trade is associated with food and medicines, particularly in asia, where the trade could be an important element in rural livelihoods and food security. in addition, translocation of wildlife can be an important tool for both conservation and animal welfare. case studies of disease threats movement of pets in february , the uk adopted the pet travel scheme (pets) to enable pets with the appropriate documentation to move between the uk and certain countries. this represented the first major change in uk quarantine regulations since the importation of dogs act. the scheme is primarily designed to prevent the importation of rabies, with secondary measures to prevent the introduction of the cestode parasite echinococcus multilocularis, which is currently endemic in continental europe and can be transmitted from canids to humans to cause potentially fatal alveolar echinococcosis. eighty-one countries now qualify under the scheme but quarantine remains the only option for non-listed countries. the pets is a model for the use of legislation to minimize risks: the risks of importing rabies under the scheme are small. however, other zoonotic diseases continue to pose a substantial risk and there are few formal checks for these under the system. figure shows the volume of dog and cat movements into the uk following the introduction of the pets. leishmania infantum is the predominant cause of both visceral and cutaneous leishmaniasis throughout the mediterranean region, including southern france, portugal, spain, italy, greece, turkey and north africa [ ] ; the main reservoir host is the domestic dog. seropositivity rates of canine leishmaniasis can be o % [ ] in the mediterranean, with patterns of infection that reflect the distribution of the sand fly vector (phelobotomus species) [ , ] . in brazil, where canine visceral leishmaniasis is an important emerging disease, canine infection in endemic areas can be as high as % [ ] . to control the disease, a mass culling of seropositive dogs has been adopted in several areas but has not been effective [ ] . a feasible alternative to culling dogs is to fit collars treated with the insecticide deltamethrin; preliminary results have shown a large drop in the number of bites by sand flies and infection rates in dogs [ ] [ ] [ ] . this might be the only way to control the increasing spread of canine visceral leishmaniasis and could be recommended as a control measure for dogs leaving and re-entering the uk under pets. quarantine is not effective in preventing the introduction of leishmaniasis due to the long incubation period of the disease. outbreaks of leishmaniasis have occurred in dogs in the usa as a result of mediterraneanbased military personnel who have returned home with their pets. in addition, there has been an apparent increase in the incidence of leishmaniasis in us hunting dogs [ ] -although the cause remains unclear, it could be a result of either animal movements or spread of the vector. the chances of leishmaniasis becoming established in domestic dogs in the uk are small because, currently, phelobotomus species are not known to be present in the uk. however, if climate change does affect air temperatures substantially, the sand fly vector could become established in the uk and, potentially, could maintain endemic leishmaniasis and other 'exotic' parasitic diseases [ ] , the introduction of which would probably result from the movement of domestic pets. in addition to leishmaniasis, diseases such as heartworm (dirofilaria immitis), babesiosis (babesia canis), ehrlichiosis (ehrlichia canis) and echinococcosis (echinococcus granulosus and e. multilocularis) are all likely to be moved with domestic pets into the uk and elsewhere [ , ] . the responsibility falls on domestic pet owners in such a situation who should ensure the use of anthelminthics -agents that destroy or expel parasitic intestinal worms -and, possibly, insecticide-treated collars. owners should also be advised about preventative measures before pet travel. e. multilocularis is common in red foxes in hokkaido, northern japan, where the prevalence of infection in foxes is as high as % and high worm burdens are recorded in some individuals [ ] . dna sequencing of parasite isolates from this area show that it was probably introduced in the s from a neighbouring island [ ] through the movement of infected foxes. this disease now presents an important public health problem, with a human incidence of . per people [ ] , or w cases annually. domestic dogs have become part of the transmission cycle and close contact between humans and their pets is a major risk factor. a recent risk analysis [ ] showed that the movement of pet animals between hokkaido and the rest of japan is likely to result in review further geographical spread of the parasite, particularly because there are few movement controls or programmes for screening and treatment. rabies is a prime example of an infectious disease in which transmission can be enhanced by animal movements. flores island in indonesia was free of rabies until [ ] ; in that year, three dogs were imported from a rabiesendemic area and this was sufficient to result in human deaths (mainly children) and the culling of almost % of the dog population in some areas as part of an unsuccessful control campaign [ ] . flores island is now endemic for rabies, which has become a major public health issue, and dealing with its introduction has incurred a high cost. translocation of raccoons from florida to virginia for hunting purposes [ ] also led to the emergence of raccoon rabies in the mid-atlantic states of the usa, with thousands of animal cases that resulted in enormous expenditure on rabies post exposure prophylaxis and oral vaccination programmes [ ] . france now has rabies-free status because terrestrial rabies has been eliminated through a long-term concerted effort to vaccinate foxes [ ] . however, the risk is ever present and, in , contacts with an illegally imported rabid domestic dog resulted in people requiring post-exposure prophylaxis [ ] . since the s, the uk has conducted regular bovine tuberculosis (btb) testing of cattle herds [ ] . herd breakdowns -the occurrence of btb in a previously btb-free herd -have become more common since and the causes of these breakdowns are the subject of intense debate. molecular evidence (analysis of spoligotype data) from btb isolates in the uk [ ] indicates an important role for cattle movements in the long-distance spread of disease and the establishment of novel strains in new geographic areas. while also highlighting environmental, wildlife and other factors in the short-range spread of btb in the uk, gilbert et al. [ ] quantified the strong association between the proportion of inward movements from infected areas and the breakdown of herds, which highlighted the strong predictive power of cattle movements for later disease distributions. the importance of contact networks in the spread of infectious diseases of livestock in the uk has been clearly highlighted [ ] ; in addition, a small proportion ( %) of farm holdings contributes to the majority ( %) of movements. if one of these high-contact farms was infected with btb, its impact on long-range spread to many other farms would be substantial. a thorough understanding of livestock contact networks at different scales is essential in predicting such spread. trypanosoma evansi (the tabanid-transmitted pathogen responsible for surra in livestock) has spread in south east asia, particularly in the philippines where there is high mortality in those areas in which the parasite has been detected. this spread has been blamed on the movements of livestock as part of herd-improvement programmes [ ] . similarly, another trypanosome species (trypanosoma brucei rhodesiense) caused an outbreak of human sleeping sickness in a previously unaffected area of uganda. this was caused by movement of the cattle reservoir of the trypanosome parasite through markets without proper screening or disease control. veterinary and public health services are now struggling to control a rapidly spreading sleeping sickness epidemic in humans [ ] . these introductions would have been entirely preventable if local authorities had been alerted to the risks and if resources were made available to administer prophylaxis or treatment to the animals. the integrated management of disease threats (e.g. the treatment of animals as a means of preventing disease spread to humans) would be a cost-effective and efficient use of resources, particularly where zoonotic diseases are concerned. in late january or early february , fmd was introduced to a pig farm in the north of england. disease spread through this pig herd probably occurred with three waves of infection and amplification. however, fmd was not notified to the authorities until it was identified during a routine meat inspection at an abattoir in the south east of the country (several hundred miles away) on february; fmd was confirmed on february [ ] . at the time of disease identification in the abattoir, the virus had already spread to a neighbouring farm where sheep and cattle were infected, probably by airborne spread [ ] . sheep from this farm were sold to a local market on february and sold again two days later at a larger market [ ] . by the time the first case was confirmed, an estimated - farms had been infected across the uk [ , ] . rapid tracing identified the pig farm but a movement ban for the whole of the uk was delayed for three days (although all international movements were stopped immediately). this three-day delay might have caused a further farms to become infected, mostly from contacts with markets [ ] . the virus spread to france through the movement of infected sheep before the ban. in addition, calves bound for the netherlands were in a vehicle next to these sheep at a stopping point and it is thought that the virus was transmitted between these two consignments -fmd was confirmed in the netherlands on march [ ] . this outbreak clearly demonstrates the risks associated with large-scale movements of animals over long distances across international borders. given modern abattoirs and chilling transport units, the question arises as to why this sort of live animal movement is necessary at all. in africa, livestock are one of the few tradable commodities available to millions of poor households, particularly those living in the more arid regions of sub-saharan africa. there are well-established trade routes across the continent that supply the large coastal populations of west and central africa and the arabian peninsula. these animal movements are of major importance for the dissemination of new strains of fmd and review other diseases such as rift valley fever [ ] . modern dna sequencing techniques make it possible to compare isolates and identify probable sources of infection. for example, these techniques enabled the tracing of outbreaks caused by the sat fmd serotype as it spread up the east african coast into saudi arabia through cattle movements from somalia and eritrea [ ] and its subsequent spread west into cameroon [ ] . in these regions, herdsmen are nomadic or semi-nomadic and move with their animals on a seasonal transhumance in search of grazing, which brings different herds into contact with one other and increases the risk of spread of fmd [ ] . the movements of domestic and wild animals are complex and profitable but are extremely risky from a disease perspective. movements can result in the introduction of exotic animal diseases or human pathogens, which might themselves have important economic and/or public health impacts. one example occurred in when humans in the usa became infected with monkey pox [ ] , which originated from the importation of wild west african rodents. minimizing such risks should be a high priority and, in some cases, this might involve preventing the animal trade altogether: from the perspective of disease introductions and animal welfare, it is difficult to justify the movement of exotic birds or mammals simply for the pet trade. attempting to ban such movement, however, is likely to drive it underground, which makes riskmitigation far more difficult. with livestock, there is a strong case for trading only in livestock products -a commodity-based approach -rather than live animals themselves [ ] but this, of course, is dependent on the appropriate investment and infrastructure being available to process animal products and to provide adequate quality assurance. for diseases linked to livestock, such as fmd, bovine tuberculosis and sleeping sickness, markets have an important role in the dissemination of infectious organisms. markets serve as contact nodes between infected herds and the ease of transportation can result in the widespread dissemination of animals that have been in contact in a market. contact nodes such as quarantine facilities, markets and ports of entry can also result in the transmission of agents between individuals and species, with rapid subsequent dissemination. better communication between scientists, livestock traders, livestock keepers and decision makers is required to avoid this. the key issue that requires most attention is to acquire a better understanding of the risks of global movements. simple risk assessments that focus on the individual country trying to protect itself from disease introductions are no longer sufficient and passive detection of disease at ports of entry is an increasingly dangerous strategy as the volume of movements increases. a holistic understanding of risk at the global level is required to understand disease risks by species and by country, supplemented by an efficient global surveillance network in which different animal species are regularly screened, particularly before moving from their source areas. control of disease threats should be dealt with locally rather than when movement has already occurred, which requires knowledge of species, volumes moved and fine-scale information on point of origin. knowledge of movement routes is the key to predicting the pattern of spread of infectious diseases of humans [ ] , and similar data could be crucial to understand animal disease risks. databases already exist [ ] [ ] [ ] that list pathogens, hosts and probable risks of emergence; additional layers of information on the movements of host species and their impacts on the risks of emergence should be added, and the information should be made publicly available. such global cooperation and international level disease control will lead to better risk management and mitigation. 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wildlifethreats to biodiversity and human health bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronaviruslike virus in chinese horseshoe bats highly pathogenic h n influenza virus in smuggled thai eagles quarantine premises in essex, department for the environment, food and rural affairs sunbleak and topmouth gudgeon -two new additions to britain's freshwater fishes emerging infectious diseases and amphibian population declines emergence of amphibian chytridiomycosis in britain giant toads bufo marinus in australia and venezuela have antibodies against 'ranaviruses' systematic review of the distribution of the major vector-borne parasitic infections in dogs and cats in europe canine leishmaniasis imported diseases in small animals host preferences of phlebotomine sand flies at a hypoendemic focus of canine leishmaniasis in central italy assessment of an optimized dog-culling program in the dynamics of canine leishmania transmission 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descriptive epidemiology from an epizootic of raccoon rabies in the middle atlantic states, - the ascension of wildlife rabies -a cause for public health concern or intervention elimination of terrestrial rabies in western european countries risque de rage en france et importation illégale d'animaux en provenance de zones d'endémie rabique investigations of cattle herd breakdowns with bovine tuberculosis in four counties of england and wales using vetnet data the population structure of mycobacterium bovis in great britain: clonal expansion cattle movements and bovine tuberculosis in great britain epidemiological implications of the contact network structure for cattle farms and the - rule trypanosoma evansi control and containment in australasia a burgeoning epidemic of sleeping sickness in uganda descriptive epidemiology of the footand-mouth disease epidemic in great britain: the first months airborne transmission of foot-and-mouth disease virus from burnside farm early dissemination 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forecast and control of epidemics in a globalized world risk factors for human disease emergence diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents acknowledgements e.m.f. and s.c. are funded by the uk department for international development (dfid), although the views expressed are not necessary those of dfid. we thank philippe ankers of vétérinaires sans frontières (vsf)-suisse for supplying some key reference material. key: cord- -butuv n authors: galvani, alison p. title: emerging infections: what have we learned from sars? date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: butuv n nan meat industry expanded recently. global warming will likely contribute to the spread of dengue beyond tropical regions (tony mcmichael, national centre for epidemiology and population health, canberra, australia). habitat fragmentation by deforestation may increase the contact between people and reservoir species. for example, hemorrhagic fever virus has been linked to deforestation in south america. containing an emerging disease depends on rapidly designing and implementing a control strategy appropriate to the epidemiology of the disease. interdisciplinary and international collaboration occurred with unprecedented rapidity during the sars outbreak. the network of laboratories in countries organized by the world health organization (who) coordinated information sharing (david heymann, who) and was instrumental in rapidly identifying the etiologic agent of sars ( ) and in fulfilling koch's postulates ( ) (albert osterhaus, erasmus university, rotterdam). as is typical of an emerging disease, no vaccines or drugs to combat sars existed, making quarantine, patient isolation, travel restrictions, and contact precautions the only means of limiting transmission. mathematic models provided a framework for evaluating alternative control measures and making predictions about the course of the epidemic ( , ) . previously, similar models had guided public health policy, for example, in halting an outbreak of hoof and mouth disease in the united kingdom in ( , ) . one of the complications in setting parameters in an emerging disease model is the difficulty in estimating epidemiologic limits from the initially small sample sizes. thus, openly sharing data and analysis of key model parameters are vital. the model must be appropriate to the nature of the disease and the accuracy of the parameter estimates ( ). stochasticity inherent in transmission dynamics will be particularly pronounced when infection prevalence is low. population heterogeneity and the network structure of human interactions will affect the spread of an emerging disease. in the sars outbreak, healthcare workers were at particular risk ( ) and acted as bridges carrying the infection from the hospital and causing community wide epidemics. high-risk "core groups" have been a major focus of hiv/aids models for years ( ) , but the movement of sars patients into the core (i.e., the hospital) adds a further complication ( ). the two waves of sars clusters in toronto (robert maunder, mount sinai hospital, toronto) highlight the need for surveillance even after an outbreak appears extinguished. management of the sars epidemic also demonstrated that public service infrastructure, which affords the greatest chance of success ( ), is essential to the rapid containment of an outbreak. in areas most affected, contact tracing was important ( ) . in guangdong, police departments tracked down contacts of infected persons, who were then followed up for days after exposure. evaluating the surge capacity of public health services and hospitals is one way to assess the preparedness of a medical system. the case-fatality rate is a key determinant of the public health impact of an emerging disease and was high for sars at approximately % ( ) . the relationship between infectiousness and onset of symptoms is also important. patient isolation has greater potential as a control strategy if the illness can be diagnosed before the person becomes infectious (roy anderson, imperial college london). in contrast, persons infected with influenza virus are highly infectious before they become symptomatic. the rapidity of pathogen turnover means that evolution in pathogen populations can occur on a time scale that is epidemiologically relevant. indeed, sars-cov evolved during the course of the sars outbreak in china ( ) . similarly, influenza is perpetuated in the human population by the evolution of new antigenic variants every year (robin bush, university of california, irvine) ( ) . even if the transmissibility of an emerging disease is initially below the threshold necessary to sustain it in a population, the potential for the organism's evolution to higher levels may exist ( , ) . thus, one should not become complacent about diseases that are repeatedly introduced through zoonosis, but teeter on the edge of sustainability within the human population. the success with which who coordinated the global collaboration in containing sars galvanized the world health assembly to grant who greater authority to verify outbreaks, conduct investigations of outbreak severity, and evaluate the adequacy of control measures. the outcome of this new authority will depend on integrating the expertise of public health officials, medical doctors, and epidemiologists worldwide with guidance from disease transmission models. the sars outbreak demonstrated that an epidemic in one part of the world is not just an individual nation's problem but a global problem. alison p. galvani* *university of california, berkeley, california, usa newly discovered coronavirus as the primary cause of severe acute respiratory syndrome koch's postulates fulfilled for sars virus curtailing transmission of severe acute respiratory syndrome within a community and its hospital transmission dynamics of the etiological agent of severe acute respiratory syndrome (sars) in hong kong: the impact of public health interventions transmission intensity and impact of control policies on the foot and mouth epidemic in great britain dynamics of the uk foot and mouth epidemic: stochastic dispersal in a heterogenous landscape uses and abuses of mathematics in biology a major outbreak of severe acute respiratory syndrome in hong kong infectious diseases of humans: dynamics and control a cluster of cases of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome: temporal stability and geographic variation in case-fatality rates and doubling times molecular evolution of the sars coronavirus during the course of the sars epidemic in china influenza vaccine-outmaneuvering antigenic shift and drift the role of evolution in the emergence of infectious diseases infectious disease dynamics: what characterizes a successful invader? an open access publishing conference was convened in atlanta, georgia, on january , , by the libraries of the centers for disease control and prevention (cdc) and emory university. open access is an emerging publishing model for peerreviewed scientific research in which authors and their publishers grant free access to their work as long as the authors are acknowledged and the publisher ensures that the work is made freely available in a digital archive ( ). the conference brought together key stakeholders including scientists, researchers, publishers, and librarians and included approximately participants with offsite registrants connecting through the simultaneous web cast.the keynote address, "the coming revolution in the publication of scientific papers," delivered by harold varmus, emphasized that ) in today's internet era, the traditional gutenberg print publishing model is outdated; ) electronic publishing has the advantages of lower costs, global distribution, content that can be linked to datasets, improved archiving, and full-text searching; and ) rigorous peer review is possible in electronic and open access formats. open access publishing challenges include engaging professional societies in this approach, building sustainable business plans, and changing academic culture so that published works are evaluated for content rather than for the journal label. open access publishing is typically financed by author fees along with a combination of philanthropic and advertising support. examples are the public library of science, journal of clinical investigation, and biomed central key: cord- - nrfipz authors: jay, taylor r.; von saucken, victoria e.; landreth, gary e. title: trem in neurodegenerative diseases date: - - journal: mol neurodegener doi: . /s - - - sha: doc_id: cord_uid: nrfipz trem variants have been identified as risk factors for alzheimer’s disease (ad) and other neurodegenerative diseases (ndds). because trem encodes a receptor exclusively expressed on immune cells, identification of these variants conclusively demonstrates that the immune response can play an active role in the pathogenesis of ndds. these trem variants also confer the highest risk for developing alzheimer’s disease of any risk factor identified in nearly two decades, suggesting that understanding more about trem function could provide key insights into ndd pathology and provide avenues for novel immune-related ndd biomarkers and therapeutics. the expression, signaling and function of trem in ndds have been extensively investigated in an effort to understand the role of immune function in disease pathogenesis and progression. we provide a comprehensive review of our current understanding of trem biology, including new insights into the regulation of trem expression, and trem signaling and function across ndds. while many open questions remain, the current body of literature provides clarity on several issues. while it is still often cited that trem expression is decreased by pro-inflammatory stimuli, it is now clear that this is true in vitro, but inflammatory stimuli in vivo almost universally increase trem expression. likewise, while trem function is classically described as promoting an anti-inflammatory phenotype, more than half of published studies demonstrate a pro-inflammatory role for trem , suggesting that its role in inflammation is much more complex. finally, these components of trem biology are applied to a discussion of how trem impacts ndd pathologies and the latest assessment of how these findings might be applied to immune-directed clinical biomarkers and therapeutics. human genetic studies have provided crucial insight into neurodegenerative disease (ndd) pathogenesis. alzheimer's disease (ad) is a prime example of how advances in genetic technology have facilitated the evolution of our understanding of the etiology of ndds. early studies using genetic linkage approaches identified familial mutations in proteins related to amyloid beta production, amyloid precursor protein (app), presenilin (psen ) and psen , as well as the late onset ad (load) risk variant apolipoprotein e (apoe ) [ ] . these studies provided important insight into amyloid as a critical factor in ad pathogenesis and prompted application of molecular approaches and animal models to understand the disease. since [ ] , case-control genome wide association studies (gwas) have identified many novel ad-associated genetic variants [ ] . though many of these individually confer only modestly elevated risk for developing ad, collectively these studies provide broad insight into the pathways and processes involved in load. many identified genetic linkages are implicated in modulating immune function [ ] , demonstrating an important role for the immune response in ad. more recently, next generation sequencing technologies have made possible the identification of rare variants, some of which may confer higher disease risk and therefore can provide important insight into genes with strong biological roles in disease [ ] . the application of whole exome sequencing [ ] and gwas with imputation based on predicted genetic associations [ ] to ad led to the identification of relatively rare variants in the gene triggering receptor expressed on myeloid cells (trem ) that are associated with a high risk for developing ad. heterozygous trem variants confer similar risk for ad as one copy of apoe . significantly, the adassociated trem variants are largely coding variants, in contrast to most of the single nucleotide polymorphisms (snps) identified in gwas [ ] , making it more straightforward to translate into in vitro and in vivo models and perhaps also into therapeutics [ ] . trem variants have now also been linked to other ndds, suggesting that trem is critically involved in shared disease mechanisms. the excitement in the field following identification of these ad-associated trem variants was also driven by its implications, providing a clear link between the innate immune system and ndd pathogenesis. while it has long been known that immune cell function is dysregulated in ad and other ndds, it was not clear whether this actively contributed to disease pathogenesis and progression or was just a secondary response to ad-related pathology. however, this debate was largely settled in favor of the former when trem variants were found to be significantly associated with risk for ad and other ndds, and to form a genetic basis of polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (plosl, also known as nasu-hakola disease). because trem is exclusively expressed on immune cells, these genetic associations were hailed as providing conclusive evidence that immune dysregulation can be a primary, causal contributor to ndd pathogenesis [ , ] . thus, ndd-associated trem variants provide a new avenue to investigate the important roles that the immune system plays in neurodegeneration [ ] . in the years since trem variants associated with ad risk were identified, many groups have developed research programs aimed at understanding trem genetics, expression, structure, signaling, function, and its relationship to ndd pathologies and applied these findings to clinical biomarkers and therapeutics. progress in these areas has clarified our understanding of the biology of the trem receptor. while it was previously thought that trem expression was decreased by proinflammatory stimuli and mediated anti-inflammatory effects, it is now clear that its roles are more complex. in vitro, inflammatory stimuli decrease trem expression but in vivo trem expression is increased in inflammatory contexts. more than half of studies report that trem has a pro-inflammatory effect, suggesting that there must be cell type-and context-dependent functions of the receptor. recent studies have also illuminated new aspects of trem biology which necessitate a reevaluation and reinterpretation of previous literature. one example is the finding that soluble trem is produced in ad in a disease progression-dependent manner [ ] and that this soluble form of the receptor may have distinct biological effects [ , ] . other fundamental aspects of trem biology are also under intense investigation, including epigenetic and posttranslational modification of trem that affect expression and function, the ontogeny of trem expressing cells in the brain, and how noncanonical signaling pathways may contribute to trem function. this review offers a comprehensive synthesis of these studies alongside previous trem literature to identify areas of consensus and emerging questions in the field. this understanding will be crucial to support informed design and interpretation of studies of trem and the immune response in ndds moving forward. diverse trem variants are associated with ndd risk there is great diversity in the trem variants that have been associated with ndds, including single amino acid substitutions, frameshift and nonsense mutations, and changes in splice sites predicted to alter the inclusion or exclusion of particular exons [ ] . and, while most of the trem variants identified are present in the coding sequence, there have also been disease-associated variants found in the 'utr [ ] , and upstream of the transcription start site [ ] . the first ndd-associated trem variants identified were w x and w x, which result in premature truncation of the protein, a variant at the consensus splice site which results in exclusion of exon , and the k n mutation, which disrupts association of trem with its obligate intracellular signaling adaptor, dap [ ] . these all likely result in a loss of trem function, and a patient with the e x nonsense variant of trem had no detectable trem transcript levels [ ] . subsequently, a host of diverse trem variants have been identified. despite their structural diversity, all of the ndd-associated trem variants identified have been suggested to confer loss of function through different mechanisms. however, whether loss of function truly unifies all of these variants is still very much an open question. trem variants are the genetic basis of plosl and some familial ftd cases trem was first identified as a genetic cause of plosl, also commonly known as nasu-hakola disease [ , ] , which is characterized clinically by bone cysts and fractures, neuropsychiatric symptoms and dementia [ ] . neuropathologically, plosl patients have axonal degeneration and white matter loss, as well as cortical atrophy [ , ] . this is accompanied by an inflammatory response consisting of increased microglial density and activation and astrocytosis [ ] . these neurological manifestations can also occur in the absence of fractures [ ] or bone cysts [ ] . paloneva and colleagues [ ] were the first to link trem variants with plosl, and since then, many studies have identified homozygous trem variants that form the genetic basis of plosl [ ] [ ] [ ] [ ] [ ] [ ] . studies in families with frontotemporal dementia (ftd) [ ] or frontotemporal lobar dementia (ftld) found that plosl-associated trem variants t m [ , ] , w x [ ] , q x and y c [ ] in either homozygosity or heterozygosity could also cause ftd [ ] [ ] [ ] (fig. ) . case-control studies were then performed to assess whether trem variants might increase risk for ftd in the general population. initial studies suggested that there was a positive association between trem variants as a whole and risk of ftd [ , ] with a significant association found between ftd risk and individual trem variants including t k and l p [ ] and r h [ ] . others failed to replicate this association with trem variants and ftd or ftld [ , , , ] . however, an association between trem variants and specific endophenotypes of ftd, including reduced white matter volume, seizures and motor symptoms has been reported [ ] . together, it is not clear whether trem variants increase risk for ftd outside of specific familial cases, but they may influence specific clinical manifestations of the disease. fig. diverse trem variants are associated with ndds. genetic variants in the trem gene (shown above) result in diverse changes in the protein structure (shown below). these variants occur in almost every exon (black boxes) and impact known protein motifs (sequences highlighted in blue) and flank many sites of known protein modifications (amino acid number and type of modification detailed inside black boxes). trem variants have been found to be significantly associated with many ndds, including ad (variants shown in yellow), ftd or ftld (pink), pd (purple) and plosl (red). the table shows genetic variants that have been found to be significantly associated with disease risk, with supporting references shown in dark green and references that provide strong counterevidence shown in red. references shown in light green did find a significant association between the trem variant and disease risk, but only in one or multiple populations they examined or only after inclusion of previously published literature into metastudy analyses. while these variants have been significantly associated with disease risk, many more studies find suggestive but not significant associations between additional trem variants and ndd risk which are not represented here [ , , , , , , , , - , - , - , - , , , , , , , , , , , , , , - ] trem variants are associated with risk for ad it was investigated whether trem variants could also confer risk for alzheimer's disease. while it was first suggested that plosl-associated genes might confer risk for ad in [ ] , a small, case-control study in failed to demonstrate a significant association with ad risk [ ] . however, larger studies in found that heterozygous expression of the trem r h [ , ] and d n variants [ ] were significantly associated with ad risk. the association of trem variants with ad has been extensively replicated [ , , [ ] [ ] [ ] [ ] and the r h variant [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] validated in neuropathologically-confirmed cases [ ] . other variants have also been consistently shown to confer ad risk, including d n [ , ] , r h [ , ] , l p and t k, and h y [ , ] (fig. ). while rare, individuals with ad homozygous for the r h [ , , ] and l p [ ] variants have also been identified. studies examining the association of trem variants with particular ad endophenotypes yielded somewhat inconsistent results, likely due to the small sample size of patients with these trem variants. some found that r h trem variant carriers had a decreased [ ] or a trend toward decrease [ ] in the age of ad onset, though others found no significant association [ , ] . additionally, some found that disease progression was accelerated in r h carriers [ , ] , though this was not observed in all studies [ ] . other variants were also found to increase [ ] or decrease [ ] the rate of ad progression. despite possible differences in ad onset or progression, the clinical presentation of ad in r h carriers is similar to non-carriers [ , ] , though there may be a higher incidence of some endophenotypes including seizures and motor symptoms [ ] . trem variants have also been assessed as potential risk factors for other neurodegenerative diseases, though the findings in these other disease contexts are less definitive. in amyotrophic lateral sclerosis (als), one study found a significant association of the r h variant and als risk, as well as an inverse correlation between trem levels in the spinal cord and survival in als patients [ ] , though this was not replicated by others [ ] . the trem r h variant has also been reported to be associated with increased risk of parkinson's disease [ ] by some [ , , ] but not all studies [ , , ] . in order to understand these differences, lill and colleagues [ ] divided their groups by ethnicity and found that the odds ratio of the r h variant was significantly higher in their northern european population compared to non-northern europeans. others identified another parkinson's disease (pd)-associated snp kb upstream of trem [ ] , though its effect on trem expression is not known. the trem r h variant has also been investigated as a risk factor for posterior cortical atrophy [ ] , multiple system atrophy [ ] , essential tremor [ ] , multiple sclerosis [ ] and creutzfeldt-jakob disease (cjd) [ ] , though these studies were not conclusive. one family has been identified in which a mutation in trem is thought to result in progressive non-fluent aphasia [ ] , though other cases will be necessary to confirm this association. so far, evidence suggests that trem variants are not significantly associated with dementia with lewy bodies [ ] , ischemic stroke [ ] or progressive supranuclear palsy [ ] . because many of these diseases share overlapping clinical features with ad and ftd, it will be important to validate any associations of trem with other ndds in neuropathologically confirmed cases. overall, the association of trem variants with these other ndds is less clear, and future studies with large sample sizes in diverse but well-matched populations will be required to definitively establish whether trem variants confer risk for ndds other than plosl, ftd and ad. importantly, the association of trem variants with multiple ndds suggests it may underlie common disease mechanisms. trem dysfunction may provide insight into mechanistic links among these diseases. epidemiologically, the prevalence of trem variants differs greatly among individuals from different genetic backgrounds [ ] . in caucasian populations, the minor allele frequency (maf) of the r h trem variant ranges from . - . % in the united states, to up to % in some specific british populations [ , , ] . while the r h variant is virtually absent in east asian individuals, [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] nine other trem variants were present in east asian populations and collectively associated with ndd risk [ ] . similarly, the maf of the r h and r h variants are much lower in african americans compared to european american populations [ , ] . however, exonic sequencing of trem revealed variants that had much higher mafs in the african american population compared to european americans, and some of these variants were significantly associated with ad risk in that population [ ] . because of the small mafs, it is not clear whether the effect sizes of these variants differ among different ethnic groups as well. the low mafs and diversity in the frequency of trem variants across populations necessitates that studies have large study populations and be well-matched for ethnicity. it will also be important to take advantage of the identification of these trem variants across diverse populations to gain a full understanding of how trem variants confer ad risk and how they might interact with other genetic differences among individuals from distinct genetic backgrounds. several groups have examined the relationship between trem and other ndd risk factors. variants in siglec- (cd ) are significantly associated with ad risk and cd levels on human blood monocytes were found to inversely correlate with surface trem levels [ ] . the ad-associated cd allele increased its surface expression, effectively decreasing trem signaling. another ad-associated gene, membrane-spanning -domain family a (ms a), was found to be co-enriched with trem in the human brain and is substantially upregulated in monocytes derived from patients with ploslassociated trem variants [ ] . an ad-associated snp in ms a correlated with altered soluble trem (strem ) levels in cerebrospinal fluid (csf), suggesting that ms a may also regulate trem expression or processing [ ] . trem levels are also increased in ad mouse models lacking pgrn expression, which models lower pgrn expression observed with an ad and ftld-associated pgrn genetic variant [ ] . components of the trem signaling pathway have also been associated with ndds, including its intracellular signaling adaptor dnax activation protein of kda (dap , also termed tyrobp). dap and trem variants produce virtually indistinguishable phenotypes in plosl patients [ , ] . however, satoh and colleagues [ ] found that dendritic cells derived from monocytes of plosl patients with trem or dap mutations had very different patterns of gene expression, suggesting that they may produce the same phenotype through different molecular mechanisms. a rare dap variant at the site of interaction with trem also confers risk for developing early onset ad [ ] and dap was found to play a central role in ad-related molecular networks [ ] . as its relationship to ndds might predict, dap deficient mice also have synaptic degeneration and reduced myelination [ ] , though recent evidence suggests that dap deficiency may be neuroprotective in an ad mouse model [ ] . the precise mechanisms underlying these changes are not yet understood. variants in additional proteins associated with the trem signaling pathway, ship and colony stimulating factor receptor (csf r), have been associated with ad risk and leukoencephalopathy with spheroids [ , ] . in addition, apoe, a putative trem ligand [ ] [ ] [ ] , is clearly established as an ad risk factor [ ] . finally, environmental risk factors for ad including traumatic brain injury [ , ] , diabetes [ ] and age [ ] , all alter trem expression in the brain. together, the identification of variants in genes involved in these common immune pathways suggest that trem , along with its interaction partners, together play an important role in modifying ndd pathology. co-regulation of trem and other members of the trem family trem is located on human chromosome in a gene locus containing several trem and trem-like genes, such as trem , triggering receptor expressed on myeloid cells like transcript (treml ) and treml , which likely originated from duplication events but now have relatively diverse sequences [ ] [ ] [ ] . many of these genes, including trem , are highly conserved between humans and mice, while others are present only in mice (trem and treml ) or humans (treml and nkp ). there may be some shared mechanisms of gene regulation across the locus. for example, there is a retinoid x receptor (rxr) binding site upstream of the entire locus that is thought to result in coordinate regulation of these genes [ ] . however, in some cases, opposing regulation of these different genes has been shown, such as between trem and trem [ ] , and between trem and treml [ , ] . it is not known what factors contribute to these inverse correlations in expression. snps within this locus can also result in changes in expression of multiple trem genes. variants in the treml [ ] and treml [ ] gene have been shown to increase brain trem and treml expression levels. chan and colleagues [ ] found that ad-associated variants in trem result in reduced trem expression on human monocytes and increased trem expression. moreover, an intronic variant in trem which decreases its expression leads to increased amyloid accumulation and cognitive decline in ad patients [ ] . however, non-ad associated variants decreased both trem and trem expression, leading the authors to suggest that the ratio of trem and trem expression rather than the absolute changes in expression may be important for disease. additionally, variants in treml increase pd risk [ ] , and other disease-related snps that alter treml levels [ ] associate with ad in gwas analyses [ ] . another variant of treml was found to be protective against developing ad [ ] . an intergenic variant associated with ad risk was shown to alter rna levels of treml [ ] . these data highlight the importance of characterizing the expression patterns of all trem members in an effort to pinpoint co-regulatory mechanisms among these genes physiologically and in the context of disease. trem expression is highly cell-type and context specific. however, the molecular mechanisms governing this highly specific regulation of trem expression are just beginning to be understood. while there are several predicted transcription factor binding sites in the utr's and promoter region of trem , only a few have been functionally validated. pu. , a master regulator of myeloid cell fate specification, is present in the trem promoter [ ] . the rxr agonist bexarotene was found to enhance rxr occupancy of known binding sites [ ] upstream of trem in mice, though this did not correlate with increased trem rna levels [ ] . however, in ad mouse models, bexarotene did enhance gene expression of trem [ ] , suggesting that rxr may effectively induce trem transcription when cells are already primed in a particular context. nkκb [ , ] , protein e [ ] , rankl, and nfat [ ] have all been shown to regulate trem expression in different cell types, but it is not clear whether these factors directly regulate expression. one group has suggested that nfκb may instead regulate trem expression by increasing levels of microrna a which, in reporter assays, decreased trem expression [ , [ ] [ ] [ ] [ ] [ ] . there are also epigenetic changes that have been shown to influence trem expression. in humans, hippocampal enrichment of -hydroxymethylcytosine ( hmc), a marker of active demethylation, at the trem transcription start site and in exon were found to positively correlate with trem mrna levels [ ] . methylation upstream of the trem transcription start site was also increased in ad, a context in which trem levels are also increased [ ] . additionally, methylation at the cpg sites in intron negatively correlated with trem mrna levels in human leukocytes, and methylation at these sites was reduced in ad patients [ ] . others have found that h kme and h kme , histone modifications which typically are associated with active gene transcription, are increased at the trem locus in db/ db mouse adipose tissue [ ] and in cultured dendritic cells and macrophages during differentiation [ ] , contexts in which trem mrna levels are elevated. finally, there are likely post-transcriptional mechanisms of regulation of trem expression which contribute to differential expression in different contexts. trem mrna stability can be dynamically regulated. the half-life of trem mrna in cultured human peripheral blood mononuclear cells went from . h to h after toll-like receptor (tlr) ligation [ ] . furthermore, hu and colleagues [ ] found that trem mrna expression strongly correlated with surface levels of trem protein on monocytes, but only weakly correlated with protein expression on granulocytes in the plasma. this suggests that, in addition to differential regulation of trem transcription through transcription factors and epigenetic markers, trem expression can be further differentially controlled at the mrna and protein level in distinct cell types through mechanisms which are not resolved. how trem variants affect trem expression is a topic currently under investigation. as discussed above, some trem variants are known to reduce trem expression. the nonsense mutations e x [ ] and q x [ ] were both found to eliminate trem protein expression. trem rna levels were reduced in patients harboring the variant t m [ ] and a splice donor mutation in intron [ ] . heterozygous expression of a variant that affects trem splicing in intron , which is associated with early onset dementia, also affected the expression of the common variant allele of trem , reducing it by more than half [ ] . it is unclear how these variants result in reduced trem transcript levels, though it has been suggested that epigenetic changes may partially account for these effects. in contrast, the r h variant either did not change [ ] or trended toward increasing trem transcript levels [ ] in individuals with ad. although there were no changes found in gene methylation upstream of trem 's transcription start site in r h carriers compared to controls [ ] , variants tend to cluster around exon where trem expression was found to be correlated with hmc enrichment [ ] . whether variants affect regulation of trem rna expression at the level of epigenetics, transcription factor binding, rna stability or altering the cell phenotype in a manner that indirectly drives alterations in trem transcription is not yet clear. at the protein level, in transfected cell lines t m and y c trem variant proteins were found to be degraded by the proteasome, leading to decreased protein expression [ ] . in ad patients with r h variants, there was a trend toward decreased trem protein levels in one study [ ] , but a trend toward an increase in another [ ] . it will be crucial to continue collecting data from trem variant carriers to gain a clearer picture of how trem expression is altered by different disease-associated variants. in plosl patients with dap mutations, there was a variable effect on dap expression levels, in some cases increasing and in others decreasing expression [ ] . it may be that trem variants also alter trem expression in distinct ways. trem is expressed on many cells of the myeloid lineage, as its name suggests, including dendritic cells [ , , , [ ] [ ] [ ] , granulocytes [ ] , bone marrow and monocyte derived macrophages [ , , , , ] , and tissue macrophages like splenocytes [ ] , kuppfer cells [ ] , alveolar macrophages [ , ] , and osteoclasts [ , , ] (fig. ) . trem is not reported to be expressed on lymphocytes [ ] . its expression on circulating monocytes remains controversial. initially it was thought that trem was only expressed after differentiation of monocytes into macrophages [ ] , and others have provided further data to support a lack of trem expression or expression on only a small subset of monocytes in humans [ , , , ] and mice [ ] . however, others have detected trem expression in whole blood [ , [ ] [ ] [ ] and specifically on human [ , ] and mouse [ ] monocytes. these disparate findings may be due to differences in sensitivity of detection or other technical factors, but it will be important to resolve moving forward. it is clear that trem is expressed in the brain and most evidence documents that trem is expressed exclusively within the brain by microglia. all published studies to date find that primary cultured microglia express trem [ , [ ] [ ] [ ] [ ] . in vivo, most studies detect trem expression in mouse microglia [ , , [ ] [ ] [ ] [ ] [ ] [ ] , though some find that it is expressed by only a subset of these cells [ , [ ] [ ] [ ] [ ] and others could detect trem rna but not protein expression in mouse microglia [ ] . furthermore, studies demonstrated that when microglia are acutely depleted from the brain through csf r antagonist treatment in vivo, [ ] through cd b-hsvtk depletion in brain slices [ ] , or chronically in pu. knockout mice [ ] , trem is no longer detectable in the cns. in humans, experimental outcomes have been more variable, with some detecting high levels of trem expression across all microglia [ ] , others finding lower levels of trem expression [ ] and others not detecting microglial expression of trem [ ] . with a few exceptions, most evidence is in strong agreement that trem is expressed, at least under normal physiological conditions, specifically in microglia within the cns. can be present as a full-length protein (shown at top) or as a soluble product. this can occur through proteolytic cleavage by adam followed by γ-secretase to produce soluble trem (strem ), a c-terminal fragment (ctf) and an intracellular domain (icd). trem can also be alternatively spliced to produce soluble isoforms. one alternative transcript has been validated in mice, while two have been validated in humans and four others are predicted to occur [ , , , , , , , ] trem expression changes throughout neurodevelopment and varies across brain regions trem expression in the cns is also regulated throughout development and its expression pattern varies across different brain regions. trem is first detectable in the mouse cns at e and continues to be expressed through adulthood [ ] . however, at p , all brain myeloid cells express trem rna, but not all of these cells still express detectable levels by p , which has also been supported in vitro [ ] . this early elevated trem expression also occurs in other organs following macrophage infiltration during organogenesis [ ] . trem expression is also regulated differentially across brain regions in humans and mice. trem was found to be highly expressed in white matter [ , ] , hippocampus [ , , ] and spinal cord [ , ] , among other regions. while this could suggest that particular microenvironmental niches induce trem expression locally within distinct microglial populations, it may also simply reflect the high density of microglia in these regions [ ] . trem expression has been shown to be dramatically altered in the contexts of inflammation, injury and disease. in vitro, application of classically pro-inflammatory molecules (tnfα [ , ] , il β [ , , ] , ros [ , ] , ifnγ [ ] , tlr agonists, including lipopolysaccharide trem is expressed in many immune cells, and is localized to microglia in the cns. trem expression has been assessed in a variety of human and mouse cell types. these data represent trem expression in these cells under homeostatic conditions, though, as discussed in the next section, trem expression can change in the contexts of inflammation or pathology. references shown in green are supportive of trem expression in the cell type listed while those in red did not detect trem expression using the listed method of detection. references in yellow provide evidence of expression, but at low levels or in a small percentage of cells assayed. the graph represents the cell types in which trem expression has been examined at a size relative to the number of studies and methods used to detect trem expression in that cell type. they are graphed along the y-axis according to the percentage of these findings which support trem expression on these cells [ , , , , , , , , , , - , - , , - , , , , , , ] (lps) [ , , , , [ ] [ ] [ ] [ ] , cpgs [ ] and other tlr ligands [ , , , ] , mitochondrial lysates [ ] and bacteria [ ] ) decreased trem expression, while anti-inflammatory molecules (vasoactive intestinal peptide [ ] and il [ ] ) increased trem expression. in contrast, in vivo, inflammation and different disease states almost universally increase trem expression. stimuli that induce an inflammatory response in the lung increase trem expression in alveolar macrophages [ , , [ ] [ ] [ ] [ ] [ ] [ ] . high fat diet [ , ] increased inflammation and trem expression in adipose tissue, liver and brain. trem is also upregulated in numerous other inflammation-related contexts, including sepsis [ ] , rheumatoid arthritis [ ] , corneal infection [ ] , macular degeneration [ ] , glioma [ ] , oral [ ] , esophaegeal [ ] , and liver [ ] cancers, following prosthetic joint implants [ ] , osteoporosis [ ] , colonic mucosal injury [ ] , colitis [ ] , gastrointestinal mucositis [ ] and muscular sarcoidosis [ ] . in the cns, trem expression is increased in the context of traumatic brain injury [ , ] , stroke [ , ] , spinal nerve transection [ ] , als [ ] , pd [ ] , prion disease [ , ] , models of demyelination [ , , [ ] [ ] [ ] and following beta-amyloid (aβ) vaccination [ ] . only one study found reduced trem expression in in vivo inflammatory contexts, following lps injection and middle cerebral artery occlusion in mice [ ] . trem has also been shown in almost all cases to positively correlate with aging, both in mouse models [ , ] and in humans [ ] . soluble trem , a product of full-length protein cleavage or alternative splicing, is detectable in csf and its levels were also positively correlated with age [ , , , ] , though this was not reflected in blood [ ] . taken together, these studies largely demonstrate that, in vitro, inflammatory stimuli decrease trem expression, while in vivo, inflammatory stimuli predominantly increase trem expression, clearly suggesting that the dogma based on early studies that trem expression is universally reduced in inflammatory contexts is not applicable to in vivo contexts. why this occurs is not clear, but may reflect differences in cell recruitment, acute versus chronic signaling, non-cell autonomous signaling pathways, or phenotypic changes in myeloid cells that occur when they leave their native environment. the most comprehensive assessment of changes in trem expression has been performed in the context of alzheimer's disease. studies in ad patient brain tissue almost exclusively show increased trem expression [ , , , , [ ] [ ] [ ] [ ] , and some [ , , ] but not all [ ] found this was also reflected in increased trem levels in monocytes from ad patients. trem levels in the brains of ad mouse models are also increased. one study reports a reduction in trem rna levels before the onset of pathology in tg mice [ ] , though after the onset of pathology, all amyloid models of ad examined have increased trem rna and protein levels [ , , [ ] [ ] [ ] [ ] [ ] . this upregulation of trem expression occurs shortly after the onset of pathology and largely seems to correlate with amyloid burden [ , , ] and the association of myeloid cells with amyloid plaques [ , ] . tau models of ad also show increased trem levels [ , ] , however, trem is only increased long after neurofibrillary tangle development in these models [ ] , consistent with trem upregulation at late stages of disease progression in postmortem ad brain tissue [ ] . several studies have sought to determine what aspect of ad pathology drives trem expression. trem is upregulated in myeloid cells associated with plaques [ , , , , , ] , and specifically, trem is highly expressed on myeloid cell processes in contact with plaques [ ] . in support of a plaque-driven upregulation in trem expression, varvel and colleagues [ ] depleted microglia from the brains of an ad mouse model and allowed the brain to repopulate with new myeloid cells. these new cells that repopulated the brain initially failed to associate with plaques, but eventually became plaque-associated, and coordinately upregulated trem expression. furthermore, stereotactic injection of beta-amyloid (aβ ) into the cortex and hippocampus of wild-type mice was sufficient to induce an upregulation in trem transcripts within h [ ] . together, these findings suggest that amyloid can increase trem expression in myeloid cells. to determine whether this effect was cell-autonomous, cultured microglia were treated with aβ, though these studies have so far produced inconsistent results with respect to trem expression [ , ] . significantly, melchior and colleagues [ ] found that there was no effect on cultured microglia treated with beta-amyloid (aβ ) on trem levels, but if they added aβ to mixed glial cultures, this did result in upregulation of trem on microglia by flow cytometry. this suggests that, at least this aβ species may drive trem expression through feedback from other cell types, though the signals that mediate aβ-induced upregulation of trem on myeloid cells are not yet known. not all myeloid cells associated with plaques express trem [ , ] . investigation of which subset of myeloid cells upregulated trem in the ad brain has yielded conflicting results. some findings suggest trem + cells may be peripherally derived macrophages rather than brain resident microglia. jay and colleagues [ ] found that trem was expressed on cd hi myeloid cells which expressed the monocyte marker ly c and not the microglial-specific marker p ry . following toxoplasma gondii infection in xfad mice, mohle and colleagues [ ] found that trem was expressed most highly by c-c chemokine receptor type (ccr ) + ly c lo f / + cells in the brain, a marker signature of peripherally derived macrophages. trem expression in other disease models, including basal cell carcinoma [ ] , sciatic nerve transection [ ] and colonic mucosal injury [ ] , was found to be upregulated coincident with the peak of macrophage infiltration into the tissue. in contrast, others have detected trem expression on "dark" microglial cells in the brain, which are d + and c − , a marker signature consistent with resident microglia [ ] . wang and colleagues [ ] also performed experiments in which age-matched cd . wt donors were parabiosed with cd . ad mouse models and were not able to detect cd . cells in the brains of the ad mice. indeed, the contribution of peripherally derived macrophages to ndd pathologies has a long and controversial history. however, if trem is expressed on these cells, it suggests they may play a key role in modulating pathology, and thus this issue should continue to be explored. the structure of full-length trem trem is a single pass transmembrane protein whose ligand binding domain includes an extracellular ig-like domain [ ] with n-linked glycosylation sites [ , , , ] , phosphorylation sites and disulfide bonds which are thought to perform important structural roles. the transmembrane domain anchors trem to the membrane and contains the intramembraneous lysine residue necessary for association with its intracellular membrane adaptor, dap . this is followed by a short cytoplasmic tail with no established function. diseaseassociated variants of trem alter many of these structural elements (fig. ). many variants are found in exon and may change the structure of its ligand-binding domain, impacting the affinity of trem for different ligands. for example, the y c variant associated with plosl and ftd is predicted to alter an important flanking sequence of the cysteine residues which form trem 's disulfide bonds [ , ] . likewise, glycosylation is affected in cells transfected with y c and t m trem variants [ ] , though not in several other variants examined [ ] . glycosylation of the r h variant was also found to be reduced [ ] , though not to the same extent as the other trem variants in vitro. in humans expressing the r h variant there were no significant differences in the level of glycosylation [ ] but there were differences in the pattern of glycosylation [ , ] . trem variants can also affect other important structural motifs of the trem protein such as those required for dap association, and overall protein folding and stability. t m and y c variants [ ] along with v g are predicted to be important for protein packing. consistent with impaired protein folding, t m and y c variants exhibit enhanced proteasomal degradation [ ] . the r h variant has been predicted to impair protein stability [ ] , but transfected r h-trem constructs actually have an increased half-life relative to wt trem and are resistant to proteasomal degradation in the endoplasmic reticulum (er) [ ] . ndd-associated trem variants located on the surface of the protein (r h, t k, d n and r h), are not predicted to substantially alter trem structure [ , ] but instead affect ligand binding [ ] . as the structure of trem and disease-associated variants continue to be resolved, we will gain better insight into the organization of structural features essential for trem function. the structure and production of soluble trem trem can also be produced as a soluble protein (fig. ) . soluble trem (strem ) has been detected in the supernatants of mouse [ ] and human cells in culture [ , ] . it has been proposed that strem could be produced by both alternative splicing and proteolytic cleavage. insertions [ ] or frameshifts [ ] preceding exon terminate the transmembrane domain and are predicted to yield a soluble product. in human brain tissue, at least three trem isoforms have been detected [ ] , with isoform , encoding the full-length protein, being the most highly expressed [ ] . meanwhile, the transcript encoding a -residue splice isoform is expressed to a lesser extent than isoform in the hippocampus of ad patients [ ] , whereas the degree of expression of the -residue splice isoform has yet to be resolved. based on rna sequencing data in ad mice, - % of transcripts were predicted to be alternatively spliced [ ] . notably, these alternative transcripts have been identified in human monocytes [ ] , and in ad brain tissue [ , ] . evidence of elevations in expression of trem exons and in advanced ad cases by microarray-based gene expression analysis [ ] is suggestive of trem alternative splicing in ad [ , , ] . the expression of full length and splice isoforms of trem are strongly correlated in ad tissue, suggesting all trem isoforms may be coordinately regulated [ ] . dna methylation within the body of the gene has been shown to impact alternative splicing [ ] , and as trem can be methylated within exon [ ] , it is possible that context-dependent changes in methylation may also result in altered splicing. however, direct evidence that alternatively spliced mrnas are translated is lacking. the sequential proteolytic processing of trem has definitively been shown to generate strem [ , , ] . in vitro inhibitor studies revealed full-length trem is proteolytically cleaved by a disintegrin and metalloproteinase domain-containing protein (adam ) resulting in shedding of the ectodomain [ , , ] . detection of trem c-terminal fragments (ctfs) within cell lines [ , , ] and human brain extracts [ ] is suggestive of a two-step proteolytic cleavage event of trem . following ectodomain shedding by adam , the remaining membrane-associated trem c-terminus undergoes intramembrane proteolysis by γ-secretase to release its intracellular domain [ ] . γ-secretase cleavage of trem results in accumulation of trem ctfs, without a corresponding increase in full-length trem at the plasma membrane upon γ-secretase inhibition [ , , ] . it is not known how this process is regulated, but it is known that trem 's adaptor protein dap is required for strem production, at least in some contexts [ ] . whether the production of strem in ad occurs by alternative splicing or ectodomain shedding is unclear. it may be cell-type and context dependent as different cell types showed either an up-(dendritic cells) or downregulation (microglia, monocytes) of the trem splice transcript levels when stimulated by lps [ , ] . il and il were also shown to enhance strem production in bone marrow derived macrophages [ ] . this is consistent with studies examining other members of the trem family as trem [ ] [ ] [ ] and tremlike transcript- [ ] which also produce cell-specific protein products. trem variants linked to ad and other neurodegenerative diseases can alter soluble trem generation. trem variants can drive novel trem splicing [ , ] such as the plosl-associated trem variant c. + t > c in which conversion of a single nucleotide at a splice-donor consensus site within intron results in the deletion of exon in addition to exon and/or exon [ ] . this is proposed to produce soluble protein products [ ] lacking either the transmembrane (tm) domain or both the tm and ectodomain. while most trem variants are expressed in all isoforms of trem , there are diseaseassociated variants expressed only in the alternatively spliced isoforms of trem [ , , , , ] . several reports have also demonstrated that trem variants t m and y c yield significant changes in strem release by cultured cells [ , ] . this was also evident in individuals homozygous [ , ] and heterozygous [ ] for the t m variant, exhibiting a loss or reduction in csf levels of strem , respectively. other case reports showed disease-associated trem variants lowered csf strem levels compared to a non-carrier group [ ] . while some have reported decreased levels of strem and trem intracellular domain (icd) production in vitro with the r h variant [ ] , the trem r h variant acted similarly to wild-type in strem production in other in vitro assays [ ] and ad patients carrying the r h variant had elevated levels of csf strem [ ] . there was a trend for higher transcript levels of a -residue trem splice variant in cortices of late-stage ad cases with the r h risk allele compared to significant elevation in this splice isoform in non-carrier ad cases [ ] . these data altogether suggest that different trem variants have distinct effects on strem levels. in addition to altering proteolytic cleavage, the localization of trem within the cell can greatly impact trem signaling, and its trafficking appears to be a highly dynamic process. in homeostatic conditions, trem seems to be primarily found intracellularly [ ] , associating with the trans-golgi network [ , , ] and in a population of exocytic vesicles [ ] . these vesicles appear to be continuously shuttled to the membrane, a process which can be rapidly induced by increases in ca + in response to ionomycin [ ] . it is not clear what other specific stimuli or disease contexts result in changes in trem localization within the cell, but this will be critical to understand trem 's functional role in these contexts. trem is recycled from the membrane in clatherin-coated vesicles in a beclin- [ ] and vps dependent manner [ , ] . vps mediates recycling of trem from the membrane via retromer complexes [ ] . when this process is blocked, trem increases its association with the lysosome and is degraded [ ] . trem variants can impact trem localization within the cell. in cells in which trem variants were transfected, t m and y c [ , , , , ] as well as other variants [ ] significantly reduced trem surface expression. these variants increased the localization of trem with the er [ , ] , which may indicate impaired protein folding. the r h variant was found to either not alter surface expression [ , ] or reduce surface expression of trem to a lesser extent [ ] . unlike the other variants, r h trem was mostly localized to the trans-golgi network rather than the er, comparable to the wt receptor [ , ] . however, yin and colleagues [ ] did find that the r h variant had reduced association with vps , resulting in increased lysosomal degradation following recycling of the receptor from the surface. overall, this suggests that variants may, in part, impact trem function by altering the localization of trem within the cell. while trem has been proposed to play a functional signaling role exclusively on the cell surface, this could also impact possible functional roles of trem in other cellular compartments. despite substantial efforts, the identity of the biological ligands of trem remains controversial (fig. ) . trem is known to modulate myeloid cell activity in response to microbial products [ ] , which led several groups to test bacteria as a possible source of trem ligands. using a trem -fc fusion protein that consists of the extracellular ectodomain of trem attached to the fc-portion of human igg, trem was found to bind to some bacteria [ , ] , including gram-positive (s. aureus) and gram-negative species (e. coli, f. tularensis), but not s. cerevisiae [ , ] , salmonella or typinmurin [ ] . trem was specifically found to bind highly anionic bacterial products [ ] and pertussis [ ] and cholera [ ] toxins. in addition to microbial products, trem was also reported to bind to high molecularweight nucleic acids [ ] and heat-shock protein [ ] , that were further shown to initiate trem signaling in reporter cell lines. while some of these interactions were found to be of relatively low affinity [ ] , recognition of many molecules like carbohydrates and glycans induce only minimal biological signaling at low densities, but at high densities when receptors are forced into closer contact, can create strong biological effects [ ] . indeed, glycosaminoglycans and specifically heparin sulfate were found to modulate trem binding [ ] and it has been suggested that this may result in clustering of trem on the membrane, thus potentially modulating trem binding to other ligands [ ] . trem signaling and function. trem has been proposed to bind to a variety of different ligands, categorized here by lipids, lipoproteins and ligands associated with damage-or pathogen-related molecular patterns. these ligands bind to the trem receptor. following ligand binding, trem can associate with dap homodimers or dap /dap heterodimers to mediate downstream signaling. this signaling requires phosphorylation of the adaptor, following which activating (shown on left in green) or inhibitory (shown on right in red) signaling components can bind. these activating components have been shown to initiate different downstream pathways that lead to cell proliferation and differentiation, survival, phagocytosis, chemotaxis and inflammation. while many other signaling components are thought to play a role downstream of trem activation, only those validated as part of the signaling pathway responsible for the listed functions have been included here. association with inhibitory components is thought to prevent activation of these downstream pathways. lipids: pe [ , ] , ps [ , , , , ] , pa [ , , ] , pg [ , , ] , pc [ , , , , ] , pi [ , , ] , cl [ , , ] , sm [ , , , ] , la [ , ] , sa [ , , , ] , apo-/lipoproteins: apoe [ , , ] , apoj [ ] , apoa- [ , ] , apoa-ii [ , ] , ldl, hdl [ , , ] , pamp/damps: [ , ] , (−) bacterial products (lps, lta) [ ] , nucleic acids [ ] , heat shock protien [ ] , apoptotic cells [ , , , , ] , macropahages [ ] , astrocytoma cells [ ] unidentified trem ligands were also detected by trem -fc binding to the cell surface of macrophages [ ] , human astrocytoma cells [ ] , dendritic cells [ ] , n a cells [ , ] , thp- cells [ ] and apoptotic cells [ ] . in support of these findings, trem deficiency partially impairs microglial recognition of apoptotic cells [ , , ] . recent studies have demonstrated that trem binding to these cells is sensitive to proteinase k, suggesting the receptor binds to protein [ ] , perhaps complexed with proteoglycans. it had previously been shown that members of the trem family recognized lipid ligands [ ] , and lipids may also help mediate the interaction of trem with ligands on the cell surface. polar lipids found on the cell surface were assessed as possible trem ligands [ , , ] , using lipid arrays [ ] and reporter assays [ , ] . while anionic lipids seemed to produce among the highest levels of response, additional factors that influence the particular lipids that trem recognizes requires further study. this lipid binding may allow trem to sense changes in the local environment as exemplified by trem -positive cells binding to externalized phosphatidylserine on apoptotic cells [ ] and myelin debris [ ] . some studies suggest that trem may bind lipids in cis with other protein-based interactions [ ] . trem has also been reported to bind to lipoproteins, including apolipoprotein a (apoa-i), clusterin (clu), and low density lipoprotein (ldl), though apoe has been most widely demonstrated to bind to soluble trem -fc. this binding seems to occur independent of apoe isoform [ ] [ ] [ ] ] and be dependent on residues - [ ] . because trem binds to lipids, the lipidation status of apoe and other apolipoproteins could dictate their binding affinity to trem . several studies [ , , ] demonstrate that trem -apoe binding is not dependent on lipid loading. however, others have found that lipidation was necessary to drive trem binding [ ] . lipid association is reported to be necessary for trem binding to apoe, apoa-i and apoa-ii from cynomolgus macaque csf and serum [ ] . apoe binding to trem was found to induce trem signaling in nfat reporter cell lines [ ] , though how its binding to trem would alter signaling in vivo remains to be determined. because apoe can bind to apoptotic cells [ ] and amyloid plaques [ , ] , it has been proposed that an interaction between trem and apoe may indirectly allow it to mediate recognition and phagocytosis of these substrates. this may be important in the recognition of ad-related stimuli by trem , because trem was found not to bind to plate-bound aβ [ ] , but did bind to areas around amyloid plaques in an ad mouse model [ ] . however, the possibility of trem binding directly to aβ, in addition to these indirect interactions, has not been fully excluded. together, these data indicate that, in addition to proteinproteoglycan complexes, trem may also bind to lipids and protein-lipid complexes. ad-associated trem variants including r h, r h, d n and t k, are found on the surface of the protein, and impact ligand binding [ ] . studies employing a trem r h-fc chimeric protein revealed the r h mutation significantly reduces trem binding to cells [ ] , apoe [ ] including all three isoforms [ ] and its lipidated form [ ] , other lipids [ , ] , apolipoproteins [ , ] and lipoproteins [ ] . trem variants at either the same residue (r a, r e and r a) [ ] or with a similar r-to-h substitution (r h) [ , ] as r h similarly disrupted trem recognition of apolipoproteins [ ] or cells [ ] . however, r h and r h variants demonstrated relatively comparable lipid detection to wt trem [ ] . other variants residing within the trem ectodomain (y c, t m, k m) effectively abolished trem binding to proposed ligands [ ] , while those located on the ectodomain proximal to the stalk region of trem (d n, t k) exhibited enhanced interactions with some ligands [ , ] , while decreasing association with others [ ] . trem variants within the stalk region (h y, e k, r w) or intracellular domain (l p) had no significant impact on ligand binding. the differences among these diseaseconferring trem variants' recognition of cells, lipids, lipoproteins and apolipoproteins may alter how they impact downstream signaling. however, it remains unclear whether the ligands identified thus far are the relevant binding partners of trem in vivo and mediate the receptor's ability to respond to damage or infection in the cns. future work establishing the full array of physiological trem ligands, and how variants impact these interactions will be instrumental in elucidating the role of trem on myeloid cells in response to different pathologic stimuli. because the major isoform of trem has only a short cytoplasmic tail, it requires the intracellular adaptor dap [ , ] to mediate several of its signaling functions [ , ] . along with trem , dap is required for signaling of other trem family members [ ] , mdl- , and siglecs and can be used as an adaptor for other receptors critical for regulating myeloid cell function including csf r and toll like receptors [ ] . in some contexts, cross-talk among these receptors has been shown to occur at the level of dap availability. in order to associate with the membrane, dap requires the presence of its receptors [ ] . indeed, dap is clustered at the same area of the membrane where trem is highly upregulated on myeloid cell processes in contact with plaques in an ad mouse model [ ] . at the membrane, dap can associate with other dap molecules to form homodimers through cysteine residues in its short extracellular domain [ ] . in some contexts, dap can also heterodimerize with dnax activation protein of kda (dap ) [ ] which can modify downstream signaling cascades. it is not known whether dap complexes constitutively associate with trem or whether this is induced upon ligand binding in vivo but recent data using a split luciferase assay found that trem -dap association in transfected hek cells was primarily driven upon trem stimulation [ ] . interestingly, the t m trem variant but not r h or s c variants enhanced the constitutive association with dap in this system [ ] . regardless, activation of trem and other dap -associated receptors results in tyrosine phosphorylation of dap within its immunoreceptor tyrosine-based activation motifs (itam) by src family kinases casein kinase ii at residues - and at residues - by pkc [ ] . this phosphorylation occurs only when dap is receptorassociated [ ] and serine / threonine phosphorylation of the itam motifs are required for signaling [ ] . these phosphotyrosine residues on the dap itam serve as docking sites for a number of molecules that initiate signaling cascades that activate an immune response. there are several immune stimulating molecules that associate with dap in response to trem activation (fig. ) . crosslinking trem , commonly used to mimic trem activation, can result in recruitment of dap , pi k or lab to the trem -dap complex. in turn, these molecules are activated through tyrosine phosphorylation, principally by itam-associated syk and go on to activate downstream signaling components, including akt, rac, vav and mapks, including erk [ , , , , , [ ] [ ] [ ] [ ] . these initiate changes in gene expression and cytoskeletal rearrangement which mediate many downstream cellular functions associated with immune cell activation [ ] . while itam domains are typically activating, they can be inhibitory in certain contexts [ , ] . when the itam motifs of dap are partially phosphorylated [ ] , inhibitory phosphatases ship, shp and the adaptor downstream of kinase (dok ) are recruited to the trem -dap signaling complex. these molecules inhibit immune activation [ , [ ] [ ] [ ] , possibly through blocking dap , pi k, and syk association with the trem -dap complex and preventing activation of erk, vav and calcium mobilization [ ] . whether signaling through dap results in activation or inhibition of the immune response seems to be receptorand stimulus-dependent. activation of trem and myeloid dap -associating lectin (mdl ) but not sirpβ enhanced association of the inhibitory ship with dap [ ] . similarly, macrophage colony stimulating factor (mcsf) alone but not mcsf and rankl induced localization of ship to dap [ ] . even different levels of the same stimulus can induce association of trem -dap with different downstream signaling components. for example, peng and colleagues [ ] found that a low dose, but not a high dose of lps resulted in association of dap with dok . this served to dampen the cellular response to lps, as dok deficiency increased downstream signaling components, cytokine production and death of mice administered an otherwise sub-lethal dose of lps. however, at high doses of lps treatment, dok did not associate with dap . ship was also shown to moderate the response of trem -induced proliferation. when ship deficient preosteoclasts were exposed to an activating trem antibody, osteoclast formation was upregulated an additional -fold [ ] . whether dap serves to activate or inhibit the immune response depends on the receptor it is associated with, the stimulus used to activate that receptor and the strength of that stimulus. it may also depend on availability of different downstream signaling components locally at the membrane, their relative expression in the cell [ ] or other environmental factors [ ] . much of the signaling data thus far was performed in cultured osteoclasts, and future studies may find that other cell types use distinct signaling mechanisms. whether trem signaling is activating or inhibitory in the context of disease is also not known, and a greater understanding of the different trem pathways that are relevant in disease will be instructive. interestingly, ship variants also confer risk for ad, and it is thought that these variants result in a change in the transcriptional start site of ship resulting in a protein that lacks its sh domain which is necessary for association with itams and immunoreceptor tyrosine-based inhibitory motifs (itims) [ ] . this suggests that these inhibitory components that associate with trem deserve attention moving forward in understanding immune-related pathways that are important in ad. there is a general consensus that trem likely acts as a homodimer or homomultimer to induce downstream signaling. this is a common mechanism of activation of other receptors with similar structures to trem in which ligand binding induces complex formation and initiates downstream signaling cascades. almost all studies examining trem signaling have provoked trem dimerization using antibody-mediated crosslinking to induce signaling [ ] . however, there is also evidence that trem can associate with other receptors, including plexina [ , ] . other studies are suggestive that trem could also bind to treml [ ] through co-ip experiments or csf r due to their close linkage in network analyses [ ] and the strong commonalities in their downstream pathways [ , ] . while these last two interactions remain to be validated, it is certainly possible that trem also acts through these alternative heteromeric complexes. whether this is regulated by the cell type or context, and whether this is an important role of trem endogenously in the context of ndds, remain to be determined. the biological roles of strem have been controversial. initially strem was postulated to act as a decoy receptor opposing full-length trem function. a soluble version of a closely related trem family member, strem , modeled by a trem -fc fusion protein, competes with its membrane-bound form to block trem signaling [ ] and produces opposing effects on inflammation and survival following lps injection in mice [ ] . soluble trem may similarly act as a decoy receptor to negatively modulate trem signaling [ , ] . in support of this, in vitro a chimeric trem -fc protein used to model soluble trem inhibited osteoclastogenesis, a process that requires trem -dap signaling [ ] . recent studies suggest that strem may have its own biological function. exogenously applied strem was internalized by cultured bone marrow derived macrophages, and promoted survival in cells lacking trem expression [ ] . however, strem failed to rescue phagocytosis in trem deficient bone marrow macrophages in culture [ ] . recent data also demonstrate that treatment of microglial cell lines with trem -fc or a hek-cell produced strem peptide increases survival, in line with full-length trem function [ ] . this study also found that strem strongly induced an inflammatory response in culture models of microglia. these data suggest that there are important biological roles of strem other than acting as a decoy for the full-length trem receptor. the mechanisms underlying strem function are not yet well understood, but interestingly, does not require the presence of full-length trem or its intracellular adaptor dap [ ] . the signaling role of trem ctfs is also starting to be explored. in the absence of γ-secretase activity, membrane-associated trem ctfs have been proposed to either promote trem anti-inflammatory signaling in response to lps [ ] , or impair trem signaling by sequestering dap from interacting with full-length receptors [ ] , reducing dap phosphorylation and downstream plcγ activation [ , ] along with trem -mediated phagocytosis [ ] . production or stabilization of ctfs on the membrane may also provide a point of cross-talk through which trem could modulate the signaling of other dap -associated receptors. it is clear that further study will be necessary to gain insight into how these soluble trem products impact signaling both of trem and other pathways critical to innate immunity. while trem expression and signaling are contextdependent, there are some commonalities in trem function that have been found across the diverse cell types and environments in which it has been studied (fig. ) , one of which is regulating myeloid cell number. the impact of trem on myeloid cell number outside of the context of disease or stimulus is not completely clear. while knocking down trem in primary microglia lead to reduced cell number [ ] , it had no effect on osteoclasts derived from peripheral blood mononuclear cells (pbmc's) from plosl trem e x carriers lacking trem expression [ ] and microglial numbers were the same up to year of age in mouse models lacking trem expression. however, crosslinking trem did promote an increase in osteoclast number in culture [ ] . what is clear is that trem has an effect on increasing myeloid cell number in response to inflammation or disease. trem deficiency was shown to prevent increases in the brain myeloid cell populations in response to traumatic brain injury [ ] , ischemia [ , ] , aging [ ] , and in the initial response to demyelination [ ] , though it did increase the number of cells in a model of sepsis [ ] . trem deficiency also prevented local increases in myeloid cells around plaques in ad. amyloid plaques are typically surrounded by a rapidly recruited [ , ] cluster of activated myeloid cells in ad human brain tissue [ , ] and in ad mouse models [ ] . recent evidence demonstrates reduced myeloid cell accumulation around amyloid plaques in trem hemizygous [ , , ] , and trem - [ , , , , ] and dap -deficient [ ] ad mouse models, as well as in postmortem ad human brain tissue from individuals harboring the trem r h variant [ ] . these data illustrate that trem , and its adaptor protein, dap , are required for myeloid cell accumulation around amyloid plaques. while wang and colleagues [ , ] found that trem deficient ad mice had a decrease in total brain myeloid cells, others found that this was primarily driven by the specific loss of plaque-associated myeloid cells [ , ] . together, this suggests that trem is important for myeloid cell expansion in response to disease. evidence suggests that in various contexts, trem is important for myeloid cell survival, proliferation and chemotaxis, all of which could lead to disease-associated increases in myeloid cell number. trem has been shown in multiple contexts to be important for cell survival. osteoclasts [ ] and bone marrow derived macrophages [ ] from trem deficient mice, and liver cancer [ ] and glioma cell lines [ ] in which trem was knocked down had increased levels of caspase , bcl- -associated x protein (bax), annexin v and tunel positivity, all suggesting that trem deficiency enhanced apoptosis. similarly, primary microglia and the bv microglial cell line with reduced trem expression had decreased survival, along with decreased levels of elements of the survivalrelated wnt/β-catenin pathway [ ] . conversely, trem activation through receptor crosslinking increased survival of monocyte-derived dendritic cells [ ] and osteoclasts [ ] . in culture, microglia derived from trem deficient mice did not show more cell death at baseline, but when levels of csf , an important factor for the maintenance of microglial survival, were reduced, trem deficient microglia were more likely to undergo apoptosis [ ] . this was also found to be the case in bone marrow derived macrophages [ ] . in an ad mouse model, trem deficiency also increased the number of plaque-associated myeloid cells which were tunel+ [ ] . taken together, these studies suggest that trem is protective against apoptosis, especially under stressful cellular conditions. trem may also increase cell number through promoting myeloid cell proliferation. in glioma cell lines [ ] , liver cancer cell lines [ ] , and primary microglia [ ] , reduced levels of trem led to cell cycle arrest. the number of proliferating myeloid cells were also decreased in vivo in response to demyelination [ ] , colonic mucosal injury [ ] and in ad mouse models [ , ] lacking trem expression. while the mechanisms of this regulation of proliferation are not clear, trem deficiency in cultured osteoclast precursors prevented csf -mediated proliferation [ ] , a process also critical for proliferation of many macrophage populations, including brain myeloid cells. it has been suggested that trem may interact with the csf- receptor to mediate these effects. in dendritic cells derived from plosl patient pbmcs expressing q x and v g trem variants, gene expression profiling identified "negative regulation of proliferation" as a genetic pathway which was significantly increased in variant carriers compared to controls [ ] . in addition to being important for directing proliferation of the cells in which it's expressed, trem might also promote a myeloid cell phenotype that directs proliferation of other cells in the surrounding microenvironment. trem is highly upregulated during organogenesis when macrophages release factors to promote proliferation of surrounding cells [ ] , in tumor associated macrophages where analogous macrophagedriven trophic support occurs [ ] , and following cns trauma where myeloid cells serve as an important source of neurotrophic support during tissue repair [ ] . interestingly, trem is strongly upregulated by neural stem cells [ ] and esc-derived oligodendroglial precursors [ ] . a relationship between trem expression and neurogenesis has not yet been explored, but given the influence of trem of proliferation on other cell types, this may warrant further examination. trem may also influence cell differentiation. differentiation was impaired in osteoclasts derived from plosl patients expressing trem variants [ ] and in raw macrophages deficient for trem through a plexina dependent pathway [ ] . however, otero and colleagues [ ] demonstrated that mouse-derived trem deficient preosteoclasts differentiated into osteoclasts faster. though the role of trem in cell differentiation is not completely clear, this step in cell phenotype determination may also contribute to the changes in cell numbers and population observed in the context of trem deficiency. another potential contributor to trem 's role in expanding the myeloid cell population in the context of disease or inflammation is by modulating chemotaxis or migration of these cells. in culture, knocking down trem reduced chemotaxis of glioma cells in a boydon chamber assay in response to serum [ ] , and in the bv microglial cell line in a scratch assay [ ] . microglia from trem deficient brain slices exhibited reduced chemotaxis into co-cultured brain tissue from old or ad mouse models [ ] . in addition, trem deficient mice had fewer microglia migrate to the site of apoptotic neuron injection in the brain and had slower process extension toward a brain laser lesion as measured using two photon microscopy [ ] . conversely, trem crosslinking increased ccr -dependent chemotaxis [ , , ] . trem was also found to be co-enriched with genes involved in purinergic signaling, a key pathway directing microglial chemotaxis in network analyses [ ] , though whether trem regulates p r-receptor mediated chemotaxis has not been examined experimentally. however, others did not see a deficit in chemotaxis in plosl patient-derived osteoclasts [ ] . kiialainen and colleagues [ ] found that pbmc's cultured from patients with plosl-associated trem variants had both up-and down-regulated components of the chemotactic response. it may be that different components of the chemotactic pathway and therefore different types of chemotaxis are differentially regulated by trem . studies have examined the effect of trem on specific chemotactic pathways that are involved in tissue infiltration by myeloid cells. wang and colleagues [ ] found that trem deficient glioma cell lines downregulated cxcl , cxcr , mmp and mmp which are all important in tissue invasion. in network analyses, trem was significantly co-enriched with dock and dock which are involved in tissue transmigration [ ] and mice deficient for trem had reduced leukocyte infiltration following experimental induction of colitis [ ] . there was also decreased neutrophil recruitment to mouse lungs in response to bacterial infection in trem deficient mice [ ] . in vitro, cells lacking trem expression had reduced chemotaxis toward ccl [ ] . mice lacking dap were found to have significantly reduced recruitment of peripheral macrophages in vivo in response to cigarette smoke or intranasal c-c motif chemokine ligand (ccl ) administration, and these dap -chemotactic deficits were found to be rescued by reintroducing a trem -dap fusion construct [ ] . trem deficient mice also had reduced levels of ccl [ ] and fewer peripheral immune cells in their brain following middle cerebral artery occlusion (mcao) [ ] . there was a trend toward a correlation between ccl and strem levels in the csf of human ad patients, which may suggest that trem plays a role in mediating ccl -mediated chemotaxis of cells in the context of ad as well. however, other studies have found no link between trem and monocyte trafficking into inflammatory tissues [ ] . future studies will be necessary to assess which chemotactic pathways are influenced by trem and whether that includes pathways related to peripheral immune cell infiltration into the cns in ndds. a well-characterized function of trem is to enhance phagocytosis. trem is expressed in a subset of myeloid cells within the cns that have high phagocytic capacity [ ] . across numerous in vitro studies, loss of trem results in reduced phagocytosis of a variety of substrates, including apoptotic neurons or neuronal cell lines [ , , , ] , bone [ , ] , bacteria and bacterial products [ , , , ] and lipids [ , ] . conversely, trem activation or overexpression enhanced uptake of these substrates [ , , ] . trem expression correlated with aβ uptake in bv cells in which trem was knocked down or overexpressed [ ] . aβ uptake was also reduced in trem deficient primary microglia [ , ] and in the n microglial cell line expressing a non-functional trem when plated onto brain slices from ad mouse models [ ] . in agreement with these findings, in vivo, trem deficient mice have reduced localization of aβ within cd + phagosomes in ad mouse models [ ] and reduced uptake of deposited aβ three hours after injection into the brain [ ] . together, these findings suggest that trem is important for aβ uptake by brain myeloid cells. however, in culture, trem expression was no longer found to correlate with aβ uptake after pretreatment of cells with lps [ ] . a similar effect was observed in a mouse model of sepsis where injection of myeloid cells overexpressing trem enhanced bacterial phagocytosis and survival, but not if the mice were pretreated with lps [ ] . these findings suggest that the mechanisms of trem -dependent phagocytosis can be modified by other signals in the microenvironment. interestingly, the other modulatory components present in the brain microenvironment change throughout the course of ndds, which could explain some of the differences in trem function at different stages of disease progression. outside of ad, trem is important for clearance of myelin in experimental autoimmune encephalomyelitis (eae) [ ] and peri-infarct tissue in mice following mcao [ ] . however, it does appear that the effect of trem on phagocytosis can be cell type specific. sharif and colleagues [ ] found that bone marrow macrophages derived from trem deficient mice had reduced phagocytosis, but trem deficient alveolar macrophages had increased uptake of bacteria in vitro and in vivo. r h [ ] and r h trem variants had impaired phagocytosis [ ] . this was also true in hek cells transfected with trem -dap fusion constructs expressing r h, t m and y c variants [ ] . interestingly, while all of these variants impaired uptake of polystyrene beads, t m and y c but not r h impaired uptake of e. coli particles [ ] , suggesting that different trem variants could affect recognition of specific phagocytic substrates as well as induce changes in basal phagocytic activity reflected in the fluid phase uptake of beads. more studies will be required to parse out the role of trem in basal phagocytosis and cargo-driven phagocytosis of specific substrates. the mechanism underlying trem -dependent uptake of various substrates is not clear. while transfection of a trem -dap construct into cho cells was shown to be sufficient for uptake of neuro a cells [ ] , it may be that trem does not have to directly bind to its phagocytic substrates, as trem binding to hsp was sufficient to increase phagocytosis of bacteria [ ] . if trem does not directly bind to these substrates, then it must interact with other phagocytic pathways. it is possible that trem impacts fluid-phase phagocytosis rather than cargo driven phagocytosis. trem may also interact with other phagocytic receptors. for example, mertk is essential for the phagocytosis of apoptotic cells [ ] and is upregulated on the same cell population as trem in ad [ ] . network analyses have also shown that trem is co-enriched with genes involved in fcγr and complement-mediated phagocytosis [ ] . in support of an association between trem and fc-dependent phagocytic pathways, stimulating cells lacking trem function with an antibody against the desired phagocytic substrate did increase internalization of the substrate, but did not rescue it back to wt levels [ ] . trem deficient alveolar macrophages were found to increase phagocytosis and this was found to be dependent on the upregulation of first component of complement q (c q) in these cells [ ] , which acts to opsonize phagocytic substrates. it may also be that strem plays a role in binding and directing phagocytosis of substrates by these other pathways, as adam inhibitors reduced strem production and decreased phagocytosis of e. coli [ ] . some have also suggested that these findings may reflect changes in degradation of phagocytic substrates rather than their uptake. forabosco and colleagues [ ] found that genes associated with lysosome activity were co-enriched with trem across the brain and in monocyte-derived macrophages. in plosl patients, there is an accumulation of large cd + myeloid cells, suggesting that phagocytic uptake by these cells may be intact [ ] . in a cuprizone model of demyelination, trem deficiency was found not to impair uptake of myelin debris, but that this debris remained in cells longer than in controls, suggesting that degradation was specifically impaired [ ] . this was also found to be true in trem deficient macrophages which were able to take up bacteria at comparable levels to cells expressing wt trem but were unable to kill and degrade them [ ] . however, jiang and colleagues [ ] found that in primary microglia in which trem was knocked down, aβ degradation was unaffected. together, the exact role of trem in phagocytosis and other means of cellular uptake and degradation of substrates from the microenvironment remain unclear, though it clearly does play an important role in these processes. trem interacts with many other inflammation-related pathways. while trem has been touted as being antiinflammatory, it seems that the interaction between trem and other inflammation related pathways is actually more complex. depending on the precise stimuli, the strength [ ] and duration [ ] over which they are presented, the cell type and the context, trem can play different roles in the inflammatory response. in support of this, network analyses found that trem was co-enriched with both classically pro-and antiinflammatory gene clusters in the brain [ ] . likewise, a microarray analysis of macrophages derived from a plosl patient pbmc's showed components of the inflammatory response and innate immune response were both up-and down-regulated, respectively, relative to controls [ ] . outside of the context of injury or disease, the transcriptional profiles of trem deficient [ ] or overexpressing [ ] myeloid cells compared to controls was fairly similar. it is in the context of disease where trem seems to heavily influence changes in inflammation-related pathways. trem has been classically described as being antiinflammatory and several in vitro and in vivo studies are supportive of an anti-inflammatory role for trem in certain contexts. knocking down trem in cell lines increases levels of proinflammatory mediators such as inos, tnfα, il β and il [ ] in response to apoptotic neuronal membrane components [ ] , tlr ligands [ ] , including lps [ , , , ] and aβ [ ] . a transient knock down of trem in the p s tau model and in the samp model of accelerated aging also increased inflammatory cytokine production [ , ] . trem deficiency also resulted in increased levels of ifnγ, tnfα and inos [ ] following colonic mucosal injury and trem knockdown or antibody-mediated inhibition increased expression of many inflammationrelated cytokines following corneal infection [ ] . moreover, overexpressing trem in cell lines, amyloid [ ] and tau models of ad [ ] reduced levels of these pro-inflammatory transcripts. together, these studies suggest that in some contexts, trem can attenuate inflammatory responses. however, many other studies also support that trem can mediate or amplify inflammatory responses. for instance, trem knockdown impaired ros production [ , ] . trem deficient microglia are more ramified in culture, a morphological signature of reduced activation [ ] . trem deficient ad mouse models have reduced levels of inflammation-related transcripts in both unbiased rna sequencing approaches [ ] and in the genes il β and il in targeted analyses [ , ] . plaque-associated cells in ad mouse models deficient [ ] or haploinsufficient [ ] for trem also had decreased cell soma size, surface area and increased process length, indicative of reduced activation [ ] . recent work using single cell sequencing approaches indicates that trem is required specifically for a second phase of the myeloid cell response in ad which allows cells to fully adopt a neurodegeneration-associated phenotype [ ] . this may be true in diverse disease contexts as pro-inflammatory cytokine levels were also reduced in trem deficient mice following traumatic brain injury [ ] , ischemia [ ] , lung infection [ , ] and demyelination [ ] , where trem deficient brain myeloid cells exhibited a less activated morphology [ ] . conversely, activation of trem in a macrophage cell line increased no release [ ] , agonizing trem following spinal nerve transection increased tnfα and il β [ ] and overexpression of trem increased expression of il , tnfα and mcp in mouse adipose tissue [ ] . because these studies examined gene expression in whole tissue, it is not clear whether these changes are due to changes in immune cell phenotype or alteration in cell number in the affected tissues. however, taken together, these findings clearly indicate that trem can also promote inflammatory responses in certain contexts. this body of data strongly opposes the often-cited descriptor of trem as an anti-inflammatory receptor. future studies will be required to delineate the molecular and environmental determinants that govern how trem contributes to the inflammatory response in different contexts. while the number of studies have been limited, trem variants associated with ndds also seem to have mixed effects on inflammatory responses. the r h variant impaired inflammatory responses in bv cells [ ] , yet r h carriers with ad had increased expression of genes related to inflammatory pathways compared to non-carriers [ ] . a variant within intron of trem , which is prevalent in african american individuals, was found to be significantly associated with levels of c-reactive protein (crp), a systemic marker of inflammation, whose expression is primarily driven by il and il β [ ] . however, it is not clear whether this represents a direct relationship between this trem variant and systemic inflammation. in addition to impacting the inflammatory responses of myeloid cells, trem also seems to be able to indirectly feedback onto the inflammatory response in other cells within the microenvironment, including astrocytes. astrocytosis, measured by glial fibrillary acid protein (gfap) levels, was reduced across all stages of pathology examined in trem deficient ad mouse models [ , ] , in areas of active demyelination [ ] and trended toward a reduction in gfap area in mice following ischemia [ ] . however, gfap levels were unchanged in trem deficient mice at acute and chronic time points following traumatic brain injury [ ] , suggesting that trem must work in tandem with context-dependent signals to alter astrocyte activation. one of the characteristic features of plosl is astrocytosis [ ] and in a plosl patient with a trem variant, gfap levels were significantly increased in frontal lobe tissue [ ] . this suggests that trem can play multiple roles in regulating astrocyte activation depending on the precise context. while regulating cell number, phagocytosis and inflammation are the best studied roles for trem , other studies have suggested additional roles for the receptor, such as regulation of synaptic pruning and monitoring of synaptic function [ ] . because of the cross-talk between trem and complement pathways and a clear role of trem and complement in phagocytosis in disease [ ] , it would be of interest to assess whether trem influences synaptic function by modulating synaptic pruning, either normally during development or aberrantly in the context of ndds. others have suggested that, due to the close apposition of trem + cells to oligodendrocyte precursors during development, they may support their function [ ] . trem has also been shown to be important for angiogenesis following stroke [ ] . because of trem 's proposed lipid-related ligands, and the strong links between lipid metabolism and ndds, it would not be surprising if trem also played roles in this pathway. in support of this, lipid metabolism was the most strongly altered pathway in trem deficient mouse brains following cuprizone-induced demyelination [ ] . how trem affects these normal functions within the brain has not been studied, but may represent important future areas of investigation. outside of the brain, studies have proposed additional roles for the trem receptor. trem has been proposed to play a role in adaptive immunity. myeloid cells expressing higher levels of trem were able to increase t cell proliferation better than those expressing lower trem levels [ ] . trem was also found to be coenriched with genes related to adaptive immunity in gene network analyses [ ] . however, others have found that activating trem through crosslinking did not upregulate molecules involved in antigen presentation [ , ] , suggesting that trem -mediated stimulation of adaptive immune responses may be indirect or require additional environmental factors. trem also seems to be important for cell maturation [ ] and in particular multinucleation of osteoclasts [ , , ] . it is not yet known how trem might mediate these additional functions. the observed changes in trem expression, signaling and function with disease-associated genetic variants ultimately translate to changes in ndd pathology. many studies have focused on how trem and diseaseassociated variants impact ad-related pathologies. studies examining loss of trem function in amyloid mouse models initially appeared to support contradictory conclusions. some groups found that trem deficiency reduced [ , ] while others found that it increased [ ] amyloid pathology. however, recent evidence has harmonized these results by demonstrating that trem deficiency has a changing role throughout ad progression, reducing amyloid pathology early but increasing it at later stages of disease [ ] . studies overexpressing trem in ad mouse models also found a temporal effect of this overexpression, which reduced pathology early in disease progression [ ] but no effect at a later time point [ ] . this is supported by korvatska and colleagues [ ] who demonstrate accelerated disease progression in r h carriers compared to non-carriers with ad. this may also explain discrepancies in human studies of r h carriers who found no association between r h carriers and non-carriers in amyloid deposition [ ] and others who found that r h carriers had significantly more plaques compared with noncarriers [ ] . this changing role for trem throughout progression of amyloid pathology may also reflect a dynamic role for myeloid cells themselves. as hickman and el khoury [ ] posit, these brain myeloid cells may be protective early through clearance of aβ, but detrimental later in disease progression when they enhance the inflammatory response without being effective phagocytes. alternatively, it could reflect how trem impacts the phenotype and abundance of distinct myeloid cell subsets, or perhaps other microenvironmental cues which change trem downstream signaling to favor alternative pathways. while mouse models so far have recapitulated several aspects of trem localization and function observed in human brain tissue, it is worth noting that there is a caveat to studying trem in mouse models of ad with psen mutations since γ-secretase is also important for trem ctf cleavage [ ] . it is not clear whether changes in γ-secretase activity would be likely to greatly alter trem function in vivo, but this should be a consideration when interpreting these studies. interestingly, there also appears to be a difference in the mechanism by which trem affects pathology early and late in disease progression. early, trem deficiency decreases the number of plaques, while later in disease progression, increase in pathology is instead driven by increased plaque size [ ] . while it's not clear exactly how trem could modulate aβ proteostasis early in disease progression to impact plaque number. trem was shown to impact app processing in a genome-wide sirna screen [ ] , though since it is not neuronally expressed, this would likely occur through indirectly altering neuronal phenotype. conversely, later in disease, the association of myeloid cells with plaques has been proposed to limit plaque growth by forming an insulated microenvironment or barrier [ , ] . trem deficiency appears to impede the formation of this barrier, and in doing so, cause a shift from compact to diffuse plaques [ ] . trem has also been studied in ad in the context of modifying neuritic dystrophy. several studies have found increased neuritic dystrophy around plaques in trem deficient mice [ , ] and in human r h carriers with ad [ ] . trem overexpression in -month but not -month-old app/ps mice had increased levels of synaptophysin, suggesting that enhanced trem expression may protect against aβ-driven synapse loss [ , ] . one possible mechanism for this lies in the larger, more diffuse plaques with high soluble aβ affinity [ ] observed in trem deficient mice late in disease progression [ ] . the relative toxicity of soluble aβ is well documented, including its roles in blocking longterm potentiation [ ] and inducing tau hyperphosphorylation and aggregation [ , ] . together, these data suggest that functional trem is necessary for microglial clustering around amyloid plaques and may thereby form a barrier around plaques which limits neuritic dystrophy. however, not all data support a protective role of plaque-associated myeloid cells on ad pathology. microglia can serve as synaptotoxic agents in ad through complement-mediated synaptic pruning [ ] . in this way, the loss of plaque-associated myeloid cells due to trem deficiency could be beneficial. others suggest that it may not be that more dystrophic neurites are formed around plaques in trem deficient mice and r h carriers, but that trem deficient myeloid cells are not as effective at clearing them [ ] . further evidence will be required to assess the formation and clearance of dystrophic neurites across stages of ad in the context of trem deficiency or trem variants to assess these possible mechanisms. the impact of trem on tau pathology in ad has also been examined. the effect of trem on phosphorylated tau (p-tau) accumulation in dystrophic neurites in ad is not clear, with some studies showing an increase [ , ] and others showing a decrease [ ] in hyperphosphorylated tau markers surrounding plaques in trem deficient amyloid mouse models of ad. these different outcomes are likely related to disease progression dependent effects on the amyloid pathology driving this accumulation. less work has been done in tau models of ad, but overexpressing trem under the cd b promoter in the p s tau model of ad resulted in reduced hyperphosphorylated tau levels, coordinate with a decrease in activation of two of the known tau kinases, cyclin dependent kinase (cdk ) and gsk β [ ] . opposite effects were observed in p s mice in which trem was knocked down [ ] . in humans, trem protein levels in the temporal cortex of ad patients correlated with tangle score and paired helical filament (phf) levels [ ] and strem levels in csf are correlated with csf tau levels early in clinical ad progression [ ] , suggesting an important relationship between trem and tau pathology in humans. r h patients had higher levels of csf p-tau [ , ] , and a variant located upstream of trem was associated with increased tau pathology in the brain [ ] . together, these findings suggest that trem variants may also have an impact on tau-related pathologies in ad, though the mechanisms governing this association are less clear. studies have also examined how trem and its variants impact neuronal and tissue loss and cognition in ad. trem protein levels in the temporal cortex of ad patients were positively correlated with cleaved caspase and negatively correlated with the presynaptic marker snap [ ] , suggestive that loss of trem could impact synapse pathology. there was also a significant reduction in neurons in layer v of the cortex in trem deficient amyloid models of ad [ ] and a substantial rescue of neuronal loss when trem was overexpressed in amyloid [ ] and tau [ ] ad mouse models. these changes in neuron number also correlated with a rescue in behavioral deficits [ , ] . interestingly, however, trem expression levels on peripheral monocytes correlated with lower mmse [ ] and moca scores as well as reduced gray matter volume [ ] . r h variant carriers with ad also had reduced gray matter volume [ ] in the temporal cortex and hippocampus [ ] . there was also a trend toward a reduction in hippocampal volume [ ] and significant decreases in other brain regions [ ] in r h carriers even in the absence of clinical ad. though no changes in cognitive function were reported in middle-aged r h carriers [ ] , the variant did correlate with cognitive deficits in older adults with r h variants [ ] . this was also true in healthy individuals heterozygous for nhd variants [ ] . these findings suggest that trem variants may have direct effects on neuronal loss, even in the absence of ad pathology. although not as extensively studied as in ad, the impact of trem on other ndd-related pathologies has also been assessed. plosl patients have severe white matter dystrophy [ ] and oligodendrocytes that survive in plosl patient white matter express markers of cell stress [ ] suggesting a role for trem deficiency in white matter degeneration. in cuprizone-mediated demyelination models, trem deficiency impaired recovery and increased levels of axonal degeneration markers [ , ] . in addition, injecting mice with trem transduced myeloid cell precursors prevented eae-induced demyelination and ameliorated motor phenotypes [ ] . in addition to affecting oligodendrocyte survival and recovery following demyelination, trem variants in plosl have also elucidated other roles of trem in ndds. plosl patients often experience seizures [ , ] , resulting in excitotoxicity. one patient with a predicted loss-of-function trem plosl mutation had a reduction in many synaptic components, including nine gaba receptor subunits, which could play a role in mediating this enhanced excitability [ ] . however, the mechanism underlying this phenomenon is not well understood and requires further study. the effect of trem on inflammation-related pathologies trem has also been shown to modify tissue loss and behavior in several neuroinflammatory contexts. trem deficiency reduced hippocampal volume loss and improved some behavioral outcomes at chronic time points following traumatic brain injury [ ] . in contrast, trem deficient mice had increased infarct volume in one mcao model [ ] , though no change in another study [ ] . treatment with a trem agonist induced pain behavior in mice in a dap -dependent manner even in the absence of nerve injury [ ] . outside of the brain, trem deficiency results in increased body weight and glucose and insulin intolerance in mice fed a high fat diet [ ] . trem variant carriers may also have increased risk of systemic infection [ ] and trem deficiency is detrimental in the context of bacterial infection [ , , , ] . overall, in the brain and the periphery, despite great advances in assessing how trem alters pathology, there is still no clear picture of how trem mediates these diverse functional impacts across inflammatory and disease contexts. this will require a greater understanding of trem expression, signaling and function and how these features change in the context of pathology. while these studies do point toward some common mechanisms by which trem might modify aspects of pathology relevant to multiple ndds, it is clear that the role of trem in ndds is not simple. since the identification of ndd-associated trem variants, and the detection of strem in the csf and plasma of ad patients, there has been much excitement about how this may translate into immune-related ndd biomarkers and therapeutics. elevated levels of strem were first detected in the csf of patients with multiple sclerosis (ms) and other inflammatory neurologic diseases [ ] and were found to be significantly elevated in ms patients [ ] . these findings served as an impetus to examine whether csf strem might also be changed in ad patients. groups have reported elevations [ ] , reductions [ ] or non-significant changes [ ] in csf strem in ad cohorts not stratified by disease stage (table ) . however, as shown in table , studies that did divide subjects by stage of disease progression, csf strem levels were found to be significantly higher in patients with ad-related mild cognitive impairment [ , , ] and mild dementia [ , ] compared to controls and ad cases [ ] . cross-sectional studies in patients with dominantly inherited ad confirmed a significant increase in csf strem starting years before expected onset of clinical dementia, but found no significant differences between ad patients and controls beyond years after symptom onset [ ] . together, these studies suggest a specific elevation in csf strem levels in the early symptomatic stages of ad. interestingly, strem was also found to be significantly elevated specifically in early stages of als pathology before returning to baseline at late stages of disease [ ] . while it's not clear whether these disease-stage dependent effects are due to a common mechanism, it will be interesting to see whether this pattern continues to be consistent across ndds. the detection and reported changes with disease progression in csf levels of strem have raised questions of its origin and biological meaning in health and disease. in ad patients, csf strem was not associated with changes in csf aβ [ , , , ] but positively correlated with csf biomarkers total tau [ , ] and phosphorylated tau [ , , ] , including in cross-sectional cohorts with dominantly inherited ad [ ] . several groups have proposed that csf strem levels may signify microglial activation in response to ad-related pathology [ , , , , ] . evidence of csf strem positively correlating with glial protein ykl- in csf [ , ] , another proposed ad immune biomarker, in addition to immunosuppressive agents causing a reduction in csf strem levels [ ] are consistent with this theory. changes in strem appear to be independent of apoe status [ ] [ ] [ ] . further work assessing how strem generation changes with microglial phenotype will be needed to definitively validate strem as an indicator of microglial activation. a recent clinical study proposes a neuroprotective role for strem , reporting higher gray matter volume in areas susceptible to ad pathology for mild cognitive impairment (mci) and ad patients with high csf strem levels [ ] though this correlation was not found in all studies. higher levels of csf strem at late stages of als also correlated with longer survival [ ] . these findings, in combination with the positive correlation between csf tau and strem , could indicate that elevated strem production occurs as a protective response to neurodegeneration, though there is no definitive consensus yet as to the biological significance of strem . the current data on strem illustrate limitations for its use as an ndd biomarker. several ndds including ms, ad and ftd, are associated with elevated strem in csf [ , ] , suggestive of a common innate immune mechanism in these distinct pathologies. it has also been suggested as a biomarker for welding fume exposure [ ] and, as discussed above, can be regulated in many other inflammation-related contexts. this lack of disease and context specificity in strem changes raises concerns for its utility as a diagnostic tool for ad. recent data show csf strem levels are altered differentially throughout ad progression. these observations raise questions about its utility as a diagnostic readout for ad disease status in diverse neurologic cohorts, though does suggest that strem levels could be helpful in identifying stage of ad in coordination with other biomarkers [ ] . moreover, heslegrave and colleagues [ ] acknowledged that strem levels in ad patients and controls, while significantly different, substantially overlap, thereby further limiting its diagnostic utility. transcriptome-based studies found dysregulation of several innate immune genes in blood from ad patients [ ] , which led others to assess whether trem expression might also be changed in blood. while studies did find a correlation between trem expression on blood monocytes and an ad diagnosis [ ] , strem in plasma was shown to not correlate with csf strem levels [ ] and plasma samples yielded non-significant differences in strem of ad and ftd patients compared to healthy controls [ , ] . while strem alone does have clear limitations as a biomarker, it may have a potential application as part of a biomarker panel to assess the immune response in ad and other neurodegenerative diseases. in addition to its potential as a biomarker, many have suggested that trem -directed therapeutics may prove to be a novel target for ndds. there are several factors to consider in developing trem therapeutics. first, while trem variants confer as strong a risk for developing ad as one copy of the apoe allele, the minor allele frequency of trem variants are substantially lower, with less than % for trem to approximately % for apoe [ , ] . thus, though some have suggested that therapeutics might want to restore wt trem function in these variant carriers as a potential therapeutic, correcting trem variants are not likely to be a broadly applicable therapeutic approach. rather, studying trem variants that confer risk for ndds will illuminate components of the immune response centrally important in immune modulation of pathology, and serve as a prerequisite to developing targeted immune-directed therapeutics. so far, the field has identified potential changing roles for immune cell function throughout progression of ad, and possibly identified a key role for peripherally derived immune cells in ad pathology, which would greatly aid in therapeutically targeting the immune cells relevant to ad pathology. common functions of trem have been identified across multiple ndds which suggest therapeutic targets could be relevant to multiple disease contexts. however, we have not found a simple explanation for what trem does across cell types and contexts. based on its disease progression dependent effects, it does not appear that simply activating or inhibiting trem would be beneficial even in the context of ad. there may also be sex-dependent effects of trem , as some [ ] but not all [ ] have shown differences in strem levels in csf between male and female subjects. likewise, a trem variant was associated with markers of systemic inflammation specifically in women not men, and was hormone-independent [ ] . with the lack of strong biomarkers to stage ndds and the variability in clinical progression among patients, it is not likely that increasing or decreasing trem will be the universal solution to ndd pathologies. rather, understanding when, where and how trem is working is more likely to provide insights into immune function that can be modulated throughout disease progression. however, the emphasis on understanding trem in ndds began just years ago and we still have a long way to go to understand trem 's expression, signaling, function and effects on these various pathologies. it will also be essential to start to dissect how the diverse array of trem variants result in ndd risk. while this understanding of trem variants may not directly translate into trem -directed biomarkers or therapeutics at this time, the insight into how the immune system actively participates in ndd pathology promises to provide many avenues for a new class of immune-directed therapeutic targets for ndds. genetics of alzheimer's disease evidence for novel susceptibility genes for late-onset alzheimer's disease from a genome-wide association study of putative functional variants alzheimer's disease risk genes and mechanisms of disease pathogenesis targeting microglia for the treatment of alzheimer's disease trem variants in alzheimer's disease variant of trem associated with the risk of alzheimer's disease genetic variations underlying alzheimer's disease: evidence from genome-wide association studies and beyond variant trem as risk factor for alzheimer's disease essential role of the microglial triggering receptor expressed on myeloid cells- (trem ) for central nervous tissue immune homeostasis how neuroinflammation contributes to neurodegeneration trem and the neuroimmunology of alzheimer's disease dominantly inherited alzheimer n. early changes in csf strem in dominantly inherited alzheimer's disease occur after amyloid deposition and neuronal injury trem promotes microglial survival by activating wnt/β-catenin pathway soluble trem induces inflammatory responses and enhances microglial survival mutation analysis of the ms a and trem gene clusters in a case-control alzheimer's disease data set investigating the role of rare heterozygous trem variants in alzheimer's disease and frontotemporal dementia genetic determinants of disease progression in alzheimer's disease mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype dap /trem deficiency results in impaired osteoclast differentiation and osteoporotic features a lipid metabolic disease-"membranous lipodystrophy"-an autopsy case demonstrating numerous peculiar membrane-structures composed of compound lipid in bone and bone marrow and various adipose tissues neuropsychiatric and genetic aspects of a new hereditary disease characterized by progressive dementia and lipomembranous polycystic osteodysplasia nasu-hakola disease: the first case reported by nasu and review variable expression of microglial dap and trem genes in nasu-hakola disease nasu-hakola disease (polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy -plosl): a dementia associated with bone cystic lesions. from clinical to genetic and molecular aspects immunohistochemical characterization of microglia in nasu-hakola disease brains polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (plosl): a new report of an italian woman and review of the literature a case of nasu-hakola disease without fractures or consanguinity diagnosed using exome sequencing and treated with sodium valproate nasu-hakola disease with a splicing mutation of trem in a japanese family absence of trem polymorphisms in patients with alzheimer's disease and frontotemporal lobar degeneration the genetic causes of basal ganglia calcification, dementia, and bone cysts dap and trem an italian family affected by nasu-hakola disease with a novel genetic mutation in the trem gene targeted sequencing approach to identify genetic mutations in nasu-hakola disease a novel mutation in trem gene causing nasu-hakola disease and review of the literature french res network ff-a. trem mutations are rare in a french cohort of patients with frontotemporal dementia homozygous trem mutation in a family with atypical frontotemporal dementia using exome sequencing to reveal mutations in trem presenting as a frontotemporal dementia-like syndrome without bone involvement variants in triggering receptor expressed on myeloid cells are associated with both behavioral variant frontotemporal lobar degeneration and alzheimer's disease heterozygous trem mutations in frontotemporal dementia investigation of the role of rare trem variants in frontotemporal dementia subtypes trem in neurodegeneration: evidence for association of the p.r h variant with frontotemporal dementia and parkinson's disease r h trem variant increases risk of typical early-onset alzheimer's disease but not of prion or frontotemporal dementia the role of trem r h as a risk factor for alzheimer's disease, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, and parkinson's disease movement disorders and genetics in frontotemporal dementia lipomembranous polycystic osteodysplasia (brain, bone, and fat disease): a genetic cause of presenile dementia alzheimer's dis neuroimaging i. gene-based rare allele analysis identified a risk gene of alzheimer's disease the rare trem r h variant exerts only a modest effect on alzheimer disease risk trem and neurodegenerative disease rare trem variants associated with alzheimer's disease display reduced cell surface expression more evidence for association of a rare trem mutation (r h) with alzheimer's disease risk trem analysis and increased risk of alzheimer's disease coding variants in trem increase risk for alzheimer's disease trem is associated with the risk of alzheimer's disease in spanish population missense variant in treml protects against alzheimer's disease assessing the role of the trem p.r h variant as a risk factor for alzheimer's disease and frontotemporal dementia assessment of trem rs association with alzheimer's disease in a population-based sample: the cache county study triggering receptor expressed on myeloid cells variant is rare in late-onset alzheimer's disease in han chinese individuals the triggering receptor expressed on myeloid cells (trem ) is associated with enhanced inflammation, neuropathological lesions and increased risk for alzheimer's dementia a rare coding variant in trem increases risk for alzheimer's disease in han chinese association study of the trem gene and identification of a novel variant in exon in iranian patients with late-onset alzheimer's disease r h variant of trem associated with alzheimer disease in a large late-onset family clinical, genetic, and neuropathological study trem and neurodegenerative disease a trem variant alters the accumulation of alzheimer-related amyloid pathology frontobasal gray matter loss is associated with the trem p.r h variant trem variant p.r h as a risk factor for sporadic amyotrophic lateral sclerosis convergent genetic and expression datasets highlight trem in parkinson's disease susceptibility trem rare variant p.r h is not associated with parkinson's disease evaluating pathogenic dementia variants in posterior cortical atrophy assessment of trem rs association with parkinson's disease and multiple system atrophy in a chinese population trem r h variant and risk of essential tremor: a cross-sectional international multicenter study no evidence for shared etiology in two demyelinative disorders, ms and plosl ariffin r: a case of trem mutation presenting with features of progressive non-fluent aphasia and without bone involvement trem p.r h substitution is not associated with dementia with lewy bodies trems in alzheimer's disease: genetic and clinical investigations genetic analysis of trem variants in chinese han patients with sporadic parkinson's disease association study of trem polymorphism rs with leucoaraiosis or parkinson's disease in the han chinese population assessment of trem rs association with amyotrophic lateral sclerosis in a chinese population triggering receptor expressed on myeloid cells variants are rare in parkinson's disease in a han chinese cohort association study of trem polymorphism rs with late-onset alzheimer's disease in chinese han population investigation of trem , pld , and unc c variants in patients with alzheimer's disease from mainland china lack of genetic association between trem and late-onset alzheimer's disease in a japanese population lack of genetic association between trem and alzheimer's disease in east asian population: a systematic review and meta-analysis trem is associated with increased risk for alzheimer's disease in african americans trem : a new risk factor for alzheimer's disease cd modulates trem : convergence of alzheimer loci cerebrospinal fluid soluble trem is higher in alzheimer disease and associated with mutation status opposing effects of progranulin deficiency on amyloid and tau pathologies via microglial tyrobp network tyrobp genetic variants in earlyonset alzheimer's disease integrated systems approach identifies genetic nodes and networks in late-onset alzheimer's disease osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in dap -deficient mice deficiency of tyrobp, an adaptor protein for trem and c receptors, is neuroprotective in a mouse model of early alzheimer's pathology mutations in the colony stimulating factor receptor (csf r) gene cause hereditary diffuse leukoencephalopathy with spheroids meta-analysis of , individuals identifies new susceptibility loci for alzheimer's disease trem binds to apolipoproteins, including apoe and clu/apoj, and thereby facilitates uptake of amyloid-beta by microglia apolipoprotein e is a ligand for triggering receptor expressed on myeloid cells (trem ) the triggering receptor expressed on myeloid cells binds apolipoprotein e the role of apolipoprotein e in alzheimer's disease triggering receptor expressed on myeloid cells deficiency alters acute macrophage distribution and improves recovery after traumatic brain injury differential detection of impact site versus rotational site injury by magnetic resonance imaging and microglial morphology in an unrestrained mild closed head injury model chronic consumption of a western diet induces robust glial activation in aging mice and in a mouse model of alzheimer's disease insights into trem biology by network analysis of human brain gene expression data the human trem gene cluster at p . encodes both activating and inhibitory single igv domain receptors and includes nkp trem and trem-like receptors in inflammation and disease trems in the immune system and beyond the active enhancer network operated by liganded rxr supports angiogenic activity in macrophages opposing roles of the triggering receptor expressed on myeloid cells and triggering receptor expressed on myeloid cells-like transcript in microglia activation missense variant in treml protects against alzheimer's disease a candidate regulatory variant at the trem gene cluster associates with decreased alzheimer's disease risk and increased treml and trem brain gene expression no association of trem rs and trem rs with alzheimer disease reply genome-wide association and population genetic analysis of c-reactive protein in african american and hispanic american women a comprehensive profile of chip-seq-based pu. /spi target genes in microglia rnasequencing reveals transcriptional up-regulation of trem in response to bexarotene treatment expression of the phagocytosis-essential protein trem is down-regulated by an aluminum-induced mirna- a in a murine microglial cell line divergent neuroinflammatory regulation of microglial trem expression and involvement of nf-κb e proteins regulate osteoclast maturation and survival regulation of itam adaptor molecules and their receptors by inhibition of calcineurin-nfat signalling during late stage osteoclast differentiation microrna- a-mediated down-regulation of the microglial-enriched triggering receptor and phagocytosis-sensor trem in age-related macular degeneration deficits in the mirna- a-regulated endogenous trem phagocytosis sensor-receptor in alzheimer's disease (ad); an update regulation of trem expression by an nf-kappa b-sensitive mirna- a aluminum-induced amyloidogenesis and impairment in the clearance of amyloid peptides from the central nervous system in alzheimer's disease over-expressed pathogenic mirnas in alzheimer's disease (ad) and prion disease (prd) drive deficits in trem -mediated a beta peptide clearance trem upregulation correlates with -hydroxymethycytosine enrichment in alzheimer's disease hippocampus increased dna methylation near trem is consistently seen in the superior temporal gyrus in alzheimer's disease brain dna methylation changes at trem intron and trem mrna expression in patients with alzheimer's disease in vivo evidence of enhanced dimethylation of histone h k on upregulated genes in adipose tissue of diabetic db/db mice genome-wide promoter analysis of histone modifications in human monocyte-derived antigen presenting cells inhibition of rank expression and osteoclastogenesis by tlrs and ifngamma in human osteoclast precursors increased expression of trem in peripheral blood of alzheimer's disease patients triggering receptor expressed on myeloid cells (trem ) promotes adipogenesis and diet-induced obesity impaired differentiation of osteoclasts in trem- -deficient individuals mutations in trem lead to pure early-onset dementia without bone cysts expression and processing analyses of wild type and p.r h trem variant in alzheimer's disease brains disease-associated mutations of trem alter the processing of n-linked oligosaccharides in the golgi apparatus molecular characterization of antigen-peptide pulsed dendritic cells: immature dendritic cells develop a distinct molecular profile when pulsed with antigen peptide transcript profiles of dendritic cells of plosl patients link demyelinating cns disorders with abnormalities in pathways of actin bundling and immune response a dap -mediated pathway regulates expression of cc chemokine receptor and maturation of human dendritic cells characterisation and trophic functions of murine embryonic macrophages based upon the use of a csf r-egfp transgene reporter empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the tlr / agonist pam( )csk( ) (blp) clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells- trem governs kupffer cell activation and explains belr genetic resistance to malaria liver stage infection dap is required for macrophage recruitment to the lung in response to cigarette smoke and chemotaxis toward ccl inflammatory response in rat lungs with recurrent exposure to welding fumes: a transcriptomic approach trem , a dap -associated receptor, regulates osteoclast differentiation and function alzheimer's disease-associated trem variants exhibit either decreased or increased ligand-dependent activation trem -mediated early microglial response limits diffusion and toxicity of amyloid plaques trem mrna expression in leukocytes is increased in alzheimer's disease and schizophrenia monocytes following knockdown of dap , a causative gene for nasu-hakola disease higher peripheral trem mrna expression levels are related to cognitive deficits and alzheimer's disease and amnestic mci trem- promotes macrophage survival and lung disease after respiratory viral infection different mechanisms of apolipoprotein e isoform-dependent modulation of prostaglandin e- production and triggering receptor expressed on myeloid cells (trem ) expression after innate immune activation of microglia analysis of the host transcriptome from demyelinating spinal cord of murine coronavirus-infected mice dap and trem , molecules involved in innate immunity and neurodegeneration, are coexpressed in the cns a role for trem ligands in the phagocytosis of apoptotic neuronal cells by microglia developmental regulation of trem and dap expression in the murine cns: implications for nasu-hakola disease triggering receptor expressed on myeloid cells- is involved in prion-induced microglial activation but does not contribute to prion pathogenesis in mouse brains distribution and signaling of trem /dap , the receptor system mutated in human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy dementia upregulation of trem ameliorates neuropathology and rescues spatial cognitive impairment in a transgenic mouse model of alzheimer's disease dual induction of trem and tolerancerelated transcript, tmem b, in amyloid transgenic mice: implications for vaccine-based therapies for alzheimer's disease trem -transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis triggering receptor expressed on myeloid cells (trem ) deficiency attenuates phagocytic activities of microglia and exacerbates ischemic damage in experimental stroke heterogeneous expression of the triggering receptor expressed on myeloid cells- on adult murine microglia trem deficiency eliminates trem (+) inflammatory macrophages and ameliorates pathology in alzheimer's disease mouse models colony-stimulating factor receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain trem protein expression changes correlate with alzheimer's disease neurodegenerative pathologies in post-mortem temporal cortices differential modulation of trem protein during postnatal brain development in mice microrna- a-mediated down-regulation of the microglial-enriched triggering receptor and phagocytosis-sensor trem in age-related macular degeneration dj- deficiency triggers microglia sensitivity dopamine toward a pro-inflammatory phenotype that is attenuated by rasagiline cutting edge: trem- attenuates macrophage activation silencing of triggering receptor expressed on myeloid cells- enhances the inflammatory responses of alveolar macrophages to lipopolysaccharide triggering receptor expressed on myeloid cells- fine-tunes inflammatory responses in murine gram-negative sepsis enhanced inhibitory effects of a novel cpg motif on osteoclast differentiation via trem- down-regulation mitochondrial lysates induce inflammation and alzheimer's disease-relevant changes in microglial and neuronal cells the triggering receptor expressed on myeloid cells inhibits complement component q effector mechanisms and exerts detrimental effects during pneumococcal pneumonia vasoactive intestinal peptide re-balances trem- /trem- ratio in acute lung injury intratracheal instillation of single-wall carbon nanotubes in the rat lung induces time-dependent changes in gene expression microarray-based analysis of the lung recovery process after stainless-steel welding fume exposure in sprague-dawley rats impact of cigarette smoke on the human and mouse lungs: a gene-expression comparison study differential regulation of dap and molecules associated with dap during host responses to mycobacterial infection adipose tissue transcriptome changes during obesity development in female dogs triggering receptor expressed on myeloid cells- protects against polymicrobial sepsis by enhancing bacterial clearance the immunoreceptor tyrosine-based activation motif (itam) -related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints trem- promotes host resistance against pseudomonas aeruginosa infection by suppressing corneal inflammation via a pi k/akt signaling pathway overexpression of trem enhances glioma cell proliferation and invasion: a therapeutic target in human glioma whole genome expression profiling in chewing-tobacco-associated oral cancers: a pilot study diagnostic marker signature for esophageal cancer from transcriptome analysis trem sirna inhibits cell proliferation of human liver cancer cell lines polyethylene particles stimulate expression of itamrelated molecules in peri-implant tissues and when stimulating osteoclastogenesis in vitro gene expression profile of the bone microenvironment in human fragility fracture bone efficient colonic mucosal wound repair requires trem signaling bacterial sensor triggering receptor expressed on myeloid cells- regulates the mucosal inflammatory response molecular characteristics of high-dose melphalan associated oral mucositis in patients with multiple myeloma: a gene expression study on human mucosa th -m immunity in lesions of muscular sarcoidosis and macrophagic myofasciitis attenuated inflammatory response in triggering receptor expressed on myeloid cells (trem ) knock-out mice following stroke trem /dap signal elicits proinflammatory response in microglia and exacerbates neuropathic pain systemic inflammation modulates fc receptor expression on microglia during chronic neurodegeneration trem sustains microglial expansion during aging and response to demyelination astrocyte-targeted production of interleukin- reduces astroglial and microglial activation in the cuprizone demyelination model: implications for myelin clearance and oligodendrocyte maturation identification of soluble trem- in the cerebrospinal fluid and its association with multiple sclerosis and cns inflammation t cells specifically targeted to amyloid plaques enhance plaque clearance in a mouse model of alzheimer's disease neuroprotective effect of trem- in aging and alzheimer's disease model triggering receptor expressed on myeloid cells knockdown exacerbates aging-related neuroinflammation and cognitive deficiency in senescence-accelerated mouse prone mice strem cerebrospinal fluid levels are a potential biomarker for microglia activity in early-stage alzheimer's disease and associate with neuronal injury markers cerebrospinal fluid soluble trem in aging and alzheimer's disease elevated trem mrna expression in leukocytes in schizophrenia but not major depressive disorder changes in the expression of genes related to neuroinflammation over the course of sporadic alzheimer's disease progression: cx cl , trem , and ppar gamma transcriptomics and mechanistic elucidation of alzheimer's disease risk genes in the brain and in vitro models neocortical and hippocampal trem protein levels during the progression of alzheimer's disease chf (csp- ) induces microglia alternative activation in plaque-free tg mice and primary glial cultures exposed to beta-amyloid viral gene transfer of apps alpha rescues synaptic failure in an alzheimer's disease mouse model a genome-wide gene-expression analysis and database in transgenic mice during development of amyloid or tau pathology trem is upregulated in amyloid plaque-associated microglia in aged app transgenic mice nuclear receptors license phagocytosis by trem (+) myeloid cells in mouse models of alzheimer's disease upregulation of trem ameliorates neuropathology and rescues spatial cognitive impairment in a transgenic mouse model of alzheimer's disease dark microglia: a new phenotype predominantly associated with pathological states silencing of trem exacerbates tau pathology, neurodegenerative changes, and spatial learning deficits in p s tau transgenic mice replacement of brain-resident myeloid cells does not alter cerebral amyloid-beta deposition in mouse models of alzheimer's disease immune hyperreactivity of aβ plaque-associated microglia in alzheimer's disease trem haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy chronic toxoplasma gondii infection enhances beta-amyloid phagocytosis and clearance by recruited monocytes acute injury in the peripheral nervous system triggers an alternative macrophage response trem lipid sensing sustains the microglial response in an alzheimer's disease model neurodegenerative disease mutations in trem reveal a functional surface and distinct loss-of-function mechanisms the alzheimer's disease-associated r hvariant of trem has an altered glycosylation pattern and protein stability trem mutations implicated in neurodegeneration impair cell surface transport and phagocytosis evidence of trem variant associated with triple risk of alzheimer's disease preparation, crystallization, and preliminary crystallographic analysis of wildtype and mutant human trem- ectodomains linked to neurodegenerative and inflammatory diseases mycobacterium bovis bcg cell wall-specific differentially expressed genes identified by differential display and cdna subtraction in human macrophages transcriptomics and mechanistic elucidation of alzheimer's disease risk genes in the brain and in vitro models sequential proteolytic processing of the triggering receptor expressed on myeloid cells- (trem ) protein by ectodomain shedding and gamma-secretase-dependent intramembranous cleavage expression and processing analyses of wild type and p.r h trem variant in alzheimer's disease brains functional involvement of gamma-secretase in signaling of the triggering receptor expressed on myeloid cells- (trem ) metalloproteinases shed trem- ectodomain from lipopolysaccharide-stimulated human monocytes a soluble form of the triggering receptor expressed on myeloid cells- modulates the inflammatory response in murine sepsis production of soluble triggering receptor expressed on myeloid cells by lipopolysaccharide-stimulated human neutrophils involves de novo protein synthesis tlt- s, alternative transcripts of triggering receptor expressed on myeloid cell-like transcript- (tlt- ), inhibits the triggering receptor expressed on myeloid cell- (trem- )-mediated signaling pathway during osteoclastogenesis a split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein trem in live cells triggering receptor expressed in myeloid cells (trem ) trafficking in microglial cells: continuous shuttling to and from the plasma membrane regulated by cell stimulation microglial beclin regulates retromer trafficking and phagocytosis and is impaired in alzheimer's disease vps -dependent recycling of trem regulates microglial function the trem receptor family and signal integration campylobacter jejuni targets immunoglobulin-like receptor lmir -o-sulfo-beta-d-galactose moiety of endogenous sulfoglycolipids is a potential ligand for immunoglobulin-like receptor lmir pattern recognition by trem- : binding of anionic ligands trem- (triggering receptor expressed on myeloid cells ) is a phagocytic receptor for bacteria the innate immune response to salmonella enterica serovar typhimurium by macrophages is dependent on trem -dap pertussis toxin targets the innate immunity through dap , fcrgamma, and myd adaptor proteins evidence for tlr and fcr gamma-card activation by cholera toxin b subunit and its direct bindings to trem and lmir receptors the surface-exposed chaperone, hsp , is an agonist of the microglial trem receptor lectins as pattern recognition molecules: the effects of epitope density in innate immunity trem -ligand interactions in health and disease cutting edge: inhibition of tlr and fcr responses in macrophages by triggering receptor expressed on myeloid cells (trem)- and dap trem- , triggering receptor expressed on myeloid cell- , negatively regulates tlr responses in dendritic cells specific lipid recognition is a general feature of cd and trem molecules the alzheimer's disease risk factors apolipoprotein e and trem are linked in a receptor signaling pathway cloning and characterization of a novel mouse myeloid dap -associated receptor family the expanding roles of itam adapters fcrgamma and dap in myeloid cells trem haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased immunoreceptor dap bearing a tyrosine-based activation motif is involved in activating nk cells trem -and dap -dependent activation of pi k requires dap and is inhibited by ship trem and beta-catenin regulate bone homeostasis by controlling the rate of osteoclastogenesis the linker for activation of b cells (lab)/non-t cell activation linker (ntal) regulates triggering receptor expressed on myeloid cells (trem)- signaling and macrophage inflammatory responses independently of the linker for activation of t cells trem- promotes macrophagemediated eradication of pseudomonas aeruginosa via a pi k/akt pathway inflammation, microglia and alzheimer's disease microglial activatory (immunoreceptor tyrosinebased activation motif)-and inhibitory (immunoreceptor tyrosine-based inhibition motif)-signaling receptors for recognition of the neuronal glycocalyx you say itam and i say itim, let's call the whole thing off: the ambiguity of immunoreceptor signalling monophosphorylation of cd a and cd b itam motifs initiates a ship- phosphatase-mediated inhibitory signaling cascade required for b cell anergy the src homology domaincontaining inositol -phosphatase negatively regulates fcgamma receptormediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors the sh -containing ′-inositol phosphatase (ship) is tyrosine phosphorylated after fcγ receptor clustering in monocytes a physical interaction between the adaptor proteins dok and dap is required to inhibit lipopolysaccharide signaling in macrophages environmental factors determine dap deficiency to either enhance or suppress immunopathogenic processes genetics ignite focus on microglial inflammation in alzheimer's disease plexin-a and its interaction with dap in immune responses and bone homeostasis ddr (discoidin domain receptor ) suppresses osteoclastogenesis and is a potential therapeutic target in osteoporosis sporadic adult-onset leucodystrophy with axonal spheroids and pigmented glia with no mutations in the known targeted genes trem- ligand expression on platelets enhances neutrophil activation trem- amplifies inflammation and is a crucial mediator of septic shock contribution of nuclear factor of activated t cells c to the transcriptional control of immunoreceptor osteoclast-associated receptor but not triggering receptor expressed by myeloid cells- during osteoclastogenesis trem -deficiency reduces the efficacy of immunotherapeutic amyloid clearance dap stabilizes the c-terminal fragment of the triggering receptor expressed on myeloid cells- (trem ) and protects against lps-induced pro-inflammatory response dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of alzheimer's disease relationship of microglia and astrocytes to amyloid deposits of alzheimer disease the complex of microglial cells and amyloid star in three-dimensional reconstruction microglial response to amyloid plaques in appsw transgenic mice altered microglial response to a beta plaques in appps - mice heterozygous for trem disease progression-dependent effects of trem deficiency in a mouse model of alzheimer's disease trem regulates microglial cell activation in response to demyelination in vivo innate immunity: the missing link in neuroprotection and neurodegeneration? microglial activation mediates host neuronal survival induced by neural stem cells immunomodulation by transplanted human embryonic stem cell-derived oligodendroglial progenitors in experimental autoimmune encephalomyelitis trem deficiency impairs chemotaxis and microglial responses to neuronal injury the signaling adapter protein dap regulates multinucleation during osteoclast development biology of the tam receptors trem modifies microglial phenotype and provides neuroprotection in p s tau transgenic mice a unique microglia type associated with restricting development of alzheimer's disease do glia drive synaptic and cognitive impairment in disease? complement and microglia mediate early synapse loss in alzheimer mouse models clearance of tissue debris by trem -transduced myeloid cells promotes recovery of experimental autoimmune encephalomyelitis trem overexpression has no improvement on neuropathology and cognitive impairment in aging appswe/ps de mice pathway-based analysis of genome-wide sirna screens reveals the regulatory landscape of app processing microglia constitute a barrier that prevents neurotoxic protofibrillar abeta hotspots around plaques naturally secreted oligomers of amyloid protein potently inhibit hippocampal long-term potentiation in vivo molecular mechanisms of amyloid oligomers toxicity tau pathogenesis is promoted by abeta - but not abeta - the effect of increased genetic risk for alzheimer's disease on hippocampal and amygdala volume investigation of triggering receptor expressed on myeloid cells variant in the wisconsin registry for alzheimer's prevention neuropsychological tests and functional nuclear neuroimaging provide evidence of subclinical impairment in nasu-hakola disease heterozygotes lc , an autophagosome marker, is expressed on oligodendrocytes in nasu-hakola disease brains a japanese case with nasu-hakola disease of dap gene mutation exhibiting precuneus hypoperfusion the genetic causes of basal ganglia calcification, dementia, and bone cysts: dap and trem trem regulates microglia activation in response to cns demyelination soluble trem- in cerebrospinal fluid from patients with multiple sclerosis treated with natalizumab or mitoxantrone increased cerebrospinal fluid soluble trem concentration in alzheimer's disease in vivo differential brain clearance and catabolism of monomeric and oligomeric alzheimer's aβ protein cerebrospinal fluid strem levels are associated with gray matter volume increases and reduced diffusivity in early alzheimer's disease a data-driven approach links microglia to pathology and prognosis in amyotrophic lateral sclerosis the apoe ε genotype modulates csf ykl- levels and their structural brain correlates in the continuum of alzheimer's disease but not those of strem csf strem : marking the tipping point between preclinical ad and dementia? perturbation of the transcriptome: implications of the innate immune system in alzheimer's disease csf biomarkers and incipient alzheimer disease in patients with mild cognitive impairment a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells trem variants and risk of alzheimer's disease: a metaanalysis a novel compound heterozygous mutation in trem found in a turkish frontotemporal dementia-like family we thank erin g reed-geaghan for providing her insight and critical feedback. her contributions substantially improved the conceptual organization and clarity of the manuscript. this work was supported by the alzheimer's association (bfg- - , to gel), nia grant rf ag (to gel), nia grant r ag (to gel), nia national research service award f ag (to trj). • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -e q mck authors: holgate, stephen t. title: genetic and environmental interaction in allergy and asthma()() date: - - journal: j allergy clin immunol doi: . /s - ( ) - sha: doc_id: cord_uid: e q mck asthma is an inflammatory disorder of the airways involving coordinate up-regulation of t(h) -type cytokines encoded in a cluster on chromosome q( - ) on t cells and inflammatory cells. there is also a requirement for local airway susceptibility factors that, together with t(h) polarization, results in hyperresponsiveness, variable airflow obstruction, and, over time, remodeling of the airway wall. asthma has strong genetic and environmental components that interact both in the induction and subsequent expression of the disease phenotypes. multiple genes are involved and probably interact. whole genome screens are beginning to identify gene-rich regions of special relevance to asthma and atopy, although a novel disease-related gene has yet to be discovered from these. by contrast, there are a plethora of candidate genes whose function in relation to disease pathophysiologic mechanisms and response to treatment are known. two examples are polymorphisms involving il- receptors and the enzymes controlling cysteinyl leukotriene production. abnormal signaling between the epithelium, which is in contact with the environment, and the underlying (myo)fibroblasts and dendritic cells indicating reactivation of the epithelial mesenchymal trophic unit, which is involved in fetal lung development and branching, provide a basis for asthma that encapsulates both t(h) polarization and airway wall remodeling. (j allergy clin immunol ; : - .) asthma is a complex disorder involving a combination of genetic and environmental interactions that culminate in a specific type of inflammation involving mast cells, eosinophils, and macrophages and polarization of t cell-mediated immunity toward enhanced production of cytokines encoded in a cluster on the long arm of chromosome . these include il- , il- , il- , il- , and gm-csf, which orchestrate the isotype switching of b lymphocytes to produce ige, maintain the persistence of t-helper (t h ) lymphocytes with their enhanced capacity to secrete cytokines of the il- gene cluster and recruit and maintain the survival of mast cells, basophils, and eosinophils. in contrast to the many animal models of t h polar-ization in the lung, which tend to be self-limiting, asthma is a chronic disorder characterized by exacerbations and remissions and an underlying bronchial hyperresponsiveness (bhr) of the airways to a wide variety of environmental factors. in established disease a major part of the bhr can be separated from the inflammatory response and most likely represents structural changes to the airways with deposition of matrix proteins throughout the airway wall and "remodeling" of the formed elements including epithelial goblet cell metaplasia, smooth muscle hypertrophy, and an increase in microvessels and nerves. whether these features occur as a consequence of or in parallel with the inflammatory response is not known, but increasingly in asthma it is becoming appreciated that many of these structural elements are themselves altered to produce cytokines, growth factors, and mediators that may contribute to the sustenance of the inflammatory response. although for therapeutic reasons it has become convenient to consider asthma as a single disease entity, this clearly is not the case, with many variants occurring. from a clinical standpoint, a minimal subdivision includes atopic asthma, cough variant asthma, brittle asthma, intrinsic asthma, aspirin-intolerant asthma (aia), and occupational non-ige-dependent asthma. when disease severity and responsiveness to treatment are added to this categorization, the number of variations becomes very large. if these manifestations are controlled by specific genes and environmental interactions, then the true complexity of this disease becomes appreciated. atopy, the propensity to generate ige against common environmental allergens, is the strongest single risk factor that has so far been identified for asthma, increasing the risk in those affected by -to -fold. although atopy and asthma interact, they are not interchangeable. thus, although a high proportion of children and young adults are atopic, even in countries with a high prevalence of atopy (eg, united kingdom, australia, and new zealand) full-blown chronic asthma will develop in only in atopic patients. moreover, in adults, especially at the severe end of the asthma spectrum, atopy diminishes as a risk factor. the phenotype of other chronic inflammatory diseases, such as crohn's disease or psoriasis, presents little diagnostic uncertainty but, in the case of asthma, there is a lack of diagnostic precision. this has led to the use of intermediate phenotypes reflecting asthma and atopy, such as bhr, serum total and allergen-specific ige, skin prick test (spt) positivity, and circulating eosinophil counts. although these provide quantitative measures, bhr, for example, can be assessed in many different ways (eg, histamine or methacholine provocation, exercise or cold air challenge, sulfur dioxide challenge, peak expiratory flow variability, etc), each measure describing different airway characteristics. in addition, there are numerous ways in which an individual can be designated atopic. the danger in using intermediate phenotypes is the assumption that their genetic basis will be the same as that of the disease state. several large epidemiologic studies have established that the presence of asthma corresponds to high serum levels of total ige. , however, when analyzed on a family basis, a tendency to be a high ige producer proved to be only one factor related to the inheritance of asthma susceptibility and, in itself, had a limited ability to predict asthma inheritance. an alternative approach has been to pool multiple measures of asthma (eg, respiratory questionnaire, spt, specific and total ige) to generate summary scores using, for example, principal component regression analysis. this method can also be used to construct a quantitative trait in the form of an asthma score in which the influence of atopy is removed (fig ) . although it has long been known that asthma and related atopic diseases cluster in families, the genetic basis for this has eluded definition. in families a striking association has been shown between asthma in parent and offspring, hay fever in parent and offspring, and eczema in parent and offspring, suggesting that, in addition to genetic factors determining allergen sensitization, there are also important genetic factors that determine the end-organ in which this was expressed, a conclusion drawn from further large epidemiologic studies. , one method that can be used to great effect in complex disorders to determine the relative contribution of genes and the environment is to study the concordance of a trait in monozygotic (mz) and dizygotic (dz) twins, in which the former have identical genotypes and the latter share on average only half their genes. thus a disease that has a strong genetic component will show a higher rate of concordance in mz than in dz twins. in a large survey of twin pairs, concordance rates for asthma, eczema, and hay fever were all substantially higher for mz than for dz twins. this observation has also been shown for bhr, spt responses, and serum total and specific ige. , in a study deliberately designed to disaggregate genetic from environmental effects, both allergic disease and partial phenotypes were compared in mz and dz twins reared together and apart. whether together or separated, mz twins showed a greater concordance than dz twins, indicative of a strong genetic contribution. this has been further strengthened by the findings in a recent , danish twin pairs with use of additive and genetic and nonshared environmental modeling that % of asthma susceptibility was genetic and a substantial part of the variation liability of asthma was the result of environmental factors. segregation analysis is a method for estimating the pattern of inheritance of a disease by observing how it is distributed through family pedigrees. several different modes of inheritance have been proposed for elevated serum total ige, including autosomal recessive, autosomal dominant, and polygenic inheritance. [ ] [ ] [ ] applica- tion of more sophisticated segregation models to asthma and its partial phenotypes have shown that it is unlikely that any single gene predominates, although for physician-diagnosed asthma in us hispanics a major autosomal codominant gene has been proposed. in contrast to single-gene disorders, which are rare, subject to severe mutations and deletions, and exert a large phenotypic effect often independent of environmental factors, complex genetic traits such as asthma and related allergic disorders are common and result from mild mutations in multiple genes each of which has a small effect on the phenotype and requires subtle genegene (epistasis) or gene-environmental interactions for optimal expression. two fundamental approaches are being used to discover susceptibility genes in asthma and atopy: linkage analysis with functional cloning and association analysis for mutations of "candidate" genes thought to be involved in disease pathogenesis. linkage analysis uses family data to follow the transmission of genetic information between or across generations to enable genes to be identified by their position on the genetic map; no prior knowledge of disease pathophysiologic features is required. linkage studies require families usually enriched with the disease, which must be accurately phenotyped. depending on the approach, families can be nuclear families (ie, children and their parents), extended pedigrees, or inbred populations. linkage analysis requires the saturation of the entire human genome at regular intervals with microsatellite markers comprising variable nucleotide tandem repeats whose precise position on the genetic map are known. whole-genome screens use to dinucleotide, trinucleotide, or tetranucleotide markers spaced to cm across the genome. the proximity of a marker to a disease-related gene is estimated by measuring the number of recombination events between them; the closer loci are, the less chance they will be separated at meiosis (fig ) . the significance of linkage is measured by the "lod score" (log of the odds ratio of the likelihood of a hypothesis of linkage to the likelihood of a hypothesis of no linkage). for example, for a lod score of , there is a % chance of a false-positive linkage. a lod score of . is suggestive of linkage, . significant linkage, and . highly significant linkage. this type of parametric linkage analysis requires an a priori knowledge of the mode of inheritance of the disease or its partial phenotype, which, for asthma and allied allergic disorders, is not known. under such circumstances nonparametric linkage analysis or allele sharing methods are more reliable but less powerful. affected sib-pair analysis is most commonly used, which involves studying affected relatives in a pedigree to see how often a particular copy of a chromosomal region is shared identical by descent (ie, inherited from a common ancestor within a pedigree). for example, siblings can share or copies of any locus. if the disease or partial phenotype is linked to a certain marker, then the affected siblings will inherit identical alleles of the marker more frequently than expected by chance alone. when assessed by a chi-square test a lod score of > . is considered significant. association studies are case control studies based on a comparison of unrelated affected and unaffected individuals from a population. although association can be performed for any random dna polymorphisms, they are most meaningful when applied to functionally significant variations in genes having a clear biologic relationship to the trait. positive association may occur under circumstances: ( ) the allele is contributing to the phenotype, ( ) population (racial or ethnic) admixture, and ( ) when alleles at loci are close together (linkage disequilibrium). an alternative type of case control is the transmission disequilibrium test, which avoids the confounding effects of an incorrectly matched control population and uses a trio design of parents and affected sibling. a number of whole-genome scans for asthma have now been completed (table i) . [ ] [ ] [ ] [ ] [ ] although there is an emerging consensus for some chromosomal regions, in different populations there exist many where linkage has not been reproduced. although this may represent true heterogeneity on the basis of racial differences, it is of concern that all the whole genome screens have been conducted on relatively small numbers of families ( - ), whereas it is predicted on statistical grounds that in excess of sibling pairs is needed to provide absolute confidence for linkage to a specific marker. one possible way around this is to pool data from different studies either by meta-analysis or through establishing a voluntary network in which separate groups pool their results in a single analysis. even when a chromosomal region has been narrowed down to - cm, the task of identifying which gene (or genes) within this stretch of dna is contributing to the disease phenotype is daunting. this requires the construction of physical maps of the region with use of overlapping genomic dna clones and techniques such as exon trapping and complementary dna selection to identify genes in a given section of dna. with the availability of integrated genetic and physical maps it is possible to obtain an inventory of genes mapping to the specified genetic interval and scan this for genes of known function (positional-candidate approach). once a specific gene and its mutations have been identified, their expression can be assessed in diseased tissue by a combination of pcr and in situ hybridization and functional studies undertaken by overexpressing and underexpressing the gene in appropriate human cell lines and transgenic mice. at the time of writing, no candidate genes for asthma have yet been reported that have emerged from whole genome screens. a large number of mutations have been described for candidate genes that influence functions relevant to known disease mechanisms in asthma and allied disorders. in all cases, altering the level of expression or function of a specific protein accounts for only a very small component of the disease phenotype. table ii displays candidate genes that have found widest acceptance in different populations. in many cases it is highly likely that several mutations involving a number of components of a particular metabolic or physiologic pathway are needed to fully manifest the effect of a candidate gene. for example, for il- a polymorphism at - involving a c→t substitution in the promoter region on chromosome q results in increased transcription of il- and therefore increased responsiveness to il- (eg, by enhanced ige production). clearly, there may be other functionally active polymorphisms influencing il- secretion. moreover, at least common polymorphisms have also been described in the coding region of the il- receptor gene on chromosome p . , of which lead to amino acid changes of the gene product (fig ) . ) decrease stat activation, whereas others appear to have no effect on il- signalling. the role of glutamine (gln) arginine (arg) is not clear. , , when or more il- r polymorphisms occur together (eg, ser prol and gln arg, stat phosphorylation is reduced and irs / phosphorylation is increased. if the level of il- secretion is also increased (eg, in the - promoter polymorphism), then any genetic effects mediated through the il- r or il- receptor are likely to be magnified. another example of gene-gene interaction may occur in the leukotriene pathway. the cysteinyl leukotrienes ltc , ltd , and lte are critical mediators of airway narrowing, microvascular leakage, mucus secretion, and eosinophilia in bronchial asthma. , the terminal enzyme for cysteinyl leukotriene (cyst-lt) synthesis is ltc synthase encoded on chromosome q . we have shown that in patients with aspirin-intolerant asthma (aia) the expression of this enzyme in mast cells and eosinophils is increased -fold in parallel with enhanced cyst-lt production. this might explain why such patients find particular benefit from being treated with cyst-lt receptor antagonists (ltras) such as montelukast. an a→c polymorphism at the - position of the ltc synthase promoter has been shown to be strongly associated with aia (odds ratio . ). this base substitution creates an extra activator protein- transcription factor binding site, leading to increased enzyme transcription. we have also shown that the a/a or a/c allele is also found more commonly in patients with severe asthma and is accompanied by a greater ability to produce ltc on ex vivo activation of the peripheral blood eosinophils and a tendency toward enhanced responsiveness to the antileukotriene drug zafirlukast. -lipoxygenase ( -lo) is the first committed enzyme in the biosynthesis of leukotrienes. a series of naturally occurring mutations have been discovered within the glucocorticoid-rich transcription factor binding region in the promoter of the -lo gene. these involve deletion of , deletion of , or addition of zinc finger (sp /egr ) binding sites. when transfected, the mutant alleles result in reduced sp /egr binding, and reporter gene transcription is reduced by % and % compared with the wild-type sequences. in patients with asthma, only those possessing mutant -lo alleles were relatively resistant to treatment with the -lo inhibitor abt- ; the mean fev improved by ~ % compared with ~ % for the wild-type and heterozygotes. thus, if there were mutations both of the cysteinyl lt synthase and -lo in favor of greater ltc production, then a subtype of "leukotriene-dependent" asthma can be envisaged. with the recent cloning and expression of the cyst-lt receptor, variation in expression, ligand binding, or transduction signaling may further the appearance of leukotriene dependence. such mechanisms may help explain the responder-nonresponder phenomenon that is emerging in clinical studies of asthma involving ltras. , although asthma and related allergic disorders are closely linked to atopy, there are other important gene-environmental interactions that are of central importance in the clinical expression of disease. in the case of asthma, exposure to oxidant air pollutants (eg, ozone, no x , particulates, tobacco smoke) has been linked to worsening disease. respiratory virus infections, especially those caused by rhinoviruses and coronaviruses, account for the majority of asthma exacerbations in children and adults. , because both these environmental factors operate by increasing the activation of proinflammatory transcription factors in epithelial and inflammatory cells (eg, nuclear factor-κb), genetic factors regulating this cascade are likely to be of considerable importance in determining susceptibility to exacerbations of continuing disease. the complex cellular events that are linked to altered stress responses at the mucosal surface on exposure to pollutants or respiratory viruses are only just being revealed, but key among them is the ability of the epithelium to protect itself from such insults by antioxidant pathways. , these include glutathione peroxidase, glutathione synthase, and xanthine oxidase. in susceptible mice genetic linkage has shown that ozone-induced lung inflammation is directed by genes encoded on chromosome , including the strong candidate tnf-α, a pleiotropic cytokine generated during oxidant-induced cell injury. there are numerous studies linking asthma with a reduced antioxidant status. , because antioxidants may also be provided in the diet (eg, vitamins a, e, and c), asthmatic subjects defective in endogenous antioxidant-generating capacity may be especially susceptible to dietary deficiencies. [ ] [ ] [ ] a similar case can be made for another dietary link to asthma. both in animals and in in vitro studies in humans omega- polyunsaturated fatty acid (pufa)-enriched diets has been shown to reduce allergen-induced inflammatory responses and cyst-lt generation, respectively. , on this basis, it has been suggested that supplementation of the diet with omega- fatty acids in the form of fish oil might protect asthmatic patients by reducing the capacity to generate cyst-lts. unfortunately, clinical trials with diet supplementation have been disappointing both with allergen provocation and in clinical asthma. [ ] [ ] [ ] however, broughton et al have recently shown that only those patients who have high urine secretions of lte responded favorably to omega- fatty pufa dietary supplementation, whereas in those who were low excretors the asthma deteriorated or was unchanged rather than improved. because urinary lte excretion is a measure of the activity of the -lo pathway, , it is possible that responders and nonresponders to this dietary intervention could be determined by prior genotyping. it has been suggested that reduced exposure to bacteria or their products during early infancy is a key factor in programming the immune response toward an allergic phenotype. thus children brought up in livestock farming communities have a substantially lower risk for development of allergies. it is suggested that exposure to bacteria or their products (eg, endotoxin) polarizes the immune response toward a protective t h phenotype by enhanced il- (or il- ) production by professional antigen-presenting cells such as dendritic cells, thereby providing a negative signal for t h polarization by enhanced ifn-γ production. the lower prevalence of allergic disease in formerly communist countries and the subsequent increase that has been observed since reunification has also been cited as supporting the "hygiene hypothesis" of allergic disease, as has the increased urban-to-rural gradient of allergic sensitization in african communities. however, what is notable about the changes in prevalence being observed in the former eastern bloc countries and in africa is that allergic sensitization has increased but asthma and bhr has not. this adds further evidence to the view that asthma and allergy are not equivalent or even linearly related and that local organ-derived factors are important. the recent description of a polymorphism of cd , the endotoxin receptor that is linked to the development of atopy in children, is of great interest and will encourage a search for other candidate gene polymorphisms linked to susceptibility to early life infection, including ifn-γ, natural resistance-associated macrophage protein- , natural resistance-associated macrophage protein- , and the mannose-binding protein. although genetic and environmental factors that operate to direct the immune response toward the t h phenotype are fundamental to understanding the origin and pathogenesis of allergic diseases, genetic and the environmental factors that direct this response to selected organs are of key importance. eczema is a strong predictor of asthma persistence and, in chronic severe disease, there is evidence for gastrointestinal epithelial permeability and inflammation. therefore the epithelium may play a particularly important role in directing the inflammatory and remodeling responses in chronic allergic disease. subepithelial deposition of interstitial (repair) collagens, impaired epithelial proliferative responses to damage, enhanced inflammatory and cytokine growth factor secretion, and evidence for altered subepithelial myofibroblast function in asthma suggest a fundamental abnormality in the epithelial-mesenchymal trophic unit that is involved in fetal lung development. , abnormal signaling between the epithelium and myofibroblast and to dendritic cells provides a basis for asthma that encapsulates both t h polarization and airway wall remodeling. whether this hypothesis can be sustained will require research at the gene by environment interface and its application to disease as it occurs in humans. cytokine networks in the pathogenesis of bronchial asthma: implications for therapy the inflammation-repair cycle in asthma: the pivotal role of the airway epithelium airways remodelling: the chronicity of asthma, quaeritur focus viii cytokine production by cell cultures from bronchial subepithelial myofibroblasts difficult asthma reversing the trend: reducing the prevalence of asthma proportion of asthma attributable to sensitisation to aeroallergens association of asthma with serum ige levels and skin-test reactivity to allergens relation between airway responsiveness and serum ige in children with asthma and in apparently normal children the relationship between parental and children's serum ige and asthma genetic analysis of atopy and asthma as quantitative traits and ordered polychotomies linkage of asthma to markers on chromosome in a sample of families using quantitative phenotype scores the familial incidence of allergic disease genetic risk for asthma, allergic rhinitis and atopic dermatitis allergy in twin pairs total and specific ige (rast) in atopic twins bronchial reactivity pattern in non asthmatic parents of asthmatics atopic disease and immunoglobulin e in twins reared apart and together genetic and environmental influence on asthma: a population-based study of , danish twin pairs a genetic study of immunoglobulin e genetics of total serum ige levels: a regressive model approach to segregation analysis detection of a recessive major gene for high ige levels acting independently of specific response to allergens analytic options for asthma genetics evidence for mendelian inheritance of serum ige levels in hispanic and non-hispanic white families genetic dissection of complex traits: guidelines for interpreting and reporting linkage results genetic dissection of complex traits transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (iddm) a genome-wide search for quantitative trait loci underlying asthma collaborative study on the genetics of asthma. a genome-wide search for asthma susceptibility loci in ethnically diverse populations genetic influences of chromosome q -q and q on specific ige responsiveness to common inhaled allergens among african american families: collaborative study on the genetics of asthma genome-wide search for asthma susceptibility loci in a founder population a genome-wide search for linkage to asthma: german asthma genetics group report on the working group on phenotype approaches workshop on genetics of asthma: methodological approaches promoter polymorphisms in the chromosome gene cluster in asthma and atopy common polymorphisms in the coding part of the il- -receptor gene ileu val variant of il- a upregulates ige synthesis and associates with atopic asthma the polymorphisms s p and q r in the interleukin- receptor * gene are associated ith atopy and influence signal transduction effect of allergy associated mutation in human il- ra (q r) on human il- induced signal transduction the association of atopy with a gain of function mutation in the * subunit of the interleukin receptor leukotriene antagonists and synthesis inhibitors: new directions in asthma therapy inflammatory mechanisms in asthma over expression of leukotriene c synthase in bronchial biopsies from patients with aspirin-intolerant asthma drug therapy: treatment of asthma with drugs modifying the leukotriene pathway leukotriene c synthase promoter polymorphism and risk of aspirin-induced asthma variant ltc synthase gene enhance in vitro ltc synthesis and clinical response to zafirlukast, international symposium: aspirin intolerance and related syndromes: a multidisciplinary approach naturally occurring mutations in the human -lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription egr- and sp interact functionally with the -lipoxygenase promoter and its naturally occurring mutants pharmacogenetic association between alox promoter genotype and the response to anti-asthma treatment characterisation of the human cysteinyl leukotriene cys lt receptor oral montelukast, inhaled beclomethasone and placebo for chronic asthma: a randomised control trial panel studies for investigating the acute health effects of air pollution community study of role of viral infections in exacerbations of asthma in school children in the community respiratory viruses and exacerbations of asthma in adults is nk-kb the sensor of oxidative stress? toxicological mechanisms underlying oxidant pollutant induced airway injury linkage analysis of susceptibility to ozone-induced lugn inflammation in inbred mice dietary antioxidant vitamin intake and lung function in the general population a prospective study of diet and adult-onset asthma effect of fresh fruit consumption on lung function and wheeze in children n- polyunsaturated fatty acids and cytokine production in health and disease effect of dietary intake of omega- and omega- fatty acids on severity of asthma in children effect of dietary fish oil supplementation on the antigen-induced late phase response in the skin dietary fish oil effects on seasonal hay fever and asthma in pollen sensitive subjects reduced asthma symptoms with n- fatty acid ingestion are related to -series leukotriene production microbial stimulation as an aetiologic factor in atopic disease prevalence of hay fever and allergic sensitisation in farmer's children and their peers living in the same rural community: scarpol team development of t-cell memory agonist inhalant allergens: risks for the future the epidemiology of childhood asthma the rising trends in asthma and allergic disease polymorphism in the -flanking region of the cd gene is associated with circulating soluble cd levels and with total serum immunoglobulin e the bronchial epithelium as a key regulator of airway inflammation and remodelling in asthma the attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit key: cord- - qdrshz authors: scully, crispian title: respiratory medicine date: - - journal: scully's medical problems in dentistry doi: . /b - - - - . - sha: doc_id: cord_uid: qdrshz ●. upper respiratory infections are commonplace, especially in young people, and are often contagious; ●. lower respiratory infections are often contagious and some are potentially fatal; ●. asthma is common and may be life-threatening; ●. chronic obstructive pulmonary disease is common and disabling; ●. tuberculosis worldwide is an important infection, affecting people with hiv/aids or malnutrition particularly; ●. lung cancer is common and usually has a poor prognosis. • upper respiratory infections are commonplace, especially in young people, and are often contagious the respiratory tract consists of the upper respiratory tract (urtnose, paranasal sinuses, pharynx and larynx; discussed in ch. ) and the lower respiratory tract (lrt): the respiratory airways (trachea, bronchi and bronchioles) and lungs (respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli), discussed in this chapter. protective mechanisms in the respiratory tracts include a mucociliary lining. particles or pathogens are trapped in the mucus and driven by ciliary action (the ciliary elevator) to the pharynx. mucociliary trans port declines with age but any effect on clinical infection has not been proved. lymphoid tissues of the waldeyer ring (adenoids, palatine and lingual tonsils) are important in developing an immune response to pathogens. however, the best respiratory defence mechanism is the cough reflex, the components of which include cough receptors, affer ent nerves, the cough centre, and efferent nerves and effector muscles. impairment of any of these -as may be seen in older patients or those with conditions associated with lowered consciousness (e.g. sedative use and neurological disease) -can weaken protection. dysphagia or impaired oesophageal motility may exacerbate the tendency to aspi rate foreign material. the alveolar defence mechanisms include mac rophages, immunocytes, surfactant, phospholipids, immunoglobulin g (igg), ige, secretory iga, complement components and factor b; many immune defects manifest with recurrent respiratory infections. lung function is vital to gas exchange -the blood absorbs oxygen and releases carbon dioxide. normal gas exchange requires adequate alveolar ventilation, normal ventilation/blood flow relationships and adequate alveolar-capillary membrane surface area. breathing (ven tilation) depends on respiratory drive, which reacts to the respiratory load. this process requires work and results in gas exchange. oxygen is transported in combination with haemoglobin in erythro cytes and a small amount dissolved in plasma. the oxyhaemoglobin dissociation curve is sigmoidal; once the oxygen saturation falls below %, the amount of o transported to the tissues and brain falls rapidly. high temperatures, acidosis, raised co and raised , diphosphoglycerate ( , dpg) levels encourage oxygen offloading, whereas fetal haemoglobin and carboxyhaemoglobin have the con trary effect. chronic hypoxaemia (e.g. at high altitudes) stimulates release of erythropoietin from the kidneys, with a rise in red cell pro duction, and raised , dpg. athletes have abused erythropoietin to gain competitive advantage (ch. ). the most common lrt disorders are asthma and chronic obstructive pulmonary disease (copd). respiratory disorders are common, and are often caused or aggravated by tobacco smoking. they may significantly affect general anaesthesia (ga) and conscious sedation (cs), since they are often a contraindica tion to use of benzodiazepines, opioids, ga agents and other respira tory depressants. impaired gas exchange leads to laboured breathing and can cause significant incapacity. features include cough, sputum production, wheeze, dyspnoea, chest pain, cyanosis, fingerclubbing ( fig. . ) , use of accessory muscles of respiration with indrawing of the intercostal spaces (hyperinflation), and abnormalities in chest shape, movements, respiratory rate and breath sounds. cough may be a feature of any respiratory problem but, if chronic, may herald serious disease -for example, copd, cancer or infec tion such as tuberculosis. mucoid or mucopurulent sputum is often a feature ( fig. . ); purulent sputum indicates acute bronchitis, bronchiectasis or lung abscess. blood (haemoptysis) or bloodstained sputum, though common in acute infections (especially in preexisting copd), bronchiectasis and pulmonary embolism, may herald an even more serious condition -for example, possibly one due to carcinoma or tuberculosis. wheezing is caused by airways obstruction and is a typical sign of asthma or copd. breathlessness (dyspnoea) is distress ing, and may be caused by respiratory or cardiovascular disease, or by anaemia, and is particularly ominous if it persists at rest. excessive resistive load, such as in asthma, copd and cystic fibro sis, impairs airflow. elastic load increases because of, for example, interstitial fibrosis, muscle paralysis and obesity. diagnosis of respiratory disorders is from the clinical features sup ported by imaging (especially chest radiography). spiral computed tomography (ct) can now scan the lungs in a quick - second breathhold and therefore, instead of producing a stack of individual ct slices, which may be misaligned due to patient movement or breathing in between slices, provides highresolution three dimensional images. respiratory function tests can measure individual components of the respiratory process. spirometry is the basic screening test for assess ing mechanical load problems, the quantification involving determi nation of the vital capacity (vc) -slow vital capacity (svc) and/or forced vital capacity (fvc) -and the speed of maximal expiratory flow (mef; fig. . ). in health, about % of a normalsized vc is expelled in second (fev ). the peak flow meter, which measures the peak expiratory flow rate (pefr; the earliest portion of forced expiration), is a simple measure of airflow obstruction, when the fev is a much smaller fraction of the vc. in lung restriction, the diminished vc can be mostly expelled in about second. serial meas urements (e.g. in asthma) provide valuable information about disease progress. the reversibility of airways obstruction is usually assessed by spirometry before and after use of a bronchodilator agent. arterial blood gas analysis yields considerable information about gas exchange efficiency. arterial hypoxaemia in adults is defined as pao below . kpa breathing room air, although it is not usually treated as clinically important unless below kpa, when oxygen saturation will be % or less (table . ). arterial carbon dioxide tension (paco ) is used as an inversely pro portional index of 'effective' alveolar ventilation. hence, a high paco is taken to indicate poor alveolar ventilation. alveolar hypoventila tion (raised paco ) with a normal ph probably represents a primary ventilatory change present long enough for renal mechanisms to compensate, as in chronic ventilatory failure. ventilation/blood flow relationships are most simply assessed by considering the size of the difference between the amounts of oxygen and carbon dioxide in the blood and in the air; the differences are small if the lungs are work ing efficiently. disparity between ventilation/blood flow ratios results in abnormally wide differences -and then alveolar-arterial po and arterial-alveolar pco gradients will be abnormal. alveolar capillary surface area is assessed by measuring the uptake of carbon monoxide -usually abnormal in diffuse interstitial inflam matory and fibrotic processes and in emphysema. assessing bronchial reactivity and the exercise response can help evaluate breathlessness. simple exercise testing provides information about overall fitness and the appropriateness of cardiorespiratory responses. radionuclide lung scanning, blood gas analysis and sputum cytology or culture are sometimes needed in addition. management can include oxygen administration by mask or nasal cannula (figs . and . ) . lrt disorders can cause significant incapacity and are often a con traindication to ga, and even to cs. asthma is common, affecting - % of the overall population; it is on the increase, particularly in childhood, with a frequency of up to % in some highincome countries. asthma usually begins in childhood or early adult life; about half the patients with asthma develop it before age years. bronchial hyperreactivity causes reversible airway obstruction from smooth muscle constriction (bronchospasm), mucosal oedema and mucus hypersecretion. there are two main types, extrinsic (allergic) and intrinsic asthma (table . ). extrinsic (allergic) asthma, the main childhood type, may be pre cipitated by allergens in animal dander, feathers or hair, drugs (e.g. nonsteroidal antiinflammatory drugs [nsaids] and some antibiot ics), food (e.g. eggs, fish, fruit, milk, nuts), house dust (mite allergens) or moulds. patients frequently have or develop other allergic diseases, such as eczema, hay fever and drug sensitivities. extrinsic asthma is associated with ige overproduction on allergen exposure, and release of mast cell mediators (histamine, leukotrienes, prostaglandins, bradykinin and platelet activating factor), which cause bronchospasm and oedema. about % of asthmatic children lose their asthma or improve by adulthood. intrinsic asthma is usually of adult onset and not aller gic, but appears rather to be related to mast cell instability and airway hyperresponsivity. triggers include emotional stress, gastro oesophageal reflux or vagally mediated responses. either type of asthma can be triggered by: infections (especially viral, mycoplasmal or fungal); irritating fumes (e.g. traffic or cigarette smoke); exercise (possibly due to cold air); weather changes; emotional stress; foods (e.g. nuts, shellfish, strawberries or milk) or additives (such as tartrazine); and drugs (e.g. aspirin and other nsaids, beta blockers and angiotensinconverting enzyme inhibitors [aceis]). in wellcontrolled patients with asthma, clinical features may be absent. during an asthmatic episode, symptoms may include dysp noea, cough and paroxysmal expiratory wheeziness with laboured expiration. the frequency and severity of attacks vary widely between individuals (table . ). patients may become distressed, anxious and tachycardic, have reduced chest expansion and be using accessory respiratory muscles to increase their ventilatory effort. nasal polyps are common, especially in aspirinsensitive asthmatics. children with asthma initially suffer from repeated 'colds' with cough, malaise and fever, often at night. asthma is typically diagnosed when the patient has more than one of the following -wheeze, cough, difficulty breathing and chest tightness -particularly if these are frequent and recurrent; are worse at night and in the early morning; occur in response to, or are worse after, exercise or other triggers, such as exposure to pets, cold or damp air, or with emotions or laughter; or occur without an association with colds. there is often: ■ a personal history of atopic disorder ■ a family history of atopic disorder and/or asthma ■ widespread wheeze, heard on chest auscultation ■ a history of improvement in symptoms or lung function in response to adequate therapy. a prolonged asthmatic attack, which is refractory to treatment, may lead to lifethreatening status asthmaticus (persisting for more than hours). failure of the patient to complete a sentence, indrawing of the intercostal muscles, a rapid pulse, a silent chest and signs of exhaustion are suggestive of impending respiratory arrest. diagnosis of asthma is from the clinical history and presentation, based on recognizing a characteristic pattern of episodic symptoms in the absence of an alternative explanation. investigations include a chest radiograph (to exclude other diagnoses, such as a pneumo thorax), spirometry (serial pefr), skin tests and blood examination (usually eosinophilia, raised total ige and specific ige antibody concentrations, which may help identify allergens). occasionally, a histamine or methacholine challenge is used if the diagnosis is unclear. in children with an intermediate probability of asthma, who can perform spirometry and have evidence of airways obstruction, assess the change in fev or pefr in response to an inhaled bronchodilator (reversibility) and/or the response to a trial of treatment for a speci fied period; if there is significant reversibility, or if a treatment trial is beneficial, a diagnosis of asthma is probable. management includes patient education, smoking cessation advice, avoidance of identifiable irritants and allergens, and use of drugs. home use of peak flow meters allows patients to monitor progress and detect any deterioration that may require urgent modification of treatment. treatment should be based on the amount by which peak flow is reduced (a pefr diary should be kept). drugs used for asthma management (table . ) include oxygen, shortacting β agonists (sabas; such as salbutamol), corticosteroids, leukotriene receptor antagonists and omalizumab (a recombinant humanized monoclonal antiige antibody that reduces the antigen specific ige). inhaled longacting β agonists (labas) may be needed ( fig. . ). deaths from asthma are usually a result of failure to recognize dete rioration or reluctance to use corticosteroids. other factors that have been studied include: ■ air pollution -there is an association between air pollution and aggravation of existing asthma ■ allergen avoidance -there is no consistent evidence of benefit ■ breast-feeding -there is evidence of a protective effect in relation to early asthma ■ electrolytes -there is no consistent evidence of benefit ■ fish oils and fatty acid -there is no consistent evidence of benefit ■ house dust mites -measures to reduce the numbers of house dust mites do not affect asthma severity ■ immunotherapy -allergenspecific immunotherapy is beneficial in allergic asthma ■ microbial exposure -there is insufficient evidence to indicate that the use of probiotics in pregnancy reduces the incidence of childhood asthma ■ modified milk formulae -there is no consistent evidence of benefit pets -there are no controlled trials on the benefits of removing pets from the home ■ tobacco -exposure to cigarette smoke adversely affects quality of life, lung function, need for rescue medications and longterm control with inhaled steroids. there is an association between maternal smoking and an increased risk of infant wheeze ■ weight reduction -there is an association between increasing body mass index and symptoms of asthma. elective dental care should be deferred in severe asthmatics until they are in a better phase; this can be advised by the patient's general practitioner. asthmatic patients should be asked to bring their usual medica tion with them when coming for dental treatment. local anaesthe sia (la) is best used; occasional patients may react to the sulphites present as preservatives in vasoconstrictorcontaining la, so it may be better, where possible, to avoid solutions containing vasoconstric tor. adrenaline (epinephrine) may theoretically enhance the risk of arrhythmias with betaagonists and is contraindicated in patients using theophylline, as it may precipitate arrhythmias. relative analgesia with nitrous oxide and oxygen is preferable to intravenous sedation and gives more immediate control. sedatives in general are better avoided as, in an acute asthmatic attack, even ben zodiazepines can precipitate respiratory failure. ga is best avoided, as it may be complicated by hypoxia and hyper capnia, which can cause pulmonary oedema even if cardiac function is normal, and cardiac failure if there is cardiac disease. the risk of post operative lung collapse or pneumothorax is also increased. halothane or, better, enflurane, isoflurane, desflurane and sevoflurane are the preferred anaesthetics, but ketamine may be useful in children. allergy to penicillin may be more frequent in asthmatics. drugs to be avoided, since they may precipitate an asthmatic attack (see later), include those listed in box . . acute asthmatic attacks may also occasionally be precipitated by anxiety; it is important to attempt to lessen fear of dental treatment by gentle handling and reassurance. even routine dental treatment can trigger a clinically significant decline in lung function in approximately % of asthmatics. acute asthmatic attacks are usually selflimiting or respond to the patient's usual medication, such as a betaagonist inhaler, but status asthmaticus is a potentially fatal emergency (ch. ). there may be complications caused by the antiasthmatic drugs (table . ). gastrooesophageal reflux is not uncommon, with occasional tooth erosion. periodontal inflammation is greater in asthmatics than in those without respiratory disease. persons using steroid inhalers may develop oropharyngeal candidosis or, occasionally, angina bullosa haemorrhagica. guidelines on the management of asthma may be found at: http://www.sign.ac.uk/guidelines/fulltext/ /index.html, http:// www.nice.org.uk/guidance/qualitystandards/indevelopment/asthma. jsp and http://www.britthoracic.org.uk/portals/ /guidelines/ asthmaguidelines/qrg % .pdf (all accessed september ). churg-strauss syndrome (css) is a rare, potentially fatal, systemic vasculitis similar to polyarteritis nodosa (pan), characterized by severe asthmalike attacks with peripheral eosinophilia, and intravas cular and extravascular granuloma formation with eosinophil infiltra tion and skin lesions in %. cardiopulmonary involvement is the main cause of death. css is diagnosed if at least of the criteria listed in box . are positive. the year survival of untreated css is %. combination treatment with cyclophosphamide and prednisolone (prednisone) provides a year survival of %. management problems relating to patients with css may include res piratory impairment and corticosteroid treatment (ch. ). chronic obstructive pulmonary disease (copd; chronic obstructive airways disease, coad) is a common, chronic, slowly progressive, irre versible disease (most frequently a combination of chronic bronchitis and emphysema), characterized by breathlessness and wheeze (airways obstruction), cough and sputum. chronic bronchitis is defined as the excessive production of mucus and persistent cough with sputum production, daily for more than months in a year over more than consecutive years. it leads to production of excessive, viscous mucus, which is ineffectively cleared from the airway, obstructs and stag nates, and becomes infected, usually with streptococcus pneumoniae, moraxella catarrhalis and haemophilus influenzae. patchy areas of alveolar collapse can result. emphysema is dilatation of air spaces dis tal to the terminal bronchioles with destruction of alveoli, reducing the alveolar surface area available for respiratory exchange. copd is now the preferred term for conditions with airflow obstruction because of a combination of airway and parenchymal damage; patients were previ ously diagnosed as having chronic bronchitis or emphysema. copd is characterized by airflow obstruction -defined as an fev / fvc ratio reduced to less than . . if fev is % or more, a diagno sis of copd should only be made if there are respiratory symptoms (e.g. dyspnoea or cough). the airflow obstruction is not fully revers ible, does not change significantly over months, and is usually progres sive in the long term. the most important causes of copd include cigarette smoking, environmental pollution, dusts, chemicals or occupational exposures to various substances. exposure to smoke from home cooking or heating fuels may contribute. deficiency of the antiproteolytic enzyme alpha antitrypsin is a rare cause of emphysema. there is often significant airflow obstruction before the person is aware of it and so copd typically remains undiagnosed until patients are in their fifties. differentiation from asthma is important (table . ). a diagnosis of copd should be considered in patients over the age of who have a risk factor (e.g. smoking) and exertional breath lessness, chronic cough, regular sputum production, frequent winter 'bronchitis' or wheeze. clinical judgment is based on history, physical examination, confirmation of airflow obstruction using spirometry (postbronchodilator spirometry) and assessment of the severity of dyspnoea (tables . and . ). copd is characterized by breathlessness and wheeze (airways obstruction), cough and an early morning mucoid sputum production. to investigate symptoms that seem disproportionate to spirometric impairment progressive dyspnoea, low oxygen saturation, carbon dioxide accumu lation (hypercapnia) and metabolic acidosis mean that patients may ultimately become dyspnoeic at rest ('respiratory cripples'), especially when recumbent (orthopnoea), and eventually develop respiratory failure, pulmonary hypertension, right ventricular hypertrophy and rightsided heart failure (cor pulmonale). two clinical patterns of copd are recognized: ■ 'pink puffers' -patients with emphysema who manage to maintain normal blood gases by hyperventilation, and are always breathless but not cyanosed; rather they are pink from vasodilatation ■ 'blue bloaters' -patients with chronic bronchitis who lose their co drive, fail to maintain adequate ventilation and become both hypercapnic and hypoxic with central cyanosis, cor pulmonale and oedema (for these patients, the respiratory drive is from the low po and thus oxygen administration is contraindicated) (table . ). the diagnosis of copd is based upon clinical history and presen tation. investigations include a chest radiograph (which may show hyperinflated lung fields with loss of vascular markings); arterial blood gases (which should be measured if pulse oximetry shows oxygen satu ration less than %); spirometry; and lung function tests. fev is reduced in all cases (fev of less than % signifies severe copd) and the flow-volume curve shows a typical pattern, with reduced flow rates at mid and lowerlung volumes. a ratio of fev :fvc of less than % confirms airways obstruction. patients with copd and their family should be educated about the disease, and about required lifestyle changes and medication. nondrug therapy includes: stopping smoking (nicotine replacement therapy or bupropion may help); exercise by pulmonary rehabilitationof proven benefit; weight loss (improves exercise tolerance); and vaccination (pneumococcal and influenza vaccines). drug therapy includes shortacting bronchodilators (anticholinergic drugs [ipra tropium bromide]) and β agonists (salbutamol) to treat the reversible component of airway disease; corticosteroids (inhaled or systemic); and antibiotics (amoxicillin, trimethoprim or tetracycline). mucolytics, such as carbocisteine, reduce acute exacerbations by almost onethird. longterm oxygen therapy (ltot) reduces mortality. people with stable copd who remain breathless or have exacerba tions, despite using shortacting bronchodilators, should be offered the following as maintenance therapy: ■ if fev is % of predicted or more: use either a longacting β agonist (laba) or longacting muscarinic antagonist (lama). ■ if fev is less than % predicted: either a laba with an inhaled corticosteroid (ics) in a combination inhaler, or a lama. offer a lama in addition to a laba plus ics to people with copd who remain breathless or have exacerbations, despite taking laba plus ics, irrespective of their fev . provide pulmonary rehabilitation for all who need it; noninvasive ventilation (niv) is the treatment of choice for persistent hyper capnic ventilatory failure during exacerbations not responding to medical therapy. the frequency of exacerbations should be reduced by appropriate use of inhaled corticosteroids and bronchodilators, and vaccinations. bronchodilators (shortacting β agonists [saba] and shortacting muscarinic antagonists [sama]) should be the initial empirical treat ment for the relief of breathlessness and exercise limitation. ics have potential adverse effects (including nonfatal pneumonia) in people with copd. offer a oncedaily lama in preference to fourtimes daily sama to people with stable copd who remain breathless or have exacerbations, despite using shortacting bronchodilators as required, and in whom a decision has been made to commence regular maintenance bronchodilator therapy with a muscarinic antagonist (see above). most patients -whatever their age -are able to acquire and main tain an adequate inhaler technique. bronchodilators are usually best administered using a handheld inhaler device (including a spacer device if appropriate). patients with distressing or disabling dyspnoea, despite maximal therapy using inhalers, should be considered for nebulizer therapy. they should be offered a choice between a face mask and a mouth piece to administer their nebulized therapy, unless the drug specifically requires a mouthpiece (for example, anticholinergic drugs). some patients with advanced copd may require maintenance oral corticosteroids when these cannot be withdrawn following an exacer bation. these individuals should be monitored for the development of osteoporosis and given appropriate prophylaxis. theophylline should only be used after a trial of saba and laba, and only to those who are unable to use inhaled therapy, as there is a need to monitor plasma levels and interactions. the dose of theo phylline prescribed should be reduced at the time of an exacerbation if macrolide or fluoroquinolone antibiotics (or other drugs known to interact) are given. there is insufficient evidence to recommend prophylactic antibiotic therapy in the management of stable copd. mucolytic drug therapy should be considered in patients with a chronic cough productive of sputum. if patients remain symptomatic on monotherapy, their treatment should be intensified by combining therapies from different drug classes, such as: ■ β agonist and theophylline ■ anticholinergic and theophylline. inappropriate oxygen therapy in people with copd may depress respiration. ltot is indicated in patients with copd who have a pao of less than . kpa when sta ble, or a pao greater than . kpa and less than kpa when stable, and one of: secondary polycythaemia, nocturnal hypoxaemia (oxygen saturation of arterial blood [sao ] of less than % for more than % of the time), peripheral oedema or pulmonary hypertension. to reap the benefits of ltot, patients should breathe supplemental oxygen for at least hours per day. to ensure that all those eligible for ltot are identified, pulse oximetry should be available in all health care settings. the assessment of patients for ltot should comprise the measurement of arterial blood gases on two occasions at least weeks apart in patients who have a confident diagnosis of copd, who are receiving optimum medical management and whose copd is stable. patients should be warned about the risks of fire and explosion and told not to smoke when using oxygen. ambulatory oxygen therapy should be considered in patients on ltot who wish to continue oxygen therapy outside the home, and who have exercise desaturation, are shown to have an improvement in exercise capacity and/or dyspnoea with oxy gen, and are motivated to use oxygen. adequately treated patients with chronic hypercapnic respiratory failure who have required assisted ventilation during an exacerbation, or who are hypercapnic or acidotic on ltot, should be referred to a specialist centre for consideration of longterm niv. advanced emphysema is occasionally treated with sur gery -excision of large acquired bullae or, rarely, lung transplantation. patients with copd who need dental care can be classified as follows: ■ patients at low risk -experience dyspnoea on effort but have normal blood gas levels. these patients can receive all dental treatment with minor modifications. ■ patients at moderate risk -experience dyspnoea on effort, are chronically treated with bronchodilators or recently with corticosteroids, and pao lowered. a medical consultation is advised to determine the level of control of the disease before any dental treatment. ■ patients at high risk -have symptomatic copd that may be end stage and poorly responsive to treatment. with these patients, a medical consultation is essential before any dental treatment is carried out. patients with copd are best treated in an upright position at midmorning or early afternoon, since they may become increasingly dyspnoeic if laid supine. it may be difficult to use a rubber dam, as some patients are mouthbreathers and not able to tolerate the additional obstruction. la is preferred for dental treatment, but bilateral mandibular or palatal injections should be avoided. patients with copd should be given relative analgesia only if absolutely necessary, and only in hospital after full preoperative assessment. cs with diazepam and midazolam should not be used, as benzodiazepines are respiratory depressants. patients should be given ga only if absolutely necessary, and intravenous barbiturates are contraindicated. secretions reduce airway patency and, if lightly anaesthetized, the patient may cough and contaminate other areas of the lung. postoperative respiratory complications are more prevalent in patients with preexisting lung diseases, especially after prolonged operations and if there has been no preoperative preparation. the most important single factor in preoperative care is cessation of smok ing for at least week preoperatively. respiratory infections must also be eradicated; sputum should first be sent for culture and sensitivity, but antimicrobials such as amoxicillin should be started without await ing results. the medical management of copd should be optimized prior to surgery. the ultimate clinical decision about whether or not to proceed with surgery should rest with a consultant anaesthetist and consultant surgeon, taking account of comorbidities, functional status of the patient and necessity for the surgery. composite assessment tools, such as the american society of anesthesiologists (asa) scoring system, and not just lung function, are the best criteria for the assessment of patients with copd before surgery. those taking corticosteroids should be treated with appropri ate precautions (ch. ). interactions of theophylline with other drugs, such as adrenaline (epinephrine), erythromycin, clindamycin, azithro mycin, clarithromycin or ciprofloxacin, may result in dangerously high levels of theophylline. ipratropium can cause dry mouth. guidelines for the management of copd may be found at: http:// publications.nice.org.uk/chronicobstructivepulmonarydisease cg (accessed september ). respiratory viruses usually spread by touch or airborne transmission and the very small particles ( - . micrometres) can avoid the upper respiratory tract defences and the mucociliary elevator to reach the lung alveoli. a range of viruses can cause lower respiratory tract infections (lrtis ; table . ). some viruses (e.g. influenza and respiratory syncytial) can spread from the upper to the lower respira tory tract via infection of the respiratory epithelium and can lead to bacterial superinfection and pneumonitis (pneumonia). mycoplasmal (atypical) pneumonia and tuberculosis (tb) may be direct infections. epidemics of a potentially fatal severe acute respiratory syndrome (sars) have been caused by a coronavirus that originated in china and spread worldwide; h n bird influenza also arose as an epidemic; and a similar epidemic, but of swine influenza (h n ), emanated from mexico (see later). bacterial infections, such as pneumonia or lung abscess, can also result from material aspirated into the lungs, and are usually unilat eral. those who aspirate more than others have, as a result, more frequent lrti and this is seen in alcohol and other drug abusers, as well as comatose patients. exogenous penetration and contamination of the lung can result from trauma (e.g. a stab wound or road traffic accident) or surgery. entamoeba histolytica can occasionally cause pneumonia -by direct extension from an amoebic liver abscess (table . ). patients with endocarditis, or septic pelvic or jugular thrombo phlebitis, may experience lrti acquired haematogenously and then it is often bilateral. immunocompromised persons (e.g. those with human immunode ficiency virus/acquired immune deficiency syndrome [hiv/aids] and transplant recipients) and people with bronchiectasis or cystic fibrosis are also susceptible to respiratory infections by a range of opportun istic microbes. pneumocystis jiroveci (p. carinii), for example, is a com mon cause of potentially fatal pneumonia in immunocompromised patients -especially those with hiv/aids (chs and ). clinical features of lrti vary according to the part of the respiratory tract mainly affected: ■ bronchiolitis causes rapid respiration, wheezing, fever and dyspnoea -but is restricted mainly to infants. ■ bronchitis causes cough, wheezing and sometimes dyspnoea. ■ pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. antimicrobial therapy is indicated, particularly for pneumonia. antivirals have not been highly effective. oxygen may be needed. pneumococcal vaccine is indicated for older people. the majority of lrtis are severe illnesses, and are contraindications to all but emergency dental treatment. ga is hazardous and absolutely contraindicated. dental treatment should be deferred until recovery, or be limited to pain relief. influenza is mainly a communitybased infection transmitted in house holds and communities. healthcareassociated influenza infections can arise in any healthcare setting, most commonly when influenza is also circulating in the community. influenza is a contagious disease caused by influenza virus types a, b or c. type a has two main subtypes (h n and h n ); it causes most of the widespread influenza epidemics and can occasionally be fatal. type b viruses generally cause regional outbreaks of moderate severity, and type c viruses are of minor significance. a person can spread influenza starting day before they feel sick and for another - days after symptoms start. influenza can be pre vented or ameliorated by vaccination each autumn; this is especially indicated for older people and those with cardiorespiratory disease. influenza attacks virtually the whole respiratory tract; symptoms appear suddenly after - days and include fever, sore throat, nasal congestion, headache, tiredness, dry cough and muscle pains (myalgia). most people recover in - weeks but infection can be lifethreatening, mainly because primary influenzal viral pneumonia can lead to sec ondary bacterial pneumonia or can exacerbate underlying conditions (e.g. pulmonary or cardiac disease). the old and very young, and those with chronic disorders, are more likely to suffer complications, such as pneumonia, bronchitis, sinusitis or otitis media. influenza has also been followed by depression, encephalopathy, myocarditis, myositis, pericarditis, reye syndrome and transverse myelitis. rest, maintenance of fluid intake, analgesics, antipyretics, and avoid ance of alcohol and tobacco help relieve symptoms. aspirin must never be given to children under the age of years who have 'flulike symptoms, and particularly fever, as this can cause reye syndrome. zanamivir (an antiviral that works against influenza types a and b) can shorten the symptoms by approximately day, if treatment is started during the first days of illness. other antiviral drugs include amantadine, oseltamivir and rimantadine; they may be helpful but their use is restricted mainly to immunocompromised persons, since they can cause adverse effects. influenza can be a severe contagious illness so all but emergency den tal treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. influenza type a subtype h n can cause an illness known as 'avian influenza' or 'bird 'flu' in birds, humans and many other animal spe cies. hpai a(h n ) -'highly pathogenic avian influenza virus of type a of subtype h n ' -is the causative agent and is enzootic in many bird populations, especially in southeast asia. it has spread globally and resulted in the deaths of over people and the slaughter of mil lions of chickens. a vaccine that could provide protection (prepandrix) has been cleared for use in the european union. h n is a more recent emergent infection, similar in many respects. swine influenza is common in pigs in the midwestern united states, mexico, canada, south america, europe (including the uk, sweden and italy), kenya, china, taiwan, japan and other parts of eastern asia. transmission of swine influenza virus from pigs to humans is not com mon, but can produce symptoms similar to those of influenza. a outbreak in humans ('swine 'flu') was due to an apparently new strain of h n arising from a reassortment produced from strains of human, avian and swine viruses. it can pass from human to human. antiviral agents such as oseltamivir may help. vaccines are now available. an outbreak of a lifethreatening febrile respiratory infection appeared in , originating from guangdong, china, and was named severe acute respiratory syndrome (sars). caused by a newly recognized coronavirus (sarsassociated coronavirus, sarscov), sars spread via close contact to many countries across the world. according to the world health organization, people worldwide became sick with sars during the course of the first recognized outbreak and died. the incubation period of - days is followed by a high fever (above . °c), malaise, headache and myalgia. some people also experience mild upper respiratory symptoms and, after - days, lower respiratory signs -a dry cough and dyspnoea, potentially progressing to hypox aemia. sars can cause a pneumonia with a mortality approaching %, particularly in older or immunocompromised people. artificial ventilation has been needed in - % of cases. antiviral agents, such as oseltamivir or ribavirin, may help. inactivated vaccines, virally and bacterially vectored vaccines, recombinant protein and dna vaccines, as well as attenuated vaccines, are under development. sars is a severe illness, and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. for all contact with suspect sars patients, careful hand hygiene is important, including handwashing with soap and water; if hands are not visibly soiled, alcoholbased handrubs may be used as an alternative to handwashing. if a suspected sars patient is admitted to hospital, infection control personnel should be notified immediately. infection control measures (www.cdc.gov/ncidod/hip/iso lat/isolat.htm; accessed september ) should include standard precautions (e.g. hand hygiene): healthcare personnel should wear eye protection for all patient contact; contact precautions (e.g. gown and gloves for contact with the patient or their environment); and airborne precautions (e.g. an isolation room with negative pressure relative to the surrounding area and use of an n filtering disposable respirator for persons entering the room). pneumonia is classed as 'primary' if it occurs in a previously healthy individual, and is usually lobar; it is called 'secondary' if it follows some other disorder, such as previous viral respiratory infections, aspir ation of foreign material, lung disease (bronchiectasis or carcinoma), depressed immunity (e.g. alcoholism or immunosuppression), or aspir ation of oral bacteria ( pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. complications can include lung abscess or empyema (pus in pleural cavity). it is important to avoid alcohol and tobacco, but use analgesics and antipyretics to relieve the symptoms. broadspectrum antimicrobi als given promptly and empirically usually include a macrolide (azithromycin, clarithromycin or erythromycin), quinolone (moxiflox acin, gatifloxacin or levofloxacin), or doxycycline for outpatients. for in patients, cefuroxime or ceftriaxone plus a macrolide is used. prophylaxis includes immunization against influenza and pneumococci. pneumonia is a severe illness and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. ventilatorassociated pneumonia (vap) is discussed later. legionellosis is a bacterial respiratory infection caused by one of the family legionellaceae, gramnegative aerobic bacilli, ubiquitous in water and soil but particularly preferring warm aquatic environments. the term legionnaire's disease was coined as a result of an outbreak of the previously unrecognized respiratory disease in an american legion meeting in philadelphia in , but it is now recognized worldwide, many infections being contracted during travel abroad, particularly to spain, turkey and some other mediterranean areas. legionella bacteria can be found in natural freshwater environments, usually in insufficient numbers to cause disease. legionella grow best in warm water, as in hot tubs, cooling towers, hot water tanks, large plumbing systems, or the airconditioning systems of large buildings. though there are over legionellaceae, most infections are caused by legionella pneumophila. disease is contracted by inhalation of contaminated mist or vapour, mainly (approximately %) through aerosolization of infected water in airconditioning systems, hotwater systems, humidifiers, nebulizers, showers and spa pools. outbreaks have mostly been linked to aerosol sources in the community, cruise ships and hotels, with the most likely sources being whirlpool spas, air conditioning units in large buildings, potable (drinking) water systems, and water used for bathing. risk factors include: ■ exposure to: recent travel with an overnight stay outside of the home (outbreaks of travelassociated legionellosis are infrequently identified but more than % of cases are thought to be associated with recent travel) whirlpool spas recent repairs or maintenance work on domestic plumbing ■ systemic illhealth: alcohol use chronic kidney disease diabetes immune defects liver disease malignancy smoking. illness mainly affects males over , smokers, heavy drinkers, older people and the immunocompromised. also vulnerable are travellers, especially middleaged and older tourists, and conference or business groups, possibly because of tiredness or age. many young people have been exposed to infection and become seropositive, but remained healthy. there is no evidence of persontoperson transmission of legionellosis. legionellosis manifests as one of two clinical syndromes (table . ). legionnaire's disease is typically a lobular type of pneumonia, which can be fatal but is fortunately rare; infection can range from discrete patches of inflammation and consolidation to involvement of whole lobes. pontiac fever is milder and usually subsides rapidly, often with out treatment. people who should be tested for legionnaire's disease include those with pneumonia in the following groups: because legionella is commonly found in the environment, clinical isolates are necessary to interpret the findings of an environmental investigation. diagnosis can be by rapid urine molecular testing for l. pneumophila antigen, and culture of respiratory secretions on selective media. sensitivity and specificity of the diagnostic tests are shown in table . . pontiac fever is a selflimited illness; most cases recover within week and few benefit from antibiotic treatment. overall mortality in legionnaire's disease may be as high as %, and over % in older people and up to % in the immunocompromised. erythromycin is standard treatment; cephalosporin is an alternative. legionella species are present in roughly twothirds of potable water samples collected from domestic and institutional taps and drinking fountains, and from a similar percentage of dental units, but water from these dental units often has higher bacterial concentrations (ch. ). there are reports of legionella infections in dental unit water lines, and antibodies and occasionally frank infection demonstrated in dental staff; at least one patient appears to have contracted and died from infection emanating from a dental practice. prevention is crucial, involving (ch. general aspects tuberculosis (tb) , an infection caused by mycobacteria, affects approxi mately onethird of the world's population ( . billion people); it is a major global health problem, some million people dying from it annu ally. tb disproportionately affects the poorest persons in both high income and developing countries. in highincome countries, most human tb arises from mycobacterium tuberculosis, transmitted from person to person through the air. tb usually affects the lungs initially (pulmonary tb) but can also involve brain, kidneys, spine and other parts. from victorian times to about the second world war, mycobacterium bovis infection from infected cows' milk (bovine or btb) was a major cause of morbidity and mortality; it was clinically and pathologically indistin guishable from infection caused by m. tuberculosis. cattletesting and a slaughter programme became compulsory in and, by the s, the incidence of tb in cattle had been substantially reduced. tuberculosis from m. bovis in cows' milk was virtually eliminated in highincome countries by the tuberculin testing of cattle and pasteurization of milk. in the developing world, many cattle still have tb, and btb is still seen. btb has also increased in highincome countries over the last two dec ades and an infection rate of up to % in badgers -and transmission to cattle -may explain this. tb is not spread by touch or by drinking glasses, dishes, sheets or clothing. it is usually transmitted by infected sputum, typically from close contacts such as family members, but is unlikely to be transmit ted between normal social contacts. tb can present an occupational risk to healthcare professionals, including dental staff. one outbreak of drugresistant tb in new york involved at least patients, most of whom contracted tb in one of hospitals; nearly % of the patients were also hivpositive, and most were young males of hispanic or african heritage. tb has been transmitted between pas sengers during longhaul airline flights. the risk of transmitting tb though air circulation is now low because the highefficiency particu late air (hepa) filters on newer commercial aircraft are of the same type as those used in hospital respiratory isolation rooms; indeed, the number of times air is cleaned each hour exceeds the recommendation for hospital isolation rooms. subsaharan africa has the highest rates of active tb per capita, driven primarily by the hiv epidemic. the absolute number of cases is highest in asia, with india and china having the great est burden of disease globally. in the usa and most western european countries, the majority of cases occur in foreignborn residents and recent immigrants from countries in which tubercu losis is endemic. immunocompromised people -such as diabetics and severely immuno deficient patients, like those with hiv/aids (about % of south africans with hiv/aids also have tb) -and patients in prisons or institutions are at risk. tb also mainly affects medically neglected persons, such as vagrants, alcoholics, intravenous drug abusers or older homeless people. the main groups at increased risk for infection therefore include people who are resourcepoor or immunoincompetent, especially: tb in developing countries is particularly widespread and is increasing, the highest rises in incidence being in southeast asia, subsaharan africa and eastern europe. in highincome countries, the incidence is also rising, probably because of worsening social deprivation, homelessness, immigration, hiv infection and intra venous drug abuse. it is now as common in london as in the devel oping world, and is seen especially in immigrants, such as those from the indian subcontinent, africa and south asia. this increase appears to be a result of the development of tb disease in individu als who may have been infected for some time and of new infections acquired in the uk, or as a result of travel to other countries where tb is common. london accounted for the highest proportion of cases in the uk in ( %), followed by the west midlands region ( %); % of these were born outside the uk and mainly originated from south asia and subsaharan africa. in , there was a rise in the number of tb cases compared to , as well as an increase in drug resistance. more information on tb, including statistics, can be found at: http:// www.hpa.org.uk/publications/infectiousdiseases/tuberculosis/ and http://www.tbfacts.org/tbstatistics.html (both accessed september ). initial infection with tb is usually subclinical. about % of those infected develop overt disease; of these, half will manifest within years (primary tb), while the remainder will develop postprimary disease. inhaled mycobacteria may cause subpleural lesions (primary lesion) and lesions in the regional lymph nodes (primary complex). body defences usually localize the mycobacteria, though these remain viable; infected persons are not obviously ill and are unlikely to know they are infected (latent ; table . ). latent tb infection (ltbi) usu ally becomes active only after many years, if body defences become weakened (box . ). however, active tb can develop shortly after mycobacteria enter the body, if body defences are impaired such as in ageing, drug or alcohol abuse, or hiv/aids. also, in massive infec tions, acute active tb can result, typically causing a chronic productive cough, haemoptysis, weight loss, night sweats and fever. erythema nodosum may be associated. extrapulmonary tb is less common; it may appear as glandular involvement in the neck or elsewhere, and is less infectious than pulmonary tb. lymph node tb may lead to lymphadenopathy, caseation of the nodes and pressure symptoms -for example, on the bronchi. postprimary tb follows reactivation of an old primary pulmonary lesion and results in features ranging from a chronic fibrotic lesion to fulminating tuberculous pneumonia. the pulmonary lesions may extend and lead to a pleural effusion. reactivation or progression of primary tb may also result in widespread haematogenous dissemina tion of mycobacteria -'miliary tb'. multiple lesions may involve the central nervous system, bones, joints, and cardiovascular, gastrointes tinal and genitourinary systems. clinical presentation in tb is thus variable, depending on the extent of spread and the organs involved. as it frequently passes unrecog nized for so long, the mortality is high. similar illnesses to tb may also be caused by atypical (nontuber culous) mycobacteria, such as m. avium complex (mac; see below). the diagnosis of tb is suggested by the history and confirmed by physical examination, a massively raised erythrocyte sedimentation rate (esr), positive tuberculin skin tests (tsts; mantoux or heaf test for a delayed hypersensitivity reaction to protein from m. tuberculosis [purified protein derivative; ppd]) and chest imaging. hypersensitivity develops with - weeks of infection and can be detected by conversion of the tst from negative to positive, but tsts are neither % sensi tive nor specific. a positive mantoux reaction indicates previous immu nization (bcg; bacille calmette-guérin -live attenuated m. bovis) or current infection -not necessarily disease. chest radiography may show scarring and hilar lymphadenopathy. computed tomography (ct) may show areas of calcification or highlight a tuberculous abscess. smears and culture of sputum, blood, laryngeal swabs, bronchoalveolar lavage, gastric aspirates or pleural fluid may be tested for mycobacteria. polymerase chain reaction (pcr) techniques have greatly acceler ated the diagnosis and speciation, though ziehl-neelsen, auramine or rhodamine microbial stains are still used. the mycobacteria growth indicator tube (mgit) system gives results as early as - days. blood assay for m. tuberculosis (bamt) may be positive by interferongamma release assay (igra). some % of people over years have a positive igra. the igra can be used in place of (but not in addition to) tst. igras measure the immune reactivity to m. tuberculosis. white blood cells from most persons that have been infected with m. tuberculosis will release interferongamma (ifnγ) when mixed with m. tuberculosis antigens. a positive test result sug gests that m. tuberculosis infection is likely; a negative result suggests that infection is unlikely. latent infection (ltbi) can be diagnosed with either a tuberculin skin test or an igra (more specific). igra gives a result within hours and should be used biological therapy is given, such as for rheumatoid arthritis or inflammatory bowel disease. prior bcg vacci nation does not cause a falsepositive igra test result. more informa tion on the igra is available at: http://www.cdc.gov/tb/publications/ factsheets/testing/igra.htm (accessed september ). active tb is diagnosed by sputum microscopy and culture in liquid medium with subsequent drugsusceptibility testing. nucleic acid people who should be tested for tb include those who have symp toms, those who have had close daytoday contact with active tb disease (family member, friend or coworker), those who have hiv infection or aids, those with lowered immunity, those who are required to for employment or school, and those about to be treated with biological agents. the top priority of tb control programmes is to identify and give complete treatment to all patients with active disease. tb is a notifi able disease and contact tracing is an important aspect of limiting spread. treatment with antibiotics is indicated for people who are sick with tb, those infected but not sick, and those who are close contacts of infectious tb cases. treatment for 'symptomatic sputumpositive' patients, which should be instituted as soon as possible, is combination chemotherapy, usually isoniazid plus rifampicin plus pyrazinamide or ethambutol for months, with continuation of daily isoniazid and rifampicin for a further months. treatment for 'asymptomatic' patients who are believed to have been infected by contacts, but are not unwell, includes isoniazid for months or isoniazid and rifampicin for months. rifapentine is a longacting rifampicin used once weekly. fluoroquinolones (moxifloxacin) may also act against tb. there may be resistance to one or more than one antibiotic. currently, given the potential risk of drugresistant tb being present, treatment is usually started with isoniazid, rifampicin, pyrazinamide and ethambutol (or a quinolone such as gatifloxacin or moxifloxacin) for months, then isoniazid and rifampicin for months. all antituberculous drugs (table . ) have potentially serious adverse effects and require careful monitoring. if patient compliance is considered to be poor, directly observed therapy (dot), where drugs are dispensed by and taken in the presence of a healthcare profes sional, may be indicated. new drugs are on the horizon. immunization using bcg is advocated for schoolchildren, highrisk individuals and healthcare professionals -although its efficacy has been questioned. new vaccines are in development. chemoprophylaxis with isoniazid and rifampicin is indicated in a number of situations (box . ) . tb can become resistant to the drugs used to treat it particularly when the drugs are misused or mismanaged. this may occur, for example, when: in some developing countries, approximately % of cases are multi ple antibioticresistant; this is termed multidrugresistant tuberculosis (mdrtb); in the uk, only a small minority currently fall into this category but the number of cases is increasing. mdrtb is defined as resistance to rifampicin and isoniazid; it may be atypical in presenta tion and the infection disseminates. more than % of people with tb worldwide have mdrtb, and eastern europe has a high prevalence. mdrtb is seen mainly in people with hiv/aids and in hiv/aids and in africans. bedaquiline, is a new antitubercular agent the first active agent against tuberculosis to be registered since . extensively drugresistant tuberculosis (xdrtb) is a rare type of mdrtb, not only resistant to isoniazid and rifampin, but also to any fluoroquinolone and at least one of three injectable secondline drugs (i.e. amikacin, kanamycin, or capreomycin). xdrtb is of special concern for immunocompromised people (e.g. with hiv/aids), who are more likely to develop tb, and have a higher risk of death if they do develop it. xdrtb is most often encountered in people from eastern europe, russia and africa. it has been transmitted in healthcare facilities and is now seen worldwide. it is essentially untreatable, though capreomycin has been used effectively to treat mdrtb in hivpositive individuals. totally drugresistant tb was reported initially in - in india, iran and italy; it is spreading, despite denials, and is most disquieting. chronic ulcers, usually on the tongue dorsum, are the main oral manifestation of tb. they result from coughing of infected sputum from pulmonary tb, including in hivinfected persons with tb, but are rare and such cases (usually middleaged males) may result from neglect of symptoms or default from treatment. occasionally, the diagnosis is made from biopsy of an ulcer after granulomas are seen microscopically. acidfast bacilli are rarely seen in oral biopsies, even with the help of special stains, so unfixed material should also be sent for culture if possible. tuberculous cervical lymphadenopathy is the next most common form of the infection and is particularly com mon among those from south asia. most tb lymphadenitis is pain less, with several enlarged, matted nodes, but systemic symptoms are present only in a minority and only about % have pulmonary mani festations on radiography (fig. . ) . diagnosis relies on tuberculin testing, which can be positive in both tuberculous and non tuberculous mycobacterial cervical lymphadenitis. any person with lymphadenop athy and recent conversion from a negative to positive tuberculin test should be suspected of having mycobacterial infection, and this should prompt biopsy (e.g. fineneedle aspiration biopsy) for culture or histo logical confirmation. pcr will improve diagnosis, as culture must wait - weeks for a result. oral complications of antitubercular therapy are rare, but rifabutin and rifampicin can cause red saliva. pulmonary tb is of high infectivity, as shown by cases of tuber culous infection of extraction sockets and cervical lymphadenitis in patients treated by an infected member of staff at a dental clinic. dental staff who themselves were hivpositive, working in a dental clinic for hivinfected persons in new york, have died from tb con tracted occupationally. transmission of mdrtb between two dental workers may have occurred in an hiv dental clinic. infection control is thus important, so staff with tb are usually precluded from their occupation until treated. management of a patient with tb depends upon the level of poten tial infectivity (table . ) . patients with open pulmonary tb are con tagious, and dental treatment is thus best deferred until the infection has been treated. treatment with appropriate drugs for weeks drasti cally reduces the infectivity of patients with pulmonary tb. if patients with open pulmonary tb must be given dental treatment, special pre cautions should be used to prevent the release of mycobacteria into the air, to remove any that are present and to stop their inhalation by other persons. reduction of splatter and aerosols, by minimizing cough ing and avoiding ultrasonic instruments, and use of a rubber dam, are important. improved ventilation, ultraviolet germicidal light, new masks and personal respirators, and other personal protective devices, such as hepa filters, are indicated ( fig. . ) . mycobacteria are very resistant to disinfectants, so that heat sterilization must be used. la is safe and satisfactory. relative analgesia is contraindicated because of the risk of contamination of the apparatus. ga is also contraindicated for dental treatment because of the risk of contamina tion of the anaesthetic apparatus and because of impaired pulmonary function. aminoglycosides, such as streptomycin, enhance the activity of some neuromuscular blocking drugs and in large doses may alone cause a myasthenic syndrome. possible drug interactions are shown in table . . other factors, such as alcoholism or intravenous drug use (ch. ), hepatitis (ch. ) or hiv disease (ch. ), may also influence dental management. mycobacteria other than tuberculosis (mott) are widely distributed in water, soil, animals and humans, and rarely cause disease. severe mott infections have been seen, however, in individuals predisposed because of defects in the interleukin (il ) and interferongamma (ifngamma) pathways. mycobacterium abscessus, a bacterium found in water, soil and dust, has been known to contaminate medications and products, including medical devices. healthcareassociated m. abscessus can cause a vari ety of infections, usually of the skin, but it can also cause lung infec tions in persons with various chronic lung diseases and is increasingly recognized as an opportunistic pathogen in cystic fibrosis (cf) patients persontoperson transmission of atypical mycobacteria is not important in acquisition of infection, except for skin infections. on rare occasions, mott skin infections have followed tattooing with contaminated tattoo inks. many people become infected with and har bour mott in their respiratory secretions without any symptoms or evidence of disease. individuals with respiratory disease from mott do not readily infect others and, therefore, do not need to be isolated. mott are generally not infectious to others. infection with m. abscessus is usually caused by injections of con taminated substances or by invasive medical procedures employing contaminated equipment or material. infection can also occur after accidental injury where the wound is contaminated by soil. there is very little risk of transmission from person to person. mac complex, m. scrofulaceum and m. kansasii are possible causes of tuberculous cervical lymphadenitis. mac may also infect the lungs (similar to tb), skin or lymph nodes. lung disease is also caused occasionally by m. kansasii, mainly in middleaged and older persons with underlying chronic lung conditions. m. fortuitum and m. chelonae may cause skin and wound infections and abscesses, frequently associated with trauma or surgery. m. marinum may cause 'swimming pool granuloma', a nodular lesion that may ulcerate, usually on an extremity. m. ulcerans may produce chronic ulcerative skin lesions, usually of an extremity. m. abscessus skin infections present with swollen and/ or painful areas that are usually red, warm and tender to the touch, and which can also develop into boils or pustules. other features of m. abscessus infection are fever, chills, muscle aches and malaise. cervical lymphadenitis due to mac, m. scrofulaceum and m. kansasii may affect otherwise healthy young children, most commonly pre school females who have unilateral cervical lymphadenopathy, typically in the submandibular or jugulodigastric nodes, and they may form a 'cold abscess'. mott is the usual cause in children under years but tb is more common in older patients. absence of fever or tuber culosis, a positive tuberculin test and failed response to conventional antimicrobials are highly suggestive of mott, but definitive diagnosis is by smear, culture or pcr of biopsy material obtained by fineneedle aspiration or removal of nodes. treatment is based on results of laboratory testing, which should identify the appropriate antibiotic. preventive treatment of close contacts of persons with disease caused by mott is not needed. most mott are resistant to standard antitubercular medication and, though it is possible that clarithromycin or clofazimine may have some effect, excision of affected nodes is the usual recommended therapy. water from dental units may contain mott species; mycobacterial proliferation in biofilms may explain the extent of this contamination (ch. ). aspiration syndromes are conditions in which foreign substances are inhaled into the lungs and which can have consequences ranging from asphyxia to infection and lung abscess. dental restorations or frag ments of teeth, plaque, gastric contents and other materials may be aspirated, especially if material enters the pharynx, and particularly if the cough reflex is impaired for any reason. most commonly, aspiration syndromes involve oral or gastric contents associated with gastrooesophageal reflux disease (gord), swallowing dysfunction (ch. ), neurological disorders and structural abnormalities, such as a pharyngeal pouch. cricopharyngeal dys function involves cricopharyngeal muscle spasm or achalasia of the superior oesophageal sphincter, and can be seen in infants who have a normal sucking reflex but have incoordination during swallowing, pos sibly secondary to delayed development or cerebral palsy. anatomical disorders, such as cleft palate, pharyngeal pouch, oesophageal atresia, tracheooesophageal fistula, duodenal obstruction or malrotation, and motility disorders, such as achalasia, may have an aspiration risk. infirm older patients are also at risk of aspiration, especially if they are bedbound or have neurological disorders. isolated superior laryngeal nerve damage, vocal cord paralysis, cerebral palsy, muscular dystrophy and riley-day syndrome (familial dysautonomia) are all associated with increased risk of aspiration. ventilatorassociated pneumonia (vap), as defined by the centers for disease control and prevention (cdc), is present when the chest radiograph shows new or progressive infiltrate, consolidation, cavitation or pleural effusion in conjunction with either new onset of purulent sputum or change in character of sputum, and an organism isolated from blood, or the isolation of an aetiological agent from a specimen obtained via suction aspiration through an endotracheal or tracheostomy tube. the major route for acquiring endemic vap is oropharyngeal colo nization by endogenous flora or by exogenously acquired pathogens from intensive care units. vap is the most commonly reported health careacquired infection in patients receiving mechanical ventilation, with prevalence rates consistently in the - % range. mortality rates in vap are at least double those in patients without vap, ranging from % to % when the infection is caused by a multidrugresistant gramnegative pathogen. the healthcare infection control practices advisory committee of the cdc has developed guidelines for the prevention of vap. these include strategies aimed at preventing aspiration of contaminated oral or gastric material (e.g. raising the head of the bed and draining subglottic secretions), and interventions to alter bacterial coloniza tion of stomach (e.g. stress ulcer prophylaxis and selective digestive decontamination) and mouth. oral hygiene, suctioning and the provi sion of moisture to lips and oral mucosa, plus toothbrushing, may be important in prevention of vap. there are also strategies for manag ing ventilator circuits (e.g. replacement of ventilator circuits, use of closed rather than open suction, and use of heat moisture exchange as opposed to heated circuit technology). lung abscess is a localized infection leading to cavitation and necro sis. while some cases result from aspiration of foreign material, most develop from pneumonia caused by infection with staph. aureus or klebsiella pneumoniae. bronchial obstruction by carcinoma is another important cause. symptoms resemble those of suppurative pneumonia. there is a risk of infection spreading locally or leading, via septicaemia, to a brain abscess. diagnosis rests mainly on the chest radiograph, which may sometimes show cavitation or a fluid level. antimicrobial chemotherapy, postural drainage and relief by bronchoscopy of any obstruction are indicated. a wellrecognized cause of lung abscess is inhalation of a tooth or fragment, a restoration or rarely, an endodontic instrument. when undertaking endodontics or cementing restorations, such as inlays or crowns, a rubber dam or other protective device should always be used to avoid the danger of inhalation. lung abscesses may also result from aspiration of oral bacteria, particularly anaerobes, especially in infirm older patients or those who are intubated. the other main dangers in dentistry are with ga, particularly if an inadequate throat pack has been used. patients who inhale tooth frag ments or dental instruments must have chest radiographs (lateral and posteroanterior) and, if necessary, bronchoscopy. loeffler syndrome appears to be an allergic reaction, usually to the parasitic worm ascaris lumbricoides, or drugs such as sulphonamides. it manifests with pulmonary infiltrates (and abnormal chest radio graph) and eosinophilia (eosinophilic pneumonia). the disease usually clears spontaneously. sarcoidosis, so named because skin lesions resembled a sarcoma, is a multisystem granulomatous disorder, seen most commonly in young adult females in northern europe, especially in people of african heritage. the aetiology is unclear but propionibacterium acnes and p. granulosum have been implicated and associations have been reported with exposure to inorganic particles, insecticides, moulds and occupations such as firefighting and metalworking. serum sam ples contain antibodies directed against mycobacterium tuberculosis antigens. sarcoidosis is associated with hladrb and dqb , and a butyrophilinlike (btnl ) gene on chromosome . thelper (th ) cells release il and ifnγ, and augment macrophage tumour necrosis factor alpha (tnfα) release. cd regulatory t cells cause a limited impairment of cellmediated immune responses (partial anergy) but no obvious special susceptibility to infection. sarcoidosis affects the thorax in %, but has protean manifestations and can involve virtually any tissue (table . ). sarcoid most typi cally causes löfgren syndrome (fever, bilateral hilar lymphadenopathy, arthralgia and erythema nodosum, especially around the ankles; figs . and . ). other common presentations may include pulmonary infiltration and impaired respiratory efficiency, with cough and dyspnoea in severe cases, or acute uveitis, which can progress to blindness. susceptibility to lymphomas has been suggested but not confirmed. because of its vague and protean manifestations, sarcoidosis is under diagnosed. in the presence of suggestive clinical features, helpful investigations include: chest radiography (enlarged hilar lymph nodes); raised serum angiotensinconverting enzyme (sace ; table . ) in acute disease (this is insensitive, nonspecific and a poor guide to therapy); positive gallium citrate or gadolinium or positron emis sion tomography (pet) scans; labial salivary gland or transbronchial biopsy (for histological evidence of noncaseating epithelioid cell granulomas) -except in löfgren syndrome, which is a classical clini cal diagnosis. fdeoxyglucose pet is helpful in identifying sites for biopsy. nonspecific findings may include mild anaemia, leukopenia, eosinophilia, hypergammaglobulinaemia, raised esr and low serum albumin. hypercalcaemia is common because of extrarenal produc tion of active vitamin d and can result in renal damage. alkaline phosphatase, 'nucleotidase, lysozyme and adenosine deaminase levels are raised in hepatic sarcoidosis. evidence of impaired delayed hypersensitivity reactions to some antigens may be useful. kveim skin tests are not now used. half the patients with sarcoidosis remit within years and about % remit by years. patients with only minor symptoms usually need no treatment but corticosteroids, sometimes with azathioprine, methotrexate, tetracyclines, hydroxychloroquine, infliximab or etaner cept, are given if there is active organ disease (ocular disease, progres sive lung disease, hypercalcaemia or cerebral involvement). biopsy of the minor salivary glands frequently shows noncaseating granulomas and association with other features of sarcoidosis, par ticularly hilar lymphadenopathy. this is an important diagnostic find ing that may obviate more invasive procedures. sarcoidosis can involve any of the oral tissues but has a predilection for salivary glands. asymptomatic swelling of the parotid glands or cervical nodes, and less frequently the lips, may accompany systemic disease. superficial or deepseated red submucosal nodules may develop intraorally and on the lips. nontender, wellcircumscribed, brownishred or violaceous nodules with superficial ulceration have also been reported. the oral and lip lesions may occasionally precede systemic involvement. there is enlargement of the major salivary glands in about % of cases; some have xerostomia, and the association of salivary and lacri mal gland enlargement with fever and uveitis is known as uveoparotid fever (heerfordt syndrome). salivary swelling may also be seen with out other features of heerfordt syndrome. the salivary gland swellings usually resolve on treatment of sarcoidosis but this may take up to years. facial palsy and other cranial neuropathies may be seen. there is also an association with sjögren syndrome, when ssa and ssb serum autoantibodies are found. rarely there is an association of thyroiditis with addison disease, sjögren syndrome and sarcoidosis (tass syndrome). there is a group of patients who have histologi cal features of sarcoid in one or more sites in the mouth, such as the gingivae, but no systemic manifestations. a few of these patients may ultimately develop other more or less systematized disease but the majority probably have isolated lesions. such cases, where no exog enous cause for the granulomatous reaction can be found, are regarded as having 'sarcoidlike' reactions (orofacial granulomatosis) and treat ment is unnecessary. however, patients should be kept under observa tion for as long as possible. management of patients with systemic sarcoidosis may include con sideration of respiratory impairment, uveitis and visual impairment, renal disease, jaundice or corticosteroid treatment. la is safe and satisfactory. cs is contraindicated if there is any res piratory impairment. ga should only be given in hospital. lung cancer is the most common cancer in highincome countries in males and most frequently affects adult urban cigarettesmokers. bronchogenic carcinoma accounts for % of all primary lung cancer and has also become increasingly common in women (because of increased tobacco use), to the extent that the mortality rate for the two sexes has become almost equal. metastases from cancers elsewhere are also frequently found in the lungs. recurrent cough, haemoptysis, dyspnoea, chest pain and recurrent chest infections are the predominant features. local infiltration may cause pleural effusion, lesions of the cervical sympathetic chain (horner syndrome), brachial neuritis, recurrent laryngeal nerve palsy or obstruction of the superior vena cava with facial cyanosis and oedema (superior vena cava syndrome). there are many nonmetastatic extrapulmonary effects of bron chogenic (or other) carcinomas -for example, weight loss, anorexia, fingerclubbing, neuromyopathies, thromboses (thrombophlebitis migrans), muscle weakness, various skin manifestations and ectopic hormone production (of antidiuretic hormone, adrenocorticotropic hormone, parathyroid hormone and thyroidstimulating hormone). metastases from bronchogenic cancer are common and typically form in the brain (which may manifest with headache, epilepsy, hemi plegia or visual disturbances), liver (hepatomegaly, jaundice or ascites) or bone (pain, swelling or pathological fracture). the diagnosis is based on history and physical examination, supported by radiography, ct and magnetic resonance imaging (mri), sputum cytology, bronchoscopy and biopsy. spiral ct appears to detect tumours at an early stage. the overall year survival rate is only %. radiotherapy is the most common treatment. only some % of patients are suitable for surgery but, even then, the year survival is only about %. chemotherapy has been disappointing, except in smallcell carcinomas. dental treatment under la should be uncomplicated. cs should preferably be avoided. ga is a matter for specialist management in hospital, as patients often have impaired respiratory function, espe cially after lobectomy or pneumonectomy. this, along with any muscle weakness (myasthenic syndrome, eaton-lambert syndrome) that can make the patient unduly sensitive to the action of muscle relaxants, makes ga hazardous. oral cancer may be associated with lung cancer, and vice versa, or develop at a later stage (ch. ). such synchronous or metachronous primary tumours must always be ruled out. metastases can occasionally affect the orofacial region and cause enlargement of the lower cervical lymph nodes, epulislike softtissue swellings or labial hypoaesthesia or paraesthesia in the jaw. soft palate pigmentation is a rare early oral manifestation. lung cancer is a fairly common cause of death in dental techni cians, but it is unknown whether this is due to smoking alone or to dust inhalation. cystic fibrosis (cf) is one of the most common fatal hereditary dis orders. inherited as an autosomal recessive trait, with an incidence of about in births, it is the most common inherited error of metabolism and is seen mainly in people of european descent. the gene responsible is on chromosome q. cf is caused by defects in the cystic fibrosis transmembrane conductance regulator (cftr), a protein that appears to be part of a cyclic adenosine monophosphate (camp)regulated chloride channel, regulating cl − and na + transport across epithelial membranes, and ion channels and intracellular fluid flow in sweat, digestive and mucus glands. the basic defect in cf is abnormal chloride ion transport across the cell membrane of nearly all exocrine glands. the blockage of salt and water movement into and out of cells results in the cells that line the lungs, pancreas and other organs producing abnormally thick, sticky mucus that can obstruct the airways and various glands, especially in the respiratory tract and pancreas. involved glands (lungs, pancreas, intestinal glands, intrahepatic bile ducts, gallbladder, submaxillary and sweat glands) may become obstructed by this viscid or solid eosino philic material. recurrent respiratory infections result in a persistent productive cough and bronchiectasis, with the lungs becoming infected with a variety of organisms including staph. aureus, haemophilus influenzae, pseudomonas aeruginosa, strep. pneumoniae, burkholderia cepacia, and sometimes mycoses or mycobacteria. mycobacterium abscessus is a nontuberculous mycobacterium increasingly recognized as an opportunistic pathogen in cf patients. viral infections, such as mea sles, can have severe sequelae. pancreatic duct obstruction leads to pancreatic insufficiency, with malabsorption and bulky, frequent, foulsmelling, fatty stools. gallstones, diabetes, cirrhosis and pancreatitis may result. sinusitis is very common. growth is frequently stunted. the mutations can also cause con genital bilateral absence of the vas deferens, so fertility is impaired in most males with cf. in women, fertility may be impaired by viscid cervical secretions, but many women have carried pregnancies to term. most patients have a high concentration of sodium in their sweat (also reflected in the saliva); a sweat test showing sodium and chloride values of more than mmol/l is considered positive, between and mmol/l equivocal, and less than mmol/l negative. physiotherapy and postural drainage are crucially important. clearance of sputum is helped by water aerosols and bronchodila tors (terbutaline or salbutamol), but mucolytics such as carbocisteine, methyl cysteine and dornase alfa are of questionable effectiveness. treatment with ivacaftor, a cftr potentiator, improves chloride transport through the ion channel. amoxicillin and flucloxacillin are effective prophylactic antimicrobi als and may be given by aerosol. vaccination against measles, whoop ing cough and influenza is important. a low fat intake, adequate vitamins and oral pancreatic enzyme replacement (pancreatin) are also necessary. doublelung or heart-lung transplantation may eventually become necessary. sinusitis is very common; most cf patients have recurrent sinusitis and nasal polyps. the major salivary glands may enlarge and hyposali vation sometimes occurs. the lowfat, highcarbohydrate diet and dry mouth may predispose to caries. enamel hypoplasia and black stain may be seen, and both dental development and eruption are delayed. tetracycline staining of the teeth was common but should rarely be seen now. pancreatin may cause oral ulceration if held in the mouth. la is satisfactory but cs is usually contraindicated because of poor respiratory function. ga is contraindicated if respiratory function is poor. lung disease, such as bronchiectasis, liver disease and diabetes, may complicate treatment. bronchiectasis is dilatation and distortion of the bronchi. causes include: ■ congenital defects, which should be considered in all patients include cystic fibrosis, kartagener syndrome, alpha antitrypsin deficiency, collagen defects (e.g. marfan syndrome) there is no identifiable underlying cause in about % of adults and % of children. the damaged and dilated bronchi lose their ciliated epithelium and therefore mucus tends to pool, causing recurrent lrtis, typically with strep. pneumoniae, haemophilus influenzae or pseudomonas aeruginosa. overproduction of sputum, which is purulent during exacerbations, a cough (especially during exercise or when lying down) and finger clubbing are typical features, with recurrent episodes of bronchitis, pneumonia and pleurisy. haemoptysis is not uncommon. in advanced bronchiectasis, chest pain, dyspnoea, cyanosis and respiratory failure may develop. complications may include cerebral abscess and amyloid disease. chest radiography and pulmonary function tests are required. high resolution ct (hrct) is useful. postural drainage is important. antimicrobials, such as amoxicillin, cephalosporins or ciprofloxacin, are given for acute exacerbations and for longterm maintenance treatment. ga should be avoided where possible and is contraindicated in acute phases. workers exposed to airborne particles may develop pulmonary disease (pneumoconiosis), which ranges from benign (e.g. siderosis) to malig nant, as in mesothelioma from asbestosis (see appendix . ), but any pneumoconiosis can cause significant incapacity. ga may be contraindicated; the physician should be contacted before treatment. berylliosis may be a hazard in some dental technical laboratories, when lung cancer is more frequent. respiratory complications following surgical operations under ga include segmental or lobar pulmonary collapse and infection. they are more common after abdominal surgery or if there is preexistent respiratory disease or smoking (see also ch. ), and can be signifi cantly reduced by smoking cessation, preoperative physiotherapy and bronchodilators, such as salbutamol. if postoperative pulmonary infection develops, sputum should be sent for culture, and physiotherapy and antibiotics should be given. the common microbial causes are strep. pneumoniae and haemophilus influenza; in this case, suitable antibiotics include amoxicillin and erythromycin. hospital infections may include other microorganisms, such as mrsa, klebsiella, pseudomonas and other gramnegative bacteria. inhalation (aspiration) of gastric contents can cause pulmonary oedema and may be fatal (mendelson syndrome); it is most likely if a ga is given to a patient who has a stomach that is not empty, has a hiatus hernia or is in the last trimester of pregnancy. prevention is by ensuring the stomach is empty preoperatively; if it is not, an anaes thetist should pass an endotracheal tube. antacids or an h receptor blocker, such as cimetidine or ranitidine, may be given by mouth pre operatively to lower gastric acidity. if gastric contents are aspirated, the pharynx and larynx must be carefully sucked out. systemic corticosteroids have been recommended but probably do not reduce the mortality. respiratory distress in premature infants may be caused by immaturity of surfactantproducing cells, when the alveoli fail to expand fully; this necessitates endotracheal intubation for many weeks. it may, in turn, result in midface hypoplasia, palatal grooving or clefting, or defects in the primary dentition. the same oral effects may be seen with prolonged use of orogastric feeding tubes. the degree to which subsequent growth corrects these deformations is currently unknown, though the palatal grooves typically regress by the age of years. using soft endotracheal tubes does not obviate this problem and, at present, the best means of avoiding palatal grooving appears to be the use of an intraoral acrylic plate to stabilize the tube and protect the palate. acute respiratory distress syndrome (ards) is a sequel to several types of pulmonary injury and some infections, including those with oral viridans streptococci. patients with endstage pulmonary disease are considered for potential transplantation, usually using a lung from a braindead organ donor. a combination of ciclosporin, azathioprine and glucocorticoids is usu ally given for lifelong immunosuppression to prevent a tcell, alloim mune rejection response. inhaled nitric oxide modulates pulmonary vascular tone via smooth muscle relaxation and can improve ventilation/perfusion matching and oxygenation in diseased lungs. early graft failure following lung transplantation has been described by various investi gators as reimplantation oedema, reperfusion oedema, primary graft failure or allograft dysfunction. pathologically, this entity is diffuse alveolar damage. see also chapter . a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. cardiopulmonary transplantation (heart and lung transplantation) is the simultaneous surgical replacement of the heart and lungs in patients with endstage cardiac and pulmonary disease, with organs from a cadaveric donor. all transplant recipients require lifelong immunosuppression to pre vent a tcell, alloimmune rejection response. see also chapter . a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. national institutes of health: national institute of allergy and infectious diseases healthcare infection control practices advisory committee guideline for the prevention of healthcare associated pneumonia nosocomial pneumonia: state of the science extensively drugresistant tuberculosis as a cause of death in patients coinfected with tuberculosis and hiv in a rural area of south africa a review of the possible role of oral and dental colonization on the occurrence of health careassociated pneumonia: underappreciated risk and a call for interventions reducing ventilatorassociated pneumonia through advanced oraldental care: a month study apic infection control and applied epidemiology: principles and practice sepp. ventilatorassociated pneumonia and oral care: a successful quality improvement project guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings a randomized trial of dental brushing for preventing ventilatorassociated pneumonia pneumonia associated with a dental unit waterline the pathogenesis of ventilatorassociated pneumonia: its relevance to developing effective strategies for prevention aspects of human disease chronic obstructive pulmonary disease (copd) aspects of human disease in vitro antibacterial activities of oral care products against ventilatorassociated pneumonia pathogens the impact of a simple, lowcost oral care protocol on ventilatorassociated pneumonia rates in a surgical intensive care unit intermittent suction of oral secretions before each positional change may reduce ventilatorassociated pneumonia: a pilot study current trends and newer concepts on diagnosis, management and prevention of respiratory tract infections key: cord- -xe pkahz authors: reinero, carol r.; masseau, isabelle; grobman, megan; vientos‐plotts, aida; williams, kurt title: perspectives in veterinary medicine: description and classification of bronchiolar disorders in cats date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: xe pkahz this perspectives in veterinary medicine article seeks to define, describe putative causes, and discuss key diagnostic tests for primary and secondary bronchiolar disorders to propose a classification scheme in cats with support from a literature review and case examples. the small airways (bronchioles with inner diameters < mm), located at the transitional zone between larger conducting airways and the pulmonary acinus, have been overlooked as major contributors to clinical syndromes of respiratory disease in cats. because the trigger for many bronchiolar disorders is environmental and humans live in a shared environment with similar susceptibility, understanding these diseases in pet cats has relevance to one health. thoracic radiography, the major imaging modality used in the diagnostic evaluation of respiratory disease in cats, has low utility in detection of bronchiolar disease. computed tomography (ct) with paired inspiratory and expiratory scans can detect pathology centered on small airways. in humans, treatment of bronchiolar disorders is not well established because of heterogeneous presentations and often late definitive diagnosis. a review of the human and veterinary medical literature will serve as the basis for a proposed classification scheme in cats. a case series of cats with ct or histopathologic evidence of bronchiolar lesions or both, either as a primary disorder or secondary to extension from large airway disease or interstitial lung disease, will be presented. future multi‐institutional and multidisciplinary discussions among clinicians, radiologists, and pathologists will help refine and develop this classification scheme to promote early and specific recognition and optimize treatment. disease. advances in understanding bronchiolar disorders in humans have been hampered by lack of a widely accepted classification scheme because of different viewpoints of clinicians, radiologists, and pathologists. in humans, bronchiolar disorders are classified by inciting cause (eg, infection, drug related, immune-mediated, or occupational or environmental exposures), clinical sequelae (eg, restrictive, obstructive, or mixed disorders), imaging, histologic features, or some combination of these. , , bronchiolar disease can be focal or diffuse, acute, or chronic, inflammatory or fibrotic, and confined to the lungs or develop as part of a variety of systemic diseases. , in primary bronchiolar disorders, disease centers on bronchioles and spares other parts of the respiratory tract. in secondary bronchiolar disorders, disease extends to small airways from either the large airways or pulmonary parenchyma (eg, interstitial lung diseases [ilds] ). bronchiolar disorders, although recognized in humans, have not yet been described as distinct clinical entities in veterinary medicine. lower airway diseases of cats (eg, asthma, chronic bronchitis, parasitic bronchitis) are common, although lower airway disease has been considered a large (ie, "bronchial") airway disorder. extension of inflammation into the bronchioles, although at times recognized histologically, is not characterized clinically. uncommonly recognized in cats, descriptions of ilds infrequently mention bronchiolar involvement. , with increasing use of computed tomography (ct), abnormalities suggestive of bronchiolar disease are being identified in cats. multidisciplinary input from clinicians providing detailed clinical descriptions, radiologists characterizing ct features, and pathologists documenting and describing bronchiolar lesions will be essential to develop a consensus classification. bronchiolar disorders may result from infection, environmental or occupational exposures, immunologically mediated disease, neoplasms, chronic aspiration, and drug-induced toxicity, among other causes. [ ] [ ] [ ] [ ] although a description of each is beyond the scope of this article, a discussion of environmental bronchiolar disorders in humans appears relevant because cats and humans share their environment and potential exposures. known or suspected exposures causing bronchiolar disorders in humans have been reviewed and include chemicals (eg, cleaning compounds, pesticides, artificial flavorings), inhalant particulates, animal or mineral dusts, and gas and smoke inhalation. previously, environmental bronchiolar disorders were thought to be an acute sequela to a severe, overwhelming exposure, but more recently, disease with an insidious onset of clinical signs without a recognized overexposure event (perhaps representing cumulative smaller exposures) or a mild single exposure has been appreciated. injury to bronchiolar epithelial cells leads to inflammation and a fibroproliferative repair response, ultimately resulting in mural fibrosis (constrictive bronchiolitis) or intraluminal fibrosis (proliferative bronchiolitis). four histopathologic types of disease have been proposed: cellular bronchiolitis (inflammatory infiltrate of the bronchioles), constrictive bronchiolitis (concentric fibrosis of the wall leading to narrowed airway caliber), proliferative bronchiolitis (polyps of connective tissue within the bronchiolar lumen), and bronchiolitis obliterans organizing pneumonia (proliferative bronchiolitis with polyps extending into the alveolar ducts and alveoli). box the importance of the secondary pulmonary lobule in humans and the lack of an analogous structure in cats superior imaging detail of computed tomography (ct) allows comparison with histologic features. in the human lung, an understanding of microscopic anatomy centering on the secondary pulmonary lobule is critical to make these correlations. • the secondary pulmonary lobule consists of polyhedral regions approximately cm in diameter bordered by interlobular septa • secondary pulmonary lobules have multiple pairs of lobular bronchioles and arterioles that accompany each other into the center of this anatomic unit (ie, the so called "centrilobular" location) • lobular bronchioles successively divide in the following manner: terminal bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli • blood flows from arterioles into sheets of capillaries in the walls of alveoli and finally are drained by venules located at the borders of the secondary pulmonary lobules (ie, in the interlobular septal regions) • lymphatics extend from respiratory bronchioles tracking back to the hilus of the lung in bronchovascular bundles and are present in the interlobular septa following venules back to the hilus; no lymphatics surround alveoli • collectively, the cross-sectional area of the bronchioles is larger than the proximal airways; although they provide little resistance to airflow in the normal lung, even mild disease of the small airways can have severe detrimental effects on lung function , in cats, there is a lack of a structure analogous to the secondary pulmonary lobule seen in humans. this has important implications in use of the same ct descriptors and between-species comparative studies. compared to humans, cats lack connective tissue septa in the lung outlining a secondary pulmonary lobule (box ). furthermore, there is no distinctive recognizable pattern of analogous bordering venules and lymphatics arranged in discrete polyhedral shapes that might comprise this unit. thus, even in disease, pathology on ct scans of cats would not reflect distribution around a pulmonary lobule. terms used in imaging of human lung such as centrilobular, panlobular, interlobular, and intralobular are not appropriate descriptors in cats. dogs are similar to cats and also lack secondary pulmonary lobules, and the pulmonary acinus has been suggested to be the important highresolution ct (hrct) unit of the canine lung. the acinus is defined as the region of the lung supplied by a single terminal bronchiole, representing the smallest functional unit of the lung. because it is smaller than the secondary pulmonary lobule that is the smallest visible structure on hrct, we believe that the pulmonary acinus is not a replaceable term as the important hrct unit of the feline (or canine) lung. instead, descriptors of collective changes of multiple acini visible on hrct will need to be developed and refined. thoracic radiography is insensitive for diagnosis of small airway (bronchiolar) disease, likely contributing to the absence of its recognition in clinical practice. thoracic radiographs in humans with bronchiolar disease may be normal or show nonspecific changes such as hyperinflation, nodules, and reticular to alveolar infiltates. , in our experience, thoracic radiographs in cats with histologic evidence of bronchiolar disease can have bronchial or bronchointerstitial patterns (with or without bronchiectasis), patchy alveolar patterns, ill-defined nodular opacities, and hyperinflation or hypoinflation. the radiographic appearance of histologically confirmed bronchiolar disease in cats does not present as a single pathognomonic radiographic pattern, but is highly variable, reflecting the different diseases encompassed in the spectrum of bronchiolar disorders. importantly, without clinical recognition of these disorders, bronchiolar disease is not considered as a differential diagnosis. in humans, hrct has been instrumental in improving diagnosis of bronchiolar disorders, establishing them as important disease entities in pulmonary medicine. with more commonplace use of ct in cats, we are recognizing some features similar to those described in humans. paired inspiratory and expiratory scans and thin section reconstruction ( . - mm slice thickness) provide optimal detail. normal bronchioles in humans and cats are below the limits of resolution on hrct, but with dilatation, mural thickening, and intraluminal plugging, small airways become visible. bronchiolar disorders have characteristic hrct features, classified as direct or indirect signs. , direct signs imply a change in the walls or lumens of bronchioles resulting in an ability to visualize them. these signs include centrilobular nodules (humans only), tree-in-bud pattern (nodular opacities connected to branching linear structures originating from a single stalk reflective of impacted debris, cells, or fluid within bronchioles), peribronchiolar ground glass opacity or consolidation, and dilatation (bronchiolectasis). , , indirect signs reflect changes to the pulmonary parenchyma distal to the diseased bronchiole and include mosaic attenuation (because of air trapping) or, in the setting of bronchiolar disorders, rarely mosaic perfusion. because air trapping may be imperceptible on inspiratory images, expiratory scans are crucial to accentuate air trapping. direct findings reflect inflammation or proliferative changes within the bronchiolar lumens, whereas indirect findings reflect fibrosis within the bronchiolar wall (eg, constrictive bronchiolitis). definitive confirmation of bronchiolar involvement requires histopathology. the microscopic anatomy of bronchioles is similar between humans and cats. distal bronchioles are lined by simple cuboidal epithelium supported on a thin lamina propria and surrounded by smooth muscle. airways end as respiratory bronchioles before the alveolar parenchyma in the cat ( figure ) and in humans. microscopic morphology has been used to categorize lesions as inflammatory or fibrotic, recognizing there are a limited number of ways airways respond to injury. inflammatory or cellular lesions often are subcategorized as acute, chronic, acute on chronic, granulomatous, or eosinophilic. fibrotic lesions reflect the site affected (intraluminal or intramural); overlap in inflammatory and fibrotic changes can be noted within the same patient. multiple wedge biopsy specimens are recommended to capture patchy and sometimes subtle lesions. a particular histologic pattern of disease may have a wide variety of causes. for example, inflammatory bronchiolitis can be caused by infection, aspiration, transplant rejection, and extension from large airway disease or systemic collagen vascular disease, among other causes. fibrosis may be a sequela to chronic inflammation so that in end-stage disease, the inflammatory etiology can be missed. thus, classification schemes based solely on histopathologic features are likely to be less clinically useful, and multidisciplinary f i g u r e normal cat lung histology (hematoxylin and eosin stain). terminal bronchioles (tb) are lined by low cuboidal epithelium and surrounded by thin smooth muscle. the airway terminates as respiratory bronchiole (rb) before entering the alveoli input should be sought to allow meaningful interpretation of histologic findings. this should not dampen enthusiasm for the central role of histopathology in an understanding of bronchiolar disorders. in fact, in humans, histopathology may be the only means to document bronchiolar disease in the subset of symptomatic patients with normal lung function (as assessed by spirometry, diffusion capacities, and cardiopulmonary exercise testing) and normal hrct scans. in humans, bronchiolar disorders often have distinct historical data, clinical presentations, ct findings, and histopathology. our proposed scheme, adapted for cats, is based on a state-of-the-art review of bronchiolar disorders in humans that focuses on discrete clinical syndromes supported by distinct ct imaging patterns and histopathologic features. of note, this scheme is not universally accepted, and there are other proposed schemes biased by specialty, in particular pathology. primary bronchiolar disorders in humans encompass disease in which the pathologic process is limited to bronchioles. they are distinct from secondary disorders in which extension to bronchioles occurs from large airway disease or from ild. analogous disorders occur in cats and will require further efforts among veterinary clinicians, radiologists, and pathologists for full characterization. herein, we describe a series of clinical cases in cats with small airway disease using a modification of the aforementioned classification scheme adopted from usage in humans (table ) . protocols for thoracic ct and histologic examination are provided in supporting information and . although this suggested classification scheme is a starting point for cats, it will need modification as awareness of bronchiolar disorders in cats increases and cases are investigated prospectively. additionally, it is critical to realize that veterinarians are likely to detect bronchiolar disorders late in the disease course for several reasons: (i) cats hide respiratory disease well, (ii) aggressive diagnostic testing, including lung biopsy, is rarely advocated unless underlying disease is serious and there are potentially treatable differential diagnoses, (iii) serious disease is associated with anesthetic risk, and (iv) advanced diagnostic tests are expensive. detection of end-stage lesions will not provide much needed clues about common underlying etiologies because the inciting factor may be gone, and there are likely many insults that share a final common pathway with similar clinical, physiological, and histologic appearances. at the present, we believe that ct provides the best minimally invasive antemortem evidence of bronchiolar disease in cats. importantly, histopathologic correlates will be required before ct alone can be considered a less invasive surrogate diagnostic test. | primary bronchiolar disorders . | constrictive/obliterative bronchiolitis subepithelial and peribronchiolar fibrosis and inflammation that externally surrounds and narrows or obliterates the lumen of bronchioles is termed constrictive bronchiolitis obliterans (cbo). in advanced stages, the bronchiolar lumen undergoes complete cicatrization and is replaced by collagenous tissue. this pattern of bronchiolar fibrosis is not to be confused with intraluminal polyps of connective tissue within the bronchiolar lumen as occur in polypoid bronchiolitis obliterans (pbo). in cats, pbo previously has been described with associated pneumonia (ie, bronchiolitis obliterans with organizing pneumonia ) and is considered an ild. disorders and exposures in humans leading to cbo include prior infection, inhalational injury (flavoring chemicals in microwave popcorn, e-cigarettes, coffee, and others [ ] [ ] [ ] [ ] , ingested drugs or toxins, autoimmune disease, and connective tissue disorders. humans present with clinical signs of an obstructive lung disorder with an insidious onset of progressive dyspnea and cough. thoracic radiography may be normal or show hyperinflation from air trapping; airway wall thickening also may be appreciated. , high-resolution ct predominantly identifies mosaic attenuation, bronchiolectasis or bronchiectasis, and air trapping accentuated on expiratory views. , most cases are progressive, poorly responsive to corticosteroids and result in respiratory failure and death. one cat in a case series had suppurative bronchopneumonia, bronchiolitis obliterans, and pulmonary fibrosis; it is unclear if this cat had a primary bronchiolar disorder or an ild with secondary bronchiolar involvement. another cat in a case series had "fibrosing bronchiolitis" and "pneumonia" as histologic descriptors but not as the clinical syndrome or final diagnosis. recently, a reversible form of acute fibrosing bronchiolitis has been excluded from the list of primary human bronchiolar disorders at this time are diffuse panbronchiolitis (described as predominantly occurring in humans of asian descent with a genetic predisposition), respiratory bronchiolitis (a smoking-related airway disorder), and follicular bronchiolitis (a lymphoproliferative pulmonary disease). b in the human classification scheme, "acute bronchiolitis" is used. this is a common disorder almost always secondary to infection in the pediatric population. we have revised the feline classification scheme to include acute and chronic infectious diseases affecting the small airways. c excluded from the list of human bronchiolar disorders as a component of ilds at this time are hypersensitivity pneumonitis (not yet recognized in cats), and respiratory bronchiolitis-associated ild/desquamative interstitial pneumonia (smoking-related disorders). identified in humans. although this acute form has not yet been identified in cats, treating with immunosuppressive drugs to prevent permanent bronchiolar fibrosis has important implications. no air trapping is visualized on the expiratory ct image as illustrated by the reduced lung volume (double headed arrow), and homogenous diffuse increased lung attenuation. pleural thickening is accentuated during exhalation (arrowhead). the right side of the patient is on the right of the ct images box challenges in discriminating primary from secondary bronchiolar disorders a diagnosis of feline asthma is frequently made based on thoracic radiography showing hyperinflation with a bronchial or bronchointerstitial pattern (with or without lobar atelectasis) and is the major obstructive airway disorder clinically described in cats. • constrictive/obliterative bronchiolitis represents an important "new" differential diagnosis, especially in cats with other unusual radiographic features, those who fail to respond to treatment with bronchodilators and glucocorticoids, or both. thoracic computed tomography (ct) should be offered in these situations. although a ct scan was not performed in case , presumptively there would have been lesions localized to small airways. pathology extending from bronchioles into the interstitium makes it tempting to speculate that primary bronchiolar diseases may in their end stages appear as an ild, analogous to ilds starting in the interstitium and progressing to a secondary bronchiolar disorder (see case ). • lesions involving both bronchioles and interstitium are challenging to determine which region suffered the initial insult before progression to other local regions. this underscores the need to take adequately large specimens from multiple regions of the lung (more and less affected) when submitting for histopathologic examination. alveolar ducts. the pathologic response to inhalation of asbestos, silica or silicates, aluminum, and coal (among others) targeting small airways is mural fibrosis with mild chronic inflammation leading to luminal narrowing. , in humans, classic pneumoconiosis leading to pulmonary parenchymal fibrosis is considered a restrictive lung disorder, whereas mineral dust airway disease may present as an obstructive airway disorder. . . | case • identification of mineral dusts can be challenging unless a careful search with polarized light is used; very small particulates will be difficult to visualize using routine microscopy. • owners should be questioned about specific environmental exposures and identified mineral dusts should be avoided. • it is unclear in cats if mineral dust airway disease in its end stages can appear as a constrictive bronchiolitis or pulmonary fibrosis, and further study is needed. ct or histopathologic evidence of bronchiolar involvement in cats. [ ] [ ] [ ] [ ] [ ] [ ] in most cases, bronchiolar involvement was overshadowed by alveolar involvement, with pneumonia being the final diagnosis. in humans, primary disorders of the bronchioles, not fitting into the aforementioned categories, currently are considered together as "other primary bronchiolar variants". diffuse aspiration bronchiolitis (dab), once thought to be a disease of the bed-ridden elderly with neurologic disorders predisposing to aspiration, is now recognized in young individuals with and without occult gastroesophageal reflux disease. [ ] [ ] [ ] depending on the type and extent of infection, these cases may be treatable with a better prognosis and/or potential for cure. of interest, the thoracic ct scan in the cat described in case was originally interpreted by the attending radiologist as consistent with usual interstitial pneumonia (ie, idiopathic pulmonary fibrosis), an as yet, untreatable disease with a grave prognosis. a -year-old fs domestic longhair cat was presented for chronic productive cough and gagging especially pronounced after eating and drinking, intermittent vomiting and anorexia, and regurgitation. on physical examination, a grade iii/vi parasternal heart murmur was heard, respiratory rate was increased ( breaths per minute), and body condition score was / . a diffuse bronchointerstitial pattern was observed on thoracic radiography. computed tomography was suggestive of dab with a multifocal distribution of lesions ( figure a-d) . specifically, nodules of similar size were equally spaced from each other and, when in the periphery, were at the same distance from the pleural surface, consistent with affected small airways. bronchoalveolar lavage cytology was unremarkable. cough improved after treatment with omeprazole. histologic evaluation of tissue was not performed. however, the supportive clinical history, including conditions predisposing to repetitive microaspiration, the bronchiolocentric ct lesions, and subsequent improvement in clinical signs after a trial of omeprazole, supports presumptive dab. a -year-old fs dsh cat was presented for potential mammary tumors. the cat had lived in south korea for . years until months before presentation. the cat had been treated for psychogenic alopecia with megestrol and methylprednisolone acetate and subsequently developed mammary masses. relevant history included exposure to "yellow dust" in south korea, a serious environmental health threat affecting air quality and leading to respiratory disease in humans. in the right cranial lung lobe, which could not easily be ascribed to asthma, lobectomy was performed. histology identified mild to moderate eosinophilic bronchitis and bronchiolitis with luminal mucus accumulation, moderate bronchial gland hyperplasia, mild subpleural air trapping consistent with asthma, but no evidence of an infiltrative disease was found ( figure ). the cat initially was treated with po, followed by inhaled, corticosteroids and a weight loss plan with marked clinical improvement. a -year-old dsh female spayed cat was presented for acute labored respiration. prior respiratory signs included wheezing, coughing, and sneezing for - months. on physical examination, a respiratory rate of /min, increased bronchovesicular sounds and morbid obesity were observed. an echocardiogram identified normal left atrial size; a probrain natriuretic peptide test was negative. on thoracic radiography, a mild to moderate bronchial pattern involving the caudal lung fields and right middle lung lobe atelectasis was observed. thoracic ct with contrast identified a tree-in-bud pattern with regions of accentuation of mosaic attenuation on the expiratory series suggestive of air trapping (figure a,b) . bronchoalveolar lavage fluid cytology identified % f i g u r e a -year-old female spayed domestic shorthair cat previously diagnosed with cellular bronchiolitis. right lateral (a) and ventrodorsal thoracic projections (b) belonging to the same cat from figure . when compared to figure a -year-old mc dsh cat was presented for chronic coughing and wheezing and chronic kidney disease. on physical examination, a body condition score of / , grade ii/vi parasternal systolic heart murmur, expiratory wheezes, and irregularly shaped kidneys were identified. thoracic radiographs disclosed a severe diffuse bronchial pattern with subtle hyperinflation. heartworm antibody and antigen testing was negative. there was no response to fenbendazole. thoracic ct identified bronchial wall thickening and thickened bronchovascular bundles, bronchiolectasis, undulated pleural margins, right middle and accessory lung lobe atelectasis, and a tree-in-bud pattern in humans, inhalational exposures may lead to hypersensitivity pneumonitis or pneumoconiosis; cigarette smoke is associated with respiratory bronchiolitis-ild and desquamative interstitial pneumonia. a single case report described "desquamative interstitial pneumonialike" histologic lesions in a cat with no mention of environmental exposure to tobacco smoke. idiopathic pulmonary fibrosis has been described in cats although the initial histopathologic characterization did not provide specific mention of lesions centered around small airways. a -year-old fs dsh cat was presented with chronic weight loss, cough, and respiratory distress. thoracic radiography performed months previously disclosed an unstructured interstitial pattern and a moderate bronchial pattern, more severe in the ventral thorax (figure a,b) . treatment for presumptive asthma with prednisolone and theophylline resulted in minimal response. on physical examination, respiratory rate was /minute and body condition score was / . on thoracic ct, multifocal areas of consolidation with air bronchograms were observed, more severe in the ventral aspect of the thorax and at the periphery • computed tomography changes affecting larger airways such as increases in peribronchovascular density, peribronchial cuffing, and thickened airway walls support feline asthma, chronic bronchitis, or parasitic bronchitis as the primary lesion. cytology of balf is key to classification of large airway disorders in cats. , antemortem lung biopsy is rarely performed as less invasive diagnostics allow for reasonable classification and treatment. • in case , lung biopsy was performed because of concern for a comorbid condition in addition to feline asthma; the lesions noted on ct were not explained by histopathologic examination. • in case , chronic bronchitis was the antemortem clinical diagnosis; however, most cats with chronic bronchitis do not present in respiratory distress. the more serious clinical signs and objective evidence of air trapping on ct scan is attributed to extension of involvement of inflammation to small airways. larger numbers of cases with large airway disease will be required to determine if cats with small airway involvement have more severe clinical signs, refractory responses to treatment, or a more guarded prognosis. euthanasia. on necropsy, severe multifocal chronic-active terminal and respiratory bronchiolitis with bronchiolar loss, parenchymal fibrosis, interstitial inflammation, and smooth muscle hyperplasia with marked locally extensive pleural fibrosis were observed (figure a, b) . the continuum between primary bronchiolar disorders and end-stage fibrosis is discussed in box . authors declare no conflict of interest. authors declare no off-label use of antimicrobials. authors declare no iacuc or other approval was needed. authors declare human ethics approval was not needed for this study. carol r. reinero https://orcid.org/ - - - megan grobman https://orcid.org/ - - - box pulmonary fibrosis is an end-stage lesion and can mask the primary disease process bronchiolocentric interstitial pneumonia or airway-centered interstitial fibrosis in humans can result from chronic small airway-terminal acinar disease with progression to encompass associated alveolar parenchyma with inflammation and fibrosis. , • the striking amounts of fibrosis and clinical picture of a restrictive lung disease is the reason the authors categorized case as an ild; however, it is likely this case may represent an advanced primary bronchiolar disorder leading to an "end-stage" lung and what most veterinary clinicians, radiologists, and pathologists would call idiopathic pulmonary fibrosis • the cat in case illustrates the need for additional study and refinement of the classification scheme for bronchiolar disorders in the cat. small airways disease and mineral dust exposure. prevalence, structure, and function small airways involvement in coal mine dust lung disease airway-centered interstitial fibrosis: etiology, clinical findings and prognosis interstitial lung diseases that are difficult to classify: a review of bronchiolocentric interstitial lung disease the computed tomographic "treein-bud" pattern: characterization and comparison with radiographic and clinical findings in cats evaluation of biomarkers in bronchoalveolar lavage fluid for discrimination between asthma and chronic bronchitis in cats advances in the understanding of pathogenesis, and diagnostics and therapeutics for feline allergic asthma imaging of small airways diseases the pathologist's approach to small airways disease bronchiolar disorders occupational and environmental bronchiolar disorders use of keyhole lung biopsy for diagnosis of interstitial lung diseases in dogs and cats: cases the pathology of interstitial lung disease in nylon flock workers bronchiolitis: classification, computed tomographic and histopathologic features, and radiologic approach bronchiolar disorders: classification and diagnostic approach bronchitis and bronchiolitis in a cat with cilia-associated respiratory bacillus-like organisms clinical, radiographic, and pathologic features of bronchiectasis in cats: cases ( - ) thoracic radiography, bronchoalveolar lavage cytopathology, and pulmonary parenchymal histopathology: a comparison of diagnostic results in cats interstitial lung diseases in dogs and cats part i: the idiopathic interstitial pneumonias respiratory syncytial virus (rsv) and its propensity for causing bronchiolitis obstructive bronchiolar disease identified by ct in the non-transplant population: analysis of consecutive cases bronchiolitis: adopting a unifying definition and a comprehensive etiological classification histologic findings in lung biopsies in patients with suspected graft-versus-host disease occupational causes of constrictive bronchiolitis obliterative bronchiolitis relationships among subgross anatomy, computed tomography, and histologic findings in dogs with disease localized to the pulmonary acini. vet radiol ultrasound imaging of small airways and emphysema imaging of small airways disease bronchiolar disorders: a clinical-radiological diagnostic algorithm constrictive bronchiolitis in soldiers returning from iraq and afghanistan centers for disease control and prevention. obliterative bronchiolitis in workers in a coffee-processing facility-texas flavoring chemicals in e-cigarettes: diacetyl, , -pentanedione, and acetoin in a sample of products, including fruit-, candy-, and cocktail-flavored e-cigarettes increased respiratory disease mortality at a microwave popcorn production facility with worker risk of bronchiolitis obliterans update on flavoring-induced lung disease adult bronchiolitis-a clinical and pathological interpretative classification pathologic manifestations of bronchiolitis, constrictive bronchiolitis, cryptogenic organizing pneumonia, and diffuse panbronchiolitis correlation between fineneedle aspiration cytopathology and histopathology of the lung in dogs and cats fibrosing bronchiolitis evolving from infectious or inhalational acute bronchiolitis. a reversible lesion classification and approach to bronchiolar diseases interspecies transmission of canine influenza virus h n to cats and chickens by close contact with experimentally infected dogs pathology and viral antigen distribution of lethal pneumonia in domestic cats due to pandemic (h n ) influenza a virus feline herpesvirus associated with interstitial pneumonia in a kitten acute bronchointerstitial pneumonia in two indoor cats exposed to the h n influenza virus influenza a virus (h n ) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts primary mycoplasma pneumonia associated with reversible respiratory failure in a cat diffuse bronchiolar disease due to chronic occult aspiration diffuse aspiration bronchiolitis: analysis of consecutive patients importance of diffuse aspiration bronchiolitis caused by chronic occult aspiration in the elderly architecture and inflammatory cell composition of the feline lung with special consideration of eosinophil counts european respiratory society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias a clinical approach to diffuse parenchymal lung disease identification and characterization of an idiopathic pulmonary fibrosis-like condition in cats radiographic and histopathologic characteristics of pulmonary fibrosis in nine cats a case of atypical diffuse feline fibrotic lung disease interstitial lung diseases in dogs and cats part i: the idiopathic interstitial pneumonias desquamative form of cryptogenic fibrosing alveolitis in a cat idiopathic bronchiolocentric interstitial pneumonia identification of spontaneous feline idiopathic pulmonary fibrosis: morphology and ultrastructural evidence for a type ii pneumocyte defect follicular bronchiolitis: a literature review intravenous adipose-derived mesenchymal stem cell therapy for the treatment of feline asthma: a pilot study perspectives in veterinary medicine: description and classification of bronchiolar disorders in cats key: cord- -ygfnkgqp authors: fujita, yu; takeshita, fumitaka; kuwano, kazuyoshi; ochiya, takahiro title: rnai therapeutic platforms for lung diseases date: - - journal: pharmaceuticals (basel) doi: . /ph sha: doc_id: cord_uid: ygfnkgqp rna interference (rnai) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. the induction of rnai relies on small silencing rnas, which affect specific messenger rna (mrna) degradation. two types of small rna molecules, i.e. small interfering rnas (sirnas) and micrornas (mirnas), are central to rnai. drug discovery studies and novel treatments of sirnas are currently targeting a wide range of diseases, including various viral infections and cancers. lung diseases in general are attractive targets for sirna therapeutics because of their lethality and prevalence. in addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. recently, increasing evidence indicates that mirnas play an important role in lung abnormalities, such as inflammation and oncogenesis. therefore, mirnas are being targeted for therapeutic purposes. in this review, we present strategies for rnai delivery and discuss the current state-of-the-art rnai-based therapeutics for various lung diseases. traditional surgery, bivas-benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. endotracheal applications are currently being used by many practitioners in the pulmonary field [ , ] ; this is useful for studying pulmonary drug delivery in mice. however, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. complications, such as vascular injury and air leakage, are possible due to the tracheotomy. (b) endotracheal route: sirnas are sprayed directly from the mouth into the lungs using a microsprayer ® aerolizer (penn-century, philadelphia, pa, usa) and a laryngoscope. it is important to maintain a clear view of the trachea during the procedure. intranasal delivery is another common method of pulmonary drug application in animal studies. in many studies, in vivo success has been demonstrated in delivering sirnas to the lungs intranasally [ , , ]. an experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. although the success in delivering sirnas intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [ ] , this approach has potential for the clinical application of sirnas. phase ii clinical trials have been initiated for the treatment of respiratory syncytial virus (rsv) infection, making use of intranasal application of naked chemically modified sirna molecules that target viral gene products [ , ] (see section . . . for details). intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. therefore, sirnas administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. in fact, it has been reported that intranasal application of unformulated sirnas resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application [ ] . the intranasal method is suitable for some lung diseases, such as upper respiratory infection by rsv, and it also has potential for systemic delivery rather than pulmonary delivery of sirnas. therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. careful choice of efficient delivery in response to the condition of lung diseases is necessary. the use of aerosols to deliver medication to the lungs has a long history. administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. there are several inhalation devices available for the delivery of drugs into the lungs. metered dose inhalers (mdis) and dry powder inhalers (dpis) are the most common modes of inhaled delivery. mdis are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (copd), and a spacer is an external device that is attached to an mdi to allow for better drug delivery by enhanced actuation and inhalation coordination. for most mdis, the propellant is one or more gases called chlorofluorocarbons (cfcs). although cfcs in drugs are safe for patients to inhale, they are harmful to the environment. therefore, further development of inhalable sirnas may not be the best way forward. dpis are devices that deliver medication to the lungs in the form of dry powder. the use of dpis has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin [ ] and low-molecular-weight heparin [ ] ; thus, it could be a better device for delivering sirnas to the lungs. the advantages of dpis are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using sirnas have relied on intratracheal or intranasal delivery. the reason could be the difficulty in formulating inhalable sirnas and maintaining the stability during the delivery process. a suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. the use of spray-drying as a technique for engineering dry powder formulations of sirna nanoparticles, which might enable the local delivery of biologically active sirna directly to the lung tissue, has been demonstrated [ , ] . in the future, the technique is desirable to estimate the in vivo study on sirna therapy for inhalation. in the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. there are two main barriers to efficient pulmonary sirna delivery to the cells of the lung. the first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells [ , ] (figure ) . a remarkable feature of the respiratory tract is its high degree of branching. airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. all of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. it is generally acknowledged that the critical factor for efficient sirna delivery depends on the properties of rnai drug particles in terms of size, charge, shape, velocity and density. for efficient pulmonary sirna delivery, the particles must be deposited in the lower respiratory tract. deposition in the airway is affected by the particle size and patient's pulmonary function. a particle size between - μm is found to be the most appropriate for deposition at the lower respiratory tract [ ] . in addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the sirna deposition to the diseased area of the respiratory tract. besides, the extracellular barriers to sirna delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. at the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. during infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance [ ] . moreover, asthma and copd are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function [ ] . the airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. figure . extracellular barriers to pulmonary sirna delivery. the anatomical feature of the respiratory tract is its high degree of branching. the mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. the deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. in the alveolar, clara cells and type ii alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. the surfactants act as the main barrier for sirna delivery because they reduce the transfection efficiency. in addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. the particles taken up into the macrophages are subsequently degraded inside the cells. these factors present major barriers to targeted pulmonary delivery. the second is the airway cell membrance and its intracellular barriers ( figure ). for efficient gene silencing in the lungs, sirnas must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. once the sirnas reach the target cells, they must be trafficked into the cytoplasm and taken up by argonaute (ago) /rna-induced silencing complex (risc), which degrades mrnas and, subsequently, suppresses the sequence-specific gene expression. for efficient endocytosis to occur, particles should be under nm in size. particles within this size range could also avoid macrophage uptake and delayed lung clearance [ ] . the physicochemical properties of sirnas also play a significant role in crossing the biological membrane. despite their small size, the negative charge and chemical degradability of sirna molecules prevent them from readily crossing biological membranes. therefore, efficient sirna delivery approaches need to overcome this limitation by facilitating cellular uptake. one of the main functions of a delivery vector is to facilitate the cellular uptake of sirnas [ ] . the electrostatic complexation of sirna molecules with cationic lipids and polymers helps to mask their net negative charge. the positively charged sirna carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis [ ] . after cellular internalization, the sirna carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. to avoid lysosomal degradation, sirnas must escape from the endosome into the cytoplasm, where they can associate with the rnai machinery. endosomal escape is a major barrier for efficient sirna delivery [ , ] . the endosomal entrapment and lysosomal degradation of sirna and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. an ideal delivery agent should protect sirnas from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. multiple approaches for the delivery of sirnas have been reported, ranging from the relatively simple direct administration of saline-formulated sirnas to lipid-based and polymer-based nanoparticle approaches and sirna conjugation and complexation approaches [ ] . the negative charge and chemical degradability of sirnas under physiologically relevant conditions make its delivery a major challenge. accordingly, the delivery of sirnas usually requires a vector or carriers for their transfection into the target cells. in general, both viral and non-viral vectors are being assessed for sirna delivery to lung cells. some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model [ ] and to induce rnai in a range of animal tissues [ ] . recently, guo et al. showed that lentivirus-mediated sirna was used to specifically knock down the expression of nuclear protein (nupr ) in vivo, which resulted in inhibited tumor growth [ ] . however, viral-based delivery has several disadvantages. the immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications [ ] . recent well-documented cases, such as the death of jesse gelsinger due to complications related with an adenoviral delivery vector, highlight this problem [ ] . in addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function [ ] . . intracellular barriers to pulmonary sirna delivery. barriers to cellular internalization are dependent on the surface properties of sirna and carriers (e.g., charge and size). after sirnas are successfully taken into the target cells by endocytosis, the main barriers for delivering sirnas to its site of action are the endosomal entrapment and lysosomal degradation of sirna and carriers. to direct target-gene silencing, the sirnas need to escape from the endosome into the cytoplasm, where they associate with the ago /rna-induced silencing complex (risc) to direct the cleavage of mrnas bearing complementary binding sites. as an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of sirnas to the lungs due to their reduced toxicity [ ] . ongoing research into the transfection of primary cells and whole organisms with sirna using non-viral transfection agents has produced some promising results. lipid-based delivery vectors are successfully used to deliver sirna in vitro and in vivo [ ] . cationic lipids are composed of positively charged head, a linker and hydrophobic. in general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. many commercial sirna transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-dharmfect [ ] , oligofectamine [ ] , lipofectamine [ ] and transit-tko [ ] . similarly, cationic polymers have also been assessed for sirna delivery to lung cells. cationic polymer polyethylenimine (pei) is widely used for sirna delivery [ , ] . pei is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. on the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses [ , ] . although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective sirna delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. an important point for sirna-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses [ , ] . interestingly, some studies have shown that it was possible to administer "naked sirnas" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response [ ] . the term "naked sirnas" refers to the delivery of sirnas without any delivery vectors. naked sirnas are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < min. thus, some studies of systemic delivery of naked sirnas have failed to achieve the downregulation of the targeted gene [ , ] . in contrast, there have also been some successes of locally delivering naked sirnas to the lungs [ , , , ] . a few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked sirnas [ , ] . indeed, in one clinical trial, the delivery of naked sirnas for the treatment of rsv has been used [ , ] . this successful evidence can be because that naked sirnas for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. although it is unclear how the naked sirnas cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked sirnas (aln-rsv ) that were administered intranasally. this explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. the active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. naked sirna delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. nevertheless, the advantage of naked sirnas over delivery vectors in the treatment of lung diseases is controversial [ , ] . further in vivo investigations about both naked sirnas and non-viral vectors are required. lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. the high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. above all, copd is the fourth-leading cause of death in most industrialized countries and is predicted to become third by [ ] . therefore, decisive action is needed to stem the rising health and economic burden this represents. chronic lung diseases, such as copd and asthma, are disorders of the airways largely related to the presence of persistent inflammation. the approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. this was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. corticosteroids are still an important therapeutic intervention. however, they are used with limitations in copd and moderate to severe asthma. likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. treatment of lung disease using cell-specific targeting as well as rnai techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. pulmonary applications of sirna in in vivo conditions are frequently studied and often result in clinical trials [ , ] . the findings of recent clinical studies of pulmonary rnai therapeutics are discussed. since the discovery of rnai, the therapeutic potential of sirnas has been rapidly recognized. in , the first human clinical trial of rnai-based therapy was initiated for the treatment of age-related macular degeneration with a sirna targeting vegf-receptor delivered intravitreally [ ] . many studies have been conducted over the past few years that involve the delivery of sirnas to the lungs for the treatment of various lung diseases. delivery to the lungs will be most important to moving sirna technology into the clinic. a number of sirna-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and rsv infection. table is a summary of clinical trials of sirna-based therapeutics [ ] . sirna shows potential for the treatment of various pulmonary viral infections, and it has been reported that sirna-based therapeutics can also be used in the treatment of influenza [ ] , parainfluenza virus [ ] , severe acute respiratory syndrome (sars) [ ] , and rsv [ ] . above all, rsv is the most promising therapeutic target of sirnas. rsv is a common cause of serious respiratory infections in infants and children. it also produces significant morbidity and mortality in adult immunocompromised or elderly populations [ ] . an rsv vaccine is not available, and the only approved antiviral therapy for rsv is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. thus, a safe and efficacious rsv therapy has long been awaited for both pediatric and adult patients. rnai-based therapy has shown promising effects in murine models of rsv infection [ ] . the sirna, aln-rsv , is directed against the mrna encoding the n-protein of rsv that exhibits specific in vitro and in vivo anti-rsv activity. it is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. aln-rsv has undergone complete phase i intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated [ ] . additionally, aln-rsv has been evaluated in a randomized, double-blind, placebo-controlled phase ii trial in lung transplant patients with rsv respiratory tract infection [ ] . the administration of aln-rsv to rsv infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. based on these results, a larger multinational, randomized, double-blind phase iib trial of aln-rsv has been initiated in lung transplant patients to confirm and extend these findings. cancer is a major target of rnai-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by rnai. lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. rnai-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. the major advantage of rnai therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. the simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving sirnas and mirnas. there have already been significant improvements in sirnas for primary or metastatic lung cancer treatment by targeting oncogenes such as akt [ ] , wilms tumor (wt ) [ ] , overexpressed genes such as the insulin-like growth factor receptor (igf- r) [ ] , nupr [ ] and ezh [ ] . some of these studies have successfully shown the efficacy of rnai-based therapy through intrapulmonary administration of sirnas with non-viral vectors. although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with sirnas is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. there are currently some clinical trials in progress estimating the safety and efficacy of sirna-based drugs for cancer treatment. atu , a sirna-lipoplex targeted against protein kinase n (pkn ), prevented lung metastasis in a phase i trial of various cancer models [ ] . pkn is a downstream effector of the phosphoinositide -kinase (pi k) signaling pathway [ ] , which regulates diverse cellular responses, including development, growth, and survival [ ] . recently, pkn has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with atu in cultured primary endothelial cells showed an essential role of pkn for endothelial tube formation and migration [ ] . atu can be considered as a potential sirna for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. inflammatory lung disease, also called copd, includes a wide range of lung ailments. these related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. they are influenced by a combination of environmental, genetic, and epigenetic components [ ] . copd is a chronic inflammatory disease of the airways. this disease is hallmarked by airflow that is not fully reversible. systemic and local airway inflammation has been implicated in the pathogenesis of copd [ ] . copd is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. smoking might cause cells to senesce due to dna damage through increased cell turnover, which in turn leads to accelerated telomere shortening [ ] . lately, a lot of studies have investigated the role of cellular senescence in the development and progression of copd [ ] . although several medication classes, including inhaled corticosteroids, are used for copd treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. current interventions that have been shown to improve mortality in copd are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. many people are developing copd, and the cause of this condition is complicated and not thoroughly understood. one key factor is genetic susceptibility. some studies have shown a large genetic contribution to the variability in pulmonary function and copd [ , ] . polymorphisms in multiple genes have been reported to be associated with copd [ ] , such as transcription factor [e.g. nuclear factor-kappa b (nfκb)] [ ] , extracellular matrix (e.g., matrix metalloproteinase- (mmp- )) [ , ] , cytokines [e.g. tumor necrosis factor (tnf)-α] [ ] , chemokines [e.g. interleukins (il)- , il- receptor and chemokine receptor (ccr) ] [ , ] , and apoptosis (e.g., caspase- and vascular endothelial growth factor (vegf)) [ , ] . many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. although several new treatments that target the inflammatory process are now in clinical development, such as tnf-α inhibitors and i-kappab kinase complex (ikk ) inhibitors [ , ] , clinical trials with sirnas have never been performed in copd. the delay of drug development for copd might be due to the relatively recent emergence of research addressing the molecular basis of copd. furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of copd and to develop monitoring techniques to support the development of rnai therapies. currently, no available treatments reduce the progression of copd or suppress the inflammation in small airways and lung parenchyma. the rnai-based approach for the key molecules also has potential implications for the treatment of copd. asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. the world health organization estimates that million people are currently affected and that, by the year , another million will be affected by the disease [ ] . inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. however, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. many target genes have been identified that participate in the pathogenesis of asthma. the most promising targets include genes coding for cytokines (il- , il , and il- ), cytokine and chemokine receptors (il- receptor and ccr ), and tyrosine kinases [spleen tyrosine kinase (syk) and lck/yes-related novel tyrosine kinase (lyn)], as well as for transcription factors [signal transducers and activators of transcription (stat ), stat , gata , and nfκb] that are involved in asthma [ , , ] . the genes that have been assessed as sirna targets for the treatment of asthma in preclinical models are reported [ ] . currently, in a clinical trial for asthma, excellair tm (zabecor, bala cynwyd, pa, usa), a sirna that targets syk, is being used. the kinase is involved in signaling from a b cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. it has been reported that antisense oligonucleotides administered by aerosol were potent to decrease syk expression, mediator release from alveolar macrophages, and syk-dependent pulmonary inflammation [ ] . moreover, inhibition of inflammatory mediators was shown in a study using sirna targeting syk in airway epithelial cells [ ] . following the successful results of the company's phase i clinical trial, a phase ii trial for its asthma drug candidate excellair tm has already been initiated. some of the current treatments for asthma and other inflammatory conditions, such as tnf-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. in contrast, sirna targeting syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. overall, recent progress of sirnas to the lungs has also improved the therapeutic feasibility of rnai for inflammatory lung diseases. the rapid progress will put sirna-based therapeutics on a fast track to the clinic. mirnas are small endogenous noncoding rnas that regulate gene expression by repressing translation or promoting the degradation of their target mrna. mirnas regulate gene expression by binding to the ′ untranslated region (utr) of their target mrnas and mediating mrna degradation or translational inhibition. in the human genome, transcripts of approximately % of all mrnas are estimated to be targeted by mirnas [ ] . according to their function, mirnas play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies [ ] . a growing number of mirnas have been shown to be involved in different lung diseases. this evidence makes mirnas a promising technology for current and future therapeutic development. we discuss the role of some mirnas in various lung diseases as well as the possible future of these discoveries in clinical applications. table shows the summary of mirnas in therapeutic development. at this point, a mirna-based therapy has already entered a phase ii clinical trial. there is evidence that upregulation or downregulation of mirnas is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. many studies have focused on the role of mirnas in inflammatory lung diseases, such as copd [ , ] , pulmonary fibrosis [ ] [ ] [ ] [ ] , and asthma [ ] [ ] [ ] [ ] (table ) . [ ] [ , ] the pathogenesis of copd is attributed to not only chronic inflammation in the airways but also systemic inflammation [ ] . cigarette smoking is the main risk factor for the development of copd. smoking has been shown to cause biological change in the gene expression of the lungs [ ] , and there are some reports about smoking-related mirnas [ , , ] . however, there are few reports that focus on the mirnas related to the pathogenesis of this disease with systemic inflammatory components. recent study on pulmonary fibroblasts of copd patients presents less expression of mir- a after stimulation with proinflammatory cytokines when compared with non-copd subjects with similar smoking histories [ ] . the downregulation of mir- a resulted in a prolonged mrna half-life of cyclooxygenase- , thus increasing prostaglandin e in fibroblasts from copd subjects. moreover, ezzie et al. researched the difference of mirna profiles expressed in the lungs of smokers with and without copd. they concluded that mir- and mir- a were the most affected mirnas in subjects with copd [ ] . yet, copd is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history [ ] . a better understanding of the complexity of the disease and potential clinical relevance of the identified mirnas is needed. pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. this disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β (tgf-β) and other cytokines produced at the lesion of active fibrosis [ ] . recently, it was reported that mirnas may play an important regulatory role in the pulmonary fibrotic change in the lungs. the downregulation of let- d in idiopathic pulmonary fibrosis (ipf) resulted in increased collagen deposition and alveolar septal thickening [ ] . in addition, liu et al. reported that the oncogenic mir- was found to be upregulated in ipf patients and in the murine lungs with bleomycin-induced fibrosis [ ] . although these mirnas may be potential therapeutic targets because their expression is related to the regulation of tgf-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. pulmonary fibrosis is also a complicated illness that can have many different causes. focus on the role of mirnas in asthma has recently increased. asthma is an inflammatory disease of the airway that is characterized by an abnormal response of t helper- (th )-type cd +t lymphocytes against inhaled allergens [ ] . in a different asthmatic mouse model, there was an observed increase in the expression of mir- in the lungs [ ] . this report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of il- , favoring the th lymphocyte response. a toll-like receptor (tlr )-induced th lymphocyte induces high expression of mir- , and selective blockade of mir- suppressed the asthmatic phenotype [ ] . in addition, airway remodeling is a characteristic feature of asthma and has important functional implications. rodriguez et al. have shown that mir- is related to the development of inflammatory infiltration into the lung and airway remodeling [ ] . thus, some studies present a functional connection between mirna expression and asthma pathogenesis and suggest that targeting mirnas in the airways may lead to anti-inflammatory treatments for allergic asthma. despite the evidence from experimental models, the expression profiling of mirnas in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in mirna expression [ ] . further investigations about the role of mirnas related to asthma pathogenesis are required. although the basic evidence of mirna biology is still providing new insights, applications of mirna-based therapy for inflammatory lung diseases are less advanced than those for lung cancer [ ] . one reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. the presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. understanding the role that mirnas play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. given the significant roles that mirnas play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of mirna-based therapies, including restoring functions of tumor suppressive mirnas or inhibiting oncogenic mirnas. the development of mirna-based therapies for lung cancer is growing prosperously with the help of new rnai technologies. compared to sirna-based therapies, which are already in clinical trials, mirnas are less toxic and have the potential to target multiple genes. the difficulty associated with mirna delivery is mainly equal to that of sirnas. the critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects [ ] . there are two strategies as the therapeutic applications of mirnas for lung cancer [ ] . one strategy is mirna replacement therapy, which involves the re-introduction of a tumor suppressor mirna mimic to restore a loss of the function. mirna mimics are synthetic rna duplexes designed to mimic the endogenous functions of mirnas with chemical modifications for stability and cellular uptake. the concept of mirna replacement therapy is most exemplified by the let- mirna. let- is a tumor-suppressor mirna in non-small-cell lung cancer that inversely correlates with the expression of the ras oncoprotein, a key cancer gene [ ] . intranasal administration of let- mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that mirna replacement therapy is indeed promising [ , , ] . another mirna that shows the value of mirna replacement is provided by mir- a [ , ] . local and/or systemic delivery of a synthetic mir- a mimic led to accumulation of mir- a in the tumor tissue and inhibition of lung tumor growth. lately, ling et al. also showed that tumor suppressor mir- exhibited anti-lung cancer activity through post-transcriptional regulation of erbb [ ] . thus, therapeutic mirna mimics have a powerful potential by attacking multiple genes relevant to several diseases. however, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of mirna mimics will lead to an accumulation of exogenous mirnas in normal cells [ ] . although the assumptions are well founded, there is still insufficient evidence for toxicity caused by mirna mimics. indeed, several in vivo studies failed to reveal side effects caused by the mirna mimics and suggested that delivery of mirna mimics to normal tissues was well tolerated [ , ] . it will be important to research mirna mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. the second strategy is directed toward a gain of function and aims to inhibit oncomirs by using anti-mirnas. chemical modifications, such as '-o-methyl-group and locked nucleic acid (lna), would increase oligo stability against nucleases [ ] . antisense oligonucleotides contained in these modifications are termed antagomirs or "lna-antimirs" [ , ] . they are oligonucleotides with sequences complementary to the endogenous mirna and inhibit the specific mirna function. an lna-antimir against mir- has been shown to effectively silence mir- in non-human primates [ ] , and the findings support the potential of these compounds as a new class of therapeutics. moreover, it has also been reported that anti-mir- delivered into lung tumor xenografts in mice led to inhibited tumor growth [ ] . relative to studies on mirna mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. this illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to rnase, and pharmacologic properties. therefore, inhibition of mirna function by chemically modified antimir oligonucleotides has become an important and widely used approach. recent data from the first phase ii study in patients with chronic hcv infection treated with the lna-modified antimir- showed that this compound was well tolerated and provided continuing viral suppression. an increasing number of studies have examined the therapeutic potential of mirnas. recently, the evidence of roles for mirnas in determining drug resistance has emerged [ ] . cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. in this situation, combining mirna mimics or antimir with chemotherapy may potentiate the efficacy of the cancer treatment in the future. in addition, mirnas related with cancer stem cells may significantly broaden the field of mirna-based therapy and suggest that mirnas can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis [ , ] . hence, the main challenge is the successful delivery and chemical modifications of the therapeutic mirnas to the target tissue without harming normal tissues. rnai-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. one of the greatest challenges in rnai-based therapy continues to be the delivery method of the therapeutic sirnas and mirnas to the target cells. pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. a realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. the advancement of pulmonary sirna delivery to the clinic illustrates that rnai-based therapy holds a central place in the future treatment of lung diseases. on the other hand, mirnas have the opportunity to target multiple genes in a fine-tuned manner, and the mirna-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. in particular, anti-mirna therapy by chemically modified antimir oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. increased evidence has indicated that mirnas fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. further understanding of the detailed mechanisms of rnai-based therapy and investigations of more effective delivery methods are required for future development. these novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients. strategies for silencing human disease using rna interference rnai therapeutics: a potential new class of pharmaceutical drugs small interfering rna inhibits hepatitis b virus replication in mice rnai suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia confirming the rnai-mediated mechanism of action of sirna-based cancer therapeutics in mice chemical modification of sirna sirna and isrna: two edges of one sword targeted delivery of antisense oligodeoxynucleotide and small interference rna into lung cancer cells poly(ester amine)-mediated, aerosol-delivered akt small interfering rna suppresses lung tumorigenesis poly(beta-amino ester) as a carrier for si/shrna delivery in lung cancer cells inhibition of non-small cell lung cancer cell proliferation and tumor growth by vector-based small interfering rnas targeting her /neu rodriguez-padilla, c. wt gene silencing by aerosol delivery of pei-rnai complexes inhibits b -f lung metastases growth inhibition of influenza virus production in virus-infected mice by rna interference using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque intrapulmonary delivery of xcl -targeting small interfering rna in mice chronically infected with mycobacterium tuberculosis effective treatment of respiratory alphaherpesvirus infection using rna interference a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus rna interference: new therapeutics in allergic diseases emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease suppression of plasminogen activator inhibitor- by rna interference attenuates pulmonary fibrosis development and preclinical efficacy of novel transforming growth factor-beta short interfering rnas for pulmonary fibrosis pulmonary delivery of therapeutic sirna the lung as a route for systemic delivery of therapeutic proteins and peptides spray drying of sirna-containing plga nanoparticles intended for inhalation design of an inhalable dry powder formulation of dotap-modified plga nanoparticles loaded with sirna silencing of fas, but not caspase- , in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis in vivo gene silencing (with sirna) of pulmonary expression of mip- versus kc results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury nonviral sirna delivery to the lung: investigation of peg-pei polyplexes and their in vivo performance intratracheal versus intravenous liposomal delivery of sirna, antisense oligonucleotides and anticancer drug attenuation of fibrosis in vitro and in vivo with sparc sirna rnai-mediated suppression of constitutive pulmonary gene expression by small interfering rna in mice intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations non-invasive pulmonary aerosol delivery in mice by the endotracheal route pulmonary vaccine delivery inhibition of respiratory viruses by nasally administered sirna noninvasive delivery of small inhibitory rna and other reagents to pulmonary alveoli in mice immunological and toxicological implications of short-term studies in animals of pharmaceutical aerosol delivery to the lungs: relevance to humans evaluation of the safety, tolerability and pharmacokinetics of aln-rsv , a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) clinical evaluation of inhaled insulin inhalable lactose-based dry powder formulations of low molecular weight heparin extracellular barriers in respiratory gene therapy nanodelivery in airway diseases: challenges and therapeutic applications expression of respiratory mucins in fatal status asthmaticus and mild asthma remodeling in asthma and chronic obstructive lung disease polymeric nanocarriers for drug delivery to the lung nonviral delivery of synthetic sirnas in vivo rapid crossing of the pulmonary endothelial barrier by polyethylenimine/dna complexes delivery of sirna therapeutics: barriers and carriers cellular sirna delivery using cell-penetrating peptides modified for endosomal escape delivering silence: advancements in developing sirna therapeutics gene silencing by adenovirus-delivered sirna effect of adenovirus-mediated rna interference on endogenous micrornas in a mouse model of multidrug resistance protein gene silencing lentivirus-mediated rnai knockdown of nupr inhibits human nonsmall cell lung cancer growth in vitro and in vivo cancer gene therapy: challenges and opportunities us gene therapy in crisis rnai-dependent and -independent antiviral phenotypes of chromosomally integrated shrna clones: role of vasp in respiratory syncytial virus growth non-viral sirna delivery to the lung lipid-based systemic delivery of sirna protection against lethal influenza virus challenge by rna interference in vivo fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by pi -kinase and nf-kappa b full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung cationic liposome-mediated delivery of sirnas in adult mice lipidic systems for in vivo sirna delivery expression profiling reveals off-target gene regulation by rnai induction of an interferon response by rnai vectors in mammalian cells lack of interferon response in animals to naked sirnas rnai-mediated gene-targeting through systemic application of polyethylenimine (pei)-complexed sirna in vivo systemic delivery of rafsirna using cationic cardiolipin liposomes silences raf- expression and inhibits tumor growth in xenograft model of human prostate cancer pulmonary gene silencing in transgenic egfp mice using aerosolised chitosan/sirna nanoparticles a combinatorial library of lipid-like materials for delivery of rnai therapeutics immunologic aspects of chronic obstructive pulmonary disease drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics suppression of ocular neovascularization with sirna targeting vegf receptor the us national institutes of health respiratory syncytial virus infection in elderly and high-risk adults rna interference therapy in lung transplant patients infected with respiratory syncytial virus down-regulation of igf-ir using small, interfering, hairpin rna (sirna) inhibits growth of human lung cancer cell line a in vitro and in nude mice ezh silencing with rnai enhances irradiation-induced inhibition of human lung cancer growth in vitro and in vivo atu , a liposomal small interfering rna formulation targeting protein kinase n , inhibits cancer progression pkn is required for malignant prostate cell growth downstream of activated pi -kinase cellular function of phosphoinositide -kinases: implications for development, homeostasis, and cancer the stability of mrna influences the temporal order of the induction of genes encoding inflammatory molecules inhaled and systemic corticosteroids in chronic obstructive pulmonary disease cigarette smoke induces senescence in alveolar epithelial cells senescence in chronic obstructive pulmonary disease identifying targets for copd treatment through gene expression analyses targeting the nf-kappab pathway in asthma and chronic obstructive pulmonary disease neutrophil elastase contributes to cigarette smoke-induced emphysema in mice pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease tumor necrosis factor-alpha gene polymorphism in chronic bronchitis characterization of cigarette smoke-induced inflammatory and mucus hypersecretory changes in rat lung and the role of cxcr ligands in mediating this effect comprehensive gene expression profiling of rat lung reveals distinct acute and chronic responses to cigarette smoke inhalation alveolar wall apoptosis causes lung destruction and emphysematous changes inhibition of vegf receptors causes lung cell apoptosis and emphysema copd: current therapeutic interventions and future approaches p mapk inhibitors, ikk inhibitors, and tnfalpha inhibitors in copd chronic respiratory disease-asthma sirna as a therapy for asthma targeting allergic airway diseases by sirna: an option for the future? antisense-and rna interference-based therapeutic strategies in allergy aerosolized syk antisense suppresses syk expression, mediator release from macrophages, and pulmonary inflammation syk tyrosine kinase participates in beta -integrin signaling and inflammatory responses in airway epithelial cells many roads to maturity: microrna biogenesis pathways and their regulation micrornas in lung diseases regression of murine lung tumors by the let- microrna development of a lung cancer therapeutic based on the tumor suppressor microrna- the microrna mir- a inhibits prostate cancer stem cells and metastasis by directly repressing cd dysregulation of micrornas after myocardial infarction reveals a role of mir- in cardiac fibrosis therapeutic silencing of microrna- in primates with chronic hepatitis c virus infection control of stress-dependent cardiac growth and gene expression by a microrna mir- family regulates postnatal mitotic arrest of cardiomyocytes microrna- promotes palmitate-induced apoptosis in cardiomyocytes by down-regulating sirt microrna- delays als progression and promotes regeneration of neuromuscular synapses in mice defective erythroid differentiation in mir- mutant mice mediated by - - zeta downregulation of the serum response factor/mir- axis in the quadriceps of patients with copd microrna expression in induced sputum of smokers and patients with chronic obstructive pulmonary disease mir- mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis inhibition and role of let- d in idiopathic pulmonary fibrosis mir- is a major regulator of genes associated with pulmonary fibrosis participation of mir- in pulmonary fibrosis requirement of bic/microrna- for normal immune function microrna- is up-regulated in allergic airway inflammation and regulates il- p expression antagonism of microrna- suppresses the effector function of th cells and the development of allergic airways disease down-regulation of mir- a contributes to up-regulation of rhoa in bronchial smooth muscle cells gene expression networks in copd: microrna and mrna regulation reduced mir- a increases prostaglandin e( )in chronic obstructive pulmonary disease fibroblasts identification of keratinocyte growth factor as a target of microrna- in lung fibroblasts: implication in epithelial-mesenchymal interactions downregulation of microrna expression in the lungs of rats exposed to cigarette smoke micrornas as modulators of smoking-induced gene expression changes in human airway epithelium copd as a lung disease with systemic consequences--clinical impact, mechanisms, and potential for early intervention transcriptomic studies of the airway field of injury associated with smoking-related lung disease copd, a multicomponent disease: implications for management asthma and allergic inflammation microrna expression profiling in mild asthmatic human airways and effect of corticosteroid therapy micrornas in inflammatory lung disease--master regulators or target practice? artificial micrornas as sirna shuttles: improved safety as compared to shrnas in vitro and in vivo the promise of microrna replacement therapy ras is regulated by the let- microrna family the let- a microrna protects from growth of lung carcinoma by suppression of k-ras and c-myc in nude mice the let- microrna reduces tumor growth in mouse models of lung cancer nanoparticles modified with tumor-targeting scfv deliver sirna and mirna for cancer therapy tumor suppressor mir- suppresses lung cancer cell progression through post-transcriptional regulation of erbb silencing of micrornas in vivo with "antagomirs lna-mediated microrna silencing in non-human primates regression of a lung cancer tumors by anti-mir- vector role of microrna in anticancer drug resistance microrna mir- inhibits human pancreatic cancer tumor-initiating cells this work was supported in part by a grant-in-aid for the third-term the authors declare that there are not conflicts of interest to report. key: cord- -y sfr c authors: nan title: scientific programme date: - - journal: pediatr nephrol doi: . /s - - - sha: doc_id: cord_uid: y sfr c nan the kidney plays an important role in ion homeostasis in the human body. several hereditary disorders characterized by perturbations of renal magnesium reabsorption leading to hypomagnesemia were described over the past years. only recently, mutations in renal ion channels and transporters have been identified in several of these diseases, following positional cloning strategies in families with these disorders. muations in the slc a gene have been identified in patients with gitelman syndrome, an autosomal recessive disorders characterized by hypokalemia, hypomagnesemia and hypocalciuria. slc a encodes the thiazide-sensitive na + , cl-cotransporter (ncc) in the distal convoluted tubule (dct), the nephron site of active renal magnesium reabsorption. the hypomagnesemia in gitelman syndrome has been shown to be secondary to the primary defect in ncc. mutations in the fxyd gene have been found in patients with autosomal dominant renal hypomagnesemia associated with hypocalciuria (idh). fxyd encodes the gamma-subunit of the na + , k + -atpase, which is expressed primarily in the kidney with the highest expression levels in dct and tal. hypomagnesemia in patients with idh can be severe (< . mm) and cause generalized convulsions. the molecular mechanism for renal mg + loss in this autosomal dominant type of primary hypomagnesemia remains to be elucidated. in patients with autosomal recessive hypomagnesemia with hypercalciuria and nephrocalcinosis (fhhnc) mutations in the cldn gene, and very recently also in the cldn gene, have been identified. these genes encode claudin and claudin respectively, which are essential components of the paracellular pathway for magnesium at the level of the thick ascending limb of henle's loop. mutations in trpm , encoding the epithelial mg + channel trpm in the apical membrane of dct cells, have been identified in patients with mg + wasting and secondary hypocalcemia (hsh). this autosomal recessive disease is due to defective intestinal and renal mg + (re)absorption, and affected individuals show neurologic symptoms of hypomagnesemic hypocalcemia, including seizures and muscle spasms during infancy. in my lecture, i will give an update on the proteins involved in magnesium reabsorption and the emerging pathophysiological understanding of hereditary disorders in which renal handling of this divalent ion is disturbed. transcriptional control of genes that regulate tissue patterning and the cell cycle is fundamental to normal renal development. the mammalian kidney arises from reciprocal inductive tissue interactions between the ureteric bud and the metanephric mesenchyme. these interactions result in growth and branching of the ureteric bud and its daughter collecting ducts, a process termed, renal branching morphogenesis, and formation of nephrogenic progenitors and mature nephrons. expression and activity of transcription factors in ureteric and metanephric mesenchyme cells determines the patterning of ureteric bud and metanephric mesenchyme derived tissue elements. this lecture will highlight transcription factors critical to renal branching morphogenesis and nephrogenesis, with particular emphasis on those factors that are mutated in humans with renal malformation. the regulation of transcription factor activity will be discussed in the context of growth factor signaling pathways downstream of wnts, bone morphogenetic proteins and sonic hedgehog and pathways such as notch that control cell differentiation. the complexity of transcriptional signaling will be highlighted in the normal and malformed kidney at the level of transcriptional factor interactions and target gene promoters that can be targeted by mutiple signaling pathways. t. benzing diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. recent studies have emphasized the critical role of the slit diaphragm of podocytes for the size-selective filtration barrier of the kidney and revealed novel aspects of the mechanisms leading to proteinuria, both in inherited and acquired diseases. several critical structural protein components of the slit diaphragm have been identified. recently, it has been shown that slit diaphragm proteins, in addition to their structural functions, participate in common signaling pathways. this talk will focus on what is known about the importance of the podocyte for the function of the glomerular filter of the kidney. it will provide a snapshot of our current understanding of the signaling properties of slit diaphragm proteins and project a framework for further studies necessary to delineate the function and dynamics of the slit diaphragm protein complex and the pathogenesis of nephrotic syndrome. long-term survival of children with end-stage renal disease (esrd) has increased in the last years but the mortality rate remains high. cardiovascular disease accounts for to % of all deaths, infectious disease about %. a prolonged period of dialysis versus having a renal graft and persistent hypertension are mortality risk factors. the prevalence of all sorts of morbidities is high among those who have reached adulthood. nearly % of all these patients suffer from left ventricular hypertrophy and life threatening vascular changes; nearly one third has clinical signs of metabolic bone disease. this accounts for both dialysis and transplanted patients. the chance of getting cancer is times increased as compared to the general population; skin cancer and non-hodgkin lymphomas are most reported. a long period of dialysis at childhood is associated with impairment of both cognitive and educational attainment in adulthood. yet, despite all these negative outcomes, the health perception of young adults with childhood onset esrd is good. unemployment under young adults on chronic dialysis with pediatric onset of disease is higher than among healthy age related peers, but much lower than among dialysis patients of the same age with adult onset of disease. an impaired social development in childhood is associated with an impaired mental health perception at adult age. research and therapy in children with esrd should focus not only on prevention of graft failure but also on prevention of co-morbidity, especially cardiovascular disease, life threatening infections and malignancies. early transplantation, more extended forms of frequent hemodialysis in those who can not be transplanted, a more rigorous treatment of hypertension, avoidance of calcium-containing phosphate binders, reduction of the chronic inflammatory state, and tailor made anti-rejection therapy after transplantation may all be targets to improve outcome in future patients. psychosocial care should be more focused on attainment of independency and preparation for adult life, i. e. schooling and job career. applying regenerative medicine to combat organ shortage laboratory of regenerative nephrology, edmond and lili safra children's hospital, sheba medical center, tel hashomer, sackler school of medicine, tel aviv university, tel aviv, israel organ transplantation has been one of the major medical advances of the past years; however, it is becoming increasingly apparent that the supply of organs is limited and will not improve with current medical practice. regenerative medicine is focused on the development of cells and tissues for the purpose of restoring function through transplantation. when facing late stages of esrd, which necessitate whole kidney transplantation, organogenesis represents an alternative to combat organ shortage. indeed, previous data pinpoints a window of time in human and pig kidney organogenesis that may be optimal for transplantation into mature recipients. "window" transplants are defined by their remarkable ability to grow, differentiate and undergo vascularization, achieving successful organogenesis of urine-producing miniature kidneys with no evidence of trans-differentiation into non-renal cell types, lack of tumorigenicity and reduced immunogenicity compared to adult counterparts. in contrast, when dealing with earlier stages of esrd or acute renal disease, which allow for individual cell replacement, both bonemarrow derived and kidney specific stem cells, might be applicable. accordingly, we show in proof-of-principle experiments that a novel population of multi-potential stem cells derived from the adult murine kidney are capable of re-populating ischemically injured tubular cells, while hematopoietic stem cells (cd +cd +) or hemangioblastic progenitor cells can give rise to peritubular endothelial cells. thus, different types of biological materials (embryonic kidney, adult kidney, adult bone marrow) offer new and powerful strategies for future tissue development and regeneration in renal medicine. k. tory, É. kis, a. szabó, g. reusz st department of pediatrics, semmelweis university, budapest, hungary it is well known that chronic renal failure significantly increases the risk of cardiovascular mortality in adults. transplantation decreases this risk; however, it still exceeds that of the normal population. as the number of children on renal replacement therapy (including transplantation) has significantly increased in recent years, there has been a growing amount of evidence regarding the increased cardiovascular vulnerability of this specific patient group. one important factor of cardiovascular mortality in adults is the dysfunction of the autonomic nervous system, previously designated as autonomic neuropathy. in a series of studies using the conventional ewing tests as well as heart rate variability (hrv) monitoring we were able to show altered sympathovagal balance in children on peritoneal-and hemodialysis, that was at least partly reversible following renal transplantation. according to our data sympathetic overactivity plays a major role in the sympathovagal disequilibrium observed. correspondingly, beta-adrenergic blockade improves the decreased hrv in the young. early, accelerated arteriosclerosis is another important, recently recognized consequence of crf in children. arterial calcification leads to increased arterial stiffness deteriorating the cushioning function of the aorta. arterial stiffness can be assessed by the reproducible and non-invasive measurement of the pulse wave velocity (pwv). in adults, it was found to be a strong, independent predictor of cardiovascular mortality. hypertension, hyperphosphatemia/elevated serum calciumphosphate product and vitamin d deficiency are the principal factors associated with pwv. recently, pwv was also shown to be increased in children on dialysis. however, the difference to the normal population may only be apparent if a control group matched for body dimensions is used, because of the dependence of pwv on body dimensions and uremic children's significant growth deficit. furthermore, increased pwv could also be shown in children following renal transplantation (tx) indicating the persistence of these lesions even following successful tx. the data on the autonomic nervous system and arterial dysfunction in young patients point to the necessity of early prevention in order to avoid the cardiovascular complications of crf. this study was supported by grants otka-t -f -f and ett / . nitric oxide (no) production is reduced in ckd due to decreased renal and widespread decreases in endothelial no production. possible causes of no deficiency are: ). substrate (l-arginine) limitation; ). increased levels of circulating endogenous inhibitors of nitric oxide synthase (particularly asymmetric dimethylarginine [adma]); ). decreased nos protein abundance/activity. decreased l-arginine availability in ckd is likely due to perturbed renal biosynthesis secondary to damage to the renal parenchyma. in addition, inhibition of transport of larginine into endothelial cells and shunting of l-arginine into other metabolic pathways (e. g. arginase) will also decrease availability. elevated plasma and tissue levels of adma in ckd are functions of reduced renal excretion (minor), reduced catabolism by dimethylarginine dimethylamino-hydrolase (ddah) and possibly increased protein methylation. an increase in adma has emerged as a major independent risk factor in many forms of cardiovascular disease as well as end-stage renal disease (and probably ckd). decreased renal nos protein abundance and activity have been reported by us in multiple models of ckd in the rat. the neuronal (nos ) isoform is always reduced in the presence of injury, and is preserved in settings where there is protection from damage (female; wistar furth rat vs. sprague dawley). generalized and nnos specific inhibition accelerates underlying renal injury and in vulnerable animals will cause injury in the absence of underlying renal disease. we also find that relaxin (rlx) mediated prevention and repair of damaged kidneys requires an intact no system in order to function. in summary, no deficiency can cause cardiovascular and renal disease, and ckd results in no deficiency, contributing to a vicious cycle that promotes progression. an intact no system is required for rlxmediated repair of kidney damage. no deficiency in ckd is likely due to many causes: decreased arginine synthesis/availability and transport; increased endogenous adma functioning as competitive inhibitor; decreased enzyme abundance and activity will all lead to reduction in no generation. in addition, the oxidative stress of ckd will further reduce nos activity, switch the nos to become oxidant generators and will scavenge no to form the harmful peroxynitrite. selected glomerulopathies due to single gene defects such as finnish type nephropathy, diffuse mesangial sclerosis alport/thin membrane disease and inherited lipid disorders and will be discussed. finnish nephropathy is due to mutations of nephrin, a major structural component of the slit diaphragm. over nephrin mutations are known associated with variably absent slit diaphragms, originally thought to be specific for constitutional nephrin mutations. however, sporadic minimal change disease and membranous glomerulonephritis may have absent slit diaphragms suggesting that nephrin may participate in the nephrotic syndrome in nonhereditary diseases. dms is one cause of congenital nephrotic syndrome, characterized by podocyte proliferationis shared by wt (denys-drash syndrome), laminin α (pierson syndrome) and galloway-mowat syndrome mutations. alport nephritis is a paradigm in which only a subset of mutations may predict disease severity (col ivα )). heterozygous alport is similar to thin membrane disease (tmd), but % of tmd harbor col (iv) α mutations and a subset has col ivα . nail patella syndrome due to heterozygous loss of lmx b gene which regulates col ivα - expression during kidney development. in classic nail-patella pathology fibrillar collagen bundles within the gbm are identical in severe and mild disease. thus the mutation does not correlate with prognosis. a mutation overlap in factor h gene in lipid disorders such as dense deposit disease (ddd) and lcat underscores glomerular pathologies that ranges from classic ribbon-like deposits in ddd, to lucent gbm deposits characteristic of lcat, or subendothelial lipid pools in lipoprotein glomerulopathy. the importance of genetics underscoring the pathology is amply demonstrated but mutations may not determine prognosis. background genes, post-translational modification and or protein/protein interactions in podocytes or the gbm may act as phenotypic modifiers. acute renal failure (arf) has many different definitions. rifle criteria distinguish "risk, injury, failure, loss and end-stage renal disease" features of this event. classic forms include pathophysiological cases of renal hypoperfusion and direct parenchymal injury, as well as postrenal anatomical obstruction. microvascular mechanism is in general the effect of disturbed balance between vasoconstriction (in response to endothelin, angiotensin ii, thromboxane a , leukotrienes and increased sympathetic nerve activity) and vasodilatation (in response to nitric oxide, pge and bradykinin). endothelial and vascular smooth muscles cells may undergo structural damage and increased leukocytes-endothelial adhesion is a cause of inflammation. cytoskeletal breakdown, loss of polarity, apoptosis, necrosis, desquamation of viable and necrotic cells with tubular obstruction are underlying mechanisms of acute tubular necrosis. ischemic and direct tubular injury dominate as causes of arf, however specific epidemiology is strictly age-related. sepsis and major surgeryrelated events are the most common causes of arf in hospitalized patients. data on genetic background of certain genes polymorphisms and susceptibility to specific risk factors in newborns and infants are conflicting. several prophylactic and therapeutic techniques are available, however not of universal value. appropriate fluid management is crucial in ischemic arf, classic hemolyticuremic syndrome and rhabdomyolysis, however fluid overload is the one of major predictors of poor outcome in children admitted to icu. neither "renal dose" of dopamine, nor loop diuretics change the outcome. involvement of extrarenal organs worsens the overall outcome, which is the poorest in patients with multi-organ failure. early introduced renal replacement therapy is one of the key modalities in icu-treated patients. continuous techniques are of major value. in specific cases, such as hepato-renal syndrome, albumin ("liver") dialysis serves as an effective bridge to liver transplantation. hyperostosis-hyperphosphatemia syndrome (hhs) is a rare recessively inherited disease, manifested by persistent severe hyperphosphatemia and self-remitting episodic bone pain with radiologic findings of periosteal reaction and cortical hyperostosis. hyperphosphatemia in this patient population is not counter-balanced by pth or vitamin d, posing a mirror image of two hypophosphatemic states which result from increased activity of fibroblast growth factor (fgf ). the two hypophosphatemic disorders which result from enhanced urinary phosphate leak are: dominantly inherited hypophosphatemic rickets and tumor induced osteomalacia. this observation was the impetus to study the role of fgf in hhs. affected individuals were found to have low levels of the full length, biologically active fgf , but markedly augmented amounts of the cleaved inactive fragments. patients were found to be homozygous for a mutation in the galnt gene encoding a peptide involved in mucin-type o-glycosylation. decreasing the expression of the galnt gene by rna interference resulted in augmenting processing of the intact fgf . our research indicates that the primary defect in hhs is a state of underglycosylation of fgf- resulting from reduced expression of ppgantase-t , due to mutations in galnt , and leading to augmented processing at the cleavage site. these changes in fgf- would abolish its phosphaturic effect and lead to severe persistent hyperphosphatemia. this study provides the pathogenetic mechanism of the first mucin-type o-glycosylation defect identified. our observation lends further credence to the primary and essential role of fgf in phosphate homeostasis through a pth-independent pathway. this was substantiated most recently by several reports which showed that mutations in the fgf gene are responsible for familial hyperphosphatemic tumoral calcinosis (fhtc) . a further study showed that hhs and familial hyperphosphatemic tumoral calcinosis are allelic disorders with a founder mutation in our region. immune responses govern the outcome of many forms of chronic kidney disease. gene therapy offers the potential to modify immune genes to improve outcome. we will discuss the potential use of gene transduction as a therapy for chronic kidney disease. background: chronic proteinuric renal disease is a major cause of end stage renal disease in man. adriamycin nephropathy is a murine model of chronic proteinuric renal disease where initial chemical injury is followed by immune and structural changes that mimic human disease. foxp is a gene specifically expressed by regulatory t (treg) cells. forced expression of the gene foxp causes transduced t cells to develop a regulatory phenotype. we hypothesised that foxp transduced regulatory t cells could protect against renal injury in adriamycin nephropathy. methods: the retroviral vectors expressing foxp and green fluorescent protein (gfp) (foxp /migr) and gfp alone (migr) were transfected into package cell lines ecopack - , which produced the two retroviruses. cd + t cells were isolated from spleen of balb/c mice and stimulated by anti-cd mab and il- for hours and then were infected with either retrovirus. expression was confirmed and phenotypic and in vitro functional assays demonstrated a regulatory phenotype. one week after infection, the gfp+ positive cells were sorted. foxp and control vector (migr) transduced t cells were administered to adriamycin (adr)-induced progressive renal nephropathy in mice. results: adoptive transfer of the foxp transduced t cells protected against renal injury. urinary protein excretion was reduced; there was less renal injury as measured by glomerulosclerosis, and interstitial infiltrates. serum creatinine, glomerular sclerosis and tubulointerstitial alterations were significantly lower in adr-foxp group, compared to those without treatment (adr) and treated with control vector (migr) transduced group (adr+migr). the foxp transduced cd t cells also showed suppressive activity in vitro. we conclude that foxp induced t reg cells may have a therapeutic role in protecting against immune injury and disease progression in chronic proteinuric renal disease. the italkid project is a prospective, population-based registry that was started in . prevalent and incident cases of chronic renal insufficiency (cri) in children and adolescents were identified throughout italy (total population base: . million children). the inclusion criteria were: i) creatinine clearance (ccr): < ml/min/ . m bsa; and ii) an age of < years at the time of registration. as to december st, a total of patients had been registered. the incidence of cri was estimated . cases per million and the (point) prevalence . per million of the age-related population (marp). the probability of kidney survival at years of age was significantly different depending on the ccr at study entry, being % in patients with mild renal insufficiency (ccr - ml/min), % in those with moderate renal insufficiency (ccr - ml/min) and % in those with severe renal insufficiency (ccr < ml/min). the patients with normal (< . ) and low ( . - . ) upr/ucr compared to those with mild (> . ), showed a significantly slower decline in ccr (deltaccr + . ± . and - . ± . vs - . ± . ml/min/yr) and a higher kidney survival ( . and . vs . %). the incidence of renal replacement therapy (rrt) was . /year/ patients and the casefatality rate on conservative treatment . %. patients showed a significantly different slope of ccr before pubertal growth spurt as compared to after: - . ± . ml/min/ . mq/yrs and - . ± . . a non-linear pattern of decline in the probability of kidney survival, with a steep decrease during pubertal and early post-pubertal age was observed: the overall probability of rrt at age was estimated . %, while . % of the patients will eventually required rrt before the nd decade of life was over. suggesting that pubertal development triggers the progression of cri in children. treatment with angiotensin converting enzyme inhibitors did not significantly modify the naturally progressive course of hypodysplastic nephropathy. ambulatory blood pressure monitoring (abpm) is a relatively new technique of blood pressure assessment in children and adolescents that offers several distinct advantages over traditional methods of blood pressure measurement, including the ability to detect white coat and masked hypertension, as well as the ability to assess nocturnal blood pressure. the role of abpm in the diagnostic evaluation of pediatric patients with elevated blood pressure is well-established, and recent surveys have demonstrated that it is used quite often by pediatric nephrologists and other practitioners during the initial evaluation of elevated blood pressure. it is less well-established, however, what role abpm might play in hypertensive children and adolescents once hypertension has been diagnosed and treatment initiated. studies in adults have demonstrated that abpm can assess whether patients on antihypertensive medications have achieved goal blood pressure, or whether their hypertension has progressed in severity. abpm can be used to follow nocturnal blood pressure in patients with chronic kidney disease (ckd) and diabetes, facilitating early identification of non-dipping, which is a known risk factor for progression of ckd or development of diabetic nephropathy, respectively. abpm can also be incorporated into clinical trials of antihypertensive medications to help assess their efficacy and safety. while additional data supporting these applications of abpm to pediatric hypertension clearly need to be generated from well-designed clinical trials, we propose that there is ample justification to utilize abpm just as frequently after the diagnosis of hypertension as before. childhood obesity is increasing globally in epidemic proportions and affects children in both industrialized and non-industrialized nations. in the last years the percentage of overweight or obese children has increased from % to almost %. if current trends continue, as many as % of children may be overweight or obese by the year . obesity predisposes to development of the metabolic syndrome, which is defined in children as the presence of three or more of the following features: abdominal adiposity (bmi > %ile), serum triglycerides > %ile, serum hdl cholesterol < % ile, fasting blood glucose > mg/dl, and/or hypertension (systolic or diastolic blood pressure > %ile adjusted for age, gender and height). the metabolic syndrome occurs in about % of children, but in - % of overweight children. the presence of the metabolic syndrome increases the risk for cardiovascular disease and chronic kidney disease almost ten-fold in adults. adipose tissue does not just store fat, but also has important endocrine and immune functions mediated through adipocytokines, including leptin, adiponectin, resistin, apelin and visfatin, and classical cytokines such as tnf-a and il- . increased leptin, decreased adiponectin and increased inflammatory cytokines, which occur in obesity, are known to induce vascular endothelial dysfunction and increase blood pressure. increase in adipose tissue also leads to infiltration by monocytes, macrophages and lymphocytes, which are stimulated to produce additional cytokines that may contribute to the systemic inflammation associated with obesity and vascular inflammation associated with hypertension. screening for hypertension and metabolic syndrome in obese children is critical to allow early identification of the metabolic syndrome and aggressive early intervention to reduce the risks for progression to cardiovascular disease and chronic kidney disease in later life. therapeutic strategies must include lifestyle changes of weight loss, healthier diet and regular physical exercise as well as treatment of hypertension, hyperlipidemia or hyperglycemia. blood pressure (bp) regulation is affected by numerous physiologic, biochemical, genetic and environmental factors. it has been suggested that exogenous conditions affecting intra-uterine growth and development may pre-program individuals for hypertension, metabolic abnormalities and cardiovascular morbidity in later life. animal data and human autopsy findings support a link between intrauterine growth, nephron endowment and postnatal hypertension. conceptually, it appears increasingly unclear whether the association of birth weight and bp in later life is mediated by intrauterine growth retardation as suggested by various animal models, whether prematurity per se affects bp programming independent of the fetus, nutritional status, or whether postnatal circumstances statistically linked to low birth weight affect this relationship. we have designed a study to evaluate the relationship between gestational age, birth weight and bp abnormalities by applying abpm in a group of children born preterm with and without intrauterine growth retardation and a local control group of children born at term with appropriate weight. this study represents the first systematic assessment of -hour cardiovascular regulation in children and adolescents born preterm. our findings indicate that a fraction of these preterm born subjects has a selective nocturnal increase in systolic bp, resulting in an elevated prevalence of non-dipping. our analysis suggests that intrauterine growth retardation, rather than prematurity per se, is the major effector of the early cardiovascular abnormalities observed in preterm children. moreover, we have found that nocturnal systolic bp was closely linked to heart rate, pointing to a possible role of sympathetic hyperactivation. while little data is available regarding a link between low birth weight and/or prematurity and sympathoadrenal function in adult humans, a role of the sympathetic nervous system in the programming of adult hypertension has been consistently demonstrated in various animal models of fetal growth retardation. in conclusion, we detected subtle abnormalities of circadian bp regulation in those preterm born children who suffered from intrauterine growth retardation and this may reflect sympathetic hyperactivation. the intrinsic tendency of kidney disorders with reduced nephron mass to progress and the quest for renoprotective strategies are an ongoing focus of renal research. in adults, hypertension is not only a marker but also a major driving force of renal failure progression. renin-angiotensin system blockers (ace inhibitors and at receptor blockers) are preferred antihypertensive drugs in ckd due to their specific renoprotective effects beyond blood pressure (bp) control, mediated by their antiproteinuric, antiinflammatory and antifibrotic properties. it is less clear whether bp reduction to low-normal values has an additional salutory effect on gfr preservation. children suffer from a markedly different spectrum of renal diseases than adults, with a preponderance of hypo/dysplastic kidney malformations. hypertension and proteinuria are common but usually moderate. to elucidate the renoprotective efficacy of ace inhibition and strict bp control in children, the escape trial was launched by a consortium of european pediatric nephrologists. a total of children and adolescents with stage ii-iv ckd received a fixed dose of ramipril and were randomized to intensified (< th percentile) or conventional bp control ( th- th pct). h-bp, proteinuria and gfr were monitored over a -year period. h mean arterial pressure (map) was reduced by ramipril from to mm hg (i. e. from . to sds) on average. subsequently, mean map was lowered further to mm hg at months in the intensified arm, and maintained around mm hg ( . sds) in the conventional control arm (p< . ). on average . antihypertensive drugs were added to ramipril in the intensified and . in the conventional treatment arm (p= . ). treatment tolerability was excellent in both arms, with less than % dropouts due to side effects. in summary, ramipril was effective and well tolerated in children with ckd. it is possible and safe to target low-normal bp in children. final results regarding renal survival will be available in summer and presented at the ipna meeting. nephronophthisis and joubert syndrome: converging on convergent extension? nephronophthisis (nphp), an autosomal recessive cystic kidney disease, leads to terminal kidney failure in adolescence. nphp may be associated with retinitis pigmentosa (rp) in senior-loken syndrome (slsn) or with cerebellar vermis aplasia in joubert syndrome (jbts). we have identified by positional cloning genes as mutated in nphp. the gene product of nphp , nephrocystin- , acts in focal adhesion signaling. by positional cloning we detected mutations in the nphp /inversin gene as causing infantile nphp (type ) with association of situs inversus or rp. we demonstrated expression of nphp , in primary cilia of renal epithelial cells, supporting a new unifying theory for the pathogenesis of cystic kidney diseases (watnick et al. nature genet : , ) , stating that the products of all genes mutated in cystic kidney disease in humans, mice, or zebrafish are expressed in primary cilia, basal bodies, or centrosomes of renal epithelial cells (hildebrandt et al. nature rev genet : , ) . identification of nphp mutations in nphp type also revealed the cause of the renal cystic disease mouse model "pcy", for which treatment has been demonstrated. positional cloning of nphp led to the demonstration that its gene product, nephrocystin- , is conserved in c. elegans and expressed together with nephrocystin- in ciliated head and tail neurons of the nematode. the role of primary cilia function for retinal-renal syndromes was confirmed by identification of the novel nphp gene. recently, several signaling pathways have been implicated in the downstream signaling pathways that connect ciliary/basal body function to the renal cystic phenotype. these include the wnt signaling/planar cell polarity pathways, and the hedgehog signaling pathway. we implicated the planar cell polarity signaling pathway in nphp by positional cloning of mutations in the gene nphp /cep as causing joubert syndrome. abrogation of nphp function in zebrafish caused planar cell polarity (convergent extension) defects and recapitulated the human phenotype of joubert syndrome. further gene identification in nphp will result in the definition of functional networks of primary cilia, centrosomes, and planar cell polarity as they pertain to the pathogenic mechanisms of nphpassociated syndromes and other cystic kidney diseases. hnf b is a transcription factor that is expressed in bile ducts, intestine, pancreas and renal epithelia. germ-line inactivation of the mouse hnf β/tcf gene is embryonic lethal (e . ) due to defective differentiation of the extra-embryonic visceral endoderm. hnf β is later expressed in endoderm derivatives and in the mesonephros. to understand the function of hnf b at later stages of development and during organogenesis, we applied a conditional inactivation based on a (cre-loxp) strategy. with the use of a cre recombinase that is expressed in renal collecting ducts and henle loops (ksp-cre) we found that hnf b inactivation leads to polycystic kidney disease (pkd). this is reminiscent of the renal phenotype of patients carrying heterozygous mutations in tcf /hnf β. these patients suffer from maturity onset diabetes of the young type (mody ) and at the same time from renal cysts (renal cysts and diabetes or rcad). this cystic phenotype is linked to the defective expression of pkhd and pkd , two genes mutated in pkd patients. the cellular and molecular mechanisms underlying pkd are still poorly understood. it was recently speculated that planar-cell-polarity signalling (wnt) could be at the basis of cyst formation. maturation of nephrons during development is accompanied by a considerable lengthening of tubules. this process involves an intense proliferative phase without any increase in tubule diameter. interestingly, we discovered that the progeny of consecutive cell divisions are adjacent one another and oriented in parallel to the axis of tubules. this suggested that upon lengthening, cells divide according the tubule axis. in addition, d image reconstructions revealed that oriented cell division is due to the alignment of the mitotic spindle with the axis of the tubule. we hypothesized that oriented cell division could play an essential role in preventing tubular dilation during the massive proliferative phase that accompanies tubular elongation. indeed, our results indicated that both in ksp-cre-kidney-specific-hnf β-deficient pups and in the pck rats, lacking the expression of pkhd , the mitotic alignments were highly distorted. our results indicate that hnf β plays a crucial role in activating the expression of genes involved in the control of tubular size maintenance during tubular elongation. emerging evidence suggests that the intravenous injection of bone marrow-derived cells improves renal function after acute tubular injury. examination of human transplant biopsies of female kidneys that had been transplanted into male recipients have shown the presence of tubular cells that co-express the y chromosome and epithelial markers. however, controversy exists as to whether the protective effect is due to engraftment of the cells in the injured tubule or an endocrine/paracrine effect of the injected cells. our studies demonstrate that intravenous infusion of whole bone marrow from male mice into female recipients results in the appearance of significant numbers of y chromosome + cells in the kidney interstitium, and rare y chromosome + cells in the tubules. the majority of the interstitial cells express leukocyte markers such as cd . in addition, we have found that i. v. or i. p. injection of bone marrow stromal cells (msc, adherent non-hematopoietic marrow cells) into mice reduced the severity of cisplatin-or ischemia-induced aki. examination of these kidneys demonstrates that mscs enhance tubular cell proliferation and decrease tubular apoptosis after injury. examination of multiple tissue sections at or days after injury failed to reveal any examples of y chromosome cells within the tubules, and only rare examples of y chromosome cells within the renal interstitium. furthermore, exposure to conditioned medium from cultured mscs (msc-cm) significantly diminished cisplatin-induced death of cultured proximal tubule cells in vitro, while i. p. administration of msc-cm in the mouse markedly diminished the rise in bun associated with cisplatin injection. thus our data suggests that hematopoietic cells and their derivatives from adult bone marrow enter the kidney in response to injury where they are primarily localized to the interstitial space as inflammatory cells. in rare instances, these cells may differentiate into, or fuse with, tubular epithelial cells. in contrast, bone marrow mscs fail to enter the kidney in significant numbers, but can protect the endogenous tubular cells from toxic injury by secreting a factor or factors that limit apoptosis and enhance proliferation. renal hypodysplasia (rhd) is characterized by a reduced nephron number, small kidney size and disorganized renal tissue. a hereditary basis has been established for a subset of affected patients, suggesting a major role of developmental genes involved in early kidney organogenesis. gene mutations with dominant inheritance causing rhd, urinary tract anomalies and defined extrarenal symptoms have been identified in tcf (renal cysts and diabetes syndrome (rcad)), pax (renal-coloboma syndrome (rcs)), eya and six (branchio-oto-renal syndrome (bor)) for the most frequent of these syndromes. a recent study on a cohort of patients with rhd and consecutive renal insufficiency demonstrated that % of them had mutations in one gene encoding for a transcription factor. the majority of mutations were identified in tcf (hnf β) (especially in the subset with kidney cysts) and pax . this study demonstrates that subtle extrarenal symptoms in syndromal rhd easily can be missed. genetic testing in children with rhd should be preceded by a thorough clinical evaluation for extrarenal symptoms, including eye, ear, and metabolic anomalies. the presence of these anomalies increases the likelihood of detecting a specific genetic abnormality. in addition, mutations in genes that are usually associated with syndromes can occur in patients with isolated rhd. the ret receptor, its ligand gdnf and the co-receptor gfra play a pivotal role during early nephrogenesis and enteric nervous development. in humans, activating ret mutations cause multiple endocrine neoplasia, whereas inactivating mutations lead to hirschsprung disease. while ret deficiency also causes renal hypodysplasia (rhd) in the mouse model, genetic abnormalities in ret have not been characterized in human isolated renal malformations to date. the ret mutations, y f and s l, reportedly predisposing to medullary thyroid carcinoma (mtc) were found in one and six patients respectively in the same rhd cohort. none of the patients or their carrier relatives had clinical evidence of mtc at the time of the study. our findings suggest that ret mutations predispose to both mtc and rhd, with a low penetrance for either disorder. interestingly, a gdnf mutation was found in addition to a ret mutation and to an eya mutation in a patient with the branchio-oto-renal syndrome suggesting an oligogenic inheritance. mutations in clcn , the gene encoding the chloride/proton exchanger clc- , underlie most forms of dent's disease, an x-linked nephrolithiasis syndrome that is always associated with low molecular weight proteinuria. clc- belongs to the clc gene family of chloride channels and transporters and, in addition to other sites, is expressed in apical endosomes of the proximal tubule. in these vesicles, it co-localizes with the proton pump and with endocytosed proteins. previously thought to be a clchannel, it is now known to mediate electrogenic cl -/h + -exchange. this notion remains compatible with a role in supporting the acidification of endosomes by providing an electrical shunt for the h + -atpase. to clarify the pathogenesis of dent's disease, we created a knock-out mouse model. it displayed low molecular weight proteinuria due to a largely reduced endocytosis by proximal tubular cells (fluid-phase and receptor-mediated). the acidification of renal cortical endosomes was reduced in the ko. the failure of the pt to endocytose pth led to an increased luminal, but not systemic concentration of this hormone. this, in turn, resulted in hyperphosphaturia due to a retrieval of the phosphate transporter napi- a from the pt plasma membrane. nephrolithiasis can be explained by altered renal handling of pth and vitamind, all of which are secondary to the impaired endocytosis. there is an increased prevalence of nephrolithiasis in individuals with obesity, type ii diabetes, and the metabolic syndrome. in particular, uric acid constitutes a much higher percentage of stones in these patients compared to the general stone-forming population. we strived to examine the pathophysiologic connection between the metabolic syndrome and uric acid stones. in the vast majority of cases, the principal abnormality in uric acid stones is not hyperuricosuria but an excessively low urinary ph. uric acid nephrolithiasis is in fact a disease of 'urinary acidification' although there is no systemic metabolic acidosis in the classical sense because acid-base balance is achieved and no excessive acid is accumulated. however, the excretion of protons using low pk closed buffers rather than the high pk open buffer ammonia dictates a low urinary ph. since protonated uric acid has a sparingly low solubility compared to ionized urate, low urine ph promotes uric acid precipitation. thus uric acid nephrolithiasis is an innocent bystander of low urinary ph. the link between the metabolic syndrome and urinary ph as a continuous variable is explored by epidemiologic, human metabolic, and laboratory studies. in population-based studies in stoneformers, low urinary ph is associated with higher body weight. urinary ph is also lower with increasing number of features of the metabolic syndrome (waist circumference, high triglycerides, low high density lipoproteins, hypetension, hyperglycemia) and within each of the features, the severity of each parameter is inversely proportional to urinary ph. in metabolic studies in humans, low urinary ph is associated with low peripheral insulin sensitivity. when studied as in-patients on identical metabolic diets, patients with type ii diabetes and uric acid stone-formers have high net acid generation than normal volunteers. this alone lowers urinary ph although the reason for the elevated acid load is not clear at present. in addition to high acid generation, uric acid stone formers tend to use buffers other than ammonia to buffer protons in the urinary resulting in unduly acidic urine ph. when challenged with an acute acid load, the ammonium excretion response is markedly blunted in uric acid stone-formers. a similar urinary abnormally of acidic urine ph and underutilization of ammonia is seen in the zucker diabetic fatty (zdf) rat compared to their lean counterpart. these animals have peripheral insulin resistance, elevated serum free fatty acid and have significant steatosis in their kidneys. one abnormality in the kidney is reduced expression and activity of the na + /h + exchanger nhe which is the major transporter that excretes ammonium into the urine and its activity is stimulated by insulin. causality is supported by the fact that treatment of the animals with a thiazolidinedione partially reversed the fat infiltration, urinary acidification abnormality and reduced expressed of nhe . the direct effect of fat on the renal proximal tubule was tested in cultured cells. provision of fatty acids beyond the oxidation capacity of the tubule leads to dose-dependent impairment of nhe , generalized dysfunction and eventually cell death. a sub-cytotoxic dose of free fatty acid did not affect baseline nhe activity and expression but reduced the ability of insulin to activate nhe . in summary, the metabolic syndrome is associated with increase acid generation that is independent of diet. this increased acid load is effectively excreted by the kidney. failure to maximally utilize the ammonium buffer system results in lower urinary ph and titration of urate to its insoluble form as uric acid and results in uric acid nephrolithiasis. part of the pathogenic mechanism maybe lipotoxicity from fat infiltration of organs. uric acid nephrolithiasis is an "innocent bystander" which is a sentinel of a more generalized alteration in acid generation and excretion. genetic hypercalciuria recurrent kidney stone production is one of the most common diseases of the bipedal human condition, occurring in up to % of the population in western societies. unlike four-footed animals who likely perish in the wild from a selection disadvantage from a painful calculus, human beings continue to function, and have the ability to express the biologic defects repetitively that result in a stone. idiopathic hypercalciuria, where a specific gene defect has not yet been established, is a shrinking subpopulation of recurrent calcium stone formers, as specific mutations and functional polymorphisms of genes intimately or distantly related to calcium homeostasis arise from the sequencing of the human genome applied to clinical stone disease. other postulated mechanisms can be sought for in this manner as well, and may await larger population-based studies. the familial nature of nephrolithiasis is clear and robust for most calcium-based stone formers, and may have a gender-specificity for that inheritance. the role of environment in its expression remains controversial. the systemic nature of genetic hypercalciuria may be seen through its associated effects on skeletal health and biology. between - % of children with genetic hypercalciuria have abnormally low bone density measured by dual-energy absorptiometry that cannot be explained by correction for height or body mass. further, normalization of urinary calcium excretion with a variety of therapies is associated with improvements in bone density values over time. a proposal for re-classifying hypercalciuria pathogenetically will be presented, and linked to observational data across the world for successful therapeutic approaches. a call for a stone registry and more careful determination of etiology will be sought through the ipna congress. the systems that regulate blood pressure are plastic during development and can be permanently reset. experiments in animals show that it is surprisingly easy to produce lifelong changes in blood pressure by minor manipulations of the mother's diet before and during pregnancy. this phenomenon has been referred to as "programming". epidemiological and animal studies show that programmed effects operate within the normal range of growth and development, and influence the risk of hypertension, coronary heart disease and stroke in later life. a clinical study of people aged years from the helsinki birth cohort showed that two different paths of growth preceded the development of hypertension. people already diagnosed as having hypertension had small body size a birth and low weight gain from birth to two years but grew rapidly after two. at age eleven years their body size was around the average. as adults they tended to be obese and insulin resistant. a second group of people had not been diagnosed but their blood pressures were classified as hypertensive under current definitions. they were short at birth, had low weight gain from birth to two years and remained small after two. at age eleven years they were short and thin. as adults they tended to be overweight and have atherogenic lipid profiles. the first path of growth is similar to that which leads to coronary heart disease in this cohort. the second path is that which leads to stroke. this paper will present data on the maternal and placental influences through which these paths originate. nephron number is a key feature of the conceptual paradigm positing that cardiovascular and metabolic diseases that arise during childhood and adulthood have their origin in events that occur during fetal life. in mammals, nephrons are derived from a subset of cells resident within the metanephric blastema. blastemal cells participate in reciprocal inductive tissue interactions with the ureteric bud. these interactions induce the ureteric bud to grow and branch. in turn, ureteric bud branches induce discrete populations of the metanephric blastema to undergo successive transitions resulting in the formation of mature nephrons. a distinct population of metanephric blastema cells in the stroma modulates branching morphogenesis and nephrogenesis. identification of genes that control both branching morphogenesis and nephrogenesis is providing insight into the molecular pathways that could be targeted by environmental, nutritional and hormonal factors that control fetal programming. this lecture will highlight the morphologic and cellular events critical to renal branching morphogenesis and nephrogenesis, and the gene networks that regulate or counter-regulate these events. these gene networks will then be considered in the light of non-genetic factors that modulate their activities. it is now accepted that early life environment can modulate adult phenotype, including the blood pressure. the likely primary mechanism is epigenetic modulation of gene expression during a sensitive period of fetal maturation, but the pathogenesis of the later development of hypertension is unclear. participation by extrarenal factors such as central regulation and peripheral vascular function has been evoked; however, a strong body of experimental evidence suggests that the "setpoint" for renal regulation of na balance and extracellular volume is altered. because both humans and many experimental models with prenatally programmed hypertension appear to have a decrease in the total number of nephrons, impaired filtration of na has been hypothesized to be an important pathogenetic factor. more recent evidence has suggested that postnatal inflammation and accumulation of reactive oxygen species in the renal interstitium may contribute to the genesis of hypertension by upregulating distal nephron na transport. furthermore, the role of renal vascular function remains to be determined. the different mechanisms are not mutually exclusive; it is conceivable that both a prenatal "priming" and a postnatal "second hit" are required for hypertension to become manifest. j. ingelfinger epidemiological studies published in the late s by barker and his group -and since replicated in many populations -provide evidence of an inverse relationship between birthweight and risk of cardiovascular disease, hypertension, and renal dysfunction in adult life. both clinical studies and animal models have been used to investigate mechanisms underlying these observations [as cited in recent reviews. the concept that changes in the intrauterine milieu affect the growing fetus resulting in alterations in physiology and general health in later life has been termed perinatal programming or developmental origins of health and disease [dohad] . yet, despite a large and burgeoning literature about this phenomenon and its relationship to cardiovascular and renal disease, involved mechanisms remain elusive. maternal malnutrition or exposure to various medications or substances leads to an adverse in utero environment that may impair nephrogenesis, evident in experimental animal studies as well as in clinical reports in humans. nephron deficit at birth persists throughout life, creating "low glomerular endowment, " an important risk factor for hypertension and esrd in adulthood. for a number of years it has been hypothesized that nephron number may strongly influence blood pressure as well as susceptibility to renal disease in later life. recently clinicopathologic observations suggest that a relationship more directly. renal morphogenesis involves complex events in which many genes interact in the formation of the final kidney. when the normal pattern of nephrogenesis is interrupted, renal abnormalities may ensue. during renal development, two major events -ureteric bud (ub) branching and mesenchymal-to-epithelial transformation -greatly impact the outcome of renal morphogenesis. renal malformation accounts for approximately percent of childhood renal failure and represents the end result of failure of fundamental embryonic processes in ub and metanephric mesenchyme (mm) lineages. this presentation will review the data concerning renal responses to perinatal challenges as these occur and later evolve during childhood. we will consider the implications and the data available concerning screening, follow-up and management of at-risk persons. generation of oxidized lipoproteins in obstructive nephropathy -atherogenic or fibrogenic? children's hospital and regional medical center, division of pediatric nephrology, seattle, united states chronic kidney disease, regardless of etiology, is characterized by a relentless progression of fibrosis that gradually destroys the normal renal architecture, particularly in the tubulointerstitium. obstructive uropathy accounts for approximately percent of pediatric patients with chronic kidney disease (ckd) and end-stage renal disease (esrd). after post-natal relief of obstruction, the optimal treatment of children with obstructive uropathies remains unknown and for many the progression of ckd to esrd is inevitable. therapeutic options are limited by an incomplete understanding of fibrogenic pathways in the kidney. the major renal fibrotic pathways identified thus far can be broadly classified into those involved in transforming growth factor beta (tgf-b) activation, macrophage-mediated inflammation, angiogenesis, and extracellular matrix production/degradation. it is becoming increasing clear that key molecules have multiple roles in several of these pathways triggering fibroinflammatory events targeting specific cell types. oxidative stress represents an intersection of many of these fibroinflammatory pathways leading to cellular activation and tissue injury. oxidized lipoproteins accumulate in the circulation and renal interstitium in both experimental models and patients with ckd and esrd. many parallels have been drawn between atherogenesis and the pathogenetic mechanisms of progressive kidney destruction by fibrosis. although ckd is clearly associated with an increased cardiovascular risk, it is not clear whether oxidized lipoproteins amplify fibrogenic pathways in the kidney. research in our laboratory suggests that hypercholesterolemia increases injury severity in obstructed kidneys. scavenger receptors mediate the cellular effects of oxidized lipoproteins during atherosclerosis and activate both inflammatory and oxidative pathways. dietary and antioxidant therapies have clear benefits in animal models but limited efficacy in patients. our studies suggest that blocking key scavenger receptors leads to a significant attenuation of both oxidative and pro-inflammatory pathways during chronic injury by obstruction in hypercholesterolemic mice. interventions targeting scavenger receptor signaling may represent an alternative strategy to attenuate both the progression of ckd and cardiovascular disease. the resolution of injury and promotion of renal repair comprises a delicate balance between cell death and destruction of tissue architecture in relation to cell differentiation, maturation and extracellular matrix (ecm) remodeling. although many studies have focused on the cellular and molecular events leading to the development of renal fibrosis, less is understood about the process of renal repair and regeneration. this is despite the fact that the kidney has a significant capacity for regeneration and cellular replacement following acute damage. the present study describes the structural, functional, and expression profile analysis of endogenous renal repair and the regenerative potential of the kidney following reversal of ureteral obstruction (r-uuo) in the mouse. days after unilateral ureteral obstruction there is renal tubular cell loss, activation of an inflammatory cascade leading to widespread cortical interstitial fibrosis and the loss of normal medullary architecture. following to weeks after r-uuo there was marked tubular repair and regeneration of medullary components, ecm remodeling and decreased inflammatory cell infiltration. the structural repair observed at weeks post-release of ureteral obstruction was associated with a - % recovery of the glomerular filtration rate (gfr). expression profile analysis was performed to visualize patterns of gene expression that were differentially expressed in the repaired and remodeling areas following r-uuo. we are also interested in the regulation of cellular recovery and the processes involved in epithelial cell re-differentiation in regenerating tubules following injury. tubular epithelial cell cilia may play potential roles in directing the orientation of cell division and epithelial differentiation during the endogenous remodeling process. our results suggest that renal cilium lengthening may be an important factor in the response to injury and subsequent recovery of renal function. these studies propose that a lengthening of renal epithelial cilia increases their sensitivity to flow and reduces damaging epithelial dedifferentiation in the injured renal tubules. a better understanding of the key events involved in endogenous renal repair and remodeling may open the way to new interventions based on their manipulations aimed at acceleration of renal regeneration and prevention of scarring. soren nielsen has kindly agreed to present this topic, however was not able to submit an abstract. the final regulation of urinary k excretion in the fully differentiated kidney is accomplished in the distal nephron, including the cortical collecting duct (ccd), where cell k passively diffuses into the urine through apical k selective channels. the prevalence of the sk/romk channel and its high p o at the resting membrane potential has led to the belief that this channel mediates baseline k secretion. less easily detected is the bk channel which is characterized by a low p o at the physiologic resting membrane potential and [ca + ] i . bk channels are activated by membrane depolarization, elevation of [ca + ] i , and/or membrane stretch, and can be selectively blocked by iberiotoxin (ibx). we have reported that flow-stimulated net k secretion (jk) in the adult rabbit ccd is (i) blocked by ibx and (ii) associated with increases in net na absorption (jna) and [ca + ] i , leading us to conclude that bk channels mediate this process. recent studies have examined the acute and chronic regulation of bk channel-mediated flow-stimulated jk. we reported that flowstimulated jk requires an increase in [ca + ] i due to luminal ca + entry and er ca + release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. channel expression is regulated long term during postnatal development and by dietary k intake. specifically, an increase in tubular fluid flow rate fails to elicit an increase in jk in the rabbit ccd until the th wk of postnatal life, coincident with appearance of immunodetectable bk channels, whereas flow-induced increases in jna and [ca + ] i in -wk-old ccds are "mature". a role for the bk channel in renal k adaptation has been suggested by the observation that dietary k loading leads to an increase in abundance of bk message in microdissected ccds with redistribution of immunodetectable channel proteins from an intracellular pool to the apical membrane. additionally, ccds isolated from k loaded animals demonstrate enhanced flow-stimulated jk compared to tubules from control fed animals. in sum, emerging evidence suggests that the bk channel plays a prominent role in distal k secretion in response to increases in urinary flow rate and dietary k intake. the late developmental appearance of this channel is compatible with the need of the growing animal to retain k early in postnatal life. recent advances in molecular genetics of hereditary hypomagnesemia substantiated the role of a variety of genes in human magnesium transport. this knowledge on underlying genetic defects helps to distinguish different clinical subtypes and gives insight into molecular components involved in magnesium transport. during the last four decades, numerous reports concerning inherited magnesium losing disorders have been published and their distinctive phenotypic features have been discussed. phenotypic characterization of affected individuals and experimental studies of appropriate animal models have contributed to a growing knowledge of renal magnesium transport mechanisms. the identification of the affected nephron segments, the different modes of inheritance and the observation of additional characteristic symptoms promoted a classification into different subtypes of inherited magnesium losing disorders. in general, primary magnesium wasting disorders are relatively rare. the prevalence of the more frequent entities, for example gitelman syndrome, has been estimated to be approximately : . . for most of the other disease entities, relatively few cases have been reported in the literature. depending on the genotype, the clinical course is sometimes mild or even asymtomatic. therefore, the disease prevalence might be underestimated for some of these syndromes. magnesium transport has been intensively studied in humans and various animal models leading to accepted concepts underlying the pathophysiology of the different forms of hypomagnesemia. however, the electrophysiological characterization of magnesium pathways has been complicated by unintentional simultaneous measurement of other cations so that the molecular correlates mediating mammalian magnesium transport components remained undefined. a different approach to study components of magnesium transport arises from genetic analysis of families affected with magnesium wasting diseases. linkage studies enabled the localization of several genes involved in hereditary hypomagnesemia and in the last decade, a number of genes have been identified by positional cloning. these genes have provided first insight into mammalian magnesium transport molecules. distal renal tubular acidosis may be inherited as an autosomal dominant or recessive trait. mutations in three genes -slc a , atp v b and atp v a -are associated with the various forms of disease, and give rise to a wide spectrum of clinical severity. in general, dominant mutations do not affect ion transport function per se and do not affect hearing, whereas recessive disease is characterized by loss of function and deafness. some unusual functional consequences of mutations in ae and a proteins are mistargeting and loss of protein-protein interaction respectively, and these will be discussed. the heart in pediatric nephrotic syndrome y. frishberg shaare zedek medical center, pediatric nephrology, jerusalem, israel in recent years, the molecular bases of several conditions which lead to steroid-resistant nephrotic syndrome (srns) have been identified. the common denominator shared by these clinical entities is that they all result from structural defects in the glomerular barrier, thus explaining their unresponsiveness to immunosuppressants. for instance, the congenital ns of the finnish type is caused by mutations in the nphs gene encoding nephrin. a recessive form of srns was found to result from mutations in nphs encoding podocin which is specifically expressed in podocytes. we have previously shown that a founder mutation in nphs (r x) is the prevalent cause of hereditary srns ( % of tested are homozygotes) among arab children in israel. interestingly, we noted that a number of patients who are homozygous for the r x mutation in podocin have a co-existing cardiac disorder. only a few case reports described an association between srns and cardiac defects. we questioned whether the glomerular-barrier disorders, which have been considered to be kidney-specific, have implications on other organ-systems. thus, we systematically reviewed the cardiac status of these srns patients at the time of diagnosis while they had normal blood pressure and preserved renal function ( ). cardiac anomalies were detected in % of children, the most common of which were cardiac hypertrophy and pulmonary stenosis. analyzing two control groups enabled us to conclude that cardiac disorders in homozygotes for mutations in nphs cannot be attributed to an association by chance or to a state of persistent ns. because human podocin mrna is expressed in fetal heart, we hypothesize that it may have a role in normal cardiac development and this will be an issue of further investigation. this is the first study showing a role for podocin in extra-renal tissues and therefore recommends early cardiac evaluation for timely medical management. cvd is the world-wide biggest obstacle to long-term survival of children and adolescents with ckd. mortality from cvd is excessively high on dialysis and continues to be a threat after renal transplantation. early diagnosis of the individual risk for cvd would enable preventive measures on an individual basis. however, prospective studies with hard end points -cardiac events -are difficult if not impossible to conduct in children and adolescents. on the other hand, the known high morbidity and mortality in elderly dialysis patients may largely result from pre-existing comorbidity (advanced atherosclerosis has been demonstrated in this age group at initiation of dialysis). for this reason, investigations in patients with childhood onset ckd may provide a diagnostic window to study the pathogenesis of cardiac and vascular changes in subjects without comorbidities. several studies using non-invasive measurements of surrogate markers for cvd have demonstrated a pattern of early systemic cvd, including changes in intima-media thickness (imt) of conduit arteries (aorta, a. carotis) and muscular arteries (a. femoralis), altered function of peripheral resistance arteries (venous occlusion plethysmography) and abnormalities in the heart (echocardiography). patients show a significant decrease in post-ischemic vascular reactivity with evidence of vascular stiffness and frequently have extraosseous calcifications often involving coronary arteries and heart valves. importantly, these studies have found little evidence for correlations with classical risk factors (except hypertension), but with abnormalities of calcium and phosphorus metabolism and their therapy, including the intake of active vitamin d preparations and calcium-containing phosphate binders. thus, patients with childhood-onset ckd are at high risk to develop systemic cardiovascular changes, which may represent a new disease originating from the survival of ckd, a previously deadly disease, and interventions for the prevention of renal osteodystrophy. this therapeutic challenge needs to be addressed with high priority to enable long-term survival of children with ckd. while their mere existence is still questioned by some investigators, lipid rafts have recently gained a large amount of attention because of their apparent involvement in various cellular processes, including signaling, membrane trafficking, polarization, and endo-as well as exocytosis of proteins as well as pathogens. membrane rafts have been defined as small ( - nm) heterogeneous, highly dynamic, sterol-and sphingolipid-enriched domains that compartmentalize cellular processes and that can sometimes be stabilized into larger platforms by protein-protein or proteinlipid interactions. rafts are of special interest for pediatric nephrologists for two reasons: . they play a critical role in immune cell activation, especially in the formation of the immunological synapse (is), and thus in many important disease processes affecting our patients, including -but not limited to -transplant rejection. beyond their participation in is formation, rafts also facilitate signaling through other immune cell receptors, such as the interleukin- receptors, where they may ascertain cytokine selectivity and specificity. . equally important, rafts serve as essential site for proper interactions between nephrin and podocin, thus establishing the integrity of the glomerular filter. general aspects of raft biology as well as their role in immune cell signaling will be reviewed in more depth in this presentation. raft involvement in glomerular filter formation and especially genetic aspects relevant to disturbances of this involvement and of the associated integrity of the filter, as seen in hereditary nephrotic syndromes, are discussed in subsequent presentations in this symposium. reports of familial forms of fsgs date back to , with the observation of an autosomal recessive disease primarily within the finnish population. it is characterized by massive proteinuria in utero, with up to to grams of protein loss per day. nphs encodes a gene product termed nephrin that localizes to lipid rafts within the slit diaphragm of the podocyte. steroid-resistant nephrotic syndrome (srns) is an autosomal recessive nephrotic syndrome and manifests between months and years of age, rapid progression to esrd, and with few cases of recurrence after renal transplantation. the gene product is podocin (nphs ), located on q - . podocin most likely functions in the structural organization of the slit diaphragm and regulation of its filtration function. it has been shown to interact in vivo with both nephrin and cd -associated protein (cd ap), a cytoplasmic binding partner of nephrin. mutations in the alpha-actinin gene (actn ), localized to chromosome q have been associated with autosomal dominant fsgs, characterized by adult onset disease of variable severity and rate of progression to esrd. fractions of the mutant protein have been shown to form large aggregates within podocytes ultimately compromising the function of the normal actin cytoskeleton, both through its abnormal function and toxic accumulation. recently, a disease-causing mutation for hereditary fsgs has been localized to chromosome q - , and identified as the transient receptor potential cation channel, subfamily c, member (trpc ). the missense mutation causes a highly conserved proline in the first ankyrin repeat of trpc to become a glutamine at position (p q). the trpc p q mutation causes increased and prolonged calcium transients in transfected cells. the mutant channel also significantly enhances cation signals triggered by at receptor activation. biotinylation and immunostaining studies reveal that the mutation also appears to cause mislocalization of the ion channel to the cell surface. whereas previously reported mutations such as nphs , nphs and actn have emphasized the importance of cytoskeletal and structural proteins in glomerular diseases, trpc related fsgs suggests an additional mechanism for renal disease pathogenesis. knowledge of trpc mediated calcium entry into cells may offer unique insights into therapeutic options for glomerular diseases. t. huber university hospital, department of nephrology, freiburg, germany the sense of touch relies on the ability of specialized sensory cells to convert mechanical stimulation into ionic currents. mechanoreceptor cells respond to external force by opening ion channels. recent findings highlight now a potential role for the mechanosensitive ion channel trpc at the glomerular filtration barrier. trpc localizes to the slit diaphragm and mutations of trpc cause familial glomerular disease. mutations of the phb-domain protein podocin are the most common cause of hereditary nephrotic syndrome and we demonstrate that podocin and mec- , the closest homologue of podocin in caenorhabditis elegans, bind cholesterol to regulate the activity of associated ion channel complexes: deg/enac channels for mec- and trpc channels for podocin. both the mec- -dependent activation of mechanosensation in c. elegans and the podocin-mediated activation of trpc channels requires cholesterol. our data suggest that the recruitment of cholesterol by podocin and mec- to ion channels plays an important role in regulating their activity. these findings promote the concept that podocin, similar to the function of mec- , may be part of a mechanosensitive protein complex at the slit diaphragm of podocytes. isidro salusky has kindly agreed to present this topic, however was not able to submit an abstract is there a role for bisphosphonates in pediatric bone disease? f. santos hospital universitario central de asturias, universidad de oviedo, pediatria, oviedo, spain bisphosphonates are being increasingly and successfully utilized to prevent bone fractures and treat bone pain in children with severe osteoporosis from different origins. a largest experience has been accumulated with the administration of cycles of intravenous pamidronate in children with osteogenesis imperfecta. in addition, to the bone resorption inhibition mediated by their effects on osteoclasts, bisphosphonates given in large doses inhibit normal and ectopic mineralization. thus, bisphosphonates have also been used in children with hypercalcemia and in the treatment of calcinosis and heterotopic ossification, bisphosphonates have been administered to renal transplanted adults to prevent or treat bone loss induced by chronic administration of glucocorticoids and might also be useful in the management of urolithiasis in selected hypercalciuric patients. the potential clinical utilization of bisphosphonates in the prevention and treatment of vascular calcification in patients with chronic renal failure is now being explored, although no data on children in this clinical setting are available. a number of questions as to the precise clinical indications to start bisphosphonates' administration, the type of bisphosphonate to be used, the duration of the treatment, the best way to monitor its effectiveness and the risk of longterm toxic effects remain to be answered. water is the solvent in which all metabolic reactions occur. body water moves between compartments with diverse compositions that are separated by semi-permeable lipid membranes. water exchanges also occur between maternal and fetal blood separated by several layers of tissue, the so-called placental membranes. the fetus appears to be dependent on placental flow and perfusion pressure for the bulk of his water requirements, and the prostanoids play a significant role in the control of ureteroplacental and umbilicoplacental blood flows. osmotic and hydrostatic forces control placental water flux. the amniotic fluid (af) appears early during gestation and its volume increases rapidly. the net af volume turnover approximates % per day. major sources of af production are represented by fetal lungs secretions, and by fetal urine. the af is initially isotonic, and becomes hypotonic when significant amounts of dilute urine are produced by the fetus. disposal of water is effected by fetal swallowing of af and by the intramembranous (im) pathway, that is the route of absorption between the fetal circulation and the amniotic cavity. this route appears to play an important role in the overall regulation of af volume and composition. the fact that water crosses the im pathway in excess of solutes suggests a role for aquaporin water channels in allowing this transport. circumstantial evidence indicates that the im water flow is regulated by aquaporin . homeostatic changes in placental permeability could thus be up or down-regulated by the number of aquaporin water channels in the membrane. systemic lupus (sle) is a multigenic and multifactorial disease, characterized by b lymphocytes polyclonal activation with decreased tolerance, autoantibodies production and immune complexes formation. dna was initially thought to be the mayor auto-antigen, however, it is not immunogen and injection of dna-anti dna does not induce lupus nephritis. the complex of dna and histones (nulceosome) is provided with a positive electrical charge which favours the binding to heparansulphates in the gbm. in sle high levels of nucleosomes are present in circulation due to accelerated lymphocytes apoptosis or defective removal of apoptotic cells. lupus nephritis (ln) is consequent to deposition of immune complexes, activation of lymphomononuclear cells and reactivity of renal cells. the who classification of lupus nephritis has been recently reviewed by the isn/rps. the new classification takes into account a distinction between forms without endocapillary hypercellularity (mesangial or subepithelial deposits) and others with endocapillary hypercellularity (involving less or more than % with segmental or global distribution). prognosis and treatment of ln need a flexible therapy, tailored on histological picture and clinical data. steroids must be given at high doses for induction therapy but have drawbacks of heavy morbidity and mortality. the addition of immunosuppressive drugs improves the therapeutic index. the nih protocol used cyclophosphamide (cyc) pulses in severe ln (monthly pulse for months followed by quarterly pulses for year). more recent studies, comparing low with high doses cyc pulses, failed to prove a significantly different effect. for maintenance therapy of ln, mycofenolate mofetil may be a good alternative to cya or aza. rituximab, a chymeric monoclonal antibody anti cd , which selectively and profoundly depletes b lymphocytes, has provided very interesting results in sle with poor response to classical therapy or in relapse. the possibility of rotating agents with different side effects may allow to lower the doses of steroids, to reduce drug-specific morbidity, and to improve the compliance of patients. efforts should be done to minimize steroids and cyc which are very effective but are the main responsible of invalidating and even lifethreatening complications. for half a century now, physicians have tried to classify vasculitic syndromes. classification of vasculitides is required to put in perspective the pathogenesis and therapeutic advances and to provide a uniform language, given the variation in the epidemiology of these diseases. the "american college of rheumatology" criteria have been used for classification and the chapel hill consensus criteria for definition purposes in children as well however they are based totally on adult criteria. taking into account the differences in children and the new developments in medicine a group of pediatricians have aimed to revise the classification of vasculitic syndromes encountered in children. the consensus group consisted of a multinational panel of experts who were pediatricians, pediatric rheumatologists and pediatric nephrologists. the delphi and nominal group techniques were used. this project has provided classification criteria for henoch-schönlein purpura, childhood polyarteritis nodosa, wegener granulomatosis and takayasu arteritis. this was an important task since appropriate classification criteria for vasculitis in children has been missing for far too long. we hope that the international and multispecialist composition of the expert group involved will facilitate the applicability of this classification for most vasculitic diseases in children seen around the world and will meet the needs of pediatricians. these criteria are now to be validated using a large registry for childhood vasculitides. anti-neutrophil cytoplasm antibodies (anca) are well established as a diagnostic marker for small vessel vasculitis, including wegener's granulomatosis and microscopic polyangiitis. there is increasing evidence that anca are directly involved in the pathogenesis of vascular inflammation in these disorders. in clinical studies, there is a clear relationship between levels of anca and the activity and extent of disease, and anca positivity confers an increased risk of relapse. rising titres of anca predict relapse, and two studies have shown that pre-emptive treatment of those with rising titres can prevent relapse. of great interest is the recent report of a newborn child who developed glomerulonephritis and lung haemorrhage after transplacental transfer of anca. in vitro experiments have shown that anca can activate cytokine primed neutrophils to release oxygen radicals, enzymes and inflammatory cytokines. this is achieved through both direct f(ab) binding and fc receptor engagement. in co-culture, anca can induce neutrophil mediated killing of endothelial cells. in flow chamber studies, anca can induce neutrophil adhesion and transmigration across endothelial cell monolayers. more recently, anca have been shown to be pathogenic in experimental models of disease. high titre anti-mpo antibodies induced by immunisation of mpo deficient mice can transfer glomerulonephritis and vasculitis to naive recipients. immunisation of rats with mpo induces anti-mpo autoantibodies which lead to crescentic glomerulonephritis. intravital microscopy in this model confirms that anca can induce leukocyte transmigration and microvascular haemorrhage in vivo. however, many questions remain unanswered. some patients may have high levels of anca without disease activity, and others may have typical disease without detectable anca. it seems likely that t cells may be involved, not only in providing help for anca synthesis, but also in mediating tissue damage. it is also possible that an additional inflammatory stimulus, for example an infection is required, to enhance the inflammatory effects of anca. greater understanding of the role of anca in vascular inflammation will hopefully lead to safer and more effective approaches to treatment. hacettepe university, faculty of medicine, department of pediatric nephrology, ankara, turkey the therapy of vasculitic syndromes poses a problem for the caring pediatrician. the treatment of anca associated vasculitides will be presented to cover microscopic polyangiitis, wegener granulomatosis and churg strauss syndrome. treatment of all anca-associated diseases are similar. steroids and cyclophosphamide are the mainstay of induction treatment. in severe patients with kidney involvement steroids can be given in the form of intravenous methylprednisolone for - days ( - mg/kg/d, max. gr), followed by daily oral corticosteroids ( . mg/kg/d, max. mg/d). cyclophosphamide may be given at mg/kg/d p. o. or monthly iv pulses . gr/sqm for at least months. for maintenance treatment there are again many different regimens for oral corticosteroids. along with corticosteroids for maintenance regimen there are a number of protocols suggesting the continuation of cyclophosphamide. the cycazarem study has shown that the replacement of cyclophosphamide with azathioprine at months was also as effective for disease control. methotrexate has also been shown to be an alternative for maintenance treatment. treatment of the childhood polyarteritis nodosa (pan) with systemic disease is similar to that of anca-related vasculitides. there are a number of non-anca associated vasculitides in childhood. the most frequent in childhood are henoch-schönlein purpura (hsp), kawasaki disease (kd) and takayasu arteritis (ta). the treatment of hsp is usually symptomatic. however, for severe kidney involvement with extracapillary proliferation and rapidly progressive disease severe immunosuppressive treatment is indicated. triple treatment with steroids, cyclophoshamide and dipyridamole have been given in various series. for kd intravenous immunoglobulin at a dose of g/kg still remains the first choice of treatment along with salicylates. for ta therapy depends on the extent of vessel invovlement: severe disease necessitates steroids and cyclophosphamide whereas for less intensive vessel involvement methotrexate and steroids may suffice. treatment period depends on the actiivty of the disease. lb. zimmerhackl haemolytic uraemic syndrome (hus) is the most common cause of acute renal failure in children. the syndrome is defined by the triad of microangiopathic haemolytic anaemia, thrombocytopenia and acute renal failure (creatinine over the th percentile). world wide hus is increasing. in a german/austrian multicenter study we follow children with hus occurring in the years to . year follow-up data are now available www. hus-online. at. from this study the following results are obvious. hus affects predominantly children of kindergarten age. the median age at onset is , years. the majority of hus is of infectious origin. shigatoxin (stx) producing escherichia coli (stec, ehec) are present in over % of patients. the predominant shigatoxin type is type ii. hus is classified into two clinical subgroups. "typical" hus usually occurs after a prodrome of diarrhoea (d+hus), and "atypical" hus (ahus), which is not associated with diarrhoea (d-hus). the majority of d+hus worldwide is caused by ehec type o : h , which is transmitted to humans via different routes. however non-o groups are emerging and are predominant in europe. transmission of disease in elder patients is predominantly through food poisoning and direct contact to farm animals. in infants and small children direct transmission from human to human seems to be more likely. currently there are no specific therapies preventing the disease course. anti-shigatoxin antibodies are being tested by several companies. if this may prevent hus is open to study. otherwise the therapy at present is symptomatic. parenteral volume expansion before hus in patients with positive stx or ehec stool culture may counteract the effect of thrombotic process before development of hus and attenuate renal injury. use of antibiotics, antimotility agents, narcotics and non-steroidal anti-inflammatory drugs should be avoided during the acute phase in particular during the prodromal phase. from our own study the prevention is best done by preventing primary ehec infection. however, patients with severe course and long term sequelae should be screened for genetic abnormalities in the complement system. if auto antibodies against complement proteins or the vwf play an relevant role is under discussion. patients under one year of age at onset have a significant worse outcome and should be kept under surveillance. patients below month of age are very likely to have an inborn error of complement or vwf and should be tested specifically. the european registry on hus and related disorders may help to determine these abnormalities: www. haemolytic-uraemic-syndrome. org. in order to improve long term outcome of these patients, increased awareness and an european (international?) task force is mandatory. in adults with chronic kidney disease, protein-energy malnutrition and inflammation are risk factors for death and accelerated cardiovascular disease. the "malnutrition-inflammation-cachexia syndrome" (mics); anorexia, increased basal metabolic rate, and loss of lean body mass is associated with low serum albumin, decreased protein intake, elevated c-reactive protein, and low serum cholesterol levels in adults. in children, mics manifests as growth retardation. clinical research focusing on the evolution of mics in pediatric ckd to understand its causes and consequences and how nutritional interventions alter its course may be the key to improving survival in ckd. baseline cross sectional data from the ongoing chronic kidney disease in children study, of children (n= ) aged - yrs (mean= . yrs) with estimated gfr ± ml/min/ . m (mean iohexol gfr ml/min/ . m ) shows substantial growth retardation in ckd with median height percentile= . greater height deficits are seen at lower gfr's. symptoms of decreased appetite and nausea are reported by % and % of those with gfr < respectively. mean ldl cholesterol is lowest in those with gfr < ( vs mg/dl in gfr ± ml/min/ . m group). serum albumin declines as serum creatinine increases (r=- . ). unlike previous reports in adult ckd, increases in crp were not associated with lower gfr at baseline. further exploration of the mics in pediatric ckd will be presented. the causes of growth failure in pediatric patients with chronic kidney disease (ckd) are multifactorial. it is an open question as to which factors play a key role in diminishing physical growth. it is also unclear which mechanisms may become pace makers for the therapeutic improvement of growth. furthermore, growth failure in children with ckd affects total body height, body proportions and composition as well as organ development. growth failure may also lead to disproportion which can only be identified by detailed anthropometric measurements. we were able to demonstrate that ckd patients have a specific age-dependent pattern of growth and distinct changes in segmental growth (trunk, arm and leg length) from birth to adolescence. leg growth in relation to other parameters of linear growth showed the most dynamic growth changes and emerged as the best indicator of growth in children with ckd. trunk growth had little synchronicity with leg growth. furthermore, we found that anthropometric measurements can be used as a diagnostic tool in distinguishing different sub-groups of ckd patients, for instance, children with syndromic ckd. in the heterogeneous group of patients with focal and segmental glomerulosclerosis, patients with schimke's disease were found to have a dramatically decreased sitting height/leg length ratio. as the disturbance in growth of ckd patients is a marker of the severity of the disease and of the quality of renal care, the annual analysis of growth failure from early childhood to adolescence should be used as a landmark information of the quality of medical care and as a helpful tool in differential diagnosis and of specific courses of ckd in sub-groups of patients. for example, patients with congenital or acquired renal diseases, dialyzed or transplanted patients and in gender differences. in addition, anthropometric measurements are able to identify specific growth patterns in children with ckd, which should be considered in the assessment of treatment efficacy such as in rhgh therapy. b. tönshoff, l. weber, b. höcker university children's hospital, st department of pediatrics, heidelberg, germany it is currently under debate whether steroid avoidance or late steroid withdrawal provides the best overall risk-to-benefit ratio in pediatric renal transplantation. late steroid withdrawal has the advantage over steroid avoidance that immunological high-risk patients and those with unstable graft function can easily be identified beforehand and be excluded from steroid-free immunosuppression. in order to further validate this approach, we performed a prospective randomized open-label multicenter trial in low-risk pediatric renal transplant recipients ( f, m; mean age . yrs; range, . to . ) on csa (target trough level - ng/ml), mmf ( mg/m per day) and methylprednisolone ( ) ( ) mg/m per day), who were randomly assigned > year posttransplant to continue steroids or to withdraw over a period of months. an interim analysis was performed at a mean observation period of mo after study entry; patients had been followed for at least mo. there were drop-outs ( reversible acute rejection episode, switch to sirolimus and to tacrolimus). transplant function as assessed by calculated ccr remained stable in both groups, no graft was lost. prepubertal children off steroids gained relative height from baseline - . ± . sds to - . ± . sds after yrs, while patients on steroids lost relative height (- . ± . sds at baseline, - . ± . sds after yrs); a comparable pattern was observed in pubertal patients. the standardized body mass in patients off steroids declined from . ± . sds at baseline to . ± . after yrs (p< . ), while it tended to increase in patients on steroids (baseline, . ± . sds; after yrs, . ± . ). the rate of adverse events, mainly infections, was comparable in both groups. patients off steroids required less frequently antihypertensive medication ( %) than patients on steroids ( %). a significant reduction of serum cholesterol (by %) and triglycerides (by %) in response to steroid withdrawal was observed. conclusions: this interim analysis indicates that late steroid withdrawal in selected pediatric renal transplant recipients on csa and mmf is safe, allows catch-up growth and ameliorates cardiovascular risk factors. at the ipna meeting, full month outcome data of all patients will be available. u. frei, j. noeldeke renal transplantation faces two major challenges: the organ shortage resulting in extended waiting times and an aging population resulting in increased death with a functioning graft. the eurotransplant senior program (esp) allocates kidneys within a narrow geographic area from donors aged > years to recipients > years regardless of hla. this analysis investigates the impact of the esp on waiting time, graft and patient survival. the esp group (n= , old to old) was compared to two groups allocated via the eurotransplant kidney allocation system (etkas) with either similar donor age [old to any (o/a), donor age > , n= ] or recipient age [any to old, (a/o), recipient age - , n= ]. all patients were transplanted between and . since initiation of the esp ( ), availability of elderly donors doubled and waiting time for esp patients decreased. local allocation led to shorter cold ischemia time ( . vs. > . hours, p< . ) and less delayed graft function (dgf, esp . % vs o/a . %, p= . ) but - % higher acute rejection rates. importantly, graft and patient survival were not negatively affected by the esp allocation scheme. the esp age matching of elderly donors and recipients is an effective allocation system for organs from elderly donors. the effect of age matching on the duration of the waiting time a. rahmel, m. slot, j. smits eurotransplant international, leiden, the netherlands in eurotransplant the proportion of children, i. e. patients aged under the age of at time of registration, with end-stage renal disease (esrd) on the renal waiting list amounts to only . %. despite this small proportion the eurotransplant kidney allocation system (etkas) specially addresses pediatric transplant candidates. in order to increase their chances of receiving a transplant in time children are assigned via etkas extra points for hla matching and receive an age bonus. in order to evaluate this allocation policy, the chances for receiving a kidney for patients in different age groups were evaluated. for the cohort of patients registered in the year , a year followup was obtained. for patients aged under at time of registration [n= ] % and % received a kidney from a post-mortem donor, within year and years after listing. for children aged to [n= ] these rates were at and years after listing % and %, and for children between age and [n= ] % and % respectively. adult patients were less likely to receive an organ: % and % of patients aged between and were transplanted within and years after listing. senior esrd patients can benefit from the eurotransplant senior program (esp), their chances for a transplant were % and % within and years, respectively. the life span of a renal allograft is limited in time. in the eurotransplant experience % of the post-mortem kidneys used for transplantation in children failed within years, compared to % for the living donor renal transplants (rtx). donor age is an important factor associated with long term renal allograft function. age matching between donor and recipient is hampered by the low number of pediatric donors. in the period - , heart beating kidney donors aged under were reported and used for rtx, donors were between and years of age, and donors were between and years old. in the same period adult donors were reported and used for transplantation. in the period - , % of the pediatric donor kidneys ultimately served a pediatric recipient. to improve the allocation of pediatric donors to pediatric recipients, et is in the process of implementing a rule giving pediatric recipients priority in case of pediatric donors. gender and sex hormones are playing a central role in the incidence and progression of different renal diseases. vascular tone, endothelial function and immune response are influenced by gender. in renal transplantation ischemic injury is always present. females are more resistant to postischemic renal failure than males. following ischemia/reperfusion injury renal vascular resistance decreases, allowing fast restitution of blood flow and oxygen supply in females. additionally, stabilization and preservation of tubular function following ischemia is achieved by the protection of na+/k+ atp-ase and heat shock protein activity contributing to the observed gender differences. posttransplant immune reactions also differ between genders. female gender predisposes to more severe acute rejection following kidney transplantation, partly due to differences in the effectivity of immunosuppressants used. ineffective immunosuppression in females, as well as different cytotoxicity profile of the drugs is contributing to more prone immune reactions shortly after transplantation resulting in more severe acute rejection. in contrast, similar to the slower deterioration of other renal diseases, progression of chronic graft failure is less pronounced in females in experimental and clinical settings. estradiol decreases profibrotic processes, preserves endothelial function and modifies influx of immune cells into the graft. the fine balance between alloantigen dependent immune reactions and alloantigen independent factors and its impact on longterm graft function is modified by gender. new evidences supporting the significance of sexual dimorphism following kidney transplantation may present the base of gender modified therapeutic approaches in the future. iga nephropathy: aetiology, incidence, and geographic distribution iga nephropathy (igan) is characterized by mesangial deposits of iga, proliferation of mesangial cells and expansion of matrix. the accumulation of iga and complement fractions within glomeruli was initially ascribed to deposition of iga immune complexes (igaic) due to mucosal immune response with predominant iga synthesis. this hypothesis has offered an explanation of the relationship between infections of the upper airways and gross hematuria. high levels of igaic have been detected in - % of patients, mostly of polymeric iga , and polymeric iga are detectable in renal deposits. this observation is consistent with hypothesis of either bone marrow or mucosal origin. however, no specific viral or alimentary antigens have been found in mesangial deposits, and the qualitative properties of polymeric iga have rather become of interest, particularly the glycosylation pattern of iga . human iga is o-glycosylated with carbohydrate chains of n-acetyl galactosamine (galnac) and galactose (gal) which may be covered with sialic acid (neu ac). patients with igan exhibit circulating iga with reduced gal and/or neu ac and increased exposure of n-galnac. such aberrantly glycosylated iga can circulate in monomeric form or participate in the formation of autoaggregates/true immune complexes. it likely escapes clearance by hepatic receptors and has a preferential renal deposition by virtue of enhanced reactivity with the mesangial matrix components. the role of a triggering event has not ruled out. in recent reports, antigens of bacterial origin and the secretory tract component have been found in renal deposits, making scientists reconsidering again the role of bacterial infections. tonsils recurrent infections may provide a suitable aetiology for igan, and the effect of tonsillectomy on the long term outcome of igan is under evaluation. the prevalence of igan varies in different areas, due to ethnic and environmental factors, being particularly frequent in mediterranean europe, northern europe, asia and australia. it is still debated whether differencies in frequency, clinical features and disease progression among patients with igan from different countries are actually due to uneven distribution of this diseases or these discrepancies are due to the different criteria for performing renal biopsy. definition. igan is characterized by the presence of dominant (or codominant) mesangial deposits of iga on immunofluorescence microscopy, frequently with c and sometimes with igg or igm. classification. the glomeruli display a broad spectrum of histologic changes, related in part to the differences in the indication for the biopsy of the referring nephrologist. a number of classification systems have been used to describe the histologic manifestations of igan. two will be referred to here: the system of m. haas (ajkd : - , ): ) minimal histologic lesion -the glomeruli exhibit no more than a minimal increase in mesangial cellularity, without segmental sclerosis or crescents. ) fsgs-like -the glomeruli display focal segmental sclerosis in a pattern resembling primary focal segmental glomerulosclerosis, with at most a minimal increase in mesangial cellularity and no crescents. ) focal proliferative glomerulonephritis (gn) - % or fewer of the glomeruli are hypercellular. the increase in cellularity may be limited to mesangial areas, or there may be obstruction of glomerular capillaries by proliferated endocapillary cells. crescents may be present. ) diffuse proliferative gn -more than % of glomeruli are hypercellular. the hypercellularity may be segmental or global, and crescents may be present. ) advanced chronic gn - % or more of the glomeruli are globally sclerotic, and/or there is % or more tubular atrophy or loss in the cortex. the system of s. emancipator (heptinstall's pathology of the kidney, lippincott-raven, philadelphia, , pp - ) : a -normal/minimal glomerular lesions b -focal mesangial proliferation c -diffuse mesangial proliferation d -focal segmental endocapillary proliferation superimposed on mesangial proliferation d -focal segmental endocapillary proliferation alone e -diffuse endocapillary proliferation f -diffuse extracapillary proliferation (crescents in > %), with or without endocapillary proliferation g -glomerulosclerosis (> % of the glomeruli are sclerotic) h -unclassifiable or combined lesions diffuse proliferative gn (c) and focal proliferative gn (d ) are the major patterns of expression of glomerular injury. indicators of a poor outcome. these include a high proportion of glomeruli with crescents, numerous sclerotic glomeruli, interstitial fibrosis and tubular atrophy, extension of the iga deposits into the peripheral glomerular capillary walls, and hyaline arteriolosclerosis. differential diagnosis. mesangial iga deposits are present in henoch-schönlein nephropathy, and may be present in lupus nephritis, chronic liver disease, coeliac sprue, certain dermatologic diseases, and some rheumatologic diseases. iga nephropathy [igan] is defined by the presence of mesangial iga, but otherwise the histopathological and clinical features are very variable. we do not yet know sufficient about pathogenic mechanisms to understand whether igan is a single disease. although traditionally called an 'immune complex' disease there is no direct evidence that mesangial iga deposition in igan occurs through classical antigen-antibody interactions. mesangial cells do carry receptors for iga, which may play a key role in iga deposition and subsequent injury, but these are not yet fully characterised. mesangial iga in igan is polymeric iga . there is no evidence of mucosal immune system overactivity, indeed there seems to be underproduction and abnormal t cell control of mucosal iga production, along with overproduction by the marrow of iga iga has a hinge region peptide structure which is a site for o-glycosylation. in igan circulating and mesangial iga both have abnormal o-glycosylation at the hinge region. the same defect is seen in henoch-schönlein purpura only when there is renal involvement. the glycosylation defect is not due to abnormal peptide structure of the hinge; the possibility that there is reduced activity of the key enzyme b , galactosyltransferase in b cells or plasma cells has not yet been confirmed. alternatively the glycosylation changes may reflect a shift in the production of mucosal type of iga to the marrow. while iga deposition is caused by disease mechanisms specific to igan, subsequent inflammatory and fibrotic events are probably driven by mechanisms common to other chronic glomerular disease. in some patients iga deposition is not followed by inflammation, in others inflammation resolves without fibrosis. cytokine and growth factor production by mesangial cells is a sufficient explanation for glomerular inflammation and fibrosis. however little is yet understood of any genetic or environmental influences which protect some patients from progressive renal injury. our recent controlled trials by the japanese pediatric iga nephropathy treatment study group demonstrated that treatment of children with severe iga nephropathy with prednisolone, azathioprine, heparin/warfarin, and dipyridamole for years early in the course of the diseaseprevents immunological renal injury and progression of the disease. the majority of patients with iga nephropathy in our series are diagnosed early in the course of the disease, and the asymptomatic period before the discovery of urinary abnormalities is short. early diagnosis and early treatment is very important in iga nephropathy. . combined therapy for severe childhood iga nephropathy (j am soc nephrol : [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . seventy-eight children with newly diagnosed iga nephropathy showing diffuse mesangial proliferation were randomly assigned to receive either the combined therapy of prednisolone, azathioprine, heparin-warfarin, and dipyridamole for two years (group ) or the combination of heparin-warfarin and dipyridamole for two years (group ). urinary protein excretion was significantly reduced in group patients, but remained unchanged in group patients. the percentage of glomeruli showing sclerosis was unchanged in group patients, but significantly increased in group patients. . steroid treatment for severe childhood iga nephropathy (clin j am soc nephrol, : - , ) . in this study we have compared the effects of prednisolone, azathioprine, warfarin, and dipyridamole (combination) with those of prednisolone alone in children with newly diagnosed iga nephropathy showing diffuse mesangial proliferation. patients were randomly assigned to receive either the combination or prednisolone alone for two years. the primary endpoint was the disappearance of proteinuria, defined as urinary protein excretion < . g/m /day. thirty-nine of the patients receiving the combination and of the receiving prednisolone completed the trial. thirty-six of the patients ( . %) receiving the combination and of the ( . %) receiving prednisolone reached the primary endpoint by the two-year follow-up point (p= . log-rank). the percentage of sclerosed glomeruli was unchanged in the patients receiving the combination, but increased in the prednisolone group (p= . ). the frequency of side-effects was similar in the two groups. long-term administration of recombinant human erythropoietin (epo) has become the most common way of treating anemia in chronic renal disease. standard amounts per unit body weight have been recommended for the initial dose. however, several authors have noted that the dose per unit body weight needed for a given response is higher in younger children than in older children or adolescents and that it is increasing with decreasing body weight. furthermore, for a given absolute dose of epo the outcome was investigated in adult hemodialysis patients, but no dependence was found in patients weighing - kg. a similar model to the hemoglobin-time data of children aged - years treated with epo for renal anemia did not find an impact of body weight on response when it was modelled in terms of absolute dose. in a similar analysis to the hemoglobin-time data children and adolescents aged - years were analyzed, in order to more definitively answer the question if, for given absolute doses the hematopoetic response to epo in children depends on body weight. neither the dose response parameter e max and ed showed dependence on body weight. the median hemoglobin response to a standard dose was similar to that reported for adults. it can be concluded that younger and smaller children need relatively more epo than older children. doses for children should be specified as absolute amounts rather than amounts per unit body weight. references: scigalla p. effect of recombinant human erythropoietin treatment on renal anemia and body growth of children with end-stage renal disease. in: baldamus ca, scigalla p, wieczorek l, et al, editors erythropoetic agents are the mainstay of treatment for renal anemia. although there are several different marketed forms of erythropoetin, they are not substantially different in their ability to stimulate erythropoesis. however, darbepoetin, which differs in one amino acid, and has additional glycosylation sites, has a longer half-life and therefore a presumed longer duration of action. this provides a theoretical advantage, suggesting that less frequent injections will be required. clinical experience and studies have confirmed that both erythropoetin (epo) and darbepoetin (dar) are effective to maintain hb values within the recommended range. also, though there is some experience with epo injected every couple of weeks, the overall evidence suggests that dar has a longer duration of action, and injections are required less frequently. for many children, particularly the pre-dialysis population, anemia is successfully controlled with dar injected every - weeks. however, this apparent advantage of dar is somewhat reduced because of the increased pain reported with dar injections compared to epo. both epo and dar have been used successfully in children of all ages. with epo, the doses required to maintain hb values appear to be increased in infants compared to older children; this may not be the case with dar, but there is much less experience with use of dar in infants. also, the administration of dar in infants is hindered by the need to inject portion of a unidose pre-filled syringe, which may introduce inaccuracies in dosing, is wasteful, and is not user-friendly. the side effect profile for each product is similar, and specifically each has been associated with development of hypertension, which is most likely with higher hb values. pure red cell aplasia has also been reported with each product. thrombocytosis has been reported with dar use. overall, there is little to recommend one product over the other. the need for less frequent injections may favor dar for use in most children beyond infancy, whereas it is easier to administer epo accurately in infants. how much iron is needed and how much is toxic? iron deficiency is the primary reason for ineffective erythropoiesis in patients with chronic kidney disease (ckd) who receive an erythropoiesis stimulating agent (esa). reasons for iron deficiency include inadequate dietary intake, blood loss from the gastrointestinal tract, frequent blood tests, loss of blood in the extracorporeal circuit of hemodialysis (hd), as well as the hepcidin related impairment of the intestinal absorption of iron and its release from the reticuloendothelial system. supplementation of iron can be by either the oral or intravenous (iv) routes, although the national kidney foundation-kidney disease outcomes quality initiative (k/doqi) strongly recommends the preferential use of iv iron in children and adult patients receiving hd. the k/doqi recommended target iron indices for all children on dialysis are a serum ferritin > ng/ml and a transferrin saturation (tsat) > %. while the serum ferritin should not regularly be > ng/ml, some such patients will achieve a higher hemoglobin value following an iv course of iron therapy. of the iv iron agents, the non-dextran products appear to be safest, and pediatric dosing recommendations exist for ferric gluconate and are currently being studied for iron sucrose. differences do exist for the clinical (e. g. proteinuria) and subclinical (e. g. oxidative injury) toxicities associated with the currently available iv iron products and should be taken into consideration when prescribed. until recently the major physiological function of erythropoietin (epo) was thought to be the induction of erythropoiesis. however, a growing body of evidence indicates that epo has tissueprotective properties and prevents ischemia induced tissue damage in several organs including the kidney. a main target of epo´s action is the endothelium, and one of the pivotal intracellular pathways mediating the beneficial effects of epo is the activation of akt, i. e. serine/threonine protein kinase b. as a result, akt phosphorylates the proapoptotic factor bad, which in turn causes inhibition of programmed cell death (apoptosis). moreover, experimental studies revelead that epo is a potent regulator of endotheial progenitor cell (epc) proliferation and differentiation. collectively, these data support the hypothesis that epo is a key molecule in the process of endothelial (vascular) repair and neoangiogenesis. treatment with rhuepo or analogues could therefore open new therapeutic strategies in regenerative cardiovascular medicine. introduction: africa as a continent is besieged by many health challenges including malaria, hiv (upwards of % of paediatric admissions), tuberculosis and malnutrition with an infant mortality rate (imr) ranging from (southern africa) to (mozambique). good health facilities are on the whole not available except in the extreme north and south of the continent with doctors/ population in south africa but in kenya only and even lower in other parts of africa. renal disease: renal disease in adults is an unknown quantity with no information being available regarding children's renal disease, where many babies do not even have access to antenatal ultrasounds. situation in south africa: in reality, there are only centres (cape town and johannesburg/ pretoria) doing paediatric dialysis and transplantation in significant numbers with small numbers interspersed in the rest of the country. this raises numerous issues: -accessibility for children to renal care -retaining minimum standards of care for children -both dialysis and transplantation -combined with adult units -central government funding in form of tertiary services grant for paediatric renal care -children not first priority on any transplant program in terms of organ allocation -private facilities vs state facilities -ethical decisions dialysis facilities: peritoneal dialysis (pd) first line therapy for acute renal failure, as can be performed in any setting with minimal equipment and expertise. in the setting at rxh, we have a greater than % survival in infants and children dialysed using pd even in a sophisticated intensive care setting. haemodialysis and chronic dialysis may not be easily available in most settings and realistically need transfer to one of the major centres. transplantation: again limited to only few centres, with at least % living related donations from family members. initial good year ( %) and year ( %) graft survival but results then deteriorating as patients enter their adolescent years often with little support and transfer to adult units in their early teens. controversial issues here include access to one kidney transplant only, no chronic dialysis if not suitable for transplant and transplantation in hiv positive recipients. conclusions: adequate resource allocation is required for paediatric renal care especially where resources are limited. immunosuppression remains the cornerstone of successful transplantation. in developing countries transplantation is mostly from living donors and transplant is thus a once in a lifetime chance. in this backdrop immunosuppression is a challenge as several issues have to be overcome. namely, non-availability of newer drugs, high costs and paucity of drug monitoring facilities. in most countries immunosuppression is based on a triple drug regimen of cyclosporin (cya), prednisolone (pred) and azathioprine (aza). in the last few years mmf and tacrolimus (tac) has been initiated in a few centres. several tailoring strategies have been employed taking into consideration costs, drug availability, tissue typing facilities and drug monitoring. firstly in hla identical transplants which constitute - % of the total, cya based regimen are used. marked reduction to - mg/kg at months and complete withdrawal at one year in rejection free transplants is safe with continuing of aza and pred. secondly several centres selectively use tac and mmf in cases with early rejection or transplants with > mismatches. it is possible to switch to cya or aza after months on individual basis. thirdly induction protocol with atg for days in transplants with > mismatches and in retransplants is cost effective. cost considerations have increased the use of generic calcinurin inhibitors with market share of - % in different countries. co administration of p competitors has reduced doses of cya by - % with considerable cost reductions. siut has been running a living related transplant program for more than years with dialysis and follow-up of recipients and donors. keeping in mind economic constraints a model of government public partnership was developed which provides all facilities including drugs free of cost. we adopted a number of tailoring strategies e. g. strict monitoring, tailoring by hla match and donor age and use of biological agents in risk groups. in first years we used parent drugs, however increasing costs necessitated use of generics after establishing bioequivalence in controlled trials. graft survival rates were maintained at % and % at and years. in conclusion, immunosuppression in developing countries require besides newer drugs, monitoring facilities, affordable costs and regular follow-up as transplantation for majority is once in a lifetime chance and failure equates with death. occurrence of infections following kidney transplantation is a major reason for hospitalization, and an important cause for renal dysfunction & mortality in developing countries. more than % renal transplant recipients get serious infections, with a - % risk of mortality. infections in the first month after transplantation are similar to those in surgical patients; opportunistic pathogens and cmv predominate between - months; and tuberculosis (tb) after months. the risk of tb in patients on maintenance dialysis & following transplantation is between - %. the onset of tb is usually within months of transplantation. clinical syndromes include pleuropulmonary tb, followed by disseminated tb, pyrexia of unknown origin and lymph node disease. demonstration of m. tuberculosis, on microscopy or culture might not be possible. nonrifampicin based treatment regimens (inh, pyrazinamide, ethambutol, fluoroquinolone for months, followed by hef for months) are used. inh prophylaxis ( - months) is advised in patients with tuberculin positivity or contact with active tuberculosis. cmv disease results in considerable morbidity & mortality; its timing is influenced by donor/recipient serological combination, state prior to transplantation, use of antilymphocyte induction therapy or preventive strategies. cmv disease is characterized by a non-specific febrile illness; features of enterocolitis, pneumonia, hepatitis, myocarditis, esophagitis, chorioretinitis & bone marrow involvement are variable; disseminated disease is rare. cmv disease exacerbates the net immunosuppression, increasing the risk for opportunistic infections. patients with cmv disease are also at risk for acute rejection and chronic allograft injury, atherosclerosis & vascular injury. diagnosis of cmv disease is based on a combination of viral serology, shell vial cultures, pp antigen assay and pcr. the cost of prophylaxis and treatment is a major limitation to the use of ganciclovir or valganciclovir. other infections in transplant recipients include malaria, p. carinii and fungi (cryptococcosis, candidiasis, mucormycosis, aspergillosis). infections are an important cause of morbidity & mortality in renal transplant recipients. their management continues to be challenging due to difficulties in diagnosis, unsatisfactory follow-up and cost of medications. ethical issues of renal replacement therapies n. orta, p. zibaoui, e. lara university of carabobo/insalud, pediatric nephrology, valencia, venezuela important ethical issues in pediatric nephrology (pn): genetic and molecular techniques, prenatal therapies for urinary anomalies, treatment of children with chronic renal disease (esrd). ecosonography can detect nephrourological anomalies and studies of amniotic fluid give information on chromosome alterations and renal function. particular situations, can lead to dilemmas related to pregnancy interruption and neonatal dialysis and transplantation (dt) possibilities. renal insufficiency (ri) presents at any age, and peritoneal, hemodialysis or hemofiltration could be applied. patients without structural abnormalities, ri secondary to toxics or isquemic nephropathies have better prognosis. these procedures are complicated and costly and increases, and may have secondary effects with ethical implications. treatment by dialysis developed in the 's and inclusion of children was not considered. this has changed with advances in dt. since 's dt programs were setup for children, but received criticisms and considered unethical, but were continued because of familiar and humanitarian demands and today it is clear that children benefit with that. scientific societies recommend: . children who receive dialysis must meet the following criteria: -diagnosis of esrd, legal authorization, possibility of transplantation, acceptable quality of life; may not be rejected for economic, social or psychological reasons, nor gender, age, race or mental conditions. biopsies are the current 'gold standard' for monitoring transplant patients, but it has been shown that even mild rejection episodes, based on pathology grading, can have poor outcomes and even biopsies with normal early pathology can progress rapidly with chronic allograft injury. there is considerable inter-observer variability of biopsy pathology readings that adds an additional confounder to this method of analysis. identification of non-invasive biomarkers in blood or other fluids would allow for the possible elimination of frequent biopsies. hence, the identification of diagnostic and predictive non-invasive genomic markers will be a worthy tool to aid in the clinical monitoring of transplant patients. the use of dna microarrays as a hypothesis generation tool for determining gene expression differences across thousands of genes in a data set is increasing. recently, the use of microarrays has been applied to the transplant field and holds great promise for unraveling the mechanisms at play in various transplant processes and for identifying new tissue specific and non-invasive biomarkers predictive of clinical outcomes. as microarrays produce large amounts of data, bioinformatics tools are being developed to determine gene expression patterns. gene clustering and class prediction tools aid in the discovery of molecular signatures in different disease processes while literature mining, gene family analysis and pathway analysis help in understanding the biological relevance of these signatures. initial studies in acute rejection and graft dysfunction have produced possible markers for risk stratification of the rejection event and have suggested underlying mechanisms at play, that may now allow us to test novel drugs for treating specific acute rejection episodes. the most exciting application of microarrays lies in our ability to predict clinical outcomes by non-invasive serial monitoring, eliminate the requirement of transplant biopsies and individualize patient management by accurately predicting the patient's sensitivity to immunosuppression-sufficient to suppress the allo-response, yet insufficient to abrogate the innate response. responsible array data handling and accessible reporting will open new doors for transplant researchers through increased use of computers and collaborations towards these kinds of novel insights and treatment options for transplant patients in the future. this talk focuses on dna microarrays, their application to transplantation, and discusses some of their limitations and recent applications, as well as some key research studies where dna microarrays are applied to understanding the molecular differences in acute transplant rejection that segregate and likely control the differences in rejection treatment responsiveness as well as decline in graft function, as well as gaining insights into the different biological processes that govern these differences. the molecular pathogenesis of vur is not well understood. uroplakins (ups) are expressed in the urothelium and are developmentally regulated by rab b and tbx genes. up ii and iiia-null mice exhibit a primary ( o ) vur phenotype. using microarrays and rt-pcr, we analyzed gene expression in surgically-discarded ureteric tissue from children undergoing reimplantation for o reflux, compared with adult living-related transplant donors as normal controls, as well as children with o reflux (megaureter, duplicated ureters, and posterior urethral valves) as age-matched disease controls. we also studied urine protein profiles using -dimensional electrophoresis ( d-page) followed by time-of-flight mass spectroscopy (q-tof-ms). rt-pcr showed partial expression of ureteric upia, upib, upii and upiiia genes in patients with o reflux. protein screening using western immunobloting confirmed that some uroplakins were undetectable. real-time rt-pcr revealed that ureteric up ia, up ib, upii, and up iiia gene expression decreased significantly, whereas upiiib gene expression increased at least -fold compared to controls. compared with patients with o vur, the decrease of upiiia expression in those with o reflux was highly statistically significant (p< . ). the expression of rab b and tbx genes also decreased significantly in patients with o and o reflux as well. total urinary protein concentration increased by -fold in the o vur patients without detectable renal scarring on dmsa scans; and increased further in patients with renal scarring, -fold for o vur and -fold for o vur patients. proteomic analyses of proteinuria revealed an overall increase of protein with mw> . kda in the pi range . to . in patients. q-tof ms identified one predominant urinary protein of ~ kda as sera-transferrin, which was confirmed by elisa quantitation. we hypothesize that the abnormal developmental expression pattern of the up, rab b and tbx genes in vur patients results in abnormal urothelial functions, which lead to leakage and/or secretion of proteins into the urine, and that this occurs in the absence of scarring from urinary infections. further identification of these protein biomarkers may lead to non-invasive diagnostic tests for vur. biomarker discovery in acute kidney injury p. devarajan cincinnati children's hospital medical center, department of pediatric nephrology and hypertension, cincinnati, united states acute kidney injury (aki), previously referred to as acute renal failure (arf), represents a common and persistent problem in clinical medicine. despite significant improvements in therapeutics, the mortality and morbidity associated with aki remain high. a major reason for this is the lack of early markers for aki, akin to troponins in acute myocardial disease, and hence an unacceptable delay in initiating therapy. fortunately, the application of innovative technologies such as functional genomics and proteomics to human and animal models of aki has recently uncovered several novel genes and gene products that are emerging as biomarkers. the most promising of these are chronicled in this symposium. these include a plasma panel (ngal and cystatin c) and a urine panel (ngal, . since they represent sequentially expressed biomarkers, it is likely that the aki panels will be useful for timing the initial insult and assessing the duration of aki. based on the differential expression of the biomarkers, it is also likely that the aki panels will distinguish between the various types and etiologies of aki. however, they have hitherto been tested only in small studies and in a limited number of clinical situations. it will be important in future studies to validate the sensitivity and specificity of these biomarker panels in clinical samples from large cohorts and from multiple clinical situations. such studies will be markedly facilitated by the availability of commercial tools for the reliable and reproducible measurement of biomarkers across different laboratories. a. watson children and young people's kidney unit, notthingham university hospitals, nottingham, united kingdom the achievement of adequate chronic peritoneal dialysis in children requires close attention to clinical, dietetic and psychosocial aspects of care. successful dialysis is dependent upon excellent peritoneal access and swan neck coil catheters with downward facing exit sites are increasingly favoured with many placed laparoscopically. automated peritoneal dialysis is employed in most developed countries but adolescents may be given the choice of capd which may be the only modality available in developing countries. training and support by committed nursing staff are of paramount importance as is regular review by a paediatric renal dietitian. growth parameters should be regularly monitored and supplemental enteral feeding introduced early. many infants are nasogastrically fed, but the preferred route for supplemental enteral feeding is via a gastrostomy which can be placed at the same time as the pd catheter. dialysis fill volumes should be assessed in terms of body surface area and intra-abdominal pressure measurements. there can be discrepancies between urea and creatinine clearances and adequate dialysis includes combining clinical parameters with regular growth measurements as well as biochemical data including phosphate, calcium and parathyroid levels. peritoneal function tests are useful in monitoring progress but the greatest challenges are in sustaining long-term dialysis, particularly in infants where transplantation is likely to be delayed. support for the families with a respite care 'package' may delay or prevent burnout and possibly reduce peritonitis rates. conventonal pd solutions are acidic, contain unphysiological concentrations of lactate and glucose and highly toxic glucose degradation products (gdp), which are substrates for advanced glycation end product (age) formation. repeated administration induces epithelial to mesenchymal cell transition, loss of the mesothel cell layer, progressive submesothelial fibrosis and angiogenesis, ultimately leading to ultrafiltration failure. meanwhile pd solutions with an improved toxicity profile are available. multi chamber pd solutions separate glucose from the buffer at a very low ph, which largely prevents gdp formation. after mixture ph is close to normal. they are equally effective with regard to solute-and water transport, reduce inflow pain and systemic age load. dialysate effluent markers indicate increased mesothelial cell mass, reduced peritoneal inflammation and reduced angiogenesis. animal studies demonstrate preservation of pd membrane morphology and function, respective human biopsy data however are pending. a european paediatric multi centre trial accomplished recently elucidates the impact of the lactate and bicarbonate buffer. a randomized cross over trial in adult patients points to an improved preservation of residual renal function; a korean registry suggests improved technique and patient survival. icodextrin solutions reduce glucose and gdp exposure, improve extracellular fluid status, left ventricular mass and stabilize membrane function in anuric adult capd patients and should be beneficial in children, too. amino acid based solutions achieve similar clearance and ultrafiltration rates, reduce glucose and gdp load and allow for a phosphate free amino acid supply. the nutritional benefit however is questionable. other, thus far experimental strategies include addition of locally active compounds to pd fluids such as gdp scavengers, inhibitors of age formation and antifibrotic agents. surface active phospholipids may increase peritoneal contact area and ultrafiltration. gene therapy appears highly promising but is still far from being clinically applied. in summary, there is substantial evidence for increased biocompatibility of the recently introduced multi chamber and icodextrin based pd solutions. they should improved long term morbidity and mortality of pd patients; this however still needs to be proven. the impact of the ippr on the treatment of peritonitis b. a. warady children's mercy hospital and clinics, pediatrics nephrology, kansas city, united states the international pediatric peritonitis registry (ippr) is an initiative that was established to evaluate the safety and efficacy of largely opinion based peritonitis treatment guidelines in which empiric antibiotic therapy ( st generation cephalosporin and ceftazidime or a glycopeptide and ceftazidime) was stratified by disease severity. forty-seven centers from countries contributed data on children and peritonitis episodes treated in accordance with the guidelines. culturenegative peritonitis accounted for % of all episodes, with a marked regional variability in the incidence of this disorder, as well as in the peritonitis causative organisms. overall, % of cases achieved full functional recovery, a portion following relapsing peritonitis ( %). in-vitro evaluation revealed only % sensitivity of gram-positive organisms to a st generation cephalosporin (eastern europe > north america) and % sensitivity of gram-negative organisms to ceftazidime. in contrast, % of gram-positive organisms and % of gram-negative organisms were sensitive to the combination of either a st generation cephalosporin or an aminoglycoside. whereas the risk of empiric treatment failure was associated with the presence of a gram-negative infection (p= . ), neither the risk factors assumed by the guidelines nor the choice of empiric therapy were predictive of the final functional outcome of the peritonitis episodes. the data collected by the ippr will serve as an important source of evidence to be incorporated into revised pediatric peritonitis treatment guidelines. ch. aufricht medical university, pediatrics, vienna, austria peritoneal dialysis (pd) is a safe, cost effective and widely used form of renal replacement therapy in patients with end stage renal failure. however, up to a third of patients on pd will suffer from technical failure during their course. identification of the patients at highest risk would be of high clinical relevance. cytotoxicity of pd fluids due to low ph, hyperosmolarity, and/or high concentrations of lactate, glucose and its degradation products causes mesothelial cell injury that ranges from minor cellular dysfunction to overt (apo)necrosis. the same physicochemical properties of pdf that cause such cellular injury also induce pathways leading to repair and recovery. this so-called cellular stress response results in a switch of the cellular machinery from routine procedures towards reaction against stressors. the complex machinery of the cellular stress responses not only counteract direct toxic injury caused by pdf but also attenuate inflammation or other potentially deleterious cellular processes. infectious, uremic and toxic injuries might converge in cellular inflammation. whereas inflammatory processes triggered by infection protect the peritoneal cavity against invading microorganism, chronic sterile smoldering 'cytotoxic' inflammation may result in aberrant healing processes and peritoneal fibrosis. recently, polymorphisms of many proteins involved in relevant cellular responses have been described and related to altered resistance and/or susceptibility to pathogenetic processes with potential influence in pd. in this review, we will focus on key effectors of stress responses, of cellular inflammation and of fibrogenesis such as heat shock proteins (hsp), cytokines (il- ), chemokines (il- ) toll-like receptors (tlr) and growth factors. given the recently shown role of these 'players' for the interplay between mesothelial injury, inflammation and cytoprotection, these polymorphisms will likely be relevant for mesothelial cell damage during pd. taken together, the ability to understand -and ultimately modify -the risk profile of a given patient will be essential for tailoring individual pd therapies. the pathologic diagnosis of chronic allograft nephropathy (can) was introduced in by the banff classification system for renal allograft injury. it originally included at least four entities that it was acknowledged could not always be distinguished by biopsy: ) chronic rejection; ) chronic calcineurin inhibitor (cni) toxicity; ) hypertensive vascular disease; ) chronic infection and/or reflux (solez, ki : ) . over the ensuing decade, the definition of can has expanded and fluctuated such that some pathologists have come to use the term to mean a specific pathologic entity comprising "…progressive graft dysfunction accompanied by chronic interstitial fibrosis, tubular atrophy, vascular occlusive changes and glomerulosclerosis. " (nankivell, nejm : , while others have suggested can should be used to describe all causes of renal allograft dysfunction involving fibrosis. the resulting confusion over terminology has in some ways hindered the growing awareness of the multiple discrete, diagnosable and often treatable diseases capable of causing chronic graft injury. these diseases include: ) chronic (primarily antibody-mediated) rejection; ) de novo and recurrent glomerular disease; ) calcineurin inhibitor toxicity; ) interstitial fibrosis and tubular atrophy without evidence of any specific etiology. to this list should be added other recognizable causes of late graft dysfunction such as polyoma virus infection and hypertension. in the new banff classification can will no longer be a diagnostic category (colvin, world transplant congress, ) . instead, the term sclerosis will be used to describe: "interstitial fibrosis/tubular atrophy, not otherwise specified, " or "if/ta nos. " the increasing use of protocol biopsies in clinically stable patients has dramatically increased awareness of the ongoing pathological changes almost every renal allograft appears to be undergoing almost from the first moments of engraftment. techniques are now available in most centers to reliably recognize the presence of chronic antibody-mediated rejection. these include: ) typical morphological findings: lamination of glomerular basement membranes, arterial intimal fibrosis, interstitial fibrosis/tubular atrophy; ) c d staining in peritubular capillaries or glomeruli; ) the presence of circulating donorspecific antibodies (takemoto ajt : . treatment of late, chronic antibody-mediated rejection is in its infancy and may include use of anti-b cell antibody (rituximab), ivig, and/or plasmapheresis. calcineurin inhibitor toxicity can be more reliably identified by focusing on blood vessels in the allograft, primarily the location of hyaline deposits in the arterioles. nodular, peripheral hyalinosis is found almost exclusively in cni toxicity, whereas sub-endothelial and transmural hyaline deposits are non-specific and can be seen in hypertension, aging and diabetic nephropathy. there is increasing concern that recent gains in short-term renal allograft survival and reductions in acute rejection rates have not resulted in improved long-term graft survival. while this is likely due to multiple factors, including infections and malignancies associated with over immunosuppression, unrecognized (sub-clinical) acute cellular rejection (moreso ajt : ), and noncompliance in some patients, much evidence points to the primary role of cni toxicity in many if not the majority of chronically failing allografts maintained on cni's. experience with conversion from cni to sirolimus in adult patients has been reviewed in a recent editorial (betard nephrol dial transplant : editorial comments) . in studies involving nearly patients, between % and % of patients with late allograft dysfunction failed to respond favorably to cni withdrawal and replacement with sirolimus. acute rejection episodes were rare, occurring in only six patients. common adverse effects included dyslipidemia, anemia and proteinuria. in one study, of patients developed proteinuria after conversion, in the nephrotic range. pediatric experience with cni withdrawal has been limited (kerecuk, pediatr nephrol : falger, pediatr transplant : , hocker, pediatr transplant . our recent experience at stanford with cni withdrawal in patients was not favorable, with % of patients experiencing an associated acute rejection episode (weintraub, wtc ) . prior history of acute rejection significantly increased the relative risk of acute rejection after cni withdrawal (rr= . ), and proteinuria was common. patients with advanced chronic graft dysfunction were at increased risk for graft loss. in summary, the use of the term can to describe all causes of chronically failing renal allografts is to be discouraged in favor of a search for specific etiologies whenever possible. antibody-mediated rejection and cni toxicity are major causes of late allograft dysfunction. protocol biopsies are helpful in identifying patients with treatable causes of late allograft dysfunction. cni withdrawal/avoidance may be successful, but optimum patient selection criteria and withdrawal/replacement strategies have not been determined. pediatric experience to date with cni withdrawal has been limited, but it appears that late withdrawal after cni injury and graft dysfunction have become well established may be associated with inferior outcomes. prospective trials in pediatric patients are needed to address these issues. which immunosuppression in pediatric transplantation? p. hoyer university children's hospital, essen, germany recent results in pediatric renal transplantation have reached one year graft survival rates better than %. current immunosuppressive drugs should be classified according to their interference with the immunesynapsis as signal , signal and signal blocking agents. the variety of immunosuppressive drugs does allow more treatment combinations than the potential number of large scale studies in children. the definition of current unmet needs should guide employment of drugs. calcineurin inhibitors (cni) are still the basis of immunosuppression. individual risk profile leads to preferences for cyclosporine or tacrolimus. while in adults cni reduction seems to be the major goal, the search for steroid sparing or avoidance protocols has attracted major interest in pediatric transplantation. growth and body configuration as well as cardiovascular risk factors should be in the focus of research. antibody induction protocols, mainly with il- receptor antibodies, are increasingly popular, but efficacy is less clear than concluded from adult studies and might depend on initial combination therapy. mmf (cellcept®) or mpa (myfortic®) are effective drugs with cni sparing potential. early adequate dosing seems to be of greater importance than the choice of the cni, but the price to pay may be an increase in infectious complications. mtor inhibitors are promising in avoiding nephrotoxic side-effects; specific side effects on male gonadal function and possible interference with growth should be considered before any recommendation can be given. fty would have been of especial interest for children because of maintaining viral infectious response, but phase- studies are on withhold. the vision of tolerance has stimulated research on regulatory t-cells; i. e. cd cd +t-reg cells and the mastergene foxp . the impact of lymphocyte depletion induction protocols with campath on operational tolerance needs further studies. newer drugs in development are isa , a cyclosporine analog without nephrotoxicity; a modified release form of tacrolimus which might improve compliance; the phosphokinase inhibitor aeb with the potential to avoid cnis; the jak inhibitor cyp , which might interfere with signal (il and il -receptor); and lea y (belatacept) with blocks costimulatory signals. according to the new european drug legislation some of these drugs will be subject for mandatory testing in pediatric patients to get marketing authorisation. the future might be a more tailored immunosuppression according to the induvidual needs of the patient. immunosuppression minimization strategies, under the umbrella of a newer generation of more powerful induction and maintenance immunosuppressants, are being increasingly applied to pediatric organ transplantation, with the greatest emphasis on minimization of steroids and calcineurin inhibitor agents. safe elimination of these steroids carry unprecedented advantages for reducing patient morbidity and chronic graft injury, but may also result in unanticipated changes in immunological homeostasis and drug pharmacokinetics, heralding closer surveillance for posttransplant infections and alterations in drug bioavailability and dosing, as well as break-through immunologic responses. in single center studies, pediatric renal transplantation appears safe without steroids. daclizumab first dose doubling and extended use for months replaces steroids effectively without evidence of over-immunosuppression, and may be the pivotal causative for the reduced acute rejection seen in the face of steroid avoidance. this pilot protocol has been tested in a prospective, multicenter randomized us and canadian study. b. maecker, c. klein department of pediatric hematology/oncology, hannover medical school, hannover, germany posttransplant lymphoproliferative disorders are severe complications of immunosuppressive therapy after solid organ transplantation causing significant morbidity and mortality. to define prognostic factors, we have analyzed pediatric solid organ graft recipients (kidney, liver, heart/lung) that were reported to the german ped-ptld registry. ptld was diagnosed at a median time of months post organ transplantation with sigifcantly shorter lagtime in liver versus heart or renal graft recipients. the five-year overall and event-free survival was % and %, respectively. stage iv disease with bone marrow and/or cns involvement was independently associated with poor survival. no differences in outcome were observed between early and late onset ptld, monomorphic or polymorphic ptld, and ebv-positive or ebv-negative ptld, respectively. patients with burkitt-like ptld and c-myc translocations had very short survival. these factors should be important to consider for future prospective interdisciplinary trials that are urgently needed to define rational treatment strategies. cystinosis is an autosomal recessive lysosomal storage disorder affecting children and adults all over the world. in cystinosis cystine is trapped in the lysosomal compartment due to a defect in its egress transport protein cystinosin encoded by the gene ctns. by mechanisms still not fully understood this leads to tubular and glomerular kidney and other organ failures (e. g., thyroid, muscles) if left untreated. kidney involvement is prominent from shortly after birth on as renal tubular fanconi syndrome with the clinical consequences of failure to thrive and rickets. cystinosis is the single most common cause of renal fanconi syndrome in childhood. therefore, any recognition of renal glucosuria, generalized aminoaciduria, phosphaturia, small molecular weight proteinuria, polyuria, and metabolic acidosis (due to renal bicarbonate loss) should lead to prompt consideration of cystinosis as possible cause. this can be done utilizing biochemical analytical methods measuring the cystine content of polymorphonuclear leucocytes. corneal cystine crystals, seen in slit lamp examination, are pathognomic as well, but may not be visible before months of age. left undiagnosed and untreated, patients will develop in addition to the existing tubular insufficiencies pronounced glomerular kidney failure often already present at diagnosis and inevitably leading to end stage renal failure typically at the end of the first decade of life. kidney failure in cystinosis presents differently from other forms of glomerular kidney failure because of the overlap of tubular and glomerular insufficiencies. kidney transplantation will be curative with respect to kidney function. specific treatment with cysteamine to lower the intralysosomal cystine content apparently is as important as before transplantation to prevent or attenuate other organ failures and therefore has to be continued after kidney transplantation. cysteamine treatment has been introduced in the s and has been approved in the s. early diagnosis and diligent treatment is able to prevent or ameliorate major organ complications. unfortunately, until now no newborn screening exists due to the biochemistry and cell biology involved. the high prevalence of a european founder mutation, i. e., a kb deletion in the ctns gene, makes molecular based methods not feasible. lowe syndrome and dent's disease: two ends of a spectrum d. böckenhauer great ormond street hospital for children nhs trust, nephrology, london, united kingdom lowe syndrome and dent's disease are x-linked disorders; the former is a systemic disorder characterized by cataracts, mental retardation and proximal tubulopathy whilst the latter was originally described as an isolated kidney disorder with tubular proteinuria, hypercalciuria/ nephrocalcinosis and progressive renal impairment. mutations in ocrl underlie lowe syndrome, whereas the majority of cases with dent's disease are caused by mutations in clcn . recently, mutations in ocrl have been identified in a subgroup of patients with dent's disease (also called dent- ). it is unclear, why some patients with ocrl mutations get dent's disease, while others develop lowe syndrome. however, careful clinical observation has revealed that dent- patients have evidence of systemic involvement. moreover, the degree of severity of symptoms in lowe syndrome is highly variable. thus, mutations in ocrl can cause a spectrum of symptoms, from tubular proteinuria and hypercalciuria to the full manifestations of lowe syndrome. here, we will review the clinical phenotype of the disorders, what is known about their pathophysiology and discuss genotype/phenotype correlation. fabry disease, the second most prevalent lysosomal storage disorder after gaucher disease, is an xlinked inborn error of the glycosphingolipid metabolic pathway and affects approximately : , live births. mutations in the gene encoding alpha-galactosidase a, lysosomal hydrolase, lead to systemic glycosphingolipid deposition, resulting in profound dysfunction of neurological, renal, cardiac, and cerebrovascular systems. the initial phase begins in childhood or adolescence and is characterized by neuropathic pain, angiokeratomas, and ocular deposits. the later phase is distinguished by progressive cardiac, cerebral, and renal involvement, leading to multi-organ dysfunction and death. few patients have historically survived past their mid s. although renal involvement usually becomes prominent in adulthood, adolescents may develop proteinuria and decreased glomerular filtration rate. timely diagnosis is critical, given that the enzyme replacement therapy likely delays progression of the serious complications of fabry disease and may have potentially preventive benefits. specifically, there is growing evidence that initiation of enzyme therapy slows progression of chronic kidney disease (ckd); it remains unknown whether enzyme therapy in the currently employed doses can prevent ckd. pediatricians have a particularly important role in making the diagnosis, since they are likely to be the first providers to encounter fabry patients and thus initiate therapy before irreversible tissue injury develops. nephrologists should consider the diagnosis of fabry disease when a patients present with ckd with nephrotic or subnephrotic proteinuria, often with skin lesions (but these may be limited in distribution), episodic extremity pain, and/or psychiatric problems. further research is required to determine the efficacy of enzyme replacement therapy to prevent organ damage in children, to identify optimal therapeutic doses and schedules, and to define efficacy of additional treatments for fabry kidney disease, include angiotensin converting enzyme inhibitors and angiotensin receptor blockers. w. van't hoff great ormond street hospital for children nhs trust, pediatric nephrology, london, united kingdom children with a complete deficiency of methylmalonyl coa mutase develop both renal tubular and glomerular dysfunction. the renal tubular dysfunction is characterised by renal tubular acidosis, defective urinary concentration and hyporeninaemic hypoaldosteronism. a chronic interstitial nephritis also develops leading to long-term glomerular renal damage. chronic kidney disaes is not evident on routine testing as muscle mass is reduced and protein intake is markedly restricted. formal measurement of gfr using chromium edta showed that of such mma patients have a gfr < mls/min/ . m ), and by years, of patients had a gfr < . most mma patients with ckd do not have excess proteinuria nor hypertension. although vit b responsive mma patients in general have a more favourable outcome, late onset renal complications have been seen. in addition to ckd, mma patients can develop cardiomyopathy, pancreatitis, gut dysmotility and chronic or acute encephalopathy. management is based on a protein-restricted, high calorie diet, allopurinol to control hyperuricaemia, supplements of carnitine and metronidazole therapy to reduce production of propionate. haemodialysis has successfully cleared plasma mma and improved the metabolic and nutritional status. liver transplantation has been performed in a number of younger children, as enzyme replacement therapy, but is associated with a significant morbidity and mortality (including late neurological deaths). renal transplantation has been reported in a small number of older patients but it is not yet clear how good the graft outcome will be. combined liver-kidney transplantation has been undertaken again in small numbers, with a high morbidity and significant mortality although there are a very few remarkable survivors. so far, there are only centre specific experiences and unfortunately reviewing the literature will not give a full picture of long-term outcome and complications, due to the bias of the reported results towards favourable outcomes. there is a clear need to share data on the management of these rare patients in order to better understand the best approach. estimations of the extent of hiv disease in sub-saharan africa are that million people are currently infected of which the total number of children is . million. worldwide it is thought that . million children are infected so it can be seen that the majority reside in africa. in south africa, we have a minimum of hiv infected children aged less than yrs of age with only on highly active anti-retroviral therapy (haart) at present. currently - % of all paediatric admissions to hospital may be hiv related. hiv associated renal disease, is not yet well documented among children -especially from africa -but now with the growing availability of haart, this information has become more important for appropriate management. hiv renal disease presents in many forms, including including hiv-assoc nephropathy (hivan) and hiv immune complex kidney disease (hivick) amongst others. questions have now been raised as to whether a screening program should be introduced as haart, despite side effects and drug interactions, has revolutionised management if hiv infection detected early enough. transplantation which was thought previously to be an absolute contraindication and potential waste of valuable resource is now possible, provided there is maintenance of haart, hiv viral load is undetectable for > mths and cd > cells/mul. there remains concern about feasibility in children with increased rejection, reduction of calcineurin inhibitors and infections such as recurrence of hepatitis c post-transplant. despite these concerns, studies in adherent adults have clearly shown that adult patients with hiv infection do better following transplantation than on dialysis. of concern, are some studies, showing that certain black patients may have a genetic predisposition to hiv infection. overall, transplantation can be successful in these patients provided the hiv disease is under control. however, hiv disease -including renal disease -remains a major problem in sub-saharan africa due to limited resources and lack of availability of drugs, where other health priorities may prevail. background. previous studies suggest that hiv- induces dysfunction and/or injury of endothelial cells, leading to the systemic release of fibroblast growth factor - (fgf- ). to date, the role that circulating fgf- may play in the pathogenesis of childhood hivan is not clearly understood. objective. here we sought to determine the potential role of circulating fgf- in the pathogenesis of hivan using wild type (wt) and hiv-tg mice. methods and results. to determine whether circulating fgf- induced ultrastructural changes in renal glomerular endothelial cells and podocytes, we injected human recombinant fgf- daily for days into fvbn wt and hiv-tg mice (n= in each group). by electron microscopy we found that fgf- induced endothelial swelling and mild fusion of the foot processes in wt mice. these lesions were more severe in hiv-tg mice, which showed enlargement of podocytes, protein reabsorption droplets, significant fusion of the foot processes, and collapsing glomerulopathy. subsequently, to confirm these findings in a different experimental model system, we used recombinant adenovirus carrying a secreted form of human fgf- (ad-fgf- ). four to five weeks old hiv-tg mice without evidence of abnormal protenuria (urine protein/creatinine ratio (up/uc) < ), and their wild type littermates were injected with x plaque forming units (pfu) per mouse of ad-fgf- or control ad-lacz vectors through the retro-orbital (r. o.) venous plexus (n= per group). all mice were followed for three weeks. all hiv-tg mice injected with rad-fgf- developed heavy proteinuria (up/uc > ). in addition, % showed elevated bun levels (> mg/dl) and renal histological lesions typical of hivan. in contrast, wt mice, developed transient and moderate proteinuria (up/uc < ), without renal failure or permanent renal damage. the number of animals with proteinuria in hiv-tg mice injected with ad-lacz group was consistent with the natural history of the renal disease progression. control wt mice injected with ad-fgf- developed mild proteinuria ( %) that returned to normal values - days after the injection. fgf- induced microcystic tubular dilatation and recruitment of mononuclear cells both in wt type and hiv-tg mice, although the changes were more significant in the later group. conclusion. taken together, these results suggest that the accumulation of fgf- in the circulation of hiv-tg mice induces glomerular endothelial and podocyte injury leading to the development of heavy proteinuria, renal failure, and the typical renal histological features of hivan. we conclude that the elevated levels of fgf- in the circulation of hiv-infected children may be a significant risk factor for the development and/or progression of hivan. human immunodeficiency virus associated nephropathy (hivan) is the third leading cause of kidney failure in young african american adults in the united states. the prevalence and natural history of the disease in children is not well documented. we have examined our experience in children, aged months to years (mean age . ± . years) with predominantly perinataly acquired hiv- infection from their mothers. since , of these children have been evaluated routinely for evidence of renal disease with quantitative assessment of proteinuria by random urine protein to creatinine ratios (upr/cr). renal functional studies included serum creatinine with estimation of glomerular filtration rate (egfr), urinalyses, renal scintigraphy, ultrasound and renal biopsy when possible. the patients were divided according to their level of proteinuria. those with nephrotic range proteinuria (upr/cr> . ) were designated as hiv-ns (n= ; %). those with persistent intermediate range proteinuria (upr/cr: . < . ) were designated as hiv-pp (n= ; %). those with no proteinuria (upr/cr< . ) were designated as having no nephropathy (hiv-non; n= ; %). the great majority of patients were treated with highly active antiretroviral therapy (haart), although those with hiv-ns had less time on treatment when their proteinuria was discovered. moreover, the virulence of the hiv infection as measured by viral load (vl) was significantly greater in those patients with proteinuria. also, viral load correlated positively with degree of proteinuria (r= . ; p< . ). mortality as shown by kaplan-meier survival curves was significantly greater in the hiv-ns group as compared to the pp and non groups. renal failure occurred only in the group with nephrotic range proteinuria. during the past years we have dialyzed children with hiv infection in our end stage renal disease (esrd) program for a total of patient dialysis months. since , all patients are managed on hemodialysis due to the high occurrence of fungal peritonitis in our initial experience. survival has improved remarkably during the past years with a median survival of months (range to months). the next stage is to begin renal transplantation in those patients with adequate control of the hiv infection. aim: to determine the spectrum of severe renal disease in our hiv infected children; excluding severe sepsis and septic shock syndromes. indications for their referral, renal biopsy and histology relating to outcome. methods: retrospective analysis of all children referred to the paediatric renal service from the general wards and hiv clinic from january to december . analysis includes age of presentation, sex, nutritional status, symptoms, renal function, histology, associated diseases including infections and follow-up. results: total of children with a mean age of . ± . years with a male: female ratio of : . . there was an overlap of symptoms but the commonest presenting symptom was haematuria in %, severe urinary tract infection and pyelonephritis in %, anasarca with or without any nephrosis in %, acute renal failure in %, chronic renal failure in %, severe electrolyte disturbances in %. renal biopsy was performed in ( . %) children. histogically % had immune complex disease (icd) of which . % had lymphoid interstitial pneumonitis (lip). . % had fsgs of which one had lip but also had icd, . % had severe interstitial nephritis, associated infectious changes in . % and atn in . %. one patient each had minimal change, mpgn, abdominal kaposi sarcoma and kidney/bladder stones. . % had or were treated for pulmonary tuberculosis. mean follow-up was ± . months. death occurred in . % ( ). all patients with fsgs with some renal dysfunction and severe sepsis demised. conclusion: despite the high burden of hiv disease, severe renal complications have a low prevalence. good correlation of icd and lip ( . %) but prognosis is good. pulmonary tuberculosis and infection is the main complications resulting in high mortality in nephrotic syndrome with fsgs. hiv associated nephropathy (hivan) is one of the most common causes of renal failure in hiv seropositive african americans. in the usa, hivan has become the third leading cause of end stage renal disease (esrd) in african americans over the age of . while the introduction of haart has decreased both the mortality and infectious complications of hiv infection, the incidence of hivan has reached a plateau and has not decreased. with reduced mortality, the prevalence of seropositive patients in the esrd program continues to increase dramatically. the histopathological findings of hivan include focal segmental glomerulosclerosis of the collapsing variant combined with microcystic tubule dilatation. the most common other diagnosis is mesangioproliferative glomerulonephritis associated with hepatitis c infection, a common comorbid condition. typical features of hivan include renal enlargement and echogenicity by ultrasound analysis. microscopic findings include coexistent microcystic tubule dilatation and glomerular involvement. usually there is mild to moderate tubulointerstitial inflammation, interstitial edema, and fibrosis as well. glomerulosclerosis is usually focal and segmental with collapse of the glomerular tuft and hypertrophy of visceral epithelial cells. hivan is caused by renal epithelial infection by hiv- in a susceptible host. clearly there are genetic factors, based on the racial predilection of this disease. patients with hivan are . times more likely to have a relative with renal failure. in susceptible individuals, hiv infection induces renal epithelial proliferation and apoptosis. the kidney represents a tissue-specific compartment in which hiv- can replicate in a previously unrecognized reservoir. while hivan is not currently considered to be an aids-defining condition, patients with hivan should be treated with haart. in some instances, haart has completely reversed the disease process, although it does not rid the kidney of virus. thus, in patients with hivan, the kidney is a true reservoir for replication competent hiv. whether this occurs in other forms of renal disease associated with hiv infection or in patients without renal disease remains to be determined. prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. renal sympathetic nerves directly innervate renal tubules and blood vessels and plays a role in the regulation of glomerular filtration rate and renal sodium excretion. we examined if renal nerves play an a role in mediating the hypertension in prenatal programming. pregnant sprague-dawley rats were injected daily with intraperitoneal dexamethasone between th and th day of gestation. renal norepinephrine concentration was measured at weeks of age. renal denervation was preformed at age weeks of age in control and prenatal dexamethasone treated rats and blood pressure was measured at age of weeks. renal norepinephrine concentration was ± ng/gr in controls and ± ng/gr in the group that received prenatal dexamethasone (p< . ). systolic blood pressure at weeks of age was ± mmhg in sham operated controls, ± mmhg denervated controls (p=ns). blood pressure was elevated to ± mmhg in dexamethasone treated sham operated group (p< . ), but was normal at ± mmhg in the dexamethasone treated denervation group. in conclusion, prenatal dexamthasone results in elevated renal norepinephrine levels. bilateral renal denervation normalized the systolic blood pressure in rats that received prenatal dexamethasone. these data are consistent with the renal nerve playing an important role in mediating the hypertension in prenatal programming by dexamethasone. objective: evaluation of clinical outcomes in surgically treated children with renovascular hypertension (rvh). methods: rvh patients treated surgically at a single centre between and were retrospectively reviewed: % were male, . - . (median . ) years of age, with systolic blood pressure (sbp) mmhg ( - mmhg). results: bilateral renal artery stenosis was present in %, midaortic syndrome(mas) in %, intrarenal disease in % and coexisting cerebral disease in % of patients. surgical procedures (n= ) included: a) nephrectomy (n= ), b) autologous surgery for both aortic reconstruction (n= ) and renal revascularisation (n= ) (renalartery reimplantation (n= ), renal bypass (n= ) and autotransplantation (n= )) and c) synthetic graft interposition for renal revascularisation (n= ), aortic reconstruction (n= ) or both (n= ). the majority ( %) of patients who received synthetic grafts had vascular anatomy too complex for autologous surgery. technical failure leading to secondary nephrectomy occurred in patients. postoperative complications were haemorrhage (n= ), septicaemia (n= ), and chylous ascites (n= ). there were no operative deaths. patients from the uk were followed up for . ( . - ) years. sbp post-surgery improved ( mmhg, range - mmhg, p< . ). outcomes were normal sbp without treatment ( %), improved ( %) or unchanged sbp ( %). reduction of sbp led to loss of contralateral kidney in patient. children required re-interventions ( angioplasties and surgical procedures) for progressive disease (n= ), narrowing of the synthetic graft (n= ) and re-stenosis of the autologous bypass (n= ). conclusion: rvh is a progressive disease of extensive nature. surgery benefited % of children when performed in conjunction with conservative therapy and, if indicated, interventional radiology. h. wong unlike adults with predominant primary hypertension (htn), the majority of children diagnosed with htn traditionally suffered from secondary forms of htn. however, substantial changes have occurred to the demographics of pediatric population over the past years. in addition, our definition, awareness and understanding of children with hypertension has also changed. we therefore retrospectively reviewed the current causes of htn in children followed in a single tertiary care pediatric nephrology referral center between january and december . patients who were either diagnosed with or treated for htn at the time of their last visit where included. gender, height, weight, age at the time of diagnosis, causer of htn and casual blood pressure were recorded. out of patients, ( . %) were diagnosed with htn. the majority were males (n= , %). median was years ( month- years) age at time of diagnosis and . years ( . months- years) at time of the last follow-up. secondary htn was the most common cause of pediatric htn (n= , %) followed by primary (n= , %) and then white coat (n= , %). renal htn was the most common cause of secondary htn ( / , %). for patients referred initially for assessment of htn (n= ), primary (n= , %) and secondary (n= , %) were the two most common diagnosis followed by normotension (n= , %) and white coat htn (n= , . %). twenty two ( . %) patients were diagnosed before the age of year. out of renal transplant patients, had htn ( %). renal htn remains the most common cause of pediatric htn overall and continues to represent a large portion of children referred for htn. all children suspected of having htn should continue to have thorough investigations of renal disease to identify the underlying cause. m. sinha , c. booth , j. simpson , n. dalton , s. qureshi , c. reid , s. rigden evelina childrens hospital, guys and st. thomas's nhs foundation trust, department of pediatric nephrology, london, united kingdom evelina childrens hospital, guys and st. thomas's nhs foundation trust, department of pediatrics cardiology, london, united kingdom king's college london, department of medicine, london, united kingdom background: hypertension (ht) is a frequent complication in paediatric tx patients. the evolution of end organ damage and its relationship to ht in these patients is not well described. aim: to study the predictive value of an abnormal -hour abp profile for end organ damage. methods: patients underwent simultaneous casual blood pressure (cbp), abpm, echocardiogram (lvh if lvmi > g/m . ) and ecg assessment, with measurement of several biochemical cardiovascular risk markers. cbp data for -months prior to study date was analysed as time averaged z-score. results: we present initial results of a -year prospective study. patients ( male) aged . y± . (mean±sd) and . y± . since tx were studied. on the day of study all patients had normal cbp. % had lvh of whom % had abnormalities on abpm. overall, % patients had both abnormal abpm and lvh. % had other findings: % abnormal abpm but no lvh; % normal abpm but no lvh; % normal abpm and lvh. data were analysed for differences between groups of patients, with and without lvh. bp alone was significantly associated with increased lvmi. time averaged cbp was normal in all patients although differed significantly between the groups: systolic bp z-score [mean (ci)] . ( . , - . ) with lvh; -. (- . , - . ) without lvh. there were significant differences by abpm criteria relating to several components of the abpm profile including mean arterial pressure, systolic and diastolic bp load. no difference was found between the groups for hb, ca*po product, ipth and cgfr. conclusions: we have found the majority of our renal transplant patients have normal cbp but have abnormal abpm in association with lvh. this suggests better control of hypertension should be achieved in patients who have abnormal abp profiles and lvh. a new group of patients with normal abpm but lvh has also been identified. background: obesity is an independent risk factor for renal failure. therefore, we compared the body composition of pediatric nephrology patients with the general child population over two decades. methods: , patients with a mean age of . ± . years were studied. in , patients ( . %), sufficient data were available to analyze body composition. body composition was measured as body mass index (bmi) z-score because of the age dependency, calculated on the basis of data from the national (usa) center for health statistics ( ) . results: enuresis ( . %), hematuria ( . %), recurrent urinary tract infections ( . %) and proteinuria ( . %) were the most common diagnoses. the bmi z-score of the pediatric nephrology patients increased significantly from . ± . in - to . ± . in - and . ± . in - . while the rate of this increase was not statistically different from that seen in the normal population, they consistently demonstrated a significantly higher bmi z-score (average + . ) over time. nephrotic edema, non-nephrotic proteinuria and hypertension were not confounding factors. conclusions: patients seen in our pediatric nephrology service over two decades had a higher bmi than the average child population. this implies that these patients are at even greater risk for development of chronic kidney disease later in life. we recommend therapeutic intervention to address this potentially modifiable risk factor. objective: b cell dysregulation is believed to be involved in the development of childhood-onset systemic lupus erythematosus (sle). there is limited evidence regarding efficacy and safety of interventions targeting b cell in children. in our study we evaluated efficacy and safety of b lymphocyte depletion therapy. methods: data of children ( % male) with sle aged . years ( . - . ) treated with rituximab in a single centre were retrospectively reviewed. biochemical parameters were evaluated before and after treatment, and the normalisation of the parameters was assessed as a primary outcome. results: prior to rituximab therapy all patients received extensive immunosuppressive agents. indications for rituximab therapy were chronic (n= ) or acute illness, in either relapsing sle (n= ) or first presentation (n= ). rituximab mg/m was intravenously administered twice within a -week period in combination with cyclophosphamide. patients were followed up for . years ( . - . ) . no serious side effects were seen, except for viral infections such as herpes zoster (n= ). all patients with high creatinine (n= ; ± mmol/l) prior to rituximab showed a decline within months (mo), achieving a stable level by mo ( ± mmol/l, p< . ). all patients with hypoalbuminaemia (n= ; . ± . g/l) improved ( mo: . ± . g/l, p< . ). low c level ( . ± . g/l) as seen in patients prior to treatment resulted in an increase up to mo ( . ± . g/l, p< . ). a decline of previously high anti-dsdna (n= ; ± iu/ml) was observed in all patients ( mo: ± iu/ml, p< . ). conclusion: rituximab is safe and effective when used in combination with standard immunosuppressive agents. further prospective studies are essential to evaluate the longterm safety of the drug. outcome of severe henoch-schönlein purpura nephritis treated with longterm immunosuppression m. shenoy, m. bradbury, l. lewis, n. webb royal manchester children's hospital, department of nephrology, manchester, united kingdom aims: to look at the long-term outcome of all children with severe henoch-schönlein purpura nephritis (hsn) treated with long term immunosuppression in a single centre over a ten year period. patients and methods: retrospective review of the records of children ( male) with iskdc grade b, , and hsn managed at our institution from / / to / / results: the mean age at presentation was . years (range . - . years). the median estimated glomerular filtration rate (egfr) at presentation was ml/min/ . m (iqr . - . ) and urine protein: creatinine ratio (up: uc) was mg/mmol (iqr - ). the indication for biopsy was nephrotic syndrome in , nephrotic range proteinuria in , sub-nephrotic range proteinuria in , acute nephritis in and nephritic-nephrotic syndrome in . a total of patients were treated with weaning dose of steroids, of whom were also commenced on long-term azathioprine (mean duration . months). of these children received an - week course of oral cyclophosphamide ( - mg/kg/day) prior to azathioprine therapy ( - mg/kg/day). outcome: after a mean follow-up period of years, ( %) have made a complete recovery, ( . %) have persistent proteinuria but normal egfr, ( . %) have persistent proteinuria and are on anti-hypertensive therapy with normal egfr and ( . %) have progressed to esrd. older age at presentation was the only independent risk factor for poor outcome ( . years vs. . years, p= . ). conclusions: despite treatment with cyclophosphamide, long-term steroids and azathioprine, a majority of children with hsn grade - b on initial biopsy have persistent renal abnormalities on long term follow-up. only older age at presentation was associated with poor outcome. background: resent advances in podocyte biology indicated that the main cause of the heavy proteinuria in nephrotic syndrome (ns) is a dysfunction of slit diaphragm. on the other hand, the classical charge selective barrier is not likely to have a place in slit diaphragm. therefore we reevaluated the charge selective barrier function in ns and chronic glomerulonephritis using recently established charge selectivity index (csi; takahashi et al, pediatr res : , ) in comparison with dent disease. patients and methods: csi is a clearance ratio of igg (stokes einstein radius - , pi . - . ) and iga (stokes einstein radius , pi . - . ) . the assay of serum and urinary igg and iga was performed using laser nepherometry and enzyme immuno-assay. in order to evaluate the csi of normal glomerular filtrate, we measured the csi of dent disease. the urine of dent disease is considered to be a concentrate of filtered protein from normal glomerulus, without having a process of tubular protein reabsorption. thirty eight patients with podocyte diseases (focal and segmental glomerulosclerosis , finnish type congenital nephrotic syndrome , steroid sensitive nephrotic syndrome ), patients with chronic glomerulonephritis (iga nephritis , henoch-schönlein purpura nephritis , mesangiocapillary glomerulonephritis , alport syndrome ) and patients with dent disease, were analyzed. results and conclusion: csi (mean±sd) of podocyte disesses, chronic glomerulonephritis and dent disease was . ± . , , ± . and . ± . respectively. the results apparently indicated that the charge selective barrier of gcw is working strongly in normal glomerulus, less strongly in podocyte diseases and not working in chronic glomerulonephritis. objectives: nphs mutations have been reported in familial and sporadic srns. we investigated the prevalence of nphs mutations among south-east asian chinese and their association with clinical outcomes. methods: genomic dna from patients with primary sporadic srns (mean age at onset . ± . years, range . - . years) and cord blood controls were screened on all exons and exonintron boundaries using direct sequencing. results: a missense heterozygous c>t mutation in exon was identified in only one patient. polymorphisms t>c and a>g in exon were detected in both groups. in the patient group, the genotypic frequency of tt, tc, cc at position was . , . and . , and aa, ag and gg at position was . , . and respectively, consistent with hardy-weinberg expectations. there was no significant difference in allele frequencies between patients and controls. using binary logistic regression analysis, individual polymorphisms did not appear as informative predictors of poor clinical outcome, defined as persistent proteinuria or renal failure (p> . ). on analyzing composite genotypes, carriers of at least a copy of the c allele were significantly associated with poor outcome (p= . , or= . , % ci: . - ), while heterozygotes for a>g were associated with good outcome (p= . , or= . , % ci: . ± . ), suggesting possible interactions between the polymorphic sites. further analysis showed that the genotypic combination of tc/cc with aa was more likely to have a poor clinical outcome. no significant linkage disequilibrium was detected between the two polymorphisms. conclusion: the concomitant occurrence of at least a copy of the c allele and the aa genotype may be associated with poor clinical prognosis in srns but larger studies are needed to confirm these findings. renal hypodysplasia (rhd) is characterized by a reduced kidney size and/or maldevelopment of the renal tissue following disturbed organogenesis. numerous deletion mouse models of developmental genes have been established presenting with anomalies of the kidneys resembling rhd, among these the knock-out of bmp and six . here, we report on the first human mutations in bmp and six identified in children with rhd, among these three different mutations in bmp in five unrelated patients (ser cys, thr ser, asn lys) and three different mutations in six also in five unrelated individuals (leu phe, pro leu, asp asn). overexpression assays in zebrafish demonstrated that ventralization and dorsalization caused by bmp and six overexpression, respectively, could be diminished after overexpression of mutant constructs expressing the human mutations identified. morpholino knock-down of zebrafish bmp and six . reveals specific roles of these genes for pronephric development, affecting the expression of wilms tumor- (wt ) and glomerular development. rna analysis of cos and hek transfected with different bmp constructs showed a lower level of mrna abundance in bmp mutants, indicating a possible negative feedback of the mutants on their own mrna expression and/or stability. nonreducing western analysis revealed that s c-bmp forms alternative protein complexes as compared to wildtype-bmp , due to the formation of extra disulfide bonds. these studies implicate six and bmp as important players in the development of the renal system, and suggest that defects in these proteins could affect kidney development at multiple stages leading to the congenital defects observed in rhd patients. apoptosis is important in normal renal development in which regulation of cell numbers is critical. studies have shown that apoptosis occurs in non-cystic tubules in pre-uremic pkd kidneys, thus providing the mechanism whereby expansion of cysts is accompanied by loss of normal nephrons. to date, it is unclear which initiating factors (intrinsic, through mitochondrial damage or extrinsic, through ligand activation of death receptors) are responsible for apoptosis in pkd and whether they are the same in ad-and arpkd. aim: to elucidate the sequence of activation of intracellular apoptotic pathway(s) in both ad-and arpkd. methods: proteins were extracted from tissues of human ad-and arpkd kidneys and normal adult (nhk) and fetal kidneys (hfk). quantitative western immunoblot analysis was carried out for markers of intrinsic and extrinsic apoptosis. immunohistochemical staining for these markers was performed on tissue sections of the same kidneys. results: markers of extrinsic apoptotic pathways, caspases and , were significantly increased in ar-and in adpkd tissue by comparison to nhk and hfk tissues. these increases were seen in early stages of adpkd and were more pronounced later in the disease. for the intrinsic pathway, caspase and bid were increased in hfk tissue, but unchanged in ad-and arpkd tissue compared to nhk tissue. staining for caspase was found in hfk, but was absent in all other kidney tissues. staining for caspase was seen in early adpkd and endstage (es) adpkd as well as in es arpkd. caspase was identified as the main executing caspase of fetal development and adpkd, but wasn't seen in arpkd, where caspase predominated. conclusion: induction of extrinsic pathways of apoptosis predominates in pkd and occurs early in the disease process in adpkd, but only later in arpkd. intrinsic apoptosis predominates during development. intrauterine growth restriction (iugr) is a risk factor for an aggravated course of renal diseases in later life. the influence of postnatal factors, eg. accelerated catch-up growth, is not well understood. we therefore analysed the influence of postnatal nutrition after iugr on the developement of later renal inflammation and fibrosis in the rat. iugr was induced by low protein diet ( % vs. %) in pregnant wistar dams. litter size was reduced to or male animals in iugr (lp , lp ) and control animals (np , lp ), respectively. animals were sacrificed on day . mean arterial blood pressure was similar in all four groups. lp -( . ± . ml/h/ g) and np animals ( . ± . ml/h/ g) showed reduction of endogen creatinine clearance by % (vs. np and lp ) (p< . ). renal mrna expression of il ( , x), tgfβ ( , x) , endothelin ( , x) und osteopontin ( , x) was significantly higher in lp than in np . lp showed the highest glomerulosclerosis-score (( . ± . ) (vs. np ( . ± . ), lp ( . ± . ) and, np ( . ± . )) (p< . ). as marker of extra cellular matrix expansion glomerular collagen-iv deposition was significantly higher in lp ( . ± . %) (vs. np ( . ± . %), lp ( . ± . %) and np ( . ± . %)) (p< . ). postnatal nutrition modifies the consequences of iugr in the kidney. increased postnatal nutrition of the individual animal is associated with aggravated renal inflammation and fibrosis after iugr. unilateral renal agenesis (ura): how intensively do we need to investigate and follow-up? s. rhodes, a. watson nottingham university hospitals, children and young people's kidney unit, nottingham, united kingdom a single kidney is one of the commonest urinary tract abnormalities in the general population. concerns remain about long-term outcomes with a reduced nephron mass and hence intensity of investigations and duration of follow-up. we identified cases with ura (ectopic and pelvic kidneys excluded) from our nephrourology database between and . ( %) occurred in males and ( %) had left ura. ( %) were detected antenatally with / ( %) recognised in the last years. median age of detection for postnatal ura was mths (range . - mths) with uti ( %) as the main indication for ultrasound scan (uss). % patients were classified as simple ura with no associated abnormality and % as complex with problems such as vesicoureteric reflux (vur)( %), hydronephrosis or scarring. all cases reviewed had uss which showed compensatory hypertrophy in / ( %). / ( %) had dmsa scan and / ( %) micturating cystogram. of those antenatally detected single kidneys with normal initial uss none had vur, scarring, hypertension or proteinuria. these patients were discharged from follow-up at the median age of mths (range - mths). all complex cases have continued under follow-up. conclusions: our data suggest that the incidence of antenatally detected ura may be increasing. investigations need to be individualised depending upon the initial uss. the value of routine dmsa and mcug in simple cases is questioned. most of these patients can be discharged after adequate documentation of compensatory hypertrophy of the normal kidney, absence of proteinuria, normal blood pressure and renal function. autosomal dominant pdk (adpkd) in childhood j. crocker, p. wornell, p. acott iwk health centre/dalhousie univeristy, division of nephrology, halifax, canada autosomal dominant polycystic kidney disease (adpkd) is the most common genetic kidney disease with in adults progressing to end-stage renal disease (esrd). a program for intervention in childhood at presentation with adpkd was developed and instituted in . our long-term objective is to modify clinical parameters that may contribute to risk of development of esrd in adulthood. seventy children with adpkd (average age . yr) were followed, of which ( %) had nephromegaly +/-cysts on antenatal ultrasound. modifiable risk factors were common including hypertension ( %), hyperlipidemia ( %), and proteinuria ( %). ace inhibitors were first line therapy for proteinuria and/or hypertension. ace inhibitors may modify cyst progression so they have been made available to non-hypertensive children as well. most patients with hyperlipidemia responded to dietary intervention with one patient developing gallstones. in the past years we have focused on renal calculi, as this is a known risk for a subgroup of adults with poor prognosis. we noted patients ( %) have glycinuria, which is a precursor to oxaluria. four patients have passed calculi with two of these being diagnosed by genetic linkage analysis for adpkd without radiographic cysts present. cerebral vascular studies of patients with severe headaches revealed two with vascular structural anomalies. all adolescent females received counseling regarding appropriate contraception and their pregnancy risks of hepatic cyst progression. an early intervention program targeting modifiable risk factors of adpkd patients during childhood and adolescence may modify adult renal failure risk in this population. background: techniques of chronic infant dialysis have evolved during the past decades but morbidity and mortality are not well described. methods: a retrospective review was performed on infants - months of age with end stage renal disease (esrd) treated with maintenance dialysis during the past years. the experience was divided into era ( era ( - (n= ) and era ( era ( - . dialysis modality, morbidity, and long term survival were assessed and compared between the eras. results: all patients were begun on peritoneal dialysis (pd). there were males and females. age at initiation of dialysis was , months. the predominant diagnoses were dysplasia/obstructive uropathy (n= ), autosomal recessive polycystic kidney disease (arpkd) (n= ), congenital nephrotic syndrome (cns) (n= ), other (n= ). overall survival is % ( / ) with current age of survivors ranging from months to years. mortality between era = % and era = % was not significantly different. fifteen ( %) survived to receive a kidney transplant. overall median survival is . years (era = . years; era = . years; p= . ). conclusions: although long term survival is possible in infants with esrd, mortality and morbidity remain high. improved technologies for automated pd should address the needs of the infant < kilograms. joubert syndrome and related disorders (jsrd) are a group of autosomal recessive conditions characterized by a complex neuroradiological malformation resembling a molar tooth on imaging. clinically, jsrd are characterized by overlapping phenotypes presenting neurological signs and variable involvement of other organs such as kidneys (mainly nephronophthisis -nph), retina and liver. seventy-nine italian jsrd patients from unrelated families were recruited in pediatric nephrology and neurology centers. medical records and brain mri were reviewed. patients without chronic renal failure (crf) underwent measurement of their glomerular and tubular function, a ddavp urine concentration test and a renal ultrasound. retinal examination was performed in most patients. seven patients younger then years had normal renal function but were not fully tested. of the remaining cases, had no evidence of renal disease, had developed crf mostly in their second decade of life, and were younger then years of age and had urinary concentration defects, of whom also had hyperechogenic kidneys by renal ultrasound. when comparing this latter group with age-matched jsrd patients, no other tubular function abnormality was detected. in multiplex pedigrees, no discrepancies in renal involvement between affected family members was observed. retinal involvement was significantly associated with renal disease. conclusions. evidence of interstitial renal disease was observed in one third of italian patients with jsrd. renal involvement should always be suspected in these patients, particularly if they have evidence of retinal dysplasia or other family members with symptoms related to nph. these patients should be assessed with a urine concentration test. we examined the course of children with primary obstructive megaureters (pom) treated in our hospital from - . pom occurred more often in boys ( %, p< . ) and on the left side ( %, p< . ). pom ( %) were only treated conservatively. four underwent immediate surgery following the first mag renography. one of these was nephrectomized after unsuccessful urinary diversion and persistent renal hypertension. of the kidneys primarily managed conservatively, needed a ureterocystoneostomy later on due to increasing obstruction. surgical correction of pom required a mean of days of hospitalization (incl. temporary urinary diversion, removal of catheters and treatment of complications). one child required surgical revision of early post-operative ureteric stenosis. urinary tract infections (uti) were a common complication (n= , mean . per patient). as utis occurred mainly in infants, hospital admission was commonly required ( %). only children never acquired a uti ( %). eight patients had a poor outcome as defined by partial kidney function < % on the affected side (n= ), atrophy on final ultrasound (n= ) or nephrectomy (n= ). the most potent predictor of reduced unilateral kidney function was a small kidney present from birth. secondary kidney growth failure occurred only in one case. the initial degree of obstruction on renography, but not the degree of hydronephrosis or size of megaureter predicted outcome. also, prenatal diagnosis of pom, surgical treatment and the occurrence of utis showed no association with outcome. in summary, the long-term prognosis of pom appears favorable. adverse outcomes were more closely related to congenital kidney size deficit than to the degree of obstruction. surgical interventions and the high uti incidence led to significant hospitalization times. r. bhimma, m. adhikari, k. asharam university of kwazulu-natal, maternal and child health, durban, south africa background: steroid resistant (sr) forms of ns have a poorer outcome in blacks compared to other racial groups. methods: children with srns - years old were analysed retrospectively for the period - . treatment schedules included oral cyclophosphamide with prednisone only (n= ); prednisone on alternate days with methylprednisolone and oral cyclophosphamide (n= ); oral prednisone on alternate days, doses of intravenous methylprednisolone on alternate days and monthly doses of intravenous cyclophosphamide (n= ) or cyclosporine adjusted to a trough level of - mg/ml (n= ). we compared the clinical, biochemical characteristics and outcome of these children using different forms of therapies. results: ( . %) underwent renal biopsy. ( . %) were indian and ( . %) were black. ( . %) had minimal change ns, ( . %) had focal segmental glomerulosclerosis (fsgs), ( . %) a proliferative form of ns, and ( . %) had other forms of ns. / ( . %) indian children biopsied were in complete remission and / ( . %) with mcd who were treated with oral cyclophosphamide and prednisone only achieved complete remission. / ( %) indian children not biopsied who received only oral prednisone and cyclophosphamide achieved complete remission. / ( . %) black children who were biopsied achieved complete remission. none of the eight black children who were not biopsied given only oral cyclophosphamide and prednisone achieved complete remission. conclusion: since % of indian children with srns responded to a trial of oral cyclophosphamide and prednisone but none of the black children did, we propose the use of oral cyclophosphamide therapy in non black children before embarking on renal biopsy. mj. kemper, c. moeller, n. rink, m. van husen, de. müller-wiefel university of hamburg, pediatric nephrology, hamburg, germany introduction: ssns is often complicated by a refractory clinical course with frequent relapses and steroid dependency despite aggressive alternative immunosuppressive regimens. since recent immunological findings suggest an alteration of not only t-but also b-cell immunity in ssns (kemper (kemper , we hypothesized that that immunological targeting of b-cells with antibodies directed against cd (rituximab) is able to maintain remission (rm) in treatment refractory patients with ssns methods: a total of six patients with complicated courses of ssns had severe steroid-dependency and toxicity. all patients had previously been treated with cyclophosphamide, two patients relapsed on maintenance therapy with cyclosporine, one relapsed on additional levamisole and one on mmf. treatment was initiated at a median age of (range - . ) years and rtx was given at steroid induced rm at a dose of x mg/m bsa within weeks. results: a complete b-cell depletion was induced for at least months in all patients. rm could be maintained in all patients and steroid treatment could be discontinued after a median of . months (range - . ) . also csa could be discontinued in the two patients on maintenance treatment. at current follow-up of . months (range - . ) the two patients after csa discontinuation relapsed after . and . months, respectively, but responded after steroid induced rm-to a second course of rtx. all other patients remained in remission off treatment so far. no significant clinical side effects were noted. conclusion: in summary and conclusion, rtx seems to be a therapeutic option in complicated ssns. long-term follow-up and future prospective studies are necessary to further define the role of rtx in the treatment of refractory steroid sensitive nephrotic syndrome. efficient control of secondary hyperparathyroidism can be achieved by calcimimetics. they increase calcium sensing receptor (car) sensitivity to extracellular calcium. in r- -treated uremic rats, proteinuria has been significantly reduced by calcimimetics. thus, we examined the potential direct effect of r- on podocytes. the car was expressed in cultured immortalized podocytes (quantative rtpcr, western blot) and in podocytes obtained from healthy and subtotally nephrectomized rats (immunohistochemistry). glomerular car abundance was increased by uremia and r- . the car colocalized with the plasma membrane and intracellular filaments in cultured podocytes, but not with the caveolin -and -rich membrane fractions. we then studied the effect of r- on the mitogen-activated protein kinase (mapk) family. jnk was not activated. p showed a biphasic response pattern, whereas erk / (and further downstream, p ribosomal s kinase) showed dose-( - nmol/l) and time-dependent phosphorylation, resulting in the activation of the transcription factor camp response element binding protein (creb). creb phosphorylation induced bcl-xl expression and bad phosphorylation, both of which have prosurvival activity. specificity was confirmed by addition of mek / inhibitor u , which completely blocked r- -induced creb phosphorylation. facs analysis revealed a significant, % decrease in puromycin-induced apoptosis in podocytes treated with r- for , and hrs. in conclusion, calcimimetics induced prosurvival gene expression in podocytes via mapk, protecting them from apoptosis. calcimimetics may have a direct renoprotective action beyond control of hyperparathyroidism in chronic kidney disease patients. we report our experience with plasma therapy in a family of three sisters from whom two are homozygous twins (patients b and c), presenting a missense mutation of the exon of the human complement factor h gene (hf- ). the factor h concentration has always remained normal and no systemic complement activation has ever been detected. in a ten years period, the followings have been observed: / no use of plasma exchange lead to immediate esrf of native kidneys (patient a); ) conventional plasma therapy ( sessions at presentation followed by repeated ml/kg plasma infusions when relapse of hemolysis and thrombopenia) could not impede esrf (patient b); ) the use of intensive pe at presentation (daily pe ( ml/kg ffp until plasma creatinine normalization followed by one pe/ w indefinitely) lead to a normal gfr years after presentation despite relapses (patient c); / no prophylactic pe for tx lead to immediate hus relapse and transplant loss (patient ); ) prophylactic pe allowed a successful tx in patient b (pl. creat mcmol/l after years); ) early and intensive pe allowed complete normalization of several hus relapses after tx (patient b). conclusions: in fh mutation-related atypical hus, ) intensive and indefinitely prolonged pe can allow a normal function of native kidney at long term; ) prophylactic pe allows successful kidney tx; ) early and intensive pe allow reversion of hus relapse after tx. the success of plasma therapy is bound to the followings: a/ the use of pe and not plasma infusion. b/ the prolongation of daily pe after normalization of hemolysis parameters, and further prophylactic pe. c/ the use of pe prophylaxis from before tx. d/ a minimum pes frequency of one/week in case of prophylactic treatment for tx. e/ immediate intensification of pe frequency in case of relapse. objectives of study: heparan sulfates (hss) are highly polyanionic sugar chains in the glomerular basement membrane (gbm) and have been reported to play an important role in the chargeselective permeability of the kidney. alterations in hs expression have been reported in a number of renal pathologies. in this study, we evaluated if degradation of hs in the gbm resulted in proteinuria in rats, using a controlled in vivo approach. methods: heparinase iii and neuraminidase were injected i. v. in -month old wistar rats at t= hr and t= hr, and kidneys were removed at t= hr. urine samples were taken at various time points. cryosections were stained for hs using specific antibodies, and for hs stubs (generated by heparinase). in addition, hs was evaluated at the electron microscopical level. neuraminic acid expression was analysed by peanut agglutinin lectin. the hs content in urinary samples was evaluated by agarose gel electrophoresis. urinary neuraminic acid was studied by an enzymatic colorimetric assay. presence of urinary albumin (proteinuria) was investigated by sds-page and by a competition elisa. results: injection with heparinase iii resulted in an almost complete absence of glomerular hs staining. cupromeronic blue staining was also greatly reduced in the gbm, further indicating that hs was largely degraded. staining for the hs proteoglycan core protein agrin was unaltered. in the urine a strong increase in hs was found, already at the first point in time of urine collection ( hr after the first injection). however, no urinary albumin or other proteins could be detected at any point in time analysed. injection of rats with neuraminidase resulted in a major increase of albumin in the urine. conclusion: in conclusion, removal of hs from the gbm does not result in acute albuminuria, whereas removal of neuraminic acids does. introduction and methods: children undergoing deceased ( ) or living ( ) donor kidney transplantation were randomized to receive tacrolimus, azathioprine, steroids and two doses of basiliximab (tas+b) or tacrolimus, azathioprine and steroids(tas). previously reported six month follow-up biopsy-proven acute rejection rates were . % (tas+b) and . % (tas). patient survival was % and graft survival % in both arms (am j transplant ; : - ) . in this investigator-driven follow-up study individual outcome data were submitted annually to the coordinating centre. results: two year follow-up data were obtained for ( . %) of the who completed the six month study. there was one death in the tas group occurring at month . there were graft losses in the tas+b arm and in the tas arm. all graft losses in the tas+b arm occurred within the first months. kaplan-meier estimates of year graft survival were . % for tas+b and . % for tas (p= . breslow generalised wilcoxon test). episodes of biopsy proven acute rejection occurred in in tas+b and in tas, kaplan-meier estimates of freedom from rejection being . % and . % respectively (p= . ). renal function did not differ significantly between the two arms; the median (iqr) plasma creatinine levels were ( - ) in tas+b and ( - ) in tas (p= . ). similarly there was no evidence of a difference in either systolic or diastolic blood pressure between the two arms. there was one case of b cell ptld in the tas+b arm at months in addition to two cases in the tas arm previously reported in the month study. conclusions: the addition of basiliximab to a regimen of tacrolimus, azathioprine and steroids does not appear to result in an improvement in either acute rejection rate or graft survival at two year follow-up. further data collection is ongoing. chronic renal dysfunction is a major complication after heart transplantation (htx). the pathophysiology is not yet fully understood but is thought to be in part due to calcineurin inhibitor (cni) toxicity, and reducing cni exposure has become one of the main strategies aimed at ameliorating renal outcome in htx recipients. we previously reported a significant improvement of renal function in htx children with biopsy-proven cni nephrotoxicity and chronic renal failure, year after the reduction of cni dosage with a concomitant replacement of azathioprine by mycophenolate mofetil. we ought to determine whether this improvement was persistent after years of follow-up, despite the histological lesions of chronic cni nephrotoxicity. gfr evaluated by annual inulin clearance had improved from a mean of . ± . ml/min/ . m (range - ) at time of the switch to . ± . ml/min/ . m (range - ) (p= . ) one year after ( % improvement), and remained stable at . ± . ml/min/ . m (range - ) (p= . ) years after the switch. maximal urinary osmolality followed the same increase profile from ± mosm/kg before the switch to ± at one year and ± after years (p= . ). meanwhile, the occurrence of serious adverse events such as infectious episodes, acute rejection and chronic allograft dysfunction as assessed by clinical examination, echocardiography and endomyocardial biopsies were not different from a control group of patients whose treatment was unchanged. no malignancy was observed in either group. in conclusion, reduction of cni dosage and replacement of azathioprine by mycophenolate mofetil lead to a safe and long-lasting improvement of renal function in children with heart transplants and cni-induced nephropathy. previous studies have demonstrated reduced bone mineral density (bmd) in some patients with idiopathic hypercalciuria (ih). reduced bmd during childhood may impact adult peak bone mass. bisphosphonates employed in adults with ih and reduced bmd resulted in conflicting outcomes. we evaluated the effects of oral bisphosphonate, alendronate (ale), in patients with persistent ih and reduced bmd. patients presented at ages , , yr, with hematuria/dysuria, had recurrent urolithiasis, and all had ih (uca > mg/kg/ hr). despite maximal traditional rx with low na/high k diet, thiazides, k-citrate, ih persisted ( . - mg/kg/ hr). at ages , and . yr, dxa showed reduced bmd, and ale , and mg/once weekly was given for , and months, respectively. after months of ale, uca decreased significantly compared to baseline (mean±sd, . ± . vs. . ± . mg/kg/ hr, p< . ). compared to baseline, bmd z scores year after starting ale improved at the spine (- . ± . vs. - . ± . , p< . ) and hip (- . ± . vs. - . ± . ) . the decline in uca during ale rx correlated with the increased bmd z scores in the spine (r=- . , p= . ) and hip (r=- . , p< . ). height z scores, serum creatinine, ca, p, electrolytes and pth remained normal throughout the rx period and no further hematuria or new stones occured. in summary, ale normalized uca previously resistant to traditional rx, eliminated urinary symptoms and improved reduced bmd in children with ih. the use of a single oral weekly dose appears adequate and safe. larger prospective studies are needed to confirm these preliminary results. in addition to its classical role in the regulation of calcium (ca) and phosphate (po ) homeostasis, vitamin d has important immunomodulatory and anti-inflammatory effects, that in turn can influence atherosclerotic vascular disease. we studied the impact of vitamin d levels and inflammation on vascular structure and function in children on dialysis. children (age . ± . yrs) on dialysis (mean duration . ± . yrs) were studied. all children received α-hydroxyvitamin d (alfacalcidol) . cumulative data on ca, po and pth, doses of po binders and alfacalcidol were recorded. , -dihydroxyvitamin d (vit d) and high-sensitivity crp (hs-crp) levels were measured by i radioimmunoassay and elisa respectively. all children had carotid intima-media thickness (cimt), pulse-wave velocity and cardiac ct for coronary calcification. ( %) children had vit d deficiency (levels< pmol/l), ( %) hadnormal levels ( ± pmol/l) and ( %) had high levels (> pmol/l). vit d positively correlated with serum ca, caxpo and alkaline phosphatase. both cimt and calcification showed a bimodal distributionpatients with vit d levels < or > pmol/l were significantly more likely to have calcification or raised imt than those with vit d levels in the normal range. hs-crp levels independently predicted cardiac calcification (p= . ) but not cimt. there was a strong inverse correlation between vit d levels and hs-crp (p< . , r=- . ). patients with vitamin d levels< pmol/l and hs-crp levels> mg/l had -fold greater calcification scores than subjects below these cutoffs. in conclusion, vit d deficiency is common despite treatment in children on dialysis. vit d may be an important mediator of vascular damage both through its hypercalcaemic and anti-inflammatory actions. severe growth failure remains one of the challenging problems in the care of children suffering from chronic renal failure (crf). although, rhgh has been proven to increase final adult height in prepubertal crf patients only limited data on its efficacy in the pubertal age-range are available. in addition, the impact of the underlying renal disease and the mode of renal replacement therapy on final height in these patients remain unclear. we report on final height data of ( female) severely growth-retarded crf patients (standardized height <- sds; database: kigs medical outcomes, pfizer). mean age at start of rhgh therapy was . ± . years, standardized height was - . ± . sds and duration rhgh therapy was . ± . years (range . to . years). at baseline % of the patients were on conservative treatment, % were on dialysis and % had a functioning renal allograft. in the whole study population mean standardized height was increased in the first treatment year and at attainment of final adult height by + . sds and + . sds, respectively (each p< . vs. baseline). prepubertal children aged less than years at start of rhgh therapy showed the best growth response (+ . sds). in pubertal patients mean increase in standardized height was + . sds, whereas growth response in patients with delayed onset of puberty (>+ sd) was significantly lower (+ . sds; p= . vs. other groups). the duration of rhgh therapy was positively associated with cumulative height gain. growth response was significantly lower in patients on long-term dialysis and in patients with nephropathic cystinosis. conclusion: rhgh therapy in severely growth retarded prepubertal and pubertal crf patients results in an increased final adult height. growth response is diminished in crf patients with markedly delayed onset of puberty. tightly regulated rankl/opg system is essential for normal bone remodelling. however, the exact roles of those osteogenic markers in uremic bone disease have yet to be defined. we therefore assessed the potential relationship of the rankl/opg system in bone biopsy proven secondary hyperparathyroidism (hpt) in dialyzed children. methods: patients aged ± years were on ccpd for ± months. s-ca, p, alk p'tase, pth, opg and rankl levels were measured. bone biopsies were obtained after double tetracycline labeling and none of the patients were treated with vitamin d for four weeks prior to biopsy. results: s-ca levels were . ± . mg/dl, p: . ± . mg/dl, alk p-tase: ± u/l, pth: ± pg/ml. bone biopsy findings revealed high turnover bone disease in patients, patients had normal bone formation rate (bfr). mean opg and rankl levels were . ± . and . ± . pmol/l, respectively (reference control=opg: . ; rankl: . ). opg correlated with bfr (r= . , p< . ) and adjusted apposition rate (r= . , p< . ), while inversely correlated with osteoid maturation time (omt) (r=- . , p< . ) and mineralization lag time (r=- . , p< . ). rankl/opg ratio was negatively correlated with mineral apposition rate (r=- . , p< . ) and positively with omt (r= . , p< . ). pth was correlated with bfr (r= . , p< . ) and resorption area (r= , , p< . ), but not with any mineralization markers. there were no correlations between pth and opg or rankl. conclusion: opg, in contrast to rankl, exerts a dual effect on the skeleton by promoting mineralization and increasing bfr. these osteogenic markers might be of benefit in characterizing the turnover, mineralization and volume of the skeletal lesions of secondary hpt as recently recommended by kdigo. "sevcan bakkaloglu was supported by tÜbitak (scientific and technological research council of turkey). this study was supported by usphs grants dk- , dk- and mo -rr . " pth values are widely used to guide therapy for renal osteodystrophy in patients treated with maintenance dialysis yet target values are based on cross-sectional studies and discrepancies between bone formation rates (bfr) and pth have been described during intermittent calcitriol rx. thus, we evaluated the relationship between serum biochemical parameters and bone turnover during treatment with intermittent vitamin d sterols. patients aged ± yrs with biochemical and bone biopsy (bbx) proven nd hpt received months of vitamin d ( , d or d ) given in twice weekly oral dosing and phosphate binders. dose of vitamin d was titrated upwards monthly to maintain st pth-ima(nichols r ) levels between - pg/ml. bbx was then repeated. s-ca, p, alk p-tase and pth by st and nd pth-imas were obtained monthly throughout therapy. baseline values were: p: . ± . mg/dl, ca: . ± . mg/dl, alk p-tase: ± iu/l, st pth-ima ± pg/ml, nd pth-ima: ± pg/ml. final values were: p: . ± . mg/dl, ca: . ± . mg/dl, alk p-tase: ± iu/l, st pth-ima: ± pg/ml, and nd pth-ima: ± . bone formation rate (bfr/bs) decreased from ± to ± um /mm /d (nl: - . ); % achieved normal bone turnover. nd pth-ima values were - % lower than st pth-ima levels; there was no difference in predictive capability of the two assays. patients achieving normal bfr/bs had st pth-ima values of ± pg/ml. the sensitivity, specificity and ppv of a st pth-ima range of - pg/ml for normal bfr/bs were % ( % ci: - %), % ( - %), and % ( - %) respectively. during therapy with intermittent vitamin d sterols, maintaining pth levels higher than currently recommended results in normal bfr/bs and prevents adynamic bone. maternal diabetes will induce abroad array of congenital malformation. consistently with these hypotheses, we observed the defects of renal development of diabetic pregnancy from late phase of embryogenesis to postnatal period in animal model (c bl/ j mice). histological analysis of phenotypes revealed decreased glomerular numbers, glomerular hypertrophy and hypercellularity, as well as renal tubular detachment in sequential late phase of kidney development in the offspring of diabetic female mice. tunel assay showed signficinatly increased cell apoptosis in fetal kidneys of hyperglycemic group. rt-pcr and fish study of kidneys of fetal and newborn mice revealed that gdnf and early growth response alpha (egr-α) are two of crucial genes inhibited in hyperglycemic ambience. in human, we presumed that children of gestational hyperglycemic mothers have the same defects of renal development as our animal model similarly, thus we measured and compared echogram of kidney/liver echoenic ratio in children born to gestational hyperglycemic mothers and health children. interestingly, our human ultrasonic study indicated that after age of months, the diabetic children had increased echogenic ratio of kidney/liver than the healthy age matched children (p< . ). we also found that . % of diabetic children had nonobstructive hydronephrosis. this simple sonographic procedure may provide a permissive was for clinicians to obtain the basis of long-lasting follow-up of these high-risk children as early as possible. keywords: diabetic embryopathy, renal development, glial cell line-derived neurotrophic factor, early growth response alpha, renal sonogram. genetic inactivation of spry in mice results in increased number of ub branches and expanded gdnf, c-ret and wnt- expression domains (basson et al., dev. cell, ) , indicating that spry is a negative regulator of the gdnf-ret-wnt pathway. ang ii induced ub branching morphogenesis, partly via stimulation of egfr tyrosine kinase activity (yosypiv et al. jasn, ) . we tested the hypothesis that ang ii stimulates the gdnf-ret pathway via repression of spry . cd mice metanephroi were dissected on embryonic (e) day e . , grown on filters in the presence or absence of ang ii ( - m, n= /group) for hours and subjected to whole-mount ish with digoexigenin-labelled spry , c-ret, wnt- and gdnf crna probes. spry mrna was expressed in ub branches and in condensing mesenchyme. c-ret and wnt- were expressed in ub tips, and gdnf-in the metanephron mesenchyme. ang ii downregulated spry expression in the ub. in contrast, c-ret and wnt- were induced by ang ii in the ub tip cells. gdnf expression in the mesenchyme was also upregulated by ang ii. in addition, ang ii stimulated ub tip cell proliferation, as determined by in vivo brdu in corporation ( . ± . vs. . ± . ; p< . ) compared to control. these findings suggest a model in which ang ii-mediated inhibition of spry gene expression releases. ret tyrosine kinase activity leading to upregulation of c-ret and its downstream target gene, wnt- . enhanced wnt- expression, in turn, induces gdnf in the adjacent mesenchyme. this causes focal bursts of ub tip cell proliferation and branching. these results support the hypothesis that abnormal collecting system development in angiotensinogen, renin, ace or at -deficient mice is at least partly due to aberrant regulation of the ub branching morphogenesis program. objectives of study: nephrogenesis requires a fine balance of many factors that can be disturbed by intrauterine growth restriction (iugr), leading to a low nephron endowment. our previous studies have shown that offspring born to mothers supplied low protein diets during pregnancy have fewer glomeruli than normal at birth in sd rats. the aim of this study was to identify the possible pathogenesis of abnormal nephrogenesis and decreased glomerular number in iugr by comparative proteomic approach. methods: iugr was induced in sd rats by isocaloric protein restriction in pregnant dams. kidney proteins were obtained from neonatal normal rats (control group) and iugr rats (iugr group) respectively. a series methods including -de, silver staining, mass spectrometry and database searching were used. the -de test was repeated three times in each group. results: after silver staining, the -de image analysis detected average ± spots in iugr group and ± spots in control group. the average matched rate was % and % respectively. the differential proteomic expression analysis found eleven protein spots were expressed only in iugr group and one in control group. seven protein spots were up-regulated more than folds and two down-regulated more than folds in iugr group compared with those in control group. these protein spots were preliminarily identified, which were structural constituents of cytoskeleton including vimentin, cytokeratin , perlecan and b-actin, transcriptional factors including splicing factor, rho gdp dissociation inhibitor alpha and cell division proteinkinase , enzymes including retinal dehydrogenase , transketolase and so on. conclusions: data from this study may provide, at least partly, valuable experimental evidence of proteins involved in the pathogenesis of abnormal nephrogenesis and decreased glomerular number in iugr. the aim of the study is to describe the natural history of tcf /hepatocyte nuclear factor- beta linked disease in children. prenatal and postnatal renal evolution and extrarenal manifestations of patients were reported. thirty one had prenatal diagnosis of developmental nephropathy: bilateral foetal hyperechogenic kidneys or a hyperechogenic kidney with a controlateral multicystic dysplastic kidney. % had cysts. the mean prenatal renal length was normal ( , sd) . intrauterine growth was normal with median birth weight of . kg (range . - . ) and % were small for gestational age. after a mean follow-up of months, the renal growth was impaired with a mean renal size of - . sd. patients with a solitary functioning kidney showed no compensatory hypertrophy. thirty one patients developed bilateral nephropathy and isolated unilateral multicystic kidney. twenty eight patients had cysts, mainly cortical bilateral microcysts. the mean gfr was . ± . ml/min/sc. sixteen patients had stage chronic kidney disease (ckd), four were classified as stage , eleven as stage or ckd and two were diagnosed with end stage renal failure. annual decline of the glomerular filtration rate was . ml/min. extrarenal manifestations included patients with diabetes, one with exocrine pancreatic insufficiency, one with pancreatic hypoplasia without clinical symptoms and with cholestasis. we found a complete heterozygous deletion of the tcf gene in patients and nine different point mutations. three patients had the g c mutation, with unilateral mcdk and with bilateral severe hypoplasia leading to early renal dysfunction. tcf gene anomalies are associated with low renal function decline but in some patients end stage renal failure appeared early in life. long term follow-up is needed to determine the incidence of extrarenal symptoms, particularly diabetes. objectives: although it has been observed more than one hundred years ago that the urinary tract is very resistant to infection, its antimicrobial mechanisms have not yet been systematically characterized. we sought to elucidate the expression and relevance of the antimicrobial peptide cathelicidin in this area. methods: in order to investigate the expression of cathelicidin in health, urine samples from healthy children, pieces of healthy renal tissue, and cell cultures were analyzed. to evaluate the expression of cathelicidin during infection, urine samples from children with urinary tract infection, a mouse model of ascendant pyelonephritis and cell culture experiments were employed. the relevance of cathelicidin production was tested by bacterial challenge of cathelicidin-deficient and neutropenic mice. in addition, we studied the in vitro effects of cathelicidin on the growth and multicellular behavior of bacteria as well as we tested sensitivity of clinical e. coli strains to the peptide. results: cathelicidin is expressed by epithelial cells of healthy urinary tract and excreted into urine in low concentrations. during urinary tract infection, the secretion of cathelicidin by epithelial cells significantly increases within minutes. invading neutrophils are the source of the second wave of cathelicidin. epithelial cathelicidin protects the urinary tract against bacterial infection while neutrophil-derived cathelicidin influences the severity of infection. cathelicidin displays multiple effects on bacteria. low concentrations of cathelicidin inhibit multicellular behavior, e. g. biofilm formation of e. coli, and high peptide concentrations kill bacteria. bacteria resistant to cathelicidin have an increased ability to invade the urinary tract. conclusion: the antimicrobial peptide cathelicidin is a key factor of the mucosal immunity of the urinary tract. the predictive value of procalcitonine (pct) plasma level for renal scarring after an acute pyelonephritis (apn) is still debated. during apn, the bacterial lipopolysaccharide of the membranes induce the release of tnf, il and il . these cytokines lead to inflammation syndrome and fibrosis sequellae resulting from cell apoptosis induced by nitric oxide (no) release that is catalysed by no-synthase. the aim of this work was to clarify the physiological role of pct in renal parenchyma apoptosis and fibrosis caused by apn. we conducted a prospective study in children with a first apn episode (fever> . c°, crp> mg/l, monomicrobial urine positive culture> . fcu/ml). we excluded patients with any concomitant infection, renal dysplasia or obstructive uropathy or grade - vur children were enrolled (age . m, median m). on admission, pct, crp and phospholipase a (pla ) were quantified in serum. scintigraphy with m tc-dmsa was performed on day and months later in case of initial abnormalities. fisher's test was performed and statistical significance was defined as p< . . results: on day , ( %) presented renal parenchyma alterations, at m underwent control scan and ( %) had renal scars. pla and pct levels were correlated with early dmsa lesion but not with renal scars at m. paradoxically, initial pct level was significantly lower in the presence of renal scars ( . vs . ng/ml, p< . ). significant pct increase was observed in favourable progress (recovery . vs aggravation . ng/ml, p< . ) and no difference between recovery and improvement evolution. these results suggest the protective effect of pct against apoptosis resulting from down-regulation of nitric oxide release; so high pct values could be interpreted with caution in apn. acute pyelonephritis (apn) may lead to renal scarring with later risk of hypertension and renal insufficiency. the aims of this study were to analyse prospectively renal scars progression with time and their impact on renal growth. methods: patients (pts), aged from to years who presented scars on dmsa at months after apn were included. in these children a second dmsa was done after years. evolution of scars was analyzed following pre-established criteria independently by observers. scar progression was classified as follows: =no change, =partial improvement, =total resolution. in addition a renal ultrasound was repeated to assess kidney growth using z-score. results: renal units ( pts: f, m) were studied. the incidence of vesicoureteral reflux (vur) was . % ( / ). per renal units, vur were observed in / ( %); ( % vur grade (g) i, ii and % g iii, iv). there were scars observed month after apn which evolution over years was as follow: = % ( / ), = % ( / ) and = % ( / ). incidence of vur was higher in children presenting scars; / ( %) against / ( %) in those with or scars. to identify factors interfering with renal growth, we analyzed potential confounding variables using robust linear regression. z-score was worsened with increased number of scars observed at month after apn p< . ) and improved with disappearance of vur ( . ci . - . ; p< . ). conclusion: between month and three years after apn, % of scars improved. in our population, the number of scars secondary to pna was the most important factor affecting renal growth. the second prognosis criterion was the correction of vur. prevention of pna and rapid treatment to avoid kidney scars seems to be the essential aim to preserve optimal kidney growth. v. smolkin , , r. halevy , , w. sakran , , y. kennes , a. koren , ha'emek medical center, pediatric b, afula, israel ha'emek medical center, pediatric nephrology unit, afula, israel ha'emek medical center, bacteriology and microbiology laboratory, afula, israel the ruth and baruch rappaport school of medicine, haifa, israel febrile urinary tract infection (uti) is a relatively common infection disease in childhood. children consider at risk for uti commonly receive prophylactic antibiotics to prevent recurrence of this urinary tract disease because of the risk of kidney scarring, which may lead to complications such as hypertension or end-stage renal disease. compliance with antibiotic prophylaxis after uti was assessed in children, using a parent questionnaire and a urine test for antibacterial substances. thirty six children ( st group) received prophylactic treatment during the period of months (range - months) and the other patients ( nd group) received antibiotic prophylaxis for a longer duration ( months, range - months). in the first group of patients, ( %) of parents reported giving the antibiotics every day in comparison with ( %) in the nd group. twenty nine ( %) of urine tests were positive for antibacterial substances in the st group but only ( %) in the second group. in the st group of patients the difference in recurrent infection between regular takers and non-takers was statistically highly significant. no significant difference was found in recurrent uti rate in takers and non-takers in the nd group. failure to understand the reason for prophylaxis and forgetfulness was found to be a main reason for non-compliance. imaging studies evaluating the kidneys and urinary tract are performed routinely after a febrile uti. evidence of their value in changing management or affecting long term outcome is limited. as part of a multicentre, rct (iris ) evaluating different antibiotic regimes in the treatment of first febrile urinary tract infections, we undertook as a secondary objective, an evaluation of the diagnostic protocol. the imaging modalities (ultrasound, dmsa scintigraphy within days and voiding cystogram within - months) were assessed for their ability to predict long term parenchymal damage. children of years of age or less at the time of investigation and who had normal renal function and antenatal ultrasound, were considered suitable for analysis of the diagnostic course. us was performed in children with ( %) normal, minor changes were noted in the remainder, apart from case requiring a change in management in the form of a pyeloplasty for pelvi-ureteric obstruction. the cystourethrogram was performed in of the children with ( %) demonstrating vur. the cystogram was a poor predictor of long term damage, being positive for reflux in only of the children who subsequently developed scarring. an acute dmsa scan performed in all children, exhibited findings consistent with acute pyelonephritis in ( %). a repeat dmsa scan at months in those with evidence of acute pyelonephritis demonstrated a scarring rate of %. the data did not demonstrate the clinical efficacy of routinely performing scintigraphy in the acute phase or a cystourethrogram. our recommendations for a reasonable diagnostic work up in small children with their first episode of uti are ) performance of a scintigram months following the acute infective episode, and ) close surveillance to identify eventual recidivists. introduction: focal segmental glomerulosclerosis (fsgs) has a high recurrence rate after renal transplantation (rtx). recurrence can lead to tx loss. disease recurrence (dr) seems to be influenced by genetic background. methods: patients with childhood onset of biopsy proven fsgs who were transplanted were evaluated. genetic investigations of the nphs gene were done in patients ( . %). results: mean age at diagnosis was . years. first renal transplantation was performed at age of . years. patients received a living related (lrd), a deceased donor (dd) graft. patients data under investigation. p ( . %) recurred after a mean time of , years, p with lrd ( . %) , p with a dd ( . %). patients ( . %) lost their graft after , years, patients due to recurrence. a second rtx was performed in patients, with dd, with lrd, patients data under investigation, with % dr. a third transplantation was performed in patients and a fourth in one patient. screening for nphs mutations revealed patients with mutation in the nphs gene, homozygot, heterozygot. all patients with a homozygote nphs mutation did not recur and only the one patient with a heterozygous mutation ( / ) recurred compared to / without a mutation (p< . ). conclusion: living related transplantation was advantageous to decreased donor transplantation. patients with nphs mutations had a lower risk of recurrence after transplantation compared to patients without mutations. patients with fsgs should be screened for nphs mutations. objective: prospective ebv surveillance post tansplantation reduces incidence of acute rejection and risks of post transplant lymphoproliferative disorders (ptld). methods: prospective screening of ebv by polymerase chain reaction (pcr) and serology in all patients transplanted between - . results: patients transplanted between may and september . were cadaveric and live-related transplants. recepient ebv serology status was known but not on donors. basiliximab was used for induction and maintenance comprised of tacrolimus/azathioprine (aza) until and mycophenolate (mmf)/tacrolimus from . all received corticosteroids. had mmf/tacrolimus, aza/tacrolimus. ebv screened within first week post transplant, weekly for a month, monthly for months. when ebv pcr lv > . , mmf/aza was withdrawn, tacrolimus levels kept between - ng/ml. pcr/ serology is checked weekly for months, monthly for , until lv falls < . and vca igg becomes positive. the mmf/aza is reintroduced. patients with ebv pcr lv < . are kept on full immunosuppression while viral load and vca igg monitored. ebv viraemia detected following prospective screening. was re-activation. were symptomatic. onset varied to . months post transplant (mean . months). were on mmf/tacrolimus received aza/tacrolimus. had pcr lv> . consequently maintained on tacrolimus an alternate day steroids for period of time. cmv patient and donor status was known and antiviral prophylaxis given to nonimmune. the use of antivirals made no impact on viral load. conclusion: no patient experienced acute rejection or ptld despite modification of therapy with our prospective screening programme. antivirals have no role in protecting or reducing viral load in already infected patients. graft attrition rate is highest in the first three months after renal transplantation. we analysed data in a recent cohort from all dutch centres for pediatric renal transplantation to determine incidence and causes of such early graft failure. methods: data from all pediatric renal transplants performed between - - and - - were analysed retrospectively. a common immunosuppressive protocol was used in all centres. thrombosis prophylaxis was given according to centre policy. early failure was determined as graft failure within months after transplantation. prevalence of possible risk factors of graft failure identified by literature search were extracted from patient charts. data were analysed with univariate and multivariate logistic regression. results: the cohort consisted of transplants. age of the recipients was - yrs (mean . ). there were early graft failures ( . %). major causes of graft failure were thrombosis ( . %) and are( . %). univariate analysis identified an association of early failure with complications during surgery (or . , p= . ), cold ischemia time (or . , p= . ), side of graft donation (or . , p= . ) and diuresis in the first hour after transplantation (or . , p= . ). multivariate regression analysis showed that complications during surgery were associated with the risk of early failure (or . , p= . ), as were side of graft donation (or . , p= . ) and diuresis in the first hour after transplantation (or . , p= . ) . only the occurrence of complications during surgery was significantly associated with early graft failure due to thrombosis (or . , p= . ). conclusion: thrombosis is the major cause of early graft failure after renal transplantation in dutch children, especially after surgical complications. prospective studies are needed to determine whether early failure can be decreased by more rigorous prophylaxis. background: interleukin (il)- is a major contributor to inflammation and cell death during ischemia-reperfusion (ir) injury and its deleterious effects are mediated mainly by nuclear factor-κb (nf-κb) activation. receptor binding and signalling of il- can be blocked by the il- receptor antagonist (il- ra). the aim of our study was to characterize the effects of il- ra administration on inflammation, apoptosis and infiltration in renal ir injury. methods: renal ischemia was induced in lewis rats (n= /group) by clamping of the left renal artery for min followed by reperfusion of h or five days respectively, when kidney were removed for histological and molecular analysis. results: treatment with il- ra ameliorated ischemic renal tissue injury and inflammatory infiltration. futhermore, the number of apoptotic tubular cells was lower in il- ra treated animals h after ischemia, which was paralleled by a bax/bcl- mrna ratio towards anti-apoptotic effect. il- ra reduced the expression of monocyte chemoattractant protein- (mcp- ) mrna h and days and that of intracellular adhesion molecule- (icam- ) expression h after reperfusion in the kidney. conclusions: our results indicate that il- ra treatment ameliorates renal ir injury and this protective effect might be mediated by reduced induction of nf-κb mediated mcp- , icam- and the decreased ratio between bax and bcl- mrna expression. k. satomura, y. santo osaka medical center for maternal and child health, pediatric nephrology and metabolism, izumi, japan many studies have reported that angiotensin-converting enzyme inhibitor (acei) and angiotensin receptor blocker (arb) show renoprotective effects in adult patients with acquired renal diseases. however, these effects have not been studied in children with renal hypoplasia or dysplasia (rhd). in this study, we explored the renoprotective effects of these drugs in children with rhd. patients and methods: participants of the study were patients with rhd aged less than years. a follow-up for more than one year was conducted in our outpatient clinic. the patients had chronic renal failure, and had not received acei or arb in the past. the change of glomerular filtration rate (gfr) per year, urinary protein/creatinine (up/cr) ratio, serum potassium levels, and blood pressure before the treatment with trandolapril or candesartan were compared with those of one year after the treatment. the estimated gfr was calculated by the schwartz formula. wilcoxon rank sum test was applied to evaluate the statistical significance. results: eleven patients were eligible for this study. the mean age of the patients was . ± . years and the estimated gfr was . ± . ml/min/ . m when trandolapril or candesartan was administered. the change in the estimated gfr for a year significantly improved after the treatment with trandolapril or candesartan compared with the pre-treatment period (- . ± . vs. . ± . ml/min/ . m /year, p= . ). the up/cr ratio significantly decreased at one year after the treatment compared with that before the treatment ( . ± . vs. . ± . , p= . ). the blood pressure and serum potassium level showed no significant changes. conclusion: these data suggest that treatment with acel or arb has beneficial effects on the renal function in children with mild to moderate renal failure due to rhd. objective of the study: postischaemic arf is influenced by sex hormones. dehydroepiandrosterone (dhea) pretreatment diminishes postischemic injury. previously we demonstrated that after renal ischaemia-reperfusion (i-r) injury the expression and activity of na, k-atpase (nka) is impaired in male rats. here we tested the impact of dhea on postischaemic survival, renal damage, mrna and protein expression of nka. methods: left renal pedicles of dhea ( . mg/kg/day) and propylene glycol, as vehicle (pg) treated male rats (g dhea and g pg , respectively) were clamped for min followed by (t ) and (t ) hours of reperfusion. survival rate, histological damage, serum creatinine (cn) and urea nitrogen (bun) were investigated. the mrna expression and protein level of nka α and β subunit were also determined. sham operated animals served as control. results: dhea treatment was associated with better postischemic survival (p< . g dhea vs. g pg ). postischaemic cn and bun were higher and renal histology showed more rapid progression vs. controls, however there was no difference between g dhea and g pg groups. mrna expression of nka α subunit was higher in g dhea vs. g pg at every time points, however it was decreased in i-r groups vs. controls (p< . ). similar changes were observed in nka α protein level (p< . , g dhea vs. g pg in controls, t , t ). mrna and protein expression of α subunit were different only at t (p< . , g dhea vs. g pg ). conclusions: our study indicates that nka is more protected from the detrimental effects of i-r injury in dhea treated animals, which might be a contributing factor of improved postischaemic renal function and could help to preserve cell and organ homeostasis even in male animals. the work was supported by otka f -t , bolyai and semmelweis grants. objective of study: recently bnp has been identified as a useful cardiac marker for risk stratification in adults. whether bnp has a similar diagnostic potential in pediatric population with ckd has to be established. the aim of the study was to assess the value of bnp to predict the cardiac dysfunction in children with ckd. methods: to test this suggestion, the relationship between plasma concentrations of bnp, echocardiographic parameters and cardiovascular risk factors (lipid profile, hypertension, secondary hyperparathyreoidism) has been evaluated. a cohort group consisted of children and young adults ( pts mean age x= . ± . years; controls mean age x= . ± . years). out of studied pts children were with ckd stage and k/doqi ( pts ckd stage ; dialyzed pts) and transplanted subjects with stable graft function (gfr= . ± . ml/s/ . m ). results: no association was found between bnp and gfr and/or s-creat (p= . ; p= . ; p= . ). the highest bnp plasma levels have been found in dialyzed children ( . pg/ml; resp. . pg/ml in tx; resp. , pg/ml in ckd stage group) as compared to controls ( , pg/ml p< , ). bnp was independently related to left ventricular mass (r= . ; p< . ) as well as to ejection fraction (r=- . ; p< . ) and preload (r= . ; p< . ). significant correlation between bnp and hypertension (r= . ; p< . ) has been observed. more over, bnp correlated with ipth (r= . ; p< , ) and diastolic dysfunction (r= . ; p< , ). plasma bnp concentrations did not significantly differ before/after dialysis procedure (p= . ). conclusions: bnp is a sensitive marker for cardiac dysfunction in ckd children. however, prospective studies in larger group of pts are needed to confirm our preliminary data. we have reviewed the data of patients with nephropathic cystinosis (nc) followed in our department since . overall, patients have died during the follow-up period. the mean follow-up was , ± , yrs (range , ) . / patients have initiated dialysis and / received a cadaveric renal transplant. univariate analysis showed that the time to reach a serum creatinine value of mg/dl was significantly associated with the patient's age at the last follow-up (o. r. , /yr; p< . ), the age at the beginning of cysteamine (mea) treatment (o. r. , /yr; p= . ), the dose of mea (o. r. , /g, p= . ) and the use of ace-inhibitors (o. r. , ; p= . ). by multivariate analysis, only the period of treatment (or , , p= . ) and therapy with ace-inhibitors (or , , p= . ) were significantly associated with better outcome. similar results were obtained when assessing the time to dialysis. there was a significant correlation between the dose of mea with intra-leucocytic cystine levels (ilc). by univariate analysis, the dose of mea expressed in mg/m correlated more with the outcome then the dose expressed in mg/kg. statural growth was significantly associated by multiple correlation analysis only with growth hormone (gh) treatment (p< . ). altogether, these results indicate that the renal prognosis of nc has been improving over the past two decades. this improvement may not be solely attributable to the introduction of mea for the treatment of nc. our data suggest that the overall management of these patients has improved, including more efficient symptomatic treatment of the fanconi syndrome and the use of medications such as ace inhibitors and gh. background: the chronic kidney disease in children (ckid) cohort study, targeted to enroll children aged - yrs with chronic kidney disease (ckd) by estimated gfr (egfr schwartz) of - ml/min/ . m aims to assess risk factors for kidney disease progression in children. objective: to describe the characteristics of the ckid cohort at study entry. design/methods: at the ckid baseline visit, gfr is measured by plasma disappearance of iohexol. children undergo physical exam, standardized bp measurements, blood and urine testing and parent and child interview. results: as of / / , children had completed the baseline visit, median age . yrs, % caucasian, % african american, % male and % hispanic ethnicity. median age-adjusted height and weight percentiles were % and %, respectively. underlying cause of ckd was urologic, cystic or hereditary in %, % had acquired glomerular disease. median egfr at study entry was . ml/min/ . m , % higher than the median iohexol-based gfr . ml/min/ . m . % of participants reported a history of hypertension, % anemia, % seizures, and % depression. symptoms of headache, nausea, loss of appetite and weakness steadily increased in prevalence with lower gfr. assessment of family history in parents and grandparents revealed: high blood pressure %, high cholesterol %, kidney disease %, dialysis %, and kidney transplant %. conclusions: children with stage iii ckd have significant height deficits, hypertension, anemia, and seizure disorders. cross-sectionally, increasing non-specific symptoms are associated with lower gfr. collection of plasma, serum and genetic material will facilitate understanding of novel risk factors and biologic mechanisms underlying kidney disease progression and associated morbidities. aim: increased glomerular filtration rate (gfr) has been implicated in the development of diabetic nephropathy. large normal interindividual variations of gfr hamper the diagnosis of renal hemodynamic alterations. we examined renal functional reserve (rfr) in children with insulindependent diabetes mellitus (iddm) to assess if hyperfiltration occurs. methods: the renal hemodynamic response following dopamine infusion was examined in iddm children ( . ± . y) with a mean duration of diabetes of . years and compared to controls. results: mean baseline gfr in diabetic children did not differ from control population ( . ± . vs. . ± ml/min/ . m ), whereas renal plasma flow was significantly lower ( . ± . vs. . ± ml/min/ . m , p< . ) and filtration fraction was increased ( ± vs. ± %, p< . ), compared to controls. the mean rfr was lower (p< . ) than in control subjects (- , ± vs. ± ml/min/ . m ). conclusion: the observation that rfr is reduced or absent suggests that all children are in a state of glomerular hyperfiltration with increased intraglomerular pressure regardless of the baseline normal values of gfr. while measurements of rfr may be helpful in diagnosing the presence of hemodynamic changes, the relevance to development of diabetic nephropathy remains unknown. only - % develops diabetic nephropathy, suggesting that glomerular hyperfiltration is only a concomitant risk factor. lp diet induces ischemic renal injury involving epithelial cells from osom. here, we tested whether hsp would stabilize renal na+k+atpase attachment to the cytoeskeleton during recovery from lp. after weaning, rats (n= ), were fed for days with a lp diet ( %), then were recovered by means of a normal protein diet ( %rp) each group had an age-matched control group ( %, np). tissues from cortex and osom were homogeneized in buffer plus . % triton x- . protein levels were measured by western blot. in vitro coincubation of solublen on cytoeskeletal and insoluble cytoskeletal-associated fractions in the presence or absence of anti-hsp antibody was performed. interaction between proteins was determined by coimmunoprecipitation. increased na+k+atpase dissociation was shown in soluble fraction from osom as a result of lp diet vs. np ( . ± . vs. ± . , p< . ). meanwhile, decreased hsp levels in the same fraction was shown (lp: . ± . vs. np . ± . , p< . ). translocation of hsp to the cytoeskeletal injured fraction associated with stabilization of na+k+atpase was shown in osom from lp, after in vitro coincubation of the cytoskeletal fraction of lp and non cytoskeletal fraction of rp. these effects were abolished by the addition of anti-hsp antibody. coimmunoprecipitation showed that the amount of hsp coprecipitating with na+k+atpase increased in lp osom, in rp results were similar to control. in cortex, absence of significant differences was shown in the na+k+atpase and hsp expression at in vivo and in vitro experiments among groups. in lp and rp cortex tissues, interaction of both proteins was similar to control. our results allow us to suggest that hsp has a critical protective role in the integrity of the cytoskeletal anchorage of na+k+atpase during recovery from ischemic lp injury in osom. tubular reabsorption of mg + occurs predominantly by paracellular flux in the thick ascending limb of the loop of henle. it is mediated by the, tight junction, mg + channel protein, paracellin- , which is encoded by the gene pcln- . cyclosporin a (csa), a widely used immunomodulatory drug, decreases tubular mg + reabsorption leading to urinary mg + wasting and hypomagnesemia. the exact molecular mechanisms of this modulation are unknown. in this study we examined the effect of csa on the paracellin- gene in renal cell lines and mouse kidney. the effect of csa on the pcln- gene promoter was examined. a plasmid, containing the human pcln- (hpcln- ) promoter ( . kb) upstream to a luciferase reporter gene, was transfected into opossum kidney (ok) and human embryonic kidney (hek ) cells prior to exposure to micrograms/ml csa for hours. mice received csa ( mg/kg/day) for up to days. at h intervals blood/urine mg + and ca + levels were determined and kidneys harvested for paracellin- mrna quantitation (real-timepcr) as well as protein quantification (western blot) and visualization (immunofluorescence). csa decreased hpcln- promoter activity by %- % in transfected ok and hek cells, compared to control cells. csa-treated animals displayed hypomagnesemia with increasing urine mg + and ca + levels paralleled by a - % decrease in pcln- mrna after and days of csa exposure. a similar effect on paracellin- protein expression inthe renal tubule was evident in response to csa treatment. csa may influence paracellin- -mediated mg + transport at the transcriptional level. involvement of the calcineurin-dependent tonebp pathway or the calcineurin-independent map kinase pathway in this response is subject to future research. this effect of csa on the paracellin- gene may play a role in the renal magnesium wasting and hypomagnesemia induced by csa. it is well established that proteinuria induces tubular injury, which is closely associated with progressive decline of renal function. megalin has an important physiological role in the reabsorption of a variety of low molecular weight proteins along the proximal tubule. in this study, we examined whether megalin mediates tubular injury secondary to glomerular diseases. we utilized megalin knockout (ko) mice, in which % of proximal tubular cells have mosaic nullmutation in the megalin gene. to induce glomerular injury, these were mated with a transgenic line, nep , in which selective podocyte injury can be induced by injection of immunotoxin, lmb . megalin ko/nep mice were injected with lmb ( . ng/g bw) and analyzed weeks later. they showed moderate proteinuria (upro/cr= , vs . before lmb ) and mild tubular injury, which were comparable to those observed in nep mice with intact megalin gene injected with the same dose of lmb . in the latter, megalin was still detectable in injured tubules. within each megalin ko/nep mouse, we compared megalin-intact (+) vs deficient (-) proximal tubular cells by immunostaining. the majority of megalin+ cells showed enhanced staining for albumin. in contrast, most megalin-cells were not stained for albumin. aquaporin- , which is highly expressed in normal proximal tubules, was diminished in . % of megalin+ cells, whereas it was diminished in only . % of megalin-cells. some proximal tubular cells expressed mcp- . in that, % of mcp- expressing cells were megalin+. the results suggested that megalin plays an important role in the reabsorption of massively filtered proteins in glomerular diseases, thereby contributing to proximal tubular cell injury. objectives of study: the hallmark of nephropathic cystinosis is lysosomal cystine accumulation, primarily leading to fanconi syndrome. although all tissues haveelevated cystine levels, it is not known why the kidney is first affected. it is postulated that decreased atp production in cystinosis results in defective proximal tubular reabsorption, a process driven by na, k-atpase. to study this hypothesis, we have monitored atp levels and viability in conditionally immortalized proximal tubular cells (ptc) of cystinosis and healthy controls. methods: urinary sediment of cystinotic patients and healthy controls was suspended ins supplemented culture medium. primary cultures were transfected with sv ts a t antigen allowing proliferation at °c and maturation at °c. to confirm the proximal origin of the cells ) expression of aquaporin- (aqp ) and dipeptidyl-peptidase iv (dpp-iv) was demonstrated using immunoblot technique and ) morphology was determined using em. ptc were matured at °c for - days, followed by cystine measurement using hplc and atp determination using luciferase assay. results are expressed as nmol/mg protein. results: colonies of cystinotic and control cell lines (n= ) with cobblestone morphology developed after weeks and expressed aqp and dpp-iv, confirming their proximal origin. in cystinotic ptc, cystine levels increased from . at °c to . after days at °c compared to . in controls. intracellular atp decreased in cystinotic ptc during days from . to . , while atp levels in controls remained stable (range . - . ). atp levels inversely correlated with cystine accumulation in cystinotic ptc (r=- . , p= . ). conclusion: decreased atp levels in conditionally immortalized ptc during days maturation suggest that alterations in atp levels are involved in tubular dysfunction in cystinotic patients. aquaporin ( aqps have been identified to date. aqp conveys % of the peritoneal water transport in pd. cell migration and angiogenesis are altered in aqp ko mice; erythrocyte pco depends on aqp . human peritoneal mesothelial cells (hpmc) from non-uremic patients and a human mesothelial cell line (met- a) were incubated with conventional (c-pdf), icodextrin (ico), bicarbonate (b-pdf) and lactate based double chamber pd solution (l-pdf). mrna was measured by real time rtpcr, and protein by western blot and immunocytochemistry. hpmc and met- a express aqp , , and . incubation of met- a and hpmc with c-pdf, ico and l-pdf did not change aqp and expression compared to medium. in contrast, incubation with b-pdf increased aqp and mrna and protein in both hpmc ( h aqp mrna: ± , aqp : ± %) and met- a (aqp : ± , aqp : ± %). the effect on aqp was in part explained by differences in ph, the effect on aqp was largely independent of ph. addition of mmol/l of bicarbonate to l-pdf increased aqp and mrna (l-pdf+bic: aqp : ± , aqp : ± %). aqp expression was suppressed by all three pd fluids (b-pdf: ± . , l-pdf: ± . , c-pdf %: ± %), aqp was up regulated by b-pdf ( ± %). b-pdf improved hpmc migration. we for the first time demonstrate the expression of four different aqps in peritoneal mesothelial cells. they are markedly regulated by pd-solutions. the upregulation of aqp , , and by bicarbonate based pdf may have important implications on long term peritoneal membrane function, wound healing and neoangiogenesis. the regulation of cx cl (fractalkine) by thromboxane a in inflammation there is marked leukocyte infiltration into the damaged tissue. chemokines recruit and direct leukocytes to the injured site. the chemokine cx cl is up-regulated in renal inflammation such as glomerulonephritis and allograft rejection. thromboxane a (txa ), a potent vasoconstrictor in the kidney, is also pro-fibrotic. txa is up-regulated early in inflammation and its effects are mediated by binding to the tp receptor. the aim of this study was to examine the effect of txa on cx cl expression. we previously showed that cx cl recycles between the cell surface and an internal compartment. cell surface cx cl is cleaved by the enzyme, tace, to yield the soluble species of the chemokine. we generated human ecv- cells stably expressing cx cl and treated cells with a tp agonist. levels of cx cl were quantitated by western blotting. cx cl decreased by - min after tp stimulation, but recovered by h. using flow cytometry, we found that cell surface levels of cx cl decreased by min, but began to recover before levels of total cx cl were replenished. to verify if early recovery of cx cl was due to redistribution from the internal to the plasma membrane compartment, we used fluorescence recovery after photobleaching (frap). after min tp stimulation decreased endocytosis of cx cl was seen. we postulated that loss of cell surface cx cl is due to cleavage by tace. accordingly, a tace inhibitor prevented early loss of cx cl after tp stimulation. in summary, txa induces rapid proteolytic shedding of cell surface cx cl by tace, but later increases expression of cx cl at the cell surface by inhibiting internalization of the chemokine. this could release the soluble chemotactic protein from the cell adhesive transmembrane protein, promoting leukocyte recruitment initially, whilst promoting leukocyte adhesion later in the time course. the response to steroid therapy is used to characterize idiopathic nephritic syndrome (ins) as steroid sensitive (ssns) or steroid resistant (srns). ssns pathogenesis has been associated with activation of t-cells and srns has been associated with activation of tgfb and progression to chronic kidney disease. our objective was to determine differences in the urinary excretion of inflammatory cytokines; opn, icam and tgfb , between ssns (n= ), srns (n= ), and controls (ctr, n= ) in an attempt to identify specific biomarkers of steroid sensitivity or lack thereof. for this purpose, we used a protein array high-throughput technique and confirmed the findings by elisa. in addition, we performed immunohistochemistry (ihc) for the above cytokines on renal biopsy samples. mean age, gender, race, body mass index, and estimated glomerular filtration rate were not statistically different among three groups. there were no statistically significant differences between ssns and srns in regard to the presence of hypertension, ace treatment, and renal histology (p= . , . , and . , respectively) . urinary excretion of icam , opn, and tgfb were statistically significantly higher in ins subjects vs. ctr. urinary icam and opn were higher in ssns than in srns (p= . and p= . , respectively). however, the urinary excretion for tgfb was similar in ssns and srns. the ihc failed to reveal differences in renal tissue expression of the studied cytokines. there was no correlation between urine and kidney tissue expression. in summary, our preliminary data suggest that urinary opn and icam may serve as biomarkers of ssns. this assumption needs to be tested prospectively. methods: this is an interim report of this single center study on proteinuria in hiv positive children. there are children aged - yrs. registered in our hiv clinic. all children attending the outpatient clinic or seen on admission are studied. questionnaires included bio and clinical data. blood pressure was measured in all children. a clean catch or bag urine is obtained from all and urine biochemistry done on an aliquot. patients found to have proteinuria + or above are referred to the nephrology unit. their urine is quantitated (timed urine collection), renal function, renal ultrasound and cd count estimated. anf, le cells, fbc including platelet count and genotype will be assayed. a renal biopsy will be performed for nephrotic range proteinuria. result: eighty children positive for hiv have been studied. there were males and females giving a ratio of . : . their mean and median ages were . yrs and . years respectively. all had normal blood pressures, renal ultrasound, and renal function. fifteen of the children ( . %) had - mg/dl proteinuria; ( . %) haematuria. their mean cd + cell count was . /mm , range - . there was no significant difference between low cd + count and the presence of proteinuria (p> . ). seventy seven of the patients had vertical transmission of hiv; two acquired the infection from blood transfusion; one from scarification marks. conclusion: there is a high prevalence of proteinuria among children with hiv infection in our region. hiv positive children should be screened and intervention instituted to avert or delay the onset of chronic renal failure. carotid intima media thickness (cimt) and brachial artery flow mediated dilatation (fmd) are novel indices of subclinical atherosclerosis. we studied these indices in children with nephrotic syndrome (ns) using high resolution ultrasonography (hrus). children with ns ( ssns; srns) of - months duration and normal sibling controls underwent cimt and fmd determination using a . mhz us probe. routine biochemistry, lipid profile and hscrp were carried out to look into associations with cimt and fmd. statistical analysis was carried out using epiinfo . . cases and controls were similar in age, sex, growth and egfr. mean maximum cimt (+- % ci) (in mm) was higher in the ns group ( . +- . ; range . - . ) vis-a-vis controls ( . +- . ; range: . - . ) (p< . ). srns and ssns cases were similar ( . +- . vs . +- . mm, p= . ). nephrotic children showed significantly lower fmd ( . +- . % vs . +- . %, p= . ). cimt and fmd varied with frequency of relapses in last months ( . + . mm, . +- . % (no relapses); . +- . mm, . +- . % (> relapses). children with ns had mean hscrp of . +- . mg/l. on univariate analysis, cimt correlated with disease profile at onset (r= . , p< . ), fmd (r=- . , p= . ), male sex (r= . , p= . ), bmi (r= . , p= . ), cyclosporine exposure (r= . , p< . ), total cholesterol (tc) (r= . , p< . ), ldl (r= . , p< . ), vldl (r= . , p< . ), triglycerides (r= . , p< . ), atherogenic index (ai) (r= . , p< . ) and creatinine (r= . , p= . ). fmd correlated with systolic blood pressure, age, bmi, weight, total cholesterol, vldl and triglycerides (p< . ). to conclude, hrus can detect early atherosclerosis using fmd and cimt as the indices in children with nephrotic syndrome with disease duration of years and more. a ten-year old girl, who presented with biopsy proven acute severe changes of type membranoproliferative nephritis (mpgn ) appears to have shown complete resolution of her disorder after nine months mycophenolate moffetil (mmf) (cellcept -roche) added to relatively rapidly reducing steroids. she presented aged nine years with incidental finding of significant proteinuria and upper tract haematuria when presenting with abdominal pain. blood pressure, chemical renal function and haematology were normal. c and total haemolytic complement were at the lower limit of normal. renal biopsy showed light microscopy (lm) changes of markedly enlarged glomeruli with significantly increased mesangial cellularity and matrix, together with 'double contours' of basement membrane. inmmunofluorescence (if) was strongly positive for igg and c . electron microscopy (em) showed mesangial cell interposition into the basement membranes. she was treated with iv pulse methylprednisolone, followed by high dose oral prednisolone (pnl). after one month, mmf mg bd was added as pnl was tailed to alternate day therapy. pnl was reduced more quickly than usual due to difficulty with side effects and excellent clinical response. within three months of mmf, proteinuria and haematuria had disappeared. blood pressure and renal function have remained normal throughout treatment. after nine months, while on . mg/kg alternate day pnl and mg bd mmf, repeat biopsy showed normal sized glomeruli on lm with no significant changes, no positive staining on if and normal em without mesangial interposition. this case suggests that mmf may be a key drug in management of mpgn . g. stringini, e. malagnino, f. emma bambino gesu children's hospital and research institute, department of nephrology and urology, rome, italy serum creatinine values may vary considerably between normal subjects. these variations are in part secondary to differences in muscle mass, body composition and creatinine tubular secretion. in addition, low nephron number, which is influenced by genetic and intrauterine factors, is associated with increased incidence of arterial hypertension, late-onset proteinuria and chronic renal failure. despite compensatory glomerular hypertrophy, autopsy data indicates that kidneys with fewer glomeruli tend to be significantly smaller. on these bases, we have hypothesized that a significant part of the variance of normal serum creatinine levels is related directly to individual differences in renal size. to test this hypothesis, we have reviewed the data of renal ultrasound examinations performed at our institution between and . patients were selected if they had conditions that should not influence renal size (low tract uti, enuresis, bladder instability, idiopathic hypercalciuria), if they had normal gfr and an ambulatory blood pressure measurement. analyses were performed after expressing renal length, serum creatinine levels and blood pressure values as standard deviation scores (sds) corrected for gender, age, height and weight, by multiple nonlinear regression analysis. a significant negative correlation was found between serum creatinine levels and renal length (p< . ). when renal length measurements were grouped in quartiles, serum creatinine sds was on average . sds higher in the st quartile (small kidneys), in comparison with the th quartile (large kidneys). no correlation was found between kidney length and single measurements of ambulatory blood pressure. these data indicate that renal size is accountable for a significant part of the variance in serum creatinine levels that is observed in the normal pediatric population. henoch-schönlein purpura (hsp) is the commonest small vessel vasculitis of childhood. henoch-schönlein purpura nephritis (hspn) is the major determinant of long term prognosis in hsp. the objective of this randomised controlled trial was to determine the effect of early prednisolone treatment on the development and progression of nephropathy in children with hsp. methods: children under years of age, with a diagnosis of hsp, presenting to secondary care centres in england and wales were randomly assigned to receive either placebo or prednisolone ( mg/kg/day (max mg) for days, followed by mg/kg/day for days (max mg)). patients, parents, paediatricians and investigators were 'blinded' to assignment of treatment or placebo. the primary outcome measure was the determination of the presence or absence of proteinuria, defined as urine protein: creatinine ratio (up: uc) > mg/mmol, months after initial presentation in treated and untreated patients. results: children with hsp were randomised. patients were assigned to receive prednisolone (group a) and patients were assigned to receive placebo (group b). patients in group a and patients in group b did not complete the study. there was no difference in the incidence of proteinuria at months, in patients receiving prednisolone ( / ) compared with those receiving placebo ( / ) or= . , % ci . - . , p= . . conclusions: this is largest prospective randomised placebo-controlled trial of the role steroids in hsp in the literature to date. this study provides compelling evidence of absence of a beneficial effect of early treatment with prednisolone in the development of nephropathy months after disease onset in children with hsp. while quality of life (qol) is generally assumed to be poor on dialysis and to improve markedly after kidney transplantation, systematic assessments of qol in children have not been performed to date. to date, we have examined general and health-related qol in children and adolescents with esrd aged - years treated by hemodialysis (hd; n= ), peritoneal dialysis (pd, n= ) or renal transplantation (tx, n= ), using a well-established instrument (kindl) comprising an ageadapted set of questionnaires (kindl). the obtained measures of overall and item-specific qol were transformed to standard deviation scores (sds). general qol was close to normal and did not differ significantly between hd (- . ± . sds), pd (- . ± . ) and tx (- . ± . ). psychological well-being was better in children on pd ( . ± . ) than after tx (- . ± . , p= . ) and on hd (- . ± . , p= . ). physical wellbeing did not differ between treatment groups (hd: - . ± . , pd: . ± . , tx: (- . ± . ). qol related to family life tended to be compromised in dialyzed (- . ± . ) vs. tx children (- . ± . , p= . ) . social interaction with friends was considered moderately impaired by all patient groups (hd: - . ± . , pd: - . ± . , tx - . ± . ). self-esteem was close to normal in all groups (hd: - . ± . , pd: - . ± . , tx . ±. ). in summary, our preliminary results suggest that subjective qol is remarkably good in children with esrd, with surprisingly small differences between treatment modalities. tx appears to be perceived beneficial with respect to the integrity of family life but provides poorer psychological qol than pd, possibly due to the constant concern with graft loss. background: gfr is determined in the ckid cohort study by plasma disappearance of iohexol (igfr). updated serum creatinine (scr)-based formulas (sgfr=k ht/scr) are needed for bedside clinical use. objective: derive formulas based on enzymatic scr to estimate gfr in the ckid study in children aged - yrs with sgfr of - ml/min/ . m . methods: from children, height (ht, meters), igfr, and scr & bun (mg/dl) by bayer advia were obtained. gfr was estimated from regression models: a(ht/scr) b ( /bun) c where a is gfr if ht=scr and bun= ; b and c are exponents. results: % (n= ) were male; median age= , scr= . , bun= and igfr= . formulas ranged from an updated schwartz formula (a= . , b= , c= ; r = . ), to sex-specific (a= . , b= . , c= in girls, and a= . , b= . , c= in boys), which yielded r = . , to the most complex (a= . , b= . , c= . in girls, and a= . , b= . , c= . in boys), which yielded r = . , higher (p< . ) than the r of the updated schwartz formula. by random selection of of subjects in independent trials, we assessed the predictive ability of each formula on the other observations. sex-specific formulas produced unbiased results and increasing precision with increasing complexity. the updated schwartz underestimated igfr in boys by % (p< . ) and overestimated igfr in girls by % (p< . ). conclusions: scr, ht, sex, and bun together provided unbiased estimates of igfr and explained % of its variability in children with igfr of to ml/min/ . m . extrapolation to ht/scr of (normal body habitus and kidney function) and bun of yielded predictions of ml/min/ . m for gfr in girls and boys. including cystatin c and broadening the gfr range to include normal levels are data being collected by ckid to extend the formulas and provide wider clinical utility. f. hussain, m. mallik, a. watson nottingham university hospitals, children and young people's kidney unit, nottingham, united kingdom considerable variation exists between units in terms of techniques used for renal biopsy. the bapn agreed an audit using published standards and a questionnaire was sent to all uk paediatric nephrology centres. agreed to a prospective audit between / / and / / . the survey revealed information leaflets are sent pre-biopsy in ( %) centres with only one using play preparation. ( %) routinely perform biopsies as daycase procedures (dc); ( %) use general anaesthetic (ga). realtime ultrasound is the favoured method in ( %) of centres. biopsies are performed by nephrologists only in ( %), nephrologists with radiologists in ( %) and radiology alone in ( %). of biopsies ( native), ( %) were performed as a dc with ( %) being done under ga. the mean age of patients was yrs (range . ± . yrs). the standard for the number of passes of native kidneys (< in %) was achieved in . % with no significant difference between grade of operator or nephrology/radiology speciality. standard number of passes in transplanted kidneys (< in %) was achieved in . %. adequate tissue was obtained for diagnosis in . % (standard > %). the significant complication rate (macroscopic haematuria and/or delay indischarge) was higher than the set standard of < % at . %. there was no significant difference in complication rates whether the biopsy was performed as a dc or inpatient procedure (p= . ) or whether ga or sedation was used (p= . ). conclusions: the survey highlights significant variation in practice with limited use of preparation materials and dc. the results may enable individual units to reflect upon their techniques and complication rates and has stimulated constructive debate about indications and training issues. objective: to characterize bp in children enrolled in the ckid study, an observational cohort study of children - y old with schwartz estimated gfr of - ml/min/ . m . design/methods: bp's were obtained using an aneroid sphygmomanometer. gfr was measured by iohexol disappearance. bp was classified according to the th report on bp in children. hypertension (htn) was defined as bp- th percentile (uncontrolled), or as self-reported htn plus current treatment with antihtn meds. pre-htn was defined as bp th- th percentiles. results: children (mean age ± y; % male) were studied. mean gfr was ± ml/min/ . m and mean ckd duration was ± y. for sbp, / ( . %) subjects had htn of these, subjects ( %) had uncontrolled htn. additional subjects ( %) had pre-htn. of subjects with systolic htn, only % had measured sbp< th percentile, and among the subjects with measured sbp> th percentile, only ( %) were taking antihtn meds. for dbp, / subjects ( . %) had htn of these, subjects ( %) had uncontrolled htn. subjects ( %) had pre-htn. of subjects with diastolic htn, just % had measured dbp < th percentile, and among the subjects with measured dbp> th percentile, only ( %) were taking antihtn meds. among children with gfr< ml/min/ . m , the prevalence of systolic or diastolic htn appeared to increase with decreasing gfr. conclusions: a significant proportion of children enrolled in the ckid study suffer from elevated bp. nearly % of children with ckd had measured bp> th percentile, and more than % of these children were not receiving antihypertensives, indicating that htn in pediatric ckd is frequently under-or even untreated. long-term follow-up of the ckid cohort should reveal the effect of elevated bp on ckd progression. background: we recently demonstrated elevated excretion of the putative urinary biomarkers tgf-β and et in a large cohort of children with ckd. tgf-β excretion was highest in kidney disorders associated with marked tubulointerstitial fibrosis, such as obstructive uropathy, nephronophthisis and pkd. aim: to investigate the course and predictive value of urinary tgf-β and et excretion in children with ckd undergoing ace inhibition (acei). methods: to date, patients have been followed for years and patients for years in a prospective, interventional trial investigating the nephroprotective efficacy of acei w/out intensified blood pressure control. results: average bp was reduced to the mid normal range. proteinuria initially decreased by %, but gradually reincreased to baseline levels within years. urinary tgf-β excretion, which was initially elevated -fold above healthy controls, continuously decreased during treatment from . ± . ng/g creatinine to . ± . , . ± . , and . ± . after , , and years, respectively (p< . ). in contrast, et excretion increased by % within the first years of follow-up. neither baseline nor the change in tgf-β and et excretion during treatment predicted the short-or long-term antihypertensive, antiproteinuric response to acei or ckd progression rate. conclusions: the marked reduction of urinary tgf-β excretion probably reflects the antifibrotic effect of acei but, at least over yrs of observation, does not predict the early and late course of proteinuria and ckd progression. hence, urinary tgf-β appears to be a biomarker of tubulointerstitial disease activity rather than of global disease progression. the surprising increase of et excretion may reflect the induction of compensatory hemodynamic mechanisms during acei. cardiovascular complications are the most important cause of death in pediatric patients with endstage renal disease. therefore early diagnosis and treatment are very important. brain natriuretic peptide (bnp) is released in response to volume overload or conditions that cause ventricular stretch. the aim of the study was to investigate whether bnp can be used for early diagnosis of cardiac complications in pediatric patients with esrd. twenty-four patients on peritoneal dialysis (mean age . ± . years), patients on hemodialysis (mean age . ± . years) and sex and age matched healthy children (mean age . ± . years) were included the study. plasma bnp levels were significantly higher in the patient group than those in the control group ( . ± . vs . ± . pg/ml, respectively, p< . ), but there was no significant difference between hemodialysis and peritoneal dialysis patients. in patients with hypertension, bnp levels, left ventricular systolic and diastolic diameters were significantly higher than those in the patients without hypertension. in patients with higher crp levels, bnp levels were significantly higher than those in the patients with low crp levels. bnp levels had a positive correlation with left ventricular mass index (r= . , p= . ) and a negative correlation with ejection fraction (r=- . , p< . ) and shortening fraction (r=- . , p< . ) significantly. there was no significant relation between bnp levels and anemia, dialysis duration, and dialysis modality. in conclusion, high plasma bnp level is significantly correlated with dilated left ventricle and it may be useful as a biochemical marker for identification of pediatric dialysis patients with cardiac dysfunction. we observed a high prevalence of left ventricular hypertrophy (lvh) and impaired lv contractility in children with stage ii-iii chronic kidney disease (ckd) (jasn , jasn . in a prospective, open-label assessment in children receiving ace inhibition (ramipril mg/m /d) with or without additional antihypertensive medication, we evaluated by echocardiography lv mass (lvm), geometry and myocardial mechanics at baseline and after (n= ) or mos (n= ) of treatment. lvh was defined by lvm index> g/m and concentric geometry by relative wall thickness> . ( th normal percentile). lv systolic function was assessed at the midwall level by circumferential shortening (ms). normalized h mean arterial blood pressure (bp) was reduced from . ± . at baseline to . ± . and - . ± . sds after and mos respectively. lvmi was reduced significantly after (from . ± . to . ± . g/m . , p< . ) and mos (from . ± . to . ± . , p< . ). of those patients presenting with lvh at baseline, lvm regressed to the normal range in / ( %) after mos and in / ( %) after mos. the prevalence of concentric lv geometry remained unchanged (baseline: %, mos: %, mos: %). age and afterload-corrected myocardial function increased from ± % to ± % at mos (p< . ). the changes in lvm and myocardial performance were independent of the randomized bp target and gfr. change in lvm was correlated with change in hemoglobin level (r= . , p< . ) and change in myocardial function with change in bp level (r= . , p< . ). in conclusion, fixed-dose ace inhibition and tight bp control induce regression of established lvh in the majority of children with stage ii-iii ckd. this is associated with a normalization of myocardial contractility. objectives of the study: periods with insufficient erythropoietic activity may occur during the erythropoietin (epo) treatment in chronic haemodialysis (hd). we determined the effects of a short-term suspension of epo therapy on various oxidative stress parameters during a -week follow-up in hd patients. methods: the antecedent epoetin beta (eb) treatment was suspended for days. after that, patients received eb two times a week and patients received darbepoetin alfa (da) once weekly. concentrations of whole blood oxidized and reduced glutathione (gssg, gsh) and various haematological parameters were determined weekly. erythrocyte malondialdehyde (e-mda) and the activities of erythrocyte superoxide dismutase, catalase and glutathione peroxidase were determined at weeks , and . results: the ratio gssg/gsh was increased in both groups after continuation of the suspended epo therapy (p< . and p< . week vs. the baseline in the da and eb group, respectively) and also at week (p< . and p< . vs. the baseline in the da and eb patients, respectively). the activities of the antioxidant enzymes were increased at week in both groups (p< . vs. the baseline for da and p< . vs. the baseline for eb), and returned to their week levels by week . the e-mda level decreased in both groups (p< . week vs. the baseline for da and p< . weeks and vs. the baseline for eb). conclusions: a short-term suspension of epo therapy caused characteristic time-dependent changes in the oxidative stress. the ratio gssg/gsh increased at weeks and . activities of the antioxidant enzymes were elevated at week , resulting in an improvement in lipid peroxidation. these results might have implications in certain conditions with transient alterations in the erythropoietic activity in hd patients. rrf has been associated with better nutritional status both in adults and children on peritoneal dialysis and in adults on chronic hd. there are no data on the influence of rrf on nutrition in children on chronic hd. three-days dietary reports and simultaneous urea kinetic monitoring of children, adolescents and young adults on chronic hd (age: . - . years) were retrospectively analyzed. protein catabolic rate, an index of nutrition adequacy, was normalized by body weight (npcr). in patients with rrf, total kt/v (kt/vtot) was calculated summing hd kt/v(kt/vhd) and rrf (evaluated by residual urea clearance ku-). in all patients, npcr and dietary protein intake (ndpi) were significantly correlated (p< . ). kt/vtot was correlated with npcr(p< . ) while correlation between kt/vhd and npcr was not significant (p: . ). in patients with rrf, ku resulted significantly associated with npcr (p< . ) while kt/vhd was not (p: . ). npcr was significantly higher in patients with rrf ( . ± . vs . ± . g/kg/day; p< . ). patients on recombinant growth hormone (rhgh) treatment showed npcr higher than those without rhgh ( . ± . vs . ± . g/kg/day; p< . ). however, in a multiple regression model including age, the use of rhgh, rrf, kt/vtot and kt/vhd, npcr resulted significantly associated only with rrf (b: . ; p< . ). inconclusion: in children, adolescents and young adults on chronic hd treatment, rrf is associated with better nutrition. rrf positively affects nutrition independently from hd efficiency and rhgh effects. possible hypothesis are a more selective (although decreased) depuration and the positive influence of water excretion maintenance on food intake. more efforts have to be made in order to maintain rrf in children on chronic hd. during the past three years, six women have undergone chronic haemodialysis during pregnancy at the pediatric renal unit of the adelaide women's and children's hospital. five have delivered normal infants at between and weeks gestation; one is presently at weeks gestation, with an apparently normal fetus. four were already receiving chronic haemodialysis at the time of conception; the others began dialysis at weeks gestation. the protocol includes six days per week dialysis for at least two hours per treatment. a number of practical and emotional issues have arisen, with major potential psychosocial hazards, including: unplanned pregnancy due to ignorance of fertility; precipitation onto the dialysis program; acceptance of increased dialysis time; concerns regarding effects of drugs and dialysis/kidney failure on the fetus; the likelihood of prematurity; the perceived difficulties of motherhood while on a chronic dialysis program; loss of income, social networks and independence. the program has been successful due to the cooperative approach from the multidisciplinary team consisting of nephrologists, obstetricians, obstetric physicians dialysis nurses, midwives, dieticians, physiotherapists, psychiatrists and social workers. the social worker has provided counselling and coordinated transport and assistance with housework, childminding and other day to day tasks. although the program has had overall success, there have been a number of pitfalls worth discussing, including those arising from the complex interactions between the members of the various disciplines, and those involved in maintaining the balance of clinical and psychosocial needs of the women. (median . ) , weight from . to . kgbw (median ). the vascular access was a central catheter (kt) in children, an arteriovenous fistula (avf) in , avf and kt alternatively in patients. duration of hd ranged from to m (median . ). dual lumen tunnelled cuffed kt were inserted in children through the internal jugular vein [ijv]), surgically in , percutaneously in . all kt have had an immediate good function. nine kt were exchanged over a guide wire for dysfunction. kt infections with positive blood culture were successfully treated in cases. duration of kt ranged from to m (median ). at the end of the follow-up, kt were still in function, removed for a mature avf, after renal transplantation (rt), for improved gfr and failed. patency of the venous network after withdrawal of kt was assessed in children (doppler , mri ) and showed normal patency in , ijv thrombosis in , brachiocephalic thrombosis in and stenosis in . thirty avf were created in children, distal in ( %). immediate patency was obtained in all cases except . the median blood flow ranged from to ml/min/m (median ). following the primary surgery, repeated surgical procedures included a superficialization of the vein in , refection for stenosis or thrombosis in , reduction of overflow in , nd avf creation in and ligation after rt in . percutaneous transluminal angioplasty was performed in children. duration of patency ranged from to m (median . ). in conclusion, hd is feasible in small children. nevertheless, kt are associated with risk of venous occlusion and obtention of a reliable avf frequently need several interventions, altogether leading to a limitation of hd indications in young children. - , ( %) were < years of age. the underlying disorders were congenital nephropathies in % (malformations %, hereditary %) and acquired diseases in %. living related donation (ld) was performed in % and preemptive tx in %. immunosuppressive (is) protocol varied considerably between the countries and over time. -year graft survival (gs) was % in ld and % in grafts from deceased donors (dd). gs improved significantly for dd grafts with time. the number of acute rejections (ar) during the first year posttx was significantly lower in ld recipients, in tac-treated children and during the second half of the study period. this improvement over time was also seen in separate analysis of cya-treated children. the proportion of rejection-free patients increased in all countries. median height sds at tx was - . (- . to + . )(boys - . , girls - . ). height sds increased to - . at years posttx. conclusions: gs results were excellent and the frequency of ar low, especially in children with ld grafts and tac treatment. interesting differences between the countries concerning donor source, preemptive tx, is and use of protocol biopsies were found. n. marcun varda, a. gregoric maribor teaching hospital, department of pediatrics, maribor, slovenia objectives: essential hypertension (eh), identifiable in children, is associated with cardiovascular (cv) diseases in adulthood. the aim of our study was to evaluate the presence of some traditional and non-traditional cv risk factors in our children and young adults with eh in the search for additional cv risk. the prevalence of metabolic syndrome (ms) was also investigated. methods: a total of children and young adults, diagnosed with eh, were included in our study. they were compared with a control group of healthy children, matched for sex and age, with regards to specific aspects in the history, body mass index (bmi), waist: hip ratio, full blood count, crp, serum cholesterol, hdl cholesterol, ldl cholesterol, triglycerides, uric acid, glucose, insulin, fibrinogen, homocysteine, apolipoprotein a , apolipoprotein b and lipoprotein (a). in addition, the prevalence of ms was calculated. ms was defined as having three or more ms components according to the national cholesterol education program's adult treatment panel iii criteria, tailored for children. results: the differences in values of bmi, crp, platelets, triglycerides, uric acid, apolipoprotein a , apolipoprotein b and homocysteine between the hypertensive patients and the controls were statistically significant. in all hypertensives positive family history of hypertension in the first or second generation was revealed. overweight (bmi > th percentile for age and sex) was identified in %, obesity (bmi > th percentile) in %, abnormal glucose homeostasis in %, high serum triglycerides in % and low hdl in % of the hypertensives. ms was present in % of these children and in % of our controls. conclusion: we demonstrated that children and young adults with eh differ from the population of healthy children in some specific cv risk factors, and are therefore at an increased cv risk. background: rtd is a rare autosomal recessive disease of differentiation of fetal kidneys with poorly developed proximal tubules. fetal and neonatal findings include oligohydramnios, preterm birth and neonatal death due to pulmonary hypoplasia, anuria and hypotension. mutations in genes in the renin-angiotensin system (ras), encoding for angiotensinogen, renin, angiotensin converting enzyme and angiotensin ii receptor type , have been revealed. we report the first rtd patients surviving the neonatal period and still being alive. both patients had affected siblings demised in utero or neonatally. case : oligohydramnios, birth at weeks, grams. neonatal course: pulmonary hypoplasia, pneumothorax, hypotension and anuria with ventilation ( weeks) and dialysis ( days) . current findings at years: creatinine umol/l, normal blood pressure. genetic and functional analysis: homozygous mutation of angiotensinogen gene ( g-a); very low angiotensinogen concentration ( . ng/ml; normal - ), absent plasma renin activity (normal . - . ng/ml/h) despite very high active renin ( pg/ml; normal - ). case : oligohydramnios, birth at weeks, grams. neonatal course: mild respiratory distress, hypotension and anuria with oxygen ( day) and dialysis ( months) . current findings at years: renal transplantation at age years, good graft function. genetic and functional (as neonate) analysis: homozygous mutation of renin gene ( g-a); very low active renin (< . pg/ml; normal - ). conclusions: rtd is caused by inactivating mutations in genes encoding the ras resulting in chronic low perfusion pressure of the fetal kidney. genetic and functional analysis of ras contributes to diagnosis of rtd. this observation extends the rtd phenotype from a uniformly fatal to a more favourable disease. objectives of study: to evaluate the relationship between serum uric acid (ua), new onset essential hypertension (eh) and endothelial dysfunction (ed) in the youth. methods: subjects with abpm proved new onset eh (aged , ± , years, bmi , ± , kg/m ), overweight/obese normotensive youth (oo) matched for age and bmi, and age matched healthy normotensive controls (nt) with normal weight were enrolled. ua, total cholesterol, hdl, ldl, triglycerides and creatinine were analyzed in blood, glomerular filtration rate (gfr) was calculated according to schwartz formula and endothelial function was assessed using flow-mediated dilation (fmd, %). results: new onset eh was associated with overweight/obesity in % of subjects. serum ua levels were significantly higher in eh than oo ( ± vs. ± umol/l, respectively, p< , ), in eh than nt ( ± vs. ± umol/l, respectively, p< , ), and in oo than nt ( ± vs. ± umol/l, respectively, p< , ). total cholesterol, hdl, ldl and triglycerides were significantly higher in eh and oo compared with nt (p< , ). no significant differences were found in lipidograms between eh and oo. serum creatinine and gfr did not differ significantly between the groups. fmd was significantly lower in eh comparing with oo and nt ( , ± , vs. , ± , vs. , ± , %, respectively, p< , ) and in oo comparing with nt ( , ± , vs. , ± , %, respectively, p< , ). conclusions: new onset eh in the youth is associated with overweight/obesity, higher serum ua and ed. ua may play a causal role in the pathogenesis of eh and commonly with eh and proatherogenic serum profile contributes to ed in overweight/obese hypertensive youth. objective of study: fibromuscular dysplasia (fmd) is a systemic arteriopathy of the small and medium sized vessels. it is the first cause of renal arterial stenosis (ras) in childhood. the aim of this study was to describe the natural history of fmd in children. we analysed all the data of children with fmd. results: mean age at diagnosis was years months old. hbp was discovered fortuitously in cases, after symptoms of malignant hypertension in . in all cases bp values corresponded to severe hypertension (mean bp of / mmhg). extra renal localizations were found in half of the patient, and the most frequent pathological arteries were the superior mesenteric and the carotida. the mean number of arterial stenosis at diagnosis was per patient. seven patients had a primary fmd with familial history for patient. in the others fmd were associated with polymalformatif syndromes: reported by grange ( ), moya-moya disease ( ), neurofibromatosis type i ( ). after a median follow-up time of months the number and severity of stenosis increase at arteries per patient. these lession, although to a lesser extend, were already observed as soon as months of follow-up. percutaneous transluminal renal artery angioplasty were proposed in cases when the bp was uncontrolled indeed a multi antihypertensive therapy with severe renal stenosis (> %) or when renal growth was impaired. prognosis is severe with cardiologic complications ( / ) and death. conclusions: intimal form of fmd accounts for the majority of multivisceral fmd. at diagnosis several hypertension is almost present. vascular ultrasonography imaging techniques is usefull in follow-up. a conservative treatment has to be privilegied. this disease is evolutive with increase of pathological arteries number, aggravation of stenosis degree and sometimes renal function impairment. objective: the effect of over weight on blood pressure elevation (bp) is more frequently present in childhood. several studies have demonstrated the efficacy of angiotensin-converting enzyme inhibitors (acei) therapy in obese hypertensive adults, but data on children are very limited. methods: obese (bmi: z score > . sd) primary hypertensive (systolic ordiastolic blood pressure > th) children (aged - years) were enrolled to this single centre prospective study. patients received ramipril ( . - . mg/bwkg/day) once daily. office and ambulatory bloodpressure measurements and serum biochemical analysis were performed at start and after and months of treatment. patients ( , %) hadimpaired glucose tolerance (igt) on oral glucose tolerance test (ogtt). results: ( , %) patients completed the six months ramipril therapy. reduction in -hour mean arterial pressure (map) was , mmhg (- , sd) after month and , mmhg (- , sd) months treatment respectively. bp was reduced with equal efficacy during day-and night time. / ( , %) patients were lost during the follow-up. ( , %) patients with high uric acid levels also were treated with allopurinol. eleven patients ( %) received second antihypertensive medication because of the blood pressure remained uncontrolled. ( pts metoprolol, pts amlodipine) ( , %) patients suffered recurrent cough, but otherside effect has not observed. the serum glucose and insulin levels havenot changed significantly during the follow-up. conclusion: oncea day given ramipril significantly decreases the blood pressure inobese hypertensive children. it is effective, tolerable and safe bloodpressure lowering monotherapy in childhood. further studies and longer follow-up are necessary to prove its long term beneficial effect in the childhood metabolic syndrome combined with hypertension. hw. zhang, j. ding, f. wang, yf. wang peking university first hospital, department of pediatric nephrology, beijing, people's republic of china objectives of study: females with x-linked alport syndrome (xlas) have variable phenotypes, from microscopic hematuria to chronic renal failure, which can not be clarified solely by mutation features of col a gene. x-inactivation has been suspected to be one of the responsible reasonsfor this phenomenon, but no definite correlation has been demonstratedso far. in order to confirm whether the phenotypes of xlas femalescorrelate with x-inactivation, we analyzed the xinactivation patternsin peripheral blood cells in xlas females and in skin fibroblasts in xlas females. methods: the x-inactivation analysis was performed using hpaiipredigestion of dna followed by polymerase chain reaction (pcr) of thehighly polymorphic cag repeat of the androgen receptor (ar) gene. results: results showed that the average x-inactivation levels of the mutant allele decreased while the degree of proteinuria increased, there was anegative correlation between the degree of proteinuria and thex-inactivation ratios of the mutant allele in blood cells (r=- . , p= . ). however, there was no correlation between the degree of proteinuria and the x-inactivation ratios of the mutant allele in skin fibroblasts (r=- . , p= . ). though of patients ( . %) had the similar x-inactivation ratios in both blood cells and skin fibroblasts, there was also no correlation between the x-inactivation ratios of the mutant allele in skin fibroblasts and that in peripheral blood cells (r= . , p= . ). conclusion: we concluded that the x-inactivation ratios in blood cells correlated with the degree of proteinuria, which might explain partially the diverse phenotypes in female xlas patients. more studies, including post-transcription regulation, environmental factors, and so on, are still needed. objective: to report frasier syndrome (fs) with wt mutation and abnormal expression of podocyte molecules which is the first case in mainland china. methods: peripheral blood cells were analyzed for chromosome karyotype and wt gene mutation. the ratio of +kts/-kts isoforms was quantified with genescan and genescan software. expressions of podocyte molecules were detected by immunohistochemical staining. result: the patient presented with steroid-resistant fsgs and male pseudohermaphroditism. the wt ivs + g>a mutation was found in one allele in the proband, but not in her parents. the ratio of +kts/-kts was . in the proband, and was . and . in her mother and father, respectively. podocyte molecules expression altered in normal-and abnormal-appearing glomeruli. wt expression showed diffuse nuclear staining with less obvious speckles compared with that in controls. wt (antibody against c-terminal) displayed strong, normal, faint and negative stained podocyte nuclei within the same glomerulus. the staining intensity of wt (antibody against the n-terminal) was very faint. conclusion: taking clinical data, pathology, karyotype analysis and genetic testing together, we diagnosed the first case of fs in mainland china, which prompts there might be more cases underdiagnosed. wt displayed diffuse nuclear staining with less visible speckles compared to controls, supporting the view of the differential nuclear localization of kts isoforms. our study further confirmed that wt mutation resulted in abnormal expression of podocyte molecules in glomeruli of fs, though we did not know whether this phenomenon directly or indirectly resulted from loss of wt regulation. dent disease is an x-linked disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, calcium nephrolithiasis and the development of renal insufficiency after the middle age. so far, two genes responsible for the development of dent disease have been identified, i. e., clcn and ocrl . ocrl was originally identified as a causative gene for lowe syndrome. in japan, igarashi et al. described for the first time that idiopathic tubular proteinuria in japan is identical to dent disease by mutational analyses. most of japanese patients with dent disease are identified by annual urinary screening as chanced proteinuria and several clinical phenotypes, such as incidence of nephrocalcinosis and long term outcome of renal function remains to be elucidated. furthermore, identification of ocrl as a second causative gene for dent disease has made the understanding of dent disease more complex. here we report patients with dent disease phenotype with the results of genetic analyses and clinical features. out of patients in different families, mutations ( %) in clcn were identified. in the patients with no clcn mutations, genetic analysis in ocrl is ongoing, and at the moment different mutations in ocrl ((i stop, r c, r w and r h) were identified. the remaining patients are now beeing investigated. among the patients in whom clcn mutations were detected, hypercalciuria are not always present. in several elderly patients, mild renal function impairment is present. there are similar clinical phenotypes between patients with clcn or ocrl mutations, but serum levels of ldh and ck are likely to be higher in those with ocrl mutations. in summary, we will present genotype and phenotype heterogeneity in japanese dent disease, and will discuss the clinical spectrum of dent disease. a. gulati , s. sethi , j. lunardi , m. kabra , n. gupta , p. hari , a. bagga all india institute of medical sciences, st department of pediatrics, new delhi, india hôpital de la tronche, genetics, grenoble cedex. france, france objectives: to study the clinical features and genetic basis of patients with lowe syndrome and identify female carriers. methods. case records of patients with lowe syndrome presenting to this hospital, between - yr old, were reviewed. the clinical features were recorded and glomerular and tubular functions assessed. a detailed genetic analysis was performed on dna extracted from peripheral blood of patients and their mothers, which involved sequencing of all exons of the ocrl gene. results: all patients showed failure to thrive, bilateral congenital cataracts, refractory rickets, delayed motor and language milestones and proximal renal tubular acidosis with fanconi syndrome. the mean schwartz gfr was ml/min/ . sq. m. genetic analysis showed distinct mutations of ocrl gene in all patients studied. we report new mutations having identified the variants c. a>g and c. g>t in exon , the variant c. _ ins t in exon and c. - a>g in intron in independent patients. we also found previously described mutations involving the region c. _ del tg and c. c>t in exon . four of mothers were heterozygous carriers. all genotypically proven carriers showed characteristic lenticular opacities. conclusion: the identification of mutations in the ocrl gene provides confirmation of the diagnosis of lowe syndrome. the new mutations described in north indian children expand the range of mutations that give rise to this condition. these observations have important implications for molecular diagnosis and genetic counseling in families with lowe syndrome. juvenile nephronophthisis (nph), an autosomal recessive nephropathy, is the most common genetic cause of chronic renal failure in childhood. in - % of known cases, nph is associated with joubert syndrome (js), a neurological disorder described in patients with cerebellar ataxia, mental retardation, hypotonia and respiratory dysregulation. mutations in three genes, ahi , nphp and nphp have been identified in patients with nph and js. as nphp mutations usually cause isolated nephronophthisis, the factors which predispose to the development of neurological symptoms in some patients are poorly understood. to determine such genetic factors and to assess the mutation rate of ahi , nphp and nphp in nph and js, a cohort of families with nph and at least one js-related neurological symptom was screened for mutations in these genes. thirteen ( %) and ( %) unrelated patients were homozygous or compound heterozygous for nphp and nphp mutations, respectively. in patients ( %) without nphp , nphp or ahi mutations, mutations in a novel gene (nphp /cors ) encoding a ciliary protein have been identified. interestingly, of the patients with nphp mutations carried either a heterozygous truncating mutation in nphp ( patient), a heterozygous missense mutation in ahi ( ) or the ahi variant r w ( ) . the variant r w affects an amino acid conserved in vertebrates and predicted to be 'possibly damaging' by polyphen software. in conclusion, nphp , nphp or nphp mutations can be found in % of patients with nph and js in our cohort. our finding that half of all patients with nphp mutations carry a mutation or a damaging variant in nphp or in ahi strongly supports the notion that epistatic effects provided by these genes contribute to the appearance of neurological symptoms in patients with nphp mutations. molecular cytogenetic techniques such as array-based cgh have been instrumental in the identification of microimbalances associated with syndromic phenotypes. we investigated patients with unclear syndromic nephropathies (e. g. urinary tract malformations, focal segmental glomerulosclerosis, and persistent hematuria/proteinuria) and additional clinical features, such as mental retardation, heart defects or growth abnormalities. array-cgh analysis was performed with a whole-genome array with large insert clones providing an average resolution of < . mb. results: in one -year old female patient presenting with microhematuria, proteinuria, mental retardation including severe speech impairment, senso-neuronal hearing loss, and recalcitrant focal epilepsy, we detected a microdeletion in chromosomal bands xq . -q . this deletion was verified by fish, found to be uniallelic, . - . mb in size, and not to be inherited from the mother. electron microscopy of kidney biopsy showed splitting of the lamina densa and a thin basal membrane, which is diagnostic for alport syndrome. high-resolution cranial magnetic resonance imaging including white fibre tracking revealed a severe neuronal migration disorder with subcortical band heterotopia (double cortex syndrome), i. e. a second band of cortical neurons within the white matter below the true cortex. conclusions: in patients with unclear syndromic nephropathies, we identified a female with a contiguous gene syndrome at xq . -q . the microdeletion includes the x-linked alport syndrome gene col a and the lisx gene associated with subcortical heterotopia, mental retardation and epilepsy. thus, the phenotype observed in our female patient combines features of the amme-complex (alport syndrome, mental retardation, midface hypoplasia, elliptocytosis) with x-linked lissencephaly. ( f, m; . ± . yrs, hemoglobin . ± . gr/dl, ferritin ± mg/dl) were enrolled. lipid profile, acute phase proteins (apps) were measured. renal tubular functions, plasma vegf level, urinary nag/cre ratio were determined. abpm and imt measurements were performed. the results were compared with healthy controls. results: mean -hour, day and night systolic-blood pressure (sbp), diastolic-bp (dbp) and mean arterial pressure values were comparable to that of control group. dipping in dbp was less in tm ( . % vs . %; p< . ). the ratio of patients with less than % dipping in sbp (non-dippers) was higher in tm ( . % vs . %, p< . ). mean plasma vegf level was . ± . pg/ml [ . - . ] in tm, was within normal range (< pg/ml) in controls. apps were normal. imt of common carotid artery (cca) was . ± . mm in tm group and . ± . mm in controls, (p< . ); imt of internal carotid artery (ica) was . ± . mm in tm group and . ± . mm in controls (p< . ). positive correlations were found between vegf and microalbuminuria, b -microglobulin and homosistein; between nag/cre and microalbuminuria; between cca-imt and age, and a negative correlation between cca-imt and ferritin. conclusions: renal tubular damage, abnormalities in abpm, increase in vegf and increase in cca-imt and ica-imt occur when the patients are asymptomatic and routine laboratory test are normal. optimal hemoglobin levels and deferoxamine therapy do not prevent the development of renal and vascular damage. l. sylvestre , e. santos , p. granzotto , e. siqueira , l. moreira , l. rispoli , n. mendes , r. meneses hospital pequeno principe, pediatric nephrology, curitiba, brazil centro universitario positivo unicenp, curitiba, brazil introduction: hypertension is frequently underdiagnosed in children. early diagnosis and a planned follow-up is helpful in detecting and preventing the harmful long-term consequences of hypertension. therefore, we created a specific outpatient clinic to have a better follow-up of these patients. material and methods: we reviewed the files of patients from the outpatient clinic of hypertension, from the dept. of pediatric nephrology in hospital pequeno principe, curitiba, brazil, followed for more than months, from march to december . we analyzed demographic and anthropometric data, diagnosis, staging of hypertension, presence of target-organ damage and treatment. results: patients were eligible, boys, mean age at the first visit . years old, mean follow-up of months. mean bmi= . ( % overweight and obese). secondary hypertension was present in % of the cases, predominantly due to parenchymal renal disease; essential hypertension associated to overweight and obesity in patients ( %) and there was no established diagnosis yet in other patients. fifty-five patiens performed at least one abpm of hours, showed hypertension. twelve from patients showed left ventricular hypertrophy and from patients had abnormalities of the retinal vasculature associated to hypertension. the most frequent drugs used to treat hypertension were ace inhibitors ( ) and calcium channel blockers ( ). conclusion: our data are in accordance that secondary hypertension is frequent in children, mostly associated to renal disease. furthermore, we could detect a large number of obese hypertensive children and adolescents. target-organ abnormalities are not as frequent as in adults but need to be monitored. intrauterine growth retardation (iugr) is characterized by low nephron number with or without reduced kidney size. leptin, an important hormone in the regulation of body fat massand weight, is decreased in fetuses with iugr. in the present study, weexamined the effects of leptin in a metanephric mesenchymal cell line ms . ms is generated from the metanephroi of embryonic day . homozygous mouse transgenic for h- kb-tsa. incubation of ms withleptin , , , or ng/ml for h did not affect either [ h]-thymidine incorporation or cell number. on the other hand, [ h]-leucine incorporation was significantly increased by leptin in a dose dependent manner ( ± %, ± %, ± %, and ± % of control by , , , and ng/ml, respectively). protein/dna was also increased . -fold by leptin ng/ml. leptin ng/ml activated both erk and p . the levels of phosphorylated-erk (p-erk) and phosphorylated-p (p-p ) , as assessed by western blot analysis, started to increase at min, peaked at min ( . -and . -fold increase respectively) remaining elevated at , , and h, and began to decrease at h returning to the baseline level at h. the levels of p-erk and p-p at min were increased by leptin in a dose dependent manner ( . -, . -, . -, and . -fold increase by , , , and ng/ml respectively). the levels of total erk and p remained unchanged. increase in [ h]-leucine incorporation stimulated by leptin ng/ml was completely inhibited by coincubation with a mek inhibitor pd μm or a p inhibitor sb μm. these results demonstrate that leptin induces hypertrophy of metanephric mesenchymal cells via erk and p . the hypertrophic effect of leptin may play a role in normal renal development and may explain reduced kidney size in a hypoleptinemic state, iugr. objectives of study: to investigate the mechanism of nephron deficit in rat model of intrauterine growth retardation (iugr). methods: a rat model of iugr was built by maternal low-protein ( %) dietthroughout pregnancy. newborn male pups were chose as our studyobjects. the proliferation and apoptosis in kidney was showed by ki- detection and tunel method. expression of wt , bcl- , bax, and p mrnas in renal tissue were examined by real-time pcr, and expression of wt and bcl- gene products in renal tissue were examined by immunohistochemistry and western blot. the final number of glomeruli was determined at weeks of age when nephrogenesis has finished. results: at weeks postnatally, iugr offspring had fewer glomeruli per kidney than those in controls (p< . ). in iugr newborns, tunel positive cells were more numerous in the nephrogenic zone. renalwt and bcl- mrna levels were significantly reduced in newborn iugrpups, and the bcl- mrna/bax mrna ratio was also decreased, but therewas no change in the expression of p mrna. in iugr newborns, the wt and bcl- protein expressions were significantly decreased, and the irimmunostaining were also suppressed in the nephrogenic zone. conclusions: these results suggest that reduction of nephron number in iugr rat may be associated with enhanced apoptosis in kidneydevelopment. decreased wt and bcl- expressions and reduction of the bcl- /bax ratio may contribute to the molecular mechanisms behind these findings. objective of the study: the aim of this study was to identify risk factors for urinarytract infection (uti) during follow-up of children with isolatedantenatal hydronephrosis. methods: between and , patients were diagnosed with isolated fetal renal pelvicdilatation (rpd) and were prospectively followed. after initialclinical and imaging evaluation, us scans, clinical examination, andlaboratory reviews were scheduled at -month intervals. the event ofinterest was time until occurrence of first episode of uti. cox's regression model was applied to identify variables that wereindependently associated with the uti. results: a total of patients were included in the analysis ( boys and girls) themedian fetal rpd was mm (iq range, . ± ) and patients ( . %) presented bilateral rpd. seventyeight ( %) infants presented urinarytract anomaly. the most frequent detected uropathy was upjo ( ), followed by primary vur ( ), and megaureter ( ). median follow-up timewas months (iq range, - months). during follow-up, uti occurred in ( %) of the children. the incidencerate of uti was episodes per person-month. the incidence rate of uti has decreased from . episodes per person-month in the first year of life to . in the second year, and to . after the third year. by survival analysis, the risk of uti for the whole series was estimated in . % at months, % at months, and % at months of age. after adjustment, two variables were independent predictors of uti during follow-up: female gender (rr= . , % ci, . - . , p= . ) and presence of uropathy (rr= . , % ci, . - . , p= . ). conclusion: according to findings, in a cohort of antenatal hydronephrosis, girls with vur or urinary tract obstructionhad a higher risk of uti during follow-up. objective of the study: to identify predictive factors of resolution of fetal renalpelvic dilatation (rpd) in a cohort of medically managed children. methods: between and , patients were diagnosed with isolated rpd and were prospectively followed. of infants, ( %) were clinically managed. after initial clinical and imaging evaluation, us scans, clinical examination, and laboratory reviews were scheduled at -month intervals. the event of interest was rpd resolution, regardedas renal pelvis < mm on two consecutive renal sonograms. cox's regression model was applied to identify variables that were independently associated with the event. results: a total of patients were included in the analysis and uropathy was diagnosed in ( %) infants. median follow-up time was months (interquartile range, to months). during follow-up, ( %) patients presented rpd resolution. by survival analysis, the estimate of rpd resolution for the whole series was % at months, % at months, and % at months of age. the median time for rpd resolution was estimated at months ( % ci, - ). in the survival analysis, three variables were found to be significantly associated with resolution of rpd during follow-up: mild fetal rpd, grades - (sfu grading system), and presence of uropathy. after adjustment, only absence of uropathy remained as an independent predictor of rpd resolution (rr= . , % ci, . - . , p< . ). conclusion: according to these findings, it was estimated that rpd resolution occurs in about half of the patients without uropathy at years of age. objective: to study clinical and pathological characteristics of antineutrophil cytoplasmic autoantibody (anca)-positive glomerulonephritis(gn). methods: clinical data of thirteen patients during years, with anca-positive gn were analyzed. results: of patients with anca-positive gn with an average age of . ± . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) years, patients were female and were male. the average course was . ± . months. cases onset between december and february. there was , , cases whose chief complaint on admission was anaemia, swollen and hematuria respectively. the main clinical symptoms were: anaemia ( %), hematuria/proteinuria ( %), renal functional lesion ( %), edema ( . %), oliguresis ( . %), hypertension ( . %), arthralgia ( . %), rash ( . %), abdominal pain ( . %), fever ( . %). laboratory tests: bun, scr and esr were high while hemoglobin was low in all patients. mpo-anca was positive in cases c was normal in cases and low in cases. pathology features: all glomeruli are affected and show different degree of segment and glomus sclerosis. there was different degree of capsula glomeruli thickening. glomerulus capillary loop was necrotic, nuclear leukocytes and cell debris could be seen. inflammatory cell interstitial infiltration with lymphocyte and plasmocyte. endotheliocyte of small vessels of interstitial was swelling and vessel wall was edema, necrosis and glassy degeneration. immunofluorescence showed no or small immune complex deposition and showed pauci-immune gn. conclusion: onset of patients with anca-positive gn was hiding and there were nonspecific clinicalsymptoms in the early stage that reduced late diagnoses until esrd. female patients was predominant. renal pathology showed segment necrotic nephritis interstitial inflammation and polyangitis. the disease had unfavourable prognosis. background: renal function maturation isn't achieved at birth. vlbw infants exposed to intensive care have an increased risk of developing renal function impairment. moreover, we showed that ibu was associated with a significant impairment in renal function at one week of life in vlbw infants. objective: to evaluate renal function development in infants treated with ibu for pda closure as compared to infants not exposed to ibu, during the first month of life. methods: multicentric prospective cohort study of to weeks gestation (ga) infants exposed or not exposed to ibu within the first days of life. infants presenting with renal impairment at birth, urinary tract malformation, or contraindication to ibu were excluded. infants exposed to ibu were paired to controls according to ga, centre and crib score. creatinine clearance (ml/min/ . m ) was measured for glomerular filtration rate evaluation. fractional excretion of sodium (fena%), micro-albuminuria (mg/l) and alpha -microglobinuria/creatininuria ( /ucr: mg/mmol) were also measured once a week up to month of life results: infants were studied, half exposed to ibu. birth weight was ± g (mean±sd), and ga ± wks. results are presented in the table: *p= . ;**= . ;***< . . j. rouillard lafond, mp. morin, c. girardin centre hospitalier universitaire de sherbrooke, département de pédiatrie, sherbrooke, canada objectives of study: many studies have focused on the negative effects of low birth weight, especially caused by intra-uterine growth restriction, on adult renal function. however, few have addressed the impact of extreme prematurity (< weeks) on renal function of these children during their later childhood and adolescence. the aim of the present study is to estimate the renal outcome of this population, by determining blood pressure, glomerular filtration rate, fractional excretion of sodium and microalbuminuria in children ( girls and boys) aged to years old (mean age ) born between - weeks of gestation (mean: weeks). methods: during one encounter, height, weight and blood pressure were measured for each subject. blood tests were conducted to quantify creatinine, electrolytes and cystatin c. microalbumine, creatinine and electrolytes were dosed in one micturition. pertinent risk factors of renal damage in their perinatal history were noted. results: renal insufficiency, defined by a clearance of less than ml/min/ , m , was present in subjects ( %) when estimated by schwartz formula ( , - , ml/min/ , m ) and in ( %) when estimated by cystatin c ( , ml/min/ , m ). furthermore, ( %) children presented an elevated fractional excretion of sodium ( , - , %) . finally, children ( %) presented microalbuminuria, with albumin/creatinine ratio greater than , mg/mol ( , - , mg/mol) . those children presented more episodes of hypotension during neonatal period (p= , ) and have a tendency to have had neonatal asphyxia more than the others (p= , ). conclusions: these results suggest that children born extremely preterm may present renal insufficiency and sign of tubulopathy as early as adolescence, with microalbuminuria possibly announcing upcoming glomerulopathy. organogenesis isregulated by epithelial-mesenchymal interactions that take place in theembryonic kidney between the metanephric mesenchyme (mm) and theepithelium of the ureteric bud (ub). the mm expresses signals thatregulate ureteric branching while the ub signaling leads to inductionof nephrogenesis. as a response to the ub signals the mm cellscondense, aggregate, epithelialize and undergo simple morphogenesis togenerate segmented nephrons during kidney organogenesis in connectionto ureteric bud branching. we reported earlier that mutagenesis of fgf function from the whole embryonic mesoderm leads to kidney failure. activation of wnt- gene expression encoding another essential signal for nephrogenesis also depends on fgf function (perantoni et al., ) . given the important role of fgf in kidney development we targeted fgf roles in urogenital system (ugs) development with an ugs specific cre line pax cre. pax credeletion was expected to recombine the floxed genome in the nephronprecursors, ureteric bud and the wolffian duct derivatives. in crossesbetween fgf c/c and fgf n/+ ; pax cre mouse lines fgf gene was deleted successfully. as a result the whole ugswas affected. the kidney was severely reduced in size. newborn nullmice were born alive but died within hours likely due to kidneyfailure. in the deficient kidney the organization of theproximal-intermediate segments of the developing nephron was disturbed. marker analysis with in situ hybridization was consistentwith serious defects in nephrogenesis. we conclude that fgf functionis involved in the control of wolffian duct development andsegmentation of the assembling nephron. the zellwegerspectrum disorders (zsds) are characterized by a generalized loss of peroxisomal functions caused by deficient peroxisomal assembly. clinical presentation and survival are heterogeneous. although most peroxisomal enzymes are unstable in the cytosol of peroxisomedeficient cells of zsd patients, a few enzymes remain stable among which alanine: glyoxylate aminotransferase (agt). its deficiency causes primary hyperoxaluria type (ph , mim ), aninborn error of glyoxylate metabolism characterized by hyperoxaluria, nephrocalcinosis, and renal insufficiency. despite the normal level of agtactivity in zsd patients, hyperoxaluria has been reported in several zsdpatients. we aimed to determine the prevalence of hyperoxaluria in zsds and tofind clinically relevant clues that correlate with the urinary oxalate load. methods: we reviewed medical charts of dutch zsd patients with prolonged survival (>one year). results: urinary oxalate excretion was assessed in and glycolate in patients. hyperoxaluriawas present in ( %), and hyperglycolic aciduria in ( %). renal involvement with urolithiasis and nephrocalcinosis was present in five of whichone developed end-stage renal disease. the presence of hyperoxaluria, potentially leading to severe renal involvement, was statistically significant correlated with the severity of neurological dysfunction. discussion: zsd patients should be screened by urinalysis for hyperoxaluria and renal ultrasound for nephrocalcinosis in order to take timely measures to preventrenal insufficiency. menkes disease is a very rare x-linked recessivedisorder of copper metabolism. the frequency is : . live births. mutations in the atp a gene are described, which encodes for aintracellular copper-binding membrane protein. pathogenetically a defect in copper absorption is responsible for inadequate synthesis of copper requiringenzymes and causes multisystemic manifestations. the clinical picture ischaracterized by early neurodegenerative symptoms like muscular hypotony andcerebral seizures. the patients also present with the so called kinky hair, hyperelastic skin, and anomalies of the kidneys and the urinary tract. wereport on a patient of non consanguineous parents of german origin. theprenatal ultrasound did not show any malformations. birth at weeks ofgestation. postnatally a softening of the cranial bones and a vesicoureteralreflux iv° with dilatation of the renal pelvis combined with a subpelvicureteral stenosis had been observed, which were operated at the nd month of life. at the age of months the patient presented with seizuresand abnormal hair structure. within the diagnostic course menkes diseasehas been suspected and the nonsense mutation parg x has been identified inthe atp a gene. the mother is not a conductor, a mutation in thegermline cannot be excluded. conclusion: the combination of urinary tract malformations and neurodegenerative symptoms should let you think of the very rare menkes disease. introduction: primary hyperoxaluria type (ph ) is an inborn error of glyoxylate metabolism due to the deficient activity of the hepatic peroxisomal enzyme agt (alanine: glyoxylate aminotransferase). it leads to excessive endogenous oxalate production. patients develop urolithiasis and renal insufficiency. the contribution of specific precursors in the pathway leading to endogenous oxalate synthesis is not known. this is warranted to design appropriate treatments. we aimed to test the contribution of different precursors to oxalate synthesis. methods: wild type mouse hepatocytes were incubated with different potential precursors of glyoxylate, either in the presence or absence of alanine. in the absence of alanine flux through agt is deficient thereby mimicking agt deficiency. similar experiments were also performed in hepatocytes from agtdeficient mice. results: oxalate production was found to be highest with glyoxylate as substrate in the absence of alanine, whereas oxalate production was lower with glycolate, hydroxypyruvate, glycine, fructose, and ethylene glycol. the results obtained in wild-type hepatocytes incubated in the absence of alanine were comparable to those obtained in hepatocytes from agt-deficient mice. upon addition of alanine to wild-type mouse hepatocytes, however, resulted in % lower rates of oxalate production, in contrast to hepatocytes from agt deficient mice. discussion: hepatocytes derived from agt deficient mice represent a good model to study the contribution of different precursors to oxalate production in ph . s. grisaru , c. geary-joo , f. snider , j. cross university of calgary, department of pediatrics, calgary, canada university of calgary, department of biochemistry and molecular biology, calgary, canada gcm (glial cell missing), is a transcription factor necessary for the formation of placental syncytiotrophoblast in mice. gcm mutant mice die before nephrogenesis at embryonic day (e) . during early murine development gcm expression is limited to the placenta. however, immediately after birth, gcm is increasingly expressed in the kidney in proximal tubular cells in the outer medulla. we recently reported successful rescue the gcm null phenotype using a tetraploid aggregation approach (jasn vol. , ) . since our previous report, further analysis of the aggregation products confirmed only homozygous mutants ( males and a female) obtained from six hundred and twenty five transferred aggregate embryos resulting in live pups. abnormal cortico-medullary patterning was demonstrated by histology analysis of adult gcm null mice kidneys. this abnormality was further defined by immunohystochemical detection of known nephron segment-specific markers (aquaporin- , aquaporin- and tamm-horsfall protein) in gcm null kidney sections. to define the expression of gcm in human kidneys, commercially available anti-human gcm polyclonal antibodies were used to detect gcm protein in tissue sections of newborn kidneys obtained from autopsies. gcm was detected by immunohystochemistry in the renal cortex in tubular structures with cells having a brush border suggestive of proximal tubules. gcm signal was not detected in the renal medulla. conclusion: in humans, gcm is expressed in renal proximal tubules at birth whereas in adult mice its mutation is associated with abnormal renal cortico-medullary ultrastructure. this effect may represent a primary role for gcm in late renal development and patterning, or structural changes occurring postnatally secondary to alterations of tubular physiology caused by gcm inactivation. objectives: lupus nephritis (ln) in singapore children treated with cyclophosphamide and/or azathioprine has a poor prognosis with a reported -year renal survival of %. this study examined the long-term outcome of children with lupus nephritis using a new protocol comprising pulse intravenous methylprednisolone, mmf ± cyclosporine. method: twenty-one children with ln (age range at start of treatment . - . years) who were treated between the years to were included in this retrospective study. mmf dose was mg/m /day. mean duration of follow-up was . ± . (range . - . ) years. treatment outcome was defined by systemic lupus erythematosus disease activity index (sledai), renal function, proteinuria and serologic markers. effect of steroids on growth was assessed by height standard deviation score (htsds). statistical analysis was performed using wilcoxon signed rank test. results: at presentation, % had nephritic-nephrotic syndrome, % had nephrotic syndrome, while % had renal failure requiring dialysis. renal biopsy classification (who) was ii in %, iii in %, iv in %, and v in %. comparing pre-mmf treatment and current follow-up parameters respectively, sledai ( . ± . objectives of study: to understand the effects of response gene to complement (rgc- ) in tgf-β induced epithelial-mesenchymal transition (emt) on human renal proximal tubular epithelial cells (hptecs). methods: constructed rgc- expression plasmids and rgc- sirna hairpin plasmids, transient transfected them into hptecs in vitro, and then treated hptecs with tgf-β ( ng/ml) or vehicle alone for indicated time ( , min, h, h, h) . rt-pcr and western blot were used to determine the expression of a-sma, ecm (col-i, fn ). the mrna expressions of e-cadherin and sm a were detected by rt-pcr. results: ( ) the promoting effects of rgc- on emt. instead of stimulation with tgf-β , the hptecs, those overexpressed rgc- gene, de novo obtain the ability to produce markers of myofibroblast phenotype (a-sma, col-i and fn) and sm a gene, as well as lost the capability of expressing e-cadherin gene. ( ) the eliminating effects of rgc- sirna on emt that induced by tgf-β . after stimulation of tgf-β for hours, the expression of a-sma, col-i, and fn as well as sm a gene in hptecs, those rgc- genes were interfered with rgc- sirna, were significantly decreased than that in controls. conclusions: rgc- was an important regulator for tgf-β and its downstream signalling smad proteins on emt. background: low birth weight is associated with a low nephron endowment. this may predispose to hyperfiltration and cascading proteinuria particularly if obesity develops. our report relates to an emerging population of children with proteinuric kidney disease in our multiethnic community. methods: forty-two obese children (mean age ± years) with proteinuric kidney disease (kd) were studied. twenty-four were of normal birth weight (nbw> grams) and were of low birth weight (lbw< grams). there was a female ( / = %) and an ethnic predominance ( african, hispanic). degree of proteinuria was determined by the random urine protein (pr) and albumin (alb) to creatinine (cr) ratios (upr/cr and ualb/cr). renal function (egfr) was estimated from the schwartz formula. body mass index was used as a measure of obesity (> % centile). insulin resistance was measured by the homeostatic model assessment (homa). kidney tissue was obtained in of the patients for pathology and histomorphometry. results: average bmi was ± % tile. fasting insulin and homa scores were not significantly different in the obese nbw versus obese lbw children. renal biopsy specimens revealed focal glomerulosclerosis (fsgs) in the majority of patients ( / = %). progression to end stage kidney disease was significantly greater in lbw compared to nbw children with a median renal survival of years, p< . . glomerulomegaly as measured by glomerular diameter was similar in obese patients and significantly greater than non-obese controls with fsgs. conclusions: obesity appears to be a confounding factor in the development of glomerulosclerosis and progression of kidney disease in children. low birth weight and concomitant low nephron endowment may contribute to disease progression, especially in those of african and hispanic descent. objective: to determine long term outcome and prognostic factors of iga nephropathy in a large single center cohort of pediatric patients. patients and methods: we have reviewed the medical charts of patients with biopsy proven igan that have been followed at our institution from to , with a minimal follow-up of years. follow-up data, including proteinuria > mg/ h or the need for ace-is therapy, chronic renal failure (crf) and hypertension were analysed after years and years of follow-up. data from patients with follow-up longer than years were also available (mean follow-up . years, range - years). % of patients received therapy (cyclophosphamide in patients and/or steroid±ace-is in the remaining). clinical features at the onset, histology class (lee and haas) and treatment during the first years were analysed by multivariate analysis against the above mentioned dependet variables. results: the average follow-up was . years (range - years). presentation symptoms included macroscopic hematuria in % of patients. at the end of follow-up, renal function was normal in % patients, patients have reached end-stage renal disease and had chronic renal failure. proteinuria or the need for aceis at years was significantly associated with the age of onset (or: . [ . - . ] ) and proteinuria at the onset (or: . [ . - . ] ). crf was significantly associated with familial igan (or> ). hypertension at onset was significantly associated with persistent hypertension during follow-up (or: . [ . - ] . conclusion: taken together, these data indicate that the overall prognosis of igan is good during childhood and that the worst prognostic factor for development of crf is familial igan. overall, histological classification had a poor correlation with the outcome of the disease. this study was designed to compare three urinary protein expert systems for profiling proteinuria (pu) in children with kidney diseases. freshly voided urine was collected from children with glomerular diseases, children with tubular diseases and healthy children aged - years. out of children with renal disease had a glomerular filtration rate (gfr) < ml/min/ . m . the urinary protein expert systems were . albumin/total protein ratio (apr), . alpha- microglobulin/alpha- -microglobulin + albumin algorithm (aaa), and . the complex upes algorhithm (using serum creatinine, urinary total protein, alpha- -microglobulin, albumin, igg, alpha- -macroglobulin and dipsticks). apr correctly identified glomerular pu in of ( %) children with glomerular diseases, tubular pu in of ( %) children with tubular diseases and normal pu in of ( %) healthy children. aaa correctly identified glomerular pu in all ( %) children, tubular pu in of ( %) children, and all healthy children were characterized as having no pathological pu. upes differentiated the type of pu in children with glomerular diseases into glomerular ( / patients) and mixed glomerulo-tubular ( / patients). tubular pu in children with partial or complete renal fanconi syndrome was identified in / patients and described as mixed glomerulo-tubular pu in / patients. mixed glomerulo-tubular pu was only found in children with ckd stages - of glomerular and tubular diseases. in conclusion, urinary protein expert systems may be used to distinguish between glomerular and tubular pu. the aaa algorithm had the highest reliability when compared with the two other expert systems and the accuracy was not negatively influenced by a decrease of gfr. however, upes provided additional information on mixed glomerulo-tubular pu in patients with a low gfr. background: the three lmw proteins cystatin c (cys), β -microglobulin (β -m) and β-trace protein (β-tp) are useful markers of gfr. cys is particularly well suited for the detection of incipient renal failure. however, corticosteroid medication has been shown to stimulate cys production. aim of the study: analysis of the effect of corticosteroid therapy on the correlation between gfr and the three lmw markers. patients: patients ( f, m; median age . years, range . to . ) with malignant (n= ) or nephrological (n= ) diseases underwent a single-shot inulin clearance. the respective lmw proteins were measured by particle-enhanced immuno-nephelometry. children received corticosteroids (prednisone or dexamethasone) in a mean dosage of mg/m /d of prednisone equivalent (pred/bsa). multiple linear regression analysis was performed between the lmw markers as dependent and both gfr and steroid-dose as independent variables. results: mean gfr was . ± ml/min/ . m , mean cys . ± . mg/l, β -m . ± . mg/l and β-tp . ± . mg/l. cys was highly correlated with the reciprocal of gfr (p< . ) but not with corticosteroid-dose (p= . ), whereas both β -m and β-tp were highly correlated (p< . ) with both the reciprocal of gfr and the reciprocal of pred/bsa. discussion: using gold-standard gfr measurements, we cannot confirm earlier reports indicating an increase in serum cys during corticosteroid medication. by contrast, steroids significantly lowered both β -m and β-tp serum concentrations. we conclude that at least in patients with mild renal insufficiency cys -unlike β -m and β-tp -appropriately reflects gfr also during steroid therapy. this further supports the concept of cys being a superior marker of incipient renal failure. objective: to repeatedly follow kidney function since onset of type diabetes and evaluate whether gfr can predict development of micro-or macroalbuminuria or end stage renal disease. design: observational cohort study. methods: since , all diabetic patients undergo renal function tests every nd to rd year from onset. children, boys, have done clearance studies. healthy children and young adults, - years of age, served as controls. gfr was evaluated by clearance of inulin during water diuresis and continuous infusion. results: gfr during the first years after onset of diabetes was significantly higher than that of controls (mean - vs. ml/min/ . m ). at onset, and years after, boys had significantly higher gfr than girls (mean , , vs. , , ml/min per . m ) but after years no differences were found between sexes. the occurrence of microalbuminuria, albuminuria during the first - years was analysed and mean of gfr of , and years, and years, , and years and all those gfrs separately were compared between patients still normoalbuminuric, microalbuminuric and macroalbuminuric after and years resp. no significant differences were found between the groups. moreover the change in gfr from to , to and to and to years were also compared between the groups and no significant differences were found. young adults reached end stage renal disease (esrd) after - (median ) years and comparing their gfrs during the first - years with those still normoalbuminuric after years, no significant difference was found. conclusions: hyperfiltration is found in children with type diabetes during the first - years from onset and hyperfiltration was equally seen during the first - years in those children who in the future developed normo-, micro-, albuminuria and/or esrd. background: hiv associated nephropathy (hivan) remains an important entity despite the use of highly active anti-retroviral therapy (haart). our objectives were to determine the prevalence and severity of renal manifestations in a cohort of hiv infected children during the haart era. methods: a retrospective analysis was conducted on children infected with hiv. renal assessments included quantitation of proteinuria, radiologic abnormalities, and renal function. persistent proteinuria (pp) was defined by urine protein to creatinine ratio (upr/cr) > . detected on at least two measurements month apart. renal sonography and mag renal scintigraphy were categorized according to the presence of bilateral increased echogenicity and/or nephromegaly, and cortical retention and/or diffuse parenchymal disease, respectively. hivan was considered in those children that had pp associated with any radiological abnormalities. results: of the children, . % were perinatally infected. eighty-five ( . %) had pp. of these, had pp alone, while ( . %) developed hivan. the mean age of onset of hivan was . ± . years. overall mortality at the time of analysis was . % and it was highest in those with hivan. viral load (vl) > , copies was significantly associated with hivan. creatinine clearance was significantly decreased in patients with hivan. conclusions: the prevalence of pp in our population of perinatally infected children remains high ( . %), with at least half of them showing evidence of hivan. persistently high vl (> , copies) was associated with the presence of hivan. a spectrum of renal related disorders is a frequent occurrence in hiv infected children and should be sought with periodic urinalysis, quantitation of proteinuria and renal function, and imaging and/or histopathological studies. mmf has shown to be effective in adult ln, whereas only anectodical data are reported in childhood. we evaluated mmf in children with ln, f/ m, mean age: . ± . yrs, proteinuria > g/day, decreased c and increased anti-dsdna serum levels, normal renal function. renal biopsies, before mmf, showed the following classes (weening) : iv in cases, iii in , ii in , vi in . before mmf: patients have received i. v. cyp; more received aza and csa but were in flare-up of disease; the remaining were newly diagnosed patients. each patient received three i. v. metilprednisolone pulses and thereafter mmf (plus oral prednisone(p): mg/kg/day) was administered (mean dose: ± . mg/kg/day; through level: . ± . μg/ml). outcome was monitored by sledai score, renal function, proteinuria. in children p was tapered and, after . ± . month mean time, stopped; children were receiving p ( . mg/kg/day). the mean followup is ± months. sustained clinical remission was observed: proteinuria was absent in all, in patients an increase of serum c and c and a decrease of anti-dsdna levels was seen. significant steroid sparing effect was obtained: hypercorticysm dramatically improved. of the patients achieved years of mmf treatment and in them, at this time, a serial second renal biopsy was performed: histopathological activity indices reduced ( . ± . vs. . ± . , p< . ), whereas chronicity indices did not change ( . ± . vs. . ± . ). no haematological and/or gastrointestinal side effects were observed. our pilot study suggests that mmf represents a good alternative to traditional therapy in the treatment of sle in children, and in controlling disease activity and as steroid sparing agent without significant side effects over the entire period of therapy. mmf has shown to be effective, during treatment, in mantaining remission of childhood sd and csad ns, but few data are available on the mmf long term effectiveness after drug stopping. we report the results of two years mmf treatment in children with sd and csad ns. the characteristics of sd and csad groups were, respectively: patients, boys, mean age . yrs vs. pts, boys, mean age: . yrs (p< . ); first episode ns mean age: . yrs vs. . yrs; ns mean duration: . yrs vs. . yrs (p< . ); mean steroid therapy duration: . yrs vs. mean csa therapy duration: . yrs; histologic features: fsgs , mc vs. fsgs , mc . in both groups mmf was started after remission was achieved with prednisone administered at the last relapse. mmf treatment: lenght: months; mean dose: . ± , mg/kg/day; plasma through level: . ± . mcg/ml; non responder: patient presenting a ns relapse during mmf. in sd group ( %) and in csad ( %) subjects were responders. in patients of sd group and patients of csad group p could be withdrawn over a mean period of . months, so that ns remission was sustained just by mmf; remaining patients were receiving p at a mean dose of . mg/kg/a. d. two years mmf treatment was accomplished in / ( %) patients. at . ± months mean followup since mmf withdrawal, ( %) patients ( of sd and of csad group) relapsed after . months ( . - . ) mean time no haematological or gastrointestinal side effects were observed. our results demonstrate that two years mmf treatment in sd and csad ns children is effective not only in maintaining remission during therapy, but also in achieving persistent remission after withdrawal of drug in a significant rate (> %) of patients; the side effects and the rate of mmf dependence are negligible with respect to those of steroids and csa. information on long-term renal function following treatment for wilms tumor (wt) are relatively scanty. previous studies reported a worrying late development of microalbuminuria (uma), hypertension (hpt) and even reduction in glomerular filtration rate (gfr). the aim of the present study was to evaluate the long-term renal outcome in a cohort of patients who underwent uninephrectomy for wt. glomerular function (as creatinine clearance by cockroft-gault formula) was calculated and uma (as uma/ucr ratio) as well as urinary b microglobulin excretion were detected. -hours ambulatory blood pressure monitoring was also recorded. fifteen patients ( f) with a median age at wt diagnosis of . yrs (range . - . ) were studied. the median follow-up was . yrs ( . ± . ). eight patients had been classified as wt stage and as wt stage ii. all patients had been treated with unilateral total nephrectomy and chemotherapy. two of the children had also been addressed to radiotherapy. the primary disease did not recur in any of the patients. the median age at time of investigation was , yrs (range: , - , ). none of them had a gfr below normal limit (mean gfr was . ± . ml/min/ . m ). urinary b microglobulin excretion was normal (mean ub /ucr: . ± . ) in all of the patients. the mean uma/ucr ratio, was . ± , with only patient exhibiting higher then normal values (uma/ucr ratio: , ). the hr blood pressure was normal in all patients with a mean systolic and diastolic blood pressure sds of - . ± . and - . ± . , respectively. we conclude that as far as renal function, unilateral total nephrectomy combined with chemotherapy for low-stages wt can be look at as a safe treatment although it might be wise to monitor renal function at -year interval. classicaly, patients are divided into monosymptmatic enuresis (mne) and non-monosymptomatic enuresis, however, there is upcoming evidence that this subtyping might be artificial. the aim was to register the characteristics of nocturnal diuresis-rate and bladder volume in both subtypes. methods: retrospective analysis of consecutive patient-files, age - , primary consulting for enuresis in a tertiary center. registration of incontinentia diurna (id) and maximal functional bladder volume (vmax), hours urine-collections in day and nighttime-collections with uosmol and diuresis-volume (dv) and-rate. patients are divided into a mne and nmne-group. results: ) vmax is significantly lower than bladder volume for age, ) nocturnal polyuria is only present in / patients, ) nocturnal diuresis is >vmax, ) there is a significant linear correlation between nd and the nocturnal/daytime-diuresis-ratio, indicating fluid intake dependency. ) there is a negative correlation between nd and urinary osmolality. ) but the positive correlation between total nocturnal osmotic excretion and nd is much stronger. this unexpected observation cannot be explained by the classical primary vasopressin-theory. conclusion: our data show (almost) no statistical difference between the mne and nme-groups, suggesting a continuum instead of separate identities. both groups have a significant low vmax and nocturnal polyuria. the observation of the extremely strong correlation of nocturnal polyuria with the high osmotic excretion and high h urine-production suggests that nocturnal diuresis-rate is highly fluid-and nutrition-dependent, and therefore more attention should be given to this part of the urotherapy. a. deguchtenaere, a. raes, j. dehoorne, r. mauel, e. vanlaecke, p. hoebeke, j. vande walle there is increasing evidence that a subgroup of patients with nocturnal polyuria may have an abnormal circadian rhythm of tubular sodium which may result in vasopressin resistance. the pathogenesis of this phenomenon remains to be elucidated. however if the increased sodiumexcretion overnight results in the ddavp-resistance, decrease of the sodium-excretion-overnight may respond in subsequent ddavp response. aim of the study: retrospective study on the circadian rhytm of diuresis-rate and osmotic excretion in basal condition and subsequent during introduction of ddavp, diet and furosemide. results + discussion: ) baseline-values show significant lower uosmol and higher diuresis-rate overnight compared to controls. striking is the > % part of electrolytes to explain the high osmotic excretion. ) introduction of ddavp results in a normalization of nocturnal uosmol, but despite a significant decrease of uosmol overnight, nocturnal polyuria persists. ) protein-and sodium-restriction results only in slight differences, but of course we do not have data on the compliance. ) furosemide in the morning results in a significant increase of daytime diuresis, osmotic and sodium-excretion, but as compensation decreased nighttime diuresis, osmotic and sodiumexcretion. ) in / cases the antidiuretic effect results in an anti-enuretic effect. conclusion: this pilot study clearly demonstrates that introduction of early morning furosemide results in significantly lower nocturnal diuresis. because the urinary osmolality remains high, this correlates with decreased nocturnal osmotic excretion associated with increased osmotic excretion (sodium) during daytime. background: the elasticity of the vessel walls decreases with age, this process is dramatically speeded up by uremia. as an early indicator of arteriosclerosis pulse wave velocity (pwv) increases along with arterial stiffness. aim: to establish normal values for pwv in healthy children; to compare it with children on dialysis. patients, methods: pwv was measured with a pulsepen device in healthy children and young adults (age range - years) as well as in uremic children ( , ± years) (crf) treated by hemodialysis (n= ) or peritoneal dialysis (n= ). two control groups of - childrens were formed using the database of healthy children: one matched for age (a-c) and one adjusted for height and weight (h/w-c). blood pressure, heart rate, serum calcium (ca), phosphate (p) , and parathyroid hormone levels were also determined. results: a significant linear correlation was found between pwv and age (r= , ), height (r= , ), weight (r= , ), (p< , ), systolic blood pressure (r= , ) and heart rate (r=- , ) (p< , ). crf patients were smaller by , cm than a-c (p< , ), and younger than h/w-c by , years (p< , ). pwv in crf ( , ± , m/s) did not differ significantly from a-c ( , ± , ), however it was elevated in comparison to h/w-c ( , ± , p< , ). serum p, caxp and pth was increased in crf (p< , ) conclusion pwv is higher in children with crf as a sign of increased arterial stiffness. controls matched for height and weight should be used in states of severe growth retardation. a number of established risk factors potentially responsible for arterial dysfunction are present in crf. ngal has been identified as an early marker of acute renal failure (arf). sepsis in very low birth weight (vlbw) infants is associated with arf more often than recognized. the aim of this study is to determine whether ngal represents a marker of renal impairment in vlbw infants affected by sepsis. samples of urine of vlbw infants were prospectively collected for weekly measurement of ngal. after evaluation of the clinical course, groups were identified: group sepsis includes infants affected with septic events associated with some degree of renal impairment and group normal includes uncomplicated vlbw infants. a mouse model of sepsis was created in neonatal mice by intra-peritoneal introduction of salmonella lipopolysaccharide. kidneys were harvested hours after the challenge, and ngal mrna was quantified by real time pcr. ngal values of the normal group did not differ with gestational age or post-natal age of the neonates. the upper bound of the th percentile confidence interval was ng/ml. the median value of ngal in the sepsis group at days before the septic event was ng/ml and during sepsis was ng/ml: these values were not significantly different, but both were significantly higher (p< . ) than the median of the normal group ( ng/ml). once sepsis had been treated, the median value of urinary ngal was similar to normal ( ng/ml p= . ). changes in urine ngal concentration paralleled changes in serum creatinine. sepsis induced animal models showed a dramatic increase of ngal mrna in kidney tubules that paralleled acute renal failure. these neonatal animal and human data suggest that ngal may be an early marker of renal impairment in septic vlbw babies. further investigation is necessary to more exactly define the temporal relationship between the onset of sepsis/arf and rise in urine ngal concentration. we report cases of acute renal failure (arf) associated with orellanus syndrome, a cortinarius mushroom poisoning. grand-father, mother, father and son presented with arf week after gastrointestinal symptoms and weeks after repetitive ingestion of wild mushrooms. critically arf was observed for the year-old boy: anuria, severe metabolic disorders (hyponatremia mmol/l, hyperkaliemia mmol/l, serum creatinine mmol/l, blood urea mmol/l). renal biopsy was performed for the grand-father, father and son (day , and respectively after presentation) and showed similar lesions: severe tubulointerstitial nephritis (tin) with tubular necrosis and interstitial fibrosis. renal replacement therapy was necessary for the father and the son. the mother recovered completely in two weeks without dialysis while renal function improved slowly for the two men. the boy is still hemodialyzed months later. his main problem is uncontrolled hypertension. the diagnosis was confirmed weeks later since fungal spores of cortinarius orellanoides were observed in the contaminated meal by light microscopy. the severity of the disease seems to be related to the toxin quantity: the kg boy ate mushrooms as much as his father and grand-father, the mother ate much less. cortinarius spp poisoning is an exceptional paediatric cause of arf. gastrointestinal disorders are the main symptoms of the initial phase of the poisoning appearing days after the mushrooms ingestion. the renal phase is delayed (median . days) characterized by arf secondary to tin. the toxin effects are dose-related, explaining the severity of the boy's symptoms. the prognosis is severe with % of end stage renal failure. currently no treatment is available. although rare, mushroom poisoning should be considered in the differential diagnoses of arf with tin. l. mendels, ah. bouts, j-c. davin, j. groothoff emma children's hospital/academic medical center, pediatric nephrology, amsterdam, the netherlands background: inchronic dialysis, tertiary hyperparathyroidism (th) is clinically revealed by persistent combined high parathyroid hormone (pth), normalor high total serum calcium (tca) and normal phosphate levels. sincethe introduction of bicarbonate containing dialysate in peritoneal dialysis (pd), we have observed combined high tca and pth level sunusually early after onset of dialysis therapy. in most of the secases, ionized calcium (ica) levels were low. we aimed to investigate the extent of this discrepancy and its association with the mode of therapy. methods: serum ica, tca, pth, bicarbonate and capillary ph were assessed over years in pediatric pd, hemodialysis (hd) and in transplanted (tx) patients. associations between tca, pth and ica were analyzed. results: comparedto tx patients, we found in pd and hd patients a lower mean ica/tcaratio (both p< . ), an increased mean tca-ica (p< . and p < . , respectively), and a higher number of combined normal/increased tca and decreased ica and increased pth values ( . % and . %, respectively for pd and hd, vs. % for tx). alow ica/tca was associated with a high capillary ph (r=- . ; p< . ), a high venous bicarbonate (r=- . , p= . ) and a lowage (r= . , p= . ). conclusions: ica levels are warranted for monitoring calcium phosphate homeostasis in dialysis patients, especially in young pd patients. the use of only tca levels might lead to an inadequate treatment with vitamin d, and henceinduce the development of autonomous hyperparathyroidism in these patients. preterminal renal failure (prf)and end-stage renal disease in children and adolescents are associatedwith an increased risk of atherosclerosis and cardiovascular disease. oxidative stress is one of the pathogenetic factors that could possiblybe influenced by therapeutic interventions. we investigated biomarkers of oxidative stress in children (median age . years) with prf (median gfr ml/min/ . m , range - ) andin children (median age . years) under peritoneal dialysis (pd)and healthy age-matched controls (c). plasma samples wereinvestigated for malondialdehyd (hplc) and carbonyl groups in proteins(elisa) as biomarkers for oxidative stress as well as the plasmaantioxidative substances vitamin c (photometric), vitamin e (hplc), ubichinols (hplc), sulfhydryl groups in proteins(photometric), erythrocyte resistance to radicals and the total radicaltrapping antioxidant capacity (trap). in both patient groups prf and pdwe found a depletion of sulfydryl groups and ubichinol- and a reducedresistance of erythrocytes to radicals. malondialydehyd (p< . ) andcarbonyl groups (p< . ) were elevated in the pd group compared tocontrols. conclusion: from these studies we concludethat in children under peritoneal dialysis biomarkers of oxidativestress are elevated. moreover antioxidative defenses in preterminalrenal failure as well as under peritoneal dialysis are impaired. significant acute renal failure due to non-steroidal anti-inflammatory drugs: inpatient setting in united states non-steroidal anti-inflammatory drugs (nsaid) are freely available over-the counter. many children routinely use them without medical supervision. fourteen inpatients mean age of . ± . years ( males, females), were referred to nephrology for acute renal failure. based on history, biochemistry, imaging and urinalysis the diagnosis of acute renal failure due to nsaid was made. all patients admitted to taking ibuprofen and six also consumed naproxen. the exact doses of either could not be scientifically determined as none were prescribed by a physician. none of the patients had underlying renal diseases at the time of admission. nine patients had proteinuria and had hematuria (including one with gross hematuria). one patient had nephrotic syndrome but resolved spontaneously without steroids and has remained in remission for years. two patients required dialysis. only one of the dialyzed patient required steroid therapy for recovery of renal function. all data are expressed as mean±sd. the mean duration of hospitalization was ± . days. the mean serum creatinine at the peak of renal failure was . ± . mg/dl (range . - . ). all patients recovered renal function with normalization of serum creatinine to . ± . mg/dl (range . - , p< . ). however, the duration from onset to normalization of serum creatinine was ± days; indicating many patients had abnormal renal function for aprolonged period. in conclusion, nsaids pose significant risk of renal failure forsignificant duration and as an entity may be under recognized. objectives: treatment with growth hormone (gh) improves growth retardation of chronic renal failure. cdna microarrays were used to investigate gh-induced modifications in gene expression in the growth plate of uremic young rats, the organ where longitudinal growth takes place. methods: rna was extracted from the tibial growth plate from two groups (n= ) of young rats: uremic (nx) and uremic treated with . mg/kg/day of intraperitoneal gh for one week (nxgh). after reverse transcription, agilent technology was used to analyze differential gene expression by microarrays containing , rat probes (four hibridizations were performed). most expressed genes were detected using linear models and bayesian methods. to confirm gene expression changes shown by the chips, some genes known to play a physiological role in growth plate metabolism were analyzed by real-time quantitative polimerase chain reaction (qpcr). the ribosomal protein l (rpl ) expression did not show changes in the array and was used as the housekeeping gene. results. gh modified the expression of genes, being upregulated and down-regulated. the assay was validated by the qpcr results, which confirmed the sense of expression modification found in the arrays for insulin like growth factor i (down), insulin like growth factor ii (up), collagen alpha (down) and proteoglican type (up) . conclusions: this study shows for the first time the profile of growth plate gene expression modifications caused by gh treatment in experimental uremia. the further analysis of selected individual genes, whose expression is differentially modified by gh will contribute to explain the mechanism of the stimulating effect of gh on growth in chronic renal failure. objectives of study: children with chronic renal failure have an increased risk of cardiovascular disease. this is associated with endothelial dysfunction, a key pathophysiological factor in atherosclerotic disease. circulating endothelial progenitor cells (epcs) have the potential to repair endothelial damage and promote angiogenesis. in adults, the number of epc in peripheral blood correlates with endothelial function and reduced epc levels are associated with a higher incidence of cardiovascular events. we aimed to investigate if children on long-term hemodialysis (hd) therapy have reduced epc levels. methods: we quantified circulating epc in pediatric hd patients before a midweek hd session and healthy age-matched controls. epc are a subfraction of the haematopoeietic stem cells (hsc) expressing both hsc-marker cd and the vegf-receptor- kdr. using flow cytometry, epcs were identified as cd +kdr+ cells and quantified relative to the number of granulocytes in the sample. results: the number of epcs in the peripheral blood was significantly reduced in hd patients ( . ± . vs . ± . / granulocytes, % reduction; p= . ). the total number of circulating hsc also tended to be lower in hd patients ( . ± . vs . ± . / granulocytes, % reduction; p= . ). conclusion: the number of circulating epcs is significantly reduced in children on long term hd. reduced epc levels may contribute to endothelial dysfunction and accelerated atherosclerosis in children on long term hd. future studies are needed to identify the cause of this deficiency and to evaluate if increasing epc levels provides therapeutic benefit. objectives: darbepoetin alfa (aranesp ® ) is a novel erythropoiesis stimulating protein that has been shown in adult trials to have safety and tolerability equivalent to recombinant human erythropoietin. however, to date there is only limited published data on the use of aranesp inpaediatric patients. the objective of this study was to determine the safety and efficacy of darbepoetin in children with chronic and endstage kidney disease. methods: from to , children with either chronic or end stage kidney disease were enrolled in a prospective observational study. the initialdose of darbepoetin was . mcg/kg weekly (either iv or sc) and subsequent dose was titrated to achieve haemoglobin (hb) between and g/dl. results: data analysis to date includes patients ( male : female) whose agesranged from month to years (mean years). hb improved significantly with darbepoetin treatment from mean g/dl (range - ) at start of treatment to g/dl (range - , p< . ) at completion. the mean starting dose was . mcg/kg/week (range . - . ) which was not significantly different to the dose atthe end of the study ( . mc/kg/week, range . - . ). however, there was a significant change in the frequency of administration, with % commencing on weekly treatment, but only % still on weekly treatment at the end of the study (p< . ). the most common treatment interval in stable patients was fortnightly ( %) but a significant number tolerated even longer intervals ( % dosed every weeks or longer). injection pain was common, but there were no other significant adverse events. conclusions: darbepoetin alfa is a safe and effective therapy for anaemia associated with kidney disease. the majority of children will maintain satisfactory haemoglobin at a dosing interval of every weeks orgreater. high prevalence ( %) of left ventricular hypertrophy (lvh) and impaired systolic myocardial function in children with mild-to-moderate chronic renal failure (crf) were observed in previous studies (jasn , jasn ). in adult patients with uncomplicated arterial hypertension, lv mass (lvm) exceeding compensatory value for body size and cardiac workload (inappropriate or ilvm) is associated with poor prognosis, independently of lvh. we tested in crf children if increased lvm compensates or exceeds the expected values for individual cardiac load and if ilvm is associated with impaired cardiac function. complete anthropometrics, biochemical profile and doppler echocardiograms were obtained in children (age . ± . yrs; gfr . ± . ml/min/ . m ). ilvm was defined above % of the value predicted for individual body size, gender, and stroke work and lvh was defined as lvm/m . > g/m . . patients showed ilvm. children with ilvm had higher mean age and lower heart rate as compared to patients with appropriate lvm (both p< . ), without differences in blood pressure, bmi and gfr. after controlling for differences in age, gender distribution and presence of lvh, patients with ilvm showed similar cardiac geometry and diastolic function parameters compared to children with compensatory lvm (p=ns). in contrast, presence of ilvm was associated with lower lv ejection fraction ( . ± . % vs . ± . %) and lower midwall fractional shortening ( . ± . % vs . ± . %)compared to children with compensatory lvm (both p< . ), indicating impaired lv chamber performance and reduced systolic myocardial function. in conclusion, in . % of children with mild-to-moderate crf, lvm is inappropriately increased for individual cardiac workload and body size. presence of ilvm is associated with reduced systolic function, independently of age, gender and presence of lvh. pediatric nephrology centers were enrolled. age groups of the patients were as follows: . % newborn, . % - months, . % months - years, . % - years. underlying diseases were prematurity ( . %), malignancy ( . ), congenital heart diseases (chd, . ), urologic disorders ( . %). low fluid intake was noticed in % of cases. . % of cases developed arf after they have been hospitalized. time to diagnose arf was longer in the surgery department ( . ± . days) compared to pediatrics ( . ± . days), p< . . thirty-nine percent of patients were on mechanical ventilation (mv) before the diagnosis of arf, an additional . % needed mvl after the diagnosis of arf. arf was prerenal, intrinsic and obstructive in %, % and % respectively. hemodialysis and peritoneal dialysis was performed in . % and . % of cases. mortality was . %; and it was secondary to non-arf related causes in . % of cases; presence of mv, intrinsic arf, prematurity, chd, malignancy and being in intensive care unit were poor prognostic factors. conclusion: our nationwide data suggest that nephrologist, intensivist and pediatrician should focus on risk groups to prevent and to diagnose arf earlier. appropriate fluid intake and earlier consultation to a nephrologist are simple but may be effective measures to prevent arf.. hemolytic uremic syndrome is characterized by the triad of hemolytic anemia, acute renal failure, and thrombocytopenia. recent studies have shown that shiga-toxins (st) may stimulate apoptotic cell death in renal tubular cells, but the underlying molecular mechanisms remain to be elucidated. in the present study, confluent llc-pk cells were exposed to st and cell death was studied with morphological and biological assay. in llc-pk cells st was found to induce apoptotic cell death in a dose-and time-dependent manner. the expression of calpain and bax were significantly up regulated by st, while the expression of bcl- was down regulated. cell death was completely inhibited by a specific calpain inhibitor, but not by a broad caspase inhibitor, zvad-fmk, implicating a caspase-independent pathway via calpain. moreover, we found that serum factors could trigger a survival signal against st-induced cell death through pi k/akt pathway. in conclusion, activation of calpain mediates st-induced renal proximal tubular cell death, and the expression of bcl- and bax were oppositely altered. stimulation of pi k/akt signalling protects cells against death. verocytotoxin (vt)-producing e. coli (vtec) infection represents the main cause of hemolytic uremic syndrome (hus) in children. a nationwide surveillance system of hus was introduced in italy in may to follow the trend of vtec infections. for each patient, epidemiological and clinical information was collected by a standardized questionnaire. laboratory diagnosis of vtec infection was based on the detection of vtec and free vt in stools and of antibodies to the lipopolysaccharide (lps) of serogroups, o , o , o , o , and o in the sera. the immuno-detection of vt on circulating neutrophils was also performed on some patients. as of december , cases have been notified, accounting for a mean annual incidence of . x , in the - age group with a significant difference among the regions (from . to . ); median age of patients: months, % males. most cases ( %) occured in summer from june to september. seventy-nine per cent of the cases had prodromal symptoms such as bloody diarrhea ( %) and non-bloody diarrhea ( %). five patients ( . %) died from the disease. stools and/or sera were collected from cases. evidence of vtec infection was observed in cases ( %). the vtec serogroups most commonly detected were o ( % of the vtec-positive patients), followed by o , o , o and o . the number of cases associated with non-o infections increased over time: from the o -associated cases are the most frequent. during the surveillance-period epidemic clusters have been registered: -lombardia, -veneto, and campania. the role of vesico-ureteral reflux (vur) as a predisposition for acquired renal scarring with urinary tract infections (uti) has been questioned in recent years. few studies have investigated baseline factors associated with chronic nephropathy in severe reflux. we aimed to evaluate dmsa scans in children having any degree of primary vur associated with uti in order to identify variables that are predictors of the presence and/or development of renal scar. data of patients with proven uti who have primary vur were evaluated retrospectively. patients and renal units were classified as scar (+) and scar (-) by dmsa results. the following parameters were assessed with respect to their relation to presence of renal scarring: sex, age at diagnosis, grade (g) of reflux, number of subsequent utis (on new renal scars). there were patients (m/f: / , median age months) and refluxing units. variables increasing the likelihood of scar detection were: male gender ( / vs. / , p= . ; or . ), > months of age (for girls only; / vs. / , p= . ; or . ), g iv-v reflux (or . vs. g i-iii reflux and . vs. no reflux). all boys having g iv-v reflux and girls over months of age having g iv-v reflux had very high rate of scarring compared to the rest ( / vs. / , p= . , or . and / vs. / , p= . , or . , respectively). however, variables increasing the likelihood of new/progressive scar development were only the presence of previous scar (or . ) and uti number > (or . ). neither uti number nor new/progressive scar development was affected by vur grade. in conclusion, the most predictive variables for the presence of renal scarring among children presenting with a uti were male gender, age (being > months; only for females) and grades iv-v reflux, while new/progressive scar development was associated with presence of previous scar and uti number. d. hothi , e. harvey , c. goia , d. geary hospital for sick children, department of pediatric nephrology, toronto, canada hospital for sick children, educational, toronto, canada introduction: adequate ultrafiltration (uf) is necessary for good health in dialysis dependent patients. however uf can be hindered by development of intradialytic symptoms and hypotension. objectives: to determine whether sodium ramping, uf profiles and mannitol could improve uf without increasing intradialytic morbidity in children. method: a standardized hd practice was instituted in our unit. we prospectively analysed dialysis treatments from chronic patients with routine scheduled hd, hrs x - /wk. results: uf volumes between . to . % of the dry weight were achieved. mannitol reduced the risk of developing intradialytic symptoms by % (p< . ) without altering the risk of hypotension, with a mean uf volume of . % of the dry weight. a linear sodium ramp ( - mmol/l) increased the odds of intradialytic symptoms (p= . ) and hypotension (p< . ), with no difference in the mean uf volume. all uf profiles increased the risk of intradialytic symptoms but the effect was not statistically significant except with profile (stepwise reduction of uf during procedure). achievement of dry weight was least likely with uf profile (p< . ); there was no statistical difference in the mean uf volume between them all. conclusion: uf volumes higher than the traditional recommendations of % of the dry weight can be achieved in children. the use of mannitol increased the uf volumes and reduced symptoms without increased hypotensive episodes. objective: innate immunity and urinary tract response play a central role int he development of urinary tract infection (uti), in which heat shock protein (hsp) and toll-like receptor have a key position. patients and methods: hspa b a( )g and tlr a( )g genotypes were determined using allele-specific polymerase chain reaction in patients treated with recurrent urinary infection. allelic prevalence was related to reference values of healthy controls. clinical data were also reviewed and statistically evaluated. results: hspa b ( )gg genotype and hspa b ( )g allele occurred more frequently in uti patients versus controls (p= . ) and both were associated with a higher risk of renal scarring (p= . and . , respectively). tlr ( )ag genotype and tlr ( ) g allele had also higher prevalence among uti patients than controls (p= . and . , respectively). the combination of carrying ag genotype at both sites meant the greatest risk for uti (p= . ). conclusion: our data indicate an association between the carrier status of hspa b ( )g and tlr ( ) one of the major goal of hemodialysis adequacy is to achieve the fixed endsession body weight, so called dry weight, in order to limit overhydration and thereby cardiovascular risks. the prescribedultrafiltration, can induce hypotensive episodes and thereby limit thedry weight achievement. on line equipments offer the assessment of theblood volume (bv) and its relative variation. the bv is derived fromdirect measurement of the hematocrite. weroutinely use such an equipment (fresenius a c) over all thesessions since february , conducting to a clinical experience of bvm curves. these registered curves could be related to thehemodialysis prescription parameters (uf, nad, td, kt/v) to the dryweight, the blood pressure and to the clinical dialytic symptoms. thisexperience conducts us to define the normal bv curve over a sessionand its variations. starting dialysis induces an acute initial ( to min) decrease of bv, to %, mostly asymptomatic: extra corporeal circuit filling; this initialdecrease is a sign of normality. there after the normal bv curve should be flat; uf rate/amount being compensated by the plasma refilling rate: iso osmotic dialysis, no symptoms, no cramps, no hypotension, no vomiting. incase of no bv decrease over the dialysis session, the patient isoverloaded: reduce his dry weight. in case of a decrease of the bvhigher than %, there is a hypotensive risk: uf rate/amount, dryweight, sodium dialysate, sodium temperature and kt/v urea (cellular water shift) should be individually adapted conducting to arefilling curve. the effectiveness on the bv of these individual changein dialysis prescription can be directly, on line attested by the bvmcurve: plasma refilling capacity test. the bv changes reactivity is rapid, to min. renalscarring following acute pyelonephritis (apn) in children is a frequentcomplication which may impair renal growth. its pathophysiology includes host response, bacterial virulence, associated malformationand/or renal dysplasia. we prospectively studied virulence factors of e. coli isolates from children with a first episode of apn (fever > . c°, crp > mg/l, monomicrobial e. coli positive culture > * cfu/ml). we excluded patients with anyconcomitant infection, renal dysplasia or obstructive uropathy (us examination, renal length < sd) or grade - vur. renal scarring was evaluated by dmsa scan performed to months after apn. patients were included in a multicentre prospective randomized study comparing short vs long i. v. treatment with ceftriaxone as a first line antibiotic treatment [in press]; out of them fulfilled criteria for virulencestudy. six virulence genes were investigated by multiplex pcr (pap, sfa, afa adhesine genes; cnf , hly toxin genes, aeraerobactin gene) and the k capsular antigen was researched by latextest. we identified distinctive virulence profiles; % of e. coli strains had one or more virulence factors; % expressed the aer genewith at least one adhesin or one cytotoxic factor. renal scars occurredin % of cases and low grade ( - - ) vur was detected in %. statistical analysis did not show any correlation between the presenceof scars and e. coli virulence pattern. in addition, scarring was not correlated with the antibiotoc regimen but was correlated with grade vur (p . ). most e. colistrains associated with apn in children show several virulence factors, mainly adhesins and cytotoxins, but their profile was not correlated with renal scarring. background: acute lobar nephronia (aln), a severe renal parenchymal inflammatory disease, ranging between acute pyelonephritis (apn) and frank abscess formation, has been diagnosed with increasing frequency due to the advancement of non-invasive diagnostic modalities and the development of systematic diagnostic schemes. e. coli is the most common bacterialpathogen isolated from the urine samples of aln patients and the associated percentage is significantly higher than those among the patients with first time urinary tract infections. this prospective study was conducted to elucidate and differentiate the bacterialvirulence factors associated with aln and apn in pediatric patients. methods: patients included in the present study were those suspected of anupper uti and underwent a systematic scheme of ultrasonographic, ct and tc m-dmsa evaluation for the differential diagnosis of aln andapn. exclusion criteria were any evidence of underlying diseases orurinary anatomical anomalies except vur. the e. coli isolates from the urine samples of patients were screened with pcr analysis for various urovirulence genes. pulsed-field gelelectrophoresis was used to analyze the genetic association of theisolates. results: a total of patients were enroled. forty-six patients were diagnosed as aln, while the other cases were apn. diverse genotypes were found among the e. coli isolates in either group. among the pathogenetic determinants examined, multivariate logistic regress analysis indicating that a papg ii allele was the only significant urovirulence factor associated with aln (p< . ; odds ratio, . ). conclusions: while no specific genetic lineage was identified among the e. coli isolates studied, a papgii gene was found strongly associated with the cause of aln among pediatric patients without underlying disease other than vur. -< yr during - were sent to leading paediatric nephrologists of asian countries/regions. those having national renal registry were to use the registry data. results: data from countries/regions were returned (incl. national registries), namely china, hong kong sar, india, indonesia, japan, malaysia, pakistan, philippines, singapore, south korea & thailand. a total of esrd patients were reported: on peritoneal dialysis (pd), on haemodialysis (hd), & transplant (tx) of %, % & % respectively. chronic pd: capd and automated pd (apd) were the main modes of pd at ratio of . to . only countries had apd morethan capd. peritonitis rate ranged from episode in to patient-months, and seemed less common in those having more apd. chronic hd: hd comprised of % chronic dialysis, mostly adolescents. a-v fistula wasused in %, and permanent catheter % for vascular access. background: current data suggest the role of chronic inflammation and lipid disorders in atherogenesis. the aim of the study was to evaluate established and new markers of atherosclerosis in apd and hd pediatric patients and to assess whether the method of dialysis has an impact on those factors. methods: soluble(s) e-selectin, il- and il- concentrations were evaluated by elisa in sera of apd patients on, hd patients and controls. hscrp levels were assessed by nephelometry. the lipid profile (total cholesterol (chol), hdl-chol, ldl-chol, triglycerides (tgl)) was also estimated. results: se-selectin concentrations in dialyzed patients were higher than in controls (apd p< . ; hd p< . ) and in apd were increased vs. hd (p< . ). there were no differences in median values of il- , il- and hscrp between examined groups. chol levels were increased only in apd vs. controls (p< . ). hdl-chol concentrations were decreased in all dialyzed patients when compared to controls (apd p< . ; hd p< . ), without difference between apd and hd. ldl-chol in apd and hd were higher than in controls (apd p< . ; hd p< . ), but failed to differentiate between two dialysis modalities. tgl levels behaved in the same way. conclusions: the elevated se-selectin concentrations in all patients show the role of endothelium in atherogenesis in ckd children. thus, the se-selectin augmentation may serve as an early marker of endothelial activation, appearing prior to inflammation (unchanged hscrp, il- ). increased seselectin and cholesterol levels in apd patients prove that children on peritoneal dialysis are more prone to atherosclerosis than those on hemodialysis. background: end-stage renal disease (esrd) is associated with an increased risk of cardiovascular morbidity and mortality. according to recent data, arterial stiffness measured by aortic pulse wave velocity (pwv) and augmentation index (aix) is a strong independent predictor of cardiovascular mortality in adult esrd patients. few studies have been reported regarding arterial stiffness in the paediatric renal population. methods: aortic pwv and aix (difference between the first and the second systolic peaks on the aortic pressure waveform divided by the pulse pressure) were determined in haemodialysis children ( boys; age ± years) by applanation tonometry using a sphygmocor device. seven of the hd patients ( boys; age ± . years) received a renal transplant (tx) and were restudied ( ± months post tx). the immunosuppressive regimen included basiliximab induction, cyclosporine, mycophenolate mofetil, and steroids. results: in the hd population, aortic pwv ( . ± . m/s) was correlated with age (p= . ), weight (p= . ), height (p= . ) and systolic blood pressure (p= . ). in the transplanted cohort aix decreased in six children out of seven after transplantation ( . ± . % on hd versus - . ± . % after tx). no significant change was observed for aortic pwv ( . ± . m/s on hd versus . ± . m/s after tx). objective: to study the pathogenic role of host and escherichia coli virulence factors in the development of e. coli febrile urinary tract infection (uti) in children with acute cystitis (ac), acute pyelonephritis (apn) and renal scar. materials and methods: isolates recovered from children consecutively admitted to the hospital with e. coli febrile uti that diagnosed as ac (n= ) or apn (n= ) were retrospectively enrolled into this study. virulence genes of e. coli, that included papg genes (classes i-iii), aer, cnf , fimh, hlya, afa, sfa/foc, iha, usp, irone and ompt, were detected by polymerase chain reaction analysis. results: young age (=< months), male sex were more frequently associated host factors for patients with apn, but old age (> months) and female sex were more frequently associated with renal scar formation. after multilogistic regression analysis, with regard to e. coli virulence factors, the papg class ii gene might play a more important role in the development of e. coli apn. however, iha was significantly higher in young children with acute pyelonephritis. afterwards, age, gender, duration of fever before admission, and crp level were considered as potential confounders for the further multivariate analyses, specifically estimating the relative risks of e. coli genotypes to the incidence of acute pyelonephritis and renal scar by age group and the existence of vesicoureteral reflux. odds ratios with % confidence intervals for each variable were utilized to estimate the relative risk of acute pyelonephritis and renal scar. in addition, there were no differences between young children and old children, if we excluded the factor of vesicoureteral reflux (vur). conclusion: both host and e. coli virulence factors contribute to the development of febrile uti, apn and renal scar. since / we have started an acute peritoneal dialysis (duration - days) in infants (age days to months) with the use of the baxter acute set for children under constant warming of the complete dialysate inflow tract to °c (barkey system). as dialysate solutions we chose a glucose/bicarbonate/lactate solution (physioneal ) in all patients and in patients a mixture of this solution with a . % amino acid solution (nutrineal) in a : ratio. the body weight before the renal insufficiency was . to . kg. in patients the renal failure followed cardiothoracic surgery. the handling of the system was easy. because of obstruction by omentum and fibinous layers, respectively, the dialysis catheter had to be cleared surgically in patients. body temperature could be kept constant and in the normal range, even with low body weight and intensive dialysis (as measured by dialysate volume per kg body weight and day). in the infants dialyzed with the glucose/amino acid-mixture a decreased loss of albumin through the dialysate (as measured by the necessary intravenous albumin substitution), a better glucose homeostasis (less episodes of hyperglycemia and less need for insulin infusions) as well as a better acid-base control (less episodes of metabolic alkalosis) could be found. the detected tendency to a better homeostasis (concerning body temperature, serum albumin, blood glucose and acid-base) with this dialysis system and the used dialysate solutions could help to increase the survival chances of infants with renal failure. this will be evaluated prospectively. objectives: varicella-zoster-virus (vzv) infection can cause significant morbidity and mortality in the immunocompromised patient. since there is no clear correlation between antibody titers and protection monitoring after vzv vaccination is unclear. patients and methods: serum samples of nineteen pediatric transplant recipients were investigated for vzv igg antibody titers and avidity (elisa test). a relative avidity index (rai) < % showed evidence for low-avidity antibodies, an rai > % high-avidity antibodies, borderline avidity inbetween. the control group consisted of healthy children. had suffered from varicella infection after wild-virus contact, had undergone varicella vaccination. as there was no difference between diseased and vaccinated controls both subsets were treated as one group. results: median vzv igg antibody titers were u/ml (range - ) for transplanted children and u/ml (range - ) for control subjects (n. s.). median rai was % for transplant patients and % for controls (p= , ). there was no correlation between rai and antibody titers in either group. rai increased significantly with time after vaccination or infection in both groups. despite protective antibody titers after vaccination and an rai of % one transplant recipient showed a moderate vzv infection that required antiviral treatment. postinfectious course showed an increase of rai up to nearly %. conclusion: vzv igg antibody avidity might be a pathbreaking parameter regarding decision making for a second vaccination before transplantation or preemptive treatment in a transplanted patient in case of exposure. in japan, almost half of children with esrd received renal transplantation and more than % of those were living kidney transplants from their parents. burden of esrd during childhood frequently causes psychosocial problems in their families, including parental relationship problems. we investigated how parental divorce or death affected the choice of renal transplantation in children. patients and methods: in children younger than years old who started renal replacement therapy in our hospital, percentages of the children losing a parent or parents by divorce or death were investigated. we compared renal transplantation rates and percentages of fathers as kidney donors between those living with both parents and those without. results: in observation periods ( . ± . years), ( %) received renal transplants from living and deceased donors. nineteen children ( %) lost a parent or parents by divorce (n= ) or death (n= ). in all divorced families, mothers got parental authority. ten parents divorced during ckd period, after start of dialysis, and after transplant. of children living with both parents, ( %) transplanted with kidneys from fathers, mothers, and deceased donors. of children whose parents divorced, ( %) transplanted from fathers ( just before and after their divorce), mothers and other family members. however, in children at least one parent died, only ( %) transplanted from a deceased donor. conclusion: a high parental divorce rate from ckd period was observed in children with esrd, suggesting burden of the disease on their families. renal transplantation was preferred even in divorced families and divorced fathers still were willing to donate kidneys to their children. objective of study: to determine the importance of gastrointestinal evaluation in pre-transplantation phase in pediatrics with end stage renal disease (esrd). methods: twenty four children with esrd ( female, male) mean age . (± . ) years on maintenance hemodialysis were included in this study. upper gastrointestinal endoscopies were performed and four gastric antral and duodenal biopsy specimens were obtained for urease test and histological study for all patients. serum gastrin levels were measured in all patients, too. a control group was chosen to compare the rate of h. pylori infection using student's t. test. results: gastrointestinal symptoms were present in ( %) of patients. seventeen ( %) patients had abnormal upper gastrointestinal endoscopic findings. h. pylori was detected in % of patients and % in control group (p< . ). in symptomatic patients % had abnormal endoscopic findings and % had positive urease test for h. pylori infection. while, in asymptomatic cases these rates were % and %, respectively. seventy one percent of patients with gastrointestinal lesions and % of patients with normal endoscopic examination were infected. high serum gastrin levels in infected and non-infected patients were detected in % and . %, respectively (p< . ). conclusion: we demonstrated a significant number of patients with peptic ulcer diseases and h. pylori infection and secondary hypergastrinemia. this study showed that, clinical symptoms are not a reliable predictor of gastrointestinal problems. our results emphasize the importance of periodic, and also pre-transplant gastrointestinal evaluation in these patients to find out their problem and manage appropriately. key words: renal failure, hemodialysis, peptic ulcer disease, helicobacter pylori, hypergastrinemia. background: preservation of a stable allograft function in children following renal transplantation (rtx) depends on various factors including genetic variability. the gene of the angiotensin i converting enzyme (ace) has been shown to influence allograft function. we therefore analysed various polymorphisms of the renin-angiotensin system in children following rtx and kidney donors and associated genotypes with loss of renal function. patients and methods: children and adolescents ( male, female, mean age at transplantation . ± . years) with stable renal function and observation period exceeding months were included. mean follow-up time was . years ( . to years). dna was extracted from all recipients and donors and genotyped using rflp. the following polymorphisms were studied: renin g/a; ace i/d; angiotensinogen (agt) met/thr and angiotensin ii receptor type- (at r) a/c. the slope of glomerular filtration rate (gfr) was determined by linear regression analysis and correlated with the genotype. results: allelic frequencies were not different from healthy controls. genotypes of renin, agt and at r showed no significant association with the slope of gfr but patients homozygous for the ace-d-allele had a significantly steeper decline of gfr when compared to homozygous carriers of the ace-i-allele (slope dd: - . ± . vs. ii: - . ± . ; p= . ). the dd-genotype was also present in out of donors and in four cases a dd-recipient received a kidney from a dd-donor. those four patients showed a more pronounced decline of gfr (- . ± . ; p= . ). in addition, dd-recipients had a significantly increased systolic and diastolic blood pressure before rtx. conclusions: the dd-genotype is associated with a faster, non-immunological loss of graft function which has to be evaluated in prospective studies. year large single-center review b. warshaw, l. hymes, l. greenbaum, s. amaral from - , children received renal transplants at emory university/children's healthcare of atlanta of whom ( %) had nephroticsyndrome (ns) as their primary diagnosis ( fsgs, minimal change). all children received calcineurin-based immunosuppression. thirteen children ( %) developed recurrent ns within the first week post-transplant and received plasmapheresis (pp) - times weekly. nine ( %) had complete resolution of proteinuria. two who responded to pp suffered graft loss fromlate recurrences of ns at years. ns did not resolve with pp in children who suffered either delayed function ( ) or early cessationof function ( ); each of the latter lost their grafts within the first months. patients did not have recurrences of ns. comparison of these groups showed significantly increased riskfor recurrence with younger patient age (p< . ), interval < years from onset of ns to esrd (p< . ), and living donor source ( / = % ld vs / = % dd; p< . ). no differences were seenfor hla match, donor age, gender, or african american race. actuarial graft survival for children with recurrence was % at year and % at years vs. % & % for patients without recurrence. conclusions: ns recurred in % of children with ns as their primary cause of esrd. risk factors for recurrence included younger age, interval < years from onset of ns to esrd, and living donors. most recurrences responded to pp ( %); failure to respond was associated with delayed or early cessation ofrenal function & early graft loss. the incidence of recurrence was strikingly high ( %) among living donor recipients, suggesting a need to explore prophylactic strategies such as preemptive pp in this group. methods: a single-centre retrospective case-controlled study including children < yr (g ) and matched kidney tx recipients older than yr ofage (g ). patients were matched for donor type and tx period. kaplan-meier method was used for patient and graft survival. results: tx were performed using deceased donors in both groups. median recipient age, weight and height at tx in g were . [ . - . ] yr, . [ . - . ] kg and . [ . - . ] cm, respectively, while median age in g was . [ . - . ] yr. hypoplasia-dysplasia wasmore frequent in g ( vs %). median hla-dr mismatch, time ondialysis and number of blood transfusion before tx were not different. in g , kidneys were placed intraperitoneally and most of vascular anastomoses were done on distal aorta ( %) and inferior vena cava ( %). median follow-up was . [ . - . ] yr and . [ . - . ] yr ing and g , respectively. patient survival was % at yr and % at yr in g ; patients died in g ( ptld, recurrence of primary disease), none in g . fiveyear graft survival was % in g and % in g . acuterejection episodes occurred in % in g and in % in g (p . ). chronic rejection led to late graft losses in g ( . to . yrafter tx) vs in g . renal function did not differ between the groups during the first yr post tx. the average height gain was better in g at yr post tx (+ . sds vs. - . ). primary disease recurrence was observed in cases ( in g ) causing graft lossin cases. two arterial thromboses were observed in g causing earlygraft-loss. conclusion: the outcome after cadaveric kidney transplantation is as good in children under yr of age at transplantation as in older recipients in our experience. c. garcia, v. bittencourt, d. malheiros, a. tumelero, j. antonello, a. oliveira, v. garcia cancer is an increasingly recognized problem associated with immunosuppression. recent reports, however, suggest that sirolimus (srl) has anti-cancer properties that could address this problem. aim: to report a retrospective analysis of preliminary results of patients who received srl because of post-transplant de novo malignancies in a consecutive cohort of pediatric kidney recipients. patient and methods: we retrospectively evaluated the efficacy and safety of srl in pediatric renal transplantation recipients, who were ± years when converted to srl. the post-transplant de novo malignancies were: gall bladder and hepatic leiomyoma (n= ), wilms tumor in the native kidney (n= ), ptld (n= ) and hpv-associated neoplasia. the immunossupressive regimen at the malignancy diagnosis was tacrolimus/cyclosporin, mmf/azathioprin and prednisone. all were converted to double immunossupression with sirolimus at a standard dose of mg/day (tl - ng/ml) and prednisone. patients with ptld were also treated with rituximab, and the patients with wilms tumor received chemotherapy. mean follow-up after srl conversion is ± months. results: all patients were maintained with srl and pred without rejection, with good renal function and no cancer recurrence (follow-up to months). the patients with wilms tumor are still on chemotherapy. follow-up control after srl conversion in the patients: conclusion: although the intraperitoneal group had characteristics associated with increased surgical risk (they tended to be younger and smaller, with a higher incidence of aortic anastomoses and a higher incidence of multiple vessels), surgical complications were significantly lower than expected in the extraperitoneal group % v. %. objective of study: proteinuria is a frequent complication in adult patients after renal transplantation (r-tx) and is associated with poor graft survival. in children, there are no studies focusing primarily on proteinuria after r-tx. the aim of this study was to investigate the prevalence of proteinuria in children after r-tx and to evaluate changes of proteinuria during a -yr study on intensified antihypertensive therapy. methods: protein excretion was measured in -hr urine and proteinuria defined as > mg/m /day. proteinuria was investigated at baseline, and years after intensifying of the antihypertensive therapy in children with uncontrolled hypertension at baseline (i. e. additional antihypertensive drugs given to children with blood pressure > . pc). children ( . ± . yrs) out of from our center fulfilled inclusion criterias (> months after r-tx, no acute rejection (ar) in the last months, no recurrent fsgs). results: the prevalence of proteinuria was % at baseline, % after year (ns) and it decreased to % after years (p< . ). the mean protein excretion was ± mg/m /day at baseline, ± after year (ns) and it decreased to ± after years (p< . ). mean number of antihypertensive drugs increased from . ± . drugs/patient to . ± . after years (p< . ). mean nighttime bp decreased significantly after years. the number of patients on ace-inhibitors increased from % at baseline to % after years (p< . ). conclusions: this is the first study on proteinuria in children after r-tx. it showed that proteinuria is a frequent finding in transplanted children and that intensified long-term antihypertensive treatment using ace-inhibitors can decrease not only bp but also proteinuria in these patients. allograftrejection involves t cell activation and proliferation and multipleinflammatory components. monocyte chemotactic peptide- (mcp- ) is achemoattractant and activating factor for monocytes. interleukin- (il- ) may contribute to monocyte recruitment, results intubulointerstitial damage. cytotoxic lymphocytes induce target cell death by ligation of the fas-fasligand. allelic polymorphisms in recipient genes coding for them reported to beassociated with variations of outcome in renal transplantation. the aim was to investigate impact of mcp- - a/g and il - g/c, fas- a/g polymorphisms on acute allograft rejection (ar). there were males and females, ± . years meanly; of the graftscame from living-related donors, were from cadavers. the controlgroup consisted of unrelated, healthy individuals with similar ageand sex. ar group was composed by patients experienced at least one ar episode within the first months of transplantation. the non ar group was comprised by kidney transplant patients without ar. there was no significant difference between renal transplant patients and healthy controls in genotype distribution of allelic frequencies of il- , fas and mcp- polymorphisms. while il- and fas gene polymorphisms had no effect on the incidence of ar episodes, there was significant association with mcp- . the distribution of the genotypes for mcp- - a/g in ar group were aa/ag/gg , %, , %, , % respectively. the distribution of the genotypes for mcp- - a/g was aa/ag/gg %, %, % in nonar group respectively. the carriage of g allele at - position of mcp- gene has a significant association with ar (or: , , %, ci: , - , ). the il- and fas gene polymorphisms had no effect on the incidence of ar. mcp- - g allele carriage increases the ar risk in turkish renal transplant patients. the high recurrence rate of focal segmental glomerulosclerosis (fsgs) in transplanted kidney recipients suggests the hypothesis that such patients have a circulating factor that changes glomerular capillary permeability. serum from patients with fsgs increases glomerular permeability to albumin, and this permeability factor was partially identified as a protein. the removal of this protein by plasmapheresis (pp) decreases proteinuria. object: the aim of this paper is to provide data about the therapeutic effect of pp in fsgs children with recurrence in the transplanted kidney. methods and results: twenty eight pediatric kidney transplant recipients had fsgs as cause of renal failure from to in our center, confirmed by biopsy pre-transplant. seventeen of these ( . %) had a recurrence (proteinuria > g/m per day associated with hypoalbuminemia). the mean age was ± . years, , % were caucasians and , % were performed with living donor. since , patients who presented fsgs recurrence were treated with cycles of pp ( cycles/weekly), initiated immediately post-recurrence (n= ). immunosuppression comprised of cyclosporin in high doses (c levels of - ng/ml) or tacrolimus (tl= mg/dl), mycophenolate sodium or mofetil (until azathioprine was used) and prednisone. among patients who received pp (n= ), ( . %) achieved a complete remission. there were no cases of remission among those six patients who were not treated with pp. those who achieved remission after pp had no recurrence. the patients treated with pp had infectious complications: one patient had cytomegalovirus disease and two patients had varicella. conclusion: pp appears to be effective in treating recurrent fsgs following kidney transplantation. it should be started as soon as possible. because the calcineurin inhibitors (cni) cyclosporin a (csa) and tacrolimus (tac) are drugs with a narrow therapeutic index, individualization of cni dosage by therapeutic drug monitoring is indisputable. however, the optimal strategy for monitoring cni therapy is currently under debate. dosing of cnis according to the molecular effect of the drug on its target cells could optimize immunosuppressive therapy with cnis. for this purpose, we developed a reliable, precise, and robust whole blood assay based on the measurement of the expression of three nfat-regulated genes (il- , ifng and gm-csf) in pma/ionomycin-stimulated lymphocytes before and . (tac, c . ) or hrs (csa, c ) after oral drug intake. the inhibition of genes in this assay is independent from other commonly used immunosuppressive drugs and reflects calcineurin inhibition expressed as residual nfat activity. in a pilot study, patients (mean age yrs, mean time period posttransplant mo.) were analyzed. in csa-treated patients (n= ), a mean c concentration of ng/ml (range, - ng/ml) corresponded to a mean residual nfat-activity of % (range, - %) ; the correlation between individual residual nfat-activity and c was moderate (r= , , p< . ). at a csa-c of ng/ml, the residual nfat-activity varied between % and %. in tactreated patients (n= ), a mean c . concentration of ng/ml (range, - ng/ml) corresponded to a mean residual nfat-activity of % (range, - %); the correlation between individual residual nfat-activity and c . was also only moderate (r= , , p< . ). conclusion: these data indicate that there is a considerable inter-patient variability of residual nfat-activity at a given maximal cni blood concentration. ongoing studies are validating this assay regarding clinical outcome criteria of immunosuppression such as acute rejection and infections. hus due to antibodies against factor h (husafhab) is a very rare disease for which a limited experience in its management is available. we present a case of husafhab who was transplanted but lost her graft after only mos. in aug a -mos old child was admitted for a d-hus which did not go into remission and required chronic peritoneal dialysis. no fh and mcp gene mutation were detected nor adamst- activity was decreased. afhab were not searched at that time. in apr. the child underwent cadaver renal transplant (rtx). the immunosuppressive regimen was basiliximab-prednisone-cyclosporine-mycophenolate mofetil. fifteen days after rtx, following surgery for urinoma, the child exhibited an hus relapse with thrombocytopenia, haemolytic anemia and increased serum creatinine (scr). high afhab were detected ( au/ml). four plasma exchanges (pe) were performed over wks with a drop of afhab to control level ( au/ml) and a remission of hus. until the end of aug, afhab remained low (< au/ml) and the child was well (scr range: . - . mg/dl). in early aug, the patient was shifted to tacrolimus for severe hypertrichosis with a fluctuation of its plasma level (lower recorded value: . ng/ml) signs of acute rejection developed and metilprednisolone (mtp) pulses brought scr to baseline level. in oct. , following an urti, the child presented with severe proteinuria (upr/ucr: , ) without other clear signs of hus recurrence. since afhab were increased ( au/ml), pe was restarted but an acute hemorrhagic complication (hemotorax) occurred and pe had to be interrupted. septicemia followed and the child became anuric. the renal biopsy performed days later showed signs of glomerular and vascular thrombotic microangiopathy. renal function did not recover despite mtp pulses and pe. in nov the child was back to pd and in dec the graft was removed. background & aims: recently, the role of nitric oxide (no) in the pathogenesis of idiopathic nephrotic syndrome (ins) has been intensively investigated. however, its rapid turnover has made us impossible to investigate the quantity and the source. as we have developed a novel method for quantitative analysis for no by a new fluorescent indicator, , -diaminofluorescein (daf- ), its amount produced by both t and b lymphocytes in ins was studied. methods: five children with steroid-sensitive ins (mean age: . y) were included in this study, together with children with other renal diseases (mean age: . y) such as chronic glumerulonephritis and alport syndrome with significant amount of proteinuria, and healthy adults (mean age: . y) for the control. no production from cd + cell and cd + cell was investigated by a flow cytometry using daf- . results were expressed as mean fluorescence intensity and were compared among the groups. results: the amount of no produced by cd + cell and cd + cell in children with ins was significantly greater than those in children with other renal diseases and healthy adults (cd + cell: . ± . [mean±sd], . ± . , and . ± . , respectively; cd + cell: . ± . , . ± . , and . ± . , respectively, p< . ). additionally, both cd + cell and cd + cell during nephrotic relapse produced more no than in nephrotic remission (p< . ). discussion: patients with relapsing ins showed increased production of no by both t and b lymphocyte. these findings indicate that no plays some role in the pathogenesis of ins and suggest that an abnormal immune system may exist not only in t but also in b lymphocytes. a. bagga, a. sinha, s. menon, p. hari aim: the treatment of patients with srns is challenging. based on suggestions that b-lymphocytes are crucial in the pathogenesis of nephrotic syndrome, we examined the efficacy of rituximab (rtx) in patients with srns refractory to standard therapies. methods: six patients ( with initial, late resistance), - yr old, were included; biopsy showed minimal change & focal segmental glomerulosclerosis in each. all had previously received iv high-dose steroids, alkylating agents & calcineurin inhibitors (cni) for - yr with periods of partial (pr; urine - +) or complete remission (cr; urine trace/negative). all now had srns refractory to -months treatment with cni. rtx ( mg/sq. m) was infused iv every week for weeks. therapy with cni and/or alternate-day prednisolone, & cotrimoxazole prophylaxis was continued. patients were monitored for proteinuria and renal functions. results: at a median interval of wk following the last rtx dose, cr was seen in & pr in patients. remission was sustained in patients, despite tapering doses of steroids & cni. one case had relapse of nephrotic syndrome -months later, which responded to steroid treatment. at median follow-up of wk, cr, pr and recurrence of nephrotic proteinuria ( - +) were seen in , and patients respectively. mean urine albumin-to-creatinine ratio was . at baseline and . at followup; respective blood levels of albumin were . and . g/dl & cholesterol and mg/dl (all p< . , anova); difference in leukocyte counts and levels of igg were not significant and none had serious infections. conclusions: this is the first report on the efficiacy of rtx in sustaining remission in patients with srns. therapy with this agent appears promising for difficult srns, with a better risk/benefit profile than other medications. quantitative or functional deficiency of factor h results in uncontrolled complement activation and is an important cause of familial hemolytic uremic syndrome (ahus). factor h-related proteins (fhr) constitute a protein family which share structural and most likely functional similarities to factor h. we here describe complete deficiency of fhr- /- as novel cause of ahus. factor h and fhr- /- were quantified by elisa and were further analyzed by western blot using specific antibodies. complement activation was determined by measuring c and c . serial hgb (g/dl), platelet, creatinine (mg/dl), and ldh (u/l) were measured. a year old girl presented with a day history of lethargy, pallor, vomiting and hypertension. hgb was g/dl, platelets x /l and creatinine was . mg/dl. initial therapy consisted of packed red cell and platelet transfusion, followed by steroid therapy. representation occurred weeks later with hypertension, edema, persistent anemia thrombocytopenia and renal dysfunction (creatinine . mg/dl). renal biopsy demonstrated features of chronic thrombotic microangiopathy. low c levels indicated activation of the complement system. while western blot analysis showed normal factor h level, fhr- /- was absent. by repetitive plasma infusion and plasmapheresis, the cycle of hemolysis and thrombocytopenia could be disrupted. chronic periodical plasma infusion q days resulted in regression of renal impairment to a degree (current baseline creatinine . mg/dl). in conclusion, fhr- /- are thought to have co-factor activity and play a role in complement activation in ahus. deficiency of fhr- /- may lead to a subclinical form of ahus such that patients may initially present with features of chronic renal failure. however, factor replacement therapy may lead to regression of renal impairment. s. choudhry objectives of the study: the aim of the study is to investigate the long term prognosis ofsevere childhood iga nephropathy after years combined therapy. patients: we examined patients who had entered thejapanese pediatric iga nephropathy treatment study group between and , had been treated with years combined therapy (combinationof prednisolone, azathioprine, dipyridamole and heparin/warfarin) andhad been followed for more than years after the therapy. significantproteinuria is defined as more than . g/m /day. results: mean age at onset was . years ( - years), proteinuria at diagnosis was . ± . g/m /day, proteinuria after the combined therapy was . ± . g/m /day and mean follow-up period after the combined therapy was . years ( . - . years). proteinuria improved in all patients during thecombined therapy (p< . ). the final prognoses were as follows: patients ( %) showed no proteinuria, ( %) showed proteinuria withnormal renal function and ( %) proteinuria with decreased renalfunction. we compared the clinical and pathological parameters betweenpatients with proteinuria (n= ) and those without proteinuria (n= ) at the last observation. period between disease onset and start of treatment, glomeruli showing crescents before the combined therapy (%) and glomeruli showing pathological changes after the combined therapy (%) were the significant risk factors for the proteinuria at the last observation. logistic multivariable analysis revealed that glomeruli showing pathological changes after the combined therapy (%) was theonly independent risk factor for the proteinuria at the last observation. conclusions: pathological activity of nephritis at the end of the combined therapy might correlate well with the final proteinuria and the long term prognosis. s. arun, a. bagga, s. bhatnagar, p. hari, s. menon, s. saini aim: relapses in ssns of ten follow minor infections and are associated with perturbed t-cell function. based on data that zn supplements modulate t-cell function and reduce risk of infections, we examined its efficacy in reducing relapses in patients with ssns. methods: in this double blind rct, consecutive patients with ssns - yr old, stratified into frequent (fr= ) and infrequent (ifr= ) relapsers, were randomized to -months therapy with zn ( mg daily) or placebo. patients with fr also received long-term, alternate day prednisone. relapse and infection rates were monitored monthly. blood levels of zn, sil r, il and interferon (ifn) were measured at baseline, relapse andend of study. results: patientsin the zn (n= ) and placebo (n= ) groups had similar baseline clinical & laboratory features. the former showed % lower frequency of relapses (difference in means - . ; % ci - . , . ) with trend towards reduction from -months onward. a higher proportion ( . %) of patients in the zn group had sustained remission compared toplacebo ( . %). reduction in relapse rates was higher in fr receiving zn vs. placebo (- . ; ci - . , . ); respective sustained remission was seen in % and % patients (relative risk . ; ci . , . ; p . ). no differences were found in infection rates, and levels of zn, sil r & il ; levels of ifn were higher in those receiving zn compared to placebo (p= . ). conclusions: zn supplementation for -yr was effective in maintaining remission and reducing relapse rates. the effect was mediated not by reducing infections but perhaps through effect on th cytokines. while zn therapy appears promising, these results need confirmation in patients with frequent relapses and perhaps at a higher dose. background: n-glycosylation process in the endoplasmic reticulum (er) is tightly regulated and orchestrated by many factors such as chaperones and energy systems. we showed that adequate nglycosylation is crucial for nephrin to assemble to the plasma membrane (jasn, ) . additionally we recently demonstrated that the er stress evoked by glucose starvation induces hypoglycosylated nephrin retained in the er, which is rescued by dexamethasone (dex) (ki, ) and immunosuppresant: mizoribine (mzr) (submitting). in the present study, we tested whether other er stress inducer, hypoxia, could also interfere the alteration of nephrin n-glycosylation system and whether dex and immunosuppressants rescue its defective process. methods: nephrin-expressing cell line was cultured either in % o or % o for hours in the presence or absence of dex, mzr and cyclosporin a (csa), followed by western blot analysis with nephrin, cytochrome c. intracellular atp concentrations were measured by the highperformance liquid chromatography. protein expression of cyclophilin d (cyp-d), a component of mitochondrial permeability transition pore (mptp), was tested in the samples from human glomeruli and cultured podocyte. results: hypoxia induced hypoglycosylated nephrin. csa, but not dex and mzr, inhibited the formation of this hypoglycosylated nephrin and rescued mature form. csa inhibited the increase of cytochrome c in the cytoplasm caused by hypoxia. in addition, csa partially rescued the decrease of intracellular atp. cyp-d was distinctly observed in human glomeruli and located in podocytes. conclusion: csa inhibit the formation of hypoxia-induced hypoglycosylated nephrin through protecting the mptp opening via selectively binding to cyp-d in the mitochondria, resulting in a recovery of atp. csa may exert direct action on the alteration of nephrin biogenesis induced by the er stress. background and objectives: in two previous randomized controlled trials (rcts) we showed that treatment of severe childhood iga nephropathy (iga-n) with diffuse mesangial proliferation using prednisolone, azathioprine, heparin-warfarin, and dipyridamole reduced immunologic renal injury and prevented any increase of sclerosed glomeruli. in one of the two rcts we also showed that treatment with prednisolone alone did not prevent a further increase of sclerosed glomeruli. accordingly, the immunosuppressant is considered to play an important role in the combination therapy. often however, we were unable to complete the azathioprine regimen due to its severe side effects. therefore we considered that a different, but effective immunosuppressant would be worth trying. mizoribine, like azathioprine, is an antimetabolite that exerts its immunosuppressant effect by inhibiting lymphocyte proliferation. design, setting, participants, and measurements: in this pilot study, we administered mizoribine instead of azathioprine as part of the combination therapy for treatment of children with severe iga-n and evaluated the efficacy and safety of the regimen. results: eighteen patients reached the primary endpoint (urinary protein/creatinine ratio < . ) during the two-year treatment period. the cumulative disappearance rate of proteinuria determined by the kaplan-meier method was . %. mean urinary protein excretion was reduced from . g/m /day to . g/m /day (p< . ). after treatment, the mean percentage of glomeruli showing sclerosis was unchanged in comparison with that before treatment. no patients required a change of treatment due to side effects. the efficacy and safety of the mizoribine combination seems to be acceptable for treatment of children with severe iga-n. objectives of study: alport syndrome is a hereditary renal disease which is normally diagnosed by either histopathological studies or a genetical analysis. we attempted to diagnose alport syndrome by means of immunofluorescence staining of cultured cells with collagen type alpha chains obtained from voided human urine specimens. methods: the cells were cultured from the voided human urine of patient with x-linked alport syndrome, patient with sporadic alport syndrome, and patient with autosomal-dominant alport syndrome. for comparison purposes, controls cells were cultured from the voided human urine of patient with iga nephropathy, patient with fsgs, and patient with purpura nephritis. the cultured cells were stained by immunofluorescence techniques with collagen type alpha , , , , and chains. results: in the cultured cells of the controls staining for collagen type alpha and chains were observed both in the extracellular matrix and in the cytoplasm while for the staining of collagen type alpha , , and chains were observed in the cytoplasm. in cultured cells of xlinked and sporadic alport syndrome staining of collagen type alpha chain was lost or attenuated. in the cultured cells of autosomal-dominant alport syndrome the staining patterns of collagen type alpha , , and chains were observed. the staining patterns of the collagen type alpha chain in cultured cells of alport syndrome correlated with that observed in renal biopsies obtained from alport syndrome patients as reported previously. hence, the staining of cultured cells with the collagen type alpha chain obtained from voided human urine may therefore be a potentially useful means of diagnosing alport syndrome in a non-invasive manner. the aim of our study: was to examine zeta chain expression in cd +, cd + t cells and nk cells from ins children in active phase of the disease and in remission and to assess the effect of h and h anti-cd + ril- stimulation on zeta chain expression. we enrolled ins children in relapse before initiating the therapy, ins children in complete remission of proteinuria on prednisone for - weeks ( mg/kgbw/d) and age-matched controls. zeta expression was determined by flow cytometry as mean fluorescence intensity (mfi). results: cd +cells: mfi in relapse was higher than in remission and in controls. anti-cd + ril- stimulation had no impact on zeta expression in acute phase and in cotrols, but increased mfi values after h in pts with remission. cd +cells: zeta expression stayed unchanged irrespective of the examined group or antibody stimulation. nk cells: there were no differences between mfi values before stimulation and in relapse after stimulation. in remission and in controls antibody stimulation decreased zeta expression. in controls, mfi values for nk cells were higher than those for cd + and cd + populations, but this preponderance disappeared after h stimulation. in remission, that nk cell predominance vanished after h stimulation. in relapse, mfi values in all cells were comparable and stimulation had no impact on lymphocyte proportions. urinary protein, creatinine, and scd were measured. scd was measured using a elisa kit. scd was expressed as ng/g of urine creatinine. data were analyzed using anova and spearman rank correlation (src) tests. ) scd urinary concentrations in patients with mlns in relapse were higher than patients in remission, healthy controls and other patients with proteinuria (anova p . ) but similar to those seen in sle. ) in patients with mlns relapse, scd concentration did not correlate with urine protein/creatinine ratio (src, r: . , p: . ) ) in patients with mlns, serial urine scd concentrations were measured over a period a time. scd was increased at the time of relapse and decreased toward normal range weeks before patients went into remission. conclusion: urinary scd is elevated in mlns patients in relapse. this increased urinary concentration is not due to the proteinuria, since scd was not elevated in other patients with glomerulopathies and proteinuria. upregulation of podocyte scd during relapse may play a role in the pathogenesis of proteinuria in mlns. aim: patients with mpgn type ii/dense deposit disease (ddd) and atypical haemolytic uremic syndrome (ahus) secondary to defective complement control are found to have a high prevalence of y h polymorphism of factor h gene (cfh), which is linked to age-related macular degeneration (amd). however, no studies have looked into an ocular phenotype of children with complement based renal diseases, yet. methods: in two pediatric nephrology centres all patients with mpgn ii/ddd and ahus were identified and screened prospectively for the presence of y h polymorphism and drusen maculopathy. all patients have been on immunomodulatory therapy at the time of screening. results: four children were identified (male:female= : ) with mpgn ii/ddd and ahus. there were two children in either group. the median (range) age at examination was . years ( . to . years). of the four patients, all were found to have the y h polymorphism. none of the patients had evidence of drusen at the time of examination. two patients with mpgn ii/ddd and one patient with ahus had additional factor h mutations, which were thought to be responsible for the renal disease. conclusion: in our study of children with mpgn ii/ddd and ahus we found a % prevalence of the y h polymorphism of factor h gene, which puts these patients at higher risk for drusen maculopathy. therefore, all children with either mpgn ii/ddd or ahus should be screened for y h polymorphism; and if found positive have regular follow-up ophthalmologic examination. in the future, should y h be proven to be of functional significance with respect to complement control, there would be a role for plasma infusion or replacement of purified factor h for the treatment of both the renal and the ocular phenotype. we previously demonstrated that angiotensin-( - ) is an endogenous ligand for the g-protein coupled receptor mas. the aim of this study was to evaluate the role of angiotensin-( - ) mas receptor in kidney structure and function by using transgenic mice with genetic deletion of the receptor mas. mas -/-(knockout) mice were compared to mas +/+ (wild type) mice regarding renal function parameters and kidney histology. the animals were housed in metabolic cages to obtain -hour urine samples for measuring urinary volume, osmolality and microalbuminuria. at the end of the experiment, mas -/-and mas +/+ mice were sacrificed by decapitation to harvest the kidneys for histological analysis and immunofluorescence of collagen types iii and iv. the quantification of collagen expression was determined by confocal microscopy (zeiss lsm ). urinary volume was significantly reduced in mas -/-mice as compared to mas +/+ animals ( . ± . vs. . ± . ml/ hs in mas +/+ mice, p< . ). this change was associated with an increase in urinary osmolality ( ± vs. ± mosm/kg in mas +/+ mice, p< . ) and albumin excretion ( . ± . vs. . ± . mg/ hs in mas +/+ mice, p< . ). the histological analysis showed a significant reduction in the glomerular diameter in mas -/-mice as compared to mas +/+ animals ( . ± . vs. . ± . μm in mas +/+ mice, p< . ), where as any significant change was observed in tubular diameter. the immunofluorescence of mas -/-kidneys revealed a marked increase of the expression of collagen types iii and iv in periglomerular region as well as in external and internal medulla. collagen iv was also augmented in mesangial area of mas -/-mice. these results suggest that the receptor mas is critical for the regulation of renal structure and function. a circadian rhythm in calciuria is evident with a peak after : h, a lower excretion overnight and a nadir in the early morning. during the peak period both ca/creat ratio (figure) and calcium output frequently surpass the reference values of . mg/mg and mg/kg/day respectively. conclusion: a significant circadian variation in calciuria is evident in normal school age children, with peaks surpassing commonly used reference values for hypercalciuria. this peak excretion is independent from urine output, glomerular filtration and intake. the average h excretion of mg/kg is rarely surpassed despite this peaks. the management of children with secondary hyperparathyroidism is complicated and should start early in the course of renal insufficiency. in spite of an optimal management hyperparathyroidism is sometimes uncontrolled and calcimimetics like cinacalcet hydrochloride which directly stimulates calcium sensing receptors and potently suppress pth secretion are an alternative to parathyroidectomy. they are very promising agents, but paediatric experience is lacking. a years old girl with a bardet biedl syndrome without medical care came last year with end stage renal failure. thanks to daily hemodialysis, serum phosphate level was kept within normal limits and phospho-calcic product remains below . as recommended. in spite of this treatment, serum pth level increased to ng/l. this toxic hyperparathyroidism with optimal monitoring of serum calcium, phosphate, vitamin d levels led us to start calcimimetics. a , mg/kg cinacalcet dose was administrated and induced a decline of pth level to ng/l one month later. a years old boy with recessive polycystosis and chronic renal failure was treated with calciumbased phosphate binders and sevelamer hydrochloride but with a poor compliance and a free diet which led him to hyperparathyroidism (pth to ng/l). in addition, a parathyroid gland hyperplasia with two adenoma was individualized. start of calcimimetics treatment (less than mg/kg) led to a rapid decline in pth level from ng/l to ng/l. the two adenoma decreased in size. in case of hyperparathyroidism, with close monitoring of serum calcium, phosphate, alkaline phosphatase and vitamin d levels, calcimimetics are an excellent alternative to surgery, with safe use and low dose in these cases. preliminary favourable experience has been reported in children but paediatric experience is lacking. side effects result from chronic administration of steroids. the aims of this study are to analyze graft function and metabolic effects of low dose and withdrawal of steroid therapy. this is a single center pilot, one arm and prospective study. methylprednisone (mp) was decreased to . ± . mg/kg/day (low dose) after months. we studied changes in graft function, height velocity, lipid profile, body composition and bone mass after year of low dose mp and after a nd year following mp withdrawal. patients received daclizumab induction; tacrolimus and mycophenolate mofetil. the inclusion criteria was st living related graft and pra < %; patients were enrolled and total follow-up was . years. age at transplantation was . - years, females, prepubertal, postpubertal patients. patients and graft survival were %, acute rejection (ar) occurred after months of mp withdrawal in / ( %) prepubertal patients (banff , b). in prepubertal patients, at the moment of steroid withdrawal and year later creatinine clearance was . ± . and . ± . ml/min/ . m , p< . ; height velocity . ± . and . ± . cm/year, p: ns, with a height increment of . ± . sds, p< . during the last year of follow-up. graft function did not change in postpubertal patients. in all patients at the moment of steroid withdrawal and year later lipid profile was normal; fat body mass . ± . and . ± . kg, p: ns; lean body mass . ± . and . ± . kg, p< . ; total skeleton bmd - . ± . and - . ± . sds, p: ns and lumbar spine bmd - . ± . and - . ± . sds, p< . . this study demonstrates that low dose and steroid withdrawal allow catch up growth, normal lipid profile with no fat accumulation. steroid withdrawal prevents bone loss with increment of lean body mass, but with a concerning rate of ar and graft function deterioration in prepubertal patients. children with x-linked hypophosphatemic rickets (xlh) usually present with progressive disproportionate stunting. we therefore conducted a year randomized controlled trial on theeffect of rhgh therapy on body anthropometry ( parameter) in prepubertal children with xlh (each patients in rhghandcontrol-group). age at baseline was . ± . yrs (mean±sd), height was - . ± . sd, calcitriol dosage was ng/kg x day, and phosphate dosagewas mg/kg x day (each p> . rhgh-vs. control-group). results: within the whole study population longitudinal bodydimensions were more affected than transversal body dimensions aswell as circumferences (extremities/trunk). leg length (- . ± . sd; p< . ) was the most impaired longitudinal parameterexplaining % of the overall variability of total body height, whereassitting height (- . ± . sd) was the best preserved longitudinalparameter. longitudinal body dimensions were significantly increased after months rhgh-treatment (height + . ± . sd; sitting height + . ± . sd, leg length + . ± . sd; arm length + . ± . sd; each p< . ). in contrast, progressive stunting was noted in control patients (height - . ± . sd; sitting height - . ± . sd; leg length - . ± . sd; arm length - . ± . sd; each p< . vs. rhgh-group). rhgh treated patients showed a significant increase in all transversal body dimensions (each p< . ), as well as a decrease in skin folds (range - . up-to - . sd; each p< . ). one year rhgh treatment in severely growth retardedprepubertal children with xlh leads to improvement of longitudinal bodydimensions, harmonization of body (i. e. transversal body dimensions & body proportions), and decrease of body fat. thalassemia is an hemolytic anemia characterized by decreased production of β globin. the chronic hemolysis requires frequent bloodtransfusions that caused hemosiderosis, including iron deposition in the kidney. removal of excess iron via iron chelators, the most commonof which is desferal produce iron excretion in urine and stool. few studies have been published, the studied patients, mostly adults, exhibited proteinuria, aminoaciduria, low urine osmolarity and excess secretion of the proximal tubular function markers, n-acetyl-d-glucoseaminidase (nag) and β microglobulin. the iron accumulation may causes lipid oxidative peroxidation and this oxidative process cause's tissue damage. in this study we examined thalassemia major patients treated with desferal. the degree of hemosiderosis was determined by measuring serumiron and ferritin. children without iron metabolism disorders orrenal disease serve as controls. the renal and tubular function was analyzed. no differences in creatinine clearance, blood hco , na and k fe, tmp/gfr was found. serum uric acid was equal in the two groups but itsurine excretion was significantly higher in the thalassemic group probably due to persistent hemolysis. the nag level and its ratio to creatinine were significantly higher compared to the control group. the results of our work showed that most of the thalassemic patients have sub clinical disturbances in tubular function and high nag in theurine. these results raise the question of treating thalassemic patients with oral chelators which removes iron from cells preventing the oxidative process; moreover, and add antioxidants which may prevent the deposition of iron in tissues. in light of these findings, it would be advisable to routinely check glomerular and tubular function as part ofthe follow-up in these patients. objectives of study and methods: little information is available on the long-term follow-up in patients with biallelic mutations in the two genes slc a and kcnj , encoding the bumetamidesensitive na-k- cl cotransporter and the in wardly rectifying renal potassium channel, respectively. we evaluated the long-term follow-up (> years) in patients with these two forms of bartter syndrome. the slc a and kcnj genes were screened by dhplc and sequencing techniques. results: the long-term follow-up period was to years, median , after diagnosis, in patients with mutations in slc a ( homozygotes, compound heterozygotes) and patients with mutations in kcnj ( homozygotes, compound heterozygotes) genes. medical treatment with indo methacin at last follow-up control including supplementation with potassium in patients and medical treatment with indomethacin in patients (meandose . mg/kg/day). in two patients (one with slc a and another one with kcnj mutations) growth hormone (gh) deficiency was detected with specific tests. both patients were treated with recombinant human gh. at the end of follow-up, body height was < ° percentile for agein of the patients, of whom with gh deficiency and body weightwas < ° percentile for age in patients (none of them with gh decifiency conclusions: these data demonstrate that some patients with biallelic mutations inthe slc a and kcnj genes tend to present slight impaired glomerular kidney function after a median follow-up of years and that growth retardation and gh deficiency are often present in these patients. the cytosolic c-terminus of nhe does not mediate its apical localization or baseline activity -implications for blood pressure and ph homeostasis the epithelial sodium proton exchanger, nhe , is expressed in the apical membrane and subapical endosomes of the proximal tubule. apical localization is fundamental to proximal tubular na+, water and hco -absorption, a process necessary for the maintenance of plasma ph and blood pressure. moreover the redistribution of nhe between these compartments alters its activity. for example, acute hypertension leads to an increased endomembrane accumulation of nhe and pressure natriuresis. nhe is composed of a n-terminus with transmembrane helices and a cytosolic c-terminus. the former is necessary for sodium-proton exchange while the latter regulates activity. the goal of our studies was to evaluate the contribution of the cytosolic c-terminus to apical localization and therefore to nhe function. to this end we generated a series of nhe constructs with sequential deletions in the cytosolic c-terminus. all constructs contained an extracellular epitope tag to facilitate the delineation between cell surface and endomembrane nhe . stable renal epithelial cell lines were generated expressing each construct. surprisingly, even when the entire c-terminus was deleted nhe was still detectable at the apical membrane. we proceeded to evaluate the activity of the truncated exchangers and found they retained activity. finally, we assayed the attachment of nhe to the actin cytoskeleton (a process thought to be mediated by the c-terminus), by measuring their solubility in the weak detergent triton x- and by measuring their mobility in the plane of the plasma membrane. both assays confirmed that exchangers lacking the c-terminus retained their association with the plasma membrane. in conclusion, the cytosolic c-terminus of nhe is not necessary for apical localization nor baseline nhe activity. further studies will be needed to confirm its role in specific regulatory processes. background: ibu is used as a safer alternative to indomethacine for pda treatment. however, safety/efficacy balance has not been extensively studied. animal and a few clinical studies suggested that ibu might also have significant side effects. objective: to evaluate renal function at one week of life in infants treated with ibu as compared to infants not exposed to ibu, within the first days of life. methods: multicentric prospective cohort study of to weeks gestation (ga) infants exposed or not exposed to ibu. infants presenting with renal impairment at birth, urinary tract malformation, or contraindication to ibu treatment were excluded. infants exposed to ibu were paired to controls according to ga, centre and crib score. creatinine clearance (ml/min/ . m ) was measured for glomerular function evaluation; fractional excretion of sodium (fena) and microglobinuria/creatininuria ( /ucr) for tubular function evaluation. results: infants were studied: exposed to ibu for pda closure with . % efficiency and controls. birth weight was ± g (mean+sd), and ga . ± . wks. glomerular filtration rate (gfr) significantly decreased on day in ibu exposed infants. noteworthy, diuresis remained in the normal range, but was significantly lower after ibu treatment (given on day ) as compared to controls. antiresorptives, particularly bisphosphonates (bp), offer a promising treatment inpediatric bone disease. bp have been successfully used to treat childrenwith osteogenesis imperfecta (oi), secondary osteoporosis, fibrousdysplasia, hypercalcemia. concerns exist regarding bone mineralizationand bone growth and transient adverse reactions. according to available literature, bp were successfully used in ~ children, intravenous pamidronate (pm) being the most frequently used one (> children, mostly with oi), followed by alendronate (al) (~ patients with oi, secondary osteoporosis, hypercalcemia), risedronate, etidronate, zoledronate, clodronate, olpadronate. oi treatment with bp resulted in an increase in bmd, improved growth, relief from pain, mobility improvement, improved quality of life and a drop in fracture rate there are sparse data comparing oral and i. v. bp. oral and i. v. bp seem to have a similar effect in children with oi. daily al therapy seems to be safe and effective in children with oi, and daily or weekly administration of al led to increase in bmd in patients with secondary osteoporosis. there is scarcity of randomized, double blind, placebo-controlled or active comparator trials with bp in children. currently, double-blind, placebo-controlled study with risedronate andactive comparator trial with intravenous zoledronate in children with oi are planned. bp therapy should be used in context of a well-runclinical program with specialist knowledge in the management of pediatric metabolic bone disorders. other emerging antiresorptive agents include denosumab, glucacon-likepeptide- and calcitonin tablets. these drugs are tested in phase iii trials in adults. their applicability to children is likely to be discussed in the coming years. the current kdigo recommendations on classification or renal osteodystrophy recommend assessment of three areas of bone histology: turnover, mineralization, and volume. while lesions of bone turnover are prevalent in children treated with dialysis, little is know about the prevalence of mineralization defects and their response to therapy with vitamin d sterols and phosphate binders. we evaluated the skeletal mineralization (osteoid thickness (o. th.) and osteoid maturation time (omt)) in patients ages ± years treated with maintenance dialysis who were not receiving vitamin d sterol therapy. serum biochemical markers were: ca: . ± . mg/dl, p: . ± . mg/dl, pth: ± pg/ml, alk p'tase: ± iu/dl. % of patients with high turnover bone disease ( % ci: - %) and % ( - %) of patients with normal or adynamic bone had abnormal skeletal mineralization as reflected by both a prolonged omt and widened o. th. subsequently, a subset of patients underwent treatment with calcitriol and calcium carbonate. while serum pth levels decreased by % (p< . ) and bone formation rate decreased by % (p< . ) with therapy, there was no change in o. th or omt from baseline. indeed, % ( - %) of patients had abnormal mineralization after therapy. we conclude that mineralization defects are prevalent in children treated with dialysis and are not affected by current therapeutic options. further studies are needed to determine the pathophysiology and optimal treatment of these defects. introduction: clara cell secretory protein (cc ) is a protein synthesized primarily by non-ciliated bronchiolar epithelial cells, the clara cells. like other low-molecular size proteins, plasma cc is rapidly eliminated by glomerular filtration and reabsorbed by proximal tubular cells. this protein has been rarely studied in the paediatric age. the sensitivity of cc in urine was compared to that of b -microglobulin (b m) and n-acetyl-b-d-glucosaminidase (nag conclusions: cc is a good marker of the proximal tubular function. cc is related better with the urinary b m elimination, since both are low-molecular size proteins that are reabsorbed by the proximal tubule. sd. kim, bs. cho kyunghee university hospital, pediatrics, seoul, south korea background: many studies have demonstrated that arb prevents renal progression in patients with glomerular nephritis or diabetic nephropathy by inhibition of hmc proliferation and reduce of extracellular matrix expansion and glomerulosclerotic changes. however, the molecular effects of arb in cultured hmc have not been completely defined. we investigated differential gene expression by arb treatment on cultured hmc according to time sequence using cdna chip (affymetrix). methods: mc was grown in dmem with % fbs and then arb was treated on cultured mc. rnas of hmc at different time points ( , , and h after arb treatment and no treatment) were compared using affymetrix cdna chip. to validate the patterns of gene expression analyzed by the microarrays, some genes were selected and semi-quantitative rt-pcr was performed. results: among genes, humoral immune response, cytokine activity, il- receptor binding, chemotaxis, cell cycle arrest, and morphogenesis associated genes were down-regulated for , , and h after arb treatment. they also showed different clustering according to their changing patterns. conclusion: the present study demonstrates profile of gene expression as time goes by after arb treatment on proliferation of human mc. gene expression by arb treatment on cultured hmc showed sequential changes. our results showed that chemotaxis and immune response associated genes were suppressed by arb treatment on hmc. further evaluation of individual genes will be conducted to elucidate molecular mechanism. podocin is the major component of the slit diaphragm, the site responsible for size and charge selectivity of filtration. mutations in the nphs gene, which encodes podocin can lead to steroidresistant nephrotic syndrome (srns). in this work we studied whether genetic changes of podocin might predispose to the development of sporadic non-familial srns in childhood. we screened for podocin mutations children ( m/ f; mean age . ± . years) with sporadic srns and healthy controls. renal biopsy in srns patients revealed mesangial proliferative glomerulonephritis (n= ), focal segmental glomerulosclerosis (fsgs; n= ), membranoproliferative glomerulonephritis (n= ) and membranous nephropathy (n= ). the mean age of onset of srns was . ± . years. all exons of podocin gene of patients with srns and controls were amplified and direct dna sequencing was performed. there is one novel nonsense heterozygous mutation of c. g>t p. glu>stop was identified in the st exon of nphs gene in srns child with fsgs who has renal transplantation at the age of years without recurrence of fsgs in her graft. we found one kind of single nucleotide polymorphism c. + a>g in the th exon of nphs gene in % ( / ) srns patients ( with fsgs; with membranoproliferative glomerulonephritis) and % ( / ) children in controls. allele frequency of this polymorphism of the nphs gene in srns patients and controls was not different significantly. our results indicate that mutation-detected rate in the nphs gene in russian children with sporadic srns was %. the low mutation-detected rate and identified polymorphism c. + a>g in nphs gene unlikely predispose to the development of sporadic srns in russian children. further determining the slit diaphragm genetic profile of children with sporadic srns is warranted in order to improve disease classification and tailoring of treatment. for diagnose of essential hypertension is necessary except all causes of secondary hypertension (sh). case: family history of -year-old female patient was unremarkable for hypertension, incl. renal diseases. during last months she had often headaches with intermittent vertigo. she was not anywhere examined. at admission on intensive care unit patient had headache, her pulse rate was /min, respiratory rate /min, bp (right arm) was / mmhg and the lower extremity bp / mmhg. other physical examination was normal, incl neurological exam. fundoscopy of the eyes did not show any evidence of hypertensive retinopathy. cardiac ultrasound showed mild left ventricular hypertrophy. abnormal laboratory parameters (without medication) were: hypokalemia ( . mmol/l), metabolic alkalosis (ph . , hco . mmol/l), low urinary output of sodium ( mmol) and chloride (< mmol), high urinary output of potassium ( mmol). renal function tests were normal and no abnormalities were detected by renal ultrasonography, incl. doppler exam of renal blood flow. by large hormonal analyse were detected high plasma renin activity (pra; . ng/ml/hod; n.r. . - . ) and very mild increased serum aldosterone (ald; . nmol/l; n.r. - . ). during the ct renal angiography was found solid mass in the upper part of the left kidney. a partial left nephrectomy was performed and histological exam revealed juxtaglomerular cell tumor (so-called reninoma, re). after the resection we stopped subsequently patients antihypertensive therapy. in a -month follow-up our girl was normotens. re is a very rare cause of sh. re typically present during adolescence or young adulthood. this cause of severe sh should be considered along renal artery stenosis in adolescents presenting with secondary hyperaldosteronism or in causes with renin-secreting tumor of non-renal origin. we report our preliminary molecular findings in twelve turkish cystinosis patients. the patients were to years; male/female ratio was : . all presented initially with severe failure to thrive, polyuria and polydipsia. cystinosis was diagnosed at age month to years. six of the patients reached end stage renal failure at ages ranging from . to years necessitating renal replacement therapy; are currently on hemodialysis, one is on capd, and two were transplanted. while three of remaining have renal fanconi syndrome with proteinuria, had kidney failure in varying degrees. molecular analyses involve an initial multiplex pcr, checking for the presence or absence of kb founder deletion, and subsequent sequencing of the coding exons of ctns. interestingly, none of the nephropathic cystinosis patients carried the kb deletion. instead, one patient had a new homozygous kb deletion of exons to of ctns. one patient was homozygous for a known bp deletion in exon , i.e., c. del gact. two patients were homozygous for new missense mutations in exons and , i.e., c. g>a (r g) and c. a>g (y c), respectively. the most common mutation in our turkish patients was a new exonic splice site mutation in exon , i.e., c. g>a (e e). of alleles studied, carried this mutation, which is expected to disrupt proper splicing. two patients were compound heterozygous for one of the above-mentioned mutations and a known missense mutation in exon , i.e., c. g>a (g r). in two patients, we could not find any disease-causing mutation; in two patients, we could find only one disease-causing mutation. in summary, cystinosis patients of turkish ancestry show ctns mutations different from those of western european patients. these findings will be of relevance for molecular-based screening and diagnostic methods for cystinosis. introduction: the association of hypertension with bell's palsy in adults has been reported from - %, but there is few data in children. the presence of bell's palsy in children requires a complete evaluation for hypertension, solid tumors, leukemia, neurofibroma, and trauma. hypertensive children usually present with clinical manifestations of underlying disease, but with substantial elevation, symptoms of hypertension develop. although headache, dizziness, blurred vision and seizure are common neurologic symptoms of hypertension, but facial paralysis is not a wellrecognized presenting feature of hypertension in children. case report: this paper describes two severely hypertensive children who referred to children's hospital of tabriz with periferal hemifacial nerve paresis and initial diagnosis of bell's palsy. case : a years old girl who admitted with blood pressure of / mmhg on admission. renal ultrasound study revealed left small size kidney and renal scintigraphy followed by angiography confirmed renovascular hypertension. case : a years old boy with blood pressure of / mmhg on admission. sonography of kidneys was normal except for a small size ( mm) stone in left kidney. renal scintigraphy and angiography were normal. all other evaluations for etiology of hypertension including cardiac and endocrine investigations were negative. treatment of hypertension with antihypertensive drugs resulted in complete recovery of facial paresis in both cases within - months. conclusion: unilateral peripheral facial nerve paresis is a rare presentation of hypertension in children. unawareness of this presentation may result in delay in diagnosis of hypertension which may increase further with steroid therapy for bell's palsy. to uncover the frequency and the spectrum of nphs mutations in egyptian children with non familial steroid-resistant nephrotic syndrome (srns), patients were screened by pcr-singlestrand confirmation polymorphism analysis of nphs gene followed by direct sequencing. nphs mutations were evident in patients ( . %) who were bearing five novel mutations including two frame shift mutations ( - insg and - insg), two missense mutations ( a>c and t>a) and a silent mutation ( a>t). there were no phenotypic or histological characteristics of patients bearing nphs mutations, apart from the earlier onset of the disease, compared to those who were not bearing mutations. in conclusion, nphs mutations are prevalent in egyptian children with non-familial srns and this may in part explain the less favorable prognosis reported in these patients. , which is an inherited systemic disease of connective tissue primarily affecting the skin, retina, and cardiovascular system, also leads to rvh. these symptoms of pxe are usually apparent in adulthood and rarely observed in children. here, we describe a very rare pediatric case of rvh caused by pxe. case report: a -year-old boy was noticed to have severe hypertension ( / mmhg) when he was admitted to our hospital for an operation for exotropia. he had no previous or family history of pxe or hypertension. laboratory examination showed that plasma renin ( . pg/ml) and aldosterone ( . ng/dl) concentrations were markedly elevated. his systolic and diastolic blood pressure (bp) decreased . % and . % from baseline after administration of captopril, respectively. a computerized tomographic scan of his abdomen showed multiple calcified vessels in kidneys and spleen. yellowish papules on the bilateral axillary regions and inguinal area were detected. characteristic histological changes of pxe such as elastic fiber mineralization and calcification were noticed in the biopsy of affected skin lesions. conclusion: pxe is characterized clinically by high heterogeneity in the age of onset and extent and severity of organ system involvement. although its symptoms usually appear in the second or third decade of life, gastrointestinal bleeding and acute myocardial infarction have been reported in childhood. we should also consider pxe as one of the causes of rvh in children, because its prognosis depends largely on the extent of extracutaneous organ involvement. the clcnkb gene is rarely reported as having a large deletion mutation, but all cases reported previously were large homozygous deletions and a large heterozygous deletion is impossible to detect by direct sequencing. patients and methods: this report concerns a genetic analysis of japanese patients with type iii bs. to identify the mutations in the clcnkb gene, we used polymerase chain reaction (pcr) and direct sequencing to investigate all exons and exon-intron boundaries in the patients and their family members. to detect large heterozygous deletion mutations of the clcnkb gene, we conducted semi-quantitative pcr amplification using capillary electrophoresis. results: four mutations were identified, comprising novel bp deletion mutation (c. _ delct), an entire heterozygous deletion and a heterozygous deletion mutation of exon and of the clcnkb gene. the nonsense mutation w x in the clcnkb gene was detected in all patients ( of them were homozygous and were heterozygous) and this finding indicates that this nonsense mutation is likely to constitute a founder effect in japan. two patients had large heterozygous deletion of the clcnkb gene, which was proved by semi-quantitative pcr amplification. conclusions: capillary electrophoresis is a new method and extremely useful for detecting large heterozygous deletions, and should be employed to examine type iii bs cases in whom only a heterozygous mutation has been detected by direct sequencing. obesity defined as body mass index (bmi) above th percentile for age and gender is regarded as a risk factor for cardiovascular (cv) target organ damage (tod) in children and adults with ph. however, when using percentile-based definition of obesity one may miss children at risk of cv complications. the aim of the study was to compare sensitivity of traditional definition of obesity (bmi> th pc) and bmi thresholds (tbmi) for cv complications described by katzmarzyk et al. ( ) majority of children with idiopathic nephrotic syndrome (frequent relapsers, steroid dependent and steroid resistant) require adjunctive therapy. the response to cyclophosphamide (cp) in these children is variable and difficult to predict. there may be an effect of polymorphic expression of gst on the remission with cp. in this study, we have tried to evaluate the correlation of gst polymorphism and response to cp therapy in these children we studied gst polymorphism in consecutive children ( males, females) with steroid sensitive (n= ) and steroid resistant (n= ) nephrotic syndrome, receiving cp therapy. we evaluated the inter-relationships between gstm , gstt , and gstp genotypes and correlated it with the response to cp therapy in these children. the mean age of onset was . ± . years. out of children, % children responded to cyclophosphamide therapy. the null genotype of gstm and gstt was observed in . % and . % respectively while val genotype of gstp was seen in % children. there was no significant correlation among the various individual genotypic combinations. however, a trend was seen towards remission in children with a combination of gstp polymorphism with gstt null genotype (p= . ) or combination of gstp polymorphism with gstm wild type genotype (p= . ) another important finding in our study was that there was a significantly higher frequency of val allele of gstp in responders to cyclophosphamide as compared to nonresponders (p= . ). the results indicate that the presence of the val allele correlates with the response to cyclophosphamide in these children. gst gene polymorphism may be a significant therapeutic tool in the management of children with idiopathic nephrotic syndrome receiving therapy. objectives of study: primary vesicoureteral reflux (vur) is a common pediatric disease that may lead to reflux nephropathy and end-stage renal disease. the renin-angiotensin system (ras) was proposed to be associated with primary vur. the objective of this study was to investigate whether the gene polymorphisms of the ras are involved in primary vur and correlation with the severity of vur in taiwanese children. methods: we studied the angiotensin ii type receptor (at r) c a gene polymorphism for association with susceptibility to primary vur and disease severity in vur children and healthy controls. fifty four of the vur patients had low-grade vur (grade i-iii) and had high-grade vur (grade iv and v). to analyze the polymorphisms in the c a of at r gene, the snp genotyping assay were performed. the genotypic frequency and allele frequency for four ras genes were analyzed to detect the correlation between the patients with mild, severe vur, and healthy control. results: we found that the c a of at r gene was associated with the development and severity of vur. significantly higher c and lower a allele frequencies were presented in vur patients (c allele . and a allele . ) compared with controls (c allele . and a allele . , p< . ). the similar results were observed in both mild (c allele . and a allele . ) and severe (c allele . and a allele . ) vur compared with controls (p< . ). the cc genotype was higher in vur patients compared with controls (p< . ). conclusions: at r c a gene polymorphism was associated with the development and the severity of vur in taiwanese children. it raises the possibility to utilize the genotype of at r as a risk factor to evaluate the development and severity of primary vur. results: stone analysis led to diagnosis of cystinuria in patients ( % of all), but only were children ( . % of the paediatric stone patients). age at diagnosis in three patients was - years and in four - years. time from first manifestation (pain x, gross haematuria x, urinary retention x, uti x) until diagnosis was - years in seven, - in three and even and years in two. renal function was impaired in patient (ckd stage ii). the urinary cystine test was positive in all patients with cystine stones examined, but was negative in family members thus excluding cystinuria in them. family history of urinary stones was positive in two; the brother of one had died of renal failure due to bilateral urolithiasis. conclusions: delays in diagnosis of cystinuria in armenia are unacceptable. production of tablets containing nickel/dithionite which is non-toxic in contrast to brand test (cyanide/nitroprusside) is planned and could allow screening of all patients with stones of unknown origin. background: classically, childhood hypertension has always been recognized as secondary and frequently demanded an exhaustive etiological investigation. however, in the last two decades, many studies have been shown that pediatric patients also present primary hypertension and, sometimes, the adult disease probably begins during childhood. the aim of this study was to analyze a cohort of patients followed-up at a single tertiary unit (belo horizonte, brazil). methods: in this retrospective cohort study, the records of diagnosed with arterial and followed at our unit between and were analized. a data base was used for statistical analysis. results: of patients, . % were male and . % female. the distribution of patients according to the age at diagnosis was: - . years, ( . %), yr- . yr, ( . %), yr- . yr, ( %), and yr, ( . %). the causes for hypertension were: renal diseases in ( %), primary hypertension in ( %), renovascular disease in ( %) and ohers in patients ( %). the comparison between primary hypertensive subjects and patients with renal diseases showed that, although blood pressure was similar at admission, a better control was achieved in the first group (p< . ). the frequency of overweight and/or obesity was higher in primary than in secondary hypertension ( nephrnophthisis (nphp) is a very rare cause of crf in korea. identification of five genes mutated in nphp subtypes - has linked the pathogenesis of nphp. ten percent of affected individuals have retinitis pigmentosa, constituting the senior-löken syndrome. we experienced juvenile-onset crf with leber's amaurosis in two sibilings. case : a -year-old boy presented with pale appearance. he had severe renal impairment and visual disturbance, but no symptom of polyuria and polydipsia. his past annual school screening urinalysis was normal. his growth and development were normal except opthalmological findings and pallor. case : his -years-old sister also had a visual disturbance and was found to have crf. there was no specific problem during perinatal period. opthalmologic findings were similar to her brother's. nphp and on chromosome q was analyzed by direct sequencing. sequencing of mitochondrial dna-mbol digestion for mutation was analyzed by pcr-sequencing and rflp. electroretinogram revealed a decreased in amplitude of a and b waves. on the kidney biopsy, some of glomeruli were globally sclerotic. remaining showed no cellular proliferation or capillary wall thickening. interstitium was diffusely fibrotic and in which were scattered lymphocytes. most tubules were preserved well but a few dilated tubules were intermingled. no positive reaction for immunoglobulines or complements in if. by em, tubular membranes showed thickening, splitting and disintegration. nphp and genes were not identified. there was no transition mutation at mitochondrial dna nt. , , , . late-onset senior-löken syndrome were diagnosed in these cases even though there were no polyuria and polydipsia of initial symptoms of nphp and nphp and were not identified. we need to identify other known or novel mutation genes. nail-patella syndrome (nps) is an autosomal dominant disease characterized by classic tetrad of dysplastic nails, absent or hypoplastic patellae, elbow dysplasia, and iliac horns. some patients manifest nephropathy and adult-onset glaucoma. nps is associated with mutations in the lmxib gene. there is marked inter-and intrafamilial variability in the phenotypes. in this study, phenotype-genotype correlation was analyzed in unrelated korean children with nps. the probands were boys and girls. they manifested dysplastic nails ( / , %), absent or hypoplastic patellae ( / , %), elbow dysplasia ( / , %), iliac horns ( / , %) and nephropathy ( / , %) . four missense mutations/ in the lim-b domain (h q and l p) and in the homeodomain (r q and a p)/and frame-shifting deletion (c, dela) were identified in the lmxib gene.r q and a p are known to be common mutations, and r q was detected in patients in this study. autosomal dominant inheritance was identified in patients by phenotype and genotype analysis of the family members and in patients by phenotype analysis only. remaining patient had de novo r q mutation. one patient and her mother with r q mutation developed nephrotic syndrome, which progressed to end-stage renal disease (esrd). another patient with h q mutation had asymptomatic proteinuria with microscopic hematuria, and her father had esrd. galucoma was not detected in any patients or family members affected. there were inter-and intrafamilial variability of the phenotypes, but no genotype-phenotype correlation was identified. this is the first study to characterize mutations in the lmx b gene in korean patients with nps. r q is a common mutation in korean, also. the mechanism underlying the phenotypic variations and predisposing factors to the development nephropathy remain unknown. the pathological mechanism which would be responsible for higher production of digitalis similar compounds in essential arterial hypertension (ah), would be a genetically defect kidney, which causes difficulty in na + excretion. higher intake of salts can be an etiological factor inessential ah only patients with inherited abnormalities of thetransport mechanisms in the cell membrane.the objective of the studywas to establish the incidence and type of ah and frequency of genetic factor in the investigated population of school children (age - years). in children with essential and borderline ah we evaluated the activity of erythrocyte membrane na+, k+-atpase in the presence ofvarying atp concentrations. atp, adp and amp levels and lipid peroxides (as tbars) in the erythrocytes and tbars in plasma were measured. our data have shown that the prevalence of ah is lowest in - years oldchildren, while it is the highest in - years oldchildren. essential ah was established in ( . %) and borderline in ( . %) children. genetic factor were found in . % of children with essential ah and in . % of children with borderline ah. but, statistical analyses of the first and second degree relatives of children with essential and borderline ah suggested normal prevalence of hypertension. atp, adp and amp levels, as well as, lipid peroxides were not significantly altered compared to healthy children. kinetic profileof erythrocyte plasma membrane na+, k+-atpase activity revealed the presence of noncompetitive (allosteric) inhibition of enzyme activityin the children with essential and borderline ah. the differences in the kinetic properties of na+, k+-atpase between two investigated groups of children suggested that this dynamic model could be used as potential biological marker for early diagnosis and differentiation of these two type of ah. majority of these patients were obese and with increasing body weight nighttime hypertension became prominent in these patients. objectives: it has been shown that weight gain is directly related to increase inblood pressure. the objective of this study was to analyze the frequency of overweight/obesity in children/adolescents with primary hypertension (ph) and the relationship between the overweight/obesityand the stage of hypertension (h). methods: we analyzed the data of patients aged - . years (m ; f ) diagnosed with primary hypertension. overweight/obesity was defined asperc. of bmi - / c. the stage of hypertension was determined according to the th report on the diagnosis, evaluation,and treatment of high blood pressure in children and adolescents of the nhlbi working group. results: out of pts, prehypertension was found in pts ( . %), stage i h in ( . %) and stage ii h in pts ( . %). stage ii h was more frequentin boys ( % vs. % in girls). combined systolic/diastolic h was found most frequently, in pts ( . %), isolated elevation in systolic blood pressure (bp) was found in pts ( . %), while only pts ( . %) had isolated elevation in diastolic bp. sixty-one pts ( . %) were found overweight/obese; of which ( . %) above c bmi. theobese children were predominantly found to have stage ii h: . % ofthe obese children. none of the obese children had prehypertension. contrary to the general perception, boys were found more frequently overweight/obese ( . % boys vs. . % girls). conclusion: our data shows that stage ii ph is not rare also in theteen-age group. frequently it is accompanied with overweight/obesity, especially in boys. we can conclude that the roots of ph in the adultage are already set from the childhood. our efforts should focus onearly diagnosis of ph and include prevention of obesity as the possible contributing factor for the development of ph. objectives of study: alport syndrome (as) is a progressive renal disease characterized by hematuria, progressive renal failure, high-tone sensorineural hearingloss and ocular lesions. xlinked dominant alport syndrome (xlas) isthe major inheritance form, accounting for almost % of the cases, caused by mutations in col a genes. there are no good cures available for as at present, but there are some patients who requestfor prenatal diagnosis and genetic counciling. this study performed thefirst prenatal diagnosis in chinese as family. methods: the entire coding sequence of col a mrna of peripheral blood lymphocytes was amplified using nest pcr to screen mutations in a chinesexlas family. mutation analysis of the fetus was performed on both cdna-based level and dna-based level of amniocytes. fetus sex was determined by pcr amplification of sry as well as karyotypes analysis. maternal cells contamination was excluded by linkage analysis. results: mutation screen showed that there was a g to a substitution at position in exon of col a gene (c.g a), it was subsequently confirmed at genomic dna level by pcr amplification of exon of col a gene. the mutation was identified in the index case, family members aswell as the pregnant lady. the prenatal diagnosis showed that fetus didnot carry the same mutation as the mother detected by both cdna-basedand dna-based mutation analysis. pcr amplification product of sryas well as karyotypes analysis revealed a male fetus. linkage analysis of the three x chromosome markers showed that there was no contamination of maternal cells in amniocytes. conclusion: after mutation identification of col a gene in a large chinese as family, a successfully prenatal diagnosis was performed in one of the female members in this family based on both cdna as well as genomic dna level of amniocytes. objectives of study: alport syndrome (as) is a clinical and genetic heterogenousdisease. autosomal recessive as (aras) is caused by mutations in col a and col a genes, accounting for % of the cases. thin glomerular basement membrane nephropathy (tbmn), is also caused by mutations in col a and col a genes, inherited as an autosomal dominant trait. differentiation between early stages of aras and tbmn may be difficult in clinic, itdepends on gene mutation analysis of col a or col a . methods: the whole entire coding regions of col a and col a mrna of peripheral blood lymphocytes were analyzed using reverse transcription polymerase chain reaction (rt-pcr) and direct sequencing to screen mutations in three chinese aras families, the corresponding exon with flanking intronic sequence was further amplified to confirm the abnormality. results: both the entire coding regions of col a and col a mrna were successfully amplified and completely sequenced by seven overlapping pcr products, respectively. results showed that there were variations of col a and variations of col a , respectively. among the variations of col a ,three were snp reported previously, four were new snp, one nonsense mutation, one small insertions and one splicing mutation, the latter three were the pathogenic mutations for the three families, respectively. all the four variations of col a were snp reported previously. we concluded that rt-pcr and direct sequencing using peripheral blood lymphocytes rna is a practical, sensitive and feasible approach for analysis col a and col a gene variants in autosomal recessive alport syndrome patients, which offers a useful, inexpensive and timesaving approach for systematic gene analysis in patients with aras. objectives of study: gitelman syndrome (gs) is an autosomal recessive tubular disorder characterized by metabolic alkalosis, hypokalemia, hypomagnesemia and hypocalciuria. the majority of gs patients carry inactivating mutations of the slc a gene encoding the sodiumchloride cotransporter located in the distal convoluted tubule. this study aimed to detect mutations in a chinese family with gs. methods: two brothers presenting muscle weakness, hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria were clinically diagnosed as gitelman syndrome. the two brothers, as well as their healthy brother and parents, were detected for mutations in slc a gene by direct pcr for each exon and then sequencing on genomic dna extracted from peripheral blood cells. results: the two patients were found to have the same compound heterozygous mutations (c. c>t and ivs - t>c) in the slc a gene. the two mutations were also detected in paternal and maternal genomic dna, respectively. the unaffected brother had one mutation (c. c>t) only. conclusion: a novel compound heterozygous mutation on the slc a gene was revealed in a chinese family with gs. the objective: to determine the clinical value of ambulatory blood pressure monitoring (abpm) in pediatric kidney disease. methods: patients with common kidney diseases aged from - yrs were enrolled. -hour abp were performed by welch allyn abpm . the number of cases whose ccr> mmol/l/ . m , ccr - mmol/l/ . m , ccr - mmol/l/ . m , ccr - mmol/l/ . m and ccr< mmol/l/ . m is , , , and , respectively. seven patients only took fosinoprilto control blood pressure did abpm again more than one weeks after therapy. healthy children performed in german in was used asthe normal data. casual bp was measured by sphygmomanometer. children took echocardiography to calculate left ventricular mass index (lvmi). results: the incidence of nocturnal hypertension was significantly higher than that of diurnal hypertension (p< . ).theincidence of non-dipper in children with kidney disease was . %. nocturnal dipping rate in the patients was significantly lower than that in the healthy children (p< . ). the incidence of masked hypertension and white coat hypertension inchildren with kidney disease were . % and . % respectively. nocturnal systolic and diastolic dipping rate of the children with lupus nephritis and acute glomerulonephritis were lower than those of the children with henoch-schönlein purpura nephritis (p< . ). lvmi hadpositive correlation with -hour diastolic blood pressure load (r= . , p= . ), but it had no correlation with casual bp. after taking fosipril, -h, diurnal and nocturnal average abp decreased significantly (p< . ). nocturnal dipping rate increased, but did not reach statistical difference (p> . ). conclusions: abpm was an effective tool for diagnosis and management of hypertension in childhood with kidney disease. identification of novel mutations in the col a gene of japanese patients with x-linked alport syndrome background: alport syndrome consists of nephritis, often progressing to renalfailure, and sensorineural hearing loss. x-linked phenotype (omim# ) is the result of mutation in the gene for the alpha- chain of collagen iv (col a ). objectives of study: to find mutations causing x-linked alport syndrome in japanese patients. patients and methods: diagnosis criteria of alport syndrome were proteinuria, familial history of renal failure andchanges of gbm in electron microscopy. to identify the mutations in the col a gene. results: for the time being we finished genetic analysis of patients with suspected x-linked alport syndrome, males and females. we identified mutations in of patients; of them were novel mutations. they comprise: nonsense mutations, missense mutations and mutations of splicing acceptor site resulting in exon skipping or truncation, and establishing of premature stop codon. in previous reports of x-linked alport syndrome, mutation detection rate was around %. the present study provides a detection rate of . %, although our number of examined cases is limited. conclusions: direct sequencing of rt-pcr and pcr products is efficient method of finding mutations in col a gene. we found mutations in high percentage of investigated patients. objectives of study: to make a genetic diagnosis of juvenile nephronophthisis for a chinese boy. methods: analyze the presentation, family history, laboratory investigations, and histological features of a patient suspected of juvenile nephronophthisis, make a clinical diagnosis. to confirm the diagnosis on genetic level by pcr amplification of satellite markers located within the known homologous deletion of nphp ,including del- , del- , del- , del- -( ) , del- and markers outside the deletions (ranbp / and d s )as control. results: the boy was nine years old, he was admitted becauseof renal failure for months. this case manifested by school age, negligible onset, anemia, polyuria, renal failure at years old,without hypertension and abnormal urine analysis. his sister presented with anemia at year old, and died of uremia at years old.laboratory investigations showed no proteinuria and hematuria. thekidney size is normal, and the cortical medullary boundary is obscure.the histological features consist of the disintegration of tubularbasement membrane, the atrophy and dilation of renal tubules, theinterstitial cell infiltration and fibrosis. he was suspected asjuvenile nephronophthisis. by pcr amplification, we found the missingof the satellite markers of del- , del- , del- -( ) and del- , which indicate that there is the common large homologous deletion ( kb) in the child's nphp . conclusions: nephronophthisis is the major hereditary cause ofchronic renal failure. the boy was suspected of nephronophthisis because of chronic renal failure, family history, normal kidney sizeand hitological features. according to the age of renal failure, he was suspected as juvenile nephronophthisis. the diagnosis was confirmed by gene analysis. it is the first juvenile nephronophthisis case for chinese confirmed by gene analysis. cutaneous small-vessel vasculitis is a rare condition in children and is commonly associated with a wide spectrum of systemic inflammatory conditions, malignancies, infections or drug hypersensitivities. enalapril is anangiotensin-converting enzyme inhibitor, commonly used for the treatment of hypertension. cutaneous vasculitis due to enalapril has very rarely been reported and only with adults. we report a case of an -yea-old boy presented with a pruritic eruptionover his lower legs that started hours after he had initiated treatment with enalapril at a dose of . mg/kg/daily. he had a history of hypertension due to a shrieked right kidney since infancy and he wason treatment with nifedipine and propranolol for the last three years. this medication was discontinued because it was ineffective, the day before initiation of enalapril. clinical examination revealed palpable purpuric lesions involving lower legs and ankles. he was otherwise well. a skin biopsy of the lesions showed leucocytoclastic vasculitis of small vessels. abnormal investigations were elevated c-reactive protein, esr and leucocytosis. further laboratory tests including creatinine, liver function tests, urineanalysis, antinuclear antibody, cryoglobulins, viral serology, complement levels, were normal or negative. enalapril was discontinued and he was started oral prednizolone and ceterizine over the next ten days. the skin eruption regressed rapidly. cutaneous vasculitis induced by enalapril is a rare adverse effect and it is essential a prompt recognition and withdraw of the suspicious drug. objectives of study: barttin, the gene product of the bsnd gene involved in bartter's syndrome with sensorineural deafness, is an essential b-subunit for clc-ka and clc-kb chloride channels, and it is expressed in the kidney as well as in the inner ear. one patient affected by deafness and renal bartter phenotype without bsnd mutations has been previously reported with simultaneous mutations in both clcnkb (responsible for classic bartter syndrome) and clcnka genes. we report here on a new case of a -months-old boy presenting such a disease. a severe polyhydramnios was detected during the pregnancy. he presented also polyuria, growth retardation, nephrocalcinosis and sensorineural deafness. laboratory studies revealed metabolic alkalosis (plasma hco , mmol/l), hypokalemia ( , mmol/l), hypercalcuria (cau/cru , mg/mg) and elevated plasma renin ( pg/ml). the aim of the study was to identify the cause of the severe renal salt wasting and sensorineural deafness in this patient. methods: dna sequencing analysis was performed on the bsnd, clcnkb, and clcnka genes. results: no mutation was detected in the bsnd gene. we identified a reciprocal partial homozygous deletion of exons - for the clcnkb gene and theloss of exons - for the clcnka gene. conclusions: the disruption of both clc-ka and clc-kb chloride channels leads to a syndrome clinically not distinguishable from bsnd, characterized by severe salt wasting and deafness. this digenic disorder is due to simultaneous mutations on the two genes clcnka and clcnkb respectively. the tight topology of the highly homologous clcnka genemight predispose to an unequal crossing over leading to partial orcomplete deletions of the clcnkb gene. we hypothesize that thischimaeric resulting gene interferes with the correct function of both the channels clc-ka and clc-kb, and leads to a bsnd-like phenotype. familiary hypomagnesemia with hypercalciuria and nephrocalcinosis (fhhnc) is a nautosomal recessive tubular disorder that is frequently associated with progressive renal failure. mutations in cldn which encodes the renal tight junction protein paracellin- were identified as the underlying genetic defect. in this work we present a gipsy family with fhhnc in which the mother and daughter both showed homozygous mutations in the cldn gene. a three-year-old girl was investigated after acute pyelonephritis and was found to have medullary nephrocalcinosis, hypomagnesemia ( , mmol/l), hypercalciuria ( , mg/kg/d), hypermagnesuria (femg %) and incomplete distal renal tubular acidosis. her renal function was normal. her mother demonstrated also bilateral medullary nephrocalcinosis, hypomagnesemia ( . mmol/l) with high femg ( %), moderate renal failure (creatinine umol/l) and high ipth levels ( pg/ml). as a child she had afebrile seizures, but serum mg levelwas not measured. index patient's uncle had history of recurrent passage of calculi in childhood, bilateral medullary nephrocalcinosis, normocalcemic hypercalciuria but his serum mg was not determined. he developed obstructive uropathy due to impaction of the calculus in urethra and eventually progressed to terminal uremia and later was transplanted. mutational analysis confirmed that the index case, motherand uncle were homozygous for the common mutation in the cldn gene (leu phe), while the father was heterozygous. we present a new family from macedonia with fhhnc with unusual presentation over two generation. mutational analysis confirmed also the diagnosis of fhhnc in the uncle although as a child he was considered to have idiopathic hypercalciuria. thus, serum mg should be routinely checked in children with nephrolithiasis/nephrocalcinosis. m. pan ' czyk-tomaszewska, j. s ' ladowska, j. so l /tyski, m. roszkowska-blaim arterial hypertension is one of the complications of reflux nephropathy. the aim of the study was to assess the risk factors of arterial hypertension in children with primary vesicoureteric reflux (vur). we studied children aged from to years mean ± . , with unilateral and with bilateral vur. in all children ambulatory blood pressure monitoring (abpm) and m tc dmsa renal scanning (dmsa) were performed and z score body mass index (z-score bmi) were calculated. the following criteria were examined as predictive risk factors: age, gender, age at diagnosis andat resolution of vur, period between diagnosis and resolution of vur,grade of vur, type of vur (unilateral or bilateral), treatment modality (medical, surgical), z-score bmi, grade of dmsa and birth weight. results: i grade of dmsa scan was diagnosed in children, iigrade in , iii grade in and iv in patients. hypertension (hp) was diagnosed in children ( %). the mean value of z-score bmi in children with hypertension was significantly higher in comparison withchildren with normal blood pressure ( . ± . and . ± . ). the multivariate discriminate analysis showed that zscore bmi and grades of dmsa scars were significant risk factors for developing arterial hypertension in children with vur. both parameters had the same influence on development of hypertension (standardized coefficient of discriminate analysis , and , respectively). conclusion: the development of hypertension in children with vur is associated with higher bmi and higher grades of renal scars in dmsa scan. grange syndrome comprises arterial stenoses with hypertension, brachy syndactyly, bone fragility, learning disability, and cardiac defects and it has been reported in the members of two families up to now. we report here a -year-old boy with hypertension, multiple arterial stenoses, microcephaly and brachysyndactyly. severe hypertension ( / mmhg) was detected on his routine control for acute rheumatic carditis and he was hospitalised for investigation. he had no complaints. his parents were first degree relatives. his physical examination revealed microcephaly, bilateral brachysyndactyly between the forth and the fifth fingers of hands and the second and the third fingers of feet. complete blood count, electrolytes, renal, thyroid, and hepatic functions and acute phase reactants were all in normal limits. echocardiography showed aortic and mitral insufficiency and left ventricular hypertrophy. renal and cerebral magnetic resonance angiographies demonstrated stenoses at bilateral renal, internal carotid, superior cerebral and posterior cerebellar arteries. association of hypertension with microcephaly and finger abnormalities and multiple arterial stenosis suggested grange syndrome. chromosomal analysis showed xy karyotype. hypertension persisted despite triple antihypertensive agents and percutaneous renal angioplasty was performed leading to a reduction of antihypertensive medication. with this case report we wanted to remind the recognition of this extremely rare syndrome in a hypertensive child with multiple morphological abnormalities and percutaneous angioplasty as a treatment modality in cases with persistent severe hypertension. hypertension is usually indicative of an underlying disease process in children. the combination of severe hypertension with hyponatremia is called hyponatremic hypertensive syndrome (hhs). in this report we present a case of hhs. the boy was referred to our hospital at the age of years old with generalized tonic clonic convulsion. his past history was insignificant and positive findins of the patients were as follows: hypertension ( / mm/hg), agressive behaviors after convulsive period, metabolic alkalozis (blood gases, ph: , , hco : , , pco : , ), hypocloremia ( meq/l), hypokalemia ( meq/l), hyponatremia ( meq/l), low urine density, polyuria and high serum aldosterone levels ( pg/ml, range: - ). the serum renin, cortisol, tyroid and antidiuretic hormones were within normal range. abdominal color doppler ultrasound were revealed double renal arteries in both kindneys. cranial and abdominal tomography examinations and cebrospinal-fluid examination were normal. the diagnosis of hhs was suggested based on these clinical and laboratory findins and therapy of nifedipine was started. the arterial pressure improved with this therapy. in hss due to renal ischemia, activation of renin angiotensin system which induce pressure natriuresis and thus, thence hyponatremia. we think that presence of renal arterial anomaly might be a cause of an ischemic condition and activation of renin-angiotensin-aldosterone system in our patient. also, activation of aldosterone could enhance with hyponatremia and it could be suppressed of renin secretion in our patient. [hadtstein et al. hypertension ] . the aim of this study was to analyze whether children with renal scars have altered rhythms of mean arterial pressure (map) and heart rate (hr). study design: ambulatory -hour blood pressure profiles of untreated patients with renal scars associated with recurrent urinary tract infections and vur were investigated and compared to healthy controls. results: pre-pubertal, but not pubertal patients with renal scars displayed a number of significant differences to controls. before puberty, the mesors of map and hr were significantly elevated, and the peak-trough difference of the h curve was blunted by . mm hg for map and . bpm for hr. the amplitudes of h and h map rhythms were blunted by . and . mmhg, and those of h and h hr rhythms by . and . bpm (all p< . ). pubertal patients did not display any abnormalities of bp or hr rhythms. we did not find any correlations of the degree of renal scarring, bmi or gfr with the abnormalities in cardiovascular rhythms. conclusion: in summary, pre-pubertal children with renal scarring due to vur show blunted circadian and ultradian rhythms of bp and hr. this phenomenon may reflect subtle alterations of autonomous nervous signaling in children with damaged kidneys; it remains to be addressed whether such abnormalities constitute an independent cardiovascular risk factor. mutations in the eya gene are associated with bor/bo syndrome and rarely with an isolated renal phenotype. we present a family from macedonia displaying a novel eya gene mutation. a six-year-old female was found to have bilateral grade iii vesicoureteral reflux (vur) after an episode of acute pyelonephritis. mutational analysis detected a novel eya gene mutation [an inframe bp deletion (c. _ delttg), resulting in l del of eya a]. she had no clinical stigmata for bor/bo syndrome. the paternal grandmother died due to end stage renal failure. all first grade relatives underwent detailed physical and ophtalmological examination, audiometry, ultrasound screening of the urinary tract and mutational analysis of the eya gene. the father and older sister were mutation carriers and both had normal ultrasound findings. the physical examination did not reveal abnormalities suggestive for bor/bo syndrome, except the sister had esotropia/ hypermetropia and horizontal nystagmus. the male sibling was found to have mild hydronephrosis on the left side. he was not a mutation carrier and cystographic study was not performed at that time. on repeat ultrasound examination he demonstrated dilatation of the right prevesical ureter. the cystography revealed presence of right sided vur grade iii. in conclusion: we present two siblings with vur; although eya gene mutation was found in one of them, it can not be incriminated for the renal phenotype in this family, as co-segregation of the mutation with the vur does not occur in all affected members of the family. decision to perform cystographic study should be still based on clinical grounds. associations asymmetric dimethylarginine (adma) is a novel predictor of cardiovascular (cv) outcomes in adults. aim: to evaluate adma in children with hypertension. subjects: children ( girls/ boys; median age: yrs) with primary (n= ) and secondary (n= ) hypertension. methods: adma levels were analyzed by elisa method. in study subjects antropometrical data, ambulatory blood pressure monitoring (abpm), left ventricular mass index (lvmi), carotid artery intimamedia thickness (cimt), -hour urinary albumin excretion (uae) and serum biochemical markers (lipids, uric acid) were also evaluated. results: adma concentration was positively correlated to serum uric acid (r= . , p< . ) and uae (r= . , p< . ). when analyzed in boys separately, more apparent correlation was found for uae (r= . ) and a tendency for adma to be associated with lvmi r= . , p= . ). interestingly, when analysis restricted to older children (above the median age), adma correlated to lvmi more significantly (r= . , p< . ). no relation was found of adma to cimt, lipids, and abpm parameters. moreover, patients with left ventricular hypertrophy ( %) and microalbuminuria ( %) had a tendency for higher adma values compared to those without these abnormalities ( . ± . vs . ± . μmol/l, p= . and . ± . vs . ± . μmol/l, p= . , respectively). when data analyzed on the basis of adma values, patients in the top quartile showed the worst profile. when compared to the lowest quartile, they had higher lvmi (p< . ), higher uae (p< . ; inter-quartile comparison p= . ), and higher uric acid concentration (p= . ). conclusions: the associations of adma with known factors of cv damage found in this study provide evidence that adma is closely linked to the early cv dysfunction in hypertensive children. nephrogenic diabetes insipidus (ndi) is a disease characterized by unresponsiveness of distal tubule and collecting duct to vasopressin. although ndi usually presents with polydipsia and polyuria, most infants do not have any of these symptoms and presentation is with features of dehydration like fever, constipation, vomiting, failure to thrive and developmental delay. so, the diagnosis is usually delayed until hypernatremia is noted. almost all infants go through a period of hypogammaglobulinemia at approximately the th- th months of age. at this time, the serum ig g level reaches its lowest point, and many normal infants begin to experience recurrent respiratory tract infections. the clinical findings of ndi and transient hypogammaglobulinemi of infancy (thi) may be seen at the same period. here, we report a five-month old boy with ndi. on his history, he was admitted because of recurrent fever attacks and was diagnosed as thi when he was three-months-old. on his follow-up, hypernatremic dehydration was detected and he was diagnosed as ndi. at the time of diagnosis, he had intracranial calcification secondary to delayed diagnosis of ndi. in infants with ndi, recurrent fever attacks due to dehydration may occur and incorrectly lead to think as an infectious disease. we think that this report can be an important warning for clinicians. we analyzed nphs , nphs and neph gene in japanese cns patients independent of the patients in previous report (k.i. ) from different japanese group which suggested that the mutation of nphs was not a major cause of cns in japanese patients. we extracted genomic dna from leukocytes and analyzed all exons and exon-intron boundaries for nphs , nphs and neph using polymerase chain reaction and direct sequencing after informed consent had been obtained. the study was approved by the ethics committees of okayama university medical school. results: we found compound heterozygous mutations of nphs in patients and homozygous mutations in patients. one of these homozygous mutations was already reported in paper from europe and was not found in more than healthy japanese. another one cause defection of trafficking to the membrane as we reported before (k.i. (k.i. , ajkd . the other patients have only heterozygous mutation in nphs that healthy parent also has. we could find any mutations in neither nphs nor neph in the patients with heterozygous mutation. one of these patients has mild form of cns. another one has neither expression of nephrin nor podocin protein on kidney tissue by immunohistochemistry. interestingly, patients out of have the same mutation in nphs as nt (delc). parents who have this mutation heterozygously were from neighboring prefectures. all mutations including this mutation but one have never been reported out of japan which was isolated from continent. all amino acids substituted by missense mutations which seem to be causative were preserved among mouse, rat and human. conclusions: our studies clearly demonstrated that nphs is a major cause of cns even in japanese patients. moreover, nt (delc) is a common mutation in japanese cns patients like fin major or minor. long-term survival of childhoodonset ckd is mainly limited by the manifestation of cardiovasculardisease (cvd). the development of early stages of cvd can be assessedby non invasive methods, e.g. flow mediated vasodilation (fmd) ofperipherial arteries and measurement of intima media thickness (imt) of the common carotid artery. wetherefore performed fmd and imt investigations in children and adolescents (mean age . ± . years) suffering from ckd (mean gfr ± ml/min/ . m ) on conservative treatment (n= ), dialysis treatment ( hd, pd) and after renal transplantation (n= ), and in sex-and age-matched healthy controls. ckdpatients showed significantly decreased fmd ( . ± . % vs . ± . %, p< . ), where as imt did not significantly differ between patients and controls ( . ± . mm vs . ± . mm, p= . ). within the ckd group the presence of arterial hypertension tended to be associated with increased imt ( . ± . vs . ± . mm, p= . ). in contrast, duration of ckd, mode of renal replacement therapy and degreeof renal dysfunction was not significantly associated with fmd and imt findings. conclusion: children and adolescents suffering from ckd show decreased arterial elasticity irrespective of the mode of renal replacement therapy, the rebycontributing to increased long-term cardiovascular morbidity and mortality. the genetic researches have shown the connection between the essential hypertension and angiotensinogen gene. the researches of preeclampsia also showed the connection with angiotensinogen gene. according to established connection of angiotensinogen gene with essential hypertension and also preeclampsia the aim of our research was to determine by the help of m t, g- a anda- c polymorphisms of angiotensinogen gene whether there is a genetic disposition for essential hypertension in those children whose mothers had preeclampsia. at the same time we wanted to establish whether the polymorphisms of angiotensinogen gene can be connected with preeclampsia in women in our population. two groups of children were studied: children of mothers who had preeclampsia and children of mothers without hypertensive disease in the pregnancy. we also studied two groups of mothers: mothers who had preeclampsia and mothers without hypertensive disease in the pregnancy. m t, g- a and a- c polymorphisms of angiotensinogen gene were performed using the pcr method. in investigating the differences between the two groups of children no statistically significant differences were found in m t,g- a and a- c polymorphisms of angiotensinogen gene. in investigating the differences between two groups of mothers no statistically significant differences were found in genotype distribution. the results of our study failed to confirm that with help of m t, g- aand a- c polymorphisms of angiotensinogen gene we might establish genetic disposition for essential hypertension in those children whose mothers had preeclampsia. we did not confirm the association between m t, g- a and a- c polymorphism of angiotensinogen gene and preeclampsia either. ( ) previously treated also with cyclosporine. before start of fosinopril treatment all had proteinuria . - . g/ h and gfr > ml/ . m /min. all active steroid and immunosuppressive treatment were discontinued. eleven children also were treated with calcium channel blockers to control hypertension. after twelve months of fosinopril ( . - . mg/kg) treatment a dna analysis for ace-gene polymorphism was performed. three patients carried ii, -id and -dd gene polymorphism of ace gene. no significant differencein proteinuria at start of fosinopril treatment between all polymorphism types was observed. all patients with ii polymorphism had a good antiproteinuric effect of fosinopril with more than -fold decrease in proteinuria in comparison with just , fold decrease in id polymorphism and no response in dd. renal function remained stable in all children except of with id and with dd gene polymorphism who demonstrated decrease in gfr. we suggest that ii polymorphism of ace gene may be associated with better antiproteinuric effect of acei while dd predicts poor response. wider study is required to confirm genetic predisposition for ace blockade efficacy in proteinuric diseases. introduction: dent disease is x-linked recessive proximal tubulopathy, due to mutations in the clcn gene. it is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and progresive renal failure. aim: case report of patient with short stature and proximal tubulopathy where dent disease is determined by gene analysis. method: seven-year-old boy is refered after endocrinological exemination where abdominal ultrasound showed nephrocalcinosis. there are anamnestic data neither of oedema, macrohaematuria, nor poliuria or hypertension. there are also no data of chronic renal failure in the family. we describe detailed diagnostic procedures performed in the boy and his family. results: we determined: proteinuria ( , g/day), elevated urinary excretion of beta- microglobuline, microscopic hemathuria, hypercalciuria ( - mg/kg/day), nephrocalcinosis, decreased tubular reabsorption phosphate ( - %). values of grow hormone, parathormone on thyroid hormone are normal. except hypercalciuria, which is registered in patient's mother, all other analysis performedin family members are betwen referent values. diagnosis is finalized by mutation analysis, which has showed s l substitution on cncl . mutation carrier is mother with normal fenotype. conclusion: dent disease is rare x-linked nephrocalcinosis. definitive diagnosis ofthis proximal tubulopathy which leads to progressive renal demage, is not possible without evidence of gene mutation in renal chlor channel. introduction: the segmental renal infarction is a rare disease, allthough it is more frequent in children than in adults and can be clinically veryrelevant. objective: we describe the clinical course of two children with idiopaticsegmental renal infarction who suffered severe arterial hypertension. cases description: the typical clinical picture of this disease, as seen in our twopatients, is characterized by metabolic alkalosis, hyponatremia, hypokaliemia, hyper-reninemia, hyperaldosteronism and renal salt lossfrom the contralateral enlarged kidney. hypertension was diagnosedduring the study of haematuria in the first case and due to ahypertensive emergency in the second case. the ethiology was found tobe renovascular in both patients, involving the occlusion of small renal arteries causing segmental renal infarction. our first patient was treated with partial nephrectomy, and the second patient wastreated with antihypertensive medication given the impossibility ofremoving the renal infarcted area. conclusions: in the clinical picture, the sodium depletion with increased urinary volume of the contralateral healthy kidney can be explained by the goldblatt model. the gold standard test for the diagnosis is theselective renal arteriography, which is the most sensible and specifictest for diagnosing renovascular hypertension secondary to a segmental renal infarction because it helps identifiing the segmentary artery ofthe infarcted area, this being the source of increased focal renin production. the definitive treatment is the surgical segmentectomy. if segmentectomy is not feasible because of the localization of the infracted area as inour second patient, a medical treatment is needed. conclusion: although an inverse association between birth weight/blood pressure has been suggested in several large studies, interpretation is still controversial. in our study, we have found only a weak inverse correlation between birth weight and pad (abpm): no statistical significance has been found among other variables, although graphs show a trend of low birth weight children towards hypertension. probably a long term follow-up is necessary. he took longer to have improvement, but his calcemia, phosphatemia and alkalinephosphatase are normal. the coding region of the vdr was amplified by pcr and directly sequenced. results: three mutations (two novels) were identified. two patients had anovel mutation in exon , changing the amino acid glutamine to glutamicacid at position (q e). one patient had a novel mutation in exon changing glycine to valine at position (g v). those mutations are in the ligand binding domain and belong to the patients with better control of the disease. one had mutation in exon , in the dna binding domain, changing arginine to a stop codon at position (r x) and it was from the patient with worse response to the treatment. conclusions: two novel mutations are presented in the vitamin d receptor and it could be possible to do a correlation between the clinical evolution and the localization of the mutations. we aimed to evalute the effects of intrauterine growth retardation (iugr) on blood pressure (bp) in small for gestational age (sga) children. sga children ( f, m) aged . ± . y ( preterm and term) and ( f, m) agematched control with appropriate for ga (aga) were included in the study. -hr ambulatory blood pressure monitoring (abpm)was performed using an oscillometric device (mobilograph). th centile limit was set according to published data sets, nocturnal threshold was % less than awake limits. bp was classified as normal if the mean sbp is < th percentile and sbp load is > %. nocturnal bp dip for sbp and dbp were evaluated. in sga and in aga case, office sbp were above th%, all dbp values were normal in both groups. according to the mean of abpm records, systolic, diastolic ht were determined in sga group, and systolic, no diastolic ht were in aga group. there were sga and aga children whose sbp load over %, and only of them remarked as hypertensive according to mean sbp values. children considered as hypertensive had significantly higher sbp loads than those that were not hypertensive( % vs %, p< . ). abpm records were not different between preterm and term sgas. sga group had lower nocturnal bpdip for sbp and dbp in comparison to aga group( . % vs . % for sbp, . %vs . %for dbp, p: . ). although it was not statistically significant, the frequency of non-dipper children was higher in sga group ( . %) than those in aga group ( . %). except the lower nocturnal bp dip and higher sbp loads in sga group which may be a marker for further hypertension, no clear association between being sga and hypertension could be found in our study population. pres is a rare clinico-radiological entity associated with acute hypertension, renal insufficiency and immunosuppressive therapy. it is typically reversible but cases with irreversibility had rarely been reported. we report contrasting cases of pres associated with renal disease, one with full recovery and the other subsequently had epilepsy. first patient had steroid sensitive nephrotic syndrome with acute hypertension, fluid overload and acute renal failure while the second patient had severe flare of lupus nephritis with malignant hypertension, renal failure and thrombotic thrombocytopenia. both had acute onset of drowsiness, blindness and seizures and mri findings of subcortical oedema in the parietal-occipital-temporal regions. while the second patient had full recovery, the first patient developed temporal epilepsy and repeat mri showed mesial temporal sclerosis. pres is attributed to capillary leak from functional cerebral vascular changes of hypertension, rather than infarction. prolonged vascular disruption may lead to ischemia resulting in neurological sequelae and chronic epilepsy. using mri, pres can be readily diagnosed and normalisation of blood pressure is imperative in patients at high risk of pres. efforts have been done to describe the significance of various genetic polymorphisms (snps) in acute renal failure (arf). available reports do not investigate the predictive value of snps in disease forecasting, because so many interacting factors influence arf that classical statistical methods become unstable. high-dimensional nonparametric methods such as random forest technique overcome this problem and help to identify each clinical and genetic factor that possibly contributes to disease. we tested the classification value of basic clinical data available at birth and snps in arf of preterm infants. we determined the relative importance score (ri) of each parameter in classification. low birth weight and gestational age had the highest ri; just few snps had medium ri, while the majority of snps had small ri. then we created variable-patterns and searched for pattern with the highest accuracy of classification. for each complication, the accuracy was . solely basic clinical data were considered. if snps were incorporated, the accuracy of classification for arf was not improved. in contrast with previous observations these findings suggest that the snps do not provide additional information about arf-risk of the general preterm population. conclusions: srbd in children with elevated blood pressure is higher than that of the general pediatric population. the prevalence appears highest in patients with pre-hypertension and may be higher in patients with essential and secondary hypertension than in patients with white-coat hypertension. the renal phenotype in lowe syndrome is similar to dent's disease a -month-old girl presented with heart failure due to severe hypertension ( / ). doppler ultrasonography (us) revealed a right renal artery stenosis. biological findings showed normal serum creatinine (scr) ( mmol/l), hyponatremia ( mmol/l) and hypokalemia ( mmol/l). aldosterone plasma level was increased as were epinephrine ( pg/ml n< ) and norepinephrine ( pg/ml n< ) plasma levels measured times. renal arteriography confirmed the presence of a nearly obstructive right renal artery stenosis with a normal left kidney. pta failed to improve signifcantly the renal artery flow. in the following hours after pta, her scr level increased, up to mmol/l. doppler us showed an unchanged right kidney but hypoperfused area in the left kidney confirmed by the tc -dmsa scan. both aspects were strongly in favor of focal ischemic events. secondarly, her renal function improved but hypertension remained difficult to control autotransplantation of the right kidney was then done, but unfortunately, thrombosis of the right renal artery occured in the following hours. six months later, her plasma creatinine level is of mmol/l. her cardiac function is normal but she has to remain on antihypertensive drugs. what exactly happened during arteriography in her left kidney, in order to provoke such ischemic injury, knowing that the left renal artery was not catheterized during arteriography and pta? one hypothesis is that arterial vasospasms occured, explained by a high vasospastic predisposition due to enhaced cathecolamines secretion. such increased has already been drescribed in patients with renal artery stenosis. this case raised the question of preparing patients in this condition with efficient antispasm therapy agents before radiological vascular investigations are done. rtd is a cause of oligohydramnios leading to potter's sequence and neonatal anuria. it is characterized by absent or hypoplasic proximal tubules. these lesions can be secondary to non genetic conditions involving renal hypoperfusion. genetic forms of rtd have autosomal recessive transmission. mutations of genes encoding for the renin-angiotensin system have been reported. evolution is most often severe with in utero or neonatal death. case report: this boy was born at weeks of gestation with oligohydramnios, arthrogryposis, large fontanels with poor calvarian ossification. he was referred to intensive care unit for respiratory distress, low blood pressure (bp) unresponsive to vasopressors and anuria (plasma creatinine μmol/l at day ). renal ultrasound (us) showed enlarged kidneys with bilateral cortical hyperechogenicity. weaning from ventilation and vasoactive drugs was possible at day . glomerular filtration recovered (plasma creatinine μmol/l at day ). at age , the child is well with normal growth and a mild chronic renal insufficiency (creatinine clearance ml/min/ , m ). bp is in the low normal ranges with plasma renin x over the upper range of normal values and normal plasma aldosterone. on renal us, kidney volume normalized with persistent cortical hyperechogenicity. genetic study revealed compound heterozygous mutation in the gene encoding angiotensin converting enzyme. one is a nonsense mutation (r p), the other is a truncating mutation (k fsx ). it is possible that the first mutation is less deleterious than the other and this could partly explain the unusually favourable evolution of this rtd. in conclusion, this is a rare report of a child with autosomal recessive rtd surviving after neonatal period. further functional studies are needed to explain this unusual phenotype. data on hbp in children are very limited, and it is unclear how much it is specific in pediatric patients. the objective of our study was to determine the reproducibility of hbp in children and adolescents. automatic omron -cp devices, which have been validated for use in children and adolescents, were provided to all families. hypertensive children measured blood pressure (bp) every minutes, three times consecutively in the morning or early afternoon, and three times in the evening during days. the reproducibility of hbp was quantified using repeated measures analysis of variance test and the sd of differences between average hbp values of consecutive days. confidence interval (ci) was calculated. we studied patients ( girls, boys); mean age was , year, range - years. there were measurements of systolic blood pressure (sbp) and diastolic blood pressure (dbp). in the morning or early afternoon the mean sbp and dbp were , mmhg (sd , ) ( % ci , - , mmhg) and , mmhg (sd , ) ( % ci , - , mmhg), respectively. in the evening the mean sbp and dbp was , mmhg (sd , ) ( % ci , - , mmhg) and , mmhg (sd , ) ( % ci , - , mmhg). the mean average difference between the daily measurements of sbp and dbp were analyzed with each period and were not statistically significant (p> . ). on the basis of these results, we conclude that self-monitoring of blood pressure at home in children has good reproducibility and is not influenced by white coat effect. case report: a -year-old caucasian girl presented with history of severe polyuria and polydipsia of few months duration. past history was significant for head injury and possible seizure activity at age yr. there was no history of headaches, vomiting, or visual problems. family history negative for di. physical examination was essentially normal. basal plasma and urine osmolality, and plasma vasopressin were and mosm/kg, and < . pg/ml, respectively. at the conclusion of water deprivation test these readings were and mosm/kg, and . pg/ml, respectively. however, urine osmolality increased to mosm/kg after desmopressin injection, confirming the diagnosis of cdi. mri scan revealed absence of the bright spot on t weighted images. genetic analysis detected avp gene mutation a t, where the normal alanine at amino acid position is changed to threonine. elevated levels of plasma vasopressin after water deprivation were misleading and intriguing. we believe that elevated plasma vasopressin, although immunoreactive was devoid of biological activity. conclusion: molecular genetic evaluation should be performed in all children with cdi without an identifiable cause, even in the absence of a positive family history of cdi. mutations of the nphs gene encoding podocin lead to autosomal recessive corticoresistant nephrotic syndrome, focal and segmental glomerulosclerosis (fsgs), and early end-stage renal disease (esrd). in mice, podocin inactivation by homologous recombination results in diffuse mesangial and glomerular sclerosis, esrd and death within the first weeks of life. this early demise precludes extensive study and elucidation of the molecular pathways engaged by podocin in the mature kidney. we have, therefore, generated a novel mouse model in which a tamoxifeninducible creer-loxp system allows for conditional inactivation in a temporal fashion in podocytes. following tamoxifen administration in nphs flox/-, cre+ mice, cre expression was noted in % of glomeruli and in - % of podocytes. two weeks after cre induction, podocin expression is decreased in podocytes correlating with the appearance of selective albuminuria at - days. this progresses to massive, nonselective proteinuria by weeks, along with high blood pressure and impaired renal function. optical microscopy of kidneys showed no lesion at week and fsgs progressively developed by weeks along with tubular lesions. however electron microscopy revealed a partial foot process effacement at week with no significant albuminuria that extends by weeks along with abnormalities of the basement membrane and development of albuminuria. no mesangial or vascular lesion has been noted, differentiating it from our previous podocin null model in which renal development may be affected by podocin loss or nephrotic syndrome. this model will allow a better understanding of the mechanisms underlying the development of nephrotic syndrome and the role of podocin in the mature kidney, and will be crucial to test new therapeutic approaches. glomerulonephritis is a group of diseases with complex etiology, pathogenesis, morphological features and clinical course. the renin-angiotensin and coagulation systems genes are important group of candidate genes involved in pathogenesis of chronic renal diseases. the purpose of our study was to analyze the association of genetic polymorphisms of these genes with glomerular kidney diseases. the study population consisted of patients with immunological glomerular kidney diseases and patients with renal failure with glomerulonephritis as primary disease. the control group consisted of healthy subjects. by means of the polymerase chain reaction (pcr) the following polymorphisms were evaluated: insertion/deletion (i/d) polymorphism in intron of the angiotensin-converting enzyme gene (ace), g/ g polymorphism of the plasminogen activator inhibitor- (pai- ). no significant association was found between the ace and pai- allele and genotype frequencies and the disease. more progressing of glomerulonephritis current was marked at patients with simultaneous has dd genotype of ace gene and g/ g gene pai- (c = , ; p= , ). our results suggest that ace i/d and pai- g/ g polymorphism is an important modifying gene in the progression of glomerulonephritis. captopril objectives: secondary causes of hypertension such as renovascular hypertension are more abundant in children unlike adult population. the objective of this retrospective study is to assess the use of captopril renography (cr), which provides a non-invasive approach in the differential diagnosis of hypertension. patients and methods: clinical, radiological and scintigraphic data of a total of patients were analyzed. there were girls and boys (mean age: ± years). none of the patients had parenchymal renal disease or reduced renal function. all patients were orally hydrated before scintigraphic study. cr was performed hour after orally captopril administration and iv furosemide injection was done simultaneously with - mci tc m-mag . when post-captopril study was normal, baseline scintigraphy was not performed. computed tomography angiography (cta) was performed in and gadolinium-enhanced magnetic resonance angiography (mra) was performed in children in addition to routine renal doppler ultrasonography (dus). results: nineteen out of patients had other comorbid diseases as follows: obesity n= , previosly-diagnosed fibomuscular dysplasia n= , n= neurofibromatosis, n= demyelinating diseases, n= bartter and n= turner syndrome. cr was normal in patients. in children abnormal cr findings in correlation with radiological methods were reported. correlation with radiological methods could not be done due to suboptimal technique in patients, of whom dus in obese children could not be interpreted. in the other patient, although cr was abnormal, radiological methods could not confirm scintigraphy. conclusions: captopril renography is a useful and simple-to-perform imaging modality in children suspected of having renovascular hypertension. takayasu's arteritis (ta) is a chronic, inflammatory, large-vessel vasculitis affecting the aorta and its main branches, which causes stenosis, occlusion, rarely aneurysm and distal ischemia. the disease is most common in young women, it is rare in inviduals before the age of years. clinical presentation may be heterogeneous. in this report, we present a pediatric patient with ta who had hypertension as the sole manifestation of multipl critical arterial involvement but no other symptoms. a -year-old boy was admitted with hypertension. the acute-phase reactants were moderately elevated with an erythrocyte sedimentation rate mm/h, and a c-reactive protein value of mg/l. serologic tests including ana, anti-ds dna, c and panca, complement c , and c were negative and other laboratory data were normal. mr angiography showed multiple severe stenosis or occlusions of the thoracic and abdominal aorta together with bilateral renal arteries, and saccular aneurysm in the abdominal aorta. immunosuppressive treatment including pulse steroid and methotrexate was prescribed. the patient underwent angioplasty of bilateral renal arteries and suprarenal aorta, and a stent was placed in the right renal artery. ta should be kept in mind in the differential diagnosis of hypertension in children, even if they do not have other associated symptoms of the disease. human urotensin ii is the most potent vasoconstrictor which circulates in the plasma of healthy individuals. it was suggested that it has an endocrine role in sodium handling and even in metabolic syndrome. the aim of this study was to investigate the role of u-ii in obese adolescents with hypertension. fourteen obese adolescents with essential hypertension (group ) were compared with thirteen age-and sex-matched obese adolescents with white-coat hypertension (group ). they underwent twenty-four hour abpm and echocardiographic investigation, complete physical examination, including adiposity indexes. plasma and urinary levels of u-ii were measured by ria. obese adolescents in group have significantly higher blood pressure measurements than those in group confirmed by abpm. there was no significant difference in left ventricular mass index between two groups. no significant difference was found in plasma u-ii concentrations (pg/ml) between group and group ( . ± . and . ± . , respectively), whereas mean urinary u-ii level (pg/mg urinary creatinine) was significantly higher in group than that of group ( . ± . , . ± . , respectively). considering the renal synthesis and vasoactive role of u-ii, these results suggest that u-ii may have a role in adolescents with white-coat hypertension. distal renal tubular acidosis (drta) and deafness is a rare autosomal recessive disease characterized by severe metabolic acidosis in childhood and inappropriately high urinary ph along with sensorineural hearing loss. the disease is caused by defects in the atp v b gene. the aim of the present study was to determine the molecular basis for drta with deafness in eight patients from four families (a-d) in israel. molecular testing was done by sequencing the coding exons of the atp v b gene in one affected child from each family. a population screen was performed for mutations found in family a. the results yielded a different mutation in the atp v b gene, as follows: families a and d: missense mutation, c>g (p r), in addition to a single nucleotide polymorphism ( t>c) in the first codon; family b: insertion mutation, - insc (i fs); family c: a novel nonsense mutation, c>t (r x). in conclusion, the phenotype of drta and deafness concurs with mutations in the atp v b gene. in the present study, four families of different origins with the same phenotype had three different genotypes, indicating that there is no single common mutation in israel. these findings have implications for genetic counseling during pregnancy and testing of families. the fact that all the patients that were examined have harbor mutations in the atp v b gene, pointed for the specific clinical phenotype. making correct and early clinical diagnosis is a fundamental step in finding the molecular basis of this rare disorder. delta f is the most common cystic fibrosis (cf) mutation worldwide. the prevalence is approximately % in caucasian and - % in asian while f l is demonstrated in only . %. homogenous delta f mutation is recognized as the most common genotype however there are small numbers of cf patients having delta f /f l. in the present study we report a year-old thai boy, originated from north india, presented with recurrent episodes of febrile illness, hyponatremia, hypokalemia and metabolic alkalosis since months of age. he was transferred to our hospital for further investigation. blood chemistry revealed serum electrolytes: sodium , potassium . , chloride . , bicarbonate . meq/l, and urine electrolytes: sodium< , potassium . , chloride< meq/l. after intravenous fluid administration, hyponatremia and metabolic alkalosis improved. dna sequencing analysis of his blood demonstrates compound heterogenous mutation for delta f and f l in cftr gene. t to g transversion at nucleotide and g to a transversion at nucleotide are found without altering amino acid encoding gene. in conclusion, we report a rare case of cf with delta f /f l genotype presented with recurrent hyponatremia and metabolic alkalosis. awareness of electrolyte abnormalities during febrile illness, proper genetic counseling and long-term follow-up are necessary in this patient. objectives of study: about % of families with congenital nephrogenic diabetes insipidus (ndi) have mutations in the aquaporin gene (aqp ), which codes for the vasopressin-sensitive water channel. only seven aqp mutations are known to cause a dominant form of the disease. in this study we performed genetic and clinical studies in a generation family with autosomal dominant inheritance of a partial di phenotype. methods and results: the proband (man, yrs old) was initially diagnosed with partial central diabetes insipidus due to antidiuretic effect of ddavp. his daughter ( . yrs old) also showed polyuria and polydipsia which was responsive to high doses of intranasal ddavp. the pedigree was consistent with an autosomal dominant inheritance pattern. sequence analysis of the entire coding region of the avp gene and aqp gene was performed in the proband and his affected daughter and in unaffected family members. the avp gene was normal in all subjects. in the two affected patients but not in the healthy subjects we identified a novel missense mutation in one allele of exon of the aqp gene (c. c>t) which predicts a substitution in the c-terminal part of the aqp protein (p.r w). conclusion: partial ndi can be caused by heterozygosity for a p.r w substitution of the aqp water channel. the substitution is likely to significantly alter the c-terminal tail of aquaporin which is important for proper trafficking to the apical plasma membrane. the preservation of some residual antidiuretic function indicates that some aqp tetramers are processed correctly. the study further illustrates the importance of molecular diagnostic tools in establishing a correct differential diagnosis in familial cases of di. a background and aim: dent's disease is a renal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. mutations in the clcn gene encoding the chloride/proton exchange transporter clc- cause this disease. we have described an alu insertion in clcn that leads to exon skipping in the patient's mrna. in this study, we investigated the consequence of this mutation on the function of two putative exonic splicing enhancers (eses) in exon and the role of alu '-end sequences on splicing inhibition. methods: minigenes were constructed by inserting exon and exon -alu with their intronic flanking sequences into the exon trap vector. artificial mutations were introduced by site-directed mutagenesis. plasmids were transfected into cos cells by electroporation. pre-mrna splicing analysis was performed by rt-pcr. the ese finder web resource was used to predict the mutagenesis effects on the eses. results: alteration of one of the putative eses, the predicted binding site for srp , induced exon skipping. restoration of this site within the alu sequence promoted the incorporation of exon -alu in the mrna. however, restoration of both eses leaving the alu sequence intact resulted in exon skipping. furthermore, substitution of the eses for the first seven nucleotides of the alu element, and insertion of this sequence next to the eses increased exon skipping. deletion or modification of the alu '-end enhanced exon inclusion in the mrna. in the patient carrying the clcn alu insertion, dent's diseases is caused by disruption of an ese and by inclusion of splicing inhibitory sequences leading to exon skipping. this work was supported by grant pi from fondo de investigación sanitaria. factor h-associated hemolytic uremic syndrome (fhd-hus) is a rare disease with incomplete penetrance. it is not clear why and how carriers of the same mutation will eventually develop or not develop the disease and this uncertainty is sometimes responsible for anxiety in the carriers badly affecting their quality of life (particularly of parents with children carrying the mutation). in the attempt of estimating disease risk in subjects with factor h gene mutation, the families of fhd-hus patients (all of which had a documented mutation on the scr ) currently being treated at our center, were screened to identify the carriers after having been informed on the purpose of the analysis. twenty-eight subjects (age range - yrs), out of analyzed, revealed heterozygous mutations in the carboxy-terminal region of fh gene ( %). among subjects carrying the mutation, ( %) had exhibited the disease at a mean age of years (range . - ), of which ( %) before age , whereas the remaining subjects (mean age of yr -range - ) were healthy at time of the study. the figure shows the probability of carriers to be hus-free by age (no event occurred after age yrs). in the meantime that predisposing factor are identified, the presented survival curve (fig. ) can be an useful tool to estimate the residual risk of hus in individual carriers according to their specific age. cerebello-oculo-renal syndrome (cors) (also called joubert syndrome (js) type b) and meckel (mks) syndrome belong to the cilliopathy group of developmental autosomal recessive disorders associated with primary cilium dysfunction. nephronophthisis (nphp), the most frequent genetic cause of renal failure in children and young adults, is associated with retinal degeneration and cerebellar vermis aplasia in cors. mks is characterizd by renal cystic dysplasia, central nervous system malformations and hepatic developmental defects. these syndromes are genetically heterogenous: mutations in ahi and nphp can cause cors and mutations in mks can lead to mks, while mutations in mks and nphp are found in both syndromes. using snp mapping, we identified missense and truncating mutations in a novel genee, nphp , in both cors and mks. interestingly, all sequence changes were either nonsense or frameshift mutations in fetuses with mks whereas patients with cors had either only one or no truncating mutation. we then show that inactivation of nphp mouse orthologue recapitulates the cerebral and renal defects of cors/mks. we further demonstrate that nphp protein co-localizes at the basal body/centrosomes with nephrocystin- and nephrocystin- , the protein products of both nphp and nphp , known disease genes for nphp. in addition, missense mutations of nphp protein found in cors patients diminishes its interaction with nephrocystin- . taken together. our findings demonstrate that mutations of this novel ciliary gene can cause the multiorgan syndromes of either cors or mks, which therefore represent a continuum of the same underlying disorder. severe antenatal bartter's syndrome ga + , bw: g). both had classical signs of antenatal bs including very poor thrive, polyuria ( - ml/kg b.w./h) and responded to indomethacin treatment. direct sequencing was performed on pcr-amplified genomic dna. the entire coding region of both genes was analyzed. the kcnj gene was normal but both children were homozygous for a single base substitution ( t>a) in exon of the slc a gene predicting a premature stop codon (y x). the parents of both children and two siblings were heterozygous for the same mutation. by restriction enzyme analysis, the t>a substitution was absent among chromosomes from healthy subjects. conclusion: two children with severe antenatal bs were identified and the clinical phenotype was characterized. we identified in both children a novel mutation causing a premature stop codon of the bumetanide-sensitive-sodium-potassium-chloride co-transporter (nkcc ). despite large potassium supplements and indomethacin treatment, both children show persistent polyuria, vomiting, and insufficient thrive. background: mutations of nphs encoding the transmembrane slit diaphragm protein nephrin cause congenital nephrotic syndrome of the finnish type, characterized by massive proteinuria and the development of nephrotic syndrome before months of age. about different nphs mutations have been described. a large number of these are missense mutations resulting in single amino acid replacement mostly located in the extracellular domain. some nphs mutations have been associated with mild phenotypes responding to angiotensin-converting enzyme and prostaglandin synthesis inhibition. patient history: we report on a son of consanguineous turkish parents with a novel nphs alteration and steroid-resistant congenital nephrotic syndrome. after normal pregnancy and delivery at weeks of gestational age the boy presented with edema and proteinuria at the age of months. he responded to angiotensin converting enzyme inhibitor treatment reducing his proteinuria from a maximum of g/g creatinine to g/g creatinine and allowing him to develop normally until the age of months when cardiovascular insufficiency in the course of a hyperpyretic infection led to hypoxic encephalopathy. genetic testing revealed a homozygous in-frame -bp insertion in exon of nphs . both parents were found to be heterozygous. no mutations were found in nphs or in exons and of wt . the alteration results in an insertion of a glycine in an extracellular immunoglobulin domain of nephrin. the pathogenicity of this alteration is unclear. nephrin may be misfolded and mislocalized as in some missense mutations of the extracellular domain. alternatively the alteration might lead to an altered extracellular homo-and heterodimerization of nephrin. functional studies are currently underway to determine the effect of this novel alteration in nphs . objectives of study: interleukin receptor antagonist (il- ra) gene polymorphism has been found to affect disease susceptibility and activity in several inflammatory diseases. we investigated the association between il ra gene polymorphism and childhood nephrotic syndrome (ns) in a turkish population. methods: we analyzed the genetic polymorphism of il- ra gene in patients with childhood ns and healthy controls by using pcr. five alleles of the il- ra gene were identified and designated as il- ra* , il- ra* , il- ra* , il- ra* , and il- ra* , according to the variable number of tandem repeats in intron . results: in the ns group, the allele frequencies of il- ra* , il- ra* , il- ra* , il- ra* , and il- ra* were . , . , . , . , and . % compared with %, %, %, %, and % in the controls. a high allele frequency of il- ra* (x = . , p= . ) and a lower allele frequency of il- ra* (x = . , p= . ) were found in childhood ns. the other polymorphisms were not significantly different from normal controls. conclusions: a high allele frequency of the il- ra* allele may affect the disease susceptibility in childhood ns. objectives of study: congenital anomalies of the urinary tract (caut) are common in humans, and the incidence is increasing with recent advances in prenatal ultrasonographic examinations. interstitial fibrosis, which correlates with infiltration of inflammatory cells is common finding in the kidney with long-term ureteral obstruction. up-regulation of monocyte chemoattractant protein- (mcp- ), may be a common regulatory pathway involved in the progressive renal damage with any etiologies leading to interstitial fibrosis. mcp- gene polymorphism - a/g had been suggested to influence circulating mcp- levels and gene expression thus, might be one of the genetic markers for progression of renal damage. our aim was to investigate the frequency of mcp- genotypes and allele - g in patients with caut. objectives of study: congenital urological anomalies are well recognized as causing renal dysfunction. on the other hand, patients without congenital anomalies but with urinary bladder dysfunction (bd) could also develop renal parenchymal damage. it has been reported that angiotensin converting enzyme (ace) i/d polymorphism was a risk factor for renal parenchymal damage in certain renal diseases including vesicoureteral reflux. the aim was to determine i/d polymorphism as a potential risk factor for renal parenchymal damage in patients with congenital obstructive uropathy and to compare them to patients born with normal anatomy and innervation of bladder, sphincter and pelvic floor but which could develop upper urinary tract abnormalities. objectives od study: to determine the frequency of pyelocaliceal dilation (pcd) in asymptomatic infants and its connection with presence of other urinary tract anomalies (uta) and urinary tract infection (uti). methods: ultrasonographic screening (us) of urinary tract (ut) was performed on unselected population of healthy infants ranging between days and months of age ( boys and girls). kidneys were divided into two groups according to grade of pcd. group i consisted of kidneys with pcd, whose anterio-posterior diameter was - mm, while patients from the group ii had a-p diameter wider than mm.patients were followed up (ranging - months) by serial ultrasound examinations and with other methods for presence of uti and uta if necessary. results: in examined population , % had uta, and , % had uti. pcd was found in infants ( , %). in the group i, consisted of infants ( males and females) pcd increased during the time in , % infants, remained unchanged in , %, decreased in , %, and disappeared in , % patients. uta was found in ( , %) infants, and uti in ( , %) infants. in the group ii, consisted of male infants, pcd has not changed in ( , %) infants, decreased in ( , %), while dilation disappeared in ( , %) patients. uta was found in ( , %), and uti in ( , %) infants. conclusions: us of ut is useful and valuable method for detecting pcd. our results indicate that mild pcd in infants increases the risk for uta approximately times and uti times, while severe pcd raises the risk for uta approximately times, and uti times. early diagnosis and early treatment of uta and alertness for possible uti should be the final goal of the kidney damage prevention. therefore we recommend that us of ut should be done in all children in the first months of life. in order to determine factors involved in kidney development, the spatial and temporal expression pattern of intermediate filaments (cytokeratins, vimentin) , epithelial growth factor (egf) and transforming growth factor alpha (tgf-α), was investigated in developing kidneys (mesonephros and metanephros) of - week human embryos. immunohistochemistry for detecting specific antibodies was used on paraffin sections. in the - week human mesonephros, vimentin was moderately expressed in all mesonephric structures, while cytokeratins were seen only in the mesonephric nephrons. moderate to strong expression to egf and tgf-α detected in all mesonephric structures, decreased with advancing development. in the - -week metanephros, vimentin was mildly expressed in all metanephric structures. later on, its expression increased in collectin system and interstitium. in the th developmental week, first to appear were cytokeratins and in the ureteric bud and ampullae, while from the th week onward, both cytokeratins showed increasing expression in the collecting system and nehrons. at early stages, egf and tgfá showed moderate to strong reactivity in the collecting system, the metanephric mesenchyme and cups. from the th week onward, expression of both factors decreased in differentiating nephrons. expression of all investigated developmental factors was in line with subsequent mesonephric degeneration. expression pattern of intermediate filaments in the metanephros might be associated with mesenchimal to epithelial transformation of developing nephrons. some mutations of cytokeratins are lethal, while others might lead to some multifactorial disorders. egf and tgf-α expression patterns of the metanephros indicate their role in induction, proliferation and growth of metanephric structures. their disturbed expression might cause reduction in kidney growth. we have demonstrated that renin-angiotensin system (ras) and mitogen-activated protein kinase (mapk) family constribute to the renal development. growing evidences indicate that aldosterone, a final element or ras, is an independent and powerful mediator of various renal disease. purpose of this study was to evaluate the role of endogenous aldosterone in renal development including cell proliferation and apoptosis, and the expression of mapk family. newborn rat pups were treated with spironolactone ( mg/kg/d) in olive oil or vehicle for d. to identify cellular changes, kidneys were examined for proliferating cell nuclear antigen (pcna) by immunohistochemical (ihc) stain, and apoptotic cells by tunel stain. immunoblot, ihc stain and rt-pcr for mapks, phospho-mapks, and p gene were performed. spironolactone treatment resulted in decreased body weight, decreased pcna-positive proliferating cells and increased tunel-positive apoptotic cells, especially renal cortical epithelial cells (p< . ). in the spironolactone-treated group, c-jun n terminal kinase (jnk)- and phospho-jnk- protein expressions were significantly increased, whereas extracellular signal -regulated kinase (erk)- and p protein expressions were sigificantly decreased compared with the control group (p< . ) in immunoblot and ihc stain. expressions of erk- , phospho-erk- and , and p sere not changed by spironolactone treatment. in rt-pcr, erk- and p mrna expressions were significantly increased in the spironolactone-treated group (p< . ). we conclude that aldosterone inhibition in the developing rat kidney decreases cellular proliferation and increases apoptosis, and modulates the expressions of jnk- , erk- and p . mapk family expression may be implicated to differentially participate in aldosterone-related intracellular signaling pathways in the developing kidney. objectives: early detection of anomalies of the kidney and urinary tract (ut) helps to prevent complications but is hampered in moldova by diagnostic and logistic problems. to assess the extent of late diagnosis we studied the clinical data of all children referred to us for suspected ut infection (uti) in / and found to have renal or ut anomalies. methods: children ( males; age months- years) found to have anomalies of the kidney and ut and treated conservatively were studied (newborns and infants < months were seen elsewhere). work-up included ultrasonography in all, voiding cysto-urethrography (vcug) in %, urography in % and scintigraphy in % of patients. results: reasons for referral were febrile uti ( %); abdominal pain ( %), diarrhea and vomiting ( %), enuresis ( %) and dysuria ( %). uti was confirmed by urine culture in two thirds of cases. age at diagnosis of renal or ut anomaly was < year in %, - years in %, - years in %, - years in % and > years in %. renal hypo/dysplasia was found in , solitary kidney in and a horseshoe kidney in children. anomalies of the ut included hydronephrosis due to ureteral obstruction ( up and uv-obstruction), vesico-ureteral reflux ( ), duplex systems ( ) and bladder anomalies or mild infravesical obstruction ( ). serum creatinine was in the normal range in all children. urolithiasis was found in patients. conclusions: anomalies of the kidney and ut were diagnosed in nearly half beyond the age of years, thus with considerably delay. based on the results of this study a new strategy of renal ultrasound screening in newborns including prenatal diagnosis and of closer co-operation with referring physicians has been implemented. objective of the study: the purpose of this study was to report the outcome of infants with antenatal hydronephrosis. methods: between may and june , all patients diagnosed with isolated fetal renal pelvic dilatation (rpd) were prospectively followed at asingle tertiary renal unit. inclusion criteria were presence of rpd equal to or greater than mm on prenatal ultrasound after weeks gestation, at least six-months of follow-up, and at least two postnatalus scans. the events of interest were presence of uropathy, rpd resolution, urinary tract infection (uti), and hypertension. rpd was classified as mild ( - . mm), moderate ( - . mm) and severe (> mm objective of the study: the aim of this study was to compare the accuracy of ultrasound renal parameters to discriminate between significant uropathy and idiopathic renal pelvis dilatation. methods: neonates who were found to have isolated fetal renal pelvis dilatation (rpd) underwent systematic investigation and were prospectively followed. a us scan was performed after the first week of life and all infants underwent vcug. neonates with rpd larger than mm were examined by renal scintigraphy. receiver-operating characteristic (roc) plots were constructed to determine the accuracy of three indexes: fetal rpd, postnatal rpd, and sfu grading system. results: a total of infants were included in the analysis. ninety-five fetuses ( %) presented bilateral renal pelvis dilatation. seventy-nine infants ( %) presented urinary tract anomaly, corresponding to renal units. the most frequent detected uropathy was ureteropelvic junction obstruction ( ), followed by primary vesicoureteral reflux ( ), and megaureter ( ) conclusions: our data support the view that fetal and postnatal us renal parameters are useful markers to identify infants with clinically significant uropathies. there was no significant difference in performance among the indexes. objective of the study: the aim of this study was to evaluate the diagnostic accuracy of ultrasound (us) renal parameters to identify vur in infants withisolated antenatal hydronephrosis. methods: neonates who were found to have isolated fetal renalpelvis dilatation (rpd) underwent systematic investigation and wereprospectively followed. a us scan was performed after the first week of life and all infants underwent vcug. neonates with rpd larger than mm were examined by renal scintigraphy. receiver-operating characteristic (roc) plots were constructed to determine the accuracy of three indexes: fetal rpd, postnatal rpd, and sfu grading system. results: a total of infants were included in the analysis. seventeen infants ( . %) presented vur, corresponding to renal units. to discriminate between renal units with and without vur areaunder the curve (auc) was estimated by the roc curve, which was . ( % ci= . - . ), . ( % ci, . - . ), and . ( % ci, . - . ) for fetal rpd, postnatal rpd, and sfu grading system, respectively. there was no statistically significant difference between the indexes. the optimal threshold for fetal rpd was mm with a sensitivity of % ( % ci, - ) and specificity of % ( % ci, - ), for postnatal rpd the respective figures were: % ( % ci, - ) and % ( % ci, - ) for the cut-off of . mm. conclusions: our data shown that the magnitude of rpd is a poor predictor of presence of primary vur. there was no significant difference in performance among the indexes. objetive: we investigated the prevalence of renal calcification in children with autosomal ressecive polycystic kidney disease (arpkd) and studied the metabolic changes thats could cause this complication. methods: patients with arpkd. girls/ boys; range age m to y. criteria inclusion presence typical imaging findings: enlarged kidney and diffusely increased renal echogenicity and poorly defined renal margins on sonography; suggestive imaging features with positive results renal or liver biopsy, results patients, girls and boys, range of age - y, had ct scan renal clacification bilaterally. without renal clacifications < year. children renal calcifications, sistemic hypertension, portal hypertension and gastrointestinal bleeding and one renal colic. renal insufficiency patients, it was mild in (gfr > ml/min/ , mt ), moderate in (gfr - ), and severe in one (gfr< ). all with renal insufficiency had distal tubular acidosis. hypocitraturia urine patients. urinary calcium, uric acid, oxalates, and cystines normal in all. tuberoussclerosis (tsc) -an autosomal dominant inherited genetic disorder -is characterized by development of hamartomatous growths in many organs.two causative genes, tsc (chromosomal locus q ) and tsc (chromosomal locus p . ) have been identified. tsc gene is adjacent to pkd , the major gene for autosomal dominant polycystic kidney disease (adpkd) on chromosom p and contiguous germline deletion of both genes results in severe polycystic kidney disease phenotype at birth. at months of age bilateral abdominal masses were occasionally palpatedin a previously healthy girl. ultrasonography demonstrated enlarged (approximately cm) kidneys with multiple large cysts resembling those seen in adpkd. renal function and blood pressure was normal. suspicion of tuberous sclerosis was raised due to numerous hypopigmented cutaneous macules on the trunk and extremities. echocardiography demonstrated rhabdomyomas in the left ventricle with no hemodynamic significance. an isolated juxtapapillary astrocytoma was found in the left eye. her psychomotor development was normal with no history of seizures. cerebral magnetic resonance imaging revealed multiple subependymal nodules with noobstruction of the cerebral fluid pathways. by multiplex ligation-dependent probe amplification (mlpa) a large dna deletion was identified spanning from tsc exon to pkd exon permitting the diagnosis of tsc -pkd contiguous gene syndrome. cardiac rhabdomyoma and cutaneous manifestations were found in her father as well but no renal changes. at years of age the girl is doing fine with ace blockers against hypertension. renal function is still normal. the size of the cardiac rhabdomyomas is diminishing while the cerebral and ocular hamartomasare unchanged. this additional report focuses tsc in an infant presenting with polycystic kidneys and cutaneous lesions. improvements in ultrasound technology and the appropriate timing of antenatal ultrasound has led to refined prenatal diagnosis and enhanced accuracy of diagnosis of fetal renal anomalies and makes it possible to treat obstructive and/or refluxing uropathies before the onset of clinical symptoms.a retrospective review of patients; girls ( , %) and boys ( , %) admitted to our clinic between january -december with antenatal urinary anomalies were investigated to determine the urinary tract anomalies, and the follow-up results are presented. routine prophylaxis was started at admision and the imaging studies were performed. the mean gestational age at detection was , ± , weeks. the mean age of admittance was , ± , days and the average follow-up period was , ± , months. antenatal ultrasonoghrapy examination showed anomalies in / renal units. of these antenatally observed renal units, had postnatal urinary tract anomalies. of these postnatally observed renal units, urinary tract anomalies were detected (multiple urinary tract anomalies in patients) ( table ) . fifty-two ( , %) children had surgical interventions such as; ureteroneocystostomy, pyeloplasty, nephroureterectomy, puv resection. eighty-one ( %) of our patients had urinary tract infection during follow-up and renal scar was detected in ( %) patients. acute renal failure developed in patients and chronic renal failure developed in patients and three patients died. we conclude that all infants with fetal urinary abnormalities should be evaluated, so that we can recognize and treat congenital anomalies that may affect renal function or cause urinay tract infection, renal scarring. the majority of patients with fetal urinary anomalies can be managed safely with close conservative follow-up. fetal urinary tract obstruction at days gestation (e ) produces small kidneys with cysts, whereas obstruction at days (e ) generates large kidneys with cysts. in the present study, we investigated the mechanism for the generation of small kidneys by urinary tract obstruction at an earlier gestational age, e . fetal lambs underwent urethral and urachal ligation at e (n= ) or e (n= ). fetal lambs were delivered by c-section , , days after obstruction, or at term ( days gestation). unoperated kidneys of e , e , e , and at term served as controls. the percentage cystic area of kidneys obstructed at e and e was not different days after obstruction ( ± % vs ± %). after days, however the percentage of cystic area became larger in kidneys obstructed at e ( ± % vs ± %), and was significantly larger days after obstruction ( ± % vs ± %). proliferating cells, detected by pcna staining, were found in cysts and tubules of obstructed kidneys increasing toward term, and were more abundant in kidneys obstructed at e . on the other hand, pcna-positive cells in the nephrogenic zone were reduced in obstructed kidneys. the decrease was more prominent in kidneys obstructed at e . apoptotic cells, identified by tunel staining, were detected in the inner medulla of obstructed kidneys equally in kidneys obstructed at e and e during the fetal stage. at term, tunel-positive cells were rarely present in normal kidneys or kidneys obstructed at e , but were found abundantly in the interstitium and occasionally in cysts and tubules of kidneys obstructed at e . in conclusion, urinary tract obstruction at an earlier gestational age produces small kidneys by inhibition of mesenchymal cell proliferation, which may be due to compression by cysts. a. results: detrusor hyperreactivity, most commonly in i and iv or v grade was found in . % of vur children. the maximum detrusor pressure was above cm h . detrusor-external dyssymetry was found in % of children, most frequently in grade i and iv or v grades, and detrusor-internal dyssymetry was recognized in . % of children with vur, most frequently in grade i and iv or v. in . % of children with i-iii grade of vur cystometric capacity was reduced but child with v grade had increased capacity of the urinary bladder. glomerular filtration according to schwartz equation was normal and independent of vur grade. decrease in osmolality below mosm/kg h in nocturnal urine was only detected in the group of children with iv and v grade of vur,. there was no correlation between detrusor tension and osmolality of urine and glomerular filtration rate. conclusions: ). dysfunction of the lower urinary tract, with detrusor hyperactivity was detected, as the most frequent dysfunction in % of children below year old with i-v degree of vur, ). the maximun detrusor pressure in the voiding phase was highest in grade i and iv iv-v reflux children. hypomelanosis of ito was first described as a disorder characterized by unusual unilateral or bilateral macular hypopigmented whorls, streaks and patches. subsequently, neurologic, skeletal and ocular involvement were described. kidney involvement has only been exceptionally reported. herein, we describe a case of a male infant with hypomelanosis of ito and renal involvement. the patient was born at weeks of gestation by cesarian delivery. the ultrasound scan at weeks of gestation revealed bilateral enlargement of kidneys, decreased corticomedullary differentiation and cysts located in the cortical and subcapsular regions. these findings were confirmed at two months of life by ct scan. skin examination showed hypopigmented linear and round diffuse lesions located on the right leg and arms. the ophthalmological examination showed anterior capsular and posterior subcapsular cataract of the left eye. as previously reported in other cases of hypomelanosis of ito, the patient presented a transient leucocytosis (max . /mm ) during the first months of life.the renal biopsy showed a classic picture of glomerulocystic kidney disease, whereas the skin biopsy confirmed the clinical diagnosis of hypomelanosis of ito.three other cases of kidney disease in hypomelanosis of ito have been reported. of these, one case presented abnormalities of the glomerular basement membrane, and one case presented with polycistic kidney disease. the third case had renal cystic dysplasia with a histological picture containing glomerular cysts. alltogether these reports suggest that genes involved in hypomelanosis of ito are important for normal renal development and may be implicated in cystogenesis, when mutated. here we report on a male newborn (birth weight g, length cm) who presented with progressive edemas, oliguria and failure to thrive during the first week of live. on clinical examination he showed bilateral microcoria and decreased muscle tone. laboratory work-up revealed large proteinuria ( g/g creatinine), hypoalbuminemia ( g/l) and renal failure ( objectives. to investigate the incidence, nature, and management of associated genitourinary malformations in children with multicystic dysplastic kidney (mcdk). methods. in this retrospective study, we analyzed the medical records and imaging studies of children with mcdk. in children ( %) anomalies of the urinary tract were suspected prenatally in ultrasound studies. in the remaining children the diagnosis of mcdk was made postnatally. results. the male/female sex ratio was : . the left side was involved in ( %) children. voiding cystourethrography was done in ( %) children, the isotopic m tc-dmsa scan of the kidney in ( %). urogential anomalies were present in ( %) children. among them, contralateral urologic anomalies were found in patients (vesicoureteral reflux in and hydronephrosis in ), and ipsilateral in (vesicoureteral reflux in , ureterocele in , and hydroureter in ). genital abnormalities such as uterine didelphys and hydrocele were found in children. fourteen ( %) patients underwent follow-up examinations with ultrasonography (mean follow-up . years, range months to years). compensatory hypertrophy of the contalateral kidney was found in most children and decreased size of ipsilateral dysplastic kidney was found in out of children with follow-up. no cases of hypertension or tumor developed during the follow-up. conclusion. ultrasound can be used safely to diagnose unilateral mcdks and associated genitourinary malformation. although the risk of hypertension and development of malignancy is low, follow-up evaluation of contralateral renal function and genitalia will be needed. in cases of hydronephrosis and/or urinary tract infection, voiding cystourethrography is necessary and possibility of association with genital anomalies should be considered until the puberty. a. background: ectopic ureter is a rare anomaly. its incidence is at least four times higher in females and also more than % of the ectopic ureters drain duplicated systems in females. the most common presenting symptoms of an ectopic ureter are urinary tract infection and incontinence. diagnosis is often delayed and may remain undiagnosed until adulthood. case report: a -month-old girl was admitted to our hospital with the complaints of fever and discomfort. her mother recognized intermittent dribbling of urine while changing her napkin. physical examination revealed fever ( . °c), diaper dermatitis and intermittent dribbling of urine. urinalysis revealed leukocytouria, acute phase reactants were elevated (crp: . , esr: ) and renal function tests were normal. the patient was hospitalized with the diagnosis of acute pyelonephritis. tc m dimercaptosuccinic acid (tc-dmsa) scan revealed an increase in the size of the right kidney and decreased uptake in the upper half of the same kidney. ultrasound was performed with the suspicion of an ectopic ureter and it showed right duplicated kidney with marked dilatation of the upper collecting system. the ureter was also dilated in its whole length and ended ectopically distal to the bladder. contrast-enhanced magnetic resonance urography (mru) demonstrated right obstructed duplicated system with vaginal ectopic insertion of upper pole ureter. discussion: this case was presented to underscore the role of careful physical examination in the diagnosis of this rare anomaly that is by paying attention to the complaints of the family. ultrasound is the initial, important diagnostic modality in these patients especially if done by experienced radiologists. the diagnosis can be confirmed with mru by depicting the exact insertion of the ectopic ureter. objectives of study: to evaluate the occurrence and severity of vesicoureteral reflux (vur) in young infants with a history of mild prenatal hydronephrosis. the usefulness of voiding urosonography (vus) in the diagnosis of vur was also evaluated. methods: forty seven infants ( males, females) with a history of mild prenatal hydronephrosis, diagnosed between st to th week of gestation, were enrolled in the study. postnatal ultrasound was performed within the first month of life. voiding cystourethrography (vcug) and at the same time, contrast-enhanced harmonic vus was performed at the age of . - . months. results: the prenatal ultrasound revealed an anterior-posterior pelvic diameter of - mm in fetuses and - mm in . postanatal ultrasound showed an anterior-posterior pelvic diameter of - mm in infants and - mm in . vur was found in of ( . %) infants (grade i: , ii: , iii: ). the vur was detected by both vcug and vus in of children, only by vcug in and only by vus in of infants. the vur that was missed by vcug was more severe (grade ii and iii), compared with this one missed by vus (grade i). conclusions: even though prenatal hydronephrosis was associated with a quite important occurrence of vur, this was of mild or moderate severity. comparison between the two imaging modalities showed that the vur missed by vus were with no clinical significance (grade i), whereas the vur missed by vcug were more severe. although further study is needed, vus could be an alternative method, mainly in girls, in whom the imaging of the urethra is not necessary, thus avoiding the radiation exposure. early treatment with indomethacin in a neonate with a bartter syndromea case report neonatal bs is a rare genetic disorder characterized by sodium, potassium and chloride urinary wasting, hypokalemic metabolic alkalosis with hyperreninemia and hyperaldosteronism in the absence of hypertension and high level of urinary prostaglandins. indomethacin therapy is controversial because of toxicity for gut and kidney. a premature boy of unrelated couple was born by cesarean section at weeks because of early rupture of membranes and fetal distress. birth weight was g, length cm and apgar score / / . pregnancy was complicated by severe polyhydramnios. baby was mechanically ventilated for first hours and treated with antibiotics because of suspected sepsis. marked polyuria and dehydration were present from st week. metabolic parameters revealed hypokalemia, hyponatremia and hypochloremic metabolic alkalosis. serum creatinine was slightly elevated and gfr in normal ranges. blood pressure was normal with raised plasma renin activity and aldosterone level. sodium excretion via urine and level of renal and systemic prostaglandins were increased. nephrocalcinosis was detected on us from week. additonally to electrolyte supplementation indomethacin was started on the th day at a dose of mg/kg/day. in first months child experienced septic episodes, candida pyelonephritis and was operated because of bowel obstruction. dna analysis of affected child found new mutations in romk gene: p l and q x in herited from parents who carried one mutation each. at months height and weight are at and head circumference at percentile with slightly retarded psychomotoric development. he continues on . mg/kg/day indomethacin therapy. blood electrolyte profile is normal without supplements. us shows no progression of nephrocalcinosis. early treatment with indomethacin may prevent life-threatening complications and reduce the development of nephrocalcinosis. nimuselide, a cox inhibitor is widely used in india for relief of pain and fever. we describe cases of fetal renal abuse leading to neonatal renal failure due to maternal ingestion of nimuselide in the third trimester of pregnancy. results: all patients were diagnosed as having renal failure in the first few days of life.there were boys and one girl.. all the mothers had normal pregnancies except for oligohydramnios that was detected during the last months of pregnancy in all cases. children presented with anuria from birth. the remaining two had non-oliguric renal failure with metabolic acidosis as the presenting feature in one and poor feeding and lethargy in the other. usg revealed normal sized kidneys. one patient also showed increased echogenicity. out of the anuric children underwent peritoneal dialysis for a period varying from to weeks without recovery of renal failure. the remaining were managed conservatively. two of these patients are now in chronic renal failure at ages and years. only one patient recovered completely after days of anuria. all the mothers had taken nimuselide in the last trimester for periods varying from few days to several weeks for relief of pain or fever. some of them had taken multiple courses of short duration. the mother of the child who recovered completely had taken nimuselide in the last days before delivery. renal biopsy done in one baby revealed renal tubular dysgenesis. conclusion: nimuselide intake in the last trimester of pregnancy can be associated with oligohydramnios and neonatal renal failure that can be irreversible. renal tubular dysgenesis may be the underlying pathology. objectives of study: pkd is the most common inherited renal disease. aim of ours study was to analyse clinical and laboratory features of the different types pkd. patients and methods: we described the clinical presentation of children with pkd ( boys, girls) diagnosed in pediatric nethrology department between and . the patient's age range was from months till years. we retrospectively studied the family histories, clinical and biochemical data (physical examination, level of arterial blood pressure, blood and urine creatinine levels, serum levels of urea, glomerular filtration rate), ultrasonography, scintigraphy. results: the analysis of the family histories revealed adpkd in patients ( boys, girls), arpkd in ( boys, girls) and nondifferential pkd in ( boys, girls) children. pkd diagnosed by antenatal ultrasound in cases ( adpkd, arpkd). the mean follow-up adpkd were , year (range - years), arpkd - , year (range - , years). conclusion. patients with arpkd demonstrated the early beginning of an arterial hypertension and progressing chronic renal failure. one girl with neonatal form of arpkd died. chronic renal failure developed in ( , %) cases of pkd. objectives: this prospective study was to answer the question on the need of long-term follow-up and correlation of renal functions with the age at surgery and grade of o.u. prior to surgery in patients after surgery of obstructive uropathy (o.u.). methods: selected biochemical markers of glomerular and tubular functions and ultrasonographic findings in patients ( boys) who underwent surgery due to uni-or bilateral o.u. of grade iii. and iv. (age at surgery < months) in - were examined at mean age of . ± . years. the results were compared to healthy controls and/or to reference values according to age. consequently, patients were devided into groups according to the age at surgery ( - months, - months, - months) and grade of o.u. prior to surgery. results: serum concentration of cystatin c (s-cysc) was significantly higher in patients when compared to control group (p< . ). while s-creatinine was within reference interval in all patients, s-cysc was increased in . % when compared to reference interval. decreased tubular resorption and concentration ability was found in % and % patients, respectively. non-specific aminoaciduria was detected in . % patients. on ultrasound, . % kidneys after surgery had residual dilatation of renal pelvis. the differences in renal functions in patients according to their age at first surgery were not significant except for u-nag activity with significant negative linear trend with higher age at surgery. the grade of o.u. prior to surgery did not have significant influence on renal functions. conclusions: mild tubular dysfunction and slightly reduced gfr in the part of patients make longterm nephrologic follow-up reasonable. our results support the trend of postponing early postnatal surgical intervention in patients with positive ultrasound screening of o.u. and normocreatininemia. objectives of study: to evaluate, during almost five years of follow-up, the changes among preoperative and postoperative renal function in infants ( m/ f) with prenatal severe hydronephrosis (hn) and upj obstruction. methods: upj obstruction was diagnosed by a mag renal scan, performed at - weeks of age to establish baseline differential renal function; surgery was made if there was evidence of obstruction injury, and/or progression of hn and/or symptoms. the group was re-imaged months after surgery, after months, year and then annually. results: initial differential renal function was moderate in . % of males and in % of females and good in . % and in % respectively. also, % of kidneys required surgery because of declining function, with mean differential renal function in the affected kidney of % that improved to % already at months after pyeloplasty. there was no significant functional improvement, in the kidneys that underwent correction because of increased hn or symptoms and final renal function was > %. after pyeloplasty t was > min. in % and - min. in % of cases (p< . ) there was no statistically significant correlation between initial grade of hn and initial renal function. surgical treatment was performed between - months of age and there was no significant difference in postoperative results, ascribed to patient age at surgery. ma values, greater than controls at diagnosis, reduced at months after surgery in all, but % of children. during the follow-up, the mean ccr and bp values were in normal range for age in all children. conclusions: our findings showed improvement also in kidney with preoperative uptake less of %. this may be to explain in according to an inverse correlation between degree of renal dysplasia and gestational age. objective of study: is to determine the postnatal course and follow-up of children with fetal hydronephrosis. methods: in years period ( ) ( ) ( ) ( ) ( ) ( ) ( ) we followed infants with antenataly detected hydronephrosis. all infants were submitted to ultrasonographic examination of kidneys and bladder. if indicated, the isotope renography, micturating cysto-urethrography, i.v.urography and mr were performed. results: the diagnosis of hydronephrosis was established during - th weeks of gestation by obstetritian. first postnalat ultrasound investigation was performed during neonatal period in most children ( %). in ( , %) infants we diagnosed idiopathic hydronephrosis, in ( , %) vesicouretheral reflux (vur) grade ii-iv, in ( , %) hypofunction of one kidney, in ( , %) ureteropelvic junction obstruction (upjo), in ( , %) ren duplex and ureterocele, in ( , %) ampular pelvis and in ( , %) afunction of one kidney. after - months we found normal ultrasound in ( , %) children. the ultasound results were stable in ( , %) children and in ( , %) there was progression of hydronephrosis. four ( , %) infants underwent immediate surgery. conclusions: in a group of infants with antenataly detected hydronephrosis the diagnosis was confirmed postnataly in % infants. more than % of infants required long term follow-up. in , % the immediate surgey was required. this data support the need for antenatal detection of hydronephrosis. in the same period we followed up infants with urologic abnormalities which were not detected antenataly.the fetal ultrasound is reliable screening method in detection of urologic abnormalities. the considerable number of anomalies which were not detected antenataly are the result of insuffitient use of fetal ultrasound investigation. hemolytic uremic syndrome (hus) is defined by acute renal failure, microangiopathic hemolytic anemia, and thrombopenia. perinatal asphyxia (pa) may cause renal failure after birth and is often associated to disseminated intravascular coagulopathy (dic) with platelet consumption. however, no biological investigation permits to distinguish clearly neonatal hus from dic. we report three neonates with renal failure due to different degrees of pa. they presented biological features compatible with hus such as fragmentocytes ( %), thrombopenia (< , /mm ), anemia (< g/dl). serum creatinine on day was , , mmol/l respectively, requiring peritoneal dialysis in one patient. haptoglobin was undetectable for all three patients. factor h and i were in the lower normal range; components of the complement system (c , c ) and adamt activity were decreased. two patients received daily fresh frozen plasma infusions over the first weeks. renal function improved in two patients until day ; one patient has chronic renal failure. all other parameters suggestive for hus were normal on day , , and respectively. no severe neurological consequences were noted for either of them. pa may be responsible for multiorgan damage via ischemic lesions. ischemia may result in endothelial cell injury, the crucial event for the development of thrombotic microangiopathy. we hypothesize that endothelial cell damage concomitant with pa may lead to a vicious circle resulting in consumption of platelets and plasma factors involved in hemostasis and/or fibrinolysis. early use of fresh frozen plasma may correct these alterations. renal biopsies might have been useful but are technically difficult in newborns. in conclusion, pa and neonatal hus are difficult to distinguish and endothelial cell damage may be a common pathyphysiological aspect and might requirespecific treatments. a. medynska, m. nalesniak, k. kilis-pstrusinska, d. zwoliñska congenital posterior urethral valves (puv) are the most common cause of lower urinary tract obstruction in male neonates. we aimed to review our experience with puv children ( boys) in respect to retrospective analysis of the clinical course of disease. we analyzed: ultrasound during pregnancy, age of disease onset, clinical symptoms at admittance to hospital, outcome. average lenght of follow-up was , years, varying between month and years.obstruction of urinary tract was suspected by prenatal ultrasound in patients. the initial presenting symptoms were as follows: urinary tract infection boys, failure to thrive patients, increased abdominal circumference - , abdominal pain , enuresis - , acute renal failure , and chronic renal failure children. in association with puv renal dysplasia/hypoplasia in patients, undescended testis , bladder trabeculation were found. the diagnosis of puv was confirmed by voiding cystourethrograms and/or cystoscopy. primary vesicouretral reflux was documented in pts. hydronephrosis and/or megaureter were observed the most often in boys. the diagnosis of puv was established in pts at the age of less month, in pts between - months, and in between - years. surgery was performed in pts in neonates period including primary valve surgical ablation and/or cutaneostomy vesicostomy. chronic renal failure was diagnosed in boys in first year of live. of them progressed to end-stage renal disease. globally during the follow-up pts developed end-stage renal disease. pts have done a graft. only boys survive without progression to chronic renal failure. the presentation of puv is variable and currently antenatal detection is the most common mode. outcome boys with puv is poor. patients need nephrologic assesment from birth. background: furosemide is among the most frequently used drugs in the neonatal unit but few studies analyze the beneficial effect and complications in this patient group. objectives of the study: to analyze the therapeutic clinical effect and to document side effects of furosemide therapy in extremely preterm infants born < weeks gestational age (ga). methods: twelve infants born < weeks ga were prospectively included during the fall . the following parameters were documented prior, during and after furosemide administration: clinical status, serum/urine electrolytes, creatinine, albumin, blood gases and furosemide exposure. ultrasound of the kidneys and a wrist radiograph were performed at - weeks to rule out osteopenia/rickets and nephrocalcinosis respectively. no statistical analysis were done due to the small study size. results: general oedema, respiratory rate, apneic spells and oxygen supplementation decreased. arterial/venous pco decreased and partial oxygen saturation increased indicating improved lung function. hco increased and ph decreased. urinary excretion of sodium, potassium, chloride and calcium increased while phosphate excretion decreased. serum sodium and chloride decreased and potassium increased initially. six infants had electrolyte disturbances and metabolic alkalosis. one infant died during the study period. in the remaining infants, of had worsening of their patent ductus arteriosis, had osteopenia or rickets and had nephrocalcinosis. the total side effect score was increased in the infants with the highest furosemide exposure. conclusions: this small study suggests that furosemide is beneficial in extremely preterm infants born < weeks ga and that the associated side effects correlate to the total drug exposure. we recommend caution for long term administration of furosemide. conclusion: although children in our study suffered significant neonatal hie resulting in arf, glomerular and tubular function recovered sufficiently to cope with increasing body mass and metabolic needs. unlike reported studies, we did not find any significant evidence of cri in the survivors of neonatal hie and arf. one with marginal microalbuminuria will need further observation. c was born after a monozygotic monoamniotic twin pregnancy (gestational age= weeks). she presented with twin-twin transfusion syndrome with hypotrophy (birth weight= g, other twin= g), severe hypovolemia, anemia and acute renal failure. she required a blood transfusion, mechanical ventilation ( days), and peritoneal dialysis ( days). she recovered without bronchodysplasia but with chronic renal failure (creatininemia at months= μmol/l). the following months were uneventful but, as usual in tts, she exhibited growth retardation and slight mental delay compared to her twin. she reached terminal renal failure at years of age and was successfully kidney transplanted. after age of years, she presented with increasingly frequent and severe pulmonary infections predominantly in lower lobes of both lungs. after extensive explorations, the diagnosis of bilateral bronchiectasis was made. the search for classical aetiologies of such pathology was negative. the symmetric aspect and the absence of other etiologies lead to the consideration of bronchiectasis as a congenital pathology. numerous publications demonstrate the role of the renin-angiotensin system (ras) in renal and cerebral damages in tts. authors demonstrate the role or ras in development of vasculature in fetus but also of cartilage and muscle in different organs including lung. associations between tts and lung pathologies need to be further investigated. urofacial ( the urofacial (ochoa) syndrome (ufs) is a rare disease that occurs in both sexes and is more frequent when the parents are closely related. it has both urinary and facial abnormalities. ufs is a rare autosomal recessive disorder and a potential gene has been mapped to chromosome q -q . they present a bladder voiding dysfunction due to impaired neural communication between the bladder and the spinal cord, resulting in incomplete emptying of the bladder. this usually results in enuresis, urinary tract infection, hydronephrosis and in some severely affected patients, end-stage renal disease developed. the facial abnormality is a characteristic expression that, when these patients smile, their facial musculature inverts and they appear as if they are crying. we report a year-old girl who has inverted facial appearance, voiding dysfunction and vezicoureteral reflux. she had constipation and did the intermittan uretral cathaterization for five years. after a detailed evaluation, she was diagnosed as ochoa syndrome due to inverted facial expression. we report this case, because early diagnosis of ufs is very important for early assessment and management of urinary problems to prevent development of chronic renal failure. we think that only a smile can give a strong high light for this unusual 'inverted' facial expression and patients can be screened earlier for severe voiding dysfunction. tar syndrome is a congenital malformation syndrome characterized by bilateral absence of the radii and a thrombocytopenia. the known urinary anomalies with tar are duplex ureter, dilatation of renal pelvis, horseshoe kidney, and functional problems like vesicouretheral reflux and pyelonephritis. case: nine years old patient with tar syndrome was admitted with a complaint of bright red urine repeated three times. the microscopic hematuria without dysmorphism of erythrocytes accompanied with no proteinuria were determined in repeated urine sample microscopy. iga, ana, anti dna serology were negative. urine culture was clean. stone formation ( x mm) in the upper pole of the right kidney was established by the abdominal ultrasonography. postoperative chemical analysis of the stone, revealed that it was consisted of oxalate monohydrate and dihydrate. but the patient discontinued his follow-ups afterwards for a year. in this period, he had macroscopic hematuria attack once. when he applied for the second time, he reported macroscopic hematuria. cystoscopy was done for etiology. many tortuous and engorged vessels were seen by this evaluation in the bladder mucosa ( figure ). there was no active bleeding point. result: in this report, kidney stone and telangiectasia found co-incidentally in the bladder of a patient with tar syndrome during the examination of hematuria are discussed as there is no case report demonstrating nephrolithiasis and telangiectasia in tar patients in the literature. figure : many tortuous and engorged vessels in the cystoscopy. antenatal hydronephrosis (anh) is one of the common fetal abnormalities detected on ultrasound (us). the long-term renal prognosis for infants with mild to moderate postnatal hydronephrosis (hn) is unclear and controversial. a systematic review of the published literature was performed to determine an evidence based approach in infants with antenatally diagnosed hn and to identify those at risk of significant post natal nephro-uropathy (pnu). key questions were identified. does anh predict renal tract pathology in neonates? what is the value of prophylactic antibiotics in infants with anh? can postnatal us diagnose significant pnu? when is the optimal time to screen infants postnatally? which imaging modalities are necessary to diagnose the cause of the hn? how many neonates with anh would need to be investigated to prevent one case of esrf/crf? a search strategy was formulated. out of titles only seven studies met the validity criteria for inclusion. the results indicate that antenatal us is a valid screening test for pnu (sensitivity %, specificity %), but is not a predictor of pnu. the detection rate of hn by us is the same whether it is done early or late in neonates. two normal ultrasounds over a minimum of one month are required to screen for pnu. infants with a renal ap diameter over mm are at risk of a significant pnu and should be investigated further. us is not a substitute for cystourethrogram or dynamic isotope studies to determine the cause of hn. none of the studies addressed the role of prophylactic antibiotics. there was insufficient data to calculate the total number of cases that would need to be investigated to prevent one unfavourable outcome (esrf/crf). high quality population based cohort studies with long term follow-up into adulthood are required to determine the optimum postnatal management of mild to moderate hn in infants with anh. jeune asphyxiating thoracic dysplasia: a case report jeune asphyxiating thoracic dysplasia (jatd) is one of the congenital hepatorenal fibrocystic syndromes. it is characterized by renal, hepatic, pancreatic abnormalities with associated skeletal abnormalities including a long and narrow thorax, metaphyseal irregularities, and shortness of the ribs and long bones. this report describes a pediatric patient with jatd developed end-stage renal failure. a -year-old boy was admitted with complaint of vomiting and pallor. he had dysmorphic appearance including trigonocephaly, short stature, long thorax and short limbs and fingers, polydactyly and fascial dysmorphism. laboratory findings revealed severe anemia (hb . g/dl), high bun and creatinine levels ( mg/dl and . mg/dl respectively) and normal liver tests. abdominal usg showed a severe intrahepatic biliary tract dilatation and intraparenchymal cysts in liver, pancreas and kidneys. mr pancreatocholangiography was consistent with caroli's disease. jatd should be considered when caroli's disease exists with skeletal abnormalities. follow-up antenatal hydronephrosis: one centre experience the most common renal abnormality detected antenatally is hydronephrosis ( % to % of all pregnancies). in this paper, we report follow-up our patients with antenatal hydronephrosis (ah) between and . diagnosis of hydronephrosis was made > mm of antero-posterior diameter (ap) of the renal pelvis. ah was detected in patients on antenatal ultrasound examination. of the patients with ah, ( . %) were found to have hydronephrosis postnatally and ( . %) postnatal scans were normal. in of these patients ( . %) had bilateral and ( . %) of these patients had unilateral hydronephrosis. in these patients with ah, uretero-pelvic junction obstruction (upj) ( . %), reflux ( . %), uretero-vesical junction obstruction ( . %), posterior urethral valves ( . %) and mega-ureter ( . %) were identified. in follow-up period, ( . %) patients with reflux, ( . ) patients with upj were treated with surgery (p< . ). in conclusion, upj was most important cause of ah and most of them were treated with surgery. hyponatremia and renal tubular acidosis in severe vesicoureteral refluxa case report sahlgrenska academy, department of pediatric, gothenburg, sweden case report: st child to healthy parents without heredity for kidney or metabolic disease. antenatal bilateral hydronephrosis. postnatal examination showed bilateral vur grade v, normal urethra. antibiotic prophylaxis was started, no urinary tract infection (uti) occurred. normal s-creat and s-na + at weeks of age. the electrolytes remained normal during the next months. normal growth. the boy was thirsty with high urine volumes. at month of age feeding problems began with retarded weight gain. no vomiting or diarrhoea. at admission to the hospital the child was dehydrated, s-na + mmol/l, s-cl - mmol/l, s-ph . , bicarbonate mmol/l, s-creat μmol/l. crp mg/l, urine culture negative. u-na + mmol/l, u-ph . , u-osmolality mosm/kg. the anion gap was normal and there was no lactacidosis. the s-aldosterone was elevated , nmol/l, s-cortisone normal. treatment included intravenous rehydration, na + supplement and oral bicarbonate. blood chemistry normalised and the child's general condition improved rapidly. conclusions. children with severe reflux are at high risk for uti but may also develop impaired tubular function with diabetes insipidus, renal tubular acidosis and hyponatremia. the mechanisms include down-regulation of vasopressin receptors and impaired distal tubular function. close clinical monitoring of these children with regular blood chemistry and weight controls is important. purpose: we aim to prospectively study the natural history of minimal to severe grades of antenatal hydronephrosis (anhn) in our local chinese population and correlate the renal pelvic diameter (rpd) with the outcome. patients and methods: cases of anhn were prospectively followed up along a predefined protocol using us, mag and mcu in all. obstruction (pujo or vujo) was defined as a need for surgery based on symptoms and deteriorating function, not on mag drainage time. results: neonates were followed up for minimum of years. eighty percent had normal or minimal hn on postnatal scans (rpd - mm), . % had mild ( - mm) or moderate ( - ) hn and . % severe hn (> mm). seventy-eight percent infants had benign course; . % had partial obstruction which improved; . % had vesicoureteric reflux (vur) one of which required surgery. another . % required surgery for obstruction. the roc curve for obstruction requiring surgery showed optimal cut-off point of . mm (sensitivity %, specificity . %). prolonged diuretic t / was not predictive of surgery. severity of hn did not correlate with presence or grade of vur. fifteen patients developed uti despite antibiotic prophylaxis and had focal scars, all occurred in association with high grade vur or obstruction. the prognosis of infants with minimal or mild anhn is good. however rpd is poorly correlated with vur. the chances of obstructive lesions requiring surgery are high when the rpd is above mm. in those with high grade reflux or obstruction, urinary tract infection may lead to renal scars. objectives of study: cystinosis is a rare disease presented initially with renal fanconi syndrome, and renal glomerular failure develops later in childhood. without cysteamine treatment, patients affected with cystinosis uniformly died during childhood in the absence of renal replacement therapy (rrt). cysteamine is not available here and in some other areas of the world. the aim of this paper is to describe a beneficial effect of acacia gum in a patient with cystinosis and chronic renal failure. method: years old girl with cystinosis presented with symptomatic uremia as she didn't receive cysteamine. serum creatinine . mg/dl, blood urea mg/dl. the girl was hospitalized and vomiting controlled with intravenous fluid and pyridoxine. chronic dialysis was not available for her and the parents refused treatment with intermittent acute peritoneal dialysis. the girl was treated with a new therapeutic regimen (therapy ; ( ): ) combining the traditional conservative management of crf (dietary and pharmacologic) with addition of acacia gum (ag) g/day as an urea lowering agent aiming at improving her condition without dialysis. results: treatment was associated with amelioration of the uremic symptoms and improved general well being. after weeks of treatment, serum creatinine . mg/dl, blood urea mg/dl. during months of follow-up she continued in experiencing improved well being and urea levels was kept below mg/dl without dialysis. conclusion: it was possible to improve the health of patient with cystinosis despite the nonavailability of cysteamine and the appropriate rrt. objectives of study: the pattern of renal tubular disorder (rtds) has been infrequently reported in the literature, and the pattern of rtds in iraqi and arab children is not known. methods: from june to august , it was possible to evaluate children with suspected rtd to determine the type of their tubular defect. there was evidence of rtd in only patients; males ( %) and females ( %). their ages at referral ranged between months and years (mean . years). in patients with oculo-cerebro-renal syndrome, there was no evidence of rtd and one patient had hyperoxaluria which not a rtd. results: seven types of rtds were identified. the three most common disorders were: idiopathic hypercalciuria ( %), cystinosis ( %) and renal tubular acidosis rta ( %). four of the patients with rta have proximal rta, and four have distal rta. four of the patients with hypercalciuria have also significant hyperoxaluria > mg/kg/day. conclusion: the pattern of rtds in iraqi children differs from the previous studies: in germany the three most frequent disorders were cystinosis, xlhr, and idiopathic hypercalciuria. objectives of study: few literatures reported the incidence of ocular abnormalities in chronic renal failure (crf). the aim of this paper is to determine the incidence of ocular abnormalities in childhood crf. methods: from january to december , patients with crf (at the university hospital in al kadhimiyia) were examined to determine the presence of ocular abnormalitites. fifty patients were males ( . %) and ( %) were female. their age at referral ranged from months to years (mean year). they were followed for a period ranged from days to f years. results: corneal cystine crystals were the most common ocular abnormalities associated with childhood crf observed in patients with nephropathic cystinosis ( . %). congenital cataract & glaucoma were observed in patients ( . %) with oculo-cerebro-renal syndrome (ocrs). congenital cataract & chorioretinal hypoplasia were present in patient with ocrs. hypertensive retinopathy occurred in patients. acquired cataracts occurred in one patient with hinman syndrome in association with hypocalcaemia and non-compliance with calcium and onealphacalciferol supplementation. retinitis pigmentosa in one patient with laurence moon biedl syndrome. bilateral optic atrophy in one patient with familial nephropathy associated with club feet. proptosis in one patient with membranoproliferative glomerulonephritis. conclusion: ocular abnormalities are relatively common in childhood crf occurring in approximately %. objective: hypocalcaemia has been reported as a complication of phototherapy especially in neonates. we studied the relation between serum calcium level and urine calcium to creatinine ratio in neonates under phototherapy. method: icteric newborns ( males and females) treated by phototherapy entered into study by non accidental sampling. the consent was taken from parents on admission. all were breastfed healthy newborns. weight was checked and serum samples for calcium and bilirubin and urine aliquots for calcium, creatinine and osmoloality were sent on arrival (group i), after hour of starting (group ii) and hour after discontinuing phototherapy (group iii). hypercalciuria was defined by uca/ucr > . , hypocalcemia was defined by serum calcium < mg/dl in the term and < mg/dl in the premature. chi , anova, wilcoxon rank test and spearman were used to compare frequency, means, median and correlation. p< . was considered significant. two groups were designed, pateints whose therapy were finished at least months (group ) and those either on therapy or less than months passed from the last protocol of cytostat (group ). demographic data, cumulative dosages of anticancer drugs, history of other nephrotoxic agents, nephrectomy, radiotherapy and acute renal failure were recorded. we used ctc ( ) to evaluate renal function. chi and mann whitney u test and biniary logistic regression were used to compare percentage, scoring and correlation respectively. p value less than . was considered significant. result: out of patients were in group and ones were in group . the mean of age was . years (± . sd). the median (range) of therapy and termination was months ( - months) and month ( - month) in group and month ( - ) and months ( - months) in group respectively. the percentage of reversible renal failure, proteinuria, abnormal serum calcium and magnesium, metabolic acidosis and urinary concentration defect was higher in group . (table ) these differences were statistically significant (p< . ). we found no correlation between ctc score and dosage of drugs, age, sex, history of radiotherapy or nephrotoxic agents (p> . ). conclusion: mild to moderate tubular dysfunction has been observed in survivors of leukemia. routine follow-up of renal functions is recommended. v. tramma, k. giourtzis, v. fotoulaki, k. nousia-arvanitaki aristotle university, th pediatric clinic, thessaloniki, greece although cftr is expressed in the kidney, patients with cystic fibrosis (cf) have not been reported to have major renal abnormalities with the exception of urolithiasis. the aim of this study was to determine renal function and the potential risk factors for renal stone formation in cf patients older than years of age. the findings of metabolic evaluation of cf patients having confirmed urolithiasis (mean age: , ± , ) were compared with those of cf patients without urolithiasis (mean age: , ± , ) and those of healthy volunteers (mean age: , ± , ). evaluation included plasma sodium (na), potassium (k), chloride, bun, creatinine, uric acid, calcium (ca), phosphorus (p), magnesium (mg) and parathormone (pth). twenty-four hour urine collection for creatinine, uric acid oxalates, ca, mg, k + , na + and microalbuminuria was also performed. glomerular filtration rate (gfr) was calculated and fresh urine samples were examined for the presence of crystals, erythrocytes, glycosuria and microorganisms. patients with cf and urolithiasis showed significantly increased values of bun (p: . ), pth (p: . ) and gfr (p: . ), very low urine mg levels (p: . ) and microalbuminuria (p: . ) as compared to cf patients without urolithiasis. there was no correlation of urolithiasis with hypercalciuria and hyperoxaluria. furthermore, all cf patients showed significantly increased pth levels (p: . ), very low urine mg levels (p: . ) and microalbuminuria (p: . ) as compared to healthy volunteers. conclusions: renal dysfunction was demonstrated in older cf patients, probably, secondary to the primary defect of renal chloride channels. extracellular volume regulators, such as hormones, may also be implied. urolithiasis may be the result of renal dysfunction. conclusions: the morbidity of hsp had obviously increased in recent years. the familial cases, the initial symptoms of no palpable purpura at onset and the cerebral, pulmonary, cardiac and pancreatic involvement should be paid attention. objectives of study: systemic lupus erythematosus (sle) is an autoimmune disease affecting multiple organs and tissues including central nerve system, cardiovascular system and kidney. although etiologic mechanisms of sle are incompletely known, overproduction of immunoglobulin g autoantibody may contribute to onset of this disease. while still incompletely understood, the etiology of systematic sle is considered to involve both genetic and environmental factors. we encountered two boys with severe sle from unrelated families and analyzed polymorphisms of the gene that encodes cytotoxic t-lymphocyte associated (ctla)- , a protein important in t-cell activation and immune tolerance. abnormal function of the gene may participate in causation of autoimmune disease including sle, resulting in production of immunoglobulin against various self-antigens. case report: in family , a boy showed serious cardiovascular complications associated with heart failure while his mother also had clinically active sle including nephritis. a boy in family developed severe renal complications and peripheral vasculitis accompanied by disseminated petechiae in the lower extremities; his paternal grandfather had died from fibrinous pneumonia caused by sle. results: analysis of the ctla- gene indicated that the boy in family and his mother possess a gg genotype in ctla- exon at + together with a -bp fragment length of the ' untranslated region (utr) in exon . the boy in family also showed gg at + . no association with disease activity was found for polymorphism of the promoter region in exon at - in either family. conclusions: disorders of the ctla- gene, especially a gg genotype in exon at + and/or bp fragment length of the 'utr in exon , may be involved in early development of sle in japanese children such as the boys described here. this disorder is transmitted mainly as x-linked trait, and is caused by mutations in the col a gene encoding α chain of type iv collagen. in some families, x-linked as is associated with diffuse leiomyomatosis. we present clinical, pathologic and molecular-genetic findings in japanese family with this inheritance mode of as in association with leiomyomatosis. case report and results: as was diagnosed in a one-year-old boy with recurrent aspiration pneumonia caused by esophageal stenosis from leiomyomatosis. he had macroscopic hematuria and bilateral cataracts. diagnosis was confirmed by electron microscopy coupled with type iv collagen chain subtype staining in a renal biopsy specimen. thickening and irregular contours of the glomerular basement membrane (gbm) and splitting of the lamina densa were evident by electron microscopy. immunofluorescent staining for type iv collagen chains failed to show staining for α (iv), α (iv), or α (iv) in the gbm, associated with lack of α (iv) and α (iv) staining in the bm of bowman's capsule. his mother, who exhibited esophageal leiomyomatosis and is heterozygous for as, showed a discontinuous staining pattern for α (iv) along the epidermal bm. genetic analysis in the boy revealed the deletion of the first two exon of col a together with deletion of the ' end of col a . conclusion: identification of an as patient during infancy is extremely rare. clinical manifestations, including macrohematuria, cataracts and leiomyomatosis caused by the large deletion involved col a to col a , led to early presentation with as. functional voiding disorders of the bladder occur in the absence of any anatomic/neurological abnormality and present with wetting. invasive urodynamic studies are discomforting, not easily available in emerging countries and costly. this study aims to validate non-invasive urodynamics. children below years, with possible voiding disorders evaluated prospectively. non-invasive evaluation included history, examination, frequency volume charting, ultrasonography, urinalysis and renal functions. micturating cystourethrogram was carried out for children with urinary tract infection. all children underwent invasive urodynamic studies (uds) and the significance of association of the parameters of non-invasive assessment with invasive urodynamics was determined. the chi square test was used for statistical analysis using the epi software. children underwent invasive uds. the commonest abnormality was detrusor instability (di) in ( . %). dysynergic voiding (dv) noted in ( . %), lazy bladder in and an occult neurogenic bladder in . the study was normal in . repressing the disease progression may be . mg/kg/day or more background: henoch-schoenlein purpura is classified into the small vessel vasculitis. there may be no reliable indicator of the disease activity. steroid treatment ( mg/kg/day of prednisolone) has been thought of as a means with which alleviate abdominal pain. however, this dose seemed to be not effective to intervene the disease progression to nephritis. objectives: forty-three japanese children with henoch-schoenlein purpura were enrolled in this study. fibrinogen degradation product e-fraction (fdp-e) value was measured once or twice a week in the patients. coagulation factor xiii was simultaneously measured in of the patients in the early phase of illness. with an aim to alleviate abdominal pain, . - . mg/kg/day of prednisolone had been administered to of the patients. results: at presentation, only of the patients had low factor xiii activity. on the contrary, of them had elevated serum fdp-e value. longitudinal fdp-e measurement revealed that patients whose fdp-e value normalized within the second weeks of illness had minimal risk of nephritis. in this group, of had microhematuria. in the other patients group with prolonged (after fourteen days of illness) elevation of fdp-e values, of had nephritis. furthermore, of had proteinuria after three months of illness. these patients who had received prednisolone therapy with less than . mg/kg/day in the early phase of illness. the other patients with . mg/kg/day or more prednisolone therapy had no nephritis. summary: the disease activity of hsp and hspn might reflect the duration of elevated serum fdp-e. more than . mg/kg/day of prednisolone may repress the disease progression to nephrits. background: all major organs are involved more or less in thalassemia and most of them have been studied thoroughly in previous literature. renal system involvement has not been scrupulously scrutinized yet. method: in a randomized prospective study, renal findings of children and young adults, aged - years, with thalassemia major (group ) were compared to other cases of thalassemia intermedia (group ). blood urea nitrogen, serum creatinine, uric acid, calcium, phosphate, urinalysis, and sonographic findings were evaluated. results: mean age was ± . years in group and ± . in group . mean serum ferritin level was ± ng/ml in group vs. ± . ng/ml in thalassemia intermedia group (p< . ). % of subjects of group had received or was on hydroxyurea at the time of evaluation. serum uric acid was significantly higher in patients with thalassemia intermedia ( conclusion: significant renal involvement is not a frequent complication in children and young adults suffering from thalassemia. hyperuricemia and microscopic hematuria is more common in thalassemia intermedia than thalassemia major. case: a -year-old female patient was born at term (weight g, lenght cm) and for aspiration of amniotic fluid required resuscitation and mechanical ventilation for days. perinatal hypoxia was a cause of her acute renal failure but dialysis was not needed. patient's follow-up during next yrs showed mild form of chronic renal failure (crf) without hypertension: serum creatinine (scr; range during follow-up [rdf] - μmol/l), glomerular filtration rate (gfr; rdf - . ml/min/ , m ) and p (rdf - mg/ h). at the age yrs we performed renal biopsy (rb) because girl's p and scr increased ( mg/ h, μmol/l, respectively) and gfr decreased ( ml/min/ . m ). rb showed c q-nephropathy (c qn) with focal glomerular sclerosis and hyalinosis. c qn is a rare disease and as a first diagnostic step is differentiation against lupus nephritis. progression of c qn to crf is infrequent. probably two renal diseases were a cause of crf in our patient -hypoxic renal damage during neonatal period and c qn. objective: renal dysfunction has been reported in survivors of neoplastic disease. early diagnosis of renal damage may decrease the morbidity in those with partial or complete remission. we studied the frequency of nephrotoxicity in pediatric patients whose therapy were completed. method: pediatric cancer patients ( f, m) who were at least one year off therapy, enrolled in a prospective cross sectional study from to in oncology department of ali asgar children hospital. demographic data, cumulative dosages of anticancer drugs, history of other nephrotoxic agents, nephrectomy, radiotherapy were recorded. fasting blood and urine samples were collected to calculate fractional excretion of mg, ca, p, upr/ucr, clcr, urine osmolality and blood gas analysis. result: the mean of age was . years. out of patients had lymphoproliferative malignancies (group ) and had solid tumors (group ). the mean of therapy was . month. treatment was discontinued for . month in average. the median of blood ca, p, mg, bicarbonate,and cr were . mg/dl, . mg/dl, . mg/dl, . meq/l and . mg/dl, respectively. the median of fractional excretion of ca was . % this rate was . % for p excretion and . % for mg excretion. clcr was . ml/min/m in median. the medians of urine osmolality was mosmol/kg/h o. the median of urine protein to urine creatinine ratio was . mg/mg. these values were not different between two groups (p> . ) but urine concentration was defective in solid tumors group (p= . ). mild to moderate nephrotoxicity was seen in . % of cases. using binary logistic regression we found no correlated factor (p> . ). conclusion: mild to moderate tubular dysfunction has been observed in survivors of chemotherapy. routine follow-up of renal functions are recommended. the study is to discuss the treatment of hemolytic uremic syndrome (hus) after acute stage. methods: there were children who lived through acute stage of hus then continued treating. besides angiotensin converting enzyme inhibitors (acei) and early restriction of protein intake, the study was to use therapeutic schedule according to clinical classification, response to prednisone and pathological manifestation, which referred to clinical classification diagnosis and treatment of child with glomerulus disease (the program) established by nephrology group in pediatric branch of chinese medical association (cma). results: after months to years follow-up mild type children maintained the normal blood pressure and renal function and urine examination, except for recurrence. in gravis type children maintained the normal blood pressure and renal function and urine examination, another children who manifested as durative abnormality of urine examination developed into end stage renal failure (esrd) and died in the th, th and th month at last. another gravis type children untreated after stage of hus died in the th day to th day of the course. conclusion: it could improve the prognosis of children after acute stage of hus evidently to use therapeutic schedule according to clinical classification, pathological manifestation. objective: to find the prevalence of hematuria in patients with thalassemia major. methods: of total patients with thalassemia major under regular blood transfusion, cases were randomly selected. history was reviewed and physical examination was done. urinalysis was performed in all the patients. in those with hematuria ( or more rbc/hpf) or suspicious to hematuria ( - rbc/hpf) second urinalysis was done at the next transfusion time. more investigations were done in those with persistent hematuria. results: the patients had age range of months to years and male to female ratio was . . hematuria was detected in ( %) and suspicious in patients ( . %). sixty four percent of the patients with hematuria were female and it was persistent in urinalysis in % of cases. in % of the patients with hematuria, blood transfusion was started before the end of first year. in those with hematuria or suspicious to hematuria, % had sterile pyuria and % had proteinuria (these figures were . % and . % respectively, in those without hematuria). hypertension was not detected, but patients had secondary diabetes mellitus. conclusion: hematuria is not uncommon in patients with thalassemia major and is more prevalent in girls and in those with early transfusion. background: it has been widely recognized that cyclosporine a (cya) is a useful immunosuppressive drug in renal transplantation. although it has been also accepted that cya is an effective drug for pediatric nephrotic syndrome in the past two decades, the effective serum concentrations are not revealed. the functional roles of cya has been reported that cya inhibits the production of interleukin (il- ) in vivo and in vitro. aim: in this study, we investigated the correlation of serum concentrations of cya levels with il- levels in pediatric nephrotic syndrome cases. methods: seven children ( boys and girl, mean age . ± . years) with minimal change nephritic syndrome were enrolled in this investigation. cya (mean dose, . ± . mg/kg/day) was administrated in two divided doses before meal with or without administration of predonisolone. blood samples were collected just before, , , and hours after administration of cya. the serum concentrations of cya and il- were measured immediately. results: the peak blood concentrations of serum cya were observed at hour after administration. the concentrations of serum il- levels reduced at hour after administration of cya, and kept the same levels during hours afterwards. the serum concentrations of cya which inhibited more than % of the serum concentrations of il- required ng hr/ml. conclusions: we confirmed that cya inhibited the production of il- in children with nephrotic syndrome. these findings suggest that the necessary serum concentrations of cya to maintain the sufficient suppressive rate were more than ng hr/ml in pediatric nephrotic syndrome. this study aimed to evaluate the circulating angiotensins in female adolescents with type diabetes (dm ) and to compare with the results obtained in healthy age-matched adolescents to disclose possible changes in plasma peptide concentrations that could be related to microalbuminuria and metabolic control. patients were divided as female adolescents with dm (n= ) and adolescent age-matched controls (n= ). diabetic patients were evaluated at our endocrinology center and healthy adolescents were selected from our primary care unit. plasma levels of angiotensin (ang) i, ang ii and ang-( - ) were determined by radioimmunoassay. glycohemoglobin and microalbuminuria were also measured. results were expressed as medians or means and standard deviation. kruskal wallis was used for median comparisons and t-test for means. the level of significance was p< . . adolescent dm patients exhibited high levels of glycohemoglobin ( . ± . %). microalbuminuria was detected in ( %) patients with a disease duration of . ± . years. angiotensin concentrations were significantly increased in dm patients (p< . compared to controls) and ang-( - ) levels were -fold higher than control values. on the other hand, the levels of ang-( - ) in microalbuminuric patients were significantly lower than in non-microalbuminuric diabetic adolescents (p< . ). the comparisons between ratios of ang-( - )/ang i and of ang ii/ang i suggested a predominance of ang ii formation rather than ang-( - ) in microalbuminuric diabetics when compared to normoalbuminuric patients. our results showed an overall increase of angiotensins in a young female diabetic population, and further suggested a pathophysiological role for angiotensin-( - ) in dm . the pediatric nephrologist is often faced with the difficulty of determining adequate iron supplementation in children with chronic kidney disease (ckd). soluble transferrin receptor (stfr) and the stfr to log(ferritin) ratio (stfr-f index) have been proposed as markers of iron deficiency (id) independent of inflammation; however, their relationship with c-reactive protein (crp) and their age dependency have not been established. we therefore embarked on a prospective study of healthy children undergoing minor surgery to determine reference ranges for stfr (dade behring n-latex stfr analyser, dade behring bn prospec) and stfr-f index. we studied the relationship between crp and ferritin, transferrin, stfr and stfr-f index. we also compared the relationship between mean corpuscular volume (mcv) and ferritin, stfr and stfr-f index in children. results: for ages . . , . ) with mcv, which we used as a marker of id in the absence of a non-invasive gold-standard; however, only stfr-f index, but not ferritin, transferrin and stfr, was independent of crp. this study shows that ferritin, transferrin, and stfr, but not stfr-index, are dependent on acute phase reactions. it is therefore hypothesized that stfr-f index provides a more useful marker for monitoring the iron status in ckd patients. conclusions: low osmolality is a crucial factor to facilitate water absorption at least in the rat small intestine, while the absorption of sodium may be influenced by the concentration of sodium and glucose. (definition iccs). standard treatment consisted of general advice on voiding and drinking habits, alarm treatment and occasionally vasopressin. constipation was diagnosed on clinical considerations (history, stool charts, physical examination, occasionally x-ray). all patients received general instructions according to bowel habits. laxatives were prescribed when the patient was diagnosed as constipated. treatment goal was daily bowel movements. treatment results were evaluated months and years after discharge. results: patients were included. mean follow-up was . yrs. overall success rate (full response) was . % ( months) and . % ( years). laxative use: . % (n= ) of the patients received laxatives, % (n= ) did not. in patient information was lost. there was no significant difference in success rate between the laxative group compared with the non-laxative group (p= . , chi-square). treatment modality: . % (n= ) received general advice only without laxatives, all but one had a full response. . % (n= ) were treated with advice and alarm, ( . %) of them received laxatives. response to alarm treatment was . %. no significant difference in success rate between the laxative and non-laxative group (p= . , chi-square). patient was dry after vasopressin and laxatives. conclusion: the majority of patients with mse can achieve nocturnal continence without laxatives. constipation treatment with laxatives may be supportive, but is not essential in the treatment of mse. aim: hypocalcemic tetany is a known complication of plasmapheresis. we studied the changes in ionised and total calcium, and magnesium concentrations during plasmapheresis, with and without supplementing the replacement fluid with calcium and magnesium. methods: plasmapheresis was carried out by using . % human albumin solution (has) with or without supplements for the first % of the exchange, and fresh frozen plasma (ffp) for the last %. we measured ionised and total calcium and total magnesium at the beginning and end of the has, and after minutes of ffp infusion. results: we undertook pairs of plasmapheresis runs with and without supplements in children who had a variety of renal conditions. during the exchange with unsupplemented has, the total calcium fell from . to . mmol/l (ci . - . , p< . ), the ionised calcium fell from . have raised significant problems in minor surgeries. but the developmental mechanism of ponv is not clear until now. previously, we have experienced a case with ihn and ponv who showed extremely high plasma antidiuretic hormone (adh) level at the onset of ihn and that elevated adh level induced by minor surgery was supposed a trigger of ihn and ponv. in this study we investigated various values including plasma adh in cases taking kidney biopsy in order to clarify the mechanism of ihn and ponv. methods: fifteen patients taking percutaneous kidney biopsy were study subjects (mean age . years). plasma adh, serum electrolytes and osmolality were measured before and - hours after kidney biopsy. urine samples were collected to measure electrolytes and osmolality. results: high plasma adh level ( . ± . pg/ml) was observed in out of subjects ( %). serum sodium level dropped significantly in these cases. six of cases showed ponv, we divided all cases into groups: ponv group and non-ponv group. the result was that plasma adh level was significantly high in ponv group. conclusion: our study make it clear that elevated plasma adh level is frequently seen in children taking kidney biopsy and suggest that hydration with hypotonic saline solution after surgery is inappropriate because of the risk of developing ihn. it also become clear that high plasma adh level might lead to ponv as the same mechanism seen in motion sickness. it is suggested that adh secretion by stress after minor surgery is associated with not only ihn but also the onset mechanism of ponv. polyarteritis nodosa (pan) occurs more commonly in patients with familial mediterranean fever (fmf) and visceral hematomas are seen in almost half of the patients. we report here a year-old girl with pan and fmf presented with multiple visceral hematomas. the patient was on colchicine therapy for four years because of fmf but uncompliant to therapy. she addmitted with the complaints of fever, malaise, abdominal pain and artralgia lasting for two months. she was pale and extremely cachectic with atrophic muscles of extremities. she had fever, hypertension, hepatosplenomegaly and arthritis. she had anemia with normal renal and hepatic function tests, albumin levels, and electrolytes. multiple hypoechogenic mass lesions were detected on liver and bilateral kidneys on ultrasonography and computerised tomography and diagnosed to be hematomas by laparascopic examination. urinalysis, hematological tests for bleeding and blood marrow examinations were normal. bacterial cultures and serological tests did not reveal any infectious agent. serum complement levels were normal with negative antinuclear antibody, anti-dna, p and c antineutrophil cytoplasmic antibodies. renal angiography showed multiple aneurysms in bilateral renal arteries leading to the diagnosis of pan. she was successfully treated with intravenous pulse methylprednisolone followed by oral perdnisolone and oral cyclophosphamide together with colchicine and antihypertensive agents. she has been followed up for four years without any complaints and normal laboratory and radiological findings except multiple scar formations on kidneys on dmsa-renal scanning. results: the stone-free rate was % after one eswl session. the above rate increased to % and % after the second and the third session respectively. regarding surgical treatment with pcnl, the overall stone-free rate was %. in children initially treated with pcnl, an eswl session was performed later successfully, for residual calculi. open surgical removal was required in children with structural anomalies. the patients with staghorn calculi underwent nephrolithotomy combined with eswl in cases of residual fragments. patients underwent ureterscopic procedures to address ureteral stones and complete fragment removal was obtained. no major sideeffect were observed, during the above procedures. conclusions: it seems that the advances in instrument technology provide a variety of safe and effective methods in the management of paediatric urolithiasis. the incidence of open surgery has thus fallen. minimally invasive methods must form the first choice of treatment, while open surgery should be undertaken mainly in cases of coexisting congenital abnormalities. in all children the following parameters were estimated: a) timetable of ne, b) feeling/volume chart (frequency and biggest quantity of urination=functional capacity of bladder), c) ultrasound of urinary tract (size of kidneys, bladder capacity, bladder wall thickness, postvoiding residual urine) and d) urodynamic parameters (uroflowmetry and water cytometry). the ud bladder parameters were then correlated with the us and voiding diary findings. results: all children had sufficient data registered to allow reliable analysis. ud studies showed that children with mild pne had normal urodynamics findings, us parameters and voiding diary findings as well. ud studies reveal a relatively high incidence of instability in children with moderate and mainly in those with serious ne. conclusions: in children with pne, urodynamics did not have a significant additional value compared to baseline diagnostics and it should be avoided. on the contrary, findings from urodynamic studies in children with serious ne show that it has a useful role in this type of enuresis evaluation and management. objectives of study: hyperlipidemia, especially when started during early childhood will increase the risk of atherosclerosis. it is also a major risk factor for allograft nephropathy and post-transplant hyperlipidemia, so its diagnosis and treatment is highly suggested. in this study we have evaluated the effect of hemodialysis on the lipid profile of children with end stage renal disease (esrd). methods: twenty-two children with esrd who were on maintenance hemodialysis in shiraz pediatric hemodialysis unit were studied. they were asked not to take greater than % of their total daily calories as fat at least month before sampling. after a -hour overnight fasting and before starting dialysis, blood samples were taken for lipid profiles. for each patient with total cholesterol, tg or ldl-c levels more than th percentile for age and gender or hdl-c level less than mg/dl, was defined as dyslipidemia. results: nineteen out of children ( . %) had abnormal lipid profiles. atherogenic factor of tg/hdl-c ratio more than as a major risk factor for cardiovascular disease was in %. conclusion: dyslipidemia is common in hemodialysed children. so, hemodialysis set-up change and antilipimic medication, and replacement of l-carnitinine is recommended for correction of dyslipidemia in this group of patients. objectives of study: bipolar renal length measurement is an integral part of the assessment of urinary tract in childhood, and is routinely performed on ultrasonography and renal scintigraphy investigation. correlation between kidney size measurement on ultrasonography and dimercaptosuccinic acid (dmsa) scintigraphy is not well recognized. the purpose of this study was to comparison renal size measured by dmsa scintigraphy and ultrasonography to find if there is acceptable agreement between renal lengths by these two methods. methods: as cross sectional retrospective study, patients enrol in this study and their dmsa scan results and kidney ultrasonography reports compared. the agreement between renal size measured by two methods for left and right kidneys were evaluated separately using bland-altman plot. pearson's correlation coefficient was used to examine their correlation. statistical significance was calculated by paired-student t-test. the same tests were used to for kidneys with normal dmsa scans. results: correlation coefficient showed close correlation between kidney length measured by ultrasound and dmsa scan, but there were significant differences between two methods (paired t-test, p< . ). comparison between renal size measured by ultrasonography and dmsa scan using the methods of bland & altman plot in all patients and the group with normal kidneys showed a systematic bias of about + . mm for left and + . mm for right kidneys. conclusion: despite of close correlation between dmsa scan and ultrasonography for kidney length measurement; kidney size is overestimated by about percent in dmsa scan study and this matter must be considered in practice. medical treatment of cystinuria is often considered disappointing. patients undergo frequent surgery which is often followed by early relapse. the aim of our study was to prospectively evaluate, in a paediatric population, the efficacy of a conservative medical approach for long-term treatment of cystinuria, to prevent the formation of new renal stones and reduce the number and dimension of pre-existing stones. twenty-one stone former cystinuric patients were treated with a combined approach which included cystine-binding drugs. free and bound urine cystine levels were routinely measured every four months. drug dosage was adjusted in order to maintain a steady free urine cystine level below mmol/mmol creatinine (a three fold increase of normal level). in the patients who completed the study, renal stone episodes were reduced from . to . episodes/year, and in several patients the number and dimension of pre-existing renal stones were reduced. during the entire follow-up, percutaneous lithotripsy to remove an obstructive stone was required in only one subject. no relapse was observed months after treatment. the dosage required to achieve target levels was very closely correlated to patient body weight: older children required a lower dose to achieve target levels. in conclusion: medical management of cystinuria is feasible. the treatment must be personalised, at least in pediatric age. the amount of required drug is strictly depending from body size. it is mandatory to obtain a low free urine cystine level before any invasive procedure to reduce the risk of early relapse. objective: to study the pathophysiology of nutcracker syndrome (ns) and to assess the role of the upright position imaging and superior mesenteric artery (sma) angle measurement in the diagnosis. methods: doppler us findings in children with ns and in healthy control subjects were compared. the mesenteric angle, peak velocity (pv) and anteroposterior (ap) diameter of the left renal vein (lrv) at the hilar and aortomesenteric portions were measured in both the supine and upright positions. the means ±sd of the sma angle, ap diameter and pv ratio between the two portions were calculated and cut-off levels for the diagnosis of ns were established. results: the diameter and pv ratios were significantly different between the patient and control groups both in the supine and upright positions (p< . ). differences (d) between the supine and upright positions were also significant for the diameter of the lrv at the aortomesenteric portion, diameter ratio and sma angle in both groups. upright position imaging revealed comparatively narrower sma angles and more pronounced entrapment findings in patients with ns. the sma angle measurement had a sensitivity of . % and a specificity of . % in the supine position and . % and . % in the upright position when the cut-off values were set to less than ° and °, respectively. the upright position has significant effects on the lrv hemodynamics and angle of sma both in patients and healthy subjects. sma angle measurement may be a useful adjunct parameter in the diagnosis of ns. ). in addition, a statistically higher rate of pathological abnormalities on renal biopsy was noted in the group with microscopic hematuria combined with proteinuria and also in cases with more severe hematuria. conclusions: school urinary mass screening has greatly contributed to diagnosing chronic renal diseases. continuous medical observation is required when abnormal urinalysis is observed, and a more aggressive medical approach such as renal biopsy should also be performed if necessary. this study compared the outcome of children with proliferative ln (who class iii/iv) using a new protocol comprising pulse intravenous methylprednisolone, mmf +/-cyclosporine, with standard prednisolone and cyclophosphamide/azathioprine. method: twenty-three children with proliferative ln (age range at diagnosis . - . years) who were followed up for . - . (range . - . ) years, were included in this retrospective study. group i (n= ) received prednisolone with cyclophosphamide and/or azathioprine. group ii (n= ) received the combined mmf protocol with mmf dose of mg/m /day. poor outcome was defined as death or chronic renal failure. survival analysis was performed using the log-rank test. effect of treatment on growth at last follow-up was assessed using height standard deviation score (htsds). differences between the groups were analyzed using the mann-whitney and fisher's exact test. results: at last follow-up, significantly more group i compared to group ii patients had higher serological activity as defined by low serum complement c ( % vs % respectively, p= . ). in addition, -year actuarial survival was higher in group ii ( %) compared to group i ( %). all the group ii patients achieved complete remission of proteinuria compared to group i ( . ± . vs . ± . g/d/ . m respectively, p= . ). group ii patients also had lower htsds on long-term follow-up compared to group i (- . ± . vs - . ± . respectively, p= . ). conclusion: combination immunosuppressive protocol involving mmf +/-cyclosporine resulted in better renal outcome in children with proliferative ln without compromising on growth. this regimen allowed steroid tapering to alternate day dosing without increasing lupus activity. background: a full dose of corticosteroid is required to induce complete remission (cr) in steroidsensitive nephrotic syndrome (ssns), unless it is possible to taper and discontinue along with the course after cr. however, the mechanism of this change in steroid sensitivity remains unknown. p-glycoprotein (pgp) has a function to eliminate given corticosteroids from cytoplasm, which results in inducing corticosteroid resistance. therefore, we analyzed a drug delivery perspective using the real-time polymerase chain reaction (pcr) of multiple drug-resistant gene (mdr ; encoding pgp) messenger rna (mrna) expression. patients and methods: fourteen patients with steroid-sensitive nephrotic syndrome (ssns; male/ female: / , age: - years old; mean . ) were enrolled in this study. mdr mrna gene expression of peripheral blood nucleated cells (pbnc), before and after cr (a total of nineteen sets of blood samples), were quantified using real-time pcr and then carried for analysis. results: the mdr mrna levels before cr were variable in each patient. however, there was an apparent decrease in the mdr gene expression of pbnc after cr (p< . ). the results suggest that pgp may play a role in the ability to taper corticosteroids after cr in ssns. : week prednisone mg/m /day + weeks mg/m every other day). all other patients (b) received daily prednisone . - mg/kg/day for weeks and - % of initial dose for week, followed by alternate day steroids ( week) with tapering by . mg every - weeks down to . - mg. "frequent relapses" (less than months after discontinuation of initial steroid therapy or of first relapse therapy) were treated with chlorambucil . - . mg/kg/day for weeks and half of this dose for - months. results: seven patients (with long treatment) were lost to follow-up and were studied. six of ( %) of a had a relapse . ± . months after the end of initial therapy; became infrequent and frequent relapsers. of ( %) of b relapsed . ± . months after the end of initial therapy; became infrequent and frequent relapsers. frequent relapsers ( % in a and % in b) were treated with chlorambucil and all but one achieved long remission (> year). conclusions: first relapse occurred later after onset of ssns in patients with long ( weeks) as compared to short ( weeks) initial steroid therapy but the time interval between the end of initial therapy and the first relapse and the proportion of relapsers were similar. longer initial therapy may result in a lower number of frequent relapsers. nearly all patients had long remissions periods that were, however, achieved at the expense of early administration of chlorambucil. conclusions: the medium age of pts with metabolic stones was found to be higher than the medium age of pts with infectious stones. the familial occurrence of kidney stones was found to be important %. the ultrasonographic examination is the most important one. the stones composed by calcium oxalate and calcium phosphate were found to have the highest percentage. metabolic abnormalities were found in % of patients and hypercalciuria was the most common disorder. hypocitraturia is considered to be a risk factor the calcium stones. in an attempt to explore the new treatment for the childhood-onset intractable steroid-dependent nephrotic syndrome (sdns), we have recently performed the treatment with high-dose mizoribine (mzr), the inhibitor of inosine monophosphate dehydrogenase, and suplatast tosilate dimethylsulfonium std), a selective th cytokine inhibitor, which were both made in japan. mzr has been commonly used for the treatment of frequently relapsing sdns in japan at a dose of - mg/kg/day (maximally mg/day) divided into two doses (kidney int : , ). we used high-dose mzr (mean: . mg/kg/day) once before morning meal for adolescent patients with frequently relapsing sdns who had been treated with long-term cyclosporine (csa) resulted in moderate to severe csa nephropathy. with this treatment for years, seven out of patients weaned off csa and experienced less relapses without apparent adverse effects by high-dose mzr. std is a both il- and il- inhibitor and commonly used for childhood asthma. we used std at a dose which is for the treatment of asthma for children with sdns (mean . years) without previous csa treatment. after one year follow-up with std treatment, the relapse rate of nephrosis was decreased from . ± . to . ± . per year (p= . by wilcoxon signed-ranks test), where as the dosage of orally given predonisolone was also decreased from . ± . to . ± . mg/kg/day (p= . by wilcoxon signed-ranks test). objectives of study: to evaluate the efficacy and safety of long-term cya treatment for pediatric sle patients. pediatric sle patients in their teens suffer from many relapses and severe side effects caused by steroid and cyclophosphamide. there have hardly been any reports on the long-term cya treatment in children. methods: we retrospectively compiled cases of childhood-onset sle female patients (mean: . years) admitted to our department from to . the initial treatment was methylprednisolone pulse therapy followed by prednisolone (psl). at the onset, patients had class lupus nephritis and showed class . after several relapses, cya was added and used for to months. the dose of cya ranged from . to . mg/kg/day, and the target trough level from to ng/ml. results: under this low-dose cya treatment, patients had no relapse while had a relapse after months. in all patients, psl was reduced to alternate day treatment (mean: . mg/ days), and patients under years gained the target height. of all patients, developed hypertrichosis, gingival hyperplasia, transient elevation of s-cr with acute gastro-enteritis. although case had elevated s-cr after months of cya treatment, it returned to normal level within months after the cessation of cya. five patients had second biopsy after years, and showed mild tubulointerstitial (t-i) changes. two had third biopsy after years and both showed mild t-i changes only. the presence of t-i changes had no relation to s-cr, u-beta mg and u-nag. conclusion: low-dose cya treatment might be an effective and safe second line treatment for sle patients with many relapses in teens. it is also important to perform renal biopsy periodically to detect cya-induced renal damage which hardly shows any abnormalities in blood or urinary tests. hypersensitivity to inulin is rare; two cases of food allergy and some cases of allergy after inulin infusion have been reported. an -year-old boy suffering from severe iga nephropathy (igan) is reported with both anaphylactic reaction and concomitant relapse of his nephropathy due to inulin infusion, used for measuring gfr years after first symptoms. pruritus, sibilants and cough were observed during a first renal function test. prick and intradermal tests were negative for inulin. the patient presented with pallor and asthenia during a second inulin infusion performed under dexchlorpheniramin, leading to immediate infusion stop. he was read mitted because of fatigue and nausea; acute renal failure was diagnosed days after inulin infusion. a drug-induced acute interstitial nephritis was first suspected. however, due to the presence of macroscopic hematuria and proteinuria, a renal biopsy was performed and showed acute proliferative relapse of igan. few data are available about inulin-induced hypersensitivity. chandra described anaphylaxis and cardiorespiratory arrest immediately after administration of sinistrin. a retrospective study of all recorded cases of hypersensitivity associated with renal function tests was performed by our pharmacovigilance unit. , tests using inulin clearance were realized both in adults and children; patients experienced side effects which were divided into groups: respiratory symptoms, rash and general signs. most side effects were minor and no life threatening complication occurred. the underlying mechanism of inulin hypersensitivity is not well known. although % of patients with inulin-associated hypersensitivity underwent a first renal function test, we can speculate that presensitization with food inulin may occur, sometimes leading to severe problems such as in our patient with iga-mediated immunological dysregulation. . we have previously demonstrated a composite heterozygous nphs mutation of both v x and r h in a chinese patient with srns. however, it is not clear the molecular mechanisms of mutant podocinlead to proteinuria. some evidences proved the possible interaction between podocin and trpc . this study explored the effects of mutant podocin on the free cytosolic ca + and apoptosis of podocyte in order to clarify the possible causative mechanism of mutant podocin. methods: . the pdsred n -wild/mutant podocin was constructed by using site-directed mutagenesis. . mouse podocyte clone was cultured and transfected with pdsred n -wild/mutant podocin. . free cytosolic ca + was measured using the fluorescent indicator, fluo -am. results: the low level of free cytosolic ca + was detected in normal podocyte and the transfected podocytes with r h mutant podocin. the v x mutant podocin increased the free cytosolic ca +more evidently than the over-express podocin in transfected podocytes. podocyte apoptosis were not detected in the blank-vector (just pcdna . ) transfected podocytes and normal podocyte. the v x and r h mutant podocin increased the podocyte apoptosis more evidently than the over-express podocin in transfected podocytes. conclusions: the v x mutant podocin might induce podocyte apoptosis via the increment of free cytosolic ca +. however, whether the increment of free cytosolic ca + is induced by trpc and the involved signal pathway should be further investigated. y. xing, q. fan, j. ding objectives of study: podocytes slit diaphragm (sd) associated molecules (nephrin, podocin and cd ap) play a critical role on maintaining the integrity of glomerular filter. vegf is produced by podocyte, and acts on endothelium and podocyte itself. but, it is not clear whether there are some relationships between vegf and sd associated molecules. our study detected the expression of sd associated molecules and vegf in adriamycin (adr) nephrotic rats. methods: the adr rat was established by adr injection. distributions of sd molecules and vegf were detected by immunochemistry. the mrna and protein of sd molecules and vegf was examined by real-time pcr and western, respectively. nephrin phosphorylation were detected by immunoprecipitation. results: distribution of nephrin, podocin and cd ap changed evidently, and the staining intensity of vegf decreased evidently. nephrin mrna increased at day , and returned to the normal at day and ; podocin and cd ap mrna constantly increased from day until day . the protein of nephrin increased at day until day ; podocin was dramatically upregulated at day , and thereafter recovered again, but was downregulated at day ; cd ap prominently increased at day and day . tyrosine phosphorylation of nephrin was decreased evidently at day , and vegf mrna did not show significantly changes at any time points observed. however, vegf protein reduced significantly from the th day, and also reduced evidently at days and days . conclusion: the abnormality of nephrin, podocin and cd ap may be one of the mechanisms that lead to proteinuria in adr-induced nephrotic rats. the occurrence of proteinuria in adr rats may be also associated with the reduced vegf protein, which may be related with the reduction of nephrin phosphorylation. these results suggested there may be some relationship between vegf and sd molecules. the objectives of study: mutations in genes encoding structural proteins of slit diaphragm can lead to nephritic syndrome. just recently, another gene trpc mutation was identified in autosomal dominant fsgs. trpc encodes ion channel protein trpc , whose expression has not been clarified completely in kidney. this study aims to explore the expression and distribution of trpc in normal human, mouse and rat renal tissue and the mouse podocyte clone (mpc ). methods: distributions of trpc in normal human, mouse and rat renal tissue and cultured podocyte was observed with immunochemistry staining. the mrna expression of trpc , , , , and was detected by using rt-pcr. the protein expression of trpc in human, mouse and rat renal cortex and differentiated mpc was detected with western. results: trpc showed weak staining in glomeruli and strong in renal tubules and vessels in human kidney, however, strong in glomeruli and was mainly distributed along the capillary loops and mesangium in mouse and rat kidney. the staining of trpc was observed in differentiated mpc , which is distributed evenly on the cell membrane. the specific pcr band of trpc , , , , and was detected in mouse kidney and differentiated mpc . the sequence of the amplified pcr products is same as that published in genebank. the specific kda protein band of trpc was detected in normal human, mouse renal cortex and differentiated mpc . conclusion: the expression of trpc was verified in normal human, mouse and rat kidneys and in differentiated mpc . these results will benefit for further screening the possible mutation of trpc in acquired nephrotic syndrome, and investigating the relationship between trpc and the proteinuria-related podocyte molecules. methods: twenty children with kd ( boys and girls, aged from to months) were enrolled in our study. kidney sizes (including kidney length and kidney volume) were measured during acute stage in these patients. twenty age-and sex-matched healthy children and febrile children served as healthy controls and fever controls. left kidney length and age were used for correlation analysis and analysis of covariance. results: kidney lengths in patients with kd were significantly larger than those of healthy children (p< . ). the mean sd score of kidney length was . ± . for these patients (p< . , vs - . ± . in normal control). kidney volume analysis yields the similar result ( . ± . cm vs . ± . cm , p= . ). there was no kidney enlargement in the fever controls. up to % of the children with kd have absolute nephromegaly (>mean+ sd). this incidence is as frequent as that of lymphadenopathy and extremities change, the diagnostic criteria of kd. conclusion: these results confirm the presence of large kidneys in the children with kd and also provide another useful indicator for kd diagnosis if the diagnostic criteria is not yet well established. during renal inflammation macrophages infiltrate the renal parenchyma, and their number correlates with the intensity of inflammation. macrophage migration inhibitory factor (mif) was described originally to be a product of t-cells and macrophages. mif plays an important role in renal tissue injury. to our knowledge, the studies that assessed the role of macrophages in acute renal infection were few and the role of mif was not evaluated. the aim of this study was to assess mif in uti and compare the urinary excretion of mif in pyelonephritis, cyctitis and also control group to find a non-invasive and sensitive method to differentiate them. in this prospective case-control study pediatric patients with uti ( patients with acute pyelonephritis, patients with acute cystitis) and healthy children were recruited. urine mif concentration was quantitated by elisa and corrected for urine creatinine. the mean ratios of urine mif/cr were calculated as . (sem= . ) pg/μmol creatinine in acute pyelonephritis, . (sem= . ) in acute cystitis and . (sem= . ) in healthy individuals. urine mif/cr ratio was significantly higher in pyelonephritis than the ones in acute cystitis (p= . ) and control (p= . ). roc analysis was demonstrated that urine mif/cr ratio could considered potentially useful index to detect acute pyelonephritis [p= . , area under curve (auc)= . ]. the optimal cut-point of . pg/μmol creatinine for urine mif/cr ratio could potentially separates acute pyelonephritis patients from healthy individuals (sensitivity and specificity of % and . %, respectively). the underlying histopathological characteristics in biopsied renal diseases are of great importance in determining the long-term prognosis and provides useful information in clinical practice. ethnicity seems to play a critical role in the epidemiology of biopsied renal diseases the aim of this study is to provide data of clinical manifestations of biopsy-proven native renal diseases in iranian children. in this retrospective study, iranian children who were diagnosed as renal disease between and january , were evaluated. diffuse and focal mesangial proliferative glomerulonephritis was present in . % of all biopsies performed. mpgn, fsgs and mcd were observed in . %, . % and . % the most common clinical syndrome at any age is nephrotic syndrome ( . %), followed by nephritic syndrome ( . %), nephrotic-nephritic syndrome ( . %), recurrent macroscopic hematuria ( . %), asymptomatic urine abnormalities (aua) ( . %) and azotemia was seen in . % of patients. mesangial proliferatiove gn ( / = . %), poststreptococal gn ( / = . %) are the most frequent pathologies with acute nephritic syndrome presentation. the most frequent causes of aua were mesangial proliferative gn and hsp. thrombotic microangiopathy (hus) was the most prevalent cause of arf. inthis study, chronic tubulo interstitial nephritis ( . %) and alport ( . %) were the most common causes of crf presentation in our patients. in conclusion, mpgn remains the most common histopathological subtype in children with renal biopsied disease. the incidence of fsgs continues to be high in iranian children. the aim of this study is to assess postnatal kidney volume development and to compare the intrauterine and extrauterine kidney growth curves in premature infants. one hundred neonates were enrolled in this study. all infants had their kidney volumes measured by renal ultrasound examination. group ga consisted of neonates whom were evaluated within hours after birth, and their gestational ages were used in the analysis. group ca included premature infants born before weeks of gestation and was evaluated - days after birth, and their conceptional ages were used in the analysis. left kidney volume, body weight, body height and age were used in the correlation analysis. kidney volumes in group ca infants were significantly larger before weeks of age, but smaller after weeks of age than those of group ga infants (p= . ). there was a significantly better growth in body weight (p= . ) and body height (p< . ) in group ga infants. however, a larger kidney volume was noted in group ca infants with the same body weight (p< . ). conclusion: chart of postnatal growth of normal kidney volume before weeks of conceptional age in premature infants is presented. our data suggests that intrauterine growth may have a regulatory influence on kidney growth, and the reduced kidney volume in the premature infants may start from the early extrauterine period. objectives of study: to illuminate the role of prohibitin (phb), a tumor suppressor which inhibit cell proliferation by repressing e f-mediated transcription, in tubulointerstitial fibrosis (tif). methods: renal biopsy specimens were obtained from children with primary glomerulonephritis. phb and α-sma proteins expression were detected by immunohistochemistry. subcellular location of phb in nrk- f was detected by confocal microscope. changes of phb protein and mrna expression in cells upon tgf-β stimulation were detected. after transfected with phb plasmid, cell cycle and α-sma protein and mrna expression in cells treated with or without tgf-β were detected. results: phb protein was found at normal renal tissues, with a positive distribution in interstitial cells and tubular epithelial cells. phb was down-regulated in tissues with tif and negatively correlated with tif degrees (p< . ). phb is majority located at cytoplasm as well as at nucleus in nrk- f. phb protein and mrna expression in cells were decreased when treated with tgf-β , and the effects were both time-dependent and dose-dependent. extraneous phb inhibited cells proliferation induced by tgf-β , and phb over-expressing cells failed to enter the cell cycle compared with non-transfected cells (p< . ). α-sma was not expressed in control cells while de novo expression of α-sma in cells upon tgf-β stimulation was increased. overexpression of phb did not affect basic α-sma expression but dramatically repressed tgf-β -initiated α-sma expression (p< . ). conclusions: extraneous phb suppresses renal interstitial fibroblasts proliferation and cell phenotypic change induced by tgf-β , which indicates phb as a potential target to halt tif progression. results: the prevalence of urine abnormalities of first screening was over . %, and that of the second screening was about . %. the prevalence was different with various methods. the specificity of method b was higher than method a. testing two urine samples for each child had higher specificity. the direct cost of method a and b was - . and - . rmb, respectively. for screening twice, the corresponding cost was no more than - . and - . rmb, respectively. using method a to screen twice for each child was convenient and economical, which also reduced the false positive rate effectively. the prevalence of urine abnormalities of junior highschool children was significantly higher than that of elementary school-children in xh and the peak point was seen at the point of years old. however, there was no significant difference between children in ja and yp. more than months of follow-up diagnosed cases of iga nephropathy. conclusions: urine abnormalities of school-children could be detected through urine screening at school. for shanghai, method a with screening twice was convenient, economical, and could reduce the false positive rate effectively. objectives: angiopoietin-like protein (angptl ) is involved in lipid metabolism and angiogenesis. the present study was to examine angptl expression in human kidneys with proteiuria, in adramycin rats (adr), and in puromycin induced podocyte damage. methods: immunohistochemistry was performed on kidney biopsies from children with mcd, mn, fsgs, tbmn. in adr, angptl expression was determined by quantitative real-time rt-pcr in glomeruli and tubuli dissected from frozen section of kidneys with laser microdissection system. in mpc , a conditionally immortalized mouse podocyte cell line in vitro, angptl , perlecan and agrin were detected through real-time pcr with the induction of puromycin. detachment assay was performed in podocytes tranfected by angptl -pcdna . . results: in human kidneys, co-labeling showed angptl expressed in the cytosol of wt positive cells. quantitative computerized analysis showed that angptl in glomeruli in mcd and mn were significantly higher than that of tbmn, fsgs respectively (p< . ). in adr, angptl in glomeruli increased significantly at st or th day (p< . ) after adriamycin injection compared with control. and the expression of angptl in glomeruli was correlated with h urinary protein (r= . , p< . ). in mpc both protein and mrna expression of angptl on podocytes were up-regulated with the induction of puromycin. in podocytes transfected by angptl -pcdna . the expression of perlecan or agrin increased significantly compared with control (p< . ). the attachment ratio was shown . %± . % hs after puromycin treatment on podocytes transfected by angptl compared with . %± . % on normal podocytes, and . %± . % on untransfected podocytes. conclusions: angptl is predominantly expressed in podocytes which could be involved in podocyte damage and the development of proteiuria. ( ), iv ( ) and iii ( ), respectively. only one patient had microhematuria. we found that of them had a very low c serum levels. clq and c deposits were all strong positive in renal tissues. our findings suggest that biopsy should be strongly considered in this patient population. the significant renal involvement (class iii, iv, or v ln) could be found in sle patients with very lower proteinuria with or without hematuria. patients in bfb group received computer-assisted biofeedback program while those in ddavp group took minin. both therapies were carried out for month and then months follow-up was taken. parameters of follow-up included enuresis diary-urine flow rate and aqp in urine. results: pne patients were recruited ( boys, and girls), whose mean age was ( . ± . ) years. at the end of treatment and three months later, total effective rates in bfb group were significantly higher than those in ddavp group. uroflowmetry findings showed that in bfb group maximum flow rate, voided volume and ratio of coordinative detrusor-sphincter contraction increased after treatment. ratio of normal flow curve increased at second follow-up (p< . ). in ddavp group voided volume and voiding time decreased after treatment. ratio of normal flow curve and coordinative detrusor-sphincter contraction had no change after treatment. two bands of aqp ( and ) were detected in the morning urine. density of patients bands was significantly lower than that of the controls. density of bands in ddavp group after treatment were significantly higher than that before, but there was no difference between datas before and after treatment in bfb group. conclusions: bfb and ddavp are both effective therapies for pne in children. bfb is helpful in correcting voiding dysfunction and ddavp can increase aqp protein in the urine. with higher effective rate within four month, bfb is strongly recommended. objective: to describe the clinical course of non-parasitic chyluria in a thai pediatric case. this is the first report in children. results: the -year-old boy presented milky urine lasting for one year. urine tests showed heavy proteinuria (protein to creatinine ratio . mg/mg), lipiduria (triglyceride mg/dl). the proof of a pronounced hypertriglyceriduria led to the diagnosis of chyluria. his renal function was normal. numerous red cells and lymphocytes were observed in the urine, and postprandial cystoscopy revealed milky cloudy urine emanating from right ureteral orifices. retrograde pyelography demonstrated pyelolymphatic backflow. serum immunoglobulin g for wuchereria bancrofti and circulating filarial antigen in the peripheral blood were negative. chest x-ray, abdomen computed tomography and intravenous urography did not demonstrate abnormal mass or malformation. proteinuria and lipiduria ceased before sixth week of a medium-chain triglyceride-rich diet. there was no recurrent chyluria after weeks of mct-rich diet were completed. conclusion: in non-parasitic chyluria with unknown etiology, the low-fat diet with mct supplementation alone is effective. the prognosis is excellent. there was a significant improvement of waz comparing data at admission and at the end of follow-up (p< . ). there was also a significant improvement of whz comparing data at admission and at the end of follow-up (p= . ). only ( . %) patients presented with a whz less than - . at the end of the follow-up. conclusion: children with primary vur presented an improvement in somatic growth with medical management. objective: the aim of this study is to investigate the clinical practical value of using doppler ultrasound to detect renal blood flow in renal parenchymatous diseases of children. methods: the renal arteries, segmental arteries and interlobar arteries were detected by doppler ultrasound. the parameters were peak systolic velocity (vmax), minimum velocity in diastole period (vmin), vmax/vmin (s/d), resistive index (ri) and pulsatility index (pi). there were cases of healthy children, cases of acute poststreptococcal glomerulonephrits, cases of primary nephrotic syndrome and cases of chronic renal failure. results: the doppler renal blood flow in normal school children was high velocity and low resistant type. typical cases of acute nephritis with edema and oliguria appeared low velocity and high resistant type, ri, pi and s/d of all renal arteries were significantly increased, vmin are significantly decreased (p< . ). after to weeks all parameters returned to normal. during edema period and convalescence, the renal blood flow of primary nephrotic syndrome is low resistant type, ri, pi and s/d of segmental arteries and interlobararteries were significantly decreased (p< . ). the feature of low circulating blood capacity was not alleviated even though edema was vanished and urine output was increased. the doppler in chronic renal failure was high resistant and low velocity type. ri, pi and s/d were all significantly increased, vmin were significantly decreased (p< . ). when ri was great than . , the extent of damage in kidney function was serious and the prognosis was bad. conclusion: renal blood flow provided a new non-invasive method for clinic diagnosis and evaluation of the prognosis in children renal parenchymatous diseases. we concluded that the dms is an important cause of congenital nephrotic syndrome. the outcome of our patients was poor and most of our patients died before years old. objectives: the antiphospholipid syndrome is defined by the association of arterial/venous thromboses or obstetrical fetal loss with the presence of antiphospholipid antibody. this syndrome may be primary or secondary, particularly in association with systemic lupus erythematous. this study is to examine the frequency of anticardiolipin antibodies and the association between anticardiolipin antibodies with some symptoms in children with lupus nephritis. methodology: twenty-five children with lupus nephritis from / to / in department of nephrology, children's hospital o were included in the study. we find the relationship between anticardiolipin antibodies with hematologic and renal involvement. results: anticardiolipin antibodies was positive in patients ( %), for anticardiolipin igm antibody ( %), for anticardiolipin igg antibody ( %). there was a positive correlation between the presence of anticardiolipin antibodies and thrombocytopenia. in patients with positive anticardiolipin antibodies, patients had mta on renal biopsy. conclusion: anticardiolipin antibodies are associated with thrombocytopenia and mta. aim: the methodologies for quantitating urinary calcium excretion have not been standardized. the aim of this study was to compare urinary calcium/osmolality (uca/osm) ratio with calcium/creatinine (uca/cr) ratio and to assess the correlation of both ratios with daily urinary calcium excretion for the diagnosis of hypercalciuria in children. patients and methods: children aged - years (mean . ± . years) were included in the study. they were randomly selected from previous study's larger patient population. non-fasting, second morning urine samples were collected from all children. children were divided into two main groups: ) children with uca/cr < . (mg/mg) and ) children with uca/cr < . . -hour urine samples were collected from the second group, who were further divided into two subgroups: a) children with daily calcium excretion < mg/kg/day and b) hypercalciuric children (daily calcium excretion > mg/kg/day). results: mean uca/osm ratio was significantly lower in the first ( ) group than the second ( ) group ( . ± . vs . ± . mg/l/mosm/kg, p< . ); but there was no difference between a and b subgroups. the correlations of both uca/osm and uca/cr ratios with -hour calcium excretion were poor (r= . for both). conclusion: uca/osm ratio correlated with spot uca/cr ratio. but its superiority on uca/cr ratio in the diagnosis of hypercalciuria could not be shown. interestingly, values of -hour calcium excretion as a definite diagnosis test of hypercalciuria; did not correlate mathematically with those ratios of hypercalciuric or non-hypercalciuric children. using uca/osm ratio as a screening test would not separate hypercalciuric children. background: microalbuminuria is a biomarker of renal damage. the presence of microalbuminuria in patients with a solitary kidney has been described, but the pathophysiology leading to its occurrence is poorly understood. it is postulated that microalbuminuria is the early result of hyperfiltration. methods: we concomitantly measured inulin clearance, filtration fraction (ff) and microalbuminuria in children with a single kidney. correlation between the occurrence of microalbuminuria and a high filtration fraction was done. microalbuminuria was defined as an albumin/creatinine ratio (acr) > g/mol for boys and girls. normal filtration fraction was defined as < %, and normal inulin clearance as > ml/min x . m . during the same study, we also measured microalbuminuria in children with severe grade iii to v vesico-ureteral reflux (vur). results: children with a single kidney were evaluated. patients ( %) had a normal ff, and only one ( %) in that group had an abnormal acr. patients ( %) had elevated ff, and ( %) had an abnormal acr. the presence of an abnormal acr was highly correlated with an abnormal ff (p= . ). the mean gfr between the groups with normal or abnormal microalbuminuria did not differ significantly ( ± ml/min x . m vs. ± ml/min x . m , respectively). there was no significant association between microalbuminuria and a high ff in patients with severe reflux (p= . ). discussion: we found the presence of microalbuminuria to be significantly associated with an elevated ff in children with a single kidney. this finding goes in line with the pathophysiology of a reduced nephron mass, leading to hyperfiltration, and ultimately to glomerular sclerosis. the benefit of renin-angiotensin-aldosterone blockade in these patients remains to be proven. chyluria is the excretion of chyle from the urinary tract and indicates the presence of an abnormal communication between intestinal lymphatics and the urinary tract. it can be of parasitic or nonparasitic etiology. southern brazil is not an endemic region for filariasis. aim: report a case of a -yrs caucasian adolescent girl referred to our out-patient clinic. history: yrs before, she started to pass milky urine with white clots. no edema. normal blood pressure. she was investigated in another hospital and underwent a renal biopsy, that was normal. a diagnosis of nephrotic syndrome was made. she was treated initially with steroids and after changed to cyclosporin, lisinopril and simvastatin. conclusion: chyluria, although a rare conditione specially in children and adolescent in nonendemic areas, should beconsidered in the differential diagnosis of nephrotic syndrome. macroscopic examination of the urine, that is milky and cloudy, is simple and very helpful. also, triglycerides are found only in the urine of patients with chyluria. these simple tests will avoid unnecessary treatment, which is not without side effects. low-density lipoprotein apheresis (ldl-a) has been tried in the treatment of patients with steroidimmunosupression resistant nephrotic syndrome (ns) due to focal segmental glomerulosclerosis (fsgs). we would like to report a child case study of fsgs with ns and renal insufficiency due to mitochondrial abnormality treated by ldl-a and to clarify the therapeutic effects of this treatment. a -year-old boy was referred to our hospital with complaints of heavy proteinuria and edema. a routine examination revealed proteinuria of . g/day, serum albumin (alb) of . g/dl and creatinine clearance (ccr) of . ml/min. renal biopsy specimen showed fsgs and perceptive deaf nass was recognized, necessitating a hearing aid. the a g point mutation in mitochondrial gene was detected by using genomic dna isolated from peripheral blood leukocytes and by the molecular analysis using an allele-specific polymerase chain reaction (pcr). oral prednisone ( mg/kg/day for eight weeks), intravenous methyl-prednisone pulse therapy ( . g/day,three times a week on the consecutive days for three weeks) and oral cyclophosphamide ( mg/day for eight weeks) were not effective to reduce proteinuria. a protocol of ldl-a was designed for treatment twice-a-week for four weeks and then once-a-week for six weeks. following treatment by ldl-a, serum total cholesterol and ldl were markedly changed form to mg/dl and from to mg/dl, respectively. a small but significant increase in alb from . to . g/dl and a remarkable decrease in proteinuria from . to . g/day were also successfully obtained. conversely, no marked changes in ccr were detected. the results of the present study indicate that a rapid decrease in proteinuria and an excellent increase in alb by ldl-a provide a possible therapy for drug-resistant ns due to fsgs with mitochondrial abnormality. glomerular filtration rate (gfr) can be estimated in children by various formulas based on body height and serum creatinine (s cr ) measurements such as the schwartz formula (egfr sch =kxbh/s cr ). we evaluate the performance of egfr sch in estimating gfr in a pediatric cohort when compared to i-iothalamate clearance (igfr), used as the reference standard for measuring gfr. between and , we obtained igfr and egfr sch on subjects. for subjects who had more than one igfr, the first measurement was used for analysis. mean age was ± (range - , % age= ), % male. mean s cr was . mg/dl (median . ), mean igfr ± ml/min/ . m and mean egfr sch ± ml/min/ . m . figure shows a scatter plot of the data with a line representing perfect agreement. figure shows a residual plot comparing the difference between estimated and measured gfr to egfr sch . pearson r correlation between the two variables was . (ln scale). accuracy of egfr sch within % and % was % and %, respectively. the median difference between igfr and egfr sch was . ml/min/ . m (median % difference %). for igfr > , - , - and < ml/min/ . m , egfr sch overestimated gfr by %, %, % and %, respectively. however, the median difference between igfr and egfr sch for the same groups was , , and ml/min/ . m , respectively. in conclusion, agreement between egfr sch and igfr is poor. egfr sch overestimates igfr at all levels of gfr, but bias of egfr sch vs. igfr increases progressively with higher gfr levels. in clinical instances when an accurate estimation of gfr is critical for patient management, the use of egfr sch should be reconsidered. until a more applicable estimation equation is developed, isotope measurement of gfr remains the ideal method to determine gfr in this population. background: immunosuppressive therapies other than corticosteroids, potentially associated with serious adverse effects, are urgently required for children with frequently relapsing nephrotic syndrome (frns). this study evaluated the efficacy and safety of long-term treatment with a moderate dose of cyclosporine (cya) in children with frns. methods: in this prospective, open-label multicenter trial, patients, from to years old, were randomly divided into two groups. for the first months, both groups received cya (sandimmune) in a dose that maintained the whole-blood trough level between to ng/ml. during the next months, the dose of cya was adjusted to maintain a trough level between and ng/ml in group a, while group b received a fixed dose of . mg/kg per day of cya. the primary end point was the rate of sustained remission. results: at months, the rate of sustained remission was % in group a (n= patients), as compared with only % in group b (n= ) (p= . ). the hazard ratio for relapse was . ( % ci, . to . ) in group a as compared with group b (hazard ratio= . ). at months, the rate of progression (to frns)-free survival was % in group a and % in group b (p= . ). mild arteriolar hyalinosis of the kidney was found in ( . %) of patients in group a and ( . %) of in group b; no patient had striped interstitial fibrosis or tubular atrophy. conclusion: cya given for years in a dose producing a trough level between and ng/ml for the first months, followed by a trough level between and ng/ml for the next months is an effective and relatively safe treatment for children with frns. with this regimen, about % of patients are expected to remain relapse-free during years of treatment, without the most critical adverse effect of cya, i.e., interstitial changes of the kidney. renal stone disease has been regarded as an uncommon problem in children especially in the first year of life. we evaluated clinical findings and metabolic examination of children with urinary tract stone presenting in the first year of life. there were boys ( %) and girls ( %), the mean age of admittance was , ± , months. the average follow-up period was , ± , months. urolithiasis was diagnosed during evaluation for uti and incidentally. positive family history for urolithiasis was reported in ( , %) patients. in / ( , %) patients urinary metabolic examination was not normal (table ). in of patients ( %), stones were located in kidneys which was bilateral in ( %) patients and one patient had passing stone which had never seen in ultrasonographic examination. stones were examined in subgroups. in ( %) patients stones were measured mm or smaller (group ), in patients ( %) they were between - , mm (group ) and in only patient the stone (cystin) was larger than mm (group ). stones measuring mm and larger were found highly associated (in of children, %) with abnormal ultrasonographic findings mainly hydronephrosis. in group , stones disappeared spontaneously in / ( %) patients. urinary tract infections (uti) were present in ( %) patients. one fourth of cases had associated genitourinary tract abnormalities mainly vesicoureteral reflux in ( %) patients. we conclude that the presenting symptoms of urolithiasis in the first year of life show a wide spectrum so that high index of suspicion is important for early detection. stones measuring mm and smaller may have great chance to disappear. we also emphasize the importance of screening for uti in patients with urolithiasis under year of age. background: long term complications of glycogen storage diseases (gsds) include delayed puberty, hepatic adenoma and renal disease. in the present study we aimed to detect renal involvement in children with glycogen storage disease and to determine the most accurate laboratory test to be the gold standard for early detection of this renal dysfunction. methods: twenty-seven children known to have gsd were included in this study. fifteen healthy age-and sex-matched children were also included as controls. routine urine analysis, urinary β microglobulin and microalbumin were done for all patients and controls. renal function tests, serum electrolytes, alkaline phosphatase, urinary calcium, blood and urine ph, urinary and plasma aminogram, in addition to calculation of glomerular filtration rate (gfr), bone x-ray to detect rachitic manifestations and abdominal ultrasound to measure renal size were done for all patients. results: twenty-one patients had one or more renal abnormality. the most common was increased urinary β microglobulin ( / ) followed by abnormal gfr whether low or high ( / ) and microalbuminuria ( / ). sonographically there was nephrocalcinosis in one case and renal stone in another one. the auroc curve for β microglobulin was . , (p= . ) and . for urinary microalbumin/creatinine ratio (p= . ). the best cutoff level to predict renal abnormality for urinary β microglobulin was . mg/l with % sensitivity and % specificity and the best cutoff value for urinary microalbumin/creatinine ratio was . with % sensitivity and % specificity. in conclusion: renal abnormalities are common in patients with gsd. urinary β microglobulin can be considered the gold standard for early detection of renal dysfunction in these patients. the aim of this study was to investigate the role of neutrophil activation, protein oxidation and ceruloplasmin in the pathogenesis of hsp, which has been not investigated previously. serum activities of myeloperoxidase (mpo) and arylesterase (aryl) and levels of free thiol, ceruloplasmin (clp) and total oxidant status (tos) were measured in children with hsp ( boys, girls; mean age . ± . years) at the onset of the disease and during remission in comparison with matched healthy subjects. patients at active stage had significantly higher mpo activity ( ± vs. ± u/l, p< . ), higher clp ( ± vs. ± mg/dl, p< . ) and tos values ( . ± . vs. . ± . μmol h o /l, p< . ) than controls. patients had significantly lower aryl activity ( ± vs. ± u/l, p< . ) and lower free thiol levels ( ± vs. ± μmol/l, p< . ) than controls. there were patients with gis involvement, with joint and with renal involvement. no significant differences were found in the oxidant stress markers between patients with or without organ involvement (p> . ). significantly positive correlations were found between tos and mpo (r= . , p= . ), and tos and clp (r= . , p= . ) at the disease onset; while a negative correlation was found between mpo and thiol (r=- . , p= . ) during remission. in conclusion, protein oxidation and neutrophil activation may play important roles in the pathogenesis of hsp. gastrointestinal system, joint and/or renal involvements were not together with different magnitude of oxidant stress. further studies are required to identify oxidizing substances and to develop therapeutic strategies to reduce oxidant stress in hsp. , ) . if the first remission occurred after days, the median time to relapse after discontinuation of steroid therapy was significantly lower than in children with shorter remission time ( . vs . months; p< , ). in conclusion children who fail to achieve a prompt remission after a first episode of ns are more likely to have frns or sdns. these retrospective data provide the rationale for individualizing the initial steroid treatment of mcns according to the time to obtain a remission. a prospective study is needed to validate this approach. the aim of this study was to determine the influence of osmolality of the first morning urine (ofmu) to efficacy of the desmopressin therapy in enuretic (pne) children and to compare the values of ofmu in enuretic and non-enuretic children. methods: we investigated ofmu in group of children with pne and in group of control non-enuretic children. pne group was divided into subgroup i (ofmu < mosm/kg h o) and subgroup ii (ofmu > mosm/kg h o). additionally, we measured ofmu months after the initiation of desmopressin therapy and recorded the number of wet nights. regarding the number of wet nights we divided pne group to subgroups: subgroup a (< wet nights/month), subgroup b ( - wet nights/month), and subgroup c (> wet nights/month). results: the statistically significant difference between control group and pne group regarding ofmu was not found (p= . ). all children from subgroup i had < wet nights/month during desmopressin therapy. children from subgroup ii had < wet nights/month, and had > wet nights/month during desmopressin therapy. the difference between those two groups was statistically significant (x = . , p= . ). in children from subgroup a the difference between ofmu-s during and before treatment was mosm/kg h o, in children from subgroup b it was mosm/kg h o and in children from subgroup c it was , mosm/kg h o. there was the statistically significant difference among those subgroups. conclusion: children with pne had usually similar ofmu like non-enuretic children. low ofmu is a good prognostic factor for desmopressin therapy of pne, especially in patients whose ofmu is < mosm/kg h o. children with bigger difference of ofmu before and during therapy had better response to desmopressin therapy. we can conclude that ofmu can help in choosing the appropriate therapy for pne in children. yh. ng , kl. chan kk women's and children's hospital, pediatric nephrology, singapore, singapore singapore general hospital, neonatology, singapore, singapore aim: to evaluate the clinical course and outcome of primary vesicoureteric reflux (vur) in patients with antenatal hydronephrosis in a neonatal unit. method: a prospective observational study of neonates with antenatal hydronephrosis born between january and december in the neonatal unit of singapore general hospital. neonates with significant hydronephrosis postnatally underwent micturating cystourethrography (mcu). records were reviewed with regards to the clinical course and outcome of primary vur. results: of neonates with antenatal hydronephrosis, ( %) had significant hydronephrosis postnatally and underwent mcu. . % ( / ) were diagnosed with primary vur at median age of weeks. there were more male (n= ) than female infants with primary vur. ( %) infants had bilateral vur. % (n= ) of the renal refluxing units (rru) had low grade vur and % had high grade vur with the majority ( %) being grade iii vur. repeat mcu for rru at years showed that % (n= ) had spontaneous resolution of vur, % had improved vur grade and % had similar vur grades as before. infants develop vur in the contralateral kidney which was previously normal. infants ( rru) underwent dmsa with renal scarring noted in infants. all infants were noted to have renal scarring without a history of urinary tract infection (uti). interestingly, male siblings were found to have grade iii vur with renal scarring with subsequent spontaneous resolution. none of the study subjects underwent surgery. median age of follow-up was . years (range . - . years). conclusion: unlike neonates with vur detected after uti, infants with primary vur were predominantly male, had higher grade of vur with spontaneous resolution in the majority. early diagnosis of primary vur may provide the opportunity for reduced incidence of reflux nephropathy. t. neveus , g. läckgren , j. wahlberg , n. wahlin uppsala university children's hospital, uppsala, sweden uppsala university hospital, department of transplantation surgery, uppsala, sweden objectives and methods: loin pain hematuria syndrome is a rare entity consisting of recurrent macroscopic hematuria with debilitating loin pain. it has only been described in adults, etiology is unclear and treatment is controversial but the therapy with best recorded success is to remove the kidney and reposition it in the pelvis. our objective was to show that the condition exists in childhood as well. results: aj, a previously healthy -year-old girl, was admitted because of recurring cystitis-like symptoms with microscopic hematuria but without bacteriuria. ultrasound and urography were normal. the episodes continued during the following years with increasing hematuria, now macroscopic, and increasing loin pain that was somewhat exercise-dependent. a renal ct scan was normal, as was cystoscopy, urography and ultrasound, but during cystoureteroscopy dilated vessels were noted in the mucosa of the right renal pelvis. antegrade pyelography, high resolution renal ct angiography, invasive renal angiography, mag renogram were all normal, gfr . nephrological evaluation, including kidney biopsy and coagulation tests were also normal and during cystoscopy blood could be seen emerging from the right ureteral orifice. by this time the patient was years old and was dependent on opioids in order to be able to go to school. after long discussions with the nephrologist, urologists, pain specialist and transplantation surgeon, the family opted for autotransplantation as a last resort. this was performed january and the girl became almost momentarily pain-free. nowadays she does not need any analgesics, but after prolonged exercise (like several days of horseback riding) she may experience slight pain in the left loin and/or hematuria. conclusions: idiopathic loin pain hematuria syndrome exists in childhood and may possibly be treated with renal autotransplantation. j. van der deure, a. ockhuijsen, m. sondaar deventer ziekenhuis, st department of pediatrics, deventer, the netherlands objective: enuresis is a common pediatric problem. psychosocial factors (psf) influence the results of enuresis treatment in children. aim: to determine the short and long term effects of psf on enuresis treatment in a general pediatric population. methods: we reviewed the data of our enuresis patients treated from - . relevant contributing psf were categorized. initial follow-up was at months after training. a written questionnaire was sent years after training. treatment success was defined as > % improvement in dry nights. results: pts were included. in pts ( . %) contributing psf were recognised. categorized problems: family related n= ( %), behavioural problems n= ( %), motivation/support n= ( . %), learning disabilities n= ( . %). overall success rate was . % at months and . % at yrs (overall resp quest . %, psf . %, psf %, > psf %). success rate in the psf group was . % ( psf . %, > psf . %) at months and . % ( psf %, > psf %) at yrs. statistics: success rate in the group with psf is significantly lower as compared to no psf at months (p< . (chi-square), or . , % ci . - . ) and at yrs (p= . (chi-square), or . , % ci . - . ) success rate at months is significantly lower in pts with > psf, compared with psf. (p= . (chi square), or . , % ci . - . ) . no significance could be demonstrated at yrs (p= . , chi-square) but this may be due to the variety in response rates. t. papalia, r. greco, r. bonofiglio hospital annunziata, nephrology, cosenza, italy actually a new litholitic therapy includes the phytotherapy agents as phyllantus niruri (pn), a plant used for years in brazil to treat urinary stones. in this work we estimate the effect of pn intake (uristone gr/die) in children ( m/ f, ± years old) with urolithiasis. the pn has been administered for short term (from to months) in children wih caox urinary stones and for months in with struvite stones. besides all children treated with dietary intervention: high fluid intake, sodium restriction, normal calcium intake and a diet low in animal protein. urinary and plasma analysis, body weight, map, ph, creatinine clearance, urinary excretion of mg and citrate were determined at baseline, month and at the end of the study. the patients were studied by renal ultrasonography at baseline, , , months. nobody of them had been undergone extracorporal shock wave lithotripsy. there were no differences in the mean values of urinary and plasma parameters before and after pn intake, except for a significant reduction in the mean urinary calcium in hypercalciuric pts ( ± , vs , ± mg/kg/die). in this follow-up n° patients showed a faster stone clearance after a regular intake of pn and the others showed a smaller stone diameter. previous reports showed pn has a potent inhibitory effect on caox crystal adhesion and/or endocytosis by renal tubular cells and inhibitory effect on crystal growth, which might be related to the higher incorporation of gags into the calculi. our results suggest pn appears to represent a nontoxic and a low cost alternative for the prevention and treatment of stone disease, especially in the children. further studies are necessary to validate these preliminary findings. d. weitzel, c. schäfer, k. hohenfellner, u. pfeffer, m. neukirch german clinic for diagnostic, pediatrics, wiesbaden, germany objective: does sonographic quantification of the renal parenchyma allow estimation of isotopic renal function? method: sonographic kidney images of patients (age to months; mean ) were measured retrospectively. in all images of both kidneys taken from dorsal the volume on the base of length, width and depth was calculated. the parenchymal area (pa) in the longitudinal and cross section was calculated by planimetry. the distribution of renal function via mag was compared with sonographic values as volume and pa of each kidney in relation to whole kidney volume and pa respectively. patients with reduced global kidney function and time space of more than months between isotopic study and sonography were excluded. results: interrater variability regarding planimetry of pa in longitudinal section (from dorsal taken images) was as good as measurement of kidney length (correlation coefficient (k)= , - , and , - , respectively). all sonographic parameters correlated significantly with the isotopic parameters of renal function. the latter correlated best with the pa in longitudinal section (from dorsal taken images) k , . the combination with planimetry in cross section did not improve correlation (k , ). difference of the proportional pa of the left kidneys (in correlation to whole kidney pa) in comparison to isotopic proportional renal function lead to mean difference of - , % with a standard deviation , %. if only kidneys with split function of - % the mean difference of proportional pa was - , % and the standard deviation , %. conclusion: the distribution of total pa of both kidneys correlates significantly with the distribution of renal function (left and right) in isotopic studies. if sonographic planimetry might change the indication for isotopic studies in respect of renal function needs to be proofed in prospective studies. background: childhood incontinence is a common important urologic problem. especially daytime incontinence is often neglected by the parents until it turns out to be a significant clinical problem. the aim of this study was to evaluate the clinical characteristics of the patients with incontinence that were followed in our nephrology clinic. study design: patients were followed between the dates of . . - . . and they were admitted solely due to incontinence or with concomitant urinary tract infections were enrolled. results: the study comprised patients ( m, f; mean age . ± . years). fourty-two patients had only nocturnal enuresis (ne) ( primary, secondary). twelve patients had daytime incontinence (di) ( primary, secondary) and had both ne and di ( primary, secondary and both primary ne and secondary di). all, except two (neurogenic bladder), had functional incontinence. twelve patients had additional fecal incontinence and had constipation. sixty-two percent of the patients had one or recurrent urinary tract infections (uti) in their past history, % had accompanying vesicoureteral reflux and % had urinary stones. ultrasound revealed unilateral or bilateral dilatation in % and other anomalies in % of the patients. nineteen patients had abnormal dimercaptosuccinic acid scintigraphy findings. timed voiding schedule and double voiding were recommended to all patients with daytime incontinence, % of the patients received anticholinergic treatment and % received antimicrobial prophylaxis. discussion: overall, approximately / of our patients had associated uti and / had abnormal dmsa findings. therefore every patient with uti should be questioned about urinary incontinence and be treated carefully if present. the aim of the study was to determine early parameters of ultrasound and dmsa scanning diagnostics of reflux nephropathy (rn) in children. we examined children with rn and vesicoureteric reflux (vur). all children were comparable on gender and age. all patients underwent color doppler ultrasound (cdus), x-ray and dmsa scan. they were divided into two groups: ) children with unilateral rn a according to classification of smellie j. et all, (n= ) ; ) children with vur without renal damage (n= ). we established that data of cdus (diastolic velocities (vd) , ± , mm/sec, systolic velocities (vs) , ± , mm/sec, resistive indices (ri) , ± , , pulsatility indices (pi) , ± , ), dmsa scanning (time of the maximal accumulation , ± , sec, maximal activity , ± , sob/sec, mean velocities of accumulation , ± , mm/sec, the contribution to the common accumulation , ± , %) are characteristic for patients with rn a. data of cdus (vd , ± , mm/sec, vs , ± , mm/sec, ri , ± , , pi , ± , ), dmsa scanning (time of the maximal accumulation , ± , sec, maximal activity , ± , sob/sec, mean velocities of accumulation , ± , mm/sec, the contribution to the common accumulation , ± , %) are characteristic for patients with vur without renal scars. the ranges of cdus and dmsa scanning were significantly different between children from comparing groups (p< , ). our result suggest that data of cdus (vd, vs, ri, pi), dmsa scanning (time of the maximal accumulation, maximal activity, mean velocities of accumulation, the contribution to the common accumulation) can be used to early diagnostics of scarring in children with vur. the purpose of this study was to determine normal reference values for urinary uric acid/creatinine ratios in healthy turkish children. in this study, random urine specimens from children ( boys, girls) aged month to years were analyzed for uric acid and creatinine, and urinary uric acid/creatinine ratios were determined from each sample. uric acid/creatinine ratios were the highest in children aged - months and showed a significant decrease with age (p< . ). uric acid/creatinine ratios were not significantly different between the sexes except - years. girls between - years had higher urinary uric acid/creatinine ratios when compared with boys (p< . ). there was no correlation between urinary uric acid/creatinine ratios and protein intake. our results show that urinary uric acid/creatinine ratio changes with age. when assessing the urinary uric acid/creatinine ratio, a child's age should be considered. we provided normal reference values of urinary uric acid/creatinine ratio for using in our region. the aim of the study was to investigate microbiological marker of activity of uti. e. coli and s. aureus p were isolated from urine of children with uti. the children were divided into groups: . with pyelonephritis in the acute period (n= ); . with pyelonephritis in the period of remission (n= ); . with cystitis in the acute period (n= ); . with cystitis in the period of remission (n= ) healthy children consists the group of control. definition of bactericidal activity of urine (bau) was carried out by our original method. the essence of the method consisted in measuring of the optical density (od) of the bacteria after their contact with urine (experience) and isotonic solution of nacl (control) after minutes of endurance in meat peptone mediums with c during - hours. bau was calculated under the formula: bau (%)=(odc-ode)/odc* %, (odc -control group, ode -experience group). we established that the level of bau did not correlate with urine ph (r= , ), osmolality (r= , ), lysozymuria (r= , ), lysinuria (r=- , ). we established that the low level of bau was marked in children of control group ( - %). the patients in active period of uti had high level of bau (> %). the parameters of bau didn't depend from the level of uti (pyelonephritis or cystitis). the level of bau reduced in the period of remission of uti. we established that the level of bau correlated with bacteriuria (r= , ), leucocyturia (r= , ). the level of bau didn't depend from the degree of urine dissemination (r=- , ). so, the level of bau is correlated with laboratory parameters of uti and can be used as new additional microbiological marker of diagnostics of activity of uti. the evolution of the alport syndrome in brazilian children vesicoureteric reflux (vur) is common in children with urinary tract infections (uti). if vur coexisting with uti there is a high risk of progression to end-stage renal disease (esrd). the correct diagnosis is important. we observed children ( girls and boys) aged mo to yrs at the time they have been diagnosed as having vur. the follow-up period was mo up to yrs after the diagnosis. all children with vur grade have been operated. after antireflux operation incidence of uti dramatically decreased even this cannot prevent progressive kidney damage in some patients. children with less severe vur have been put on prophylaxis. controlled mcu was performed usually after year later. if vur disappeared medication have been stopped. vur grade - had a tendency of resolution under conservative treatment in . % of the patients. in children associated urinary tract malformations were found: duplicated system, dysplastic kidney, kidney agenesia, dystopic kidney, urethral stenosis and bladder outlet obstruction. in patients nonfunctioning kidney have been found. dysfunctional voiding was common finding. blood pressure and physical development have been controlled. kidney size, function and scar formation have been followed by dmsa scan. we observed kidney growth at mos intervals. during the follow-up period infants have had reversible renal insufficiency. one patient with bilateral vur grade went into renal failure at the age of yrs. conclusions: vur is still one of the most common leading causes to esrd in childhood. even the existing controversy concerning treatment modalities it is obvious that low grade vur does not need operative treatment. it is indicated in high grade vur to prevent repeated and severe uti but it cannot preserve progression of the disease because of high incidence of coexisting kidney dysplasia. results: from forty children with nephrolitiasis, ( %) were boys and ( %) were girls. the mean age was . ± . months. the youngest age was months old. the most common clinical presentation was abdominal discomfort ( %), followed by uti ( %), microscopic hematuria ( . %), macroscopic hematuria ( %), spontaneous urinary calculi ( %), flank pain ( %). nine of children were presenting with chronic renal failure (crf). statistical analysis showed that age had correlation with the present of crf in children with nephrolitiasis (p= . ). the clinical presentations of nephrolitiasis were varied. abdominal discomfort and uti were the major signs and symptoms. there was correlation that age may influence the present of crf in children with nephrolitiasis. objective: renal involvement is one of the most frequent and serious manifestations of sle. we analyzed the treatment and renal outcome of patients with lupus nephritis. methods: seventy-seven identified patients were retrospectively analyzed from jan. to dec. . the outcome was divided as complete remission ( -hour proteinuria < . g, plasma creatinine level normal and sledai < ), partial remission (abnormal renal damage index improved > %, -hour proteinuria > . g, sledai < ) and no response, respectively. results: fifty-four patients were biopsy proven ln ( %). fifty-seven patients followed up more than month. all the eleven patients with class i or ii achieved remission, using prednisolone together with either hcq, or tripterygium or mmf or cyclophophamide (ctx). in forty-three patients with class iii or iv or v, they were given prednisolone together with mmf or ctx. we found that remission was in cases, part remission cases and no response in cases. associations between methylenetetrahydrafolate reductase (mthfr) c t polymorphisms and several vascular diseases have been reported. this is a clinical study designed to investigate the possible effects of (mthfr) c t polymorphisms on the developement of henoch-schönlein purpura (hsp), renal involvement, and clinical course. fourty-one patients with hsp ( m/ f) mean age ( , ± , years) were included in the study. eighteen of the patients had renal involvement. the control group consisted of healthy children. blood samples were obtained for mthfr c t transition, homocysteine, folic acid and vitamine b in the patients and controls. the genotype frequencies (cc/ct/tt) of mthfr in the hsp group were , / , / , and , / , / , in the control group, respectively (ns). the genotype frequencies (cc/ct/tt) were , / , / , in the patients without renal involvement and , / , / , in those with renal involvement, respectively (ns). homocysteine levels were , ± , in the hsp patients and , ± , μmol/l in controls (ns). vitamine b levels were , ± , pg/ml in the hsp patients and , ± , pg/ml in the control group (ns). folic acid levels were , ± , in the hsp and , ± , ng/ml in the control group (p< , ). no significant relationship was present with the mthfr genotype and plasma homocysteine, folic acid and vitamine b levels. no association with mthfr gene polymorphism and homocysteine plasma levels could be detected in patients with hsp and hsp nephritis. although mthfr gene polymorphisms have been found to be associated with several vascular diseases, the results of this study indicate that other mechanisms should be operative in the developement of hsp and hsp nephritis. we report the symptoms, signs and laboratory values at onset and during month-follow-up of hsp in a prospective study of children ( girls, boys) with mean age of . years ( . - . y). the first sign of hsp was purpura in ( %), oedema or other joint symptoms in ( %), abdominal pain in ( %) and melena in ( %) patients. petechiae appeared on the average days after the first symptoms ( - d) if purpura was not the first sign (n= ). % of the cases were diagnosed between september and march. the mean delay from the first symptoms to the diagnosis was days ( - d our results based on an unselected and prospective patient material demonstrate that renal symptoms in hsp children develop early, are common and should be followed up at least months. the kidney is a metabolically active organ, so any alteration in kidney function might affect nutrient utilization. objective: analyze the nitrogen balance (nb) as a marker for adequate food consumption in children with chronic renal insufficiency (cri). material and methods: patients ( boys, girls) diagnosed and managed in the nephrology and nutrition departments. they were placed in two groups depending on age: group a: patients aged ± . years, follow-up ± . years, glomerular filtration rate (gfr) estimated by cr-edta: ± ml/min/ . m ; group b: patients aged . ± years, follow-up . ± . years, gfr estimated by cr-edta: ± ml/min/ . m results: group a had a worse weight and height evolution: weight: - . ± . sd in group a vs . ± sd in group b; height - . ± . sd vs - . ± . sd (respectively). group a showed a significant increase in tnf blood levels (p< . ) that was inversely related with weight and height. bn was significantly greater in group a ( . ± . gr/day) than in group b (- . ± gr/day) (p< . ) and this was related with higher calorie (p< . ) and protein (p< . ) intakes. there was no difference in alimentary nutritional breakdown. nb improved significantly over the follow-up (p< . ). there was no relation between nb and gfr. there was a significant increase in triglyceride levels and significantly lower blood urea levels in group a (p< . ). we conclude that nitrogen balance depends on protein and calorie consumption and is independent of the severity of renal affectation. we present a case of a months old boy, who was delivered to our intensive care unit because of high fever, acute renal failure and dilatation of the urinary tract. his hemoculture and urinary culture were positive for e. coli and a severe urosepsis was diagnosed. his urethral catheterization was unsuccessful, so a suprapubic puncture was performed to relieve the urinary system and provide sufficient urinary flow. after the urinary sepsis was cured with adequate board spectrum antibiotics, a voiding cystourethrography (vcug) was performed to reveal the suspected anatomical abnormality, vesicoureteral reflux (vur) respectively. meanwhile a continuously growing nodular tumor of the penis was observed. the vcug showed the signs of urethral stenosis, but posterior urethral valve and vur was excluded. the doppler ultrasound found a solid vascularised tumor, x . cm in its diameter. during the surgical operation the tumor couldn't been totally removed, as it was infiltrating the surrounding tissues and the urethra. the histopathological examination of the biopsy specimen confirmed a juvenile xanthogranuloma (jxg). this is an uncommon, benign, non-langerhans cell histiocytosis, primarily seen in infancy as a solitary cutaneous lesion, predominantly in males. systemic form of the disease is rarely seen. usually it resolves spontaneously without any further treatment, but the differentiation from a malignant neoplasm is essential. according to the authors' search, this is the nd reported case in the literature and the first pediatric report of the jxg of the penis. urine examination using x-ray diffractometry background and goal: x-ray powder diffraction analysis is widely used in chemistry and pharmacology and for other industrial purposes. in medical science it is used for analyzing kidney stones and investigating retained crystals in tissue sections. in the department of mineralogy and petrology we investigated urine samples, at first diagnostically to detect urinary amino acids, glucose and compounds and, secondly, to detect calcium oxalate hydrate, which can be employed for early detection of renal tubular injury when no significant differences in renal function values exist. materials and methods: after sedimentation and dehydration, authors investigated more than urine samples of children using x-ray diffractometry. results: of them were glucosuric due to diabetes mellitus; in these cases glucose could be detected in each of their urine samples. in cases different amino acids due to aminoacidopathy were detected. the urine samples of children -kidney stone problems in history -were examined, in one case struvit, in the other cases ca oxalate crystals were identified. also, samples of children were examined, hours after at least hours long anesthesia, ca oxalate hydrate appeared in their urine referring to renal tubular injury due to inhalational anesthetic agents. in cases urine samples of children treated in the intensive care unit were analyzed, in % ca oxalate crystals could be detected. in cases healthy children's urine were investigated, as control ones. conclusion: x-ray diffractometry, as a highly sensitive method can be used efficiently in clinical measurements. further investigations are needed in order to determine its place in clinical trials. authors emphasize the importance of collaboration of different sciences, as well. drug intake in the background of sudden death? microalbuminuria was significantly more in patients more than years of age as compared to younger patients on bivariate analysis (p= . ). on logistic regression analysis, though microalbuminuria was more in patients more than years of age, it was not statistically significant. the association between microalbuminuria and urinary specific gravity levels of < . was statistically significant (p= . ), similar results were seen on logistic regression analysis. there was no correlation between microalbuminuria and hospitalizations, crises, previous blood transfusions, hemoglobin electrophoresis and serum creatinine levels. conclusion: identification of risk factors for microalbuminuria may allow earlier intervention to prevent renal complications in patients with ssd. in developing countries at primary health care level urinary specific gravity should be done routinely in patients with ssd to identify cases at risk of microalbuminuria. m. bak, e. serdaroglu, y. bicilioglu aim: the aim of the present randomized-controlled study was to compare desmopressin (dp), alarm and combined treatments in nocturnal enuresis. the study included children ( boys and girls) with nocturnal enuresis. the mean age was . ± . years (ranged - years) and the mean wet-nights was . ± . day per month before treatment. the patients were followed for one month before treatment and randomized to dp ( patients), alarm ( patients) and combined ( patients) treatment groups. the dp group was received μg orally one-hour before sleep and alarm group was used wet-stop bedwetting alarm device after education of parents. the patients were followed months in treatment period and months after discontinuation of treatments. results: wet-nights per months was significantly reduced between before treatment and last month of treatment in dp ( . ± . to . ± . , p< . ), alarm ( . ± . to . ± . , p< . ) and combined treatment ( . ± . to . ± . , p< . ) groups. treatment success (> % decreasing in wet-nights) and complete response ( % dry) rates was %, %, % and %, %, % in dp, alarm and combined treatment groups respectively. the more rapidly decreasing in wetnights was observed between dp used and only alarm treatment group but this effect disappeared after months. relapse rates was %, % and % in dp, alarm and combined treatment groups respectively between successfully treated patients (p= . ). conclusion: alarm treatment is the best intervention with low relapse rates and no potential adverse effect in nocturnal enuresis. dp group has higher relpse rate but adding to dp may achieve more rapid decreasing in wet nights especially in patients and parents expecting rapid result. aim: it is often difficult to collect urine from infants. use of specifically designed urine collection pads gives reliable results for routine biochemistry tests in adult urine. their use for routine and metabolic tests in paediatric urine has not been investigated. the aim of this study was to evaluate whether the pads give reliable results for routine and metabolic biochemistry tests in paediatric urine. methods: urine collected by bag or clean-catch from infants < y without metabolic disorders was divided into two aliquots, one of which was added to a collection pad, incubated for min at °c, then recovered by aspiration. routine and metabolic analyses were performed on pad/non-pad aliquots. additionally, selected metabolic analyses were performed on pad/non-pad urine from patients with diagnosed inborn errors and urine spiked to simulate metabolic disorders. for quantitative analyses, pad/non-pad results were compared using bland-altman bias plots, passing and bablok regression and paired t-tests. results: routine tests (urea, electrolytes, creatinine, osmolality, calcium: creatinine, phosphate: creatinine, magnesium: creatinine, urate: creatinine, n= ) showed close concordance with no clinically significant pad/non-pad differences. in infants without metabolic disorders, aminoacids (n= ), organic acids (n= ) and mucopolysaccharides (n= ); and in patients with metabolic disorders -phenylketonuria (n= ), mucopolysaccharidoses ii (n= ) and iii (n= ), inborn errors of organic acid metabolism (n= ) and cystinuria (n= ), all showed excellent pad/non-pad concordance. sugar chromatography in urine spiked with glucose/galactose/fructose showed identical staining intensity in pad/non-pad samples. conclusion: urine collection pads give reliable results for a wide range of routine and metabolic tests in paediatric urine. a post-translational modification of arginine residues in proteins and subsequent proteolysis result in release of symmetric dimethylarginine (sdma). sdma is considered an end product of metabolism, excreted primarily by the kidney. several previous studies have reported a significant relationship between glomerular filtration rate (gfr) and plasma sdma. to determine the potential value of sdma in the assessment of gfr in children we have measured sdma in samples taken during routine measurement of glomerular filtration rate (gfr) using plasma clearance of inutest. patients ( male) requiring routine gfr measurement were studied. median (range) age . y ( . - . ) , height (ht) cm ( - ), and surface area . m ( . - . ). gfr was measured using the plasma clearance of inutest, and plasma sdma and creatinine (pcr) by liquid chromatography stable isotope dilution electrospray mass spectrometry-mass spectrometry method. estimated gfr (egfr) was calculated from the formula x ht (cm)/pcr (μmol/l). median gfr was ml/min/ . m ( - ), plasma sdma . μmol/l ( . - . ), pcr . μmol/l ( . - ), and egfr ml/min/ . m ( - ). as expected both plasma sdma and pcr increased with a decline in gfr. compared to gfr the correlation with /sdma, r= . (p< . ) was better than for /creatinine, r= . and similar to that for egfr, r= . . comparing sdma with inutest gfr for detection of gfr < ml/min/ . m the area under the roc curve was . (p< . ). the equivalent areas for pcr and egfr were . and . , respectively. in conclusion plasma sdma is an endogenous marker of gfr in children and is superior to pcr because it appears to be independent of body size. since the calculation of egfr requires accurate measurement of height, plasma sdma may provide a practical alternative for assessment of gfr in children. thrombotic microangiopathy (tma) consists of thrombocytopenia, microangiopathic hemolysis, and thrombi in the microvasculature of vital organs. broad categories of causes of tma include infectious (verotoxin-induced hus), hematologic (ttp), complement based (atypical hus), immune-mediated (sle, anti-phospholipid antibody syndrome), and drug-induced (cyclosporine). thorough investigation is required to detremine the underlying etiology in order to provide specific therapy and information about prognosis. a year girl presented with short stature and was found to have hypertension, proteinuria, renal failure (creatinine umol/l), and anemia (hgb g/l). platelets were normal. she also had spondylometaphyseal dysplasia, scoliosis, lymphopenia, mild pulmonary hypertension and aortic stenosis. on pulmonary function testing, her dlco was decreased. chest ct demonstrated small micronodules. a ventilation-perfusion scan was normal. renal biopsy showed features of tma. she was treated with dialysis and underwent renal transplantation one year later. there has been no disease recurrence months post-transplant. genetic and immunologic workups were negative, including anti-cardiolipin antibodies. a thrombophilia workup was negative apart from a heterozygous mutation in mthfr. c levels were mildly reduced. anti-neutrophil antibodies were negative. complement system studies, including factor h, and smarcal analysis for schimke immuno-osseous dysplasia are underway. although the descriptive diagnosis of tma can be applied here, the underlying pathophysiologic diagnosis has still to be defined. it is of particular importance that further efforts be made to identify the etiology given the potential risk of disease recurrence in the renal graft. background: long term outcome of renal functions after liver transplantation (lt) in wd is not studied yet. aim: the aim of this study was to determine the long term outcome of renal functions in children receiving lt for wd. patients and methods: renal functions were examined in (f/m: / ) liver transplanted patients for wd before and long after lt and compared with renal functions of patients (f/m: / ) with lt for a hepatic disease other than wd. the mean age of subjects was . ± . years in the patients group and . ± . years in the controls. the mean duration of follow-up was at least years. glomerular and tubular functions were assessed using the conventional equations for measured creatinine clearance (gfr), tubular phosphate reabsorption (tpr), daily protein and calcium excretion in both groups. results: mean gfr before lt was . ± ml/min/ . m in the study group and . ± . ml/min/ . m in the controls (p= . ). the mean tpr before lt was found to be . %± . % in the study group and %± . % in the controls (p= . ). daily protein excretion rate before lt was found to be high in both groups, as well as urinary calcium excretion. an increase in gfr was observed in the study group after lt (p> . ), while it was slightly decreased in the controls (p> . ). tpr increased significantly in the study group after lt ( . %± . %) (p= . ) and although it was found to be significantly lower in the study group than the controls before lt, in the long term follow-up the difference between the groups was disappeared (p= . ). conclusion: tubular dysfunction is frequent in patients with wd. liver transplantation for hepatic failure secondary to wd is a lifesaving procedure. it corrects the underlying hepatic defect as well as renal defects and leads to long-term survival. rather conflicting results are available regarding the neurocognitive development of children with ckd, due to small sample sizes, cross-sectional study designs, differing methodological approaches and historical trends in patient selection for renal replacement therapy. we prospectively examined in a standardized, multi-center effort children with ckd aged . - years. children were treated either conservatively (n= , gfr < ml/min/ . m ) or by dialysis (hd: n= , pd: n= ). a sub-sample of children underwent repeated testing after - months. bayley and snijders-oomen developmental tests were performed and the measured values normalized to standard deviation scores (sds). general cognitive development averaged at - . (± . ) sds. % of the patients scored <- . sds, i.e. below the th percentile of the normal population. no significant differences were observed between pre-dialysis (- . + . ) and dialyzed patients (- . + . ). impaired neurocognitive function was marked in infants (- . + . sds), whereas school children showed a distribution similar to healthy children ( . + . sds, p< . ). the global neurocognitive sds remained unchanged in the longitudinal sample (t =- . ± . ; t =- . ± . ). in summary, our preliminary results demonstrate a high prevalence of neurocognitive impairment in infants with ckd. we assume that this finding reflects the improved survival of children with complex disorders affecting not only the kidney but also brain development. the poor performance of this age group highlights the importance of close neurocognitive follow-up and early developmental interventions. objectives of study: to make a diagnosis of a girl with kidney subcapsular hydrops, abnormal urinanalysis and hypertension. methods: physical examination and laboratory investigations were analyzed. results: a years old girl was admitted because of kidney subcapsular hydrops, proteinuria without edema, and hypertension ( / mmhg) for days. no trauma or familial hypertension history were provided. no hydrops was found before the age of year. urinary rbcs were - /hp and protein was mg/kg/ hr. the serum albumin was normal. ultrasound examination revealed normal sized kidneys, increased echogenicity in both kidneys, and subcapsular hydrops on the upper pole of the right kidney connected with an old renal fissure. ucg and fundus examinations were normal. gfr of the right kidney was slightly decreased as compared to the left ( ml/min vs. ml/min, by dtpa scan). by puncture of hydrops, yellow clear fluid was drained, the analysis showed similar composition to that of original urine, so subcapsular urinoma was diagnosed. urine collection from two kidneys separately was performed by cystoscopy; nonselective proteinuria of + was found in urine from the right and + from the left kidney. analysis revealed urea . mmol/l, potassium . mmol/l, creatinine . mmol/l in the right kidney urine compared to urea . mmol/l, potassium . mmol/l, and creatinine . mmol/l in the left, which suggested that the right kidney function was compromised. according to proteinuria from both kidneys with microscopic hematuria, without edema and hypoalbuminemia, glomerulonephritis was diagnosed. the girl was diagnosed with glomerulonephritis and subcapsular urinoma. it was a rare case because of their co-incidence. reasons for the hypertension, if caused by the glomerulonephritis or the pressure by subcapsular urinoma, as well as reasons for subcapsular urinoma need to be clarified during the follow-up. the aim of this study was to detect factors that could interfere with the results of des treatment. methods: fifty-six patients . to . years old with des without improvement by previous therapies were randomly distributed into two voiding training programs: group (g ): patients submitted to kegel exercises training sessions for three months; group (g ): patients submitted to biofeedback sessions over a two month period. both groups adhered to a voiding and drinking schedule, received adequate toilet posture instructions and were reinforced through the maintenance of voiding diaries. clinical evaluation was carried out before each programs initiation and , , and months after end of the program. all patients were submitted to renal and dynamic ultrasound before and months after each program's conclusion. the following variables were analyzed: gender, age at diagnosis, treatment group type, vesico-ureteric reflux, constipation, urinary tract infection, asymptomatic bacteriuria, bladder wall thickening and post void residual (pvr) urine. the logistic regression model was applied to identify independent variables associated with response to treatment. results: urinary continence was improved after completion of either training program. success in diurnal urinary incontinence varied from . to % in g and from . to . % in g . success in nocturnal urinary incontinence varied from . to . % in g and from . to . % in g . in multivariate analysis three variables remained independently associated with bad response to treatment: constipation with soiling, bladder wall thickening and pvr urine (p< . ). conclusion: studies using multivariate regression analysis to identify predictors of response to behavioral therapy are important for the development of selection criteria for prescribing these therapies to children. we report the case of a four-year-old child born to consanguineous parents, who presented first at two months of age with respiratory failure. during the admission he developed panyctopenia, hypertension and nephrotic range proteinuria. the metabolic workup revealed methylmalonic academia and aciduria along with homosystinuria; highly suggestive of cobalamin deficiency. the renal biopsy showed chronic thrombotic microangiopathy (tma). the muscle biopsy showed the presence of nemaline rods on electron microscopy. cobalamin c deficiency was confirmed by genetic analysis as the patient was homozygous for the mutation c / . at the age of one, he was noted to be visually inattentive and developmentally delayed. on ophthalmology examination there was evidence of bilateral maculopathy and dysfunction of rods and cons on electroretinogram. he subsequently went on to develop bilateral bovine maculopathy. since his initial presentation he has been maintained on hydroxycobalamin, betaine and folate; with no further relapse of proteinuria or panyctopenia. however, despite adequate doses of hydroxycobalamin, the maculopathy progressed. to our knowledge, this is the first report of a child with both tma and bovine maculopathy. the aim of the study was to assess the rate of vesicoureteral reflux (vur) in patients with lower urinary tract dysfunction (lutd) of nonneurogenic origin. dysfunctional voiding may result in lower urinary tract symptoms in children and is commonly associated with urinary tract infections and vur. we investigated patients with voiding dysfunction during last three years: boys and girls, a mean age of . years, with a mean follow-up of months, all with normal renal function. mean pre and post treatment symptom scores were . and . respectively. vur was detected in % of the children with unstable bladders and . % of the children with stable bladders as detected by video urodynamic investigation, % of the children with urinary tract infection (uti) on admission, and % of the children with no history of uti on admission. the co-existence of vur in our group with voiding dysfunction was . % ( patients). all patients with vur had low grade reflux (grades i-iii) and of them bilateral, the remaining unilateral. renal us was performed in patients and revealed hydronephrosis in patients, while the remainder showed no abnormalities. vur was found to be present in % of the children with abnormal us findings, while only % of the children with normal us findings were affected. furthermore, the patients that had spontaneous resolution of reflux showed a markedly greater improvement in symptom scores. a significant portion of patients with lutd ( . %) have low grade vur. in detection of vur in patients with lutd, hydronephrosis is a good indicator of the presence of reflux, while utis and urodynamic findings were not found to be significant indicators. the overall spontaneous resolution rates of vur in patients with lutd and stable bladder following treatment was found to be . % and . %, respectively. - g) were grouped: group (n: ) was the sham group. group , and (n: for each) received mg/kg twice daily ptx intraperitoneally (i.p.), mg/kg/day gen i.p. and both ptx and gen at the same dosages for consecutive days, respectively. the rats were weighed at the beginning and than weekly during the study. after the last dose -hour urines were collected. then, rats were sacrificed and blood samples were obtained from the abdominal aorta. bun, serum creatinine (scr), creatinine clearance (ccr), renal superoxide dysmutase (sod), catalase (cat) and thiobarbituric acid reactive substances (tbars) levels were determined. all the parameters were compared between the groups. results: body weights were not different between the groups either at the beginning or during the study. bun levels were significantly higher in group than the other groups (p< . ). scr and ccr levels were similar between the groups and , but the levels were higher than those of groups and (p< . ). sod and tbars levels were similar between all groups. the levels of tubular cell apoptosis and caspase- expression were significantly higher in group than the other groups (p< . ). conclusion: we may conclude that ptx administration significantly reduced the apoptosis in gentoxicity, but we could not demonstrate any evidence of ptx-related reduced oxidative stress. the lack of evidence for the widespread use of antimuscarinics and holding exercises in mne prompted us to design a randomized controlled trial comparing interventions in four groups of children with mne: placebo (a) or oxybutynin chloride (b) in combination with a daily regimen of standardized fluid intake and holding exercises or as monotherapy (c) and (d). a fifth group, to be treated with alarm only, was planned as control. randomization was stratified for participating hospital, sex, age, tanner stage, family history of mne, previous treatment, and bladder capacity class, being the largest from either the maximum voided volume (mvv) from a h frequency volume chart or the volume after baseline holding exercises (hev). after weeks intervention with holding exercise (a and b) , hev had increased, both with oxybutynin ( ± (sd) ml to ± (sd) ml, p< . ) and placebo ( ± (sd) ml to ± (sd) ml, p< . ). without holding exercises, only oxybutynin (d) increased hev ( ± (sd) ml to ± (sd) ml (p< . ). mvv increased also in groups a and b (holding exercises), not c and d. cure rate (less than wet night in the last for weeks) was low: a / , b / , c / and d / . control group with alarm had / cure. cure was not related to hev, mvv or delta hev and mvv. this questions the relevance of increasing bladder capacity in mne. background: sickle cell disease (scd) is an inherited disorder of beta-globulin synthesis of haemoglobin, resulting in a tendency for haemoglobin polymerisation and consequent vasoocclusion, tissue hypoxia, and ensuing organ damage. the kidney is a particularly sensitive to hypoxia and renal failure is a major cause of morbidity and mortality in scd. children with scd commonly hyperfiltrate, the glomerular filtration rate (gfr) then typically falls back towards normal in adulthood. routine estimation of gfr in children is primarily derived from the height and plasma creatinine (pcr) measurements using k a constant dependent on the creatinine analytical method. this formula has reduced accuracy in children with a gfr > ml/min/ . m . it has never been validated in hyperfiltration or children with scd. recently, new gfr markers have been proposed including symmetrical dimethylarginine (sdma) that may be independent of body size. in this pilot study we tested the hypothesis that estimated gfr and/or sdma allow a reliable estimation of gfr in scd. methods: hbss patients, age range - yrs (mean age yrs) attending the evelina children's hospital were studied. the patients were on regular blood transfusions for stroke management and undertook a formal gfr measurement using plasma clearance of inutest. the plasma samples were also used for the measurement of pcr and sdma by stable isotope dilution mass spectrometry. egfr was calculated using k= . results: inutest gfr ranged from - ml/min/ . m . there was significant inverse correlation between sdma and inutest gfr (p< . )). there was no significant correlation between either pcr or egfr and inutest gfr. conclusions: this early data suggests that sdma might prove valuable in monitoring gfr in children with scd. introduction: the aim of this study was to identify the risk factors for renal scarring in children with lower urinary tract dysfunction (lutd) by using data available at the time of patient admission to the interdisciplinary management program of lutd at hospital das clínicas. material and methods: medical records of patients were assessed retrospectively concerning gender, presence of vesicoureteric reflux (vur), bladder capacity, detrusor overactivity, residual urine, urinary tract infection (uti), asymptomatic bacteriuria, constipation, detrusor-sphincteruncodination (dsu), high detrusor pressure at maximal cystometric capacity, low compliance, thickness and trabeculation of the bladder wall. renal scarring was diagnosed by dmsa scan. statistical analysis was performed by univariate and multivariate analysis. a p value < . , % confidence interval was considered significant. results: renal scarring was detected in patients ( %). abnormal bladder capacity, detrusor overactivity, residual urine, asymptomatic bacteriuria, constipation, dsu, high detrusor pressure and low compliance were not associated with renal sccaring. vur, uti, decreased bladder capacity, urinary residue, trabeculated and thick bladder wall were associated with scarring at univariate analysis. multivariate analysis showed vur (p< . ) and female gender (p= . ) as independent risk factors for renal scarring. thickness of the bladder wall was a marginal risk factor (p= . ). conclusion: urodynamic parameters didn´t predict renal damage in this study. uti was not a risk factor for renal scarring; however, it was associated with vur (p= . ). although vur was the main risk factor in our analysis, renal scarring was probably due to multifatorial causes as vur was associated with itu. results: comparison of test a and b demonstrates that a mild fluid load h before the administration of ddavp nasal spray ( puffs) results in major significant differences. ). maximal concentrating capacity (cc) is reached later than h after administration (p< . ). in / % cc at sleeping time is < % of max, thereby resulting in a persistent np in the first hour of the night. ) uosmol is significantly ( %) lower in the overnight collection (u ), correlating with a higher diuresis-rate ( %) ). the duration of the ddavp effect is shorter leading to an increase in diuresis-rate and to a decrease in the u osmolality in / children in the urine-collection between - h in the morning. this proves that in up to % of the patients the ddavp-effect does not cover the full night. conclusion: test b demonstrate sthat fluid intake prior to the ddavp-administration influences significantly the antidiuretic effect of ddavp (onset, maximum and duration). this might explain the partial response, suggestive for insufficient pd (and pk) effect of the spray. test a proves that fluid restriction h before ddavp administration, significantly reduces the nocturnal diuresisvolume. m. schmidts, c. schnakenburg, k. häffner, c. jacobi, k. schwab, m. pohl university hospital, center for pediatrics and adolescent medicine, freiburg, germany background: enteropathic hemolytic uremic syndrome (d+hus) is responsible for % of all hus cases. in addition to the nephrological and neurological complications, pancreatic damage resulting in diabetes mellitus is also possible and appears to be associated with more severe cases and elevated mortality. case report: a -year-old girl suffering from d+hus with severe colitis, acute renal failure, dyskinesia, dysarthria and agitation provoked by shiga-like toxin positive ehec infection in july, . the severe affection was characterized by partial thalamic infarction, prolonged leukocytosis (max. g/l) and the necessity of weeks of dialysis. days after the disease onset, hyperglycemia (max. mg/dl glucose) was noted. c-peptide was found to be low indicating reduced insulin secretion. months after hus the patient continues to be insulin dependent. clinically apparent exocrine pancreatic insufficiency has resolved spontaneously. gfr had recovered to ml/h/ , m , but then kidney function deteriorated and dialysis had to be resumed after months. kidney histology at this time showed severe nephron loss compatible with chronic changes after hus and ruled out other unrelated kidney disease. conclusion: in addition to the kidney damage, chronic pancreatic damage can occur in hus. therefore blood glucose levels should be monitored in all hus patients. it is tempting to speculate that patients after childhood hus might be at risk for diabetes mellitus later in life. objective: childhood nephrotic syndrome (ns) is characterised by a relapsing course resulting in significant corticosteroid burden or prescription of cytotoxic immunosupressive therapy. this randomised controlled study was carried out over years at a single centre in sri lanka to compare the efficacy and safety in children with steroid dependant ns treated with intravenous cyclophosphamide or intravenous vincristine therapy. methods: thirty-nine sequential children with steroid dependant ns with evidence of steroid toxicity were randomly allocated to receive either intravenous cyclophosphamide ( mg/m monthly for months) or vincristine ( . mg/m weekly doses followed by doses monthly). both groups received an identical tapering regimen of oral prednisolone for months. all children were reviewed on monthly basis for one year focusing on recurrence of proteinuria and side effects of therapy. finding of +++ proteinuria for consecutive days was diagnostic of relapse. results: there were ( m: f) children in the cyclophosphamide group (mean age . years) and ( m: f) in the vincristine group (mean age . years). during one year of follow-up / ( %) in the cyclophosphamide group suffered a relapse while / ( %) suffered a relapse in the vincristine group. p= . (comparison of proportions using standard error. ci . to . ). no serious adverse effects were encountered in either group. conclusion: in steroid dependant ns, intravenous cyclophosphamide therapy is superior to intravenous vincristine therapy in maintaining sustained remission. we present the clinical and biochemical features of a patient with antenatal bartter syndrome who was found to have a novel romk mutation. the patient presented antenatally with severe polyhydramnios. polyuria, hyponatraemia and hyperkalaemia were evident soon after birth. she had marked hypercalciuria and developed medullary nephrocalcinosis in early infancy. failure to thrive was evident from months. hypokalaemia was a late feature, developing gradually from months. serum chloride levels were consistently - mmol/l whilst urinary chloride levels were consistently < mmol/l, only reaching higher levels after treatment was commenced. alkalosis was not present and the patient did demonstrate some response to furosemide implying some functional capability of na-k- cl. renin and aldosterone levels were persistently elevated. treatment with indomethacin, nacl and kcl produced a good clinical response. mutational analysis revealed compound heterozygous mutations in kcnj , the gene encoding romk. both mutations, m t and l r are in the terminus of the protein thought to have a role in channel gating. similarities and differences from the classically described presenting features of antenatal bartter syndrome highlight the clinical heterogeneity in this condition. this relates to the different identified kcnj mutations which are likely to affect romk function in different ways. the m t mutation has been described, but past electrophysiological studies in sf cells transfected with the m t mutant have not identified any differences in k + conductivity from wild type romk. given that the mutation appears to be clinically relevant in this case, further functional studies are indicated. l r is to our knowledge a novel mutation. expression of these mutations in oocytes is now planned to enable evaluation their effects on channel function and regulation. a -year-old boy was admitted because of nephrotic syndrome. renal biopsy, performed after weeks of prednisone ( mg/m od) + methylprednisolone pulses ( mg/kg each), showed focal segmental glomerulosclerosis. prednisone was tapered (to mg/m od) and enalapril was introduced, without any significant improvement. two weeks later, the patient had transient hypoperfusive acute renal failure which required acei discontinuation. due to the persistence of proteinuria, cyclosporine was started ( mg/kg/day). proteinuria gradually decreased and ceased within the subsequent two weeks. in the meantime the boy had started to complain of low-grade fever without other symptoms and with normal physical examination. laboratory tests only showed leucocytosis (wbc x /ml) and increased c-reactive protein ( mg/l). a chest x-ray revealed an upper mediastinal enlargement and a total body ct scan confirmed the presence of several enlarged mediastinal lymph nodes, whose biopsy led to the diagnosis of hodgkin disease (hd) nodular sclerosis subtype, cd +; stage was iia. cyclosporine and the residual steroid treatment were discontinued and the patient was given six cycles of abvd (doxorubicin, bleomycin, vinblastine, dacarbazine), followed by radiation therapy. presently, months after stopping chemotherapy, both hd and nephrotic syndrome are still in remission. once again, this case points out both that fsg can be secondary to a lymphoproliferative disease and that chemotherapy for hd might have been effective to keep fsgs in long-lasting remission. several treatment methods including increased fluid intake and dietary modification, medical therapies such as potassium citrate and use of extracorporeal shockwave lithotripsy (eswl) and finally surgery methods are used for treatment of urolithiasis. the aim of this study was to evaluate the etiological and clinical characteristics and level of response to medical therapy with polycitra in children with urolithiasis. one hundred thirty-four patients had urolithiasis of which cases followed thetreatment instructions and fulfilled the inclusion criteria for this study. struvite stone were excluded from the study. all other patients who had an initial ultrasonography showing stone inurinary tract were treated with polycitra-k (potassium citrate grams, citric acid . grams in liter of distilled water) irrespective of the cause of the stone. at the end, complete resolution or passage or a decrease in the size of stone in later sonography was defined as response to treatment. hypercalciuria and hyperuricosuria were found to be etiological factor in % and % of patients respectively. the stone analysis was found that % of them were ca-oxalate. stone disease was more common between the age of - years. the most common complaint was hematuria ( %). eswl was performed in % of patients who did not respond to polycitra and had surgically active stones. calcium-oxalate stones were the most frequent stone which responded to polycitra. the response rate in girls and boys was equal and in different age groups the response rate was almost equal. methods: mmcs were expanded in culture and immunocytochemistry was used to characterize the cells. after gentamicin-induced atn, fluorescently-labeled cells were transplanted and traced in kidney tissues by fluorescence microscopy. kidney pathology was studied by hematoxylin-eosin staining, apoptosis was examined by the tunel assay, and ki- and bcl- expression were examined by immunohistochemistry and reverse transcription-polymerase chain reaction, respectively. results: ( ) mmcs (rimm- cells) were successfully expanded in culture. the phenotype of the cultured cells were vimentin-positive and kreatin-negative by immunocytochemistry. ( ) in the mmcs-treated group: the mortality rate decreased; renal function clearly improved; damage to the cell-treated kidneys was reduced and histopathologic lesion scores were lower; proliferation of renal tubular epithelial cells was improved; the apoptosis of renal tubular epithelial cells was reeuced; and the expression of bc - mrna and protein was upregulated. the subcapsular transplantation of mmcs could ameliorate renal function and repair kidney injury. atn is the most common reason for arf, and there is still an absence of effective therapies. this study was done to observe the effect of mobilizing bone marrow cells with stem cell stimulating factor (scf) and gm-csf on recovery from gentamicin-induced acute tubular necrosis (atn) in rats. atn was included in male sprague-dawley rats with five daily high dose intraperitoneal injections of gentamicin. subcutaneous injections of scf and gm-csf were administered simultaneously and these cytokines was observed at day , , , , and . leukocyte numbers, percent venous blood cd + cells, mortality rate, and concentration of the urine proteins, urine nag, bun, scr, and ccr, histopathogic lesion scores were determined. twelve hours after bone marrow ablation (bma) by lethal x-ray radiation, gentamicin-induced spf atn rats were given five daily injections of scf and gm-csf. bun, scr, and histopathogic lesion scores were evaluated at day , and . the effects and mechanism of scf and gm-csf on atn was observed. the number of leukocytes and the cd + cell percentage increased significantly in atn rats between and days after scf and gm-csf injection. in addition, mortality rates dropped, the peak value of renal function increased, renal function were rapid ameliorated and histopathologic lesions were reduced. there was no significant effect on atn rats after bma. this study demonstrates that scf and gm-csf effectively mobilized bone marrow stem cells in atn rats. rapidly improving renal function and decreasing mortality rate. these results suggest that bone marrow stem cell mobilization may be an effective therapy for atn. key words: acute tubular necrosis, bone marrow stem cells, stem cell factor, granulocytemacrophage colony stimulating factor, irradiation. objective of the study: the response to recombinant human erythropoietin (rhuepo), unit/kg twice weekly was studied prospectively in children and adolescents with end stage renal failure who were either transfusion dependent or had hematocrits (hct) < %. methods: rhuepo was given to haemodialysis (hd) patients and patients on conservative treatment, with mean age ( . ± . ) years, males and females with mean hct ( . ± . ). blood pressure, haematocrit, iron-indices, serum potassium, calcium, phosphorus, alkaline phosphatase, urea nitrogen and intact parathyroid hormone (ipth) were monitored serially. results: serum aluminum was measured randomly in patients, results were normal ranging from - ug/l. when serum ferritin was < ng/ml during therapy, they received iron supplementation. according to the response, patients were divided into groups, the non-responders group with hct< , mean age ( . ± . ) years, males and females with mean ipth ( . ± . pg/ml) and group of responders with hct > with mean age ( . ± . years), males and females with mean ipth ( increase of fatty acids such as nonesterified fatty acid (nefa) and triglyceride (tg) with oxidative stress and production of cytokine/chemokine occured during cisplatin (cp) toxicity. statin ( hydroxy- -mthylglutaryl coenzyme a reductase inhibitors) have been postulated to have pleiotrophic effects. we examined whether statin, pravastatin, would ameliorate renal damage induced by cp. male wistar rats (weight - g) fed standard chow were divided into groups: control-rats received tap water alone for days; cp treatment-rats that received cp ( mg/kg, i.v.) on the th day of the study; cp+pravastatin treatment-rats that received pravastatin ( mg/kg/day) in their drinking water for days and cp injection as indicated in the preceeding group. blood and urine samples were collected and the kidney were removed days after cp treatment. urinary excretions of protein and -hydroxy-deoxyguanosine ( -ohdg), serum levels of creatinine and fatty acids were measured. histology was evaluated by light microscopy with immunohistochemistry for pentosidine, n-carboxymethyllysine (cml), and heme oxygenase (ho)- . expression of ho- mrna in the kidney was also measured. pravastatin decreased urinary excretions of protein and -ohdg and ameliorated renal function diminishing areas of tubular damage and positive staining of pentosidine and cml compared with those of cp treatment alone. however, positive staining area and mrna expression of ho- were not significantly changed by pravastatin treatment. although pravastatin did not influence serum levels of total and low-density lipoprotein cholesterol, serum tg level decreased and was equivalent to that of control group. these findings suggest that pravastatin treatment partially protects against cp-induced nephrotoxicity in rats, through its antioxidant as well as lipid-lowering effect. n. bresolin, v. fernandes, f. carvalho, j. goes, l. araujo, m. simon, g. gamborgi, m. zanin hospital infantil joana de gusmo, nephrology pediatric department, florianópolis, brazil objectives: the objective is a report case of acute renal failure (arf) in a child contact with lonomia obliqua caterpillar in southern brazil. the accidents with lonomia oblique has increased in south of brazil in recent years. this increase can be related with reduction of caterpillar natural predators and deflorestation. the venom of lonomia caterpillar provokes hemorrhagic syndrome resembling disseminated intravascular coagulation (dic) and hemolytic anemia. arf could be an important complication of hemoglobinuria that has been recently described in adults. methods: a case report. results: the present is a first case described of arf in a child after contact with lonomia obliqua caterpillar. conclusions: the arf can occur in any age, however, the contributing factor to the development of arf remains obscure. there are mechanisms pointed out: fibrin deposition in glomerular microcirculation, ischemia in hemorrhagc shock with hypotension and venom direct action. the present article related a case of lonomia obliqua accident in a child who revealed coagulation disorder, hemolytic anemia, arf and always, she was hemodynamic stable. the treatment included antilonom serum, urine alkalinization, hyperhydration and peritoneal dialysis for four days. she was treated and followed per year when she recovered her normal renal function. introduction: secondary hyperparathyroidism develops in chronic kidney disease as a consequence of impaired phosphate, calcium and vitamin d homeostasis. the treatment includes phosphate binders and vitamin d analogues, but sometimes ineffective. we report two cases of refractory secondary hyperparathyroidism treated successfully with cinacalcet. case : a -month-old boy with end stage renal disease (esrd) due to posterior urethral valve started on peritoneal dialysis at months of age. he developed secondary hyperparathyroidism with serum parathyroid hormone (pth) level reaching pg/ml. serum calcium had been in the range of . to . mg/dl and serum phosphorus in the range of . to . mg/dl. despite treatment with phosphate binders and vitamin d analogues, pth levels kept increasing to pg/ml. x-ray showed the cupping and fraying of the distal ends of radius and ulna. we started cinacalcet mg daily and increased the dosage up to mg daily. eight months later, pth level decreased to pg/ml and bone changes resolved. case : a -year-old boy with esrd due to primary hyperoxaluria started on peritoneal dialysis at years of age. he developed secondary hyperparathyroidism with serum pth level of pg/ml. serum calcium had been in the range of . to . mg/dl and serum phosphorus in the range of . to . mg/dl. despite treatment with phosphate binders and vitamin d analogues, pth level kept increasing to pg/ml. x-ray showed the distal radius and ulnar fracture of left arm and right femur fracture. we started cinacalcet mg daily. five months later, pth levels decreased to pg/ml. conclusion: cinacalcet is effective in the secondary hyperparathyroidism resistant to phosphate binders and vitamin d analogues in children with chronic renal failure. rota virus (r) is a common pathogen as the cause of gastroenteritis in childhood. we experienced some cases with r infection who had the renal failure induced by uroammoniac calculi as well as dehydration. we examined the clinical feature and laboratory findings of the cases with viral acute gastroenteritis in r and non-rota virus (nr) groups for - . with rapid diagnosis test, we checked the patients that needed the hospitarization medical treatment from newborn to five-years old. in the cases of r group and cases of nr group, we campared the clinical findings, blood chemistry test and urinalysis of r groups in acute and convalescent a tage with those of nr group. r group had significantly (p< . ) lower in ph (vein blood gas test) and higher in blood uric acid (bua) compared with those of nr group. these findings suggest that the elevation of bua and acidosis in r group may induce the formation of renal calculus resulting in postrenal failure. our patient is white male y/o. at year of age, he was dx. with acute lymphocytic leukemia and was induced to remission on pog ; he presented tumor lysis syndrome during induction, acute renal failure with no recovery in renal function and rrt was started (ccpd) and continue on the same therapy. during the last years, he presented chronic pancreatitis, ards, sepsis. he had episodes of major vessel thrombosis probably due to central lines (several femoral lines and vascath placed in his thorax) and l-asparaginase. our patient did receive years of chemotherapy and radiation; and he is on complete remission. on / , , for pre-kidney transplant evaluation, a mri test was requested using gadolinium ml/kg for evaluating his vascular system (abdomen and pelvis) with inconclusive results. another mri test was requested months later using gadolinium . ml/kg. weeks later, his mother noticed brawny hyperpigmented, shiny, tightly bound-down, indurate plaques of his bilateral lower extremities, (more significantly on his lateral and posterior calves, and in his anterior and lateral thighs) a punch skin biopsy showed increasing number of fibroblastic cells and widened space between septa in the deep reticularis dermis compatible with nfd. nfd is a fibrosing disorder identified among patients with renal disease with cutaneous finding of skin thickening in extremities and trunk, very similar to those of systemic sclerosis. etiology, pathogenesis and clinical course remain unknown. the majority of cases have been reported in dialysis or kt patients and gadolinium has been identified as a trigger of nfd. in our case, there appears to be a link between the use of gadolinium and the developed of nfd. background: chronic renal failure (crf) is an independent cardiovascular risk factor. changes in calcium-phosphate homeostasis not only affect the quality of bones but also constitute the biochemical base for vascular calcification. aim: to find a method better describing factors of calcification using routine laboratory examinations and computed evaluation of complex equilibria. methods, patients: data of crf and of transplanted (tx) children were compared to a healthy control group of . tx children's parameters were taken before and and month following transplantation. three different strategies were used to analyse factors of calcification: ca x p product, ca-p activity value and the concentration of cahpo . the ca x p product and the ca-p activity value were not informative, because they didn't represent the direction of change in the complex chemical equilibrium. the cahpo concentration was increased in the crf and the tx group (before transplantation) ( . ± . mmol/l) compared to the healthy control group ( . ± . mmol/l) (p< . ). after and months to transplantation the cahpo concentration was in the normal range in the tx group. a negative correlation was found between the concentration of ica + (ionic calcium) and pth (parathyroid hormone) in the dialysed children (r= . ), but not in the transplanted group (r= . ). conclusion: according our findings the force driving calcification is better represented by the concentration of cahpo , the base compound of all primary calcification, than by measuring ca and p separately. follow-up study is needed to establish the predictive value of determination of cahpo . this study was supported by grants otka-t and ett / , the national office for research and technology (nkth) and szentágothai j. knowledge centre. introduction: acute renal failure is a rare complication of nephrotic syndrome and its cause is still unknown. several investigators reported that the most important factor for renal failure was acute tubular necrosis (atn); however, some cases did not have laboratory findings of tubular dysfunction paradoxically. patients and methods: we reviewed cases of nephrotic syndrome with acute renal failure (nsarf) since at metropolitan kiyose children's hospital. seven of cases had calcium deposition in the distal tubules predominantly. we analyzed the clinicopathological findings in these cases. results: the pathological diagnoses of these cases were as follows: minor glomerular abnormalities, mesangial proliferative glomerulonephritis (without iga deposition), and focal segmental glomerulosclerosis. interstitial nephritis was not documented in any case. the common characteristics of these cases were calcium deposits in the distal tubules. some cases had focal simplification of tubular epithelium. all cases were an initial episode of nephrotic syndrome, and cases were steroid resistant. almost all cases had hypertension. in addition, the range of urinary β microglobulin and fena were to and . to . %, respectively. discussion: in these cases, there was enhanced na reabsorption and urinary β microglobulin was only mildly elevated at the time of renal failure. these findings were not consistent with atn. additionally, pathological finding of severe tubular damages was not found on biopsy. contrarily, tubular obstructions due to ca depositions were consistent with these clinicopathological findings. conclusion: our findings suggest that tubular obstruction due to calcium deposition plays an important role in the etiology of nsarf except for atn. background: natriuretic factor was found in urine of chronic uremia for more than years. n-terminal pro-b-type natriuretic peptide (prontbnp) is postulated to be the natriuretic factor owing to its elevation in chronic kidney disease (ckd). however, salt wasting and non-saltwasting chronic kidney disease haven't been partitioned as different entities before. this study is designed to clarify whether prontbnp is different in salt wasting and non-salt wasting ckd and if it is the main causative factor of natriuresis. methods: patients with salt wasting ckd, which are mainly composed of congenital renal structure abnormalities, and patients with non-salt wasting ckd, which are mainly composed of glomerulonephritis, were collected. all of them are non-dialysis-dependent and without heart failure. serum prontbnp in these two groups and normal controls were sampled. fractional excretion of sodium (fena) and estimated clearance of creatinine (ccr) were also checked. results: prontbnp was elevated in salt wasting group compared to normal controls (p= . ). moreover, prontbnp was even significantly higher in non-salt wasting group than in salt wasting one (p< . ). in salt wasting ckd, prontbnp and fena were correlated (r= . , p= . ), but the same result was not observed in non-salt wasting group. prontbnp was significantly correlated to ccr and age using multiple regression analysis (p< . ). conclusion: prontbnp elevation was different between salt wasting and non-salt wasting ckd. volume expansion explained the difference and prontbnp elevation in salt wasting ckd resulted from other mechanisms. since salt wasting ckd, in which the influence of volume expansion was eliminated, had a good correlation between prontbnp and fena, prontbnp may be one of the major contributing natriuretic factor in chronic uremia. background: cidofovir is a new antiviral drug that has a broad spectrum of activity against a number of dna viruses. several observations reported its efficacy as topical treatment of resistant warts. we herein report a systemic complication of acute renal failure associated with such topical therapy. casereport: girl aged , received a renal transplant in and for end-stage renal failure secondary to haemolytic uremic syndrome. her initial immunosuppressive regimen consisted of prednisone, tacrolimusand mycophenolate. five years post transplant she developed chronic allograft nephropathy with mild renal impairment. as a consequence of long standing immunosuppression she also developed invalidating, widespread periungueal warts. a well conducted, aggressive, conventional management of her warts including reduction of immunosuppression failed to improve the symptoms. she subsequently received an intralesional injection of a mg/ml cidofovir solution. forty eight hours after the treatment she developed local swelling, inflammation and pain along with acute alteration of renal function. although cidofovir seric dosages performed on day , , and postinjection were negative we attributed this acute renal failure to a potential nephrotoxic side effect of cidofovir. renal biopsy showed no evidence of rejection but mild alterations of the tubes compatible with tubulo-interstitial nephritis. spontaneous recovery of renal function occurred within months. conclusion: we describe an acute renal failure as a consequence of topical cidofovir treatment of warts in the context of renal transplant with mild preexisting renal failure. topical administration of cidofovir needs as carefull use and monitoring as its parenteral administration, especially in patients with altered renal function. the hemolytic uremic syndrome (hus) is a disease characterized by microangiopathic hemolytic anemia and variable organ impairment with a predominat feature of renal failure in children. the aim of this study was to investigate the presence of plasma lipid peroxidation products in the acute phase of hus. we have analyzed the levels of lipid peroxidation, determined fluorometrically as thiobarbituric acid-reactive substances (tbars), in the plasma of patients (aged - months with a median of ) diagnosed with the shiga toxin-associated form of the disease. in all cases, tbars determinations were performed at hospital admission, during treatment and upon discharge. tbars values averaged . ± . and . ± . mm sem at admission and discharge respectively (reference value < . mm). of the patients examined, presented conserved diuresis during the course of the syndrome, while the other were anuric and required dialysis. maximum tbars values resulted significantly higher (p< . by anova followed by newman-keuls test) in the dialysis-requiring patients as compared to those that had conserved diuresis ( . ± . vs . ± . mm respectively) we also investigated a possible correlation between tbars and plasma creatinine, urea, lactate dehidrogenase (ldh) and platelet count. positive and highly significant correlations (r= . and . , p< . ) were observed when tbars values were plotted as a function of plasma creatinine and urea values respectively. no significant correlations were detected for tbars and plasma ldh values or platelet count. thus, patients affected by shiga toxin-associated hus exhibit increased levels of oxidative stress. also, there is a positive correlation between the magnitude of oxidative stress and the severity of renal failure. c. soares, j. diniz, e. lima, g. oliveira, c. camargos, b. sousa, e. oliveira objective of the study: the purpose of this retrospective cohort study was to report the clinical course of children and adolescents with chronic kidney disease (ckd) enrolled in a pre-dialysis program. methods: the records of patients with ckd admitted to an interdisciplinary pre-dialysis program from to were retrospectively analyzed. the program consisted of conservative management of children and adolescents with ckd and was conducted by an interdisciplinary team including pediatric nephrologists, pediatricians, nurses, psychologists, nutritionists, and social workers. the patients were admitted with a glomerular filtration rate (gfr) equal to or below % of the value expected for their age according to normal reference data. demographic, clinical and laboratory data at entry and at the end of the follow-up were analyzed. renal survival analysis was performed using the kaplan-meier method. results: the median age at admission was . years (interquartile range: . - . yr). the most frequent primary renal disease was congenital urological anomaly in patients ( %), following by glomerular diseases in ( %), cystic diseases in ( %), and others in children ( %). at admission, patients ( %) had ckd stage , ( %) had ckd stage , and ( %) presented ckd stage . median follow-up time was . years (iq range, . - . ). at the end of the follow-up, children presented ckd stage or ( %), patients were in stage ( . %), in stage ( %), and children were in stage ( %). it was estimated by survival analysis that the probability of ckd stage was % at years, % at years, and %at years after admission to the pre-dialysis program. conclusion: the long-term overall renal survival is relatively poor in a cohort of children and adolescents with ckd. objective: characterize the association between proteinuria and gfr in a cross-sectional study of children with mild to moderate chronic kidney disease (ckd). methods: first morning urine protein to creatinine ratios (up/c) and gfr (measured by iohexol blood-disappearance) for children enrolled in the ckid cohort study were examined using loglinear regression of proteinuria and gfr, adjusted for age, sex, body surface area (bsa), and ckd cause. conclusions: in children with mild to moderate ckd, there was a log-linear relation between proteinuria and gfr. the prospective cohort design of ckid will assess the risk of gfr decline associated with level of proteinuria in children with glomerular vs non-gn causes of ckd. objective: to study the clinical characteristics of chronic renal failure (crf) in children. the clinical data of children with crf in the last fifteen years were retrospectively analyzed. results: the first main causes of crf in children were still glomerular disease, and congenital deformities of urinary system and hereditary renal disorders were the second main causes. the initial age of children with crf which were caused by congenital deformities of urinary system and hereditary renal disorders were relatively younger than children with crf which were caused by glomerular disease. the symptoms of some crf in children were not typical. conclusions: glomerular disease were still the first main causes of crf in children. urine screening test, early renal function examination and kidney b-mode ultrasonic were attribute to early diagnosis and proper management of crf in children. the aim of the study was the evaluation of influence of clinical and biochemical parameters upon degree of impairment of cardiac function in dialysed children. methodology: chronically dialysed ( hd, pd) children participated in the study ( m, f), aged - , yrs (x= , ± . yrs). echocardiography examinations were carried out with a hp device. diastolic and systolic left ventricular (lv) dimension, ejection fraction (ef) and lv mass index (lvmi) were evaluated. mean values of estimated parameters in months preceeding echo were calculated. results: on the basis of echo examinations groups were singled out: a (n= ) of normal heart function, b (n= ) of impaired systolic and diastolic heart function and c (n= ) of normal systolic and impaired diastolic heart function. no differences between groups according age, bmi, dialysis and renal insufficiency duration were found. in group of children with severe cardiac lesion (b group) a higher lv mass (a vs b vs c: , vs , vs , g/m) and statistically significant lower ejection fraction ( , vs. , vs. , %) were ascertained. these children were anuric ( vs. vs. ml/d), their systolic ( , vs. , vs. , ) and diastolic ( , vs. , vs. , ) blood pressure were significantly higher, so was the number or hipotensive medications ( , vs. , vs. , ) . bioimpedance analysis showed overhydration in group b (tbw% vs vs . ; ecw/icw . vs . vs . ). conclusions: the vast majority of chronically dialysed children demonstrate an impairment of cardiac function mainly of diastolic parameters. anemia, malnutrition, hypervolemia, anuria and hypertension stand for a significant risk factors of cardiac impairment in dialysed children. we report here the clinical findings and prognosis of patients ( girls, boys) with infantile oxalosis diagnosed between june and december in order to remind this rare autosomal recessive metabolic disorder as the potential cause of acute renal failure in infancy. the mean age of the patients was . months (range: . - months). all patients had the complaint of vomiting since birth, had irritability, and had convulsions. seven of them had parental consanguinity ( %) and family histories revealed urolithiasis in four patients and infantile deaths in one. two patients were cousins without any other family history. all patients presented with anemia (hemoglobin: . - . g/dl), renal failure (creatinine levels: . - . mg/dl) and metabolic acidosis. abdominal x-rays showed bilateral nephrocalcinosis, renal ultrasonographies revealed normal sized kidneys with diffuse and intensive hyperechogenity and loss of corticomedullary differentiation in all patients. crystal deposition was demonstrated in fundoscopic examination of patients, bone marrow aspiration of patients, and renal tissues of patients that underwent renal biopsy. besides supportive therapy, peritoneal dialysis was performed on patients, but only one patient accepted to continue the therapy. four patients died within few months and were lost to follow-up, probably died. two patients have been followed up for months one on continuous ambulatory peritoneal dialysis. in conclusion, this rare disease with fatal outcome should be remembered and investigated in infants with renal failure and bilateral nephrocalcinosis since early combined renal and liver transplantation may be life saving if it can be performed. acute renal failure following halothane anesthesia in young child? case report we present here the case of renal failure and hepatocellular lysis that developed h after the exposure of halothane anesthesia during eye examination. previously a healthy months old boy had a cardiac arrest, during the above mentioned diagnostic procedure, therefore cpr was applied which all happened in another hospital. after a few hours he was transferred to icu at our hospital, consciousness, hemodinamic stabile. in the following hours he developed acute non-oliguric renal failure (maximal level of urea mmol/l, creatinine mmol/l), as well as hepatocellular lysis (alt u/l, ast u/l). plasmaferesis and continuous venovenous hemodiafiltration were immediately applied followed by conservative measures. the level of serum transaminase returned to the normal values within a week and the level of creatinine and urea within two weeks. fully recovered boy was released home. toxic effects of halothane anesthesia in children are reported in only few cases in the literature. objective of study: many cases of chronic aristolochic acid nephropathy (caan) in adults had been reported, but it had rarely been described in children. we reported children with caan to investigate its clinical and pathological characteristics. methods: three cases were studied retrospectively and relevant literatures were reviewed. results: three children received traditional chinese herb medicine containing aristolochic acid to months for different basal diseases including chronic aggressive hepatitis b, secondary hydrocephalus and purpura nephritis and suffered from caan from mouths to years after they began receiving the chinese medication. the main manifestations were renal failure of various degree accompanied with proximal and distal tubular dysfunctions. two of them began with anemia and suffered from serious renal failure. the onset of another case was glucosuria with renal function impaired mildly, and she presented secondary fanconi's syndrome simultaneously. their pathological characteristics were diffuse paucicellular interstitial fibrosis and marked tubular atrophy with mild glomerular impairment. after withdrawal of the chinese herb medicine, renal failure in two patients attenuated progressively. the clinical features of caan in children are progressive renal failure and renal tubular disfunction. its pathological characteristics are diffuse paucicellular interstitial fibrosis and marked tubular atrophy. therefore, we emphasize the recognition of the nephrotoxicity of traditional chinese herb medicine and prevent caan from happening in children. objective of study: recombinant human erythropoietin (rhuepo) treatment may cause pure red cell aplasia (prca), but it had been rarely described in children. in order to acquaint ourselves with this disease, we reported a boy with chronic renal failure developed prca following rhuepo treatment. methods: one case was studied retrospectively and relevant literature was reviewed. results: a -year-old boy suffered from chronic renal failure combining with renal anemia and received rhuepo treatment. two weeks later, his hemoglobin (hb) increased from to g/l, and maintained to g/l for weeks. subsequently his hb declined suddenly at a rate of g/l/day, despite rhuepo increased in dose. his reticulocyte count reduced to . /l without other cytopenias in peripheral blood. a series of laboratory examinations were performed, including bone marrow cell smear and culture to confirm his prca. because various of other factors such as parvovirus etc. induced prca was excluded, we considered the boy's prca was due to rhuepo treatment, although we didn't detect anti-epo antibodies for lacking of the reagent. the boy received erythrocyte suspension transfusion to correct his anemia and was waiting for renal transplant in the following period. conclusions: during the treatment of rhuepo, sudden and rapid reducing of hb might be a hint of rhuepo induced prca. the diagnosis should be based on the clinical presentation, the absence of red cell precursors in bone marrow and the detection of anti-epo antibodies. renal transplant and immunosuppressive therapy might be effective. m. zaniew, b. mroziñski, a. warzywoda, e. stefaniak, a. siwiñska, j. zachwieja among ambulatory blood pressure monitoring (abpm) parameters, pulse pressure (pp) provide an information on cardiovascular (cv) status. recently, a novel parameter i.e. ambulatory arterial stiffness index (aasi) has been proposed. aim: to investigate pp and aasi in relation to left ventricular (lv) geometry and carotid intima-media thickness (cimt) in children with chronic renal failure (crf). subjects: children ( f/ m; median age: yrs) with crf treated conservatively (n= ) and with dialysis (hd; n= /pd; n= ). methods: we studied demographic data, echocardiography data [left ventricular mass (lvm), left ventricular mass index (lvmi), interventricular septum (ivs), left ventricular posterior wall (lvpw), left ventricular end diastolic diameter (lvedd), relative wall thickness (rwt)], cimt and abpm. from abpm, we calculated pp [difference between systolic blood pressure (sbp) and diastolic blood pressure (dbp)] and plotted dbp against sbp (regression slope). aasi was defined as minus this regression slope. results: a positive correlation was found between aasi and pp (r= . , p< . ). aasi was correlated to lvedd (r= . , p< . ) and rwt (r=- . , p< . ). pp was related to: weight (r= . , p< . ), body surface area (r= . , p< . ), body mass index (r= . , p< . ), and lvm (r= . , p< . ), lvmi (r= . , p< . ), lvpw (r= . , p< . ), lvedd (r= . , p< . ), rwt (r=- . , p< . ). neither aasi nor pp was associated to cimt. children with lv hypertrophy had higher pp than without this alteration (p< . ). when data analyzed across quartiles of pp, children in the upper quartile showed higher lvm (inter-group comparisons p< . ), lvmi (p< . ), lvpw (p< . ) and lvedd (p< . ). conclusions: pp is a stronger predictor than aasi of lv geometry in children with crf.. acute renal failure in newborn period may be caused by prenatal, natal and postnatal factors. among them, obstructive lesions of the genital tract (e.g. imperforate hymen and vaginal atresia) are very rare. children with hydrometrocolpos due to distal vaginal atresia may present with severe obstructive uropathy and its consequences. hydrometrocolpos is the result of vaginal obstruction and can become an emergency in newborn period. acute renal failure associated with distal vaginal atresia appears to be rare, with only one report in the paediatric literature. here we report a -dayold infant with a hydrometrocolpos causing life-threatening renal failure. percutaneous drainage did result in dramatically improved clinical and laboratory findings of the patient. objectives of study: sympathetic overactivity is currently considered as an important mechanism of both development and progression of chronic renal failure. however, this statement was mostly based on the researches in which the participants were adult patients with terminal renal failure. little information is available on autonomic changes in pediatric patients with mild renal insufficiency. our aim was to determine whether there is sympathetic activation in children with chronic pyelonephritis in a stage of mild renal insufficiency. methods: patients met the inclusive criteria were selected and assigned into two groups according to the creatinine clearance. group i had patients with creatinine clearance between and ml/min/ . m while group ii have patients with normal creatinine clearance. baseline of age (from - years old), gender and diagnosis between the groups are comparable. time domain analysis of heart rate variability in short-term recordings of minutes was performed in both groups. as well vain questionnaire for assessment of autonomic state was performed in all participants with their parents help. results: the outcomes of heart rate variability analysis showed sympathetic overactivity of . % patients in group i vs . % in group ii, and the difference is statistically significant (t= . , p< . ). significant difference was also found in results of vain questionnaire: . % of patients in group i were estimated as "sympathetic", while only . % in group ii (t= . , p< . ). conclusions: based on the consistent findings of the two methods used in this study, we conclude that sympathetic overactivity may be found in children with even mild renal insufficiency, and it should be considered as an early event in the development of pediatric renal failure. the aim of this study was to describe the prevalence of myocardiopathy in pediatric patients with different stages of ckd (stages to ).methodology: inclusion criteria -gfr < ml/ . m , ckd treatment > months, age < years old. echocardiograms were performed using standard techniques. left ventricular mass (lvm) was measured by two-dimensional directed m-mode echocardiography according to the american society of echocardiography criteria. lvm index was assessed and when > g/m . it was considered severe hypertrophy. the relative wall thickness (rwt) was measured to assess the lv geometric pattern. age, high blood pressure (hbp), anemia, time and type of treatment were compared to the echocardiographic findings. results: we evaluated patients, mean age years old, % on pre-dialysis, % on hemodialysis (hd) and % on peritoneal dialysis (pd). patients on hd were evaluated in the interdyalitic period. twenty-seven patients ( %) had myocardiopathy, clvh in ( %), elvh in ( . %) and cr in ( %). severe hypertrophy ocurred in pacients ( . %). there was no significant difference in relation to age and high blood pressure. patients with clvh were on hd for longer time and had a lower hematocrite when compared to the patients without clvh ( ± vs ± months; p< . ) and ( ± vs ± ; p< . ) respectively. there was a significant correlation between hematocrite and left ventricular mass (r = . ). conclusion: we observed a high prevalence of myocardiopathy in this study population. the risk factors associated to clvh were anemia and time on hd. larger and prospective studies are needed to analyze the impact of myocardiopathy in the cardiovascular mortality in children as well as the results of interventions applied to correct these risk factors. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . urological problems such as vesicoureteral reflux ( , %), obstructive uropathy ( , %) and neurogenic bladder ( , %) were the leading underlying conditions causing cri with a total of , %. while majority of the patients were having gfr levels less than ( , %) or between to ml/min/ , m ( , %), gfr level was between - in only . %of the patients. patients with pth levels between - and more than pg/ml comprised the majority ( , and % respectively). the gfr levels correlated positively with hemoglobin/hematocrit and calcium levels and negatively with phosphorus and pth levels (p< . ). renal replacement therapies were initiated in , % of the patients. the most striking result of this study is the predominance of vur or related urological problems that are relatively preventable as the underlying causes of cri in children. early diagnosis and appropriate medical or surgical management of these conditions should be emphasized together with achieving broader coverage of the pediatric cri population in terms of supportive treatment. twin-twin transfusion syndrome (ttts) complicates up to % of monochorionic twin pregnancies. shunting of blood between twins through common placental blood vessels can cause growth restriction, oligohydramnios and anaemia in the donor twin. renal impairment in the donor twin has been reported as a transient phenomenon with full recovery. we describe a case series of three children with chronic renal failure due to ttts. all cases were monochorionic diamniotic twins. in cases and , growth discrepancy was noted on prenatal scanning. in the case anomaly scans were normal. gestation at birth ranged between and weeks. in all cases there was a significant birth weight discrepancy between twins. post-natal ultrasound appearances were variable: case had normal scans within the first month but echogenic kidneys at months; case had initial increased echogenicity but features consistent with cortical necrosis at weeks; case had small kidneys with no cortico-medullary differentiation. all children became dialysis dependent within the first year of life. age of commencement ranged from days to months. case was successfully transplanted at years. case had an unsuccessful transplant at years and remains on dialysis at years. case remains on dialysis at years. cases and are now at mainstream school with only mild learning difficulties; case has some gross motor delay but other wise normal development. with advances in neonatal intensive care and improved survival of low birth weight babies, this presentation may become increasingly common. even severe renal involvement can occur in the absence of other significant hypoperfusion injury. the management of survivors of ttts with established renal failure may present unique challenges and opportunities. introduction: endothelial heparan sulfate proteoglycans (hspg) play an important role in various biological processes in the renal glomeruli. it is involved in the inflammatory process by acting as a ligand for l-selectin. furthermore, leukocytes are able to interact with chemokines bound to hspg (examples are il- , rantes and mcp- ). this will lead to activation of the integrins on leukocytes and result in more stable leukocyte-endothelial wall adhesion. in this work, we have studied the effect of a subtoxic dose of shiga-like toxin (stx) and on the hspg and the possible implications on the pathogenesis of the hemolytic uremic syndrome (hus). methods: to study this effect, primary human umbilical venous endothelial cells (huvec) and primary human glomerular microvascular endothelial cells (gmvec) were incubated for hours with a subtoxic dose of stx or . then, cells were treated with the enzyme heparinase (which cleaves off hspg). non-treated cells were used as controls. after treatment with the enzyme, the endothelial cells were exposed to flowing human leukocytes in a parallel plate flow chamber. the amount of adherent leukocytes was calculated. -: not treated with heparinase , +: treated with heparinase conclusion: stx and cause an upregulation of the amount of adhering leukocytes on both endothelial cell types. treatment of the endothelial cells with heparinase decreases markedly this upregulation. the effect can not be detected without stx-incubation. this can be explained by a possible upregulation of hspg on endothelial cells by stx, resulting in a higher level of bound chemokines or an increased binding of released chemokines to hspg. this will lead to an increased adhesion of leukocytes and will induce a more severe inflammatory process in the renal endothelium. this effect will contribute to the devastating pathogenesis of the hus. c. soares, j. diniz, e. lima, g. oliveira, c. camargos, b. sousa, m. almeida, e. oliveira objective of thestudy: the purpose of this study was to identify clinical, nutritional and laboratory factors associated with the rate of progression of chronic kidney disease (ckd) among the children and adolescents admitted to a pre-dialysis interdisciplinary program. methods: one hundred eight children and adolescents aged months to years with ckd in conservative management were prospectively followed up from to . renal survival was analyzed by the kaplan-meier method and cox's regression model. a multivariate model was developed from the admission until occurrence of ckd stage (glomerular filtration rate < ml/min). the following data were obtained at admission: gender, race, age at admission, weight z score, renal primary disease, blood pressure, and laboratory data (serum creatinine, serum urea, glomerular filtration rate, hr urinary protein excretion, hematuria). results: median follow-up time was . years (iq range, . - . ) and patients ( %) progressed to ckd stage . in the univariate analysis the following variables were significantly associated with the event: non-white race (p= . ), age > years (p= . ), ckd stage at baseline (p= . ), renal primary disease (p< . ), severe proteinuria (p< . ). after adjustment, variables remained as independent predictors of ckd stage : severe proteinuria (rr= . , % ci= . ± . ), glomerular disease as renal primary disease (rr= . , % ci= . ± . ), and ckd stage at baseline (rr= . , % ci, . ± . ). conclusion: the combination of three factors -severe proteinuria, glomerular disease, and ckd stage at admission -was an independent indicator of adverse outcome in children and adolescents in a pre-dialysis interdisciplinary program. background: allogenic hematopoetic stem cell transplantation (allo-hsct) has gained world wide acceptance as a treatment for various diseases. renal dysfunction is one of the major complications that influences transplant related mortality (trm) rates following hsct. in this prospective study, we aimed to investigate the effect of allo-hsct on renal function in children. methods: renal ultrasonography, dmsa scintigraphy, analysis of renal and tubular function tests were performed before, shortly and -year after hsct. acute renal toxicity (art) was classified according to bearman criteria. grade =increase in creatinine up to twice the baseline; grade =increase in creatinine above twice baseline; grade =increase in creatinine above twice baseline and need for dialysis. chronic renal insufficiency (cri) was defined as gfr< ml/min/ . m and failure (crf) as need for dialysis. results: between april and june , children (median age: . years) underwent allo-hsct because of hematologic disorders (malignancy, ; hemoglobinopathy, ; aplastic anemia, ). all patients except one received nontbi conditioning regimen. six patients ( . %) were died because of trm but none of these patients had primary art. during the first days, patients ( . %) had grade art (csa nephrotoxicity in , vod in patients). grade art was defined in patients ( . %) (vod in , sepsis in , csa nephrotoxicity in patient). eleven patients had structural renal abnormalities before hsct, of them persisted and new patients had pathologic results one year after hsct. in long term period, ( . %) of evaluable children had cri and no patient had crf requiring dialysis. conclusion: renal dysfunction was found to be a frequent complication after allo-hsct in children. therefore, renal functions should be followed carefully in these patients. . years and at the end of follow-up, and compared between the three groups. there were no significant differences between groups in so far as gender, underlying disease, age at diagnosis, proteinuria, hypertension, hyperparathyroidism, use of ace inhibitors or renal size. erithropoietin use was significantly higher (p< . ) in group . gfr improved in all three groups during their first two years of life. the cut-off point on the roc curve indicating worse gfr long term evolution was ml/min/ . m at two years of life (sensitivity %, specificity %). g. zilleruelo, am. onder, j. chandar, o. nwobi, c. abitbol background: catheter-related bacteremia (crb) is a common complication of tunneled-cuffed hemodialysis catheters. our objective was to investigate the effectiveness of tissue-plasminogen activator-tobramycin locks (tpa-abl) in preventing crb in pediatric patients. methods: a retrospective analysis of pediatric hemodialysis patients was performed. patients with > crb/ catheter days (cd) were designated as high risk. those with < crb/ cd were of average risk. three eras of consecutive months were studied. in era , high risk patients were detected. during era , the high-risk group was placed on times weekly prophylactic tpa-abl. in era the high-risk group was given once weekly tpa-abl. results: there was a significant decrease in the rate of crb with prophylactic tpa-abl which was more pronounced when given three times/week. conclusions: the use of prophylactic tpa-abl times weekly significantly reduces the rate of crb in patients at high risk. prophylaxis once weekly may be less effective than thrice a week. l. was born after a normal pregnancy without a special personal or familial history, was seen at the age of years, after a days long ordinary oro-pharyngeal viral infection treated by symptomatic treatments. he presented with inflammation of the left cheek with slight fever ( °) and a sole purpuric lesion of the left leg ( cm) and some petechiae on the thorax. blood count showed haemolytic anemia (haemoglobin= g/dl, schizocyte= %, increased ldh), white cells ( % neutrophils) and platelets. no germs were found in hemoculture, lumbar punction, stools or urines cultures. creatininemia was μmol/l. in the following hours, despite immediate antibiotics administration and without any shock sign, several purpuric extensive necrotic lesions appeared, renal insufficiency increased (creatininemia= μmol/l), platelets count decreased to and markers of diffuse intravascular coagulation dramatically increased. in the following days, proteinuria, macroscopic hematuria and hypertension appeared. after days on anticoagulation therapy, renal function remained stable while anemia, thrombopenia and coagulation disorders persisted. coagulation factor tests demonstrated a heterozygote deficiency of factor v leiden (also found in the father) and an acquired protein s deficiency secondary to streptococcal infection. after protein s infusion and plasma exchanges, his state improved and necrotic lesions ceased. this initial hematologic and renal presentation could have suggest a hus but the large purpuric lesions remain unusual in such pathology. a in children on chronic peritoneal dialysis malnutrition is being diagnosed with an objective combined nutritional score (abn score) based on anthropometry and bioimpedance analysis indices (nephrol dial transpl ; : - ) . aim of this study was to investigate the prevalence of malnutrition and the main factors associated with it in children with ckd - . we planned a cross-sectional study of children with ckd - , mean age . ± . years, who underwent controls in our institution between september and december , . the data of abn score, age, primary renal disease, standard blood and urinary tests, and estimated gfr (schwartz formula) were collected.the prevalence of malnutrition (abn score < . ) in the whole population was %. the abn score was positively correlated with age, height sds, serum hemoglobin, total protein and albumin (p value from < . to < . ), while a negative correlation was found with serum phosphate and proteinuria (p< . ). patients with steroid-resistant nephrotic syndrome had lower abn score values than those with other primary renal diseases ( . ± . vs . ± . ; p< . ). the percentage of children with malnutrition in the different stages of ckd increased in parallel with the decrease of gfr, from % in the ckd group to . % in the ckd group. in conclusion, the prevalence of malnutrition in children with ckd - is not negligible. low hemoglobin, total protein and albumin levels and high serum phosphate and urinary protein excretion, particularly in small children with growth impairment, strongly suggest the need for an in depth nutritional assessment, in order to diagnose malnutrition and treat it accordingly. e.c. developed atypical hus at months of age. a heterozygous factor h gene mutation was found. despite plasma-exchange treatment numerous relapses led the child to ckd stage : creatinine clearance (ccr) . ml/min/ . sqm according to schwartz formula. at yrs we started a programme of fresh frozen plasma (ffp) infusions, - ml/kg bw. the child was poliuric and hypertensive, notwithstanding the treatment with ramipril, amlodipine, clonidine and furosemide. in the first months he received ffp every days. mean ccr during this period was . ± . ml/min/ . sqm. haptoglobin (hap) was still < mg/dl in / tests ( . %), ldh was increased ( . ± . u/l), hemoglobin and platelet count were always in the normal range. the treatment schedule was then changed to ffp every days for the next months. during this period, ccr was significantly higher ( . ± . ; p< . ) and ldh significantly lower than in the first months ( . ± . ; p< . ); haptoglobin was always > mg/dl. no differences between the two periods were found for hemoglobin, platelet count, proteinuria, microalbuminuria and blood pressure values. both in the first and the second period, ccr was negatively correlated with ldh (r . , p< . ) and with the bioimpedance analysis (bia) measure of resistance, which is an index of body hydration. in conclusion: . the only signs of disease activity in atypical hus can be minor abnormalities in laboratory tests, such as increased ldh and decreased haptoglobin levels; . ffp infusions are useful in maintaining hus remission and preserving kidney function, provided that the treatment schedule is optimized; . bia is useful in monitoring hydration status of children with ckd, especially those with poliuria and under ace-inhibitors, as it allows for the correct interpretation of serum cr values. analgesic-antipyretic agents are commonly used medications worldwide for the treatment of pain and fever in children. acute renal failure is commonly seen in adults after treatment with analgesicantipyretic agents. this complication has rarely been reported in children. two patients, ages and years were admitted with a diagnosis of acute, non-oliguric renal failure. one had symptoms of upper respiratory tract infection, and the other had been suffering from vomiting and abdominal pain. appropriate evaluations including detailed history especially the history of drug ingestion, physical examination, and measurement of serum creatinine concentrations were performed in the emergency department. both patient had ingested analgesic-antipyretic agents (mefenamic acid, and paracetamol) before the onset of acute renal failure. none of the patients had previous history of renal disease or concomitant treatment with other drugs. none had oliguria or anuria, dehydration, abnormal serum electrolyte concentrations, or evidence of glomerulonephritis. microscopic hematuria and leukocyturia were found in one patient. serum creatinine was . and . mg/dl at presentation. both of them recovered completely within a week. we emphasize that clinicians must be aware of renal toxicity of analgesic-antipyretic drugs with low doses. with the increasing use of over-the-counter analgesic-antipyretic agents, this association may become more prevalent. cardiovascular disease is a main cause of morbidity and mortallty in patients with chronic kidney disease (ckd). the pathophysiology of cardiovascular disease (cvd) in ckd remains uncertain but nowadays sympathetic hyperactivity is recognized as an important mechanism involved. this observational and transversal study of patients from five to years old, submitted to dialysis or at least four months after kidney transplantation (ktx), without signs of transplantations rejections, with definite ckd and creatinine clearance of ml/min or less. the subject (median age= years; . % female) were classified accordingly with treatment modality: conservative (n= ), peritoneal dialysis (capd) (n= ), hemodyalisis (n= ) and renal transplantation (n= ) submitted to l-metaiodobenzilguanidine ( l-mibg) planar and tomography scintigraphy and heart rate variability (hrv). comparisons among groups were made using anova and the association between variables was assessed using spearman's correlation coefficients and bonferroni correction was used during multiple comparisons testes. a p value < . was considered significant. hemodialysis patients presented increased cardiac washout (p= . ), heterogeneous pattern of l-mibg distribution (p= . ) and lower values of low frequency component of hrv (p= . ). capd subjects had reduced lung washout (p= . ). the cardiac washout had positive correlation with pth and negatives correlation with creatinine clearance. there was a significant negatives association between the rr interval in low frequency (lf) and cardiac washout. the uremic cardiac disautonomia might be characterized by decreased low frequency component of hrv and increased l-mibg washout and heterogeneous distribution pattern by left ventricular walls; these abnormalities subsided after ktx. d+hus is the main cause of acute renal failure in children. extrarenal manifestations are associated in more than % of the cases. hus causes toxin mediated endothelial cell damage, resulting in thrombotic microangiopathy and intraluminal thrombosis of small vessels, with subsequent tissue ischemia and necrosis of involved organs. a -year-old boy has been admitted for d+hus associated with escherichia coli o diarrhea. he presented with renal failure and hypertension requiring hemodialysis for days, hemolytic anemia ( g/dl, schizocytes %) requiring blood transfusions, severe thrombopenia ( g/l) and hyperleucocytosis ( /mm ). severe hemorragic colitis with duodenitis required prolonged parenteral nutrition. at days after onset, the child presented with confusion, slurred speech followed by loss of consciousness associated with major hyperglycemia ( mmol/l) and elevated corrected natremia ( mmol/l) without ketosis requiring transfer in intensive care unit (icu). continuous hemofiltration associated with insulin therapy ( . ui/kg) was then established with slow decrease of natremia and serum glucose within hours. neurologic condition rapidly improved. serum amylase and lipase were normal. insulinemia at the same time as the highest hyperglycemia was low ( , ui/ml), and search for human insulin, islet cell and glutamic acid decarboxylase antibodies were negative. insulin therapy could be discontinued within days. at the last follow-up, months later, neurologic examination, serum glucose and glomerular filtration rate were normal. in conclusion: / hyperglycemic hyperosmolar coma is a severe complication yet never reported in d+hus; / continuous hemofiltration with constant monitoring of biologic parameters could avoid permanent lesions due to rapid correction of this major metabolic unbalance. chronic renal failure (crf) can interfere with the regulation and time dependent secretion of multiple hormones. adrenocortical function in children with crf is examined in a few studies with a limited number of patients, and the results are controversial. in this study our aim is to evaluate adrenocortical function in basal and stimulated conditions, and to determine the relationship with the glomerular filtration rate (gfr), etiology and duration of crf in a larger group of patients. sixty-one patients with crf ( f, m; mean age . ± . years) were studied. the patients were grouped according to etiology [structural abnormalities (n: ), glomerulonephritis (n: ), others (n: )], duration of crf [ - (n: ) , - (n: ) and > months ( )] and gfr [ - (n: ), - (n: ), < ml/min/ . m (n: )]. cortisol levels were measured at : a.m. (basal cortisol) and : p.m. low dose acth test ( . micg/m synacthen iv) was performed. delta cortisol was calculated as peak cortisol minus basal cortisol during the acth test. diurnal rhythm is accepted to be preserved if : a.m. cortisol/ : p.m. cortisol is greater than two. basal cortisol levels and peak cortisol response to low dose acth is similar in all groups. median levels of delta cortisol found to be higher in the gfr< ml/min/ . m group; p= . . diurnal rhythm seems to be more preserved in the gfr - ml/min/ . m group (% ) compared to gfr< group (% ); p= . . no correlation was found between the basal, peak and delta cortisol, gfr and duration of crf. our preliminary results have shown that there is no difference in the basal and peak cortisol levels between all groups. this is the first study in children showing that adrenocortical function is preserved in groups with gfr levels between - ml/min/ . m . objective: this study was we evaluated bk virus and jc virus in urinary after renal transplantation. methods: because these polyoma viruses are excreted in urine, these patients ( females and males, . ± . years old) was analyzed by polymerase chain reaction. all patients were living donor renal transplantation from a parent. results: two patients have detected bk virus in urine. as the type of immunosuppressive treatment, one had tacrolimus and mycophenolate mofetil, and one more had methylprednisolone and tacrolimus. seventeen percent of the patients had quantifiable bk virus-dna in urine. thirty-three percent of the patients had quantifiable jc virus-dna in urine. there was non significant relationship between these polyoma viruses in urine and the type of immunosuppressive treatment. no patients developed interstitial nephritis during the study. conclusions: the activation of bk virus and jc virus does not seem to be related to the type of immunosuppressive treatment. the pathogenetic role of polyoma virus infection in renal transplantation recipients further researches are needed. background: urinary tract infection (uti) due to neurogenic bladder, secondary to large spina bifida as myelomeningocele, is well known, but the association of small occult spina bifida (sbo), having normal physical examination, with that of infection has not been reported. we studied the frequency of spina bifida occulta in children with urinary tract infection. method: the kub of voiding cystouretrography in patients ( f, m) with average of age year (± . sd), who referred to radiology department of ali asgar children hospital for uti, were observed for sbo. all patients had normal physical examination. chi were used to find the frequency of the level of sbo and the differences respectively. p< . was considered significant. result: out of patients had sbo. the order of frequency of the level of sbo was s ( %), l ( , %), s s ( , %), l s ( , %), and l s s ( %). patients had vesicoureteral reflux (bilateral in cases, at left and at right side). the severity of vur was % grade (g) ii, , % g iii, . % g iv, and . % g i. spina bifida occulta was detected in out of patients with vur. this difference was not statistically significant (p= . ): conclusion: the high frequency of lumbosacral sbo in the patients with urinary complaint may imply some lower urinary tract dysfunction in these patients although we need a control study in normal children. we evaluated interleukin- and interleukin- levels in the urine of children with renal scarring, with vesicoureteral reflux ( / , group a ) and without vesicoureteral reflux ( / , group a ) and in the urine of healthy children (group b). none of the children had urinary tract infection for the last ten months. interleukin- and interleukin- were determined using a commercially available human enzyme-linked immunosorbent assay kit. results: urinary interleukin- levels were below the lower detection limit in all samples. interleukin- levels were detectable in all urine samples of children with renal scarring and below the detection limits in the urine samples of healthy children. there were no statistically significant differences between urinary interleukin- levels in children with and without vesicoureteral reflux. there is a relationship between the grade of renal scars, the time past from their development and the urinary levels of il- . the introduction of mycophenolate mophetil (mmf) was an important advance in immunosuppressive therapy but its use is associated with gastrointestinal intolerability (gi) requiring dose reductions or drug interruptions. enteric coated mycophenolate sodium (ec-msp) was designed to improve mycophenolic acid (mpa)-related gi by delaying the release of mpa until reaching the small intestine. at present, its immunosuppressive activity in pediatric renal transplant with gi has not been clarified. we studied trough levels of active metabolite mpa (c ), changes in kidney function, mixed lymphocyte culture, cytotoxic antibodies as well as cytotoxic response before and after months of the conversion from mmf to ec-mps in renal transplant recipients with gi. in the immunosuppressive protocol used: mmf, tacrolimus and corticosteroid, mmf ± mg/m /day was replaced by ec-mpa ± mg/m /day. after months of treatment with ec-mpa, the incidence of gi decrease to . %. the levels of mpa were about % higher on ec-mps ( . ± . ug/ml) compared to mmf administration ( . ± . ug/ml). serum creatinine, creatinine clearance and urinary protein excretion did not change ( . ± . to . ± . mg/dl, . ± . to . ± . ml/min/ . m and . ± . to . ± . gr/ hr, respectively). also, during ec-mpa, proliferative response and cytotoxic antibodies showed no significant change. the release of il- was striking augmented with mmf or ec-mps therapy, but interferon-γ and tnf were low under both treatments. our data indicate that conversion from mmf to ec-mps leads to an improvement in gi without altering key elements of immunosuppression. however, the monitoring of mpa before and after conversion should be appropriated to the optimization of therapy efficacy. re. munarriz, ju. arakaki, ce. munarriz pediatric clinic, pediatric nephrology, buenos aires, peru objective: to describe the results of a program of chronic ambulatory peritoneal dialysis (capd) in children in peru by means of conventional indicators. methods: children ( , % male) were included in a longitudinal descriptive study. the average age was , ± , years (rank of , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . % were from lima (the capital city) and % came from other cities of the country. primary glomerulonephritis ( , %) was the main cause of the renal insufficiency. we evaluated the program from to . results: the average weekly kt/v was , ± , . the average dose of erythropoietin (epo) was ui/kg/week. % of the patients had average annual albumin of more than , gr./dl. the average annual protein catabolism index (icp) was , ± , gr/kg/d. the weight/age, height/age, weight/height z scores at the beginning and at the end were . / . , - . / . , - . / , - . / . , . / . - . / . , respectively. the average hematocrit was . ± . %. the rate of peritonitis adjusted for the period was , episodes/patients-year ( episode every months) and the mortality for the same period were %. conclusions: the indicators evaluated in our patients are according to the results of the international literature. is repeated urine culture essential after antibiotic therapy in uti in thai children? background: in , the american academy of pediatrics recommended for uti treatment, that urine culture be repeated only in children < years with fever > hours, since the chance of a positive urine culture in other children is very low. objective: to evaluate the cost-effectiveness of repeated urine culture after antibiotic therapy in childhood uti. patients and methods: a retrospective review of the records of children diagnosed with uti in songklanagarind hospital from jan -dec , . results: there were patients total, of which were excluded due to no repeated urine culture. patients with uti episodes were analyzed ( boys and girls). ( . %), ( . %) and ( . %) patients aged < , - and > years respectively. ( . %) had a repeated urine culture with significant growth. multivariate analysis showed that age < year, etiologic agents psuedomonas aeroginosa or enterococci spp., fever > hours and kub anomalies were the most significant risk factors for positive repeated culture, while vur and recurrent uti episodes were not significant risk factors. if we consider that a child who has at least one of the above risk factors should have a repeat urine culture, then cases ( . %) will require repeating urine culture and x . =$us . will be saved and ( . %) positive repeated urine culture will be missed. conclusion: our study in a group of thai children indicates that repeated urine culture after antibiotic therapy is still recommended. our aim was to find out if there are any signs of renal scarring and reduced renal function without recurrent uti in patients with obstructive pyelonephritis. there were investigated children ( - y) with - years passed after diagnosis of obstructive pyelonephritis. these patients were investigated during years long period without uti. all the children had a treatment with nitrofurantoin during season viral infections. we investigated excretion with urine of the collagen product (hydroxyproline) and activity of lisosomal enzymes (b n-acetilglutamase, elastase, pseudocholiesterase). as a control group, healthy children of the same age were investigated. our result demonstrates the decrease of the level in urine of collagen product and lisosomal enzymes and normalization of tubular and glomerular functions during years remission of pyelonephritis. prevention of recurrent uti and maintenance of the remission of pyelonephritis leads to the decrease of sclerosing processes on kidney. regular dialysis induces insecurity and special psychological problems associated with staff-, machine-and artificial material-dependence. the more severe the child's physical problems are the more probable is developing of emotional disorders, a sense of loneliness and an exaggerated dependence on the parents and staff. a psychological and social study of children on regular dialysis was performed. there were girls and boys, aged - (mean: ) ys. two children ( %) are exclusively treated with haemodialysis, ( %) are on peritoneal dialysis and in ( %) both methods interchanged. somatic concomitant disorders are present in ( %) of children. among psychological disorders, an inclination to unsociability, autoaggression or aggression towards the immediate environment has developed in children ( %), ( %) do not like talking about disease while ( %) are communicative and sociable. psychosocial characteristics showed: emotional difficulties (anxiety, mild depression) in %, feeling of being physically different from peers (short stature, less physical ability) in %, lowered self-esteem and social isolation as a result of school absenteeism in %. overprotection of parents was presented in %. different psychological changes were present in some children. of the children are of school age: ( %) attend special schools and the remaining follow regular education programs. with the help from their teachers, children on dialysis can master regular school programs, in spite of the time spent on dialysis. a good and continuing co-operation of dialysis staff and sick children and their parents as well as a more intensive co-operation with psychologists and teachers are necessary to reduce psychological disorders and promote a better adaptation to the life of their healthy coevals. we report a -year-old girl with syndrome frasier who developed b cell lymphoma within nine month after live related kidney transplantation. in induction therapy we applied atg up to days, mycophenolate mofetil and cyclosporine. routine abdominal ultrasound revealed multifocal hypoechogenic changes in liver and spleen. computed tomography showed diffuse focuses of changed liver tissue in length up to . cm, precontrast density of - hu and postcontrast of - hu. in spleen there were three similar changes (up to . cm). after surgical biopsy of liver, patohistological examination confirmed diffuse large cell b lymphoma (cd positive, moderate risk). pcr ebv was positive. we immediately started with ganciclovir intra venously and decreased cyclosporin and mmf (within two weeks) up to completely exclusion from therapy. in the same time we increased prednisolon on mg daily. after weeks from beginning ganciclovir she treated with rituximab, one dose of mg every week (five weeks). repeated abdominal ultrasound and two controls computed tomography showed markedly regression of tumorous lesions. after . months of last rituximab doses scan showed normal spleen and liver findings. all lesions were resolved. in two occasions she developed severe leucopenia without any infection complication. she still has got moderate lymphopenia due to continual ganciclovir therapy. during this period ( months) of illness course she treated with ganciclovir intravenously due to repeated ebv reactivation (positive pcr). despite enormous reduction in immunosupressive therapy renal function remain stabile without episodes of acute rejection. - . years) . the most of them, % ( patients) received graft from live related donor. % of patients had preoperative dialysis and % were preemptive. the mean waiting period for transplantation was months (range to months). congenital anomalies of kidney and urinary tract were the most common underlying diseases ( patients), then nephrotyc syndrome ( patients), hereditary nephritis ( patients), polycystic kidney disease ( patients) and others. daclizumab was the most commonly used as immunosuppressive induction agent and the maintenance immunosuppressive therapy consisted of azathioprine or mycophenolate mofetil (mmf), cyclosporine or tacrolimus and prednisone. two patients had immediate postoperative surgical complication and graft loss due to thrombosis of a. renalis and donor tubular necrosis respectively. three patients had delayed graft function; two of them underwent cadaveric transplantation. one patient had recurrence of primary kidney disease only few days after transplantation and graft loss after one year. other patient lost the graft after years due to noncompliance and chronic cellular rejection. after one year of follow-up graft survival rate is % and patient survival rate %. at the end of follow-up (mean range . months), patients had normal, patients slightly decreased renal graft function. catch-up growth was seen during the first year after transplantation from mean height sds of - . to . . the mean goal of our further intention is improvement of cadaver renal transplantation. j. lee, y. shim, s. lee objectives; although the variable risk factors of urinary tract infection (uti) in children such as virulence factors of the pathogenic bacteria and vesicoureteral reflux are already well known, the role of normal flora clononizing the intestinal tract and genitourinary tract is not fully understood. the change in colony forming units (cfu) of lactobacillus in chidren with uti is primarily investigated to explore the role of lactobacillus, one of the human normal flora, in development of uti. methods; lactobacillus was cultured from stool, urine, and periurethral or vaginal discharge of febrile infants with uti. those with confirmed uti by the suprapubic urine culture were classified to uti group (n=- ), and those with negative urine culture and confirmed viral illness were classified to control group (n= ). lactobacillus was anaerobically cultured in dico rogosa sl agar (becton, dickinson and company, usa) for hours at °c. results: the cfu of lactobacillus in stool was correlated with those in periurethra, vagina and urine (p< . ). the cfu of lactobacillus in stool was significantly lower in uti group than in control group ( , ± , vs , , ± , , , p< . ) . the cfu of lactobacillus in periurethra or vagina was significantly lower in uti group than in control group ( , ± , vs , ± , , p< . ). the cfu of lactobacillus in urine was significantly lower in uti group than in control group ( ± vs , ± , , p< . ). the cfu was distributed mostly at low level in uti group, which was significantly different from that in control group (p< . ) conclusions; the cfu of lactobacillus in stool, periurethra, vagina and urine is low in infants with uti. it is suggested that lactobacillus has a role in the development of uti. pediatric small bowel transplantations are associated with pronounced electrolyte inbalances in the posttransplant period. we investigated the sources of possible electrolyte losses. our hypothesis was that electrolyte inbalances after intestinal transplantation might be augmented by fk (tacrolimus) mediated renal toxicity rather than short bowel syndrome alone. we retrospectively reviewed eleven living-related small bowel transplantations between october and december . the data collected included frequent serum and urine electrolyte profiles, renal function parameters, enterostoma electrolytes, and fk levels in the postoperative period up until either discharge or graft loss. we analyzed pearson's correlations between fk levels, serum electrolytes, renal function parameters, and also fractional excretions (fe). in order to investigate possible delayed nephrotoxic effects of fk , we correlated all values of the same day as well as with fk levels of , , and hours earlier. furthermore, we analyzed our data stratified by fk dosing ranges. our results show decreased gfr and prominent increase of renal sodium, phosphorus, and magnesium losses along with rising fk levels, suggesting this pathway as a major contributor to their imbalances. furthermore, fk levels were associated with serum calcium and phosphorus decline, though urinary calcium excretion was not significantly changed. signs of renal toxicity are apparent within the first hours of fk challenge. our data suggest that fk mediated nephrotoxiticy is a significant contributor to electrolyte imbalances observed after small bowel transplantation. objectives of study: urinary tract infections (uti) are common in children and c-reactive protein (crp) in serum is often used to evaluate the severity of the renal inflammation. recently it was demonstrated that crp can be produced locally in the kidney. we therefore measured the crp concentration in both serum (s-crp) and urine (u-crp) from children with uti to evaluate the extent of local production and the usefulness of measuring u-crp for diagnosis of inflammatory kidney disease. methods: children ( boys) with uti were studied (median age . years, range days- years). children had fever . °c or more. the uti was caused by e.coli in patients. all children were examined within a few days of diagnosis by dmsa scan for evaluation of renal function. as controls were used children with respiratory infection (pneumonia in most) and elevated s-crp in whom uti was ruled out by negative urine culture. u-crp was measured in a commercial hscrp elisa. normal value was < mg/l results: in the uti patients, the median s-crp was mg/l (range - ) and u-crp mg/l (range - ). there was a significant correlation between the s-crp and u-crp concentrations (< . ). dmsa scans were abnormal in ( %) uti patients. the proportion of abnormal scans increased significantly with the crp concentration in both serum (< . ) and urine (< . ). all control patients had increased s-crp concentrations (median mg/l, range - ) but the median u-crp was . mg/l (range - ). the u-crp in the patients with uti was significantly higher than in the controls with other infections (< . ). conclusions: our data strengthens the concept that crp can be produced locally in the kidney during uti. the usefulness of measuring u-crp to evaluate inflammatory kidney disease needs to be tested further. s. paunova, a. kucherenko, i. smirnov, g. serova, i. donin, l. revenkova, n. goltsova in order to reveal the role of cytokines in renal tissue damage in infants with urinary tract infection (uti) patients aged from to months were examined. in all of them inflammatory markers (esr, crp, leukocyte count), urine concentration of tumor necrosis factor-α (utnf-α) and transforming growth factor-β (utgf-β) standardized to urinary creatinine concentrations were evaluated. two groups of patients were determined: ) with uti and normal urodynamics (n= ), ) with uti and urodynamic disorders (vur, hydronephrosis) (n= ). healthy infants were used as controls. as a result, all the patients demonstrated significantly elevated utnf-α, utgf-β creatinine ratio in comparison with controls (p< , ) with no difference between groups. but children with normal urodynamics (from the st group) presented with severe uti ( st -a) showed urine tnf-α tgf-β creatinine ratio in , times higher than other patients of st gr and close to nd gr. data ( st gr.-utnf-α/ucr= , ± , , utgf-β/ucr= , ± , , st gr.-utnf-α/ucr= , ± , , utgf-β/ucr= , ± , ; nd gr -utnf-α/ucr= , ± , , utgf-β/ucr= , ± , , p , - < , ) . to conclude, tnf-α and tgf-β are responsive for renal tissue impairement in infants with uti. elevated tnf-α tgf-β creatinine ratio in a part of infants with normal urodynamics suggests that renal damage begins early in infection mostly due to inflammation. one may suspect a predisposition of that subgroup of patients to fibrogenic process and subsequent scar formation. prompt diagnosis and localization of pyelonephritis are of great importance in treatment and prognosis of the patients. the urinary excretion of enzymes, in particular n-acetyl-beta-dglucosaminidase (nag), is considered a relatively simple and non-invasive method in the detection of renal tubular function under various conditions such as pyelonephritis. this study was performed to determine the diagnostic value of urinary nag in acute pyelonephritis and to compare it with other indices traditionally used for this purpose in children. this is a quasi experimental study conducted from april to may on children with pyelonephritis. diagnosis of pyelonephritis has been based on standard criteria. seventy-two patients between month and years were recruited. fresh random urine samples were obtained on the admission time and at th hour of treatment and were tested for nag and creatinine. there was a significant difference in pre and post-treatment urinary nag/creat ratio (p< . ) and the sensitivity and specificity of urinary nag in diagnosis of pyelonephritis were % and % respectively. there was a significant correlation between urinary nag level and kidney ultrasonography results. patients whose ultrasonography showed hydronephrosis, had the highest level of urinary nag and patients who showed urinary stone in their ultrasonography had the lowest level of urinary nag. in addition, there was a reverse correlation between urine culture results and urinary nag level; patients who had negative urine culture had higher level of urinary nag in comparison with patients with positive urine culture. we conclude that urinary nag is elevated in children with pyelonephritis especially in the presence of urinary tract abnormality and the absence of renal stone. type- hyperoxaluria (ph- ) is an autosomal recessive disorder caused by the impaired activity of the hepatic peroxisomal alanine-glyoxilate aminotransferase. the disease leads to end stage renal disease (esrd) and combined liver-kidney transplantation (clkt) is required. we report cases diagnosed with ph- who received clkt. case- : she had attacks of dark urine without any pain and renal stones were determined on sonography when she was . years old. she was diagnosed with ph- and received peritoneal dialysis (pd) at the end of the first year and cadaveric clkt was performed when she was yearsold. at the age of , she had chronic allograft nephropathy and began to have hemodialysis (hd). recently, . year after hd, cadaveric renal transplantation (tx) was performed. she is well after the second tx. case- : he was admitted with polyuria, polydypsia, stomachache and renal stones were determined on sonographic examination. he had esrd and pd was started when he was years old. at the age of , cklt was performed from his mother. his liver and renal functional tests are well months after cklt. case- : he was evaluated because of having an older brother diagnosed with ph- when he was . years old. he had no complain, but sonography showed multiple calculi. within years he experienced flank ache, hematuria attacks and anuric phases due to obstruction and esrd appeared and he received hd and clkt was performed from his full-match sister at the age of . . he is doing well years after tx. here, we present the favourable clinical outcomes of our patients with cklt to indicate the validity of this treatment of choice in ph- . tenascin (tn) is a glycoprotein component of extracellular matrix (ecm) which is not present in the normal kidney tissue. tissue plasminogen activator inhibitor- (pai- ) regulates fibrinolysis and the plasmin mediated matrix metalloproteinase activation and it is also not expressed in the normal kidney. recent studies focus on the pathogenesis of advanced renal diseases. in this study we evaluated tn and pai- staining in the renal tissues of pyelonephritic rats using immunohistochemistry (ihc) as to understand if these markers may be served as histological parameters of pyelonephritis like fibrosis, tubularatrophy or vascular changes. seven rats were injected . ml solution containing e. coli atcc cfu/ml into left renal medullae. seven rats were designed as sham group and were given . % nacl. rats were sacrificed days after injections. renal tissues were studied histopathologically by hematoxylin and eosinand scored for the parameters of pyelonephritis. tn and pai- expressions were studied semiquantatively by ihc by tenascin novocastra (ncc-tenas-c) and pai- (h- ) santa cruz biotechnology. both tn and pai- expressions increased in the pyelonephritic groups. we observed acorrelation between tenascin and fibrosis, vascular changes and tubular atrophy and pai- expression showed a correlation with only fibrosis. we conclude that increase in renal tn and pai- expression shown in experimental pyelonephritis are the responsible factors for the fibrotic changes of persistent renal damage. introduction: urinary beta microglobulin (β mg) urinary excretion is a good index of proximal tubular cell dysfunction. objective: to determine β mg excretion significance in determining the outcome in respect to scar and renal insufficiency. patients and methods: urinary β mg and creatinine (cr) was measured in urine samples of whom proceed to do dmsa renal scan at the time of admission and months later to detect scar. β mg was measured using radioimmunoassay method using β mg -test kit (radim company). twenty children had various grades of renal scars. results were compared with ratios of children with low uptake and normal scanning and normal children. student t test, anova, and unpaired t-test was used for analysis and differences considered significant if p< . . results: the mean urinary β mg/cr was significantly higher in the scarring group ( . ± . ) than in the normal group ( . ± . ) and in low uptake group ( . ± . ) (p< . ). patients with grade iii had higher values ( . ± . ) than grades i ( . ± . ) and ii ( . ± . ) (p< . ). patients without renal scar had β mg/cr ratio below . microgram/mg. the mean β mg/cr was higher in patients with vur grades and ( . ± . ) than patients with vur grades to ( . ± . ) (p= . ). maximum β mg/cr was detected in patients with grade vur ( . ) and the minimum was zero in non-refluxing patients and normal children. two patients with high grade vur and the highest levels of β mg/cr ratio ( . and ) progressed to renal failure in years time, the first patient was treated by hemodialysis and the second underwent renal transplantation. conclusion: measurement of urinary β mg is useful in the early detection of tubular damage in patients with renal scars. introduction: chronic allograft nephropathy refer to the progressive decline of renal function seen in some renal transplant recipients in association with alloantigen-dependent and alloantigenindependen factors. hyperlipidemia is a risk factor for chronic allograft nephropathy in adult pts, where no data exist in pediatric transplant population. methods: in this cross sectional study, patients ( can/ non-can) that aged - yr and - mo (mean: mo) after transplantation, were evaluated for lipid profile and renal function tests. results: hyperlipidemia has high prevalence in our patients both pre and posttransplantation and hypercholesterolemia and increased ldl had significant correlation with chronic allograft nephropathy (p= . ) and p= . respectively. conclusion: in pediatric patient as in adults hyperlipidemia and particularly hypercholesterolemia has significant correlation with chronic allograft nephropathy and as adults may need specific therapy. results: pre-transplantation renal replacement therapy time ranged from to months. eleven children underwent pre-emptiverenal transplantation. / transplants were from living related donors. donor age ranged from days to years. grafts were from donors < year of age and of these grafts were transplanted en-block. hla mismatches ranged from to antigens. primary disease was: focal segmental glomerulosclerosis in , rapidly progressive glomeluronephritis in , reflux nephropathy in , nephronophthisis in , iga nephropathy in , congenital nephrotic syndrome in , dysplasia-hypoplasia in , idiopathic membranous glomerulonephritis in , henoch-schönlein purpura in , hemolytic-uremic syndrome in , nephroblastoma in , polycystic kidney disease in and of unknown origin in children. patient survival at five years was %. allograft survival with living related transplants at one, two and five years was %, % and % respectively and with cadaveric transplants at the same periods was %, % and % respectively (p< . ). regarding en-block grafts, they functioned immediately and satisfactory and presented excellent graft function years later. most kidneys were lost due to acute or chronic rejection (n= ). other causes were renal artery thrombosis (n= ), infections (n= ), withdrawal of immunosupressive regime (n= ). conclusions: results of this single center series of pediatric renal transplants are encouraging from the standpoint of patient and allograft survival. conclusion: in infants with hn, the incidence of uti was higher especially in those with ou, hn of higher sfu grade or hun. the antibiotic prophylaxis may be recommended during year after birth in infant with hn because the incidence of uti was high in these period. results: the underlying diseases were: sepsis with mods ( . %), septic shock ( . %), severe intoxication ( . %), trauma with sirs ( . %), drowning ( . %), abdominal compartment syndrom ( . %) and inborn metabolic disorders ( . %). children ( %) had acute renal failure, ( %) patients met non-renal crrt criteria. cvvh was performed in ( %) children, cvvhdf in ( %) children. crrt duration was to hours (median . hours). dynamics of blood urea, creatinine, c-reactive proteine (crp) and white blood cells (wbc) were evaluated. significant decline (p< . ) of creatinine along the treatment with cvvh as well as during cvvhdf was observed. blood urea levels showed significant decrease only in children treated with cvvhdf (p< . ). significant decrease of wbc and crp was observed only using cvvh. children from the study group survived (overall mortality . %). in non-survivors was time from crrt initiation to its termination compared to time interval from crrt initiation to the death of children with - organs failure significant (p< . ) where as in non-survivors with - failured organs it was not. conclusion: cvvh is efficient at removing urea and creatinine as well as inflammatory mediators (crp, wbc). cvvhdf is more potent to blood urea elimination. authors suggest preferring cvvh to cvvhdf in critically ill children to affect basic inflammatory parameters. to analyse hd and pd prescription (pr) adopted in chronic dialysis children, were viewed data of pd regimens in patients ( . - years) and hd regimes in patients (age . - years) treated in pediatric centres in . pd patients were on automated pd: -nightly intermittent pd (nipd; pr): . ± . ( - ) hrs; . ± . ( - ) cycles/night; dwell volume (dv) ± ( - ) ml/m bsa; -tidal pd ( pr): . ± . ( - ) hrs; . ± . ( - ) cycles/night; dv ± ( - ) ml/m ; tidal volume . ± . ( - )%; -continuous cycling pd (ccpd; pr): . ± . ( - ) hrs; . ± . ( - ) cycles/night; dv ± ( - ) ml/m ; daytime dv ± ( - )% of night dv. in % of pr dialysis fluid (df) glucose concentration was > . %, and in % buffer was bicarbonate. df of daytime dwell was . % glucose ( pr) or icodextrin ( pr). patients with residual diuresis were . % of those on nipd, % on tidal pd, and . % on ccpd. hd was performed as bicarbonate hd ( %), hemodial filtration ( %) and acetate-free biofiltration( %). patients received sessions/week in % of cases, /week in %, and /week in % of cases; oxalosis patients were on daily hd. session duration was hrs in pr, hrs in , and . hrs in . dialyser membrane was: polysulfone ( %); hemophane ( %); polyamide s ( %); cellulose acetate ( %); polyacrylonitrile ( %); cellulosediacetate ( %); cellulose triacetate ( %); polymethylmetacrylate ( %); polyether/carbonate ( %). ratio between dialyser surface area and patient bsa was . ± . ( . - . ) and was - . in %, . - in %, > . in %, and < . in % of cases. isoniazid (inh) is widely used in most prophylactic and therapeutic anti-tuberculosis regimens because of its effectiveness and low cost. acute intoxication by isoniazid is known to cause symptoms of seizures, metabolic acidosis, coma, and even death. we present a case of acute isoniazid poisoning in a seven years old patient who ingested tablets ( mg) of isoniazid. she was admitted unconscious, with ventilatory insufficiency and convulsions. renal and liver function tests were in normal ranges. she was intubated and mechanically ventilated. despite parenteral midazolamand pyridoxine (vitamine b ) treatments convulsions went on. then hemodialysis was performed and after hemodialysis convulsions and ventilatory insufficiency were disappeared and the patient was conscious and she was extubated. hemodialysis may be an effective treatment alternative for the patients who doesn't respond pyridoxine treatment. the aim of this study is to analyse children under two years of age, with their first febrile urinary tract infection (uti), identifying bacteriological etiology, antimicrobial resistance, urologicalabnormalities and renal damage. this is a prospective study including children ( % girls) with their first febrile uti. mean age was months ( - ), ( %) patients were younger than months ( % of them were boys). urine was obtained by suprapubic aspiration ( . %) or transurethral catheterization ( . %). % had positive nitrite on urinalysis and . % had leukocyturia. they were submitted to ultrasonography (usg), dmsa scan (within months) and voiding cystourethrography (vcug). the most frequent microorganism found in urine culture was escherichia coli, ( . %). in this study high bacterial resistance to antimicrobials was observed in relation to the following antibiotics: ampicillin ( . %), first generation cephalosporyn ( . %), sulfamethoxazole/ trimethopin ( . %). resistance to second generation cephalosporyn, aminoglycoside, nitrofurantoin and nalidixic acid was lower than . %. renal ultrasound was abnormal in . % of the infants. vesico-ureteral reflux (vur) was observed in . %, although only . % had vur grade iii or more. the dmsa scan showed that ( %) patients had renal parenchymal damage. fourteen of these ( . %) had normal esr. there were ( . %) reinfections within a months period, even under prophylactic treatment, and the presence of vur grade iii or more was the only one with a significant relationship. conclusion: there were high levels of bacterial resistance to frequent used antimicrobials. this finding points toward a need for reviewing criteria of choosing initial blind therapy. investigation with dmsa scan is important in the detection of renal parenchymal scars, irrespective of the reflux grade. purpose: urinary tract infection (uti) has a risk of renal damage and is associated with vur. vur is investigated only by vcu. however, vcu is an invasive, painful study and many patients hesitate to be taken the study. we studied the correlation of vur in vcu and defect of dmsa scan and investigated the possibility of substitution of vcu by dmsa scan. material and methods: from to , the medical records were searched for children admitted to cheongju st.mary's hospital with the first uti who had been evaluated with both dmsa scan and vcu within months of the infection. the value of several clinical signs, laboratory findings, the resultsof dmsa scan and vcu were investigated. bacteriuria was defined as , or greater colony-forming units in urine from a bag, midstream or catheter sample. results: there were patients underwent both studied at the first uti. mean age of the patients was months old. the male patients were ( %). the vur was found in of the patients ( %), grade i-ii in and grade iii-v in patients. there was no significant correlation with the presence of vur in sex, fever duration, blood white cell count and the level of serum creactive protein (crp). but the patients with vur grade iii-v were significantly older than the patients with grade i-ii reflux or without vur. there were abnormal dmsa scan findings in of ( %). of these patients, were without vur, with vur grade i-ii and with vur grade iii-v. the abnormal dmsa scan was correlated with the presence and severity of vur. but vur was found in % of patients with normal dmsa scan. conclusions: abnormal dmsa scan is correlated with the presence of vur, so the patients with abnormal dmsa scan require vcu. in order to prevent missing a quarter of patients with vur shown normal dmsa, vcu should be recommended in all children with first febrile uti. cuba is the largest of the carribean islands with its , millions inhabitants. cuba is considered as a developing country and is classified in the group of: "lower middle income countries and territories". despite low financial resources, cuba has succeeded to develop an efficacious health care system with comparable results to those of west europe and usa (who data) ccl: ) hd is the most prominent form of pediatric dialysis (automated night pd is in progress); ) the no of transplantations is relatively low because of no participation to an international transplantation network; ) high no of pediatric nephrologists; ) high quality of patients care. background: inborn errors of metabolism in neonates are often characterised by hyperammonaemic coma within the first days of life and require prompt diagnosis and specific treatment such as toxin removal and nutritional support. cvvhd seems to be the optimal modality for extracorporal ammonium detoxification, however, little experience with small numbers of children has been accumulated. patients and methods: from to , patients with hyperammonaemia ( male, female) were admitted for dialysis treatment: neonates (mean age . ± . days, range - days) with a mean birth weight of ± g and infants (mean age . ± . years, range to years). in neonates and infants we inserted venous double lumen shaldon catheters (predominately femoral or jugular vein) for cvvhd treatment while neonates and infant underwent capd treatment. results: plasma ammonia levels (range - μg/dl before dialysis and - μg/dl after dialysis) were reduced by % within . ± . h by cvvhd and within . ± . h by capd (p< . ). total dialysis time was . ± . h for cvvhd patients. no major mechanical complications were observed with cvvhd and stable blood flows ( - ml/min) and dialysate flows ( - ml/m /h) were achieved. due to severity of underlying disease, out of neonates ( %) undergoing cvvhd died on day - while out of neonatal capd patients ( %) died on day and one infant patient died after days of capd treatment. twelve out of neonates ( %) survived with no or moderate mental retardation. conclusions: cvvhd is an effective modality to eliminate plasma ammonia within few hours. however, vascular access and blood flows are critical restrictions. mental retardation has to be evaluated in larger scale studies. r. vilalta, e. lara, a. madrid, s. chocron, j. nieto hospital materno-infantil vall de hebron, nefrologia pediatrica, barcelona, spain background: transplant nephropathy is the main cause of renal failure in kidney transplanted children. until this situation is proved by biopsy, sometimes the progressive raise of creatinine leads to raise the anticalcineurinic (cni) drugs with added nephrotoxicity. sirolimus (sir) plus an anticalcineurinicin less dose and mycophenolate (mmf) could offer in kidney-transplanted children an immunosuppressive regime with less toxicity and even an improvement of renal function. methods and patients: paediatric kidney-transplanted patients developed biopsy-proved chronic allograft nephropathy (age - y, mean ) a follow-up post-transplant of y and exhibit also tubular involvement and acute cni toxicity. sir was added in all patients as a rescue therapy at . mg/kg/d. results: after a follow-up period of months, creatinine level diminished (p< . ) in patients ( in group tac, in group cya, with no significant differences). creatinine level did not show a significant change in the other patients ( group tac, group cya, basal creatinine . mg/dl. serum cholesterol changed from ± mg/ ml to ± (ns) and serum tryglicerides from ± mg/ ml to ± (ns). proteinuria also did not show changes ( ± to ± mg/m /h (ns). conclusions: a poly-drug approach with less dose of anticalcineurinic added to the antiproliferative effect of sirolimus and the inhibition of purine synthesis based on mycophenolate mofetil could suppose an improvement of the renal function in children graft nephropathy an even in the graft survival. steroids have been a cornerstone in renal transplant immunosuppression. several strategies have been used to minimize their side effects. new immunosuppressive drugs have allowed steroid withdrawal or total avoidance. aim: to evaluate a new protocol with steroid-free maintenance immunosuppression in pediatric renal transplant. patients and methods: a prospective, non-randomized study in consecutive first renal allograft recipients, followed-up to months. patients received prednisone the first days, two-dose basiliximab induction and maintenance therapy with tacrolimus (tac) and mycophenolate mofetil (mmf). no steroids were given after d posttransplant. controls were historical-matched steroid-based children receiving basiliximab, tac and mmf. all patients gave informed consent. anthropometric, biochemical variables, acute rejection and cmv infection were compared using student-t test and regression analysis. results: a better growth pattern was seen in steroid-free maintenance group reaching a normal growth at months. gfr and serum glucose were similar in both groups. total cholesterol levels were significantly lower in the study group. the incidence of acute rejection was . % in steroidfree maintenance vs . % in steroid-based group, no differences in cmv infection and blood pressure were observed. hematocrit levels were lower during the first months after transplant in the steroid-free group, increased after months post-transplant. patient and graft survival was % at two-yr post transplant in the two groups. conclusions: this steroid-free maintenance immunosuppressive protocol was efficacious, safe, with a lower incidence of acute rejection, not increased risk of infection, preserving optimal growth, renal function and reducing cardiovascular risk factors. objectives of study: to evaluate the lipid profile and its possible implications in children with end stage renal disease (esrd) or renal transplantation. methods: children ( boys, girls) aged from . to years, on peritoneal dialysis (group i) and with renal transplantation (group ii) were studied. in all children were examined: serum creatinine, total cholesterol, triglycerides, high density lipoproteins (hdl) and low density lipoproteins (ldl). a cardiac and liver ultrasonography were also performed. the body mass index (bmi) and blood pressure were evaluated in all children. / children received also a triplex carotid study. the median values of blood creatinine, cholesterol, triglycerides, hdl, ldl as well as the number of children with bmi over th percentile in both groups were shown in table i . all children had normal findings in triplex carotid study. cardiac ultrasonography was abnormal in child of group i and in children of group ii. only child presented lipoid invasion in liver ultrasound. / children of group i and / children of group ii presented hypertension, well controlled, with antihypertensive therapy. conclusions: frequent evaluation of lipid profile is recommended in all children with esrd or renal transplantation independently their bmi. in our study, children with renal transplantation presented better lipid profile compared with children on peritoneal dialysis. group i with < months (n= ; , ± , months) and group ii > months (n= ; , ± , months). results: serum albumin, serum lipids values and the distribution of the categories of the peritoneal membrane did not present significant differences between the groups. hypertension, renal residual function (p= , ), the renal urea kt/v (p= , ) and the weekly renal ccr (p= , ) were significantly higher in group i, while the weekly peritoneal ccr (p= , ) and the total weekly ccr (p= , ) were significantly higher in group ii. catch-up was not observed in any group. control of the cholesterolemia, trigliceridemia and albuminemia were maintained with the dialysis time in both groups. the goals of adequacy of the doqi for kt/v and ccr were gotten respectively in , % and , % of the group i and in , % and , % of the group ii. the longer time in dialysis was associated with the lowest values of renal residual function, renal kt/v and renal ccr. the capacity of transport of the membrane was similar in both groups. objectives of study: to explore the characterize of peritoneal transport in chinese children with chronic peritoneal dialysis. methords: pet was carried out times for six children (mean ages . ± . , aged from to years) who were maintained by capd, and the infusion volume of dialysate was ± ml ( ml/m ). the peritoneal solution transport rate was evaluated by the standards of twardowski's and ppdsc's criteria. results: in our study, the initial pet was performed at . ± . days following initiation of pd, the -hours of peritoneal creatinine clearance ( h-d/p) and glucose absorption ( h-d/d ) was . ± . and . ± . , respectively. according to the standards of twardowski's and ppdsc 's criteria, the peritoneal transport categories were divided into high transport (h) ( / ), high average transport (ha) ( / ), low average (la) ( / ) for peritoneal solution transport, and h ( / ), ha ( / ), la ( / ), low transport ( / ) for glucose absorption. no low transport type of solution was uesd in our patients. the coincidence rate of peritoneal creatinine and glucose transport types were % and % between the twardowski's and ppdsc's criteria, respectively. the different changes of peritoneal transport type were found in two patients with continuous pet. the value of h-d/p increased after peritonitis episodes. our results showed that the pet in % of capd children fall into high and high average transport categories elevated by ppdsc's and adult standards. the peritoneal solute clearance was adequacy in the children, but net water ultrafiltration was lower. standard pediatric pet and its criterie are consistent with the adult criteria. the capability of peritoneal solute transport increased after peritonitis episodes. verapamil (vp) is known to alter cyclosporine (csa) bioavailability. the impact on immunoregulators (il- , tgf-β , and tgf-β ) in allograft recipients remains unresolved. a prospective open study to examine the impact of vp on peripheral blood cell mrna encoding il- , tgf-β , and tgf-β and serum il- , tgf-β , tgf-β protein levels was performed. parental written informed consent was obtained in all cases. children with stable renal allograft function (< months), and receiving immunosuppression (csa, pdn, either with aza or mmf) were included. in the first visit, a clinical examination, two-point ( and h) csa pharmacokinetic profile, serum creatinine, serum for il- , tgf-β , and tgf-β protein levels (by elisa) were obtained; peripheral mononuclear cells were collected for measurement of transcripts for s rrna (house keeping gene) and mrna for il- , tgf-β , and tgf-β (by real time quantitative pcr assay). after the visit one, patients were either withdrawn of vp (if the subject was already receiving vp) or started on vp mg/kg/day (if the subject was not receiving vp). two weeks after, a repeat clinical evaluation and blood collection, as in the first visit, were performed. pediatric recipients of renal allografts were included ( were from ld, mean post-transplant time . years, mean csa dose . mg/kg/day). the c h and calculated auc - h were significantly higher in those receiving vp, but there was no difference in csa trough levels. protein and mrna levels of il- tgf-β , and tgf-β were not different. were previously seen by a nephrologist. logistic regression was performed on anemia (hgb< . g/dl) and showed relative risk in blacks was . vs. whites. relative risk in those who did not receive epo was . vs. those who did. of black patients, were anemic and previously seen by a nephrologist. of white patients, were anemic and previously seen by a nephrologist. in summary, blacks and patients not receiving epo at the time of dialysis initation were more likely to be anemic. despite being seen previously by a nephrologist, nearly % of patients were anemic when starting dialysis. further analysis is needed to determine causality to improve anemia control in incident dialysis patients. of the avf were in whites with in a black patient. the avg was in a black patient, with a cvc distribution of whites and blacks. patients with cvc had been previously followed by a nephrologist and of these had been followed for > months. in summary, incident pediatric hemodialysis patients are primarily having cvc as initial access type. with . % having been previously seen by a nephrologist and % of these for greater than months, the reasons behind not having an avf or avg as primary access need to be explored and improved upon. this high incident cvc use is consistent with data reported in the united states, but not with other european and asian countries. an effort to have a permanent avf or avg in incident pediatric hemodialysis patients needs to be made by the patient's nephrologist. to find the preventive measures for recurrent uti in infants with first febrile uti and normal urinary tract (ut), the incidence of recurrent uti and its risk factors were investigated. method: from june, to june, under months of age (- mon: , - mon: ), who were diagnosed as the first febrile uti and proved to have normal ut, were enrolled to the retrospective study. for all infants with nonretractile prepuce, topical application of hydrocortisone for - weeks and physiotherapy was recommended. during the following year, the incidence of recurrent uti and the well-known risk factors such as female, young age, phimosis, vaginal reflux, and initial mtc-dmsa(+) pyelonephritis were evaluated. result: the incidence of recurrent uti in infants with normal ut was . % and recurrent uti episode was . /patient-year. the recurrent incidence in male infants was . %, which was not significantly different from . % in female infants (p= . ). the recurrent incidence in younger infants was significantly higher than in older infants [- mon: . %, - mon: . %, p= . ]. this age-related difference was significant in male infants [- mon: . %, - mon: . %, p= . ], but not in female infants (p= . ). in infants with persistent nonretractile prepuces, recurrent uti developed in . %, which was higher than . % in infants with retractile prepuces (p= . ). the presence of the vaginal reflux (p= . ) or initial mtc-dmsa(+) pyelonephritis (p= . ) showed no significant difference in the incidence of recurrent uti. conclusion: in uti infants with normal ut, younger infants under months of age and nonretractile prepuces of male infants were the risk factors for recurrent uti. objective: vascular endothelial growth factor (vegf) appears to play a central role in the process leading to peritoneal angionesis and increased level of vegf may conrtibute to high peritoneal small-solute transport rate (ptsr) in continuous ambulatory peritoneal dialysis (capd) patients in adult. vegf-c is related to lymphogenesis, but its role in peritoneal solute transport rate is not known. in this study, we evaluated possible relationship between dialysate vegf and vegf-c levels and pstr in children. method: twelve children with no apparent inflammation process or disease, who had been on capd, were enrolled. standard peritoneal equilibration test (pet) was done to evaluate pstr. d/pcreat and d/d gluc were calcualted at hr of pet. overnight dialysate levels of vegf and vegf-c were measured using commercial elisa kit. correlation between dialysatevegf (or vefg-c) and d/pcreat (d/d glu) was analyzed. results: mean peritoneal dialysis duration was . ± . months. mean overnight dialysate vegf and vegf-c level were . ± . pg/ml and . ± . pg/ml, respectively. a significant correlation was noted between the dialysate vegf-c and vegf level (r= . , p= . ). dialysate vegf level had negative correlation with d/d glu of hr pet (r=- . , p= . ). vegf-c had no correlation with d/d glu or d/p creat. conclusion: there was significant relationship between dialysate vegf and vegf-c levels in children and significant correlation was also noted between dialysate vegf and ptsr. it seems that vegf contribute to high ptsr also in children on capd. m. feldkötter, l. stapenhorst, b. beck, u. bangen, b. hoppe we currently use sirolimus as a second line medication in transplanted patients with a distinct nephrotoxicity of calcineurin-inhibitors. as our short term experiences were not as positive as expected, we performed a short term meta-analysis in our renal transplant recipients under sirolimus treatment: we give an account of seven kidney transplant patients who were either directly started or were switched to a medication with sirolimus during september to february . the reasons for this action taken were calcineurin-inhibitor side effects like severe arteriolopathy with lossof gfr, atypical haemolytic-uraemic-syndrome, seizures after the first dosages of cya and a tacrolimus induced exanthema. in four of seven patients switched to sirolimus we observed severe side effects, exaggerating those of the calcineurin-inhibitor and hence, in three patients the latter treatment was installed again. findings were distinct proteinuria in two patients, hyperlipidemia in three patients, wound healing disorders and, most strikingly, treatment resistant severe pancytopenia in one patient and severe interstitial pulmonary fibrosis in another patient, both with amelioration after termination of the medication, but still the need of oxygen therapy in the latter patient. in addition we noticed a slightly faster reduction of the gfr calculated with the schwartz formula in five patients compared to the previous immunosuppressive regimen. based on these findings we strongly feel that a more critical discussion of each case is necessary before changing the immunosuppressive medication. also, the question arises on whether sirolimus can really be valued as an equivalent alternative to a calcineurin-inhibitor based immunosuppressive regimen in pediatric kidney transplantation. y. kovalski, r. cleper, i. krause, m. davidovits schneider children's medical center of israel, nephrology and dialysis unit, petah tiqwa, israel background: despite significant technical improvements, haemodialysis in infants with end-stage renal disease (esrd) is still associated with significant morbidity and mortality. methods: the files of patients weighing less than kg with esrd who were treated with haemodialysis at our institute between and were reviewed for background and treatment characteristics, morbidity and outcome. results: the study group included patients aged - months (mean . months) weighing . - . kg (mean . kg). mean duration of dialysis was . months. vascular access posed the major problem. ten patients were dialysed through a central venous cuffed catheter and one through an arteriovenous fistula. an average of three different vascular accesses was required per patient (range - ). mechanical difficulties were the most common cause of central line removal ( . %), followed by infections ( . %). major complications causing significant morbidity were intradialytic haemodynamic instability, hyperkalemia, coagulation within the dialysis set, anaemia, hypertension, inadequate fluid removal and recurrent hospitalisations. analysis of outcome revealed that patients underwent successful transplantation, one returned for haemodialysis after . years due to graft failure, and died. conclusion: haemodialysis is a suitable option for low-weight paediatric patients with esrd awaiting transplantation, when performed in highly qualified centers. the importance of antibiotic prophylaxis in management of vur vesicoureteral reflux (vur) cause urinary tract infection (uti) and renal scarring is a common condition in children. the detection and treatment of vur before renal scarring is vital. recently, optimal management of low grade vur is controversial. the aim was to explore the kidney outcome in a cohort of patients with vur. the patients were divided into five subgroups according to vur grades. all of them were treated with low dose prophylactic antibiotics until the age of years. urine culture was repeated monthly. background: anemia is a common complication in patients on hemodialysis. treatment of anemia with recombinant human erythropoietin (rhuepo) may lead to iron deficiency. intravenous sodium ferric gluconate complex (sfgc) therapy improves iron stores. objectives of study: aim of our study was to assess effects of maintenance sodium ferric gluconate therapy in pediatric patients on hemodialysis on mean hemoglobin (hb), hematocrit (hct), transferrin saturation (tsat), serum ferritin and rhuepo dose, as well as safety of therapy with sfgc. methods: intravenous sfgc therapy was administered for months in mean dose of . mg/kg/week to eight pediatric patients on hemodialysis. patients were from to years old ( males and females, aged . ± years). all patients were prescribed rhuepo before start of study. results: sfgc therapy was successful in maintenance of mean hb (increased from . to . g/dl), mean hct (improved from % to %), mean tsat (from to %) and mean ferritin level (from to ng/ml). high ferritin levels in two patients were due to inflammatory disease rather than the sign of iron overload. the mean weekly rhuepo dose decreased from to iu. no significant adverse event due to intravenous sfgc therapy occurred. conclusions: intravenous maintenance sfgc use in pediatric patients on hemodialysis was safe and successful in maintenance of iron indices, thus allowing reduced use of rhuepo. the the viral hepatitis b still remains a serious problem, especially actual in patients with end-stage renal disease (esrd) on renal replacement therapy (rrt). the high frequency of hbv infection transmission in hemodialysis units and immunodeficiency modify hepatitis clinical course and outcomes and worsening vaccination results and renal graft survival. we have analyzed the results and influence on transmission of hb -infection of hepatitis b vaccination in children aged from to years with esrd on chronic hemodialysis. majority of children were boys ( . %) older than years ( . %). an assessment of hbs-ag has been conducted prior and during the vaccination (engerixb) by scheme - - months. after first vaccination hbv infection was detected there after in . % of children, after second vaccination in . %. in all patients ( ) which have received three tours of vaccination, an active immune response was developed. thus, vaccination against viral hepatitis b is effective and prevents hbv infection in children with end stage renal disease on chronic hemodialysis. renal transplantation (tx) represents the best treatment for the patient with crf. scientific advance has been able to optimize the immunosuppressive treatment however the adherence to treatment has been not maintained. aims: to identify the factors that influence in non-adherent behavior with the purpose of designing effective educational strategies. methods: the qualitative focus was carried out through patients and tutors interviews. the quantitative aspect applies for epidemic variables, time post-tx, percentages and frequency of the sentences coming from the analysis of the interviews. nurse, psychologist and a social worker were incorporated with the purpose of elaborating an instrument based on seven questions related to the transplantation, risk and/or loss of the graft; besides the events happened as consequence of this, allowing that interviewed manifested with freedom their opinions. the interview was recorded in a microcassette and later transcribed. analysis was determined by categories containing the answers of each question granting the agreement sentences according to the frequency which was repeated in each interview. informed consent was obtained. results: tx ( - ; non-adherent, % of them were interviewed. mean age: . ys. loss the graft: %, time post-tx: . months, dd: % ld: %. the lack of supervision in the taking of medications, numbers/schedules medications, family conflicts and the poor communication with the parents/medical team seem to be the main factors for non-adherence. conclusion: it is necessary to modify the pattern of the patient's attention transplanted under the pattern of chronic suffering that allows the sick person's and their family active incorporation to the process in an integral way to the multidisciplinary group. infantile results: patients ( females, males) < kg, / -< kg at pd start were treated. they consisted % of our center's pd patients ( pts). age at pd start: . ± months (median ), / pts < mo. pd therapy duration: - mo (median ), pts > mo. esrf cause: congenital nephrotic syndrome pts, dysplastic kidneys , cortical necrosis . pts were fed by gastrostomy, pts received gh (growth hormone). / pts had hypertension (ht) treated with > drugs and - cv events. pd type: / cycler-assisted, / capd (continuous ambulatory pd), / both. pd adequacy targets (kt/v> . ) were reached in / . peritonitis: . episodes/patient-month, pts had > episodes. / pts had > pd catheters and / > pd-related surgery. outcome: / -kidney transplantation, / switched to hd for infections or uncontrolled ht. height ±sds median , weight ±sds median + . . conclusions: small infants with esrf can be successfully treated by pd despite high rate of infectious, cv and surgical complications. pd therapy main target is optimal growth towards kidney transplantation. hyperlipidemia is a well recognised complication of renal transplantation. it is a fairly common problem in the paediatric renal transplant population. its prevalence ranges from % to % though the mechanism is not clear. steroids, calcineurin inhibitors and rapamycin are the main culprits in inducing hyperlipidemia, which is a potential risk factor for cardiovascular heart disease and graft dysfunction. long term effects of these immunosuppressive drugs in children have not been adequately studied. of the calcineurin inhibitors cyclosporine (csa) was found to induce hyperlipidemia compared to tacrolimus (tac). post-ransplant hyperlipidemia is well described in adults; the same cannot be said in children. in adults, post-transplant hyperlipidemia increases risk of cardiovascular disease to fold. screening and management of hyperlipidemia has therefore become an important part of current long term management of transplant patients. there is a limited data on prevalence of hyperlipidemia in renal transplant in children and even more so locally here in south africa. most of the known studies have been conducted in the first world, there was therefore need to determine prevalence locally. this information would ultimately assist in the overall management of our renal transplant recipients. majority of the patients had normal lipid profile. % of the patients had high cholesterol levels, while % of the patients had high tg levels. / ( %) of the patients on csa had hypercholesterolemia compared to only / ( %) on tac (p= . ). / ( %) of the patients on csa had high tg compared to / ( %) on tac (p= . ). the study concluded that the prevalence of hypercholesterolemia and hypertriglyceridemia in renal transplant pts is high, comparable to other studies and that there is a tendency towards having more lipid abnormalities in transplant pts on csa. grade vur in ( %) and . the incidence of abnormal findings was significantly higher in children with uti and vur than in those with uti without vur ( . % vs %; p< . ). in children with no vur, grades - vur, grade vur and grades - vur, renal scarring rates were %, %, % and %, respectively. the patients with higher grades vur tended to have more than scars on their dmsa scans (p< . ). our findings suggest that renal scarring resulting from uti is mostly related to vur, but sometimes is caused by the infection itself. we can conclude that vcu is essential for diagnosis of vur, but mtc-dmsa scan shouldn't be avoid in the management of children with uti. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] years. seventythree ( . %) of them were on triple immunosuppressive therapy, one on double therapy, and one didn't use any medication. overall, subjects ( %) had at least one episode of uti. twenty-four episodes of urinary tract infection occurred in children: episodes in two girls with neurogenic bladder (ngb), episodes in two boys with posterior urethral valve (puv), four episodes in an obese girl with laurence-moon-biedl syndrome, episode of uti in girl with unknown primary renal disease and episodes in girl, one with polycystic kidney disease and one nephronophthisis. conclusion: uti following kidney transplantation was more common in children with known lower urinary tract abnormalities. key words: urinary tract infection, kidney transplantation. background: in japan, the severe shortage of cadaveric kidneys led clinicians to attempt performing abo-incompatible living kidney transplantation (tx). some reports demonstrate successful results of the combination of plasmapheresis (pp), strict immunosuppression and splenectomy. however, we should concern about a great risk of surgical invasion and postoperative serious infection in younger patients who underwent splenectomy. recently, a few reports suggested anti-cd monoclonal antibody (rituximab) can be an alternative to splenectomy. patient and method: a years old boy with bilateral hypoplastic kidneys had been treated with peritoneal dialysis for years. since his blood type o was incompatible with paternal blood type a, we arranged to perform tx using pp and rituximab without splenectomy. a single pp was performed on days - , - , and - to reduce anti-a antibody (ab) titer. rituximab was administered in a single dose of mg/m on day - . basiliximab, tacrolimus, mycophenolate mofetil and methylprednisolone were used for immunosuppression. results: before tx, the anti-a ab titer was reduced from : to : , and cd level was suppressed from % to %. tx was performed without splenectomy and he had excellent initial graft function. we observed anti-a ab titer rose up to : on day + , but it decreased spontaneously to : . there were no side effects and severe infections during the perioperative period, but the neutropenia treated by gcsf appeared months after rituximab administration. the protocol biopsies were performed in month and months, which revealed no signs of rejection. conclusions: abo incompatible tx using pp and rituximab without splenectomy can be a therapeutic option in children to avoid a invasive surgery and infectious risks. further establishment of optimal protocol for children are necessary to obtain excellent outcomes safely. . . , . . , and . . , . . , in hivan, and od, p= . , p= . , respectively. serum p levels were . . , . . , and cap were . . , and . . , in hivan and od, p= . , p= . the aim of this study is to identify the outcome of the physically or socially handicapped children with end stage renal disease (esrd) receiving chronic peritoneal dialysis (cpd). among patients commenced on cpd, handicapped children with esrd receiving cpd were identified in our unit during the period between november and february . age at cpd initiation ranged from . to . years (median age: ; girls, boys). underlying diseases were neuropathic bladder and vesicoureteral reflux (in patients), chronic pyelonephritis (in patients), vesicoureteral reflux (in patients), amyloidosis (in patients), and alport syndrome (in patient). causes of handicapped status against cpd were inadequacies of indoor resources (in patients), cerebral palsy (in patients), down syndrome (in patient), inadequate psychosocial status (in patient), surgically corrected rectovesicale fistula and ectopic anus (in patient), blindness (in patient), ventriculoperitoneal shunt and paraplegia (in patient), colostomy (in patient). all catheters were implanted percutaneously by the same pediatric nephrologist. median duration of dialysis was (range - ) months. during a total of dialysis months, episodes of peritonitis ( episode/ . patient-month), episode of exit-site infection, and episode of tunnel infection occurred in of children. except for an inguinal hernia in patient, we did not observe any mechanical complication related to catheter. cpd was terminated in children (death in , renal transplantation in , switch to hemodialysis in ). before initiation of renal replacement therapy, some negative baseline factors may not be really contraindications for cpd. the socioeconomic and geographic factors greatly influence the prevalence and outcome of renal disease in children. the subspecialty of pediatric nephrology in sudan was established few years ago and the facilities for the management of renal problems are limited. the aim of this study is to review the demographic profile, complications and outcome of capd after nineteen months of treatment. all children who underwent capd from june to january were studied. there were children ( males), the mean age was years (range from months to years). the majority of children has undetermined cause of esrd ( children). the most common complication was peritonitis (peritonitis rate was / . patient/months. patients had refractory peritonitis that necessitate catheter removal, exit-site infection was documented in children and catheter block in children. there were drop out of the program were due to deaths, changed modality and one family withdraw treatment. in conclusion this analysis has stimulated improvement in nurses training and supervision as well as attempts to improve catheter survival and microbiology monitoring. the - ) , looking for symptoms of hypovolemia (cramps, abdominal pain or headache) during or at the end of hd treatment. bioimpedance measurements were performed at the end of each session according to the khz tetrapolar technique; resistance and reactance values were plotted on the age and gender specific th , th and th percentiles of the vector distribution in the healthy population (reference tolerance ellipses) as a resistance-reactance graph. hypovolemia (hv) was indicated by a vector shifted to the upper pole, out of the % tolerance ellipse; normovolemia (nv) by a vector inside the ° ellipse. patients complained of one or more of the above-mentioned symptoms in % of hd sessions, while biva suggested hv in . % of the sessions. symptoms were significantly more common (p< . ) in sessions with hv ( / cases; positive predictive value . %) than in those with nv ( / cases; negative predictive value . %). biva suggested hv in / sessions with symptoms (sensitivity . %) and nv in / sessions without symptoms (specificity %). no significant differences in the accuracy of biva were found between patients either younger vs older than years, or with height sds <- vs >- , or taking vs not taking antihypertensive drugs. in conclusion, biva can be useful in assessing dry weight in children and young adults on hd: since patients with a vector shifted to the upper pole, out of the reference % tolerance ellipse, are at high risk of hypovolemia during the next hd treatment, the increase of the dry weight is then indicated chronic antibody-mediated rejection can occur as a de novo complication in renal allograft recipients and is associated with c d deposition in peritubular capillaries in the renal graft and positive circulating anti-hla antibodies, although the sensitivity and specificity of positive c d staining for chronic humoral rejection requires further study. renal outcome appears to be worse in c d positive patients. current treatment strategies to manage c d-positive chronic humoral rejection are poorly defined. various protocols with enlarged doses of tacrolimus, mycophenolate mofetil, plasmapheresis, ivig and rituximab have been reported in adult patients. we investigated four pediatric patients (mean age . yrs; range to yrs) after renal transplantation that developed c d positive chronic rejection. in of patients, maintenance immunosuppression with calcineurin inhibitors had previously been minimized because of severe toxicity. in of patients, an elevated anti hla class ii antibody titre could be detected; donorspecific antibodies were positive in patients. all patients experienced a progressive deterioration of graft function. treatment with repeated intravenous immunoglobulin (ivig) ( g/kg body weight per week over four consecutive weeks) followed by a single dose of rituximab ( mg/m ) was therefore initiated. three of four patients showed an improvement of graft function with a mean increase of gfr by %. one patient with advanced chronic transplant nephropathy lost his graft after months. this pilot study demonstrates that the combination of high-dose ivig and rituximab can stabilize or improve transplant function in chronic antibody-mediated rejection without major side effects. the use of ivig and rituximab appears to reduce the active immunologic process, but larger trials are needed to support these observations. cardiovascular diseases are some of the most important causes of morbidity and mortality in children with end stage renal disease (esrd). chronic inflammation has been suggested to be a risk factor for cardiovascular diseases. the aim of this study was to investigate the relation between crp and cardiac changes in children on hemodialysis. this study was conducted on patients ( patients were hypertensive & were normotensive), males ( %) and females ( %) on regular hemodialysis due to esrd. their ages ranged from to years (mean . ± . ). sixty age-and sex-matched controls were also included. significantly higher velocity of circumferential fibre shortening (vcfs), tei index, interventricular septum thickness in diastole (ivsd), left ventricular mass index (lvmi) and isovolumetric relaxation time (ivrt) and significantly lower e/a ratio were found in all patients as well as in hypertensive & in normotensive groups as compared to the controls. significantly higher ivsd and lvmi were found in hypertensive patients than normotensive patients. significantly higher high sensitivity crp (hs-crp) & crp latex were found in all patients as well as in hypertensive & in normotensive groups when compared to the controls. crp was significantly higher in both study groups with cns symptoms and cardiac symptoms in comparison to those without. it was also significantly higher in patients with increased lvmi & than in those with abnormal e/a ratio. hdlc showed a significantly direct negative effect on crp. s. ca + , se. p and ca x p had a significantly direct positive effect on it. we can conclude that the cardiac affection in children with esrd appears in the form of lv hypertrophy with early diastolic affection. crp could be correlated to these changes and to cns symptoms and cardiac symptoms in these patients. is. lim, hs. lee, dw. kim, wh. choi chung-ang university, pediatrics, seoul, south korea purpose: urinary tract infections are common clinical problems occurring in infants and pediatric patient groups, most frequently caused by uropathogenic e. coli. urinary pathogens almost always infect the host through ascension from the rectum, vagina to the urethra and bladder. recurrent urinary tract infection is a disorder involving repeated or prolonged bacterial infection of the bladder or lower urinary tract. in this study, we examined the substitusion effect of probiotics in the high risk group of recurrent urinary tract infection. objectives & methods: patients diagnosed as recurrent urinary tract infection were administered probiotics for six months, and urine cultures were checked during the period. probiotics in this study were selected among the products commercially saled in korea, namely lactobacillus acidophillus, bacillus subtilis, and bifidobacterium infantis. single blind study was done for selection of probiotics for patients. result: the separated bacteria from the urinary tracts of the patients were the same as administered probiotics in some patients. conclusion: in recurrent urinary tract infection, there seemed to be a substitution effect of probiotics for uropathogenic bacteria, and it is reasonable to administer probiotics for long period in the high risk group of recurrent urinary tract infection. renal insufficiency therapy in children: quality assessment and improvement: the rich q study objectives: outcome studies in children on chronic renal replacement therapy (crrt) have revealed a -time increased mortality and % co-morbidity in adult survivors. information on the quality of care of treatment centers and on the impact of advised quality indicators on outcomes in children are lacking. no data exist on the impact on these outcomes of the different treatment modalities, peritoneal dialysis, hemodialysis & transplantation either. until now, no structural corporation and consensus on general guidelines with respect to crrt exist between the dutch (nl) and belgium (b) centers for pediatric crrt. aim of the study: . assessment of the current quality of treatment crrt in children (qt) in nl & b and of the effect of recurrent peer review of the achieved outcomes on the qt. . the assessment of the effect of different treatment modalities on outcomes. . the creation of a format for multicenter trials. methods: all prevalent patients on chronic dialysis aged < years at onset of the study & all incident patients during the study period with onset of crrt< years of age, from b & nl will be included. treatment characteristics and quality indicators of crrt with respect to physical and psychosocial outcomes will be collected of all patients. operational data collection and management will be performed by members of the dutch institute for quality care in dialysis patients (hans mak institute). each months, all data will be revealed and actively discussed by representatives of all centers (peer review). the effect of registration and peer review on the qt will be analyzed after & years. comparison will be made between the effects of cumulative periods of different rrt models on outcomes. the study will be performed between august & . on estimation, patients will be analyzed. renal renal transplantation in patients with lower urinary tract dysfunction (lutd) of different origin is a challenging issue in field of pediatric transplantation. we report our single centre experience to evaluate the patient and graft survival as well as risks of the surgery and immunosupressive therapy. among pediatric transplant patients patients had severe lower urinary tract dysfunction. videourodynamic test was performed in all patients preoperatively and postoperatively. the cause of urological disorders was secondary to neurogenic bladder (n: ) and valve bladder (n: ). clean intermittent cathatetization (cic) was needed in patients to empty the bladder. pretransplant augmentation ileocystoplasty was created in four patients and gastrocystoplasty in one patient to achieve low-pressure reservoir with adequate capacity. three of the patients received kidneys from cadaveric and of them from living donors. the mean age at transplantation was ± . years. the median duration of transplantation was months (range - months). at their last visit median creatinin levels were . mg/dl ( . - . ) . three patients had recurrent symptomatic urinary tract infections who had augmented bladder and on cic. one of them had creatinine levels of . mg/dl. one patient with ileocystoplasty who developed urinary leak and ureteral stricture in early postoperative period was treated by antegrade j stent. severe lutd reserves high risks for graft kidney. however our data suggests that renal transplantation is safe and effective treatment modality if the underlying urologic disease properly managed during the whole course of transplantation period. since surgery and follow-up of these patients is more complicated, patient compliance and experience of transplantation team will have significant impact on the outcome. r. meneses, l. sylvestre, j. sousa, d. ribeiro hospital pequeno principe, pediatric nephrology, curitiba, brazil introduction: in july , we started a systematic evaluation program of each patient on chronic pd. the aim of this study was to analyze the long-term outcome of children on pd program. material and methods: we evaluated all the patients on pd between july and may , who performed complete protocols, with a minimal interval of months between them, consisting of: anthropometric measurements, blood pressure and cardiological status, standardized laboratorial evaluations, pet test, clearance and kt/v, measurement of the intra-peritoneal pressure (ipp), occurrence of infections, hernias or constipation and need to change the catheter. we then compared all the evaluations using the graphpad prism software, a p value < . was considered significant. results: out of patients were eligible, mean age ± years old at the first evaluation, % boys, primary renal disease: % uropathies, % glomerulopathies, % tubulopathies and % other causes. there was an improvement on bmi and weight/height z-scores and worsening of height/age z-score, but none was significant. there was also no significant decrease in residual renal function (p= . ), adequacy parameters remained stable: clearance (p= . ) and kt/v (p= . ). most patients were converted from capd to ccpd and nipd, and some had to increase daytime dwells (p= . ).constipation and the number of infections improved but not significantly. laboratorial evaluations, peritoneal membrane characteristics, ipp, need to change the catheter and occurrence of hernias did not change over the time. conclusion: a long-term maintenance of children in peritoneal dialysis program is possible, but reaching a satisfactory clinical condition is a great challenge. several points need to be checked for planning a better adequacy and survival of dialysis technique in children waiting for a graft. a rigorous follow-up protocol seems helpful in precocity of prescription strategy modifications. we observed a stable long-term outcome observing these adequacy tools. outcome the recurrence of primary disease in transplants is a well-known problem. we report our single centre experience to assess the frequency of the recurrence of primary glomerulonephritis in children after renal transplantation. medical reports after of children with primary glomerular disease were evaluated. the grafts were nine from living related and four from cadaveric donors. eight of them were diagnosed as fsgs, of them mpgn and of them pan. the mean age was . ± . years. however the median transplantation duration was months, one of the fsgs patient had hyperacute rejection. five years later she had second graft with the serum creatinine . mg/dl at th year of second transplantation. and all recipients were immunosuppressed with either cyclosporin a or tacrolimus, azothioprine or mmf and steroid based regimens. mutational analysis was available in two patients, they had homozygous podocin mutations. post transplant recurrence of fsgs was confirmed in one patient. glomerular tip lesion was the only histologic abnormality on graft biopsy. he has treated with plasmapheresis with no improvement of proteinuria. two of the fsgs patients had thromboses after transplantation. one of them had cardiac thrombosis with heterozygote mthfr mutation and one of them had renal artery thrombosis and loss of graft with prothrombin a mutation. both of them have had additional risk factors for thrombosis. they have all functioning grafts except one. we have not observed any recurrence in patients with pan and mpgn. although the number of our patients quite small, renal and patient survival seems to be more favourable in our experience but we strongly recommend the evaluation of all risk factors of thrombosis and give appropriate anticoagulation. skin involvement in factor h deficiency (fhd) associated to hemolytic uremic syndrome (hus) has never been reported. we describe the case of a young adult on regular hemodialysis (hd) for fhd-hus who developed microangiopatic skin lesions and was successfully treated with plasma exchange (pe). the patient developed end stage renal disease secondary to fhd-hus (scr ) in , when she was . after one year of hd she complained of severe night pain in the perimalleolar areas, followed by skin lesions which evolved into superficial ulcers (fig ). in august , due to the worsening of the skin lesions, the patient started hd and pe ( litres of fresh frozen plasma per session twice a wk) based on the hypothesis that skin lesions were expression of thrombotic microangiopathy. after wks of pe there was a skin improvement (fig. ) and a pain relief. pe was discontinued. wks later she started to complain of the usual pain in the right foot. pe program combined to hd was restarted and the symptoms ceased again. pe was gradually discontinued and she was addressed to regular plasma infusion of . litre per wk. so far, after months the pain and the skin lesions did not show up again. m. belingheri, s. cristino, p. basile, v. bianchi, a. leoni, s. testa, l. ghio, a. edefonti, g. ardissino ospedale maggiore policlinico irccs, mangiagalli e regina elena, pediatric nephrology, milan, italy background: in rapidly growing children on hemodialysis (hd), the determination of dry weight still remains troublesome. bioimpedance analysis (bia) is potentially helpful in quantifying the fluid to be removed but its specific role, in routine clinical practice, is not yet clearly set. the aim of the present study was to test the feasibility of prescribing ultrafiltration (uf) exclusively based on bia parameters. methods: differences in body weight, resitance (rx) and reactance (x-c) between pre-and post-hd were calculated in order to derive the equivalence between uf and bia parameters in a years old girl over a period of months. for consecutive hd sessions, uf was prescribed exclusively based on the derived uf-bia equivalence. this period was compared with hd sessions where uf was prescribed by the conventional approach. results. xc correlated with ultrafiltration better (r: . ) than rx (r: . ). bia-based compared with weight-based uf prescription showed a significantly lower number of hd sessions complicated by hypotension ( % vs. %), need of fluid reinfusion ( % vs. %) and a better quality of the hd sessions ( % vs. %). conclusion. prescription of uf solely based on xc is feasible and provides a better outcome compared to the conventional modality of uf prescription. we believe that this approach could be useful for any patients with low tolerance to uf or with problems in setting the correct dry weight. aim: the aim of the present prospective study was to determine the incidence of urinary tract infection (uti) and related abnormalities in children ages between - years. material and methods: all children between - years old whose admitting to first step health offices (routine controls and immunization) was screened for uti with urine dipstick test after education of minimum two persons from first step health offices according to protocol with two tertiary child care center and health directorate of izmir province in turkey between july and july . all patients with urine dipstick test abnormalities were referred to tertiary child care centers for evaluation. urine microscopic evaluation and urine cultures and other further investigations were performed in tertiary care centers after obtaining urine with urinary catheterization. results: . children ( % boys) were screened with urine dipstick test. the children's mean age was . ± . mo (median mo). screening test was found normal in . ( %) children. of ( %) of referred child were admitted to tertiary care centers and evaluated for uti. uti was demonstrated in children ( . % of screened and % of evaluated children's). uti incidence was found . % in girls and . % in boys. urinary tract abnormalities were found in children ( . % of screened and . % of evaluated children's). the most common urinary abnormality was vesicoureteral reflux ( patients). conclusion: the uti incidence was . % in children ages between - years, uti more common in girls than boys in this age group and only small group of children has urinary tract abnormality which is determined with routine urine screening. knowledgement: thank you for this opportunity to health directorate of izmir province. we describe ds post-peldrt in children with no known neurologic problems and discuss potential predisposing factors. a . kg girl with renal dysplasia was started on a calcineurin inhibitor (cni) one week pre-t and when her blood urea nitrogen (bun) was mg/dl. on admission for t, the bun had increased to , and her serum sodium (na) was mmol/l. post-t, she remained intubated and paralyzed to permit generous volume supplementation, including : replacement of her vigorous urine output (uop), initially with . % nacl in water. five hours post-t, her bun was and her na . after modification of uop replacement, her na normalized. on the morning of post-t day , paralysis was discontinued, but she did not awaken and had sluggish pupillary reactions. computed tomography of the head (cth) revealed diffuse cerebral edema, and brain death occurred. a kg adoloescent with polycystic kidneys was started on a cni days before peldrt. his bun and na then were and , respectively, and had not changed on the day of t. post-t, the patient was immediately extubated. with uop replacements as described above, his bun and na decreased from to and to , respectively, over hours despite adjustments in the na content of his intravenous fluids based on urine na levels. the patient then had a -second tonic-clonic seizure, followed by a -hour post-ictal state. cth was negative, and the patient recovered completely. we conclude that ds, caused by a rapid decrease in serum bun and thus osmolality, may complicate peldrt in settings even with older pediatric recipients or without excessive elevation of pre-t bun. other factors contributing to this ds may include relatively mild hyponatremia and cni effects on both pre-t uremia and seizure threshold. results: patients, predominantly males, ages between months and years old. the mean incidence of peritonitis was . episodes/patient months. fifty-seven patients ( %) had at least one episode of peritonitis.there were peritonitis, % percent from all episodes began at home, % caused by gram negatives, % by gram positives, % by fungus, % had a negative culture and in less than % it was not performed. the mean treatment time was days, % had a good response to initial empiric antibiotics (cefazoline and amicacine). the interval between the beginning of dialysis and the first peritonitis episode varied from to days, occurring in the first months in % of the patients. successful treatment occurred in % of the cases, % were transfered to hemodialysis, % had a consecutive peritonitis episode, and patient died due to mesenteric artery trombosis. conclusion: peritonitis occurred early in our patients. even though most of them have a good initial response, there is still a great amount that have complications leading to technique failure. continuous education for the patients and health team, aiming early diagnosis and treatment, are useful to preserve the technique and decrease morbidity and mortality associated to peritonitis. d. davis, j. emancipator, x. zhu, c. rosen objective: to assess for sd in p chronic kidney disease (ckd) patients before and after rtx. methods: we assessed symptom (sx) domains of sleep disorders: ) sleep-disordered breathing (sdb); ) insufficient sleep (is) (shortened sleep time or nap); ) excessive daytime sleepiness (eds); and ) restless leg syndrome (rls) using a set of standardized questionnaires in patients with ckd (age - yrs) including non-d non-tx (ndntx) (n= ), d (n= ), rtx (at least months post-tx) (n= ), and age-matched sibling controls (c) without known ckd. the presence of an overall sd was defined by positive responses in any of the sx domains. results: mean age (se) ranged from . ( . ) to . ( . ) in the pt groups (p> . ) without significant differences in gender, race, or congenital ckd. estimated mean gfr (ml/min/ . m ) (se) was significantly higher in the rtx group [ . ( . ) methods: in a prospective design, renal transplanted children, who had renal transplantation at least months before, at namazee hospital, were enrolled in our study. immunosuppressive regimen consisted of cyclosporine and prednisolone plus mycofenolat mofetil or azathropin. data regarding gfr, serum creatinine, electrolytes, lipids and c and c levels was collected at beginning, in one-month, and five-month intervals. cyclosporine was adjusted to - ng/ml based on c level. patients were divided into two c (< and > ng/ml) and two c (< and > ng/ml) subgroups. discussion: similar creatinine levels, drug dosage, and complications of c and c subgroups may be due to dependence of renal function to several factors other than cyclosporine dosage. regarding coefficient of variation, c was more accurate and reliable than c level. as there was no significant difference in mean c and c levels, and renal function at beginning and the end of the study, there seems to be no need to check c levels after renal transplantation. purpose: preparation is necessary in order to effectively meet the critical needs of the post-operative pediatric kidney transplant patient upon their arrival to the icu following transplantation. the increasing number of children requiring liver transplantation services has made it evident that it is important to have guidelines in place for their initial and often specialized post-operative care. methods: the main goal is to provide the child with appropriate post-operative care and to recognize and quickly address complications. therefore the icu nurse will: · monitor the patient continually and conduct full assessments a minimum of time/hour (airway, breathing, ventilation, perfusion, neurological status, etc) . · observe the incision for signs of bleeding, evisceration, and dehiscence. · treat post-operative pain. · update family with findings, etc. · see that appropriate post-operative studies (ultrasound, laboratory studies, etc) are completed. outcomes: nurses in the icu monitor the pediatric post-operative kidney transplant patients very closely as outlined. this allows for quick recognition of problems and immediate intervention. it is the practice of these nurses to be fully aware of the patient's status as well as any changes that might be problematic. conclusions: nurses are prepared to care for pediatric kidney transplant patients and very carefully follow established guidelines for assessment. following guidelines for assessing and caring for pediatric kidney transplant patients upon admission to the icu has proven to be affective in allowing nurses to quickly recognize complications and notify the appropriate clinician. background: uremia is an independent cardiovascular risk factor. transplantation increases life expectations of patients with crf, however there is still an increased risk of accelerated arteriosclerosis. the pulse wave velocity (pwv) is a non-invasive marker of arterial distensibility, it increases along with arterial stiffness, as an early indicator of arteriosclerosis. aim: to evaluate pwv values of transplanted (tx) children. patients, methods: pwv was measured with a pulsepen in tx (age , ± , years). two control groups were formed using a database of healthy children ( - years): one matched for age (a) and one adjusted for height and weight (h/w). blood pressure, heart rate, serum calcium (ca), phosphate (p) , and pth were also determined before transplantation and at the time of the pwv measurement. results: tx patients were smaller by , cm (p< , ) than a and younger by , years than h/w (p< , ). pwv in tx ( , ± , m/s) did not differ significantly from a ( , ± , ) , however it was elevated in comparison to h/w ( , ± , p< , ). serum p, caxp and pth was increased before transplantation, all the values returned into the normal range except for creatinine ( ± micromol/l) at the time of the study. there was no correlation between pwv and the actual values of ca, p and pth. conclusion: pwv is higher in transplanted children as a sign of increased arterial stiffness. controls matched for height and weight should be used in states of severe growth failure. although a number of established risk factors potentially responsible for arterial dysfunction were present before transplantation, they were normal at the time of the study. the long lasting effect of uremia before transplantation could be in part responsible for the increased pwv in children after transplantation. supported otka-t -fo -f and ett / . d. derakhshan , h. jalaeian , a. derakhshan department of pediatric nephrology, shiraz, iran shiraz organ transplantation center, nemazee hospital, shiraz, iran backgrounds: bartter syndrome is an inherited recessive autosomal tubulopathy characterized by hypochloremia, hypokalemia, metabolic alkalosis associated with potassium renal leakage, and normal blood pressure despite increased plasma renin activity. patients with this syndrome may have proteinuria or hematuria, but most of them have normal gfrs. here we report on a child with bartter syndrome who developed esrd (end stage renal disease) and underwent successful cadaveric kidney transplantation. case presentation: a -year-old girl presented to the pediatrics nephrologist with failure to thrive, severe hypokalemia, hypochloremia, metablolic alkalosis, and normal blood pressure and the diagnosis of bartter syndrome was considered for her. however, due to poor compliance, she did not receive any medications, did not give consent for kidney biopsy and did not attend her opd follow-up visits for about years, when she developed esrd and went on chronic hemodialysis ( /weeks) . her little sibling also was diagnosed to be suffering from bartter syndrome at this time. after months, she received a cadaveric renal allograft. afterwards, her kidney function, serum electrolytes, and growth have improved dramatically. discussion: in this case, we postulate that long-term hypokalemia due to bartter syndrome led to chronic interstitial nephritis and renal dysfunction. successful renal transplantation, even after the onset of esrd, for severe clinically bartter syndrome results in correction of metabolic abnormalities and excellent graft function. we propose that bartter syndrome should be considered as a possible cause of esrd and an indication for early renal transplantation, a procedure that results in a cure for the underlying disease and significant improvements in patient's quality of life. h. jalaeian , a. derakhshan , d. derakhshan , m. fallahzadeh , z. bazargani , m. basiratnia shiraz organ transplantation center, nemazee hospital, shiraz, nemazee hospital, shiraz, iran fasa university of medicine, pediatrics, fasa, iran shiraz university of medical sciences, pediatric nephrology, shiraz, iran background: obesity is a major issue in the end stage renal disease population. while studies evaluating the effect of obesity on transplant outcomes in adults have yielded varying results, this issue is even still more controversial in children. methods: in a cross-sectional design, pediatric recipients, aged - at transplantation and with normal graft function for at least months after transplantation, were evaluated. we grouped the data with regard to the body mass index (bmi) percentiles as group i (bmi > th), group ii (bmi < th), group iii (bmi > th), group iv (bmi < th). we compared the clinical and laboratory findings between groups i and ii and between groups iii and iv. obesity was defined as bmi > th and being overweight was defined as bmi > th. results: there were children ( males, females) with mean age at time of transplantation of . ± . years (range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and mean follow-up of . ± . years. . % of children were overweight and . % were obese. no difference was found regarding age, height, duration of pretransplantation dialysis, or age at transplantation between groups i and ii and between groups iii and iv. (p> . ). further more, no difference was found in regard to serum creatinine, bun, glomerular filtration rate, and -year graft survival rates among obese and/or overweight and other children. no correlation was found between bmi and gfr (p> . ). conclusion: obese and overweight recipients can have excellent graft function and survival rates that are comparable to their non-obese counterparts. denying patients access to renal transplantation on the basis of obesity per se does not appear to be justified. d. derakhshan , h. jalaeian , a. derakhshan , b. sabet , m. fallahzadeh nemazee hospital, shiraz, iran shiraz organ transplantation center, nemazee hospital, shiraz, iran introduction: tb is an important cause of morbidity and mortality in renal transplant recipients, especially in developing countries. this study was done to identify the incidence of tuberculin test positivity before transplantation as well as the influence on outcome of graft function and patient survival in children who receive renal allografts. methods: all children with esrd who received a renal allograft between and were evaluated. as a routine pre-operative measure, a tb test was administered, using ppd. the ppdpositive recipients were compared with ppd-negative subjects, regarding age, gender, graft function, graft outcomes and patient survival rates. patients were divided into < mm versus > mm induration. results: the mean age of recipients was . ± . years (range, - ) with a male/female ratio of . : . the majority of children were on chronic dialysis with mean duration on dialysis of . ± . months. the tuberculin test was positive in . % of children; all of them received isoniazide prophylaxis on diagnosis of latent tuberculosis. overall, the -year, -year, and -year survival rates were . ± . %, . ± . %, and . ± . %. three year survival rate was not different among ppd positive or ppd negative individuals. ( . ± . vs. ± . %; p> . ) in addition, no difference was found for -year or -year graft survival rates (p> . ). also serial serum creatinine levels at -month, -month, -year, -year, and - year interval after transplantation was not statistically different (p> . ). conclusions: detection of latent tuberculosis infection is an important step in the control of tuberculosis among asymptomatic pediatric kidney transplant. with proper management, latent tb does not affect transplantation outcome among children. h. xu, q. shen, ss. ruan, yl. bi, yq. lu, x. wang children's hospital of fudan university, department of nephrology, shanghai, people's republic of china objectives of study: we have started the first pediatric renal transplantation project in children hospital in china from . survival of patients and grafts for the patients are %. the clinical features were analysed and specific problems related to drugs and infections were reviewed. methods: children ( ~ years old) underwent rtx. the duration of follow-up was months to months (average of . months). results: all the patients were on automated peritoneal dialysis prior to rtx. the transplanted kidneys came from cadaveric donors (one were a -year-old brain-dead boy). patients received il- receptor antibodies as induction therapy and the other one with alg due to high level of population reactive antibody. after the rtx, all the patients were on triple immunosuppressive treatment (prednisolone, mmf, fk or csa) . no patient developed postoperative complication and delayed graft function occured. during the follow-up, case suffered from calcineurin inhibitor renal toxicity and changed to rapamycin treatment, from acute respiratory distress syndrome due to infection and from elevated liver enzymes owing to drugs. one had acute rejection at months after the operation. a severe anemia appeared on him after the rejection recovered. the cause of the anemia was found by the positive of serum anti-parvovirus b igm and completely recovered from the ivig treatment. at latest follow-up, the mean serum creatinine level of the patients was . ± . umol/l and egfr was . ± . ml/min/ . m . some patients received a support from "shanghai child renal failure trust fund". conclusions: the improvement of surgical technique, adequate dialysis prior to rtx, rational use of medicine, financial support and regular follow-up are all important for improving the outcome. h. bunker-wiersma , , j-c. davin the area under the curve (auc) of cyclosporine is strongly related to the efficacy and toxicity of the drug and its variability is mainly determined by the absorption phase. close therapeutic drug monitoring (tdm) is warranted to optimise therapy, using more appropriate methods to estimate the auc, than trough concentration measurement. from july we started auc guided monitoring of cyclosporine therapy based on two concentration measurements, c and c . the results of this method are reported. methods: all paediatric renal transplant recipients treated with cyclosporine were included in the analysis. bayesian, model based estimation of auc values was performed at each out-patient visit or during hospital admission. calculated pharmacokinetic parameters, treatment efficacy data and side effects were collected over the month period after introduction. target auc values were derived from previous studies in adult patients: ng/h/ml in the first three months after transplantation and ng/h/ml after three months. results: early or stable post renal transplant patients were evaluated, divided in two groups (group i: < months after transplantation; group ii: > months after transplantation). in patients with trough concentrations below the therapeutic range, more than % auc's were in the therapeutic range in both groups. conclusion: auc guided monitoring of cyclosporine after kidney transplantation in children using c and c is practically feasible and presents the major advantage that c has not to be determined precisely hours after csa administration. it is more closely related to the total drug exposure as compared with trough concentration monitoring and isolated c . this method may facilitate the use of lower doses of cyclosporine and by this way limitside-effects. objectives: there is no satisfying data about reproductive functions after kidney transplantation in adolescence who have end stage renal disease (esrd) during childhood. we analysed the reproductive functions of kidney transplanted male adolescences. patients and methods: nine patients who followed between - were enrolled in the study. except one preemptively transplanted patient, all were on hemodialysis/peritoneal dialysis before transplantation. mean dialysis duration was ( - ) months. their ages ranged between - years (mean . ) at transplantation. at the urologic examination, their mean age was years. all patients had normal renal functions. results: all patients had normal testicular volume, libido and erectil functions. except one all patients had normal serum levels of lh, fsh, total and free testosterone. seven of the patients semens were available for analysis. / patients had normal sperm parameters. transplantation had been performed before adolescence period in these patients. one of these patients had been treated with intensive cyclophosphamide before. oligospermia was detected in , defective morphology in , low sperm motility in / patients. conclusion: although adult transplanted patients mostly have normal semen profiles; male children with end stage renal failure would not have normal spermatogenesis at the adolescence period; even after successful renal transplantation. in our study only patients had normal semen profile, even hormon levels were normal. renal transplantation age seemed to be more crucial than the duration of esrd, of primary diagnosis or previous cyclophosphamide usage. r. vilalta, j. nieto, e. lara, a. madrid, s. chocron hospital materno-infantil vall de hebron, department of pediatric nephrology, barcelona, spain background: inhibition of il- receptors by basiliximab is irreversible and extended in time (mean days). basiliximab (anti cd receptor) is used usually in the induction regime in our first cadaveric-donor kidney transplants. its re-use when chronic allograft nephropathy (can) develops could be useful. however some concern could exist related to possible adverse reactions (anaphylactic shock) linked to re-exposure to this drug because is an heterologous protein. less adverse reactions as lymphokine release syndrome has been described with the use of other monoclonal antibodies as the anti-cd receptor rituximab. objective: to describe our experience in the treatment with basiliximab of seven children with banf ii can. patients: seven children ( - years old (means . y.), boys, girls) showed biopsy-proved banf ii can. its period post-transplant ranged from to years (mean . y.) and their creatininine level from to mg/ ml (mean . ). all of them had been received at the transplant time basiliximab, tacrolimus or cyclosporine, mycophenolate and tapered steroids to reach . mg/kg/day in the third month post-transplant. when can developed, sirolimus was used in two patients, but was withdrawn due to increase of proteinuria. results: one dose of basiliximab ( mg/ . m ) was administered after steriod pulses ( mg/kg/day) in all patients. their basal immunosuppression was not changed. plasma creatinine diminished by % in four patients in the second week post-treatment and this improvement was sustained in two patients after one year follow-up. proteinuria did not change in any patient. in the course of this treatment no adverse reactions were observed. conclusion: ) use of basiliximab in can is safe and possibly useful. ) exposure to different monoclonal antibodies in paediatric kidney transplantation could be usual in the future; in the induction time, in the treatment of humoral rejection if exists (rituximab) and in the treatment of can. ) it is necessary to establish that exposure and re-exposure to different antibodies is safe and without major adverse effects as our limited experience supports. r. vilalta, e. lara, a. madrid, s. chocron, j. nieto hospital materno-infantil vall de hebron, department of pediatric nephrology, barcelona, spain background: there are limited knowledge of kinetics and pharmacodynamic effect of sirolimus in paediatric renal transplantatation. provided that sirolimus is effective and safe in combination with tacrolimus and mycophenolate (mmf), the initial dose needed, the evolution of blood levels and the steady state should be studied in order to optimise its clinical use. objective: to establish a possible correlation between dose/level ratio of rapamune and other parameters as age, gender or puberal state. patients and methods: between and , paediatric patients ( girls, boys) received a cadaveric kidney transplant. age ranged from to years (mean y.), and all of them received mmf and steroids. sirolimus were used from . to . mg/kg/day, to obtain levels between to ng/ml. results: dose/level ratio obtained allowed us to describe three types of patients: an infant-type i dose-level patient (age - y), a prepuberal type ii (age - y) and an adult-type iii dose-level patient (age - y). type i needed sirolimus between . and . mg/kg/day (sd± . ), type ii between . and . mg/kg/day (sd± . ) and type iii between . and . mg/kg/day (sd± . ) to obtain all of them a constant blood levels between and ng/ml. the same positive correlation was obtained regarding the puberal status. no correlation were observed regarding the gender. introduction: developing of diabetes mellitus after renal transplantation is one of the determining factor in the survival of the patient and the graft. in present study we assessed the carbohydrate metabolism status of ntx. methods: we analyzed patients' data about recently developed carbohydrate metabolism failure after ntx. children underwent ntx between - were investigated. thirty-nine children ( girls/ boys) underwent ogtt, who had no ptdm. we analyzed the incidence of ptdm/igt, the combination of immunsuppressive therapy, the number of transplants, the proportion of cadaver/living donor, hcv, blood pressure, lipid metabolism, bmi, graft function parameters and the time since ntx. results: ptdm developed in children ( %). four of patients required insulin therapy. we diagnosed igt in of with ogtt investigated patients ( %). all ptdm/igt patient got tacrolimus and continous steroid therapy. the dose of steroid was . mg/day in the ptdm/igt group vs. . mg/day no ptdm/igt (p< . ). during ogtt the trough level of tacrolimus was higher in the ptdm/igt group . ng/ml vs. . ng/ml (p< . ). in the other parameters we did not find any significant differences between ptd/igt and no ptdm/igt patients. discussion: the most important reasons in the development of ptdm and igt after transplant are steroid therapy and higher tacrolimus trough level. in transplant children we recommend the regular fasting glucose and ogtt examimation, the reduction of steroid and tacrolimus in case of stable graft function. otka f- , otka-t , ett / , ett / the introduction: measurement of plasma bnp is a novel noninvasive approach in the assessment of cardiovascular status. in our study we investigated the role of bnp in the monitoring of cardiovascular status of children with crf or renal transplant (ntx). methods: we examined children with crf (n= , boys/ girls, age: , year ( , - )) or ntx (n= , boys/ girls, age: , ) ). patients underwent echocardiographic investigations (ivs, lvedd, lvesd, pw and fs) and their bnp levels were measured (age matched normal values were used). other cardiovascular risk factors, such as hgb, htk, ca, p, creatinine and blood pressure were also evaluated. a correlation between bnp and echocardiographic results was calculated. results: the values of lvesd, fs and bnp levels of renal transplant patients were significantly better than those of crf patients (p< , ). the other parameters did not show significant differences. bnp levels were significantly higher in all age groups of crf patients as compared to the normal levels. in younger ntx patients this value was within normal limits. in older ntx patients, and in those that had their transplants a long time ago we measured higher bnp levels, which correlated significantly with graft function as well (p< , ). bnp showed a significant positive correlation with lvesd and a significant negative correlation with fs only in crf patients. the elevated bnp levels showed the worsening of cardiac function even when the echocardiographic parameters were still normal. the hgb, htk, ca, p and creatinine values were significantly better in ntx patients and showed no correlation with bnp. summary: bnp is an early, easily usable marker in diagnosing and following decreased cardiac function of both crf patients and after ntx. otka f- , otka-t , ett / , ett / c. garcia , v. bittencourt , s. vitola , e. didone , e. guerra , f. pires , a. tumelero , d. malheiros , v. garcia department of pediatric nephrology, porto alegre, brazil complexo hospitalar santa casa, department of nephrology and kidney transplantation, porto alegre, brazil complexo hospitalar santa casa, department of surgery, porto alegre, brazil the objective is relate the results of consecutive kidney transplants carried out in children in a single center. patients and methods: analysis of kidney transplants performed in patients less than years old, carried out from may to december . results: kidney transplants were performed. % of the patients were female, % were caucasian and % were african-brazilian. the mean age at the transplant was . ± . years. the most frequent etiology of renal failure was vesico-ureteral reflux/obstructive uropathy ( %), followed by glomerulopathy ( %). the donor was deceased in % and living related in % (parents %). the initial immunosuppression was cya+aza+pred in . %, cya+mmf+pred in , %, tac+aza+pred in . %, tac+mf+pred in . %, tac + mf without pred in . %. sirolimus was employed initially in cases. induction with okt /atg occurred in patients and received anti-il receptor antibody. the graft losses during years of follow-up were secondary to chronic allograft nephropathy in ( %), vascular thrombosis in ( . %), acute rejection in ( . %), recurrence of original disease in ( . %). there were transplants in patients with focal segmental glomerulosclerosis, ( . ) had a recurrence after transplant. eight were treated with plasmapheresis and % obtained a total remission. the survival of graft in the first, fifth and tenth year was: %, % and % respectively. the graft survival in the th year according the immunosuppression was % using azathioprin and prednisone, % with cya/aza or mmf and % with tac/aza or mmf. the patient survival in the first, fifth and tenth year was: %, % and % respectively, infection was the main cause of death. j. feber , p. geier , b. chaudry , h. wong , g. filler children's hospital of eastern ontario, division of pediatric nephrology, ottawa, canada london health science center, departments of pediatrics, london, ontario, canada successful pediatric renal transplantation (tx) should fully correct the metabolic abnormalities of end-stage renal failure. however, ckd may persist because of only half of the normal nephron endowment and other factors (ischemia, nephrotoxocity etc). height, bmi and blood pressure (bp) z-scores, cystatin c-gfr, hemoglobin (hb), serum pth, hco , cholesterol, mycophenolic acid (mpa) and sirolimus (sir) levels were analyzed retrospectively in tx recipients ( males, age . ± . years) at months post tx (t ) and at . ± . years (median . ) post tx (t ). data are expressed as mean±sd. height z-scores remained significantly lower than controls (t : - . ± . ; t : - . ± . , ns), growth failure occurred in % of pts at t and % of pts at t . bp z-score did not change from t to t , but hypertension was diagnosed in % pts at t and % pts at t . gfr (ml/min/ . m ) was . ± . at t and . ± . at t (ns), mean decline of gfr was . ± . %/year. hb z-score remained below normal at - . ± . at t and - . ± . at t (ns) and anemia was diagnosed in % and % of pts at t and t respectively, despite trough levels of both mpa ( . ± . mg/ml, pts) and sir ( . ± . mg/ml, pts) that would be considered adequate. hypercholesterolemia was detected in . % pts at t and % pts at t , whereas only . % of pts at t and . % of pts at t were labeled as obese. bone disease was diagnosed in . % pts at t and . % pts at t . we observed suboptimal growth, hypertension, hypercholesterolemia, bone disease and persistent anemia in a significant proportion of tx children despite iron supplementation, adequate mpa and sir levels and good kidney function. these ckd complications require careful monitoring and intervention. a. al midani , g. koffman , j. john , s. stephen , s. suzanne , r. lord royal free hospital, transplantation, london, united kingdom great ormond street hospital for children nhs trust, transplantation, london, united kingdom objectives: to document factors predisposing towards surgical complications over years in a single pediatric renal transplant centre. methods: we retrospectively analysed consecutive renal transplants between jan and dec . patients were divided into group , without complications, and group , with complications. we compared variables previously identified as risks for surgical complications between the two groups: live/deceased donor, donor and recipient age, gender and weight, side of organ donation, cold ischaemia time, single/multiple vessels, intraperitoneal/extraperitoneal approach, anastomosis to aorta/iliac vessels, thrombosis prophylaxis (changed from heparin to aspirin in oct ). results: / ( %) were complication free; % patients developed one or more surgical complication: wound infection / ( . %), wound dehiscence ( . %), prolonged ileus ( . %), lymphocoele ( . %). patients were re-explored: ( . %) for bleeding, ( . %) for graft repositioning. we observed ( . %) cases of renal artery stenosis. overall, ( . %) graft loss occurred secondary to thrombosis, % ( / ) prior to changing our prophylaxis from heparin to aspirin ( . % on aspirin). urological complications occurred in ( . %): ureteric leaks and ureteric stenoses. the variables between group and group were as follows: under kg: % v %, less than yrs old: % v %, intraperitoneal approach: % v %, anastomosis onto the aorta: % v %, no aspirin prophylaxis: % v %, other variables were the same in both groups. conclusions: % of patients developed surgical complications. a higher rate of surgical complications was seen in recipients under , using the intraperitoneal approach onto the aorta. the introduction of aspirin prophylaxis reduced graft loss due to thrombosis from % to . %. other variables did not affect the complication rate. m. medeiros , v. sharma , r. ding , s. valverde , am. hernández , p. garcía , y. fuentes , m. suthanthiran hospital infantil de mexico federico gomez, departamento de nefrologia, mexico, mexico weill cornell medical college, immunogenetics and transplantation center, new york, ny, united states the forkhead transcription factor foxp is highly expressed in cd +cd + regulatory cells (tregs). the foxp +cd +cd + cells play a central role in immune tolerance and tgf-β is reported to induce foxp expression in vitro. whether there is an in-vivo association between foxp and tgf-β is not known. we investigated the hypothesis that there is a positive association between foxp and tgf-β in children with stable renal graft function. parental written informed consent was obtained before enrollment in all cases. children with stable renal allograft function for a minimum of months were studied. a complete clinical examination was performed; peripheral mononuclear cells were collected for measurement of transcripts for foxp , tgf-β , tgf-β , and s rrna (house keeping gene) using by real time quantitative pcr assay. correlation between transcript levels was performed using pearson r. results: pediatric recipients of renal allografts were studied. tgf-β and foxp were highly expressed in peripheral blood mononuclear cells, and there was a highly significant and positive correlation between levels of mrna for foxp and tgf-β (r= . , p< . )), whereas no significant correlation was found between tgf-β vs. tgf-β (and tgf-β vs. foxp ). conclusion: foxp expression in vivo is strongly correlated with tgf-β expression in peripheral mononuclear cells of stable renal transplant recipients. introduction: studies suggest that pre-emptive lamivudine therapy improves survival in hbv renal transplants. however, long-term outcome is not well established. method: four chinese adolescents with chronic hbv infection were transplanted. they were put on cyclosporin a, mycophenolate mofetil and prednisolone. prophylactic lamivudine was given just before transplantation and was continued afterwards. hbv status and liver enzymes were monitored serially. results: four patients were transplanted at the age of . ± . ( . - . ) yrs old. they were followed up for . ± ( - ) months and no mortality was reported. alanine transaminase (alt) was only transiently elevated in the first months post-transplant in all cases and became normal afterwards. there was no hepatitis flare and liver function was normal at the last follow-up. hbeag and hbv dna were positive in patient before transplantation and remained positive at the latest follow-up. mutation in the ymdd motif of the hbv genome was detected in the same patient and undeterminable in the other three due to low virus load. this patient remained clinically stable with normal liver function except there was a rise of viral load from baseline. all grafts were functioning and there was one late acute cellular rejection which responded to treatment and there was no hepatitis flare. latest mean serum creatinine was ± ( - ) umol/l. conclusion: ymdd mutation and resistance to lamivudine treatment may happen but appear to have little clinical significance. our long-term results showed that renal transplant seems feasible and safe in this population up to yrs follow-up. there are no studies in mexican children (mx) . the aim of the study was to determine tacrolimus pharmacokinetics (pk) in mexican renal transplant children and compare it wih reported pk in aa and ca. methods: a seven point pharmacokinetic profile ( , . , , , , and h) was performed in ten children receiving tacrolimus as part of the immunosuppressive therapy, mean age was . ± . years, mean post transplant time . ± months. c and cmax were obtained directly from experimental points, the auc and t / was obtained with a non-compartmental model using winnonlin version . . results: in cyclosporine (csa) is widely used for immunsuppression in transplant recipients and for treatment of srns. however, patients can develop csa associated cutaneous side effects, e. g. hypertrichosis, skin cancer, and viral warts due to human papillomavirus infection. here we report on a -yearold boy suffering from a microdeletion syndrome ( q-) and srns (histology: minimal change nephropathy) starting at the age of years. since the initial combination therapy with corticosteroids and cyclophosphamide was associated with severe side effects (sepsis, leukopenia) and did not lead to sustained remission csa therapy was initiated. csa-treatment resulted in rapid clinical remission. however, after months the patient developed viral warts (hands, trunk and head), although csa trough levels were kept below μg/l. therefore, immunosuppression was switched to mmf ( x mg/day) resulting in sustained remission of srns and rapid disappearance of viral warts within months. conclusion: conversion to mmf may be a usefull treatment strategy in srns showing csa associated side effects like viral warts. - . were live related (lrd) and the remaining cadaveric (cad). were pre-emptive(pet). all received basiliximab induction hrs prior to surgery. in addition, induction immunosuppression consisted of tacrolimus and methylprednisolone. in all but patients, basiliximab was re-administered at day . patients, aged and yrs (one cad and other lrd respectively) developed acute noncardiogenic pulmonary oedema - hrs after transplantation. both children had renal dysplasia as primary cause of renal failure. both required delayed ventilation and were ventilated for to days respectively. there was a rapid rise in c reactive protein in both patients. both grafts had primary function, but the cad transplant subsequently developed acute tubular necrosis, and was eventually lost within weeks due to thrombotic micro angiopathy and severe acute antibody mediated rejection despite immunosuppression with sirolimus, mycophenylate, steroids and plasma exchange therapy. conclusion: we report a rare but serious side effect of basiliximab. to our knowledge, this is the first report of basiliximab induced non-cardiogenic oedema so early post transplantation and in such young children. early recognition and aggressive appropriate supportive therapy is vital for patient and where possible, graft survival. ( ); cmv seroconversion ( ); seizures ( ); hypertension ( ), uti ( ), adverse reaction to basiliximab ( ), delayed graft function ( ), acute rejections ( ), chronic allograft nephropathy ( ). patients had well matched kidneys ( or less mismatches), were poorly matched. grafts were lost from latter group, both were cad, had acute tubulointerstitial nephritis and tacrolimus toxicity and the other thrombotic microangiopathy and eventually acute antibody mediated rejection. chronic allograft nephropathy (can) is a complex phenomenon caused by underlying kidney disease and superimposed by environmental and genetic factors. we investigated the association of polymorphism in the genee nos with the can. nitricoxide is synthesized from l-arginine in vascular endothelial cells by nitric oxide synthase. endothelial nitric oxide plays an important role in endothelial dysfunction and involved in the inflammation. the gene encoding enos maps to chromosome q q . . a missense variant of the enos gene in exon shows a transversion of g to t at nucleotide position (g t) that results in a replacement of glu by asp at amino acid residue (glu asp). the aim was to investigate the association between can and g t polymorphism of the endothelial nitric oxide synthase gene. the g t mutation at exon of the endothelial nitric oxide synthase gene, enos gene polymorphism, was analyzed in turkish children with renal transplantation. the g t polymorphism of the endothelial nitric oxide synthase gene was determined by polymerase chain reaction and restriction fragment length polymorphism. were grouped according to stages of chronic kidney disease (ckd) as estimated by the calculated glomerular filtration rate (gfr, schwartz formula). measurements of structural and functional surrogates for cardiovascular disease (cvd) included intima-media thickness (imt) of the common carotid artery (cca), pulse wave velocity (pwv) and augmentation index (aix). aix and pwv reflect the degree of arterial stiffness and were calculated from pulse wave recordings at the arteria carotis and arteria femoralis (sphygmocor device). results: patients and healthy control subjects had a mean age of years. imt was not significantly different in patients and controls. significant differences were found in the aix, which was increased by %: the mean aix was in healthy subjects was - , % and in transplanted subjects - , %. pwv was increased by % ( , m/s vs. , m/s). both aix and pwv increased in parallel with the degree of renal impairment (stages of ckd). table . discussion: weight gain post transplant is multifactorial, like cultural, psychological and associated to steroids. weight gain was observed in general, patients with overweight or in risk of overweight didn't loose weight postransplant even they were aware of the consequences. introduction: tacrolimus is metabolized by cytochrome p a and has a narrow therapeutic range. we report a kinetic interaction between tacrolimus and amlodipine, a potent cytochrome p inhibitor, resulting in anuric acute renal failure. case report: a -year-old male renal transplant recipient received amlodipine, a calcium channel blocker as antihypertensive treatment while he was on tacrolimus ( . mg/kg per day). he presented first with diarrhea and developed subsequently, dizziness and fatigue, related to acute anuric renal failure, requiring hemodialysis for days. tacrolimus trough levels were in the desired therapeutic range ( - ng/ml) until recently. three days after introduction of amlodipine, tacrolimus trough levels increased to a toxic level of . ng/ml. after discontinuation of amlodipine, tacrolimus levels returned to the normal range in seven days and renal function recovered progressively. no polymorphisms in the expression of cyp a and p-glycoprotein were detected. discussion: tacrolimus is known to be a substrate of p-glycoprotein, responsible for drug secretion into the intestinal lumen and metabolized by enterocytic cyp a. amlodipine is a competitive inhibitor of cyp a. as no abnormalities of cyp a and p-glycoprotein were found, we suspect that drug interaction due to competitive inhibition of tacrolimus metabolism by amlodipine was responsible for these toxic effects. concomitant diarrhea might have played an additional role for increased tacrolimus serum levels, presumably in relation to diarrhea associated dysfunction of enterocytic cyp a and p-glycoprotein. conclusions: amlodipine and diarrhea increase tacrolimus blood concentration by inhibiting its metabolism. amlodipine should not be used in patients on tacrolimus. careful monitoring of tacrolimus blood levels is recommended in case of concomitant diarrhea. urinary tract infection (uti) remains a significant cause of infectious complications in renal transplant recipients. the aim of the study was to determine the frequency of uti following renal transplantation in our center. the records of patients (f/m: / ) who underwent renal transplantation were evaluated retrospectively. among them patients (f/m: / ) were found to have at least one episode of uti during follow-up. the records were examined for the age, sex, primary disease, and duration of chronic renal failure, donors, posttransplant follow-up and recurrence of uti. biochemical analysis of blood for renal functions, complete blood count, creactive protein and sonographic examination of patients were also recorded and results were compared with renal transplanted patient who did not develop any episode of uti (group ). the mean age of the group was . ± . years, while it was . ± years in the group . mean duration of post-transplant follow-up was . ± . years for group and . ± . for group . four patients ( . %) in group and patients ( . %) in group had vesicoureteral reflux (p> . ). five patients had single uti while patients had more than one uti. though we did not find any difference between girls and boys in terms of presence of vesicoureteral reflux, frequency of uti in girls was found to be significantly higher than in boys (p= . ). ultrasonographic examination of patients during uti in group revealed pyelonephritis in and hydronephrosis in patients. the most frequent microorganism causing uti was e. coli. age, donor source and etiology of chronic renal failure did not influence the incidence of urinary tract infection. our data suggests that urinary tract infection remains a frequent but mostly benign complication in the pediatric transplant population, especially in female gender. the growing population of transplanted patients requires the consideration of the potential side effects of the different treatment regimens. experience of the last decade with calcineurin and nucleoside reverse transcriptase inhibitors revealed important renal side effects. we describe a years old girl who was known to have liver failure related to wilson's disease (wd). she had orthotopic liver transplantation from her mother years ago and treatment with tacrolimus and mmf was initiated. although she was known to have proximal renal tubular acidosis secondary to wd, renal tubular functions were found to be normal within the three months of transplantation. two years after the transplantation lamivudine was initiated because of de novo hepatitis b infection in transplanted liver. a couple of months later she developed renal fanconi syndrome with metabolic acidosis, hypophosphatemia, glycosuria and aminoaciduria. she needed high doses of sodium bicarbonate and phosphate supplementation. tacrolimus was suspected to be the cause of late post transplant renal acidosis and was replaced by sirolimus. however, months later, at the th month of lamivudine treatment, she was hospitalized because of metabolic acidosis, mild hyperglycemia and inability to walk. electromyographic examination revealed myopathic changes while liver biopsy was normal with a normal tissue copper level. renal biopsy showed findings of karyomegalic nephropathy which could be the result of the action of antimitotic agents. we suspect that our patient's tubular dysfunction, myopathy and hyperglycemia may have resulted from mitochondrial dysfunction which is triggered by tacrolimus and augmented by lamivudine. however, randomized and prospective studies with large groups of patients are needed for definite results about mithocondrial side effects of these drugs. recombinant factor viia (rfviia, novoseven) is a new hemostatic agent that was initially indicated in hemophiliac patients. recently it has been used successfully for the treatment of bleeding in patients with thrombocytopenia, and acquired and congenital platelet dysfunction. epstein syndrome, also known as alport-like syndrome, is a rare autosomal dominant disease characterized by proteinuria, chronic renal failure, hearing loss, and thrombocytopenia with giant platelets. our group previously reported functional alterations of giant platelets of boy with epstein syndrome, who rapidly progressed to end stage renal disease during adolescence. the first nonheartbeating kidney transplantation at age was failed because of the severe postoperativebleeding irresponsible for traditional therapy (packed red cells, platelets, and fresh frozen plasma), result in immediate graft failure and the need for transplant nephrectomy. the second kidney transplantation was years later, after a single intravenous bolus injection μg/kg body weight rfviia, which was repeated one and hours after the surgery. rfviia successfully controlled the bleeding in the peri-and postoperative phase and no side effect and thrombotic complication occurred and his graft function is still stable after years. recombinant factor viia may have a potential role in the treatment of phenotypic bleeding associated with chronic kidney disease. cyclosporin a (csa) and mycophenolic acid (mpa) have a wide interindividual variability in their pharmacokinetics (pk). among others, intestinal p-glycoprotein (p-gp) expression and cyp a activity have been held to be responsible for that variability. in adult kidney transplant (rtx) patients, an influence of these gene polymorphisms has not been shown; however, there are no data in pediatric patients. we reasoned that such an influence might be masked in adults by confounding environmental factors accumulating over the decades of life. we therefore investigated a possible influence of gene polymorphisms of p-gp and cyp a on defined dose-adjusted pk-parameters in children with rtx (age . ; range, . - . yrs). pk parameters (auc, c ) were assessed , , , and weeks after rtx. real-time, rapid-cycle pcr methods were used for genotyping. the allele frequencies for the mdr c t allele (expression and in vivo activity of p-gp) of % and for the cyp a -v allele of % were comparable to those reported for caucasian populations. dose-adjusted pk parameters of csa and mpa were not significantly different in patients with and without the cyp a -v allele or patients with different mdr c t genotypes. along with that finding, neither of the polymorphisms investigated into was associated with renal function or the incidence of acute rejection episodes. we studied how the il- r β-chain becomes enriched in lipid rafts of activated human t cells, isolated by ficoll gradient and sheep red cell rosetting, and how its tyrosine phosphorylation, which requires its heterodimerization with the common cytokine r β-chain (βc), occurs there. imunoblots (ibs) of sucrose gradient fractions of cell lysates obtained during a -hour activation with phytohemeagglutinine (pha) showed the gradual, largely selective, translocation of the β-chain into rafts. as dimerization or lipid modification can be mechanisms underlying raft enrichment, we assessed lysates of pha-activated cells in ibs under non-reducing versus reducing and crosslinking conditions but did not see evidence of β-chain dimerization. however, exposure to cycloheximide to interfere with post-translational acylation, or to the palmitic acid analogue -bromohexadecanoic acid substantially diminished raft enrichment of the il- r β-chain. we next performed ibs of il- r β-chainand βc-immunoprecipitates from raft and non-raft fractions of activated t cells before and after il- treatment. we found that il- exposure triggers the translocation of small amounts of βc, accompanied by il- r β-chains, into rafts, resulting in its heterodimerization with the il- r β-chain and their tyrosine phosphorylation. all of these processes were attenuated in the presence of the il- r β-chain-blocking antibody daclizumab. we conclude that the raft enrichment of the il- r β-chain requires palmitoylation and provides the focal point for the formation of the highaffinity il- r via il- r β-chain-mediated "chaperoning" of few βcs into these domains, establishing novel raft-dependent mechanisms underlying cytokine r specificity and selectivity in human t cells. iga nephropathy (igan) is an immunecomplex disease resulting from a defect in mucosal iga response. food antigens have been implicated in the pathogenesis. gut permeability to antigenic substances is immature at birth and its maturation is delayed by early administration of antigenic foods while breast feeding accelerates this process. we aimed to evaluate if exposure to antigenic foods in early life is associated with a predisposition for igan in childhood. three groups including children with igan (group , n= ), primary non-iga glomerulopathies (group . n= ) and healthy controls (group , n= ) were formed. their parents filled a questionnaire regarding the age at diagnosis, gestation time, birth weight, feeding by breast milk, formula, cow's milk and complementary foods. all groups were similar for age, sex, gestation period, birth weight and the rate and duration of breast feeding. in addition, the rate of formula feeding was also similar in all groups. however, cow's milk consumption rate was higher in group and than in group . introduction of formula was earlier in groups and than in group . in addition, the children in group were younger than the other groups at the onset of feeding by cow's milk and weaning. roc curves predicted . , . and . months as the best cut-off age values for formula feeding, cow's milk feeding and weaning for predicting the presence of igan, respectively. ors for igan with respect to these cutoff levels were ( % ci: - ), . ( % ci: . - . ) and . ( % ci: . - . ), respectively. the results of this preliminary study indicate that early introduction of antigenic foods might increase the risk of future primary igan. results: they were males and females, with a male: female ratio of : . their ages ranged from months to years (mean . years), with a peak age of - years. the common presenting complaints were generalised oedema ( %); oliguria ( %) and hypertension ( % we report nine patients (three males) with mesangiocapillary glomerulonephritis (mcgn) from a single paediatric nephrology centre. the average age at presentation was . years (range . to . ). all had nephrotic syndrome. seven had mcgn type and two had mcgn type ii. six of seven tested had positive c nephritic factor. three patients responded well to steroids and ace inhibitors and received no further therapy. five had a good response initially but relapsed when steroids were tapered and one patient had a poor response to steroids. these six patients received calcineurin inhibitor (ci) therapy. four responded well with resolution of proteinuria. one patient relapsed when tacrolimus was withdrawn after months of therapy but proteinuria resolved after re-introduction of therapy. two patients had a poor response to ci therapy. one remains stable but with heavy proteinuria. the second patient (with mcgn type ii) initially had a complete remission of proteinuria on steroids but relapsed months after presentation while on prednisolone mg on alternate days. repeat biopsy showed % crescents. treatment with pulsed intravenous steroids, cyclosporin and mycophenolate mofetil was ineffective and she progressed rapidly to end stage renal failure. we conclude that ci treatment might be useful in mesangiocapillary glomerulonephritis. prospective, randomised controled trials are required to determine their place in the management of this disease. iga nephropathy (igan) is caused by a primary defect in mucosal iga response leading to increased antigenic stimuli reaching to bone marrow. enteric flora is important for mucosal and systemic immunity, and probiotics regulate specific and innate immunity by maintaining microbial balance in the gut. saccharomyces boulardii (s.boulardii), a probiotic, increases intestinal siga production, protects enteric infections and also prevents atopic and immunoinflammatory diseases. we aimed to evaluate the effect of s.boulardii on experimental igan, induced by oral polio virus vaccine (opv) administration to the mice. four groups of male balb/c mice (n= for each) were formed. groups i and ii were immunized enterally by opv at the onset, nd and th weeks. group ii was also given s.boulardii in drinking water throughout the study. group iii was given only s.boulardii, while group iv received no treatment. two weeks after the last opv dose, all the animals were sacrificed to obtain their kidneys for histopathological evaluation and all four groups were compared with respect to the severity of histopathological changes. while there was mild to moderate mesengial proliferation and widening, tubular atrophy, interstitial inflammation and fibrosis in group i, no remarkable histological changes in the other groups were noted. immunofluorescence microscopy revealed universal deposition of iga and some c in group i, while there was no iga or c deposition in the other groups. electronmicroscopy revealed mesengial proliferation along with matrix expansion, focal basement membrane thickening and electron-dense deposits in the mesengial area in only opv group and the other groups were normal. in conclusion, enteral s.boulardii administration prevented experimental igan development in mice. the aim was to assess the correlation of renal histopathological findings with clinical diagnosis in order to recognize the pattern of kidney diseases in our pediatric population. methods: a total of renal biopsies performed on children who presented to the surgical kidney hospital in damascus during a period of years were retrospectively reviewed. results: nephrotic syndrome alone accounted for % of all cases, followed by hematuria in %, mild to moderate renal impairment including allograft dysfunction in %, nephritic syndrome in %, and hsp in %. the most common histologic lesion was mcd in ( %). fsgs was the second most common lesion ( %) followed by mesangial gn ( %), mpgn ( %), post-infectious gn ( %), iga nephropathy ( %), membranous gn ( %), cns of finnish type ( %), alport syndrome ( %), interstitial nephritis ( %), nephronophthisis ( %), hsp ( %), acute rejection ( %), chronic rejection ( %), nephrocalcinosis ( %), crescentic gn of undetermined origin ( %), and lastly, % were completely within normal limits. familial and inherited diseases were encountered in %. histopathologic diagnosis was mostly useful in nephrotic cases. while in hematuria cases, the usefulness of the histologic findings in terms of therapeutic and/or prognostic point of view was definitely less. one of the reason for that in our series is perhaps because we still do not have facilities to perform electron microscopic evaluation of the renal tissue. however, controversy about the usefulness of renal biopsy in such cases is still there. conclusion: this study provides an important data on the pattern of pediatric renal diseases in our center and highlights the usefulness histologic findings in guiding the therapeutic plan especially for nephrotic children. aim: the aim of this study was to determine the efficacy of tacrolimus in the management of sr fsgs in children. study design: this was a prospective study of children with sr fsgs treated with tacrolimus ( . - . mg/kg per day in divided doses over hours adjusted to a trough level between - ng/ml) for months in combination with low dose steroids. other therapies included angiotensin converting enzyme inhibitors, folic acid, multivitamins and lipid lowering agents. results: the mean age at study entry was . years (range . - . ). the mean duration of nephrotic syndrome before initiation of tacrolimus therapy was . years (range . - . ). at the end of the treatment period ( %) children were in complete remission, ( %) children were in partial remission and ( %) failed to respond. the average period of follow-up following cessation of tacrolimus treatment was . months (range . - . ). at last hospital follow-up ( %) of children were in complete remission, ( %) in partial remission and ( %) in relapse. children demised from dialysis related complications following cessation of tacrolimus treatment. adverse events included sepsis ( ), nausea ( ) diarrhea ( ), anaemia ( ) and worsening of hypertension ( ) . conclusion: tacrolimus is a safe and effective treatment for sr fsgs. however, like cyclosporine some children tend to relapse following cessation of treatment. it has been rarely reported in association with graves-disease. now we present a previously healthy -year-old japanese girl who had proteinuria due to stage i mn and graves disease. patient: she was found to have + proteinuria and a goiter at her school medical examination simultaneously. serum free thyroxine was . ng/dl (normal range . ~ . ), thyroid-stimulating hormone (tsh) less than . microu/ml ( . ~ . ), anti-microsomal antibody t (~ ), anti-thyroglobulin antibody t (~ ), and tsh-receptor antibody % (~ ) consistent with graves' disease. the electron microscopy finding of her renal biopsy specimen showed the presence of electoron-dense deposits located in the subepitherial and intramembranous spaces. with immunofluorescence microscopy, the bright granular staining of igg along the gromerular capillary wall was found. these findings were characteristic of mn. objectives of study: to investigate whether graves disease caused mn in this patient. methods: we examined the presence of thyroid microsome and thyrogrobulin in glomeruli by immunofluorescence study using anti-thyroid microsomal antibody and anti-thyrogrobulin antibody. result: glomerular granular staining of thyroid microsomal antigens was demonstrated corresponding to igg granular deposits, but that of thyrogulobulin was absent. conclusion: mn in this patient is presumed to be caused by immunecomplexes mediated by thyroid microsomal antigens. objective: to explore the role of oxidative stress reaction on the injury of glomerular podocyte slit diaphragm molecular barrier. methods: thirty-two male spraque-dawley (sd) rats were randomly divided into control, low dose ( . mg/kg), nephrotic ( . mg/kg), overdose ( . mg/kg) groups by the dosage of adriamycin (adr) injection.the levels of malondialdehyde (mda) glutathione peroxidase (gsh-px), hydroxy radical ( . oh) and superoxide dismutase (sod) in renal cortex were measured; the expression of podocin was measured with immunohistochemistry. results: ( ) compared with control group, the levels of mda in renal cortex and -hour urinary protein were increased, the levels of sod in renal cortex was decreased in adr-treated groups, especially in nephrotic group (p< . ). ( ) in control group, podocin staining was a sable linearlike pattern along the capillary loops of glomerulus; in nephrotic group, podocin staining was a light tan discontinuous punctiform or short linear-like pattern along the capillary loops of glomerulus. compared with control group, the score of podocin immunohistochemical staining was decreased in adr groups, especially in nephrotic group (p< . ). ( ) there were some significant negative correlations between the score of podocin immunohistochemical staining and the levels of mda in renal cortex. there were some significant positive correlations between the score of podocin immunohistochemical staining and the levels of sod in renal cortex. conclusion: ( ) there was close relationship between podocin and the development of proteinuria. ( ) there were significant correlations between the reduction of podocin in glomerular podocyte slit diaphragm and oxidative stress reaction, especially lipid peroxidation. lupus nephritis (ln) remains an important problem in patients with sle. to evaluate the clinical course, histopathology and the efficacy and safety of high-dose pulse cyclophosphamide (ctx) in children with ln. retrospectively, children with ln were studied; all patients underwent renal biopsy and were followed up for at least years. the clinical and serologic data at the time of renal biopsy were recorded. five of them were excluded because of short period of follow-up or defective laboratory data. based on renal biopsy (who classification for ln), patients were treated with the following regimens: one patient (class i) with low-dose prednisolone (pred), (class ii, iii) with high-dose of pred, (class iv) with high-dose pred and received intermittent intravenous (iv) ctx pulses (monthly for months, then every months) followed by mycophenolate mofetil (mmf) as maintenance therapy. there were girls and boys. the mean age at the time of diagnosis of sle was . years. eighteen patients were more than years old. sixty percent of the patients were presented as nephritic-nephrotic syndrome. there was with class i, with class ii, with class iii, with class iv and none with class v based on biopsy. eighty-five percent of cases went in remission, one was hemodialytic and died due to renal failure and cns involvement. among cases with class iv, responded to pred and iv ctx pulses. there was no evidence of side effects. it seems that iv pulse ctx does induce remission of clinical and renal disease in the majority of early diagnosed children with severe ln. furthermore, it appears that mmf is an appropriate drug for maintenance therapy. however, this study was based on a small number of subjects. further studies to confirm the long-term efficacy and safety of ctx pulse therapy on larger numbers of patients are needed. forming group i were compared with children on short course steroid therapy (iskdc regime) (group ii). children were examined for steroid side-effects and underwent blood tests, ophthalmologic evaluation and radiological examination. results: though remission was achieved in < weeks by % in group ii against % in group i, the total dose received ( - mg/kg) was lower in group i ( % vs %). forty-six % had - relapses, % had - relapses and % had - relapses. the proportion of children having > relapses was much higher in group ii ( % versus %). mean relapse/patient/ year was . (sd . ) in group i against . (sd . ) in group ii. delayed bone age ( %), radiological evidence of osteoporosis ( %). cushingoid facies ( %), posterior subcapsular cataract ( %), decreased growth velocity ( %) and hypertension ( %) were the side effects and were almost equally distributed in the two groups. more patients from group ii received a higher cumulative dose/ kg/year of > mg ( %versus % in group i) and these had higher risk for hypertension, delayed bone age and osteoporosis. conclusion: alternate day, prolonged therapy (soyka regime) compared to short course, daily therapy (iskdc) resulted in lower cumulative dose to the patient. acute side effects and severity of infections were less. mean relapse/patient/year were lower. group ii patients receiving higher cumulative dose had osteoporosis and delayed bone age. objetive: to assess urinary protein excretion decrease in patients with primary srns treated with ec-mps methods: cohort of patients, mean age: years with primary srns. inclusion criteria: primary sncr with of focal segmental glomerulosclerosis (fsgs). exclusion criteria: glomerular filtration rate (gfr) % than baseline level, leukopenia, absence of decrease of proteinuria excretion by month of treatment with ec-mps. mean time after the initial diagnosis of ns until the introduction of ec-mps was calculated in patients who decreased urinary protein excretion below nephrotic range and in non-responsive patients and the glomerular damage, interstitial fibrosis and tubular atrophy were classified as: absent, mild, moderate, severe ( to , risk grade> ). ec-mps dosing: - mg/m /day. complete response was considered a reduction in the urinary protein excretion lower than or equal to mg/m /hour; partial response: urinary protein excretion ranging from to mg/m /hour and absence of response: urinary protein excretion in the nephrotic range. laboratory monthly assessments: serum creatinine, urea, hemoglobin and blood cell counts, lipids, serum proteins, amilase, hours urinary protein excretion. . no significant differences in the frequency of both alleles were observed among patients with different grades of hypertension or proteinuria. in conclusion, drb * , and possibly alleles confer susceptibility to psagn. however the severity of the disease is not determined by these two alleles. methods: fifty children who diagnosed as biopsy-proven fsgs were studied retrospectively by medical records. response to treatment and pathologic slides, we compared normal renal function group (n= ) and decreased renal function group (n= ), assessed the factors affecting renal survival and progression to renal failure. results: the mean age at onset was / years, gender ratio m: f was . : and the mean duration of follow-up was / years. the overall renal survival rate was % at years, % at years. five-year survival rate was % in normal renal function group, but % in decreased renal functin group. between the two groups, there were no significant differences in age at onset, gender ratio, amount of proteinuria, incidence of hematuria, hypertension and mesangial hypercellularity. decreased renal function group showed higher serum creatinine level, poor response to treatment, higher percent of glomeruli with sclerosis, moderate to severe tubulointerstitial change and vascular change (p< . ). the prognostic factors of renal survival rate were same as above (p< . ). there were no significant factor has shown relations with the progression rate to renal failure. conclusion: we reviewed the factors affecting long-term outcome of fsgs. serum creatinine level, steroid responsiveness and the degree of glomerulosclerosis were significant prognostic factors. but, age at onset, gender, amount of proteinuria, incidence of hematuria and hypertension were not considered as a prognostic factor. a background: several studies have suggested that cyclosporine (csa) and methylprednisolone pulse therapy (mpt) may be effective for idiopathic steroid-resistant nephrotic syndrome (isrns) in children; however, the optimal regimen has yet to be established. the present study evaluated the efficacy and safety of years' treatment with csa (neoral) combined with mpt and prednisolone (psl) in such patients. methods: in this prospective study, children with biopsy-proved isrns were enrolled. all patients received csa and psl ( mg/kg every other day). patients who had focal segmental glomerular sclerosis (fsgs) additionally received mpt (methylprednisolone at a dose of mg/kg per day for consecutive days at weeks , , , , and ) . the dose of csa was adjusted to maintain a trough level from to ng/ml for the initial months, followed by to ng/ml for months to , and to ng/ml for months to . results: twenty-six patients were enrolled. their mean age was . ± . years. two-year follow-up was completed in patients. histological examination at study entry revealed minimal changes in patients, diffuse mesangial proliferation in , and fsgs in . at the end of the therapy, patients had complete remission, including who had occasional relapses of steroid-sensitive nephrotic syndrome, and had partial remission; the remission rate was . %. nephrosis persisted in patient. disease progressed to end stage renal failure in patient. serial renal biopsy at the end of the study showed mild signs of csa-related renal toxicity, including tubulointerstitial fibrosis in ( . %) of patients. conclusion: combination therapy with csa, mpt, and psl for years was clearly effective and produced a high remission rate without serious csa-related renal toxicity in children with isrns, in contrast to previous reports. objectives: patients with hemolytic uremic syndrome who do not require dialysis in acute stage usually have a good prognosis. however the spectrum of renal compromise is wide. we believed non-anuric patients with higher creatinine values in acute stage could have different evolution when compared to patients with lower values. aims: ) to analyze the outcome after a year and ) to determine if peak serum creatinine values in acute stage would be a prognostic marker. methods: patients, aged . months at hemolytic uremic syndrome, were analyzed. they were classified into groups: group i, complete recovery; group ii had subgroups: iia, microalbuminuria, and iib, proteinuria and/or high blood pressure, both with normal renal function; group iii, chronic renal failure; and group iv, end stage renal disease. peak creatinine value was definided as the highest value of at least determinations in acute stage. these data were available in patients and they were divided in those with creatinine equal to or higher than , mg/dl ( patients) and those with lower values ( patients). the relationship between creatinine and final outcome was analyzed. we applied fisher's test. results: after a mean follow-up of years, patients were in group i, in group iia, in group iib ( with hypertension, with proteinuria and with both) and in group iii. eight out of patients ( %) with creatinine equal to or higher than . mg/dl in acute stage and out of ( . %) with lower values were in groups iib and iii in the last visit (p= . ). conclusions: ) after years, % developed proteinuria, high blood pressure or chronic renal failure and % microalbuminuria. ) peak creatinine values in acute stage were a prognostic indicator. objective of study: the aim of this study was to assess the serum concentration of hs-crp in children with nephrotic syndrome (ns) treated with prednisone and cyclosporine a (cya). methods: patients were divided into groups: i - ns children ( - years) in relapse, examined twice: a -before treatment and b -after proteinuria regression (a - week course of prednisone therapy), ii - children with steroid-dependent or steroid-resistant ns, treated with cya, also examined twice: d -before treatment with cya, e - months after therapy. control group (c) consisted of healthy children. serum hs-crp level was determined using a nephelometric method with behring nephelometer analyzer, dade behring. results: it was shown that median hs-crp concentration was the highest in children with relapsing steroid-sensitive ns before treatment (ia). after proteinuria regression (ib), the hs-crp level decreased and did not differ from healthy controls (c) (p> . ). in group ii, before cya administration (iid) the level of hs-crp was normal, but increased after months of treatment (iie) up to a level six times higher that of the control group (p< , ). conclusions: in children with steroid-sensitive nephrotic syndrome in relapse, the serum hs-crp level is increased but returns to normal values after a - week glucocorticoid treatment course. in children chronically treated with cya due to ns, serum hs-crp level increases significantly during the therapy. slit diaphragm connecting adjacent foot processes of podocyte is the final barrier of glomerular capillary wall to prevent proteinuria. both podocytes and neuronal cells are terminally differential cells and they share many common features. nurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation, and they have been understood to be specifically expressed in neuronal tissue. we found that neurexins are expressed not only in neuronal cells but also in several organs including kidney. our immunofluorescence study shows that neurexins are restrictedly expressed in glomeruli in kidney. dual-labeling immunofluorescence studies show that neurexins are localized close to a cd ap. we also detected some portions of neurexin staining are coincident with that of rab a, a synaptic vesicle associated molecule. we found that a single splice variant of neurexin- is expressed in glomeruli. the staining intensity of neurexin in the glomeruli clearly reduced and their staining pattern shift to more discontinuous patchy pattern in puromycin aminonucleoside nephropathy and anti-nephrin antibody induced nephropathy. the alteration of neurexin in these models was detected more clearly and rapidly than nephrin. we confirmed that neurexin is expressed in podocyte also in human kidney section. these observations suggest that neurexin is involved in the development of proteinuria and that neurexin could be an early diagnostic marker of podocyte injury. to further elucidate the clinical relevance of t-cell abnormality in minimal change nephrotic syndrome (mcns), and to predict the consequences of mcns, we studied t-cell receptor (tcr) diversity by analizing cdr size distribution and the frequency of v repertoire usage. thirty-six pediatric patients with mcns were enrolled. eighteen were frequent relapsers and/or steroiddependent (fr/sd) and were non-frequent relapsers (nfr). the study was performed to analyze serial changes of tcr v repertoires in the two groups of patients. frequencies of v repertoire usage were determined by flowcytometry, and tcr cdr length distribution was analyzed by genescan. in nfr patients, abnormalities in the distribution of v repertoires were few in both cd + and cd + t cells. in fr/sd patients the patterns were normal in cd + t cells, while selected v repertoires were significantly increased in cd + t cells in some patients. furthermore, tcr diversity was significantly reduced in both cd + and cd + t cells in fr/sd patients as shown by marked skewing of cdr size distributions. it is noteworthy that in some fr/sd patients the initially abnormal tcr diversity improved as the clinical symptoms improved such that they became nfr over the years. analysis of tcr diversity may delineate the subgroup of patients with fr/sd and provide a rationale for early intervention with immunosuppressive therapy for these patients. background: transforming growth factor-β is known to play a role in the interaction between metabolic and homodynamic factors in mediating accumulation of extracellular matrix in the diabetic nephropathy. tgf-β gene polymorphism was associated with circulating tgf-β levels and influenced the pathogenesis of fibrotic diseases including diabetic nephropathy. in this study, we examined the relationship between tgf-β gene codon polymorphism and type diabetic nephropathy with more than -year history of disease. methods: we conducted a case-control study, which enrolled type diabetes. a total of patients with diabetic nephropathy were compared with patients without diabetic nephropathy. tgf-β codon genotyping was determined using polymerase chain reaction with sequence specific primers method. results: distribution of tgf-β codon genotype in the patients either with nephropathy or without nephropathy is confined to hardy-weinberg equilibrium. methods: nzb/w f female mice were distributed into three experimental groups (n= per group) according to age: , . and months. at specific time-point for each group, -hour urine and blood samples were collected to determine proteinuria, osmolality and creatinine levels. after sacrifice, kidneys were removed to measure chemokines and cytokines levels by elisa (pg/ mg of tissue). results: urinary flux was significantly lower at . than at and months. a significant reduction in creatinine clearance and an increase in proteinuria were detected at . and months when compared to months. no significantly changes were observed in serum and urinary osmolality. regarding inflammation, mcp- significantly increased at . ( . ± . ) and remained elevated at months ( . ± . ) when compared to months values ( . ± . ). kc was also higher at than at months ( background: nephrotic syndrome (ns) is related to immunological factors and renal inflammatory mechanisms. many studies showed that inflammatory mediators, especially interleukin- (il- ) and monocyte chemoattractant protein- (mcp- ), have a role in kidney injury. changes in their urine concentration were found in lupus nephritis and iga nephropathy. thus, the aims of this study were to evaluate il- and mcp- in serum and urine samples of pediatric patients with primary ns and to verify the relation between these measurements and protein excretion. methods: patients were divided according to current -hour proteinuria into two groups: lower than mg/ hours (group , n= ) and higher than mg/ hours (group , n= ). blood and -hour urine samples were collected and stored at - °c. il- and mcp- were measured by elisa standard methods. results: blood il- and mcp- did not differ between the groups. urinary il- levels (pg/mg of creatinine) were significantly elevated in patients of group when compared to group ( . ± . vs. . ± . , p< . ). although group also exhibited higher values of urinary mcp- ( . ± . pg/mg creatinine) than group ( . ± . pg/mg creatinine), they did reach statistical difference. conclusion: the inflammatory process in ns seems to be a local phenomenon, since blood levels of these chemokines were similar in all groups. moreover, our findings showed a relation between il- and the presence of proteinuria and suggested a role for this local inflammatory mediator in disease activity. the characteristics of iga nephropathy detected in school urinary screening iga nephropathy (igan) is one of common types of glomerulonephritis in children. however, progression to esrd in patients with igan is not as rare as originally thought. in korea, school urinary screening (sus) program, an useful tool to find out abnormal urinary findings, initiated in . igan was the most common histopathological change in children with isolated hematuria and/or proteinuira in sus. we studied to clarify the clinical and pathologic characteristics of iga nephropathy detected by sus in korea. we investigated patients (symptomatic group= vs sus group= ) had been diagnosed with igan following renal biopsy at the yeungnam univ. hospital between may and may . these patients were analyzed clinical nature, laboratory data and histopathologic findings (haas classification in lm) and progress, retrospectively. their mean age were . ± . and . ± . , respectively, at the time of kidney biopsy. gross hematuria and edema apt to be common in symptomatic group. there were no significant difference in serum iga level, estimated ccr, -hours protein amount, light microscopic class and electron microscopic findings between two groups. mesangial iga deposition was significantly more intense in symptomatic group with gross hematuria. in addition to iga deposition in capillary and immune dense deposition in intramembrane is significantly common in symptomatic group with nephrotic range proteinuria. however, progression to chronic renal failure was not noted in both groups during . ± . and . ± . months respectively. also there were no difference in outcome according to treatment modalities. a longer follow-up period is needed to obtain more information on progression of igan with nephritic range proteinuria by disclosing iga deposition in capillary and immunedense deposition in intramembrane. outcome of srns is uncertain and especially patients unresponsive to treatment have a high risk to develop esrd. prognosis has improved with the introduction of csa, however but long-term follow-up data are scarce and response to csa in patients with genetic forms of srns is uncertain. we report on patients with srns, that was diagnosed at a median age of . (range . - . ) years. treatment with csa was initiated on concomitant prednisone therapy, however steroids were discontinued after due course in all patients. median follow-up is . ( . - . ) years. patients had fsgs on renal histology and patients had mcns. complete remission (cr) was defined as reduction of proteinuria < mg/m /d. partial remission (pr) was defined as reduction of proteinuria of at least % and cessation of edema with serum albumine levels > g/l. results: patients ( fsgs, mcns) reached cr with csa treatment. in of these ( fsgs, mcns) csa could be tapered and discontinued successfully after a median time of ( . - . ) years. eight patients showed ( fsgs) a pr while patients ( fsgs) showed no repsonse (nr). of patients going into esrd had podocin mutations (pm). only one patient with pm showed partial remission on csa. our data indicate a positive effect of csa treatment in srns, especially in sporadic cases. in patients with cr tapering and even discontinuation of csa is possible. prognosis of srns has improved with csa treatment. objective: we planned to investigate the effects of rsv on the proteinuria and glomerular structure of rats and to explore the role of rsv in the pathogenesis of minimal change nephrotic syndrome. methods: sd rats were inoculated with , , and pfu rsv respectively, and sacrificed on days , , , and postinoculation (rsv , rsv , rsv , rsv , rsv ). renal histology was observed by light microscopy and electron microscopy. meanwhile, the proteinuria and serum parameters were measured. the rsv rna and rsv titer were determined by in situ hybridization and plaque assay respectively. immune complex deposits were detected by immunofluorescence microscopy. results: after inoculation, the urinary protein excretion was increased, especially in pfu rsv , , (p< . ). the serum albumin of pfu rsv , , and different-titer rsv decreased, but no significant differences in cholesterol, urea nitrogen and creatinine were found among all. slight hypercellularity in minority glomeruli and swelling of partial tubular epithelial cells were observed in rsv , of different-titer, whereas a relief of the above changes and no abnormalities were detected in rsv and rsv respectively under a light microscopy. extensive foot process effacement was observed in pfu rsv , , under an electron microscope. rsv rna signal and rsv titer of renal and pulmonary tissues, depending on the dose of inoculum, reached their climax on day postinoculation, especially in pfu rsv . no immune complex deposits were detected in the renal tissues. conclusion: our study reports for the first time that rsv can lead to nephropathy in rats on day - postinoculation, especially in pfu rsv-inoculated rats, which may be a new exploration of the pathogenesis of mcns. objectives of study: podocytes play an important role in maintaining the glomerular filtration barrier structure and functions, which associates with several podocyte-specific proteins. our previous study indicated the antiproteinuric effects of dexamethasone were achieved by changes the expression of certain podocyte molecules in vivo. the extracellular signal-regulated kinases regulates a wide range of cellular processes. the aim of the present study is to analyze the potential effects of dexamethasone on podocytes in vitro and to investigate its associated signal transduction pathway. methods: immortalized mouse podocyte clone was divided into three groups: the dexamethasonetreated group (dex-group), the dexamethasone with puromycin aminonucleoside-treated group (dex-pangroup), and control. in dex-group, cultured podocytes were treated with dexamethasone, while in dex-pan group, cells were treated with dexamethasone for min first, then added puromycin for different time periods. changes of the protein expression of podocyte-specific proteins and phosphorylation of erk were analyzed by western blotting analysis. result: compared with the control group, in dex-group, the expression of nephrin, podocin, cd ap, α-actinin proteins and vegf protein started to elevate at hr, while neph showed no obviously change. erk signaling pathways was activated. increased phosphorylation of erk was marked but transient, which increased from min to min, then decreased. at min the level of phosphorylation of erk returned to the baseline. the phosphorylation of erk level was significant raised in dex-pan group, and lasted to min. conclusion: dexamethasone alters the expression of certain podocyte-specific proteins not only in vivo but also in vitro, and in part, through the activation of the erk signal pathway. kh. kim minimal lesion in the renal biopsy of idiopathic nephrotic syndrome (ns) patients (pts) generally predicts a benign course. fsgs lesions in nspts, on the other hand, are associated with increased risk of steroid resistance and progression to renal failure. fsgs may be observed in follow-up biopsies despite being initially undetectable. whether this represents sampling error or the true natural history of this condition, earlier markers of steroid resistance and poorer prognosis could prove helpful. renal morphometric analyses was performed in ns pts, ages to years, initially diagnosed with minimal lesion, with (n= ) or without(n= ) subsequent progression to fsgs or end stage renal disease (esrd), along with controls ages to years. the age of patients in progressive and non-progressive group and controls were similar. gbm width in patients with minimal lesion ( ± nm) who subsequently progressed, was significantly greater than that in non-progressive group ( ± nm, p< . ). gbm width in both groups was not significantly different compared with the controls ( ± nm). average foot process width was increased in patients both with progression and non-progression as compared with controls, but there was no significant difference between progressive and nonprogressive group. the length density of podocyte detachment per gbm surface was not significantly different between progressive, non-progressive groups and control groups. there were no significant differences in the mean glomerular volume and cortical interstitial volume fraction between progressive and non progressive group. in conclusion, gbm width may help to differentiate between progressive and non-progressive groups in minimal lesion nephropathy. this may be a pathogenetic clue and needs further study. objective: our study is to investigate the correlation between clinical features and pathological characteristics on henoch-schönlein purpura nephritis (hspn) and the therapeutic methods. methods: fifteen boys and five girls aged - years (median . years) with hspn were analyzed retrospectively. the clinical characteristics, laboratory data, pathological findings and therapeutic methods were investigated. results: the patients with isolated hematuria and isolated proteinuria, the pathologic patterns were lighter then gradeii b ; eight patients with hematuria and proteinuria and seven patients with nephrotic syndrome, the pathologic patterns of injury is more severe then gradeii b , whose pathologic patterns injury exceeded gradeii b is . %. twelve patients with nephritic-range proteinuria (> mg/kg.d) and nephrotic syndrome received corticosteroids, cyclophosphamide, heparin and dipyridamole treatment. nine of twelve patients received intravenous pulse methylprednisolone (mp) and pulse cyclophosphamide (ctx). fourteen of twenty patients obtained stable remission after months to years, and five of twenty patients have asymptomatic microscopic hematuria, another one only has minimal proteinuria. conclusions: if the clinical features showed nephritic-range proteinuria or nephrotic syndrome, the renal pathologic changed markedly as well. for patients whose pathologic exceeded grade iii b or have renal tubule and interstitial damage, our study suggests that mp pulse therapy have satisfied curative effect. materials and methods: in experiment , higa mice, c bl/ mice and balb/c mice were inoculated intravenously with live cb and inactivated cb once a month from to mo of age. in experiment , higa mice, c bl/ mice and balb/c mice were inoculated intravenously with live cb and inactivated cb once at wk of age. mice in the control group were inoculated with vehicle. the kidneys were extirpated from mice of each group killed with time after inoculation for histological evaluation. experiment : we examined prostaglandin (pg) synthetic activity of cultured human mesangial cells. results: the scores for mesangial iga deposition, pcna-positive and matrix scores at weeks were higher in higa mice with live cb than in higa mice with inactivated cb or without cb . on em examination, swelling and detachment of endothelial cells from hours after inoculation and increase of serum ifn-gamma concentration were found in mice with live cb . the scores for mesangial iga and igg depositions, pcna-positive and matrix scores at weeks were more frequently found in balb/c mice with live cb . production of pge and txb significantly increased in cultured human mesangial cells damaged by live cb . conclusion: these results suggest that cb provokes exacerbation of renal pathological findings in higa mice via endothelial injury and ifn-gamma production, and may play important roles in igalike glomerulonephritis pathogenesis. rapidly progressive glomerulonephritis (rpgn) is a rare occurrence in alport syndrome (as). this report describes the case of a -year-old malewith as who developed rpgn and considers the cause of rpgn in this patient. he had a history of persistent hematuria and proteinuria since birth. at the age of years, he was diagnosed with as based on a renal biopsy. he developed nephrotic syndrome at the age of years. before administration of cyclosporine (cya), repeated renal biopsy was performed at the age of years. the biopsy specimen showed pathologic lesions characteristic of as without crescents. using immunofluorescence (if) staining, the expression of alfa- chains of collagen iv was found to be absent in the glomeruli. therefore, cya was administered for eight months. although he recovered from nephrotic syndrome, the effect of cya was limited. after the cessation of cya, his renal function slowly exacerbated. at the age of years, the administration of angiotensin receptor blocker was started. no subsequent anti-proteinuric effect was noted and his renal function improved to cr . mg/dl. however, by the age of years, he showed macrohematuria and acute deterioration in renal function to cr . mg/dl. subsequently, he underwent a third renal biopsy. on light microscopy, the biopsyspecimen showed diffuse cellular crescentic glomerulonephritis. if findings indicated pauci-immune type and electron micrography showed a few subepithelial deposits. a serological study revealed negative results for mpo-anca, pr -anca, and anti-glomerular basement membrane antibody. despite immediate treatment with pulses of methylprednisolone, cyclophosphamide, and plasma exchange, he progressed to end stage kidney disease. the patient reported here presents either a super imposition of rpgn upon a preexisting case of as or a new morphologic and clinical presentation for as. the wt gene encodes a zinc finger transcription factor involved in kidney and gonadal development. mutations of the wt gene have been shown to cause denys-drash syndrome (dds) and frasier syndrome (fs). the association of early onset nephrotic syndrome progressing to renal insufficiency, xy pseudohermaphrodism and wilms' tumor characterizes dds. renal biopsy shows diffuse mesangial sclerosis (dms). fs is also characterized by xy pseudohermaphrodism and nephropathy, but patients have delayed kidney failure characterized histologically by fsgs. this report examines three girls with nephrotic syndrome related to mutations in the wt gene, but with normal female karyotype and development. two girls with early-onset steroid-resistant nephrotic syndrome presented classical wt mutations coding for an amino acid change (d n) and (r w) at exon that is typical of dds. both children were phenotypically and genotypically females. they developed end stage renal failure within years. one girl had a wilms' tumor on the right kidney. the third child was identified with heavy proteinuria at years of age. laboratory investigations revealed a protein level of . g/dl ( - g/dl), albumin . g/dl ( . - . g/dl). proteinuria worsened to g/ h and she failed to respond to prednisone. renal biopsy demonstrated fsgs in % of glomeruli. the splice site mutation ivs + g >a, known to be associated with fs, was found in this patient. karyotype was , xx and she had normal uterus and adnexae on ultrasound. angiotensin-converting enzyme inhibitors were prescribed but she still has heavy proteinuria without renal failure. the classical clinical presentation of dds and fs is out dated. pediatric nephrologists need to consider the possibility of these genetic syndromes in evaluation of females with steroid-resistant nephrotic syndrome. we aimed to compare the effects of cyclosporine (csa) or mmf with or without combination of vitamin a,d,e or n-acetyl-l-cysteine (nac). the study included rats in eight groups: control, nephrotic syndrome without treatment, treatment with csa, mmf, vitamin a,d,e, combination of csa and vit ade, combination of mmf and vit ade and combination of nac and vit ade. all rats except the control group were given adriamycin. blood samples were drawn and -h urine were collected on day and at weeks and . at the th week, -h urinary protein excretions in the treatment groups were higher and serum albumin levels were lower than that of th week and control groups (p> . ). at the th week, urinary protein excretions in group csa&ade was lower than of the groups of csa, mmf, mmf&ade, ade and nac&ade with non-significance (p= . , p= . , p= . , p= . , p= . ) . serum albumin in group mmf&ade and group ade were significantly higher than of control group (p= . , p= . ). serum albumin in group ade was significantly higher than that of groups of csa and csa&ade (p= . , p= . ). serum triglyceride in group mmf&ade was significantly lower than that of groups csa and csa&ade. serum creatinine in group mmf&ade was lower than that of the groups of csa, csa&ade, mmf, ade and nac&ade and was significantly lower than that of control group (p= . ). serum creatinine in group csa was significantly higher than of groups of csa&ade, mmf, mmf&ade (p= . , p= . , p= . , respectively). in group nas+ade, total oxidant was significantly higher and total anti-oxidant was significantly lower than other treatment groups (p< . ). we showed that the better effect on proteinuria, serum triglyceride and albumin and lower serum creatinine by adding vitamin a, d, e in the treatment of experimental nephrotic syndrome with csa or mmf. conclusion: ht is associated with known risk factor of progression of biopsy-proven gn such as s-cr or proteinuria. the crb is an important tool for studies focusing on the epidemiology of gn in the czech republic and serves as a basis for cooperation in this field. ( ), lupus-nephritis ( ), iga-nephropathy ( ), minimal change disease and membranous nephropathy (by ) were observed. the distinction of morphological variants of the fsgs was started recently (last months). among the patients with fsgs, which were biopsied during this short period ( ), all have had a tip-lesion. these patients were started cyclosporine a mg/m /day with complete ( ) and partial ( ) remission achievement after - months. thus, the focal and segmental glomerulosclerosis is the most frequent cause of the nephrotic syndrome in children. the focal and segmental glomerulosclerosis with tip-lesion is characterized by favourable course and good response to therapy with cyclosporine a during the short-time period. objectives of study: molecules of monocyte chemoattractant protein- (mcp- ), β-catenin and cytokeratin (ck ) express increased in cellular crescent, which is a severe pathological change in renal diseases. however, it is unknown whether these molecules in urine correlate to the number or extent of cellular crescents. methods: urinary molecules mentioned above were detected in healthy subjects and patients with renal diseases by elisa. the expressions of these molecules and macrophage (cd positive) in biopsy specimens were also investigated. results: significant higher levels of these molecules in urine were demonstrated in all patients with renal diseases compared with healthy subjects (p< . ), but the highest level in patients of the cellular crescent group. the significant correlations were revealed between these molecules in urine and the index of cellular crescents (r= . , r= . , r= . , p< . ), between these molecules in glomeruli and the index of cellular crescents (r= . , r= . , r= . , p< . ), and between these molecules in glomeruli and in urine (r= . , r= . , r= . , p< . ). conclusions: this study suggested that the detection of urinary mcp- , β-catenin and ck might become potential biomarkers for clinical diagnosing cellular crescent lesions and assessing cellular crescent extent in renal diseases. this study aims to evaluate the benefits and harms of levamisole in steroid dependent (sd) and frequent-relapsing (fr) nephrotic children. material and methods: a total of steroid-sensitive nephrotic children were recruited prospectively from january to december . females and males, ( %) fr, ( %) sd. mean age at the beginning of levamisole was , years. twelve didn't receive any alternative drug before, received cyclophosphamide. renal biopsy was perfomed in patients, had minimal change disease (mc), one patient had mc at the first biopsy and fsgs in the second. the patients were divided in two groups according to their steroid response: sd and fr. levamisole was started at a dose of , mg/kg/ h as a second line drug in order to try to prolong periods of remission. clinical and laboratorial controls were performed monthly. patients were evaluated as: total responders: when steroid withdrawal was possible, partial responders: when steroid reduction was possible, non-responders: no steroid modification in months. the responders used levamisole for one year. results: the response of frequent-relapsing patients was: % of total response, % of partial response and % of non-responders. steroid-dependent patients had % of total response, % partial and % non-responsers. only one patient developed leukopenia. now % ( ) are out of levamisole and in remission, % ( ) are still using levamisole(alone or low-dose of prednisone), % ( ) are using cyclosporin, one used cyclophosphamide and one is using low-dose prednisone. conclusion: levamisole is a promising alternative drug for nephrotic syndrome. the major advantage of levamisole is its steroid-sparing effect with minimal toxicity. conclusions: hypertension and renal insufficiency were less frequently seen in chinese children with fsgs, isolated hematuria as unique clinic presentation was common in fsgs. all pathological variants had tubular-interstitial lesions, but vascular lesions were rarely seen. most fsgs children with nephrotic syndrome were sensitive to steroid at initial stage, and easy to develop frequent relapse gradually, immunosuppressive agent may be helpful to elevate remission rate. the aim of the study was to assess whether the serum index iga/c can be a usefull marker of activity igan and hsn in children. twenty children with igan (mean age . ± . years) and children with hsn (mean age . ± . years) were retrospectively analysed. in all children urine analysis, gfr were checked and the levels of serum iga and c were measured before therapy, serum iga/c was than calculated. at the onset of illness, igan and hsn was diagnosed based on the renal biopsy in mean time . ± . years. changes in light microscopy were graded i-v according to the classification of who (cwho). all biopsies were scored for activity and chronicity index (ai max score , ci max score ) (pediatr.nephrol. , , heparan sulfate proteoglycans are present both as structural components of the gbm and as modifiers of growth factor signaling on the cell surface. in mcns the presence of certain hs epitopes inthe gbm is decreased. hsulf and hsulf are recently identified endosulfatases, that can remodel the sulfation pattern of hs and thereby control the availability and presentation of factors such as fgf and hgf to their high affinity receptors. sulfatase activity requires posttranslational modification by formylglycine-generating enzyme or sulfatase modifying factor (fge/sumf ) that is counteracted by its paralogue pfge/sumf . we demonstrated that podocyte, endothelial and tubular epithelial cell lines express mrna for sulf , sulf , sumf and sumf . we investigated the in vivo distribution patterns of these enzymes in human kidney specimens by in situ hybridization. in histologically normal kidneys (n= ) expression of hsulf mrna was largely restricted to peritubular and glomerular endothelial cells, where as hsulf mrna was weakly expressed in all glomerular resident cell types. expression of sumf and sumf mrna was present in a minority of mesangial cells and podocytes, as well as in avariable number of glomerular and peritubular endothelial cells and invascular smooth muscle cells. in mcns (n= ), the glomerular expression patterns of hsulf , sumf and sumf mrna did not differ significantly from that in controls. in contrast, hsulf mrna expression was increased in podocytes. increased hsulf expression by podocytes is a novel factor to be considered in the pathogenesis of mcns. the onset and duration of ins, histopathological changes in renal biopsy, results of corticosteroid therapy and proteinuria selectivity reflecting the alteration of glomerular capillary wall were analysed. materials and methods: children with ins aged - , followed up to month, and healthy children were studied. in all patients stage of ckd were diagnosed. ins children were divided in two groups regarding to serum cystatin c levels as a marker of gfr: group i (n= )children with unchanging gfr, ii (n= ) -patients with impairment renal function over the study period. serum total protein, albumin, cholesterol, cystatin c, creatinine and immunoglobulin igg, and urinary protein, albumin, igg and creatinine were measured. results: serum cystatin c levels were higher in both groups of ins patients compared to healthy children (gr. i: . ± . , gr. ii: . ± . vs . ± . mg/l; p< . ). in group ii amount of -hour proteinuria was significantly higher than in group i ( . ± . vs . ± . g/l; p<. ), however other biochemical parameters (including igg excretion, albuminuria, selectivity index) were not different. in group ii higher age of onset was found. in this group mesangial proliferation and focal segmental glomerulosclerosis more often were observed. conclusions: the age of onset, histopathological diagnosis and proteinuria can be considered as important markers of ckd progression in children with ins. probably, longer follow-up of children with ins is necessary to find other prognostic factors. k. kilis-pstrusinska, k. fornalczyk, d. zwoliñska cyclosporine a (csa) has been used as a therapeutic option for steroid-dependent and steroidresistant nephrotic syndrome (ns). the aim of the study was to assess the effects of long term csa treatment in children with ns. methods: we performed retrospective study to evaluate safety and efficacy of csa therapy in children with ns ( girls and boys), aged - years. results: before introducing csa, in patients steroid-dependent ns and in -steroid-resistant ns were observed. children presented symptoms of steroid toxicity. pre-treatment renal biopsy was performed in patients ( children without biopsy because of renal agenesia). minimal change disease was diagnosed in ( %) children, focal segmental glomerulosclerosis (fsgs) in ( %), mesangial glomerulonephritis (gn) in ( %) and membranoproliferative gn in ( %) cases. all children were taking csa (target blood trough levels - ng/ml) more than months, mean months (range - ). complete remission was achieved in ( %) children, partial in ( %). in ( %) patients csa treatment was continued with mild dose of steroids. patients ( %) did not respond to the therapy and one of them end stage renal failure developed. following side effects have been observed: hyperuricuria ( % of patients), hyperuricaemia ( %), hypomagnesaemia ( %), hypertension ( %), hypertrichosis ( %), gingival hyperplasia ( %), hepatotoxicity ( %), gastritis and ileitis symptoms ( %). in patients control renal biopsy was performed after - months of csa therapy. in patients progression to fsgs was seen. only in one case histological findings of csa nephrotoxicity occurred. conclusion: long-term csa treatment in children with steroid-dependent and steroid-resistant nephrotic syndrome can be consider as an effective and safe therapy. introduction: membranous nephritis represents a rare disease in childhood with an incidence of . to % in renal biopsy specimens among various types of glomerulonephritis. although in many cases the disease is considered as idiopathic the association of membranous nephritis and infectious agents it is also well known. aim: we report a case of a months old baby girl of asian origin, presented with macroscopic haematuria of glomerular origin, proteinuria, - gr/ hours, hyaline and granular casts, maculopapular rash on the legs and microcephaly. methods: renal function tests in terms of plasma urea and creatinine were normal. the renal biopsy showed membranous nephritis. tests for infectious diseases (torch screen) showed a primary cmv infection. this diagnosis was based on the presence of high level cmv specific igm antibodies, increased igg antibodies and low avidity of cmv specific igg antibodies. a real-time-pcr of the renal biopsy specimen was positive for cmv as well, confirming this virus as the causative agent of membranous nephritis in the presented case. treatment with gancyclovire per os was introduced and it was most impressive since proteinuria disappeared in the following two to three weeks. two years after the diagnosis the child remains well and asymptomatic. in summary: to our knowledge, among various infectious agents, there is no case of congenital or secondary nephropathy described so far due to cmv infection in children. objective of study: pulse methylprednisolone therapy (pmt) has been shown effective in proteinuric renal diseases. but its exact effect in children with steroid-sensitive relapsing nephrotic syndrome still has no definite conclusion. to evaluate the effect and adverse effects of pmt, we performed a retrospective study. methods: there were cases of steroid-sensitive relapsing nephritic syndrome received pmt. they all had been treated with oral prednisone in similar condition previously. a self control design was used to compare the effect of pmt and oral prednisone. results: the average age was . ± . years. ten cases attained complete remission after the first course of pmt, and the average duration of pmt until remission was . ± . days. one case attained complete remission after the second course of pmt. compared with the effect of oral prednisone previously, seven cases attained complete remission more rapidly. paired-samples t-test was performed to compare the effects of pmt and oral prednisone in six cases with very similar state, however, the difference between them was not significant (p= . ). during the treatment of pmt, adverse effects were found in cases. conclusion: compared with oral prednisone, the superiority of pmt had not been definitely confirmed, and adverse effects might appear during the treatment. therefore we should be very strict with administering pmt in children with steroid-sensitive nephrotic syndrome. the study we performed was retrospective, so it was necessary to design a prospective clinical randomized controlled trial to further evaluate its effect. objective of study: pulse methylprednisolone therapy (pmt) has been shown effective in proteinuric renal diseases. but the exact effect of pmt in children with steroid sensitive nephrotic syndrome (ssrn) still has no definite conclusion. to evaluate the effect and adverse effects of pmt, we performed a prospective study. methods: a prospective clinical randomized controlled trial was conducted to compare the effect and adverse effects of pmt with oral prednisone (op) in children with ssrn. thirty-one children were enrolled with only suitable for evaluation including patients in the pmt group and in op group. results: there was no significant difference between both groups in complete remission rate. the average durations of therapy until remission were . ± . days in pmt group and . ± . days in op group, respectively. complete remission was more rapidly attained in pmt group (p= . ; < . ). during the treatment and the following months, no significant difference of adverse effects was found between both groups. there was no significant difference between both groups in relapse rate during months follow-up. conclusion: compared with oral prednisone, pmt could induce complete remission more rapidly and did not company with more adverse effects in children with ssrn. pmt had no effect on the reduction of relapse rate in the following months after administration. outcome of childhood henoch-schönlein purpura nephritis with nephroticrange proteinuria in a single center objective: the majority of children with henoch-schönlein purpura nephritis (hsn) only with hematuria and/or mild proteinuria have good chances for recovery. however, it is still unclear how we should treat hsn with persistent massive proteinuria. the aim of this study is to evaluate the outcome of childhood hsn with nephrotic-range proteinuria treated with angiotensin converting enzyme inhibitor (acei) and corticosteroids. methods: patients with henoch-schönlein purpura ( boys, girls) ranging from . to . ( . ± . ) years old at onset visited our hospital between april and december . thirty seven ( . %) developed hsn. mean age of ( boys, girls) hsn patients with nephroticrange proteinuria (> mg/h/m ) at the time of diagnosis was . ± . years old. two developed nephrotic syndrome, but none had renal insufficiency at onset. one patient suffered moderate proteinuria and renal dysfunction years after the onset. renal biopsies were done in cases and showed for grade i, for grade ii and for grade iii respectively (classification by international study of kidney diseases in children). results: eleven of the patients received acei and no patients were treated with immunosuppressive agents except for corticosteroids and methylprednisolone pulses. after a mean follow-up of . years, patients showed complete remission, had mild proteinuria or microscopic hematuria, only one postpartum patient presented with nephrotic-range proteinuria, and none developed renal insufficiency. conclusions: our results suggested that the short-term outcome of childhood hsn with nephroticrange proteinuria but not renal insufficiency was relatively favorable. thus, the degree of proteinuria is not a sole prognostic factor for childhood hsn. therefore, the indication for corticosteroid therapy should be clarified. recent observations determined a r x specific exon mutation in the gene encoding nephrin (nphs ) which evidenced an unexpectedly mild finnish-type congenital nephrotic syndrome (cns) phenotype. the long-term follow-up of patients with this mutation is actually known only in a few patients. we reported the long term follow-up of a girl originary of sicily (italy) presenting such a mutation. the first years of her life were previously reported (s.guez et al pediatr nephrol ). in brief the child was pre-term born from related parents and she presented proteinuria ( - g/day) from birth. renal biopsy was consistent with the diagnosis of cns and molecular evaluation demonstrated a homozygous exon r x nonsense mutation in the nphs gene. in the first years she was treated by human albumin without a regression of proteinuria that decreased up to . - . g/day with enalapril treatment ( . mg/kg/day); clearance of creatinine at years of age ranged between and ml/min/ . mq. during the following years (at the last control the girl was year old) she had continued enalapril treatment ( . mg/kg/day). creatinine clearance was ml/min/ . mq/day, serum total protein g/l; serum albumin g/l and proteinuria ranged between . - . g/day. both height and weight were at th for age and pubertal stage was normal; blood pressure was / mmhg. in conclusion, this is the first reported long-term follow-up in an italian patient affected by finnishtype cns with the specific r x nonsense mutation in the nphs gene: even if proteinuria persisted there was no worsening in gfr. serum total protein, albumin and growth were normal confirming the milder phenotype in comparison with the typical finnish forms. long-term enalapril treatment may also have contributed to the prognosis in this specific form of congenital nephrosis. atypical hemolytic uremic syndrome (ahus) frequently results in end-stage renal failure and can be lethal in many cases. recently, it has been recognized that many cases of ahus are associated with a defective control of the complement activation cascade. more than sixty different mutations in complement factor h gene (cfh) have been reported so far. guidelines for treatment modalities are yet to be established although plasma infusions and exchanges are often advocated. we describe a patient who presented at months of age with ahus (anemia, thrombopenia, acute renal failure requiring hemodialysis) associated with heterozygous combined de novo complement factor h mutations (s l and v a) on the same allele. laboratory investigations showed normal levels of complement c , c and factor h. during the first episode, daily plasma exchanges (pe) using cryosupernatant (cr) as replacement fluid resulted in a resolution of hemolysis and complete normalization of renal function. two ahus recurrences were successfully treated with daily pe and subsequently pe were weaned to twice weekly. one year after the first episode, pe were stopped and pi regimen ( ml/kg twice weekly and weekly thereafter) was started. at the present time, the patient has been receiving weekly pi ( ml/kg) for one year. transitory falls in haptoglobin levels or platelet counts are observed periodically and successfully treated by intensification of pi ( ml/kg) twice weekly for one or two weeks. renal function remains normal.although our observation demonstrates the effectiveness and good tolerance of large volumes of pi ( ml/kg), long-term efficacy of such a therapy remains to be evaluated. because of the possibility of secondary failure of plasma therapy, it is important to investigate alternative approaches such as combined liver-kidney transplantation. common variable immunodeficiency (cvid) is characterized by reduced serum immunoglobulin levels and recurrent bacterial infections. there have been only previous case reports of renal granulomas in cvid and only one of them was associated with immune complex glomerulonephritis. we present a case of renal granuloma and glomerulopathy in a patient with cvid. a -year-old girl, with a past history of uveitis, presented with cardiac tamponnade and bilateral pleural effusions. investigations revealed nephrotic syndrome (serum albumin of (normal - ) g/l and proteinuria > g/l), microscopic hematuria and reduced serum igg levels ( . g/l, normal . - . g/l). lupus nephritis and serositis were first suspected and corticosteroids were initiated. no serum anti-nuclear or anti-dna antibodies, nor complement activation were detected. a renal biopsy was performed and showed global glomerular endocapillary proliferation. intratubular calcium deposits were present. in the interstitium, a noncaseating epithelioid granuloma was found. immunofluorescence studies showed significant mesangio-parietal igm deposits with few c and c q. as igm-immune complex glomerulonephritis has been reported in sarcoidosis, this diagnosis was highly suspected. further investigation revealed normal lung parenchyma and mediastinum, normal serum angiotensinconverting enzyme, . oh d , calcemia and calciuria. the nephrotic syndrome gradually improved, but serum igg levels remained persistently low ( . g/l). the diagnosis of cvid was confirmed and iv immunoglobulins were initiated. this is the first report of concomitant renal granuloma and igm-immune complex (without igg) glomerulonephritis in a cvid patient. in summary, cvid must be included in the differential diagnosis of renal granuloma and should be differentiated from sarcoidosis to ensure appropriate therapy. o. nwobi, c. abitbol, j. chandar, w. seeherunvong, g. zilleruelo background: rituximab, an anti-cd antibody, has been proposed as therapy for refractory systemic lupus erythematosus (sle), although its use in children remains anecdotal. we present our initial longterm experience on the safety and efficacy of rituximab for treatment of sle in children. methods: pediatric patients with sle refractory to standard treatment protocols were treated with rituximab for - doses ( mg/m ). all had proliferative nephritis and systemic manifestations of vasculitis. clinical disease activity was scored using the sle-disease activity index (sledai). proteinuria is reported as the urine protein to creatinine ratio (upr/cr). patients have been followed an average of . ±i. years. results: b cell depletion occurred within month and remained suppressed for up to months. clinical course, renal function and proteinuria improved in the majority of patients as summarized below: objective: to assess efficiency and safety of treating crns patients with srl. subjects: patients, mean age . years old ( yrs- yrs) ( females) affected by crns. inclusion criteria: histological diagnosis of focal and segmental glomerulosclerosis, glomerular filtration rate (gfr) above ml/min/ . m , negative plasma pregnancy test, signed child and parents informed consent. exclusion criteria: secondary nephrotic syndrome, white blood cells (wbc) count below /mm , chronic hepatopathy, coagulopathy, tumoral or infection processes. discontinuation criteria: sustained decrease in gfr by more than % of the initial rate, decrease of the wbc count to less than /mm , occurrence of lymphoprolipherative and tumoral processes, severe infection, alterations in coagulation parameters, positive pregnancy test or inflammatory process or lack of changes in proteinuria or its increase after four-month treatment. methods: srl dose and dosage: dose of to mg/m /day (up to mg/day) administered once a day. expected dosage range: to ng/ml (hplc-uv method). treatment duration: months. results: patients showed nephrotic syndrome remission; average srl dose: mg/m /day; average srl dosage: . ng/ml (range . - . ). average proteinuria prior to treatment: mg/m /hour (range - ); average proteinuria after treatment: mg/m /hour (range . - ); average time of remission: months and days, none of patients showed adverse events that would have lead to the treatment discontinuation. if these data are representative of the universe of patients we are dealing with, the confidence interval (p= . ) of the percentage of patients with not proteinuria after treatment is going to be between % and % if patients are assessed. conclusions: srl caused crns remission in patients who were refractory to traditional immunosuppressive treatments. background: retinoic acid-inducible gene-i (rig-i) may play an important role on inflammatory and immune processes by regulating the expression of various genes, and has been reported to be expressed in various inflammatory diseases. we studied the expression of rig-i in human lupus nephritis and evaluated the correlation between its expression and the histological activity of the renal disease in these cases. methods: expression of rig-i in the glomeruli was assessed by indirect immunofluorescence method; frozen sections of ten kidney tissue specimens obtained from eight patients with lupus nephritis were stained with a monoclonal antibody for rig-i. kidney tissue specimens from eight patients with minimal-change (mc) disease were used as controls. results: expression of rig-i was detectable as granular immunofluorescence in a mesangial and capillary distribution in all the patients with lupus nephritis, but was absent or only trace-like in the patients with mc disease. the glomerular immunoreactivity for rig-i was correlated with the histological activity and severity of the renal disease in the patients with lupus nephritis. conclusions: rig-i expression occurs at levels detectable by indirect immunofluorescence and may be potentially useful as a parameter reflecting the renal histological activity in cases of lupus nephritis. cyclophosphamide pulse therapy for crescentic, proliferative iga nephropathy in children iga nephropathy is one of the most common glomerular kidney diseases in europe, up to % of affected adults need dialysis after years. therapy of crescentic, proliferative iga nephropathy in children is not well examined. between / and / seventeen children (main age years ( - ), male/female= / ) with biopsy-proven mesangioproliferative glomerulonephritis as manifestation of iga nephropathy were enrolled. nine patients (male/female= / ) had severe clinical manifestations (renal failure, nephrotic proteinuria or cerebral vasculitis) with extracapillary glomerular proliferations (crescents). this children were treated by intravenous methylprednisolone pulses ( / / / mg/m body surface area over days) followed by monthly intravenous cyclophosphamide (cph) pulses ( x mg/m body surface area) with gradually tapered oral prednisolon ( - mg/m body surface area/ h over months) and aceinhibitor (enalapril . - . mg/kg/d). after months of treatment a significant reduction of proteinuria ( . ± . to . ± . g/m /d; p< . ) and improvement of kidney function (gfr ± . vs. ± ml/min/ . m , p< . ) was observed. no notable adverse events were recorded. six patients had a second renal biopsy after completing cph-therapy; only crescent was found in examined glomeruli (initial findings: crescents in glomeruli). after follow-up of ( - ) months all children have unaltered renal function, further episodes of macrohematuria or gross proteinuria did not occur. intravenous cph pulse therapy seems to be an effective therapeutic option in paediatric patients suffering from crescentic iga nephropathy. eye involvement in children with primary fsgs distinct eye abnormalities have been described in children with nephrotic syndrome, particularly in diffuse mesangial sclerosis (pierson syndrome). the aim of the study is to investigate whether there are any associated ocular anomalies in childhood primary focal segmental glomerulosclerosis (fsgs). demographic characteristics, age at onset, drug therapy and duration of the disease were defined in patients ( girls, boys, mean age . ± . years) with biopsy proven fsgs. standard steroid therapy was prescribed to the patients. a detailed ophtalmological examination was performed in all patients. the median age at diagnosis was years ( - years). mean followup time was months ( - months). eleven patients ( . %) reached to chronic renal failure during follow-up period. overall, patients ( %) showed various eye abnormalities. nuclear opacity was found inone child and posterior subcapsular opacities probably due to corticosteroid therapy were present in two cases. two patients had myopic astigmatism and two had exotrophia. importantly, patients had anisometropic ambliyopia, had mittendorf spots and had pigmentary changes in macula, which had never been described in the literature. mutational analysis for nephrin, podosin, wt and lamb genes are still going on. we don't know whether particular mutations are related to particular eye findings like pierson syndrome, yet. however our findings emphasize that ophtalmological evaluation should be performed in all patients with primary fsgs at the time of diagnosis. regardless of the underlying disease, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. to investigate whether high glucose and advanced glycosylation endproduct (age) induce podocyte phenotypical changes, including quantitative and distributional changes of zo- protein, we cultured rat glomerular epithelial cells (gepc) under ) normal glucose ( mm,=control) or ) high glucose ( mm) or ) age-added or ) high glucose plus age-added conditions. high glucose plus age-added condition could increase the permeability of monolayered gepcs and induce ultrastructural separation between confluent gepcs. zo- moved from peripheral cytoplasm to inner actin filaments complexes by both age-added and/or high glucose condition in cutured gepc by confocal imaging. high glucose plus age-added condition also decreased zo- protein amount and its mrna expression to statistically significant level compared to normal glucose or osmotic control conditions. we could also confirm the activation of pkb/akt signaling pathway in gepc by age, high glucose and insulin in pi k-dependent manner. in addition, an inhibition of pi kinase by ly was able to prevent quantitative and distributional changes of zo- protein induced by high glucose and age. these findings suggest that both high glucose-and age-added condition induce the cytoplasmic translocation and suppresses the production of zo- at transcriptional level and these changes may be mediated by pi k/akt signaling. this pathway could explain the role of zo- in the phenotypic changes of podocytes in diabetic conditions. henoch-schönlein purpura (hsp) is common in the pediatric population, while wegener's granulomatosis (wg) is rare. although both diseases are classified into the vasculitis syndrome, their clinical symptoms, the treatment and the prognosis are considerably different. the classic clinical triad of wg consists of upper and lower airway disease, renal involvement and small vessel vasculitis. we present a rare childhood case in whom hsp-like symptoms were developed prior to wg symptoms. a -year-old girl developed ankle joint pain and swelling and purpura on bilateral legs and hands in the absence of abdominal pain. she was diagnosed as hsp and treated with oral prednisolone (psl) therapy. although high dose psl temporarily rescued these symptoms, purpura and joint swelling and pain reappeared in parallel with a reduction of psl dose. at this moment, because microscopic hematuria, mild proteinuria and hoarseness were also noticed for the first time, serum proteinase- antineutrophil cytoplasmic antibody (pr- anca) was evaluated and detected to be high. moreover renal pathology showed necrotizing pauci-immune glomerulonephritis, leading to the final diagnosis as wg. after methylprednisolone pulse therapy followed by oral psl combined with cyclophosphamide, her clinical symptoms with wg were resolved together with the reduction of serum pr- anca titer. taken together we emphasize that pr- anca should be evaluated even in the patients who only develop hsp symptoms. background: short-term efficacy of steroid therapy for pediatric patients with iga nephropathy (igan) has been reported. however, there are a number of cases in whom their igan recurs after stopping the steroid therapy. recent japanese study indicated that tonsillectomy had a significant impact on renal outcome. also, another japanese study showed mizoribine was effective for igan in children. in this study, we examined the effects of a treatment regimen consisting of tonsillectomy and steroid pulse therapy followed by mizoribine (tx) for chronic relapsing igan in children. method: ten cases who showed chronic relapsing igan were included as the subjects (mean age at onset: . yrs, mean age at initiation of tx: . yrs). they were divided into two group, a normal renal function group (group a, n= ) and an impaired renal function group (group b, n= ). the changes in hematuria, proteinuria, renal function, and adverse events were prospectively examined for more than months. result: a negative of hematuria was observed in % of group a and % of group b. a negative of proteinuria was seen in % of group a. in addition, in group b, deterioration of renal function was not observed during the observation periods. there were no serious adverse events associated with this treatment regimen. conclusion: a treatment regimen consisting of tonsillectomy and steroid pulse therapy followed by mizoribine treatment seems to be effective to control of acute inflammation coexisting with chronic glomerular lesions, and can be a valuable addition to therapeutic options for treating patients with chronic relapsing igan in children. objective of study: to present a case of silent stenosing ureteritis in a boy with henoch-schönlein purpura (hsp). case report: a -year-old boy was admitted with a typical hsp. urine findings were normal on admission. a gastrointestinal bleeding on the th day of illness suggested corticosteroid treatment. the ultrasound on the th and th day revealed a normal urinary tract. on the th day he developed non-painful macroscopic haematuria, followed by microscopic haematuria and proteinuria, which reached the nephrotic range on the th day. thus, the boy was treated with methylprednisolone pulses and cyclophosphamide for weeks. renal biopsy was not performed because of his parent's refusal. microscopic haematuria and proteinuria gradually subsided, with complete disappearance at the th month. during this time he was asymptomatic, with no episodes of macroscopic haematuria or colicky flank pain. at the th month of illness a new ultrasound revealed a major left hydronephrosis. computer tomographic urography showed a complete ureteropelvic junction obstruction. the mtc-dmsa scan revealed a % relative function ipsilaterally. a left pyelostomy was performed. during the next four months after draining, the urine volume of the affected kidney was . - . ml/kgr/hour, the creatinine clearance - ml/min/ . m and the mtc-dmsa scan showed a % relative function. based on the above findings a nephrectomy was decided. conclusions: although rare, stenosing ureteritis should be considered in hsp. the typical clinical presentation with haematuria in association with colicky flank pain may not always occur, as in the present case, or may be confused with the symptoms of hsp itself. thus, the repetition of an ultrasound during the process of the disease may be necessary, in order for this complication to be diagnosed and treated early, preventing serious renal outcome. mitochondrial diseases are either due to sporadic or inherited mutations mainly in mitochondrial dna located genes. with regard to renal manifestations, tubular dysfunctions are common; however, the existence of solitary glomerulopathy has recently become apparent. in such case, the pathomechansim of the glomerular proteinuria is still obscure. wepresent a year-old girl who was found to have asymptomatic proteinuria (up/cr . ) in the absence of hematuria, azotemia, tubular dysfunctions, lacking any neurological manifestations. her family history showed maternal inheritance with mild proteinuria of grandmother and renal insufficiency of young uncle. light microscopy revealed glomeruli of normal appearance and of global sclerosis. electron microscopy showed swollen mitochondria in podocyte of normal appearance glomerulus. pointmutation rate of mitochondrial dna, a g, was detected as less than % in grandmother, % in mother and % in the patient examined byperipheral blood cells. the proteinuria completely disappeared months after treatment with combined therapy of arb and acei. to determine the responsible molecule for the pathomechanism of proteinria, immunostaining followed by conforcal microscopy with slit diaphragm associated molecules (sdm) (nephrin, podocin), gbm associated molecules (type iv collagen alpha chains, laminin isoforms, perlecan) and podocalyxin was studied and compared to the controls. interestingly distinct decrease of expression with sdm was observed even in normal appearance glomerulus of the patient. taken together, a g mutation itself may lead to depletion of atp and/or increase of free radicals in podocyte, which predominantly affect the biogenesis of sdm, result in pathological glomerular proteinuria. the mechanism of antiproteinuric effect of arb/acei therapy should be evaluated by serial biopsy specimen. objectives: to report the effectiveness of pulse cyclophosphamide induction therapy in children with diffuse proliferative lupus nephritis. to identify predictors for unresponsiveness to the treatment. methods: thai children under years of age with biopsy-proven diffuse proliferative lupus nephritis who were admitted to chiang mai universityhospital between and were retrospectively studied. responsiveness to treatment, defined as urinary protein to creatinine ratio of less than . , was assessed at the end of induction period. the clinical characteristics and laboratory data including gender, age at diagnosis of sle, duration of disease before treatment, hypertension, clinical nephrotic syndrome, amount of proteinuria, serum creatinine, creatinine clearance, serum c level and presence of crescentic formation in renal biopsy were compared between the two groups who responded and did not respond to the treatment. results: a total of patients ( % female) with the mean age at diagnosis of sle of . ± . year were studied. nineteen patients ( %) achieved remission at the end of induction therapy. there were no significant differences in all parameters studied between responsive and nonresponsive groups. despite the indifference in the amount of proteinuria, the proportion of patients with nephrotic-range proteinuria was higher in unresponsive group. conclusions: pulsecyclophosphamide is an effective regimen for induction therapy in children with diffuse proliferative glomerulonephritis. although no definite predictor was detected in this study, higher proportion of patients with nephrotic-range proteinuria in the unresponsive group wasnoted. born small for gestational age, but not early postnatal weight gain aggravates the course of idiopathic nephrotic syndrome in children clinical and animal studies have shown a higher risk of an aggravated course of renal disease in childhood after birth small for gestational age (sga). fast catch-up growth after sga seems to support the development of later disease. in a retrospective analysis of cases with idiopathic nephrotic syndrome treated between and in a university centrefor paediatric nephrology we identified children as sga and asappropriate for gestational age (aga). we related the course of disease to birth weight and catch-up growth. median age of manifestation in sga was . ( . - . ) years vs. . ( . - . ) years in aga children. in all sga children renal biopsy was performed, while only % of the aga children underwent renal biopsy showing nodifference in renal histology. in the sga group, % patients developed steroid resistance (vs. % aga, p< . ). the number of relapses was not different. % sga children needed antihypertensive treatment inthe course of the disease compared to % of aga children. catch-up weight gain between birth and months of age did not influence the course of disease. in conclusion we could find evidence for an aggravated course of idiopathic nephrotic syndrome in former sga children, but weight gainduring the first two years of life did not influence the course of disease. the mechanisms of perinatal programming in later renal disease need further investigation. wilson's disease(wd) is a disorder of copper metabolism that affects numerous organsystems including kidneys. besides renal tubular dysfunction as a result of excessive storage of copper, renal manifestations due to therapeutic complications can also develope specially with d-penicillamin. in this study we investigated the frequency and spectrum of renal manifestations during d-penicillamin therapy in wd. of patients receiving d-penicillamin for wd, patients( . %) ( boys, girl) developed findings of glomerulopathy within month to year after initiation of therapy and all were histologically diagnosed to have membranoproliferative glomerulonephritis (mpgn). the patients were between - years old, and they had normal urinalysis and renal function tests in their first presentations. two siblings developed hematuria and proteinuria below nephrotic range while the other two developed nephrotic syndrome. one of these latter patientsalso had acute renal failure needing temporary peritoneal dialysis. three patients had low complement (c) levels, had antinuclearantibody (ana) positivity, two had c-antineutrophil cytoplasmic antibody (anca) and one p-anca positivity. d-penicillamin therapy replaced by zinc sulphate in all patients. all renal findings improvedin patients within - months with normal renal functions and complement levels, and negative ana, p and c-anca tests. after years all were clinically in remission of mpgn confirming the role of d-penicillamin in development of renal disease. objectives of the study: the use of tacrolimus in steroid-resistant (sr) focal segmental glomerulosclerosis (fsgs) has been reported in single and small series. the aim of this report is to exhibit experienceon the management of children with sr fsgs in whom tacrolimus had been started on due to the therapy resistance. methods: fk combined low-dose oral steroid was started on three male patients ( , , -yearold) with sr fsgs who had been following for three years. all of them had failed various cyclosporin a, cyclophosphamide and steroid regimens prior to treatment with fk . the application was . mg/kg/day in two divided doses over h adjusted to a trough blood level between and ng/ml for months. other therapies included angiotensin-converting enzyme inhibitors, vitamin d and calcium analogues, and lipid-lowering agents. results: a reduction in proteinuria to normal levels was noted between - weeks following the initiation. the remission was achieved overall during the treatment. the relapse was recorded following cessation of tacrolimus in - weeks. the drug was generally well tolerated with no sideeffects and adverse reactions. the ratio of infectious events did not differ from the former regimens. conclusion: tacrolimus may be effective in controlling the proteinuria of patients with srfsgs during the therapy. there is a trend of relapse following cessation of treatment. the duration for drug uptake is a topic of debate. further study in larger population is warranted. introduction: histological features of focal segmental glomerulosclerosis are found in % of pediatric patients with steroid resistant nephrotic syndrome. upto % (between - %) children with fsgs progress to esrd. objective: to study the clinical course of childhood fsgs and determine the possible predictive factors of chronic kidney disease. method: case records of children who had biopsy proven fsgs and had presented to the sindh institute of urology and transplantation between - , were retrieved. clinical and laboratory parameters at baseline, response to steroids and cyclosporine, development of crf (as defined by a gfr of < ml/min/ . m ), and histopathological details were analysed. result: a cohort of children with a mean age of . ± . years and a m: f ratio of : was identified. after a mean follow-up time of years, / ( . %) developed crf. on univariate analysis, male sex ( % vs %, p- . ), > years age at onset ( % vs. %, p- . ), hypertension ( % vs. %, p- . ) and microhematuria at presentation ( % vs. %, p- . ) were significantly associated with risk of developing crf. steroid resistant course ( % vs. %, p= . ) was more prevalent in those who developed crf. the crf group was also more likely to have an elevated creatinine at baseline ( . % vs. %, p< . ). moderate tubular atrophy and a high percentage of segmentally and globally sclerosed glomeruli were found in those who developed crf. patients progressing to crf were more likely to have a partial response to cyclosporine ( % vs. %, p= . ) conclusion: factors such as age, microhematuria, hypertension, elevated baseline creatinine, steroid resistance, tubular atrophy, percent global sclerosis and partial response to cyclosporine are likely predictive of progression to chronic kidney disease in children with fsgs presenting to our center. chronic glomerular nephropathies in children are marked by an often unfavourable evolution, so that the establishing of a prognosis at the time of the diagnosis is both a professional and a moral duty for the pediatric nephrologists. purpose: the estimation of the current practice renal survival prognosis in children with chronic glomerular nephropathies, by using clinical and laboratory elements in different histological forms of primitive chronic glomerulonephritis (cgn), with a minimum period of observation of one year. we analyzed parameters that may intervene in the duration of renal survival: type of cgn, age at the debut of the illness, histological scores of activity and chronicity, the presence of tubular atrophylesions and that of interstitial fibrosis, renal failure (rf) installment time, in cases with normal renal function at the beginning, the time until the initiation of dialysis in cases with esrf, respectively. the statistic analysis of data has been carried outwith epi soft (fishcer test). the results have been as follows: unfavourable evolution has been taken into consideration in the cases which have presented fixed nitric retention or which required the initiation of dialysis. the initiation of dialysis was necessary in cases ( %), out of which ( %) having associated between and of the considered risk factors. if the histological type (sfgs, dgs, mpgn) is added to the obtained score, the accuracy of the estimation increases to %. in conclusion: the usage of prognosis scores composed of current elements of diagnosis that have proven to have statistical significance, as far as the renal survival prognosis is concerned, may allow the invoking of a medium-term prognosis in the evolution of children with cgn. introduction: the srns can lead to a progressive deterioration of the renal function and therefore needs an aggressive therapy. one of the alternatives of treatment is the association described by mendoza et al which consists of prolonged use of methylprednisolone with cyclophosphamide (cp), which has a remission rate of %. the objective of this study is to evaluate retrospectively the clinical evolution of children with srns treated with mendoza's protocol. method: between and , children, male and female, with srns were subjected to a renal biopsy and later treated with mendoza's protocol. cp was used in children that had not responded to pulses of methylprednisolone and presented a relapse. all of the patients received supplemental calcium. the clinical evaluation included stature, weight, ophthalmic fundus examination, proteinuria in urine recollection of hours and/or protein to creatinine ratio and blood chemistry. results: eleven patients ( %) had fsgs. nine ( %) presented complete remission, two ( %) had partial remission, four ( %) did not respond to treatment, of which evolved to terminal kidney disease. nine patients received cp. of the complications secondary to treatment with steroids, % had a linear growth suppression and an increase in their bmi and patient presented cataracts with visual impairment. conclusion: the prolonged treatment with boluses of methylprednisolone and cyclophosphamide is a good alternative in patients with srns. the prolonged use of high doses of steroids can cause linear growth suppression and other adverse reactions, so it is advisable torealize a genetic study in all of the patients with srns to be able to exclude the patients that have a genetic mutation and so avoid unnecessary treatment. objectives: to study the clinicopathological profile and outcome of lupus nephritis (ln) in indian children. methods: clinical and histopathological features and outcome of children with ln was retrospectively reviewed. patients were included if they fulfilled the acr criteria for the diagnosis of sle and had either of persistent proteinuria, active urinary sediments or renal dysfunction. outcome was analyzed at years and at last follow-up. results: ofthe children studied, were boys. the mean age (sd) at diagnosis was . ± . (median . , range - . ) yr; children were youngerthan years of age. the mean age at presentation and renal biopsy was . ± . (median . , range . - ) years. the mean duration of follow-up was . ± . (median . , range . - . years) years. . % patients were followed for more than years. commonest clinical manifestations were fever ( %), hypertension ( %) and malar rash ( %). and % of patients presented with nephrotic and nephritic syndrome respectively before the diagnosis of sle. the commonest pathology was class iv nephritis ( %) followed by class ii ( %). hypertension, hematuria, nephrotic syndrome and decreased egfr were significantly associated with class iv ln. at last follow-up . , . and % patients were in ckd stage ii, iii, and iv respectively. the patient survival rate at year and at last follow-up was and % respectively, while no patient developed esrd at years. infections were seen in % cases that resulted indeath in patients; died of hepatic encephalopathy. conclusion: sle nephritis has a varied presentation and high morbidity. a significant proportion of patients developed infections during the course of disease. clinical, pathological features and outcome in our study do not differ markedly from those in most pediatric series. background: atypical hus has a frequently relapsing course and a poor renal prognosis. low c plasma concentration suggests alternate complement pathway regulatory abnormalities: factor h (fh), factor i (fi) and membrane cofactor protein (mcp). case report: we report an year old girl with atypical hus due to acquired fh deficiency caused by anti-fh antibodies (abs). hus was diagnosed on the basis of acute renal failure, microangiopathic hemolyticanemia and thrombocytopenia. past history: recurrent fever with culturenegative pharyngitis (suspected pfapa syndrome). decreased c ( mg%, normal> ) with normal c . adamts activity was depressed, no cleaving protease abs. hemodialys (hd) and plasma exchange (pex) were started days later. severe urticaria and angoiedema during pex was treated with methylprednisolone and chlorpheniramine. hematological and renal improvement were observed after the rd pex session. relapses occurred in months: -controlled by pex; -(with suspected pfapa) with single dose corticosteroids, -with prednisone therapy, terminated with single dose ivig mg/kg. elevated anti fh ab titer- au with decreased fh functional activity were found in pre-pex plasma. fh, fi, factor b and c antigenic levelsnormal. hematological remission and renal function improvement without hus relapse ensued while tapering corticosteroids. two years after presentation, onprednisone mg/day anti fh titer dropped significantly with restoration of functional fh activity, gfr ~ ml/min/ . m . conclusions: in cases of atypical hus, active search for anti fh ab iscrucial for implementing specific and effective therapy: plasma exchange, iv ig and steroids for improving course and longterm prognosis. research centre for child health rams, department of pediatric nephrology, moscow, russian federation children aged , - years with idiopathic biopsy-proven steroid-resistant focal and segmentary glomerulosclerosis (fsgs) were treated with cyclosporine a (csa) , - , mg/kg as initial dosage, oral prednisolone , mg/kg every other day tapered to the -th month and methylprednisolone pulses (mp) - mg/kg every other day for the first - weeks in of patients. serum creatinine level was controlled once a month. after months of csa treatment complete or partial remission of proteinuria was in ( %) of children, no effect in ( %). serum creatinine level increased in % on an average in the group remission of proteinuria. in the group of non responded patients the creatinine elevation was significant same - %. after year of csa treatment complete or partial remission observed in ( %), no effect in ( %). elevation of serum creatinine level in children with remission was , % (without significant difference compared to month's csatreatment). increasing of the creatinine level more than % in two patients leaded to double tapering csa dose resulted in normalization of serum creatinine level. in the group of csa non responders the significant increasing of serum creatinine level ( %) was revealed (compared to month's csa treatment). in cases the elevation ofcreatinine level was more than % and these patients turned into esrd eventually. in all of non-responders csa was discontinued. we concluded that serum creatinine level in csa respondrers was stable without significant elevation during the year of treatment. csa therapy for year without any effect influenced on renal function decreasing. clinical objective: to describe the clinical course and outcome of pediatric patients with cgd treated with iv mpt ( mg/kg x doses monthly for months, then dose monthly for the next months) methods: patients' medical records were reviewed. pre, post-treatment and follow-up hr urineprotein, creatinine and gfr were compared using paired t-test; and proteinuria, hematuria and blood pressure using mcnemar's chi square. outcome measures were analyzed usingmean ± sd and frequency distribution. results: patients were included, male, female. mean age at disease onset is . ± . years. mean duration of follow-up is . ± . months. % achieved complete remission after a mean of . cycles, % partialremission after a mean of . cycles and % were treatment failures. mean relapse rate is . ± . on treatment and . ± . at follow-up. renal survival rate is %. hour urine protein and the proportion of patients with proteinuria, hematuria and hypertension significantly decreasedafter treatment and remained stable at follow-up. serum cr/gfr were also stable pre, posttreatment and at follow-up. no serious side effects were noted. conclusion: this protocol induced a high and early remission rate ( % after . cycles)among the patients. majority demonstrated stable renal function and blood pressure over time. relapse rates were low and treatment is generally safe. recommendation: the protocol can be offered to oral-steroid or alkylating agent-resistant patients with satisfactory remission rates. objectives of study: nestin, an intermediate filament protein which has a role in regulating cellular cytoskeletal structure, is restrictedly expressed in the podocytes of human kidneys. in the present study nestin expression was investigated in biopsy specimens of children with focal segmental glomerulosclerosis (fsgs). methods: kidney biopsy specimens taken from children with diagnosis fsgs were investigated. diagnosis was performed on light microscopy, immunofluorescence microscopy, taking into account clinical data. for immunomorphology monoclonal anti-nestin antibody from mouse (sc- , clone c . a , santa cruz biotechnology) diluted : , was applied on cryostat or paraffin sections using labeled streptavidinebiotin (lsab+ dako) method. visualization was performed by dako aec substrate. kidney biopsies of patients without nephrotic syndrome, mainly with mesangioproliferative gn were used for control staining. results: the mean age at the time of biopsy was . ± . years, and all patients had nephrotic syndrome. half of them revealed some focal tubulointerstitial changes: tubular atrophy, slight interstitial fibrosis and lympho-monocytic infiltration. in two cases mutation of wt gene and in one case mutation of nphs gene was detected. four cases had familial character of fsgs. nestin expression was variably present in different cases of fsgs. decreased expression was detected in glomeruli with segmental mesangial sclerosis and capsular adhesions. conclusion: fsgs revealed heterogenity concerning nestin expression. nestin expression was diminished in affected glomeruli. background: renal effects of altered ob/ob-r pathway may contribute to obesity, and diabetesassociated proteinuria. in the kidney ob/ob-r stimulates collagen type i and iv synthesis and upregulates tgfβ and tgfβ receptors. objective: to determine ob/ob-r and its downstream (jak/stat/socs) pathway expression in nephrotic syndrome (ns) and fsgs. design/methods: microarray analysis of kidneys of -week ( w) and -month ( m) old transgenic mice (tg) and controls (ctr) was performed, and confirmed by quantitative pcr. kidney sections were analyzed by immunohistochemistry (ihc) and western blot analysis (wb). urinary ob/ob-r of children with ns classified as steroid sensitive (ssns) or steroid resistant (srns), were measured by elisa. results: ob, ob-r, and jak , , mrna expressions were not statistically different at w and m between ctr and tg. stat and socs mrna were increased . -fold (sem ± . ), and . fold (sem± . ) at m in tg, p= . and p= . respectively. ihc and wb (p= . ) of kidney sections showed no significant difference between the groups. we examined ctr, ssns, and srns patients with comparable bmi, age, race and gender. urinary protein to creatinine ratio in ssns and srns was . (sem± . ) and . (sem± . ) respectively, p= . . urinary ob (p= . ), ob-r (p= . ), and tgfβ (p= . ) were not statistically significantly different between the groups. conclusions: ob/ob-r was not upregulated in tg at the onset of proteinuria and fsgs. however, advanced fsgs ( m tg) showed significant activation of socs , an ob/ob-r negative regulatory pathway. pediatric patients with early srns had no significant increase in urine ob/ob-r. this data suggests the role for ob/ob-r regulatory pathways in the development of advanced fsgs. primary fsgs presents clinically with steroid-resistant (srns) or steroid-dependent (sdns) nephrotic syndrome or proteinuria. the data shows, that % patients with fsgs progress to esrd in years follow-up. objectives: the aim of the study was to analyze the long-term outcome of patients with primary fsgs diagnosed in kidney biopsy. material: the study group consisted of children ( males, females) followed from . all patients were treated with immunosupression and renoprotection. the clinical data were analyzed after follow-up lasting for mean ± , years ( , - years) . children presented with nephrotic syndrome ( srns and sdns) and with proteinuria. the age ranged from , - years mean , ± , at the time of diagnosis. more then kidney biopsy was performed in children -in progression from mcd (n= ) and mes (n= ) to fsgs were observed. results: the clinical remission was observed in / patients ( %) and was not correlated with initial proteinuria (mean , ± ). in / patients crf was observed, of them progressed to esrd ( were successfully transplanted). among patients, who had fsgs as their initial glomerular lesion (n= ), the percentage of glomerular sclerosis was significantly higher in a group in which remission was not obtained after long-term follow-up ( , vs. %, p< , ). in out of patients with follow-up over years progression to esrd was observed ( , %), / were transmitted to adult centers with persistent proteinuria. conclusions: immunosuppression and renoprotection in patients with fsgs can prevent the progression of crf. extensive glomerular sclerosis is a predictor of unfavourable outcome. further clinical and genetical studies are needed to establish the effective therapy modalities. mycophenolate and who had previously undergone a renal biopsy, recieved mg twice daily (maximum gram twice daily) during six months. prednisone was concurrently prescribed at at dosage of mg/kg/every other day, during weeeks and . mg/kg/every other day during the subsequent weeeks. results are expressed as mean±sd. results: seven chidren, boys and two girls were enrolled. oncet of their ns was at age . ± , yr (range - yr) and mmf was initiated at . ± . yr ( . - . yr) . six were sr and one sd. two patients had previously recieved cyclosporine, two patients cyclophosphamide and one chlorambucil. renal histology displayed: focal segmental glomerulosclerosis (n= ), minimal change (n= ), mesangial proliferation (n= ) and membranous glomerulonephritis (n= ). at the end of the follow-up: three patients were in partial remission, two were in complete remission and two had no response to mmf. initial and final serum creatinine concentration . ± . vs . ± . mg/dl), estimated gfr ( . vs. ± ml/min/ . m ) and serum albumin ( idiopathic nephrotic syndrome is the most frequent glomerular disease in childhood. most patients are steroid responsive but half of them relapse and often become steroid-dependent. they are exposed to long term steroid complications on the one hand and relapses due to insufficient disease control on the other hand. our aim is to determine predictive risk factors for high degree steroid dependence. in france, steroid-resistance is defined as persistent proteinuria after one month of daily oral prednisone ( mg/m ) and pulses of methylprednisolone (mpn) ( g/ . m ). we included steroid responsive children with disease onset between and . the mean age at diagnosis was . years (range . - ). all patients initially received prednisone mg/m per day. the following parameters were analysed: age at onset, gender, days to remission with initial steroid therapy, mpn pulses, numbers of relapses, steroid dependency, immunosuppressive drugs. twenty of the patients were steroid-dependent; among the steroid dependent patients, received mpn pulses. % of those patients ( / ) were treated by cyclosporine during follow-up. on the other hand, only % ( / ) of the patients who did not receive mpn required cyclosporinebased therapy during follow-up (chi-square test, p= . ). interestingly, there was no correlation between treatment days until remission during the initial prednisone course and the risk for later steroid dependence. conclusion: the need for mpn pulses, but not the time interval until remission helps to predict steroid dependence. patients, necessitating mpn pulses to obtain remission are at risk to require cyclosporine for disease control. by identifying these children, we could eventually / avoid multiple relapses by earlier use of adequate immunosuppression and / avoid side effects related to long term high dose steroid therapy. membranous nephropathy (mn) with antitubular basement membrane antibodies is a rare condition. relapse of tubular dysfunction in renal transplant recipients has been published in one case, but relapse of mn in the renal graft has not yet been reported. a -year old boy presented first with steroid resistant nephrotic syndrome associated to tubular dysfunction. renal biopsy revealed mn associated to interstitial fibrosis and granular deposits of iga, igg, and c along the tubular basement membrane. indirect immunofluorescence (if) revealed circulating anti-tubular basement membrane antibodies. he received a renal allograft at the age of years. an acute rejection episode on day required three steroid pulses and okt- . renal biopsy revealed the presence of interstitial and vascular rejection (banff iii) and relapse of tubular basement membrane deposits. renal function normalized within days and remained stable (gfr estimated by schwarz formula= ml/min per . ). proteinuria remained negative and urinalysis normal over years under the immunosuppressive regimen including fk, azt, and prednisone. at the age of years, proteinuria increased progressively over weeks reaching . g/day, whereas serum creatinine remained stable. renal biopsy revealed the presence of granular deposits along the glomerular basement membrane suggesting a late relapse of mn in the transplant. rituximab therapy (day , , , and ) followed by switch from azathioprine (aza) to mmf resulted in a complete biological remission with negative proteinuria. indirect if revealed progressive decrease of antitubular basement ab level during rituximab treatment and a negative signal was obtained months after the switch from aza to mmf. this is the first report of glomerular relapse of mn with anti tubular basement antibodies in a renal transplant recipient. nephrotic children are at risk for severe pneumococcal infections. the best moment for antipneumococcal vaccination is controversially discussed. we investigated the serologic response after pneumo vaccination in children ( girls) with nephrotic proteinuria and hypoalbuminemia, immediately after initialisation of prednisone therapy at mg/m (group ) and in children after tapering down of prednisone to < . mg/kg eod (group ). there was no difference in both groups concerning antibody (ab) response, relapse frequency, or number of steroid dependent forms. in group , pneumo ab levels at presentation (m ) were . ± . (mean±se) . at m antibody levels increased -fold to . ± . (p< . ). serum levels at m were . ± . . one year after vaccination ab levels ( . ± . ) decreased compared to m (p< . ), but remained increased compared to m (p< . ). there was no increased delay until remission in both groups compared to a retrospective control group. severe hypoalbuminemia (< g/l) at the time of vaccination was not related to a lower serological response on m . during relapses, antibody levels decreased significantly compared to levels before relapse (p< . ), but increased again once remission was obtained. even during relapses, ab levels remained higher (> -fold) than pre-vaccination levels. conclusion: nephrotic children on high dose glucocorticoid therapy respond to anti-pneumococcal vaccination and their ab levels remain elevated during relapses. vaccination at disease onset may be beneficial as those patients with relapses during the tapering down of steroids already have increased anti pneumo ab at the time of relapse. crescentic glomerulonephritis with isolated c deposits associated to complement abnormalities: a new entity? introduction: several progressive renal diseases present proteinuria, as a result of glomerular and tubulointerstitial injuries. thus, some studies prove that proteinuria is an important predict factor for progression of renal failure. angiotensin converting enzyme inhibitors (acei) are efficient in reducing proteinuria and preserve renal function in patients with diabetic or non-diabetic nephropathy. the purpose of this study was to evaluate the efficacy and security of acei in children. material and methods: the acei (enalapril) was used in normotensive and hypertensive patients with chronic renal disease, with microalbuminuria/proteinuria. results: we studied patients ( girls), for at least months, mean age was . ± . years ( month to years). / patients had glomerulopathy, / chronic pyelonephritis, / systemic disease, / renal hypoplasia/ dysplasia, / cystic renal disease and / arterial hypertension. the mean dose of enalapril was , mg/kg/d ( , to , ) and it was used during , months (mean). we observed a normalization of proteinuria/microalbuminuria in , % of cases. in seven patients, the drug was discontinued due to: / irregular use, / vertigo, / hypercalemia and acute renal failure recovered after withdrawn the drug. during the use of enalapril we did not observe significant difference in potassium or creatinine serum levels, as well as blood pressure measurements. conclusions: the use of enalapril in pediatric patients with renal disease and proteinuria/ microalbuminuria showed security and efficient. therefore, we suggest it as a antiproteinuric and renoprotective agent in children. a. filleron, al. adra-delenne, l. ichay, f. dalla-vale, h. valette, d. morin chu montpellier, pediatric nephrology, montpellier, france in order to evaluate the long-term efficacy of oral cyclophosphamide (cp) in children with sdns, the outcome of patients ( girls) treated in our unit for steroid sensitive ns were studied retrospectively. median age at diagnosis was . years (range . to ). initially, they received oral prednisone (p) : mg/m /d for weeks and p dosage was then tapered for the next weeks (totale dose p: mg/kg). relapses of proteinuria were treated with p ( mg/m /day) and, in case of steroid dependency (sd), p was maintained on an alternate day regimen. one single child had no relapse, while had a relapse rate of less than /year. the remaining frequently relapsed and received oral cp - mg/kg/day for weeks, totale dose mg/kgbecause of their high relapse rate with steroid toxicity. median duration of p treatment was . years (range . to ) before cp was given. in one case cp had to be stopped because of hemorragic cystitis. follow-up after cp treatment was, at least, years. p was stopped in the following months after cp in / children, but has to be continued in the remaining because of early relapse of proteinuria. among those children, only one had no more relapse years after cp. in children, relapse of proteinuria occured . ± months after cp. in those patients, / had to receive another steroid sparing drug such as cyclosporine or mycophenolate mofetil (mmf) because of recurrence of sd. in our experience, cp treatment in ns with steroid toxicity is associated with a significant change in the relapse rate in only / children and in the remaining , improvement was transient. our results suggest that alternative treatment, such as mmf, has to be evaluated as first-line steroidsparing agent in those patients with sdns and steroid toxicity. interleukin - the aim of our investigation is to compare the concentration of total ige, specific ige, interleukin- (il- ) and gamma-interferon (gamma-ifn) in serum of children with initial and relapsed steroid-sensitive mcns and of children with atopic dermatitis at the age from to years. the concentration of total ige was measured by immunoenzymatic method and il- , gamma-ifn by immunoassay technique using monoclonal antibodies. the result showed that % of children with atopic dermatitis had the increased concentration of total ige; specific ige was increased to alimentary allergens- , %, household- , %, inhaled- , %. the concentracion of il- was , ± , pg/ml, of gamma-ifn was ± , pg/ml. the result showed that % of children with mcns had increased concentration of total ige; specific ige was increased to alimentary allergens- , %, household- , %, inhaled- , %. the concentration of il- was , ± , pg/ml, of gamma-ifn was , ± , pg/ml. according to our investigation, the concentration of il- and gamma-ifn in children with mcns were not significantly different then in children with atopic dermatitis. conclusion: the fact that there were not significant differences in serum total ige and specific ige, il- and gamma-ifn in children with mcns and atopic dermatitis gives us a reason to suppose that these diseases have identic mechanisms of pathogenesis with ige reaction i-type with activation of t-limfocyte. to clarify the pathogenesis of mcns, comprehensive studies for these cells would be worthwhile. there are several lines of evidence that the slit diaphragm (sd) not only serves as a structural framework for filtration barrier but also has an essential role as a signaling platform. nephrin is tyrosine phosphrylated by src-family tyrosine kinase, fyn. phosphorylated nephrin recruits nck to sd, and regulates assembly of actin filament. the crucial roles of tyrosine phosphorylation in podocyte is also indicated by renal malfunction observed in fyn knockout mice. neph has a longer cytoplasmic domain and a larger number of tyrosine residues in its cytoplasmic region than nephrin. but knowledge about tyrosine phosphorylation of neph is limited. here we characterize neph as a substrate of fyn. fyn interacted with and phosphorylated cytoplasmic domain of neph in vitro and in cultured cells. peptide mass fingerprinting of neph cytoplasmic domain phosphorylated by fyn in vitro identified at least five tyrosine phosphorylation sites. site-directed mutagenesis confirmed that these tyrosine residues were indeed phosphorylated in cultured cells. in pull-down analysis with neph from rat glomerular lysate, neph specifically bound to an adaptor protein grb and a tyrosine kinase csk in a phosphorylation-dependent manner. coimmunoprecipitation experiments revealed phosphorylation of y and y were crucial in neph -grb binding. furthermore, tyrosine phosphorylated neph suppressed erk activation elicited by fyn, and also inhibited fyn-induced ap- transcriptional activation. these inhibitory effects required the intact binding motif of the grb sh domain, and both y f and y f mutants failed to inhibit erk activation. these results indicate that fyn orchestrates a wide spectrum of protein-protein interactions at sd by phosphorylating neph as well as nephrin, and neph modulates downstream signaling by phosphorylation-dependent association with adapter proteins. celiac disease (cd) is a common disorder in southern europe and has a protean clinical presentation. hla class ii aplotypes dq and/or dq are present in % of cd patients and in % of the normal population. the observation of three patients with both cd and nephritic syndrome (ns) prompted us to study hla class ii aplotypes in our patients with ns. in all children with ns admitted to our unit we determined the presence of dq /dr , dq /dr , dq /dr e dq /dr aplotypes and anti-transglutamidase antibodies (ab-httg). hla typing was done by dna extraction and pcr amplification and electrophoresis in agarose; ab-httg determination was made by elisa. as control groups we examined children with cd and first degree relatives (of theirs). in so far we have studied children with ns ( males, females, age ranged - years); are steroid sensitive (ssns), steroid resistant (srns). a renal biopsy was done in and showed minimal lesions in , focal and segmental sclerosis in , membranoproliferative gn in , membranous gn in and iga deposition in . corticosteroids or other immunosuppressant were administered in when blood was drown. dq and/or dq aplotypes were present in out of patients ( . %), in out of ssns ( . %), in out of cd relatives ( %) and in all cd patients. dq /dr combination was present in a smaller percentage of ns compared to control groups. ab-httg were detected in one patient out of ( . %). purpose: to investigate activity of antithrombin and a protein c at children with mcns: at active period (proteinuria more g/m /d; hypoalbuminemia < g/l), at in incomplete remission (the third day of absence of proteinuria, hypoalbuminemia < g/l), at in proof remission. methods: activity of natural anticoagulants in blood was defined by a clotting method with use of reactants "roche" and "behring". results: activity of antithrombin in blood in the active period of disease sharply decreased ( , ± , %, p< , ), and already in the period of incomplete remission came back to norm ( , ± , %), characteristic for the period of full remission ( , ± , %). activity of a protein c in blood in the active period of mcns was high ( , ± , %, p< , ), during incomplete remission decreased ( , ± , %, p< , ), in the period of proof remission was in norm ( , ± , %, p< , ). at children with mcns dependence of decrease in activity antithrombin from weight hypoalbuminemia (r= , , p< , ), hyperfibrinogenemia (r=- , , p< , ), hypercholesterolemia (r=- , , p< , ) and hyper-lipoproteinemia (r=- , , p< , ) is established. authentic distinctions of factor of the attitude of activity of protein c/ activity of antithrombin depending on the period (the active period - , , incomplete remission - , , proof remission - , ) are received. conclusion: at children with mcns changes in system of natural anticoagulants: decrease in activity antithrombin below % and increase of factor of a parity of anticoagulants (more than , ) testify to hypercoagulation and risk of thrombosis. varicella objectives: the pattern of steroid responsiveness of nephrotic syndrome may change during the course of the disease in children with steroid sensitive nephrotic syndrome (ssns) and/or in different populations. patients and results: a prospective cohort study was conducted in centers. patients who were initially diagnosed as ssns in and followed for five years were included. standard questionary forms from children( boys) with a mean age of . years ( months- years) at presentation were submitted for entry to data coordinating center. / patients who showed initial steroid sensitivity with a follow-up period of at least one year ( - years) were included in the study. seventy three ( . %) children remained in sustained remission at year; nine patients showed steroid resistance. / patients were followed for years, whose clinical course were sustained remission in ( %) and steroid resistance in ( %). steroid response rate from to years remained stable ( - %). eight children out of totally patients who were steroid sensitive initially, became steroid resistant in the first year. the remainder showed steroid resistance at the nd year ( ), at th year ( ) or at th year ( ) . renal biopsy was performed in children who developed steroid dependency or steroid resistance. nine patients revealed fsgs, minimal change disease, mesangioproliferative gn, membranoproliferative gn, one igm nephropathy. only two patients who had minimal change nephropathy in initial biopsy progressed to fsgs after and years. conclusion: steroid response rate was between - % and steroid resistance was - % in years follow-up. secondary steroid resistance within the first year of presentation seemed to be predictive for their subsequent courses. the need of biopsy was not high. ssns seemed still as a relatively benign condition in our population. the aim of this study was to asses the changes in coagulation/fibrinolysis system in chronic renal disease (crd) by measuring plasma levels of von willebrand factor (vwf) and plasminogen activator inhibitor - (pai- ). we studied children ( - years old) with nephrotic syndrome (ns): minimal change disease (n= ), focal segmental glomerulosclerosis (n= ), mesangioproliferative glomerulonephritis (n= ), membranoproliferative glomerulonephritis (n= ). relapse of the disease was observed in patients. healthy age matched children served as controls. serum levels of pai- : ag and vwf were measured by elisa. results. pai- and vwf levels were elevated in all morphological forms of ns in relapse and remission compared with controls (p< , ) except the mcd remission in which they were the same as controls (p> , ). the highest levels of pai- and vwf were discovered in relapse of proliferative forms (mespgn, ± ng/ml and , ± , me/ml, respectively; mpgn, , ± , ng/ml and , ± , me/ml, respectively) compared with nonproliferative (mcd , ± , ng/ml and , ± , me/ml, respectively; fsgs , ± , ng/ml and , ± , me/ml, respectively, p< , ). conclusion. these data suggest activation of coagulation/fibrinolysis system in relapse of ns and the absence of normalization in the remission phase. our results confirmed that more severe fibrin formation via activation of intraglomerular coagulation and fibrin accumulation is characteristic for mpgn, likely by deficiency of the fibrinolysis system. introduction. several recent case reports suggest that rituximab (rtx) could be an effective treatment for idiopathic nephrotic syndrome. in a retrospective study, data were collected from patients (mean age: . years) treated with rtx for steroid dependent nephrotic syndrome (mean duration of the disease: months). four of were treated during a remission period. eight of were treated in association with one or several other immunosuppressive (is) treatments (prednisone, anticalcineurin, mycophenolate mofetil). rtx efficacy was admitted when the previous is treatment was withdrawn or significantly tapered-off, or when the proteinuria disappeared with no other change than rtx treatment. a complete b-cell depletion was confirmed in all patients when assessed ( / ) even when rtx was infused during a period with nephrotic proteinuria. rtx was considered to be effective in cases especially when given in association with other immunosuppressive treatment during a period with remission of proteinuria ( / success, follow-up to months). conversely rtx failed to induce remission among patients who were treated during a proteinuric period with no other immunosuppressive drug ( / failures). finally rtx was considered to be effective among of patients treated in association with other is drugs during a proteinuric period (follow-up and months). there was no significant side effect during rtx infusion. delayed side effects were observed for patients: case of neutropenia and pneumocystis pneumonia and case of hypogammaglobulinemia. conclusion. rtx is an effective treatment in a subset of patients with severe steroid dependent nephrotic syndrome. further prospective data are necessary to determine if rtx could become an alternative to other immunosuppressive drugs in patients with toxic side effects. infections are leading causes of death in lupus patients. disseminated histoplasmosis has been commonly documented in immunocompromised patients including lupus patients. we report a case of lethal cerebral histoplasmosis in a child originating from french guyana. lupus disease was revealed by typical malar rash. she developed a class ii lupus nephritis treated with prednisone and azathioprine. then she developed restrictive lung disease, recurrent arthritis and pericarditis. later on, nephrotic syndrome revealed a class iii lupus nephritis treated with methylprednisolone pulses and mycophenolate mofetil. four years following the onset of the disease, she was admitted because of febrile seizures and five months later for a febrile coma. repeated lumbar punctures displayed hypercellularity with depressed levels of glucose and elevated protein concentrations but sterile cultures. according to the presence of high titers of lupic specific antibodies and cerebral mri suggesting vasculitis, neurological flare of lupus was considered and immunosuppressive treatment was increased (methylprednisolone and cyclophosphamide pulses, plasma exchanges). a repeated lumbar puncture evidenced presence of histoplasma capsulatum. despite antifungic treatment the child died. our report emphazises the difficulty to discriminate opportunistic infections from the wide spectrum of lupus clinical features. symptoms of infection may mimic those of lupus, or conversely, may be masked by immunosuppressive drugs. infection screening should take in account clinical feature as well as endemic context. our report is the first case of isolated cerebral histoplasmosis in a child with systemic lupus. renal manifestations of mitochondrial cytopathies have been described, but nephrotic syndrome with respiratory chain disorders (rc) was described extremely rarely in infancy. we report a months-old boy with a mitochondrial cytopathy preceded by months history of steroid-resistant nephrotic syndrome. on admission his clinical condition was deteriorating rapidly with gross oedema, ascites, hypertension and oliguria. fundoscopic examination revealed salt-pepper sign which was thought to be consistent with intrauterine infection (iui) at that time. however, serologic and microbiologic investigation of iui was inconclusive. a sensorineural hearing loss was found to associate his findings. podocin mutation was negative. a percutaneous renal biopsy was undertaken and revealed diffuse mesengial sclerosis. a significant decrease in mitochondria was observed on electron microscopic examination. the child progressed to end stage renal failure and was successfully managed by peritoneal dialysis. during his follow-up a fine tremor was observed in his hands and cranial mri revealed demyelinisation in left thalamus and occipital lobe. steroid resistant nephritic syndrome, sensorineural hearing loss, ocular and neurologic findings has led us to be suspicious about mitochondrial cytopathy and muscle biopsy was done. though muscle biopsy was normal, the results of biochemical analysis showed a deficiency of the respiratory chain complex iv (cytochrome c oxidase) (rc iv). the clinical phenotype and the deficiency of respiratory complex iv thought to be compatible with deficiency of the cytochrome c oxidase deficiency protein cox . nephrotic syndrome with rc disorder were described extremely rarely in infancy. based on these observations, we suggest that rc disorders should be considered in patients with early onset nephritic syndrome. human parvovirus b (hpvb ) was identified as the cause of a self-limited childhood febrile illness with rash, namely erythema infectiosum. most of hpv-b infections are usually mild or asymptomatic, but in some cases infection is associated with serious systemic complications. renal involvement in patients with hpvb infection was discussed in recent, mostly anecdotal, case reports. the majority of these reports were described in adults, whereas only a few cases of childhood were defined whom presented with mesangiocapillary proliferative glomerulonephritis, fsgs or tubulointerstitial nephritis. a literature search revealed no cases of acute endocapillary proliferative glomerulonephritis in childhood. a -year-old girl was admitted with fever, cough, maculopapuler rash, hemoptysis, dark-colored urine, multiple lymphadenopathies, hepatosplenomegaly. she developed acute nephritic syndrome during the course of this complex clinical features. laboratory data showed proteinuria, hematuria, hypocomplementemia, the presence of igm and igg antibodies to hpvb and positive reaction of serum hpvb dna using a polymerase chain reaction. renal biopsy showed acute endocapillary proliferative glomerulonephritis with coarse granular c depositions in a "starry sky pattern" which is more peculiar to poststreptococcal glomerulonephritis. electron microscopy revealed subendothelial and hump-shaped subepithelial dens deposits. there was no evidence of either a mycobacterial or a streptococcal infection and the diagnosis of goodpasture syndrome and connective tissue disorders were excluded by clinical and laboratory investigations. based on the literature review, this case represents, to our knowledge, the first time that a direct relationship between parvovirus infection and acute endocapillary proliferative glomerulonephritis has been demostrated in a child. objective: the purpose of this retrospective cohort study was to report the clinical course of children and adolescents with focal segmental glomerulosclerosis (fsgs) according to steroid response. methods: the records of patients with biopsy-proven fsgs admitted between and were retrospectively reviewed. demographic, clinical and laboratory data at entry and at the end of the follow-up were analyzed. the patients were classified according to the initial prednisone response into two groups: group (g ): response (complete or partial remission) (n= ) and group (g ): non-response (prednisoneresistant) (n= ). renal survival analysis was performed using the kaplan-meier method. results: the median age at admission was . years (iq range: . ± . yr) in g and . years (iq range: . ± . yr) in g . seventeen patients ( %) of g , and patients ( %) of g presented with hematuria at admission, and ( %) children of g and ( %) of g presented blood pressure levels above the th percentile. g presented a higher h proteinuria ( . mg/ h) at admission when compared to g ( . mg/ h, p= . ). median follow-up time was . years in g and . years in g . the renal survival rate was % at years and % at years in g , % at years and % at years in g . conclusion: progressive renal insufficiency was more frequent in patients with fsgs who have initial resistance to prednisone therapy. objectives of the study: adults with chronic kidney disease (ckd) show impaired immune status. in this study, the profile of mononuclear cell subsets was related to the age and actual gfr in children and compared to healthy controls. methods: the expression of lymphocyte surface antigens was evaluated on peripheral blood (pb) mononuclear cells using three-color flow cytometry in children with ckd (stage - ) on conservative treatment. we analyzed absolute and relative numbers of total leukocytes, total lymphocytes and subsets: cd +, cd +, cd +cd +, cd +cd +, cd -cd / +, cd +hla-dr+, cd +cd +, cd +, cd +αβ+, cd +γδ+, cd ra+, cd ra+cd +, cd ra+cd +, cd ro+, cd ro+cd +, cd ro+cd +, cd +cd +. results: in younger ckd children (below years) absolute numbers of cd +, cd +cd +, cd +cd +, αβ+t, γδ+t cells and cd /cd ratio was higher, the percentage of cd +cd +, cd ra+cd +, cd ro+cd +, cd ro+cd +, αβ+t cells and the absolute number of cd ro+cd + cells was lower than in the oldest group. in children with the lowest gfr (below ml/min) the percentage of cd +, cd + was lower, the absolute number of cd +cd +, cd +, and the percentage of nk-cells, cd +cd +, cd +, cd ro+cd + cells was elevated as compared to ckd stage group. the absolute number of cd +, cd +cd +, cd ra+cd +, αβ+t, γδ+t cells and percentage of total lymphocytes, cd +, cd +cd +, cd + was lower in ckd children than in controls. conclusion: impaired immune status is observed already in early stages of ckd. progressive disturbances in pb lymphocytes percentage mostly in the naive and memory t cells and the shift in the cd /cd balance were found in pre-dialysis children with ckd. with progressive loss of renal function, we observed an increased expression of activation markers on t cells such as cd or cd . introduction: relapse of steroid resistant nephrotic syndrome (srns) after renal graft occurs iñ % of the pediatric patients. medical management is based on increased immunosuppression with the use of iv cya and plasma exchanges (pe). however, this strategy fails in ~ % of the treated patients. new immunosuppressive agents may improve the outcome of relapsing srns post transplant. case report: a -year-old boy with srns reached esrd and received a cadaveric kidney transplant after two years on hemodialysis. the immunosuppressive regime was cya, mmf and steroids. seven days post transplant gross proteinuria ( g/day) reoccurred. iv cya was administered over two months (blood level: ~ ng/ml) associated to pefloxacine and pe (n= ), and followed by oral cyclophosphamide (cyp), resulting in partial disease control. cyp was discontinued due to haematological toxicity after one month. proteinuria increased again from to g/day within months, despite high dose oral cya ( mg/kg/day) and mmf. etanercept (a tnf blocking agent) was introduced at a dose of mg twice weekly (combined with three steroid pulses) and maintained over two months: proteinuria decreased to . g/day over days. etanercept was discontinued followed by a relapse of the ns and re-introduced eight months later, with, again a significant decrease of proteinuria to a baseline level of . g/day. conclusion: treatment with anti-tnf agents in nephrotic children has been reported once in a boy with high degree steroid dependent ns; a spontaneous decrease of disease activity over time cannot be excluded in this patient and might jeopardize data interpretation. our case is the first report of successful antiftreatment despite a constantly high activity of the nephrotic syndrome, demonstrated by relapse after discontinuation of etanercept while the patient was on post transplant immunosuppression. fournier´s gangrene (fg) is defined as a specific, quick and progressive form of synergic necrotizing fasciitis of multi-bacterial origin that affects perineum muscular fascia, genital region and surrounding areas with skin gangrene due to thrombosis of subcutaneous blood vessels. it describes the clinical case in a male preschooler of four years of age with idiopathic nephrotic syndrome (ns) that subsequent presented fg of the scrotum. broad-spectrum antibiotics, intravenous albumin and surgical cleaning of the scrotal necrotic tissues were indicated. pseudomona aeruginosa was isolated from necrotic tissue cultures. the later evolution was satisfactory with healing of the affected area and remission of the ns subsequent to the steroidal treatment. fg is an uncommon in children, rapidly progressive infection of the genital, perineal and perianal regions. it is characterized by a synergistic necrotizing fasciitis leading to thrombotic occlusion of small subcutaneous vessels and development of gangrene. until now few cases have been report fg in children, and still less associate to the kidney diseases. et all ( ) described a -year-old boy presenting with steroid resistant ns developed fg of the scrotum so that to our knowledge, this patient seems be the second case reported in medical literature with both pathologies. high index of suspicion, prompt diagnosis, broad spectrum antibiotics followed by wide debridement is the key to successful treatment. objectives of study: to evaluate a long term experience on iga nephropathy (igan) presenting in childhood and investigate clinical and histological factors that may act as early markers of renal disease progression. methods: retrospective review of data from children and adolescents with biopsy proven igan in the last years. demographic and clinical data at presentation and severity of renal histological involvement were recorded and related to renal dysfunction markers identified at the last review. results: twenty-five patients were studied ( m/ f) with median age at onset of ( - ) and follow-up of ( - ) years. on presentation recurrent macroscopic hematuria was present in patients, microscopic hematuria (mh) in , proteinuria in ( nephrotic), hypertension in and transient acute renal failure in . renal histology findings were focal mesangioproliferative in , focal proliferative in , diffuse proliferative in and focal sclerosing glomerulonephritis in . six patients showed tubulointersticial and extraglomerular vascular lesions (tevl) with glomerular crescents in . on follow-up, patients remitted ( spontaneously, with ace inhibitors). of the remaining, were kept on ace inhibitors due to proteinuria ( ), hypertension ( ) or both ( ). one patient (with focal glomerulosclerosis and tevl) developed esrd within a year after diagnosis, despite treatment. at last review, patients presented progressive renal disease with a mean decrease of ml/min/ , m in gfr per year. these ( m/ f) showed mainly mh and proteinuria at onset and tevl. conclusions: early renal function impairment in childhood igan can occur and may be associated with mh and proteinuria at presentation and with focal glomerulosclerosis and tevl on renal histology. proteinuria persistence in a number of patients emphasizes the need for long term followup into adulthood. adhesion molecules, il- +p and cd +cd + and cd +cd + lymphocyte subsets in childhood nephrotic syndrome background: parathyroid hormone (pth) can modulate t cell activation and proliferation through as yet incompletely identified mechanisms. since the pth receptor (pthr) is a g protein-coupled receptor and thus a candidate for association with lipid rafts, and since pth has been shown to alter membrane phospholipid metabolism, we explored the relationship of the pthr with lipid rafts in human t cells. methods and results: we found by flow cytometry that neither physiologic nor pathologically elevated concentrations of pth affect the up-regulation of the raft marker gm- or of the partially raft-associated activation marker cd in purified t cells stimulated with phytohemagglutinin (pha). moreover, we detected the pthr exclusively in non-raft fractions of these cells after sucrose gradient separation. conclusions: these data indicate that in human t cells, the pthr does not associate with lipid rafts and that pth does not modulate these domains. accordingly, other mechanisms underlying the actions of pth on human t cells need to be sought. the direction and magnitude of potassium transport in nephron segments depend on the sitespecific distribution of transporters in tubule cell membranes. potassium depletion has been demonstrated to be associated with altered sodium reabsorption in renal tubule segments. we examined whe her potassium transporters protein expression is associated with altered abundance of major renal na + transporters, that may contribute to the development of hypokalemia in lp. after weaning rats (n= ) were fed days with lp diet ( %), then they were recovered with a normal protein diet ( %, rp), each group had a control group ( %, np). we examined the changes in the abundance of the na + /h + exchanger (nhe ), na + k + atpase, na + k + clcotransporter (bsc- ), na + clcotransporter (tsc), epithelial sodium channel (enac) subunits and romk in kidneys of lp, np, rp rats. controls were normalized to . results reduced clcreat (ml/min) in lp vsnp ( . ± . vs . ± . ), hypokalemia ( . ± . vs . ± . meq/l) and increased fe k+ ( . ± . vs . ± . %) were demonstrated. immunoblotting revealed that the abundance of nhe in cortex was severely decreased. the amount of bsc- ( . ± . , p< . ) and tsc ( . ± . , p< . ) protein levels were enhanced in the inner stripe (isom) and outer stripe of the outer medulla (osom), respectively. romk protein levels were increased in lp ( . ± . , p< . ), the protein abundance of the enac subunits α, β and γ was increased near . fold each in response to lp. na + k + atpase protein levels showed no differences in cortex and osom. after rp, na + transporters expression returned to control values. conclusion: increased expression of bsc- , tsc, enac subunits and romk, contributing to distal potassium secretion was shown in hypokalemia from lp. a role of aldosterone may be suggested. v. belostotsky , mz. mughal , j. berry , n. webb royal manchester children's hospital, pediatric nephrology, manchester, united kingdom st. mary's hospital, pediatrics, manchester, united kingdom manchester royal infirmary, vitamin d laboratory, manchester, united kingdom aims: to describe the prevalence of vitamin d deficiency in south asian and white uk children with renal disease. to establish how decreased levels of vitamin d affect pth in patients with a normal gfr. methods: children aged - years were enrolled in the study: were of white uk, of s asian and of other ethnic origin. were on dialysis, had chronic renal failure, had various renal disorders with normal gfr (> ml/min/ . m ), had a transplant ( with anormal gfr). blood samples were collected to establish the levels of -vitamin d ( -ohd); pth; creatinine. -ohd concentration < ng/ml was defined as vitamin d deficiency; levels between - ng/ml as vitamin d insufficiency. serum pth of . - . pmol/l was defined as normal. results: the prevalence of vitamin d deficiency/insufficiency was higher in s asian ( %) than white ( %) children (p< . ). ofthe ( s asian, white and other) children with normal gfr / s asian and / white children had pth concentrations > . pmol/l. of these / s asian, / white children had low levels of -ohd (p= . ). of transplant patients with reduced gfr, of with a high pth had low -ohd levels, compared with of with a normal pth (p= . ). conclusions: many s asian children attending our renal clinic are vitamin d deficient/insufficient and the prevalence of this problem is significantly higher than that in the white population. high pth values in the setting of a normal gfr can often be explained by vitamin d deficiency and should result in serum -ohd levels being measured. nephronophthisisis a rare recessive autosomal disease which may be either limited to progressive chronic tubulointerstitial nephritis or associated with extrarenal involvement (eye, liver, central nervous system, etc.); mutations/deletions have been found in at least nphp genes. fibrous dysplasia is a benign skeletal lesion due to an activating mutation inthe gene that encodes the α subunit of stimulatory g protein and occurs after fertilization in somatic cells; it involves one or several bones and may be part of mccune albright syndrome. we report on a boy with fibrous dysplasia of bone diagnosed at yrs of age, who underwent protocol renal function tests at yrs of age in the follow-up of pamidronate treatment. inulin clearance was ml/min/ . m and potassium reabsorption rate was . % where as there was neither urinary phosphate wasting nor hypercalciuria. serum magnesium was decreased ( . mmol/l) without reabsorption abnormality and serumuric acid progressively increased with age. in addition, due to increasing microalbuminuria, a treatment with acei was started at yrs of age. renal ultrasonography at yrs of age showed hyperechoic reduced-sized kidneys with few microcysts. a renal biopsy (light and electron microscopy) was performed at yrs of age, which showed nephronophthisis-like lesions, i.e., diffuse interstitial fibrosis and focal thickening of tubular membrane basement. dna analysis revealed no nphp gene deletion but is still under investigation.nephronophthisis has been reported in association with skeletal involvement (coneshapedepiphyses) and fibrous dysplasia with hypophosphatemic rickets or fanconi syndrome. however no association between fibrous dysplasia of bone and nephronophthisis-like lesions has been described and may be a new picture of the nephronophtisis/medullary cystic kidney disease complex. m. bald, m. holder, h. leichter olgahospital, pediatric nephrology, stuttgart, germany puumalaviruses belong to the group of hanta viruses and are transmitted by inhalation of aerosolized particles of the red bank vole (cletriomonysglareolus) which is endemic in the alb-danube region of southern germany. infections with puumala virus were first described as "nephropathia epidemica" in scandinavia with the clinical symptoms of fever, thrombocytopenia and acute renal failure. over the last seven years three boys with acute renal failure were admitted to our hospital after vacationing in the region endemic for puumalavirus. all three presented with high fever, influenza like symptoms aswell as pronounced abdominal or flank pain. they showed a decreased gfr ( , and ml/min/ . m , respectively) with hematuria and proteinuria. cbc revealed no leucocytosis or anemia, but thrombocytopenia in of the patients. they had no oliguria, but patients had marked polyuria in the recovery phase of renal function. arenal biopsy in the boy with the most severe presentation showed diffuse tubular damage. puumala virus infections were confirmed in all patients by serological tests, and renal function normalized within - weeks. nephropathia epidemica due to puumala virus infections have to be included in the differential diagnosis of acute renal failure in patients from endemic regions. severe abdominal or flank pain are common symtoms in these patients; renal failure is transient and the general prognosis is good. aims: the objective of this study is to determine the relationship of urinary calcium excretion (uca) with sodium and protein intake in a pediatric population of families with low income. methods: children, f and m, ages to years from families with income <$ /month were studied. protein intake was estimated with a -day dietary record. a nonfasting urine sample was collected for dipstick, calcium, creatinine, sodium, potassium, urea and uric acid. urinarycalcium/creatinine (ca/cr), sodium/potassium (na/k), uricacid/creatinine (au/cr) and urea/creatinine (u/cr) ratios were calculated. children with a urinary ca/cr > . mg/mg, were submitted to a day period of high sodium foods restriction after which a second urine sample was collected. results: mean (x) and standard deviation (sd) for ca/cr, na/k, au/cr and u/cr ratios were: . ± . mg/mg, . ± . meq/meq, . ± . mg/mg and . ± . mg/mg respectively. the th percentiles for ca/cr, na/k, au/cr and u/cr were . , . , . and . respectively. x±ds for protein intake was . ± . g/kg/day. the incidence of hypercalciuria was . % in the initial urine sample and . % in the second. correlation was significant between ca/cr ratio and na/k ratio (r: . , p< . ), acu/cr ratio (r: . , p< . ) and u/cr (r: . , p< . ), not significant between ca/cr ratio and protein intake. conclusions: the incidence of hypercalciuria in this serie is lower than previously reported values in venezuela for the general pediatric population and decreases further when sodium intake is controlled. although no correlation was found between uca and protein intake, we could speculate that protein intake near to the daily recommended requirements of g/kg/day, could be a possible reason for the lower incidence of hypercalciuria in this population. -year old girl presented with rapid onset of muscular weakness and a short history of severe dysphagia, dysphonia nad significant wasting. on examination, she was dystrophic (bmi , ) and had signs of myopathy. laboratory findings confirmed myopathy (cpk , ukat/l, ast , ukat/l, myoglobin ug/l). there was striking hypokalemia (s-k , mmol/l) suggesting hypokalemic paralysis. diagnosis of distal renal tubularacidosis (drta) was based on confirmation of hyperchloremicmetabolic acidosis, severe hypokalemia, high urinary ph and positive value of urinary anion gap (s-cl mmol/l, ph , , be , urinary ph , ). there was evidence of other signs of renal tubular impairment (urinary beta- -microglobulin mg/l, glomerulo-tubular proteinuria , g/ h). autoimmune tests (high positive rheumatoid factor, anf, ena ss-a/ro, ss-b/la, high circulating immunocomplexes) and low values of sialometric measurements ( , ml/ x minutes) revealed primary sjogren´s syndrome as the underlying cause of drta. the renal biopsy confirmed chronic tubulo-interstitial nephritis compatible with this diagnosis. full recovery of muscle weakness and laboratory findings of hypokalemia and acidois followed potassium and alkali replacement. corticosteroids were administered with subsequent addition of cyclosporine a because of disease activity. conclusion: primary sjogren´s syndrome is a rare diagnosis in childhood and adolescence and should be considered in patients presenting with hypokalemic paralysis due to drta. m. caletti, h. lejarraga, s. caíno, a. jiménez introduction: ndi is a chronic, genetic disease caused by an inability to effectively conserve urinary water, due to a lack of response of distal renaltubule to antidiuretic hormone. the main symptom is polyuria, polydipsia and growth impairment. objective: to evaluate long term growth in height and weight of children with ndi. patients and methods: sixteen patients with ndi attending hospital for a median period of . years (range . / . yrs) were studied. treatment consisted of indometacine, hydroclorotiazide and amiloride (iha). height and weight was measured with standardized anthropometric techniques. z scores(sds) for all measurements were calculated according to national standards. results: all children responded favourably to treatment. mean birth weight sds was not different from zero; mean height and weight at diagnosis was ± . and ± . sds respectively, and at the end of follow-up was ± . and ± . respectively. the majority of patients´s growth curves evolved below the th centile. ten out of children experienced some catch up in height (mean height gain: . sds (r: - . / . )). mean weight gain during follow-up was . sds (r: . / . ) . mean gain in body mass index was . sds (range . / . ). in the two patients who attained adult height, adolescent growth spurt was normal, and final height was within normal limits for standards and for parental height. correlation coefficient between gain in height andage at diagnosis was ± . . conclusion: although mean height at follow-up was below the expected normal value, combined therapy with iha is compatible with some catch up growth in height and weight. the lower the initial height, the greater the height gain. adherence to treatment is essential for normal growth in children with ndi. body growth of children with steroid-responsive idiopathic nephrotic syndrome m. noer, i. irwanto, n. sumiarso, m. chalim soetomo hospital, school of medicine airlangga university, department of child health, surabaya, indonesia objectives of study: the present study was designed to evaluate the statural growth of children with steroid-responsive idiopathic nephrotic syndrome, attending the pediatric nephrology unit department of child health, school of medicine airlangga university, soetomo hospital, surabaya, indonesia, with a minimum follow-up of years. methods: anthropometrice valuation included weight, height, and growth velocity expressed as mean and standard deviation scores (sds), relative to the normal population (nchs/cdc ). these indices were analyzed at admission and then every months of follow-up. all patients were treated with prednisone, according to indonesian consensus of management of idiopathic nephrotic syndrome in children. results: of children ( boys and girls), patients ( boys) aged / years to years (mean . years) were analyzed. initial mean height and z score (height for age) were . ± . cm and - . ± . , respectively. mean height and z score (height for age) of the last follow-up were . ± . cm and - . ± . , respectively. mean growth velocity were . ± . cm/year where boys ( . %) had growth velocity less than cm/year. total cumulative dose of steroid during years of follow-up were . ± . mg or . ± . mg/kgbw. conclusions: the cumulative dose of steroid up to . mg/kg body weight in children with nephrotic syndrome during years of treatment did not influence their growth velocity. background: recently it has been reported in adult patients (pts) that deterioration of renal function was associated with the lost of nocturnal blood pressure (bp) dip and enhanced urinary sodium (una) and protein (uprt) excretion during night. objectives of study: to investigate the circadian rhythms of bp, una and uprt in children with chronic kidney disease stage i (ckd i). methods: in pts ( boys) aged . ± . years with ckd i (chronic glomerulopathy confirmed by renal biopsy in % pts), hour bp was monitored during daytime (d) and nighttime (n) and urinary samples for uprt, urinary creatinine (ucr), and una, were collected for both periods. results: serum creatinine-based gfr was ± ml/min/ . m , uprt ranged from to (median ) mg/ h, and una from to (median ) mmol/ h. in general we found a highly significant nocturnal decrease in systolic bp (from to mmhg), diastolic bp ( to mmhg), mean arterial pressure ( to mmhg), heart rate ( to /min) urinary output (uo), una and uprt. the regression equations were as follows: uod (ml/m /h)= + . xuon (ml/m /h); unad (mmol/l)= . + . xunan (mmol/l); uprtd (mg/m /h)= . + . xuprtn (mg/m /h) and urinary osmolality (us) d= + . xusn. nocturnal decrease of uo correlated with nocturnal decrease of ucr and uprt, and nocturnal decrease of uprt correlated with nocturnal decrease of uo. more than half of the patients were classified as non dipper. they differ significantly from dipper only in night/day changes of us. conclusion: night/day changes of uo, una and uprt in pts with ckd i may be calculated from the given regression equations. these changes are not correlated with nocturnal bp decrease. non dippers have greater nocturnal change of us compared to dippers. follow-up of these parameters will clarify their importance in progression of ckd. autosomal autosomal dominant proximal renal tubular acidosis (prta) it is described l. g. brenes ( ) at seven members of one family. we diagnosed seven members of the afghani family with prta: mothers and children ( girls, boy from , till years) with hyperchloremic metabolic acidosis. pedigree analysis suggested an autosomal dominant inheritance pattern. observable patients did not have ricket and nephrocalcinosis. deafness and ocular abnormalis are absent. the plasma hco concentration is decrease in the range of , to , mm/l, minimal urine ph is < , . parameters of blood creatinin and glomerular filtration rate were normal. urine calcium excretion was normal. therapy strategy of prta at observable patients provides high dozes of citrates/bicarbonates - mmol/kg per h. all india institute of medical sciences, division of nephrology, department of pediatrics methods: retrospective case-search. data of previously reported prospective trial (n= ) was also included. results: all except patients had previously been treated with both levamisole and cyclophosphamide. forty-two cases qualified for the study and were administered mmf for a mean duration of . months ( % ci, . , . /patient in the first months of treatment and . episodes (n= ) in next six months of mmf treatment (p< . ), an average reduction of % ( % ci, . , . ) from the pre-mmf phase. nine ( . %) patients had no relapses while on mmf therapy we present sibling cases of as with heavy proteinuria at early childhood. a boy ( year-old) and a girl ( yearold) were diagnosed as x-linked as. since a boy developed persistent heavy proteinuria (up/cr . ) with macrohematuria andresistant to arb/acei therapy, we treated him with cyclosporina (csa) that could lead to complete remission. to investigate pathomechanism of proteinuria, we tested immunostaining for slit diaphragm associated molecules (nephrin, podocin), gbmassociated molecules (laminin, perlecan, agrin) and podocalyxin using frozen sections from his firstand second biopsy and girl's one (up/cr . ). in the specimens from boy's first biopsy and girl's one, light microscopy revealed mild mesangial proliferation and no differences ofthe expression with perlecan, agrin and podocalyxin compared with controls. however, when determined laminin isoforms, fetal type laminin (alpha beta gamma ) wasdistinctly observed in the gbm, whereas that was localized only inmesangium with controls. interestingly when compared mature laminin isoform (alpha beta gamma ), beta chain was specifically less expressed in the gbm. however there were no differences of expression of these molecules in the specimens between pre-and post-treatmentwith csa. in boy's second biopsy, % glomeruli were detected to becollapsed. together with the recent report showing that laminin beta mutation causes congenital nephrotic syndrome factor v leiden mutation and steroid resistant membranous glomerulonephritis: a case report m. buyukcelik , m. karakok turkey renal compications of d-penicillamin therapy in wilson's disease sami ulus children's hospital, department of pediatric nephrology sami ulus children's hospital, department of pediatrics sami ulus children's hospital hass classification), treatment and outcome. thirty-nine patients; boys ( . %) and girls ( . %) time of the last examination (median months, min< year) after the admission as a long term follow-up. clinically, group i; while microscopic hematuria was detected in patients, patients had repeated attacks of hematuria, had isolated mild proteinuria/hematuria, group ii; patients had nephritic, had nephrotic syndrome and had both. biopsy grades in the patients: %, % had grade i, . %, % had grade ii, %, % had grade iii, %, % had grade iv in group i and ii, respectively. group i and ii patients recovered completely (no hematuria and proteinuria) . %, . % as well as %, %, short-term, longterm, respectively. while recovery rates in fish-oil and/or ace-inh treatment group was . % and . %, in corticosteroids group, it was %, % short-term, long-term,respectively. no patients who received immunosuppressive treatment had improved. however, ( . %) patients would suffer from esrd. initial presentation, severity of renal involvement and type of the treatment were not found to have a prognostic value (p> . ). in children, igan is characterized by extreme pathogenetic, clinical and histological polymorphisms radojevic university of belgrade, faculty of medicine, institute of pathology, belgrade, serbia institute of mother and child health of serbia, belgrade, serbia university children's hospital, department of nephrology, belgrade, serbia celiac disease hla aplotype in children with nephrotic syndrome s i (if) or mcp or with anti-cfh autoantibodies. varicella hasn't been described as a triggering event of ahus. we report two cases of ahus associated with complement dysfunction revealed after varicella infection. case . a five year-old boy presented with non post-diarrheal hus, days after varicella. serum creatinine was μmol/l, hemoglobin . g/dl, schizocytes %, platelets g/l. glomerular filtration rate normalized within days. search for shiga toxin-producing e coli in the stools and serum anti-lipopolysaccharides antibodies were negative. plasma c , cfh and if levels were normal. no mutations of cfh and if were found. mcp cell-surface expression was decreased and a c f mutation in mcp exon was demonstrated. case . a four year-old girl had ahus days after varicella at that time, complement system study showed normal c level ( mg/l, normal to mg/l), normal cfh level ( %, normal - %), but the presence of anti-cfh autoantibodies. no mutations of cfh, if or mcp were found. in conclusion, these cases outline that varicella can be the triggering event of ahus associated with complement dysregulation about ( . %) children had relapses after initial remission. various infections were responsible for relapsed with in / ( . %) who had relapses. about children ( . %) relapsed with out cause where as poor compliance was observed in ( . %). overall infection and relapse rate was . and . /pt/yr respectively. among children with infections, most common types of infections were acute respiratory infections (ari), diarrhea and uti seen in ( . %), ( . %) and ( . %) of cases respectively. other types of infections like malaria, peritonitis, skin infection and pulmonary tuberculosis were seen serum pai- and tgf-beta levels in profliferative forms of glomerulonephritis in children russian federation research centre for child health rams, department of pathology, moscow, russian federation we aimed to investigate serum levels of plasminogen activator inhibitor - (pai- ) and transforming growth factor-beta (tgf-beta ) in children with proliferative forms of glomerulonephritis (gn). children were examined ( - years old) with gn (steroidresistent ns, n= ; steroidsensitive ns, n= , isolated haematuria, n= ) and healthy age matched controls. mesangioproliferative gn (mespgn) was detected in patients, membranoproliferative gn (mpgn) in cases. serum levels of pai- : ag and tgf-beta were measured by elisa. results. the highest levels of pai- : ag and tgf-beta were observed in relapse of mpgn: , ± , ng/ml and these results confirm prosclerotic effects of pai- and tgf-beta via increased fibrin deposits and extracellular matrix accumulation in the renal tissue and promotion of disease progression rituximab treatment for idiopathic nephrotic syndrome: a retrospective study of cases v. guigonis , a. dallocchio , m. dehennault urinary and serum annexin v levels in children with steroid sensitive and steroid-resistant nephrotic syndrome hôpital robert debré-aphp, pediatric intensive care unit indonesia this study was aimed to evaluate the efficacy of pulse dose of cyclophosphamide in children with srns admitted in child health department faculty of medicine university of indonesia/cipto mangunkusumo hospital jakarta between - . -month period, one child died, and the rest ( children) did not complete the regimen. five out of children who finished the treatment had remission, while others were still experiencing heavy proteinuria. remission was achieved in various time, children were in remission after the first dose, in children it was achieved at the third and sixth dose. in further follow-up time; one child remained in remission, one child had relapse when still receiving cpa, children got relapse one month after stopping cpa, and one child had relapse after months ceasing cpa. nausea and vomiting were found in children indonesia this study is to evaluate the anthropometric measurements of children with nephrotic syndrome methods: a descriptive retrospective study at child health department, cipto mangunkusumo hospital, jakarta. data were collected from medical records of nephrotic syndrome %) irns; body height and body weight of <p was found in none of frns/sdns, / ( , %) srns, and / ( , %) irns; bmi >p was found in / ( , %) frns/sdns, / ( , %) srns, / ( , %) irns. conclusion: the percentage of children with frns/dsns and srns with body height <p il- ) during active stage (as: urinary protein > mg/m per hour, serum albumin < . g/dl) and remission stage (rs) in ss-mcns. a total of patients with ss-mcns ( of patients with as and of patients with rs) and healthy children were recruited for studies. the mean±sd of serum il- , se-selectin and sicam, levels were significantly higher in patients with as than in patients with rs ( ± . / . ± pg/ml; . ± . / . ± . ng/ml and . ± . / . ± . ng/ml respectively, p< . ). in spite of, higher levels of il- , se-selectin and sicam in patients with as than controls, difference was not statistically important. the percentage of cd +cd + lymphocyte subsets were statistically grater in patients with as severe proximal renal tubular acidosis in pearson syndrome birth weight was g. pallor was initially noted during the neonatal period and referred to our hospital with anorexia, vomiting, diarrhea, weakness, and increased pallor at wk of age. on the physical examination she was pale and the other systems were unremarkable. investigation showed hypoplastic anemia, and bone marrow examination showed cytoplasmic vacuolization of both myeloid and erythroid precursors, and maturation arrest of granulopoesis. family history was negative for hematological disease. the diagnosis ps was considered on the basis of early severe refractory anemia associated with vacuolization of bone marrow precursor cells and ring sideroblasts. treatment was started consisting of vitamine b and folic acid. she was followed with growth retardation, moderate anemia and leucopenia up to age . . at that age, the girl was readmitted with severe vomiting and dehydration. on admission, she had moderate metabolic acidosis, hypokalemia, high plasma lactate, and hypophosphatemia. further investigations showed tubuler proteinuria, glucosuria, aminoaciduria, and defective bicarbonate reabsorbtion in the proximal tubule. she developed refractory metabolic acidosis resulting in a cardiac and respiratory failure and death. to confirm the diagnosis of ps, molecular studies were performed bp common deletion was found medial calcification in intact human arteries from children with chronic kidney disease is associated with apoptosis and osteogenic differentiation -clinical and laboratory correlations r using intact human vessels we studied the phenotypic changes in medium sized arteries ex vivo and in vitro after exposure to ca and po . arteries were retrieved during insertion of pd catheters or at transplantation from children: dialysis (n= ), ckd stage iv (n= ) and compared with mesenteric vessels from controls. vessel rings were incubated with graded concentrations of ca and po upto days. calcium and alkaline phosphate (alk) were measured by cresolphtalein complexone and colorimetry. immunohistochemistry for bone marker proteins, inhibitors of calcification and apoptosis were performed. laboratory findings were related to patient's clinical and biochemical parameters, carotid intimamedia thickness (cimt) and coronary calcification. vessels from ckd or dialysis patients had increased baseline vessel wall ca compared with controls (p= . ). when exposed in vitro to ca and/or po , dialysis vessels showed greater calcification than those from controls or ckd patients (p< . ). in the presence of elevated po even a small increase in ca increased calcification (p< . ). calcification was associated with apoptosis (tunel +) and could be inhibited using apoptosis inhibitor zvad. alk in ckd and dialysis vessels and along with upregulation of bone-markers suggests an osteogenic conversion of vsmcs using our unique in-vitro model, we have shown for the first time that vascular damage induced by elevated ca-po as well as factors specific to dialysis primes vessels for rapid progression of medial calcification altered expression of major renal na + and k + transporters c. ruete , p. vallés , university of cuyo, department of pathology turkey the effect of corticosteroid therapy on bone metabolism in nephrotic syndrome was examined. sixty-nine patients with idiopathic nephrotic syndrome (age: . ± , years) and healthy controls (age: . ± , years) were divided into groups: group : patients who were on remission but still receiving steroid therapy, group : patients who were on remission and free of steroids within the last year and group : patients with active nephrotic syndrome and receiving steroid therapy. serum total calcium, ionized calcium, phosphorus, alkaline phosphatase, magnesium, parathyroid hormone, (oh)d, serum cystatin c, urine protein, urine creatinine and urine cystatin c levels measured in all patients including the control group. in addition, lumbar spine bone mineral density z scores were measured in the patient group objectives of study: we evaluated the clinical, laboratory and urinary tract echosonographic findings in patients with ah and rh. methods: there was prospective clinical study, included patients (mean age . ± . ) with ih (normocalcemic, normophosphatemic, with hours urinary calcium excretion greater than mg/kg/day) all analyzed parameters (dysuria, positive family history of urolithiasis, microscopic hematuria, urinary tract microlithiasis) showed low sensitivity and specificity, and none of the parameters could be considered reliable in differentiating ah versus rh. average value of una/cr was greater in patients with rh conclusion: none of the analyzed clinical parameters, laboratory and echosonographic parameters except values of urinary excretion after calcium deprivated diet could be considered reliable in differentiating ah versus rh. patients with rh have higher level of hours urinary calcium excretion than patients with ah. patients with rh have significantly greater excretion of urinary sodium compared with patients with ah idiopathic hypercalciuria (ih) is defined as hypercalciuria with no detectable cause. low bmd with increased fracture rate and tendency to short stature has been reported in ih patients. we aimed to perform calcaneus qus in children with ih and relate to u-ca, body height and number of prevalent fractures (fx). children ( girls, boys; patient. body height was recorded and qus was measured on both heels with cuba clinical. the -h u-caexcretion (u-ca/ h) was assessed and calculated in mmol/kg/ h. results were expressed as z-scores ±sd. czech anthropometric parameters from a survey and previously obtained qus values of the healthy czech pediatric population served as reference data. qus results were also calculated as height adjusted values with the use of heightmatched standards. u-ca/ h was matched to healthy european paediatric population values we found no correlations between fx and bua (either age-related orheight-adjusted) or fx and vos (age-related or height-adjusted). neither were there any correlations between u-ca and fx, or u-ca/ hand bua or vos, respectively. in conclusion, children with ih had normal height, normal values of bua and low vos (fc) (p) (p) (mr) (fc) (fc) (op) (p) alpay h. (p) (p) amann k. (fc), (p), (p) amanullah f. (p) (op) amaro a. (op) (op) amore a. (sy) (p) anarat a. (fc) (mr) (p) (op) (fc) (op) (p) (p) bael a. (op) (p), (p), (p) (fc) (fc) (p) balat a. (p) (p) (fc) bayazit ak. (p) (p) (p), (p), (p) (p) (p) (p) (p) bensman a. (op) (sa), (p) (p) (fc) bereczki cs. (op) (fc) (op), (fc) (op) (sy) bi yl. (p) (fc) (p) (op) (p) biyikli n. (p) (p) (fc) bocanegra v. (fc) (fc) (p) boh m. (fc) bökenkamp a. (op) (fc) (fc) (op) (op) (op) bourrier t. (p) bouts ah. (op), (fc) (op) (p) bubic-filipi lj (p) (op), (fc) (op) (p) (p) caropreso m. (op) (fc), (p) (fc) (p), (p) chartapisak w. (p) (p) (p) chaves a. (op) (p) chemodanova m. (p) (p) chen j. (op) (p) (p) (p) (p) (op) (p) (p), (p), (p) clermont mj (p) (fc), (p) (sy) codoceo a. (p) coelho g. (op) (sy), (fc) (sy) (p) (fc) (fc) (p) (fc) (p) coutinho s. (p) coviello d. (p) (fc) craig j. (op), (p) cransberg k. (fc) (p) (p) cristino s. (p) (fc) (p) cross j. (op) cruz mr (fc) (p) (fc), (p), (p) (sy) deanfield j. (fc) (p) decena-galvez a. (p) (fc) (p) (p) deguchtenaere a. (op) (p) (p) (sa), (p) (p) delucchi a. (p) (p) (p) (p) (p) (p) (fc), (p) (fc) dinda a. (p) (p) (p) (p) dittrich k. (fc) (fc) (p), (p) dötsch j. (fc), (p), (p) dragon-durey ma (p) (fc) (p) (fc) (p) (p) (p) (p) dursun i. (p) (p) (p) edefonti a. (p) (p) eke f. (fc), (p) (p) (fc), (fc), (p), (p) (fc) (p) (p) (p) evim m. (p) f faerch m. (p) fallahzadeh mh. (p) (p) (sy) feig d. (p) (p) (p) fella a. (op) feneberg r. (op) (op) (p) filler g. (fc) (p) (op) fischbach m. (op) sy) fitoz s. (p) (fc) (p) (p) fujita h. (op) (p) fujita t. (p) (p) fukuhara d. (p) (op) (p) (p) (op) (op) garnier a. (p) (p) (op) (sy) geary-joo c (fc) (fc) (op) (sy) (fc) (p) gross ml. (fc) (p) (p) (fc) (fc) (p) (p) guo w. (op) (p) (op) (p) haberal m. (p) (fc) (op), (fc) (fc), (p) (fc) (fc), (p) (fc) (p) (mr) (p) (p) (op) (op) hernández am. (p) (op) (op) (fc) (op), (fc) (p) hou jr. (p) (p) (op) (p) (fc) (fc) (p), (p) iharada a (op) (op) (p) (fc), (fc) (p) (p) (sy) (p) kathiravelu a. (fc) (op) (op) (op), (fc) (p) (p) (p) (p) (p) (p) (mr) (p) (op) (p) kondo y. (fc) (p), (p) kosuljandic-vukic d. (p) (fc) kovalski y. (p) (op) (p) (p) (p) kru èic d. (p) krylova-olefirenko a. (p) (p) (p) (p) (sy) (fc) (p) (p) kurayama r. (op) (p) (fc) lalatta f. (op) (p) (sy), (p) (op) (p) lau yw (op) (op) (p) llanas b. (op), (fc), (p) llewellyn-edwards a. (fc) (fc) (p) (fc) luis-yanes m (p) (p) (sy) maeda a. (p) maekawa k. (p) (p) (p) (p) (p) (p) (p) mak r. (sy) (op) (op) (p) (fc), (fc) (fc) (p) (sy) (fc) (p) (p) (p) medynska a. (p) (p) mehls o. (fc) (p) (fc), (p) (op) (op) (op) (fc), (p) (p) (fc) (p) molnár-varga m. (p) (fc) (fc) (p) montini g. (fc) (p) (p) (op) (p) (p) morimoto t. (op) (p) (op) (p) morita t. (p) (p) mortazavi f. (p) (fc) moscaritolo e. (p) (p) mosig d. (p) (op) (fc) (p) moxey-mims m. (fc) (op) (p) mudun a. (p) (fc) mughal mz. (p) (p) müller t. (op) müller v. (fc), (sy) müller-esterl w. (p) müller-wiefel de. (fc) (sy), (fc) (op) fc) (op) (fc) (p) nghia h. (p) niaudet p. (mr) (p) (p) nuzzi f. (op), (op), (p) nwobi o. (p) (op) (op) (p) (op) (p) (sy) (p) ostalska-nowicka d. (p) (p) otukesh h. (p) (p) (p) (p) ozen a. (p) (sy), (p) (p) (p) paik kh. (p) (p) pan'czyk-tomaszewska m. (p) (p) (fc) paripovic d. (p), (p), (p) (fc) pasqualini t. (op) fc) pastori a. (p) pászka d. (op) (op) (p) pereira a. (op) (fc) (fc), (p) (p) fc) (op), (p) raes a. (op), (op), (fc) (fc) (p) rigden s. (fc) (fc) ring e. (p) rink n. (op) (op) ristoska-bojkovska n (fc), (p) roszkowska-blaim m. (p) (p) (p) ruffo gb. (p) ruhlmann a. (op) ruiter j. (op) rumeau r. (fc) rusai k. (fc) rusnak f. (p) rüther u. (p) rutjes n. (p) rybarova s. (p) rychlik i. (p) ryckewaert-dhalluin a (p) sabasiñska a. (p) (p) sabolic-avramovska v. (p) (p) (op) safouh h. (p) saha a. (p) sahin h. (p) (op) saint-cyr c. (p) saito a. (p) (fc) sakalli h. (p), (p), (p) sakalli ercoban h. (p) (sy) (p) salusky i. (fc) (op) (p), (p) sarkissian a. (p) (p) (fc) (p) (fc) (op) sayili a. (p) schäfer b. (p) (fc), (fc), (fc), (sy), (fc), (fc), (fc), (fc) (fc) schmidt-gayk h. (mr) schmidts m. (p) schmitt cp. (fc) (op) (p) (fc) schneider-maunoury s. (p) (p) (sy), (fc) (fc) (op) (p) seeherunvong w. (fc) (p) (op) (p) semama d (op) (p) (p) (p), (p) shin yh (p) (p) (fc) (p) (p), (p), (p) siwiñska a. (p) (fc) (op) (p) spasojevic b. (p), (p), (p) spasojevic-dimitrijeva b stanic m. (p), (p), (p) stankovic a. (p) (p) (mr) (fc) (op) (mr) (p) (p) (p) (p) (p) (p) szteblich a. (op) t (fc) (p) taha g. (p) (p) taheri derakhsh n. (p) (p) (p) (fc), (p), (p) tan ph (p) (p) (fc) (p) (op) (p) (p) (p) tizard e. (fc) (p) toenshoff b. (fc) (p) tönshoff b. (fc) (p) (op) (fc) (fc) (op) (op) (p) (op) (fc) urdaneta-carruyo e. (p), (p) urdaneta-contreras a (fc) (p) (p) valverde s. (p) (p) van den heuvel l. (fc) (p), (p) van't hoff w. (sy), (p) (p) (fc), (p) (sy) (sy) (fc), (fc) (p), (p), (p) waters a. (op) (fc), (fc), (fc) (mr) (op) (fc) (fc) (op) (fc), (p) (fc) wingen a. (op) (sy) (fc) (sy), (fc) (fc), (p) (fc) (sy) (p) (fc) (p) (p) (p) (op), (p) (fc) (p) yap hk. (fc) (p) yata n. (p) (op) (p) (p) yilmaz a. (p) (op) (op) (p) (p) (p) zhou jh (fc) (op), (fc), (sy) zingg-schenk a (p) (fc) zurowska a. (fc) (op) the il (- g/c) polymorphism is associated with initial peritoneal transport status in children commencing chronic pd acknowledge: this study was from iran university of medical science. atypical hemolytic uremic syndrome: an unsolved case of complement dysregulation (p) aim: we have shown that rats overexpressing il- gene developed a mcn with proteinuria, hypoalbuminemia and hypercholesterolemia. this study aimed to determine the role of il- on hypercholesterolemia in this model. methods: recombinant rat il- gene in a mammalian expression vector was transfected into quadriceps of wistar rats by in-vivo electroporation. serum il- , albumin, cholesterol, creatinine and urine albumin were measured serially. after sacrifice on day , hepatic gene expression of hmg-coa reductase (hmg-coar), acat , cholesterol- a-hydroxylase (ch ah), ldl-receptor (ldlr) and il- receptor subunits were determined by rt-pcr. results: compared to control rats (n= ), il- -transfected rats (n= ) showed significant albuminuria ( . ± . vs . ± . mg/day, p< . ), hypoalbuminemia and hypercholesterolemia ( . ± . vs . ± . mmol/l, p< . ) at day . serially, this rise in serum cholesterol preceded the increase in proteinuria and fall in serum albumin. serum cholesterol correlated significantly with il- levels (r= . , p< . ). in of the il- -transfected rats with serum cholesterol> . mmol/l, hepatic gene expression (mean±sem) was significantly upregulated compared to controls for hmg-coar ( . ± . vs . ± . , p= . ), acat ( . ± . vs . ± . , p= . ), ch ah ( . ± . vs . ± . , p= . ) ldlr ( . ± . vs . ± . , p= . ) and il- ra ( . ± . vs . ± . , p< . ). conclusion: the increased cholesterol synthesis in il- -induced mcn was associated with increased hepatic gene expression of hmg-coar and acat , which are important enzymes for cholesterol synthesis. associated increased hepatic gene expression of ch ah and ldlr involved in cholesterol metabolism could be a negative feedback response. e. bordador, f. anacleto philippine general hospital, pediatric nephrology, manila, philippines general objective: to formulate a clinical scoring system that will predict the presence of glomerular crescents in patients with severe nephritis. specific objectives: ( ) to describe the profile of children with acute nephrotic-nephritic syndrome ( ) to correlate clinical parameters with renal histopathology. methods: this is a retrospective study. twenty six charts from the philippine general hospital, admitted between january -march were retrieved. included in the study were children with acute nephritic -nephritic syndrome who underwent kidney biopsy. excluded were patients with small/contracted kidneys on renal ultrasound. statistical tool used were univariate analysis, pearson's product moment method and chi -square test. results: profile variables of the subjects were analyzed. afterwhich, each clinical parameter was tested using univariate analysis, if significantly correlated with the renal biopsy result using pearson's product moment method at critical value= . at p< . . out of parameters, only parameters were noted to be significant, these were serum creatinine, blood urea nitrogen and glomerular filtration rate. further analysis was made by separately treating male from female population using a chi-square test with critical value x ( ,. ) = . . the test identified another three clinical features among female which were significantly associated with renal biopsy results, these were blood pressure, anemia and hematuria. from these significant parameters associated with renal biopsy results, a clinical scoring system was then conceptualized in order to identify patients with high probability of crescents on renal biopsy. conclusion: cresent maybe use as an accurate screening tool to predict presence of glomerular crescent in patients with severe nephritis. heterogeneous effect of acei therapy in children with proteinuric nephropathies b. kranz, s. diepenbruck, u. vester, r. buescher, a. wingen, p. hoyer university of duisburg-essen, pediatric nephrology, essen, germany background: chronic proteinuric nephropathies are at high risk of developing progressive renal insufficiency. the renin-angiotensin-aldosteron-system blockade is a well documented strategy to reduce proteinuria in adult patients. the efficacy and risks of renal-protective therapy with angiotensin-converting-enzymeinhibitors (acei) in children with proteinuric kidney diseases is of concern. method: in this retrospective study the efficacy of acei as antiproteinuric therapy in children with chronic proteinuric nephropathies is evaluated. patients: sixty-three children (mean age . ± . years) have been treated with acei for a mean of . ± . years (range . ± . years) because of proteinuria (hus n= , alport syndrome n= , psh n= , others n= ). results: proteinuria in all patients declined from a median of . g/d to . g/d after years (p= . ). there was a drop out of patients because of end stage renal failure during the followup. one-third of patients showed a continuous reduction in proteinuria from . g/d to . g/d (median) within years while a second third had a transientimprovement followed by a reincreasing proteinuria later. patientswith hus showed a good response (decrease of proteinuria from . g/d to . g/d) in contrast to patients with alport syndrome who developed increased proteinuria ( . g/d to . g/d) despite of acei and patients with psh who had no change in proteinuria.interpretation: it has to be discussed whether biological compensation mechanisms may bypass the ace inhibition in single patients and whether the underlying illness may predict the efficacy of acei.aims: to determine the clinicolaboratory renal manifestations; glomerular, extra-glomerular histopathologic lesions; renal tubular dysfunction (rtd) frequency, and outcome of a short-term renal follow-up in nigerian children with systemic lupus erythematosus (sle). methods: a non-randomized prospective study of consecutive cases of childhood-onset sle with nephropathy. baseline/follow-up clinicolaboratory data were collected. each patient was followedup for months. results: seven of children studied were girls (f:m, . ). median age at diagnosis was . years. median diagnosis time interval ( . years) and median time of renal disease onset ( . year) were similar. hypertension, nephrotic syndrome, and acute renal failure occurred in . %, . % and . % of the patients, respectively. the glomerular lesions were non-proliferative lupus nephritis (ln) in . % (class ii ln); focal (class iii ln) and diffuse (class iv ln) proliferative ln (pln) in . % and . %, respectively. tubulointerstitial nephritis (tin, . %), and rtd ( . %) were common. arf (p= . ) and rtd (p= . ) were significantly associated with severe tin. complete renal remission rate at end-point was . %. relapse and renal survival rates were . % and . %, respectively. rtd was persistent in . %. conclusion: renal function disorders, diffuse pln, and extra-glomerular lesions were frequent. significant association of arf and rtd with severe tin in this series suggests the need for early renal tubular function (rtf) assessment in our sle patients. deranged rtf may be marker of severe tin in sle warranting early confirmatory renal biopsy and aggressive interventional treatment.we report two cases of children with crescentic glomerulonephritis (gn) associated to isolated c deposits. patient . a year old girl was admitted for macroscopic hematuria, nephrotic range proteinuria (proteinuria/creatininuria= mg/mmol) and renal failure (serum creatinine μmol/l). anca and other antibodies were negative. c , c and ch activity were normal but c nef was detected. the patient was treated by prednisolone and cyclophosphamide pulses followed by oral corticotherapy. renal function normalized, but proteinuria persisted (proteinuria/creatininuria= mg/mmol). a relapse occurred ten months later. corticotherapy and cyclophosphamide pulses were reinitiated and were successful, followed by mmf maintenance therapy. patient . a year old girl was admitted after viral infection for macroscopic hematuria and fever. proteinuria was of nephrotic range (proteinuria/creatininuria= mg/l). the search for autoimmunity was negative. c nef was detected but c , c and ch activity were normal. serum creatinine increased to μmol/l. after three pulses of prednisolone followed by oral prednisone, renal function normalized. histological examination of the two renal biopsies revealed endo-and extracapillary gn with numerous granulocytes in the capillary lumen. cellular crescents involved % of the glomeruli. immunofluorescence demonstrated isolated c deposits in the mesangium and along the glomerular basement membrane (humps). c q, igg, iga and igm staining were negative. background: the risk of end stage renal disease (esrd) is low in unilateral wilms tumor, although patients with wagr or associated genitourinary anomalies are at higher risk. esrd is attributed mostly to hyperfiltration in the remnant kidney. immune complex glomerulonephritis (icg) has not been previously reported in wilms tumor patients. objectives: to report cases of icg in unilateral wilms tumor. methods: retrospective chart review. patient # a boy with cryptorchidism, diagnosed with unilateral wilms tumor at y of age. patient # a girl with wagr and unilateral wilms tumor diagnosed at y of age. both had chemotherapy after tumor resection. results: patient # : within mo of tumor resection, a proteinuria of . g/ hrs and rising creatinine were noted. renal biopsy was consistent with icg. within mo of surgery the patient developed esrd requiring chronic hemodialysis. patient # : within y of tumor resection, the patient developed progressive, asymptomatic proteinuria up to g/ hrs. the renal biopsy revealed changes typical for icg. the patient was treated initially with enalapril and prednisone. due to no response, mycophenolate mofetil (mmf) was added and prednisone was weaned. after mo of therapy, her proteinuria improved to . g/ hrs. her serum creatinine continued to be normal at . mg/dl, with calculated gfr of ml/min/ . m . conclusions: this is the first report of icg in patients with unilateral wilms tumor with rapid progression to esrd in the first patient, but successful response to mmf in the second patient. despite low risk for progressive proteinuria in wilms tumor patients, it is prudent to monitor these patients for proteinuria and perform a renal biopsy if proteinuria is progressive. mmf therapy may be attempted to decrease proteinuria and to delay the onset of esrd.aim of the study: to present our first results of rhgh treatment mainly in children on hemodialysis. patients and methods: sixteen children, aged . - . years (mean age . ± . ) with height below - . standard deviation score (sds) for age or height velocity below - . sds for age, were selected to receive rhgh therapy at our nephrology and hemodialysis department. most of them were on hemodialysis ( children) with mean spent time . ± . years ( - years) before an initiation of rhgh therapy. one half of patients were prepubertal ( children) and second half were in early puberty (testicular volume between and ml for boys and breast development b or b in girls). all received - iu/ml per week by daily subcutaneous injection for months to years. the year before rhgh therapy served as a control period. results: during the first year of treatment, mean height velocity in hemodialysis patients increased from . cm/year to . cm/year (p< . ) and in the second year was . cm/year (p= . ). the mean height sds in hemodialysis children did not improved significantly during the first year of rhgh treatment (from - . sds to - . sds, p= . ). neither weight, nor the body mass index varied compared with the pretreatment period. no change was observed in glucose, total proteins, albumins, cholesterol and triglycerides levels. the mean increment in bone age was . years. pubertal status had no influence on response to rhgh. conclusions: therapy with rhgh improved height velocity in children with esrd. no significant side effects were observed in children during the . treatment years. two patients developed secondary hyperparathyroidism but the relationship with rhgh remains uncertain. in our treatment group rhgh therapy was safe. a. waters , a. trautmann , p. zipfel , e. harvey , ch. licht hospital for sick children, department of pediatric nephrology, toronto, canada university children's hospital, department of pediatric nephrology, heidelberg, germany atypical hemolytic uremic syndrome (ahus) is characterized by the absence of a diarrheal prodrome, the tendency to relapse and a poor outcome. functional and quantitative deficiency of complement regulatory proteins or inhibiting autoantibodies result in uncontrolled complement activation, which eventually causes ahus. -we report a case of ahus with complement dysregulation associated with a progressive refractory response to plasma infusions. factor h and factor h-related proteins (fhr) were quantified by elisa and were further analyzed by western blot. complement activation was determined by measuring c . serial hgb (g/dl), platelet, creatinine (mg/dl), ldh (u/l) were measured. initial presentation was at months of age with thrombocytopenia, hemolytic anemia and increased creatinine. ahus was suspected as e coli infection was ruled out. disease remission followed several plasma exchanges. monthly plasma infusion maintained remission. therapy intervals exceeding month promoted relapses. nine years later, the relapse interval decreased and over the subsequent years, thrombocytopenia persisted as plasma infusion requirements increased. a concomitant decline in renal function (creatinine . mg/dl) occurred with the development of persistent proteinuria and hypertension. at years of age, she deteriorated acutely with hypocomplementemia and thrombopenia. quantitative factor h was normal. autoantibodies to platelets and factor h were negative. intravenous immunoglobulin combined with oral steroids resulted in normalization of platelet count. complement dysregulation is associated with ahus. hereditary defects can be treated with factor replacement therapy. refractory responses may subsequently arise due to the development of autoantibodies against complement regulatory proteins. complement dysregulation requires further analysis in our patient. objectives: angiopoietin-like protein (angptl ) is involved in lipid metabolism and angiogenesis. the present study was to examine angptl expression in human kidneys with proteiuria, in adramycin rats (adr), and in puromycin induced podocyte damage. methods: immunohistochemistry was performed on kidney biopsies from children with mcd, mn, fsgs, tbmn. in adr, angptl expression was determined by quantitative real-time pcr in glomeruli and tubuli dissected from frozen section of kidneys with laser microdissection system. in mpc , a conditionally immortalized mouse podocyte cell line in vitro, angptl , perlecan and agrin werer detected through real-time pcr with the induction of puromycin. detachment assay was performed in podocytes tranfected by angptl -pcdna . . results: in human kidneys, co-labeling showed angptl expressed in the cytosol of wt positive cells. quantitative computerized analysis showed that angptl in glomeruli in mcd and mn were significantly higher than that of tbmn, fsgs respectively (p< . ). in adr, angptl in glomeruli increased significantly at st or th day (p< . ) after adriamycin injection compared with control. and the expression of angptl in glomeruli was correlated with h urinary protein (r= . , p< . ). in mpc both protein and mrna expression of angptl on podocytes were up-regulated with the induction of puromycin. in podocytes transfected by angptl -pcdna . the expression of perlecan or agrin increased significantly compared with control (p< . ). the attachment ratio was shown . %± . % hs after puromycin treatment on podocytes transfected by angptl compared with . %± . % on normal podocytes, and . %± . % on untransfected podocytes. conclusions: angptl is predominantly expressed in podocytes which could be involved in podocyte damage and the development of proteiuria. purpose: we present cases of a previously undescribed pattern of membranoproliferative glomerulonephritis, in children with neuroblastoma on chemotherapy. the pattern of injury shows unusual focal capillary loop proteinaceous deposits possibly related to toxic chemotherapeutic drugs. the resultant hypertension and renal impairment made bone marrow transplant a challenging prospect. method: case series results: case : this boy with stage neuroblastoma, developed severe renal impairment and hypertension during treatment. thus there were difficulties in administration of chemotherapy and he required early surgery. at tumor resection a nephrectomy was necessary. he received an autologous stem cell transplant. he became unwell at transplant and required haemofiltration. he made a good renal recovery and did not relapse. case : he was diagnosed at months with stage s neuroblastoma. he completed treatment but later relapsed. during treatment his gfr reduced and he developed severe hypertension. this lead to restrictions in chemotherapy. renal biopsy was carried out at tumor resection. at bone marrow transplant he was very unwell and required haemofiltration. he has chronic hypertension and low gfr. light microscopy findings in both -global diffuse membranoproliferative pattern of injury -large numbers of proteinaceous resorption droplets -features of a protein deposition disease electron microscopy findings common to both -large number of differently sized protein droplets in the endothelial cells -no obvious immunecomplexes/deposits -protein deposition disease with a membranoproiferative pattern of injury conclusion: both cases showed deposits in the kidneys which may be tumor protein in origin and the resultant glomerulonephritis, hypertension and renal impairment lead to challenges in transplant. the long term consequences are yet to be revealed. methods: establish the cultured glomerular mesangial cells of rat in vitro, ~ generations of cells were used in the experiment after identification. the experiment included five groups: ctrl, lps, high, middle and low dose fos groups. gmc proliferation were detected by mtt. the changes of ln, fn and col protein secretion were detected by the elisa. the changes of lnbeta mrna expression were detected by semi-quantitative real-time pcr. results: ( ) fos can inhibit the effect of proliferation induced by lps. ( ) mesangial cells can secrete some ecm protein in normal culturing medium, ecm protein secreted by mesangial cells was significantly higher in lps group than that in ctrl group (p< . ), while ecm protein was significantly lower in all fos groups than that in lps group (p< . ). ( ) lnbeta mrna expression was significantly higher in lps group than that in ctrl group, while that in fos group was significantly lower than in lps group. conclusions: lps can induce the increase of secretion of the ecm, including ln, fn, col fos can inhibit the secrection of ecm in gmc as dose-dependent manner at the mrna and protein levels. the conclusion supplies the theoretical evidence for the renal protection of fos. h. hong, w. na, y. li, w. qiang guangzhou first municipal people's hospital, department of pediatrics, guangzhou, people's republic of china objective: to observe the effects of fosinopril (fos), the new generation angiotensin-converting enzyme inhibitor (acei), on protein and mrna expression of tgf-β of rat glomerular mesangial cell (gmc) induced by lps; to demonstrate the preventive mechanism against glomerular sclerosis by applying fos. methods: culture rat gmc in classic way. the cultured cell were divided into groups, namely ( ) control group, ( ) lps group, ( ) lps+fos group. detect tgf-β concentration in gmc supernatant fluid by elisa; estimate tgf-β mrna expression by semiquantitative real-time rt pcr. results: lps group is obviously higher than control groups in tgf-β secretion and mrna expression, while lps+fos group drops distinctively in tgf-β secretion and mrna expression compared with lps group. conclusions: fos has obvious inhibited on tgf-β expression of rat gmc both at protein level and mrna level, which reveals that it might be an important mechanism by fos on restraining the development of glomerulosclerosis. r. kahn , n. akbari , j. wieslander , w. müller-esterl , a. christensson , k. westman , t. hellmark , d. karpman lund university, pediatrics, lund, sweden wieslab ab, lund, sweden institute of biochemistry ii, frankfurt, germany lund university, nephrology, lund, sweden vasculitis is an inflammation with neutrophil influx in and around blood vessels. patients may have elevated plasma levels of neutrophil-derived proteinase and anti-neutrophil cytoplasmic antibodies (anca) directed to proteinase , suggested to be involved in the pathogenesis of disease. we have previously shown that the kallikrein-kinin system (kks) is activated in vasculitis. in vivo the kks is activated on endothelial cells and neutrophils when high-molecular-weight kininogen (hk) is cleaved by kallikrein thus liberating bradykinin. bradykinin is a potent mediator of inflammation. in the present study we investigated if neutrophil-derived proteases, and proteinase in particular, could induce activation of the kks and bradykinin release. purified neutrophils from ten vasculitis patients ( adults, children) and thirteen controls were treated with triton-x to induce lysis. proteinase was immunoadsorbed from the neutrophil extracts. bradykinin and proteinase levels were measured by elisa. hk proteolysis was detected by immunoblotting. proteinase incubated with purified hk induced physiological breakdown of hk and bradykinin release. this was inhibited by preincubation of proteinase with anti-proteinase . triton-x treated neutrophil extracts from both patients and controls induced hk proteolysis and bradykinin release whereas the neutrophil extracts from which proteinase had been immunoadsorbed did not. levels of proteinase in the neutrophil extracts from patients and controls did not differ. these findings suggest that neutrophil derived proteinase can proteolyse hk in a physiological manner thus liberating bradykinin, thereby initiating kallikrein-independent activation of the kks. introduction: this is a prospective study to evaluate the safety and efficacy of tacrolimus in consecutive children with steroid-resistant nephrotic syndrome (srns). methods: all of them were subjected to kidney biopsy. tacrolimus was given in dose of . - . mg/kg/day in two divided doses to attain trought levels of . - . ng/l. these patients were followed-up every weekly initially for the first month, followed by monthly visits. urine spot protein creatinine estimation was done at each visit. besides blood glucose, serum creatinine, urea, electrolytes, albumin, and complete blood count were done once a month. results: the mean age of onset was . ± . yrs. of the children, had mcd, had fsgs and another had dmh on histopathology. tacolimus had to be withdrawn in children: of the rest children who received adequate therapy, complete remission was seen in ( %) children, ( %) attained partial remission and was non responsive. the mean time to achieve remission was . + . days and the mean dose of tac was . + . mg/kg. the mean urine spot protein/creatinine ratios were significantly lower ( . ± . vs . ± , p= . ) and mean serum albumin significantly greater ( . ± . vs . ± . , p= . ) as compared to those prior to tac. of the children who attained complete remission, patients are off steroids and tac and in sustained remission, while the rest are still on tac therapy. conclusions: this is the largest study so far on the safety and efficacy of tacrolimus therapy in children with srns. we conclude that tacrolimus is a useful therapeutic alternative in children with srns who are unresponsive to cyclophosphamide and cyclosporine. objectives: to describe hiv infected paediatric patients from our centre with pathology proven renal disease. methods: retrospective review of biopsy data base and case notes of patients with hiv referred with renal problems. results: patients were identified who had biopsy confirmed renal disease. the mean age of the patients was , yrs (range: months to years). twelve of the patients were african and two were of mixed race. renal pathology was divided into three groups: ) hiv associated nephropathy (hivan): five patients. ) mesangioproliferative nephropathy: patients ) other: acute pyelonephritis in , mesangial proliferation plus interstitial nephritis in , renal tuberculosis in , hiv immune complex disease (hivick) in one. conclusion: there is a high degree of variability of renal pathology in children with hiv and renal disease which upholds the need accurate diagnosis when confronted with these patients. background: acute poststreptococcal glomerulonephritis (apsgn) is the most common glomerular disease of children in our country. it has not been studied well in this region yet. here, we report our experience with psgn in a tertiary referral center during a five-year period. method: hospital records of all children who had been admitted from mar. to mar. to nemazee hospital with diagnosis of acute glomerulonephritis (agn) were reviewed. all demographic, clinical, paraclinical data and consumed medications were obtained. results: among children diagnosed as agn, ( %) had apsgn. other ( %) children had mpgn (n= ), mespgn (n= ), iga nephropathy (n= ), lupus nephritis (n= ), rpgn (n= ), and fsgs (n= ). mean age in children with apsgn was ± . (range, years. children ( %) developed apsgn following a sore throat or upper respiratory infection while ( %) cases developed after impetigo. ninety-five ( %) patients developed apsgn during the cold seasons of the year. periorbital edema was found in children ( . %), hypertension in ( %), gross hematuria in ( %), oliguria in ( %), generalized edema in ( %), azotemia (bun> ) in ( %), and nephrotic range proteinuria in ( . %). aso titer was high in ( %). low c was detected in ( %) and low c in ( %). dilutional anemia in ( . %), hyponatremia in ( %), and hyperkalemia in ( %) children among whom, required hemodialysis. regarding medications, patients had received only furosemide, cases took furosemide and nifidipine and for patients furosemide+nifidipine+another antihypertensive medication was prescribed. conclusion: acute psgn is the most common type of glomerulonephritis in this region. it follows sore throat in the majority of cases. it usually has an uneventful course. y. guo, zh. wang, x. liu west china second university hospital, sichuan university, department of pediatrics, chengdu, people's republic of china objective: we planned to explore the mechanism of glomerular basement membrane (gbm) damaged by rsv, through investigating the effects of lmwh on proteinuria and glomerular structure of rsv nephropathy. methods: sd rats were inoculated with pfu rsv and lmwh iu/kg. group a: rsv was given in the first days, and lmwh was given for the following days; group b: the mixture of rsv and lmwh was given in the first days, then lmwh was given for other days; group c: lmwh was continuously given throughout days and on the th - th day rsv was also given; group rsv and the control were respectively inoculated by rsv and dmem for days. renal histology, urinary protein excretion and serum parameters were observed. rsv rna in renal and pulmonary tissue was determined by in situ hybridization. results: there was no significant increase in urinary protein excretion of the lmwh-treated groups (a . ± . , b . ± . , c . ± . , mg/ h) compared with the control, but that of group rsv ( . ± . mg/ h) gradually increased after rsv inoculation. there was just a decrease in albumin ( . ± . g/l) and an increase in urea nitrogen ( . ± . μmol/l) of group rsv only. no change of the glomeruli detected in all lmwh-treated groups, while congestion and swelling in glomeruli of group rsv were observed significantly. glomerular microstructures of the lmwh-treated groups were almost normal, while extensive foot process effacement was observed in group rsv. rsv rna signal expressed weaker in the lmwh-treated groups than in group rsv. conclusion: rsv damages hs on gbm by electrostatic interaction. lmwh, as the analog of hs, charged with anion, competes with hs to combine with rsv to keep gbm from being destroyed, and then reduce the proteinuria. s. zhai, zh. wang west china second university hospital, sichuan university, department of pediatrics, chengdu, people's republic of china objective: the study is to explore the relevance among gags, hpa and ela in the steroid responsive nephrotic syndrome (srns). methods: ( ) children with srns were selected, including the active (n= ), the convalescent (n= ), the remissive (n= ). purpuric nephritis and healthy children were served as the control.( ) using the improved whiteman process detected the urinary gags. ela activities in plasma were determined by the amount of -nitroaniline released per unit time. immunocytochemistry and image analysis method were used to detect the expression of hpa of peripheral blood leukocyte. results: . gags of the active were the highest ( . ± . ) of all (p< . ). in contrast with the healthy, the active and the convalescent ( . ± . , p< . ) were significant difference, but the remissive no difference ( . ± . , p> . ). . all of ns showed higher level of hpa than the healthy (p< . ). comparing with the healthy, hpa was significant difference both in the active (iod . ± . , p< . ) and in the convalescent (iod . ± . , p< . ),but no difference in the remissive (iod . ± . , p> . ). by contrasting the active and the purpuric, their difference of hpa was no statistic significance (p> . ). . all of ns showed higher levers of ela than the healthy (p< . ). the healthy was strikingly different, contrasting with the active ( . ± . ) and the convalescent ( . ± . , p< . ), but no difference with the remissive ( . ± . , p> . ). .there was a significant correlation among the urinary protein, urinary gags, hpa and ela with simple linear regression analysis. conclusion: in the srns, proteinuria may be resulted by the spallation of hpa and ela for gags on gbm. ( ), combination withhaematuria, hypertension and/or renal insufficiency ( ), extrarenalsymptoms ( ), and familial mediterranean fever (fmf, ) . of the non-nephrotic patients, had extrarenal symptoms, were nephritic, had rapidly progressive glomerulonephritis (gn), renal failure and isolated urinary abnormalities. biopsy samples were evaluated by light microscopy in yerevan and zurich and by electron microscopy (except for amyloidosis) and immunohistochemistry (last ) in zurich. results: the most common histological lesion was renal amyloidosis ( %), followed by focal segmental glomerulosclerosis (fsgs, %), lupus nephritis ( %),systemic vasculitis/hus and minimal change disease (mc, . % each),mesangioproliferative gn/iga-nephropathy and membranous nephropathy(mn, % each), hereditary nephritis and membrano-proliferative gn typei ( % each), acute postinfectious gn (apgn, %), and dense deposit disease (ddd, %). the miscellaneous group includes,apart from interstitial nephritis ( %), unclassified or inadequate biopsies and specimens with mostly sclerosed glomeruli( %). the majority of patients with amyloidosis of fmf, fsgs/mc andmn were nephrotic, but % of patients with amyloidosis had non-nephrotic proteinuria. conclusions: several glomerular lesions were considerably more frequent than in other studies, particularly amyloidosis of fmf, mn and lupus nephritis, and to lesser extent membranoproliferative gn type and ddd. apgn is underrepresented because less than % of all patients (> ) had a biopsy. this study would not have been possible without international collaboration. henoch-schönlein purpura in children: an epidemiological study amongst dutch pediatricians on incidence and diagnostic criteria j. aalberse , , k. dolman , g. ramnath , r. rodrigues pereira , j-c. davin the aim of the present study on the incidence of henoch-schönlein purpura (hsp) in dutch children is not only to give some insight in the epidemiology of hsp in the netherlands but also to record the diagnostic criteria used by dutch pediatricians and to evaluate the accuracy of the latter using the presence of iga in the skin when biopsies are available. methods: in , all dutch pediatricians received monthly a card asking to mention new diagnosed hsp. pediatricians reporting one or more new patients with hsp were sent a list of questions concerning symptoms, blood and urine parameters, skin biopsy, diagnostic criteria and follow-up duration needed. results: two hundred and thirty-two patients from - of age ( . / ) were reported as having started hsp in . twenty nine % presented with renal symptoms. in accordance with the classification criteria of the american collegeof rheumatology (acr), eighty percent of pediatricians consider that isolated purpura (without hematological abnormalities) is sufficient to allow the diagnosis of hsp in children. from the skin biopsies performed, only ( %) presented with iga deposits. the follow-up duration considered as necessary was longer in case of renal symptoms at presentation. however, % of patients without renal symptoms would be followed for more than one year. conclusion: considering the recent ( ) eular/pres endorsed consensus criteria for the classification of childhood vasculitides, hsp should have been diagnosed in only of the patients of our study. the use of isolated non-thrombocytopenic purpura as only criterian to diagnose hsp in children might therefore lead to over diagnosis and unnecessary follow-up. noteworthy, the eular/pres criteria remain to be validated by a prospective study. the clinical presentation and response to therapy of childhood pan in johannesburg, south africa. method: retrospective record review of twelve children with a clinical diagnosis of pan treated between and . results: there was unequal number of males and females; average age at presentation was . ± . - . years, all were black children. eight children had more than acr criteria and sufficient clinical criteria (eular/pres consensus criteria). musculoskeletal and cardiac diseases were the commonest finding at presentation ( %), cutaneous, hypertension ( %), renal and gastrointestinal disease ( %), central nervous system disease ( %) and constitutional features ( %). two children had bone involvementwith periosteal reactions on plain x-ray. angiographic abnormalities were found in ( %), and ( %) had positive histology (skin/renal biopsies). tuberculosis was diagnosed in ( %), and had positive streptococcal titers. all patients were ana and hepatitis b negative, but there were five patients anca positive ( =p-anca, =c-anca, =both) ( %). the crp was elevated in / ( %), esr also in / , while % had both elevated. all patients received oral glucocorticoids, methylprednisolone ( - pulses- mg/m ), ivi cyclophosphamide ( - pulses, mg/m ), oral azathioprine, and required i. conclusions: children with pan in johannesburg present at a younger age with multi-organ disease. they require aggressive therapy with both glucocorticoids and cytotoxic therapy to ensure good outcomes. objective of the study: in order to evaluate the predictive factors of chronic kidney disease (ckd), the records of children with biopsy-proven mesangioproliferative nephrotic syndrome (mpns) admitted between and were retrospectively reviewed. methods: renal survival was analyzed by the kaplan-meier method and cox's regression model. two multivariate models were developed: ( ) from the onset of symptoms to the occurrence of ckd and ( ) from the time of renal biopsy to ckd. the following data were obtained at admission an dat the time of renal biopsy: gender, race, age at the onset ofnephrotic syndrome symptoms, age at admission, blood pressure, laboratory data (serum creatinine, serum urea, glomerular filtration rate, -hr urinary protein excretion, hematuria). patients were classified according to the response to the initial course of prednisone: ( ) a complete response was defined as a proteinuria < . g/day; ( ) a partial response was defined as urinary protein excretion of < g/day and > . g/day, and ( ) no response was defined as urinary protein excretion of > g/day. results: median follow-up time was years (iq range, . ± . ) and patients ( %) progressed to ckd. at baseline, after adjustment variables remained as independent predictors of ckd: creatinine > . mg/dl (rr= . , ci %= . ± . ) and non-response to steroids (rr= . , ci %= . ± . ). at the time of renal biopsy, after adjustment variables remained as independent predictors of ckd: age> . yr (rr= . , ci %= . ± . ) and creatinine > . mg/dl (rr= . , ci %= . ± . ). conclusion: serum renal function at baseline and initial response to prednisone were strong predictors of progression to ckd in our cohort of children with mesangioproliferative nephrotic syndrome. methods: an illustrative case history of a boy with sle and vhd in the absence of antiphospholipid antibodies was described. results: a -year-old boy with a history of sle for about five years was admitted to our hospital due to intermitted arthralgia, facial erythema, increased serum creatinine and oliguresis in october . he had been treated irregularly with prednisone and immunosuppressants. however,the disease was not always controlled well. during thishospitalization, the problems of hypertension, renal failure and anemia had been resolved and maintained, but the problem of intolerance to increased circulation volume was obvious, and three times echocardiography (ecc) showed moderate to severe mitral valve insufficiency. reviewed his history, suspected mitral leaflets vegetations was revealed by ecc four months after onset, he complained of chest pain, chest distress and breathholding several times without causes since eighteen months after onset and manifested with heart failure once, and ecc showed moderate to severe mitral valve regurgitation four months ago and twenty days ago respectively. ruling out the possibility of infective endocarditis, rheumatic heart disease and congenital heart diseases, vhd were considered secondary to sle. since the vhd becomes hemodynamically significant, valve surgery is needed. conclusions: vhd is generally asymptomatic and omitted easily, so routine cardiac evaluation of children with sle using electrocardiography, echocardiography and chest x-ray is recommended to early detect and treat cardiac abnormalities, which may lead to better survival. objective: we have previously reported that deleted in esophageal cancer (dec ), a potent tumour suppressor gene, was specifically upregulated in cd + cells of patients with relapse of mcns, using differential display rt-pcr. this study aimed to further characterize the potential function of dec in mcns. methods: semi-quantitative rt-pcr was used to verify the dec gene expression in children with relapse and remission of mcns. jurkat cells were transfected with plasmid containing dec gene or vector alone. gene expression was regulated by tet-on/off system. the effect of dec on jurkat cell proliferation was assessed by h-thymidine incorporation. cell cycle analysis was performed following propidium iodide staining. protein localization was determined by immunofluorescent staining with anti-dec antibody. results: we confirmed that dec gene expression was significantly increased in children with mcns in relapse ( . ± . ), as compared to remission ( . ± . , p< . ), normal controls ( . ± . , p< . ), patient controls with viral infections ( . ± . , p< . ) and nephrotic patients with other forms of glomerulonephritis ( . ± . , p< . ). in dec -transfected jurkat cells, cell proliferation was inhibited by . %, compared with vector control. cell cycle analysis indicated that dec arrested jurkat cell cycle progression by blocking its entry into the g /m phase. immunofluorescent staining with anti-dec antibody suggested that dec was a cytoplasmic protein, which was in agreement with psort. conclusion: dec gene expression was significantly upregulated in children with relapse of mcns. our results showed that dec acts as a t-cell proliferation suppressor and arrests cell cycle progression, and thus may be important in mediating the number or function of cd + cells during relapse of mcns. objective of study: the aim of the study was to assess plasma and urine concentrations of vascular endothelial growth factor (vegf) in nephrotic syndrome children (ns) depending on the total dose of glucocorticoids (gc) and the percentage of lymphocytes with glucocorticoid receptor expression (cd /gcr). methods: we examined children ( - years) , allocated to three groups: group i: children with the first ns onset, group ii: children with ns relapse, group c: healthy children. the ns patients were examined: a: before treatment and b: - weeks after prednisone administration at a dose of mg/m / h. plasma and urinary vegf levels were determined using the immunoenzymatic elisa method. flow cytometry was applied to assess cd /gcr expression. results: higher plasma and urinary vegf concentrations were noted in ns children before treatment (a), as compared to control subjects (c). following prednisone therapy (b), vegf level was reduced but it was still higher than in the control group. positive correlation was observed between vegf and protein in the urine (group i r= . , p< . , group ii r= . , p< . ) and a weak positive correlation between vegf in plasma and urine (group i r= . , p< . , group ii r= . , p< . ). cd /gcr expression was lower in group ii. in both groups, the correlation between plasma vegf and cd /gcr was positive (p< . ). conclusions: . plasma and urinary vegf levels increase during nephrotic syndrome onset. . glucocorticoid treatment reduces plasma and urinary vegf levels in ns children. objective of study: the aim of the work was to determine the expression of p-glycoprotein (p-gp) on peripherial lymphocytes (cd ) in children with steroid-dependent nephrotic syndrome (sdns) during cyclosporine a (cya) and ace-inhibitor (ace-i) treatment. methods: the study group (i) consisted of children with sdns aged - years, with a subsequent proteinuria relapse at the time of prednisone dose reduction. all ns children were examined three times: a: at proteinuria relapse, before cya treatment, b: after months, c: after months of cya administration. control group (ii) consisted of healthy children. cd /p-gp was measured using a flow cytometry assay. serum cya level was assessed by means of the immunofluorescence method. results: the expression of cd /p-gp in ns relapse, prior to cya+ace-i administration was much higher (median . %, range . - . %) when compared to healthy controls ( . % range . - . %). the absolute number of cd /p-gp in this examination was almost times higher when compare to healthy controls (p< . ). after months of cya+ace-i therapy the expression of cd /p-gp decreased dramatically and was similar to the controls. similar results were obtained after -months of treatment. when analysing the correlation between cd /p-gp and serum cya concentration a strong negative correlation was found in both examinations. the correlation was stronger in group ib (during the treatment with higher cya doses): (r=- . , p< . ) than in ic (after reduction the cya dose: (r=- . , p< . ) conclusions: the results of our studies indicate that cya in sdns inhibits the expression of p-gp. cya is an alternative therapy that may lead to optimization of glucocorticoid doses, thus reducing the risk that goes along with treatment. backround: hemolytic uremic syndrome is considered as the main cause of acute renal failure in childhood. it is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. the epidemiology of the disease varies between counties. aim of the study: to describe demographic and epidemiologic aspects of hus, presented in a years period at the biggest children's hospital in the country. material-methods: patients aged days - years, mean age . years, ( boys, girls) were diagnosed with the syndrome. of them , . %, presented as d+ cases, with preceeded respiratory system symptoms and with no preceeded symptoms. treatment consisted of fresh frozen plasma (ffp) and whole blood transfusions in all cases. peritoneal dialysis was necessary in cases, haemodialysis in , while plasmapheresis was performed in cases. three children, all boys, suffered from recurrent type of syndrome. a girl required treatment with peritoneal dialysis for months followed by recovery of renal function. one-five years follow-up: one baby months old died, children received renal transplantation, in renal function remained mildly reduced (gfr - ml/min/ . m ) and suffered of hypertension. the rest recovered fully. summary: hus represents a rather minor public health problem with low mortality rate in greece. methods: three children with pauci-immune ln were retrospectively reviewed. results: the reported cases fulfilled the american rheumatism association criteria for sle. at admission, all had a -month history of glomerulopathy without intensive therapy. case presented with nephrotic proteinuria, macroscopic hematuria and progressively deterioration of renal function. laboratory data included a positive antineutrophil cytoplasmic antibodies (anca) on myeloperoxidase immunoassay. she was positive for lupus anticoagulant (la) but negative for anti-cardiolipin (acl) and anti-beta- glycoprotein i (β gpi) antibody. renal biopsy showed pauciimmune necrotizing and crescentic ln. case manifested with proteinuria, microhematuria and hypertension. anca was negative. case had nephrotic proteinuria, microhematuria and hypertension. the case had positive la, acl and anti-β gpi antibody. anca was negative. the biopsy findings of both case and case indicated pauci-immune mesangial proliferative ln. electronic microscopy revealed segmental and diffuse foot process effacement respectively. all three ln remitted following the treatment of steroid and immunosuppressive agents. conclusions: these atypical ln were considered to be associated with the distinct pathogenesis, rather than immune complex-mediated glomerular injuries. we supposed the possibility of ancaassociated necrotizing and crescentic glomerulonephritis for the first case. for the others, primary or secondary podocytes lesions might alter the glomerular permeability. the nephrotic syndrome is a clinical picture that characterized by proteinuria, hypoproteinemia, oedema and hyperlipidemia. although the primer nephrotic syndrome is the most common type of nephrosis in children, it may also develop during the course of infections including hepatitis b and c virus infections, whereas hepatitis a virus infection related nephrotic sendrome is very rare in children. in this paper, we present a case of who had suspected diagnosis hepatitis a virus infection related nephrotic syndrome. the boy at the age of years was referred to our hospital due to vomiting, abdominal distension and oliguria. physical examinations were revealed palpebral and tibial oedema, hepatomegaly, decreased breath sounds and mat percussion on right hemithorax. icter were not detected. laboratory examination were showed proteinuria, hyperlipidemia, hypoalbuminemia, high alt and ast levels with normal levels of bilirubin, alkaline phosphatase, complement c /c , urea and creatinine. in viral serologic examinations, hepatitis b and c related antibody were found negative, but anti-hav igm was found positive. chest radiography and ultrasonography examination were revealed right pleural effusion and abdominal ultrasonography examination was revealed ascites. based on these findings, we thought that the nephrotic syndrome could be developed due to anicteric hepatitis a virus infection in our patient. low dose steroid treatment was started (prednisolon mg/kg/day, month). edema was improved, negative urine protein was seen, and liver functions were not deteriorated during this therapy in our patient. hepatitis a virus may be cause of nephrotic syndrome by forming of immune-complexes. in conclusion, although hepatitis a virus infection is rare cause of nephrotic syndrome in children, it should be investigated as a seconder casuse. membranoproliferative glomerulonephritis (mpgn) is a rare, chronic glomerulonephritis, and its prognostic factors and outcome are not known very well in childhood. in this retrospective study, we reviewed the clinical, laboratory and histopathological features of children with primary mpgn. thirty-three children ( boys, girls) presented at a mean age of . years (range - years). they were followed for a median of months (range - months). the clinical presentation in children was nephritic-nephrotic syndrome in children ( . %), nephritic syndrome in ( . %) and nephrotic syndrome in ( . %). nine patients had renal failure at presentation. at the end of study, patients ( . %) had complete remission and patients had abnormal findings ( proteinuria, microscopic hematuria, hypertension, esrd). all of patients were treated with steroid. eight patients were given another immunosuppressant drug (cyclophosphamide , azathioprine ) in addition to steroid therapy. the interval between appearance of symptoms and admission, durations of microscopic hematuria and proteinuria, and systolic blood pressure at presentation were higher in children with abnormal findings when compared with children with complete remission. there were no significant differences in clinical presentation type or histopathological features between children with abnormal findings and children with complete remission. our results showed that delayed detection and treatment, uncontrolled hypertension and unresponsiveness to steroid in early period were poor prognosis predictors. we did not determine any correlation between histopathological findings and outcome. we need further investigations including larger patient population. partial lipodystrophy (pl) is a rare condition of unknown etiology, with childhood onset. it is characterized by progressive loss of subcutaneous fat of face, neck, trunk and upper extremities together with c hypocomplementemia. usually, patients do not have clinically evident renal disease or abnormalities until they have had the disease for or more years. but, the case we reported here, firstly presented with membranoproliferative glomerulonephritis (mpgn), and during follow-up pl was observed. a six years old girl was presented with sore throat, vomiting and loss of appetite for the last fifteen days. her development was normal and the child was asymptomatic till years of age. the parents were relatives of the third degree. there was no history of similar cases in the family. the physical examination of the patient was normal. urinalysis revealed hematuria and pyuria. proteinuria was not present. renal function tests were normal. laboratory examination revealed low serum complement c but normal serum c . the lipid profile was also normal. patient was followed with the diagnosis of poststreptococcic glomerulonephritis. because of persistent low complement c and nephrotic range proteinuria renal biopsy was performed at the rd month of follow-up. renal histology revealed mpgn consistent with type . c nephritic factor was negative. at the same time it was observed that the face took on a cadaverous look with prominent malar bones, chin and zygoma because of the loss of buccal fat. according to these findings, partial form of lipodistrophy, which is frequently associated with mpgn, was considered in the patient. during two years of follow-up she had two recurrences of nephrotic range proteinuria and she is now in remission with prednisolone therapy. as far as we know this is the first example of pl which is developed during follow-up of mpgn. central nervous system abnormalities in children with acute post-streptococcal glomerulonephritis (apsgn) are rare and are considered to be secondary to acute severe hypertension, electrolyte imbalances and uraemia. cerebral vasculitis associated with acute post-streptococcal glomerulonephritis has been rarely reported in pediatric literature. a -year-old girl with a severe headache, vomiting, edema and macroscopic heamaturia is presented. she had history of upper respiratory infection before two weeks. so, the patient was diagnosed as apsgn. on admission, she was normotensive and biocemically well balanced. two hour later, she experienced a grand mal seizure. mri examination of brain showed not only multiple areas of increased density in white and gray matter, and cerebellum but also subarachnoid bleeding, consistent with vasculitis. during follow-up, abducens nerve palsy was detected. histopathological features on renal biopsy specimen, an elevated antistreptolysin level and fallen c complement level were compatible with apsgn. all clinical and laboratory abnormalities improved with steroid therapy (pulse and oral methylprednisolon). in conclusion, children with apsgn may present central nervous system abnormalities without hypertension, uraemia and electrolyte disturbances. results: hsp was diagnosed in patients, median age years old. all had the skin manifestations, . % abdominal pain and % arthritis. patients developed hsp nephritis ( . %), mean presentation time was . months after phs diagnosis. renal biopsy was performed in patients, and the most common histopathological finding was hsp nephritis grade iii a. age of onset older than years was statistically significant for nephritis development (chi square < . ). chronic renal insufficiency incidence was . %. conclusions: the main complication of hsp is nephritis. follow-up should include evaluation by a pediatric nephrologist. age of onset older than years is an important risk factor for hsp nephritis. objectives of study: acquired abnormalities of coagulation and fibrinolysis in nephrotic syndrome have been implicated in the pathogenesis of deep vein and arterial thrombosis. resistance to activated protein c due to amutation in the gene for factor v, the commonest inherited risk factor for venous thrombosis, could contribute to risk of both arterial and deep vein in patients with nephrotic syndrome. we report an arterial thrombosis in a young girl with idiopathic membranousglomerulonephritis (mgn) and factor v leiden mutation. case report: a year-old girl was admitted to our hospital with swelling of both legs and cyanosis of her toes. her mother had history of abortion in the second trimester of gestation, and parents were second-degree relatives. physical examination showed peripheral edema in both extremities and peripheral cyanosis in toes. laboratory study showed the nephrotic range proteinuria. serum albumin was . g/dl, and cholesterol was mg/dl. creatinine, electrolytes, prothrombin/partial-thromboplastin times, c , c , fibrinogen, protein s, c, antithrombin iii and homosistein levels were within normal limits. ana, anti-dsdna, p-anca, and c-anca, antiphospholipid igg/igm, anticardiolipin igg/igm were all negative. genetic study showed the heterozygote mutation of factor v leiden (fv/g a). in arteriography, there was complete occlusion on the posterior tibiaartery of left leg, occlusion of mid portion of right femoral artery and the posterior tibia artery of right leg. renal biopsy findings werefelt to be compatible with mgn. conclusions: we postulate that patients with concurrent nephrotic syndrome and factor v leiden mutation may have an increased risk of thrombosis. screening for factor v leiden mutation may be indicated in patients with idiopathic nephrotic syndrome. introduction: hus (d+) is the nd cause of chronic renal failure (crf) in children in argentina. proteinuria is the main predictor of progression to crf. patients with renal failure, when treated with restricted proteinin take show slower progression to end-stage of rcf. proteinuria appeared in patients with hus sequelae subject to an overload of protein intake. objective: to evaluate the effect of a controlled protein intake on proteinuria in patients with renal disease due to hus and normal renal function (> ml/min/ . m ). patients and methods: within a multicenter, randomized, double blind, controlled trial, carried out inorder to study the effect of iace on the development of renal disease*, proteinuria before and after a controlled diet was measured in patients with proteinuria due to hus and normal renal function. protein intake was indicated according to rda for age. protein intake was estimated with a questionnaire on former hours, and with hrs urea excretion. proteinuria was measured in hours urine collection at the beginning of the study and at , and days after onset of the study. results: median age at onset was . months (r: . - . ); mean of length of follow-up after hus onset was . months (r: . - ). in sixty five ( , %) patients, proteinuria reduced to normal values; in the remaining ( , %) there was no change. median of initial and final proteinuria in the children whose proteinuria became normalised, was . mg/k/day (ds . ) and . (ds . ) respectively < . . conclusion: controlled protein diet (rda) normalizes proteinuria in patients with significant proteinuria secondary to hus and normal renalfunction. * a trial financed by roemmers laboratory, argentina. differential diagnosis of hematuria is the significant task of any nephrologist while most common reasons for hematuria demand histopathological diagnosis. iga-nephropathy is the progressive glomerular disease which is known to be a common reason for hematuria. the diagnosis ofiganephropathy is often missed because of variability of clinical presentations, long-term asymptomatic course and therefore waiting tactics in prescribing renal biopsy. the aim of the study was to evaluate the incidence of iga-nephropathy and to define its characteristic clinical and morphological features in children in belarus. we present results of immunohistochemical staining for iga mesangial deposition of kidney biopsy preparations. the mean age of the patients enrolled in the study was , ± , years. at the moment of biopsy patients clinically presented isolated hematuria ( %), hematuria with proteinuria ( %), nephrotic syndrom ( %), nephrotic syndrom with hematuria and hypertension ( %), isolated proteinuria ( %) and others in less than % each. iga mesangial deposition was detected using immuno histochemical staining with polyclonal rabbit anti-human iga. we found diffuse mesangial deposition of iga in patients ( % of all). two of them had nonnephrotic isolated proteinuria, one nephrotic syndrom. vast majority of patients were hematuric (either isolated or combined with proteinuria). iga nephropathy incidence among hematuric patients was %. in histopathological examination we found mild segmental mesangial hypercellularity in patients, mild to moderate global and segmental mesangial proliferation in (in two of this patients we also found cellular and fibrous crescents in less than % glomeruli). one patient had focal-segmental glomerulosclerosis secondary to diffuse mesangial proliferative glomerulonephritis and clinically presented heavy proteinuria. we analyzed nphs mutations in chilean pediatric patients with srns due to fsgs, diffuse mesangial sclerosis (dms) and minimal change disease (mcd). nphs mutation analysis was performed in patients (sporadic cases n= , familial cases n= ). nphs exons and were amplified by pcr and subjected to automatic sequencing or enzyme restriction analysis using clai (c. g>a) and hin i (c. c>t) respectively. patients median age at disease onset was months (range , % of which were male. histological diagnosis were fsgs (n= ), dms (n= ) and mcd (n= ). ten of patients ( . %) had mutations in nphs ( / fsgs, / dms, / mcd). eight of patients bearing mutations were sporadic cases. seven patients were compound heterozygous for r q and a v. patients with mutations were significantly older than those without mutations (median age versus months, p< . ). resistance to cyclosporin was observed in of cases with mutations and of cases without mutations (ns). we studied the frequency of r q and a v in healthy volunteers with similar ethnic background. only one control individual was heterozygous for r q. no a v mutation was detected. our study demonstrated that nphs mutations are a major cause of srns in chilean pediatric patients. since most of the srns patients bearing nphs mutations were cyclosporin resistant, it is advisable to perform nphs mutation screening before starting immunosuppressives. in this study we aimed to investigate the long-term prognosis of henoch-schönlein nephritis (hsn) in childhood. between and , patients with hsn were investigated retrospectively. there were males and females with a mean age of . years. they were graded according to the degree of renal involvement. grade : isolated microscopic hematuria (n: ); grade : hematuria and mild proteinuria (n: ); grade : acute nephritic syndrome (n: ); grade : nephrotic syndrome/hematuria (n: ); grade : acute nephritic and nephrotic syndrome (n: ). renal biopsy was performed on patients in grade and . twenty patients had extensive crescent formation (> %) on renal biopsy, and were given triple therapy [iv pulse methylprednisolone (mpz) mg/kg/d for days, followed by oral prednisolone (op), oral cyclophosphamide- mg/kg/day for - months and dipyridamole]. the other patients with < % crescent formation were given mpz followed by op and dipyridamole. the patients in grade and were given op and dipyridamole. grade and were not given any immunosupressive agent. during the follow-up period (mean ± . ; range - months), patients in grade , patients in grade , patients in grade , patients in grade and patients in grade showed complete remission ( %). of the patients with extensive fibrosis on renal biopsy, had persistent nephropathy ( %) and developed endstage renal failure ( %). the remaining patients showed near-complete recovery with minimal urinary abnormalities ( %). in conclusion, although initial presentation of renal involvement determines the prognosis in hsn, intensive treatment with triple therapy appears to be effective in severe renal disease especially if started before the development of fibrotic changes in crescents and tubulointerstitial tissue. aim: the bacterial infection in nephrotic syndrome (ns) is still the big problem and is one of the leading death causes in ho chi minh city, vietnam. we did this study with aims to overview this complication in children with ns. methods: our study population consisted of children with nephrotic syndrome during one-year prospective cohort. twenty seven out of patients who developed severe bacterial infection including pneumonia, peritonitis, urinary tract infection, bacteremia or cellulitis were recorded. results: from june to june , there were children with nephrotic syndrome recruited to the study. the severe bacterial infection stood at . % (n= ). in these children, children were first ns, children were relapsing ns. the percentages of pneumonia, urinary tract infection, cellular infection and peritonitis were . % (n= ), . % (n= ), % (n= ) and . % (n= ), respectively. no patients with bacteremia were recorded. in patients with uti, e.coli wasin patients, proteus in and enterococus in . ns children with uti were asymptomatic. ns children with peritonitis had typically clinical manifestations of fever, abdominal pain, tenderness. one out of four patients with peritonitis cultured streptococcus pneumoniae inperitoneal fluid. some factors associated with severe bacterial infection were increase in weight ( . vs . %, p= . ), very low serum albumin ( . vs g/l; p= . ), rise in serum a globulin level ( . % vs . %; p= . ) and hyperfibrinogenemia ( . vs . g/l; p= . ). conclusions: bacterial infection has still been a common problem in children with nsin ho chi minh vietnam. it is important to investigate and manage this complication early to reduce mortality rate. primary nephrotic syndrome (ns) is the most common glomerular disease in children which mainly responds to corticosteroids. however, > % of children experience a relapse with recurrent episodes. the aim was investigation of outcome in ns patients, retrospectively. clinical, histological data and treatment responses of children presenting with ns from to were reviewed. all patients were treated with steroid treatment firstly and classified as steroid sensitive ns (ssns) and steroid resistant ns (srns) according to clinical or laboratory responses. there were patients, male and female, had followed-up for . ± . months. age at onset ranged from - (median ) months. seven patients were admitted with hematuria and ns ( mpgn, mgn) that excluded from this study. patients were treated by only classical steroid treatment. thus, ( . %) patients were ssns and classical steroid therapy was successful in this group. of all ssns patients were evaluated with biopsy ( fsgs, dmp) because of frequently recurrent ns. in srns group ( patients) with one cytotoxic agent, complete and stable remission was induced in patients ( in fsgs, in mlh, in dmp) while patients ( in fsgs) who responded to more than one cytotoxic agents had partial remission with symptomatic relief. five children ( in fsgs) were refractory to all cytotoxic therapies. cyclophosphamide (cp) was used as the first cytotoxic agent in patients and induced complete remission in ( . %). the patients who relapsed following cp and patients who failed to respond were treated with further cytotoxic therapies such as cyclosporine a (cs) or mmf. in patients, cs was used as the first cytotoxic agent and induced remission in patients ( . %). steroid must be the initial drug in childhood ns. cp could be used successfully as an immunosuppressive agent in srns patients. aim: annexin v has a molecular weight of - kda and has been reported to possess anticoagulant activity, inhibition of phospholipase a , regulation of membrane transport, proliferation and signal transduction. it is reported that urinary annexin v concentration may be an indicator of apoptosis and acute renal injury related to the urinary protein level. the aim of this study is to define the role of urinary annexin v, serum annexin v concentrations as new prognostic tools and follow criteria in children with steroid sensitive and resistant ns. methods: annexin v concentrations were measured in serum and hour urine samples in steroid sensitive nephrotic syndrome (ns) patients in both relaps and remission periods (group and respectively) and in steroid-resistant ns (group ) and sex and age matched healthy controls (group ). total protein, albumin, cholesterol concentrations and hour urinary excretion of protein and creatinine were measured in all groups. results: in steroid resistant ns group (group ), median of urinary annexin v/creatinine ratio was significantly higher than all the other groups ( . ng/g creatinine (min-max: . - . ) vs . ng/g creatinine (min-max: . - . ) in group (p: . ); . ng/g creatinine (min-max: . - . ) in group (p: . ), and . ng/g creatinine (min-max: . - . ) in group (p: . )). serum annexin v concentration was significantly higher in group (median . ng/ml) than in group (median . ). no significant correlation was found between urinary protein excretion and urinary annexin v/creatinine ratio. conclusion: remarkably increased urinary annexin v/creatinine ratio could be used as a determinant factor in children with steroid resistant ns, and it may be a prognostic factor in these children. v. baudouin , f. bernaudin , a. garnier , t. kwon , m. peuchmaur , g. deschenes , c. loirat hôpital robert debré-aphp, service de néphrologie pédiatrique, paris, france chic, service de pédiatrie, créteil, france cgvhd is the most common late complication of hsct. clinical manifestations mimic lupus disease but renal involvement is unusual. a year-old boy underwent hsct from an hla-identical sibling donor for sickle cell disease. he received myeloablative therapy (cyclophosphamide, busulfan, antithymocyte globulin). prophylaxis of acute gvh consisted in prednisone and mmf until month . moderate skin and mucosa cgvh appeared at month so that prednisone and mmf were restarted. at month prednisone was stopped and mmf decreased to half dose. at year, while clinical features of cgvh intensified, fortuitous diagnosis of ns was done. glomerular filtration rate and blood pressure were normal. biopsy showed membranous glomerulonephritis and mesangial hypercellularity. immunofluorescence confirmed granular immune deposits of igg and c treatment consisted in prednisone ( . mg/kg/d for month, progressively tapered to . mg/kg/e.o.d at month ) and cyclosporine ( mg/kg/d, trough levels - ng/ml). proteinuria decreased to < . g/l in months. cyclosporine was progressively stopped between month and without relapse of proteinuria. ns associated with cgvhd had been reported in about patients ( patients < yr-old). retrospective studies estimate occurrence around % of patients with cgvhd. it was secondary to membranous glomerulonephritis in % of cases, minimal changes disease in %. strengthening of immunosuppression led to complete remission in % of patients with minimal changes disease and % of those with membranous glomerulonephritis ( % partial remission). usual treatment consisted in prednisone ( %) and cyclosporine ( %). this manifestation of cgvhd is probably underestimated and can occur at the same time or later than other clinical features. early detection of proteinuria in patients with cgvhd is recommended to adapt immunosuppressive treatment. objectives: to see if cyclosporine a (csa) is safe and effective in reducing proteinuria in children with the iga nephropathy (igan) or the henoch-schoenlein purpura nephritis (hspn). methods: the biopsy proven patients ( with igan, with hspn) who showed increased proteinuria (> +) for longer than months were included. the blood level of csa, serum chemistries, urine analysis and complete blood cell counts were carried out every other month along with the physical exams. results: csa was given at an amount of . . mg/kg/day for . . months in average. complete remission of proteinuria in ( . %) and partial remission in ( . %) were achieved by csa treatment. five ( . %) non-responders were discontinued for csa treatment in the middle of the trial. the ration of urine protein to creatinine was initially . . and reduced gradually with time to . . , . . , . . at , , months after csa treatment, respectively. twenty eight patients showed hypertrichosis, three experienced transient elevation of serum creatinine, and two complained difficulties in taking the medication due to severe nausea. for . months after completion of the csa treatment, patients redeveloped proteinuria and had to receive the nd csa trial. no clear difference was observed in the pharmacokinetic profiles of csa attributable to the non-response or recurrence. follow-up renal biopsies were carried out in patients after completion of the csa therapy and no csa toxicity was found. there was no alteration of linear growth pattern. conclusions: this study has a limitation of lacking the control group but the csa treatment is assumed to be very effective and a safe method to attain the remission of proteinuria in pediatric patients with the igan or hspn. j. madrigal , e. fernandez , p. noguera , p. carranza the aim of this retrospective study was : ) to correlate the histopathological diagnosis with steroid response,persistent hematuria, hypertension and or abnormal renal function tests (gfr) ) to evaluate the response of the patients with srns and sdns to the oral cytotoxic drugs , in a period of consecutive years. ) to correlate the response of this group of patients to the oral cytotoxic drugs with their histopathological diagnosis ) to observe the incidence of fsgs in costarrican children during a period of years. ) based on these observations, reevaluate the indications of the kidney biopsy in our patients . we reviewed all the clinical records of patients with the diagnosis of ns and in whom a kidney biopsy have been done. patients with incomplete data were excluded. two consecutive reviews were made: the first included all patients diagnosed between january and december (group a) and the second, between january and december (group b). a total of medical records were analyzed; patients were excluded, and the remaining were studied. results: all patients had been referred for edema or new onset nephrotic syndrome before treatment had been initiated. in our patients the steroid response was also the most important factor to predict the histological diagnosis and the response to the treatment with cytotoxics. the presence of hematuria and abnormal serum creatinine at the time of diagnosis were a predictive factor for the steroid response but not for the histological diagnosis. arterial hypertension achieved statistical significance only between mcns and fsgs but it was useful as a predictive factor for the hystological. in our group of patients with srns treated with cytotoxics, . % with mcns responded, versus . % of the patients with dmgn p< , . a. guersoni, v. mello, v. benini, s. laranjo children with srns are exposed to prolonged and high doses of steroid therapy and other immunosupressants that can lead to a variety of serious side effects. these include statural growth impairment, obesity, osteoporosis, cataract, hypertricosis and psychological disturbances.we carried out a prospective single-center study to evaluate the efficacy of mycophenolate in children with steroid-resistant disease, female and male, aged . ± . years. histological findings were: minimal change disease (mcd) in children, focal segmental glomerulosclerosis (fsgs) in and membranous nephropathy in one. all patients had been treated with at least one course of cyclophosphamide, metil-prednisolone in , while had also been treated with cyclosporine before mmf. the initial dose of mmf was mg/m per day, together with a minimal reduction dose of corticosteroids associated with angiotensin ii receptor blockers (arbs) and sinvastatin.three patients went into complete remission, into partial remission and showed no remission. partial remission was described as loss of edema and improvement in symptoms, despite persistence of significant but improved proteinuria, that was classified either as moderate or low proteinuria according to the level. side effects were: diarrhea (n= ), neutropenia (n= ), infectious disease (n= ). mmf is an important new therapeutic option when associated with angiotensin ii receptor blockers (arbs) and sinvastatin for srns with mcd or fsgs, providing improvement in edema and symptoms despite persistence of proteinuria, with no compromise of physical appearance or risk of nephrotoxicity. background: primary focal segmental glomerulosclerosis (fsgs) that is resistant to steroids and other immunosuppressive agents has a guarded long-term prognosis. patients who fail to respond to current treatment may benefit from therapies that inhibit renal fibrosis and retard progressive loss of kidney function. objective: the font study (novel therapies in resistant fsgs) is a phase i clinical trial designed to test the safety, tolerability, and pharmacokinetics (pk) of novel agents that reduce renal fibrosis in patients with resistant fsgs. methods: patients, age - yr and egfr > ml/min/ . m , with resistant fsgs who fail treatment in the nih supported trial evaluating cyclosporine vs. dexamethasone plus mycophenolate mofetil or who are screen failures due to prior exposure to these drugs are eligible. patients are assigned to receive rosiglitazone mg/m per day po or adalimumab mg/m every other wk sc. the treatment phase is wk and patients undergo an initial (wk ) and steady state (wk ) pk assessment. results: patients have been screened and have been randomized to each test therapy. patients have completed rosiglitazone therapy and have completed adalimumab. four serious adverse events have occurred in patients receiving rosiglitazone, none related to the study drug. fatigue ( %), gastrointestinal complaints ( %), and headache ( %) were the most common adverse events in patients given rosiglitazone. the outcomes in the adalimumab group have not been analyzed. conclusion: these preliminary results indicate the feasibility of performing phase i assessments of novel agents that can target renal fibrosis in patients with resistant fsgs. the findings will be used to design phase ii randomized clinical trials in this cohort of patients at high risk of progression to end stage renal disease. supported by grant niddk r . object: to study the effect of fty on glomerulosclerosis and expression of cell cycle regulatory proteins in subtotal nephrectomized rats. methods: rat were divided into sham-operation group, glomerulosclerosis model group and fty treated glomerulosclerosis group, rats in each groups. the rats in later two groups were subjected to / nephrectomy. after operation, the treat group was fed with fty for weeks. the expression of collagen, fibronectin, and cycline, p , p were determined by immunohistochemistry methods. results: after treatment with fty , up began to decrease from w after operation, significantly lower than in model group (p< . ). the model group showed higher level of scr from w, which was much higher than in control group (p< . ). in fty treated group, scr level were much lower than in model group. fty could obviously inhibit the expressionof col-and fn in glomeruli and attenuate the extent of glomerulosclerosis. moreover, fty could upregulate glomerular expression of p and downregulate glomerular expression of p and cycline. the expression levels of p and cycline were significantly lower in treatment group than in model group (p< . ), but still higher than in control group (p< . ). p expression in glomeruli was stronger in treatment group than in model group (p< . ), and lower than in normal group but without significant statistic difference. conclusion: fty can diminish urine protein excretion and prevent glomerulosclerosis in subtotal nephrectomized rats. this protective effect is presumed to be associated with its role in downregulation of cycline expression and upregulation of p expression in glomerular cells, and inhibition of extracellular matrix accumulation in glomeruli. the authors illustrate severe side effect of steroid therapy ulcerative gastroduodenitis-and rare complication (multiple cerebral thrombo-embolism) in the case of a year old girl with steroid resistent nephrosis. in childhood occurence of thrombosis in steroid sensitive nephrosis syndrome is . %, while it comes up to . % in steroid resistant cases. on admittance she presented the classic symptoms of nephrotic syndrome. one month after the initiation of steroid therapy haematemesis, melaena occurred, and after appropriate therapy was cured. due to the progression of the nephrotic syndrome, steroid shot and later immunesuppressive therapy was started. eight weeks after onset she became unconscious for a shortwhile and had a transient episode of right hemiparesis. at the same time ct and mri disclosed bilateral parieto occipital ischemic territorial vascluar lesions, with relative sparing of the cortical ribbbon. following icu observation her state rapidly improved and after a two week period she became free of symptoms. renal biopsy disclosed the pattern of minimal change nephrosis with diffuse mesangial hypercellularity and a slight amount of igm positivity. immunological evaluation-with the capacitiy to reveal systemic immunological diseases remained negative. having been put on an evidence based protocol the patient's present nephrological state was unremarkable, with proteinuria less then g/day. in the present work the authors discuss the factors predisposing to thrombo-embolism with special emphasis on the possible preventive measures and therapy. igg autoantibodies to c q (antic q) have been reported to play a pathogenic role in immunecomplex mediated diseases (sle, apsgn, membranoproliferative gn, etc). the occurrence of antic q in adult patients with sle has been shown to correlate with disease activity and some immunological parameters (hypocomplementemia, anti-dsdna) and may be useful in the early diagnosis of lupus nephritis (ln) or even as a predictor of renal flares. the presence of antic q in children with apsgn was associated with more severe disease manifestations and a lack of spontaneous recovery. associations between antic q, c and c complement levels and disease manifestations in children with gn were investigated and compared with healthy controls. antic q were measured by elisa and c and c by immunoturbidimetry, respectively. of patients with gn were positive for antic q compared to / healthy controls. antic q were associated with active ln and hypocomplementemia: / patients with sle were found to be antic q-positive. nine of these had active renal disease at the time of blood sampling compared to / being antic q-negative. / antic q-positive patients had low c level and / had low c level. in children with apsgn, / were positive for antic q. antic q positive patients had significantly higher proteinuria, more often hypertension and c -hypocomplementemia. all patients in which apsgn did not resolve spontaneously were antic q-positive. antic q were associated with active nephritis and hypocomplementemia in patients with sle. in children with apsgn antic q-positive patients have more severe disease and stronger c -hypocomplementemia then those being antic q-negative. m. zahrane , l. fawaz , l. nesseim cairo university hospital, pediatric nephrology, cairo, egypt cairo university hospital, pediatrics, cairo, egypt cairo university hospital, cell pathology, cairo, egypt objective of the study: growth hormone (gh) and insulin-like growth factors are essential for normal growth and development. chronic renal failure (crf) results in major changes in the circulating growth hormone/insulin-like growth factor (igf) system. our aim is to study clinical and laboratory parameters of growth and osteodystrophy including igf and igfbp as part of the somatotropic hormone axis in egyptian children suffering from crf on conservative therapy. methods: egyptian children ( boys and girls) with a mean age of . y ( . to . y) suffering from crf on conservative therapy and controls were included in the study. ht, wt and tsf were measured and followed up for a period of months. at the end of the follow-up period serum for igf and igfbp , renal function, electrolytes, ca, p and alkaline phosphatase and acid base balance were measured and an x-ray of the left hand and wrist was done to determine their bone age by tanner and whitehouse. results: our study shows that children suffering from crf in egypt on conservative therapy have growth retardation with a mean ht of . sds, a mean wt of - . sds. tsf mean was - . sds. on the average the patients had a delay of . y (± . ) in their bone age. their height was retarded more than their bone age with a height age/bone age of . (± . ). alkaline phosphatase as a markers of renal osteodystrophy is significantly correlated to the height, height age, bone age and to the ph. the mean igf sds (- . ± . ) did not differ from that of controls while the mean igfbp sds ( . ± . ) was significantly higher in patients with crf than in controls. height and weight were significantly correlated to igf but not igfbp . there is a significant correlation between igfbp level and the glomerular filtration rate. conclusions: the imbalance between normal insulin-like growth factor-i (igf-i) and markedly increased igfbp plasma levels plays a pathogenic role for growth retardation in children with chronic renal failure. the lower the gfr the higher the igfbp level. the latters inhibitory action may provide hope for improving growth in cases of crf by reducing the level of igfbp or displacing igf from it. s. sultana , h. rahman , m. hossain bangladesh medical college, pediatric department, dhaka, uttara, bangladesh bangabandu sheikh mujib medical university, pediatric nephrology, dhaka, bangladesh objective: to find out the impact of different etiology of chronic renal failure on growth in children. methods: this prospective study was carried out in the department of pediatrics, bangabandhu sheikh mujib medical university (bsmmu), dhaka, bangladesh, from october to october . fifty children of both sexes under years of age with clinical and biochemical evidence of chronic renal failure (crf) with creatinine clearance (ccr) of < ml/min/ . m were included in the study. on the basis of underlying causes of crf, the children were divided into congenital (n= ) and acquired (n= ) groups. all patient's height and weight were measured. radiographs of hands, digits, ankle and knee joints, lumbar spine & skull were obtained to evaluate the presence of renal osteodystophy (rod) and for assessment of bone age. serum intact parathormone (ipth) level was also assayed in all patients. growth parameters and presence of radiographic and biochemical features were evaluated in two groups. results: crf children due to congenital anomalies had stunting and wasting in ( . %) and ( . %) cases respectively and the difference between two groups of crf patients was highly significant (p< . ). alkaline phosphatase ( . ± . u/l) and ipth ( . ± . pg/ml) were also significantly higher than acquired group (p< . and p< . respectively). radiographic features of rod were present in ( %) cases in congenital group in comparison to ( %) in acquired group and the growth zone lesion was the commonest type of rod in congenital group ( . %). conclusion: all efforts should be made to diagnose the presence of crf as early as possible, especially in infants and in children with early onset crf who seem to lose growth potential. introduction: in patients with thalassemia major the most important cause of morbidity and mortality is organ failure due to iron deposits, desferioxamine toxicity and anemia. this study was designed to define renal abnormalities associated with thalassemia and to find early marker (markers) of renal dysfunction. patients & method: thlassemic children ( female and male) with mean age of . ± . yr. were studied. all of them were received desforioxamine. age and sex matched healthy children were involved in the study. blood and timed urine sample were obtained for hematologic and biochemical tests. the results were compared between case and control group. results: mean value of bun, serum creatinine, creatinine clearance, serum electrolytes, urine osmolality, fractional excretion of sodium and potassium were not statistically different between two groups. level of urinary nag (n-acetyle-beta-d-glycosaminidase) was significantly higher in patients than in controls (p: . ). there was a positive relation between urinary nag and duration of disease (p: . ). the was no statistically significant relation between urinary nag and serum ferritin. tubular function was not altered by hypertransfusion. conclusion: proximal tubular dysfunction is found in thalassemic patients. measuring urinary nag can guide the physician to find the early tubular abnormality in patients without frank renal dysfunction. severity of the abnormalities is correlated with the duration of disease. the present study aimed to investigate the effects of isolated ma and ma associated with mild renal function impairment on fracture healing in rats. ma was induced by chronic ingestion of % ammonium chloride solution as the unique source of liquid and renal dysfunction was produced by unilateral nephrectomy. thirty male holtzman rats ( - g) were divided into six groups: control group (c,n= ) non-operated rats receiving tap water, acidotic group (ac,n= ) non-operated rats ingesting % ammonium chloride; sham water (s,n= ) sham-operated animals receiving tap water; sham acidotic (sac,n= ) sham-operated rats ingesting % ammonium chloride; nephrectomy water (n,n= ) nephrectomized rats ingesting tap water; and nephrectomy acidotic (nac,n= ) nephrectomized rats ingesting % ammonium chloride. after one week, blood samples were obtained to measure ph and gases, and a fracture of the right tibia was manually produced. four weeks later, fracture healing was evaluated by radiological and histological parameters. blood ph and gases, serum electrolytes and creatinine were also determined. data were compared by anova followed by newman keuls or fisher's exact test. fracture healing in nac, ac, sac animals was significantly altered as compared to c group. there was an additive effect of metabolic acidosis and unilateral nephrectomy in fracture healing process as shown by the comparison of sac and ac rats using radiological and histomorphometrical parameters. there was no difference between electrolytes and creatinine levels in all groups at the end of the experiment. this study showed a higher frequency of delayed fracture healing and nonunion in the presence of ma, which is worsened by unilateral nephrectomy. our data indicated an important interaction between bone and kidney in acid base homeostasis. introduction: dent disease, an x-linked recessive tubulopathy, is historically characterized by lowmolecular weight proteinuria, hypercalciuria, nephrocalcinosis/lithiasis and slowly progressive renal failure. most cases are caused by mutations in the clcn gene (dent disease , omim # ), some patients with dent like phenotype have defects in the lowe syndrome gene ocrl (dent disease , omim # ). patients: male patients from families with urinary findings resembling dent disease are reported. in patients, mutations in the clcn gene were found, in patients ocrl mutations. all children have increased values of urinary alpha- -microglobuline, but also unselective glomerular proteinuria. / have mild hypercalciuria, / demonstrate mild renal insufficiency. amost all patients have increased echogenicity of renal parenchyma, but mild medullary hyperechogenicity is found only in of patients. metabolic acidosis or renal phosphate wasting is not found. interestingly, of children have increased values of creatinine kinase of unknown origin, clinically asymptomatic and independant of clcn or ocrl mutations. conclusion: the phenotype and genotype of dent disease is very heterogeneous. diagnostic criteria of dent disease and of lowe syndrome should be discussed. e. sahpazova, d. kuzmanovska, l. spirevska, n. ristoska bojkovska pediatric clinic, nephrology, skopje, macedoniathe nutritional condition of children ( males and females) mean age . ± . (range - years) with moderate renal failure have been followed for three year. glomerular filtration rate (gfr) was measured by creatinine clearance calculated with schwartz formula and was ranged from . to ml/min per . m . the nutritional condition was determined by anthropometrics and nutritional measurements. the patients were divided in four groups depending of their protein intake, primary disease, ages and glomerular filtration rate. all patients were following an -ad libidum -diet. nutritional intake was determined by minimum of two -day prospective dietary diaries. % of children received significantly lower protein intake. the mean protein intake (% of who recommendation) determined by dietaries of patients with -sub-optimal intake -was . % vs. . % in patients with -adequate protein intake -(p< . ). all patients have a calorie intake of at least % of the who recommendations. the relative distribution of calories was . % from proteins, . % from carbohydrates and . % from lipids. nitrogen balance in patients was positive and correlated most significantly with increasing energy intake (r= . ). average values for height, weight, triceps skin fold, mid-arm muscle circumference, and body mass index were within sd of the mean of the normal population. the protein intake, primary disease and age of the children did not have any effect on growth and development. only patients with more advanced renal disease showed small score for height and growth velocity. key words: chronic renal failure; uremic children; nutritional status; nutritional intake; u. aslanova , t. morimoto , e. farajov , n. kumagai , n. sugawara , a. ohsaga , y. maruyama , s. tsuchiya , s. takahashi , y. kondo tohoku university, pediatrics, sendai, japan tohoku university, medical informatics, sendai, japan tohoku university, physiology, sendai, japan nihon university school of medicine, pediatrics, tokyo, japan the extracellular calcium-sensing receptor (casr) located in either luminal or basolateral cell membranes of various types of renal tubules including proximal tubules, henle's loop and collecting ducts has been thought to play a fundamental role in electrolyte metabolism. to further identify the physiological roles of the casr, we examined the effects of ca + and calcimimetics neomycin (neo), gentamicin and gadolinium chloride gd + on the intracellular ph (phi) of in vitro microperfused mouse medullary thick ascending limb (mtal) cells of henle's loop, by loading the cells with fluorescent ph indicator ', '-bis-( -carboxyethyl)- -(and- )-carboxyfluorescein and measuring the ratio of fluorescence emission at nm after exciting the dye at and nm. in a steady-state condition in hepes-buffered solution, the phi in the mtals was . ± . (n= ). a concentration of micromol/l neo in the basolateral side decreased the phi after min by - . ± . (n= , p< . ). the other calcimimetics showed similar effects on phi, whereas none of these calcimimetics in the lumen affected phi. na + removal or the inhibition of na + and proton transport with amiloride, bumetanide, or bafilomycin did not eliminate the effect of neo on phi. on the other hand, clremoval clearly eliminated the neo-induced phi decrease (- . ± . vs - . ± . in clremoval, n= , p< . ). thus, we have demonstrated for the first time that the casr is involved in the regulation of the phi in the mtal and requires clto exert its effect. background: paediatric nephrologists are often consulted for atypical rickets of renal or non-renal origin. the comeback of vitamin d deficient (classical) rickets in armenia and elsewhere is not only a public health problem but also a new diagnostic challenge for nephrologists. the aim is to analyse all paediatric patients seen in with bone deformities and suspected rickets at the arabkir hospital in yerevan. patients and methods: patients with bone deformities came spontaneously or were referred by one of us (gk). routine serum chemistry was done in yerevan. further investigations, if needed, and urine chemistry were performed in zurich. patients with rickets due to renal insufficiency were excluded. results: in we have seen patients ( males) with rachitic bone deformities aged - months (mean ± . ) at diagnosis. of these, patients had florid vitamin d deficient rickets and had sequelae of rickets but were radiologically and biochemically cured. these children with classical rickets had to be distinguished from patients with other forms of rickets: x-linked hypophosphataemia (xlh; ), vitamin d dependant rickets type ( ), and renal fanconi syndrome ( ) due to fanconi-bickel syndrome and idiopathic. conclusions: (i) the rising number of vitamin d deficient rickets is of concern and due to neglected prophylaxis, (ii) children with classical rickets came late and were in the same age range as patients with atypical rickets, (iii) hence, and because of the larger number of rachitic children, an increased awareness of nephrologists -including a full diagnostic work-up -is needed in order not to overlook rare forms of rickets, (iv) polar vitamin d metabolites should not be used before the precise type of rickets is known. b. spasojevic-dimitrijeva, m. kostic, a. peco-antic, d. kruscic, d. paripovic, m. stanic university children's hospital, nephrology department, belgrade, serbia t. porowski , w. zoch-zwierz , j. konstantynowicz , k. taranta-janusz medical university of bialystok, st department of pediatrics, bialystok, poland medical university of bialystok, department of pediatrics and auxology, bialystok, poland there are no published data on calcium oxalate (caox) crystallization and therewith associated kidney stone disease in children. the aim of this study was to determine bonn risk index (bri) in children with urolithiasis in relation to healthy age-and sex-matched controls and to assess possible associations between bri values and the size of renal stones. methods. in this cross-sectional study, we compared bri in caucasian children and adolescents ( girls, boys) aged - years (median: . ) in whom kidney stones were newly diagnosed and healthy age-and sex-matched controls ( girls, boys) without urolithiasis. urinary ionized calcium [ca + ] was measured using a selective electrode, while the onset of the spontaneous crystallization was determined using a photometer and titrating with mmol/l ammonium oxalate (ox ). the calculation of bri value was based on [ca + ] to (ox ) ratio. high resolution renal ultrasonography was done to estimate the size of renal stones. results. our results showed that bri values in children with renal stones were greater compared with healthy children without stones. bri was -fold greater, bri/kg body weight - -fold greater, bri/per . m b. s. - -fold, whereas bri/bmi was even -fold greater in cases with stones than in controls. no significant association was observed between bri and the size of stones. interpretation. children with kidney stones demonstrate increased bone risk index compared with healthy subjects without urolithiasis. an increased bri during growth, although unrelated to renal stone size reflects the risk for crystallization of calcium oxalate and may suggest early metabolic disorders leading to urolithiasis. this simple method appears to be accurate and cost-effective, thus bri may be widely used for discrimination between stone-formers and healthy children. m. dixit, n. dixit florida children's hospital, florida children's kidney center, orlando, united states acute tubulo-interstitial nephritis (atin) is an important cause of acute renal failure resulting from a variety of insults including rare immune complex-mediated tubulo-interstitial injury. drugs including non-steroidal anti-inflammatory drugs (nsaids) are far most frequent cause of atin, but overall as an entity it remains under-diagnosed as symptoms resolve spontaneously if the medication is stopped. we present a -year-old male who developed acute renal failure weeks after aortic valve surgery. he was put on aspirin following surgery and took ibuprofen for fever for nearly a week prior to presentation. he then presented to the emergency department feeling quite ill and was found to have bun of mg/dl, creatinine of . mg/dl and serum potassium of . meq/l. dialysis therapy was immediately initiated. a kidney biopsy showed inflammatory infiltrate consistent with atin. however, very intense tubular basement membrane (tbm) granular deposits of polyclonal igg and c were noted. he needed dialysis for nearly two weeks and was treated successfully with steroids. his renal recovery and disappearance proteinuria took almost a year. in conclusion, we present an unusual case of tbm immune complex-mediated atin due to nsaids with severe but reversible renal failure. the effect of corticosteroid therapy on bone metabolism in nephrotic syndrome background: nephropathic cystinosis is characterised by lysosomal cystine accumulation leading to generalized fanconi syndrome. defective tubular reabsorption of proteins, mainly by the multi ligand receptors megalin and cubilin, is considered to be the cause of proteinuria in cystinosis. whether increased glomerular permeability contributes to proteinuria in cystinosis is investigated in this study by evaluating ) urinary protein pattern in cystinotic patients and healthy controls ) expression of megalin, cubilin and their ligands transferrin, albumin, a -microglobulin (a m) and b -microglobulin (b m) in renal tissue. methods: urine of cystinotic patients and controls (n= ), aged - , were immunoblotted using antibodies against megalin, cubilin, transferrin, albumin, a m and b m. additionally, urinary levels of igg, albumin, a m and creatinine were measured. results are expressed as mg/mmol creatinine (median, range). presence of proteins in semithin paraffin sections from cystinotic and control kidneys was evaluated using antibodies mentioned before. results: cystinotic patients had increased urinary excretion of , p < ) and all tested ligands of megalin and cubilin. immuno histochemistry showed comparable expression of megalin, cubilin and their ligands in convoluted proximal tubules (pt), while the ligands in straight pt were only present in cystinotic patients. conclusion: a selective proteinuria with high molecular weight protein excretion such as igg indicates increased permeability of glomerular filtration barrier in cystinosis already at an early age. the presence of the megalin and cubilin ligands in endocytic vesicles suggests functional endocytosis. however, the enhanced staining of the ligands in cystinotic straight pt may be a result of incomplete reabsorption in convoluted pt. background: dent's disease and lowe syndrome are the most frequent x-linked tubulopathies. dent's is characterized by lmw proteinuria, hypercalciuria and nephrocalcinosis. in ca. %, this phenotype results from mutations in the clcn -gene. lowe syndrome (congenital cataract, mental retardation and generalized fanconi syndrome) is due to mutations in the ocrl gene. stunted growth is another typical finding in lowe patients. recently, in a subgroup of dent patients ocrl gene mutations have been demonstrated (dent- disease). aim of the study: comparison of the growth pattern of patients with clcn and ocrl mutations. patients: boys with proven mutations in clcn (n= , mean age . ± yrs) were compared with those with dent- disease (n= , ± yrs) and those with ocrl mutations and a lowe phenotype (n= , . ± . yrs). comparison of z-scores for height, weightand bmi. results: clcn positive boys had a significantly higher height-sds (- . ± . ) than ocrl positives (dent- : - . ± . /lowe: - . ± . ). there were no significant differences in bmi-sds and weight-sds. the difference between weight and height sds as a parameter for obesity in these small-statured children was higher in lowe than in classical dent patients, with intermediate values being found in dent- . discussion: although the renal phenotype of dent- patients is identical with classical dent, the former are more stunted. therefore, the abnormal growth pattern in dent- patients cannot be ascribed to renal dysfunction. taken together with other findings (elevated ck and ldh, mild mental retardation) our findings illustrate that dent- is indeed a mild variant of classical lowe syndrome. quantitative ultrasonometry of the calcaneus in children with idiopathic hypercalciuria lesch-nyhan syndrome is a very rare x-linked recessive disorder characterized by mental retardation, spasticity resembling cerebral palsy, choreoathetosis, self-mutilation and hyperuricemia. self-mutilation behavior is a hallmark of the disease. hyperuricemia leads to hyperuricuria and uric acid nephrolithiasis. we report on a -year-old boy with lesch-nyhan syndrome with no self-mutilation behavior who was erroneously diagnosed as having athetotic cerebral palsy. besides, he had no renal stones, the only renal abnormality detected were hyperechoic renal medullary pyramids, sonographicaly indistinguishable from medullary nephrocalcinosis. bone disease is frequently observed in children with homozygous beta-thalassemia (thal). we have observed an increased prevalence of renal stones in these patients. in order to understand the cause of this predisposition to renal stone formation we investigated markers of bone metabolism in our thal children. we studied thal children (age range - years; females and males) with no eviodence of renal stones. thal was diagnosed with haemoglobin hplc study and genetic typing. all received blood transfusion and iron chelants on a regular schedule. serum levels of pth, osteocalcin, telopeptide c-terminale (cross-laps) were determined with eclia technique; serum vitamin d ( ohd ) with elisa technique. serum calcium, phosphate, uric acid, bicarbonate, creatinine, alkalin phosphatase and sodium, calcium, oxalate, citrate and creatinine in hours and fasting urine were determined with common methods. as controls we studied children with comparable age. serum pth and vitamin d were increased in % of our patients, serum osteocalcin in % and telopeptide c-terminale (cross-laps) in %. hypercalciuria was observed in %, hyperoxaluria in % hypocitraturia in %. significant correlation were found between pth and osteocalcin (p< . ) cau/cru (p< . ); osteocalcin and cross-laps (p< . ) and vit d and cau/cru (p< . ). bone disruption due to bone marrow expansion may produce an increase in vit d and pth production with hypercalciuria producing renal stones. losartan is an angiotensin ii subtype (at ) receptor antagonist used for controlling blood pressure and urinary protein excretion in patients with hypertension and chronic renal disease. there are a few reports about the clinical implications of at- receptor antagonism that may interfere with the kidney's defense against an acid load and may thereby exacerbate metabolic acidosis in the literature. the suggested mechanism is that at- blockade by losartan exacerbates acidosis by inducing a distal-tubular acidification defect.we observed metabolic acidosis and hyperkalemia in five patients ( females/ males, age ranged - years) whom were given losartan. during the second week of the therapy all patients revealed a metabolic acidosis with (ph; ranged , to , and hco ; ranged , to mmol/l) hiperkalemia (ranged , to , mg/dl). the etiologies of chronic renal disease in the patients were focal segmental glomerulosclerosis (fsgs) in one, lupus nephritis in one and three had undergone renal transplantation according to different etiologies. glomerular filtration rate was higher than ml/sec/ , m in all patients. immune suppressive regimen of renal transplanted patients was based on tacrolimus in two patients and on cyclosporine a in one. both renal transplanted patients and other two patients with fsgs and lupus nephritis were all receiving steroid, enalapril and mycophenolate mophetil at the same time during the losartan therapy. metabolic acidosis and hyperkalemia were recovered within a week following the exclution of the losartan. in conclusion, we think detailed and controlled studies are necessary to determine the pathogenesis of the metabolic acidosis due to losartan and patients must be followed up very closely for the adverse effects of metabolic acidosis and hyperkalemia during the treatment. objectives of study: bartter's syndrome is a rare renal tubular disorder characterized by hypokalemia and metabolic alkalosis. it is also known to be effectively treated with potassium supplement, potassium sparing diuretics and indometacin. we experienced two sibling cases whose problems were incompletely solved with above mentioned conventional treatment, but rather completely with adjunctive therapy of regular hemodialysis with dialysate of low bicarbonate concentration. case : this male patient was diagnosed of bartter's syndrome when he was months old. he was treated with potassium supplement, aldactone and indometacin with marked improvement. but he still had some problems of retarded growth, severe headache and episodes of marked hypokalemia which needed repetitive admission. when he was years old, we put him weekly hemodialysis with dialysate of low bicarbonate concentration ( meq/l). with hemodialysis, he has been in good condition for years with stable blood ph and serum potassium levels. case : this female patient, the elder sister of case , was diagnosed of bartter's syndrome at one year of age. with those therapy on conventional medications, she seemed to grow out of failure to thrive, but needed repeated admissions due to episodes of dehydration and hypokalemia. after start of weekly hemodialysis with low bicarbonate dialysate, for one year she has been on good control of blood ph and serum potasium level, and was never admitted with those episodes. conclusion: regular hemodiaysis with dialysate of low bicarbonate concentration can be considered as effective adjunctive therapy in intractable bartter's syndrome. a. deguchtenaere, a. raes, j. dehoorne, c. vande walle, r. mauel, j. vande walle university hospital gent, pediatric uro-nephrologic center, gent, belgiummonosymptomatic enuresis nocturna (mne) may be associated with nocturnal polyuria (np) and low urinary osmolality during the night. besides vasopressin, recent studies have stressed the possible role of renal sodium-handling, hypercalciuria, prostaglandins and/or osmotic excretion.the aim: was to study circadian rhythm of gfr and diuresis in a highly selected group of children with persistant np. methods: population existed of children with mne and np, age - y, males. controls n= children, - y with mne, but no np. (=b). renal function during h concentration-prophyle, with timed urine samples, and measurement of p and u for na, k, osmol, creatinin. calculation of gfr by creatinine, uosmol, feosmol, diuresis vol (ml/min), fena, fecl, fek, u k /u k +u na %. statistics: paired t-test p< . between d and n, unpaired t-test, between the groups. conclusion: children with nocturnal polyuria have not only lost their circadian rhythm of diuresis and sodium-excretion but also of gfr. another observation for a reanl involvement in mne. sarcoidosis is a systemic disorder of unknown etiology, rare in children, characterized by the presence of noncaseating granuloma in affected organs. we report a -year-old boy of french african origin who presented with left hearing loss followed by bilateral deafness within months. a history of bilateral uveitis was secondarily unveiled. mild renal insufficiency (creatinine level μmol/l ; clearance: mmol/min) was diagnosed prior to cochlear implant surgery. a percutaneous renal biopsy evidenced a granulomatous interstitial nephritis with widespread interstitial fibrosis. complementary explorations showed elevated lysozyme activity at μg/l (normal < ) with elevated cd /cd ratio in bronchoalveolar lavage specimen. pulmonary function test was notable for mild diffusion impairment. cerebral mri demonstrated abnormal enhancement involving the periventricular white matter and the intracanalicular portions of both viii cranial nerves. cerebrospinal fluid showed abnormal hyperlymphocytosis ( lymphocytes per mm ) while protein was normal. three weeks after admission, bilateral uveitis recurred and was cured by local steroid therapy. despite intensive treatment with intravenous prednisone g/ , m /j per day, successive days per month associated with oral prednisone during months, glomerular filtration rate did not improve. in conclusion, sarcoidosis may apparently be revealed by acute bilateral deafness, and prompt diagnosis is needed to avoid permanent lesions. gfr cystcin= . cystc - . , formgfr= *height (cm)/s-creat (mmol/l). d difference between c inulin and tested method. conclusion: none of the tested methods seems to reveal hyperfiltration in type diabetes patients as clearance of inulin. the best correlation was found to clearance of iohexol and second best gfr estimated by /cystatin c. creatinine clearance overestimates and formula clearance underestimates gfr in diabetic patients without nephropathy. objective: to study the effects and mechanism of fty on the renal interstitial fibrosis in unilateral ureteral obstructic rats. methods: fouty-five males sd rats were randomly divided into sham-operated (sham), unilateral ureteral obstruction (uuo) and uuo treated with fty (uuo+fty ) group. . mg.kg - .d - of fty or vehicle was administrated through daily gavage and begun from two days before the operation till being sacrificed. -hour urine protein, blood urea nitrogen and plasma creatinine were determined. the renal tubular interstitial fibrosis lesion and the expression of α-sma,col-i, cd , ed were scored semi-quantitively. results: the amount of hours urine protein was much lower than that in uuo group, (p< . ). serum creatinine in fty treated group were significantly lower than those in uuo group (p< . ). the scores of renal interstitial fibrosis were lower in fty group than that in uuo group. α-sma expression was limited to vessels in sham group, but extended to renal tubule and interstitium in uuo and fty treated group, while relatively weaker expression was observed in fty group than in uuo group. some collagen expression was found in sham group, which was much enhanced in uuo group and mainly distributed in renal interstitium, the expression in fty group was also increased compared to sham group, but much lower than that in uuo group. obvious lymphocyte and macrophage infiltration were found in tubular interstitial area in uuo group but significantly less in fty treated group (p< . ).conclusion: novel immunomodulator fty can obviously inhibit renal interstitial lymphocyte and macrophage infiltration, renal tubule cell transdifferentiation, and interstitial fibrosis, thus prevent renal disease progression. background: the mesangial cell, especially as a fundationtal component in normal mature glomeruli, is essential to keep glomerular capillary lumen open and to maintain efficient ultrafiltration. loss of mesangial cells due to pathologic conditions such as glomerulonephritis leads to impaired renal function. the exact developmental origin of mesangial cells is unknown. it has been established that mesenchymal stem cells, which are derived from bone marrow, have a potential to differentiate into different lineages in response to different environments. the purpose of the study is to examined the effect of platelet-derived growth factor (pdgf) in the differentiation of bone marrow-derived cells into mesangail cells. method: isolated bone marrow cells were cultured in the medium containing collagen type i within hours, and then transferred to collagen type i.-coated dishes. the cells attached to collagen type i. in the following days were maintained in the differentiation medium containing % horse serum, μmol/l, and μmol/l of pdgf and all-trans retinoic acid. results: after cultivation under the above condition, approximately % of cells expressed β actin and desmin, which highly resembled cultured mesangial cells in rat. the induced cultured cells changed into a wide range of shapes from spindle to stellate. the results indicate that bone marrow-derived stem cells could differentiate into mesangial-like cells in vitro. key: cord- -cfci lef authors: freites nuñez, dalifer d; leon, leticia; mucientes, arkaitz; rodriguez-rodriguez, luis; font urgelles, judit; madrid garcía, alfredo; colomer, jose i; jover, juan a; fernandez-gutierrez, benjamín; abasolo, lydia title: risk factors for hospital admissions related to covid- in patients with autoimmune inflammatory rheumatic diseases date: - - journal: ann rheum dis doi: . /annrheumdis- - sha: doc_id: cord_uid: cfci lef objectives: to describe patients with autoimmune inflammatory rheumatic diseases (aird) who had covid- disease; to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to covid- . methods: an observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus ( march to april). all patients attended at the rheumatology outpatient clinic of a tertiary hospital in madrid, spain with a medical diagnosis of aird and with symptomatic covid- were included. the main outcome was hospital admission related to covid- . the covariates were sociodemographic, clinical and treatments. we ran a multivariable logistic regression model to assess risk factors for the hospital admission. results: the study population included patients with aird and covid- . of these, patients required hospital admission related to covid- . the mean age on admission was . ( . ) years, and the median time from onset of symptoms to hospital admission was ( – ) days. the median length of stay was ( – ) days. a total of patients died ( %) during admission. compared with outpatients, the factors independently associated with hospital admission were older age (or: . ; p= . ) and autoimmune systemic condition (vs chronic inflammatory arthritis) (or: . ; p= . ). no statistically significant findings for exposure to disease-modifying antirheumatic drugs were found in the final model. conclusion: our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission, whereas disease-modifying antirheumatic drugs were not associated with hospital admission. severe acute respiratory syndrome coronavirus (sars-cov- ) causes a myriad of clinical signs and symptoms, together with typical laboratory abnormalities, that manifest as the disease covid- . since the confirmation of the first patient infected with sars-cov- in spain in january , the current covid- outbreak has had a considerable impact, especially in the madrid region, where the highest incidence of covid- cases has been recorded, with more than patients admitted to the hospital until the first week of may. the incidence and severity of covid- disease seem to be higher in patients with risk factors, such as advanced age and associated comorbidities, mainly hypertension, diabetes, heart disease and previous respiratory diseases. it is not clear whether patients with rheumatic diseases are more susceptible to sars-cov- infection, or, when they are infected, whether they have more severe disease or a poorer outcome. previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk, although some patients are candidates for a higher number of infections owing to their rheumatic disease (predominantly systemic) or the treatment they are receiving for rheumatic diseases. preliminary experiences in patients with show that those with chronic arthritis treated with synthetic conventional or targeted synthetic/biologic disease-modifying antirheumatic what is already known about this subject? ► the epidemiological scenario is changing daily. there is little evidence for risk factors of poor outcome with covid- specific to autoimmune inflammatory rheumatic diseases. ► patients with an autoimmune systemic condition have a higher risk of hospital admission related to covid- compared with those with chronic inflammatory arthritis. ► disease-modifying agents were not associated with a higher risk of hospital admission related to covid- . how might this impact on clinical practice or future developments? ► our data show that, in a real-world setting, a high percentage of patients with autoimmune inflammatory rheumatic diseases and covid- required hospital admission. the patients were mainly elderly, with comorbidities and a systemic autoimmune condition. epidemiology drugs (dmards) do not seem to be at a greater risk of respiratory or life-threatening complications from sars-cov- than the general population. the epidemiological scenario is changing, and evidence on the risk factors of poor outcome with covid- specific to inflammatory rheumatic disease is scarce. in addition, there are little data on how the hospital admissions of these patients with severe covid- infection have evolved. the aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases (aird) who had covid- during the pandemic peak. we also explored possible risk factors associated with hospital admission related to covid- disease in patients with aird from a tertiary hospital in madrid, spain. the study was performed in a public tertiary hospital, hospital clínico san carlos (hcsc), in madrid, spain. the catchment area is home to almost people. we performed a prospective observational study from march (when our health area had the first hospital admission related to to april . we preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department (hcr penelope). the inclusion criteria were age > years, a medical diagnosis (according to international classification of diseases (icd- )) of inflammatory rheumatic disease and symptomatic covid- disease assessed by medical diagnosis or confirmed with a positive sars-cov- pcr diagnostic test. patient data were obtained during routine clinical practice. the study was conducted in accordance with the declaration of helsinki and the principles of good clinical practice and was approved by the hcsc ethics committee (approval number / -e-bs). the primary outcome was admission to hospital with a medical diagnosis of covid- and/or a positive pcr result between march and april compared with outpatients with symptomatic covid- disease. the covariables recorded were as follows: ( ) sociodemographic baseline characteristics including sex, age and rheumatic disease duration. ( ) type of aird, including systemic autoimmune conditions (polymyalgia rheumatica, mixed connective tissue disease, systemic sclerosis, sjogren's syndrome, vasculitis, raynaud phenomenon, polymyositis, polychondritis, sarcoidosis, antiphospholipid syndrome, autoinflammatory syndromes and systemic lupus erythematosus) and chronic inflammatory arthritis (rheumatoid arthritis, inflammatory polyarthritis, juvenile idiopathic arthritis, psoriatic arthritis, axial spondyloarthritis, uveitis and inflammatory bowel disease). ( ) baseline comorbid conditions, including hypertension, dyslipidaemia, depression, diabetes mellitus, smoking habit, kidney disease, chronic liver disease, respiratory diseases (chronic obstructive pulmonary disease and interstitial lung disease), thyroid disease, heart disease (valve disease, arrythmias, cardiomyopathy, heart failure and pericarditis), ischaemic vascular disease (stroke, cardiovascular and peripheral vascular disease), venous thrombosis/lung embolism and cancer. ( ) treatment for inflammatory rheumatic disease: (a) glucocorticoids, (b) nonsteroidal anti-inflammatory drugs (nsaids), (c) conventional synthetic disease-modifying antirheumatic drugs (csdmards): antimalarials (hydroxychloroquine and chloroquine), azathioprine, cyclophosphamide, cyclosporine, colchicine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid and sulfasalazine; (d) targeted synthetic/biologic dmards (ts/ bdmards) including: ( ) antitumour necrosis factor (tnf)alpha drugs (infliximab, adalimumab, etanercept, certolizumab and golimumab); ( ) other biologics: anti-interleukin (il)- (tocilizumab and sarilumab); rituximab; abatacept; belimumab; anti-il- / ; anti-il- (ustekinumab, ixekizumab and secukinumab); ( ) janus kinase (jak) inhibitors (tofacitinib and baricitinib). treatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy, glucocorticoids, sulfasalazine, nsaids or colchicine. regarding csdmards and ts/bdmards, treatment had to start at least month before the beginning of the study and continue until at least st march, the end of the study or hospital admission. in the case of rituximab, the last infusion had to be at least in january. patient sociodemographic, clinical, laboratory and data on treatment of rheumatic disease were obtained through hcr penelope. patients with covid- were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation. other infected patients were detected through their sick leave forms for covid- . the results of sars-cov- pcr diagnostic assays were obtained from the microbiology/ infectious service of hcsc. in addition, our hospital central services registered all medical admissions to hcsc. this information was provided from march to april. the researchers carried out an exhaustive review of the clinical histories of admitted patients to identify covid- cases and rule out patients admitted for other reasons. once the covid- cases were identified, we collected clinical, laboratory and treatment data during admission until the end of admission (either discharge or death) in order to describe the progress of the disease. the review was performed until th april in order to include follow-up data from patients admitted to the hospital with covid- . patient characteristics are expressed as mean and sd or median and iqr for continuous variables; categorical variables are expressed as percentages. statistical tests were performed to compare characteristics between patients admitted with covid- and those without hospital admissions. continuous variables were analysed using the mann-whitney test or t-test, and discrete variables were analysed using the χ or fisher exact test. univariable logistic regression analyses were performed to assess differences between hospital admissions related to covid- risk and covariates. multivariable logistic regression models (adjusted for age, sex and comorbidity) were run in a stepwise manner to examine the possible effect of sociodemographic, clinical and therapeutic factors on hospital admissions related to covid- the model also included csdmards and all other variables with a p< . from the simple regression analysis. the results were expressed as the or with its respective % ci. all analyses were performed in stata v. statistical software (stata corp). a two-tailed p value < . was considered to indicate statistical significance. a total of patients with aird with symptomatic covid- disease were included in the study (table ) . the tests were performed as an exploratory measure of the association between a variable and the outcome. most of the patients were women, with a mean age of . ( . ) years and a mean time since diagnosis of . ( . ) years. the main diagnosis was rheumatoid arthritis ( . %), followed by axial spondyloarthritis ( . %). many patients had at least one baseline comorbid condition, the most prevalent being hypertension, dyslipidaemia and lung disease. most patients were taking csdmards ( . %). half of the patients were taking glucocorticoids ( . %), a quarter were taking nsaids ( . %) and . % were taking ts/bdmards, of which adalimumab was the most frequently prescribed ( . %), followed by rituximab ( . %). only one patient was taking a jak inhibitor. interestingly, . % of the patients taking ts/bdmards were taking the drug in combination with a csdmard. a total of patients had to be admitted to the hospital because of covid- . of these, were evaluated in the hcsc emergency department ( were admitted to hcsc and were transferred to the institucion ferial de madrid (ifema) support hospital owing to the lack of capacity in our hospital at that time). the remaining three patients were evaluated and admitted to other hospitals in the autonomous community of madrid. table presents data for the patients admitted to hcsc. of the patients admitted to our hospital, . % were women, with a mean age at admission of . ( . ) years and median lag time from the onset of symptoms to the admission of ( - ) days. the median length of stay was ( - ) days (table ) . at admission, the median haemoglobin was . ( . - . ) g/dl and the median total lymphocyte count was ( - ) ng/ml. the median d-dimer value was ( - ) ng/ml. in % of patients, median interleukin (il)- levels were ( - ) pg/ml. patients received various antibiotics (mainly azithromycin, levofloxacin and third-generation cephalosporins). most patients were treated with hydroxychloroquine during admission ( %). about half received glucocorticoids ( %). eighteen were treated with lopinavir/ritonavir and received the anti-il- r antibody tocilizumab (table ) .feder a total of patients ( %) developed relevant complications during admission, the most frequent being myocarditis, thrombosis and kidney failure. only two patients were admitted to the intensive care unit during admission. the first was a patient in s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia. the patient was treated with antibiotic therapy, lopinavir/ritonavir, hydroxychloroquine and β-interferon. finally, the patient was extubated days later and is recovering. the other was a young adult patient with systemic lupus erythematosus treated with methotrexate, rituximab, hydroxychloroquine and glucocorticoids, who, days after being diagnosed with covid- (pcr+), developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening (elevated d-dimer values). the patient was treated with methylprednisolone, heparin and a cephalosporin. a few days later, the patient's condition improved and he recovered completely at discharge. of the patients admitted to hcsc, were sent to another care centre (converted-hotel hospital/ifema support hospital) when their condition improved. a further patients ( . %) were discharged home to continue self-isolation after improvement. at the end of the study, five patients remained in hospital ( . %). a total of patients died ( %) during admission ( men and women), with a median age of ( . - ) years. of the patients who died, % had relevant comorbidity (diabetes mellitus, pulmonary disease, ischaemic vascular disease, hypertension, venous thrombosis/lung embolism, lung disease and or liver disease). the main diagnoses were rheumatoid arthritis ( ), followed by spondyloarthritis ( ), polymyalgia rheumatica ( ), vasculitis ( ) and sjogren's syndrome ( ) . the results of the univariable analysis are shown in table . older age, systemic autoimmune conditions (vs chronic inflammatory arthritis) (or: . ; % ci . - . , p= . ), hypertension, diabetes mellitus, lung disease, heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital. female sex, nsaids and anti-tnf drugs (vs non-use) were associated with a statistically significant lower risk. the differences reported for the remaining variables did not reach statistical significance. the multivariable analysis was adjusted for gender, age and comorbidities related to covid- . these comorbidities were diabetes mellitus, pulmonary disease, ischaemic vascular disease, hypertension, venous thrombosis/lung embolism, lung disease and/or liver disease ( our study aims to shed light on rheumatologists' concerns regarding their patients. we found that, in a real-world setting, % of patients with aird and covid- required hospital admission. these were mainly elderly patients, with more comorbidities and systemic autoimmune conditions. our data show that patients exposed to disease-modifying agents do not seem to be at higher risk of hospital admission related to covid- . of the patients included in the study with covid- , required hospital admission. comparison of the characteristics of patients admitted to hospital because of covid- and those who did not require hospital admission were as follows: admitted patients had a median age close to years, that is, more than years older than patients who were not admitted. moreover, those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions. as for therapy, admitted patients were less frequently exposed to antimalarial and anti-tnf-alpha agents. the median lag time from onset of symptoms to admission was days, and almost % of patients had pneumonia at admission. the baseline laboratory results for admitted patients in our study are consistent with those published elsewhere [ ] [ ] [ ] [ ] and are characterised by lymphopenia and elevated acute-phase reactants. in fact, % of the patients had elevated d-dimers (normal, < ) and elevated il- (normal, < pg/ml). treatment during admission varied widely as the disease proved epidemiology table baseline demographic and clinical characteristics of patients with aird and with covid- (admitted vs no admitted at the hospital) aird, autoimmune inflammatory rheumatic disease; anti-tnf, tumour necrosis factor-alpha; copd, chronic obstructive pulmonary disease; csdmard, conventional synthetic disease-modifying antirheumatic drug; ild, interstitial lung disease; jaki, jak inhibitor; ts/bdmards, target synthetic/biologic disease-modifying antirheumatic drug. challenging for specialists, who prescribed various combinations of drugs based on little published evidence. in this sense, the anti-il- r antibody tocilizumab has proven to be beneficial in patients with covid- . treatment may also be successful in the early stages of cytokine release syndrome, if it can effectively block the signal transduction pathway of il- ; therefore, tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe covid- . in our study, almost % of the patients were treated with tocilizumab. the patients who eventually died had a median age of > years. this finding is in line with data for the general population, where over % of deaths occurred in persons > years and more than % of all deaths were in people aged ≥ years. the multivariable regression model showed that only age (increasing by % per year) and systemic autoimmune conditions continued to be risk factors for hospital admission related to covid- . as for the association between sex and risk of hospital admission, we did not find a higher risk of admission in women, despite the fact that rheumatic diseases are more prevalent in this group. the type of diagnosis seems to play an important role in the probability of hospital admission, and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritis. as it has been reported elsewhere, comorbidities play an important role in the risk of hospital admission. clinical outcomes are poorer in patients with covid- with a comorbid condition than in those without, and a greater number of comorbidities correlates with poorer clinical outcomes. diabetes is a major comorbidity in covid- , and patient's history of diabetes is an independent risk factor for morbidity and mortality in this condition. diabetes has been associated with admissions to epidemiology the intensive care unit due to covid- in recent series and has been shown to increase mortality. therefore, we adjusted for comorbidity in the multivariable analysis. treatment with glucocorticoids lost its statistical significance in the multiple regression model. however, the dose was not reported in our data, and in the case of these agents, the risk could be dosedependent. in a recent publication from a european registry, the authors found that exposure to > mg/day was associated with a greater probability of hospitalisation. the exposure to dmards, regardless of whether they were biological or synthetic, does not seem to be associated with a higher hospital admission related to covid- . although we have to consider the limited number of patients in our study, our results are in concordance with data reported elsewhere. our results should be interpreted taking into account other limitations. first, patients were included from a single centre. second, of all the patients with covid- who did not require admission, one-third contacted the rheumatology service to report the disease and the remainder were detected through the covid- discharge reports sent to their primary care physician. elderly persons and homemakers who did not contact us can be considered missing. consequently, there may be some selection bias between those admitted and those not admitted, although this problem was addressed by adjusting for confounders in the multivariable analysis. third, while it is acknowledged that clinical suspicion must be confirmed by pcr assay, almost % of patients admitted did not undergo pcr owing to the lack of tests or the extreme healthcare overload. nevertheless, all cases included were clinically compatible and managed as covid- . the key strength of our study is that it was performed in real-life conditions during then pandemic peak, with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history, including thorough hospital admission data such as laboratory abnormalities and covid- treatment data from the hospital computer services. consequently, this has allowed us to analyse the risk of hospital admission related to covid- adjusted for confounders, thus avoiding possible bias. although we are unable to modify the factors reported here, knowing them can help rheumatologists to treat and advise their patients during this new and challenging period. results provided by our study are preliminary and should be corroborated with other real-life studies, but we consider our findings helpful to increase the knowledge in the management of patients with aird and covid- . twitter benjamín fernandez-gutierrez @fergutbe comorbidities including the presence of at least one of the follows: hypertension, heart disease, vascular disease, diabetes mellitus, venous thrombosis/lung embolism, chronic kidney disease, liver disease and lung disease (ild/copd). aird, autoimmune inflammatory rheumatic disease; ;copd, chronic obstructive pulmonary disease; ild, interstitial lung disease. covid- with pulmonary involvement. an autoimmune disease of known cause informe de situación del de mayo epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study are my patients with rheumatic diseases at higher risk of covid- ? covid- infection and rheumatoid arthritis: faraway, so close! clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical course of covid- in a series of patients with chronic arthritis treated with immunosuppressive targeted therapies covid- in immune-mediated inflammatory diseases -case series from new york lymphocyte subset (cd +, cd +) counts reflect the severity of infection and predict the clinical outcomes in patients with covid- hematological findings and complications of covid- clinical features of patients infected with novel coronavirus in wuhan, china the rheumatologist's role in covid- into the eye of the cytokine storm facing the sars-cov- (covid- ) outbreak with il- r antagonists risk factors of critical & mortal covid- cases: a systematic literature review and meta-analysis comorbidity and its impact on patients with covid- in china: a nationwide analysis plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars binding of sars coronavirus to its receptor damages islets and causes acute diabetes clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china covid- in critically ill patients in the seattle region -case series characteristics associated with hospitalisation for covid- in people with rheumatic disease: data from the covid- global rheumatology alliance physician-reported registry the authors would like to thank ana m perez for her help with data collection. they would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious way.contributors bf-g, ll, jaj, lr-r and la conceived and designed the study. ddfn, jfu, amg, jic and ll collected data. la and ll performed the data analysis and interpreted the data. all of the authors were involved in the drafting and/or revising of the manuscript. funding this work was supported by the instituto de salud carlos iii (isciii), ministry of health, spain (cp / ; pi / ; and rd / / ) and cofunded by el fondo europeo de desarrollo regional (feder). the funders had no role in study design, data collection, analysis, manuscript preparation or decision to publish.competing interests none declared. patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. ethics approval the study was approved by the hospital clínico san carlos institutional ethics committee (approval number / -e-bs). this study was conducted according to the principles of the declaration of helsinki.provenance and peer review not commissioned; externally peer reviewed. this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. leticia leon http:// orcid. org/ - - - luis rodriguez-rodriguez http:// orcid. org/ - - - key: cord- -c wp m authors: morens, david m.; fauci, anthony s. title: emerging infectious diseases: threats to human health and global stability date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: c wp m nan historical information as well as microbial sequencing and phylogenetic constructions make it clear that infectious diseases have been emerging and reemerging over millennia, and that such emergences are driven by numerous factors (table ) . notably, to percent of new human infections likely originated in animals, disproportionately rodents and bats, as shown by the examples of hantavirus pulmonary syndrome, lassa fever, and nipah virus encephalitis [ ] [ ] [ ] . most other emerging/reemerging diseases result from human-adapted infectious agents that genetically acquire heightened transmission and/or pathogenic characteristics. examples of such diseases include multidrug-resistant and extensively drugresistant (mdr and xdr) tuberculosis, toxin-producing staphylococcus aureus causing toxic shock syndrome, and pandemic influenza [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although precise figures are lacking, emerging infectious diseases comprise a substantial fraction of all consequential human infections. they have caused the deadliest pandemics in recorded human history, including the black death pandemic (bubonic/ pneumonic plague; - million deaths) in the fourteenth century, the influenza pandemic ( million deaths), and the hiv/aids pandemic ( million deaths so far) [ , ] . two major categories of emerging infections-newly emerging and reemerging infectious diseases-can be defined, respectively, as diseases that are recognized in the human host for the first time; and diseases that historically have infected humans, but continue to appear in new locations or in drug-resistant forms, or that reappear after apparent control or elimination [ ] . emerging/ reemerging infections may exhibit successive stages of emergence. these stages include adaptation to a new host [ ] , an epidemic/ pathogenic stage, an endemic stage, and a fully adapted stage in which the organism may become nonpathogenic and potentially even beneficial to the new host (e.g., the human gut microbiome) or stably integrated into the host genome (e.g., as endogenous retroviruses). although these successive stages characterize the evolution of certain microbial agents more than others, they nevertheless can provide a useful framework for understanding many of the dynamic relationships between microorganisms, human hosts, and the environment. it is also worth noting that the dynamic and complicated nature of many emerging infections often leaves distinctions between emerging and reemerging infections open to question, leading various experts to classify them differently. for example, we describe as ''reemerging'' new or more severe diseases associated with acquisition of new genes by an existing microbe, e.g., antibiotic resistance genes, even when mutations cause entirely new diseases with unique clinical epidemiologic features, e.g., brazilian purpuric fever [ ] . similarly, we refer to sars as an emerging disease a decade after it disappeared, and apply the same term to the related mers (middle east respiratory syndrome) b coronavirus which appeared in saudi arabia in late [ ] . the most salient modern example of an emerging infectious disease is hiv/aids, which likely emerged a century ago after multiple independent events in which the virus jumped from one primate host to another (chimpanzees to humans) and subsequently, as a result of a complex array of social and demographic factors, spread readily within the human population. aids was not recognized as a distinct entity until [ , ] , after its initial detection among certain risk groups, such as men who have sex with men, recipients of blood products, and injection drug users. it was soon apparent, however, that the disease was not restricted to these groups, and indeed, the bulk of hiv infections globally has resulted from heterosexual transmission that has been heavily weighted within the developing world, particularly sub-saharan africa where a number of factors were responsible for this rapid spread; chief among these were human movement along truck routes accompanied by a high level of commercial sex work, inadequate public health infrastructures, poverty, and social inequality. this is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. the work is made available under the creative commons cc public domain dedication. funding: both authors are employees of nih and as such our work is funded by nih from operating funds rather than grants or other awards. competing interests: the authors have declared that no competing interests exist. other examples of disease emergences [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] include sars, which emerged from bats and spread into humans first by personto-person transmission in confined spaces, then within hospitals, and finally by human movement between international air hubs. nipah virus also emerged from bats and caused an epizootic in herds of intensively bred pigs, which in turn served as the animal reservoir from which the virus was passed on to humans. the h n pandemic influenza virus emerged from pigs as well, but only after complex exchanges of human, swine, and avian influenza genes [ ] . h n influenza emerged from wild birds to cause epizootics that amplified virus transmission in domestic poultry, precipitating dead-end viral transmission to poultryexposed humans. additional examples are many [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; however, the variables associated with emergences are unique for each and typically complex. most of the important reemerging infectious disease agents first appeared long ago, but have survived and persisted by adapting to changing human populations and to environments that have been altered by humans. dengue virus and west nile virus (wnv), distantly related flaviviruses, serve as good examples. they have been spread by geographic movement of humans in association with the mosquito vectors for the diseases. for example, dengue came to the americas in association with the slave trade of earlier centuries. in this regard, slaves infected by mosquitoes in africa presumably brought the infection to the americas by seeding the mosquito population upon arrival [ ] . similarly, wnv came to the united states in when an infected human, bird, or mosquito came by air travel from the middle east to the western hemisphere, providing a source for introduction of infection to new world mosquitoes and birds. pathogenic strains of dengue have also spread back from southeast asia to the western hemisphere, as has a major mosquito vector, aedes albopictus. unlike most arboviruses, which are partly or completely host-restricted, wnv has become adapted to multiple mosquito and avian species, a major factor in increasing its opportunity to infect humans. the lack of additional hosts undoubtedly drove the mosquitoes that are the vectors of dengue and the dengue virus itself to favor adapting to humans and to their behaviors and environments. the association of dengue with aedes mosquitoes that live in and around human habitations mean that crowding, poor sanitation, and poverty provide ideal environments for transmission to humans [ ] . host immunity factors are also thought to be involved in the severe/fatal form of dengue known as dengue shock syndrome [ ] . other non-arboviral examples of emerging infections abound. for example, cholera has repeatedly reemerged over more than two centuries in association with global travel, changing seasons, war, natural disasters, and conditions that lead to inadequate sanitation, poverty, and social disruption. emergences of disease caused by community-and hospital-acquired clostridium difficile and methicillin-resistant staphylococcus aureus (mrsa) have been driven by increased and/or inappropriate use of antibiotics, and some hospital-acquired organisms such as mrsa have now moved into community transmission. the global emergence of plasmid-spread ndm- (new delhi b-lactamase) gram-negative pan-resistant organisms, linked to global antibiotic use and inadequate antibiotic stewardship, medical tourism, economic globalization, and other aspects of modern life, has prompted calls for development of international control mechanisms [ ] that are applicable to a number of emerging bacterial diseases in the developing and developed world. drug resistance mutations have also caused the reemergences of certain pathogens such as multidrug-resistant and extensively drug-resistant tuberculosis, drug-resistant malaria, and numerous bacterial diseases such as vancomycin-resistant enterococci. fungi have made significant contributions to disease emergence as well. in africa, cryptococcal disease has already surpassed tuberculosis as a leading cause of death [ ] . other examples of fungal emergence include comorbidities in hiv-infected individuals ( ) , cryptococcus gattii epidemics in predominantly healthy persons in the u.s. [ , ] , and a u.s. nationwide epidemic of exserohilum rostratum infections associated with contaminated pharmaceutical products [ ] . while it has become possible to eradicate certain infectious diseases (smallpox and the veterinary disease rinderpest), and to significantly control many others (dracunculiasis, measles, and polio, among others), it seems unlikely that we will eliminate most emerging infectious diseases in the foreseeable future. pathogenic microorganisms can undergo rapid genetic changes, leading to new phenotypic properties that take advantage of changing host and environmental opportunities. influenza viruses serve as a good example of emerging and reemerging infectious agents in their ability to rapidly evolve in response to changing host and environmental circumstances via multiple genetic mechanisms. new ''founder'' influenza viruses [ ] appear periodically, cause a pandemic, raise widespread population immunity, and then, in table . some major factors that underlie disease emergence and reemergence [ , ] . response to human immune pressures, evolve and persist for decades using multiple genetic evolutionary mechanisms to sustain continual immune escape. the influenza pandemic virus is one example: over the past years, its descendants have evolved continually by antigenic drift, intra-subtypic reassortment, and antigenic shift, the latter producing new pandemics in and [ ] . even the genetically complex pandemic h n influenza virus is a descendant of the virus [ ] . such continuous genetic hyper-evolution forces us to develop new influenza vaccines containing new antigens on an annual basis. in the meantime, new human diseases keep emerging. as noted, in late the novel mers coronavirus emerged in saudi arabia [ ] , and in early a new h n avian influenza virus became epizootic in eastern china, causing spillover infections of humans (as of june , ), with percent case fatality [ , ] . its pandemic potential, if any, remains to be determined. whether or not such outbreaks become more widespread, they nonetheless attract global attention and require significant international effort to monitor and contain. microbial advantages can be met and overcome only by aggressive vigilance, ongoing dedicated research, and rapid development and deployment of such countermeasures as surveillance tools, diagnostics, drugs, and vaccines. we appear to be entering a new era in which several important emerging, reemerging, and stable infectious diseases are becoming better controlled (e.g., hepatitis b, rabies, haemophilus influenzae type b, and even to some extent hiv/aids). however, our success in stopping the many new emerging diseases that will inevitably appear is not assured. we have many tools in our armamentarium, including preparedness plans and stockpiles of drugs and vaccines. but each new disease brings unique challenges, forcing us to continually adapt to ever-shifting threats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . the battle against emerging infectious diseases is a continual process; winning does not mean stamping out every last disease, but rather getting out ahead of the next one. the perpetual challenge of infectious diseases emerging infections: microbial threats to health in the united states factors and determinants of disease emergence ecology of zoonoses: natural and unnatural histories the challenge of emerging and reemerging infectious diseases emerging infections: a perpetual challenge prediction and prevention of the next pandemic zoonosis emerging infectious diseases in : years after the institute of medicine report the world must build on three decades of scientific advances to enable a new generation to live free of hiv/aids world health organization. global alert and response (gar) crossspecies virus transmission and the emergence of new epidemic diseases tracing phylogenomic events leading to diversity of haemophilus influenzae and the emergence of brazilian purpuric fever (bpf)-associated clones genome characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans the persistent legacy of the influenza virus dengue research opportunities in the americas the emergence of pan-resistant gram-negative pathogens merits a rapid global political response estimation of the current global burden of cryptococcal meningitis among persons living with hiv/aids genome variation of cryptococcus gattii, an emerging pathogen of immunocompetent hosts the triple threat of cryptococcosis: it's the body site, the strain, and/or the host multistate outbreak of fungal infection associated with injection of methylprednisolone acetate solution from a single compounding pharmacy -united states reconstruction of the influenza virus: unexpected rewards from the past preliminary report: epidemiology of the avian influenza a (h n ) outbreak in china drivers, dynamics, and control of emerging vector-borne zoonotic diseases key: cord- -hayhbs u authors: gonzalez, jean-paul; souris, marc; valdivia-granda, willy title: global spread of hemorrhagic fever viruses: predicting pandemics date: - - journal: hemorrhagic fever viruses doi: . / - - - - _ sha: doc_id: cord_uid: hayhbs u as successive epidemics have swept the world, the scientific community has quickly learned from them about the emergence and transmission of communicable diseases. epidemics usually occur when health systems are unprepared. during an unexpected epidemic, health authorities engage in damage control, fear drives action, and the desire to understand the threat is greatest. as humanity recovers, policy-makers seek scientific expertise to improve their “preparedness” to face future events. global spread of disease is exemplified by the spread of yellow fever from africa to the americas, by the spread of dengue fever through transcontinental migration of mosquitos, by the relentless influenza virus pandemics, and, most recently, by the unexpected emergence of ebola virus, spread by motorbike and long haul carriers. other pathogens that are remarkable for their epidemic expansions include the arenavirus hemorrhagic fevers and hantavirus diseases carried by rodents over great geographic distances and the arthropod-borne viruses (west nile, chikungunya and zika) enabled by ecology and vector adaptations. did we learn from the past epidemics? are we prepared for the worst? the ultimate goal is to develop a resilient global health infrastructure. besides acquiring treatments, vaccines, and other preventive medicine, bio-surveillance is critical to preventing disease emergence and to counteracting its spread. so far, only the western hemisphere has a large and established monitoring system; however, diseases continue to emerge sporadically, in particular in southeast asia and south america, illuminating the imperfections of our surveillance. epidemics destabilize fragile governments, ravage the most vulnerable populations, and threaten the global community. pandemic risk calculations employ new technologies like computerized maintenance of geographical and historical datasets, geographic information systems (gis), next generation sequencing, and metagenomics to trace the molecular changes in pathogens during their emergence, and mathematical models to assess risk. predictions help to pinpoint the hot spots of emergence, the populations at risk, and the pathogens under genetic evolution. preparedness anticipates the risks, the needs of the population, the capacities of infrastructure, the sources of emergency funding, and finally, the international partnerships needed to manage a disaster before it occurs. at present, the world is in an intermediate phase of trying to reduce health disparities despite exponential population growth, political conflicts, migration, global trade, urbanization, and major environmental changes due to global warming. for the sake of humanity, we must focus on developing the necessary capacities for health surveillance, epidemic preparedness, and pandemic response. infectious diseases have swept the world, taking the lives of millions of people, causing considerable upheaval, and transforming the future of entire populations. every year pathogens cause nearly million deaths worldwide, mostly in developing countries. more than infectious diseases have emerged between the s and [ ] . also among the known arboviruses, only are known to be human pathogens, while the others only infect wild animals and/or arthropods. to anticipate an epidemic one must identify the risk, prepare an appropriate response, and control the disease spread by first identifying the vulnerabilities of the population and circumscribing the potential space into which a disease will extend. when the epidemic expansion risk is identified, adequate information must be communicated to decision makers. ultimately, an appropriate response will depend on biosurveillance, prevention, sustained data processing, communication, strategic immunization campaigns, resilience, and mitigation strategies. the viral hemorrhagic fevers (vhfs) are a diverse group of human illnesses caused by rna viruses including approximately species of the arenaviridae, filoviridae, bunyavirales, flavi viridae, and rhabdoviridae (table ) . despite the efforts placed on early detection, viruses like dengue, ebola, lassa, crimean-congo hemorrhagic fevers continue to threaten the health of millions of people, mostly in areas where demographic changes, and political and socio-economic instability interrupt vaccination campaigns [ ] . however, the threat of vhf to global health is increased by intercontinental travel and global trade. moreover, because of the high case fatality rate of some of these pathogens, such concerns extend to the potential use of these viruses by bio-terrorists [ ] . global expansion of several diseases is exemplified by the spread of yellow fever from africa to the americas, the spread of dengue fever across continents, and recently, the spread of ebola virus from the democratic republic of the congo to western africa. the concept of an epidemic, as a disease affecting many persons at the same time and spreading from person to person in a locality where the disease was not previously prevalent, was not enunciated until when john snow produced his admirable demonstration of the emergence of an infectious disease in an urban area: the emergence of a cholera epidemic in london. at that time, none could clearly comprehend the mechanisms of emergence and spread since the existence of microbes had just been demonstrated by louis pasteur in the late s and microbe transmission modes were more speculative than based on medical or scientific facts, until when robert koch demonstrated that bacteria can be transmitted and responsible for diseases. nowadays, it is extremely difficult to make a retrospective diagnosis of historical pandemics, there are currently species in the orthohantavirus genus. the pathogeny of most of them is unknown during times when clinical descriptions were rare or lacking accuracy, and the extent of an epidemic was extremely subjective. thus, it is common to note that the first outbreak described in the western world was that of the plague of athens for which thucydides rather precisely reported the symptoms; today this epidemic has often been attributed to typhus through its clinical picture and epidemic profile [ ] . the first historically recorded outbreaks due to viral agents date to antiquity when the roman armies were returning from distant countries bringing with them "exotic" diseases. indeed, the rise of a "new" virus is an extremely rare event. most often, in terms of pathogen emergence, a virus adapts through mutation and selection pressure to a human host causing disease. presumably, smallpox, measles, and influenza were among the plagues that struck the ancient latins in gusts of epidemics more or less severe. the antonin plague that extended from to ad in much of western europe, when the troops of emperor lucius verus returned from war against the parthians, is often attributed to a smallpox pandemic by historians. in the middle ages, it seems that smallpox made a return around ad to france, germany, belgium, and the british islands [ ] . the acute respiratory infections reported during the winter of - ad accompanying the return of the carolingian armies from italy have been attributed by historians to a flu epidemic. many soldiers of charlemagne died then. the disease returned regularly and fiercely in and ad to the western european peninsula [ , ] (table ) . from the plague (sensu lato, including all transmissible diseases) of antiquity, to the severe acute respiratory syndrome that emerged on the eve of the third millennium, pandemics have followed in the history of mankind. as noted by mirko grmek, a historian of medicine, it seems that one pandemic will drive in another. if several diseases circulate concomitantly, one of them will take precedence over the other, an epidemic over the previous, and it is more likely that a pandemic will prevail [ ] . plague temporarily replaced the leprosy that appeared in eurasia for over , years; during the first millennium, plague was manifested by successive pandemics that crossed continents. during the first half of the past millennium, syphilis started its expansions, crossed oceans, and became global. tuberculosis originated in europe more than , years ago, but it was only at the turn of the seventeenth century that it was considered a pandemic; smallpox was also manifest as epidemics and then was pandemic at its peak in the late nineteenth century, then smallpox persisted until the jenner area. although early medical records of smallpox are available (egypt, china, india), large and devastating epidemics were only identified in the late fifteenth century of the millennium. smallpox was introduced into the americas by spanish settlers in the caribbean island of hispaniola in and arrived in mexico in . on hispaniola island, one third of a million of the inhabitants died of smallpox in the following years. smallpox devastated the native amerindian population and was an important factor in the conquest of the aztecs and the incas by the spaniards [ ] . in , children died in goa, india, from a smallpox epidemic. in europe, smallpox was a leading cause of death in the eighteenth century, killing an estimated , europeans each year [ ] . during the twentieth century, it is estimated that smallpox was responsible for - million deaths. the last known natural case of smallpox occurred in somalia in [ ] . it is only at the end of the first millennium that all these pathologies were better understood and their infectious origins elucidated. the first pandemic of the twentieth century was attributed to the h n spanish flu that emerged in kansas in . however, this "flu pandemic" is now thought to have had subepidemic circulation earlier in france or germany or even prior emergence in china in or [ ] , and to be exascerbated by concurrent bacterial infections. although it burned out quickly by , it has been estimated that one third of the world's population was afflicted; million people died, half of them in the first weeks of the outbreak. since the s, the frequency and magnitude of dengue fever epidemics increased dramatically as the viruses and the mosquito vectors have both expanded geographically in pandemic proportions [ ] largely extending the pandemic to all the intertropical zone. in the early s, human immunodeficiency viruses (hiv- and hiv- ) spread as an acquired immunodeficiency syndrome (aids), a pandemic that continues to take its terrible toll at the global level. since the emergence of aids, organization updates, as of june only million people were accessing antiretroviral treatment and among them, seven of ten pregnant women received treatment. in , a severe acute respiratory syndrome, sars, inaugurated the twenty-first century as a first pandemic of the millennium, involving more than countries with secondary epidemic chains in asia, europe, north america, south america, and a total of cases [ ] . ultimately, one of the major characteristics that defines today's pandemics, apart from the introduction of the disease within several continents or the rapid expansion across the administrative borders of countries, is the initiation of locally active transmission of the pathogen. although, the first ebola virus disease outbreak of western africa was considered a pandemic and witnessed several exported cases with secondary epidemic chains in distant countries of the african continent (i.e., nigeria, mali), outside of africa, exported cases rarely sparked local transmission. emergence from a sporadic case to an outbreak, to an epidemic, and ultimately to a pandemic depends upon effective transmission among nonimmune hosts, host availability (density), characteristics of the vector (natural or human made) that would enable it to circumvent distances, and the pathogen infectiousness. all these dynamics are essential for an effective disease transmission and spread. an outbreak is a sudden increase in occurrences of a disease in a particular time and place, more localized than an epidemic. an epidemic occurs as the disease spreads to a large number of people in a given population within a short period of time. to spark an epidemic chain of transmission depends on factors like immune population density, virus infectiousness, promiscuity, vulnerability, etc., while the efficiency of such transmission depends on how many persons will be infected by one person (i.e., the reproductive ratio or r ). an epidemic event will therefore expand in space (beyond the first cluster of cases) and time (rapid spread). a pandemic is essentially spatial, and represents an epidemic of infectious disease that has spread through human populations across a large region, extensively across two or more continents, to worldwide. however, all these typologies harbor the same fundamentals: emergence from one index case, transmission from one host to another, and spatial expansion. altogether, an epidemic and a pandemic are respectively a local and a global network of inter connected infectious disease outbreaks (i.e., epidemic chains). ultimately, understanding how disease (i.e., pathogens) spread in the social system is fundamental in order to prevent and control outbreaks, with broad implications for a functioning health system and its associated costs [ ] . also, after the last case occurs at the end of an epidemic, the goal is to control the risk of transmission for a -day time period. this three-week period represents an incubation when the infected subject does not transmit the virus and remains asymptomatic. the " days" is based on experimental methods use in virology to detect virus replication: influenza virus infected eggs should hatch in days, there is a -day limit for an arbovirus to infect a living model (suckling mice, mice, rats, guinea pigs, cell lines). moreover, most viral infectious diseases have a maximal incubation period of days, with few exceptions (e.g., hiv, and rabies). ultimately, such -day periods multiplied by the potential of a carrier to travel will produce the risk area for the emergence of secondary cases (from a walking distance to the long distances covered by commercial jets). however, it is important to clarify that many vhf including ebola virus can be carried by an asymptomatic host for several months [ , ] . the mode of transmission profiles the epidemic pattern of a transmissible disease. it is extremely helpful when a disease emerges to rapidly surmise the mode of transmission and how to respond (e.g., water-borne disease, arthropod-borne disease, human-tohuman transmission). pathogen transmission can be interspecific or hetero-specific, direct or indirect. direct transmission occurs by close contact with infected biological products (e.g., blood, urine, saliva). indirect transmission occurs with intermediate hosts such as arthropod vectors (e.g., mosquito, tick) or mammalian vector/reservoir (e.g., rodent, chiropteran) or from infected environmental means (e.g., soil, water, etc.). mobility and transportation are the main factors for diseases dispersion, as an emblematic example, one can simply show how the - evd outbreak of western africa expanded due to the transportation of patients during their -day incubation periods, first by foot-paths, then by motorbike, then taxis and public transportation, finally becoming a global concern with patients traveling by boat or commercial airline [ , ] . host population density and promiscuity, crowded places (like schools, markets, mass transportation system) also play an important role in the efficiency of transmission as well as the level of herd immunity (e.g., annual pandemic flu), altogether this gives us the level of population susceptibility (i.e., vulnerability). environmental factors can also be major drivers of pathogen expansion, for example the emergence of nipah encephalitis. the nipah virus, when it emerged for the first time in malaysia in , was transported by its natural host, a frugivorous chiropteran. a year earlier, an immense forest fire affecting several indonesian islands had forced the escape of disease-carrying bats that took refuge in malaysian orchards, planted to nurture newly developed pig farms. both pigs and farmers became infected and nipah virus was discovered for the first time. another classical example, more associated with human environment and behavior, is the old story of the spread of dengue virus via the used tires carrying infected aedes aegypti eggs and transporting dengue across oceans and continents [ ] . understanding the mechanisms of transmission and expansion of disease vectors with respect to the typology (epidemic pattern) of a disease is the ultimate challenge for controlling and preventing disease. typologies from human-to-human transmission, zoonotic diseases, arboviruses, water-borne diseases, and others play different roles in the rate of disease spread and need to be clearly understood. finally, while an epidemic pattern is driven intrinsically by the virus and its vector, the host population, the mode of transmission, and even the human environment (e.g., population density, urbanization, agricultural practices, health system, public health policies) as well as physical environment (season, meteorology, climate changing, latitude, altitude) factor into the rate of disease spread. with respect to pandemic risk (the rapidity and area covered by disease), the main characteristics of a virus are found in its environmental persistence while remaining infectious. environmental persistence depends on: virus structure, enveloped viruses are more sensitive than the naked viruses; its mode of entry into the body of the susceptible subject (transdermal, oral, respiratory); its ability to diffuse out of the body for a sufficient period of time which will, in turn, enable transmission to a greater number of subjects (r ). altogether these intrinsic factors link to the infectivity of the virus, indeed, viruses transmitted by aerosol possess certainly the most efficient way to spark an epidemic that increases with population density and vulnerability as well as with the resistance of the virus to environmental factors outside the host cells. the cycle of transmission shapes the epidemic in time and spatial dispersion. for example, animal to human zoonoses are dictated by chance encounters between host (population density, animal farming, pets, hunting) and, eventually transmission such as that observed between human and nonhuman primates [ ] . vectortransmitted diseases (i.e., arthropod-borne diseases) depend on the vector ecology (ability to transmit, length of the intrinsic cycle of the virus, trophic preferences, vector density, seasonality, reproduction, breeding sites, food abundance for hematophagous arthropods). mobility of hosts/vectors that are part of the natural cycle will also play a role in the potential for disease expansion (e.g., mosquito-flying distance, cattle transhumance, human migration). also, other factors associated with the hosts will render a more efficient transmission: human behaviors like fear/social responses, nosocomial infections, super-spreaders); viruses having multiple natural hosts (vicariates) or vectors; vectors with multiple trophic preferences (e.g., biting cattle, birds, and primates); the incubation period in the vertebrate hosts as well as the intrinsic replication in the arthropod vectors will also intervene; ultimately subclinical infection is also an underestimated factor of virus dispersion and transmission that modifies the epidemiological pattern of disease. one can distinguish also a typology of communicable diseases that reflects the spatial and temporal mode of transmission including arthropod-borne transmission, human-to-human transmission, human-to-animal (and vice versa) transmitted diseases (i.e., zoo- predicting hfv pandemics noses) including vector and nonvector transmitted diseases, and some other types of environmentally transmitted diseases. all of them represent unique types of transmission and risk of spread with a variable path of time, and also dependent on multiple factors (environment, climate, behavior, etc.). we have to consider territories as spaces where disease can potentially expand and that can be characterized by the fundamental factors of emergence and spread: the vulnerability of the population, the level of favorable transmission factors, and the probability for the population to be exposed to the virus. vhf are exemplary for their epidemic patterns of expansion dependent on the above reviewed factors (i.e., fundamentals of emergence) and their epidemiological characteristics (i.e., virus, host, environments). for example, let us consider the control of arenavirus spread by their strong host-species association. on a geological time scale, arenaviruses such as the agent for argentinian hemorrhagic fever (ahf) coevolved with their natural rodent host and then spread according to the expansion of the rodent host. one host-one virus ultimately produces a localized endemic cycle, the distribution of the disease overlaps the distribution of the rodent host while enzootic patterns appear naturally limited to an ecosystem (e.g., local rodent populations, behaviors, and environmental factors). hantaviruses also appear as a global complex, resulting from the coevolution of virus and rodent hosts and a global dispersion of generally localized enzootic diseases [ ] [ ] [ ] . as for the pandemic risk associated with a natural virus reservoir, chiropterans are unique flying and migratory mammals that have been associated with filoviruses and other viruses of major public health importance [ ] , their potential as vectors will eventually favor the spread of these viruses into new territories. also because there is potential for a long coevolution, epidemiological patterns are also dependent on virus-host spillover, host vicariate, and other environmental factors (e.g., climate change and man-made changes in land use). other arboviruses such as yellow fever virus, dengue virus, as well as west nile, chikungunya, or zika viruses show a pandemic risk associated with the existing distribution of their respective arthropod vector, vector density, and ability to transmit virus. investigating the fundamental factors of transmission and favorable territories for disease emergence are necessary to evaluate the risk, respond to the epidemic, and control its expansion from an index case to a pandemic. ultimately, when the fundamentals are understood and epidemic/pandemic risk identified, suitable emergency funding needs to be identified and made available in endemic areas to insure political willingness and community participation. ultimately, a suitable response will improve biosurveillance, data processing, communication, strategic immunization campaigns, and research for future risk prevention. several emblematic vhf and their original "epidemiological engineering" are presented in herein. vhf such as ebola virus disease, lassa fever, rift valley fever, or marburg virus disease are highly contagious and deadly diseases, with potential to become pandemics. remarkably, vhf are essentially caused by viruses of eight families; arenaviridae, filoviridae, hantaviridae, nairoviridae, peribunyaviridae, phenuiviridae, flaviviridae, and more recently rhabdoviridae [ ] (table ) . hemorrhagic fever viruses (hfv) have been classified as "select agents" because they are considered to pose a severe threat to both human and animal health due to high mortality rate, human-to-human transmission, and, in some cases, the potential to be aerosolized and used as bioweapons [ ] . each of these hfv shares some common features that define the nosology of the vhf group, from virus structures to the clinical and epidemiological characteristics of their diseases. -hfv spread person-to-person through direct contact with symptomatic or asymptomatic patients, body fluids, or cadavers. -vhf can have a zoonotic origin, as when humans have contact with infected livestock via slaughter or consumption of raw meat, unpasteurized milk, bushmeat, inhalation or contact with materials contaminated with excreta from rodents or bats. -hfv can be vector-borne, i.e., transmitted via rodents, mosquitos, and ticks. -vhf are zoonotic diseases. accidental transmission from the natural host to humans can eventually lead to human-to-human transmission, human infection, and sporadic outbreaks. -with a few noteworthy exceptions (i.e., ribavirin), there is no cure or established drug treatment for vhf, while limited vaccines could be available, including yf, ahf, and rvf (the latter is for animals only). -vhf have common features: they affect many organs, they damage blood vessels, and they affect the body's ability to regulate itself. clinical case definitions describe vhf with at least two of the following clinical signs: hemorrhagic or purpuric rash; epistaxis, hematemesis, hemoptysis, melena, among other hemorrhagic symptoms without known predisposing host factors for hemorrhagic manifestations. in fact, during an epidemic, all infected patients do not show these signs and a specific case definition needs to be defined in accordance with the suspected or proven viral etiology of the disease [ ]. also, vhf pathogenesis encompasses a variety of mechanisms including: ( ) alteration of hepatic synthesis of coagulation factors, cytokine storm, increased vascular permeability, complement activation, disseminated intravascular coagulation. moreover, severe pathogenic syndrome is often supported by an ineffective immunity, high viral loads, and severe plasma leakage and co-infection with other pathogens [ ] . the present chapter will mainly focus on the factors that can specifically and eventually contribute to a pandemic risk and how did we learn from historical spread of the vhf. the yellow fever disease pandemic is thought to have originated in africa, where the virus emerged in east or central africa and spread to western africa. in the seventeenth century, it spread to south america through the "triangular" slave trade, after which several major outbreaks occurred in the americas, africa, and europe [ , ] . the yellow fever vaccine is a fantastic gift from pioneering vac- cinology; it is efficient, affordable for developing countries, and protects for at least a decade or even life-long. however, yf remains a particular concern at the global level and the number of cases has unexpectedly increased this past decade. nowadays, yfv causes , infections and , deaths every year, with nearly % occurring in africa. nearly a billion people live in an endemic area [ ] . although yfv is common in tropical areas of south america and africa, it has never been isolated in asia [ ] . ultimately, the pandemic risk is there, from the uncontrolled epidemic as for example in the inland remote area of the brazilian mato grosso state, to the recent burst of epidemics in west and central africa including angola, drc, as well as imported cases in kenya and china [ , ] . indeed, the risk of a pandemic exists if any imported case goes to an area where the fundamentals of emergence are present (i.e., aedes aegypti and a nonimmune human population). for years it has been stressed that yf coverage needs to be exhaustive in the endemic area, and the who international health regulations (ihr) need to be strictly respected when peoples are crossing frontiers to or from an endemic area [ ] . even though the virus was known to actively circulate in asia, north america, and africa years ago, a global pandemic of dengue fever began in southeast asia in the s [ , ] . dengue virus (denv) expansion was followed by the emergence of a dhf pandemic that occurred in the late twentieth century (see above, the "tire-mosquito larvae connection"). by the end of the century, dhf emerged in the pacific and the americas, and extended to all asian continents [ ] . lately, in the s, epidemic dengue fever occurs in africa, with a predominant activity in east africa, while sylvatic denv circulation was described in western africa [ ] . the different dengue virus serotypes spread also independently to all continents. while it is remarkable that infection with one serotype does not provide cross-protective immunity against the others, epidemics caused by multiple serotypes became more frequent, and highly pathogenic denv were identified [ ] . dengue fever to date has a global distribution with an estimated . billion people at risk. yearly, hundreds of thousands of dhf cases occur [ ] . altogether, the requirements for a dhf pandemic are globally present [ ] : the highly competent aedes aegypti and aedes albopictus denv vectors, the globally distributed denv serotypes and highly pathogenic strains, and finally, climate change that opens new breeding opportunities for these mosquitoes to expand and eventually transmit imported denv into new populations and territories [ ] . mankind will have to live with this pandemic until the new denv vaccines can be implemented. in , an unknown disease was reported by a group of laboratory workers in west germany and former yugoslavia [ ] . over the course of months, cases and seven deaths occurred. conclusions made by treating physicians at the time (and published shortly thereafter) highlighted the following: high fatality rate, risk of relapse; risk of sexual transmission [ ] . a connection was made to infected african primates, chlorocebus aethiops, when laboratory workers were exposed to their imported tissues. it took years to effectively connect marburg virus, marv, to a bat, rousettus aegyptiacus, as a natural marv reservoir in central africa [ ] . marv is considered to be extremely dangerous for humans, is classified as a risk group pathogen, and also is listed as a select agent; however, the pandemic risk cannot be assessed because only four epidemics have occurred. although marv expansion appears to be limited to a few countries in africa, the recent emergence (estimated at a few decades ago) of a second human pathogenic marburgvirus known as ravn virus, and the widely distributed old world rousette fruit bats (rousettus spp.) serving as reservoir for both viruses [ ] , are two factors that favor pandemic risk. although more than years after its emergence from a remote area on the ebola river in the central african rain forest, ebola virus (ebov) remained hidden in a cryptic natural cycle. then a series of outbreaks occurred in the large congolese rain forest of central africa [ ] . the epidemic risk was always considered to be localized and circumscribed [ ] . then, suddenly without warning, in the late months of , ebov emerged for the first time in a remote area of western africa and sparked an outbreak more massive than ever witnessed before. more than , people were infected, ten countries recorded cases (transmitted or imported), the pandemic risk raised fear, and who declared it as an inter national health emergency that requires a coordinated global approach [ ] . besides the lack of preparedness of national and international public health systems, the other major factor that played an immense role for the dispersion of evd in western africa was the extreme mobility of village populations. they followed the kissidougou forest foot-paths to the towns in guinea using motorbikes, cars, and other public transportation, then later evd traveled by plane to the global level. the evd epidemic went from outbreak to pandemic risk. like marburg virus, another member of the filoviridae, ebola virus, shares bats as a potential virus reservoir, human and nonhuman primates are highly sensitive to the virus, and inter-epidemic periods play an important role since the epidemic silences tend to diminish the attention of health services and increase epidemic risk. in this way, the first western african evd epidemic is exemplary for showing the hidden risks contained in the natural cycle of a virus, and the sudden emergence followed by an unprecedented velocity of spreading. in the absence of biosurveillance, a pandemic risk remains. hemorrhagic fever with renal syndrome, hfrs, appears first as a global concern of one virus family, several human pathogenic viruses of the genus orthohantavirus, multiple clinical presentations, and different epidemiological patterns [ ] . hantaviruses and hfrs were first described in asia [ ] ; nowadays, hantaviruses are the cause of zoonoses that are expanding worldwide. indeed, since when a previously unknown hantavirus was implicated in the first hantavirus pulmonary syndrome (hps) outbreak in the united states, several other hantavirus infections were reported in western europe, and then hantaviruses were described in south america. ultimately, after an early suspicion of the presence of the hanta viruses in africa [ ] , a novel hantavirus, sangassou virus, was isolated in in guinea [ ] . altogether we observed the emergence of the hantaviridae in the western hemisphere, from the old world to the new world, and recently discovered its first tentative steps on the african continent. with respect to the orthohantavirus genus, a real pandemic exists even when multiple viruses are involved. ultimately, as for the arenaviridae, hosts are specific and certainly the major vectors of virus dispersion. the arenaviridae includes different viral species grouped as old or new world arenaviruses [ ] , each is maintained by rodents of individual species as natural reservoir host and as vector for the viruses that are human pathogens. the rodent hosts are chronically infected without obvious illness and they pass virus vertically to their offspring. de facto, the distribution of the virus covers that of its natural hosts but is isolated in an ecosystem generally limited by natural barriers, e.g., mountains, river. a phenomenon in which rodent lineages are naturally infected by a virus and remain in such a limited environment is called "nidality" [ ] . this is what it is observed for argentinian hf, venezuelan hf, bolivian hf, and lassa hf. regarding the pandemic risk of any of these hf, arenaviruses because of their strict association with their natural hosts, like the hantaviruses, have their expansion potential limited by their natural hosts even though the latter are widely spread and could certainly be infected. such risk lies in an unexpected encounter between infected and noninfected populations under the pressures of (as yet unknown) factors that favor their migration from enzootic to non-enzootic areas. in that matter, lymphocytic choriomeningitis virus, another member of the arenaviridae, has a worldwide distribution through its domesticated natural host, the ubiquitous house mouse, mus musculus. although crimean-congo hemorrhagic fever, cchf, is a widespread disease endemic to africa, the balkans, western asia, and asian countries south of the th parallel north, it is generally transmitted by ticks to livestock or humans and therefore geographically limited to regions where tick vectors feed on humans. although the competent ixodid vector is limited, as is the abundance of their natural hosts, climate change modifies the distribution and abundance of tick hosts (i.e., tick abundance) [ ] . additionally the cchfv pandemic risk is limited by low mobility, geographical repartition, and seasonal activity, although its main natural hosts are widely dispersed from africa, to asia and europe [ ] . ultimately, human-to-human transmission occurs from close contact with the blood, secretions, or other biological fluids of infected persons but these remain rare events with a r < . altogether, a cchf pandemic risk remains hypothetical but underlined by the risk of human-to-human transmission [ ] . as for cchf, rift valley fever, rvf, is first a disease of cattle and illustrates a unique subcontinental zoonotic spread along the path of traditional herders. rvf became a transcontinental risk with trade and transportation when the virus spread from north east africa to western africa, and even to madagascar [ ] . if one considers its pandemic risk, with respect to rvf epidemiology as a mosquito-transmitted disease, two factors have to play concomitantly: the presence of infected cattle (i.e., nonimmune) and competent mosquito abundance, both considered hazards, while concretizing the risks from human vulnerability (nonimmune; mosquito bite; direct exposure to infected blood). in order to streamline the prevention and the actions to reduce epidemic risk, the various elements involved in an outbreak are here considered from a systemic point of view, considering the risk as the convergence of a hazard and vulnerability: -the presence of the threat (or "hazard" pathogen, i.e., vector, virus reservoir) is considered to be a necessary-but not sufficient-condition for the development of a disease. it is often known only in terms of probabilities, sometimes very low and therefore often subject to significant random variability in time and space. we often seek to evaluate the spatial and temporal differences of this probability, trying to measure its significance. sometimes, it only uses one character necessary to the presence of the pathogen or vector (e.g., the presence of water, a minimum temperature, a type of vegetation). -the susceptibility of the host (which is essentially linked to individual characters, genetic, biological, such as immune status or age) is individual, and often given by a probability. -direct exposure of the host to the hazard is an element of active vulnerability, depending on the behavior of the host that increases the likelihood of contact between host and hazard by exposing it to an environment conducive to his presence (e.g., travel and contacts, professional activities). it also includes all the known "risk" behaviors that increase the likelihood of direct exposure to the hazard. -passive vulnerability of the host, which is not directly dependent on the pathology, is not even necessary nor sufficient for pathology, but influences the exposure of the host to the hazard or to protection from the pathology. this protection consists of prophylaxis, access to care, availability of care. it is independent of the real presence of the hazard; the host can be vulnerable without being exposed to the threat. the vulnerability is often defined by several levels (individual, context). it is very often "spatial" as linked to phenomena of segregation or spatial concentration. this is an area primarily studied by geography. ultimately, this vision can differentiate what is active, often subject to high variability, random in time and space (the emergence or the presence of hazards is often difficult if not impossible to control) from what is passive, generally situated among more stable population levels (sensitivities, exhibitions, behaviors, and vulnerabilities). this allows for better public health preventive actions, and also to understand rationally crisis situations by preemptively targeting the most important elements of the system in terms of vulnerability, and secondly by optimizing risk reduction (elimination of vectors, vaccinations, quarantine, etc.). in all cases, these actions must be adapted to social contexts to have a real impact on risk behaviors and vulnerabilities that they generate, hence the increasing role of anthropology in the field of health. to prevent or reduce the epidemic risk, it is necessary to act on each component of this system: -reducing the susceptibility of the host (e.g., immunization, vaccination, prophylaxis). -reducing host exposure to the pathogen (e.g., vector control, quarantine, exclusion zone). -eliminating the pathogen directly (e.g., animal slaughter, disinfection, hygiene), or indirectly (e.g., suppress transmission). -reducing host vulnerability (e.g., socio-economic, behavioral, access to health care system). -reducing host exposure to emergency condition (e.g., realtime data collection, warning systems for emergency, crisis management, implementation of treatment). the rapid detection of emergence is the key to controlling the spread of an epidemic. it requires comprehensive monitoring to trigger alerts and all other risk-reducing actions, in particular, reducing the exposure of the host to the pathogen and, if possible, the elimination of the pathogen. in parallel to the monitoring and warning systems, protocols must always take into account local characteristics of political power and decision-making bodies that could otherwise render ineffective year-long action plans or warning systems (for example, the management of the chikungunya epidemic in reunion island was largely impacted by bottlenecks related to local political system) [ ] . biosurveillance and efficiency in data collection and management will be the technical keys for prevention (early detection of epidemic risk) and forecasting epidemic emergence and spread (i.e., analyzing the data in near real time taking into account the vulnerability of a given population). also, this can be achieved only by exhaustive capacity building (human and technical) mostly in the more vulnerable developing countries but also where the most advanced technology needs to be developed. networking biosurveillance systems are a major undertaking from regional to global, involving politics and diplomacy. taking in account the local characteristics of political structures and decision systems is fundamental. despite our current recognition of the risks posed by emerging and re-emerging infectious diseases to global public health and stability, reliable structured data remains a major gap in our ability to measure (and therefore manage) globally infectious diseases. who has long served as an information hub for infectious disease events worldwide; however, extracting quantitative data from who information bulletins (weekly epidemiological record and the more recent disease outbreak news alerts) proves to be a time-consuming effort with limited results in terms of operability, and exists more for the record and future analysis. the current proliferation of geospatial information tools (i.e., geographical information system, gis) and stepwise advances in data extraction capabilities have made it possible to develop robust, systematic databases facilitating anomaly detection (like clusters), infectious disease models (and model evaluation), and apples-to-apples comparisons of historic infectious disease events worldwide. however, biosurveillance capabilities-the key to global prevention and health securityremain inadequate to support true early detection and response. increased access to technology, rapidly developing communications infrastructures, smartphone usage for suspected-case reporting, and global networks of (formal and informal) disease surveillance practitioners provide an explosive opportunity to patch and improve surveillance networks. the challenge is to leverage all these developments, implement technical and capacity building where needed, before the next epidemic with global impact emerges. several organizations have developed systems to collect epidemic information and facilitate rapid response: who has the department of pandemic and epidemic diseases (ped) that develops mechanisms to address epidemic diseases, thereby reducing their impact on affected populations and limiting their international spread. among them some have self-explanatory titles: the battle against respiratory viruses (brave); early warning and response systems for epidemics in emergency (eware); emerging and dangerous pathogens laboratory network (edpln); international coordinating group for access to vaccines for epidemics (icg); global infection prevention and control network; (gipcn ); global influenza surveillance and response system (gisrs); global leptospirosis environmental action network (glean); meningitis environmental risk information technologies (merit); weekly epidemiological record (wer); emerging diseases clinical assessment and response network (edcarn). global commitment to these efforts will insure their readiness in times of need. most certainly and most importantly, any preparedness and response requires emergency funding [ ] . it has been estimated that if the ebola virus disease response started months earlier, it could have reduced the total number of deaths by % in liberia and sierra leone [ ] . we learned from this last evd epidemic that in march , the african union's minister of finance requested the african risk capacity (arc) agency to help member states to better plan, prepare, and respond to devastating outbreaks by developing new applications for financial tools, like insurance, that can significantly improve the speed of funds to affected countries and shorten the time between event and response. the agency is now developing an outbreak and epidemic insurance product primarily based on responsibly and timely budget reallocation; however, viruses do not wait. moreover, the world bank's pandemic emergency facility is designed to finance surge capacity and support international government partners to actively participate to the response. ultimately, epidemics are not one-off events, but rather demonstrate financial patterns similar to other natural catastrophes. as natural catastrophes, large epidemics can be insured by creating financial mechanisms to facilitate the movement of critical resources within affected countries and ultimately manage the spread of disease and minimizing macroeconomic impact [ ] . classical tools and strategies for predicting epidemics encompass human disease surveillance (e.g., public health and hospital statistics) and, sometimes, environmental surveys (e.g., climate, el niño, earthquake, tsunami); also more recently complying with one health concept, human and veterinary health as well environmental risk factors have been reunited in a comprehensive approach of public health risk (i.e., outbreak, epidemic risks). however, this heuristic approach of health remains limited to specific diseases and territories and does not apply as a global predictor of pandemics. first, historical data is the only available objective view of past epidemics and pandemics, needs to be collected, formatted, corrected, and analyzed. this will be the foundation of the different tools and strategies described below. in that matter, with respect to the depth of the past data available, time series of disease observation, modern tools such as internet search data have actually led to the development of several specific sites (e.g., google flu and dengue) [ ], whose search-term reports have correlated strongly with incidence estimates in several public health reports in europe, asia, and the u.s. however, even though such tools can complement classical disease surveillance, most of these sites are geographically limited and cannot be used for live monitoring of epidemic risk and for neglected tropical disease surveillance [ , ] . however, from such historical and live-collected data, health alert systems can be implemented, and prediction models can be developed. moreover, thanks to the spatial analyses, combining multiple data sources will provide the ultimate tools for livemapping an outbreak, which will lead to an efficient response when tools and strategy have been specifically identified (i.e., sufficient and available in-country heath system resources and funding; identifying variations in pathogen sequences that contribute to ro and pathogenicity; monitoring population movement; etc.). the amount of data being digitally collected and stored is exponentially accumulating. it is estimated that, as of september of , the world wide web reached . billion pages containing eight zettabytes of accessible data, and the accumulation of information is growing around % every year [ ] . this situation has generated much discussion about how to use the unprecedented availability of information and computational resources and the sophistication of new analytic and visualization algorithms for decision-making to reduce the impact of infectious diseases. in fact, it is argued that the paradigm of "big data" will change not only the way business and research is done, but significantly improve the understanding of factors leading to the emergence of infectious diseases. big data could lead to the implementation of a decentralized biosurveillance enterprise allowing organizations and individuals to take full advantage of a large collection of disparate, unstructured qualitative, and quantitative datasets. with the proper integration and the right analytics, big data could find unusual data trends leading to better pathogen detection systems, as well as therapeutic and prophylactic countermeasures. however, the impact of these analyses and forecasts depends not only on how the data is collected, ingested, disambiguated and processed, but also on how it is relayed in different operational contexts to users with different backgrounds and understandings of technology. while impressive in data mining capabilities, real-time content analysis of social media data misses much of the factual complexity. quality issues within freeform user-provided hashtags and biased referencing can significantly undermine our confidence in the information obtained to make critical decisions about the natural versus intentional emergence of a pathogen. risk factors associated with a health event in a population are often linked to environmental factors (fig. ) . they are also linked to spatial relationships between individuals, especially for infectious diseases. the geographical distribution of these phenomena reflects spatial relationships. beyond "classic" epidemiology mainly based on statistical analysis, using the location and spatial distribution is essential in the understanding of health events and analysis of their mechanisms. spatial analysis in epidemiology is a method to help determine the location (georeferenced) of risk factors. it allows one to identify the spatial and temporal differentiation in the distribution of events, using their location in time and space. when the location is available, with precision for each studied object (i.e., individuals or geographical units), it is possible to: -characterize the overall spatial distribution, using synthetic indices on the absolute position of an object, on the average spatial arrangement of objects or their values (grouping/ fig. mapping environmental factors that have a major impact on insect vector population (i.e., mosquitoes and ticks). this map of laos constitutes the basis of a risk map showing part of the hazards contributing to virus vector density that could be matched with human density and pathogen prevalence leading to a risk map (spatial risk) and eventually extended through seasonality (temporal risk). mean temperature and mean rainfalls are interpolated as climatic conditions, as environmental factors influencing the presence of mosquitoes dispersion, spatial dependence, variogram measure of autocorrelation space). -look for characteristics of the overall shape of the phenomenon (tendency, shape), and search for a theoretical spatial distribution, or for a process to model the observed spatial distribution. -look for unusual places (geographical centers and source sites; aggregates; exclusions; hot spots, cold spots), and to study the spatial relationships at the individual level. -conduct spatiotemporal analysis: search index cases, reconstruction of paths, diffusion models, models of extinction, etc. -spatial analysis allows the development of applications for modeling epidemics, preparing warning systems, as well as crisis management systems, risk prevention and analysis systems, and vaccination campaigns. many tools for biomonitoring and prevention of epidemic risk have been developed (fig. ) , as well as software tools to: (a) visualize spatial distributions. (b) synthesize and analyze position and spatial relationships between events (continuity, consolidation, attractionrepulsion, shape, centrality, displacement, diffusion processes). (c) to analyze the relationship between spatial distribution of attributed values and environmental characteristics of the phenomenon (environmental correlations). (d) to model the phenomena of emergence, dissemination, extinguishment of an epidemic. cluster detection, space-time analysis, and spatial integration with environmental and demographic data are widely used in such warning systems. multiple and complex factors are associated with the emergence and impact of pathogens in a given geographical area. therefore, public health analysts are confronted with the task to identify the likely, and unlikely, consequences and alternative critical outcomes of a given vhf outbreak. this requires the ability to monitor in near real time the dynamics of the geographical dissemination of these viruses in villages, cities, countries, continents, or the globe using new analytical techniques within the emerging field of genomicbased biosurveillance. this concept integrates microbial genotyping, next generation sequencing, metagenomics, big data and database analytics, and contextualized visualization to identify, characterize, and attribute known and unknown pathogens and generate estimates of how different contingencies will affect their impact [ ] . a genomic-based biosurveillance system includes powerful microbial genomic characterization to rapidly identify a pathogen [ ] . this characteristic makes a genomic-based biosurveillance a useful approach not only for public health but serves as a deterrence tool for intentional biological weapon development and deployment. the initial step consists of integration of signals generated by molecular-based assays and next generation dna sequencing and unbiased microbial characterization for pathogen source tracing, attribution and forensics. while each of these techniques has been discussed in the literature in detail [ ] , the integration of this information can yield a more extended view of the scale of a pathogen outbreak. the development of high-throughput the exemplary case of the highly pathogenic avian influenza virus h n in thailand. from the emergence of one imported case (red-filled circle), the pathway direction (arrowed green lines) of h n infection in farms (yellow points) is reconstituted, using dates of infection and distance between farms. results show local spread with time-to-time medium distance jumps dna sequencing technologies (i.e., dna and cdna forms of rna viral genomes) is allowing the genomic characterization of previously unknown pathogens without relying on prior reference molecular information [ , ] . this information is available within days, and even hours, of sample collection, and well before the development of animal infection models. because of their portability, this technology will become widely used in the next years in routine clinical settings. however, to be clinically and epidemiologically relevant, dna sequences must be rapidly and effectively translated into actionable information defining pathogen characteristics (i.e., virulence or drug resistance), it must point to a source of origin, and discriminate a natural event from a manmade release [ ] . while some government agencies are considering use of genomic information to develop next generation level- and level- detection/surveillance devices [ , ] , there is no reference database where researchers can retrieve standardized genomic signatures and motif fingerprints to develop primer-, probe-, and antibody-based detection technology using reference moieties. the impact of genomic-based biosurveillance in public health and biodefense will not be fully realized until addressing the current impracticality of transferring the terabytes of genomic data generated by dna sequencing devices to a centralized architecture performing analysis operations, as that might take hours or even days. therefore, a new paradigm could emerge from encouraging the development of decentralized algorithms that first determine in situ the presence of pathogen-specific genomic signatures or motif fingerprints, summarize and relay the results into an operational biosurveillance metadata format for contextualized decision support. the localized data management, time, and space required for spatial analysis is performed by geographic information systems (gis). these are computer systems that manage large volumes of data and easily use the location to perform spatial analysis. most gis are not limited to data management functions, but also integrate multiple analysis tools, data transformation, and cartographic representation. these are for the most part complex applications with enormous features. the "gis" designation covers a wide variety of software projects built according to different technical options, functionality, and diverse performances. a gis is essentially a management tool (structure, organization, entry, storage), an analytical tool (statistical and geographical treatment, spatial analysis), and a communication tool (data visualization, descriptive mapping, thematic mapping, atlas). it is also a tool that allows the use of a spatial model for the simulation of a process, such as the development of an epidemic. gis facilitates the interface between modeling and simulation program, and the geographic database, and can ultimately take over the whole of access to spatial information needed by the modeling program. the gis should thus be at the heart of organizing the collection and processing of monitoring data. to ensure the management of this system, it is important to set up a body specifying all the collection, validation, processing and dissemination of information and results (alerts, risk modeling, near real-time dissemination of results). this body must be proposed and validated by political authorities, preemptively, to avoid further blockage and to ensure effectiveness in situations of epidemic crisis. mathematical modeling is a mathematical formulation of a parameter or risk; it depends on identified or hypothesized risk factors whose coefficients are determined by a statistical or heuristic analysis from historical or observed data with the use of r , as a basic reproduction rate, to timely and spatially predict the spread-speed of an emerging outbreak. spatial-temporal modeling of health events can be seen as the final stage of the analysis. it is different from statistical modeling. despite using risk factors, it considers the epidemic phenomenon as a whole, taking into account the spatial relationships between agents (hosts, vectors, reservoirs, and pathogens), between individuals, and relationships between individuals and their environment. this model is thus useful for understanding and anticipating the epidemics, and can be generally used to classify individuals in different states (susceptible, infected, sick healed, immune) and to model the major phenomena that can change the state of an individual. however, when a model takes into account many phenomena, it can quickly become very complex. the vast majority of models are simplifications of assumed reality. two broad categories of methods are usually developed in modeling: -a deterministic approach, based on differential equations whose coefficients are adjusted from observed data, or monitoring data from epidemics. in this model, one can introduce stochastic types of components in the coefficients, studying the variability of observed data. taking no account of spatial relationships is difficult in these models, which deal in general populations, not individuals. -a nondeterministic approach, which is based on agents whose behavior is described by expertly determined rules (multiagent models). the status of each agent is calculated at each time step, from its behavior, environment, and relations between the agent and all other agents. these models take into account a more realistic description of the phenomenon, near the complex system finely describing reality. they allow us to consider spatial relationships in each time step. these models require intensive calculation, and their use is made possible by development of the power of computer calculations. let us first honestly address the fundamental questions about epdimeics and preparedness: what did we learn from all the past epidemics, what will we remember in times of need? are we prepared for the worst of these hypothetic pandemics abundantly illustrated in the cinema and unfortunately sometimes overwhelmed when reality goes beyond fiction? certainly, we are not "globally" prepared, unfortunately, at that scale, the immense natural and human disparities do not permit it, but we do our best in our own societies. the concept of disease emergence, born only at the end of the twentieth century, is a societal marker, our desire to be on alert, understand and predict epidemics. ultimately, there are a few, but necessary and difficult goals to reach for the prevention and control of any epidemic, also these goals are part of the development of our societies, as well as for education, they become part of the wellbeing for all: first, beyond understanding transmission, is needed a clear understanding of the epidemiological pattern and the spread of a given disease, before it is too late; then, which is certainly one of the more complex and costly things to achieve, is having an efficient health system to respond to an epidemic and an operational network to respond at the regional and global levels; and last but certainly not a least, having identified funding for any public health emergency will be crucial to changing our world. perhaps, in a shrinking global community, after too many ebola virus disease outbreaks, we will learn and be prepared for future epidemic challenges? the progress made, mostly by computer sciences in the overall analysis of health data, should serve as a tool in the prevention of major epidemics. let us ultimately use our predictions of pandemic risk to meet and unite beyond the current frontiers of political and social wills. epidemic predictions in an imperfect world: modelling 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in sierra leone: stalking the virus in the threatening wake of emergence perspectives on west africa ebola virus disease outbreak aedes alpobictus and the world trade in used tires, - : the shape of things to come? men, primates, and germs: an ongoing affair seewis virus: phylogeography of a shrew-borne hantavirus in siberia coevolution of rodent and viruses: arenaviruses and hantaviruses the arenavirus and rodent coevolution process: a global view of a theory bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae a novel rhabdovirus associated with acute hemorrhagic fever in central africa federal select agent program: select agent and toxins list pathogenesis of the viral hemorrhagic fevers viruses, plagues, and history: past, present and future yellow fever fact sheet n° . world health organization yellow fever in africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data the changing epidemiology of yellow fever and dengue resurgence of yellow fever in angola international health regulations in practice: focus on yellow fever and poliomyelitis yellow fever in africa: a disaster waiting to happen dengue/dengue hemorrhagic fever: the emergence of a global health problem delaporte ( ) rapp. au ministre de la marine the global epidemiology of infectious diseases, global burden of disease and injury series epidemiology of arthropod-borne viral disease the xxth century dengue pandemic: need for surveillance and research emergence of epidemic dengue/dengue hemorrhagic fever as a public health problem in the americas zur Ätiologie einer unbekannten, von affen ausgegangenen menschlichen infektionskrankheit isolation of genetically diverse marburg viruses from egyptian fruit bats virological and serological findings in rousettus aegyptia cus experimentally inoculated with vero cellsadapted hogan strain of marburg virus ebola virus circulation in. africa: a balance between clinical expression and epidemiological silence ebola and marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for africa the ebola epidemic: a global health emergency hantavirus infections : endemic or unrecognized pandemic hantaviruses: history and overview serological evidence for hantaan-related virus in africa sangassou virus, the first hantavirus isolate from africa, displays genetic and functional properties distinct from those of other murinae-associated hantaviruses past, present, and future of arenavirus taxonomy natural nidality in bolivian hemorrhagic fever and the systematics of the reservoir species driving forces for changes in geographical distribution of ixodes ricinus ticks in europe seroepidemiological studies of crimean-congo hemorrhagic fever virus in domestic and wild animals crimean-congo hemorrhagic fever virus: new outbreaks, new discoveries a systematic review of rift valley fever epidemiology - the chikungunya epidemic on la réunion island in - : a cost-of-illness study global health risk framework: pandemic financing: workshop summary african risk capacity. executive perspective: outbreak and epidemic insurance, new solution to an old problem. the rockefeller foundation from the ebola river to the ebola virus disease pandemic: what have we learned? using web search query data to monitor dengue epidemics: a new model for neglected tropical disease surveillance evaluation of internetbased dengue query data: google dengue trends biosurveillance enterprise for operational awareness, a genomic-based approach for tracking pathogen virulence bioinformatics for biodefense: challenges and opportunities biosurveillance of emerging biothreats using scalable genotype clustering optimizing biosurveillance systems that use threshold-based event detection methods biodefense oriented genomic-based pathogen classification systems: challenges and opportunities biosurveillance observations on biowatch generation- and other federal efforts: testimony before the subcommittees on emergency preparedness, response, and communications and cybersecurity, infrastructure protection, and security technologies, committee on house homeland security, house of representatives congress. house ( ) committee on homeland security. subcommittee on emergency preparedness response and communications., united states. congress. house. committee on homeland security. subcommittee on cybersecurity infrastructure protection and security technologies., united states. government accountability office: biosurveillance observations on biowatch generation- and other federal efforts: testimony before the subcommittees on emergency preparedness, response, and communications and cybersecurity, infra structure protection, and security technologies, committee on house homeland security, house of representatives acknowledgments w.a. valdivia-granda has been funded by the department of homeland security and the department of defense. we are greatfull to sarah cheeseman barthel, director, data acquisition & management metabiota, inc., for her review and input of the section on "global surveillance and data collection." key: cord- - a i h authors: bittle, james l.; muir, susie title: vaccines produced by conventional means to control major infectious diseases of man and animals date: - - journal: advances in veterinary science and comparative medicine doi: . /b - - - - . - sha: doc_id: cord_uid: a i h publisher summary this chapter reviews the development of some of vaccines and their use in controlling such major diseases as diphtheria, rinderpest, newcastle disease, smallpox, pertussis, yellow fever, rabies, etc. park–williams number (pw ) strain is used to make diphtherial toxoid for vaccines. as a source of toxin, it is rendered nontoxic by incubation with formalin under alkaline conditions. the product's retention of antigenicity, enabling it to induce antitoxin antibodies, makes it an excellent pediatric vaccine. vaccine against rinderpest virus was developed by koch in by administering bile from infected cattle. animals that survived were permanently immune. formalin- and chloroform-inactivated vaccines were developed using tissues from the infected animals. for the control of newcastle disease, a number of attenuated live-virus vaccines have been developed which are widely used to control the disease. the bl strain, the lasota strain, and the f strain are used to immunize birds of all ages by different routes, including by addition to drinking water and by spraying. protection against rabies correlates with sn antibody, which can be assessed by a number of tests. pasteur's classical vaccine, developed from infected spinal cord tissue dried at room temperature for – days, was given in a series of – inoculations beginning with material dried the longest and progressing through material dried for only days. the first parvovirus shown to be a filterable agent was feline panleukopenia virus (verge and christoforoni, ) , which was not characterized until much later (johnson, a,b; johnson et al., ) . the virus affects most members of the family felidae, causing enteritis and bone marrow hypoplasia that results in severe leukopenia. the virus infects kittens in utero and postnatally causes cerebellar hypoplasia and ataxia (kilham and margolis, ) . immunization. the first panleukopenia vaccines were produced by infecting susceptible cats and harvesting their tissues. filtrates from these tissue suspensions were treated with chemical inactivants such as formalin. two inoculations of this type of vaccine produced longlasting protection (leasure et al., ; enders and hammond, ) . however, subsequent vaccines, produced in primary feline kidney cell culture and inactivated with formalin, proved safer and more effective (bittle et al., ; davis et al., ). an attenuated live-virus vaccine was also very effective in inducing immunity and protection (slater and kucera, ) . concern for the wide-scale use of an attenuated live-virus vaccine centers on the premise that shedding may spread the virus to other species, possibly allowing the emergence of pathogenic variants in these species. attempts to immunize cats orally with attenuated live-virus vaccines have not been successful, but intranasal aerosol application has been effective (scott and glauberg, ; schultz et al., ) . three members of the parvovirus family are known to infect dogs: minute virus of canines, a defective canine adeno-associated virus, and the pathogenic canine parvovirus. canine parvovirus is related to feline panleukopenia virus and may have originated from one of the mammalian parvoviruses. in young dogs, the virus causes leukopenia and severe intestinal disease with necrosis of crypt epithelium in the small intestine. myocarditis occasionally develops, causing sudden heart failure in young dogs. in an epizootic of this disease occurred in the united states, causing high mortality (eugester, ) . immunization. because of the close relationship between feline panleukopenia virus and canine parvovirus, inactivated feline panleukopenia vaccine initially was used to protect susceptible dog populations (appel et al., a) . later, inactivated and attenuated vaccines produced with the canine virus proved to be much more effective (pollack and carmichael, ; appel et al., b) . these parvovirus vaccines have been formulated with other vaccines, including canine distemper and canine adenovirus vaccines, and are administered routinely to young dogs. porcine parvovirus has a distant serologic relationship to other parvoviruses, but it causes disease only in swine. the virus, which is widespread, causes abortions, fetal death, and infertility in sows infected early in gestation. immune sows reinfected during gestation give birth to normal piglets. immunization. numerous reports have shown the effectiveness of inactivated porcine parvovirus vaccines in swine (suzuki and fujsaki, ; mengeling et al., ; wratthal et al, ; fujisaki et al., ; joo and johnson, ) . for example, mengeling et al. ( ) demonstrated the value of a vaccine in which the virus was inactivated with acetylethyleneimine. an attenuated live-virus vaccine has been described (paul and mengeling, ) in which a porcine isolate was attenuated by - passages in a swine testicular cell line. this vaccine effectively induced antibodies and protected challenged animals. the vaccine virus did not cross the placenta; however, it did kill fetuses when inoculated in utero. the virus was shed in feces, and so could be transmitted to unvaccinated animals. the inactivated vaccines now used in united states have been effective in controlling porcine parvovirus infection. vaccination is recommended for gilts and sows before breeding. adenoviruses cause significant disease in dogs, foxes, and man, but have also been isolated from cattle, swine, goats, sheep, horses, turkeys, and chickens, where they produce mild infections, mainly associated with the respiratory and intestinal tracts. there are at least different adenoviruses, of which occur in man. each adenovirus has a narrow host range. there are two canine adenoviruses. canine adenoviruses (cav- ) causes infectious canine hepatitis, which at one time was widespread, but now has been controlled by vaccination. infected dogs also develop corneal opacities following this infection, as a result of the formation of immune complexes and uveitis within the anterior chamber of the eye (carmichael et al., ) . in foxes, cav- produces a rapidly fatal encephalitis. canine adenovirus (cav- ) causes respiratory disease in dogs, but neither hepatitis nor encephalitis in dogs or foxes. the respiratory disease varies depending on the strain of virus and bacterial superinfection. cav- may be transmitted by aerosol, whereas cav- is spread by other direct means such as contact with urine or saliva from infected animals. cav- and cav- are oncogenic in experimentally infected hamsters (sarma et al., ; dulcac et al., ) . immunization. dogs that recover from natural cav- infection are immune for a long period. the first cav- vaccines were produced by formalin inactivation of tissue homogenates from infected dogs. cav- was first adapted to tissue culture by cabasso et al. ( ) and by fieldsteel and emery ( ) . the latter modified the virus by serial passage in porcine and canine tissue cultures; the resulting vaccine immunized dogs and did not produce clinical signs of infection except for occasional corneal opacity similar to that caused by natural infection. dogs immunized with cav- vaccines are also protected against cav- (appel et al., ) . the immunity produced by the attenuated live-virus cav- vaccines is long lasting and has drastically reduced the incidence of the canine disease. although cav- is closely related to cav- , when it is inoculated parenterally into dogs, it does not cause disease, although the virus is shed from the respiratory tract (appel et al., ) . such dogs become immune to both cav- and cav- . an attenuated live-virus vaccine containing cav- is now being widely used in place of older cav- vaccines (bass et al., ) ; this has resulted in a much lower incidence of corneal opacity in recipients. however, because of the oncogenicity of adenoviruses in other hosts, the respiratory shedding of cav- virus should be a concern. in man, adenoviruses mainly produce disease of the respiratory tract which varies in severity depending on the virus and the age of infected individuals. the viruses cause acute pharyngitis in infants and children, pharyngitis and conjunctivitis in children, and acute respiratory disease (ard) in military recruits and institutionalized young adults. pneumonia may also occur, expecially following ard. a vaccine consisting of adenovirus types , , and , grown in monkey kidney cell culture and inactivated with formalin, was introduced in for use in u.s. military recruits. the vaccine was effective in reducing ard in this population (sherwood et al., ) . however, the vaccine was withdrawn from use in because of concern for possible oncogenicity of the adenoviruses, and of the sv virus present in the monkey kidney cell culture. a subsequent adenovirus, type , was passed in human tissue and was, therefore, devoid of sv genomic material. this virus, encapsulated in enteric-coated capsules, proved to be a safe and effective vaccine edmonson et al., ). an adenovirus vaccine, prepared in the same way and given simultaneously with adenovirus vaccine, was equally effective (top et al., ) . the administration of a vaccine with only one of these viruses was not effective in controlling ard caused by any of several different adenoviruses, but formulation of a vaccine with both viruses proved to be broadly cross-protective and have had a major influence in controlling ard in u.s. military recruits. over herpesviruses produce disease in man and animals. these viruses have an affinity for epithelial tissues and nervous system tissues. these tropisms lead to specific disease syndromes involving the respiratory and urogenital tracts, and the central and peripheral nervous systems. the viruses often cause persistent infections, which can be latent and can be reactivated. herpesvirus disease are generally much more severe in young humans and animals. some herpes-viruses, including epstein-barr virus (ebv) in humans and marek's disease virus in fowl, are associated with malignancies. the presence of specific antibodies may prevent or modify the clinical disease but does not prevent infection. vaccines have been developed for those herpesviruses causing major diseases in animals; however, despite the seriousness of human herpesvirus diseases, including those caused by herpes simplex virus, ebv virus, cytomegalovirus, and varicella virus, progress has been slow in developing vaccines for humans. this stems from concern over possible persistence and oncogenicity of vaccine viruses. in the past few years, several attenuated live-virus varicella vaccines have been tested and found safe and efficacious (takahashi et al., ; asano et al., ; arbeter et al, ) . since the initial recognition of infectious bovine rhinotracheitis (ibr) in the early s and the later recognition of another manifestation of the disease, infectious pustular vulvovaginitis, this bovine herpesvirus has been acknowledged as a major problem in livestock. the respiratory disease varies from mild to severe, and herd mortality can be as high as % in an acute outbreak. the virus may cause abortions in pregnant cattle, and the genital disease results in a chronic vulvovaginitis but not abortions, apparently due to a lack of viremia. immunization. attenuated live-virus vaccines were developed by serial passage of field isolates in bovine kidney cell cultures. such vaccine viruses have reduced virulence when administered intranasally and do not produce disease when administered parenterally york et al., ) . vaccine virus does not spread from vaccinated to unvaccinated contact animals. the widescale use of such vaccines has controlled this disease effectively. when administered by the intranasal route (todd et al., ) , these vaccines had the advantage of producing more rapid protection, but long-term protection was no greater than with perenterally administered vaccines (mckercher and crenshaw, ) . because of the possibility that the attenuated live-virus vaccines given parenterally might cause abortion, their use has been restricted to nonpregnant animals. a formalin-inactivated vaccine has also been developed, but requires multiple inoculations, and the serum neutralizing (sn) antibody response is low (matsuaba et al., ) . however, inactivated vaccines are used especially in dairy cattle because of concern that the attenuated live-virus vaccines may cause abortion. four herpesviruses affect horses: equine rhinopneumonitis virus (ehv- ) causes abortion which may be epizootic, and also, occasionally, rhinopneumonitis; ehv- is cytomegalovirus found in buffy coat cells of most horses, but its role in causing disease is unknown; ehv- causes equine coital exanthema, a urogenital tract disease; and ehv- is the main cause of equine rhinopneumonitis. ehv- and - are related antigenically, whereas types and are distinct (sabine et al., ; studdert and blackney, ) . immunization. the first vaccine for equine rhinopneumonitis was developed by doll and bryans ( ) , who adapted an ehv- isolate to suckling hamsters. this vaccine produced a mild disease, but induced protective immunity against the more serious respiratory disease and abortion that occurs in older animals. however, this vaccine sometimes caused abortions, and the virus was known to spread from vaccinated to unvaccinated horses. a more attenuated strain of ehv- has been used widely and is considered to be reasonably effective (mayr, ) . this strain was attenuated by passage in hamsters and in pig kidney cell culture before adaptation to an equine cell line (gerber et al., ) . this vaccine induces low levels of sn antibodies, and protects against respiratory disease but not against abortion. a formalin-inactivated vaccine, emulsified in an oil adjuvant, has also been found safe and effective in preventing respiratory disease. this vaccine, in controlled field trials, lowered the abortion rate significantly (bryans, ; bryans and allen, ) . the attenuated live-virus and the inactivated vaccines contain only ehv- , but apparently there is enough cross-protection induced by repeated vaccinations to protect against the ehv- respiratory disease. this herpesvirus is unusual in that it occurs naturally in many species-cattle, sheep, goats, swine, dogs, cats, rats, and mice. it produces a fatal disease in all of these species except adult swine, in which the disease is mild. in each species except swine the primary sign is intense puritis resulting in the animal biting the affected area. infection rapidly spreads to the central nervous system, leading to paralysis and death. in adult swine, the signs of infection are mild, usually of a respiratory nature, but abortions follow in approximately % of pregnant sows. in young pigs, especially neonates, the infection may be fatal. since the disease is prevalent only in swine, this is the only animal for which a vaccine has been developed. in examining a virulent strain of pseudorabies virus, bartha and kojnok ( ) found two plaque sizes. the small plaque size variant, called k, occurred naturally and had reduced virulence for rabbits. further studies with this strain passaged in chick embryo or calf testicle cell culture produced a safe vaccine for swine. one dose induced partial immunity and a second dose yielded good immune responses in all recipients. mcferran and dow ( ) adapted the Κ strain to vero cells and showed that one dose of a vaccine prepared from this passage material was protective. pigs vaccinated with this vaccine did not shed the virus. although the vaccine did not prevent infection, it prevented clinical disease. this attenuated live-virus vaccine is used widely. feline rhinotracheititis virus, which was first isolated in , produces a widespread respiratory disease in cats (crandell and maurer, ) . the virus may also cause fetal death. immunization. an attenuated live-virus vaccine was developed by passage of a field isolate in feline kidney cells (bittle and rubic, ) . the vaccine is given parenterally and is safe and effective (scott, ) . low levels of sn antibodies persist for at least months. vaccinated cats are resistant to intranasal instillation of virulent virus; they may be reinfected, but do not develop clinical disease (bittle and rubic, ; kahn et al., ) . the vaccine has controlled this important respiratory pathogen and, when combined with a feline calicivirus vaccine, has drastically reduced the incidence of respiratory disease in this species. this herpesvirus is highly contagious and causes lesions in the larynx, trachea, and bronchi of infected fowl. the infection causes the formation of an exudate that produces the characteristic respiratory distress and rattling in severely affected birds. birds that recover from this disease are immune for a long period, but may remain as carriers and a source of virus for reinfection of flocks. immunization. the earliest method of immunization was developed by beaudette and hudson ( ) , who applied virulent virus from tracheal exudate to the mucosa of the bursa of fabricius and the cloaca with a stiff brush. this produced a local infection and a solid systemic immunity. the use of fully virulent virus caused occasional outbreaks of disease, particularly when the scarification was not properly done or insufficient virus was present, and birds did not become immune. the virus was propagated on the chorioallantoic membrane of embryonated eggs by burnet ( ) , and this became a source of vaccine material. other methods of vaccination involved intranasal instillation (benton et al., ) and feeding in drinking water (zamberg et al., ) . attenuated strains of ltv have been developed by serial passage in cell culture (gelenczei and marty, ) and by feather follicle passage (molgard and cavett, ) . attenuated strains isolated from outbreaks or selected from passage are now used in preference to virulent virus. marek's disease virus causes lymphoproliferative disease in chickens, occurring in three forms: neural, ocular, and visceral (the latter mainly in young birds) (sevoian and chamberlain, ; biggs and payne, ) . sevoian was the first to provide evidence that marek's tumors were caused by a virus and were transmissible. the virus has been established as a gamma herpesvirus (churchill and biggs, ; soloman et. al., ) . immunization. fatalities from marek's disease caused major economic losses in the poultry industry until a vaccine was developed for its control. this was accomplished by churchill et al. ( ) , who attenuated by serial passage a virus isolated from chickens that is parenterally administered at day of age. thereafter, okazaki et al. ( ) selected a herpesvirus from turkeys (hvt) that was relatively avirulent in chickens, and zander et al. ( ) and schat and calnek ( ) , selected a natural avirulent strain from chickens. these three vaccines are effective, but the hvt strain has been more widely used because it can be obtained from infected cells and can be lyophilized (calnek et al., ) . the vaccines are given parenterally to chicks at hatching and produce good protection ( - %) against virulent virus challenge (purchase et al., ) . viruses of the family poxviridae infect most domestic animals and man. from the standpoint of immunoprophylaxis, the most important poxviruses are: orthopoxvirus, smallpox (variola), mousepox (ectromelia); avipoxvirus, fowlpox, pigeonpox; capripoxvirus, sheeppox, goatpox; leporipoxvirus, myxomatosis virus; parapoxvirus, contagious pustular dermatitis virus. all these poxviruses cause serious disease in their primary host species and some may infect other species. each of the poxviruses causes characteristic vesicular lesions on the skin and mucous membranes, with the exception of myxomatosis virus which produces hyperplastic lesions in the form of myxomas and fibromas. ectromelia (mousepox) is caused by a virus closely related to vaccinia virus and produces a serious disease of laboratory mice. vaccination with vaccinia apparently will reduce the morbidity and mortality of mousepox in a colony, but it will not prevent infection and may act to maintain a silent reservoir of virus (buller and wallace, ) . sheeppox is one of the most serious pox diseases, occurring in europe, the middle east, and africa, but it is controlled by vaccination (aygun, ; sabban, ) . goatpox occurs mainly in the middle east and africa; a goatpox vaccine has been reported to also immunize against sheeppox (rafyi and ramyar, ) . contageous pustular dermatitis virus is unrelated to sheeppox, but causes a pox-like disease in young lambs in which vesicles form around the skin of lips, nostrils, and eyes. boughton and hardy ( ) showed that animals could be protected by scarification with dried contageous pustullar dermatitis virus material similar to that use with vaccinia. myxomatosis virus causes a fatal disease of domestic rabbits and may be spread by direct contact or by blood meals of insects such as mosquitos and fleas (myers et. al., ) . mckercher and saito ( ) developed a safe and efficacious attenuated live-virus vaccine by passage of the virus in rabbit kidney cell culture. this virus, once the cause of epidemics that decimated entire cities, has now been eradicated. the control was brought about by worldwide vaccination and isolation of infected persons. another factor in the control of smallpox was that variola virus had no other host except man. immunization. material from lesions of smallpox-infected individuals had been used for centuries to infect susceptible persons so they would develop a mild form of the disease and become resistant. this variolation was dangerous but much safer than natural exposure to the smallpox virus. jenner ( ) practiced an improved form of this method by using cowpox virus (vaccinia) to inoculate susceptible persons, as described in chapter . most vaccinia vaccines were produced by scarifying the skin of a calf with infected material and harvesting the lymph from the crusted lesions as aseptically as possible. this was stored in % glycerol to stabilize the virus and preservatives were added to destroy bacteria. sheep and rabbits were also used similarly for vaccine production. vaccinia virus was also adapted to grow in embryonated eggs (goodpasture et al., ) . vaccinia virus is very stable, can be produced at a low cost, and is simple to administer. these factors played a major role in allowing the wide-scale use of vaccinia for the eradication of smallpox. fowlpox virus occurs mainly in chickens and produces pox lesions on the wattles, comb, mouth, nostrils, and eyes. the disease is spread mainly by direct contact with infected birds and blood-sucking parasites such as mosquitoes. although it is a fairly resistant virus, it is not otherwise very transmissible. immunization. fowlpox vaccine was originally made by scarifying cockrel combs with virulent virus and harvesting the exudate. johnson ( ) demonstrated that dried exudate would produce immunity when scarified in the wing web or applied to the thigh skin free of feathers. fowlpox virus was later cultivated on the chorio allontoic membrane by goodpasture et al. ( ) and used as a source of vaccine material. later the virus was adapted to tissue culture. an attenuated live-virus fowlpox vaccine produced in cell culture may be used in -day-old chicks (siccardi, ) . another vaccine, an attenuated strain (hp- ) developed by mayr et al. ( ) , is given orally to -day-old chicks, then repeated after to months. hepadnaviridae is a new-found family of viruses containing hepatitis Β virus of man as well as three similar but distinct viruses that infect woodchucks, beechey ground squirrels, and pekin ducks. these viruses have many of the same ultrastructural, molecular, and biological features and their surface antigens cross-react to a small, variable degree. the host range appears to be specific for each virus. hepatitis Β infects man and certain higher primates, including the chimpanzee and gibbon. infection with these hepadnaviruses results in subacute hepatitis, which often becomes progressive and chronic. the most important of these viruses is hepatitis Β virus, which produces a chronic disease in man (blumberg et al., ; prince, ) . hepatitis Β virus is transmitted by blood, saliva, and semen, but also from mother to offspring, the latter route accounting for as much as one-third of persistent infections. the disease is usually selflimiting, but in - % of patients the infection becomes chronic, with the virus persisting in a carrier state. there are over million chronic carriers of this virus worldwide. a late sequela in chronic carriers is hepatocellular carcinoma. it is estimated that - % of malignancies in africa are the result of hepatitis b-induced oncogenesis. immunization. the development of a vaccine for hepatitis Β was hampered by the difficulty of growing the virus in cell culture. krugman et al. ( ) was the first to show that hepatitis Β virusinfected serum could be heat-inactivated and retain its antigenicity. they also showed that this inactivated serum given parenterally could protect subjects exposed to virulent hepatitis Β virus. this led to the use of plasma from infected but healthy virus carriers as the source of antigen. carriers produce large quantities of the hepatitis Β virus, along with its outer coat protein. by purifying and inactivating the coat protein, a safe and effective vaccine was developed (hilleman et al., ) . the coat protein, naturally formed into -nm particles, was purified by ammonium sulfate concentration, isopycnic banding, rate zonal separation and enzymatic digestion. the purified protein particles were then inactivated with : formalin. the particles induce good levels of protective antibody when given in a series of three injections (symuness et al., ) . however, the high cost of this vaccine has limited its use. newer vaccines produced by recombinant dna methods are now being used, as described in other chapters. the four genera in the family picornaviridae are: aphthovirus, rhinovirus, enterovirus, and cardiovirus. viruses in the first three genera cause important diseases in domestic animals and man, whereas viruses in the fourth infect rodents. the picornaviruses in general have a primary affinity for superficial tissues especially of the digestive and respiratory tracts. viruses in the first three genera also have an ability to mutate, thus yielding many serotypes. rhinoviruses cause clinical disease in man, horses, and cattle. no vaccines have been developed for the infections of humans because of the multiplicity of viral serotypes. the number of serotypes in horses (three) and cattle (two) is fewer; nevertheless, no vaccines are available. over enteroviruses exist; of these, vaccines have been developed only for poliomyelitis viruses, avian encephalomyelitis, and duck hepatitis viruses. other viruses in this group either are of low pathogenicity or the number of serotypes is so large as to preclude the development of vaccines. the exception is human hepatitis a virus, which causes a serious disease and has one serotype; the development of both inactivated virus and attenuated live-virus vaccines is in progress (hilleman et al., ; provost et al., ) . fmd was the first animal disease shown to be caused by a virus (loeffler and frosch, ) . fmd viruses cause one of the most economically important diseases of animals and its control is critical to the world's supply of animal protein. the viruses are widespread and occur in many cattle producing regions of the world. the viruses also affect other cloven-footed animals including sheep, swine, and goats. fmd virus infection produces vesicles on oral mucous membranes, including the tongue, gums, and dental pads, but also on the skin including the interdigital spaces and teats. these vesicles on the mucous membranes coalesce and erupt, leaving large denuded areas. the mucous membrane and skin lesions can incapacitate an animal for weeks, thus severely disrupting its productivity. the viruses are highly contagious and persist for long periods in infected animals. animals that recover from natural infection are immune for approximately one year. vallee and carre ( ) showed that there was more than one fmd virus; seven serotypes with over subtypes have now been identified, making the development of effective vaccines difficult. immunization. the first fmd vaccine for cattle was reported in and consisted of a formalinized emulsion of vesicular epithelium (vallee et al., ) . a similar but improved version called the schmidt-waldmann vaccine followed and contained vesicle material from the tongue epithelium of infected cattle. this material was treated with formalin and used with aluminum hydroxide (schmidt, ; waldmann and klobe, ) . another advance was described by frenkel ( ) , who infected superficial layers of bovine tongue epithelium in culture and inactivated the newly replicated virus with formalin to produce a more uniform product. although this method is used today in some areas of the world, most fmd vaccines are now produced by growing the virus in baby hampster kidney cells (mac-pherson and stoker, ; mowat and chapman, ; capstick et al., ) . the imines replaced formalin as an inactivant in most fmd vaccines after brown et al. ( ) showed that viral inactivation was more complete, and safer vaccines could be produced by this process. all inactivated fmd vaccines contain more than one serotype, including the serotypes most common in the area in which the vaccines are to be used. although inactivated vaccines that are produced and used properly can effectively lead to the control fmd, their stability could be improved, thereby lowering their cost. this is discussed further in the chapter by brown. attenuated live-virus fmd vaccines have been developed (henderson, ) but are not used because of the fear that the virus might persist in animals and in meat and milk products from animals (hyslop, (hyslop, - . there are three polio viruses and minor variants of each. the viruses infect man by entry into the upper alimentary tract, infecting cells, and spreading via the blood to the central nervous system, producing neuronal destruction in the medulla and spinal cord. the degree of paralysis that follows infection depends on such factors as virus strain and virus tropism. the vast majority of persons infected with wild polio viruses show no apparent clinical disease. paralysis occurs only in an estimated % of infected individuals; polio virus is responsible for at least % of cases. immunization. early attempts to develop inactivated poliovirus vaccines were hampered by not knowing that there are three distinct viruses. the differentiation of the three viruses by bodian ( ) and kessel and pait ( ) was a major step toward controlling the disease. enders et al. ( ) found that poliovirus would grow in extraneural tissues of human origin and thus laid the groundwork for the development of poliovirus vaccines. the first vaccine (salk) contained all three polio viruses grown in monkey kidney cell culture and inactivated with formalin (salk et al., ) . this vaccine, introduced in , reduced the incidence of paralytic poliomyelitis - % where it was used; however, multiple doses were required and intestinal tract infection was not prevented, thus allowing the virus to continue to spread. there was an intense effort in the s to develop an attenuated live-virus vaccine that could be administered orally, and could protect the intestinal tract, thus breaking the chain of transmission. koprowski, sabin, and cox each developed vaccine strains of reduced neurovirulence that underwent extensive laboratory and field studies (koprowski et al., ; sabin, ; cox et al., ) . the strains developed by sabin were finally licensed in the united states; they produced rapid immunity as well as protection of the intestinal tract while preventing spread to unvaccinated, susceptible persons in contact with vaccinées. this improved the overall level of immunity in communities. with the widescale use of oral poliomyelitis vaccine, the incidence of paralytic disease in the united states has dropped to less than cases per year. the occasional reaction to the attenuated vaccine is discussed in the chapter by hogle and filman. avian encephalomyelitis was first discribed and shown to have a viral etiology by jones ( jones ( , . the virus is widespread and affects young chickens ( - weeks old). characteristic clinical signs are ataxia and tremors of the head and neck. extensive neuronal degeneration occurs in the anterior horn of the cord and in the medulla and pons. the virus may affect older laying birds, causing a drop in egg production. flocks that have survived an outbreak or subclinical infection during the growing period are resistant to further infection (schaaf and lamoreux, ) . moreover, infected chickens - weeks of age undergo only mild disease, providing an opportunity for vaccination (schaff, ) . calnek and taylor ( ) successfully immunized immature birds with an attentuated live-virus vaccine delivered in drinking water. a number of vaccines have been developed including the strain (calnek, ) , the nsw- strain (westbury and senkovic, ) , the philips duphar strain (folkers et al., ) , and a strain grown in chicken pancreas cell culture (miyamae, ) . inactivated ae virus vaccines have been developed for use in susceptible breeding flocks that are in production (schaaf, ; calnek and taylor, ; butterfield et al., ; macleod, ) . two viruses in the family caliciviridae cause significant disease in animals, vesicular exanthema virus in swine and sea lions and feline calicivirus. caliciviruses have also been isolated from humans, calves, reptiles, nonhuman primates, birds, dogs, and fish, but do not produce significant disease in these animals. vesicular exanthema virus caused a disease in swine closely resembling fmd (traum, ) . eradication of this disease followed the discovery that the main source of contagion was uncooked infected meat in garbage fed to swine, and the consequent enforcement of garbage cooking laws. fastier ( ) first isolated a calicivirus from a domestic cat and showed that it produced an upper respiratory tract infection. a large number of viral isolates, with different neutralization patterns, were made from clinically ill cats (crandell et al., ; bittie et al., ) . these serotypes were later shown to have a common antigen and are now considered a single serotype (povey and ingersoll, ) . this virus is widespread, having been isolated from cats in many countries. the virus produces a disease that is generally mild, but, if allowed to progress, the lesions may extend from the upper respiratory tract into the lung causing pneumonia and death (kahn and gillespie, ) . immunization. cats that recover from natural infection and have neutralizing antibodies can be reinfected, but they do not have recurrent clinical disease. an attenuated live-virus calicivirus vaccine has been prepared by serial passage at low temperature; this vaccine virus produces only mild clinical signs in recipients (bittle and rubic, ) . this attenuated live-virus vaccine is administered parenterally; it induces high levels of neutralizing antibody and protects vaccinated cats challenged intranasally with both homologous and heterologous strains (kahn et al., ; scott, ) . immunity from vaccination persists for at least months and probably longer. the calicivirus is combined with feline rhinotracheitis and feline panleukopenia to make a multivalent vaccine that is routinely used in domestic cats (bittle and rubic, ) . inactivated vaccines have also been licensed and are used in multivalent vaccines. the family reoviridae is divided into three genera: reovirus, rotavirus, and orbivirus. infections cause by member viruses are common in mammals and birds. reoviruses are commonly isolated from dogs, cats, sheep, cattle, horses, mice, rats, rabbits, birds, and man. only in birds is the disease serious enough to warrant control with vaccines. the reoviruses are commonly found in sewage, and the mode of transmission is thought to be the fecal-oral route. avian reovirus. in chickens and turkeys, reoviruses produce a widespread disease called viral arthritis (tenosynovitis). this disease of the synovial membranes, tendon sheaths, and myocardium was first recognized by dalton ( ) and by olsen and solomon ( ) . viral arthritis occurs primarily in meat-producing birds, and in acutely affected flocks there is a high rate of condemnation. there are at least five avian reovirus serotypes, but they are antigenically unrelated to the mammalian reoviruses. immunization. vaccination of breeding stock is an effective way to control viral arthritis. van der heide et al. ( ) used an attenuated live-virus vaccine and cessi and lombardini ( ) used an inactivated vaccine in laying hens to protect chicks with maternal antibody. this eliminated transmission and protected susceptible day-old chicks. rotaviruses produce acute gastroenteritis in many species, especially in newborns, including newborn calves, foals, lambs, piglets, puppies, monkeys, and humans. the clinical signs are similar in all species; in each there is acute diarrhea followed by dehydration and rapid loss of weight. secondary bacterial infection may exaggerate the symptoms and also cause pneumonia. the viruses infect epithelial cells of villi, causing desquamation and loss of absorptive function, resulting in diarrhea, rapid dehydration, and emaciation. secretory antibody is very important in protecting the epithelial surface of the small intestine (snodgrass and wells, ) ; antibody contained in colostrum is protective when in high titer. a. bovine rotavirus. bovine rotovirus causes a rapidly spreading disease in neonatal calves (mebus, ) . the antigenic relationship of the bovine rotavirus and other rotaviruses isolated from children, calves, piglets, mice, and foals is very close (woode et al., ) . immunization. an attenuated live-virus vaccine developed by mebus et al. ( ) is administered in two doses to cows prior to calving. this is meant to stimulate colostral antibody, which is passed on to the nursing calves. this vaccine has also been combined with an attenuated live-virus coronavirus vaccine and entero-toxigenic e. coli vaccine to prevent calf scours. b. porcine rotavirus. leece et al. ( ) isolated a rotavirus from piglets with fatal diarrhea. additional reports of this disease showed that it was widespread and warranted the development of a vaccine for its control. immunization. early attempts to immunize pigs by oral administration of a bovine attenuated live-virus rotavirus vaccine were unsuccessful (leece and king, ) . presently an attenuated porcine rotavirus vaccine containing two serotypes, a and a , is licensed in the united states and is being used in combination with a transmissible gastroenteritis (tge) vaccine. the vaccines are administered to pregnant sows by both parenteral and oral routes. at least two doses of vaccine are given orally, and weeks before farrowing, and one dose is given parenterally week before farrowing. this induces antiviral colostral antibody for the protection of suckling piglets. the member viruses of this genus replicate in arthropods as well as in vertebrates. the most important viruses are the bluetongue viruses, and african horse sickness viruses. colorado tick fever virus, the only virus in this genus that infects man, is transmitted by the bite of infected ticks. the disease is usually benign, and infection produces long-lasting immunity. a. bluetongue virus. bluetongue viruses infect ruminants and are transmitted by culicoides gnats. the most serious disease is in sheep, which develop fever, depression, oral lesions, pneumonia, and lame-ness. mortality can be high, especially in lambs. ewes infected early in gestation may produce lambs with hydrocephalus and other congenital deformities. although cattle rarely have clinical bluetongue disease, in utero transmission can occur, resulting in congenital deformities. of the distinct bluetongue viruses, occur in the united states. infection with one bluetongue virus confers resistance to reinfection with that same virus for several months, but cross-protection against infection with other viruses is minimal. a multivalent attenuated live-virus vaccine developed in south africa by serial passage of several different viruses in sheep proved difficult to standardize. a more uniform vaccine was later developed by alexander et al. ( ) ; it contained strains of at least five viruses, attenuated by passage in chick embryos, and gave broad protection against the multiple viruses seen in the field. a similar vaccine was developed by mckercher et al. ( ) , who isolated bluetongue virus in the united states and also attenuated a strain of serotype by serial passage in chick embryos (mckercher et al., ) . recently mcconnell and livingston ( ) have been attempting to incorporate more bluetongue virus strains into multivalent attenuated live-virus vaccines. inactivated vaccines for bluetongue would have the advantage of greater safety than attenuated live-virus vaccines. they would eliminate the possible chance of reversion to virulence, and the chance of vaccine-associated abortion and birth defects. such vaccines are under development (stott et al., ) . . african horse sickness virus. african horse sickness virus causes an acute disease in equine animals in africa, the middle east, and asia. it was shown to be caused by a virus (mcfadyean, ) and has been more thoroughly characterized by breeze et al. ( ) . the viruses are transmitted to horses by culicoides species and affect principally the vasculature of the respiratory tract causing edema of the lungs, head, and neck. the viruses also cause cardiac lesions. immunization. some animals that recover from natural infection may be reinfected, so immunity is not permanent. a spleen pulp vaccine inactivated with formalin was made by dutoit et al. ( ) and administered in multiple doses. later, an attenuated live-virus vaccine was developed by serial intracerebral passage of a field isolate in mice (alexander and dutoit, ) . however, because there are nine african horse sickness viruses, it has been necessary to adapt each to mice and to combine them in a polyvalent vaccine. such vaccine has been effective in protecting horses. the virus that causes ibd was first isolated by winterfield and hitchner ( ) using embroyonated eggs. it causes a disease of chickens in commercial poultry-producing areas. the virus affects mainly young birds - weeks of age, with clinical signs of diarrhea and dehydration. the lesions arise in lymphoid tissues such as the bursa of fabricius, thymus, and spleen, producing immunosupression with the associated opportunistic infections. immunization. both attenuated live-virus and inactivated vaccines have been developed to control ibd. the vaccines are used mainly for immunizing breeder flocks to confer passive immunity through the yolk sac of the egg. maternal antibody protects chicks for - weeks. if breeder flocks are boosted with oil-adjuvant-inactivated vaccines, maternal antibody may last longer. there are several types of attenuated live-virus vaccines with varying degrees of virulence. these vaccines are administered in water, etc., to chicks from day to - weeks of age in broiler-breeder flocks, followed by vaccination with an inactivated product at approximately - weeks of age (lukert and hitchner, ). the four genera in the family togaviraidae are: alphavirus, pestivirus, rubivirus, and arterivirus. each of these genera contain important pathogens. the alphavirus genera include: all cause encephalitis in horses and humans. in horses, the mortality rate of eee in over %, and that from wee is about - %. the main mode of transmission is by culicine mosquitoes; however, vee has been transmitted from horse to horse by contact with body fluids. immunization. infections with togaviruses produce viremia, longterm humoral antibody responses, and immunity. early vaccines were made from formalin-inactivated infected animal brain tissue. the cultivation of both wee and eee in the chick embryo by higbee and howitt ( ) made possible the development of successful inactivated vaccines (beard et al., ) . more recent vaccines for wee and eee are produced in tissue culture. an attenuated live-virus vee vaccine, first developed for humans, is also used for horses in endemic areas (berge et al., ; mckinney et al., ) . the vaccine virus is grown in primary chick embryo cell cultures; it induces long-lasting immunity. an inactivated vee vaccine has also been developed and is combined with wee and eee vaccines in a trivalent formulation. a. hog cholera (hc) virus. hog cholera virus and bovine virus diarrhea (bvd) virus antigenically are closely related pestiviruses, but are specific in the diseases they cause in swine and cattle, respectively. hc virus produces an acute febrile disease marked by multiple hemorrhages, necrosis, and infarcts in internal organs. lethargy, vomiting, and encephalomyelitis are seen in a high percentage of infected animals during an outbreak and mortality is high. immunization. passive protection with convalescent swine serum from swine has been used effectively for short-term control of outbreaks for many years (dorset et al., ) . antiserum and either virulent or partially attenuated virus strains were also used to establish active immunity. although there is only one antigenic type of hc virus, variant biotypes have arisen that are more difficult to protect against with standard vaccines. the presence of neutralizing antibody correlated well with protection. an inactivated virus vaccine prepared from defibrinated swine blood taken during the acute phase of the disease and treated with crystal violet or phenol was safe, but required multiple doses (mcbryde and cole, ) . attenuation of hc virus was first accomplished by passage in rabbits (baker, ; koprowski et al., ) . tissue culture attenuated live-virus vaccines eventually replaced the rabbit vaccine; the latter produced a rapid and long-lasting immunity. a large number of different attenuated live-virus hc vaccines with different characteristics have been used over the years, but residual pathogenicity, shedding, and spread of vaccine viruses have remained problems. a vaccine containing bvd virus was tested in swine, based on evidence that this virus could block replication of hc virus (sheffy et al., ) . however, the bvd vaccine did not protect against all strains of hc virus (tamoglia et al., ) . formerly, control of hc was difficult because hc virus persists in infected meat scraps fed to swine in uncooked garbage. however, since no vaccines have been used in the united states. by controlling the transport of swine and cooking all garbage used as feed, the disease has been eradicated from the united states, and several european countries. b. bovine virus diarrhea (bvd) virus. bvd virus causes a widespread disease of cattle, especially in young stock. clinical signs, which vary in severity, include scouring, ulcerations of the oral cavity, and abortion. young animals that recover often remain stunted and unproductive for long periods. bvd viral strains differ in their cytopathic effects in tissue culture; cattle infected with noncytopathogenic strains during the first months of gestation can transmit the virus to the fetus, which may be born viremic and immunologically tolerant. later exposure to cytopathogenic strains, naturally or by vaccination, can cause offspring to develop the more severe form of the disease (bolin et al., ) . a cytopathogenic strain of virus isolated from a calf and designated oregon (c v) strain (gillespie et al., ) became less pathogenic for calves after passages in bovine kidney tissue culture . this has been the standard vaccine strain and has been used widely for many years. for cattle never exposed to bvd antigen, this vaccine strain is safe and effective; however, persistently infected cattle may react strongly to vaccination with the cytopathic strain of bvd virus, causing a mucosal disease syndrome. it is important to identify and eliminate persistently infected cattle from herds. rubella virus. in man, rubella virus causes a generally mild exanthematous disease, with malaise and respiratory symptoms. complications include arthritis, thrombocytopenia purpura, and encephalitis. gregg's observation ( ) that rubella virus produces fetal abnormalities if infection occurs early in pregnancy emphasized the destructive effects of this virus and the need to develop a means to protect against infection. immunization. natural exposure to rubella virus evokes nasopharyngeal antibody, which is important in preventing reinfection. antibody, especially igg antibody in mother's plasma, is important in preventing fetal infection. two groups isolated rubella virus in tissue culture (parkman et al., ; weller and nova, ) , allowing the first attempts to develop vaccines. both inactivated and attenuated live-virus vaccines were tried before the latter evolved as superior products. the attenuated live-virus vaccines were developed using different cell culture systems, including monkey kidney (parkman et al., ) , duck embryo (buynak, ), rabbit kidney (peetermans and huygelen, ) , canine kidney (musser and hilsabeck, ) , and human diploid cells (plotkin, ) . the vaccines now being used are more than % effective in inducing protective levels of antibody that persist for at least years. the annual incidence of rubella in the united states has dropped from , reported cases in to less than in . equine arteritis virus. equine arteritis virus was first isolated by doll et al. ( ) . the disease caused by this virus is characterized by edema of limbs, stiffness, and swelling in the tissues surrounding the eye, and abortion. immunization. horses that recover from infection develop longlasting immunity. an effective attenuated live-virus vaccine was developed by serial passage of bucyrus field strain virus in primary equine and rabbit kidney cell culture and an equine dermal cell line (doll et al., ; wilson et al., ; mccollum, ). the vaccine has been shown in challenge trials to protect recipients for as long as months (mccollum, ) ; it does not cause any clinical manifestations and it is not spread to susceptible horses in contact with vaccinated recipients. yellow fever virus causes acute hepatitis and hemorrhagic fever in man, characterized by jaundice, shock, and renal damage. transmission is by mosquitoes belonging to the aedes genus throughout tropical areas of south america and africa. the virus is maintained in a transmission cycle between mosquitoes and monkeys, with man being infected when he enters a territory in which the monkey-mosquito cycle exists. immunization. an attenuated live-virus yellow fever vaccine was developed by passage of the virulent asibi strain in mouse brain and cell culture until it had lost its pathogenicity for monkeys and man (theiler, ) . the vaccine virus, termed d, is propagated in embryonated eggs. the vaccine, given as a single dose, is extremely safe and efficacious, providing immunity for at least years. the family orthomyxovirus comprises the influenza viruses, the cause of acute, highly contagious respiratory disease in man, horses, swine, and birds. two structural viral proteins (nf and m) divide this family into three distinct genera: a, b, and c. the viruses, especially influenza a virus, undergo genetic réassortaient, which allows variant viruses to emerge. two viral proteins, the hemagglutinin and neuraminidase, both located on the surface of virions, are important in inducing immunity. vaccines have been widely used for controlling influenza with reasonably good success. since immunity is more closely related to local secretory iga antibody than to serum antibody, it is difficult to stimulate and maintain protection with the presently used inactivated vaccines. with human infections, the type a viruses undergo occasional antigenic shifts and drifts, after which the antigens in the vaccine may not be representative of those viruses found in the field. this potential antigenic variability, as well as animal reservoirs for this virus, are responsible for the pandemics associated with this virus. as an example, the acute respiratory disease of swine caused by a type a strain of virus was first recognized in during the human influenza epidemic and is believed to have been transmitted from swine from humans. both swine and humans are susceptible to the swine virus. however, the disease in swine occurs sporadically and has not been enough of a problem to warrant the use of vaccines in that species. influenza is a respiratory infection with systemic manifestations that include fever, chills, muscular aches, etc. the severity of the disease depends on the virus strain and the susceptibility of the population. persons that have recovered from an influenza infection are usually immune to rechallenge with the homologous virus. however, the change of a few amino acids in hemagglutinin may give rise to antigenic drift and reinfection of populations. immunization. because of the epidemic threat of influenza viruses, careful surveillance for new strains is carried out in many parts of the world. the strains that public health officials predict will be the cause of the next winter's epidemics are then scheduled for vaccine production. for vaccine production, virus is grown in the allantoic cavity of embryonated eggs and is purified and concentrated by zonal centri-fugation. the virus is inactivated with formalin, ß-propiolactone, or irradiation. the quantity of viral antigen per dose is standardized before use. the vaccine usually contains several type a viruses and a type Β virus. these inactivated whole virus vaccines produce protective levels of antibody in approximately % of primed recipients and in % of the unprimed recipients. antibody levels are maintained for approximately year in primed individuals. attenuated live-virus influenza vaccines have been used extensively in the ussr with varying results. problems of adverse reactions, inconsistent potency, and questionable appropriateness of strains make it difficult to evaluate the effectiveness of these vaccines. more recent attempts to develop attenuated live-virus vaccines involve genetic reassortment, a method that offers considerable promise (reviewed by wright and karzon, ) . influenza in horses resembles the disease in man and swine. the two type a influenza viruses of importance in the horse are: a/equi l/prague/ type and a/equi /miami/ type . the disease spreads rapidly through susceptible horses, and those that recover are protected for only a short time. recovery from infection with one virus type does not provide immunity against the other. immunization. vaccines for equine influenza are produced in essentially the same manner as human influenza vaccines. formalinactivated vaccines contain both equine type and viruses and one of several adjuvants, as described by bryans et al. ( ) . vaccines of this type are widely used and effectively control equine influenza. the family paramyxoviridae contains several viruses that cause significant disease in animals. the family is composed of three genera that include the following viruses for which vaccines have been developed. these viruses are transmitted by the respiratory route and are antigenically rather stable. parainfluenza viruses infect humans, rodents, swine, dogs, and cattle. these viruses, by themselves, cause mild upper respiratory tract disease, but when combined with other viral and bacterial pathogens may cause a more severe syndrome. parainfluenza types , , , and a/ b infect humans, especially young children. type is considered the most pathogenic, causing a bronchitis and pneumonitis. vaccines for parainfluenza of rodents (sendai virus infection), dogs (canine parainfluenza), and cattle (bovine parainfluenza) have been developed. there is no licensed parainfluenza vaccine for man. a. sendai virus. sendai virus, first isolated during attempts to recover human respiratory viruses in mice (kuroya et al., ) , is a parainfluenza type virus that causes respiratory disease in mice, rats, hamsters, and swine. the disease occurs either in an acute-short duration form or a chronic-persistent, clinically inapparent form. spread is by either direct contact or by aerosol. in mouse colonies, this disease is difficult to control because the virus is so highly infective. immunization. formalin-inactivated vaccines have been effective in controlling the disease in mice and rats (fukumi and takeuchi, ; eaton et al, ; tsukui et al, ) . additionally, a temperature-sensitive mutant strain of sendai virus has been used as an aerosol-delivered vaccine in mice. it suppresses virus replication, but the vaccine virus spreads throughout the colony and makes it difficult to monitor for wild virus strains (kimura et al, ) . b. canine parainfluenza virus. outbreaks of mild respiratory disease in laboratory dogs have been attributed to parainfluenza type virus (binn et al, ; crandell et al, ) . when other respiratory agents such as mycoplasma and bordetella bronchiseptica were given intranasally after exposure to this parainfluenza virus, more severe respiratory signs occurred (appel and percy, ) . this encouraged efforts to develop a vaccine for canine parainfluenza virus. immunization. an attenuated live-virus parainfluenza vaccine has been shown to protect dogs against aerosol challenge with virulent virus (emery et al, ) . the vaccine produces no untoward effects and has been combined with canine distemper and canine adenovirus vaccines in multivalent formulations. c. bovine parainfluenza virus. a parainfluenza type virus isolated from cattle can also cause mild respiratory disease (reisinger et al, ) . the virus, when combined with other respiratory pathogens including pasteurella and infectious bovine rhinotracheitis virus, causes the severe pneumonia syndrome, called "shipping fever." immunization. an attenuated live-virus parainfluenza type vaccine administered parenterally induces good levels of antibodies and affords protection against experimental challenge (mohanty and lillie, ; thorsen et al., ) . this vaccine has been combined with infectious bovine rhinotracheitis virus and bovine virus diarrhea vaccines in multivalent formulations. an inactivated vaccine, requiring two inoculations, induces high hemagglutination-inhibition titers and lessens the severity of the disease in cattle challenged with the same virus (gale et al., ) . mumps virus causes an acute infection in man with parotitis as the main clinical manifestation, although the central nervous system and other organs including the testes and ovaries can be affected. mumps virus has a limited host range; in addition to man, only certain monkey species and laboratory rodents can be infected. immunization. recovery from natural infection with mumps virus confers long-term immunity. early experiments with formalininactivated virus derived from infected parotid glands of monkeys showed that monkeys and humans could be immunized (enders et al., ; stokes et al., ) . habel ( ) found that chick embryo grown virus could be inactivated with ultraviolet light or formalin and would induce protection in monkeys. a similar vaccine was later shown to induce protection in man (habel, ; henle et al., ) . poor antibody responses to multiple inoculations of this type of vaccine encouraged the search for a more effective vaccine. a mumps virus strain ( jeryl lynn) has been attenuated by passage in chick embryos (weibel et al., ) . the vaccine is immunogenic in - % of subjects, and neutralizing antibodies persist for at least years. the annual incidence of mumps in the united states has been reduced from , cases in to less then , in by application of this vaccine. as presently used, mumps vaccine is combined with measles and rubella vaccines in a pediatric formulation called mmr. newcastle dibcctse is one of the most serious widespread diseases affecting poultry. the disease was first described by kranevelt ( ) and shortly thereafter by doyle ( ) , who named it after the area in england where an outbreak occurred and showed that its cause was a filterable virus. the disease has several forms causing mainly respiratory, enteric, and central nervous system manifestations. the morbid-ity and mortality vary depending on the virus strain. burnet ( ) described the hemagglutinating property of the virus, which has been very helpful in its quantitation and immunodiagnosis. immunization. a number of attenuated live-virus vaccines have been developed which are widely used to control the disease. the bl strain (hitchner and johnson, ) , the lasota strain (winterfield et al., ) , and the f strain (asplin, ) are used to immunize birds of all ages by different routes, including by addition to drinking water and by spraying. vaccines containing inactivated virus do not produce long-lasting immunity but may be used in certain situations when only short-term immunity is needed, such as when boosting immunity is needed in laying flocks. increasing the antigen content and using oil-emulsion adjuvants improves the quality of these inactivated vaccines (stone et al, ; zanella and marchi, ) . measles is a highly contagious disease of humans, occurring mostly in children, causing exanthemata and sometimes more serious manifestations including encephalomyelitis. the virus has two principal immunogens, the hemagglutinin and the fusion protein (norrby et al., ) . the immunity produced by natural infection is long lasting. immunization. the growth of measles virus in chick embryo fibroblasts by enders and peebles ( ) paved the way for the development of vaccines. a formalin-inactivated measles virus vaccine was shown to induce partial immunity. however, some vaccinated children later exposed to measles virus, either naturally or as attenuated live-virus measles vaccine, developed atypical measles (atypical rashes, edema of hands and feet, and respiratory disease). it was later found that formalin-inactivated vaccine failed to stimulate antibody to the fusion protein: consequently the virus could spread from cell to cell, causing the atypical manifestations of disease (norrby et al., ). an attenuated live-virus vaccine was developed from the edmonston strain of measles virus by passage first in human cell culture, then in the amnionic sac of embryonated hen's eggs, and finally in chick embryo cells (milovanovic et al., ) . this vaccine (enders passage level b) was effective in inducing immunity but produced some adverse effects (katz and enders, ; stokes et al., ) . further attenuation by growth at lower temperature yielded an equally effective vaccine that produced fewer side effects (schwarz, ; hilleman et al., ) . attenuated live-virus measles vaccine has been combined with mumps and rubella vaccines. measles vaccine usage has reduced the incidence of measles in the united states from , cases in to , in . canine distemper virus affects most carnivores, causing respiratory, gastrointestinal, and central nervous system disease. the mortality rate in dogs is about %. dunkin and laidlaw ( ) first described the disease in detail and confirmed the viral etiology proposed earlier by carre ( ) . immunization. dunkin ( , a,b) prepared a vaccine by treating virus derived from spleens of infected dogs with formalin. initial administration of this vaccine, followed weeks later with a small dose of virulent virus, usually produced only a mild disease with solid immunity. this approach was replaced with inactivated virus vaccines, given in multiple doses, which served as the main means of controlling the disease from to . green ( ) serially passaged canine distemper virus in ferrets and produced the first attenuated live-virus vaccine; however, this vaccine caused disease in some dogs. the adaptation of canine distemper virus to the chorioallantoic membrane of embryonated eggs by haig ( ) was a major step in developing an attenuated live-virus vaccine. cabasso and cox ( ) applied this method and after passages showed that the virus lost virulence for ferrets but retained its immunizing property for dogs. rockborn ( ) adapted a strain of canine distemper virus to cell culture, and this method is now widely used to produce attenuated live-virus vaccines. rinderpest is an acute highly contagious disease of ruminants characterized by erosions and necrosis of the intestinal mucosa. the disease is epizootic in parts of africa and asia, causing great losses of cattle and buffalo. immunization. koch in developed one of the first means of immunizing cattle against rinderpest by administering bile from infected cattle. animals that survived were permanently immune. formalin-and chloroform-inactivated vaccines were developed using tissues from infected animals. these vaccines were safe, but required two or three doses and protection lasted less than a year (walker et al., ) . rinderpest virus has been adapted to several foreign hosts, including goats (daubney, ) and rabbits (nakamura et al., ) and has been attenuated by passage in these animals. tissues of these animals have been used to produce vaccines in many countries. however, on continued passage, the seed strains tend to lose their immunogenicity, and vaccines become contaminated with adventitious agents from the foreign host. the kabete strain of rinderpest virus was adapted to grow on the chorioallantoic membrane of embryonated eggs, becoming attenuated for cattle after passages . this vaccine and another containing a lapinized strain of virus adapted to embryonated eggs have been used widely in africa (nakamura and miyamoto, ) . more recently, the kabete strain was adapted to bovine kidney cell culture and after passages became avirulent for cattle. this vaccine strain is safe and efficacious in most cattle breeds (plowright and ferris, ) . protection persists for at least years. over million doses of this vaccine have been used in africa, with good success (maurer, ) . bovine rsv produces a rhinitis and catarrhal bronchiolitis in cattle (mohanty et al., ; jacobs and edington, ; paccaud and jacquier, ) . the virus appears to be widespread having been isolated in europe, the united states, and asia. human and bovine virus are related antigenically (paccaud and jacquier, ) ; however, the cattle virus is not known to infect man. immunization. nasal secretory antibody is protective but the disease may occur in the presence of serum antibody. an inactivated bovine rsv vaccine is combined with vaccines for infectious bovine rhinotracheitis, bovine virus diarrhea, and bovine parainfluenza components in a multivalent formulation. the efficacy of the rsv component in this formulation is unclear. of the viruses in the family rhabdoviridae that cause disease in man and domestic animals, the most important is rabies virus. others include vesicular stomatitis viruses (vsv) and bovine emphemeral fever virus (befv). vsv occurs sporadically and epizootically, affecting horses, cattle and swine in the united states. there is a formalininactivated vaccine (gearhart et al., ) , but it is used rarely. befv is an arthropod-transmitted disease of cattle occurring mainly in africa, but also in asia and australia; it is controlled by immunization with attenuated live-virus vaccines (van der westhuizen, ; inaba et al, ; spradbrow, ; theodoridis et al, ) . rabies is an infection of the central nervous system; the disease can occur in most mammals and is usually fatal. there are only a few documented cases of human survivors. after isolating the virus, pasteur ( ) developed a vaccine for its control. historically rabies virus has been considered as a single serotype. but now shared antigens have been found in other viruses in africa of which two, mokola and duvenhage, may be associated with human disease (shope et al. y ) . mokola virus causes a rabies-like disease in dogs and cats in zimbabwe (foggin, ) . immunization. protection against rabies correlates with sn antibody, which can be assessed by a number of tests. pasteur's classical vaccine, developed from infected spinal cord tissue dried at room temprerature for - days, was given in a series of - inoculations beginning with material dried the longest and progressing through material dried for only days (pasteur ) . even though the last inoculum was virulent enough to cause rabies, the earlier inoculations conferred sufficient immunity to protect the recipients. this method of producing a vaccine was successful in most instances but caused the disease occasionally and was eventually replaced by chemically inactivated vaccines prepared from infected brain tissue. although effective, these vaccines gave rise to undesirable side-effects because they contained a myelin-related encephalitogen present in brains from mature animals. substitution of brain tissue from immature animals such as suckling mice, rats, and rabbits with their lesser myelin antigenic content greatly reduced these post-vaccination reactions. the adaptation of the flury strain of rabies virus to growth in chick embryos led to the development of attenuated live-virus vaccines produced in this tissue (leach and johnson, ; koprowski and cox, ; black, , ) . the growth of rabies virus in tissue culture has further improved rabies vaccines (kissling, ; cabasso et al., ; emery et al., ; brown et al., ; fenje, ; abelseth, ). yet, despite their benefits, the attenuated rabies vaccines occasionally caused rabies, particularly in cats. therefore, suckling mouse brain and tissue culture again became the substrates of choice to produce inactivated rabies virus vaccines for animals. in humans the requirement for a safe substrate is more exacting than in animals. for this reason, duck embryos proved better than brain tissue to produce rabies virus (peck et al., ) . after inactivation with ß-propriolactone (bpl), this virus was an improved product for humans, although the allergenic effects from duck embryo tissue still present a problem. therefore the adaptation of the pm rabies virus strain to human diploid cells and inactivation with bpl (wiktor et al., ) was a further improvement. this vaccine is less reactive and more effective for pre-and post-exposure use in humans than any other yet made (bahmanyar et al., ) . a similar vaccine produced in bhk cells is also beneficial in animals. retroviruses have an rna genome, a portion of which encodes the unique enzyme reverse transcriptase. this enzyme imparts to retroviruses the ability to make rna-directed dna copies of their genome, which can then act as a transposable element and can be integrated into the host cell dna. thus, once a cell is infected, it may escape immune surveillance and destruction and the host animal may be infected for life. the retroviruses thus constitute a considerable challenge to traditional vaccine approaches, as discussed further in the chapters by arlinghaus and by nathanson and gonzalez-scarano. many retroviruses infect mammalian species, from mouse to man. most notable are the c-type retroviruses, including the primate, murine, and feline leukemia viruses, as well as human t-cell leukemia viruses types i and ii; the b-type retroviruses, particularly mouse mammary tumor virus; and the lentiviruses, including caprine infectious anemia virus, visna virus, equine infectious anemia virus, feline immunodeficiency virus, bovine immunodeficiency virus, and human immunodeficiency viruses (hiv- and hiv- ). in recent years, as hiv has become a major threat, massive efforts have been directed to developing an efficacious vaccine. so far, all attempts have met with failure. in fact, there are only two retrovirus vaccines that have been proven effective: a formalin-inactivated whole virus preparation of the primate saids type d retrovirus, which is capable of protecting monkeys from a lethal challenge (marx et al., ) , and the commercially available vaccine for feline leukemia. felv commonly infects cats in urban areas, usually by the oralnasal route. kittens under months of age are particularly susceptible. about % of infected cats develop persistent anemia from which myeloproliferative disease and hypoplastic anemia may follow. the immunosuppression caused by felv infection may predispose to severe chronic opportunistic infections. because cats that develop neutralizing antibody are usually immune to infection, vaccines have been developed and tested with that goal in mind. immunization. the problem in developing a vaccine for feline leukemia was to find immunogens that could be used without exposing animals to oncogenic materials. early studies with inactivated whole virus were unsuccessful (yohn et al., ) . although attenuated live-virus vaccines induce sufficiently high levels of neutralizing antibodies to be protective ( jarrett et al., ; pedersen et al., ) , their oncogenic potential makes them unacceptable. efforts to develop vaccines containing only viral proteins, such as envelope protein, have had variable results. however, cultivation of felv in fl transformed cells, followed by treatment to release viral and cell proteins, yields a vaccine that stimulates antibodies to both viral and cell membrane components. a commercial vaccine using this method of antigen production has been approved for use in the united states; it is based on studies done by olsen and lewis ( ) . subsequently, the efficacy of this vaccine has been disputed (pederson and ott, ) . viruses in the family coronaviridae cause important diseases including avian infectious bronchitis, transmissible gastroenteritis of swine (tge), feline infectious peritonitis (fip), and human coronavirus infections. other coronaviruses may cause disease in calves, dogs, mice, rats, turkeys, horses, and parrots, but the diseases are of less importance. coronavirus diseases usually follow a similar pattern, except for fip. fip is a chronic debilitating disease manifested as fibrinous peritonitis and pleuritis. the infection may be inapparent, but is fatal in a small proportion of infected cats. the immune response to fip virus seems to mediate the disease; the immune response is not protective and antibody levels are higher in diseased animals. immune complexes have also been demonstrated in renal glomeruli of cats with fip. bovine coronavirus causes acute diarrheal disease in neonatal calves (mebus et al., ). an attenuated live-virus vaccine is being used in combination with an attenuated live-virus rotavirus vaccine to control calf diarrhea. the vaccine is administered to pregnant cows near the end of gestation and stimulates colostral antibodies that offer protection to nursing calves. with the exception of avian infectious bronchitis, most coronavirus infections have been difficult to control with vaccines. perhaps this is because primary lesions are in mucous membranes of the respiratory and gastrointestinal tracts, sequestered from immune reactivity. coronaviruses produce about % of common colds in man, second only to rhino viruses. there are two groups of human coronaviruses that are antigenically distinct. ibv is a highly contagious respiratory infection of young chickens. the virus may also infect older birds, causing a decrease in egg production. the disease was first shown to be caused by a virus by bushnell and brandley ( ) . beaudette and hudson ( ) propagated the virus in chick embryos, making possible the quantification of the virus and the means for attenuation. there are a number of serotypes of ibv, making the development of an effective vaccine difficult. immunization. immunity following natural infection may last up to year, depending on the serotype and the severity of challenge. van roekel et al. ( ) first developed an immunization procedure; he used a field strain of virus to infect -to -week-old birds before they start to lay, inoculating a few of the birds and allowing infection to spread naturally through the flock. today, there are a number of attenuated live-virus vaccines licensed in the united states. there is good protection ( - %) against homologous virus strains and about % against heterologous strains (hofstad, ) . reduced pathogenicity may be associated with reduced immunogenicity, so a balance must be maintained. the attenuated live-virus vaccines are administered by the usual labor-saving devices of spraying, dusting, or placing the vaccine in drinking water. the wide-scale use of ibv vaccines has significantly reduced the economic loss caused by this disease. tge is an often fatal disease of pigs under weeks of age. the main lesion is enteritis, resulting in malabsorption, diarrhea, and dehydration. tge virus is serologically related to fip virus, but the diseases have entirely different characteristics. there is one serotype of tge virus and one serotype of fip virus. high levels of maternal tge antibody in sows' colostrum protect piglets if fed continuously. immunity of this type has been produced by feeding sows tissues containing virulent tge virus several weeks prior to gestation. the effects of this virus are relatively mild in older animals. attenuated live-virus vaccines administered parenterally to pregnant swine in the latter part of the gestation period produce colostral antibodies. apparently, in sows previously exposed to tge, this vaccine produces sufficient immune responsiveness to be of value. the vaccine is also used orally in pigs - days old to induce local immunity. the effectiveness of this use of the vaccine has not been thoroughly demonstrated. the genus corynebacterium is a heterogeneous grouping with its species placed together largely on the basis of similar cell wall components (goodfellow and minnikin, ) . these species share a basic cell wall chemistry (barksdale, ) of which the mycolic acids (silva and ioneda, ) , especially trehalose dimycolate, are frequently used as potent adjuvants in immunization protocols. two corynebacteria-c. pyogenes and c. pseudotuberculosis-are important in veterinary medicine. the former is frequently associated with ruminate suppurative conditions and abscesses, but it rarely affects man. infections with this organism are sporadic, because it is an opportunist that gains entry through wounds and abrasions. it may also be seen as a secondary invader in devitalized tissues; e.g., vaccination site abscesses. the efficacy of vaccines, toxoids, and antisera against c. pyogenes is equivocal; little is known about immunity to the bacterium. corynebacterium pseudotuberculosis causes caseous lymphadenitis of goats and sheep. it is recognized as a worldwide problem and a serious cause of economic loss to the goat industry (burrell, ; ashfaq and campbell, ) . as with c. pyogenes, the responsible bacterium, c. pseudotuberculosis, is primarily an opportunist entering wounds or abrasions, where it causes local inflammation before settling in the regional lymph nodes. immunization. cell-mediated immunity is necessary for acquired resistance and protection against c. pseudotuberculosis (jolly, ; tashjian and campbell, ; irwin and knight, ) . killed and autogenous vaccines and a toxoid vaccine have been used in attempts to immunize against the bacterium (cameron, ; brogden et al., ; nairn et al., ; burrell, ; anderson and nairn, ; brown et al., ) ; however no one vaccine has proven highly efficacious. diphtheria, characterized by the formation of a tightly adherent pseudomembrane on the pharyngeal mucous membranes of the throat and trachea, is a highly contagious disease of man caused by the bacterium c. diphtheriae. the bacterium can also be isolated from the pharyngeal mucosa of normal individuals. the organism produces a lethal protein exotoxin (gill and pappenheimer, ; collier and kandel, ) . immunization. successful immunization against c. diphtheriae actually protects against the diphtherial exotoxin. because diphtheria toxin is produced in high yield by the park-williams number strain (pw ), pw is used to make diphtherial toxoid for vaccines. as a source of toxin it is rendered nontoxic by incubation with formalin under alkaline conditions. the product's retention of antigenicity, enabling it to induce antitoxin antibodies, makes it an excellent pediatric vaccine. it is commonly utilized in combination with antigens from c. tetani and b. pertussis. the most important species of the bacillus genus, b. anthracis, is the organism responsible for the disease anthrax in both man and animals. anthrax was the first bacterial disease ever to be reported, being described by davaine in . koch in reproduced the disease via animal inoculation and in pasteur successfully vaccinated against anthrax. in animals, natural infection usually occurs by ingestion of spores that germinate in the mucosa of the esophagus or the intestinal tract. herbivorous animals, especially cattle, horses, sheep, and goats, are highly susceptible to the disease, usually the result of grazing in infected pastures or consuming infected foods. in man, anthrax is manifested in three forms: cutaneous (malignant carbuncle), pulmonary (woolsorter's disease), and gastrointestinal with cutaneous being the most common. death results from the combined effects of an extracellular toxic protein complex (vodkin and leppla, ) comprised of three components: edema factor, protective antigen, and lethal factor (leppla, ; stephen, ) . effective vaccines require all three components. immunization. the attenuated pasteur vaccine has been supplanted in veterinary medicine by stable spore vaccines, carbo-zoo vaccine, or stern vaccine (jackson et al., ) prepared from avirulent, nonencapsulated variants of b. anthracis. the viable bacterial spores are suspended in % saponin. immunity is attributed to the development of antibodies to the toxins released from growing bacteria. vaccines of killed bacteria provide little immunity, since no bacterial toxins are produced; hence, no antitoxin antibodies are generated. purified protective antigen (complex toxin) is both antigenic and immunogenic and has been used as a vaccine for humans. it is prepared by aluminum potassium sulfate precipitation of sterile b. anthracis culture filtrates and has proven highly efficacious. erysipelothrix is found in soil, water, and decaying vegetative material and carcasses. the major species of interest is e. rhusiopathiae, which has serotypes (norrung, ) . the bacterium, most notable for causing swine erysipelas, is capable of invading the tissues of both man and animal. fatally affected animals develop welt-like, discolored cutaneous lesions, and numerous hemorrhagic lesions in thoracic and abominai viscera; chronic debilitating arthritis predominates in surviving animals. immunization. the principal vaccines used to control erysipelas are formalin-killed, alum-adsorbed, whole-cell culture vaccines. these combinations of soluble bacterial glycoprotein and whole killed bacterial cells are usually produced from strains of serotype , which possess highly antigenic soluble cell wall glycoproteins. animals immunized with cell-free culture fluids develop agglutinins to the whole bacteria (white and verway, ) . such vaccines are highly effective in controlling swine erysipelas. the pathogenic clostridia invade both man and many animal species of veterinary interest, in which they cause such diseases as tetanus (c. tetani), gas gangrene (c. perfringens, c. septicum, c. oedematous), botulism (c. botulinum), enterotoxemia, and dysentery (c. perfringens). the clostridia are widely distributed in soil and water and are common inhabitants of the intestinal tracts of animals and humans. additionally, the bacteria can often be isolated from infected wounds. vaccination is not routinely practiced against all clostridial organisms, notably c. botulinum. the toxins of c. botulinum, which exert their effects upon the nervous system (schantz and sugiyama, ) , are as potent as those of c. tetani. the lethal dose of the toxin, however, is less than that required to induce an antibody response. clostridium perfringens has five serotypes, a-e, classified according to the production of lethal exotoxins. types a and c are pathogenic for man, whereas all five serotypes can affect animals (see table i ). immunization. the exotoxins of c. perfringens are antigenic proteins that can be detoxified for use in vaccines. the existence of common capsular antigens, which elicit cross-reactions between the serotypes, demonstrates the considerable heterogeneity of this group. ewes and lambs are frequently vaccinated against c. perfringens enterotoxemias. effective vaccines employ type-specific alum- precipitation or formalinized toxoids (smith and matsuoka, ; kennedy et al, ) . clostridium tetani elaborates potent neurotropic exotoxins (tetanospasmin and tetanolysin) that may be lethal for susceptible species such as man, horses, mules, swine, cattle, and sheep. birds are not naturally susceptible to the bacterium. tetanus toxin is one of the most poisonous toxins known. it acts only on the nervous system and its effect characteristically causes spastic paralysis and generalized convulsions. immunization. protective antitoxin blood levels are obtained by immunizing both humans and susceptible animals with alumprecipitated or absorbed tetanus toxoid (chodnik et al., ) . ramon and lemetayer ( ) first introduced the concept of active immunization against tetanus when they used formalinized tetanus toxoid precipitated with alum to vaccinate horses. clostridium tetani vaccines are very effective at inducing long-lasting immunity in both man and domestic animal species. a serious, often fatal disease has been successfully controlled with these vaccines. clostridium novyi possesses four antigenic types, a, b, c, and d; type a is the most common clinical pathogen. types a and Β are responsible for gas gangrene both in man and animals (elder and miles, ) . in areas where sheep simultaneously carry a heavy liver-fluke infestation, exposure to c. novyi is often associated wtih hepatic necrosis and subcutaneous edema. migrating flukes produce foci of hepatic necrosis suitable for the germination of spores and the subsequent elaboration of lethal toxins (williams, ) . immunization. effective vaccinations for c. novyi in animals employ chemically inactivated, detoxified, and adjuvanted suspensions of alum-precipitated formalinized whole broth cultures. clostridium chauvoei, which is the etiologic agent for the disease "blackleg" and is pathogenic for animals only, occurs primarily in ruminant species. protective antigens and toxins with hemolytic and necrotizing activity are formed in susceptible animals ( jayaraman et al., ) . the necrotizing toxin may effect fatal toxemia with degenerative foci of myonecrosis. immunization. immunity to c. chauvoei can be produced via vaccination with its alum-precipitated formalinized cultures (chandler and gulasekhuram, ) . clostridium septicum, in contrast to c. chauvoei, is pathogenic for both man and animal. in man, it is associated with gas gangrene and in affected animals, primarily ruminants, it is the agent most closely identified with the diseases malignant edema and braxy. the organism produces four lethal necrotizing, hemolyzing toxins that cause an increase in capillary permeability and myonecrosis. immunization. immunity to c. septicum is induced with injection of formalinized bacterial cultures. the antitoxin provides homologous protection and additionally protects against c. chauvoei. animals are often vaccinated with mixtures of clostridial species; i.e., novyi, chauvoei, septicum, perfringens, and sordelli in one combination vaccine. these are highly efficacious vaccines and are routinely used in veterinary medicine. although infections with mycobacterium tuberculosis primarily occur through inhalation of the tubercle bacillus, ingestion of large numbers of the bacilli in contaminated milk or infectious sputum can readily produce disease in susceptible species. the bacterium is pathogenic in man, but can also cause disease in monkeys, pigs, and occasionally in cattle, dogs, and parrots. the disease may be asymptomatic or produce severe, debilitating pulmonary lesions. if infection is not restricted by the immune system, the disease may be fatal (comstock, ; bloom and godal, ) . since bacteriocidal mechanisms of the normal macrophage prevent m. tuberculosis from multiplying intercellularly (goren, ; goren et al., ) , protective immunity depends on cell-mediated immunity (lagrange, ) . mycobacterium bovis, closely related to m. tuberculosis, and m. avium causes disease primarily in cattle and birds. they can, however, be contagious to man, sheep, and pigs. immunization. immunoprophylaxis for tuberculosis is based on vaccination with an attenuated, relatively avirulent strain of m. bovis that does not produce lesions. the strain is known as bcg or the bacillus of guérin ( , ) . worldwide, this is one of the most widely used human vaccines, as it has proven efficacious in controlling a severe disease. additionally, bcg has been used for nonspecific enhancement of resistance against tumors and other infec-tions. the cell wall of m. tuberculosis is a potent immunostimulant when used in freund's adjuvant. although streptococci may be normal inhabitants of the gastrointestinal tract, they may also be pathogenic for both man and animals. on the basis of characteristic cell wall components, the streptococci are traditionally divided into lancefield groupings (lancefield, ) . streptococcus agalactiae, a streptococcus group Β organism, causes severe mastitis in the bovine species and has been identified as a major cause of serious neonatal infections in man (eickoff et al., ) . capsular antigens form the four major type-specific antigens (la, lb, ii, and iii), with type iii organisms being most commonly associated with neonatal meningitis. in infants, early-onset disease occurs within the first days of life and is characterized by sepsis and pneumonia. the mortality rate is high. late-onset disease occurs around month of age and is characterized by meningitis (einstein et al., ) . immunization. there is a direct correlation between the absence of maternal igg antibody to type iii antigen and the incidence of neonatal infection. thus, susceptibility to the bacterium is related to the absence of significant levels of maternal serum antibody being transferred transplacental^ to the fetus. current vaccine developments are directed toward maternal immunization with type iii antigen (einstein et al., ) . streptococcus pneumoniae, the etiologic agent of pneumococcal pneumonia in human infants and adults, may cause septicemia, meningitis, and inner ear infections. aerosol transmission of the bacterium, often in association with viral upper respiratory infections, is the major mode of transmitting s. pneumoniae infections. streptococcus pneumoniae possesses a capsular polysaccharide capable of deterring phagocytosis, thus enhancing the virulence of the bacterium. of over types of the bacterium identified, serotypes are most frequently associated with the disease. a polyvalent pneumococcal vaccine prepared from soluble purified capsular polysaccharides of the most predominant s. pneumoniae serotypes has proven effective in adults. the capsular polysaccharides are well-tolerated and highly immunogenic; signifi-cant rises in protective serum antibody titers are achieved following vaccination (kasper et al., ) . however, vaccination of infants has not proven beneficial, because they develop no higher antibody titers to the bacteria than do unvaccinated infants (ginsburg, ) . enterotoxigenic pathogenic strains of escherichia coli may cause severe, potentially fatal, diarrheal disease in both man and domestic species, particularly neonatal cattle and swine. the capsular (k) antigens are cell-surface proteins and/or polysaccharides associated with virulence. the k antigen mediates adhesion to the microvillus of intestinal epithelial cells; production and release of enterotoxin follow (bywater, ; lonroth et al., ) . escherichia coli neonatal enteritis of newborn calves is also a serotype-specific disease (myers and guinée, ) . ail important colostral antibodies in both swine and cattle are anti-k antibodies (usually k ) (myers and guinée, ; moon et ah, ) . immunization. vaccination of gilts, sows, heifers, and cows with vaccines prepared from the k or other pilus-associated antigens has reduced morbidity and mortality from e. coli nec natal enteritis of newborn piglets and calves (nagy et ah, ; rutter, ; kohler et ah, ; childrow and porter, ; myers and guinée, ) . to prepare the porcine vaccines, bacterial strains specific for the herds to be vaccinated are used to immunize animals weeks prior to parturition, thereby generating specific, protective colostral antibodies. recombinant dna technology, discussed in the chapter by collett has introduced the potential to construct e. coli vaccine strains that would afford considerably better protection than those currently available. salmonella species are a major cause of invasive enteric infections in humans and domestic animals, with domestic poultry constituting the largest reservoir of salmonella organisms in nature. normally, infection occurs through the oral route. salmonella is a facultative intracellular pathogen; therefore, cell-mediated immunity is more important than humoral immunity in resistance to the disease, salmonellosis (fields et ah, a,b; dougan et ah, ; woolcock, ) . salmonella typhi, the only salmonella species that has a capsular polysaccharide (vi antigen), is the etiologic agent of typhoid fever, a serious and common disease in underdeveloped areas (edelman and levine, ) . this pathogen infects humans only; there is no suitable animal model for typhoid fever. immunization. few vaccines have been developed for salmonella, and most are of low efficacy with undesirable side-effects. live vaccines are more effective than killed ones in promoting better immunity (levine et ah, ; dougan et ah, ; roantree, ) . with respect to s. typhi, vaccines containing the inactivated bacteria offer only limited and transient protection with undesirable side-effects (levine, et ah, ) . the attenuated strain of s. typhi, ty- a, requires multiple doses to achieve - % protection (hirschel et ah, ) . consequently, typhoid fever has not been controlled by immunization, although the vi antigen has recently been hailed as the agent of a preventative vaccine (robbins and robbins, ) without adverse side-effects (acharya et ah, ) . yersinia pestis is the etiologic agent of plague or "black death" in man, a highly fatal disease with fever and purulent lymphadenopathy. although not a disease of domestic animals, rats, ground squirrels, and other rodents may be affected. the bacterium is spread by the rat flea, xenopsylla cheopis, from rat to rat and from rat to man. immunization. the most widely used vaccine for the prevention of y. pestis infection is haffkine's vaccine, first developed in . this vaccine is prepared from heat and phenol-killed virulent cultures. formalin-killed virulent bacteria are also successful, as are living avirulent strains (grasset, ) . the is no evidence that any vaccine protects against pneumonic plague, the most contagious and fatal form of the disease. furthermore, vaccine protection is only recommended for plague research workers. disease control is primarily dependent upon eradication of rodent carriers of y. pestis. pasteurella multocida and p. haemolytica are common commensals of the mucous membranes of the respiratory tract and oropharynx of healthy cattle, sheep, swine, dogs, and cats. when the bacteria multiply unchecked, they can penetrate the oral and/or respiratory mucosa, where they quickly grow and overpower the host's defense systems. pasteur first described this bacterium as the etiologic agent of fowl cholera; it is also associated with bovine pneumonia, swine plague, and an epizootic hemorrhagic septicemia in ungulates. the bacterium's heat-stable antigens have been used as serologic indicators in the gel diffusion precipitin test to define its serotypes (brogden et al., ) . immunization. pasteur's successful bacterial vaccine to fowl cholera, and the first vaccine ever used, consisted of avirulent cultures of the p. multocida attenuated by prolonged growth on artificial medium. killed p. multocida vaccines are prepared from virulent immunogenic strains of the bacterium. the organisms are suspended in formalinized saline, incorporated into an adjuvant, and injected subcutaneously (heddleston et al., ) . these vaccines induce substantial immunity to fowl cholera. additionally, live vaccines for oral administration have been developed for use in the poultry industry (dougherty et al., ; heddleston et al., ; oison, ; bierer and derieux, ) . pasteurella multocida is usually mixed with modified live or killed bovine rhinotracheitis virus, parainfluenza virus, bovine viral diarrhea virus, and p. hemolytica bacteria in combination vaccines to protect against pasteurella pneumonia in cattle. bovine pneumonic pasteurellosis (shipping fever) is a severe fibrinous pneumonia of feedlot cattle usually associated with biotype a, serotype infections with this organism. immunization. administration of either killed or live vaccines has been of limited efficacy in controlling shipping fever. partial protection from experimental disease follows immunization of cattle with either live p. haemolytica by aerosol or parenteral routes confer et al., ) or lyophilized p. haemolytica vaccines consisting of chemically altered, streptomycin-dependent, or modified live organisms given intramuscularly or intradermally (confer et al., ) . humoral antibody responses appear to correlate strongly with protection against experimental disease (confer etat., ; mckinney et al., ) . for example, purified p. haemolytica lipopolysaccharide stimulates specific antibody formation and has protected calves challenged with the bacterium from developing the disease (hilwig et al., ) . these obligate parasites, restricted to respiratory and pharyngeal mucous membranes, cause important diseases in porcine (h. pleuropneumonia, h. suis, andi/. parasuis), equine (h. equigenitalis), bovine (h. somuns), and avian (h. gallinarum, h. paragallinarum) hosts. most hemophilus species require two factors, hemin (x) and nicotinamide adenine dinucleotide (v), for growth. antigens associated with protection and virulence have been described fori/, paragallinarum (yamamoto, ) , the etiologic agent of infectious coryza in chickens. birds that have recovered from natural infection possess varying degrees of immunity to re-exposure (page et al., ) ; immunity is serotype-specific. adjuvanted vaccines containing multiple bacterial serotypes are prepared from chicken embryos or formalinized bacterial broth cultures and are effective vaccines in preventing infectious coryza in chickens. this bacterium is the etiologic agent of the porcine disease, contagious pleuropneumonia, which is characterized by severe multifocal, necrotizing pneumonia with venous thrombosis and associated serofibrinous pleuritis (didier et al., ) . the disease is of considerable economic importance to the swine industry, being most prevalent in situations where swine are raised under intensive management conditions. hemophilus pleuropneumoniae possesses major and minor antigens that are both common and serotype specific (gunnarson et al., ; gunnarson, ; mittal et al., ) . since high antibody titer apparently provides little protection from the disease, cell-mediated immunity may be important in protection from infection (rapp and ross, ; rosendal et al., ) . immunization. no adequate immunoprophylaxis against contagious pleuropneumonia is currently available, although many vaccines have been tried (nielsen, ; henry and marstellar, ; christensen, ; masson et al., ) . prior infection with one serotype provides protection from heterologous serotypes (nielsen, ) . the bacterial strains used in vaccines are serotype specific and, while not preventing the disease, can reduce its severity (christensen, ) . hemophilus somnus is the cause of infectious meningoencephalitis, a disease with low morbidity but high mortality in cattle. whole or sonicated bacterial cells and bacterial protein are immunogenic (noyer et al., ) and efficacious bacterins foster protective immunity in calves (williams et al., ) . the bacterins of h. somnus, adjuvanted with aluminum hydroxide, are prepared from highly immunogenic strains of the bacterium and grown in serum-free media for use as vaccines. the species in this genera, b. pertussis, b. bronchiseptica, and b. parapertussis, can be either parasites or, as in swine and dogs, common inhabitants of the upper respiratory tract. these small, serologically related bacilli produce a dermonecrotic toxin. infection is by aerosol transmission with bacteria adherent to tracheal cilia (bemis et al., ) . local, not serum, antibody concentration is important in clearance of the infection. the etiologic agent of whooping cough, Β. pertussis, produces two distinct hemagglutinins, leukocytosis-promoting factor-hemagglutinin (lpf-ha) and filamentous hemagglutinin (fha), and various toxins (pertussis toxin [pt] and dermonecrotic toxin). fha is involved in bacterial adherence to the respiratory mucosa, whereas pt is believed to be the major protective (sato and sato, ) and pathogenic antigen (steinman et al., ) . immunization. although efficacious, the safety of the human vaccines currently in use, suspensions of killed whole cells containing protective antigens, is open to question (robinson et al., ) . undesirable side-effects such as screaming, collapse, encephalopathy, and other serious neurological complications have been reported in association with b. pertussis vaccinations (dick, ) . the potencies of whole cell vaccines correlate with the antigenic content of pt (reiser and germanier, ) . bordetella bronchiseptica is an obligate parasite of the upper respiratory tract of both dogs and pigs. in dogs the bacterium frequently invades the lungs as a sequela to canine distemper (caused by a morbillivirus), causing an often fatal bronchopneumonia. the bacterium is also associated with mild to severe tracheobronchitis, "kennel cough," in dogs (bemis et al., ) . in pigs, a deformation of the bony structures of the nasal area (atrophic rhinitis) and reduction of the total volume of nasal turbinates commonly follow the bacterial infection (ross et al., ) . degenerative changes in the osteoblasts and osteocytes may be caused by elaboration of a dermonecrotic toxin (dnt), which is released from b. bronchiseptica after colonization or multiplication of the organisms on the nasal mucosa (nakai et al., ) . the release of dnt from p. multocida type d is thought to exacerbate the disease. immunization. vaccines to control canine b. bronchiseptica infections are commonly incorporated into combination packages containing attenuated live-virus canine distemper, and canine adeno and parainfluenza viruses. to control atrophic rhinitis, avirulent live, or inactivated organisms alone or in combination with p. multocida, erysipelothrix rhusiopathiae and!?, coli have been utilized in vaccines. the organisms in this group, b. abortus, b. suis, Β. melitensis, Β. canis, and b. ovis, cause the disease brucellosis in domestic animals and man. the bacterium may localize in the reproductive tract which, in the female, can lead to fetal death with subsequent abortion. brucellosis, due to b. abortus or b. melitensis, is a zoonotic disease, readily transmitted from animal to man. the potentially severe consequences of brucellosis, fetal death and abortion, in the pregnant cow and epididymitis and sterility in the bull result in significant economic loss to the cattle industry. the primary source of infection is infected animals, whose mammary and/or genital secretions may contain the bacterium. calves can become infected in utero; however, the main portals of infection are oral mucosa, nasopharynx, and conjuctiva of exposed animals. immunity to b. abortus is dependent upon cell-mediated immunity, as the presence of serum antibodies, although a significant indicator of infection, does not correlate with the immune status of the host (fitzgeorge et al., ; kaneene, et al., ; swiderska et al., ; montaraz and winter, ) . most humans who contract brucellosis have been exposed either to b. melitensis, the etiologic agent of malta or mediterranean fever, or b. abortus. brucella melitensis, found in the milk of infected sheep and goats, may produce fatal disease when ingested by humans. brucellosis of sheep and goats mimics the disease as it is seen in cattle, with fetal death and abortion occurring in ewes and does and epididymitis in rams and billies. brucella ovis infects sheep, causing late fetal death and abortion in pregnant ewes and epididymitis in rams, such as b. abortus does in cattle. immunization. brucella abortus (strain ) is currently used as the vaccine of choice for control of brucellosis of cattle in the united states. this is a viable, smooth strain that, while posing virtually no threat for cattle, may cause disease in man. the major objections to the vaccine are this pathogenicity for humans and the difficulty of differentiating vaccinated from naturally infected animals since persistent serum antibodies are induced by the vaccine. killed b. melitensis in adjuvant or live avirulent strains have been used for vaccines to induce a high degree of immunity in sheep and goats. brucella ovis bacterins in adjuvant, as well as b. melitensis vaccines, have been used to protect animals from the disease b. ovis causes, since the antigens of these two pathogens are cross-protective (diaz et al., ) . pathogenic members of this genus are associated with venereal disease, fetal death, and abortion in cattle. this bacterium is transmitted to uninfected cattle by coitus or artificial insemination and is an obligate parasite of the genital tract. immunization. stimulation of opsonizing antibodies of the igg class by systemic vaccination with adjuvanted vaccines is effective in preventing natural infection in bulls (bouters et al., ) and infertility in cows (corbeil et al., ; hoerlin and kramer, ) , and prevents the carrier state (wilkie and winter, ). unlike c. fetus venerealis, this bacterium is contracted by ingestion and is not transmitted venereally. both sheep and cattle can be infected; however, the disease is most severe in pregnant ewes, which undergo a high percentage of abortions or premature births within a flock. immunization. in sheep, vaccination with polyvalent adjuvanted vaccines is efficacious in preventing disease (thompson and gilmour, ) . the most important organism of this genus, v. cholera, causes severe, acute enteritis in humans and nonhuman primates. the etiologic agent of cholera in humans, v. cholera, causes a potentially fatal diarrheal disease. infection results in the production of a powerful enterotoxin in the small intestine, which stimulates an increase in cyclic amp in intestinal epithelial cells and causes a profuse outpouring of isotonic fluids. vibrio possesses immunogenic heat-labile flagellar (h) protein and heat-stable (o) lipopolysaccharide somatic antigens. the cholera toxin is immunologically and functionally similar to the heat-labile enterotoxin of e. coli (yamamoto et al., ) . it consists of six light subunits (l) that assist in toxin adherence to intestinal cell receptors and one heavy subunit (h), which is the toxic entity. immunization. present cholera vaccines are administered by parenteral or oral routes. parenteral vaccines consist of formalin/phenolinactivated bacteria, whereas oral vaccines employ killed or live bacteria. vaccines given by either route provide protection for approximately - months. the predominant immune mechanism is antibacterial rather than antitoxic (levine et al., ) ; antibacterial antibodies prevent attachment of the bacterium, whereas antitoxic antibodies inhibit toxin adherence to cell receptors. because current vaccines often produce adverse side-effects (feeley, ) , synthetic and semi-synthetic vaccines are currently under investigation. for the latter, a nonpyrogenic, bivalent cell-surface protein-polysaccharide conjugate is being investigated (kabir, ) . h. leptospira spp. the pathogenic genera of this family can penetrate the gastrointestinal mucosa and abraded epidermis. leptospires are transmitted through contact with the urine of animal carriers, either directly or in contaminated water or soil. rodents are the primary reservoir of the bacteria which, due to its ability to synthesize urease, colonizes the renal nephron and subsequently is shed into the urine. leptospirosis causes economically serious disease in cattle and swine by causing fetal death, abortion, and infertility. recovery from infection with one serotype lends immunity only to that serotype. this immunity is predominantly humoral, since agglutinins (igm) are responsible for the initial clearance of the bacteria; neutralizing antibodies (igg) are also protective (hanson, ; negi et al., ) . canine leptospirosis infections may be severe, occurring more commonly in male dogs than in females. man is a dead-end host for leptospires; infection in man is accidental and usually related to occupational exposure. immunization. killed, multivalent, leprospira vaccines protect against clinical disease in cattle and swine; however, in pigs, immunity does not protect against renal colonization (stalheim, ) . dogs can be vaccinated successfully with formalin or phenol-killed vaccines that contain antigens from the two most common infecting serotypes, l. canicola and l. icterohemorrhagica (kerr and marshall, ) . vaccines for humans, prepared from chemically inactivated cells of leptospires, have been used extensively in certain areas of the world. neisseria meningitidis neisseria meningitidis is a frequent cause of endemic purulent meningitis in human infants and adults, although the incidence of the disease is substantially higher in young infants (hoffman, ) . bacterial invasion of the meninges is usually hematogenous from the upper respiratory tract and is a life-threatening affliction. neisseria meningitidis has been classified into at least nine groups (a, b, c, w- , x, y, z, l, -e) on the basis of its capsular polysaccharides (morse, ) . immunization. protection against meningococcus meningitis results primarily from the presence of antibodies against the capsular polysaccharide of n. meningitidis (frasch et al., ) . group a and c polysaccharide vaccines are especially effective against disease in children over two years of age and in adults. epidemic typhus fever has afflicted mankind since ancient times. it is an acute highly infectious disease with the potential for explosive epidemics in man. significant outbreaks of the disease have been intimately associated with war and famine. the disease is characterized by sustained high fevers, headache, panencephalitis, a diffuse maculopapular skin rash, and toxic vascular damage. the fatality rate may be high. the etiologic agent, r. prowazeki, is transmitted from person to person by the human body louse pediculus humanus corporis. infection is established by inoculating infected louse feces into the skin by scratching. immunization. although no etiologic relationship has been demonstrated between r. prowazeki and the bacterial strain proteus x , these two species share a common polysaccharide antigen (castaneda, ) . the sera of infected typhus patients agglutinates proteus x and this test is now standard (weil-felix reaction) for diagnosis of the acute disease. additionally, r. prowazeki has two major antigenic components-one heat labile and the other heat stable (craigie et al., ; topping et al., ) . typhus fever vaccine contains killed organisms propagated in the yolk-sac membranes of developing chick embryos (cox, ) . this vaccine not only diminishes the symptoms of typhus in immunized persons, but it also greatly reduces the mortality rate (gilliam, ) . the usefulness of the attenuated (madrid Ε strain) vaccine is hampered because, under appropriate conditions, the strain may revert to virulence (brezina, ) . the prokaryotes proc. world vet. congress, th proc. annu. conf. aust. vet. assoc tuberculosis in bacterial infections in humans: epidemiology and control symposium on rickettsial diseases vm/sac new trends and developments in vaccines vaccines ' : modern approaches to new vaccines principles and practice of cholera control vaccines ' proc. natl. acad the prokaryotes proc. natl public health rep viral hepatitis viral hepatitis proc. west. states food anim. conf vaccination against foot and mouth disease an inquiry into the cause and effects of the vanidae vaccinae vaccines ' a system of bacteriology the mycobacteria: a source book proc. natl. acad. sei. u.s.a. diseases of poultry bovine medicine and surgery proc. int. conf. equine infect. dis proc. annu. conf. aust. vet. assoc proc. natl. acad. sei. u.sa. proc. conf. res. work. anim. dis vaccines ' proc. th annu. meet. u.s. livestock sanit. assoc proc. natl. acad proc. th annu. meet. u.s. livestock sanit. assoc the virus in yellow fever proc. int. vet. congr vaccines ' diseases of poultry were useful references in writing this chapter. key: cord- -lrbi authors: childs, james e. title: pre-spillover prevention of emerging zoonotic diseases: what are the targets and what are the tools? date: journal: wildlife and emerging zoonotic diseases: the biology, circumstances and consequences of cross-species transmission doi: . / - - - - _ sha: doc_id: cord_uid: lrbi the uneven standards of surveillance, human- or animal-based, for zoonotic diseases or pathogens maintained and transmitted by wildlife h r s, or even domestic species, is a global problem, readily apparent even within the united states, where investment in public health, including surveillance systems, has a long and enviable history. as of , there appears to be little scientific, social, or political consensus that animalbased surveillance for zoonoses merits investment in international infrastructure, other than the fledgling efforts with avian influenza, or targeted nontraditional avenues of surveillance and research. individual humans sickened or killed by an unknown infectious cause potentially indicate a zoonotic disease emergence has occurred, but, by themselves, are insufficient to document any instance of emergence. incident cases of a new zoonotic disease must come to the attention of local authorities and then be the target of clinical, epidemiologic, and microbiologic research prior to any determination that an outbreak was caused by an emerging or reemerging pathogen. satisfactory fulfillment of koch's postulates is a daunting process, involving the diagnosis of human disease, i.e., the isolation of the infecting pathogen in cell culture; the molecular and antigenic characterization of pathogens obtained from human or animal tissues; and establishing the novel pathogen's causal role as etiologic agent ). these endeavors link forever an instance of emergence with a single time point and place, a pinpoint and date on a map [fig. . in institute of medicine ( ) ]. such an accounting system is necessary, but belies the dynamic ongoing process of disease emergence. as with the invading species that perishes on a foreign shore before being identified and labeled by a knowledgeable biologist, countless cases of zoonotic disease go unrecognized and uncatalogued. these missing data limit comparative analyses of the qualities of successful invading species to the far larger outgroup of pathogens for which there are limited or negative, i.e., not detected, data (daszak et al. ; cleaveland et al. ; dobson and foufopoulos ; kolar and lodge ; see the chapter by cleaveland et al., this volume) . irrespective of the limitations of such studies, coherent trends and suites of plausible traits associated with successfully emerging pathogens have been derived from comparative studies (dobson and foufopoulos ; cleaveland et al. ; see the chapters by cleaveland et al. and holmes and drummond, this volume) , but offer little guidance on how and where to focus attention (but see the chapters by daszak et al. and merianos, this volume) . zoonotic viral emergences surprise even the scientists who are most knowledgeable within a subject area. witness the identification of a novel hantavirus causing fatal disease in the southwestern united state, after decades of search for pathogenic hantaviruses in the united states (leduc et al. ) , and the discovery of a novel lyssavirus causing a disease indistinguishable from rabies, in supposedly rabies-free australia (hooper et al. ) . although the process of zoonotic pathogen emergence often begins with identification of a case or cluster of human disease, surveillance and monitoring systems are ill equipped to detect and then characterize the unknown (see the chapters by merianos and by stallknecht, this volume) . once a new zoonotic disease is identified and a case definition is established, the systematic collection of information on incident cases of human disease is used to generate information in a usable form, through appropriate data analytic and publication processes conducted through personnel working through a central repository. when the information is disseminated back to health professionals, from the federal government to individual practitioner level, a surveillance system is established. the country of occurrence, the morbidity and mortality, and the preexisting public health infrastructure, mixed with a good portion of serendipity, influence the likelihood of detecting a newly emerged zoonosis. surveillance for zoonotic pathogens is largely based on detecting illness or infection in homo sapiens (see the chapters by merianos and by stallknecht, this volume); humans serve as the sentinel species for zoonotic agents maintained in transmission cycles in which, fortunately, they rarely play other than an incidental role as a dead-end host. a variety of surveillance systems and data sources have been successfully, if sometimes unintentionally, employed to monitor existing zoonotic diseases or to detect new diseases (table ). an example of a serendipitous outcome stemming from syndrome-based surveillance for a specific disease occurred in new york city in , with the implementation of a system to detect bioterrorism-related cases of anthrax (centers for disease control and prevention ; buehler et al. ; paddock et al. ) . the putative anthrax case definition included a febrile illness accompanied by either a rash or eschar. rickettsialpox, caused by rickettsia akari , had been an endemic, legally mandated reportable disease in new york city since the mid- s (huebner and jellison ; huebner et al. ), but since the s the median number of annual cases reported was approximately . the classical presentation of rickettsialpox includes a fever and one or more eschars at the bite sites produced by the infected mite vector transmitting r. akari . over an -month interval, cases of rickettsialpox were diagnosed through the syndromic-based anthrax-surveillance system in new york city; tissue biopsies from patients yielded the first isolates of r. akari from the united states in more than years . although rickettsialpox was a known entity, anthrax surveillance highlighted the underappreciated level of disease caused by this endemic zoonosis. surveillance systems designed to detect and monitor a specific animal disease have also uncovered novel zoonotic pathogens. in the united states, two previously unknown rhabdoviruses have been isolated from dead birds collected for monitoring and forecasting wnv activity (eidson et al. b (eidson et al. , c mostashari et al. ; garvin et al. ; travassos da rosa et al. ) . while in australia, laboratory workup of a sick pteropid bat collected in conjunction with hendra virus (hev) investigations following an outbreak of disease affecting horses and humans in halpin et al. ) yielded a new lyssavirus , australian bat lyssavirus (abl), closely related to rabies virus (fraser et al. ; gould et al. a) . within months of the isolation of abl, this virus was demonstrated to be the cause of fatal encephalitis in humans (gould et al. b) ; until this time no rabies had been reported from australia. effective, but informal, surveillance systems can be implemented rapidly following the identification of a novel zoonotic disease emergence within countries with a highly developed public health infrastructure. the interplay of factors influencing initial detection and later development of systematic surveillance are illustrated by the outbreak of hantavirus pulmonary syndrome (hps) in the southwestern united states in may . an indian health service physician noted a temporally and spatially linked cluster of cases of a severe, kitala et al. b often fatal, respiratory disease, affecting previously healthy, young-adult navajo indians residing on a reservation (duchin et al. ) . the physician notified local authorities and subsequently cdc was invited by state officials to help investigate the growing number of fatalities. testing of patient sera at cdc revealed the presence of antibodies reactive with hantaviral antigens . facilitated by epidemiologic knowledge of hantaviruses and hantaviral diseases occurring in eurasia, rapid progress was made in uncovering the natural history of this mysterious new disease. in a matter of weeks, investigators confirmed the disease was clinically distinct from eurasian disease (moolenaar et al. ) , that the etiologic agent was a new hantavirus , sin nombre virus (nichol et al. ) , and the reservoir host (h r ; for definition of terminology see the chapter by childs et al., this volume) was a species of new world rodent, peromyscus maniculatus (childs et al. ) . a relatively crude but effective national surveillance program, capitalizing on media interest in the hps outbreak, was established by june . six months later, private citizens or their physicians had reported and submitted clinical specimens for diagnostic testing from persons; confirmed hps cases were identified from states outside of the four-state region where the initial outbreak was localized (tappero et al. ) . this impromptu surveillance system was highly successful in rapidly identifying the widespread geographic distribution and sporadic incidence of hps cases throughout much of the western united states. once a zoonotic disease is characterized, formal, systematic surveillance efforts can be initiated at the state or national level in countries possessing the requisite infrastructure. national surveillance programs coordinated through cdc, with rare exceptions, focus on the systematic collection of data on human disease. national surveillance and the global network for monitoring influenza a activity among humans is the outstanding example of a system integrating epidemiologic data with the collection and characterization of influenza viral subtypes circulating throughout the world (centers for disease control and prevention d; cox et al. ) . the unquestioned value of the global influenza surveillance program rests with the vaccines produced. each year's new influenza vaccines are based on determinations of the currently circulating influenza subtypes and divining which subtypes should be incorporated into next season's vaccine cocktail. a global early warning system to detect zoonotic pathogens transmitted to humans was launched in july by the un food and agriculture organization (fao) and the world health organization (who) in collaboration with the world organization for animal health (formerly the office of international epizooties or oie) (http://www.who.int/mediacentre/news/new/ /nw /en). specifically mentioned as examples are bse and sars; data from infected and diseased humans and animals will be gathered and assessed jointly. plans to develop a global animal-based influenza surveillance program exist (centers for disease control and prevention d; stohr ) . it remains unclear if animal-based influenza surveillance will extend beyond domestic poultry and livestock to wild waterfowl and shorebird h r s, although this latter activity is strongly endorsed melville and shortridge ; see the chapter by webby et al., this volume). surveillance for zoonotic diseases among wildlife, as opposed to domestic animals and livestock, falls through the cracks of both veterinary and human health practices (see the chapter by stallknecht, this volume). reviews of animal health monitoring systems mention wildlife disease surveillance only in passing and largely in reference to the difficulties of establishing population estimates (denominator data) for defining rates, such as disease incidence, or the obstacles to developing systematic surveillance programs coordinating with human disease surveillance (ingram et al. ; see the chapters by daszak et al., merianos, and by stallknecht, this volume) . most regional or state systems collecting information on wildlife diseases are passive surveillance systems. passive surveillance in the united states, as defined by public health professionals, is the systematic collection of data on human diseases, reportable through legal mandate in most states, obtained within specified time frames on conditions listed by national notifiable disease surveillance system (nndss) (teutsch ) ; data are reported to cdc by electronic submissions via the national electronic telecommunications system for surveillance (netss) (teutsch ) . international regulations require reporting on quarantinable conditions, such as plague, yellow fever, cholera, and sars (teutsch ; centers for disease control and prevention b) . diseases covered by the nndss are established through collaborations of the council of state and territorial epidemiologists (cste) with the cdc and the nationally reportable diseases are reviewed at -year intervals, at which time case definitions are established or modified (centers for disease control and prevention ). by virtue of the population estimates provided by the us census, human surveillance data collected via nndss are population-based. summary statistics on nationally notifiable disease are published weekly in morbidity and mortality weekly report (mmwr) and summarized in annual reports (centers for disease control and prevention c). in contrast, for wildlife and domestic animal diseases, the oie, situated in paris, france, determines diseases reportable by its member counties (thiermann ) . the diseases are divided into two lists: list a diseases are of major importance in international trade of animals or animal products and have the potential for very serious and rapid spread irrespective of national borders; list b diseases are of public health importance within counties (thiermann ; http://www.oie.int). within the united states, mandated reporting of animal diseases varies by state, and voluntary reporting by professionals is a major component of data collection (salman ) . at the federal level, information is collected by the animal and plant health inspection service (aphis) of the department of agriculture (usda). given the lack of accurate population estimates for many domestic animals and livestock, passive veterinary surveillance is not population-based. surveillance for wildlife diseases exists at some level in most developed countries. as with human, surveillance, the infrastructure for receiving, typing, and storing animal specimens and the diagnostic laboratory capacity for establishing diagnoses are minimal prerequisites (see the chapter by stallknecht, this volume). within north america, the canadian cooperative wildlife health center (ccwhc), supported by the four canadian veterinary schools, was established in to promote nationwide surveillance of wildlife diseases. in canada, disease detection is carried out by a wide range of professional and voluntary field personnel, including hunters, and specimen diagnosis is conducted at provincial and federal veterinary laboratories. the central repository for data is the ccwhc, which disseminates surveillance information to persons responsible for wildlife programs and policies, and to the public (leighton et al. ). in the united states, states have often taken the lead in monitoring wildlife diseases, such as wnv among dead birds, arboviral infections among sentinel bird flocks (mostashari et al. ; eidson et al. a; komar ) , and transmissible spongiform encepahlopathy (tse) associated with elk and whitetailed deer (williams and miller ) . in several states, notably california and florida, surveillance for arbovirus activity using sentinel flocks of birds have documented trends in the enzootic activity of western equine encephalomyelitis (wee), st. louis encephalitis (sle), and eastern equine encephalomyelitis (eee) linked to climatic and local weather patterns (reeves ; shaman et al. ; day ; barker et al. ) . surveillance for viruses transmitted from wildlife h r s to domestic poultry and livestock, such as avian influenza a, subtypes of which infect and cause disease in humans (kermode-scott ; fouchier et al. ) , is conducted through the usda. additionally, the usda conducts mandated surveillance for zoonotic infections of livestock, such as bse, anthrax, and bovine tuberculosis (tb) (anonymous b; myers et al. ) . regional activities monitoring wildlife diseases, especially among game animals, such as white-tailed deer ( odocoileus virginianus ), exist through cooperative efforts involving research and educational institutions, state fish and game departments, and hunters. a successful example is the southeastern cooperative wildlife disease study (scwds) maintained at the university of georgia, where programs collect regional data on wildlife, ectoparasitic and endoparastic infestations, and microbiologic and serologic evidence of past or current infections. historical collections and independently funded research programs through scwds recently led to the rapid elucidation of the natural history of emerging tick-borne zoonoses caused by bacteria in the genera ehrlichia and anaplasma (davidson et al. ; little et al. ; lockhart et al. lockhart et al. , ; see the chapter by paddock and yabsley, this volume) . wildlife disease monitoring in sweden and northern europe has existed since the s, relying heavily on the cooperation and interest of hunters in the collection and submission of samples from game animals (mörner ; mörner et al. ) . surveillance for wildlife diseases in the uk and ireland has included bovine tb maintained by badgers (see the chapter by palmer, this volume); current plans call for increased surveillance of wildlife, notably birds for wnv, in england and wales (griffin et al. ; gormley and costello ; crook et al. ; duff et al. ; see the chapter by palmer, this volume). short-term studies of wildlife h r s are the most common survey methods employed in response to specific instances of emergence or spread of zoonotic disease. following an outbreak of human monkeypox in several us states (centers for disease control and prevention a; see the chapter by regnery, this volume), local populations of indigenous north american rodents were captured and examined for infection from areas around animal-holding facilities housing african rodents imported for the pet-trade and implicated as the source of monkeypox virus (cunha ; check ) . native american ground squirrels, coincidentally housed in the same buildings with the african rodents and purchased as pets, were implicated as the source of monkeypox virus transmitted to humans (guarner et al. ; see the chapter by regnery, this volume). short-lived studies identifying rabid raccoons were undertaken in ohio, following the first reported case of raccoon-variant rabies in that state (stefanak et al. ) . testing of trapped and road-killed raccoons helped define the geographic extent of the enzootic area of raccoon rabies in the state in preparation for the deployment of an oral rabies vaccine (orv) in an effort to prevent the westward expansion of epizootic raccoon rabies into ohio and west to other states (kemere et al. ; foroutan et al. ; aphis wildlife services factsheet ) . long-term prospective studies of zoonotic pathogens circulating within wildlife h r s are critical to understanding factors mediating irregular increases and declines within animal populations, which can drive the risk of spillover to humans. the varying population dynamics of zoonotic pathogens and their h r s are, in some instances, as with rabies virus, driven by pathogen-induced host mortality (anderson et al. ; childs et al. ; coyne et al. ) ; the risk of rabies virus spillover to domestic animals is closely, but not perfectly, mirrored by the temporal dynamics within the wildlife h r (gordon et al. ) . examples of systematic wildlife disease studies that have exceeded several years in duration are few. one ongoing example is the investigations of the population dynamics of rodent h r s and snv and other hantaviruses in the southwestern united states, which were established in the mid- s following the outbreak of hps. replicated and coordinated studies among universities in several states, using similar methodologies for population sampling, virological testing, and data management (mills et al. ) , have provided a wealth of information critical for unraveling aspects of the transmission and maintenance of hantaviruses (mills et al. a (mills et al. , b . the knowledge base established by these efforts allowed increasingly elaborate hypotheses developed from field observations to be tested. the modalities of hantaviral transmission were assessed by application of microsatellite markers to genetically identify familial relationships among individual mice; related male p. maniculatus were more likely to be snv-infected (root et al. ) , providing clues to the chain of transmission events contributing to the male bias in hantaviral infection documented by several descriptive studies (mills et al. a ). ongoing research is providing clues as to the critical h r population size required to sustain hantavirus transmission and is exploring the phenomenon of snv disappearance and reemergence in h r populations , possibly through snv maintenance within refugia of a special nature . these ongoing studies spanning more than years, have been sufficient to capture occurrences and effects of environmental drivers, such as el niño southern oscillation (enso), which occurs at semi-predictable intervals of approximately - years (chen et al. a ). enso is a principal indicator of global climate which modifies local weather patterns; increasing rainfall associated with enso is hypothesized to drive a trophic cascade of events (polis et al. ) , ultimately leading to increases in local h r populations and increased risk of hps (glass et al. ; hjelle and glass ) . remote sensing and gis techniques, coupled to a household-based case-control methodology assessing rodent abundance around residences of hps cases , predicted where p. maniculatus would be more abundant at future case houses. analyses of annual satellite images to detect local environmental conditions supportive of rodent hr population growth has proven an effective tool for predicting the qualitative level of risk (low, moderate, high) for hps over a sizable region of the southwestern us (glass et al. ) . educational recommendations and field trials of rodent-proofing methods were incorporated into the long-term investigations (glass et al. ) , to provide readily available control measures in anticipation of increased risk of hps ). animal-based surveillance is a process inherently different from human-based surveillance (table ) . with the exception of surveillance efforts targeting livestock and poultry, run through the center for animal health surveillance of the usda (king ) , no formal sampling methodology exists for estimating animal population sizes at the regional or continental level (see the chapter by stallknecht, this volume). wildlife population estimates at the continental scale are few and generally restricted to tractable populations associated with conservation efforts, with the possible exception of national waterfowl surveys , or national hunter-or road-killed indices of white-tailed deer populations (hayne ). targeted ecologic studies directed at species that are endangered or threatened have in several instances provided population-based information complementing the objectives of wildlife disease research. the most notable examples involve species that are relatively easy to observe or for which population-based indices exist, such as carcass, nest, or scat counts (leroy et al. ) . where estimates of animal numbers have been enumerated, the impact of fatal zoonotic viruses indicate certain wildlife species could serve as sentinels for monitoring viral activity; species conservation activities can provide leverage to any additional surveillance investment (see the chapter by daszak et al., this volume) . examples include great apes killed by ebola virus (leroy et al. ; walsh et al. ; see the chapters by gonzales et al., this volume), and rabies induced mortality among african wild dogs (kat et al. ; gascoyne et al. b; burrows ) , and ethiopian (whitby et al. ; sillero-zubiri et al. ) and artic wolves (ballard and krausman ; weiler table key differences in the terms "passive surveillance" and "active surveillance" and methods of data collection as used and defined by veterinary and human health professionals surveillance system or manner of data collection veterinary health a human public health b passive "the passive collection of data involves the reporting of clinical or subclinical suspect cases to the health authorities by health care professionals at their discretion." "a passive surveillance system is one in which a health jurisdiction receives disease reports from physicians, laboratories, or other individuals or institutions as mandated by state law." legally mandated, systematically collected within specified time frames, voluntarily reported to cdc not population-based specified by state and federal officials within the national notifiable disease surveillance system (nndss). population-based by virtue of the us census active "an active collection of data for any monitoring and surveillance system (moss) is the systematic collection or regular recording of cases of a designated disease or group of diseases for a specific goal of monitoring or surveillance." "in contrast, an active surveillance system is established when a health department regularly contacts reporting sources (e.g., once per week) to elicit reports, including negative reports (no cases)." key characteristics not necessarily mandated by law not necessarily mandated by law population-based population-based collects negative data a quoted from salman ( ) b quoted from birkhead and maylahn ( ) et chapman ) . for other wildlife, the lack of population estimates precludes estimation of basic epidemiologic parameters, including rates such as incidence or mortality; these capabilities are beyond those of any existing surveillance system for a wildlife zoonosis. novel animal-based surveillance and control programs are being planned for zoonotic agents, such as bse, sars-cov and influenza a subtypes which have realized or potential pandemic importance to humans or domestic animals (http://www.who.int/mediacentre/news/new/ /nw /en). the ultimate h r s for these agents includes domestic and wild animal species. for example, the h r s for influenza a subtype h ni are among wild waterfowl and shorebirds, and perhaps other avian types, although, domestic chickens and other poultry serve as both the first secondary host (h s ) or intermediate host (h i )(see the chapter by childs et al., this volume, for description of terms) and can develop as a novel h r (see the chapter by webby et al., this volume). experts within the who and elsewhere, acknowledge a need ". . . to get rid of the natural reservoir of h n , but we need to do it safely" (quote attributed to klaus stohr, project leader of who's global influenza program; cited in abbott and pearson [ ] ). however, even rough plans of how such an immense undertaking will be designed and integrated into the countries of greatest significance in asia are lacking. even when infection within an animal h r or h s is relatively detectable, national surveillance programs for monitoring morbidity and mortality among wildlife and establishing the etiologic cause of infection through a system of diagnostic laboratories are rare (see the chapter by stallknecht, this volume). if the zoonotic agent is a pathogen of domestic livestock, formal surveillance can target abattoirs or production facilities where food animals are processed, as is the major emphasis of bse surveillance conducted both in the united states by the usda (kellar and lees ; anonymous b ) and within european countries ). among wildlife, animal rabies is the only disease within the nndss for which time-series data of reasonable duration, more than years, quantity and quality has been systematically collected from all us states and territories . animal-based surveillance for pathogens causing emerging zoonotic diseases in humans is often hampered by the lack of clinical signs in infected individuals of the h r (table ) . where zoonotic viruses cause fatal disease among wildlife and domestic animal h r s, h s s, or h i s, tracking the spread of these agents is a simpler matter, although this remains a formidable challenge within countries lacking basic surveillance infrastructure. tracking the spread of influenza a subtype h n of domestic chickens, ducks, and some wild waterfowl in southeastern asia (chen et al. b; li et al. ; lu et al. ; see the chapter by webby et al., this volume), wnv in north america (garvin et al. ; guptill et al. ; walsh et al. ; larkin ) , ebola virus in central africa (leroy et al. (leroy et al. , walsh et al. ; see the chapter by gonzales et al., this volume), and rabies virus in north america, europe, and southern africa (sabeta et al. ; childs et al. ; gordon et al. ; see the chapter by nel and rupprecht, this volume) has been facilitated by the mortality these viruses cause in wildlife and domestic species. national surveillance for animal rabies is a model public health activity. as the cdc is charged with promotion of human health and disease prevention and control, animal-based rabies surveillance data are well integrated into national, state, and local human and veterinary public health programs . a brief examination of the objectives, types of data collected, and the practical use of the information disseminated through the national animal rabies surveillance program is illustrative of the potential benefits accrued from an animal-based surveillance system. surveillance for animal rabies collects information on the current status and level of rabies activity among wildlife and domestic animals at the county level within individual states. monthly counts of rabid animals, and from some states the tally of negative results, designated to the level of animal species or taxonomic group, are submitted to the cdc ). surveillance information is analyzed, summarized, and disseminated back to the data providers in a timely manner through publications ) and additional communications, which are updated annually, such as the compendium of animal rabies prevention and control (centers for disease control ). surveillance data on animal rabies are sufficiently detailed and accurate to allow human and veterinary health professionals to anticipate levels of rabies activity at the county or regional level, permitting some future planning for preventative activities, including procurement of human vaccine and human rabies immunoglobulin (hrig) for postexposure treatment of potentially exposed persons (centers for disease control and prevention b; advisory committee on immunization practices ); increasing vaccination levels of dogs and cats; and initiation of targeted control efforts to vaccinate wildlife using orv kemere et al. ) . several species of terrestrial carnivore, raccoons ( procyon lotor ), red foxes ( vulpes vulpes ), and striped skunks ( mephitis mephitis ) serve as h r s for particular genetic variants of rabies circulating in the continental us; numerous rabies virus variants are also associated with different species of bats (messenger et al. ) . rabies virus variants can be differentiated by limited sequence analysis or monoclonal antibody methods and the enzootic area where rabies variants overlap the geographic range of their terrestrial mammalian hosts can be reasonably determined . time-series surveillance data on wildlife rabies, analyzed by statistical algorithms defining and demarcating intervals of increased (epizootic) or diminished (interepizootic or enzootic) rabies activity, provide results concordant with predictions and outcomes based on numerical solutions to mathematical models of the population dynamics of rabies virus within a single h r species anderson et al. ; coyne et al. ). time-series analyses have defined the temporal dynamics of disease in a wildlife h r guerra et al. ) and demonstrated the close association of this relatively predictable process to the risk of rabies spillover to domestic animals (gordon et al. ). furthermore, these data can inform epidemiologic simulations and models predicting epizootic rabies spread (russell et al. , and have been modified to forecast the savings accrued by preventing rabies spread through the application of orv (gordon et al. ) . additionally, local data have provided the raw material to explore formal methodologies for demonstrating and assessing the impact of long-distance translocations (ldts) of infected animals on the rate and pattern of rabies spread in heterogeneous environments . the availability of remotely sensed or digitized maps, coupled with gis-assisted partitioning of landscapes into habitats of varying quality, allow explorations of the impact of landscape heterogeneity on the characteristics of epizootics and the pattern of epizootic wavefront spread (jones et al. ; smith et al. ) . such analyses have been used to assess where remedial prevention activities should be focused when breaches in orv barriers occur and where active surveillance might be considered as a complement to passive data collection where fine-scale knowledge of the presence of rabies is needed to guide interventions . however, rabies surveillance reveals several inherent difficulties to conducting any form of wildlife-based disease surveillance and offers a sobering view of the hurdles to be overcome when considering such programs in other locations for other diseases. animal rabies surveillance was implemented to provide humans with a measure of rabies risk in their communities and, other than relative species counts over years, there is no information on the incidence or impact of rabies in any animal community. the nature of the human-animal interactions required by an animal-based surveillance system provides a distorted image of rabies as a community process (fig. ) . biases inherent to data collected by animal rabies surveillance at the national level stem from the requirement of human participation in each step of the process culminating in a rabies diagnosis in an animal ( fig. a ; gordon et al. ) . the impact of human demography, measured as absolute population size per county, on the surveillance process is sufficient to account for fully % of the variation in total animal specimens tested for rabies (fig. b; childs et al. ) . total county expenditure is almost as strong a predictor, accounting for % of the variation in total animal tests performed. pathobiologic features of rabies, human behavior, and the expense associated with diagnostic testing of specimens skew the types of animals observed, harvested, and tested for rabies. medium-to-large-sized mammals are more likely to be observed by humans and reported to wildlife control officials. in a typical surveillance year, small terrestrial mammals, predominantly rodents, but some insectivores, weighing less than kg account for less than . % of the total animals tested and diagnosed as rabid (real and childs ) , although small mammals provide the greatest species diversity and the overwhelming abundance of individuals and biomass of many mammalian communities (bourliere ) . rodents are fully susceptible to rabies infection and are capable of transmitting the virus to other species (childs et al. ; winkler et al. ) ; in some countries, rodents have been implicated in natural maintenance cycles of the virus (summa et al. ; verlinde et al. ) . a major sampling bias occurs at the level of the rabies diagnostic laboratory where, in an effort to save money on personnel time and diagnostic reagents, rabies testing is typically restricted to specimens from animals directly involved fig. a , b (continued) data integrating test outcome with information on the type of animal and date and place of origin produced at the state level and submitted to cdc. b although data on each of the events partitioned in (a) are unavailable, a surrogate value of population size is used to measure the importance of human interaction in generating surveillance data, assuming that increasing numbers of humans increase the likelihood of many of the events in (a) occurring. there is a strong association between the absolute numbers of humans resident in the smallest surveillance unit (us census figures), a county within a state, and the total numbers of animals tested for rabies from that surveillance unit. the relationship is a power function in which human population size accounts for % of the variance in median total tests conducted for rabies conducted over a decade from counties in a region affected by the raccoon variant of rabies virus. " " " " " " " " " " h o x h k xh n x fig. a, b the process of wildlife-and animal-based surveillance is interactive, involving multiple, and frequently independent, interactions between humans and wildlife to generate a single datum captured. panel a depicts some examples of these interactions, which could be assigned a probability if information were available, between private citizens and local and federal agencies in the route to generating a datum on animal rabies. each process involves some interaction with an animal, a tissue sample taken from the animal, test material derived from the sample, an outcome derived from the sample at the diagnostic laboratory, and the in the potential exposure of humans or domestic animals to rabies virus (fig. a) ; other specimens go untested (torrence et al. ; wilson et al. ). many of these limitations and biases will be generic problems confronting any effort to monitor wildlife species anywhere in the world. the most widespread approaches to zoonotic disease control completely ignore the ecology of wildlife and pathogen maintenance and transmission and, therefore, the potential for interrupting pathogen transmission prior to human spillover. instead, prevention and control strategies focus on defensive measures for the human h s . national institutions charged with strategic planning for emerging diseases or intentional releases of zoonotic agents have emphasized improving diagnostic capabilities for detecting human infections, modifying the immune status of human or domestic animals through vaccines, producing better antiviral or antibacterial drugs, and enhancing human-based surveillance as an early warning system (fauchi ; centers for disease control and prevention ). with the possible exception of extensive human vaccination, each of these approaches target post-spillover events and none of these avenues of research will have the slightest impact on reducing the risk of additional emergence of viruses or other pathogens from wildlife. the current fixation on human vaccines, human diagnostics, human drugs, and human-based surveillance is the legacy of past successes. landmark achievements for zoonotic disease prevention include vaccines for yellow fever and rabies, and other vaccines of human or veterinary importance exist, or are being developed, for tick-borne encephalitis, rift-valley fever, arboviral encephalitides, sars, ebola hemorrhagic fever, hps, and many others (chang et al. ; cox et al. ; lau ; custer et al. ; matsuoka et al. ; nalca et al. ; warfield et al. ; hjelle ; tomori ; tesh et al. ; stephenson ; monath et al. ; huang et al. ) . new antiviral drugs can be designed, created, and screened with far better efficiency than at any time in the past and novel candidates and methodologies for improving the delivery of drugs to infected cells are in development (oxford et al. ; duzgunes et al. ; wu et al. ; pastor-anglada et al. ) . additionally, traditional measures of case isolation, contact-tracing, and quarantine of exposed persons, banning of public gatherings, or curtailing individual access to international travel have proved highly effective in controlling the spread of zoonotic diseases with pandemic potential, as with sars (zhong ; anderson et al. ; speakman et al. ; see the chapter by wang and eaton, this volume) (fig. ) . but sars-cov is not influenza a. methods relying on increasing social distance are unlikely to prevent the spread of humanadapted pandemic influenza a mills et al. ) . aerosol transmissibility of influenza virus in the subclinical patient precedes clinical signs by h (mills et al. ; fraser et al. ) , unlike the coincidence of clinical disease with the onset of infectiousness with sars-cov . influenza a vaccine production capacity and antiviral medication stockpiles to combat influenza spread are insufficient even in wealthy developed countries (mills et al. ). can we continue to prepare and respond to such pathogens by strictly defensive measures aimed at the human h s ? so given the proven record of achievement of a medical or technological approach to defending humans from invasion by infectious organisms, is there much to be gained by examining processes, antecedent to human spillover, for potential vulnerabilities and as intervention targets, as a complement to ongoing efforts to improve human-based disease prevention activities? the answer is yes, but a qualified yes. simply saying we need such systems glosses over the myriad of obstacles in developing programs. designing and implementing wildlife-based surveillance and targeted interventions will not be achieved in the short term and establishing the infrastructure to support these efforts would be difficult and expensive (see the chapter by merianos, this volume). the maintenance and transmission cycles of zoonotic viruses within wildlife h r s offer many of the same targets for control as do human-based interventions, with the notable exception that population culling can be exploited for control of animal reservoirs, intermediate host populations and arthropod vector species. the ultimate prevention strategy for zoonotic agents affecting humans is to abrogate or greatly reduce cross-species transmission by disrupting transmission and maintenance cycles of zoonotic viruses within the h r . (introduction of refractory genes into h v s, endosymbionts) physical/temporal barriers table ). animal quarantine, isolation of animals exposed to a pathogen, and legal bans to trade in animals or animal products originating from countries with enzootic disease act to increase social distance and decrease the likelihood of contacts between infected and susceptible hosts. immunocontraception of h r s to reduce population size or genetic modification of h v s to render vector populations refractory to infection may play a role in prevention in the future. human vaccination, treatment, and the prophylactic use of drugs are defensive measures that may prevent or reduce spillover and post-spillover spread, but will not reduce the likelihood of contact between infected h r and individual humans. (modified from childs ) however, rarely has the full force of human scientific creativity and funding been directed at understanding and interrupting vulnerable infectious processes prerequisite to, but intermediate from, the immediate circumstances leading to human infection. the most widespread approach to zoonosis control is the culling or killing of individuals of h r s, h v s, or h i s, either through selected culling (largely restricted to domestic animals) or indiscriminate population reduction (wobeser ). the most common example of culling is the use of insecticides to control h v populations and nuisance populations of mosquitoes (thier ; leprince and lane ; mount et al. ) ; however, issues related to human and environmental health have limited enthusiasm for this type of control in many circumstances. culling of wildlife h r populations has been adopted, or is planned, to curtail transmission of several viral and bacterial zoonotic pathogens to humans or domesticated livestock, although the record of population control as an effective prevention strategy limiting spillover is mixed (wobeser ; see the chapter by palmer, this volume). targeted reduction of specific h r populations for control of rabies virus variants has been employed in europe and north america. on both continents, programs have targeted red foxes (muller ; debbie ) and in north america raccoon and skunk populations have been targeted (rosatte et al. ; debbie ) . efforts are ongoing in central and south america to reduce vampire bat populations in an effort to curtail the enormous economic loses sustained from vampire-bat transmitted rabies to cattle. anticoagulants applied topically or systemically by direct inoculation into livestock are the major methods of vampire bat control (crespo et al. ; fornes et al. ; thompson et al. ). however, wildlife culling to control rabies has been deemed largely unsuccessful or unnecessary given the intensive use of orv to vaccinate susceptible h r s (centers for disease control and prevention b; macinnes et al. ; aubert b; brochier et al. ; slate et al. ) . however, mathematical modeling of different control strategies frequently identifies a combination of vaccination and targeted culling as the optimal strategy for rabies control (smith and wilkinson ; anderson et al. ) . culling has recently been halted as a control measure for badgers serving as h r s for bovine tb in england (roper ; gormley and costello ; see the chapter by palmer, this volume), although in ireland data suggest badger culling is an effective measure in reducing the incidence of tb in cattle herds (griffin et al. ) . the removal of some , badgers in england from to failed to curb bovine tb spread among cattle (delahay et al. ) . vaccination of badgers against tb is now being investigated as a part of an integrated control program that includes targeting specific sites for control and different herd management practices for high-risk regions (white and benhin ) . china initiated culling of live captured and breeding stocks of several species of carnivores, the masked palm civet ( paguma larvata ), the raccoon dog ( nyctereutes procyonoides ), and the chinese ferret badger ( melogale moschata ), implicated in the transmission of sars-cov to humans (watts ; zhong ) . the who questioned the appropriateness of culling wildlife species (parry ) and it is now appears that wild carnivores are not the actual h r for sars-cov. current information suggests that bats of the genus rhinolophus are the h r for ancestral coronaviruses giving rise to sars-cov capable of infecting wild carnivores and humans ; see the chapter by wang and eaton, this volume). irrespective of the culling of farm-raised animals, the enormous illegal trade in wildlife will continue to stock the wet markets of china, vietnam, and other southeastern asian countries, with meat and other animal products from wild carnivores and other wildlife species prized for their culinary and medicinal properties (bell et al. ; yiming and dianmo ) . control of emerging zoonotic agents circulating among domestic poultry, livestock, and companion animals is often more finely targeted at specific infected subpopulations or demographic cohorts than methods applied to wildlife. for example, the mass elimination of seropositive dogs in brazil has been used in control programs for zoonotic visceral leishmaniasis; although evidence suggests dog control has failed to reduce the number of human leishmaniasis cases (moreira et al. ) . different culling strategies have been used for the control of bse. herd culling involves destroying entire herds of cattle from which an index case of bse originated; birth cohort culling targets the subpopulation of cattle born during a specific interval of time and considered at greatest risk for having acquired bse before the prohibition of feed containing cattle-derived offal; maternal culling destroys offspring borne to high-risk cows as the risk of vertical transmission of bse is approximately % (anonymous ) ; a final subpopulation considered to be at high risk, but difficult to identify operationally, is the feeding cohort. in the uk selected culling of birth cohorts (years - ) and maternal cohorts have been the major methods employed (donnelly et al. ) , involving destruction of more than , animals (anonymous ) . france, portugal, and ireland have employed mainly herd culling, with some maternal culling in france, with the destruction of approximately , , , , and , cattle, respectively (anonymous, ) . additional culling methods may be employed as surveillance data accumulate . switzerland and belgium have adopted both herd and birth cohort culling, with , and , animals destroyed as of , respectively (heim and murray ; anonymous ) . culling of domestic poultry is the primary means of control for pathogenic influenza a subtypes, some considered to have pandemic potential as human viruses. millions of chickens and other poultry were killed in hong kong in an attempt to prevent the spread of influenza a subtype h n (watts a; tam ) , and in over million chickens were killed in eight southeast asian nations as the threat of a human pandemic looms (watts a; abbott and pearson, ; see the chapters by merianos and webby et al., this volume). in april , canada ordered the killing of million chickens and other poultry to contain an outbreak of influenza h n ; year earlier, the netherlands culled million chickens to control an outbreak of a related influenza subtype, h n (stegeman et al. ) . livestock culling resulting in major economic losses accompanied the outbreak of niv affecting swine and humans in malaysia in (stegeman et al. ; paton et al. ; see the chapter by merianos, this volume), where more than million swine were culled (lam and chua ; uppal ) . nipah virus has since re-emerged in malaysia, precipitating new rounds of culling (ahmad ) . export bans and culling have enormous economic impacts and emerging zoonotic viruses, such as influenza h n , niv, wnv, and sars-cov, confront the stake holders in a global economy with unprecedented new risks (james ; von overbeck ) . in the future, population reduction by immune contraceptive programs could be used among certain populations of h r s or h i s (ferro ; miller and killian ; lurz et al. ) (fig. ) . there are ethical and practical limits as to how culling is, and will be, employed, as populations of game species and other wildlife species considered ecologically and esthetically important will be off limits, even if the species serves as h r for a zoonotic pathogen. exceptions occur where species overabundance becomes a nuisance problem or threatens vulnerable environments, as with white-tailed deer ( odocoileus virgineanus ) in suburban environments or feral horses on barrier islands or federally controlled lands. in such instances, immune contraception may become the population reduction method of choice (kirkpatrick et al. ) . where critical species within a community become environmentally destructive when overabundant, as with elephants within the confines of protected game reserves, controlled culling through hunting could generate income for indigenous peoples, but plans to use immune contraception may present a more acceptable choice (fayrer-hosken et al. ; delsink et al. ) . the second major approach to zoonotic pathogen control is through vaccination of individuals in the target h r or h i populations. wildlife vaccination is currently limited to few species, although new vaccines are under development (table ) . japanese encephalitis virus (jev) transmission to humans often requires mosquito vectors which initially obtain a viremic bloodmeal from a swine h i , alternatively referred to as an amplifying host (daniels et al. ) ; vaccination of domestic swine to interrupt jev transmission has been attempted (daniels et al. ; ueba et al. ) . similarly, vaccines for chickens serving as the h r of influenza a virus subtypes are being employed to remove the intermediary avian host most closely associated with virus transmission to humans (lee et al. ; ellis et al. b) . intermediate or amplifying vertebrate h s s, once infected by contact with a h r , can directly transmit zoonotic viruses to the humans h s , as occurred with hev and niv transmission from pteropid bats initially to horses and swine (hooper et al. ; selvey et al. ; field et al. ; uppal ; see the chapters by daniels et al. and field et al., this volume) (fig. ) . however, the wildlife vaccine with the widest distribution and greatest proven effectiveness is orv for red foxes and raccoons. the orv most commonly in use for rabies control targeting wild carnivores is a recombinant vaccinia virus vaccine expressing the rabies virus glycoprotein gene (v-rg) (rupprecht et al. (rupprecht et al. ; orv was the first live-recombinant vaccine to be released in the field ). the vaccine is distributed in plastic sachets, often covered with a polymer containing additives designed to preferentially attract the target h r (linhart et al. (linhart et al. , , although nontarget species find these vaccine-laden baits attractive (olson and werner ) . millions of orv doses have been delivered to control red fox rabies in europe and raccoon rabies in the united states (aubert a (aubert , b slate et al. ); orv has eliminated or reduced red fox rabies in many countries in western europe aubert b) . in the united states, deployment of orv to reduce enzootic levels of rabies, such as gray fox-associated rabies in texas (steelman et al. ) , or to develop immune barriers to the spread of raccoon variant rabies and coyote/ dog variant rabies, in ohio, west virginia, and pennsylvania (the ohio barrier), and in texas, respectively (foroutan et al. ; farry et al. ; slate et al. ) , have established zones where herd immunity is sufficiently high that rabies virus transmission is interrupted. the ohio barrier was effective in preventing or reducing raccoon rabies cases west of the vaccination border to a sporadic few, but after - years of success, a serious breach of the ohio barrier, km west of the vaccine zone, sparked what appears to be a new epizootic focus anonymous. a) . rapid and extensive remedial vaccination was employed and will be essential to contain this new focus from rapidly expanding into a full-blown epizootic .this long-term approach to rabies control is expensive and demands sustained public commitment (kemere et al. ; foroutan et al. ; gordon et al. ) ; however, the alternative public health activities required should raccoon rabies become enzootic, are perhaps more expensive and also require sustained support (gordon et al. ) . although the risk for human exposure to vaccinia virus in orv exists, relatively few instances of human exposure have been reported (gordon et al. ) . in the united states, a case of systemic vaccinia occurred in a pregnant women after she was bitten by her pet dog while trying to remove a vaccine sachet from the dog's mouth . if ever fully developed and employed, genetic manipulation of h v populations, or endosymbionts of h v populations to establish vector refractoriness to infection by a zoonotic pathogen (scott et al. ; rasgon et al. ; olson et al. ; blair et al. ) , will theoretically disrupt the transmission chain leading to human infection (fig. ) . if refractory gene penetrance into a h v population is complete, a pathogen could suffer extinction; if partial, the effect would be a mirror image to partial vaccine coverage of humans. both strategies would reduce the probability of contact (see the chapter by real and biek, this volume) between an infectious vector and a susceptible human host, one reducing the proportion or number of infected vectors, the other decreasing the number or proportion of susceptible humans. as yet genetic engineering methods have no proven practical value in zoonotic disease control. quarantine of animals arriving into a country from foreign countries, where certain diseases are enzootic, has a long history (gensini et al. ) . for example, dogs traveling from the united states to the uk were subject to a -month quarantine as part of the uk's rabies prevention law; proof of vaccination and a positive serologic test now suffice (shaw et al. ; fooks et al. ) . national legislation can attempt to reduce within-country movement of species recognized to be h r s of zoonotic viruses. laws pertaining to translocations of rabies h r s were passed following the outbreak of a coyote/dog variant of rabies virus in florida following importation of infected coyotes from texas (centers for disease control and prevention ). the cdc imposed a ban on the importation of african rodents destined for the us pet trade after the introduction of monkeypox virus and the outbreak of human monkeypox that resulted from transmission of virus through an indigenous north american rodent h i infected by virus spillover where housed in the same building with the african rodents (centers for disease control b; see the chapter by regnery, this volume). on the same day as the cdc ban was announced, the food and drug administration initiated regulatory control of interstate transport of prairie dogs in an effort to limit further spread of monkeypox to humans and potentially other susceptible species (see the chapter by regnery, this volume). in a similar attempt to control the transmission of sars-cov, china passed laws prohibiting trade in certain carnivore species following the outbreak of sars (zhong ) . international laws pertaining to facilitating animal trade, while reducing the risk of exporting diseased animals or animal products, were established by the sanitary and phytosanitary measures, the sps agreement, coincident with establishment of the world trade organization (wto) in (zepeda et al. ) . the international standards are set by the oie (oie ) . national prohibitions have been instituted by various nations, as exemplified by bans on importing cattle or cattle products from countries where bse has been detected, listed, and updated on the usda website (http://www.aphis.usda.gov/lpa/issues/bse/trade/ bse_trade_ban_status.html), and bans to importing poultry from countries with enzootic avian influenza (hall ) , also listed on the usda website (http:// www.aphis.usda.gov/lpa/issues/ai_us/ai_trade_ban_status.html). public health professionals have lamented the years of budgetary neglect that have weakened our federal and state infrastructure for conducting surveillance (bryan et al. ) . national capacities to collect surveillance data of quality, which can inform prevention and intervention planning, are not developed over a year or even a decade. any diminishment in support for human-based surveillance activities is a poor prognostic for implementing novel activities, such as designing and implementing regional programs to study zoonotic pathogens within their wildlife h r s, as any of these efforts require the same long-term, continuous support. the united states has already lost much of its capacity to train scientists whose interests span field biology and laboratory sciences; the calls for increased training is a shrill mantra falling on deaf ears (institute of medi-cine , medi-cine , , centers for disease control and prevention ) . even the emergences of sars-cov, hiv, wnv, influenza a subtype h n , snv, and niv have generated little movement toward training, encouraging, or promoting our professional capacity to explore the intricacies by which such pathogens have evolved and are maintained within their wildlife hosts; but by in large, the national response has been a handful of ro s and a few training grants in vector-borne diseases and disease ecology. additionally, there has been little success at cross-training of public health and veterinary professionals at the doctoral level; schools of public health tend to have few veterinarians as full-time faculty members, although at the postdoctoral level programs such as the epidemiologic intelligence service (eis) at cdc recruit veterinarians with each class. as of july , a joint and coordinated effort to establish an international surveillance network for the monitoring of animals and humans for zoonotic pathogens, or diseases caused by them, has been announced by the who and faso in collaboration with the oie (http://www.who.int/mediacentre/news/ new/ /nw /en). the nature of this effort and details concerning program implementation in countries lacking adequate surveillance infrastructure have yet to be announced; any assessment of such a program designed to provide an early warning system for zoonotic pathogen emergence may be years in coming. the role of veterinary medicine and veterinary epidemiology in support of the sps agreement is severely hampered by the inequality of services available among nations (zepeda et al. ) . developing nations face an enormous challenge to develop surveillance and monitoring systems, diagnostic laboratories, and the coordinating infrastructure to assure the validity and quality of the process for any domestic animal and livestock disease, much less emerging zoonoses (zepeda et al. ) . the bias toward human-based surveillance and post-spillover treatment of infected humans is firmly institutionalized, and too often the mission-boundaries of federal agencies preclude coordinated advancement toward any integrative policy. as an example of the problems inherent to different federal agencies' ability to cross traditional boundaries to promote integration of human and veterinary epidemiology is illustrated by a report issued by cdc in morbidity and mortality weekly reports in response to the discovery of bse in cattle in the united states: "the occurrence of bse in the united states reinforces the need for physicians to be aware of the clinical features of variant creutzfeldt-jakob disease (vcjd) and to arrange for brain autopsies in all decedents with suspected or probable cjd to assess the neuropathology of these patients" (centers for disease control a). although efforts of the usda to trace the origins of the infected animal were briefly alluded to in this report, the final recommendation focusing on the human consequences of bse missed an opportunity to re-emphasize the critical component of veterinary surveillance. perhaps a report, written in collaboration with the usda, could have highlighted the means by which bse surveillance in cattle was to be enhanced. research focusing on wildlife h r s and the human-wildlife interface is most often funded through year-to-year contracts or limited grants to research institutions, which often lack the infrastructure to preserve data, specimens, and, too often, trained investigators for durations exceeding the length of a grant. in addition, if there are no programs in place to disseminate and use the information generated by disparate research efforts, the results from such studies will remain within the confines of some academic journal, rather than translated into recommendations to prevent or reduce the risk of human disease. currently, any products or recommendations stemming from such studies have little chance of diffusing into the public health culture (childs , in press) . the same problem exists with theoretical or mathematical approaches to infectious disease epidemiology. once mathematical models are developed and validated by use of existing data sets (russell et al. coyne et al. ; childs et al. ) , the route to integrating insights gleaned from mathematical approaches into public health practice or specific control activities is unclear. mathematical modeling as an aid to assist policy decisions has come under severe criticism from practicing veterinary professionals operating on the front lines of disease control. the disparate interpretations of the success of mathematical models in forming an effective control policy for an animal-disease disease outbreak are clearly illustrated by postcrisis reviews of the foot-andmouth-disease (fmd) outbreak in the uk in . proponents and authors of models saw the utility and predictions of models validated (woolhouse ) , while some veterinary practitioners and epidemiologists saw little to no benefit in the models as applied in a real-time crisis (salman ) . the serious and widening gulf between mathematical modeling and public health practice requires a systematic and purposeful effort on both sides to bridge these differences (childs , in press ). if communications fail, the danger exists for one class of professional to dismiss the efforts of the other as either irrelevant or hopelessly unsophisticated. whose problem is it? national and international long-range translocations of infected animals have played an extensive role in the emergence of viral zoonoses. the phenomenon is so common that it must be considered in conjunction with any control strategy based on legal restrictions to animal movement, bans to trade in wildlife, or when constructing vaccination barriers to limit pathogen spread. instances of transcontinental zoonotic viral spread reinforce the significance of ldts and the recommendation that contingencies for their occurrence should be included in any strategic plan for zoonotic disease control. in , sars spread around the world in a matter of months, eventually affecting countries on every populated continent (heymann ) . in , monkeypox was introduced into the united states along with a shipment of african rodents destined for the pet trade (cunha ; centers for disease control and prevention a; see the chapter by regnery, this volume). in , wnv was recorded in the new world for the first time, introduced into new york city by an infected vector or human host (lanciotti et al. ; kilpatrick et al. ) . in , singapore experienced outbreaks of niv infection among abattoir workers after importing swine from malaysia (chew et al. ; see the chapter by field et al., this volume) . the impact of a within-country ldt is well illustrated by the spread of raccoon rabies from a focus identified in the late s along the virginia-west virginia border, a focus likely seeded by the translocation of raccoons incubating rabies from an enzootic region of raccoon-associated rabies virus in the southeastern united states (nettles et al. ) . the resulting rabies epizootic, as the disease spread into mid-atlantic and northeastern states, was one of the most extensive and intensive wildlife epizootics recorded (childs et al. ; . a rabid bat stowaway onboard a ship originating from the west coast of the united states was discovered in hawaii, which is a rabies-free state (centers for disease control and prevention ); other instances of ldts of rabid bats, some transcontinental, have been reviewed (constantine ) . at a finer scale, quantitatively defined instances of raccoon rabies epizootic foci developing in advance of the epizootic wavefront in connecticut indicate local translocations influenced the spatial pattern of raccoon rabies spread through that state . the instance of a rabies virus variant of coyotes/domestic dogs from texas being introduced into florida with transported coyotes was described previously (centers for disease control and prevention ). without adequate compensation for losses accrued through culling or exportation bans, countries attempting to implement animal-based surveillance programs for domestic species, much less wildlife, are likely to encounter problems with voluntary reporting (see the chapter by merianos, this volume). in some instances, the mere threat of culling, as with swine in areas of malaysia affected by niv, can promote epidemic spread as farmers disperse valuable animals to protect their livelihood (chua ; see the chapter by field et al., this volume) . in addition to the enormous economic losses facing individuals whose animals are killed or whose products cannot be sold, the consequences of reporting an outbreak of a new zoonotic disease can be politically unattractive, inviting delays in reporting, as may have occurred with sars in china (enserink ) . other hidden costs associated with zoonotic disease outbreaks may persist through the burden of surveillance and animal testing (bradley and liberski ) and the loss to veterinary services (bennett and hallam ) . before implementation of any control activity, such as culling or vaccination, it is essential that the target species has been accurately and irrefutably identified as the h r or h i of importance. identification of a h r requires establishing epidemiologic plausibility using definable criteria, such as the temporal and spatial association of putative h r s to pathogen spillover, and molecular epidemiologic data linking virus recovered from a h s to virus circulating among h r s (haydon et al. ; childs ) . china initiated culling of some species of carnivores and other wildlife intended for human consumption (watts b) , although no sars-cov has yet been isolated from wild civets obtained directly from the field (bell et al. ; guan et al. ) . in , a putative h r for coronaviruses ancestral to those isolated and characterized from humans and palm civets was identified among three species of bats of the genus rhinolophus ; see the chapter by wang and eaton, this volume). molecular sequencing of sars-cov from bats, palm civets, and humans indicates a common ancestor with rapid positive selection for virulent viral subtypes infecting humans and civets (song et al. ; see the chapter by wang and eaton, this volume). removing carnivores near the top of ecological food chains can have many unforeseen, and in certain circumstances, potentially disastrous, consequences. by diluting, or severing important links in community processes, culling of top-level carnivores can cause changes in species richness and diversity in communities and increases in prey populations (ostfeld and holt ; ostfeld and keesing ) , including wild rodent h r s of other potentially dangerous zoonotic agents, such as borrelia burgdorferi and the arenaviruses and hantaviruses (logiudice et al. ; mills and childs ) . use of methods designed to control one species, such as anticoagulants topically applied to cattle to reduce vampire bat populations, can reduce populations of ecologically important species of bats unintentionally dosing themselves when roosting with vampire bats in confined spaces (mayen ; martinez-burnes et al. ) . contrast the purposeful and highly successful surveillance for animal rabies with activities targeting other known or potential pandemic zoonotic threats with wildlife h r s. subtypes of hiv i and hiv ii have emerged independently from primate sivs on at least eight independent occasions (hahn et al. ; b. hahn, personal communication to jec) . the number of sivs described among nonhuman primates in africa, as of , was approximately (apetrei et al. ). rapid replication, high mutability, and the elevated rates of recombination of lentiviruses (zhuang et al. ; see the chapter by holmes and drummond, this volume) virtually assures that new strains of siv-hiv will make the journey out of africa. there appears to be little systematic effort to enhance or build the basic infrastructure in regions of west africa that could begin to conduct surveillance for new emerging hivs at the human level or monitor the dynamics of transmission of diverse and genetically chimerical sivs transmitted among nonhuman primates. detection of spumaviruses among hunters, although uncommon (~ %), signify the extent to which humans are exposed and infected with diverse primate retroviruses (wolfe et al. ) . although some of the countries of importance are war zones and politically unstable, it is unclear that given an improving situation, surveillance for sivs spilling over to humans would be regarded as a priority among funding institutions concentrating on hiv vaccine trials. how are we surveilling and preparing for the next pandemic of influenza? currently influenza a subtype h n has a limited capacity for cross-vertebrate class transmission from birds to mammals, although infection is frequently fatal to humans once spillover succeeds sturm-ramirez et al. ; claas ; tran et al. ; see the chapter by webby et al., this volume). monitoring avian h n subtypes has been, and continues to be spotty, and largely limited to domestic poultry in which infection is often fatal to chickens and to a lesser extent ducks ). recombination of waterfowl influenza viruses within a domestic duck h r may have been the origin of highly pathogenic subtypes of h n for chickens (chen et al. b; tumpey et al. ; guan et al. guan et al. , , and successive isolates of h n from domestic ducks over time indicate increasing virulence for mammals (chen et al. b; guan et al. a guan et al. , b . domestic geese may serve a role as an independent h r for recombinant wild waterfowl-goose influenza h n subtypes and help drive the rapid evolution of highly pathogenic viruses of ducks and chickens chen et al. b ). yet the ultimate origin of h n and other influenza subtypes, h n and h n , occurring among domestic poultry and representing human threats choi et al. ) , is the diverse species of waterfowl, shorebirds, and possibly other avian types in which these various influenza subtypes circulate, often with minimal morbidity (see the chapter by webby et al., this volume). surveillance for influenza subtypes among wild waterfowl and other migratory birds is spotty (krauss et al. ; campitelli et al. ; de marco et al. ; hatchette et al. ) and largely restricted to local or regional populations, as occurs in north america and italy (krauss et al. ; slemons et al. ; hatchette et al. ; campitelli et al. ; de marco et al. ; ellis et al. a; stallknecht et al. ). the who has proposed establishing an animal influenza network to develop and coordinate research on the ecology and molecular biology of animal influenza viruses and integrate these animal-based activities with the global surveillance program for human influenza (stohr ) ; presumably emphasis will be placed on wild waterfowl and other migratory birds, in addition to domestic poultry and livestock. the uneven standards of surveillance, human-or animal-based, for zoonotic diseases or pathogens maintained by wildlife h r s, or even domestic species (zepeda et al. ) , is a global problem, readily apparent even within the united states, where investment in public health, including surveillance systems, has a long and enviable history (thacker ) . as of , there appears to be little scientific, social, or political consensus that animal-based surveillance for zoonoses merits investment in international infrastructure. however, this trend may be changing with the recent announcement of the proposal to develop a global early warning system for certain zoonotic agents or disease to be coordinated by the who, fao, and oie. technologically advanced solutions to addressing vector-borne or zoonotic disease transmission, such as genetic manipulation of mosquitoes or immunocontraception aimed at target vertebrate hosts, may involve good science, but whether these approaches represent good public health is highly debatable (scott et al. ; furguson et al. ) . novel schemes of preventing spillover of human pathogens from animal h r s can only spring from improving our understanding of the ecological context and biological interactions of pathogen maintenance among h r s. there are no easy solutions to preventing spillover and there is no reason to expect we will ever predict the wheres and whys of new emergences of zoonotic diseases (see the chapters by cleaveland et al. and daszak et al., this volume) . inevitably, the major issue arises of where surveillance and research efforts should focus, and there are many areas worthy of consideration. where the intent exists to improve global surveillance for specific zoonoses of animals, such as influenza a, every possible effort should be made to bring in new ideas and to set a standard of excellence that will encourage additional forays into these areas. as a speculative example, the ability to genetically modify plants to produce viral antigens of potential vaccine quality (castle and dalgleish ) may provide a tool to reach wild waterfowl that gather in vast numbers in specific staging areas during migration. could influenza a subtype h n genes be introduced into corn (tacket et al. ; lamphear et al. ), a favorite food of virtually all waterfowl and poultry, and would such a vaccine immunize sufficient numbers of waterfowl to reduce the susceptible population if widely dispersed among migratory staging areas? would there be a payoff from large investments to improve surveillance and knowledge of known or potential zoonotic pathogens circulating among wildlife h r populations? no one knows, but the alternative is to continue to rely on disease detection among sentinel humans. our ongoing experience with hiv, the looming threat 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hot spots key: cord- -d ll ka authors: alotaibi, badriah m.; yezli, saber; bin saeed, abdul-aziz a.; turkestani, abdulhafeez; alawam, amnah h.; bieh, kingsley l. title: strengthening health security at the hajj mass gatherings: characteristics of the infectious diseases surveillance systems operational during the hajj date: - - journal: j travel med doi: . /jtm/taw sha: doc_id: cord_uid: d ll ka background: hajj is one of the largest and the most ethnically and culturally diverse mass gatherings worldwide. the use of appropriate surveillance systems ensures timely information management for effective planning and response to infectious diseases threats during the pilgrimage. the literature describes infectious diseases prevention and control strategies for hajj but with limited information on the operations and characteristics of the existing hajj infectious diseases surveillance systems. method: we reviewed documents, including guidelines and reports from the saudi ministry of health’s database, to describe the characteristics of the infectious diseases surveillance systems that were operational during the hajj, highlighting best practices and gaps and proposing strategies for strengthening and improvement. using pubmed and embase online search engines and a combination of search terms including, ‘mass gatherings’ ‘olympics’ ‘surveillance’ ‘hajj’ ‘health security’, we explored the existing literature and highlighted some lessons learnt from other international mass gatherings. results: a regular indicator-based infectious disease surveillance system generates routine reports from health facilities within the kingdom to the regional and central public health directorates all year round. during hajj, enhanced indicator-based notifiable diseases surveillance systems complement the existing surveillance tool to ensure timely reporting of event information for appropriate action by public health officials. conclusion: there is need to integrate the existing hajj surveillance data management systems and to implement syndromic surveillance as an early warning system for infectious disease control during hajj. international engagement is important to strengthen hajj infectious diseases surveillance and to prevent disease transmission and globalization of infectious agents which could undermine global health security. the number of pilgrims participating in the hajj religious mass gathering has increased significantly over the years, with million pilgrims attending annually in the last years compared with in . , unhygienic practices and close contacts between pilgrims in overcrowded situations during the hajj rituals, as well as international travel, increase the risks of outbreaks and the spread of infectious diseases among pilgrims. the risk of infectious diseases transmission may extend to the local saudi population and to the home population of returning pilgrims after hajj. , this could strain the public health services in saudi arabia and may threaten global health security. historically, several outbreaks of infectious diseases have been reported at the hajj. these include an outbreak of cholera during the hajj that caused an estimated deaths among pilgrims and a number of international hajj-related outbreaks of meningococcal diseases in , and . the introduction of a number of pragmatic public health preparedness strategies for hajj, including vaccination and chemoprophylaxis and improved food safety and waste management, ensured that no outbreaks of cholera and meningococcal meningitis occurred during the event in recent years. however, both diseases remain a priority for public health control as do other infectious disease with global significance such as tuberculosis and zika virus disease. , in addition, new and emerging corona and influenza viruses, such as influenza h n virus, severe acute respiratory syndrome coronavirus and the middle east respiratory syndrome coronavirus (mers-cov) remain an ever present threat to mass gatherings such as hajj. as yet, no confirmed cases of mers-cov were reported during hajj. however, given the current outbreak of the disease in the kingdom, mers-cov continues to be a major risk during the event. effective health information management and dissemination allow the formulation of appropriate strategies to prevent and/ or control outbreaks and the international spread of diseases. the use of appropriate surveillance systems during mass gatherings ensures the timely collection, analysis and interpretation of health data for effective planning and response to infectious diseases threats. additionally, public health surveillance systems play a substantial role in providing reassurance of the absence of a deleterious public health event to mass gathering organizers and political office holders during an international mass gathering. in the context of hajj and saudi arabia, mers-cov is a case in point. thus, an effective infectious diseases surveillance system (idss) during hajj should be highly sensitive to detect infectious diseases events in a timely manner and to minimize the threats to the safety and well-being of pilgrims and their contacts after the mass gathering. in practice, several idsss are operational during hajj. a regular idss is applicable kingdom-wide and generates routine reports from the health facilities to the regional and central public health directorates of the ministry of health (moh) all year round. during hajj, this system is complemented by enhanced idsss to ensure timely reporting of event information for appropriate action by public health officials. however, there is little documentation of the components and operations of each system, their advantages and disadvantages as well as their efficiency in terms of timeliness of alerts and channels of reporting. here, we describe the characteristics of the idsss that were operational during the hajj, highlighting best practices and gaps and propose strategies for strengthening and improvement. the main hajj rituals take place on day - th of dhu-al hijjah (hajj month in the islamic calendar). the saudi moh collaborates with other organizations, such as the municipality and the ministry of hajj to ensure food and water safety, vector control, waste management and to provide other public health services during hajj. to that end, the moh conducted various risks assessments in preparation for the hajj, including an international health regulations (ihrs) public health core capacity assessment at the points of entry and disseminated the updated pre-travel advice and health requirements for pilgrims and workers involved in the hajj. the latter includes details of the vaccination requirement for meningococcal meningitis, yellow fever, seasonal influenza and polio. in line with ihr , pilgrims arriving from certain countries in africa and south and central america were required to present a valid yellow fever vaccination certificate on arrival. all pilgrims were required to present valid certificate of vaccination with quadrivalent (acyw ) meningococcal vaccine, and those arriving from countries in the african meningitis belt, were given mg of oral ciprofloxacin as chemoprophylaxis to lower meningococcal carriage rate among these pilgrims. oral ingestion of ciprofloxacin was directly observed by healthcare workers to ensure adherence. all pilgrims travelling from polio risk countries received dose of oral polio vaccine at borders points on arrival in saudi arabia regardless of age and vaccination status. the moh also recommended the administration of seasonal influenza vaccine to all pilgrims before arrival for hajj, particularly pilgrims at risk of developing severe complications of seasonal influenza, including pregnant women, elderly individuals, children aged months to years, pilgrims with coexisting medical conditions and healthcare workers. the command and control centre (ccc) is a special moh unit created in the aftermath of the mers-cov outbreak in to coordinate an appropriate response to infectious diseases outbreaks in saudi arabia. as the crisis management arm of the moh, the ccc coordinated the outbreak response plans of the moh during the hajj, establishing clearly defined interfaces between various moh departments and international organizations to ensure appropriate and timely response to outbreaks of infectious diseases. the ccc created three situation rooms at key locations in hajj sites; the health directorate of makkah region, almahbat mina and the mina emergency hospital. these sites were selected because pilgrims spend most of their time in makkah and mina, performing hajj rituals which potentially impacts on their safety and well-being. overall, pilgrims, including saudis and non-saudis from countries participated in the hajj. within makkah city and the holy areas, the saudi government provided free healthcare services through primary healthcare centres and hospitals including, seasonal health facilities only operational during hajj. the hospitals had a combined bed capacity of beds. this indicator-based idss is implemented country-wide in saudi arabia for routine facility-based notification of infectious diseases events all year round, including during hajj. each regional public health directorate is made up of - administrative sub-units known as 'health sectors'. these sub-units receive and review infectious disease surveillance data from the health facilities within specified geographical areas in the region for reporting to the regional public health directorates. the surveillance teams at the regional-level collate data pooled from the health sectors on disease-specific excel sheets for monthly reporting to the central directorate of public health at the moh headquarters. despite limited data management capabilities, this surveillance system was proven effective in detecting and triggering timely responses to outbreaks of measles and scabies in the kingdom in / and , respectively. enhanced idsss are activated during the hajj season to ensure early detection and prompt response to infectious diseases outbreaks. for the hajj, the enhanced surveillance became operational from the first dhul-qa'dah (islamic calendar month preceding hajj) with the arrival of the first batch of pilgrims, and continued until the end of moharam (first month of the islamic year following hajj) after the departure of the last group of pilgrims. this system is active at three main points: key points of entry to the kingdom, healthcare facilities in the hajj areas and medical office for pilgrims (formerly known as medical missions). each of these is described later. effective surveillance at the points of entry is required to prevent and control the international spread of diseases during mass gatherings, including the importation of infectious agents to the host country. during the hajj, public health surveillance teams trained to detect and report public health threats and to monitor the compliance of arriving pilgrims with the health requirements for the hajj were deployed at the kingdom's hajj entry points. these teams consisted of personnel at jeddah airport, at jeddah seaport and at medina airport. approximately % of international pilgrims arrive through these three points of entry. at each point of entry, the surveillance teams reviewed the vaccination status of arriving pilgrims by checking their vaccination cards, reported any cases of unvaccinated pilgrims or those with unverifiable vaccination status and recommended appropriate actions for these cases. the surveillance teams were also responsible for identifying and managing ill pilgrims, as well as the notification and transfer of suspected cases of infectious diseases. hospital-based surveillance teams were operating in each hospital within the hajj areas in . these were hospital staff trained to rapidly detect and report manually and electronically cases of infectious diseases presenting to the hospitals. suspected cases of infectious diseases identified at primary health centres were referred to pre-specified hospitals for confirmation of diagnosis, further management and notification to the ccc. the hospital teams were reinforced by fixed and mobile surveillance teams from the regional directorates to ensure -h active surveillance during the hajj. whereas each fixed team consisted of medical doctor and health inspector, each mobile team was made up of medical doctors (a male and a female), health inspector and driver. these personnel were drawn from different regional health directorates, across the country and mobilized for a -day refresher course, accredited by the saudi commission for health specialties. among other relevant topics, the refresher course was focused on discussing the current trends in the management and control of infectious diseases as well as the reporting formats and tools for infectious diseases surveillance during hajj. the fixed surveillance teams were assigned to each hospital operating in the hajj areas and reviewed admission logbooks for cases with clinical features of infectious diseases and followed up on cases admitted into the wards to identify and report suspected cases of notifiable diseases to ensure no cases were missed by the hospital surveillance teams. the mobile surveillance teams were tasked to conduct field investigations for reported cases of infectious diseases. these included active case finding and safe transfer of suspected cases to designated facilities, contact tracing, risk communication and liaison with medical office for pilgrims (see later) to facilitate case reporting and effective follow-up of contacts of cases. additionally, supervisory units composed of epidemiologists and infectious diseases specialists were established by the regional directorates to monitor the activities of the mobile and fixed surveillance teams and to serve as an intermediary between these surveillance teams and the regional directorates ( figure ). the supervisory units were also responsible for the isolation of suspected cases, follow-up of laboratory investigations and clinical status of hospitalized cases, as well as monitoring to ensure the implementation of the appropriate infection prevention and control procedures for patients, healthcare personnel and visitors. although the surveillance teams investigated all suspected infectious diseases cases, the following diseases were listed as high priority during hajj, and clear guidelines were provided for reporting suspected cases of these disease: mers-cov, ebola virus disease (evd), cholera, meningococcal meningitis, yellow fever, polio, rift valley fever, crimean fever, dengue fever, malaria, influenza and food poisoning. of all suspected cases detected by the idsss in the hajj, cases of malaria, cases of influenza h n , cases of food poisoning, cases of dengue and cases of non-meningococcal meningitis were confirmed after investigations. there were no confirmed cases of mers-cov illness, evd or cholera during the hajj season. hajj medical office for pilgrims refers to the healthcare representatives of some countries which send pilgrims for the hajj and accompany pilgrims during the event. they may set up clinics or hospitals within the hajj areas and provide healthcare services for their own pilgrims in compliance with the saudi moh rules and regulations. the composition of the medical office for pilgrims varies from country to country; however, it is recommended that a minimum of % of their staff should have a public health background. a memorandum of understanding is established between the saudi authorities and the medical office for pilgrims for effective coordination and communication of the standard public health requirements for the hajj. the medical offices are required to comply with the standard sanitary requirements for food preparation and handling, to educate pilgrims on personal hygiene and proper waste disposal and to submit a valid contract with an accredited firm for medical waste management to the regional directorates. additionally, they are required to provide daily reports on notifiable diseases, to establish isolation areas for suspected cases and to coordinate with the public health supervisory teams for the safe transfer of these cases when necessary. of the country medical offices operational during the hajj only ( %) were found to be compliant with the standard requirements of the saudi moh. the inconsistency of some medical representatives, with regards to compliance with the requirements of the moh and the frequent change of medical teams by countries sending pilgrims, often soon after the hajj limits sustainable partnership between the moh and the medical offices for pilgrims. locations. , overall, two electronic surveillance systems were operational during the hajj: the health electronic surveillance network (hesn) and the electronic statistical system for hajj referred to as citrex. hesn is a web-based electronic solution, introduced by the saudi moh to improve communication among public health professionals involved in outbreak management as well as to provide quality health data for planning and effective allocation of resources. hesn was initially implemented as a pilot in makkah region of saudi arabia in . by january , a country-wide implementation was initiated to control the outbreak of mers-cov in the kingdom. during the hajj, in addition to the traditional data capture and reporting tools, the hospital surveillance teams also collated and entered infectious diseases data directly into hesn once a notification was received from the laboratory, emergency rooms, isolation wards and other departments in hospitals. the uploaded data were immediately displayed on electronic dash boards in the ccc's situation rooms. data were analysed and reports generated in real-time that could be immediately accessed by public health officials and decision makers or disseminated through phone messages to responsible persons for immediate action. citrex is a web-based electronic solution that predates hesn and was used in the preceding hajj seasons. in hajj, this system was operational alongside hesn. unlike hesn which is implemented country-wide, citrex is used only during hajj to manage infectious diseases data captured inreal time from the health facilities in the holy areas (makkah, medina, arafat and mina). although the hospital surveillance teams handled data entry into hesn, the fixed surveillance teams captured the same health data into citrex for analysis and notification on distinct electronic dashboards at the ccc. over the years, the saudi government has allocated substantial resources to protecting public health during the hajj. this contributed to the development of modern surveillance systems for the hajj, evolving from the paper-based reporting tools to a more efficient web-based electronic surveillance systems. enhanced idsss were introduced to complement the conventional surveillance system in addressing the increased risks of infectious diseases transmission and outbreaks during the hajj. existing electronic surveillance systems (hesn and citrex) automatically generates reports and have the advantage of timeliness, as public health personnel at different locations can access and synchronize information management once data is captured at the reporting sites. however, the implication of having parallel systems capturing and interpreting the same health data has some potential implications, including duplication of work, depletion of already limited resources during hajj and uncertainty of the accuracy of the data. therefore, there is a need to conduct operational studies to assess the feasibility of integrating the diverse surveillance systems utilized during hajj into one efficient tool. prioritizing systems that remain operational for routine surveillance after hajj may promote the most efficient use of resources. furthermore, there is need to sustain the enhanced surveillance system and other public health interventions at key locations in the kingdom, including the points of entry, after the hajj, as a prevention and control strategy for the international spread of diseases during other mass gatherings with international dimensions, principally the umrah pilgrimage. it is estimated that over million pilgrims arrive to the kingdom yearly to participate in the umrah, which occurs nearly all-year round. syndromic surveillance could complement the existing notifiable disease surveillance systems, as an early warning system for public health threats during the hajj and umra mass gatherings. various risk assessments have shown that case-based notification systems do not meet the surveillance requirements for international mass gatherings, in terms of timeliness and coverage of possible risks groups. , quite often, time-consuming laboratory processes required for making diagnosis may stall the disease notification process, and hence prolong the time for initiating an intervention to a potential threat. symptomatic pilgrims who prefer 'quick-to-access' pharmacies may not present to the health facilities, eluding the current notifiable disease surveillance systems operational during hajj. syndromic surveillance uses aggregated data of symptom groups from a wide range of sources that precedes clinical diagnosis to set thresholds for responding to a threat. this kind of surveillance is also useful for dispelling or confirming rumours of outbreaks, based on changes in the reported number of aggregated cases in an area. the potential benefits of syndromic surveillance during mass gatherings were reported during previous olympic games. for example, the syndromic surveillance system implemented during the beijing olympic games improved the detection and response time to potential outbreaks during the games. additionally, the daily syndromic surveillance data captured during the london olympics and paralympic games reassured public health officials and political office holders of the absence of outbreaks, which substantially impacted on planning, and boosted the legacy of the event. thus, the moh through the global centre for mass gatherings medicine (gcmgm) and the saudi field epidemiology training programme is setting up a syndromic surveillance system for the hajj and umra mass gatherings to complement the enhanced idss, as an early warning system for public health threats. this system may become operational during the hajj. ensuring the health and safety, security and well-being of pilgrims are top priorities for the kingdom. achieving this is a collective responsibility that needs to be shared by saudi arabia and each country that sends pilgrims to the hajj. this is because the hajj experience is not limited to the few days pilgrims spend performing the hajj rituals. rather, it starts well before they arrive to the kingdom and lasts long after they have returned to their home countries. prevention of importation and exportation of infectious agents in hajj is key for global health security and effective infectious disease surveillance both in the kingdom during hajj, as well as in the countries of origin of pilgrims, is crucial in achieving this. therefore, it is apparent that there is a great need for the development of a well-structured, harmonized and effective collaboration, data collection and information sharing network involving the saudi health authorities and representatives from all countries sending pilgrims to the hajj. such a network would be crucial in strengthening infectious disease surveillance, preventing illnesses and responding to outbreaks during hajj, minimizing disease transmission as well as strengthening global health security through adherence to the ihrs, including notifiable diseases reporting to the who. for these reasons, the gcmgm in collaboration with the who intends to create this global network by the appointment of a hajj and umra focal point in each country which sends pilgrims to saudi arabia for these mass gatherings. considering the potential diplomatic and practical hurdles that may stall the implementation of such an international system, both organizations aim to prioritize countries sending the largest number of pilgrims to the hajj, and to retrain existing who ihr country focal point or who country office staff to function as focal point and to coordinate the activities of the network in each country. these focal points shall engage in public health preparedness activities such as dissemination of health education messages, monitoring pilgrims' health status and compliance with the hajj and umra health requirements, as well as routine surveillance for public health emergencies of international concern. additionally, they shall develop and maintain a database on pilgrims' demographics and health information as well as on public health threats including disease outbreaks in pilgrims' home countries. this database will allow the focal point to generate periodic and on-request reports on infectious disease to the who, the gcmgm or the local health authorities as required, facilitating the monitoring of disease patterns and trends globally and strengthening the kingdom's public health hajj preparedness and response capabilities. during the recently concluded hajj, the ministry of hajj introduced an electronic wrist bracelet, which pilgrims were urged to wear at all times during the pilgrimage. the bracelet captured salient demographic information for each pilgrim, including age and nationality, and was global positioning system (gps) enabled to track pilgrims' location and inform crowd control and risk communication priorities during the hajj. the moh is already exploring ways of incorporating vital health information, such as comorbidity, blood type and known allergies, in the electronic device to provide relevant data for health planning and improved health services delivery during future hajj. the saudi authorities have invested significant resources in developing model idsss for the hajj to ensure the safety and wellbeing of pilgrims, the saudi population and the population of countries sending pilgrims for the hajj. existing surveillance systems operating during hajj would be complemented by syndromic surveillance systems to ensure timely response to potential public health threats. since the hajj experience is not limited to the short time pilgrims spend performing the hajj in saudi arabia, there is a need for sustainable international collaborations between the saudi authorities, countries which sends pilgrims for the hajj and international organizations to strengthen infectious diseases surveillance and to prevent disease transmission and globalization of infectious agents which could undermine global health security. conflict of interest: none declared. the pilgrimage and its implications in a regional 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pilgrimage to mecca (hajj) manual of notification of infectious diseases world health organization. public health for mass gatherings: key considerations. geneva; who ministry of health. documentation of the hajj infectious diseases surveillance capacity meeting of heads of medical missions on public health preparedness for hajj enhanced surveillance of infectious diseases: the fifa world cup experience ministry of health. all-hazard health risk assessment for ministry of health world health organization. the health legacy of the beijing olympic games: success and recommendations. manila: who regional office for the western pacific key: cord- -rlvk bcx authors: balloux, francois; van dorp, lucy title: q&a: what are pathogens, and what have they done to and for us? date: - - journal: bmc biol doi: . /s - - -z sha: doc_id: cord_uid: rlvk bcx microbes are found on us, within us and around us. they inhabit virtually every environment on the planet and the bacteria carried by an average human, mostly in their gut, outnumber human cells. the vast majority of microbes are harmless to us, and many play essential roles in plant, animal and human health. others, however, are either obligate or facultative pathogens exerting a spectrum of deleterious effects on their hosts. infectious diseases have historically represented the most common cause of death in humans until recently, exceeding by far the toll taken by wars or famines. from the dawn of humanity and throughout history, infectious diseases have shaped human evolution, demography, migrations and history. a pathogen is defined as an organism causing disease to its host, with the severity of the disease symptoms referred to as virulence. pathogens are taxonomically widely diverse and comprise viruses and bacteria as well as unicellular and multicellular eukaryotes. every living organism is affected by pathogens, including bacteria, which are targeted by specialized viruses called phages. the number of viruses and bacteria on earth is staggering and they occupy essentially every environment. a liter of surface seawater typically contains in excess of ten billion bacteria and billion viruses. the number of viruses on earth is estimated to be around , which corresponds to roughly ten billion times the number of stars in the universe [ ] . an average human is made up of about trillion cells but carries a similar number of bacteria, mostly in the gut [ ] . the vast majority of viruses and bacteria we are exposed to have no negative effect and some can even * correspondence: f.balloux@ucl.ac.uk ucl genetics institute (ugi), darwin building, gower street, london wc e bt, uk be beneficial, though a tiny fraction of these can severely affect our health. specifically, about one in a billion microbial species is a human pathogen. indeed, approximately human pathogens have been described, whereas it has been estimated that there are one trillion microbial species on earth, the vast majority of which remain uncharacterized [ ] . pathogens can be divided into two main categories, namely facultative and obligate pathogens, reflecting how intimately their life cycle is tied to their host. facultative pathogens are organisms for which the host is only one of the niches they can exploit to reproduce. facultative pathogens are primarily environmental bacteria and fungi that can occasionally cause infection. they include many of the most problematic hospital-acquired bacteria involved in the antimicrobial resistance pandemic. a distinction is sometimes made between facultative and accidental pathogens, with the latter representing those which only occasionally infect weakened or immunocompromised hosts. typical examples of 'accidental' pathogens include neisseria meningitidis or escherichia coli. obligate pathogens require a host to fulfil their life cycle. all viruses are obligate pathogens as they are dependent on the cellular machinery of their host for their reproduction. obligate pathogens are found among bacteria, including the agents of tuberculosis and syphilis, as well as protozoans (such as those causing malaria) and macroparasites. some obligate pathogens require multiple different hosts to fulfil their life cycle. the definite host, which supports the adult form of the pathogen, is often a vertebrate and the intermediate host (referred to as a vector) is generally an arthropod or a mollusc. this alternation of vertebrate and invertebrate hosts is found in viruses (for example the zika virus), bacteria (for example lyme disease) and protozoa (malaria). trematodes (parasitic flatworms) go even further and some exhibit among the most baroque life cycles. digenetic trematodes have a basic three-host life cycle, and for some species a fourhost life cycle. for instance, halipegus occidualis sequentially has to infect a freshwater snail, an ostracod, a dragonfly nymph and ends its cycle after the dragonfly is eaten by the green frog rana clamitans, where it resides under its tongue [ ] . what is the host range of pathogens? some pathogens are limited to infecting a single host species, whereas others can infect a multitude of host species. host ranges can feel highly idiosyncratic if not outright puzzling. for example, leprosy in humans is caused by two related intracellular bacteria mycobacterium leprae and mycobacterium lepromatosis, which are essentially restricted in the wild to humans, as well as armadillos in the americas and red squirrels in scotland [ ] . conversely, yersinia pestis, another intracellular obligate bacterium and the agent of plague, has a natural life cycle involving alternating infections of rodents and fleas, but can infect essentially any mammalian host. an interesting twist in the case of plague is that y. pestis is not well adapted to the human host. with the exception of uncommon occurrences of human-to-human transmissions, referred to as pneumonic plague, plague epidemics (bubonic plague) are caused by plagueinfected fleas biting humans. somewhat ironically for a pathogen that is possibly the biggest killer in human history, bubonic plague is a complete evolutionary disaster. the human host is at a very high risk of dying, the flea cannot reproduce on a meal of human blood and the bacterium is stuck in an evolutionary dead-end as it cannot transmit to another host. there is no obvious predictor for the host range of different pathogens. intuitively, it may be tempting to predict that pathogens with a more intimate relationship with their host are more closely adapted to their host, and thus have a more restricted host range. however, there is no obvious pattern suggesting that viruses (that rely on the host cells' machinery for reproduction) have a narrower host range than bacteria. also, intracellular bacteria do not seem to have a markedly narrower host range than extracellular ones, despite being more intimately tied to their host. we know relatively little about the underlying genetic changes required for a pathogen to infect a new host, though, interestingly, only a few mutations can be required for a host jump. for example, avian influenza is only around five mutations away from being able to transmit in mammals [ ] , and a single amino acid change was sufficient for the humanadapted bacterium staphylococcus aureus to become a pathogen of rabbits [ ] . obligate pathogens tend to be highly adapted to their hosts, with sophisticated mechanisms to synchronise their life cycles with that of the host, and the ability to manipulate the host's immune system, metabolism and sometimes even behaviour. genes encoding proteins specific to pathogenicity are referred to as virulence factors, which include a variety of molecules required for colonization of the host, immunoevasion and immunosuppression, scavenging nutrients within the host, and entry into and exit out of cells for intracellular pathogens. in bacteria, virulence factors are often found in groups of genes on pathogenicity islands, which can be transferred horizontally by plasmids or other transposable elements. for example, one of the defining features of the plague bacterium y. pestis from its less virulent closest relative yersinia pseudotuberculosis, is the inclusion, early in its evolution, of two plasmids carrying genes involved in pathogenicity [ ] . while acquisition of novel genes and repurposing of existing ones is essential in the evolution towards pathogenicity, a general feature during the evolution towards pathogenicity is genome reduction through the inactivation and loss of genes. this can be primarily explained by the fact that a host represents a fairly stable and resource-rich environment where some metabolic pathways required in the environment are not necessary. genome reduction is a general trend accompanying the evolution towards pathogenicity and is observed in mycobacterium tuberculosis, pathogenic e. coli strains and in the ongoing adaptation of klebsiella pneumoniae lineages to cystic fibrosis patients. the most extreme example is leprosy (m. leprae and m. lepromatosis), which has shed nearly half the genes found in their environmental relatives [ ] . another interesting tendency of many bacterial pathogens is the secondary loss of the ability to undergo genetic recombination [ ] . pathogens cause illness to their hosts through a variety of ways. the most obvious means is through direct damage of tissues or cells during replication, generally through the production of toxins, which allows the pathogen to reach new tissues or exit the cells inside which it replicated. bacterial toxins are among the deadliest poisons known and include famous examples such as tetanus, anthrax or botulinum toxin, known as botox in its commercial application. however, the damage to the host is often self-inflicted through a strong or sometimes excessive immune response that indiscriminately kills infected and uninfected cells and damages host tissues. typical examples of maladaptive over-reaction of the immune system include cirrhosis and liver cancer in hepatitis b [ ] , or the - influenza epidemic, where the toll was highest amongst the young and healthy possibly because they mounted the strongest immune response and as such died from a 'cytokine storm' in the lungs leaving patients literally drowning in their own body fluids [ ] . some pathogens benefit from the hosts' immune reaction to spread within an infected host or increase their transmission to uninfected hosts. influenza transmits mainly through aerosols created through the sneezing and coughing it causes. vibrio cholerae triggers a strong inflammatory response in the gut mucosa, leading to watery diarrhoea and ensuring its release in the environment and thus infection of further hosts. pathogens greatly vary in the severity of their symptoms from a mild inconvenience to assured death. it is sometimes assumed that the deadliest pathogens represent recent host jumps where the pathogens' virulence is maladapted to the new host, and that co-evolution between host and pathogen will lead to more benign symptoms over time. however, this is only true in the case of strict vertical transmission (such as from mother to child), where survival and transmission of host and pathogen are intimately linked. in the case of horizontal transmission, the situation is more complex and there is no straightforward way to predict the evolution of future virulence, as it will depend on a variety of factors, including the population structure of the host and the correlation between virulence and transmission [ ] . a textbook example for reduction in virulence is the introduction of myxomatosis into the european rabbit population in australia and france in and , respectively. upon introduction, the virus initially killed about % of infected rabbits but over a few years mortality went down to %, following the emergence of attenuated virus strains and genetic resistance in the rabbit population. while the virulence went down, which translated into higher transmission rates, the current % mortality rate remains exceedingly high and there is no evidence for further reduction in the short term [ ] . bdgpl, the global lineage of the amphibian fungal pathogen batrachochytrium dendrobatidis (bd), is the only known pathogen to have extirpated entire host species. yet, over the three decades since its discovery, it shows no sign of evolving lower virulence. the primary reason there is little selective pressure on bd for virulence attenuation is that it can infect a very vast host range so that the extinction of any particular host species has limited impact on its fitness. even worse, some host species, such as the widely introduced african clawed frog, xenopus laevis, and the american bullfrog, rana catesbeiana, carry the disease asymptomatically, fuelling the global bd pandemics and limiting any shortterm prospect for significant decrease in virulence [ ] . these are just examples of the evolution of virulence but both illustrate that there is no simple pattern of decrease in pathogenicity with time. how old are the major human pathogens? apart from a few putative ancestral pathogens, including helicobacter pylori [ ] , that might have co-speciated with their human host, the infectious diseases afflicting us were acquired through host jumps from other wild or domesticated animal hosts or sometimes from the wider environment. the timing of these events and the original source remains unclear in many cases. the traditional view has been that many human pathogens emerged during the neolithic revolution. the main arguments for an origin of human pathogens linked to agriculture are based on the proximity between traditional farmers with their livestock and the emergence of higher human population densities in stable settlements enabled by agricultural subsistence. high population density is indeed required by some epidemic diseases which could not have maintained themselves on scattered groups of hunter-gatherers [ ] . this argument, however, neglects the fact that pathogens can evolve fast. also, while the proximity of humans and livestock is conducive to host jumps, humans transmitted more diseases to domestic animals than they acquired, with tuberculosis in particular having probably jumped from humans to cattle rather than the other way around [ ] . finally, this argument also neglects the high burden of pathogens in wild populations, including in the great apes. ancient direct evidence is scant for pathogens, and historical records rarely allow unambiguous attribution of described symptoms to a disease. that being said, recent progress in sequencing technology and in particular the ability to generate sequences, if not complete genomes, from ancient samples has greatly improved our understanding of the age of the major human pathogens, often leading to unexpected results. figure summarises the current knowledge on the age of the seven current 'major killers' , as well as plague, which was included due to its major impact in the past. while some of these estimates may need to be updated in the future after the emergence of new evidence, it is unlikely the general pattern will change much. some human diseases are old (for example plasmodium falciparum malaria) and others recent, such as hiv or, more surprisingly, measles. there is also no obvious pattern pointing to the neolithic revolution as a strong driver for the emergence of human pathogens. where are the resistance-conferring genes in the human genome? infectious diseases have killed well over half of all humans who have ever lived on earth. pathogens, such as childhood diseases, that affect their host prior to reproduction, through death or reduced fertility, have exerted enormous selective pressures. yet, scans of the genome for signatures of pathogen-driven selection have identified only a few variants with clear effects. similarly, genome-wide association studies (gwas) for infectious disease resistance/susceptibility have identified only few loci impacting on infectious disease susceptibility [ ] , despite their success in identifying thousands of variants involved in chronic diseases and phenotypic traits such as height. even for diseases that have affected us for a long time, for example tuberculosis, we know of no obvious protective genetic variant. given the high selective pressure pathogens must have exerted, it is reasonable to ask where all the resistance genes are. strongly protective variants may have reached fixation, rendering them undetectable unless the pathogen has a highly heterogeneous distribution range. an interesting case for regional selective pressure is the duffy negative antigen mutation protective against plasmodium vivax that is found at close to % in sub-saharan africa but virtually absent anywhere else. another situation where a resistance gene does not reach fixation arises when the protective variant is deleterious when homozygous, as in sickle cell anaemia. we might also speculate that the evolutionary potential and high genetic diversity of most pathogens limits our ability to detect protective variants in the human genome, particularly so if these were only effective against a subset of lineages within a pathogenic species. in addition to the few variants protective against specific pathogens, we also know of genomic regions involved in immunity against a wide spectrum of pathogens, such as interleukin genes or the major histocompatibility complex (mhc) system. the very high genetic diversity of the mhc is believed to have been shaped by exposure to different pathogen species [ ] . also, following the recent development of techniques to sequence ancient dna, it has been suggested that immunity genes such as those encoding toll-like receptors have been acquired following hybridization with archaic humans and are over-represented in the current gene pool of anatomically modern humans relative to genes not involved in immunity [ , ] . infectious diseases had a massive impact on our history, leading to the rise and fall of civilizations, both through the toll they took on human life but also through economic and societal collapse following epidemics. more military campaigns have probably been lost or won due to infectious diseases than due to the tactical acumen of the armies' commanders. thucydides reports in his history of the peloponnesian war, written in the th century bc, how the plague of athens devastated the citystate of athens in ancient greece during the second year [ ] ; influenza [ ] ; hiv [ ] ; tuberculosis [ ] [ ] [ ] [ ] ; p. falciparum malaria [ , ] ; hepatitis b [ ] ; measles [ ] ; plague [ , ] of the peloponnesian war ( bc) when it was on the cusp of victory against sparta, ending the golden age of pericles and athenian predominance in the ancient world. the eventual fall of the roman empire was also largely down to another epidemic, the justinian plague in - ce, which precluded the emperor justinian recovering lost territories in the western part of the empire [ ] . infectious disease played an equally important role in past human migrations. the conquistadores' sweeping conquests of large swaths of the americas in the th century was greatly aided by the diseases they brought with them, such as measles and smallpox, to which the indigenous populations had limited immunity [ ] . conversely, one of the possible reasons europeans managed to colonize africa was that they used quinine, an antimalarial drug derived from the bark of the cinchona tree [ ] . history has been shaped not only by pathogens infecting humans, but also those affecting domestic animals and crops. for example, it has been suggested that the islamic conquest of the th and th centuries did not extend to sub-saharan africa because the horses and camels of the islamic armies were dying from trypanosma spread by tsetse flies [ ] . conversely, pathogens were at other times the drivers of large migration. around one million irish people died and another million migrated to the us to escape the famine caused by phytophthora infestans destroying potato harvests between and [ ] . at least in the developed world, the leading causes of human mortality are no longer infectious diseases but instead age-associated disorders such as cancer, heart disease and diabetes. numerous countries have undergone an epidemiological transition, starting some years ago in some developed countries and less than years ago for developing countries. diseases that once devastated human populations, such as smallpox, are now eradicated. others, such as the plague or leprosy, are largely under control with the exception of a few hotspots. the current situation is, however, one of new challenges. globalization and increased mobility, particularly air travel, have facilitated the transmission of diseases not just locally but between continents. the recent outbreak of zika in the americas, for example, has been attributed in part to an increase in air travel from infected areas into brazilian airports, extending both the incidence and geographic range of the virus [ ] . the outbreak of severe acute respiratory syndrome (sars) and recurrent ebola crises in central africa highlight the ability of new and existing diseases rapidly to become significant international health threats. in addition, our ability to combat infectious diseases is also challenged by the widespread emergence of pathogen drug resistance. the global antimicrobial resistance (amr) crisis is increasingly limiting our resources to combat disease through antimicrobial therapy. thus, in spite of the global health narrative supporting a decline in the number of deaths caused by infectious disease, the complexity of our interactions with diseasecausing agents are as significant now as through history. infectious diseases continue to be a major cause of mortality globally, responsible for between a quarter to a third of all deaths and nearly half of all deaths in people under the age of , with most of these in principle avoidable. microbiology by numbers revised estimates for the number of human and bacteria cells in the body the role of damselflies (odonata: ztgoptera) as paratenic hosts in the transmission of halipegus eccentricus (digenea: hemioridae) to anurans red squirrels in the british isles are infected with leprosy bacilli airborne transmission of influenza a/h n virus between ferrets a single natural nucleotide mutation alters bacterial pathogen host tropism yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis insight into the evolution and origin of leprosy bacilli from the genome sequence of mycobacterium lepromatosis microbial diversity and the genetic nature of microbial species immunobiology and pathogenesis of viral hepatitis the influenza pandemic: insights for the st century infectious diseases of humans myxoma virus and the leporipox viruses: an evolutionary paradigm emerging fungal threats to animal, plant and ecosystem health age of the association between helicobacter pylori and man myths and misconceptions: the origin and evolution of mycobacterium tuberculosis evolution, revolution and heresy in the genetics of infectious disease susceptibility pathogendriven selection and worldwide hla class i diversity genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes introgression of neandertal-and denisovan-like haplotypes contributes to adaptive variation in human tolllike receptors cooling and societal change during the late antique little ice age from to around ad insights from paleomicrobiology into the indigenous peoples of pre-colonial america -a review selected papers from the first international conference 'camel cultures: historical traditions, present threats and future prospects'. london: rn books after the famine: plant pathology, phytophthora infestans, and the late blight of potatoes zika virus in the americas: early epidemiological and genetic findings th century variola virus reveals the recent history of smallpox pandemic influenza -including a risk assessment of h n the early spread and epidemic ignition of hiv- in human populations detection and molecular characterization of -year-old mycobacterium tuberculosis from a neolithic settlement in the eastern mediterranean armed conflict and population displacement as drivers of the evolution and dispersal of mycobacterium tuberculosis pre-columbian mycobacterial genomes reveal seals as a source of new world human tuberculosis eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in europe plasmodium falciparum accompanied the human expansion out of africa genomes of cryptic chimpanzee plasmodium species reveal key evolutionary events leading to human malaria dating the origin and dispersal of hepatitis b virus infection in humans and primates origin of measles virus: divergence from rinderpest virus between the (th) and (th) centuries early divergent strains of yersinia pestis in eurasia , years ago historical variations in mutation rate in an epidemic pathogen, yersinia pestis the authors are grateful to liam shaw for discussions on the manuscript and for financial support from the mrc, bbsrc, nerc and esrc (grants ne/m / and mr/p / ). lvd and fb wrote the manuscript. both authors read and approve the final version. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -cts n j authors: tam, john s; barbeschi, maurizio; shapovalova, natasha; briand, sylvie; memish, ziad a; kieny, marie-paule title: research agenda for mass gatherings: a call to action date: - - journal: lancet infect dis doi: . /s - ( ) -x sha: doc_id: cord_uid: cts n j public health research is essential for the development of effective policies and planning to address health security and risks associated with mass gatherings (mgs). crucial research topics related to mgs and their effects on global health security are discussed in this review. the research agenda for mgs consists of a framework of five major public health research directions that address issues related to reducing the risk of public health emergencies during mgs; restricting the occurrence of non-communicable and communicable diseases; minimisation of the effect of public health events associated with mgs; optimisation of the medical services and treatment of diseases during mgs; and development and application of modern public health measures. implementation of the proposed research topics would be expected to provide benefits over the medium to long term in planning for mgs. a mass gathering (mg), as defi ned by who, is "any occasion, either organized or spontaneous, that attracts suffi cient numbers of people to strain the planning and response resources of the community, city or nation hosting the event". mgs can be spontaneous or organised and include sports events, social or cultural functions, gatherings of displaced populations due to natural disasters or war, and political or religious congregations. [ ] [ ] [ ] [ ] major mgs are likely to involve communities from diff erent parts of the world. participants and host and home communities face health risks that are of international concern. therefore, the control of infectious diseases and provision of medical services for non-communicable dis eases and other risks to health are increasingly important in the planning for mgs. risks commonly associated with mgs include health systems having to accom modate a surge in capacity; challenges to existing health interventions; introduction and transmission of non-endemic diseases during and after mgs; diffi culties associated with risk communication to participants from diff erent cultures; and those arising because of the high profi le of some events, such as security risks. international mgs can amplify the transmission of infectious diseases, and infections can spread from the home to the host community during travel to and from the event and from the host to the home community on return. such events can pose substantial risks to global health security and present challenges for surveillance of the spread of such diseases to new ecological settings and vulnerable populations. in addition to risks of infectious diseases during mgs, non-communicable risks include cardiovascular diseases, environment-related heat injury, fi re-related injury, illnesses related to use of drugs and alcohol, occupational injuries, trauma or crush injuries associated with stampedes, exacerbation of respiratory diseases, and crowd safety. large mgs can also provide opportunities for terrorist activities. in planning for mgs, international and local health authorities need to ensure that the strengthened public health systems and rapid responses to health risks are integrated with other important components of the overall event management. eff ective public health policy should be based on evidence. the organisation of international mgs generally requires provision of huge amounts of resources by the host country and the dividend of such high visibility is called legacy. this legacy should be measured not just in terms of absolute improvements (eg, new roads and ambulances) but also in terms of improvements to the public health system and society as a whole. for example, the legacies of strengthened integration of clinical or laboratory services, early warning or syndromic surveillance, and fi eld epidemiology or response adopted for the past summer olympics (sydney, australia, athens, greece, and beijing, china) [ ] [ ] [ ] are used routinely in the health systems. the development of eff ective methods for, and improvement of, planning and handling of the health risks associated with mgs will strengthen global health security, prevent excessive emergency health problems and associated economic loss, and mitigate potential societal disruption in host and home communities. such development requires credible evidence to support activities that can reduce the global eff ect of infectious diseases and address local public health issues related to morbidity and mortality resulting from noncommunicable diseases during mgs. however, there are gaps in knowledge about many public health issues that contribute to eff ective planning. , , therefore, a robust knowledge about illnesses, from basic scientifi c understanding to societal eff ects of infections and noncommunicable diseases, is essential for modern public health practices and policy development related to the planning for mgs. several reports [ ] [ ] [ ] [ ] [ ] and who planning and guidance documents , - have drawn attention to the importance series of research into public health issues associated with mgs and identifi ed those that need immediate attention. despite these eff orts, an overarching research agenda based on public health to address the gaps in knowledge in mg health has not yet been developed. moreover, international coordination to prioritise and enable the funding and implementation of such an agenda has been lacking. the recent recognition and rapid development of mg health can provide focus on such issues. previously identifi ed research priorities for mgs tended to focus on logistical issues relating to site security and emergency management, crowd control, and surge in the need for medical services. , other specialties tend to focus on each of the specifi c public health topics that are associated with mgs. much research into existing and emerging infectious diseases is devoted to the development of rapid diagnostic methods, surveillance and response, and treatment and vaccines. although a research agenda based on public health must be underpinned by basic science, applied science and operational research are areas of particular interest to the organisers of mgs and the decision makers for public health, especially those in poorly resourced countries. the modern day idea of mg health has developed from the notion that "mass gatherings medicine is concerned with the provision of emergency medical care at organized events with > people in attendance" to include several specialties (fi gure). the intricate interactions between the diff erent specialties is essential for the planning and success of mgs. new advances or interventions undertaken in other specialties should be integrated with research into mgs. the proposed research agenda is a broad strategy for research into public health, with a focus on issues related to mgs. it is not intended to be an exhaustive compilation of all possible research questions about the strategic planning for and operation of mgs. instead the agenda is an outline of key research into methods to control public health and policy. the results of this research can provide an evidence-based platform for policy decisions and practices to reduce the risks and eff ects of mg-associated health issues and global security risks to public health. the principal objectives of this research agenda are to identify topics for research and underpin and prioritise their importance in achieving interventions for the control of public health; provide a research framework to gather evidence to address health issues associated with mgs and global security risks to public health; ensure focus on less well addressed issues such as operational and implementation research, particularly for under-resourced regions; provide a platform to enable co ordination, discussion, and interaction among organisers of mgs, public health professionals, and researchers; and encourage a multidisciplinary approach to address gaps in knowledge about health risks associated with mgs and their control. the proposed research agenda is organised as a framework of fi ve major public health research directions. although many public health emergencies associated with mgs are not predictable, much can be done to prevent and minimise their eff ects. continuous monitoring of participants' vulnerability to health risks at mgs and understanding trends in risks that are associated with specifi c events (religious, sports, or concerts) can be used to predict what might happen in the future. they are also essential for successful preparedness and management of risk reduction and strengthening the response capacity of host and home communities. recognition and analysis of the changing risks and vulnerabilities during mgs are starting points for raising awareness and communication of pending risks. building global capacity for health intelligence for noncommunicable and infectious diseases is important for the elucidation of the risks associated with mgs. most of this information is available through networks such as the emerging infectious disease networks , and the who global non communicable disease network. importantly, the information can be used to implement strategies for risk assessment and mitigation in planning for specifi c mgs (panels , ). morbidity and mortality at mgs can be mitigated through the assessment and management of risks associated with pre-existing non-communicable diseases. mitigation methods such as the provision of essential drugs and information about their availability at the mg can be initiated during pretravel medical care and advice. , incidence of trauma and heat-related illness at a site can be reduced with the provision of advice and installations to combat the eff ects of weather, and eff ective crowd control. models for the prediction of the spread of infections and occurrence of other emergency health issues during mgs have yet to be validated. many of the diffi culties in restricting the spread of emerging communicable diseases [ ] [ ] [ ] are not new and have proven diffi cult to resolve. there is also uncertainty global clinical and laboratory surveillance systems for communicable diseases, such as those for seasonal and pandemic infl uenza, are well established. the establishment of country-level surveillance systems for infectious diseases that can be adapted to diff erent epidemiological settings for mass gatherings (mgs) could also provide alerts for the occurrence of non-communicable diseases, such as radiation-related or chemical-related illnesses or those caused by extreme environmental temperatures (eg, program for monitoring emerging diseases). an important component of the alert and response strategy is an integrated event management system that provides a platform for rapid dissemination of devices and procedures required for the management of health risks. research into their development and implementation is needed. syndromic surveillance can potentially provide rapid initial information about the occurrence of both non-communicable and infectious diseases. however, its establishment and assessment of eff ectiveness during mgs might require further assessment. further work is also needed to identify appropriate parameters for assessment of the eff ectiveness of such surveillance systems during mgs. surveillance of non-communicable diseases is a formidable but necessary step for the improvement of the health of the global community. an estimated % of global mortality in was attributable to non-communicable diseases and % of such deaths occurred in low-income and middle-income countries. age-specifi c and sex-specifi c profi les of non-communicable diseases by country allow host countries of mgs to estimate possible risks of non-communicable diseases in participants from specifi c countries and plan for mitigation strategies. although the challenges for the organisers of mgs and the eff ects of infectious diseases at such events have been summarised, , there are many gaps in our understanding of emerging communicable diseases. integration of information from local and international surveillance of infectious diseases is important for strengthening the intelligence about the global threats before, during, and after mgs. the risks of non-communicable and infectious diseases during mgs are proportional to the probability of occurrence of risk factors during the event. the identifi cation of these risks factors for the diff erent types of mgs will provide a scientifi c basis for planning eff ective prevention. although major risk factors associated with non-communicable and communicable diseases are likely to be similar worldwide, factors specifi c to the type of mg might lead to health problems. systematic risk assessment helps identify potential risks of outbreaks and guides the establishment of eff ective risk management solutions. systematic assessment will also identify potential or deliberate health security risks that require assistance from other authorities and government agencies. the leading causes of morbidity and mortality during the hajj are heat-related illnesses and trauma-related injuries. identifi cation of such risks allowed event planners to instigate preventive measures and rapid response strategies. provision of shaded areas can reduce the incidence of heat-related illnesses and eff ective crowd control reduces the risk of a stampede. drug and alcohol use were identifi ed as health risks for other types of mgs; therefore, restriction of their use can mitigate the associated illnesses. the types and magnitude of health risks associated with spontaneous mgs due to natural disasters and confl icts are diff erent from those of organised mgs. objectives for risk management at such events are focused on facility-based health-care provision in addition to prevention. the potential for importation and subsequent global spread of infectious diseases during mgs are well understood. many emerging human infections are recognised as zoonotic diseases (eg, severe acute respiratory syndrome [sars], infl uenza a h n , nipah virus infection). the emergence of novel or rare pathogens in home communities and their subsequent spread to the host community and beyond can be amplifi ed during mgs. planning for the potential risks and hazards that are associated with mgs is essential to ensure success. many reports and manuals are available for planning mgs; , - however, their use should be tempered by the results of the risk assessment. importantly, planning should maximise the legacy of the mg. the conceptual model of a lasting public health legacy as a framework for the relation between planning inputs, implementation, and public health outcomes was put forward by who and the international olympic committee to ensure sustainable, positive health eff ects for the host communities after the olympic games. legacy planning should also include passing the knowledge gained to future hosts of similar mgs. series about how observations pertaining to particular pathogens, population groups, or settings can be used to develop public health policies for planning diff erent types of mgs. the development of evidence-based strategies for non-pharmaceutical inter ventions is urgently needed to address infection control and mitigate spread in the absence of available drugs and vaccines. additionally, such strategies are of particular concern for countries that do not have adequate access to pharmaceutical interventions such as vaccines and antimicrobial drugs. in some instances, available data for planning mgs might not have been assimilated in the best way for policy. a balance between basic scientifi c research and operational research is essential to inform the implementation of prevention strategies, best practices, and public health decision making (panel ). research into how to contain the spread of infectious diseases should have the broadest possible applicability in diff erent settings and at diff erent resource levels. however, some results might not be generalisable to the planning for mgs, such as those from studies of pathogen transmission in health-care settings. eff ective management of health risks for noncommunicable and infectious diseases during mgs requires planning in advance. it is an integral part of planning that consists of risk identifi cation, communication, analysis, assessment, prevention, and monitoring. , , many of the processes for risk management of non-communicable and infectious diseases during mgs are common. however, each can be specifi c to the type of mg and needs to be addressed accordingly. , high visibility of mgs complicates risk management and can lead to political and media pressure and thereby aff ect the decision-making process. prevention of the occurrence of non-communicable and infectious disease at mgs requires coordinated risk assessment and management before, during, and after the event as shown in the planning for the hajj. [ ] [ ] [ ] [ ] [ ] ideally, the primary prevention of human infections with emerging communicable diseases is the eff ective control of pathogens at their source. since at-source elimination of all emerging pathogens is not possible, secondary interventions (eg, pharmaceutical or nonpharmaceutical) are needed to mitigate the spread of infection during mgs. however, the eff ectiveness of such interventions has not been established. , in addition to reduction of the rates of morbidity and mortality associated with human infections during mgs, reduction of both the circulation of pathogens and human exposure might lessen the global health security risks. the eff ect of emergencies and crises on health can be substantially reduced if home and host communities are well prepared and are able to reduce their risks. the main challenge during mgs is the existence of systematic operative capacities such as risk assessment plans, coordinating mechanisms and standard procedures, institutional capacities, legislation and budgets, skilled vaccination is highly eff ective in the prevention of infectious diseases. however, many countries, particularly those with insuffi cient resources, have not developed strategies for vaccinating their populations at risk and people travelling to mgs. the reason is partly related to the lack of information about the transmission of infectious diseases (eg, infl uenza) and the social, economic, and health eff ects to the host and home communities. public health authorities need to decide how to eff ectively prioritise vaccine use on the basis of available information about disease burden and severity, epidemiology, and vaccine eff ectiveness and safety for vaccine-preventable infections associated with mgs. a failure to promote and implement the polio vaccination programme caused the re-emergence of poliovirus in nigeria and subsequent international spread, , emphasising the risks associated with insuffi cient vaccine coverage for participants at mgs. an outbreak of neisseria meningitides serogroup a (originating from africa) during the hajj in was later successfully controlled with the introduction of mandatory pretravel vaccination and use of fl uoroquinolones among african pilgrims. however, there are infectious diseases, including some of the most important and most dangerous, for which there are no vaccines. risk assessment and management during the planning for mgs can enable the development of eff ective health policies. strategic risk assessments are used to gather, coordinate, and analyse data that are necessary to identify existing risks, anticipate potential diffi culties, establish • enhance applications of existing vaccines against possible infectious diseases that are associated with mgs • assess the global vaccine supply and production to improve the processes of rapid response, surge in capacity, and rapid deployment and tracking of vaccine use for planning mgs • develop innovative clinical trial methods to study the eff ectiveness and safety of novel vaccines before and after licensing • develop new vaccines, platforms, and formulations that are safe with enhanced immunogenicity, especially in children and elderly people series priorities, and provide the basis for enacting targeted policies and implementation of corrective interventions. a system is needed for the measurement of the eff ect of public health policy and estimation of the probability of success. these interlinking processes are well described for regularly organised events such as the hajj and olympic games. , , an example of the eff ective development of health policy is the organisation of the hajj-such as a smoke-free environment for the prevention of fi re, structural changes to prevent crowding, and recommendations for vaccination of pilgrims to prevent transmission of infectious diseases (eg, infl uenza, meningitis, poliomyelitis). however, such eff ective policies are not possible for spontaneous mgs such as population displacement as a result of natural disasters or confl icts. development of vaccines for emerging infectious diseases presents substantial challenges and can take many years for diseases that are caused by novel pathogens such as severe acute respiratory syndrome (sars; panel ). even if a vaccine exists, it might need to be regularly updated, clinically assessed for safety and effi cacy, and promptly produced for immediate use (eg, infl uenza vaccines). the effi cacy and eff ectiveness of a vaccine are dependent on the immune responses that are determined by the age of the recipient and composition of the vaccine (eg, conjugated or adjuvanted). improvements to vaccines and formulations that can provide longer-lasting and broader activity aff ord better protection, increase the applicability of vaccines, and reduce the frequency of vaccination. during an outbreak, the important factors are the rapid production and equitable distribution of vaccines to countries in need. ensuring rapid and eff ective management of patients and prevention of diseases requires robust health services at mgs. providers of emergency services play an important part in ensuring public safety during such events. knowledge and monitoring of medical service provision during mgs has been rapidly increasing in the past decade. , , however, a lack of consolidated data for diff erent types of mgs means that organisers are not able to plan accordingly for the emergency medical services that might be needed. improved and targeted clinical management and infection control can substantially reduce the incidence and transmission of infectious diseases during mgs. optimum clinical management must be based on an improved under standing of the pathogenesis of these infections, advances in laboratory diagnosis, development and application of eff ective antimicrobial drugs, and other treatment modalities (panel ). there are many gaps in our basic understanding of how many of the pathogens that are associated with mgs cause disease in people and what factors aff ect severity of illness. host immune responses, underlying comorbidity, age, and the properties of the infecting pathogen can all contribute to severity. the clinical presentation of many infections, such as infl uenza, is not specifi c, which makes diff erential diagnosis and early treatment to reduce further transmission and severe outcomes diffi cult. for example, antibiotics can help control severity and further spread of travellers' diarrhoea caused by bacteria. rapid and reliable diagnostic testing can expedite the initiation of timely and appropriate treatment and infection control. increase in and optimisation of the repertoire of antimicrobial drugs immunomodulator drugs, immunoglobulins, and natural products) that are applicable in low-resource areas and in fi eld conditions (such as availability, whether licensed or not, acceptance, and effi cacy in diff erent ethnic, sex, and age groups) and are easy to administer in paediatric-care and emergency-care settings • optimise management of people who are at risk of severe disease and complications, including emergency-care practices that are applicable across a range of resource settings health-care capacity and response • assess the eff ectiveness of global, national, and local responses to outbreaks of communicable diseases and develop new methods for assessment • undertake operational studies to investigate the surge capacity needs, particularly in host countries for mgs, including development of triage schemes in diff erent health-care and resource settings, and surge planning to maintain adequate resources • undertake studies to identify evidence-driven clinical-care pathways and principles that optimise health-care delivery in a range of resource settings • undertake studies to develop principles and practices for rapid assessment and introduction of new interventions during health emergencies, including systems for collation, sharing, and assessment in real time of clinical data series and development of clinical research to assess effi cacy of putative adjuvant treatments such as immunomodulator drugs, passive immuno therapy, and traditional medicine that are suitable for use in under-resourced areas would be most benefi cial in the preparation for mgs (panel ). the availability and quality of health services contributes to the eff ect of infectious diseases in the source and home countries (panel ). the same pathogen that might have a small eff ect on the rates of morbidity and mortality in countries with well organised health-care systems can be devastating in countries where health-care systems are suboptimum. new public health methods need to be harnessed to help reduce the eff ect of health problems during mgs. use of innovative communication channels, such as the internet and mobile phone networks, have the potential to aid surveillance, rapid risk assessment, and dissemination of accurate information. , , mathematical modelling and risk communication have potential applicability in all aspects of research into health risks associated with mgs. some countries and mg organisers use state-of-the-art approaches for early detection and monitoring of diseases such as syndromic surveillance. , in some countries computerised health-care and laboratory-based infor mation systems are used for planning mgs and these systems can be adapted for monitoring large-scale outbreaks. other innovative technologies such as mobile phones can be used in remote areas or countries that lack the resources to gather and transmit health-related data in real time, provide rapid feedback, and train health-care workers. , applicability and use of these modern methods of monitoring in diff erent settings and contexts require further investigation, with special attention to issues related to integration and interoperability of initiatives for infection control during mgs (panel ). evidence-based public health decision making in planning and mitigating health risks requires rapid access to information. however, such information is often incomplete, evolving, and derived from an increasingly complex array of sources such as basic science researchers, epidemiologists, social and political scientists, and economists. modelling is useful in that it can incorporate diverse data to inform public health policy and decision making. , advances in mathematical modelling for public health are expected to include computational structural biology; integration of epidem iological and geographical data into phylogenetic models; within-host and population-level susceptibility models; behavioural modelling; and assessment of the eff ects of climate change on disease transmission and the use of novel datasets on contact patterns and population mobility. [ ] [ ] [ ] [ ] [ ] communication is a key strategy in risk management in planning for mgs. the sars outbreak in reinforced the idea that a timely and transparent public information policy could help reduce excessive and inappropriate public health responses and minimise the social disruption and economic consequences of a fast-moving global epidemic. , increased investment in identifying eff ective approaches and developing and assessing new communication methods will benefi t risk prevention and control eff orts. the specifi c challenge is to provide clear, credible, and appropriate communication to meet the needs of diverse communities and retain public trust in a dynamic yet unknown process. , some of the main research topics in this specialty include the link between communication and behaviour change models; development and assessment of methods that can be quickly accessed and used in mgs; and assessment of best practices, challenges, and barriers in risk research in early detection and monitoring of disease • identify, develop, and adapt modern technologies for early detection of outbreaks of communicable diseases and their application in disease surveillance during mgs • integrate and continuously assess innovative approaches and channels for disease surveillance and monitoring • develop effi cient mechanisms to address the global challenges to sharing information, data, and details about pathogens identifi ed during outbreaks at mgs in terms of local, ethical, legal, and research perspectives • defi ne the timeliness and quality of data required for early detection of disease from local to district, regional, national, and global levels • assess the application of modelling to understand and estimate key parameters for risk management • investigate the role of modelling to assess eff ect of public health policies for diff erent mgs • assess modelling in public health policy planning and strategic decision making in mg planning research into health issues related to mgs is at an early stage. research directions outlined here should contribute to the evidence that can be used to formulate risk management guidelines and assist event planning and health-care policy makers. the research agenda presented here is not intended to be restricted to specifi c aspects of health research but rather to encourage a multidisciplinary approach focused on mg health and to help gain more knowledge. in the future, the focus should be on strategies directed towards developing common research frameworks and defi nitions. additionally, the knowledge generated by use of the multidisciplinary approach to research ought to be assessed for direct relevance to mgs in terms of their capacity for integration in legacy building and systemic sharing of information. jst wrote the text. mb planned the outline for the review, and provided and consolidated who policies and guidelines about mgs. zam provided the concept for the review and planned the content with the team. ns provided the outline and wrote the section about risk communication. sb provided information about outbreak control and research agenda for infl uenza. m-pk contributed information about health research directions and vaccine development. all authors reviewed and provided advice on drafting the review. we declare that we have no confl icts of interests. we identifi ed references for this review by searching pubmed, medline, and the internet for articles published in english from january, , to june, , by using the search terms "mass gatherings", "research", "infectious diseases", "communicable diseases", "non-communicable diseases", "public health", "alert and response", "mass gatherings planning", "legacy", "outbreak", "surveillance", "prevention", "treatment", "olympic games", and "hajj". we reviewed the articles and information found during these searches. additional references cited in the articles were also reviewed. communicable disease alert and response for mass gatherings the hajj: communicable and non-communicable health hazards and current guidance for pilgrims public health surveillance for mass gatherings a literature review of the health and safety risks associated with major sporting events: learning lessons for the london olympic and paralympic games health risks at the hajj global perspectives for prevention of infectious diseases associated with mass gatherings crowd and environmental management during mass gatherings non-communicable health risks during mass gatherings public health legacy: experiences from vancouver and sydney olympic and paralympic games mass gatherings and public health: the experience of the athens olympic games who western pacifi c region. the health legacy of the beijing olympic games: successes and recommendations mass gathering medicine: a review of the evidence and future directions for research the development of conceptual models for mass gathering health preparing for infectious disease threats at mass gatherings: the case of the vancouver olympic winter games the quest for public health security at hajj: the who guidelines on communicable disease alert and response during mass gatherings asia pacifi c strategy for emerging diseases: technical papers mass-gathering medical care: a review of the literature global capacity for emerging infectious disease detection networks and the epidemiology of infectious disease global noncommunicable disease network (ncdnet): report of the first global forum convened by the world health organization who global technical consultation: global standards and tools for infl uenza surveillance infectious disease surveillance and modelling across geographic frontiers and scientifi c specialties who. global status report on noncommunicable diseases who. noncommunicable diseases country profi les emergence of medicine for mass gatherings: lessons from the hajj safe and healthy mass gatherings: a health, medical and safety planning manual for public events who. global forum on mass gatherings communicable disease alert and response for mass gatherings: key considerations who. interim planning considerations for mass gatherings in the context of pandemic (h n ) infl uenza federal emergency management agency, usa. special events contingency planning manual federal emergency management agency, usa. special events contingency planning: job aids manual world health organization writing group. non-pharmaceutical interventions for pandemic infl uenza, international measures non-pharmaceutical public health interventions for pandemic infl uenza: an evaluation of the evidence base nigerian states again boycott polio-vaccination drive global poliomyelitis eradication: status and implications epidemic group a meningococcal disease in haj pilgrims meningococcal disease and travel measuring emergency services workloads at mass gathering events automated vocabulary discovery for geo-parsing online epidemic intelligence use of unstructured event-based reports for global infectious disease surveillance mass gathering medicine: a predictive model for patient presentation and transport rates the impact of mass gatherings and holiday traveling on the course of an infl uenza pandemic: a computational model modeling the impact of global warming on vector-borne infections computational procedures for optimal experimental design in biological systems deterministic epidemic models on contact networks: correlations and unbiological terms how to make predictions about future infectious disease risks modeling and public health emergency responses: lessons from sars sars revisited: managing "outbreaks" with "communications responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management perceived risk and effi cacy beliefs as motivators of change: use of the risk perception attitude (rpa) framework to understand health behaviors eff ective media communication during public health emergencies. a who handbook communicating uncertainty-how australian television reported h n risk in : a content analysis key: cord- -l oecoot authors: pirofski, liise-anne; casadevall, arturo title: immunomodulators as an antimicrobial tool date: - - journal: curr opin microbiol doi: . /j.mib. . . sha: doc_id: cord_uid: l oecoot the spectrum of infectious diseases has shifted in the past years to include those caused by microbes that cause disease predominantly in immunocompromised individuals. this phenomenon has underscored the dependence of microbial virulence on the immune status of the host. the limited efficacy of the available antimicrobial armamentarium in immunocompromised individuals, combined with increasing resistance to these agents, has led to an urgent need for new therapies for infectious diseases. immunomodulation represents a novel approach to antimicrobial therapy that depends on bolstering host immunity, rather than direct antimicrobial activity. immunomodulators can be divided into those that are specific to pathogens (pathogen-specific) and those that are not specific to pathogens (non-specific). however, to date only a few immunomodulators have been evaluated for their efficacy as antimicrobial tools. liise-anne pirofski , and arturo casadevall , the spectrum of infectious diseases has shifted in the past years to include those caused by microbes that cause disease predominantly in immunocompromised individuals. this phenomenon has underscored the dependence of microbial virulence on the immune status of the host. the limited efficacy of the available antimicrobial armamentarium in immunocompromised individuals, combined with increasing resistance to these agents, has led to an urgent need for new therapies for infectious diseases. immunomodulation represents a novel approach to antimicrobial therapy that depends on bolstering host immunity, rather than direct antimicrobial activity. immunomodulators can be divided into those that are specific to pathogens (pathogen-specific) and those that are not specific to pathogens (non-specific). however, to date only a few immunomodulators have been evaluated for their efficacy as antimicrobial tools. introduction: immunomodulation in the context of the damage-response framework immunomodulators are usually products of the immune system [ ] . as such, it is useful to consider immunomodulation approaches to infectious diseases in the context of microbial pathogenesis. in contrast to microbe-centric views, in which microbial pathogenesis and virulence are considered to reflect singular microbial functions, the damage-response framework provides a flexible construct that accounts for the contribution of the host, as well as the microbe, to these entities [ ] . the damage-response framework considers host damage to be the common denominator in microbial pathogenesis. based on this tenet, host damage can be plotted against the host immune response as a u-shaped curve, whereby the maximal host damage resulting from a given host-microbe interaction occurs both when the immune response is weak and when it is overly strong (figures and ) . the inherent flexibility afforded by this curve lies in its ability to account for the fact that certain microbes only cause disease in certain hosts, a phenomenon that cannot be explained by views of microbial pathogenesis that consider virulence to be a singular microbial trait [ ] . a logical corollary of the damage-response framework is that infectious diseases only occur in susceptible hosts. host immune mechanisms protect against infectious diseases by preventing or reducing the damage that can result from host-microbe interaction. the relationship between host immunity and microbial pathogenesis is clearly exemplified in immunocompromised hosts, by diseases that are caused by commensal microbes, such as candida albicans and staphylococcus epidermidis, and fungi, such as cryptococcus neoformans and pneumocystis jurevecci, and the success of immune reconstitution in preventing hiv-associated diseases caused by these microbes. the phenomenon of immune reconstitution disease that follows antiretroviral therapy with highly active anti-retroviral therapy, or haart, in patients with aids (acquired immunodeficiency syndrome) illustrates how rebounding immunity can produce disease (discussed in [ ]). because the success of antimicrobial therapy is a function of its ability to ameliorate disease, and disease is a manifestation of host damage, the damageresponse framework provides a useful construct to consider approaches to treating infectious diseases that reduce host damage resulting from the host-microbe interaction. the crisis in antimicrobial therapy, which has stemmed from antibiotic overuse, misuse and the limited number of new antimicrobial drugs on the near horizon is well documented [ ] . however, another area that limits the utility of antimicrobial drug-based therapy is that antimicrobial agents are frequently ineffective in individuals with impaired immunity, often despite being highly active in vitro or in individuals with intact immunity. this underscores the crucial relationship between host immunity and microbial virulence and provides a powerful rationale for approaches to antimicrobial therapy that regulate the immune response to reduce, ameliorate or prevent host damage. approaches to immunomodulation can be divided into those that are specific to pathogens (pathogen-specific) and those that are not (non-specific). pathogen-specific immunomodulators include antibody reagents and vaccines. with the exception of the rabies and varicellazoster vaccines, currently licensed vaccines are administered to prevent acute infectious diseases rather than for therapy and are not discussed further here. non-specific immunomodulators include cytokines, antimicrobial peptides, certain antimicrobial drugs and microbes such as probiotics. at present, clinical experience with nonspecific immunomodulators as antimicrobial tools has been predominantly limited to cytokines. there are powerful historical precedents for the use of antibody-based therapies to treat infectious diseases (reviewed in [ ] ). the first era of antimicrobial therapy, early in the th century, was based on serum therapy with antibody preparations. hence, the inaugural antimicrobial agents were immunomodulators [ ]. first generation antibody reagents were abandoned because of their toxicity, which was a result of their impurity and derivation from non-human species, and the arrival of antimicrobial drugs that acted directly on the microbe. nonetheless, there was evidence for synergism between antibiotics and serum therapies [ ] . serum therapy was validated in animal models before being administered to humans [ ]; however, the mechanism by which it ameliorated infectious diseases or enhanced the efficacy of antimicrobial drugs was largely unknown. for most of the th century, the mechanisms of antibody action that were thought to influence antibody efficacy included their ability to neutralize, promote opsonization and the possible effects of ifn-g therapy in two patients with cryptococcosis in the context of the damage-response framework. patient is an individual with aids-related cryptococcosis, where susceptibility to infection is associated with a profound defect in th -type immunity as a result of cd t-cell deficiency. in this patient, the administration of ifn-g is pro-inflammatory and the increased inflammatory response might facilitate control of the infection, thus reducing damage and symptoms of disease. by contrast, patient is an individual with cryptococcal disease following immune reconstitution with haart. in this patient, administration of ifn-g might be detrimental, as cryptococcal disease is caused by an exuberant inflammatory response. hence, the outcome of ifn-g therapy depends on the immune status of the host. illustration of the dichotomous requirements for immunomodulation in patients with different immune status. patient has an infectious disease that reflects the outcome of a weak immune response, such as hiv-associated histoplasmosis or aspergillosis following stem cell transplantation. in this individual enhancement of the inflammatory response with a pro-inflammatory immunomodulator could facilitate microbial clearance, thus reducing damage and symptoms of disease. by contrast, patient has an infectious disease that reflects the outcome of an overly exuberant immune response, such as mediastinal fibrosis from histoplasmosis or allergic aspergillosis. in this individual, an anti-inflammatory immunomodulator could dampen the host the inflammatory response, thus reducing damage and symptoms of disease. notably, the immune response of this individual could have already resulted in microbial clearance. these patients illustrate that the kind of immunomodulator that would be beneficial is probably to be influenced by the immune status of the affected individual. phagocytosis or antibody-dependent cell-mediated cytotoxicity (adcc) and/or to activate complement [ ] . however, a significantly more robust menu of potential mechanisms of antibody action, which includes direct antimicrobial action, immunomodulation and generation of oxidative species, has emerged over the past decade [ ] . for example, the efficacy of a pneumococcal capsular polysaccharide-specific antibody was associated with modulation of the cellular response to pneumococcus in the lungs of mice with pulmonary infection [ ] . this finding suggested that the well-documented ability of serum therapy to ameliorate fever and other clinical symptoms of pneumococcal pneumonia [ ] could have reflected a downregulation of the host inflammatory response (or damage control). perhaps the observation that pneumococcal pneumonia was only responsive to serum therapy with the homologous capsular polysaccharides-specific antiserum in the first three days of symptoms indicated that its capacity to mediate immunomodulation was limited to the early stages of disease. antibodies to other microbes, including c. neoformans and histoplasma capsulatum are able to modulate the cellular immune response to pulmonary infection (see [ ] ). studies of antibody action have shown that the older dichotomous view that antibody immunity was only effective against extracellular pathogens, whereas cellular immunity was responsible for immunity against intracellular pathogens has been deconstructed by evidence that antibody reagents can be effective against classical intracellular pathogens, such as mycobacterium tuberculosis, c. neoformans, h. capsulatum and scores of viruses [ ]. currently, there is only one antibody reagent licensed for use against an infectious disease in the united states -palivizumab. licensed in , palivizumab is a neutralizing, humanized monoclonal antibody (mab) to protein f on respiratory syncytial virus (rsv) that reduced hospitalization for rsv in premature and other high-risk infants when given as prophylaxis [ ] . because the antiviral activity of palivizumab was associated with a reduction in inflammatory mediator release in a murine model of rsv [ ], its mechanism of action probably involves immunomodulation. despite ongoing controversies about the cost and target population of palivizumab, its success in reducing the risk of rsv in high-risk infants promoted the development of second generation reagents and vaccine candidates [ ] . recently, mycograb, a human recombinant antibody fragment was shown to significantly improve the response to amphotericin b in patients with invasive candidiasis [ ] . patients who received mycograb and amphotericin b showed a higher rate of complete overall response on day of therapy, a significantly better mycological response and less candida-attributable mortality than patients who received amphotericin b and a placebo. mycograb was safe and well-tolerated; however, episodes of hypertension occurred more frequently in patients who received mycograb than those who received a placebo. mycograb is a recombinant antibody fragment lacking an fc region, and is produced from a human anti-hsp (heat-shock protein ) cdna library with an epitope that inhibits fungal hsp , nilkvirknivkk [ ] . the development of this antibody was driven by the observation that recovery from invasive candidiasis was associated with the appearance of antibodies to a kda determinant [ ] that was found to be a fungal homolog of human hsp . mycograb was tested in patients in comparison to standard therapy. as such, the question of whether or not its efficacy in vivo depends on synergy with antifungal drugs is unanswered. nonetheless, the in vitro activity of mycograb (with amphotericin b and other antifungal agents) against resistant candida and other fungal species [ , ] suggests it could hold promise as a broadly active antifungal agent. the first mab used to treat a fungal disease in humans was the mouse mab b , which binds to the cryptococcal capsular polysaccharide glucuronoxylomannan [ , ] . extensive preclinical testing revealed that b augmented host defense mechanisms against c. neoformans, in vitro and in vivo (reviewed in [ ] ). in the clinical trial, administration of a single mg kg À dose of b to hiv-infected patients treated for cryptococcal meningitis was well-tolerated and was associated with a reduction in serum glucuronoxylomannan levels [ ] . the tolerability and promising effect of this reagent in hiv-infected patients bolsters the prospect that immunotherapeutic interventions have the potential to augment host immune mechanisms in the treatment of infectious diseases in immunocompromised individuals. now, mabs have been developed against a myriad of microbes responsible for emerging infectious diseases and/or those that cause disease in the setting of immune impairment. a human mab to bacillus anthracis toxin has recently successfully completed phase i trials and stands as a potentially useful therapeutic in the event of an anthrax biological attack [ ] . several human mabs to the sars (severe acute respiratory syndrome) coronavirus have been developed that might be useful if the disease reappears [ ] . remarkably, these mabs were developed to the point that clinical use was possible in less than five years. human mabs were highly effective against experimental shiga-toxin producing escherichia coli in piglets [ ] . studies in experimental models have revealed that the efficacy of certain mabs depends on intact cellular immunity (see [ , ] ). as such, the use of mabs in immunocompromised patients could depend on whether its efficacy requires the immune function (a subset or element of) that is lacking in the relevant patient(s). mabs have the advantage of homogeneity and high specific activity. although there is concern that mabs could have limited usefulness for microbes that demonstrate high antigenic variation and mutability, combinations of mabs have shown promise in overcoming this limitation [ ] . another type of antibody-based therapy for infectious diseases consists of polyclonal immunoglobulin-based agents, including intravenous immunoglobulin (ivig) and specific immune globulins (sigs, sometimes called hyperimmune globulin). treatment and prevention of rabies depends entirely on the combination of two immunomodulators: rabies vaccine and rabies immune globulin cytokine-based therapies contrast with antibody reagents in that they are not pathogen-specific. the rationale for the use of cytokines as adjunctive immunomodulators for infectious diseases is based on the concept that replacement or augmentation of natural mediators of host defense should enhance the antimicrobial effect of host immune mechanisms and/or antimicrobial agents. despite the logical basis for this concept, the potential antimicrobial power of these agents has been difficult to harness clinically. currently, there are only a few examples of the use of adjunctive cytokines against infectious diseases. notable exceptions are the use of recombinant a-interferons and nucleoside analogs for hepatitis b virus (hbv) and pegylated interferons and ribavirin for hepatitis c virus (hcv) [ ] . the efficacy of interferons against hcv has been attributed to the induction of th immunity [ ] . a side effect of interferon-based therapies that limits their use in certain patients is depression [ ] . the effecacy of adjunctive interferon-gamma b (ifn-g b) with amphotericin b was studied in a phase ii, double-blind placebo-controlled trial for aids-associated cryptococcal meningitis [ ] . there was a trend towards mycological response and clinical improvement among interferon recipients, with % showing improvement, compared to % of placebo-controlled subjects. although this difference did not reach statistical significance, the trend towards a beneficial effect of adjunctive interferon is encouraging, calling for further, larger scale studies and studies to identify those patients in whom adjunctive immunotherapy could be beneficial. the rationale for interferon therapy for cryptococcosis has a strong basis in preclinical studies in mice [ ] and a human study showing an association between cerebrospinal fluid levels of ifn-g and treatment in hiv-infected patients with cryptococcal meningitis [ ] . in light of the established benefit of interferon therapy for the prevention of bacterial diseases in patients with chronic granulomatous disease [ ] , adjunctive interferon could hold promise as an adjunctive agent for hivassociated cryptococcal meningitis. however, the absence of surrogate markers that can predict the patients who would benefit from interferon therapy underscores the potential pitfalls in study-design and patient selection for clinical trials. this is particularly problematic for cryptococcal meningitis, a disease that can occur in the case of weak or reconstituted immunity in patients with hiv infection [ ] . the failure to demonstrate the effect of a pro-inflammatory immunomodulator could reflect the induction of an excessive inflammatory response that promotes disease. figure provides a schematic interpretation of the use of ifn-g therapy in the context of the damageresponse framework. depending on the immunological status of the affected patient, adjunctive ifn-g therapy could be beneficial or detrimental. preclinical data, demonstrating the importance of th helper t-cell responses in protection against fungi, in experimental models has led to the proposal that adjunctive cytokines be used with antifungal agents for invasive fungal infections [ ] . the rationale for the use of colony stimulating factors (csfs), derived from granulocytes (g-csf) or macrophages (gm-csf), is in part based on their ability to alleviate neutropenia [ ] . data on the clinical use of adjunctive csfs for fungal diseases is limited to small studies or case reports (see [ ] for review). nonetheless, administration of gm-csf with antifungal agents in patients with invasive fungal infections was associated with decreased patient mortality or better response rates compared to a placebo [ ] or antifungal therapy alone [ ] . in view of the small number of affected patients and medical and ethical considerations in the design of randomized, double-blind placebocontrolled studies, it is possible that safe, well-tolerated candidate immunomodulators that are beneficial in preclinical studies will find their way into the antimicrobial armamentarium through compassionate use protocols and salvage therapy [ ] . the rationale for the use of adjunctive pro-inflammatory cytokines and certain antibodies for treating infectious diseases is to enhance the host response. however, a relatively underappreciated principle of microbial pathogenesis is that the damage resulting from host-microbe interaction can be the result of an overly exuberant host response [ ] . hence, there is a rationale for use of therapeutic interventions that dampen or reduce, as well as those that enhance or augment, the inflammatory response. it has been proposed that the beneficial effect of ivig against inflammatory diseases involves engagement of the inhibitory fc receptor, which downregulates the inflammatory response [ ] . probiotics, which are live bacteria derived from the human gastrointestinal tract, have been used as therapy for inflammatory bowel diseases, including antibiotic-associated diarrhea [ ] . probiotics remain outside the established antimicrobial armamentarium and are fraught with the potential for harm in immunocompromised hosts [ ] . nonetheless, it is logical to postulate that replacement and/or enhanced activity of the human microbiota could be beneficial for treatment of infectious diseases that reflect a failure of mucosal surfaces or of the innate mechanisms they bolster to protect against microbe-mediated damage (see [ ] the ability of macrolides to suppress the release of inflammatory mediators from phagocytes in vitro has been recognized for some time, but the clinical relevance of this phenomenon remains uncertain. nonetheless, it has been hypothesized that the benefit of low-dose macrolide therapy in chronic pulmonary diseases, could be as a result of macrolide-induced reductions in levels of inflammatory cytokines, neutrophil recruitment and biofilm formation, which have been observed in animal models and/or in vitro [ ] . in contrast to macrolides, amphotericin b [ ] and penicillin [ ] induce the release of inflammatory mediators in vitro through toll-like receptor (tlr )-mediated stimulation. the inflammatory properties of these drugs raise the question of whether their immunomodulatory properties have an independent contribution to their therapeutic efficacy. the future use of adjunctive immunomodulators for infectious diseases requires a better understanding of microbial pathogenesis and the relative need for immune activation versus immune modulation in the context of the immune response of the affected individual. in light of the fact that certain infectious diseases reflect an insufficient response, whereas others reflect an overly exuberant response, different types of interventions are likely to be required, depending on the immune status of the patient (figure ) . the damage-response framework can be a useful tool when considering the type of intervention that might be successful, but such predictions require experimental validation to be translated to the bedside and clinic. papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest . committee on new directions in the study of antimicrobial therapeutics: immunomodulation: treating infectious diseases in a microbial world: report of two workshops on novel antimicrobial therapies. washington, dc: national academies press; . this national academies report details the potential for modulating innate and acquired immunity and the microbiota to enhance the potential of immunomodulation for the treatment of infectious diseases. casadevall a, pirofski l: a reappraisal of humoral immunity based on mechanisms of antibody-mediated protection against intracellular pathogens. adv immunol , : - . this article deconstructs the older view that antibody immunity is limited to extracellular organisms and details newer mechanisms of antibody action, including their ability to function as pro-inflammatory and antiinflammatory immunomodulators as regulators of cellular immunity. . impact-rsv group: palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. pediatrics , : - . mejias a, chavez-bueno s, rios am, saavedra-lozano j, fonseca am, hatfield j, kapur p, gomez am, jafri hs, ramilo o: anti-respiratory syncytial virus (rsv) neutralizing antibody decreases lung inflammation, airway obstruction, and airway hyperresponsiveness in a murine rsv model. antimicrob agents chemother , : - . this study evaluated the effect of palivizumab on virological and immunological parameters and lung inflammation in a mouse model of rsv pneumonia. the results showed that palivizumab reduced the viral load in lung whether given before or up to h after rsv infection, but that mab administration before infection was associated with lower levels of inflammatory mediators and less lung damage. an important implication of this study is that the timing of mab administration is a key determinant of its immunomodulatory activity. interferon-induced depression in chronic hepatitis c: a review of its prevalence, risk factors, biology, and treatment approaches this article describes the etiologies of depression in patients with hepatitis c, including the contribution of interferon therapy. this review serves as an important reminder that immunomodulation can have toxicity and that the therapy for infectious diseases recombinant interferongamma b as adjunctive therapy for aids-related acute cryptococcal meningitis this study compared the efficacy of adjunctive ifn-g or placebo with amphotericin b in patients with hiv-associated cryptococcal meningitis. the trend towards a better outcome in interferon-treated patients demonstrated the feasibility of immunotherapy in immunocompromised patients and calls for further studies to define the patients who enhancement of antifungal chemotherapy by interferon-gamma in experimental systemic cryptococcosis ifngamma at the site of infection determines rate of clearance of infection in cryptococcal meningitis this study evaluated the csf response to c. neoformans infection and therapy in patients with hiv-associated cryptococcal meningitis. the results suggested that ifn-g levels were positively correlated with the response to treatment. the significance of the study is that it provided a real time evaluation of the immune response in patients that was consistent with animal models and in vitro studies a controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. the international chronic granulomatous disease cooperative study group cytokines and fungal infections treatment of acute myeloid leukemia with cytokines: effect on duration of neutropenia and response to infections malignancy: granulocyte colony stimulating factor increases the efficacy of conventional amphotericin in the treatment of presumed deep-seated fungal infection in neutropenic patients following intensive chemotherapy or bone marrow transplantation for haematological malignancies immunomodulation with interferon-gamma and colony-stimulating factors for refractory fungal infections in patients with leukemia this work was supported by grants from the national institutes of health ai (lp), ai (lp), ai (lp), ai (ac), ai (ac), and hl (ac). this study showed the benefit of cytokines and csf for invasive fungal disease in a small number of patients with leukemia. the promising results of this study support the use of immunomodulators as salvage therapy in desperately ill patients, although their benefit has not been established in large scale placebo-controlled trials. key: cord- - kozsv z authors: dewidar, bedair; kahl, sabine; pafili, kalliopi; roden, michael title: metabolic liver disease in diabetes – from mechanisms to clinical trials date: - - journal: metabolism doi: . /j.metabol. . sha: doc_id: cord_uid: kozsv z abstract non-alcoholic fatty liver disease (nafld) comprises fatty liver (steatosis), non-alcoholic steatohepatitis (nash) and fibrosis/cirrhosis and may lead to end-stage liver failure or hepatocellular carcinoma. nafld is tightly associated with the most frequent metabolic disorders, such as obesity, metabolic syndrome, and type diabetes mellitus (t dm). both multisystem diseases share several common mechanisms. alterations of tissue communications include excessive lipid and later cytokine release by dysfunctional adipose tissue, intestinal dysbiosis and ectopic fat deposition in skeletal muscle. on the hepatocellular level, this leads to insulin resistance due to abnormal lipid handling and mitochondrial function. over time, cellular oxidative stress and activation of inflammatory pathways, again supported by multiorgan crosstalk, determine nafld progression. recent studies show that particularly the severe insulin resistant diabetes (sird) subgroup (cluster) associates with nafld and its accelerated progression and increases the risk of diabetes-related cardiovascular and kidney diseases, underpinning the critical role of insulin resistance. consequently, lifestyle modification and certain drug classes used to treat t dm have demonstrated effectiveness for treating nafld, but also some novel therapeutic concepts may be beneficial for both nafld and t dm. this review addresses the bidirectional relationship between mechanisms underlying t dm and nafld, the relevance of novel biomarkers for improving the diagnostic modalities and the identification of subgroups at specific risk of disease progression. also, the role of metabolism-related drugs in nafld is discussed in light of the recent clinical trials. finally, this review highlights some challenges to be addressed by future studies on nafld in the context of t dm. nonalcoholic fatty liver diseases (nafld) is currently defined by lipid deposition that exceeds more than % of hepatocytes, as assessed from liver biopsy, and/or by more than . % hepatocellular fat content per liver weight, as assessed from magnetic resonance (mr) methods, in the absence of significant alcohol consumption and other causes of fatty liver [ , ] . nafld, which affects about % of the population [ ] , comprises a broad range of abnormalities ranging from simple fatty liver (steatosis) to non-alcoholic steatohepatitis (nash), characterized by inflammation, necrosis, and hepatocellular ballooning, and progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma (hcc) [ ] . some gene variants promote risk of nafld by altering lipid droplet formation and de novo lipogenesis (dnl), such as variants of patatin-like phospholipase domain-containing protein (pnpla ) and glucokinase regulatory protein [ ] , or by decreasing very-low-density lipoproteins (vldl) export as shown for a missense mutation (e k) in transmembrane superfamily member (tm sf ) [ ] . nevertheless, nafld is tightly associated with common acquired metabolic diseases such as obesity and type diabetes (t dm). the mutual relationship between both diseases is illustrated by several epidemiological data. the prevalence of steatosis and nash has been estimated to be and %, respectively, in t dm [ ] . the age-adjusted relative risk of nafld is about . fold higher in t dm compared to healthy humans [ ] . t dm is also an emerging risk factor for nash progression to advanced fibrosis, cirrhosis and hcc [ , ] . diabetes was even a better predictor for hcc development in people with nash and cirrhosis compared to other metabolic risk factors such as hyperlipidemia, body mass index (bmi) and hypertension [ ] . recently, a consensus panel has proposed to rename nafld a metabolic-dysfunction-associated fatty liver disease (mafld) based on the presence of overweight/obesity, t dm and evidence of so-called "metabolic dysregulation" [ ] . future will tell, if this will help to better understand the multiple relationships between nafld and t dm. in this context, nafld per se associates with more than double risk of incident diabetes pointing to specific liver-related mechanisms [ , ] . moreover, multicenter studies in skandinavia and germany have recently found that diabetes can be stratified into subtypes j o u r n a l p r e -p r o o f the circulation. on the contrary, a recent study demonstrated that obesity-induced insulin resistance preceded inflammation in adipose tissues of mice [ ] . indeed, adipose tissue inflammation might be a protective feedback mechanism as its local inhibition in mice induced ectopic lipid accumulation in liver, glucose intolerance, and systemic inflammation [ ] . besides released inflammatory cytokines and ffa, adipose tissue could still communicate with the liver and muscle through secretion of different adipokines such as adiponectin and leptin. persons with nash have lower serum adiponectin compared to those with nafld with or without normal liver enzymes [ ] . by contrast, the circulating levels of leptin were higher in people with nafld and t dm, probably due to increased leptin resistance, and were associated with disease severity [ ] . increased ffa influx to skeletal muscle promotes accumulation of intramyocellular lipid (imcl). reduced mitochondrial oxidation contributes as well to imcl as shown in aging and insulin-resistant humans [ ] . consequently, skeletal muscle exhibits insulin resistance, which often precedes the onset of t dm and insulin resistance in the liver [ ] . lipid intermediate metabolites, in particular sn , diacylglycerols (dag), link imcl to skeletal muscle insulin resistance through activation of protein kinase c-theta (pkcθ) resulting in its translocation from cytoplasm to the plasma membrane [ ] . muscles of insulin resistant humans with obesity and t dm showed increased dag content and pkcθ activity as compared to healthy humans [ ] . mutation studies highlighted serine amino acid residue (ser ) of irs to be a substrate for activated pkcθ [ ] . as a consequence of skeletal muscle insulin resistance, postprandial energy storage shifts from glycogen synthesis in the muscle into triacylglycerol (tag) in the liver, promoting nafld development [ ] . gut microbiome is increasingly recognized as a modulator of liver pathogenesis through what is called the "gut-liver axis" [ ] . distinctive alterations of gut microbiome were reported in humans having nash and t dm [ ] . the intestinal microbiome alters host metabolism by modulating the production of short-chain fatty acids (scfa) e.g. butyrate, acetate, and propionate, which have beneficial effects on insulin sensitivity, lipid and glucose metabolism [ ] . also, intestinal dysbiosis could associate with increased intestinal permeability permitting translocation of bacterial lipopolysaccharide (lps) into the systemic circulation, j o u r n a l p r e -p r o o f which could induce fat deposition in the liver, nash progression, weight gain, and diabetes [ ] . moreover, intestinal microbiome could suppress the expression of fasting-induced adipocyte factor (fiaf) in intestinal epithelium, which functions as an inhibitor of circulating lipoprotein lipase, resulting in increased tag storage in the peripheral tissues [ ] . also, ethanol-producing microbiome could increase blood alcohol concentration in nash, which is metabolized in the liver generating high levels of reactive oxygen species (ros). the last mechanism could explain the histological similarity between nash and alcoholic steatohepatitis [ ] . furthermore, microbiota metabolize liver-derived primary bile acids into secondary bile acids. the latter are reabsorbed into bloodstream and may act as signaling molecules via a variety of receptors including farnesoid x receptor (fxr), which regulates the transcription of different metabolic genes involved in bile acid synthesis, transport, lipogenesis, and glucose homeostasis [ ] . insulin resistance in both skeletal muscle and adipose tissues initiates liver steatosis by providing precursors and substrates for dnl and mitochondrial β-oxidation e.g. glucose, ffa and glycerol [ ] . although reesterification of ffa derived from diet and adipose tissue is the dominant contributor to tag pool in the liver ( %), it did not increase in people with nafld. on the other side, dnl-derived ffa contribute by about %, which is severalfold higher as compared to individuals without nafld ( %) [ ] . later, insulin resistance of the liver develops, resulting in increased gluconeogenesis and elevation of endogenous glucose production (egp) from the liver, which contributes to fasting hyperglycemia in individuals with t dm [ ] . insulin signaling inhibits typically hepatic gluconeogenesis through akt-induced phosphorylation of forkhead box (foxo ) and induces lipogenesis through activation of sterol regulatory element-binding proteins (srebp c) and mammalian target of rapamycin complex (mtorc ) pathways. during hepatic insulin resistance, insulin does not suppress gluconeogenesis efficiently, while dnl is preserved or even increased. to explain this discrepancy, pathway-selective hepatic insulin resistance was postulated, which means that only one arm of insulin signaling is defective i.e. akt/foxo leading to reduced insulin-mediated suppression of gluconeogenesis, whereas insulin-activated srebp- c/mtorc pathway remains intact and activates lipogenesis [ ] . actually, dnl was reduced after induction of hepatic insulin resistance by feeding mice with j o u r n a l p r e -p r o o f a high-fat diet (hfd) [ ] , which challenges the concept of selective hepatic insulin resistance and suggest the existence of alternate mechanisms that contribute to increased dnl and gluconeogenesis in insulin-resistant liver, for example, hyperinsulinemia, insulinindependent re-esterification of adipose tissue-derived ffa, and increased acetyl coa generation from β-oxidation of ffa, which allosterically activates pyruvate carboxylase enzyme, leading to enhanced gluconeogenesis [ ] . in addition, increased blood glucose level can activate carbohydrate response element-binding protein (chrebp) signaling pathway, thereby stimulating expression of several glycolytic genes, which provide additional metabolic precursors for dnl [ ] . in line, chrebp overexpression induced stearoyl-coa desaturase (scd ) expression, an enzyme responsible for the biosynthesis of monounsaturated fatty acids (mufa), resulting in increased liver fat content [ ] . pkc epsilon (pkcε) is crucial in mediating hepatic insulin resistance and once activated by dag, it translocates to the cell membrane, and phosphorylates specific threonine residue (thr in human and thr in mouse) on insulin receptor leading to destabilization of the active configuration of insulin receptor kinase and inhibition of its tyrosine kinase activity [ ] (figure ). in general, both hyperglycemia and toxic lipids such as ceramides, dag, ffa, and cholesterol can induce deleterious effects on liver cells (glucolipotoxicity), which might initiate nafld progression from simple steatosis to nash and fibrosis via various mechanisms, including cell death, oxidative stress, endoplasmic reticulum (er) stress and mitochondrial disorders [ ] . alterations in the activity and abundance of oxidative phosphorylation (oxphos) proteins and antioxidant enzymes were described in various animal models of nafld [ ] . impaired hepatic mitochondrial function was evident as well in t dm and nash [ , ] . in a mouse model of choline-deficient diet-induced nafld, mitochondrial oxphos was increased at weeks but lost at a later time point [ ] . also, the higher maximal respiration rate of liver mitochondria was severalfold higher in insulin-resistant obese individuals with fatty liver as compared to lean individuals [ ] . these studies highlight the flexibility of mitochondria to adapt to increased metabolic inputs to keep energy homeostasis and to protect against the harmful effects of increased ffa and tag in the liver. in nash, mitochondrial flexibility was lost, which was associated with increased ros production and exhaustion of protective antioxidant enzymes [ ] (figure ). whether loss of mitochondrial flexibility is a cause or consequence for nafld progression remains obscure. depletion of atp due to j o u r n a l p r e -p r o o f mitochondrial disorders, together with hyperglycemia and lipid overload could be inducers for another signaling pathway, termed unfolded protein response "upr", which is adaptive response to resolve unfolded or misfolded proteins and to restore er homeostasis [ , ] . prolonged upr stress can activate jnk and nf-kb signaling pathways, which are involved in insulin resistance, liver steatosis, and inflammation [ ] . furthermore, er stress could increase insulin resistance through induction of lipin- expression, which is a phosphatase enzyme that catalyzes biosynthesis of dag leading to activation of dag/pkcε axis [ ] . interestingly, upr could also increase liver steatosis through activation of srebp- c signaling pathway [ ] . elevated ros and upr are well-identified pathways that could induce hepatocytes death in nash. hepatocytes apoptosis and necroptosis are the main forms of cell death in human steatohepatitis and diet-induced mouse models of nash [ ] . the key fibrogenic liver cells, hepatic stellate cells (hscs), usually exist in a quiescent state and get activated by engulfment of apoptotic bodies, the inflammatory milieu, or damage-associated molecular patterns (damps) released from stressed and dying hepatocytes [ ] . interestingly, ffa-mediated lipotoxic effects stimulate hepatocytes to release extracellular vesicles (evs) with distinctive micrornas (mirna) profile that increase the expression of fibrogenic genes in the surrounding hscs [ ] (figure ). free cholesterol could directly sensitize hscs to the action of tumor growth factor (tgf)-β, a potent fibrogenic cytokine [ ] . treatment of human and rat immortalized hscs cell lines with saturated fatty acid increased the expression of various profibrogenic genes [ ] . macrophages aggregate as well around dead hepatocytes forming a crown-like structure, a phenomenon that exists only in persons with nash but not in those with simple steatosis [ ] . recruitment of more inflammatory cells from systemic circulation is facilitated by "find me" signals that are released from dead cells [ ] . inhibition of inflammatory monocytes recruitment via inhibition of c-c chemokine receptors type and type (ccr /ccr ) suppressed fibrogenesis and steatohepatitis in murine nash [ ] . macrophages can modulate also hepatic insulin resistance and favor tag accumulation in hepatocytes through secretion of il- β that downregulates peroxisome proliferator-activated receptor (ppar) α, a key transcriptional pathway involved in fatty acid oxidation [ ] . liver sinusoidal cells (lsecs) are fenestrated cells that exist in close vicinity to hepatocytes, j o u r n a l p r e -p r o o f macrophages and hscs, which, under physiological conditions, regulate lipid transport, maintain the quiescence of kupffer cells, resident liver macrophages, and hscs [ ] . at early stage of nafld, lsecs lose their fenestrae, a process termed capillarization, which could favor liver steatosis and initiate hscs activation [ ] (figure ). during nash, lsecs display a pro-inflammatory phenotype that promotes steatohepatitis [ ] . autophagy is a self-degradative process which is used by the cells to remove misfolded proteins and damaged organelles [ ] . singh et al. showed that the cells can use autophagic process as lipolytic mechanism to mobilize lipids from intracellular lipid store, which is termed in this case "macrolipophagy" [ ] . various in vitro and in vivo studies showed that autophagy was decreased in fatty hepatocytes [ ] . impairment of autophagy by palmitic acid in macrophages induces inflammatory il- β production [ ] . on the other side, selective loss of autophagic activity reduced liver fibrogenesis in cultured hscs [ ] and protected against diet-induced insulin resistance [ ] . the results highlight that the net effect of autophagy on nafld might depend on the tissue or type of the cells that show autophagic dysfunction and the stage of nafld. the liver biopsy is considered the gold standard for nafld diagnosis, especially for distinguishing steatosis from inflammation and fibrosis [ ] . nevertheless, this technique has several limitations not only resulting from the invasive procedure and rare post-interventional complications, but also due to the small tissue volume, which might not be representative for the whole liver and may not take into account inhomogeneous distribution of nafldassociated alterations. novel imaging modalities such as ultrasound-or mr-based techniques are of increasing value, as recently reviewed [ ] , but are still not generally available, so that there is an urgent need for noninvasive screening tools. non-invasive detection of nash and fibrosis remains challenging today. biomarker-based panels such as aspartate aminotransferase (ast)/platelets count ratio index, fibrosis- (fib- ) index, fibrotest, fibrospect ii, and nafld fibrosis score (nfs) offer variable diagnostic efficacy for assessing different stages of liver fibrosis [ , ] . although combination of these panels could enhance their predictive values [ ] , they still suffer from limited accuracy even compared to the alanine aminotransferase (alt) and ast, particularly in t dm [ ] . in this regard, transient elastography looks like a promising alternative in diabetes clinics for detection of liver fibrosis in nafld [ ] . numerous biomarkers have been developed to specifically track features of nash progression and fibrosis. cytokeratin (ck) , an intermediate filament protein that is cleaved during cell death to ck m and ck m , has been intensively investigated as a surrogate of nash-associated liver cell damage, but a recent meta-analysis of studies reported a maximum sensitivity of . for nash diagnosis [ ] and suboptimal diagnostic value was shown in t dm [ ] . the ecm turnover marker, type iii procollagen, can offer a diagnostic efficacy of . and . to detect moderate and advanced liver fibrosis in t dm, respectively [ ] . in , the european associations for the study of the liver, obesity and diabetes (easl-easo-easd) jointly released recommendations for diagnosis and monitoring of disease severity in persons with suspected nafld and metabolic risk factors [ ] . people with metabolic risk factors, such as t dm, should undergo assessment by abdominal ultrasound, serum transaminases and fibrosis markers (e. g. fib- , nfs). elevated transaminases or steatosis plus abnormal fibrosis test shall require referral to a specialist. this strategy has raised the question of a possible overreferral when adhering to these guidelines [ ] . however, recent analyses show that a refined strategy of specific combining indices such as fli and fib- could reduce the number of people with t dm for further diagnostic work up to a reasonable size [ ] . this retrospective analysis also found that certain non-invasive biomarkers are consistently associated with different patterns of diabetes-related complications. analyses of the plasma metabolomic of insulin-resistant individuals with nafld suggested that bile salts, e. g. glycocholate, taurocholate, and glycochenodeoxycholate allow to detect nash in one study [ ] , while another study reported an association with insulin resistance j o u r n a l p r e -p r o o f but not with liver necroinflammation [ ] . also amino acids, specifically a glutamate-serineglycine index, was associated with hepatic insulin resistance and transaminases and discriminated individuals with fibrosis grade - from those with grade - independently of bmi [ ] . the diagnostic performance of a serum-based lipidomic analysis was substantially lower for nafld detection in t dm compared to healthy individuals [ ] . nevertheless, certain sphingolipid species were recently found to be increased in insulin-resistant nash and to correlate with hepatic oxidative stress and inflammation [ ] . one circulating small noncoding rna, mirna- , was found to be more than . fold in steatosis and further doubled in nash [ ] . this mirna was also higher in t dm with nafld than in those without nafld and provided better prediction of nafld when combined with ldl and waist-to-hip ratio [ ] . moreover, extracellular vesicles (ev), which among other cargo also transport mirna, may be promising biomarkers for nafld as shown by higher serum levels in people with nafld, obesity and diabetes [ ] . recently, metagenomics data derived from gut microbiota alterations allowed to detect advanced fibrosis in nafld patients, of whom % had t dm, with a robust diagnostic accuracy (auroc: . ) [ ] . the current guidelines recommend only lifestyle modification and -for certain groupsbariatric or metabolic surgery to treat nafld [ , ] . although the numerous activities in this field, no pharmacological treatment is currently approved or expecting approval for the use in nafld with or without concomitant t dm (table and ), except for obeticholic acid (oca). marketing authorization application for oca has been submitted to the u.s. food and drug administration (fda) and european medicines agency (ema), awaiting fda decision by june [ ] . against this background, current guidelines and recommendations primarily advise lifestyle modification (healthy nutrition and exercise) and weight loss in overweight/obese persons. the easl-easo-easd guidelines recommend to consider pharmacological treatment in people with nash when combined with fibrosis and in those with less severe disease, but high risk conditions for disease progression such as t dm [ ] . the american association for the study of liver diseases (aasld) recommends to limit pharmacological treatment to those with biopsy-proven nash and fibrosis [ ] . j o u r n a l p r e -p r o o f weight management and physical exercise are key to the treatment of both nafld and t dm. a proof-of-concept study showed that a hypocaloric diet with weight loss of ~ kg within weeks not only normalized fasting egp and thereby hyperglycemia, but decreased hepatic tag content down to normal concentrations in obese t dm humans with nafld [ ] . subsequent studies in larger cohorts extended these results by demonstrating that a very low-caloric diet with weight loss of about % rapidly normalized liver fat content in in dividuals with t dm, which persisted for one year if weight loss was maintained [ ] . interestingly, mediterranean, low-saturated fat and high plant-based protein diets also improve steatosis in nafld combined with t dm despite minor or no relevant weight loss [ , ] . in addition, physical activity and exercise training interventions can also decrease liver fat content, which may not be exclusively depending on concomitant weight loss [ ] . although the beneficial effects of structured behavioral treatment to improve histological endpoints, likely extend beyond reduction of hepatic fat content to ameliorating the grade and stage of inflammation and fibrosis [ ] , only a minority of the people manages to adhere to dietary weight loss and exercise programs, which raises the issue of other therapeutic approaches [ ] . weight-loss inducing drugs could be an attractive option for persons with nafld with a bmi > kg/m or > kg/m in the presence of at least one metabolic comorbidity, as an adjunct treatment to lifestyle modifications [ , ] . currently, the most-popular weight-loss inducing medications associated with at least % body weight decrease in one year as compared to placebo are orlistat, the fixed combination of phentermine and topiramate or naltrexone and bupropion, and the glucagon-like peptide- (glp- ) receptor agonist (glp- ra), liraglutide [ ] . of these drugs, orlistat treatment failed to improve liver histology when compared to placebo [ ] , while no reports are available for topiramate, naltrexone, bupropion and phentermine regarding hepatic endpoints in humans with nafld [ ] . only bariatric or metabolic surgery is a therapeutic option to induce sustained weight loss partricularly in people with combined nafld and t dm. in obese humans, bariatric surgery resulted in % resolution of nash within one year [ ] and in % complete remission of t dm [ ] . surgical weight loss improves glucose metabolism and insulin sensitivity by different mechanisms such as increased glp- secretion [ ] and epigenetic modification [ , ] . several antihyperglycemic drug classes were or are currently being investigated in clinical trials and preclinical models to evaluate their efficacy for people with nafld and with or without t dm. metformin reduces body weight, hepatic gluconeogenesis -by yet unclear mechanisms [ ] , and the risk of macrovascular complications, which is still controversially discussed due to lack of optimally designed clinical trials [ ] . despite its action on hepatic metabolism, former small-scale studies reported conflicting results [ , ] (table ) and recent metaanalysis failed to demonstrate any effect of metformin on liver histology [ ] . nevertheless, epidemiological, observational and preclinical studies suggest that metformin may reduce the risk of hcc and also in t dm, possibly by promoting apoptosis, but controlled clinical trials are not available [ ] . dpp degrades incretins such as glp- so that dpp- i treatment increases the postprandial levels of endogenous glp- , which leads to lower glucose peaks after meals [ , ] . in individuals with nafld with prediabetes or recent onset diabetes, sitagliptin did not improve liver steatosis or liver fibrosis compared to placebo as assessed by mr-based techniques [ ] . in line, another recent trial reported no benefits for sitagliptin versus placebo on liver fibrosis or steatohepatitis in t dm [ ] (table ) . in contrast, a moderate reduction in liver fat content was observed with vildagliptin in individuals with t dm [ ] . j o u r n a l p r e -p r o o f the actions of endogenous glp- are mimicked by glp- receptor agonists (glp- ra), which effectively reduce blood glucose levels and also reduce the risk for cvd in t dm [ ] . in individuals with nafld and t dm, liraglutide in combination with metformin reduced liver, subcutaneous, and visceral fat [ ] . also, liraglutide improved hepatic steatosis measured by mr-based methods as well as resolved biopsy-proven nash without worsening of fibrosis [ , ] (table ) . however, a recent subanalysis did not detect an effect of liraglutide on liver fat content quantified by h-mrs [ ] (table ) . respective trials with semaglutide and dulaglutide are still waiting for results (nct , nct ). in an animal model of t dm, exenatide also counteracted hcc development by suppression of stat -regulated genes [ ] . glucose-dependent insulinotropic polypeptide (gip) represents the second important incretin with proposed beneficial effects on peripheral energy metabolism [ ] . tirzepatide, a novel dual agonist for gip and glp- receptors with probably greater efficacy than glp- ra [ ] , decreased transaminases and surrogate markers of liver injury paralleled by increased circulating adiponectin levels in people with t dm [ ] . a clinical trial on nash resolution is currently ongoing in persons with nash with or without t dm (nct ). sglt i inhibit reabsorption of glucose in the proximal renal tubule resulting in glucosuria and calory loss, thus effectively reducing blood glucose level and body weight in t dm. moreover, sglt is show beneficial effects on cardiovascular risk and progression of nephropathy [ , ] . most, but not all recent randomized controlled trials (rcts) showed that sglt i treatment resulted in reduction in liver fat content compared to placebo [ ] [ ] [ ] [ ] [ ] (table ) . in an open-label pilot study in liver-biopsy proven cohort of nash with t dm, treatment with empagliflozin for months reduced liver steatosis, ballooning and fibrosis and induced nash resolution in approximately half of those persons [ ] . animal models further suggested anti-inflammatory, anti-oxidant and pro-apoptotic actions of sglt i with cardio-protective effects as well as inhibition of nash progression and tumor growth of hcc [ , ] . the dual sglt / i, licogliflozin, is expected to block both intestinal and renal glucose (re)absorption [ ] and currently investigated to evaluate its efficacy on resolution of nash as monotherapy and as combination therapy with the fxr agonist, tropifexor, in people with nash and fibrosis (nct ). first results hint at improvement of transaminases and reduction of steatosis by licogliflozin in nash [ ] . ppars is a family of nuclear receptors composed of multiple isoforms with wide tissue distribution [ ] . the ppar-γ agonist pioglitazone had convincing efficacy on nash resolution in individuals with and without t dm, but with conflicting results on fibrosis [ ] [ ] [ ] (table ) . of note, pioglitazone has been withdrawn in many european countries because of its disadvantageous safety profile [ ] . however, pioglitazone exerts beneficial effects on cardiovascular outcomes in persons with t dm and a history of cvd [ ] . it has been proposed that non-ppar-γ dependent mechanisms, as the inhibition of the mitochondrial pyruvate carrier (mpc), might mediate the beneficial effects of pioglitazone on nafld [ ] . however, a novel drug with ppar-γ sparing effects and mpc binding activity did not improve histological components of nash in individuals with t dm compared to placebo [ ] . elafibranor is a dual agonist for ppar-α and ppar-δ without ppar-γ stimulation [ ] . based on a post-hoc analysis, nash was resolved without worsening of fibrosis in a higher percentage of people with and without t dm in the elafibranor group as compared to the placebo [ ] and a follow-up study on these findings has been initiated ( table ) . recent announcements on an interim analysis state that elafibranor treatment failed to resolve nash and improve fibrosis when compared to placebo [ ] . saroglitazar is another dual ppar-α/γ agonist with higher affinity for ppar-α. in a mouse model of nash, saroglitazar reduced liver steatosis, inflammation and prevented fibrosis development and a respective clinical trial is ongoing [ ] ( table ) . lanifibranor is a pan-ppar agonist for ppar-α, β, and γ receptors focused on treatment of liver fibrosis and nash (nct ). in an animal model of liver fibrosis, lanifibranor decreased hepatic collagen deposition [ ] . several other strategies for pharmacological targeting of nash have emerged over the last few years; however, these strategies are not specifically designed for t dm, but for the whole nafld collective. they comprise antiinflammatory drugs, but also modulators of other pathways, which are briefly summarized in the following sections. j o u r n a l p r e -p r o o f fxr can be activated by bile acids in a negative feedback mechanism to suppress bile acid synthesis [ ] . oca is a potent semisynthetic and selective fxr agonist approved for treatment of primary biliary cholangitis [ ] . oca treatment resulted in histologic improvement of nash in people with and without t dm compared to placebo [ , ] ; however, concerns were raised about oca-induced changes in plasma lipoprotein profile [ ] . tropifexor, another potent fxr agonist [ ] , is currently being tested in combinatorial approaches of nash treatment (cenicriviroc and licogliflozin; nct ). oltipraz is a synthetic dithiolethione with antisteatotic effects by inhibiting the activity of lxr-α. it activates adenosine monophosphate activated protein kinase (ampk) and inactivates s k , affecting lxr-α thus reducing lipogenesis and increasing lipid oxidation [ ] . a recent -week phase clinical trial found decreased steatosis measured by h-mrs with oltipraz compared to placebo treatment [ ] and a respective phase clinical trial is ongoing ( table ). chemokine receptors type (ccr ) and type (ccr ) are expressed on various inflammatory and fibrogenic cells [ ] . cenicriviroc is a ccr /crr dual antagonist that reduced insulin resistance, liver inflammation and fibrosis in diet-induced models of nash [ ] . in recent rcts in a nash cohort with fibrosis, cenicriviroc treatment did not improve nas but may reduce liver fibrosis [ , ] . currently, there is an ongoing clinical trial to evaluate the effects of cenicriviroc on fibrosis in nash ( table ) . both people with obesity, metabolic syndrome or t dm as well as those with nafld are at increased risk for dyslipidemia. statins, inhibitors of -hydroxy- -methyl-glutaryl-coenzyme a (hmg-coa) reductase, are generally safe and have unmet efficacy to decreased serum ldl and prevent cardiovascular outcomes, despite slightly increasing the risk of t dm [ ] . use of statins associated with a % lower relative risk of hepatic decompensation and mortality in cirrhosis and a trend towards lower fibrosis progression in non-cirrhotic liver diseases [ ] . however, the data on liver histology ist limited [ ] so that statins are not currently recommended for the management of nafld [ ] . the inhibitor of intestinal j o u r n a l p r e -p r o o f cholesterol absorption, ezetimibe, decreased the histological nas, but not consistently liver fat content in an analysis of the few available studies [ ] . fenofibrate, a ppar-α agonist, does not affect or even increase liver fat content or volume [ , ] . nevertheless, the combination of statins with certain anti-nash drugs, such as oca, could be beneficial to counteract drug-related increases in ldl during long-term treatment. inhibition of dnl in nafld may be achieved by inhibition of acc as this enzyme catalyzes the conversion of acetyl coa into malonyl coa, which acts as a substrate for fatty acid synthesis and inhibitor for fatty acid β-oxidation [ ] . in a recent clinical trial in individuals with nash with and without t dm, treatment with the dual acc and acc inhibitor gs- decreased liver steatosis without improvement of fibrosis [ ] . the major concern of this strategy is that decreased dnl in nafld might channel lipids towards other harmful directions, e.g. increased blood lipids [ ] . scd is a key enzyme for hepatic lipogenesis that catalyzes the conversion of saturated fatty acids to mufa and its downregulation protected mice from high carbohydrate-induced liver steatosis [ ] . a clinical trial with aramchol, an inhibitor of scd , showed a reduction in liver fat content, resolution of nash and improvement of liver fibrosis in persons with nafld with prediabetes or t dm [ ] . these results paved the way for a respective phase clinical trial ( table ) . mitochondrial uncoupling describes any process that uncouples the electron transport from atp synthesis in mitochondria [ ] . , -dinitrophenol (dnp) was widely used for the treatment of obesity before its discontinuation due to life-threatening serious adverse events [ ] . to overcome the side effects of dnp, improved formulas of dnp were recently developed to decrease the toxic to effective dose ratio (dnp-methyl ether (dnpme) and controlled-release mitochondrial protonophore (crmp)) [ , ] . oral crmp targets mainly the liver due to the first-pass effect and decreased hepatic insulin resistance, hepatic steatosis and liver fibrosis in a methionine-choline deficient rat model of nash [ ] . crmp was also effective in nonhuman primates with diet-induced nafld for reduction of hepatic steatosis and egp [ ] . thyroid hormone deficiency has been associated with nafld development [ ] . thyroid hormone analogues decreased liver fat, liver transaminases, and inflammatory and fibrosis markers in animal models of nash [ ] . resmetirom is a liver-targeted highly selective thr-β agonist which showed efficacy in reducing liver fat content in nash with and without t dm [ ] . currently, there is ongoing clinical trial for evaluation of long-term outcomes of resmetirom and its efficacy on nash resolution ( table ). vitamin e is a potent antioxidant [ ] . in nash without t dm, vitamin e improved nas without worsening of fibrosis [ ] . despite concerns regarding adverse effects of long term vitamin e usage, treatment with vitamin e for ≥ years reduced the risk of liver failure in a nash cohort with advanced fibrosis and with or without t dm [ ] . therapeutic manipulation of intestinal microbiome is still in its infancy. rodent data showed efficacy for fecal microbiota transplantation in improving nafld in a diet-induced nash model [ ] . also, modulation of the intestinal microbiota by antibiotic treatment reduced liver transaminases in nafld [ ] . the use of prebiotics and probiotics in obese nafld/nash is currently not supported by high-quality clinical studies with mr-or biopsy-based endpoints [ ]. the evidence for shared pathophysiological mechanisms between t dm and nafld will help to develop strategies for detecting and treating both diseases and preventing their leading complication, cvd. altered lipid and energy metabolism, insulin resistance, low-grade inflammation and intestinal dysbiosis represent key targets. in addition to weight loss by lifestyle modification or bariatric surgery, glp- ra and sglt i are promising antihyperglycemic concepts with beneficial effects on nafld and cvd. in addition, many j o u r n a l p r e -p r o o f metabolism-based drugs are currently studied comprising ppar agonists, endocrine dual coagonists to modulators of hepatic metabolism or microbiota. nevertheless, several roadblocks need to be overcome to reduce the burden of nafld in t dm. first, there is still lack of preclinical animal models that encapsulate essential features of human nash and diabetes. second, available biomarkers lack diagnostic efficacy to identify nafld progression. innovative strategies such as cluster analysis already enabled detection of a diabetes subtype (sird) with high risk of nafld [ ] . combination with computational integration of multiomics data shall identify specific disease signatures and pave the way to precision medicine and targeted management of t dm and nafld. finally, studies on so-called endpoints are scarce, which may be due to the need of long-term studies to evaluate liver-related mortality, to the current neglect to accept cvd morbidity and mortality as nafld outcome and to ongoing discussions on the relevance of surrogate markers. the research of the authors is supported in part by grants from the german federal ministry phase iii registered interventional trials on "clinicaltrials.gov" for nafld as accessed on th april . bl, baseline; ccr / , c-c chemokine receptors type and type ; fxr, farnesoid x receptor; hba c , glycated haemoglobin; lxr, liver x receptor; mpc, mitochondrial pyruvate carrier; na, data not available; nafld, non-alcoholic fatty liver disease; nfs, nafld fibrosis score; ppar, peroxisome proliferator-activated receptor; nash, non-alcoholic steatohepatitis; scd, stearoyl-coa desaturase; sglt, sodium-glucose cotransporter; thr, thyroid hormone receptor; t dm, type diabetes. *only placebo-controlled, randomized clinical trials are listed, except for saroglitazar, a -arm active-controlled study. the diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the american association 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total fecal microbiota transplantation alleviates high-fat diet-induced steatohepatitis in mice via beneficial regulation of gut microbiota efficacy of rifaximin on circulating endotoxins and cytokines in patients with nonalcoholic fatty liver disease key: cord- - qzpo l authors: adalja, amesh a.; watson, matthew; toner, eric s.; cicero, anita; inglesby, thomas v. title: characteristics of microbes most likely to cause pandemics and global catastrophes date: - - journal: global catastrophic biological risks doi: . / _ _ sha: doc_id: cord_uid: qzpo l predicting which pathogen will confer the highest global catastrophic biological risk (gcbr) of a pandemic is a difficult task. many approaches are retrospective and premised on prior pandemics; however, such an approach may fail to appreciate novel threats that do not have exact historical precedent. in this paper, based on a study and project we undertook, a new paradigm for pandemic preparedness is presented. this paradigm seeks to root pandemic risk in actual attributes possessed by specific classes of microbial organisms and leads to specific recommendations to augment preparedness activities. the recent global experience with severe infectious disease epidemics has triggered much interest in understanding the broader pandemic threat landscape. a substantial proportion of pandemic and biological threat preparedness activities have focused on list-based approaches that were in part based on pandemic influenzas of the past, historical biological weapon development programs, or recent outbreaks of emerging infectious diseases (e.g., sars, mers, ebola) (centers for disease control and prevention ; casadevall and relman ) . but such an approach inherently fails to account for agents not currently known or those without historical precedent. for that reason, preparedness activities that are limited to these approaches may hamper preparedness and lessen resilience. the purpose of this study was to analyze the characteristics of pathogens that could be capable of causing a global catastrophic biological risk (gcbr). these would be events in which biological agents-whether naturally emerging or reemerging, deliberately created and released, or laboratory engineered and escaped-could lead to sudden, extraordinary, widespread disaster beyond the collective capability of national and international governments and the private sector to control. if unchecked, gcbrs would lead to great suffering, loss of life, and sustained damage to national governments, international relationships, economies, societal stability, or global security (schoch-spana et al. ) . given the severe potential public health consequences of pandemic events, there needs to be a vital interest in developing and maintaining a flexible, rapid, and robust response capability. anticipating the forms of microbial threats that might cause future pandemics can help strengthen preparedness and response capacities. this paper proposes a framework for considering future pandemic threats and provides recommendations for how this framework should inform pandemic preparedness. review of the published literature and previous reports: the project team surveyed the current biomedical literature on the topic of emerging infectious disease characteristics, the pathogenic potential of microbes, and related topics. the literature review was microbe-and species-agnostic, encompassing all classes of microorganisms and host species. the literature review was accomplished with extensive pubmed searches on these subjects. relevant us government policy and strategy were reviewed. interviews: the project team interviewed more than technical experts who work in and are intimately knowledgeable about this field. interviewees were drawn from academia, industry, and government. our goal was to ascertain the experts' views about the essential traits needed for a pathogen to become a gcbr, to contextualize historical outbreaks in light of these traits, and to determine which currently known infectious disease agents possess such characteristics. pandemic pathogen meeting: the project team completed a preliminary analysis that synthesized the results of our literature review and expert interviews. those findings were used to design and facilitate a meeting held on november , , that included many of those who had been interviewed for this project. the meeting was held at the johns hopkins center for health security in baltimore, md. the purpose of the meeting was to gain additional insight and input into the project analysis, examine assumptions, and test possible recommendations. participants included representatives of us and foreign academic institutions, the federal government, and other independent subject matter experts. this paper is based on the findings of the project and is modification of the project report (johns hopkins center for health security ). when a pathogen has the capacity to cause a pandemic, it will possess several attributes that other microbes, capable of causing only sporadic or limited human infections, will lack. these traits can be divided into several categories: spread via respiratory transmission; capable of spread during incubation period prior to symptom onset; no preexisting host immunity; and other possible intrinsic microbial characteristics. many of these characteristics have been captured and are reflected, in equation form, by casadevall (casadevall ) . microbes have varied routes of transmission, ranging from blood and body fluids to vector-borne to fecal-oral to respiratory (airborne and respiratory droplet). while each mode of transmission is capable of causing large outbreaks if sustained human-to-human transmission is possible and left unchecked, certain modes of transmission are more amenable than others to intervention. for example, the transmission of an infectious disease caused by blood and body fluid transmission can be halted with infection control measures such as gloves or gowns. of the various modes of transmission, the respiratory route is the mechanism most likely to lead to pandemic spread. this is chiefly due to the fact that interventions to interrupt this method of spread are more difficult to implement when the simple and universal act of breathing can spread a pathogen. the prolific spread of influenza, pertussis, measles, and rhinoviruses is testament to this fact (herfst et al. ) . by contrast, although pathogens spread by the fecal-oral route, such as vibrio cholera and the hepatitis a virus, can generate explosive outbreaks, even a modicum of sanitary infrastructure can quench the outbreak. vector-borne outbreaks are a special case of a non-respiratory-spread agent. indeed, the only postulated extinction of a mammalian species by an infectious organism, the christmas island rat, was caused by a vector-borne trypanosome (wyatt et al. ) . for most of the agents that use this class of transmission, the spread is limited by a geographically and climatologically restricted vector habitat. humans can protect against vectors, and they can change where they live, but the christmas island rat could not. these factors have generally served to limit the pandemic potential of microbes that are spread by vectors. exceptions to this general limitation of vector-borne viruses include microbes spread by anopheles and aedes mosquitoes. pathogens spread by these mosquitoes have higher pandemic potential, given the geographic breadth of their spread. for example, most of sub-saharan africa is hospitable to the malaria-transmitting anopheles mosquitoes, while residents in % of us counties-as well as half the world's population-are regularly exposed to aedes mosquitoes that serve as vectors for high viremia flaviviruses and alphaviruses. such phenomena are borne out by the prolific spread of dengue, chikungunya, and zika (sinka et al. ; centers for disease control and prevention ). the onset and duration of the period when a person is contagious during an infection also play a major role in spread. diseases that are contagious during a late stage of infection, when infected people are very sick and therefore have more limited opportunities for spread, may be delimited in their spread. on the other hand, diseases that are contagious prior to symptom development, during the incubation period, or when only mild symptoms are present have greater opportunities for spread as infected individuals are able to conduct their activities of daily living with little or no interruption. modeling studies with simulated outbreaks have shown that the presence or absence of this timing of transmission factor can be decisive in whether an outbreak can or cannot be controlled. if a microbe is contagious before a person is seriously ill while the disease is still incubating, then there is higher potential for pandemic spread. historical examples reinforce this idea, as the only human infectious disease to be vanquished from the planet-smallpox-was one that was not contagious during the incubation period (fraser et al. ) . by contrast, a microbe such as the influenza virus, which is contagious prior to symptom development and has a wide range of clinical severity, is able to infect widely and is not amenable to control (brankston et al. ). microbial pathogenicity cannot, in reality, be separated from host characteristics. as elucidated by pirofsky and casadevall's host damage framework, disease is a complex interplay between a host immune system and a microbe (pirofski and casadevall ) . in congruity with this paradigm, host features and microbial pathogenicity are discussed together. for a microbe to cause a gcbr-level pandemic, it will be necessary for a significant proportion of the human population to be immunologically naïve to the agent so that the microbe would have a high number of susceptible humans to infect. additionally, large quantities of a sufficiently effective countermeasure (vaccine or antimicrobial agent) would not be available. immunologic naïveté would be expected with a zoonotic pathogen. the microbe, correspondingly, would have to possess the ability to evade the host immune response through virulence factors, immunological camouflage, or other features that allow a productive infection to ensue. additionally, human receptors that are utilized by a pandemic-causing microbe would likely be widespread in the population, facilitating permissive infection in the majority of humans. receptors may also provide target organ tropism for the agent, allowing severe disease to occur (e.g., lower respiratory tract and central nervous system). case fatality rates (cfrs) need not be inordinately high to cause a gcbr-level event, as evidenced by the . % cfr reported for the influenza pandemic-the event closest to an actual human gcbr in the modern era (taubenberger and morens ) . a low but significant cfr adheres to the host density threshold theorem. according to this commonly held theorem, a microbe that kills too many of its hosts will run out of susceptible hosts and be extinguished (cressler et al. ) . while this may be true of pathogens that are closely linked to one host species, it is not applicable to sapronotic diseases such as amebic encephalitis and cholera (in certain contexts), which can infect and kill without jeopardizing future transmission or survival. indeed, many extinction-level amphibian infectious diseases are sapronotic in nature, such as the chytrid disease of salamanders and frogs (fisher ) . additionally, a gcbr-level event may not confer direct mortality. reproductive effects (i.e., in the manner of rubella or zika) or carcinogenic effects (e.g., htlv- ) could, in many ways, be highly detrimental to the future of humanity, as they could lead to significant curtailment of lifespans and diminishing birth rates, which could ultimately result in significant population collapse (rasmussen et al. ; tagaya and gallo ) . given the right context, any microbial organism could evolve or be engineered to be a gcbr. however, the most likely cause of a gcbr presently is a virus, with rna viruses being the most probable (woolhouse et al. ). historically, bacterially caused infections such as plague have had incredible impacts on the human species (raoult et al. ). however, the development of antibacterial therapies, beginning with the sulfonamides in and then penicillin in , has severely limited the ability of this class of microbes to cause a gcbr-level pandemic. in addition, the relatively slower speed of replication and accumulation of mutations also disadvantages this class over viruses. for example, a human infected with the hepatitis c virus (an rna virus) produces trillions of virions per day, whereas the doubling time of yersinia pestis, the cause of plague, is . h (neumann et al. ; deng et al. ) . the public health crisis of multiple-drug-resistant bacteria, such as carbapenem-resistant enterobacteriaceae (cre) and others, is very alarming (logan and weinstein ) . the spread of these bacterial agents, for which few if any treatments exist, threatens the entire practice of modern medicine, from cancer chemotherapy to joint replacement therapy. however, these organisms, which have variable attributable mortality, tend to be unable to efficiently infect human hosts that are not compromised or hospitalized. as such, the risk to the general public is constrained. large outbreaks of cholera and plague have represented true public health emergencies in yemen and madagascar, but their spread reflects severe infrastructure deficiencies caused by war and supply constraints rather than true global pandemic risk (qadri et al. ; roberts ). fungi represent prolific pathogens outside of the mammalian species. outbreaks of chytrid fungal disease in frogs and salamanders as well as snake fungal disease represent true existential threats to affected species (fisher ) . however, fungi are largely thermally restricted, and only limited members of this class of microbes can infect warm-blooded organisms such as mammals (casadevall ) . indeed, a fungal filter is hypothesized to have existed and may be partly responsible for mammalian warm-bloodedness. the success of the mammalian-adapted fungus that causes white-nose syndrome in bats is facilitated by the lower body temperature that occurs during their hibernation (foley et al. ) . human infections with fungi tend to be severely damaging only in an immunocompromised host. the human innate immune system contends with countless fungal spores that are present in every breath of air. as such, many endemic fungal diseases, such as histoplasmosis or coccidioidomycosis, do not cause harm in the majority of immunocompetent humans infected. even newly emerging fungi such as candida auris and cryptococcus gattii are largely subjected to this limitation (chowdhary et al. ; centers for disease control and prevention ). one of the most widespread fungal outbreaks-the exserohilum fungal meningitis outbreak-was abetted by direct injection of a contaminated medical product into the spinal region of humans, which is not a usual mechanism of infection (casadevall and pirofski ) . without thermal adaptation (which might be feasible with deliberate manipulation), fungi, many of which are sapronotic and do not rely on or need mammalian hosts, will not constitute a pandemic threat to humans. prions-transmissible infective proteins-are one of the most fascinating and understudied of infectious agents. these agents, which are responsible for diseases such as kuru and new variant creutzfeldt-jakob disease (vcjd, the human form of "mad cow disease") in humans, cause scrapie, chronic wasting disease, and bovine spongiform encephalopathy in other mammalian species (chen and dong ) . though highly damaging to humans and other species they infect, prions require specific conditions for spread. new variant creutzfeldt-jakob disease was to date the most highly publicized outbreak of a human prion disease; it resulted in human cases tied to the consumption of beef products primarily in england in the s and the s (hilton ) . other modes of transmission of cjd tied to iatrogenic spread via contaminated surgical instruments or cadaveric hormone products ceased once protective measures were put in place (bonda et al. ). kuru, a geographically restricted prion disease, was spread via human cannibalism in papua new guinea, and the outbreak abated once that practice was ended in the s (liberski et al. ) . the transmission characteristics of prion diseases are such that very extraordinary circumstances, on a par with human cannibalism or massive food contamination, must be present for a gcbr-level risk to be present for humans. additionally, and almost by definition, such an event would be slow-moving (prions were once known as "slow viruses"). protozoal organisms have the distinction of being the only infectious disease to have caused the extinction of a mammalian species. the christmas island rat, unable to outrun its vector, was felled by a vector-borne trypanosome (t. lewisi) during the early twentieth century on the australian island (wyatt et al. ) . human forms of trypanosomiasis have not risen to such a level of concern. human protozoal infections have exerted tremendous pressure on the species, and it is hypothesized that half of all humans who have lived died of malaria, which still kills approximately half a million humans annually (world health organization ). however, the development of antimalarial compounds and vector avoidance strategies has proved successful when they are able to be employed appropriately, and they have relegated malaria to a pathogen whose impact is amenable to control. nonetheless, one aspect of malaria is of particular concern: the development and spread of artemisinin-resistant forms, which render treatment extremely challenging with little to no effective antimalarial agents left for use. largely confined to specific regions of asia, such as cambodia and myanmar, this organism poses severe treatment challenges and, if artemisinin-resistant forms were to spread to africa, could represent a continent-wide catastrophic biologic risk (haldar et al. ). ameba, ectoparasites, and helminths all have limited pandemic risk, as they are constrained by pathogenicity, transmissibility, or both. clonally transmissible tumors-such as the notable devil facial tumor disease in tasmanian devils-are rare occurrences in humans, with restricted modes of transmission (maternal-fetal and organ transplantation). space-adapted organisms (e.g., salmonella that originates on earth but spends time in the space station before coming back to earth) can exhibit enhanced virulence; however, they still are susceptible to antibiotic treatment and normal control measures: there is no evidence they pose greater epidemic risk than normal salmonella (wilson et al. ). an alien microbe species that is obtained on mars or meteorites and brought back to earth, one of the focuses of the planetary protection program at the national aeronautics and space administration (nasa), was not deemed by our interviewees and meeting participants to be likely to pose a threat. and if such a species were found, it would be unlikely to be adaptable to an earthlike planet environment, as adaptations to its home planet's markedly different environments would likely preclude adaptations to earth. even though the chances of serious biological risk posed by such a sample return are deemed to be low, there are many uncertainties, and the highest level biocontainment procedures are being considered for specimens that might harbor such non-earth-based organisms (national research council ). traditionally, viruses have been ranked at the highest level of pandemic risk, and dedicated preparedness efforts often focus solely on viruses. a disproportionate focus on viruses is justified, however, based on several aspects unique to the viral class of microbes. the high rate of replication of viruses-for instance, over trillion hepatitis c virions are produced per day in a human infection-coupled with the mutability inherent in such short generation times gives viruses an unrivaled plasticity. this plasticity allows for host adaptability, zoonotic spillover, and immune system evasion. the lack of a broad-spectrum antiviral agent-like ones available for bacterial and even fungal organisms-also confers a special status on viruses. with no off-the-shelf treatment available to contain a viral outbreak, and likely no vaccine, containment efforts, at least in the early stages, will likely need to be made in the absence of a medical countermeasure (zhu et al. ) . there is a strong consensus that rna viruses represent a higher pandemic threat than dna viruses (kreuder johnson et al. ) . this assessment is derived from the fact that the stability of rna as a genomic material is less than that of dna, giving more genomic pliability to the rna viruses. dna viruses such as smallpox do challenge this assumption, and concern exists surrounding the related risks of monkeypox viruses, which are increasingly spreading in the absence of a smallpox vaccine campaign (kantele et al. ) . as monkeypox outbreaks continue to occur with longer chains of transmission, employing smallpox vaccines in target populations might be considered. another aspect of viral characterization is the location of replication. viruses with greater capacity for widespread have been shown in studies to be more likely to replicate in the cytoplasm of a cell (pulliam and dushoff ; olival et al. ) . this is postulated to be due to the higher affinity a virus must have for a particular type of host in order to be permitted entry into its nucleus, and this greater affinity would limit its zoonotic potential because it would be likely to be strongly tied to its usual host. in general, it is dna viruses that tend to have a nuclear replication cycle, while rna viruses have a cytoplasmic cycle. strikingly, smallpox-a dna virus with proven ability to cause pandemics-is a cytoplasmic replicator, while influenza-an rna virus with proven ability to cause pandemics -has a nuclear replication cycle. the exceptions to these rules argue against any overly strict adherence to them. other factors that may increase a virus' potential to cause a global catastrophic risk include a segmented genome (as exemplified by influenza viruses), a comparatively smaller genome size, and high host viremia (e.g., vector-borne flaviviruses). for example, the flu virus' segmented genome makes novel genetic assortment an eventuality, while a large genome may prevent nimble mutations. however, with each characteristic it is impossible to find a general rule, as exceptions abound. among currently studied viruses, the influenza a viruses are widely judged to pose the greatest pandemic risk based on historical outbreaks and viral characteristics (silva et al. ; imai et al. ). analysis of influenza risks is made in the centers for disease control and prevention (cdc)'s influenza rapid assessment tool (irat) which ranks h n as the most concerning influenza virus strain (centers for disease control and prevention ). there are several viral groups other than the orthomyxoviruses (which include the h n strain of influenza a) that are spread by respiratory routes, possess rna genomes, and merit enhanced attention: paramyxoviruses (especially these three genera: respirovirus, henipavirus, and rubulavirus), pneumoviruses, coronaviruses, and picornaviruses (especially these two genera: enterovirus and rhinovirus). based on our analysis and their inherent characteristics, these viral groups are the most likely source of a gcbr-level threat. there are efforts under way to construct viral catalogs of as many viruses as possible. the explicit aim of these projects is to reduce the uncertainty of outbreaks by extensively cataloging as many viral species as possible, so that a virus that causes a disease is less likely to be truly unknown. at the meeting and interviews for this project, a number of experts expressed concern that, while efforts to catalog and broadly sequence viruses in the animal world would provide new scientific discovery, we should not expect that it will identify the source of the next pandemic or that it can change the work being done for pandemic preparedness. broad viral sequencing would uncover many novel viruses. however, the vast majority of discovered viruses will not have the ability to infect humans let alone the prospect of widespread in the population. only a few viruses possess this ability. this work should be pursued with the objective of fundamental viral scientific discovery, rather than the goal of near-term improvement in pandemic preparedness. in the clinical practice of medicine, syndromic diagnosis-that is, making a nonspecific diagnosis, such as "sepsis," "pneumonia," or "viral syndrome," with little to minimal laboratory testing-is the norm. specific diagnosis (i.e., sending patient samples for definitive laboratory diagnosis) is often eschewed if it does not affect clinical management, is costly, and is not revealed with routine tests, and/or if the patient recovers. this practice has become enshrined not only in resource-poor areas in which access to diagnostic testing may be limited, but also in resource-rich areas, like north america and western europe, where specific diagnoses are viewed as superfluous. however, the yield from pursuing an etiologic diagnosis in infectious syndromes such as atypical pneumonia, sepsis, encephalitis, meningitis, and clinically significant fevers of unknown origin may be considerable, as it will provide important insight into the ongoing torrent of threats posed by the microbial world. by causing an infection with enough severity to come to medical attention, the culpable microbes have already established that they are damage-causing pathogens to humans-a feat that only a sliver of the microbial world can accomplish (woolhouse et al. ). many of these microbial diagnoses cannot be made through the routinely ordered diagnostics. therefore, a special effort would need to be made to get to a microbial diagnosis. if that were to be done more frequently and at a more strategic level around the world, it would provide an opportunity to develop new situational awareness regarding which microbes are circulating and infecting humans-information that is clinically valuable in its own right and more attuned to uncovering gcbr-level pathogens than broad viral cataloging. such efforts should not be limited to exotic "hot spots" of disease emergence but should be practiced in localities that are broadly representative of where these conditions occur. particular hot spots of emergence due to the presence of unique risk factors may be higher yield overall, but they should not be the sole sites of investigation. infectious disease emergence can occur anywhere, as evidenced by the h n pandemic, which was first recognized as the etiology behind a mild pediatric upper respiratory infection in california and west nile fever emerging in cases of undifferentiated encephalitis in the new york city metropolitan area in the late s (centers for disease control and prevention ; nash et al. ) . such a program would have significant cost and infrastructure implications in resource-constrained regions, so it would be most logical to set up sentinel or strategic sites for pursuing this level of microbial diagnosis in ways that are broadly representative. in developed nations such as the usa, these programs are available but underutilized because of lack of awareness or perceived lack of value by clinicians, for whom it will often not likely change therapeutic decisions. many participants in the project voiced the view that any microbe's pandemic potential could be substantially enhanced by human factors and poor preparedness, which could exacerbate a pathogen's spread or damage-causing potential. specific issues identified included gaps in hospital preparedness, medical countermeasure manufacturing capacity, medical countermeasure manufacturing locations, impacts on critical workforce members, and cascading effects on vital programs such as food production. for example, concentration of intravenous fluid manufacturing plants in puerto rico created massive shortages after a hurricane took the plants offline in (wong ) . the inability of hospitals to surge to meet enhanced patient needs for ventilators or icu beds is another potential constraint. human factors could also take the form of mistaken actions that are based on political considerations but are not supported by an evidence-based medical rationale, or scientific mistakes based on human error, such as misidentifying a microbe or misinterpretation of scientific or epidemiologic data. for example, early in the sars outbreak, mistakes regarding the etiology of the viral agent occurred, and the west african ebola outbreaks were initially thought to be cholera, delaying response efforts for months (world health organization ). some participants in this study were of the view that such factors as these could outweigh any intrinsic property possessed by a microbe or any physiologic vulnerability possessed by a human. magnification by human error could cause delays in response or awareness, allowing a pathogen to spread wider and deeper into the population and rendering containment more difficult, sowing panic, and severely stressing the healthcare infrastructure of a region. the majority view, however, was that intrinsic microbial characteristics are the main driver of a microbe's ability to cause a pandemic. pandemic preparedness should place a high priority on preparing for rna viral threats, given their frequent spread by respiratory route, cytoplasmic replication, and high mutability. surveillance, science, and countermeasure development programs and efforts should logically allocate significant resources to this class of microbes. except for influenza and certain coronaviruses, there are not major preparedness efforts being made for other viruses in this class of microbes. while rna viruses were at the top of the list of concerns, other classes of microbes, such as bacteria, fungi, and protozoa, should not be completely dismissed given characteristic that pose special concerns. cultivating and maintaining expertise in the epidemiology, surveillance, and pathogenicity of all classes of microbes, with explicit incorporation of a one health approach-which incorporates and integrates information from infectious diseases of plants, amphibians, and reptiles-will help foster the broad capacities needed for emerging pandemic and global catastrophic biological risks. pathogen-based lists, both usa and global, based on influenza precedents, historical biological weapon programs, and emerging infectious diseases were responsible for galvanizing early activities in the field of pandemic preparedness and have helped drive many important contributions. but these lists could create a sense of confidence regarding the prediction of future pandemic threats. lists can become frozen in the minds of those in the field and may be viewed as exhaustive rather than as starting points. additionally, inclusion in lists could also be sought for political (and not epidemiologic) reasons if inclusion carries with it the prospect of enhanced funding for a long-neglected endemic problem. one of the chief rationales behind this project was to attempt to move away from a strict list-based approach when considering pandemic threats and to develop a framework grounded in the facts of a microbe's biology and epidemiology. we recommend that risk assessment be rooted in the actual traits that confer pandemic or global catastrophic biological risks as opposed to a pathogen's presence on some earlier developed list. as respiratory-borne rna viruses have been identified as possessing heightened pandemic potential, it is important to strengthen surveillance activities around these viruses where they currently exist and establish them where they are not yet in place. currently, of the respiratory-borne rna viruses, only influenza and certain coronaviruses receive high priority for surveillance. while some efforts to understand coronaviruses, in the wake of sars and mers, exist, there is no systematic laboratory surveillance of coronavirus infections in humans. similarly, no such program exists for rhinoviruses, parainfluenza viruses, rsv, metapneumoviruses, and similar viruses. since this class of viruses is most likely to hold the future pandemic pathogen, constructing an influenza-like surveillance approach that better characterizes the prevalence, patterns, and geographic distribution of these viruses should be a priority. such an approach would focus on human infections, characterizing the epidemiology, virologic features, antiviral susceptibility (if applicable), and clinical manifestations in a fashion that mimics the extensive influenza surveillance conducted by the cdc and other international entities. currently, outside of anti-influenza antivirals, there is only one fda-approved antiviral for the treatment of respiratory-spread rna viruses (ribavirin). of the six fda-approved influenza antivirals-amantadine, rimantadine, baloxavir, zanamivir, oseltamivir, and peramivir-all target influenza viruses specifically and have no activity outside influenza, with two influenza a-specific agents (amantadine and rimantadine) rendered virtually obsolete because of resistance. the other antiviral agent (inhaled ribavirin) is approved for the treatment of respiratory syncytial virus (rsv) but has very limited use due to poor efficacy and major toxicity concerns for both rsv and parainfluenza viruses. there are currently no approved antivirals for any other respiratory-spread rna viruses in the world. prioritization of antiviral compounds against this group of viruses may lead to acceleration of drug development and (government and nongovernment) incentivizing programs. such antiviral compounds would have an advantage over many other emerging infectious disease countermeasures: these viruses exact a considerable toll in the form of community infections each year, providing a basis for a traditional pharmaceutical market as well as one for emerging infectious disease. pursuing not only broad-spectrum rna antivirals, but also those specifically targeted to specific viruses such as rsv, would increase the likelihood of yield. nontraditional molecules, such as monoclonal antibodies and immunomodulators, should also be investigated for a role in the treatment and prevention of rna virus respiratory infections (walker and burton ) . such adjunctive treatments may lead to improved clinical outcomes. to date, only one virally targeted monoclonal antibody is fda-approved: pavalizumab for prevention in high-risk infants. as with the above discussion regarding antivirals, the need for vaccines against respiratory-borne rna viruses should also be prioritized. currently, aside from influenza, for which a moderately effective but technically limited vaccine exists, there are no other vaccines for respiratory-borne rna viruses. experimental vaccines targeting rsv have made it into late clinical development only to fail. several important initiatives in this realm do exist and could be augmented to move beyond specific targets that have already been recognized. for example, the coalition for epidemic preparedness innovations (cepi) has selected a coronavirus (mers-cov) and a paramyxovirus (nipah) for vaccine development incentivizing (røttingen et al. ) . such a program could, in potential future initiatives, select additional vaccine targets from this group of viruses and even encourage the development of broadly protective vaccines against groups of viruses-for example, a vaccine that protects against all four strains of human parainfluenza viruses, both mers and sars covs, and both hendra and nipah viruses. additionally, the heightened interest at the national institutes of health (nih) in a universal influenza vaccine in the wake of the moderately severe - influenza season should be channeled to provide significantly increased resources to this endeavor (paules et al. ) . as certain avian influenza viruses are of the highest threat tier, a universal influenza vaccine (even one that just protects against a strains) could substantially hedge against an influenza virus attaining gcbr status. as was evident during the influenza pandemic and subsequent influenza seasons, the treatment of influenza is suboptimal, despite evidence-based guidance. the status of the treatment for other respiratory viruses is even less defined. while there currently is not a robust antiviral armamentarium against these viruses, there are important clinical questions that occur with their treatment that merit further study. for example, what adjunctive therapies are useful? what coinfections may be present? at what stage of illness are rescue oxygenation devices warranted? as many of these viruses are highly prevalent in the community and are frequently encountered by clinicians in both outpatient and inpatient settings, finding answers to these questions would render clinicians more adept at dealing with pandemic versions of these viruses. with respect to influenza, there is a growing literature on the use of antiviral agents in combination with anti-inflammatory agents such as nonsteroidal anti-inflammatory agents (nsaids) and macrolide antibiotics (hung et al. ) . untangling the nuances of these treatment effects in order to develop robust guidance would have an impact on the ability to cope with an influenza-driven gcbr. because of the higher likelihood that a gcbr-level threat might emerge from the group of rna viruses with respiratory-spread, special attention to research on these agents is warranted if such research could increase pandemic risks. while much research on this class of viruses would be low risk and managed by appropriate approaches to biosafety, experimentally engineered antiviral resistance, vaccine resistance, or enhanced transmission, for example, would raise major biosafety and biosecurity concerns. the appearance of the h n influenza a strain was thought to have resulted from laboratory escape (zimmer and burke ) . it is important to understand the kinds of work being performed with these agents and, in particular, to know of experiments that are being done or are being proposed that would result in increased pandemic risks. those experiments should have their own special review and approval process that is consistent with the risks and assesses the risks and benefits of this work before approval or funding of this work. as unknown infectious syndromes abound in all locations, and any given infectious syndrome may have as its etiology a potentially unknown or unappreciated microbe, specific diagnosis should be a routine endeavor. atypical pneumonias, central nervous system infections, and even upper respiratory infections often are treated without any etiologic agent being identified. as diagnostic technologies and devices improve in breadth, speed, and ease of use, the increasing uptake of these devices will provide a new opportunity to enhance situational awareness of an infectious syndrome in any location where they are deployed. such devices are currently being used in research projects in the developing world. the more routine use of devices, such as multi-analyte molecular diagnostic devices, has the capacity to provide a fuller picture of the microbiological epidemiology of any given syndrome, illuminating what has heretofore been biological dark matter (doggett et al. ; kozel and burnham-marusich ) . coupled with heightened surveillance of respiratory-borne rna viruses, the ability to capture an early signal of a potential pandemic pathogen will be greatly enhanced. to date, certain considerations have limited the uptake and use of these devices: cost, perceived lack of clinical impact, and constraints on hospital resources such as isolation beds. impacts on hospitals might be noted in laboratory testing volume as well as costs. however, when these devices are viewed in the context of pandemic preparedness, the cost-effectiveness calculation should change. these considerations could be moderated if they are considered part of a hospital's emergency preparedness activities and not exclusively as clinical (they also have benefit for antibiotic stewardship activities in both inpatient and outpatient settings). in fact, the use of these devices should be considered on a par with mechanical ventilators, vaccines, antivirals, and antibiotics in the context of pandemic preparedness. pilot projects demonstrating the feasibility of procuring such devices for infectious disease emergency preparedness could be conducted. understanding the microbial characteristics most importantly regarding the risks of pandemic or global catastrophic biological threats can help strengthen pandemic preparedness activities. while rna viruses pose the greatest risks, there are characteristics of other microbial classes that cause special concerns and are important to consider in scientific research agendas and in public health preparedness efforts. this analysis leads to a series of recommendations related to disease surveillance, antiviral and vaccine development, clinical research, and research oversight. taken together, assessment of key microbial class characteristics plus the focused actions that follow this assessment can broadly help improve preparedness for pandemic and global catastrophic risks. human prion diseases: surgical lessons learned 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vectors the exceptional oncogenicity of htlv- influenza: the mother of all pandemics passive immunotherapy of viral infections: 'super-antibodies' enter the fray space flight alters bacterial gene expression and virulence and reveals a role for global regulator hfq hospitals face critical shortage of iv bags due to puerto rico hurricane. the guardian rna viruses: a case study of the biology of emerging infectious diseases assessing the epidemic potential of rna and dna viruses ground zero in guinea: the ebola outbreak smouldersundetected-for more than months historical mammal extinction on christmas island (indian ocean) correlates with introduced infectious disease broad-spectrum antiviral agents historical perspective-emergence of influenza a (h n ) viruses key: cord- -u slrjmk authors: jiménez, ma Ángeles; sánchez, belén; pérez alenza, ma dolores; garcía, pilar; lópez, jose vicente; rodriguez, alejandro; muñoz, Álvaro; martínez, fernando; vargas, astrid; peña, laura title: membranous glomerulonephritis in the iberian lynx (lynx pardinus) date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: u slrjmk the iberian lynx is the most endangered felid species in the world, confined nowadays to two isolated metapopulations in the southwest of spain, where less than individuals survive. little is known about the diseases that affect these animals in the wild or in captivity. kidney samples from necropsies of iberian lynxes, wild and captive, were examined by histopathology, immunohistochemistry (igg, igm, iga, laminin, type iv collagen, and fibronectin), electron microscopy (n = ) and immunogold labelling for igm, igg and iga in one case, in order to characterize the glomerulopathy prevalent in this species. urinalyses from records were available for of the necropsied animals and blood and urine samples from free ranging and captive iberian lynxes were prospectively obtained in order to evaluate the renal function of the living population. a focal, diffuse membranous glomerulonephritis (mgn) that progressed with age was diagnosed in all but one of the animals in different stages not associated to concurrently known infectious diseases. positive immunoexpression of igm and igg was observed in the glomerular capillary basement membranes and intramembranous electron-dense deposits, compatible with immune complexes (ics) were seen with electron microscopy. the immunogold labelling was also positive for igm and igg in the electron-dense areas. the serum biochemistry and urinalyses also revealed signs of mild chronic kidney disease in of the animals evaluated. in conclusion, the membranous glomerulopathy affecting the iberian lynx is a progressive disease of immune origin. we postulate a possible genetic predisposition towards the disease, enhanced by inbreeding and a possible connection to an immune-mediated systemic disease. the iberian lynx is considered to be the most endangered felid species in the world (nowell and jackson, ) . its situation is critical due to habitat loss and fragmentation, being confined nowadays to two isolated metapopulations in the southwest of spain (gaona et al., ; ferreras, ; rodriguez and www.elsevier.com/locate/vetimm veterinary immunology and immunopathology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] abbreviations: bun, blood urea nitrogen; ckd, chronic kidney disease; fcv, feline calicivirus; fcov, feline coronavirus; felv, feline leukaemia virus; fiv, feline immunodeficiency virus; ics, immune complexes; iris, international renal interest society; malt, mucosa-associated lymphoid tissue; mgn, membranous glomerulonephritis; pas, periodic acid schiff reagent; pbs, phosphate buffer solution; rt, room temperature; tbs, tris buffer solution; usg, urine specific gravity. delibes, ) . today, less than animals are estimated to be left (palomares et al., ; guzmán et al., ) . the remaining population presents signs of inbreeding and allele dropout (johnson et al., ; godoy, ) . very little is known about the diseases that affect these animals in the wild or in captivity. bovine tuberculosis has been reported to be the cause of death of some iberian lynxes during the years and that probably acquired the disease by eating on infected fallow deer (dama dama) or wild boars (sus scrofa) of the area (briones et al., ; aranaz et al., ; peña et al., ) . a recent histopathological study of our group, evaluating the status of peripheral lymph nodes, spleen and malt (mucosa-associated lymphoid tissue) in iberian lynxes necropsied between the years and , has reported a generalized immune depletion in these animals, apparently not related to infectious agents or malnutrition (peña et al., ) . routine histological examination of kidneys obtained from the necropsy of animals between the years and , including the in the abovecited study, revealed a high prevalence of glomerulonephritis (peña, unpublished data) . this disease is common in the domestic cat (felis cati), especially the membranous type, and is usually of immune origin (grant maxie, ; glick et al., ) . glomerulonephritis can be associated to viral infections caused by feline coronavirus (fcov), feline leukaemia virus (felv) and the feline immunodeficiency virus (fiv) (glick et al., ; slauson and lewis, ; grant maxie, ; newman et al., ) . these pathogens are known to affect a wide variety of wild felids such as the cheetah (acinonyx jubatus), the african lion (pantera leo), the puma (puma concolor), the canada lynx (lynx canadensis), etc. (roelke et al., ; kennedy et al., ; biek et al., ) . also, many non-specific parasitic infections that can affect domestic animals are associated with immunemediated glomerulonephritis (newman et al., ) . a case of glomerulonephritis associated to heartworm infection has been described in the black-footed cat (felis nigripes) (deem et al., ) . furthermore, a non-immunogenic glomerulosclerosis has been reported in captive cheetahs (acinonyx jubatus) (munson, ; bolton and munson, ; munson et al., ) . rare diseases of uncertain origin are frequently described in endangered animals, usually related to poor genetic diversity or to captive conditions. the study of these diseases is important because of their relevance in endangered species conservation programs. a good example of this is the cheetah, considered a paradigm for disease vulnerability (munson et al., ) . this is the first report of glomerulonephritis in the iberian lynx. the main objectives of this study were to characterize by means of histology, immunohistochemistry, and electron microscopy, the type of glomerulopathy detected in the iberian lynx, to determine its pathogenesis, and to evaluate serum biochemical and urinary parameters related to renal function in wild and captive iberian lynx surviving in the southwest of spain. the membranous glomerulopathy affecting the iberian lynx is a progressive disease of immune origin. we postulate that the iberian lynx has a likely genetic predisposition towards the disease, enhanced by inbreeding, and is potentially connected to a possible immune-mediated systemic disease. twenty-seven necropsies were performed on iberian lynxes between the years and . twenty-one animals came from the wild and six from captivity with ages that ranged from days to years. there were females and males. the main cause of death were car accidents (n = ), followed by tuberculosis (n = ), traumatisms (other than car accidents, n = ), fights (n = ), squamous cell carcinomas (n = ), and other diseases (n = ). serology and pcr results for various infectious agents that included those related to glomerulonephritis (fcov, feline calicivirus (fcv), fiv, and felv,) were provided. with the exception of one animal that was positive for felv in serology, and two that were pcr positive for fcov (without histological signs of feline infectious peritonitis), the remaining animals were negative for these agents. right and left kidney samples from necropsies were fixed in formalin, paraffin embedded and sectioned mm wide for hematoxilin-eosin, pas (periodic acid schiff reagent), masson's trichromic, congo red and methenamine silver stains. in order to quantify the lesions in the kidneys, each glomerulus was classified according to the severity of lesions in three categories: ( ) healthy or mild, ( ) moderate and ( ) severe glomerulonephritis or glomerulosclerosis. the percentages of each category of glomeruli were established in all samples. kidneys were then classified as stage or apparently healthy when over % of the glomeruli belonged to category (unaltered or presenting slight changes). the remaining kidneys were classified considering the most prevalent category of glomerular lesion (stage , moderate kidney affection; stage , severe kidney affection). the streptavidin-biotin peroxidase method was used on dewaxed kidney sections in order to detect the expression of fibronectin, laminin, and type iv collagen, components of the glomerular basement membrane. this technique was also used for iga, igm, and igg, markers of the immunoglobulin fraction of immune complexes. antigens were unmasked with trypsin (sigma . %, c, min) for the detection of laminin, fibronectin, iga, igm, and igg while a buffer citrate (tri-sodium citrate panreac , . %; ph ) heated in a microwave oven for min was used to unmask type iv collagen antigen. endogenous peroxidase was deactivated in all cases (h o . % in methanol) at room temperature (rt), for min. unspecific staining of laminin and fibronectin was blocked incubating the slides with normal swine serum (dako , x , dilution / , min, rt) and with normal rabbit serum (dako , x , / ) for iga, igm, and igg). the primary antibodies used (all incubated at c overnight) were polyclonal rabbit anti-laminin (dako , z , / ), polyclonal rabbit anti-fibronectin (dako , a , / ), monoclonal mouse anti-type iv collagen (dako , clone civ , / ), polyclonal goat anti-feline iga (serotec , aa , / ), polyclonal goat anti-feline igm (serotec , aa , / ) and polyclonal goat anti-feline igg (serotec , aa , / ). biotinylated secondary antibodies swine anti-rabbit (for laminin and fibronectin, dako e , / ,), rabbit anti-goat (for iga, igg, and igm, pierce , , / ) and rabbit anti-mouse (for type iv collagen, dako e , / ) were used (incubated min at rt) after washing. slides were then washed and incubated with a streptavidin peroxidase conjugate (zymed - , / , min rt). a chromogen solution containing - diaminobenzidine tetrachloride (sigma chemical co. d ) and h o in distilled water was used for developing, and finally, slides were counterstained with hematoxylin (sigma gh - - ). all washes were done with tbs (tris buffer solution ph . ). the glomerular capillary basement membranes served as internal controls for type iv collagen, laminin and fibronectin, while lymphoid tissues with plasmatic cells from iberian lynx were used as positive controls for igm, igg and iga. negative controls were used substituting the primary antibodies with tbs. selected kidney samples fixed in formalin (n = ) and in . % glutaraldehyde (n = ) from animals without concurrent diseases or detected viruses were processed for electron microscopy. after fixation, the samples were washed in a phosphate buffer (pbs, ph . ) and included in % osmium (diluted in pbs) for h at room temperature. they were then washed in distilled water and dehydrated in acetones of increasing percentage ( , , , , and %) . the samples were gradually infiltrated in a müllenhauer mixture resin and solidified at c for h. the resin blocks were then processed and studied in the electron microscopy center of the complutense university of madrid, spain. one additional kidney sample fixed in formalin was selected for immunogold labelling of igm, igg and iga. the sample was thoroughly washed in pbs at c until clear of formalin. it was then dehydrated in alcohols of increasing percentage ( , , , , and %) while the temperature was lowered gradually (from c to À c for alcohols at and %, respectively) until reaching À c. resin blocks were obtained by gradually infiltrating the sample with lowicryl resin at À c. tissue samples were obtained from the resin blocks and placed in grids for immunogold labelling. polyclonal goat anti-feline igm, iga and igg (serotec , aa , aa , aa ; / ) were used as primary antibodies. the grids were incubated at c overnight and later washed with . m glycine in tbs. then they were incubated for h with nm colloidal gold-affinipure donkey anti-goat igg (jackson immunoresearch , - - , / ). the grids were washed again with the glycine solution. they were stained with % uranyl acetate and reynolds lead citrate, washing with distilled water after both and finally dried and observed by electron microscopy. urinalyses from records were available for nine of the necropsied animals. these were retrieved and evaluated. a prospective study of free ranging and captive iberian lynxes was carried out (table ). blood samples (n = ) and urine samples (n = ) were collected from anaesthetized iberian lynxes (table ) . serum biochemistry parameters related to renal function (total proteins, albumin, blood urea nitrogen (bun), creatinine, calcium, and phosphorus) were determined. the analytical results have been evaluated based on published values for the canada lynx (felis lynx canadensis) (weaber and johnson, ) , the european lynx (lynx lynx) (ryser-degiorgis, personal communication), and the domestic cat (dibartola and rutgers, ) . the urinalysis included urine specific gravity (usg), albumin, glucose, leukocytes, blood, and examination of the urine sediment. the protein-to creatinine ratio was also measured in order to quantify proteinuria. chronic kidney disease (ckd) was categorized in four stages using serum chemistry concentrations and considering the presence of markers of kidney damage according to the recommendations of the international renal interest society (iris) (polzin, ) . the markers of kidney damage used for the iberian lynx were: elevated bun, hypoalbuminemia, elevated serum creatinine concentration, hyperphosphatemia, hypocalcemia, impaired urine concentrating ability (usg was considered normal when ! ), proteinuria, and renal hematuria. these must be confirmed to be of kidney origin and at least one must be present to identify the existence of renal disease. stage (non-azotemic) was considered when markers of kidney damage were present and serum creatinine concentration was < . , stage (mild renal azotemia) presented markers and creatinine values between . and . mg/dl. in stage (moderate renal azotemia) creatinine values must be . - . mg/dl, and in stage (severe renal azotemia), creatinine values exceed mg/dl. the stages were further subcategorized using the protein-to-creatinine ratio as proteinuric (ratio > . ), non-proteinuric (ratio < . ) or borderline proteinuric (ratio . - . ). the associations of epidemiological, pathological and immunohistochemical data were studied using computer software spss. categorical variables were analyzed by pearson and yates chi-square tests. pearson correlation coefficients were used to study continuous variables. levene f-tests were used to analyze the homogeneity of variances. if variances were equal, f-tests or pooled t-tests were chosen to evaluate the variables. if variances were not equal, then welch tests or separate variance t-tests were selected. values p < . were considered significant. non-parametric statistical tests (kruskal-wallis and median test) have been performed when necessary. epidemiological variables included: age as a numerical variable in months and as a categorical variable: (young, - months; young adult, - months; and adult/senile, > months), gender, origin (sierra morena or doñana), and free or captive. in order to evaluate the kidney lesion, four histopathological variables were used: (a) percentage of glomeruli in category , (b) percentage of glomeruli in category , (c) percentage glomeruli in category and (d) stage of kidney affection (stage , none or minimal lesion; stages and , moderate and severe) (categorical). other variables were considered as present or absent such as interstitial nephritis, peritubular fibrosis, and hyaline cylinders. immunohistochemical variables concerning normal or increased expression of laminin, iv collagen, and fibronectin and the presence or absence of igg, igm, and iga were also included. a focal, diffuse membranous glomerulonephritis (mgn) was diagnosed in all of the animals, regardless of their age, sex, and whether they came from captivity or from the wild, except a -day-old cub that did not present any changes in the glomeruli. sclerotic glomeruli were focally scattered in the cortex, and stained blue in the mesangium with masson's trichromic (fig. ) . bowman's capsules appeared focally thickened. the kidneys were classified according to the percentage of altered glomeruli. ten animals were found to have unaltered or slight changes in over % of the glomeruli (stage ), presented moderate mgn (stage ) and only one animal had a severe mgn (stage ). interstitial fibrosis was seen in animals and chronic (lympho-plasmacytic) interstitial nephritis in (not including the granulomas found in the kidneys of the three animals with tuberculosis). other renal lesions such as calcifications and hyaline cylinders in tubules were seen in seven animals. the congo red stain was negative in all of the animals. severity of mgn was found to increase significantly with age: high percentages of normal and minimally altered glomeruli were significantly associated to young animals (mean . % ae s.d. . , median . %) while lower percentages were associated to young adults (mean . % ae sd . , median . % of unaltered glomeruli) and adult/senile animals (mean . % ae sd . , . %) ( p = . anova test). the non-parametric kruskal-wallis ( p = . ) and the median tests ( p = . ) were also significant for this association. there was also a positive correlation between the age (in months) and the percentage of severely affected glomeruli ( p = . ). the presence of interstitial lympho-plasmacytic infiltrates (chronic interstitial nephritis) was significantly associated to the advanced grade of mgn ( p = . ). interstitial fibrosis was evident in advanced mgn ( p = . ). a slight increase of the three components of the glomerular basement membrane, laminin, fibronectin and type iv collagen was observed in glomeruli at beginning or middle stages of glomerulonephritis, while in glomeruli presenting glomerulosclerosis, laminin and fibronectin would always appear intensely expressed, in a focal or diffuse matter, and type iv collagen could either be intensely expressed (fig. ) or negative. laminin expression was significantly associated with the advanced grade of mgn ( p = . ). a positive immunoexpression of igm and igg was observed in the thickened glomerular capillary basement membranes in early stages of glomerulonephritis (figs. and ) , while in glomerulosclerosis, the expression of these two immunoglobulins was negative. iga expression was negative in all cases. electron microscopy revealed irregularly thickened glomerular capillary basement membranes (n = / ) and effacement of foot processes (n = / ) (fig. ) . electron-dense deposits, compatible with immune complexes (ics), were located intramembranously (n = / ) (fig. ) . some electron-lucent areas also were seen in the basement membranes, compatible with areas where immune complexes were being reabsorbed (n = / ). in glomerulosclerosis, the capillary basement membranes were thickened and wrinkled; foot processes were effaced and sometimes lost. electron-dense deposits were not found in this stage. the immunogold labelling for igm and igg stained positive the electron-dense structures that were compatible with (ics) (fig. ) , while iga was not detected. the urinalyses available from nine of the necropsied animals presented low urine specific gravity (usg) in five cases (usg < ), three of which presented proteinuria qualitatively measured (albumin ! mg/ dl). two other animals presented proteinuria (albumin ! mg/dl) with normal specific gravity. haematuria was seen in six animals and glycosuria was present in one. serum biochemistry parameters of the live iberian lynxes were: total proteins (range . median . ), creatinine (range . - . mg/dl; mean . ae s.d. . ; median . ), calcium (range . - . mg/dl; mean . ae s.d. ; median . ), and phosphorus (range . - . mg/dl; mean . ae s.d. . ; median . ). twelve animals had hyperphosphatemia (phosphorus > . mg/dl), presented hypocalcemia (calcium < . mg/dl), animals had uremia (bun > mg/dl), presented elevated creatinine (creatinine > . mg/dl). nine lynxes presented decreased usg (< ) and proteinuria was detected qualitatively in animals. according to the iris ckd classification system, five animals did not present any signs of kidney damage, two presented only slight hyperphosphatemia and uremia that could not be attributed to the kidney so were considered healthy also, six animals were classified in stage (non-azotemic), seven in stage (mild-azotemia), and three animals in stage (moderate renal azotemia). one animal in stage was subclassified as proteinuric and three animals were subclassified as borderline proteinuric in stages , and (table ) . membranous glomerulonephritis (mgn) in different stages was the main lesion described in the kidneys of the iberian lynxes. this disease is characterized by an irregular increase of the glomerular capillary basement membranes due to the deposit of circulating immune complexes, or specific antibody attachment to components of the glomerulus (franklin et al., ; valaitis, ; newman et al., ) . the immunohistochemistry revealed basement membranes to be thickened by specific components such as laminin, fibronectin and type iv collagen. the detection of immunoglobulins g and m in the capillary basement with immunohistochemistry along with the intramembranous electron-dense deposits seen with the electron microscopy, confirm the presence of ics in our samples and an immune origin of the gn. the immunogold labelling results matched the immunohistochemistry since both igm and igg were detected over the electron-dense deposits and iga was absent. signs of complete and partial reabsortion of ics were also detected with electron microscopy, but not when there were histological features of sclerosis. immune complexes are usually present during initial stages of the disease and are progressively reabsorbed by the basement membrane, which thickens after the process. repeated injury on the basement membranes can impair their capacity to reabsorb immune complexes and regenerate, and therefore sclerosis of the membranes occurs as it loses its structural detail and capacity to eliminate ics (valaitis, ) . the most severe lesions, including the glomerulosclerosis, were significantly related to the age of the animals, which corresponds with a progressive degenerative disease. the other lesions found in many of the kidneys such as chronic interstitial nephritis and tubular fibrosis were less frequent and related to the severity of the mgn, and therefore were considered secondary to the disease. the presence of hyaline cylinders indicates some degree of filtration impairment of the tubuli, as consequence of the glomerular injury (newman et al., ) . the study of the urinalysis available from some of the necropsied animals was important to determine if there were analytical signs of renal dysfunction that corresponded with the histological findings. unfortunately, only nine urinalyses of the necropsied animals were available. nevertheless, the presence of proteinuria and low urine specific gravity in five animals, suggests that filtration might have been impaired to some degree. glycosuria and hematuria were also present in some cases. these are unspecific signs of renal damage and there was no information available about existing extrarenal infections at the time of sampling, therefore their interpretation should be taken with caution. blood and urine sampling of the living population was important to evaluate if there was a correspondence between the prevalent histopathological findings and the existence of chronic kidney disease (ckd) in the live animals. there is one study published on serum biochemical characteristics of the iberian lynx done in wild iberian lynxes (beltrán and delibes, ) , and recently another study is being done with a larger number of animals by members of this team, describing the parameters observed in the population regardless of the presence of infection or other diseases. due to this situation the iris staging system is useful since it permits the diagnoses of ckd even when reference ranges are not very accurate (polzin, ) . according to the results obtained with the iris classification system, out of animals presented some signs of chronic kidney disease, though only three presented moderate azotemia and none of them were found in the most severe stage. the presence of renal disease seems to exist among the living population, both captive and wild, and this would correlate with the histological changes detected in the necropsied animals. another common feature of renal failure is hyperphosphatemia and therefore was included as a marker of kidney disease. it appears when the glomerular filtration rate declines around % below normal (grauer and dibartola, ) and it was detected in of the animals. in two of them it was not considered since the urinary analyses were not available and it could have been due to recent ingestion. hypocalcemia was present in four of the animals with hyperphosphatemia. the main consequence of hyperphosphatemia is the development of secondary renal hyperparathyroidism (nagode and chew, ; kates et al., ; barber and elliot, ) . it is usually associated to hypocalcemia, though hypercalcemia or normal serum calcium concentrations accompanying hyperphosphatemia can also be found due to the compensation of the parathyroid hormone (pth) (barber and elliot, ) . the consequences of a secondary hyperparathyroidism are completely unknown in the iberian lynx, but it may be interesting to explore animals during sampling or in future necropsies for signs of pain or osteodistrophia seen in other species (barber and elliot, ) . membranous glomerulonephritis is a common glomerular affection in cats that can be associated to viral infections (felv, fiv, fcov, etc.) , neoplasms, intoxications (methyl mercury), familial diseases, autoimmune pathologies, or in many cases it is considered idiopathic (glick et al., ; enestrom and hultman, ; newman et al., ) . membranous glomerulonephritis can also be secondary to other systemic immunological diseases such as systemic lupus erythematosus or immune complex polyarteritis in dogs and cats (newman et al., ) . the only iberian lynx without signs of membranous glomerulonephritis was the -day-old female cub that had died in a sibling quarrel. with the exception of the few animals with neoplasias or tuberculosis, or the three lynxes that were pcr positive for viral detection (seven animals altogether), features that could explain the presence of mgn were not found in the rest of the animals. other vascular and metabolic diseases like hypertension and diabetes mellitus are non-immunogenic causes of glomerular disease (alpers, ) and the glomerular capillary basement membranes can also appear thickened due to hypertension or sclerosis (buckalew, ; newman et al., ) . hypertension, probably associated to stress, genetic and dietary factors (a high protein diet), was postulated to be the cause of the glomerulosclerosis described in a high percentage of captive cheetahs . the pathogenesis of the glomerular injury caused by these diseases lacks an immune component and therefore is different to the one seen in the iberian lynx. however, the iberian lynx is also a carnivore, and high protein ingestion can cause hypertension and accelerate progressive renal failure, which leads to glomerulosclerosis (buckalew, ) . therefore, this could be contributing to the progression of the disease, as occurs in the cheetah, even though the main cause of the glomerulopathy is immunogenic. inherited glomerulopathies have been described in many breeds of dogs and there are reports in cats, these are primary glomerular diseases (grauer and dibartola, ; casal et al., ) . it may be interesting to take these into consideration since the iberian lynx displays very low genetic diversity for a felid, with two remnant populations that have undergone a recent founder effect or population bottleneck (godoy, ) . recent studies have found that it possesses a deficiency of heterozygosity possibly due to inbreeding and some degree of allele loss (johnson et al., ) . this could encourage the inheritance of a certain glomerular disease among the population. animals as young as or months presented glomerulonephritis suggesting there may be a predisposition for the disease. also, the cause of the generalized immune depletion described in of the animals (peña et al., ) included in this study remains undetermined and a possible relationship with the membranous glomerulonephritis should be taken into consideration. as with many species in danger of extinction, with the handicap of low genetic diversity that seem to lead to uncommon diseases, the importance of a detailed study of the diseases that may affect the iberian lynx is evident. in conclusion, the membranous glomerulopathy that affects the iberian lynx is a progressive disease of immune origin. we postulate that the iberian lynx may be genetically prone to the disease, which could be enhanced by inbreeding and also may have a connection with an immune-mediated systemic disease. future studies are necessary. nevertheless, none of the animals are known to have died from chronic renal failure yet, though the results indicate that it may be widely extended, considering that the remaining population is of less than individuals. because of the critical state of this endangered species, signs of progression of this disease should be watched for, especially in the wild population. robbins and cotran pathologic basis of disease bovine tuberculosis (mycobacterium bovis) in wildlife in spain feline chronic renal failure: calcium homeostasis in cases diagnosed between and hematological and serum chemical characteristics of the iberian lynx (lynx pardina) in southwestern spain serologic survey for viral and bacterial infections in western populations of canada lynx (lynx canadensis) glomerulosclerosis in captive cheetahs (acinonyx jubatus) bovine tuberculosis and the endangered iberian lynx pathophysiology of progressive renal failure familial glomerulopathy in the bullmastiff heartworm (dirofilaria immitis) disease and glomerulonephritis 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cd depletion comparative pathology of glomerulonephritis in animals renal glomerular disease hematologic and serum chemistry values of captive canadian lynx this research was supported by the spanish ministry of environment, research project / in agreement with the environmental council of the ''junta de andalucía'', ''dirección general de investigación'' project cgl - /bos and ''red nacional de parques nacionales'' project / . we thank all the veterinarians and biologists involved with the field work, management and sampling of the iberian lynxes at doñana national park and sierra morena. we also thank dr. marie-pierre ryser-degiorgis for the serum biochemical reference values of the european lynx. we are grateful to ana vicente, agustín fernández and dr. maría luisa garcía for their assistance processing the electron microscopy samples and to pedro aranda for his histotechnology assistance, and to pedro cuesta for his assistance in the statistical analysis. key: cord- -kvv fx n authors: barratt, ruth; shaban, ramon z.; gilbert, gwendoline l. title: clinician perceptions of respiratory infection risk; a rationale for research into mask use in routine practice date: - - journal: infection, disease & health doi: . /j.idh. . . sha: doc_id: cord_uid: kvv fx n abstract outbreaks of emerging and re-emerging infectious diseases are global threats to society. planning for, and responses to, such events must include healthcare and other measures based on current evidence. an important area of infection prevention and control (ipc) is the optimal use of personal protective equipment (ppe) by healthcare workers (hcws), including masks for protection against respiratory pathogens. appropriate mask use during routine care is a forerunner to best practice in the event of an outbreak. however, little is known about the influences on decisions and behaviours of hcws with respect to protective mask use when providing routine care. in this paper we argue that there is a need for more research to provide a better understanding of the decision-making and risk-taking behaviours of hcws in respect of their use of masks for infectious disease prevention. our argument is based on the ongoing threat of emerging infectious diseases; a need to strengthen workforce capability, capacity and education; the financial costs of healthcare and outbreaks; and the importance of social responsibility and supportive legislation in planning for global security. future research should examine hcws' practices and constructs of risk to provide new information to inform policy and pandemic planning. abstract outbreaks of emerging and re-emerging infectious diseases are global threats to society. planning for, and responses to, such events must include healthcare and other measures based on current evidence. an important area of infection prevention and control (ipc) is the optimal use of personal protective equipment (ppe) by healthcare workers (hcws), including masks for protection against respiratory pathogens. appropriate mask use during routine care is a forerunner to best practice in the event of an outbreak. however, little is known about the influences on decisions and behaviours of hcws with respect to protective mask use when providing routine care. in this paper we argue that there is a need for more research to provide a better understanding of the decision-making and risk-taking behaviours of hcws in respect of their use of masks for infectious disease prevention. our argument is based on the ongoing threat of emerging infectious diseases; a need to strengthen workforce capability, capacity and education; the financial costs of healthcare and outbreaks; and the importance of social responsibility and supportive legislation in planning for global security. future research should examine hcws' practices and constructs of risk to provide new information to inform policy and pandemic planning. preventing the transmission of infectious diseases in healthcare settings, and in society more broadly, is a core goal of contemporary public health and infection prevention and control (ipc). in recent years outbreaks of emerging infectious diseases caused by respiratory viruses have drawn considerable global attention, in particular severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and pandemic influenza a, h n (table ) . consequently, global and national planning for pandemic diseases is grounded in the expectation that a novel respiratory infection is most likely to be responsible for the next pandemic or infectious disease emergency [ ] . respiratory infectious diseases are transmitted via contact, droplet and/or airborne modes, necessitating healthcare worker (hcw) use of surgical masks or respirators and other personal protective equipment (ppe) together with appropriate hand hygiene. hospital-based transmission of respiratory infectious diseases of high consequence, such as influenza, can be minimised by limiting the part hcws play as vectors or victims of disease. hcws may continue to work with mild respiratory illness (presenteeism), which can be serious or life-threatening if transmitted to vulnerable patients, but they also may suffer serious effects from occupationally-acquired respiratory infections, leading to increased staff absenteeism, which will compromise patient care during epidemics. while policies and protocols for optimal use of ppe and other transmission-based precautions exist in the majority of healthcare facilities, hcw compliance with them is typically limited, particularly in non-outbreak situations or in the early stages before an outbreak is recognised [ , ] . in particular, hcws' use of protective masks when caring for patients with respiratory infections is an important and well-documented ipc measure [ ] . yet hcw use of protective masks, and ppe in general, during routine care is often suboptimal and can result in healthcare-associated acquisition of infection [ ] (table ) . while hcw compliance with the use of protective masks during infectious disease outbreaks has been well reported [ ] , there has been limited examination of hcw behaviours with respect to protective mask use during routine clinical care [ ] . consistent routine use of protective masks, based on relevant clinical indications, is important in preventing or delaying transmission from an unrecognised initial/index case [ ] . the appropriate use of ppe, including respiratory protection, and hand hygiene in routine care is critical to minimising pathogen transmission to staff and other patients; sub-optimal use exposes both hcws and patients to infection. compliance of hcws with wearing a protective mask may be related to their perception of risk and their risk-taking behaviours. the existing ipc literature primarily focuses on this topic in the context of sars or other pandemic respiratory diseases, with few papers investigating risk constructs for healthcare workers in routine care. the first and classic response to suboptimal behaviour is educative, with the provision of in-service and other training. we argue that the factors that lead to suboptimal use go far beyond knowledge and education, as well a subsequent national outbreak resulted in healthcare associated cases within the first month with over one fifth of these cases being hcws. one reason for so many hais has been attributed to sub-optimal use of routine protective equipment by hcws and the potential for infected hcws to act as vectors of infection [ ] . documented in other behaviours such as hand hygiene [ ] . interventions, and the research efforts used to generate evidence to support them, must take account of individuals' constructs and perceptions of risk and risk-taking behaviour. these perceptions are necessarily heterogeneous and vary between individuals and clinical settings. therefore, an understanding of the perceptions and behaviours regarding ppe use in different contexts is needed to inform successful behaviour change interventions [ ] . the importance and urgency of addressing suboptimal mask use by hcws is, in our view, based on a range of interconnected reasons all of which are critical to global health and security. these are as follows: the continuing burden of emerging infectious diseases for many centuries, since the age of the plague and smallpox epidemics to the th century outbreak of hiv/ aids, human infectious diseases of high consequence have presented a significant global public health challenge. these pandemics have resulted in deaths and disability of millions of people across the world, as well as causing social and economic disruption. despite improvements in communicable disease prevention and control, including effective sanitation, vector control, vaccines, and the international health regulations developed by the world health organization (who) [ ] , the new, emerging infectious diseases continue to threaten the well-being and economic stability of society and impose a significant burden on healthcare. although some infectious diseases, such as plague or smallpox, no longer present an active global pandemic threat, this century has seen both new and re-emerging infectious diseases give rise to widespread outbreaks. of particular current concern is re-assortment of rna in viruses such as influenza a which contributes to emerging pandemic influenza strains [ ] . furthermore, several zoonotic viral diseases that have infected humans through animal-to-human contact have also demonstrated human-to-human transmission, such as nipah virus [ ] . antecedents for the increasing burden of infectious diseases include a global population boom, changes in the use of land and environment, loss of wild life habitat, increased contact between wild and domestic animals and humans, the expansion in travel, an ageing population and developments in medical interventions. the latter two have led to an increase in the number of immunecompromised people who are susceptible to significant disease from emerging infections. many of these people attend, or are frequent inpatients of, healthcare facilities and therefore are at risk of healthcare-associated infections (hais). cheaper, easier and faster modes of travel, particularly by air, have enabled emerging infectious diseases to disperse more widely in short periods of time, than ever before. a clear example of this was sars, which spread from one "super-spreader" in a hotel in hong kong to numerous other countries via international guests who were infected, by contact, while staying in the same hotel [ ] . similarly a large outbreak of mers involving cases resulted from a single traveller returning to south korea from the middle east and attending several hospital emergency departments after he became unwell [ ] . the number of active outbreaks that are present around the world will vary on any given day; however at time of writing there were traveller alert notices for at least twelve different infectious diseases in more than countries [ ] and, on average, global infectious diseases emergencies are notified via the who each day [ ] . the use of protective respiratory masks has a human resource impact in healthcare organisations. clinicians are at a higher risk of acquiring influenza and other respiratory diseases than adults working in non-healthcare settings [ ] . sub-optimal protective mask use can increase this risk, which is exacerbated during high-risk periods such as the winter respiratory virus season. staff illness from respiratory infections has a direct impact on the workforce resulting in loss of productivity and associated economic burden within the healthcare setting, particularly with influenza [ ] . other respiratory viral diseases, such as the common cold, also contribute to a reduced work output [ e ] . productivity is affected if workers take leave to care for family members who are ill or children, because schools have been closed. although annual influenza vaccination is widely promoted as a means to reduce staff illness, average uptake by hcws is poor, unless is it mandatory. seasonal vaccine efficacy varies from year to year because of variable matching between vaccine and circulating strains, but is generally less than e % [ ] . even when hcw flu vaccine uptake is high the risk remains, because of vaccine mismatch with circulating strain, limited vaccine efficacy and/or mild or subclinical (but transmissible) infection in vaccinated subjects [ ] . consequently, hcws should still use respiratory protection when caring for patients with respiratory symptoms and/or patients at high risk of infection during outbreaks or high levels of respiratory infections in the community. not wearing a protective mask increases the risk of occupationally-acquired respiratory disease. hcw absenteeism due to influenza increases on average by two days per hcw, both during pandemic and a seasonal virus outbreaks [ ] . ip et al. [ ] examined overall sickness absences including sick leave due to acute respiratory infection (ari) for four distinct influenza periods between and including the influenza a(h n )pdm pandemic in hong kong. results showed that the daily hcw absenteeism rate for ari increased from the pre-pandemic in september a uk healthcare worker contracted monkeypox after caring for a patient with the disease prior to diagnosis. in a eurosurveillance report (add in ref) about the case, public health officials said that some hcws had been exposed as they were not wearing optimal personal protective equipment. baseline by . % and . % during the epidemic and pandemic periods respectively [ ] . similarly in canada, researchers demonstrated a significant increase in the rate of sick hours between the pre-influenza and / influenza period with only % of staff having zero sick hours productivity losses related to the common cold [ ] . a study examining the effect of influenza vaccination on emergency department workers' absentee rates reported that % of vaccinated and % of non-vaccinated workers required sick leave for influenza-like illness [ ] , although significant absenteeism during the h n influenza pandemic was not noted in the australian emergency workforce [ ] . staff illness compromises the quality and safety of patient care by loss of continuity of care through the requirement to employ agency staff in place of regular staff, who may be unfamiliar with the specialism of the clinical setting [ ] . staff absenteeism during outbreaks of emerging or high consequence infectious diseases, may also be due to hcws fear of acquiring the infection [ ] . similarly, presenteeism, or coming to work when ill, also results in a loss of productivity due to staff not working at full capacity [ ] . the health and safety of other staff are put at risk by hcws who continue to work while ill, while patient safety may be compromised through impaired clinical judgement. in a study undertaken in a children's hospital in philadelphia, ( %) of medical staff who were surveyed, reported that they would work with significant respiratory symptoms, despite acknowledging the infection risk to their co-workers and patients [ ] . in another study over % of us hcws who were surveyed worked with symptoms of influenza-like illness [ ] . whilst it is important to avoid presenteeism, it may be occasionally unavoidable e.g. because of significant or specialised staff shortages. if so, the risk may be mitigated by appropriate mask use. the hcws work capability may also be impaired by any physical and psychological consequences of wearing a mask, such as claustrophobia, respiratory distress, discomfort and skin irritation. the financial costs to society for respiratory infectious diseases can be significant. a us study estimated the annual economic burden of influenza, in , to be around us$ billion [ ] , while lost productivity due to influenza in france and germany was estimated at us$ e billion per year [ ] . sub-optimal mask use is likely to be associated with an increase in financial costs for individuals, the healthcare system and subsequently the wider society. although existing research has not examined the direct costs of not wearing a protective mask, van buynder et al. ( ) estimated the financial cost of hcws absenteeism due to influenza-like-illness to be greater than can$ million during the / winter season in a health district in british columbia [ ] . in addition, there are sick leave payments for staff and the costs incurred to replace them with casual staff. workers compensation fees may be driven up by hcws who take risks by not wearing masks. furthermore, there are significant monetary costs associated with patients acquiring a healthcare associated respiratory infection. the probability of a patient acquiring an influenza-like-illness increases when exposed to an infectious hcw, with one study reporting a relative risk of . when compared to no documented exposure [ ] . expenses for a hai include the overall cost of care for any additional inpatient bed days as a result of the infection, antiviral medication, other supportive therapy, radiology, laboratory and direct costs associated with the use of isolation and ppe measures. a korean study reported an average medical cost for a patient hospitalised with influenza in / was us$ . ae . [ ] . when a higher level of ipc measures is required e.g. mers or other emerging infectious disease, these costs can be excessive. veater et al. ( ) calculated an additional cost of pounds sterling per person per day, mainly due to staff time and ppe costs [ ] . third, sub-optimal mask use is associated with reductions in cost effectiveness of training methods in the use of ppe. effective training in ppe use is resource intensive and thus expensive to execute, whether delivered as demonstration learning by experts or technology-based education. inadequate training in ppe protocols is cited as one of the causes for poor compliance with ppe [ ] . these findings question the cost-effectiveness of current training methods. there is also a financial cost attached to the incorrect choice or unnecessary use of a mask, particularly in the case of the more expensive particulate respirator mask, or during a global outbreak event where stocks may be limited. the knowledge and skills of hcws are factors that affect protective mask use, therefore investigating how knowledge and cognition impacts on the hcw decision-making for mask use can inform the delivery of education and how policies are implemented. some of the aspects of knowledge related to mask use that may influence hcw behaviour include the source of knowledge, the indications for mask use, which type of mask to choose, how the mask functions to provide protection and how to put on and remove the mask safely. in the context of an emerging infection and limited available information, personal experience can influence hcws' perceptions of risk and behaviours related to protective mask use [ , ] . in contrast, a study undertaken in an outpatient paediatric setting, demonstrated that the use of ppe was not influenced by infectious risk perception [ ] . prior education and training will provide some of the essential information and skills required for optimal mask use but, in practice, routine training in the use of ppe is often cursory or non-existent. in a survey of healthcare workers in the us, % of doctors reported having received ppe training only as students (including clinical rotations) or not at all (c.f. % of nurses) [ ] . despite prior education, hcws may not apply their knowledge to the workplace [ ] . the method of training is therefore an important consideration for effective retention of knowledge and skills over time. several studies argue for improving the evaluation and training of hcws using ppe for infectious diseases and examining the effectiveness of various teaching approaches [ , ] . the recent ebola virus disease (evd) outbreak instigated intensive ppe training around the world, with a focus on donning and doffing protocols to maximise hcw safety. unsafe use of ppe has been blamed for some hcws becoming infected with evd or sars; subsequently several research studies have reviewed the effectiveness of different training techniques for the safe donning and doffing of ppe [ ] . these have included interactive online courses, and classroom teaching that incorporates fluorescent dye or harmless bacteriophages as surrogate markers of contamination [ ] . video-reflexive ethnography (vre) has been used as an interventional methodology to improve ipc practices [ ] . this method allows the hcw to view video footage of themselves making decisions around and subsequently using protective masks in every-day complex work. the clinicians can then reflect on their behaviour and suggest ways in which their own and colleagues' mask use can be optimised. although the techniques taught for donning and doffing protective masks as part of routine ppe are generally heterogeneous around the world, there are variations in mask design which may affect skills. there is also a lack of standardisation between and within institutions as to which clinical indications warrant a n or surgical mask. within society in general, individuals are not only motivated to protect themselves from infectious disease but often demonstrate a moral responsibility to protect others if they themselves are infectious [ ] . during periods of high-risk for respiratory infectious disease, such as the annual influenza season or a novel influenza pandemic, health departments have, and may, encourage or mandate the use of a protective respiratory mask by the general public to minimise the transmission from symptomatic people to others [ ] . in healthcare facility waiting rooms it is recommended that symptomatic patients be given a respiratory mask to wear to protect others as part of respiratory hygiene [ ] . this social behaviour may alter the perception of risk for staff towards mask use in two ways, particularly in the emergency department. firstly, hcws may take a view that it is the patient's, not their own, responsibility to abide by these infection prevention measures and purposefully choose not to wear a mask on the basis of responsibility. secondly, they may not perceive a risk of becoming infected if a patient is wearing a mask and so will not use one. there are several risks for hcws adopting this behaviour. the patient may not wear the mask correctly or remove it at any time, especially if they are kept waiting for long periods, thus exposing other patients and hcws. additionally, the patient may not be able to tolerate a mask for long if unwell and will then remove it. clinical examination may put the hcw at higher risk of exposure, even if the patient is wearing the mask correctly when they enter the room or cubicle and certain procedures, such as taking a swab for influenza testing, collection of an induced sputum specimen or intubation, require removal of the patient's mask. if an hcw fails to adequately explain why they are wearing a mask it can erect a social barrier between the hcw and patient. patients may feel stigmatised if staff wear a mask to care for them [ ] while staff may feel that wearing a mask in the ed can inhibit empathy and rapport with a sick patient [ ] . hcws working in paediatric units have expressed concern that ppe may frighten their patients [ ] . social interactions within the workplace can influence the health-related behaviour of workers. the safety climate and group norms at hospital unit level have been shown to influence the risk-taking behaviour associated with facial protective equipment [ ] . the use of protective masks in the healthcare setting is governed locally by policies in health and safety and ipc. as indicated earlier, adherence to such policies and guidelines is often poor. similar to other types of ppe and ipc measures, there is no strong culture of enforcement of policy relating to protective masks in the healthcare setting. this raises questions about the efficacy of mask policies, their awareness by hcws and how they are judged by clinical staff. in some countries, state-wide legislation mandates the use of a protective mask for various categories of clinical staff during the annual influenza season, if they have not received the influenza vaccination [ ] . this enforced measure has been resisted by some clinical staff because of its impact on personal choice [ ] and by others as illogical when considering the risk from all respiratory pathogens [ ] . although many countries provide national occupational safety and health policy direction, few enforce protective mask use in healthcare settings. nevertheless, sub-optimal mask use reinforces poor behaviour in the workplace and contravenes workforce health and safety responsibilities of employees [ ] . the behaviours of hcws towards protective mask use can affect the progression of a respiratory infectious disease outbreak and, if inappropriate, facilitate a pandemic. the consequences of a pandemic on a global scale are significant, with substantial negative societal effects. ease of access to international travel has been a significant factor in the worldwide spread of recent pandemics such as pandemic influenza a h n , sars and mers, therefore international travel and trade are often restricted [ ] . personal freedom of movement is also affected by public health quarantine measures and the prohibition of public gatherings. education is disrupted through school closures which results in parents taking time off work as a consequence. in addition to the consequences described above, the provision of healthcare to the general population can be disrupted. in , the influenza a h n pandemic impacted severely on the normal functioning of emergency departments in australia [ ] . more than three times the number of patients were seen, most with non-serious influenza symptoms. staff reported that heavy workloads, lack of infection control facilities and distraction from their core business compromised the care of non-flu patients. large numbers of patients requiring care will lead to bed shortages and hospital admission gridlock, probable loss of critical care beds which are blocked with long stay respiratory patients and the cancellation of routine surgical lists [ ] . furthermore, there will be fewer hcws available to provide the care due to their own illness or having to look after family members. in this paper we detail why we need to know more about hcws' decision-making and risk-taking behaviour in relation the use of masks for protection against infectious respiratory diseases. we argue that the value of such research would be its potential impact on the ongoing threat of emerging infectious diseases, workforce capability, capacity and educational needs, the financial costs of healthcare and outbreaks and the importance of social responsibility and appropriate legislation in planning for global security. specifically, research is required to determine whether hcws' perception of risk as it relates to the protection of themselves and others against transmission of infection influences their behaviour towards the use of a protective mask. there is also a need to determine the personal, professional and contextual factors that impact on hcws' perceptions of risk and their use of protective masks for infectious diseases. an exploration of the practices and constructs of risk by hcws will therefore provide valuable information to inform policy and pandemic planning. the sub-optimal use by hcws of protective masks for respiratory diseases has a significant impact at individual, organisational, societal and global levels. furthermore, the consequences of poor mask use will be exacerbated during a widespread outbreak or pandemic of a novel infectious respiratory disease, when pharmacological agents or vaccination are unavailable. minimising the transmission of respiratory disease through protective mask use leads to better outcomes for healthcare, workforce capability and economic stability. this paper has presented the background and justification for research into the attitudes and behaviour of hcws towards protective mask use for respiratory infectious diseases during non-outbreak situations so as to optimise the use of masks when indicated in every day practice. the research can provide insight into perceptions of risk and risk-taking behaviour in respect of mask use for respiratory infectious diseases and help to bridge the gap between theory and practice (see table ). rb and rs originated the concept for the paper and rb drafted the manuscript. glg and rs had critical review and input into the preparation of the manuscript. all authors approved the final version of the manuscript. rzs is a senior editor and glg a section editor of infection, disease and health but neither had a role in peer review or editorial decision-making of the manuscript. the authors declare no other conflict of interest. this work is supported by the australian partnership for preparedness research on infectious diseases emergencies (apprise) of which author glg is a chief investigator and author rb is recipient of a doctoral scholarship. this research presented in this article is solely the responsibility of the authors and does not reflect the views of apprise. not commissioned; externally 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international health regulations: explaining the use of trade and travel barriers during disease outbreaks pandemic (h n ) influenza and australian emergency departments: implications for policy, practice and pandemic preparedness the practical experience of managing the h n influenza pandemic in australian and new zealand intensive care units key: cord- - yb b g authors: hammerschmidt, sven; hacker, jörg; klenk, hans-dieter title: threat of infection: microbes of high pathogenic potential – strategies for detection, control and eradication date: - - journal: international journal of medical microbiology doi: . /j.ijmm. . . sha: doc_id: cord_uid: yb b g abstract infectious diseases due to microbes of high pathogenic potential remain a constant and variable threat for human and animal health. the emergence of new diseases or the re-emergence of diseases that were previously under control complicates the situation to date. infectious disease research, which has undergone a dramatic progress in understanding disease mechanisms such as host–pathogen interactions, is now focusing increasingly on new strategies for prevention and therapy. significant progress has been achieved in the development of delivery systems for protective heterologous protein antigens and in veterinary vaccinology. a landmark of infectious diseases research is the chemical synthesis of genomes, a major new field of research referred to as “synthetic biology”, that to date has resulted in the chemical synthesis of the poliovirus and of phage φx genomes and their expression as infectious viruses. on the molecular level the evolution of pathogens and mechanisms of genome flexibility, which account for several pathogenic properties of infectious agents, have received increased attention. bacterial toxins are an additional threat to human health and their interference with host cells and cellular functions is receiving more attention. this report highlights some of the lectures that were presented during the international symposium 'threat of infection: microbes of high potential -strategies for detection, control and eradication' in july in wu¨rzburg (germany). this meeting was organized by the german academy of natural scientists leopoldina (deutsche akademie der naturforscher leopoldina, halle, germany), the acade´mie des sciences (paris, france) and the research center for infectious diseases of the university of wu¨rzburg together with other scientific societies (germany). the speakers and two poster sessions covered many aspects of infectious diseases, including political and social aspects. at the molecular and cellular levels, the action of virulence factors and toxins, the potential applications of rna interference and mechanisms of genome flexibility were discussed. the reservoirs and transmission of pathogens, novel detection methods and the exploitation of mechanisms of host-pathogen interaction were further discussed for the development of novel preventive and therapeutic approaches. a comprehensive summary with papers of each of the speakers who had been invited for an oral presentation during this symposium will be published soon in the series 'nova acta leopoldina' (http://www.leopoldina.uni-halle.de). infectious diseases are the biggest killer of children and young adults, accounting for more than million deaths a year and as many as one in two deaths in developing countries. almost % of deaths from communicable diseases are caused by six diseases: acute respiratory and diarrhoeal infections, aids, tuberculosis, malaria, and measles. david l. heymann (who, geneva) reminded that many deaths occur as a result of preventable diseases such as measles, hepatitis b, diphtheria, whooping cough and, most often, because of failure to implement vaccination programs. the burden of infectious diseases is not restricted to developing countries, since previously controlled infectious diseases such as tuberculosis and diphtheria have re-emerged in europe and other developed areas of the world. high mortality due to infectious diseases is alarming but so too is the fact that large populations in remote areas of the developing world are at risk of disabling diseases, such as polio, leprosy, lymphatic filariasis, and onchocerciasis. as further reported by david l. heymann these diseases cause a double economic burden by reducing the work force and undermining the financial security of already impoverished families. most infectious diseases can be prevented with drugs or vaccines that are already available, but inadequate funding of health care in developing countries or the inability to deliver the antimicrobial drugs and antibiotics to the entire population contributes to the devastating situation ( table ) . the emergence of severe acute respiratory syndrome (sars), which is one of diseases newly identified in recent decades, clearly demonstrated that every country is vulnerable and that the economic consequences, exaggerated by public fear of the unknown, can be felt around the world. today, diseases can be transported from one continent to another in a matter of hours. for these reasons public health care systems have received more attention. david l. heymann stated that global partnerships and strategies are required that increase the power of epidemic control such as real-time electronic communications. in considering novel vaccination strategies against microbes of high pathogenic potential, one has to distinguish between the case of bio-warfare and newly emerging agents with high pathogenic potential. we have to consider that, in the latter case, mass vaccination or even vaccination of high-risk individuals would be unrealistic especially since the overall risk of side effects will exceed the risk of disease. rapid protection of individuals at risk has to be given high priority. this causes various problems, including rapid diagnosis as well as prompt induction of a highly efficacious host response, both in naive and in infected individuals, in addition to prompt chemotherapy. therefore, classical vaccination strategies need to be complemented by generic vaccination strategies and immunomodulatory interventions. such strategies must include both innate and acquired immunity. in his presentation, stefan h. e. kaufmann (max-planck-institute for infection biology (mpi), berlin) suggested that the following vaccination strategies against intracellular bacteria deserve consideration: (i) attenuated viable strains, (ii) naked dna encoding protective antigens and (iii) protective antigens expressed by recombinant viable vectors (bacteria or viruses). extracellular bacteria are controlled by antibodies -typically opsonizing antibodiesand, hence, vaccination strategies are based on killed vaccines or subunit vaccines. protection against intracellular bacteria depends on t-lymphocytes rather than antibodies (winau et al., ) . although recent advances in genomics, basic immunology and molecular biology provide the guidelines for constructing novel vaccine candidates in research programs involving stretching from basic science to applied field studies. werner goebel (microbiology, wu¨rzburg, germany) highlighted the development of live virulence-attenuated bacteria as delivery systems for protective heterologous protein antigens. two different strategies were used: one that allows the release of the antigen from gramnegative bacterial carriers via type secretion system (t ss) (gentschev et al., ) and one that introduces antigen-determining plasmid dna or rna into mammalian cells including antigen-presenting cells via intracellular bacteria (dietrich et al., ; pilgrim et al., ; schoen et al., ) . a self-destructing variant of the gram-positive bacterium listeria monocytogenes, which activates a phage-derived lysine and lyses upon internalization, was used for efficient release of dna or mrna into the cytosol of host cells (pilgrim et al., ; schoen et al., submitted) . transfer of dna and rna was highly efficient not only into epithelial and fibroblast cell lines, but also into phagocytes including dendritic cells. the a-hemolysin (hlya) secretion system of e. coli was exploited as a t ss to deliver heterologous antigens. hlya can be genetically fused to almost any protein antigen without drastically affecting secretion, e.g. by virulence-attenuated salmonella strains including s. typhi ty (gentschev et al., ) . exploitation of type iii and auto-transporter secretion systems of gram-negative bacteria for recombinant vaccine delivery into the cytosol of mammalian cells has recently been demonstrated (ru¨ssmann, a, b; rizos et al., ) . werner goebel emphasized that these approaches might not only be useful in combating infectious diseases but might also open up new avenues in anti-tumor therapy by either designing anti-tumor vaccines or by delivering therapeutic agents. infected animals are not only a major economic factor in animal husbandry but also have a high impact as reservoirs of human pathogens. veterinary vaccines have to be cost-efficient, easy to administer and efficacious within a short period of time after, preferably, one immunization. this implies a rather exclusive reliance on live vaccines. fox rabies, a major threat for human health, will serve as an example. initiated by swiss researchers, oral immunization schedules have been devised which were initially based on the use of baits composed of chicken heads filled with blisters containing live-attenuated rabies vaccine (steck et al., ) . further research led to the development of industrially produced bait casings that are attractive enough to be eaten by foxes (schneider, ) . bait distribution over large areas eradicated rabies or drastically reduced its incidence in several european countries (po¨tzsch et al., ) . the successful oral immunization against rabies in foxes was followed by a similar approach against classical swine fever (csf) in wild boar. csf is a highly contagious disease of high pathogenic potential that is contained within the european countries by elimination of all pigs on infected holdings. most of the sporadic occurrences of csf in domestic pig can be epidemiologically linked to wild boar. therefore, elimination of csf virus from the wild boar population is a major prerequisite for protecting domestic pig farms. a corn-based bait that contains a blister filled with the live-attenuated csfv c-strain vaccine has all but eliminated csfv, which once was quite common in the german wild boar population (kaden et al., ) . veterinary vaccinology has recently achieved spectacular success by introducing the first genetically engineered live viral vaccines and marker technology able to differentiate between infected and vaccinated animals (diva or marker concept). thomas c. mettenleiter from the friedrich-loeffler-institute (insel riems, germany) illustrated the success of disease control by applying the diva concept. the diva concept (van oirschot, ) has been successfully employed for the elimination of aujeszky's disease (ad) in pigs caused by the alphaherpesvirus pseudorabies virus (prv; mettenleiter, ) . based on the initial finding that several live-attenuated prv vaccine strains lack a major surface antigen (glycoprotein e, ge) which is invariably present in all field strains (mettenleiter et al., ) , a simple elisa system has been developed that is able to specifically detect the presence or absence of anti-ge antibodies in the animal (van oirschot et al., ) . based on this concept, eradication programs allow the use of ge-deleted ('marker') vaccines combined with sero-surveillance using detection of anti-ge antibodies. using this approach, germany has been declared free of ad by february , (mu¨ller et al., ) . it is important to mention that during the ad eradication campaign, genetically modified live vaccines were used for the first time in europe on a wide scale. a similar eradication program has recently been started to eliminate bovine herpesvirus infections in cattle (beer et al., ; kaashoek et al., kaashoek et al., , . the less complex rna viruses generally do not specify a protein product like the glycoprotein e of prv and, therefore, require different diva approaches (scahaw (scientific committee on animal health and animal welfare), ). the diva concept is also applicable to the control of foot-and-mouth-diseases virus (fmdv), a picornavirus of the genus aphthovirus. only four virally encoded structural proteins are present within the mature virion, whereas in infected cells (and animals), non-structural proteins also act as antigens. thus, infected animals produce antibodies against both structural and non-structural proteins, whereas animals vaccinated with the inactivated virions will only produce antibodies against virion components. a recombinant swine fever virus (csfv) vaccine (de smit, ) with an antigenetically different e glycoprotein (from bovine viral diarrhea virus) induces excellent protection against csfv challenge (reimann et al., ) . likewise, a diva strategy is based on differential detection of antibodies against a heterologous neuraminidase in avian influenza. these examples, presented by thomas c. mettenleiter, illustrate the success of the diva concept in cost-efficient control and eventual elimination of specific infectious agents in livestock. a virgin population is particularly threatened by unintentional or intentional re-introduction of eradicated pathogens, such as variola virus. release of smallpox virus, the only virus to be successfully eradicated, would be catastrophic, since % of the population is susceptible to the disease and nobody under the age of years has ever been vaccinated (fenner et al., ) . the last natural case of smallpox occurred in somalia in . routine vaccination programs were stopped and vaccine producers ceased making vaccines. approximately million died from the disease prior to its eradication. the eradication strategy comprised widespread vaccination of at least % of the population, and surveillance for the early detection of cases and control of outbreaks. smallpox virus officially still exists in two laboratories of the who, one in atlanta, georgia, usa and one in novosibirsk, russia (and possibly unofficially in others). in these laboratories, research is continuing. the former deputy director of the soviet union biological weapons program declared in that smallpox would be developed as a weapon and that an annual stockpile of ton variola virus would be maintained (alibek and handelman, ) . bioterrorism has considerably dampened the hopes of positive medical and economic benefits resulting from the global eradication of a disease. in his presentation, reinhard kurth (robert-koch institute, berlin, germany) asked whether we can really eradicate infectious diseases. the development of a new vaccine or improvement of the old vaccine face challenges: the clinical efficacy of a new vaccine cannot be tested in humans (rosenthal et al., ) and the side-effects of the old one will be not accepted by the community. most countries, however, have no vaccine and will be dependent on others in the case of emergency as reported by donald l. henderson (johns hopkins center for civilian biodefense studies, baltimore, usa). poliomyelitis is the only disease worldwide that is currently being seriously targeted for eradication with a possibility of success. the number of cases decreased from over , cases in countries to cases in six countries between and . although in the majority of regions poliovirus, a human enterovirus of the picornaviridae, has already been eliminated (rasch et al., ) , the global eradication campaign will be in danger in those african countries which have for economic reasons reduced the vaccination campaigns after achieving polio elimination. the termination has led to an increase of susceptible persons. ninety percent of polio infections are asymptomatic. transmission can, therefore, easily occur without detection and even new outbreaks are at high risk. this problem is present in africa where sudden new cases are seen after many years of being polio-free. new problems will arise in the posteradication period, including those posed by the new threat of bioterrorism. the live attenuated virus used as a vaccine can be transmitted and recombine with other enteroviruses, most likely c-cluster coxsackie viruses. the result can be a vaccine virus -vaccine-derived poliovirus (vdpv) -with increased virulence and transmissibility. long-term circulation of such vdpv has, in the past, occurred in countries with suboptimal oral polio vaccine (opv) vaccination rates (kew et al., ) . this occurred independently in egypt ( ( - ( ), hispaniola ( , philippines ( ), and madagascar ( - . the live attenuated (sabin) opv was developed in the s. to minimize the circulation of vaccine viruses in, e.g. immunodeficient or unvaccinated individuals, it is essential that the who initiates an early and synchronized stop of live attenuated (sabin) opv vaccinations (maclennan et al., ) . after the confirmed global eradication it is up to the individual countries to stop vaccinating or to switch to using inactivated (salk) polio vaccine (ipv). at least for those countries in which ipv is produced, the manufacturers have to maintain large quantities of the wild-type virus and the corresponding seed virus stocks. even after the cessation of vaccination it will be essential to conserve the wild-type virus in case of emergency to fall back on these vaccines (henderson, ) . the problems with the containment of wild-type poliovirus would be reduced if the production of ipv could be successfully based on strains derived from the sabin vaccine virus (s-ipv, doi et al., ) . most of the basic mechanisms of poliovirus pathogenesis are still obscure. this is true for the replication of poliovirus in the gastrointestinal tract after oral infection and the mechanism by which the virus spreads to the central nervous system (cns). another mystery is the cunning preference of the virus to destroy motor neurons in the cns, especially in the spinal cord, a specificity leading to paralysis and possibly deathpoliomyelitis. a major determinant of poliovirus pathogenesis is the cd receptor (koike et al., ; mendelsohn et al., ) . analysis of human and primate specimens of the gastrointestinal tract revealed strong expression of cd in enterocytes, in follicularassociated epithelial cells and m-cells of peyer's patches (pps), which represent an important site of replication during first stage of infection (minor, ; iwasaki et al., ) . endothelial cells in the interfollicular region of pps were also found to express cd , an observation that leaves open the possibility that poliovirus may enter the bloodstream via vessel damage in these tissues (iwasaki et al., ) . entry into the cns can occur by passage through the blood-brain barrier or at neuromuscular junctions, followed by retrograde axonal transport to the motor neuron cell body. the latter is the route of cns invasion in ''provocation poliomyelitis'', e.g. injury enhanced disease progression (gromeier and wimmer, ) . a cargo protein of the dynein motor, tctex- , was discovered to have affinity to the c-terminal intracellular domain of cd . therefore, a new model of retrograde transport was proposed: once the virus has been internalized at the neuromuscular junction, it is transported to the cell body of motor neurons in cargo vesicles along axonal microtubules, driven by the dynein motor complex . the chemical synthesis of poliovirus in the absence of natural template (cello et al., ) indicated that a virus can no longer be considered extinct when its genome sequence is known. a poliovirus-specific double-stranded dna (''cdna'') that was assembled from synthetic desoxy-oligonucleotides was transcribed with an enzyme of bacteriophage t (t rna polymerase) yielding poliovirus rna. this rna was identical to the genomic rna of nucleotides except for the end (cello et al., ; van der werf et al., ) . the sequence of the synthetic poliovirus (spv) cdna was designed such that it contained ''silent'' mutations as identity markers (cello et al., ) . the spv rna was incubated in a cell-free extract of uninfected hela cells that directs virus specific translation, genome replication and virion assembly (molla et al., ) . this via synthetic cdna outside living cells generated polio virus has a plaque phenotype similar to that occurring in nature. the spv is growing in tumor cells but is highly attenuated (att) in cd transgenic mice (cello et al., ) . the first biologically active cdna synthesized was that of rna phage qb (taniguchi et al., ) . this landmark accomplishment heralded the birth of reverse genetics in virology. in contrast to cello et al. ( ) who synthesized virus from chemical information only, naturally occurring rna were required as templates for the enzymatic reactions preparing qb cdna. this is fundamentally different and, as proof of principle, has far reaching consequences as stated by eckard wimmer (stony brook, ny, usa). the chemical synthesis of genomes of viruses or bacteria is no longer a dream. this research referred to as ''synthetic biology'' has already become a new hot field. consequently, a paper by smith et al. ( ) described the synthesis of a chemical mixture of phage fx , bp long, in only weeks. the re-programming of microorganisms by existing functional modules (''lego biology'') may yield novel microbes capable of functioning as highly specific and efficient mini-factories (ferber, ) . genetic flexibility is a phenomenon that accounts for the pathogenic properties of many infectious agents. therefore, one focus of genomic research has been the characterization of mechanisms that are involved in genomic variability and evolution of bacterial pathogens. a new type of genomic entity -known as pathogenicity islands -has been shown to contribute to the rapid evolution of bacterial pathogens by horizontal gene transfer (for review, see dobrindt et al., ) . another flexible genetic element is represented by integrons, which were first identified as the primary mechanism for antibiotic resistance gene capture and dissemination among gram-negative bacteria. studies on yersinia and bartonella have further shown that numerous gene rearrangements, massive gene inactivations and loss of gene functions are at least as important determinants of pathogenicity as gene acquisitions. siv g.e. andersson (uppsala, sweden) presented that the deterioration of the a-proteobacterial human pathogen genomes, which could mean eliminations of a few thousand genes, shifted the bacterial lifestyle to intracellular animal environments and vector-mediated transmission pathways. a computational approach was used to infer ancestral gene sets and to quantify the flux of genes along the branches of the aproteobacterial species tree (boussau et al., ) . the analysis suggests that rickettsia and wolbachia represent the earliest diverging lineage in the tree, followed by caulobacter (boussau et al., ) . the analysis indicated a loss of more than genes prior to the divergence of rickettsia and wolbachia, followed by another loss of about genes prior to or associated with the emergence the typhus pathogen. r. conorii contains more than genes not present in r. prowazekii, the causative agent of epidemic typhus. eighty percent of the genes are orphans and not identified in any other species. genomic analysis suggests further a massive loss of more than genes immediately prior to the divergence of bartonella and brucella. this is followed by yet another major loss of some genes prior to the divergence of b. henselae and b. quintana. the genome sequence of the louseborne b. quintana comprises , , bp, and that of the zoonotic agent b. henselae comprises , , bp (alsmark et al., ) . a genome comparison elucidated a high degree of overall similarity between the two genomes. b. quintana and b. henselae, which cause trench fever and cat-scratch disease, respectively, are unique among bacteria in their ability to induce tumorlike lesions of the skin (bacillary angiomatosis) as well as the liver and spleen (bacillary peliosis) of immunocompromised individuals. both are transmitted by insect vectors, using mammalian reservoirs, and infecting similar cell types. however, b. quintana is a specialist, using only humans as a reservoir, whereas b. henselae is more promiscuous. the difference in gene content is mainly due to the presence of several genomic islands in b. henselae that are not present in b. quintana. around % of the unique genes in b. henselae were attributed to a prophage region of kb and three genomic islands of , and kb, respectively. the prophage is an evolutionary mosaic with genes of different origins that are interspersed with genes showing sequence similarities to a putative phage in wolbachia. potential virulence features located on the b. henselae genomic islands are multiple copies of hecb and fhab putatively coding for a hemolysin transporter and a filamentous hemagglutinin, respectively. these two proteins form a two-partner secretion system, where the hecb gene product may mediate the transport of filamentous hemagglutinin that is required for attachment to the host cell (alsmark et al., ) . the largest unique island in b. quintana is only kb and contains a gene with sequence similarity to yopp in yersinia enterocolitica and yopj in yersinia pseudotuberculosis. sequences in b. quintana located at positions corresponding to islands in b. henselae contain remnants of phage and island genes, suggesting that these were present in the common ancestor of the two species. in total, the siv g.e. andersson's group has identified pseudogenes and extensively degraded gene sequences in b. henselae (including tandem duplication remnants) as compared to fragmented genes in b. quintana. the rearrangements flank islands uniquely present in b. henselae, indicative of a link between rearrangement and insertion/deletion events. remnants of genes present in the b. henselae islands have been identified at the breakpoint positions in b. quintana. as reported by siv g. e. andersson both of the two bartonella genomes contain numerous scars of putative phage and plasmid integrations, indicative of exposure to genetic parasites in the evolutionary past. however, the human pathogen specialist b. quintana has lost most of these sequences and seems no longer to accumulate genetic parasites. thus, both of the two human pathogen specialists, r. prowazekii and b. quintana, have experienced an enhanced frequency of genome degradation as compared to their closest relatives with other host-vector reproduction strategies. interestingly, the high pathogenic potential of yersinia pestis is not solely attributable to the presence of two additional plasmids as shown by elisabeth carniel (institut pasteur, paris). a comparative analysis with the ancestor y. pseudotuberculosis revealed chromosomal genes in addition to the plasmids that represent the new genetic material in y. pestis. in contrast, other pseudogenes and genes absent from y. pestis were detected, indicating that as many as % of y. pseudotuberculosis genes no longer function in y. pestis (chain et al., ). it appears that loss of function is at least as important as gene acquisition in the evolution of y. pestis. high genetic variability is particularly dangerous if pathogens originate from animal reservoirs. an example is pandemic influenza which is a zoonotic disease caused by the transfer of influenza a viruses or virus gene segments from aquatic bird reservoirs to humans and domestic animals. in their natural reservoir these viruses are in evolutionary stasis -they are in equilibrium with their natural host and cause no disease. but after transfer to new hosts they evolve rapidly posing a constant threat to human and animal health as told by robert g. webster (st. jude children's research hospital, memphis, usa). widespread infections of poultry with h n viruses in asia have caused increasing concern that this subtype forms the basis for human-to-human spread and interspecies transmission. multiple opportunities for the successful mammalian transmission of h n influenza viruses are provided by their continuing evolution in asia, their propensity for re-assortment, the generation of multiple genotypes of h n viruses (matrosovich et al., ) , antigenic drift in the ha gene of h n viruses, and the acquisition of high pathogenicity for aquatic birds. if an opportunity for re-assortment with human influenza strains occurs, then the likelihood of successful transmission between humans is high. marburg and ebola viruses, family filoviridae, reemerge periodically from an unknown animal reservoir. they cause severe hemorrhagic fever in humans and non-human primates. disturbances of the blood tissue barrier, primarily controlled by endothelial cells, and immune suppression seem to be the key pathogenic factors of the disease as reported by heinz feldmann (national microbiology laboratory, winnipeg, canada). the endothelium is affected in two ways: directly by virus infection leading to activation and lytic replication and indirectly by a mediator-induced inflammatory response. these mediators originate from virus-activated cells of the mononuclear phagocytic system which are the primary target cells. immune suppression may result from lytic infection of circulating and sessile cells of the mononuclear phagocytic system, inactivation of neutrophils by secreted viral proteins, necrosis of antigen-presenting cells, and lymphoid depletion. in addition, the virion structural protein (vp ) has been identified as an interferon antagonist (basler et al., ) . despite being clearly immunosuppressive, there is good evidence for protective immunity during filovirus hemorrhagic fever. in contrast to survivors and asymptomatic cases that show igm and igg antibody responses to viral antigens, fatal infections usually end with high viremia and little evidence of a humoral immune response. neutralizing antibodies are directed against the transmembrane glycoproteins, which are responsible for virus entry. however, infectivity-enhancing antibodies directed against the transmembrane glycoprotein have also been described. the transmembrane glycoprotein can be used to provoke a protective immune response in animal models including nonhuman primates. several distinct approaches including dna vaccination or replication-deficient and replication-competent recombinant viruses can achieve this. thus, the transmembrane glycoprotein serves as the most promising vaccine candidate for filoviral hemorrhagic fever (feldmann et al., ) . yellow fever virus and lassa virus, an arenavirus, are zoonotic viruses that both cause , - , clinically apparent infections per year in west africa, carrying a mortality of - %. while the transmission, epidemiology and immunology of rodent-borne lassa fever is different from mosquito-borne yellow fever, the current re-emergence of both diseases is influenced likewise by the ecology of the viral reservoirs as well as the immunity of the susceptible human populations. herd immunity to yellow fever virus which was achieved in the s through mass-vaccinations is generally fading, since immunization programs have been discontinued. humoral cross-immunity due to heterologous flavivirus infections can mitigate severe disease, but cannot prevent yellow fever epidemics, which are again threatening the urban centers of west africa. no vaccine has ever been developed against the mainly t-cell-controlled lassa virus, but outbreaks of the disease have been rare because of focally restricted contact with infected rodents in west africa. migration of susceptible human populations and severe changes in the habitat of the rodent reservoir have now led to increased transmission of the virus and export of lassa fever cases to european countries (j. ter meulen, institute of virology, university of marburg, germany). the flavivirus family comprises about different viruses, most of which are transmitted to their vertebrate hosts by mosquitoes or ticks. several flaviviruses are important human pathogens -including the yellow fever, dengue, japanese encephalitis, west nile, and tick-borne encephalitis viruses -and some of them have proven to have the characteristics of newly emerging pathogens. as pointed out by franz x. heinz (institute of virology, medical university of vienna, austria) enormous progress has been made in recent years in the elucidation of the molecular structure of flaviviruses, which has greatly enhanced our understanding of specific functions during entry and assembly, viral pathogenicity, antigenic relationships, and immunogenicity. tick-borne encephalitis virus is endemic in many european countries and large parts of asia, including northern china and northern japan. three closely related subtypes can be discerned (european, siberian, far eastern), which circulate in their natural foci between different mammalian species and ticks (mainly ixodes ricinus and ixodes persulcatus). humans are infected only accidentally and do not play any role for maintaining the natural cycle of the virus. both the virus and the geographic distribution of natural foci are remarkably stable and exhibit relatively little variation, suggesting relative evolutionary stasis of the virus and a delicate balance of environmental factors required for this maintenance in nature. a highly purified formalininactivated whole virus vaccine has been in widespread use in europe and proven to be an excellent means for the prevention of tick-borne encephalitis in humans. the recent introduction of west nile virus to northern america is a dramatic example of the potential of flaviviruses to conquer new territories within a short period of time. although the same virus sporadically occurs in europe and also causes rare infections in humans, it has never been able to establish endemicity similar to what has occurred in america, where the virus has apparently found ideal ecological conditions for its maintenance and spread. so far human vaccines are not yet commercially available but are being tested in human trials. the four types of dengue viruses can cause severe forms of dengue fever (hemorrhagic fever and shock syndrome) and pose an enormous health threat to tropical and subtropical countries around the globe. these severe forms of the disease are apparently promoted during secondary infections with a certain dengue virus type by immunological enhancement phenomena triggered through the pre-existing immunity against a different type. this phenomenon has severely hampered the development of dengue vaccines, because of concerns that vaccination could induce a similar immunopathological state in vaccinated individuals. bacterial toxins represent important and highly efficacious virulence determinants of pathogens and are, therefore, a threat to human health in connection with infectious diseases (bradberry et al., ; o'loughlin and robins-browne, ; . elucidating the mode of action of toxins is important to understand pathogenicity and will help to prevent and treat diseases (karmali, ) . substantial progress has been made in elucidating the mechanisms by which bacterial toxins, such as tetanus, botulinum, anthrax and shiga toxin, invade cells and interfere with vital cellular functions. kirsten sandvig (institute for cancer research, norwegian radium hospital, oslo) introduced the pathways used by shiga toxin. the bacterial shiga toxin produced by shigella dysenteriae, shiga-like toxins (stxs) produced by escherichia coli, cholera toxin (vibrio cholerae), and certain plant toxins such as ricin act on cells after entry of an enzymatically active part of the molecule into the cytosol. in general, the toxins have one moiety which binds to cell surface receptors and another, enzymatically active moiety, which enters the cytosol and exerts the action of the toxin. studies of toxin entry into cells also provide us with basic knowledge about endocytosis mechanisms, intracellular transport and membrane translocation of proteins in cells (iversen et al., ; lauvrak et al., ; llorente et al., ; . shiga toxin was the first molecule found to move all the way from the cell surface to endosomes, and then retrogradely to the golgi apparatus and the er (sandvig et al., ) . a similar trafficking has been shown also for toxins such as cholera toxin, ricin and others . on the other hand a number of protein toxins travel a much shorter distance before being translocated to the cytosol (sandvig, ) . endocytosis is mediated via both a clathrin-dependent and -independent mechanism. the experiments with ricin indicated that the endocytosis can be independent of lipid rafts and can even under some circumstances operate without gtp hydrolysis (garred et al., ) . shiga toxin can also enter cells by clathrin-independent mechanisms (lauvrak et al., ; nichols et al., ; saint-pol et al., ) . however, the fraction of toxin exploiting the different pathways is likely to be cell-type dependent. similarly, cholera toxin can be endocytosed by different mechanisms (shogomori and futerman, ; singh et al., ; torgersen et al., .) and most probably cell-type-dependent differences exist. shiga toxin and ricin seem to be transported from the early endosomes by a rab -independent process, either directly to the golgi or via the perinuclear recycling compartment (iversen et al., ; johannes and goud, ; sannerud et al., ) . for shiga toxin rab is implied in the transport and clathrin is required for efficient endosome-to-golgi transport (johannes and goud, ; lauvrak et al., ; saint-pol et al., ) . both molecules are not required for ricin transport (iversen et al., ) . after arrival in the trans-golgi network the toxins are transported to the er. neither shiga toxin nor ricin contain a kdel sequence that could facilitate retrograde transport by the kdel receptor and the copi-coated vesicles. the kdel receptor-dependent transport has been shown for pseudomonas exotoxin a (jackson et al., ) . in the case of shiga toxin there is evidence for a rab , copi-independent pathway, but not all details are known (johannes and goud, ; sannerud et al., ) . in summary, kirsten sandvig indicated that more work is required to understand the retrograde transport and that the answers are important and might help to exploit the toxins in medical therapy. despite significant progress was made in controlling infectious diseases and understanding disease mechanisms with special emphasis on the molecular level, we face to date several new threats due to infectious agents. vaccination programs aimed to eradicate high pathogenic microorganisms with high mortality rates. nevertheless, the emergence of newly identified diseases such as sars and the re-emergence of diseases has become a great public health concern worldwide. a further threat is the dramatic increase of antimicrobial resistance of pathogens. antimicrobial resistance against antibiotics and other drugs has a deadly impact on the control of diseases such as tuberculosis, malaria, cholera, dysentery, and pneumonia. the basis to control infectious diseases and epidemics is the installation of worldwide accessible communication platforms by public health care systems. the interchange and storage of our current know-how and experience of successfully employed eradication programs and disease treatments is required to combat infectious diseases. a landmark in modern molecular microbiology and combinatorial chemistry is the chemical synthesis of microorganisms. this provides not only the possibility to construct novel delivery systems or vaccines but may also lead to an improved understanding of key molecules and events in the development of diseases. on the other hand, this so-called ''lego biology'' may represent a tool for bioterrorism in terms of constructing infectious agents with high pathogenic potential. the microbial evolution and genomic variability is accompanied with horizontal gene transfer. however, novel studies have indicated that not only gene acquisition but also loss of gene functions represents important determinants of pathogenicity. comparative genome analysis of microorganisms with special emphasis on intracellular pathogens will provide insights whether the evolution of these microorganisms is also associated with genome reduction by gene loss and resulted in a highly specified pathogenic potential. the developments in vaccinology in veterinary medicine highlight the potential and application of vaccine research. the successful disease control in animals by veterinary vaccination campaigns offers new perspectives to implement vaccination programs in low-or middleincome countries or even after bioterrorism attacks. veterinary vaccines have to be cost-efficient, easy to administer and efficacious within a short period of time. a single immunization is preferred. to reach and maintain high vaccination rates, novel vaccines have to fulfil these requirements. these vaccines should be ideally not based on live vaccines as used for veterinary vaccinology. instead, preferable oral immunizations with subunit vaccines and novel antigen-delivery systems have to be implemented in order to reduce the burden of infectious diseases worldwide. biohazard. random house the louse-borne human pathogen bartonella quintana is a genomic derivative of the zoonotic agent bartonella henselae the ebola virus vp protein functions as a type i ifn antagonist marker diagnostic for the eradication of bovine herpesvirus type : possibilities and limitations computational inference of scenarios for aproteobacterial genome evolution ricin poisoning chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template insights into the evolution of yersinia pestis through whole-genome comparison with yersinia pseudotuberculosis laboratory diagnosis, epizootiology, and efficacy of marker vaccines in classical swine fever: a review delivery of antigen-encoding plasmid dna into the cytosol of macrophages by attenuated suicide listeria monocytogenes genomic islands in pathogenic and environmental microorganisms progress with inactivated poliovirus vaccines derived from the sabin strains ebola virus: from discovery to vaccine smallpox and its eradication. who synthetic biology. microbes made to order reconstitution of clathrin-independent endocytosis at the apical domain of permeabilized mdck ii cells: requirement for a rho-family gtpase delivery of protein antigens and dna by attenuated intracellular bacteria use of the alpha-hemolysin secretion system of escherichia coli for antigen delivery in the salmonella typhi ty a vaccine strain mechanism of injuryprovoked poliomyelitis countering the posteradication threat of smallpox and polio endosome to golgi transport of ricin is independent of clathrin and of the rab -and rab -gtpases formation of clathrin-coated pits with long dynaminwrapped necks upon inducible expression of antisense to clathrin immunofluorescence analysis of poliovirus receptor expression in peyer's patches of humans, primates, and cd transgenic mice: implications for poliovirus infection the kdel retrieval system is exploited by pseudomonas exotoxin a, but not by shiga-like toxin- , during retrograde transport from the golgi complex to the endoplasmic reticulum facing inward from compartment shores: how many pathways were we looking for? a conventionally attenuated glycoprotein e-negative strain of bovine herpesvirus type is an efficacious and safe vaccine an inactivated vaccine based on a glycoprotein enegative strain of bovine herpesvirus induces protective immunity and allows serological differentiation oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in germany prospects for preventing serious systemic toxemic complications of shiga toxin-producing escherichia coli infections using shiga toxin receptor analogues circulating vaccine-derived polioviruses: current state of knowledge the poliovirus receptor protein is produced both as membranebound and secreted forms efficient endosome-to-golgi transport of shiga toxin is dependent on dynamin and clathrin induction of direct endosome to endoplasmic reticulum transport in chinese hamster ovary (cho) cells (ldlf) with a temperature-sensitive defect in epsilon-coatomer protein (epsilon-cop) failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man the surface glycoproteins of h influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily aujeszky's disease (pseudorabies. virus: the virus and molecular pathogenesis -state of the art pseudorabies virus avirulent strains fail to express a major glycoprotein poliovirus cell-free, de novo synthesis of poliovirus interaction of the poliovirus receptor cd with the dynein light chain tctex- and its implication for poliovirus pathogenesis eradication of aujeszky's disease in germany rapid cycling of lipid raft markers between the cell surface and golgi complex effect of shiga toxin and shiga-like toxins on eukaryotic cells bactofection of mammalian cells by listeria monocytogenes: improvement and mechanism of dna delivery summarizing the rabies situation in europe - from the rabies bulletin europe worldwide eradication of poliomyelitis an avirulent chimeric pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus autodisplay: efficacious surface exposure of antigenic urea fragments from helicobacter pylori in salmonella vaccine strains developing new smallpox vaccines yersinia outer protein e, yope. a versatile type iii effector molecule for cytosolic targeting of heterologous antigens by attenuated salmonella bacterial type iii translocation: a unique mechanism for cytosolic display of heterologous antigens by attenuated salmonella clathrin adaptor epsinr is required for retrograde sorting on early endosomal membranes transport of toxins across intracellular membranes membrane traffic exploited by protein toxins retrograde transport of endocytosed shiga toxin to the endoplasmic reticulum pathways followed by ricin and shiga toxin into cells retrograde traffic in the biosynthetic-secretory route: pathways and machinery diagnostic techniques and vaccines for foot-and-mouth disease, classical swine fever and avian influenza rabies control by oral vaccination of wildlife bacterial delivery of functional messenger rna to mammalian cells cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raftindependent mechanism selective caveolin- -dependent endocytosis of glycosphingolipids generating a synthetic genome by whole genome assembly: phix bacteriophage from synthetic oligonucleotides oral immunization of foxes against rabies qb dnacontaining hybrid plasmids giving rise to qb phage formation in the bacterial host internalization of cholera toxin by different endocytic mechanisms synthesis of infectious poliovirus rna by purified t rna polymerase diva vaccines that reduce virus transmission differentiation of serum antibodies from pigs vaccinated or infected with aujeszky's disease virus by a competitive enzyme immunoassay apoptosis paves the detour path for cd t cell activation against intracellular bacteria the authors wishes to express their appreciation for the helpful contributions and permissions of the speakers on (parts of) this report which gives an insight of presentations held during the symposium ''threat of infection'' in july in wu¨rzburg, germany. the authors are grateful to dr. anthony p. pugsley (paris) for helpful discussions and suggestions. key: cord- -dv xq k authors: davis, eric m.; ramani, chintan; quigg, mark title: neurologic manifestations of systemic disease: sleep disorders date: - - journal: curr treat options neurol doi: . /s - - -z sha: doc_id: cord_uid: dv xq k purpose of review: sleep is intimately involved in overall health and wellbeing. we provide a comprehensive report on the interplay between systemic diseases and sleep to optimize the outcomes of systemic disorders. recent findings: spanning the categories of endocrinologic disorders, metabolic/toxic disturbances, renal, cardiovascular, pulmonary, gastrointestinal, infectious diseases, autoimmune disorders, malignancy, and critical illness, the review highlights the prevalent coexisting pathology of sleep across the spectrum of systemic disorders. although it is rare that treating a sleep symptom can cure disease, attention to sleep may improve quality of life and may mitigate or improve the underlying disorder. recent controversies in assessing the cardiovascular relationship with sleep have called into question some of the benefits of treating comorbid sleep disorders, thereby highlighting the need for an ongoing rigorous investigation into how sleep interplays with systemic diseases. summary: systemic diseases often have sleep manifestations and this report will help the clinician identify key risk factors linking sleep disorders to systemic diseases so as to optimize the overall care of the patient. all earth's species maintain a solar -h cycle of rest and activity, and disrupting the cycle affects adaptation and homeostasis. sleep's quotidian "normalness" means that analogous to fish not knowing about water until it is dry, sleep is not commonly thought about until it is disrupted. for example, about % of the adult population complain of transient insomnia, and about % experience chronic insomnia that disrupts daytime function [ ] . patients with chronic insomnia experience less work productivity, more absenteeism, more accidents, and more hospitalizations, leading to direct treatment costs of approximately $ b annually [ ] . considering the potential widespread reach of comorbid sleep disorders, evaluating sleep in the neurological patient is important. this review will introduce the accepted organization of sleep disorders, review important features in history taking and evaluation, and survey the systemic diseases that have important comorbidities with particular sleep disorders. an abridged listing of sleep disorders from the american academy of sleep medicine (table ) provides an overview of the current classification [ ] . insomnia is a chronic dissatisfaction with sleep duration and quality that is associated with daytime dysfunction. although pharmacologic treatment is often pursued for chronic insomnia management, outcomes are often better addressing underlying factors with the early use of cognitive-behavioral therapy for insomnia (cbt-i) [ ] . sleep-related breathing disorders involve dysfunction of the respiratory system during sleep, usually resulting in daytime hypersomnia. obstructive sleep apnea (osa), central sleep apnea (csa), and respiratory effort related arousals are classified under this category. treatment options including continuous positive airway pressure (cpap), positional therapy, mandibular advancement devices, healthy weight loss, and even a novel cranial nerve stimulator which protrudes the tongue forward during sleep [ ••] . central hypersomnias are defined as a primary dysregulation of sleep resulting from dysfunction of the central nervous system that causes daytime hypersomnia. often treatment addresses the underlying cause and may include use of strategic napping and wake-promoting medications. circadian disorders consist of various lesions or external disruptions of the circadian timing system that desynchronize the brain's clock from the external solar light-dark cycle, resulting in hypersomnia or insomnia in a clockdependent fashion. treatment of circadian rhythm disorders involves adjusting life around the patient's desired sleep time or augmenting factors that entrain the body's clock. parasomnias represent disorders of faulty inhibition of waking behaviors that arise inappropriately during sleep and are divided into those that occur during nonrem sleep, rem sleep, or state transitions. rem sleep behavior disorder is a parasomnia characterized by loss of muscle atonia during rem sleep that usually occurs in patients with neurodegenerative disorders. it is often treated effectively addressing other sleep disturbances and treating with clonazepam or melatonin [ ] . sleep-related movement disorders consist of fragmentary, often repetitive body movements that can disrupt sleep or, sometimes worse, disturb the sleep of bed partners. periodic limb movement disorder (plmd) and restless legs syndrome (rls) both fall under this category and are treated with repletion of iron stores and consideration of dopaminergic agonists [ ] . a sleep history helps a patient disclose sleep findings and helps the physician organize it into categories of hypersomnia, sleep habits and scheduling, sleep characteristics, environmental issues, and sleep interrupters ( table ) . the epworth sleepiness scale quantifies the degree of hypersomnia [ ] . most adults require - h of daily sleep [ ] and prefer it organized into either a monophasic, nocturnal schedule or in a biphasic pattern augmented with an afternoon "siesta." the sleep pattern characterizes the presence and severity of sleep-onset insomnia, sleep maintenance insomnia, or terminal insomnia (insomnia distributed within the last half of the sleep period). "catch-up" sleep, a phenomenon of prolonged sleep on a free day, is a classic sign of sleep deprivation. habitual early-phase advances ("morning larks"), late-phase delays ("night owls"), or a chaotic, irregular schedule can be a sign of circadian disorders. one also must inquire about common sleep disruptors including leg movements, snoring, witnessed apneas, and environmental factors. the sleep diary, often available through standardized forms or even websites or smartphone apps, consists of - weeks of self-reported sleep times. the overnight polysomnography (psg) is the gold-standard measurement of sleep architecture, respiratory disorders such as osa, and parasomnias. in the case of osa, the unattended (home) sleep study has had an the multiple sleep latency test (mslt) consists of a series of daytime naps from which sleep onset is calculated. the test, in combination with psg performed the night before, is the gold standard in measuring hypersomnia, especially in the evaluation of narcolepsy. wrist actigraphy provides measurements of long-term patterns of rest and activity as proxies for sleep and wakefulness. such patterns can help to corroborate histories of sleep duration and timing. popular smartphones and other ambulatory devices with physiological monitoring capabilities may transform the evaluation of sleep. however, a recent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries or standard psg [ ] . the overall conclusion is that, at the beginning of , wearable devices are not ready for reliable quantification of sleep across individuals. although serial recordings confined to a single individual may hold some value, these measurements have yet to be validated. considering the various sleep disorders and diagnostic tools afforded by a good sleep history and sleep testing, understanding the relationship between sleep disorders and systemic diseases has far-reaching implications in optimizing the care of the patient. the following sections will address sleep manifestations of various neurological disorders arising from systemic disease based on organ system. almost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep, choking, hypersomnia, or fatigue [ ] . osa is present in approximately % [ ] . a unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airway's soft tissues and dilator muscles even though myxedema can be absent [ ] . larger goiters can also cause osa by external compression of the airway [ ] . on the other side of the thyroid spectrum, hyperthyroidism is most closely associated with insomnia, occurring in % of patients [ ] . arousal disorders-specifically, sleep walking-also occur, especially in the setting of thyrotoxicosis [ ] , proposed to arise from frequent arousals and impairment of attaining slow-wave sleep as the direct result of thyroid hormone. beyond the treatment of the specific sleep disorder, sleep problems usually remit following appropriate treatment of the underlying thyroid disorder [ ] . sleep disorders affect high proportions of those with type diabetes mellitus (dm): surveys of patients with dm compared with those of controls show a nearly -fold propensity for insomnia, fourfold higher use of sedative-hypnotics, and a -fold higher rate of hypersomnolence [ ] . osa is highly prevalent in dm, and many are undiagnosed [ ] . contributors to a multifactorial series of sleep disruptors include periodic limb movements and restless legs syndrome (rls), diabetic neuropathy, and fluctuations in blood glucose [ ] . dm presents an excellent model by which to demonstrate the reciprocal effects of sleep disruption on the primary disease. first, sleep disturbances affect the regulation of the neuroendocrine control of appetite. sleep deprivation promotes overeating through hyperactivity of orexin system [ ] and activates the hypothalamic-pituitary-adrenal system to increase cortisol secretion resulting in impaired glucose tolerance [ , ] . these multiple mechanisms support clinical observations that untreated osa may be reason for the ineffective treatment of dm, and that accordingly, treatment with cpap leads to improvements in glycemic control in some patients [ ] . sex hormones and gender affect the distribution and susceptibility to a variety of sleep disorders. men, on the basis of relative airway collapsibility, have approximately a twofold increased risk of osa compared with women ( - % in males and - % in females) [ ] . a potential side effect in the treatment of hypoandrogenism is the facilitation of osa given the impact testosterone has on upper airway collapsibility [ ] . testosterone levels may affect the propensity for chronic insomnia. men with hypoandrogenism demonstrate reduced sleep efficiency, increased nighttime awakenings, and reduced deep sleep compared with the normaltestosterone-level controls, although it is not clear whether these features improve with testosterone therapy [ ] . women experience higher rates of chronic insomnia (risk ratio of . for women versus men) which becomes even more pronounced in the elderly [ ] . despite sleeping longer, overall sleep quality is often lower in women than men [ ] . the distribution of sleep disorders in women varies with reproductive lifespan. younger women are more susceptible to restless legs syndrome (rls), mainly on the basis of menses-associated iron-deficiency. during pregnancy, women are at significantly increased risk for the development of rls with an overall prevalence exceeding % of all pregnant patients [ ] . treatment of rls in pregnancy involves iron supplementation with a goal ferritin level mcg/l. often, oral iron repletion is adequate although there are reports of intravenous iron therapy in severe cases of pregnancy-related rls and iron-deficiency [ ] . pregnancy is also associated with an increased prevalence of osa (up to % of pregnant patients during the third trimester) which is associated with increased risks of complications including gestational hypertension, gestational dm, and pre-eclampsia [ ] . although not a particular systemic neurological disease, pharmacological effects on sleep form an important aspect of neurological sleep medicine, since many medications that are used by neurologists may affect sleep. table shows common medications that provoke insomnia, hypersomnolence, respiratory suppression, parasomnias, and rls/periodic limb movement disorder. sleep disturbances are highly prevalent in patients with chronic kidney disease (ckd) spanning the broad spectrum of sleep disorders including hypersomnia, insomnia, sleep-related breathing, and rls. the prevalence of osa in ckd ranges from to %, rates that are not explained solely by overlapping comorbidities common to both osa and ckd [ ] . the co-occurrence of both ckd and osa is associated with increased cardiovascular events and all-cause mortality [ ] [ ] [ ] . usually, osa develops in patients with ckd independent of underlying renal dysfunction, but some evidence shows that ckd can cause or exacerbate osa and central sleep apnea. proposed mechanisms for this causal relationship include uremic neuropathy, altered chemosensitivity, and hypervolemia [ ] . accordingly, renal replacement therapy and fluid removal [ ] may improve obstructive or central sleep apnea. conversely, treatment of sleep apnea with pap may improve renal function in those with borderline renal impairment [ ] . rls is a common and debilitating symptom in patients with ckd, occurring in up to % of patients on hemodialysis compared with that in approximately % of the general population [ ] . although rls symptoms generally follow a circadian rhythmicity with increased symptoms occurring at night, rls symptoms can occur during the long periods of daytime inactivity during hemodialysis [ ] . treatment is primarily focused on ensuring adequate iron stores then considering medical therapy, as per routine care of rls. sleep disorders and infectious diseases have few specific associations. in general, acute infection is associated with mild encephalopathy that masquerades as hypersomnolence and fatigue. pro-inflammatory cytokines are implicated in the development of these constitutional symptoms. some infections, however, directly affect regulatory centers of the sleep-wake system. encephalitis lethargica is a historical, pandemic cause of hypersomnolence of renewed interest since this review is being written in the middle of the covid- pandemic. also known as von economo's encephalitis, it occurred in association with the spanish flu pandemic of [ ] . an estimated million were affected worldwide. the most common subtype, the somnolentophthalmoplegic form, developed after flu-like symptoms of fever and malaise and consisted of subsequent ophthalmoplegia accompanied by long periods of hypersomnia. despite the appearance of deep sleep, patients could be easily awoken and sometimes maintained memories of activities that had transpired around them while "asleep." this state of acute akinetic psuedosomnulence could be followed by the development of chronic postencephalitic parkinsonism. the pandemic associated with the severe acute respiratory syndrome coronavirus (sars-cov- ; i.e., covid- ), occurring during the writing of this review, features evolving literature. the first reports centered on respiratory symptoms. although the involvement of the nervous system now appears prevalent [ ] , sleep disorders have yet to be specifically reported. however, the psychological responses to social distancing, change in schedules, and other features of an active pandemic have caused a wave of anxiety and depression, which in turn have been associated with poor sleep quality. for example, a survey of chinese health care workers showed prevalences of depression at %, anxiety at %, and insomnia at % [ ] . postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy. in , certain vaccinations in europe for the h n pandemic caused narcolepsy at a risk of : , in pediatric patients [ ] . fortunately, the risk of postvaccination narcolepsy appeared confined to specific vaccine formulations. the incident, however, has led to ongoing research in the immunological etiology of narcolepsy. african trypanosomiasis, or sleeping sickness, remains important in the developing world. it is a parasitic infection spread by the tsetse fly that is endemic in sub-saharan africa. the first symptoms include fever, headaches, and lymphadenopathy. once the parasite enters the central nervous system, disordered, fragmented sleep ensues often with inversion of the circadian sleep-wake cycle. the world health organization outlines treatment with a regimen of antiparasitic medications once symptoms have started [ ] . non-alcoholic fatty liver disease (nafld) consists of idiopathic hepatic steatosis with a prevalence of to % of the general population with increased frequency in individuals with obesity or dm [ ] . given these co-associations, osa is common. untreated osa may exacerbate liver injury because of oxidative stress and systemic inflammation [ ] and is a risk in conversion from nafld to liver fibrosis [ ] . trials with cpap have shown inconsistent results in markers of liver injury following treatment of osa [ ] . the symptoms of gastroesophageal reflux disease (gerd) worsen during sleep, particularly if sleep occurs soon after a meal [ ] . the lower esophageal sphincter that normally prevents reflux may be compromised by the increase in thoracic pressure in the setting of the upper airway obstruction [ ] . patients with symptoms of gerd should be screened for osa, and conversely, interruption of sleep in absence of osa may improve with treatment with a proton pump inhibitor (ppi) [ ] or by simply elevating the head of the bed. inflammatory bowel disease (ibd) has bilateral interactions with sleep [ ] . given the relationship between sleep deprivation/fragmentation on cytokine regulation and immune dysfunction, it is hypothesized that poor sleep quality worsens overall symptoms of ibd [ , ] . additionally, the pro-inflammatory state disrupts the circadian rhythm [ ] . subjective and objective measurements of sleep quality and timing should be considered in patients with ibd particularly in those who have frequent inflammatory flares despite otherwise adequate management. an algorithmic approach to sleep assessment in ibd patients has been proposed by canakis et al. [ ] . systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders, with a prevalence of sleep disturbances of greater than % [ ] . the mechanisms of sleep disturbances, as well as the reciprocal relationship in the contribution of poor sleep to worse autoimmune status, are thought to be similar to those described above with ibd [ , ] . the specific sleep disorders prevalent in this group are osa and periodic limb movement disorder (plmd) (both with greater than % prevalence) [ , ] . as seen above, hypersomnolence and activity-limiting fatigue arise from specific sleep disorders, pain, and medication side effects well as the primary effects of the primary pro-inflammatory status [ , ] . often treating the underlying autoimmune disorder improves associated fatigue. however, if sleepiness persists, then evaluating for a comorbid sleep disorder such as obstructive sleep apnea is indicated. one syndrome with possible autoimmune origins is chronic fatigue syndrome. sleep disturbances, insomnia, and unrefreshing sleep are common symptoms, yet patients rarely report relief despite appropriate identification and treatment of comorbid sleep disorders [ ] . cognitive-behavioral therapy (cbt) and graded exercise therapy are commonly pursued treatment approaches [ ] . obstructive lung diseases (most commonly asthma, chronic obstructive pulmonary disease (copd), and less common disorders such as cystic fibrosis (cf) or bronchiolitis obliterans) may affect nocturnal ventilation. osa and copd often overlap given shared body habitus and other mutual risk factors; estimates of comorbid osa and copd range from to % [ ] . patients with severe copd treated with nocturnal noninvasive ventilation (nippv; a more advanced form of positive airway pressure) experience an absolute risk reduction of % of the risk of hospital readmission or death at months, compared with those treated with standard care and without nippv [ •] . insomnia is another common complaint among patients with copd. circadian bronchial constriction may cause nocturnal wheezing, dyspnea, or other symptoms of asthma prompting the patient to awaken [ ] . in addition, the hyperadrenergic response to beta agonist inhalers used in treatment for acute dyspnea impairs sleep onset (see table ). the growing success in treatments for cf patients means that sleep disorders arising from their intrinsic obstructive lung disease are now coming to the attention of caregivers. many factors contribute to sleep disruption including chronic cough, frequent infections, abdominal discomfort, reflux, frequent stools, medication side effects, and psychological disease [ ] . in addition to sleep disruption, patients with cf are susceptible to hypoventilation that worsens with disease progression. use of nippv in high-risk patients with hypercapnia has been shown to improve physiologic parameters and at times can positively impact symptoms, particularly in patients who have severe disease while awaiting lung transplant [ ] . restrictive lung diseases (defined by a reduced total lung capacity) include those with parenchymal damage such as idiopathic fibrosis, hypersensitivity pneumonitis, or other interstitial pneumonias. alternatively, lung parenchyma is normal in restrictive diseases such as obesity hypoventilation syndrome, hemi-diaphragm paresis, or neuromuscular disorders (muscular dystrophies, amyotrophic lateral sclerosis). restrictive lung disease patients, as seen above with obstructive disease patients, are susceptible to nocturnal hypoventilation, subsequent co retention, and compensatory sleep fragmentation. use of nippv in patients with severe restrictive lung disease (spanning obesity hypoventilation syndrome to muscular dystrophies and als) has had positive impacts on survival and quality of life [ , ] . over % of patients with congestive heart failure (chf) have comorbid osa mainly on the basis of mutual risk factors of dm, hypertension, obesity, and older age [ , ] . in addition, insomnia in those with chf may arise from a variety of factors including diuretic medications (and subsequent nocturia), positional heart failure symptoms, increased adrenergic status, or psychosocial factors [ ] . treatments addressing comorbid osa and insomnia improve sleep quality but demonstrate mixed results in terms of long-term cardiovascular outcomes [ , ] . patients with acute myocardial infarction (ami) experience both acute and chronic sleep disorders. due to the circadian variability of adrenergic hormones and cardiac and systemic vasculature [ ] , the timings of ami, sudden cardiac death, and arrhythmia occur with increased frequency at night [ ] . cardiac ischemia may present a series of nocturnal symptoms including paroxysmal dyspnea, chest pain, agitation, or insomnia. surviving patients are at risk for chronic sleep disorders such as insomnia and sleep-disordered breathing with or without the co-occurrence of anxiety or depression [ ] . retrospective, longitudinal data demonstrate that those with osa and who are adherent with cpap experience improved cardiovascular morbidity and mortality over non-adherent patients [ ] . however, these findings have not been clearly supported by prospective, randomized trials. the sleep apnea cardiovascular endpoints trial (save trial) has called into question the causal link between the treatment of osa and cardiovascular outcomes. with a mean follow-up of . years, those randomized to pap experienced no significant improvements in study endpoints of death from cardiovascular causes, ami, stroke, and hospitalization for unstable angina, chf, or transient ischemic attack compared with controls [ ••] . because of possible insufficient cpap use and because of the lack of main indications for cpap treatment (such as severe sleepiness), interpretation of the findings of this large trial remains controversial. in practice, these authors often pursue cpap treatment for patients with osa and cardiovascular risk factors (even in the absence of sleepiness) at least for a trial period to assess adherence to treatment and to determine if there are subjective and objective improvements to sleep quality. with a prevalence range of - %, osa is common in patients with atrial fibrillation and other arrhythmias [ ] . accordingly, the sleep heart health study showed a two-to fivefold higher risk of arrhythmia in patients with severe osa compared with that in controls [ ] . retrospective series show that, in patients with atrial fibrillation and untreated osa, the risk of atrial fibrillation recurrence following cardioversion is % compared with % in patients who are adherent to cpap [ ] . however, a prospective randomized control trial called retrospective findings into question [ ] . similar in design to the save trial, patients with atrial fibrillation were randomized to cpap versus usual therapy from a cohort in which sleepiness was specifically excluded. this small trial ( total) assessed the primary outcome of time to arrhythmia recurrence. both arms had recurrence rates of %. although the trial showed that cpap itself provides no specific benefit to those with atrial fibrillation, the outcomes for treatment of those with both disorders remain unclear. although the above studies centered on associations between cardiac disease and osa, patients with chf, ami, and atrial fibrillation experience high rates of central sleep apnea (csa) as well, exceeding % in patients with mild symptomatic chf as an example [ ] . cheyne-stokes respiration, a cyclical form of csa, results when circulatory impairment perturbs the normal responsiveness in respiratory control resulting in alterations in "the loop gain" in modulating changes in carbon dioxide and oxygen levels in the bloodstream [ ] , analogous to overly aggressive adjustments to a thermostat in response to changing temperature. the presence of csa has been considered a marker of increased mortality in patients with chf, although aims to resolve the treatment of csa (with cpap or more advanced modalities) have not clearly demonstrated an improvement in cardiovascular outcomes [ ] . estimates of the prevalence of sleep disturbances across cancer patients range widely from to % [ , ] . insomnia is the most common disorder with prevalence levels ranging from to % [ , ] . patients with cancer who undergo psg have shorter total sleep times, longer times in bed, low sleep efficiency, and proportionately less deep sleep than controls [ ] . insomnia in patients with cancer is driven by a multitude of factors including pre-existing socioeconomic and psychiatric disorders, fatigue, age, rls, pain, and medication effects [ , ] . treatment follows that for the general population. although sedative-hypnotics are most commonly prescribed, no evidence exists for specific pharmacologic interventions for sleep disturbances in this population [ ] . cognitive-behavioral therapy is currently the recommended first-line treatment for chronic insomnia [ ] . because the rarity of trained psychologists makes finding a provider difficult in some circumstances, the electronic delivery of cognitive-behavioral therapy has been sought as an alternative to face-to-face therapy [ , ] . the bilateral interactions between sleep and critical illness form a rapidly changing area of investigation which is made particularly challenging given the difficulties in measuring sleep in critically ill patients [ , ] . lack of sleep-or its encephalopathic analog-may affect outcomes in critical illnesses. for example, a lack of scorable rem sleep correlates with longer ventilator weaning time compared with controls with intact rem [ ] . failure rates on noninvasive ventilation are impacted by sleep continuity [ ] . delirium, a common neurobehavioral syndrome seen in upwards of % of patients in the icu [ , ] , is associated with significantly worse outcomes in the icu including longer hospital stay and increased mortality [ ] . in turn, disrupted circadian rhythms are a strong risk factor for the development of delirium [ ] . ongoing work may establish that the optimization of circadian rhythmicity can directly improve icu outcomes. the most widespread technique is to provide hospital environments that enable entrainment to a stable circadian rhythm. exposure to daylight, mitigation of noise, stimulation, and lights during "quiet periods" have been established as good practice in many icus [ ] . other investigations have centered on pharmacological interventions. melatonin, a hormone with important regulatory interactions with the main clock, has been employed to optimize sleep and circadian rhythm in the icu. however, a small randomized trial of nights therapy with melatonin versus placebo did not alter behavioral sleep or the need for sedation for delirium [ ] . perhaps more promising is the use of dexmedetomidine, a potent selective alpha- adrenergic receptor agonist, which provides what appears to be adequate sedation without obliteration of underlying sleep-wake cycles. a prospective study of patients on mechanical ventilation with delirium was randomized to receive dexmedetomidine or placebo in addition to standard medical care [ ] . patients in the treatment arm had a significant increase in the number of ventilator-free hours at days and shorter duration of delirium when compared with the placebo. although this trial suggests a role for dexmedetomidine in the treatment of delirium in mechanically ventilated icu patients, those in the treatment arm also required reduced amounts of other sedatives, and therefore, its benefit may be secondary rather than primary. nevertheless, dexmedetomidine may serve as a less "deliriogenic" sedative agent in the icu. other: agrypnia excitata some disorders specifically disrupt the thalamic regulation of sleep, present with primary sleep disturbances, and have a variety of systemic or hereditary causes and thus are difficult to classify in our system-based organization used above. agrypnia excitata is a syndrome of unrelenting insomnia, autonomic hyperactivity, agitated delirium, and oneiric stupor (dream-like movements and hallucinations) [ ] . polysomnography of those with agrypnia excitata will show the absence of slow-wave sleep and sleep spindles with fluctuating, poorly sustained wake, stage n , and rem sleep stages. fatal familial insomnia is an autosomal dominant disorder caused by a mutation of the prion protein. patients experience progressive, subacute dementia, delirium, and insomnia. treatment is supportive. morvan's syndrome is a rare autoimmune disorder that is caused by antibodies to voltage-gated potassium channels. patients present with acute onset of insomnia along with myokymia and diaphoresis that progresses at variable rates and usually is fatal without treatment. most have high levels of immunoglobulin and respond to plasmapheresis. finally, an unfortunately common syndrome is delirium tremens that arises acutely within h of cessation of alcohol in abusers. the onset of ophthalmoparesis and ataxia denotes wernicke's encephalopathy. unless prophylactically treated with thiamine and appropriate inhibitory alcohol analogs (such as benzodiazepines), permanent sequelae in the form of hippocampal degeneration may occur (korsakov's syndrome: profound and permanent anterograde amnesia). a sleep history is the first step in the evaluation of a patient with systemic disease who presents with "trouble sleeping." patients with hypersomnia that is not easily attributable to insufficient sleep should be evaluated with overnight polysomnography, or in certain patients with isolated risk factors for osa-home sleep testing. the mainstay treatment for osa is positive airway pressure, especially if underlying systemic disease prevents modification of the underlying risk factors of obesity. insomnia is best treated with cognitive behavioral therapy that 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with insomnia atypical sleep in ventilated patients: empirical electroencephalography findings and the path toward revised icu sleep scoring criteria altered circadian rhythmicity in patients in the icu impact of sleep alterations on weaning duration in mechanically ventilated patients: a prospective study sleep continuity: a new metric to quantify disrupted hypnograms in non-sedated intensive care unit patients delirium in the postoperative cardiac patient: a review comparison of propofol and dexmedetomidine infused overnight to treat hyperactive and mixed icu delirium: a protocol for the basel prodex clinical trial pharmacologic prevention and treatment of delirium in critically ill and non-critically ill hospitalised patients: a review of data from prospective, randomised studies delirium: a disturbance of circadian integrity? med hypotheses prevention and management of delirium in critically ill adult patients in the intensive care unit: a review based on the padis guidelines a double-blind placebocontrolled randomised pilot study of nocturnal melatonin in tracheostomised patients dexmedetomidine vs. haloperidol in delirious, agitated, intubated patients: a randomised open-label trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations papers of particular interest, published recently, have been highlighted as:of major importance key: cord- -r rkrya authors: harcourt-brown, frances title: chapter clinical pathology date: - - journal: textbook of rabbit medicine doi: . /b - - . - sha: doc_id: cord_uid: r rkrya nan as rabbits are used extensively for toxicological and physiological studies, there are many scientific papers about the effects of experimental infections, drugs and toxic substances on haematological and biochemical parameters. there is also information about diseases of commercial rabbits, which are mainly investigated by post-mortem examination. in contrast, there is a dearth of literature about the effects of clinical diseases on the blood picture of rabbits or the use of blood tests as diagnostic or prognostic indicators. it is not always possible to extrapolate from other species, especially carnivores such as dogs and cats to the herbivorous rabbit with its specialized physiology. at the present time, much of the information that is available on the haematology and biochemistry of pet rabbits is anecdotal, although it can be helpful and is better than no information at all. the collection of blood and urine samples is covered in section . . parameters such as glucose, creatine kinase and aspartate aminotransferase (ast) can be altered by stress associated with handling and restraint, or tissue damage that has occurred during sample collection. for example, potassium results appear less reliable in samples taken with a plastic cannula as opposed to a hypodermic needle . rabbit blood haemolyses easily and clots quickly (perry-clark and meunier, ). small clots affect haematology results and haemolysis affects certain biochemistry results, especially potassium and serum inorganic phosphorus that are released from erythrocytes. rapid clotting can affect the performance of some analysers and heparinized syringes and needles are required. it is not possible to take a guaranteed fasting sample from a rabbit because they ingest caecotrophs. parameters such as blood glucose are affected by digestion. some parameters such as bile acids, cholesterol and urea follow a diurnal rhythm that also affects the total and differential white cell count (fox and laird, ; fekete, ; loeb and quimby, ) . stress associated with car journeys or a period in unfamiliar surroundings will increase blood glucose and alter haematological parameters such as the distribution of neutrophils and lymphocytes. pregnancy affects parameters such as protein, haematocrit, cholesterol, alkaline phosphatase, triglycerides (viard-drouet et al., ) , glucose, sodium, calcium, phosphate and red cell indices (palm, ) . serum cholesterol is the parameter that is most affected and can be up to % lower in pregnant than in nonpregnant animals (palm, ) . anaesthesia affects some blood parameters such as potassium . the effect of anaesthesia on biochemical parameters is minimized by taking samples within minutes of induction. intravenous or intraosseous fluids will also affect haema-clinical pathology tological findings (ros et al., ) and blood samples should be taken prior to treatment. there are a number of published reference ranges for haematological and biochemical parameters in rabbits. conversion factors for the variety of units that are used in reference ranges are given in table . . differences in analytical techniques between laboratories can lead to disparity in results. laboratory data are often derived from populations of rabbits of the same breed, sex and age that are not genetically diverse. in contrast, the pet rabbit population is made up of a variety of breeds, cross breeds and is composed of rabbits of all ages. significant breed and sex differences have been noted for some parameters in laboratory rabbits (kozma et al., ) . published reference ranges for pet rabbits are either derived from sets of data from laboratory rabbits or from data collected by the author. some reference ranges are an amalgamation of other references ranges and result in a range so wide that almost any result will fall within it. for example, the reference range for serum albumin concentra-tions of pet rabbits is given as - g/l (malley, ) based on four published sets of data. gillett ( ) gives an even wider range of - g/l for laboratory rabbits based on five sets of data. for some parameters there are big differences between published reference ranges. an example is blood calcium. gillett ( ) gives a range of . - . mmol/l in comparison with . - . mmol/l by harkness and wagner ( ) . differences in calcium content of the diet between the groups of rabbits or differences in analytical technique could explain these discrepancies. different laboratory methods can result in large differences between reference ranges. an example is alkaline phosphatase. collins ( ) gives a reference range of . - . iu/l in comparison with - iu/l (gillett, ) or - iu/l (harkness and wagner, ) . automated flow cytometers are designed to measure numbers of human blood cells. rabbit erythrocytes are smaller in diameter than human erythrocytes and also vary in diameter. these differences cause problems with some automated analysers. automated differential white cells counts cannot be relied upon for rabbit blood and accurate results can only be obtained using manual counting methods (kabata et al., ) . clinical pathology some morphological characteristics of rabbit blood cells are different from other species. the red blood cells vary in diameter within a range of . - . µm (sanderson and philips, ) . this variation in diameter (anisocytosis) is a feature of blood smears from rabbits and is not a significant finding (see plate ). the red cell distribution width (rdw) is a measurement of the variation in size of erythrocytes and is higher in rabbits ( - , idexx reference range) than in dogs and cats ( - ; bush, ) . in dogs and cats, anisocytosis is indicative of the presence of reticulocytes and a regenerative anaemia. in rabbits, - % of circulating erythrocytes may be reticulotytes. polychromasia and reticulocytes in rabbit blood smears have been attributed to the short life span and high turnover of erythrocytes (kraus et al., ) . nucleated red cells and howell-jolly bodies can also be found occasionally (mclaughlin and fish, ) . rabbit neutrophils have an almost colourless cytoplasm and contain two types of granules. the smaller granules stain pink giving a pinkish colour to the cytoplasm. larger granules stain a deeper pinkish-red. the overall colour of the neutrophils varies according to the proportion of large and small granules. the granular appearance of the cytoplasm has led to different nomenclature. rabbit neutrophils may be called heterophils, pseudoeosinophils, acidophils or amphophils depending on the text (sanderson and philips, ; benson and paul-murphy, ) . neutrophils measure - µm in comparison with eosinophils that measure - µm (sanderson and phillips, ) . small lymphocytes are seen more commonly than large lymphocytes. the average cell diameter for small lymphocytes is - µm (cooke, ) . the lymphocytes are round cells with the typical morphology described for other species. an occasional large lymphocyte may have a few azurophilic granules in the cytoplasm (jain, ) . monocytes are large nucleated cells measuring - µm (cooke, ) . the nucleus has a diffuse lacey chromatin pattern that lightly stains a purple blue. the vacuolar cytoplasm stains light blue. eosinophils can be distinguished from neutrophils by their greater size and large acidophilic granules. in contrast to other laboratory species, basophils are frequently found in the circulation of rabbits in small to modest numbers (jain, ). a reference range for haematological parameters is given in table . . the haematological picture gives an indication of the general health status of a rabbit. stress and a range of diseases will alter haematological parameters. hinton et al. ( ) analysed the haematolog-• rabbit blood clots quickly and haemolyses easily • food deprivation does not guarantee a fasting blood sample as rabbits ingest caecotrophs • stress associated with transport or handling can affect parameters such as blood glucose and the distribution of neutrophils and lymphocytes • pregnancy, anaesthesia, blood collection techniques and intravenous fluid therapy will influence some blood results • time of day can influence blood results as many parameters follow a duirnal rhythm in common with many physiological processes in rabbits • laboratory reference ranges are often derived from animals of the same breed and strain. pet rabbits come from a more genetically diverse population • reference ranges for pet rabbits are often an amalgamation of laboratory reference ranges that can be so wide that almost any result will fall within them • different analytical techniques can result in disparity between laboratory reference ranges • automated flow cytometry is not suitable for differential white cell counts in rabbits. accurate results can only be obtained by manual counting methods. ical findings in healthy and diseased rabbits and found that blood cellularity was a good indicator of disease especially with regard to erythrocyte and lymphocyte counts. these findings are in agreement with studies of experimental infections in rabbits krueger, , ) and in a clinical study by harcourt-brown and baker ( ) . in this study, significantly higher red cell counts, haemoglobin values, haematocrits and lymphocyte counts were found in rabbits kept outside with unlimited access to grazing and exercise. a comparison was made with rabbits kept in hutches and those suffering from dental disease (see figure . ). reference ranges for packed cell volumes (pcv) vary between sources with values between % and % (malley, ) . pet rabbits tend to have pcv values at the lower end of the range, typically between and % (harcourt-brown and baker, ) . values greater than % are indicative of dehydration, especially in rabbits suffering from gut motility problems. values of less than % indicate anaemia that can be classi-fied into non-regenerative and regenerative in a similar manner to other species. a regenerative anaemia is associated with chronic blood loss, e.g. due to heavy flea infestation or from a site that intermittently bleeds such as a uterine endometrial aneurysm. uterine adenocarcinomas are a common finding in middle-aged does. lead poisoning can result in a regenerative anaemia with the presence of nucleated erythrocytes, hypochromasia, poikilocytosis and cytoplasmic basophilic stippling (fudge, ) . a nonregenerative anaemia is caused by diseases such as lymphoma or chronic renal failure. autoimmune haemolytic disease has not been described as a clinical phenomenon in pet rabbits although there are reports that it occurs. autoimmune haemolytic anaemia has been reported in laboratory rabbits in association with lymphosarcoma (weisbroth, ) . chronic debilitating disease such as dental disorders or abscesses often cause a mild anaemia in pet rabbits (harcourt-brown and baker, ) (see figure . ). nucleated red blood cells can be associated with acute infectious processes although a few may be present in normal blood films. experimentally, infections with escherichia coli and staphylococcus aureus cause an increase in clinical pathology number of circulating nucleated red blood cells during the septicaemic phase of the disease krueger, , ). there is a natural diurnal variation in total white cell count with lowest counts occurring during the late afternoon and evening (fox and laird, ) . the white cell count also varies with age (jain, ) . it is higher in young rabbits of approximately months of age and in adults over year old. the first peak in leucocyte count is due to an increase in the number of lymphocytes. the second peak is due to an increase in the number of neutrophils. in other species, an increased white blood cell count is seen in response to bacterial infection or in response to endogenous or exogenous corticosteroids. rabbits do not develop marked leucocytosis after either acute infectious challenge or the intramuscular injection of cortisone acetate (toth and january, ) . in two studies by krueger ( , ) controlled experimental infections with staphylococcus aureus, streptococcus pyogenes, escherichia coli and candida albicans resulted in fever, increased plasma cortisol concentrations, neutrophilia and lymphopaenia but no significant increase in total white blood cell count. high white cell counts can be found in rabbits with suffering from lymphosarcoma (mclaughlin and fish, ) . low white blood cell counts can be found in association with chronic disease (hinton et al., ) . clinical pathology figure . . comparison of some blood parameters of pet rabbits. as part of an investigation into the relationship of metabolic bone disease with dental disease, blood samples were taken from pet rabbits presented for veterinary treatment or for health checks. at the time of sampling the rabbits were assigned to one of four groups: add (advanced dental disease), edd (early dental disease), h (healthy rabbits kept in hutches), fr (rabbits kept in free-range conditions in a large enclosure with unlimited access to exercise and natural daylight all year round). no rabbits in the free-range group (fr) showed signs of dental disease; all the rabbits in the add, edd and h groups were kept in hutches. blood results from rabbits that were found to be suffering from other conditions, such as renal disease or neoplasia, were not included in the statistical analysis. the add group consisted of rabbits that were presented for veterinary treatment for dental disorders such as acquired malocclusion or abscesses. all the other rabbits in the edd, h and fr groups were presented for neutering, health checks, vaccination or euthanasia. rabbits in the edd group had signs of early dental disease, such as horizontal ribs on the incisors, swellings along the ventral border or the mandible or epiphora related to elongation of the roots of the upper primary incisors. the investigation was conducted to compare blood parameters related to calcium metabolism in rabbits with or without dental disease. the healthy free-range group was used as a control. during the course of the study, differences in haematological pictures and albumin values emerged among rabbits kept under the different husbandry regimens. complete blood counts from free-range rabbits were comparable with laboratory reference ranges whereas there were significantly lower red cell and lymphocyte counts in rabbits suffering from advanced dental disease. the low lymphocyte counts of rabbits with dental disease suggest they suffered from chronic stress. serum albumin values were significantly higher in rabbits kept in free-range conditions than in those suffering from advanced dental disease or those unaffected by dental disease but kept in hutches. total serum calcium concentrations were highly correlated with serum albumin levels. rabbits kept in hutches showed trends towards anaemia and lymphopaenia. plasma parathyroid hormone (pth) concentrations were higher and total serum calcium concentrations were lower in hutch-kept rabbits with advanced dental disease in comparison with rabbits kept in free-range conditions. these results indicated that acquired dental disease of pet rabbits is related to husbandry and is associated with alterations in calcium metabolism. reprinted from harcourt-brown and baker ( ) with kind permission from the journal of small animal practice. although the total white cell count of rabbits seldom alters in diseased rabbits, the differential white cell count may show a number of changes due to the redistribution of white blood cells. a feature of many diseases in rabbits is an alteration in the ratio of neutrophils to lymphocytes and a reduction in blood cellularity (hinton et al., ) . the neutrophil: lymphocyte ratio has been suggested as a method of predicting whether a rabbit is normal or abnormal (mclaughlin and fish, ) . jain ( ) described a physiological variation in the neutrophil: lymphocyte ratio according to the age of the rabbit. the ratio changes from : in the second month of life to : in rabbits over year of age. stress and increased cortisol levels can affect this ratio as well as disease. . . . effect of stress on the differential white cell count stress alters the differential white cell count in any species. rabbits are particularly susceptible to the effects of stress. a car journey to the surgery, a period in the waiting room next to a barking dog or the excitement of handling can be reflected in the blood picture. adrenaline and cortisol affect the distribution of lymphocytes throughout the body. administration of exogenous adrenaline to rabbits results in redistribution of lymphocytes from spleen and bone marrow to peripheral blood, lungs and liver (toft et al., a) . conversely, exogenous corticosteroid administration results in a redistribution of lymphocytes from the peripheral blood, bone marrow and spleen to the lymphatic tissue in rabbits (toft et al., b) . prolonged periods of stress cause neutrophilia and lymphopaenia. marked changes in white cell distribution with a relative neutrophilia and lymphopaenia were found in a study of the cortisol levels and haemograms of rabbits after transport, either by air or by lorry. the changes in white cell distribution lasted for - h and were correlated with increased cortisol levels (toth and january, ) . disease is stressful as well as having a direct effect on the production and distribution of white cells. rabbits with experimental infections exhibit a neutrophilia and lymphopaenia in comparison with control rabbits handled and sampled in exactly the same manner but inoculated with heat killed cultures (toth and krueger, ) . rabbits inoculated with a heat-killed culture do not experience the same rise in plasma cortisol concentrations as those inoculated with a live culture, indicating that the stress response is initiated by disease rather than by handling. therefore the stressful effects of a long car journey to the surgery or a morning spent in a kennel next to a barking dog is more likely to affect the neutrophil:lymphocyte ratio than the excitement response of taking blood. neutrophils function primarily as phagocytes and are important in infectious conditions and in inflammation. in other species, a neutrophilia occurs in response to inflammation, especially bacterial infection. an increase in the number of circulating neutrophils causes a rise in total white blood cell count. this response is not marked in rabbits. however, a change in the distribution of white cells can occur in response to infection with a relative neutrophilia and lymphopaenia but no alteration in total white cell count (toth and krueger, ) . a mature neutrophilia accompanied by an increase in plasma cortisol can also be associated with stress (toth and january, ). lymphocytes are involved in immunological responses and are distributed throughout the body in various tissues including blood, bone marrow, lymph nodes, spleen and gut-associated lymphoid tissue. the number of lymphocytes in the blood reflects a balance between cells leaving and entering the circulation and does not necessarily reflect a change in lymphopoiesis. increased cortisol levels cause a lymphopaenia and increased adrenaline levels cause lymphocytosis (toft et al., a, b) . in rabbits, lymphopaenia is a feature of a variety of clinical diseases (hinton et al, ) . marked lymphopaenia has been reported as a feature of differential white cell counts of pet rabbits especially those suffering from dental disease (see figure . ) (harcourt-brown and baker, ) . lymphoma is a relatively common tumour of rabbits and atypical lymphocytes can be found in the peripheral blood of these patients. the main function of eosinophils is detoxification either by inactivation of histamine, or histamine-like toxic materials. eosinophils are important in the allergic response and are capable of phagocytosis (kerr, ) . chronic eosinophilia can be seen in diseases of tissues that contain large numbers of mast cells such as the skin, lungs, gastrointestinal tract and uterus. eosinophilia can be associated with parasitism, especially when parasites are migrating through tissue. mild eosinophilia has been associated with experimentally induced chronic ascarid parasitism in rabbits (gupta and trivedi, ) . however, heavy worm burdens are rare in pet rabbits. encephalitozoonosis does not appear to cause an eosinophilia. slight to moderate elevations in eosinophil counts can be observed after traumatic wound repair in rabbits (fudge, ) . although eosinopaenia can be a significant finding in other species, low eosinophil counts or a zero count are not unusual in rabbits. basophils are similar to neutrophils but have dark blue cytoplasmic granules. although basophils are rare in blood films from species such as the dog, they may be seen commonly in rabbit blood (kerr, ) . basophil counts as high as % have been reported in clinically normal animals (benson and paul-murphy, ). in other species, monocytosis is associated with chronic disease, particularly chronic inflammatory conditions. in rabbits, increased monocyte counts can be associated with chronic bacterial infection. hinton et al. ( ) noted increased monocyte counts in rabbits with subcutaneous abscesses, mastitis and 'labyrinthitis'. however, monocyte counts within the laboratory reference do not signify the absence of chronic infection. rabbits with chronic osteomyelitis due to dental disease can have monocyte counts within the laboratory reference range (harcourt-brown, unpublished data). a reference range for biochemical parameters is given in table . . clinical pathology key points . • rabbit erythrocytes vary in diameter and anisocytosis can be a normal finding in rabbit blood films • polychromasia and small numbers of reticulocytes and nucleated red cells may be seen on normal blood films • rabbit neutrophils have a granular cytoplasm and may be mistaken for eosinophils • there are several different terms for the rabbit neutrophil. some authors use terms such as heterophil, pseudoeosinophil, acidophil or amphophil instead of neutrophil • basophils are frequently found on blood films from rabbits • low blood cellularity, i.e. anaemia and lymphopaenia, is a non-specific feature of disease in rabbits • high numbers of nucleated red blood cells may be associated with infectious disease • the neutrophil:lymphocyte ratio should be approximately : in adult rabbits. alterations in the ratio can be associated with stress or disease • adrenaline causes a shift of lymphocytes from the spleen and bone marrow to blood. cortisol causes a shift of lymphocytes away from the bloodstream to the spleen and lymphatic tissue • an increase in total white cell count is unusual in rabbits even in the presence of infection • a neutrophilia with a left shift occurs in response to infection • monocytosis can be seen in association with chronic infection. herbivores, such as rabbits, differ from carnivores in their carbohydrate metabolism. carnivores eat periodically and have sudden large intakes of nutrients that must be stored for utilization during the fast between meals. herbivores graze for long periods of the day and are continually absorbing nutrients from the digestive tract. in rabbits, volatile fatty acids are produced from bacterial fermentation in the caecum and are continually absorbed as an energy source. a fasting sample is difficult to obtain from a rabbit. withholding food does not prevent caecotrophy and the digestion of caecotrophs provides a source of glucose. blood samples taken after h of food deprivation may show no alteration in blood glucose levels (kozma et al., ) . hyperglycaemia is a relatively common finding in rabbits and can be accompanied by glycosuria. handling alone can cause an increase in blood glucose to the order of . mmol/l experimentally (knudtzon, ) and mmol/l or more anecdotally. diabetes mellitus has not been described in pet rabbits and there is some difference of opinion about its importance as a clinical disease (hoefer, ; jenkins, ; rosenthal, ) . herbivorous animals withstand the absence of insulin more readily than carnivorous ones (bentley, ) and are therefore not so susceptible to diabetes mellitus. diabetes mellitus has been induced in laboratory rabbits by the administration of alloxan. it has also been described as total protein - g/l e urea . - . mmol/l b vitamin a (plasma) - µg/ml ± < µg/ml indicates deficiency (liver levels of < µg/g liver denote deficiency) b vitamin e (plasma -tocopherol) > µg/ml (< . µg/ml indicates deficiency) a hereditary disease in rabbits. a laboratory strain was selectively bred as an animal model of human diabetes mellitus. (roth and conaway, ) . affected animals were polydipsic, polyuric and polyphagic with severely impaired insulin release. elevated glycosylated haemoglobin values of . % were observed in the overtly diabetic animals in comparison with . % in normal animals. increased glycosylated haemoglobin levels did not correlate with plasma glucose concentrations (cannon and conaway, ) . histologically there was hypergranulation of ␤-cells of the islets of langerhans. obesity and ketoacidosis were not features of diabetes mellitus in the laboratory rabbits. the hyperglycaemia was in the region of - mg/dl ( - . mmol/l) and there was marked glycosuria. roth and conaway ( ) described the maintenance of one diabetic individual on insulin at a dose of up to units per day for years. ketonuria was not observed. in pet rabbits, a diagnosis of diabetes mellitus cannot be made on a single blood sample and requires serial blood and urine sampling to confirm the diagnosis. in view of the physiological factors that can increase blood glucose levels it is advisable to take repeat blood samples from hyperglycaemic rabbits with time of day, phase of digestion, anaesthesia, influence of handling or car journeys in mind. mild glycosuria is not a significant finding. hyperglycaemia can be seen in the terminal stages of gut stasis and is a poor prognostic sign (harcourt-brown, personal observation). it is associated with fatty degeneration of the liver at post-mortem examination. marked hyperglycaemia is also seen in association with painful conditions such as acute intestinal obstruction. blood glucose levels can rise to - mmol/l and return to normal once the condition is resolved (harcourt-brown, unpublished observation). experimental haemorrhagic or traumatic shock results in hyperglycaemia proportional to the severity of the condition. hyperthermia also results in hyperglycaemia (mclaughlin and fish, ) . diseases that elevate serum glucose levels in other species, such as hyperadrenocorticism or acute pancreatititis have not been reported in pet rabbits, although they could occur. hypoglycaemia is a significant finding in rabbits and is associated with anorexia, starvation or disturbances in the digestion and absorption of carbohydrates. it can be a sign of hepatic dysfunction. a drop in blood glucose leads to mobilization of free fatty acids from adipose tissue and contributes to the development of ketoacidosis and fatty degeneration of the liver. measurement of serum glucose is of value in moribund rabbits as a basis for the selection of appropriate fluid therapy. other causes of hypoglycaemia such as addison's disease or insulinomas have not been reported in pet rabbits although such conditions could occur. interpretation of total protein concentrations is similar to other mammals. artefactual increases in protein concentrations can result from excessive venous stasis during blood collection. fluid and small molecules leave the plasma, resulting in a relative increase in proteins. this situation can occur in rabbits, especially in miniature breeds with small veins. an increase in total protein indicates dehydration, chronic and immune-mediated disease. in rabbits dehydration due to water deprivation or gastrointestinal disturbances commonly occurs. examination of the haematocrit and albumin and globulin fractions can assist differential diagnosis. liver disease, chronic enteropathy, starvation or malnutrition may result in reduced protein levels. glomerulonephropathy or protein-losing enteropathy are uncommon conditions that could cause low total protein levels in rabbits. a decrease in both albumin and globulin may be associated with haemorrhage or exudative skin lesions such as fly strike. the liver is the sole site of albumin synthesis and hypoalbuminaemia is feature of advanced liver disease in all species. in rabbits, heavy parasitism is a cause of liver disease. eimeria steidae causes hepatic coccidiosis (see section . . . ). cysticercus pisiformis, the larval stage of taenia pisiformis, migrates through the liver and results in the development of fibrous tracks and necrotic foci (see section . . ). severe infestations can result in low albumin levels. non-hepatic causes of low serum albumin include glomerulonephropathy, protein-losing enteropathy, malabsorption and cardiogenic ascites. laboratory reference ranges for serum albumin levels in rabbits can be wide and vary between sources. sex differences have been reported in laboratory rabbits. one study showed that female new zealand white rabbits had higher serum albumin levels than males, although other studies have found no sex differences (kozma et al., ) . in rabbits, hypoalbuminaemia is most likely to be associated with nutritional factors such as abnormal caecotrophy, incorrect diet, starvation or malnutrition associated with dental disease. primary or secondary hepatic neoplasia occasionally occurs in pet rabbits. hepatic coccidiosis is a cause of low serum albumin levels, especially in young rabbits that have been kept in colonies. a high serum albumin concentration is not a feature of any specific disease, although an increased albumin level in conjunction with a raised pcv is indicative of dehydration. in a study by harcourt-brown and baker ( ), pet rabbits kept in free-range conditions had significantly higher serum albumin concentrations than rabbits that were suffering from advanced dental disease. they were also significantly higher than rabbits kept in hutches that were not suffering from dental problems (see figure . ). the difference in albumin levels was attributed to differences in diet and husbandry. caecotrophs are a source of amino acids for rabbits and normal caecotrophy is an important element of their protein metabolism. low fibre diets, obesity, dental disease or skeletal abnormalities can prevent rabbits ingesting caecotrophs from the anus and reduce the available amino acids for protein synthesis. rabbits that are kept in hutches are more likely to be eating a low fibre diet and to suffer from obesity and skeletal problems than rabbits living outside with unrestricted access to natural vegetation and exercise. plasma globulins are made up of a range of proteins including carrier proteins and immunoglobulins or antibodies. the types of globulin can be separated into five fractions by electrophoresis. the ␥-globulin fraction is almost entirely composed of immunoglobulins. some globulins can be synthesized in the liver but immunoglobulins are synthesized exclusively in lymphoid tissue. acute inflammation, chronic disease or immune-mediated disease can cause an increase in globulin levels. myeloproliferative disease results in abnormal levels of immunoglobulin production. there are few published data on the significance of globulin concentrations in rabbits. lipaemia can artefactually elevate protein levels with some analytical methods. experimental infections with rabbit coronavirus result in hypergammaglobulinaemia. analogies have been made between coronavirus infection in rabbits and feline infectious peritonitis in cats (digiacomo and mare, ) . coronavirus occurs in laboratory rabbits but is an unlikely diagnosis in the pet rabbit. cholesterol is synthesized in the liver or absorbed from the diet. it is a metabolic precursor of steroid hormones. cholesterol is broken down in the liver and excreted in bile. in other species, elevated cholesterol levels are indicative of a variety of metabolic disorders such as hypothyroidism, hepatopathy, diabetes mellitus, and hyperadrenocorticism. low levels can occur in association with impaired hepatic function. changes in serum triglyceride levels reflect a similar range of diseases. blood levels of triglycerides increase after a meal, especially if it is a fatty meal. in rabbits, there are some physiological factors that affect cholesterol levels. male rabbits have lower cholesterol levels than females and there is a diurnal variation with higher levels occurring during the late afternoon (loeb and quimby, ) . large variations in blood cholesterol and triglyceride values can occur between individual rabbits (yu et al., ) . a fasting blood sample is required for cholesterol and triglyceride assay. in rabbits, it is difficult to obtain a fasting sample because of caecotrophy. abnormal cholesterol or triglyceride levels are most likely to be associated with dietary factors or hepatic impairment. in anorexic rabbits, especially obese ones, a lipaemic sample is a poor prognostic indicator as it signifies impaired fat metabolism and the presence of hepatic lipidosis (see section . ). a rise in triglyceride levels has been found in association with experimentally induced chronic renal failure in rabbits (tvedegaard, ) . in other species, amylase is found in the pancreas and to a lesser extent in the salivary glands, liver and small intestinal mucosa. amylase has a short half-life and is rapidly removed from the circulation. it is excreted by the kidney. elevated levels indicate pancreatic disease or renal insufficiency. in rabbits, amylase is present in pancreatic tissue in high concentrations. low concentrations are found in the salivary glands and none is produced by the liver (jenkins, ) . amylase is also produced by caecal microorganisms and is present in caecotrophs aiding conversion of glucose to lactic acid during digestion in the stomach and small intestine. serum amylase levels are lower in rabbits than other species (mclaughlin and fish, ) . pancreatic duct obstruction or pancreatic disease can result in a rise in blood amylase values. rabbits can survive experimental ligation of the pancreatic duct (brewer and cruise, ) . the rabbit secretes a large amount of bile, approximately seven times as much as a dog on a weight basis (brewer and cruise, ) . the rabbit also differs from other species in the excretion of breakdown products of haemoglobin. the rabbit has low biliverdin reductase activity (fekete, ) and only % of biliverdin is converted to bilirubin. bilirubin values can be affected by fasting. glucose administration to rabbits lowers serum bilirubin concentrations by modifying hepatic conjugation and increasing biliary secretion (mclaughlin and fish, ) . in rabbits, biliary obstruction results in jaundice and raised serum bilirubin values. in young rabbits, hepatic coccidiosis is the most usual cause of jaundice. in older rabbits, bile duct obstruction from neoplasia is more likely. aflatoxicosis from the ingestion of mouldy feed can result in hepatic fibrosis and jaundice (krishna et al., ) . viral haemorrhagic disease (vhd) causes acute hepatic necrosis with elevated bilirubin concentrations in association with dramatic increases in ast and alt concentrations. vhd is invariably fatal, although some rabbits may survive long enough to develop jaundice before they die. there is little on information on haemolytic disease as a cause of jaundice in pet rabbits. haemolytic anaemia has been reported in association with lymphosarcoma in laboratory rabbits (weisbroth, ). in other species, alt is used as an indicator of hepatocellular damage, especially in dogs and cats. alt is also in found in other tissues such as muscle and red blood cells. an increase in alt signifies cell damage, although the degree of the increase does not correlate with the severity of hepatic disease and is not a prognostic indicator (willard et al., ) . in rabbits, liver alt activity is lower than in other species and there is less organ specificity (rosenthal, ) . alt is also present in cardiac muscle. the half-life of alt in the rabbit is approximately h. in the dog the half-life is - hours (jenkins, ) . hepatic coccidiosis due to eimeria steidae is a cause of increased blood alt concentrations especially in conjunction with an increase in alkaline phosphatase, bilirubin and gamma gt. elevated alt values have been found in asymptomatic house rabbits and have been attributed to the effects of organic solvents in wood shavings used as litter material. other liver diseases such as neoplasia can cause a rise in alt but sometimes not until the condition is advanced (mclaughlin and fish, ) . low doses of aflatoxin caused a significant rise in alt concentrations in a group of laboratory rabbits (fekete and huszenicza, ) . hepatic lipidosis will elevate alt levels. in other species ast is widely distributed throughout the body. in particular, it is found in skeletal muscle, cardiac muscle, liver and erythrocytes. like alt, ast is an indicator of tissue damage. it is sometimes used as an indicator of liver disease, especially in horses in which alt is not liver specific. in rabbits, ast is found in liver, heart, skeletal muscle, kidney and pancreas with the highest activity in the liver and skeletal muscle (benson and paul-murphy, ) . physical exertion or tissue damage during blood collection can elevate results. raised ast levels can be found in association with liver disease. ggt is found in liver and kidney tissue. in other species, ggt is used as an indicator of hepatobiliary disease especially in horses and ruminants where it is associated with longterm liver damage. although ggt is found in high concentrations in renal tubular cells, kidney disease does not lead to elevated blood levels, probably because the enzyme is excreted in the urine (bush, ) . in the rabbit ggt is located predominantly in the renal epithelium with low activity in the liver. liver ggt is present primarily in bile duct epithelial cells and is therefore an indicator of hepatobiliary disease rather than hepatocellular damage (mclaughlin and fish, ) . in cases where there is renal tissue damage, urine ggt may be increased in addition to plasma concentrations. alkaline phosphatase consists of a group of several isoenzymes that hydrolyse phosphates at an alkaline ph (kerr, ) . it is one of the most widely distributed enzymes in the body. alkaline phosphatase is found particularly in bone, liver and intestinal wall. different isoenzymes are produced from each site. increases in plasma activity are usually due to the isoenzymes derived from liver and bone. higher concentrations are found in young animals with high osteoblastic activity. in rabbits, alkaline phosphatase is present in nearly all tissues. it is found in association with cell membranes and especially in intestinal epithelium, renal tubules, osteoblasts, liver and placenta. the rabbit has three ap isoenzymes -the intestinal form as well as two isoenzymes present in both liver and kidney (mclaughlin and fish, ) . there is a wide variation between laboratory reference ranges for ap values for rabbits. examples include: . - . iu/l (collins, ) ; - iu/l (gillett, ) ; - iu/l (harkness and wagner, ) . different analytical techniques could account for these variations. in a survey of blood parameters relating to calcium metabolism in pet rabbits, serum alkaline phosphatase values varied widely even in apparently healthy individuals (harcourt-brown and baker, ) . increased levels of alkaline phosphatase are seen in biliary obstruction, e.g. neoplasia or hepatic coccidiosis. experimental ligation of the common bile duct results in increased levels of alkaline phosphatase up to iu/l (mclaughlin and fish, ) . enteric disease can also elevate alkaline phosphatase values (jenkins, ). bile acids are derived from cholesterol and are secreted into the intestine to aid fat digestion. from the gut, they are reabsorbed into the circulation and transported to the liver to be resecreted in the bile. impaired hepatic function results in increased concentrations of bile acids in peripheral blood. there are physiological variations in circulatory bile acid concentrations in association with the digestion of food and stimulation of the gall bladder to release bile into the small intestine. in most species, a fasting sample should have a low concentration of bile acids of less than µmol/l (kerr, ) . impaired hepatic function can result in marked rises in fasting serum bile acid concentrations. in rabbits, the production of bile acids shows a circadian rhythm (fekete, ) . there is a problem in obtaining a fasting sample from rabbits due to the ingestion of caecotrophs. bile acids are not included in published reference ranges for rabbits at the present time. bile acid levels in excess of µmol/l have been found in association with hepatic coccidiosis in comparison with levels that are generally less than µmol/l (harcourt-brown, unpublished data). urea is a nitrogenous waste product that is formed in the liver as the end product of deamination of amino acids. it is transported in the blood to the kidney where it is excreted in the urine. in other species, high blood urea concentrations are indicative of impaired renal function that may be due to renal disease or poor perfusion due to circulatory disorders or cardiac disease. low blood urea levels can reflect hepatic dysfunction. in rabbits, many physiological factors influence the concentration of urea in the blood. dietary protein concentrations and quality, withholding food and natural diurnal rhythms can all affect blood urea concentrations. higher levels occur in the late evening (loeb and quimby, ) . the rabbit's urea metabolism is further complicated by urea utilization by caecal microflora during catabolism or during periods of dietary excess. therefore small fluctuations in serum urea concentrations are difficult to interpret. laboratory reference ranges apply to animals that are fed a standard diet and have usually been bled at a specific time of day. pet rabbits are subject to greater fluctuations in blood urea values due to the variation in diet and other factors, and can have values slightly higher than laboratory reference ranges. prerenal azotaemia associated with poor renal perfusion occurs during periods of dehydration. the rabbit has a limited capacity to concentrate urea and a greater volume of urine is required when urea load increases (brewer and cruise, ) . increased blood urea values were recorded in a study by licois et al. ( ) of young rabbits with diarrhoea experimentally induced with coccidiosis. the authors suggested that the blood urea values rose as a result of intense nitrogen catabolism during weight loss associated with the disease. water deprivation can lead to high blood urea values as high as mmol/l in association with creatinine values in excess of µmol/l (harcourt-brown, unpublished data). water deprivation can be due to a lack of available drinking water, caused either by an oversight by the owner or by a faulty mechanism on the drinking bottle. in rabbits, dehydration can cause urea and creatinine values that would signify renal disease in the dog and cat. high levels usually return to normal once the animal is rehydrated. therefore, urea and creatinine values should be checked before making an absolute diagnosis of renal failure. as in other species, elevated blood urea values in rabbits are associated with renal insufficiency. nephrolithiasis is a cause of kidney disease in the rabbit (see section . ). abdominal radiography is indicated in rabbits with raised urea and creatinine levels. encephalitozoon cuniculi can cause low-grade kidney disease in rabbits with mild elevations in blood urea. most cases are subclinical. e. cuniculi infection causes granulomatous lesions in the kidneys that become pitted and scarred with fibrotic areas. the parasite has been associated with chronic renal failure with blood urea values in the region of . mg/dl ( . mmol/l) and creatinine of . mg/dl ( . µmol/l) in a study by ewringmann and göbel ( ) . affected rabbits were anaemic with low haemoglobin and red cell counts and had elevated serum potassium concentrations. neoplasia, interstitial nephritis, nephrotoxicity also occur in rabbits and cause renal disease. low blood urea values in association with impaired hepatic function and the use of anabolic steroids have been described (benson and paul-murphy, ). creatinine is a nitrogenous waste product that, like urea, is transported in the blood to the kidney where it is excreted in the urine. creatinine is not the product of amino acid breakdown but of creatine which is a substance present in the muscle and is involved in high energy metabolism (kerr, ) . the slow catabolism of creatine results in a slow inflow of creatinine to the plasma at a rate which is directly proportional to the individual's muscle mass but is unaffected by any change in muscular activity or muscle damage. any changes in blood creatinine concentrations are due to changes in excretion and are a reflection of renal function. concentrations rise quickly at the outset of renal disease and decrease when an improvement of renal function takes place. creatinine deteriorates in plasma and readings from old samples (> h) cannot be relied upon. there is interference from a variety of other substances such as bilirubin (which decreases creatinine) or cephalosporins (which increase creatinine). the rabbit's complex digestive physiology and the compromised renal capability of correcting acid-base disorders make the rabbit a prime candidate for electrolyte imbalances (see section . ). dietary deficiency of electrolytes such as sodium and potassium is unlikely in the herbivorous diet of rabbits. instead, electrolyte problems are more likely to be associated with abnormal losses. although rabbits do not vomit, water and electrolyte absorption and secretion are affected by gastrointestinal disease. if facilities are available, electrolyte assays, especially potassium, can be a valuable part of the diagnostic workup for critically ill rabbits. in general, changes in sodium concentrations reflect the osmolality of extracellular fluid rather than the total body sodium content. increased blood sodium concentrations (hypernatraemia) can be the result of water deprivation or the loss of low sodium fluids. decreased sodium concentrations (hyponatraemia) may occur as a result of chronic renal failure when the kidney cannot concentrate urine and fast urine flow through the renal tubules prevents effective sodium/ potassium exchange. lipaemia or hyperproteinaemia can artefactually reduce affect sodium concentrations if certain laboratory methods are used. at the present time, there are few data available on clinical conditions that affect sodium concentrations in rabbits. about % of the total body potassium is intracellular, so measurement of extracellular potassium in blood samples does not give a true reflection of the potassium status of the patient. the balance between intracellular and extracellular potassium is regulated by aldosterone, insulin and catecholamines and is affected by blood ph. aldosterone stimulates renal excretion of potassium. insulin promotes the movement of potassium into cells. the effects of these hormones prevent large diet-induced changes in plasma potassium concentrations. potassium is an important ion in the maintenance of membrane potential. abnormally high or low potassium concentrations can have life-threatening consequences due to impaired electrical activity of cells. high blood potassium concentrations can result in cardiac arrest. alterations in blood potassium levels can be due to alterations in dietary intake and textbook of rabbit medicine key points . • stress of handling can cause a marked elevation of blood glucose levels in rabbits. levels as high as mmol/l can occur in association with handling. higher levels (> mmol/l) may be seen in association with stressful or painful diseases such as intestinal obstruction • there is debate about the occurrence of diabetes mellitus in domestic rabbits. it has been induced in laboratory rabbits and a genetically susceptible laboratory strain has been bred • large individual variations in blood cholesterol and triglyceride values can occur in rabbits • jaundice is unusual in rabbits but may be seen in association with cholestasis in diseases such as hepatic coccidiosis or neoplasia • there is a wide variation between laboratory reference ranges for alkaline phosphatase values • blood urea and creatinine values can be high in cases of prerenal azotaemia in rabbits and do not always signify renal failure. causes include dehydration or water deprivation. excretion, or redistribution across cell membranes. to maintain electroneutrality, potassium ions shift from intracellular to extracellular fluid in exchange for hydrogen ions. in other species, hypoadrenocorticism (addison's disease) reduces the exchange of sodium and potassium ions across the cell membrane and results in increased serum potassium and decreased serum sodium concentrations. hyperkalaemia can be the result of impaired renal excretion of potassium due to kidney disease or from tissue trauma such as crushing injuries that release large amounts of potassium into the circulation. acidosis causes a redistribution of potassium across the cell membrane. artefactually high levels of potassium can result from leakage from red cells in haemolysed samples or those that have not been separated until several hours after the blood was taken. low blood potassium can cause muscular weakness and depression. hypokalaemia can be the result of dietary potassium deficiency or as a result of potassium loss from the gastrointestinal tract. diuresis or the use of potassium-free intravenous fluids also cause hypokalaemia. alkalosis can cause redistribution of potassium and sodium across the cell membrane and result in hypokalaemia. artefactually low potassium concentrations are uncommon although they can occur secondarily to hyperlipidaemia or hyperproteinaemia (willard et al., ) . blood collection through a catheter that contains residual potassium-free fluids can lead to erroneously low results. in rabbits, the effect of blood collection methods on plasma potassium levels has been investigated. discrepancies in results were found between blood collected from the ear and from the carotid artery when the blood was collected with a plastic catheter but not with a g needle . general anaesthesia with pentobarbitone depressed plasma potassium values but sedation with chlorpromazine did not affect results. serum potassium concentrations were found to be higher than plasma and in venous rather than arterial blood . low serum potassium values have been found in unanaesthetized rabbits in conjunction with signs of muscular weakness (harcourt-brown, unpublished data). affected animals can still eat and drink but are unable to move. it is not known whether hypokalaemia is the cause of the muscular weakness. possible causes of hypokalaemia are discussed in section . . . . further investigations of serum potassium concentrations of rabbits are required to know the clinical significance of measured values and the influence of various physiological states. in horses, blood potassium concentrations can fall to . mmol/l after prolonged exercise due to potassium loss in sweat and to . mmol/l while eating hay due to potassium loss in saliva. during moderate exercise concentrations can rise to . mmol/l due to potassium release from muscle cells (kerr, ) . similar physiological variations could occur in the rabbit. calcium is an essential element that is involved in many body systems. most of the body's calcium is stored in bone in conjunction with phosphate. calcium is an essential part of the structure of bones and teeth. it is an important cation in intracellular and extracellular fluid where it is required for muscle metabolism, enzyme activation, blood coagulation and osmoregulation. calcium is found in the blood in three forms: ionized, bound to other anions (especially phosphate) and bound to protein (especially albumin). because of the protein binding capacity of calcium, total serum calcium concentrations are proportional to albumin concentrations. ionized calcium is the physiologically active component of blood and is involved in the permeability of cell membranes. hypocalcaemia is a life-threatening condition. in many species, a high demand for calcium during late pregnancy and lactation can result in hypocalcaemic tetany. there are also some metabolic disorders that can result in alterations in serum calcium concentrations in other species. examples include renal, pancreatic and neoplastic diseases. the rabbit has a different calcium metabolism from other domestic species (see section . . ). dietary calcium is readily absorbed from the intestine and total plasma values reflect dietary intake. total blood calcium levels are higher and can vary over a wider range than other species. an erroneous diagnosis of hypercalcaemia is often made because of the rabbit's high total serum calcium levels in comparison with other animals. parathyroid hormone (pth) regulates calcium metabolism in a similar manner to other animals, but a reduction in plasma pth level occurs at a higher plasma calcium concentration than in other species . the kidney plays an important role in calcium regulation and has a high fractional excretion for calcium when blood levels are high. calcium is excreted in the urine in which it forms calcium carbonate precipitate. some authors have suggested monitoring blood calcium concentrations as part of the protocol for treating 'sludgy urine'. however, high blood calcium levels are not the sole cause of urinary tract disease in rabbits (see section . . ). there are differences between published reference ranges for total serum calcium values in rabbits. variations in dietary calcium intake could account for some of the discrepancies. the peak blood level that was obtained by increasing dietary calcium intake in laboratory rabbits was . mmol/l ( . mg/dl) in a study by chapin and smith ( a) . experimental calcium restriction resulted in minimum serum concentrations of . - . mmol/l ( - mg/dl) before a rapid decline just before death in a separate study by chapin and smith ( b) . a reference range of . - . mmol/l taken from eight laboratory reference sources has been made by goad et al. ( ) . values outside this range are encountered in otherwise healthy individuals and a range of . - . mmol/l is acceptable for pet rabbits on a varied diet. total blood calcium levels in rabbits are also affected by age and reproductive status. kamphues et al. ( ) found that increased calcium intake only resulted in higher total plasma calcium concentrations in adult rabbits and not young ones of - weeks. serum calcium concentrations in growing rabbits are fixed at a value of approximately . mmol/l ( mg/dl) (kamphues et al., , gilsanz et al., . blood calcium levels decrease during pregnancy (assane et al., ) . due to the protein binding properties of calcium, albumin levels can also affect total calcium concentrations. albumin concentra-tions in pet rabbits are variable and appear to be affected by the manner in which they are kept (harcourt-brown and baker, ) . in dogs and man, total serum calcium values can be adjusted by using a mathematical formula that takes albumin concentration into account. (adjusted calcium concentration = measured serum total calcium concentration -serum albumin (g/dl) + . ). this formula is unreliable in cats (flanders et al., ) and has not been investigated in rabbits. at present, most published reference ranges for pet rabbits refer to total serum calcium concentrations. ideally, ionized calcium should be measured for an accurate assessment of calcium status but special sample handling and equipment is required that precludes its measurement in most practice situations. however, affordable equipment is now becoming available that can be used in the practice laboratory. measurements of ionized calcium have been made during experimental investigations using rabbits. warren et al. ( ) found a linear relationship between total serum calcium and ionized calcium values. a group of non-pregnant female and male rabbits were found to have ionized serum calcium levels of . + . mmol/l. kamphues et al. ( ) reported ionized calcium values of . + . mg/dl ( . + . mmol/l) in adult rabbits on an average dietary calcium intake ( . %). hypocalcaemic tetany has been reported in lactating does (barlet, ) . hypercalcaemia is seen in rabbits with chronic renal failure and impaired calcium excretion (see section . . . ). in a study by tvedegaard ( ) , experimentally induced chronic renal failure resulted in total serum calcium concentrations of . + . mmol/l. levels in excess of . mmol/l have been recorded in association with neoplasia (voelkel et al., ) . inorganic phosphate is involved in many enzyme systems and is important in carbohydrate and muscle metabolism as well as forming a major component of bone. phosphate is obtained from the diet. vitamin d and pth influence intestinal absorption of phosphorus in a similar manner to calcium. pth stimulates renal excretion of phosphate and renal conservation of calcium. the ph of intestinal contents and the presence of cations such as calcium and magnesium can affect the availability of dietary phosphate. abnormalities of phosphate metabolism are complex and interdependent with many other factors. blood phosphate values can be difficult to interpret and need to be examined alongside other parameters. in other species, physiological activities such as feeding and exercise reduce serum phosphorus concentrations (aitken and allen, ) . there are drug interactions that alter serum phosphorus values. examples include phosphate binders, anaesthetic agents, bicarbonate, parenteral glucose, anabolic steroids, diuretics and tetracyclines (willard et al., ) . phosphorus can shift between the intracellular and extracellular space in response to alterations in acid-base metabolism. in addition to these problems of interpretation, phosphate values are subject to artefactual error caused by poor sample handling. haemolysis releases phosphate from erythrocytes, which results in elevated values. in rabbits, there is little information about the clinical relevance of serum phosphate concentrations. rabbit blood clots easily and good quality non-haemolysed samples can be difficult to obtain. hyperphosphataemia can be due to impaired renal phosphorus excretion due to kidney disease. hypophosphataemia may result from dietary deficiency, impaired intestinal absorption or metabolic disorders. blood lead concentrations are given in different units depending on the source. the conversion factor for µg/dl to µmol/l is . . a study by roscoe et al. ( ) evaluated three diagnostic tests for lead toxicity in rabbits. whole blood lead concentrations of greater than . mg/dl ( . µmol/l) were considered a reliable indicator of lead ingestion. measurement of urinary delta-aminolevulinic acid (␦ala) was considered unreliable. erythrocytes from rabbits that were given lead fluoresced red when exposed to light rays of - nm. the fluorescent erythrocyte test (fet) was considered a convenient and reliable test for lead ingestion. swartout and gerken ( ) described two clinical cases of lead poisoning that had blood levels of µg/dl ( . mg/dl) and µg/dl ( . mg/dl). they gave a range of - µg/dl ( . - . mg/dl) as a normal range for laboratory rabbits. pth is released by the parathyroid gland in response to both a fall in blood calcium and low serum , -(oh) d levels. it is responsible for the minute to minute regulation of calcium, due to its quick, short duration response. pth stimulates conversion of ( -oh-d) to the active from of vitamin d ( , -(oh) d ) that, in turn, stimulates intestinal absorption of calcium. pth also stimulates osteoclastic resorption of bone to release calcium, phosphorous and magnesium into the circulation. pth stimulates renal conservation of calcium but not phosphorous, which results in an increase in blood calcium without an increase in phosphorous concentrations. in animals with disturbances in calcium metabolism that result in bone demineralization, pth levels are high, and pth can be used to investigate metabolic bone disease that is often nutritional in origin. dietary calcium deficiency, or a failure to absorb calcium are reasons for metabolic bone disease. failure to absorb calcium can be due to vitamin d deficiency or unavailability of calcium due to binding with substances such as oxalates, fats or phosphates in the gut. pth assays are available in commercial laboratories that specialize in endocrinological investigations. pth assays have been performed on pet rabbits as part of an investigation of the possibility of metabolic bone disease as a cause of poor tooth and bone quality and the development of dental disease in rabbits (harcourt-brown and baker, ) . sample handling is of paramount importance as the hormone is labile and haemolysis interferes with the assay. samples require separating and freezing immediately after collection and must be shipped in the frozen state to a laboratory. sufficient, non-haemolysed blood to harvest - ml of serum or plasma needs to be collected. as pth is responsible for the minute to minute regulation of blood calcium, values can vary over a wide range making interpretation of a single result difficult. diet, age, pregnancy, lactation, diurnal rhythms cause physiological variations in results. warren et al. ( ) reported pth values of . + . pg/ml in a group of nonpregnant farm and laboratory rabbits and harcourt-brown and baker ( ) reported values of . + . pg/ml in a group of pet rabbits kept outside under free-range conditions all year round (see figure . ). values as high as - pg/ml have been recorded in baseline samples from laboratory rabbits . values in excess of pg/ml have been found in pet rabbits, one of which was found to have a liver tumour on subsequent post-mortem examination (harcourt-brown, unpublished data). in the uk, serological tests are available for encephalitozoon cuniculi, toxoplasma gondii, myxomatosis, viral haemorrhagic disease and treponema cuniculi as part of the commercial health screening of laboratory rabbits. commercial laboratories may accept individual samples from pet rabbits for serological screening. it is advisable to consult a veterinary laboratory in the first instance. in the usa, serology and a pcr test are also available to detect pasteurella multocida infection (sanchez et al., ) but these tests are not available in the uk at the present time (september, ) . serological testing for encephalitozoon cuniculi antibodies can be useful in the differential diagnosis of neurological diseases such as vestibular syndrome or paraplegia (section . ) or uveitis (section . . . ). it is also indicated in animals with mild renal insufficiency (section . . ). serological and histological tests from naturally infected rabbits have demonstrated the presence of antibodies before the organism can be seen in the kidney. lesions were not seen in the brain until at least weeks after the first detectable antibodies, suggesting that serology is a sensitive procedure for early diagnosis (cox and gallichio, ) . therefore animals with clinical signs that are seronegative are unlikely to be suffering from encephalitozoonosis, although experimental infections with encephalitozoon cuniculi have shown the presence of granulomas in the brain of animals that had become seronegative (kunstyr et al., ) . conversely, asymptomatic rabbits can be seropositive. therefore serology can only be used as a guide in the diagnosis of encephalitozoon cuniculi infection. antibody titres can be helpful in distinguishing between recent and chronic infection. simultaneous detection of igm and igg suggest recent infection. urinanalysis is summarized in table . . urine collection is described in section . . . in common with other herbivorous species, rabbits excrete alkaline urine. urinary ph is approximately - . . rabbit urine is normally turbid due to the presence of calcium carbonate that can be seen as sediment in collected samples. the urine from anorexic, pregnant, lactating or young rabbits is often clear. the colour of normal urine can vary from pale yellow, to orange, brown or red, mimicking haematuria. plant porphyrin pigments are the cause of red coloured urine and can be distinguished from haematuria with urinanalysis dipstick tests. alternatively, a wood's lamp can be used, as urinary pigments fluoresce when exposed to ultraviolet light (benson and paul-murphy, ) . in addition to the presence of calcium carbonate crystals, oxalate or ammonium magnesium phosphate crystals can also be found in normal rabbit urine. the specific gravity of rabbit urine is difficult to evaluate accurately due to the presence of mineral deposits but is approximately . - . . traces of glucose and protein can be present in normal rabbit urine. as in other species, rabbit urine can be spun and the sediment examined microscopically for the presence of crystals, red cells, inflammatory cells and bacteria. cultures can be taken to confirm bacterial infection and aid antibiotic selection. examination of urine sediment stained with gram stain can reveal encephalitozoon cuniculi spores that are oval, strongly gram-positive with a coiled filament inside (pye and cox, ; patton, ) . ketones may be detected in the urine of anorexic rabbits and is a poor prognostic sign as it is associated with the development of hepatic lipidosis. rabbits produce two types of faeces: hard dry pellets that are composed of compressed indigestible fibre and soft caecotrophs that are composed of a smooth paste rich in bacteria and other microorganisms. the first step in faeces examination is to determine which type of faeces has been collected. it is often samples of soft faeces or caecotrophs that are collected for examination because their consistency is abnormally loose or the owner has mistaken uningested caecotrophs for diarrhoea. microscopically the two types of faeces are completely different. hard faeces contain indigestible fragments of plant debris and little else. caecotrophic material contains a wide range of microorganisms including large gram-negative bacilli, bacteroides, large metachromic staining bacilli and many other bacteria including oval and fusiform rods. in some types of diarrhoea, a mixture of indigestible fragments of plant material can be seen alongside a range of typical caecal microorganisms. this signifies a failure of the proximal colon to separate the indigestible and digestible fractions of the diet. coccidial oocysts or eggs of the non-pathogenic oxyurid passalurus ambiguus can be found in both hard and soft faeces of infected rabbits. coccidial oocysts can be confused with a nonpathogenic saccharomyces, a budding sporogenous yeast that can be present in large numbers in rabbit faeces (see figure . ). clostridium spiroforme is a large grampositive, semicircular or spiral-shaped bacterium that may be seen in faecal smears from diarrhoeic rabbits or those that have died from enterotoxaemia. centrifuging faecal material at rpm for minutes and gram staining the residue after the supernatant has been removed improves the chance of diagnosis (langan and o'rourke schaeffer, ) . although the presence of semicircular bacteria in the faeces or caecal material is suggestive of clostridial enterotoxaemia, it is not a reliable diagnostic criterion. clostridia species can be present in the normal caecal flora and proliferate after death. the demonstration of the toxin and anaerobic culture of the organism is required for positive diagnosis (carman and borriello, ) . clostridium piliforme, the causative organism of tyzzer's disease, is not detected in faeces. a pcr test is available in the usa for detection of this organism. escherichia coli is not a normal inhabitant of the rabbit gut flora clinical pathology (a) coccidial oocysts and saccharomyces (ϫ ). shows large numbers of coccidial oocysts interspersed with saccharomyces guttulatus. the faecal sample was from a -week-old, thin rabbit that was suffering from diarrhoea. faecal material was emulsified with water before being passed through a fine sieve to remove the coarse debris. the homogenate was then centrifuged and the supernatent discarded. the residue was mixed with saturated salt solution and centrifuged again. after the sample had been spun, more saturated salt solution was added to the test tube until a meniscus formed. a cover slip was placed over the meniscus and the sample left for approximately minutes for the oocysts or worm eggs to float to the top of the tube. at the end of this period, the cover slip and surface film was removed from the test tube, placed on a microscope slide and examined under low power. (b) eimeria steidae (high power) (image kindly supplied by dr sheelagh lloyd, division of animal pathology, university of cambridge). several eimeria spp. affect rabbits and mixed infections occur. the species can be differentiated by the morphological characteristics of the oocysts. the most pathogenic species is eimeria steidae, which causes hepatic coccidiosis (see section . . ). e. steidae invades the epithelial cells of the bile ducts and can cause severe liver damage. oocysts may be seen in faeces from infected rabbits or in smears of bile collected from rabbits during post mortem examination. the relative sizes of coccidial oocysts and saccharomyces guttulatus can be seen in (a,b). worm eggs are larger than coccidial oocysts. the most common helminth infestation of pet rabbits is passalurus ambiguus. adult worms or ova from p. ambiguus may be seen in rabbit faeces. the ova are ovoid, slightly flattened and asymmetrical with a cap at one end. (c) saccharomyces guttulatus (high power) (image kindly supplied by idexx laboratories, wetherby). saccharomyces guttulatus is a budding yeast that is commonly found in the faeces of rabbits, chinchillas and guinea pigs. it is not believed to be pathogenic. some texts call the yeast cyniclomyces guttulatus. although small numbers may be present in some animals. enteropathogenic strains can be found in association with diarrhoea in weanling rabbits. pathogenic salmonella spp. may be isolated although post-mortem material is usually available in these cases. infectious enteritis is rare in the individual pet rabbit. plucked hair samples may be examined visually for the presence of mites that are just visible with the naked eye. under good illumination, cheyletiella mites can be seen in scale and skin debris that has been brushed out of the coat. after a few minutes, individual mites can be seen moving into the warmth of the illuminating light. egg cases and cuticles from developmental stages of the fur mite leporacus gibbus (formerly known as listrophorus gibbus) may also be seen on visual examination of hair brushings. they remain attached to hair shafts after hatching or moulting and give the fur a characteristic 'salt and pepper appearance' in heavy infestations. visual evidence of l. gibbus is seen more easily on white or light coloured areas of fur, especially if it is wet. occasionally lice may be found. microscopic examination of acetate strips applied to the skin of alopecic areas can be used to detect cheyletiella parasitovorax. all stages of the life cycle of c. parasitovorax can be seen by this method. skin brushings can also be examined microscopically. fleas, flea dirt, cheyletiella parasitovorax and leporacus gibbus can be seen in skin brushings examined under low magnification. a trichogram is useful in differentiating between conditions that cause alopecia. for this technique a sample of hair is plucked from as close to skin as possible using forceps. the hair is placed on a microscope slide, taking care to ensure the hairs remain orientated in the same direction. a drop of mineral oil and a cover slip are applied before examining the shafts of hair under the microscope. abrupt, fragmented, distal ends of the hair shaft suggest barbering by a companion. egg cases, cuticles or adult mites, usually leporacus gibbus, may be seen attached to hair shafts. the presence of fungal spores in broken hair shafts plucked from a lesion is diagnostic of dermatophytosis. dermatophyte infection can be demonstrated by the presence of mycelia or ectothrix arthospores in potassium hydroxide preparations of macerated scale. asymptomatic infections can be detected by brushing the entire body with a sterile toothbrush and incubating the brushings at °c on dermasel agar (oxoid). plates that do not show fungal growth within weeks can be considered negative (vangeel et al., ) . dermatophyte infections are usually due to trichophyton mentagrophytes that does not fluoresce under ultraviolet light. microsporum canis infections can also occur that are evident from the characteristic apple green spores that fluoresce under a wood's lamp. clinical pathology key points . • rabbit urine may be coloured red due to the presence of plant pigments. dipsticks can be used to differentiate haematuria from red urine • normal rabbit urine contains calcium carbonate sediment. small quantities of triple phosphate and oxalate crystals may be present • traces of glucose and protein can be present in normal rabbit urine • rabbits produce two types of faeces that differ in composition. the first step in faeces' examination is to determine which type of faeces has been collected • hard faeces are composed of compressed strands of indigestible fibre • soft faeces or caecotrophs are composed of a strong smelling paste rich in bacteria • some rabbits with diarrhoea excrete a mixture of hard and soft faeces signifying a failure of the proximal colon to separate the indigestible and digestible fractions of the diet • coccidial oocysts and ova from passalurus ambiguus may be seen in both types of faeces • a non-pathogenic yeast saccharomyces guttulatus may be seen in large numbers and can be mistaken for coccidial oocysts (see figure . ) • clostridium spiroforme can sometimes be seen in large numbers on a gramstained smear of faeces collected from a rabbit suffering from enterotoxaemia. exudate from crusty lesions may be examined for the presence of mites. although psoroptes cuniculi normally inhabits the ear canal, the mite can be found in other areas of the body such as the perineal skin folds and can be seen on microscopic examination of the exudate from affected areas. skin scrapings may be required to demonstrate sarcoptic mange mites in scabies cases that are characterized by intense pruritus and crusty lesions. dark field microscopy can be used to look for treponema cuniculi organisms in crusty lesions suggestive of rabbit syphilis (section . . ). the organism is a motile corkscrew-shaped spirochaete. lesions are found on the mucocutaneous junctions of the anus and genitalia or on the nose, lips and eyelids (see plate ). the lesion is abraded with a sterile saline-soaked swab. serum from the lesion is then expressed on to a slide and covered with a cover slip before being examined immediately (digiacomo et al., ) . the slide can be placed in a moisturized chamber. the differential diagnosis of exudative skin lesions in rabbits can be difficult and histopathological examination of biopsy specimens may be required. cerebrospinal fluid can be collected from the cisterna magna of rabbits in a similar manner to other animals. some normal parameters are summarized in table . . depressed glucose concentrations (< mg/dl) may be indicative of purulent inflammation (kusumi and plouffe, ) . key points . • cheyletiella parasitovorax, fur mites (leporacus gibbus), lice (haemodipsus ventricosus), fleas or flea dirt can be seen in skin brushings from affected animals • acetate tape strips can be used to detect all stages of the life cycle of cheyletiella parasitovorax • a trichogram can differentiate between dermatophytosis and barbering. mites, cuticles and egg cases may be seen attached to the hair shafts • psoroptes cuniculi, the rabbit ear mite, can sometimes be seen on microscopic examination of smears from skin lesions in other parts of the body such as the perineum • dark field microscopy may be used to detect treponema pallidum organisms. minerals and electrolytes part influence of dietary calcium/phosphorous ratio on blood calcium, phosphate and magnesium during gestation in the rabbit. (article in french, english abstract) plasma calcium, inorganic phosphorus and magnesium levels in pregnant and lactating rabbits clinical pathology of the domestic rabbit comparative vertebrate endocrinology physiology interpretation of laboratory results for small animal clinicians laboratory diagnosis of clostridium spiroforme-mediated diarrhoea (iota enterotoxaemia) of rabbits glycosylated haemoglobin levels in a colony of spontaneously diabetic rabbits (abstract) the calcium tolerance of growing rabbits calcium requirement of growing rabbits common diseases and medical management of rodents and lagomorphs clinical chemistry serological and histological studies on adult rabbits with recent naturally acquired encephalitozoonosis leucocyte subpopulations in cerebrospinal fluid of normal rabbits (abstract) clinical course and treatment of venereal spirochaetosis in new zealand white rabbits viral diseases untersuchungen zur klinik und therapie der encephalitozoonose beim heimtierkaninchen (article in german recent findings and future perspectives of digestive physiology in rabbits: a review effects of t- toxin on ovarian activity and some metabolic variables of rabbits adjustment of total serum calcium concentration for binding to albumin and protein in cats: cases ( - ) biochemical parameters of clinical significance in rabbits. ii. diurnal variations rabbit hematology selected drug dosages and clinical reference data effect of dietary calcium on bone density in growing rabbits total serum calcium concentrations in rabbits effect of ascaris suum infection on blood picture of rabbits in relation to the production of immunity (abstract) textbook of medical physiology parathyroid hormone, haematological and biochemical parameters in relation to dental disease and husbandry in pet rabbits clinical biochemistry and haematology clinical pathology values in pregnant and non-pregnant rabbits rabbit and ferret parasite testing vascular access ports for chronic serial infusion and blood sampling in new zealand white rabbits isolation of encephalitozoon cuniculi from urine samples evaluation of the effect of pentobarbitone anaesthesia on the plasma potassium concentration of the rabbit and the dog effect of intraosseous saline infusion on hematological parameters chronic plumbism in rabbits: a comparison of three diagnostic tests (abstract) interpretation of selected clinical pathology values in ferrets and rabbits ferret and rabbit endocrine disease spontaneous diabetes mellitus in the new zealand white rabbit rabbits. in an atlas of laboratory animal haematology pasteurellosis in rabbits. compendium on continuing education lead induced toxicosis in two domestic rabbits redistribution of lymphocytes after cortisol administration (abstract) the redistribution of lymphocytes during adrenaline infusion. an in vivo study with radiolabelled cells (abstract) alteration of sleep in rabbits by staphylococcus aureus infection haematological effects of exposure to three infective agents in rabbits physiological stabilization of rabbits after shipping arterial disease in chronic renal failure. an experimental study in the rabbit prevalence of dermatophytes in asymptomatic guinea pigs and rabbits changes in plasma parameters in rabbit does as a function of their physiological state and feed rationing acute phase reactants ceruloplasmin and haptaglobin and their relationship to plasma prostaglandins in rabbits bearing the vx carcinoma (abstract) regulation of calciotropic hormones in vivo in the new zealand white rabbit neoplastic diseases small animal clinical diagnosis by laboratory methods biochemical values of normal rabbit serum medicine of rabbits and rodents, th edn. williams and wilkins. hinton, m., jones d.r.e., festing m.f.w. ( ). haematological findings in healthy and diseased rabbits, a multivariate analysis. lab anim., , - . hoefer, h.l. ( ). rabbit weisbroth s.h., flatt r.e., brewer n. ( ).biology and diseases of rabbits. in laboratory animal medicine. pp - . academic press. krishna l., dawra r.k., vaid j., gupta v.k. ( ). an outbreak of aflatoxicosis in angora rabbits (abstract). key: cord- - vzntckq authors: rondeau, mark p. title: hepatitis and cholangiohepatitis date: - - journal: small animal critical care medicine doi: . /b - - - - . -x sha: doc_id: cord_uid: vzntckq • hepatitis is defined as any inflammatory cell infiltrate within the hepatic parenchyma; the term cholangiohepatitis describes the extension of that inflammation to include the intrahepatic bile ducts. • although many causes of hepatitis and cholangiohepatitis have been described in dogs and cats, the cause in many cases remains unknown. • a suspicion of hepatitis or cholangiohepatitis may be based on supportive historical, physical examination, and clinicopathologic findings that are similar for most causes of hepatic disease. a diagnosis of hepatitis or cholangiohepatitis is made ultimately via histopathologic evaluation of hepatic tissue. • the mechanisms of hepatocellular injury in animals with hepatitis and cholangiohepatitis are poorly understood. elucidation of these mechanisms may provide the basis for future therapeutic options. • successful treatment of the patient with hepatitis or cholangiohepatitis involves addressing the underlying disease or inciting cause and providing aggressive symptomatic therapy and supportive care. mark p. rondeau, dvm, dacvim (internal medicine) • hepatitis is defined as any inflammatory cell infiltrate within the hepatic parenchyma; the term cholangiohepatitis describes the extension of that inflammation to include the intrahepatic bile ducts. • although many causes of hepatitis and cholangiohepatitis have been described in dogs and cats, the cause in many cases remains unknown. • a suspicion of hepatitis or cholangiohepatitis may be based on supportive historical, physical examination, and clinicopathologic findings that are similar for most causes of hepatic disease. a diagnosis of hepatitis or cholangiohepatitis is made ultimately via histopathologic evaluation of hepatic tissue. • the mechanisms of hepatocellular injury in animals with hepatitis and cholangiohepatitis are poorly understood. elucidation of these mechanisms may provide the basis for future therapeutic options. • successful treatment of the patient with hepatitis or cholangiohepatitis involves addressing the underlying disease or inciting cause and providing aggressive symptomatic therapy and supportive care. hepatitis is defined as any inflammatory cell infiltrate within the hepatic parenchyma, and the term cholangiohepatitis describes extension of that inflammation to include the intrahepatic bile ducts. a diagnosis of these conditions is based on histopathologic examination of hepatic biopsy specimens. the histopathologic appearance gives clues regarding the duration of the inflammation. acute hepatitis is characterized by a combination of inflammation, hepatocellular apoptosis, necrosis, and possibly regeneration, but a lack of fibrosis. the relationship between the development of hepatitis and necrosis is complex, and it can be difficult to determine which abnormality was the initial lesion. chronic hepatitis, on the other hand, is identified by the presence of fibrosis, proliferation of ductular structures, and regenerative nodules in addition to an inflammatory infiltrate, apoptosis, and/or necrosis. the type of inflammatory cellular infiltrate may give the clinician some clues regarding the cause. occasionally, causative agents are identified within biopsy specimens. however, the cause remains unknown for many cases of hepatitis and cholangiohepatitis in dogs and cats. this chapter discusses the clinical presentation of animals with hepatitis and cholangiohepatitis and outlines the most commonly recognized clinical syndromes with respect to diagnosis and treatment of the specific disease. effective treatment of patients with hepatitis or cholangiohepatitis includes specific therapy of any identified inciting cause and aggressive symptomatic and supportive therapy. a discussion of symptomatic treatment and supportive therapy for the sequelae of hepatitis and cholangiohepatitis can be found in chapter . in general, the historical findings associated with hepatitis are nonspecific, as with most types of liver disease. exposure to certain etiologic agents or toxins may be ascertained from the client history and thus raise the suspicion for hepatic involvement. because of the large reserve capacity of the liver, a short duration of clinical signs does not necessarily indicate acute disease. animals with cholangiohepatitis (ch) may not show outward clinical signs until a significant portion of hepatic function is affected. presenting owner complaints for animals with hepatitis may include vomiting, diarrhea, anorexia, lethargy, polyuria, polydipsia, abdominal distention, dysuria, neurologic abnormalities associated with hepatic encephalopathy or vascular accidents, and icterus. similar to historical findings, the physical examination findings in animals with hepatitis are often nonspecific. icterus, when present in the absence of hemolytic anemia, suggests disease of the hepatic parenchyma or extrahepatic biliary system. animals with acute hepatitis are more likely to have fever and abdominal pain, and those with ch are more likely to have ascites. hepatomegaly may be present in some patients, especially those with acute hepatitis. many animals with hepatitis do not have any of these physical abnormalities present on the initial examination, and serum biochemical changes in those cases are likely to direct the clinician toward the liver as the site of disease. the pathogenesis by which hepatitis and cholangiohepatitis lead to hepatocellular necrosis and apoptosis is not understood completely. experimental studies have suggested many mechanisms of hepatocellular injury, but their specific evaluation in dogs and cats with hepatitis is lacking. mechanisms of hepatocellular injury that are not specific to hepatitis include tissue hypoxia, lipid peroxidation, intracellular cofactor depletion, intracellular toxin production, cholestatic injury, endotoxic insults, and hepatocyte plasma membrane injury. hepatocytes are especially susceptible to anoxia because the liver receives a mixture of venous and arterial blood. hypoxic damage quickly leads to plasma membrane and cytosolic organelle injury secondary to adenosine triphosphate (atp) depletion. free radicals may cause oxidative cellular injury that can result in lipid peroxidation and subsequent plasma membrane damage. cellular toxins may bind to nucleic acids and inhibit protein synthesis. cholestasis causes retention of bile acids that directly damage cellular organelles. endotoxins work via various mechanisms, most of which involve stimulation of inflammatory cells to produce inflammatory mediators (cytokines such as prostaglandins and leukotrienes) that perpetuate inflammation within the liver parenchyma. experimental work in mouse models suggests an important role for tumor necrosis factor-α (tnf-α) in the initiation and perpetuation of hepatitis. tnf-α, produced secondary to the interaction of the costimulatory molecules cd on t cells and feline cholangitis into two main categories: neutrophilic cholangitis and lymphocytic cholangitis. histologically, neutrophilic cholangitis (nc) is characterized by infiltration of neutrophils within the wall or lumen of intrahepatic bile ducts. this disease can be seen in acute and chronic stages. in acute neutrophilic cholangitis (anc), edema and neutrophilic inflammation may extend into the portal areas. in chronic neutrophilic cholangitis (cnc), a mixed inflammatory infiltrate may be noted in portal areas, along with varying degrees of fibrosis and bile duct hyperplasia. this syndrome was referred to previously as acute cholangiohepatitis or suppurative cholangitis-cholangiohepatitis. , nc can occur in cats of any age, breed, or sex. clinical signs are nonspecific and include anorexia, lethargy, vomiting, and weight loss. the duration of these clinical signs ranges from a few days to a few months and may be shorter in cats with anc than in those with cnc, but this is not a consistent finding. , physical examination findings commonly include dehydration and icterus. fever is present in % to . % of cases. , some reports suggest that fever is associated more commonly with anc than cnc, whereas others recognize no difference. , hepatomegaly is seen in fewer than half of the cases and abdominal pain is noted occasionally. , , biochemical analysis commonly reveals increased activity of alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and γ-glutamyltransferase (ggt) ranging in severity from mild to severe. however, increased liver enzyme activity may be absent in some cases. cholangitis in cats has been associated with inflammatory bowel disease (ibd) and pancreatitis, and many investigators believe that nc is the result of an ascending bacterial infection from the gastrointestinal (gi) tract. however, rates of bacterial isolation using traditional methods have varied greatly, from less than % to more than % in affected cats. , when isolated, common bacterial species include escherichia coli, enterococcus spp., clostridium spp., and staphylococcus spp. samples for aerobic and anaerobic bacterial cultures should be obtained in any cat suspected of having cholangitis; gallbladder bile is preferred to liver tissue as the culture source. treatment with a broad-spectrum antimicrobial therapy, focusing on enteric flora, is recommended pending results of culture and susceptibility testing. prognosis for cats with nc is typically good with aggressive treatment, although sequelae may include bile duct obstruction, acute necrotizing pancreatitis, sepsis, and multiple organ dysfunction. lymphocytic cholangitis (lc) is a chronic form of disease that is characterized histologically by a mixed inflammatory infiltrate (typically small lymphocytes, or lymphocytes and plasma cells) within portal areas and is associated with varying degrees of fibrosis and bile duct hyperplasia. inflammation within the walls or lumens of intrahepatic bile ducts may be present but is not a specific hallmark of the disease. lc likely includes a wide spectrum of clinical diseases with varying severity and clinical significance. lc likely includes syndromes that have been referred to previously as chronic cholangiohepatitis, nonsuppurative cholangitis-cholangiohepatitis, and lymphocytic portal hepatitis. , , , the clinical picture of cats with lc varies widely and has significant overlap with other forms of hepatobiliary disease in cats, including nc. , nonspecific clinical signs, including anorexia, lethargy, vomiting, and weight loss, may be chronic and intermittent. physical examination findings may include icterus, hepatomegaly, or ascites, but none are consistent findings. signs of hepatic encephalopathy (dullness, ptyalism, seizures) may develop in severely affected cats. definitive diagnosis is made by liver biopsy. as discussed for nc, ancillary diagnostics cd on hepatocytes and kupffer cells, stimulates hepatocyte apoptosis through the fas-fas ligand pathway. a better understanding of the complex mechanisms of hepatocellular injury in animals with hepatitis may encourage the development of novel therapeutic modalities for affected patients. box - lists the reported causes of hepatitis and cholangiohepatitis in dogs and cats. a complete discussion of all disease entities is beyond the scope of this chapter. a discussion of the most common clinical syndromes follows. the feline cholangitis complex is one of the most common hepatobiliary disorders in cats. this syndrome has been reported in dogs but is primarily a feline disease. several classification schemes have been proposed to define the various elements of this syndrome. the world small animal veterinary association (wsava) liver standardization group has proposed a classification system that divides hyperplasia of ductular structures and the absence of an identifiable underlying cause. the optimal treatment protocol for animals with cch has not been well studied, but immunosuppressive therapy is the mainstay of treatment. corticosteroids are the only class of drug shown potentially to provide benefit and their use is indicated in patients with signs of hepatic failure. other immunomodulatory drugs that may be used include ursodeoxycholic acid, metronidazole, azathioprine, and cyclosporine. colchicine may delay progression of hepatic fibrosis. copper chelation may be beneficial when copper retention is a significant contributing factor. the overall prognosis is difficult to ascertain because asymptomatic animals may have a slowly progressive course and excellent prognosis. however, once hepatic failure and/or cirrhosis develops, the prognosis is poor. the role of copper in the pathogenesis of cch is unclear. elevated hepatic copper levels have been identified in many dogs with cch, but because biliary excretion is the major mechanism of maintaining copper homeostasis, any cause of cholestasis would be expected to increase hepatic copper levels. however, it has been shown in the bedlington terrier that elevated copper levels (caused by an inherited defect in excretion) lead to chronic hepatitis and cirrhosis. however, it may be difficult to determine which came first, the copper accumulation or the hepatitis. a propensity for increased hepatic copper levels in association with cch has been described for many breeds in addition to the bedlington terrier, and these are listed in box - . a suspected primary hepatic copper storage disorder also has been reported in one cat. whether the copper accumulation is a primary or secondary event, the excessive copper is damaging to hepatocytes. copper chelation treatment has improved or resolved the hepatic pathologic findings in a group of doberman pinschers with elevated hepatic copper levels and subclinical cch. hepatic tissue should be harbored for copper quantification in any dog undergoing liver biopsy. if elevated levels are identified, a reduction of dietary copper and chelation with d-penicillamine ( to mg/ kg q h, given to hours before feeding) or trientine ( to mg/ kg q h, given to hours before feeding) are likely to be beneficial. nonspecific reactive hepatitis is a histologic diagnosis that describes the liver's response to a variety of extrahepatic disease processes. the lesion is characterized by widespread inflammatory infiltrates (usually lymphocytes and plasma cells) in the portal areas and parenchyma in the absence of hepatocellular necrosis. identification of this lesion should alert the clinician that a liver-specific problem is unlikely and that further investigation into the underlying disease process is necessary. viral hepatitis is uncommon in dogs and cats. most viral infections carry a poor prognosis. specific therapy is not available or has not been evaluated. symptomatic therapy and supportive care are therefore the primary therapeutic options. infectious canine hepatitis is caused by canine adenovirus type i. this disease has become rare because of extensive vaccination protocols using the cross-reacting adenovirus type ii vaccine. as such, the disease is seen only in young, unvaccinated dogs. the degree of antibody response determines the severity of disease, with a poor provide information to support hepatobiliary disease but are not specific for lc. activity of serum liver enzymes is increased in many but not all cases and varies in severity. abdominal radiographic and ultrasonographic findings are nonspecific but may aid in the recognition of concurrent disease. the cause of lc is unknown, although a chronic response to an ascending bacterial infection from gi flora and an association with ibd and pancreatitis (as seen with nc) has been suggested. , immunohistochemical analysis of hepatic biopsy specimens from cats with lc has shown a predominance of cd + t cells infiltrating the bile duct epithelium and periportal areas, a smaller proportion of b cells forming discrete aggregates in the portal regions, and expression of major histocompatibility complex class ii on the biliary epithelium. , these findings, combined with anecdotal response to glucocorticoid therapy and the fact that active infection has been documented rarely in cats with lc, have led to the suspicion that lc is an immune-mediated disease. treatment typically involves immunosuppressive glucocorticoid therapy in animals with no evidence of infection. treatment with ursodeoxycholic acid ( to mg/kg po q h) has anecdotal and theoretic benefits, although no clinical studies examining its efficacy in cats have been published. prognosis is typically good with appropriate management, although concurrent disease is common and may affect prognosis. although many causes of chronic hepatic inflammation in dogs have been identified, the term canine chronic hepatitis (cch) describes an idiopathic, progressive necroinflammatory disease of unknown cause that is common in the canine population. evidence supports an immune-mediated process as the perpetuating factor, , , although it is unclear whether the disease is a primary or secondary immune response. because of the chronic nature of the disease and the large reserve capacity of the liver, many affected animals are not identified until the onset of fulminant hepatic failure. however, increasing numbers of cases are now being identified at an earlier asymptomatic stage as a result of increased hepatic enzyme activity that is noted on routine serum biochemical screening. animals of any age and sex are affected, although middle-age female dogs may be overrepresented. cch is seen with increased frequency in certain breeds (box - ), suggesting a familial predisposition. no specific diagnostic findings separate cch from other causes of hepatitis. ultimately, the diagnosis is based on histopathologic examination of liver tissue revealing inflammation (usually lymphocytic and plasmacytic, occasionally neutrophilic), necrosis and/or apoptosis, evidence of regeneration, fibrosis and/or choices include azithromycin, ceftriaxone, and cefotaxime. prognosis is typically good, but patients often require intensive supportive care, including hemodialysis in animals with oliguric or anuric renal failure. pulmonary involvement worsens prognosis. bartonella species are arthropod-transmitted bacteria that have been associated with multiple clinical syndromes in veterinary medicine. bartonella henselae and bartonella clarridgeiae have been identified as causes of hepatic disease in dogs. clinical findings are similar to those of dogs with other causes of hepatitis. histologic examination of hepatic tissue from dogs with b. henselae infection has revealed peliosis hepatis and granulomatous hepatitis, both of which have been described in infected humans. diagnosis was made via identification of bartonella dna using pcr techniques on hepatic biopsy specimens. this is the preferred method of diagnosis because serologic assays impart information only regarding exposure, and granulomatous hepatitis may be caused by other agents. the cause of granulomatous hepatitis in dogs frequently is unknown, although reported causes include fungal infection, mycobacterial infection, dirofilariasis, lymphoma, histiocytosis, and intestinal lymphangiectasia. azithromycin is the antibiotic of choice for treatment of bartonellosis, although its use in dogs with hepatic disease caused by bartonella spp. has not been evaluated thoroughly. other antibiotics that may be effective include doxycycline (high dose, to mg/kg q h), enrofloxacin, and rifampin (in combination with doxycycline or enrofloxacin). an important cause of hepatitis in critically ill dogs and cats is bacterial seeding of the liver secondary to bacteremia or via translocation from the gi tract. commonly isolated aerobic bacteria include staphylococcus spp., streptococcus spp., and enteric gramnegative organisms. commonly identified anaerobes include bacteroides spp., clostridium spp., and fusobacterium spp. the diagnosis of bacteremia can be difficult in veterinary patients (see chapter ). septicemia-induced hepatitis should be suspected in critically ill animals that develop clinicopathologic evidence of hepatic disease while hospitalized, especially those in which bacterial infection or severe gi disease have been documented. treatment with broad-spectrum antimicrobials (pending sensitivity testing), along with aggressive supportive care, are vital to a successful outcome. the liver is particularly susceptible to toxic injury because it receives blood from the portal circulation. histologic changes in the liver secondary to toxic injury vary and may include no changes, hepatocellular swelling, steatosis, necrosis, cholestasis, inflammation, and/ or fibrosis. several substances reported to cause hepatotoxicity are noted in box - , but this is by no means an exhaustive list. because of the varying and nonspecific nature of histologic changes, diagnosis of hepatotoxicity often is made on the basis of clinical suspicion (biochemical alterations, such as marked increases in liver enzyme activity) with or without a history of known exposure. treatment involves removal of the offending agent and aggressive supportive care. s-adenosylmethionine (same) ( mg/kg po q h) has been effective in treating acetaminophen toxicity. , although its effectiveness against other forms of hepatotoxicity has not been evaluated, it is a logical choice for supportive care in animals suffering any hepatotoxic insult, mainly because of its ability to increase hepatic glutathione levels, which may increase antioxidant and repair abilities. response resulting in an acutely fatal syndrome. animals that mount an appropriate response may recover or develop ch. corneal edema and anterior uveitis may develop in animals that recover from acute illness. the diagnosis is made by histopathologic identification of large basophilic to amphophilic intranuclear inclusion bodies within hepatocytes and kupffer cells that are identified during the first week of infection. histopathology also reveals multifocal coagulative necrosis and a neutrophilic inflammatory infiltrate that may not be present in animals with severe acute infection. feline infectious peritonitis (fip) is caused by the feline enteric coronavirus. fip can affect any organ in the body. cats with hepatic involvement often have increased activities of alt and ast and develop hyperbilirubinemia as the disease progresses. histologic lesions include multifocal necrosis (often around blood vessels) with associated infiltration with neutrophils and macrophages. pyogranulomatous lesions may be noted on the liver capsule. immunohistochemistry can be performed on liver biopsy specimens to confirm the presence of virus. when hepatic involvement occurs, the disease is uniformly fatal. because there is no definitive treatment, supportive care is the mainstay of therapy. leptospirosis is caused by any one of several serovars of spiral bacteria belonging to the species leptospira interrogans sensu lato. the commonly isolated serovars in small animals include leptospira icterohaemorrhagiae, leptospira canicola, leptospira pomona, leptospira hardjo, leptospira grippotyphosa, and leptospira bratislava. infection in dogs most commonly results in acute renal failure, although hepatic involvement may occur in % to % of cases. , other clinical manifestations of infection include pulmonary hemorrhage, uveitis, and acute fever. infection in young animals and infection with serovars l. icterohaemorrhagiae and l. pomona are more likely to result in hepatic involvement. affected dogs may show acute hepatitis or develop chronic hepatitis with subclinical acute infection. although cats are generally resistant to leptospirosis, experimental infection with l. pomona has caused hepatic lesions in this species. patients with hepatic involvement show increased activity of hepatic enzymes (alt, ast, alp), although alp often is affected most severely. hyperbilirubinemia and signs of hepatic failure may occur. diagnosis of leptospirosis usually is based on clinical suspicion because of renal and hepatic involvement combined with serologic evidence of infection. however, antibody titers may be negative during the first week of infection, and antibody production may persist for only to weeks. suspected patients with negative antibody titers and a short duration of illness should be treated as though they have leptospirosis, and antibody titers should be repeated in weeks. histopathologic changes in the liver of affected animals may include coagulative necrosis and infiltration of lymphocytes and plasma cells with lesser numbers of neutrophils and macrophages. organisms may be identified in biopsy specimens with silver staining, but this is an insensitive diagnostic test. polymerase chain reaction (pcr) techniques to detect organisms in blood and urine samples are available. these techniques have not been well studied in dogs with clinical disease, but they are likely to make this diagnosis less challenging in the future. historically, treatment recommendations have included penicillin to eliminate the leptospiremic stage, followed by doxycycline to eliminate the carrier state. however, treatment with doxycycline alone is effective for the leptospiremic stage and carrier state ( mg/kg po/iv q h). penicillins may be used in animals that do not tolerate doxycycline. alternative antibiotic histopathologic features, immunophenotyping, clonality, and eubacterial fluorescence in situ hybridization in cats with lymphocytic cholangitis/cholangiohepatitis chronic hepatitis, cirrhosis, breed-specific hepatopathies, copper storage hepatopathy, suppurative hepatitis, granulomatous hepatitis, and idiopathic hepatic fibrosis characterization of the inflammatory infiltrate in canine chronic hepatitis effects of corticosteroid treatment on survival time in dogs with chronic hepatitis: cases ( - ) improvement in liver pathology after months of d-penicillamine in doberman pinschers with subclinical hepatitis performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases treatment and outcome of dogs with leptospirosis: cases ( - ) acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment and prevention infectious diseases of the dog and cat canine bartonellosis detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease peliosis hepatic in a dog infected with bartonella henselae granulomatous hepatitis in dogs: nine cases s-adenosylmethionine (same) for the treatment of acetaminophen toxicity in a dog s-adenosylmethionine protects against acetaminophen-induced hepatotoxicity in mice canine and feline gastroenterology morphological classification of the parenchymal disorders of the canine and feline liver acute hepatic injury: hepatic necrosis and fulminant hepatic failure cd -cd interactions drive hepatocyte apoptosis in murine fulminant hepatitis histopathologic evaluation of feline inflammatory liver disease cholangiohepatitis in a dog morphological classification of biliary disorders of the canine and feline liver the cholangitis/cholangiohepatitis complex in the cat relationship between inflammatory hepatic disease and inflammatory bowel disease, pancreatitis, and nephritis in cats wsava classification and role of bacteria in feline inflammatory hepatobiliary disease a survey of feline inflammatory hepatobiliary disease using the wsava classification clinical features of inflammatory liver disease in cats: cases ( - ) feline cholangitis: a necropsy study of cats intrahepatic biliary disorders immunohistochemical characterization of the lesions of feline progressive lymphocytic cholangitis/cholangiohepatitis key: cord- -a rd kas authors: guo, zuiyuan; he, kevin; xiao, dan title: early warning of some notifiable infectious diseases in china by the artificial neural network date: - - journal: r soc open sci doi: . /rsos. sha: doc_id: cord_uid: a rd kas in order to accurately grasp the timing for the prevention and control of diseases, we established an artificial neural network model to issue early warning signals. the real-time recurrent learning (rtrl) and extended kalman filter (ekf) methods were performed to analyse four types of respiratory infectious diseases and four types of digestive tract infectious diseases in china to comprehensively determine the epidemic intensities and whether to issue early warning signals. the numbers of new confirmed cases per month between january and december were used as the training set; the data from were used as the test set. the results of rtrl showed that the number of new confirmed cases of respiratory infectious diseases in september increased abnormally. the results of the ekf showed that the number of new confirmed cases of respiratory infectious diseases increased abnormally in january and february of . the results of these two algorithms showed that the number of new confirmed cases of digestive tract infectious diseases in the test set did not have any abnormal increases. the neural network and machine learning can further enrich and develop the early warning theory. public health emergencies caused by the continuous occurrence of emerging infectious diseases and unexplained diseases in the world such as severe acute respiratory syndrome (sars), h n influenza a, ebola haemorrhagic fever and h n highly pathogenic avian influenza have already become prominent public health issues in the past years [ ] . in addition, existing © the authors. published by the royal society under the terms of the creative commons attribution license http://creativecommons.org/licenses/by/ . /, which permits unrestricted use, provided the original author and source are credited. infectious diseases such as tuberculosis, dengue fever, malaria and influenza are also jeopardizing human health [ ] [ ] [ ] [ ] . faced with the complex situation of current infectious disease prevention, we should not only strengthen the surveillance of the epidemic information of infectious diseases but also issue early warning signals as soon and as accurately as possible to strive for the timely identification of outbreaks and epidemics in the early stages and to take rapid response measures to minimize the harm to social and economic development. since the sars outbreak, a web-based, daily infectious disease confirmed case surveillance system, which covered notifiable infectious diseases, was instituted in april in china [ ] . by , the number of monitored infectious diseases increased to types [ ] . this system is capable of collecting the number of confirmed cases of various infectious diseases nationwide in real time and greatly increases the timeliness of infectious disease reports. therefore, it establishes an important platform for surveillance of the epidemic intensity, analysing epidemiological patterns and practising the early warning model [ ] . early warning of infectious diseases involves issuing signals before or in the early stages of the infectious disease outbreak to warn that the event may occur or its extent and degree may expand. this is an important prevention measure to avoid or reduce harm to public health and social security caused by infectious diseases. currently, early warning systems of infectious diseases have already been established in many developed countries; these systems include the global outbreak alert and response network established by the who in [ ] , the global public health intelligence network established by collaboration between the who and health canada in [ ] and promed-mail established in and initiated by the international society for infectious diseases [ ]. these systems play important roles in the prevention and control of infectious diseases, particularly the prevention of emerging infectious diseases and bioterrorism attacks. the china infectious diseases automated-alert and response system developed by the chinese center for disease control and prevention began to operate nationwide in april [ ] . it is a more perfect and practical early warning system of infectious diseases established in china for the first time. it plays an irreplaceable role in the prevention and control of infectious diseases in china. the purpose of analysing infectious disease surveillance data obtained from an early warning model is to eventually determine whether the epidemic displays any abnormal increases. currently, commonly used early warning systems include simple control charts, moving average control charts, exponential weighted moving average and space scan statistics [ ] . these systems have already played positive roles in early warnings for poliomyelitis, bacillary dysentery, measles and other public health emergencies. in the twenty-first century, research on artificial neural networks continues to deepen and includes extensive applications in many fields including precision medicine and public health [ ] [ ] [ ] [ ] [ ] . in this study, we used real-time recurrent learning (rtrl) and extended kalman filter (ekf) to perform early warning research on four types of respiratory infectious diseases (measles, influenza, rubella and mumps) and four types of digestive tract infectious diseases (hepatitis a, hepatitis e, typhoid fever and paratyphoid fever, and bacterial and amoebic dysentery) that have higher incidence rates among notifiable infectious diseases in china. currently, these two algorithms have been extensively applied in nonlinear prediction and model establishment [ ] . we used these technologies in the field of epidemiology. the number of new confirmed cases per month was used as a sequential input signal and nonlinearly mapped to an output signal. if an output signal was over the threshold value, it was possible to determine the abnormal increase in the number of cases in the early stage of the disease epidemic. this study enriches the existing early warning theory of infectious diseases to further excavate the application potential of public health big data and increase the level of analysis and prediction of the condition of infectious diseases. the numbers of new confirmed cases of eight types of infectious diseases reported per month between january and december were obtained from the center for national public health scientific data (http://www.phsciencedata.cn). this center only collected data until [ ] . data between january and december were obtained by consulting the national health commission website (http://www.nhc.gov.cn/) [ ] . based on this objective fact, we selected the recurrent neural network model for analysis. the socalled recurrent network indicates that the network uses the output produced after one input as an intermediate variable, which is used as a part of an input layer together with the next input vector for continuous calculation. the recurrent neural network continuously receives input signals; therefore, it is called the dynamically driving recurrent neural network. targeting this model, we used rtrl and ekf to estimate the synaptic weights of the network [ ] . the rtrl learning algorithm indicates that the adjustment of synaptic weights of a fully connected network is real-time. figure a shows that the recurrent network was composed of q neurons and m external inputs. the network had two different layers: the concatenated input-feedback layer and the processing layer for calculating nodes. accordingly, the synaptic connections of the network were also composed of feed-forward and feedback connections. because four types of respiratory and four types of digestive tract infectious diseases were included, the value of m was . the number of neurons, q, in the processing layer was also set to . the output of the network was the last neuron in the processing layer. in addition, the numbers of new confirmed cases of the four types of infectious diseases were used as inputs in the model. because the numbers of cases of different diseases might have correlations and seasonal fluctuations [ ] [ ] [ ] [ ] [ ] [ ] , they were standardized in advance. after standardization was completed, the average value of each input variable should be approximately zero, there should be no correlation between variables, and covariances should be approximately equal. since the learning process in this study was supervised learning, the expected responses of the network should be determined. we calculated the cumulative probabilities of training samples under a multivariate normal distribution. these probabilities were arranged from low to high, and the % quantile was used as the threshold value. all neurons were set to have the same activation function, and a sigmoid odd function in the form of a hyperbolic tangent equation was used as the activation function. the parameters of this function were set to appropriate values such that the expected responses of the network were and − [ ] , which denoted over the threshold value and lower than the threshold value, respectively. furthermore, when the test samples were identified by our network, we consider that an early warning signal will be generated once the output value of the network is greater than zero. moreover, in terms of the learning rate, we selected search-then-converge scheme [ ] . respiratory and digestive tract infectious diseases were separately calculated using sets of numbers of cases between and as the training sets and sets of numbers of cases in as the test set. based on the recurrent network established in figure a , the idea of the sequential state estimation was used to divide the network state space under training into the actual state and the measurement state of the system [ ] . the former could not be directly observed; instead, a set of observation values was measured indirectly to estimate the actual state of the system. the entire set of synaptic weights of neurons was used as the actual state of the system. the value that measured whether the current condition of infectious diseases was at a normal level, i.e. expected response, was used as the observation state of the system. the vector activated by recurrent nodes of the network and the input vector used as the driving force together formed the input signal. the activation function was the same as that in rtrl. therefore, the process of mapping from the input space to the output space was also nonlinear. this task was finished using ekf. figure b displays the basic framework of this algorithm. w n indicates the synaptic weights of the network at the nth time step, which was the state vector of the system. it and dynamic noise together form the state vector of the next time step of the system. the recurrent node activation vector, input vector and state vectors in the system were mapped to the output layer under the function of the nonlinear measurement function. the output vector was influenced by multivariable white noise to form the observable state of the system. the specific model establishment process is shown in the electronic supplementary material. the above algorithms are shown in the electronic supplementary material. figure b shows the time distribution of the four types of digestive tract infectious diseases. they all showed obvious seasonal fluctuations. except for hepatitis e, which had a high incidence in the spring, the other digestive tract infectious diseases all had high incidences in the summer. in addition, except for hepatitis e, the numbers of cases of the other three infectious diseases all showed a decreasing trend year by year. the model was trained after epochs, then the synaptic weights of the network were determined, and data from was used to validate model performance. figure a shows the early warning results of respiratory infectious diseases between january and december of . the rtrl results showed that the number of cases in september was significantly higher than the historical level; although the early warning value in july was higher than zero, the increase in the number of cases was not significantly different from the historical level. the ekf results showed that the numbers of cases in january and february of were significantly higher than the historical level. figure b shows the early warning results of digestive tract infectious diseases. both algorithms showed that the number of cases throughout all of was not significantly higher than the historical level in the same period. figure shows the time distribution of the numbers of cases of the four types of respiratory infectious diseases in the same historical period when the early warning signal was issued. the results showed that the major reason for issuing the early warning was the increase in influenza and mumps cases. particularly in january and february of , a nationwide influenza pandemic occurred in china, which caused a significant increase in the number of influenza cases [ ] . analyses of surveillance data using early warning models are used to eventually make a decision on whether the epidemic is increasing abnormally. two types of calculating principles are employed for commonly used early warning models. one type is the calculation of the threshold value of statistics under a certain confidence level based on some specific statistical distributions and previous surveillance data; if the current statistical level obtained from actual measurement values is higher than the threshold value, the early warning signal is issued. the other type is the calculation of the expected number of cases based on specific statistical distributions and previous surveillance data; when the actual value is higher than the expected value, the early warning signal is issued [ ] . for example, some classical warning approaches, such as the exponential smoothing model, poisson regression and the arima model, that follow the above ideas have been effectively applied and constantly improved to increase the accuracy of early warning [ ] . the innovation of this article is that we broke through the traditional thinking framework. we did not calculate the threshold value or the expected number of cases; instead, the system itself calculated the epidemic intensity and made the decision on whether to issue early warning signals. the neural network has the characteristics of continuous optimization through self-iteration; therefore, when there are more training times on the system, its decision will be more accurate and the system will present a certain intelligent type. furthermore, this method can be used not only to consider multiple types of infectious diseases at the same time, but also to analyse multivariate data, such as the number of patients in outpatient clinics, drug consumption data in hospitals and sales data for over-the-counter drugs in pharmacies. by analysing the complex relationship among multiple factors, we can judge the law of disease development and changes comprehensively. both respiratory and digestive tract infectious diseases have different epidemic patterns in different seasons. the former mainly has epidemics in the winter and spring and the latter mainly has epidemics in the summer. therefore, the standardized processing of input vectors of the model should consider the influence of seasonal factors. according to the epidemic patterns of infectious diseases, we believed that vectors consisting of the numbers of cases of the four types of respiratory infectious diseases or the four types of digestive tract infectious diseases in every month all conformed to the multivariate normal distribution; in addition, the average vector and covariance matrix in every month also differed. after the standardized processing of all input vectors, the influence of seasonal factors on this model could be eliminated. we used month as a unit to perform analyses on the numbers of cases in this study. to improve the timeliness of early warning, week or day can be used as the unit for analysis. the average vectors and covariance matrix should also be adjusted accordingly. the level of the threshold value could be adjusted according to the actual condition. when the threshold value was higher, the number of cases necessary to issue an early warning was higher; when the threshold value was lower, the result was the opposite. the learning rate determined the convergence rate of the network model. if the learning rate was a constant number, the convergence rate was slower. if the learning rate was a constant divided by the times of iteration, although the convergence of the stochastic approximation algorithm could be ensured, fewer iteration times might have the risk of parameter amplification when the constant value was higher [ , ] . therefore, we chose the search-then-converge schedule as the commonly used learning rate annealing programme in online learning. it had the advantage of combining the expected characteristics of the model with the traditional stochastic approximation theory [ ] . in this study, the numbers of cases of some types of infectious diseases were used as the input variables in the model to make an early warning decision through comprehensive analysis. therefore, an early warning was issued only when the numbers of cases of these infectious diseases showed an overall increase and exceeded the threshold value. however, it was not necessary to issue an early warning only when all numbers of cases increased. figure shows that when the system issued an early warning, the numbers of measles and rubella cases not only did not increase but also were at lower levels compared to the historical level in the same period. the model made the decision on the high incidence of respiratory infectious diseases after the numbers of influenza and mumps cases increased. therefore, we should perform the analysis on the actual number of cases of each infectious disease based on the model result to determine which infectious disease epidemic caused the early warning. if the model did not issue an early warning, then it could not indicate that the number of any infectious disease cases did not increase significantly. thus, during the analysis of the epidemic, we should assess actual data for a detailed analysis. if the analysis was performed only based on the results of the model, it would be possible that the number of some infectious diseases already significantly increased but the model did not timely issue an early warning, thus causing delay in the timing of prevention and control. according to figure , we found that the proportion of the same results according to the two algorithms is . % ( / ), indicating that their decisions regarding the epidemic trend of diseases were similar. different results occurred in january, february, july and september of . according to figure a ,b, only the numbers of influenza cases in january and february of greatly increased compared to that in the historical periods; conversely, the numbers of cases of the other three infectious diseases all remained at lower levels. figure c shows that the numbers of mumps and influenza cases slightly increased in september . at these three time points, the results of these two algorithms differed. these results indicated that although the numbers of cases increased, the overall results were not significantly higher than that in the same historical periods. the early warning value of rtrl in july was slightly higher than zero; thus, we ignored its early warning significance. to increase the sensitivity of early warning, these two methods could be used simultaneously for analysis and determination of the epidemic intensity of the disease through a comparison of early warning results between these two algorithms. in this study, we selected eight types of infectious diseases with a higher incidence for analysis. these two models are not applicable for diseases with lower incidences such as anthrax, cholera and the plague; therefore, other methods are necessary for early warning [ ] . because the chinese notifiable infectious diseases surveillance and report system began to operate nationwide in [ ] , only disease data in the recent years could be obtained. the sample size of the training set was small, which may have influenced the calculation results for synaptic weights in the model to a certain extent. this study only references a new modelling approach and was limited in its scope in terms of validation. future work will be focused on gathering more data and cases and providing a more comprehensive model validation. furthermore, we evaluated the epidemic intensities of infectious diseases in the entire nation. however, infectious disease epidemics, such as dengue fever and malaria, might be limited to a certain area, or they may spread throughout the entire country after an outbreak in a certain area, such as sars and h n influenza a. if analysis and judgement were performed only based on the national data of these diseases, prevention and control of the epidemic would usually be delayed. therefore, this model should be implemented in every province and every city to increase the timeliness of early warning of infectious diseases all over the country. in addition, real-time multivariate analysis is currently one of the research focuses in the field of early warnings for diseases, including multistatistical process control, biological change-point detection and some others [ ] . since the application of artificial neural networks in the field of early warnings for diseases is relatively new, further research is required to achieve greater precision. ethics. since there is no experiment of humans and animals in the study, we did not require review by the ethical institutional review board or written informed consent. data accessibility. the raw data required for this study are available in the electronic supplementary material. authors' contributions. z.g. conceived of the study, designed the study, analysed the data and drafted the manuscript; k.h. provided guidance regarding the algorithm; d.x. revised the manuscript and provided comments. all authors gave final approval for publication. competing interests. all authors declare no competing interests. funding. this work was supported by the national science and technology major project (grant no. zx ) and the national key r&d program of china (grant no. yfc ). establishing a web-based integrated surveillance system for early detection of 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network-based real-time prediction of glucose in patients with insulin-dependent diabetes inferring plasmodium vivax transmission networks from tempo-spatial surveillance data dynamic mortality risk predictions in pediatric critical care using recurrent neural networks. dynamic mortality risk prediction neural networks and learning machines chinese information system for infectious diseases control and prevention seasonal pattern of influenza activity in a subtropical city modeling seasonal measles transmission in china explaining seasonal fluctuations of measles in niger using nighttime lights imagery seasonality of hepatitis: a review update seasonal dynamics of typhoid and paratyphoid fever high mean water vapour pressure promotes the transmission of bacillary dysentery a stochastic approximation method stochastic approximation methods for constrained and unconstrained systems towards faster stochastic gradient search acknowledgements. we would like to thank american journal experts (www.aje.com) for english language editing. royalsocietypublishing.org/journal/rsos r. soc. open sci. : key: cord- - pn u authors: gaddy, hampton gray title: using local knowledge in emerging infectious disease research date: - - journal: soc sci med doi: . /j.socscimed. . sha: doc_id: cord_uid: pn u emerging infectious diseases (eids) are a growing global health threat. the best research protocol to date on predicting and preventing infectious disease emergence states that urgent research must commence to identify unknown human and animal pathogens. this short communication proposes that the ethnobiological knowledge of indigenous and impoverished communities can be a source of information about some of those unknown pathogens. i present the ecological and anthropological theory behind this proposal, as well as a few case studies that serve as a limited proof of concept. this paper also serves as a call to arms for the medical anthropology community. it gives a brief primer on the eid crisis and how anthropology research may be vital to limiting its havoc on global health. local knowledge is not likely to play a major role in eid research initiatives, but the use of the incorporation of eid awareness into standard medical anthropological practice would have myriad benefits, even if no eids were discovered this way. emerging infectious diseases (eids) present a major threat to global health. they are communicable human diseases that have recently grown in their geographic and/or host range. for example, west never fever, lyme disease, and methicillinresistant staphylococcus aureus (mrsa) are eids that have that spread geographically in recent decades. eids that have recently jumped the species barrier from infecting non-humans to infecting humans are called zoonoses, and they include ebola virus disease, hiv/aids, and covid- . zoonoses comprise about % of all eids that have emerged in recent years, and they are especially feared for the toll they could take on global health over time (world health organization, ) . that toll is expected to grow. the th century paradigm of host-pathogen interaction suggests that species barriers should be very difficult for pathogens to overcome, but the stockholm paradigm, a new but well-substantiated understanding of pathogens, challenges this. it proposes that species barriers tend to be much lower than previously thought and will tend to fall significantly in the face of anthropogenic environmental change (brooks et al., (brooks et al., , . for example, climate change is already associated with many recent vector-borne disease outbreaks (reisen, ) , since the s, eids have been discovered at a rate of more than three per year (woolhouse & gaunt, ) , and that rate has been increasing since the s (jones et al., ) . as such, researchers from various disciplines view eids as a growing crisis-one that requires interdisciplinary research, especially in order to limit infectious disease emergence (parkes et al., ; goodwin et al., ; brooks et al., ) . this paper aims to contribute to that research agenda. the main research protocol for preventing infectious disease emergence is dama (documentation-assessment-monitoring-action). it proposes an integrated research initiative to isolate and categorize unknown pathogens ('documentation'), identify which are most likely to become eids ('assessment'), develop surveillance networks for those pathogens ('monitoring'), and take proactive steps to minimize human exposure to them ('action') (brooks et al., (brooks et al., , . dama is still very much on its first phase of implementation. the largest research program that has studied zoonosis emergence was predict, a ten-year effort funded by the u.s. agency for international development (usaid) and launched in . it focused on isolating pathogens from animals in likely hotspots of zoonotic emergence, i.e. low-income areas of africa and asia (kelly et al., ) . as yet, that seems to be the only tested strategy for 'documentation.' this paper suggests that utilizing local knowledge may also be a useful strategy for documenting potential eids. local knowledge refers to "the knowledge that people in a given community have developed over time, and continue to develop. it is based on experience; often tested over centuries of use; adapted to the local culture and environment; embedded in community practices, institutions, relationships and rituals; held by individuals or communities; and dynamic and changing" (food and agriculture organization, ). the concept of 'local knowledge' significantly overlaps with those of 'traditional knowledge,' 'indigenous knowledge, 'traditional ecological knowledge,' and 'situated knowledge,' each of which tends to be used in different social sciences. this paper primarily uses the term 'local knowledge,' as it is the broadest of these concepts. however, i primarily use it to refer to the experience-and community-based knowledge of indigenous people and vulnerable communities in low-income countries. borrowing from the idea of local knowledge as 'situated knowledge' (e.g. haraway, ; nazarea, ; tschakert et al., ) , i refer to these communities as 'locally-situated communities.' local knowledge is already used as a source of empirical knowledge to different extents by different disciplines. traditional ecological knowledge is a bountiful source of pharmaceutical drugs, as well as knowledge about ecology, environmental health, and the health of indigenous communities (e.g. alves & rosa, ; finn et al., ) . moreover, the study of local disease knowledge is well-established within medical anthropology. in this paper, i propose that local knowledge is also a useful source of information about potential eids that are unknown to science. local knowledge can be a problematic source of information. however, it likely contains relevant information about human diseases, animal diseases, and zoonoses that has not yet been empirically documented. i will discuss this possibility with reference to theoretical considerations and some limited case studies. there are theoretical reasons to suspect that local knowledge could be useful for identifying potential eids. as already mentioned, ethnobiological systems can surpass empirical knowledge of certain subjects. those subjects are generally distant from the purview of empirical knowledge gathering systems, e.g. geographically remote or in lowincome settings, and they are often salient to local life. human and animal diseases that exist in locally-situated communities fit both of those criteria. useful information about diseases of which the scientific community was already aware. a study of local knowledge in ghana about buruli ulcers, a poorly understood, necrotic infection caused by mycobacterium ulcerans, revealed information that is likely useful for understanding the etiology and life cycle of the disease (tschakert et al., ) . similarly, local knowledge in malaysia and latin america has been helpful in identifying neglected hideouts of the animal vectors that transmit dengue fever (dickin et al., ) and chagas disease (abad-franch et al., ) , respectively. given that such communities have independent knowledge of diseases already known to science, it is likely that they have knowledge of diseases not known to science. this is not a valid inference if science is assumed to have a complete understanding of disease, but this is not the case. accurate estimates of global pathogen diversity are very difficult to obtain, but only a small fraction of potential pathogens have been empirically documented (pedrós-alió & manrubia, ). moreover, there are ecological reasons why locally-situated communities are more likely to be in contact with animal diseases. such communities often live in highly biodiverse environments. cultural diversity correlates with biodiversity on a global scale (gorenflo et al., ) , and it is likely that this principle applies to pathogen diversity as well (guernier et al., ) . therefore, locally-situated populations are arguably in closer proximity to a higher density of animal disease than any other population just because of their geography-not to mention because of any economic or subsistencedriven needs for such communities to interact with animals. it is also likely that locally-situated communities face relatively high rates of zoonotic infection. sharing an ecosystem with a great diversity of pathogens increases the risk of disease transmission almost in and of itself, given that risk of zoonotic infection is a function of ecological connectivity (brooks et al., ) . additionally, ecological degradation is a risk factor for zoonosis (ostfeld, ; bonds et al., ) , and locally-situated communities are generally the most likely to live in degrading ecosystems. the ecosystems in which such communities live are among the most likely to face damaging anthropogenic pollution, e.g. due to poor regulation and the presence of pollution-prone industries (hoover et al., ; sapkota & bastola, ) . such communities are also least able to mitigate the effects of climate change in their environments. for arctic communities, this is especially true because warming is happening faster there than any other region of the planet (dai et al., ) . therefore, locally-situated communities can be expected to be in especially frequent proximity to human diseases, animal diseases, and zoonoses that are unknown to the scientific community. one can expect those communities to have knowledge of at least some of those diseases. zoonoses, as novel diseases, should always have a high degree of salience within communities. regarding the salience of animal diseases, indigenous communities, in particular, rely heavily on the animals in their environment for subsistence. furthermore, many such groups have a high degree of psychological connection with the other organisms around them (salmón, ) . animal diseases can be practically, economically, and spiritually relevant to indigenous and low-income communities. there is the problem that pathogens themselves are too small to be observed without formal training, and the same may apply to certain symptoms of human and animal diseases. ethnobiological theory recognizes that the size of natural features limits the perceptual salience of those features (hunn, ) . despite that, locally-situated communities do, in fact, tend to have extensive knowledge about animal disease. therefore, since there is likely a high density of potential eids in those communities and those communities have likely noticed many of those diseases, their local knowledge can be expected to be a useful source of novel knowledge about potential eids. history also tells us that this is likely the case. the most salient human and animal diseases were likely known first by ancient populations, who, of course, did not have modern methods for collecting knowledge (see hoeppli, ). there are, of course, problems with using local knowledge as a source of information about eids. translating local knowledge into empirically relevant information can be difficult at times. for example, this would be a problem if one encounters a locally described disease for which there is no biomedical name. understanding the local description of the disease may prove challenging, especially as doing so may require anthropological and/or linguistic analysis (kleinman, ) . then, empirically confirming the existence and nature of that disease may prove challenging, especially if the disease is an alleged zoonosis. i recognize that interdisciplinary integration is often difficult and costly, but the recruitment of anthropologists, linguists, and local guides can mitigate all of these challenges. furthermore, if there is a paradigm shift towards viewing local knowledge as a valued source of disease surveillance, the opportunity costs of collecting and using it may fall. incorporating an awareness of eids into medical anthropology has benefits even if no eids are discovered this way. in particular, i think that an awareness of eids can produce a more dynamic understanding of health progress and security. for example, the current covid- pandemic has shown that although traditional statistics like infant mortality rates and life expectancy are usually good markers of health progress, the fundamental mark of a strong health system is its ability to respond to an emerging threat. recognition of the fact that eid outbreaks are likely to become more common may lend new importance to the study of resilience in health systems. that said, i think that the limited case studies that follow show that there is some real promise to discover eids by using local knowledge. the literature on local knowledge and potential eids is scarce. i will discuss three useful but limited case studies below. however, it does not seem that any anthropological or ethnobiological studies have been conducted with the explicit aim of learning about diseases unknown to science. this is not that surprising. anthropology is the discipline that has studied indigenous medicine and disease to the greatest extent. however, until recent decades, medical anthropology had focused more on the ritual and psychology of local medical systems than any biological or epidemiological knowledge that could be gained from studying them (waldstein & adams, ) . there is now a slowly growing recognition within health science literature that non-scientist stakeholders can be vital in the production of empirical knowledge (e.g. catley et al., ; quinlan & quinlan, ; den broeder et al., ) . but, i found no studies of local health knowledge that engaged with the possibility that locally-situated communities might know of human infectious diseases, animal diseases, or zoonoses that are still unknown to science. most studies did not actually seem to account for that possibility in their design. i suspect that this disregard for local disease knowledge is partly because of the popular but incorrect perceptions that infectious disease emergence is an uncommon and unpredictable event (brooks et al. ) . that thought presumably leads researchers to assume that indigenous knowledge would not be helpful in discovering new diseases. for those reasons, finding proof of concept for what i propose is difficult. however, i present three somewhat promising case studies. the first comes from a study of disease knowledge held by fula-speaking pastoralists in the far north region of cameroon (moritz et al., ) . locals identified two diseases called haahaande and gawyel, which they said could infect both cattle and humans. the authors of this study dismissed this information, saying that heartwater (the scientific name for haahaande) and blackleg (the scientific name for gawyel) are not zoonotic diseases. case reports suggest that the local knowledge may have been correct. heartwater is a systemic cattle disease caused by ehrlichia ruminantium; it is characterized by increased vascular permeability that causes fatal respiratory, cardiovascular, and neurological symptoms. there are reports that e. ruminantium can infect humans (esemu et al., ) , in whom it may cause fatal encephalitis (allsopp, ) . furthermore, e. ruminantium has been shown to infect a wide variety of wild and domestic animals (peter et al., ) , suggesting that it is a plausible zoonotic agent. blackleg is a myonecrotic disease caused by species of clostridium. moritz et al. ( ) suggested that gawyel could refer to infection by c. chauvoei or the less common c. septicum. c. chauvoei is known to have infected humans in at least two cases-though it is unclear whether those cases were zoonotic (nagano et al., ; weatherhead & tweardy, ). an analysis of c. chauvoei genomes concluded that the species has too little genetic diversity to infect non-ruminants (rychener et al., ) , but recent paradigms of infectious disease emergence suggest that intraspecific genetic diversity is not necessarily requisite for pathogens to colonize new host species (brooks et al. ) . c. septicum is known to infect humans, and some human cases have been presumed to be zoonotic in origin (barnham, ) . however, neither species of clostridium has been widely recognized as a zoonotic pathogen (e.g. songer, ) . this case study suggests that local knowledge can be a source of information about e. ruminantium and clostridium zoonosis. a second case study comes from research on maa-speaking pastoralists in northern and eastern tanzania (mangesho et al., ) . these pastoralists also identified blackleg (locally called emburuo) as a zoonosis. additionally, they identified a respiratory infection called mapafu ya kikohozi ya mbuzi, which they said was contracted from goats, and they identified a fungal infection called ndororo, which they said was contracted by stepping in raw cattle remains or slurry. these two diseases were only mentioned in passing by the authors, as was emburuo, and no attempts were made to identify a biomedical name for them, nor to anthropologically or medically validate their existence. i mention this case because of how frustrating it is. ndororo and mapafu ya kikohozi ya mbuzi may refer to diseases that have already been empirically documented, diseases that are not empirically known, or they may be local constructions that do not correspond to any biomedical illness. ndororo could refer to any number of foot fungi. mapafu ya kikohozi ya mbuzi could be contagious caprine pleuropneumonia, a respiratory disease known to infect goats in tanzania, but this disease has never been reported in humans (iqbal yatoo et al., ) . in the absence of more information, there is no way to judge whether the diseases are novel or even medically real. however, there are countless similarly brief and frustrating references in existing literature, e.g. ente kurwaara omutima and okunvara ckine in katunguka- rwakishaya et al. ( ) , eyaliyal in gradé et al. ( ) , and many diseases in catley & mohammed ( ) . i propose that much of this local knowledge may be useful for identifying potential eids. a third case study comes from camel-herding pastoralists in somalia and northern kenya. surveys of these communities have consistently identified two camel respiratory diseases with distinct symptomatic and epidemiological characteristics (wako et al., ) . one is called hergeb in somali; it is characterized by frequent outbreaks that cause nasal discharge and mortality among young camels. the other is called dhuguta in somali; it is characterized by infrequent outbreaks that cause coughing and emaciation. there is no empirical knowledge about these diseases. several respiratory pathogens are known to cause disease in camels, including strains of influenza, respirovirus, mycoplasma sp., streptococcus sp., small ruminant morbillivirus, and infamously, mers-cov. however, none of these have yet been identified as hereb or dhuguta. furthermore, either might be one of the many other locally known camel respiratory diseases that have been described across africa, e.g. mhaz in volpato et al. ( ) , sonbobe in bekele ( ) , and ah and laxawgal in catley & mohammed ( ) . given the limited scientific knowledge about camel respiratory diseases and the abundance of local knowledge about them, it seems that the local knowledge of pastoralists should certainly be studied further in the future. case studies show that local knowledge can be a useful source of new information about human diseases (e.g. buruli ulcers), animal diseases (e.g. camel respiratory infections), and potential zoonoses (e.g. blackleg and heartwater). this is not surprising, given how sophisticated systems of ethnomedical and ethnoveterinary knowledge have been found to be. this is also not surprising given the apparent proximity of locally-situated communities to animal, pathogen, and zoonosis biodiversity. this review is preliminary, but the theoretical considerations and limited case studies i present suggest that locally-situated knowledge can be an important source of information about potential eids. the dama protocol is currently our best defense against the growing eid crisis, and the documentation stage, the 'd' in dama, should not fail to engage with the documentation of local knowledge. the inclusion of anthropologists in diverse public health disciplines is growing (e.g. stellmach et al., ) , and the study of eids is another area in which their expertise is needed. community participation in chagas disease vector surveillance: systematic review ehrlichia ruminantium: an emerging human pathogen? biodiversity, traditional medicine and public health: where do they meet? clostridium septicum infection and hemolytic uremic syndrome studies on the respiratory disease 'sonbobe' in camels in the eastern lowlands of ethiopia. tropical animal health and production serologic evidence for human ehrlichiosis in africa participatory epidemiology: approaches, methods, experiences. the veterinary ethnoveterinary knowledge in sanaag region, somaliland (part ii): notes on local methods of treating and preventing livestock disease arctic amplification is caused by sea-ice loss under increasing co citizen science for public health mosquitoes & vulnerable spaces: mapping local knowledge of sites for dengue control in seremban and putrajaya malaysia ehrlichia species, probable emerging human pathogens in sub-saharan africa: environmental exacerbation the value of traditional ecological knowledge for the environmental health sciences and biomedical research food and agriculture organization interdisciplinary approaches to zoonotic disease ethnoveterinary knowledge in pastoral karamoja ecology drives the worldwide distribution of human diseases situated knowledges: the science question in feminism and the privilege of partial perspective the knowledge of parasites and parasitic infections from ancient times to the th century indigenous peoples of north america: environmental exposures and reproductive justice size as limiting the recognition of biodiversity in folkbiological classifications: one of four factors governing the cultural recognition of biological taxa contagious caprine pleuropneumonia -a comprehensive review global trends in emerging infectious diseases organisation for social science research in eastern and southern africa (ossrea) one health proof of concept: bringing a transdisciplinary approach to surveillance for zoonotic viruses at the human-wild animal interface patients and healers in the context of culture: an exploration of the borderland between anthropology, medicine, and psychiatry exploring local knowledge and perceptions on zoonoses among pastoralists in northern and eastern tanzania on not knowing zoonotic diseases: pastoralists' ethnoveterinary knowledge in the far north region of cameroon human fulminant gas gangrene caused by clostridium chauvoei ethnoecology: situated knowledge/located lives biodiversity loss and the rise of zoonotic pathogens all hands on deck: transdisciplinary approaches to emerging infectious disease the vast unknown microbial biosphere ehrlichia ruminantium infection (heartwater) in wild animals ethnobiology in one health vector-borne diseases clostridium chauvoei, an evolutionary dead-end pathogen kincentric ecology: indigenous perceptions of the human-nature relationship foreign direct investment, income, and environmental pollution in developing countries: panel data analysis of latin america clostridia as agents of zoonotic disease anthropology in public health emergencies: what is anthropology good for? situated knowledge of pathogenic landscapes in ghana: understanding the emergence of buruli ulcer through qualitative analysis ethnoveterinary of sahrawi pastoralists of western sahara: camel diseases and remedies indigenous knowledge of pastoralists on respiratory diseases of camels in northern kenya the interface between medical anthropology and medical ethnobiology key: cord- -ssiggi z authors: pappas, g.; kiriaze, i.j.; giannakis, p.; falagas, m.e. title: psychosocial consequences of infectious diseases date: - - journal: clin microbiol infect doi: . /j. - . . .x sha: doc_id: cord_uid: ssiggi z historically, there has been an exaggerated fear related to infection compared to other conditions. infection possesses unique characteristics that account for this disproportionate degree of fear: it is transmitted rapidly and invisibly; historically, it has accounted for major morbidity and mortality; old forms re-emerge and new forms emerge; and both the media and society are often in awe. because, in an outbreak, the patient is both a victim and a vector, and because there exists the potential for infringement of personal rights in order to control an outbreak, infection may be viewed (and has been depicted in popular culture) as a foreign invasion. during recent outbreaks, fear, denial, stigmatization and loss have been recorded in the implicated individuals. stigmatization and discrimination may further involve ethical correlations, and attempts to adress these issues through activism may also have unwarranted effects. public health initiatives can address the public's fears by increasing health literacy, which can contribute to reducing stigmatization. a man watches the news and starts feeling anxious. his hands feel sweaty and his heartbeat increases. he experiences a sense of agitation as he hears of a possible bird flu pandemic. the media puts before him scattered images of people rushing to buy flu vaccines, discussions on the utility and potential shortage of antiviral agents, journalists reporting the death toll of the previous influenza pandemics, the hundreds of millions of birds slaughtered in southeast asia, the hundreds of millions of human victims expected worldwide, the extraordinary expense of the control of past outbreaks and the anticipated expense apparently needed to enhance preparedness. the man feels overwhelmed by the amount of information. in a nearby hospital, a nurse in the emergency department thinks of asking for a long-term leave because she wants to be absent when an outbreak emerges; she is thinking of her family and feels she is unqualified to deal with, and not secured against, morbid infection. an infectious diseases specialist is on a plane, returning from an international congress on infectious diseases; several hours earlier, he attended a lecture about the then evolving severe acute respiratory syndrome (sars) outbreak. now, a chinese passenger sits in a nearby seat; the specialist is transiently overwhelmed by fears: what if this person is a carrier; what should one do if this fellow passenger coughs? later, he manages to reassure himself. these three individuals, among many others, experience levels of fear associated with infectious diseases in their everyday lives. they share the anxiety, the uncertainty, and the potential for irrational behavior due to fear of an unknown disease. they suffer from 'germ panic' [ ] . infectious diseases have had a significant role in shaping human history, and are responsible for, through the great plagues of the past, more deaths than any other human pathology [ ] ; these outbreaks have engraved an automatic response in our subconscious of a fear of infection. in an era of major scientific progress in battling, and even eliminating, certain infections, this fear may seem unwarranted. yet 'germ panic' consistently re-emerges, in contrast to the fear related to more burdensome entities, in terms of mortality, such as cardiovascular disease. why is it that infectious diseases cause the most significant psychological unrest, both in the public and in health professionals alike? infection is: (i) transmissible, (ii) imminent and (iii) invisible. moreover, the field of infectious diseases is ever-expanding. the risk of cardiovascular disease is a recognized entity with predisposing factors that have changed little over the years. on the other hand, numerous new major threats have emerged during the last three decades; the pandemic of aids, the sars outbreak, the ominous scenarios of an avian influenza pandemic, and the threat of biological weapons are just some examples explaining the concern among health authorities, the media, and the public. the evolution of the 'global village' further enhances the fear of contracting exotic diseases that can be imported into metropolitan areas (e.g. the chikungunya virus) [ ] , diseases that can be transmitted in the context of air travel [ , ] , or simply diseases that emerge in new areas as a result of nature's peculiar ways (e.g. the west nile virus new world epidemic) [ ] . fear, in strict neuropsychological terms, is a normal reaction to an evolving threat, preparing the individual, both physically and mentally, for an acute response to possible harm. this reaction, however, is triggered both in the cerebral cortex, the outcome of a rational mental approach to the present situation, and by the amygdala, a process generated earlier than the cortical one, which is subconscious and potentially irrational, often crossing the barrier to panic. there are numerous exogenous factors that shape the nature of this subconscious response. the psychological response of both patients and the public to the threat of infection has been evaluated with respect to numerous circumstances in recent years, not only acute outbreaks such as sars, but also gradually evolving pandemics such as aids, threats with marginal risk for humans such as bovine spongiform encephalopathy (bse; mad cow disease), and even threats that are only theoretical such as avian influenza. moreover, inordinate psychological responses to infection have been recorded in the context of epidemics. for which an unidentified, readily transmissible agent with high mortality was responsible. fear, denial and frustration, which comprise three sequential stages of the rational response to fear, have been reported as predominant among patients or quarantined individuals during the sars outbreak in canada and amoy gardens in hong-kong [ ] [ ] [ ] . loss and a conflict between duty to the patient and the will to be with one's family have been recorded in health care workers quarantined during the sars outbreak in canada [ ] . anxiety extends (in the case of patients and exposed persons) beyond the physical consequences of infection, to social consequences such as stigmatization, with the latter even extending to asian populations of non-endemic regions such as new york's chinatown [ ] . a similar case of stigmatization during an acute outbreak was also racially orientated: in the us hantavirus outbreak, the native american indians were stigmatized by the term 'navajo disease', a term which ignored the fact that non-navajos were also becoming ill; as a result, 'anti-indian racism mixed with fears of disease' emerged [ ] . the potential effect of psychological reactions was also exemplified in the plague outbreak in surat, india, which led to an extended official and unofficial quarantine, with stigmatization being disproportionate to the extent of the outbreak [ ] . in the case of an unknown agent, a lack of preparedness on the part of medical authorities and misleading information reproduced by the media may further aggravate these pathological psychological responses. in the sars epidemic, both these factors have been recognized, and media miscommunications and inconsistent health policies have been highlighted as factors amplifying stigmatization in hong kong [ ] . medical authorities can also inadvertently augment a problem by initiating and recycling fear. apart from the awe-inducing isolation procedures, devices and uniforms (with the latter being reminiscent of astronauts and the concept of alien invasion), the medical disputes over preventive and therapeutic strategies may perpetuate fear when made public. psychosocial reactions in gradually evolving epidemics: the case of aids: the aids pandemic was also attributed to a hitherto unknown agent, but significant differences contributed, in part, to shaping the psychological response of both patients and the public. the aids pandemic developed over a period of years, instead of days, and it was related to sexual practices, further influencing public response. the initial stages of the disease, however, were reminiscent of the 'navajo disease', in that a marginalized population was targeted and stigmatized. however, the history of aids highlights the fact that such discrimination continues to exist, and targeted populations are marginalized through germ panic. activism here acts like a double-edged sword; it fights discrimination and augments public health literacy, but may also enhance fear [ ] . attempts to raise awareness of an issue may be subject to media misinterpretation; continuous discussion of an issue may raise awareness, but also may raise the sense of threat in individuals who are inadequately informed. although the psychological responses to some extent reflect the epidemic, the aids story exemplifies that responses also reflect the content of public education campaigns and public health efforts, as well as media and news coverage [ ] . the surgeon general's aidsrelated campaign in the usa took place in , comprising the first official nationwide effort to promote risk reduction or even explain the mechanics of hiv transmission. it is worth noting here that a pamphlet by callen and berkowitz entitled 'how to have sex in an epidemic', produced by several gay activists, was distributed in , years ahead of the surgeon general's campaign, to help sort through the confusing information concerning the new epidemic and the divergent theories regarding the cause of the syndrome [ ] . as a result, the epidemic was better understood among the gay community, regardless of the officials' silence, which left the rest of the population uninformed for a protracted period. a similar observation was made in israel in a region that was affected by poultry avian influenza; the residents of this area had a significantly greater understanding compared to residents of the rest of the country [ ] . awareness is a key issue, particularly when there is ample time for it to be enhanced. fear of forthcoming epidemics: the case of avian influenza, mad-cow disease, and more to come: fear may be a physical response leading to individual protection, but, sometimes, protective measures undertaken according to public initiative can lead to increased morbidity because of the protective measures themselves rather than the threat against which they were supposed to be protective [ ] . in the case of both bse and the, only now gradually subsiding, avian influenza pandemic scenarios, a common denominator was the climax of the threat, with the mass media capturing the public's attention, classically highlighting the subconscious, and memories of the great epidemics of the past (e.g. the spanish influenza pandemic). in the case of bse, fear rapidly extended to other countries [ , ] and continents [ ] [ ] [ ] , aided by coverage of the subject in well respected journals of medical and general interest; in the latter case, with eye-catching titles such as 'can it happen here?'. in one french study [ ] , the perceived risk of bse (which is significantly different to the actual risk) modified the public's approach towards meat consumption, although this modification of the peoples' cognitive and affective responses to hazard peaked rapidly and subsided in approximately year. in the case of avian influenza, a similar 'vaccination panic' that rapidly subsided was recorded in greece [ ] , underlining the distorted ways in which the public reacts when overwhelmed by information. the psychosocial effect of misconceptions about the disease was also demonstrated in israel, where the public had a distorted perception of the dynamics of human-to-human transmission [ ] . the way that the media and scientists present relevant information can also account for this effect [ ] . 'scare statistics' and imaginary titles in the news all contribute to arouse the subconscious perception of threat; although some have proposed the use of fear as an educational tool, behavioral effects in this case have not been demonstrated [ ] . regarding avian influenza, fear extends to hospital personnel and the public alike [ , ] , and cannot be underestimated. a study conducted in hong kong showed that the majority of the public would expect panic or other forms of stressrelated responses to emerge [ ] , as well as a potential for stigmatization [ ] . fear of infection in non-epidemic situations: people continue to use antibiotics, even when advised against doing so, for numerous respiratory tract infections of obvious viral origin. patients fear that they may develop pneumonia and overestimate the morbidity, even the mortality, related to their symptoms. infection is often considered as a social issue that indirectly leads to stigmatization, as in the case of brucellosis, where patients may express denial, because of a correlation of the infection with a lower socio-economic status (i.e. an indirect form of stigmatization) [ ] . this has been the case also for outbreak-causing diseases in the aftermath of the outbreak, as with bse, where protective measures have been dismissed by many uk farmers as potentially stigmatizing individual farmers in terms of 'bad practicing' [ ] . fear develops in public and refers to the society. its evolution is not a strict medical process of the nervous system, but the result of a complex interplay of medical and social factors and forces. fear of infection is not only engraved in our subconscious as a result of memories of former epidemics, but also because of fictional dramatizations of such potential threats. the way that infectious diseases are presented in the cinema is a typical example and can influence society's perceptions [ ] . the concept of an unseen foreign invasion, the numerous apocalyptic views of the end of the world as a result of an unknown virus, and the scenes of panic, are all derived from public fears and they concomitantly, via feedback, shape these fears. mass media is another major factor that shapes the physical and psychological response of the public to an infectious disease threat, as depicted in numerous attack scenarios in the literature [ ] [ ] [ ] . a simulation of a q fever outbreak in spain after deliberate release highlighted such potential: one journalist retrieved a medical report of person-to-person transmission of the disease; the public was already informed that such transmission is not possible; some journalists accused the scientists of hiding the truth; the public felt misinformed by the scientific community. and this was a scenario focusing on an agent of limited mortality [ ] . it would be unfair to judge the public as a homogenous group; the public is a coalition of numerous subgroups of individuals, with vastly different social, educational and economical backgrounds. one would expect these subgroups to face threats of infection in different manners. for example, a higher educational background should theoretically be related to lower levels of fear; on the other hand, it may be related to increased access to information in general and to medical advice, and thus to increased individual participation in the development of the perception of 'threat'. these differences in the perception of disease in general, and infection in particular, among individuals of different social, economical and educational status have not been adequately evaluated. a series of ethical dilemmas applies to the control of infectious diseases, and these dilemmas further serve to enhance the fear of infection. the typical ethical dilemma is the conflict between feelings and decisions [ ] ; in an outbreak, the patient is a victim, but also a vector, and isolation and quarantine practices may make stigmatization unavoidable. a recent statistical model has focused on the effect of individual psychological responses during the outbreak itself; fear induces a 'fight or flight' response, flight in this case predisposing to outbreak spread [ ] . control of a large-scale infectious disease outbreak may often demand the infringement of individual liberties and civil rights [ ] . these ethical dilemmas extend beyond the actual nature of the disease and its psychological consequences, and may implicate the means and content of public communications [ ] , from authorities and the media, during an outbreak (i.e. how much actual information can the public handle without going into panic, and where does the thin line between the right to know and panic lie in this case). these recently observed psychosocial responses are not unique. we not only have re-emerging diseases, but also re-emerging responses to disease. the equivalent of the famous plague doctor mask of the s in venice is the white surgical mask worn during recent epidemics. public health initiatives can address the public's fears by increasing education about a disease. enhanced health literacy, along with wide-ranging access to health information, can contribute to early case detection and may be useful in reducing stigma and decreasing levels of fear of an illness. the making of a germ panic, then and now emerging infections: a perpetual challenge cases of chikungunya fever imported from the islands of the south west indian ocean to transmission of infectious diseases during commercial air travel contact tracing of passengers exposed to an extensively drug-resistant tuberculosis case during an air flight from beirut to paris west nile virus the experience of sars-related stigma at amoy gardens fear and stigma: the epidemic within the sars outbreak risk perception and compliance with quarantine during the sars outbreak the psychosocial effects of being quarantined following exposure to sars: a qualitative study of toronto health care workers s chinatown: the politics of risk and blame during an epidemic of fear the coming plague: newly emerging diseases in a world out of balance stigma in the time of influenza: social and institutional responses to pandemic emergencies public health communication: evidence for behavior change fatal advice: how safe-sex education went wrong differences in public emotions, interest, sense of knowledge and compliance between the affected area and the nationwide general population during the first phase of a bird flu outbreak in israel suffocation from misuse of gas masks during the gulf war bse fears stir the swiss risk perception of the 'mad cow disease' in france: determinants and consequences fear of bse risks could hit us blood banks can it happen here? panic over mad cow had already infected europe. now it's our turn japan's first bse case fuels fears elsewhere reaction to the threat of influenza pandemic: the mass media and the public avian flu: the creation of expectations in the interplay between science and the media effects of episodic variations in web-based avian influenza education: influence of fear and humor on perception, comprehension, retention and behavior a crisis: fear toward a possible h n pandemic survey of hospital healthcare personnel response during a potential avian influenza pandemic: will they come to work? perceptions related to human avian influenza and their associations with anticipated psychological and behavioral responses at the onset of outbreak in the hong kong chinese general population health literacy in the field of infectious diseases: the paradigm of brucellosis an exploration of the drivers to bio-security collective action among a sample of uk cattle and sheep farmers infectious diseases in cinema: virus hunters and killer microbes smallpox: an attack scenario attack scenarios with rickettsial species: implications for response and management q fever in logrono: an attack scenario are there characteristics of infectious diseases that raise special ethical issues? coupled contagion dynamics of fear and disease: mathematical and computational explorations ethics and infectious disease guilt, fear, stigma and knowledge gaps: ethical issues in public health communication interventions the authors state that there were no sources of funding for the present study. the authors have no conflicts of interest to declare. key: cord- -e g u nz authors: melillo, alessandro title: rabbit clinical pathology date: - - journal: j exot pet med doi: . /j.jepm. . . sha: doc_id: cord_uid: e g u nz with rabbit patients, as in other species, analyzing blood and urine samples can be useful and informative, although interpretation of the results is sometimes challenging. this article summarizes the interpretation of laboratory results from rabbits. hematological parameters can yield information about the red blood cell population and leukocyte response to stress and pathogens. biochemistry evaluation can be used to investigate liver, kidney, and other organ function, and urinalysis results may yield additional information about kidney function and electrolyte imbalances. serological tests are available for several pathogens of rabbits, including encephalitozoon cuniculi, although the significance of positive results and antibody titers is not clear. serum protein electrophoresis aids the understanding of protein disorders and the immune response to acute and chronic inflammation. r abbits can mask signs of illness or show few or confusing clinical signs. additional information may be gained from laboratory tests, and in-house analyzers can provide a complete profile with a small volume of the patient's blood. unfortunately, most of the published data on rabbit hematology and biochemistry values are descriptions of the effects of toxins on hematological and biochemical parameters of laboratory rabbits. there is little information available that describes the effect of clinical disease on the blood parameters of companion rabbits, or on the use of blood tests as diagnostic and prognostic indicators. the lack of biochemical data for pet rabbits is changing as practitioners collect information and researchers are more cognizant of diagnostic and prognostic hematologic indicators. the blood volume of a healthy rabbit is approximately to ml/kg, and % to % of the blood volume may be safely collected. many sites are described for blood collection in rabbits. cardiac punc-ture is used in laboratory rabbits but is not recommended for pet animals. both the marginal ear vein and central ear artery are easily accessible, but they may be difficult to access in some patients. collecting a sufficient volume of blood from these sites to perform all of the desired clinical tests may also be difficult, especially from dwarf breeds with small ears. sampling from ear vessels can occasionally result in thrombosis and subsequent avascular necrosis of parts of affected pinna tissue. blood may be collected from the cephalic vein, which is straight and easily accessible, but, because of the short antebrachium, occlusion of the vessel by encircling the limb at the elbow is difficult. the cephalic vein is also small and easily collapses. the jugular veins are large and allow for ample-sized blood volumes to be collected, but jugular phlebotomy can be stressful for rabbits and may require chemical restraint. the dewlap may interfere with jugular vein visualization, especially in obese does. an accessible and efficient site for blood sampling in rabbits is the lateral saphenous vein (fig ) . most biochemical parameters of rabbits can be measured from serum or plasma. rabbit blood clots easily at room temperature and will coagulate quickly if not mixed with anticoagulant during collection. heparin is a suitable anticoagulant because it does not alter biochemical parameters, even though the anticoagulant/blood ratio is not always optimal. hemolysis can be prevented by letting the blood drop from the needle into the tube, but often, only a few drops can be collected before the blood clots. the technique can be improved by heparinizing syringes and needles through aspiration of a few drops of heparin into the syringe, then removing the excess with injection pressure. the small amount of heparin remaining in the needle prevents clotting without significant alterations in biochemical parameters. it is important to make several air-dried blood smears at the time of venipuncture before the anticoagulant in the tube and transport can modify red and white cell morphology. most of the standard blood stains work well. automated flow cytometry is reliable for analyzing most hematological parameters for rabbit patients. age, sex, breed, and circadian rhythms all affect hematological and biochemical parameters in rabbits. rabbits under weeks of age have lower red blood cell (rbc) and white blood cell (wbc) counts. the total wbc and lymphocyte counts are lowest in the late afternoon and evening, when the heterophils and eosinophil counts rise. urea and cholesterol levels tend to increase at the end of the day. stress can alter many different hematological parameters (e.g., blood glucose). prolonged stress, such as transportation, unfamiliar noises, smell, chronic pain, and poor environment, can induce heterophilia, lymphopenia, and leukocytosis. simple handling does not induce this response, but several muscle enzymes including lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase elevate after physical restraint of the rabbits, especially if they are fractious or unfamiliar with handling. sedation or general anesthesia can be helpful. isoflurane anesthesia does not appear to affect blood parameters in rabbits. hemolysis can induce several artifacts, such as decreased rbc and amylase values and increased ldh, ast, creatine kinase, total protein, and potassium levels. in-house analyzers can be very sensitive to hemolysis, thereby altering the true blood parameters of the patient. hematology results of rabbits can be difficult to interpret. most reference ranges are from experimental laboratory studies that are run on homogeneous groups of rabbits belonging to the same breed, strain, age, and environmental conditions. this can be very different from the clinician's situation of dealing with a heterogeneous population of pet rabbits. many texts amalgamate reference ranges from different sources to create ranges so wide that they include almost any result. another problem is that healthy pet rabbits are very hard to find, so samples from rabbits with an apparently acute condition may also show changes due to an underlying chronic problem, such as malnutrition, improper husbandry, or subclinical disease. for example, harcourt-brown and baker showed that rabbits that were caged, fed on commercial mixes, and suffered from dental disease had consistently lower packed cell volumes (pcv), rbc counts, hemoglobin values, and lymphocyte counts in comparison with rabbits kept outside with a more natural diet and exercise. rabbit erythrocytes are typical mammalian anucleate biconcave discs with an average diameter of . m, which is midway between cats and dogs. erythrocyte size varies between . and . m, which is often reported as a marked anisocytosis (fig ) . the short life span ( days) and high turnover of erythrocytes is reflected as polychromasia, which is not clinically significant. the presence of a few nucleated rbcs ( - ϫ leukocytes) and the occasional howell-jolly bodies (fig ) should be considered within the normal reference range for rabbits and not an indicator of cellular regeneration. the published reference range for pcv in the rabbit is % to %, but pet rabbits often have lower values of % to %. values higher than % may indicate dehydration, which is usually linked to gastrointestinal (gi) stasis. combined pcv and total protein (tp) is useful to differentiate acute conditions from subclinical chronic diseases that have suddenly deteriorated. a pcv of less than % indicates anemia, especially if the rbc and hemoglobin levels are low as well. nonregenerative anemia associated with chronic disease is common in pet rabbits. otitis media, dental disease with or without abscesses, pneumonia, pododermatitis, mastitis, endometritis and pyometra, renal disease, and osteomyelitis are all examples of chronic infections that can be associated with nonregenerative anemia in pet rabbits. regenerative anemia is manifested by significant and rapid reticulocyte production and usually indicates blood loss. causes of external hemorrhage in rabbits include trauma and severe flea infestation. common internal causes include hematuria due to kidney or bladder stones or bleeding uterine adenocarcinomas or endometrial aneurysms in does. intravascular hemolysis is an unusual cause of regenerative anemia after ingestion of leaves and stems of potato plants and possibly other solanaceae. alliums (onion, garlic, and chives) may also cause heinz body anemia. autoimmune hemolytic anemia has been reported in laboratory rabbits in association with lymphosarcoma, and isolated cases have been treated in private practice (harcourt-brown, personal communication, november, ). lead toxicosis is a cause of regenerative anemia, characterized by many nucleated erythrocytes, hypochromasia, poikilocytosis, and basophilic cytoplasmatic stippling. nucleated red cells of more than % to % of the rbc can be linked with the acute, septicemic phase of an infectious disease, although this is unusual because of the presence of a nonregenerative anemia from underlying chronic disease. a well-prepared air-dried smear is required for an accurate differential white cell count and evaluation of the cytological appearance of each cell type (figs - ). differential white cell counts and cell morphology can be used to develop a differential diagnoses list and to determine the general condition of the patient. interpretation of wbc data from rabbits is different from other domestic species including dogs, cats, and birds, in which a leukocytosis is the response to inflammation. with rabbits, although leukocytosis can be identified in cases that have been diagnosed with lymphosarcoma, it is not the usual response to inflammation. laboratory investigations have shown no increase in the total number of circulating leukocytes in rabbits injected with bacteria or yeast, in domestic carnivores, anisocytosis usually reflects the presence of reticulocytes and indicates regenerative anemia. this is not true in rabbits in which % to % of the circulating erythrocytes may be reticulocytes. the occasional howell-jolly body is not clinically significant either. rabbit clinical pathology although fever, increased plasma cortisol concentrations, neutrophilia, and lymphopenia were observed. , in clinical practice, rabbits with sepsis can show a variety of responses including a neutropenia, normal neutrophilic count, or a mature neutrophilia, accounting for more than % of wbcs. band neutrophils appear to be a rare finding in clinical infection, and the absence of a left shift does not rule out an infectious problem. an alteration of the neutrophil/lymphocyte ratio showing a relative neutrophilia coupled with lymphopenia may indicate a response to infection. this ratio is approximately : in an adult healthy rabbit, but stress can alter the neutrophil/lymphocyte ratio. transport, waiting in a room full of unfamiliar sounds and smells, or even restraint for clinical examination can change the ratio. gentle clinical examination and blood collection do not seem to affect the differential white cell count, whereas more prolonged stress like travel or exposure to barking dogs in the waiting room can induce a lymphopenia and relative neutrophilia, that may persist for up to to hours. the total wbc can be used to further characterize acute stress from chronic stress (e.g., malnutrition, improper husbandry, prolonged social stress, dental disease), as both a leukopenia and lymphopenia are eosinophil. rabbit eosinophils measure to m in diameter and have a purple bilobed or horseshoe-shaped nucleus. the cytoplasm is obscured by so many granules that the cell looks orange-pink and foamy. the abundance of granules is the main difference between the eosinophils and the neutrophils. removal of histamine and histaminelike toxins is the most important function of the eosinophils, suggesting that they therefore play an important role in controlling allergic reactions. more common with chronic stress. chronic stress is often reported as general leukopenia and lymphopenia in a rabbit's differential wbc evaluation. the primary role of the lymphocytes is to respond to those activities that stimulate the immune system. in rabbits, lymphocytes are primarily found in the blood, spleen, bone marrow, lymph nodes and the lymphatic tissues in the gi tract. the number of circulating lymphocytes is a balance between the cells entering and leaving the bloodstream and does not necessarily reflect a change in lymphopoiesis. in rabbits, it has been shown that an increase in adrenaline levels (acute stress) induces lymphocytosis, whereas raised cortisol levels (chronic stress) leads to lymphopenia. viral diseases may result in a normal or higher lymphocyte count. other causes of lymphocytosis are lymphoma and lead poisoning. eosinophilia in rabbits can occur when tissues rich in mast cells, such as the skin, lungs, gi tract, or uterus, are involved in disease. eosinophilia can indicate the presence of an abscess and may be found during wound healing. in other species, eosinophilia is linked to parasitic diseases, especially when larvae are moving through tissues, but this is rare in domestic rabbits. encephalitozoon cuniculi does not stimulate an eosinophilic response. in clinically healthy rabbits, a very low eosinophilic count, even , is a common finding. high levels of cortisol (chronic stress) can induce eosinopenia. monocytosis is linked with chronic inflammation (e.g., abscesses, mastitis, tympanic bulla empyema). however, the absence of a monocytosis does not rule out inflammation. monocyte counts within the normal range are a common finding in rabbits with osteomyelitis due to dental disease. the diameter of rabbit basophil measures to m. its nucleus is less segmented than the eosinophil or heterophil and difficult to see because of the many deep purple granules that obscure the light gray cytoplasm. as in other species, the function of the rabbit basophil is not fully understood, but these cells are often present in large numbers of rabbit blood smears. basophilia with concurrent eosinophilia has been described in rabbits with chronic skin problems (e.g., atopy, pyoderma). in rabbits, ast is widely distributed in many tissues. it is present in cardiac tissue and muscle, as well as the liver, and has a short half-life ( hours). although higher ast levels may be found in patients diagnosed with liver damage, struggling during col- lection or hemolysis of the sample also raises ast levels. creatine phosphokinase levels also increase after restraint and are purely muscular in origin. ldh is produced by muscle and liver cells in rabbits and therefore is not beneficial as a diagnostic tool for many disease evaluations. reference levels for ast, ck, and ldh can be found in table . in many mammalian species, alanine aminotransferase (alt) is a useful indicator of hepatocyte damage because of its specificity for liver tissue and its long half-life ( - hours in dogs). in rabbits, alt is not as useful as an indicator of liver damage as with other species because, like other herbivores (e.g., horses, cattle, guinea pigs), alt is not liver specific and has a shorter half-life (around hours). however, alt concentrations are not affected by restraint and therefore can be used as a diagnostic tool. slightly increased alt levels are a common finding in apparently healthy rabbits. mildly increased alt levels in healthy rabbits have been attributed to exposure to low concentration of toxic substances, such as resins in wood-based litter or aflatoxins in food. raised alt levels (with alkaline phosphatase (alp), bilirubin and glutamyltransferase [ggt]) can be associated with hepatic lipidosis or may be found in patients with hepatic coccidiosis (eimeria steidae) or torsion of a liver lobe. alp is a widely distributed enzyme; liver and bone contain the highest concentrations, but it is also found in bowel epithelium, kidney tubules, and placenta. a physiological cause of high-serum alp concentrations is osteoblastic activity in growing animals. animals with bone lesions will show raised alp levels. as a liver enzyme, alp does not increase because of hepatocellular damage but is indicative of bile stasis (e.g., hepatic coccidiosis, liver abscesses, neoplasia, lipidosis). extrahepatic causes, such as abscesses or neoplasia, can cause bile stasis by occluding the bile ducts. rabbits produce alp isoenzymes in the liver. an intestinal isoenzyme is quite abundant, so serum alp concentrations are actually the sum of these isoenzymes, which may explain why many reference ranges are vague and wide and why raised alp levels in clinically healthy animals are a common finding. alp does have a diagnostic value because it is not altered by restraint and thus is considered a good indicator of real tissue damage. reference levels for alt and alp can be found in table . ggt is a useful indicator of chronic liver disease with bile stasis in horses, cattle, and domestic carnivores; however, in rabbits, the activity of ggt is low. activity of this enzyme is high in the kidney, yet renal ggt does not reach the circulation because it is eliminated with the urine. therefore, elevated ggt levels in the rabbit are most often linked to obstructive lesions of the bile ducts, but with a lower sensitivity than that found in other species. reference levels for ggt can be found in table . bile pigments are produced during the breakdown of the heme molecule by hepatocytes and are excreted into the bowel. bilirubin levels reflect either hepatocyte or bile tract function. rabbits produce a large amount of bile for their weight, and the main compound is biliverdin, for which there is no commercial laboratory test. about % of biliverdin is converted to bilirubin, which is found in the blood in measurable amounts. the main cause of hyperbilirubinemia is bile flow obstruction. in young rabbits, hepatic coccidiosis is the most common cause of biliary obstruction, while in adult rabbits it is biliary neoplasia. cellular causes of hyperbilirubinemia, and rarely icterus, are aflatoxicosis (e.g., eating moldy food), which induces hepatic fibrosis (alt is usually raised too), and viral hemorrhagic disease, which causes acute hepatic necrosis with concurrent high levels of all hepatocellular enzymes. if the rabbit survives long enough, icterus may be seen. bilirubin may also be increased in diseases that cause hemolysis (e.g., immune-mediated hemolytic anemia). in other species, a comparison of preprandial and postprandial serum bile acid concentrations is used as an indicator of liver function. in rabbits, cecotrophy makes it almost impossible to fast a rabbit for the preprandial sample, so bile acid measurement is not a routine procedure in clinical practice, although persistently raised bile acids have been reported in association with hepatic disease. cholesterol is synthesized in the liver or obtained from the diet and is a precursor of steroids. it is metabolized by the liver and excreted in bile. in carnivores, hypercholesterolemia is linked with several metabolic diseases, such as hypothyroidism, hyperadrenocorticism, diabetes, and hepatopathies. hypocholesterolemia indicates liver failure. cholesterol and triglycerides levels peak after a meal and fasting is needed for accurate measurement, which limits their diagnostic value in rabbits because of cecotrophy. abnormal levels of cholesterol and tri-glycerides can be related to a diet rich in fats, obese patients, or hepatic disease. in anorexic patients, hypercholesterolemia carries a poor prognosis because it indicates end-stage hepatic lipidosis. hypercholesterolemia has also been linked with pancreatitis, diabetes mellitus, nephrotic syndrome, and chronic renal failure. decreased cholesterol levels in rabbits might be found in cases of liver failure, chronic malnutrition, and even pregnancy (up to % below the range). unlike other species, amylase is an almost pure pancreatic enzyme in rabbits, with little or no content in salivary glands, intestinal tissue, or liver. therefore, raised amylase levels in rabbits reflects pancreatic damage from pancreatitis, pancreatic duct obstruction, peritonitis, or abdominal trauma. renal failure can also cause hyperamylasemia because this enzyme is cleared by renal filtration. corticosteroids (exogenous or endogenous) can raise amylase values in serum, whereas hemolysis lowers it. there is little information on the function and diagnostic value of lipase in rabbits. increased lipase values may indicate cellular damage to the pancreas as it does in other species. as with amylase, lipase is artifactually elevated by corticosteroids. urea is a by-product of protein catabolism and is excreted by the kidneys into the urine. urea levels in rabbits depend on the circadian rhythm (peak in late afternoon and early evening), quantity and quality of proteins in the diet, nutritional status, liver function, intestinal absorption, urease activity of the caecal flora, and hydration status. often, small changes in urea levels are difficult to interpret. reference ranges have been determined from laboratory rabbits fed on a standardized diet and bled at the same time of the day, whereas clinicians see pet rabbits fed on a variety of foods and samples are taken at random. slight elevations in blood urea are a common finding. reference levels for urea can be found in table . creatinine is a protein catabolite that is produced from the muscle creatine and excreted by glomerular filtration at a constant rate. creatinine is a more reliable test of renal function than blood urea. in rabbits, prerenal azotemia can be caused by dehydration because rabbits have a limited ability to concentrate urine. only a few hours without drinking or losing fluids, as in cases of ileus or diarrhea, may cause an increase of urea and creatinine to levels compatible with renal failure. urea and creatinine levels rapidly return to normal once the dehydration deficit is corrected. stress can also induce shock and cardiac disease, classified as prerenal disease, causing a decrease in renal perfusion. another potential cause of prerenal azotemia is gi hemorrhage, which results in increased protein digestion. azotemia is also indicative of renal disease, usually affecting the rabbit patient in association with hyperkalemia or hypokalemia, hypercalcemia and coexisting hyperphosphatemia, nonregenerative anemia, and isostenuric urine. the most common cause of renal failure in rabbit patients is e. cuniculi, which causes granulomatous and then fibrotic lesions in the renal parenchyma. other possible causes of renal failure are chronic interstitial nephritis, glomerulonephritis, pyelonephritis, nephrolithiasis, renal cysts, and lymphosarcoma. postrenal azotemia can occur because of obstruction to urine flow as a complication of bladder sludge or urolithiasis. an abdominal radiograph is mandatory in any azotemic rabbit. blood urea levels below the reference range indicate hepatic insufficiency or muscle mass loss (e.g., dental disease). reference levels for creatinine can be found in table . glucose metabolism in rabbits is different from dogs or cats. not only do rabbits eat continuously during the day, but they also use volatile fatty acids produced by cecal flora as a primary energy source. a fasting blood sample is impossible to obtain because rabbits ingest fecal pellets. a rabbit that is not given food can continue to ingest cecotrophs. it has been shown that days of starvation does not reduce blood glucose levels in rabbits. diabetes mellitus is rare in rabbits, although hyperglycemia is a common finding and may be associated with glucosuria. reports of confirmed diabetes mellitus are from laboratory strains bred as a model for human diabetes. clinical signs commonly observed in pet rabbits with diabetes mellitus are polyphagia, polyuria, polydipsia, very high blood glucose levels (Ͼ mg/dl), and glycosuria with significantly elevated glycosylated hemoglobin and raised triglycerides; however, obesity and ketoacidosis are not observed. in clinical practice, most cases of hyperglycemia are due to stress (e.g., transport, handling, venipuncture, underlying disease). a marked hyperglycemia (around mg/dl) is reported in cases of acute intestinal blockage by a foreign body. early mucoid enteropathy may be associated with hyperglycemia. in rabbits with gi stasis, hyperglycemia carries a bad prognosis because it may indicate hepatic lipidosis. other causes of raised serum glucose levels are traumatic or hypovolemic shock and hyperthermia. acute pancreatitis could cause blood glucose abnormalities, even though the role of the pancreas in glucose metabolism in rabbits is less important than in other species. glucocorticoids and other drugs can raise blood glucose. hyperadrenocorticism has not been described in rabbits. hypoglycemia is an important finding. in anorexic patients, it indicates that the rabbit is using adipose tissue and is at risk of developing hepatic lipidosis. hypoglycemia may occur in terminal mucoid enteropathy, liver failure, or other chronic diseases. rabbits with acute sepsis may be hypoglycemic too. insulinoma has not been described in rabbits. reference levels for glucose can be found in table . the complex gi physiology and the limited ability of the kidneys to correct acid-base alterations make rabbits susceptible to electrolyte imbalances. any disturbance of serum electrolytes should alarm the clinician to possible pathology of the digestive or excretory system. anorexia can rapidly lead to metabolic acidosis. the diagnostic value of sodium for rabbit patients is low. hypernatremia can be due to dehydration or loss of fluids (e.g., diarrhea, peritonitis, burns, myiasis). hyponatremia is usually associated with polyuric renal failure (acute or more commonly chronic), when urine flow in the renal tubules is too fast to impede the sodium-potassium exchange. lipemia or hyperproteinemia can artifactually decrease sodium levels in serum. reference levels for sodium can be found in table . potassium is homeostatically important because it is essential for maintenance of membrane potential. changes in membrane potential can be lethal (e.g., impaired contractility of myocardial cells due to hyperkalemia can cause arrhythmias and cardiac arrest). intracellular and extracellular potassium levels are maintained by a complex mechanism of exchange between cells and microenvironment, and regulated by several hormones such as aldosterone, insulin, and catecholamines. hypoadrenocorticism has not been described in rabbits. instead, raised potassium levels are more often due to acute renal failure or urine flow obstruction. severe tissue damage can also cause hyperkalemia by dispersing potassium into the extracellular space. for the same reason, hemolysis (e.g., intravascular, bad sampling technique, letting the sample wait too long before separating the serum) can artifactually raise potassium levels. another indirect cause of higher potassium levels in serum is metabolic acidosis, which increases the exchange of potassium ions across the cell membrane. reference levels for potassium can be found in table . causes of hypokalemia in rabbits include dietary insufficiency and loss of fluids from the gi system (e.g., saliva, mucoid diarrhea) or the kidneys (e.g., renal failure, diuretic drugs). stress-induced increases in catecolamine levels can also cause hypokalemia. alkalosis, although rare in rabbits, decreases the blood potassium concentrations by stimulating the cellular uptake of potassium ions. hyperproteinemia and lipemia can artifactually decrease blood potassium levels that may present clinically as sensory depression and muscle weakness. a correlation between low potassium levels in blood and "floppy rabbit syndrome" has been reported. in other herbivorous species (e.g., horses), blood potassium levels fluctuate widely, depending on physical activity or even on the quantity of saliva produced during the meals. these examples of serum potassium level fluctuation are purely physiological and may occur in rabbits. calcium is found in blood either bound to serum proteins or ionized free in the serum. most laboratories report a total serum calcium value, which is the sum of bound and ionized calcium. ionized calcium is a more precise measurement but is a more difficult and expensive parameter to test. total serum calcium is influenced by dietary intake, serum protein levels, and other metabolic conditions. calcium metabolism in rabbits is different from that in other animals. blood calcium levels are influenced more by the calcium content of the diet than in the dog or the cat. rabbits absorb calcium in proportion to the concentration of the ion in the gut, and the kidney eliminates the excess. vitamin d is not important in calcium absorption if dietary levels are high, yet it does play an important role if dietary levels are low. vitamin d is also important in calcium distribution within the body. as with other species, parathyroid hormone regulates blood calcium levels, but the level at which calcium is moved from the blood to the bones is high in rabbits. consequently, blood calcium levels are higher, and the normal range is broader than that in other species. growing youngsters and pregnant does use more calcium, resulting in lower blood calcium concentrations. in these rabbits blood calcium concentrations rarely rise above mg/dl, even when fed calcium-rich diets, whereas adult rabbits on a varied diet can show calcium levels up to to mg/dl ( table ) . the urinary excretion rate of calcium is around % to % for rabbits, whereas most mammals excrete no more than % of their calcium through renal filtration. this predisposes rabbits to the formation of sludge and stones in the rabbit urinary system. although the constant excretion of calcium could be a cause of renal failure in rabbits fed unbalanced diets, hypercalcemia is also a consequence of renal disease in rabbits because of the inability of the kidney to eliminate excess calcium. measurement of blood calcium is essential to diagnose and treat renal disease in rabbits. hypocalcemia is rare but is reported in rabbits. the most frequent cause of low blood calcium levels is hypoalbuminemia due to poor nutrition. hypocalcemic seizures have been described in late-pregnant and lactating does. phosphorus is involved in many enzymatic systems in rabbits, but its main function is contributing to the proper formation of bones and teeth. because phosphorus is present inside cells, blood phosphate concentrations are easily increased by hemolysis (e.g., spontaneous or sampling problems). blood phosphate values should always be evaluated with blood calcium levels to determine the mineral balance in patients diagnosed with urinary tract stones, dental disease, or other signs of nutritional secondary hyperparathyroidism. because the kidney is the main organ involved in phosphorus balance by regulation of glomerular filtration and tubular reabsorption, blood phosphate levels can be an indirect measurement of kidney function and, in general, parallel azotemia. serum phosphorus levels can be elevated as a result of prerenal, renal, and postrenal effects. hyperphosphatemia usually indicates chronic kidney failure (a loss of more than % of nephrons) given that serum phosphorus levels are normalized by compensatory mechanisms in early-onset renal disease. hyperphosphatemia may also be an indicator of soft tissue trauma. reference levels for phosphorus can be found in table . hypophosphatemia is not rare, but its clinical significance is unknown at this time, with dietary deficiencies or reduced intestinal absorption possibly involved. total protein, consisting of the sum of albumin and globulin, is an important parameter in any species of animal. many factors (e.g., age of the animal, reproductive status, pregnancy) can affect tp levels. total serum protein levels can be artifactually raised by hemostasis at the sample site through fluid loss because of digital pressure on the vessel from which the blood is collected. reference levels for tp, albumin and globulins can be found in table . the main cause of hyperproteinemia in rabbits is dehydration. raised tp levels may also indicate a chronic infectious or metabolic process. measuring albumin and globulin fractions helps to differentiate the causes of hyperproteinemia. hypoproteinemia is usually due to chronic malnutrition or protein loss. if both albumin and globulin are low, hemorrhage or protein loss through exudative skin lesions such as burns or flystrike should be considered. other possible causes must be examined in cases of hypoalbuminemia with normal or raised globulins. because the liver is the only site of albumin synthesis, a lowered albumin level may indicate an advanced hepatic disease, such as hepatic coccidiosis (e stiedae) or scarring and necrosis due to the migrations of cysticercus (taenia) pisiformis larvae. protein-losing nephropathies (glomerulonephropathy) and enteropathies are rarely diagnosed in rabbits. a common cause of hypoalbuminemia in pet rabbits is chronic malnutrition either from poor diet or advanced dental disease. all causes of reduced cecotrophy (e.g., dental disease, obesity, back pain) can be reflected as low protein, especially albumin levels. a method of evaluating serum proteins is by electrophoresis (eph), which divides the globulins into distinct fractions. in acute disease, alphaglobulins are elevated; consequently, a rabbit with an alpha-globulin peak may have a bacterial infection or a developing abscess, and/or present febrile. the beta portion of globulins consists of several proteins classified as "acute-phase" proteins including fibrinogen. fibrinogen correlates with inflammation in rabbits, although the correlation is not as evident as in other species. plasma is preferable to serum for eph because it does include fibrinogen. gamma-globulins are mainly antibodies, and a peak of this fraction indicates a subacute to chronic inflammation, especially when associated with a bacterial infection. coronavirus infections lead to an impressive increase of rabbit globulins as feline coronavirus does in cats, but this disease is probably limited to laboratory set-tings. the correlation between eph curves and different rabbit pathologies is unknown and currently under investigation. there are serological tests for antibodies against e cuniculi, toxoplasma gondii, treponema cuniculi, myxomatosis, viral hemorragic disease, and pasteurella multocida, although their availability varies in different parts of the world. the most common serological assay used in private practice is for the antibodies to e cuniculi. laboratory studies have shown that infected rabbits start to develop a measurable serological immune response weeks after infection, weeks before e cuniculi is found in the kidney or in the urine, and at least weeks before any brain lesion. , if a serologic test for e cuniculi is negative for a neurological rabbit patient, one could assume, with confidence, that this patient does not have the disease. if the test is positive, neurological signs may be due to e cuniculi or a different disease process. the seropositive result may be due to past exposure to the pathogen, which is common within most rabbit populations. further laboratory tests may be helpful to determine a definitive diagnosis. a correlation between antibody titers and neurological signs is hard to prove. a rising titer after weeks is often considered diagnostic in suspect cases. the kidney is a target organ for e cuniculi, so evaluation of renal function and structure by biochemistry, urinalysis, and ultrasound may aid one's diagnostic overview. inflammatory changes in the cerebrospinal fluid are suggestive of e cuniculi but are nonspecific. at the present time, the definitive diagnosis of rabbit encephalitozoonosis requires histopathology or isolation of the spores from the urine by microscopy or polymerase chain reaction assay. although serology for t cuniculi can be used to screen a breeding facility, it has little value in clinical practice. at least months are needed for antibody levels to become measurable in the blood even with clinically evident dermatological lesions. this substantial lag in the development of antibody titers can lead to false-negative test results. positive titers without skin lesions can indicate subclinical disease or previous infection in which the skin lesions are missed. antibody titers fall rapidly once the rabbit is treated for t cunicoli. serological testing for p multocida is available in some countries, but its use in clinical practice is limited. a single positive response has no clinical significance because many rabbits harbor the bacteria in the nasal cavities without illness. a positive titer can also indicate previous exposure to the bacteria rather than clinical infection. repeat testing after weeks, while looking for a rising titer, can be helpful in obtaining a correct diagnostic evaluation. results from rabbits younger than months can be difficult to interpret because maternal antibodies are still present. a high antibody titer is not protective and may indicate subclinical disease. urine samples can be collected from spontaneous micturition, bladder expression, catheterization (quite difficult because sedation/anesthesia is required), or cystocentesis, with the latter being the preferred method for obtaining a sample for bacterial culture. care should be taken when performing a cystocentsis, because microtrauma to the bladder wall can stimulate local mineralization and it is possible to puncture the cecum, an enlarged uterus, or even an abscess. if bacteriology is not required, free catch from a clean litter tray is the easiest way to obtain a urine sample. manual expression of the bladder should be done carefully because the wall is thin and ruptures easily, especially if an obstruction is present or the rabbit struggles. in cases of chronic cystitis, the risk for bladder rupture is lower because the bladder is thickened. it is preferable to collect the first urine of the morning and to run the analysis as soon as possible. normal rabbit urine is dense and rich in minerals, so it should be centrifuged or filtered for biochemical analysis or examinations. clear urine indicates low calcium excretion, which may be pathological because of renal failure or physiological in the case of growing or lactating rabbits. the color may range from light yellow to reddish brown. in most cases, dark urine is caused by dietary pigments; however, the urine should be checked for hematuria, which may be caused by uroliths, urinary tract inflammation/infection, uterine problems, or anticoagulants. test dipsticks work well to evaluate blood, glucose, ketone, and ph levels in rabbit urine but are not accurate for other parameters. glucose may be found as a consequence of stress hyperglycemia; checking urine collected at home can help to rule out stress-related problems. true glucosuria indicates altered energy metabolism, such as hepatic lipidosis, or, very rarely, diabetes mellitus. ketones are always abnormal and indicate anorexia (even short duration), hepatic lipidosis, pregnancy toxemia, or diabetes. the ph of rabbit urine tends to be high ( . to ) in rabbits fed a correct diet. acidic urine indicates acidosis due to anorexia, fever, pregnancy toxemia, or hepatic lipidosis. it is possible that, because a normal kidney would eliminate acidic urine in cases of acidosis, the finding of alkaline urine in an anorexic rabbit could indicate compromised renal function. specific gravity (sg) indicates the ability to concentrate urine. a refractometer is a more reliable tool to measure sg than dipsticks. most normal rabbit urine is quite dilute, with an average sg of . (range, . - . ). prerenal azotemia is associated with raised sg (Ͼ . ), whereas true azotemia linked to renal failure is associated with dilute urine (sg Ͻ . ). urine-specific gravity is useful if it is examined alongside urine protein concentrations. protein traces in clinically normal rabbits, especially the young, are not significant, whereas a dilute urine (Ͻ . ) with proteins is very significant. proteinuria appears earlier than biochemical changes in renal disease, making this test useful in clinical practice. measuring urine proteins/urine creatinine ratio (Ͻ . is suggested as normal) may further improve the sensitivity of urine-specific gravity testing. sediment examination can differentiate normal urine that is rich in crystals from sludge. after centrifugation, normal crystals should resuspend when shaken, while sludge remains as a solid mass. cytology is not very different from that of other mammals; a small number of leukocytes are considered normal in rabbits. gram or trichrome stains can reveal e cuniculi spores. parathyroid hormone, hematological and biochemical parameters in relation to dental disease and husbandry in pet rabbits laboratory medicine: avian and exotic pets notes on rabbit internal medicine the biology of laboratory rabbit alteration of sleep in rabbits by staphylococcus aureus infection hematological effects of exposure to three infective agents in rabbits physiological stabilization of rabbits after shipping rabbit surgical pathology effects of t- toxin on ovarian activity and some metabolic variables of rabbits textbook of rabbit medicine the anatomy, physiology and the biochemistry of the rabbit spontaneous diabetes mellitus in the new zealand white rabbit veterinary laboratory medicine serological and histological studies on adult rabbits with recent naturally acquired encephalitozoonosis ferrets, rabbits and rodents clinical medicine and surgery key: cord- - v r jij authors: recht, judith; schuenemann, verena j.; sánchez-villagra, marcelo r. title: host diversity and origin of zoonoses: the ancient and the new date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: v r jij simple summary: there is a wide variety of diseases caused by bacteria, viruses, and parasites that are transmitted to humans by different routes from other animals. these diseases, known as zoonoses, represent % of new or reemerging infectious diseases. there is a considerable impact of these diseases on the economy and health at local and global levels, including zoonotic diseases caused by the ingestion of food and products derived from animals. the wide range of animal species that host these disease-causing organisms include all groups of mammals. birds are the second significant animal group to act as hosts for zoonoses. much progress has been made in understanding disease evolution and animal origin, with important contributions from fields such as paleopathology and analysis of dna, applied to ancient human bone remains. the study of ancient diseases such as brucellosis and tuberculosis benefits from these approaches. more research is needed as new diseases emerge causing pandemics and some previously eradicated reemerge in some regions. global efforts are focused, based on evidence generated by research, on the prevention of new pandemics. abstract: bacterial, viral, and parasitic zoonotic diseases are transmitted to humans from a wide variety of animal species that act as reservoir hosts for the causative organisms. zoonoses contribute an estimated % of new or reemerging infectious diseases in humans. all groups of mammals have been shown to act as hosts for transmission of different organisms that cause zoonoses, followed in importance by birds; with both wild and domestic species identified as hosts in specific cases. there has been considerable research progress leading to a better understanding of the host range, animal origin, evolution, and transmission of important zoonoses, including those caused by the ingestion of food and products derived from animals. paleopathology studies of ancient human bone lesions, in combination with ancient dna analysis of the causative pathogen, have contributed to our understanding of the origin of zoonotic diseases, including brucellosis and mycobacterial zoonoses. however, there are still knowledge gaps and new confirmed and potential hosts are reported locally with some frequency. both the economic cost and burden of disease of zoonoses are substantial at local and global levels, as reflected by recent coronavirus pandemics that spread rapidly around the world. evidence-based prevention strategies are currently a global priority increasingly recognized, especially in zoonoses-affected regions. zoonoses, or diseases transmitted to humans from vertebrate animals in both rural and urban settings where humans live, are widespread, with an estimated % of all human infectious diseases antimicrobial-resistant isolates. as the leading cause of zoonotic disease in both animals and humans, salmonella was placed in an antimicrobial resistance (amr) "serious threats" category of the usa cdc in [ ] . some strains of salmonella causing infections have developed resistance to antibiotics such as ciprofloxacin, azithromycin, and ceftriaxone, often used to treat patients with severe infections [ ] . in the us, campylobacter causes an estimated . million campylobacteriosis infections annually, of which % have decreased susceptibility to antibiotics used to treat these infections, including ciprofloxacin and azithromycin, limiting treatment options especially in low-and middle-income countries [ ] . in , a systematic review and meta-analysis of studies on interventions to reduce antibiotic use in food-producing animals compared the presence of antibiotic-resistant bacteria in animals and humans [ ] . based on this review, the world health organization (who) launched new guidelines on the use of antimicrobials in these animals, recommending to stop using antibiotics routinely to promote growth and prevent disease [ ] . here we present a review on zoonoses host diversity and animal origin of selected zoonoses, with an emphasis on brucellosis and mycobacterial zoonoses. we discuss examples of paleopathology and ancient dna studies contributing to our knowledge on disease origin and evolution, followed by viral zoonoses including recent pandemics as examples of recent and emerging zoonoses. the range of animal hosts of pathogens linked to zoonotic diseases is in most cases impressively diverse. some zoonotic disease outbreak studies have revealed new (sometimes unexpected) animal sources of human infection. hosts may include both wild and domesticated animals, some leading to foodborne zoonoses. monitoring animals for the presence of important pathogens is the focus of public health strategies. aside from pathogens shared by humans with invertebrates (vectors or intermediate hosts for disease transmission), the majority (about %) of the reservoirs known for zoonotic diseases are mammalian, followed by avian hosts [ ] . among other mammalian major taxa ("orders"), humans share the most pathogens with artiodactyls (frequently in proximity), followed by rodents, carnivorans, and primates. taxonomic identification of source groups for the emergence of zoonotic diseases could help to improve targeting of surveillance and interventions leading to prompt containment and even prevent zoonotic pandemics. however, given the taxonomic breadth reported for the many diseases, pinpointing specific groups and concentrating on them may not be justified. the information on hosts collected in tables s -s reflects how imprecisely and often very generally this is reported, and how vague our knowledge is in most cases. for example, often reports are of "rodents"-this group alone represents more than one-third of recognized mammalian species, with over of them [ ] . bats are the natural reservoirs of a significant number of important viral zoonoses, including probably the recent covid- that caused a high impact global pandemic this year ( ), still ongoing and discussed below. it has been hypothesized that bats are unique hosts in this regard, in particular as compared with rodents, the other is a particularly speciose major group (order) of placental mammals besides bats. however, the total number of zoonotic viruses identified in bats ( ) was lower than in rodents ( ) . there is a higher number of rodents than bat species ( -fold) [ , ] . a recent report on the largest dataset of the zoonotic virus-reservoir relationships built with avian and mammalian reservoir hosts of viruses aimed at determining whether some animal species are special reservoirs of zoonotic pathogens [ ] . the results did not support this view but a host-neutral variation instead; the analysis showed that the proportion of viruses infecting humans showed minimal variability across reservoir taxonomic orders and were in agreement with the number of animal species within each group (including for bats and rodents), with rare reservoir host effects restricted to one or two viral families. the camel has been highlighted in a recent review that showed zoonoses reported in this animal in iran (where raising camels is common with close contact between farmers and camels well as meat and milk consumption) including plague, q fever, campylobacteriosis, tuberculosis, salmonellosis, rabies, mers, and toxoplasmosis ( [ ] , section ). a plague outbreak in southern afghanistan in manifested as cases of acute gastroenteritis with deaths; the outbreak was linked to the consumption or handling of camel meat [ ] . birds are the main reservoir of the influenza a virus (table s ) . pigs, susceptible to infection with both avian and mammalian influenza viruses, may be acting in interspecies viral transmission as "mixing vessels", in which avian and mammalian influenza viruses recombine through a process known as "reassortment" to produce novel strains that can then infect humans [ ] . a role for wild migratory birds in the transmission of zoonoses has been acknowledged either as a reservoir host or by dispersing infected arthropod vectors. for example, west nile virus was likely introduced in the usa via infected birds, expanding in - along the atlantic seaboard, a common bird migration route, to reach southern florida to establish an enzootic cycle with mosquitoes and then from to , expanded westward possibly as a result of elliptical avian migration routes (reviewed in [ ] ). migrating birds can also carry one or more ectoparasites such as mites, ticks, fleas, and lice, all arthropods that themselves can carry pathogens; a potential role for migrating birds in dispersing ticks and associated pathogens that cause zoonotic disease has been recently highlighted [ ] . gut microbes and obligate ectoparasites such as ticks, fleas, and lice, depending on their animal hosts for transport. in this context, it is remarkable that recent geological events may have had a large impact on the dispersal capacity of both ectoparasites and gut pathogens. the megafauna (animals over . kg ( lb) body weight) decline in the late pleistocene/early holocene, which led to a decrease in seeds and nutrients dispersal, may have also caused a reduction in the movement of ectoparasites and generalist fecal microbes, including escherichia coli, to~ % of pre-extinction levels based on reductions after extinction estimated using the average home range for modern and extinct species [ ] . in this study, the distance that gut pathogens can travel between consumption and defecation, which is size-dependent, showed the largest declines in the americas and eurasia, however, whether pathogens disappeared with the previous hosts or adapted to new hosts is not known. this subject could be explored by metagenomic analyses of pathogens present in extinct mammal dung [ ] . chagas disease is an example of a successful parasitic zoonosis with a wide range (hundreds) of mammal species hosts in south america, transmitted by dozens of triatomine bug species (table ) . known as american trypanosomiasis, it also owes its name to carlos chagas, a brazilian researcher who first described it in brazil in as a disease due to the parasite trypanosoma cruzi (named after oswaldo cruz, another brazilian scientist), later shown to parasitize species of mammals from seven "orders" and triatomines from genera [ ] . a comprehensive study of reservoirs and wild hosts of t. cruzi. in brazil showed that species of several placental "orders" (artiodactyla, chiroptera, primates, carnivora, rodentia, cingulata, pilosa) and one marsupial (didelphimorphia) were involved in transmission, with four of them (primates, didelphimorphia, chiroptera, and the carnivora species nasua nasua) considered as key reservoir taxa exhibiting higher rates of parasitemia [ ] . in latin america, about million people are estimated to be infected with t. cruzi through feces or night bites of the "kissing bug" (triatomines), or through blood transfusion, maternally, or orally through contaminated food that can lead to an acute phase usually without symptoms or very mild [ ] . after - years following the acute phase, however, symptomatic chronic disease can occur, leading to irreversible damage to several organs such as the heart, esophagus, and colon [ ] . cattle, bison, african buffalo, cervids, brushtail possums, badgers, kudu can be reservoirs ingestion (unpasteurized dairy products, undercooked meat including bushmeat), inhalation, contamination of breaks in the skin * information sources: [ , , [ ] [ ] [ ] [ ] ; additional tables with information on many bacterial, viral, and parasitic zoonoses are in the supplementary material (tables s -s , respectively). trypanosoma cruzi has been identified in hosts such as armadillos and monkeys (in and , respectively, by chagas in brazil) [ ] , as well as cats and dogs. a recent report of a study lasting two decades ( - ) of t. cruzi infection in wild mammals in brazil showed that % of mammals were seropositive and % with high parasitemia, indicating infectivity potential [ ] . a recent study reported a potential effect on chagas disease transmission of oil palm plantations in colombia, where the main vector in the region has been captured and reported to have t. cruzi natural infection, based on vertebrate host analyses of blood meals from nymphs that revealed vertebrate species including pigs, house mouse and opossum [ ] . hunting dogs in indigenous mayangna and miskitu populations from nicaragua's remote bosawás biosphere reserve were shown to have t. cruzi antibodies and therefore previous exposure ( / sera screened, or %), suggesting hunting dogs as the potential zoonotic risk for chagas disease in these communities [ ] . the origin of chagas disease is unclear. bats may have been the original reservoirs hosts of t. cruzi, as postulated in the bat-seeding hypothesis, followed by a host switch to a non-volant mammal and then several switches to humans resulting in the diversity of lineages circulating in human populations. two genetic lineages of trypanosomes associated with bats were recently detected in rural areas of southern ecuador [ ] , one of which, named tcbat, is t. cruzi-related and was detected in a -year-old female in a forest area in northwestern colombia as a mixed infection of t. cruzi i and tcbat genotypes [ ] . a recent systematic review on zoonotic diseases that manifest with human febrile illness reported in malaria-endemic countries showed a wide distribution of these diseases causing febrile illness; half of them were bacterial diseases [ ] . for a list of selected bacterial zoonoses reflecting the impressive diversity of this group of diseases, see table s . next, we discuss two well-known bacteria genera-brucella and myocabacteria-as they represent ancient human diseases for which origins are still highly debated. two major diseases caused by these bacteria are brucellosis and tuberculosis. they affect the skeleton, causing bone pathologies in about - % of individuals who have the disease [ ] and can result in similar lesions in affected individuals despite being caused by very different bacterial species. paleopathology studies along with ancient dna analysis of bone lesion remains have been critical in determining the origin for each of these two diseases. the overlapping appearance of affected bones in the spine in both infections have led researchers to initially suspect a tuberculosis lesion, with molecular analyses demonstrating in some cases that the cause was brucellosis. a study that evaluated calcified nodules from a th-century skeleton found at an abandoned medieval village in northwest sardinia, italy, initially hypothesized the nodules were due to tuberculosis, but shotgun metagenomics revealed medieval brucella melitensis genome sequences instead [ ] . even today, with advanced health technology, there is overlap in the understanding of symptoms and laboratory test results for these two diseases [ ] , making differential diagnosis difficult in countries such as india, where they are both endemic [ ] . caused by bacteria of the genus brucella (mostly brucella melitensis), brucellosis is a common zoonotic infection resulting in febrile illness in humans, mainly through the ingestion of unpasteurized dairy products and direct contact with infected animals (table ). travelers to endemic areas may get infected by consumption of unpasteurized milk or other dairy products and may be the source of imported cases into their own countries (mostly from infected cheeses consumed by their families), as is the case for most of the acute brucellosis cases in north america and northern europe [ ] . re-emergence of zoonotic diseases in countries where they have been previously eradicated has been reported, calling for a need for disease-free countries to remain aware and implement regional monitoring systems. brucellosis has reemerged in bulgaria after years, probably due to the illegal import of infected animals from endemic border countries, as has occurred with bovine brucellosis in france, with possible cross-border brucellosis transmission into europe from middle-eastern countries with the highest incidences of brucellosis worldwide such as turkey and syria (reviewed in [ ] ). the exact prevalence of both animal and human disease due to brucellosis is not well known. the economic burden of brucellosis is considerably high in low-income countries in tropical asia and africa [ ] . in , out of . million estimated cases of brucellosis in kenya, , people died, % of whom were livestock keepers [ ] . brucellosis can have skeletal manifestations, with bones and joints affected being the most frequent complications occurring in up to % of cases [ ] . analysis of vertebral lesions and pathological changes described in a . - . million-year-old male skeleton of australopithecus africanus from south africa were interpreted as resulting from initial phases of brucellosis [ ] . this disease is an example of human and domestic animal paleopathology studies suggesting brucellosis in ancient bone remains, with most cases involving adult male skeletal individuals showing lumbar vertebrae and sacroiliac joints involved [ ] , evidence which combined with ancient dna analysis by pcr have confirmed the presence of brucella dna (reviewed in [ ] ). as discussed above, dna detection of brucella dna has been critical in confirming brucellosis in ancient human remains when paleopathology initially suggested tuberculosis. however, brucella bacteria do not preserve as well as mycobacteria, which have a thicker, hydrophobic and more resistant cell wall that has allowed more data to be collected for ancient infection for the latter type of disease-causing bacteria (discussed next). mycobacteria vary in epidemiology, reservoirs, and their ability to cause disease, with groups such as the mycobacterium tuberculosis complex including m. tuberculosis and m. bovis (see bovine tuberculosis below) that infect a large range of mammals including humans via inhalation of droplets containing bacteria that reach the lungs; m. leprae (see leprosy section below); "nontuberculous mycobacteria" [ ] . human tuberculosis caused by m. tuberculosis is a well-known disease of unclear origin, with a considerable impact on global health. it affects an estimated million people annually. those living with hiv are more likely to be infected with and die of tuberculosis [ ] . the origin of tuberculosis is under debate regarding whether it started as a zoonosis from cattle or not. one hypothesis states that it originated with bovid milk consumption known to occur in the early neolithic in europe [ ] . another hypothesis based on biomolecular studies proposes a new evolutionary scenario with human tuberculosis having a human origin, present in early african human populations at least , years ago and expanding with human migration, especially in the neolithic [ ] . in both scenarios, human-animal proximity starting in the neolithic was a critical factor for emergence/expansion, due to close interaction between early domesticates and humans and increased human density. human density in some areas peaked during the industrial revolution when crowded dairy cow populations in densely populated urban settings were the source of milk [ ] . although rare, tuberculosis can result in osteological lesions which can help identify, along with molecular analysis, tuberculosis infection in ancient bone remains. these approaches have been applied to materials from both humans and animals. however, molecular genetics applications in these cases have limitations, including cost. human remains from a medieval churchyard in england in which both m. tuberculosis and m. bovis were predicted due to milk and beef consumption at the time, showed only m. tuberculosis dna in human skeletons that displayed morphological symptoms of tuberculosis (reviewed in [ ] ). a particular question on ancient dna in this context relates to the origins of tuberculosis in the americas. while today european lineages of m. tuberculosis are found, morphological evidence exists that may support a pre-columbian prevalence of the disease. a recent report on sequencing and analysis of three mycobacterial genomes from peruvian human skeletons dated to - revealed the presence of tuberculosis before european contact in pre-columbian south america [ ] , a region with considerable morphological evidence of pre-columbian tuberculosis previously reported. in this study, the three sequenced mycobacterial genomes were most closely related to bacteria belonging to the m. tuberculosis complex that have adapted to seals and sea lions (m. pinnipedii) than those adapted to humans today in europe, asia, or africa. these data suggest that sea mammals may have played a role in tuberculosis transmission to humans across the ocean, an intriguing possibility for a zoonotic origin of new world tuberculosis to be further investigated. bovine tuberculosis resulting from m. bovis infection affects cattle and other mammals and, when transmitted to humans, becomes zoonotic tuberculosis (table ). its overall incidence has decreased due to cattle control and routine pasteurization of milk from cows, as infection happens mostly by consuming unpasteurized milk and dairy products or by wound contact occurring during slaughter or hunting [ ] ; occupational exposure of livestock workers may occur via inhalation of aerosol or cough from infected cattle. this form of widespread zoonotic tuberculosis can be fatal, present mainly in africa (causing approximately % of all pulmonary tuberculosis cases) and southeast asia. in , out of cases of bovine tuberculosis in kenya, people died (about %); % of the fatalities were livestock keepers [ ] . often bovine tuberculosis caused by m. bovis in humans is indistinguishable clinically from human tuberculosis resulting from m. tuberculosis infection; bovine tuberculosis is estimated to account for up to % of human tuberculosis cases in some countries [ ] . however, accurate information is lacking on the incidence of tuberculosis due to human m. bovis infection from countries with high tuberculosis and hiv prevalence and where populations have direct contact with cattle such as sub-saharan africa [ , ] . in these low resource settings, it is often assumed (and not tested) that tuberculosis is caused by m. tuberculosis, leading to underestimating the real incidence of zoonotic tuberculosis and underscoring a need to accurately diagnose and treat human tuberculosis caused by m. bovis [ ] . m. bovis has a broad host range including domestic and wild animals, with feral maintenance hosts such as badgers (meles meles) in the uk and ireland, the brushtail possum (trichosurus vulpecula) in new zealand, and the white-tailed deer (odocoileus virginianus) in michigan, usa posing a threat for livestock infection as well as disease eradication from cattle (reviewed in [ ] ). cases of bovine tuberculosis in humans linked to deer hunting and handling in an endemic white-tailed deer area have been reported in michigan [ ] . there is also epidemiological evidence in new zealand that feral ferrets (mustela furo) may be tuberculosis vectors for cattle, and can be used as sentinels for this disease as an alternative to possums (reviewed in [ ] ). mycobacterium avium, a causative bacterium of tuberculosis in birds, can also be transmitted to mammals, including livestock. the livestock industry suffers economic losses and trade restrictions due to its incidence. widespread paratuberculosis in animals (also known as jones disease) including bovine paratuberculosis, is an endemic disease especially in developing countries affecting livestock production, zoo, and wildlife animals (reviewed in [ ] ). m. avium subspecies can cause paratuberculosis in humans, manifested as inflammatory bowel disease and autoimmune diseases including asthma, insulin-dependent diabetes mellitus, sarcoidosis, rheumatoid arthritis, multiple sclerosis, celiac disease and may also be a contributing factor to crohn's disease [ ] . mycobacterium leprae, the main causative agent of leprosy in humans, a chronic disease with skin lesions and peripheral nerve damage mostly spread via a human-to-human transmission with zoonotic transmission from natural reservoirs, which are not clearly understood yet. the first animal reservoir to be discovered was the nine-banded armadillo (dasypus novemcinctus) in the southern united states [ ] [ ] [ ] . this armadillo, which can be naturally infected with m. leprae and shows similar disease presentation as humans systemically, have been used as animal models for leprosy studies by laboratory infection; they present typical plantar ulceration with foot ulcers increasing as the infection progresses (reviewed in [ ] ). this species has been recently shown to be a potential reservoir in brazil, where in some areas people hunt and eat them as a dietary source of protein [ ] . recently, m. leprae was also isolated from red squirrels on brownsea island in the southern uk [ ] . for both nine-banded armadillos and red squirrels reservoirs, related m. leprae strains were found via ancient dna studies in human skeletons from england and denmark dating to medieval times, suggesting a potential european origin of the strains present today in the reservoirs [ , ] . non-human primates in several regions have been identified as additional m. leprae reservoirs [ ] . m. leprae genomes were obtained from a chimpanzee (pan troglodytes) from sierra leone, a cynomolgus macaque (macaca fascicularis) from the philippines, and a sooty mangabey (cercocebus atys) from west africa [ ] . marmosets (callithrix jacchus) in brazil, also examined as possible hosts for m. leprae, were not positive by dna analysis although mycobacterial dna (rpob locus) was detected [ ] . in contrast to the previously discussed zoonoses, which are linked to well-known diseases, viral zoonoses are the cause of the majority of recent human pandemics, suggesting that viruses may evolve more rapidly than other pathogens to adapt to the human host, sometimes leading to the person-to-person transmission without the need for another reservoir, and with transmission enhanced in dense populations and by human travel [ ] . many of them are distributed worldwide (table s ). here we present examples of recent viral zoonoses, some resulting in widespread pandemics, to showcase the clear involvement of wild animals, which are often debated in the previously introduced cases likely due to the old nature of those diseases. in many areas of central africa in particular, wild meat (known as bush meat) is in high demand. human contact with wild animals during hunting and preparation or consumption of undercooked meat has led to important zoonoses developing. an example may be hiv/aids, which was linked to the butchering of hunted chimpanzees in africa, afterward adapting to human-to-human transmission [ ] . the human ebola epidemic in west africa in - caused by the ebola virus was linked to the hunting or handling of infected gorillas and other wild animal carcasses ( [ ] , table s ). the congo's ebola hemorrhagic fever affects gorillas and chimpanzees; about gorillas were killed in gabon and the republic of the congo in - by the ebola virus [ ] . asia is another example of wild animals trafficking for consumption, especially in china, myanmar, vietnam, and thailand, where "ye wei" ("wild taste" for wild and exotic animals) was associated with social status including in the dynastic eras as well as currently being widely sold in wet markets and restaurants, sourced legally or illegally from the wild or wildlife farms (reviewed in [ ] ). important zoonoses such as some caused by coronaviruses (table ) have also been associated with the consumption of wild meat in markets, two of them in china. the severe acute respiratory syndrome coronavirus (sars-cov) in and the middle east respiratory syndrome coronavirus (mers-cov) in each caused a large pandemic, but these were small compared to the one caused by sars-cov- , a second sars virus recently identified as the cause of covid- . the sars pandemic caused cases and deaths and mers resulted in cases and deaths [ ] , while covid- has caused, as of july and only six months after who declared it a pandemic on january, over million cases and slightly over half a million deaths [ ] . the animal sources for sars and mers were identified as the civet and dromedary camel, respectively (table ) . however, these are considered intermediate hosts, as these zoonotic diseases are thought to have originated in bats [ , ] , subsequently spilling over to intermediate hosts, and eventually jumping to humans [ ] . a very recent phylogenetic dating study based on bioinformatic approaches strongly suggests that sars-cov- emerged directly, without an intermediate host, from the same horseshoe bat subgenus of sars-like coronaviruses, with both sars-cov and sars-cov- diverging at the same time ( - years ago) from currently known extant bat virus [ ] . nonhuman primates were shown to be an effective yellow fever sentinel system in brazil. yellow fever (table s ) , a reemerging viral zoonotic disease endemic in africa and south america transmitted from vector mosquitoes, often causes outbreaks in both humans and nonhuman primates in brazil. this country has an established and successful yellow fever national surveillance program that includes postmortem nonhuman primate studies for early circulating virus detection and prompt implementation of vaccination and vector control [ ] . after a epizootic occurred in brazil's espirito santo state, deceased nonhuman primates (two howler monkeys (alouatta spp.) and not further identified) were examined with of them showing typical yellow fever features; yellow fever was diagnosed the same year for of animals tested from southern states of brazil ( % occurrence) [ ] . emerging zoonotic diseases causing epidemics have prompted scientists to focus on efforts towards identifying factors involved in disease emergence as well as possible prediction tools, including modeling approaches. recent such modeling used for spatial mapping of hotspots showed that the global distribution of the risk of emerging zoonotic diseases is higher in tropical regions with wildlife biodiversity (especially mammals) experiencing land-use changes [ ] . this study, along with previous ones [ ] , predicted a higher risk in tropical, developing countries. the authors stated that even though emerging infectious disease events have been predominantly reported in developed countries, this is probably an artifact due to stronger surveillance and reporting systems in these areas. it would be hard to test this idea. the one health multisector approach response to zoonoses threat is an appropriate one, which may need input from additional sectors, such as travel and tourism, social and political scientists, anthropologists, and economists to better plan and implement strategies related to surveillance, capacity building, and risk reduction when addressing, for example, the possible closure of wet markets in communities that rely heavily on them for income and food [ ] . there is a wide range of species reported as hosts of zoonoses, making predictions on new and potential reservoirs based on phylogenetic considerations challenging. basic information on which species serve as hosts is lacking for many regions and diseases. although considerable progress has been made in our understanding of these diseases' reservoirs, animal origin, and evolution, with contributions made by paleopathology and ancient dna detection for ancient diseases such as brucellosis and zoonotic tuberculosis. further research is needed to gain insights into mechanisms of disease emergence and transmission. in view of the recent pandemics, research on zoonoses should be prioritized towards developing evidence-based prevention strategies. these should include approaches based on sustainable living and food consumption that address inequality, as commentators across the globe currently discuss prompted by the covid- pandemic. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s : selected bacterial zoonoses, table s : selected viral zoonoses, table s : selected parasitic and other non-bacterial non-viral zoonoses. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection or interpretation of data and the writing of the manuscript or in the decision to publish the results. prediction and prevention of the next pandemic zoonosis prioritizing zoonoses for global 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and human ebola outbreaks, gabon and republic of congo ebola outbreak killed gorillas baby pangolins on my plate: possible lessons to learn from the covid- pandemic covid- has killed more people than sars and mers combined, despite lower case fatality rate origin and evolution of pathogenic coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin evolutionary origins of the sars-cov- sarbecovirus lineage responsible for the covid- pandemic surveillance for yellow fever virus in non-human primates in southern brazil outbreak of yellow fever among nonhuman primates global hotspots and correlates of emerging zoonotic diseases global trends in emerging infectious diseases this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -nquov i authors: murphy, f.a. title: epidemiology of human and animal viral diseases date: - - journal: encyclopedia of virology doi: . /b - - . - sha: doc_id: cord_uid: nquov i viral disease epidemiology is the study of the determinants, dynamics, and distribution of viral diseases in populations. the risk of infection or disease in a population is determined by characteristics of the virus, the host, and the host population, as well as behavioral, environmental, and ecological factors that affect virus transmission from one host to another. viral disease epidemiology has come to have a major role in clarifying the etiologic role of particular viruses and viral variants as the cause of specific diseases, in improving our understanding of the overall nature of specific viral diseases, and in determining factors affecting host susceptibility and immunity, in unraveling modes of transmission, in clarifying the interaction of viruses with environmental determinants of disease, in determining the safety, efficacy, and utility of vaccines and antiviral drugs, and especially in alerting and directing disease prevention and control actions. information on incidence, prevalence, and morbidity and mortality rates contributes directly to the establishment of priorities for prevention and control programs, whether this involves vaccine or drug development and delivery, environmental and hygienic improvements, enhancement of nutritional status, personal or community behavior, agricultural and food processing enhancements, reservoir host and vector control, and international cooperation and communication. evs, they encode other types of defense molecules. among these are the inhibitor of apoptosis (iap) genes. the iap of amev has been well characterized and functionally inhibits apoptosis. a related amev gene that functions to inhibit apoptosis is a homolog of the baculovirus pan-caspase inhibitor, p . another novel protein expressed by amev is a cu-zn superoxide dismutase (sod) . although a number of the orthopoxviruses encode genes with homology to this class of sods, neither the vv or myxoma virus proteins are functional in that capacity, although they are present within the virion. the sod expressed by amev is functional as an sod but is not essential for virus growth in culture. the deletion of the sod gene from amev appears to have no effect on the growth of the virus in gypsy moth larvae. it is clear that this large subfamily of the family poxviridae provides a wealth of possible information about the basic mechanisms of the poxvirus lifecycle. there appear to be a number of interesting variations on the molecular details which define this overall family of viruses. there are clear similarities to the vertebrate poxviruses in virion morphology, double-stranded dna genome, cytoplasmic life cycle, and rna expression. yet the differences between the cvs and evs are significant and represent an area of research that has not been fully explored. the data that have been obtained from genomic sequencing has been essential to identifying some of the different proteins that are present in the evs, as well as identifying potentially missing homologs of vv proteins. it is important to note that there are large differences at the dna level between the two sequenced evs, indicating that there is probably a wide variety of unique features within the evs as a group. as more sequence information becomes available, the diversity of this family of viruses may become more evident. direct contact transmission involves actual physical contact between an infected subject and a susceptible subject (e.g., kissing, biting, coitus). epidemic major increase in disease incidence affecting either a large number of humans or animals or spreading over a large area. epidemiology the study of the determinants, dynamics, and distribution of diseases in populations. fomite an inanimate object that may be contaminated with virus and become the vehicle for transmission. herd immunity the immune status of a population that affects viral transmission rates. often used in describing the elimination of a virus from a population when there are too few susceptible hosts remaining to sustain a transmission chain. horizontal transmission the transfer of infectious virus from one human or animal to another by any means other than vertical transmission. iatrogenic transmission transmission via health care procedures, materials, and workers (e.g., physicians, nurses, dentists, veterinarians). incidence rate (or attack rate) a measure of the occurrence of infection or disease in a population over time -it refers to the proportion of a population contracting a particular disease during a specified period. mathematical model (epidemiological) a means to convey quantitative information about a host-virus interaction, such as an epidemic or an emerging disease episode, by the construction of a set of predictive mathematical algorithms. nosocomial transmission pertains to infections acquired while a patient, human or animal, is in hospital. prevalence rate the ratio, at a particular point in time, of the number of cases currently present in the population divided by the number of subjects in the population at risk; it is a snapshot of the occurrence of infection or disease at a given time. species jumping (or host range extension) referring to a virus that derives from an ancient reservoir life cycle in animals, but has subsequently established a new life cycle in humans or a different animal species and no longer uses, the original animal reservoir. transmission the process by which a pathogen is shed from one host and infects the next. vector-borne transmission involves the bites of arthropod vectors (e.g., mosquitoes, ticks, sandflies). vertical or transplacental transmission occurs from mother to fetus prior to or during parturition, either across the placenta, when the fetus passes through the birth canal, or via colostrum and milk. vertical transmission transmission of virus from parent to progeny through the genome, sperm, or ovum or extracellularly (e.g., through colostrum or across the placenta). zoonosis disease which is naturally transmitted to humans from an ongoing reservoir life cycle in animals or arthropods, without the permanent establishment of a new life cycle in humans. viral disease epidemiology is the study of the determinants, dynamics, and distribution of viral diseases in populations. the risk of infection or disease in a population is determined by characteristics of the virus, the host, and the host population, as well as behavioral, environmental, and ecological factors that affect virus transmission from one host to another. epidemiology attempts to meld these factors into a unified whole. the depiction of the interaction of factors favoring the emergence of a viral disease (figure ) , called 'the convergence model', is taken from the us institute of medicine study, microbial threats to health, emergence, detection and response (national academy press, ) . at the center is a box representing the convergence of factors leading to 'the black box', reflecting the reality that many unknown interactions are important virologically and epidemiologically. the foundations of epidemiology predate the microbiological and virological sciences, starting with hippocrates, the greek physician and father of medicine, who in the fourth century bc made important epidemiologic observations on infectious diseases. john snow is called the father of modern epidemiology because he developed excellent quantitative methods while studying the source of a cholera outbreak at the broad street pump in london in . snow was followed by william farr, who in the s advanced the use of vital statistics and clarified many of the principles of risk assessment and retrospective and prospective studies. their vision is reflected in the fast-changing science of epidemiology which is now supported by advanced computer technology, sophisticated statistical methods, and very sensitive and specific diagnostic systems. by introducing quantitative measurements of disease trends, epidemiology has come to have a major role in improving our understanding of the overall nature of disease and in alerting and directing disease control activities. epidemiology is also effective in ( ) clarifying the role of particular viruses and viral variants as the cause of disease, ( ) clarifying the interaction of viruses with environmental determinants of disease, ( ) determining factors affecting host susceptibility, ( ) unraveling modes of transmission, and ( ) field testing of vaccines and antiviral drugs. the comparison of disease experience between populations is expressed in the form of 'rates'. the terms 'incidence rate' and 'prevalence rate' are used to describe quantitatively the frequency of occurrence of infection or disease in populations. 'incidence rate' (also called attack rate) is defined as the ratio of new cases occurring in a population to the size of the population during a specified period of time. prevalence rate is the ratio of the total number of cases occurring in a population to the size of the population during a specified period of time. 'seroprevalence rate' relates to the occurrence of antibody to a particular virus in a population. because viral antibodies, especially neutralizing antibodies, often last a lifetime, seroprevalence rates usually represent cumulative experience with the virus. the term 'case-fatality rate' is used to indicate the percentage of subjects with a particular disease that die from the disease. all these rates may be affected by various attributes that distinguish one individual from another: age, sex, genetic constitution, immune status, pregnancy, nutritional status, and various behavioral and medical care and patient management parameters. the most widely applicable attribute is age, which may encompass immune status as well as various physiological variables. a viral disease is characterized as 'endemic' when there are multiple or continuous chains of transmission resulting in continuous occurrence of disease in a population over a period of time. 'epidemics' are peaks in disease incidence that exceed the endemic baseline or expected rate of disease. the size of the peak required to constitute an epidemic is arbitrary and is related to the background endemic rate and the anxiety that the disease arouses (e.g., a few cases of rabies is regarded as an epidemic, whereas a few cases of influenza is not). a 'pandemic' is a worldwide epidemic. a proper description of an outbreak of disease or an epidemic must include the parameters of 'person (or subjects in the case of animals), place, and time'. such descriptive information is a necessary first step in describing the occurrence, distribution, course, threat, and anticipated action response to the initial recognition of a cluster of cases of disease. much of the initial investigation called for rests in common sense, observational acuity, and an insightful 'index of suspicion'. much of the initial investigation has been termed 'shoe-leather epidemiology'. the trigger for such initial investigation is most often an astute clinician (physician or veterinarian) or an astute pathologist. there are two basic analytic techniques used to investigate relationships between cause and effect and to evaluate risk factors of disease. these are the 'case-control study' and the 'cohort study'. in the case-control study, investigation starts after the disease has occurred -it is a retrospective study, going back in time to determine causative events. although this kind of study does not require the creation of new data or records, it does require careful selection of the control group, matched to the test group so as to avoid bias. the retrospective case-control study lends itself to quick analysis and is relatively inexpensive to carry out. in the cohort study, the prospective study, investigation entails the gathering of new data to identify cause-effect relationships. this kind of study is expensive and does not lend itself to quick analysis as groups must be followed until disease is observed. however, when cohort studies are successful, proof of cause-effect relationship is often incontrovertible. the term 'molecular epidemiology' is used to denote the use of any of a large number of molecular biological methods in support of epidemiologic investigations. for example, with herpesviruses, restriction endonuclease mapping has provided a means of identification of unique viral genotypes -in an epidemiologic study recognized as the first based upon viral molecular characterization, the source of herpes simplex virus causing disease in a hospital newborn nursery was traced to one persistently infected nurse rather than any of several other possible shedders. with rotaviruses and bluetongue viruses, polyacrylamide gel electrophoresis of the segmented viral rna has been used epidemiologically, for example, to unravel outbreaks involving multiple viral variants. panels of monoclonal antibodies have been used to distinguish virus variants for epidemiologic purposes; they have been particularly useful in elucidating host-range and geographic variants of rabies virus. today, partial sequencing has become the most commonly used molecular epidemiologic methodology; partial sequencing of poliovirus isolates recovered from patients indicates whether they are wild type (even local or introduced wild type), attenuated vaccine type, or a vaccine type that has reacquired neurovirulence during human passage. partial sequencing of foot-and-mouth disease viruses can offer the same kind of geographic information of virus movement as has proved so useful in polio control and eradication programs, but because of political sensitivities in some countries a robust international reference laboratory system has not been established that could provide the same kind of practical disease control information as has been the case with polio. thus, with many human and animal viruses molecular epidemiologic studies are flourishing, but more such studies should lead to international reference laboratory systems to guide prevention and control actions. such studies are developing rapidly today to deal with the threat of a human pandemic of avian influenza, but there are many more viral diseases, especially animal diseases, in need of this kind of development. one of the landmarks in the history of infectious diseases was the development of the henle-koch postulates which established the evidence required to prove a causal relationship between a particular infectious agent and a particular disease. these simple postulates were originally drawn up for bacteria, but were revised in by rivers and again in by evans in attempts to accommodate the special problem of proving disease causation by viruses ( table ) . in many cases, virologists have had to rely on indirect evidence, 'guilt by association', with associations based on epidemiologic data and patterns of serologic positivity in populations. today, many aspects of epidemiologic investigation play roles, especially in trying to distinguish an etiological, rather than coincidental or opportunistic relationship between a virus and a given disease. for example, early in the investigation of human acquired immunodeficiency syndrome (aids), before its etiology was established, many kinds of viruses were being isolated from patients and many candidate etiologic agents were being advanced. prediction that the etiologic agent would turn out to be a member of the family retroviridae was based upon years of veterinary research on animal retroviruses and animal retroviral diseases. this prediction was based upon recognition of common biologic and pathogenetic characteristics of aids and animal retroviral diseases. this prediction guided many of the early experiments to find the etiologic agent of aids; later, after human immunodeficiency virus (hiv ) was discovered, its morphological similarity to equine infectious anemia virus, a prototypic member of the genus lentivirus, family retroviridae, was the key to unraveling confusion over the fact that the human virus killed host lymphocytes rather than transforming them as typical oncogenic retroviruses would do. ever since, this essence of comparative medicine has been guiding hiv/ aids research in many areas, including drug design, diagnostics, and vaccine development. hiv/aids epidemiologic research has often been intertwined with research on the several simian immunodeficiency viruses (sivs). seroepidemiology is useful in public health and animal health investigations and in research to determine the prevalence or incidence of particular infections, to evaluate control and immunization programs, and to assess past history when a 'new' virus is discovered. when paired serum specimens are obtained from individuals several weeks apart, the initial appearance of antibody in the second specimen or a rise in antibody titer indicates recent infection. similarly, the presence of specific immunoglobulin m (igm) antibody in single serum samples, indicating recent infection, may be used in seroepidemiologic studies. correlation of serologic tests with clinical observations makes it possible to determine the ratio of clinical to subclinical infections. because of advanced diagnostic/serologic methods, sentinel studies can yield many valuable data in timely fashion about impending disease risks. for example, sentinel chicken flocks are set out for the early detection of the presence of arboviruses such as west nile virus in the united states. these flocks are bled and tested weekly for the presence of virus or antiviral antibody; they provide an early warning of the levels of virus amplification that occur before epidemics. the immunogenicity, potency, safety, and efficacy of vaccines are first studied in laboratory animals, followed by small-scale closed trials, and finally in large-scale open trials. such studies employ epidemiologic methods, rather like those of the cohort (prospective) study. in most cases, there is no alternative way to evaluate new vaccines, and the design of trials has now been developed so that they yield maximum information with minimum risk and acceptable cost. viruses survive in nature only if they are able to be transmitted from one host to another, whether of the same or another species. transmission cycles require virus entry into the body, replication, and shedding with subsequent spread to another host. portals of virus entry into the body include the skin, respiratory tract, intestinal tract, oropharynx, urogenital tract, and conjunctiva. in some cases, viruses use a particular portal of entry because of particular environmental or host-behavior factors and in other cases because of specific viral ligands and host-cell receptors. in many cases, disruption of normal host-defense mechanisms leads to entry that might otherwise be thwarted; for example, papillomaviruses may enter the deep layers of the skin via abrasions, acid-labile coronaviruses may enter the intestine protected by the buffering capacity of milk, and influenza viruses may enter the lower respiratory tract because a drug has dampened cilial action of the respiratory epithelium. the exit of virus from an infected host is just as important as entry in maintaining its transmission cycle. all portals used by viruses to gain entry are used for exit. the table criteria for disease causation: a unified concept appropriate for viruses as causative agents of disease, based on the henle-koch postulates, and modified by a. s. evans important elements in virus shedding are virus yield (from the standpoint of the virus, the more shedding the better) and timeliness of yield (again, the earlier the shedding the better). viruses that cause persistent infections often employ remarkable means to avoid host inflammatory and immune responses so as to continue shedding. for example, the epidemiologically important shedding of herpes simplex viruses and that perpetuates the viruses in populations requires recrudescence of persistent ganglionic infection, centrifugal viral genomic transit to peripheral nerve endings, and productive infection of mucosal epithelium, all in the face of established host immunity. virus transmission may be 'horizontal' or 'vertical'. the vast majority of transmission is horizontal, that is, between individuals within the population at risk. modes of horizontal transmission of viruses can be characterized as direct contact, indirect contact, common vehicle, airborne, vector-borne, iatrogenic, and nosocomial. vertical or transplacental transmission occurs between the mother and her fetus or newborn. some viruses are transmitted in nature via several modes, others exclusively via one mode (see table ). 'direct contact transmission' involves actual physical contact between an infected subject and a susceptible subject (e.g., kissing, epstein-barr virus, the cause of mononucleosis, biting (e.g., rabies); coitus (sexually transmitted viral diseases)). indirect contact transmission occurs via 'fomites', such as shared eating utensils, improperly sterilized surgical equipment, or improperly sterilized non-disposable syringes and needles. 'common vehicle transmission' pertains to fecal contamination of food and water supplies (e.g., norovirus diarrhea). common vehicle transmission commonly results in epidemic disease. 'airborne transmission' typically results in respiratory infections (and less typically in intestinal infections), but these infections may also be transmitted by direct and indirect contact. airborne transmission occurs via large droplets and via very small droplet nuclei (aerosols) emitted from infected persons during coughing or sneezing (e.g., influenza) or from environmental sources. large droplets (> mm in diameter) settle quickly, but droplet nuclei evaporate forming dry particles (< mm in diameter) which remain suspended in the air for extended periods. droplets may travel only a meter or so while droplet nuclei may travel over much longer distances. 'vector-borne transmission' involves the bites of arthropod vectors (e.g., mosquitoes, ticks, and sandflies). 'iatrogenic transmission' involves health care procedures, materials, and workers (e.g., physicians, nurses, dentists, and veterinarians). 'nosocomial transmission' pertains to infections acquired while a patient, human or animal, is in hospital. 'vertical or transplacental transmission' occurs from mother to fetus prior to or during parturition. certain retroviruses are vertically transmitted in animals via the integration of viral dna directly into the dna of the germline of the fertilized egg. other viruses are transmitted to the fetus across the placenta; yet others are transmitted when the fetus passes through the birth canal. another vertical transmission route is via colostrum and milk. vertical transmission of a virus may or may not be associated with 'congenital disease' (i.e., disease that is present at birth) which may be lethal (and the cause of abortion or stillbirth) or the cause of congenital abnormalities. the herpesviruses, especially cytomegaloviruses, and rubella virus cause important congenital diseases in humans, and pestiviruses, such as bovine viral diarrhea virus, in animals. enteric infections are most often transmitted by direct contact and by fomites in a 'fecal-oral cycle' that may include fecal contamination of food and water supplies; diarrheic feces may also splash to give rise to aerosols (droplets and droplet nuclei). respiratory infections are most often transmitted by the airborne route or by indirect contact via fomites in a 'respiratory cycle', that is, virus is shed in respiratory secretions and enters its next host through the nares during inhalation. the respiratory cycle is responsible for the most explosive patterns of epidemic disease in humans and all domestic animal species. perpetuation of a virus in nature depends upon the maintenance of serial infections, that is, a chain of transmission; the occurrence of disease is neither required nor necessarily advantageous. infection without recognizable disease is called 'subclinical' or 'clinically inapparent'. overall, subclinical infections are much more common than those that result in disease. their relative frequency accounts for the difficulty of tracing chains of transmission, even with the help of laboratory diagnostics. although clinical cases may be somewhat more productive sources of virus than subclinical infections, because the latter do not restrict the movement of the infected host, they can be most important as sources of viral dissemination. in most acute infections, whether clinically apparent or not, virus is shed in highest titers during the late stages of the incubation period, before the influence of the host-immune response takes effect. persistent infections, whether or not they are associated with episodes of clinical disease, also play an important role in the perpetuation of many viruses in nature. for example, prolonged virus shedding can reintroduce virus into a population of susceptibles all of which have been born since the last clinically apparent episode of infection. this is important in the survival of rubella virus in some isolated populations. sometimes the persistence of infection, the production of disease, and the transmission of virus are dissociated; for example, togavirus and arenavirus infections may have little adverse effect on their reservoir hosts (arthropods, birds, and rodents), but transmission may be very efficient. on the other hand, the persistence of infection in the central nervous system, as with measles virus in subacute sclerosing panencephalitis (sspe), is of no epidemiological significance, since no infectious virus is shed from this site. the virulence of the infecting virus may directly affect the probability of its transmission. the classic example of this is rabbit myxomatosis. in australia, mosquito-borne transmission of myxoma virus was found to be most effective when infected rabbits maintained highly infectious skin lesions for several days before death. highly virulent strains of the virus were found to kill rabbits so quickly that transmission did not occur, and naturally attenuated strains were found to produce minimal lesions that healed quickly and did not permit transmission. virus strains at either extreme of this virulence spectrum were found not to survive in nature, but virus strains of intermediate virulence have circulated for many years. with most viruses, endemic or epidemic transmission leads to a level of immunity in the host population that affects or even interrupts further transmission. the 'herd immunity' effect is countered in some cases by viral antigenic variation. for example, influenza viruses undergo genetic variations ('shift' and 'drift') such that persons immune to previously circulating virus strains are susceptible to new strains. assessing these genetic changes is the main objective of laboratory-based surveillance programs, which in turn are the basis for decisions on the formulation of each year's influenza vaccine. it is self-evident that the long-term survival of a virus requires that it be continuously transmitted from one host to another. in general, for rapidly and efficiently transmitted viruses such as many respiratory viruses, local survival of the virus requires that the susceptible host population be very large. a virus may disappear from a population because it exhausts its potential supply of susceptible hosts as they acquire immunity to reinfection with the same virus. depending on duration of immunity and the pattern of virus shedding, the 'critical population size' varies considerably with different viruses and with different host species. the most precise data on the importance of population size in acute nonpersistent infections come from studies of measles. persistence of measles virus in a population depends upon a continuous supply of susceptible children. analyses of the incidence of measles in large cities and in island communities have shown that a population of about half a million persons is needed to ensure a large enough annual input of new susceptible hosts, by birth or immigration, to maintain measles virus in the population. because infection depends on respiratory transmission, the duration of epidemics of measles is correlated inversely with population density. if a population is dispersed over a large area, the rate of spread is reduced and the epidemic may last longer, so that the number of susceptible persons needed to maintain transmission chains is reduced. on the other hand, in such a situation a break in the transmission chain is much more likely. when a large proportion of the population is initially susceptible, the intensity of the epidemic builds up very quickly and attack rates are almost % ('virginsoil epidemic'). on the other hand, when measles vaccination programs are implemented properly the virus disappears completely from the regional population. because most viruses are host-restricted, most viral infections are maintained in nature within populations of the same or related species. however, there are a number of viruses that may have multiple hosts and spread naturally between several different species of vertebrate host, for example, rabies and eastern equine encephalitis viruses. the term 'zoonosis' is used to describe multiple-host infections that are transmissible from animals to man. the zoonoses, whether involving domestic or wild animals or arthropods, usually represent important problems only under conditions where humans are engaged in activities involving close contact with animals or exposure to arthropods. many viral zoonoses are caused by arboviruses. arboviruses have two classes of hosts, vertebrate and invertebrate. over arboviruses are known, of which about cause disease in humans and in domestic animals; some of these are transmitted by ticks, some by mosquitoes, and yet others by phlebotomine flies (sandflies) or culicoides spp. (midges). arthropod transmission may be 'mechanical', where the arthropod acts as a 'flying pin', or more commonly, 'biological', involving replication of the virus in the arthropod vector. the arthropod vector acquires virus by feeding on the blood of a viremic person or animal. replication of the ingested virus, initially in the arthropod's gut, and its spread to the salivary glands takes several days; the interval varies with different viruses and is influenced by ambient temperature. virions in the salivary secretions of the vector are injected into human or animal hosts during subsequent blood meals. most arboviruses have localized natural habitats in which specific receptive arthropod and vertebrate hosts are involved in the viral life cycle. vertebrate reservoir hosts are usually wild mammals or birds; humans are rarely involved in primary transmission cycles, although the exceptions to this generalization are important (e.g., venezuelan equine encephalitis, yellow fever, and dengue viruses). humans are in most cases infected incidentally, for example, by the geographic extension of a reservoir vertebrate host and/or a vector arthropod. ecological changes produced by human activities disturb natural arbovirus life cycles and have been incriminated in the geographic spread or increased prevalence of arbovirus diseases. from the time of william farr, who studied epidemic disease problems in the s, mathematicians have been interested in 'epidemic curves' and secular trends in the incidence of infectious diseases. with the development of computer-based mathematical modeling techniques, there has been a resurgence of interest in the population dynamics of infectious diseases. there has also been a resurgence in controversies surrounding the use of models; critics say 'for every model there is an equal and opposite model'. so, the proof of the value of models lies in their practical application, and in recent years there have been more and more successes. for example, when for counterterrorism reasons universal smallpox vaccination was being considered, models that showed that vaccine could be used effectively after rapid detection of a terrorism incident led to a decision to stockpile, but not widely use vaccine. as another example, when a foot-and-mouth disease epidemic raged in the united kingdom in , a model showed that only the most vigorous stamping-out campaign could get ahead of the movement of the virus across the country. the model, seeming eminently logical now, importantly provided the kind of veracity and political will needed to accelerate the stamping-out campaign. models may be used to determine ( ) patterns of disease transmission, ( ) critical population sizes to support the continuous transmission of viruses with short and long incubation periods, ( ) the dynamics of endemicity of viruses that become persistent in their hosts, and ( ) the variables in age-dependent viral pathogenicity. computer modeling also provides useful insights into the effectiveness of disease control programs. much attention has been given to modeling the future of the aids epidemic in the united states and the rest of the world. such models usually start with historical data on the introduction of the etiologic virus, hiv , proceed to the present stage of the epidemic where the disease has become well established in many countries and in fewer countries subject to prevention and treatment strategies, and then proceed to project its course into the future. during the first years of the aids epidemic in the united states, african countries, and then in asian countries, most models underestimated developing trends; more recently models have become more accurately predictive -but in many places more and more sobering. knowledge of the epidemiology and modes of transmission of infectious diseases is critical to the development and implementation of prevention and control strategies. data on incidence, prevalence, and mortality contribute directly to the establishment of priorities for prevention and control programs while knowledge of viral characteristics and modes of transmission are used in deciding prevention strategies focusing on vaccine development and delivery, environmental improvements, enhancement of nutritional status, improvement in personal hygiene, and behavioral changes. see also: disease surveillance; viral pathogenesis; zoonoses. prevalence of the disease is significantly higher in subjects exposed to the putative virus than in those not so exposed. . incidence of the disease is significantly higher in subjects exposed to the putative virus than in those not so exposed temporally, the onset of disease follows exposure to the putative virus, always following an incubation period a regular pattern of clinical signs follows exposure to the putative virus, presenting a graded response, often from mild to severe a measurable host-immune response, such as an antibody response and/or a cell-mediated response, follows exposure to the putative virus experimental reproduction of the disease follows deliberate exposure of animals to the putative virus, but nonexposed control animals remain disease free. deliberate exposure may be in the laboratory or in the field, as with sentinel animals elimination of the putative virus and/or its vector decreases the incidence of the disease prevention or modification of infection, via immunization or drugs, decreases the incidence of the disease control of communicable diseases manual, th edn mandell, douglas, and bennett's principles and practice of infectious diseases the epidemiology of viral infections veterinary virology fields virology evolution of viral diseases virus dynamics: mathematical principles of immunology and virology emerging microbial threats to health in the st century. institute of medicine/ national academy of sciences microbial threads to health, emergence, detection and response veterinary epidemiology general features all rights reserved. the lymphocryptoviruses (lcvs) present in old world nonhuman primates, including ebv-like viruses of chimpanzees and rhesus monkeys. these viruses share homologous sequences and genetic organization, and infect the b lymphocytes of their host species, resulting in the establishment of latent infection in vivo and transformation key: cord- -u dj yw authors: hewitt, judith a.; lutz, cathleen; florence, william c.; pitt, m. louise m.; rao, srinivas; rappaport, jay; haigwood, nancy l. title: activating resources for the covid- pandemic: in vivo models for vaccines and therapeutics date: - - journal: cell host microbe doi: . /j.chom. . . sha: doc_id: cord_uid: u dj yw the preclinical working group of accelerating covid- therapeutic interventions and vaccines (activ), a public-private partnership spearheaded by the national institutes of health, was charged with identifying, prioritizing, and communicating sars-cov- preclinical resources. reviewing sars-cov- animal model data facilitates standardization and harmonization and informs knowledge gaps and prioritization of limited resources. to date, mouse, hamster, ferret, guinea pig, and non-human primates have been investigated. several species are permissive for sars-cov- replication, often exhibiting mild disease with resolution, reflecting most human covid- cases. more severe disease develops in a few models, some associated with advanced age, a risk factor for human disease. this review provides a snapshot that recommends the suitability of models for testing vaccines and therapeutics, which may evolve as our understanding of covid- disease biology improves. covid- is a complex disease and individual models recapitulate certain aspects of disease; therefore, the coordination and assessment of animal models is imperative. covid- took the world by surprise. while the disease spread around the globe, no vaccines or drugs were available to ameliorate its effects. as a result, the national institutes of health (nih) and the foundation for the nih (fnih) established the accelerating covid- therapeutic innovations and vaccines (activ) partnership in april comprised of experts in the fields of virology, public health, vaccine and drug development. this public-private partnership brings together industry, academic and government stakeholders in an unprecedented collaboration to share information and resources to impact the trajectory of the pandemic. there are four working groups: preclinical, clinical therapeutics, clinical trial capacity and vaccines (collins and stoffels, ; nih, ) . the preclinical working group (wg) was charged "to standardize and share preclinical evaluation resources and methods and accelerate testing of candidate therapies and vaccines to support entry into clinical trials" (collins and stoffels, ) . under normal circumstances, the response to emerging pathogens is a methodical linear progression of in vitro, in vivo, and clinical studies, yet the urgency of the response to the current pandemic requires these activities to be carried out in parallel. prior experience with other coronaviruses, both in vitro and in vivo models, provides a guiding foundation for current research and effectively enables interventions to the disease. activ's recommendations on accelerating preclinical in vitro studies are described in the accompanying manuscript (grobler et al). animal models help us not only to understand the pathogenesis and mechanisms of sars-cov- disease biology, but also to elucidate aspects of pharmacology, toxicology and immunology of the therapeutic and vaccine strategies. these data may provide confidence that the products being developed can prevent or treat disease; however, no single animal model fully recapitulates human covid- disease. while there is no substitute for randomized controlled clinical trials, there are great advantages to adjunctive research in animal models to explore indepth mechanisms, immunology/pharmacology, vaccine or therapeutic efficacy and discover biomarkers that may be useful in the clinic and to ensure the safety of the candidates in humans. understanding the pharmacokinetic/pharmacodynamic relationships such that effective and safe doses can be extrapolated from in vitro antiviral activity, preclinical pharmacokinetics, toxicology and clinical modeling (fda, ) can allow compounds to proceed into clinical testing without necessarily demonstrating efficacy in an animal model. although animal model data may not be required to advance to the clinic, it can provide useful comparison data, especially informative in a pandemic. the selection of appropriate animal models of infection, disease manifestation, and efficacy measurements is important for vaccines and therapeutics to be compared under activ's umbrella using master protocols with standardized endpoints and assay readouts. models of sars-cov- infection include mice (ace transgenic strains, mouse adapted virus, and aav transduced ace mice), hamsters, rats, ferrets and non-human primates (nhps). the urgency of the need to coordinate efforts to reduce duplication and cycle times also necessitated an assessment of supply and demand. specifically, resource optimization of all the models, but particularly that of the nhps, and priority setting of experimental therapeutic and vaccine interventions was immediately warranted. the national primate research centers (nprcs), established years ago with funding from the nih, serve as a major resource in this effort to provide nhp expertise, models for human diseases, and nhp resources to nih and other investigators (www.nprcresearch.org). similarly, nih funded mouse repositories stand ready to supply mice to the broader scientific community for research and testing. the activ preclinical wg efforts are facilitating a coordinated effort of the seven nprcs, which work together as a consortium to assure the highest degree of rigor and ethics, as well as to manage the relative paucity of available primates and nprc-abls facilities in which to perform sars-cov- in vivo research. the nprcs are playing a major role in activ, although the animal models in our assessment are not restricted to the nprcs or those available through nih sponsored repositories. beyond activ, operation warp speed (ows) is the us government's effort to make million doses of vaccine available by january (hhs, ) . ows is also making use of master protocols, and activ and ows are sharing information around protocols being developed. ows plans to perform animal model testing at contract research organizations under contract to the biodefense advanced research & development authority (barda) and niaid, as well as government labs in the department of defense. coordinating protocols and plans between activ and ows for the supply of non-human primates is critical to the success of all pandemic related efforts. the activ preclinical wg aims to assess the status and applicability of all animal models that are under development and to share information in real-time including: taking inventory of the resources and annotating the salient features of the models, establishing a data collection tool to assess the status and applicability, highlighting the gaps in effort and knowledge, and suggesting redirection to fill the gaps. this initial publication describes the current status of animal models, as determined from the available data in the inventory. as features of the covid- disease are described, for example the rare but serious multisystem inflammatory syndrome in children (mis-c) (rowley, ) , new animal models can help address these complications, ideally in a coordinated fashion to accelerate our understanding. additional animal model data are welcomed, particularly to address gaps in our knowledge, and we will describe how members of the scientific and medical community can submit data for inclusion. it is appropriate to understand the course of human infections in order to model disease in animals; therefore, we present a summary of human disease first. since the outbreak began in china, some of the earliest and most cited publications report numerous cases from china (chauhan, ; huang et al., ; wu and mcgoogan, ) . covid- is characterized by phases of increasing severity, generally described as asymptomatic, mild, moderate or severe disease. mild disease includes general symptoms such as fever, fatigue, cough, headache, diarrhea, sore throat, congestion, muscle or body aches, and/or vomiting; loss of sense of smell and/or taste and a dry, non-productive cough are more pathognomonic of covid- . moderate disease is characterized by mild pneumonia, shortness of breath, and/or pressure in the chest. severe symptoms include difficulty breathing, bluish lips, inability to stay awake, confusion, acute respiratory distress, septic shock and/or multi-organ failure which may lead to death. the median time from symptom onset to pneumonia is about days and to severe hypoxemia is - days (chauhan, ) . progression to severe disease is associated with advanced age and/or comorbidities . siddiqi and mehra (siddiqi and mehra, ) noted that the progression of disease was associated with transition from an antiviral response to an inflammatory response. garcia (garcía, ) posits that responses at various immunological checkpoints determine the course of disease, to include relatively weak stimulation of innate immune responses, adaptive antibody and t cell responses, and potentially strong activation of proinflammatory chemokines. garcia urges increased monitoring of immunological responses to better understand the checkpoints that prevent progression of or promote disease severity, noting that much of what we understand comes from comparing immunological findings in moderate and severe cases, with few reports following asymptomatic or mild cases. finally, ziegler, et al., recently described regulation of ace cell surface expression in upper airway cells by ifn-α, with significant upregulation in humans and nhps but not in mice (ziegler et al., ) . this highlights the need to further understand the risk/benefit of antiviral or ifn therapy treatments in infected humans. further understanding of this dynamic is critical to balancing the immune response and efficacious treatments. (ziegler et al., ) . descriptions of viral loads determined by pcr in clinical infections note that respiratory viral loads (sputum or nasopharyngeal swabs) were significantly higher, peaked later and were detected longer in severe vs. mild infections zheng et al., ) . while virus could be detected for longer periods from stool samples, there was not a difference between mild and severe disease (zheng et al., ) . a larger study of hospitalized cases demonstrated that viral load detected by pcr from nasopharyngeal swabs was a significant predictor of mortality, with mean log viral loads of . copies per ml for survivors vs. . copies per ml for those who died (pujadas et al., ) . gulati and colleagues published a comprehensive review of coronavirus disease, comparing sars-cov, mers-cov and sars-cov- , focusing not just on pulmonary disease but encompassing other organ systems as well: cardiovascular, hepatobiliary, gastrointestinal, renal, neurologic, musculocutaneous and hematologic (gulati et al., ) . the covid- reports were retrospective, clinicopathologic or case reports, and generally noted increased frequency of extrapulmonary involvement with more severe disease (icu) and comorbidities. dyspnea is predictive of icu admission, and many patients exhibited abnormal chest computed tomography, predominantly ground glass opacities. histopathology demonstrated interstitial fibrosis and inflammatory infiltrates. severe disease requiring intensive care was associated with lymphopenia, leukocytosis, neutrophilia, and increases in alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, total bilirubin, creatinine, creatinine kinase, blood urea nitrogen, troponin-i and d-dimer (gulati et al., ) . these extrapulmonary organ systems are not well understood in most animal models; hence, the primary endpoints of animal models have been testing of viral load and immune and inflammatory responses. no single animal model has ever been predictive of human infection, or exactly modelled it. human infection happens by chance, and very little is known about how long a person needs to be in contact with the pathogen nor how much pathogen is needed prior to symptoms presenting. for animal models to be useful, we need to identify the most reproducible methods for challenge that lead to infection and symptoms that emulate those seen in humans. host tropism for viruses is complicated by the need for host factors to facilitate virus entry and replication, the host's immune response and pathways by which viruses evade immune responses (douam et al., ) . models for the closely related sars-cov and mers-cov viruses have paved the way for sars-cov- infection and pathogenesis studies and have bolstered expertise in studying coronavirus vaccines and therapeutics. angiotensin converting enzyme or ace was discovered as the receptor for sars-cov and has proven to be the receptor for sars-cov- as well (zhou et al., b) . phylogenetic comparisons of ace proteins allowed for predictions of species that are susceptible to sars-cov- infection luan et al., ; qiu et al., ; wan et al., ) , as ace is a key determinant of infectivity . mice have mismatched ace receptors and two approaches have been used to solve this problem: ( ) mice that express the human ace receptor; or ( ) a sars-cov- strain that is adapted to recognize the murine ace receptor. although both approaches provide a way to model covid- infection in mice, neither is a perfect substitute for human infection. the goal for animal models is to understand and/or provide the most accurate and complete predictor of results in a variety of human clinical settings. at this time, there are several models for sars-cov- in development ( table ) . nhp models are traditionally considered the most translational models to humans. nhps bear close similarities to human genetic, neurological, cognitive, physiological, reproductive, anatomical, and immunological systems. their susceptibility to most human pathogens is not surprising, and as such, are models for many of the most intractable acute and persistent pathogens. their size and longevity make them excellent models for pathogenesis, allowing repeated sampling and imaging in vivo for longitudinal studies. however, their limited supply in the face of numerous drug and vaccine candidates makes them an even more precious resource. it is therefore imperative to prioritize agents to be tested by demonstrating tolerability and efficacy in smaller mammalian models: mice, hamsters and ferrets are currently the small animal species of choice. small animal models are presented first, as the tractable models used in early discovery and development. mice and hamsters predominate, given that there are many choices that are becoming available. guinea pigs do not appear to be productively infected as virus cannot be detected, despite presenting early (day ) pulmonary histological changes, therefore are not the best model of disease (dick bowen, personal communication). mice mice hold a prominent position in disease research. their small size, ease of use, rapid breeding, ability to be inbred as well as readily genetically modified have made the mouse the go-to model in biomedical research. mice can be used to rapidly screen vaccines, antivirals, and other therapeutics in a relatively high throughput, pipeline approach. there are several mouse models for sars-cov- infection listed in table , each with advantages and limitations. it bears emphasizing that mouse models that are not available from public repositories or commercial vendors cannot be scaled effectively to meet the high demand for animals and supply the high throughput needs of the community. those models that remain in private labs, no matter how good they may be, will be of limited value to the large-scale efforts needed to evaluate the many vaccines, antivirals and other therapeutics being investigated for the pandemic response, unless they are made available through commercial suppliers or repositories. the use of the standard laboratory mouse for infection of sars-cov and sars-cov- has been limited due to amino acid differences in the ace receptor between mouse and humans that result in reduced susceptibility to infection in this model. older balb/c mice are more susceptible to infection, but still the infectivity is modest, and the requirement of an aged animal can be a significant disadvantage in screening therapeutics (roberts et al., a; vogel et al., ) . one solution to the lack of infectivity in laboratory mice is to use an adapted virus, where multiple passages/or and selected mutations in the virus make the mice more susceptible to infection. a recent publication demonstrated a mouse adapted virus q t/p y that was not only able to readily infect mice but was used to demonstrate the effectiveness of neutralizing antibodies to reduce viral replication in vivo . a second solution to the lack of infectivity in common laboratory mice is to express the human ace gene (hace ), either by viral transduction or genetic engineering. two labs have used adenovirus transduction to express human ace in the lungs of mice, resulting in mild disease noted by viral replication and weight loss, that could be ameliorated by neutralizing monoclonal antibodies (hassan et al., ) or antivirals and convalescent sera (sun et al., a) . several transgenic mouse models carrying the human ace gene were created to study sars-cov infection. five transgenic models currently exist and are rapidly being assessed for their use in the study of covid- . the first model is a transgenic mouse developed in china by chuan qin, where the human ace cdna is under the control of the mouse ace promoter, icr-tg(ace -ace ) cqin/j. sars-cov- infection in these mice leads to weight loss and virus replication in lung with histopathology indicative of interstitial pneumonia. sars-cov and sars-cov- cause mild disease and no death was reported in this model . a second model, developed by clarence peters in , employed another transgenic construct tg(cag-ace )ac ctkt with the human ace gene expressed under the control of a ubiquitous cag promoter. these mice are highly susceptible to sars-cov infection, demonstrate weight loss, along with other clinical manifestations before reaching % mortality within days after intranasal infection (tseng et al., ; yoshikawa et al., ). these mice are currently being bred in order to determine how they respond to infection with sars-cov- (kent tseng, personal communication). two other transgenic mouse models also expressing the human ace gene using different epithelial based promoters, tg(foxj -ace ) rba and b .cg-tg(k -ace ) prlmn/j, were developed by ralph baric and stanley perlman, respectively (mccray et al., ; menachery et al., ) . both models are considered severe models of sars-cov, with significant weight loss, disease pathology, evidence of encephalitis and death by - days post infection. interestingly, they differ in severity for covid- , where the b c tg(foxj -ace ) rba model displayed a variable response to infection with sars-cov- , while the b .cg-tg(k -ace ) prlmn/j model develops a uniformly severe response. half of the infected b c tg(foxj -ace ) rba mice recovered from infection, demonstrating no significant signs of weight loss or illness; the other half of the infected b c tg(foxj -ace ) rba mice demonstrated severe illness, body weight loss and required euthanasia . the b .cg-tg(k -ace ) prlmn/j mouse develops a severe infection and lung pathology in response to sars-cov- . reports from multiple independent research groups report that upon infection, mice lose significant body weight, have a hunched posture and require euthanasia - days post challenge in % of infected mice moreau et al., ; oladunni et al., ; rathnasinghe et al., ; winkler et al., ) . importantly, these mice are not hypersensitive to infection, and lower doses of the virus result in less severe disease progression . viral loads are detected in the brain of these mice, but initial reports indicate the brain infection to be less than the severity-associated encephalitis seen with sars-cov infection in this model, with minimal histopathological changes observed (oladunni et al., ) . the tg(foxj -ace ) rba and b .cg-tg(k -ace ) prlmn/j mice are available for distribution to research scientists by the mmrrc and the jackson laboratory, respectively. another mouse model was recently developed by sun et al, c bl/ -ace em (ace )yowa . this model utilizes crispr/cas to knock-in the human ace cdna into the mouse ace locus, utilizing the endogenous mouse ace promoter. this model also results in infectivity, however, the mice recover from body weight loss post infection with sars-cov- with mild lung pathology (sun et al., b) . given the ease of genetic engineering in mice, one can expect to see a variety of new models developed in the immediate future. these models will differ in terms of random transgenesis, targeted knock ins, variations in constructs, reporter tags and new ways to deliver the human ace gene, such as through recently reported viral transgenesis (hassan et al., ; sun et al., a) . humanized mice represent another potential approach, and while there are distinct advantages, there are additional challenges that impact rigorous testing of vaccine and therapeutic efficacy, namely the required technical expertise, preparation time, immunocompromised status of mice, variability, and limited throughput (skelton et al., ) . xenografts of human lung tissue into immunodeficient mice have resulted in lung-only mice or bone marrow/liver/thymus-lung (blt-l) mice, which demonstrated infection with mers-cov, rsv, zika and cytomegalovirus and in the case of blt-l mice, produced a human immune response (wahl et al., ) . the use of nod scid gamma (nsg) mice for engraftment of human immune cells will prove to be a powerful tool in studying the interaction of the human immune system with the virus. new models incorporating the nsg genetic background and the human ace gene are underway. while the current models will be critical for high throughput screening of therapeutics, the newer and milder models will be critical to understanding how infection responds to comorbidities such as type diabetes, hypertension, obesity etc. these j o u r n a l p r e -p r o o f comorbidities can be induced, but also achieved through genetic crosses to existing mouse models engineered with mutations in key genes to produce these desired phenotypes. furthermore, differences between mice and humans can be manipulated to more faithfully represent human disease, such as exploiting differential regulation of ace by interferons (ziegler et al., ) . other applications of the mouse models include exploring the effects of genetic diversity and various genetic backgrounds, again helping to translate why some individuals are more susceptible to severe disease manifestations, while others recover quickly or are asymptomatic. golden syrian hamsters have been shown to have distinct advantages as models for diseases involving respiratory viral infections including influenza virus, adenovirus and sars-cov (miao et al., ; roberts et al., b) . following infection by the intranasal route, golden syrian hamsters demonstrate clinical features, viral kinetics, histopathological changes, and immune responses that closely mimic the mild to moderate disease described in human covid- patients (chan et al., b; imai et al., ; sia et al., ) . in this form of non-lethal disease, the clinical signs include rapid breathing, decreased activity and weight loss that is most severe by day post infection. airway involvement is evident with histopathology showing progression from the initial exudative phase of diffuse alveolar damage with extensive apoptosis to the later proliferative phase of tissue repair. micro-ct analysis of infected hamsters revealed severe lung injury with the degree of lung abnormalities related to the infectious dose. commonly reported imaging features of covid- patients with pneumonia were present in all infected animals (imai et al., ) . high dose sars-cov- infection led to severe weight loss and partial mortality while older hamsters appear to exhibit more pronounced and consistent weight loss (osterrieder et al., ) . other findings include intestinal mucosal inflammation, myocardial degenerative changes, viral rna in brain stem and lymphoid necrosis. there is a marked activation of the innate immune response with high levels of chemokines/cytokines induced by the infection (chan et al., b) . transmission of covid- from infected hamsters to naïve cage mates suggests utility of the model for studying transmission (chan et al., a; chan et al., b; sia et al., ) . in addition, passive transfer studies, with either convalescent sera (chan et al., b) or neutralizing monoclonal antibodies (rogers et al., ) show great promise for studies related to immunity and vaccine development. the golden syrian hamster model of sars-cov- infection appears to be a suitable model for the evaluation of antiviral agents (kaptein et al., ; rosenke et al., ) and candidate vaccines . hamsters carrying the hace receptor under the control of the epithelial k promoter are also being evaluated as a model. in an initial study of sars-cov- infection of hace -hamsters, clinical signs were observed including elevated body temperatures, slow or reduced mobility, weight loss and mortality ( out of animals). virus titers were detected in lungs, heart and brain tissues, with the highest titers observed in lungs on days - (> logs) (bart tarbet, personal communication). hamsters with compromised immune systems, by either cyclophosphamide treatment or rag -deficiency demonstrated more severe disease, longer in duration (cyclophosphamide induction) or resulting in mortality (rag -/-) and could be protected by human antibody given prophylactically . ferrets are considered good models for respiratory diseases as the physiology of their lung and airways are close to humans and they have been used extensively to model disease caused by many respiratory viruses including influenza (thangavel and bouvier, ) , rsv (stittelaar et al., ) and sars-cov (van den brand et al., ) . unlike rodents, ferrets cough and possess a sneeze reflex, making them a particularly useful model in the study of disease transmission. ferrets exhibit lethargy and appetite loss following infection with sars-cov- via the intranasal route, but the disease does not progress to acute respiratory disease, and the animals recover from the infection shi et al., ) . virus shedding from the upper respiratory tract (nasal washes, saliva) can persist for up to days post-infection; the length of shedding appears to be dependent on the initial viral challenge dose and can be intermittent after days. mild multifocal bronchopneumonia is observed early post-infection (day in animals receiving to logs of virus) with mild multifocal bronchopneumonia developing after one week. fever has been reported in some studies but neither coughing nor dyspnea have been observed shi et al., ) . ferrets re-challenged after days post initial infection appear to be completely protected (ryan et al., ) . sars-cov- was transmitted readily to naïve direct contact ferrets but less efficiently to naïve indirect contact ferrets (shi et al., ) and efficiently via the air resulting in a productive infection and the detection of infectious virus in indirect recipients (richard et al., ) . disease in ferrets following sars-cov- infection appears to be very mild, less severe as compared to ferrets infected with sars-cov. the use of small animals for preclinical research in the study of sars-cov- infection involves a broad spectrum of models, from infecting wild type animals with adapted viruses, to multiple methods of introducing human ace receptors. disease is typically mild although transgenic mice present with more severe disease. each model offers selected advantages that will be useful not only for the testing of therapies and diseases, but in the understanding of disease enhancement and related co-morbidities. nhps are indispensable models for evaluating medical countermeasures against infectious diseases and are considered the gold standard animal model for modeling human infectious diseases. the lack of suitable substitutes for nhp models for predicting response in humans serves as a bottleneck for the development of countermeasures against infectious disease like sars-cov- . summarized below is the progress to date to establish models for covid- in different primate species. rhesus macaques (m. mulatta) exposed to sars-cov- become infected and display a mild, non-lethal shedding disease phenotype, which includes little to no clinical observations. if j o u r n a l p r e -p r o o f clinical observations are reported they are typically transient and include reduced appetite, mild dehydration, tachypnea, piloerection and dyspnea (munster et al., ) . when reported, fever is mild and transient beginning shortly after exposure (i.e., day pe) and resolving within or days (munster et al., ) . body weight loss findings, if reported, are mild transient drop in weights followed by recovery (munster et al., ) . clinical chemistry and hematology are generally unremarkable. however, transient leukocytosis, neutrophilia, monocytosis and lymphopenia are reported (munster et al., ; singh et al., ) . imaging (radiographs or pet/ct) confirm rhesus macaques are infected, with infiltrates and ground-glass appearances in radiographs beginning early after exposure (day or ) and resolution occurring by day - post-exposure (munster et al., ; singh et al., ) . anecdotal evidence suggests that older rhesus develop a chronic infection where infiltrates persists throughout the study . when available, pet/ct images corroborate the radiograph findings (singh et al., ) . virus is detected in nasal, throat, rectal, ocular swabs and in bronchoalveolar lavage (bals) via median tissue culture infectious dose (tcid ) beginning approximately day post-exposure (pe), peaking around day / pe and decreasing after day pe (chandrashekar et al., ; chao shan, ; deng et al., ; munster et al., ; yu et al., b) . finally, exposed rhesus macaques seroconvert, as demonstrated by a sars-cov- anti-spike elisa and neutralization assays to various endpoint titers depending on the laboratory and assay format utilized, and are protected from reinfection (bao et al., a; munster et al., ) . cynomolgus macaques (m. fascicularis) have been used to study the pathogenesis of sars-cov, where aged animals were more likely to develop disease. when exposed to sars-cov- , they become infected, but show no overt clinical signs of disease . weight loss is not observed, but in some studies, infected animals have a fever on day two to three (johnston et al., ; . virus shedding from the upper respiratory tract occurs, peaking early at day one post infection in young animals and day four in aged ( - years) animals, then decreasing rapidly but still detected intermittently up to day post infection . overall, higher levels of virus shedding were measured in aged animals compared with young animals . they develop mild to moderate lung abnormalities, macroscopic lesions in the lungs including alveolar and bronchiolar epithelial necrosis, alveolar edema, hyaline membrane formation and accumulation of immune cells (finch et al., ; johnston et al., ) . while self-limiting, the disease in cynomolgus macaques does recapitulate many aspects of human covid- and could be utilized to test preventative and therapeutic strategies . african green monkeys (agms) exposed to sars-cov- as young adults display a mild, nonlethal shedding disease phenotype, which includes little to no clinical observations (hartman et j o u r n a l p r e -p r o o f al., ). if clinical observations, such as fever, are reported, they are typically transient and mild with no serious manifestations (hartman et al., ; woolsey et al., ) . body weight findings are generally unremarkable. clinical chemistry and hematology reveal mild and transient shifts in leukocyte populations, mild thrombocytopenia, and selected liver enzymes (woolsey et al., ) . a measure of acute inflammation, crp, is elevated early in infection (woolsey et al., ) . imaging (radiographs or pet/ct) confirms agms are infected with infiltrates and ground-glass appearances in radiographs beginning early after exposure (day or ) and resolving by day - pe. when available, pet/ct images corroborate the radiograph findings. finally, plethysmography suggests respiratory disease, but there is no consistent trend (hartman et al., ) . presence of sars-cov- in bal via rt-pcr and plaque assay is detected by approximately day pe and is present at least through day pe (hartman et al., ; woolsey et al., ) . finally, exposed agms do seroconvert as demonstrated by a sars-cov- anti-spike elisa and neutralization assays to various endpoint titers, depending on the laboratory and assay format utilized (hartman et al., ; woolsey et al., ) . of note, one study (hartman et al., ) used a viral isolate from munich which had the d g amino acid substitution in the spike protein that has been demonstrated to be more infectious to cells in culture and was reported to be more infectious in humans (korber et al., ) . disease in those animals was similarly mild as was seen in agms infected with the washington isolate without the d g substitution. a limited dataset suggests that age is a co-morbidity for sars-cov- disease severity in agms. investigators at tulane national primate research center infected older agms, two by multiple routes of infection including intranasal, intratracheal, and conjunctival routes, and two animals by aerosol route with sars-cov- . one of two animals from each group exhibited acute respiratory disease syndrome, ards, with radiologic and histologic abnormalities observed primarily within the right caudal lung lobes (blair et al., ) . of the two animals with severe disease, one met the criteria for euthanasia at day (aerosol) and the other at day (multiroute infection) (blair et al., ) . there did not appear to be significant differences in viral replication, or disease pathogenesis associated with aerosol versus multi-route infection (blair et al., ) . of the surviving older agms, clinical disease was mild. some transient fevers and lethargy were observed, but no serious manifestations. clinical chemistries and hematologies were generally unremarkable. radiographs confirm the older agms are infected with infiltrates and groundglass appearances in radiographs beginning early after exposure (day or ) through at least day pe. pet/ct images corroborate the radiograph findings. finally, viral shedding (rt-pcr) in pharyngeal, nasal, buccal, bronchial brush, rectal and vaginal swabs begins ~d -d / pe in exposed animals (blair et al., ) . in view of the severe disease observed in of animals as well as the observation of cytokine storm in of animals, older agms may serve as faithful if impractical models of severe disease to evaluate therapeutic strategies such as immune modulators and may also provide insights into sars-cov- pathogenesis. however, additional studies are clearly needed to corroborate these initial findings. other non-human primates finally, limited data is available describing disease presentation following sars-cov- exposure in pigtail macaques, baboons, and marmosets. post-exposure data shows these animals can be infected but they are not as widely used. baboons had more severe pathology and shed virus longer than macaques, and may be a good model for cardiovascular and diabetic comorbidities (singh et al., ) ; they have also been used for immunogenicity studies (tian et al., ) . marmosets did not exhibit fever, were difficult to monitor by radiograph, and did not mount an immune response even though they were positive for virus by nasal swabs ; pathology was reduced compared to macaques (singh et al., ) . thus far, pigtail macaques have only been used for immunogenicity studies (erasmus et al., ) . the authors recognize that the development of new and refined animal models for covid- disease is a rapidly evolving field and that this summary is not a complete review, but rather a description of the currently available data and the resulting guidance that can be drawn from it. some themes are emerging and warrant monitoring. mice expressing hace and hamsters are reasonable models for early testing of potential countermeasures, displaying mostly mild disease with reproducible endpoints of weight loss and viral burden, and in transgenic mice, severe disease resulting in lethality. intranasal inoculation is generally the route of infection, and the dose of virus varies and has an impact on the course of disease. given the availability of animals and the ability to rigorously test for statistical significance using groups of or more animals, small models should be utilized to the greatest extent possible and may be sufficient for moving into clinical studies. based on available data from multiple challenge routes (intratracheal, intranasal, oral, ocular and combinations of these, along with small particle aerosol) and multiple dose ranges ( e - e ), routes and challenge do not appear to have significant impact on disease presentation (i.e., mild yet reproducible). as the nhp model matures, focused efforts will be required to demonstrate any dose response and different disease presentation following a range of doses and routes. anecdotal evidence suggests severe disease may be more common, however, focused studies are needed to confirm. based on a fisher's exact test with alpha level . , a sample size of n= per group ensures % power to detect % vaccine efficacy in a comparison of protection between vaccinated and sham animals (measured by absence of pathology in lung, assuming that % of sham animals become infected). analysis of selected nhp studies shows that n= per group ensures % power to detect a mean difference of ~ . log viral burden between groups (as measured by genomic rt-pcr in bal on day post challenge; allan decamp, personal communication). additional data are needed to allow these calculations to be applicable to across studies and laboratories. young adult rhesus macaques have extremely mild symptoms that may be difficult to associate with symptomatic benefit for interventions, however viral load differences may prove a useful biomarker. african green monkeys may prove to be more useful and data is still being collected for cynomolgus macaques. while there may be a desire to select the best animal model for comparison across studies, in practice, all good models should be utilized to meet the demand for countermeasure testing. few models faithfully recapitulate severe disease, and further exploration is needed, including assessment of comorbidities. to understand the landscape and to facilitate rapid data sharing, activ wg members created an inventory tool to collect information on animal studies from published literature, preprints, and unpublished studies in progress. this tool started as a spreadsheet to collect the most important information about studies, such as information on the animals, the virus isolates used, the challenge process, parameters measured in the studies, and relevant observations/endpoints, along with the laboratory performing the study. the group then made assessments as to whether disease was none-to-minimal, mild, moderate or severe. a few species that are not infected are included, to prevent unnecessary duplication or perhaps even to spur enhancements, for example the development of adapted viruses. a summary of the information is available on the ncats open portal, under the animal models tab (https://opendata.ncats.nih.gov/covid /animal). as we learn more about models, this site will be updated. activ preclinical wg members have curated this information by combing peer reviewed publications and preprints, along with information that is not publicly available but is available to us. additional efforts are currently underway to develop a nonhuman primate studies covid- coordinating center (nhpsccc) that will bridge the covid- work at the national primate research centers and could be a repository for data from many animal models. recognizing that activ does not constitute the entire universe of activity in this area, we invite others to submit data to activ, particularly as it fills gaps or further informs the models we have summarized. the information on submitting such animal model data will be found on the ncats open portal animal model page when it becomes available. it is important to consider these animal models in the context of human disease. we adapted a framework for human disease from siddiqi and mehra (siddiqi and mehra, ) and placed the animal models summarized here in that framework (figure ) . this graphical framework includes the full spectrum of disease but does not indicate the frequency of severity. the authors noted that % of cases recovered after mild disease, while % progressed to the most severe form, with half of those cases succumbing to disease, for a . % mortality rate (siddiqi and mehra, ) . it is therefore not surprising and completely consistent with human disease that most animal models present with mild disease and recover. some aged mice and aged african green monkeys present with more severe disease than younger animals; additional comorbidities have not yet been explored but would be expected to model more severe disease. each of the current models in development is yielding valuable information about infectivity, routes of infection, viral persistence, reinfection, and relative level and types of pathogenesis per species. as researchers around the world seek to discover effective therapies and vaccines to treat or to prevent covid- , judicious choices will be needed to assure that the models are available for comparative studies. the complex interplay between the host and the virus means that each model is unlikely to represent every aspect of disease in humans, and thus different models may be recommended for therapeutics compared with vaccines, due to animal availability and endpoints of studies. indeed, investigators have begun testing vaccines and therapeutics as soon as species are known to get disease, more rapidly than under normal circumstances, where additional studies might be performed to better understand the disease model prior to testing countermeasures. table indicates which models could be used for testing vaccines, antivirals, neutralizing antibodies or other therapies, but does not make specific recommendations. this is based on the relevant endpoints one can measure that would inform vaccine or therapeutic efficacy: viral load (swabs, bal, etc.), body weight, body temperature, lung imaging, lung function, and cytokines. antivirals have been tested in mice (sheahan et al., ) , hamsters (kaptein et al., ; rosenke et al., ) , cynomolgus macaques (maisonnasse et al., ) and rhesus macaques (rosenke et al., ; williamson et al., ) . antibodies have been assessed in hamsters rogers et al., ) and mice . finally, vaccines have been tested for immunogenicity in small animals, but efficacy studies have largely been done in rhesus macaques van doremalen et al., ; yu et al., a) . the activ animal model pages on the ncats open portal will be updated with publications reflecting the utility of various models. master protocols currently in development will define key parameters and timepoints to follow along with group sizes, allowing comparison across studies. the severity of disease, rapid transmission and global spread of this new virus make the development of vaccines and therapeutics an urgent priority. typical development plans have been greatly accelerated by performing many activities in parallel, often at risk, shortening timelines with unprecedented speed (figure ) . this includes animal model development and testing, which can contribute to the identification and confirmation of correlates and surrogate markers for use in the clinic. the vaccines that have been chosen by ows can be compared in animal models while phase i and phase ii clinical work continues (corey et al., ) . selection of an appropriate animal model depends on the questions to be addressed. table presents our recommendations on which of the currently available animal models to select, focusing on testing classes of therapeutics and vaccines for reducing various aspects of covid- . for example, carboxylesterase activity is high in rodents but not humans (bahar et al., ; li et al., ; rudakova et al., ) , impacting the pharmacokinetics of antivirals such as remdesivir and the suitability of models for preclinical testing of specific drugs (warren et al., ) . it is clear that many models are available which faithfully reproduce the early phases of infection and lung disease, followed by recovery; recovery is a prevalent feature of covid- in humans. models of severe disease are largely determined by fatal outcomes when untreated but may not capture all signs and symptoms associated with human covid- disease, for example coagulopathies. one difference between some models and human disease is the observation of virus in the brain in mice and hamsters, which requires further exploration. there is a report of virus in csf in a single clinical case (zhou et al., a) though most neurological complications are considered to be inflammatory responses (gulati et al., ) . comorbidities have not been rigorously explored in animal models thus far, yet we know the strong association of age, hypertension, diabetes, lung and heart disease with poor prognoses in covid- . immune responses likely play a key role in determining the severity of disease yet are not as easily manipulated in animal species that are phylogenetically closer to humans as they are in mice. in order for these preclinical studies to have the most impact, their design is critically important to assure that results are rigorous and reproducible. studies will require statistical justification based on clinical and laboratory measures such that studies are appropriately powered, to prevent getting uninterpretable results that can be misleading and wasteful of animal resources. one method to enhance reproducibility that is currently under development is the adoption of shared master protocols for design and sampling. not only can experiments be compared across sites, but also controls located at different sites can be combined for increased power, while retaining contemporaneous infection controls at each site. this strategy can reduce the number of controls used overall, conserving precious resources. if assays and challenge stocks can be harmonized across experimental sites, many of the variables can be further reduced. defining animal models and their use is a prerequisite to performing studies to compare vaccines and therapeutics, so that the most promising ones advance to the next phase, whether it is testing in nhps or the clinic. the prioritization schemes for the development and testing of treatments and vaccines are beyond the scope of this manuscript. this aim of this manuscript is defining the utility of animal models to inform the requisite prioritization of animal studies, particularly in nhps where resources are limited relative to the number of candidate vaccines and therapeutics. covid- is a multi-faceted, multi-factorial, multi-systemic, highly infectious disease that evokes wide ranging responses in humans, from asymptomatic to severe disease with respiratory, gastrointestinal, circulatory, neurological and normal to hyper immune responses. no single animal model recapitulates the totality of pathogenesis or predicts interventional responses faithfully as in the human. yet as with the response to other emerging infections, animal models will play a key role, even as candidate vaccines and therapeutics are entering clinical trials at a record pace. we have presented various animal models that are being developed and the role they may play in responding to covid- . our summary table of animal models and their applications has been posted to the ncats open portal (https://opendata.ncats.nih.gov/covid /animal) and will be continuously updated as models are advanced and further interrogated. the key models appear to be mice (various), hamsters and for nhps, rhesus macaques and african green monkeys. most mimic the mild form of covid- disease, with the exception of transgenic mice, and additional research may result in development of more severe disease models. activ invites submission of information on animal models to be included in our assessment, as we work toward standardized and harmonized animal models, while balancing resources and availability. table . recommended animal models for specific stages of human covid- disease. within each medical need, the disease aspects are presented in order of increasing complexity, while animal models are presented in the order in which they should be approached. advantages and limitations are also presented to help in the selection of the appropriate animal model. agm = african green monkey; ards = acute respiratory distress syndrome; mabs = monoclonal antibodies; nhp = non-human primates; pk = pharmacokinetics species difference of esterase expression and hydrolase activity in plasma lack of reinfection in rhesus macaques infected with sars-cov- . biorxiv the pathogenicity of sars-cov- in hace transgenic mice ards and cytokine storm in sars-cov- infected caribbean vervets disruption of adaptive immunity enhances disease in sars-cov- surgical mask 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province sars-cov- : underestimated damage to nervous system a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues the authors would like to thank the activ preclinical working group for discussions and the spark for this manuscript. we would also like to thank annaliesa anderson for critical review, along with michael diamond, prabha fernandes, and tomas cihlar. we would especially like to thank kara carter and jay grobler for their review and discussions on figures and shared content with the companion manuscript. we thank katinka vigh-conrad for assistance with figures. key: cord- -itvc cv authors: hollingsworth, t déirdre title: counting down the goals for neglected tropical diseases: what have we learned from quantitative analysis and transmission modeling? date: - - journal: clin infect dis doi: . /cid/ciy sha: doc_id: cord_uid: itvc cv the control of neglected tropical diseases (ntds) has received huge investment in recent years, leading to large reductions in morbidity. in , the world health organization set ambitious targets for eliminating many of these diseases as a public health problem by , an aspiration that was supported by donations of treatments, intervention materials, and funding committed by a broad partnership of stakeholders in the london declaration on ntds. alongside these efforts, there has been an increasing role for quantitative analysis and modeling to support the achievement of these goals through evaluation of the likely impact of interventions, the factors that could undermine these achievements, and the role of new diagnostics and treatments in reducing transmission. in this special issue, we aim to summarize those insights in an accessible way. this article acts as an introduction to the special issue, outlining key concepts in ntds and insights from modeling as we approach . neglected tropical diseases (ntds) are a diverse group of infections identified by the world health organization (who) as diseases that predominantly infect low-income populations in tropical countries, causing a large burden of morbidity and some mortality, and thus perpetuate the cycle of poverty [ ] . in , the who declared ambitious targets to reduce the burden of these diseases by eliminating them as a public health problem by [ ] . in support of these aspirations, a diverse consortium of donors, pharmaceutical companies, government agencies, and others made large commitments of funding, donated treatments, and other activities for of these diseases in the london declaration on ntds [ ] . large morbidity gains have been made over recent years [ ] , and there are active discussions on how to exploit the likely synergies between the goals for ntds and universal health coverage (uhc), a sustainable development goal (sdg; target . ) [ ] , in particular how to extend these gains to the hardest-to-reach or conflict-affected communities [ ] . of these diseases, guinea worm is targeted for eradication; the remaining infections are targeted for elimination as a public health problem in some settings. the adjustment of strategies to achieve control of the infections, informed by mathematical modeling, is the focus of this special issue. infectious disease modeling has an increasing role in public-health policy, with resulting challenges and successes [ ] . appropriate analyses can provide thorough investigation and interpretation of data, as well as identify where the knowledge gaps are most acute. models can also be used to rigorize our thinking on the processes of infection and transmission and test hypotheses about the likely dynamics and epidemiology. although there has been ongoing research into modeling of ntds [ , ] , this research has sometimes been limited by the extent of biological knowledge and data on which to base these models. the availability of more extensive data, together with strong partnerships between researchers in different fields, including by the ntd modelling consortium [ ] , has led to marked improvements in these efforts. researchers have made contributions not only in informing treatment strategies but also in informing diagnostic development and the applicability of new tools or treatments and in understanding the natural history of disease. however, in comparison with other infectious diseases, we still have limited epidemiological data on ntds; thus, although we have performed formal model comparisons [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , there remain large uncertainties in processes and parameters that could have an impact on the dynamics, as highlighted below. this means that we need to be cautious about overstating our results, even when the policy need is acute. this presents us with the challenge of correctly calculating and communicating the uncertainties in these complex systems while still giving an accessible message to end users. this article acts as an introduction for a special issue that aims to increase the accessibility of the results so far by summarizing insights from ntd models and identifying key themes for the control of these diseases. it should be noted that in this special issue and in this article we have focused on epidemiological analyses and modeling and have not extended our focus to geospatial, spatial dynamic, or health economic modeling, all of which have an important part to play in developing policy for infectious diseases. we focus on the role of interventions to reach the goals for ntds. the authors of this issue are aiming to increase the repeatability of our science. the code for the models used in this special issue were previously published alongside more technical articles [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] as supplementary information or on our website (www.ntdmodelling.org). the release of raw code is not a complete answer to accessibility and reproducibility, but it is a step in the right direction [ ] . the diseases are usually divided into groups based on the methods used to control them, and we have summarized our results in this way. the first group includes those diseases that are mainly controlled by intensified disease management (idm) or increased detection, screening, and treatment of infection. the second group includes those diseases that are mainly controlled by mass drug administration (mda). although there are major epidemiological differences between diseases in these groups, they share some common uncertainties in informing control, which are discussed in each section. leprosy, the gambian form of sleeping sickness (human african trypanosomiasis), visceral leishmaniasis in the indian subcontinent, and chagas disease are four london declaration ntds which are characterized as idm infections. they have long, variable periods between infection and symptomatic disease and, generally, late diagnosis. control strategies are focused on reducing time to diagnosis and case finding with accompanying vector control, where appropriate. these diseases are characterized by long, uncertain incubation periods and an unknown degree of transmission by asymptomatic individuals (figure ). the potential role of asymptomatic individuals in transmission is well known in epidemiology. the close link between symptoms and infectiousness for smallpox and severe acute respiratory syndrome (sars) has been calculated to be crucial in controlling these diseases [ ] . that analysis explicitly considers the relative contribution of asymptomatics in terms of their contribution to the number of onward transmissions that an individual would be responsible for during a new outbreak, or the basic reproduction number, r , in a way that is implicitly included in many models, but less elegantly presented. we could adopt a similar framework for these ntds. first we would need to separate asymptomatics into presymptomatic and nonsymptomatic (figure , yellow and blue), highlighting the problems in language around asymptomatics [ ] . for the area under this curve is proportional to the expected number of onward transmissions due to different stages of infection in a wholly susceptible population. if there are many nonsymptomatic people for each symptomatic individual, they may collectively contribute substantially to transmission even if their individual contribution is low (multiple yellow areas for single blue, green areas). for the symptomatic individuals, the relative infectiousness and duration of the symptomatic phase will determine the population-level impact of diagnosing and treating cases earlier (covering more of the green or even blue area). vector control or other mass interventions could reduce both symptomatic and asymptomatic transmission by reducing all transmissions. nonsymptomatic individuals yellow, left hand plot (figure ), infectiousness may rise and fall but is generally expected to be low, based on pathogen measurements. a key question is, of course, the relationship between these pathogen measurements and transmission, which is unknown and likely to be nonlinear. the contribution of a single nonsymptomatic individual to transmission is proportional to the area under the infectivity since infection curve (yellow area, figure ), which might be large or small when compared with symptomatic infection. a similar calculation has contributed to the debate in human immunodeficiency virus (hiv) around the relative roles of the early, brief period of high infectivity when compared with the much longer period of asymptomatic infection with lower transmission rates [ ] [ ] [ ] . unfortunately, every aspect of infectivity and duration of infection ( figure ) is highly uncertain for these ntds. of course, it is not only the contribution of each nonsymptomatic individual to transmission that is important, but also the proportion of the infected population who fall into this group ( figure , yellow vs blue). it is also crucial to note that this contribution will change through the course of an epidemic and an intervention and will be dependent on the type of intervention being applied, as nicely illustrated for visceral leishmaniasis in this issue [ , ] . if the symptomatic phase is highly infectious and of sufficient duration ( figure , right-hand plot, green and red), interventions to identify cases early in the symptomatic period are likely to be highly effective. although chagas may not follow the increasing infectivity over time pattern but instead have rather high infectivity during early acute infection, diagnosis is so rare that postsymptomatic infections are often treated as asymptomatic infections, which may contribute substantially to transmission [ ] . in summary, the balance between infectivity, duration of infection, and frequency of asymptomatic versus symptomatic infection may undermine any attempt to control a disease solely by increased case finding. this can be mitigated by reducing all infectivity through vector control or other transmission-reducing interventions, which reduces the infectivity of all infected invidiuals (the height of the curve in figure ). although this theoretical framework is useful, there is much modeling work to be done to populate a more concrete discussion of the relative roles of different phases of infections for these complex diseases. the details of each infection are, of course, very important and should be considered individually. leprosy, a directly transmitted bacterial infection, was one of the first ntds to have global targets for elimination as a public health problem, leading to large declines, although these have stabilized in the last decade [ ] [ ] [ ] . leprosy exemplifies the problem with surveillance for a disease in which cases are identified if both the infected individuals seek care and the appropriate care is available for them and mathematical modeling provides methods for estimating the pool of undiagnosed infections [ ] . in addition, modeling has highlighted the need for much earlier diagnosis and suggests that targeted case finding through household contact tracing, perhaps combined with postexposure prophylaxis, could hold great potential for control (table ). in constrast with the global scope of leprosy, the gambian form of sleeping sickness (human african trypanosomiasis), which is transmitted by tsetse flies, is focused in western africa. it has a high case fatality rate and is targeted for elimination because it is thought to be an anthroponotic disease and current interventions have led to large drops in case numbers [ ] . the main method of control is through screening of populations and treatment of infected individuals. a key question for sleeping sickness is the potential contribution of vector control as a complement to screening and treating. modeling suggests that it could have a large impact, reducing transmission not only from cases but also from the uncertain quantity of asymptomatic or presymptomatic individuals, or even animal hosts [ ] . the modelers also highlight the potential to increase the impact of screening, both passively, by increased access to diagnostics, and actively, by targeting highrisk groups (table ) , to reduce the duration of infection and therefore transmission (the area under the curve in figure ) by all infected individuals. kala azar, or visceral leishmaniasis in the indian subcontinent, is a parasite transmitted by sandflies, predominantly in the poorest communities. it poses a number of challenges for control, which consists of improving case detection and indoor residual spraying [ ] . cases are falling drastically, reducing the burden of disease, but there is debate around the drivers of this decline, the size and nature of any asymptomatic pool, and the risk of resurgence [ ] . modeling acts as a tool to investigate some of the possible scenarios and evaluate different policy interventions in response to them [ , ] . in particular, the recent modeling of the different stages of the control effort suggests that there should be some accounting for underlying transmission rates when selecting interventions and that post-kala-azar dermal leishmaniasis (pkdl), a late-stage potentially highly infectious state, could undermine control and should be studied more closely (table ) . chagas disease is an anthropozoonosis caused by the protozoan trypanosoma cruzi, which is often contracted in childhood, when symptoms are rarely diagnosed; instead it is more commonly diagnosed through sequelle such as heart disease in adulthood [ ] . there are huge complexities in the zoonotic life cycle in different settings, and modeling can be used to evaluate how different vector-control interventions are likely to affect transmission [ ] . the modeling summarized in this issue highlights the value of vector control in reducing the infectiousness of all infected individuals, as well as the value of increasing diagnosis rates (table ) . across the idms, the models demonstrate how key uncertainties in life history have the potential to undermine the impact of current control long term but that intelligent intervention design may be able to overcome them. a cornerstone of large-scale ntd control, specifically for lymphatic filariasis, onchocerciasis, soil-transmitted helminths, schistosomiasis, and trachoma, is mda, sometimes in combination with vector control. an mda program requires repeated distribution of treatments to large numbers of individuals, without diagnosis. they are therefore only considered when diagnosis of infection is difficult (eg, stool-based microscopy, night-time blood samples and microscopy), there is little care-seeking by infected individuals, and there is a treatment with an excellent safety profile with a straightforward or single treatment schedule ( figure ). donation of the treatments for these diseases by the pharmaceutical manufacturers has transformed the opportunities for reducing the burden of these diseases, but single or limited course treatments it has required additional investment to deliver the treatments, as well as data to determine when and where mda should be delivered. through the course of a successful mda, increasing numbers of treatments go to those uninfected at the time (figure ). of course, these individuals are uninfected because they have been protected due to the ongoing mda program, which is a measure of the program's success. the issue of infectious asymptomatic individuals, which is a major concern for the idm diseases, is not such an issue as asymptomatic people are regularly treated as part of the mda. therefore, the key questions are who to treat (eg, which age group), how often to treat, and when treatments can be stopped [ ] . a number of things can lead to the failure of an mda, all of which have been investigated using mathematical modeling, including in this issue (figure ) . one of the important issues in program design, and which can undermine a program's success, is which parts of the population should be treated (figure ). if the wrong group is treated, you may see reductions in burden in this group, but not in the population at large. this is discussed in of the papers in this special issue. soil-transmitted helminths are transmitted through helminth eggs in feces contaminating the environment [ ] , and schistosomiasis is caused by intestinal worms that are passed in feces or urine and contaminate the water. in the case of schistosomiasis, the eggs then go on to infect snails, the parasite is amplified and rereleased into the water, and humans are infected through contact with that water [ ] . for both of these infections, current guidelines suggest that treatments should be targeted at children, with different frequencies according to prevalence. the modeling studies in this special issue suggest that the current guidelines might be altered slightly to optimize their impact, through either targeting adults or changing the thresholds for switching strategies [ , ] (table ) . the other helminths considered here, lymphatic filariasis, transmitted by mosquitoes and a risk factor for elephantiasis, and onchocerciasis, transmitted by black flies and the cause of river blindness, are also targeted for elimination through mda, sometimes accompanied by vector control [ ] . the lymphatic filariasis campaign has been particularly successful, with billions of treatments given and recent scale-back of treatment in areas where the targets have been met. the policy discussion is around how best to accelerate achievement of the goals using alternative treatment strategies and, in particular, when and where these strategies might be most appropriate [ ] and how they might be combined with vector control to slow down nonadherence figure . schematic of factors that could undermine the success of a mass drug administration program. monitoring and evaluation of programs is usually focused around a survey just prior to a round of treatment. if infection is not falling as quickly as expected, it could be due to any of the reasons outlined in the schematic, most of which cannot be detected by routine surveillance. the epidemic growth rate between rounds of mda [ ] . if the bounce back rate is too fast, or the interval between treatments is too long, this can lead to all the gains from the previous round being lost (figure ). for onchocerciasis, the programs are at the point of adapting their strategies to reach beyond the large morbidity gains achieved so far. the modeling work discusses the alternative strategies and the potential for mda combined with vector control to accelerate or achieve elimination ( table ) . all of the articles on mda policies highlight the importance of the epidemiological setting, the appropriate group being targeted, and systematic nonadherence, where particular groups either do not have access to or are refusing treatment, and note that these are often poorly measured ( figure ) [ , , [ ] [ ] [ ] . the issue of systematic nonadherence has been highlighted in modeling studies for many years but has recently become a point of focus again [ , , ] . for trachoma, a bacterial infection that can cause blindness and is transmitted through an uncertain combination of vectors and direct contact, the modelers highlight an additional aspect of mda, which is resistance to the drugs used for mass treatment (table ) [ ] . this is because the mda is a single dose of a broad-spectrum antibiotic, and so the concerns about rapid emergence of resistance have been present since the beginning, but current evidence suggests that the selection pressure from this single dose may not be as high as feared. they also highlight the risk of resurgence due to importation of cases, which is a particular concern because of the rapid epidemic growth rate of trachoma [ ] . both of these issues are relevant for the other mda campaigns ( figure ), but the longer time between generations and hence the slower epidemic growth rates for the helminths mean that both resistance and re-emergence are likely to be slower than for trachoma. however, as these campaigns have been running for decades in some cases, it is important to consider. in addition to these issues, there are outstanding questions around when and where to halt mda campaigns, which future modeling will inform. treatment has already been halted in some areas for the lymphatic filariasis campaign. issues of ongoing residual transmission, albeit with a likely slow growth rate, are being addressed and the decision to stop is being reevaluated [ ] . in this article, we have discussed main interventions for the control of ntds: idm and mda. these definitions are part of a shifting landscape that is dependent on a changing epidemiology, demography, and on the availability of new tools. for example, with an appropriate treatment with a good safety profile in uninfected and nonsymptomatic persons, a disease could move from case detection to mda or, when combined with the right diagnostic, to a screen and treat infection. similarly, as prevalence falls for mda diseases, if the right diagnostic becomes available, addressing these diseases could shift to a test-and-treat campaign or even case management. one of the roles of modeling is to evaluate the likely impact of new tools, treatments, and diagnostics; this is an active area of ongoing research that is not stressed in this issue. modeling of ntds is constrained by particularly limited data, as these articles highlight through presentation of uncertainty in predictions, sensitivity analyses, or scenario-based investigation. in contrast with many other infections, the dynamics of these diseases are also characterized by slow timescales, which mean that many qualitative behaviors are robust to these unknowns. however, it should be noted that these analyses should be viewed as a current state of our knowledge, and data from ongoing research have the potential to reduce some of these key uncertainties. across these diverse diseases, there are a number of common themes. • interventions should be tailored to the environment in which they are used, which requires more intensive data but should deliver greater gains. • reaching the right populations and ensuring uptake of screening, treatment, or mda is an essential part of any campaign, and models can indicate at what level of coverage or systematic nonadherence these campaigns are more likely to fail. • for a number of diseases, the relative contribution of sustained vector control transmission is an area of current evaluation. vector control has the potential to speed the gains due to other interventions and maintain the gains once the biomedical interventions have taken place, but it may only be needed in certain areas. despite the large number of biological unknowns or uncertainties for ntds, the slower dynamics allow us to develop our insights as data become more available. in summary, the modeling analyses in this special issue demonstrate that goals for ntds are likely to be met in a large number of areas. they also indicate what additional interventions are likely to be required in higher transmission areas or areas with particular epidemiological features. as such, this represents state-of-the-art modeling in this area and provides actionable information for policy development. acknowledgements. the author would like to thank the editors and staff of clinical infectious diseases for all their contributions in developing and delivering this special issue on behalf of all the authors in it. their support, patience, and clear guidance have been invaluable. the author also thanks members of the ntd modelling consortium for discussions regarding their contributions to the special issue and their contributions to tables and . disclaimer. the views, opinions, assumptions, or any other information set out in this article are solely those of the author and should not be attributed to the funders or any person connected with the funders. financial support. this work was supported by the bill and melinda gates foundation in partnership with the task force for global health through the ntd modelling consortium (grant no opp ) and the children's investment fund foundation (uk). supplement sponsorship. this article appears as part of the supplement "reaching the goals for nine neglected tropical diseases, " sponsored by the ntd modelling consortium. potential conflicts of interest. the author reports no conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. accelerating work to overcome the global impact of neglected tropical diseases-a roadmap for implementation available at: 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modeling understanding the transmission dynamics of leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in bihar complementary paths to chagas disease elimination: the impact of combining vector control with etiological treatment optimisation of mass chemotherapy to control soil-transmitted helminth infection soil-transmitted helminth infections human schistosomiasis investigating the effectiveness of current and modified who guidelines for the control of soil-transmitted helminth infections are we on our way to achieving the goals for schistosomiasis morbidity control using current who guidelines? lymphatic filariasis and onchocerciasis are alternative strategies required to accelerate the global elimination of lymphatic filariasis? insights from mathematical models how can onchocerciasis elimination in africa be accelerated? modeling the impact of increased ivermectin treatment frequency and complementary vector control models of trachoma transmission and their policy implications: from control to elimination effectiveness of annual ivermectin treatment for wuchereria bancrofti infection the importance of patient compliance in repeated rounds of mass drug administration (mda) for the elimination of intestinal helminth transmission a comprehensive assessment of lymphatic filariasis in sri lanka six years after cessation of mass drug administration key: cord- -i cwutg authors: mazet, jonna a. k.; wei, qin; zhao, guoping; cummings, derek a. t.; desmond, james stephen; rosenthal, joshua; king, charles h.; cao, wuchun; chmura, aleksei a.; hagan, emily a.; zhang, shuyi; xiao, xiangming; xu, jianguo; shi, zhengli; feng, feng; liu, xiuping; pan, weiqing; zhu, guangjian; zuo, liyao; daszak, peter title: joint china-us call for employing a transdisciplinary approach to emerging infectious diseases date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: i cwutg nan in the spring of , a newly pathogenic h n influenza virus emerged in people in china, likely associated with wild and domestic birds (kageyama et al. ) . marking the exact scenario that public health experts had fearedpeople were being infected, getting sick, and dying without the source of the virus being rapidly and definitively identified. in the absence of a clear understanding of the mode of transmission, early control of this epidemic proved difficult, especially since the most likely suspect reservoirs, animals being sold in markets, were not exhibiting the signs of illness that would help officials target mitigation measures and help citizens avoid exposure. strict safety measures were enacted, including closures of markets that sell live birds and culling of animals in areas where patients have been diagnosed with confirmed cases. the associated costs of control and treatment of the sick, including secondary loss of poultry, were increased because interventions could not be efficiently targeted at the source of infection or the drivers of the virus' emergence from that source. scientific and official responses to h n influenza highlight the significant advances in infectious disease management in china and around the world. who's global influenza surveillance network and the international health regulations requirements for rapid reporting have greatly increased the candidness of reporting; today there is also a greater openness of global scientific collaboration due to the previous experiences with the sars and h n epidemics. for example, genetic sequences of h n virus isolates were made publically available within days of its identification, enabling teams around the world to study the virus and immediately advancing our ability to control the disease, unlike some cases of earlier decades (osterholm and kelley ) . the virus itself was also rapidly detected as a result of the vigilance in influenza surveillance. reagents and protocols for rapid diagnosis and increased surveillance were made available through the local and international efforts, the fruit of the immense investment of many countries within this area of scientific research. h n provides one example that highlights the scientific advances of recent decades and the bolstering of international collaboration when needed, yet human infection with this strain of influenza remains a problem in as we struggle to get another yet another devastating zoonotic disease, the ebola epidemic in west africa, under control. unfortunately, despite intensive, high-quality research efforts by a talented cadre of scientists globally, we are still not able to predict which viruses, including specific influenza subtypes, will become pathogenic to people; which will cause new epidemics in animals; nor where and under what circumstances disease will emerge. the challenge for the global health community remains: efficient targeting of investment in science, prevention, surveillance, and preparedness for infectious diseases before or immediately upon emergence. to further address this challenge, the national science foundations of both china and the united states convened a small working group of infectious disease experts with experience in the ecology of microbial pathogens and disease emergence, including severe acute respiratory syndrome (sars), influenza, and a number of other diseases. in addition to the sponsors, the chinese academy of sciences, academia in both countries, private research institutes, china centers for disease control, and us national institutes of health were represented. the necessity of expanding collaborative, interdisciplinary work was clear. therefore, the two countries decided to put forth this international call for multisectoral engagement that could transcend the high-quality, but largely scientifically siloed, approach to infectious diseases that has been occurring globally. china and the us have much in common and much to gain from increased collaboration in this realm, as well as the need and ability to lead the call for such a transdisciplinary global approach to eid research, surveillance, and management. this call is not altogether altruistically motivated, however, as the two are among the countries harboring the most documented influenza diversity in the world (rejmanek et al. ) . in addition, both countries are leading global economies; have highly mobile populations that travel extensively for commerce and tourism; face internal disparities in access to healthcare; have had recent experiences with pandemic response; are facing a rapid growth in resources consumption; have major investments in pioneering technological advances and large scientific communities; and are investing heavily in emerging disease investigation and science. as evidence of commitment to respond to their own call, scientists from china and the us have been publishing together at increasing rates in the last decade. the us is the second largest producer of scientific articles ( %), and china is the third ( %), with the most growth of a developing country (up from % in to % in ) (board ). in publications primarily authored in the us, chinese scientists are currently the most frequent international partnering coauthors ( %)-an exceptional rise from just % in (board ). academically, the two countries are increasingly linked, with the number of chinese graduate students in the us in science and engineering programs growing from , to , ; in alone, , doctoral degrees were awarded to chinese nationals from universities in the us (xie et al. ) . spending on research in these two countries also demonstrates the ability and desire to further scientific knowledge and focused collaboration. in , the us spent . % gdp ($ billion) on research, that same year china was not far behind, spending . % gdp ($ billion), up from . % in and with projections for impressive monetary commitments to the advancement of science (xie et al. ) . evidenced by these investments, both us and china have pledged to advance research and have shown impressive growth in international collaborations; thus, it is critical that they continue to set an example for transdisciplinary, global collaboration in a directed and focused manner-achieving what neither could do without the other, while promoting similar efforts across the scientific community. active economies with domestic agricultural intensification, like the us and china, are especially challenged by eids, as their continued growth and development facilitates new and dynamic ecological circumstances in which potential pathogens can readily emerge or evolve (collins ) . in both countries, vast areas have been deforested for lumber production and to clear land for other uses, including agriculture and urbanization. this type of habitat change results in a rapid and dramatic loss of biodiversity and may facilitate the increase in numbers of just a few adaptable species of plants and animals (foley et al. ) . it also increases the likelihood of people coming into contact with those species, as well as changing the ways in which they encounter each other (murray and daszak ) . this process has been recognized as means to alter the ecological balance between pathogens and their hosts and has provided access to new hosts for pathogen spillover (keesing et al. ) . despite the need for such environmental modification to support growing populations and economies, the maintenance of biological diversity is increasingly demanded by the citizens of both developed and developing countries, who recognize the numerous services and benefits to quality of life that are provided through such diversity-including increasing productivity of agricultural land, resilience against adverse natural and anthropogenic events, increased capacity to provide fuel and fiber, benefits to people's welfare, and in many cases direct improvements to health outcomes (patz et al. ) . therefore, in china and the us, many degraded ecosystems are being rapidly altered for a second time, in a greening effort, to remediate previous environmental damage and protect or repair loss of biodiversity. these efforts are important for many reasons. however, as habitat recovery is most often primarily designed for vertebrate suitability, very little attention is paid to the considerations of microbial colonization or potential overgrowth of invertebrate vectors that can contribute to the transmission and spread of diseases. for example, echinococcosis, one of the most important parasitic helminth diseases of poor people worldwide, may be re-emerging in areas of northwest china after a cycle of years of land clearing and agricultural intensification. this increase may be associated with a recent ban on sheep grazing to help remediate soil erosion and the near-complete loss of natural vegetation (yang et al. ). the recent environmental efforts also include bans on rodenticides, allowing the re-colonization of rodent intermediate hosts and their predators that serve as definitive hosts of the parasite. the resulting ''recovering'' environment is therefore also conducive for increased transmission of the pathogen to hosts, including humans; an unintended consequence of greening this agriculturally intensified area. similar examples of increased pathogen prevalence and disease transmission with land use and host diversity changes have been documented, especially for malaria (patz et al. ) and lyme disease (levi et al. ). these situations illustrate that we must take care to use a holistic or one health approach to environmental conversion for both increased economic development and restorationthat we must remember that the health of people, animals, and the environment are inextricably linked. in addition, we must track and address the unintended influence on disease transmission and pathogen ecology of both economic development and environmental improvements. the coauthors met in china to discuss the ecology and evolution of infectious disease and the steps that must be considered as priorities to promote the health of the planet. as a result of the increase in successful scientific collaboration, the working group agreed that china and the us are well positioned to lead a call for ambitious and scientifically sophisticated program of work that yields relevant, highquality science, and sets examples for best practices around the world, through a collaborative and open communication framework. a general consensus was reached among experts at the workshop that, although some aspects of infectious disease transmission and emergence are being productively addressed by the current global health community, increasingly collaborative, transdisciplinary attention is needed for the development of a detailed understanding of the drivers of disease emergence (jones et al. ) and their implications and associated recommendations for infectious disease control. those drivers identified by the group of most immediate need for increased effort were as illustrated above, both agricultural and landscape intensification and remediation can have unintended disease consequences. serious health consequences also result from impaired water safety and security due to water impoundment for irrigated agriculture, hydroelectricity generation, and shared sources for consumption by both animals and humans. similarly, urbanization and downstream greening create new environments for pathogen transmission and evolution, often in situations with high concentrations of susceptible populations (li et al. ). risk are heightened as rural to urban migration brings a constant stream of new organisms to human-and pestintensified areas. international migration for employment opportunities may also bring the urban poor into circumstances ripe for pathogen transmission (liu et al. ) . increasing trade and novel business ventures have led some looking for employment to cross country borders, exposing them to biological, social, and healthcare environments that are unfamiliar and greatly increasing susceptible workers' chances of acquiring new infections through contact with wildlife, domestic animals, food, or human sources of diseases. as they are returning home, to a population naïve to the pathogen, there are increased opportunities that could facilitate virus transmission or even trigger the beginning of the next pandemic. as economies improve, the demand for animal-based protein increases (bellaver and bellaver ) . this demand facilitates livestock market chains from rural to urban areas, as well as increases the transport of wildlife and their flora to cities. as people become more affluent, their desire for traditional foods rarely decreases; instead there is an increase in their ability to purchase these items at higher prices and frequency. the resulting growing wildlife market chain is also often clandestine, and therefore suffers from poor biosecurity. the best recent example of disease emergence from the rural to urban wildlife market chain comes from the sars pandemic, which not only resulted in loss of life, but had a devastating effect on the chinese and hong kong economies (keogh-brown and smith ). the potential increase in range and spread of pathogens with climatic change and variation must be better examined. recent examples of pathogens emerging in new areas of the globe, driven by changes in wildlife host migration patterns, illustrate that environmental drivers of pathogen spread need to be included in the holistic development of disease mitigation interventions (altizer et al. ; goldstein et al. ). finally, land degradation and rural poverty combined with changes in water availability from climate variability are driving migration (sjogersten et al. ) ; these factors combined are influencing pathogen dynamics and must be considered for increased intellectual and financial investment in order to predict and prevent disease emergence and protect local and global health. research disciplines inherently work according to long-standing cultures, and their historical development is reflected in the fruit of their collaborations. for example, % of astronomy articles have international representation among coauthors, while chemistry, social sciences, and other life sciences stand at only - %. thus, some fields need a concerted effort to encourage international collaboration (board ). in addition, group efforts involving multiple disciplines facilitate outcomes that transcend what can be accomplished in isolation and are increasingly needed to approach complex global health problems. we, therefore, issue a call for the us and china to lead efforts to improve the global environment for collaborative, transdisciplinary infectious disease research using a one health approach. to successfully achieve useful outcomes, we need to have substantial advances in three areas: ( ) transdisciplinary research with a one health focus, most crucially the fields of micro and molecular biology, medicine, veterinary medicine, epidemiology, ecology, economics, engineering, genetics, mathematics, policy, systems analysis, and agricultural and environmental sciences; ( ) integration of technology for data collection, analysis, and communication, including information technology and geospatial technologies; and ( ) broader participation of scientists and motivated citizens for data collection and evaluation, individual-based decision making, and human behavioral change. we hope to see collaboration among all relevant disciplines to holistically assess the drivers of infectious disease emergence that are key to global health and economic security. the world bank estimates that economic losses from fatal animal-origin infectious diseases between and totaled at least us$ billion and that, had a severe influenza pandemic emerged, the costs could have approached us$ trillion. costs for the west africa ebola epidemic are still rising and estimated to reach more than $ billion for just the one outbreak (world bank). further, they estimate that, in addition to millions of lives, $ . billion per year could be saved globally by preventing emerging disease outbreaks (bank ) . to be most efficient and successful, a joint china-us led effort should focus on socio-ecological systems changes facilitating and forcing pathogen evolution and emergence, rather than on specific infectious agents or geopolitical regions. significant multi-lateral investment in pathogen discovery and characterization, agent-host dynamics, multi-organismal diagnostic technologies, and mathematical forecasting for risk identification and disease prevention and control are specifically encouraged. in addition to ebola, since the world has seen mers (middle eastern respiratory syndrome) spillover into the human population and increasing and spreading cases of hpai h n in birds globally. h n illustrated the necessity of international cooperation and collaboration for the global community, especially in light of the successful timely responses, yet these were reactive, not the encouraged proactive approach that the world is still missing. these leading nations have the opportunity to set an example for best practices in science by combining intellectual, technological, and financial resources to help reduce the impacts from emerging infectious diseases at every level, from families to global economies. working more closely together, the world can head off the threat of pandemics through an improved understanding of the underlying drivers of disease emergence, with benefits for science, health, ecological integrity, and economic well-being. climate change and infectious diseases: from evidence to a predictive framework people, pathogens and our planet: the economics of one health people, pathogens and our planet arlington va: national science foundation collins ae ( ) health ecology, land degradation and development global consequences of land use phocine distemper virus in northern sea otters in the pacific ocean global trends in emerging infectious diseases genetic analysis of novel avian a(h n ) influenza viruses isolated from patients in china effects of species diversity on disease risk the economic impact of sars: how does the reality match the predictions? deer, predators, and the emergence of lyme disease urban sustainability and human health in china comparison of health status and health care services utilization between migrants and natives of the same ethnic origin: the case of hong kong human ecology in pathogenic landscapes: two hypotheses on how land use change drives viral emergence mammalian-transmissible h n influenza: facts and perspective unhealthy landscapes: policy recommendations on land use change and infectious disease emergence. environmental health perspective evolutionary dynamics and global diversity of influenza a virus responses to climate change and farming policies by rural communities in northern china: a report on field observation and farmers' perception in dryland north shaanxi and ningxia china's rise as a major contributor to science and technology impact of anthropogenic and natural environmental changes on echinococcus transmission in ningxia hui autonomous region, the people's republic of china this material is based upon work supported by the us national science foundation grant no. deb- and made possible by the generous support of american people through the united states agency for international development (usaid) emerging pandemic threats program, predict project. key: cord- - f ftmh authors: aleta, alberto; moreno, yamir title: evaluation of the potential incidence of covid- and effectiveness of contention measures in spain: a data-driven approach date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: f ftmh our society is currently experiencing an unprecedented challenge, managing and containing an outbreak of a new coronavirus disease known as covid- . while china - were the outbreak started - seems to have been able to contain the growth of the epidemic, different outbreaks are nowadays being detected in multiple countries. much is currently unknown about the natural history of the disease, such as a possible asymptomatic spreading or the role of age in both the susceptibility and mortality of the disease. nonetheless, authorities have to take action and implement contention measures, even if not everything is known. to facilitate this task, we have studied the effect of different containment strategies that can be put into effect. our work specifically refers to the situation in spain as of february th, , where a few dozens of cases have been detected. we implemented an seir-metapopulation model that allows tracing explicitly the spatial spread of the disease through data-driven stochastic simulations. our results are in line with the most recent recommendations from the world health organization, namely, that the best strategy is the early detection and isolation of individuals with symptoms, followed by interventions and public recommendations aimed at reducing the transmissibility of the disease, which although not efficacious for disease eradication, would produce as a second-order effect a delay of several days in the raise of the number of infected cases the first report by the chinese authorities of the covid- outbreak appeared in december st, . ever since then, the world health organization (who) and national public health authorities have been tracing and reporting on the evolution of the outbreak. as initially feared, and despite contention measures adopted in china, with a big city like wuhan being quarantined for weeks, the disease spread beyond mainland china. as of february th, , there are , cases worldwide, of which , correspond to china [ ] . additionally, countries, including spain, have reported at least one case of covid- . two months into the outbreak, much is still unknown about the natural history of the disease and the pathogen. important from the modeling perspective, for instance, it has been claimed that a large number of cases might have gone undetected by routinely screening passengers, due to the special characteristics of this disease [ ] . admmitedly, several studies predict that only between % and % of the cases have been detected and reported [ ] [ ] [ ] [ ] . as with any other novel disease, governments, public health services and the scientific community have been working towards stopping the spreading of covid- as soon as possible and with the lowest possible impact on the population [ , [ ] [ ] [ ] . from a scientific point of view, there are two course of actions that can be followed. on the one hand, new vaccines and pharmaceutical interventions need to be developed. this usually requires months of work. therefore, on the other hand, it is important to study the large-scale spatial spreading of the disease through mathematical and computational modeling, which allows evaluating "in-silicon" what-if-scenarios and potential contention measures to stop or delay the disease. this modeling effort is key, as it can contribute to maximize the effectiveness of any protection measures and gain time to develop new drugs or a vaccine to protect the population. here, we follow the modeling path and analyze, through a data-driven stochastic seir-metapopulation model, the temporal and spatial transmission of the covid- disease in spain as well as the expected impact of possible and customary contention measures. our model allows to implement and quantify the impact of several conventional measures in spain. these policies are mostly aimed at reducing the mobility of individuals, but we also include other plausible settings like a reduction in the time for case detection and isolation. our findings agree with previous results in the literature that have reported that a reduction as large as % in traffic flow has a limited effect on the spreading of the disease. important enough and at variance with such studies, the data-driven nature of this study and the available dataset allowed us to disentangle the impact of each transportation mode in several scenarios of mobility reduction in spain. we found that while shutting down completely any transportation means does not lead to a significant reduction in the incidence of the disease, in some contexts the arrival of the peak of the disease is delayed by several days, which could eventually be advantageous. altogether, we provide evidences supporting the adoption of a mixed strategy that combines some mobility restrictions with, mainly, the early identification of infected individuals and their isolation. these conclusions agree with the latest recommendations by the who [ ]. we also highlight that although this study has been made with data from spain, our findings can also be valid for any other country given the ubiquity of mobility patterns worldwide. stochastic seir-metapopulation models are routinely used to study the temporal and spatial transmission of diseases like the covid- . here, we made use of such class of models and implement a data-driven version that allows to obtain realistic estimates for the spatial incidence of the disease as well as its temporal dynamics. more specifically, in terms of time, we feed the model with the available data as of february th, . spatially, we consider that each province (there are in spain, see appendix b) is represented by a subpopulation. furthermore, metapopulation models are composed by two types of dynamics: the disease dynamics governed by the chosen compartmental model, seir in our case, and the mobility of the individuals across the subpopulations that make up the whole metapopulation system. the latter ingredient, the mobility, connects the subpopulations and allows the disease to spread from one subpopulation to another. in what follows, we describe these two components of our model. to model the mobility of individuals we use a data-driven approach. data-driven modeling, at variance with more theoretically inspired methods, has the advantage that it allows to directly implement and evaluate realistic contention measures, thus producing scalable and actionable what-if-scenarios. to this end, we have obtained the inter-province mobility flows provided by the ministry of development of spain [ ] . therefore, the minimal spatial unit in our system is a province. using the information from the mobility matrices, that report the origin and destination (od) of individual movements, at each time-step, we sample the number of individuals on the move from each province and distribute them across the country according to the information contained in these od matrices. important enough, this dataset not only includes the total number of individuals going from province to province, but it also distinguishes the main transportation means used by the individuals, see figure . this will allow us to gauge the effect of travel restrictions on different transportation modes. furthermore, given that the epidemic started abroad, it is important to determine in which province the disease is more likely to be seeded first. given the current global spread and incidence of the epidemic, we thus take into account that the three countries with more cases are china, south korea and italy and consider that the most plausible route for an infected individual to reach spain is by plane. thus, we collected the number of passengers coming from each country to each spanish airport in from the spanish air navigation manager aena [ ] . then, we assigned each spanish airport to its corresponding province and ranked them according to their total number of operations with each country, see figure . it is worth noticing that the information provided by aena is already aggregated by country. thus, this ranking cannot take into account which airports are mostly connected to locations where the outbreak is currently concentrated − e.g., north of italy. nevertheless, the ranking constitutes a valid proxy, and a good starting point, to seed the disease. the dynamics of the disease is governed by an seir compartmental model. in this model, individuals are classified according to their health status: susceptible (s) if they are susceptible to catch the disease; exposed (e) if they have been infected but are still asymptomatic and cannot infect other individuals; infected (i) once the incubation period is over and the individuals show symptoms and could infect others, and removed (r) when they are either recovered or deceased. within each province, the transition between compartments results from the following rules, iterated at each time step, corresponding to day: where r is the reproduction number, t e is the mean incubation time, n i stands for the number of individuals in region i and i i accounts for the number of infected individuals in such region. e→i: exposed individuals become infected at a rate inversely proportional to the mean latent period, t e . i→r: infected individuals become removed at a rate inversely proportional to the mean infectious period, t i . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. similarly, the width of the links is proportional to the number of individuals using that route. note that for multimodal travels the associated mode is the one that corresponds to the largest part of the trip, which explains why there are links from the islands to provinces without ports in the matrix corresponding to maritime trips. in what follows, we parameterize the model according to the latest estimates for the disease parameters [ , ] , namely, r = . , and a generation time t g = t e + t i = . resulting from considering t e = . and t i = . (in appendix d we report that similar results are obtained for other values of t g , see figures , and ) . additionally, note that we have not explicitly included in the model the possibility of asymptomatic transmission, which should not be mistaken by transmission from undetected or unreported cases. asymptomatic spreading is still under scrutiny and the statistical evidences are scarce and not significant enough as to be taken by granted. in any case, including this possibility would be trivially possible in this kind of models. a. quantifying the spatial and temporal evolution of the disease incidence. one of the main characteristics of the covid disease is its long latency times. currently, the average incubation period has been reported to be . [ ] . thus, before proceeding with the evaluating the impact of the disease, we . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. first compare the metapopulation model employed here with a classical sir-metapopulation framework. to do so, we use the random-walk effective distance where p (j|j) is the normalized flow matrix without row and column j, p(j) is the j-column of p with element j removed and δ is a dimensionless parameter that depends on the infection, recovery and mobility rates [ ] . this quantity, defined for sir metapopulation models, gives us the expected time that it would take for the disease to reach each subpopulation of the system, also known as the hitting time. in figure , we compare the hitting time obtained from stochastic simulations of the seir metapopulation model with the theoretical distances derived for the simplified sir model. we can see that the correlation is nearly perfect, implying that the spreading itself is quite similar in both models. however, we find that the hitting times in the seir implementation are at least two times larger than the theoretical ones for the sir scenario (on its turn, stochastic simulations of the sir model agree very well with the theoretical expectations for the model, see appendix c). thus, the addition of the latent state produces a substantial delay on the spreading of the epidemic. this is in line with the fact that the epidemic is thought to have started in mid-november or early december, however, a noticeable number of cases was only reported by early january. figure shows the expected hitting time for each province when the disease starts from different locations, as well as one case with seeds in multiple places, as obtained from the seir metapopulation model. as before, the hitting times might seem long, but this is due to the long latent periods of the disease, in agreement with the evolution of reported cases in mainland china. we also note that spanish major cities are expected to be affected by the outbreak in no more than days −but often within days− from the initial time in all the situations considered. indeed, to mimic the situation in the country as of february th, , and to make projections about the evolution of the disease into the future, we considered the scenario in which the model is initialized with infected individuals in the capital of each region where there are cases of covid : madrid, tenerife, barcelona, balearic islands, zaragoza, seville and valencia [ ] . the results show that the spreading is much faster (note that the outer circle in the individual provinces represents days while in this case it represents only days) in such a situation. complementarily to figure , we present in figure further results on the temporal and spatial evolution of the disease dynamics. here we have computed, through stochastic simulations of the model, the cumulative number of infected individuals within each region assuming that the disease propagates from barcelona by initially infected individuals. the results align with the theoretical predictions, and highlight the close relationship that exists between the two biggest cities of spain (barcelona and madrid), even though they are relatively far geographically (around kilometers through the shortest path by car). again, note that the relative slow speed of the epidemic agrees with what has been observed in china. finally, it is worth remarking that all of these quantities have been obtained assuming that no control measures are taken, which is not the current case as (detected) infected individuals are being isolated and monitored by the authorities. additionally, we also stress that it is difficult to assess whether this corresponds to a worst-case projection or not, given the many unknowns that can't be taken into account, such as inflow of infected subjects from abroad, the fraction of infectious individuals that have gone undetected, etc. however, as we show next, this data-driven modeling approach do allow to evaluate the effect of customary contention measures. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint our data-driven model is particularly useful to get insights into mobility-mediated transmission dynamics and to evaluate possible countermeasures. next, we explore diverse containment measures that could be implemented aiming at stopping the large-scale spreading of the disease. first, we analyze the effect of imposing mobility restrictions by limiting traffic flow in the country. we consider six different scenarios that correspond to each transportation mode being shutdown plus another one in which a total reduction of % of the overall traffic is imposed. these measures are extreme and unless the situation gets really critical, would not be put into practice as they bear an economic cost that would be insurmountable. nonetheless, as we show below, however drastic they appear to be, these measures are useless when it comes to completely stop the disease from propagating. indeed, a significant reduction in the estimated incidence is only obtained when other actions are feasible. admittedly, in figure a it is observed that the previous measures have no effect on the final size of the epidemic. on the other hand, if we look at the time for the peak of the epidemic to arrive, figure b , we see some minor effects. in particular, although shutting down most modes of transportation have practically no effect, if all private cars were removed (i.e., they remain confined in their corresponding province), the peak of the epidemic would be delayed by about days. the most effective of the above scenarios of mobility restriction corresponds to an unrealistic % reduction of the overall traffic, when the peak would be delayed over days. this is in agreement with previous studies that have shown that the only sizable effect of travel restrictions is to delay the peak of the epidemic. for instance, it has been claimed that the travel restrictions in wuhan only delayed the peak of the epidemic by days [ ] . another possibility, instead of limiting the mobility of the overall population, is to be extremely vigilant so as to make it possible to isolate all the cases that start to appear quickly enough. to check the impact of this policy, we have simulated a scenario in which the average number of days that an individual is able to go unnoticed and infect others is reduced from . to , . and days. in figure a , we observe that this strategy is much more effective than traffic restrictions. in particular, if we were able to reduce the time since symptoms' onset to isolation below . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. each set of measures indicates when they were applied: since the beginning of the epidemic, and after or , cases are detected in the whole country. note that individuals who escape isolation remain infectious until they recover, and thus they could be thought of as asymptomatic spreaders. days, the epidemic is greatly reduced. as a matter of fact, it has been recently reported [ ] that this average number of days went down in china from . days at the beginning of the outbreak to . days, which is one of the reasons invoked to explain why the epidemic has started to decline in mainland china. in our case, these numbers would be compatible with generation times of or . (see figure ). it is also reasonable to assume that this strategy is not easy to implement in full, either because some individuals could purportedly try to avoid isolation or due to the fact that many infected subjects have mild symptoms similar to a common flu and neither go to the doctor nor report their state. therefore, we have simulated a slightly different scenario in which individuals are isolated the same day of their symptoms' onset with a certain efficacy. that is, only a given percentage of the new cases is isolated, while the others are able to roam freely. this framework would also be compatible with having asymptomatic individuals who are able to spread the disease, something that is currently under debate and not yet statistically supported. the equivalence with such hypothetical natural history of the disease in our model is such because we do not apply the prescribed percentage to the total number of infected individuals, but only to those who have just developed symptoms, thus, those that escape will remain infectious as if they were asymptomatic until they recover. in figure b we show the effect that different percentages of new isolated cases would have on the size of the outbreak. being able to hospitalize all individuals, on average, in less than day enables to effectively stop the disease. yet, the results also show that even if all infectious are not isolated, as long as more than % of the infected individuals are, the effect would be similar and the disease would be effectively eradicated. lastly, we analyzed the consequences of self-protection measures such as wearing masks, washing more frequently ones hands or avoiding crowded places. to mimic these contexts, we simply reduced the effectivity of the transmission by a certain fraction, and study the final size of the epidemic, see figure a . the results show that a large reduction of at least % is needed to contain the disease. interestingly, if we look at the time to the peak of the epidemic, represented in figure b , we observe that decreasing the transmission not only reduces the size of the outbreak but also delays the peak. hence, even if this strategy might not be sufficient to completely stop the propagation of the disease in all cases, it could certainly help for preparedness and other clinical responses by delaying the spreading. the exception is when the reduction is very large (in the figure, beyond %) as in these cases the peak might occur earlier because the disease dies out. in concluding, it is apparent from all the results obtained for the different scenarios that we have considered that the most cost-effective strategy would be the isolation or quarantine of detected infectious cases, as long as the efficacy of such measure is over %. important for the current debate about the natural history of the disease, this policy would also work if there is a fraction of asymptomatic but infectious individuals in the population. our results also show that from a practical point of view, a combination of all the analyzed contexts can have second order benefits. as already stressed, containment measures should not only be directed towards a full cutdown of the number of infected cases. their efficacy is also given by other factors, such as delaying, even if only by a few days, the spreading of the disease. such delays are always good for preparedness and to have more time for clinical research that can lead to new pharmacological treatments or vaccines. for instance, even if traffic restrictions are not effective on their own, they facilitate the control of the population and thus it would be easier to detect infected individuals and treat them. similarly, self-protection measures and other social-distancing practices delay the spreading of the disease, freeing resources that would allow for a better management of the epidemic, in turn leading to an increase of the efficacy of individual isolation. closing public places would, in practice, reduce the transmission, which again will lower the total number of infections and thus make them more manageable for the public health system. this also highlights the importance of having a coordinated response system, since simply adopting central measures like imposing mobility restrictions and closing public places is not effective in the middle-to-long term. our model has several limitations and some of them could actually be overcome in the near future. perhaps the most important one has to do with the inability of current large-scale epidemiological models to fully account for behavioral changes in the population when a disease is evolving. as it is the case for the spreading of covid- , the information −and more often than desired, misinformation− travels faster than the disease. this produces undesired effects such as a collapse in the emergency rooms at hospitals, a proliferation of information sources that do not provide sensible advices in all cases, anticipated economic loses and, in general, uncoordinated responses. therefore, it is a pressing challenge to develop more realistic ways to incorporate in models like the one employed here all these risk-averse responses and reactions. another limitation of the current study includes the relatively low spatial resolution, which is essentially determined by the data availability. the results however indicate that the level of granularity used here is enough to capture mobility patterns and the effects of possible interventions. finally, we have not considered the temporal and spatial variability of disease parameters, nor other potentially important characteristics of the host population such as the existence of super spreaders or the age structure, which seems to play a relevant role for this disease, at least in what concerns the case fatality rate. we plan to investigate on all these issues in the near future. acknowledgments ym acknowledges partial support from the government of aragon, spain through grant e - r (fenol), and by mineco and feder funds (fis - -p). aa and ym acknowledge support from intesa sanpaolo innovation center. the funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. the inter-province flow matrices include the number of individuals that move from province to province in spain for several days of october . the data also includes the main mode of transport used by those individuals, as well as other characteristics such as the period of the day when the travel started. in our case, we have collected the matrices from the last two weeks of october and averaged them. for a deeper discussion on the characteristics of these matrices we refer the reader to the original source (in spanish) [ ] . the upper level of administrative division in spain is denominated comunidad autónoma. there are of such "autonomous communities" in mainland spain, one in the islands of the mediterranean sea (balearic islands) and one for the islands in the atlantic ocean (canary islands). besides, there are two autonomous cities (ciudades autónomas) in the north of africa, ceuta and melilla. the next administrative division is the province (provincia). there are provinces in mainland spain, in the balearic islands and in the canary islands, plus the two autonomous cities making up the subpopulations considered in our model. the number of inhabitants of each province varies a lot, from over millions in madrid and barcelona to less than , individuals in ceuta and melilla. we collected the number of inhabitants of each province from the data provided by the spanish statistical office [ ] . there are airports in spain (including the heliports in algeciras and ceuta), although the majority of them only have connections to other spanish airports or european airports. as with the provinces, the traffic in these airports varies widely, from over million passengers in madrid and barcelona to less than , in albacete and huesca. the information regarding spanish airports is provided by the spanish air navigation manager (aena) [ ] . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint to ensure that this quantity is able to predict correctly the spreading in our metapopulation, we first implement an sir model and study the effect of using a different number of seeds, figure . regardless on the number of seeds, the correlation between the obtained values and the theoretical ones is really large. however, the actual value depends on the number of initial seeds. thus, without any modification this measure can help us determine the arrival order, i.e., to which provinces the disease should arrive first, but not the precise time. nonetheless, depending on the number of seeds, a small correction has to be applied. as it is not clear yet what are the final disease parameters that affect the predictions about the spreading of the coronavirus, we here provide quantitative evidences that the results reported in the main text still hold for other values of the generation time t g . a similar exercise could be done for r , but we believe that the consensus around . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . the value used here ( . ) is higher than for other parameters like the serial interval. figures , and show results obtained for t g = and t g = . . as it can be seen, the previous conclusions hold, and in most of the cases, also quantitatively in terms of relative variation with respect to the scenario in which no measures are implemented. ministry of development of spain spanish air navigation manager regions with cases of covid as of / in spain spanish statistical office key: cord- - knig g authors: thacker, s.b.; sencer, d.j. title: centers for disease control date: - - journal: international encyclopedia of public health doi: . /b - - . - sha: doc_id: cord_uid: knig g emerging from a small, wartime government program with a regional focus on malaria in , the centers for disease control and prevention (cdc) has become a global public health agency that addresses the entire scope of public health, with over employees and contractors in nearly occupations. the cdc's expertise has expanded in direct correlation with the expanding view of public health needs: it is recognized globally for its ability to respond to urgent threat related to disease epidemics and the health consequences of disaster and war. cdc programs have contributed significantly to the eradication and reduction of diseases such as smallpox, polio, and guinea worm, as well as the control of health problems such as human immunodeficiency virus (hiv), childhood lead poisoning, breast and cervical cancer, diabetes, violence, and unintentional injuries. cdc contributions in applied epidemiology, public health surveillance, risk factor reduction, and environmental risk assessment also have been critical to the practice of public health in the united states and around the world. the emerging concerns of the new century – genomics, globalization, the built environment, information technology, global warming, emerging infections, violence, and so forth – will require not only the traditional disciplines but also new expertise and new global partners, both public and private. the history of the centers for disease control and prevention (cdc) began in with the establishment of the malaria control in war areas (mcwa), under the u.s. public health service (phs). the u.s. military had suffered severely from malaria during world war i, and although the reported incidence had dropped during the s, a cyclical -to -year pattern of disease raised concern. because the disease had been endemic in the southern united states, concern was heightened because military bases and more than essential war establishment facilities were located there. mcwa was the actualization of the vision of joseph w. mountin, md ( - , an assistant surgeon general in the phs and director of the bureau of state services, who reported to the surgeon general of the phs. the headquarters was located in atlanta, georgia, with close associations with state health departments, puerto rico, and the virgin islands, as well as laboratory facilities and field stations in multiple states that worked with all the affected states and territories. although the phs provided leadership for the new program, much of the expertise in malaria had been recruited by the military; therefore, substantial training became an essential component of the multifaceted approach taken by mcwa. physicians assessed clinical malaria and parasitologists managed the laboratories; however, mosquito control was the emphasis, and engineers and entomologists dominated mcwa. field staff from the recently terminated works progress administration were recruited to continue their work of draining malaria breeding grounds and larviciding with diesel oil and insecticides (beginning with paris green but adding ddt in , which substantially changed the approach to malaria control). state laboratory staff were trained to diagnose malaria by using the most effective techniques. the program's scope expanded to the civilian population and to other vector-borne diseases such as dengue and typhus. mwca laboratory workers also responded to requests from states for assistance in epidemic investigations, a role previously left to the national institute for health (nih) (now the national institutes of health). the mcwa program was regarded as highly successful, and even before the end of the war, mountin and his staff were considering the future. with the support of surgeon general thomas parran, jr. (surgeon general, - ) , the communicable disease center was established on july , . at the time, cdc had employees and a budget of $ . million ( table ) . the legislation that created the cdc explicitly charged the new agency with responsibility for dealing directly with state health departments in the control of communicable diseases. this role was solidified in when the association of state health officials and the american public health association agreed that cdc take the lead in defining what diseases were of highest priority and should be reportable. the key figure in this expansion of the cdc role was alexander d. langmuir, md ( - ) , who was brought to the cdc in as chief of the epidemiology division. langmuir brought experience as a member of the armed forces epidemiology board, as a practicing epidemiologist at both the local and state health departments in new york, and as a professor teaching at the johns hopkins school of hygiene and public health. he also brought vision and a strong personality that helped bring the role of the epidemiologist to prominence at the cdc and in public health practice throughout the country. the cdc expanded during those first years to include field stations in missouri, colorado, and texas, and to conduct special studies in other states. influential events and decisions in the s, however, truly established the cdc as an agency with national recognition, and mountin and langmuir were forces behind the majority of these changes. in , the cold war set the tone in international affairs, and the korean conflict fueled a concern about the pointed use of biologic weapons there and in the united states. langmuir successfully championed the concept of the epidemic intelligence service (eis), which would respond to disease outbreaks as part of a program of biologic warfare defense. the eis program was established in with the recruitment of men, including physicians and a sanitary engineer. this first class became the disease detectives, the symbol of applied epidemiology practiced at the cdc and the core of epidemiologists who would come to lead the agency in future years. by , more than men and women had graduated from the program, all with different backgrounds and experiences. together they have conducted more than investigations, not only in infectious disease but also in chronic diseases, injury, and the many other health areas that the cdc has engaged in during subsequent years ( table ) . eis alumni have become public health leaders at the cdc, throughout the united states, and around the world. the event that first brought national attention to the cdc came with poliomyelitis, the crippling childhood disease. in , more than cases were reported in the united states, and polio was the leading infectious cause of childhood death in the country. in , the university of michigan conducted a national randomized trial of the killed virus vaccine developed by jonas salk on more than u.s. school children during a year when more than cases had been reported. the positive results were announced on april , , the tenth anniversary of the death of franklin delano roosevelt, and the response was dramatic. within hours, surgeon general leonard a. scheele (surgeon general, - ) announced a national vaccination program. unfortunately, however, on april of that year, a baby was reported to have contracted polio days after vaccination; this proved to be the index case of an epidemic of vaccine-associated polio that led to a decision to shut down the program. four days later, langmuir was directed to establish the national polio surveillance unit he had been advocating, and he immediately directed the eis officers to focus their efforts on this national emergency. within less than a week, daily reports were produced by the epidemiologists in the unit. rapid investigation in the field and in the laboratory clearly implicated one of the five vaccine manufacturers as the source of the epidemic (although a second manufacturer might have had problems as well). weeks later surgeon general scheele was able to announce that the problem had been identified, safety standards had been instituted, and the vaccine was now safe to distribute. the cdc's role had been critical, and the importance of public health surveillance and of the eis was recognized. national surveillance for asian influenza in - , together with the work of cdc epidemiologists and laboratory staff, cemented the national role of the agency in disease control. the second major event of the decade was less obvious to the public -the transfer of the phs venereal disease division (vdd) to the cdc in . at the time the vdd had a larger budget than the cdc and certainly a longer history. however, the effect was more important than the budget. the vdd brought with it a grant program that distributed money to states and a program management function -the public health advisor. the grant program enhanced the agency's connection and collaboration with state health departments, and the public health advisor became the primary nonscientific manager of cdc programs and of the agency itself. another program that came to the cdc with the vdd -unfortunately one that was highly negative -was the tuskegee study of the complications of syphilis, which had begun in table centers for disease control and prevention (cdc) timeline among black men in rural alabama. although the gravity of this study was little recognized in , the matter became public in and led to a formal apology by president william j. clinton in . the cdc moved into new facilities adjacent to the emory university campus in . at the time, the cdc had more than employees and a budget of $ million. the expansion of the agency mission was equally substantial. by the end of the decade, the budget had reached $ million, and the cdc encompassed programs in immunization, hospital infection control, tuberculosis, and environmental health. the journal morbidity and mortality weekly report (mmwr) was brought from the national office of vital statistics to the cdc by langmuir in . the publication rapidly became the agency's premier publication and the avenue for publishing concise, science-based articles of current events of public health interest such as epidemics, as well as current data regarding disease occurrence and death. the cdc increasingly was recognized for its responsiveness to epidemics of infectious diseases, including drug resistance among hospitalized patients, salmonella in commercially produced chicken, polio associated with oral vaccine, and for its international work in smallpox eradication and disaster assistance. the cdc also maintained its lead federal role in disaster assistance, and staff studied the immediate and long-term health effects of such disasters as hurricane camille in . possibly, the most important event for the cdc during that decade, however, occurred in geneva at the world health assembly in when the world health organization (who), under the joint leadership of the united states and the united soviet socialists republic, endorsed the plan to eradicate smallpox. this political will, together with improved technology (e.g., the jet injector gun to deliver vaccine), was the impetus for an extraordinary global program. donald a. henderson, md (eis ) , was transferred to who to help lead the international effort. a global mass-vaccination strategy was undertaken, and active surveillance documented rapid decreases in illness and death. however, the program began to founder and eradication appeared out of reach. william h. foege, md, another eis alumnus ( ) , demonstrated in west africa that an active surveillance and containment strategy was an effective complement to the mass-vaccination strategy. in , these efforts resulted in the first successful eradication of a human disease in history, years after the last case of wild-strain smallpox in somalia. the cdc's global role in disease control was founded on its active role in smallpox eradication. two future cdc directors, foege and jeffrey p. koplan, md, who had served as an eis officer ( ) in the program, and dozens of future public health leaders and hundreds of public health workers domestically and internationally were trained in this effort. the critical role of epidemiology and public health surveillance was now recognized by a much broader audience, and the importance of international, cross-disciplinary, and cross-cultural collaboration was appreciable. the s brought further change to the cdc and initiated its programmatic expansion to broader areas of public health, including environmental health, additional chronic diseases, occupational safety and health, and injury prevention and control. the cdc's expansion in part came from the transfer of programs in these areas, notably the national institute for occupational safety and health, which was transferred in . other expansions resulted from calls for assistance from states or other federal agencies. childhood lead poisoning was being reported as a result of industrial pollution or from parents bringing lead home on their work clothing. vinyl chloride-associated liver cancer was demonstrated in population studies. investigating the short-and long-term effects of exposure to radiation after the incident at the nuclear reactor at three mile island in pennsylvania was initiated at the end of the decade. a collaborative study with the national cancer institute was conducted to investigate the association of cancer with both oral contraception and estrogen therapy. all these changes provided background for major organizational changes that occurred at the end of the decade. meanwhile, the cdc continued to have active engagement in infectious diseases. the identification of salmonella among pet turtles altered that industry, as did discovery of the contamination of commercial intravenous preparations with bacteria and the subsequent documentation of a nationwide epidemic leading to a product recall. the study of the efficacy of nosocomial infection control (senic) confirmed the effectiveness of hospital infection-control practices. internationally, the cdc was involved in documenting the threat of hemorrhagic fever viruses -first the marburg virus in germany, and subsequently the lassa and ebola viruses in sierra leone, zaire, and sudan. two events in , the agency's th year, however, might have had the most enduring impact. in spring , an outbreak of influenza at a military base in new jersey was identified as being caused by a new strain of influenza a, a strain that was quite different than the strain (a/hong kong/h n ) circulating since . the new strain (h sw n ) had demonstrated pathogenicity among humans and its ability to be transmitted person to person, circumstances that were believed to always lead to pandemics. as important, this strain was believed to be closely related to the influenza virus that had led to a pandemic in - , which had killed u.s. residents and an estimated million persons globally, affecting particularly young adults. with the cdc in the lead, scientific experts were engaged during the following months to study the problem. they ultimately recommended the policy that led to the national influenza immunization program, which targeted the entire u.s. population. vigorous efforts by the president, congress, the vaccine manufacturers, and the public health system enabled the program to begin in october . within weeks, the cdc's national surveillance program, in collaboration with the states, uncovered an increased reporting of guillain-barré syndrome among persons who had been vaccinated during the program. subsequently, the program was suspended and no epidemic occurred. an association was confirmed in a national case-control study conducted by the agency; the agency was criticized harshly, and other vaccination programs were called into question. in early august , a report from pennsylvania of possible swine influenza led to an investigation of fatal pneumonias among veterans and their families who had attended a statewide convention of the american legion in philadelphia during the third week of july. the legionnaires' disease epidemic eventually brought eis officers and many other staff to pennsylvania and stayed on the front pages for weeks during this bicentennial year. by the end of august, the field team had identified more than cases -laboratory staff had ruled out all known human pathogens -and together they had investigated multiple leads in an effort to determine a toxin. terrorism was considered, and external experts were called in. however, the strongest association identified was with the hotel that had housed the conventioneers and had hosted major business and social activities. the team returned to atlanta without a definitive answer, and in the context of the mounting criticism around the swine influenza program, continued its work. finally, in december, joseph e. mcdade, phd ( -) , in the cdc's rickettsial diseases laboratory, identified the gram-negative rod that proved to be the bacterium that caused the epidemic. legionella pneumophila became the first new human bacterial pathogen identified in decades, and mcdade and his coworkers linked it to two previously unsolved epidemics in and . the s was dominated by the epidemic caused by hiv, but the cdc was also to undergo another major organizational change that reflected a new direction for the agency. however, the decade opened with two investigations of a more traditional nature that brought national visibility to the agency and brought lawyers into the public health policy arena in a dramatic fashion. in the summer of , a report from arizona in the mmwr linked reye's syndrome with using aspirin. that report was followed by similar data from ohio and michigan, which led the cdc to conclude that the association might be real. the aspirin industry aggressively attacked, but the cdc held firm and recommended that aspirin be avoided for children with chickenpox and during influenza epidemics. surgeon general julius b. richmond ( - ) soon followed with a similar recommendation. the struggle with industry did not stop, but national data demonstrated a noticeable decrease in reye's syndrome during the next few years. meanwhile, the cdc was responding rapidly to the national epidemic of toxic shock syndrome among women. within months, the disease was linked to use of tampons and by the end of , specifically to the use of the rely tampon, made by proctor and gamble. after the manufacturer voluntarily withdrew the product, the epidemic abated dramatically. major change was continuing to happen in public health, and the cdc was involved in measuring the health effects of the radiation release from the nuclear reactor at three mile island in pennsylvania, the toxic effects of dioxin at love canal (new york) and among vietnam war veterans, and the volcanic eruption at mount saint helens (washington state). the chemical release in bhopal, india, and the investigation of toxic oil syndrome in spain reflected the international recognition of the cdc in environmental health. in , the agency announced a reorganization to reflect these new public health concerns and environmental health, chronic diseases, occupational health, unintentional injuries, violence, and maternal and child health, while maintaining excellence in the prevention of infectious diseases. this reorganization enabled the programmatic growth for the next two decades. however, a brief report of an unusual pneumonia among five homosexual men in a june issue of the mmwr changed health practice at the cdc and around the world. the pandemic of acquired immunodeficiency syndrome (aids) caused by hiv crept slowly into public consciousness, but by the end of the decade, no health problem in the world generated more interest and controversy. the cdc was at the center of both. during those years, more than a third of the agency's budget was directed toward researching this illness and the virus that causes it. the agency's budget grew to approximately $ billion by the end of the decade. its constituency grew with the budget, and both domestic and global partnerships expanded considerably. at the cdc, behavioral and social scientists rapidly developed an important niche, and the move of the national center for health statistics in to what was now the centers for disease control brought a wealth of data and enhanced the role of the statistician at the cdc and in public health practice. the last decade of the twentieth century was marked by expansion of programs and budget in the newly defined priority areas at cdc, especially chronic diseases. in , the cdc's name changed to reflect its broadened mission, and it became the centers for disease control and prevention. by , the cdc budget had exceeded $ billion. the chronic disease budget was $ million that year and was dedicated primarily to development of programs for preventing breast and cervical cancer. in addition, routine data collection was being established at the state level through the behavioral risk factor surveillance system, and state-based cancer registries were implemented to provide a data baseline for defining problems and evaluating program effectiveness. emerging infections (e.g., escherichia coli o :h , the hantavirus, and west nile virus) and reemerging infections (e.g., antibiotic and drug resistance for bacteria, tuberculosis, and malaria), together with an increasing number of effective vaccines, underscored the continuing importance of infectious diseases. by the end of the century, ten major programs (then termed centers, institute, and offices) were located at the cdc, the last focusing on unintentional injuries and violence. an th center was created a year later. global health programs related to hiv and other worldwide public health concerns continued to grow. by , a global network of more than field epidemiology training programs modeled on the eis was established. on any give day, the cdc had more than persons stationed internationally on long-term assignments. nationally and globally, the science and services of the cdc were in great demand. the mmwr was available on the internet and in received . million hits. additional notable public health events marked the early years of the twenty-first century -the terrorist attacks on the world trade center and the pentagon (and the downing of a fourth airliner in a field in pennsylvania) and the deliberate anthrax poisonings in . those two major events were followed in by hurricanes katrina and rita occurring in rapid succession. all of these crises had an extraordinary effect on the country and on the cdc. the emergence of a major change in influenza made real the threat of another pandemic, and this also played out markedly at the agency. two important themes emerged from these events. first, the nation and the cdc needed to adapt their approach to public health emergencies. although the traditional approaches worked, improvement was needed. the impact on the public health system was significant and long-lasting. second, the cdc received substantial additional funding, most of which was provided to traditional partners in state and local health departments to strengthen public health system infrastructures and to adapt to new demands. more than staff are now employed at the cdc, and the budget in exceeded $ billion, $ billion of which was shared with state and local governmental partners and with nonprofit organizations. the new era stimulated a major examination of the agency and its future, and in led to a major reorganization that focused both on adaptation and response to crises but also greater collaboration across the major programs. the intent was to focus on major public health goals and not necessarily specific disease or injury programs, except in support of these goals. as with the reorganization two decades earlier, this process will take years to finalize and to assess the impact. beginning with a regional focus on a single parasite, the cdc has become the premier public health agency in the world, and its expertise has expanded in direct correlation with the expanding view of public health needs. the emerging concerns of the new century -genomics, globalization, the built environment, information technology, global warming, emerging infections, violence, and so onwill require not only the traditional disciplines but also new expertise and new global partners, both public and private. what will remain the same at the cdc is the dedication to its mission in global public health and its adherence to the core values of accountability, integrity, and respect. the new century has already proven challenging and exciting, a situation that cdc anticipates eagerly. ohio) and provides epidemiologic assistance to canada during the winnipeg flood -epidemic intelligence service (eis) established -cdc reports first case of bat rabies in united states -cdc establishes polio surveillance unit during national investigation of contaminated vaccine -asian influenza pandemic; venereal disease division (vdd) transferred to cdc -first assistance to southeast asia in response to cholera and smallpox epidemics -permanent facilities open on property adjacent to emory university campus; tuberculosis program transferred to cdc -morbidity and mortality weekly report (mmwr) moves to cdc; cooperative cholesterol standardization program established -foreign quarantine service joins cdc control; smallpox eradicated in west africa -national institute for occupational safety and health moves to cdc -mmwr reports that lead emissions in residential area are a public health threat -first field epidemiology training program established in canada -global smallpox eradication achieved (eradication certified by the world health organization in ) -first outbreak of multidrug-resistant tuberculosis (mississippi) reported -publication of healthy people established measurable public health goals for the united states for -agency for toxic substances and disease registry established; mmwr publishes first report of toxic shock syndrome associated with tampon use -mmwr publishes first report of fatal disease eventually called acquired immunodeficiency syndrome (aids) -cdc reorganized with new centers for infectious diseases cdc established violence epidemiology branch -office on smoking and health moves to cdc -national center for health statistics moves to cdc -national center for chronic disease prevention and health promotion established -phs recommends that all women of childbearing age consume mg of folic acid daily to reduce risk for spina bifida and anencephaly -national center for injury prevention and control established -cdc's prevention mission is recognized, and it becomes the centers for disease control and prevention -polio elimination certified in the americas -cdc reports measurable levels of serum cotinine in blood of % of nonsmokers in united states -cdc participates in presidential apology for the tuskegee study of syphilis treatment among black men -cdc's laboratory response network established -national center for birth defects and developmental disabilities established -director's emergency operations center opened -cdc provides global assistance for surveillance and clinical and laboratory evaluation regarding severe acute respiratory syndrome (sars) cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective cdc's th anniversary: director's perspective-david satcher cdc's th anniversary: director's perspective sentinel for health: a history of the centers for disease control legionnaire's disease: description of an epidemic of pneumonia current cdc efforts to prevent and control human immunodeficiency virus infections and aids in the united states through information and education the cutter incident: poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the united states during the spring of . i: background. ii: relationship of poliomyelitis to cutter vaccine. iii: comparison of the clinical character of vaccinated and contact cases occurring after use of high rate lots of cutter vaccine department of health and human services reflections on the swine flu vaccination program the epidemic intelligence service of the centers for disease control and prevention: years of training and service in applied epidemiology a tribute to alexander d partnerships in international applied epidemiology training and service key: cord- -se sq c authors: lamps, l w title: infective disorders of the gastrointestinal tract date: - - journal: histopathology doi: . /j. - . . .x sha: doc_id: cord_uid: se sq c gastrointestinal infections are a major cause of morbidity and mortality worldwide. infectious organisms are often recovered by microbiological methods, but surgical pathologists may play a very valuable role in diagnosis. this review will focus on infective disorders of the gastrointestinal tract with an emphasis on enterocolitides caused by food‐ and water‐borne pathogens. diagnostic histological features of selected enteric infections will be emphasized, including those that mimic other inflammatory conditions of the gut (such as ischaemia or idiopathic inflammatory bowel disease), along with available diagnostic methods that can aid in diagnosis. infective disorders of the gastrointestinal tract are a major cause of morbidity and mortality worldwide. as numbers of transplant patients and those with other immunocompromising conditions increase, and as global urbanization and transcontinental travel become more frequent, the surgical pathologist must be familiar with infectious diseases that were previously encountered only infrequently. the pathologist's goal in evaluating a gastrointestinal biopsy for infectious colitis is twofold. first, acute self-limited and ⁄ or infectious processes must be differentiated from chronic idiopathic inflammatory bowel disease (ulcerative colitis or crohn's disease). [ ] [ ] [ ] [ ] second, dedicated attempts must be made to identify the infecting organism(s). the surgical pathologist's ability to diagnose infectious processes in tissue sections has grown exponentially with the advent of new histochemical stains, immunohistochemistry, in situ hybridization and numerous other molecular methodologies. as these techniques have developed, our understanding of the correlation between histological patterns of inflammation and specific organisms or groups of organisms has also increased. the majority of enteric infections are self-limited. those patients that undergo endoscopic biopsy often have chronic or debilitating diarrhoea, systemic symptoms, or a history of immunocompromise or other significant clinical scenarios. a discussion with the gastroenterologist regarding exact symptomatology and colonoscopic findings, as well as facts including travel history, food intake (such as sushi or poorly cooked beef), sexual practices and immune status, can greatly aid in the evaluation of a biopsy for infectious diseases. as many infective disorders of the gastrointestinal tract are acquired through contaminated food, this review will focus primarily on enterocolitides caused by food-borne bacteria. in addition, infectious processes that mimic or cause ischaemia and those that mimic chronic idiopathic inflammatory bowel disease will be emphasized, along with ancillary techniques that aid in diagnosis. despite the vast number of food-borne infections that affect the gastrointestinal tract, the spectrum of histological findings produced by a given organism can be generally categorized in one of the following ways (see table ): those producing minimal or no histological changes (e.g. vibrio species and many enteric viruses). those producing a non-specific acute self-limited ⁄ infectious colitis (aslc) pattern (e.g. campylobacter jejuni); this is one of the most common inflammatory patterns in enteric infections. [ ] [ ] [ ] [ ] those producing suggestive or diagnostic histological features (e.g. pseudomembranes, granulomas, or visible organisms). acute infectious-type colitis characteristically features intact crypt architecture with neutrophilic infiltrates in the crypt epithelium. the lamina propria may be hypercellular, containing a mix of lymphocytes, histiocytes and neutrophils; plasma cells are generally not prominent, and basal plasma cells should not be seen in acute infectious-type colitis as these are a marker of chronicity. crypt abscesses and granulomas associated with damaged crypts may also be seen. there is often damage also to the surface epithelium. [ ] [ ] [ ] since patients often do not come to endoscopy until several weeks after onset of symptoms, pathologists frequently do not see the classic histological features of acute infectious-type colitis. this is important, as the resolving phase of infectious colitis is more challenging to diagnose, as one may find only occasional foci of neutrophilic cryptitis with a patchy increase in lamina propria inflammation, which may contain numerous plasma cells and increased intraepithelial lymphocytes ( figure ). as these features can also be seen in smoldering crohn's disease and lymphocytic colitis, it is important to know the patient's symptoms and, ideally, culture results as this differential diagnosis may be difficult to resolve on histological grounds alone. selected specific food-and ⁄ or water-borne gastrointestinal infective disorders escherichia coli, campylobacter, salmonella, yersinia, shigella and enteric viruses represent the most common food-borne pathogens worldwide. it is important to note, when reviewing a patient's history, that many organisms associated with food-borne illness can also be transmitted through water or unpasteurized milk. naturally occurring water-borne outbreaks of sa. typhi, enterohaemorrhagic e. coli and norwalk virus have been reported, usually due to improperly operated water treatment plants. several other organisms (including anthrax, francisella tularensis and shigella) are particularly stable in water due to chlorine tolerance. although these organisms are sometimes recovered by culture, surgical pathologists may play a very valuable role in diagnosis, particularly when clinicians have failed to obtain cultures prior to initiation of antibiotic therapy. in addition, many of the food-and water-borne gastrointestinal infective diseases discussed below mimic other entities that are commonly encountered in surgical pathology practice, such as ischaemic colitis or idiopathic inflammatory bowel disease. these gram-negative bacteria are a major cause of naturally occurring diarrhoea worldwide. campylobacter jejuni and c. coli are most frequently associated with human enteric disease. , campylobacter may contaminate poultry, beef, veal, pork, water and milk, and are also transmitted by the faecal-oral route. the infective dose is low (as few as organisms can cause symptomatic disease). patients typically present with fever, malaise, abdominal pain and watery diarrhoea that often contains blood and faecal leucocytes. [ ] [ ] [ ] symptoms generally present within - days of exposure and last for - days. most infections are self-limited, particularly in otherwise healthy patients, although relapse is common. of note, both reactive arthropathy and guillain-barré syndrome may be associated with campylobacter infection. endoscopic findings are non-specific and include friable colonic mucosa with associated erythema and haemorrhage. histological examination shows features of acute self-limited colitis, including a neutrophilic infiltrate in the lamina propria, often most prominent in the mid-to-upper crypts; cryptitis; and scattered crypt abscesses ( figure ). architecture overall is preserved, although mild crypt distortion is occasionally noted. - the histological differential diagnosis includes other infectious entities that produce the aslc pattern. the mainstay of laboratory diagnosis is culture from stool or blood. preliminary diagnosis may be made by detection of organisms in fresh stool smears by darkfield microscopy. excellent molecular techniques exist, but are not widely available for clinical use. these gram-negative bacilli are particularly prevalent in underdeveloped countries where sanitation is poor and dairy and water supplies are contaminated with the organism. however, salmonella are also an important cause of sporadic food poisoning in developed countries. they are present in water, meat, dairy products, egg products and occasionally vegetables and fruits; they may survive partial cooking. the discussion of salmonella species is historically divided into typhoid and non-typhoid species. patients with typhoid (enteric) fever, usually caused by sa. typhi, typically present with fever that rises over several days, abdominal pain and headache. abdominal rash ('rose spots'), delirium, hepatosplenomegaly and leukopenia are also fairly common. the diarrhoea, which begins in the second or third week of infection, is initially watery but may progress to severe gastrointestinal bleeding and perforation. non-typhoid species (e.g. sa. enteritidis, sa. typhimurium, sa. muenchen, sa. anatum, sa. paratyphi and sa. give) generally cause a milder, often self-limited gastroenteritis with vomiting, nausea, fever and watery diarrhoea. [ ] [ ] [ ] [ ] [ ] the infective dose is relatively low (approximately ) organisms may cause human disease). although most salmonella infections in developed countries resolve with antibiotics and supportive care, enteric infection may progress to septicaemia and death, particularly in the elderly, the very young or patients who are debilitated. delayed treatment is associated with higher mortality. any level of the gastrointestinal tract may be affected, but the ileum, appendix and right colon are preferentially involved. the bowel wall is thickened, with raised nodules corresponding to hyperplastic peyer patches. apthoid ulcers overlying peyer patches, linear ulcers, discoid ulcers or full thickness ulceration and necrosis often occur as disease progresses. perforation and toxic megacolon may be seen, as can suppurative mesenteric lymphadenitis. peyer patches become hyperplastic, followed by acute inflammation of the superficial overlying epithelium. eventually the lymphoid follicles are infiltrated and obliterated by macrophages. the histiocyte is the predominant inflammatory cell in typhoid fever; admixed lymphocytes and plasma cells are seen, but neutrophils are typically not prominent. the ulcers are characteristically very deep, with the base at the muscularis propria. [ ] [ ] [ ] [ ] [ ] architectural distortion severe enough to mimic ulcerative colitis or crohn's disease may be present ( figure ) . with non-typhoid species, the gross findings are often milder. the lesions can be focal, and occasionally the mucosa is grossly normal or only mildly hyperaemic and oedematous. the pathological features are most often those of acute self-limited colitis, although severe cases may have significant crypt distortion. cultures are the mainstay of salmonella diagnosis. the differential diagnosis includes other enteric bacterial pathogens as well as crohn's disease and ulcerative colitis. , clinically, the incubation period of salmonella infection is longer ( - days) than with similar enteric pathogens. salmonella infection often lacks significant numbers of neutrophils in comparison with other pathogens (as well as idiopathic inflammatory bowel disease), and granulomas are unusual in salmonellosis. crypt distortion is generally more pronounced in ulcerative colitis than in salmonellosis. clinical presentation and stool cultures may be very helpful in resolving the differential. shigella are virulent, invasive gram-negative bacilli that cause severe bloody diarrhoea. shigella dysenteriae is the most common species isolated, although sh. sonnei and sh. flexneri are increasingly reported in the usa. they are generally ingested from faecally contaminated water, but person-to-person transmission is also possible. the infective dose is very low (as few as - organisms in sh. dysenteriae type ). infants and small children, homosexual males and malnourished or debilitated patients are most commonly affected in developed countries. like salmonellosis, the gross and microscopic features of shigellosis may mimic chronic idiopathic inflammatory bowel disease. symptoms include abdominal pain, fever and watery diarrhoea, followed by bloody diarrhoea that is often mucoid and ⁄ or purulent. constitutional symptoms are common. , perforation and haemolytic-uraemic syndrome have been described. most studies of mortality in shigellosis were performed in underdeveloped nations, and figures range from to %. mortality is significantly higher (> %) if patients develop sepsis. the large bowel is typically affected (often the left colon most severely), but the ileum may also be involved. the mucosa is haemorrhagic, and variably ulcerated, sometimes with pseudomembranous exudates. early shigellosis has features of aslc with cryptitis, crypt abscesses (often superficial) and ulceration. apthoid ulcers similar to crohn's disease are variably present. as disease continues, mucosal destruction develops with many neutrophils (as well as other inflammatory cells) in the lamina propria. marked architectural distortion is common, leading to diagnostic confusion with chronic idiopathic inflammatory bowel disease. stool culture is essential to diagnosis, but multiple cultures may be necessary to recover the organism, for it is fastidious and dies off quickly. polymerase chain reaction (pcr), dna probes and serological studies are also available. the differential diagnosis of early shigellosis is primarily that of other infections, particularly enteroinvasive e. coli and clostridium difficile. later on in the course of the disease, it may be extremely difficult to distinguish shigellosis from crohn's disease or ulcerative colitis both endoscopically and histologically. stool cultures and clinical history may be very helpful in resolving the differential. yersinia enterocolitica (ye) and y. pseudotuberculosis (yp) are the most common causes of bacterial enteritis in western and northern europe, and increasing numbers of cases have been documented in north america and australia. yersinia have been found in chicken, fish, shellfish, swine, cattle, milk, ice cream, beef, lamb, poultry, pork, ham and water. infection with either species may cause abdominal pain, diarrhoea and symptoms and signs of appendicitis, enterocolitis or an acute abdomen. associated mesenteric lymphadenopathy is frequent. most infections are selflimited; however, immunocompromised and debilitated patients, as well as patients on deferoxamine or with iron overload, are at risk of more serious disease. yersinia preferentially involves the ileum, right colon and appendix. grossly, involved bowel has a thickened, oedematous wall with nodular inflammatory masses centred around peyer patches. apthoid and linear ulcers may be present. both suppurative and granulomatous patterns of inflammation can be seen, and a mixture of the two is common. [ ] [ ] [ ] recent studies have shown that there is significant overlap between the histological features of ye and yp infection and that either species may show lymphoid hyperplasia, epithelioid granulomas with prominent lymphoid cuffing (figure ) , transmural lymphoid aggregates, giant cells, mucosal ulceration, cryptitis and concomitant lymph node involvement. special stains are not helpful in the diagnosis of yersinia, for the organisms are small, may be present in low numbers and are difficult to distinguish from normal non-pathogenic colonic flora. culture, serological studies and pcr assays may be helpful in confirming the diagnosis. , the major differential diagnoses for yersiniosis include other infectious processes, particularly mycobacteria and salmonella. acidfast stains and culture results should help to distinguish mycobacterial infection; clinical features and the presence of greater numbers of neutrophils, microabscesses and granulomas may help to distinguish yersiniosis from salmonellosis. crohn's disease and yersiniosis may be very difficult if not impossible to distinguish from one another histologically. features that may favour crohn's include cobblestoning of mucosa and creeping fat grossly, and changes of chronicity microscopically, including crypt distortion, thickening of the muscularis mucosa and prominent neural hyperplasia. however, some cases are indistinguishable on histological grounds. aeromonas species, initially thought to be non-pathogenic gram-negative bacteria, are increasingly recognized as causes of gastroenteritis in both children and adults. the motile a. hydrophila and a. sobria most often cause gastrointestinal disease in humans. the typical presentation is bloody diarrhoea, sometimes chronic, accompanied by nausea, vomiting and cramping pain. the diarrhoea may contain mucus as well as blood. the duration of illness varies widely, ranging from a few days to several years, indicating that aeromonas infection can cause a chronic colitis. [ ] [ ] [ ] [ ] [ ] endoscopically, signs of colitis may be seen, including oedema, friability, erosions, exudates and loss of vascular pattern; the features are often segmental and may mimic ischaemic colitis or crohn's disease. a pancolitis mimicking ulcerative colitis has also been described. the histological features are usually those of acute self-limited colitis. however, ulceration and focal architectural distortion may be seen ( figure ). stool cultures are critical to diagnosis. the differential diagnosis includes other infectious processes, ischaemic colitis and chronic idiopathic inflammatory bowel disease. culture should help exclude other infections, and typical features of ischaemia (crypt withering, mucosal necrosis) are not present with aeromonas. when architectural distortion is present in a patient with chronic symptoms, it may be very difficult to resolve the issue of aeromonas infection versus crohn's disease or ulcerative colitis. [ ] [ ] [ ] [ ] [ ] some authorities recommend culturing for aeromonas in all patients with refractory chronic inflammatory bowel disease. approximately % of the world's population are infected with this parasite, and the prevalence is much higher in tropical and subtropical locations. increasingly, male homosexuals are also found to harbour this pathogen. although some patients suffer a severe, dysentery-like fulminant colitis, many others are asymptomatic or have only vague gastrointestinal symptoms (abdominal pain, diarrhoea with or without blood, and malaise). rarely, large inflammatory masses (amoebomas) may be formed. grossly, small ulcers are the early lesion, which may coalesce to form large, irregular ulcers that are often geographical or serpiginous. the ulcers often have associated inflammatory exudate or inflammatory polyps. intervening mucosa is often normal. ulcers may undermine adjacent mucosa to produce the classic 'flask-shaped' lesion. the caecum is most commonly involved, but any level of the large bowel or appendix may be involved. fulminant colitis resembling ulcerative colitis, pseudomembranous colitis resembling that caused by c. difficile, and toxic megacolon have been described. colonoscopy may be normal in asymptomatic patients or those with mild disease. histologically, the earliest lesion is a mild neutrophilic infiltrate. in more advanced disease, ulcers are often deep, extending at least into the submucosa, with undermining of adjacent normal mucosa. there is associated necroinflammatory debris; the organisms are generally found within this purulent material. invasive amoebae are occasionally seen within the bowel wall. the adjacent mucosa is usually normal, but may show gland distortion and inflammation. the organisms, which may be very few in number, resemble macrophages with foamy cytoplasm and a round, eccentric nucleus; the presence of ingested red blood cells is pathognomonic of entamoeba histolytica. in patients who are asymptomatic or have mild symptoms, histological changes may be subtle and organisms may be particularly difficult (if not impossible) to find. invasive amoebiasis is not generally seen in patients with mild or absent symptoms. the differential diagnosis most often is that of amoebae versus macrophages within an inflammatory exudate. amoebae are trichrome positive; in addition, macrophages stain with immunostains for a -antitrypsin and chymotrypsin, whereas amoebae do not. given the not infrequent presence of skip lesions and mucosal architectural distortion, amoebiasis may also be confused with crohn's disease or ulcerative colitis, as well as other types of infectious colitis. for the diagnostic pathologist, it cannot be overemphasized that yersinia (enterocolitica and pseudotuberculosis), salmonella (both non-typhoid and typhoid fever-producing strains), shigella, aeromonas and e. histolytica, in addition to the aslc pattern of inflammation, can produce both gross and histological features that mimic idiopathic inflammatory bowel disease (table ) . correlation with clinical presentation, food and water exposure, and culture results can be invaluable tools in resolving this differential diagnosis. enterohaemorrhagic e. coli the most common strain of enterohaemorrhagic e. coli (ehec) is o :h , although there are others. this pathogen gained national attention in when a massive outbreak in the western usa was linked to contaminated hamburger patties. the organism binds intestinal epithelial cells and produces a cytotoxin similar to that produced by sh. dysenteriae, although there is no invasion. contaminated meat is the most frequent mode of transmission, but infection may also occur through contaminated water, milk, produce and person-toperson contact. , diarrhoea is usually bloody, with severe abdominal cramps and mild or no fever. non-bloody, watery diarrhoea may occur, however. only one-third of patients have faecal leucocytes. children and the elderly are at particular risk of serious illness, including the haemolytic-uraemic syndrome or thrombotic thrombocytopenic purpura. , endoscopically, patients may have bowel oedema, erosions, ulcers and haemorrhage, and the right colon is usually more severely affected. the oedema may be so marked as to cause obstruction, and surgical resection may be required to relieve this or to control bleeding. the lamina propria and submucosa contain marked oedema and haemorrhage, with associated mucosal acute inflammation, cryptitis, crypt abscesses, ulceration and necrosis. crypt withering, such as that seen in other causes of ischaemia, is often seen as well. microthrombi may be seen within small vessels and pseudomembranes are occasionally present ( figure ). , infection with ehec is probably markedly underdiagnosed. routine stool cultures will not distinguish pathogenic strains from normal intestinal flora. if ehec strains are suspected, the clinical laboratory should be notified to search for them specifically. ehec are cleared rapidly from stool, often within - days, thus cultures should be taken as early as possible. culture and serotyping are the mainstay of laboratory diagnosis; although immunohistochemical stains and molecular assays for ehec have been described, they are not widely available. the differential diagnosis of ehec includes c. difficilerelated colitis, idiopathic inflammatory bowel disease, and especially ischaemic colitis. culture and food intake history may be invaluable. in general, ehec does not produce the level of mucosal distortion seen in idiopathic inflammatory bowel disease, and lacks granulomas. the histological picture may be very difficult to distinguish from c. difficile, and the c. difficile antigen test may be very useful. as the cytotoxin produced by ehec causes ischaemia, the histological features are indistinguishable from ischaemia of other causes. biopsies from patients with enteric viral infections seldom if ever cross the stage of the surgical pathologist, as they are detected in stool samples rather than biopsy specimens. some common enteric viruses known to cause diarrhoea include, but are not limited to adenovirus, rotavirus, coronavirus, echovirus, enterovirus, astrovirus and norwalk virus. rotavirus is the most common cause of severe childhood diarrhoea and diarrhoeal mortality worldwide, followed by adenoviruses. rotavirus infections occur predominantly in infants < years old. the diarrhoea is usually accompanied by fever and vomiting. biopsy changes are very non-specific and include increased inflammatory cells in the lamina propria, degenerative epithelial changes and swollen villous tips. the diagnosis of rotavirus is generally made by stool immunoassay and ⁄ or culture, and the disease is rarely biopsied. adenovirus infection is second only to rotavirus as a cause of childhood diarrhoea. however, it has also gained much attention in recent years as a cause of diarrhoea in immunocompromised patients, especially those with hiv and aids. virtually all patients present with diarrhoea, sometimes accompanied by fever, weight loss and abdominal pain. histological features of adenovirus infection include epithelial changes such as cellular disorder, loss of orientation and degeneration. characteristic inclusions may be seen, especially in immunocompromised patients, within the nuclei of surface epithelium (particularly goblet cells). useful aids to diagnosis of adenovirus infection include immunohistochemistry, stool examination by electron microscopy and viral culture. other, less common but well-recognized causes of food and water-related gastrointestinal disease include brucellosis, bacillus cereus and listeria monocytogenes. it is also important to remember that many serious food-borne gastrointestinal pathogens may produce little or no inflammatory infiltrate at all, particularly in immunocompromised patients, even in the face of grave clinical disease. these include vibrio cholerae and non-cholera vibrio infections, enteropathogenic and enteroadherent e. coli and infection with many enteric viruses (table ). surgical pathologists may play a very valuable role in the diagnosis of gastrointestinal infectious processes. once it is determined that an infectious process is in the differential diagnosis, it is very important to alert the clinician, and often the microbiology laboratory, that this is a consideration. in addition to valuable culture techniques, the surgical pathologist's ability to diagnose infectious diseases in tissue sections has grown exponentially in recent years with the advent of new histochemical stains, immunohistochemistry, in situ hybridization and pcr analysis (table ) . , , , in summary, infective (including food-and waterborne) gastrointestinal disorders are common, although they are in general underdiagnosed and under-reported. many of these infections mimic other inflammatory gastrointestinal disease processes, such as ischaemic colitis, crohn's disease and ulcerative colitis. it is important for surgical pathologists to consider infectious agents in the differential diagnosis as they evaluate inflammatory lesions. the availability of pcr assays and immunohistochemical antibodies for infectious agents is increasing, and these techniques may prove invaluable in associating specific infectious microorganisms with histological patterns of disease, thus facilitating the diagnosis of infectious processes. the role of rectal biopsy in infectious colitis surgical pathology of the infected gut the clinical significance of focal active colitis the histopathologic spectrum of acute self-limited colitis (acute infectious-type colitis) enteric infections associated with exposure to animals or animal products precautions against biological and chemical terrorism directed at food and water supplies molecular detection of campylobacter infection in cases of focal active colitis campylobacter jejuni acute colitis caused by campylobacter fetus ss. jejuni acute diarrhoea: campylobacter colitis and the role of rectal biopsy bacterial diarrheas and dystenteries salmonella including s. typhi histopathology of typhoid enteritis: morphologic and immunophenotypic findings pathology of salmonella colitis pathology of the alimentary tract in salmonella typhimurium food poisoning infections of the gastrointestinal tract morphology of rectal mucosa of patients with shigellosis distribution and spread of colonic lesions in shigellosis: a colonoscopic study yersinia enterocolitica and yersina pseudotuberculosis the role of y. enterocolitica and y. pseudotuberculosis in granulomatous appendicitis: a histologic and molecular study the pathology of yersinia enterocolitica ileocolitis the histopathology of enteric infection with yersinia pseudotuberculosis molecular biogrouping of pathogenic yersinia enterocolitica: development of a diagnostic pcr assay with histological correlation aeromonas sobria associated left sided segmental colitis the clinical significance of stool isolates of aeromonas aeromonas as a cause of segmental colitis aeromonas-associated gastroenteritis aeromonasrelated diarrhea in adults nondysenteric intestinal amebiasis: colonic morphology and search for entamoeba histolytica adherence and invasion pathology of human amebiasis the colonic pathology of e. coli :h infection escherichia coli :h -associated colitis: a clinical and histological study of cases diarrhoeal disease: current concepts and future challenges adenovirus colitis in human immunodeficiency virus infection: an underdiagnosed entity infections involving lymph nodes key: cord- - u s y authors: ten have, h.a.m.j. title: sheltering at our common home date: - - journal: j bioeth inq doi: . /s - - -x sha: doc_id: cord_uid: u s y the current covid- pandemic has reactivated ancient metaphors (especially military ones) but also initiated a new vocabulary: social distancing, lockdown, self-isolation, and sheltering in place. terminology is not ethically neutral but reflects prevailing value systems. i will argue that there are two metaphorical vocabularies at work: an authoritarian one and a liberal one. missing is an ecological vocabulary. it has been known for a long time that emerging infectious diseases are associated with the destruction of functioning ecosystems and biodiversity. ebola and avian influenza viruses have been significant warnings. obviously, this pandemic will not be the last one. as the planet is our common home, the major metaphor to explore is sheltering at this home. catastrophic risk for the future (global priorities project ). in , it was estimated that since more than thirty-five new infectious diseases have emerged in humans; one every eight months (smolinski et al. ) . since then the list has only grown (for example, the severe acute respiratory syndrome (sars) coronavirus, swine influenza (h n and other sub-types), the middle east respiratory syndrome (mers) coronavirus, ebola virus disease (evd), and the zika flavivirus). most of these outbreaks have been localized but the human immunodeficiency virus (hiv), sars, and avian influenza (h n and other sub-types) should have been warnings that globalization can easily occur. already in , the who launched its global influenza preparedness plan, urging countries to make national bio-preparedness plans, and many countries did so (who ) . furthermore, it has been known for some time that infectious diseases are promoted by environmental degradation as a result of biodiversity loss and climate change. destruction of ecosystems is shrinking the wildlife habitat and increasing contacts between wildlife and human beings. it is estimated that zoonotic pathogens cause per cent of emerging infectious diseases in humans (jones et al. ; daszak et al. ) . the global threat of pandemics therefore does not emerge spontaneously as a natural event but is the product of human behaviour. it is a consequence of the human way of life and exploitation of the planet. destruction of biodiversity creates the conditions for the emergence of new viral diseases (quammen ) . although much is unclear, studies indicate that bats are the reservoir hosts for the novel coronavirus sars-cov- causing covid- , while pangolins might be possible hosts. bats are very common mammals. they harbour around thirty different coronaviruses but in fact many more viruses, mostly unknown. pangolins are the most frequently trafficked mammals, especially in china, used as a food source and for traditional medicine. they have been traded in the wet market in wuhan where the infection emerged (lam et al. ) . live animal markets, industrial livestock farming, trade in wild animals, and consumption of wildlife meat bring virus reservoirs in close contact with human beings. the only way to prevent future zoonotic diseases is to study the origin of pandemics. instead of waiting for the next pandemic, epidemiological research should be expanded and stringent measures taken to prevent the "jumping" of viruses to humans. what is missing in the pandemic management responses so far is the ecological perspective that pandemics are related to the current economic global order which assumes a separation of humans and nature and regards nature as a resource to be exploited and commodified. the absence of an ecological perspective is highlighted in the images and words associated with covid- . sheltering in place is a notion from the cold war. when there is an imminent nuclear threat, people should take refuge in a small, interior room, lock all windows and remain indoors, or move to special shelters. this usually takes a few hours, not days or weeks. (american red cross, ) . the concept is also used for biological and chemical threats, as well as for extreme weather events. the basic idea is to wait until the worst is over; then go outside and resume normal activities. it assumes a specific view of disasters: they are sudden events with immediate but often localized impact such as tsunamis and tornadoes. pandemics, however, are gradual disasters; they have slow impact, and move across the globe, potentially affecting everyone. the difference is important for the degree of preparedness. since pandemics come in waves, not all regions and countries are simultaneously affected. this leaves time for preparation. it also means that sheltering will be extended for weeks perhaps months, not knowing how long it will provide security. during a pandemic, sheltering at home seems a more acceptable term. it avoids connotations of mandatory quarantine or isolation. it is restricting freedom of movement but appeals to the responsibility of individuals. contrary to shelter-in-place, sheltering at home is more lenient since it allows to go out for essential business and walking, keeping physical distance to other people. the notion of "home" generally has positive connotations. in distinction to "place" it is not a neutral location. it is where people live together in a space of intimacy and privacy, often regarded as a haven or refuge, a secure place to retreat and feel comfortable, a setting for caring relationships and conviviality (mallet ) . at home means more than residing in a specific place. of course, not all homes are the same (e.g. nursing homes), not all homes are safe (e.g. domestic abuse), and not everybody has a home. now that more than half of the world population is confined to their homes, philosopher gaston bachelard reminds us that beingat-home is more fundamental than activities such as working (bachelard ) . nevertheless, the emphasis on sheltering and isolation reflects an individualistic perspective, assuming that people can easily withdraw from social interactions. in many cases, connections with environing conditions cannot be severed. for numerous people, especially in low-and middle-income settings, this is not an option. also, poor people in affluent countries cannot shelter but have to expose themselves in order to subsist. the image of sheltering at home has become incorporated in popular discourse. previously used in epidemiology, new words (e.g. self-isolation, social distancing, and lockdown) are now disseminated to advance images of control and containment. they visualize the spread of the virus, what we can do to avoid infection and to become aware of the effects of our behaviour. these images are associated with the war metaphor dominating current policies. the disease is regarded as a threat, the virus as enemy, leaders as commanders, and vaccines as new weapons. today's metaphor is associated with the older discourse of bio-invasion. since the s, many countries have developed policies to protect native biodiversity against invasive species. non-native species are considered as aliens, enemies; we need to act to protect nature. the assumption is that nature and humans can be separated. nature, and wildlife in particular, is regarded as a source of danger. however, bio-invasion is impossible to prevent. when mobility and interconnectedness are the hallmarks of globalization, bio-invasion is a global phenomenon par excellence since borders are irrelevant. the same applies to new, "emerging" viruses. viral diseases have been with us since the beginning of humanity. they are now more easily disseminated through global traffic. the point is that these "new" viruses do not simply "emerge" as natural events. their impact is the result of human activities. the military metaphor is currently applied in most countries. the effect is reinforcement of boundaries. first, between inside and outside, enemies and friends. it blames people for "bringing in the virus." second, individuals and social context are disconnected. terminology is not ethically neutral but reflects prevailing value systems. when the virus moved across the world, some words (e.g. mandatory quarantines, containment zones, home confinement) were less often used in liberal societies. while the military metaphor is predominant, it is articulated in distinct policies: authoritarian and liberal ones. both appeal to different normative frameworks which are often mixed in practical approaches. for example, "lockdown" and "quarantine" emphasize state measures beyond individual rights and freedoms. on the other hand, "self-isolation" appeals to individual responsibility. the term "social distancing" is typically neoliberal; it does not recognize the communal nature of many societies. what is at stake is physical (not social) distancing, while in many countries social connections and solidarity have grown. emphasizing self-interest risks that other persons are seen as threats. many countries, especially in the west, struggle with the balance between authoritarian and liberal policies. they are concerned with the protection of human rights and civil liberties. the discussion about the use of surveillance technologies and their impact on privacy is an example (gostin, friedman, and wetter ) . since both authoritarian and liberal policies are animated by the military metaphor, they share the same urge to control. they reflect a system of governance that administers, fosters, and secures life by controlling the population and disciplining the individual. this application of "biopower" is closely linked to the ideology of neoliberalism in emphasizing internal regulation by autonomous subjects rather than external force and pressure (foucault ; kakuk ) . even in authoritarian countries, stringent policies will not succeed if they do not appeal to the responsibility of citizens. emphasizing individual responsibility and empowerment, however, neglects the social, political, and economic dimensions of human life. it narrows bioethical discourse since it is difficult to conceptualize the significance of the context in which problems arise and to develop practices based on relationality and connectedness which are articulated in global bioethics with an ecological vocabulary. interconnectedness and interdependency articulate that humans cannot be separated from the surrounding world, not only society and culture but also the natural world of animals and plants. the starting point for bioethical discourse is therefore not individual autonomy but the broader context in which individuals are embedded. self-isolation in this perspective is not merely protecting oneself but first of all protecting fellow citizens. the experience of togetherness is not restricted to humans but involves all forms of life, even viruses. regarding humans as part of an interconnected web of life means moving from an anthropo-centric to biocentric approach with a relational concept of the self, dependent on biodiversity. this shift has been advocated by many environmental ethicists as well as in indigenous worldviews (rolston ; johnson ) .the ecological perspective implies that the military language of the pandemic is distorting the human embeddedness in the natural world. emphasizing the antagonism between humans and the virus as enemy, blames nature for diseases ignoring that diseases emerge due to environmental degradation as a result of human exploitation of biodiversity. it also disregards that microbes are inhabitants of our world; they have been and will be always there. rather than eradication, cohabitation will be required. the military metaphor encourages the search for "magic bullets" that can attack and eliminate the virus. it also creates an atmosphere of secrecy, suspicion, and mistrust where facts, findings, and potential weapons are disputed and concealed. another consequence of the ecological perspective is a broader notion of health. human health, animal health, and healthy ecosystems are linked. human beings cannot be healthy when the planet is not healthy. the implication is that governance should be global. it should be focused on health as one, as a planetary concern. applying the vision of one health means monitoring and surveilling human connections with animals, specifically in the bioindustry. another implication is that attention should not merely focus on morbidity and mortality but on prevention. future pandemics will emerge if environmental destruction and loss of biodiversity are not addressed. global interconnectedness implies that citizens in one country will be exposed to diseases when they emerge in another country. the ecological perspective therefore stresses the need for solidarity. this is not just an ethical requirement, but a medical necessity. closing borders, restricting travel, and concentrating on national interests had only a limited effect on the dissemination of covid- . public health as a common good is essential for the well-being and survival of humanity. it will require cooperation and collective action, especially in the early stages of a pandemic disease when the caseload is still manageable and secondary prevention is possible. in an ecological perspective, vulnerability to infectious diseases is not confined to specific individuals, populations, or nations. while covid- and its impact will be temporary, the effect of climate change is already there and will continue, requiring sustained action for a very long time (guterres ) . both global threats are interpreted from a similar perspective: humans are not embedded in nature. the earth is a resource that can be commodified and exploited; it is not regarded as a living organism that creates and nourishes life (humans, animals, and microorganisms). the fact that many of us are now confined to our homes is hopefully an incentive to realize that we all share a common home. it demonstrates not merely interconnectedness of human beings manifested in society and culture but furthermore their embeddedness in the natural world. what affects the health of the planet will unquestionably deteriorate human health. concepts such as "one health" and "planetary health" articulate the connection between healthcare and earthcare. they make clear that the focus of bioethics should go beyond individual health and that effective policies should be based on collective action (ten have ). given the interconnection between health and biodiversity, the notion of sheltering at our common home is most appropriate in the current circumstances. the earth is our home, as stated in the preamble of the rio declaration (united nations ) . like all homes, this common home has dark sides, clearly noticeable today. but humans have no other dwelling place. our actions can destroy this place but they can also turn it into a home where everyone feels safe and secure. facing the covid- pandemic is an opportunity to make ourselves at home in the world and to preserve our common home. fact sheet on shelter-in-place the poetics of space conservation medicine and a new agenda for emerging diseases the birth of biopolitics: lectures at the collège de france global catastrophic risks. stockholm: global challenges foundation responding to covid- : how to navigate a public health emergency legally and ethically press conference by secretary-general antonio guterres at united nations headquarters indigenous jingle dress dancing goes "viral" on social media to help heal the world. cbc news global trends in emerging infectious diseases . bioethics and biopolitics. theories, applications and connections identifying sars-cov- related coronaviruses in malayan pangolins understanding home. a critical review of the literature spillover. animal infections and the next human pandemic environmental ethics: duties to and values in the natural world microbial threats to health: emergence, detection, and response united nations. . rio declaration on environment and development. united nations general assembly a / c o n f . / who global influenza preparedness plan. the role of who and recommendations for national measures before and during pandemics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - qg fn f authors: adiga, aniruddha; dubhashi, devdatt; lewis, bryan; marathe, madhav; venkatramanan, srinivasan; vullikanti, anil title: mathematical models for covid- pandemic: a comparative analysis date: - - journal: j indian inst sci doi: . /s - - - sha: doc_id: cord_uid: qg fn f covid- pandemic represents an unprecedented global health crisis in the last years. its economic, social and health impact continues to grow and is likely to end up as one of the worst global disasters since the pandemic and the world wars. mathematical models have played an important role in the ongoing crisis; they have been used to inform public policies and have been instrumental in many of the social distancing measures that were instituted worldwide. in this article, we review some of the important mathematical models used to support the ongoing planning and response efforts. these models differ in their use, their mathematical form and their scope. abstract | covid- pandemic represents an unprecedented global health crisis in the last years. its economic, social and health impact continues to grow and is likely to end up as one of the worst global disasters since the pandemic and the world wars. mathematical models have played an important role in the ongoing crisis; they have been used to inform public policies and have been instrumental in many of the social distancing measures that were instituted worldwide. in this article, we review some of the important mathematical models used to support the ongoing planning and response efforts. these models differ in their use, their mathematical form and their scope. models have been used by mathematical epidemiologists to support a broad range of policy questions. their use during covid- has been widespread. in general, the type and form of models used in epidemiology depend on the phase of the epidemic. before an epidemic, models are used for planning and identifying critical gaps and prepare plans to detect and respond in the event of a pandemic. at the start of a pandemic, policy makers are interested in asking questions such as: (i) where and how did the pandemic start, (ii) risk of its spread in the region, (iii) risk of importation in other regions of the world, (iv) basic understanding of the pathogen and its epidemiological characteristics. as the pandemic takes hold, researchers begin investigating: (i) various intervention and control strategies; usually pharmaceutical interventions do not work in the event of a pandemic and thus nonpharmaceutical interventions are most appropriate, (ii) forecasting the epidemic incidence rate, hospitalization rate and mortality rate, (iii) efficiently allocating scarce medical resources to treat the patients and (iv) understanding the change in individual and collective behavior and adherence to public policies. after the pandemic starts to slow down, modelers are interested in developing models related to recovery and long-term impacts caused by the pandemic. j. indian inst. sci. | vol xxx:x | xxx-xxx | journal.iisc.ernet.in as a result comparing models needs to be done with care. when comparing models: one needs to specify: (a) the purpose of the model, (b) the end user to whom the model is targeted, (c) the spatial and temporal resolution of the model, (d) and the underlying assumptions and limitations. we illustrate these issues by summarizing a few key methods for projection and forecasting of disease outcomes in the us and sweden. organization. the paper is organized as follows. in sect. we give preliminary definitions. section discusses us and uk centric models developed by researchers at the imperial college. section discusses metapopulation models focused on the us that were developed by our group at uva and the models developed by researchers at northeastern university. section describes models developed swedish researchers for studying the outbreak in sweden. in sect. we discuss methods developed for forecasting. section contains discussion, model limitations and concluding remarks. in a companion paper that appears in this special issue, we address certain complementary issues related to pandemic planning and response, including role of data and analytics. important note. the primary purpose of the paper is to highlight some of the salient computational models that are currently being used to support covid- pandemic response. these models, like all models, have their strengths and weaknesses-they have all faced challenges arising from the lack of timely data. our goal is not to pick winners and losers among these model; each model has been used by policy makers and continues to be used to advice various agencies. rather, our goal is to introduce to the reader a range of models that can be used in such situations. a simple model is no better or worse than a complicated model. the suitability of a specific model for a given question needs to be evaluated by the decision maker and the modeler. for epidemiology epidemiological models fall in two broad classes: statistical models that are largely data driven and mechanistic models that are based on underlying theoretical principles developed by scientists on how the disease spreads. data-driven models use statistical and machine learning methods to forecast outcomes, such as case counts, mortality and hospital demands. this is a very active area of research, and a broad class of techniques have been developed, including auto-regressive time series methods, bayesian techniques and deep learning , , , , , . mechanistic models of disease spread within a population , , , use mechanistic (also referred to as procedural or algorithmic) methods to describe the evolution of an epidemic through a population. the most common of these is the sir type models. hybrid models that combine mechanistic models with data driven machine learning approaches are also starting to become popular, e.g., . there are a number of models, which are referred to as sir class of models. these partition a population of n agents into three sets, each corresponding to a disease state, which is one of: susceptible (s), infective (i) and removed or recovered (r). the specific model then specifies how susceptible individuals become infectious, and then recover. in its simplest form (referred to as the basic compartmental model) , , , the population is assumed to be completely mixed. let s(t), i(t) and r(t) denote the number of people who are susceptible, infected and recovered states at time t, respectively. let s(t) = s(t)/n , then, the sir model can be described by the following system of ordinary differential equations where β is referred to as the transmission rate, and γ is the recovery rate. a key parameter in such a model is the "reproductive number", denoted by r = β/γ . at the start of an epidemic, much of the public health effort is focused on estimating r from observed infections . mass action compartmental models have been the workhorse for epidemiologists and have been widely used for over years. their strength comes from their simplicity, both analytically and from the standpoint of understanding the outcomes. software systems have been developed to solve such models and a number of associated tools have been built to support analysis using such models. although simple and powerful, mass action compartmental models do not capture the inherent heterogeneity of the underlying populations. significant amount of research has been conducted j. indian inst. sci. | vol xxx:x | xxx-xxx | journal.iisc.ernet.in to extend the model, usually in two broad ways. the first involves structured metapopulation models-these construct an abstraction of the mixing patterns in the population into m different sub-populations, e.g., age groups and small geographical regions, and attempt to capture the heterogeneity in mixing patterns across subpopulations. in other words, the model has states s j (t), i j (t), r j (t) for each subpopulation j. the evolution of a compartment x j (t) is determined by mixing within and across compartments. for instance, survey data on mixing across age groups have been used to construct age structured metapopulation models . more relevant for our paper are spatial metapopulation models, in which the subpopulations are connected through airline and commuter flow networks , , , , . main steps in constructing structured metapopulation models. this depends on the disease, population and the type of question being studied. the key steps in the development of such models for the spread of diseases over large populations include • constructing subpopulations and compartments: the entire population v is partitioned into subpopulations v j , within which the mixing is assumed to be complete. depending on the disease model, there are s j , e j , i j , r j compartments corresponding to the subpopulation v j (and more, depending on the disease)-these represent the number of individuals in v j in the corresponding state • mixing patterns among compartments: state transitions between compartments might depend on the states of individuals within the subpopulations associated with those compartments, as well as those who they come in contact with. for instance, the s j → e j transition rate might depend on i k for all the subpopulations who come in contact with individuals in v j . mobility and behavioral datasets are needed to model such interactions. such models are very useful at the early days of the outbreak, when the disease dynamics are driven to a large extent by mobility-these can be captured more easily within such models, and there is significant uncertainty in the disease model parameters. they can also model coarser interventions such as reduced mobility between spatial units and reduced mixing rates. however, these models become less useful to model the effect of detailed interventions (e.g., voluntary home isolation, school closures) on disease spread in and across communities. agent-based networked models (sometimes just called as agent-based models) extend metapopulation models further by explicitly capturing the interaction structure of the underlying populations. often such models are also resolved at the level of single individual entities (animals, humans, etc.). in this class of models, the epidemic dynamics can be modeled as a diffusion process on a specific undirected contact network g(v, e) on a population v-each edge e = (u, v) ∈ e implies that individuals (also referred to as nodes) u, v ∈ v come into contact main steps in setting up an agent-based model. while the specific steps depend on the disease, the population, and the type of question being studied, the general process involves the following steps: • construct a network representation g: the set v is the population in a region, and is available from different sources, such as census and landscan. however, the contact patterns are more difficult to model, as no real data are available on contacts between people at a large scale. instead, researchers have tried to model activities and mobility, from which contacts can be inferred, based on co-location. multiple approaches have been developed for this, including random mobility based on statistical models, and very detailed models based on activities in urban regions, which have been estimated through surveys, transportation data, and other sources, e.g., , , , , . • develop models of within-host disease progression: such models can be represented as finite state probabilistic timed transition models, which are designed in close coordination with biologists, epidemiologists, and parameterized using detailed incidence data (see for discussion and additional pointers). • develop high-performance computer (hpc) simulations to study epidemic dynamics in such models, e.g., , , , . typical public health analyses involve large experimental designs, and the models are stochastic; this necessitates the use of such hpc simulations on large computing clusters. • incorporate interventions and behavioral changes: interventions include closure of schools and workplaces , and vaccinations ; whereas, behavioral changes include individual level social distancing, changes in mobility, and use of protective measures. such a network model captures the interplay between the three components of computational epidemiology: (i) individual behaviors of agents, (ii) unstructured, heterogeneous multi-scale networks, and (iii) the dynamical processes on these networks. it is based on the hypothesis that a better understanding of the characteristics of the underlying network and individual behavioral adaptation can give better insights into contagion dynamics and response strategies. although computationally expensive and data intensive, network-based epidemiology alters the types of questions that can be posed, providing qualitatively different insights into disease dynamics and public health policies. it also allows policy makers to formulate and investigate potentially novel and contextspecific interventions. like projection approaches, models for epidemic forecasting can be broadly classified into two broad groups: (i) statistical and machine learning-based data-driven models, (ii) causal or mechanistic models-see , , , , , , and the references therein for the current state of the art in this rapidly evolving field. statistical methods employ statistical and time series-based methodologies to learn patterns in historical epidemic data and leverage those patterns for forecasting. of course, the simplest yet useful class is called method of analogs. one simply compares the current epidemic with one of the earlier outbreaks and then uses the best match to forecast the current epidemic. popular statistical methods for forecasting influenzalike illnesses (that includes covid- ) include, e.g., generalized linear models (glm), autoregressive integrated moving average (arima), and generalized autoregressive moving average (garma) , , . statistical methods are fast, but they crucially depend on the availability of training data. furthermore, since they are purely data driven, they do not capture the underlying causal mechanisms. as a result, epidemic dynamics affected by behavioral adaptations are usually hard to capture. artificial neural networks (ann) have gained increased prominence in epidemic forecasting due to their self-learning ability without prior knowledge (see , , and the references therein). such models have used a wide variety of data as surrogates for producing forecasts. this includes: (i) social media data, (ii) weather data, (iii) incidence curves and (iv) demographic data. causal models can be used for epidemic forecasting in a natural manner , , , , , . these models calibrate the internal model parameters using the disease incidence data seen until a given day and then execute the model forward in time to produce the future time series. compartmental as well as agentbased models can be used to produce such forecasts. the choice of the models depends on the specific question at hand and the computational and data resource constraints. one of the key ideas in forecasting is to develop ensemble models-models that combine forecasts from multiple models , , , . the idea which originated in the domain of weather forecasting has found methodological advances in the machine learning literature. ensemble models typically show better performance than the individual models. modeling group (uk model) background. the modeling group led by neil ferguson was to our knowledge the first model to study the impact of covid- across two large countries: us and uk, see . the basic model was first developed in -it was used to inform policy pertaining to h n pandemic and was one of the three models used to inform the federal pandemic influenza plan and led to the now well-accepted targeted layered containment (tlc) strategy. it was adapted to covid- as discussed below. the model was widely discussed and covered in the scientific as well as popular press . we will refer to this as the ic model. model structure. the basic model structure consists of developing a set of households based on census information for a given country. the structure of the model is largely borrowed from their earlier work, see , . landscan data were used to spatially distribute the population. individual members of the household interact with other members of the household. the data to produce these households are obtained using census information for these countries. census data are used to assign age and household sizes. details on the resolution of census data and the dates were not clear. schools, workplaces and random meeting points are then added. the school data for us were obtained from the national centre of educational statistics, while for uk schools were assigned randomly based on population density. data on average class sizes and staff-student ratios were used to generate a synthetic population of schools distributed proportional to local population density. data on the distribution of workplace size were used to generate workplaces with commuting distance data used to locate workplaces appropriately across the population. individuals are assigned to each of these locations at the start of the simulation. the gravity-style kernel is used to decide how far a person can go in terms of attending work, school or community interaction place. the number of contacts between individuals at school, work and community meeting points are calibrated to produce a given attack rate. each individual has an associated disease transmission model. the disease transmission model parameters are based on the data collected when the pandemic was evolving in wuhan; see page of . finally, the model also has rich set of interventions. these include: (i) case isolation, (ii) voluntary home quarantine, (iii) social distancing of those over years, (iv) social distancing of the entire population, (v) closure of schools and universities; see page . the code was recently released and is being analyzed. this is important as the interpretation of these interventions can have substantial impact on the outcome. model predictions. the imperial college (ic model) model was one of the first models to evaluate the covid- pandemic using detailed agentbased model. the predictions made by the model were quite dire. the results show that to be able to reduce r to close to or below, a combination of case isolation, social distancing of the entire population and either household quarantine or school and university closure is required. the model had tremendous impact-uk and us both decide to start considering complete lock downs-a policy that was practically impossible to even talk about earlier in the western world. the paper came out around the same time that wuhan epidemic was raging and the epidemic in italy had taken a turn for the worse. this made the model results even more critical. strengths and limitations. ic model was one of the first models by a reputed group to report the potential impact of covid- with and without interventions. the model was far more detailed than other models that were published until then. the authors also took great care parameterizing the model with the best disease transmission data that was available until then. the model also considered a very rich set of interventions and was one of the first to analyze pulsing intervention. on the flip side, the representation of the underlying social contact network was relatively simple. second, often the details of how interventions were represented were not clear. since the publication of their article, the modelers have made their code open and the research community has witnessed an intense debate on the pros and cons of various modeling assumptions and the resulting software system, see . we believe that despite certain valid criticisms, overall, the results represented a significant advance in terms of the when the results were put out and the level of details incorporated in the models. northeastern and uva models (us models) background. this approach is an alternative to detailed agent-based models, and has been used in modeling the spread of multiple diseases, including influenza , , ebola and zika . it has been adapted for studying the importation risk of covid- across the world . structured metapopulation models construct a simple abstraction of the mixing patterns in the population, in which the entire region under study is decomposed into fully connected geographical regions, representing subpopulations, which are connected through airline and commuter flow networks. thus, they lack the rich detail of agent-based models, but have fewer parameters, and are, therefore, easy to set up and scale to large regions. model structure. here, we summarize gleam (northeastern model) and patchsim (uva model). gleam uses two classes of datasets-population estimates and mobility. population data are used from the "gridded population of the world" , which gives an estimated population value at a × minutes of arc (referred to as a "cell") over the entire planet. two different kinds of mobility processes are considered-airline travel and commuter flow. the former captures long-distance travel; whereas, the latter captures localized mobility. airline data are obtained from the international air transport association (iata) , and the official airline guide (oag) . there are about airports world wide; these are aggregated at the level of urban regions served by multiple airport (e.g., as in london). a voronoi tessellation is constructed with the resulting airport locations as centers, and the population cells are assigned to these cells, with a mile cutoff from the center. the commuter flows connect cells at a much smaller spatial scale. we represent this mobility pattern as a directed graph on the cells, and refer to it as the mobility network. in the basic seir model, the subpopulation in each cell j is partitioned into compartments s j , e j , i j and r j , corresponding to the disease states. for each cell j, we define the force of infection j as the rate at which a susceptible individual in the subpopulation in cell j becomes infected-this is determined by the interactions the person has with infectious individuals in cell j or any cell j ′ connected in the mobility network. an individual in the susceptible compartment s j becomes infected with probability j t and enters the compartment e j , in a time interval t . from this compartment, the individual moves to the i j and then the r j compartments, with appropriate probabilities, corresponding to the disease model parameters. the patchsim model has a similar structure, except that it uses administrative boundaries (e.g., counties), instead of a voronoi tesselation, which are connected using a mobility network. the mobility network is derived by combining commuter and airline networks, to model time spent per day by individuals of region (patch) i in region (patch) j. since it explicitly captures the level of connectivity through a commuter-like mixing, it is capable of incorporating week-toweek and month-to-month variations in mobility and connectivity. in addition to its capability to run in deterministic or stochastic mode, the open source implementation allows fine-grained control of disease parameters across space and time. although the model has a more generic force of infection mode of operation (where patches can be more general than spatial regions), we will mainly summarize the results from the mobility model, which was used for covid- response. what did the models suggest? gleam model is being used in a number of covid- -related studies and analysis. in , the northeastern university team used the model to understand the spread of covid- within china and relative risk of importation of the disease internationally. their analysis suggested that the spread of covid- out of wuhan into other parts of mainland china was not contained well due to the delays induced by detection and official reporting. it is hard to interpret the results. the paper suggested that international importation could be contained substantially by strong travel ban. while it might have delayed the onset of cases, the subsequent spread across the world suggest that we were not able to arrest the spread effectively. the model is also used to provide weekly projections (see https ://covid .gleam proje ct.org/); this site does not appear to be maintained for the most current forecasts (likely because the team is participating in the cdc forecasting group). the patchsim model is being used to support federal agencies as well as the state of virginia. due to our past experience, we have refrained from providing longer term forecasts, instead of focusing on short-term projections. the model is used within a forecasting via projection selection approach, where a set of counterfactual scenarios are generated based on on-the-ground response efforts and surveillance data, and the best fits are selected based on historical performance. while allowing for future scenarios to be described, they also help to provide a reasonable narrative of past trajectories, and retrospective comparisons are used for metrics such as 'cases averted by doing x' . these projections are revised weekly based on stakeholder feedback and surveillance update. further discussion of how the model is used by the virginia department of health each week can be found at https ://www.vdh.virgi nia.gov/coron aviru s/covid - -data-insig hts/#model . strength and limitations. structured metapopulation models provide a good tradeoff between the realism/compute of detailed agentbased models and simplicity/speed of mass action compartmental models and need far fewer inputs for modeling, and scalability. this is especially true in the early days of the outbreak, when the disease dynamics are driven to a large extent by mobility, which can be captured more easily within such models, and there is significant uncertainty in the disease model parameters. however, once the outbreak has spread, it is harder to model detailed interventions (e.g., social distancing), which are much more localized. further, these are hard to model using a single parameter. both gleam and patchsim models also faced their share of challenges in projecting case counts due to rapidly evolving pandemic, inadequate testing, a lack of understanding of the number of asymptomatic cases and assessing the compliance levels of the population at large. researchers (swedish models) sweden was an outlier amongst countries in that it decided to implement public health interventions without a lockdown. schools and universities were not closed, and restaurants and bars remained open. swedish citizens implemented "work from home" policies where possible. moderate social distancing based on individual responsibility and without police enforcement was employed but emphasis was attempted to be placed on shielding the + age group. background. statistician tom britton developed a very simple model with a focus on predicting the number of infected over time in stockholm. model structure. britton used a very simple sir general epidemic model. it is used to make a coarse grain prediction of the behavior of the outbreak based on knowing the basic reproduction number r and the doubling time d in the initial phase of the epidemic. calibration to calendar time was done using the observed number of case fatalities, together with estimates of the time between infection to death, and the infection fatality risk. predictions were made assuming no change of behavior, as well as for the situation where preventive measures are put in place at one specific time-point. model predictions. one of the controversial predictions from this model was that the number of infections in the stockholm area would quickly rise towards attaining herd immunity within a short period. however, mass testing carried out in stockholm during june indicated a far smaller percentage of infections. strength and limitations. britton's model was intended as a quick and simple method to estimate and predict an on-going epidemic outbreak both with and without preventive measures put in place. it was intended as a complement to more realistic and detailed modeling. the estimation-prediction methodology is much simpler and straight-forward to implement for this simple model. it is more transparent to see how the few model assumptions affect the results, and it is easy to vary the few parameters to see their effect on predictions so that one could see which parameter uncertainties have biggest impact on predictions, and which parameter uncertainties are less influential. model background. the public health authority (fhm) of sweden produced a model to study the spread of covid- in four regions in sweden: dalarna, skåne, stockholm, and västra götaland. . model structure. it is a standard compartmentalized seir model and within each compartment, it is homogeneous; so, individuals are assumed to have the same characteristics and act in the same way. data used in the fitting of the model include point prevalences found by pcrtesting in stockholm at two different time points. model predictions. the model estimated the number of infected individuals at different time points and the date with the largest number of infectious individuals. it predicted that by july , . % ( . - . %) of the population in dalarna will have been infected, % ( . - . %) of the population in skåne will have been infected, % ( . - . %) of the population in stockholm will have been infected, and % ( . - . %) of the population in västra götaland will have been infected. it was hard to test these predictions because of the great uncertainty in immune response to sars-cov- -prevalence of antibodies was surprisingly low but recent studies show that mild cases never seem to develop antibodies against sars-cov- , but only t-cellmediated immunity . the model also investigated the effect of increased contacts during the summer that stabilizes in autumn. it found that if the contacts in stockholm and dalarna increase by less than % in comparison to the contact rate in the beginning of june, the second wave will not exceed the observed first wave. strength and limitations. the simplicity of the model is a strength in ease of calibration and understanding but it is also a major limitation in view of the well-known characteristics of covid- : since it is primarily transmitted through droplet infection, the social contact structure in the population is of primary importance for the dynamics of infection. the compartmental model used in this analysis does not account for variation in contacts, where few individuals may have many contacts, while the majority have fewer. the model is also not age stratified, but covid- strikingly affects different age groups differently; e.g., young people seem to get milder infections. in this model, each infected individual has the same infectivity and the same risk of becoming a reported case, regardless of age. different age groups normally have varied degrees of contacts and have changed their behavior differently during the covid- pandemic. this is not captured in the model. rocklöv developed a model to estimate the impact of covid- on the swedish population at the municipality level, considering demography and human mobility under various scenarios of mitigation and suppression. they attempted to estimate the time course of infections, health care needs, and the mortality in relation to the swedish icu capacity, as well as the costs of care, and compared alternative policies and counterfactual scenarios. model structure. used a seir compartmentalized model with age structured compartments ( - , - , +) susceptibles, infected, in-patient care, icu and recovered populations based on swedish population data at the municipal level. it also incorporated inter-municipality travel using a radiation model. parameters were calibrated based on a combination of values available from international literature and fitting to available outbreak data. the effect of a number of different intervention strategies was considered ranging from no intervention to modest social distancing and finally to imposed isolation of various groups. model predictions. the model predicted an estimated death toll of around , for the strategies based only on social distancing and between and for policies imposing stricter isolation. it predicted icu cases of up to , without much intervention and up to with modest social distancing, way above the available capacity of about icu beds. strength and limitations. the model showed a good fit against the reported covid- -related deaths in sweden up to th of april, , however, the predictions of the total deaths and icu demand turned out to be way off the mark. background. finally, , used an individualbased model parameterized on swedish demographics to assess the anticipated spread of covid- . model structure. employed the individual agent-based model based on work by ferguson et al. . individuals are randomly assigned an age based on swedish demographic data and they are also assigned a household. household size is normally distributed around the average household size in sweden in , . people per household. households were placed on a lattice using high-resolution population data from landscan and census dara from the statstics sweden and each household is additionally allocated to a city based on the closest city center by distance and to a county based on city designation. each individual is placed in a school or workplace at a rate similar to the current participation in sweden. transmission between individuals occurs through contact at each individual's workplace or school, within their household, and in their communities. infectiousness is, thus, a property dependent on contacts from household members, school/workplace members and community members with a probability based on household distances. transmissibility was calibrated against data for the period march- april to reproduce either the doubling time reported using pan-european data or the growth in reported swedish deaths for that period. various types of interventions were studied including the policy implemented in sweden by the public health authorities as well as more aggressive interventions approaching full lockdown. model predictions. their prediction was that "under conservative epidemiological parameter estimates, the current swedish public-health strategy will result in a peak intensive-care load in may that exceeds pre-pandemic capacity by over -fold, with a median mortality of , ( % ci , to , )". strength and limitations. this model was based on adapting the well-known imperial model discussed in sect. to sweden and considered a wide range of intervention strategies. unfortunately the predictions of the model were woefully off the mark on both counts: the deaths by june are under and at the peak the icu infrastructure had at least % unutilized capacity. forecasting is of particular interest to policy makers as they attempt to provide actual counts. since the surveillance systems have relatively stabilized in recent weeks, the development of forecasting models has gained traction and several models are available in the literature. in the us, the centers for disease control and prevention (cdc) has provided a platform for modelers to share their forecasts which are analyzed and combined in a suitable manner to produce ensemble multi-week forecasts for cumulative/incident deaths, hospitalizations and more recently cases at the national, state, and county level. probabilistic forecasts are provided by teams as of july , (there were models as of june , ) and the cdc with the help of has developed uniform ensemble model for multi-step forecasts . model it has been observed previously for other infectious diseases that an ensemble of forecasts from multiple models perform better than any individual contributing model . in the context of covid- case count modeling and forecasting, a multitude of models have been developed based on different assumptions that capture specific aspects of the disease dynamics (reproduction number evolution, contact network construction, etc.). the models employed in the cdc forecast hub can be broadly classified into three categories, data-driven, hybrid models, and mechanistic models with some of the models being open source. data-driven models. they do not model the disease dynamics but attempt to find patterns in the available data and combine them appropriately to make short-term forecasts. in such data-driven models, it is hard to incorporate interventions directly; hence, the machine is presented with a variety of exogenous data sources such as mobility data, hospital records, etc. with the hope that its effects are captured implicitly. early iterations of institute of health metrics and evaluation (ihme) model for death forecasting at state level employed a statistical model that fits a time-varying gaussian error function to the cumulative death counts and is parameterized to control for maximum death rate, maximum death rate epoch, and growth parameter (with many parameters learnt using data from outbreak in china). the ihme models are undergoing revisions (moving towards the hybrid models) and updated implementable versions are available at . the university of texas at austin covid- modeling consortium model uses a very similar statistical model as but employs real-time mobility data as additional predictors and also differ in the fitting process. the carnegie mellon delphi group employs the well known auto-regressive (ar) model that employs lagged version of the case counts and deaths as predictors and determines a sparse set that best describes the observations from it by using j. indian inst. sci. | vol xxx:x | xxx-xxx | journal.iisc.ernet.in lasso regression . is a deep learning model which has been developed along the lines of and attempts to learn the dependence between death rate and other available syndromic, demographic, mobility and clinical data. hybrid models. these methods typically employ statistical techniques to model disease parameters which are then used in epidemiological models to forecast cases. most statistical models , are evolving to become hybrid models. a model that gained significant interest is the youyang gu (yyg) model and uses a machine learning layer over an seir model to learn the set of parameters (mortality rate, initial r , postlockdown r) specific to a region that best fits the region's observed data. the authors (yyg) share the optimal parameters, the seir model and the evaluation scripts with general public for experimentation . los alamos national lab (lanl) model uses a statistical model to determine how the number of covid- infections changes over time. the second process maps the number of infections to the reported data. the number of deaths is a fraction of the number of new cases obtained and is computed using the observed mortality data. mechanistic models. gleam and jhu models are county-level stochastic seir model dynamics. the jhu model incorporates the effectiveness of state-wide intervention policies on social distancing through the r parameter. more recently, model outputs from uva's patchsim model were included as part of a multi-model ensemble (including autoregressive and lstm components) to forecast weekly confirmed cases. types we end the discussion of the models above by qualitatively comparing model types. as discussed in the preliminaries, at one end of the spectrum are models that are largely data driven: these models range from simple statistical models (various forms of regression models) to the more complicated deep learning models. the difference in such model lies in the amount of training data needed, the computational resources needed and how complicated the mathematical function one is trying to fit to the observed data. these models are strictly data driven and, hence, unable to capture the constant behavioral adaptation at an individual and collective level. on the other end of the spectrum seir, meta-population and agent-based network models are based on the underlying procedural representation of the dynamics-in theory, they are able to represent behavioral adaptation endogenously. but both class of models face immense challenges due to the availability of data as discussed below. ( ) agent-based and seir models were used in all the three countries in the early part of the outbreak and continue to be used for counter-factual analysis. the primary reason is the lack of surveillance and disease specific data and hence, purely data-driven models were not easy to use. seir models lacked heterogeneity but were simple to program and analyze. agent-based models were more computationally intensive, required a fair bit of data to instantiate the model but captured the heterogeneity of the underlying countries. by now it has become clear that use of such models for long term forecasting is challenging and likely to lead to mis-leading results. the fundamental reason is adaptive human behavior and lack of data about it. ( ) forecasting, on the other hand, has seen use of data-driven methods as well as causal methods. short-term forecasts have been generally reasonable. given the intense interest in the pandemic, a lot of data are also becoming available for researchers to use. this helps in validating some of the models further. even so, realtime data on behavioral adaptation and compliance remain very hard to get and is one of the central modeling challenges. were some of the models wrong? in a recent opinion piece, professor vikram patel of the harvard school of public health makes a stinging criticism of modeling: crowning these scientific disciplines is the field of modeling, for it was its estimates of mountains of dead bodies which fuelled the panic and led to the unprecedented restrictions on public life around the world. none of these early models, however, explicitly acknowledged the huge assumptions that were made, a similar article in ny times recounted the mistakes in covid- response in europe ; also see . our point of view. it is indeed important to ensure that assumptions underlying mathematical models be made transparent and explicit. but we respectfully disagree with professor patel's statement: most of the good models tried to be very explicit about their assumptions. the mountains of deaths that are being referred to are explicitly calculated when no interventions are put in place and are often used as a worst case scenario. now, one might argue that the authors be explicit and state that this worst case scenario will never occur in practice. forecasting dynamics in social systems is inherently challenging: individual behavior, predictions and epidemic dynamics co-evolve; this coevolution immediately implies that a dire prediction can lead to extreme change in individual and collective behavior leading to reduction in the incidence numbers. would one say forecasts were wrong in such a case or they were influential in ensuring the worst case never happens? none of this implies that one should not explicitly state the assumption underlying their model. of course our experience is that policy makers, news reporters and common public are looking exactly for such a forecastwe have been constantly asked "when will peak occur" or "how many people are likely to die". a few possible ways to overcome this tension between the unsatiable appetite for forecasts and the inherent challenges that lie in doing this accurately, include: • we believe that, in general, it might not be prudent to provide long term forecasts for such systems. • state the assumptions underlying the models as clearly as possible. modelers need to be much more disciplined about this. they also need to ensure that the models are transparent and can be reviewed broadly (and expeditiously). • accept that the forecasts are provisional and that they will be revised as new data comes in, society adapts, the virus adapts and we understand the biological impact of the pandemic. • improve surveillance systems that would produce data that the models can use more effectively. even with data, it is very hard to estimate the prevalence of covid- in society. communicating scientific findings and risks is an important topical area in this context, see , , , . use of models for evidence-based policy making. in a new book, , radical uncertainty, economists john kay and mervyn king (formerly governor of the bank of england) urge caution when using complex models. they argue that models should be valued for the insights they provide but not relied upon to provide accurate forecasts. the so-called "evidence-based policy" comes in for criticism where it relies on models but also supplies a false sense of certainty where none exists, or seeks out the evidence that is desired ex ante-or "cover"--to justify a policy decision. "evidence-based policy has become policy-based evidence". our point of view. the authors make a good point here. but again, everyone, from public to citizens and reporters clamor for a forecast. we argue that this can be addressed in two ways:(i) viewing the problem from the lens of control theory so that we forecast only to control the deviation from the path we want to follow and (ii) not insisting on exact numbers but general trends. as kay and king opine, the value of models, especially in the face of radical uncertainty, is more in exploring alternative scenarios resulting from different policies: a model is useful only if the person using it understands that it does not represent the "the world as it really is" but is a tool for exploring ways in which a decision might or might not go wrong. in his new book the rules of contagion, adam kucharski draws on lessons from the past. in and , during the zika outbreak, researchers planned large-scale clinical studies and vaccine trials. but these were discontinued as soon as the infection ebbed. this is a common frustration in outbreak research; by the time, the infections end, fundamental questions about the contagion can remain unanswered. that is why building long-term research capacity is essential. our point of view. the author makes an important point. we hope that today, after witnessing the devastating impacts of the pandemic on the economy and society, the correct lessons will be learnt: sustained investments need to be made in the field to be ready for the impact of the next pandemic. the paper discusses a few important computational models developed by researchers in the us, uk and sweden for covid- pandemic planning and response. the models have been used by policy makers and public health officials in their respective countries to assess the evolution of the pandemic, design and analyze control measures and study various what-if scenarios. as noted, all models faced challenges due to availability of data, rapidly evolving pandemic and unprecedented control measures put in place. despite these challenges, we believe that mathematical models can provide useful and timely information to the policy makers. on on hand the modelers need to be transparent in the description of their models, clearly state the limitations and carry out detailed sensitivity and uncertainty quantification. having these models reviewed independently is certainly very helpful. on the other hand, policy makers should be aware of the fact that using mathematical models for pandemic planning, forecast response rely on a number of assumptions and lack data to over these assumptions. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. epideep: exploiting embeddings for epidemic forecasting real-time epidemic forecasting: challenges and opportunities real-time forecasting of infectious disease dynamics with a stochastic semi-mechanistic model forecasting the impact of the first wave of the covid- pandemic on hospital demand and deaths for the usa and european economic area countries an arima model to forecast the spread and the final size of covid- epidemic in italy (first version on ssrn march) accuracy of realtime multi-model ensemble forecasts for seasonal influenza in the us structure of social contact networks and their impact on epidemics computational epidemiology the structure and function of complex networks deep learning based epidemic forecasting with synthetic information | vol xxx:x | xxx-xxx | journal.iisc.ernet.in control of severe acute respiratory syndrome social contacts and mixing patterns relevant to the spread of infectious diseases optimizing influenza vaccine distribution multiscale mobility networks and the spatial spreading of infectious diseases the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak assessing the international spreading risk associated with the west african ebola outbreak optimizing spatial allocation of seasonal influenza vaccine under temporal constraints spread of zika virus in the americas generation and analysis of large synthetic social contact networks modelling disease outbreaks in realistic urban social networks containing pandemic influenza at the source episimdemics: an efficient algorithm for simulating the spread of infectious disease over large realistic social networks enhancing user-productivity and capability through integration of distinct software in epidemiological systems fred (a framework for reconstructing epidemic dynamics): an open-source software system for modeling infectious diseases and control strategies using census-based populations modeling targeted layered containment of an influenza pandemic in the united states pancasting: forecasting epidemics from provisional data influenza forecasting in human populations: a scoping review near-term forecasts of influenza-like illness: an evaluation of autoregressive time series approaches a systematic review of studies on forecasting the dynamics of influenza outbreaks a framework for evaluating epidemic forecasts forecasting covid- impact on hospital bed-days, icudays, ventilator-days and deaths by us state in the next months covid- cases and deaths forecasts tdefsi: theoryguided deep learning-based epidemic forecasting with synthetic information calibrating a stochastic, agent-based model using quantile-based emulation epidemic forecasting framework combining agent-based models and smart beam particle filtering individual versus superensemble forecasts of seasonal influenza outbreaks in the united states forecasting a moving target: ensemble models for ili case count predictions modelling the pandemic the simulations driving the world's response to covid- strategies for mitigating an influenza pandemic critiqued coronavirus simulation gets thumbs up from code-checking efforts high resolution global gridded data for use in population studies oag official airline guide code for simulating the metapopulation seir model the effect of human mobility and control measures on the covid- epidemic in china basic prediction methodology for covid- : estimation and sensitivity considerations estimates of the number of infected individuals during the covid- outbreak in the dalarna region, skåne region, stockholm region, and västra götaland region robust t cell immunity in convalescent individuals with asymptomatic or mild covid- covid- healthcare demand and mortality in sweden in response to non-pharmaceutical (npis) mitigation and suppression scenarios arbo-prevent: climate change, human mobility and emerging arboviral outbreaks: new models for risk characterization, resilience and prevention intervention strategies against covid- and their estimated impact on swedish healthcare capacity managing covid- spread with voluntary public-health measures: sweden as a case study for pandemic control cdc. covid- forecasthub projections for firstwave covid- deaths across the us using social-distancing measures derived from mobile phones policy implications of models of the spread of coronavirus: perspectives and opportunities for economists evaluating science communication mathematical models to guide pandemic response infodemic and risk communication in the era of cov- radical uncertainty: decision-making beyond the numbers the rules of contagion: why things spread-and why they stop. basic books the authors would like to thank members of the biocomplexity covid- response team and network systems science and advanced computing (nssac) division for their thoughtful comments and suggestions related to epidemic modeling and response support. we thank members of the biocomplexity institute and initiative, university of virginia for useful discussion and suggestions. this work was partially sup- research associate at the nssac division of the biocomplexity institute and initiative. he completed his phd from the department of electrical engineering, indian institute of science (iisc), bangalore, india and has held the position of postdoctoral fellow at iisc and north carolina state university, raleigh, usa. his research areas include signal processing, machine learning, data mining, forecasting, big data analysis etc. at nssac, his primary focus has been the analysis and development of forecasting systems for epidemiological signals such as influenza-like illness and covid- using auxiliary data sources. bryan lewis is a research associate professor in the network systems science and advanced computing division. his research has focused on understanding the transmission dynamics of infectious diseases within specific populations through both analysis and simulation. lewis is a computational epidemiologist with more than years of experience in crafting, analyzing, and interpreting the results of models in the context of real public health problems. as a computational epidemiologist, for more than a decade, lewis has been heavily involved in a series of projects forecasting the spread of infectious disease as well as evaluating the response to them in support of the federal government. these projects have tackled diseases from ebola to pandemic influenza and melioidosis to cholera. professor in biocomplexity, the division director of the networks, simulation science and advanced computing (nssac) division at the biocomplexity institute and initiative, and a professor in the department of computer science at the university of virginia (uva). his research interests are in network science, computational epidemiology, ai, foundations of computing, socially coupled system science and high-performance computing. before joining uva, he held positions at virginia tech and the los alamos national laboratory. he is a fellow of the ieee, acm, siam and aaas. scientist at the biocomplexity institute & initiative, university of virginia and his research focuses on developing, analyzing and optimizing computational models in the field of network epidemiology. he received his phd from the department of electrical and communication engineering, indian institute of science (iisc), and did his postdoctoral research at virginia tech. his areas of interest include network science, stochastic modeling and big data analytics. he has used in-silico models of society to study the spread of infectious diseases and invasive species. recent research includes modeling and forecasting emerging infectious disease outbreaks (e.g., ebola, covid- ), impact of human mobility on disease spread and resource allocation problems in the context of key: cord- -ccb coz authors: subiakto, yuli title: aviation medicine capacity on facing biological threat in indonesia airports date: - - journal: infect dis rep doi: . /idr. . sha: doc_id: cord_uid: ccb coz airports need high security procedures, especially for preventing outbreaks of infectious diseases spread by passenger and carried goods. outbreaks of disease form real threat to national defense that can endanger national sovereignty, territorial integrity and national security. biological agents that are dangerous sources of outbreaks infectious diseases can be spread by criminal and terrorists for biological warfare. based on data, the spread of diseases in indonesia came from abroad, such as sars from china, mers-cov from the middle east, avian influenza from china, hiv from africa etc. indonesia has a population of more than million peoples, , islands, and climate conditions that allow microorganisms to grow well. in domestic flights transported , , persons and international flights , , persons, we need to prevent the spread of diseases in indonesia entering through the airports. efforts to prevent the entry of dangerous biological agents in indonesia were carried out by quarantine officers and port health officers. the development of threat outbreak disease in air transportation is real in the future, so all indonesia airports must have action plans to prevent the spread of infectious diseases. the air force must act as guardian of sovereignty by having medical personnel on the spot for role interoperability with the personnel port health office for prevent the entry of dangerous biological agents. capacity building need for be enhanced for prevention, detection, identification and response through a training of the personnel, procurement facilities for readiness prevent, detect and respond when facing biological threat. the threat of an infectious disease outbreak is real threat to indonesia's national defense, because this is considered to endanger national sovereignty, territorial integrity and national security. the inci-dence of diseases outbreaks will increase with conditions of malnutrition, poor sanitation, poor health services, especially during natural and non-natural disasters. conditions that support disease outbreaks make indonesia vulnerable to outbreaks in certain areas, because there are still groups of people who are experiencing malnutrition, low sanitation behavior, and health services that have not been fully covered. in this regard, a comprehensive step is needed to prevent the entry and spread of potential biological agents, and need for steps to prevent, detect and identify, respond, handle the victims and overcome the infectious diseases among humans. as is known, indonesia is hit by all kind of disasters starting from earthquakes, tsunamis, floods, landslides, eruptions, technology failure etc. where such disasters often cause public health problems in form of infectious diseases, especially if the victim does not get good health services, sanitation is not repaired, or environmental damage is not immediately repaired. spread of infectious disease is also helped by indonesia's climate conditions that allow microbes grow and develop easily, so that infectious diseases can spread quickly, with impacts ranging from mild, moderate and severe. in the current global era, air transportation is choice for people for moving fast or transport goods from one place to another quickly, and between regions and countries there are no limits. in addition to providing benefits community, these conditions also have an impact on spread of infectious diseases with both passenger and goods. airports, as a place where people gather, can act as center of spread of disease and can be used as a target of terrorism by using biological agents. so that if not comprehensively anticipated it will pose a threat that if not handled well will lead to consequences that can be detrimental in terms of economy, politics and defense and state security. deliberate and accidental biological threat is using of biological weapons presents a significant challenge to our national security, our population, our agriculture, and the environment. airport is strategic place and potential target for biological threat, because is place where population will meet and travel. that are areas on land and/or flying with certain limits that are used as a place for aircraft landing and taking off, boarding passengers, loading and unloading goods, and intra-and inter-mode transportation, which are equipped with flight safety, security facilities, basic facilities and other supporting facilities. in this regard, it can be stated that airports are a gathering point for people and goods that are vulnerable to spread of diseases which can reduce public health risks. indonesia has a population of around , , persons, it spreads over , islands, and has climatic conditions that make microorganisms grow well. , seeing these conditions in context of moving people and goods more quickly make aircraft needed from city to another city, besides as a country of muslims, umrah and pilgrimage activities continue. airport is strategic installation, where both domestic and international passengers gather, goods, aircraft, ground crew, air crew and other flight equipment that require high security both physical and non-physical. the aviation industry was among the first major industries to define organizational safety, and to embrace risk management systems as an essential component to reduce the frequency and impact of aviation accidents. as showed in table , movement of people through airports domestic flight departure in indonesia in last five years ( - ) increased by . % per year, loading cargo grew by . % on average per year, and loading baggage grew . %. as showed in table , passenger from international flight on average had grown by . % per year, followed by growth of unloading cargo and baggage of . % and . %. in indonesia established a port health office that had inspected . . passenger aircraft, . . persons, of which . pilgrims, and umrah persons per day. the increase in the number of flights carrying humans and goods both domestically and internationally, in addition to providing benefits to the indonesian economy also has the potential for disease transfer, whether carried by passengers or with goods, through cargo or baggage. the number of infection transmissions that occurred across the entire aircraft cabin was strikingly large. infectious diseases and pandemics are primarily spread through aviation as a mode of travel. so we need to take action before creating impacts that can threaten human health in indonesia as a result of the spread of diseases that are not controlled: it is necessary to prevent the spread of diseases through air transportation. base on decree mohroi no. /menkes/per/iv/ , port health office in airport has the task of carrying out prevention of entry and exit of diseases, potential disease outbreaks, epidemiological surveillance, quarantine, control of environmental health impacts, health services, supervision and security of new diseases and re-emerging diseases, bioterrorism, biological elements, chemicals and radiation security in airport areas. port health office was implemented by the international health regulation (ihr- ) for prevention occurrence public health of international concern (pheic) in point of entry. port health office has capacity to reduce public health impacts, especially the spread of infectious diseases. it has carried out vector control guidance, supervised sanitation of transport equipment, improved clean and healthy living behavior and general health and improved occupational safety and health. many biological agents that cause potential outbreaks come from outside of indonesia with newly emerging, reemerging/resurging and delicately emerging species, so that to reach indonesia they require a series of trips that are quite far like sars from china, mers cov, from the middle east, avian influenza from china, hiv from africa etc. based on these incidents, it can be said that airports act as one of the entry points for spread of diseases in indonesia that are spread by passengers and goods contaminated with dangerous biological materials, or deliberately distributed by certain groups to cause fear or threaten state security. the spread of diseases in a country can occur not only from its own agents but also there are several factors that influence it, namely bad sanitation, bad nutrition, presence of diseases, irrational use antibiotic, antibotic uncontrolled distribution, uncovered vaccination and rejected vaccination, bad environmental situations, dis-eases coming from wildlife, terrorism, fast moving personnel from the area to another area, lack of biosafety in livestock. main important properties for biological agent intentional deliberate are route of entry, environmental stability, disease severity, stability against disinfection, ease of large scale production and infectivity. the spread of biological agents in airports that occurs deliberately for the purpose of criminal acts by using a biological agent known as biocrime. biocrimes is activating by using biological agents, for murder, for profit, distribution of fake vaccines or antibiotics. some biocrime activities are attacks on specific ethnic population groups within a country, who are perceived to be opposition to terrorist goals; sabotage of specific food groups, such as contamination of imported food products with toxins, pathogenic bacteria, or poison and attacks directed at one of the country's institutions, agencies, or military departments, a stock market, or a major communications center. biocrime actions carried out through airports that have occurred have been delivery of antibiotics or substandard vaccines for advantages. bioterrorism is intentional use of microorganism or toxin derived from living organism to cause death or disease in humans or the animal and plans. , , , bioterorrism might include such deliberate acts as salad contamination with salmonella, injection of hiv virus, anthrax spread by mail, spreading a virulent disease among animal production facilities, poisoning water, food and blood supplies. use biological agents for threats is becoming increasingly possible for terrorist groups. developing microorganism with increasing lethality for terrorist attacks and the rise of religious terrorism call for political, theological, tactical and strategic disincentive to using as weapons and perpetrating acts of mass destruction. beside that perception use of weapons undermine their political support; motivation religious terrorist for increase threat of mass destruction. , [ ] [ ] [ ] until now, acts of terrorism through airports in indonesia have never happened, but in the future airport can be target of acts of terrorism cause airport is place, when many people from domestic or international gather together for travel. if terrorism attacks happen it will impact reducing international credibility and influence economy, politics, and security conditions. during war use of biological agents is known as biowarfare, which is an action using microorganisms (bacteria, viruses) or toxins found in weapons for war, with the intention of reducing abilities, causing illness, and killing. [ ] [ ] [ ] [ ] [ ] open biological warfare until now has never happened, it is estimated that air transportation is the most effective means for biological warfare. using biological agent intentionally as weapons has advantages: they often are low cost of producing many biological agent, small quantities may have dramatic effects, deadly or incapacitating effect on a susceptible, they are easily disseminated, difficulty in diagnosing index cases, can result in fear, panic and social disruption and symptoms can mimic endemic, naturally occurring disease. [ ] [ ] [ ] [ ] [ ] [ ] some of key factors for pathogenic biological agents being deliberately distributed rise from several considerations: availability of production in sufficient quantity, ability to cause either lethal or incapacitating effects are achievable and deliverable, appropriate particle size in aerosol, ease of dissemination, stability (while maintaining virulence) after production in storage, weapons and environment, victims of nonconsensual performance of friendly forces. countermeasures of spread biological agent are based on characteristic of pathogenicity of the organism, mode of transmission and host range, availability of effective preventive measures and availability of effective treatment. according on global health security countermeasure bio-logical agent spread are prevent, detect, respond and ihr related hazards and point of entry. where prevent and reducing the likelihood of outbreak and other public health hazards and event defined by ihr is essential, detecting the threat early can save lives, while rapid and effective response requires multisectoral, national and international coordination and communication, ihr capacities are required at points of entry and during chemical events and radiation emergencies. indonesia has law to regulate the impact of advances in transportation technology and the era of free trade that could risk causing health problems and new emerging diseases or re-emerging diseases that reappear with faster spread and the potential to cause public health emergency. at present momenti, prevention of infectious diseases in the airport is carried out by the ministry of agriculture or by the quarantine service, passengers who experience illness suspected of contracting an infectious disease are carried out by the port health office, but not all airports have quarantine officers and port health offices. at present based on data from the ministry of health, the international port health office amounts to international airports, so still many airport aren't have port health office. the current condition between health airport and aviation medicine at the tni base is not well established, so that if there is a threat of disease outbreaks coming through the airport and at the airport there is no port health office, there will be a delay in the initial handling of victims. in this regard, role of aviation health, both doctors qualified as flight surgeons and flight nurses in air force bases need to be empowered, so that there is no spread of infectious diseases in the environment. aviation medicine have authority to examine, determine diagnoses, and evaluate the results of pilots' health checks, where the legal product is in the form of aviation health certificate. part of the duties and functions of aviation medicine in air force is providing health support and health services in flight. flights surgeons at air bases have role of carrying out preventive, curative activities on flight crews the role of aviation health in airports in an effort to prevent spread of infectious diseases originating from passengers and goods is to take preventive actions by conducting detection and identification, handling victims and carrying out referral actions. besides that, it cooperates with the occupational health and safety department and conducts checks and prevention. the collective effort is aimed at improving defenses against biological attacks. within these efforts are programs and agencies working towards increasing data collection, analysis, and intelligence gathering. the intelligence is applied to mitigating the effects of bioweapons by developing vaccines, therapeutics, and detection methods to increase the defensive posture, ultimately, biodefense initiatives protect the military forces and citizens from the effects of biological attacks. some steps that must be considered in increasing capacity of aviation medicine in air force, such as institutionalize & build regional and sub-regional mechanisms. the ramp up implementation of international frameworks make timely and relevant policies shifts to the address of disease risk drivers, regular capacity building initiatives with focus on preventing, strengthen surveillance and response, laboratory strengthening and networking, strength risk communication, invest in public health research, and map success stories from the region. aviation medicine in air force is expected to have ability in preventive activities, detection and identification and handling of victims of infectious diseases in the context of overcoming the spread of infectious diseases at airports. preventive epidemics include natural outbreaks and intentional outbrakes or leakage accidents (releases). types of preventive measures include ( ) preventing emergence and spread of antimicrobial drug resistant and emerging zoonotic diseases and governing international regulatory frameworks for food safety. ( ) promoting national biosafety and biosecurity systems and ( ) reducing number and magnitude of infectious disease outbreaks. the strategy for prevention of biological incidents includes strength of biological and toxin weapons convention, control of dual use equipment, improve control of pathogens and genetic material, improve international cooperation, improve scientific research and improve backups, check experts working in class ¾ labs. detection and identification activities carried out by laboratory, military institutions, moh by implementing detect threat early. conduct early detection of threats which include: ( ) launching, strengthening and linking global networks for realtime biosurveillance ( ) emergencies of international concern ( ) developing and deploying novel diagnostics and strengthening laboratory systems ( ) training and deploying workforce effectively for biosurveillance. , response activities were carried out by military hospitals, public hospitals, hospital moh which included decontamination of personnel, equipment (thermos scanner), facilities and infrastructure, health care for victims, responding rapidly and effectively in dealing with biological threats as international attention. ( ) developing an interconnected global network of emergency operations centers and multisectoral responses to biological incidents. encourage the strengthening of emergency operations centers: multisector emergency response teams that are trained, know how to function, have access to real time information systems and capacity to strengthen information on occurrence of outbreaks. ( ) improving global access to medical and non-medical countermeasures during health emergencies. enhancing capacity of aviation medicine in the face of the threat of the spread of dangerous biological agents is by measures on prevention, detection, and response can be done by increasing capacity of personnel and equipment. enhance capacity building ( ) personnel by carrying out procurement, force structure and unit design, ( ) equipment by making systems and equipment (thermos scanner, decontamination), facilities. ( ) readiness by implementing training education, training table top exercise (ttx), field top exercise (ftx) simulation, socialization of code of conduct and operational, equipment maintenance, supply stocks. interoperability beside stage holder national, regional and international to detect and respond biological threat on laboratory collaborating center, quarantine and observation personnel, treatment of the victim. some capacity-building activities that must be increased in stages and are sustainably are training to improve military medicine capacity in dealing with infectious diseases carried out by the ministry of defense and the tni, ministry of health, and ministry of agriculture, ministry of transportation, ministry of law and human right in form exercise are ttx and ftx activities nationally. training in order to increase the ability to overcome infectious disease outbreaks at the asean level and international. the prevailing who recommendations for the contact tracing of air travelers should be modified to include all possible sources of infection. the who should intensify its existing successful collaboration with organizations such as icao, iata, and aci in order to promote procedures for the effective management of highly contagious and life-threatening diseases. airports need high security procedures, especially preventing outbreaks of infectious diseases spread by passenger and carried goods. outbreaks of diseases form real threat to national defense that can endanger national sovereignty, territorial integrity and national security. biological agents are dangerous sources of outbreaks infectious diseases that can be spread by criminal and terrorists with biological warfare intentions. aviation medicine has a strategic role in preventing, mitigating and rehabilitating the threat of dangerous biological agents. increasing role of field health under the air force medicine has an important role in overcoming the threat of hazardous biological agents, role of the hospital as a support to victims in the field, and the role of the air force pharmaceutical institutions in the identification of biological agents. interoperability is needed between air force medicine, port health office, quarantine office, immigration, authority airport to prevent, detect and respond outbreak of disease in airport. increased flight health capacity in the face of threat of spread of biological agents by increasing the number of personnel who have the ability with multilevel and continuous training, fulfillment of facilities and infrastructure which include means of decontamination, prevention, detection and identification and handling of victims, training and operational, equipment maintenance, stock operating costs. peraturan menteri pertahanan ri nomor tahun tentang buku putih pertahanan indonesia undang-undang ri nomor tahun tentang penanggulangan bencana peraturan menteri kesehatan ri nomor: tahun tentang penyelenggaraan pelabuhan dan bandar udara sehat badan pusat statistik-statistic indonesia. statistical yearbook of indonesia air transportation statistics laporan kegiatan kantor kesehatan pelabuhan indonesia tahun pada pertemuan audiensi menteri kesehatan dengan kepala kkp seluruh indonesia screening for infectious disease at international airport : the frankfurt model peraturan menteri kesehatan nomor: /menkes/per/iv/ tentang organisasi dan tata keja kantor kesehatan pelabuhan responding to infectious disease outbreaks. nih cbrn protection: managing of threat of chemical, biological, radiological and nuclear weapons containing and preventing bilogical threats the changing face of terorrism: how real is the threat from biological, chemical and nuclear weapons biological weapons: recognizing, understanding and responding to the threat biological, chemical, and radiological terrorism, emergency preparedness and response for the primary care physician an overview on biological weapons and bioterorrism joint external evaluation tool: international health regulation undang undang ri nomor tahun tentang kekarantinaan kesehatan peran dokter penerbangan dalam pelaksanaan kewajiban pemeriksaan kesehatan bagi penerbang untuk keselamatan penerbangan, soepra biological incident operation: a guide for law enforcement laboratory biorisk management: biosafety and biosecurity key: cord- -j u ga authors: sapey, elizabeth; bafadhel, mona; bolton, charlotte emma; wilkinson, thomas; hurst, john r; quint, jennifer k title: building toolkits for copd exacerbations: lessons from the past and present date: - - journal: thorax doi: . /thoraxjnl- - sha: doc_id: cord_uid: j u ga in the nineteenth century, it was recognised that acute attacks of chronic bronchitis were harmful. years later, it is clearer than ever that exacerbations of chronic obstructive pulmonary disease (ecopd) are important events. they are associated with significant mortality, morbidity, a reduced quality of life and an increasing reliance on social care. ecopd are common and are increasing in prevalence. exacerbations beget exacerbations, with up to a quarter of in-patient episodes ending with readmission to hospital within days. the healthcare costs are immense. yet despite this, the tools available to diagnose and treat ecopd are essentially unchanged, with the last new intervention (non-invasive ventilation) introduced over years ago. an ecopd is ‘an acute worsening of respiratory symptoms that results in additional therapy’. this symptom and healthcare utility-based definition does not describe pathology and is unable to differentiate from other causes of an acute deterioration in breathlessness with or without a cough and sputum. there is limited understanding of the host immune response during an acute event and no reliable and readily available means to identify aetiology or direct treatment at the point of care (poc). corticosteroids, short acting bronchodilators with or without antibiotics have been the mainstay of treatment for over years. this is in stark contrast to many other acute presentations of chronic illness, where specific biomarkers and mechanistic understanding has revolutionised care pathways. so why has progress been so slow in ecopd? this review examines the history of diagnosing and treating ecopd. it suggests that to move forward, there needs to be an acceptance that not all exacerbations are alike (just as not all copd is alike) and that clinical presentation alone cannot identify aetiology or stratify treatment. in the nineteenth century, it was recognised that acute attacks of chronic bronchitis were harmful. years later, it is clearer than ever that exacerbations of chronic obstructive pulmonary disease (ecopd) are important events. they are associated with significant mortality, morbidity, a reduced quality of life and an increasing reliance on social care. ecopd are common and are increasing in prevalence. exacerbations beget exacerbations, with up to a quarter of in-patient episodes ending with readmission to hospital within days. the healthcare costs are immense. yet despite this, the tools available to diagnose and treat ecopd are essentially unchanged, with the last new intervention (non-invasive ventilation) introduced over years ago. an ecopd is 'an acute worsening of respiratory symptoms that results in additional therapy'. this symptom and healthcare utility-based definition does not describe pathology and is unable to differentiate from other causes of an acute deterioration in breathlessness with or without a cough and sputum. there is limited understanding of the host immune response during an acute event and no reliable and readily available means to identify aetiology or direct treatment at the point of care (poc). corticosteroids, short acting bronchodilators with or without antibiotics have been the mainstay of treatment for over years. this is in stark contrast to many other acute presentations of chronic illness, where specific biomarkers and mechanistic understanding has revolutionised care pathways. so why has progress been so slow in ecopd? this review examines the history of diagnosing and treating ecopd. it suggests that to move forward, there needs to be an acceptance that not all exacerbations are alike (just as not all copd is alike) and that clinical presentation alone cannot identify aetiology or stratify treatment. 'next to avoiding a fatal issue, our efforts must be directed to prevent the case going on to chronic bronchitis, especially in those who have had previous attacks'. r douglas powell, london ( ) an exacerbation of chronic obstructive pulmonary disease (copd) is defined as 'an acute worsening of respiratory symptoms that results in additional therapy'. the word exacerbation has a latin root; stemming from the verb exacerbare meaning 'to provoke to anger' and the oxford english dictionary defines an exacerbation as 'the process of making a problem, bad situation, or negative feeling worse'. this accurately reflects the negative impact copd exacerbations (ecopd) have on patient quality of life, lung function decline and mortality. in the uk, national audit data highlight the high mortality and readmission rates (and thus healthcare costs) associated with ecopd. exacerbations impact on patients' quality of life and even a single exacerbation is associated with an increase in mean annual forced expiratory volume (fev ) decline. the early identification, provision of appropriate treatment and subsequent prevention (or ideally, primary prevention) of exacerbations has to be a central strategy for copd care. we use clinical symptoms to diagnose an exacerbation of copd, based on the triad of increased breathlessness, increased sputum volume and/ or increased sputum purulence. these criteria are essentially unchanged over the last years, finessed with clinical investigations such as a chest radiograph, arterial blood gas, ecg, a full blood count and sputum culture (all available since - ). [ ] [ ] [ ] [ ] [ ] in stark terms, our diagnostic approach to copd exacerbations has not fundamentally changed for almost years. we have no copd-specific biomarkers and the diagnosis is often one of exclusion. this is in contrast to many other acute presentations of chronic diseases, such as a myocardial infarction (mi) in ischaemic heart disease, where specific and sensitive diagnostic toolkits including biomarkers, imaging and interventions have revolutionised care pathways and patient outcomes. such disparity in advancement raises the question of why copd is so far behind other common, debilitating and progressive chronic diseases which are associated with acute flares of symptoms. why do we not have a better diagnostic and treatment toolkit for ecopd? perhaps to move forward, we need to examine the past. in , douglas powell identified cold weather, upper respiratory tract infections and pollution as an important causes of (acute) bronchitis, observing that 'dusty employments…dusty winds (and) irritating fogs' bring on typical attacks. today, the most important listed triggers of exacerbations of copd include viral and/ or bacterial tracheobronchial infection and inhalation of environmental irritants. in a study of hospitalised (and thus severe) exacerbations of copd, bacteria or viruses were identified in % using quantitative culture and pcr. bacteria were present in % of patients, most commonly haemophilus influenzae, streptococcus pneumoniae, moraxella the variability of inflammation in sputum in one patient with copd. a spontaneous sputum sample was collected over hours post waking and following mouth rinsing procedures daily for days (visits - ), and then twice weekly for further weeks (visits , ; , ; , ) in a patient with moderate severity copd in the stable state who had been an ex-smoker for years. a differential cell count was performed and cytokines were measured in sputum sol phase. each marker is the concentration of that mediator on the visit day. neutrophil (pmn) / ml (red circles), myeloperoxidase (mpo) mg/ml (green square); tnfα pm (cerise triangle): il nm (cyan triangle); ltb nm (black diamond). adapted from sapey et al. copd,chronic obstructive pulmonary disease; tnf, tumour necrosis factor. catarrhalis; staphylococcus aureus and pseudomonas aeruginosa, in descending order of prevalence. viruses were found in %, with rhinoviruses, influenza viruses, respiratory syncytial viruses, parainfluenza viruses and coronaviruses most commonly identified, again in descending order of prevalence. viral infections are important in copd, associated with frequent exacerbations, a higher total symptom burden at presentation and a longer period before symptom recovery, perhaps reflecting the lack of specific therapies available. coinfection (bacterial and viral) is common (seen in % of severe exacerbations), associated with increased lung and systemic inflammation, longer hospitalisation and more severe lung disease. the role of air quality is of increasing interest. short-term exposure to major air pollutants (trioxygen (o ), carbon monoxide, nitrogen dioxide, sulphur dioxide, particulate matter (pm) and pm . ) is associated with respiratory risk but a recent systematic review concluded that these pollutants were also associated with risk of exacerbation. other identified triggers and/or risk factors for exacerbation include discontinuation and poor adherence with medications, poor nutritional and lower socioeconomic status and dynamic hyperinflation. these causative triggers and predisposing factors have been consistently identified across the literature, but studies also highlight alternative pathologies which might account for symptoms, including thromboembolic disease and mi (identified in % and % of suspected copd exacerbations, respectively), suggesting comorbidity is important. the treatment of exacerbations has remained short-acting bronchodilators and corticosteroids with or without antibiotics for years, almost irrespective of the underlying cause. however, with global concerns about antibiotic use and an increasing number of clinical trials of new or repurposed therapeutics given at the time of exacerbation, identifying the exacerbation trigger has never felt more relevant. can clinical evaluation alone help identify the cause? from the late s onwards, a body of evidence supported the concept that the presence of purulent sputum at exacerbation presentation was indicative of a bacterial exacerbation (eg stockley et al, ) and this has been used ever since as a potential biomarker for bacterial infections. however, there are concerns about the ability of patients to self-report sputum colour without training or a colour chart to refer to daniels et al and in some studies, sputum colour could not differentiate between a viral or bacterial aetiology. a landmark study in suggested that exacerbations could be stratified using inflammatory profile. here, % of exacerbations were associated with bacteria, % with a virus, most commonly rhinovirus, % with a significant sputum eosinophilia and % with no significant inflammation (termed pauci-inflammatory). of note, these groupings did not reflect differences in symptom burden, clinical presentation or sputum colour, which could not differentiate between causes, meaning clinicians could not predict what the cause or inflammatory profile of the exacerbation was using standard clinical evaluation alone. this has important but perhaps predictable implications for clinical practice. moving forward, as with other acute presentations of chronic disease, we should not rely on clinical symptoms, signs or non-specific investigations to direct a stratified approach to exacerbation treatment. given copd is an inflammatory disease, the immune response may provide aetiological insight. in engel hypothesised that the structural lung damage described in chronic bronchitis and emphysema might be caused by repeated infections, with multiple acute insults leading to long-term lung damage. in , morgan suggested that there were differences in the acute and chronic inflammation seen in the bronchial tree and surmised that these differences may influence patient outcome and require different treatments. fifty years on from this observation, how far has our understanding of the inflammatory basis of acute exacerbations of copd progressed? there is a substantial and convincing body of evidence that airway inflammation is prevalent in stable copd and is fundamental to its pathogenesis with studies suggesting relationships with disease severity and inflammatory burden. however, pulmonary inflammation varies greatly between individuals and within individuals with copd even when clinically stable and this heterogeneity has proven challenging in biomarker evaluation or inflammation-targeted therapeutic intervention. figure provides an example of the variability of the inflammatory profile of spontaneous sputum inflammation day to day in one patient with copd. as shown, although some mediators share a common pattern of change (if one mediator is up, others are up and vice versa), not all do (eg, on visit , tumour necrosis factor (tnf)α has increased, but leukotriene b (ltb ) and interleukin- (il ) have decreased) and this suggests that the variability in mediators does not only reflect dilution of the sample, but true variability in inflammatory pattern. there appears to be a further amplification of inflammation during exacerbation in many (but not all) patients. once an insult (bacterial, viral or environmental) sufficiently activates the resident immune cells of the airways, it appears to trigger a cascade of inflammatory mediators. this in turn recruits a wave of activated immune cells to the airways, which are predominantly neutrophils but also include eosinophils, monocytes and cd +t cells and these cells have the potential to cause significant disruption and damage when they enter tissue en masse. for example, activated neutrophils release proteinases during migration through complex tissues, degranulation, frustrated where local host defences are overwhelmed, non-resident immune cells, predominantly neutrophils, but also t cells, b-cells and eosinophils are recruited into the lung tissue, following chemokines secreted by epithelial and endothelial cells and resident immune cells. fibroblasts may be activated by growth factor releases from macrophages and epithelial cells. . recruited and resident immune cells are able to release cytotoxic granular contents, reactive oxygen species and proteinases into the tissue and these have been causally associated with the development of mucus secretion, but also emphysema and small airways remodelling, leading to progression of the underlying copd. phagocytosis and neutrophil extracellular traps (net) formation. the concentration of proteinases initially far exceed and thus overwhelm their inhibitors leading to degradation of structural lung proteins including elastin and collagen causing bystander tissue damage and cleavage of enzymes, cytokines, receptors and opsonins including components of the complement cascade and immunoglobulins. while tissue damage is heightened in exacerbations of copd, tissue repair is blunted, effecting the structural integrity of the airways. this inflammatory cascade also results in systemic inflammation, with increases in acute phase proteins such as fibrinogen and c reactive protein (crp). relationships have been described between the degree of pulmonary and systemic inflammation in some studies, a potential link between the multimorbid diseases associated with copd and copd exacerbations (see later). these processes are illustrated in figure . copd severity (definable by a number of measures, but commonly by fev in one second (fev ) at one timepoint) is not synonymous with copd activity (the trajectory of lung function decline or exacerbation frequency). some patients have mild copd by fev which is rapidly progressing or with frequent exacerbations and vice versa. while hypothetically attractive, studies have not consistently linked disease activity (such as exacerbation frequency) to the presence of increased airways inflammation in the stable state. however, the presence of potentially pathogenic bacteria on sputum culture is associated with exacerbation frequency and there is a clear relationship with bacteria and inflammation which supports the concept of inflammatory burden increasing the susceptibility to exacerbations. it is likely that some studies have been underpowered to assess differences in inflammation or have failed to include patients with high exacerbation frequencies, which is understandable given the challenges of recruiting these unstable patients to research studies. there is an association between inflammation and exacerbation outcome. symptom resolution corresponds to abating inflammation and continuation of symptoms or recurrence of exacerbation corresponds to sustained inflammation, suggesting a causal relationship between inflammatory load and host experience. while it is attractive to assume that all exacerbations of a certain aetiology might share the same inflammatory profile and burden, the complexity of host, environment and exacerbation trigger interactions within copd are likely to produce patterns with greater subtlety than that. however, just as with stable copd, within copd exacerbations there might be phenotypes or 'treatable traits' which could help focus therapeutic choices. immune cell function might provide mechanistic insight. it has been proposed that some frequently exacerbating copd patients might experience a 'triple innate immune system hit' which could increase their susceptibility to bacterial exacerbations. first, the frequent exacerbator phenotype has been associated with a reduced ability of airway macrophages to phagocytose bacteria. theoretically, this would lead to increased neutrophil recruitment and in this group neutrophilic inflammation is commonly described. second, studies suggest the accuracy of neutrophil targeting is impaired in copd and associated with heightened bystander tissue damage. third, airway macrophages and monocytes from the frequent exacerbator phenotype are less able to clear dead and dying neutrophils (and eosinophils via efferocytosis, resulting in cell necrosis and localised inflammation and tissue damage). neutrophilic inflammation is corticosteroid resistant in copd but promisingly, studies have identified potential therapeutic targets to improve impaired cellular functions. nrf activators increase macrophage phagocytosis and pi k inhibitors have been shown to increase neutrophil migratory accuracy in vitro as well as reducing inflammation with pi k inhibitors under assessment in early phase studies as a potential therapy during copd exacerbations. due to advancements initially in asthma care, trials of therapies in those copd patients with an eosinophil signal are well underway (with studies currently listed on the clinicaltrials. gov website). results to date suggest that this trait is associated with a good treatment response to oral steroids at exacerbation and inhaled steroids in the stable state, with studies of specific antieosinophil therapies (including mepolizumab) showing promise in selected patient groups. furthermore, studies of community-treated exacerbation suggest that there is no advantage in treating adults without an eosinophil signal with oral prednisolone, as this provides no symptomatic benefit and an increase risk of harm. in hospitalised ecopd, studies suggest that oral corticosteroids and shorter courses appear adequate, with no benefit using high-dose intravenous therapy. excessive use of oral corticosteroids is associated with harm, which is especially clear in studies of patients on long-term maintenance but also potentially raises concerns about uncontrolled and/or unsupported use of 'rescue packs'. of note, a recent cochrane review concluded that there was no evidence of benefit from self-management interventions (including rescue packs) to reduce all-cause hospital admission, all-cause hospitalisation days, emergency department visits, general practitioner visits, dyspnoea scores, the number of copd exacerbations or all-cause mortality although more research was needed. however, the provision of a rescue pack for patients with exacerbations remains a recommendation from the national institute for health and care excellence in the revised guideline published in december (based on expert opinion). these studies begin to highlight that there are different types of copd exacerbations, with different responses to treatment and that a 'one size fits all' approach for both treatment and prevention is overly simplistic. to further advance inflammation-based treatments, a toolkit is needed to match exacerbation aetiology with host response and therefore treatment. in other words, we need to phenotype exacerbations. the value of phenotyping exacerbations of copd is to derive patterns for treatment response or to enhance our understanding of underlying mechanisms. a frequently used, yet rudimentary classification of an exacerbation phenotype is the categorisation as 'infective' or 'non-infective' exacerbations of copd. this is commonly used to direct treatment with antibiotics and systemic corticosteroids, respectively but does not inform underlying mechanisms, likely treatment response or if the exacerbation severity and outcomes are the same. recent advances which exploit developments in biomarker identification, mediator discovery and molecular diagnostics, for example in microbial detection, have furthered our understanding of the exacerbation event. there has been great interest in studying systemic plasma samples in copd to provide insight into the pathogenesis of exacerbations. one such study included exacerbating patients (unselected, of any aetiology), assessing preselected inflammatory mediators. of these, crp, interleukin- , myeloid-progenitor inhibitory factor , pulmonary and activation-regulated chemokine, adiponectin and soluble intracellular adhesion molecule- were significantly elevated at the exacerbation event. however, no plasma mediator alone provided a robust predictive tool for diagnosing an exacerbation event. crp combined with a major symptom (dyspnoea, sputum purulence or sputum volume) improved diagnostic accuracy but no mediator/symptom combination predicted clinical severity or recovery. this result may reflect that the study included 'all comers' with an exacerbation (all aetiologies) and therefore might have been underpowered to find predictive biomarkers if the biomarkers varied by aetiology or host response. to address this, further studies utilised differing approaches to identify phenotypes of exacerbations. the first attempts to investigate biomarkers in virus-associated exacerbations as a specific phenotype were made from the east london cohort. in this study, human rhinovirus (hrv) infection was examined in healthy controls and copd patients at stable state and during exacerbation. baseline cxcl (interferon gamma inducible protein ) was higher in copd than controls, but at exacerbation, there was an increase in serum cxcl in hrv positive exacerbations, correlating with sputum hrv virus load, and no increase in hrv negative exacerbations. a combination of 'cold' symptoms and serum cxcl at exacerbation was associated with a roc of . in predicting an hrv-associated exacerbation of copd. the studies described so far tested preformed hypotheses to identify associations between inflammatory profiles and exacerbation. in the first study of its kind, the beat-copd study employed cluster analysis using mediators sampled from the airways to determine biologically distinct exacerbation groups. four biological exacerbation phenotypes were described, mapping on to inflammation, independent of each other but clinically indistinguishable. sputum interleukin β (il β) was found to be most sensitive for bacteria-associated exacerbations (proinflammatory cluster, receiver operating characteristic curve (roc) . ), serum cxcl was (again) most sensitive for virus-associated exacerbations (th cluster, roc . ) and peripheral blood eosinophils (th cluster, roc . ) was the most sensitive for sputum eosinophilic-associated exacerbations. an independent validation cohort of subjects confirmed that sputum il β, serum cxcl and peripheral blood eosinophils these studies highlight four potential exacerbation phenotypes which might provide robust treatment pathways in time. . bacterial in origin, il- β as a biomarker, neutrophilic inflammation. . viral in origin, with cxcl as a biomarker. . eosinophillic in origin and as a biomarker. pauci-inflammatory. these appear to be biologically different even when clinically indistinguishable. however, while our understanding of each of these phenotypes needs to be improved, we understand very little at all about the so-called pauci-inflammatory exacerbation. indeed, it is unclear whether this represents copd at all or the acute presentation of a related comorbidity which may also cause or exacerbate breathlessness and a cough. the recognition and gravitas of comorbidities in copd has built over the last decade or more. whether the presence of comorbidities is based on self-report or systematically sought, they are common and affect mortality. exacerbations represent a period with multiple insults to both the lung and systemically. such insults include the aetiological factor itself (pathogen or environmental), lung physiological changes and additional work of breathing, hypoxia, periods of inactivity (which can effectively be prolonged periods of 'bed rest' during an in-patient admission), with a study suggesting that an acute medical admission is associated with a median step count of per day (iqr - ), dehydration, malnutrition, the therapies prescribed and their side effects (eg, oral corticosteroids and hyperglycaemia and antibiotics and gastrointestinal disturbance) and then the sequelae of these factors including systemic inflammation, hypo and hypernatraemia/kalaemia and altered sympathetic drive. figure summarises the complex relationships between comorbidity and exacerbations in copd. there is a significant and complex interplay between the exacerbation and the comorbid condition including the impact of comorbidities on the exacerbation itself; how an exacerbation contributes to comorbid disease; the prognostic role of comorbid disease and the subclinical presentation of a comorbid condition at the time of an exacerbation. cardiovascular disease highlights the interplay and is the most studied comorbidity in this context. patients presenting to hospital with a copd exacerbation have a host of comorbid conditions and the presence of a comorbid condition and the systemic manifestation of that figure comorbidity and copd exacerbations. there are many stressors during copd exacerbations which can predispose to or exacerbate comorbidities and the multimorbidity patients experience. this figure is a schematic of some of these factors, but is not exhaustive and each stressor can influence the other, irrespective of placement in the figure. stressor include the direct effects of infection and inflammation, dyshomeostasis including hypo and hypernatraemia, kalaemia and glycaemia, hypoxia and hypercapania. organ dysfunction is common, especially of cardiac and renal origin. patients are placed on short courses of oral corticosteroids and physical activity is reduced (and can equate to bed rest in some patients), both contributing to sarcopenia and osteopenia. copd, chronic obstructive pulmonary disease. comorbidity increases the duration of an exacerbation. coexistent ischaemic heart disease leads to far greater number of symptomdays per year, while an increased blood glucose in hospitalised patients leads to a longer stay and is associated with a higher risk of death. mi is more likely in the period following presentation with an exacerbation [ ] [ ] [ ] and there is evidence of increase platelet aggregation, increased arterial stiffness as well as myocardial injury as evidenced by cardiac biomarkers at the time of a copd exacerbation. the fact that comorbid disease may present subclinically at the time of the exacerbation is also important to consider, be it as a differential or as a further contributing factor to the symptoms and challenges of managing the condition. in a prospective case series, one in patients presenting to hospital with an exacerbation of copd had criteria that would meet diagnosis of a mi. impaired cognitive function is evident, if assessed, in a large proportion of patients at the time of discharge from a hospitalised exacerbation, with no evidence of recovery months later. prognostically, comorbidities present a greater risk of hospitalisation, particularly in the presence of lower lung function, as well as increased all-cause readmissions related to multimorbidity and older age. in the eclipse study (evaluation of copd longitudinally to identify predictive surrogate end-points), the best predictor of exacerbations was a former history of them. in addition, however, a history of reflux and heartburn was a further independent factor. the presence of acute kidney injury and lower limb muscle cross-sectional area at the time of exacerbation requiring hospitalisation are both prognostic of death. the prognostic copd exacerbation score such as the validated decaf score ("dyspnoea, eosinopenia, consilidation, acidaemia and atrial fibrillation" score predicting in-patient mortality) and the pearl score ("previous admissions, emrcd score, age, right-sided heart failure and left sided heart failure" score predicting -day readmission and mortality) include cardiac comorbidity in their calculations. patients deemed as frequent exacerbators are more likely to be depressed or have coexistent cardiovascular disease or osteoporosis. it is unclear if some events labelled exacerbations are actually a presentation of a comorbid condition (and studies suggest that clinicians are less likely to diagnose mi or pulmonary embolus (pe) if there is a concomitant diagnosis of copd, ) perhaps the so-called pauci-inflammatory exacerbations, or whether the comorbidity is exacerbating the copd. there remains a role for more timely identification of comorbid disease and addressing the contributing factors. the role of systematic identification of certain comorbidities and of preventative strategies, both pharmacologically and lifestyle-based are topics for ongoing discussion and research. in the meantime, opportunity exists to ensure optimal treatment for those with identified comorbid disease, such as ensuring beta-blockers are prescribed in those who meet the criteria or that hyperglycaemia or hyperlipidaemia are adequately addressed. despite a greater understanding of the biology and complexity of copd exacerbations, this has not (yet) translated into novel therapies to treat exacerbations. there has been no new intervention to treat copd exacerbations since the widespread adoption of non-invasive ventilation to treat exacerbations with hypercapnoeic respiratory failure in the early s. from the first introduction of guidelines such as gold in , the therapy for an exacerbation is unchanged. as described below, despite being commonly used, there remain significant research knowledge gaps in determining which exacerbations do and do not require treatments with antibiotics and corticosteroids. systemic corticosteroids were first used in rheumatological disease during the late s. despite evidence in the late s that many hospitalised patients were being treated with systemic corticosteroids, it was only at the turn of the millennium that small randomised clinical trails (rcts) first documented clinical efficacy, suggesting benefit on lung function and outcomes such as length of hospital stay. around the same time, the first small outpatient trials of steroids at exacerbation reported, with modest benefits confirmed in a larger rct. later it was defined that short course ( days) treatment was as effective as longer day courses, and without the need to taper dose. with a greater emphasis on exacerbation phenotyping, more recent studies have documented the ability to safely withhold steroids in exacerbations without an eosinophil signal. however, the practicality of achieving this at point-ofcare, and the optimal blood eosinophil cut-off to guide steroid therapy remain to be determined, and there are ongoing trials in the area. given the toxicity associated with repeated courses of corticosteroids, the need for effective novel anti-inflammatory agents is also great. disappointingly, there is no evidence of benefit with the anti-tnf agent entanercept or roflumilast, for example. anthonisen's rct demonstrated the superiority of antibiotics over placebo in exacerbations presenting with at least two of the three cardinal symptoms of increased breathlessness, sputum volume and sputum purulence. importantly, this had been conducted in patients with copd, rather than just those with chronic bronchitis. however, the placebo response rate was high, likely reflecting viral pathogens as a common cause of exacerbation, and more recent studies have not shown a benefit of antibiotics in other outcomes such as prolonging the time to next exacerbation. biomarkers such as sputum colour and procalcitonin have been suggested as strategies to better guide antibiotic therapy, but there remains unmet need to better define which exacerbations do and do not benefit from antibiotic therapy. it is also notable that there are no effective interventions to treat (or prevent) rhinovirus infections, thought to be the single the most common cause of a copd exacerbation. salbutamol has been available since the late s, with ipratropium following in the s. these replaced the non-selective β adrenoreceptor agonist isoprenaline. there are no good data on long-acting bronchodilator drugs at the time of exacerbation. the s audit referred to above highlighted the widespread use of theophyllines (in % of patients), and use of respiratory stimulants such as doxapram in the management of hospitalised exacerbations. use of theophylline has reduced, while respiratory stimulants have been replaced by non-invasive ventilation for the management of hypercapnoeic respiratory failure in the respiratory ward environment, following initial studies in the early s . models of care have changed, with the recognition that earlier access to treatment for exacerbations can be associated with faster recovery and reduced risk of hospital admission. however, the risks and benefits of patient-held rescue packs remain to be definitely established. research to develop new interventions at exacerbation of copd is hampered by robust outcome measures to assess exacerbation recovery. changes in lung function are not patient centred, and changes in symptoms scores not validated. 'clinical recovery' and 'treatment failure' are subjective constructs, while studies have also examined effects of exacerbation treatment on the time to the next event given that exacerbations cluster in time, with a high-risk period for a second event in the period following recovery from a first. we have at least made progress in prevention of exacerbations, though even when used optimally there seems to be a ceiling of reduction at around %. effective interventions (outlined in table ), alone and in combination, include non-pharmacological approaches such as pulmonary rehabilitation, and pharmacological approaches the mainstay of which remains long-acting bronchodilators with or without inhaled corticosteroids and, in selected cases, prophylactic antibiotics. for patients remaining hypercapnic following a hospitalised exacerbation, domiciliary non-invasive ventilation significantly reduces the risk of rehospitalisation (with an absolute risk reduction of % in a recent landmark study). similar to strategies to better target exacerbation treatment, there is also now emerging evidence on how better to target exacerbation prevention interventions, including the optimal use of inhaled corticosteroids. thus, while exacerbation prevention strategies are incompletely effective, the challenge here is rather selecting the right combination of interventions for the right patient at the right time, rather than the absence of effective prevention strategies. in , r douglas powell advised that our management aims should be to save life and prevent further episodes of then acute and chronic bronchitis, now copd. we still have a long way to go to achieve this. exacerbations of copd are still associated with significant mortality, morbidity, readmission and poor life quality. there have been no real advancements in routine care since the s. there is considerable unmet need for novel strategies to identify, treat and prevent exacerbations, and a pressing need to better use existing therapies. this remains a major challenge. how do we move forward? copd as a disease concept has always raised the question of 'lumping or splitting'; is this one disease or many? innovation in asthma care has provided a path which perhaps copd should follow. asthma phenotypes and now endotypes provide clinically blurred but biologically distinct clusters with an emerging arsenal of treatments for those with the most difficult to manage symptoms. the concept of 'treatable traits' has gained considerable momentum in stable copd, and perhaps now the same concept should be tested further in exacerbations. we are beginning to see some differences in biological signals across exacerbation aetiologies and host responses. to build on this, we need to continue with more stratified studies of ecopd, learning from the fruitful experience of focusing on those with an eosinophilic signal, but this time using poc testing to characterise and test treatments in (eg) viral or pauci-inflammatory exacerbations. this will provide more information about aetiology, but to personalise treatment, this must be incorporated into a holistic understanding of the impact of the hosts comorbidity and immune responses. from these data, we could build our ecopd toolkit, which we hypothesise might include poc identification of bacterial or viral pathogen (ensuring that the correct antibacterial or viral therapy is used and thus reducing redundant therapy), blood biomarkers to identify or exclude an eosinophil (corticosteroid use or avoidance) or cardiac (acute coronary syndrome, heart failure) or neutrophilic treatment pathway and a measure of acuity and respiratory compromise. by exploring these ideas, we may be able to introduce a stratified approach to treatment and prevention, which might, finally, really impact on these debilitating and costly events, to the benefit of our patients. contributors all authors wrote the manuscript and all contributed to the manuscript equally. funding e sapey was funded by the medical research council, grant number mr/ r / . other authors received no specific grant for this work from any funding agency in the public, commercial or not-for-profit sectors. no, there are no competing interests for any author. global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease effect of exacerbations on quality of life in patients with chronic obstructive pulmonary disease: a year follow up study acute exacerbations and lung function loss in smokers with and without copd the impact of exacerbation frequency on mortality following acute exacerbations of copd: a registry-based cohort study copd: who cares when it matters most? national chronic obstructive pulmonary disease (copd) audit programme: outcomes from the clinical audit of copd exacerbations admitted to acute units in effect of a single exacerbation on decline in lung function in copd a treatise on the disease of the chest and on mediate auscultation. (translated by j forbes) de la numeration des globules rouges du sang ueber die form des menschlichen electrocardiogramms the determination of gases in blood and other solutions by vacuum extraction and manometric measurement. i notes on xrays ueber die isolierte farbung der schizomyceten in schmitt-und trockenpraparaten on consumption, on certain diseases of the lungs and pleura infection in the pathogenesis and course of chronic obstructive pulmonary disease infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease major air pollutants and risk of copd exacerbations: a systematic review and meta-analysis adherence to inhaled therapy, mortality and hospital admission in copd socioeconomic status, race and copd health outcomes physiological changes during symptom recovery from moderate exacerbations of copd venous 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acute ventilatory failure due to chronic obstructive airways disease early therapy improves outcomes of exacerbations of chronic obstructive pulmonary disease temporal clustering of exacerbations in chronic obstructive pulmonary disease effect of home noninvasive ventilation with oxygen therapy vs oxygen therapy alone on hospital readmission or death after an acute copd exacerbation: a randomized clinical trialeffect of home niv on outcomes after acute copd exacerbationeffect of home niv on outcomes after acute copd exacerbation small copds": copd should be an orphan disease asthma phenotypes: the evolution from clinical to molecular approaches global initiative for chronic obstructve lung disease. global strategy for prevention, diagnosis and management of copd key: cord- -zhmuzv z authors: stetzenbach, l.d. title: airborne infectious microorganisms date: - - journal: encyclopedia of microbiology doi: . /b - - . - sha: doc_id: cord_uid: zhmuzv z inhalation exposes the upper and lower respiratory tracts of humans to a variety of airborne particles and vapors. airborne transmission of pathogenic microorganisms to humans from the environment, animals, or other humans can result in disease. inhalation is an important route of exposure as the lung is more susceptible to infection than the gastrointestinal tract. ingested microorganisms must past through the acidic environment of the stomach before they can colonize tissue while inhaled microorganisms are deposited directly on the moist surfaces of the respiratory tract. inhalation of microbial aerosols can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease. following inhalation, infectious viruses, bacteria, and fungi can establish in host cells of the respiratory tract. some are translocated and infect the gastrointestinal tract and other tissues. this chapter discusses human viral, bacterial, and fungal diseases transmitted via aerosols. viral diseases presented are influenza, severe acute respiratory syndrome (sars), norwalk-like viruses (nlvs) and hantavirus disease, measles, and varicella. bacterial diseases are legionnaires’ disease, tuberculosis, and nontubercule mycobacterial disease. exposure to some gram-negative and gram-positive bacteria, endotoxin, and actinomycetes when dispersed through the air can result in disease following inhalation. fungal diseases included are histoplasmosis, coccidiomycosis, blastomycosis, cryptococcosis, and aspergillosis. the threat of bioterrorism with airborne infectious agents is also briefly presented. glossary aspergilloma pulmonary fungus ball consisting of a solid mass of hyphae growing in a lung cavity. aspergillosis disease caused by the inhalation of some species of the genus aspergillus resulting in growth of the fungus in the lungs. bioaerosol an airborne suspension of biological particles and microbial by-products. blastomycosis a symptomatic infection caused by the inhalation of the fungus blastomyces dermatitidis. chickenpox disease caused by the virus varicellazoster. coccidioidomycosis disease caused by the inhalation of coccidioides immitis arthroconidia (also called san joaquin valley fever). cryptococcosis disease caused by the fungus cryptococcus neoformans. endotoxin a lipopolysaccharide component of the gram-negative bacteria cell released during active cellular growth and after cell lysis that can cause respiratory distress when inhaled. fomite an inanimate object that transmits infectious agents to a new host. h n , h n , h n , and h n strains of avian influenza a viruses that have been linked to respiratory disease in humans. histoplasmosis disease caused by the infectious fungus histoplasma capsulatum that can manifest as mild flu-like symptoms to chronic lung disease that resembles tuberculosis. hps hantavirus pulmonary syndrome. legionnaires' disease a severe respiratory disease resulting from the inhalation of legionella pneumophila. measles term for the disease caused by the virus of the same name. mycobacterium tuberculosis the causative agent of human tuberculosis. nlvs norwalk-like viruses. pontiac fever a mild flu-like syndrome following inhalation of nonviable legionella pneumophila. rubeola another name for the disease measles. sars severe acute respiratory syndrome. sars-cov severe acute respiratory syndrome associated coronavirus. sin nombre virus causative agent of the majority of hantavirus pulmonary syndrome cases in the united states. hantavirus pulmonary syndrome hvac heating, ventilation, and air conditioning nlvs norwalk-like viruses severe acute respiratory syndrome sars-cov sars-associated coronavirus wmd weapons of mass destruction introduction inhalation exposes the upper and lower respiratory tracts of humans to a variety of airborne particles and vapors. airborne transmission of pathogenic microorganisms to humans from the environment, animals, or other humans can result in disease. the lung is more susceptible to infection than the gastrointestinal tract as ingested microorganisms must past through the acidic environment of the stomach before they can colonize tissue, while inhaled microorganisms are deposited directly on the moist surfaces of the respiratory tract. inhalation of microbial aerosols can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease. a bioaerosol is an airborne collection of biological material. bioaerosols can be comprised of bacterial cells and cellular fragments, fungal spores and fungal hyphae, viruses, and by-products of microbial metabolism. pollen grains and other biological material can also be airborne as a bioaerosol. microbial aerosols are generated in outdoor and indoor environments as a result of a variety of natural and anthroprogenic activities. wind, rain and wave splash, spray irrigation, wastewater treatment activity, cooling towers and air handling water spray systems, and agricultural processes such as harvesting and tilling are examples of activities that generate bioaerosols outdoors. indoors bioaerosols are generated and dispersed mechanical and human activity. industrial and manufacturing practices and biofermentation procedures can generate high concentrations of microbial aerosols. heating, ventilation, and air conditioning (hvac) systems, water spray devices (e.g., showerheads and humidifiers), and cleaning (e.g., dusting, sweeping, vacuuming, and mopping) result in the transport of microbial materials in the air. talking and coughing generate bioaerosols from individuals, some of which may be infectious. facilities with medical, dental, or animal care practices can generate infectious microbial aerosols. the individual particle size of particulate material in bioaerosols is generally . - mm in diameter; larger particles tend to settle rapidly and are not readily transported in the air. virus particles are nanometer in size, bacterial cells are approximately mm in diameter, and fungal spores are > mm. these microorganisms can be dispersed in the air as single units, but are often present as aggregate formations. the larger aggregates have different aerodynamic properties than single-cell units; therefore their dispersal may be different than singleunit particles. aggregates of biological material also afford protection from environmental stresses such as desiccation, and exposure to ultraviolet radiation ozone and other pollutants in the atmosphere. often bacterial cells and virus particles are associated with skin cells, dust, and other organic or inorganic material. during agricultural practices (e.g., during harvesting, and tilling), fungus spores are released from plant surfaces and the soil and raft on other particulate matter. this 'rafting' affects the aerodynamic characteristics and the survival of the cells in the bioaerosol. when biological material is dispersed from water sources (e.g., splash, rainfall, or cooling towers and fountains), it is generally surrounded by a thin layer of water. this moisture layer also changes the aerodynamic properties and aids in the survivability of the microorganisms while airborne. airborne particulate will remain airborne until settling occurs or they are inhaled. following inhalation, large airborne particles are lodged in the upper respiratory tract (i.e., nose and nasopharynx). particles < mm in diameter are transported to the lung where the - mm particles have the greatest retention in the alveoli. the average person inhales approximately m of air per day, which may result in an infective dose for some airborne pathogens. diseases resulting from inhalation of bioaerosols include allergic reaction, irritation, inflammation, infection, and respiratory disease. the by-products of microbial metabolism and foreign proteins of microorganisms and fragments of cells can result in allergic reactions and irritant responses. the inflammatory response and allergic reactivity can be debilitating to sensitized individuals in indoor and outdoor environments, and it has been demonstrated following exposure to airborne bacterial fragments present in airborne dust. however, the greatest adverse health impact following exposure to airborne microorganisms is associated with inhalation of infectious agents. these infectious agents include viruses, bacteria, and fungi. the following sections introduce representative pathogens of each grouping that are associated with the airborne route of exposure. table provides a summary of the pathogens presented in this chapter. viruses are noncellular units consisting of either dna or rna, but not both. they do not have self-directed biosynthesis and, therefore, require a living host cell for replication. following inhalation, infectious viruses can establish in host cells of the respiratory tract. some are translocated and infect the gastrointestinal tract and other tissues. influenza is a contagious respiratory illness caused by flu viruses a, b, and c. there are subtypes of influenza type a viruses based on two proteins on the surface proteins of the virus, hemagglutinin (h) and neuraminidase (n). many animals carry influenza a viruses, while influenza b viruses circulate among humans. both influenza types a and b viruses cause epidemics of disease almost every winter. influenza type c infections cause a mild respiratory illness and are not thought to cause epidemics. while symptoms can be mild, influenza may result in severe illness and death. the centers for disease control and prevention reports that there are more than hospital admissions and deaths per year in the united states due to influenza. all ages can have serious illness from influenza, but those more likely to have complications are people aged years and older, people with chronic medical conditions, and very young children. transmission of influenza viruses is from person to person in respiratory droplets expelled during coughing and sneezing. the droplets are propelled usually less than ft through the air and deposited on people in close proximity. fomites contaminated with settled infectious droplets can also serve as a vehicle for transmission of influenza. avian influenza a viruses usually do not infect humans. however, cases of human infection with avian influenza viruses have been reported since . illness caused by several strains of avian influenza a viruses (e.g., h n , h n , h n , and h n ) have been reported since . the majority of human avian influenza infections have resulted from inhalation during direct contact with infected poultry or contaminated surfaces such as avian cages, or from the ingestion of uncooked blood of infected birds. therefore, this influenza has been popularized as 'bird flu'. the illnesses resulting from avian influenza infection in humans range from typical mild influenza-like symptoms (e.g., fever, sore throat, cough, and muscle aches) and conjunctivitis to more serious cases of pneumonia, acute respiratory distress, and other severe and life-threatening complications. to date, there have been no sustained humanto-human transmission of avian influenza a viruses, but because of the potential for these viruses to gain the ability to spread easily between people, monitoring for human infection and person-to-person transmission is important. severe acute respiratory syndrome (sars) is a newly reported respiratory illness. it was first reported in february in asia. since then, it has also been reported in north america and europe. it is a viral respiratory illness caused by a coronavirus, termed sars-associated coronavirus (sars-cov). exposure to sars-cov results from close personal contact with infected individuals. transmission is generally the result of touching the skin of an infected person or objects contaminated with infectious droplets and then transferring the virus to the new host's eyes, nose, or mouth. coughing or sneezing by an infected individual also releases droplets into the air that are propelled a short distance (< m) and deposited on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. it is possible that sars can be spread further through the air by very small particles, but investigations to date suggest that this type of spread is unusual. hand hygiene, use of gown, gloves, and eye protection when in contact with an infected individual, and use of an appropriate respirator minimizes the spread of sars. norwalk-like viruses (nlvs) are a group of highly infectious viruses first reported following an outbreak of gastroenteritis in . the infectious dose is estimated at - particles. outbreaks of gastrointestinal infection resulting from exposure to nlvs are characterized by diarrhea and sudden projectile vomiting. while transmission of nlvs is primarily associated with the fecaloral route, it has been estimated that over million virus particles can be liberated during vomiting. therefore, airborne transmission within close contact is highly likely. hantavirus pulmonary syndrome (hps) is a life-threatening disease caused by rodent-borne hantaviruses. it is characterized by severe pulmonary illness and a mortality rate of - %. the sin nombre virus causes the majority of hps cases in the united states, and peromyscus maniculatus (the deer mouse) is its predominant reservoir. the virus found in the rodent urine, saliva, and feces becomes aerosolized as mist from urine and saliva or dust from feces. inhalation is the most common route of infection. however, touching the mouth or nose after handling contaminated materials or being bitten by an infected rodent also result in infection. hantavirus is not spread from person to person. classic symptoms of disease include fever, fatigue, and muscle aches. symptoms progress in - days to cough and shortness of breath. the disease measles is contracted by inhalation of infectious droplets expelled as respiratory secretions by an infected individual. the measles virus is also known as rubeola and the only known reservoir is humans. all strains of this virus belong to a single antigenic type and immunity is long lasting following infection. fever, malaise anorexia, and respiratory symptoms mimic other respiratory infections, but the appearance of koplik's spots precedes a rash on the face that proceeds down the body to the soles and palms. severe complications may occur including a chronic degenerative neurologic disease that occurs many years after a measles infection. measles is worldwide, but the success of vaccination programs in developed countries has limited the number of cases of childhood measles. however, there are still sporadic cases of measles in the united states as a result of immigration and travel of foreign visitors who spread the infection to unvaccinated or unprotected persons. chickenpox is a highly contagious disease caused by the varicella-zoster virus. chickenpox is usually a self-limited disease lasting - days with fever, malaise, and a generalized vesicular rash of blister-like lesions. the rash covers the body, but it is usually more concentrated on the face, scalp, and trunk. serious complications including bacterial infections of skin lesions, pneumonia, cerebellar ataxia, and encephalitis may occur, but are rare. disease is spread by aerosol dissemination of the virus during coughing and sneezing by an infected person or it may become airborne directly from the skin lesions. incidence of chickenpox is highest among children - years of age, but the licensing of a vaccine in and vaccination requirements for daycare and school children greatly reduced the impact of this virus in the united states. following varicella infection, the virus becomes latent in sensory nerve ganglia and may reactivate later in life resulting in shingles. unlike viruses that require a host cell, bacteria can colonize and grow on a variety of surfaces and in liquids when physical and nutritional factors are suitable. therefore, bacteria can be released into the air from a variety of natural and anthroprogenic environments. when infectious bacteria are released into the air, they can be readily transported via bioaerosols to susceptible individuals. the first report of legionnaires' disease followed the outbreak of respiratory illness among american legion conventioneers in philadelphia in . currently an estimated - cases of legionnaires' disease are reported in the united states per year. the causative agent, legionella pneumophila, is a vegetative gram-negative bacterium that is ubiquitous in freshwater environments worldwide. it survives as an intercellular parasite of protozoa thereby resulting in protection for the bacterium while in the environment. infective aerosols are generated from contaminated water spray devices such as showerheads, evaporative condensers, humidifiers, and fountains. following the inhalation, l. pneumophila replicates in host macrophage resulting in severe pneumonia. while l. pneumophila accounts for the majority of disease cases caused by this genus, other legionella species are also human pathogens, often causing disease in immunosuppressed patients. legionella bacteria that cannot replicate in the lung are cited as the cause of pontiac fever, a mild flulike syndrome. often these organisms are released in aerosols from whirlpool tubs. pontiac fever results in many more cases of illness, but fewer deaths; legionnaire's disease has a much higher mortality, but lower morbidity. no human-to-human transmission has been documented. for an outbreak of legionnaires' disease to occur, a series of events must happen. a reservoir of infectious bacteria must be present, the environmental conditions must be right for the concentration of organisms to increase, the organisms must be infectious and the infectious bacteria must be dispersed resulting in human exposure, organisms must be deposited in the appropriate site in the human host, and the host must be susceptible to infection by the legionella. mycobacterium tuberculosis is recognized for its role as the causative agent of human tuberculosis. infected individuals can spread pathogenic bacilli when coughing, talking, speaking, laughing, and sneezing. overcrowding and low-socioeconomic status are often contributing factors to the spread of tuberculosis as individuals are in close association and hygiene conditions are usually poor. the confined spaces of airplane cabins have been implicated in transmissions to passengers and flight crew members. infectious aerosols resulting from the operation of suctioning instruments during medical treatment, manipulation of tuberculosis ulcers, drainage of necrotic tissue, and exposure during autopsy have also been cited as causes of nosocomial m. tuberculosis infections. also, nontuberculosis mycobacterial species can be transported via the air and result in disease. respiratory infection in immunocompromised patients has been associated with members of the mycobacterium avium complex. mycobacterium leprae bacilli have been reported from the nose blow of an untreated person with lepromatous hansen's disease (leprosy), and mycobacterium terrae isolated from a waterdamaged building demonstrated inflammatory responses in mouse lung during laboratory experiments. escherichia coli, pantoea (enterobacter) agglomerans, pseudomonas spp., and acinetobacter spp. commonly isolated in cow barns, pig houses, and poultry barns are among the airborne gram-negative bacteria associated with animal facilities that can result in human disease. disease-causing gram-negative bacteria are also dispersed into the air from wastewater treatment plants, vegetable and herb processing, and recycling facilities. these organisms can cause a variety of health problems, especially to the young, elderly, and immunocompromised persons. endotoxin is a lipopolysaccharide component of the gram-negative bacteria cell and is released during active cellular growth and after cell lysis. while it is not an infectious particle, endotoxin is biologically active material derived from bacteria that can affect many human organ systems and disrupt humoral and cellular host mediation systems. symptoms of exposure to airborne exposure include chest tightness, cough, shortness of breath, fever, and wheezing. obstruction of airflow and exacerbation of asthma has been related to airborne endotoxin concentrations, and atopic and nonatopic asthma have been associated with endotoxin exposure because of its ability to induce inflammatory reactions. repeated inhalation exposure has been shown to induce allergenspecific airway inflammation. airborne endotoxin exposure is a concern in dusty occupational facilities such as cotton processing facilities and agricultural settings, and in industrial environments that utilize water spray components (e.g., machining fluids). indoor airborne endotoxin is a concern with humidified mechanical air conditioning systems. the presence of dogs, moisture, and settled dust increase the likelihood of airborne endotoxin in the home. numerous gram-positive bacteria are commonly disseminated from the skin, oral and nasal surfaces, and hair of humans. commonly staphylococcus spp. are among the nonendospore-forming gram-positive bacteria isolated in indoor air samples. the presence of these cells in the air increases the likelihood of nosocomial infections in healthcare settings. nosocomial infections by airborne pathogens and with antibiotic-resistant bacteria in hospital environments are transmitted among patients through aerosol dispersal from patients, staff, surfaces, and aspiration from respiratory instrumentation. airborne dispersal of pathogens is also a concern in home care and assisted living environments. additionally, the gram-positive actinomycetes have been associated with illness in occupants of waterdamaged buildings. often they are associated with fungi that colonize on surfaces and building materials following water intrusion. fungi are ubiquitous in nature. they utilize organic matter for their metabolism depending on the genus they can readily disperse spores, cellular fragments, or cells into the air. infectious fungi can cause serious mycoses of the respiratory tract and some are disseminated to the bone and other systems in the body. inhalation of spores of the fungus histoplasma capsulatum can result in histoplasmosis. this disease is not transmitted from an infected person or animal to someone else but is the result of airborne transport from environmental surfaces. histoplasmosis primarily affects a person's lungs, and its symptoms vary greatly. infected people are generally asymptomatic or they experience mild flu-like symptoms not requiring medical attention. chronic lung disease resulting from infection with h. capsulatum resembles tuberculosis, and can worsen over months or years. the most severe and rarest form of histoplasmosis occurs when it is disseminated involving spreading outside the lungs. if untreated, disseminated histoplasmosis is fatal. h. capsulatum is a dimorphic fungus with a mold (mycelial phase) when it is growing in soil at ambient temperatures, and a yeast phase after being inhaled by humans or animals. the mold spores are either microconidia ranging from to mm in diameter or macroconidia ranging from to mm. the yeast cells of h. capsulatum are oval to round in shape with diameters ranging from to mm. inhalation of airborne coccidioides immitis arthroconidia may result in the disease coccidioidomycosis (also called san joaquin valley fever). within - h after inhalation and deposition in the lung, the arthroconidia change into spherules and develop numerous endospores. when spherules rupture, they release endospores which have the capacity to develop into a mature spherule. approximately % of those who become infected are symptomatic with flu-like illness with fever, cough, headaches, rash, and myalgia. some individuals fail to recover and suffer chronic pulmonary infection. others will develop widespread disseminated infection that may affect the soft tissues, joints and bone, or meninges. coccidioidomycosis meningitis may lead to permanent neurologic damage. the immunocompromised and hivinfected persons suffer severe pulmonary conditions and diffuse lung disease. c. immitis grows in the soil of semiarid areas. it is primarily found in the lower sonoran life zone and is endemic in the south-western united states; it is also found in parts of mexico and south america. exposure to airborne arthrospores occurs after disturbance of contaminated soil by humans or natural disasters (e.g., dust storms and earthquakes). persons in areas with endemic disease who have occupations exposing them to dust (e.g., construction or agricultural workers, and archeologists) are at risk. african-americans and asians, pregnant women during the third trimester, and immunocompromised persons have a higher risk than others. blastomycosis is a symptomatic infection caused by the fungus b. dermatitidis. this fungus grows in moist soil that is enriched with decomposing organic debris, and it is endemic in parts of the southcentral, southeastern, and mid-western united states. it is also present in some areas of central and south america and parts of africa. exposure is via the inhalation of airborne spores, generally after disturbance of contaminated soil. generally, individuals who frequent wooded areas with endemic disease (e.g., farmers, forestry workers, hunters, and campers) are the most exposed ones. while there are only or cases per population in areas with endemic disease, infection results in a flu-like illness with fever, chills, productive cough, myalgia, arthralgia, and pleuritic chest pain. however, some infected individuals fail to recover and develop chronic pulmonary infection and permanent lung damage. other individuals develop widespread disseminated infection that may involve the skin, bones, and genitourinary tract, and occasionally the meninges of the brain are affected. the mortality rate for blastomycosis is approximately %. c. neoformans var. neoformans causes most cryptococcal infections in humans. infection with this yeast-like fungus can cause disease in apparently immunocompetent and immunocompromised persons, but most people do not get sick with cryptococcosis. patients with t-cell deficiencies are the most susceptible to infection, and in the united states % of cases occur in hiv-infected persons. while the initial pulmonary infection is usually asymptomatic, cryptococcosis can cause serious symptoms of brain and spinal cord disease, such as headaches, dizziness, sleepiness, and confusion. disseminated infection, especially meningoencephalitis, occurs in most patients. c. neoformans var. neoformans is found worldwide, and its habitat includes debris around pigeon roosts and soil contaminated with decaying pigeon or chicken droppings. exposure generally occurs when dried bird droppings are disturbed and dust containing cryptococcus disperses in the air. the exact nature of the infectious particles of c. neoformans is not known, but they are likely to be dehydrated yeast cells or basidiospores of the appropriate size range to be deposited in the lungs. once in the lung, the yeast cells become rehydrated and acquire the characteristic polysaccharide capsule. virulence of this organism is associated with the ability to produce the large capsule and shed great amounts of capsular material into the body fluids of the host. aspergillosis is an invasive pulmonary infection with fever, cough, and chest pain. a localized pulmonary infection occurs for immunocompetent persons who have an underlying lung disease. the fungus infection may disseminate to other organs, including brain, skin, and bone. exposure may also result in allergic sinusitis and allergic bronchopulmonary disease. a variety of aspergillus species have been implicated. aspergillus fumigatus and aspergillus flavus most often have been cited, but aspergillus terreus, aspergillus nidulans, and aspergillus niger have also been associated with the disease. these fungi are ubiquitous in the environment, and their conidia (spores) are disseminated from soil, compost piles and other decomposing plant matter, household dust, building materials, and ornamental plants. water-damaged buildings can also be a source for exposure, and infections have been associated with dust exposure during building renovation or construction. the threat of purposeful transmission of airborne of pathogenic microbial and microbial toxins as weapons of mass destruction (wmd) has increased the awareness of the importance of bioaerosols. the centers for disease control and prevention and the department of health and human services have listed several pathogenic microorganisms as select agents. of these, francisella tularensis, yersinia pestis, bacillus anthracis, and smallpox, the causative agents of tularemia, plague, anthrax, and smallpox, respectively, are ones that can be dispersed via aerosol and, therefore, are a concern for inhalation infection. continued researches and awareness by public health professionals are needed to recognize these diseases and minimize the risk of exposure of the public. see also: aeromicrobiology/air quality; biological warfare; emerging infections; influenza; respiratory viruses; smallpox, historical; surveillance of infectious diseases bioaerosols in the environment bacillus anthracis aerosolization associated with a contaminated mail sorting machine legionella and legionnaires' disease evaluation of exposure to airborne bacterial endotoxins and peptidoglycans in selected work environments infection due to legionella species other than l. pneumophila key: cord- -uy f f o authors: nara, peter l.; nara, deanna; chaudhuri, ray; lin, george; tobin, greg title: perspectives on advancing preventative medicine through vaccinology at the comparative veterinary, human and conservation medicine interface: not missing the opportunities date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: uy f f o abstract vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life—resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past years, even in the past years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para- and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it’s melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the st century. vaccination has historically and remains one of the most cost-effective and safest forms of medicine today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives and economies in both rich and poor countries by its direct impact on human and animal life-resulting in the eradication of small pox, huge reductions in the burden of previously common human and animal diseases such as polio, typhoid, measles in human medicine and contagious bovine pleuropneumonia, foot-and-mouth disease, screwworm and hog cholera and the verge of eradicating brucellosis, tuberculosis, and pseudorabies in veterinary medicine. in addition vaccination along with other animal production changes has provided the ability to produce otherwise unaffordable animal protein and animal health worldwide. the landscape however on which vaccinology was discovered and applied over the past years, even in the past years has and is undergoing continuous change. for vaccination as a public health tool to have its greatest impacts in human and veterinary medicine, these great medical sciences will have to come together, policy-relevant science for sustainable conservation in developing and developed countries needs to become the norm and address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the well-being of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the preventative sciences and medicine lead to sustainable cultural and cost-effective public health and economic changes of the future is never more evident than today. the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-, political/economic problems. the single profession that is best positioned to impact vaccinology is veterinary medicine. it's melding with human medicine and their role in future comparative and conservation-based programs will be critical to the successful application of vaccines into the st century. published by elsevier ltd. the new millennium did not bring the anticipated global internet technology shutdown however, it has brought with and heralded a time of significant change, opportunity and challenges. i and my co-authors goal in this overview are to celebrate, provocate, instigate innovate and activate those in society who are in interested to contributing to the betterment of human and animal health through vaccination. for vaccination to have its greatest chance of working policy-relevant science for sustainable conservation in developing countries needs to address poverty (including lack of basic health care) in communities affected by conservation, and to consider costs and benefits (perceived or not) affecting the wellbeing of all stakeholders, from the local to the multinational. the need to return to and/or develop new education-based models for turning the tide from the heavily return-on-investment therapeutic era of the last century into one where the investment into the pre-ventative sciences and medicine lead to sustainable cultural public health and economic changes of the future is never more evident than today. if the article gets the attention of researchers, educators/teachers, funders, policy makers, economists and the general public in both developed and developing countries to become involved in finding collaborative solutions to the conservation crisis than we will consider it a success. vaccination has and remains one of the most cost-effective and safest forms of medicine toward improving health today. along with basic understanding of germ theory and sanitation, vaccination, over the past years, has transformed lives in both rich and poor countries-resulting in the eradication of smallpox and huge reductions in the burden of previously common diseases such as polio, typhoid and measles. immunization is particularly well suited to all countries including those with weak health systems, because it requires relatively less training and equipment and does not depend on skilled diagnosis, long-term drug regimens or extensive medical care. immunization and sanitation remain as the most important public health modality responsible for improving the gnp of developing countries through additional gains in healthier children who are better educated and grow up to impact on their productivity. like schoolchildren, healthier workers have better attendance rates and are more energetic and mentally robust. workers in healthy communities, moreover, need to take less time off to care for sick relatives. body size, which is greatly influenced by one's health during childhood, has been found to have large impacts on long-term productivity. recent economists [ ] have calculated that a -year increase in life expectancy improves labor productivity by %. despite the weakness of health systems in many developing countries, three-quarters of the world's children now receive a standard package of childhood vaccines through the who/unicef expanded program on immunization to protect them against diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis [ ] . these vaccines currently save an estimated million lives a year -almost , lives a day -and protect millions more from illness and permanent disability, thus providing as mentioned above a healthier cohort of people to contribute to the economic development of the nation. the full package of basic vaccines (diphtheria, tetanus, pertussis, polio, measles and neonatal tuberculosis) costs less than $ per year of life saved in poor countries. "life-years" and "year of life" consistently refer to disability-adjusted life-years (dalys). interventions are generally considered extremely costeffective if the cost per year of life is less than $ . by comparison, antiretroviral treatment for hiv/aids-an intervention that donors widely support in the developing world costs up to five times as much at $ to $ per life-year saved; by way of comparison, in the us and the uk medical interventions are considered cost effective at $ , to $ , per life-year saved [ , [ ] [ ] [ ] . the week during the conference in amsterdam it was reported that "vaccine-preventable deaths reach new low in u.s." as reported in a federal report released tuesday, november , people readily associate the role of veterinarians with private veterinary practice focused on pets and farm animals, but the true dimensions and contributions of veterinary medicine are much broader and reflect expanding societal needs and contemporary challenges to animal and human health and to the environment [ ] . veterinary medicine has responsibilities in biomedical research; ecosystem management; public health; food and agricultural systems; and care of companion animals, wildlife, exotic animals, and food animals. the expanding role of veterinarians at cdc reflects an appreciation for this variety of contributions. veterinarians' educational background in basic biomedical and clinical sciences compare with that of physicians. however, unlike their counterparts in human medicine, veterinarians must be familiar with multiple species, and their training emphasizes comparative medicine. veterinarians are competent in preventive medicine, population health, parsitology, zoonoses, and epidemiology, which serve them well for careers in public health. the history and tradition of the profession always have focused on protecting and improving both animal health and human health [ ] . since , a total of diseases have been eliminated from equine, poultry, and livestock populations in the united states [ ] . the elimination of these livestock diseases, along with outstanding research in animal health, is key to the remarkable gains in the efficiency of u.s. animal production [ ] . partly as a consequence, u.s. residents spend only approximately % of their disposable income on food, whereas residents in other countries pay three or four times more [ ] . although this achievement is recognized to have added billions of dollars to other parts of the u.s. economy, its success in allowing the u.s. public access to a nutritious, affordable, and sustainable food supply -also important for the public's health and well-being -is far less appreciated. the success of the national brucellosis and tuberculosis elimination campaigns has benefited not only the u.s. livestock industries but also human health by substantially reducing these zoonotic threats in animals. additional public health contributions can be attributed to the food safety and inspection service of the u.s. department of agriculture (usda), which has substantially reduced the burden of food-borne illnesses, improved food safety, and eliminated other zoonotic threats. over the years, cdc has worked closely with usda and the food and drug administration to improve the safety of u.s. foods and reduce antimicrobial resistance in pathogens that infect both humans and animals. veterinary scientist and veterinarians within the health and human services serve in many critical capacities. veterinary officers in the commissioned corps work throughout the u.s. department of health and human services and in other federal agencies. most veterinary officers are assigned to the cdc, nih, fda, usda, epa, ogha, ndms and state department. other veterinarians and veterinary scientists function as medical research scientists post-doctoral nih/nci fellows, principal investigators, some specializing in lab animal medicine and providing critical lab animal health infrastructure and support, design of animal models for human disease in most of the hhs institutes. some are part of the hhs national disaster medical services and were deployed as the veterinary medical assistant teams (v-mats) and supported the search and rescue in the world trade disaster. additionally, since , the avma and the college of american pathologists have been working together to create a standard nomenclature that would allow veterinarians, physicians, and other medical professionals to create electronic medical records that use a common language. the systematized nomenclature of medicine, snomed, was initially created by the college for human medicine but has sincethrough the partnership with avma -expanded to include veterinary terms. on july , , the department of health and human services announced it would make snomed available nationally at no charge, a step toward instituting a standardized electronic medical records system. the hhs signed a -year, $ . million contract with the college to license snomed and make it available nationally. the national library of medicine is administering the program. prior to the hhs's agreement with the avma, practitioners in areas of practice nationally on the front line of surveillance would have had to pay a $ to $ annual registration fee to access snomed. the cdc has expanded the role, scope, and influence of veterinarians and veterinary scientists and epidemiologists in public health since its inception in [ ] . early in the history of cdc, veterinarians in the u.s. public health service and the cdc veterinary public health division helped reduce zoonotic diseases, especially rabies and food-borne illnesses [ ] . today, veterinarians serve throughout cdc in positions that address not only infectious diseases but also the entire spectrum of public health challenges: environmental health, chronic diseases, human immunodeficiency virus infection and acquired immunodeficiency syndrome, injuries, immunizations, laboratory animal medicine, global health, migration and quarantine, health education, and bioterrorism. veterinarians contribute as epidemiologists, laboratory scientists, policymakers, researchers, and surveillance experts and in environmental and disease prevention and control programs both domestically and globally. at cdc, veterinarians have participated in the epidemic intelligence service since [ ] . forty-one states now have state veterinary public health officials. in , almost students and faculty attended the first veterinary student day at cdc; in april , cdc will co-host an inaugural conference with the association of schools of public health and association of american veterinary medical colleges. in addition, cdc has been recognized as a world association for animal health collaborating center for emerging and re-emerging zoonoses. the cdc publication, emerging infectious diseases, has highlighted zoonotic diseases in nearly every issue to zoonotic diseases and has devoted an annual issue in each of the previous years. the cdc has provided an important scientific forum for zoonotic disease research and programs both domestically and globally and should serve as a template for the nih, as will be discussed later in this paper (section . ), for moving these highly trained and broad-based medical skill set professionals from a more decentralized setting into a central institute at the nih. benjamin franklin's famous quote, "an ounce of prevention is worth a pound of cure" was actually fire-fighting advice-he founded the first fire fighting organization in philadelphia, its obvious application to medicine, although obvious, has not been a mainstay of heavily invested research and development in human health practices. many of the leading causes of death and disability in the united states can be prevented [ ] . primary prevention can prevent or arrest the disease process in its earliest stages by promoting healthier lifestyles or immunizing against infectious disease [ ] . secondary prevention, by detecting and treating asymptomatic risk factors or early asymptomatic disease, can substantially reduce subsequent morbidity or mortality. the human and veterinary clinician plays a pivotal role in both primary and secondary prevention. health professionals deliver vaccinations, screen for modifiable risk factors such as high blood pressure and high cholesterol, counsel patients about smoking and other behavioral risk factors, provide screening tests for early detection of cancer and other chronic conditions, and advise patients about the benefits and risks of preventive therapies such as postmenopausal hormone replacement therapy. the preventative health care landscape has changed in some regards in the years since the u.s. preventive services task force (uspstf/task force) was first established in to provide advice about prevention for health professionals. prevention became more of an integral component of primary health care [ ] . delivery of clinical preventive services such as immunizations, mammograms, and cholesterol screening has risen steadily over the past two decades (national center for health statistics [ ]). roughly % of employers now include well-child visits, childhood immunizations, screening tests, and adult physical examinations among covered health benefits, compared to less than half that did so in [ ] . interest in prevention grew significantly among the public, clinicians, educators, employers, and policymakers [ ] and health plans and individual clinicians were increasingly being held more accountable for the quality of the preventive care they provide to their patients [ ] . at the close of the th century, health care costs in the united states continued to rise steadily, accounting for . % of the gross domestic product in [ ] , and debate on health care funding for the aging american population intensified. no doubt fueled by the incredibly imbalanced historic spending in preventative healthcare of cents for every cents spent for curative treatment [ ] . numbers are harder to come by for recent years, but given the spiraling costs of treatment since it is likely that this ratio has gone down considerably since then-possibly grossly estimated to be closer to : today. in this environment, preventive services often compete with one another and with diagnostic-and treatment-oriented care for increasingly constrained resources [ ] . while preventive services are often believed to save costs, delivery of most preventive services, with few exceptions (e.g. some immunizations), incurs net costs [ ] . evidence that us society clearly favors the cure (or treat) approach to disease over prevention can be shown in the following ways. first, though there is a shortage of preventive medicine specialists (public health, general preventive medicine, occupational medicine, and aerospace medicine physicians), in the us the number of residents in training in was less than . % of all residents, not sufficient for replacement or to fill the expanding demand for the specialty's skills and talents [ , ] . second, preventive medicine residencies and subspecialties in human and veterinary medicine are generally found in only graduate medical education programs not financed by cms or mainstream academic training programs. third, we believe that our preventive acts are only statistical, whereas our curative acts are certain. this mistaken belief perhaps derives from our sense that we have more control over cure outcomes than prevention outcomes-we think that we do cure, whereas we only facilitate prevention. this notion of doing vs. facilitating is an important one, because if we believe that our curative actions are more effective than our preventive ones then we will more likely act toward the more effective. the editor of the british medical journal, fiona godlee, expressed this well when she states, "because it is acted on healthy people, preventive medicine needs even stronger supporting evidence on benefits and harms than therapeutic interventions" [ ] . thus substantial gaps in the delivery of effective preventive care in the united states remained, however, because clinicians continued to face many of the same barriers that originally spurred the formation of the first uspstf. identifying effective interventions were and are difficult in prevention, where prospective controlled trials are often difficult to conduct. these studies come from the field of epidemiology which has changed remarkably during its growth in the past quarter century. one of those changes has been a mixed blessing of ever-increasing specialization among its practitioners at the cost of the generalist. this phenomenon has shaped the field and a partial explanation for this trend is found in the decline in the availability of training funds not focused on specific and general disease areas. without returning to the training of general conservation-medicine based epidemiologists, the needed trend associations and study designs that are needed to show the economic and public health returns related to preventative practices field will not be realized and in addition lose some of its ability to quickly respond to new and expected emerging public health challenges. conflicting recommendations from different organizations, further exacerbated by the advocacy positions of some groups, leave many clinicians uncertain about what to do. clinicians facing increasing time pressures in practice may question the value of some routine preventive interventions, as may employers and other payers struggling with accelerating health care costs. although more prevention information is reaching the public, the messages conveyed are often inconsistent and increasingly colored by commercial self-interest. clinicians may feel compelled to provide unproven or ineffective services because patients demand them or they fear being sued, but patients may find that insurance coverage for individual preventive services, especially new technologies, is inconsistent. the importance of clarifying what we know and do not know about the effectiveness of specific preventive services is as important in as it was in . although the uspstf was disbanded in with the release of the guide, the need to keep pace with the rapid growth in scientific evidence led to convening a second panel in . the second uspstf was smaller, with only members, eight of whom were primary care physicians. it refined the previous group's methods for reviewing evidence and making recommendations, and expanded the scope of topics. it adopted policies for disclosure of conflicts arising from financial interests, funding sources, or other affiliations. the work of the second uspstf was marked by strengthened ties with both federal and nongovernmental partners, including primary care subspecialty societies. the work of the second uspstf culminated in the publication of the second edition of the guide in , which covered over interventions in areas. by the time the second edition of the guide appeared, the environment for preventive medicine and evidence-based medicine had changed dramatically. managed care organizations, which had emerged as a dominant paradigm for delivering and paying for health care, included some preventive care among basic covered services more commonly than had traditional fee-for-service insurance. at the same time, the heightened competition spurred by managed care brought increased attention to costs and value of treatments with less attention given to prevention. the guide was frequently cited by health plans and systems of care in defending their health maintenance programs and benefits packages, and its recommendations informed many of the health plan employer data and information set (hedis) quality measures developed by the national committee on quality assurance for evaluating health plan performance but not integrated into cost saving practices by the insurance companies. the rapid progress towards universal vaccination coverage in the s and s has slowed in recent years. unicef funding for vaccination fell from $ million to $ . million between and [ , ] . global coverage of the diphtheria, tetanus, and pertussis (dtp ) vaccine has been at around % since [ ] . fifty-seven developing countries have yet to eliminate neonatal tetanus, and , babies died of the disease in . ten developing countries reported cases of polio in june , despite the massive (and largely successful) global effort to eradicate the virus [ , , ] . sixty-two percent of countries, meanwhile, had still not achieved full routine immunization coverage in , with gavi estimating that at least . million additional infants need to be reached to achieve full coverage. there are several factors behind this loss of momentum. although dramatic progress has been made in increasing worldwide vaccination coverage from below % to above %, the task has inevitably become harder now that the easiest-to-reach populations have been vaccinated. because these communities are more elusive, the average cost per vaccination has increased, and it may be that other apparently cheaper health interventions have become more attractive. there are many practical problems impeding vaccine delivery. delivering vaccines to patients requires functioning freezers and reliable transport to move the vaccines from port to clinic; clinics refrigerators (which in turn require a constant supply of energy); good roads and with access to people who need to be immunized; parents who know the value of vaccination; trained medical staff to deliver the dose; and sterile syringes. only % of vaccineimporting countries could guarantee vaccine safety and quality [ ] , while a further study of developing countries found that at least half of injections were unsafe [ , [ ] [ ] [ ] [ ] . the third factor behind the lack of progress in recent years is political. political disruptions have affected coverage in some areas. in somalia and congo, for example, where vaccination rates have fallen rapidly in the past decade, war and social breakdown have impeded public health campaigns, despite "vaccination days" in congo that temporarily halted fighting. gauri et al. have found that the quality of institutions and governance are positively correlated with vaccination coverage [ , ] . politics in the developed world have also played a part. according to a report by the us institute of medicine, in the us vaccine industry was forced to stop offering low-price vaccines to develop-ing countries following congressional hearings that "savaged" the industry for "allegedly subsidizing vaccines for the poor children of the world by charging high costs to us families and taxpayers". as the institute of medicine points out, this move was based on a flawed premise, as the us vaccines would have been developed anyway to protect american children and travelers. public perceptions of vaccination change-as coverage spreads through a community and it reaches a point at which those who are unvaccinated are highly unlikely to catch a disease because herd immunity has set in. at this juncture, it may be more rational for an individual to refuse vaccination in order to avoid any risk of side effects. with the oral polio vaccine, for example, there is a one in a million chance of paralysis, and in societies where mass vaccination has eliminated the disease, the risk of paralysis is greater than that of catching polio itself. what had once been a public and private good is now a public good but a private risk. as more and more people choose to avoid this risk, of course, overall coverage rates decline, and the community is once again exposed to the threat of the disease. public perceptions have been influenced by vaccine scares. controversy and the attendant bad publicity about the safety of vaccines have been abetted by incidents such as the withdrawal of half the us supply of flu vaccines in due to contamination at the manufacturer [ ] . in addition, alarms over the safety of vaccines such as that for measles, mumps and rubella (mmr), which some believe to cause autism, have further fanned the anti-vaccine movement's flames [ ] . in the us, disputes continue to rage about the scientific basis of such claims, but the preponderance of the evidence, according to the us centers for disease control (cdc), says that the mmr vaccine is safe [ ] . in response to these types of controversies in the us, the institute of medicine has called for independent oversight of vaccine safety studies to ensure the fairness and openness of the vaccine safety datalink program, which is overseen by the cdc. as one can see there are many complex factors that have to be considered when bringing vaccination programs into existence. the impact of vaccination of animal diseases on agriculture is typically assessed in quantitative terms-lost revenues; costs of eradication, decontamination, and restocking; and the numbers of affected farms, animals and humans. this approach can be applied universally to all outbreaks in all countries because it normally reflects the hard data supplied by large commercial operations and the estimates by relevant governmental agencies of small farmer impact [ ] . when used exclusively, however, it fails as a barometer, because it does not and cannot factor in the multi-dimensional character of major disease events-and the accompanying societal effects that often get lost when it comes to assessing the damage in developing countries. the quantitative approach must also be interpreted, and cannot be used "as is" for comparing impacts in developed and developing countries. further, while export trade losses in a developing country may be small in terms of the dollar amount, the impact upon its pre-epidemic market share is inevitably greater and more persistent. other impacts such as effects on human health and community stability tend to be more visible and last longer in developing countries, particularly at the village level where animal are husbanded primarily for the benefit of the immediate family, and often in impoverished circumstances [ ] . the consequences of animal diseases in domesticated birds and livestock can be complex and generally go well beyond the immediate effects on affected producers. these diseases have numerous impacts, including: • productivity losses for the livestock sector (e.g. production losses, cost of treatment, market disturbances). • loss of income from activities using animal resources (in such sectors as agriculture; energy; transportation; tourism). • loss of well-being of human beings (morbidity and even mortality rates; food safety and quality). • prevention or control costs (production costs; public expenditure). • suboptimal use of production potential (animal species, genetics, livestock practices). the most direct economic impact of animal diseases is loss of production and/or productivity, and ensuing income losses for farmers [ ] . however, if the economy depends on one or some of the vulnerable products, the impacts can be serious, and local food security can be threatened. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. if the farm economy is diversified or if there are other opportunities to generate income, the impacts can be mitigated. the economic impact also depends on response strategies adopted by farmers and possible market adjustments. the loss of the farmer's "well-being" will generally be lower than the value of the lost product, except where the farmer has few alternatives or is wholly dependent on the affected product, which is quite often the case in developing countries. direct losses are the result of the disease itself (they may be very high when mortality rates are between and %), or from animal health measures (stampingout policies) [ , ] . in vietnam, one of the countries most seriously affected by the avian flu, almost million birds - % of the country's poultry population -had to be destroyed at an estimated cost of us $ million ( . % of gnp) [ ] . the smaller scale producers lost the least in absolute terms, but the most in relative terms, as the outbreak resulted in losses equivalent to upwards of times their daily income (from us $ a day or less). in africa, abortions caused by the rift valley fever virus not only affect birth rates, but also push human consumption of milk downward in the year following an outbreak [ ] . in the dairy farming sector in kenya, it is estimated that losses in milk production accounted for % of all losses caused by an outbreak of foot-and-mouth disease in the s. direct costs are generally well below the indirect costs of animal diseases and are directly linked to the rapid containment of outbreaks: case studies have shown that early detection and the implementation of appropriate measures in the event of an outbreak are essential to help minimize direct losses as much as possible. conversely, inappropriate control and eradication measures are at the root of such endemic situations, which are much more difficult, and infinitely more costly, to keep under control or eradicate. the livestock sector plays a significant role in the economic development of many countries and vaccination can serve as one of the most important means of assuring its health. as such the cost of not developing new and important or properly applied vaccines can have tremendous economic consequences. the production of meat and other animal-based food items generates income, jobs, and foreign exchange for all stakeholders in the animal industries. consequently, an epizootic which could have been otherwise mitigated by vaccination can affect the industry's upstream (inputs, genetic resources) and downstream activities (slaughterhouses, butchering operations, processing, marketing) in terms of jobs, income for the stakeholders in the industry, or market access. a survey by the food and agriculture organization of the united nations (fa ) on avian flu revealed that in the most seriously affected regions of indonesia, % of permanent workers at industrial or commercial farms lost their jobs [ ] . similarly, an outbreak of contagious bovine pleuropneumonia in botswana led to the destruction of more than , animals in the most seriously affected province, and the immediate closure of the export slaughterhouse, which employed persons. owing to the catalyst role of livestock raising in the rural economy as a whole, the costs of the indirect effects of these measures were later estimated to be seven times higher than the costs caused by direct losses [ ] . in vietnam, % of the poorest segment of the population, for which poultry farming accounts for - % of household income, is particularly vulnerable to income losses caused by avian flu. the fao and world organization for animal health (oie) estimate that between one-third and one-half of the populations living in the most seriously affected areas of southeast asia depend on poultry farming for at least a part of their income [ , ] . in france, the leading european poultry producer, it is estimated that farmers affected by the crisis lost % of their income in months (between january and march ). the effects of the production losses are also linked to price variations, which are caused by supply and demand (im)balances. depending on the market, prices can rise sharply (consumer product on the domestic market) or plummet (product banned for export but cleared for consumption on the domestic market, product deemed too dangerous for human consumption or perceived as such). in brazil, where % of products are exported, the price of a day-old chick, an early indicator of a possible change in production, reportedly fell by %. and even in cases where the country is not infected, market uncertainties and the fall in prices prompted the largest producers to cut back production by % this year. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. in / , the rift valley fever outbreaks in east africa seriously affected pastoral economies in somalia, with a decline of more than % in exports (which generate more than % of foreign exchange in "somali land"), following an embargo declared by saudi arabia on all animal products from the horn of africa [ , ] . conversely, the world bank has reported that eradication of certain major diseases to facilitate access to "high value" export markets can provide considerable benefits. loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. uruguay is a good example of a country that gained access to a lucrative market after eradicating foot-and-mouth disease. beef exports increased in volume by more than % and in value by % after the oie declared uruguay to be officially foot-and-mouth disease-free without vaccination in . access to the u.s. market (where prices are double those of the domestic market) provides uruguay with additional revenue to the tune of us $ million each year. a medium-term analysis showed that access to "pacific rim" markets would generate additional revenue of us $ million each year, and yet, before the disease was eradicated, uruguay had been spending (only) us$ million to us $ million each year on vaccines to combat foot-and-mouth disease. in this case, control costs would account for less than % of the revenue generated by exports alone [ ] . animal diseases that could be controlled by vaccination can have major effects on food availability and quality for poor communities. it is well known that agriculture plays an important role in the generation of income and jobs in other sectors but the closeness of this interdependence became particularly obvious during recent epizootics. for pastoral societies, animal husbandry contributes directly and indirectly to food security and to nutrition as a source of quality proteins, vitamins and trace elements, traction, and com-mercially tradable products [ , ] . certain diseases could have significant repercussions on food supply and the nutrition of poor communities that do not have readily available substitute products, which could therefore lead to famine (rinderpest for example). poultry meat is the primary animal protein in africa (which has little to begin with) and the indispensable source of discretionary income for the survival of millions of small farmers. the high mortality rates as a result of avian flu, which is extremely pathogenic, and the sanitary slaughter of poultry would therefore have a negative impact on the food available to the entire population, as well as on rural revenue. furthermore, developing or transition countries which generally have poor public health systems are particularly at risk from zoonoses making vaccination against these diseases particularly important to target. in / a major rift valley fever epidemic in egypt resulted in , human cases and fatalities [ , ] . twenty years later, a new epidemic affected over , persons in east africa, and persons succumbed to the hemorrhagic form of the disease. but zoonoses also affected industrialized countries with high health standards as was the case with the bovine spongiform encephalopathy crisis in europe [ ] . food-borne diseases (over have been classified) are a major source of acute gastroenteritis (which costs the netherlands us $ million per year) and the cause of major morbidity with fatalities among children in the third world [ ] . in the specific case of a pandemic, most of the economic loss is caused by the increase in morbidities and fatalities in the human population and its repercussions on the world economy. the most recent estimates suggest that the "spanish" influenza in caused the death of million persons, that is, . % of the population at the time. the most obvious economic losses were the reduction in quantity and productivity of the workforce, and according to the experts, in the case of a pandemic could represent times more than all the other losses combined [ ] . another category of economic impact is linked to individual strategies to avoid contamination-or to survive possible contamination. the example of the severe acute respiratory syndrome (sars) clearly shows the sharp drop in demand in the services sector (tourism, public transport, retail trade, hospitality and food services) resulting from the combined efforts of individuals to avoid any close contact [ ] . based on the experience with severe acute respiratory syndrome in south-east asia, the world bank thinks that an avian flu pandemic could result in a % loss of the world's gross domestic product and cost the world economy us $ billion in the space of year. the losses are difficult to calculate and would undoubtedly be much more significant in light of the extremely high mortality rates in developing countries which do not have good health care systems. the impact of animal diseases on the tourism and leisure sectors could also be quite significant. the negative effect of foot-and-mouth disease in the united kingdom on these two sectors amounted to us $ billion because of restrictions on access to rural areas and represented more than half of the total cost of the disease [ ] . the federation of american scientists' animal health/emerging animal diseases (ahead) project proposed a major program in sub-saharan africa to detect and document the extent of infectious diseases shared by farm and wild animals, and to supply treatment, prevention and control services to remote communities that have previously been neglected by other programs, both national and international. this program, international lookout for infectious animal disease (iliad), was implemented in south africa [ ] . at the core of iliad is the need for a permanent and sustainable regional program of in situ surveillance designed to detect, monitor, treat, prevent and control infectious diseases with the goals of increasing livestock production in remote farming communities, protecting the health of wild species, building indigenous physical and professional resources, and introducing communications and epidemiology information technologies. transmission of infectious diseases is rampant in remote communities in the sub-saharan region, just as they once were in the united states and as they always are wherever poverty and farming co-exist. diseases shared by wild, farmed and captive/bred animals, and by animals and humans, suppress food production, frustrate species preservation efforts and greatly affect public health. detection, prevention and control of these diseases are an essential element in expanding trade, improving nutrition, exploiting ecotourism and ensuring food security. iliad is structured in the investor mode-an international consortium of donor groups providing short-term developmental assistance with program direction and oversight provided by veterinary diagnostic, public policy and epidemiology experts representing the sub-saharan africa partnership members-the renowned onderstepoort veterinary and exotic disease institutes (ovi) and tuskegee university (tu), and fas-ahead. given positive assessments of the benefits of the program after years, national or provincial institutions will integrate some or all of the activities into their official veterinary and agricultural activities. it is difficult to calculate the cost of the public's loss of confidence in animal industries in their countries, or of an importer country towards the veterinary services of the exporter country. animal diseases can have major effects on food availability and quality for poor communities. consumers' obsessive fear of bovine spongiform encephalopathy (mad cow disease), fed by the media and which a good communication strategy could have prevented, would have tremendous social repercussions on a europe still reeling from long term economic repercussions. in italy, the baseless perception of a food risk related to avian flu coupled with low confidence in public health services eventually resulted in a % reduction in the consumption of poultry and eggs. the loss of confidence by an importer country can trigger a lasting embargo and major economic and social repercussions (arabian peninsula embargo on the horn of africa, affected by the rift valley fever virus). loss of access to, or the opportunity to access, regional and international markets generally have more significant economic implications than just production losses. animal diseases might also have indirect long-term impacts, affecting deferred productivity. this is the case for example of the reduction in the fertility rate of long-cycle species, the effects of which span periods of - years [ ] . in short, the long-term costs of a slow response are rarely taken into account. economic analyses focus primarily on the effects of the outbreaks and rarely take into account the long-term effects of an endemic situation (characterized by less virulent outbreaks which recur for several years). this is the case of classic swine fever in haiti where recurrent outbreaks reduced the usage rate by %, which for pig farmers meant a loss of revenue of us $ . million per year [ ] [ ] [ ] . with major crisis, long-term impacts would make themselves felt, since the additional costs of financing prevention and control measures would lead to an equivalent reduction in savings and investments. for example, the analysis of the global impact of the avian flu crisis in europe was complicated by outbreaks of foot-and-mouth disease in brazil, the largest global exporter of beef and poultry. it is therefore easy to imagine what the combination of these two events would mean in terms of the upward push of prices of all meats, similar to what occurred in with north american beef and bovine spongiform encephalopathy. the european union, a net importer of beef, especially from brazil, would see an increase in the price of beef in its internal markets stemming from the embargo imposed on brazilian beef because of the foot-and-mouth disease. it must be pointed out that the crises could have a cumulative impact, particularly since they are amplified by the effects of globalization the following example therefore illustrates the ripple, spillover and remote effects: in the united states, where % of oleaginous and cereal production is geared towards animal production. an epizootic which reduces animal production by % would have the immediate consequence of the loss of , jobs, a surplus of . ton in cereals and oleaginous products, a % reduction in world trade and, crises in other producing countries. in , nearly americans out of every , died each year of infectious disease. laurie garrett, author of "the coming plague: newly emerging diseases in a world out of balance", writes that in the postwar environment, powerful medical weaponry (antibiotics, vaccines, water treatment, anti-malaria drugs) gave scientists confidence that they could significantly control and/or eradicate infectious disease from viral, bacterial or parasitical sources. in the late s, the surgeon general of the usa, william h. stewart, said that ". . .it was time to close the book on infectious diseases and pay more attention to chronic ailments such as cancer and heart disease." a measure of that success came towards the end of the s, when the world realized that smallpox had become the first disease to be eradicated from the human species. such halcyon days from the s to the early s are but a memory. by , the numbers were down to per , . the "health for all" accord, signed in , set a goal of the year for eliminating many international scourges. but amid all this optimism, the numbers started rising. in , people per , died and we know the rest of the story, . . . or do we? the grandiose optimism rested on two false assumptions; that microbes were biologically stationary targets, that for the most part human and other animal diseases were for the most part limited to those species and geographically sequestered. scientists have witnessed an alarming mechanism of microbial adaptation and change, anything but stationary, microbes and the insects, rodents and other animals that transmit them are in a constant state of biological, ecological flux and evolution [ ] . according to the u.k. centre for tropical veterinary medicine, - % of all the known species of infectious organisms that affect human health (causing a quarter of the world's deaths) can be transmitted by animals. approximately of these infectious organisms are linked to diseases that have only recently emerged, or have increased in severity (and geographic distribution) in recent years. who averages outbreak investigations every year, and around will require an international response. more than new and highly infectious diseases have been identified in the last years. furthermore, known strains of diseases such as tuberculosis, many species of gram positive and negative bacteria as well as many parasites, e.g. malaria, food animal coccidia have developed resistance to various classes of antibiotics, while old diseases have reappeared, such as cholera (in angola, with deaths), yellow fever (new cases recently reported in guinea, sudan, mali, and senegal), plague, dengue fever, meningitis, hemorrhagic fever, measles, mumps, rubella and diphtheria. there are emerging diseases just among marine life, reports the book conservation medicine, and these include tuberculosis in fur seals and chlamydiosis in sea turtles. more staggering than these cited numbers in living animals including humans are those coming out of the microbial genetic sequencing and diversity studies involving the oceans. recent data in this field suggest that the oceans of the world contains approximately ( ) phage particles or virions (cf. million metric tons), much of it turning over once per day and most likely be a regular source for current and future zoonotic and human infections. this vast mutation engine, even if one assumes a minimal mutation rate, generates the equivalent of hundreds of new complete human genomes per day. a -l sample of surface seawater was concentrated; ∼ × ( ) viral particles, the dna once randomly sheared and cloned yielded , fragments for sequencing. data analysis showed that most of the sequences were from previously unknown viruses. approximately . % of the total sequence samples overlapped, suggesting that the marine viral community was highly diverse. a unique mathematical analysis further suggested that approximately ( ) different new viral types may be present. it is obvious from this most recent description that complex and confounding zoonotic interactions can be expected to occur as ecosystems become concentrated and/or diluted during the upcoming environmental and ecological change. this will require a new breed of medical scientists that have foregone the days of super-and sub-specialization and are now grounded in both depth and broad general eco-and bio-medical systems training. few people recognize the broad range of clinical and basic veterinary research and its many important contributions to society in the realms of public health and food safety, vaccination, fertility, drug and vaccine development, surgical techniques and biodegradable materials, space medicine, animal health and welfare, and comparative medicine [ ] . opportunities in veterinary research include comparative studies with animals that shed light on human health problems; the development of tools to better detect, prevent, and control zoonotic diseases (that spread from animals to humans); the establishment of scientifically based policies for the humane treatment of animals; and the development of measures to secure and protect the nation's food supply and farm-animal economy from a potential act of bioterrorism [ , ] . the new complex problems of the new millennium will require new educational models that train para-and professional people for thinking and solving complex inter-related biological, ecological, public-economic problems. the single profession that is most centered on the new paradigm is veterinary medicine. the three major disciplines within veterinary medicine and research -public health, comparative clinical and basic medicine, and animal health -are closely intertwined [ , ] . for example, research in comparative medicine contributes to animal health through the development of preventive medicine and treatment. the study of wildlife diseases contributes not only to wildlife health and conservation, but also to public health because many animal diseases can spread to humans. therefore, collaborative and interdisciplinary research is crucial in translating scientific advances from one traditional discipline to another. however, interdisciplinary research is in many cases hampered by administrative, funding, and cultural barriers between institutions. furthermore, agencies that support veterinary research have specific missions. funding to support proposed interdisciplinary research can be difficult to obtain when it is partially related to the mission of several agencies but does not perfectly fit the mission of any one agency. the future requires the veterinary research community to encourage research funders to develop a long-term interagency strategy for veterinary research [ , , ] . in , rudolph virchow, the father of comparative medicine, stated, "between animal and human medicine there are no dividing lines-nor should there be. the object is different but the experience obtained constitutes the basis of all medicine" [ ] . sir william osler ( - ), considered the best-known physician in the english-speaking world at the turn of the century, called the "most influential physician in history" is quoted as saying "veterinary medicine and human medicine complement each other and should be considered as one medicine." dr. calvin schwabe, a retired uc davis professor of veterinary medicine who pioneered the use of human disease tracking techniques in the study of animal illnesses, a global authority on animal diseases that are communicable to human beings and was an early visionary in a field that today is marked by the emergence of pathogens such as avian influenza, mad cow disease and sars. in , dr. schwabe established the department of epidemiology and preventive medicine at the uc davis school of veterinary medicine-the first of its kind in the world at a vet school. the author of more than publications, he promoted the concept of "one medicine," which attempts to bring the fields of human and animal health care together. to this goal in june of the american veterinary medical association (avma) announced that in partnership with the american medical association (ama) there was an adopted resolution calling for collaboration on a one health initiative. the two national, medical organizations will work collaboratively on areas of mutual medical interest, such as pandemic influenza, bioterrorism risks, biomedical research and will be charged with developing strategies to promote collaboration among the various health science associations, colleges, government agencies and industries. a quote from a ama board member, duane m. cady, md "new infections continue to emerge and with threats of cross-species disease transmission and pandemic in our global health environment, the time has come for the human and veterinary medical professions to work closer together for the greater protection of the public health in the st century," the avma policies supporting the concept of "one health" include: furthermore to strengthen this initiative and give it a renewable funding foundation, it would be recommended to move toward, the creation of: ( ) a specific focus at the national institutes of health (nih) on integrated veterinary research via the roadmap initiativemore specifically to create or combine existing potions of institutes into an institute of comparative medicine (icm); ( ) a joint interagency collaborative programs could also be established to enhance interdisciplinary collaborative research and have either re-routed an/or new congressionally supported intra-mural and extra-mural funding program; and ( ) while working out and introducing the legislative changes for the icm the nih should create a veterinary liaison with all the current institutes like the veterinary-medicine and public-health liaison at the centers for disease control and prevention (cdc) in which a veterinarian, dr. lonnie j. king, was selected to head up the agency's national center for zoonotic, vector-borne, and enteric diseases-a relatively recent creation, the center is dedicated to understanding infectious disease ecology and will help to ensure integration of veterinary and human medical research. the american veterinary medical association, with information provided by many other organizations and institutions, conservatively estimates a current deficit of public health veterinarians (e.g. usda food safety and animal disease control, homeland security, research on domestic and foreign animal diseases, wildlife disease control, laboratory animal care and research) and is expected to increase possibly to , by as the human population increases without intervention. comparably the health resources and services administration (hrsa) in the u.s. department of health and human services (dhhs) released a report in , projecting a shortfall of approximately , physicians in [ ] . if current trends continue, the full time equivalent (fte) physician supply is projected to grow to , by , while demand for physicians will increase to , due to the growth and aging of the u.s. population (physician workforce policy guidelines, ). the report projects shortages will be in greatest in non-primary care specialties. it has been over years since the federal government has allocated funds to increase the number of veterinarians and physicians that graduate each year. to begin to alleviate this problem the veterinary workforce expansion act (s. /h.r. ), which is currently being considered by the united states house of representatives and senate would provide an average of $ million per year for the next years in the form of competitive grants to help increase the number of veterinarians entering public practice. if passed and funded, this bill would allow the nation's veterinary schools and other institutions training public health veterinarians to apply for competitive grants to increase capacity in the form of classrooms, teaching laboratories, research facilities, and administrative space. this bill would be vital to the nation's ability to protect human and animal health, as veterinarians are often the first line of defense for both. the text of the bill can be found at: http://thomas.loc.gov by searching by the bill numbers listed above. a list of co-sponsors to the bill can also be found via that site by selecting the link entitled "bill summary & status". over the past century, humanity has had a devastating impact on the earth's wildlife and ecosystems. we are in fact living through the largest mass extinction since the end of the dinosaurs million years ago. unless effective solutions are found, this new century will see the demise of countless more species and pristine ecosystems, particularly in the tropics. the global society, and what surrounds and influences it, are in profound change. these changes will have very significant impacts on future veterinary medicine and veterinary medical education. there are major demographic, political, environmental, disease, technological, and economic influences, all forcing changes onto society. a few examples illustrate the point [ ] . • with immigration into north america accelerating, combined with a declining birth rate, the ethnic diversity in society will continue to increase, with the associated impact on values. • in , for the first time in history, urban people will outnumber rural people. • political destabilization, inflamed by bio-terrorism and religious fanaticism, is expected to increase. • changes in the atmosphere are causing powerful shifts in the environment (melting of the ice caps, rising sea levels) and in the climate (hurricanes, flooding). • global water shortages, especially in heavily populated areas, will soon approach critical levels. to information and service (http://www.jvmeonline.org/cgi/ content/full/ / / #b #b ). • consumer spending power in emerging economies will go from $ trillion to $ trillion by , but the gap between rich and poor is increasing. how will these changes alter the needs of society? how must academic veterinary medicine adapt to prepare veterinarians to respond to these new needs? clearly, humanity has yet to find a way to live on planet earth in a potentially sustaining manner where by stabilizing flora and fauna remain intact to promote healthy ecosystems diversity for all species including our very ownhumans. for example, it is estimated that the equivalent of six earths would be needed to sustain the current world's population if people everywhere consumed natural resources at the rate we do in the united states. it is interesting to think that vaccination directly or indirectly impacts six of the seven united nations millennium goals. understanding and coping effectively with an emerging crisis may sometimes require the birth of action-oriented "crisis disciplines." conservation medicine: ecological health in practice brings together an impressive group of experts from diverse specialties medicine, veterinary science, conservation biology, epidemiology, parasitology, public health, and others) to examine the links among human health, wildlife health, and ecosystem health and begin to address questions like: • how factors such as climate change, endocrine disruptors, and toxic microalgae affect wildlife and human health; • the importance of biodiversity for human health (as medical models, sources of medicines, factors in the ecology of infectious diseases, and indicators of environmental quality), with a review of biodiversity-related biomedical research projects funded by the national institutes of health from to ; • how the health of rainforest-dwelling peoples depends on such diverse factors as forest integrity, floods, seasonality, community organization, education, gender dynamics, national budgets, and global markets; • how wildlife health relates to environmental security; • the health hazards of ecotourism; • the causes and impacts of emerging infectious diseases of humans and wildlife; • how the health of terrestrial and marine animals and ecosystems are monitored, and descriptions of innovations using stool dna and retrovirus evolution as markers of animal population dynamics, stool hormones to indicate species stress, and animal behaviors as proxies for the health of ecosystems; • how habitat fragmentation and reduced biodiversity can increase the risk of lyme disease infection; • how land use changes such as deforestation and water projects influence the ecology of malaria and other vector-borne infections; • how ecological health and wildlife disease are managed in national parks; • the role of zoos in the recovery and conservation of endangered species; • how reducing the burden of infectious disease among park workers in africa could prevent a devastating epidemic among the world's remaining mountain gorillas; • how efforts to control livestock diseases are affecting wildlife health and ecosystems in botswana; • teaching ecosystem health in an undergraduate medical curriculum. more than one billion poor people in asia and africa are closely linked with animals for their livelihoods and they pay a really high tribute to different animal diseases-one can easily demonstrate that improving animal-health mechanisms at the national and local level, and decreasing this weight of animal diseases, will lead very quickly to the alleviation of poverty for this class of people. the world organization of animal health (oie) brings together chief veterinary officers from countries in an effort to create global standards of animal health and animal welfare robust enough to withstand the daunting challenges of worldwide commerce. with an annual budget of around $ million, it is a significantly smaller organization that its human-health counterpart, the world health organization, which has some $ billion a year expenditure on its programs. last year, over billion food animals were produced worldwide to help feed a population of billion people, resulting in trillions of pounds of animal products distributed worldwide and projections for show that demand for animal products will increase by %, especially in developing countries. this poses unprecedented risks for human safety and will require a closer linking of the two agencies. along with those core concerns, oie also addresses farming practices, analyzes import risks, and mediates bilateral trade disputes among its member countries. among its most important achievements in recent years, has been the eradication of rhinderpest, a centuries-old intestinal disease also known as "cattle plague in some countires." in the early s, rhinderpest wiped out upwards of $ million worth of african livestock, contributing to widespread human famine. in general professional students of veterinary and human medicine are exposed more likely to a more scientifically exposed than a politically exposed culture. animal and human health policies must be science-based the rallying cry that is heard from animal and human health professionals around the world. the second verse of this mantra is often 'science, not politics', as if science is unquestionably 'good' and politics 'evil'. antipathy toward politics is worn as a badge of honor. the crowning moment frequently comes with the proclamation, 'i am a scientist, i want nothing to do with politics. ' the phrase 'science-based' infers that the underlying justification of animal/human health policy is derived from knowledge gathered through the systematic observation of, or experimentation with, phenomena. scientific knowledge implies the compilation and analysis of data by individuals with advanced education in specific disciplines. scientists seek facts, the fundamental truths which explain the world around us. at face value, the 'science, not politics' paradigm has a great deal of appeal. however, the very notion of fundamental truth is illusory, as scientific knowledge changes frequently with new observations and experiments. furthermore, conjecture and refutation characterize the scientific method, with disagreement and debate the recognized features of scholarly pursuit. scientists often reach conflicting interpretations of observational and experimental data. consequently, individual scientists may champion different, even diametrically opposed, sets of ideas and principles, so that any number of alternatives may be justified as 'science-based'. finally, animal health professionals typically consider only the biological and physical sciences as 'true sciences', dismissing the social sciences. politics reflect the human need for organization of authority, whether in public or private life. politics exist whenever two or more people come together. the terms 'office politics' and 'family politics' are recognized as clearly as the collective activities surrounding local or national governance. politics exist even in science, affecting scientific organizations, refereed publications and academe. indeed, politics are inescapable. all public courses of animal/human health action adopted by governments emerge from the interplay of science and politics. the policy-making process is governed by rules and regulations, affected by the organizational culture of the government agencies involved, and constrained by legal authorities, political correctness and resource availability. the animal/human health policy-making process involves consideration of current biological and physical scientific knowledge. policy decisions also consider social science factors including ideologies, economics and public opinion. hence the etiology of animal/human health policy is multifactorial. the current older traditional schools of veterinary and human medicine and future schools of "one medicine" will need to include leadership, economics and local, state, national and international governances courses and better training in mechanisms of public policies and rule making. epidemiologists accept the concept of multifactorial etiology as a basic tenet. we discuss disease in terms of agent, host and environment interactions. the practice of applied epidemiology demands a breadth of knowledge and the ability to work in interdisciplinary teams. the more complex the problem, the greater the demand for additional knowledge and insights. veterinary and human epidemiologists study risk factors for disease in animal/human populations and develop strategies for health promotion and disease control [ ] . unfortunately these are done frequently in a vacuum when in fact the two are inter-related and contributing to the observed and at the time undiagnosed disease spectrum however, animal/human health problems cannot be resolved by consideration of biological and physical factors alone. the veterinary and human (someday to become one and the same) epidemiologist working with field problems soon recognizes the critical role played by people in animal/human health issues. applying epidemiological principles to animal/human disease prevention requires consideration of social issues such as attitudes toward animals, cultural and religious mores, and individuals' willingness and capability to implement the prevention strategies. implementing prevention on a national scale brings additional factors into the equation such as availability of resources, adequacy of veterinary services, and animal health infrastructure, among others. therefore, animal health policy development and imple-mentation require attention to macroepidemiology, the study of all of the economic, social and political inputs which affect the distribution and impact of animal or human disease at the national level [ , ] . the low hanging fruit of yesterdays fields of microbiology, zoonotic and emerging infectious diseases, immunology, antibiotic sensitivity, vaccine development, oncology, anti-viral drugs, public health, ecology, environment, human and animal health, to name a few have been picked. today comparative and interdisciplinary research is critical to translating scientific advances from one discipline or species to another and providing new insights into human health problems. scientific fields such as laboratory animal medicine, pathology, immunology, biophysics, mathematics, bioinformatics, genetics, molecular biology and toxicology, when combined with veterinary medicine, have proven especially relevant to success in biomedical research. veterinarians also have contributed to public health through the care of companion animals. fifty-seven percent of all u.s. households own a dog, cat, or both. in addition, millions of exotic animals, birds, and reptiles are kept as pets [ ] . although pets enrich the lives of humans, they also potentially can threaten public health. veterinarians help educate the public about prevention of zoonoses; vaccinate large numbers of pets for zoonotic diseases, such as rabies and leptospirosis; and reduce the level of ecoparasites that can transmit human diseases and intestinal worms, such as roundworms and hookworms, which can cause serious health problems in humans. the , private-practice veterinarians in the united states form a valuable front line for detecting adverse health events, reducing zoonotic diseases, and delivering public health education. because veterinarians work at the interface of human, animal, and environmental health, they are uniquely positioned to view this dynamic through the lens of public health impact. significant changes in land use, expansion of large and intensified animal-production units, and microbial and chemical pollution of land and water sources have created new threats to the health of both animals and humans [ ] . because animals share human environment, food, and water, they are effective sentinels for environmental, human, and public health problems, including bioterrorism. concerns are increasing about antimicrobial resistance of pathogens, waste and nutrient management, and potential runoffs into streams, rivers, and oceans. food animal and wildlife populations are inextricably linked to some environmental problems. together these have led to creation of a new scientific discipline called conservation medicine and ecosystem health, and veterinarians are assuming a leadership role in the field [ ] . several decades ago, special factors came together to create a new epidemiologic era characterized by increases in emerging and reemerging zoonoses [ ] . humans, animals, and animal products now move rapidly around the world, and pathogens are adapting, finding new niches, and jumping across species into new hosts. in , approximately billion food animals were produced to help feed a world population of . billion persons; the united nations' food and agriculture organization estimates that demand for animal protein will increase by % by , especially in developing countries [ ] . the lessons learned from severe acute respiratory syndrome, west nile virus, monkeypox, and avian influenza are reminders of the need to view diseases globally; integrate animal and public health surveillance, epidemiology, and laboratory systems; and create new strategic partnerships among animal, human, and public health professions [ , ] . veterinarians are essential to the detection and diagnosis of and response to these threats and are integral to first-line defense and surveillance for bioterrorism agents. the convergence of human and animal health drove creation of the newly proposed national center for zoonotic, vector-borne, and enteric diseases. plans are being completed to establish several multidisciplinary state-level zoonosis research and development centers. the veterinary profession has recently gained a important foothold on the health and human services government research institutes at the cdc and evolved in prominence as a member of the health professions and has established its importance and usefulness to human and public health. it is hoped and expected that with the developing visions and challenges outlined in this overview we will see the continued melding of veterinary and human medicine to create new educational programs and tools for developing future leaders for solving globally some of the most challenging public and animal health, ecosystem, and conservation problems of the st century. the 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and monitoring systems for animal health programs and disease surveys committee on assessing the nation's framework for addressing animal diseases, national research council. animal health at the crossroads: preventing, detecting, and diagnosing animal diseases. washington sustainable agriculture solutions (sas)-action report the annual report the annual report understanding and improving health and objectives for improving health. vols an agenda for action: veterinary medicine's crucial role in public health and biodefense and the obligation of academic veterinary medicine to respond investing in health immunization at a glance immunization at a glance world development indicators polio eradication website, global case count measuring the national economic benefits of reducing livestock mortality the primary author wishes to acknowledge and dedicate this paper to the late dr.'s jonas salk and maurice hilleman whose lifetime experiences and contributions are unprecedented in medicine and vaccinology and whom personally encouraged and instilled in me during numerous public and private discussions and meetings the importance of working in and helping to advance the field of vaccinology for the betterment of global human and animal health. key: cord- -q if li authors: simpson, ryan b.; zhou, bingjie; alarcon falconi, tania m.; naumova, elena n. title: an analecta of visualizations for foodborne illness trends and seasonality date: - - journal: sci data doi: . /s - - -x sha: doc_id: cord_uid: q if li disease surveillance systems worldwide face increasing pressure to maintain and distribute data in usable formats supplemented with effective visualizations to enable actionable policy and programming responses. annual reports and interactive portals provide access to surveillance data and visualizations depicting temporal trends and seasonal patterns of diseases. analyses and visuals are typically limited to reporting the annual time series and the month with the highest number of cases per year. yet, detecting potential disease outbreaks and supporting public health interventions requires detailed spatiotemporal comparisons to characterize spatiotemporal patterns of illness across diseases and locations. the centers for disease control and prevention’s (cdc) foodnet fast provides population-based foodborne-disease surveillance records and visualizations for select counties across the us. we offer suggestions on how current foodnet fast data organization and visual analytics can be improved to facilitate data interpretation, decision-making, and communication of features related to trend and seasonality. the resulting compilation, or analecta, of visualizations of records and codes are openly available online. disease surveillance systems worldwide face increasing pressure to maintain and distribute data in usable formats with clearly communicated visualizations to promote actionable policy and programming responses . decade-long efforts to sustain surveillance systems improve early outbreak detection, infection containment, and mobilization of health resources [ ] [ ] [ ] [ ] and create adaptive, near-time forecasts for disease outbreaks , . web-based platforms provide access to more accurate, timely, and frequent surveillance data. the world health organization's (who) flunet, for example, provides time-referenced data on worldwide influenza . publicly available downloads increase the flexibility for analyses and enables adaptive research due to frequent and timely reporting. the pandemic of novel coronavirus disease (covid- ) serves as a vivid demonstration of how limited access to publicly available high-quality data can stymy research. as the quantity and diversity of data available for processing, synthesizing, and communicating increases, new visual analytics, including complex multi-panel plots, must be considered to monitor trends, investigate seasonality, and support public health planning . these visualizations, and the methodologies used to generate them, must be standardized to enable comparability across time periods, locations, at-risk populations, and pathogens. however, current surveillance systems, including foodborne disease surveillance in the united states, often compress time series records to simplistic annual trends [ ] [ ] [ ] [ ] [ ] and describe seasonality by the month(s) with the highest cases per year or the first month of outbreak onset [ ] [ ] [ ] [ ] [ ] [ ] . visualizations using these annual trends or broad assessments of seasonality fail to utilize the full complexity of surveillance data and in some cases may be misleading. more specifically, these visualizations fail to provide detailed examination of how long-term trends change over time, how seasonality estimates vary by year or across locations, or how peak timing and amplitude estimates could change over time. the cdc foodborne disease active surveillance network (foodnet) provides preprocessed population-based foodborne-disease surveillance records and visualizations via foodnet fast, a publicly available data portal , . the foodnet fast platform contains rich demographic data, including age group, gender, and ethnic group, valuable for a broad spectrum of analyses. the visualizations aim to aid users in identifying trends of nine laboratory-confirmed foodborne diseases in select counties from ten us states and nationally. however, in the present form and due to substantial data compression, the available data and visualizations provided are limited in scope preventing the foodnet fast allows data download and visualization of these diseases for a user-specified time period. data downloads include information on the incidence of confirmed cases, monthly percentage of confirmed cases, distribution of cases by pathogen, and totals of cases, hospitalizations, and deaths. for multi-year periods, the portal aggregates totals and monthly percentages into single statistics for the full time period selected rather than showing individual years. this aggregation ensures case anonymity but monthly time units minimize the refinement of trend and seasonality analyses. to calculate monthly percentages of confirmed cases for all diseases in one year and one location, we had to download each state-year combination individually, for a total of files in ms excel format. to create a time series of total monthly cases by pathogen and location, we used data from two tables in each data download: annual counts of confirmed cases (long format) and monthly percentage of confirmed cases (wide format). we transposed the monthly percentages of confirmed cases from wide to long format and then multiplied them by the annual counts of confirmed cases (supplementary figure s ). since the provided monthly percentages are rounded to digit in the data download, calculated counts slightly under-or over-estimate annual totals. we did not round non-integer cases in our calculated time series to best preserve the monthly distribution of cases from the original data download. a monthly time series of confirmed cases of hospitalizations or deaths could not be reconstructed as described because no information is provided on their monthly percentages. we next calculated disease rates using confirmed monthly cases and annual population data. rates are preferred over counts since changes in counts could be a direct result of changes in the population catchment area of a surveillance system. the number of counties and states monitored in foodnet increased between and and has remained constant to date since (supplementary table s ). we downloaded county-level population estimates from the , , and us census bureau interannual census reports, which provide annual population estimates [ ] [ ] [ ] . we then estimated state-level foodnet population catchment area by adding all mid-year (july st ) populations of surveyed counties monitored in each year. next, we calculated the united states population catchment area by adding all state-level estimates for all surveyed counties for each year. finally, we developed a time series of monthly rates per , , persons for each pathogen and location by dividing monthly counts by annual population estimates and multiplying this quotient by , , . in addition to monthly rates, we calculated yearly rates by adding all monthly counts each year, dividing by the annual population, and multiplying this quotient by , , . modeling trends and seasonality. we estimated trend and seasonality characteristics using negative binomial harmonic regression (nbhr) models, which are commonly used to analyse count-based time series records with periodic fluctuations [ ] [ ] [ ] . these models include harmonic terms representing sine and cosine functions, which allow us to fit periodic oscillations. the regression parameters for these harmonic terms serve as a base for estimating important characteristics of seasonality: when the maximum rate occurs (peak timing) and the magnitude at that peak (amplitude). we calculated peak timing, amplitude, and their confidence intervals from nbhr model coefficients using the δ-method, which allow us to transform the regression coefficients of the model to seasonality characteristics based on the properties of the basic trigonometric functions (supplementary table s ) , . to estimate annualized seasonality characteristics, we applied a nbhr model for each study year and location with the length of the time series set to to represent the months of the year. we also estimated seasonality characteristics for the full time period. to show average trends across the entire -year period, we fit a nbhr model with three trend terms (linear, quadratic, and cubic) where the length of the time series varied according to when foodnet began surveying that location from to months. the selection of three polynomial terms was driven by the clarity of interpretation as a monthly increase and the potential for overall acceleration or deceleration, although other ways of assessing the trend such moving averages and spline functions could be also explored. plot terminology. we develop multi-panel visualization techniques using the best practices of current data visualization resources , and our own research , , . a multi-panel plot, as defined by our earlier work, "involves the strategic positioning of two or more graphs sharing at least one common axis on a single canvas . " these plots can effectively illustrate multiple dimensions of information including different time units (e.g. yearly, monthly), disease statistics (e.g. pathogens, rates, counts), seasonality characteristics (e.g. peak timing, amplitude), and locations (e.g. state-level, national). we use the following common, standardized terminology across visualizations to ensure comprehension: • disease -each of the nine reported foodnet infections, including campylobacteriosis (camp), listeriosis (list), salmonellosis (salm), shigellosis (shig), infection due to shiga toxin-producing escherichia coli o and non-o (ecol), vibriosis (vibr), infection due to yersinia enterocolitica (yers), cryptosporidiosis (cryp) and cyclosporiasis (cycl) • monthly rate -monthly confirmed cases per , , persons • yearly rate -total confirmed cases in a year divided by the mid-year population of all surveyed counties in that location (cases per , , persons) • frequency -the number of months reporting the disease rates in the same range • peak timing -the time of year according to the gregorian calendar that a disease reaches its maximal rate; for monthly time series, peak timing ranges in [ , [, i .e. from . (beginning of january) to . (end of december) • amplitude -the mathematical amplitude, or the midpoint of relative intensity; for nbhr models, the amplitude estimate reflects the ratio between the disease rate at the peak (maximum rate) and the disease rate at the midpoint (median rate) • foodnet surveyed county -the counties under foodnet surveillance as of • non-surveyed county -all remaining counties within a surveillance state as of . we present our analecta of visualizations allowing to describe trend, examine seasonal signatures, curves depicting characteristic variations in disease incidence over the course of one year, and understand features of seasonality, such as peak timing and amplitude across locations and diseases. we illustrate all visualizations using salmonellosis for the united states from - . the full analecta with time series data and code are available on our website (https://sites.tufts.edu/naumovalabs/analecta/) with data and code also available on figshare . describing trend. the interpretability of trends in a time series plot is greatly affected by the length and units of the time series. foodnet fast aggregates data annually, as shown in supplementary figure s , which provides clear, concise information on annual rates. in this example, the rate of salmonellosis remains largely unchanging over time with distinct outbreaks seen in and . as expected, by compressing data to annual rates, supplementary figure s masks within-year trends of disease rates. foodnet reports and publications similarly tend to show only inter-annual changes in disease counts or rates [ ] [ ] [ ] [ ] [ ] , . without more granular within-year variations, the viewer cannot determine if increased yearly rates are driven by erratic outbreaks in a specific month or higher rates across all months of the year. to capture within-year trends, we propose a multi-panel plot that combines information on monthly rates, inter-annual trends, and the frequency distribution of rates by utilizing the shared axes of individual plots (fig. ) . the right panel of fig. provides a time series of monthly rates with a nbhr model fit with three trend terms (linear, quadratic, and cubic). the inclusion of polynomial terms allows us to capture long-term trends (linear term) and their acceleration and deceleration over time (quadratic and cubic terms). the predicted trend line is shown in blue and its % confidence interval is in grey shades. the estimated median monthly rate is shown in red. the left panel depicts a rotated histogram of rate frequencies indicating the right-skewness of the monthly rate distribution. the histogram shares the vertical monthly rate-axis with the time series plot and is essential for connecting two concepts: the distribution of monthly counts on the base of their frequency and the distribution of monthly counts over time. two pictograms refer to the selected pathogen and location. figure shows the stability of seasonal oscillation in salmonellosis over time series with increased rates from - followed by a gradual decrease in rates through . while preserving the within-year seasonal fluctuations, the plot provides additional information. alternating background colours help distinguish differences in the shape of seasonal curves between adjacent years. an increasingly darker hue for the monthly rate values distinguishes more recent data from more historic data. contrasting background colours mixed with a gradual intensity of line hues, saturation, brightness, and transparency allow for greater focus and attention to trends in the data [ ] [ ] [ ] . the rotated histogram in the left panel of fig. shows the distribution of monthly rates and its degree of skewness due to months with high counts. we include the red median line to provide the most appropriate measure of central tendency for the skewed distribution. the shared vertical axis helps readers track those high values to a www.nature.com/scientificdata www.nature.com/scientificdata/ specific month in the time series. the distribution also justifies the use of negative binomial regression models to evaluate temporal patterns. by supplementing the time series plot with the distribution of monthly rates, we show a visual rationale for using appropriate analytical tools (negative binomial model, in this case) for calculating inter-annual trends. to better understand annual differences in seasonal behaviors, we propose a multi-panel plot that incorporates annual seasonal signatures, summary statistics of monthly rates, and radar plots (fig. ). given varying visual perceptions of these three ways of presenting seasonal patterns, we offer side-by-side comparisons that aim to increase comprehension. the top-left panel provides an overlay of all annual seasonal signatures, a set of curves depicting characteristic variations in disease incidence over the course of one year, where line hues become increasingly darker with more recent data and a red line indicates median monthly rates, as in fig. . the bottom-left panel provides a set of box plots for each month that aggregates information over the study period and provides essential summary statistics, including the median rate values and the measures of spread. the shared horizontal axis allows the two plots to be compared across the years using identical scales. to provide visual context, background colours were used to indicate the four seasons (winter, spring, summer and autumn). the right panel provides overlaying monthly rates using a radar plot where time is indicated on the rotational axis and rates are indicated on the radial axis. the radar plot emphasizes the periodic nature of seasonal variations in one continuous line with graduating colours. the colour hue of the lines, background colour, median line colour and the axis scales are uniform across all three panels. we also repeat the pictograms to refer to the selected pathogen and location. for salmonellosis, disease rates are highest in the summertime (with peaks in july and august) and lowest during the wintertime (with a well-defined february nadir). rate increases and decreases during equinox periods indicate bacterial growth rates due to more and less favourable climate conditions, respectively. the www.nature.com/scientificdata www.nature.com/scientificdata/ confidence interval (whisker), and outliers or potentially influential observations (markers) over the -year period. measures of distribution spread provide an insight for the dispersion of rates in each month: the variability of salmonellosis rates decreases in winter months closer to the february nadir but increases in summer months of july and august closer to the seasonal peak. unusually high values are indicative of erratic behavior characterized by spikes in specific months and years. the right-hand panel of fig. further emphasizes the periodic nature and the positioning of the seasonal peaks and nadirs. radar or spider plots describe time using a rotational axis where the radial distance from the centre of the plot depicts rate magnitude [ ] [ ] [ ] [ ] . radial axes, compared to perpendicular axes, show annual fluctuations as a continuous flow. this more clearly demonstrates declines of salmonellosis rates during nadir months (november to march) without the visual discontinuity of left panel visuals. to capture the advantage of a multi-panel plot (fig. ) , we incorporate the boxplot from fig. (lower left panel) with a calendar heatmap containing monthly rate values. in the heatmap, information for each individual year is shown as stacked rows of width (for each month of the year) where cell colour intensity represents the magnitude of monthly rates. like fig. , the heatmap illustrates the highest rates (shown as the darker cells) are in july and august. compared to stacked line plots, however, fig. provides an individual row for each year of the time series, allowing for greater decomposition, differentiation, and comparison of seasonal signatures across years. in this plot, seasonal changes are shown horizontally from left to right -from january to december and the yearly trend transition can be observed in a vertical view from bottom to top-from year to in the right panel. while fig. provides the annual variability of seasonal patterns, monthly rate values for each year are difficult to ascertain. instead, the emphasis is placed on similarities and differences of the seasonal curvature over time. in fig. , the attention shifts to comparing the intensity of rates per month of the year across years. here, we evaluate which months of the year are most intense across years using the intensity of each cell's colour hue to describe the intensity of rates. the fig. panel integrates information on both trends and seasonality along with the individual monthly values unlike any of the previously shown visualizations. yearly rates provide a bar graph for comparing fluctuations in inter-annual rates while the adjacent heatmap indicates the month(s) driving these fluctuations. in doing so, the calendar heatmap identifies whether inter-annual changes are driven by sporadic outbreaks or increased seasonal magnitude of rates. at the same time, the shared axis box plot provides an overview of the average seasonal signature for the entire time series, as emphasized in fig. . understanding seasonal features. detailed characterization of the timing and intensity of seasonal peaks requires a standardized estimation of peak timing and amplitude. this standardization improves upon implemented techniques of comparing months with the highest cases in a given year by applying the δ-methods to www.nature.com/scientificdata www.nature.com/scientificdata/ nbhr model parameters , . average seasonality characteristics can be estimated across the full time series while annual estimates allow for more granular comparisons between years. to depict point estimates and confidence intervals of seasonality characteristics, we use forest plots -a technique commonly used in meta-analyses , , . we develop a multi-panel forest plot to depict annual peak timing, annual amplitude, and their joint distribution, to better understand the relationship among the seasonal features and how it changes over time (fig. ) . figure is a multi-panel plot that incorporates two forest plots (one each for annual peak timing and amplitude estimates) and one scatterplot (for peak timing and amplitude) to describe seasonality features. the top-left www.nature.com/scientificdata www.nature.com/scientificdata/ panel shows peak timing estimates (as month of the year, ranging from . (beginning of january) to . (end of december) -horizontal axis) for each study year (vertical axis). the bottom-right panel shows amplitude estimates where the horizontal axis indicates the study year and the vertical axis shows the amplitude (ratio between the disease rate at peak and the median rate). the bottom-left corner shows the scatterplot of peak timing (horizontal axis) and amplitude (vertical axis) with markers representing each pair of annual estimates. measures of uncertainty ( % confidence intervals) are reflected in error bars of each marker; dashed red lines show median peak timing and amplitude estimates. forest plots in fig. provide a compact, clear, and comprehensive visual describing the stability of peak timing and amplitude, even without showing the entire seasonal signature. for example, salmonellosis peak timing and amplitude vary little each year indicating strong, stable seasonal peaks in july and august. consistent peak timing means practitioners could time preventive strategies, increase awareness for foodborne illnesses to prevent transmission, and inform food retailers of when food safety inspections should be in higher demand within their supply chains. consistent amplitude estimates show that the intensity of salmonellosis varies little over time, suggesting that federal food safety regulations have not greatly influenced the number of salmonellosis cases annually. this type of information is likely to benefit foodnet fast users. supplementary figure s provides an example of how a sporadic outbreak behavior can be depicted by forest plots of peak timing and amplitude estimates for shigellosis in ny. the lack of seasonality for shigellosis is shown by the broad confidence intervals for peak timing, spanning the entire year and beyond. figure s provides an example bar chart showing differences in the average annual incidence of salmonellosis for the ten foodnet-surveyed states. as with other foodnet visualizations, data has been compressed to show only average annual estimates. like in fig. , annual rates mask within-year seasonal variations, calling into question if differences in states are driven by single year outbreaks. the alphabetical organization of the horizontal axis makes states ranking and comparison more difficult than if they were ordered from highest to lowest rates. to ease comparisons of a single disease across www.nature.com/scientificdata www.nature.com/scientificdata/ geographic locations, we generated two multi-panel plots (figs. and ). these plots mirror the same techniques shown above but include multiple shared axes and multiple locations to draw spatial comparisons. supplementary figure s follows the same design as fig. ; we replicate this design for salmonellosis in all foodnet-surveyed states. we present all states in one plot in a descending order by the sum of yearly rates in each state and display all available data so that state level patterns can be compared. the box plot in the top panel provides an overview of the seasonal signature for the entire us. the bottom panel disaggregates the entire us by states. as shown, all states share similar peak timing in july and august for almost every surveillance year from - . for some states, like ga and ca, rates are densely concentrated from july to september with rapid decline from september to february and gradual incline from february to july. for other states, like ny and or, seasonal peaks are much less pronounced and rate differences are smaller between months. clear indication of missing data provides additional information on differences in reporting completeness not captured by previous figures. while heatmaps provide information on seasonal signatures, yearly rate bar graphs (right panel) capture state-level trends over time. states are stacked in the order of total cases from - , showing differences in the intensity of salmonellosis infection across states. comparisons within states between years help identify inter-annual rate changes over time. for example, while md and ca have generally declined in annual rates over the -year period, ga rates increased from - and steadily declined from - . in combination with heatmaps, yearly rates also allow for detailed assessment of sporadic outbreaks. for example, erratic outbreaks came from two monthly spikes in april and june for ct in while for nm in a multi-month outbreak lasted from may to july. by using shared horizontal and vertical axes, this plot eases the comparison of disease rates across months, years and states. it also helps to determine hotspots and detect potential co-occurrences of infection in different states. moreover, the plot can be periodically updated by adding new information, offering a sustainable approach to make consistent comparisons between historical data and data captured in the future. to compare seasonality features across locations, we designed a multi-panel plot similar to fig. to show average peak timing and amplitude estimates over the -year period for each state. in fig. the top-left panel plots peak timing estimates ordered from the earliest (or) to latest (ga) peak timing while the bottom-right panel plots amplitude estimates in order of magnitude. marker and line colours are used to differentiate the seasonality feature estimate and its measure of uncertainty between states. the bottom-left panel shows the relationship between peak timing and amplitude across states. figure s provides an example of foodnet fast bar chart showing differences in the total confirmed infections for each of the nine surveyed pathogens in the us from - . the visual shows that infections due to campylobacter and salmonella have the highest cumulative counts of infections while cyclospora has the lowest counts. while depicting these differences clearly, this visual lacks sufficient specificity for drawing more intricate comparisons between infections. how are counts or rates distributed by year? what are the within-year variations of rates by pathogen? how do seasonal signatures and their variability differ by pathogen? can axes be reordered or recalculated for easier comparisons between pathogen counts or rates? we propose two multi-panel plots (figs. and ) that improve the comparisons of multiple diseases for a given geographic location. figure replicates the plot design of fig. but emphasizes comparisons between pathogens for a single location. instead of a seasonal signature box plot, the top panel provides a scatterplot to illustrate the peak timing and amplitude of each pathogen. in combination with the heatmap in the bottom panel, these plots illustrate the strong seasonality of salmonellosis, campylobacteriosis, and stec in july and august and cryptosporidiosis in august. these seasonal peaks are consistent across almost all years suggesting a stable seasonal periodicity and strong alignment between infections. in contrast, infections caused by yersinia enterocolitica, vibriosis, listeriosis, and cyclosporiasis have much less pronounced seasonality and monthly rates much lower than salmonellosis or campylobacteriosis. yearly rates, shown in the right panel, indicate erratic outbreak behaviors for cyclosporiasis. given sizable differences in rates across diseases we applied a high-order calibration colour scheme. we also provide the same multi-panel, shared-axis visualization design seen in fig. for comparisons across pathogens. figure includes a forest plot of peak timing by disease pathogen (top-left panel), a forest plot of amplitude by pathogen (bottom-right panel), and a scatterplot between peak timing and amplitude estimates (bottom-left panel). as in fig. , average peak timing and amplitude estimates are calculated using nbhr models for the entire -year time series. comparisons between diseases allow for understanding the alignment of seasonal processes across pathogens as well as shared relative magnitudes in a specific location. in our case, most of the pathogens peak during the summertime except cyclosporiasis. however, if the selected diseases peak during winter months, we recommend adjusting the starting and ending months to center these peaks in the figure. in this study we offered ways of thinking on how public data platforms can be improved by using visual analytics to provide a comprehensive description of trends and seasonality features in reported infectious diseases. we emphasize the utility of multi-panel graphs by showing side-by-side different methods of depicting trends over time and features of seasonality, including disease peak timing and amplitude. we provided visual tools to show trends (fig. ) , examine seasonal signatures (figs. and ) and their characteristics (fig. ) , compare diseases across locations for trends (fig. ) and seasonal signatures (fig. ) , and drawing comparisons across pathogens for trends (fig. ) and seasonal signatures (fig. ) . we also provide guides on how to explore and compare trends and seasonality between multiple diseases and geographic locations using foodnet fast data. given varying visual perceptions, we offer side-by-side comparison of different tools aiming to increase comprehension and faster adoption of efficient graphical depictions. www.nature.com/scientificdata www.nature.com/scientificdata/ we developed a time series of monthly rates by reconstructing a time series of monthly counts (see fig. ) then dividing counts by the sum of all foodnet-surveyed counties' mid-year populations per state per , , persons. in this calculation, we recognize that average monthly percentages are rounded in the raw data file and do not sum to % annually for downloaded years. this rounding resulted in obtaining non-integer counts within our time series. to prevent modification of raw data files, we did not round counts to integers before or after calculating rates. no information is provided on the foodnet fast website for the definition of confirmed cases, and data downloads provide no metadata for distinguishing cases from hospitalizations and deaths. although the case definition is provided on the cdc website as "laboratory-confirmed cases (defined as isolation for bacteria or identification for parasites of an organism from a clinical specimen) and cases diagnosed using culture-independent methods" , it forces the user to assume that a confirmed case is any person with laboratory confirmed cultures of a specific pathogen who may or may not have been hospitalized or died from infection. foodnet also collects information on hospitalizations and deaths, but does not provide information on the monthly percentage of hospitalizations or deaths, so users are unable to reconstruct a monthly time series for deaths or hospitalizations. the foodnet fast platform states all confirmed diseases as "incidence" calculations. technical documentation on the foodnet website shows that the term incidence reflects cases per , persons (used interchangeably with a disease rate) with no distinction of whether these are newly introduced within the population (i.e. incidence) or the total persons diagnosed with a disease (i.e. prevalence) . we found that monthly rates can similarly be calculated by multiplying annual incidence rates and the monthly percentage of confirmed cases for each disease-state pair. differences between our calculations and this alternative method are no more than ± %. we suspect that rounding errors of average monthly percentages and differential population catchment areas for rate calculations cause these differences. as shown in supplemental table s , population of the surveillance catchment area is changing over time. oftentimes, publicly available surveillance datasets, including foodnet fast, do not include location-and year-specific population catchment area estimates, which are needed for calculating rates from diseases counts. as foodnet does not provide population catchment areas for calculating rates, it www.nature.com/scientificdata www.nature.com/scientificdata/ www.nature.com/scientificdata www.nature.com/scientificdata/ forces the user to assume that foodnet surveillance reaches the total population of a surveyed county (likely an overestimate), yet such oversight is easy to fix. three collaborators confirmed our monthly rate calculations for quality control. we applied the negative binomial harmonic regression nbhr models, commonly used in the time series analysis of counts and cases. while the use of nbhr models, specifically the inclusion of trigonometric harmonic oscillations, is similar to existing works on foodborne illnesses, these studies often incorporate harmonic oscillators only to adjust for or remove seasonal oscillations [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we have extended the use of harmonic terms and develop the tools to estimate peak timing and amplitude , , . the developed δ-method provides a systematic calculation of confidence intervals for peak timing and amplitude estimates based on the results of harmonic regression models. in the proposed approach, we present the amplitude as the ratio of seasonal peak to seasonal median, which offers robust estimation even for rare or highly sporadic infections. these features are not available when traditional models, like auto-regressive integrated moving average (arima), are applied . measures of uncertainty enable formal testing and comparisons across diseases in the same location or locations for the same disease. in our previous works, we have demonstrated the broad utility of the δ-method and applications of peak timing and amplitude estimation in the context of epidemiological studies , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we evaluate each state's cases individually as well as all national cases as the sum of all states' cases. our analysis evaluated all cases reported to foodnet fast irrespective of demographic factors such as age group, sex, or ethnic group. future analyses can consider conducting analyses using demographic factors available on the foodnet fast platform such as age group (< , - , - , - , - , - , - , - , + years), sex (male and female), and ethnic group (american indian and alaskan native, asian and pacific islander, black, multiple, white). to incorporate this information, our methodology for data extraction would need to be repeated for each subcategory or combination of categories desired (e.g. download files for males and files for females). www.nature.com/scientificdata www.nature.com/scientificdata/ future analyses can also consider differences in pathogen strain, which can only be obtained if extracting data for each pathogen-location-year combination (e.g. files for each of the diseases for each of locations or , files). foodnet fast, like many global disease surveillance databases, has no metadata describing missing data. foodnet fast reports missing counts using "n/a" for years when pathogens or locations were not under surveillance. however, there are also years when foodnet surveillance was live in a state, but a pathogen is missing from the data download. we believe that this missing data comes when, for a given year, a pathogen has total cases. however, we cannot specify whether absences of surveillance reporting came due to a breakdown in reporting or annual counts. without specification, we have set any year with "n/a" as missing due to no reported case information. when calculating peak timing and amplitude using the δ-methods, we applied nbhr models adjusted for harmonic seasonal oscillators and three trends (linear, quadratic, and cubic). we selected the polynomial terms as an example, yet researchers can consider alternative techniques for measuring seasonality such as splines, nonparametric regression, arima models, or their extensions. additionally, the cdc recommends using a mixed effects model when conducting time series analyses on foodnet fast data to account for differential population catchment areas and laboratory culture confirmation techniques pre-and post- , , . we focus on the analysis of individual states and diseases and adjust for population catchment variations by calculating monthly rates using county-level population estimates. future analyses could include detailed assessments between peak timing and amplitude across diseases, locations, and time periods. such analyses will help determine whether a synchronization of outbreak peaks occurs or if social, economic, or environmental factors influence peak timing and amplitude. future applications. this analecta of visualizations intends to communicate detailed information on foodborne outbreak trends and seasonality suitable for a general audience, public health professionals, stakeholders, and policymakers. future applications would involve the development of an interactive web-based platform allowing users to select the outcome, timeframe, and location of interest for educational training and research purposes. for example, public health researchers and practitioners could use this tool to generate insights related to long-term trends, changes in disease dynamics, or changes in populations at risk . information on when and where outbreaks are most common enable producers, distributors, and retailers to improve food safety practices to prevent these outbreaks. finally, this platform could aid policymakers in shaping public understanding of outbreak dynamics and using scientific evidence to refine public health policies. the analecta of our time series of monthly rates, data visualizations, and code used for all calculations and visualizations are available on our website (https://sites.tufts.edu/naumovalabs/analecta/). data and code can be directly downloaded from the website while visualizations are linked on the website to an external visualization repository. time series data and code are also available on figshare . visualizations on our website are provided in the same order as presented here: describing trends (fig. ) , examining seasonal signatures with the three standard techniques: line graphs, boxplots, and radar plots (fig. ) and heatmaps (fig. ) , characterizing features of seasonality (fig. ) , drawing comparisons across locations for trends (fig. ) and seasonal signatures (fig. ) , and drawing comparisons across pathogens for trends (fig. ) and seasonal signatures (fig. ) . file downloads are available for trend, seasonal signature, and annual time series visualizations. for images examining a single disease in a single location, downloads are formatted where the prefix abbreviates the location and the suffix abbreviates the pathogen (see supplementary table s ). for visualizations comparing multiple locations or diseases, the prefix "loc" indicates comparisons across locations while the prefix "dis" indicates comparisons across pathogens (see supplementary tables s ,s ). all statistical analyses were conducted using stata (se . ) software. all visualizations were created using r version . . and tableau professional . software. all software code is open access on our website (https:// sites.tufts.edu/naumovalabs/analecta/) and figshare, and is available for public reuse with proper citation of this manuscript . web-based infectious disease surveillance systems and public health perspectives: a systematic review detecting influenza epidemics using search engine query data innovation in observation: a vision for early outbreak detection use of unstructured event-based reports for global infectious disease surveillance algorithms for rapid outbreak detection: a research synthesis influenza seasonality: underlying causes and modeling theories flunet. global influenza surveillance and response systems (gisrs) visual analytics for epidemiologists: understanding the interactions between age, time, and disease with multi-panel graphs incidence and trends of disease with pathogens transmitted commonly through food -foodborne diseases active surveillance network preliminary incidence and trends 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petting zoo animals for use in exposure assessments global prevalence of asymptomatic norovirus disease: a meta-analysis foodnet fast: pathogen surveillance tool faq assessing the impact of environmental exposures and cryptosporidium disease in cattle on human incidence of cryptosporidiosis in southwestern ontario do contamination of and exposure to chicken meat and water drive the temporal dynamics of campylobacter cases? review of epidemiological studies of drinking-water turbidity in relation to acute gastrointestinal illness seasonality and the effects of weather on camylobacter diseases increase in reported cholera cases in haiti following hurricane matthew: an interrupted time series model complex temporal climate signals drive the emergence of human water-borne disease association between community socioeconomic factors, animal feeding operations, and campylobacteriosis incidence rates: foodborne diseases active surveillance network (foodnet) climate, human behaviour or environment: individual-based modelling of campylobacter seasonality and strategies to reduce disease burden temperature-driven campylobacter seasonality in england and wales rotavirus seasonality: an application of singular spectrum analysis and polyharmonic modeling mystery of seasonality: getting the rhythm of nature seasonal synchronization of influenza in the united states older adult population geographic variations and temporal trends of salmonella-associated hospitalization in the u.s. elderly, - : a time series analysis of the impact of haccp regulation hospitalization of the elderly in the united states for nonspecific gastrointestinal diseases: a search for etiological clues assessing seasonality variation with harmonic regression: accommodations for sharp peaks intelligence advanced research projects activity (iarpa), via - . the views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of odni, iarpa, or the u.s. government. the u.s. government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. the research was in part supported by the national science foundation (nsf) innovations in graduate education (ige) program, via grant award and by the united states department of agriculture (usda) national institute of food and agriculture (nifa) cooperative state research, education, and extension service fellowship meghan hartwick for editorial and technical assistance r.s. contributed to data extraction, formal analysis, and writing. b.z. contributed to data validation, conceptualization of visual aids, and visualization creation. t.m.a.f. contributed to data validation, review, and editing. e.n.n. contributed to methodology development, review and editing, supervision, project administration and funding acquisition. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - -x.correspondence and requests for materials should be addressed to e.n.n.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - oa hfb authors: bourgonje, arno r.; abdulle, amaal eman; timens, wim; hillebrands, jan‐luuk; navis, gerjan j.; gordijn, sanne j.; bolling, marieke c.; dijkstra, gerard; voors, adriaan a.; osterhaus, albert d. m. e.; van der voort, peter h. j.; mulder, douwe j.; van goor, harry title: angiotensin‐converting enzyme‐ (ace ), sars‐cov‐ and pathophysiology of coronavirus disease (covid‐ ) date: - - journal: j pathol doi: . /path. sha: doc_id: cord_uid: oa hfb angiotensin‐converting enzyme‐ (ace ) has been established as the functional host receptor for severe acute respiratory syndrome coronavirus (sars‐cov‐ ), the virus responsible for the current devastating worldwide pandemic of coronavirus disease (covid‐ ). ace is abundantly expressed in a variety of cells residing in many different human organs. in human physiology, ace is a pivotal counter‐regulatory enzyme to ace by the breakdown of angiotensin ii, the central player in the renin‐angiotensin‐aldosterone system (raas) and the main substrate of ace . many factors have been associated with both altered ace expression and covid‐ severity and progression, including age, sex, ethnicity, medication and several co‐morbidities, such as cardiovascular disease and metabolic syndrome. although ace is widely distributed in various human tissues and many of its determinants have been well recognised, ace ‐expressing organs do not equally participate in covid‐ pathophysiology, implying that other mechanisms are involved in orchestrating cellular infection resulting in tissue damage. reports of pathologic findings in tissue specimens of covid‐ patients are rapidly emerging and confirm the established role of ace expression and activity in disease pathogenesis. identifying pathologic changes caused by sars‐cov‐ infection is crucially important as it has major implications for understanding covid‐ pathophysiology and the development of evidence‐based treatment strategies. currently, many interventional strategies are being explored in ongoing clinical trials, encompassing many drug classes and strategies, including antiviral drugs, biological response modifiers and raas inhibitors. ultimately, prevention is key to combat covid‐ and appropriate measures are being taken accordingly, including development of effective vaccines. in this review, we describe the role of ace in covid‐ pathophysiology, including factors influencing ace expression and activity in relation to covid‐ severity. in addition, we discuss the relevant pathological changes resulting from sars‐cov‐ infection. finally, we highlight a selection of potential treatment modalities for covid‐ . this article is protected by copyright. all rights reserved. introduction microbial ecology. in fact, transplantation of gut microbiota from ace -knockout mice resulted in an increased propensity to develop severe colitis [ ] . previously, we investigated the immunolocalization of ace in healthy human organs [ ] . ace was highly expressed on lung alveolar epithelial cells and small intestinal epithelial cells, consistent with potential routes of viral transmission of sars-cov- , as both respiratory and gastrointestinal systems share interfaces with the external environment. additionally, ace was present on vascular endothelial cells and smooth muscle cells in all organs studied. in the kidney, ace was strongly expressed in the brush border of proximal tubular cells and moderately or weakly in parietal epithelial cells and podocytes, whereas ace staining was weak or negative in glomerular endothelial cells and mesangial cells. ace was also present in the basal epidermal layer of the skin and in the oral and nasal mucosa. in contrast, ace was absent in lymphoid tissues and hepatobiliary structures [ ] . the intense staining on various epithelial cells (small intestine, kidney, skin) strongly suggests raas-independent functions of ace . these findings trigger alternative hypotheses regarding ace involvement in viral transmission pathways. furthermore, we previously noted that endothelial ace was upregulated in the glomerular and interstitial capillaries in kidney diseases independent of the initial trigger, indicating that ace may also be viewed as a damage marker [ ] . summarising, ace is widely expressed in human tissues, both in principal target organs of sars-cov- and in organs that play a seemingly less important or even unknown role in covid- pathophysiology. this article is protected by copyright. all rights reserved. recently, ace was unequivocally established as the functional host receptor for sars-cov- (figure ) [ ]. binding kinetics revealed a - -fold higher binding affinity as compared to the sars-cov- virus [ , ] . these findings may partially explain the apparently easier transmissibility of sars-cov- and that increased ace expression may confer increased susceptibility to host cell entry of sars-cov- . it was previously shown that a specific region within the sars-cov- spike protein interacts with ace , leading to fusion with the host cell membrane [ , ] . an experimental animal study in ace -knockout mice further underlined the importance of this receptor in the pathogenesis of sars caused by sars-cov- [ ] . the authors hypothesised that infection with sars-cov- results in ace downregulation through its internalisation, induced by binding of sars-cov- to ace , as a mechanism contributing to the severity of lung pathologies [ ] . consequently, this would lead to impairment of the protective effect of ace on the severity of acute respiratory distress syndrome (ards). this, as well as a harmful effect of ang ii, was previously demonstrated in several animal models of ards [ ] [ ] [ ] [ ] . the interaction between ace and sars-cov- and with sars-cov- , and further downstream effects, exhibit a high level of similarity between each other [ ] . during hypoxia, ang ii-induced pulmonary vasoconstriction occurs, aimed to restore the ventilation-perfusion mismatch, but simultaneously inducing adverse pro-fibrotic effects, which both are ameliorated by concomitant upregulation of ace [ ] . under similar circumstances, sars-cov- -induced downregulation of ace could impair clearance of ang ii and hence lead to aggravation of this article is protected by copyright. all rights reserved. tissue damage. on the other hand, one may speculate that ace downregulation by sars-cov- results in decreased opportunity for further viral cell entry, thereby limiting viral spread. however, as one may hypothesise that sars-cov- infects ace -expressing cells with greater efficiency compared to sars-cov- , presumably through exploiting cellular factors promoting viral attachment and entry, it is likely that sars-cov- viruses would need less ace to enable viral spread. taken together, the role of ace in sars-cov- cellular infection is complex and not yet defined, which makes it interesting to study if and how sars-cov- interferes with ace expression and/or its regulation, and how this influences viral replication. genetic factors ace is encoded by a gene located on chromosome xp and consists of at least exons and introns, amounting to approximately kb of genomic dna [ ] . the genetic architecture closely resembles the structure of the ace gene and may lead to a variety of alternative rna transcripts. the ace gene is characterised by a number of polymorphisms, which have been associated with diversity of raas-system pathologies, such as essential hypertension [ ] . however, the genetic background of ace expression and functionality across different populations in relation to sars-cov- is largely unknown. comparative systematic analysis of coding-region variants and expression quantitative trait loci (eqtl) variants of ace across different populations, showed higher allele frequencies of eqtl variants associated with higher ace tissue expression levels in east asian compared to this article is protected by copyright. all rights reserved. european populations. this may imply a differential susceptibility to sars-cov- infection across different populations. however, no evidence supporting potential s-protein bindingresistant ace mutants was obtained [ ] . structural modelling and superimposition analyses of the native ace -and ace -s-protein complex were used to study changes in ace variants and the intermolecular interactions with the s-protein. most ace coding variants showed high structural similarity and highly similar binding affinity with the s-protein of sars-cov- . however, two allelic variants were identified that demonstrated considerable variation in intermolecular interaction with the s-protein, showing varying spatial orientation of key interacting residues of ace [ ] . these ace genetic variations may provide a basis for relative or complete potential resistance against sars-cov- infection. ace expression in the lungs and sars-cov- viral load have been suggested to increase with age, which might provide an explanation to the higher disease severity observed in older patients with covid- [ ] . advancing age is increasingly recognised as one of the strongest predictors for severe covid- [ ] . older adults (aged above years) are at increasing risk of contracting severe covid- with higher complication and case fatality rates [ ] . similar to influenza and other respiratory viral infections, gradually decreasing innate and adaptive immune responses may be expected to play an important role in this agerelated increased susceptibility. accumulating data also show the existence of a genderassociated predisposition to covid- , with men being more prone to develop severe disease than women [ ] . ace expression may be a contributing factor to this association as single-this article is protected by copyright. all rights reserved. cell transcriptomics demonstrated that ace expression was higher among asian men than asian women [ ] . observational data indicated higher frequencies of males among critically ill patients [ , ] . in line, males appeared to be more frequent among deceased patients compared to recovered patients [ ] . possible explanations of male predominance among covid- patients may be differences in exposure, smoking behaviour, other lifestyle factors, differences in chromosomal ace expression, ace expression in testicular tissue, sex hormone-driven immune system regulation, or gender differences in raas regulation [ , [ ] [ ] [ ] . interestingly, in two independent cohorts of patients with heart failure, plasma concentrations of ace were higher in men than in women [ ] . obese patients with covid- may have an increased risk of icu-admission and mortality. although obese patients frequently present with mechanical hypoventilation (leading to hypercapnic respiratory failure), those with covid- present with hypoxic respiratory failure. this led to discussions about a potential role of fat tissue in covid- pathogenesis in relation to ace expression. granting that obesity predisposes to developing chronic disease, obesity could also be an independent risk factor for covid- [ ] . bmi is significantly higher among covid- patients with critical disease requiring icu-admission compared to less severe cases [ ] . likewise, the proportion of patients with bmi > kg/m was significantly elevated in deceased patients compared to survivors. a chinese multi-centre study reported significantly higher bmi values among patients with severe disease compared to patients having only mild disease [ ] . in other emerging large case series, obesity remains this article is protected by copyright. all rights reserved. common and may be a risk factor for respiratory distress, eventually requiring mechanical ventilation [ , ] . these observations are analogous to other respiratory viral infections, for instance the h n influenza virus infection. during that pandemic, obesity also emerged as an independent risk factor for hospitalisation and death [ , ] . this could be attributed to obesity-induced impairment of the immune response, as has been welldocumented for h n influenza [ ] . mechanistically, adipose tissue-derived inflammation in obesity leads to substantial metabolic disturbances that could eventually lead to complications such as dyslipidaemia, hypertension, diabetes, cardiovascular disease (metabolic syndrome or syndrome x) and chronic respiratory failure [ ] . visceral fat tissue can induce pro-inflammatory effects, which are regulated by adipokines and ang ii. interestingly, ace is abundantly present on visceral adipocytes [ , ] . ace on adipocytes exerts systemic effects on the cardiovascular system and experimental studies demonstrated interactions between gender, adipocyte ace and complications of obesity, e.g. hypertension [ ] . of note, leptin is one of the most important adipokines driving these pro-inflammatory effects and higher leptin availability has been associated with increased ang ii levels as well as decreased ace expression and activity [ ] . in addition, high leptin levels have been associated with accumulation of alveolar fluid and increased inflammation upon hypoxia and ards [ ] . therefore, it may be hypothesised that excess visceral adipose tissue in patients with covid- may drive disease progression -whether or not affected by genderespecially through aggravating the cascade of hyperinflammatory reactions in the disease [ ] . ultimately, this 'cytokine storm' may lead to multiple organ failure in patients with this article is protected by copyright. all rights reserved. a recent meta-analysis of , patients diagnosed with covid- reported that severe disease was associated with hypertension, chronic respiratory disease and cardiovascular disease [ ] . in another report including over , patients with confirmed covid- , hypertension, chronic respiratory disease, diabetes mellitus, cardiovascular disease and cancer emerged as the most common comorbidities [ ] . many of these comorbidities are characterised by either increased or decreased ace expression and/or activity, as well as a shift in ace/ace balance in both directions. this could be related to underlying conditions and/or to treatment with raas inhibitors (discussed in: 'pathogenesis and treatment options for covid- '). however, the relative contribution of each of these underlying conditions to disease severity and mortality remains undetermined. many of the currently available reports were unadjusted for potential confounding factors, including age, sex, and lifestyle factors such as smoking and diet. similarly, many studies were uncontrolled, had relatively short follow-up periods, or were likely affected by inaccurate scoring or under-diagnosis [ ] . in general, it is advised that patients using immunosuppressive drugs should not preemptively stop their medication, because there is still much unknown about potential risks or benefits. for instance, transplanted patients frequently use ciclosporin, which has been shown to have antiviral activity against sars-cov- [ ] . patients with chronic immune-mediated inflammatory diseases (imids, e.g. rheumatoid arthritis [ra] this article is protected by copyright. all rights reserved. [ibd]) who are treated with cytokine inhibitors (e.g. tnf-antagonists, anti-il r therapy) do not seem to be at an automatically increased risk of developing severe covid- [ ] . although at first sight these treatments may seem to lead to immune suppression and may therefore be considered potentially harmful in the context of covid- , they specifically target individual inflammatory cytokines or mediators instead of a broad panel of immune system components. in fact, cytokine inhibitors potentially attenuate the hyperinflammatory state associated with covid- and may therefore exert beneficial effects. this concept is supported by observations that the pro-inflammatory cytokines induced in covid- seem to be more crucial for the host inflammatory response compared to those involved mainly in viral clearance [ ] . patients with solid malignancies treated with immune checkpoint inhibitors (icis), including anti-programmed death(-ligand)- (pd- /pd-l ), anti-cytotoxic t-lymphocyte-associated protein- (ctla- ) and chimeric antigen receptor (car) t-cell therapy for certain b-cell related haematologic malignancies, frequently experience t-cellengaging immunomodulatory effects [ ] . well-known immune-related adverse events include cytokine release syndrome (crs) and pneumonitis [ , ] , which in theory might render patients more vulnerable to infections [ ] . interestingly, these complications resemble the clinical presentation of advanced covid- and respond well to anti-il r therapy [ ] , providing a strong rationale for anti-il r therapy in covid- [ ] . as described, ace expression and activity is ubiquitously present within the human body, but many of its determinants on tissue level dynamics are unknown. however, in covid- this article is protected by copyright. all rights reserved. pathophysiology, there is seemingly huge spatiotemporal heterogeneity in organ involvement, presumably because multiple pathophysiological mechanisms may be causally involved in the observed tissue damage. both sars-cov- infection, directly mediated by ace expression and activity, and superimposed disease triggers may be responsible for the observed pathological findings. detailed pathological study of tissue specimens is therefore urgently needed to improve our understanding by disentangling the potential origins of tissue damage. the initial clinical presentation of covid- consists of respiratory symptoms such as fever, dry cough, shortness of breath, rhinitis, and, additionally, chest pain, myalgia and/or fatigue [ ] [ ] [ ] [ ] . in more severe cases needing hospitalisation, viral pneumonia develops with progressive ground-glass opacities on chest computed tomography (ct). in clinically critical cases, this is accompanied by further complications including ards, cardiac pathology and secondary infections. given the similarities between sars-cov- and sars-cov- , lung pathology shows considerable equivalence [ , ] . hitherto, there are limited reports of mainly autopsy cases describing lung pathological findings [ ] [ ] [ ] [ ] . similar to sars, covid- -associated pathological changes in the lungs generally constitute extensive diffuse alveolar damage with bilateral oedema, proteinaceous or fibrin alveolar exudates and diffuse reactive hyperplasia of type ii pneumocytes (figure a-b) . in more advanced pathology, hyaline membrane formation was observed with thickened alveolar septa caused by interstitial fibroblast proliferation consistent with fibrosis ( figure b ). in addition, variable presence of patchy mainly interstitial infiltration of mononuclear cells has been reported ( figure a) , and in some cases, multinucleated giant cells in alveoli with associated viral changes. in contrast to sars, there is seemingly more thrombo-embolic pathology observed in specimens from patients with covid- (discussed in 'thrombo-embolic risk'). also, intra-alveolar deposition of neutrophilic granulocytes was reported in a few instances, most likely due to superimposed bacterial infection. another case report showed immunostaining of the rp np protein from sars-cov- , which was prominently expressed on alveolar epithelial cells, as well as in cell debris within the alveolar space [ ] . along the respiratory tract, ace has been observed in nasal and bronchial epithelial cells. in addition, ace is abundantly expressed on the surface of alveolar type ii pneumocytes, which also co-express several other genes that are involved in the regulation of viral reproduction and transmission, including tmprss [ , ] . type ii pneumocytes usually produce surfactant, maintain their self-renewal and exert immunoregulatory functions. importantly, these cells share the same basement membrane with closely apposed capillary endothelial cells, also expressing high ace levels. these data indicate that type ii pneumocytes together with the related capillary endothelium may be a primary site of sars-cov- entrance, resulting in damage to those cells and the alveolo-capillary membrane and ongoing reactive hyperplasia of type ii pneumocytes. as type ii pneumocytes remain targets of viral entry and replication, this may lead to a vicious circle of continuing alveolar wall destruction and repair, eventually culminating in progressive severe diffuse alveolar damage. ace upregulation has also been described in airways in patients with chronic respiratory disease who are smokers, which, together with disturbed ciliary movement and abnormal this article is protected by copyright. all rights reserved. mucus viscosity, may increase disease vulnerability [ ] . however, clinical evidence may indicate that smoking does not necessarily lead to increased vulnerability [ ] . recently, it was suggested that the virus could also exploit goblet cells and ciliated cells in the nasal epithelia as entry portals, a plausible primary infection site in many patients [ ] . although covid- is primarily a severe respiratory illness, acute myocardial injury is frequently observed, manifested by increased levels of high sensitivity cardiac troponin i (ctni) or cardiac troponin t (ctnt) in up to % of laboratory-confirmed covid- patients [ , ] . the presence of myocardial injury was associated with worsened outcome, with -to -fold increased mortality rates. the highest mortality rates were observed in patients with both elevated tnt levels and pre-existing cardiovascular disease. reciprocally, pre-existing cardiovascular disease predisposes for sars-cov- -induced myocardial injury and covid- -associated mortality. whereas the relation between myocardial injury (associated with myocardial infarction, heart failure and ventricular arrhythmias) and mortality is evident, the aetiology of acute myocardial injury in response to sars-cov- infection is still unresolved. however, several potential mechanisms have been proposed, including sars-cov- -induced myocarditis, cytokine-mediated injury (i.e. a systemic cardiotoxic cytokine-storm), microvascular injury, or stress-related cardiomyopathy or myocardial infarction [ , ] . virus-induced myocarditis due to infection of cell populations residing in the heart has also been proposed, though this is still unproven [ ] . scattered individual cardiomyocyte necrosis was observed in cardiac tissue from deceased covid- patients, however without clear this article is protected by copyright. all rights reserved. signs of myocarditis [ ] . given the critical role of ace for sars-cov- cell entry, resident ace -expressing cell populations in the heart can be potentially infected. single-cell rna sequencing of discarded donor hearts revealed that pericytes, but not cardiomyocytes, express highest ace levels [ ] . this suggests that cardiac pericytes form a potential sars-cov- target cell, which may cause capillary endothelial cell dysfunction upon infection culminating in myocardial injury. so far, only one case report has been published on the presence of sars-cov- in the heart and demonstrated viral particles in interstitial cytopathic cells, most likely macrophages, but not cardiomyocytes or endothelial cells [ ] . direct cardiotoxic effects and presence of sars-cov- in the heart needs to be confirmed in larger series. as ace is abundantly expressed by endothelial cells throughout the body, it loses its ability to prevent thrombosis upon cell entry of sars-cov- [ ] . in human umbilical vein endothelial cell cultures in vitro, ace has been shown to have a role in protection of endothelial function and inhibition of the inflammatory response [ ] . in experiments with spontaneously hypertensive rats, ace activation reduced thrombus formation and platelet attachment to vessels, while these effects were reversed by inhibition of ace [ ] . putatively, direct infection of endothelial cells by sars-cov- could result in systemic impaired microcirculatory function in different vascular beds. in fact, sars-cov- has recently been shown to directly infect engineered human blood vessel organoids in vitro [ ] . the permissiveness of endothelial cells in vivo for sars-cov- was demonstrated in renal glomerular endothelial cells by electron microscopy [ ] . however, since no immunohistochemistry or immune electron microscopy was performed, it remained difficult this article is protected by copyright. all rights reserved. to distinguish between intracellular viral inclusions and normal subcellular organelles, as the latter may masquerade as viruses [ ] . furthermore, covid- was associated with endotheliitis in various organs such as lung, liver, heart, kidney and small bowel [ , ] . this suggests that direct infection of endothelium and/or perivascular inflammation may result in endothelial dysfunction, tissue oedema and a pro-coagulant state culminating in microvascular pathology, in particular in patients with pre-existing endothelial dysfunction. thrombo-embolic risk covid- patients are at particular risk for developing coagulopathy reminiscent of disseminated intravascular coagulation (dic) which was associated with mortality, possibly due to both venous and arterial thrombosis [ ] . arterial thrombosis includes ischaemia of extremities, cerebral infarctions and myocardial infarctions [ ] . after initial reports of an increased rate of venous thromboembolism (vte), including deep venous thrombosis (dvt) and pulmonary embolism (pe), a recent dutch study demonstrated a vte incidence of % and ~ % arterial thrombosis in covid- patients admitted to the icu [ ] . in this study, the vast majority ( %) of patients with vte suffered from pe. pe could be an important factor in abrupt worsening of respiratory failure in patients with advanced covid- [ ] . furthermore, several autopsy studies showed thrombi in the pulmonary vessels, which can be proximal large emboli but are most frequently identified as microthrombi. this microvascular thrombosis is predominantly observed in an environment of marked inflammatory changes including mononuclear cell infiltrates, virally infected cells and diffuse alveolar damage [ ] . this article is protected by copyright. all rights reserved. in clinical studies, strongly elevated levels of circulating biomarkers of endothelial activation have been reported [ ] . also, a clear picture of hypercoagulability is observed, with elevated d-dimers being most strikingly elevated in patients with severe disease [ , [ ] [ ] [ ] [ ] . d-dimer is a fibrin-degradation product that develops after a blood clot is degraded by fibrinolysis. moreover, d-dimer levels at hospital admission predict a worse clinical outcome [ , ] . although d-dimers are a biomarker for thrombosis, they are also known as strong acute-phase reactants. however, high d-dimer levels seem to persist in advanced covid- patients in whom inflammatory markers such il- have already decreased, stressing that their elevation is not solely secondary to systemic inflammation [ ] . furthermore, as covid- patients generally present with normal to slightly elevated platelet levels, strongly increased fibrinogen and normal to only slightly prolonged prothrombin and activated partial thromboplastin time [ ] , thromboembolic events in these patients do not seem to be a result of an hypofibrinolytic consumptive diffuse intravasal coagulation as generally observed in sepsis [ ] . interestingly, strongly increased levels of antiphospholipid (anticardiolipin and anti-β glycoprotein i) antibodies have been reported in covid- patients with venous and arterial thromboembolisms, which is a feature of the antiphospholipid syndrome (aps) [ , ] . patients with systemic lupus frequently present with aps and limb ischaemia caused by vasculopathy. in a clinical study in systemic lupus, anti-ace antibodies were found to be elevated in almost every patient and correlated with the relative activity of serum ace [ ] . furthermore, systemic lupus patients overexpress ace as a result of hypomethylation, and their vascular complications respond very well to hydroxychloroquine this article is protected by copyright. all rights reserved. treatment, being circumstantial evidence of a speculative link between ace and vascular complications in covid- [ ] . in summary, these observations underline that the hypercoagulable state in covid- may be of a systemic nature, and not limited to pe [ ] . gastrointestinal (gi) symptoms are commonly observed in patients with covid- . in a meta-analysis of , patients, pooled prevalence of gastrointestinal symptoms was . % [ ] . moreover, viral rna has been repeatedly detected in stool samples [ , ] : in the aforementioned study, the pooled prevalence of positive samples was . %. commonly observed gi symptoms include anorexia, diarrhoea, vomiting, and abdominal pain [ ] . in this study, diarrhoea as initial disease symptom was reported in % of patients, but seemingly no bloody diarrhoea. in addition, patients with digestive symptoms seemed to have a longer time from disease onset to hospital admission and presented with evidence of prolonged coagulation and elevated liver enzyme levels [ ] . theoretically, sars-cov- could directly invade the gastrointestinal epithelium via ace . in a single-cell transcriptome study, ace was found to be highly expressed in oesophageal upper and stratified epithelium, as well as in absorptive enterocytes derived from both ileum and colon [ ] . in addition, ace was co-expressed with the tmprss prime protein in absorptive enterocytes and upper oesophageal epithelial cells. in our previous study from , we found ace to be expressed in enterocytes of all parts of the small intestine, including duodenum, jejunum, and ileum, but not in colonic enterocytes [ ] . more specifically, ace was densely stained at the villous brush border, but also deeper into the intestinal wall, particularly in smooth muscle this article is protected by copyright. all rights reserved. previously, proteomics analyses demonstrated that ace protein is increased in ibd [ ] . furthermore, ace activity and elevated angiotensin( - ) concentrations were described in patients with ibd [ ] . in that study, it was shown that ang ii and angiotensin ( - ) influence colonic myofibroblast proliferation and collagen secretion, and the use of aceinhibitors (aceis) and angiotensin-receptor blockers (arbs) associated with improved disease outcome in ibd patients [ ] . until now, there is no evidence for increased susceptibility for covid- in patients with ibd. the implications of covid- for immunomodulation in ibd have recently been reviewed [ ] . previously, viral rna in faeces could be detected after viral rna in the respiratory tract became negative and evidence for gastrointestinal infection of sars-cov- was documented recently, i.e. infectious virus could be isolated from the stool [ , ] . however, another recent study did not find evidence for the presence of infectious virus in rna-positive stool samples [ ] . altogether, these observations suggest that sars-cov- actively infects and replicates within the gi tract, implying a possible role for a faecal-oral viral transmission route. liver manifestations have also been reported in covid- patients. biochemical signs of mild-to-moderate liver injury are frequently observed, including elevated liver function tests (ast, alt, γ-gt and alp), hypoalbuminaemia, prolonged prothrombin time, and increased crp, ldh and hyperferritinaemia, which may be reflective signs of acute-phase inflammation [ ] . liver damage may be primarily attributed to direct viral infection this article is protected by copyright. all rights reserved. causing hepatitis, but may also be interpreted as drug toxicity by administration of high-dose antiviral medications, antibiotics or steroids [ ] . ace is expressed in the liver, mainly on cholangiocytes instead of hepatocytes, and it has been suggested that ace might be upregulated by compensatory hepatocyte proliferation upon cholangiocyte injury [ ] . to date, however, little is known about direct viral infection of the liver by sars-cov- . one study on liver biopsy specimens showed moderate microvascular steatosis and mild lobular and portal activity, though it was unclear whether this was caused by sars-cov- infection or by drug toxicity [ ] . another study observed mild lobular infiltration by small lymphocytes, patchy necrosis and centrilobular sinusoidal dilation [ ] . interestingly, a recent single-cell transcriptomics study found high ace expression on cholangiocytes, suggesting that sars-cov- may also lead to damage of intrahepatic bile ducts [ ] . taken together, one may hypothesise that hepatobiliary involvement in covid- primarily results from biliary infection, with secondary injury to hepatocytes. recent evidence points towards significant involvement of the kidney in covid- . whereas initial studies reported a relatively modest risk for acute kidney injury (aki), subsequent studies reported an incidence rate up to % [ ] . occurrence of aki in covid- patients is associated with higher disease severity in icu-admitted patients, and is an adverse prognostic sign for survival [ ] . small studies of covid- patients have reported signs of proteinuria and haematuria in about % of hospital-admitted patients [ ] . ace expression has been confirmed on the brush border of proximal tubular cells and on this article is protected by copyright. all rights reserved. podocytes, whereas glomerular endothelial and mesangial cells were weakly positive or negative for ace [ ] . in the previous sars outbreak, renal involvement was a rare phenomenon, although, if present, aki was often a fatal disease complication [ ] . further research provided evidence that this renal involvement, in the form of aki, may be more attributed to processes behind multi-organ failure rather than active viral replication of sars viruses [ , ] . for instance, crs or cytokine storms have been reported as prior events leading to severe renal damage [ ] . more recently, sars-cov- viral antigens have been detected in post-mortem specimens, specifically in kidney tubules [ , ] . in another recent study, histopathological analysis of post-mortem findings revealed diffuse acute tubular injury (ati) with loss of brush border, non-isometric vacuolar degeneration and even necrosis, as well as prominent erythrocyte aggregates occluding the capillary lumina with resulting endothelial damage (figure c-d) [ ] . in line with the tissue distribution of ace in the kidney, coronavirus-like particles were identified in tubular epithelium and in podocytes. based on these recent findings, it is suggested that sars-cov- directly targets the kidney parenchyma, especially the renal tubular epithelium and podocytes, with secondary endothelial injury that may induce aki and lead to proteinuria and elevated serum creatinine levels in these patients. moreover, sars-cov- infections seem to be more frequently associated with aki compared to sars-cov- [ ] . the increased binding affinity of sars-cov- to ace may explain this phenomenon, as it would allow for greater renal infectivity. this article is protected by copyright. all rights reserved. in skin, ace has been demonstrated in the basal epidermal layer and eccrine sweat glands [ ] . however, reports on skin involvement started to emerge only recently. the extent and origin (reactive, direct viral damage, thrombosis, vasculitis) of skin involvement in covid- , and the relation to severity of covid- , remains to be established. following two large covid- cohort descriptions only mentioning 'skin rash' without further details in a minority of patients [ , ] , several case reports have emerged reporting abnormalities ranging from erythematous rash, urticarial plaques, purpura, to chickenpox-like vesicles, without information on histopathology [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, recalcati et al. reported skin alterations similar to the aforementioned case reports in of ( . %) medication-naive covid- patients [ ] . again, no histopathology was available. very recently, one case report linked covid- to the occurrence of immune thrombocytopenic purpura [ ] . additionally, another study reported purpura and livedo racemosa in several severely affected covid- patients with small vessel thrombosis with co-localization of complement and sars-cov- spike proteins on histopathology [ ] .this indicates direct viral infection of the small skin vessels. however, the diversity of skin features reported in covid- patients suggests other pathogenic mechanisms as well. in healthy skin, the layers above the ace expressing stratum basale, including the stratum corneum, likely provide a barrier to the virus. however, the clear expression of ace in skin suggests that if sars-cov- gets the chance to reach its receptor there, for example through damaged skin, it may be able to render itself a porte d'entrée in keratinocytes. given that sars-cov- was previously found in sweat [ ] , this raises the question whether sars-cov- could be excreted in sweat, thereby adding to its transmission potential. in addition, it raises the question whether sars-cov- is this article is protected by copyright. all rights reserved. able to infect through binding to ace in the eccrine sweat glands of palmar skin, where they are abundantly expressed. pregnancy is a unique physiological state in which a semi-allogeneic fetus (and placenta) is accepted by the maternal immune system, whilst at the same time this system has to maintain the protective capacity for defence against pathogens. due to the necessary adaptations in the immune system and a variety of physiological adaptations (e.g. increased oxygen consumption, mucosal oedema of the respiratory tract), pregnant women are generally characterised by increased susceptibility to respiratory pathogens, and consequently, severe pneumonia. although there is no evidence that pregnant women are more susceptible to sars-cov- infection, they may be at increased risk of developing severe illness when contracting sars-cov- infection. currently, there is limited evidence regarding the possibility of mother-fetal intra-uterine vertical transmission in covid- . most descriptions of sars-cov- infected pregnant women reported infections during the third trimester of pregnancy [ ] [ ] [ ] [ ] . however, uncertainty prevails about whether vertical transmission of covid- may occur in any phase of pregnancy [ ] [ ] . placentas, amniotic fluid samples or newborns (directly after delivery) with positive rt-pcr results have not been described, which means that there is no virological evidence of intra-uterine infection at the maternal-fetal interface [ , [ ] [ ] . neonatal covid- has been reported, but infection could have occurred through other routes as there was no direct evidence for intrauterine vertical transmission [ , , , , ] . in a small case series, fetal growth restriction this article is protected by copyright. all rights reserved. (fgr) has been described in sars-cov- -positive women, but no details of the placental histopathological lesions were described [ ] . there is one report that described seven placentas that were evaluated histopathologically after maternal infection with sars-cov- . placentas from infection in the first trimester were normal (n= ). increases in intervillous and subchorionic fibrin deposition were observed once delivered in the acute stage of infection (n= ) which is possibly not sars-cov-specific, but rather related to disturbances in maternal placental blood flow due to hypoxic respiratory disease. third trimester convalescent infection resulted in extensive fetal thrombotic vasculopathy (ftv) with sharply demarcated zones of avascular fibrotic villi resulting in fgr (n= ). the aetiology of the ftv might be related to thrombotic tendency due to sars-cov infection or placental hypoxia [ ] . ace ould also play a role in this process. however, although placental ace expression is found on both the fetal site (umbilical cord, placental villi in the syncytiotrophoblast, cytotrophoblast, vascular endothelium and smooth muscle cells) and on the maternal site (e.g. in the invading and intravascular trophoblast and decidual cells), regulation of placental ace expression has not yet been described in relation to sars-cov- infection [ , ] . sars-cov- has been implicated to have neurotropic potential in covid- [ ] . indeed, some patients presented with symptoms that could be attributed to neurological involvement, such as headache, confusion, anosmia, dysgeusia, nausea and vomiting [ , ] . previous research showed that sars-cov- and mers-cov infect the central nervous system with significant involvement of the brainstem [ ] . it has been suggested that neuroinvasion of this article is protected by copyright. all rights reserved. the brainstem may be at least partially responsible for respiratory symptoms in covid- patients, by compromising neurons within the respiratory centres and chemosensitive neural cells involved in respiratory and cardiovascular regulation [ ] . ace may play a role in sars-cov- neuroinvasion, as it is expressed in the brain on neurons and glial cells, particularly in the brainstem and cardiovascular regulatory areas, including the nucleus tractus solitarius, paraventricular nucleus and the rostral ventrolateral medulla [ , ] . in addition, ace is expressed in the cerebral vascular endothelium, which could lead to endothelial damage subsequently leading to viral access to the brain [ , ] . in an experimental animal study, it was demonstrated that the sars-cov- enters the brain primarily via the olfactory bulb, followed by transneuronal spread of the virus [ ] . this could explain the underlying pathophysiology of covid- -associated anosmia. however, detailed neurological investigation of covid- autopsies could further clarify the occurrence and underlying neurological pathology characteristic for sars-cov- infection. initially, sars-cov- may pass through either the mucous membranes in the upper respiratory tract, primarily the nasal and pharyngeal epithelia, or directly entering the lower respiratory tract and infect bronchial and alveolar epithelial cells [ ] . the main symptoms of respiratory infection are fever and cough. in this initial phase, the virus can enter the peripheral bloodstream via the lungs and may result in viraemia [ ] . the virus may then proceed to affect other organs expressing ace , such as the heart and blood vessels, the kidneys and the gi tract. however, the gi tract may also have direct infection by the oral this article is protected by copyright. all rights reserved. route. patients with an increased risk of developing severe disease may experience severe pulmonary involvement resulting in systemic inflammation [ ] . the massive inflammatory process at that time results in a severe cytokine storm also affecting other organs in the body. this seemingly occurs in line with other blood-derived viruses entering organs via ace on activated endothelium causing, for example, renal or gi problems. in the vasculature, this coincides with red blood cell aggregation and thrombosis. the clinical phase progresses from the initial viraemia to an acute phase (pneumonia), followed by either recovery or severe disease (including ards, aki and eventually multi-organ failure) requiring icu admission [ ] . the distinction would depend on patient comorbidity, obesity-induced pre-existent inflammation, immune function and ace/ace balance in already affected organs. each phase demands its own treatment regimen ranging from virus entry and replication inhibition in the initial phase to anti-inflammatory and anti-thrombotic medication at later stages. in the following paragraphs, we aim to highlight some of the most commonly advocated treatment strategies being explored to combat covid- . hydroxychloroquine (hcq) and chloroquine (cq) are two widely used antimalarial, antiviral and anti-rheumatic drugs. recently, in vitro results and small clinical studies emerged that demonstrated antiviral activity of these drugs against sars-cov- infection [ ] [ ] [ ] [ ] [ ] [ ] . beneficial effects were presumed to arise from blockage of viral host cell entry by increasing endosomal ph and interference with glycosylation of ace [ ] . two studies from france this article is protected by copyright. all rights reserved. reported that hcq could lead to viral load reduction within six days, especially when combined with azithromycin. however, these studies were impaired by several methodological constraints [ ] . similarly, two chinese trials were performed: one study reported no significant difference in nasopharyngeal viral carriage between hydroxychloroquine treatment and standard supportive care, whereas the other study demonstrated shorter clinical recovery time for patients receiving hcq compared to placebo [ , ] . for the latter study, however, it was not possible to extrapolate to critically ill patients, which is crucially important because this subgroup of patients may be of particularly increased risk of serious adverse effects upon treatment with hcq/cq, such as ventricular arrhythmias, hepatic failure and cardiac toxicity [ , ] . indeed, recent studies reported concerns about potential safety hazards as higher dosages were associated with higher mortality and excessive qt interval prolongation, especially when taken concurrently with azithromycin and oseltamivir [ , ] . another large observational study indicated that hcq/cq may not help in critically ill patients as its administration was not associated with either a significantly lowered or an increased risk of a composite endpoint of intubation or death [ ] . thus, currently available data on hcq/cq treatment for covid- are inconclusive, but appear far from promising. therefore, upcoming prospective randomised clinical trials will have to determine if treatment with hcq/cq would be a reasonable therapeutic strategy for covid- patients and what would be the most suitable timing within the disease course to initiate treatment. this article is protected by copyright. all rights reserved. remdesivir, a rna polymerase inhibitor, was demonstrated to be effective against sars-cov- and mers-cov. for instance, remdesivir improved disease outcome and reduced viral load in sars-cov- infected mice [ ] . in hospitalised patients with covid- , improvement of clinical status was observed in after receiving at least one dose of remdesivir [ ] . furthermore, a recently conducted randomised controlled trial evaluated the role of lopinavir and ritonavir in covid- patients, treated with lopinavir/ritonavir while received standard treatment [ ] . the authors concluded that patients treated with lopinavir/rotinavir did not demonstrate any significant improvement in hazard ratio for earlier clinical improvement or reduction in mortality at days. in contrast to the primary outcome, patients treated with lopinavir/ritonavir demonstrated clinical improvement day earlier than the control group and were discharged days earlier from the icu. although large clinical trials investigating the therapeutic effect of these antiviral therapies in covid- are lacking, it can be hypothesised that the available studies possibly included patients with severe disease alone, and, therefore, future studies may consider evaluating the role of these antiviral drugs earlier in the course of covid- [ ] . the worldwide growth of sars-cov- infections raised serious concerns about the widespread use of antihypertensive drugs, i.e. aceis and arbs, which are also used in treatment of cardiovascular diseases, chronic kidney disease and diabetes mellitus [ ] . discussions emerged about whether these drugs may exert beneficial or deleterious effects in covid- . many opinion papers have been published recently that predominantly state that this article is protected by copyright. all rights reserved. there is no scientific evidence to change the prescription of aceis or arbs for the management of hypertension in the context of preventing or treating sars-cov- infection. the use of aceis and arbs as risk factors for developing or aggravating covid- has been suggested because of their capacity to upregulate ace [ ] [ ] [ ] . however, others have advocated beneficial and protective effects of these drugs in the development of covid- [ , ] . in some animal studies, aceis or arbs increase ace levels, whilst other studies failed to demonstrate such shifts in ace [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although shifts in ace/ace balance were noted [ ] . therefore, it remains relevant to question whether raas blockers actually increase susceptibility to sars-cov- infection by increasing ace . ace is protective against severe lung injury in animal models and ace blockade or genetic ace knockouts result in extensive lung damage and decreased survival after respiratory syncytial virus infection [ ] . similarly, at r blockade by losartan attenuates lung injury in mice administered with the spike glycoprotein of sars-cov- [ ] . although few human studies have been performed investigating potential effects of raas therapy on ace expression and/or activity, it was recently reported that aceis and arbs did not increase plasma ace concentrations [ ] . similarly, others reported no clear direct effects of aceis on ace activity (as evaluated by angiotensin( - ) levels) [ , ] . several hypotheses exist about how increased tissue ace expression may be protective rather than harmful during sars-cov- infection [ ] . for example, increased ace expression may lead to enhanced sequestration of sars-cov- , but does not imply automatic activation of further downstream processes essential for viral entry, such as this article is protected by copyright. all rights reserved. involvement of tmprss , which is required for spike glycoprotein priming, or adam metallopeptidase (adam ), which is required for cleavage of the ace ectodomain resulting in increased ace shedding. furthermore, arbs lead to competition with ang ii for at r, resulting in increased ang ii to be processed by ace . this increases ang( - ) levels, which results in vasodilating and anti-fibrotic effects, providing crucial protection during coronavirus infections [ ] . furthermore, increased binding of ace to circulating ang ii could induce a conformational change resulting in less favourable binding of sars-cov- to its receptor and decreased internalization of the virus when bound to ace [ , ] . we previously observed a positive shift in plasma ang( - )/ang ii balance in favour of the beneficial ang( - ) peptide, particularly in circumstances of low sodium intake [ ] . importantly, however, plasma ace levels may be less indicative of the risk of sars-cov- infection or membrane-bound ace activity, as ace shedding by adam appears to be regulated separately [ ] . interestingly, however, plasma ace concentrations appear to be higher in older men with heart failure independent of raas inhibition [ ] . clinical trials investigating the potential (side-)effects and safety of aceis and arbs on ace expression and activity in covid- are ongoing. from a clinical perspective, it may be preferable to await these results instead of discontinuing raas inhibitors, which may lead to clinical derangement especially in patients at high-risk for covid- [ ] . since currently available evidence indicates that aceis and arbs significantly reduce mortality in cardiovascular disease, reduce progression of ckd, and are crucial in treatment of heart this article is protected by copyright. all rights reserved. failure and hypertension, most clinicians tend to maintain these regimens for their patients, regardless of sars-cov- [ ] . immunomodulating drugs or biological response modifiers alter the host immune system by interacting with a specific target crucial for disease pathogenesis [ ] . many of these compounds enrich the therapeutic armamentarium of several malignancies, autoimmune disorders, transplantation rejection, as well as infectious diseases. especially since vaccine development is time-consuming and antiviral drugs may have a limited therapeutic window, targeted immunomodulators are attractive alternatives. furthermore, these therapies may be crucial to control the hyperactivation of host inflammatory responses and 'cytokine storm' as has been described for covid- [ ] . however, caution should be taken towards this therapeutic strategy as it will remain challenging to target immune system components without compromising the host defence mechanisms necessary to fight sars-cov- infection. in this respect, targeting specific or limited effector mechanisms (e.g. production of pro-inflammatory cytokines or reactive oxygen species), should be preferred over blockage of more proximal immune targets (e.g. pattern recognition receptors) that play a more significant role in regulating host immune defence [ ] . the current hypothesis is that a cytokine storm can induce or further aggravate sars-cov- infection, and thereby suggests that blocking cytokine pathways could attenuate the disease this article is protected by copyright. all rights reserved. course. among these, interleukin- (il- ) is thought to play a prominent role. il- is a cytokine with both anti-and pro-inflammatory effects. it can be produced by almost all stromal and immune system cells (monocytes, lymphocytes, macrophages, endothelial cells, mast cells, dendritic cells) and is believed to play a central role in the development of cytokine storm [ , ] . in line with this reasoning, anti-il r therapy is a potential therapeutic option in covid- . currently available humanised monoclonal antibodies against the interleukin- -receptor (tocilizumab and sarilumab) are being tested in covid- . a small study demonstrated that tocilizumab ameliorated the increased crp in all patients, which is a direct effect of its pharmacological action. moreover, in critically ill patients with elevated il- levels, repeated doses of tocilizumab could be beneficial. however, objective clinical endpoints were not reported [ ] . although others have shown comparable results, data on the use of tocilizumab are still preliminary and larger randomised controlled trials are needed [ ] [ ] [ ] . whether anti-il r therapy should be started early in the course of the disease or restricted to patients with signs of a cytokine storm is still of debate [ ] . in addition, other cytokines such as il- , ifn-γ and tnf-α are abundant in the cytokine storm, and blocking these pathways with appropriate biological being investigated [ ] . inhibition of the jak-stat signalling pathway has also been suggested as a potential targeted therapy for covid- and several clinical trials are ongoing [ , ] . inhibitors blocking jak , such as fedratinib, have been suggested to block viral entry and combating the th -component of the host inflammatory cytokine storm, without altering interferon this article is protected by copyright. all rights reserved. signalling [ ] . sars-cov- enters host cells via ace -mediated endocytosis, which is controlled by upstream regulators including ap -associated protein kinase (aak ) and cyclin g-associated kinase (gak). one of several high-affinity inhibitors of these regulators is the jak inhibitor baricitinib, which may limit viral host cell entry and intracellular assembly of viral particles through disrupting aak and gak. baracitinib may be of particular value during the hyperinflammatory phase, in which high levels of cytokines occur that signal through the jak-stat pathway. however, the optimal time to administer cytokine inhibitors still needs to be determined and results from the aforementioned clinical trials should be awaited. the association between obesity and the progression to hypoxic respiratory failure in patients with covid- , requiring mechanical ventilation, has led to the assumption that leptin and adipokines may play a key role in this subpopulation of sars-cov- infected patients. resveratrol, an antioxidant and food supplement, has been suggested to be of potential therapeutic value because of a triple action. first, in some studies, resveratrol reduces leptin levels [ ] . second, resveratrol could suppress ang ii, which might reduce inflammation [ ] . third, antioxidant effects in the lung may reduce oxidative stress-induced lung damage [ ] . this food supplement is safe in its use (up to - grams per day) and should be studied in covid- patients as an additive to other treatments. this article is protected by copyright. all rights reserved. as a result of the increased risk of thrombotic events in covid- , guidelines currently advocate liberal use of prophylactic systemic anticoagulation [ ] . the international society on thrombosis and haemostasis recently recommended that all hospitalised covid- patients, even those not admitted to icu, should receive prophylactic-dose low-molecularweight heparin (lmwh) unless they have contraindications (active bleeding and platelet count < x /l) [ ] . however, a recent study showed that despite adequate treatment with prophylactic low-dose lmwh, covid- patients admitted to icu were still at a substantial risk for pe [ ] . this has made the dutch federation of internists decide to recommend a double dose of lmwh in icu patients with covid- , when bleeding risk allows this strategy [ ] . other guidelines advocated prophylactic systemic anticoagulation with unfractionated heparin rather than lmwh [ ] , which may be needed in high dosages because of heparin resistance [ ] . however, it is unlikely that anticoagulant treatment has a direct disease-modifying effect and it should be stressed that the initial viral load, as well as the systemic inflammatory response, needs to be attenuated since these are the driving forces for vte in covid- [ ] . future studies are warranted to determine the most suitable approach for thrombosis prophylaxis in covid- . scientific findings about ace and its role in covid- pathophysiology, it is crucial to maintain integration of available pathological and molecular evidence to establish the definite role of these potential modulating factors. unraveling the pathologic basis of covid- is essential for our understanding of the pathophysiology of the disease. unsurprisingly, severe pathological findings are mainly observed in specific target organs of sars-cov- , such as the lungs and kidneys. in severe cases, this may lead to ards and multi-organ failure not directly related to ace expression and activity. this review focused on the role of widespread ace tissue expression, which may become a reasonable therapeutic target together with its effector pathways, for example through implementation of recombinant human ace (rhace ) therapy or by targeting bradykinin metabolism in the lungs. however, it will also be important to focus on additional mechanisms that may be involved in cellular infection and may regulate the interaction between sars-cov- and ace . future studies featuring higher numbers of patients are warranted to reliably assess potential differences in ace expression, activity and regulation under a variety of physiological circumstances, such as present or lacking interaction with co-receptor or coactivating molecules, as well as in the context of commonly observed underlying conditions, including cardiovascular disease, hypertension, diabetes, obesity, smoking and respiratory disease. in particular, pathological studies of larger series of autopsy findings, probably in human and non-human primate models alike, are required to more accurately determine the relative contribution of each pre-existent co-morbidity and to discriminate between specific sars-cov- -associated pathology and superimposed pathological changes. furthermore, the this article is protected by copyright. all rights reserved. development of appropriate animal and in vitro models could help to learn more about the sars-cov- infection process itself and, most importantly, the disease progression pattern observed in humans. in any case, it is indisputable that devoting scientific efforts to analyse aspects of ace in relation to covid- pathophysiology is paramount to fuel the development and augmentation of future therapeutic strategies. the current covid- pandemic is a major challenge for public health and clinical medicine. for public health, reduction or prevention of virus transmission as well as reduction of predisposing lifestyle factors need to be implemented. for clinical management in the foreseeable future, we should strive to adopt a personalised medicine approach aimed to provide individually tailored treatment in patients affected by covid- . as highlighted in this review, this should take into account individual patient differences in mutual ace -sars-cov- interactions with their consequences for covid- pathophysiology. to achieve this, it is of cardinal importance to carefully register the individual patient phenotype and to integrate this with diagnostic (e.g. laboratory and imaging results) and therapeutic information (e.g. drug toxicity and side-effect profiles). mainly because of low patient numbers in individual studies, currently ongoing trials are challenged to take into account between-subject differences or cohort heterogeneity, which may be considered likely to explain the majority of variation in disease outcome. however, detailed phenotypical stratification of individual patients during their disease course will provide us with the necessary input for sophisticated clinical algorithms to be used for predictive modelling. consequently, these will allow us to identify rational therapeutic strategies tailored to a patient's clinical status. as such, we would distance ourselves from the "one size fits all" characteristics of and important lessons from 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events and apparent heparin resistance in patients infected with sars-cov- thromboinflammation and the hypercoagulability of covid the authors would like to express their gratitude towards else koning for her valuable help in the graphical design of the figures and martin bourgonje for critically proofreading the manuscript. in addition, the authors would like to thank dr. jan von der thüsen (department of pathology, erasmus medical center, rotterdam, the netherlands) and dr. hua su (department of nephrology, union hospital, wuhan, china) for kindly providing us with histological images. key: cord- - pyx r authors: grout, andrea; howard, natasha; coker, richard; speakman, elizabeth m title: guidelines, law, and governance: disconnects in the global control of airline-associated infectious diseases date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: pyx r international air travel is increasingly affecting the epidemiology of infectious diseases. a particular public health, economic, and political concern is the role of air travel in bringing infectious passengers or vectors to previously non-endemic areas. yet, little research has been done to investigate either the infection risks associated with air travel or the empirical evidence for the effectiveness of infection control measures on aircraft and at borders. we briefly review the interface between international and national legislation, policy, and guidelines in the context of existing infection risks and possible scenarios. we have found that public health guidance and legislation, which airlines are required to follow, are often contradictory and confusing. infection control measures for air travel need to be underpinned by coherent and enforceable national and international legislation that is based on solid epidemiological evidence. we recommend further research investment into more effective on-board vector control, health screening, and risk communications strategies, and the development of enforceable and harmonised international legislation. low air fares and a multitude of social and economic factors have resulted in increased air travel. the number of journeys taken by passengers each year has grown from approximately million in to more than · billion in . the epidemiology of infectious diseases associated with air travel and the challenges of infection control are important public health concerns, yet they are scarcely discussed in the literature. aircraft can now travel to almost any part of the world within h, and can enable spread of infection either by inflight infection transmission or by transporting infectious passengers or vectors-eg, malaria-infected mosquitoes-from endemic to non-endemic regions, thus putting populations in destination countries at risk. the combination of rising passenger numbers, new travel destinations, and on-board transmission events can influence transmission patterns of several imported diseases, including severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebola virus disease. for example, the ongoing transmission of zika virus disease is believed to have been introduced to the americas by air travel. management of these risks requires knowledge of the dynamics of infectious disease transmission and the potential effectiveness of control measures, suggesting that frontline employees (eg, airline staff) would need appropriate training to handle suspected disease cases. as a result of experiences with sars, the international air transport association (iata) issued the emergency response plan and action checklist, which consists of guidelines and best practices for aircrews during public health emergencies. to reduce the risk of on-board disease transmission, the us centers for disease control and prevention (cdc) provides cabin crews with information on general infection control measures and guidelines to identify ill and potentially infectious passengers. passengers with certain conditions-for example, someone recovering from measles-might require medical clearance; however, guidelines for these conditions vary between countries and can be subject to individual airline policy. the effectiveness of infectious disease response strategies largely depends on prompt identification of cases. current measures, such as entry and exit screening, isolation, quarantine, and travel health information might not be feasible or sufficient to control infectious disease transmission. for example, the value of entry screening measures has been questioned, , while an evaluation of border entry screening measures in several countries concluded that a combination of communication methods (eg, in the form of pre-flight health information, in-flight videos, and clinical guidance) for passengers and clinicians might be a more effective strategy for global infectious disease control. collectively, the unique dynamics and interactions at play in an aircraft environment require a distinct response to infectious disease control. in this personal view, we consider the disconnects between global health law, national jurisdictions, organisational guidelines, and aircrew compliance by discussing existing risks and presenting two infection scenarios based on current airline practice. although the risk of disease transmission exists whenever people congregate in confined spaces, aircraft are unique in having individuals from often diverse geographical regions, with differing population immunity and exposure risks, interacting with aircrews and each other. infection can occur via direct transmission through contact with skin, blood, or other bodily fluids (eg, ebola virus), or via indirect transmission without person-to-person contact. indirect transmission on an airplane can occur through infectious droplets (eg, influenza virus), through contaminated surfaces or objects (eg, meticillin-resistant staphylococcus aureus), or long-distance air travel in particular exposes passengers to several factors that could affect disease transmission. the transmission characteristics of a pathogen, ambient climatic conditions, time spent on board, and aircraft type can affect quantification of the general transmission risk. absolute figures for the risk of in-flight disease transmission are therefore not readily available and the evidence base is inadequate. mangili and gendreau reported in-flight transmission of influenza, sars, tuberculosis, measles, smallpox, and other pathogens. on a h flight from hong kong to beijing in , of passengers were infected with the sars virus by a single ill passenger, while modelling has demonstrated the possibility of in-flight transmission of mers coronavirus. protective measures are in place in modern aircraft, but these measures are not necessarily as robust as assumed. for example, commercial aircraft use highefficiency particulate air (hepa) filters to restrict exposure to small airborne particles. however, there are no regulations requiring that hepa filters be in place, and little testing has taken place to assess the effectiveness of these filters. in , ebola virus disease was brought to the usa, the uk, and nigeria by undiagnosed infected people aboard aircraft. brownstein and colleagues demonstrated the effect of air travel on the global spread of seasonal influenza, noting that decreased air traffic following the terrorist attacks of sept , , was associated with a delayed influenza season. maloney and cetron documented the air-travel-associated transmission of meningococcal disease. global air travel could spur the spread of epidemics by bringing viruses and parasites to new areas. infected mosquitoes on intercontinental flights are believed to have contributed to the global spread of malaria. , west nile virus is widely suspected to have been spread to the usa by an infected mosquito carried by plane. similarly, the introduction of zika virus to the americas coincided with an upsurge of air travel to brazil from endemic countries in . management of the risk of transporting infected passengers requires knowledge of transmission dynamics and the potential effectiveness of airport entry and exit screening measures, the ability to appropriately isolate or quarantine individual passengers on an aircraft, and adequately trained aircrew who are able to identify signs of infection and take appropriate measures. for example, who maintains that the risk of vector-borne diseases being transmitted aboard aircraft is low, but recommends aircraft disinsection (a public health measure involving insecticide treatment of aircraft interiors and holds), stating that "there have been frequent instances of insects of public health importance being introduced from one country to another, with occasional dire consequences". however, the effectiveness of disinsection is unclear. minimisation of the risk of inadvertently carrying insect vectors requires consistent use of effective control measures, including insecticides that are safe for frequent aircrew and passenger exposure. public health measures for international air travel include a range of national and international legislative tools, policies, and guidelines. globally, countries signed the legally binding international health regulations (ihrs), with the aim of controlling global disease spread. however, the only ihr provision relating to air travel is the requirement that all chief pilots provide a brief aircraft general declaration on passenger health to ground staff before disembarkation. the international civil aviation organization (icao) and iata coordinate with who and provide recommendations, but specific controls are left to the discretion of individual countries. national guidance and legislation are uncoordinated across countries, and-with no strong evidence underpinning control measures-they are often inconsistent. following the sars epidemic, iata recommended that all air carriers create an emergency response plan for public health emergencies, but these are only guidelines and legislative powers lie with national authorities. airlines face conflicting obligations, since they must comply with infectious disease controls in both origin and destination countries. airlines owe a duty of care to three different groupspassengers, aircrew, and destination country populations-and these duties sometimes conflict. for example, the us environmental protection agency prohibits usage of some insecticides because of potential risks to aircrew, whereas national laws in australia and new zealand require usage of these insecticides. us airlines flying to these countries must purchase insecticides at stopovers, and airline unions have raised serious concerns about the "inconsistent and inappropriate application", toxicity, and potential adverse health effects of these agents. other airlines have reported difficulties in aircraft storage of aerosol insecticides that were either banned or prohibited from import in some destination countries. additionally, doubt exists as to the effectiveness of disinsection, with research identifying increasing resistance of mosquitos to insecticides. although the icao encouraged more research into non-chemical disinsection procedures in , procedures have not changed and airplane disinsection policies and implementation remain inconsistent worldwide. airlines and national authorities can refuse to transport passengers they consider to be a health risk. the us air carrier access act states that carriage can be refused www.thelancet.com/infection vol april e personal view where a passenger presents with a disease that "is both readily transmitted in the course of a flight and which has serious health consequences (eg, sars, but not aids or a cold)". this rule applies to all flights of us carriers and flights to or from the usa, but it clearly requires any disease to be diagnosed before the flight. considerable debate continues about the effectiveness and practicality of screening passengers at entry, exit, or both. further research must be prioritised before national and international legislation can take a consistent, evidence-informed approach to screening, because flight duration and pathogen transmission dynamics are just two important factors that challenge one-size-fits-all recommendations. enforcement of national laws is highly variable, with non-compliance carrying financial penalties and criminal sanctions in some countries, but little evidence of enforcement in others. countries are signatories to the montreal convention, which imposes obligations to protect passengers. however, although this convention enables compensation claims to be made, proving an airline's liability for a passenger contracting an infectious disease during the flight can be challenging evidentially. even if transmission time can be proven, airlines can defend the extent to which they should have been expected to identify the risk. they can argue that liability should lie with the infectious passenger who took the flight without notifying the airline or health authorities. although industrial injury claims have been brought on behalf of aircrew for alleged adverse reactions to constant insecticide exposure in aircraft, these claims have been defended on the basis that airlines were following who guidelines. , the montreal convention does not apply to individuals in a destination country who could become infected by a passenger or imported vector. although regulatory liability might still exist and personal litigation against an airline could be undertaken, proving causal transmission will again be very difficult, particularly if the disease in question did not become symptomatic until some time after the flight had landed. two hypothetical scenarios are provided to show the potential occurrence and wider implications of disease transmission on aircraft. ebola is an infectious and often fatal disease marked by fever, nausea, vomiting, and-less frequentlyhaemor rhaging, and spread through infected bodily fluids. on a flight from frankfurt to washington, dc, a -year-old passenger started complaining of a severe headache, abdominal pain, nausea, and sweating. he recalled no specific symptoms before boarding, but claimed he had been feeling generally unwell since his arrival from abuja, an interim stopover on his itinerary that had originated in kampala days earlier. about h into the flight, his symptoms worsened and the cabin supervisor requested medical assistance. as there was no doctor on board, a nurse examined the passenger and, suspecting he might be infectious, advised the crew to "isolate him as a precautionary measure". the passenger was taken to a seat near the galley and looked after by two crew members for the remainder of the flight. meanwhile, he had violent bouts of vomiting and became increasingly disoriented. the cabin supervisor notified the chief pilot of a sick passenger, but did not communicate the severity of his condition. the pilot assumed the situation was controlled and did not contact us health authorities. upon landing, the passenger's condition had deteriorated and an ambulance was requested. after h, the passenger was determined to be positive for ebola virus. this scenario demonstrates an absence of communication between crew members and between aircrew and ground staff in the destination country. this miscommunication delayed notification of a potentially severe health risk from infected bodily fluids, such as vomit; moreover, an ambulance with infection control facilities should have been requested while the plane was airborne. this represents non-compliance with iata guidance and a potential criminal breach of us health and quarantine laws. us laws are enforceable against both individuals and organisations, with penalties including fines and imprisonment. , vector-borne diseases (eg, malaria, yellow fever, and zika virus) are transmitted by mosquitoes or other vectors to human beings, and contribute to a substantial proportion of the global infectious disease burden. mosquito ecology suggests that aircraft are associated with a higher risk of introducing a live infected mosquito than are sea or road transport. following national requirements, disinsection was carried out by aircrew during descent into mumbai airport. the flight had originated in london. a passenger who regularly travelled on this route objected to being sprayed with insecticide, pointing to potentially dangerous adverse health effects. he added that, having travelled with different airlines, he had not witnessed any in-flight spraying for years. on the return flight, several passengers complained about the presence of mosquitoes in the cabin before take-off. the aircraft had been parked on the apron of mumbai airport, with cabin and cargo doors open during baggage loading and passenger embarkation. passengers demanded protection from mosquitoes and wondered why spraying was done upon entering india, but not upon departure. this scenario reflects inconsistencies in, and in adequate monitoring of, disinsection policy. indian national law requires disinsection on inbound flights, but india is itself personal view a reservoir of vector-borne diseases. guidance from who and iata uses permissive rather than mandatory language on disinsection, and thus national policies determine whether countries choose to implement disinsection consistently for all arriving aircraft or only require the process on selected aircraft. policies are not always clear, and it is necessary to balance the fears of health risks from both insecticides and mosquitoes. to be effective, infection control measures for air travel need to be underpinned by coherent and enforceable national and international legislation that is based on solid epidemiological evidence. since aircrew are not infectious disease specialists and would not normally have medical training, recognition of potential disease cases and adequate communication of an in-flight illness remains challenging and ad hoc. the dynamics of existing, emerging, and re-emerging infectious pathogens mean that infectious diseases will always challenge control efforts as pathogens exploit novel evolutionary niches. incoherent guidelines and inconsistently applied laws unnecessarily hinder disease control efforts, and the evidence base underpinning control measures for airlineassociated infectious diseases needs to be strengthened considerably. public health involves balancing the rights of the majority against those of the individual, and issues related to air travel require particular review and improvement by the global health community. first, a systematic review should be done to appraise the evidence supporting control measures for transmission of infectious diseases via air travel. second, airlines and the global health community need to invest in research to identify better, non-toxic insecticides, or non-chemical means to control insect vectors. third, additional research and investment into airport health screening measures is required to better identify infectious passengers. disease transmission can be minimised if passengers take appropriate precautions before or during a flight, or refrain from flying altogether when ill. current education and communication strategies (and refund policies for missed flights) therefore warrant improvement. fourth, these measures cannot be implemented in the absence of enforceable and harmonised international legislation and governance. achieving this goal will be a major challenge, but a starting point could be for international or regional bodies-such as who or the european union-to produce model legislation or standards for the guidance of member states. close consultation with iata and icao would be required to develop such legislation or guidance. enforceability might be encouraged by treating this as a security issue, similar to ensuring the mechanical safety of aircraft. in the context of regular global air travel and evidence of dangerous non-endemic diseases appearing in new, vulnerable populations, the risks of airline-associated infection are growing. potential costs or inconvenience to passengers and aircrews might arguably be a lesser evil than transmission of potentially fatal infections to vulnerable populations. however, without concerted efforts from the global health community, the threat can be expected to worsen. ag developed the scenarios and drafted the manuscript with ems, who wrote on legal aspects. nh contributed to writing and interpretation. rc provided interpretation and critical review. all authors approved the version for submission. we declare no competing interests. air transport, passengers carried: international civil aviation organization, civil aviation statistics of the world and icao staff estimates transmission of infectious diseases during commercial air travel 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vector-borne diseases: overview evidence in australia for a case of airport dengue key: cord- - eh gzd authors: thiemann, alexandra k.; sullivan, rebekah j.e. title: gastrointestinal disorders of donkeys and mules date: - - journal: vet clin north am equine pract doi: . /j.cveq. . . sha: doc_id: cord_uid: eh gzd a review of common gastrointestinal disorders of donkeys and mules is presented. clinically relevant aspects of donkey behavior, anatomy, and physiology are highlighted. diagnosis, management, and treatment of conditions affecting the gastrointestinal tract from stomach to rectum, including liver and pancreas, are discussed. donkeys are kept as companions or used as working or production animals, whereas mules are mainly used for working purposes. gastrointestinal diseases are common in these animals worldwide. awareness of their stoic behavior is paramount when interpreting the severity of clinical signs. donkeys are desert-adapted animals with unique physiology and metabolism compared with horses. knowledge of their particular behaviors and physiology is the key to successful handling and management of gastrointestinal disease. donkeys with colic frequently show milder clinical signs than horses, despite the potential severity of the pathologic condition. major presenting signs include dullness, inappetence, self-isolation, and recumbency. hyperlipemia is a frequent finding in donkeys with colic. triglyceride concentrations should be measured regularly in inappetent animals and treatment instituted. the presence of hyperlipemia reduces the prognosis. it is possible to perform a rectal examination in most donkeys; it is preferable to use spasmolytics before rectal examination. ultrasound evaluation is useful if rectal examination is not possible and can be an adjunct to those cases whereby rectal examination alone has not been diagnostic. the presence of dental disease and parasitism increases the risk of colic; donkeys and mules should be included in routine preventative dental care and anthelmintic use. because of their unique physiology, donkeys differ in their response to dehydration and drug metabolism compared with horses. hematological and biochemical reference ranges are available for donkeys and mules. for many clinicians, the challenge when dealing with donkeys and mules is recognizing behavioral differences compared with horses. donkeys have a highly developed prey species behavior and can mask low-to moderate-grade and chronic pain. common clinical signs in association with gastrointestinal disease include dullness, behavior changes, lack of appetite or sham eating, recumbency, head and neck held below withers height, ears less mobile or backwards/sideways pointing and unresponsive to stimuli, self-isolation away from companions, and weight loss in chronic disease. donkeys show signs of acute colic, such as flank watching, kicking the abdomen, lying down, and rolling, which are less evident than in horses (fig. ) . mules show pain response characteristics of both parent species, being less evident than in horses, but more overt than in donkeys. their physiologic parameters fall between the donkey and horse. in the united kingdom and united states, donkeys and mules are mainly considered companion animals and often reach advanced age, whereas in developing countries, these animals are used for work in agriculture, transport, pulling, and tourism, often lack owner and veterinary care, and have a shorter lifespan. the significance is that donkeys, and to a lesser extent, mules, often present with signs of gastrointestinal disease later than horses, tend to be more systemically ill when signs are noted, and often are afflicted with multiple comorbidities. in addition to a complete history, a clinical examination should be performed in donkeys and mules with gastrointestinal disease. this clinical examination should include an assessment of mentation, general health and body condition, rectal temperature, pulse and respiration, evaluation of mucous membrane color and moisture (hydration status), auscultation of all quadrants for increased or decreased intestinal sounds, a rectal examination, nasogastric intubation in animals with abdominal pain, fecal evaluation for consistency, presence of endoparasites, and poorly digested foodstuffs. in animals with diarrhea, frequency, estimated volume, consistency, and appearance of feces, full dental examination with a mouth speculum, and development of a list of differentials that could lead to abdominal pain and dullness are recommended. normal parameters for adult donkeys include a rectal temperature of . c to . c ( . f to f), a heart rate of to beats/min, a respiration rate of breaths/min (range - ), and moist mucous membranes with a capillary refill time of less than seconds. interpretation of digital pulses in donkeys can be challenging. general points to consider are as follows: because of their pair bonding behavior, donkeys being transported for evaluation ideally should be accompanied by a bonded animal to reduce stress. preventative care (anthelmintic use, dental care, regular vaccination) is often inadequate, in particular, in developing countries. weight loss may be the only indicator of chronic gastrointestinal disease in donkeys and mules. because of the large colon enhanced water absorption capacity, diarrhea is rarely seen with acute or chronic gastrointestinal disease, including colitis. rectal examination should be performed in all, except in miniature animals. intravenous spasmolytics (hyoscine/scopolamine) at standard equine doses facilitate rectal examination. in unhandled donkeys and mules where rectal examination may be dangerous, heavy sedation and the use of stocks are indicated. mules may require between one-third to one-half extra dose of sedatives compared with a similarly sized pony or horse. if rectal examination is not possible, information about the nature of the feces and hydration status is useful. hyperlipemia is frequent in colicky and dull donkeys and can worsen the prognosis, and triglyceride measurement is indicated in these animals. as donkeys evolved in dry and arid environments with limited water access, they can better tolerate dehydration and hemoconcentration than horses. transabdominal ultrasonography may be challenging in obese animals. clipping their thick coat is recommended to facilitate image acquisition. abdominocentesis can be unrewarding in donkeys because of significant fat deposits on the linea alba; a teat cannula or catheter may be indicated, preferably after ultrasound evaluation. a foal-or pony-sized tube can be used for evaluation of gastric reflux and nasogastric therapy. in countries where rabies is endemic, the possibility of infection with this zoonosis must be considered, in particular when performing oral or rectal examination. donkeys without tetanus prophylaxis are susceptible to this condition and develop signs similar to other equids. the donkey and mule have some anatomic differences from horses that are relevant to management and treatment. the head is larger in proportion to body size and gastrointestinal disorders of donkeys and mules supported ventrally by the strong cutaneous coli muscle. this muscle is highly developed in donkeys and can obscure the jugular vein in the midneck region, making jugular venipuncture more challenging. donkeys have a narrower mandible and accentuated mandibular curve of spee, which can lead to dental overcrowding, with rostral and caudal enamel overgrowths. recognition and treatment of dental problems are critical in the management and prevention of many gastrointestinal disorders (fig. ) . see joã o b. rodrigues and gemma lilly's article, "dental disorders of donkeys," in this issue. donkeys have narrow nasal meati; hence, for nasogastric intubation, it is better to use a foal-or pony-sized tube with adequate lubrication and restraint to reduce the risk of hemorrhage. the stomach of a -kg donkey can accommodate approximately l of fluid. the length of the small intestine is around m per kg (vs m for the horse); the transverse colon is short ( cm), and the small colon is approximately m. other aspects of the internal anatomy of donkeys and mules are comparable to the horse. the spinal cord in the donkey terminates at the second sacral vertebra, and the dura may extend to the second coccygeal vertebrae. epidural injections are best done in the second intercoccygeal space at an angle of to the horizontal, space further back and at a shallower angle than in the horse because of the lack of musculature over the rump and tail head. correct placement of the epidural injection is relevant to manage conditions such as rectal prolapse, reproductive manipulation, or orthopedic pain. the donkey, and to a lesser extent the mule, is highly efficient in water compartmentalization and conservation; it is reported that they can lose % to % of body weight and recover faster once fluid is provided. plasma volume is maintained even with % dehydration. under normal conditions, they have a lower urinary output than horses when water is unrestricted. these studies were done on a limited number of heat-adapted animals and should be viewed with caution when assessing companion donkeys in temperate climates. donkeys may show few clinical and hematologic signs of volume depletion until % to % dehydration is reached. skin tenting is an unreliable sign of fluid loss in donkeys and mules (see erin l. goodrich and erica behling-kelly's article, "clinical pathology of donkeys and mules"; and elena barrio and colleagues' article, "clinical evaluation and preventative care in donkeys," in this issue). the hindgut acts as a water reservoir similar to the forestomach in ruminants; donkeys have an enhanced cecal capacity for fluid retention when faced with dehydration, which is evident by the scant diarrhea with colitis. donkeys and mules have a slower gut transit time than horses and digest fiber more efficiently. per body weight, they require less dry matter and energy intake than resting and working horses. clinically, this is reflected by their ability to gain weight easily and develop large fat deposits on the neck, back, rump, around internal organs, and along the linea alba. the donkey intestinal microbiome has not been extensively evaluated, but one can assume that there are differences with the horse and pony. dysbiosis from different conditions (diet, antimicrobials, stress) can be a complication or potential cause of gastrointestinal disease. the risk factors for gastrointestinal disease in donkeys and mules are similar to those in horses, influenced by the management and environment in which they are kept. in donkeys and mules in arid regions and marginal communities with poor access to preventative care, colic may occur because of inadequate water access, fibrous or indigestible feed provision, poor dentition, foreign object ingestion (eg, plastic bags), or heavy endoparasitism (fig. ). parasites such as strongylus vulgaris have higher prevalence in developing countries because of poor management and minimal access to anthelmintics. ingestion of moldy feed or excess seasonal lush grass can result in tympanic colic, whereas grazing low in poor grasslands may contribute to sand colic. donkey studies in the united kingdom have identified impaction colic as having high morbidity and mortality. the pelvic flexure was the main site of impaction, but mortality was higher for cecal impactions. colic impaction is also frequent with dental disease and aging. multiple diastemata, painful periodontal disease, absent molars, and loss of masticatory function increase the incidence of colic. this can be reduced by dietary modifications, including a short chop, high-fiber feed, and provision of regular high-quality dental care. note that diastemata and periodontal disease are not necessarily confined to geriatric patients, and younger animals should also have regular dental examinations. musculoskeletal disorders have also been associated with colic in geriatric donkeys, mainly due to reduced physical activity and water intake. in the united kingdom, donkeys bedded on paper or cardboard have higher rates of impaction colic, because they regard the bedding as a fiber source and ingest large quantities. donkeys are natural browsers on a wide range of feedstuffs, and this exploratory behavior, while advantageous in some environments, can cause problems. hyperlipemia can be a cause or consequence of gastrointestinal disease. it can induce ileus, hepatic failure, and gastric ulceration, whereas colic, enteritis, colitis, peritonitis, inappetence, and anorexia often lead to hyperlipemia. other causes of colic (eg, grass sickness, neoplasia, enteroliths, peritonitis) have been reported in a small number of donkeys and mules with similar signs and treatments to horses. the etiology and epidemiology of colitis in donkeys have not been fully elucidated, but is likely influenced by geography and management. in the united kingdom, individual cases and outbreaks of diarrhea have been documented, with parasites, infectious agents, and husbandry changes proposed as the main causes. an extensive discussion of donkey parasitology is provided elsewhere. , relevant gastrointestinal parasites are listed in table . gastric ulcers have been identified during endoscopic evaluation and at postmortem examination, confirming that donkeys are at risk of this pathologic condition. donkeys with gastric ulcer syndrome (dgus) do not usually exhibit the clinical signs displayed by horses with equine gastric ulcer syndrome, likely because of their stoic nature. for example, study of donkeys without symptomatic evidence of gastric disease found lesions in the squamous gastric mucosa in % of animals, whereas only . % had glandular disease. postmortem data from the donkey sanctuary identified that most chronic gastric ulcers were located in the squamous area adjacent to the margo plicatus, whereas chronic active or acute ulcers affected both the glandular and the squamous part of the stomach in few animals (g. paraschiou, personal communication, ) . these findings are similar to those reported in italy. acute gastric ulcers are rarely documented in donkeys, but this may be a reflection of infrequent diagnostics rather than true pathologic condition. concerns related to stress and risk of hyperlipemia from prolonged fasting for gastroscopic evaluation are valid. however, in study where donkeys were fasted for hours before the procedure, no adverse metabolic effects were noted. it is the authors' opinion that donkeys undergoing gastroscopy should have triglyceride concentrations measured before fasting and after the procedure. if hyperlipidemia is identified, intravenous dextrose or oral administration of glucose or sugar-rich drenches is recommended to prevent clinical hyperlipemia. suggested risk factors for dgus include high starch diets, chronic stress, hyperlipemia, long-term corticosteroid and nonsteroidal anti-inflammatory drug (nsaid) use. prophylactic use of omeprazole is recommended when there are concerns. prophylaxis and treatment protocols for squamous and glandular ulcers with omeprazole have been extrapolated from horses. pharmacologic information on injectable omeprazole is lacking in donkeys. gastrointestinal transit time is longer in donkeys compared with horses and ponies, and there is suspicion that this could be in part due to delay gastric emptying, may have clinical relevance when planning gastroscopy as previously discussed or when withholding food for diagnostic testing purposes. gastric impaction occurs sporadically, primary or secondary to other conditions (liver disease, strictures, ileus). gastric foreign bodies are more common in working donkeys because of their scavenging habits (eg, plastic bag ingestion). affected donkeys are usually dull and inappetent and may display varying degrees of abdominal pain. fecal output may be reduced. diagnosis may be tentatively made on ultrasound examination; where ultrasound is not available, diagnosis may be made based on the presence of reflux or a marked discomfort on administration of enteral fluids. treatment of suspected gastric impaction is as for other equids. pathologic condition of the small intestine is similar to horses, although there appears to be a reduced incidence of strangulating lipomas. small intestinal pathologic condition may be harder to identify in donkeys because of limitations in rectal examination. equine grass sickness is rarely reported in donkeys, but should be kept as a differential diagnosis of weight loss, inappetence, and ileus in the united kingdom. lack of clinical recognition may be the main reason for its apparent low incidence. diagnosis and management are similar to other equids. ultrasonographic abdominal examination is recommended whereby acute or chronic small intestinal lesions are suspected from clinical signs or the results of other diagnostic tests. the oral glucose absorption test may be used in donkeys suspected of small intestinal malabsorption, but careful consideration of the risks of prolonged food withholding should be made. one of the most common causes of colic in donkeys and mules is pelvic flexure impaction from poorly masticated feedstuffs or foreign bodies (eg, plastic bags), followed by impactions of the transverse colon and cecum. typically, these animals present with dullness but will continue to eat until the impaction is severe, when they show signs of abdominal pain or stand apart from the herd. there may be reduced intestinal motility. because of the delay in presentation for evaluation, there may be secondary hyperlipemia, which contributes to their higher mortality compared with large colon impaction in horses. transabdominal ultrasonography can be helpful in patients too small for rectal examination and can be also used to measure intestinal wall thickness and edema. impactions due to dehydrated feces or fibrous material can be managed via enteral fluids and osmotic laxatives (eg, magnesium sulfate or sodium sulfate), whereas impactions due to plastic bags or foreign matter may respond better to mineral oil as a nonosmotic lubricant. nutritional support via nasogastric or intravenous routes, and small amounts of grass or short chop, can be used to stimulate appetite and encourage gastrointestinal motility, but also to prevent hyperlipemia. in dehydrated working donkeys and mules, nsaids should be administered at half the indicated dose to reduce the risk of renal toxicity. geriatric donkeys with dental disease, especially with absent molars, are more prone to large colon impaction and require short chop diets and grazing to reduce risk. typhlitis and colitis in donkeys can be challenging to diagnose. animals could be found dead from peracute colitis, whereas most cases show signs of intestinal inflammation and systemic inflammatory response syndrome, including dullness, fever, and occasionally diarrhea. ventral edema and weight loss are common findings in chronic cases associated with protein-losing enteropathy and hypoalbuminemia. as with colitis in other equids, achieving a definite diagnosis can be challenging, but may be linked to emerging cyathostomins and infectious agents (fig. ) . salmonella spp and clostridium spp are occasionally isolated. information on coronavirus and gastrointestinal disease in donkeys is limited. feed contamination with mycotoxins has been implicated. rapid dietary changes that alter the microbiota could be an aggravating or risk factor. again, hyperlipemia is a frequent finding in donkeys with colitis. therapeutic protocols for colitis in donkeys and mules are similar to horses. rectal prolapse is seen more commonly in working donkeys, where exhaustion and parasitism are common. it has been associated with gasterophilus nasalis in ethiopia, which causes irritation and constant straining when present on the rectal mucosa. rectal prolapse has been reported secondary to rectal tumors and penetrating injuries (fig. ) . rectal prolapse from acute colitis is unusual compared with horses. treatment will depend on the size of prolapsed tissue and the severity of the damage. simple cases can respond to osmotic reduction using sugar and lubrication to replace the prolapse, combined with anti-inflammatory drugs (see fig. ). animals with severe lesions or that do not respond to medical management may require a purse-string suture or surgical resection. sedation, epidural anesthesia, and local anaesthetics are required. the purse-string suture using thick suture material or sterile umbilical tape is often successful. the string must be loose enough to allow defecation and should be removed within hours. depending on the inciting cause, a soft diet or a laxative may be helpful. rectal tears are a serious complication of rectal examination, and antispasmodic drugs with good restraint are recommended for rectal examination. rectal tears have been reported when stallions attempt to cover geldings. liver disease has major morbidity and mortality in the uk donkey population. clinical signs are often insidious, and liver pathologic condition may only be found because of investigation into other diseases. clinical signs of liver disease include colic (dysmotility, gastric impaction), weight loss, fever, depression, photosensitization and other skin diseases, icterus, and neurologic signs (including behavioral changes). weight loss is the most consistent finding but is not pathognomonic. hepatic disease is frequently subclinical and often detected based on serum biochemistry evaluation. liver dysfunction from excessive fat infiltration from a negative energy balance, stress, or insulin insensitivity may occur with severe hyperlipemia. liver disease has been found in donkeys with elevated adreno cortico trophic hormone (acth) and assumed poorly controlled pars pituitary intermedia disorder (ppid), but firm data on any association are lacking at present. evaluation of serum chemistry should be an early diagnostic step to investigate liver function. reference values for liver specific parameters are available in erin l. goodrich and erica behling-kelly's article, "clinical pathology of donkeys and mules," in this issue. ultrasonography is useful to assess size, echogenicity, architecture, and the presence of focal or diffuse lesions, if there is suspicion of severe acute or chronic liver pathologic condition. a biopsy may provide the most meaningful diagnostic and prognostic information. there is no validated hepatic histopathology scoring system for donkeys, and the grading system for horses is the one used. hemosiderin accumulation is a common histopathological finding in donkey hepatic tissue, but its significance is unknown. it is important to note that liver biopsy carries risks, in particular, bleeding, which can be exacerbated from hepatic disease. it is highly indicated to have another donkey available as a companion, but also as a potential blood donor. close monitoring for signs of colic and bleeding for the next hours after the procedure is recommended. treatment of liver pathologic condition will be dictated by the clinical and diagnostic findings. liver fibrosis is frequently found in the uk donkey population. use of corticosteroids and nutritional management are the mainstays of therapy. repeat biopsies would be ideal to monitor response to treatment, but it is often impractical, costly, or hard to justify. serial blood evaluation of markers of liver disease and function may be the most practical method. in animals with minor serum abnormalities of liver function, monthly chemistry evaluations are indicated. pyrrolizidine alkaloid toxicity has been documented in donkeys, with similar histopathological findings and prognosis as horses. antimicrobials should be reserved for cases where there is evidence of a bacterial hepatitis or cholangiohepatitis. hydatid cysts are occasionally found as an incidental finding on ultrasound examination. treatment or monitoring is advised as for other equines. the incidence of liver fluke (fasciola hepatica) is increasing among the uk donkey population. this parasite should be considered in donkeys inhabiting wetter environments where the presence of lymnaeid intermediate hosts (lymnaea spp; galba spp) has been documented. flukes may increase liver enzymes because of pathologic condition of the bile ducts. some animals will present with weight loss and marked elevation in liver enzymes from severe cholangiohepatitis. treatment of all animals in the herd is advised. there are no licensed fluke treatments for donkeys, and therapy is prescribed using the cascade system. the donkey sanctuary uses triclabendazole ( mg/kg orally) with follow-up fecal egg analysis to days after treatment to assess efficacy. when triclabendazole is ineffective, closantel ( mg/kg orally) may be considered, keeping in mind that it is only effective against adult flukes and redosing is required to weeks later. closantel can cause anorexia, ataxia, and blindness if overdosed. fencing off wet, marshy environments is advised to reduce exposure, and cattle or sheep screened and treated for liver flukes if they graze the same pasture as donkeys. measurement of amylase and lipase activities in dull donkeys with nonspecific abdominal pain may be suggestive of pancreatitis, but a definitive diagnosis is rare, and very few cases have been confirmed at postmortem examination. many veterinary laboratories do not measure amylase and lipase as routine tests, and results should be interpreted with caution. in horses, migrating strongylus spp and parascaris spp larvae have been proposed as a potential etiology, but their role in donkey pancreatic disease remains to be determined. hyperlipemia may further the risk of pancreatitis in donkeys. indications for surgery in donkeys are similar to those in horses, with the caveat that their demeanor and physiology may mask deteriorating parameters. pain level and dehydration are harder to assess in donkeys. before embarking on exploratory laparotomy, a thorough clinical examination is recommended to ascertain that factors that could compromise a successful outcome (eg, dental disease, malnutrition, laminitis, osteoarthritis) are taken into consideration. again, routine rectal examination and nasogastric intubation should be performed when feasible. blood and peritoneal fluid lactate values can be used in a similar way as for horses. normal blood and peritoneal fluid lactate values for donkeys have not been established; however, rising lactate concentrations suggest the presence of a surgical lesion or the presence of bacteria in the peritoneal fluid. success rates for abdominal surgery in donkeys tend to be lower than values reported for horses for several reasons, including lack of medical and surgical experience with the species, presentation at an advanced stage of disease, delayed referral due to reduced veterinary experience, lack of knowledge regarding appropriate analgesia and donkey-specific pharmacology, and finances. hospital stall accommodation may need to be adapted for the donkey; stall doors that are too high to permit visibility may need to be replaced with a hurdle or gate. it is vital that the clinician has knowledge of how to estimate and measure donkey weight and condition score before giving any medications. few drugs are licensed for use in donkeys and must be selected on the basis of the legislation of each country. in the united kingdom, prescribing guidance to avoid animal suffering where no approved drug is available is based on the cascade system, whereas in the united states, the animal medicinal drug use clarification act permits the extralabel use of certain approved animal drugs under some conditions. it is important to mention that for several drugs, there are pharmacologic differences between donkeys and mules with horses, and clinicians should use caution when extrapolating doses (see francisco j. mendoza and colleagues' article, "clinical pharmacology in donkeys and mules," in this issue). it is essential to monitor the response to analgesia given for gastrointestinal pain and be prepared to dose at more frequent intervals. phenylbutazone and flunixin meglumine can be given twice daily at standard equine doses. multimodal pain relief using opioids and other drugs should be considered, as for other equines. further information on sedation and general anesthesia in donkeys is described elsewhere (see nora matthews and johannes p.a.m. van loon article's, "anesthesia, sedation and pain management of donkeys and mules," in this issue). abdominal surgery increases the risk of hyperlipemia, and preemptive measures to prevent and manage this complication should be in place. postoperative food withholding should be weighed against the risk of hyperlipemia and other complications. triglycerides should be monitored regularly, and parenteral support may be required to maintain normal levels (see francisco j. mendoza and colleagues' article, "metabolic and endocrine disorders in donkeys," in this issue). postsurgical monitoring is essential to reduce stress. in donkeys that require prolonged stall rest, enrichment methods may be necessary. the following link provides information on enrichment for donkeys (https://www.thedonkeysanctuary.org.uk/ what-we-do/knowledge-and-advice/for-owners/environment-enrichment). there are no known published guidelines on fluid therapy for donkeys and mules, and recommendations are based on equine principles, taking into consideration that there are differences in body water partitioning. enteral fluid therapy is suitable for gastrointestinal disorders, such as large colon impaction and mild dehydration, as long as there is no gastric distension or ileus, and when resources are limited. nasogastric intubation might be the only option for working donkeys. commercially available isotonic fluids should be the first option before considering homemade recipes. supplementation with dextrose should be done in most cases. for enteral fluids, do not exceed l of fluid for a -kg donkey. the frequency of enteral fluid administration will depend on the metabolic status, the animal response to therapy, and feasibility. an indwelling nasogastric tube may be a more practical method for intermittent or continuous fluid administration. enteral fluids should not be used in animals in which reduced intestinal perfusion is suspected. intravenous fluid therapy is indicated in animals with evidence of volume depletion, when electrolyte and metabolic abnormalities are present, for those in which enteral fluid therapy is not an option, or in surgical cases. the approach follows the equine guidelines. when administering as a bolus, be aware that the advised crystalloid rate of to ml/kg over to hours equates to . to . l for a -kg donkey. in animals with colitis, high intravenous fluid rates have been associated with mural edema and a poorer prognosis (a. thiemann, personal communication). to reduce complications from overhydration, it is recommended to monitor urine output, respiratory rate, peripheral edema, and body weight. a full discussion of intravenous fluid therapy is beyond the scope of this article, and the reader is referred to equine fluid therapy reviews. use of rectal fluids has been investigated, but, in general, has been considered impractical and risky. gastrointestinal disorders are common in donkeys and mules. knowledge of these conditions, including their etiology, pathophysiology, and epidemiology can assist in their successful diagnosis and management. awareness of donkey-and mulespecific behavior, anatomy, and physiology will improve the prognosis. adequate pain management to reduce stress and understanding metabolic complications from gastrointestinal disease are essential for success. behavioural assessment of pain in horses and donkeys: application to clinical practice and future studies relationships between behaviour and health in working horses, donkeys and mules in developing countries anatomical and morphometric study of gastrointestinal tract of donkey (equus africanus asinus) sedation, anaesthesia and analgesia shifts in body fluid during dehydration in the burro, equus asinus intake, digestion and gastrointestinal transit time in resting donkeys and ponies and exercised donkeys given ad libitum hay and straw diets colic in the donkey epidemiology of impaction colic in donkeys in the uk common helminth infections of donkeys and their control in temperate regions equine gastric ulcer syndrome in adult donkeys: investigation on prevalence, anatomical distribution and severity necropsy survey of gastric ulcers in a population of aged donkeys: prevalence, lesion description and risk factors successful treatment of chronic grass sickness in a donkey asinine typhlocolitis; 'scouring' the literature for diagnostic and aetiological clues gastrointestinal disorders of donkeys and mules gasterophilosis: a major cause of rectal prolapse in working donkeys in ethiopia development and application of a scoring system for prognostic evaluation of equine liver biopsies acute pancreatitis in a horse-a case report outcome following exploratory laparotomy in donkeys: a retrospective multicentre study. beva conference proceedings appendix , body weight estimator pharmacology and therapeutics in donkeys anaesthesia and analgesia of the donkey and mule nutritional management of hyperlipaemia practical fluid therapy and treatment modalities for field conditions for horses and foals with gastrointestinal problems crystalloid and colloid therapy evaluation of the rectal route of fluid administration in horses. eceim conference proceedings key: cord- -ugy kys authors: neulight, nina; kafai, yasmin b.; kao, linda; foley, brian; galas, cathleen title: children’s participation in a virtual epidemic in the science classroom: making connections to natural infectious diseases date: - - journal: j sci educ technol doi: . /s - - -z sha: doc_id: cord_uid: ugy kys this study investigated students’ understanding of a virtual infectious disease in relation to their understanding of natural infectious diseases. two sixth-grade classrooms of students between the ages of and ( students) took part in a participatory simulation of a virtual infectious disease, which was integrated into their science curriculum. the results from our analyses reveal that students perceived the simulation as similar to a natural infectious disease and that the immersive components of the simulation afforded students the opportunity to discuss their understandings of natural disease and to compare them to their experiences with the virtual disease. we found that while the virtual disease capitalized on students’ knowledge of natural infectious disease through virtual symptoms, these symptoms may have led students to think of its transfer more as an observable or mechanical event rather than as a biological process. these findings provide helpful indicators to science educators and educational designers interested in creating and integrating online simulations within classroom environments to further students’ conceptual understanding. despite advances in medicine and technology, the prevalence of certain infectious diseases still persists, as does the fear of contracting infectious diseases. in more than , people were infected and over people died because of the severe acute respiratory syndrome (sars) (centers for disease control and prevention). similarly, in , there was national panic when the initial demand for the flu vaccine exceeded its supply and many people who wanted to be vaccinated could not (cbs news, ) . research has shown that children and adults have a difficult time understanding how an individual contracts an infectious disease, what causes these diseases to spread, and how diseases can be prevented kalish, ; sigelman et al., a) . for these reasons, the national science education standards (nrc, ) include the study of infectious disease at every grade level from grades - to increase understanding and help prevent disease spread. there have been a number of instructional approaches that include textbooks, hands-on classroom experiments, and educational technologies to teach students about infectious diseases (centers for disease control and prevention; science education partnership award program). more recently, researchers have started investigating various forms of participatory simulations as a way to teach students about infectious diseases (colella, ; hug et al., ; wilensky and stroup, ) . several features of participatory simulations are of instructional relevance for learning about infectious disease. for example, in some participatory simulations learners can create online representations of themselves, also called avatars. also, since some participatory simulations have hundreds of thousands of registered users and thousands of concurrent users, learners can simulate in real time the spread of a disease for a similar duration of a natural disease outbreak. this paper investigates the integration of a multi-user virtual environment (muve), called whyville, within classroom curriculum about infectious disease. in this study, two classes with a total of sixth-grade students became members of whyville and were able to access the website at home and during science class where they learned about infectious diseases. the students created avatars in whyville, which experienced the outbreak and spread of a virtual epidemic called whypox during a -week period. when an avatar had the disease, two important aspects of online participation were affected-the avatar's appearance and the ability to chat with other whyville participants. the feature of having the avatar's appearance change allows users to experience diseases without direct physical harm to the participant, which would be difficult to replicate in real life due to ethical considerations. our study investigated the integration of a virtual infectious disease called whypox within science classroom curriculum and its relationship to students' understanding of natural infectious diseases. we wanted to understand in which ways students perceived whypox as a natural disease and thus isolate design features that might create more effective integrations and simulations of disease in the future. lastly, we wanted to know how students draw on their participation within a virtual disease simulation in order to expand on their knowledge of natural infectious diseases. the following literature review situates the participation in a virtual epidemic within the larger body of participatory simulations before addressing how participation in a participatory simulation might enhance children's understanding of natural infectious disease. in educational settings, students have been taught about infectious diseases through a variety of instructional approaches such as textbooks, hands-on classroom experiments, and educational technologies (centers for disease control and prevention; science education partnership award program). some of these interventions used infectious disease as a medium to teach about science inquiry (hug et al., ) , while other studies focused on teaching about aspects of infectious disease such as the biology of germs , latency and immunity (colella, ) , or the probability of getting a disease (wilensky and stroup, ) . individually these studies tapped specific aspects of how children understand infectious disease and how to better teach about these aspects of infectious disease. a more recent development to teach about infectious disease is the use of participatory simulations (colella, ; hug et al., ; wilensky and stroup, ). an educational simulation has been defined as a model of some phenomenon or activity that users learn about through interaction with the simulation (alessi and trollip, ) . however, simply modeling or imitating the central features of a situation does not render an activity a simulation-in a simulation the user must have the experience of playing a genuine role and experience consequences of one's actions (gredler, ) . in a computer-supported participatory simulation, students can experience a phenomenon such as disease spread repeatedly because the teacher or designer can experimentally vary the disease. participatory simulations have differed from one another in three dimensions: the virtual reality component, which refers to how faithfully a real-life environment is replicated visually, functionally, aurally, and sometimes kinesthetically (alessi and trollip, ) ; the scale, which refers to the number of participants that can participate concurrently in a single simulation and ranges from a single classroom to hundreds of thousands of participants; and the type of platform, which at the time of this study referred to two distinct types-wearable or hand held computers (colella, ; hug et al., ; krajcik et al., ) and computer-based online multi-user environments (aschbacher, ; foley and la torre, ) . two features of participatory simulations have been salient to the study of infectious diseases-the ability to increase the user's immersiveness or feeling of presence in the simulation and the integration of the simulation into the classroom curriculum. for example, in one group of studies in which high school students used wearable computers called thinking tags to learn about latency of a virus, probability for infection, and immunity, the students felt that they had experienced a disease (colella, ) . each thinking tag had an infrared transmitter and receiver that exchanged information with the other tags in the simulation and then displayed to the user information such as how many people the thinking tag had met that were sick. the thinking tags were used within a single classroom and each simulation lasted minutes. findings from this study showed that with the thinking tags, students made references to themselves as being infected and were able to communicate naturally with each other, which seemed to encourage communication about disease. a limitation in this research was that specific assessments of students' learning were not conducted in the study. also, participatory simulations have been successfully integrated into classroom curriculums about infectious disease. for example, an eight-week curriculum called ''can good friends make you sick?'' included the use of thinking tags by a class of eighth graders learning about the biology of communicable diseases (hug et al., ) . while the curriculum in that study included infectious disease, only students' levels of inquiry and engagement were assessed. a relevant finding to the present study was that as students became more familiar with how the computer devices operated, they began to engage in more sophisticated levels of science inquiry. while the thinking tags were designed for a single classroom to use, other participatory simulations termed multi-user virtual environments (muves) have been used to access a virtual world on the internet. at the time of this study, there were two such environments that included the study of infectious disease-river city and whyville. the main difference between these two muves was the role of the student's avatar or online representation. in river city (dede et al., ) students learned about how infectious disease spread and the process of scientific inquiry by examining the sanitary conditions and disease spread in the th century. students used their avatars to interview the city's virtual inhabitants and collect data about sanitary conditions within the city but the avatars did not experience the diseases themselves. however, in whyville, students through their avatars experienced firsthand the outbreak of a virtual epidemic called whypox. for our study, we combined aspects of the approaches discussed above. we used an existing, thriving participatory simulation that was a muve, called whyville, and integrated the technology use within a classroom curriculum on infectious disease. the main difference between our study and the studies on participatory simulations with wearable or handheld computers (colella, ; hug et al., ) was scale. while these previous studies were conducted in single classrooms where an outbreak lasted only a minute, in whyville a single outbreak of whypox could last up to two months depending on the population of whyville and the chosen disease vector at the time of the outbreak. we believe that the large scale would allow for students to become more fluent with the technical aspects of the medium in addition to providing students with more time to both participate in a virtual disease epidemic and observe its impact. our study differed from previous research on whyville in that we wanted to examine how the participation in a virtual disease related to students' understanding of natural disease. prior research on whyville focused on how the introduction of the virtual disease and related disease activities on the whyville site had increased user online discussion about the spread and cure of whypox (foley and la torre, ) . to understand the extent to which students related whypox to natural infectious disease, we examined classroom discussion between the teacher and the students. prior research has shown how classroom discourse can provide opportunities for science inquiry (gallas, ; kafai and ching, ; lemke, ) . in line with this research, we looked at how class discussions might afford connections of a virtual infectious epidemic to natural infectious diseases. we also examined students' understanding of how disease spreads and how to prevent disease from occurring. researchers have shown that children have difficulty understanding these concepts kalish, ; obeidallah et al., ; siegal, ; siegal and peterson, ; sigelman et al., b; solomon and cassimatis, ) . of particular interest to the present study was au and romo's ( ) model of children's conceptualization of disease because their assessment tools evaluated students' reasoning about what caused disease. au's research team developed a four-point rubric that was used to assess children's understanding of four biological phenomena: food contamination, illness, genetics, and infectious disease. children's understanding of each phenomenon was evaluated according to how students reasoned about a scenario. if students included a biological causal explanation, (e.g., a person got sick because germs grew, reproduced, and attacked cells), their response was given a higher score than a response that included only a non-biological cause (e.g., a person got sick because he was not wearing a coat). like other research (parmelee, ), au's focus on the explicit teaching to children about the causes of infectious disease was based on the assumption that without science instruction, children and adults did not automatically reason coherently about biological phenomena such as the spread of infectious disease. in line with au's research, children who did not have this science instruction tended to reason about biological phenomena by applying their knowledge of people and everyday observable behaviors to explain the cause. participants were sixth-grade students, an equal number of boys and girls, in two classes taught by the same science teacher. the students attended a laboratory school that is affiliated with a large, urban university and comprised of a diverse ethnic sample: % latino, % african-american, % asian, and % caucasian. two-thirds of the families receive tuition assistance based on a sliding scale. over % of these students have computer and internet access from their homes (kafai and sutton, ) . all students received parental consent and provided assent for participation in the study. the classroom teacher had over years of experience working in elementary schools and teaching science. classroom activities and discussions were videotaped. the videotaped whole-class discussions were used to determine how students and teachers used whypox in their discussions about natural infectious disease. two surveys were developed to assess students' understanding of natural infectious diseases and their perceptions of whypox as a natural disease. one video camera was used to record whole-class discussions that took place throughout the study. during the whole-class recordings, a microphone was placed near the teacher. this resulted in approximately hours of videotaped whole-class discussions focused on whyville activities. the use of whyville was integrated into a week teacher-led curriculum about infectious diseases. some of the activities that students participated in as part of their science curriculum included: watching videos about specific diseases and the nature of germs; examining cell structures under the microscope; doing hands-on experiments that simulated the spread of an infectious disease; completing worksheets about cells, bacteria, and viruses; and using online tools to research specific diseases. these activities took place throughout the study, even after whyville was introduced to the students in the third week of the study when students started to log on to whyville for at least minutes every science class. students had access to the whyville website during school hours and after-school hours. each member of whyville was represented by a screen name and avatar, which was a member's online headshot that appeared on the screen after a member logged on to the website. a member could augment his or her avatar by buying or creating face parts. to travel through whyville, a member selected a destination from a drop down menu that was always accessible to the members or by clicking on the screen with the computer mouse. each destination offered a different type of activity such as: science-related activities about infectious diseases; recreational games like checkers; the center for disease control, where members could read about past outbreaks of whypox authored by children and science educators; and the whyville times, the website's online newspaper that included participant-authored articles about the site. members could communicate with other participants synchronously by having a cartoon chat box appear above their avatar face or members could communicate asynchronously through ymail, an internal mail system, and a bulletin board system (see figure ). during weeks three to five, students explored a variety of recreational and science-related activities on whyville, as instructed by the teacher. when whypox hit whyville during week five, the teacher facilitated whole-class discussions to discuss what was happening on whyville (see figure ). these discussions occurred approximately twice a week for about minutes each until the end of the study. in these discussions, the teacher and students discussed a graph that they had created in class that displayed on one axis the number of whypox infections in both classes and displayed on the other axis the date of infection. the teacher and students used this graphing activity as a springboard to discuss how whypox was affecting participation in whyville. a similar graph that displayed the entire whyville population was available online (see figure ). the students analyzed these online graphs with teacher guidance. the teacher and students also discussed technical issues about using whyville such as how to chat and how to participate in activities. in addition to the discussions about whypox, the teacher guided students in exploring the disease-related activities on whyville's cdc (see figure ). at the cdc students read about past cases of whypox and posted predictions about causes and cures in addition to using tools that simulated outbreaks of diseases by manipulating variables such as the duration of a disease (see figure ). to determine students' conceptual understanding about the causes of natural infectious disease, we developed a survey that asked students about the cause, spread, and prevention of natural infectious diseases. in addition, we included scenario-based questions and used their corresponding coding rubrics from au's studies ). an example of the infectious disease scenario with its corresponding questions is as follows: ''cathy went over to see her friend who was sick. some bad germs got inside her body. she felt okay for a day. but then the next day she started to feel sick all over her whole body. her head ached and her stomach hurt and her throat hurt-all at the same time. (a) why did it take a whole day for her to feel sick after the germs got inside her body? (b) how did the germs make her feel sick in so many parts of her body at the same time?'' the disease survey was administered at week , the beginning of the study, and week , the end of the study. to determine whether students connected the virtual disease to natural disease, we developed a survey to examine students' understanding of the virtual disease whypox and asked students to compare their understanding of whypox to natural infectious diseases and to report about their online experiences with whypox. the whypox survey was administered at week ten, the end of the study. before and after the curriculum unit on infectious diseases, the infectious disease survey was given to all students by the researchers. the whypox survey was administered only at the end of the unit. all classroom sessions were videotaped. one to two researchers of the team of five researchers observed each science class throughout this study. the research team consisted of university faculty, a postdoctoral fellow, and graduate students. the researchers administered the evaluation instruments and videotaped class discussions. the first part of analysis examined the wholeclass discussions about whypox using transcripts of the videotaped classroom discussions while the second part of analysis coded students' survey responses about natural and virtual infectious disease. video segments of whole-class discussion were examined in order to determine the efforts used by the teacher and the students to connect their whypox experience with what they had learned about natural infectious diseases. the video segments were selected from one of the classes only because there were no performance differences between the two classes on students' survey responses. the video segments we wanted to analyze occurred after the outbreak of whypox and contained discussions about whypox. after viewing these seven segments, four video segments, a total of h, met the above criteria and were transcribed completely. conversations, but not behaviors, were transcribed. after reading the video transcriptions, we developed thematic categories that were derived from theory, that classroom discourse can provide opportunities for science inquiry (lemke, ) . the video segments were coded using three thematic categories: ( ) the use of infectious disease terminology in relation to whypox (e.g., ''whypox was like an epidemic''; ''we should have quarantine in whyville''); ( ) the mapping of whypox to a natural disease in which a natural disease was referenced by mentioning symptoms of natural diseases, the virology of natural diseases, or the consequences of natural diseases; and, ( ) the immersive component of whypox in which students and teacher included experiential, social, and causal aspects of someone having whypox. we also analyzed students' answers to openended and scenario-based questions about disease causality from the disease survey using au's coding scheme (au and romo, ) . the original coding scheme that we adopted from au's research team (au and romo, ) was on a scale of to . in this coding scheme, code represented the most sophisticated understanding of disease spread, a biological causal mechanism, while codes through represented a pre-biological understanding with category representing the least sophisticated understanding of disease spread. our modification to au's coding scheme was that during data analyses we collapsed the four-point coding rubric into a binary rubric. we used a binary rubric because in this study we were interested in whether students reasoned with the most sophisticated mechanism of transfer, a biological causal mechanism, or a pre-biological understanding. we were not interested in the degrees of pre-biological understanding that were represented in codes through of au's original coding scheme. we applied the terms 'biological' and 'pre-biological' to these two codes (see table i ). to test inter-rater reliability on all of the questions we first selected a random sample of % of the surveys and found representative student answers for each code. then we selected another random sample of % of the surveys and one coder coded these using the coding key we had developed as a research team. a second coder also coded the same sample using that coding key. we considered items reliable if they shared % accuracy between the two coders. discrepancies for each item were discussed and coding descriptions for each question were revised as needed. we investigated the ways in which students perceived features of whypox. our goal was to determine how faithful the simulation was to natural infectious diseases. in developing our coding scheme for the students' reasoning of the causality of whypox, we followed au's research team but included the unique features of the virtual disease (see table i ). the coding scheme for the other questions about whypox's relationship to natural infectious disease and whypox as a learning tool as viewed by students will be listed in the results section. for the inter-rater reliability we followed the same procedures as outlined in the disease survey and accepted only items that had at least % agreement. during and after the whypox outbreak, several classroom discussions examined both whypox and its connection to natural infectious disease. we identified three ways in which the students and the teacher discussed their understandings of natural disease and compared them to their experiences with the virtual epidemic: ( ) application of terms used to explain whypox to terms used to explain natural diseases; ( ) mapping of whypox to a natural disease in which a natural disease was referenced by mentioning symptoms of natural diseases; the virology of natural diseases; or the consequences of natural diseases; and ( ) immersive component of having whypox in which the students and teacher referred to someone experiencing whypox and included experiential, social, and causal aspects of being immersed in whypox. there were many instances in the selected video segments in which more than one code applied to a single student or teacher reference. in the first way of referencing whypox with a natural disease, students and the teacher mentioned, if not discussed, major terms and concepts of infectious disease. these terms and concepts included: contagious, exposure, symptoms, infection, incubation period, epidemiologists, epidemic, quarantining, and immunity. often the students or the teacher mentioned one of these concepts to describe an aspect of their whypox experience. however, beyond the mere mention of the concept, once mentioned these concepts often became woven into conversations. for example, as the class discussed how they thought they or others got whypox, a student used the term contagious to paraphrase the teacher's comments of getting whypox through virtual contact. then, in a subsequent comment another student used the term contagious to describe how whypox was affecting her. this conversation allowed for the students to use the term contagious in an authentic situation to hopefully understand what it really meant. the second way of referencing whypox was through the creation of analogies between whypox and natural disease, and vice versa. some of the analogous natural diseases the students and the teacher referenced were sars, the plague, and the common cold. in these references, of which there were in the selected video segments, the student and the teacher often compared symptoms, preventions, or aspects of whypox to those experienced in their everyday lives or from their previous studies on natural infectious diseases. these links were used to help students make sense of what was happening with whypox. in the following example, the teacher responded to a student who thought that she had these links of whypox to natural infectious diseases, aside from emphasizing a point, may have helped students to think about the virtual infectious disease in ways similar to natural infectious disease. for example, during a class discussion about how students thought they got whypox, the teacher linked whypox to natural infectious diseases by making the comparison that the avatars in whyville were grouped like bacteria. celine: there were these heads around me and i couldn't move away from them. they were all bunched up in these colonies. teacher: colonies of bacteria around you. huh? celine: like a wall. this link may have helped students to think about the causes of whypox with a more sophisticated causal mechanism than mere contact since students had learned through other instructional methods that bacteria or viruses were the biological causal agent of natural infectious diseases, not the contact itself. lastly, students and the teacher used whypox to understand natural infectious disease through immersiveness. recall that immersiveness refers to students creating their own personal representations that students created on whyville. these references, of which there were episodes in hours of selected video segments, occurred frequently throughout the discussions, which could be an indication that students genuinely bought into the idea that they and others had the virtual disease. in this study, immersiveness appeared in three forms: experiential, social, and causal. in one form, the students and the teacher referenced themselves or others as experiencing an aspect of whypox, such as ''i saw them with whypox'' and ''i had it two days ago and it got worse.'' another way was the social dimension, which involved students noticing others who had whypox, as illustrated by this student's remarks: ''i was checking there were five people who had whypox and they were in a room alone.'' the social dimension was rarely seen in isolation of the final form of immersiveness, in which the students and the teacher speculated the causes and spread of whypox. in the following example, a student described the social aspect of being immersed as the ability to infect others with whypox, while also speculating that his getting close to the people was the cause for their whypox. lee: i wanted to infect other people. so, i went close to them and then i went away and then they got it. linking causality to proximity, a social aspect, is repeated in this next example: ''jon said that jon and mike got it first and since we're usually on the computers at the same time, maybe that we were together and we were at the same room or something.'' these last examples illustrate students' attempts to make sense of what was happening with whypox. we asked students the question ''in which ways was whypox like a real infectious disease?'' the answers show that students perceived several features of the virtual infectious disease as features inherent in natural infectious diseases. these features included being contagious ( % of the responses, n = ), having symptoms ( . %, n = ), and being like a specific other disease ( %, n = ). we asked students ''how do you think whypox spread through the community?'' all of the students' explanations for virtual disease transfer included a pre-biological causal mechanism that included activities such as contact, chat, and sneezing. no student attributed a biological causal explanation that included transfer of whypox through a piece of embedded code. we believed that computer code embedded within the muve of whyville was the biological equivalent of a natural infectious disease. we found that while the majority of students still reasoned with pre-biological causal explanations (see table ii ), there was a significant change in students' responses between pre and post from pre-biological to biological explanations (t = ) . , df = , p = . ; t = ) . , df = , p = . ), showing that twice as many students reasoned about natural infectious disease with a biological reasoning at the end of the study. these findings were consistent with the two open-ended questions about the causes of disease spread: ( ) what are some of the causes of infectious diseases? ( ) what are some things that will increase the spread of the disease? pairwise t-test analyses showed that there was significant change in students' responses to the first question (t = ) . , df = , p = . ) and second question (t = ) . , df = , p = . ). as tables iii and iv reveal, students were statistically more likely to provide a response to these questions at the end of the study than at the beginning of the study. however, the majority of these causal explanations were still pre-biological. this study identified benefits and challenges in using muves such as whyville in a classroom curriculum about natural infectious diseases. one of the main differences between the whypox disease simulation and previous participatory simulations (colella, ; hug et al., ; krajcik et al., ) was scale. we speculated that the large scale of whyville affected students in ways that prior smaller scale simulations of infectious disease could not. for example, because whypox lasted several days rather than minutes, students were faced with the reality that whypox was not going away any time soon. as evidenced by classroom discussions, students interpreted this as a problem since their faces and ability to chat were negatively affected. because face building and chatting were important activities on whyville having these activities hampered may have motivated students to look for a cure and ponder possible causes as they did in this study. in addition, students were able to track and compare the spread of the epidemic not only in their two classrooms but also in the whole online community. these observed differences led students to discuss frequency and length of visits in whyville and forms of interactions with other community members as possible reasons for contracting the disease. by using avatars in whyville students also experienced a disease without physical harm to their actual self, a form of immersiveness absent from traditional science curricula of instruction from textbooks, videos, and laboratory experiments. with this immersive component, students started referring to themselves as having the virtual disease, a phenomenon experienced in previous participatory simulations of infectious diseases (colella, ; krajcik et al., ; soloway et al., ) . this participatory component, in addition to the fact that whypox shared features similar to diseases with which the students had familiarity such as chicken pox and the common cold, allowed students to connect whypox to natural infectious diseases. this was apparent in their whole-class discussions, where the students and the teacher used whypox as a vehicle to talk about some of the causes and preventions of natural infectious diseases as well as terminology and concepts of infectious diseases. while the analysis of pre and post survey questions showed significant changes in students' reasoning about the causes of natural diseases, a large majority still provided pre-biological explanations. this finding reinforces past research indicating that the concepts of natural infectious disease are difficult for students to learn, even with instruction. the participation in the classroom curriculum on infectious disease might have been responsible for this though our research design does not allow us to identify particular causes for this change. students perceived similarities in the virtual epidemic and the spread of natural infectious diseases, with one major exception-the level of reasoning about the causes of virtual disease was less sophisticated than the reasoning about the causes of natural infectious disease. however, students did perceive features of whypox as similar as those of natural infectious disease such as being contagious and having symptoms. we thought that it was important to know how students viewed the causes of the virtual disease in case their reasoning could be used as a springboard to discuss the equivalent of natural infectious disease. our findings showed that students' reasoning about the causes of the virtual disease did not go beyond observable explanations such as touching someone or sneezing. students did not think that the cause of whypox was an internal process, such as through a computational mechanism. it is possible that the immersiveness feature of whypox presents benefits and challenges to learning about natural disease. while whypox allowed students to show symptoms and have contact with other avatars, the types of contact possible in this virtual epidemic were limited. for example, avatars could not engage in sexual intercourse, an activity that has been linked to the spread of many natural infectious diseases. also, avatars did not have exposure to unsanitary conditions, which represents another cause for many natural infectious diseases. this reduced set of experiences that an avatar could have had may have constrained how students thought about the spread of whypox. also, while whypox capitalized on students' prior knowledge of natural infectious disease through the symptoms of sneezing and dots on the face, these virtual reality symptoms may have manipulated students into thinking of disease more as an observable event. for instance, touching someone rather than the reproduction of germs in the body was seen as the cause. our results confirmed this supposition. no student attributed what we termed the computational causal explanation to whypox. this result contrasted with students' overall increase in biological causal explanations for natural infectious diseases. although the similarity of whypox to familiar natural diseases in which children and adults have naı¨ve understandings might have accounted for some of this discrepancy, a contributing reason for this discrepancy might be a missing curricular piece. this missing connection might be an introduction to computer viruses, what they are and how they function. biological virus and computer virus alike contain a piece of dna or code, which has their instructions. students could have examined the similarities and differences between biological and computational viruses. such background information might have helped students to understand possible mechanisms of whypox. we believe that the science teacher played an important role in integrating the muve of whyville within the classroom environment. this teacher engaged students in discussions about the technical aspects of the muve, how whypox was affecting the virtual community, the appearance of whypox, and connections about whypox with natural diseases. the teacher allowed students to explore whyville on their own during class time. she too was knowledgeable about the site and the various activities available. this study contributes to the existing and growing literature on participatory simulations on infectious diseases. based on our findings, we learned that having an integrated curriculum around the participatory simulation stimulated teacher-student discussions about the causes and spread of virtual and natural diseases. from these discussions, we also learned that students did not reason about the causes of virtual diseases in ways similar as natural diseases. one possible explanation that students did not reason about the causality of virtual diseases in a sophisticated way was because we did not link the two types of diseases sufficiently. specifically, because whypox took place on a computer, we thought that including computational viruses in future curricula might effectively bridge natural and virtual diseases. in such a curricular addition, students could investigate different forms of computer viruses and the ways they are transmitted. the additional study of computational viruses might help students more effectively link the causes of whypox to the causes of natural disease. finally, the role of the teacher cannot be overlooked. in the present study, the teacher engaged students in discussions about whypox and other disease-related activities on whyville. she facilitated students' use of certain features of whyville such as tools to model disease and the newspaper, whyville times, to read about past episodes of whypox. during whole-class discussions, she facilitated students to make connections to concepts and terminology used in reference to natural infectious diseases. in future implementations of whypox into the science classroom, the role of the teacher might also affect the potential impact of this intervention. the writing of this paper was supported in part by a grant of the national science foundation (nsf- ) to the second author. the views expressed are those of the author and do not necessarily represent the views of the supporting funding agency or the university of california, los angeles. multimedia for learning: methods and development gender differences in the perception and use of an informal science learning website. grant funded by national science foundation building a coherent conception of 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computer supported collaborative learning key: cord- -echu zmf authors: aich, palok; potter, andrew a; griebel, philip j title: modern approaches to understanding stress and disease susceptibility: a review with special emphasis on respiratory disease date: - - journal: int j gen med doi: nan sha: doc_id: cord_uid: echu zmf studies in animals and humans link both physical and psychological stress with an increased incidence and severity of respiratory infections. for this manuscript we define stress as the physiological responses an individual undergoes while adjusting to a continually changing environment. it is known that stressors of various types (psychological/physical) can alter the physiological levels of certain hormones, chemokines and cytokines. these alterations send information to the central nervous system to take necessary action which then sends messages to appropriate organs/tissues/cells to respond. these messages can either activate or suppress the immune system as needed and failure to compensate for this by the body can lead to serious health-related problems. little is known how stress affects disease susceptibility, yet understanding this mechanism is important for developing effective treatments, and for improving health and food quality. the current review focuses on (a) the effects of psychological stressors in humans and animals, (b) various methodologies employed to understand stress responses and their outcomes, and (c) the current status of the attempts to correlate stress and disease with respiratory disease as model system. the methodologies included in this review span traditional epidemiological, behavioral and immunological studies to current high throughput genomic, proteomic, metabolomic/metabonomic approaches. with the advent of various newer omics and bioinformatics methodologies we postulate that it will become feasible to understand the mechanisms through which stress can influence disease onset. although the literature in this area is limited because of the infancy of this research area, the objective of this review is to illustrate the power of new approaches to address complex biological questions. these new approaches will also aid in our understanding how these processes are related to the dynamics and kinetics of changes in expression of multiple genes at various levels. from the preceding discussion it is clear that stress can be defi ned in many different ways depending upon the objectives or perspective of the researcher. all these defi nitions, however, share a common component of adaptive physiological responses following challenges to homeostasis. the adaptive reactions to stressors may involve mobilization of a wide variety of physiological responses including the immune response. stress responses usually include physical perturbations that can encompass either the entire body or specifi c cellular compartments. considering the volume of work in various areas of stressors and their effects the main objective of the current review is to focus on one type of stress, which includes psychological stressors. for our purposes, stress can be defi ned as a psychologically perturbing condition occurring in response to adverse external infl uences capable of affecting physical health. transportation, fear (ie, fright and fl ight response), overcrowding and weaning in the form of social reorganization are a few of the important types of psychological stressors identifi ed in the literature. these stressors have been linked to many conditions including immune suppression, disease susceptibility, hypertension and reproductive dysfunctions. [ ] [ ] [ ] stress is a major concern because it is ubiquitous, recurring in nature and has detrimental effects on health. in this review we will primarily deal with important psychological stressors and their infl uence on respiratory disease which is one of the most widely studied models. for many years, psychological stress has been shown to signifi cantly increase disease susceptibility. , until years ago, researchers investigating the psychological factors contributing to human disease focused primarily on coronary heart disease and cancer and neglected studies on infectious diseases. however, interest in this area started to shift with the publication of evidence that psychological factors infl uenced immune function. furthermore, there was an increasing awareness that stress and other psychological factors played a role in the onset and progression of acquired immunodefi ciency syndrome (aids). these studies demonstrated a signifi cant role for psychological stressors in compromising immunity and interest in the effects of stressors in other diseases was initiated. considerable emphasis has been placed on respiratory diseases in understanding the onset and severity of the disease as a result of psychological stress. increased risk of fatal bacterial respiratory infections following a primary viral infection has been observed in a wide variety of species. this phenomenon is called viral-bacterial synergy and was fi rst established following human infl uenza epidemics when a variety of secondary bacterial respiratory infections were associated with increased mortality. studies have also linked a variety of psychological stressors with an increased incidence and severity of respiratory infections in humans , and animals. , it is known that respiratory disease has a huge economic impact in the areas of human health care, animal welfare and the food industry. , to focus the review, we will restrict our discussion to research related to a comparative analysis of the effects of psychological stress on respiratory disease in humans and cattle. there are reports which have shown direct connections between stress and immune system function. similarly, other studies have shown that social stressors could also increase the risk for upper respiratory infection. a viral challenge study provides the strongest evidence for a link between stress and susceptibility to the common cold. other studies have extended these results by considering a wider range of psychosocial factors. the effects of stress on health are often mediated by a number of psychological factors. social support can often act as a buffer against the effects of stress as cohen and colleagues showed that social support reduced viral replication rate and increased mucociliary clearance of infection. , in another report, they examined the effects of stress and social support in a routine study of upper respiratory tract illnesses. under low levels of stress, high levels of social support were associated with a decreased risk of infection, whereas social support had no effect when levels of stress were high. a separate study was done to examine the associations between psychosocial factors (stress, social support, fl uctuations in mood) and viral exacerbations of asthma. the study involved naturally occurring illnesses rather than experimentally-induced infections but it maintained several important features of the methodology used by cohen and colleagues. , for example, stress was measured at the start of the study by measuring the immune responses in terms of leukocytes present in peripheral blood in order to determine the extent to which stress could predict subsequent illness. in addition, effects of personality, smoking status, and alcohol consumption were also studied as possible predictors of susceptibility to respiratory viral infections. before considering the effects of psychosocial factors on respiratory virus-induced exacerbations of asthma, it is essential to have strong evidence that these viruses play a direct role in asthma. until recently, it appeared to be only a weak association between asthma and upper respiratory tract infection in adults. , the absence of a stronger association in these epidemiological studies of adult asthmatics could, at least in part, have been due to diffi culties in isolating human rhinoviruses and coronaviruses. indeed, results from a study using enzyme-linked immunosorbent assays for antibodies to human coronavirus and semi-nested reverse transcriptase polymerase chain reactions for detections of rhinovirus suggested a stronger association between these viruses and asthma in adults. in summary, this study confirmed that psychosocial factors and health-related behaviors were associated with increased susceptibility to colds, which then led to an exacerbation of asthma. this conclusion was made in the context of a study where both diseases (cold and asthma) were verifi ed using objective measures. the well established buffering effect of social support was observed in the high stress group and possible confounders such as demographics, health-related behaviors or personality could not account for this effect. alcohol consumption, personality and demographic factors were also shown to be important predictors of susceptibility. in contrast smoking was related to illness severity but not disease susceptibility. these results show that one must consider a range of psychosocial factors, personality traits, demographic factors, and health-related behaviors in studies of individual differences in susceptibility and severity of upper respiratory tract infections. while studies have correlated stress-related behavior with disease susceptibility, research has also been performed to determine the immunological basis of the relationship between stress and disease. exposure to viral agents that cause upper respiratory disease provokes illness in some individuals but not others. moreover, the severity of clinical symptoms among those who develop illness can vary substantially. evidence from prospective epidemiological studies , , and from experimental viral-challenge studies , show that individuals reporting greater psychological stress have both a higher incidence and a greater severity of illness. however, past attempts to identify the behavioral patterns and biological responses linking psychological stress with upper respiratory viral illness have been unsuccessful. , , other studies reported that psychological stressors acutely activate the production of interleukin- (il- ), a pro-inflammatory cytokine. il- release in response to infection is thought to be mediated by glucocorticoids, thus providing a hypothetical pathway by which stressors (via the induction of glucocorticoid production) could control cytokine release. in addition, il- triggers additional release of glucocorticoids, possibly exacerbating the stress response through positive feedback. at least one source of il- is epithelial cells as evidenced by their production of il- in vitro and in vivo when exposed to rhinovirus. because a local increase in the concentration of this pro-infl ammatory cytokine precedes the development of acute signs and symptoms of illness, it has been implicated as a mediator in the pathway for symptom expression. in fact, il- concentrations in nasal secretions were associated with upper respiratory tract symptoms among persons infected with infl uenza a virus (a/texas and a/kawasaki) and rhinovirus (strain hanks' and type ). cohen and colleagues addressed the hypothesis that il- production in response to infl uenza a virus infection represents a viable pathway through which psychological stress infl uences the severity of illness. to achieve that goal, they measured levels of psychological stress in a group of adult subjects before experimentally infecting them with influenza a virus. self-reported respiratory symptoms, mucus weights, and local il- concentrations were then measured on the day before and for seven days after virus exposure. the data collected supported the conclusion that the level of psychological stress predicts the severity of illness and also the magnitude of the cytokine response. the data were then examined for evidence that il- mediated the association between stress and illness. these analyses suggested that most of the effects of psychological stress on clinical symptoms and mucus weights could be accounted for by changes in il- . however, it is possible that il- itself is not the causal link but rather just a marker (covariate) for other pro-infl ammatory cytokines which were elevated during the course of experimental infection. for example, in another related study reported that interferon α and il- levels (but not tumor necrosis factor [tnf], il- , il- , or il- ) increased early in the course of infection and both correlated with viral titers, increases in body temperature, mucus production and symptom scores. there is also an issue regarding the correlational nature of the mediational analysis. although consistent with the hypothesis that the association between stress and illness was mediated by il- , the data do not permit causal inference. for example, this pattern of data is also consistent with increases in il- occurring in response to tissue damage associated with illness symptoms. even with these reservations, this was the fi rst aich et al study to provide evidence consistent with the hypothetical model that psychological stress infl uences upper respiratory infections through a biological pathway. there are two parts to the stress response: sympatheticadrenal-medullary (sam) and the hypothalamic-pituitaryadrenal axis (hpa). the hpa is the core stress axis in mammals and together with the sam system co-ordinates response to the diverse range of stressors from psychological to physical. there is considerable interplay between both neuronal systems especially between the noradrenergic nucleus locus ceruleus which provides central regulation of the sam and the parvocellular neurones which regulate the hpa. the sam, by triggering catecholamine release, provides the acute stress response whilst the hpa governs longer term stress defence mechanisms. together those systems regulate energy utilisation and metabolic activity throughout the body. the sam system produces the immediate shock response by acting on the hypothalamus, which activates the adrenal medulla and the sympathetic autonomic nervous system (ans) (figure ). the sam produces the "fi ght-or-fl ight" response which increases alertness, blood fl ow to muscles, heart rate, blood pressure, respiration rate, etc. and might decrease activity in the digestive system. the hpa system regulates release of the hormone crf to activate the anterior pituitary and uses another hormone, adrenocorticotropic hormone (acth), to activate the adrenal cortex to release a group of hormones including cortisol ( figure ). cortisol and other glucocorticoid hormones have various effects such as conservation of glucose for neural tissues, elevation or stabilization of blood glucose levels, mobilization of protein reserves, conservation of salts and water, suppression of wound healing and the immune system. according to seyle's general adaptation syndrome (gas) theory the general adaptation syndrome divides the stress response into three stages: stage : alarm reaction (sam and hpa activity increases and result in the "fi ghtor-fl ight" response); stage : resistance (hpa activity takes over, bodily resources are at maximum and if the stress is experienced for short term the body returns to normalcy); stage : exhaustion (with very prolonged stress, bodily systems are ineffective. sympathetic ans action reappears. adrenal cortex damage causes parasympathetic action, omics approaches in understanding stress and disease susceptibility eg, energy storage failure. the immune system collapses, and stress-related diseases increase). while it has been assumed that psychological (psychogenic) and physical (neurogenic) stressors are most closely aligned with depression, the suspicion has arisen that systemic stressors, including immune alterations, may also act in such a provocative capacity. communication occurs between the immune, endocrine, autonomic and central nervous systems such that psychological events that affect central neurochemical processes may affect immune activity. conversely, immune activation may affect hormonal processes and the activity of central neurotransmitters. thus, by virtue of the neurochemical effects imparted, immune activation may affect behavioral outputs and may even be related to behavioral pathology such as depressive illness. the hypothesis that altered immune activation may occur as a result of various stressors emerged over time. the initial theory came from hans seyles' gas, which was derived from observation and experimentation on laboratory animals. using a variety of stressors (ie, pain, thermal extremes and starvation), seyle described a common nonspecifi c stress response pathway. after initial perception of a stressor, the animal mounts an emergency alarm or fi ght-or-fl ight response. this catecholamine-driven reaction results in increased cardiovascular function and an overall increase in metabolism. if the stressor persists, the resistance phase or 'conservation withdrawal reaction' is initiated, which is a physiological coping reaction to the increased demands of maintaining homeostasis. chronic stress leads to the exhaustion phase and may lead to pathology. seyle's theory unifi ed the stress phenomena because it provided a common response pathway to all the varied stressors encountered. this pathway, the hpa axis, involves perception in the brain with release of hypothalamic corticotropin-releasing factor (crf) and vasopressin which stimulates the anterior pituitary to secrete acth ( figure ). circulating acth causes the adrenal cortex to produce glucocorticoids (gc). glucocorticoids cause gluconeogenesis with conversion of lipid to glucose for the central nervous system (cns) and other functions. thus, gas allowed the identifi cation of stressors (and presumably the status of well being of the animal) by measurement of gc levels. it was an attractive theory because gc levels were relatively easy to measure. unfortunately the gas theory has proven to be too simplistic. mason's experiments with rhesus monkeys aich et al exposed to different types of stressors revealed a disparity in neuroendocrine responses provided by specifi c stressors. for instance, monkeys subjected to emotional stress had elevated serum gc levels but those subjected to a heat-stress regime failed to show gc elevation. in addition to gc, other neuroendocrine mediators were characteristically produced in response to specifi c stressors. emerging information on the response to stressors suggests that there are at least four different avenues of neuroendocrine responses. these involve the autonomic nervous system, the hpa axis, neuropeptides, neurotransmitters and neuroimmunological peptides and receptors. interactions between the immune and nervous systems play an important role in modulating host susceptibility and resistance to infl ammatory disease. neuroendocrine regulation of inflammatory and immune responses as well as onset of disease can occur at multiple levels: (a) systemically through the anti-infl ammatory action of gcs released via hpa axis stimulation, (b) regionally through production of gcs within the affected tissue as well as by sympathetic enervation of immune organs such as the thymus and (c) locally at sites of infl ammation. estrogens also play an important role in immune modulation and contribute to the approximately -to -fold higher incidence of autoimmune/infl ammatory diseases in females of all mammalian species. during infl ammation, cytokines from the periphery activate the central nervous system through multiple routes. this results in stimulation of the hpa axis which in turn, through the immunosuppressive effects of the glucocorticoids, generally inhibits infl ammation. recent studies indicate that physiological levels of glucocorticoids are immunomodulatory rather than solely immunosuppressive causing a shift in patterns of cytokine production from a th -to a th -type pattern. interruptions of this loop at any level and through multiple mechanisms, whether genetic, or through surgical or pharmacological interventions, can render an infl ammatory resistant host susceptible to infl ammatory disease. over-activation of this axis, as occurs during stress, can also affect severity of infectious diseases through the immunosuppressive effects of the glucocorticoids. these interactions have been clearly demonstrated in many animal models using a variety of species and infectious agents. the results from these models are also relevant to human infl ammatory, autoimmune and allergic illnesses including rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, allergic asthma and atopic skin disease. while many genes and environmental factors contribute to susceptibility and resistance to autoimmune/infl ammatory diseases, a full understanding of the molecular mechanisms by which a combination of neuropeptides, neurohormones and neurotransmitters can modulate immune responses is essential for effective design of future interventions. it is clear from the previous discussion and review of previous research that the relationship between stress and disease susceptibility is very complex and intertwined with cascades of events. in order to understand or characterize stress-induced disease susceptibility one needs to identify various biological or functional pathways and interaction networking involved at any given time. to follow the cascade of molecular events happening over time one needs to employ methodologies which are holistic in nature and can provide global information related to the kinetics of multiple changes in gene expression and multiple biomolecular interactions. with the advent of various high-throughput genomic approaches it has become possible to explore complex biological processes and in one step obtain information about gene expression at the transcriptional (genomics) and translational (proteomics) level, as well as to identify metabolites arising from these responses (metabolomics/metabonomics). these methodologies together are often referred to as 'omics' approaches. although these methodologies are still in their infancy they have started showing promise in understanding systems' biology and various disease processes. work employing omics approaches to understand mechanism of stress and disease susceptibility is limited at this time. there are reports which describe the effects of oxidative stress on cellular apoptosis but there are no reports on the effects of psychological stressor and disease susceptibility in animals using holistic methodologies such as various omics approaches. the literature in this area is very scarce because these methodologies are very new and few results are currently available. the main studies to correlate stress and disease susceptibility was done either in plant systems or a correlation between oxidative stress and infectious disease. the review by hirai and saito established, using liquid chromatogram based mass spectrometric proteomic and metabolomic and cdna microarray based transcriptomic analyses, that these omics studies can reveal the genomic networking involving several pathways related to oxidative stress response and key metabolic pathways. bioinformatics and statistical approaches specifi c for detailed transcriptomics or cluster analyses of expressed genes have been developed and are useful for extracting information on various functional genomic responses. [ ] [ ] [ ] it is important, however, to understand the translation of transcriptomic information into protein expression and modifi cations since it is the protein that acts as biomarkers for various disease processes or conditions. several proteomic tools have been developed. these include gel-based two-dimensional ( d) gel electrophoresis (ge), d fluorescence difference gel electrophoresis ( dige) or gel-free multidimensional protein identifi cation technology (mudpit), , isotope-coded affinity tag (icat™), surface-enhanced laser desorption-ionization time-of-flight (seldi-tof) ms , or isobaric tag for relative and absolute quantitation (itraq) methodologies. while these technologies are being matured more appropriate biological questions can be addressed. in d-ge whether traditional or newer dige system, proteins are fi rst focused (fi rst dimension) in terms of their pi values followed by sds-page (second dimension). in contrast to traditional d-ge, dige system utilizes the fl uorescence labeling of protein samples and two-to-three different samples can then be run on a single gel which makes it superior to traditional d-ge in terms of eliminating gel to gel variation and comparative analysis. although d-ge is a very good way of separating complex protein mixtures, it is limited in terms of high-throughput analysis and sensitivity. because of low sensitivity it is mostly limited to the analysis of high abundant proteins of a system. efforts have been made to improve the sensitivity and analysis capacity of proteomics techniques. as a result, other methodologies such as d-hplc, mudpit, icat, and itraq have been developed and are gaining popularity. although various bioinformatic and data analyses approaches have been developed to analyze gel images and mass spectrometric based protein characterization a highthroughput methodology in this area has yet to be developed. functional genomic techniques of transcriptomics and proteomics and available bioinformatic and statistical analyses promise unparalleled global information during the analyses of complex biological responses. however, if these technologies are used in isolation, the large multivariate data sets produced are often diffi cult to interpret and have the potential to ignore key metabolic events to understand the true biology. high resolution h nmr spectroscopy used in conjunction with pattern recognition provides one such tool for defi ning the dynamic phenotype of a cell, organ, or organism in terms of a metabolic phenotype. in a recent review the benefi ts of this metabonomics/metabolomics approach to problems in toxicology have been discussed. one of the major benefi ts of this approach is its high-throughput nature and cost-effectiveness on a per sample basis. using such a method, the consortium for metabonomic toxicology (comet) is currently investigating approximately model liver and kidney toxins. this investigation will allow the generation of expert systems where liver and kidney toxicity can be predicted for model drug compounds, providing a new research tool in the fi eld of drug metabolism. the review also included how metabonomics may be used to investigate co-responses with transcripts and proteins involved in metabolism and stress responses such as during drug-induced fatty liver disease. by using data integration to combine metabolite analysis and gene expression profi ling, key perturbed metabolic pathways can be identifi ed. detailed omics-based bioinformatics studies, to correlate stress-dependent disease susceptibility, have yet to analyze this complex biological response. a few preliminary studies have been reported in the literature. a recent study has shown the adverse effects of using mechanical ventilators for respiratory support. this acute lung injury because of mechanical stretch can lead to high mortality and this study has utilized recent advances in bioinformatic-intense candidate gene searches to correlate the lung injury and gene expression profi les in a rodent model analyzed with microarrays. the authors used , mouse/rat orthologous genes identifi ed on rg_u a and mg_u av arrays and the expression profi les were simultaneously analyzed by signifi cance analysis of microarrays. this combined ortholog and signifi cance analysis of microarrays approach identifi ed up-and downregulated ventilator-induced stress-related candidate genes. results were validated by comparable expression levels obtained by either real-time or relative rt-pcr for randomly selected genes. k-mean clustering of candidate genes clustered several well-known lung injury associated genes (il- , plasminogen activator inhibitor type , ccl- , cyclooxygenase- ) with a number of stressrelated genes (myc, cyr , socs ). the only unannotated member of this cluster (n = ) was riken_ f est, an ortholog of the stress-related gene /mig- gene. the authors speculated that the ortholog-signifi cant analysis of microarray approach is a useful, time-and resourceeffi cient tool for identifi cation of candidate genes in a variety of complex disease models such as ventilator induced lung injury. using microarray-based genomic approaches work has also been initiated to identify hypertension-related genes in rat model. though it is a common belief that the stressors mentioned earlier can compromise host immune responses and enhance susceptibility to various diseases, very little work has been done in this area to understand the mechanism of stress dependent disease susceptibility in mammals. kelley in identifi ed eight stressors that typically occur in modern livestock production units: heat, cold, crowding, mixing, weaning, controlled-feeding, noise and restraint. all these stressors were shown to alter components of the immune system in animals and these changes in immune function may ultimately explain the physiological basis of diseaseenvironment interactions. another study in showed that neurotransmitters and neuroendocrine hormones can modify the function of immune cells. conversely, cytokines produced by immune cells can alter brain homeostasis. this connection is further manifested by experimental studies showing a relationship between stress and resistance to infection. human subjects under high stress were shown to be more susceptible to infection with common cold viruses. furthermore, a diversity of experimental animal models confi rmed that laboratory stressors such as forced exercise, avoidance learning, restraint, isolation and cold exposure made animals more susceptible to primary infection with a variety of viruses and bacteria. the cellular and molecular basis for the observed modulation of host resistance is not fully understood but involves altered functioning of both t-lymphocytes and cells of the hypothalamic-pituitary adrenal axis. also involved is the altered production of cytokines and hormones produced by the immune system and brain. emau and colleagues in showed that the onset of immunodepression by stress or viral infection in the pathogenesis of bovine pneumonia permits super infection of the lungs with mannheimia haemolytica (formerly called pasteurella haemolytica) which results in exudative fi brinous pneumonia. although these studies provide a preliminary basis of stress-induced bovine respiratory disease (brd), there was a lack of necessary information to determine the molecular basis of such dependence, which can be found by studying changes at the level of gene expression (transcriptional and proteomic) and metabolites. a recent study compared proteomics of epithelial lining fl uid from lungs of weaned and nonweaned cattle. the study was done over a shorter period of time ( h) with a combination of stressors (weaning, transportation) and important questions such as to determine specifi city and duration (stability) of the protein biomarkers are yet to be addressed. the group also studied the proteomic profi le of bronchoalveolar lung lavage fl uid following treating the calves with dexamethasone to determine the effects of glucocorticoids, which is elevated following induction of stress. , more studies are however needed (a) to fi nd biomarkers associated with stressors and infection, (b) to determine the duration of the biomarkers so that these are sustainable enough to identify and predict stressed animals (which might be more susceptible to infection in real life situation such as in feedlot cattle). disease models have been developed to study the molecular mechanisms underlying the viral-bacterial synergy which results in fatal brd infections. [ ] [ ] [ ] we have confi rmed with the infectious brd model that the stress of weaning significantly enhances the viral-bacterial synergy leading to fatal bacterial respiratory infection. the major stressors young omics approaches in understanding stress and disease susceptibility cattle experience include maternal separation or weaning, dietary changes, transportation, social reorganization and other environmental effects (figure ). with this in mind, we focused our initial studies to understand the role of weaning on the clinical response to brd. fifteen suckling calves were removed from their mothers h prior to viral infection (abruptly weaned/stressed, aw). a second group of calves was weaned wks prior to viral infection (pre-conditioned/ control, pc); wks was chosen as an appropriate interval to eliminate psychological and physiological effects associated with breaking the maternal/nutritional bond and to adapt to the dietary change and social re-organization that follows weaning ( figure ). all calves were transported to vaccine and infections disease organization vido, and aerosol challenged with bovine herpesvirus- (bhv- ) followed by m. haemolytica; this combined viral-bacterial infection induces fatal pneumonia in %- % of calves. our clinical analyses revealed a signifi cant difference in brd clinical disease when comparing freshly weaned calves ( % mortality) versus pre-conditioned calves ( % mortality). , increased mortality associated with fresh weaning was characterized by a decrease in both survival time post-infection and, interestingly, decreased lung pathology which suggested a systemic reaction. contrary to expectations, transportation induced a signifi cant cortisolemia in pre-conditioned but not freshly weaned calves ( figure ). transportation is known as an important stressor which increases blood cortisol level. [ ] [ ] [ ] [ ] however, the stressor combination of weaning and transportation may have different physiological markers than commonly expected. cortisol is a potent regulator of pro-infl ammatory cytokine transcription by acting as a negative regulator of nfκb activation, and can be used as an indicator of stress. fatal bacterial respiratory infection in calves was usually associated with an accelerated decline in serum cortisol levels. therefore, we hypothesized that increased respiratory disease susceptibility was associated with enhanced pro-infl ammatory responses in freshly weaned calves. elevated body temperature and interferon-gamma (ifn-γ) levels in nasal secretions during bhv- infection of freshly weaned calves were similar to previous reports, , supporting this hypothesis. these immune responses were signifi cantly increased despite no signifi cant difference in virus shedding. furthermore, increased il- expression, during bhv- infection of pre-conditioned calves, was consistent with reduced pro-infl ammatory responses. we conducted bovine microarray analyses of rna isolated from blood mononuclear cells to determine if changes in gene expression correlated with either stress or the severity of brd infection; results support the conclusion that differential regulation of pro-infl ammatory responses is a major mechanism contributing to increased disease susceptibility. conserved responses included an enhanced potential to aich et al respond to pathogen-associated molecular patterns through increased expression of toll-like receptors (tlrs). tlr induction of innate immune responses is mediated primarily through activation of nuclear factor kappab (nfκb). we also observed differential expression of β-defensin (a host defense peptide induced by tlr signaling) in freshly weaned calves, consistent with reduced cortisol levels. in addition, when we compared expression of select innate immune genes (eg, tlr , tlr , ifn-γ and ' ' o-adenylate synthetase) between day (post bhv- infection) and day (prior to bhv- infection) for aw-and pc-groups, results revealed a trend of increased expression of the select genes on day irrespective of the stress situation (table ). this trend of activation of select immune genes clearly revealed that following bhv- infection the innate immune response was enhanced. this observation contradicts the current hypothesis that the primary viral enhances the secondary bacterial infection by compromising the immune system. , our studies revealed that the anti-infl ammatory gene il- was upregulated on day in pc groups while β-defensin was activated on day in aw groups (table ) . il- which acts as an anti-infl ammatory gene should be activated on day to control pro-infl ammatory responses as a result of bhv- infection in either aw or in both groups; instead it was only over expressed in control (pc) groups. on a similar token β-defensin on day should also be observed in both groups to control bhv- infection. these results are particularly interesting to explore further to understand the detailed mechanism of stress-induced disease correlation. although advances have been made in understanding various disease processes, successful intervention often depends upon the stage at which disease is detected. thus, identifying markers for disease or its cause, as well as understanding the mechanism of disease onset and susceptibility, are very important. systems biology approaches such as omics (genomic, proteomic, metabonomic) and multivariate analysis to understand mechanisms and to identify biomarkers provide potential tools to address these questions. , [ ] [ ] [ ] it is particularly important to employ a combination of molecular approaches such as omics to understand complex processes such as stress and its correlation with disease susceptibility. with such goals in mind, we identifi ed and characterized a group of protein, metabolite and elemental biomarkers using serum samples from bhv- infected as well as from stressed animals. the trend of each group of biomarkers distribution was analyzed to correlate with the groupings based on stress condition or infection. multivariate analysis revealed distinct differential trends in the distribution profi le of proteins, metabolites and elements following a stress response both before and after primary viral infection. a group of acute phase proteins, metabolites and elements could be specifi cally linked to either a stress response (decreased serum amyloid a and cu, increased apolipoprotein ciii, amino acids, low-density lipoprotein [ldl], p and mo) or a primary viral respiratory infection (increased apolipoprotein a , haptoglobin, glucose, amino acids, ldl and cu, decreased lipid and p). thus, combined omics analysis of serum samples revealed that multimethod analyses could be used to discriminate between the complex biological responses to stress and viral infection. there are reports which suggest that transport can be an important stressor which alters blood leukocyte populations and sets the stage for brd. , currently we are conducting experiments in which transportation of the animals is eliminated, such that stressors related to a new environment are removed. it was documented that abrupt weaning (separation of suckling calves from their dams) causes a prolonged psychological stress response. this stress response manifests as an increase in vocalization by both calves and cows and a significant increase in time spent walking by the calf. consequently, time spent eating and resting also decreases for abruptly weaned calves. statistically, these changes in behavior return to baseline values after four to fi ve days of weaning. as is evident from the review of the literature that traditionally effects of stress have been studied by attempting to understand behavioral patterns of the subject and cellular immune response of the host. it is, however, clear that amount of data to understand such a complex condition (such as stress and stress-induced diseases) is not adequate and enough. behavioral studies may not always be correlated to a particular stressor as there might have been other parameters affecting the observations. attempts have also been made to use cortisol as a unique biomarker for stress, but cortisol is diurnal hence cannot be a reliable marker. while it is important to understand cellular immunology to establish the mechanism of stress-induced disease susceptibility, however without identifying and establishing the immune markers specifi c for stressor it will not lead into any meaningful inference. systems biology approaches using various omics and bioinformatics methodologies can identify a group of biomarkers at various levels of host immune response, gene expression and metabolism. association of the identifi ed biomarkers and the patterns in changes in their expression level above or below a determined threshold level with specifi c stressor(s) could then be used to defi ne stress situation, identif ication of individuals susceptible to stressinduced disease. moreover, establishing a pattern in changes of biomarkers is more reliable than a single biomarker. duration of identifi ed biomarkers can further strengthen the methodology for applying in real life situation. the current review established the importance of psychological stress and its effects on infectious disease severity and susceptibility with emphasis on respiratory disease. as evident from the literature, initial work focused mainly on understanding the roles of various stressors on physiology and behavior. theories emerged as a consequence of these studies which implicated the hpa axis and the sympathetic nervous system (sns) in altered immunity. however, physiological responses studied in a laboratory may not necessarily be consistent with real life phenomena, especially when attempting to analyze heterogeneous populations such as cattle or humans. experiments with human subjects cannot 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haemophilus somnus and bovine respiratory syncytial virus proteomic characterization of the interstitial fl uid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery unlocking the mysteries of virus-host interactions: does functional genomics hold the key? a comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in escherichia coli cells effect of transport stress on respiratory disease, serum antioxidant status, and serum concentrations of lipid peroxidation biomarkers in beef cattle effect of transportation on blood serum composition, disease incidence, and production traits in young calves. infl uence of the journey duration this project was funded by the saskatchewan agriculture and food rural revitalization (safrr), genome bc and genome prairie for the 'pathogenomics of innate immunity' research program and the ontario cattleman's association (oca). this manuscript is published with permission of the director of vaccine and infectious disease organization as article number . a.a.p. is the holder of an nserc senior industrial research chair. key: cord- - nchg h authors: rampling, tommy; page, mark; horby, peter title: international biological reference preparations for epidemic infectious diseases date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: nchg h recent years have seen unprecedented investment in research and development for countermeasures for high-threat pathogens, including specific and ambitious objectives for development of diagnostics, therapeutics, and vaccines. the inadequate availability of biological reference materials for these pathogens poses a genuine obstacle in pursuit of these objectives, and the lack of a comprehensive and equitable framework for developing reference materials is a weakness. we outline the need for internationally standardized biological materials for high-threat pathogens as a core element of global health security. we also outline the key components of a framework for addressing this deficiency. although several hundred centers contribute to who collaborative studies, only centers produce and hold irps. these centers are the national institute for biological standards and control in england, the center for biologics evaluation and research in the united states, and the paul-ehrlich institute in germany. the role of these institutions is to produce, store, and distribute irps and differs from those that distribute repository materials, such as the us centers for disease control and prevention, the international reagent resource, and the national institute of allergy and infectious diseases biodefense and emerging infections research resources repository. in the aftermath of the west africa ebola virus disease (evd) outbreak, there has been considerable focus on expediting research and development to ensure we are better prepared for future disease outbreaks. in , who launched the research and development blueprint for action to prevent epidemics, a global strategy and preparedness plan that seeks to build upon the successes and address the gaps identified during the evd outbreak by focusing on severe, emerging diseases with the potential to create a public health emergency and for which inadequate treatment and preventive options are currently available ( , ) . the blueprint is organized such that operations fall into clusters of activities: ) improving coordination, ) accelerating research and development processes, ) developing norms and standards, and ) streamlining operational research and development response during outbreaks. furthermore, the second cluster, which focused on accelerating research and development processes, is subdivided into distinct areas of work: ) assessing epidemic threats and defining priority pathogens; ) developing research and development roadmaps to accelerate evaluation of diagnostics, therapeutics, and vaccines; and ) outlining appropriate regulatory and ethical pathways. central to the research and development blueprint is a list of priority diseases, first published in november , with subsequent revision through a tailored prioritization method in and (table ) ( , ) . for each priority disease on the list, a research and development roadmap is to be produced that encompasses diagnostics, vaccines, and therapeutics. the first research and development roadmap to have been developed and published is for middle east respiratory syndrome coronavirus (mers-cov) ( ) , which serves to inform the roadmap generation process for other diseases on the priority pathogen list. within the mers-cov roadmap, development of reference reagents has been identified as a priority, but the major operational and ethical issues associated with sourcing and obtaining bulk starting material for such reagents have not been discussed. an allied initiative is the coalition for epidemic preparedness and innovation (cepi), whose aim is to accelerate development of vaccines for high-threat pathogens ( ) . the necessity for reference materials in development and evaluation of new diagnostics, vaccines, and biotherapeutics has been recognized by cepi by the formation of a working group on standards and assays, but they are not explicitly considered in the who research and development blueprint. before the evd epidemic, no irps existed for any diseases on the research and development bueprint. expedited development led to production of an interim ebola antibody reference reagent in october , and the first international standard was endorsed in october ( , ) . although this accelerated production represents an impressive collaborative feat, the lack of availability of pcr, antigen, and antibody irps at the start of this evd outbreak, and for subsequent outbreaks of infection with mers-cov and zika virus, likely hampered development of accurate diagnostics and vaccines ( ) . in addition, the exceptional circumstances and unprecedented duration of the evd epidemic enabled a major proportion of irp development to occur while the outbreak was ongoing. development of irps from sample acquisition to who endorsement usually takes - years, which is considerably longer than most outbreaks of diseases on the research and development blueprint. the availability of irps for often rare and sporadic diseases with epidemic potential before, or soon after, occurrence of outbreaks would facilitate diagnostics and research activities, such as seroepidemiologic studies and immunogenicity assessment of experimental vaccines. at the present time, with the exception of zaire ebolavirus antibody, antigen, and nucleic acid extensively characterized following international collaborative studies enable activity of biological preparations to be expressed in the same way globally, most commonly in international units international reference reagents less extensively characterized than international standards. not assigned international units. reference reagents are interim and not intended to be replaced when they expire or are depleted. group of reference materials established to collectively aid evaluation of assays or diagnostic tests. comply with requirements for who reference standards/reagents. working or secondary standard reference standards established by regional or national authorities, or by other laboratories, that are calibrated against, and traceable to, the primary who materials and are intended for use in routine tests. *who, world health organization. irps and a who-endorsed zika virus rna and antibody standards, no irps exist for any other disease on the research and development blueprint. the availability of irps for these diseases would facilitate development of essential tools in epidemic preparedness, including diagnostics, vaccines, and therapeutics. accurate and accessible diagnostic tools are vital in limiting the public health effect of disease outbreaks, in addition to providing reliable data for clinical and epidemiologic studies. a coordinated diagnostic product development effort for epidemic disease threats has been initiated by the foundation for innovative new diagnostics under the auspices of cepi ( ) . this initiative seeks to build on collaborations with key diagnostic partners to enable adequate provision of diagnostic capabilities during outbreaks. who states that irps are major resources for ensuring the reliability of in vitro biological diagnostic procedures used for diagnosis of diseases ( ) . the recent epidemics caused by mers-cov, ebola virus, and zika virus have highlighted complexities in prompt development of sensitive and specific assays, particularly those that would be accessible in resource constrained settings ( ) ( ) ( ) . availability of international reference standards that encompass genetic material, antigens, and antibodies would enable harmonization and calibration of tests. these standards would facilitate development and validation of diagnostic assays, which include nucleic acid amplification tests, serologic assays, and antigen-based point-of-care tests. no vaccine or therapeutic is currently licensed by regulators in europe or the united states for use for any disease on the research and development blueprint. on august , , who declared the ebola outbreak in west africa to be a public health emergency of international concern. this declaration led to subsequent concerted efforts from the international scientific community to accelerate development of ebola vaccines, with funding, regulatory and ethical review, expert advice, and manufacturing support being initiated at extraordinary speed ( ) . although vaccine development was considered to be a relatively successful component of the ebola research and development efforts, and the recombinant vesicular stomatitis virus-zaire ebola virus showed high efficacy in a ring vaccination trial ( ), this development came too late to have any major effect on the course of the west africa epidemic. other candidate vaccines also underwent clinical evaluation, and encouraging safety and immunogenicity data were generated from phase and trials of several candidates. however, studies were performed by different groups with a variety of immunogenicity assays and reference reagents being used, making comparison of immunogenicity results complicated ( ) ( ) ( ) ( ) ( ) . a set of biological standards that are common to all assays would enable calibration and harmonization of assay data between trials, and could prove vital in determining correlates of protection and clinical outcomes. these standards would also enable better selection of candidate vaccines to transition from the preclinical stage to clinical evaluation. there has been considerable interest in biological therapies for emerging epidemic threats ( ) . convalescent-phase plasma and monoclonal antibodies have been evaluated as treatment for ebola ( , ) and have been considered as therapies for zika virus ( ) and mers-cov ( , ) , forming a key focus of the mers-cov research and development blueprint roadmap ( ) . availability of †any disease identified before the next review by using the blueprint decision instrument will be included in the list. irps would enable characterization of the potency, purity, and identity of complex biological materials, such as antibodies, which would facilitate not only research of immunotherapies but also the standardization of preparations postlicensure. antigen and nucleic acid reference materials can be synthesized through recombinant techniques as long as necessary genomic sequence data are available ( ) ( ) ( ) , but for some newly emerging pathogens, acquisition of live pathogens might be required. simple blood draws can complicate field operations in the midst of an epidemic, and logistical issues regarding processing and storage of acute, potentially infectious samples can also present many obstacles. however, the ideal starting material for production of antibodybased reference preparations is serum or plasma from convalescent-phase patients. novel methods for generating fully human immunoglobulin for use as reference materials have been explored when human plasma is not readily available, such as inoculation of transchromosomal cattle and subsequent isolation of antibodies ( , , ) . however, this technology is still relatively new, and convalescent-phase serum is considered superior because it will most closely represent a clinical sample and has a polyclonal range of antibody specificities that can be further optimized by pooling samples. although many previous clinical studies of the research and development blueprint priority diseases have resulted in collection and storage of plasma from convalescent patients, several obstacles would largely preclude these samples being repurposed to generate reference materials. these issues include sample volume, receipt of appropriate consent, appropriate records, documentation of sample provenance, and willingness of researchers to share samples. a more suitable alternative to repurposing old samples would be to initiate clinical studies with objectives that include acquisition of blood samples for production of reference materials for each of the research and development blueprint priority diseases. however, such studies would have several challenges. many pathogens on the who priority pathogens list cause outbreaks that are difficult to anticipate temporally and geographically, and outbreaks are often limited in numbers of cases and duration. these factors complicate prospective acquisition of appropriate clinical samples for development of reference materials. in addition, these pathogens most commonly cause outbreaks in resource-constrained environments, where poor healthcare infrastructure, limited diagnostic capability, and suboptimal disease reporting systems would limit retrospective identification of suitable patients ( ) . for development of ebola antibody irps, plasma was obtained either from recovered evd patients in sierra leone who were enrolled in a convalescent-phase plasma trial or as donations from countries not directly involved in the outbreak (italy, norway, the united kingdom, and the united states) ( , ) . however, it would be inappropriate to use this example as a model for sample acquisition because it required occurrence of a large and devastating outbreak for a concerted international response to be successful. focused efforts to obtain material for the generation of irps should be made for high-threat epidemic diseases, and methods to identify and sample patients from previous outbreaks should be explored. in addition, preparation for promptly identifying and sampling patients prospectively given a diagnosis in future outbreaks should be made. this preparation would optimize the likelihood of obtaining high-quality samples for rare and sporadic disease pathogens, such as nipah virus or rift valley fever virus, but would require a systematic and coordinated approach that brings together local investigators with international partners in a transparent and equitable framework. global initiatives, such as the international severe acute respiratory and emerging infections consortium, exist to facilitate the rapid response to emerging infectious disease and epidemic threats by aiding the sharing of clinical research tools, such as open-access clinical study protocols. initiatives such as this consortium would be well placed to bring together international partners and coordinate a collaborative framework to outline processes for acquisition of samples for generation of reference materials. preemptive identification of study sites and preparation of study documents, including template protocols and clinical agreements detailing methods for identifying and recruiting potential donors, sample collection, and processing, and roles and responsibilities would optimize the likelihood of success in this regard. in situations in which access to existing samples and appropriate consent for use in reference preparations are unavailable, the process of acquiring new samples for development of reference materials should be registered as a unique study for each prioritized epidemic disease. this process would require ethics approval being sought through appropriate channels, thus enabling appropriate scrutiny by relevant ethics and regulatory bodies in countries coordinating activities in disease-endemic countries where samples are to be collected, and in the countries of international partners. these studies would involve minimal risk to participants because they would not involve administration of an investigational medicinal product and in most instances, only a single venesection would be required. serum, plasma, and plasma-derived products contain insufficient genetic material to be universally considered as human tissue under legislation. however, they must be considered human biological material. therefore, informed consent is an indispensable requirement for donations. the donor should receive information concerning all aspects of the study, with emphasis placed on the fact that participation is entirely voluntary. the intended use of the blood components for the generation, storage, and international distribution of reference materials must be explained, in addition to the potential beneficiaries and the procedures involved. appropriate measures should also be taken to ensure protection of personal data and confidentiality, and these issues must be explained to the donor. such information should be contained in a detailed participant information sheet that should be provided to the donor in advance of providing consent. consent processes should consist of all participants signing and dating an informed consent form before any study-specific procedures are performed. a collaborative international framework could include template participant information sheets and informed consent forms that could be readily adapted for disease-and country-specific studies when appropriate. sharing of biological materials is a necessity for rapid research progress, but recent experiences during epidemics have highlighted that concerted efforts to establish acceptable processes for obtaining and sharing reference materials are needed. at the present time, only a small number of institutions manufacture and provide reference materials to who for use as irps. therefore, export of samples to these institutions is an essential step in the production of irps for epidemic diseases. initiatives exist to facilitate sharing of benefits arising from the use of, and access to, human and nonhuman genetic material. an example of such an initiative is the pandemic influenza preparedness (pip) framework launched by who in . the pip framework seeks to address concerns of low-and middleincome countries that sharing of influenza virus specimens with who was not matched with assurances that benefits derived from such sharing would be equitably distributed ( ) . concerns about inequitable sharing of benefits were further exacerbated during the influenza a(h n ) pandemic because of unequal access to vaccines ( ) . despite the example set by the pip framework, more recent epidemics of other infectious diseases have continued to raise issues with regard to sample sharing. for example, local export restrictions in brazil prevented sharing of well-characterized samples during the recent zika virus epidemic, thereby presenting an obstacle to rapid development and assessment of diagnostics ( , ) . further generalized guidance on fair and equitable sharing of benefits arising out of use of genetic biological resources is in the nagoya protocol ( ) . this protocol is a supplementary agreement to the convention on biological diversity and contains detailed guidance divided into the following objectives: access obligations, benefit sharing obligations, and compliance obligations. no similar guidance exists for plasma-derived products, such as antibody reference materials. however, many facets of the nagoya protocol would remain pertinent to plasma-derived products and could be adapted to inform procedures. acceptance that human-derived products should not be an object of commercialization or source of profit is enshrined in several key documents ( , ) that would continue to apply to development of irps. who distributes irps either free of charge to national control laboratories or with small handling charges and shipping costs to other organizations. negotiations between relevant parties and preemptive interaction between researchers and donor country authorities could lead to clearly defined, mutually agreed upon processes for acquisition and export of samples. these processes could subsequently be specified in legally binding clinical study agreements, and thus ensure compliance with principles of equitable benefit sharing and provide solutions to complex issues, such as intellectual property, product ownership, and access rights to irps. most diseases on the research and development blueprint priority disease list are caused by category a pathogens, and handling of potentially infectious materials often requires biosafety level laboratories. in recent years, the threat of bioterrorism has resulted in strict rules and requirements, such as us federal select agent regulations, which can result in increased cost, limited research, and reduced collaboration between institutions ( ). however, there is an encouraging increase in the number of biosafety level laboratories globally, and initiatives now exist that seek to harmonize practices and facilitate collaboration between laboratories with the ultimate goal of positively contributing to global health ( , ) . the safe acquisition, processing, exporting, and importing of samples from recovered emerging high-threat disease patients would require careful planning. requirements would be based on natural history of a specific disease, study site, and patient characteristics, such as period of convalescence since the acute illness. liaison with national authorities in donor and recipient countries with regard to exportation and importation regulations and requirements would be necessary. in addition, most of these diseases occur primarily in low-and middle-income countries and often in remote or rural settings, where access to accurate medical records and diagnostic tools will be limited. clear specifications regarding method of diagnosis, patient clinical details and disease course, sample collection date, and storage details would be required. biological irps for infectious diseases with epidemic potential are a major global asset that will support timely and efficient development of vaccines, therapeutics, and diagnostics. a critical barrier to development of reference materials for epidemic infections is acquisition of suitable source material. we have highlighted a series of key issues that need to be addressed systematically in a framework that is acceptable to all parties. work should begin to develop and agree to such a framework and to generate irps for these diseases so that drugs, diagnostics, and vaccines are available for future outbreaks. dr. rampling is an infectious diseases clinician and academic clinical fellow at the hospital for tropical diseases and university college london, london, uk. his research interests are virology, epidemic preparedness, and clinical evaluation of vaccines. developing biological standards for vaccine evaluation world health organization. recommendations for the preparation, characterization and establishment of intenational and other biological reference standards. geneva: the organization the antitoxin treatment of diphtheria: m. roux at the pasteur institute the contributions of paul ehrlich to pharmacology: a tribute 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data from brazil nagoya protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their utilization to the convention on biological diversity convention for the protection of human rights and dignity of the human bing with regard to the application of biology and medicine: convention on human rights and biomedicine final report to the european commission on the project basic ethical principles in bioethics and biolaw negative impact of laws regarding biosecurity and bioterrorism on real diseases biosafety level- laboratories in europe: opportunities for public health, diagnostics, and research status, quality and specific needs of ebola virus diagnostic capacity and capability in laboratories of the two european preparedness laboratory networks emerge and evd-labnet key: cord- -wmhdw qs authors: nishtala, krishnatej; pahuja, natasha; shetty, rohit; nuijts, rudy m. m. a.; ghosh, arkasubhra title: tear biomarkers for keratoconus date: - - journal: eye vis (lond) doi: . /s - - - sha: doc_id: cord_uid: wmhdw qs keratoconus is a progressive corneal thinning, ectatic condition, which affects vision. recent advances in corneal topography measurements has helped advance proper diagnosis of this condition and increased research and clinical interests in the disease etiopathogenesis. considerable progress has been achieved in understanding the progression of the disease and tear fluid has played a major role in the progress. this review discusses the importance of tear fluid as a source of biomarker for keratoconus and how advances in technology have helped map the complexity of tears and thereby molecular readouts of the disease. expanding knowledge of the tear proteome, lipidome and metabolome opened up new avenues to study keratoconus and to identify probable prognostic or diagnostic biomarkers for the disease. a multidimensional approach of analyzing tear fluid of patients layering on proteomics, lipidomics and metabolomics is necessary in effectively decoding keratoconus and thereby identifying targets for its treatment. keratoconus is a progressive, asymmetric corneal ectasia. it is characterized by the thinning and protrusion of the cornea leading to irregular astigmatism and myopia thereby affecting the visual performance [ ] . the disease is clinically diagnosed by corneal thinning and steepening along with the presence of iron oxide hemosiderin deposits in corneal mid periphery (fleischer's ring). around % of subjects exhibit vogt's striae, which are fine stress lines caused by stretching and corneal thinning [ ] . although clinical signs are helpful for the diagnosis, keratoconus is typically confirmed by corneal topography or tomography [ ] . although keratoconus is bilateral, in certain scenarios, the clinical and topographic signs are present in only one eye (unilateral keratoconus) wherein the other eye is then diagnosed as form fruste keratoconus [ , ] . the onset of the disease occurs at puberty and progresses up to the third decade of life [ , ] . hence, a severity grading has been proposed for keratoconus to differentiate a normal eye from a keratoconic eye based on the clinical signs and corneal topography indices [ , ] . keratoconus has been reported to affect in to in individuals and has a greater prevalence in asians indicating the role of ethnicity [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . both genetic and environmental factors contribute to the disease pathology [ ] [ ] [ ] . although atopy, eye rubbing, ocular allergies, down's syndrome and tapetoretinal degeneration have been associated with keratoconus [ ] , the etiology and pathogenesis still remains unclear. despite the association of various genetic loci in twin and familial studies, [ ] the general consensus remains that the disease is polygenic and is dependent on ocular surface and tear molecular expression changes [ ] . tear fluid has been an important source of information in understanding ocular physiology [ ] . a large number of proteases and protease inhibitors have been identified in tears [ ] . zhou et al., have identified over proteins in the tear fluid majorly involved in carbohydrate catabolism, proteolysis, protein transport besides immune response and regulation of apoptosis [ ] . disease specific molecular signature from tear fluid analysis can help in understanding the etiology of the disease and to help in prognosis. in addition, tear fluid can serve as an optimal source of molecular targets for treating ocular disease conditions [ ] . in this review, we attempt to discuss, in a concise manner, the molecular markers for keratoconus that have been recently discovered and the importance of tear film as a source of these biomarkers in understanding the etiopathology as well as in prognosis of the disease. tear film is essential in maintaining ocular homeostasis and monitoring ocular surface conditions [ ] . the tear fluid secreted by the lacrimal glands is a complex matrix comprising of an inner mucin layer, middle aqueous layer and the outer lipid layer and is composed of lipids, proteins, peptides, proteases, protease inhibitors and metabolites. the lipid layer provides lubrication, stability to the tear film and plays a protective role by preventing the cornea from drying and shields against pathogens [ , , ] . tear fluid helps in nourishing the corneal epithelium and anterior stroma by delivering nutrients and metabolic products [ ] . constitution of the tear film influenced by disease specific changes in the molecular markers can be of diagnostic value. another facet of the tear fluid analysis is the lipidome. analysis of the tear fluid lipidome by rantamaki et al. shows polar lipids, and the majority of tear lipids being phospholipids; the composition of tear lipids differs distinctly from those of the meibomian gland [ ] . a more recent and comprehensive analysis of the human tear fluid identified more than lipid species belonging to major lipid classes [ ] . besides lipidomics, a metabolome analysis of tears from healthy subjects using mass spectrometry revealed tear metabolites of different classes [ ] . tear biomarker discovery tear fluid as a source of biomarkers proteins lipocalin (lcn), lysozyme (lyz), lactoferrin (ltf), serum albumin (albu), proline rich repeat proteins (prr) constitute the most abundant proteins in the tear fluid [ ] . inflammatory proteins were observed in tears of patients on long term glaucoma medication [ ] and tear fluid proteins were also considered as a means for screening diabetic retinopathy [ , ] . tear proteins have been reported to be altered in patients with keratoconus with and without contact lens wear when compared to controls [ ] . tear composition has been shown to be altered in various inflammatory diseases like dry eye syndrome (des) [ ] , keratoconus [ ] , primary open angle glaucoma [ ] and grave's ophthalmopathy [ , ] . tear fluid has been studied as a source of biomarkers in the diagnosis of systemic conditions such as breast cancer [ , ] , diabetes [ , ] , multiple sclerosis [ ] , and severe acute respiratory syndrome (sars) [ ] . these reports highlight the importance of tears not only for the study of ocular conditions but also for readouts of systemic conditions. similar to the tear proteome, the tear lipidome and metabolome can also undergo changes in keratoconus as observed in other ocular disease such as des where reduced levels of wax esters and saturated fatty acyl moieties in des patients were observed [ ] . besides lipids, the tear metabolome has also been studied to understand disease pathology. using nuclear magnetic resonance (nmr) spectroscopy, levels of about metabolites were found to be varying in the tears of des patients compared with healthy subjects [ ] . advances in technologies such as mass spectrometry and nmr have helped in studying and understanding molecular changes in the tear proteome, lipidome and metabolome relating to an ocular disease condition. storage and maintenance of the collected tear fluid under proper temperature conditions is critical for successfully discovering a biomarker. tear fluid from patients can be non-invasively collected either using a glass capillary or schirmer's strip [ ] . however, the schirmer's strip method of collecting tears is considered the most effective with minimum discomfort to the patient and is often part of the des ocular surface disease test that patients already undergo in the clinic. since tear composition is complex with various proteolytic enzymes, cytokines, and metabolites, the tear fluid should be stored at sub-zero temperatures i.e., − to − °c. a reduction in total tear protein concentration was observed when stored at room temperature for - h [ ] . genetic variation is one of the factors influencing the incidence of keratoconus [ ] . genetic predisposition to keratoconus in familial and monozygotic twins has been well established [ , ] . single nucleotide polymorphisms (snps) in hepatocyte growth factor rab gtpase activating protein (rab gap ), interleukin β (il b), cadherin (cdh ), negative regulator of ubiquitin like protein (nub ), collagen type xxvii a (col a ) and lysyl oxidase (lox) were identified by genome wide association studies (gwas) as risk factors for keratoconus [ ] . however, the molecular functions associated with these snps are not well understood. gene expression signatures of keratoconus have been reported and offer new insights into the disease process [ ] . differential gene expression analysis in debrided corneal epithelia of patients with keratoconus showed dysregulations of lox and collagens -collagen i alpha (colia ) and collagen iv alpha (coliva ) and an elevated expression of matrix metalloproteinase (mmp ) [ ] . a positive correlation between the reduced collagen expression with clinical severity of keratoconus was observed indicating their role in structural deformity in keratoconic corneas [ ] . these genes were also associated with elevated inflammatory cytokine il- in the corneal epithelium and in the tears of keratoconus patients. however, cyclosporine a (cya) treatment of tumor necrosis factor α (tnfα) stimulated corneal epithelial cells and inhibited the expression of il- , tnfα and mmp [ ] . cultured human keratocyte cells (hkcs) stimulated with three different isoforms of transforming growth factor β (tgfβ i-iii) showed deregulation of smad and smad , indicating altered tgfβ signaling in the progression of keratoconus [ ] . similarly a significant reduction in levels of alcohol dehydrogenase (class ) betapolypeptide (adh b) in cultured keratoconic corneal fibroblasts suggested its possible role in keratoconus [ ] . reduced levels of beta actin, a protein essential for cell survival and growth, along with human antigen r (hur) was observed in keratoconic and normal corneas both at the transcriptional and translational level suggesting the deregulation of these molecules as a possible trigger for keratoconus [ ] . similar reduction in gene expression of beta actin and alpha enolase was also observed in superficial keratoconus epithelial cells compared with normal corneas, suggesting the degradation of these proteins in keratoconus [ ] . nielsen et al. performed a proteomic analysis using d-gel electrophoresis followed by mass spectrometry from keratoconus patient epithelia. compared to controls, the patient epithelium overexpressed gelsolin (gsn), alpha enolase (enoa), s a and cytokeratin (krt ), which suggests a plausible role of these proteins in the pathogenesis of keratoconus [ ] while possibly associating the role of structural proteins and enzymes in keratoconus. these molecules can act as prognostic or diagnostic biomarkers and may aid in treatment of the disease. a list of the molecular markers identified in cultured corneal cells and human corneas are summarized in table . the genomic studies thus far have remained inconclusive in terms of a molecular explanation for the phenotypes observed and do not strongly correlate with the molecular expression data from patient corneas. however, the gene and protein expression data from the keratoconic cornea do show deregulation of important factors and pathways such as collagen synthesis, inflammation, extracellular matrix, structural genes, tgfβ pathway and wnt signaling, to name a few. however, in a clinic, it is difficult to obtain corneal tissue for diagnosis and molecular profiling. this makes profiling tears of patients an attractive prospect. keratoconus is associated with tissue degradation that involves extracellular matrix remodeling, collagen deficiency [ ] and more recently, increased roles of proinflammatory cytokines, cell adhesion molecules and matrix metalloproteinases [ ] . enzyme linked immunosorbent assay (elisa) analysis of capillary collected tears in [ ] , [ ] and [ ] patients with keratoconus in three different studies showed elevated levels of inflammatory markers il- , tnfα and mmp . thus, a variety of matrix metalloproteinases (mmp) - , - , - , - , interleukins (il) - , - , - , - , and tumor necrosis factor (tnf) -α and -β are elevated in keratoconus tears [ ] . studies also demonstrate higher gelatinolytic and collagenolytic activities in keratoconus [ ] . hence, patients with elevated mmp levels, when treated with . % cya for months, showed improved corneal topography characterized by reduced disease progression, localized flattening and low tear mmp [ ] . in another study where tears from keratoconus patients were analyzed by elisa, secreted-frizzled related protein (sfrp ), a protein associated with the wnt signaling pathway, was observed at lower levels compared to age-matched controls, indicating a novel signaling pathway alteration in keratoconus [ ] (table ) . tear cytokine analysis performed to determine the effect of collagen cross-linking on tear fluid composition showed alterations in the tear cytokine levels [ ] . these discoveries have significant clinical importance in aiding the [ ] . furthermore, pre-existing information based on different experimental methodologies enhances the reliability of pre-selected markers. the high specificity and sensitivity of target specific biochemical assays make them reliable and clinically relevant for detecting molecular changes both in a qualitative and quantitative manner. mass spectrometric analysis for protein biomarkers has a dual advantage of surveying the unknown species thereby discovering novel molecular changes from a repertoire of proteins, and quantifying the changes in a multiplexed manner [ ] . using a nano-lc tandem mass spectrometry approach, tears of keratoconus patients were compared with healthy controls that identified the elevation of cytokeratins, matrix metalloproteinase (mmp ) and mammoglobin b (sgb a ) [ ] . additionally, lipocalin (lcn), lysozyme c (lyz), immunoglobulin alpha & kappa (igka & igkc) and precursors to prolactin were deregulated in keratoconus [ ] . tear analysis using a -de/ms approach, identified zinc-α glycoprotein (azgp ), immunoglobulin kappa chain, and lactoferrin (ltf) to be reduced in keratoconus [ ] . using complimentary proteomic approaches of de and lcms e (mass spectral data acquired in a data-independent acquisition mode, ms e ), acera et al. have shown a significant decrease in the levels of cystatins and a higher tear lipocalin- in patients with keratoconus [ ] . moreover, balasubramanium et al. demonstrated reduction in total tear protein in keratoconus patients [ ] . protein levels of gross cystic disease fluid protein- / prolactin-inducible protein (pip) and zinc-alpha- glycoprotein have been found to be elevated in tears of patients by proteomic analysis, suggesting their application as prognostic markers for keratoconus [ ] ( table ). one of the major challenges in tear proteomics is the wide dynamic range of tear proteins. as discussed earlier, certain proteins [ ] due to their high abundance, are able to mask the identification of low abundant proteins. a comparison of the levels of abundant proteins in tears and serum showed tear fluid to be similar in its protein abundance to serum [ ] . though fractionation by de helps in resolving the abundance and complexity, it is limited by other factors such as protein isoelectric point and hydrophobicity [ ] . a gel-free approach overcomes such limitations and coupled with fractionation methods such as cation exchange chromatography, it aids in resolving sample abundance and complexity [ ] . metabolic changes as a consequence of disease can also be diagnosed from tears. tear metabolome analysis by lcms among subjects in clinically defined groups (healthy, keratoconus patients with rgp lens and keratoconus with no correction) identified different metabolites among which more than metabolites associated with glycolysis, gluconeogenesis and the urea cycle showed significant changes in keratoconus tears [ ] . thus, the changes in underlying molecular signaling and secretion pathways due to keratoconus could be disease specific, affecting numerous biological processes. metabolite analysis by targeted mass spectrometry performed on d and d cultured human corneal keratocytes (kcks), human corneal fibroblasts (hcfs) and hkcs detected about metabolites, the majority of which are involved in the oxidative stress pathway, implying its importance in keratoconus [ ] . tear proteins lyz, ltf, lcn were also reported to be downregulated in des. besides, tear cytokines il - , - and - , and tnfα, which were elevated in keratoconus, were also shown to have similar expressions in des [ , ] , indicating certain similar molecular changes among the two disease conditions. hence, it is imperative to practice caution while considering tear molecular changes as biomarkers for a specific disease. albeit, recent studies have shown that matrix metalloproteinases mmp and , azgp , sfrp , igkc and cell adhesion molecules icam- and vcam- to be specifically regulated in keratoconus [ , ] , indicating their probable exclusive role in keratoconus. in addition, the reduced [ ] and levels of sfrp [ ] are specific to the disease and highlight the molecular pathways that can be targeted for disease correction. since collagen cross-linking is the current surgical treatment available for keratoconus [ ] , reduction in the natural collagen cross linkers [ ] is perhaps expected. the molecular pathways leading to the elevation of collagenolytic factors [ ] presents another attractive target for therapy. since these large sample studies [ , ] indicate a broad range in the expression of such factors, it is imperative to tune future treatment modalities based on specific molecular targets to the correct pathways. this could be possible through tear-based biomarker profiling. it is now understood that inflammation is one of the primary drivers of keratoconus [ , [ ] [ ] [ ] ] , therefore, it is not surprising that some of the markers between keratoconus and des are similar. however, the amplitude of the deregulation of these markers such as il and mmp , in correlation with disease phenotype can be distinct amongst the different disorders with inflammatory drivers. large sample cohort studies [ , ] would further validate the role of these proteins as specific and potential biomarkers for keratoconus. therefore, accumulated information about these markers should be considered for application in diagnosis e.g., elevated mmp and il [ ] along with a reduction in markers such as lox, sfrp [ , ] in tears can give a specific diagnosis of keratoconus. many of these factors are deregulated at the corneal tissue (epithelium and stroma) level as well as the tears, further validating their importance as biological processes specific to this disease as established by different groups. with the advent of high throughput technologies such as multiplex cytokine bead array [ ] and quantitative mass spectrometry by multiple reaction monitoring (mrm) assays, multiple candidate markers can be validated in a relatively shorter time and over a larger cohort of patients. zhou et al. have demonstrated quantitation of tear proteins in a mrm assay [ ] . multiplexed validation also establishes a unique molecular fingerprint of the disease and a more appropriate way of diagnosing and treating keratoconus. tear fluid as a source of biomarkers in ocular and systemic conditions has been well studied and is shown to have translational potential. moreover, the non-invasive way of collecting the tears makes it an optimal biological fluid to study with minimal to no discomfort to the patient. studies performed on tear fluid in patients of keratoconus provided insights into the pathology of the disease and has revealed probable prognostic as well as diagnostic biomarkers for the disease. more importantly, the recent studies and data from tear analysis establishes the definitive role of inflammation as a driver of corneal collagen loss and deformity in keratoconus patients. elevated cytokines move in tandem with elevated matrix degrading enzymes to reduce levels of collagens and collagen crosslinking enzymes resulting in the disease. we term this as "keratoconus inflammaxis", a unique molecular signaling fingerprint identified with this disease and its related pathology. the advances in technology such as high resolution high sensitivity mass spectrometry have enabled mapping of the tear fluid in patients and also the heterogeneity of keratoconus pathology. a multi-omics approach integrating data from proteomics, lipidomics and metabolomics is the need of the hour for studying tear fluid as an important source of biomarkers in keratoconus to lead to effective prognosis and treatment of the disease. the pathogenesis of keratoconus imaging modalities in keratoconus incidence and characteristics of unilateral keratoconus classified on corneal topography keratoconus and related noninflammatory corneal thinning disorders keratoconus in asian eyes at a tertiary eye care facility a new method for grading the severity of keratoconus: the keratoconus severity score (kss) correlation of corneal elevation with severity of keratoconus by means of anterior and posterior topographic analysis estimation of the incidence and factors predictive of corneal scarring in the collaborative longitudinal evaluation of keratoconus (clek) study baseline findings in the collaborative longitudinal evaluation of keratoconus (clek) study a -year clinical and epidemiologic study of keratoconus influence of ethnic origin on the incidence of keratoconus and associated atopic disease in asians and white patients incidence and prevalence of keratoconus in denmark prevalence and associations of keratoconus in rural maharashtra in central india: the central india eye and medical study does ethnic origin influence the incidence or severity of keratoconus? genetics of keratoconus: where do we stand? the genetics of keratoconus genetic and genomic perspective to understand the molecular pathogenesis of keratoconus proteomic and gene expression patterns of keratoconus unraveling the molecular repertoire of tears as a source of biomarkers: beyond ocular diseases identification of proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors in-depth analysis of the human tear proteome tears as a source of biomarkers for ocular and systemic diseases complexity of the tear film: importance in homeostasis and dysfunction during disease tear analysis in ocular surface diseases innate defence of the eye by antimicrobial defensin peptides in vivo tear-film thickness determination and implications for tear-film stability human tear fluid lipidome: from composition to function extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles characterization of the human tear metabolome by 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exploring the human tear fluid: discovery of new biomarkers in multiple sclerosis the severe acute respiratory syndrome coronavirus in tears lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome a metabolomic approach to dry eye disorders. the role of oral supplements with antioxidants and omega fatty acids schirmer strip vs. capillary tube method: non-invasive methods of obtaining proteins from tear fluid effect of storage on protein concentration of tear samples the genetics of keratoconus the genetics of keratoconus genomewide association studies and assessment of the risk of disease attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity elevated expression of matrix metalloproteinase- and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine a keratoconus in vitro and the key players of the tgf-beta pathway marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts downregulation of β-actin gene and human antigen r in human keratoconus molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study inflammatory molecules in the tears of patients with keratoconus subclinical keratoconus and inflammatory molecules from tears inflammatory response to contact lenses in patients with keratoconus compared with myopic subjects effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus tear levels of sfrp are significantly reduced in keratoconus patients alterations of tear mediators in patients with keratoconus after corneal crosslinking associate with corneal changes sensitivity and specificity of a point-of-care matrix metalloproteinase immunoassay for diagnosing inflammation related to dry eye quantitation of human tear proteins using high resolution multiple reaction monitoring (hr-mrm) based-mass spectrometry urinary matrix metalloproteinase- and interleukin- and renal manifestations of primary sjogren's syndrome proteomic analysis of the tear film in patients with keratoconus gross cystic disease fluid protein- /prolactin-inducible protein as a biomarker for keratoconus disease power and limitations of electrophoretic separations in proteomics strategies tear metabolite changes in keratoconus in vitro model suggests oxidative stress involved in keratoconus disease seldi-tof-ms proteinchip array profiling of tears from patients with dry eye analysis of inflammatory cytokines in the tears of dry eye patients corneal collagen cross-linking for keratoconus we thank the narayana nethralaya foundation for funding to ag, kn and np.authors' contributions kn and np wrote the manuscript. rs and rmman helped conceptualize and advised on the manuscript. ag designed and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - o ru zz authors: migliaccio, raffaella; tanguy, delphine; bouzigues, arabella; sezer, idil; dubois, bruno; le ber, isabelle; batrancourt, bénédicte; godefroy, valérie; levy, richard title: cognitive and behavioural inhibition deficits in neurodegenerative dementias date: - - journal: cortex doi: . /j.cortex. . . sha: doc_id: cord_uid: o ru zz disinhibition, mainly caused by damage in frontotemporal brain regions, is one of the major causes of caregiver distress in neurodegenerative dementias. behavioural inhibition deficits are usually described as a loss of social conduct and impulsivity, whereas cognitive inhibition deficits refer to impairments in the suppression of prepotent verbal responses and resistance to distractor interference. in this review, we aim to discuss inhibition deficits in neurodegenerative dementias through behavioural, cognitive, neuroanatomical and neurophysiological exploration. we also discuss impulsivity and compulsivity behaviours as related to disinhibition. we will therefore describe different tests available to assess both behavioural and cognitive disinhibition and summarise different manifestations of disinhibition across several neurodegenerative diseases (behavioural variant of frontotemporal dementia, alzheimer’s disease, parkinson’s disease, progressive supranuclear palsy, huntington’s disease). finally, we will present the latest findings about structural, metabolic, functional, neurophysiological and also neuropathological correlates of inhibition impairments. we will briefly conclude by mentioning some of the latest pharmacological treatment options available for disinhibition. within this framework, we aim to highlight i) the current interests and limits of tests and questionnaires available to assess behavioural and cognitive inhibition in clinical practice and in clinical research; ii) the interpretation of impulsivity and compulsivity within the spectrum of inhibition deficits; and iii) the brain regions and networks involved in such behaviours. "all the movements of our body are not merely those dictated by impulse or weariness; they are the correct expression of what we consider decorous. without impulses, we could take no part in social life; on the other hand, without inhibitions, we could not correct, direct, and utilize our impulses". (maria montessori) defining cognitive and behavioural inhibition is a complex task. in , bari and robbins ( ) published a large and comprehensive review on inhibition and impulsivity and they listed types of "inhibition" belonging to different levels of "analysis". these types ranged from more fundamental aspects, such as synaptic inhibition like neuronal hyperpolarisation mediated by gaba fixation for example, to more complex inhibition mechanisms, such as social inhibition. from a neuropsychological and clinical point of view, two major types can be defined: cognitive and behavioural inhibition (aron, ) . they are both part of the 'cognitive control' system, an umbrella term to define a range of inhibitory mechanisms from the inhibition of prepotent tendencies, to the updating of working memory contents and consequential shifting between tasks (miyake et al., ) . this division may seem artificial, but it reflects the need to distinguish the observed phenomena from a clinical standpoint. in agreement with such a division, in obsessive compulsive disorders, for example, failures in cognitive or behavioural inhibitory processes lead to different syndromic behaviours: obsessions and compulsions respectively (chamberlain et al., ) . from a neurodevelopmental perspective, behavioural inhibition, such as the ability to control movements, appears to develop before cognitive inhibition, which acts on mental processes (wilson & kipp, ) . both cognitive and behavioural inhibition appear to then become increasingly efficient with age (mischel et al., ; olson, ; harnishfeger, ) . in an attempt to keep this distinction, cognitive inhibition is defined as the ability to resist an exogenous or endogenous interference, inhibit cognitive contents or processes previously activated and suppress inappropriate or irrelevant responses (wilson & kipp, ) . it can be intentional and conscious (such as thought suppression), or unintentional and unconscious (such as the gating of irrelevant information from working memory during memory processing) (harnishfeger, ) . behavioural inhibition, on the other hand, refers to the control of emotional and social behaviours in a social context. it is the control of overt behaviours, the ability to adapt actions to environmental changes, and suppress impulsions which violate norms (harnishfeger, ) . for some authors (e.g., rascovsky et al., ) , impulsivity is a subcomponent of the larger syndrome of disinhibition. conversely, for other authors (e.g., rochat et al., ) , disinhibition, referring to the lack of control in a specific social context, is a subdimension of the broader concept of impulsive behaviours. while impulsivity was historically associated with risk-seeking and compulsivity with harm-avoidance, it is progressively recognised that the two concepts share neuropsychological mechanisms involving dysfunctional inhibition of thoughts and behaviours. impulsivity is a multidimensional construct defined as "a predisposition toward rapid, unplanned reactions to internal or external stimuli with diminished regard to the negative consequences of these reactions" (chamberlain & sahakian, ) . it is measured through questionnaires as a trait (e.g., barratt impulsiveness scale (bis- ) (patton et al., ) ) or through tasks (e.g., go/nogo task). this reflects the different facets of impulsive behaviour, which is characterised as the (i) inability to stop automatic responses, (ii) tendency towards quick choices and decision-making (iii) difficulty to delay gratification (averbeck et al., j o u r n a l p r e -p r o o f ). impulsivity and compulsivity share the feeling of "lack of control" (fineberg et al., ; stein & hollander, ) . while impulsivity describes quick unplanned actions, compulsivity is defined as persistent non-goal orientated actions and is typically assessed through tasks measuring an inability to flexibly adapt behaviours and/or switch attention between stimuli (ahearn et al., ; fineberg et al., fineberg et al., , . for each test and questionnaire, we have described the instructions, the ability to identify cognitive or behavioural inhibition disorders and to differentiate dementia syndromes. table summarises this information. in neuropsychological practice, there are a number of tests that are performed routinely. the stroop, the hayling, the trail making and the wisconsin card sorting tests are amongst the most used (rabin et al., ; o'callaghan et al., b) . the stroop colour word interference test (stroop, ) , usually named stroop test, is a test in which colour words are written in a different coloured ink to the colour they refer to. participants are asked to identify and name the colour in which words are written, suppressing the automatic response to read the words themselves (matías-guiu et al., ) . behavioural variant of frontotemporal dementia (bvftd) and alzheimer's disease (ad) patients perform significantly less well on the stroop test than controls, but there is a poor differentiation between these two diseases (perry&hodges, ; collette et al., ) . one of the most sensitive standard tests for measuring inhibition is the hayling sentence completion task (hsct; burgess & shallice ; o'callaghan et al., b) , often referred to as hayling test. this test measures response initiation and inhibition of response by a sentence completion task. in the first part (part a), subjects must complete a j o u r n a l p r e -p r o o f sentence in an automatic condition, with an appropriate word (e.g.: for « the rich child attended a public … », the correct answer is « school»). in the second part of the test (part b), subjects must complete a sentence in an inhibition condition, with an inappropriate word, requiring inhibition of the automatic answer (e.g.: for « london is a very lively… », « city » is considered an incorrect answer, but « banana » would be considered correct). errors in part b are recorded and form an error score, while times to complete part a (initiation time) and part b (initiation + inhibition time) are also recorded. together, these measures can be computed to form an overall score of cognitive inhibition (burgess & shallice , santillo et al., matías-guiu et al., ) . impaired performances are demonstrated in bvftd and ad patients (hornberger et al., ; collette et al., ) , with poorer performances in bvftd patients (hornberger et al., ) , as well as in pre-symptomatic c orf carriers with high risk of developing bvftd (montembault et al., ) . both the stroop and hayling tests rely on the suppression of prepotent verbal responses and resistance to distractor interference. however, in healthy subjects, there is a positive correlation between the hayling test and the stroop test for response initiation but not for the inhibitory component (jantscher et al., ; santillo et al., ) . these tests therefore measure a different dimension of inhibitory capacity (santillo et al., ) . it also appears that the hayling test could be more discriminative than the stroop test when comparing bvftd and ad (o'callaghan et al., b) . another classical but less used test to assess cognitive inhibition specifically is the trail making test (tmt; ) . this is a quick and easy test in which participants are asked to connect numbers from to in ascending order in part a. in part b, participants must perform the same thing but this time, alternating between series, a number series and a letter series ( -a- -b- -c…). part b therefore requires inhibitory skills in order to switch from one series to another (amieva et al., ). thus, errors or slow performance in part b may be j o u r n a l p r e -p r o o f indicative of cognitive inhibition difficulties. a study showed that ad patients made more errors than healthy subjects, with mostly perseveration errors, due to impairments in flexibility and inhibition (amieva et al. ). finally, the wisconsin card sorting test (wcst; berg, ) is particularly used in the clinical field to measure working memory, planning skills and, more particularly, shifting abilities (coulacoglou & saklofske, ) . participants are asked to sort cards according to standard stimuli (their colour, shape or number) one at a time, but without any rules concerning the sorting criteria. the investigator chooses a classification criteria (sorting the cards according to their colour, form or number) but this is only revealed to participants by giving them feedback ("right" or "wrong") on each trial. regularly, the classification criterion is changed without any warning and the participant must notice according to the feedback received and adapt their sorting to the new sorting rule (silva-filho et al., ) . thus, this test assesses the ability to shift from one task to another and the principal measure of this ability is the number of perseveration errors, which assesses whether the subject can inhibit a routine response. this test allows differentiation between bvftd and ad patients, with a higher number of perseveration errors in bvftd patients (gregory et al., ) . another way to approach inhibition deficits on a more fundamental level is to explore its motor aspects through motor response inhibition tests. the go/no-go and the stop signal tasks were developed to measure motor response inhibition and therefore assess the underlying process required to cancel an intended movement (o'callaghan et al., b) . trials of the go/no-go task (donders, ) assess processing speed, in the go trials, and response inhibition, in the no-go trials. in this task, one or several stimuli are designated as targets or "go trials" and one stimulus is designated as a nontarget or "no-go trial". stimuli can be visual targets on a screen or motor behaviours performed by an experimenter for example. participants are asked to respond as fast and as accurately as possible for a go trial j o u r n a l p r e -p r o o f (pressing a button or performing the motor behaviour) but to inhibit their response when it is a no-go trial (coulacoglou & saklofske, ) . reaction times and number of errors are recorded. depending on the go/no-go task used, reaction times have been found to discriminate bvftd and ad patients from control subjects, whereas other studies have found that bvftd patients show similar (collette et al., ) or even better (castiglioni et al., ) performances compared with ad patients. a simple go/no-go task is found in the frontal assessment battery (fab, dubois et al., ) , which is commonly used in clinical practice. in this version of the task, participants are trained to give a particular response to a specific stimulus (e.g., tapping one time when the examiner taps twice), followed by learning a new association (e.g., not tapping when the examiner taps twice) during three more training trials. the go/no-go subtest of the fab has shown good discrimination of bvftd patients from age-matched controls. however, it was not able to discriminate between bvftd and ad patients (bertoux et al., ; castiglioni et al., ). the stop signal task, developed by the work of logan and cowan ( ) , is a variant of the go/no-go task and is widely used to measure inhibition of prepotent responses. participants must respond as quickly as possible to predetermined stimuli, the "go trials". on some trials, the go stimulus is followed by a stop signal (auditory signal for example) after a variable stop signal delay (ssd): in that case, participants are asked to inhibit their already initiated responses. speed and accuracy on the go trials are measured, as well as the stop signal reaction time (ssrt), (friedman & miyake, ; terry mc morris, ; gervais et al., ) . using this test, amieva and collaborators showed that ad patients show greater impairment in the stop signal task than in the go/no-go task (amieva et al., ) . finally, the graphic alternating patterns test consists in copying graphic alternating patterns and measures the number of perseverations. patients are asked, for example, to copy j o u r n a l p r e -p r o o f four double loops and a line with alternating peaks and plateaus. frequency of perseveration in alternating lines and loops was higher in bvftd and ad, in comparison to healthy subjects (matías-guiu et al., ) , as well as the derived rule violations score (possin et al ) . standardised caregiver questionnaires used in the behavioural assessment of neurodegenerative diseases often contain subscales related to disinhibition. behavioural questionnaires to measure disinhibition are an important contribution to the clinical interview and, in some cases, allow discrimination between neurodegenerative diseases. neuro-psychiatric inventory (cummings et al., ) consists in an interview with the caregiver to assess behavioural disturbances occurring in dementia patients, including disinhibition, agitation/aggression, aberrant motor activity and appetite disturbances (cummings et al., ; boutoleau-bretonnière et al., ) . the frequency, the severity and the impact on the caregiver are determined. this scale is mostly used in ad (vercelletto et al., ) but is also useful in discriminating between ftd and ad, with ftd patients showing higher scores on the disinhibition subscale (levy et al., ; boutoleau-bretonnière et al., ) . another clinical and behavioural assessment tool is the middleheim frontality score (mfs; de deyn et al., ) constituted of items including disinhibition, impaired emotional control or stereotyped behaviours (de deyn et al., ) . this quick and easy tool reliably discriminates ftd from ad patients, however, as it only considers the presence or absence of a behaviour, it does not allow the follow-up of patients (boutoleau-bretonnière et al. ). the frontal behavioural inventory (kertesz et al. ) is a questionnaire designed for ftd patients to capture both behavioural and personality changes and includes items j o u r n a l p r e -p r o o f specific to the disease. twelve items represent positive symptoms involving diminished inhibition such as perseverations, excessive jocularity, poor judgment, inappropriateness, aggressiveness, impulsivity or hyperorality and each item is scored on a four-point scale (kertesz et al., ; santillo et al., ) . the fbi is more specific for "prefrontal" disturbances than more general behavioural rating scales such as the npi (santillo et al., ) and allows good discrimination between ftd and ad patients (kertesz et al., ; vercelletto et al., ) . another and more recent tool specialised in the assessment of bvftd patients is the daphne scale, consisting of ten items spread across six domains; disinhibition, apathy, the cambridge behavioural inventory (cbi; bozeat et al., ) is an -item questionnaire completed by a caregiver, divided into subsections scored on a four-point scale (wedderburn et al., ) . this classic tool is useful to distinguish ad and ftd, particularly using items of disinhibition-related behaviours. the occurrence of behaviours is assessed rather than the intensity, which is in contrast to the npi which includes both measures (boutoleau-bretonnière et al., ) . this scale is very complete but is also lengthy. wear et al. ( ) successfully developed a shorter version of items in which is still able to differentiate bvftd and ad from parkinson's disease (pd), huntington disease (hd) and healthy subjects. a less used test is the frontotemporal lobar dementia modified clinical dementia rating scale (ftld-modified cdr scale, knopman et al., ) . this scale is a modified j o u r n a l p r e -p r o o f version of the cdr scale (morris, ) but with two additional domains: "language", and "behaviour, comportment, personality". this last domain assesses socially inappropriate behaviours on a four-point scale (knopman et al., ) . this scale is a reliable tool for defining disease severity in ftd (johnen & bertoux, ) but the behavioural assessment of disinhibition remains rather succinct (boutoleau-bretonnière et al., ) . finally, the frontal system behavioural scale (frsbe; grace & malloy, ) is a item behaviour rating scale designed to measure behaviours associated with damage to the frontal system with subscales measuring apathy, disinhibition and executive dysfunction in approximately minutes (grace & malloy, ; malloy et al., ) . behaviours are assessed before and after the illness, which is useful to quantify changes over time. fifteen items are included as representative of disinhibition and are scored on a five-point scale. some studies have found different neuroanatomical correlates of disinhibition in ftd by using the npi (rosen et al., ; hornberger et al., ) or the frsbe (zamboni et al., ) (see also subsection on brain correlates), which may reflect differences in sensitivity between these two tests for disinhibition measurement (o'callaghan et al., b) . overall, studies using questionnaires such as npi, cbi or frsbe have found that behaviours related to disinhibition (especially inappropriate behaviours, impulsive motor/verbal actions and ritualistic routines) were significantly higher in ftd patients compared to ad patients (levy et al., ; bozeat et al., ; wedderburn et al., ; o'callaghan et al; b). the first obvious limitation in assessing disinhibition deficits in clinical practice concerns the administration of questionnaires to the caregiver. given the typical anosognosia j o u r n a l p r e -p r o o f in patients, especially those with bvftd, caregivers are often asked to provide insight into behavioural changes of patients. however, this presents a non-negligible bias. the second limitation is the fact that these tools for diagnosis, evaluation, and follow-up assessments are mostly written tests which lack ecological validity and sometimes are even limited in their cultural validity in non-western populations (see for example kohli&kaur, , on wcst). there is therefore an important gap within this field of research which requires reliable and effective methods to assess behavioural symptoms, for example through performance-based testing (johnen and bertoux, ) . in this vein, we very recently proposed a semi-ecological paradigm to objectively identify and quantify another behavioural and cognitive symptom typical of dementia; apathy (batrancourt et al., ) . the study of disinhibition could also benefit from a similar approach, which is undoubtedly less biased in comparison to the aforementioned more "traditional" methods. j o u r n a l p r e -p r o o f in bvftd patients, cognitive and behavioural inhibition is the most frequent and early impaired domain, reported in . % of cases (lindau et al., ) . according to clinical criteria for bvftd (rascovsky et al., ) , disinhibition, especially behavioural, is a core feature of the clinical syndrome. its global frequency ranges from (lindau et al., ) to % (rascovsky et al., ) . it results in three main alterations: socially inappropriate behaviour, loss of manners or decorum, and impulsive, rash or careless actions. socially inappropriate behaviour can manifest itself by "staring, inappropriate physical contact with strangers, inappropriate sexual behaviour, verbal or physical aggression". loss of manners or decorum consists in "lack of social etiquette, insensitive or due comments, preference for crass jokes and slapstick humour, inappropriate choice of clothing or gifts". impulsive, rash or careless actions are represented by "new gambling behaviour, driving or investing recklessly, overspending, gullibility to phishing/internet scams (convery et al., ) . some symptoms, including undue familiarity, disorganised behaviours, and sexual acting out, could be related to impaired mechanisms of cognitive control (hornberger, geng, & hodges, ) . in this context, law violations, as a possible surrogate of behavioural disinhibition, are frequently observed in bvftd. these consist in "theft, traffic violations, physical violence, sexual harassment, trespassing, and public urination, thus reflecting mainly disruptive, impulsive actions" (diehl-schmid et al., ; liljegren et al., ; shinagawa et al., ) . patients with bvftd commit law violations up to five times more often than ad patients. more rarely, disinhibition can also manifest itself as inappropriate/disinhibited sexual behaviours (ahmed et al., ; mendez&shapira, ; mendez&shapira, ) . paholpak ( ) suggests that disinhibited behaviours result from at least two mechanisms, some based on impaired social cognition (e.g. saying embarrassing things) and others associated with a more generalised impulsivity (e.g. laughing or crying too easily). a lack of social cognition could lead to disinhibited behaviour in social conditions. bvftd patients have impaired emotion recognition (bertoux et al., ) , particularly for anger and disgust (lough et al., ) , and are impaired in tests of theory of mind (tom) (gregory et al., ) . this may partly explain abnormalities in interpersonal behaviour (like offensive comments or behaviours towards others), and difficulties to identify social violations. social inappropriateness is thus often the first clinical sign of a neurodegenerative process (desmarais et al., ) . finally, disruptive symptoms considered as the result of behavioural disinhibition are major predictors for caregiver distress in bvftd (cheng, ) . behavioural disinhibition is found to worsen until intermediate stages of the disease and is then followed by a tendency for improvement in later phases, as shown in a recent study on patients followed up a year assessed and rated through the npi and fbi j o u r n a l p r e -p r o o f (cosseddu et al., ) . this is particularly relevant for the evaluation of outcomes in bvftd therapeutic trials' design: behavioural disinhibition cannot be a good marker of therapeutic efficacy given its progression. patients with bvftd often present with deficits in executive functions, a term that encompasses various complex cognitive functions including cognitive inhibition (perry and hodges, ; slachevsky et al., ; hornberger et al., ; krueger et al., ; torralva et al., a,b) . errors in different cognitive tests (e.g. perseveration or rule violations) are considered representative of cognitive disinhibition and such performance assessments can aid in the diagnosis of bvftd (kramer et al., ; thompson et al., ; libon et al., b; carey et al., ) . generally, decreased error sensitivity or insensitivity (frequency of uncorrected errors) in cognitive inhibition tasks is considered a highly sensitive index of bvftd (ranasinghe et al., ) . however, if we look for clear discriminative abilities, most tests used in clinical practice, such as the stroop test, are not good enough (hutchinson & mathias, ; perry & hodges, ; matías-guiu et al., ) . only the hayling test seems to be a good candidate to reliably distinguish between bvftd and early ad patients, at least at the individual level (hornberger et al., ; hornberger et al., ; ramanan et al., ) . among the different cognitive tests available, the hayling test is more fitted to real-life inhibitory demands, with the ability to suppress inappropriate words being a part of many social interactions. in bvftd patients, it is found to be highly sensitive to cognitive inhibition impairments (hornberger et al., ; santillo et al., ; matías-guiu et al., previous studies have reported that behavioural disinhibition is more characteristic of bvftd than of ad (levy et al., ; mendez et al., ; hirono et al., ; kertesz et al. ; bathgate et al., ; nagahama et al., ) . however, neuropsychiatric symptoms such as apathy, anxiety, and disinhibition can be core aspects of ad patients (kumar et al., ) , especially at middle/late stages (kumfor et al., ) . approximately % of patients with ad show inappropriate social behaviours typically within - months of diagnosis (craig et al., ) . disinhibited behaviours range from impulsive decisions and hypersexual comments or actions, to disproportionate jocularity and incongruous approach of strangers. although uncommon as first symptoms, among alterations of social behaviour, disinhibition has been reported in . % of cases, compared to % and % of social awkwardness and apathy at the beginning of the disease (lindau et al., ) . social disinhibition seen in ad appears to be multifactorial and secondary to impaired social cognitive abilities (desmarais et al., ) , as already discussed for bvftd (see subsection . ). although ad patients are not impaired in tests of tom (gregory et al., ) , abnormalities in recognition of basic emotions are frequently reported (weiss et al., ) , which could contribute to abnormal and inappropriate behaviours. more rarely and at very early stages, ad patients may present with an atypical, bvftd-like clinical profile. however, this presentation is characterised by a milder and more restricted behavioural profile than in bvftd, with a high co-occurrence of memory dysfunction and dysexecutive abnormalities , typical ad atrophy-pattern mainly centred around temporoparietal regions (ossenkoppele et al., ) and cerebrospinal fluid biomarkers or post-mortem confirmation of ad pathology. regarding the association of such symptoms with ad severity, one of the most common tools for social/behavioural evaluation in dementia, the npi (cummings et al., ) , has been used in combination with the cdr scale (morris, ) , which is a disease severity scale. several npi dimensions, but in particular apathy and disinhibition, are found to correlate with the cdr score (kazui et al., ) . inhibitory mechanisms play a crucial role in various domains of cognition, such as working memory, selective attention and shifting abilities, usually impaired in ad. in ad, a discrepancy in performance amongst different tests can be found, according to the type of inhibitory mechanisms affected. for example, controlled inhibition processes measured by the stroop task appear to be affected, while the more automatic inhibition of return is relatively preserved, suggesting that inhibitory deficits are not the result of a general breakdown of inhibitory function (amieva et al., ) . substantial effects of ad on tasks such as negative priming (sullivanet et al., ; amieva et al., ) , which are not cognitively complex but do require controlled inhibition, support this hypothesis. more recently, kaiser and collaborators ( ) comparing two indexes, the response time and error rates, they found large differences between ad patients and controls in the basic inhibition conditions, with ad patients being slower and making more errors. however, similarly large differences were also present in j o u r n a l p r e -p r o o f many of the baseline control-conditions and a derived inhibition score (i.e., control-condition score -inhibition-condition score) suggested only a small difference for errors and not for the response time, with high variability across tasks and ad severity. inhibition tasks (especially the error rate) can discriminate ad patients from controls well, suggesting a specific deterioration of inhibitory-control skills. however, further processes such as a general reduction in processing speed and other attentional processes contribute to ad patients' performance deficits observed on a variety of inhibitory-control tasks. the hayling test shows a great potential to discriminate patients, including ad patients, from controls (nash et al., ) . another commonly used clinical test, the trail-making-test part b, is able to show cognitive inhibition deficits and can discriminate between ad and bvftd patients (ranasinghe et al., ) . in summary, disinhibition is frequent in both bvftd and ad, with greater social disinhibition in bvftd and comparable generalised impulsivity in both diseases (paholpak et al., ) . however, using the barratt impulsivness scale (bis- ), mariano and collaborators ( ) showed higher scores of impulsive behaviour in bvftd patients than in ad patients and controls, but not with neuropsychological tests. dealing with these behaviours is a hard and heavy challenge for caregivers, with cases of personal neglect or reduced self-care (diogenes syndrome) (finney&mendez, ), sexists comments, overt aggression, and in more extreme situations, they can result in criminal charges or pathological gambling (tondo et al., ) . few studies have analysed the occurrence of disinhibition in autopsy-confirmed cases. prior studies were conducted without distinguishing among patients who exhibited j o u r n a l p r e -p r o o f disinhibition alone or a combination of apathy and disinhibition symptoms leger&banks, ) . some authors highlighted the occurrence of disinhibition as a clinical presentation of bvftd, but also of some ad cases and "phenocopy" patients with psychiatric disorders (e.g., addictive disorders, gambling disorder and kleptomania) similar to bvftd (miki et al., ) . the "in vivo" differentiation of "true" bvftd cases with ftld pathology from mimicking cases is not always easy. thus, these authors proposed the following features (among others) as markers of underlying ftld pathology: a) "socially inappropriate behaviours can be frequently interpreted as contextually inappropriate behaviours prompted by environmental visual and auditory stimuli, in the framework of environmental dependency syndrome (lhermitte, ) . taking a detailed history usually reveals various kinds of such behaviours in various situations in everyday life rather than the repetition of a single kind of behaviour (e.g., repeated shoplifting); b) a correlation between the distribution of cerebral atrophy and neurological and behavioural symptoms is usually observed, and the proportion of ftld cases with right side-predominant cerebral atrophy may be higher in a psychiatric setting ("behavioural patients") than a neurological setting ("cognitive patients") ". this last element highlights the so-called "right hemisphere bias" (see specific subsection . , page ) in the generation of disinhibited behaviours. in parkinson's disease (pd), disinhibition is often reported under the term of "impulsivity" (see also section ) and occurs in . % of treated patients (weintraub et al., ) . it can manifest itself by pathological gambling, hypersexuality, compulsive shopping and binge eating, which have significant implications for patients and their families (potenza et al., ; voon and fox, ) . some studies have also reported social disinhibition in pd in progressive supranuclear palsy (psp), the pathology affecting the brainstem and basal ganglia can extend to the prefrontal cortex, resulting in a prefrontal-subcortical syndrome (rosen et al., ; donker kaat et al., ; williams&lees, ). this results in an apathetic phenotype mostly, but can include disinhibited behaviours, albeit less frequently as only described in a third of patients (litvan et al., ; bak et al., ) . assessment using the npi reported that behavioural disinhibition in psp was significantly higher than in pd, with both groups of patients under dopaminergic treatment (aarsland et al., ) . another study showed that psp and pd patients had impaired performance on the go/no-go task compared to healthy subjects, but there were no differences between patients (zhang et al., ) . finally, in a very recent study, psp patients performed similarly to controls on the hayling test, but they presented "positive" disinhibition-related symptoms on the fbi which were less severe than in bvftd (santillo et al., ) . this finding is in agreement with previous results using the cbi (bak et al., ) . inhibition impairments are also reported in . % of patients suffering from huntington's disease (hd; paulsen et al., ), manifesting as impulsivity, hyperactivity, "acting out" and emotional lability (duff et al., ) . these behaviours can include speaking out of turn, embarrassing remarks (like inappropriate sexual remarks) and childish behaviours (eddy et al., ) , which are particularly troubling for caregivers. sometimes, as mentioned above in previous diseases, disinhibition can also lead to illegal behaviours such as stealing (johnson&paulsen, ) . hd patients are also impaired in social cognition, with difficulties in recognising emotions and theory of mind impairments (snowden et al., ; snowden et al., ; bodden et al. ). these are found to lead to disinhibited behaviours in social situations (eddy et al., ) . some studies showed that disinhibition is negatively correlated with age (paulsen et al., ) , and more severe in younger mutated gene carriers (duff et al., ) . basal ganglia and caudate nucleus represent the epicentre of brain damage. the occurrence of inhibition disorders in these patients underlines the role of such structures in the genesis of such pathological behaviours (see also section ). excessive impulsivity and compulsivity are common in neurodegenerative diseases, in particular in bvftd for which they are among the discriminative diagnostic criteria (rascovsky et al., (rascovsky et al., , . in ad, impulsivity (including agitation, irritability, aggressivity, disinhibition) (rochat et al., ) tends to increase with the severity and progression of the disease (bidzan et al., ; rochat et al., ) but both impulsive and compulsive behaviours are generally less frequent than in bvftd (miller et al., ; rascovsky et al., ) . it therefore appears that in these neurodegenerative diseases, at least, impulsive and compulsive behaviours co-occur with disinhibition. j o u r n a l p r e -p r o o f here, we report major imaging and physiological results available in the literature of the considered neurodegenerative dementias. figure summarises the main hemispheric areas. as mentioned above, disinhibition, both cognitive and behavioural, is a symptom of several neurodegenerative diseases. although the literature remains relatively sparse, progress has been made in relating brain and neurodegeneration to disinhibition and its severity, often combining cognition and behaviour. one in more recent studies targeting ftd patients and using more specific measures of disinhibition as well as advanced imaging analysis methods, the orbitofrontal cortex seems to be the most important region implicated in disinhibition. in particular, atrophy of the medial part of orbitofrontal cortex is found to be associated with scores on disinhibition items of the j o u r n a l p r e -p r o o f npi (massimo et al., ) . in a study comparing subgroups of bvftd patients having as first symptom either apathy or disinhibition, it was found that the latter group showed greater atrophy in frontotemporal regions compared with the former (santamaria garcia et al., ) . multivariate analyses confirmed that brain areas including right orbitofrontal, but also right dorsolateral prefrontal, and left caudate were enough to distinguish these patient subgroups (santamaria garcia et al., ) . the importance of orbitofrontal cortex in disinhibition has also been corroborated in other neurodegenerative diseases. a study spanning a large cohort of several neurodegenerative diseases (including ad, bvftd, sv-ppa, nf-ppa, psp, cbd and mci) found that although npi-measured disinhibition correlated with various brains regions, only the orbitofrontal cortex significantly predicted disinhibition when entered into a hierarchical regression (krueger et al., ) . the frontal lobes are also associated with npi-measured disinhibition in ad patients alone, in particular with the right frontal pole cortical thickness (finger et al., ) . however, studies have demonstrated that disinhibition neural correlates also extend to the temporal lobe. in a study of bvftd and ad patients, atrophy in orbitofrontal/subgenual, medial prefrontal cortex and anterior temporal lobe areas covaried with the total error score of the hayling test of disinhibition (hornberger et al., ) . similarly, atrophy in both orbitofrontal cortex and temporal pole brain regions correlated with the npi disinhibition frequency score in this same study (hornberger et al., ) . this convergent evidence from two different behavioural measures suggests that behavioural disinhibition involves the orbitofrontal but also temporal cortices. temporal lobe involvement has been corroborated in studies using other measures of behavioural disinhibition. in a recent study comparing subgroups of ftd patients ranging from "primary severe apathy" to "primary severe disinhibition" measured on the cbi-revised j o u r n a l p r e -p r o o f questionnaire, authors found that those having isolated or additional behavioural disinhibition presented with a specific atrophy of right temporal gyri (o'connor et al., ) . this is in agreement with a pioneering study on anatomical correlates of disinhibition which found a correlation between the severity of disinhibition, quantified by using the fsbd subscale, and atrophy in the right superior temporal sulcus, right mediotemporal limbic structures and right nucleus accumbens (zamboni et al., ) . the relationship between disinhibition and right mediotemporal limbic structures has also been identified in another study in which bvftd patients having the behavioural disinhibition as first symptoms were more atrophic in mediotemporal limbic structures, compared with those presenting with apathy (santamaria garcia et al., ) . these findings support the view that successful execution of complex social behaviours also relies on the integration of reward attribution and emotional processing, represented in mesolimbic structures. taken together, these findings suggest that behavioural disinhibition stems from the breaking down of connections between the temporal lobe and orbitofrontal regions rather than a consequence of the loss of specific functions represented separately in the temporal or frontal lobes (bakchine, ) . it has been suggested that temporolimbic structures and the nucleus accumbens are part of the same circuit including the orbitofrontal cortices and that behavioural disinhibition may be due to a loss of inhibition by the frontal monitoring system on the limbic system (zamboni et al., ) . this is enhanced by the findings that atrophy of insula (part of the limbic system) correlates with behavioural disinhibition, and that in humans the anterior insula integrates emotional and visceral information into representations of present moment context that guide socially appropriate behaviours (wiech et al., ) . moreover, atrophy in the anterior insula is one of the earliest structural biomarkers of behavioural symptoms in ftd (seeley, ) . frontolimbic disconnection through the j o u r n a l p r e -p r o o f anterior insula is therefore a strong candidate mechanism for explaining symptoms of behavioural disinhibition in ftd. finally, a recent paper, using statistical classification approaches, identified four subtypes of bvftd based on distinctive patterns of atrophy defined by selective vulnerability of specific networks (ranasinghe et al., ) . the frequency of disinhibition and obsessive behaviours were the highest in the group which showed semantic appraisal networkpredominant degeneration which included the anterior temporal lobe and subgenual cingulate (ranasinghe et al., ) . thus, it may be possible to divide disinhibition into different subforms and these could be associated with different brain regions and networks. more efforts to build a consensus and refine disinhibition definitions and measures will be required in order to investigate the underlying neural correlates more precisely. in addition to the hemispheric areas, recently we have found that performance on the hayling test was correlated with grey matter volume in the cerebellum (i.e. lower cerebellar volumes are associated with lower performance) in individuals carrying a c orf mutation (montembault et al., ) . connectivity studies have shown that the cerebellum is extensively connected with the prefrontal cortex via the thalamus (behrens et al., ) , which are both key regions in ftd. the neural connectivity between these brain regions might explain the correlation between cerebellum integrity and cognitive inhibition. more specifically, brain-behaviour relationships between specific posterior cerebellar regions and cognitive inhibition, or executive tasks suggestive of cognitive inhibition, have also been reported, especially in patients affected by spinocerbellar ataxia type . thus, stroop test performance has been associated with grey matter volume in the vermis crus ii , go/no-go task performance with grey matter volume in the vermis lobule vi and finally, wisconsin card sorting task performance with grey matter volume in the vermis viib . j o u r n a l p r e -p r o o f the relationship between white matter fibers integrity and behavioural disinhibition has been investigated in recent years using diffusion tensor based imaging methods. powers and collaborators ( ) found a correlation between fractional anisotropy in the right corona radiata and npi-measured disinhibition in bvftd. however, this could be explained because of the widespread involvement of this projection fibers tract within the brain and may therefore also reflect the overall pathology severity rather than specific severity of behavioural disinhibition. a more detailed study in bvftd and ad patients found that fractional anisotropy of the uncinate fasciculus (connecting the frontal with the superior and middle temporal gyri) and of the forceps minor (from the genu of the corpus callosum to the frontal pole) correlated with scores on the hayling test (hornberger et al., ) . as these tracts all enable the connection among frontal subregions and between frontal and temporal regions, this study points to networks within these regions being implicated in inhibitory functioning. the role of right uncinate fasciculus in disinhibition has been confirmed in another study on bvftd and psp patients, using a different measure, that of the hayling total error score (santillo et al., ) , along with the right anterior cingulum and forceps minor. the authors concluded that their results support an associative model of inhibitory control, within a distributed network including medial temporal lobe, insula and orbitofrontal cortex, and connected by the intercommunicating white matter tracts. another white matter tract related to disinhibition is the superior longitudinal fasciculus (slf) which connects the frontal, occipital, and temporal lobes. bilateral slf integrity is associated with disinhibition scores measured on the frsbe in bvftd patients (sheelakumari et al., ) . however, another study found a relationship among bilateral slf and a large battery of cognitive and neuropsychological tests which also included tests of disinhibition such as the tmt (borroni et al., ) . these latter findings therefore suggest that slf may not in fact be a specific neural correlate of disinhibition but of overall cognitive abilities. the white matter tracts seemingly involved in disinhibition are numerous. this may be due to the fact that disinhibition is a complex behaviour which reflects the disconnection of multiple areas of the brain within the frontal, temporal and limbic regions, but also with the parietal lobes as demonstrated by the involvement of slf shows (see also section "neurophysiological correlates" for a complementary interpretation). the interpretation of these findings is also limited by the diverse measures used to assess disinhibition in different studies, with scales focusing more on cognitive disinhibition while others focus on behavioural disinhibition. finally, although bvftd patients' disinhibition can appear as a prominent and dominant behavioural dysfunction, it is not often isolated from other behavioural dysfunctions, such as apathy for instance. this makes the investigation of the neural correlates of disinhibition more difficult and studies should consider including measures of other behavioural symptoms as covariates in their analysis. the study of brain metabolism has provided further evidence in the investigation of the neural correlates of disinhibition in neurodegenerative diseases. a strong correlation between behavioural disinhibition, measured by the npi, and reduced metabolic activity in the posterior orbitofrontal cortex in bvftd patients has been demonstrated (peters et al., ) . however, other studies with similar results in orbitofrontal cortex also found a relationship with other limbic structures including hippocampus, amygdala, caudate, accumbens and insula in patients with ad, ftld and subjective cognitive impairments, in agreement with j o u r n a l p r e -p r o o f structural studies (schroeter et al., ; franceschi et al., ) . this was also the case in a study which used a clustering approach to identify two major variants of cerebral hypometabolism in bvftd patients, "frontal" and "temporo-limbic", and which found that isolated disinhibition assessed on the npi and fbi was only present in the latter sub-group (cerami et al., ) . however, this neuropsychological data was available for very few participants. more recently, a study found hypometabolism in the bilateral medial and basal frontal cortex to be related to disinhibition in bvftd patients presenting behavioural disinhibition (morbelli et al., ) . interestingly, hypometabolism exceeded grey matter atrophy in terms of both extension and statistical significance in all comparisons. disinhibition in these patients was assessed according to clinical examination and unstructured interviews and so is difficult to define precisely. some of these studies paved the way for discussion about a potential right hemisphere bias with respect to behavioural disinhibition especially. previously, this has been attributed to the involvement of the right hemisphere in complex social behaviours. in a recent study this point was addressed dividing disinhibition items from the frsbe into person-based versus non-person-based items. person-based items were related to social aspects, and non-person-based items were related to generalised impulsivity (paholpak et al., ) . using grey matter volumes from tensor-based morphometry, paholpak and colleagues ( ) found that severity of person-based disinhibition in both bvftd and early-onset ad patients significantly correlated with the left anterior superior temporal sulcus, whereas generalised-impulsivity correlated with the right orbitofrontal cortex (ofc) and the left anterior temporal lobe. this study therefore suggests that behavioural disinhibition can be j o u r n a l p r e -p r o o f dissected into subcategories corresponding to different brain areas highlighting the need for further defining of behavioural disinhibition. not many studies have used functional approaches to relate behavioural and cognitive disinhibition with functional networks in neurodegenerative diseases. one study published in found that prefrontal hyperactivity unique to bvftd was marginally associated with their behavioural disinhibition scores on the fbi (farb et al., ) . moreover, frontolimbic disconnection associated with lower disinhibition scores and stereotypy (measured on the stereotypy rating inventory) was correlated with elevated default-mode network connectivity in particular within the right angular gyrus node. the authors suggested that in ftd patients, observed frontolimbic disconnection may lead to unconstrained prefrontal cortex hyperconnectivity which may represent a compensatory response to the absence of affective feedback during the planning and execution of behaviour. conversely, hyperconnectivity can also be interpreted as in some psychiatric conditions. in schizophrenia, for example, diseaseinduced impaired connectivity may lead to isolation of some brain systems, which can then demonstrate hyperconnectivity because they become less susceptible to influence from other systems (van den heuvel et al., ) . it is increasingly clear that more studies investigating networks functionally involved in behavioural disinhibition are required in order to confirm such claims. j o u r n a l p r e -p r o o f (averbeck et al., ; brown et al., ; matsuo et al., ; paholpak et al., ) . impulsive and compulsive processes are thus thought to be related to cortico-striatal circuits modulated by different neurotransmitters (t. w robbins, ) , with one striatal component (ventral striatum for impulsion/dorsal striatum for compulsion) driving impulsive and compulsive behaviours and one prefrontal component (vmpfc for impulsion/ofc for compulsion) restraining them (fineberg et al., ) . more research will clarify to what extent these regions may ressemble those underlying disinhibition and shed light on the possible relationship between impulsivity, compulsivity and disinhibition. in , hughes and colleagues found changes in cortical oscillatory dynamics as well as in frontal connectivity (specifically, cross-frequency coupling changes at the inferior frontal gyrus, pre-supplementary motor area, and motor cortex) in patients with bvftd during a well characterised response inhibition task (hughes et al., ) . more interestingly, these brain measures of motor inhibition were correlated with everyday disinhibited behaviours in patients, as measured by go/no-go response inhibition paradigm. other authors have investigated recursive social decision-making behaviour which requires flexible, context-sensitive long-term strategies for negotiation which is highly sensitive to inhibition deficits (melloni et al., ) . they found that oscillatory measures could track the subtle social negotiation impairments in bvftd. risky offers during an ultimatum game moderated classic anticipatory alpha/beta activity in controls, but these effects were reduced in bvftd patients. source analysis demonstrated fronto-temporoparietal involvement in long-term social negotiation that was also affected in bvftd. this neurophysiological evidence is in agreement with evidence from structural and functional imaging pointing to similar regions. in brief, both studies suggest that brain oscillation patterns associated with behavioural control provide a neuropathological pathway of the different sources of impaired control in bvftd. within this framework, agustín ibáñez has proposed three alternative neuroanatomical frameworks of inhibitory control and social-behavioural inappropriateness. the first one is the canonical response inhibition network which includes motor, pre-motor and the inferior frontal gyrus. the second one, termed "bvftd general disinhibition model", is a network which takes into account multiple dimensions of non-adaptive behaviour and includes lateral temporal cortex, posterior and dorsal-anterior cingulate cortex, and posterior parietal cortex (lansdall et al., ) . the involvement of posterior parietal cortex could partially explain the correlation of some measures of disinhibition with the slf, a major white matter bundle connecting parietal and frontal cortices. this bvftd general disinhibition model also includes other components associated with impulse control depending on the inferior frontal region, anterior cingulate cortex but also with basal-temporal involvement (zamboni et al., ) . this is in line with the occurrence of disinhibition in neurodegenerative diseases primarily affecting basal ganglia as pd and hd. finally, the third framework is the "social context network model" (scnm) (ibañez and manes, ; baez et al., ; ibáñez and garcía, ) which explains the bvftd deficit as a general contextual deficit of social and cognitive processes and involves several brain regions, including the frontal pole, the orbitofrontal and temporal cortices, the insula, and the anterior cingulate j o u r n a l p r e -p r o o f cortex. within these regions, for example, reduced fronto-posterior oscillations in bvftd are associated with impaired social coordination (melloni et al., ; ibáñez et al., ) , and complex social emotions (exacerbated counter-empathic dispositions) are related with disinhibition and extended fronto-temporo-parietal atrophy (santamaria-garcia et al., ) . these proposed neurophysiological models of inhibition deficits mirror very closely structural, metabolic and functional correlates reported (figure ). the discussion of treatment strategies to diminish general behavioural disinhibition or impulsive behaviours in dementia patients is mainly based on bvftd. no work has investigated this in other patients, such as ad (keszycki et al., ) . trieu and collaborators ( ) have just published a complete and systematic review of the literature through searching on pubmed, embase, and psycinfo databases for reports on pharmacological interventions for individuals with bvftd (trieu et al., ) . studies were included only if the efficacy of the intervention in alleviating bvftd symptoms was provided as an outcome, and were measured with behavioural scales, such as the npi (cummings, ) or the fbi (milan et al., ) . the authors collapsed several clinical signs and symptoms under the term of "disinhibition", such as general disinhibition, agitation, aggression, irritability, and abnormal risk-taking behaviours. in this study, consistently with our review approach, interesting results for general disinhibition and abnormal risk-taking behaviour were reported. it was found that five studies reported a positive effect of therapeutic intervention on general disinhibition. the most interesting were those administrating dextroamphetamine , or citalopram ( other studies have shown positive effects on disinhibition behaviours by using diverse selective serotonin reuptake inhibitors (ssris-namely, paroxetine, sertraline, and fluoxetine) (swartz et al., ; lebert and pasquier, ; herrmann et al., ) . some ssris, such as citalopram, seem to reduce sexual disinhibition in patients with dementia (tosto et al., ) . generally speaking, neurochemistry of bvftd is characterised by serotoninergic network disruption, with decreased serotonin levels and corresponding receptors in frontotemporal regions and neuronal loss in the raphe nuclei (franceschi et al., ; huey et al., ) . also, the use of antipsychotic medications for the treatment of severe neurobehavioural symptoms has been suggested, particularly when ssris are not successful (miller & llibre guerra, ) . within this category and concerning behavioural disinhibition, aripiprazole has shown good control of sexual disinhibition in a patient with bvftd (nomoto et al., ) . in general, olanzapine, quetiapine, and risperidone have inconsistent evidence of benefit. overall, the available studies suggest that physicians should use the smallest effective dose for the shortest possible duration to minimise adverse effects, and only in cases of refractory or severe behavioral symptoms (reese et al., ) . recently, brexpiprazole has been investigated for the treatment of agitation in ad, a largely unmet area of importance. overall, well-tolerated brexpiprazole expands the armamentarium of treatment options available for these patients (stummer et al., ) . one could speculate that behavioural disinhibition could also benefit from this novel drug. old case studies also suggest that the anticholinesterase inhibitor rivastigmine might be helpful for this symptom (alagiakrishnan et al., ) , like antiepileptics such as gabapentin and carbamazepine (miller, ; alkhalil et al., ; freymann et al., ) , whereas donepezil might increase sexual disinhibition (alagiakrishnan et al., ; lo coco and cannizzaro, ) and socially disinhibited behaviour (mendez et al., ) . finally, in the case of disinhibition, particular attention must be paid to all the drugs stimulating the dopamine system. particular concern for these drugs is their potential adverse effects involving increased behavioural disturbances such as disinhibition, as well as risktaking behaviours, and hallucinations. such symptoms have been observed in patients receiving dopamine replacement therapy for pd. routine use of dopamine drugs in ftd patients is not currently recommended (tsai & boxer, ) especially if disinhibited. most studies on non pharmacological interventions in neurodegenerative diseases have focused on reducing aggression and agitation, with no, or very few, studies focusing on the treatment of impulsivity or disinhibition (keszycki, et al., ) . however, some case examples have suggested that the engagement in previous hobbies or games may reduce socially inappropriate disinhibited behaviours in ftd patients. therapeutic activities targeting other symptoms (e.g. agitation and aggression) can have a similar impact through the reduction of monotony and inactivity (ikeda et al., ) . anecdotally, we reported a comparable experience with an ftd patient during the covid- pandemic lockdown during which exacerbated disinhibition was attenuated by combining a small dose of atypical antipsychotic and doing recreational/social activities, like for example writing to family and friends to ask for news (migliaccio & bouzigues, ) . some literature regarding non-pharmacological methods to mitigate sexual disinhibition suggests either ignoring the inappropriate situations by avoiding the problem such as "substituting staff who are less likely to trigger it" and wearing clothing that opens from the j o u r n a l p r e -p r o o f back (kamel and hajjar, ) or confronting it by expressing its inappropriateness (keszycki, et al., ) . this review highlights that disinhibition is an important symptom of bvftd and ad, as well as in some other pathologies. however, the definition of inhibition across studies remains rather vague. although a division between cognitive and behavioural aspects exists in the literature, only a few studies highlighted a double dissociation (olson et al., ; harnishfeger, ) . the division has been largely based on the different types of tests that exist, with some assessing cognitive inhibition and others behavioural inhibition. thus, it is difficult to know if these are part of one same process or if these could be truly subdivided. moreover, disinhibition and impulsivity/compulsivity often co-occur, share common measurement methods and underlying neuropsychological mechanisms. however, the precise mechanisms of inhibition deficits underlying disinhibited, impulsive and compulsive behaviours are still unclear. it would be of great interest to investigate the relationships between these concepts, neuropsychological mechanisms and anatomofunctional networks specific to each subcomponent to understand whether or how these could be used in the diagnosis of neurodegenerative diseases. concerning the brain correlates of these behavioural symptoms, more efforts are required to build a consensus and refine the definition of inhibition and disinhibition as well as how these are assessed. although imaging and neurophysiological studies have put forward some frameworks, it is only after such refining that future studies will be in a position to suggest models of the mechanisms underlying inhibition deficits in 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disorders in parkinson disease: a cross-sectional study of patients impairment in emotion recognition abilities in patients with mild cognitive impairment, early and moderate alzheimer disease compared with healthy comparison subjects anterior insula integrates information about salience into perceptual decisions about pain progressive supranuclear palsy: clinicopathological concepts and diagnostic challenges the development of efficient inhibition: evidence from directed-forgetting tasks apathy and disinhibition in frontotemporal dementia: insights into their neural correlates different decision deficits impair response inhibition in progressive supranuclear palsy and parkinson's disease raffaella migliaccio is supported by "france alzheimer" and "philippe chatrier" foundations, and by "rosita gomez association". delphine tanguy is supported by École normale supérieure paris-saclay. arabella bouzigues is supported by "fondation vaincre alzheimer". raffaella migliaccio, richard levy, valérie godefroy and bénédicte batrancourt are supported by "fondation pour la recherche medicale". the authors declare no competing interests. key: cord- -xe lljz authors: overgaauw, paul a.m.; vinke, claudia m.; van hagen, marjan a.e.; lipman, len j.a. title: a one health perspective on the human–companion animal relationship with emphasis on zoonotic aspects date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: xe lljz over time the human–animal bond has been changed. for instance, the role of pets has changed from work animals (protecting houses, catching mice) to animals with a social function, giving companionship. pets can be important for the physical and mental health of their owners but may also transmit zoonotic infections. the one health initiative is a worldwide strategy for expanding collaborations in all aspects of health care for humans, animals, and the environment. however, in one health communications the role of particularly dogs and cats is often underestimated. objective: evaluation of positive and negative one health issues of the human–companion animal relationship with a focus on zoonotic aspects of cats and dogs in industrialized countries. method: literature review. results: pets undoubtedly have a positive effect on human health, while owners are increasing aware of pet’s health and welfare. the changing attitude of humans with regard to pets and their environment can also lead to negative effects such as changes in feeding practices, extreme breeding, and behavioral problems, and anthropozoonoses. for the human, there may be a higher risk of the transmission of zoonotic infections due to trends such as sleeping with pets, allowing pets to lick the face or wounds, bite accidents, keeping exotic animals, the importation of rescue dogs, and soil contact. conclusions: one health issues need frequently re-evaluated as the close human–animal relationship with pet animals can totally differ compared to decennia ago. because of the changed human–companion animal bond, recommendations regarding responsible pet-ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conforming the biology of the animal are required. education can be performed by vets and physicians as part of the one health concept. the one health initiative or concept is a worldwide strategy that recognizes that public health is connected with animal health and the environment. it concerns multidisciplinary collaboration between physicians, veterinarians, environmental scientists, public health professionals, wildlife experts, and many others [ , ] . with a multisectoral and transdisciplinary approach, public health threats can be better monitored and controlled. the resulting synergism enhances the knowledge of how diseases, known as zoonotic diseases, can be shared between animals and people with the goal of this article is based on an existing post-graduate course for veterinarians, vet technicians, family doctors, midwifes, and specialists such as pediatricians which explores healthy human-animal relationships. the existing evidence-based knowledge contained in this course has been actualized by performing a literature search to add new relevant publications. a literature search was conducted through march , using the national library of medicine's pubmed for the terms "one health" and "companion animals"; "pet ownership"; "households" and "pets"; "dogs" or "cats" or "pets" and "mental" or "physical health" or "children"; "animal assisted therapy"; "dogs" or "cats" and "nutritional problems" or "overweight" or "obesity" or "homemade" or "raw meat diets"; "dogs" or "cats" and "behavior problems" or "aggression" or "fear" or "anxiety" or "abnormal repetitive behavior"; "dogs" or "cats" and "breeding" or "genetic problems"; "dogs" or "cats" and "zooanthroponoses"; "pets" and "anthropomorphism"; "dogs" or "cats" or "exotic animals" or "rescue dogs" or "soil" and zoonoses. for some topics the internet was accessed and used as reference if additional information was not available as scientific publication. the authors selected articles that described pivotal and novel insights in the different topics. all searches were carried out without filters. the titles of all found articles were screened for relevance to the topic, and appropriate titles were assessed and selected based on their abstracts. if a selected article was a review, it was read and relevant citations were used to find primary literature on the subject. additional studies were found using the bibliographies of selected articles. occasionally, reviews were directly used as sources, mostly to convey background information that is not in the core focus of this article. original articles in english and different national languages (dutch, german, french, spanish, if available) were included. specific searches were made for citations dated after the year to ensure more recent literature on the topic had not been missed. a pet or companion animal is an animal that lives in or around the house and is fed and cared for by humans. until the s, pets were mainly kept as utility animals, for example as draft dogs or watch dogs or for pest control when it comes to cats. due to major changes that have taken place in society since the second world war, such as increased leisure time and prosperity, but also individualization of humans, animals are nowadays kept as pets and are regarded by many owners as valued family members, e.g., over % in the united kingdom [ ] . pet ownership is still increasing in many industrialized countries and these animals are more often considered a member of the family [ ] . even in china, a country where pets were banned in urban areas until , pet ownership has grown quite rapidly in the major cities. the rate of pet ownership of all usa households increased from . % to % in . dogs continue to dominate in popularity among american households. approximately % of households nationwide owned a dog, bringing the population of pet dogs to nearly million, while % of households owned cats, with a total population of million [ ] . in , an estimated million european households owned at least one pet animal; % of households owned dogs and % owned cats [ ] . there are million pet dogs and million pet cats in europe which is a % increase of dogs within years ( million dogs in ) and a % increase of cats ( million in ) [ ] . pet cats in europe thereby are more popular than dogs. an explanation of the higher popularity of cats may be the number of single-person households in the eu that rose on average by . % per annum between and to . %, and the growth of two-family households grew by % to % in this period [ ] . also, dual-earner families are widespread as a result of quite a steep growth in female employment over the past two decades [ ] . when animals live with humans, they too benefit from human interaction. over the past decades, animal welfare has evolved to recognize that animals are sentient beings capable of experiencing positive and negative emotions. the social and ethical dimensions of animal welfare, which are concerned with how human society morally regards and treats non-human animals, are also increasingly being recognized [ ] . in dutch law, the intrinsic value of kept animals is expressly incorporated and used as a guiding ethical principle that forms the basis of any further legislation. the intrinsic value is thereby defined that animals are sentient beings that can feel pain and discomfort. therefore, they should be kept free of stress, pain, disease, hunger, thirst, and should be able to show natural behavior known as the five freedoms of r. brambell [ ] . in industrialized countries animal keepers, their owners, are legally required to provide such circumstances and can be prosecuted if they infringe the law. of course, in the field there is animal abuse, negative animal welfare conditions, and animal diseases. however, in general, caretaking for companion animals is nowadays performed at a high level. new insights into animal behavior has had its influence on the general public. for example, owners are aware or are told by vets and pet shops that rabbits should not be kept alone but at least in pairs due to their need for social contact [ ] . there are various reasons to keep pets, such as love, warmth, and companionship. companion animals have an important emotional value, and promote the socialization of the lonely elderly because they facilitate additional contact with people. pets form a goal in life, reduce stress, and ensure that the owner keeps physically active. around % of dog owners and % of cat owners expressed that owning their pet makes them happy and % of owners selected this as one of the reasons they got their pet in the first place [ ] . however, the function of companion animals consists of more than just providing a socializing being. studies show other benefits of having a pet, such as the positive effect on individuals' mental and physiological health status. most research addressing the health benefits of pet ownership show reductions in distress and anxiety, decreases in loneliness and depression, and increases in physical condition [ ] . the positive benefits to human health from interacting with animals, focusing on the companion animal, have also be described with the term "zooeyia" [ ] . in fact, % of owners agreed that having a pet makes them physically healthier, with dog owners more likely to agree, most probably because dog owners exercise more ( % agreeing, compared to % of cat owners). besides, % of owners agreed that having a pet makes them mentally healthier. expressed reasons are the non-judgmental nature of their pets, their playfulness, or physical contact [ ] . another demonstrated positive influence is the blood pressure and heart rate lowering effects that occurs when stroking a friendly-looking dog or even being in the presence of a friendly animal, while it is not necessary to own a pet to obtain these stress-moderating benefits [ ] . many studies have demonstrated the association between pet ownership and cardiovascular health and dog owners appear to have a significantly greater chance of survival after a heart attack compared to people without pets [ ] [ ] [ ] . pets can therefore play an important role in reducing absenteeism and visits to family doctors or the hospital [ ] . it has been estimated that pet ownership saved australia $ billion in [ ] , while it may reduce the use of the national health service (nhs) in the uk to the value of £ . billion per year [ ] . dogs also play an increasing role as co-therapist or as supporter for people with psychological or physical disabilities. the benefits of these animal-assisted activities are improved mood, decreased physiological distress, depression, dementia, and loneliness [ , ] . examples include resident or visiting dogs in prisons, nursing homes [ ] , mental institutions, and hospitals where they can reduce patient anxiety in a hospital emergency department [ ] , reduce pain perceptions in children after surgery [ ] , or calm young patients at a pediatric dental clinic [ ] . since dogs have extremely sensitive noses, they are used for several purposes such as tracking, bomb detection, and search and rescue. in recent years, canine olfaction has also been more recognized as a diagnostic tool for identifying pre-clinical disease status, such as diabetes (ketones), different forms of cancer, and infections from biological media samples [ ] . animal-assisted therapies can act as co-therapies to facilitate psychotherapy or to provide specific types of therapeutic interventions such as improving motor skills or behavior [ ] . such interventions were effective in improving the state of children or adults with or at risk of developing mental disorders such as attention deficit hyperactivity disorder (adhd), post-traumatic stress disorder (ptsd), or autism spectrum disorder (asd) [ ] [ ] [ ] , and for the treatment of ptsd in military veterans [ , ] . assistance or service animals are trained to perform tasks for the benefit of individuals who have disabilities such as hearing loss, physical disabilities, emotional disabilities, seizure disorders, or diabetes [ ] . finally, a wide range of emotional health benefits from childhood pet ownership has been identified, particularly for those suffering from low self-esteem and loneliness. there is evidence of an association between pet ownership and educational and cognitive benefits, increased social competence, social networks, social interaction, and social play behavior [ , ] . significantly less absenteeism from school through sickness among children who live with pets has also been reported [ ] . having a dog or cat in the house during the first year of life may protect against childhood asthma and allergy [ , ] . it can therefore be concluded that companion animals contribute significantly toward the public health, but also increasingly, the health of individually challenged persons through animal-assisted interventions [ ] . providing companion animals with feed by humans, has been considered an advantage for companion animals in their relationship with humans. however, the feeding practices can also have a negative impact on companion animals [ , ] . obesity in cats and dogs is a disease which is rapidly increasing with significant and lifelong implications for animal welfare. although no universally accepted definition of canine and feline obesity exists, the american veterinary medical association defined obesity being more than % above the ideal weight of an animal. overweight is defined as %- % above the ideal weight. using body condition scores, it has been estimated that in the united states, % of dogs and % of cats are obese or overweight. a study in the united kingdom reported % of adult dogs and % of juvenile dogs as being obese or overweight [ , ] . overweight dogs are more likely to be diagnosed with, e.g., urinary tract diseases. obese and overweight dogs are at risk developing orthopedic disorders and hypothyroidism [ , ] . obese cats are at higher risk for developing urinary tract disease, diabetes mellitus, and neoplasia [ , ] . other diet-related problems in companion animals can be caused by the changed feeding behavior of humans, e.g., by providing companion animals with bone and raw feed (barf) or vegan diets. risks for companion animals associated with barf or vegan diets are the presence of microbial hazards, insufficient nutrition, and in raw meat diets the presence of risk materials like thyroid tissue. through contact with their animals, it is possible that risks could even develop for owners. there could be an increased risk of human salmonellosis because of the presence of salmonella spp. in the diet which can spread to humans through diet leftovers or by contact with animal feces. recently a review was published on the risks of barf feeding [ ] . the authors concluded that the data for the nutritional, medical, and public health risks of raw feeding are fragmentary, but they are increasingly forming a compelling body of formal scientific evidence. publications were found reporting the presence of escherichia coli o , salmonella typhimurium, campylobacter spp., and antibiotic resistant bacteria in the feed. nutritional problems, such as calcium/phosphorous imbalances and specific vitamin deficiencies [ ] are also reported. moreover, homemade diets are inherently susceptible to nutritional imbalances and deficiencies [ ] . awareness about climate change, public health and animal welfare has incited a major change in dietary choices among many individuals. the number of vegans in the world keeps growing, even quadrupling from , to , individuals between and in affluent countries such as the uk [ ] . the popularity of veganism goes beyond the scope of the human diet, as more people are interested in the possibility of feeding their companion animal a vegan diet than ever before. to create animal-free complete cat food requires replacing nutrients in animal-based materials with plant-based materials. different sources are used such as corn, rice, peas, soy, potato, and different oils and seeds. any further nutrients that are missing from plant-based materials, such as taurine and carnitine, are replaced with synthetically produced versions [ , ] . feeding trials using vegan animal food are either not performed due to testing costs or kept private due to the highly competitive vegan pet food market [ ] . additionally, they reported testing vegetarian diets for cats and dogs and found that one was lacking protein and six did not meet all amino acid concentration requirements. vegan animal food may not contain meat, but it does contain grains, soy, and corn. plant-based products, such as grains, can be a source of health problems because of the presence of mycotoxins, for example [ ] . warm, humid storage conditions can lead to the formation of mycotoxins such as aflatoxins, produced by the fungi aspergillus flavus and aspergillus parasiticus. many regular animal feeds also contain plant-based products, therefore the negative impact of feeding vegan diets to companion animals, especially obligate carnivores such as the cat or the ferret, seems therefore more related to diet insufficiency than to microbial health risks. additionally, addressing behavioral problems, the "free" provision of food might fulfil the consumptive part of feeding behavior of our companion animals but does not fulfil the appetitive phase. especially this phase of feeding patterns can have consequences for the companion animals' mental health and may result in behavioral problems if appetitive physical and mental challenges remain chronically absent in the human-animal relationship. in the human-companion animal bond, pets may develop abnormal behavior, including excessive aggression, fear and anxiety, or even abnormal repetitive behavior. abnormal repetitive behavior (arbs) were first noticed in zoo-, shelter-, and laboratory animals: all animals housed under stimulus-poor conditions and with limited space. however, companion animals can develop arbs as well, if the individual's adaptive capacity is exceeded due to, e.g., a lack of social contact, physical exercise, mental challenges, and in uncontrollable and unpredictable environments (e.g., separation, mistreatment, or inadequate application of cages). arbs can either be classified in stereotypies or compulsive disorders [ ] with stereotypies generally defined as unvarying repetitive behavior patterns with no obvious goal or function [ ] . the terminology of compulsive disorders is preferably chosen for repetitive behavior patterns that are goal-directed and show variability in the repetitive (motor) patterns [ , ] . under chronic conditions without possibilities to adapt (cope), companion animals may develop stereotypies or compulsive disorders like, e.g., tail chasing, polyphagia, compulsively self-directed licking and/or biting the coat [ ] , or feather pecking in parrots [ ] . self-directed patterns can result in serious degrees of alopecia, lick granuloma, or even self-inflicted injuries (auto-mutilation) with a risk of infection. two main reasons underlie the development of arbs in our companion animals. first of all, a lot of companion animals are social species eager for social contact. in the human-companion animal bond, the need for social contact with either conspecifics and/or humans [ , ] can remain unfulfilled if owners work from nine to five, five days a week with the pet staying alone at home on a daily basis. on the other hand, a cat which is originally a solitary hunter with a complex dynamic social structure may start overgrooming or house soiling in the presence of another cat in the territory. such situations might occur in multi-cat households, in the presence of neighboring cats, and in in-stable grouped housing conditions in shelters [ , ] . secondly, most companion animals are species that are eager for mental and physical challenges on a daily basis. the lack of foraging opportunities, the appetitive phase of feeding behavior [ ] might be another reason for the possible development of arbs in companion animals. foraging is often regarded as a high priority behavior [ , ] , i.e., an internally motivated behavioral pattern that should be performed, or otherwise may induce a state of chronic stress, which may result in behavioral pathology like arbs as described in many other animal species [ ] [ ] [ ] . foraging patterns may include walking, running, jumping, nose pushing, digging, and overseeing the area, all active patterns that imply the daily need for physical exercise and mental challenges in most of our companion animals. nonetheless, our pets mostly, if not always, get their food for free with minimal foraging challenges, except for going out - times a day. for some individuals (and especially some dog breeds, e.g., malinois, border collies, and pit bull terriers) [ , ] , situations and contexts with limited challenges can make them more vulnerable to the development of arbs. as well as arbs, other problematic behavior may develop in our companion animals, and the prevalence of some of this behavior is even higher than that of arbs, for example excessive interspecific and/or intraspecific aggression, fear, and anxiety. at what moment, and which type of problem behavior may develop, depends on the intermingled factors of, e.g., genetics, early life experiences (maternal-child bonding, weaning, socialization [ ] ), daily environment, and multiple factors in and around the human-companion animal bond. the history of breeding animals goes back to a time when humans and animals shared each other's habitat. dogs originally have been selectively bred to support human needs, such as hunting, herding, obedience, guarding, rescuing, and for companionship. this artificial selection has generated a large number of dog breeds, displaying a large variation of behavior, size, head shape, coat color, and coat texture [ , ] . unfortunately, in the last years, intensive selection for extreme looks and a narrow gene pool of many breeds has interfered in the genetic make-up of dogs, leading to unfavorable anatomy (extreme large, or extreme "teacup" small), and genetic predisposition to numerous health, welfare, and behavioral problems [ ] . over inherited disorders and traits have already been described in the domestic dog [ ] . one type of dog with a distinct dysmorphology is the brachycephalic dog. brachycephalic dogs are characterized by a large head and round face due to a shortened muzzle, a high and protruding forehead, and widely spaced large eyes. these facial features fit the concept of baby schema ("kindchenschema") proposed by konrad lorenz [ ] . infantile (cute) faces are biologically relevant stimuli for rapidly and unconsciously capturing attention and eliciting positive or affectionate behavior, including the willingness to care [ ] . the appeal of brachycephalic animals has led to specimens that are the so-called "over-typed" dogs and cats with a too short nose, excessively protruding eyes, too straight angulations, etc. breeding animals with this type of severe skull and muzzle abnormalities leads to physical and physiological hardship and limits their natural behavior [ , ] . this violates their integrity and is a big risk for their welfare. selectively breeding animals in order to express specific traits does not only alter existing animals, but also creates new ones, turning animals into an instrument for human use [ ] . the bambino sphynx cat is an example of so called "mutant breeding", where breeders deliberately stack in two steps the recessive inheriting mutations, which leads to hairlessness in sphynx cats, on the dominant inheriting (lethal) mutation responsible for the shortened legs of the munchkin cat. the lack of hair in combination with short legs interferes with the normal physiology of the cat with regard to the manner of movement, thermoregulation, and skin health. one may argue that artificial selection in exchange for money, status, or aesthetic reasons violates the animal's dignity and integrity [ ] . conclusively, artificial selection for excessive traits can have direct consequences for individual health and welfare, may obstruct and prevent a pet from fulfilling its behavioral needs, and conflicts with the current moral way of thinking on animal dignity and integrity. pet animals are not only perceived in %- % as family members or partners but are almost treated like humans. in one study, up to % of owners agreed with the statement "my dog is more important to me than any human being" [ ] . this kind of behavior is the result of the attribution of human cognitive processes and emotional states to animals, such as feelings of happiness, love, or guilt. people believe that animals have awareness, thoughts, and feelings. this behavior is called anthropomorphism, personification, or humanization and can also be applied to plants, gods, or objects. anthropomorphism appears to be caused by the perceived similarity between humans and animals and the extent to which people have developed an affectionate bond with their dogs and cats [ ] . human empathy provides the basis for the attribution of empathy to other animals, as well as attributions of the communicative ability of other animals [ ] . anthropomorphistic behavior can be harmless, such as talking to pets, which many owners do and one of the reasons for this may be the unique ability of humans to recognize facial expressions. talking to pets is also found to be linked to social intelligence [ ] . however, it can lead to animal welfare problems when the feelings of owners no longer match the needs and the intrinsic values of their animal. examples of this are designer dog clothes, animal perfumes, and jewelry, thought the use of protective coats in colder climates for small, short-haired breeds, e.g., chihuahuas, is considered useful. the large number of obese pets can also be partly attributed to anthropomorphism. studies from north america, europe, and australia to determine what proportion of animals, mainly dogs, are overweight or obese reported prevalences of between %- % [ ] . in , an estimated % of cats and % of dogs in the usa were overweight or obese [ ] . when the owner takes a treat with coffee, it is believed that the dog should also get it. even chocolate treats are given, when these are potentially fatal for dogs and cats. this also applies to a good meal that is shared with the pet. dog owners who did not consider obesity to be a disease, maybe because the facial features of their pets fit the concept of baby schema [ ] , were more likely to have obese dogs [ ] . an often-unrecognized risk for pets is reverse zoonotic disease transmission, the so-called zooanthroponosis. a review on this subject reported articles dealing with human to animal disease transmission [ ] . most of the articles dealt with bacterial pathogens but also viral, parasitical, and fungal pathogens were studied in these publications. animals reported to have been infected or inoculated with human diseases included wildlife, livestock, companion animals, and other animals or animals not explicitly mentioned. the majority of the studies focused on human to wildlife transmission with an emphasis on mycobacterium spp. for companion animals, mrsa-infection was especially reported but m. tuberculosis, influenza a, and candida albicans were also discussed. for all groups of animals, microsporum spp. and trichophyton spp. were identified as infectious agents originating from humans [ ] . recent publications report a different kind of zooanthroponosis: the transmission of high-risk, multidrug-resistant pathogens from humans to animals [ ] . a major issue mentioned is the transmission of high-risk clones of extended-spectrum beta-lactamase (esbl) producing bacteria including escherichia coli, enterobacter cloacae, and klebsiella pneumonia [ , ] . the transmission of carbapenem-resistant ndm- producing e. coli from previously hospitalized humans to dogs has also been suggested [ ] . transmission of hospital acquired antibiotic resistant bacteria from human patients to their pets has been confirmed, such as the vim- producing pseudomonas aeruginosa st strain in brazil [ ] . this increased transmission of high-risk multidrug-resistant pathogens from humans to animals was related to the closer relationships between humans and companion animals. some authors doubt the generalized pet-effect on human mental and physical health because of conflicting results that are prevalent in this area of science and the lack of publication of negative results [ , [ ] [ ] [ ] . the majority of research evidence was also considered inconclusive due to methodological limitations such as reliance on self-reports, small sample sizes that may not be representative of the general population, homogeneous populations, varying research designs, narrow range of outcome variables that were examined, and the use of cross-sectional designs that do not consider long-term health outcomes [ ] [ ] [ ] . other studies found for example that pet ownership was associated with a higher incidence of heart attacks and readmissions in heart attack patients instead of a lower incidence [ ] or that pet owners had higher diastolic blood pressure than those without pets [ ] . müllersdorf ( ) showed that pet owners had better general health but suffered more from mental problems such as anxiety, insomnia, and depression, than those who did not own pets [ ] . other studies failed to support earlier findings that pet ownership is associated with a reduced use of general practitioner services [ ] or psychological or physical benefits on health for community dwelling older people [ ] . negative effects of pet ownership include dog and cat bites or scratches, the spreading of disease (zoonoses), and fall injuries, caused by falling or tripping over dogs and cats [ ] . allergic reactions may be a consequence of animal contact and affect %- % of individuals (often genetically) predisposed [ ] . allergies relating to more uncommon pets such as fish, birds, and amphibians seem to be increasing in prevalence [ ] . other studies prove that pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged - years. therefore, advice to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given [ ] . there are also less-positive effects that pets can have on health. more excessive forms of anthropomorphism became clear in our study for the presence of zoonotic parasites in healthy dogs and cats. fifty percent of owners allow pets to lick their faces. sixty percent of the pets visit the bedroom; - % (dogs-cats) are allowed on the bed, and - % (dogs-cats) sleep with their owner in bed. six percent of pets always sleep in the bedroom. of the cats, % are allowed to jump onto the kitchen sink [ ] . this means that in addition to the detected zoonotic parasites (the hazard), there was a significant potential exposure to these pathogens. in addition to parasites, other pathogens such as bacteria, viruses, and fungi can also be transmitted by animals by direct contact through biting, licking, scratching, sneezing or coughing, handling pets or their body fluids or secretions and by indirect contact through contaminated bedding, food, water, or bites from an arthropod vector [ ] . not every individual will develop symptoms after being infected with a zoonosis. this is the result of various factors such as the causative pet species, housing, the degree of contact and contamination, the ability of a micro-organism to cause disease in humans and animals, but especially due to the degree of immunity of the recipient. in order to assess the risk of disease transmission from pets it is important that the nature and frequency of contacts between pets and their owners or other people are evaluated [ ] . we traditionally know that young children (age < years), the elderly (age ≥ years), patients with an impaired immunity, and pregnant women that carry a fragile fetus are at more than average risk of becoming ill after an infection. moreover, they may have more severe disease, have symptoms for a longer duration, or develop more severe complications compared to other patients. young children (notably those aged - years) and some people with developmental disabilities often have suboptimal hygiene practices or higher risk contact with animals which further increases risk [ ] . in children, hand-to-mouth behavior is part of their natural development and they mouth their fingers and other objects. in a meta-analysis, the average indoor hand-to-mouth frequency ranged from . to . contacts/hour and the average outdoor frequency ranged from . to . contacts/hour. the lowest value was attributed to the -to -year-olds and the highest to the -to < -month-olds [ ] . fifteen percent of dog owners and % of cat owners always wash their hands after contact with the animals [ ] . in addition, due to improved healthcare in recent decades, the group of immunocompromised patients has increased sharply. this includes, for example, patients with diabetes, post-splenectomy, after placement of implants and patients being treated with chemotherapy or immunosuppressants. the risk groups are also referred to as yopis (young, old, pregnant, and immune suppressed). patient surveys and epidemiological studies suggest that the occurrence of pet-associated zoonotic disease is low overall [ ] . many of these pathogens are not reportable and presumably underdiagnosed or not recognized by family doctors due to the general, mostly flu-like, symptoms. therefore, any reported frequency of such infections is likely underestimated. to get a better picture of zoonotic risks, a risk analysis is required where a risk score can be calculated using exposure, contagiousness of the infection, and its consequences in the human. in addition, the disease burden of an infection for the population can be calculated and expressed in disability adjusted life years (dalys). this quantifies the health loss based on two components: the life years lost due to premature death and secondly the proportional loss of quality of life as a result of the disease. there is a trend towards closer physical contact between owners and their pets or their environment which poses an increased risk of transmission of zoonotic pathogens. these trends (a general direction in which something is developing or changing) will be explained. our publication [ ] showed that a high percentage of pets were allowed in the bedroom and in bed, with % always sleeping in bed with their owner. is that a problem? already from a hygienic point of view, it is not advisable to sleep with animals or take them into bed. they do not pay attention to where they are walking outside and do not wipe their feet after arriving home. dogs like to roll in carcasses and both dogs and cats regularly lick the anus and thereafter the fur. in a pilot study with healthy dogs and healthy cats that slept with their owners, we tested % of the dogs ( ) and % of the cats ( ) positive for enterobacteriaceae on the fur or footpads. fleas and flea larvae were found on % of pets [ ] . as a result of our publication, chomel investigated in whether transmission of infections by sleeping with pets and licking the face could be found in literature. he reported that also in the usa, france and the uk, a relatively large number of pets slept in bed with their owner ( %- % of dogs and %- % of cats) [ ] . similar results of sleeping with pets were also reported from canada ( % of pets slept with children), the czech republic ( %), and qatar ( . %) [ ] [ ] [ ] . chomel found bacterial infections such as yersinia pestis (plague), bartonella henselae (cat scratch disease), methicillin-resistant staphylococcus aureus, and sometimes fatal bite wound infections such as capnocytophaga canimorsus and pasteurella multocida. furthermore, parasite infections such as cheyletiella spp. were reported. feline cowpox is a rare viral infection, but it can be transferred to the human after direct contact. both animals and humans reveal local exanthema on arms and legs or on the face. in most cases the disease is self-limiting, but immunosuppressed patients can develop a lethal systemic disease resembling smallpox [ ] . it can be concluded that, although uncommon with healthy pets, the risk of transmission of zoonotic agents by close contact between pets and their owners through bed sharing is real and has even been documented for life threatening infections such as plague [ , ] . although pets do not transmit arthropod-borne diseases to people (e.g., lyme borreliosis, ehrlichiosis, anaplasmosis), they do bring zoonotic disease vectors such as ticks and fleas, in close proximity to people, e.g., when they are sleeping with their animals [ ] . while fleas are considered a vector of bartonella henselae (the causative agent of cat scratch disease) tickborne diseases are reported as increasing as ticks expand their ranges [ , ] . with an estimated , cases a year, lyme borreliosis is responsible for the largest disease burden of any vector-borne disease in the european union [ ] . another increased risk associated with close contact with fur is when it is contaminated with zoonotic parasite eggs. especially with echinococcus multilocularis (fox tapeworm) or e. granulosus (hydatid worm, or dog tapeworm). these eggs are immediately infective and may cause serious health problems in the human, many years post infection [ , ] . despite a low prevalence of infectious (embryonated) eggs of toxocara spp. on dog's fur, the potential zoonotic risk should not be disregarded [ ] . the same risk is applicable for the persistence of sporulated toxoplasma gondii oocysts in dogs' fur [ ] . in relation to this, it is noteworthy that many publications report striking increases of ringworm, a common zoonotic fungal skin infection in mainly children caused by microsporum spp., trichophyton spp. or arthroderma spp., where the presence of pets is always mentioned [ ] . however, nowhere has it been suggested that close contact with infected pets in bed increases the infection risk [ , ] . rodents or rabbits are mainly infected with t. mentagrophytes, while m. canis is primarily found in dogs and cats. infection occurs by direct or indirect contact with infected hair, scales, or materials. infected animals may be asymptomatic carriers without clinical signs. examples are % m. canis carriage in a study of european cats [ ] , % in suspected brazilian cats [ ] , and the isolation of t. mentagrophytes dermatophytes from % of clinically healthy rabbits and % of guinea pigs in dutch pet shops [ ] . licking the face of humans by mainly dogs is an expression of their naturally submissive, positive social behavior. the owner is recognized by the dog as the dominant superior in the ranking. in a pack of dogs, submissive dogs lick their dominant counterparts at the corners of the mouth from a typical submissive attitude [ , ] . owners apparently allow this as a token of affection from their pet. such behavior is more common in young animals and has been considered as attention-seeking or care-soliciting gestures. it indicates to the owner the strength of the social bond between dogs and people [ ] . licking or nudging of veterans by service dogs may help take their mind off any negative thoughts, emotions, or memories that they might be experiencing [ ] . on the internet, many images can be found of mainly dogs, but also cats and even rats licking their owner's face. various studies show that around %- % of owners allow this [ , ] . the question is whether this is harmful due to the potential transmission of infections. the review by chomel ( ) shows in the literature that infections, especially pasteurella spp. and capnocytophaga canimorsus, were reported to have transmitted to humans by dogs, cats, kittens, and rabbits [ ] . pasteurella multocida meningitis has been reported in infants where % had been exposed directly or indirectly to the oropharyngeal secretions of household dogs or cats through licking or sniffing [ ] . zoonotic transmission via this route is also assumed for various other pathogens such as gastric helicobacter spp. [ ] , periodontal pathogens [ ] , and bartonella henselae the etiological agent in cat scratch disease. the b. henselae bacteria may cause ocular complications, including parinaud oculoglandular syndrome, a severe eye infection. the route of infection is unknown, although direct conjunctival inoculation, most likely with infected flea feces, seems to be most plausible [ ] . knowing, however, that b. henselae is present in up to % of cat saliva [ ] , it is more plausible that salivary fluid could be rubbed directly into the eye from the skin after been licked by a cat. there are several anecdotal reports of infections in mainly young children that were transmitted by being licked. one recent example is an -month-old baby that presented with fever and preseptal cellulitis with purulent discharge. the causative agent was surprisingly corynebacterium bovis, a bacterium that is normally found in bovine mastitis. it became clear that the dog was frequently allowed to lick the baby's face and was fed on raw meat [ ] . there is an ineradicable belief among a large part of the public that the licking of human wounds by dogs can disinfect them and that the saliva thereby has healing properties [ ] . in addition, it is regularly reported that a dog's tongue is believed to be sterile. this is of course not the case and the oral flora of a dog comprises hundreds of species (including pathogenic) bacteria, fungi, and viruses [ , ] . various wound healing saliva components have indeed been demonstrated in human and animal studies [ , ] . the bactericidal effects of male and female dog saliva facilitate the hygienic function of maternal licking of the mammary and anogenital areas by protecting newborns from fatal coliform enteritis caused by e. coli and neonatal septicemia caused by streptococcus canis. however, the saliva is only slightly, and non-significantly, bactericidal against wound bacteria such as coagulase positive staphylococci and pseudomonas aeruginosa [ ] . capnocytophaga canimorsus and pasteurella multocida are common commensals in the oral cavity of dogs, cats, and other species [ , ] . transmission has been reported after the licking of mucous membranes or open wounds [ ] [ ] [ ] . in patients at high risk, severe wound infections, sepsis, disseminated intravascular coagulation, or death can occur. patients with immunodeficiency, splenectomy, or alcohol dependence are at a particularly increased risk of infection with c. canimorsus [ ] . even immunocompetent persons who have been licked by a dog can develop fatal sepsis [ ] . a further negative effect of companion animal ownership is, of course, accidents inflicted on humans by these animals. these accidents can involve tripping over a cat or being dragged by an enthusiastic dog, but in literature, most evidence points towards biting and scratching incidents. dog biting and cat scratching incidents can cause physical health problems both at the time of infliction but also afterwards by triggering trauma-related secondary infections. dog biting incident reports are numerous, and numbers vary from country to country. in the uk, . dog bites per population per year were reported [ ] , while a commission in the netherlands reported in , , bite accidents per year in a population of million ( events per inhabitants) [ ] . children are especially vulnerable to dog bites. the majority of dog bites occurred in children years of age or younger ( . %) and almost all ( . %) of the dogs were known to the children [ ] . recently, a systematic review has been published that analyzed more than , bites from the literature of the past years about the risk of bites relative to specific breeds of dogs, combining bite incidence with bite severity [ ] . the analysis by breed revealed that pit bulls were responsible for the highest percentage of reported bites across all studies ( . %), followed by mixed breed ( . %), and german shepherds ( . %). dog bite incidents can result in medical treatment, hospitalization and even death. in the netherlands it was calculated that from the , dog bite victims per year, around , seek medical attention and are hospitalized [ ] . between % and % of dog bites become infected and complications become more severe when infection occurs. more than species of bacteria have been isolated from bacterial infections of dog bites, suggesting that most oral flora of dogs have the potential to be pathogenic [ ] . the top pathogens found are pasteurella, staphylococcus, and streptococcus. in literature, specific attention is given to wound infections caused by capnocytophaga canimorsus, because this bacterium is seen as the relatively deadliest pathogen. it was suggested that only % of dog bite wounds contained capnocytophaga spp. [ ] while others reported infection percentages of . % [ ] . wound infection with this bacterium can lead to severe complications like septicaemia, meningitis, osteomyelitis, peritonitis, endocarditis, pneumonia, purulent arthritis, and disseminated intravascular coagulation. c. canimorsus septicaemia has been associated with % mortality. the true number of c. canimorsus infections is probably largely underestimated due to the fastidious growth of the organism. however, infected dog bites in predisposed persons should be taken seriously especially after splenectomy [ ] . cat bite incidents occur less frequently. in only %- % of reported bite incidents in australia, cats are to blame. the long incisor teeth inflict less severe superficial wounds but because of the penetrating effect, joint and tendon infections more easily occur. in a review it is reported that %- % of cat bites become infected mostly by pasteurella multocida [ ] . the bacterium bartonella henselae can also be transmitted by cats through biting incidents but the transmission of this bacterium is much more related to a cat scratch accident. even less frequently reported animal biting incidents are those inflicted by rodents ( % of cases in australia). these bites have an infection rate of approximately %. this could result in rat bite fever in humans, an infection with streptobacillus monoliformis or spirillum minus, characterized by the triad of fever, rash, and arthritis [ ] . cat scratch disease (csd) was first described in a french boy in and is a common, often self-limited, disease that usually presents as tender lymphadenopathy caused by bartonella henselae [ ] . the cat is considered the primary reservoir for this bacterium, with infected fleas and ticks serving as vectors and humans and dogs as accidental hosts. vector transmission of this bacterium occurs via two primary routes: inoculation of bartonella contaminated arthropod feces via animal scratches, most often cat scratches, or by self-inflicted contamination of wounds induced by the host scratching arthropod bites [ ] . immunocompromised human hosts (kidney transplant patients or patients with hiv) are especially susceptible to infection. in these individuals, the disease may be present as a more disseminated form with hepatosplenomegaly, meningoencephalitis, or angiomatosis [ ] . an increasing number of pocket pets and exotic pets are kept by humans. numbers of ornamental birds in europe are estimated as million, fish tanks . million ( million ornamental fish), small mammals million, and reptiles million [ ] . these animal species can also be a source of many zoonotic diseases, especially in young children and immunocompromised individuals. most cases of these conditions are not serious, and deaths are very rare but some of these diseases can be life threatening, such as rabies, rat bite fever infections, and plague [ ] . however, there is also a trend to keep more unusual exotic animals, legally or illegally. these are "wild" animals that are kept in the home such as bats, foxes, skunks, raccoons, meerkats, prairie dogs, kinkajous, sloths, monkeys, apes, prosimians (mammals), parrots, mynah birds, finches (birds), crocodiles, turtles, tortoises, lizards, snakes (reptiles), frogs, toads, newts, salamanders (amphibians), fish, eels, rays (fish), crabs, crayfish, snails, insects, spiders, and millipedes (invertebrates) [ ] . even fruit bats are kept as pets [ ] and it is known that bats harbor a higher proportion of zoonoses than all other mammalian orders, including rabies-like viruses that are highly pathogenic for people. [ ] . another example of a zoonosis is monkeypox following the importation of prairie dogs in the usa [ ] . most reptiles and amphibians such as turtles, lizards, and frogs carry salmonella bacteria in their gut, in most cases without visible signs of infection. the infection may cause symptoms of sickness, diarrhea and fever in humans [ ] . zoonotic transmission of salmonella infections causes an estimated % of salmonellosis annually in the united states. in cases involving pet turtles, almost half ( %) of infections occurred in children younger than years [ ] . salmonella infections are often transferred by feeder rodents [ ] and outbreaks highlight the importance of improving public awareness and education in countries who receive imported reptiles [ ] . it is advised to exclude reptiles, amphibians, rodents, exotic species, baby poultry, and raw animal-based pet food items from the households of patients at high risk. in lower risk households, an understanding of the risk of salmonellosis and other pet-associated zoonoses and preventive hygiene measures is needed. the pet trade in general, with its high turnover and diversity of available species, creates a reservoir for pathogens originating from all over the globe. reptiles account for approximately % of live animal shipments imported to the united states [ ] . importation of live reptiles and amphibians for commercial purposes is for a great part unregulated at eu level except cites and customs regulations [ ] . in a risk assessment study, the five pathogens with the highest public health risk caused by the import of exotic animals were salmonella spp., crimean-congo hemorrhagic fever virus, west nile virus, yersinia pestis, and arenaviruses. the risk via legally imported animals was considered low, but substantial for illegally imported animals due to the unknown health status of the animals [ ] . avoiding exposure to exotic pet pathogens in the home is difficult and best achieved by not keeping them in the first place. otherwise, it is advised to always wash the hands immediately and thoroughly after contact with exotic pets and after handling raw (including frozen or defrosted) mice, rats and chicks. children should be supervised so that they do not put their mouths close to or kiss exotic animals. reptiles and other animals should be kept out of rooms in which food is prepared and eaten. the number of abandoned and homeless dogs and cats in europe is estimated to be over million. countries with more than million abandoned animals are italy, romania, russia, and ukraine [ ] . there is an increasing trend to rescue and import dogs from countries with stray animal problems, in europe often from southern or eastern europe and in the us from puerto rico, the dominican republic, mexico, the middle east, turkey, china, and korea [ ] . many charities and independent groups are involved to rescue dogs and seek adoption in more animal-friendly countries [ ] . new owners primarily choose to adopt from abroad based on a desire for a particular dog they had seen advertised and on concern for its situation, while some were motivated by previously having been refused dogs from local rescues [ ] . in the usa % of all household dogs were neutered and today there are no longer enough dogs being born in the usa annually to replace the approximately million dogs that die each year. developing countries have hundreds of millions of street dogs available for export, for example egypt has an estimated million, india, million, and afghanistan, million [ ] . in , more than a decade ago, the centers for disease control (cdc) estimated annual dog imports at around , . today the number of imported dogs is estimated to be more than a million a year [ ] . imported dogs are reintroducing diseases and parasites that were previously eliminated in the usa [ ] . in the usa new and lethal strains of distemper and canine influenza as a result of imported rescue dogs were reported as well as canine brucellosis, rabies, and vector-borne diseases like ehrlichiosis, heartworm, babesiosis, and leishmaniasis [ , ] . in many southern european countries, there is also a risk of exposure to diseases not encountered in the northern, importing, countries. animals could be infected with anaplasma phagocytophilum, babesia canis, brucella canis, borrelia burgdorferi, dirofilaria immitis (heartworm), dirofilaria repens (subcutaneous worm), echinococcus multilocularis (fox tapeworm), echinococcus granulosus (hydatid or dog tapeworm), ehrlichia canis, hepatozoon canis, leishmania infantum, linguatula serrata (tongue worm), onchocerca lupi, rabies, rickettsia conorii, strongyloides stercoralis, and thelazia callipeda. except b. canis, e. canis, and h. canis, the infections are zoonotic and regularly reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most of these are transmitted by ticks, sand flies, or mosquitoes that are non-endemic in the receiving countries, but a reservoir of infections has been created and the risk is that vectors will become present as result of climate change. [ ] . animals that are infected with rabies or echinococcus spp. may infect people directly. the importation of dogs from endemic, predominantly mediterranean, regions to northern europe, as well as travelling with dogs to these regions carries a significant risk of acquiring an infection. pet owners are therefore advised not to travel with dogs and to seek the advice of their veterinarian prior to importing a dog from an endemic area or travelling to such areas [ ] . for this reason, esccap developed maps on their website featuring european countries and regions with advice on endemic parasites, diseases and recommended treatments when travelling with dogs [ ] . a three-year european union funded project entitled callisto (companion animal multisectorial interprofessional and interdisciplinary strategic think tank on zoonoses), has investigated zoonotic infectious diseases transmitted between companion animals and humans and food producing animals [ ] . the committee advised that special attention should be given to stray cats and dogs. stray dogs, in particular, may pose serious health and welfare problems for humans and animals [ ] , including the transmission of zoonotic diseases such as rabies. consideration should be given to controlling companion animal movement between areas of the eu endemic for particular zoonoses and areas that are not currently endemic for that disease. reliable figures are not available, probably because of the fact that many voluntary organizations are responsible for saving and transporting these rescue animals. such data are essential in order to be able to quantify the actual risks of zoonotic diseases attributable to companion animals and to develop sustainable interventions to prevent transmission to humans and livestock [ ] . as long as there are no official guidelines, to prevent the spread of (zoonotic) diseases to new countries, the time, expense, disease risk, and the implications of adopting a dog from abroad should all be carefully considered before importation. as an alternative, the conditions for native dogs could be improved by supporting local charities to organize neutering campaigns and rehoming programs, to build local animal shelters and to improve attitudes towards dogs and their living conditions [ ] . cats and dogs harbor the enteric nematodes toxocara canis and toxocara cati, and cats are the final host for the protozoal parasite toxoplasma gondii. these parasites can be transmitted to humans because they have an oral-fecal transmission cycle. humans can be infected by ingestion of infective toxocara spp. eggs or toxoplasma oocysts from contaminated soil (gardens, sandpits, and playgrounds) [ , ] . a recent meta-analysis of data from published records indicates that public places are often heavily contaminated with a pooled global prevalence of toxocara eggs of % [ ] . both parasites are considered by cdc as part of five neglected parasitic infections. these infections are considered neglected because relatively little attention has been devoted to their surveillance, prevention, and/or treatment. the diseases that they cause have been targeted as priorities for public health action based on the number of people infected, the severity of the illnesses and the ability to prevent and treat them [ ] . tens of millions of people worldwide are estimated to be exposed to, or infected with, toxocara spp. and recent findings suggest that the effect of toxocarosis on human health is increasing in some countries. almost one fifth ( %; . billion individuals) of the world's human population is seropositive to toxocara. the highest seroprevalence rates were found in africa (mean: . %) and the lowest in the eastern mediterranean region (mean: . %) [ ] . toxocara larvae migrate into the body of the human to several organ systems with a preference for the central nervous system (brain, eyes). human toxocarosis can manifest itself as syndromes known as visceral larva migrans, ocular larva migrans, neurotoxocarosis, and covert or common toxocarosis [ ] . asthma is one of the most common chronic respiratory diseases worldwide, with a negative impact on the quality of life and socio-economic status of patients. two decades ago, the first evidence was published that suggested that toxocara infection is a neglected risk factor for childhood asthma [ ] . the finding that children infected with toxocara spp. are more likely to have asthma compared to non-infected children was recently confirmed in a systematic review and meta-analysis [ ] . cognitive or developmental delays in children or young adults who become infected is of particular concern. toxocarosis appears to be associated with decreased cognitive function [ , ] . the annual toxoplasma oocyst burden measured in community surveys has been reported as up to oocysts per square foot ( per square meter) and is greater in areas with loose soil, that cats like to use to cover their feces in gardens, children's play areas, and especially sandboxes, also called sandpits and sand piles [ ] . because a single oocyst can possibly cause infection, this oocyst burden represents a major potential public health problem. an estimated one third of the world's population harbor anti-toxoplasma antibodies. due to keeping pigs indoors, more education and awareness, the prevalence of the disease in the usa and europe declined by % over the last decades [ ] . during an acute invasion of toxoplasma parasites there is mild to major tissue damage without clinical symptoms (latent toxoplasmosis). the most important form is congenital toxoplasmosis when a woman receives her first exposure to toxoplasma during pregnancy. in early pregnancy, this can lead to abortion or to malformations that are not compatible with life shortly after birth. congenital infections may also be characterized by mental retardation and ocular defects. acquired infection after birth may result in clinical symptoms such as lymphadenitis, fever, and malaise and probably leads to a clinically symptomatic disease state more frequently than the congenital condition, with an estimated incidence of % of all ocular toxoplasmosis cases [ ] . playing in a sandbox is also found to be a predominant risk factor for s. typhimurium salmonellosis in children aged - years [ ] . this can be the result of fecal contamination of the sand by dogs and cats that have been fed raw meat (see section . . .). young children are especially at risk as they put their hands or other objects in their mouths every - min [ ] . it has also been reported that children ingest a median of mg of soil per day and that one child consumed - g of soil per day on average [ ] . it is therefore advisable not to let children play in public places or on playgrounds with loose sand, but only in sandboxes that can be covered. furthermore, washing hands after playing outside is important and fingernails must be trimmed to prevent sand being left behind. in this context, a strange trend can be observed as young children play in mud baths on june "as a way to connect and celebrate the natural joys of playing in the mud". this international mud day originated in and was initiated by an australian pedagogue who had observed this phenomenon during a visit to nepal [ ] . if the origin of the soil needed for producing the mud is unknown, there is of course an infection risk for the above discussed parasites. to prevent the transmission of zoonotic diseases from pets, risk analysis is of great value. this starts with an assessment of the potential zoonoses in an area, depending on the endemicity (the hazard, h). hazard characterization also includes prevalence in animals (the reservoir), virulence for man, transmission routes, and survival of the agent in the environment. the second step is exposure assessment (e). who is exposed to the potential hazard and for how long or how often? how much of the potential pathogen is needed to become a health risk? this inevitably is directly related to human behavior in relation to their pets. the third step is to assess the impact of getting infected (i). how serious is the disease, what is the chance of complications, and what economic consequences may be expected (e.g., labor hours lost)? each of the parameters can be ranked in classes from negligible to the most serious possibility. ranking is based on literature data, own observations (measuring), or experts' opinions. the final risk assessment can be achieved by multiplying the outcome of hazard characterization, exposure assessment and impact (h × e × i = a number). the outcome can be compared with other zoonotic agents and a ranking of significance can be made [ ] . there is one important parameter to reduce the risk of contracting a zoonotic infection that can directly be influenced, which is exposure. recommendations are particularly targeted to households with very young children, the elderly, pregnant women, or immunocompromised patients. they are based on reducing exposure to hazards and involve four categories of advice (table ) . table . recommendations to prevent the transmission of zoonotic pathogens from pets [ , ] . • washing the hands thoroughly -after animal contact, at least before eating and drinking and before preparing food or drinks -after handling raw pet food -after handling pet habitats or equipment -after cleaning up feces -after removing soiled clothes or shoes reflecting on the changed human-companion animal bond, it can be concluded that pets undoubtedly have a positive effect on human health. conversely, the human-pet bond seen nowadays is facing many challenges, putting pet welfare under more pressure due to issues such as anthropomorphism, which mainly results in obesity, breeding on extreme appearance rather than health, behavioral problems connected to unfulfilled species specific mental and physical needs, and the provision of inadequate food because owners mistakenly think they feed more naturally. with regard to the negative effects of pets this article attempts to give an impression of increasing trends in the human-companion relationship that can be observed in society, which appears to increase the risk of transmission of infection between pets and humans. it is mainly a consequence of the increasing contacts between humans and pets and with pathogens secreted by animals in the shared environment. more than out of every known infectious diseases in people can be spread from animals, and out of every new or emerging infectious diseases in people come from animals [ ] . the recent pandemic of the covid coronavirus (sars-cov- ), that may be originated from bats, is a good example of a recent emerging zoonotic infectious disease. a few cats and dogs have tested positive but are not considered as a source of infection for people. the proportion of zoonotic human disease that is attributable to pets is largely unknown. reports about the frequency of such infections are likely underestimated [ ] ; however, the risk of infection is relatively small for many zoonoses and the severity of the disease is often limited. a person's age and health status may affect their immune system, and thereby increase his or her chances of getting a disease from animals. pregnant women should avoid contact with pet rodents, reptiles, cat feces, and raw meat to prevent infection of the unborn child, abortion, or birth defects. if symptoms occur in immunocompetent, non-pregnant persons between and years of age, these are mainly of a general nature such as diarrhea or flu-like symptoms. physicians do not regularly ask about the presence of pets or pet contact, nor do they discuss the risks of zoonotic diseases with patients, regardless of the patient's immune status, which means that many cases of zoonotic diseases go undiagnosed. the general public and people at high risk of pet-associated disease are not aware of the risks associated with high risk pet practices or recommendations to reduce them. since unfamiliarity with hazards reinforces fear, communication plays an important role in this. veterinarians play a key role in education regarding risk reduction by giving advice about responsible pet ownership and the required preventive hygiene. healthcare providers such as family doctors, school doctors, and pediatricians can also provide information about safe pet ownership. physicians should ask as part of the medical history about eventual contact with pets, particularly with patients at high risk [ ] . the "one health" initiative aims to reduce this professional gap between vets and physicians [ ] . when giving recommendations to prevent zoonotic transmission, one of the often-made remarks is that people nowadays are already too hygienic. it is assumed that there is a protective influence of postnatal infection and that it might be lost in the presence of modern hygiene. this belief is based on the hygiene hypothesis that was formulated in [ ] . it was observed that hay fever in young adults was inversely related to the number of siblings in their family. this hypothesis focused exclusively on allergic conditions as a result of the modern way of life and the assumption that modern hygiene was reducing contact with bacteria. another view is that some chronic inflammatory disorders have increased over the last decades as a result of decreased frequency of infections due to pathogenic organisms [ ] . another related theory is the "old friends" mechanism that is based on the positive influence of gut parasites, non-pathogenic environmental bacteria (saprophytes, pseudocommensals), and gut commensals or microbiota. however, decreased exposure to these microorganisms is not the only reason for the increasing frequency of allergies and chronic inflammatory disorders in industrialized countries. nowadays there is more information about the immunological roles of skin, oral mucosa, and gut microbiota as well as helminths and the influence these have on the immune system [ ] . gut flora may be modified due to diet, obesity, hygiene, antibiotics, but also to psychological stress, vitamin d deficiency, and pollution [ ] [ ] [ ] . pollution also has a significant effect on the development of several respiratory problems and diseases. not only due to outdoor pollution such as fine dust, harmful solids, liquids, or gases [ ] , but also due to indoor molds as result of insufficient ventilation in energy efficient homes [ ] . finally, there is a clear increase in the allergen production of house dust mites and pollen leading to more exposure and sensitization in susceptible individuals [ ] . all together it must be clear that there is much more known about other causes of increasing allergies worldwide than simply excessive cleanliness as suggested in the hygiene hypothesis. regarding field infections with helminths such as trichuris trichiura in early life, these are associated with a reduced prevalence of allergies later in life and infants of helminth-infected mothers have a reduced prevalence of eczema. hookworm infections in developing countries are associated with a reduced prevalence of asthma [ ] . the rate of eczema in such countries was found to be about five times higher in infants whose mothers had never had helminths compared with persistent helminth-infected mothers [ , ] . helminths are nowadays even used under controlled conditions to stimulate immunity. examples are trichuris suis therapy for crohn's disease and necator americanus larvae to treat crohn's disease and other autoimmune disorders [ ] . there are no clinically apparent childhood infections found to be associated with protection from allergic disorders [ ] . it can even result in an opposite effect in the case of toxocara infection by children after soil contact. a recent meta-analysis showed that children infected with toxocara spp. are more likely to have asthma compared to non-infected children [ ] . this parasite and asthma both have elevated immunoglobulin e (ige) levels and eosinophilia in common. that means that precautions should be taken in children to prevent soil contact not only to prevent toxocara infection, but also to prevent acquired ocular toxoplasmosis. there is no need, therefore, to stimulate the contraction of pathogenic bacteria or helminths to achieve a healthy gut microbiota and to reduce allergic conditions. recommendations based on the hygiene hypothesis should preferably be based on results from controlled studies to prevent unintentional negative effects. it is both humans and companion animals who experience negative effects of a changed human-companion animal bond. the education given by vets to their clients should therefore also focus on preventing these negative health and behavioral effects. for instance, by giving science-based advice on feeding practices. in general, regulating authorities should encourage the development of enforcement criteria for breeding dogs and cats to reduce health and welfare risks. pets undoubtedly have a positive effect on human health and well-being, while owners are increasingly aware of pet health, welfare, and well-being. anthropomorphism, also resulting in behavioral problems and breeding on appearance rather than health, and trends such as keeping exotic animals and importing rescue dogs may result in an increased risk of contracting zoonotic infections. recommendations regarding responsible pet ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conform the biology of the animal, are key in preventing such negative aspects of the human-animal bond. there is no need to stimulate unhygienic practices following the hygiene hypothesis. education can be performed by vets and physicians as part of the one health concept. the brewing storm one health initiative. available online: www.onehealthinitiative.com 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pollution: a review. front. public health , , abridged version of the awmf guideline for the medical clinical diagnostics of indoor mould exposure house dust mite allergy under changing environments interactions between helminth parasites and allergy the impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection an update on the use of helminths to treat crohn's and other autoimmune diseases infections presenting for clinical care in early life and later risk of hay fever in two uk birth cohorts this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -uazc lyg authors: hedrick, stephen m. title: the imperative to vaccinate date: - - journal: the journal of pediatrics doi: . /j.jpeds. . . sha: doc_id: cord_uid: uazc lyg nan stephen m. hedrick, phd the disease legacy of civilization h uman beings are almost certainly the most diseased species on earth. by one accounting, there are at least human pathogens, including bacteria, fungi, prions, protozoa, viruses, and worms, and of these, - appear capable of causing human epidemics. , even this is likely to be an underestimate, as new and sensitive sequencing techniques continue to uncover new viruses at a steady rate. we human beings are remarkable in many ways, but why are we remarkable for playing host to so many infectious agents? why is it that we must maintain high levels of vaccine coverage to prevent infectious agents from sickening or even killing large swaths of the population? the answers lie in the story of human disease epidemics, and it begins with human cultural and technological ascendance and what we now understand to be its inevitable consequences for pestilence and death. it is about our ingenuity, which has caused the retreat of many infectious diseases, but highlights a central tension in human existence-immediate self-interest vs long-term collective welfare. the concept is not just academic; there are realworld implications that we can resolve with an understanding of human disease ecology. the notion is that we are not only culturally connected or genetically connected through a common ancestry. rather, there is another fundamental concept that is, perhaps, not widely accepted or even understood. we are biologically connected, in the present, through our exchange of infectious agents and our common susceptibility to disease. to understand modern human disease prevalence, we have only to look to the most basic principles of epidemiology. a simplified version is that diffuse or small host populations cannot sustain an acutely infectious agent, meaning one in which infection is followed by clearance and long-term immunity. as the number of people with immunity increases, the density of susceptible hosts decreases, and with the corresponding decline in transmission, the infectious agent is not maintained in the population. this principle described our preagricultural ancestors-a few thousand individuals congregated in groups but spread out over an enormous area. small or low-density populations can only sustain a certain type of infectious agent, one that persists, usually for the life of the host. , once infected with herpes viruses, such as herpes simplex virus, cytomegalovirus, or epstein-barr virus, we are infected for life, and such viruses have infected us since even before we became human beings. [ ] [ ] [ ] [ ] to some extent, this was the primordial state of disease in diffuse bands of preagricultural hunter-gathers: persistent viruses, bacteria (eg, mycobacterium tuberculosis), intestinal protozoa, worms, and fleas. our paleolithic ancestors were not disease-free, but they almost certainly did not experience periodic and devastating epidemics. , conversely, large populations that live at high density, such as modern human beings, can sustain a much greater diversity of infectious agents, including those that the immune system is able to clear. transmission from person to person is rapid enough and continuous, such that there is little selective pressure for persistence. large and dense urban populations can maintain acutely infectious agents indefinitely due to a constant source of newly susceptible hosts in the form of immigration or births. these agents often share an ability to be transmitted by casual contact such as respiratory droplets produced by a cough or a sneeze, and as evidence of the success of this pathogen strategy, there are more than different viruses from at least different virus families (adenovirus, coronaviruses, influenza virus, parainfluenza virus, respiratory syncytial virus, and rhinovirus) that cause "cold" symptoms: sneezing, coughing, and runny nose. the dawn of agriculture and the domestication of animals, especially herd animals, allowed the emergence of permanent human settlements and the growth of large situated communities. the world's population increased approximately -fold from the beginning of the agricultural revolution to the end of the th century, and most importantly, settlements eventually grew into a huge massing of humanity. simultaneously, we domesticated animals and ourselves, and we sampled all of the viruses and bacteria existing in cows, horses, pigs, sheep, goats, and birds. those that could replicate in human beings and spread from person to person by respiratory propulsion (or other means, such as fecal-oral) became established evermore in the human population. this is the answer to why we are the most diseased species on earth. we are the only species to so profoundly and rapidly change the way in which we interact with each other and other animals; in other words, we invented for ourselves an entirely new ecosystem. so, in addition to the endless parade of cold viruses that circulate among us, we acquired a great many deadly infectious agents, such as those that cause diphtheria, influenza, measles, meningitis, mumps, plague, rubella, smallpox, typhus, whooping cough, and others. each disease has its own history and severity, but all rely on large, high-density populations for continued propagation. these newly acquired infectious agents not only caused severe or deadly disease, they shaped the population. many are known as childhood diseases because they infect susceptible children who either recover from the disease and retain immunity or die. in a population in which a disease like measles existed, everyone contracted the virus exactly once, such that almost all surviving adults were immune. what does the world look like in the face of measles? from to , before the availability of a vaccine, an average of cases of measles were reported each year in the us, along with an average of measles-related deaths, encephalitis cases (often with permanent brain damage), and respiratory complications. the measles vaccine was licensed in and the measles, mumps, rubella (mmr) vaccine was licensed in . for the years between and , the number dropped to cases of measles in children younger than years of age; died, contracted encephalitis, and contracted pneumonia. since , there has been less than case per million population in the us. the global burden of measles in was an estimated deaths that were reduced through a world-wide vaccination campaign to an estimated deaths in . those who survive measles without lasting effects still have worries. one is that measles infection depresses the immune system for up to years, making children more susceptible to other infections, and a second is the possibility of developing subacute sclerosing panencephalitis, a usually fatal neurologic degenerative disease caused by reactivation of latent measles virus. the assessed risk is on the order of in measles cases and as much as -fold greater for children who contract measles before the age of months. for children who are immunocompromised, such as those being treated for leukemia, an actual measles infection is severe, extended, and often fatal. although measles is possibly the world's most infectious human virus, it was not the most devastating of the childhood infectious diseases. the smallpox death toll for just the th century has been estimated at upwards of million people, similar to the entire population of the present-day us. smallpox caused more deaths than all the wars in history. for centuries before vaccination, most urban families could count on losing multiple children to smallpox, diphtheria, scarlet fever, or whooping cough. with widespread vaccination, combined with targeted vaccination to insulate the last few cases, smallpox was eliminated as an infectious disease on earth. smallpox eradication was our first and thus far only complete victory over a human disease-causing agent, made possible by universal, global vaccination, and intensive surveillance. after tortuous millennia of epidemic disease and hundreds of millions dead, who would argue that this was not a most wonderful gift given by humankind to itself? but that gift was not without cost, and the cost was a tincture of personal independence and the acknowledgement that each of us is inextricably tied to the entire human community. it took the idea of community out of the realm of philosophy and placed it as a fundamental property of life. smallpox eradication itself was a physical enactment of the tension between personal freedom and the authority of society. in on liberty, in chapter iv, john stuart mill asks, "what then is the rightful limit to the sovereignty of the individual over himself? where does the authority of society begin? how much of human life should be assigned to individuality, and how much to society?" mill's inquiries can be answered by biology, but first consider the concept of community protection (often referred to as "herd immunity"). as the density of susceptible (unvaccinated or disease naïve) hosts declines, so does the incidence of disease. below a certain threshold, the incidence of disease (frequency of new infections), even in unimmunized people, approaches zero. this is community protection, and it follows directly from basic epidemiology. vaccination effectively reduces the number and density of the disease-susceptible people, making acutely infectious agents unsustainable in the population. conversely, because vaccine protection is sometimes imperfect, a vaccinated individual living within a diseasesusceptible population is at substantial risk. the risk of disease for any individual is thus most importantly dependent on the collective immunity of the population, especially those most susceptible to infection, usually the youngest children and oldest adults. in this regard, disease ecology does not equivocate; in the world as it exists today, our health and our very being depend on the immune status of the rest of humanity. the rightful limit to the sovereignty of the individual over him or herself stops at the boundary of disease immunity. as long as one case of smallpox could be found on earth, billions were at risk. even without considering the imperative of contagious disease, mill came to the same conclusion, "as soon as any part of a person's conduct affects prejudicially the interests of others, society has jurisdiction over it. . .." two centuries before on liberty and before the enlightenment, this was expressed after a fashion in john donne's meditation xvii, "now this bell tolling softly for another, says to me, thou must die," written while he was convalescing from a near-fatal disease, possibly typhus. although this meditation was ostensibly concerned with god as the author of every person and every death, we might equally apply it in a way that donne could not-we are each of a network, a medium for disease that transcends us as individuals. the death of one of us portends many more. we can rage against this injustice, but it is literally a fact of life. in this context, the famous line from meditation is relevant, "no man is an island, entire of itself." community protection is a fundamental concept with no strict definition. the threshold is sharp but varies with each infectious agent. it protects vaccinated and unvaccinated people alike. it is the most powerful force in disease prevention but exists only in the immunity status of the entire population network. considering the difficulty of this concept, it is no wonder that as a society and as a people we do not have a consensus the journal of pediatrics • www.jpeds.com volume • october concerning the responsibility of individuals to vaccinate their children. one way to understand vaccination decisions is as an exercise in game theory played out over the entire human population of the earth. in this case, each individual is defined narrowly in economic terms, acting as if he or she balances costs against benefits to maximize personal advantage. if most everyone cooperates (vaccinates), then everyone enjoys the benefits of being disease free. in contrast, the decision to cooperate may be perceived to have a cost, and individuals looking to maximize personal advantage will choose noncooperation at a certain probability. when no one is vaccinated and everyone is in danger, that probability is close to zero-everyone is incentivized to vaccinate or risk the possibility of deadly disease. this must have been the dominant sentiment in the time of smallpox. as universal vaccination is approached, danger diminishes with or without vaccination, and the probability of noncooperation increases. for measles, the threshold for community protection is calculated to be . %, that is, when . % of the population has received doses of mmr vaccine, the community is protected from disease. under such conditions, some parents may decide not to vaccinate and thus avoid even very rare adverse effects. the consequence of this is that the rate of vaccination drops below the threshold, and the community is no longer protected. in other words, as we proceed toward elimination of a disease by vaccination, as we are for poliomyelitis, the invisible hand of the market pulls defeat from the jaws of victory. from this reasoning, elimination of a disease on a purely voluntary basis has been proposed to be unlikely, and the thought is that compliance to protect the population or eradicate a disease can only be achieved by a mandatory vaccination policy. in the western hemisphere, we have all but eliminated measles and rubella, in one sense moving us backward in time to the pre-columbian rarity of acutely infectious diseases. however, should we lapse in our vaccination vigilance, within one generation we could replay the disease devastation of the th century that included death of more than one-half of the native inhabitants of the western hemisphere. we are part of, what watts and strogatz called, a small-world network -with no more than degrees of separation connecting the entire + billion human beings on earth. like the spread of middle east respiratory syndrome from the middle east to korea, we can consummate those connections, wherever they may be, with a day's travel. a glimpse of a future with poor vaccine adherence occurred not too long ago, with an outbreak of measles originating in anaheim, california. the infecting person (patient zero) almost certainly arrived from abroad, but most of the infected individuals were unvaccinated us residents. the decline in mmr vaccination compliance began with a medical report in the journal lancet. andrew wakefield and colleagues published an analysis of children claiming to show a connection between mmr vaccination and the onset of a newly described "pervasive developmental disorder" that they summarized as a "chronic enterocolitis in children that may be related to neuropsychiatric disorder." at face value, this is a tiny sample size with no controls. it linked common conditions with mmr vaccination based on parent's recollections. multiple large studies subsequently showed no such association, and the paper was retracted by of the authors and by the journal after a prolonged study by the general medical council in the united kingdom; however, it was not just a case of flawed science, it turned out to be fraud driven by avarice. in articles published by the british medical journal, the investigative journalist brian deer revealed how wakefield had been hired to attack the mmr vaccine by a lawyer, richard barr. wakefield and colleagues were paid to contrive the existence of a syndrome, he initially called, "autistic enterocolitis" for the express purpose of bringing a class action lawsuit against vaccine manufacturers; this occurred before initiation of the disgraced study. another apparent moneymaking scheme was ironically to market vaccines. in a press conference given after the publication of his lancet paper, wakefield said he could not support the triple mmr vaccination and called for vaccination to each disease separately. he had previously patented a single measles vaccine. the british general medical council revoked his license to practice medicine, and he was asked to leave the royal free hospital in london. the wakefield study has no basis in reality, but its publication corresponded to a substantial drop in mmr coverage in the united kingdom, europe, and the us. in response, some countries or states within the us have made vaccination a mandatory condition for entrance into schools. for example, california senate bill no. , signed into law june , requires vaccination for all children attending any public or private elementary or secondary school, childcare center, day nursery, nursery school, family daycare home, or development center. exemptions for described medical conditions are permitted, but those based on personal or religious beliefs were to be phased out. because california requires vaccination records for all schools, the effects of the bill could be tracked-and the effects were dramatic. in , more than % of school children lived in california counties with vaccination rates less than %, and % of children lived in counties with less than a % vaccination rate. interestingly, the counties with the lowest rates of vaccination were of types, rural counties largely located in the most northern part of the state and counties that include the tony urban communities surrounding san francisco and los angeles. by , more than % of children were from counties with greater than % vaccination. nonetheless, the law only requires vaccination records for students as they enter each grade span (kindergarten, seventh grade, etc), and it recognizes personal exemptions previously on file. as such, there are still schools in which a single case of measles could spark a local epidemic. contrast the vaccination rate in california, where comprehensive vaccination is required to attend school, with that of oregon, where exclusions based on personal beliefs are allowed with only a requirement for completing an informational module online. the proportion of the population in oregon counties with kindergarten vaccination rates greater than % has gone from almost % in to just % as of . in addition to community health, the notion of not vaccinating seems to deny short-term self-interest. even with a low disease incidence brought about by community protection, pertussis vaccination is a small price to pay for the prevention of whooping cough. beyond that, universal vaccination protects children with immunodeficiencies that arise either from congenital or acquired conditions and their treatments. it can eliminate a disease from the world for all time, saving all future generations, but at what cost? what is the safety of vaccination? each vaccine from each manufacturer is reviewed by the food and drug administration for safety before licensing, and after licensing, the centers for disease control and prevention and food and drug administration maintain a nationwide monitoring system, the vaccine adverse event reporting system, a signal detection system to identify rare events not found in prelicensing reviews. the program allows anyone to report an adverse reaction online, by fax, or by mail. the centers for disease control and prevention also operates the vaccine safety datalink in conjunction with us care organizations that track data from more than million people. these data provide the means to monitor the safety of current and recently introduced vaccines nearly in real-time as they are administered to people across the country. the data from the vaccine safety datalink are used to devise the vaccine regime for children and assess the frequency of complications as they arise. there also exists a national vaccine injury compensation program (vicp), run by the health resources and services administration. this program receives reports of adverse vaccine reactions, studies each claim, and makes of determinations: ( ) an adverse reaction occurred "more likely than not"; ( ) the individual is compensated, although the panel does not concede that there occurred a vaccine-related adverse reaction; and ( ) the case is adjudicated by a court within the us court of federal claims. because the vicp is the only avenue for vaccine-related compensation in this country, the number of filed cases is one measure of the number of adverse reactions severe enough to incite a claim. for the years of through , there were approximately . billion vaccine doses distributed in the us. the total number of cases brought before the vicp was , and the number ultimately compensated was . that is, about in a million vaccine doses was associated with some sort of adverse reaction severe enough to bring a patient to the vicp. importantly, this is but an average for all vaccines and all groups of people, but it highlights the overall rarity of vaccine-associated, severe adverse events. considering the benefit to the individual and to society, this would seem to be a reasonable risk. aside from sober risk assessment, sticking an infant with a needle to induce an immune reaction might feel unnatural. but it isn't so. the immune system is naturally engaged and constantly fighting many potential infections on a continuous basis. for example, in people with an acquired immunodeficiency, such as those low numbers of t lymphocytes, previously benign bacteria, fungi, and viruses become deadly: cytomegalovirus, candida, pneumocystis carinii (now pneumocystis jirovecii), toxoplasma, and other environmental agents can cause sickness or death. regardless of the presence of actual disease-causing agents, without the constant activity of our immune system, we perish. another concern is that multiple vaccinations might "overload" the immune system, causing children to be more susceptible to unvaccinated diseases. however, in a study of nonvaccine-targeted infections recorded from emergency department visits, there was no significant correlation with the number of vaccines given to children - months of age. medical studies are difficult to evaluate, even for professionals. the wisdom of one moment is often replaced in the next. a reasonable course of action with respect to new clinical findings is to wait and act conservatively. however, we now have a century's worth of experience in vaccinating billions of people. we have witnessed the regression or elimination of many infectious diseases in the face of vaccination. and we have studied the short-and long-term effects of vaccination. this is now established science. we can work to make vaccines even safer and more effective, but we cannot as a society regress to some past era in which we count hundreds of thousands of measles or polio cases per year. infectious diseases are a major, and almost certainly permanent, part of human existence. the growth of civilization with the addition of animal domestication made the appearance of epidemic diseases inevitable, but human inventiveness has allowed us to find countermeasures that relieve at least some of our collective misery. furthermore, the experience of humankind over the past several millennia has shown that we have no choice; our place in the network of hosts susceptible to human pathogens gives lie to our notions of complete personal independence. even the most atavistic society would not choose for their children a path of immune naiveté (at least not for long). perhaps this is an instructive irony. it takes deadly infectious diseases to see that we are all of a one species, biologically connected, and isolated on earth. ■ submitted for publication jan , ; last revision received jun , ; accepted jun , risk factors for human disease emergence ecological origins of novel human pathogens search strategy has influenced the discovery rate of human viruses the meaning of human existence network structure and the biology of populations measles endemicity in insular populations: critical community size and its evolutionary implication epidemiology meets evolutionary ecology molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses epstein-barr virus infection review of cytomegalovirus seroprevalence and demographic characteristics associated with infection evolutionary origins of human herpes simplex viruses and infectious diseases in primitive societies genomics, the origins of agriculture, and our changing microbe-scape: time to revisit some old tales and tell some new ones the common cold plagues and peoples historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states vaccines. long-term measles-induced immunomodulation increases overall childhood infectious disease mortality subacute sclerosing panencephalitis: the devastating measles complication that might be more common than previously estimated human immunology of measles virus infection smallpox and its eradication. geneva: world health organization vaccination and herd immunity to infectious diseases why does measles persist in europe vaccination and the theory of games : new revelations of the americas before collective dynamics of 'small-world' networks measles outbreak linked to disney theme parks reaches five states and mexico wakefield's "autistic enterocolitis" under the microscope how the case against the mmr vaccine was fixed after a debacle, how california became a role model on measles vaccination rate jumps in california after tougher inoculation law safety monitoring in the vaccine adverse event reporting system (vaers) the vaccine safety datalink: successes and challenges monitoring vaccine safety opportunistic infections in patients with and patients without acquired immunodeficiency syndrome association between estimated cumulative vaccine antigen exposure through the first months of life and non-vaccine-targeted infections from through months of age key: cord- -yp ofhi authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: chapter animal models of human viral diseases date: - - journal: animal models for the study of human disease doi: . /b - - - - . - sha: doc_id: cord_uid: yp ofhi abstract as the threat of exposure to emerging and reemerging viruses within a naive population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. by using animal models in this endeavor, the response to viruses can be studied in a more natural context to identify novel drug targets, and assess the efficacy and safety of new products. this is especially true in the advent of the food and drug administration's animal rule. although no one animal model is able to recapitulate all the aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to be considered before an animal study are the route of challenge, species of animals, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand the disease progression, pathogenesis, and immunologic responses in humans. furthermore, to test vaccines and medical countermeasures, well-developed animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease. a good animal model of viral infection should allow many parameters of infection to be assayed, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations, and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for initially testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for the pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most experiments performed in small animal models when possible, and nhps only used to fill in remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen-free (spf), transgenic, and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables larger or more frequent blood samples to be collected, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains; thus, greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic animals are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically the closest species to humans; thus, disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns of experimentation on nhps, along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure that the least amount of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, route of exposure and the dose should be as close as possible to the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are also often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on the viruses for each family that are the greatest concern for public health worldwide. poliovirus (pv) is an enterovirus in the picornavirus family and causes poliomyelitis. humans are the only natural host for the virus, but a number of nhp species are also susceptible. all three serotypes of pv cause paralytic disease, but it is relatively rare with only - % of infected individuals ultimately developing paralysis. humans typically acquire and transmit the virus by the oral-fecal route, although transmission by aerosol droplets may also be possible. the virus replicates in the oropharyngeal and intestinal mucosa, made possible by the resistance of pv to stomach acids. cd expression in peyer's patches and m cells suggest that these cell types may be important during initiation of infection. replication at extraneural sites . in vivo models of viral diseases affecting humans precedes invasion into the central nervous systems (cnss), when it occurs. two effective vaccines, the salk killed polio vaccine delivered by the intramuscular route and the sabin live attenuated polio vaccine delivered orally, have been used very successfully to eliminate the disease from most parts of the world. the world health organization has led a long and hard-fought global polio eradication campaign, with much success, but full eradication has not yet been achieved. since , between and cases of pv infection are reported worldwide each year. thus, animal models are also needed to test new vaccine approaches that could be used toward eradication of polio in the areas where it still persists. additionally, the recent focus of work with pv animal models has been fraught with urgency, as to gain understanding of pv pathogenesis before the eradication effort is complete and work with this virus ceases. animal models for the study of pv consist of nhp models and mouse models. mice are susceptible to certain adapted pv strains: p /lansing, p /lsb, and a variant of p /leon. mice infected intracerebrally with p /lansing develop disease with some clinical and histopathological features resembling that of humans. wild-type mice are not susceptible to wildtype pv; however, the discovery of the pv receptor (cd ) in led to the use of hcd transgenic mice as a model of pv infection. these mice are not susceptible to pv by the oral route and must be exposed intranasally or by intramuscular infection to induce paralytic disease. interestingly, hcd mice that have a disruption in the interferon (ifn)-a/b receptor gene are susceptible to oral infection. this finding has given rise to speculation that an intact ifn-a/b response may be responsible for limiting infection in the majority of individuals exposed to pv. thus, mouse models have proven to be very useful in gaining a better understanding of pv disease and pathogenesis. rhesus macaques are not susceptible to pv by the oral route, but they have been used extensively to study vaccine formulations for safety and immunogenicity, for monitoring neurovirulence of the live attenuated sabin vaccine, and in the past for typing pv strains. bonnet monkeys are also susceptible to oral inoculation of pv, which results in the gastrointestinal shedding of virus for several weeks, with paralysis occurring in only a small proportion of animals. consistent paralytic disease can be induced in bonnet monkeys (macaca radiata) through exposure to pv by infection into the right ulnar nerve (at the elbow), resulting in limb paralysis that resembles human paralytic poliomyelitis both clinically and pathologically. as such, bonnet monkeys can be used to study pv distribution and pathology and the induction of paralytic poliomyelitis or provocation paralysis. hepatitis a virus causes jaundice, which is a public health problem worldwide. the incubation period lasts from to days with an average of days. transmission between humans occurs by the oral-fecal route, person-to-person contact, or ingestion of contaminated food and water. hepatitis a virus causes an acute and self-limited infection of the liver with a spectrum of signs and symptoms ranging from subclinical disease, to jaundice, fulminant hepatitis, and in some cases death. , the disease can be divided into four clinical phases: ( ) incubation period, during which the patient is asymptomatic but virus replicates and possibly transmits to others. ( ) prodromal period, which might last from a few days to a week with patients generally experiencing anorexia, fever (< f), fatigue, malaise, myalgia, nausea, and vomiting. ( ) icteric phase, in which increased bilirubin causes characteristic dark brownish colored urine. this sign is followed by pale stool and yellowish discoloration of the mucous membranes, conjunctiva, sclera, and skin. most patients develop an enlarged liver, and approximately - % of the patients have splenomegaly. ( ) convalescent period, with resolution of the disease and recovery of the patient. rarely, during the icteric phase, extensive necrosis of the liver occurs. these patients show a sudden increase in body temperature, marked abdominal pain, vomiting, jaundice, and the development of hepatic encephalopathy associated with coma and seizures, all signs of fulminant hepatitis. death occurs in - % of patients with fulminant hepatitis. experiments showed that hepatitis a causes disease only in humans, chimpanzees, several species of south american marmosets, stump-tailed monkeys, and owl monkeys via the oral or intravenous (iv) routes. [ ] [ ] [ ] [ ] it is known that cynomolgus macaques are infected with hepatitis a virus in the wild. amado et al. used cynomolgus macaques (macaca fascicularis) for experimental hepatitis a infections. the animals did not exhibit clinical signs of disease, but viral shedding was observed in saliva and stool as early as h postinoculation (pi) and days pi, respectively. although mildto-moderate hepatic pathology was observed in all macaques, seroconversion and mildly increased alanine aminotransferase (alt), an enzyme associated with liver function, were observed in some of them. because this study had a very small group of animals (four macaques), the data should not be considered as conclusive, and more studies are needed to better define the cynomolgus macaque model. although hepatitis a virus is transmitted by the oralfecal route, studies in chimpanzees and tamarins showed that the iv route was much more infectious than oral route was. there was no correlation between dose and development of clinical disease for either species or experimental routes, and similar to cynomolgus macaques, none of these species showed clinical signs of disease. inoculation of common marmosets (callithrix jacchus) with hepatitis a virus did not produce clinical signs of disease as seen in other nhp models. , liver enzyme levels increased on day pi, and monkeys had measurable antihepatitis a antibodies by day pi. an experimental study with cell culture-adapted hepatitis avirus in guinea pigs challenged by oral or intraperitoneal routes did not result in clinical disease, increase in liver enzymes, or seroconversion. viral load was detected in stool and serum between days and and and days, respectively. liver pathology showed mild hepatitis. furthermore, histopathology indicated that virus replicated in extrahepatic tissues such as spleen, regional lymph nodes, and intestinal tract. in summary, none of the animal models for hepatitis a infection is suitable for studying pathogenesis of the virus because all clinical and most of the laboratory parameters remain within normal range or only slightly increased after the infection. one possibility is to test the safety of vaccines against hepatitis a virus in those models with demonstrable viral shedding. noroviruses, of which norwalk is the prototypic member, are responsible for up to % of reported food-borne gastroenteritis cases. in developing countries, this virus is responsible for approximately , deaths annually. a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants. , symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation normally is for - days, with symptoms enduring for - days. viral shedding is indicative of immunocompromised status within an individual with the elderly and young having a prolonged state of shedding. transmission occurs predominately through the oral-fecal route with contaminated food and water being the major vector. a major hindrance to basic research into this pathogen is the lack of a cell culture system. therefore, animal models are used not only to determine the efficacy of novel drugs and vaccines but also for understanding the pathogenesis of the virus. therapeutic intervention consists of rehydration therapy and antiemetic medication. no vaccine is available, and development of one is expected to be challenging given that immunity is short lived after infection. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus can be monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity are measured exclusively by assay of viral shedding. it was determined that more virus was needed to create an infection when challenging by the oral route than when challenging by the iv route. chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route. although none of the animals developed disease symptoms, viral shedding within the feces was observed within - days postinfection and lasted anywhere from days to weeks. viremia never occurred, and no histopathological changes were detected. the amount and duration of viral shedding were in line with what is observed upon human infection. a recently identified calicivirus of rhesus origin, named tulane virus, was used as a surrogate model of infection. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever days postinfection. viral shedding was achieved for days. the immune system produced antibodies that dropped in concentration within days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease. a murine norovirus has been identified and is closely related to human norwalk virus. however, clinically, the viruses present a different disease. the murine norovirus does not induce diarrhea nor vomiting and can develop a persistent infection in contrast to human disease. [ ] [ ] [ ] porcine enteric caliciviruses can induce diarrheal disease in young pigs and an asymptomatic infection in adults. gnotobiotic pigs can successfully be infected with a passaged clinical noroviruses isolate orally. diarrheal disease developed in % of the animals, and % were able to shed virus in their stool. no major histopathological changes or viral persistence was noted. calves are naturally infected with bovine noroviruses. experimentally challenging calves with an oral inoculation of a bovine isolate resulted in diarrheal disease [ ] [ ] [ ] the link between equine cases and the human disease was confirmed in by observing cases of fatal encephalitis in children living in the same area as the equine cases. during this outbreak, eeev was isolated from the cnss of these children as well as from pigeons and pheasants. eeev primarily affects areas near salty marshes and can cause localized outbreaks of disease in the summer. the enzootic cycles are maintained in moist environments such as coastal areas, shaded marshy salt swamps in north america (na), and moist forests in central america and south america (sa). birds are the primary reservoir, and the virus is transmitted via mosquitoes. furthermore, forest-dwelling rodents, bats, and marsupials frequently become infected and may provide an additional reservoir in central america and sa. despite known natural hosts, the transmission cycles in these animals are not well characterized. reptiles and amphibians have also been reported to become infected by eeev. eeev pathogenesis and disease have been studied in several laboratory animals. as a natural host, birds do not generally develop encephalitis except pheasants or emus, in which eeev causes encephalitis with - % mortality. young chickens show signs of extensive myocarditis in early experimental infection and heart failure rather than encephalitis is the cause of death. besides the heart, other organs such as pancreas and kidney show multifocal necrosis. additionally, lymphocytopenia has been observed in the thymus and spleen in birds. eeev causes neuronal damage in newborn mice, and the disease progresses rapidly, resulting in death. similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, whereas inoculation via the subcutaneous route causes a pantropic infection eventually resulting in encephalitis. , guinea pigs and hamsters have also been used as animal models for eeev studies. , guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed and resulted in brain lesions in these animals. subcutaneous inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis and death. in addition, parenchyma necroses were observed in the liver and lymphoid organs. intradermal, intramuscular, or iv inoculations of eeev in nhps cause disease but does not always result in symptoms of the nervous system. intracerebral infection of eeev results in nervous system disease and fatality in monkeys. the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in monkeys when they are infected by the peripheral route. intranasal and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but less drastic than those caused by intracerebral injections. the aerosol route of infection also progresses to uniformly lethal disease in cynomolgus macaques. in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed, and nhps became moribund and were euthanized between days and days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals. similar clinical signs and pathology were observed when common marmosets were infected with eeev by the intranasal route. both aerosol and intranasal nhp models had similar disease progression and pathology as those seen in human disease. a common marmoset model was used for comparison studies of sa and na strains of eeev. previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected intranasally with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized - days after the challenge. although sa strain-infected animals developed viremia, they remained healthy and survived the challenge. epizootics of viral encephalitis in horses were previously described in argentina. more than , horses died from western equine encephalitis virus (weev) in the central plains of the united states in . weev was first isolated from the brains of horses during the outbreak in the san joaquin valley of california in . although it was suspected, the first diagnosis of weev as a cause of human encephalitis occurred in , when the virus was recovered from the brain of a child with fatal encephalitis. in horses, the signs of disease are fever, loss of coordination, drowsiness, and anorexia, leading to prostration, coma, and death in about % of affected animals. weev also infects other species of birds and often causes fatal disease in sparrows. weev infection occurs throughout western na and sporadically in sa as it circulates between its mosquito vector and wild birds. chickens and other domestic birds, pheasants, rodents, rabbits, ungulates, tortoises, and snakes are natural reservoirs of weev. , weev has caused epidemics of encephalitis in humans, horses, and emus, but the fatality rate is lower than that for eeev. predominately young children and those older than years demonstrate the clinical symptoms of the disease. severe disease, seizures, fatal encephalitis, and significant sequelae are more likely to occur in infants and young children. , typically, the disease progresses asymptomatically with seroprevalence in humans being fairly common in endemic areas. species used to develop animal models for weev are mice, hamsters, guinea pigs, and ponies. studies with ponies resulted in viremia in % of the animals - days pi. fever was observed in of animals, and six exhibited signs of encephalitis. after subcutaneous inoculation with weev, suckling mice started to show signs of disease by h and died within h. in suckling mice, the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on days - postinfection. mice were severely ill by day and appeared hunched and dehydrated. death occurred between days and , and both brain and mesodermal tissues such as heart, lungs, liver, and kidney were involved. , intracerebral and intranasal routes of infection resulted in a fatal disease that was highly dependent on dose, while intradermal and subcutaneous inoculations caused only % fatality in mice regardless of the amount of virus. studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases. cns involvement was evident with intracerebral, intraperitoneal, and intradermal inoculations. weev is highly infectious to guinea pigs. intraperitoneal inoculation of weev is fatal in guinea pigs regardless of virus inoculum, with the animals exhibiting signs of illness on days - , followed by death on days - (nalca, unpublished results). very limited studies have been performed with nhps. the intranasal route of infection causes severe, lethal encephalitis in rhesus macaques. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model would be useful for testing of vaccines and therapeutics against weev. venezuelan equine encephalitis virus (veev) is maintained in nature in a cycle between small rodents and mosquitoes. the spread of epizootic strains of the virus to equines leads to high viremia followed by a lethal encephalitis, and tangential spread to humans. veev can easily be spread by the aerosol route making it a considerable danger for laboratory exposure. in humans, veev infection causes a sudden onset of malaise, fever, chills, headache, and sore throat. , , symptoms persist for - days, followed by a -to -week period of generalized weakness. encephalitis occurs in a small percentage of adults ( . %); however, the rate in children may be as high as %. neurologic symptoms range from nuchal rigidity, ataxia, and convulsions to the more severe cases exhibiting coma and paralysis. the overall mortality rate in humans is < %. laboratory animals such as mice, guinea pigs, and nhps exhibit different pathologic responses when infected with veev. the lymphatic system is a general target in all animals infected with cns involvement variable between different animal species. the disease caused by veev progresses very rapidly without showing signs of cns disease in guinea pigs and hamsters. mortality is typically observed within - days after infection and fatality is not dose dependent. veev infection lasts longer in mice, which develop signs of nervous system disease in - days and death - days later. lethal dose in mice changes depending on the age of mice and the route of exposure. in contrast to guinea pigs and hamsters, the time of the death in mice is dose dependent. mortality is observed generally within - days after infection and fatality is not dose dependent. subcutaneous/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans. virus begins to replicate in the draining lymph nodes at h pi. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis. , aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure do. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death. , , intranasal challenge of c h/hen mice with high dose veev caused high morbidity and mortality. viral titers in brain peaked on day postchallenge and stayed high until animals died on day - postchallenge. protein cytokine array done on brains of infected mice showed elevated interleukin (il)- a, il- b, il- , il- , monocyte chemoattractant protein- (mcp- ), ifng, mip- a, and regulated and normal t-cell expressed and secreted levels. this model was used successfully to test antivirals against veev. xi. viral disease veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at - h after infection and lasted < h. secondary fever generally began on day and lasted - days. veev-infected nhps exhibited mild symptoms such as anorexia, irritability, diarrhea, and tremors. leucopenia was common in animals exhibiting fever. supporting the leucopenia, observed microscopic changes in lymphatic tissues such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection. the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day postinfection during the secondary febrile episode. cynomolgus macaques develop similar clinical signs including fever, viremia, lymphopenia, and encephalitis upon aerosol exposure to veev. chikungunya virus is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics mainly within africa and southeast asia. the virus is transmitted by aedes mosquitoes. given the widespread endemicity of aedes mosquitoes, chikungunya virus has the potential to spread to previously unaffected areas. this is typified by the emergence of disease for the first time in in the islands of the southwest indian ocean, including the french la reunion island, and the appearance in central italy in . , the incubation period after a mosquito bite is - days followed by a self-limiting acute phase that lasts - days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported. individuals typically develop a stooped posture due to the pain. for approximately % of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions, including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of and years have an increased risk of developing neurological manifestations. there is no effective vaccine or antiviral. wild-type c bl/ adult mice are not permissive to chikungunya virus infection by intradermal inoculation. however, it was demonstrated that neonatal mice were susceptible, and severity was dependent upon age at infection. six-day-old mice developed paralysis by day , and all died by day , whereas % of nine-day-old mice were able to recover from infection. by days, mice were no longer permissive to disease. infected mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications. , an adult mouse model has been developed by injection of the ventral side of the footpad of c bl/ j mice. viremia lasted - days accompanied by foot swelling and noted inflammation of the musculoskeletal tissue. , adult ifna/br knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within days. this model was useful in identifying the viral cellular tropism for fibroblasts. imprinting control region (icr) cd mice can also be used as a disease model. neonatal mice subcutaneously inoculated with a passaged clinical isolate of chikungunya virus developed lethargy, loss of balance, and difficulty in walking. mortality was low, % and % for newborn cd and icr mice, respectively. the remaining mice fully recovered within weeks after infection. a drawback of both the ifna/ br and cd mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised. long-tailed macaques challenged with a clinical isolate of the virus developed a similar clinical disease to humans. initially, the monkeys developed high viremia with fever and rash. after this period, viremia resolved and virus could be detected in lymphoid, liver, meninges, joint, and muscle tissue. the last stage mimicked the chronic phase in which virus could be detected up to two months after infection, although no arthralgia was noted. dengue virus is transmitted via the mosquito vectors aedes aegypti and aedes albopictus. given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to dengue virus. this results in approximately million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia. it is estimated that there are , deaths each year caused by dengue hemorrhagic fever (dhf). there are four serotypes of dengue virus, numbered - , which are capable of causing a wide spectrum of disease that ranges from asymptomatic to severe with the development of dhf. incubation can range from to days, with the average being - days. the virus targets dendritic cells and macrophages after a mosquito bite. typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure. thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis. severe disease has a higher propensity to occur upon secondary infection with a different dengue virus serotype. this is hypothesized to occur due to antibodydependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in developing an animal model, it is important to note that mosquitoes typically deposit - pfu, and is therefore the optimal range to be used during challenge. a comprehensive review of the literature regarding animal models of dengue infection was recently published by zompi et al. several laboratory mouse strains including a/j, balb/c, and c bl/ are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis. [ ] [ ] [ ] a higher dose of an adapted dengue virus strain induced dhf symptoms in both balb/c and c bl/ . , this model can also yield asymptomatic infections. a mouse-adapted (ma) strain of dengue virus introduced into ag mice developed vascular leak syndrome similar to the severe disease seen in humans. passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased, and animals died due to vascular leak syndrome. another ma strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability. intracranial injection of suckling mice with dengue virus leads to death and has been used to test the efficacy of therapeutics. scid mice engrafted with human tumor cells develop paralysis upon infection, and are thus not useful for pathogenesis studies. , df symptoms developed after infection in nod/scid/il rgko mice engrafted with cd þ human progenitor cells. rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and laboratory-adapted strain of dengue virus . a passaged clinical isolate of dengue virus type was recently used to create a model in immunocompetent adult mice. interperitoneal injection in c bl/ j and balb/c caused lethality by day - postinfection in a dose-dependent manner. the first indication of infection was weight loss beginning on day followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, aspartate aminotransferase (ast) and alt, were also observed. viremia was established by day . this model mimicked the characteristic symptoms observed in human dhf/dss cases. a novel model was developed that used infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of dengue virus type . infected mosquitoes then fed upon the mouse footpad to allow for the transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the amount of mosquitoes it was exposed to, with four to five being optimal. detectable viral rna was in line with what is observed during human infection. severe thrombocytopenia developed on day . this model is intriguing given that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus. nhp models have used a subcutaneous inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection. the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes. throughout these preliminary studies, no clinical disease was detected. to circumvent this, a higher dose of dengue virus was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed. further development would allow this model to be used for testing of novel therapeutics and vaccines. although primates do not develop disease upon infection with dengue, their immune system does produce antibodies similar to those observed during the course of human infection. this has been advantageous in studying ade. sequential infection led to a crossreactive antibody response, which has been demonstrated in both humans and mice. this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was -to -fold higher than was previously reported; however, no clinical signs were apparent. the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. japanese encephalitis virus ( jev) is a leading cause of childhood viral encephalitis in southern and eastern asia and is a problem among military personnel and travelers to these regions. it was first isolated from the brain of a patient who died from encephalitis in japan in . culex mosquitoes, which breed in rice fields, transmit the virus from birds or mammals (mostly domestic pigs) to humans. the disease symptoms range from a mild febrile illness to acute meningomyeloencephalitis. after an asymptomatic incubation period of - weeks, patients show signs of fever, headache, stupor, and generalized motor seizures, especially in children. the virus causes encephalitis by invading and destroying the cortical neurons. the fatality rate ranges from % to %, and most survivors have neurological and psychiatric sequelae. , jev virus causes fatality in infant mice by all routes of inoculation. differences in pathogenesis and outcome are seen when the virus is given by intraperitoneal inoculation. these differences depend on the amount of virus and the specific viral strains used. the biphasic viral multiplication after peripheral inoculation is observed in mice tissues. primary virus replication occurs in the peripheral tissues and the secondary replication phase in the brain. hamsters are another small animal species that are used as an animal model for jev. fatality was observed in hamsters inoculated intracerebrally or intranasally, while peripheral inoculation caused asymptomatic viremia. studies with rabbits and guinea pigs showed that all routes of inoculation of jev produce asymptomatic infection. serial sampling studies with -day-old wistar rats inoculated intracerebrally with jev indicated that jev causes the overproduction of free radicals by neurons and apoptosis of neuronal cells. following a study in by the same group, showed that although cytokines tumor necrosis factor (tnf)-a, ifn-g, il- , il- , il- , and chemokine mcp- increased gradually and peaked on days pi with jevin rats, the levels eventually declined, and there was no correlation with the levels of cytokines and chemokines and neuronal damage. intracerebral inoculation of jev causes severe histopathological changes in brain hemispheres of rhesus monkeys. symptoms such as weakness, tremors, and convulsions began to appear on days - , with indicative signs of encephalomyelitis occurring on days - postinfection for most of the animals followed by death occurring on days - postinfection for most of the animals followed by death. although intranasal inoculation of jev results in fatality in both rhesus and cynomolgus monkeys, peripheral inoculation causes asymptomatic viremia in these species. , west nile virus west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early s , and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia. the virus first reached the western hemisphere in the summer of , during an outbreak involving humans, horses, and birds in the new york city metropolitan area. , since , the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from to days. the mortality rate after neuroinvasive disease ranges from % to %. , [ ] [ ] [ ] the most severe complications are commonly seen in the elderly, with reported case fatality rates from % to %. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. although many early laboratory studies of wn encephalitis were performed in nhps, mice, rat, hamster, horse, pig, dog, and cat models were used to study the disease. [ ] [ ] [ ] [ ] [ ] [ ] [ ] inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e. asymptomatic, fever, encephalitis). thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subcutaneous inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia. , wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and intraperitoneal routes, resulting in encephalitis and % mortality. intradermal route pathogenesis studies indicated that langerhans dentritic cells are the initial viral replication sites in the skin. , the infected langerhans cells then migrate to lymph nodes, and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases. [ ] [ ] [ ] [ ] tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared normal during the first days, became lethargic at approximately day , and developed neurologic symptoms by days - . many of the severely affected animals died - days after infection. viremia was detected in the hamsters within h after infection and persisted for - days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the above-mentioned monkey experiments by pogodina et al., persistent wnv infection was found in the brains of hamsters. the etiologic agent of severe acute respiratory syndrome (sars), sars-coronavirus (cov), emerged in as it spread throughout countries in a period of months, infecting > people and causing nearly deaths. , the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to days and can be detected in stool, suggesting other modes of transmission are also possible. although other members of the family usually cause mild illness, sars-cov infection has a % case fatality with the majority of cases in people over the age of years. , after an incubation period of - days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough. in some sars, cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within weeks and coincides with the induction of high levels of neutralizing antibodies. in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses. infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection. virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues. sars-cov can replicate in many species, including dogs, cats, pigs, mice, rats, ferrets, foxes, and nhps. chinese palm civets, raccoon dogs, and bats are possible natural hosts. no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (w %), viral replication, and pathology. in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models use mice, hamsters, ferrets, and nhps (table . ). mouse models of sars-cov typically are inoculated by the intranasal route under light anesthesia. young, -to -week old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinates, with a peak on day and clearance by day postexposure. there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis). intranasal sars-cov infection of c bl/ (b ) also yield reduced weight gain and viral replication in the lungs, with a peak on day and clearance by day . in contrast, balb/c mice - interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. s mice and b mice show outcomes to sars-covinfection similar to those observed for balb/c mice but have lower titers and less prolonged disease. one problem is that it is more difficult to obtain large numbers of mice older than year. a number of immunocompromised knockout mouse models of intranasal sars-cov infection have also been developed. svev mice infected with sars-cov by the intranasal route develop bronchiolitis, with peribronchiolar inflammatory infiltrates, and interstitial inflammation in adjacent alveolar septae. viral replication and disease in these mice resolve by day postexposure beige, cd À/À, and rag À/À mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs. signal transducer and activator of transcription- (stat ) ko mice infected intranasally with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths. the stat ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. syrian golden hamsters (strain lvg) are also susceptible to intranasal exposure of sars-cov. after the administration of tcid (tissue culture infective dose), along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis. there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. some mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred. damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge. clinical disease has been observed in several studies. sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day postexposure as well as mild alveolar damage in - % of the lungs, occasionally accompanied by severe pathology within the lungs. with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or intratracheal routes generally results in a very mild infection, which resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness, but others have rash, lethargy, and respiratory signs and pathology. rhesus have little to no disease and only have mild findings upon histopathological analysis. agms infected with sars-cov have no overt clinical signs, but dad and pneumonitis have been documented. viral replication has been detected for up to days in the lungs of agms; however, the infection resolves and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were thought to be involved in the sars-covoutbreak. intratracheal and intranasal inoculation of civets with sars-covresults in lethargy, decreased aggressiveness fever, diarrhea, and conjunctivitis. leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. common marmosets have also been shown to be susceptible to sars-cov infection. vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine have used live-attenuated, killed, dna and viral-vectored vaccine strategies. an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease. as such, immune mice had th -type immunopathology upon sars-cov challenge. severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported. additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance. mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. ma sars and human ace transgenic mice are available. all mammals experimentally or naturally exposed to rabies virus have been found to be susceptible. this highly neurotropic virus is a member of the lyssavirus genus and is transmitted from the bite of an infected animal to humans. the virus is able to replicate within the muscle cells at the site of the bite, and then travel to the cns. once reaching the cns by retrograde axonal transport, the virus replicates within neurons creating inflammation and necrosis. the virus subsequently spreads throughout the body via peripheral nerves. a typical incubation period is - days, and is highly dependent upon the location of the bite. proximity to the brain is a major factor for the onset of symptoms. the prodromal stage lasts from to days and is when the virus initially invades the cns. flu-like symptoms are the norm in conjunction with pain and inflammation at the site of the bite. subsequently, there are two forms of disease that can develop. in % of cases, an individual develops the encephalitic or furious form. this form is marked by hyperexcitability, autonomic dysfunction, and hydrophobia. the paralytic, or dumb form, is characterized by ascending paralysis. ultimately, both forms result in death days after the onset of symptoms. once the symptoms develop, there is no proven effective therapy. in the developing world, death is caused by the lack of access to medical care including postexposure prophylaxis. in na, fatal cases result because of late diagnosis. syrian hamsters have been challenged with rabies virus intracerebrally, intraperitoneally, intradermally, and intranasally. all animals died as a result of the exposure, although intracerebral and intranasal inoculation led to only the furious form depicted by extreme irritability, spasms, excessive salivation, and cries. the virus used had been isolated from an infected dog brain and passaged in swiss albino mice. animals inoculated by intracerebral injection develop disease within - days, whereas all other routes of entry develop disease within - days. this model has been used to study and test novel vaccine candidates. mice have been extensively studied as an animal model for rabies. it was shown that swiss albino mice intracerebrally injected with a virus isolated from a dog developed only the paralytic form of disease days after the initial challenge. balb/c mice are universally susceptible to intracerebral injection of rabies virus within days. disease symptoms include paralysis, cachexia, and bristling appearing - days before death. a more natural route of infection via peripheral injection into masseter muscles was tested on icr mice. these mice developed neurological signs including limb paralysis, and all died within - days. icr mice have been instrumental in analyzing novel vaccines and correlates of protection. this line of mice was also used to assess the value of ketamine treatment to induce coma during rabies infection. another mouse line used is the p neurotrophin receptor-deficient mouse. this mouse developed a fatal encephalitis when inoculated intracerebrally with the challenge virus standard. bax-deficient mice have also been used to determine the role of apoptotic cell death in the brain during the course of infection. a viral isolate from silver-haired bats can also be used in the mouse model. this strain is advantageous given that it is responsible for the majority of deaths in north na. early death phenomenon is typified by a decrease to time of death in a subset of individuals and animals that have been vaccinated and subsequently exposed to rabies. this trend has been demonstrated experimentally in swiss outbred mice and primates. , cynomolgus and rhesus were both infected with passaged rabies virus to create an nhp model. a high titer of virus was needed to induce disease, but exposure was found to not be universally fatal. the animals that survived beyond weeks within the experiment did not develop clinical disease nor succumb to infection. primates that did develop disease refused food and had progressively less activity until death. this lasted from h up to days, with all animals with symptoms dying within weeks. bats have been experimentally challenged with rabies. vampire bats, desmodus rotundus, intramuscularly injected with a bat viral isolate displayed clinical signs including paralysis in half of the population of the study animals. of those who did develop disease, the duration was days, and incubation period ranged from to days. regardless of disease manifestation, % of challenged animals died. skunks can be challenged intramuscularly or intranasally with either challenge virus strain or a skunk viral isolate. interestingly, the challenge virus strain more readily produced the paralytic form, whereas the street form of rabies developed into the furious form. however, the challenge strain virus resulted in a shorter incubation period of - days in comparison to - days seen with the street virus. filoviridae consists of two well-established genera, ebola virus and marburg virus (marv) and a newly discovered group, cuevavirus (table two other ebola viruses are known; taï forest (tafv; previously named cote (circumflex over the 'o') d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. the disease in humans is characterized by aberrant innate immunity and a number of clinical symptoms such as fever, nausea, vomiting, arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, anorexia, and numerous others. approximately % of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.). natural transmission in an epidemic is thought to be through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, intramuscular, intraperitoneal, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures. because filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type ifn response. however, wild-type inbred mice are susceptible to filovirus that has been ma by serial passage. balb/c mice, which are the strain of choice for intraperitoneal inoculation of ma-ebov, are not susceptible by the aerosol route. for aerosol infection of immunocompetent mice, a panel of bxd (balb/ c  dba) recombinant inbred strains were screened, and one strain, bxd , was shown to be particularly susceptible to airborne ma-ebov, with % lethality to low or high doses (w or pfu). these mice developed weight loss of > % and succumbed to infection between days and postexposure. the aerosol infection model uses a whole-body exposure chamber to expose mice aged - weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately . mm and a geometric standard deviation of approximately . for min. another approach uses immunodeficient mouse strains such as scid, stat ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by intraperitoneal or aerosol routes. mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection. liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps. although most mouse studies have used ma-ebov or ebov, an intraperitoneal ma marv model is also available. ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds. hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models. an intraperitoneal ma-ebov infection model has been developed in syrian hamsters. this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, uses male -to -week-old syrian hamsters that are infected with ld of ma-ebov. virus is present in tissues and blood collected on day , and all animals succumbed to the disease by day . detailed accounts of this model have been presented at international scientific meetings by ebihara and feldmann et al. but have not been reported in a scientific journal at the time of writing this chapter. guinea pig models of filovirus infection have been developed for intraperitoneal and aerosol routes using guinea pig-adapted ebov (gp-ebov) and marv (gp-marv). , guinea pigs models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent (hourly) intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. hartley guinea pigs exposed to aerosolized gp-marv or gp-ebov become moribund at times comparable to that of nhps, generally succumbing to the infection between and days postexposure. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses ( or pfu target doses) but lethality drops with low (< pfu) presented doses of airborne gp-marv, and more protracted disease is seen in some animals. weight loss of between % and % is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day , occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day postexposure. blood chemistries (i.e. alt, ast, alkaline phosphatase (alkp), and blood urea nitrogen) indicating problems with liver and kidney function are also altered late in the disease course. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans. old world primates have been primarily used for the development of intraperitoneal, intramuscular, and aerosol models of filovirus infection. uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, in agms and marmosets. [ ] [ ] [ ] [ ] [ ] low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. prominent features of the infections are onset of fever by day postexposure, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. clinical disease parameters may have a slightly delayed onset in aerosol models. petichial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species. dyspnea late in infection is a prominent feature of disease after aerosol exposure. a number of pronounced pathology findings include multifocal necrosis and fibrin lesions, particularly within the liver and the spleen. lymphocytolysis and lymphoid depletion are also observed. multilead, surgically implanted telemetry devices are useful in the continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection. the most recently developed telemetry devices can aid in plethysomography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate. outbreaks caused by nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra outbreaks have yet to be reported outside of australia. , hendra was the first member of the genus to be identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from human to human or bat to human. nipah has the distinction of being able to be transmitted by humans, although the exact route is unknown. the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized to be needed for successful transmission. both viruses have a tropism for the neurological and respiratory tract. hendra virus incubation period is - days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting. disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure. nipah virus has an incubation period of days to weeks. much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within - h. survivors of infection typically make a full recovery; however, % suffer permanent sequelae, including persistent convulsions. at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because observed human cases are all at terminal stages. the best small animal representative is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died xi. viral disease . in vivo models of viral diseases affecting humans within - days postinfection. the progression of disease and timeline are highly dependent on dose and route of infection. intranasal inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas intraperitoneal resulted in tremors and paralysis within h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, while the brain was the most affected organ. this model has been used for vaccination and passive protection studies. [ ] [ ] [ ] the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge. , inoculation with hendra virus via the subcutaneous route leads to a generalized vascular disease with % mortality. clinical signs were apparent - days postinfection with death occurring within days of cns involvement. higher inocula have been associated with the development of encephalitis lesions. intradermal and intranasal injections do not lead to disease, although the animals are able to seroconvert upon challenge. inoculum source does not affect clinical progression. nipah virus challenge only develops disease upon intraperitoneal injection and results in weight loss and transient fever for - days. virus was shed through urine and found to be present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder. ferrets display the same clinical disease as seen in the hamster model and human cases. , upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within - days including fever, coughing, and dyspnea. lesions do develop in the ferrets' brains, but to a lesser degree than seen in humans. cats have also been used as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is - days and leads to respiratory and neurological symptoms. , this model has proven to be useful in a vaccine challenge model. squirrel and agms are representative of the nhp models. within the squirrel monkeys, nipah virus is introduced by either the intranasal or iv route and subsequently leads to clinical signs similar to that in humans, although intranasal challenge results in milder disease. upon challenge, only % animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in the lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge. agms have been found to be a very consistent model of both viruses. intratracheal inoculation of the viruses results in % mortality, and death within . and - days postinfection for hendra and nipah, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis. , the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue and nipah virus can be located in urine. pigs have been investigated as a model as they develop a respiratory disease upon infection with both nipah and hendra. [ ] [ ] [ ] oral inoculation does not produce a clinical disease, but subcutaneous injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as days postinfection. nipah can also be transmitted between pigs. nipah was able to induce neurological symptoms in % of the pigs, even though virus was present in all neurological tissues regardless of symptoms. within the pig model, it seemed that nipah had a greater tropism for the respiratory tract, while hendra for the neurological system. horses also are able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within days. , animals died within days postexposure and were found to have interstitial pneumonia with necrosis of alveoli. , virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but found to be nonpermissive to infection. , suckling balb/c mice succumb to infection if the virus is inoculated intracranially. embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within - days postinfection. respiratory syncytial virus is responsible for lower respiratory tract infections of million children under the age of years, which in turn results in three million hospitalizations and approximately , deaths. within the united states, hospital costs alone amount to > million dollars. outbreaks are common in the winter. the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and further spreads to the lower respiratory tract. incubation for the virus is - days. respiratory syncytial virus is highly virulent leading to very few asymptomatic infections. disease manifestations are highly dependent upon the age of the individual. primary infections in neonates produce nonspecific symptoms including the overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting the virus at this age results in an increased chance of developing childhood asthma. young children develop recurrent wheezing, whereas adults exacerbate previous respiratory conditions. common clinical symptoms are runny nose, sneezing, and coughing accompanied with fever. mortality rates in hospitalized children are - % with the greatest burden of disease seen in - -month-olds. there is no vaccine available, and ribavirin usage is not recommended for routine treatment. animal models were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated respiratory syncytial virus vaccine (fi-rsv). this vaccineinduced severe respiratory illness in infants who received the vaccine and were subsequently infected with live virus. mice can be used to model disease, although a very high intranasal inoculation is needed to achieve clinical symptoms. , strain choice is crucial to reproducing a physiological relevant response. age does not affect primary disease manifestations. however, it does play a role in later sequelae showing increased airway hyperreactivity. primary infection produces increased breathing with airway obstruction. , virus was detected as early as day and reached maximum titer at day postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. cotton rats are useful given that it is a small animal disease model. the virus is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after intranasal inoculation. , it has been reported that viral replication is -to -fold greater in the rat model than in the mouse model. the rats develop mild-to-moderate bronchiolitis or pneumonia. although age does not seem to factor in clinical outcome, it has been reported that older rats tend to take longer to achieve viral clearance. viral loads peak by the fifth day, dropping to below the levels of detection by . the histopathology of the lungs seems to be similar to that in humans after infection. this model has limited usage in modeling the human immune response to infection as challenge with the virus creates a th response, whereas humans tend to skew toward th . [ ] [ ] [ ] fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally via intranasal inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is thought to be useful in studying mucosal immunity during infection. chimpanzees are permissive to replication and clinical symptoms of respiratory syncytial virus including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms nor high levels of viral replication. bonnet monkeys were also tested and found to develop an inflammatory response by day with viral rna detected in both bronchial and alveolar cells. the chimpanzee model has proven to be useful for vaccine studies. , sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine respiratory syncytial virus. lambs were also found to be susceptible to human respiratory syncytial infection. , when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within days. during the course of disease, viral replication peaked at days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features between them and humans. , the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; ha and na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within days. the rate of infection is estimated at % in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately , people in the united states die each year from seasonal influenza. thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing. influenza virus replicates in the upper and lower airways, peaking at approximately h postexposure. infection can be more severe in infants and in children under the age of years, people over the age of years, or immunocompromised individuals in whom viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis. pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyrogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza itself, accounting for approximately % of all influenza-associated fatalities. death, often due to ards can occur as early as days after the onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation. the h n avian strain of influenza has lethality rates of approximately % (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus, the potential for global spread is a major concern. the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. lethality rate can vary with the virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, superinfection and sepsis, severe with ards, and neurologic manifestations. also, models can use seasonal or avian strains and models have been developed to study transmission, important for understanding the potential for more lethal strains such as h n for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines. inoculation is by the intranasal route, using approximately ml of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with an mmad of < mm. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c bl/ j mice. males and females have equivalent disease, but influenza is generally more infectious in younger -to -week-old ( - g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. mice lack the mxa gene, which is an important part of the human innate immune response to influenza infection. the mouse homolog to mxa, mx , is defective in most inbred mouse strains. weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs. for more virulent strains, mice may require euthanasia as early as h postexposure, but most mortality occurs from to days postexposure accompanied by decreases in rectal temperature. pulse oximeter readings and measurement of blood gases of oxygen saturation are also used to determine the impact of influenza infection on respiratory function. virus can be isolated from bronchial lavage fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild-tomoderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha- -acidglycoprotein and lung weight are also frequently present. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa , predominates over sa , which is why h , h , and h subtypes usually need to be adapted to mice and h n , h , h , and h viruses do not require adaptation. to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. however, h n and the pandemic influenza virus can cause lethal infection without adaptation. h n infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia. as in humans, the virulence of h n is attributable to damage caused by an overactive host immune response. additionally, mice infected with the h n influenza produces severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia. in superinfection models, a sublethal dose of influenza is given to mice followed days later by intranasal inoculation of a sublethal dose of a bacterial strain such as s. pneumoniae or s. pyrogenes. morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to weeks. at least one transmission model has also been developed in mice. with h n influenza, transmission rates of up to % among cage mates can be achieved after infection by the aerosol route and cocaging after h. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans. [ ] [ ] [ ] ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa , and sa , ) within the airways similar to that of humans. influenza was first isolated from ferrets infected intranasally with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments. when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by intranasal. inoculation of . - . ml containing approximately - egg id dropwise to each nostril. influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts - days for mildly to moderately virulent strains. ferrets are more susceptible to influenza a than to influenza b strains and are also susceptible to avian influenza h n strains without adaptation. virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirror that seen in humans. nonadapted h n , h n , and h n have mild-to-moderate virulence in ferrets. strains of low virulence have predominant replication in the nasal turbinates. clinical signs and other disease indicators are similar to that of humans with a mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia. replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract ferrets correlates with severe disease and lethal outcome. h n -infected ferrets develop severe lethargy, greater ifn response, transient lymphopenia, and replication in respiratory tract, brain, and other organs. old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models, which are deficient for h n vaccine studies because there is a lack of correlation with hemagglutination inhibition. of old world primates, cynomolgus macaque (m. fascicularis) are most frequently used for studies of vaccines and antiviral drug therapies. , h n and h n infections of cynos are very similar to those in humans. cynos develop fever and ards upon intranasal inoculation of h n with necrotizing bronchial interstitial pneumonia nhps are challenged by multiple routes (ocular, nasal, and tracheal) simultaneously  pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h n results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the h n caused a very high mortality rate due to an aberrant immune response and ards and had > % lethality (humans only had a - % lethality). ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains such as h n . influenzainfected rhesus macaques represent a mild disease model pathogenesis for vaccine and therapeutic efficacy studies. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates such as squirrel and cebus monkeys. rats (f and sd) inoculated with rat-adapted h n developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes. additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets. cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies. , cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted. , nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at h postexposure. virus was cleared from the lung by day and from the nares by day , but animals had bronchial and alveolar damage, and pneumonia for up to weeks. there is also a s. aureus superinfection model in cotton rats. coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic pig influenza models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h n . , although pigs infected with influenza may have fever, anorexia, and respiratory signs such as dyspnea and cough, mortality is rare. size and space requirements make this animal difficult to work with, although the development of minipig (ellegaard gottingen) models may provide an easier-to-use alternative. incubation period, animals exhibit signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers. mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans. , after - days of incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever. [ ] [ ] [ ] depending on the severity of the disease when the symptoms start, - % of the hospitalized patients might die in - days or - days after the disease onset. hepatic failure, renal failure or disseminated intravascular coagulation (dic), and encephalitis are demonstrated within patients during postmortem examination. mice are one of the most susceptible animal species to rvfv infection. subcutaneous or intraperitoneal routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice. , mice start to exhibit signs of decreased activity and ruffled fur by day - postexposure. immediately after these signs are observed, they become lethargic and generally die - days postexposure. ocular diseases or hemorrhagic form of the disease has not been observed in mice models so far. increased viremia and tissue tropism were reported in mice with increased liver enzymes and lymphopenia observed in sick mice. rats and gerbils are also susceptible to rvfv infection. rats' susceptibility is dependent on the rat strain used for the challenge model. there was also noted an age dependence in susceptibility of rats. although wistar-furth and brown norway strains and young rats are highly susceptible to rvfv infection, fisher , buffalo and lewis strains, and old rats demonstrated resistance to infection. , similar pathologic changes such as liver damage and encephalopathy were observed in both rats and mice. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils. similar to the rat model, the susceptibility of gerbils was also dependent on age. so far, studies showed that rvfv does not cause uniform lethality in an nhp model. intraperitoneal, intranasal, iv, and aerosol routes have been used to develop the nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort. monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to those seen in humans such as pathological changes in liver and hemorrhagic disease. there was no ocular involvement in this model. recently, smith et al. compared iv, intranasal, and subcutaneous routes of infection in common marmosets and rhesus macaques. marmosets were more susceptible to rvfv infection than were rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subcutaneous routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of intranasally challenged marmosets. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, seems to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans. incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts - days. during the prehemorrhagic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients. , over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. [ ] [ ] [ ] [ ] until recently, the only animal that manifests disease is the newborn mouse. infant mice infected with cchfv intraperitoneally caused fatality around day postinfection. pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day ) and the brain (day ). the recent studies using knockout adult mice were successful to develop a lethal small animal model for cchfv infection. , bente et al. infected stat knockout mice by the intraperitoneal route. in this model, after the signs of fever, leucopenia, thrombocytopenia, viremia, elevated liver enzymes, and proinflammatory cytokines, mice were moribund and succumbed to disease in - days of postexposure. the second model was developed by using ifn-alpha/beta (ifna/b) receptor knockout mice. similar observations were made in this model as in the stat knockout mouse model. the animals were moribund and died - days after exposure with high viremia levels in the liver and spleen. other laboratory animals, including nhps, show little or no signs of infection or disease when infected with cchfv. butenko et al. used agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day postinfection, the animals did not exhibit signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. in , fagbami et al. infected two patas monkeys (cercopithecus erythrocebuserythrocebus) and one guinea baboon (papio papio) with cchfv. although all three animals had low level viremia between days and after inoculation, only the baboon serum had neutralizing antibody activity on day postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia, and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes. shepherd et al. infected species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. although scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (tatera brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and m. coucha), and syrian hamsters were negative. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed the onset of viremia earlier than those infected by the subcutaneous or intraperitoneal routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather by rodents. rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by the inhalation of infected rodent saliva, feces, and urine. human infections can normally be traced to a rural setting with activities such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention. the viruses have a tropism for endothelial cells within the microvasculature of the lungs. there are two distinct clinical diseases that infection can yield; hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses. hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul. incubation lasts two to three weeks and presents as flulike in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops, which can further progress to shock in approximately % patients. the overall mortality rate is %. infection with dobrava and hantaan viruses are typically linked to the development of severe disease. hps was first diagnosed in within the southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus. this virus is still the leading cause within na of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay. , the first report of hps in maine was recently documented. andes virus was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans. the fulminant disease is more lethal than that observed for hfrs with a mortality rate of %. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation. incubation typically occurs - days after exposure. unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted. vaccine development has been hampered by the vast diversity of hantaviruses and the limited number of outbreaks. syrian golden hamsters are the most widely used small animal models for hantavirus infection. hamsters inoculated intramuscularly with a passaged andes viral strain died within days postinfection. clinical signs did not appear until h before death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged sin nombre isolate. no hamsters developed any symptoms during the course of observation. although an antibody response to the virus that was not dose dependent was determined via an enzymelinked immunosorbent assay. hamsters infected with andes virus were found to have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day and lasted up to days postinfection. infection of hamsters with andes virus yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver. lethal disease can be induced in newborn mice but does not recapitulate the clinical symptoms observed in human disease. adult mice exposed to hantaan virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease. , knockout mice lacing ifn-a/b were found to be highly susceptible to hantaan virus infection. in a study looking at a panel of laboratory strains of mice, c bl/ mice were found to be most susceptible to a passaged hantaan viral strain injected intraperitoneally. animals progressed to neurological manifestation including paralyses and convulsions and succumbed to infection within - h postinfection. clinical disease was markedly different than that observed in human cases. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported. , cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease. , challenge with andes virus to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within days postinfection. aerosol exposure led to of monkeys and of iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all these viruses share common clinical manifestations. lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april. this virus is responsible for , - , infections per year, leading to approximately , deaths. outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases sprung up in germany, the netherlands, the united kingdom, and the united states due to transmission to travelers on commercial airlines. transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex. humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease can occur in % of individuals. the incubation period is from to days, and the initial onset is characterized by flulike illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease, which is permanent for a third of those affected. the overall fatality is about %; however, of those admitted to the hospital, it is between % and %. there is no approved vaccine, and besides supportive measures, ribavirin is effective only if started within days. , the primary animal model used to study lassa fever is the rhesus macaque. aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus more closely mirrored the disease course and histopathology observed in human infection. iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt. disease was dose-dependent with iv, intramuscular, and subcutaneous inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be used, and mimic a more natural route of infection. within this model, the nhp becomes viremic after - days. clinical manifestations were present by day , and death typically occurred within - days. , intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns. this pathogenicity is seen in select cases of human lassa fever. , a marmoset model has recently been defined using a subcutaneous injection of lassa fever virus. virus was initially detected by day and viremia achieved by day . liver enzymes were elevated, and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases. mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is completely dependent upon the major histocompatibility complex (mhc) background and age of animal along with the route of inoculation. guinea pig inbred strain was found to be highly susceptible to lassa virus infection. the outbreed hartley strain was less susceptible, and thus, strain has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus. infection with pichinde virus that has been passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death. , the guinea pig is an excellent model given that it not only results in similar disease pattern as humans but also the viral distribution is similar along with the histopathology and immune response. , infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig model. the virus was injected intraperitoneally resulting in the animals becoming lethargic and anorexic within - days. virus was first detected at days and reached maximum titers within days. neurological symptoms began to appear at the same time, and all the animals died by day . pneumonitis, pulmonary hemorrhage, and edema were also present. these results were recapitulated with a nonadapted pichinde virus. [ ] [ ] [ ] globally, diarrheal disease is the leading cause of death with rotavirus being one of the main etiological agents responsible. according to the world health organization, rotavirus alone is responsible for a third of all hospitalization related to diarrhea and , - , deaths per year. the virus is very stable due to its three-layer capsid, which allows it to be transmitted via the oral-fecal route, depositing itself in the small intestine. rotavirus is highly contagious, and only viruses are needed to cause symptomatic disease. the host determinant with the greatest influence on clinical outcome is age. neonates typically are asymptomatic, which is suggested to be due to the existence of maternal antibodies. hence, the most susceptible age group is months to years, coinciding with a drop in these protective antibodies. within this age range, children will develop noninflammatory diarrhea. virus replicates in the intestinal villus enterocytes resulting in their destruction and malabsorption of needed electrolytes and nutrients. symptoms of disease include watery, nonbloody diarrhea with vomiting, fever, and potentially dehydration that lasts up to a week. there is a short episode of viremia during the course of infection. mice can be used as both an infection and disease model depending upon age at challenge. mice < days old develop disease, whereas older mice are able to clear the infection before the onset of symptoms. this halts the study of active vaccination against disease in the infection model. in the adult mouse model, the course of the infection is monitored via viral shedding within the stool. infant mice, specifically balb/c, receiving an oral inoculation of a clinical strain of virus developed diarrhea within h postinfection, and % of those exposed developed symptoms within h postinfection. symptoms lasted from to days with no mortality. viral shedding was at its peak at h and lasted up to days. there were noted histopathological changes within the small intestine localized to the villi that was reversible. within the adult mouse model, oral inoculation of a mouse rotavirus strain showed viral shedding by days lasting up until days postinfection. these mouse models have been used to study correlates of protection and therapeutic efficacy including gastro-gard Ò . , , rats can also be used as disease models depending upon the strain of rat. , suckling fischer rats were exposed to a simian strain of rotavirus orally. the rats were susceptible to diarrheal disease till they were days old with age determining the length of viral shedding. rats have mainly been used to study the correct formulation for oral rehydration. these rodents are large enough to perform in situ intestinal perfusions. within these studies, -day-old rats were infected with a rat strain of rotavirus by orogastric intubation. within h postinfection, the rats developed diarrhea, at which point the small intestine was perfused to compare differing solutions of oral rehydration. gnotobiotic pigs are also used given that they can be infected with both porcine and human strains. they are susceptible to developing clinical disease from human strains up to weeks of age. they allow for the analysis of the primary immune response to the virus given that they do not receive transplacental maternal antibodies and are immune competent at birth. another advantage of this model is that the gastrointestinal physiology and mucosal immune system closely resemble that of humans. this model has been useful in studying correlates of protection. gnotobiotic and colostrum-deprived calves have also been used as an experimental model of rotavirus infection. they are able to develop diarrhea and shed live virus. gnotobiotic lambs can also develop clinical disease upon oral inoculation with clinical strains. infant baboons, agms, and rhesus macaques have all proven to be infection models with severity measure by viral shedding. , retroviridae human immunodeficiency virus type the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects . % of the world's population. a greater proportion of infections and deaths occur in sub-saharan africa. worldwide, there are approximately . million deaths per year with > , being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv- and hiv- , with hiv- having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv- . hiv targets t-helper cells (cd þ), macrophages, and dendritic cells. acute infection occurs - weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd þ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd þ t-cells decline to < cells per microliter; thus, cell-mediated immunity becomes impaired, and the person is more susceptible to opportunistic infections and certain cancers. humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of intact and functional human innate and adaptive immune responses. the scidhu hiv infection model has proven to be useful, particularly in screening antivirals and therapeutics. a number of different humanized mouse models have been developed for the study of hiv, including rag À/ÀgcÀ/À, rag À/ ÀgcÀ/À, nod/scidgcÀ/À (hnog), nod/ scidgcÀ/À (hnsg), nod/scid blt, and nod/ scidgcÀ/À (hnsg) blt. cd þ human stem cells derived from the umbilical cord blood or fetal liver are used for humanization. hiv- infection by intraperitoneal injection can be successful with as little as % peripheral blood engraftment. vaginal and rectal transmission models have been developed in blt scidhu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv- viremia occurs within approximately days pi. in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans. importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hiv-infected humanized mice, and at least some mechanisms of pathogenesis that occur in hiv-infected humans also occur in the hiv-infected humanized mouse models. the advantage of these models is that these mice are susceptible to hiv infection, and thus, the impact of drugs on the intended viral targets can be tested. one caveat is that although mice have a "common mucosal immune system," humans do not, due to the differences in the distribution of addressins. thus, murine mucosal immune responses to hiv do not reflect those of humans. there are a number of important nhp models for human hiv infection. simian immunodeficiency virus (siv) infection of macaques is widely considered to be the best platform for modeling hiv infection of humans. importantly, nhps have similar, pharmacokinetics, metabolism, mucosal t-cell homing receptors, and vascular addressins to those of humans. thus, although the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through the use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero, or through breast milk. [ ] [ ] [ ] there are also macaque models to study the emergence and clinical implications of hiv drug resistance. these models most routinely use rhesus macaques (macaca mulatta), cynomolgus macaques (macaca fasicularis), and pigtailed macaque (macaca nemestrina). animals of all ages are used, depending on the needs of the study. for instance, the use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently used. studies are performed in bsl- animal laboratories, and nhps must be of simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied. challenges may be through a single high dose. iv infection of rhesus macaques with tcid of the highly pathogenic siv/deltab induces aids in most macaques within - months (mean of months). peak viremia occurs around week . aids in such models is often defined as cd þ t-cells that have dropped to < % of the baseline values. alternatively, repeated low-dose challenges are often used, depending on the requirements of the model. , because nhps infected with hiv do not develop an infection with a clinical disease course similar to that in humans, siv or siv/hiv- laboratory-engineered chimeric viruses (simian-human immunodeficiency virus or shiv) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease, which progresses to aids and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv- and is more closely related to hiv- . however, the polymerase region of siv is % homologous to that of hiv- , and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors; thus, hiv- rt is usually put into siv for such studies. sivmac is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt or integrase inhibition. nhps infected with sivmac have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd þ t-cell depletion. additionally, sivmac uses the cxcr chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry. nhps infected with shiv strains may not develop aids, but these models are useful in testing vaccine efficacy. for example, rt-shivs and env-shivs are useful for the testing and evaluation of drugs that may target the envelope or rt, respectively. one disadvantage of the highly virulent env-shiv (shiv- . p) is that it uses the cxcr coreceptor. of note, env-shivs that do use the cxcr coreceptor are less virulent; viremia develops and then resolves without further disease progression. simian-tropic (st) hiv- contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for three months, followed by clearance. a number of routes are used for siv or shiv infection of nhps, with iv inoculation being the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than does the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) one month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase the susceptibility to infection by atraumatic vaginal instillation. upon vaginal instillation of tcid of shiv- p , peak viremia was seen around days postexposure with > copies per milliliter and dropping thereafter to a constant level of rna copies per milliliter at days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac . an example of an intrarectal model used juvenile ( - year-old) pigtailed macaques, challenged intrarectally with tcid s of siv mne to study the pathogenesis related to the virulence factor, vpx. here, viremia peaked at approximately days with > copies per milliliter. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistently high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of highly active antiretroviral therapy on virus and t-cells in the male genital tract, adult ( -to -year old) male cynomolgus macaques were intravenously inoculated with aid s of sivmac , and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization. pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course. importantly, mother-to-infant transmission models have also been developed. pregnant female pigtailed macaques were infected during the second trimester with mid shiv-sf p by the iv route. four of nine infants were infected; one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission and the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and terminal-repeat retrotransposon in miniature (trim) alleles), differences between challenge strains, and challenge routes. nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the longterm clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade. unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study that used an adenovirus-based vaccine approach. vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv is more difficult to protect than mucosal and is used as a "worst-case scenario." however, efficacy at one mucosal route is usually comparable to that at other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect. there are > human papillomaviruses (hpvs), with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately a third of these are transmitted sexually. of these, virulent subtypes such as hpv- , hpv- , hpv- , hpv- , and hpv- place individuals at high risk for cervical and other cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist that use animal papillomaviruses in their natural host, or a very closely related species. , these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which are a very closely related species. in this model, papillomas can range from cutaneous squamous cell carcinomas on the one end of the spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) cause florid warty lesions in the mucosa of the oral cavity within - weeks postexposure in experimental settings. the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv- and hpv- make this a useful model. these lesions typically spontaneously regress - weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged weeks to years, with no history of copv, are typically used for these studies. infection is achieved by the application of a ml droplet of virus extract to multiple . cm scarified areas within the mucosa of the upper lip of anesthetized beagles. bovine papillomavirus (bpv) has a wider host range than do most papillomaviruses, infecting the fibroblasts cells of numerous ungulates. bpv- infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques, rhesus papillomavirus, is very similar to hpv- and is associated with the development of cervical cancer. mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, but they are often used to look at immunogenicity of vaccines. , herpesviridae please see chapter . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including squirrels. , mpxv was discovered during the pox-like disease outbreak among laboratory java macaques in denmark in . no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until in zaire (the present drc) with the continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of , a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and the western hemisphere. among reported cases, human cases were laboratory confirmed during an outbreak. , it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-toperson spread occurs by large respiratory droplets or direct contact. most of the clinical features of human monkeypox are very similar to those of ordinary smallpox. after a -to -day incubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash. , typical maculopapular rash follows the prodromal period generally lasting - days. the average size of the skin lesions is . - cm, and the progress of lesions follows the order macules through papules, vesicles, pustules, umblication then scab and desquamation and lasts typically - weeks. fatality rate is % among the unvaccinated population and death generally occurs during the second week of the disease. , mpxv is highly pathogenic for a variety of laboratory animals, and so far, many animal models have been developed by using different species and different routes of exposure (table . ). because of the unavailability of variola virus to develop animal models and resulting disease manifestations in humans that are similar, mpxv is one of the pox viruses that are used very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat -deficient c bl/ mouse, prairie dogs, african dormice, ground squirrels are highly susceptible to mpxv by different exposure routes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cast/eij mice, one of the inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose-dependent mortality after intranasal exposure to mpxv. studies with the intraperitoneal route of challenge indicated a higher susceptibility to mpxv with an almost -fold less ld when compared to the intranasal route. scid-balb/c mice were also susceptible to the intraperitoneal challenge route, and the disease resulted in mortality day postinfection. similarly c bl/ stat À/À mice were infected intranasally with mpxv, and the infection resulted in weight loss and mortality days postexposure. mice models mentioned here are very promising for screening of therapeutics against pox viruses, but testing in additional models will be required for advanced development. high doses of mpxv by intraperitoneal or intranasal route caused % mortality in days postexposure and days postexposure, respectively, in ground squirrels. the disease progressed very quickly, and most of the animals were lethargic and moribund by day postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv in ground squirrels by subcutaneous route resulted in systemic disease and the mortality in - days postexposure. the disease resembles hemorrhagic smallpox with nose bleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. because in the us outbreak the virus was transmitted by infected prairie dogs, this animal model has recently been studied much further and used to test therapeutics and vaccines compared to other small animal models. , , , studies using intranasal, intraperitoneal, and intradermal routes of exposure showed that mpxv was highly infectious to prairie dogs. by using the west african mpxv strain, the intraperitoneal route caused a more severe disease and % mortality than challenge by the intranasal route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to the intraperitoneal route, the intranasal route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in intraperitoneally infected animals. recent studies by hutson et al. used intranasal and intradermal infections with west african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and generalized pox lesions. therefore, this model can be used for testing therapeutics and vaccines against pox viruses. the african dormouse is susceptible to mpxv by the foodpad injection route or intranasal route. mice exhibited decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of and pfu provided % mortality with a mean time to death of days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and hemorrhage in lungs were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. considering the limited availability of ground squirrels and african dormice, lack of reagents to these species, and resemblance to hemorrhagic smallpox disease, these models are not very attractive for further characterization and vaccine and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal models for mpxv. , , , , during our studies by using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day postexposure. complete blood count and clinical chemistries showed abnormalities similar to those of human monkeypox cases with leukocytosis and thrombocytopenia. whole-blood and throat swabs had viral loads peak around day , and in survivors, gradually decrease until day postexposure. because doses of  ,  , or  pfu resulted in lethality for % of the animals, whereas a dose of  pfu resulted in % lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, before animals can develop pox lesions, they succumbed to disease. with the low challenge dose, pox lesions were observed, but they were few in comparison to the iv model. mpxv causes dose-dependent disease in nhps when given by the iv route. studies showed that with  pfu iv, challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an intratracheal infection model deposits virus into the trachea, delivering directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation by skipping the upper respiratory system. fibrinonecrotic bronchopneumonia was described in animals that received pfu of mpxv intratracheally. although a similar challenge dose of intratracheal mpxv infection resulted in a similar viremia in nhps than with the aerosol route of infection, the timing of the first peak was delayed by days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately -fold lower. this suggests that local replication is more prominent after aerosol delivery compared to that after intratracheal delivery. an intrabronchial route of exposure resulted in pneumonia in nhps. delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and the intratracheal route of infection models, the number of pox lesions was much less than in the iv route of the infection model. a major downside of the iv, intratracheal and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus into the blood stream or into the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human variola virus (varv) infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is decided to be used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b is one of the most common infections worldwide with > million people chronically infected and , cases per year of liver cancer due to infection. the virus can naturally infect both humans and chimpanzees. hepatitis b is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture. the age at which one is infected dictates the risk of developing chronic disease. acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms last for a few weeks before resolving. after this acute phase, life time immunity is achieved. of those infected, < % will develop the chronic form of disease. chronicity is the most serious outcome of disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is times more likely to develop in a chronically infected individual than in a noncarrier. the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepadna virus suggest that x protein may be responsible. many individuals are asymptomatic until complications emerge related to chronic carriage. chimpanzees have a unique strain that circulates within the population. , it was found that - % of all wild-caught animals from africa are positive for hepatitis b antigen. natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease. to date, the use of chimpanzees provides the only reliable method to ensure that plasma vaccines are free from infectious particles. this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially attuned to these studies given that their immune response to infection directly mirrors humans. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hepatitis b, none develop hepatic lesions or liver damage as noted by monitoring of liver enzymes. mice are not permissible to infection, and thus, numerous transgenic and humanized lines that express hepatitis b proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease. recently, the entire genome of hepatitis b was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection. hepatitis b can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses. the woodchuck hepatitis virus was found to induce hepatocellular carcinoma. within a population, - % of all neonatal woodchucks are susceptible to chronic infection. a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within years of the initial infection in woodchucks, whereas human infection takes much longer. the acute infection strongly resembles what occurs during the course of disease in humans. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage. challenge with virus in neonates leads to a chronic infection, while adults only develop the acute phase of disease. a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. it was found to develop an acute hepatitis with a productive infection. hepatitis d is dependent upon hepatitis b to undergo replication and successful infection in its human host. there are two modes of infection possible between the viruses: coinfection in which a person is simultaneously infected or superinfection in which a chronic carrier of hepatitis b is subsequently infected with hepatitis d. coinfection leads to a similar disease as seen with hepatitis b alone; however, superinfection can result in chronic hepatitis d infection and severe liver damage. both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d. a recently published report demonstrated the use of a humanized chimeric mouse to study the interactions between the two viruses and drug testing. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus be a low passage clinical isolate; thus, animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that which occurs in natural disease. to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the above characteristics cannot always be satisfied, however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for 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preclinical drug evaluation key: cord- -ckg mf t authors: b, liu; qf, han; wp, liang; xy, shi; jj, wei title: decrease of respiratory diseases in one social children welfare institute in shanxi province during covid- date: - - journal: j public health (oxf) doi: . /pubmed/fdaa sha: doc_id: cord_uid: ckg mf t background: to assess the impact of disinfection measures on the incidence of common diseases in children welfare institute during the epidemic of corona virus disease (covid- ), and provide a basis for the daily disinfection management of children welfare institute. methods: this study surveyed and analyzed common diseases among children under the age of in one social children welfare institute in shanxi province from january to may in – by the year-on-year method. results: the prevalence rate of respiratory diseases in was a significantly negative growth compared with and . there was no obvious pattern of changes in digestive diseases group. conclusion: in view of the above anti-epidemic measures, it indicates that the children gathering institutions should strengthen effective personal protection and public health management to reduce infectious disease among children. in the middle of december , the corona virus disease as the third zoonotic human coronavirus (co v) of the century was reported from wuhan, hubei, china, and spread rapidly worldwide within a short time, which was declared finally as the controllable pandemic disease by world health organization. , in january , the national health commission of the people's republic of china classified o cially covid- as the class b infectious diseases according to 'the law of the people's republic of china on the prevention and control of infectious diseases,' and took powerful measures to prevent and control as class a infectious diseases. notwithstanding epidemic situation is relatively mild in shanxi, the local government initiated firstlevel response to major public health emergencies at the first time, and formulated a series of measures including quarantine home, closing the educational institutions and entertainment venues to contain the contagion of the epidemic and protect public health. children in the welfare institute as one of the social vulnerable groups is the key object to protect, however, no detailed study on them facing the epidemic has been conducted to date. we selected an o cial and comprehensive social children welfare institute in shanxi province as subject to complete this survey. up to now, nearly people have been adopted including more than minors and less than adults. besides, there are about permanent employees and more than hired employees. in order to resist the epidemic of covid- outbreak, this welfare institute actively responded to government exhortation and carefully planned many urgent measures according to 'guidelines on the prevention and control of pneumonia epidemic in new corona virus infections in service organizations of children's welfare (first edition)' released by the o ce of the ministry of civil a airs, for examples, disinfection and sterilization, isolation and age-based partitioned management, as well as monitoring body temperature. so far, there has been no case of infection in this welfare institute. at the same time, we find that the prevalence rate of respiratory diseases for children significantly reduced compared with the same period of previous years. this will highly remind us that whether we can strengthen the daily prevention and control for diseases to reduce the incidence of respiratory diseases for children. this study collected the number of patients under the age of in one social children welfare institute in shanxi province during the epidemic of covid- and compared with the same period of and by the year-on-year method. all data of this study have been reviewed and approved by the ethics review committee of shanxi medical university. the patients were divided into respiratory diseases group, digestive diseases group and other groups. respiratory diseases group includes upper respiratory tract infections, colds, pneumonia, flu, etc. digestive system diseases group includes diarrhea, gastroenteritis, etc. other groups include congenital diseases, surgical diseases, etc. all groups include the number of inpatients and outpatients. we mainly analyzed the number of patients by investigating the original records from the a liated hospital of this welfare institute. table and figure show that the number of patients will show a peak in january and gradually decrease from february to may each year. in from march to may, the number of patients with respiratory diseases decreased significantly. the year-on-year growth rate of respiratory diseases group in was lower than that in , which was a significantly negative growth, and there was no obvious pattern of changes in digestive diseases group. according to the data analysis of the same time period from to , the anti-epidemic measures lead to that the prevalence rate of respiratory diseases reduced significantly among children of the social children welfare institute in compared with and . however, the prevalence rate of digestive diseases did not change. respiratory diseases are the most common diseases among children, especially those children living in groups who are more likely to su er from epidemic infective diseases. the decrease in the incidence of respiratory diseases suggests that the level of disease prevention and control in this social children welfare institute has increased significantly. compared with other institutions, the children welfare institute has the following features: (i) the children living in the welfare institute, a relatively closed environment, belong to a fixed population. most children will live in welfare institutions for a long time. (ii) the impact on children's sports here during the epidemic period is negligible because most of these children su er from basic diseases or disabilities which make them carry out fewer outdoor activities than ordinary children. during the epidemic of covid- , the diet and the sport areas of the children has not changed. (iii) the welfare institute is equipped with an a liated hospital for sick children. all diseases will be diagnosed and treated by the a liated hospital. (iv) during the epidemic of covid- , the welfare institute has taken some measures such as personal protective measures, public environmental measures, reducing social visits and age-based partitioned management. compared with adults, children are more likely to su er from respiratory or digestive diseases because of their weaker immunity, especially children in the welfare institutes. although these two types of infectious diseases are selflimiting, they can spread rapidly in semi-closed environments such as schools and the welfare institutes, which can a ect seriously the health of children. based on the survey analysis, we can clearly conclude that the actions taken during the epidemic of covid- have strikingly resulted in the prevalence rate transform of respiratory diseases for the children, which suggests that the disinfection and management in welfare institutions administration should be strengthened daily. the following are the experiences during the epidemic of covid- . face masks on the one hand, the use of masks can protect us from transmission by preventing the inhalation of respiratory pathogens and reducing the hand-to-face contact, on the other hand, mask-wearing when sick may reduce the transmission of influenza virus to protect others. , the caregivers and medical sta s should wear the mask to protect themselves and reduce the possibility of diseases transmission by a mobile person. it is favorable for the children with a poor physical condition. hand hygiene some studies have shown that contaminated hands are the main mode of transmission of infectious diseases. hand hygiene is important for reducing the transmission of some gastrointestinal diseases and respiratory diseases. , simplified three-step hand-washing in s can e ectively remove the hand bacteria and meet the requirements of sanitary hand disinfection, considering children have limited attention. children can use soap and water, or some waterless hand disinfectants to clean their hands before meals, after using the washroom and after playing outside. some studies have proved that exposures of alcohol hand sanitizer may lead to harmful health e ects including ocular irritation, vomiting and so on, by swallowing it deliberately or unintentionally. therefore, alcohol hand sanitizer should be used under adult supervision, and make sure it is placed out of reach for children in daily life. cough etiquette children should be taught cough etiquette like covering the nose and mouth with a tissue or a mask when coughing or sneezing to prevent the spread of bacteria, if tissues are unavailable, using the upper sleeves or elbow, not hands, is also useful. , , , therefore, we recommend highly that managers should take cough etiquette information as a part of regular science and health courses in order to make children understand the healthy benefits of cough etiquette. at the moment, we suggest encourage and monitor children to develop favorable habits by singing motivational songs, posting pictorial flipcharts and painting cue cards in daily life. surface disinfection baths, washbowls, toilets, seats, handles and such 'high-touch' surfaces are highly recommended to use disinfectant or % medical alcohol to wipe. when using disinfectant, the sta must realize that it is corrosive so be sure to wipe with clean water after minutes of action. besides, all biocides have some toxicological risks to human health and/or the environment. , for children, passive exposure to cleaning bleach may have adverse e ects in increasing the risk of respiratory and other infections. therefore, the sta members should make sense of the potential toxicological hazards before using disinfection procedures. toys disinfection the bigger toys used by all children and the smaller toys used by a certain circle make the virus spread faster. one randomized controlled trial (rct) demonstrated that toys disinfection biweekly can decrease the detection of multiple viruses, including adenovirus, rhinovirus and respiratory syncytial virus in the environment, so tools disinfection is essential in the welfare institute. natural ventilation we should use natural ventilation such as opening doors and windows as much as possible. the air conditioning system should have su cient fresh air input under the safe working situation. when not using air conditioner, the air channel should be closed. due to banning the visit from a social person is unachievable, the children welfare institute should set up a special area for an external person to prevent children from being completely exposed to the infection source. previous and newly enrolled children live in di erent areas according to age and gender. once infectious diseases breaking out, it can limit the scope to children in the same group and area as soon as possible to prevent the spread of diseases. likewise, air pollution plays a role in respiratory diseases. , factory closed led to the air quality better during the epidemic of covid- . according to the related report, air pollution index evidently improves. this suggests us to reduce the outdoor activities during the period of severe air pollution. regrettably, we cannot guarantee that there is no flow of personnel within years, which leads respondents are unfixed. besides, our study is limited to only one child welfare institute in china, so the sample size is relatively small and poorly representative. by analyzing the data, it is found that the prevalence rate of respiratory diseases was decreased significantly for the children in the social children welfare institute during the epidemic of covid- . it demonstrated that normalized prevention and management are the preferential measures to reduce prevalence rate of infectious diseases. so, we make the following suggestions. first, in the view of the children welfare institutes, they should establish and improve the prevention and control system of infectious diseases, which covers tertiary prevention and specific details of daily work including disinfection methods, disinfection frequency, disinfection ranges and so on. in addition to countermeasures of severe epidemics, the children welfare institutes must also pay more attention to implement the normalized prevention and management of common diseases among children. second, stand on the government's point to consider, it should enhance fund endorsement and policy priority for the children welfare institutes to make sure that they are able to safeguard and raise the life quality of children. the children welfare institutes can replace life facilities, recommend the advanced disinfection equipment, enrich dietary diversity, etc., under the support of the government. at the same time, in order to protect rights of children further, related laws and regulation are also replenished sooner rather than later. third, start from the view of the society, the most people lack the understanding and knowledge of the children welfare institutes, thus, relational society institutes like non-profit organizations should strengthen propaganda of children welfare to improve the social awareness of the charity. for example, conducting lectures, organize charity activities, release information about the children welfare institutes on the internet and so on. most of children in welfare institute are orphan or the disabled, so the society and government should take care of them as much as possible to guarantee a favorable living and educational environment. their healthy growth still demands our joint e orts further in the future. the authors received no financial support for the research, authorship and/or publication of this article. the authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. the data underlying this article were provided by this social children welfare institute by permission. data will be shared on request to the corresponding author with permission of the social children welfare institute. a comprehensive review of animal models for coronaviruses: sars-cov- , sars-cov, and mers-cov pathological findings of covid- associated with acute respiratory distress syndrome national health commission of the people's republic of china. notice of the national health council of the people's republic of china shanxi province initiated first-level response to major public health emergencies isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak the implications of covid- for the care of children living in residential institutions republic of china. the ministry of civil a airs issued an emergency notice novel coronavirus pneumonia epidemic prevention and control work of a service organization in the field of children welfare institute should be done with all e orts e ectiveness of hand hygiene 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respiratory hygiene/cough etiquette in healthcare settings e ectiveness of personal protective measures in reducing pandemic influenza transmission: a systematic review and meta-analysis pilot of an elementary school cough etiquette intervention: acceptability, feasibility, and potential for sustainability national health commission of people's republic of china. guidelines for public protection against novel coronavirus infection does disinfection of environmental surfaces influence nosocomial infection rates? a systematic review disinfection and the prevention of infectious disease: no adverse e ects? domestic use of bleach and infections in children: a multicentre cross-sectional study e ect of cleaning and disinfection of toys on infectious diseases and micro-organisms in daycare nurseries roles of sunlight and natural ventilation for controlling infection: historical and current perspectives the influence of particulate matter on respiratory morbidity and mortality in children and infants impact of air pollution on the burden of chronic respiratory diseases in china: time for urgent action environmental perspective of covid- key: cord- - vlxa i authors: williamson, e. d.; westlake, g. e. title: vaccines for emerging pathogens: prospects for licensure date: - - journal: clin exp immunol doi: . /cei. sha: doc_id: cord_uid: vlxa i globally, there are a number of emerging pathogens. for most, there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti‐microbial resistance (amr) acquisition by micro‐organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard, encompassing phase iii clinical trials for efficacy. an alternative is to develop such vaccines to request us emergency use authorization (eua), or equivalent status in the united states, canada and the european union, making use of a considerable number of regulatory mechanisms that are available prior to licensing. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. globally, there are a number of emerging and re-emerging pathogens. some of these cause endemic disease in regions of the globe, where they are maintained in zoonotic reservoirs and transmitted to man either by direct or indirect contact. for most of the emerging pathogens there are no licensed vaccines available for human use, although there is ongoing research and development. however, given the extensive and increasing list of emerging pathogens and the time and investment required to bring vaccines into clinical use, the task is huge. overlaid on this task is the risk of anti-microbial resistance (amr) acquisition by micro-organisms which can endow a relatively harmless organism with pathogenic potential. furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease. vaccination provides a defence against these emerging pathogens. however, to date, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard. while approval of vaccines for diseases caused by such pathogens would clinical and experimental immunology review article series editor: e diane williamson make a significant impact on disease outbreaks, taking niche vaccines into clinical development, including phase iii clinical trials for efficacy, requires a large investment in time and money. an alternative is to develop such vaccines to request us emergency use authorization (eua), or an alternative status in the united states, canada and european union (eu) making use of a considerable number of alternative regulatory mechanisms that are available prior to licensing, so that the products are deployable at the first indications of a disease outbreak. this review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve eua or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. pathogens which are classed as emerging or re-emerging are identified through global surveillance programmes and organizations such as the world health organization (who). although labelled 'emerging', most of these pathogens are not new and have either been quiescent in the environment until the conditions are opportune to emerge or have evolved from a parent organism to adapt to the prevailing conditions. thus, there is an intricate relationship between the environment, the climate, wildlife and human existence and lifestyle. the coronaviruses exemplify this point: the ancestral virus possibly existed approximately years ago [ ] . coronaviruses have a wide species range infecting birds, bats, chickens, pigs, dogs, cats and rodents [ ] . however, the first human coronavirus was described only in the s [ , ] , and the coronavirus causing severe acute respiratory syndrome (sars) was discovered only in [ ] [ ] [ ] , while that causing middle east respiratory syndrome (mers) first emerged in [ ] . it is likely that warm-blooded flying birds and bats have co-evolved with the coronaviruses to aid dissemination [ ] . for example, sars is thought to have first infected old world bats, then spreading to horseshoe bats [ ] , civets and finally to man [ ] . in the outbreak of sars in china and adjacent countries, phylogenetic analysis suggested that the virus spread from bats to humans, possibly through the intermediary civet species. neither bats or civets showed any clinical signs of infection, and it is thought that bats are the main zoonotic reservoirs for the virus [ ] . organizations such as the who, national institutes for allergy and infectious diseases (niaid) and the us centers for disease control (cdc) publish lists of emerging pathogens which may be viral, bacterial or rickettsial in nature. the who priority list contains viruses which have been prioritized as the most likely to cause epidemics and for which the who will establish a blueprint programme for accelerated research and development (r&d) [ ] . the list published in february is shown in table . niaid [ ] and cdc [ ] also publish lists of pathogens of priority which comprise bacteria and viruses, but categorize these into three groups depending on pathogenicity, accessibility and the availability of vaccines and therapies. all the viruses listed by the who also occur on these lists, alongside bacterial pathogens of concern. one of these is yersinia pestis, causative of bubonic and pneumonic plague, which is recognized by all three bodies (who, cdc, niaid) as a current priority following the exceptionally large and serious outbreak between september to april in madagascar [ ] , where the disease is endemic. in addition, niaid recognizes the added threat to human health posed by the acquisition of amr by pathogens and who has also published a priority list [ ] of bacterial species for which r&d is required to develop new antibiotics (table ) . globally, there are many regions of endemic disease which are maintained by reservoirs of zoonotic pathogens in the local wild animal species. these pathogens comprise viral, bacterial and rickettsial species and some have complex lifecycles, infecting an environmental (e.g. standing water) or animal reservoir and then being transmitted either by direct contact with man or indirectly, via an insect (e.g. mosquito) or mammalian (e.g. bat, civet, camel) vector, to man, from which they may be spread by human-tohuman transmission. (fig. ). an example of a serious and widespread bacterial infection is mosquito-transmitted plasmodium falciparum, causing malaria in many tropical regions. malaria causes significant morbidity and co-morbidity in these regions in which it is endemic. due to the complex life cycle of the causative bacterium, it has been challenging to achieve a vaccine for malaria. the most advanced candidate is the rts,s/as vaccine [ ] , which is undergoing a pilot implementation in three countries in sub-saharan africa, with a view to determining its impact on disease prior to a wider implementation [ ] . despite the availability of approved vaccines [ , ] , typhoid fever and cholera remain significantly debilitating enteric diseases in regions of the world where hygienic living conditions are poor and there is little access to health care. both infections are caused by bacteria (salmonella typhi and vibrio cholera, respectively) which exist in contaminated water and food. tuberculosis (tb) is a respiratory infection which is widespread globally. caused by mycobacterium tuberculosis, and transmitted to man from zoonotic reservoirs (badgers, cattle) with a high potential for subsequent human-to-human transmission, tb is prevalent in susceptible individuals living in overcrowded conditions [ ] . although the bcg vaccine has been in routine use for many years, variable efficacy has been reported, depending on region of use [ , ] with - % reported in the united kingdom but lower levels in equatorial countries. the emergence of multi-drugresistant tb (mdr tb) in recent years raises the bar for treatment of this disease and makes a high level of vaccine efficacy even more important [ ] . examples of zoonotic reservoirs which maintain endemic viral diseases are numerous and are summarized in table . in particular, viral endemic disease is caused by the coronaviruses (mers and sars) in saudi arabia and the eastern mediterranean countries; and by the lassa arenavirus, which is endemic in west african countries and caused a serious outbreak of lassa fever in nigeria in [ ] ; other viruses which are endemic and cause viral haemorrhagic fever include dengue, which is widespread in tropical and subtropical regions worldwide [ ] , the filoviruses (ebola and marburg) which have a zoonotic reservoir in bats in sub-saharan africa [ ] ; and yellow fever virus, which is prevalent in africa, central and south america and the caribbean [ ] . interestingly, the same mosquito (aedes aegypti) which spreads yellow fever virus also transmits the dengue, chikungunya and zika viruses [ ] . chikungunya virus has a widespread distribution in africa, asia, india and south america and with occasional cases in europe, and causes a debilitating, but rarely fatal, disease [ ] . zika virus emerged in brazil in [ ] , although it was first detected in monkeys in uganda as early as [ ] , and the first documented human case occurred in nigeria in [ ] . the large brazilian outbreak of zika viral disease culminated in , with thousands of cases reported [ ] . rift valley fever virus (rvfv) is another zoonotic virus which is endemic in sub-saharan africa and primarily infects cattle, sheep and goats, but can be transmitted to man by mosquito bite to cause an acute fever [ ] . an example of a bacterium which has been classed as a re-emerging pathogen and which is endemic in global regions is y. pestis, causative of plague. bubonic [ ] [ ] [ ] . over thousands of years, y. pestis has evolved away from the enteric yersinia species to become a lethal flea-transmitted bacterium [ ] . it is transmitted to man, typically from a zoonotic reservoir in infected rats or other rodents (e.g. ground squirrels or prairie dogs), by flea-bite to cause bubonic plague [ ] (fig. ). if not detected and treated, this can develop into either septicemic plague or the most serious form of all, a secondary pneumonic plague. in turn, individuals with pneumonic plague can transmit this by aerosol droplet to others, to establish a primary pneumonic plague infection. each year, a few cases of plague are also reported in the southwestern united states, where the disease has been endemic in the rodent population since the late s [ ] . as well as infecting the rodent population, infected fleas can spread y. pestis to other wildlife species (e.g. rabbits/hares or, rarely, domesticated animals [ ] ) which, in turn, raises the potential of transmission to man by inhalation to cause a primary pneumonic plague. in endemic areas, outbreaks of plague are often associated with seasonal environmental changes, causing rodents to stray closer to human habitation. the plague outbreak in madagascar during / was particularly serious, with an estimated cases and deaths ( · % fatality rate) [ ] . this outbreak was approximately sixfold greater than usual, with an unusual predominance of pneumonic, rather than bubonic plague. although y. pestis is susceptible to antibiotics, such as the aminoglycosides (gentamycin, streptomycin), the fluoroquinolones (e.g. ciprofloxacin) or tretracyclines (e.g. doxycycline) [ ] , these need to be administered very early to a suspected plague case, and ideally before symptoms emerge. additionally, there have been reported instances of antibiotic resistance including to multiple antibiotics [ , ] . hence, there is a clear and increasingly urgent need for an efficacious vaccine. other bacterial endemic diseases include melioidosis and glanders which, although not caused by zoonotic pathogens, are endemic in southeast asia where the bacteria reside in soil and are transmitted to humans through occupational exposure, e.g. working in paddy fields [ ] . another bacterial disease which is endemic in the northern hemisphere is tularaemia, caused by the bacterium francisella tularensis, which has zoonotic reservoirs in the rabbit, hare and rodent populations in the south, central and western united states and is transmitted to man by ticks and biting flies [ ] . rickettsial species, such as coxiella burnetii, causative of q-fever, comprise bacteria which exist within another cell, and as such q-fever is contracted when humans are exposed to aerosolized droplets from the urine, milk, faeces or birth detritus from infected sheep, goats and cattle [ ] . for all these pathogens, there is a requirement for efficacious approved vaccines to curtail or prevent regular disease outbreaks. for some of these pathogens (e.g. rvfv, cchf) there are vaccines [ ] , but these are not widely available [ ] or have been used and withdrawn for safety or regulatory reasons (e.g. the live vaccine strain for tularaemia) [ ] or they are not universally suitable, requiring a screening test prior to administration due to the potential for a hypersensitivity response (as for the vaccine for q-fever) [ ] . there is no readily available licensed plague vaccine, although a series of killed whole cell vaccines (kwcv) has been produced and used in the past, mainly in the biodefence context (reviewed in [ ] ). additionally, live attenuated plague vaccines, derived from an attenuated mutant strain as the ev series, have been used in the former ussr (fussr) and are still used in asia, notably in russia and china [ , ] . ev , the most commonly cited of these, provides protective efficacy against plague, but is licensed for human use only in countries of the fussr and is documented to cause serious adverse effects in non-human primates and malaise and adverse effects in human vaccinees [ ] . whatever the context, all these diseases would be positively impacted by the availability of efficacious and approved vaccines. however, to a greater or lesser extent they are all niche diseases with no major commercial incentives to drive vaccine development programmes. this is a space that non-governmental organizations (ngos) such as the coalition of epidemic preparedness innovations (cepi) [ ] and global vaccine alliance (gavi) [ ] have entered and they are supporting vaccine efforts for some of the diseases listed above. additionally, philanthropic funders such as the gates foundation are supporting vaccine r&d efforts [ ] . in the united kingdom, and subsequent to the ebola outbreak in west africa, vaccine networks for human and veterinary vaccines have formed to prioritize vaccine efforts in these respective contexts [ ], while the department of health, together with innovate uk, has supported r&d of vaccine candidates for the prioritized pathogens [ ] . as a result of these global initiatives, a number of candidate vaccines are being developed for emerging bacterial and viral pathogens, examples of which, although by no means exhaustive, are cited here [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . who reports ongoing global vaccine r&d efforts [ ] and also tracks the progress of clinical trials for emerging pathogens [ ] . here it may be worth drawing a distinction between prophylactic vaccination, i.e. general use prophylaxis (gup), given routinely and not necessarily in the face of specific, predicted outbreaks, in contrast to post-exposure prophylactic (pep) vaccination, to be given after a suspected exposure to a pathogen, or to ring-fence an outbreak or, indeed, post-exposure therapeutic (pet) vaccination, to be given after an actual exposure. in the pep and pet contexts, the benefit of vaccination vastly outweighs the risk of disease (i.e. the risk : benefit ratio is low), while in the prophylactic context the risk : benefit ratio may be greater than, or equal to, · ). for infections with short incubation times, e.g. less than h, as for pneumonic plague, pep or pet vaccination may only be useful if administered under antibiotic cover. in the united states, once a vaccine candidate has been thoroughly tested for safety in non-clinical models, and in an escalating-dose, statistically powered, phase i design in the clinic, from then on it may be possible to pursue approval for an eua, rather than pursue the full-length pathway to biological licensing authorization (bla). this can enable the earlier availability of vaccines for use in endemic regions. however, it should be noted that eua is only available through the food and drug agency (fda) in the united states. some alternative regulatory mechanisms that may be considered prior to licensing are outlined below. from the discovery phase to the clinic, vaccine development requires the completion of a series of steps represented as a generic outline in fig. . the regulatory agencies lay down specific guidance for these steps [ , ] and this review presumes no authority in this regard, but seeks to give a generalized overview of a generic vaccine development process. exit from the discovery and preclinical phases requires substantive data demonstrating immunogenicity and efficacy in at least one suitable animal model(s). where possible, efficacy in the animal model should be demonstrated by direct exposure to the pathogen concerned. technology transfer for manufacture under good manufacturing practice (gmp) will require a demonstration of known provenance of all essential materials required in the manufacture of the vaccine candidate. this includes seed stocks of cell lines from which recombinant proteins may be expressed, e.g. escherichia coli, human embryo kidney cells, baculovirus, tobacco mosaic virus; or seed stocks of attenuated vaccine vectors, e.g. viral vectors such as adenovirus, vesicular stomatitis virus, modified vaccinia ankara; or bacterial vaccine vectors, e.g. salmonella, listeria; the genetic constructs cloned into the cell line in question; all culture media and components and all formulations and excipients. transfer of the manufacturing process to gmp may include scale-up and conversion to, for example, fermentation conditions, or to plant-based, mammalian cell line or insect cell line expression on an expanded scale. this transfer will probably require the demonstration of consistency between consecutive batches at gmp, which will also enable the development of scaled-up downstream processing methodology. vaccine components (the drug substance) from these batches may be formulated (the drug product) as required and used in safety/toxicology studies and for immunogenicity/efficacy in an appropriate second animal model, which will be as representative as possible of the human response. the second animal model is often, but not necessarily, a non-human primate, and the selection of this second model will depend entirely on the vaccine indication. there will also be a requirement to generate sufficient stability data on both the drug substance and the drug product and to demonstrate that the drug product is stable for at least the duration of the intended phase i clinical trial under prevalent conditions in that location. clearly, extended stability testing under a range of conditions, including accelerated conditions (high ambient temperature and relative humidity), will also be required to progress the vaccine through development. as well as determining its stability, both the drug substance and product will require characterization for properties such as identity, purity, isoelectric point, osmolality, endotoxin levels and potency, to demonstrate consistency between batches and to allow for release of these for clinical trials. it is essential to ensure safety of the drug product before entering a clinical trial, and the drug product may be tested in suitable small animal models for the absence of adverse effects under conditions of repeated dosing at the anticipated human dose level, as well as in the intended human schedule, but at multiples of the anticipated human dose-level. the use of rodent models (mouse or rat) for this testing allows for sufficient statistical powering of such safety/toxicological testing. if the clinical trial is to be conducted in women of child-bearing age, it may be necessary to carry out reproductive toxicology testing of the drug product; in this case it may be necessary also to use a sensitive rabbit model and to administer the vaccine to pregnant rabbits to screen for adverse effects in the mother and potential teratogenic effects in the f generation. the national regulatory authority will expect to review the existing safety data and outlines of further protocols to be used. a review of all the data pertaining to the candidate vaccine will be required by the regulators in order to proceed to a clinical trial. depending on the specific requirements of the regulatory authorities in the country of origin/manufacture of the vaccine and also the intended location of the clinical trial, this may take the form of an investigator's brochure, protocol and summaries of manufacturing, non-clinical and clinical data in a clinical trial application (for the european medicines agency, ema) or an investigational new drug (ind) application (e.g. us fda). other international regulators (e.g. in canada, japan and china) will have variations on the documentation required. in the united states, the alternative approaches to full marketing approval that are available for vaccines under development and which may be considered through the fda are to either request an eua or to request expanded access (ea), sometimes called compassionate use. the eua is issued in support of potential and actual public health, military and domestic emergencies involving chemical, biological, radiological and nuclear agents (cbrn), including emerging infectious diseases, e.g. pandemic influenza. such fda-approved medical products which may be stockpiled for use in emergencies are referred to as medical countermeasures (mcm) and include biological products, e.g. vaccines, drugs and devices. specifically, the eua authority is separate and distinct from use of an investigational medical product held under an ind and must be able to treat serious or life-threatening diseases or conditions. in contrast, ea submissions are for products used under an ind or a protocol (treatment plan), or submitted as a protocol amendment to an existing or new ind. the ea categories cover use under either an individual patient ind or protocol, individual emergency ea use, or ea for an intermediate-size population or for widespread use (large populations). other us regulatory programmes for biologicals and drugs to treat serious or life-threatening conditions include fast-track designation, breakthrough therapy designation, accelerated approval pathway and priority review designation. in canada, access to drugs through special access programmes (saps) exists for serious and life-threatening conditions, when marketed alternative products are not available or unsuitable and evidence supports the intended use. priority review of marketing submissions and 'notice of compliance with conditions' for such products may also be considered and granted by health canada. in the european union, the ema supports early patient access to new medicines and which are eligible for a marketing authorization application under the centralized procedure and either target unmet medical needs or those which have a major public health interest. the ema has launched a priority medicines (prime) scheme to facilitate early dialogue and support the development of medicines that target unmet medical needs and which promotes an accelerated regulatory assessment. in addition, the prime scheme is intended for seriously debilitating or life-threatening diseases, compassionate use of unauthorized medicines for patients with an unmet medical need (when no satisfactory treatment is available in the european union) and also provides conditional marketing authorization. the regulation and rules of access to compassionate use programmes varies between national government authorities across eu member states. in the united kingdom, the early access to medicines scheme (eams) gives patients with life-threatening or debilitating conditions access to innovative medicines without marketing authorization, but for which there is a clear medical need. a two-stage evaluation process initially considers promising innovative medicine (pim) designation before an eams scientific opinion. also in the united kingdom, the potential supply of unlicensed medicinal products (specials) may be considered by the medicines and healthcare products regulatory agency (mhra) for the importation of medicines that have marketing authorization status from countries outside the united kingdom and european union. where there is an unmet need of priority to the who for a new vaccine, the who may establish a blueprint programme [ ] . as vaccine candidates for the specific blueprint programme advance through the development process, and particularly when they have attached clinical data, the who may run a prequalification check to determine whether the candidate meets their requirements on behalf of un agencies that will ultimately purchase vaccines [ ] . to do this, who will engage with subject matter experts to draft and publish a target product profile (tpp) for a vaccine requirement. who will use its scientific advisory group of experts (sage) to review vaccine candidate performance against the tpp and to prequalify a vaccine candidate(s) which fulfils the tpp. assuming that the preclinical safety and toxicology testing has been satisfactory, a protocol for phase i testing of the vaccine candidate may be submitted to the regulatory authorities for approval. the primary objective of the phase i clinical trial is to test the vaccine candidate for safety in informed and consenting adult volunteers who will be selected according to pre-agreed inclusion/exclusion criteria. as this is the first time the candidate is being used in man, the phase i trial is typically small and cautious. if a range of dose-levels is being tested, it may be necessary to dose a sentinel group of single subjects from each arm of the study to assess safety through week of immunization and starting at the lowest level before proceeding to the next level. the data from the sentinel group would be reviewed before proceeding with the main study. then the first cohort would be dosed at the lowest level before proceeding to the next level, and so on. volunteers will be closely monitored for adverse effects in the clinic at each dosing time-point and will typically maintain a diary at home to record any symptoms arising between time-points. an independent safety monitoring panel, including medically qualified personnel, will be required to monitor the reporting of adverse effects. volunteers may also be blood-sampled to assess vaccine immunogenicity from baseline and serial samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. saliva/stool samples. all volunteers may be followed up for a pre-agreed period on completion of the study, to check for latent adverse events and to monitor, e.g. for maintenance of circulating antibody titres or memory response to the vaccine. the second phase of clinical trials typically allows for an enlarged study to assess vaccine safety and to monitor immunogenicity. during this phase, significantly expanded cohorts of volunteers may be dosed with vaccine at doselevel(s) selected as optimum from the phase i trial and can be monitored for safety and a more detailed immunogenicity assessment, which may include assays of both serological and cellular memory responses. once again, volunteers may be blood-sampled for baseline and serial serum/plasma samples, and depending on vaccine type may be required to supply additional samples which can be collected non-invasively, e.g. whole blood/saliva/stool. in the event that a phase iii trial for efficacy is not feasible, for ethical or practical reasons, it may be necessary to plan a blood-sampling regimen in the volunteers which will enable sufficient sample volumes to be available for animal rule studies (discussed below). if sufficient safety data are gained from a phase ii trial, the regulatory authorities may be able to consider the vaccine for eua. the third phase of clinical trials is typically designed to assess efficacy. however, where this is not feasible for either practical/logistical or ethical reasons, phase iii could be regarded as a further, significantly enlarged trial for vaccine safety with adequate follow-up of vaccinated volunteers. a phase iii field trial of vaccine efficacy may be feasible in the event of an anticipated seasonal outbreak, in which case vaccination could be given prophylactically and well in advance. as long as effective biosurveillance programmes are in place to instigate a rapid response to new infections in an unvaccinated placebo cohort, the latter group may be included in a prophylactic efficacy trial. conversely, reactive mode vaccination in the context of an actual outbreak is likely to be more complicated, as it would be unethical to omit anyone at risk of infection from the vaccination programme. hence, sufficient safety data on the candidate vaccine would be required in case of need to administer it to pregnant/nursing mothers, children and elderly people as well as to the general adult population. additionally, it may be necessary to administer an adjunct to the vaccine, such as an appropriate antibiotic, to both cohorts (vaccinated and placebo). whatever the context, careful consideration of the trial design needs to be made and approved by the regulators to achieve adequate statistical powering and to determine the need to provide supplementary antimicrobial therapy, and also the need for a placebo cohort. after the anthrax letters incident in the united states in in which anthrax was released through the postal system, resulting in five fatalities and widespread anxiety [ ] , the fda issued the animal rule (fda cfr) [ ] to expedite the development of vaccines and therapies for biothreat agents for which it is neither feasible nor ethical to carry out phase iii efficacy studies in man. in this situation, the animal rule makes provision for the substitution of animal efficacy data for human efficacy data. the animal efficacy data should demonstrate a reasonable likelihood that the candidate vaccine or therapy would be efficacious in man [ ] . the animal efficacy data should be provided ideally from more than one animal model, or from one model only if that model is well-characterized and authentically represents the human disease syndrome [ ] . in addition, the fda may require supporting data to include pharmacokinetic/pharmacodynamic (pk/pd data) and a determination of the pathophysiological mechanism of action of the biothreat agent and a 'reasonable understanding' of the protective mechanism of the proposed vaccine or therapy. thus, if the candidate is a vaccine, a reasonable understanding of the protective mechanism(s) being invoked by it requires an identification of the immune correlates of protection. immune parameters which correlate with protection in the selected animal models may include the total titre of circulating antibody induced to the vaccine and the determination of a minimum cut-off titre required to confer protection. alternatively, the titre of functional or neutralizing antibody within that total may provide the correlate, particularly where the neutralization of specific virulence factors produced by the biothreat agent is identified and quantifiable [ ] . for some vaccines, the induction of a cellular memory response instead of, or in addition to, circulating antibody will provide the correlate. this is measurable by the ex-vivo recall response of peripheral blood mononuclear cells (pbmcs) on stimulation with the vaccine antigen(s) [ ] . in the absence of direct efficacy testing in the human vaccinee, the immune correlate identified above provides a surrogate marker of efficacy. if this is a functional antibody, the titre induced in man needs to be compared with the titre required in the animal model(s) to provide protection. this can be achieved, for example, by the in-vitro neutralization of a specific virulence factor, or by a competitive enzyme-linked immunosorbent assay (elisa), where the test antibody is competed with a known neutralizing antibody for binding to the target antigen or by the passive transfer of antibody from a human vaccinee into a naive animal, followed by pathogen challenge [ ] . whatever the immune correlate may be, its effect would be expected to follow the pattern shown in fig. , where as the value increases, the likelihood of death in the vaccinee decreases [ ] . thus, vaccine efficacy = relative reduction in risk of death = − (% of vaccinated subjects at < protective level) (% of unvaccinated subjects at< protective level) . although a number of immunotherapies and pretreatments have been licensed under the animal rule, the first vaccine to be licensed in these circumstances is 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safer world about gavi, the vaccine alliance vaccine delivery:bill and melinda gates foundation dual route vaccination for plague with emergency use applications immunogenicity and safety of subunit plague vaccine: a randomized phase a clinical trial rift valley fever mp- vaccine phase clinical trial: safety, immunogenicity and genetic characterisation of virus isolates development of vaccines against cchf virus a monovalent chimpanzee adenovirus ebola vaccine boosted with mva effect of dengue serostatus on dengue vaccine safety and efficacy non-neutralising antibodies elicited by recombinant lassa-rabies vaccine are critical for protection against lassa fever chadox and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice vaccine and therapeutic options to control chikungunya virus chikungunya vaccines in the pipeline a bacteriophage t nanoparticle based dual vaccine against anthrax and plague who: health products in the pipeline for infectious diseases world health organization. who vaccine pipeline tracker yjurcmg xwo kvuyedybmzdcxqbyjgdczm/pubhtml# fda guidances for vaccine development vaccines for emerging pathogens: from research to the clinic. part regulatoryinformation/guidances/vaccines/default.htm ema guideline for vaccine development who guidance for clinical evaluation of vaccines who vaccine standards: vaccine prequalification. available at the anthrax vaccine and research: reactions from postal workers and public health professionals code of federal regulations (cfr) cfr . - . evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible predictive models and correlates of protection for testing biodefence vaccines first vaccine approval under the fda animal rule the authors declare no competing interests. key: cord- - s ysih authors: honigsbaum, mark; méthot, pierre-olivier title: introduction: microbes, networks, knowledge—disease ecology and emerging infectious diseases in time of covid- date: - - journal: hist philos life sci doi: . /s - - -x sha: doc_id: cord_uid: s ysih this is an introduction to the topical collection microbes, networks, knowledge: disease ecology in the twentieth century, based on a workshop held at queen mary, university london on july – . more than twenty years ago, historian of science and medicine andrew mendelsohn asked, “where did the modern, ecological understanding of epidemic disease come from?” moving beyond mendelsohn’s answer, this collection of new essays considers the global history of disease ecology in the past century and shows how epidemics and pandemics have made “microbes complex”. in , the historian of science and medicine j. andrew mendelsohn posed a deceptively simple question. "where," he asked, "did the modern, ecological understanding of epidemic disease come from?" helpfully, mendelsohn provided an answer. arguing that it was difficult to see how "the fledgling ideas and methods of upstart population ecology, or the premises of parasitology could have conquered bacteriology," he suggested these perspectives were already present in medical microbiology at its inception but re-emerged more strongly in the interwar and post-war period, often from within the very institutions most closely associated with germ theory and the reductionist pursuit of vaccines and chemotherapeutic drugs (mendelsohn , pp. - ) . in particular, mendelsohn chided the australian immunologist and nobel prize winner frank macfarlane burnet for having "left out half the story" when in he had claimed in biological aspects of infectious disease that the new "ecological point of view" had been due to the ascendance of other scientists over "bacteriologists who had been trained as medical men" (burnet , pp. - ) . coming in the wake of renewed scholarly interest in the - influenza pandemic (honigsbaum (honigsbaum , -an outbreak that according to mendelsohn had posed "complex" new challenges for public health-mendelsohn's answer struck many as persuasive at the time, but in the two decades since it has become clear that his answer was incomplete. in his pioneering essay on the "natural histories of disease," the historian of medicine warwick anderson offered a partial riposte to mendelsohn by showing how burnet's ideas had been fashioned by his interactions with animal ecologists in the s and s, and the disease challenges that confronted microbiologists working in colonial and postcolonial settler societies ( ; see also anderson ) . since then case-studies of particular scientific figures and institutions that echo anderson's findings have been accumulating (anderson ; honigsbaum honigsbaum , a jones ; way ; méthot ) . nevertheless, it remains to be explained how scientists came to postulate an "evolutionary time scale" and to develop "integrative models" in place of a more static view of the relation between environment, health, and disease, to use anderson's words ( , p. ) . adding to the complexities about the multiple intellectual origins of this nonreductionist perspective, terms such as "virulence," "pathogen," and "infection" have been historically defined in different ways in biology and medicine, creating semantic confusion about the nature of biological processes in host-parasite interactions (méthot and dentinger ) . furthermore, local scientific traditions and research styles differ from one context to another, but scholarship on the history of disease ecology has mostly concentrated on dominion science and especially on american, australian, and british contexts, even though similar concepts flourished elsewhere, for instance in russia, as the study by susan jones and anna amramina in this collection demonstrates (jones and amramina ). last but not least, in writing the first draft of the natural history of disease, prominent scientists like macfarlane burnet and joshua lederberg have selectively identified prestigious forerunners of their ecological ideas and suggested key influences on their work, while leaving out other potentially important sources and antecedents (anderson ) . thus, a global picture of this multi-site tradition, of its actors and their personal and professional relations, and of its particular place within twentieth-century biomedical science is still wanting. if disease ecology emerged in the s and formed a specific "analytic framework for understanding the interactions of microorganisms and macrobial hosts" (anderson , p. ) , it still remains to be characterized with respect to other trends in the biomedical sciences in the past century, including the rise of experimentalism and the earlier development of medical geography, as well as the intellectual exchanges that helped shape the field of disease ecology. for instance, what role did transnational scientific networks play in the propagation and circulation of ecological ideas, and how and to what extent did microbiologists forge alliances with researchers in allied fields? reconstructing these "ecologies of knowledge," to invoke charles rosenberg's useful phrase, and the borders between them, is no easy task. as anderson points out, since historians of science and medicine took up mendelsohn's question in earnest they have described multiple influences on the field's formation and composition. these include not only parasitology and animal ecology, but veterinary pathology, medical geography, and institutions concerned with the control of agricultural pests. in the process, medical researchers came to adopt a very different language to that employed by microbe hunters, one that eschewed the old bacteriological metaphors of a "war" or "struggle" with microbes in favour of a more dynamic, interactive, and cooperative vision of health (see lederberg ) . whether or not it was couched in explicitly ecological language, it was a vision that tended to see disease as the result of temporary imbalances or disturbances of biological relationships, rather than as something that could or ought to be conquered or eradicated. and it was a vision that was explicitly sympathetic to darwinian perspectives and that was prepared to embrace long evolutionary time frames. in so doing, it could not help but provoke deep philosophical questions about what the french bacteriologist charles nicolle ( ) termed the "birth, life and death of infectious diseases" and the waxing and waning of epidemics in different historical epochs (see méthot a, this issue) , and what the rockefeller researcher rené dubos called the "symbiosis between humankind and earth" (see honigsbaum a, b) . in the summer of we invited a diverse group of scholars to re-interrogate mendelsohn's question at a workshop at queen mary, university london. most of us were historians of science, medicine and technology, but with a view to opening up interdisciplinary perspectives the group also included anthropologists, biologists, and epidemiologists specializing in emerging infectious diseases (eids). as the latter reminded us, in recent years a succession of epidemics of sars, ebola, and zika, has driven home the importance of ecological perspectives in pandemic planning. yet, while it is now widely recognised that the disturbance of natural habitats can result in zoonotic pathogens being dislodged from their ecological niches and infecting human populations, we still lack a comprehensive account of when and how disease ecology entered the medical mainstream. if anything, the need for such an account has become even more urgent following the emergence in december of the novel coronavirus (sars-cov- ) and the pandemic of covid- that followed. although at time of writing the source of sars-cov- has not been established, the fact that it shares % of its genes with a coronavirus previously isolated from wild bats, and that the earliest human cases occurred in or a near a wild animal market in wuhan, in central china, points strongly to the pandemic's zoonotic origins and that the world should be prepared for further pandemics triggered by "spillover" events. as three of the leading experts on zoonoses and pandemics put it in a paper in the new england journal of medicine, "we have created a global, human-dominated ecosystem that serves as a playground for the emergence and host-switching of animal viruses" (morens et al. (morens et al. , p. . back in , in the aftermath of the sars epidemic, a research team from the university of hong kong warned the scientific community that "the presence of a large reservoir of sars-cov-like viruses in horseshoe bats, together with the culture of eating exotic animal in southern china, is a time bomb" (cheng et al. , p. ) . sadly, that call was not taken sufficiently seriously. we can only hope that the present pandemic of covid- has finally put to rest macfarlane burnet's and david o. white's complacent and dangerous assumption that "the most likely forecast about the future of infectious disease is that it will be very dull" (burnet and white , p. ) . in our view the failure to provide a comprehensive account of the field lies partly with medical historians who have applied the term "disease ecology" avant la lettre, reading it back onto historical texts from the s and s, and perhaps even before. in fact, as jon arrizabalaga shows, the term first appeared in a article entitled studies in disease ecology by the french medical geographer jacques may. however, years earlier, may had already published on the ecology of human disease and in he had proposed replacing the phrase "medical geography" with "ecology of health and disease," so clearly the ideas were present in his thought at an earlier date. may's ideas were to influence the croatian physician and historian of science and medicine mirko d. grmek in the s (e.g. "pathocenosis"), who developed his ideas of disease ecology further when writing the first history of aids and introducing a taxonomy of the concept of "emerging disease" in the early s (arrizabalaga , this issue; see also méthot b) . inspired by the teaching of nicolle, the french biologist and physician hervé harant also promoted the science of "medical ecology" in the montpellier area in the mid-twentieth century. however, instead of drawing a sharp boundary between medical geography and disease ecology he insisted on their being continuous with one another. drawing on the writings of geographer maximilien sorre and the concept of a "pathogen complex", harant relied on the then emerging ecological terminology, but this understanding of ecology had more to do with the natural history tradition than with population studies as such. furthermore, neither may nor harant referred to what burnet called the "ecological point of view" or cited the writings of their fellow frenchman, the rockefeller microbiologist rené dubos. this despite the fact that by the s dubos was fast developing a reputation as an apostle of the burgeoning environmental movement and was increasingly employing horticultural metaphors to express his unease with short-term medical fixes, such as antibiotics, and to distance himself from eradicationist perspectives. indeed, though towards the end of his career dubos acknowledged that he had had few occasions to use the word ecology before the s, he claimed that ever since , when he had embarked on a career as an experimental biologist, he had "looked at things from an ecological point of view" (honigsbaum a, b, p. ) the result was that rather than take pasteur's "pure culture" road, dubos decided it would be more profitable to take the road that "lead to physiological and ecological studies" (honigsbaum b, p. ). dubos's assertion points to a fundamental definitional and methodological problem: namely, what exactly does it mean to have an "ecological point of view" and is such a viewpoint contiguous with both "disease ecology" and "medical ecology?" furthermore, if, as may and harant appear to have believed, disease ecology is closely related to medical geography, what distinguishes one from the other and does it matter if the person invoking ecological perspectives considers themselves primarily a medical researcher or a geographer of disease? and more importantly: what did "disease ecology" mean to scientists such as dubos, meyer, and burnet? as historians, we are confronted with the issue of anachronism when using the phrase "disease ecology" retrospectively since ecology was not a full-fledged scientific discipline until the s (tilley ) . the phrase has multiple meanings, and not only refers to a particular scientific field but to various political movements with different agendas. as the papers collected in this thematic issue make clear, scientists-when they did provide a definition-adapted the term ecology to fit their own particular projects. indeed, although each of these ecologically-oriented medical researchers tended to represent themselves as singular, and, as anderson has observed, "as the sole author of the idea, and rarely cited others, even those linked to him by education or friendship" ( , p. ), none of them worked in a vacuum but were connected by networks of scientific exchange and shared intellectual passions. a good example comes from the two papers with which we open the collection. at first glance, rené dubos's investigations of the tubercle bacillus at the rockefeller institute in new york in the s would seem to have little do with frank fenner's classic study of myxomatosis in australia in the s, but, as honigsbaum and anderson show, both researchers were fascinated with the phenomenon of virulence and the environmental and ecological conditions governing host resistance to infection and disease. moreover, in , fenner, at burnet's suggestion, had spent months at dubos's laboratory at the rockefeller institute. there, he had assisted dubos in his investigations of the chemical and morphological properties of the tubercle bacillus and the role of metabolic processes in converting latent tubercular infections into full-blown cases of the disease. the result was that by the time fenner returned to australia in and renewed his association with burnet he was primed to apply these holistic perspectives derived from the laboratory study of mycobacteria-what might be termed physiological ecology-to host-parasite interactions in the field. his opportunity came in when he was invited to lead an "ecological study of mosquitoes" with a view to explicating their role in the transmission of the myxoma virus, which had been introduced to australia a few years earlier in an effort to curb rabbit populations. fenner's study, which drew on what anderson calls "local ecologies of knowledge," in particular scientific networks operating through australia's council of scientific and industrial research, was the first to trace the coevolution of host resistance and parasite virulence in a natural setting in real-time. in the process, fenner showed how natural selection served to regulate the virulence of the myxoma virus and stabilize host-parasite interactions in the wild. little wonder that dubos, who followed the progress of fenner's study closely, hailed it as an example of "host-parasite ecology" and that fenner later described dubos and burnet as his two most important intellectual influences (honigsbaum a, b, this issue, p. ) . perhaps more significantly from the point of view of the trajectory of ecological ideas in the post-war period, anderson shows how fenner's study "offered techniques that could be applied equally well to biological warfare among humans," thereby underlining the "congruence of the ecological mindset with prevailing cold-war thought styles" (anderson , p. ) . another example of the operation of transnational scientific networks in fostering ecological ideas was the pioneering plague research conducted by the russian zoologist evgeny pavlovsky and his soviet colleagues in the s. best known for his concept of "natural nidality"-a term derived from the russian word "ochag", meaning "hearth" or "home"-pavolvsky appears to have had a much wider influence that was previously supposed, and not just on thinkers sequestered behind the iron curtain. on the contrary, susan jones and anna amramina maintain that there was "a surprisingly vigorous knowledge exchange with western ecologists and biomedical scientists even under the most difficult conditions" (jones and amramina , this issue). for instance, pavlovksy was in regular contact with charles nicolle in tunis, and even visited the pasteur institute in tunisia on the eve of the first world war (see méthot a, this issue). the two men exchanged a number of letters over the years, together with books and laboratory materials, and shared their ideas about the nature of infectious processes. while medical geography relied on the two-dimensional mapping of disease ranges onto places, the soviet "natural focus" framework located disease explicitly in what jones and amrarmina describe as "spaces of interaction between components of non-human assemblages in nature." it was an order of analysis in which humans were de-centred and secondary. instead, pavlovksy and his colleagues focused on the mutual influences between the key constituents of plague systems-rodents, microorganisms, fleas-and factors such as landscape, climate, and human cultures. in so doing, soviet ecologists were able to actively incorporate ideological concepts into their plague studies, particularly in the soviet borderlands where ecology provided a philosophical and material framework for furthering ideological goals of "sanitizing" colonial landscapes. in this way, jones and amramina argue, the natural focus framework was "entangled in political as well as material ecologies of knowledge and practice." indeed, to the extent that the framework "provided scientific validation and guidance for stalinistera colonization and development projects," this was a key basis of its success. these ecological perspectives had particular appeal to microbiologists working in tropical or settler environments-hence burnet's "ecological point of view"-but for many years lacked a unifying term. instead they found expression in the language of "host-parasite interactions," "equilibrium states", "immune balance", "latent infections" and changes in virulence and pathogenicity. to complicate the picture, throughout the period when these ideas were making inroads into microbiology, the laboratory continued to be the place where scientific breakthroughs and the reputations of medical researchers were made. it was the era of "microbe hunting" and the search for specific chemotherapeutic agents-one in which medical scientists cultivated an image as slayers of germs and held out the promise that medicine would triumph over the microbes of infectious disease and bring an eventual end to epidemics. yet as christoph gradmann argues, this approach entailed a rather static view of microbes, or one in which evolution of resistance to antibiotics occurred over long darwinian time periods, although by the s ecological perspectives were also making inroads into infection medicine as hospitals became "hothouses" for drug-resistant microbes (gradmann , this issue) . a key implication of our study is that there was nothing inevitable about the adoption of ecological perspectives by medical researchers in the interwar period and the integration of these ideas into laboratory research and public health in the decades after the second world war. rather, such ideas often ran counter to the dominant bacteriological paradigm and institutional and commercial research agendas, including cold war biological warfare research, aimed at the manufacture of vaccines and chemotherapies. indeed, as dubos' early warnings about the dangers of antibacterial resistance illustrate, it took many years for these insights to find their way into medical schools and to be taken up by medical authorities. furthermore, while for some medical thinkers a focus on the ecological, environmental and social factors governing host-pathogen interactions and the evolution or resistance fostered scepticism about short-term technological 'fixes' for medical problems, for others these ecological insights were seen as an adjunct to disease control and systems designed to enhance pandemic prevention. in this respect, the study has important implications for current policy debates around polio eradication and the monitoring of pathogens, such as influenza, that have long been considered pandemic threats, as well as other putative pandemic threats listed by the world health organization in its research and development blueprint. our study also provides much needed historical context for current policy debates around "emerging infectious diseases". this concept is closely associated with a report by the institute of medicine and the molecular biologist joshua lederberg. a former student of dubos, lederberg drew on ecological insights associated with dubos's and burnet's writings in proposing his own view of a natural history of disease (see méthot and fantini ) . however, while dubos' and burnet's ideas exerted a huge influence over lederberg and other medical researchers who came of age in the s and s, we do not know precisely how these ideas found their way into epidemiology and public health teaching. nor can we say precisely what became of these ideas in the s and s-periods marked by the legionnaires' disease and swine flu outbreaks, and the advent of hiv/aids-much less why the eid concept took precisely the form it did in the early s (but see weir and mykhalovskiy ). to answer this question, and provide a more comprehensive understanding of why it is that modern practices of disease control and preventive medicine are crafted in the way they are, more work needs to be done to fill in developments in the period from to . to conclude, mendelsohn's claim that modern ideas of disease ecology were already present in bacteriological epidemiology at the inception of medical microbiology is only true if one adopts a narrow and self-limiting definition of disease ecology. instead, we argue that the modern notion of disease ecology rests on the notion that microbial pathogens reside in ecological niches in equilibrium states and that it is the disturbance of these harmonious natural environments that trigger epidemics and pandemics. it is only by broadening our understanding of the historical origins of mendelsohn's "modern, ecological understanding of epidemic disease" and the medical researchers and scientific networks responsible for the integration of such perspectives into medical microbiology and public health, that historians will arrive at a more definitive account of the intellectual origins of these ideas. this is not merely an intellectual exercise. as the debate over the role of ecological causation in sars-cov- demonstrates, the history of modern ideas of disease ecology can not only help us make sense of the past, it can also illuminate current scientific debates around emerging infectious diseases, and the interaction between biological, economic, and cultural factors in current pandemic emergencies, as well as others that may be lurking beyond the horizon. 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d'histoire des sciences ecology and infection: studying parasite interactions at the interface of biology and medicine escaping pandora's box-another novel coronavirus naissance, vie et mort des maladies infectieuses africa as living laboratory: empire, development, and the problem of scientific knowledge, - the invisible and indeterminable value of ecology: from malaria control to ecological research in the american south global public health vigilance: creating a world on alert key: cord- -ys s l e authors: gaspari, valeria; neri, iria; misciali, cosimo; patrizi, annalisa title: covid‐ : how it can look on the skin. clinical and pathological features in twenty covid‐ patients observed in bologna, northeastern italy date: - - journal: j eur acad dermatol venereol doi: . /jdv. sha: doc_id: cord_uid: ys s l e global public health is currently dealing with the explosive spread of the novel coronavirus disease (covid‐ ) [ ]. this new type of viral pneumonia, spread from its first focus in wuhan (hubei, china) to across all the world, until a pandemic condition was declared. clinically. the most common symptoms of the disease are cough and fever. more than % of patients have asymptomatic to moderate disease, but about % get severe pneumonia and % develope a multi‐organ failure [ ]. global public health is currently dealing with the explosive spread of the novel coronavirus disease (covid- ) [ ] . this new type of viral pneumonia, spread from its first focus in wuhan (hubei, china) to across all the world, until a pandemic condition was declared. clinically. the most common symptoms of the disease are cough and fever. more than % of patients have asymptomatic to moderate disease, but about % get severe pneumonia and % develope a multi-organ failure [ ] . the diagnosis of covid is based on a multifactorial approach including clinical symptoms, vital parameters, radiological and laboratory findings the virus isolation, necessary to confirm the diagnosis, is obtained through nasopharyngeal and oropharyngeal swab. italy is one of the most involved countries in this pandemia with . total cases till now [ ] . there are only a few reports concerning the skin manifestations in covid patients. until now twenty skin manifestations in covid patients came to the attention of the dermatology unit of the city of bologna, in emilia romagna, the third italian most affected region. of the twenty patients observed, eighteen of the cases were related to the disease, and two to the devices used for the ventilation assistance, one developing a severe sebopsoriasis of the face, and one a facial herpes. among the eighteen cases related to the disease nine presented exanthematic rashes ( figure a-b) , six presented acral vasculitic eruptions (figure c-d), two a polymorpholike urticaria (figure e) and one a varicelliform eruption. the median age of the patients was years; seventeen were male, three were female. with regards the cases related to the disease, in two the signs were present at the onset, while in the other sixteen they appeared later. we excluded a iatrogenic origin, as these patients had not this article is protected by copyright. all rights reserved assumed any drugs potentially involved in skin reactions over the previous days. other viral etiologies were excluded by performing serology for the viral infections associated with cutaneous manifestations, for example parvovirus b and enterovirus. most of them reported itching and burning sensation. only two of them referred pain. they were variably symptomatic for the respiratory tract, but none of them had such a severe lung involvement as to require intubation. one of them was completely asymptomatic, and only the acral vascular manifestation led us the suspicion of coronavirus infection. in six of the patients showing exanthematic rashes a punch biopsy for histological examination was obtained ( figure f) , showing features of perivascular dermatitis and vasculitis, which are compatible with that of a viral exanthem. it is known that exanthematic rashes can occur during viral infection [ ] . we can say that erythematous rashes during coronavirus infections may have the same origin as the other viral rashes [ ] . instead, the vasculitic eruptions could be due to the vascular changes observed in these patients. degeneration of the endothelium, and vascular damages including both formation of thrombus and congestion in small vessels, were observed in organs other than the lung in autopsies from skin. indeed, while the -ncov is mainly distributed in the lung, the damage caused by the infection also involvesthe vessels, with the possibility of ischemic and embolic damages [ ] . the clinical patterns of the rashes described in covid- patients till now include urticaria, acral ischemia, morbilliform, livedo reticularis, vesicular, and petechial [ ; - ] . as regards the histological patterns, perivascular dermatitis, and transient acantholytic dermatosis are those described till now [ ]. we are presenting this paper to share our cases of skin involvement during the coronavirus disease. undoubtedly no certain association can be established between covid- and skin eruptions, and further studies are needed. coronavirus disease (covid- ) situation report - clinical characteristics of coronavirus disease in china ministero della salute. coronavirus disease (covid- ) parvovirus b : a dna virus associated with multiple cutaneous manifestations cutaneous manifestations in covid- : a first perspective a pathological report of three covid- cases by minimally invasive autopsies a case of covid- pneumonia in a young male with full body rash as a presenting symptom. clin pract cases emerg med covid- can present with a rash and be mistaken for dengue a dermatologic manifestation of covid- : transient livedo reticularis key: cord- - yxurnev authors: green, manfred s; leduc, james; cohen, daniel; franz, david r title: confronting the threat of bioterrorism: realities, challenges, and defensive strategies date: - - journal: lancet infect dis doi: . /s - ( ) - sha: doc_id: cord_uid: yxurnev global terrorism is a rapidly growing threat to world security, and increases the risk of bioterrorism. in this review, we discuss the potential threat of bioterrorism, agents that could be exploited, and recent developments in technologies and policy for detecting and controlling epidemics that have been initiated intentionally. the local and international response to infectious disease epidemics, such as the severe acute respiratory syndrome and west african ebola virus epidemic, revealed serious shortcomings which bioterrorists might exploit when intentionally initiating an epidemic. development of new vaccines and antimicrobial therapies remains a priority, including the need to expedite clinical trials using new methodologies. better means to protect health-care workers operating in dangerous environments are also needed, particularly in areas with poor infrastructure. new and improved approaches should be developed for surveillance, early detection, response, effective isolation of patients, control of the movement of potentially infected people, and risk communication. access to dangerous pathogens should be appropriately regulated, without reducing progress in the development of countermeasures. we conclude that preparedness for intentional outbreaks has the important added value of strengthening preparedness for natural epidemics, and vice versa. the biological weapons convention prohibits the manufacture and use of biological weapons. it came into force in , and has undergone periodic reviews, the last being in . to date, countries are signatories to the convention. unfortunately, terrorist groups or rogue governments are unlikely to feel bound by international agreements. the potential for bioterrorism is of particular concern, since it can cause disease, death, and panic-in great disproportion to the resources expended. there have been a few well documented cases of bioterrorism. in , a religious sect in the usa deliberately contaminated restaurant salad bars with salmonella typhimurium, intending to disrupt local elections. the attack resulted in several hundred cases of salmonellosis and no deaths. the anthrax letters incident in in the usa resulted in cases of inhalation anthrax, with five deaths, and another cases of cutaneous disease. extensive circumstantial evidence strongly suggests that the perpetrator was a civilian employee of the us military. however, no evidence of a clear motive was found. thousands of workers received prophylactic or post-exposure therapy, and affected buildings were decontaminated at huge expense. , in , a cult in japan carried out an attack using anthrax spores with no physical casualties, but later, evidence of post-traumatic stress syndromes was found in victims of the attack. the perpetrators were apparently planning to use other agents such as q fever bacteria, botulinum toxin, and ebola viruses, but they were detained before they could implement further attacks. in this review, we discuss the threat of bioterrorism, potential perpetrators, and general preparedness principles. we examine the special characteristics of biological agents that could potentially be used for bioterrorism, advances in prevention and treatment of diseases caused by these agents, and the remaining deficiencies in the management and control of possible bioterrorist outbreaks. in all respects, the ways in which the resources developed for bioterrorism preparedness could be used for controlling naturally occurring epidemics remain a guiding principle. • preparedness for intentional outbreaks will strengthen the response to naturally occurring epidemics • high level leadership should be maintained with responsibility and authority • health-care providers should maintain awareness of biological agents with bioterrorism potential and consider the presence of unknown pathogens • emergency room and community physicians should be updated regularly about the clinical manifestations of diseases caused by potential bioterrorist agents and emerging infectious diseases. • personal protective equipment should be improved to become more user friendly • improved surge capacity (the ability to rapidly gear up the health system to cope with a sudden, large increase in patients with a serious, contagious disease) is required, particularly in peripheral areas • the capacity of general and reference laboratories should be increased, to keep developing faster, more reliable diagnostic tests • new and improved vaccines (pre-exposure and post-exposure) and treatment regimens should be developed • clinical and environmental surveillance needs to increase • syndromic surveillance systems can be maintained to register suspicious or confirmed cases reported by physicians, and the data can be used to improve risk communication programmes and to monitor the progress of an outbreak • an adequate stockpile of vaccines and medications should be maintained, both nationally and internationally • to improve preparedness for natural and bioterrorist outbreaks, international cooperation should include joint exercises involving multiple countries and constant improvement in the exchange of information on potential bioterrorism threats and management following the breakup of the former soviet union, there was concern that loss of control of their biological weapons programme could allow terrorist groups to gain access to both the weapons and scientific expertise. additionally, in the past few years, developments in the field of microbial genetics have heightened concern about the possible abuse of new technologies. since there are so many unknowns, it is extremely difficult to assess the risks and threats of bioterrorism. , the most likely perpetrators could be disgruntled individuals, terrorist organisations, or rogue countries that are believed to support international terrorism. whereas individual attackers are unlikely to cause mass casualties, terrorist organisations could pose a substantial threat if they gain access to sophisticated biological weapons, materials, or scientific expertise. although regulations and safeguards for securing dangerous pathogens in research laboratories now exist in most countries, the scope of these regulations and the extent of the safeguards vary. rogue countries have the necessary capabilities for a bioterrorist attack but might be restrained by the threat of the response of a unified global community. knowledge gained from legitimate research that could also be applied to bioterrorism is considered dual-use. as a result, the regulation of legitimate research on infectious diseases has increased. there will always be a risk of the "insider threat", , which typically involves a single individual, so it is important to assure that new regulations truly increase security and have minimal negative effect on legitimate research. the cost of regulations applied to research on infectious diseases, in terms of missed opportunities for international collaboration, exchange of pathogens, and sharing of novel agents, is often intangible and overlooked. it is essential to promote healthy organisational cultures to enhance both safety and security in laboratories. since a bioterrorist attack is a low-risk, high-impact event, effective and sustained preparedness is an essential component in both the deterrence and management of an attack. a bioterrorist attack has a lot in common with naturally occurring public health emergencies resulting from infectious diseases. however, there are some important differences. since it is a deliberate act to cause harm, there are the obvious security considerations. the resulting outbreak differs in some important ways from naturally occurring epidemics-for instance, it is more likely to be a point source outbreak initiated by simultaneous exposure to many people. the infectious agent used is likely to be uncommon and possibly not endemic to the region, might have been modified genetically to make it resistant to current medications and vaccines, and produced in a way that enhances its transmission or virulence. therefore, early clinical symptoms and signs after infection with a bioterrorist agent might be unusual, complicating both recognition and management of the disease. these factors could create greater public panic. despite the many similarities with naturally occurring outbreaks of infectious disease, preparedness for bioterrorist attacks is more complex. in many aspects, a bioterrorist attack has the characteristics of a mass casualty event, and thus preparedness involves strengthening of the specialised infrastructure that is required for treatment of seriously ill patients over a very short period of time. new prophylactic and treatment regimens for unusual diseases are required, to ensure their accessibility when needed, along with clear standards for the handling and study of dangerous pathogens. when the proportion of available resources poor international preparedness • delay before the who declared a public health emergency of international concern • delay in implementing coordinated international assistance • logistic challenges in delivering support to assist epidemic response • shortcomings in who's regional and country-level capacity exposed • lack of global plans to address an epidemic of a high-risk pathogen in the least developed urban centres • evaluation of promising vaccine and therapeutic interventions came too late and ebola virus. the local and international responses to the west african ebola virus epidemic revealed shortcomings that could allow highly contagious epidemics of infectious disease to spread widely before they are terminated (panel ). during the cold war, agents that could potentially be used as biological weapons were identified on the basis of the following characteristics: pathogenicity for humans, animals, or plants; ability to cause disability or death; stability and infectivity as small particle aerosols; and capability of being readily and rapidly produced and weaponised in munitions or delivery systems. more characteristics have been added, to include other features of biological agents such as the relative ease of medical prevention or treatment and the likelihood of harm to the perpetrator. the us centers for disease control and prevention (cdc) identified bacteria, viruses, and toxins that could potentially be weaponised (panel ). in , they categorised them into three groups-a, b, and cdepending on ease of dissemination, severity of illness caused, and ability to cause death. biological agents can be infectious and contagious, infectious but not usually contagious, or toxins if they are neither. category a agents were considered the greatest risk to public and national security. the more recent classification of tier select agents and toxins is similar to the category a classification (table ) . other agents, such as naturally occurring pathogens, produce diseases that are considered of intermediate risk to the public (eg, brucellosis, glanders, q fever). they are moderately easy to disseminate, and include emerging and re-emerging infectious diseases. however, genetic modifications could make them more virulent, produce uncharacteristic clinical signs, increase their resistance to treatment and vaccines, and even change their transmissibility or host range. genetic modifications could be made using the tools of synthetic biology; such activities might be an example of dual-use research. , for instance, in , the spanish influenza pandemic virus was reconstructed, and the poliovirus was synthesised nearly years ago. the addition of an immuno-modulatory gene to the mousepox virus genome in , rendered a mousepox vaccine ineffective, and this technology could potentially be applied to the smallpox virus. the recent synthesis of the extinct horsepox virus has been a reminder that the smallpox virus could be reconstructed, and that the regulations that have been put in place to prevent the misuse of powerful, cheap, and globally available tools must be reconsidered. this possibility has also raised the issue of whether research results should sometimes be censored, or even refused publication, if the potential to cause harm is too high. although bioterrorist agents could be disseminated through multiple routes, the aerosol route would likely maximise exposure. contagious agents could produce a large number of second and later generation cases, depending on the number of people initially exposed, the series average number of people who acquire the disease from one infected individual (r ), and the disease generation time in humans. for instance, the r of pneumonic plague has been estimated to be around · , whereas the r for smallpox is likely to be around . for diseases that are not contagious, such as inhalational anthrax, the number of cases of disease will depend almost entirely on the size of the population exposed and the timing of post-exposure antibiotic prophylaxis. aerosolised agents remain the threat of most concern, but safety and security of food [ ] [ ] [ ] and water supplies are also important components of primary prevention (panel ). new methods to detect toxins in food, such as antibody based assays, are being developed. rapid diagnostics take on additional urgency in a bioterrorist event, because of both health and security concerns. since the anthrax letters, there have been major advances in diagnostic capabilities. some of the greatest advances in the past decade have been in the speed and reduced cost of sequencing capabilities. , highly sensitive and specific pcr-based systems, coupled with modern sample preparation technologies, have enabled sequencing technologies to become less costly, more portable, and multiplexed. with fieldable patient-side diagnostics and sequencing outputs directly connected via cloud-based networks, health-care providers globally can make decisions more rapidly and respond more quickly for individual care or outbreak detection. a rapid, cartridge-based assay for francisella tularensis has been developed for use at point of care. a system that uses a sensitive microsphere technology to detect both antibodies and antigens is now available to diagnose infections with ebola virus and lassa virus. although diagnostic elisa tests are available for anthrax antibodies, , a compact system (genexpert) that includes both sample processing and pcr amplification can produce a result in about minutes. a rapid and sensitive method to detect smallpox virus has been developed for use at point of care, based on antibody immuno column for analytical processes (abicap) immunofiltration, that produces results in about minutes. however, diagnostic electron microscopy is also still considered a fast and efficient method to identify smallpox and other viral agents. ebola virus was rapidly sequenced during the outbreak in sierra leone to link sporadic cases with the transmission chains. advanced proteomics are also being developed as reference assays and a new method for simultaneous immunodetection of anthrax, plague, and tularaemia from blood cultures has recently been reported, using multiplexed suspension arrays. next generation safety and security of food [ ] [ ] [ ] and water supplies are important components of primary prevention: • intentional contamination of food should be considered, particularly during a large foodborne epidemic with a common source • salmonella and shigella species, enterohaemorrhagic escherichia coli (all serotypes), vibrio cholerae, cryptosporidium parvum, and noroviruses are all potential candidates for intentional contamination of food • contamination of water with biological agents should still be considered, even though it is unlikely to be the major target of bioterrorism, due to chlorination, dilution, and the need for large quantities of the agent to cause a substantial outbreak • c parvum and noroviruses are more resistant to chlorination than other agents, so can be a threat to the water supply • food-borne or water-borne dissemination of these biological agents might lead to higher rates of morbidity and case fatality than previously observed, if the population has been exposed to substantially higher infectious doses • algorithms could be developed to measure the likelihood that outbreaks of disease were a consequence of intentional contamination of food or water, using descriptive, analytical, and molecular epidemiologic tools (none are known to be available so far) increased networking and collaboration of laboratories will also improve the response to intentional outbreaks. effective global surveillance of infectious diseases is essential to control both intentional and naturally occurring epidemics. surveillance data can be used to monitor the progress of an outbreak, and for risk communication. to obtain information rapidly, the ongoing collection of health-related data (termed syndromic surveillance) has been introduced, to monitor patterns of symptoms and signs that are suggestive of an outbreak. [ ] [ ] [ ] [ ] although it was hoped that syndromic surveillance would be a more sensitive method for early detection of an epidemic, frequent reports of unusual increases in incidence of non-specific illnesses can desensitise and paralyse the system. in fact, early detection will depend largely on alert, prepared clinicians. for example, when the anthrax attacks occurred, an astute clinician identified the index case. emergency room and community physicians should be updated regularly on the clinical signs and symptoms associated with the most common bioterrorist agents. the syndromic surveillance system would be more useful after suspicious or confirmed cases have been reported by physicians. a focused analysis of the surveillance data against non-specific, background disease rates could detect changes and provide information about the dynamics of the disease. special legislation might then be necessary, to gain access to medical records. the internet facilitates other potential forms of surveillance and communication about infectious diseases. , one such system is promed, which was established by the user community and has proven effective in connecting clinicians and scientists around the world; it has already served as an early warning system standard contact and airborne precautions should be taken. supportive therapy and antibiotics can be provided for secondary infections. there is some evidence of the potential efficacy of thiosemicarbazones. cidofovir has shown in vitro efficacy against variola, and has shown efficacy against other diseases caused by human orthopoxviruses, notably diseases caused by vaccinia viruses. it has also shown efficacy in animal models of orthopoxvirus infections. since , the us food and drug administration (fda) has approved drugs and biologic agents developed under the animal rule. this rule allows for approval of a drug that cannot be tested for efficacy in humans, but is effective in animals and safe in humans. the first drug approved under this rule was the monoclonal antibody raxibacumab for treatment of inhalation anthrax. tecovirimat is a drug that inhibits all orthopoxviruses tested in vitro. it was found to be highly effective in treating monkeypox and rabbitpox in animals and is considered safe in humans. tecovirimat is being considered by the fda for approval for use in humans to treat smallpox under the animal rule. standard and droplet precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline have been approved for the treatment of pneumonic plague. streptomycin and gentamicin have been found to be effective in treatment, although there is some evidence of the development of multiple resistance. , tularaemia isolation of the patients is not necessary and standard precautions should be taken. ciprofloxacin, levofloxacin, and doxycycline are all approved for the treatment of tularaemia. streptomycin and gentamicin have been found to be effective. , haemorrhagic fevers standard contact and airborne precautions should be taken until diagnosis is confirmed. subsequently, droplet precautions can be considered. supportive care and treatment of secondary infections can be provided. ribavirin is now approved for treatment of lassa fever and it also appears to be effective against new world arenaviruses and crimean-congo haemorrhagic fever. protective n respirators and clothing should be provided to health-care personnel. clothing of patients should undergo decontamination and thorough handwashing. supportive therapy is available, with antibiotics such as ciprofloxacin, doxycycline and ampicillin. if the bacteria are resistant to some of the antibiotics, the treatment regimen will depend on sensitivity testing. the regulations require immediate reporting of serious health risks by all member countries. additionally, who has established the global outbreak alert and response network, and the european union has a programme called bichat to improve cooperation between member states in preparedness and response to biological and chemical attacks. they operate the early warning and response system for outbreaks of communicable diseases. the world organisation for animal health has developed plans to identify and deal with a bioterrorism attack on populations of food-producing animals. canada has established the global public health intelligence network for worldwide monitoring of threats to public health. a major benefit of a less formal global collaboration is the development of networks of trust among knowledgeable scientists and clinicians, who are considered early warning posts for both natural and intentional outbreaks. the one health initiative encourages collaboration between health professionals and is as important for bioterrorism preparedness as it is for management of emerging infectious disease and the global spread of antimicrobial resistance. the management of patients that have been infected during incidents of bioterrorism can be challenging. precautions and treatment regimens for several bioterrorist agents are summarised in panel . although supportive care serves as the basis of management for all agents, treatments for some of the relevant diseases have substantially progressed. the management of inhalation anthrax has advanced since the attack, , , with improvements in critical care, and in treatment of acute lung injury and acute respiratory distress syndrome, severe sepsis, and septic shock. pleural effusions are routinely drained and there are more options for antimicrobial therapy. antibiotics are still recommended for days after exposure or diagnosis, together with the anthrax vaccine. if the vaccine is given concurrently with antibiotic treatment, the period of treatment could be shortened. antibiotics for treatment of other bacterial infection are usually given for shorter periods, since the causative agents do not sequester spores. tularaemia is treated with ciprofloxacin or doxycycline. for smallpox, antivirals such as cidofovir or a related acyclic nucleoside phosphonate analogue appear to be more effective than post-exposure vaccines in preventing mortality, according to experiments in non-human primates infected with monkeypox virus. this suggests that antivirals might play an important role when preparing for a smallpox outbreak. for viral haemorrhagic fevers, ribavirin might have some efficacy in post-exposure prophylaxis. a small-molecule antiviral drug, gs- , has been developed that appears to be effective in treating ebola virus infection. for intentional and other sudden outbreaks of contagious disease, isolation of patients and controlling risks to health-care workers remains extremely challenging, as was noted in the mers coronavirus, sars, ebola virus disease, and avian influenza epidemics. hospital units adequately equipped for isolation are needed, similar to those equipped to care for filovirus-infected patients, with negative pressure air filtration. if facilities are too small for the number of patients, a lower level of isolation with strict barrier nursing should be implemented, and in the event of a very large outbreak, there might be a need to set up isolation facilities in public buildings. in regions with poor infrastructure, it might be necessary to consider treating patients in their homes. people who have died should be regarded as infectious and handled with the same precautions used for patients. burial procedures might have to be modified, but every effort should be made to respect religious practices and traditions of the local culture. quarantining of people who might have been exposed to the infectious agent can be problematic, as was the case in the sars and west african ebola virus epidemics. since the quarantined population includes both people who were exposed and people who were not, the risk of disease transmission is higher. during the ebola virus outbreak in west africa, suspect cases were held until they could be cleared as negative, which took about days pending the pcr results. on a national level, reducing the movement of populations is a sensitive issue, potentially interfering with commerce. closure of schools is an important means of achieving social distancing to reduce spread. whether the public should use masks during an outbreak of a contagious disease is less clear, and the efficacy of the masks is extremely variable. the most important reasons for variable efficacy are differences in facial shape, incorrect application, and duration of use. a substantial proportion of the cases and fatalities in the sars and ebola virus epidemics were among healthcare workers. clear guidelines specific to each agent are available to health-care personnel, public health workers, and emergency workers for the use of masks and personal protective equipment. the national ebola virus training and education center has been established in the usa to train health-care workers and assist hospitals in preparing for patients infected with high hazard virus in the usa and other countries. laboratory workers must be trained to work with dangerous pathogens and wear protective gear. designated threat pathogens must be stored, handled, and transported under a different set the role of vaccines in pre-exposure and post-exposure prophylaxis measures should be in place to protect the population from biological agents likely to be used in an attack before an incident occurs. however, since a bioterrorist incident is likely to be caused by biological agents not covered by routine immunisation, pre-exposure prophylaxis is generally confined to vaccines for military forces, health-care workers, and emergency response personnel. to the majority of the population, only postexposure prophylaxis is relevant. post-exposure prophylaxis for an intentional outbreak would include both those people known to be exposed during the incident and those people who were infected by others. the prophylaxis itself might consist of both vaccines and antimicrobials. when using live, attenuated vaccines, the relatively large proportion of the population who has some form of immunodeficiency has to be taken into account. , monoclonal antibody preparations are now being considered for prophylaxis in select, high risk groups. table summarises pharmacological prophylaxis for tier pathogens. currently, the vaccines that would most likely be used for pre-exposure or post-exposure prophylaxis are the smallpox and anthrax vaccines. since routine vaccination against smallpox was stopped in the s, less than % of the world's population has been vaccinated, and antibody titres usually decline markedly after to years. residual cellbased immunity can persist for many years. , postexposure prophylaxis involves ring vaccination, which requires intensive tracing and vaccination of primary contacts, followed by vaccination of secondary contacts, and finally, vaccination of all people in a defined affected region. for post-exposure prophylaxis of those directly exposed during the incident, vaccination can be effective if given within to days of exposure. since it might take that amount of time to detect the first cases after exposure, the vaccine will generally only be effective for secondary and subsequent contacts. immune-boosting adjuvants and toll-like receptor agonists have the potential to improve the immune response to post-exposure vaccination. serious side-effects are relatively rare , but can affect com pliance. in those cases, lower doses of vaccine might be administered, to provide adequate protection with fewer side-effects. newer smallpox vaccines are in development, including those that could immunise people who have atopic dermatitis. because it is produced in small quantities by collecting antiserum from immunised humans, there might be a shortage of vaccinia immune globulin, used to treat people who would have serious side-effects with the vaccine. one possible solution to this shortage would be to use antibodies against other poxviruses, such as cowpox and monkeypox, because of their cross-protective properties. since naturally occurring inhalation anthrax is extremely rare, there have been safety and immunogenicity profiles of the anthrax vaccine in humans, but the efficacy has only been tested in animal models, and not in clinical trials. [ ] [ ] [ ] the current anthrax vaccine is made from culture filtrates of a toxigenic, avirulent, nonencapsulated mutant of the bacillus anthracis vollum strain, and is administered in five intramuscular doses, followed by annual boosters. the protective, antigen specific memory b cells persist for many years after vaccination and are associated with humoral immunity. serum igg response to the vaccine has been % after the fourth dose. for post-exposure prophylaxis in unvaccinated people, the vaccine should be administered as a three-dose subcutaneous series (at , , and weeks), in conjunction with a -day course of appropriate antimicrobial drugs. it has been given to thousands of us military personnel, and notable adverse events have been rare. the anthrax vaccine is not recommended for pregnant women, although one study with women in the us military, inadvertently vaccinated during pregnancy, did not show evidence of an increase in birth defects. in this study, women received the vaccine in the first trimester and in the second and third trimeseter. more effective anthrax vaccines that require fewer doses are constantly being tested, , such as the neat protein anthrax vaccine, a dual purpose influenza vaccine that protects against anthrax, and a combined anthrax-plague vaccine. apart from the d yellow fever live attenuated vaccine and the junin virus vaccine, no vaccines for haemorrhagic fevers have been licensed. since the west african ebola virus epidemic, new ebola virus vaccines that have been long under development are being used successfully in the epidemic in the democratic republic of the congo. essentially no other licensed vaccines are available for other tier select agents. the previously licensed, formalin-inactivated, whole-bacilli plague vaccine has not proven effective against primary pneumonic plague in non-human primate models, series us military. the vaccine has not been used widely for preexposure prophylaxis and has no place in post-exposure prophylaxis. new vaccines against tularaemia are under development , including one that might provide crossprotection between plague and tularaemia. the investigational pentavalent (abcde) botulinum toxoid vaccine was provided by the cdc for laboratory workers at high risk of exposure to botulinum toxin and it has also been given to military members that are at risk. the botulinum toxoid vaccine produces effective immunity after several months and has no value for postexposure prophylaxis because of the short latent period of the toxins. this vaccine was discontinued in , because of declines in immunogenicity and adverse events. new recombinant botulism vaccines are being developed, in addition to vaccines against glanders and rift valley fever. [ ] [ ] [ ] efforts are ongoing to greatly shorten the time required to develop and produce new vaccines and other immune approaches. improved technologies are important for rapid scale-up and production of new treatment regimens, particularly following an attack with a contagious agent. the largely unpredictable nature of an epidemic initiated intentionally is likely to increase uncertainty and reduce public trust in the authorities. public education and effective risk communication are essential to increase public confidence and improve cooperation and compliance with recommended medical counter-measures. the anthrax vaccine in military populations has caused considerable scepticism regarding the need for, safety, and efficacy of the vaccine. [ ] [ ] [ ] clinicians and public health personnel should have access to up-to-date information, and the general public should be provided with nontechnical information and simple instructions on how to act during an emergency. sandman has proposed that "one should not over-reassure, ack nowledge uncertainty, and share dilemmas". this behaviour would only cause overreaction or panic when new information about the risk is made public. risk communication will be necessary at all stages: before a bioterrorist incident occurs, when an incident is suspected, when it is confirmed, while it is taking place, and in the aftermath. credible and trusted spokespersons, including respected clinicians, scientists, and public servants for a country, should be adequately informed before an incident. during an outbreak, there could be unexpected events, such as atypical presentation of cases, varying responses to treatment (including unusual side-effects), and false positive and false negative diagnoses. the public might lose trust in the authorities if apparently unexposed people become ill. the advent and global distribution of social networking increases the risk of the dissemination of false or misleading information. lastly, a major infectious disease incident will also require flexibility and possible changes of established government policy. environmental detection of biological agents is another area of research that should be developed. to date, most systems of environmental detection have focused on anthrax, as a result of the anthrax attacks. , however, a sensitive and specific set of recombinase polymerase amplification assays for fast screening, detection, and identification of b anthracis in a field setting has recently been developed. the rare occurrence and likely small effect of an aerosol bioterrorist attack limits the practical use of environmental detection to special event venues, public transportation systems, and possibly some government buildings thought to be likely targets. many countries have national stockpiles of drugs and vaccines, for use in the event of a biological or chemical attack, or for serious outbreaks that might achieve epidemic proportions. the usa, for instance, maintains a strategic national stockpile of vaccines and other medical countermeasures. global stores of smallpox vaccines are held by who, in addition to stores held by individual countries. some countries have undertaken active vaccination programmes against smallpox and anthrax in the military and first responder populations. preparedness for bioterrorist incidents requires constant re-evaluation of policies. although there is no evidence that the h n influenza pandemic or the - ebola virus epidemic in west africa were initiated intentionally, the local and international responses revealed strengths and weaknesses in the current state of preparedness for bioterrorist incidents. the ebola virus epidemic spread to a number of countries, with more than cases reported worldwide and a case fatality rate of more than %. imported cases of ebola virus disease were identified in the usa and spain. locally, in the affected countries in west africa, % of the people who died because of ebola virus disease were health-care workers. various shortcomings in the response to the epidemic have been identified since (panel ). accurately predicting the intentional misuse of a biological agent to cause harm is difficult without intelligence data, but several attempts have been made to rationally predict the categories of risk: man-made, natural, accidental, contagious, and non-contagious. series risks. the risk of bioterrorism has called into question some of the dogmas related to eradication of diseases such as poliomyelitis and measles. for example, if polio is successfully eradicated, universal vaccination might have to continue because of the risk of poliovirus being used as a bioterrorist agent. [ ] [ ] [ ] emerging and reemerging infectious diseases will continue to be a threat, but preparedness for bioterrorism is, in many ways, similar to preparedness for naturally emerging disease. all countries should collaborate to address the root causes of terrorism, and develop appropriate preventive strategies. effective preparedness is, in itself, a deterrent to bioterrorism, since it reduces the incentive to use biological weapons by making a country or region a hard target. it is also the cornerstone of consistent and effective responses to naturally occurring epidemics. the abuse of biological agents can be further reduced or discouraged with reliable intelligence and an effective response if it does occur. national and regional resources and capabilities will vary, but all will require infrastructures that are capable of recognising and dealing with a variety of biological agents. the needs of specific populations, such as the paediatric population, pregnant women, elderly people, and people with immunological disorders, must also be addressed. funding for biodefence is crucial to adequate preparation and response to bioterrorist threats. international preparedness for bioterrorism has the dual benefit of strengthening the infrastructure for responding to naturally occurring epidemics of highly pathogenic organisms. lessons from the west african ebola virus epidemic show that health-care providers must always be watchful for unusual presentations of disease, and new and improved approaches must be developed for early detection and response. health-care providers need more effective means of isolating infected patients, and better methods to control the movement of potentially infected people outside of the affected areas. personal protective equipment should be inexpensive and effective, and available to use with minimal training and under harsh environments. protection of health-care workers against infection remains particularly prob lematic, and should be a focus of research and development. the ebola virus epidemic has highlighted the importance of improving the logistics of moving human and material resources in areas with relatively poor infrastructure. risk communication and public education before and during an outbreak need to be improved. more clinical trials should be fast-tracked during development of new vaccines and antiviral drugs. preparedness for a low-risk, high-impact event that is bioterrorism should be monitored constantly, tested in tabletop exercises, , and integrated into the routine functioning of the health system. here it would serve the dual purpose of ensuring that countries are prepared to meet the challenges of controlling epidemics of emerging and re-emerging infectious diseases. msg designed the review, did the literature search and was responsible for writing the manuscript. jld assisted in the literature search, revised the manuscript, and supplied technical expertise. dc assisted in the literature search and revised the manuscript. drf helped design the review, contributed source material, did the literature search, and helped write and revise the manuscript. we declare no competing interests. we searched pubmed and google scholar using the terms "bioterrorism", "sustainable bioterrorism preparedness", "all-hazards infectious disease preparedness", "biological threat agents", "bioterrorism preparedness", "emerging infectious diseases", "smallpox", "anthrax", "plague", "tularemia", "botulism", "hemorrhagic fevers", and "risk communication". we searched national and international reports from the who and us centers for disease control using the terms "bioterrorism", "bioterrorism preparedness", and "emerging infectious diseases". we also completed web searches for "disease surveillance", "infectious disease diagnostics", "medical countermeasures", "emergency healthcare delivery", and "risk management". we focused on academic literature in english and restricted most of our searches to documents published since , with an emphasis on those published after , and included mainly those published since . federal funding for health security in fy a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, : epidemiologic findings remediation of bacillus anthracis contamination in the u.s. department of justice mail facility total decontamination cost of the anthrax letter attacks bacillus anthracis incident post-traumatic stress disorder symptoms in victims of tokyo subway attack: a -year follow-up study biological warfare and bioterrorism: a historical review the threat of bioterrorism: identifying the unknown. ffi focus biodefense in the st century department of health and human services (hhs). possession, use, and transfer of select agents and toxins-addition of bacillus cereus biovar anthracis to the hhs list of select agents and toxins. interim final rule and request for comments destruction of microbial collections in response to select agent and toxin list regulations implementing the select agent legislation: perfect record or wrong metric? gaps remain in china's ability to detect emerging infectious diseases despite advances since the onset of sars and avian flu health system resource gaps and associated mortality from pandemic influenza across six asian territories ebola: lessons learned and future challenges for europe public health assessment of potential biological terrorism agents responsible conduct by life scientists in an age of terrorism mitigating the risks of synthetic biology. council on foreign relations characterization of the reconstructed spanish influenza pandemic virus chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox labmade smallpox is possible, study shows the de novo synthesis of horsepox virus: implications for biosecurity and recommendations for preventing the reemergence of smallpox epidemiologic determinants for modeling pneumonic plague outbreaks risk assessment and risk communication strategies in bioterrorism preparedness. nato security through science series a: chemistry and biology estimating time and size of bioterror attack threat of a biological terrorist attack on the us food supply: the cdc perspective a large food-borne outbreak of group a streptoccocal pharyngitis in an industrial plant: potential for deliberate contamination german outbreak of escherichia coli o :h associated with sprouts water and bioterrorism: preparing for the potential threat to u.s. water supplies and public health a proteomics assay to detect eight cbrn-relevant toxins in food index case of fatal inhalational anthrax due to bioterrorism in the united states rapid outbreak sequencing of ebola virus in sierra leone identifies transmission chains linked to sporadic cases real-time pcr to identify variola virus or other human pathogenic orthopox viruses pocket dna sequencers make real-time diagnostics a reality sensitive detection of francisella tularensis directly from whole blood by use of the genexpert system comparison of magpix assays and enzyme-linked immunosorbent assay for detection of hemorrhagic fever viruses the early humoral immune response to bacillus anthracis toxins in patients infected with cutaneous anthrax specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin g antibodies to anthrax toxin protective antigen rapid detection of bacillus anthracis bloodstream infections by use of a novel assay in the genexpert system rapid and sensitive point-of-care detection of orthopoxviruses by abicap immunofiltration rapid viral diagnosis of orthopoxviruses by electron microscopy: optional or a must? striking against bioterrorism with advanced proteomics and reference methods simultaneous immunodetection of anthrax, plague, and tularemia from blood cultures by use of multiplexed suspension arrays the changing face of pathogen discovery and surveillance public health surveillance and infectious disease detection evaluation of a syndromic surveillance system using the wsare algorithm for early detection of an unusual, localized summer outbreak of influenza b: implications for bioterrorism surveillance syndromic surveillance during pandemic (h n ) outbreak internet-based surveillance systems for monitoring emerging infectious diseases digital surveillance for enhanced detection and response to outbreaks if syndromic surveillance is the answer, what is the question? clinical recognition and management of patients exposed to biological warfare agents public health. ethics and the conduct of public health surveillance digital disease detectionharnessing the web for public health surveillance factors influencing performance of internet-based biosurveillance systems used in epidemic intelligence for early detection of infectious diseases outbreaks the internet and the global monitoring of emerging diseases: lessons from the first years of promed-mail social and news media enable estimation of epidemiological patterns early in the haitian cholera outbreak geneva: world health organization the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health with the changing biological threat…smart international engagement policy would lower cost and increase national security animals as sentinels of bioterrorism agents clinical management of potential bioterrorism-related conditions wisconsin department of health services. infection control and prevention-standard precautions oral tecovirimat for the treatment of smallpox plague: recognition, treatment, and prevention the sanford guide to antimicrobial therapy new therapeutic approaches for treatment of tularaemia: a review centers for disease control and prevention expert panel meetings on prevention and treatment of anthrax in adults antitoxin treatment of inhalation anthrax: a systematic review consequences of delayed ciprofloxacin and doxycycline treatment regimens against francisella tularensis airway infection a single cidofovir treatment rescues animals at progressive stages of lethal orthopoxvirus disease therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu implementing a negative-pressure isolation ward for a surge in airborne infectious patients large-scale quarantine following biological terrorism in the united states: scientific examination, logistic and legal limits, and possible consequences ebola and fda: reviewing the response to the outbreak, to find lessons for the future assessment of the potential for international dissemination of ebola virus via commercial air travel during the west african outbreak transmission of ebola viruses: what we know and what we do not know estimating the size of the u.s. population at risk of severe adverse events from replicating smallpox vaccine influence of population immunosuppression and past vaccination on smallpox reemergence next-generation monoclonal antibodies: challenges and opportunities analysis of historical data suggests long-lasting protective effects of smallpox vaccination duration of neutralizing antibody persisting in thai individuals after childhood vaccination against smallpox a model for a smallpox-vaccination policy can postexposure vaccination against smallpox succeed? immune-boosting adjuvants tlr and tlr agonists improve postexposure vaccination efficacy of live smallpox vaccines risks of serious complications and death from smallpox vaccination: a systematic review of the united states experience adverse events following smallpox vaccination with acam in a military population factors associated with healthcare worker acceptance of vaccination: a systematic review and meta-analysis reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses comparing new-generation candidate vaccines against human orthopoxvirus infections long-term safety of replication-defective smallpox vaccine (mva-bn) in atopic eczema and allergic rhinitis cross-neutralizing and protective human antibody specificities to poxvirus infections randomized, double-blind, placebo-controlled, safety and immunogenicity study of formulations of anthrax vaccine adsorbed plus cpg (av ) in healthy adult volunteers select human anthrax protective antigen epitope-specific antibodies provide protection from lethal toxin challenge protective antigen-specific memory b cells persist years after anthrax vaccination and correlate with humoral immunity lethal factor antibodies contribute to lethal toxin neutralization in recipients of anthrax vaccine precipitated serum igg antibody response to the protective antigen (pa) of bacillus anthracis induced by anthrax vaccine adsorbed (ava) among u.s. military personnel an overview of adverse events reported by participants in cdc's anthrax vaccine and antimicrobial availability program safety of inadvertent anthrax vaccination during pregnancy: an analysis of birth defects in the u.s. military population progress and novel strategies in vaccine development and treatment of anthrax host immunity to bacillus anthracis lethal factor and other immunogens: implications for vaccine design progress toward the development of a neat protein vaccine for anthrax disease a dual purpose universal influenza vaccine candidate confers protective immunity against anthrax a bivalent anthrax-plague vaccine that can protect against two tier- bioterror pathogens, bacillus anthracis and yersinia pestis experimental treatment of ebola virus disease with tkm- : a single-arm phase clinical trial plague vaccines: current developments and future perspectives plague vaccines: status and future intranasal delivery of a protein subunit vaccine using a tobacco mosaic virus platform protects against pneumonic plague protective immunity against lethal f. tularensis holarctica lvs provided by vaccination with selected novel cd + t cell epitopes francisella tularensis live vaccine strain deficient in capb and overexpressing the fusion protein of igla, iglb, and iglc from the bfr promoter induces improved protection against f. tularensis respiratory challenge yopp-expressing variant of y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia notice of cdc's discontinuation of investigational pentavalent (abcde) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins recombinant botulinum neurotoxin hc subunit (bont hc) and catalytically inactive clostridium botulinum holoproteins (cibont hps) as vaccine candidates for the prevention of botulism burkholderia pseudomallei and burkholderia mallei vaccines: are we close to clinical trials? safety and immunogenicity of a mutagenized, live attenuated rift valley fever vaccine, mp- , in a phase dose escalation and route comparison study in humans bioterrorism risk communication policy why do uk military personnel refuse the anthrax vaccination? a longitudinal study of uk military personnel offered anthrax vaccination: informed choice, symptom reporting, uptake and pre-vaccination health notes from the field: compliance with postexposure prophylaxis for exposure to bacillus anthracis among u.s. military personnel-south korea rapid detection of bacillus anthracis spores using immunomagnetic separation and amperometry rapid detection of viable bacillus anthracis spores in environmental samples by using engineered reporter phages sensitive and specific recombinase polymerase amplification set of assays for fast screening, detection and identification of bacillus anthracis in a field setting the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences pandemic preparedness and response-lessons from the h n influenza of global catastrophic biological risks: toward a working definition expert views on biological threat characterization for the u.s. government: a delphi study the risk of bioterrorism re-analysed risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication eradicating polio: a balancing act vaccination against polio should not be stopped vaccines should be kept even if polio is wiped out ebola and zika: cautionary tales medical countermeasure development since : a long way yet to go preparing for biological threats: addressing the needs of pregnant women safety of live vaccinations on immunosuppressive therapy in patients with immune-mediated inflammatory diseases, solid organ transplantation or after bone-marrow transplantation-a systematic review of randomized trials, observational studies and case reports biosecurity: assessing the bioweapons threat a plague on your city: observations from topoff shining light on "dark winter key: cord- -fsj dg authors: patterson, grace t.; thomas, lian f.; coyne, lucy a.; rushton, jonathan title: moving health to the heart of agri-food policies; mitigating risk from our food systems date: - - journal: glob food sec doi: . /j.gfs. . sha: doc_id: cord_uid: fsj dg our food systems are progressively more industrialized and consolidated with many modern food value chains involving multiple countries and continents, and as such being associated with changes in risk profile and impacts of emerging and re-emerging diseases. disease outbreaks that sweep through a single region can have massive impacts on food supply, while severe outbreaks of human pathogens can disrupt agricultural labor supply or demand for products perceived as ‘unsafe’. market pressures have generally rewarded production of cash crops for fuel and energy dense, low nutrient processed foods over production of fruits and vegetables for local consumption. climbing rates of food-related ncds and pre-existing conditions leave the population increasingly susceptible to infectious diseases that are often driven by or arise from the food system. therefore disease and diet from our food systems cause impacts on human health, and human health issues can impact on the functioning of the food system. the covid- outbreak is the most recent example of food system driven disease emergence and of massive supply and demand shocks in the food system, experienced as a direct and indirect result of this disease. the effects of the food system on disease spread (and vice versa) must be addressed in future plans to prevent and mitigate large scale outbreaks. health policies must acknowledge the food system as the base of our health system, as must agri-food policy recognize the pre-eminence of human health (directly and indirectly) in decision making. the health of the global population is underpinned by our food systems, yet these systems are often only nominally included in health policy. in recent times, attention has been given to reformulation and marketing policies in moderate attempts to reduce climbing rates of obesity and non-communicable diseases (ncds) (oecd, ) . others focus on reducing foodborne pathogens by regulating processing and enacting trade restrictions (trienekens and beulens, ) . many of these policies focus on downstream actors and processes in the food system and neglect the foundations of how our food is produced. while such interventions are easier to enact, they have limited effects on health and are generally reactionary and insufficient to reverse the trajectory of increasing health burdens. in this manuscript we review some of the major human health externalities driven by our current food systems, with an emphasis on livestock-systems and their linkages to the wider food system, the impacts of our response to these externalities and outline key areas in which better integrated health & agri-food policy may mitigate these factors. although the interactions between food systems and our health are many and complex, we outline here four key health externalities arising from these systems of concern across the globe; ) the dual burden of malnutrition, ) foodborne disease, ) antimicrobial resistance and ) emergence of novel pathogens. despite an increasing ability to produce sufficient calories for the growing human population, distributional inequalities have produced a situation where we face the dual burden of chronic under-nutrition (black et al., ) and a global pandemic of overweight and obesity and related ncds, often within the same geographical locality (shrimpton and rokx, ) . diet related risks are estimated to be responsible for million ( - mn) disability adjusted life years (dalys) a year globally, and dietary improvements could potentially prevent one in every five global deaths (afshin et al., ) . diet related risks also monopolize large portions of many national health-care budgets (phe, ) . from - , it is estimated that an average of ⋅ % of healthcare expenditure in the organisation for economic co-operation and development (oecd) member countries will be spent treating obesity-related diseases, equivalent to us dollars (usd) purchasing power parity billion per year (oecd, ) . current food systems exploit demand for affordable, convenient, and tasty food to encourage consumption of nutritionally poor, ultraprocessed foods in both high, middle and low income countries. as economic markets incentivize expanded food value chains, they inevitably produce food with more and more processing to improve taste, texture, and longevity (swinburn et al., ) . the processed state of these foods, not necessarily the macronutrient content or energy density, induces increased calorie intake and weight gain associated with exposure to ultra-processed foods (hall et al., ) . marginalized groups in high income countries are often reliant on these highly processed, energy-dense, nutrient poor foods to meet their energy needs. ultra-processed foods can be aspirational in low and middle income countries (lmics) and are becoming more affordable and convenient (swinburn et al., ) . these foods (and the value chains that produce and market them) are among the most impactful determinants of the growing global burden of food-related ncds. food-related ncds also increase susceptibility to infectious disease. for diseases such as zika, west nile, and dengue death is rare in the absence of any pre-existing conditions (badawi et al., ; chan-yeung and xu, ) and preliminary analysis suggests that the likelihood of adverse outcomes in sars-cov- are significantly higher in patients with hypertension and diabetes (zhou et al., ) . this increased risk may be driven by upregulation of the sars-cov- receptor ace or heightened inflammation in people with these comorbidities (pinto et al., ) . these diet-related co-morbidities are visible symptoms of underlying structural inequalities which result in marginalized communities bearing disproportionate disease burdens (garg et al., ; icnarc, ) . whilst the quantity and nutritional quality of our global diets gains increasing attention, foodborne diseases are also associated with a substantial human health burden, with approximately million dalys lost annually through of the major foodborne diseases, the burden of which are disproportionately borne by lmics (jaffee et al., ; li et al., ) . thirty-five percent of this burden is attributable to pathogens from animal source foods (asfs) (li et al., ) , the increasing demand for which, particularly across lmics, is a major driver in an increasing foodborne disease burden (quested et al., ) . specific aspects of livestock value chains associated with an increase foodborne disease risk may differ across 'informal' and 'formal value chains. high prevalence foci of the zoonotic tapeworm taenia solium, etiological agent of neurocysticercosis, are found where free-ranging pig husbandry practises coincide with low levels of sanitation and insufficient implementation of meat inspection . informal value chains, which supply - % of food needs in sub-saharan africa, and the poorly regulated slaughter, processing and retail practises associated with them can result in extensive microbial contamination of products, which may not be sufficiently mitigated by consumer food preparation practises (jaffee et al., ) . the formalisation of livestock value chains does not completely mitigate all foodborne disease risks. longer value chains with increasing number of processing or handling steps between 'farm and fork' provide opportunities for cross-contamination, adulteration or spoilage without sufficiently enforced regulation. the progressively intensified systems of livestock production seen increasingly across the globe may also exacerbate the risk posed by microbial hazards. intensive systems bring animals into close proximity under circumstances which induce metabolic and psychological stress (humphrey, ; martínez-miró et al., ) , increasing the contact opportunities and susceptibility of these populations for disease transmission, including those pathogens which have important implications for food safety. this is particularly evident in monogastric systems, where intensive production contributes to over half of the global pork production and over % of global poultry production (steinfeld et al., ; thornton, ) , and where campylobacter and salmonella spp. have been increasingly prevalent (carrique-mas et al., ) . in order to mitigate the effects of high pathogen challenge, which also have production-limiting effects, intensive livestock systems have been heavily reliant on antimicrobials for prophylactic, treatment and growth promotion reasons . resistant bacteria reside in humans, animals, food and the environment and there are no barriers to the transmission of resistance genes between these sites or amongst bacterial species (holmes et al., ) . pathways for the spread of resistant organisms, from within animal populations to humans, are well recognized and result from the selective pressures from antimicrobial use in livestock. consequently, antimicrobial use in food-producing animal species represents an important driver of antimicrobial resistance (amr) (marshall and levy, ) . the human health burden from amr is forecasted to reach trillion usd by with a worldwide mortality around million (o'neill, ) . in response to the amr crises, there is growing international pressure to reduce the use of antimicrobials within livestock systems and to prohibit the use of antimicrobial growth promoters (agps) in livestock. trade-offs exist, however, between the risks of antimicrobial use and the pressure to ensure global food security. a ban on agps could be associated with a reduction in the value of global meat production of between and billion usd and restricting the use of antimicrobials for prophylactic and therapeutic use whilst retaining current intensive husbandry practices will impact both productivity and animal welfare. lastly, disease emergence and transmission are of growing concern within our intensive, increasingly homogenous and interconnected food systems. lack of genetic diversity has become commonplace in our global food systems, within both livestock and crop production (bennett et al., ; khoury et al., ) , and is a risk factor for heightened susceptibility to outbreaks of plant and animal disease. this 'monoculture effect' is best illustrated in agronomy, where non-diverse cropping can lead to large scale losses, such as those suffered through rice blast (zhu et al., ) and the panama disease epidemic of bananas in the s (ordonez et al., ) . similar genetic susceptibilities exist within livestock. porcine reproductive and respiratory syndrome virus, a major disease burden to the global pork sector, has led to exacerbated losses within genetically homogenous herds as compared to herds with a wider genetic pool (lunney et al., ; halbur et al., ) . as well as the emergence of production limiting pathogens within livestock systems, the emergence of novel, zoonotic pathogens from our food systems is at the forefront of the global consciousness, of which sars-cov- is only the latest example in an increasing frequency of such events. novel zoonoses have emerged most commonly from wild mammals such as rodents and bats, which have either adapted to anthropogenic habitat changes, or have increased contact with humans or livestock through agricultural incursions into habitats or establishment of bush-meat value chains (johnson et al., ) . examples of direct pathogen spillover from wildlife directly to humans, predominantly associated with formal or informal bushmeat value chains, include hiv and ebola (hahn et al., ; kock et al., ) . there is also a potential threat to human health from the transmission of disease from wildlife into livestock populations and then further propagation through intensive production (wilcox et al., ) . in cases of the novel febrile encephalitis, nipah virus (niv), emerged amongst pig farmers in malaysia. the intensification of mango and pig production within a localised geographical area are believed to have provided a pathway for the virus, circulating in fruit bats, to infect an intensively managed commercial pig population and subsequently spread to farmers (pulliam et al., ) . zoonotic and non-zoonotic disease outbreaks and our responses to the presence or risk of these pathogens can destabilize food systems, leading to increased food insecurity and downstream health and economic effects. fig. illustrates several of the key disease outbreaks of the previous three decades which have had profound impacts on human health and food-security. in europe, outbreaks of bovine spongiform encephalitis (bse) and food and mouth disease (fmd) led to large scale supply and demand shocks in the beef and dairy industries. the use of meat and bone meal in cattle feed was associated with the mid 's emergence of the degenerative prion disease bse, which was later found to cause newvariant crutzfeldt-jacob disease (nvcjd) in humans (anderson et al., ) . this led to a raft of mitigation measures with short and long-term costs, including large scale culling programmes, new harvesting and processing regulation, a ban on animal by-product use in animal feeds and restrictions on beef and other animal products (including milk, semen and embryos) export from affected countries (kimball and taneda, ; thiermann, ) . the effects on the food system and health from the fmd outbreak in the uk also extended beyond production losses (knight-jones and rushton, ) . trade restrictions between fmd free and fmd affected countries were sometimes extended to unrelated products. culls produced public outrage against the livestock industry that outlasted the outbreak itself, which together likely contributed to the observed spike of depression and suicide among farmers (thomas et al., ) . in areas where a large proportion of the population is dependent on agriculture for both income and food supply, such as ethiopia, endemic fmd has direct effects on the food security of farmers by reducing milk production and oxen availability for cropping (knight-jones and rushton, ) . pork is now the most frequently consumed meat per capita worldwide and is responsible for over half of meat consumption in asia (ritchie and roser, ) . introduction of african swine fever virus (asfv) into china, home of approximately % of the global herd (ritchie and roser, ) , resulted in rapid spread throughout the naive pig population. over % of pigs have died or been culled (fao, ), causing an estimated $ billion in direct economic losses (moore, ) and reverberations through industries including feed mills, veterinary services, restaurants, and tourism. pork prices soared to a peak of % year-on-year (moore, ), increasing demand for and price of other meat products and leaving poorer households at increased risk of food insecurity and malnutrition (rocha et al., ) . similar patterns were seen across the same region less than a decade before in response to outbreaks of highly pathogenic avian influenza where aggressive containment measures drove increases in meat prices and heightened local food insecurity (burgos et al., ) . lmics, particularly those in southeast asia, are the site of the majority of industrialized poultry farming. large scale flocks are more likely to contract hpai (h n ) than small, personal flocks (otte et al., ) and experience the greater share of economic effects. yet smallholders, traders, slaughterers, and transporters are at greatest risk of personal negative health outcomes as a result of such diseases, as they often belong to poor households that are unable to weather economic shocks and are sensitive to food insecurity and malnutrition. the west african ebola outbreak of provides an example of severe, localized effects of an outbreak on the food system due to travel restrictions and market closures (mann et al., ) . panic buying caused shortages of some items and dramatic increases in prices. labor shortages resulted in decreased food production and loss of income across communities, particularly in rural regions (de la fuente et al., ) . nutrition operations were a low priority among humanitarian response groups and were inadequately supplied (kodish et al., ) . they also neglected to consider the needs of communities that did not have ebola outbreaks but had become food insecure from food production losses. after the outbreak, many ebola disease survivors were denied the opportunity to purchase food due to lingering fear of infection. on a global scale, the ongoing covid- outbreak has also led to massive perturbations in food systems. lockdowns and retail closures directly cause food insecurity among vulnerable populations, national stockpiling has prompted some countries to halt or reduce exports of staple grains and legumes (reuters, ) , and travel restrictions have inhibited farmers across europe from hiring seasonal migrant workers to harvest fruit and vegetable crops (carroll et al., ) . workers in food retail and transport that remain employed are experiencing unexpected health risks, severely reducing the ability of some sectors of our food-systems to operate as has been particularly noticeable in the vulnerability of meatpackers and subsequent covid- -related shutdown of some meat processing plants in usa (apostolidis, ) . workers provided with little protection or compensation have in some instances organized strikes to protest their lack of economic and health protection (mcginnis, ) and lengthy screening proceedures or social-distancing measures have the potential to dramatically reduce production capacity in some sectors (hailu, ) . once social distancing protocols are lifted, medium to large enterprises will likely be best poised to recover and many small businesses integral to local food systems will be unable to bounce back, producing economic knock-on effects on wholesalers, processors, and producers across the supply chain. while food insecurity surges as a result of control measures, food waste is also increasing. it is estimated that over a third of all food produced for human consumption is wasted (fao, ), a figure which will grow as shopping patterns change from multiple small shops per week to larger, more infrequent shops (cranfield university, ). food systems lack the flexibility needed to adjust to rapidly evolving situations within disease outbreaks, causing still more waste (evans, ) . restaurant and coffee shop closures have decreased demand for milk and dairy farmers have been told to pour milk down the drain, yet supermarkets have restricted purchase of milk and other core products (keane, ) . closures of large meat processing plants due to worker illness will result in large scale culling of animals across the usa. meanwhile, emergency assistance agencies are running low on supplies to support the deluge of the newly food insecure (power et al., ) . despite the undeniable link between our food systems and health, the sustainable development goals (sdgs) do not explicitly link these areas other than within the context of malnutrition (un, ) . public health policies must pay greater consideration to the role of food systems as the baseline of population health, while agri-food policies should consider human health as their raison d'être. agri-food policies can promote health in direct and indirect waysglobally % percent of employment is in agriculture, this rises to % in low income countries (world bank, ). sustainable agriculture supports economic prosperity, environmental wellbeing, and social equity (i.e. the triple bottom line), all of which further support stable access to healthy diets and healthy environments. well executed, integrated health-agri-food policy should improve baseline health, mitigate infectious disease risks and increase the resilience of our food systems to protect food security, particularly for the most vulnerable. herein we highlight some key examples of these policies in action, where further strengthening is required, and the dynamics that complicate enactment of health-centered agri-food policy (fig. ) . evidence-based health-agri-food policies that support access and uptake of healthy diet and exercise, particularly in communities with high inequality, can help reverse the growing trend towards obesity and minimize morbidity and mortality from infectious disease. a multipronged approach is needed to set healthy food preferences at an early age, change the environment to encourage healthy choices (particularly fig. . integration of health and agri-food policy. current and recommended agri-food and health policies to reduce the frequency and impact of disease outbreaks. at the point of purchase), and reduce barriers to expression of healthy preferences (hawkes et al., ) . policies that limit consumer exposure to less healthy foods by reducing portion sizes, reformulating foods, and regulating advertising are often blocked or weakened by lobbying groups (swinburn et al., ) . there is little immediate financial motivation to adopt more environmentally and health friendly standards, though social demand is increasingly reorienting markets to value transparency and social and environmental aspects of corporate performance. policy interventions that rely on behavioral change, such as consumer awareness campaigns and front of pack labeling, are relatively easier to enact but have limited longevity or effectiveness in the presence of other food stimuli or under stress (hill, ; leng et al., ) . the dynamics surrounding the adoption, implementation, and effectiveness of regulatory health policy are exemplified by the high fat, salt, and sugar (hfss) taxes that have been adopted across diverse nations with a wide range of outcomes (world health organization regional office for europe, ). most slightly reduced consumption of hfss foods but it is unclear what affect, if any, these taxes have on other health indicators in isolation. low income consumers that rely on affordable, energy-dense foods may be unduly burdened by hfss taxes but benefit from alternative price interventions such as subsidization of fruit and vegetables (neff et al., ) . while current regulatory interventions in isolation have minimal effect on downstream health indicators, they provide precedent for future regulatory policy and encourage action surrounding the role of large companies in shaping the food environment. significant improvements in global population health will require cooperation of people and companies involved in food systems to fundamentally change how they operate, but some communities have overhauled their local food environments with a ground-up, systemic approach that bypasses many of the barriers encountered on a national level and inspires awareness and innovation. in baltimore, usa, policies have been enacted to re-familiarize consumers with the systems and people that produce their food (department of planning, ), incentivize wholesaler produce distribution to small convenience stores, and encourage grocery chains to move into low income communities. city government also supports local ownership of food retail outlets in underserved neighborhoods, cultivating economic growth, a sense of community, and the agency to improve the health issues people see in their neighborhood. baltimore's food policy initiatives demonstrate how to mobilize community members to incorporate agri-food policies across city operations and government, business and economic development, and community organizations to combat the multifactorial roots of poor dietary health. even prior to the covid- pandemic the international community had been aware of the increasing threat from emerging zoonotic pathogens, the 'dual burden' imposed by endemic zoonoses on livestock productivity and human health, the biological and chemical hazards present in our food and the looming spectre of a post-antibiotic world. the protection of consumers from zoonoses and foodborne diseases acquired from the consumption of diseased animals or via unhygenic slaughter and processing has been a concern for centuries and formal meat inspection, still broadly recognisable today, was instigated in europe in the 's (edwards et al., ) . the multi-faceted nature of infectious disease risks within our food-systems and the plethora of public and private actors working within them, with differing roles and responsibilities has driven calls for an integrated 'one health' approach, endorsed at the highest level by the who, oie, fao tripartite and the world bank. despite growing acceptance of this concept internationally and the establishment of several regional and national one health units, there are still major challenges in operationalization. power struggles between ministries for 'ownership' of the movement, poorly integrated policies, lack of interoperability in data systems, and resource constraint for frontline services have all been identified as key barriers, and the multiplication of one health initiatives, specifically those with single issue focus, may risk undermining the strength of the moment (spencer et al., ) . this splintering and 'siloisation' of one health can be illustrated by kenya, where four separate one health bodies now exist with separate remits for zoonoses, amr, aflatoxins and pesticides but with no over-arching co-coordinating mechanism across these areas, and with other one health issues such as foodborne disease lacking an inter-ministerial 'home' (kimani et al., ) . although challenges exist, the one health approach has been repeatedly employed to mitigate animal and human disease threats, as exemplified by the response to colistin resistant e. coli (crec) in china (wang et al., ) . colistin is used predominantly in the livestock sector but is an important antimicrobial of last resort for human disease. following the discovery of widespread distribution of colistin resistance gene mcr- in e. coli, the chinese ministry of agriculture and rural affairs banned use of colistin as a growth promoter in livestock. three years on, significant reduction was observed in the relative abundance of mcr- in pigs, crec carriage in pigs and chickens, human carriage of mcr- positive e. coli, and human infection with crec. a clearly defined problem and solution, strong political support, and effective inter-agency enforcement contributed to the success of this policy and provides guidance for future approaches to threats to animal and human health. the one health approach will only be as strong as its constituent parts; environmental stewardship is often missed completely, whilst veterinary and human public health systems have often been chronically under-funded in favor of support to primary food production and curative human medicine. strengthening these capacities and meeting countries' commitments under the international health regulations and the oie pathway for veterinary services programmes is integral to the global health security agenda (belay et al., ) . the capacity building activities within the ghsa thus far have relied predominately on external donor funding. ultimately, the sustainability of these activities will require resource commitments by national government and a greater acknowledgement of the role of private actors across food systems (kelly et al., ) . there an increasing number of private standard setting organizations to enhance global food safety such as the global-good agricultural practises standard (king et al., ) . private standards can be excessively high, which improves food safety but may be unattainable for smallholders or farmers in lmics, effectively shutting them out of global markets and further highlighting the need for a collaborative, multi-sectoral approach to food safety. responses to emerging pathogens and outbreaks are often fear driven and reactionary. during the swine flu (h n ) outbreak, egypt culled their entire pig population, despite pigs not transmitting the virus to humans (atlani-duault and kendall, ). traceability systems have become more sophisticated and widespread and can support narrower outbreak responses, yet retailers and governments often issue blanket recalls and import restrictions during health crises (van der vorst, ) . similar to other components of food safety programs, development of and compliance with better traceability systems largely rest with private companies and may not be harmonized with standards set by public health bodies. to improve response time and accuracy during outbreak events, quality traceability systems across the value chain must be combined with transparency and communication with decision makers. prior to such events, governments should develop and train networks of communication that connect food systems agents, trustworthy spokespeople, and consumers to minimize panic and reactionary measures (who et al., ) . national outbreak preparedness plans and risk reduction strategies concerning diseases that originate outside the food system typically do not include considerations for protecting food systems but should be a key factor in planning potential response efforts (ortu et al., ) . localities can contribute to outbreak preparedness by conducting food system vulnerability assessments using fault tree analysis across a range of emergencies (biehl et al., ) . even when food systems are considered in planning exercises, insufficient follow through, lack of resources, or unwillingness to allocate available resources may render such exercises meaningless. policymakers must prioritize action following planning exercises, as emerging infectious disease and food safety events are increasingly likely as food systems become more globalized. corporate concentration of agricultural sectors both horizontally and vertically has resulted in centralizing control of large swaths of the food system among a few companies, reducing the resilience of food system to internal and external shocks (howard, ) . these oligopolies have vast lobbying power, making it difficult to enact structural change. positive change at this level will require market incentives and non-governmental accountability systems similar to what is seen in current efforts to combat climate change (heymann et al., ) . reliance on food imports also increases vulnerability to food insecurity. an estimated / th of the global population in were dependent on food imports for their basic dietary needs (fader et al., ) , leading to an unequal power dynamic between net-exporters & net-importers and undermining the development of locally appropriate value-chains. at the production level, agri-food policies which support local farmers and smallholders can strengthen food system resiliency by increasing the diversity of local food systems and protecting against economic shock. one example is the uk government department for the environment food and rural affairs' farm diversification grant scheme, which helped farmers in the uk develop a diverse portfolio of income sources (turner et al., ) for this reason. support for agricultural diversification among smallholders in lmics can improve food security, reduce poverty, and strengthen resilience to climate change and market instability, resulting in long term increases in productivity (joshi et al., ; world bank, ) . however, there are several obstacles to farm-level adoption of diversified farming systems, including start-up costs, potential initial productivity losses, lack of access to technical guidance or support, and market disencentives such as commodity crop subsidies and insurance (harvey et al., ) . some farm level barriers can be overcome with an institutionally organized systems or landscape approach that incorporates multiple stakeholders to achieve a collective diversification goal. policymakers should work to increase accessibility of diversification tactics, support research to provide further evidence and guidance for diversification, and ensure follow through on these commitments. while the countries in the african union have pledged to enhance financial investment in agriculture, only nine are on track to reach agricultural research spending goals (vilakazi and hendriks, ) . at the consumer level, stable access to food during periods of crisis can be ensured by policies that support emergency food access and flexibility within supply chains. the covid- outbreak underscores the need for policies to support and improve access to food assistance agencies, which not only supply emergency food but also help those in need access benefits, job training, and medical resources (environmental audit committee house of commons, ). food systems have wide and far-reaching impact on health, economy, and society locally and globally, while health issues and our response to them have major impacts on how food systems operate. a sustainable and healthy food system supports fair wages for individuals across the food value chain, provides affordable, nutritious, diets that are culturally acceptable, operates in an environmentally sustainable manner, and is resilient and adaptable. foodborne disease and emerging infectious disease events should naturally decrease as food systems approach these ideals. this vision is far-removed from the current reality but provides a set of priorities through which agri-food policies should be developed. major challenges exist concerning the reorientation of market incentives and food systems standards, heightened accountability, changes in land use, and cooperation between countries and corporations. yet, consumers are increasingly aware of the broader effects of current food systems and are beginning to widely challenge the status quo. the severity of the current covid- outbreak and its unusually far-reaching impacts on food systems will hopefully provide the stimulus and opportunity to for both high level decision makers and the wider consumer base to more seriously focus on restructuring our food systems to better support the health of the global population. lft is supported by the university of liverpool wellcome trust institutional strategic support fund and is a soulsby foundation one health travelling fellow. transmission dynamics and epidemiology of bse in british cattle meatpackers are deeply vulnerable to covid- . expect a reckoning for us workers | lse covid- . london sch. econ. polit. scienve covid- blog influenza, anthropology, and global uncertainties 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addressing zoonotic diseases in countries role of urbanization, land-use diversity, and livestock intensification in zoonotic emerging infectious diseases productive diversification of african agriculture and its effects on resilience and nutrition, productive diversification of african agriculture and its effects on resilience and nutrition world health organization regional office for europe emergence of african swine fever in china genetic diversity and disease control in rice the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -erdwejd authors: diaz, j. h. title: global climate changes and international trade and travel: effects on human health outcomes date: - - journal: encyclopedia of environmental health doi: . /b - - - - . - sha: doc_id: cord_uid: erdwejd there is now near-unanimous scientific agreement that greenhouse gas emissions generated by human activities have increased global temperatures and changed the earth's climate. there is, however, no universal agreement on how rapidly, regionally, or asymmetrically the earth will warm; or on the true impact of global warming on infectious disease outbreaks and natural disasters and their inevitable public health outcomes. in addition, many other factors influence the emergence and reemergence of infectious diseases in a changing environment including international trade and travel, exotic eating habits, lifestyle and residential choices, host susceptibility, and microbial adaptation. the ultimate effects of climate changes and the increased distribution of pathogens by international trade and travel will not be limited to infectious disease outbreaks in immunologically naïve populations but will also impact world food production and quality, air quality, drinking water availability and quality, immigration, urban relocation, and civil unrest. despite the uncertainties in outcomes and their magnitudes, the active responses to climate changes in a global economy must include combinations of environmental, political, regulatory, socioeconomic, and public health measures. the ultimate effects of global warming on rainfall, drought, and tropical cyclone activity will have farreaching human health impacts, not only on weatherrelated infectious disease outbreaks but also on world food production and supplies, access to safe drinking water, and mass population relocations. a more frequent drought-monsoon cycle supports outbreaks of malaria transmitted by water surface ovipositing anopheles species mosquitoes and dengue and chikungunya fever outbreaks transmitted by container-breeding aedes species mosquitoes. as nighttime temperatures increase and glaciers and permafrost retreat into the highlands, the geographic distribution ranges of mosquito and tickborne diseases extend to higher altitudes and to new regions in formerly disease-free areas with competent insect vectors. this article will explore the influences of free trade and international travel on public health outcomes in a warming world with a global economy. the interrelationships of climate change, international commerce, free trade, and international travel on observed human health consequences many factors will influence the onset of emerging and reemerging infectious disease outbreaks including climatic and ecosystem changes, new vector-pathogen relationships, human health behaviors, and human host susceptibilities. emerging infectious diseases may be defined as those that have recently appeared in human populations, have expanded their ranges of distribution, or threaten to increase their prevalences and distribution ranges in the near future. today most emerging infectious diseases arise in the natural environment as zoonoses, such as hantavirus pulmonary syndrome and severe acute respiratory syndrome (sars); or adopt a competent, new insect vector, such as aedes albopictus, a new mosquito vector for dengue and chikungunya viruses ( figure ). reemerging infectious diseases may be defined as those whose pathogenesis, clinical manifestations, and treatment strategies are well known but have reemerged as public health threats, often with increased antimicrobial drug resistances, such as multidrug resistant tuberculosis and methicillin-resistant staphylococcus aureus. in addition to climatic, ecologic, and microbial factors, other significant factors that influence the emergence and reemergence of infectious diseases include international trade and air travel, the globalization of agriculture and food production, exotic eating habits, lifestyle, and residential choices. the worldwide spread of the asian tiger mosquito, a. albopictus, by imported tire shipments on container ships from southeast asia has introduced a new secondary (to aedes aegypti) vector for dengue fever into the tropical americas and chikungunya fever in india and indian ocean islands (figure ). accessible airline connections now permit infected individuals to travel anywhere in the world in less than h, delivering human reservoirs of malaria, dengue, west nile virus, and chikungunya fever to new temperate areas for autochthonous or local transmission by new and adaptable mosquito vectors, often recent air or sea arrivals themselves. west nile virus was most likely imported to the united states in through air by the arrival in new york city of either an infected passenger or an infected culex species mosquito from an endemic region of east africa or the middle east. by , competent local culex vectors had initially established a mobile reservoir for west nile virus in wild birds in wet, warming ecosystems that began to move the virus rapidly across the united states from new york to the west coast. the initial wild animal reservoir for introduced west nile virus in the united states was so specific that it targeted only birds of the family corvidae, especially crows and jays. by , west nile virus infections were reported in other wild and domestic animals and humans across the continental united states and had caused over cases of meningoencephalitis with deaths (case fatality rate (cfr) ¼ . %). the major mosquito vectors of emerging and reemerging infectious diseases are featured in table . there were many historical examples of the international transport of human pathogens or their vectors by world exploration and trade that preceded any significant impact from recent climatic changes by centuries. prominent examples included the introduction of syphilis in the americas by european explorers, the spread of bubonic plague to europe by genoans returning from wars in the middle east, and the introduction of yellow fever carriers and mosquito vectors in the americas by west african slave traders. some recent examples of the international transport of pathogens or their vectors include the expanded global distribution ranges of plasmodium falciparum and p. vivax malaria and neuroangiostrongyliasis (nas) caused by the rat lungworm, angiostrongylus cantonensis. the most common reasons for malaria to occur in the industrialized nations of north america and europe where malaria was once endemic are also related to international air travel and include airport malaria and imported malaria. although similar subtropical ecosystems will support malaria reintroduced into formerly endemic regions such as the southern united states and the northern mediterranean, the exact impact of warming temperatures and greater precipitation on expanded malaria distribution ranges worldwide remains uncertain. nevertheless, malaria has become endemic at higher alpine altitudes in east africa and in south america. airport malaria is defined as the intercontinental transfer of malaria through the introduction of an infective anopheline mosquito vector into a nonendemic disease area with a changing ecosystem that supports the vector-pathogen relationship. however, imported malaria is defined as the intercontinental transfer of malaria by the movement of a parasitemic person to a nonendemic disease area with locally competent anopheline vectors in a welcoming ecosystem. airport malaria is acquired through the bite of an infected tropical anopheline mosquito within the vicinity of an international airport. the malaria-infected mosquito vector is a new arrival on an international flight from a malaria-endemic region. climate change has now expanded the geographic distribution of malaria-endemic regions and extended the length of seasonal malaria transmission cycles in endemic regions, so more arrivals of malaria-carrying mosquitoes are anticipated. how often do infected mosquitoes travel by air from tropical disease-endemic nations to capital cities in industrialized nations with disease-supporting warming ecosystems? in , random searches of arriving airplanes at gatwick airport in london found that of airplanes from tropical countries contained mosquitoes, some of which were female anopheles species capable of transmitting malaria. after the female anopheles species mosquito leaves the aircraft, she may survive long enough, especially during temperate periods, to take a blood meal and transmit malaria pathogens, usually in the vicinity of an international airport. after one or more figure the female aedes albopictus, or asian tiger mosquito, has been disseminated in coastal temperate zones worldwide by global trade and has genetically adapted to become a competent new vector for dengue and chikungunya viruses. reproduced from cdc image, available at http://www.dpd.cdc.gov/dpdx. blood meals, female mosquitoes seek a water surface to lay their eggs. all female mosquitoes lay their eggs in standing water, either on the surface or just below. the anopheline vectors of malaria prefer to lay eggs in drainage ditches, marshy areas, and puddles. the culicine vectors of west nile virus, dengue, and chikungunya fever prefer to lay their eggs in containers that trap freshwater, such as flower pots and even discarded tires. climate changes, particularly warming nighttime temperatures and increased precipitation, offer several selective advantages to all mosquito species including ( ) a longer reproductive life and a prolonged breeding season, ( ) opportunities for more blood meals during gestation, ( ) plenty of standing water surfaces for egg laying, and ( ) a faster egg hatch over days and not weeks. as international air travel between malaria-endemic nations and malaria nonendemic nations increases, cases of airport malaria have increased. in , two cases of p. falciparum malaria were diagnosed in persons without histories of travel to malaria-endemic regions living and km from gatwick airport. hot, humid weather in britain may have facilitated the survival of imported, infected anopheline mosquitoes. during the summer of , six cases of airport malaria were diagnosed in the vicinity of charles de gaulle airport near paris. four of the patients were airport workers, infected at work, and the others were residents of villeparisis, a small town approximately . km away from the airport. to reach villeparisis, the infected anopheline mosquitoes were thought to have hitched a car ride with airport workers who lived next door to two of the patients. in addition to airport malaria transmitted by infected mosquito air travelers, many countries throughout the developed world are reporting an increasing number of cases of imported malaria because of the great increase in long-distance air travel by infected passengers. malaria cases imported from africa to the united kingdom rose from in to in . in , an italian woman was infected with malaria following a bite from a local, malaria-competent vector, anopheles labranchiae. this species had been a common malaria vector in italy until the country was declared malaria free in . the local mosquito responsible for transmitting malaria was thought to have acquired the parasite after biting a parasitemic girl who had recently arrived in italy from india. airport malaria was ruled out in this case because of the great distance from the nearest international airport. this case illustrated the ease with which imported malaria may be reestablished in a formerly endemic nation with a warming climate, competent local anopheline vectors, and a humid and wet ecosystem that supports vector-host-parasite relationships. in the united states, recent outbreaks of presumed local or autochthonous mosquito-borne malaria transmission have been reported in california, following the immigration of agricultural workers from malaria-endemic areas of mexico. in , a p. vivax malaria outbreak resulted in cases, in mexican migrant workers, over a -month period. epidemiological and microbiological investigations later confirmed secondary spread from infected immigrants to other immigrants and local residents transmitted by local malaria-competent, anopheline vectors. prevention and control strategies for airport and imported malaria should include early case definition, case confirmation, and treatment; strengthened vector surveillance to detect the potential for autochthonous transmission; and drainage of potential mosquito breeding and egg-laying surface water sites. although the relationships among infected vector importation, index case immigration, reclaimed disease ecosystems, and malaria transmission are complex, future attempts to control and eradicate airport and imported malaria should be based on an understanding of disease transmission mechanisms and an appreciation that climate and ecosystem changes can support reemerging local mosquito-borne infectious diseases, especially malaria, dengue, chikungunya fever, and west nile virus (table ) . in addition to commercial, business, and recreational international air travel, infectious diseases may also be transmitted by sea from endemic to nonendemic nations with warming ecosystems that will support host-pathogen relationships. infected arthropod vectors, infected animal hosts, especially rodents, and even virulent microbes travel well at sea, especially in hot and humid ship cargo holds and in cargo containers. recent outbreaks of cholera in ecuador and peru have followed increased shipping trade with southeast asia, where vibrio cholerae is endemic in coastal estuaries. traceback investigations have demonstrated that container vessels pump in contaminated saltwater ballast into their hulls in their homeports for smoother transoceanic sailing and then discharge the ballast before unloading in distant ports with warming estuaries. such practices can effectively import cholera bacteria, marine viruses, and harmful algae to new, warming marine ecosystems and fisheries causing microbial and algal toxin contamination of shellfish beds and regional fisheries. the exotic pet trade, exotic cuisines, adventure travel, and emerging infectious diseases in the developed world the helminthic infections that can cause eosinophilic meningitis (em), neuroangiostrongyliasis (nas) and gnathostomiasis (gns), and chagas disease, an arthropod-borne zoonosis, share many of the characteristics of emerging infectious diseases supported by free trade in a warming world. nas is now endemic in hawaii and some coastal us cities following the us importation of nas by stowaway rodent hosts on cargo ships from china and southeast asia of the causative parasite, a. cantonensis, or rat lungworm. the intentional introduction of an intermediate a. cantonensis host, the giant african land snail (achatina fulica), to control insect pests on us farmlands and to serve as exotic pets for home terrariums also imported the unwelcome parasites to us ecosystems by paratenic or transporting hosts. gnathostoma spinigerum-induced gns has been recently recognized as an emerging imported disease in the united kingdom. since g. spinigerum is endemic in central and south america, most notably in mexico, gns may soon become another emerging potential cause of em in north america, given the adventurous and exotic eating habits of north americans abroad. the reduviid insect vectors of chagas disease have now moved from the rural areas of latin america into cities and coastal resort areas frequented by international travelers. the vectors have also migrated northward into the temperate areas of the midsouthern united states and have established a trypanosoma cruzi zoonosis among wild animals and some domestic animals kenneled outdoors as far north as virginia. the exotic pet trade and the importation of infectious diseases: monkeypox and neuroangiostrongyliasis in addition to a. cantonensis-infected african giant snails, the exotic pet trade has also imported monkeypox to the united states in pet rodents. in , cases of monkeypox were reported among three midwestern us states (illinois, indiana, and wisconsin). fortunately, there were no deaths, but patients were hospitalized for supportive treatment, including a child with encephalitis. the monkeypox virus, a smallpox-like orthomyxovirus, was first isolated in the congo river basin of west africa in . after an incubation period of - weeks, monkeypox is characterized by a prodrome of headache, fever, fatigue, and backache, followed by a characteristic rash. the monkeypox rash is similar to smallpox with evolving macules, vesicles, and pustules that crust over and heal within - days (figure ) . unlike smallpox, pronounced lymphadenopathy is usually present and complications may include pneumonia and encephalitis. although person-to-person transmission may occur, infection is usually transmitted by contact with contaminated animals. the cfr ranges from % to %, with higher cfrs in young children. traceback investigations of the us monkeypox outbreak revealed that all patients had had contact with pet animals: patients had contacts with pet prairie dogs, with a pet rabbit, and with a pet imported giant gambian rat, cricetomys gambianus. the source of the monkeypox virus introduced into the united states was later identified as infected giant gambian rats imported from ghana to texas and sold to an illinois pet distributor, who housed the animals together before sale to pet shop owners and others. although the monkeypox virus is endemic in west african river basins, including the gambia and congo, the humid river bottomland ecosystems of the mississippi-missouri-ohio river basins of the central united states will support the transmission of the virus from imported rodents to domestic rodents, especially prairie dogs and squirrels, and to rabbits (figure ) . the close contact between pet owners and their pets permitted the transmission of the zoonotic infection, for which there is no specific treatment, to humans. regulations now prohibit the importation of african rodents into the united states, and sentinel monitoring systems of local rodents for monkeypox infections have now been initiated. like the rat lungworm, the monkeypox virus may become an unwelcomed, but established, zoonosis in the united states as a result of relaxed free trade regulations, especially of the exotic pet trade, and a welcoming, warming riverbottomland ecosystem. a. cantonensis, the rat lungworm, was first described in china in , living in the pulmonary arteries of rats. the first human infection was reported from taiwan in . the life cycle is complex and involves a rodent definitive host and an appropriate mollusk intermediate host, usually land snails or slugs (figure ) . adult worms mature in rat brains, enter the central circulation, and mate in the pulmonary arteries producing eggs. the eggs become first-stage larvae that penetrate pulmonary vessels to access the respiratory tree, where they are coughed up, swallowed, and excreted in feces. these second-stage larvae must be consumed by land snails or slugs to mature into infective third-stage larvae, be eaten by rodents, and maintain the parasite's life cycle. man becomes a dead-end host by consuming raw intermediate mollusk hosts, or food items contaminated by their slime, or by consuming raw, crustacean (shrimp, crabs, fish, and frogs) transport, or paratenic, hosts that consumed infected mollusks. in man and paratenic hosts, the neurotropic larvae migrate to the cns (neural larva migrans) seeking to mature into young adults as in rat brains but eventually die causing em. a. cantonensis is enzootic throughout southeast asia, most indian and pacific ocean islands, including the hawaiian islands, many caribbean islands, and has even been reported in new orleans, louisiana. the global spread of the parasite resulted from international trade, parasite-infested rat stowaways disembarking container ships, and the intentional introduction of giant african land snails as biological controls and exotic pets. in , a. cantonensis was isolated from % of norway rats, rattus norvegicus, trapped in new orleans, louisiana, between april and february . in , a case of a. cantonensis-induced em was reported from new orleans in an -year-old male who presented with a -week history of headache, stiff neck, low-grade fever, and myalgias. he admitted eating a raw snail from the street on a dare a few weeks earlier. a second presumed autochthonous case of a. cantonensis infection was reported from south louisiana in in a -year-old man who presented with neck and backaches, myalgias, and paresthesias. he admitted having consumed, on a dare, two raw legs from a green tree frog, hylidae cinerea, days before symptom onset. a. cantonensis was responsible for an outbreak of nas in us travelers returning from jamaica to chicago in , who had consumed romaine lettuce. the lettuce food vector was actually imported to jamaica from the united states and presumably contaminated somewhere in between with snails or slugs or their secretions containing infective a. cantonensis larvae. in summary, there is now ample clinical, epidemiological, parasitological, and immunological evidence that an a. cantonensis zoonosis has been established in the continental united states and in the caribbean in rats, mollusks, and paratenic frog hosts as a direct result of international commerce. although cases of nas are rarely confirmed by the identification of a. cantonensis larvae or adults in the cns, most cases can now be confirmed serologically and epidemiologically; the reported cfr in us cases is relatively low ( . %); and most patients recover completely, even without specific antihelminthic treatment. the most effective prevention and control strategies for nas include ( ) educating citizens and travelers in endemic areas that snails, slugs, freshwater fish and shrimp, frogs, and crabs must be cooked, not marinated or refrigerated, before being eaten; ( ) washing all vegetables thoroughly before eating them uncooked; ( ) washing hands thoroughly after handling pet african land snails or cleaning out their terrariums; ( ) reducing and controlling the definitive host rodent populations with rodenticides; ( ) reducing and controlling snail and slug paratenic host populations with molluscicides; and ( ) policing and restricting the exotic pet trade. originally confined to southeast asia and japan, gns is acquired by eating raw or undercooked foods, infected with third-stage larvae of the roundworm, g. spinigerum. g. spinigerum is a common roundworm of wild and domestic cats, dogs, and other carnivores that coils within submucosal tumors in the stomach of definitive hosts, mates, and releases eggs in the host's feces. the eggs embryonate into first-stage larvae in fresh or brackish water ecosystems and are ingested by small crustacean intermediate hosts, which become prey for larger humans are incidental hosts, passage of larvae in humans has never been documented, and humans do not transmit either a cantonensis or a. costancensis causes eosinophilic meningitis, a meningoencephalitis characterized by eosinophils in the cerebrospinal fluid (csf). common in parts of southeast asia and pacific islands, africa and the canbbean. causes eosinophilic enteritis, an eosinophilic inflammation of the mesenteric arterioles of the ileocecal region of the gastrointestinal tract that mimics appendicitis. common in parts of central and south america eggs hatch in the lungs, and first-stage larvae are passed in rodent feces (a. cantonensis) third-stage larvae are ingested by rats first-stage larvae infect snails and slugs. slugs and snails are intermediate hosts, and after two molts, the larvae reach the infective (third) stage. humans become infected through food containing third-stage (infective) larvae. food items may include uncooked or undercooked snails or slugs, infected paratenic hosts (i.e., crabs, freshwater shrimp), and raw vegetables contaminated with snails or slugs. eggs hatch in the lungs, and first-stage larvae are passed in rodent feces (a. cantonensis) figure the life cycle of the rat lungworm, angiostrongylus cantonensis, which causes eosinophilic meningitis (a), is compared to the life cycle of angiostrongylus costaricensis, which causes eosinophilic enteritis (b). reproduced from cdc image, available at http:// www.dpd.cdc.gov/dpdx. predators including fish, shrimp, crabs, crayfish, frogs, and snakes ( figure ). the larvae mature into infective thirdstage larvae in these transport or paratenic hosts, encyst in tissues, but do not develop into adults, unless the paratenic hosts are consumed by definitive carnivorous hosts. once infective larvae are consumed by predators, they will mature into adults in the stomach and restart the parasite's life cycle ( figure ). since humans are not the natural definitive hosts, infective larvae consumed by humans in raw foods will not develop into adults but will penetrate the gastrointestinal tract and migrate hematogenously causing cutaneous or visceral larva migrans in any organ system. typically, the most common foods containing infective larvae have included fish, shrimp, crab, crayfish, frog, snake, and chicken. however, most human cases have followed consumption of raw or citrus-marinated fish (ceviche) or shellfish. in , gns caused by g. spinigerum was first recognized as an emerging imported helminthic infection in the united kingdom in a case series of patients treated over a -month period. in this series, the median incubation period was months; peripheral eosinophilia was present in ( %) of the patients, and was not a reliable screening tool; and cases presented with a myriad of symptoms ranging from migratory cutaneous swellings (also known as yangtze edema in asia, or nodular eosinophilic migratory panniculitis in the united states) to eosinophilic gastritis. today, gns remains relatively common in southern china, thailand, and bangladesh; is becoming more common throughout latin america and the caribbean; and is most often described in the united states in southeast asian immigrants. a diagnosis of gns should now be considered for all patients with a history of travel to endemic regions and migratory cutaneous swellings, eosinophilic gastritis, or a combination of cutaneous swellings with any manifestation of neural larva migrans, especially eosinophilic meningoencephalitis and migratory radicular pain or radiculomyelitis. neural gns has also caused radiculomyeloencephalitis and subarachnoid hemorrhage. most fatal cases of gns have been associated with neural larva migrans and em, with eosinophils comprising over % of the cerebrospinal fluid (csf) cell count. prevention and control strategies for gns include ( ) educating citizens and travelers in endemic areas that fish, shrimp, crayfish, frogs, crabs, chicken, and snakes must be cooked thoroughly first and not eaten raw, marinated, or refrigerated and ( ) seeking medical care immediately for evaluation of persistent nonspecific gastrointestinal illnesses or migratory subcutaneous swellings. regional warming in northern latitudes and adventure travel in the americas: t. cruzi and chagas disease (american trypanosomiasis) chagas disease, or american trypanosomiasis, is an arthropod-borne protozoan infectious disease endemic throughout most of the americas, caused by the trypanosome, t. cruzi, and transmitted to man by reduviid, or kissing, bugs ( figure ) . the life cycle of t. cruzi is depicted in figure . reduviid bugs, specifically the triatomines (phylum insecta, order hemiptera, family reduviidae, subfamily triatominae), transmit several strains of wild animal t. cruzi among many nonspecies-specific wild mammalian reservoir hosts throughout the americas (figure ). there are also many competent species of reduviid vectors capable of transmitting zoonotic wild strains of t. cruzi to domestic animals and man throughout the americas (figure ) . like malaria and west nile virus, chagas disease may also be transmitted congenitally and by blood transfusion and organ transplantation. unlike malaria and west nile virus, however, chagas disease was recently found to be transmitted by the ingestion of infected triatomines, which poses special risks to international travelers who eat or drink unpasteurized foods made from palm oils or raw sugarcane. in its world health report, the world health organization (who) noted that chagas disease caused more deaths from parasitic disorders than any other parasitic disease in latin america, and that t. cruzi was responsible for the third greatest number of parasitic infections in the world following malaria and schistosomiasis. chagas disease is now the most common cause of myocarditis worldwide, and chagasic heart failure has become an increasingly common indication for heart transplantation in the americas. since reduviid bites occur at nighttime and are either painless, possibly from combinations of salivary local anesthetics and anticoagulants, or associated with pruritus. the localized pruritus only serves to induce rubbing and scratching by sleepy victims, effectively dispersing infective trypomastigotes across bite-damaged epidermal surfaces or adjacent mucoepidemal junctions. since there is no vaccine to prevent chagas disease and current chemotherapy is limited to only two drugs, most efficacious only in the earliest stages of acute or reactivated t. cruzi infections, the best preventive strategies for chagas disease in travelers to the americas should be directed at ( ) the education of travelers to t. cruzi-endemic areas of the americas in the transmission risks of chagas disease; ( ) a recommendation for sleeping under pyrethroid-impregnated insect nets, especially when staying overnight in thatched and mudwalled huts or unmortared cabins; and ( ) a recommendation to travelers to drink only bottled, boiled, or pasteurized beverages; to avoid all local brews, especially those made from local palm trees and sugarcane; and to avoid chewing on unwashed sugarcane stems or palm hearts and avoid using unwashed sugarcane stems as swizzle sticks for beverages. several components of climate change, particularly warming temperatures and more frequent drought-rainy season cycles, have supported the success of new vectorpathogen relationships, as in airport and imported malaria transmission. some insect vectors, particularly mosquitoes, have been given selective advantages by climate change, free trade, and air travel. introduced pathogens from tropical regions, such as the monkeypox virus and the rat lungworm, have found new animal reservoirs in warming ecosystems north of the equator. how should humankind respond to climate change and its inevitable impact on biological systems and the quality and safety of human life? the united nations, through its agencies and panels, such as the who and ipcc, has taken the lead in directing appropriate international responses that will include combinations of environmental, political, regulatory, socioeconomic, and public health measures. such measures must include limiting anthropogenic greenhouse gases, fostering renewable energy resources, improving natural disaster forecasting, developing drought and disease-resistant food crops, recognizing the disease potential of introduced pathogens in a warming world, instituting sentinel monitoring for infectious diseases in animals and man, and developing primary prevention strategies for climate change-related infectious disease outbreaks and extreme weather events. primary prevention strategies for emerging infectious diseases should include new vaccines for avian influenza, sars, and west nile virus. primary injury prevention strategies for extreme weather events figure triatoma infestans, a common reduviid vector of trypanosoma cruzi among animals and humans in the americas. reproduced from cdc image, available at http:// www.dpd.cdc.gov/dpdx. should include early warning systems for heat waves, floods, tornadoes, tsunamis, and tropical cyclones. developing global climate anomalies suggest potential disease risks for chikungunya outbreaksfthe globalization of vectorborne diseases wildlife, exotic pets, and emerging zoonoses climate change and human health how environmental exposures influence the development and exacerbation of asthma climate change: the public health response climate change: synthesis report, ar syr summary for policymakers wildlife as a source of zoonotic infections world health assembly : climate change and health global environmental change and health: impacts, inequalities, and the health sector climate change and human health: present and future risks dengue and hemorrhagic fever: a potential threat to public health in the united states improved surface temperature prediction for the coming decade from a global climate model what makes ticks tick? climate change, ticks, and tick-borne diseases emerging tick-borne infections: rediscovered and better characterized, or truly new? emerging infectious diseases, an online journal published by the united states centers for disease control and prevention financial support was provided by departmental and institutional sources. the author has no conflicts of interest to disclose. key: cord- - ebve k authors: rohr, jason r.; dobson, andrew p.; johnson, pieter t.j.; kilpatrick, a. marm; paull, sara h.; raffel, thomas r.; ruiz-moreno, diego; thomas, matthew b. title: frontiers in climate change–disease research date: - - journal: trends ecol evol doi: . /j.tree. . . sha: doc_id: cord_uid: ebve k the notion that climate change will generally increase human and wildlife diseases has garnered considerable public attention, but remains controversial and seems inconsistent with the expectation that climate change will also cause parasite extinctions. in this review, we highlight the frontiers in climate change–infectious disease research by reviewing knowledge gaps that make this controversy difficult to resolve. we suggest that forecasts of climate-change impacts on disease can be improved by more interdisciplinary collaborations, better linking of data and models, addressing confounding variables and context dependencies, and applying metabolic theory to host–parasite systems with consideration of community-level interactions and functional traits. finally, although we emphasize host–parasite interactions, we also highlight the applicability of these points to climate-change effects on species interactions in general. the notion that climate change will generally increase human and wildlife diseases has garnered considerable public attention, but remains controversial and seems inconsistent with the expectation that climate change will also cause parasite extinctions. in this review, we highlight the frontiers in climate change-infectious disease research by reviewing knowledge gaps that make this controversy difficult to resolve. we suggest that forecasts of climate-change impacts on disease can be improved by more interdisciplinary collaborations, better linking of data and models, addressing confounding variables and context dependencies, and applying metabolic theory to host-parasite systems with consideration of community-level interactions and functional traits. finally, although we emphasize host-parasite interactions, we also highlight the applicability of these points to climate-change effects on species interactions in general. the climate change-disease controversy global climate change and the unprecedented rate of infectious disease emergence represent two of the most formidable ecological problems of our time [ ] [ ] [ ] [ ] [ ] . several high-profile papers assert that climate change will increase the global distribution and prevalence of infectious diseases to the detriment of human health, biodiversity and ecosystem services (see glossary), which has placed climate change-disease interactions at the center of scientific, political and public agendas [ ] [ ] [ ] . indeed, there is compelling evidence that climate affects many diseases, including malaria, cholera, dengue and plague in humans [ ] [ ] [ ] [ ] , bluetongue in livestock [ ] and diseases of amphibians, turtles and corals [ , [ ] [ ] [ ] . however, the notion that climate change will generally increase diseases has been challenged recently in several papers demanding greater rigor and a better appreciation of the complexity of climate change-disease interactions [ , [ ] [ ] [ ] [ ] . these papers emphasized the presence of potentially confounding factors in many climate change-disease studies, calling into question whether climate change will, in fact, cause widespread increases in human and wildlife diseases. moreover, papers published on the absence of disease are scarce relative to those on its presence and, thus, there is likely to be a publication bias against climate preventing disease outbreaks. this controversy surrounding climate change-disease interactions underscores the need for a clearly defined research agenda. here, we outline key gaps in data, theory and scale that point to the frontiers in climate change-infectious disease research. we hope that this will help resolve this controversy, synthesize knowledge and advance understanding. we emphasize the interdisciplinary nature of the problem, encouraging collaborations among epidemiologists, disease ecologists, climatologists, modelers, geographical information system (gis) specialists, sociologists, economists and policy and management practitioners. amplifying host: a host that contributes positively to pathogen transmission, as opposed to a diluting host whose presence causes an overall decrease in transmission. complex life-cycle: a parasite life-cycle requiring more than one host species. dilution effect: a decrease in disease risk owing to an increase in host species diversity. direct transmission or life-cycle: a parasite life-cycle requiring only one host species. ecosystem service: processes and characteristics of ecosystems that benefit humans. emerging disease: a disease that is increasing in incidence or in its host or geographic range. ensemble modeling: an approach that integrates the forecasts of several climate change models. free-living stage: a stage of a parasite that lives outside of its host or hosts. herd immunity: the resistance of a whole group of hosts to an infectious agent, owing to the resistance to infection of a proportion of the group members. metabolic theory: describes how the rate at which organisms take up, transform and expend energy and materials (i.e. metabolic rate) controls ecological processes at all levels of organization, from individuals to the biosphere. prevalence: the proportion of hosts infected with a given parasite. secondary extinction: an extinction caused by the extinction of another species. corresponding author: rohr, j.r. (jasonrohr@gmail.com) gaps in data, models and their integration null models some of the controversy surrounding the effects of climate change on disease stems from questionable null models that can lead to erroneous conclusions. for example, although researchers frequently assume that pathogens will experience range expansions as they move pole-ward, tropical range contractions might also occur [ ] . it is presently unclear whether range shifts, contractions, or expansions are most likely and, thus, a neutral hypothesis of no change in the geographic ranges might be the most defensible null expectation [ , ] . similarly, the need to shift distributions pole-ward or to higher altitudes as the planet warms, coupled with species variation in dispersal abilities, has stimulated the hypothesis that climate change will cause phenological mismatch between parasites and their hosts [ ] . singer and parmesan [ ] , however, recently pointed out that evidence in support of this hypothesis is based on the null assumption of perfect synchrony, despite phenological mismatch being common before anthropogenic climate change, at least for some insect-host plant interactions. clearly, historical baseline data are needed to generate appropriate null models and test climate change-disease hypotheses properly. multiple variables, confounded variables and context dependencies predicting the impact of climate change on disease requires determining the net impact of numerous effects, including those that have opposing directions. a wellstudied example is the effect of temperature on the transmission of vector-borne pathogens, such as malaria. at cooler temperatures (e.g. c), an increase in temperature is expected to increase not only biting rates, parasite replication within mosquitoes and mosquito development, but also mosquito mortality, making the net effect of increasing temperatures difficult to determine. mathematical models provide a powerful tool for integrating these complex interactions, but model validation requires welldesigned laboratory experiments (box ) and field data sets that are long and detailed enough to enable fitting of the relevant parameters. disease control efforts can also make it challenging to determine effects of climate change on disease. for instance, if climate change causes range shifts of parasites from tropical to temperate countries, this might result in an overall reduction, rather than increase, in human diseases because temperate countries often have superior health infrastructures [ ] . regardless of the outcome of climate change on diseases, integrating control efforts into projections should improve predictions of future disease risk for humans and wildlife. furthermore, given that control measures could obscure increases in transmission, incorporating control measures into models could reveal underlying increases in disease risk that might otherwise be missed. similar to disease control measures, intrinsic factors, such as temporal variation in herd immunity, pathogen spread and parasite evolution, can co-vary with changing climate [ ] . intrinsic factors can give rise to oscillations in disease whose frequencies might differ from those of extrinsic drivers, making it difficult to identify the contributions of each to temporal population patterns [ ] . for instance, the effects of climate on cholera dynamics became more evident after controlling for cycles in temporary immunity because climate has fewer impacts on cholera when a large fraction of humans are resistant to the bacterium [ ] . as another example, effects of el niñ o southern oscillation (enso) events and climatic variability on disease-related amphibian declines were only revealed after controlling for a multidecadal pattern in extinctions that was probably caused by the spread of the pathogenic chytrid fungus [ ] . observational studies can identify the best climatic predictors of disease-related response variables, but manipulative experiments are crucial for testing whether climatic factors truly have causal relationships with disease. here, we present some common issues with climate-change experiments and suggest improvements that should enhance the quality of data obtained from future studies. issue: climate-change researchers commonly treat samples within a single environmental chamber as independent replicates of temperature (e.g. [ ] ) (i.e. pseudoreplication), which can confound temperature with anything else that might differ among chambers (e.g. light, humidity or air circulation). have true and adequate replication of temperature treatments, for instance by building a large number of independent incubators out of styrofoam, heat tape and thermostats. replicate the effect of temperature in time (i.e. temporal blocks). place multiple experimental units within each incubator and analyze the data using appropriately nested mixed-effects models that treat the chamber as the level of replication for testing temperature effects (e.g. [ ] ). such analyses require a minimum of four chambers to compare two temperature treatments. including more than two levels of temperature and treating temperature as a continuous predictor. this can enable the detection of nonlinearities and is required to provide functional relationships for integrative and/or predictive models [ ] . conduct meta-analyses of independent tests of temperature effects [ ] . issue: in many experiments, all individual organisms are initially held at a single temperature and then a subset are transferred to higher or lower temperatures and experimentally infected with a pathogen. in this design, temperature is confounded with the magnitude of the temperature shift that occurs at the start of the experiment, making it unclear which is driving any observed effect of the temperature treatment. improvement: adequately acclimate study organisms to the temperature of interest before applying treatments. issue: field experiments often provide more ecologically relevant data than do laboratory experiments, but it can be challenging to manipulate climate in the field. improvements: use creative ideas for manipulating climate in field experiments. heating coils or continuous co input chambers can be used to replicate climate or climate-associated treatments. increase temperatures via the greenhouse effect by enclosing small, open-top plots in clear plastic [ ] . trends in ecology and evolution june , vol. , no. unconsidered components of climate are also potential confounders of disease-climate interactions. many hosts and pathogens are influenced by the interactive effects of multiple abiotic and climatic factors, such as moisture, temperature and co [ , ] . imagine the hypothetical scenario in which a region experiences increases in temperature and precipitation that have opposite effects of a similar magnitude on a disease. a univariate analysis might erroneously suggest that the host-pathogen system was insensitive to the climate change. despite the obvious need to understand how climate components interact to affect disease, there is a paucity of studies that do so. also problematic is the likelihood that diseases are affected by interactions between climate and other natural enemies [ , ] or environmental changes (e.g. land-use change) [ , ] . nonlinear effects can generate important and surprising climate impacts on host-pathogen interactions. for instance, the fitness of most organisms decreases in either direction away from the optimal climate (although more complex nonlinear functions can also occur). hence, changes in climate should often generate nonlinear effects on fitness, which contrasts with the frequent expectation that there will be consistent increases or decreases in host or parasite fitness with climate change. these expectations are probably only justified for small climatic shifts that do not cross the optima of an organism. however, most studies have insufficient variation in climate to detect nonlinearities and few generate reliable parameter estimates for modeling (box ). one example of a nonlinearity is given by threshold responses, which transpire when large shifts in variable response (e.g. transmission intensity) occur over narrow windows of change in a crucial parameter, such as temperature [ ] [ ] [ ] . climatic threshold responses have been suggested to be important in biodiversity losses [ ] and human, coral and plant disease outbreaks [ , , ] . for instance, evidence suggests that coupling between cholera dynamics and climate is transient, occurring only during strong enso events. this observation is consistent with a threshold response, whereby climate is only an important driver of cholera during climatic extremes [ ] . threshold responses are difficult to capture with standard linear statistical models and challenges associated with stochasticity, finite population sizes, time-lags and covariates present additional obstacles to detecting thresholds [ ] . more sophisticated statistical approaches that allow for nonlinearities [ ] , as well as other techniques, such as scale-dependent correlation analysis [ ] , the significant zero crossings (sizer) model [ ] , or models that allow for flexible treatment of regime shifts [ ] , might be necessary to detect climatic threshold effects on disease. improved data and data-model integration this review of null models, confounding variables, context dependencies and nonlinearities in climate-disease interactions underscores the need for: (i) better data on baseline interactions and intrinsic and extrinsic factors affecting disease; (ii) long-term data sets that can effectively parse out how variation is explained by different factors [ , ] (e.g. control efforts, host immunity); (iii) improved use and development of statistical and mathematical models to reveal adequately relationships between climate and disease dynamics, ideally in conjunction with a more thorough integration of field and experimental data (box ); and (iv) the need to consider both climate change and alternative hypotheses as drivers of disease (using information theoretic approaches; e.g. [ , , ] ) ( figure ). ideally, both experiments and models should take into account the substantial uncertainty associated with climate projections, for instance by using model-averaging or ensemble-based approaches [ ] . whenever possible, model assumptions should be made explicit and models should be effectively validated [ ] . improving data collection and modeling efforts will require collaborations among epidemiologists, disease ecologists, statisticians, modelers and gis specialists. furthermore, judicious decision-making will require effectively communicating scientific results to sociologists, economists and policy and management practitioners, who must carefully weigh scientific findings, economic costs and public perceptions. as data on climate change-disease interactions accumulate, the time is ripe for the maturation of predictive theories on climate change-disease interactions. we suggest three areas of theoretical development ( figure ): (i) theory to predict the outcomes of specific host-parasite interactions as a function of climate; (ii) theory to predict where on the earth climate change will have the greatest impact; and (iii) theory to predict which host-parasite systems might be most sensitive to climate change (box ). the metabolic theory of ecology has been useful in describing biological patterns from organismal to macroecological levels [ ] . although metabolic theory is often too coarse to predict accurately outcomes of fine-scale differences among metabolic rates of organisms, it captures broad variation among organisms that differ substantially in size [ ] . given that most parasites are orders of magnitude smaller than their hosts, metabolic theory might be useful in predicting and understanding the outcome of climate-dependent host-parasite interactions [ ] . this should be especially true for parasites with free-living stages and for poikilothermic hosts, whose body temperatures fluctuate with environmental temperatures [ ] . as an example, global climate change is expected to increase climatic variability [ , ] and metabolic theory offers predictions for how host-parasite interactions might respond to this climate change. first, owing to their faster metabolisms, parasites should acclimate to temperature shifts more quickly than will their hosts [ ] , perhaps providing them with a temporary advantage in host-parasite interactions. second, smaller organisms have fewer cells and processes requiring adjustment following temperature shifts and, thus, generally withstand greater temperature extremes than do larger organisms [ ] . finally, owing to their shorter generation times, parasites opinion trends in ecology and evolution june , vol. , no. should evolve more quickly than their hosts to changes in climate. nevertheless, most research on climate change and disease has neglected evolutionary processes, despite evidence highlighting the importance of evolution in disease processes on ecological timescales [ ] and in mitigating against the impacts of climate change [ ] . in support of these predictions derived from metabolic theory, recent empirical and theoretical evidence suggests that increased variability in temperature can alter hostparasite interactions. for instance, temperature variability appears to be important in amphibian-chytrid fungal interactions [ , , ] (figure c ), avian influenza outbreaks [ ] and malaria epidemics in the east african highlands [ ] . additionally, diurnal fluctuations around low mean temperatures accelerated the growth of plasmodium parasites (the causative agent of malaria) relative to an equivalent constant mean temperature (figure a) , whereas fluctuations around high mean temperatures slowed parasite growth (figure b) [ , ] . why temperature shifts sometimes benefit the pathogen and other times the host remains unclear, but further research on the impacts of climatic means versus variances will be needed if one is to predict accurately the impacts of climate change. locations where climate change will have the greatest impact identifying the geographic regions of the earth that are most vulnerable to climate change will greatly assist in targeting disease management and monitoring efforts. there are two general schools of thought on determining where climate change will have the greatest impacts. the more traditional notion is that organisms in temperate and polar regions will be most affected by climate change because temperatures are increasing disproportionately in these regions relative to the tropics [ , , ] . recent work, however, suggests that tropical hosts and parasites might be as impacted by increasing temperatures as those at higher latitudes, despite the smaller increases in tropical temperatures [ , ] . because tropical climates are less variable, tropical organisms are adapted to much narrower temperature ranges than are temperate and arctic species and, thus, are expected to be more sensitive to small changes in climate [ ] . additionally, because metabolic rate increases exponentially with temperature, organisms in the warmer tropics experience a greater change in metabolism with each unit increase in temperature than do organisms in temperate and polar better theory • can metabolic theory predict climate effects on parasitism in different locations and systems? • can functional traits and control measures be used to predict climate-induced changes in range, incidence, or severity of parasitic diseases (fig. dilutio tank treatm chem conce bd categ quant a y y a c. n n . c a n n a a n n a c. y y . c a y y a a y y a better data and models • gather higher-quality data over longer time periods • conduct improved experiments to help inform predictive models (box ) • incorporate new factors into models: means and variances of interacting climate components, justified null models, uncertainties, nonlinearities, disease control measures (box ), intrinsic factors, and alternative hypotheses for disease dynamics regions [ ] . indeed, when considering both recent global warming and the exponential relationship between metabolic rate and temperature, dillon et al. [ ] estimated that organisms in tropical and northern temperate zones are experiencing the largest absolute increase in metabolic rates and, thus, are being impacted most by climate change. although metabolic theory has the potential to help predict host-parasite outcomes and geographic regions where organisms might be most affected by climate change, we suspect that several issues will need to be addressed before its predictive abilities can be validated. first, it is unclear whether parasites will follow the same metabolic 'rules' as box . a risk matrix for predicting host-parasite systems sensitive to climate change determining which host-parasite systems are most sensitive to climate change will also help to target management and monitoring efforts. parasites with poikilothermic hosts, vectors and free-living stages, or that live at high latitudes or elevations, have greater exposure to variable climatic conditions and, thus, might be more likely to respond directly to changes in climate than will parasites with endothermic hosts and direct transmission [ ] . although these intrinsic properties of host-pathogen systems might determine 'fundamental' sensitivity to direct effects of climate change, the 'realized' effects of climate change will be determined by behavioral adjustments (e.g. microclimate selection by vectors), extrinsic adaptation and evolution by hosts, vectors and pathogens, as well as disease control measures. hence, we suggest that a functional, trait-based approach, which addresses direct sensitivity to climatic factors, coupled with an understanding of control measures and the other confounding factors mentioned in the section ''gaps in data, models and their integration'' might prove valuable for determining the overall significance of climate change for different diseases. this risk matrix results in six general disease categories, where overall risk is the product of direct sensitivity to climate and management difficulty ( figure i) . some diseases, such as those restricted to high elevations or latitudes or that prefer cooler temperatures, might experience range contractions with climate change (categories , ). for example, several fungal entomopathogens of insects are expected to decline [ ] (category ). similarly, white pine blister rust, cronartium ribicola, which costs more to control than any other conifer disease, is expected to decrease if conditions get warmer and drier [ ] (category ). indeed, many pests of crops are expected to decrease under warmer and drier conditions [ ] . category diseases show limited direct responses to climate and have good options for control. an example is measles, which is directly transmitted and has a highly effective vaccine. category diseases are also relatively insensitive to direct effects of climate change, but have less effective mitigation measures. this category might apply to numerous wildlife and zoonotic viruses for which options for management or control are limited (e.g. sars corona virus, hendra virus, nipah virus and ebola virus). other diseases are more directly sensitive to climate-change impacts, but might be countered by effective control measures (category ). for example, malaria is sensitive to climate change but high capacity exists for mitigation in developed regions, such as europe and the usa [ , ] , although it may be a category disease in other regions where resources are more limited [ , ] . category diseases, which are both sensitive to climate and difficult to control, would also include many wildlife diseases, such as chytridiomycosis in frogs [ , ] and various diseases of coral [ , ] . this risk matrix emphasizes the direct sensitivity of hosts and parasites to climate change, but host-parasite systems can also be affected by climate through more subtle indirect mechanisms. it is therefore important to quantify how climate change modulate hostparasite interactions both directly and indirectly. direct impact of climatic change free-living organisms. second, the metabolic approach does not yet explicitly incorporate species interactions, such as parasitism [ ] , and accounting for these interactions often improves predictions of climate-change impacts [ ] . third, the emphasis of metabolic theory has been on effects of mean temperature, but changes in other climatic components, such as precipitation and climatic variability, also could impact species interactions (figure ), especially for parasites with life stages outside the host. finally, understanding of how abiotic factors influence host immunity remains in its infancy [ , ] , but will probably have an important role in predicting the outcome of host-parasite interactions (e.g. [ ] ). until more of these knowledge gaps are filled, it will remain unclear whether climate change will have the biggest impact on host-parasite interactions at mid-high latitudes, in the tropics, or at difficult-topredict locations scattered throughout the world (figure ). addressing these gaps will require collaborations among physiologists, immunologists, community ecologists, climatologists and modelers. gaps in scale: a community-and biodiversity-based perspective contemporary research has uncovered the importance of community dynamics to parasite transmission and vice versa [ , , ] and how biodiversity buffers communities against both disease [ ] [ ] [ ] [ ] and climate change [ ] . nevertheless, most disease research has emphasized single host-single parasite interactions [ ] . thus, under-standing of climate change impacts at the scale of whole communities of hosts and parasites remains early in development. in particular, there is an apparent paradox, at the level of communities, which has not been explicitly mentioned in the climate change-disease literature. evidence is mounting that climate change will reduce biodiversity [ , ] , including parasite diversity [ ] . indeed, parasites might be more sensitive to secondary extinctions than might nonparasitic species [ ] . this expected loss of parasite diversity, however, seems to be at odds with the notion that climate change will generally increase diseases [ ] [ ] [ ] . to shed light on this apparent paradox, researchers must understand the patterns of climate-induced parasite declines and the dilution effect, the hypothesis that biodiversity generally reduces wildlife and human diseases [ ] [ ] [ ] [ ] (figure ). this will only occur with collaborations among epidemiologists, theoreticians and community ecologists. climate-driven patterns in declines of parasite species if climate change causes parasite extinctions rather than just range shifts [ ] , the probable non-random nature of these declines [ ] could influence disease severity. for instance, relative to generalist parasites, parasites that specialize on one or a few hosts should be more likely to go extinct as their hosts decline [ ] . furthermore, we predict that climate change will cause more extinctions of parasites with complex life-cycles than of those with direct trends in ecology and evolution june , vol. , no. transmission, because there is a greater chance that at least one of their necessary host species will go extinct with climate change. we also expect a greater fraction of human parasites to go extinct in tropical than temperate regions because a higher percentage of tropical human diseases specialize on a vector species ( % tropical versus % temperate) and/or a wild animal reservoir ( % tropical versus % temperate) [ ] . although parasite extinctions might reduce wildlife and zoonotic diseases, the severity of the remaining diseases could increase or decrease. if, for instance, rare or less pathogenic parasites go extinct more so than abundant or highly virulent parasites, then the impact of parasite extinctions on overall disease incidence and severity might be small. however, the loss of many rare parasite species could be substantial. furthermore, range shifts could expose hosts to novel parasites, which might lead to more severe disease than in disease-endemic areas. this is a concern for potential climate-induced range shifts of human malaria [ ] . in addition, generalist parasites, which might be more likely to persist with climate change (see above), can be more challenging to control because they can be maintained by multiple host species and can therefore persist with higher virulence to a subset of their host species relative to specialist parasites [ ] . increased temperatures might also increase the frequency and intensity of transmission by lengthening the 'growing season' of parasites that survive climate change [ ] . however, the same changes might sometimes drive decreases in transmission if host immunity is enhanced at higher temperatures or when temperatures exceed the optimum for parasite transmission. finally, global warming is generally increasing temperature minima more than maxima and this might be more likely to move temperatures for parasite and vector performance towards their optima than beyond it [ ] . the severity of disease is also likely to be altered by climate-driven changes to host composition. in some host-parasite systems, the most abundant and resilient species are also hosts that amplify transmission, whereas other species might decrease disease risk [ ] . in some of these systems, such 'amplifying' hosts increase in abundance as the density of less resilient, 'diluting' hosts decline [ ] . if extinctions caused by climate change are biased towards these rarer hosts, as we suggest, and these are indeed disease-diluting hosts, climate change might reduce the disease-buffering capacity of biodiversity and increase prevalence and severity of diseases that persist with climate change [ ] [ ] [ ] . however, the relative contributions of individual species to transmission are poorly known for most pathogens and, thus, the significance of any loss in host species remains uncertain. accounting: determining net effects most importantly, the net effect of any anthropogenic factor on disease requires careful accounting [ , ] . researchers must balance the loss of parasites against the loss of the buffering capacity of biodiversity, changes in disease severity, impacts of emerging co-infections (e.g. effects of hiv emergence on malaria [ , ] ) and the ability of humans to enact control measures. regardless of what this accounting reveals, researchers would be remiss to ignore impending changes to parasite, host and non-host diversity when forecasting the effects of climate change on host-parasite interactions. understanding climate change-disease interactions is a formidable problem because of its interdisciplinary nature and the complexities of hosts, parasites and their interactions with the multiple factors that can co-vary with climate change. effective forecasting of climate-change impacts on disease will require filling the many gaps in data, theory and scale (figure ). although this 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of a pesticide on amphibians? partitioning effects on exposure and susceptibility to pollution knowing when to draw the line: designing more informative ecological experiments climate warming strengthens indirect interactions in an old-field food web climate change and forest diseases we thank mercedes pascual for comments on this paper and the ecological society of america for sponsoring the symposium on climate change and disease that helped develop the ideas presented here. key: cord- -f uk m authors: fraser, douglas d.; slessarev, marat; martin, claudio m.; daley, mark; patel, maitray a.; miller, michael r.; patterson, eric k.; o’gorman, david b.; gill, sean e.; wishart, david s.; mandal, rupasri; cepinskas, gediminas title: metabolomics profiling of critically ill coronavirus disease patients: identification of diagnostic and prognostic biomarkers date: - - journal: crit care explor doi: . /cce. sha: doc_id: cord_uid: f uk m objectives: coronavirus disease continues to spread rapidly with high mortality. we performed metabolomics profiling of critically ill coronavirus disease patients to understand better the underlying pathologic processes and pathways, and to identify potential diagnostic/prognostic biomarkers. design: blood was collected at predetermined icu days to measure the plasma concentrations of metabolites using both direct injection-liquid chromatography-tandem mass spectrometry and proton nuclear magnetic resonance. setting: tertiary-care icu and academic laboratory. subjects: patients admitted to the icu suspected of being infected with severe acute respiratory syndrome coronavirus , using standardized hospital screening methodologies, had blood samples collected until either testing was confirmed negative on icu day (coronavirus disease negative) or until icu day if the patient tested positive (coronavirus disease positive). interventions: none. measurements and main results: age- and sex-matched healthy controls and icu patients that were either coronavirus disease positive or coronavirus disease negative were enrolled. cohorts were well balanced with the exception that coronavirus disease positive patients suffered bilateral pneumonia more frequently than coronavirus disease negative patients. mortality rate for coronavirus disease positive icu patients was %. feature selection identified the top-performing metabolites for identifying coronavirus disease positive patients from healthy control subjects and was dominated by increased kynurenine and decreased arginine, sarcosine, and lysophosphatidylcholines. arginine/kynurenine ratio alone provided % classification accuracy between coronavirus disease positive patients and healthy control subjects (p = . ). when comparing the metabolomes between coronavirus disease positive and coronavirus disease negative patients, kynurenine was the dominant metabolite and the arginine/kynurenine ratio provided % classification accuracy (p = . ). feature selection identified creatinine as the top metabolite for predicting coronavirus disease -associated mortality on both icu days and , and both creatinine and creatinine/arginine ratio accurately predicted coronavirus disease -associated death with % accuracy (p = . ). conclusions: metabolomics profiling with feature classification easily distinguished both healthy control subjects and coronavirus disease negative patients from coronavirus disease positive patients. arginine/kynurenine ratio accurately identified coronavirus disease status, whereas creatinine/arginine ratio accurately predicted coronavirus disease -associated death. administration of tryptophan (kynurenine precursor), arginine, sarcosine, and/or lysophosphatidylcholines may be considered as potential adjunctive therapies. objectives: coronavirus disease continues to spread rapidly with high mortality. we performed metabolomics profiling of critically ill coronavirus disease patients to understand better the underlying pathologic processes and pathways, and to identify potential diagnostic/prognostic biomarkers. design: blood was collected at predetermined icu days to measure the plasma concentrations of metabolites using both direct injection-liquid chromatography-tandem mass spectrometry and proton nuclear magnetic resonance. setting: tertiary-care icu and academic laboratory. subjects: patients admitted to the icu suspected of being infected with severe acute respiratory syndrome coronavirus , using standardized hospital screening methodologies, had blood samples collected until either testing was confirmed negative on icu day (coronavirus disease negative) or until icu day if the patient tested positive (coronavirus disease positive). interventions: none. measurements and main results: age-and sex-matched healthy controls and icu patients that were either coronavirus disease positive or coronavirus disease negative were enrolled. cohorts were well balanced with the exception that coronavirus disease positive patients suffered bilateral pneumonia more frequently than coronavirus disease negative patients. mortality rate for coronavirus disease positive icu patients was %. feature selection identified the top-performing metabolites for identifying coronavirus disease positive patients from healthy control subjects and was dominated by increased kynurenine and decreased arginine, sarcosine, and lysophosphatidylcholines. arginine/kynurenine ratio alone provided % classification accuracy between coronavirus disease positive patients and healthy control subjects (p = . ). when comparing the metabolomes between coronavirus disease positive and coronavirus disease negative patients, kynurenine was the dominant metabolite and the arginine/kynurenine ratio provided % classification accuracy (p = . ). feature selection identified creatinine as the top metabolite for predicting coronavirus disease -associated mortality on both icu days and , and both creatinine and creatinine/ arginine ratio accurately predicted coronavirus disease -associated death with % accuracy (p = . ). conclusions: metabolomics profiling with feature classification easily distinguished both healthy control subjects and coronavirus disease negative patients from coronavirus disease positive patients. arginine/kynurenine ratio accurately identified coronavirus c oronavirus disease (covid ) is caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), which continues to spread rapidly worldwide ( , ). diagnosis of covid typically requires polymerase chain reaction for sars-cov- genes or immunoassay for sars-cov- antigens. covid primarily affects lungs, but dysfunction of other organs, such as heart and kidneys, has also been reported ( ) ( ) ( ) ( ) . the severity of covid may involve the excessive release of inflammatory mediators ( - ) together with microvascular thrombi formation secondary to endothelial injury/activation and glycocalyx degradation ( ) . critically ill covid patients are admitted to the icu, where the mortality rate is reported to be - % with standardized icu care ( , ) . although a number of protein mediators have been identified that predict covid -associated death ( , ) , a further characterization of covid -associated processes and pathways is essential for the identification of novel diagnostic/prognostic biomarkers and for improving covid patient outcomes. metabolomics measures a person's metabolite profile (chemicals with an molecular weight < , da), including amino acids, organic acids, biogenic amines, acylcarnitines, glycerophospholipids, sphingolipids, sugars, and many other compounds ( ) . metabolites fall downstream of genetic, transcriptomic, proteomic, and environmental events, thus providing a cohesive measure of a subject's recent phenotype. two complementary analytical methods for metabolomics are proton nuclear magnetic resonance ( h nmr; μm range) spectroscopy and mass spectrometry (ms; nm-pm range). previous studies have demonstrated the diagnostic and prognostic potentials of metabolomics profiling in selecting patient populations (e.g., traumatic brain injury [ ] ). metabolomics profiling of critically ill covid patients over the first days of their icu stay was the overall aim of this exploratory study, thereby identifying potential metabolite candidates and/or combinations as diagnostic/prognostic biomarkers. our specific objectives were: ) to determine/compare the metabolomes between covid positive (+) icu patients and either healthy control subjects or covid negative (-) icu patients, ) to determine specific metabolites that most accurately differentiated covid + from either healthy control subjects or covid -icu patients, and ) to determine whether specific metabolites can predict covid outcome shortly after icu admission. this study was approved by the western university, human research ethics board (hreb). given the unprecedented pandemic situation and the restricted hospital access for substitute decision makers, waived consent was approved for a short, issued march , ) . in keeping with the society for critical care medicine statement on "waiver of informed consent in emergency situations" ( ), the following criteria were considered relevant for hreb approval of waived consent: the subjects were admitted to the icu with a life-threatening condition; the subjects had impaired decisional capacity; the research staff encountered significant obstacles and delays when attempting to contact the absent substitute decision makers; the study risk was minimal; the research knowledge gained on this new, lethal disease offered an eventual chance of benefit; and community consultation had been implemented. given the pandemic circumstances and the waived consent model applied, no further attempts were made to contact the surviving patients and/or substitute decision makers. the last patient enrolled under waived consent was may , . we enrolled consecutive patients who were admitted to our level academic icus at the london health sciences centre (london, on, canada) and were suspected of having covid based on standard hospital screening procedures ( ) . blood sampling began on icu admission for up to days in covid -patients or up to days in covid + patients, with an additional blood draw occurring on day for covid + patients who have not been discharged. covid status was confirmed as part of standard hospital testing by nasopharyngeal swab detection of two sars-cov- viral genes on polymerase chain reaction ( ). patient baseline characteristics were recorded at admission and included age, sex, comorbidities, medications, hematologic labs, creatinine, arterial-partial-pressureto-inspired-oxygen ratio, and chest x-ray findings. we calculated multiple organ dysfunction score ( ) and sequential organ failure assessment score ( ) for both covid + and covid patient groups to enable objective comparison of their illness severity. both patient groups were characterized as having confirmed or suspected sepsis diagnosis using sepsis . criteria ( ) . we also recorded clinical interventions received during the observation period including use of antibiotics, antiviral agents, systemic corticosteroids, vasoactive medications, vte prophylaxis, antiplatelet or anticoagulation treatment, renal replacement therapy, high-flow oxygen therapy, and mechanical ventilation (invasive and noninvasive). final participant groups were constructed by age-and sex-matching covid + patients with covid -patients and healthy controls without disease, acute illness, or prescription medications that were previously banked in the translational research centre, london, on, canada (reb id# e; reissued march , ; "repository of control biological specimens from healthy volunteers for future research purposes"; directed by dr. d. d. fraser; https://translationalresearchcentre.com/) ( , ) . standard operating procedures were used to ensure all samples were treated rapidly and equally. blood was obtained from critically ill icu patients via indwelling catheters in the morning and placed immediately on ice. if a venipuncture was required, research blood draws were coordinated with a clinically indicated blood draw. in keeping with accepted research phlebotomy protocols for adult patients, blood draws did not exceed maximal volumes ( ) . once transferred to a negative pressure hood, blood was centrifuged and plasma was isolated, aliquoted at μl, and frozen at - °c. all samples remained frozen until use and freeze/ thaw cycles were avoided. a targeted quantitative metabolomics approach was used to analyze the samples using a combination of direct injection (di) ms with a reverse-phase lc-ms/ms custom assay. this custom assay, in combination with an absciex qtrap (applied biosystems, foster city, ca/mds sciex, foster city, ca) mass spectrometer, can be used for the targeted identification and quantification of up to different endogenous metabolites including amino acids, acylcarnitines, biogenic amines and derivatives, uremic toxins, glycerophospholipids, sphingolipids, and sugars ( , ) . the method combines the derivatization and extraction of analytes, and the selective mass-spectrometric detection using multiple reaction monitoring pairs. isotope-labeled internal standards and other internal standards were used for metabolite quantification. the custom assay contained a -deep-well plate with a filter plate attached with sealing tape, and reagents and solvents used to prepare the plate assay. the first wells were used for one blank, three zero samples, seven standards, and three quality control samples. for all metabolites except organic acid, samples were thawed on ice and subsequently vortexed and centrifuged at , × g; µl of each sample was then loaded onto the center of the filter on the upper -well plate and dried in a stream of nitrogen. subsequently, phenyl-isothiocyanate was added for derivatization. after incubation, the filter spots were dried again using an evaporator. extraction of the metabolites was then achieved by adding µl of extraction solvent. the extracts were obtained by centrifugation into the lower -deep-well plate, followed by a dilution step with the ms running solvent ( . % formic acid in water, . % formic acid in acetonitrile for biogenic amines and amino acids, and . % formic acid in methanol for all other classes of metabolites). for organic acid analysis, µl of ice-cold methanol and µl of isotope-labeled internal standard mixture were added to µl of serum sample for overnight protein precipitation at - °c, followed by centrifugation at , × g for minutes. a total of µl of supernatant was loaded into the center of wells of a -deep-well plate, followed by the addition of -nitrophenylhydrazine reagent. after incubation for hours, butylated hydroxytoluene stabilizer ( mg/ml) and water were added before lc-ms injection. mass spectrometric analysis was performed on an absciex qtrap tandem ms instrument (applied biosystems/mds analytical technologies, foster city, ca) equipped with an agilent series uhplc system (agilent technologies, palo alto, ca). the samples were delivered to the mass spectrometer by an lc method followed by a di method. data analysis was done using analyst . . (foster city, ca). plasma samples contain a significant concentration of large-molecular-weight proteins and lipoproteins, which affects the identification of the small-molecular-weight metabolites by nmr spectroscopy. a deproteinization step, involving ultrafiltration as previously described ( ) , was therefore introduced in the protocol to remove plasma proteins. prior to filtration, -kda cutoff centrifugal filter units (amicon microcon ym- , burlington, ma) were rinsed five times each with . ml of h o and centrifuged ( , × g for min) to remove residual glycerol bound to the filter membranes. aliquots of each plasma sample were then transferred into the centrifuge filter devices and spun ( , × g for min) to remove macromolecules (primarily protein and lipoproteins) from the sample. the filtrates were checked visually for any evidence that the membrane was compromised, and for these samples, the filtration process was repeated with a different filter and the filtrate inspected again. the subsequent filtrates were collected and the volumes were recorded. if the total volume of the sample was under µl, an appropriate amount from a -mm kh po buffer (ph ) was added until the total volume of the sample was . µl. any sample that had to have buffer added to bring the solution volume to . μl was annotated with the dilution factor and metabolite concentrations were corrected in the subsequent analysis. subsequently, . µl of a standard buffer solution ( % d o: % . -mm kh po ph . v/v containing sodium trimethylsilylpropanesulfonate (dss) [ . -mm , -dimethyl- silcepentane- -sulphonate, . -mm -chloropyrimidine- carboxylate, and . % nan in h o]) was added to the sample. the plasma sample ( µl) was then transferred to a -mm samplejet nmr tube for subsequent spectral analysis. all h-nmr spectra were collected on a -mhz avance iii (bruker, billerica, ma) spectrometer equipped with a -mm hydrogen, carbon, nitrogen z-gradient pulsed-field gradient cryoprobe. h-nmr spectra were acquired at °c using the first transient of the nuclear overhauser effect spectroscopy (noesy) presaturation pulse sequence (noesy dpr), chosen for its high degree of quantitative accuracy ( ) . all free induction decays were zero-filled to k data points. the singlet produced by the dss methyl groups was used as an internal standard for chemical shift referencing (set to ppm). for quantification, all h-nmr spectra were processed and analyzed using an in-house version of the magnetic resonance for metabolomics (magmet)-automated analysis software package using a custom metabolite library. magmet allows for qualitative and quantitative analyses of an nmr spectrum by automatically fitting spectral signatures from an internal database to the spectrum. each spectrum was further inspected by an nmr spectroscopist to minimize compound misidentification and misquantification. typically, all of visible peaks were assigned. most of the visible peaks were annotated with a compound name. it has been previously shown that this fitting procedure provides absolute concentration accuracy of % or better ( ) . medians (interquartile ranges [iqrs]) and frequency (%) were used to report icu patient baseline characteristics for continuous and categorical variables, respectively; continuous variables were compared using mann-whitney u tests (or kruskal-wallis tests, as appropriate), and categorical variables were compared using fisher exact chi-square, with p values of less than . considered statistically significant. receiver operating characteristic (roc) curves were conducted to determine sensitivity and specificity of individual metabolite ratios for predicting a binary outcome. area-under-thecurve (auc) was calculated as an aggregate measure of metabolite ratio performance across all possible classification thresholds. all analyses were conducted using spss version (ibm, armonk, ny). covid analyte data were visualized with a nonlinear dimensionality reduction on the full data matrix using the t-distributed stochastic nearest neighbor embedding (t-sne) algorithm ( ). t-sne assumes that the "optimal" representation of the data lies on a manifold with complex geometry, but with low dimension, embedded in the full-dimensional space of the raw data. for feature selection, the raw data for each subject were ingested within each feature, across subjects. a random forest classifier was trained on the variables to predict covid status or covid outcome ("scikit-learn" module for python . . open source). a random forest is a set of decision trees, and consequently, we were able to interrogate this collection of trees to identify the features that have the highest predictive value. feature selection was not performed in preprocessing. during training, the random forest classifier performed an implicit feature selection; the top features are those that appear highest ranked in the most trees. to reduce overfitting, the number of trees and maximum depth of each tree was limited ( ) ; thus, covid status was determined using a six-fold cross validation with a random forest of trees, whereas patient outcome was determined using a three-fold cross validation with a random forest of trees and a maximum depth of . to remain conservative and to limit the risk of overfitting further, no hyperparameters were tuned or optimized by design and intent. furthermore, to validate the results and ensure no overfitting occurred, a simple linear support vector machine classifier was used to compare the predication accuracies with excellent concordance. we investigated covid + patients (median years of age = . , iqr = . - . ), age-and sex-matched covid -patients (median years of age = . , iqr = . - . ), and ageand sex-matched healthy controls (median years of age = . , iqr = . - . ; p = . ). baseline demographic characteristics, comorbidities, laboratory values, and chest x-ray findings are reported in table . the covid -patients had significantly higher unilateral pneumonia, whereas covd + patients were more likely to have bilateral pneumonia. sepsis was "confirmed" by infectious pathogen identification in only % of covid patients, whereas sepsis was "suspected" in the remaining % ( ) . a mortality rate of % was determined for covid + patients. we measured a total of plasma metabolites using both di-lc-ms/ms and h nmr. in the event of metabolite repeats measured with both techniques ( metabolites), the h nmr metabolite repeat measurements were deleted from the combined metabolite database, yielding a final number of metabolites analyzed. figure a shows a t-sne plot illustrating that the icu day covid + patient metabolome was distinct and easily separable from age-and sex-matched healthy control subjects. in fact, classification accuracy was % when comparing the two metabolomes. we then identified the top eight metabolites underlying these differences between the cohorts, which are shown in figure b with their associated % importance. in the covid + cohort, relative to the healthy control subjects, kynurenine increased . fold whereas arginine decreased . -fold, sarcosine decreased . -fold, and lysophosphatidylcholines (lysopcs) all decreased . -fold on average. the least number of metabolites that were required to maintain a % classification accuracy between the cohorts was then determined, with only arginine (cutoff ≤ . μm) and kynurenine (cutoff ≥ . μm) required. the excellent predictive ability of an arginine/kynurenine ratio for discriminating a covid patient from a healthy control subject (cutoff ≤ . ) is shown with roc analysis in figure c (auc = . ; p = . ). a comparison of covid + and covid -patient cohorts revealed distinct metabolomes. feature classification again identified kynurenine as one of the leading metabolites underlying the differences between the covid + and covid -cohorts ( fig. a) . we then determined that an arginine/kynurenine ratio again showed an excellent discriminative ability to determine covid status on icu day (cutoff ≤ . ) via roc analyses (auc = . ; p = . ; fig. b ). figure c shows an arginine/ kynurenine ratio time plot for the covid + and covid patients over icu days. the cohorts' ratios were significantly different on icu days and (p = . ). figure a shows a t-sne plot for covid + patients that either survived or died, and demonstrates that the outcomes were distinct and separable. to optimize outcome prediction in covid + patients, the number of metabolites was narrowed using feature selection (fig. b) . creatinine was the leading metabolite and could predict death with % accuracy on both icu days (cutoff > μmol/l) and (cutoff > μmol/l). to improve the variation in patient creatinine values, we then tested the ability of a creatinine/arginine ratio to predict death; the corresponding time plot is shown in figure c . death could be predicted with % accuracy on both icu days (cutoff ≥ . ) and (cutoff ≥ . ), as the creatinine/arginine ratios were significantly different between the covid patients that lived or died at both time points (p = . ). the creatinine/arginine ratios normalized by icu day , regardless of eventual outcome. there were no deaths during the icu days. in this study, we measured metabolites in plasma obtained from icu patients, both covid + and covid -, as well as age-and sex-matched healthy control subjects. given the number of metabolites measured, we analyzed the data with the stateof-the-art machine learning. our exploratory data indicate the presence of a unique covid plasma metabolome dominated by changes in kynurenine, arginine, sarcosine, and lysopcs. additionally, we identify that either creatinine alone or a creatinine/arginine ratio predicted icu mortality with % accuracy. despite the exploratory nature of our study, the data generated suggest that these three metabolites (kynurenine, arginine, and creatinine) could be considered for further investigation as continuous data are presented as medians (interquartile ranges) and categorical data are presented as n (%). vte prophylaxis represents the number of patients receiving venous thromboembolism prophylaxis with regular-or low-molecular heparin; new antiplatelets represents the number of patients who were started on aspirin or clopidogrel during icu stay; new anticoagulation represents the number of patients who were started on therapeutic anticoagulation with regular-or low-molecular heparin, or novel oral anticoagulants potential diagnostic and prognostic biomarkers for covid and that they may be useful for patient stratification in clinical interventional trials. our covid + icu patients were similar to those reported in earlier cohorts ( ) ( ) ( ) ( ) with respect to demographics, comorbidities, and clinical presentation. in contrast to covid -icu patients, our covid + icu patients had a higher frequency of bilateral pneumonia. previous work by our study group in these same patients has determined a unique inflammatory profile characterized by elevated tumor necrosis factor and serine proteases ( ) , and a thrombotic profile associated with endothelial activation and glycocalyx degradation ( ). by employing targeted proteomics, figure . a, subjects plotted in two dimensions following dimensionality reduction in their respective metabolites by stochastic neighbor embedding. green dots represent healthy control subjects, whereas orange dots represent age-and sex-matched coronavirus disease positive (covid +) icu patients (icu day plasma). the dimensionality reduction shows that based on the plasma metabolites, the two cohorts are distinct and easily separable. the axes are dimensionless. b, feature classification, demonstrating the top eight plasma metabolites that classify covid + status versus healthy control subjects with their % association. c, receiver operating characteristic analysis of healthy control subjects versus covid + patients, using an arginine/kynurenine ratio, demonstrates an area-underthe-curve (auc) of . (p = . ). the cutoff value is . . the diagonal broken blue line represents chance (auc . ). we also identified six novel protein immune biomarkers that accurately predict covid associated death ( ) . taken together with the data from this study, covid represents a severe illness with a unique pathophysiological signature, as well as a high mortality rate. indeed, in our cohort of covid patients, icu death was % with standardized icu care. our study has identified a unique metabolome in covid + icu patients that is hypothesis-generating for future diagnostic/ prognostic studies. not only have we provided a rank order listing of metabolites important for covid status, we also identified metabolites that accurately determined covid icu outcome. the former represents diverse metabolites that influence immune function and survival, whereas the latter represents compromised renal function early in icu care. importantly, the metabolites required for covid diagnosis (arginine/kynurenine ratio) and outcome (either creatinine alone or creatinine/arginine ratio) can be easily measured using only ms or immunoassay, making their use as covid biomarkers affordable and easily available. point-of-care analyses for these metabolites could be rapidly developed, such as a lateral flow immunochromatographic assay. furthermore, our study raises the possibility that dietary supplementation of tryptophan, arginine, sarcosine, and lysopcs as adjunctive therapies may aid covid outcome. covid status relied heavily on increased plasma kynurenine. the essential amino acid tryptophan is metabolized to elevate the energy-producing cofactor nicotinamide adenosine dinucleotide, with kynurenine as the first stable intermediate to be formed ( ) . increased degradation of tryptophan, with a consequential increase in kynurenine, occurs during an immune response and is driven by the release of interferon-gamma from the activated t-cells. covid caused intense t-cell activation ( , ) with an approximate -fold increase in plasma interferon-gamma in critically ill covid patients when compared with healthy control subjects ( ) . although plasma kynurenine effectively discriminated covid + patients from healthy control subjects, determination of covid status in icu patients required further specificity that was optimally provided by an arginine/kynurenine ratio. arginine, an amino acid precursor for nitric oxide, was significantly depressed in covid + patients. arginine depletion is likely secondary to the intense requirement for nitric oxide signaling and antiviral activity ( ) , as well as consumption by the enzyme arginase that represents a macrophage immunoregulatory mechanism ( ) . as arginine is essential for tissue repair ( ) , its depletion could potentially delay and/or compromise icu recovery. sarcosine, an amino acid that helped discriminate covid + patients from healthy control subjects, was also significantly depressed. although not superior to the arginine/kynurenine ratio for diagnosing covid status, sarcosine sequestration may have a critical role in covid pathology. sarcosine enhances the activity of antigen presenting cells ( ) and activates autophagy ( ) , or the body's removal of damaged cells and their immunostimulatory debris. as a protective catabolic process during covid , autophagy is critical to the antiviral response by direct elimination of virus, the presentation of viral antigens, and the inhibition of excessive inflammation ( ) . sarcosine levels decrease with age ( ) , and the elderly are most susceptible to covid morbidity and mortality. depressed plasma lysopcs also helped discriminate covid + patients from healthy control subjects. the partial hydrolysis of the dimensionality reduction shows that based on the plasma metabolites, the two cohorts are distinct and easily separable. the axes are dimensionless. b, feature classification, demonstrating the top eight plasma metabolites that classify covid + icu patient outcome as alive or dead with their % association. plasma creatinine was the leading outcome predictor metabolite. c, a time plot, demonstrating the creatinine/arginine ratio for covid + icu patients over icu days that either survived (blue dots) or died (orange dots). the two cohorts are significantly different on icu days and (**p = . ). healthy control range values are represented by green shading. phosphatidylcholines by phospholipase a produces lysopcs, which are subsequently implicated in endothelial activation ( ) and phagocytosis of cellular debris ( ) . decreased plasma lysopcs has been observed in sepsis ( ) , where lysopcs may aid pathogen elimination, and therapeutic replacement has been suggested to improve sepsis outcome ( ) . acute renal dysfunction is strongly associated with high mortality in icu patients ( ) . plasma creatinine, a marker of renal dysfunction, was an excellent discriminator for covid patients that either lived or died. in our covid + cohort, two patients had chronic kidney disease and two patients required renal replacement therapy. the angiotensin-converting enzyme receptor that is essential for sars-cov- uptake is highly expressed on tubule epithelial cells ( ) . acute kidney injury is reported to occur in up to % of covid patients ( ) and is secondary to acute tubular injury from direct viral infection ( ) . our data suggest that covid diagnosis (arginine/kynurenine ratio) and outcome (creatinine alone or creatinine/arginine ratio) can be determined with point-of-care measurements of kynurenine, arginine, and creatinine, and that this rapid and affordable biomarker approach may be complimentary to the more expensive and time-consuming diagnostic tools currently employed (e.g., polymerase chain reaction and antigen immunoassay). furthermore, our study raises the possibility that dietary supplementation of tryptophan (kynurenine precursor), arginine, sarcosine, and lysopcs may aid covid outcome as adjunctive therapies. despite the novelty of the metabolite biomarkers discovered, our study has several limitations. first, we only studied critically ill patients and we cannot determine the full metabolome changes associated with icu admissions. second, although our covd study population was limited, we still identified strong associations between the individual metabolites and outcomes and we fulfilled an urgent need for exploratory data to focus future hypothesisdriven studies on larger cohorts. third, we report only mortality as our primary clinical outcome. future studies with larger sample sizes can explore whether reported changes in specific metabolites correlate with additional clinical outcomes such as functional status in survivors. finally, our analyses employed a cross-validation methodology in which the classifier was trained multiple times, each time on a different subset of the data, with the remainder of the data withheld for use only in testing. the reported accuracy is the mean accuracy of all such trials. this is a standard, accepted, technique in the machine learning literature, but should be validated on a larger testing set that is used only once. overfitting was minimized by using a very small number of trees with a limited depth ( ) , and the results verified by training a simple linear vector machine and by identifying concordance between the results. in summary, we report a unique metabolome in covid + icu patients, with identification of three metabolites that appear to be accurate diagnostic/prognostic biomarkers for future studies. given the rapid spread of covid and the critical need for rapid and affordable diagnostics, our data may be invaluable for future testing. in addition, our exploratory data may be useful for guiding resource mobilization and/or goals of care discussion, but only after validation in larger covid + cohorts. furthermore, patient stratification is critically important for future covid interventional trials. world health organization: who director-general's 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mediate lysophosphatidylcholine-induced endothelial cell activation apoptotic cells induce migration of phagocytes via caspase- -mediated release of a lipid attraction signal lipidomic profiling of plasma and erythrocytes from septic patients reveals potential biomarker candidates product inhibition of secreted phospholipase a may explain lysophosphatidylcholines' unexpected therapeutic properties renal failure in the icu: comparison of the impact of acute renal failure and end-stage renal disease on icu outcomes kidney and lung ace expression after an ace inhibitor or an ang ii receptor blocker: implications for covid- acute kidney injury in patients hospitalized with covid- ultrastructural evidence for direct renal infection with sars-cov- key: cord- -et lfl authors: mifflin, lauren; ofengeim, dimitry; yuan, junying title: receptor-interacting protein kinase (ripk ) as a therapeutic target date: - - journal: nat rev drug discov doi: . /s - - -y sha: doc_id: cord_uid: et lfl receptor-interacting serine/threonine-protein kinase (ripk ) is a key mediator of cell death and inflammation. the unique hydrophobic pocket in the allosteric regulatory domain of ripk has enabled the development of highly selective small-molecule inhibitors of its kinase activity, which have demonstrated safety in preclinical models and clinical trials. potential applications of these ripk inhibitors for the treatment of monogenic and polygenic autoimmune, inflammatory, neurodegenerative, ischaemic and acute conditions, such as sepsis, are emerging. this article reviews ripk biology and disease-associated mutations in ripk signalling pathways, highlighting clinical trials of ripk inhibitors and potential strategies to mitigate development challenges. (ripk ) is a master regulator of the cellular decision between pro-survival nf-κb signalling and death in response to a broad set of inflammatory and pro-death stimuli in human diseases , . ripk kinase activation has been demonstrated in post-mortem human pathological samples of autoimmune and neurodegenerative conditions [ ] [ ] [ ] [ ] , and inhibition of ripk kinase activity has shown efficacy in a wide range of animal models of human diseases. as tnfr -mediated ripk activation is the most comprehensively characterized paradigm, ripk inhibitors were originally considered to primarily offer a small-molecule alternative to anti-tnf antibody therapies for tnf-driven autoimmune conditions. however, as researchers continued to delve into the mechanisms governed by ripk , it has become apparent that ripk inhibitors may offer key therapeutic options that anti-tnf therapies do not: first, ripk inhibitors are safe in the central nervous system (cns) as ripk kinase does not signal through tnfr which has a protective role in the cns ; second, ripk participates in a broader set of pro-inflammatory activities than those restricted to tnf ; third, ripk is regulated by a distinct set of signalling molecules that are genetically implicated in human autoimmune and autoinflammatory diseases, as discussed below, and thus patient stratification may be important in conducting clinical trials of ripk inhibitors. necrostatin- s (nec- s) was the first small-molecule inhibitor of ripk kinase to be developed and has been widely used to investigate the role of ripk in mechanistic studies and animal models of human diseases , [ ] [ ] [ ] [ ] . broad therapeutic applications of ripk inhibitors for the treatment of a wide range of human diseases are being investigated in clinical trials. the peripherally restricted gsk′ is being developed for peripheral autoimmune diseases, including psoriasis, rheumatoid arthritis (ra) and ulcerative colitis - . the brain-penetrant dnl is in human clinical trial phase ib/iia for amyotrophic lateral sclerosis (als) , . these trials have laid the groundwork for advancing clinical applications of ripk inhibitors. the important role of ripk in driving cell death and inflammation, the established safety of inhibiting ripk kinase activity in humans and the ability to develop selective small-molecule kinase inhibitors of ripk due to the presence of a unique kinase-regulating allosteric pocket are the major factors that have contributed to ripk 's prominence as a therapeutic target. in this review, we outline the current understanding of ripk biology in activating cell death and nf-κb signalling, systematically review monogenic and polygenic variants of known ripk regulators and discuss how these mutations may contribute to disease pathology. the involvement of ripk in sepsis and acute ischaemic conditions is also discussed. we postulate that the improved understanding of genetic and mechanistic data may be beneficial in segmenting patients in clinical trials, particularly for neurodegenerative and inflammatory diseases. finally, we summarize the current state of ripk inhibitors in the clinic, including disease indications, small-molecule chemotypes, as well as ripk target engagement and pharmacodynamic biomarkers. distinct kinase and scaffold functions of ripk ripk is a -kda protein with an amino-terminal (n-terminal) kinase domain, a carboxy-terminal (c-terminal) death domain and an intermediate domain with a rhim (receptor-interacting protein homotypic interacting motif) that can bind to other nf-κb (nuclear factor κ light chain enhancer of activated b cells). a protein complex whose pathway, which can be activated in response to cytokines, free radicals, viral or bacterial antigens and other stressors, mediates the transcription of genes required for pro-survival and pro-inflammatory signalling. (tumour necrosis factor receptor ). a type i membrane receptor that contains an intracellular death domain that, when activated, can recruit receptor-interacting serine/threonine-protein kinase (ripk ) and tradd to mediate inflammatory and pro-survival functions through the nf-κb pathway, as well as cell death mediated by ripk kinase activity. rhim-containing proteins , . whereas the c-terminal death domain mediates homodimerization as well as heterodimerization with other death domain-containing proteins, such as fadd, tnfr and fas, the n-terminal kinase domain mediates autophosphorylation in trans to promote its own activation , . the scaffold function of ripk is essential for mediating pro-survival nf-κb signalling and mouse perinatal survival: ripk -/mice are born normally but die at postnatal day - (ref. ). ripk deficiency reduces nf-κb-mediated transcription of pro-survival proteins, such as cellular flicelike inhibitory protein (cflip), ciap and a (ref. ). the perinatal lethality of ripk -/mice can be rescued by inhibition of both apoptotic and regulated necrotic cell death (necroptosis) in ripk -/-fadd -/-ripk -/-, ripk -/-casp -/-ripk -/or ripk -/-fadd -/-mlkl -/mice [ ] [ ] [ ] [ ] [ ] . in contrast to the phenotype of ripk -/mice, mice harbouring ripk knock-in kinase dead mutations, including d n and k a, or a k r death domain mutation that blocks the dimerization-mediated activation of ripk are normal , , . in fact, these mutant mice are resistant to various inflammatory and degenerative conditions in mouse models of disease. interestingly, both genetic and pharmacological inhibition of ripk kinase activity, such as treatment with nec- s or gne (refs , ), offer complete resistance against the tnf-induced animal model of septic shock , , . thus, the kinase activity of ripk promotes cell death and inflammation whereas the scaffold function of ripk supports postnatal survival. activation of ripk kinase mediated by tnfr signalling promotes most of the deleterious effects activated by tnf in human disease . in tnf-stimulated cells, the activation of ripk is regulated in a transient multimeric complex associated with the intracellular domain of tnfr , known as complex i (fig. ) . the recruitment and activity of these proteins determines the balance between inhibited ripk kinase activity, where nf-κb pro-survival signalling is activated, and activated ripk kinase activity, which leads to inflammation and cell death. mutations that impact the recruitment to complex i or activity of these proteins leads to aberrant ripk kinase activity in disease, most commonly autoimmunity or inflammation (box ). ripk and tradd, another death domaincontaining adaptor protein, are rapidly recruited into complex i by binding to the death domain of tnfr . activation of ripk in complex i, as measured by the well-established phosphorylation of s , can enact either apoptotic or necroptotic forms of cell death , . in complex i, the activation of ripk is determined by a code including complex ubiquitylation, phosphorylation and other modification events on ripk , which include those directly organized by tradd and those mediated by proteins transcribed and translated downstream of the nf-κb pathway that are also recruited into the complex (for example, a ) . this post-translational ripk code, which may be both cell type-specific and stimulus-specific, modulates the extent of ripk kinase activation, which in turn determines the mode of cell death. there is an extensive body of literature identifying cell type-specific roles for ripk activity using conditional mouse models that we and others have summarized previously, and so do not cover in detail in this article , . for example, in certain cell types, such as oligodendrocytes, tnf stimulation alone may promote the activation of ripk and cell death , ; in other cell types, such as fibroblasts, sustained activation of ripk kinase can only be achieved when tnf stimulation is combined with chemical inactivators of ripk repressors, such as ciap / (inhibited by sm- ), tak , tbk or ikks, that are both mediators of the nf-κb pathway and inhibitors of ripk kinase activation , [ ] [ ] [ ] . ubiquitylation of ripk in complex i, which is organized by tradd, is essential for suppressing the aberrant activation of ripk kinase. tradd recruits traf and the e ubiquitin ligases ciap and ciap into complex i to mediate ripk k ubiquitylation. k ubiquitylation of ripk , in turn, mediates the recruitment and activation of tak kinase through the polyubiquitin binding adaptors tab and tab (ref. ). k ubiquitylation of complex i facilitates the recruitment of the lubac complex containing hoip, hoil and sharpin, which in turn performs m ubiquitylation of ripk and tnfr . m ubiquitylation of complex i is important for the recruitment of the trimeric ikk complex through the polyubiquitin-binding adaptor subunit ikkγ/nemo , , as well as other ubiquitin binding proteins such as abin (a binding and inhibitor of nf-κb- ) and the kinase tbk (refs , ). the activation of ripk is suppressed by direct inhibitory phosphorylation mediated by tak , ikkα/β and ΤΒΚ (ref. ). ciap may also mediate k ubiquitylation of ripk to promote its degradation by the proteasome . in addition, tnfaip , which is transcriptionally induced by the nf-κb pathway, encodes the ubiquitin editing enzyme a , which controls the activation of ripk by modulating its ubiquitylation pattern . tnf stimulation of fibroblasts derived from either wild-type mice or healthy patients does not lead to cell death owing to the suppression of ripk activation in complex i by inhibitory ubiquitylation and phosphorylation. stimulation with tnf in combination with factors that reduce the inhibition of ripk leads to necroptosis, ripk -dependent apoptosis (rda) and inflammation (box ). in experimental paradigms, ripk kinase activity and necroptosis can be activated by treatment with a combination of tnf, cycloheximide (chx), which blocks the activation of nf-κb-mediated transcription and translation, and zvad.fmk, which blocks the activation of caspases . inhibition of caspases strongly sensitizes to ripk activation, as the cleavage of ripk by casp provides an important inhibitory mechanism to block the overactivation of ripk (refs [ ] [ ] [ ] ). rda can be activated with a combination of tnf and the tak inhibitor ( z)- -oxozeanol (known as z ), or tnf and sm- , which blocks inhibitory phosphorylation and ubiquitylation of ripk , respectively, and blocks activation of pro-survival nf-κb signalling . as detailed in this review, these experimental manipulations mimic some aspects of human genetic deficiencies tnf (tumour necrosis factor). a pro-inflammatory cytokine typically secreted by macrophages/monocytes and microglia. tnf signals through two receptors: tnfr , which mediates cell death and nf-κb signalling; and tnfr , which mediates non-canonical nf-κb signalling. a transient complex associated with the intracellular domain of tnfr upon tnf stimulation that includes ripk and many other regulators of nf-κb activation and cell death. www.nature.com/nrd in various regulators of nf-κb activation that lead to aberrant ripk kinase activation and provide guidance for possible indications where ripk inhibitors may be efficacious (table ) . mutations in the genes that encode ripk and multiple proteins that regulate ripk signalling can lead to immune and autoinflammatory diseases. these clinically identified mutations highlight the important role of ripk in regulating the innate immune response and provide mechanistic insights into the functional role of ripk in disease. the spectrum of immune and autoinflammatory diseases presents as a continuum between autoimmune disorders involving primarily the adaptive immune system and autoinflammatory conditions involving primarily the innate immune system, all of which can be found in diseases involving ripk . complex i: stimulation of tnfr by tnf promotes the formation of an intracellular signalling complex associated with the death domain of trimerized tnfr that recruits two death domaincontaining proteins: adaptor protein tradd and receptor-interacting serine/ threonine-protein kinase (ripk ). tradd recruits e ubiquitin ligases ciap / and xiap to perform k ubiquitylation of complex i, including ripk k , which in turn recruits the lubac complex, comprised of hoip, hoil and sharpin. lubac mediates linear (m ) ubiquitylation of ripk . deubiquitinase cyld and its adaptor protein spata modulate m /k ubiquitylation of ripk . m deubiquitinase otulin activates lubac. k ubiquitin chains on ripk recruit tab / and tak . m ubiquitin chains on ripk recruit the nemo-iκb kinase (ikk) complex, tbk , a , abin (a binding inhibitor of nf-κb- ) and optn. a in complex i suppresses the activation of ripk kinase. nf-κb activation: activation of tak and the ikks promote nf-κb pathway activation to mediate transcription of both proinflammatory and pro-survival genes, including a , which modulates the ubiquitylation of ripk to control its activation, and cellular flice-like inhibitory protein (cflip), which modulates activation of caspase (casp ). ripk -dependent apoptosis (rda): activation of tnfr under a , abin , ciap / , nemo, tbk , ikk or tak -deficient conditions leads to ripk kinase activation. activated ripk binds to fadd and casp to form complex iia and promote activation of caspases and apoptosis. increased levels of a promote the activation of ripk in complex iia. necroptosis: inhibition of casp mediated cleavage of ripk promotes ripk dimerization via the c-terminal death domain, which leads to its activation and the subsequent formation of the necrosome (complex iib) comprised of ripk , fadd, casp , ripk and mixed-lineage kinase domain-like pseudokinase (mlkl), which in turn executes necroptosis. ripk -independent apoptosis: when the nf-κb pathway is inhibited, tnf stimulation can promote the formation of a cytosolic complex with fadd and casp to mediate apoptosis independent of ripk . p, phosphate; ripk i, ripk inhibitor; ub, ubiquitin. adapted from ref. , springer nature limited. a form of apoptosis enacted by activated (cleaved) caspase that is independent of ripk kinase activity. rare mutations leading to loss of function (lof) and gain of function (gof) of ripk have been identified in individuals with immunodeficiencies and autoinflammatory diseases (table ) . rare biallelic lof mutations in ripk , including missense, nonsense and frameshift mutations, have been identified in patients with combined immunodeficiency and inflammatory bowel disease (ibd) , . whereas ripk -/mice die perinatally, patients with lof mutations in ripk exhibit paediatric onset of primary immunodeficiency characterized by an increased susceptibility to infections and early-onset ibd , , . at the cellular level, the loss of ripk in human skin fibroblasts impairs activation of the nf-κb pathway, mapks and jun in response to tnf or poly(i:c) and increases the activation of necroptosis mediated by ripk and mixedlineage kinase domain-like pseudokinase (mlkl), but not apoptosis , . the production of pro-inflammatory cytokines, including tnf, il- and il- , in response to lps stimulation is severely impaired in peripheral blood mononuclear cells (pbmcs) from patients with ripk lof compared with control pbmcs . dysregulation of cytokine production and the host defence response towards the gut microbiome likely play a key role in promoting early-onset ibd and progressive polyarthritis in these patients. casp inactivates ripk by cleaving human and mouse ripk after residues d and d , respectively, which separates the ripk kinase domain from the intermediate and death domains . mice with a d a knock-in mutation that prevents cleavage by casp die embryonically, and can be rescued by cis-inactivation of ripk by d n mutation, the loss of tnfr , or inactivating both necroptosis and apoptosis by double knockout of ripk and fadd, or mlkl and fadd , , . rare variants of ripk , such as d v and d h, that block cleavage by casp have been identified in individuals with an auto somal dominant autoinflammatory disease , . in contrast to the phenotype observed in patients with ripk lof mutations, patients with heterozygous noncleavable ripk mutations develop autoinflammatory disease characterized by recurrent fevers and lymphadenopathy. marked increases in pro-inflammatory cytokines and chemokines, such as il- , tnf and interferon-γ (ifnγ), were found in sera from patients. impaired cleavage of ripk d variants by casp hypersensitized patient pbmcs to ripk activation, including both apoptosis and necroptosis induced by tnf, which can be blocked by the ripk inhibitor nec- s (ref. ). although pbmcs from patients with non-cleavable ripk mutations are more susceptible to inflammatory stimulation, fibroblasts from one such patient showed resistance to necroptosis and ferroptosis and reduced expression of pro-inflammatory cytokines in response to stimuli . considerable changes in the gene expression patterns were found in these fibroblasts, including downregulated expression of ripk , ripk and tnfr , as well as elevated levels of the anti-oxidative glutathione and genes that offer resistance to ferroptosis. such compensatory gene expression might be necessary to overcome the deleterious effects of non-cleavable ripk and allow for patient survival, although these findings need to be expanded to additional patients. together, these data suggest that autoinflammatory disease caused by non-cleavable ripk variants may represent a canonical human ripk hyperactivating disease that can respond to ripk inhibitor treatment. dysregulation of ripk signalling is involved in a heter ogeneous group of monogenic immune and autoinflammatory diseases that can present with either episodic or chronic symptoms (table ) . as many of these disease-associated genes are also involved in regulating nf-κb signalling , some amount of disease patho logy can be attributed to altered nf-κb signalling. interestingly, these genes, including tnfaip (encoding a ), tnip (encoding abin ), ikbkg (encoding nemo), otulin and members of the lubac complex, are also direct regulators of ripk activation (table ) . thus, exogenous triggers that lead to transient inflammation in healthy subjects may promote sustained inflammation and cell death involving different tissues and organs in individuals with aberrant ripk regulation. traditional treatment for autoimmune diseases has focused on managing immune hyperactivity by dampening nonspecific inflammatory responses and immune cell proliferation. however, this approach renders patients vulnerable to opportunistic infections that can be life-threatening. understanding the inflammatory box | conditions that sensitize to ripk kinase activation multiple genetic and non-genetic conditions may promote receptor-interacting serine/threonine-protein kinase (ripk ) activation with tnf signalling. • genetic loss-of-function mutations in tnfaip as well as changes in the levels of a encoded by tnfaip , which can lead to various autoimmune and inflammatory diseases , [ ] [ ] [ ] . • inactivation of apoptotic mediators, such as caspase (casp ) or cellular flice-like inhibitory protein (cflip), which decreases suppression of the ripk -mediated necroptotic pathway , . • rare variants of ripk , such as d v and d h, which block the cleavage of ripk by casp and lead to an autosomal dominant autoinflammatory disease , . • reduction of tbk , which directly performs inhibitory phosphorylation on ripk and leads to neuroinflammation and neurodegeneration in amyotrophic lateral sclerosis (als)/frontotemporal dementia , . • reduction of lubac activity in autoimmune and inflammatory diseases by mutations in hoip, hoil , sharpin and otulin, which leads to reduction of m ubiquitylation of ripk important for suppressing its kinase activity , , [ ] [ ] [ ] [ ] , . • increased accumulation of ripk after optn loss, which promotes degeneration of oligodendrocytes and axonal demyelination and neurodegeneration in als , . • increased levels of reactive oxygen species, which result in the formation of disulfide bonds that promote ripk activation . • ageing-related reductions in suppressors of ripk -regulated pathways, the best characterized of which is the reduction of tak seen in ageing human brain samples , . • inhibition of proteasomal or lysosomal degradation of ripk or other members of cell death complexes, such as ripk (refs , , ). • ischaemic conditions, which lead to increased activation of ripk and deleterious downstream pathways by both apoptosis and necroptosis , . www.nature.com/nrd mechanisms regulated by ripk may help to develop therapies that can specifically target the disease pathology in these rare diseases. furthermore, understanding the contribution of ripk in these rare diseases may also help to elucidate roles for ripk in autoimmune and inflammatory diseases that are not genetically linked to ripk . a deficiency. a , encoded by the tnfaip gene, is an inducible ubiquitin-editing enzyme that restricts both toll-like receptor (tlr) and tnf-induced inflammatory responses by regulating the ubiquitylation of key signalling proteins, including ripk , traf and nemo . mouse models with cell lineage-specific a deficiency phenocopy different human inflammatory diseases, suggesting an important role for a in restricting ripk activation in multiple tissues ( showed increased levels of pro-inflammatory cytokines, such as tnf, il- β and il- , and demonstrated clinical improvement after treatment with anti-tnf or anti-il- β therapy. in addition, low-penetrance coding and non-coding variants in tnfaip have been suggested to underlie multiple autoimmune diseases, including crohn's disease, psoriasis, ra, type diabetes mellitus and susceptibility to allergy and asthma [ ] [ ] [ ] [ ] . reduced a expression may even contribute to atopic eczema (atopic dermatitis) , one of the most common inflammatory skin disorders, affecting up to % of adults and % of children globally. these studies suggest that dysregulated a may be a common underlying factor in the pathogenesis of inflammatory diseases. nemo deficiency syndrome. nemo, the scaffolding subunit of the ikk holocomplex comprising ikkα and ikkβ, is critical for regulating activation of the nf-κb pathway during canonical inflammatory responses . nemo contains an n-terminal dimerization domain involved in binding with ikkα and ikkβ, and two distinct ubiquitin binding domains, including a uban domain that preferentially binds to m over k ubiquitin chains . nemo deficiency syndrome is a complex disease caused by lof or hypomorphic mutations in the x-linked ikbkg gene, which encodes nemo, and includes clinical definitions of incontinentia pigmenti and anhidrotic ectodermal dysplasia with immunodeficiency. a case study demonstrated that anti-tnf therapy (infliximab) successfully treated inflammatory colitis in an infant with nemo deficiency . nemo -/mice and epidermis-specific knockout of nemo (nemo eko ) reproduced incontinentia pigmenti phenotypes, including embryonic lethality in males and inflammatory skin lesions in heterozygous females , . the skin inflammation of nemo eko mice can be delayed by crossing them with tnfr -/- , suggesting that ripk kinase may be involved in mediating inflammatory response in incontinentia pigmenti. mutant mice with specific loss of nemo in intestinal epithelial cells (iecs) (nemo iec-ko mice) show apoptosis of paneth cells and colonocytes, and microbiotadriven chronic colitis . the colitis of nemo iec-ko mice was blocked by genetic or pharmacological inhibition of ripk kinase activity, suggesting that the pathology in this mutant line may be driven more by ripk dependent cell death and inflammatory mechanisms than failure to activate nf-κb , . taken together, these studies suggest that ripk kinase activity may play an important role in colitis and excessive inflammation in nemo syndrome. thus, ripk kinase inhibitors may be beneficial for the treatment of colitis and other inflammatory issues in patients deficient in nemo. however, as patients with nemo deficiency syndrome have an inability to mount a successful immune response and are hypersensitive to infection, reminiscent of patients deficient in ripk , the inability to properly activate the nf-κb pathway may also contribute to the symptoms of nemo deficiency syndrome. otulin deficiency. otulin is the only known deubiquitylating enzyme that specifically removes m ubiquitin chains . patients with biallelic hypomorphic mutations in otulin develop a severe form of autoinflammatory disease, known as otulipenia or otulin-related autoinflammatory syndrome (oras) , . in otulin-deficient cells, levels of m ubiquitylation on multiple target proteins, such as nemo, tnfr and ripk , are generally increased. as activity of the lubac activity complex is negatively regulated by m ubiquitylation, otulin-deficient cells have reduced lubac activity . cells derived from patients with otulin deficiencies have a strong inflammatory signature including overproduction of pro-inflammatory cytokines, such as il- β, il- , il- and ifnγ, in response to lps, tnf or il- β. these otulin-deficient cells are also sensitized to tnfinduced cell death . consistent with the important role of tnf in this disease, these patients responded well to treatment with anti-tnf antibodies (infliximab) . otulin -/mice and otulin c a catalytically inactive knock-in mice die during embryonic development , . embryonic lethality of the otulin c a mice can be delayed by ripk d n/d n or tnfr -/-, whereas combined loss of ripk and casp prolongs survival to the perinatal stage, suggesting that necroptosis is activated when otulin is not functional. the lubac complex is composed of hoip, hoil and sharpin. patients identified with lof or hypomorphic mutations in hoip or hoil develop immunodeficiencies and autoinflammatory disease characterized by frequent viral and bacterial infections and multi-organ autoinflammation [ ] [ ] [ ] . these hypomorphic mutations in hoip and biallelic biallelic lof immunodeficiency and multi-organ autoinflammation ibd, inflammatory bowel disease; lof, loss of function; oras, otulin-related autoinflammatory syndrome; ripk , receptor-interacting serine/threonine-protein kinase . lof mutations in hoil lead to a reduction in levels of the lubac complex driven by a loss of stability. lubac-deficient cells show reduced levels of ikk phosphorylation and compromised nf-κb activation. lubac is critical for suppressing ripk activation, and, as a consequence, enhanced ripk activity may be responsible for autoinflammatory symptoms in patients harbouring lubac mutations . mice deficient for the gene encoding the lubac regulatory subunit sharpin, also known as chronic proliferative dermatitis mice (cpdm) (sharpin cpdm/cpdm mice), suffer from severe multi-organ inflammation, particularly in the skin with similarity to atopic dermatitis and psoriasis in humans , . interestingly, the skin inflammation of cpdm mice can be blocked by reducing or knocking out tnf , which points to a key role for this signalling node in the cpdm phenotype. inhibition of ripk by gne or ripk -d n protects against dermatitis in cpdm mice , , . polygenic human diseases, including several autoimmune and inflammatory diseases, are associated with mutations or risk variants in multiple genes, which can include regulators of ripk . when developing ripk kinase inhibitors for the treatment of these diseases, patient stratification may be important to identify those with pathogenic activation of ripk . crohn's disease and ulcerative colitis are the two primary forms of ibd that are both characterized by intestinal inflammation and epithelial cell loss. genome-wide association studies (gwas) of crohn's disease and ulcerative colitis have identified more than loci that are above the thresholds of significance . however, non-genetic factors likely play a major role in conferring susceptibility as the concordance rate in monozygotic twins is only % in ulcerative colitis and - % in crohn's disease . tnfaip , the gene encoding a , is a recognized risk factor for both crohn's disease and ulcerative colitis. both increased and decreased expression of a has been associated with ibd, suggesting the importance of balancing ubiquitin editing activity in complex i. whereas mice with enterocyte-specific a deletion are hypersensitive to experimental colitis and tnf-induced epithelial apoptosis , excessive a can dimerize, which sensitizes iecs to undergo ripk -dependent apoptosis when stimulated by tnf (table ) . iecs with elevated a in the mucosa of patients with ibd are associated with casp activation. inhibition of ripk has been shown to ameliorate pathology in both a -deficient and elevated conditions , . as specific single-nucleotide polymorphisms (snps) in tnfaip have been found to be correlated with primary therapeutic response to anti-tnf (infliximab) therapy , changes in intestinal a expression may be used as a biomarker for ripk dependent pathology to stratify patients with ibd for treatment with ripk inhibitors. gwas have also identified atg l and other autophagy-related genes as risk factors for ibd . atg l is a component in the macromolecular complex that lipidates lc /atg to promote the formation of the canonical double-membrane autophagosome. atg l t a , a risk allele associated with ibd susceptibility, introduces a caspase cleavage site that destabilizes the atg l protein product and reduces autophagy in the presence of tnf , . atg l -deficient intestinal organoids show increased sensitivity to tnf-induced necroptosis, which can be effectively inhibited by nec- s (ref. ). both atg l iec conditional knockout mice and a iec conditional knockout mice show increased severity of hypothermia induced by tnf, which can be blocked by the ripk inhibitor gne (ref. ). psoriasis. gwas identified more than disease susceptibility regions, including the tnfaip , tnip and rela loci in psoriasis . reduced levels of tnfaip nature reviews | drug discovery mrna and a protein have been found in the skin of patients with psoriasis, which can be directly correlated to disease severity . furthermore, specific snps in tnfaip have also been found to predict the response to anti-tnf therapies , suggesting the predictive value of tnfaip in identifying patients likely to respond to a ripk inhibitor treatment for psoriasis. tnip , encoding the abin protein, is one of the highest scoring non-mhc genes associated with various autoimmune and autoinflammatory diseases, including psoriasis, psoriatic arthritis, systemic sclerosis and systemic lupus erythematosus (sle) across multiple gwas . abin , a ubiquitin binding protein with a uban domain that binds preferentially to linear ubiquitin chains, interacts with a to negatively regulate the activation of mapk and nf-κb-mediated gene transcription downstream of tnfr and tlrs [ ] [ ] [ ] . abin , which is recruited into the tnfr signalling complex by binding linear ubiquitin chains, in turn promotes the recruitment of a (ref. ). abin -deficient cells and a -deficient cells are both sensitized to ripk activation and necroptosis. tnip -/mice die embryonically owing to liver necrosis that can be rescued by ripk -/or ripk d n/d n . tnip heterozygosity sensitizes cells to a hyperactive antiviral response mediated by an nf-κb-dependent and ripk -independent mechanism that promotes the expression of pattern recognition receptors, such as tlr , rig-i and mda (ref. ). inhibition of ripk blocks the increased production of pro-inflammatory cytokines in tnip +/mice in response to viral stimuli. thus, abin regulates the innate immune response through the nf-κb pathway, utilizing both ripk -dependent and independent signalling pathways. inhibition of ripk may reduce the exaggerated inflammatory response in patients with tnip mutations without affecting their host defence responses. rheumatoid arthritis (ra) is a common inflammatory autoimmune disease characterized by chronic synovial inflammation, joint destruction and systemic complications. the hla-drb alleles are the primary genetic risk factor associated with ra. in addition, gwas have identified ~ non-hla loci, including tnf, associated with ra. gwas data also suggest the involvement of multiple immune cell types, including t cells, b cells, natural killer cells, dendritic cells and monocytes, as well as the pro-inflammatory cytokines produced by these cells, in mediating ra pathogenesis . interestingly, snps and reduced levels of tnfaip have been associated with ra . inhibition of ripk by gne improved pathology in a rodent model of collagen-induced arthritis . is an autoimmune disease of the cns resulting in neurodegeneration that involves the loss of myelinating oligodendrocytes that normally insulate the axons of neurons. tnf is strongly linked with the aetiology of ms , . oligodendrocytes can undergo necroptosis in the presence of tnf stimulation alone . in lesions of brain samples from patients with ms or mouse models of ms, activation of ripk , ripk and mlkl and formation of the necrosome were observed . cflip is transcribed in response to nf-κb activation and can suppress the formation of apoptotic signalling complexes . in the lesions from patients with ms, levels of cflip were found to be decreased, which may be the result of chronic neuroinflammation. decreased cflip and reduced levels of activated casp in patients with ms, and thus decreased casp inactivating cleavage of ripk , likely explain the increased ripk kinase activity and necroptosis seen in ms. genetic (ripk d n/d n ) and pharmacological (nec- s) inhibition of ripk kinase ameliorated disease pathology, improved animal behaviour, attenuated the production of pro-inflammatory cytokines and decreased recruitment of immune cells in two animal models of ms . another inhibitor of ripk kinase developed by takeda also showed efficacy in an animal model of ms . the contribution of peripheral immune cells and microglia to ms has been highlighted by a recent genetic association study using , ms cases and , controls . this study identified putative ms susceptibility genes that are highly expressed in cell types involved in adaptive immunity and innate immunity, including tnfaip , which encodes a . as ripk is highly expressed in macrophages and microglia in experimental autoimmune encephalomyelitis lesions, inhibition of ripk may directly reduce inflammatory responses as ripk activity is involved in mediating the inflammatory responses in these cells , . thus, the beneficial effects of inhibiting ripk may include modulation of both inflammation and cell death in susceptible oligodendrocytes, microglia and neurons. this raises the intriguing possibility that ripk may act in both a cell-autonomous and a non-cell autonomous manner in ms. as the existing approved ms drugs all target adaptive immunity and do not directly address neuroinflammation that drives progression in ms, ripk inhibition may represent a novel therapeutic approach that targets multiple aspects of disease pathology. responses. sle is a chronic condition that affects multiple organs and tissues. multiple independent snps in the tnip and tnfaip genes are found to be associated with sle [ ] [ ] [ ] . assembly and activation of the necroptotic machinery, including ripk , has been implicated in netosis, a programmed suicide of neutrophils in tissues involved in mounting normal immune responses against pathogens [ ] [ ] [ ] . netosis results in the formation of neutrophil extracellular traps (nets); this leads to the release of autoantigens from neutrophils and contributes to autoimmune pathology . in several diseases in which nets drive toxicity, inhibiting ripk and thereby blocking other components of the necroptosis pathway has shown efficacy in disease models, including autoimmune vasculitis, venous thrombosis and sle [ ] [ ] [ ] . more broadly, toxicity induced by neutrophil www.nature.com/nrd extravasation and net formation has been implicated in widespread diseases, ranging from autoimmune conditions to neuro degenerative diseases where an impaired blood-brain barrier results in aberrant net formation in the brain parenchyma [ ] [ ] [ ] . the canonical and alternative complement pathways are well-characterized and essential contributors to the pathogenesis of diseases such as sle. the activation of complement in disease results in complementdependent cytotoxicity. complement can activate ripk -ripk -mlkl-dependent necroptosis as a contributor of complement-dependent cytotoxicity, which can be attenuated by ripk inhibitors . furthermore, in some instances of autoimmunity where complement activation drives disease, the complement pathways can be activated by neutrophil netosis, which is also regulated by ripk activation . taken together, these findings suggest that ripk may be involved in the pathogenesis of neutrophil and complement-mediated autoimmune conditions. given that ripk activation and necroptosis have now been genetically and mechanistically linked with human neurodegenerative diseases, there has been a keen interest in developing cns-penetrant ripk inhibitors to alter disease progression for diseases such as als, frontotemporal dementia (ftd), ad and other ageing-related neurodegenerative diseases , , (fig. ) . beyond these indications where several independent groups and clinical data have confirmed a role for pathogenic ripk kinase activation, limited data suggest that ripk activation may also play a role in the pathology of huntington disease and duchenne muscular dystrophy; however, the mechanisms underpinning this have not yet been defined , . als is a progressive neurodegenerative disease that affects motor neurons in the brain and the spinal cord. ftd is a group of related neurodegenerative diseases that lead to the progressive degeneration of neurons in the temporal and frontal lobes of the brain, with consequences on social, emotional and language abilities. lof mutations in two genes, optn and tbk , lead to familial als/ftd , . loss of optn, a monogenic cause of als, resulted in activation of ripk and necroptosis in the spinal cords of optn -/mice, which was rescued by genetic and pharmacological inactivation of ripk kinase . optn -/cells and the spinal cords in optn -/mice show increased levels of total ripk protein, due to decreased proteasomal degradation of ripk protein. furthermore, gain-of-function mutations have been found in p (encoded by sqstm ), another causal als factor that associates with optn; p mutants have been found to interact with ripk to shift the balance between necroptotic and apoptotic death , . tbk haploinsufficiency is a monogenic cause of als/ftd , . mice lacking both copies of tbk show profound ripk activation, resulting in embryonic lethality that is rescued by genetic inactivation of ripk (ref. ). furthermore, tbk is responsible for directly inhibiting ripk by phosphorylation at t ; thus, loss of tbk activity increases susceptibility to ripk activation. age-dependent reduction of tak expression in human brains was shown to cooperate with heterozygous loss of tbk to promote late-onset als/ftd-like pathology mediated by decreased ripk inhibition , . the activation of ripk , ripk and mlkl has been found in post-mortem spinal cord samples from patients with als . an ex vivo human co-culture system of astrocytes and motor neurons from patients harbouring a sod familial als mutation showed necroptotic death of motor neurons, which could be rescued with nec- (ref. ). in addition, a sod g a mouse model of als treated orally with nec- s showed improvements in survival, motor activity and axonal pathology. taken together, these observations suggest a potential approach to genetic stratification of patients in a disease where ~ % of patients exhibit idiopathic disease without known genetic identifiers. ad is a form of progressive dementia that leads to the irreversible loss of neurons involved in cognitive and executive functions. the neuropathology of ad includes the presence of amyloid plaques and neurofibrillary tangles. chronically sustained neuroinflammation plays an important role in mediating neural dysfunction and neurodegeneration in ad. similar to what is observed in the insoluble fractions of post-mortem human cns samples from patients with ms and als , , increased activation of ripk , ripk and mlkl was also found in the insoluble fractions of sporadic ad post-mortem brains , . a recent study reported the detection of all three activated components of the necrosome machinery, pripk , pripk and pmlkl, in granulovacuolar degeneration lesions in degenerating neurons in ad and preclinical stages of ad pathology . interestingly, the presence of activated necrosome components correlated with neuronal loss in ad-affected brain regions, such as the hippocampal ca region and the frontal cortex layer iii, and tau pathology, but not aβ pathology. ripk may play an important role in driving neuroinflammation in ad . the activation of ripk was shown to mediate the transcriptional induction of cst , which encodes an endosomal/lysosomal cathepsin inhibitor named cystatin f, in microglia of mouse models of ad. cst is a biomarker for disease-associated microglia present in spatial proximity to aβ plaques found in both post-mortem human ad brain samples and in an ad mouse model . inhibition of ripk suppresses the expression of a set of genes, such as ch h, which are altered in microglia in two animal models of ad, in the sod g a model of als and in ageing microglia, which enhance the ability of microglia to uptake and degrade aβ . ch h is one of the cholesterol metabolism-related genes associated with ad . treatment with nec- s has been shown to be efficacious in reducing neuro inflammation and cognitive deficits in the app/ps aβ-driven mouse model of ad . these results suggest that ripk may control a neuroinflammatory pathway involved in multiple neurodegenerative diseases and in ageing. (also known as complex iib). a complex comprising activated ripk , receptor-interacting serine/threonine-protein kinase (ripk ) and mixedlineage kinase domain-like pseudokinase (mlkl) that enacts necroptosis, a regulated form of necrotic cell death. similar to the loss of oligodendrocytes seen in ms, ripk and necroptosis may also drive white matter loss, which has been found to be among the earliest brain pathological changes in ad, preceding the formation of neural fibrillary tangles and amyloid plaques . in addition, age-related changes in brain white matterwhich include the reduction of myelinated fibres and myelin sheath degeneration co-localized with increased ubiquitin deposits -coupled with the presence of neuroinflammation may mediate the age-related cognitive decline seen in elderly individuals without ad . the neuroinflammation and oligodendrocyte degeneration present in normal ageing human brains suggests the possibility of modulating ripk kinase to support cognitive function in ageing. in addition, ageing is associated with mitochondrial dysfunction and the subsequent production of reactive oxygen species, the latter of which has shown responsiveness to ripk inhibition in animal models , . although this area requires significantly more exploration, one elucidated mechanism by which ageing sensitizes to ripk activation is through the reduction of tak expression in the cns resulting from cerebral hypoperfusion in ageing human brains , fig. | mechanisms of ripk in neurodegenerative diseases. activation of receptor-interacting serine/threonineprotein kinase (ripk ) in neurodegenerative diseases may promote the death of neurons and oligodendrocytes cell-autonomously, and inflammation in microglia and astrocytes that acts non-cell-autonomously to promote neurodegeneration. neurons and oligodendrocytes: amyotrophic lateral sclerosis (als)/frontotemporal dementia (ftd)associated loss-of-function (lof) mutations in tbk and optn and ageing-related reduction in tak can promote ripk -dependent apoptosis and necroptosis, which can be inhibited by ripk inhibitors. parkinson disease (pd)associated mutations in opa and drp , als-associated mutations in sod and the chemical stressor mptp lead to mitochondrial dysfunction and the production of reactive oxygen species (ros). cytosolic ros can modulate cysteine residues on ripk , which promotes activation. microglia and astrocytes: activation of ripk leads to the production of pro-inflammatory cytokines in microglia. activation of ripk in alzheimer disease (ad) leads to increased production of cst and ch h, both of which are associated with the disease-associated microglia phenotype. pd/lysosomal storage disorder (lsd)-associated genes, such as gba and npc , also result in lysosomal dysfunction, which can promote ripk activation, likely by promoting the accumulation of ripk . als-associated mutations in sod are also known to promote mitochondrial dysfunction and ros production, which may promote glial activation of ripk in disease. mlkl, mixed-lineage kinase domain-like pseudokinase; p, phosphate; rda, ripk -dependent apoptosis; ripk i, ripk inhibitor. www.nature.com/nrd ripk at s may provide a biomarker for ripk activation in ageing brains. a non-synonymous variant in sharpin has been identified as a risk variant for late-onset ad . as sharpin-deficient cpdm mice develop tnf-dependent severe dermatitis and multi-organ inflammation that can be blocked upon inhibition of ripk kinase , , , the reduction of sharpin activity might contribute to the risk of developing late-onset ad by reducing m ubiquitylation of ripk to promote its activation. parkinson disease and lysosomal storage disorders lrrk , the most common monogenic cause of parkinson disease (pd), which is also upregulated in sporadic disease, has been identified as a sensitizer to ripk -dependent apoptosis , . additionally, in vitro and in vivo models of these diseases suggest that ripk can be activated by mitochondrial damage or lysosomal dysfunction. induced pluripotent stem cell-derived dopaminergic neurons from patients harbouring opa mutations that sensitize to mitochondrial fragmentation undergo necroptosis, which can be inhibited by ripk inhibitors. treatment with nec- can also rescue in vivo loss of dopaminergic neurons in mice treated with mptp, a rodent model of pd . as mitochondrial dysfunction is known to produce high levels of reactive oxygen species, it is possible that increased reactive oxygen species are directly responsible for ripk activation in pd . consistent with ripk activation downstream of lysosomal inhibition seen in ad models, lysosomal dysfunction caused by niemann-pick disease, type c and gaucher's disease also results in ripk -dependent activation of necroptosis , . sepsis is a life-threatening condition caused by immune hyperreactivity to viral, bacterial and fungal infections . sepsis is characterized by dysregulated production of pro-inflammatory cytokines (known as a cytokine storm), lymphopenia, coagulopathy, increased vascular permeability and eventual organ failure and death. both genetic inhibition and pharmacological inhibition of ripk in animal models have been shown to block tnf-induced sepsis , . kinase inhibition of ripk leads to robust and highly reproducible protection against sepsis, including attenuation of hypothermia and complete rescue of lethality. these observations suggest that the kinase activity of ripk is critical in propagating immune hyperreactivity following infection, which may happen in septic conditions associated with severe pathogen infection. the efficacy of ripk inhibitors in sepsis is mechanistically attributable to the reduction of pro-inflammatory cytokines and circulating damage-associated molecular patterns, rescue of increased intestinal or vascular permeability and activation of the clotting cascade in the vascular endothelium compartment , , . interestingly, the uncontrolled cytokine storm, which occurs in both patients and animal models, is thought to be driven by the interplay of inflammatory signalling and inflammatory cell death . this deleterious cycle could be attenuated through ripk inhibition, not only through a blockade of inflammatory response but also by inhibiting necrotic cell death; the ripk inhibitor nec- has been shown to protect against lung injury in a neonatal model of sepsis induced by bacteria-driven caecal slurry . patients with severe covid- caused by the novel sars-cov- infection exhibit multiple hallmarks of sepsis with increased plasma pro-inflammatory cytokines, including il- , ifnγ, ccl , il- , g-csf and tnf, coagulation dysfunction and lymphopenia, which has been suggested to be a predictor for the severity of covid- (refs , ). activation of ripk has been shown to promote the production of proinflammatory cytokines, including il- and tnf, as seen in patients with non-cleavable ripk mutations . ripk has also been shown to promote the production of il- , tnf, il- and gm-csf in mice in response to lps or poly(i:c) injection, which mimic bacterial or viral infections, respectively , . in addition, ripk -mediated necroptosis of hiv-infected cd + t cells has been implicated in the depletion of t cell populations in patients , suggesting that, more broadly, lymphopenia seen in viral infections such as hiv and sars-cov- might be attributable to the activation of ripk . activation of necroptosis in severe sepsis and septic shock was also supported by the elevated levels of ripk in the plasma of these patients . taken together, these data suggest that an ripk inhibitor may present a novel therapeutic option to reduce the aberrant hyperinflammatory response and sepsis in the context of both viral and bacterial infections. nec- was shown to be protective in the middle cerebral artery occlusion mouse model of stroke , which was the first example of the role of necroptosis in animal models of human diseases. subsequently, multiple in vivo studies confirmed that ripk kinase inhibition shows efficacy in middle cerebral artery occlusion models [ ] [ ] [ ] [ ] [ ] , acute brain trauma and ischaemia-reperfusion injuries in the brain, retina, heart, kidneys and liver , [ ] [ ] [ ] [ ] [ ] . widespread ripk activation has been seen in ischaemia-reperfusion models of stroke, with rapid activation of necroptosis in neurons and endothelial cells, which may transition to apoptosis driven by the reduced tak levels as the result of cerebral hypoperfusion . although there are compelling in vivo data that ripk inhibition is effective at rescuing pathological and behavioural attributes following acute cerebral insults, a ripk inhibitor has yet to be advanced into clinical trials for these conditions, which is largely due to broad challenges seen in conducting clinical trials for acute cerebral insults . however, since a ripk inhibitor may reduce cell death under both ischaemic and haemorrhagic stroke conditions, the ability to administer a ripk inhibitor to stroke patients without differential diagnosis in an ambulance is an advantage that should be considered to reduce neuronal loss and preserve vascular function in the limited time window. involvement of ripk has also been suggested in mediating acute kidney injuries induced by cisplatin, gadolinium contrast, sepsis or ischaemia-reperfusion , [ ] [ ] [ ] [ ] [ ] and hepatitis induced by ethanol, lps, acetaminophen or concanavalin a [ ] [ ] [ ] [ ] . interestingly, a polymorphism in the tnf promoter at position (known as the tnfa allele) leading to increased levels of tnf is associated with insulin resistance and is prevalent at higher rates in patients with nonalcoholic fatty liver disease . similarly, patients with nonalcoholic steatohepatitis and mice fed high-fat diets also showed increased levels of tnf, ripk and mlkl . finally, ischaemia-reperfusion injuries are commonly seen following organ transplant. in mouse models of lung, kidney and heart transplant, inhibition of ripk or necroptosis in the transplanted tissue improved allograft survival [ ] [ ] [ ] . clinical development of ripk inhibitors ripk contains a unique hydrophobic pocket that allosterically regulates the activation of its kinase activity , (fig. ). all ripk inhibitors described to date bind to this pocket. a phenotypic screen for small molecules that can block necroptosis led to the identification of the first specific inhibitors of ripk , -( h-indol- -ylmethyl)- -thiohydantoins and -( h-indol- -ylmethyl)hydantoins known as nec- s , (table ) . a subsequent family of benzo xazepinone ripk inhibitors was published by glaxo smithkline (gsk), which included gsk′ and the clinical candidate gsk′ (refs , ), which was followed by the identification of a dihydropyrazole chemotype of ripk inhibitors including gsk′ and gsk′ (refs , ). published and patented compounds generated by takeda, genentech and rigel are all from the same benzoxazepinone family as the gsk′ series [ ] [ ] [ ] . fig. . gsk′ was the first ripk kinase inhibitor to enter phase ia clinical trials, and has subsequently progressed through phase ia, ib and ii clinical trials , , . this peripherally restricted compound is currently being tested for peripheral autoimmune diseases. following completion of the initial phase ii clinical trials with gsk′ in ulcerative colitis, ra and psoriasis, phase ib trials in psoriasis were reinitiated at a much higher dose ( mg versus mg daily) . gsk′ has continued to show an excellent safety profile in a phase iia clinical trial in mild to moderate plaque psoriasis, but additional data may be needed to demonstrate efficacy in this indication . dnl (denali therapeutics) was the first brainpenetrant ripk inhibitor advanced into a phase ia clinical trial; although this programme was later discontinued owing to limited post-dosing liver toxicity deemed unrelated to ripk inhibition, this study demonstrated the safety of inhibiting ripk in the cns . denali therapeutics subsequently initiated a phase ia clinical trial for dnl , another brain-penetrant ripk inhibitor, followed by phase ib/iia trials of this compound in als and ad . as of june , phase ib/iia clinical trials for dnl are ongoing for als; however, denali announced that dnl , a brain-penetrant back-up compound, will be entering phase ia trials in early (ref. ). due to the progressive, chronic nature of ad and that > % ripk kinase inhibition may be required for this disease, dnl was deemed to have potential dose-limiting toxicities that were not seen preclinically with dnl , although no toxicity has been seen in the clinic to date. sanofi has partnered with denali on ripk inhibitors and completed a phase ia clinical trial of the peripherally restricted dnl for peripheral autoimmune indications, including ms , , . rigel has also initiated a phase ia clinical trial targeting autoimmune diseases with r , a benzoxazepinone presumed to be hydrophilic hydrophobic receptor-interacting serine/threonine-protein kinase (ripk ) contains a unique hydrophobic pocket located between the n terminus and c terminus of the kinase domain, which allosterically regulates kinase activation. all ripk inhibitors discovered to date, such as necrostatin- s (nec- s) shown here, bind to this pocket and stabilize ripk in an inactive conformation (pdb: ith). this pocket is created owing to the outward movement of the αc-helix, resulting in the loss of an ionic pair between catalytic lys and glu of the αc-helix. the other side of the pocket is formed by the dlg motif in the inactive dlg-in conformation (catalytic asp facing away from the active centre) and the activation segment, which immediately follows the dlg motif. ser residue in the activation segment forms a critical hydrogen bond with the indole of nec- s. gsk′ and other benzoxazepinones also extend into the atp binding pocket, which may contribute to the increased affinity. adapted with permission from ref. ad in neurodegeneration; psoriasis, ulcerative colitis and ra in autoimmune disease; and pancreatic ductal adenocarcinoma in oncology [ ] [ ] [ ] [ ] [ ] . phosphorylation of s ripk has been established as a biomarker of ripk target engagement , . in phase ia clinical trials of dnl , ex vivo assay-based phosphorylation of s ripk in human pbmcs was used to assess the inhibition of ripk kinase , suggesting clini cal feasibility to determine the activation of ripk in blood samples for target engagement studies. in the phase ia study of gsk′ , target engagement was also measured in pbmcs ex vivo using a novel immunoassay based on a conformational change that occurred following inhibitor binding to ripk (ref. ). production of several pro-inflammatory cytokines and chemokines regulated by ripk kinase activity can also be used as biomarkers of ripk activation . of these rapidly inducible, secreted factors, gsk selected the chemokines macrophage inflammatory protein α (mip α; also known as ccl ) and mip β (also known as ccl ) as pharmacodynamic biomarkers downstream of ripk activation . gsk′ dose-dependently reduced mip- α and mip β production, which corresponded to their target engagement data. denali presented data indicating that il- β, il- and mcp (also known as ccl ) were also ripk responsive, demonstrating that dnl dose-dependently reduced il- β in primary human cells . taken together, these studies demonstrate that ripk preclinical biology has provided clinically validated biomarkers of ripk activation in blood samples, which may also provide value as inclusion criteria to identify patients who will most benefit from ripk inhibitor therapy. lof or gof mutations in the gene encoding ripk itself, as well as in those encoding multiple regulators of the nf-κb pathway and ripk activation, such as tak , nemo, a , abin , otulin and the lubac complex, have been identified in human inflammatory and ageing-related diseases. in addition to these monogenic diseases, dysregulation of ripk , such as that which occurs on down-regulation or up-regulation of a , may also be involved in mediating polygenic diseases involving ripk . the activation of ripk -mediated neuroinflammation may provide a common basis for the role of ripk in human neurodegenerative diseases. although a specific set of genetic associations between ripk activation and neurodegenerative diseases has been discovered, the presence of activated ripk , ripk and mlkl in post-mortem human pathological samples seen in ms, als and ad demonstrates the direct relevance of this pathway even in sporadic cases. notably, the role of a and cflip, which are transcriptional targets of nf-κb, in suppressing the activation of ripk suggests that defects in activation of the nf-κb pathway, in genetic and sporadic settings, may promote the activation of ripk from dysregulated a -mediated ripk ubiquitylation modification as well as inactivation of casp due to dysregulated cflip expression. phosphorylation of ripk s , as well as downstream events (such as pripk and pmlkl) and the increased production of specific pro-inflammatory cytokines (such as ccl , mip α/ccl , mip β/ccl , il- and tnf), can be used as biomarkers of ripk activation. however, a brain-penetrant positron-emission tomography imaging probe for ripk kinase activity would be particularly helpful in the development of ripk inhibitors for the treatment of neurodegenerative diseases. as pathways downstream of ripk , including cell death, pro-inflammatory cytokines and nf-κb activation, can be modulated directly by disease pathogenesis, it is important to include biomarkers that specifically measure ripk activation when considering a clinical study. it is also important to remember the cell type specificity of ripk activation and the genetic context of the diseases, as sustained activation of ripk in specific cell types, such as fibroblasts, can only be achieved in certain settings. clinical trials of ripk inhibitors in patients are still in their early days, and, to date, limited chemotypes of ripk inhibitors with appropriate in vivo properties have been developed. development of selective, potent and safe small-molecule inhibitors of ripk , biomarkers to reliably measure the clinical efficacy of ripk kinase inhibition and patient stratification remain the key challenges facing future clinical development. published online xx xx xxxx this review summarizes what is currently known about the role of ripk in neurological diseases regulation of rip kinase signalling at the crossroads of inflammation and cell death this paper provides the first evidence for the role of ripk in a chronic autoimmune disease with neurodegeneration -ms this paper provides the first genetic connection of ripk kinase and necroptosis with als necroptosis activation in alzheimer's disease ripk mediates a diseaseassociated microglial response in alzheimer's disease essential protective role of tumor necrosis factor receptor in neurodegeneration targeting ripk for the treatment of human diseases identification of rip kinase as a specific cellular target of necrostatins this paper isolates multiple necrostatins, including nec- , and uses necrostatins to define necroptosis as a regulated necrotic cell death mechanism that broke the traditional dogma activity and specificity of necrostatin- , small-molecule inhibitor of rip kinase rip: a novel protein containing a death domain that interacts with fas/apo- (cd ) in yeast and causes cell death this paper demonstrates that the decline of tak levels in human ageing brains sensitized the activation of ripk to promote neuroinflammation and degeneration and the onset of als/ftd in individuals heterozygous for tbk death-domain dimerization-mediated activation of ripk controls necroptosis and ripk -dependent apoptosis the death domain kinase rip mediates the tnf-induced nf-κb signal this paper shows that the scaffold function of ripk is involved in mediating nf-κb activation necroptosis in development and diseases ripk blocks early postnatal lethality mediated by caspase- and ripk rip suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition ripk regulates ripk -mlkldriven systemic inflammation and emergency hematopoiesis the pseudokinase mlkl and the kinase ripk have distinct roles in autoimmune disease caused by loss of death-receptor-induced apoptosis ripk maintains epithelial homeostasis by inhibiting apoptosis and necroptosis cutting edge: ripk kinase inactive mice are viable and protected from tnf-induced necroptosis in vivo cutting edge: rip kinase activity is dispensable for normal development but is a key regulator of inflammation in sharpindeficient mice this paper demonstrates ripk activation in human ra and psoriasis samples by ps immunochemistry, efficacy of a ripk inhibitor in animal disease models of ibd, ra and skin inflammation, and effect of ripk inhibitor for pancreatic cancer metastases rip kinase-dependent necrosis drives lethal systemic inflammatory response syndrome cutting edge: ripk kinase inactive mice are viable and protected from tnf-induced necroptosis in vivo spata regulates the activation of ripk by modulating linear ubiquitination regulation of ripk activation by tak -mediated phosphorylation dictates apoptosis and necroptosis tbk and ikkε prevent tnf-induced cell death by ripk phosphorylation serine phosphorylation inhibits ripk kinase-dependent cell death in models of infection and inflammation tab and tab activate the nf-κb pathway through binding to polyubiquitin chains holding ripk on the ubiquitin leash in tnfr signaling abin- regulates ripk activation by linking met ubiquitylation with lys deubiquitylation in tnf-rsc ubiquitin-mediated regulation of ripk kinase activity independent of ikk and mk a -a bipartite ubiquitin editing enzyme with immunoregulatory potential mutations that prevent caspase cleavage of ripk cause autoinflammatory disease a dominant autoinflammatory disease caused by non-cleavable variants of ripk ) (ref. ), this paper reports the discovery of a dominant autoinflammatory human disease caused by non-cleavable ripk activity of caspase- determines plasticity between cell death pathways biallelic ripk mutations in humans cause severe immunodeficiency, arthritis, and intestinal inflammation human ripk deficiency causes combined immunodeficiency and inflammatory bowel diseases cleavage of the death domain kinase rip by caspase- prompts tnf-induced apoptosis cleavage of ripk by caspase- is crucial for limiting apoptosis and necroptosis ripk can mediate apoptosis in addition to necroptosis during embryonic development nf-κb pathway in autoinflammatory diseases: dysregulation of protein modifications by ubiquitin defines a new category of autoinflammatory diseases loss-of-function mutations in tnfaip leading to a haploinsufficiency cause an early-onset autoinflammatory disease haploinsufficiency of a impairs protein-protein interactome and leads into caspase- -dependent enhancement of nlrp inflammasome activation analysis of autoimmune diseaseassociated variants in type diabetes identifies q / tnfaip as a susceptibility locus sequencing of tnfaip and association of variants with multiple autoimmune diseases genome-wide scan reveals association of psoriasis with il- and nf-κb pathways farm dust and endotoxin protect against allergy through a induction in lung epithelial cells keratinocyte expression of a / tnfaip controls skin inflammation associated with atopic dermatitis and psoriasis regulation and function of ikk and ikk-related kinases specific recognition of linear ubiquitin chains by nemo is important for nf-κb activation infliximab therapy for inflammatory colitis in an infant with nemo deficiency nemo/ikkγ-deficient mice model incontinentia pigmenti female mice heterozygous for ikkγ/ nemo deficiencies develop a dermatopathy similar to the human x-linked disorder incontinentia pigmenti skin lesion development in a mouse model of incontinentia pigmenti is triggered by nemo deficiency in epidermal keratinocytes and requires tnf signaling nemo prevents rip kinase -mediated epithelial cell death and chronic intestinal inflammation by nf-κ-dependent and -independent functions otulin antagonizes lubac signaling by specifically hydrolyzing met -linked polyubiquitin the deubiquitinase otulin is an essential negative regulator of inflammation and autoimmunity biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease otulin limits cell death and inflammation by deubiquitinating lubac otulin deficiency in oras causes cell type-specific lubac degradation, dysregulated tnf signalling and cell death human hoip and lubac deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia second case of hoip deficiency expands clinical features and defines inflammatory transcriptome regulated by lubac immunodeficiency, autoinflammation and amylopectinosis in humans with inherited hoil- and lubac deficiency cell death and inflammation -a vital but dangerous liaison a spontaneous mutation characterized by chronic proliferative dermatitis in c bl mice linear ubiquitination prevents inflammation and regulates immune signalling rip kinase activity is critical for skin inflammation but not for viral propagation host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease epidemiology of inflammatory bowel disease in a german twin cohort: results of a nationwide study enterocyte-specific a deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis elevated a promotes tnf-induced and ripk -dependent intestinal epithelial cell death a prevents inflammasomedependent arthritis by inhibiting macrophage necroptosis through its znf ubiquitin-binding domain a controls intestinal homeostasis through cell-specific activities crohn disease: a current perspective on genetics, autophagy and immunity atg l t a variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense a crohn's disease variant in atg l enhances its degradation by caspase autophagy protein atg l prevents necroptosis in the intestinal epithelium large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants low tnfaip expression in psoriatic skin promotes disease susceptibility and severity tnfaip gene polymorphisms are associated with response to tnf blockade in psoriasis abin- is a ubiquitin sensor that restricts cell death and sustains embryonic development de-ubiquitination and ubiquitin ligase domains of a downregulate nf-κb signalling molecular recognition of m -linked ubiquitin chains by native and phosphorylated uban domains abin- heterozygosity sensitizes to innate immune response in both ripk -dependent and ripk -independent manner integrative analysis of genome-wide association study and expression quantitative trait loci datasets identified various immune cell-related pathways for rheumatoid arthritis functional evaluation of tnfaip (a ) in rheumatoid arthritis selective modulation of tnf-tnfrs signaling: insights for multiple sclerosis treatment tnf receptor genetic risk mirrors outcome of anti-tnf therapy in multiple sclerosis ref. ), this paper provides the first insight that genetic variants in the tnfr /ripk signalling pathway modulate response to anti-tnf therapy in autoimmune disease the flip side of life discovery of -oxo- , , , -tetrahydro- h-pyrazolo[ , -c]pyridine derivatives as potent, orally available, and brain-penetrating receptor interacting protein (rip ) kinase inhibitors: analysis of structure-kinetic relationships international multiple sclerosis genetics consortium. multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility caspase blockade induces rip -mediated programmed necrosis in toll-like receptoractivated microglia necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression a large-scale replication study identifies tnip , prdm , jazf , uhrf bp and il as risk loci for systemic lupus erythematosus association of tnfaip interacting protein , tnip with systemic lupus erythematosus in a japanese population: a case-control association study association of a functional variant downstream of tnfaip with systemic lupus erythematosus challenges in the characterization of neutrophil extracellular traps: the truth is in the details the pseudokinase mlkl activates pad -dependent net formation in necroptotic neutrophils necroptosis and neutrophil-associated disorders a role for receptor-interacting protein kinase- in neutrophil extracellular trap formation in patients with systemic lupus erythematosus: a preliminary study necroptosis controls net generation and mediates complement activation, endothelial damage, and autoimmune vasculitis activated platelets induce mlkl-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis neutrophil extracellular traps (nets) in autoimmune diseases: a comprehensive review neutrophils promote alzheimer's disease-like pathology and cognitive decline via lfa- integrin loss of the tight junction proteins occludin and zonula occludens- from cerebral vascular endothelium during neutrophilinduced blood-brain barrier breakdown in vivo receptor-interacting protein kinases and , and mixed lineage kinase domain-like protein are activated by sublytic complement and participate in complementdependent cytotoxicity necrostatin- ameliorates symptoms in r / transgenic mouse model of huntington's disease necroptosis mediates myofibre death in dystrophin-deficient mice mutations of optineurin in amyotrophic lateral sclerosis haploinsufficiency of tbk causes familial als and fronto-temporal dementia sqstm mutations in familial and sporadic amyotrophic lateral sclerosis the autophagy machinery controls cell death switching between apoptosis and necroptosis exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways necroptosis drives motor neuron death in models of both sporadic and familial als necrosome complex detected in granulovacuolar degeneration is associated with neuronal loss in alzheimer's disease a unique microglia type associated with restricting development of alzheimer's disease cholesterol -hydroxylase on chromosome q is a susceptibility gene for sporadic alzheimer's disease oligodendroglial cells in alzheimer's disease what's behind the decline? the role of white matter in brain aging inhibiting ripk limits neuroinflammation and alleviates postoperative cognitive impairments in d-galactose-induced aged mice inhibiting rip improves chronic stressinduced cognitive impairments in d-galactose-induced aging mice sequential activation of necroptosis and apoptosis cooperates to mediate vascular and neural pathology in stroke a rare functional variant of sharpin attenuates the inflammatory response and associates with increased risk of late-onset alzheimer's disease lrrk activation in idiopathic parkinson's disease regulation of a distinct activated ripk intermediate bridging complex i and complex ii in tnfα-mediated apoptosis pharmacological inhibition of necroptosis protects from dopaminergic neuronal cell death in parkinson's disease models rip autophosphorylation is promoted by mitochondrial ros and is essential for rip recruitment into necrosome necroptosis in niemann-pick disease, type c : a potential therapeutic target ripk as a potential therapeutic target for gaucher's disease sepsis: inflammation is a necessary evil rip kinase -dependent endothelial necroptosis underlies systemic inflammatory response syndrome inhibition of necroptosis attenuates lung injury and improves survival in neonatal sepsis clinical features of patients infected with novel coronavirus in wuhan lymphopenia predicts disease severity of covid- : a descriptive and predictive study ripk and ripk kinases promote cell-death-independent inflammation by toll-like receptor necroptosis takes place in human immunodeficiency virus type- (hiv- )-infected cd + t lymphocytes necroptosis regulated proteins expression is an early prognostic biomarker in patient with sepsis: a prospective observational study synergistic protective effects of humanin and necrostatin- on hypoxia and ischemia/ reperfusion injury necrostatin- prevents necroptosis in brains after ischemic stroke via inhibition of ripk -mediated ripk /mlkl signaling necrostatin- ameliorates intracerebral hemorrhage-induced brain injury in mice through inhibiting rip /rip pathway necrostatin- improves long-term functional recovery through protecting oligodendrocyte precursor cells after transient focal cerebral ischemia in mice inhibition of necroptosis rescues sahinduced synaptic impairments in hippocampus via creb-bdnf pathway necrostatin- reduces histopathology and improves functional outcome after controlled cortical impact in mice role of necroptosis in autophagy signaling during hepatic ischemia and reperfusion the role of necroptosis in cardiovascular disease necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model phenytoin inhibits necroptosis ripk deficiency or catalytically inactive ripk provides greater benefit than mlkl deficiency in mouse models of inflammation and tissue injury challenges in acute ischemic stroke clinical trials programmed necrosis in acute kidney injury rip (receptor-interacting protein kinase ) mediates necroptosis and contributes to renal ischemia/reperfusion injury the rip -kinase inhibitor necrostatin- prevents osmotic nephrosis and contrast-induced aki in mice necroptosis and ferroptosis are alternative cell death pathways that operate in acute kidney failure necrostatin- attenuates sepsisassociated acute kidney injury by promoting autophagosome elimination in renal tubular epithelial cells absence of receptor interacting protein kinase prevents ethanol-induced liver injury necrostatin- protects against d-galactosamine and lipopolysaccharide-induced hepatic injury by preventing tlr and rage signaling necrostatin- protects against reactive oxygen species (ros)-induced hepatotoxicity in acetaminophen-induced acute liver failure ripk protects from tnfα-mediated liver damage during hepatitis tumor necrosis factor α promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis inhibition of regulated necrosis attenuates receptor-interacting protein kinase -mediated ischemia-reperfusion injury after lung transplantation ripk -mediated necroptosis promotes donor kidney inflammatory injury and reduces allograft survival ripk -mediated necroptosis regulates cardiac allograft rejection structural basis of rip inhibition by necrostatins discovery of a first-in-class receptor interacting protein (rip ) kinase specific clinical candidate (gsk ) for the treatment of inflammatory diseases dna-encoded library screening identifies benzo[b][ , ]oxazepin- -ones as highly potent and monoselective receptor interacting protein kinase inhibitors characterization of gsk′ : a structurally distinct, potent and selective inhibitor of rip kinase rip kinase drives macrophagemediated adaptive immune tolerance in pancreatic cancer discovery of -oxo- , , , -tetrahydro- h-pyrazolo[ , -c]pyridine derivatives as potent, orally available, and brain-penetrating receptor interacting protein (rip ) kinase inhibitors: analysis of structure-kinetic relationships potent and selective inhibitors of receptor-interacting protein kinase that lack an aromatic back pocket group rip inhibitory compounds and methods for making and using the same randomized clinical study of safety, pharmacokinetics, and pharmacodynamics of ripk inhibitor gsk in healthy volunteers ) (ref. ), this paper outlines pharmacokinetic and pharmacodynamic biomarkers used in clinical trials for ripk inhibitors response to inhibition of receptorinteracting protein kinase (ripk ) in active plaque psoriasis: a randomized placebo-controlled study dnl , a centrally penetrant ripk inhibitor, inhibits rip kinase phosphorylation in a randomized phase i ascending dose study in healthy volunteers denali therapeutics announces positive clinical results with its lead ripk inhibitor molecule and intention to initiate patient studies in multiple indications in collaboration with sanofi denali therapeutics provides broad update on its ripk program partnered with sanofi denali therapeutics announces that its partner sanofi has commenced dosing of dnl in a phase study isoxazolidine derived inhibitors of receptor interacting protein kinase (ripk ) rigel pharmaceuticals provides business update prior to investor & analyst call identification of a rip kinase inhibitor clinical candidate (gsk ) for the treatment of pancreatic cancer necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression develop, defeat degeneration an association between asthma and tnf- g/a polymorphism: meta-analysis germline mutations in the extracellular domains of the kda tnf receptor, tnfr , define a family of dominantly inherited autoinflammatory syndromes traf signaling in human health and disease xiap deficiency is a mendelian cause of late-onset ibd xiap deficiency syndrome in humans association of peli polymorphisms in systemic lupus erythematosus susceptibility in a chinese population haploinsufficiency of tab causes congenital heart defects in humans whole-genome sequencing reveals important role for tbk and optn mutations in frontotemporal lobar degeneration without motor neuron disease infantile onset intractable inflammatory bowel disease due to novel heterozygous mutations in tnfaip (a ) multiple polymorphisms in the tnfaip region are independently associated with systemic lupus erythematosus novel heterozygous c y a / tnfaip gene mutation is responsible for chronic inflammation in autosomal-dominant behcet's disease negative regulation of the nlrp inflammasome by a protects against arthritis characteristics of a gene polymorphisms and clinical significance in patients with rheumatoid arthritis association of tumor necrosis factor α-induced protein interacting protein (tnip ) gene polymorphism (rs ) with lupus nephritis in egyptian patients a deficiency in b cells enhances b-cell proliferation and results in the development of autoantibodies a (tnfaip ) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis association of two independent functional risk haplotypes in tnip with systemic lupus erythematosus abin dysfunction as a genetic basis for lupus nephritis genetic correlation between amyotrophic lateral sclerosis and schizophrenia genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci x-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired nf-κb signaling genomic rearrangement in nemo impairs nf-κb activation and is a cause of incontinentia pigmenti. the international incontinentia pigmenti (ip) consortium ref. ), this paper identifies mutations in nemo as the cause of incontinentia pigmenti and anhidrotic ectodermal dysplasia with immunodeficiency atypical forms of incontinentia pigmenti in male individuals result from mutations of a cytosine tract in exon of nemo (ikkγ) ikk-related genetic diseases: probing nf-κb functions in humans and other matters caspase- deficiency presenting as late-onset multi-organ lymphocytic infiltration with granulomas in two adult siblings the ubiquitin-modifying enzyme a restricts ubiquitination of the kinase ripk and protects cells from necroptosis a deficiency causes spontaneous neuroinflammation in mice keratinocyte-specific ablation of the nf-κb regulatory protein a (tnfaip ) reveals a role in the control of epidermal homeostasis a protects cells from tnf-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms discovery of small molecule rip kinase inhibitors for the treatment of pathologies associated with necroptosis discovery of a highly potent, selective, and metabolically stable inhibitor of receptorinteracting protein (rip ) for the treatment of systemic inflammatory response syndrome e , a highly selective inhibitor of receptor-interacting protein kinase , protects cells against cold hypoxia-reoxygenation injury discovery of potent necroptosis inhibitors targeting ripk kinase activity for the treatment of inflammatory disorder and cancer metastasis structure guided design of potent and selective ponatinib-based hybrid inhibitors for ripk structure-activity relationship analysis of a novel necroptosis inhibitor, necrostatin- structure-activity relationship study of a novel necroptosis inhibitor, necrostatin- a novel necroptosis inhibitor-necrostatin- and its sar study denali therapeutics announces first-in-human dosing of its ripk inhibitor clinical program catalytic activity of the caspase- -flip(l) complex inhibits ripk -dependent necrosis ciaps block ripoptosome formation, a rip /caspase- containing intracellular cell death complex differentially regulated by cflip isoforms genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in alzheimer's disease proteasome inhibition blocks necroptosis by attenuating death complex aggregation lysosomal damage after spinal cord injury causes accumulation of ripk and ripk proteins and potentiation of necroptosis ischemic insults induce necroptotic cell death in hippocampal neurons through the up-regulation of endogenous rip studies in the authors' laboratories (j.y.) are supported by nih grants r -ag , r -ag and rf -ag . the authors thank q. zhou of zhejiang university, j. lewcock of denali therapeutics and d. vucic of genentech for helpful comments. the authors thank b. toure of nido biosciences for assistance with figures. j.y. conceived the review topic. l.m. and j.y. wrote the manuscript with support from d.o. j.y. is a consultant for denali therapeutics and sanofi. d.o. is an employee of sanofi. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -oe yv y authors: dutta, ritaban title: replacement management in cattle: health management date: - - journal: reference module in food science doi: . /b - - - - . - sha: doc_id: cord_uid: oe yv y replacements are the future of the dairy industry. focusing on improving health management of replacements will yield tremendous returns through decreased losses of animals with the greatest genetic potential on the dairy, decreased costs of medication, improved growth rates, improved feed efficiency and earlier entry into the milking herd. health management begins before replacements are born with attention to the nutrition of lactating and dry cows, the vaccination of lactating and dry cows, control of length of the dry period and both control of the disease status of the dams and the cleanliness of the calving environment. greater attention must be paid to animal and environmental biosecurity to prevent introduction of diseases into the herd and to digestive disorders such as diarrhea, internal parasites and appropriate vaccination programs for the calves. health management of replacements is often overlooked because producers do not see the immediate returns for their efforts. common sense management in cattle, historical facts, experience based practice cultural and social aspects, combined with research, would depict that having adequately optimised balanced diets for the replacements, without producing excessive body conditions, could achieve a production of healthy replacements with superior levels of milk production. continual video monitoring of the herd, modern thermal infrared imaging of the dry cows and calves body parts to identify early symptoms, and overall animal health and biosecurity risk analysis could achieve a sustainable and efficient replacement management practice in cattle industry. replacements are the future of the dairy industry. focusing on improving health management of replacements will yield tremendous returns through decreased losses of animals with the greatest genetic potential on the dairy, decreased costs of medication, improved growth rates, improved feed efficiency and earlier entry into the milking herd. health management of dairy replacements begins before the replacements are born. several factors, such as nutrition of lactating and dry cows, vaccinations of lactating and dry cows, length of dry periods, cleanliness of the calving environment and disease status of the dams, will ultimately affect disease resistance and health of replacements. it is important to note, however, that cows that are overfed tend to have difficulty calving because of being excessively over conditioned. cows that are underfed, which results in mineral or vitamin deficiencies or lack of body condition, may produce inferior and low-volume colostrum. they also may experience difficulty calving. protein deficiency in cows during the dry period may lead to low birth weights, low metabolic rates and poor vigor of calves, resulting in poor survivability. some research also indicates that inadequate protein and energy nutrition of the dam results in poor absorption of immunoglobulins from colostrum by the calf. cows that lose condition during the dry period are also at greater risk of experiencing calving difficulty. calves that experience difficult births require more time before being able to stand, experience an increase in the time to voluntary suckling and have a decreased ability to absorb immunoglobulins. all these problems result in decreased transfer of passive immunity from the dam to the calf and increased risk of disease in calves. as the degree of calving difficulty increases, the risk of mortality for calves increases. proper nutrition of dairy cows during lactation and the dry period will help decrease disease risks for replacements. in recent times researcher are employing electronic cattle tags to remotely monitor cows' general health conditions correlated with the movements of the head and other body parts. some farming communities apply more direct video surveillance mechanism to monitor milking herd to prevent any possible disruption in milk production by taking care of early animal health symptoms and unexpected animal behaviours. intelligent processing of video stream, combined with in-situ visual observations by a farmer could provide a better management culture in replacement management. low cost thermal imaging cameras could also be deployed to monitor the cows and calves body temperature, environmental conditions, and any unusual spots in a herd that has significantly higher temperature than normally expected. early prevention of any bacterial and viral disease spread is extremely important for precalving and calving management. this issue has become ever more important in today's world, where market accessibility for a local producer of milk and dairy products, could be global. bigger market access and business proposition comes with a significantly higher level of responsibility. zero tolerance attitudes towards animal and food biosecurity are key to greater market access and business sustainability. greater awareness is needed to achieve a standard biosecurity management in cattle industries. informed decision on planned vaccinations of the dams will also impact disease resistance of dairy replacements and a better preventive management. proper vaccination of the dairy herd will increase the concentration of antibodies (immunoglobulins specific for diseases) in colostrum. dams may be vaccinated during the dry period against pathogens that are common causes of diarrhea in calves, such as escherichia coli, rotavirus and coronavirus. vaccination of the dams increases the concentration of antibodies against these pathogens in colostrum, thus providing increased protection for calves, resulting in decreased incidence or duration of diarrhea. vaccination of the dams during the dry period is more effective for prevention of disease in calves than vaccination of calves at an early age. the immune system of neonatal calves is unable to respond quickly to a vaccination or an infection because the immune system of the newborn is immature at birth. both numbers and effectiveness of antibody-producing cells are lower in calves at birth than in adult cattle. therefore, it is important for calves to obtain antibodies against common diseases of calves by consumption of colostrum rather than from an attempt to vaccinate calves at an early age. vaccination of the dams against pneumonia may also help to decrease the incidence or severity of this disease in replacements. another important factor that may affect the health of replacements is the length of the dry period of the dam. a dry period that is too short, i.e. less than weeks, may not provide enough time for involution of the mammary gland and preparation for the next lactation. cows with shortened dry periods produce small quantities of colostrum that may also have low concentrations of immunoglobulins. it is important for health of replacements, therefore, that cows have at least a -week dry period for production of high-quality colostrum. management of the calving environment has a tremendous impact on the health of replacements. it is important for calves to be born in a clean, dry environment. wet, sloppy stalls provide a perfect environment for growth of bacteria. calving on a grass lot may be the best alternative when the climate is dry and mild. if a maternity barn is used, it is important to clean stalls thoroughly between calvings to prevent transfer of disease. maternity stalls should only be used for calving and never for housing sick cows. maternity pens and sick pens should be kept in separate facilities in order to prevent transfer of disease to highly vulnerable neonates and periparturient dairy cows. it is also important for the cows to be as clean as possible at calving in order to prevent calves from contracting disease organisms when suckling or attempting to suckle their dams. preferably, calves should be separated from dams prior to suckling in order to prevent the calf from ingesting pathogens present on the legs, belly, flanks or udder of the cow as the calf attempts to nurse. separating the calf from the dam and feeding colostrum by bottle also ensures adequate intake of colostrum for transfer of passive immunity from the dam. finally, it is important to know the disease status of cows prior to calving. diseases such as johne's disease, milk fever (hypocalcaemia) close to calving, bovine viral diarrhea (bvd), and bovine leucosis virus (blv) may be passed in utero or through colostrum. calves should only be fed colostrum from cows known to be free of these diseases. it is important, therefore, to maintain a supply of frozen, high-quality colostrum from cows free of such diseases. today's cost could be tomorrow's gain, so conservativeness towards deploying new technologies in replacement management could be addressed by community based farming, to try new practice, to tackle ever growing biosecurity related threats in dairy industries. the importance for baby calves of adequate consumption of immunoglobulins from colostrum has been reviewed elsewhere. mortality resulting from lack of consumption of adequate amounts of immunoglobulins is commonly greater than % and has been reported to be as high as %. others have indicated a -fold increased risk of mortality when calves do not consume colostrum. % of the hypocalcaemia cases occur within day of calving, because extremely high rate of drain calcium drainage (and other substances) from the blood during the milk and colostrum production, which cattle bodies are unable to replace quickly enough, bring severe health risk and subsequent economic loses. along with economic losses from high mortality rates as a result of lack of colostrum consumption, there are also increased costs associated with increased medication and poor feed efficiency. transfer of passive immunity (absorption of immunoglobulins from colostrum) can be determined using commercial kits that measure immunoglobulins in the blood. for adequate protection of calves, blood immunoglobulin concentrations should be at least mg ml À . serum protein concentrations in calves are also highly correlated with the concentration of immunoglobulins in blood and can be used to determine adequate transfer of passive immunity. a hand-held refractometer can be used to measure serum protein; levels greater than . g ml À by h of age indicate adequate consumption of colostrum. the similar technology could be used to measure 'specific gravity of urine' and 'refractive index of serum', as researchers are aiming to combine these factors for better diagnosis. the use of colostrum substitutes and replacers may help improve disease resistance in calves when high-quality colostrum is not available. it's also important to have a better feeding management plan for the dry cows weeks before the calving, as this effects the amount of calcium available to replace blood calcium, and how efficiently body can utilize the available calcium at a very critical period. the most prevalent health problem of calves on most farms in the united states is diarrhea. organisms such as cryptosporidium parvum, rotavirus and coronavirus that cause diarrhea will not respond to antibiotic treatment. for cryptosporidiosis, the only means of prevention is sanitation, which includes controlling flies. for rotavirus and coronavirus, the most effective prevention is vaccination of the dam to increase antibodies in the colostrum against these organisms. other organisms, such as e. coli and salmonella sp., may be resistant to many of the commonly used antibiotics. producers often give antibiotics to calves during episodes of diarrhea in order to prevent secondary infections; however, this practice often does more damage than good, killing beneficial gut microflora and damaging the gut lining. the first step in caring for calves with diarrhea is to provide fluids for hydration and electrolytes for mineral loss, while continuing to provide milk for protein and energy. an electrolyte solution can be fed from min to h after each feeding of milk or milk replacer until feces return to normal. secondly, the organism causing diarrhea should be identified to determine whether antibiotic treatment is needed. pneumonia is the second most prevalent health problem of replacements, especially for replacements raised indoors. research has shown that calves raised in individual hutches (plastic, fiberglass or wooden structures providing individual housing) perform very well and have fewer health problems, especially pneumonia, than calves raised in closed buildings. open-front housing for older heifers should also help prevent pneumonia. adequate, draught-free ventilation is important for prevention of pneumonia. hutches, pastures and open-front housing for replacements provide optimal ventilation. in addition, hutches can be moved from location to location, giving producers the opportunity easily to remove old bedding and to break disease cycles. no matter what type of housing is used for replacements, cleanliness, dry bedding and adequate ventilation are essential to decrease incidence of disease. another important factor for controlling disease in replacements is grouping of heifers. most producers in the united states house young calves individually. in other areas, housing calves in groups and using mob-feeders is an efficient method of rearing calves during the liquid feeding phase. after weaning, calves should be housed in small groups of or fewer until they have successfully made the transition from liquid feed to dry feed and the transition from individual housing to competing for food. the grouping could be done better by multi-sensory historical recording of individual cow behavioural patterns, body temperature, provenance of any previous disease records, long term eating patterns from the young age, and any obvious known symptoms at the time of the grouping. additionally, by housing in small groups (rather than mixing large groups of animals at one time), producers can limit the exposure of calves to disease organisms and match calves more closely by size. as calves age, they can be housed in increasingly larger groups; however, animals should be grouped so there is not more than kg difference in size of animals up to months and not more than kg difference in size for older animals. biosecurity is an outcome of a human systemso people are at its core. all dairy producers must actively institute biosecurity measures to prevent introduction of disease into the herd and to minimize spread of disease within the herd. for replacements, it is extremely important to prevent exposure of younger animals to older animals that may have johne's disease. exposure is not limited merely to animal-to-animal contact, but also includes articles of transmission, such as manure on hands, clothing and boots of workers, manure from older animals on equipment for feeding and handling replacements, or water that has been contaminated by older animals. in addition, flies can transfer diseases from older to younger animals. producers must determine whether to have a closed herd or to allow introduction of new animals to the farm. if new animals are brought to a farm, the producer should work closely with a veterinary surgeon to determine which vaccinations animals should receive prior to coming to the farm. once new animals arrive on the farm, or animals return to the farm from contract-growers or exhibitions, they should be quarantined for at least days. this will allow time to determine if the new animals are likely to become ill and to allow the new animals to be exposed more slowly to any disease organisms currently on the farm. airborne disease spread in cattle farming is rear but when it happens, severity of damage is significantly higher. prions, the proteins that cause mad cow disease and creutzfeldt-jakob disorder could be spread through the air, rather than just through contaminated food, as recent studies have reported. foot-and-mouth disease (fmd) is a severe, highly contagious viral disease of cattle and swine, primarily get spread under favourable weather conditions. the disease may be spread considerable distances by over a route, within a short time period, directed by wind speed, air pressure and temperature. given the fact that airborne spread is probably the one of the most serious issue in animal biosecurity management, a predictive meteorological service for national level biosecurity risk pathway analysis is essential, for prevention of large-scale livestock disease control. in recent time researchers have reported interesting results and applications of a data driven predictive model to track a probable route of an airborne disease, an alerting system to provide better biosecurity decision support for efficient management of cattle health. other potential sources of disease entry into replacements are visitors, vehicles removing dead animals, feed-delivery vehicles, wild and domestic animals, and birds. within the herd of replacements, diseases can be transferred by using needles on more than one animal or using the same glove to palpate more than one animal. producers must identify potential sources for transfer of disease-causing organisms within the herd and from outside the herd and institute a management plan to control them. digestive disorders can occur in dairy replacements, resulting in problems such as acidosis and overeating diarrhea. overeating diarrhea is found in replacements during the liquid feeding phase and may be prevalent in systems using accelerated feeding programs. this form of diarrhea can be treated by decreasing the amount of dry matter offered to calves in the liquid diet until the consistency of the feces returns to normal. care should be taken to determine whether increased fluidity of the feces is caused by overeating or by disease organisms. if caused by disease organisms, treatment should include administration of an electrolyte solution and may require use of antibiotics. acidosis can occur in replacements if they consume large amounts of grains. forages comprise the basis for diets for replacements after months of age. animals that gain access to fields of maize or bags of feed by accident will often suffer acidosis leading to laminitis (founder) or even death. animals that are affected will generally have severe diarrhea. they can be treated by withholding grain until feces return to normal, followed by gradual reintroduction of grain into the diet. several types of internal parasites are found in dairy replacements. perhaps the most common problem is coccidiosis. coccidiosis causes diarrhea, which may be severe, resulting in weight loss, dehydration and anemia. animals can be treated with a coccidiostat, such as amprolium, for severe coccidiosis. coccidiostats such as decoquinate or lasalocid may be included in grain rations or even in milk replacers to help control coccidiosis. another common internal parasite of calves is cryptosporidium parvum. this organism causes diarrhea in young calves at - days of age that lasts approximately a week. there are no cures for cryptosporidiosis and no means of prevention other than sanitation to decrease the pathogen load. treatment involves electrolyte solutions along with continued milk feeding. replacement animals are very vulnerable to internal parasites (especially worms) during their first grazing season. deworming of heifers yields economic returns in growth rates and feed efficiency. producers should consult their veterinary surgeon to determine the most effective method of treating internal parasites both to decrease the parasite load in the animals and to prevent shedding of eggs onto pastures. depending on geographical location, different deworming strategies are needed to control internal parasite populations. producers should be aware that cold temperatures cause larvae to undergo arrest, even when ingested into the host. during this arrested stage, the larvae are resistant to most deworming agents. many external parasites, including various species of flies, affect health and growth of replacements. several species of blood-sucking flies affect replacements. horn-flies, face-flies, stable-flies, ticks, lice and mite can be a major problem for cattle. they can cause substantial blood loss, transmit diseases including mastitis to replacements and decrease growth rates. use of forced back rubs is probably the most effective method of decreasing populations of horn-flies. additionally, removal of manure, which is the major breeding habitat for horn-flies, helps decrease populations. another type of fly, the stable-fly, breeds in wet feed. severe infestations of stable-flies can cause up to a % decrease in milk production. counts of flies per animal cause economically important losses in milk production and growth. removal of waste feed from under feed troughs and other areas to decrease breeding areas is the most important mechanism for control. horse-flies and deer-flies are also blood-sucking flies and may be responsible for spread of several diseases but are impractical to control. common house-flies are not blood-sucking insects but feed on muzzles, eyes and open wounds. they can be contaminated with more than viruses and bacteria, as well as other disease-causing organisms. the main form of control for common houseflies is sanitation and removal of breeding material because many house-flies are resistant to insecticide sprays. cattle grubs are another parasite common in north america. the main damage to cattle is caused by the migration of the grubs through host tissues and production of cysts on the animals' backs. growth rates can be adversely affected with infestations of cattle grubs. appropriate insecticide treatment will kill grubs; however, care must be taken not to administer insecticides when large numbers of grubs may have accumulated in the spinal canal. killing of large numbers of grubs at once can lead to anaphylaxis in cattle. other external parasites that may affect dairy cattle include fleas, lice, ticks and mites. itchiness and formation of scabs should be examined by a veterinary surgeon who can prescribe appropriate forms of treatment. many disease occurrences can be prevented or at least minimized by appropriate vaccination programs. the program that is appropriate, however, will vary from region to region, and even farm to farm. establishment of a vaccination program requires knowledge of diseases prevalent in the area, history of diseases on the farm, history of diseases in the herd, vaccinations used previously in the herd and an assessment of the risk of contracting economically important diseases based on management of the herd (open or closed). producers should, therefore, consult their veterinary surgeon to develop a vaccination program appropriate for their animals, management and location. timing of vaccinations is important for replacement animals. if the colostrum consumed by the calf contained antibodies against the disease organism present in the vaccine, the vaccine will not generate a sufficient immune response in the replacement animal. maternal antibodies obtained from colostrum may be present up to months of age, preventing an adequate response to vaccinations. it may be beneficial to wait until months of age or greater for many initial vaccinations in calves in order to avoid interference from maternal antibodies. additionally, many vaccines are not effective in neonatal calves because their immune system is not sufficiently developed to generate a protective response. common mistakes made in vaccination programs are lack of booster vaccinations at the appropriate time and lack of frequent vaccinations. if the vaccination protocol calls for an initial vaccination followed by a booster vaccination within - weeks, maximum protection will not be achieved without the booster vaccination. essentially, the money spent for the first injection is wasted. the second problem, lack of frequent vaccinations, is seen especially with leptospiral vaccines. leptospiral vaccines should be administered every months to achieve adequate protection. it is also important for heifers to start receiving leptospiral vaccinations at months of age so that they have received two vaccinations by the time they are used for breeding. health management of replacements requires attention to many different areas, ranging from nutrition and management of late lactation and dry cows to vaccinations of replacements. a better-informed management plans for biosecurity risk and analysis is essential for the next generation replacement cattle management. recent development of low cost sensory technology and an introduction to infectious disease control on farms: biosecurity. american feed ingredients association arlington. bovine alliance on management and nutrition large dairy herd management internal parasites of dairy cattle integrated manure management to reduce environmental impact: ii. environmental impact assessment of strategies dynamic cattle behavioural classification using supervised ensemble classifiers vaccines and vaccination programs application of an integrated outbreak management plan for the control of leptospirosis in dairy cattle herds nutrient and immunity transfer from cow to calf pre-and postcalving optimal management of replacement heifers in a beef herd: a model for simultaneous optimization of rearing and breeding decisions replacement management in cattle: health management. encyclopedia of dairy sciences key: cord- - x os m authors: crowe, j.e. title: human respiratory viruses date: - - journal: encyclopedia of virology doi: . /b - - . -x sha: doc_id: cord_uid: x os m viruses are the leading causes of acute lower respiratory-tract infection in infancy. respiratory syncytial virus (rsv) is the most common pathogen, with hmpv, piv- , influenza viruses, and rhinoviruses accounting for the majority of the remainder of acute viral respiratory infections. humans generally do not develop lifelong immunity to reinfection with these viruses; rather, specific immunity protects against severe and lower respiratory-tract disease. respiratory virus infections of humans are the most common and frequent infections of man. hundreds of different viruses can be considered respiratory viruses. viruses that enter through the respiratory tract include viruses that replicate and cause disease that is restricted to the respiratory epithelium, and other viruses that enter through the mucosa but also spread by viremia causing systemic disease. an example of the latter is measles virus. sars coronavirus is another. in general, viruses that do not cause viremia are capable of reinfecting the same host multiple times throughout life. in contrast, infections with systemic viruses induce lifelong immunity. probably, the high rate of reinfection of mucosally restricted viruses reflects the difficulty and metabolic cost of maintaining a high level of immunity at the vast surface area of the mucosa. virus-specific iga levels are maintained at high levels generally only for several weeks or months after infection. the anatomy and the cell types of the respiratory tract dictate to a large degree the type of disease observed during respiratory virus infection. the demarcation between the upper and lower respiratory tracts is the vocal cords. the structures of the upper respiratory tract, which are all interconnected, include the nasopharynx, the larynx, the eustachian tube and middle ear space, and the sinuses. significant collections of lymphoid tissue reside in the upper respiratory tract, the tonsils and the adenoids. the lower respiratory tract structures include the trachea, bronchi, bronchioles, alveoli, and lung tissue. the cell types that line the respiratory tract are complex, and exhibit different susceptibilities to virus infection. the predominant cell types are ciliated and nonciliated epithelial cells, goblet cells, and clara cells. smooth muscle cells are prominent features of the airways down to the level of the bronchioles, and the lung possesses type i and ii pneumocytes. the disease syndromes that are associated with respiratory viruses generally follow the anatomy of the respiratory tract. different viruses appear to have tropisms for different cells or regions of the respiratory tract; therefore, there are particular associations of viruses with clinical syndromes. the clinical diagnoses for infections with disease manifestations in the respiratory tract are rhinitis and the common cold, sinusitis, otitis media, conjunctivitis, pharyngitis, laryngitis, tracheitis, acute bronchitis, bronchiolitis, pneumonia, and exacerbations of reactive airway disease or asthma. clinical syndromes with more systemic illness due to respiratory viruses include the influenza syndrome, measles, severe acute respiratory syndrome (sars), and hantavirus pulmonary syndrome (hps). the principal causes of acute viral respiratory infections in children became apparent through large epidemiologic studies conducted soon after cell culture techniques became available. the landmark studies of association of viruses with clinical syndromes were performed in the s and s. recent studies have increased our understanding of the causes of viral respiratory infection in infants, especially because of the advent of molecular tests such as the polymerase chain reaction (pcr), which is more sensitive than cell culture. respiratory syncytial virus (rsv), parainfluenza viruses (pivs), adenoviruses, and influenza viruses were identified initially as the most common causes of serious lower respiratory tract disease in infants and children. more recently, human metapneumovirus (hmpv) was identified as a major cause of serious illness. in the last years, a number of additional viruses have been associated with respiratory illness, as discussed below. however, still, infectious agents are not identified in - % of clinical illnesses in large surveillance studies, even using sensitive diagnostic techniques such as viral culture on multiple cell lines, antigen detection assays, and rt-pcr based methods. it is not known if these illnesses are due to identified pathogens that are simply not detected due to low titers of virus in patient samples or if there are novel agents that are yet to be identified. reactivation of latent viruses, such as the herpesviruses hsv and cmv, and adenoviruses occurs in immunocompromised humans, particularly subjects with late-stage hiv infection, those with organ transplantation, and patients with leukopenia and neutropenia caused by chemotherapy. cmv is the most frequently recovered virus from diagnostic procedures such as bronchoalveolar lavage in immunosuppressed patients with pneumonia. these patients also suffer more frequent and more severe disease including mortality with common respiratory viruses, including rsv, hmpv, piv, influenza viruses, rhinoviruses, and adenoviruses. nosocomial transmission including large unit outbreaks is not uncommon, and can result in high frequency of transmission. picornaviridae a wide variety of picornaviruses cause respiratory disease, including rhinoviruses, the enteroviruses a to d including coxsackieviruses a/b, echoviruses, non-polio enteroviruses, and parechoviruses - . enterovirus infections occur most commonly in the summer months in temperate areas, which differs from the season of many of the other most common respiratory viruses such as paramyxoviruses and influenza virus. rhinovirus infections occur year-round. rhinovirus is a genus of the family picornaviridae of viruses. rhinoviruses are the most common viral infective agents in humans, and a causative agent of the common cold. there are over serologic virus types that cause cold symptoms, and rhinoviruses are responsible for approximately half of all cases of the common cold. rhinoviruses have single-stranded positive-sense rna genomes. the viral particles are icosahedral in structure, and they are nonenveloped. rhinovirus-induced common colds may be complicated in children by otitis media and in adults by sinusitis. most adults, in fact, have radiographic evidence of sinusitis during the common cold, which resolves without therapy. therefore the primary disease is probably best termed rhinosinusitis. rhinovirus infection is associated with exacerbations of reactive airway disease in children and asthma in adults. it is not clear whether rhinovirus is restricted to the upper respiratory tract and induces inflammatory responses that affect the lower respiratory tract indirectly, or whether the viruses spread to the lower respiratory tract. in the past, it was thought that these viruses did not often replicate or cause disease in the lower respiratory tract. however, recent studies discern strong epidemiological associations of rvs with wheezing and asthma exacerbations, including episodes severe enough to require hospitalization. likely, rhinoviruses can infect the lower airways to some degree, inducing a local inflammatory response. another possibility is that significant local infection of the upper respiratory tract might induce regional elaboration of mediators that causes lower airways disease. association of rhinovirus infection with lower respiratory tract illness is difficult to study because cell diagnosis by cell culture is not sensitive. rt-pcr diagnostic tests are difficult to interpret because they are often positive for prolonged periods of time and even asymptomatic individuals may have a positive test. comprehensive serologies to confirm infection are difficult because of the large number of serotypes. nevertheless, most experts believe rhinoviruses are a common cause of lower respiratory tract illness. these viruses cause oral lesions and often are associated in children with a disease syndrome termed 'hand-footand-mouth disease'. the pharyngitis associated with this infection often is marked by the very characteristic findings of herpangina, a clinical syndrome of ulcers or small vesicles on the palate and often involving the tonsillar fossa associated with the symptoms of fever, difficulty swallowing, and throat pain. outbreaks commonly occur in young children, in the summer. non-polio enteroviruses are common and distributed worldwide. although infection often is asymptomatic, these viruses cause outbreaks of clinical respiratory disease, sometimes with fatal consequences. studies have associated particular types with clinical syndromes, as enterovirus with wheezing and enterovirus with pneumonia. the term 'echo' in the name of the virus is an acronym for enteric cytopathic human orphan, although this may be an archaic notion since most echoviruses are associated with human diseases, most commonly in children. there are at least echovirus serotypes. echoviruses can be isolated from many children with upper respiratory tract infections during the summer months. echovirus has been associated with laryngotracheitis or croup. epidemiology studies also have associated echoviruses with epidemic pleurodynia, an acute illness characterized by sharp chest pain and fever. these viruses have been assigned a new genus of the family picornaviridae because of distinctive laboratorybased molecular properties. the most common member of the genus parechovirus, human parechovirus (formerly echovirus ) is a frequent human pathogen. the genus also includes the closely related virus, human parechovirus (formerly echovirus ). human parechoviruses usually cause mild respiratory or gastrointestinal illness. most infections occur in young children. there is a high seroprevalence for parechoviruses and in adults, and a few clear descriptions of neonatal cases of severe disease. respiratory syncytial virus rsv is a single-stranded negative-sense nonsegmented rna genome virus of the family paramyxoviridae, genus pneumovirus. it is one of the most infectious viruses of humans and infects infants at a very young age, often in the first weeks or months of life. it is the most common viral cause of severe lower respiratory tract illness in children and one of the most important causes of hospitalization in infants and children throughout the world. there is one serotype, but circulating viruses exhibit an antigenic dimorphism such that there are two antigenic subgroups designated a and b. reciprocal cross-neutralization studies using human sera showed that the antigenic groups are about % related. reinfection is common and can be caused by viruses of the same subgroup. yearly, epidemics of disease often peak between january and march in temperate regions. rsv infection causes mild upper respiratory tract infection in most infants and young children, but results in hospitalization in . - % of infants. most children have been infected by the age of years. there is an association of rsv infection early in life and subsequent asthma, although a causal relationship is controversial. most hospitalized infants are otherwise healthy, but some groups are considered high risk for severe disease such as premature infants especially those with chronic lung disease and infants born with congenital heart disease. immunocompromised patients of any age are at risk of severe disease. these viruses constitute a group of four distinct serotypes (types - ) of single-stranded rnaviruses belonging to the family paramyxoviridae. when considered as a group, they are the second most common cause of lower respiratory tract infection in young children. piv is the most common cause of severe disease. repeated infection throughout life is common. first infections are more commonly associated with lower respiratory tract disease, especially croup, while subsequent infections typically are limited to the upper respiratory tract. pivs are detected using cell culture with hemadsorption or immunofluorescent microscopy, and rt-pcr. in , investigators in the netherlands described a new human respiratory virus, hmpv. evidence of near universal seroconversion was found in the general population by years of age, suggesting ubiquitous infection in early childhood. this virus, a member of the genus pneumovirus with rsv, differs from rsv in that it lacks the ns and ns nonstructural genes that counteract host interferons and it possesses a slightly different gene order. studies of the role of hmpv in pediatric lower respiratory tracts infection (lri) in otherwise healthy children in the united states, using a prospectively collected -year database and sample archive representing about children, revealed that nearly % of lri in children was associated with a positive hmpv test. this and similar studies suggested that the virus is one of the major respiratory pathogens of early childhood. the clinical features of hmpv lri were similar to those of other paramyxoviruses, most often resulting in cough, coryza, and a syndrome of bronchiolitis or croup. interestingly, hmpv seemed to be clinically intermediate between rsv and piv in that it tended to cause bronchiolitis with similar frequency to rsv but more frequently than piv, while causing croup less often than the latter. studies in subjects with conditions predisposing to increased risk of respiratory illness suggest that hmpv plays a significant role in exacerbations of asthma in children and adults, lri in immunocompromised subjects, and in the frail and elderly. measles virus, a paramyxovirus of the genus morbillivirus causes infection with systemic disease, also known as rubeola. the virus is spread both by direct contact/fomite transmission and by aerosol transmission, and therefore is one of the most highly contagious infections of man. the classical symptoms of measles include or more days of fever that is often quite high and a clinical constellation of symptoms termed 'the three cs': cough, coryza, and conjunctivitis. a characteristic disseminated maculopapular rash appears soon after onset of fever. transient mucosal lesions in the mouth of a characteristic appearance (koplik's spots) are considered diagnostic when identified by an experienced clinician. the virus causes a number of systemic effects and can be complicated by severe pneumonia, especially when primary infection occurs in an unvaccinated adult or immunocompromised person of any age. mortality in developing countries is high, especially when infection occurs in the setting of malnutrition. these emerging pathogens that are grouped in their own new genus henipaviruses may not be respiratory pathogens in a conventional sense, but they are paramyxoviruses that probably infect humans by the respiratory route. nipah virus is a newly recognized zoonotic virus, named after the location in malaysia where it was first identified in . it has caused disease in humans with contact with infectious animals. hendra virus (formerly called equine morbillivirus) is another closely related zoonotic paramyxovirus that was first isolated in australia in . the viruses have caused only a few localized outbreaks, but their wide host range and ability to cause high mortality raise concerns for the future. the natural host of these viruses is thought to be a certain species of fruit bats present in australia and the pacific. pigs may be an intermediate host for transmission to humans in nipah infection, and horses in the case of hendra. although the mode of transmission from animals to humans is not defined, it is likely that inoculation of infected materials onto the respiratory tract plays a role. the clinical presentation usually appears to be an influenza-like syndrome, progressing to encephalitis, may include respiratory illness, and causes death in about half of identified cases. influenza is a single-stranded segmented negative-sense rna genome virus of the family orthomyxoviridae. there are three types of influenza viruses: influenza virus a, influenza virus b, and influenza virus c. influenza a and c infect multiple species, while influenza b infects humans almost exclusively. the type a viruses are the most virulent human pathogens among the three influenza types, and cause the most severe disease. the influenza a virus can be subdivided into different subtypes based on the antibody response to these viruses. the subtypes that have been confirmed in humans in seasonal influenza, ordered by the number of known human pandemic deaths, are: h n which caused the pandemic, and h n which caused the pandemic of avian influenza that originated in china, h n which caused the pandemic of . currently, h n , h n , and b viruses cause annual seasonal epidemics. in addition, h n virus infection of humans occurred during an epizootic of h n influenza in hong kong's poultry population in . the disease affected animals of many species and exhibited a high rate of mortality in humans. the virus is spreading throughout asia, carried by wild birds. human-to-human transmission does not occur efficiently at this time; however, there is widespread current concern about the potential for an h n pandemic if the virus acquired transmissibility among humans. the h n avian virus also has unusual zoonotic potential. in this virus caused an outbreak in humans in the netherlands associated with an outbreak in commercial poultry on several farms. one death occurred and people were confirmed to have h n influenza virus infection. h n virus appears to endemic in pigs and humans. h n , h n , h n , and h n human infections have been reported. influenza b virus is almost exclusively a human pathogen, and is less common than influenza a. it mutates less rapidly than influenza a, and there is only one influenza b subtype. in humans, common symptoms of influenza infection and syndrome are fever, sore throat, myalgias, headache, cough, and fatigue. in more serious cases, influenza causes pneumonia, which can be fatal, particularly in young children and the elderly. influenza pneumonia has an unusually high rate of complication by bacterial superinfection with staphylococcal and streptococcal bacterial pneumonia occurring in as many as % of cases in some clinical series. viruses of the family adenoviridae infect both humans and animals. adenoviruses were first isolated in human lymphoid tissues from surgically removed adenoids, hence the name of the virus. in fact, some serotypes establish persistent asymptomatic infections in tonsil and adenoid tissues, and virus shedding can occur for months or years. these double-stranded dna viruses are less than nm in size, and have nonenveloped icosahedral morphology. the large dsdna genome is linear and nonsegmented. there are six major human adenovirus species (designated a through f) that can be placed into immunologically distinct serotypes. human respiratory tract infections are mainly caused by the b and c species. adenovirus infections can occur throughout the year. sporadic outbreaks occur with many of the serotypes, while others appear to be endemic in particular locations. respiratory illnesses include mild disease such as the common cold and lower respiratory tract illness, including croup, bronchiolitis, and pneumonia. conjunctivitis is associated with infection by species b and d. there is a particular constellation of symptoms called 'pharyngoconjunctival fever' which is very frequently associated with acute adenovirus infection. in contrast, gastroenteritis has been associated most frequently with the serotype and virus of species f. immunocompromised subjects are highly susceptible to severe disease during infection with respiratory adenoviruses. the syndrome of acute respiratory disease (ard), especially common during stressful or crowded living conditions, was first recognized among military recruits during world war ii and continues to be a problem for the military following suspension of vaccination. ard is most often associated with adenovirus types and . members of the genus coronavirus also contribute to respiratory illness including severe disease. there are dozens of coronaviruses that affect animals. until recently, only two representative strains of human coronaviruses were known to cause disease, human coronavirus e (hcov- e) and hcov-oc . a recent outbreak of sars-associated coronavirus (sars-cov) showed that animal coronaviruses have the potential to cross species to humans with devastating effects. there has been one major epidemic to date, between november and july , with over cases of the disease, and mortality rates approaching %. sars-cov causes a systemic illness with a respiratory route of entry. sars is a unique form of viral pneumonia. in contrast to most other viral pneumonias, upper respiratory symptoms are usually absent in sars, although cough and dyspnea occur in most patients. typically, patients present with a nonspecific illness manifesting fever, myalgia, malaise, and chills or rigors; watery diarrhea may occur as well. recently, investigators reported the identification of a fourth human coronavirus, hcov-nl , a new group coronavirus. evidence is emerging that hcov-nl is a common respiratory pathogen of humans, causing both upper and lower respiratory tract illness. human coronavirus (hcov) hku was first described in january following detection in a patient with pneumonia. several cases of respiratory illness have been associated with the virus, but the infrequent identification suggests to date that this putative group coronavirus causes a low incidence of illness. several herpes viruses cause upper respiratory infections, especially infection of the oral cavity. herpes simplex pharyngitis is associated with characteristic clinical findings, such as acute ulcerative stomatitis and ulcerative pharyngitis. herpes simplex virus (hsv- ) and herpes simplex virus (hsv- ), also called human herpesvirus (hhv- ) and human herpesvirus (hhv- ), respectively, cause oral lesions, although over % of oral infections are caused by hsv- . primary oral disease can be severe, especially in young children, who sometimes are admitted for rehydration therapy due to poor oral intake. a significant proportion of individuals suffer recurrences of symptomatic disease consisting of vesicles on the lips. epstein-barr virus (ebv) mononucleosis syndrome is often marked by acute or subacute exudative pharyngitis; in some cases, the swelling of the tonsils in ebv pharyngitis is so severe that airway occlusion appears imminent. most of the viruses of the family herpesviridae can cause severe disease in immunocompromised patients (especially hematopoietic stem cell transplant patients), including cytomegalovirus (cmv), ebv, varicella-zoster virus, herpesvirus , herpesvirus , and herpesvirus . a new virus was identified recently in respiratory samples from children with lower respiratory tract disease in sweden. sequence analysis of the viral genome revealed that the virus is highly related to canine minute virus and bovine parvovirus and is a member of the genus bocavirus, subfamily parvovirinae, family parvoviridae. this virus was tentatively named human bocavirus (hbov). hbov has been identified as the sole agent in a limited number of respiratory samples from children hospitalized with respiratory tract disease. it remains to be seen whether the virus is causative of or merely associated with disease in these preliminary studies. over cases of hps have been reported in the united states. the disease was first recognized during an outbreak in . about a third of recognized cases end in death. the four corners area outbreak is well known; however, cases now have been reported in states. patients with hps usually present with a febrile illness beginning with symptoms of a flu-like illness. physical examination is not specific, often only with findings of fever, and increased heart and respiratory rates. in addition to the respiratory symptoms, abdominal pain and fever are common. diagnosis is often delayed until a severe illness occurs requiring mechanical ventilation. rotaviruses are dsrna enteric viruses that are the most common cause of severe viral infectious gastroenteritis in children. clinical series suggest that some children with gastroenteritis suffer upper respiratory symptoms during the prodrome of disease manifestation, and virus can be recovered from respiratory secretions. some reports suggest that rotavirus infection is associated with lower respiratory tract illness, although this association is unclear. these dsrna viruses (named using an acronym for respiratory enteric orphan virus) are not clearly associated with respiratory disease, but seroconversion rate is high in the first few years of life, and they probably cause minor or subclinical illness. pharyngitis occurs with primary hiv infection and may be associated with mucosal erosions and lymphadenopathy. polyomaviruses are small dsdna genome nonenveloped icosahedral viruses that may be oncogenic. there are two polyomaviruses known to infect humans, jc and bk viruses. eighty percent or more of adult us subjects are seropositive for these viruses. jc virus can infect the respiratory system, kidneys, or brain. bk virus infection causes a mild respiratory infection or pneumonia and can involve the kidneys of immunosuppressed transplant patients. given the overlap in the winter season of these viruses in temperate areas, it is not surprising that co-infections with two or more viruses occur. in general, when careful studies using cell culture techniques were used for virus isolation, more than one virus was isolated from respiratory secretions of otherwise healthy subjects with acute respiratory illness in about - % of cases in adults and - % in children. there is little evidence that more severe disease occurs during co-infections, although there is insufficient evidence on this point to be definitive. the incidence of two molecular diagnostic tests being positive (generally rt-pcr, for these rna viruses) is expected to be higher than that of culture, because molecular tests can remain positive for an extended period of time after virus shedding has ended. respiratory viruses generally have two main modes of transmission, large particle aerosols of respiratory droplets transmitted directly from person-to-person by coughing or sneezing, or by fomites. fomite transmission occurs indirectly when infected respiratory droplets are deposited on hands or on inanimate objects and surfaces with subsequent transfer of secretions to a susceptible subject's nose or conjunctiva. most respiratory viruses, unlike measles virus or varicella zoster virus, do not spread by small particle aerosols across rooms or down halls. therefore, contact and droplet precautions are sufficient to prevent transmission in most settings; handwashing is critical in healthcare settings during the winter season. ribavirin is a nucleoside antimetabolite pro-drug that is activated by kinases in the cell, resulting in a triphosphate nucleotide form that inhibits rna replication. the drug was licensed in an aerosol form in the us in for treatment of children with severe rsv lower respiratory tract infection. the efficacy of aerosolized ribavirin therapy remains uncertain despite a number of clinical trials. most centers use it infrequently, if ever, in otherwise healthy infants with severe rsv disease. intravenous ribavirin has been used for adenovirus, hantavirus, measles virus, piv, and influenza virus infections, although a good risk/benefit profile has not been established clearly for any of these uses. a humanized mouse monoclonal antibody directed to the f protein of rsv, 'palivizumab', is licensed for prevention of rsv hospitalization in high-risk infants. it is efficacious in half or more of high-risk subjects. a more potent second-generation antibody is being studied in clinical trials. experimental treatment of both immunocompetent and immunocompromised rsv-infected subjects has been reported but the efficacy of this approach is not established. there are four licensed drugs in the us for treatment or prophylaxis of influenza. 'amantadine' and 'rimantadine' are two of the drugs that interfere with the ion channel activity caused by the viral m protein of influenza a viruses, which is needed for viral particle uncoating following endocytosis. the other two drugs, 'oseltamivir' and 'zanamivir', are neuraminidase inhibitors that act on both influenza a and b viruses by serving as transition state analogs of the viral neuraminidase that is needed to release newly budded virion progeny from the surface of infected cells. the cell surface normally is coated heavily with the viral receptor sialic acid. resistance to the ion channel inhibitors arises rapidly during prophylaxis or treatment, and in resistance levels became so common in circulating viruses that the cdc no longer recommends use of these drugs. 'interferon-a' has been shown to protect against rhinovirus infections when used intranasally. this biological drug causes some side effects, such as nasal bleeding, and resistance to the drug developed during experimental use, so the molecule is no longer being developed for this purpose. 'pleconaril' has been tested for treatment of rhinovirus infection, as it is an oral drug with good bioavailability for treating infections caused by picornaviruses. this drug acts by binding to a hydrophobic pocket in the vp protein and stabilizing the protein capsid, preventing release of viral rna into the cell. the drug reduced mucus secretions and other symptoms and is being further examined. 'acyclovir' and related compounds are guanine analog antiviral drugs used in treatment of herpes virus infections. hsv stomatitis in immunocompromised patients is treated with 'famciclovir', or 'valacyclovir', and immunocompetent subjects with severe oral disease compromising oral intake are sometimes treated. these compounds have also been used prophylactically to prevent recurrences of outbreaks, with mixed results. intravenous acyclovir is effective in hsv or varicella zoster virus pneumonia in immunocompromised subjects. 'ganciclovir' with human immunoglobulin may reduce the mortality associated with cmv pneumonia in hematopoietic stem cell transplant recipients and has been used as monotherapy in other patient groups. 'cidofivir' is a nucleotide analog with activity against a large number of viruses, including adenoviruses. intravenous cidofovir has been effective in the management of severe adenoviral infection in immunocompromised patients but may cause serious nephrotoxicity. there are licensed vaccines for influenza viruses. in the us, both a trivalent (h n , h n , and b) inactivated intramuscular vaccine and a live attenuated trivalent vaccine for intranasal administration is available. the efficacy of these vaccines is good when the vaccine strains chosen are highly related antigenically to the epidemic strain. antigenic drift caused by point mutations in the ha and na molecules leads to antigenic divergence, requiring new vaccines to be made each year. the influenza genome is segmented, which allows reassortment of two viruses to occur during co-infection, which sometimes leads to a major antigenic shift resulting in a pandemic. pandemics occur every - years on average. there is current concern about the potential for an h n pandemic, and experimental vaccines are being tested for this virus. to date, h n vaccines have been poorly immunogenic compared to comparable seasonal influenza vaccines. vaccines were developed for adenovirus serotypes and , and these were approved for preventing epidemic respiratory illness among military recruits. essentially, these were unmodified viruses given by the enteric route in capsules, instead of the respiratory route, which is the natural route of infection leading to disease. inoculation by the altered route resulted in an immunizing asymptomatic infection. all us military recruits were vaccinated against adenovirus from to with near complete prevention of the disease in this population, but the sole manufacturer of the vaccine halted production in and supplies ran out years later. since , adenovirus infection has reemerged as a significant problem in the military with approximately % of all recruits suffering illness due to adenovirus infection during basic training; some deaths have occurred. live attenuated vaccine candidates are under development and being tested in phase i and ii clinical trials for rsv and the pivs. mutant strains with reduced pathogenicity were isolated in the laboratory, tested, and sequenced. now, vaccine candidates are being optimized by combining mutations from separate biologically derived viruses into single strains using recombinant techniques for generating rnaviruses from cdna copies, a process called reverse genetics. subunit vaccines have been developed for rsv, but there are safety concerns about their use in young infants because formalin inactivated vaccine induced a more severe disease response to infection in the s. there are no vaccines against rhinoviruses as there is little or no cross-protection between serotypes, and it is not feasible to develop a vaccine for over serotypes. efforts to develop coronavirus vaccines are in the preclinical stage. viruses are the leading causes of acute lower respiratory tract infection in infancy. rsv is the most common pathogen, with hmpv, piv- , influenza viruses, and rhinoviruses accounting for the majority of the remainder of acute viral respiratory infections. humans generally do not develop lifelong immunity to reinfection with these viruses; rather, specific immunity protects against severe and lower respiratory tract disease. risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level viral infections in relation to age, atrophy, and season of admission among children hospitalized for wheezing parainfluenza viruses what have we learned from the tucson children's respiratory study? epidemiology of respiratory syncytial virus infection in washington, dc. part ii. infection and disease with respect to age, immunologic status, race, and sex characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season fields virology human t-cell leukemia viruses: general features m yoshida, university of tokyo, chiba, japan elsevier ltd. all rights reserved.glossary px region htlv sequence between the env and ltr, encoding tax, rex and other small regulatory proteins. rex trans-modulator of viral rna splicing and transport. tax pleiotropic regulator activating viral and cellular replication interacting with cellular transcription factors, tumor suppressor proteins, and cell cycle checkpoints. human t-cell leukemia virus (htlv- ) is the first established tumorigenic retrovirus of humans; exogenous to humans this virus is classified as the species human t-cell leukemia virus, in deltaretroviridae, within the family retroviridae. htlv- infection is associated with leukemia and neural disease, adult t-cell leukemia (atl) and htlv- -associated myelopathy/tropical spastic parapasis (ham/tsp), respectively. the genomic structure of the virus with genes for nonstructural proteins established a distinct viral genus that includes bovine leukemia virus. htlv- has no oncogene, but nevertheless transforms t cells rather efficiently and is identified as the etiologic agent of atl. htlv- has unique regulatory proteins, tax and rex, and tax has been identified as a critical molecule not only in regulation of viral replication but also in induction of atl. after long and enormous efforts to identify a retrovirus in human tumors, htlv was described in t-cell lines as a convincing human retrovirus. the first report of the virus (htlv) was from a patient with mycosis (mf) in the us, and another (adult t-cell leukemia virus (atlv)) was from a patient with atl in japan. subsequently, the mf case was characterized as atl and the two isolates were established to be the same following a comparison of their genomes.a prototypical retroviral genome contains the gag, pol, and env genes encoding the virion proteins including core proteins, reverse transcriptase, and surface glycoprotein, respectively. acute leukemia viruses generally have an oncogene acquired from cellular genes that substitutes a part of the gag, pol, and env sequences. in contrast to these genomes, htlv has additional genes in an extra px region between env and the ltr (ltr -long terminal repeat). this unique genomic structure classified htlv as a member of a distinct genus of the retroviridae, which includes htlv- , and- , bovine leukemia virus (blv), and simian t-cell leukemia viruses (stlv- , - , and - ). htlv- was isolated from a patient with hairy t-cell leukemia and its genome similarity to the type is about %.stlvs have been isolated from various species of old world nonhuman primates, including the japanese macaque, african green monkey, pig-tailed macaque, gorilla, and chimpanzee. their genomes share - % homology.blv infects and replicates in b cells of cows and sheep and induces b-cell lymphoma. key: cord- -gj mfzxz authors: de sanctis, vincenzo; ruggiero, leopoldo; soliman, ashraf t; daar, shahina; di maio, salvatore; kattamis, christos title: coronavirus disease (covid- ) in adolescents: an update on current clinical and diagnostic characteristics date: - - journal: acta biomed doi: . /abm.v i . sha: doc_id: cord_uid: gj mfzxz the current outbreak of infections with sars-cov- is defined as coronavirus disease (covid- ). the clinical symptoms of covid- include fever, fatigue, cough, breathing difficulty that may lead to respiratory distress; a small population of patients may have diarrhea, nausea or vomiting. the highest infection rate occurs in adults; however, neonates, children, and adolescents can also be infected. as the outbreak continues to spread worldwide, attention has switched toward determinants of clinical manifestations and disease severity. the situation surrounding the outbreak is rapidly evolving and the information and recommendations are changing as new information becomes available. this paper summarises the current findings (april , ) from a systematic literature review on the current knowledge of covid- in adolescents ( - years according to the who definition) and reports the preliminary epidemiological data stated by the italian national institute of health. (www.actabiomedica.it) the coronaviruses (covs) belong to a family of viruses that may cause various symptoms, such as cough, fever, breathing difficulty, and lung infection, mainly pneumonia ( ) . these viruses are common in animals worldwide, but, in a few instances, are known to infect humans ( ) . human coronaviruses (hcovs) were first described in the s in patients with common cold. since then, a number of hcovs have been discovered ( e, oc , nl , and hku ), including sars-cov causing the severe acute respiratory syndrome and mers-cov, the middle east respiratory syndrome; both are highly transmissible and pathogenic viruses that emerged in humans at the beginning of the st century ( ) . these hcovs are associated with lower respiratory tract syndromes, spread from personto-person via close contact; they have high morbidity and mortality caused by the progression to acute respiratory distress syndrome (ards). it was recently discovered that dromedary camels in saudi arabia harbor three different hcov species, including a dominant mers hcov lineage that was responsible for outbreaks in the middle east and south korea during ( ) . on january , a novel coronavirus, -ncov, was officially identified as the cause of an outbreak of diffuse pneumonitis in the city of wuhan in hubei province, china. the epidemic has progressed very quickly in the following weeks, and an increasing number of cases have occurred daily in many countries ( ) . the disease was named covid- by the world health organization (who) on february ( ) , and the virus, sars-cov- by the international committee on virus taxonomy on the same day. on march , who classified the outbreak as a pandemic ( ) . currently sars-cov- has now spread to all continents excluding antarctica. on april , over million patients with covid- have been reported globally, with approximately , cases having recovered and over , deaths, according to the data compiled by the center for systems science and engineering at johns hopkins university. the us has the highest reported number of patients with covid - worldwide, accounting for approximately one quarter of all global cases. after the us, italy, spain, germany, china, france, iran, and the uk have the higher number of patients. italy still has the largest number of deaths globally, accounting for nearly one third of all global fatalities (tables and ) , but it appears that the rate of new infections has slowed in the last few days. according to the who, case and contact definitions are based on the current available information and are revised daily as new information accumulates. countries may need to adapt case definitions depending on their local epidemiological situation and other factors. all countries are encouraged to publish definitions used online and in regular situation reports, and to document periodic updates to definitions which may affect the interpretation of surveillance data. as the outbreak continues to spread worldwide, attention has switched toward determinants of clinical manifestation and disease severity. this paper summarises the findings of a systematic literature review on the current knowledge of covid- in adolescents ( - years, according to the who definition) and reports the preliminary epidemiological data on adolescents in italy. literature was identified by searching the following online databases: pubmed, google scholar, me-drxiv, biorxiv, who, centres for disease prevention and control (cdc), european centre for disease prevention and control, italian national institute of health (iss), centre for evidence-based medicine (cebm), worldometer, and health authorities. the searches concluded on april . the following search terms were used: " novel coronavirus, -ncov, wuhan coronavirus, wuhan pneumonia, covid- in children and adolescents". articles were screened by title, abstract, and full text. google searches were also used to provide access to government and international institutes. in our review we preferentially included the relevant peer-reviewed scientific publications written in english. the mean incubation period is brief, reported as . days, with the th percentile of the distribution at . days ( % confidence interval: . - ) ( ) . overall, these estimates will be refined as more data become available. available evidence indicates that human transmission of sars-cov- occurs via close contact with respiratory droplets produced when a person exhales, sneezes, or coughs, or via contact with fomites. the virus has been detected in blood, saliva, tears, and conjunctival secretions ( ) ( ) ( ) ( ) . sars-cov- rna was also detected in stool specimens but according to who-china report, fecal-oral transmission did not appear to be a significant factor in the spread of infection (report of the who-china joint mission on coronavirus disease ,covid- . february [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . all ages are susceptible to this viral infection ( , ) . high viral loads have been detected in nasal and throat swabs soon after symptoms onset; it is thought that the viral shedding pattern may be similar to that of patients with influenza. pharyngeal viral shedding is high during the first week when symptoms are mild or prodromal, peaking on day , suggesting an active virus replication in the upper respiratory tract tissues. other studies have also shown a higher viral load in the nasal cavity as compared to the throat, with no difference in viral burden between symptomatic and asymptomatic people. patients can be infectious for as long as the symptoms last and even after clinical recovery ( , ) . up to now, no cases of transmission via the faecal-oral route have been reported for sars-cov- , which might suggest that infection via this route is unlikely. detailed epidemiological information based on a larger sample of covid- patients is needed to determine the infectious period of sars-cov- , as well as whether transmission can occur from asymptomatic individuals during the incubation period ("pre-symptomatic" period). however, such sources of infection cannot be effectively identified due to the absence of symptoms. so far, research evidence is lacking, although there are a few studies suggesting that pre-symptomatic or asymptomatic carriers may cause viral transmission ( , ) . asymptomatic carriers can also be a source to propagate the outbreak ( ) . a study in singapore identified . % of patients among seven clusters of cases in which presymptomatic transmission was likely to have occurred to days before symptom onset ( ) . these patients are not easy to detect when initially infected but might have abnormal symptoms later. in a small number of case reports and studies, a familial cluster of infection associated with sars-cov- has been reported, indicating possible personto-person transmission during the incubation period ( , ) . the best evidence so far comes from the diamond princess cruise ship, which was quarantined with all passengers and crew members repeatedly tested and closely monitored. a modelling study found that approximately people with confirmed infection ( %) were asymptomatic ( ) . pregnant women and their fetuses represent a high-risk population during disease outbreaks. until march , the outcomes of pregnant women infected with sars-cov- and neonates have been reported with no definite evidence of vertical transmission ( ) . prenatal complications may include premature labor and fetal distress. however, vertical transmission cannot be ruled out. there have been reports of infection in neonates born to mothers with covid- ( ) , and virusspecific antibodies have also been detected in neonatal serum samples ( ) .therefore, it is crucial to screen pregnant women and implement strict infection control measures, quarantine of infected mothers, and close monitoring of neonates at risk of covid- . in conclusion, several properties of this virus make prevention difficult namely: the non-specific features of the disease, the infectivity before onset of symptoms during the incubation period, the transmission from asymptomatic people, the long incubation period, the tropism for mucosal surfaces such as the conjunctiva, the prolonged duration of the illness and the transmission after clinical recovery. many reports provide descriptions of the clinical signs associated with covid- in wuhan and other cities in china. the clinical symptoms range from mild cough and fatigue to severe acute respiratory distress and respiratory failure. in adults, the main prevalent clinical manifestations include: sore throat, cough, high fever, tachypnea/dyspnea, and chest tightness/ pain. the presence of fever is significantly higher in adults compared to children. in adults and adolescents with severe pneumonia the respiratory rate is > breaths/minute and the spo is ≤ % on room air. the leading cause of death in patients with covid- is respiratory failure from acute respiratory distress syndrome ( , , , ) . a wide range of diagnostic tests are commercially available for sars-cov- , some of which have received authorization for use by various national regulatory agencies. countries have implemented different testing strategies, reflecting the availability of equipments, diagnostic reagents, and capability of their national health system. the cdc recommends collection of a nasopharyngeal swab specimen to test for sars-cov- ( ). a positive test generally confirms the diagnosis of covid- , although false-positive results may occur. negative rt-pcr tests on nasopharyngeal specimen swabs despite ct findings suggestive of viral pneumonia have been reported in some patients who ultimately tested positive for sars-cov- ( ) . based on the findings of chest radiographs in , respiratory distress syndrome (ards) in cov-id- patients, only . % had abnormal findings ( ) . in contrast, ( . %) had abnormal and diverse chest ct images, in which ground-glass opacity (ggo) was the most common abnormality ( . %), followed by local patchy shadowing ( . %), and interstitial abnormalities ( . %). in addition, . % patients had bilateral patchy shadowing ( ) . as the disease progress, follow-up ct scans may show enlargement and consolidation of single ggo, an enlarged fibrous stripe, and solid nodules. generally, two types of abnormal radiographic presentations are seen in children and adolescents: multiple opacities and patchy opacities ( ) . in the liao et al study ( ) , only ( . %) adolescent patients showed ground-glass opacity at chest ct scanning, compared with ( . %) young adults. rodriguez-morales et al ( ) performed a systematic literature review with meta-analysis, using three databases to assess clinical, laboratory, imaging features, and outcomes of covid- confirmed cases. articles were retrieved for the time frame ( / / - / / ). after screening, articles were selected for full-text assessment, being finally included for qualitative and quantitative analyses. decreased albumin ( . %), high c-reactive protein ( . %), high lactate dehydrogenase (ldh) ( . %), lymphopenia ( . %), and high erythrocyte sedimentation rate (esr) ( . %), were the most prevalent laboratory findings. in short, confirmation of infection requires nucleic acid testing of respiratory tract samples (e.g., throat swabs). however, nasopharyngeal specimens may miss some infections and a deeper specimen may need. alternatively, repeated testing can be used because, over time, the likelihood of the sars-cov- present in the nasopharynx increases. serologic tests, once generally available, should be able to identify patients who have either current or previous infection but a negative pcr test. clinical diagnosis may be made based on symptoms, exposure, and chest imaging. chest ct may plays a key role in detection or diagnosis of cov-id- infection with some typical ct features while the initial rt-pcr result is negative. consequently, at present, exposure history, clinical manifestations, chest ct features and laboratory results, should be taken in consideration to diagnose covid- infection. at present, information regarding the epidemiology and clinical features caused by covid- in adolescents are scarce in the literature. as a first step, we reviewed the epidemiological characteristics of -ncov. from february-april , the reported prevalence of infection in adolescents in china ( ), korea ( , ) , usa (children and adolescents < years) ( ) , and italy ( ) was equal to %, %, . %, and . %, respectively. tables and , and figure illustrate the available data in adolescents compared to children ( - years) in china ( ) and italy (ruggiero l, data calculated from ref ) . notably, a higher number of positive cases were reported in the north of italy ( ) . in the chinese report, the time period from the peak of the symptom-onset-based epidemic curve (around january ) to the peak of the diagnosis-based epidemic curve (february ) was about days ( ). the proportion of chinese children who developed severe or critical covid- illness with breathlessness, acute respiratory distress syndrome (ards), and shock was much lower ( %) than among chinese adults ( %). in the united states, among , covid- cases in children aged < years, nearly one third, cases ( %) occurred in children, aged - years, and ( %) in children aged - years ( ) . no admissions in intensive care units or deaths were reported in patients younger than years up to april . compared to the adult population, cai et al. ( ) observed a longer period of incubation in adolescents with a mean of . days (range - days) from the time of exposure to symptoms. ludvigsson ( ) , in a systematic literature review, reported that the transmission could emerge rapidly between younger patients and their family members or close contacts. a detailed study on epidemiology, transmission patterns, and clinical characteristics in adolescents and young adults with covid- has been recently published ( ) ( ) ( ) . in general, common symptoms on admission were similar to those in adults, namely: dry cough ( . %), fever ( . %), and expectoration ( . %). lung auscultation may reveal rales and crackles. the less common symptoms included headache, fatigue, sore throat, chest pain, anorexia, myalgia, dizziness, diarrhoea, nausea, and shortness of breath ( ) ( ) ( ) . as far as we know, anosmia/hyposmia or ageusia/ dysgeusia has not been reported in adolescents. the american academy of otolaryngology -head and neck surgery (march , internet publication) has proposed adding anosmia and dysgeusia to the list of screening items for potential infection in adults and recommends that clinicians consider testing and self-isolation of these patients in the absence of other respiratory diseases such as rhino sinusitis or allergic rhinitis. over % of patients were asymptomatic, or with mild, or moderate clinical symptoms ( ) . however, whether the limited number of covid- in children is due to lower susceptibility or milder presentation leading to missed detection remains unknown ( ) . the mild nature of covid- in pediatric cases is likely to be multifactorial. cai et al. ( ) reported that while covid- viral shedding in respiratory specimens lasts longer in children than adults, and was also observed in the stool specimens, the virus was not detected in serum samples. the absence of viremia has likely contributed to the lack of severe illness in children cases ( ) . severe or critical illness was reported in . % of children aged to years, . % in children aged to , . % in the to age group, and . % in adolescents years and older. one -year-old child in this cohort died ( ) . compared with young adults, no severe cases and higher odds of asymptomatic cases ( . % vs . %) were observed in adolescent patients ( ) . patients with chronic diseases are at greater risk of infection. data on the management of comorbidities in patients with covid- is limited ( ) and should be tailored to the patient's chronic disease ( , ) . there are no vaccines available and there is little evidence to date on the effectiveness of potential therapeutic agents. management strategies for children and adolescents are absent because of the limited number of patients in this age group with covid- . therefore, treatment has been focused on symptomatic and respiratory support (oxygen inhalation, fluid management, and the use of broad-spectrum antibiotics to cover secondary bacterial infection). apart from the above, the use of interferon-a nebulization, anti-viral drugs (such as, remdesivir lopinavir/ritonavir), and chloroquine have been reported ( ) ( ) ( ) . in summary, compared with elderly patients, adolescent and young adult covid- patients have a longer incubation period, a shorter serial interval, higher odds of being asymptomatic and a lower mortality rate. larger epidemiologic studies are needed to confirm the lower susceptibility and milder clinical presentation of covid- in adolescents. the commonest clinical and laboratory findings in adolescents with covid- are summarized in table . the who, on january , declared that the new coronavirus sars-cov- outbreak is a public health emergency of international concern. pediatric covid- cases, including adolescents, are rapidly increasing around the world. early recognition and rapid diagnosis are essential to prevent transmission and provide supportive care in a timely manner. the cdc is working closely with state and local health partners to develop and disseminate information to the pub lic on general prevention of respiratory illness, including the -ncov. this includes everyday preventive actions such as: washing hands, cover- table . summary of the commonest clinical and laboratory findings in adolescents with covid- infection (from references: - ). young adults is longer than in older patients. . compared to young adults, adolescents were less likely to be overweight/obesity, to smoke and drink alcohol. . no severe cases and higher odds of asymptomatic cases ( . % vs . %) were observed in adolescent patients . the adolescent and young adult patients with covid- have different patterns of symptoms and lower incidence of abnormal laboratory findings. the inflammatory markers of c-reactive protein (crp) and procalcitonin (pct) were elevated in . % and . % of cases, respectively. . ground-glass opacity at chest ct scanning was found in % of adolescents compared with . % of young adults. . compared to young adults, adolescent patients received less oxygen inhalation therapy and had lower number of days with persistent fever. . lower number of adolescents developed severe complications. extended follow-up is needed to provide more detailed information on the potential risk factors interfering with clinical outcomes. ing the mouth and nose when coughing or sneezing, and staying home when ill. thus, everybody should follow the national guidelines around screening, testing, containment, care and practice social distancing. additional information and resources for this outbreak are available on the cdc website (https://www.cdc. gov/coronavirus/ -ncov/ index.html). considering that all age groups are susceptible to sars-cov- infection, it is of critical importance also, that the youngest subjects, who may be asymptomatic or have milder symptoms, should comply with the self-isolation procedures in order to prevent virus diffusion. additionally, personal protective equipment (ppe) is crucial when care providers look after an infected person. as more is learned about this novel virus and outbreak, more data will emerge to facilitate the diagnostic and preventive measures for containing and minimizing the covid- infection. at present, we suggest that in the presence of active community transmission, subjects presenting with a fever of unknown aetiology or with a fever in the presence of common cold or pneumonia symptoms should be tested for covid- , and influenza a and b viruses to rule out possible co-infection considering the seasonal overlap between influenza and covid- . in helping to prevent influenza, all persons aged ≥ months should receive influenza vaccine annually ( ) . according to the united nations educational, social and cultural organization (unesco), countries have implemented nationwide school closures, impacting over , million children and youths. at this moment, we do not have strong evidence to guide decisions on duration of school closures and how duration may affect public health. extending school closure will support the overall effectiveness of social distancing and thus aid in lowering the peak of the epidemic curve. however, prolonged periods of school closures and movement restriction may lead to additional emotional unrest and anxieties ( ) . education, social support mechanisms and access to health services need to be maintained, with parents and/or guardians playing a key role ( , ) . further relevant aspects should be also taken in consideration (table ) . this review has numerous limitations; few articles are focusing on the clinical features of covid- in adolescents. some clinical and epidemiologic risk factor data might be incomplete, as they were collected by multiple teams under protocols that, by necessity, changed as the situation progressed. data on outcomes, including hospitalization, were missing and this likely resulted in an underestimation of outcomes. no comprehensive data are available on the impact of covid- on adolescents with an underlying condition, especially those with respiratory system morbidity, but it is reasonable to consider that they might be at increased risk for severe disease. in conclusion, all clinicians should keep themselves updated about recent developments including global spread of the disease. we encourage coordinated efforts between national and international health agencies to develop effective surveys in this age group. we express our sincere condolences to the patients and their families who had covid- around the world. we greatly respect the efforts of all the hospital employees and their families, who are working tirelessly during this outbreak, and the collaborative partnership at all levels of individuals in the table . the united nations population fund recommendations (unfpa: www.unfpa.org/ sites/default/files/resource; modified) . many vulnerable young people are at greater risk of contracting covid- , such as young migrants, young refugees, homeless young people, those in detention, and young people living in crowded areas and in poverty. . if caregivers are infected, quarantined, or die, protection and psychosocial issues for adolescents need to be addressed. . with prolonged stress on the health system to address covid- , a disruption of the normal delivery of sexual and reproductive health services and information to young people will need to be addressed. . the need for mental health services and counselling is paramount, as many people, including young people, who are facing high levels of anxiety and stress related to cov-id- . . adolescents and youths, especially adolescent girls 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b xy objective: response to the coronavirus disease (covid‐ ) pandemic has resulted in shelter‐in‐place orders and major changes to individuals’ daily lives. the impact of such stressors on disease activity in individuals with axial spondyloarthritis (axspa) is unclear. the aim of this study is to examine whether stress, anxiety, and depression are associated with patient‐reported disease activity, after accounting for important factors. methods: we administered a survey to an axspa cohort from a single center with well‐defined demographic and disease characteristics. we included questions about job status changes, exercise, medication use, disease activity (by the bath ankylosing spondylitis disease activity index [basdai]), and psychological factors (stress, depressive symptoms, and anxiety). separate multivariable linear models examined the associations between perceived stress, anxiety, and depression with the basdai. results: after adjustment for potential confounders, those with higher levels of stress had a statistically significant . ‐point higher basdai, on average, compared with those with lower levels of stress ( % confidence interval [ci]: . , . ). those with higher levels of anxiety also had a statistically significant higher basdai, on average, compared with those with lower levels of anxiety (β: . , % ci: . , . ). the association between depression and basdai was not statistically significant. we did not find differences in these associations among subgroups of age, job status, or county of residence. conclusion: individuals with axspa with higher levels of stress and anxiety had significantly higher disease activity levels, although with a difference below clinical importance. further planned studies will evaluate the trajectory of disease activity. severe acute respiratory syndrome coronavirus is a novel coronavirus that causes coronavirus disease (covid- ) , which the world health organization declared a pandemic in ( ) . by early april , there were over confirmed cases in the united states, with global cases climbing past a million ( ) . in response to rising case numbers, san francisco, california, was placed under a shelter-in-place order on march , , with many cities, counties, and states subsequently doing the same ( ) . prior literature in other rheumatic diseases has been conflicting in terms of how major natural disasters, such as hurricanes and earthquakes, impact disease activity ( ) ( ) ( ) ( ) ( ) ( ) . stress is implicated as an important factor that affects disease activity, as has been demonstrated in rheumatoid arthritis (ra) ( ) ( ) ( ) . however, there have been few studies of axial spondyloarthritis (axspa) that have directly examined the association of stress with disease activity ( ) , and none them have evaluated the association of major stressors such as natural disasters or pandemics with disease activity. as a shelter-in-place order impacts the entire community and may result in occupational changes (eg, working from home or loss of income), limitations on physical activity (eg, closure of nonessential businesses including gyms), and medication changes (eg, in light of concerns of increased infection risk with immunosuppression), covid- represents a unique situation to study the impact of stress on disease activity in axspa. the purpose of this study is to study the impact of this major event, particularly in light of local shelter-in-place ordinances and major economic changes, on disease activity in an existing natural history cohort of individuals with axspa. the primary aim is to examine whether stress, anxiety, and depression are associated with a change in disease activity, after accounting for important factors. study population. the natural history of axspa study is an ongoing prospective cohort of subjects at the university of california, san francisco (ucsf). adults who met either the modified new york criteria for ankylosing spondylitis (as) or the assessment of spondyloarthritis international society (asas) criteria for nonradiographic axspa were enrolled from . the purpose of the cohort study is to explore potential mechanisms responsible for disease, risk factors for the development and progression, associated comorbidities, treatments and treatment complications, and the natural history of spondyloarthritis. patients are followed at cohort study visits every months. data collection and variables. for the current study, subjects were emailed a link to a survey hosted online through the hipaa-compliant platform qualtrics that was specifically focused on issues surrounding the past months during the covid- pandemic. survey objectives, risks, benefits, and study team contact information were provided to patients before questions were initiated. this study received internal review board (irb) approval from ucsf following a modification to the cohort irb. the university of washington determined that further irb review was not needed for the analysis of this study. medication use. patients were asked about baseline nonsteroidal anti-inflammatory drug (nsaid) and biologics use, and whether there was any change over the prior two months (increased, decreased, stopped, stayed the same, switched, or did not use at baseline). the interval of months (february to april ) was chosen to capture potential changes that patients may have instituted on account of the pandemic. exercise. patients were asked about baseline ("six months ago") exercise frequency, duration, and specific type. then they were asked about change over the prior months (increased, decreased, stopped, stayed the same, or did not exercise at baseline). job status. patients were asked for details about their current employment status, including whether they were unemployed, and their current work location if employed. covid- . patients were asked whether they had received a nasal swab or other form of testing for the diagnosis of covid- and whether the results were positive. if testing had been performed, they were asked about symptoms and any treatment received. they were also given the opportunity to provide any concerns they had regarding covid- in an open-ended question. disease activity. the bath ankylosing spondylitis disease activity index (basdai) was administered as an assessment of disease activity over the prior week. the basdai is a composite measure of as disease activity and consists of six questions that address five major symptoms in as: fatigue, spinal pain, peripheral joint pain and swelling, localized tenderness, and morning stiffness ( ) . scores range from to , with higher scores reflecting greater disease activity. psychological factors were measured by the following instruments: the perceived stress scale is a -item, self-reported instrument that measures perceived stress in relation to life events over the past month ( ) . scores range from to , with higher scores indicating greater stress. the center for epidemiologic studies -depression (ces-d) scale is a -item, self-reported measure of depression symptoms with an emphasis on depressed mood in the past week ( ) . scores range from to , with higher scores indicating more severe symptoms. a score of or greater is suggestive of possible depression and a score of or greater of probable depression. the patient reported outcomes measurement information system (promis) anxiety short form is a six-item, selfreported measure of anxiety over the past days ( ) . scores are converted to a standardized t-score, with a population mean of and standard deviation of . cutoff scores to define levels • few studies have evaluated the relationship of major stressors and disease activity in axial spondyloarthritis (axspa), and none have examined this association in the setting of a natural disaster or pandemic. • in april , during the peak of the coronavirus disease (covid- ) pandemic in the united states, we surveyed patients with axspa from a single center cohort regarding changes in job status, exercise, medication use, disease activity, and psychological factors including perceived stress and anxiety. • after adjustment for important confounders, including baseline disease activity, those with higher levels of perceived stress had significantly higher disease activity compared with those with lower levels of stress. the same association was seen for anxiety levels and disease activity. • these findings highlight stress and anxiety as independent predictors of patient-reported disease activity among individuals with axspa during the covid- pandemic, particularly while shelterin-place measures were in effect. | of anxiety have been suggested as none/slight (< ), mild ( . - . ), moderate ( . - . ), and severe (≥ ) levels of anxiety ( ) . baseline measures, including demographics, axspa disease characteristics, medication use, and comorbidities were obtained from either the cohort database (in the case of time-invariant characteristics) or from the most recent study visit prior to march . disease activity was recorded using the basdai. nsaid use was recorded as none, low dose, or high dose ( ) . biologic use, including tumor necrosis factor inhibitors and interleukin- inhibitors, was recorded as a binary variable (using, yes/no). comorbidities were extracted using information available in the electronic medical record problem list and medication list, as appropriate. analysis. we performed descriptive statistics for baseline characteristics and survey responses, which included calculation of pairwise correlation coefficients for stress, anxiety, and depression scales. we used multiple imputation with chained equations with iterations to impute missing values for the basdai at baseline ( ) ( ) ( ) . we included the following variables as predictors in the imputation model: age, gender, race, survey basdai, baseline basdai, change in nsaid use, change in biologic use, change in exercise, change in job status, total promis anxiety score, and total ces-d score. complete case analyses were performed as a secondary analysis. because stress, anxiety, and depression may be closely related, we examined correlations among these scores. for the first primary analysis, we compared the survey basdai score between those with higher versus lower levels of stress (perceived stress scale dichotomized at the median). we used multivariable linear regression and adjusted for the following potential confounders identified a priori: age (years), gender, race (white versus other), baseline basdai, decrease or cessation of nsaid use, decrease or cessation of biologic use, decrease or cessation of exercise, and whether there was a change in job status. for the second analysis, we used multivariable linear regression to compare the survey basdai between those with higher versus lower levels of anxiety (promis anxiety score dichotomized at the median) after adjusting for the same confounders as in the first analysis. for the third analysis, we compared the survey basdai between those with higher versus lower scores on the ces-d scale (dichotomized at the cutoff of ) using multivariable linear regression with adjustment for the same confounders as in the previous two models. in exploratory analyses, we tested interactions (α = . for statistical significance) in the primary analysis model between perceived stress level and ) age greater than years, ) job change, and ) residence outside of san francisco. all analyses were conducted in stata software version (statacorp) using robust standard error estimates and an α level of . (with the exception of tests of interaction). as of april , there were patients in the cohort. the survey was initially sent on april , . several reminders, including emails, messages through the electronic medical record, and telephone calls, were sent. as of april , , the final response rate was % with complete responses. overall, survey respondents had a mean age of . ± . years, % were male, and % were white. baseline data are described, by stress level based on the perceived stress scale, in table . groups by stress level were similar, with the exception of a lower proportion of males in the higher stress group ( % versus %) and a higher proportion with diagnoses of depression in the higher stress group ( % versus %). table . groups were similar in terms of gender, as classification, and treatment for axspa. nonrespondents were older and a higher proportion were white as compared with respondents. nonrespondents had a numerically higher basdai at baseline but also a higher proportion of missing data for the basdai. survey outcomes are shown in table , stratified by stress level. the majority of respondents did not alter their nsaid or biologic dosing ( % and %, respectively). however, % reported decreasing or stopping their exercise as compared with months prior, and % reported a change in their job. nine received testing for covid- , but none reported a positive test. there were more people who reduced or stopped their nsaids (but not biologics) in the high-versus low-stress group; and more people who had job changes and were not currently working in the high-versus low-stress group. the scores for ces-d, anxiety, and basdai were higher in the high-stress group compared with the low-stress group. stress, anxiety, and depression scores were highly correlated (pairwise correlation coefficients ranged from . to . , table ), so separate multivariate regression analyses were conducted for each factor. there were missing data for the baseline basdai for respondents, which were imputed using multiple imputation with chained equations. after adjustment for potential confounders and baseline disease activity, those with higher levels of stress had a statistically significant . -point higher basdai, on average, compared with those with lower levels of stress ( % confidence interval [ci]: . , . ). those with higher levels of anxiety had a statistically significant . -point higher basdai, on average, . note. continuous variables are reported as mean ± sd and median (interquartile range) and categorical variables as n (%). to assess differences between high-and low-stress level groups, we used χ tests for categorical variables and mann-whitney u tests for continuous variables. values missing for the following: as classification (n = nonrespondents), biologic use (n = nonrespondents), nsaid use (n = nonrespondents), baseline basdai (n = respondents, n = nonrespondents). abbreviations: as, ankylosing spondylitis; basdai, bath ankylosing spondylitis disease activity index; nsaid, nonsteroidal antiinflammatory drug. a total cohort of . not included in this table: declined survey participation and who were deceased. | compared with those with lower levels of anxiety ( % ci: . , . ), after adjustment for potential confounders and baseline disease activity. the average difference in the basdai was not significantly different compared with those with higher versus lower scores on the ces-d scale (β: . , % ci: − . , . ). results were similar using complete case analysis (data not shown). in exploratory analyses, the association of stress with disease activity did not differ across subgroups of age, job status, or county of residence (p = . , . , and . , respectively), nor did the association of anxiety with disease activity differ by these subgroups (p = . , . , and . , respectively). in this study, higher levels of perceived stress and anxiety, but not depression, were significantly associated with higher disease activity levels among individuals with axspa. we did not find differences in this association among subgroups of age, job status, or county of residence. to our knowledge, this is the first study to evaluate patient-reported disease activity in relation to psychological factors among individuals with axspa during a major pandemic. although the estimated average difference in the basdai comparing between stress and anxiety levels was less than the minimal clinically important difference (mcid) of . reported by pavy et al ( ) , the differences between groups may become more apparent upon longitudinal follow-up of this cohort as the pandemic evolves. stress is related to disease activity in rheumatic disease ( ) ( ) ( ) . however, whether stress is an independent predictor of elevated disease activity has not been definitively demonstrated in axspa. jiang et al examined psychological status, sleep quality, and stress due to life events over the prior months in patients with as and found an association between anxiety/depression and disease activity ( ) . previously published studies have evaluated patients with ra, systemic lupus erythematosus (sle), and inflammatory bowel disease (ibd) following natural disasters ( ) ( ) ( ) ( ) ( ) ( ) , with inconclusive findings. wallace et al followed patients with ra and with sle for months after the northridge earthquake, but no flares were documented ( ) . a study in japan surveyed women with ra who had experienced a natural disaster (predominantly typhoons, tornados, and torrential rain) between and . surveys were administered and months following the event, with the finding that % experienced deterioration of functional status, whereas % experienced a worsening of self-rated health status ( ) . in contrast, a study in taiwan compared patients with sle inside and outside of the disaster zone following the major earthquake of september . the researchers found that months following the disaster, neither the exposed nor the comparator group had experienced a significant change in the clinical symptoms of sle ( ) . factors affecting short-term and long-term impact can vary. two studies examined the frequency of disease relapse in ibd immediately following the great east japan earthquake of march , and then with follow-up data and years postdisaster. the authors found that the factors that influenced long-term relapse were different from those that influenced short-term relapse ( , ) . however, many of these studies were small, some did not have adequate comparators, and there was high potential for unmeasured confounding. the covid- pandemic represents a unique universal stressor on our patient population, as the shelter-in-place orders affected multiple cities, counties, and entire states in the united states, with impacts on job status, the ability to exercise, and access to health care. information on covid- risk for people living with rheumatic disease was also scarce in the early days of the pandemic, as these comorbidities were not reported in the initial large case series published from china, europe, or the united states ( ) ( ) ( ) ( ) ( ) ( ) . larger reports from a global rheumatology registry are forthcoming ( ) . at the end of march , michaud et al surveyed a large us registry of patients with rheumatic disease (for-ward) and found that common themes relating to covid- were concerns over risk of infection and how best to manage immunosuppressive medications ( ) . the strengths of this study include the use of a well-defined axspa cohort with detailed baseline data on important confounders as well as baseline data on disease activity. we were able to administer a survey with disease-specific questions, capturing a fairly homogenous population of rheumatic disease. the survey had a moderate response rate of %. additionally, we used validated measures of anxiety, stress, and depression with mean levels in our cohort that were similar to those in other rheumatic disease cohorts ( , ) . there are limitations of this study that we must acknowledge. first, this is an observational study limited to two time intervals: before and after the pandemic. there is potential selection bias regarding survey responses, as those with lower stress levels and disease activity may be more inclined to respond to the survey. generalizability may be limited, as all of the patients in this study were under the care of one rheumatologist. during the survey period, most of the patients were living in the bay area of california, which had shelter-in-place implementation that differed from other areas of the country. unmeasured confounding and measurement error are also possible, as we were limited in the granularity of detail that we could include in our survey. we did not have stress or anxiety measures in prior surveys, so we could not measure changes in these variables. finally, our use of the basdai as a patient-reported measure of disease activity is limited by its use of subjective, rather than objective, questions. our survey-based study of an axspa cohort at a single us center found that stress and anxiety were significantly associated with patient-reported disease activity, independent of confounding factors. although the average difference in basdai was below the mcid, these findings suggest that the covid- pandemic may have had an impact on axspa disease activity through increased stress and anxiety. we will continue this study with further iterations of the survey as a repeated measure. this will allow us to look at both population-and subject-level trajectories over time. world health organization. coronavirus disease (covid- ) pandemic san francisco department of public health. order of the health officer no. c - b can an earthquake cause flares of rheumatoid arthritis or lupus nephritis? the impact of hurricane hugo and the san francisco earthquake on a sample of people with rheumatoid arthritis impact of natural disasters on the functional 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index perceived stress scale the ces-d scale: a self-report depression scale for research in the general population patient reported outcomes measurement information system (promis) promis anxiety scoring manual asas recommendations for collecting, analysing and reporting nsaid intake in clinical trials/epidemiological studies in axial spondyloarthritis introduction in multiple imputation for nonresponse in surveys flexible imputation of missing data using the outcome for imputation of missing predictor values was preferred establishment of the minimum clinically important difference for the bath ankylosing spondylitis indices: a prospective study clinical features of patients infected with novel coronavirus in wuhan, china characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention clinical characteristics of coronavirus disease in china baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region clinical characteristics of covid- in new york city presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the rheumatic disease and covid- : initial data from the covid- global rheumatology alliance provider registries experiences of patients with rheumatic diseases in the us during early days of the covid- pandemic association between pain sensitization and disease activity in patients with rheumatoid arthritis: a cross-sectional study determinants of sleep problems in patients with spondyloarthropathy all authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. dr. liew had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. study conception and design. liew, castillo, katz, haroon, gensler. acquisition of data. castillo, gensler. analysis and interpretation of data. liew, zaccagnino, katz, haroon, gensler. key: cord- - m xscs authors: severance, emily g.; yolken, robert h. title: chapter role of immune and autoimmune dysfunction in schizophrenia date: - - journal: handbook of behavioral neuroscience doi: . /b - - - - . - sha: doc_id: cord_uid: m xscs abstract in this chapter, we review data in support of the concept that immune system dysregulation is the most plausible explanation that reconciles gene by environmental interactions in schizophrenia. early investigations of this topic demonstrated aspects of aberrant activation of humoral immunity, including autoimmunity, associated with schizophrenia, whereas current research efforts have expanded this theme to include elements of innate immunity. advances in our understanding of inflammation and molecules of both the adaptive and innate immune system and their functional roles in standard brain physiology provide an important context by which schizophrenia might arise as the result of the coupling of immune and neurodevelopmental dysregulation. schizophrenia is a debilitating and complex brain disorder of unknown etiology. complicating our understanding of the causes and pathophysiology of schizophrenia is the likelihood that what we call schizophrenia is actually a heterogeneous assemblage of etiological conditions across a broad spectrum (arnedo et al., ) . reigning evidence supports a schizophrenia etiopathogenesis arising from and perpetuated by a multisourced genetic by environmental interaction (demjaha, maccabe, & murray, ; modinos et al., ; van os et al., ; tsuang, ) . although schizophrenia is highly heritable, the disease is polygenic, and gene studies to date have identified an enormous number of susceptibility loci (kavanagh, tansey, o'donovan, & owen, ; schizophrenia working group of the psychiatric genomics consortium, ). thus, the disease is thought to manifest when one or more of many possible genetic predispositions co-occurs with exposure to one or more of many possible environmental factors. relevant environmental factors can derive from a diversity of sources including exposures to infection, food-derived antigens, stress, smoking, cannabinoids, pollutants, and other toxins (allen, liu, pelkowski, et al., ; allen, liu, weston, et al., ; fine, zhang, & stevens, ; fineberg & ellman, ; fraga et al., ; severance, yolken, & eaton, ; suarez-pinilla, lopez-gil, & crespo-facorro, ; zhang et al., ) . if these exposures coincide with critical periods of fetal and neonatal brain maturation, there is the potential to aberrantly impact important brain processes including neural migration, synaptogenesis, myelination, and synaptic pruning. coinciding with these neurodevelopmental landmarks are events crucial for the instigation and maturation of innate and adaptive immunity. a possible role for immune system dysregulation in schizophrenia etiopathogenesis would reconcile both genetic and environmental hypotheses. a number of genetic loci found to associate with schizophrenia involve immune functions directly or implicate biological pathways that can influence immune function. for example, a consistently replicated locus for association with schizophrenia is the p chromosomal region that houses the major histocompatibility (mhc) locus and human leukocyte antigens (corvin & morris, ; purcell et al., ; stefansson et al., ) . the mhc/human leukocyte gene family functions to identify self and nonself entities and any dysfunction of these genes can render susceptibility to infectious disease, graft rejection, cancer, and autoimmunity. environmental triggers that show consistently replicated associations with schizophrenia are also those that result in immune activation. exposures to infectious pathogens, food antigens, and autoantigens have been especially well-studied risk factors for the development of schizophrenia, and special consideration is afforded to the timing, intensity, and type of immune activation elicited by these exposures (jones, mowry, pender, & greer, ; kirch, ; knuesel et al., ; meyer, ; muller, ; rothermundt, arolt, & bayer, ; torrey & peterson, ; . the focus of this chapter is to review some of the evidence in support of an immune and autoimmune dysfunction in the etiology, pathogenesis, and ii. neurobiology of psychotic disorders pathophysiology of schizophrenia. from a historical perspective, a recurrent immune theme predominated the early literature with a particular emphasis on schizophrenia-associated immunoglobulins and antibrain antibodies. these ideas still formulate the basis of current immune topics in schizophrenia, but over the years the scope has widened beyond the adaptive immune system to encompass also innate immunity. advances in our understanding of inflammation and mediators of both the adaptive and innate immune system and their functional roles in standard brain physiology provide an important context by which schizophrenia might arise as the result of the coupling of immune and neurodevelopmental dysregulation. the focus of this review on immune system aberrations in schizophrenia requires a review of basic knowledge of the major molecules and cells involved in the highly regulated balance of interacting innate and adaptive immune pathways. the function of the immune system is to protect the organism from disease and allow distinction between self and nonself entities, a process that is generally classified into the innate (nonspecific, always present) and adaptive (specific, triggered) immune systems. the innate immune system is composed of physical epithelial barriers, monocytes/ macrophages, dendritic cells, natural killer cells, and circulating plasma proteins. microbial invaders or compromised cells interact with recognition receptors found on monocytes/macrophages and dendritic cells. pattern recognition receptors can be cytoplasmic, membrane-bound, and secreted and include toll-like receptors, complement receptors, nucleotide-binding oligomerization domain (nod)-like receptors, pentraxins, and c-reactive protein. the adaptive immune system is composed of two immune response types: humoral (antibody) immunity and t-lymphocytemediated immunity. during activation of the adaptive immune system, binding of the invading antigen to b lymphocytes precipitates its differentiation into plasma cells that produce immunoglobulin antibodies specifically targeted to the invading antigen (alberts, ; rothermundt et al., ). the complement system acts in conjunction with the humoral immune system to form immune complexes with the antibody bound antigens and clear these from the body (walport, a (walport, , b . upon binding to monocytes/macrophages, pathogenic and other antigens also trigger the t-cell cascade, where t cells differentiate into cytotoxic t cells, t-helper cells, and natural killer cells. the lysis of cells containing the invading antigen is accompanied by the production of pro-and anti-inflammatory cytokines, signaling proteins that function in immune regulation (alberts, ; rothermundt et al., ) . dysregulation of any of these molecules, proteins, or cells at any stage of these pathways irrespective of a genetic or environmental origin can result in disorders of the immune system, which generally can take the form of inflammatory diseases, immunodeficiency, autoimmunity, or some forms of neoplasia. for complex psychiatric disorders such as schizophrenia, it is also necessary to understand how perturbations of these immune processes might impact the brain. because schizophrenia is thought to originate as a result of aberrant neurodevelopment, it is important to note that for a number of these classic immune factors, including complement, mhc, toll-like receptors, and pentraxins, additional functions in the developing brain are continuously being identified (benoit & tenner, ; bialas & stevens, ; boulanger, ; fourgeaud & boulanger, ; frodl & amico, ; garate et al., ; nagyoszi et al., ; pribiag & stellwagen, ; stephan et al., ; stevens et al., ; trotta, porro, calvello, & panaro, ) . it is also becoming increasingly evident that circulating endogenous peripheral immune entities may directly access the central nervous system (cns) as a result of directed regulation or compromised endothelial barriers. at the same time, it is possible that invading or resident pathogens or their products could directly exert detriment to the cns by similarly penetrating these barriers. as such, the spectrum of psychiatric dysfunctions known as schizophrenia may be the compilation of different stages of an immunoneurological intersection gone awry from both external and internal pathological molecules and pathways. early observations prepared a foundation for the studies of today where the role of immune activation is no longer questioned but understood to be the most parsimonious etiological explanation that encompasses a gene by environment landscape of schizophrenia. in this section, we will review the history of these immune associations and especially illuminate adaptive humoral immune system dysregulation because immunoglobulin abnormalities were the focus of early investigations (kirch, ; rothermundt et al., ) . although many of these early studies are inconsistent regarding the impact of any single infectious pathogen or autoimmune reaction against brain tissue, these investigations offer snapshots of how the immune process might be relevant to and influence brain function. importantly, they bring to light issues that are still relevant today and that are now studied without previous restrictions such as unrecognized disease heterogeneity, constricted study designs, and limited laboratory technologies. activation of the adaptive immune system and specifically of humoral immunity generally is manifested by changes in the levels of immunoglobulin antibodies with respect to the disease state. schizophrenia-associated changes in the levels of plasma and cerebrospinal fluid (csf) proteins were repeated findings that implicated immunoglobulins and solidified the idea that in schizophrenia, either an infectious or an autoimmune process might be occurring (amkraut, solomon, allansmith, mcclellan, & rappaport, ; bock & rafaelsen, ; burian, kubikova, & krejcova, ; durell & archer, ; fessel, a fessel, , b gammack & hector, ; hendrie, paraskevas, & varsamis, ; selecki, todd, westwood, & kraus, ; solomon, allansmith, mccellan, & amkraut, ; strahilevitz & davis, ) . of particular interest were reports that people with schizophrenia who had elevated immunoglobulin levels were also the least likely to show clinical improvement over the course of hospitalization compared with those with lower immunoglobulin levels (amkraut et al., ) . an infectious disease component contribution to psychotic mental disorders is often first attributed to esquirol ( ) , who suggested that the dissemination of psychoses unfolds similarly to an epidemic-like process (esquirol, ) . this observation was followed by other reports of psychotic epidemics in the decades following world war i and the influenza epidemic (kirch, ; menninger, menninger, , torrey & peterson, . the possible role of an antigen derived from a pathogenic organism such as a virus or bacteria took root in various forms and the early years of the viral hypothesis of schizophrenia is well-reviewed by peterson ( , ) and kirch ( ) , with exposures to neurotropic viruses such as herpes simplex virus , measles, and rubella figuring prominently (kirch, ; torrey & peterson, . there was also an extensive literature base primarily from the s to s that describe a variety of antibody reactions in people with schizophrenia including the rosenow antibody-antigen skin reaction. this reaction was based on a hypothesis that several brain diseases such as epilepsy and schizophrenia were the result of alpha-hemolytic streptococci as measured by a cutaneous reaction to a streptococcal antibody or antigen that was obtained and cultured from nasopharynx samples (rosenow, ) . results from these studies were varied, with some showing greater immune response (cutaneous reaction) associated with schizophrenia and others showing no difference (gurassa & fleischhacker, ; rosenow, ) . we will revisit this idea of a pathogen-derived viral or bacterial source of immune activation in schizophrenia in its current form in a later section, because it is still a relevant hypothesis that is being explored with the benefit of modern tools such as high throughput sequencing. meanwhile, early literature on the topic of autoimmunity received similar effort and attention. one very early study of postmortem brain tissue identified the presence of autoantibodies to brain proteins and launched the idea that schizophrenia and other psychoses may have an autoimmune basis (lehmann-facius, ) . this theme continued in later decades when the role of autoantibodies to brain proteins was actively studied and disputed (boehme, cottrell, dohan, & hillegass, ; durell & archer, ; fessel, a fessel, , b heath, ; heath & krupp, ; heath, krupp, byers, & lijekvist, a , b jones et al., ; kirch, ; mellsop, whittingham, & ungar, ) . in some of these studies, the observation again came that levels of antibrain antibodies seemed to correlate with the intensity of psychotic symptoms and were generally higher during the early disease state and during acute attacks (glebov, ; gurevich, ; stamboliev, ; stoimenov, ) . dysregulation of the adaptive immune system and especially of humoral immunity still figures prominently in today's literature examining immune-based hypotheses for schizophrenia. speculation that medication is behind changes in immune marker levels is unavoidable; however, studies of patients who are antipsychotic naive or who have a recent onset of the disease support specific immune activation early in the course of disease, even before medication is administered (beumer et al., ; drexhage et al., ; drexhage et al., ; leonard, schwarz, & myint, ; miller, mellor, & buckley, ; mondelli & howes, ; severance et al., b; steiner et al., ; stojanovic et al., ) . next we describe some current evidence available regarding schizophrenia-specific immune responses to external antigens and autoantigens. exposure to infectious disease pathogens during the pre-and postnatal period as defined by an antibody response is significantly associated with the future development of or current status of schizophrenia (arias et al., ; brown & derkits, ; buka, cannon, torrey, & yolken, ; fellerhoff, laumbacher, mueller, gu, & wank, ; mortensen et al., ; niebuhr et al., ; xiao et al., ; yolken et al., ; . we include both pre-and postnatal exposure references in this section and in a later section will review the implications on neurodevelopment of strictly maternal-occurring immune activation from a variety of sources including pathogens. pathogenic microorganisms are relevant to schizophrenia pathophysiology because they or their products can be neurotropic as well as cytotoxic or because the process of immune system activation is pathogenic in schizophrenia. certain viruses known to be neurotropic include the herpes simplex viruses, cytomegalovirus, and epstein-barr virus; these viruses are also of interest because their life cycle can contain a latent state from which they can be periodically reactivated (kirch, ; torrey & peterson, . to date, the strongest association of an infectious disease agent with schizophrenia is toxoplasma gondii, a neurotropic parasite, and this relationship is well-reviewed in numerous analyses and meta-analyses (arias et al., ; monroe, buckley, & miller, ; torrey, bartko, lun, & yolken, ; torrey, bartko, & yolken, ) . other pathogens that have shown significant associations with schizophrenia and psychoses also include epstein-barr virus, measles, polio, influenza, coronaviruses, human herpesvirus , borna disease virus, human endogenous retrovirus, and chlamydophila spp (arias et al., ; brown, begg, et al., ; dickerson, stallings, origoni, copp, et al., ; karlsson et al., ; karlsson, schroder, bachmann, bottmer, & yolken, ; khandaker, stochl, zammit, lewis, & jones, ; mednick, machon, huttunen, & bonett, ; perron et al., ; prasad, shirts, yolken, keshavan, & nimgaonkar, ; severance et al., ; suvisaari, haukka, tanskanen, hovi, & lonnqvist, ) . of note, exposure to the process of infection may be as or more important than the virulence or neurotropism of any single pathogen. a large study of the swedish national birth registry suggested that exposure to viral cns infections during childhood could result in the later development of schizophrenia (dalman et al., ) . unlike other investigations, this study did not support a link of bacterial infections with the development of subsequent psychoses. a different study, however, found that urinary tract infections (likely of bacterial origin) were found to occur with increased prevalence in schizophrenia and associated with acute relapse of psychosis (graham, carson, ezeoke, buckley, & miller, ; miller et al., ) . other conditions typically characterized by bacterial infection (sinusitis, tonsillitis, and pneumonia) were associated with the development of schizophrenia in the prenatal exposure scenario, as were genital and other reproductive infections (babulas, factor-litvak, goetz, schaefer, & brown, ; sorensen, mortensen, reinisch, & mednick, ) . it is expected that if schizophrenia in some people is the result of a specific virus or parasite, then evidence in the form of dna sequences would be found in the brain. these data, however, have thus far been elusive. the ability to efficiently search for this needle in a haystack came several years ago with the advent of highthroughput sequencing. the infancy of this field has not yet uncovered evidence for a causative pathogen, but ongoing investigations have brought about findings in unexpected places, including microbes associated with the gut microbiome. the connection between food sensitivity and propensity for schizophrenia was pioneered by f. curtis dohan, who hypothesized that wheat glutens and bovine milk caseins were broken down into bioactive exorphins that could penetrate through gut barriers, enter systemic circulation, and have access to the cns. his work was based on observations of celiac disease overlap with schizophrenia, with strong correlations of hospitalization rates for schizophrenia with wheat availability during wartime and improvement of psychotic symptoms following removal of wheat and dairy products from the diet (dohan, (dohan, , (dohan, , (dohan, , dohan, harper, clark, rodrigue, & zigas, ) . a recent resurgence in this field is exemplified by the numerous antibody studies that confirm an increased immune response directed at these food antigens, including a role for maternal antibodies to food antigens and the possible presence of an antigen-specific immune reaction up to years before diagnosis of the disease (cascella et al., ; dickerson, stallings, origoni, vaughan, et al., ; jackson et al., ; karlsson et al., ; lachance & mckenzie, ; niebuhr et al., ; samaroo et al., ; severance et al., ; . the presence of food-derived exorphins or antibodies against them have been documented in the csf of individuals with a variety of psychoses including schizophrenia and coupled with a propensity for blood-brain and csf-brain barrier defects might implicate a neurotropic role of these peptides in the etiology or pathophysiology of the disease (axelsson, martensson, & alling, ; bauer & kornhuber, ; kirch et al., ; lindstrom, besev, gunne, & terenius, ; lindstrom et al., ) . autoimmune disease epidemiology and schizophrenia have been strongly linked for some time, with the first vestiges of the association coming in the form of findings suggestive of an inverse correlation between rheumatoid arthritis and schizophrenia (benros, eaton, & mortensen, ; eaton, hayward, & ram, ; . observations of a co-occurring psychosis with a number of autoimmune diseases including celiac disease, multiple sclerosis, systemic ii. neurobiology of psychotic disorders lupus erythematosus, autoimmune thyrotoxicosis, autoimmune hepatitis, and psoriasis also lent credence to the idea of an interrelated component of autoimmunity and the brain (benros et al., ; eaton et al., ) . celiac disease perhaps provides the strongest association with schizophrenia and reinforces the idea that for some, immune activation and autoimmunity have roots in the gut (baldwin, ; dohan, dohan, , dohan, , eaton et al., ) . celiac disease is a disease whereby the ingestion of wheat gluten launches an immune reaction that damages the epithelial lining of the small intestine through an autoimmune attack on tissue transglutaminase that breaks down the gluten peptide (alaedini & green, ; green et al., ; guandalini & assiri, ) . in the same way that the type of pathogen infection is probably not as important as the infectious process itself in causing brain pathologies such as schizophrenia, the specific type of autoimmune disease may not be the primary determinant of brain pathology. instead, the occurrence of a state of autoimmunity and its association with schizophrenia is rather likely to be a suggestion of the pathophysiology or faulty mechanism that is at work, perhaps as a disjunctive operation of an immune system pathway that has failed to function. large danish population-based studies, in fact, confirm that individuals or first-degree family members who had any history of an autoimmune disease have a % increased relative risk for schizophrenia (eaton et al., ) . the autoimmune link with schizophrenia was further solidified in an even larger investigation of this registry, and interestingly, this risk was further elevated in those with a history of an infection (benros et al., ) . this finding is not surprising given the fairly established literature base supporting the idea that exposure to infectious agents generates an autoimmune response (ercolini & miller, ) . as mentioned in a previous section, documenting and characterizing autoantibodies directed at brain proteins has been intriguing researchers for decades with generally mixed results. among the many autoantigens analyzed for an association with schizophrenia and psychosis are n-methyl-d-aspartate (nmda) receptors (deakin, lennox, & zandi, ; ezeoke, mellor, buckley, & miller, ; jones et al., ; masdeu et al., ; muller, ; pearlman & najjar, ; steiner et al., ; steiner et al., ) . this nmda receptor antibody quest was fueled by findings that antibodies to the nmda receptor were elevated in women with ovarian teratoma and psychoses-related encephalitis (dalmau et al., ) . other targets of autoimmune investigations include neuregulin- , human endogenous retroviruses, cholinergic muscarinic receptors, nicotinic acetylcholine receptors, dopamine d receptors, mu-opioid receptors, serotonin receptors, α-amino- -hydroxy- -methyl- isoxazolepropionic acid receptors, gamma-aminobutyric acid receptors, glutamic acid decarboxylase, potassium channel receptors, cardiolipin, dna, histones, and mitochondria (deakin et al., ; ezeoke et al., ; jones et al., ; masdeu et al., ) . an increased understanding of the underlying immunopathological processes and an improved characterization of reactive epitopes involved in disease pathogenesis might improve the predictive value of autoantibody assays and provide for reliable markers of disease susceptibility. a movement away from schizophrenia as a solely brain-centric disease is an active one in psychiatric research circles where an increasing awareness of the importance of the gastrointestinal (gi) tract, the body's largest immune organ, may share a bidirectional pathway with the brain. the strong association between food-based sensitivities and schizophrenia implicates the gi tract as an important site to search for immunological dysfunction. food antigen sensitivity is but one of a number of risk factors for schizophrenia that are related to gut inflammation, and this immunoglobulin g (igg) sensitivity joins other gut-related risk factors such as endothelial barrier defects, celiac disease, and exposure to t. gondii . research at this interface has shown in translational models that intestinal inflammation is a significant comorbidity of schizophrenia, and markers of this inflammation correlate with antibodies to food antigens such as gluten and casein at heightened rates in people with schizophrenia . it has been demonstrated in rodent models that the schizophrenia-associated pathogen t. gondii has many effects on the gut and during infection allows the passage of gluten peptides to translocate into circulation and provoke an antibody response (severance, kannan, et al., ) . in the presence of compromised epithelial and endothelial barriers, not only do foodbased peptides but also bacteria and other related harmful substances cross into the systemic circulation and generate more inflammation and propagate autoimmunity. markers of bacterial translocation are elevated in schizophrenia and also found to correlate with the antibody response to food antigens (severance et al., a) . thus gut-based inflammation can be added to the growing list of studies that implicate both peripheral and cns inflammatory pathways associated with schizophrenia (dickerson et al., ; drexhage et al., ; fillman et al., ; fillman, sinclair, fung, webster, & shannon ii. neurobiology of psychotic disorders weickert, ; gibney & drexhage, ; leonard et al., ; linderholm et al., ; miller, buckley, seabolt, mellor, & kirkpatrick, ; miller et al., ; monji et al., ; muller, ; muller, myint, & schwarz, ; torrey et al., ; . the burgeoning field of gut brain axis analyses is the subject of investigations directed at the understanding of how gut microbes might impact neuronal connections in the cns. importantly, the gut microbiome functions to regulate the immune system. the ability of intestinal epithelial cells to actively respond to microbes is mediated by innate immune pattern recognition receptors (toll-like receptors), nod-like receptors, and helicases expressed on cell surfaces. during times of mucosal stress, gut homeostasis becomes disrupted (stockinger, hornef, & chassin, ) . although there are numerous reports of autism-related altered communities of the intestinal microbiome (adams, johansen, powell, quig, & rubin, ; finegold et al., ; finegold, downes, & summanen, ; kang et al., ; parracho, bingham, gibson, & mccartney, ; williams et al., ; williams, hornig, parekh, & lipkin, ) , studies of the microbiome in schizophrenia are scant. preliminary clinical studies report altered pharyngeal and intestinal microbiomes in individuals with schizophrenia as compared to controls (yolken & dickerson, ) . some insight can be gleaned from rodent studies, where manipulations of gut microbiota do in fact result in behavioral, biochemical, and molecular changes (collins, surette, & bercik, ; foster & mcvey neufeld, ; hsiao et al., ; stilling, dinan, & cryan, ) . diaz-heijtz et al. ( ) , for example, illustrated that behavioral effects accompanied changes in synaptic markers, synaptophysin and psd , in the striatum (diaz heijtz et al., ) . in these rodent studies, animal phenotypes were recovered with manipulations of gnotobiotic (germ-free) animals, vagotomy, probiotics, and/or antibiotics. the ability of an extrinsically or intrinsically derived microbe, cell, protein, or other product normally found in peripheral circulation to enter to the cns renders discussion of epithelial and endothelial barriers an important topic. barrier permeability of the gut, blood-brain barrier, or blood-csf barrier (axelsson et al., ; bauer & kornhuber, ; kirch et al., ) can arise from a variety of environmental factors or from genetic mutations in the many biological pathways that impact this cellular architecture. barrier structures are composed of tight junctions (zonula occludens) that occur between the epithelial cells of the gi lumen of the gi tract; similar tight junction structures comprise the blood-brain barrier (deli, ; jong & huang, ) . the csf-brain and csf-blood barrier are slightly different, but these interfaces at the choroid plexus and arachnoid membrane are also relevant areas of access to the brain from the csf (laterra, keep, betz, & goldstein, ) . for schizophrenia, cns barrier dysfunction has been evaluated in studies of csf dynamics and is often attributed to a low-grade, systemic inflammation (bauer & kornhuber, ; bechter, ; bechter et al., ; kirch et al., ; severance, gressitt, alaedini, et al., ) . in conjunction with analyses of plasma and csf protein dynamics, it has been possible to detect evidence for barrier defects or restricted flow, as is particularly evident by the high prevalence of plasma-derived albumin. abnormal measures of plasma-derived albumin in the csf are noteworthy because the cns does not synthesize albumin and its elevation would require transport across the blood-brain or blood-csf barrier (tibbling, link, & ohman, ) . an increased albumin ratio can be indicative of either an anatomical barrier defect or a decreased csf flow rate, a dysfunction with numerous physiological causes (reiber, ; whedon & glassey, ) . the presence of pathological cns structures such as choroid plexus calcification, arachnoid cysts, and decreased brain volume all can disrupt csf flow patterns and all of these conditions have been previously associated with psychoses and schizophrenia (arango et al., ; kuloglu, caykoylu, yilmaz, & ekinci, ; laterra et al., ; marinescu, udristoiu, & marinescu, ; narr et al., ; reiber, ; rimol et al., ; sandyk, ; shiga et al., ; veijola et al., ; whedon & glassey, ) . although a systemic state of inflammation that might impact barrier integrities is most likely the result of immune activation from an environmental source, cellular barrier proteins and related biological pathways may also be the result of genetic associations. specific barrierrelated genes that have been significantly associated with schizophrenia include the tight junction protein claudin- , cytoskeletal elements such as actin, haptoglobin, and nitric oxide synthetase (burghardt, grove, & ellingrod, ; hall, trent, thomas, o'donovan, & owen, ; horvath & mirnics, ; maes et al., ; sun et al., ; wan et al., ; wei & hemmings, ; yang et al., ; ye et al., ; zhao et al., ) . the etiology and pathogenesis of schizophrenia likely stem from aberrant neurodevelopment (lewis & levitt, ; piper et al., ; rapoport, giedd, & gogtay, ) . perinatal-occurring environmental disturbances such as maternal stress, infection, or obstetric complications may interact adversely in genetically predisposed offspring to impact neural migration, synaptogenesis, myelination, and synaptic pruning (knuesel et al., ) . epidemiological and preclinical studies clearly indicate that exposure to maternal immune activity is associated with pathological brain development and thus maternal immune activation has become a strong risk factor for the development of schizophrenia (bauman et al., ; brown & derkits, ; canetta et al., ; garbett, hsiao, kalman, patterson, & mirnics, ; meyer, ; ii. neurobiology of psychotic disorders shi, smith, et al., ) . specifically, maternal exposure to cytomegalovirus, herpes simplex virus type , influenza, rubella, t. gondii, and wheat glutens have all been documented to increase the risk of development of psychosis or schizophrenia brown, begg, et al., ; brown, cohen, greenwald, & susser, ; brown, hooton, et al., ; buka et al., ; ellman, yolken, buka, torrey, & cannon, ; karlsson et al., ; mortensen et al., ; pedersen, stevens, pedersen, norgaard-pedersen, & mortensen, ; xiao et al., ) . this repertoire was recently expanded to include exposure to general inflammation and innate immunity based on measures of c-reactive protein and complement c q (canetta et al., ; severance, gressitt, buka, cannon, & yolken, ) . in this section, we will review the timelines of brain and immune development and review the evidence where these trajectories might intersect and result in brain disorders (figure ) . neural development is a highly regulated process and since molecules and proteins of the immune system are continually being found to participate in mechanisms of normal brain development, any immune overactivation, or failure of the immune system to activate will impact brain circuitry. the immune environment during pregnancy is a complex balance aimed at preserving immune protection of both sides of the maternal-fetal interface. several good reviews are available of how this interface is skewed maternally toward inhibiting fetal immunity and regulating and maintaining a protective th environment over the pro-inflammatory cytotoxic th immune response needed to fight infectious disease (belderbos, levy, meyaard, & bont, ; morein, blomqvist, & hu, ) . maternal immunity is antibody based and functions to maintain immune tolerance in the fetus and breast-feeding neonate. as a result, all antibodies including autoantibodies are passed to the offspring during this period. furthermore, while under maternal immune protection, the antigen recognition system of the fetus is immature. once maternal-derived immune factors are depleted, the immune system of the neonate must be redirected to become competent, including a more active th component. maturation of the innate and adaptive immune systems is a process that occurs from the fetal stage through adulthood (belderbos et al., ; knuesel et al., ; morein et al., ) . molecules and proteins of the immune system are intrinsically intertwined with important brain processes during development. these processes include initial proliferation of glia and neurons, consequent migration, programmed cell death, formation of synapses, figure developmental timelines of the brain and the immune system. complex disorders such as schizophrenia are thought to arise when one or more neurodevelopmental processes are interrupted because of genetic and/or environmental factors. various immune molecules, proteins, and cells such as c q and major histocompatibility complex function in the brain during neurodevelopment, suggesting that any disruption in the immune system during pregnancy or postnatally has the ability to compound synaptic misconnections. compiled from belderbos et al. ( ) , dietert et al. ( ) , kneusel et al. ( ) , and morein et al. ( ) . ii. neurobiology of psychotic disorders myelination, and synapse pruning with the overall endpoint to establish functional neuronal circuits (knuesel et al., ) . here, we present the case of complement c q as an example of an immune molecule that is highly active in the developing brain and that is also implicated in schizophrenia-associated gene and environmental studies. in the developing immune system, relevant processes include immune cell appearance, colonization, expansion, and maturation. complement c q and mhc were some of the first immune molecules identified to function in synapse development and pruning in the brain (boulanger, ; fourgeaud & boulanger, ; huh et al., ; shatz, ; stevens et al., ) . complement pathway-related genes that have been associated with schizophrenia include the c qb gene, complement control-related genes, and complement surface receptor gene cd (havik et al., ; zakharyan et al., ) . biologically, complementcontaining circulating immune complexes were elevated in individuals with schizophrenia compared to controls and a primary antigenic component of these immune complexes was often found to be casein or gluten boyajyan, khoyetsyan, tsakanova, & sim, ; mailian, boiadzhian, sogoian, sim, & manukian, ; mayilyan, weinberger, & sim, ; vetlugina, logvinovich, maslennikova, & vasil'eva, ) . finally, elevated levels of maternal c q igg have been found to increase the odds for psychosis in offspring (severance, gressitt, buka, et al., ) . given that maternal igg antibodies begin transfer to the fetus at weeks' gestation and approach maternal levels at time of birth (malek, sager, kuhn, nicolaides, & schneider, ; simister, ) , this study introduces the interesting possibility that autoantibodies to c q present in the mother might interact with fetal c q during critical periods of brain development. specifically, if the process of normal c q-mediated synapse formation and pruning is interrupted, synaptic connections will presumably be permanently altered in the developing brain either through overpruning or through underpruning. other studies have connected the presence of maternal autoantibodies and with the development of autism spectrum disorders where maternal autoantibodies have been found to recognize brain proteins critical to the neurodevelopmental process (braunschweig et al., ; brimberg, sadiq, gregersen, & diamond, ) . this chapter provides an introduction into some of the mechanisms by which the immune system might be involved in the development of schizophrenia. if schizophrenia has an immune component, and if evidence indicates a primary rather than secondary role in disease pathogenesis, then interventions that target the immune system are warranted. toward this end, one purpose of this review was to emphasize the very diverse and multiple ways in which the immune system might impact schizophrenia. its etiology, pathogenesis, and pathophysiology may not just be a function of exposure to an infectious agent or food antigen or dysfunctional innate immunity. therefore, designing a treatment strategy to an extraordinarily heterogeneous disease is difficult. it is extremely important to be able to identify the subsets of people who have immune-related conditions and fully characterize what kind of immune anomaly is present. only in this manner can tailored treatments be evaluated. in the future, therapeutic strategies might involve monoclonal or monospecific antibodies to antagonize or inactivate antigenic or other protein targets or use of other immunosuppressive treatments. the rapid advance in the use of monoclonal antibodies for the treatment of autoimmune disorders provides hope that such therapies can also have a major impact on schizophrenia as well. dietary interventions have been successful in some instances clinically, and developmental compounds aimed to normalize gut function and endothelial barriers in other capacities appear promising (freeman, ; jackson et al., ; kristoff 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population prenatal xenobiotic exposure and intrauterine hypothalamus-pituitaryadrenal axis programming alteration transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder key: cord- -qwh inx authors: mendoza, jose luis accini; estrada, victor hugo nieto; lópez, nelly beltrán; bolaños, elisabeth ramos; franco, daniel molano; castell, carmelo dueñas; moreno, albert alexander valencia; amaya, iván camilo alarcón; flórez, john serna; valencia, bladimir alejandro gil; camilo pizarro, g; polo, yulieth maría zabaleta; meza, carmen lucia chica title: actualizacion de la declaraciÓn de consenso en medicina critica para la atenciÓn multidisciplinaria del paciente con sospecha o confirmaciÓn diagnÓstica de covid- date: - - journal: nan doi: . /j.acci. . . sha: doc_id: cord_uid: qwh inx antecedentes y objetivos: la enfermedad por coronavirus de (covid- ) es una enfermedad ocasionada por el nuevo coronavirus del síndrome respiratorio agudo grave (sars-cov- ). se identificó por primera vez en diciembre de en la ciudad de wuhan, en los meses siguientes se expandió rápidamente a todos los continentes y la organización mundial de la salud (oms), la reconoció como una pandemia global el de marzo de . la mayoría de los individuos son asintomáticos pero una baja proporción ingresan a cuidados intensivos con una alta morbilidad y mortalidad. este consenso tiene como objetivo actualizar la declaratoria inicial emitida por la asociación colombiana de medicina crítica (amci) para el manejo del paciente críticamente enfermo con covid- dentro de las áreas críticas de las instituciones de salud. métodos: este estudio utilizó dos técnicas de consenso formal para construir las recomendaciones finales: delphi modificada y grupos nominales. se construyeron preguntas por la estrategia pico. grupos nominales desarrollaron recomendaciones para cada unidad temática. el producto del consenso fue evaluado y calificado en una ronda delphi y se discutió de forma virtual por los relatores de cada núcleo y los representantes de sociedades médicas científicas afines al manejo del paciente con coid- . resultados: expertos nacionales participaron en la actualización del consenso amci, especialistas en medicina critica y cuidados intensivos, nefrología, neurología, neumología, bioeticistas, medicina interna, anestesia, cirugía general, cirugía de cabeza y cuello, cuidados paliativos, enfermeras especialistas en medicina crítica, terapeutas respiratorias especialistas en medicina crítica y fisioterapia, con experiencia clínica en la atención del paciente críticamente enfermo. la declaratoria emite recomendaciones en los ámbitos más relevantes para la atención en salud de los casos de covid- al interior de las unidades de cuidados intensivos en el contexto nacional de colombia. conclusiones: un grupo significativo multidisciplinario de profesionales expertos en medicina crítica emiten mediante técnicas de consenso formal recomendaciones sobre la mejor práctica para la atención del paciente críticamente enfermo con covid- . las recomendaciones deben ser adaptadas a las condiciones específicas, administrativas y estructurales de las distintas unidades de cuidados intensivos del país. background and objectives: the coronavirus disease (covid- ) is caused by the new severe acute respiratory syndrome coronavirus (sars-cov- ). it was first identified in december in wuhan, china. in the following months it spread quickly to all continents and was recognised as a global pandemic by the world health organization (who) on march th, . most cases of infection remain asymptomatic, while a low proportion require intensive care, experiencing high morbidity and mortality. this consensus aims to update the initial statement issued by the colombian association of critical medicine (amci) for the management of the critically ill patient with covid- within the critical areas of health institutions. methods: this study used two formal consensus techniques to construct the final recommendations: modified delphi and nominal groups. questions were constructed using the pico strategy. recommendations for each thematic unit were developed by nominal groups. the consensus product was evaluated and qualified in a delphi round, and was discussed virtually by the speaker of each nucleus, as well as the representatives of scientific medical societies related to the management of the patient with covid- . results: a total of national experts participated in the update of the amci consensus, all specialists in critical and intensive care medicine, nephrologists, neurologists, chest physician, bioethicists, internal medicine specialists, anaesthetists, general surgeons, head and neck surgery, palliative care, nurses specialised in critical medicine, respiratory therapists specialised in critical medicine and physiotherapy, with clinical experience in the care of critically ill patients. this update issues recommendations in the most relevant areas for health care of covid- patients within the intensive care units, contextualised for colombia. conclusions: a significant multidisciplinary group of professionals, who are experts in critical medicine, reviewed and issued recommendations on best practice for the care of critically ill patients with covid- through formal consensus techniques. recommendations must be adapted to the specific, administrative, and structural conditions of the different intensive care units in the country. para la actualización de la declaratoria se utilizaron dos técnicas para el desarrollo de consensos de tipo formal, técnica delphi modificada y grupos nominales. un consenso formal permite integrar las opiniones de un colectivo de expertos que están expuestos a un tema específico (experto afectado) con la mejor evidencia científica disponible, utilizando técnicas que permitan reducir los sesgos de subjetividad. la técnica delphi es una metodología que plantea enviar cuestionarios a un grupo de expertos, para que califiquen una serie de recomendaciones en rondas reiteradas con retroalimentación de los resultados y respuestas anónimas, la técnica delphi empleada fue modificada, variante a la versión original propuesta por la corporación rand en , pero se mantuvo las ventajas de la técnica, la iteración y retroalimentación para reflexión de las propias opiniones. los grupos nominales es una técnica que reúne a un grupo de expertos bajo la coordinación de un facilitador para evaluar y calificar información o preguntas ( , ) . para la actualización se convocó grupos nominales con expertos multidisciplinarios cada uno con un líder o jefe de núcleo. los grupos construyeron las preguntas por metodología pico y desarrollaron progresivamente las recomendaciones hasta las versiones finales. el proyecto se desarrolló en fases, fase : formulación del problema y socialización; fase : elaboración de las preguntas, fase : formulación de las recomendaciones y ronda de calificación. las estrategias de búsqueda se desarrollaron en bases de datos especializadas (medline, embase, lilacs, central), en las circunstancias donde no se encontró evidencia directa, se utilizó y se adaptó evidencia indirecta del tópico relevante en el paciente críticamente enfermo general. expertos con un promedio de años de experiencia en la atención del paciente crítico evaluaron y calificación las recomendaciones en la metodología delphi mediante un cuestionario distribuido por medio de correo electrónico, respetando la política de privacidad de datos vigente. recomendaciÓn se recomienda que los prestadores de servicios de acuerdo con su infraestructura física y la disponibilidad de recursos (tecnológico, humano, de interdependencia y apoyo) definan su modelo de atención para pacientes con covid- en estado crítico basado en principios de factibilidad, efectividad, seguridad y la relación entre la demanda (momento epidemiológico) y capacidad/capacidades de respuesta:  modelo . atención de pacientes con sospecha o confirmación diagnóstica covid- . este enfoque permite concentrar, optimizar y racionalizar recursos y reducir el riesgo potencial de contagio al equipo de atención, de apoyo y de pacientes.  modelo . atención mixta, de pacientes con y sin diagnóstico de covid- , en escenarios que cuentan con unidades de aislamiento normatizados (presión negativa y > -renovaciones completas de aire por hora) soportado en el documento institucional de gestión organizacional y operativo del servicio de cuidados intensivos, descrito en los procesos prioritarios. amci ® menos accidentalidad y violencia). la reducción de procedimientos quirúrgicos complejos electivos es una opción razonable condicionado a las posibilidades del paciente. sin embargo, situaciones como la progresión y descompensación de las patologías crónicas asociadas a las medidas de restricción social puede plantear un efecto bumerang con mayor demanda de camas de uci. con base en las predicciones simuladas de las tasas esperadas de ingreso a uci de pacientes con covid- contrastado con el déficit de servicios y camas de cuidados críticos en el país a partir de la capacidad instalada se han planteado fases de desarrollo cuyas características en términos de servicios, recursos y cronología se aprecian en la ilustración y .  fase (para el gobierno nacional: ampliación de la capacidad instalada*). parte de la liberación de camas de cuidados intensivos destinadas para atención covid- bajo el modelo y/o . la liberación de camas y servicios con mínima adaptación es la fase más inmediata y resolutiva que debe acogerse a la exigencia normativa (resolución de ), en la que se espera menos mortalidad, morbilidad y tasa de complicaciones asociados con la atención de pacientes con covid- en estado crítico.  fase (optimización para el gobierno nacional*) representan el reordenamiento de las camas de cuidados intermedios adultos en camas de cuidados intensivos y de hospitalización en intermedio. de adultos. el gobierno nacional toma en cuenta la ampliación de la capacidad instalada hospitalaria descrita en el plan territorial. para esta fase se necesitan equipos de ventilación mecánica (excluidos por la norma de estos servicios), monitoreo básico y avanzado y un número mayor de talento humano multidisciplinario competente. amci ® acuerdo con las etapas ( - ) y los perfiles requeridos priorizados como primera, segunda y tercera línea de respuesta*. en la ilustración podemos observar que avanzar de las fases a se va a necesitar mayor intervención en términos de organización, planeación operativa, formación por competencias y apoyo por telesalud (teleapoyo o teleexperticia). el personal no especializado o especializado de servicios hospitalarios diferente a urgencia, quirófano o uci pueden constituir grupos de apoyo para la gestión administrativa y de índole humanitaria (líneas de respuesta) la integración de estos requerimientos adaptativos se ha puesto de manifiesto en la experiencia del centro médico new york -presbyterian weill en la ciudad de nueva york, en donde la demanda de camas de unidades de cuidados intensivos (uci) y ventilación mecánica excedió su capacidad.( ) se recurrió a los quirófanos y de recuperación las cuales no estaban en uso porque los procedimientos electivos habían sido pospuestos. se hicieron adecuaciones físicas para garantizar la vigilancia continua de los pacientes y la seguridad del equipo de atención. se capacito a todo el personal de cuidado perioperatorio disponible y fueron distribuidos en las áreas recién configuradas. las enfermeras familiarizadas con las máquinas de anestesia asumieron como terapeutas respiratorios y los intensivistas de anestesia supervisaban estas unidades. recomendaciÓn se recomienda la adopción de un modelo simulado de predicción (basado en el cociente de fatalidad, tasa de ingreso a uci y el número de reproducción ro) para proyectar, de manera anticipada las necesidades de recurso físico, tecnológico y humano de cuidados críticos en fase de preparación de la pandemia por covid- . las tomas de decisiones relacionadas con el fortalecimiento de la capacidad/capacidades de la oferta de cuidados críticos (habilitadas o adaptadas) pueden deducirse mediante la aplicación de modelos matemáticos que intentan, desde la dinámica epidemiológica, establecer el efecto simulado de las medidas de mitigación o supresión adoptadas sobre la tasa de contagio a través del tiempo. con ello se busca planear el uso de recursos e implementar acciones de prevención y de distanciamiento social más eficientes, así como establecer las necesidades en materia de cama de cuidados críticos (intensivos e intermedios) habilitadas o adaptadas en áreas de expansión y con ello los recursos resolutivos como tecnologías, interdependencias y talento humano capacitado. la capacidad predictiva del modelo resulta de establecer: la información en términos de casos proyectados (población susceptible), el modelo de transmisión, el impacto de las amci ® intervenciones no farmacológicas (mitigación y supresión) para lograr disminuir el número de reproducción r (tasa de contagio), la distribución de la gravedad de la enfermedad y con ello el porcentaje esperado de casos críticos el decreto del . . ( ) , por el cual se declara un estado de emergencia económica, social y ecológica en todo el territorio nacional proyectó para una tasa de contagio de . y cerca de millones de casos, la ocurrencia de mil casos de pacientes con covid- en estado crítico ( . %) y la necesidad de incrementar las camas de cuidados intensivos en un % que con una estancia promedio de días tendría un costo de mil millones de pesos. colombia cuenta con cerca de camas de cuidados intensivos ( / parte son intermedio) de las cuales el % podría ser utilizada para la atención de pacientes covid- ( ). con base en el modelo matemático ha proyectado la necesidad de camas de uci indicando por deducción la necesidad de camas a partir de un plan adaptativo de expansión y extensión. colombia reporta a la fecha actual , casos confirmados y nuevos casos de covid- confirmados y cerca del % ocupan una cama de cuidados intensivos (esta cifra es mayor si se tomaran en cuenta los casos sospechosos). de este modo, los modelos matemáticos permiten predecir el comportamiento epidemiológico de la enfermedad y con esto anticiparse a proyectar el plan de fortalecimiento hospitalario (incluyendo camas, tecnologías, talento humano) y los recursos financieros para respaldar la expansión del cuidado críticos. estos modelos deben ser predictivos y no reactivos al comportamiento epidemiológico de la enfermedad y solo debe des escalarse hasta después de reducirse a menos de . el índice ro. ( - ) recomendaciÓn se sugiere la implementación de una estrategia de telesalud (teleapoyo o teleexperticia) en el marco de la pandemia covid- , cuando no se cuente con un intensivista presencial, que, mediante una tecnología adecuada complemente la atención en las áreas de cuidados críticos realizado por personal capacitado. aún cuando es considerada ventajosa sus implicaciones en términos de resultados clínicos, económicos y de riesgos legales no se ha demostrado. en situaciones donde se declara una pandemia los sistemas de salud pueden tener dificultades para hacer frente a una demanda exponencial y fuera de control. esto puede ser así en el marco pandemia covid- que prevé un % de pacientes en condición crítica y que amerita reorganización y/o adaptación de su capacidad de respuesta. incrementar la disponibilidad de camas y servicios de cuidados críticos mediante una estrategia de expansión supone retos asociados a la insuficiencia que se puede presentar en talento humano especializado específicamente de especialistas en medicina crítica y cuidado amci ® intensivo, escenario que se puede complicar en la medida que intensivistas sean separados o aislados en el curso de la epidemia. en este contexto se hace necesaria la implementación de modelos ágiles de telesalud (ts) para el acompañamiento de las unidades de cuidado crítico en expansión y de instituciones prestadoras de servicios de salud de baja y mediana complejidad para la regulación con los equipos de referencia y contrarreferencia de pacientes que pueden necesitar atención en cuidados intensivos. ( ) ( ) ( ) ( ) ( ) ( ) el decreto ley de ( ) plantea la adopción de medidas en el sector salud para garantizar la prestación de los servicios de salud y para facilitar la implementación de modelos de atención que incluyan la telesalud y la prestación de los servicios en la modalidad de telemedicina se determinan algunas medidas temporales para: i) adecuar temporalmente un lugar no destinado a la prestación de servicios de salud, intra o extra mural. ii) prestar servicios en modalidades o complejidades diferentes a las habilitadas dentro de las cuales puede estar la telemedicina iii) prestar servicios de salud no habilitados. en este decreto también se establecen condiciones temporales para la implementación de plataformas tecnológicas para la telesalud. en complemento, la resolución de ( ) (estándares de habilitación) plantea la modalidad de la telemedicina (prestador remisor-prestador de referencia) para las unidades de cuidado intermedio e intensivo. la telesalud se puede prestar de dos maneras: teleapoyo y teleexperticia. tabla . teleapoyo (ta) soporte solicitado por un profesional de la salud a otro profesional de la salud a través de tic siendo responsable de la conducta quién solicita el apoyo. no requiere habilitación y por tanto no requiere autorización transitoria relación a distancia con comunicación sincrónica o asincrónica utilizando tic entre dos profesionales de la salud, uno de los cuales atiende presencialmente al usuario y otro atiende a distancia. el primero es responsable de las decisiones/recomendaciones entregadas al paciente y el segundo es responsable de la calidad de la opinión que entrega y debe especificar las condiciones en las que se da dicha opinión, lo cual debe consignarse en la historia clínica. requiere autorización transitoria (decreto ) ( ) la telesalud y la prestación de servicios de salud en esta modalidad son estrategias seguras y efectivas para guiar, a distancia, el diagnóstico y el tratamiento del paciente hospitalizado y en estado crítico. sus ventajas generales se presentan en la tabla . asociaciÓn colombiana de medicina crÍtica y cuidados intensivos. amci ® tabla . ventajas generales de la telesalud decreto legislativo de .  facilita la viabilidad de aplicación modelos organizativos que favorecen la continuidad y la integridad asistencial y la atención centrada al entorno del paciente, aplicando conceptos de globalidad e interoperabilidad a las organizaciones sanitarias, dando lugar a nuevas formas de organización y trabajo en red.  mejora de la calidad asistencial, ya que facilitan el acceso y la disponibilidad de servicios asistenciales en condiciones de calidad.  mejora calidad de vida del paciente por la disminución de desplazamientos para la atención ya que permite la atención o monitorización remota con tic en su domicilio.  mejora la oportunidad y la resolutividad de la atención.  facilita la equidad en el acceso a los servicios de salud independientemente de la localización geográfica (acerca la atención especializada a toda la población).  mejora la atención integral y seguimiento tanto de los pacientes crónicos, como los de las enfermedades de baja prevalencia.  reduce los tiempos de espera (tanto en la realización del diagnóstico como en el tratamiento), evitando complicaciones por no atención oportuna.  posibilita realizar atención remota de mediana y alta complejidad en la baja complejidad, reduciendo el número de remisiones.  disminuye la posibilidad de infección cruzada entre usuarios de los servicios de salud y el personal de salud.  incide en la formación y competencia del talento humano en salud.  facilita la educación de pacientes en medicina preventiva y salud pública.  descongestiona servicios de urgencias y consulta externa.  contribuye a la reducción de movilidad de personas en la ciudad.  responde a las necesidades inmediatas en salud de la comunidad.  es un medio de racionalización de costos en salud. puede abarcar otros servicios de gestión administrativa como entrega de fórmulas o facturación. amci ® se recomienda la implementación de un modelo de cuidados críticos covid- liderado por intensivistas, en áreas habilitadas o adaptadas, con el beneficio preponderante de disminuir la mortalidad, tiempo de estancia y optimización de recursos. se recomienda una cobertura por intensivistas de al menos horas diarias y un cociente intensivista/paciente cercano a intensivista por cada - pacientes, basado en la alta complejidad de la enfermedad critica covid- con un alto porcentaje de pacientes en ventilación mecánica, largos tiempos de estancia y alto riesgo de mortalidad. fuerte a favor fundamento un intensivista es un profesional médico capacitado en medicina crítica y de cuidados intensivos conforme a los estándares establecidos por una institución de educación superior debidamente reconocida ante el ministerio de salud. este especialista debe liderar y tomar todas las decisiones con respecto al cuidado de los pacientes críticos, incluyendo admisiones y egresos, qué médicos consultar, estándares de atención, gestión de la calidad y seguridad, gestión humana y ética, interacción con la familia e implementación de un programa de investigación y de formación continua para mejorar capacidades y competencias del equipo de atención, control de conflictos, entre otras.( ) existe una enorme validez conceptual y una preponderancia de evidencia que sugiere que ser atendido por un especialista en cuidados críticos (intensivista) es "bueno" para los pacientes de la uci. la mayoría de los estudios demuestran el impacto positivo de un uci dirigida por intensivistas los modelos de personal médico de la uci más ampliamente estudiados difieren en el nivel al cual los intensivistas están involucrados en el manejo de los pacientes. las uci de alta intensidad son aquellas donde un intensivista de tiempo completo u obligatorio maneja a la mayoría de los pacientes diariamente. las uci de baja intensidad no tienen participación intensivista u ofrecen consultas intensivistas electivas. un metaanálisis mostró que un modelo de alta intensidad en comparación con uno de baja intensidad estuvo asociado con una menor mortalidad en la uci, menor mortalidad hospitalaria, y una reducción significativa en la duración de la estancia hospitalaria. un modelo de alta intensidad por la noche se asoció con menor mortalidad solo cuando durante el día era de baja intensidad. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) asociaciÓn amci ® forzosa" por condición de riesgo siempre en consonancia con los términos establecidos en el decreto ley de ( ). según la normatividad vigente todo miembro del equipo de atención, especialista no intensivista o no especialista (enfermeras, fisioterapeutas/terapeutas respiratorias) y auxiliares de enfermería deben tener constancia de asistencia a acciones de formación continua y/o capacitación en atención covid- en cuidados críticos las cual puede ser parte de un programa institucional de capacitación liderado por el intensivista coordinador o titular del servicio o a partir de cursos respaldados por instituciones académicas acreditadas o los ofrecidos por la asociación colombiana de medicina crítica y cuidados intensivos (amci). las tablas y nos muestra como dentro de este proceso adaptativo por estado de emergencia y atribuido a un desequilibrio entre la oferta y la demanda, otras especialidades, profesionales de la salud y personal en formación pueden hacer parte de los equipos de atención bajo las siguientes premisas: . la supervisión, coordinación y liderazgo del intensivista es necesaria y . el intensivista establece los roles y competencias del th no intensivista y no normatizado, de acuerdo a sus perfiles, y delegada acciones asistenciales (vía aérea, accesos vasculares, reanimación cardiopulmonar, pronación), administrativas (ordenes médicas, notas clínicas) o de naturaleza humanitaria (comunicación con la familia, apoyo emocional al th, etc.). es necesario considerar los roles de las especialidades que formarán parte de la gestión asistencial y/o administrativa de pacientes con covid- en áreas críticas habilitadas o adaptadas de manera transitoria en colombia tabla . integridad de interdependencia (norma / ) integralidad e interdependencia (adaptiva) gfa integridad de interdependencia (norma / ) integralidad e interdependencia (norma / ) integridad de interdependencia (norma / ) obligatorio en gestión asistencial integralidad e interdependencia(norma / ) obligatorio gestión asistencial apoyo a tomas de decisiones(gfa) apoyo a tomas de decisiones (gfa) a:formacion continua + covid- (curso virtual); b: requiere capacitación covid- (curso virtual); c: gfa: grupo focal asistencial, sistema alerta-acción, rcp, pronación, accesos vasculares, ingreso a uci. d: gfad: grupo focal administrativo: consentimiento, notas de evolución. ts: telesalud. se recomienda la aplicación de la escala news- por parte de un equipo de respuesta rápida, para establecer el lugar de atención de pacientes con diagnóstico definitivo o sospecha de infección por covid- que encuentran en los servicios de urgencias o de hospitalización. se recomiendan escalas como el qsofa y el curb- para apoyar la decisión tomada con base en la escala news- . sin embargo, un qsofa mayor o igual a puntos (mínimo / ) y un crb- mayor o igual puntos tienen baja sensibilidad (alta incidencia de falsos negativos) para identificar pacientes que puedan requerir ingreso a uci. se recomienda el score de riesgo covid- -gram para identificar el riesgo de desarrollar un estado de la enfermedad crítico en pacientes con covid- y como herramienta complementaria a la escala news- en escenarios de alta demanda y escasez de recursos, de manera que la decisión de ingreso a uci se haga sobre aquellos pacientes que realmente se beneficiarán de la misma en términos de vidas salvadas y número de años salvados. la escala news fue construida y validada en paciente con infección por el virus de la influenza a/h n y se recomienda como una herramienta objetiva para decidir nivel de atención, incluyendo ingreso a uci o situaciones terminales que requieren acompañamiento familiar y medidas de cuidado paliativo. esta escala incluye variables fisiológicas que son: frecuencia respiratoria, saturación arterial de oxígeno (spo ), uso de oxígeno suplementario, presión arterial sistólica, frecuencia cardiaca (pulso), temperatura y nivel de conciencia. es de mencionar que esta escala no contempla la edad del paciente. en un reporte del acute medicine task force of the royal college, london, uk, recomendó la utilización de la escala news en los servicios de urgencias ( ) . un estudio que evaluó una base de datos con . signos vitales obtenidos de . pacientes demostró que esta escala tiene una buena capacidad para discriminar pacientes en riesgo para un desenlace combinado de paro cardiaco, ingreso no anticipado a uci o muerte dentro de las primeras horas de atención; de esta manera genera una gran oportunidad para el establecimiento temprano de una intervención clínica que cambie el pronóstico del paciente ( ) . la escala news- tampoco contempla la edad, pero sí incluye la presencia de hipercapnia bajo diferentes niveles de spo y oxígeno suplementario. esta escala es la que ha sido estudiada como herramienta para identificar pacientes en riesgo de desarrollar un estado de enfermedad crítica por covid- con un valor ≥ puntos ( / ) ( ) . con base en la escala news- se establece el grado de riesgo, el tipo de alerta, y la intensidad de monitoreo requerido; y de acuerdo con el puntaje arrojado, se define claramente el nivel de atención que requiere el paciente con diagnóstico de infección por covid- , incluso ingreso a uci (tabla ). esto se establece a través de una escala de puntuación así: -score puntos: manejo domiciliario bajo aislamiento y signos de alarma. -score punto: manejo domiciliario y seguimiento clínico en casa. -score - puntos: manejo en salas de hospitalización. -score - puntos: manejo en uci, área covid- . -score ≥ puntos sin condición extremadamente grave o irreversible y con alta posibilidad de recuperación: traslado a uci, área covid- . amci ® -score ≥ puntos con condición extremadamente grave y con datos de irreversibilidad o enfermedad terminal: no ingresa a uci y se traslada a salas de hospitalización con acompañamiento familiar y consulta a experto en bioética y cuidados paliativos. la ilustración establece un flujograma de conductas basado en el puntaje del news- . tabla . news- score. el qsofa con un valor ≥ puntos es otra herramienta recomendada para decidir qué pacientes que ingresan a uci. esta herramienta fue recomendada por la tercera definición de consenso (sepsis- ) para identificar pacientes con alto riesgo de muerte o estadía prolongada en uci entre aquellos con sospecha de infección ( ) . en este score, un punto es asignado para variables así: frecuencia respiratoria ≥ /min, presión arterial sistólica ≤ mmhg y escala de coma de glasgow (ecg) < . el score curb- y su versión simplificada, el crb- se utilizan para evaluar la severidad de enfermedad en personas hospitalizadas con neumonía adquirida en comunidad (nac). ambos scores han sido adoptados por la sociedad británica de tórax para predecir la necesidad de soporte respiratorio o vasopresor intensivo (srvi) en pacientes con covid- ( ) . el score crb- asigna un punto para variables así: confusión de reciente inicio, frecuencia respiratoria ≥ /min, presión arterial sistólica < mmhg o presión arterial diastólica ≤ mmhg y edad ≥ años. pacientes con un score ≥ puntos necesitan hospitalización ( ) . en un estudio clínico observacional realizado sobre los primeros pacientes ingresados en un hospital de noruega con diagnóstico confirmado de covid- , se evaluó la utilización de sistemas de score clínicos al momento del ingreso: news- , qsofa, crb- y sirs, con los puntos de corte previamente mencionados ( ) . la enfermedad se clasificó como enfermedad severa y enfermedad crítica. solo pacientes ( %) se clasificaron como enfermedad crítica. al evaluar los scores con sus puntos de corte, pacientes presentaron un qsofa ≥ [ con enfermedad severa ( %) y con enfermedad crítica ( %)], solo pacientes presentaron un crb- ≥ [ con enfermedad severa ( %) y con enfermedad crítica ( %)] y pacientes presentaron un news- ≥ [( con enfermedad severa ( %) y con enfermedad crítica ( %)]. la mediana del score news- para pacientes con enfermedad severa fue de . [riq: - ] vs . [riq: . - ] para pacientes con enfermedad crítica. los autores concluyen que el qsofa y el crb- se comportan similar y con una baja capacidad para la identificación de enfermedad crónica en pacientes con covid- ; por otro lado, los datos indican que el news- podría ser una herramienta más útil para identificar pacientes con riesgo de un curso más agresivo de la enfermedad ( % vs. %) ( ) . amci ® un estudio de cohorte retrospectivo realizado en un hospital en liverpool (uk), el puntaje qsofa se comportó como el más específico ( %; % ic: % - %) pero el menos sensible ( %; % ic: % - %) en comparación al puntaje sirs y la escala news como predictor de mortalidad hospitalaria en un grupo de paciente admitidos por sepsis al servicio de emergencias ( ) . otro estudio retrospectivo de un único centro comparó el rendimiento de sistemas de score, qsofa, curb- y crb- para predecir la necesidad de soporte vasopresor o respiratorio intensivo (svri) en pacientes ingresados con diagnóstico confirmado de covid- a un hospital de wuhan (china) ( ) . un total de pacientes ( . %) necesitaron svri durante su estadía en el hospital. la tasa de mortalidad hospitalaria en esta cohorte fue de pacientes ( . %). se evalúo el rendimiento de las escalas con análisis de curva roc (auc), puntos de corte óptimo, sensibilidad, especificidad y valores predictivos. el punto de corte óptimo del crb- para predicción de srvi fue de puntos, con una sensibilidad del % y una especificidad del . %. el valor auc del score crb- para predecir la necesidad de svri fue significativamente más alto que el del qsofa ( . ± . vs. . ± . , p= . ). los valores de auc fueron similares entre crb- y curb- para predecir svri ( . ± . vs. . ± . , p= . ). los autores concluyen que el crb- podría ser mejor que el qsofa para identificar paciente con covid- en riesgo de necesitar svri. su et al consideran que fue la inclusión de la edad ≥ años dentro del score crb- lo que le dio un mayor grado de superioridad sobre el qsofa ( ) . el crb- puede ser una herramienta de puntuación útil para covid- debido a su simplicidad en la aplicación, especialmente en emergencias y condiciones de escasez de recursos. finalmente, el score de riesgo covid- -gram fue descrito por un grupo de investigadores en covid- quienes reunieron datos de pacientes en hospitales en china ( ) . esta fue una cohorte retrospectiva multicéntrico en la que se recogieron un total de variables entre demográficas, médicas, clínicas (signos y síntomas), imagenológicas y de resultados de laboratorios. utilizando la metodología de regresión lasso (least absolute shrinkage and selection operator) construyen un modelo de regresión multivariable resultando en un score de riesgo predictivo para desarrollar enfermedad critica en pacientes con covid- confirmado al momento de la admisión. de las variables iniciales fueron predictores independientes estadísticamente significativos para el desarrollo de enfermedad crítica. estas variables fueron: anormalidad en los rx tx (or: . ; %ic: . - . recomendaciÓn se recomienda la aplicación de un algoritmo basado en una evaluación dinámica del score news que involucre una escala de evaluación funcional, para priorizar el ingreso a uci con transparencia científica y ética con equidad social, y de ser posible respaldado por un comité de priorización clínica (cpc) integrado por expertos de cuidado intensivo y un representante del comité de ética hospitalaria durante la pandemia por covid- . se recomienda un modelo determinado por prioridades para definir criterios de ingreso a uci, permite establecer rápidamente qué pacientes se benefician de ingreso uci y qué pacientes deben permanecer en servicios de hospitalización, o con acompañamiento familiar y cuales con medidas de cuidado paliativo. fuerte a favor page amci ® fundamento la pandemia covid- nos ha enseñado que la disponibilidad de camas de uci puede ser insuficiente y el plan estratégico diseñado para ampliar la capacidad de respuesta debe ir de la mano con la implementación rigurosa de un protocolo de triaje y de priorización de ingreso a cuidados intensivos, como medida extraordinaria para optimizar los recursos, mitigar y controlar los efectos de la pandemia sobre el balance oferta (efectiva y resolutiva) y la demanda. los protocolos de triaje y priorización están diseñados para asignar los limitados recursos de una manera justa y transparente donde, por definición, algunas personas serán excluidas del acceso a la atención orientado a aumentar la disponibilidad de camas de cuidados intensivos. sin embargo, es necesario enfatizar que la disponibilidad de camas no es un fin en sí mismo. la intención implícita y explícita de los protocolos de clasificación debiera ser el «bien público» de maximizar la supervivencia de la población. pero es incorrecto suponer que este bien público se logra al maximizar la supervivencia entre los que reciben cuidados intensivos. si bien muchos protocolos de triaje reconocen esto al tratar de excluir a los pacientes que no lo necesitan absolutamente (el «demasiado sanos») y los que tienen menos probabilidades de beneficiarse (él «demasiado enfermo»), no prestan suficiente atención a las diferencias entre grupos en términos de la duración de los cuidados intensivos necesarios para lograr resultados. si el objetivo del triage es mejorar la supervivencia de la población con un recurso escaso, entonces el recurso escaso no son camas, sino días de cama; no son ventiladores, sino tiempo de ventilación. de ello se deduce que el triaje no será efectivo si en la valoración, no se discrimina adecuadamente y se considera de manera equívoca que la gran mayoría de las personas que requieren cuidados intensivos tienen una probabilidad similar de supervivencia y una duración de estadía anticipada similar. ( ) de este modo, el ingreso a uci debe acogerse a los criterios habituales, científicos y éticos, bajo el rigor de "idoneidad clínica" tomando en cuenta parámetros como la gravedad de la enfermedad, la presencia de comorbilidades (severidad, clase funcional), potencial de recuperabilidad, deseo del paciente (o la familia), de equidad distributiva y el uso de las escalas validadas de severidad y de predicción de ingreso a cuidados intensivos. los pacientes con covid- tienden a progresar después del inicio de los síntomas dentro de los a días a una forma grave con síndrome de dificultad respiratoria aguda (sdra) o falla multiorgánica órgano. la identificación temprana y simple de pacientes que requieren respiración intensiva o el soporte vasopresor sería de gran valor durante el brote covid- . ( ) ( ) ( ) ( ) la implementación de un algoritmo "dinámico" que vincule uno o más de las escalas fisiológicas (news con o sin qsofa y/o crb- ≥ ), una escala de predicción de ingreso a uci (covid- -gram) y un puntaje de fragilidad (vipi) puede informarnos sobre el estado actual y evolutivo de la enfermedad y a priorizar el ingreso de pacientes a uci permitiendo un uso óptimo de los recursos y tomar decisiones éticas, transparentes y centradas en la dignidad de los pacientes y el bien público ilustración - . se recomienda no usar escalas de severidad de enfermedad (criterios objetivos) para definir el traslado de pacientes de uci hacia un nivel de menos complejidad de atención, ya que estas escalas no han sido validadas para este uso. se recomienda en la atención por covid- en cuidados intensivos utilizar los mismos criterios de egreso que se emplean para el traslado desde uci hacia una unidad de menor complejidad de pacientes sin infección por covid- . se recomienda contar con áreas de bajo nivel de complejidad asignadas solo a la atención de paciente con infección por covid- , las cuales serán las áreas hacia donde se realiza el de-escalamiento gradual de los pacientes basado en su evolución clínica. cuando se habla de criterios objetivos, se hace referencia a escalas de severidad de enfermedad que ayuden a tomar decisiones más racionales y no basadas en consideraciones tradicionales de resolución de cuadros clínicos. no hay una recomendación definida sobre el uso de escalas de severidad de enfermedad para definir el de-escalamiento de la atención para pacientes críticos. los sistemas de evaluación de severidad de enfermedad generales y específicos pueden identificar una población específica de pacientes en alto riesgo de deterioro clínico luego del traslado fuera de la uci( ); sin embargo, su valor para evaluar que tan preparado está un paciente individual para ser trasladado a un nivel inferior de cuidado no ha sido evaluado( ). los criterios que recomienda el colegio americano de cuidado intensivo( ) para definir el traslado desde uci a un nivel de menor complejidad (unidad de cuidados intermedios o sala de hospitalización) se basan en principios:  cuando el estado fisiológico del paciente se ha estabilizado y ya no es necesario monitoreo y tratamiento en uci.  cuando el paciente cumpla con los criterios de admisión del nivel de menor complejidad, teniendo en cuenta la disponibilidad actual del recurso, el pronóstico del paciente y la presencia de intervenciones activas en curso. específicamente para los pacientes con alto riesgo de muerte y reingreso a uci en quienes se decide no hacer intervenciones adicionales (alta severidad de enfermedad, inestabilidad fisiológica, soporte orgánico), el colegio americano de cuidado intensivo( ) siguiere pasarlos a una unidad de menor nivel de atención o a un hospital de cuidado agudo de largo plazo; siempre con un formato escrito donde se deje claro la decisión para reducir la tasa de reingreso a uci( ). específicamente en situaciones de pandemia y escasez de recursos, la sociedad española de medicina intensiva, crítica y unidades coronarias (semicyuc) recomienda la realización de la escala sofa al menos cada horas para identificar pacientes con evolución tórpida y progresiva a un fallo multiorgánico luego de la iniciación de tratamientos de soporte vital. esto permitirá la adecuación y reorientación de medidas terapéuticas a un objetivo más paliativo, incluyendo la transferencia del paciente a un nivel más bajo de complejidad y la consulta al servicio de cuidado paliativo para que brinde la atención respectiva( ). finalmente, se debe mencionar que no se ha establecido una diferencia en los criterios de egreso de pacientes con covid- para su traslado desde uci hacia una unidad de menos complejidad, con respecto a los utilizados para pacientes sin infección por covid- ( ). todo paciente hospitalizado en uci sea covid- o no, debe ser valorado diariamente para establecer en qué momento su condición clínica permite que sea trasladado fuera de la uci hacia una unidad de menor complejidad. en caso de tratarse de pacientes con sospecha o diagnóstico de covid- , este traslado puede hacerse a una unidad de cuidados intermedios dispuesta como área covid- o una sala de hospitalización con igual asignación( ); esto es lo que se conoce como de-escalamiento gradual de la atención hasta el final egreso del paciente a casa. se recomienda para pacientes con covid- que evolucionan hacia la mejoría, utilizar los criterios clásicos de de-escalamiento del nivel de atención (unidad de cuidados intermedios o sala de hospitalización) que propone el colegio americano de cuidado intensivo( ) . se recomienda aplicar la escala sofa al menos cada horas sumado al criterio de fragilidad y años de vida saludables salvados estos criterios podrían ser válidos en pacientes con fallo terapéutico a las medidas óptimas y orientar decisiones de deescalamiento de medidas y de nivel de complejidad de atención cuando la demanda potencial supera la oferta (capacidad y capacidades), estas decisiones deben ser tomadas idealmente en junta médica. amci ® se recomienda generar procesos administrativos más eficientes para el traslado de pacientes fuera de la uci hacia niveles de menor complejidad, estos ayudarán a liberar recursos para otros pacientes. se recomienda no utilizar el resultado de la rt-pcr positiva para decidir el egreso de la unidad de cuidados intensivos. la literatura no ha definido unos criterios de flexibilidad en uci para el traslado de pacientes con sospecha o diagnóstico confirmado de covid- ; y los criterios que definen la posibilidad de egreso de uci y que aplican a todos los pacientes, incluso aquellos ingresados por una condición crítica en relación con infección por covid- , están claramente definidos por el colegio americano de cuidado intensivo en su documento-guía del .( ). los principios fundamentales han sido revisados en los fundamentos de la pregunta . es importante implementar una estrategia de identificación temprana para aquellos pacientes con soporte vital avanzado que evolucionan progresivamente a fallo multiorgánico y pocas probabilidades de recuperación; el de-escalamiento de medidas y su posterior traslado fuera de la uci, liberará espacio para otros pacientes en situaciones de desborde de la demanda. la sociedad española de medicina intensiva, crítica y unidades coronarias (semicyuc) recomienda que en caso de complicaciones o que se prevea una mala evolución tanto clínica como funcional, se plantee el retiro terapéutico por futilidad y se inicie un manejo con medidas de soporte paliativo, tal como ha sido considerado en los fundamentos de la pregunta ( ) . para el grupo español es claro que, adecuar procesos administrativos más eficientes para traslado de pacientes fuera de la uci a los usualmente utilizados en condiciones normales de práctica clínica, ayudaría a ser más eficientes en la gestión de la demanda ( ) . finalmente, la persistencia de una rt-pcr positiva no es una contraindicación para el traslado del paciente fuera de la uci siempre y cuando se aseguren condiciones de aislamiento por contacto y aerosol en aquellas áreas de menor complejidad de atención; estas incluyen zonas de expansión o área del hospital específicamente acondicionadas para el manejo de pacientes estables o pacientes con limitación de esfuerzo y manejo paliativo de su condición ( ). recomendaciÓn se recomienda que la disposición final de cadáveres de personas con sospecha o diagnóstico de covid- se haga preferiblemente por cremación. en tal caso, las cenizas pueden ser objeto de manipulación sin que suponga ningún riesgo. amci ® se recomienda que la disposición final del cadáver se haga por inhumación en sepultura o bóveda individualizada cuando no se cuente con instalaciones para cremación en el territorio donde ocurrió el deceso o la disponibilidad de esta tecnología desborda la capacidad económica de las personas. se recomienda realizar siempre el aislamiento del cadáver en el lugar del deceso, siguiendo las recomendaciones del ministerio de salud y la protección social (msps). se recomienda que en los casos que se requiera necropsia médico legal y estuviese indicada la cremación, esta deberá contar con la orden del fiscal del caso. se recomienda que la institución establezca en sus procesos prioritarios un protocolo humanizado de despedida bajo estrictos criterios de bioseguridad. no existe una evidencia fuerte que recomiende hacer una disposición de cadáveres de pacientes fallecidos con sospecha o diagnóstico confirmado de covid- con un acto de cremación o con inhumación y sepultura en féretro; sin embargo, siempre y cuando la manipulación y manejo del cadáver hasta su disposición final se haga manteniendo todas las medidas de precaución para evitar la diseminación del virus y siguiendo la normatividad legal vigente, ambas formas son aceptadas. la infección por covid- es una enfermedad con gran variabilidad en presentación clínica, alta tasa de contagio y para la cual no existe en el momento un tratamiento definido. el riesgo de contagio al personal que ejecuta autopsias o procedimientos de tanatopraxia y la probabilidad de diseminación de la enfermedad por la manipulación de cadáveres no se conoce, pero se considera que puede ser alto, teniendo en cuenta que, en ausencia de la aplicación de un método de diagnóstico masivo, todo caso debe considerarse potencialmente positivo. por tanto, el manejo de cuerpos de personas fallecidas con diagnóstico confirmado, sospechoso o probable de sars-cov- (covid- ), debe realizarse con la mínima manipulación posible( ). el cadáver debe ser transferido lo antes posible al depósito y entregado al servicio funerario antes de horas luego del fallecimiento( ). el transporte, la cremación o inhumación, según sea el caso, se efectuará en el menor tiempo posible, con el fin de prevenir la exposición de los trabajadores y comunidad general al virus sars-cov- (covid- ). se debe evitar la realización de rituales fúnebres que conlleven reuniones o aglomeraciones de personas( ). el alistamiento del cadáver será realizado en el ámbito hospitalario del mismo sitio del deceso. las personas que accedan a la habitación donde se encuentre el cadáver, deben tomar las precauciones de transmisión por contacto y gotas, y para ello deben contar con todos los elementos de protección personal (epp) y seguir los procedimientos de amci ® bioseguridad de acuerdo con lo establecido en el protocolo del msps.( ) para hacer el alistamiento del cadáver, se debe cubrir todos los orificios naturales con algodón impregnado de solución desinfectante y se deberá envolver en su totalidad sin retirar catéteres, sondas o tubos que puedan contener los fluidos del cadáver, en tela antifluido o sábana; luego se deberá envolver en dos bolsas plásticas biodegradables que cumplan con las características técnico-sanitarias de impermeabilidad y resistencia a la presión de gases en su interior( ). se debe rociar el interior y el exterior de ambas bolsas con solución desinfectante de hipoclorito sódico que contenga . ppm de cloro activo( ) (exceptuando los casos asociados de covid- y muerte violenta). una vez el cadáver esté adecuadamente dispuesto en las bolsas, se podrá movilizar sin riesgo hacia el depósito de cadáveres siguiendo la ruta intrahospitalaria dispuesta para este traslado. luego el cadáver podrá será entregado al personal del servicio funerario para su depósito en ataúd o contenedor de cremación o inhumación y posterior traslado al sitio de destino final (horno crematorio y/o cementerio), luego de completar toda la documentación necesaria. cuando deba practicarse necropsia médico legal, el cuerpo será entregado a los servidores del sistema judicial quienes asumirán la custodia( ). si se han seguido correctamente todas estas indicaciones, se asume que no hay ninguna diferencia entre disponer del cuerpo enviándolo al crematorio o colocarlo en ataúd para llevarlo al tanatorio y realizar el entierro. si se opta por lo primero, las cenizas pueden ser objeto de manipulación sin que supongan ningún riesgo.( ) se recomienda que el trabajador de la salud conozca a través de la institución donde labora, los riesgos éticos, de salud y seguridad a que se expone por la atención en el paciente covid- , evitando así conflictos e incertidumbres que afecten la atención. se recomienda que las instituciones prestadoras de salud a través de los líderes de atención médica, guíen y orienten a los trabajadores, para ofrecer una mejor atención médica y menor daño emocional durante la pandemia. se recomienda dar a conocer las directrices institucionales sobre el nivel de atención (uci vs. sala médica); inicio del tratamiento de soporte vital (incluyendo rcp y soporte de ventilación); retirada del tratamiento de soporte vital; y derivación a cuidados paliativos (centrados en la comodidad) en la atención médica de los pacientes durante la pandemia, esto genera una atención caracterizada por alivio del sufrimiento, no abandono, respeto a amci ® los derechos y preferencias de los pacientes, igualdad moral de las personas y la equidad en la distribución de riesgos y beneficios en la sociedad. el apoyo de la práctica ética es necesario integrarlo al cuidado de la salud y al bienestar de la fuerza laboral del cuidado en salud. reconociendo los desafíos especiales a que se enfrentan al responder al covid- . esto forma parte del liderazgo en la atención médica y del servicio del deber cívico. berlinger n. el de abril del . en su artículo "respondiendo a covid- como un desafío regional de salud pública pautas preliminares para la colaboración regional que involucra hospitales". refiere que los trabajadores del equipo médico tienen el deber de conocer la gestión asistencial de los "desafíos éticos" previsibles durante la emergencia de salud pública (pandemia covid- ). que los desafíos éticos surgen cuando existe incertidumbre acerca de cómo "hacer lo correcto" es cuando los deberes o valores en los trabajadores entran en conflicto. estos desafíos afectan a la fuerza laboral (carga moral y emocional ante una decisión no prevista) en la atención médica. así como la operatividad en la atención médica (falta de epp y recursos que pueden limitar el buen desempeño por temor a infectarse).( ) los líderes de atención médica tienen el deber de guiar a los trabajadores de atención médica que experimentan condiciones laborales exigentes, mayor riesgo de daños ocupacionales, incertidumbre ética y angustia moral durante una emergencia de salud pública.( ) chih chen a, t. el de abril del . en su editorial ¿cómo deben prepararse los sistemas de salud para la evolución de la pandemia de covid- ? sugiere un apoyo emocional adecuado para el personal y horas razonables de exposición al riesgo para evitar el agotamiento, ya que los profesionales de la salud luchan por cuidar a los pacientes y proteger sus vidas y sus familias. se refiere que a medida que aumenta el número de casos, los médicos y los trabajadores de la salud en la primera línea deben reducir al mínimo su carga de trabajo clínico. las instituciones de atención médica deben reasignar al personal realizar tareas no clínicas, incluidos el papeleo y la recopilación de datos, tanto como sea posible. los hospitales deben tomarse su tiempo para capacitar al personal para implementar eficazmente las precauciones de contacto y los procesos de flujo. ( ) jick j.l. el de marzo del . en relación con la obligación de planificar la atención médica, considera que: los líderes de atención médica tienen el deber de planificar la gestión de los desafíos éticos previsibles durante una emergencia de salud pública. la planificación de los desafíos éticos previsibles incluye la identificación de posibles decisiones de triage, herramientas y procesos. en una emergencia de salud pública que presenta una enfermedad respiratoria grave, es posible que se deban tomar decisiones de clasificación sobre el nivel de atención (uci vs. sala médica); inicio del tratamiento de soporte vital (incluyendo rcp y soporte de ventilación); retirada del tratamiento de soporte vital; y derivación a cuidados paliativos (centrados en la comodidad) si el tratamiento de soporte vital no se iniciará o se suspenderá. es posible que también se deban tomar decisiones de clasificación en relación con la escasez de personal, espacio y suministros. el deber de cuidado fundamental requiere fidelidad al paciente (no abandono como una obligación ética y legal), alivio del sufrimiento y respeto a los derechos y preferencias de los amci ® pacientes. el deber de cuidado y sus ramificaciones son el enfoque principal de la ética clínica, a través de los servicios de consulta de ética clínica a pie de cama, el desarrollo de políticas institucionales y la educación y capacitación en ética para los médicos. deberes de promover la igualdad moral de las personas y la equidad (justicia en relación con la necesidad) en la distribución de riesgos y beneficios en la sociedad. estos deberes generan deberes subsidiarios para promover la seguridad pública, proteger la salud de la comunidad y asignar de manera justa recursos limitados, entre otras actividades. estos deberes y sus ramificaciones son el foco principal de la ética de la salud pública. no se puede emitir una recomendación a favor o en contra acerca del uso de los medicamentos y dispositivos "prototipos" utilizados en el manejo del covid- denominados de uso compasivo o fuera de etiqueta, se considera sin embargo que no pueden ser utilizados por fuera de ensayos clínicos o protocolos institucionales estandarizados de evaluación del perfil de riesgo/beneficio y bajo la aplicación de consentimiento informado. se entiende como uso compasivo la utilización, en pacientes aislados y al margen de un ensayo clínico. dichos medicamentos experimentales no han sido aprobados aún por la fda, ( ) y no se ha demostrado su seguridad y eficacia. es importante recordar que el medicamento médico puede tener efectos secundarios inesperados y graves, y que los pacientes deben considerar los posibles riesgos cuando procuran acceder a un producto médico experimental. hay que tener en cuenta que, para utilizar un medicamento bajo las condiciones de uso compasivo, se requerirá el consentimiento informado por escrito del paciente o de su representante legal, un informe clínico en el que el médico justifique la necesidad de dicho tratamiento. la regulación de la utilización de medicamentos por la vía del uso compasivo se ha realizado dentro de un texto legal sobre la realización de ensayos clínicos. una interpretación común del uso no indicado en la etiqueta y el uso compasivo de medicamentos es que, si el paciente murió, murió de la enfermedad, pero si el paciente sobrevivió, sobrevivió debido al medicamento administrado. recomendaciÓn se recomienda en la comunicación inicial con los familiares del paciente adulto con sospecha o diagnóstico de covid- críticamente enfermo incluir de forma clara y transparente los aspectos relacionados con el derecho al final de la vida que incluye: proporcionalidad en el tratamiento, adecuación del esfuerzo terapéutico, documento de voluntad anticipada, adecuación del esfuerzo terapéutico y la atención paliativa. situaciones estas que se pueden presentar durante la evolución hospitalaria y que requieren de una decisión conjunta entre el médico y el familiar del paciente. se recomienda dar una información específica, y adecuada a los familiares del paciente con sospecha o diagnóstico de covid- , para que firmen el consentimiento informado, generando esta información confianza y comprensión en el familiar. la información del consentimiento que recibe el familiar debe constar dentro de la historia clínica. fundamento el ministerio de salud y protección social del de marzo del a través del documento de "recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid- ". ante la posible circunstancia de pacientes sin capacidad para la toma de decisiones, por deterioro del estado general o requerimiento de aislamiento, en el cual no se puede contactar a su representante, conduzca la toma de decisiones teniendo en cuenta la prioridad de no hacer daño y la modificación en las condiciones de disponibilidad de los recursos en caso de presentarse deterioro. recuerde que esta situación debe preverse y debe ser informada a los representantes desde la admisión del paciente". durante toda la atención debe darse información sobre la posibilidad de que se presenten limitaciones de acceso a los soportes necesarios incluido al personal de salud , lo anterior puede limitar los derechos individuales o preferencias, esto debe ser informado al paciente y su familia, para que les permita entender que bajo la emergencia, puede presentarse una circunstancia que en condiciones habituales pudiera ser reversible de ser tratada pero en el contexto actual los recursos pueden verse trágicamente limitados, sin que esto implique abandono en el cuidado. el documento se refiere a la información que debe recibir el paciente o su familiar sustituto durante su evolución o fallecimiento. ( )la información durante la evolución también debe incluir: la información sobre el ejercicio de derechos al final de la vida incluyendo la adecuación de los esfuerzos terapéuticos y la suscripción de documentos de voluntad anticipada la consulta y revisión de existencia de este en todos los casos. se recomienda tener un consentimiento informado al ingreso hospitalario del paciente covid- , se deben tener en cuenta las circunstancias del paciente al ingreso hospitalario, si la capacidad para la toma de decisiones está limitada por su estado clínico o incapacidad mental. de ser estas las circunstancias se dará la información al familiar en primera línea de consanguinidad quien asume por el paciente el consentimiento de la información (consentimiento sustituto). se recomienda tener el consentimiento informado en situaciones de excepción o urgencia ante la pandemia por covid- , debe ser universal, en el que se informe el ingreso a la uci, o a cualquier otra área hospitalaria, realización de procedimientos, administración de tratamientos, posibles riesgos, beneficios durante su hospitalización. con esto se respeta el derecho a la autonomía personal en el paciente competente. en caso contrario el familiar tomará la información y asume el consentimiento a la información dada. es importante que el familiar esté informado de las decisiones que se vayan tomando durante la evolución hospitalaria (realización de procedimientos, inicios o cambios de tratamientos, movilización dentro del área hospitalaria. etc.) fuerte a favor fundamento el ministerio de salud y protección social, el de marzo del ha elaborado un formato de "consentimiento informado para acompañante de casos probable/confirmado de covid- ". en que se expresa: "que de manera detallada se me ha suministrado información completa, suficiente, con un lenguaje sencillo y claro. el profesional de la salud me ha explicado la naturaleza de la enfermedad, acerca del significado de caso sospechoso o confirmado del coronavirus covid- en cuanto a su presentación clínica, modo de contagio, medidas para contenerla, posibilidad de sufrir la enfermedad, complicaciones o muerte, mientras permanezca como acompañante del paciente". este documento se firma al ingreso por el acompañante o familiar quien asume las decisiones durante su estancia hospitalaria. ( ) el consentimiento informado en los pacientes covid- será un consentimiento sustituto para su ingreso a la uci y para los procedimientos que en la uci se realicen (colocación de tubo orotraqueal, diálisis, colocación de catéteres, reanimación cardiopulmonar, ecmo, etc.). previa información y autorización del familiar. en circunstancias normales el consentimiento debe ser firmado por el paciente quien en su autonomía acepta la información sobre su manejo y tratamiento. feld ad. recomendaciÓn se sugiere ante la pandemia del covid- , si es posible, que el grupo de expertos en bioética y/o comité de ética institucional sean consultados y estén informados por el médico responsable para la orientación o consejo en la toma de directrices ante decisiones difíciles. se sugiere en lo posible que el médico tratante no asuma solo la responsabilidad moral de la decisión y que la decisión sea institucional y documentada en la historia clínica e informada a los familiares. en caso de no contar con un comité de bioética y/o ética hospitalaria el médico responsable podrá tomar la decisión fundamentada en principios éticos y derechos del paciente o convocar a una junta médica u otro comité relacionado con su dilema o consultar un apoyo externo en bioética. débil a favor fundamento el ministerio de salud y protección social. ( ) el de marzo del en el documento "recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid- establece: "que en caso de que la institución cuente con un comité de bioética y/o Ética, con el servicio de bioética o Ética clínica, o consultoría clínico-ética, se debe definir una ruta de consulta para los casos que de forma concreta puedan superar las recomendaciones generales. de igual forma establece que en las circunstancias actuales que se viven el actuar ético es parte integral del profesionalismo del cuidado. teniendo en cuenta que los profesionales de la medicina serán los llamados a tomar decisiones de alto estrés moral, al tener que adherirse y promover conductas concretas basados en las circunstancias que les rodean para dar o retirar tratamientos de las personas infectadas, quienes esperan confirmación del diagnóstico y de las personas que acuden a la atención en salud por razones diferentes a la infección por covid- . partiendo de lo antes referenciado, se recomienda que las acciones emprendidas, busquen siempre poder responder a: a. no hacer daño b. beneficiar c. actuar con justicia sobre la persona en el contexto de la emergencia frente a la justicia sanitaria de la población d. mantener la integridad profesional" el comité de bioética y/o comité de ética institucional en tiempos normales o en tiempos de pandemia deberá mantener actualizadas las directrices de toma de decisiones. que apoyaran al médico responsable en la decisión. de no contar el médico con dicho comité o directrices institucionales y deba tomar una decisión que no permite interconsultar, deberá justificar en la historia clínica fundamentado en los principios éticos y derechos del paciente las razones que lo llevaron a tomar la decisión e informar y dialogar con los familiares del hecho. esto es importante que siempre quede documentado en la historia clínica la acción moral y ética de la decisión y el diálogo con la familia. en caso de que el médico responsable no cuente con un comité de bioética y/o comité de ética institucional, ni con el apoyo externo de expertos en bioética. y no quiera tomar la amci ® decisión a título personal podrá consultar a otro médico de la institución su decisión y entre ambos definir la acción a seguir. esta decisión conjunta debe ser documentada en la historia clínica e informada al grupo de trabajo y a los familiares como junta médica. si los familiares después de recibir la información no quedan satisfechos ellos tienen el derecho a la segunda opinión. los comités de bioética y/o comités de ética hospitalaria son entes administrativos, consultores, orientadores, asesores y consejeros de las situaciones que tienen que ver con el respeto y cumplimiento de los principios éticos, deberes y derechos de los pacientes, sus recomendaciones no son vinculantes, apoyan y orientan la decisión médica. aconsejando la mejor decisión ante una situación que genera un dilema moral o ético en el médico responsable. las decisiones médicas son tomadas por el consultor en bioética, quien es médico. los comités institucionales fuera de un comité de bioética y/o ética que podría dar apoyo al médico responsable y que serían otras instancias consultivas serian el comité de humanización, comité de historias clínicas, comité de bioseguridad, comité de infectología, comité de mortalidad hospitalaria, comité de paliativos o un comité de gerencia. pues todos los mencionados tiene que ver con el bienestar del paciente y la seguridad del médico ante una decisión. las decisiones especiales deben ser tomadas inicialmente a través de la realización de un comité (pueden ser los mencionados), en su defecto una junta médica. una vez se tenga la decisión esta debe ser consultada a la familia como una decisión institucional respaldada por el comité o junta médica realizada. se recomienda que todo ensayo clínico que se realice en la institución debe ser presentado, revisado y aceptado por un comité de investigación local o un comité de investigación externo nacional o internacional. se recomienda que todo paciente que se incluya en un ensayo clínico debe contar con un consentimiento informado el cual garantiza la aceptación voluntaria a participar y la comprensión de los objetivos, riesgos, beneficios, derechos y responsabilidades que tiene dentro de la investigación. se recomienda el consentimiento informado en todo ensayo clínico, el cual debe ser debe ser individual en tiempos de normalidad como en tiempos de pandemia por covid- . solo el comité de ética en investigación podrá establecer en tiempos de normalidad o de pandemia las condiciones de dispensa o excepción al requisito de obtener el consentimiento informado. refiere que el consentimiento informado tiene sus raíces en el código de núremberg de y la declaración de helsinki de y ahora es un principio rector para la conducta en la investigación médica. en el consentimiento informado para investigaciones clínicas es claro que los participantes deben entender ampliamente los componentes del consentimiento. ( ) thanh tam, n. et al. el de enero . mediante una revisión sistemática de pubmed, scopus y google scholar y revisando manualmente las listas de referencias para publicaciones hasta octubre de . realizó un metaanálisis de los resultados del estudio utilizando un modelo de efectos aleatorios para tener en cuenta la heterogeneidad. evaluó la proporción de participantes en ensayos clínicos que entienden los diferentes componentes del consentimiento informado. encontrando que los participantes en ensayos clínicos deben comprender los componentes fundamentales del consentimiento informado como: la naturaleza y los beneficios del estudio, la libertad de retirarse en cualquier momento y la naturaleza voluntaria de la participación, así como la comprensión de otros componentes, como la aleatorización y el placebo. la proporción de participantes en ensayos clínicos que comprendieron diferentes componentes del consentimiento informado varió de . % a . %. esto asegura que la toma de decisiones de los participantes es significativa y que sus intereses están protegidos. ( ) la red de américa latina y el caribe de cnb-unesco, ( de marzo de ) que agrupa a las comisiones y consejos nacionales de bioética cuya finalidad es la de asesorar sobre los problemas éticos relativos a las ciencias de la vida y la salud humana expresa su preocupación ante la realización de investigaciones biomédicas en relación con la pandemia de enfermedad infecciosa por coronavirus covid- . reconociendo lo siguiente en relación con el consentimiento informado: que la investigación con seres humanos durante las emergencias debe contar con garantías éticas mayores, no menores, que en las situaciones ordinarias. que en situación de excepción o emergencia los participantes deben seleccionarse en forma justa y proporcionar una justificación adecuada cuando se escogen o excluyen determinadas poblaciones, distribuyendo en forma equitativa las posibles cargas y beneficios de participar en esa investigación. "que se debe obtener el consentimiento informado individual de los participantes incluso en una situación de excepción o alarma, a menos que se cumplan las condiciones para la dispensa del consentimiento informado. las cuales el comité de revisión ética solo puede decidir dar la dispensa al requisito de obtener consentimiento informado: a) si no es factible obtenerlo; y si además los estudios: b) tienen un importante valor social y científico, c) solo suponen riesgos mínimos para los participantes, d) no implican agravio comparativo con otros grupos en situación o no de vulnerabilidad; y si e) se garantiza que no se privará a la población investigada de acceder en forma preferencial al beneficio derivado. de otorgarse un consentimiento informado amplio, éste debería ser única y exclusivamente para los procesos asociados con covid- ".( ) amci ® se recomienda considerar la transición del cuidado intensivo al cuidado paliativo en todo paciente con sospecha o diagnóstico de covid- sin mejoría a pesar de las intervenciones óptimas, con empeoramiento progresivo de su pronóstico vital y ante un evidente deterioro; aplicando medidas generales en control de síntomas ( manejo de secreciones -tratamiento del dolor -tratamiento de la disnea -sedación paliativa), así como apoyo espiritual, siempre acompañando al paciente y nunca abandonarlo en el final de la vida. fuerte a favor fundamento la sociedad española de anestesiología, reanimación y terapéutica del dolor en su documento: "marco Ético pandemia covid- " madrid, de marzo de refiere: la sedación paliativa en pacientes hipóxicos con progresión de la enfermedad no subsidiaria de tratamiento debe considerarse como una expresión de buena práctica clínica y debe seguir las recomendaciones existentes. si se prevé un período agónico no corto, se debe proporcionar una transferencia a un entorno no intensivo.( ) se recomienda la utilización de guías establecidas previamente a la pandemia por el ministerio de salud y sociedades científicas para orientar las decisiones que se tomen al final de la vida en pacientes con sospecha o diagnóstico de covid- . estas guías deben ser divulgadas al equipo de atención y aplicadas en los pacientes en casos de: adecuación del esfuerzo terapéutico (aet), orden de no reanimar (onr), consentimiento sustituto, voluntades anticipadas, cuidados paliativos. se recomienda fundamentar las decisiones del final de la vida individualizadas a cada paciente y a cada situación sin llegar a tomar decisiones apresuradas sin fundamento científico o ético, solicitando de ser posible una valoración por medicina paliativa para el manejo de síntomas. se recomienda indagar durante la hospitalización de todo paciente con sospecha o diagnóstico de covid- , si en tiempos de salud hizo válida su autonomía y realizó un documento de voluntad anticipada, teniendo en cuenta que esta será equivalente al consentimiento informado. amci ® terapéuticos y la suscripción de documentos de voluntad anticipada.( ) el inicio de sedación paliativa con reubicación del pacientede ser necesario el des escalonamiento por deterioro clínico. en situación de pandemia covid- cuando se refiere a situaciones del final de la vida se relaciona a la adecuación del esfuerzo terapéutico, la sedación paliativa la cual será la maniobra terapéutica que se utilizará en pacientes no recuperables y que no son candidatos a cuidados intensivos por covid- ,( ) que evolucionan desfavorablemente y tienen mal pronóstico a corto plazo, así como la disnea refractaria y la limitación del esfuerzo terapéutico. el delirium o síndrome confusional por fallo cerebral agudo, es un problema habitual en situaciones de alteración orgánica severa, y ha sido descrito como uno de los síntomas neurológicos presente en los pacientes que sufren infección por el covid- ( ). wilson c. de abril del . en su artículo "la crisis golpea al final de la vida" se refiere a que el brote de coronavirus está obligando a las personas a enfrentar dilemas en torno a la cantidad de atención médica que se debe brindar al final de la vida y apresurar decisiones controvertidas sobre rechazar ciertos tratamientos. dicen los expertos que esto ha alentado a más personas a tomar decisiones de tratamiento anticipadas relacionadas con la rcp y la ventilación,( ) haesen s. el de mayo de . en su artículo "dirigir a los ciudadanos a crear directivas anticipadas" las voluntades o directrices anticipadas son para las personas que quieran asumir plenamente su papel de ciudadanos responsables tomando decisiones proactivas. la decisión de redactar directivas anticipadas marca un cambio del enfoque actual de "aceptación" a un escenario de "exclusión voluntaria".( )al emitir una directiva de tratamiento anticipado, una persona autónoma puede expresar formalmente qué tipo de tratamiento desea y no desea recibir en caso de que se enferme o se lastime y no pueda decidir de manera autónoma sobre su tratamiento. ( ) ministerio de salud y protección social en su documento de voluntades anticipadas que es el documento en el que toda persona capaz, sana o en estado de enfermedad, en pleno uso de sus facultades legales y mentales y como previsión de no poder tomar decisiones en el futuro, declara, de forma libre, consciente e informada su voluntad sobre las preferencias al final de la vida que sean relevantes para su marco de valores personales.( ) se recomienda que el paciente crítico con covid- que no es candidato para ingresar o continuar recibiendo cuidados intensivos y que presente deterioro rápido con mal pronóstico a corto plazo, se le brinde una adecuación del esfuerzo terapéutico orientada a acompañamiento al final de la vida, alivio del sufrimiento y control de síntomas. dependiendo de la disponibilidad de recursos se sugiere dentro del plan de atención hospitalaria contar con un área destinada a la atención del final de vida con el recurso físico, humano y de procesos necesario. marzo del . refiere: en situación de adecuación terapéutica, retirada de medidas y/o mala evolución es adecuado derivar al paciente a un área de menor complejidad para establecer el plan de cuidados paliativos. consultar al servicio de cuidados paliativos para procurar la continuidad de cuidados de los pacientes en los que se haya acordado la limitación de tratamientos y aliviar su sufrimiento, incluyendo la sedación paliativa en los casos en los que sea precisa.( ) schmidhauser tf. el de abril . considera en su publicación que: los cuidados paliativos durante la pandemia de covid- deben adaptarse a un estilo de" cuidados paliativos de emergencia" ya que los pacientes pueden deteriorarse rápidamente y requieren decisiones rápidas y planes de tratamiento claros. estos deben ser seguidos fácilmente por los miembros del personal de salud que atienden a estos pacientes. además, los cuidados paliativos deben estar a la vanguardia para ayudar a tomar las mejores decisiones, atender a las familias y ofrecer apoyo espiritual.( ) se recomienda como estrategia de protección personal en las unidades de cuidado intensivo sin presión negativa y cubículos abiertos utilizar de forma continua el respirador n o fpp , adicional a otros elementos de protección para prevención del contagio por covid- . la atención segura en áreas crítica para todas las modalidades de atención se fortalece a través de las medidas de precauciones estándar en el manejo de todos los pacientes, establecidos en el "manual de medidas básicas para control de infecciones en prestador servicios de salud" ley de , por la cual se dictan medidas sanitarias. resolución de , numeral y numeral . epp. el respirador, n o fpp , puede utilizarse de manera continua por a horas, o desecharlo antes si está visiblemente contaminada o si está húmeda. luego de colocar la n se debe verificar prueba de ajuste antes de ingresar a la unidad para atención de pacientes con covid- de la siguiente manera: mascarillas sin válvula de exhalación: cubra la totalidad de la mascarilla con ambas manos y exhale con fuerza. si nota fugas de aire por sus bordes, reajuste la posición del respirador. mascarillas con válvula de exhalación: cubra el respirador con ambas manos e inhale con energía. deberá sentir una presión negativa dentro de la mascarilla. si detecta alguna pérdida de presión o entrada de aire, reajuste la posición del respirador. no se puede emitir una recomendación a favor o en contra acerca de la efectividad de bioseguridad del uso extendido, continuo o intermitente de los respiradores n ó ffp . sin embargo, se considera que puede ser una alternativa, bajo la adopción de un protocolo riguroso, cuando se debe optimizar el uso de los epp en el contexto de un acceso limitado. el reúso no está permitido en colombia. la duración máxima del uso continuo de la n es de a horas, siguiendo las recomendaciones del manual de medidas básicas para control de infecciones en ips de minsalud. pero en la vida real, ningún trabajador tolera a horas continuas con un respirador. por esto, su uso continuo en el sitio de trabajo dependerá de la necesidad de pausar para comer, para ir al baño, etc. en este caso, se guardará en una bolsa de papel para su nueva colocación, si tiene menos de horas, o desechará si está visiblemente contaminada o se torna húmeda. el reúso de la n dependerá de la casa del fabricante, de si contiene o no celulosa en su estructura del respirador. por ejemplo, la recomendación de desinfección para los respiradores sin celulosa es con peróxido de hidrógeno vaporizado al % por minutos (tabla y ). los respiradores n de uso industrial tienen mayor contenido de celulosa que los de uso médico por lo tanto para procesos de esterilización, solo los n de uso médico podrán ser esterilizados mediante de peróxido de hidrógeno vaporizado (sterrad®) ( - ) . ¿existe superioridad en términos de protección personal para el personal sanitario y de apoyo dentro de las unidades de cuidados intensivos con la utilización del overol frente a la bata manga larga con antifluido durante la atención del paciente con covid- ? no se puede emitir una recomendación a favor o en contra si los overoles ofrecen mayor protección por cobertura corporal frente a otros elementos como vestidos largos, batas y delantales. resulta intuitivo que su uso genera una mayor protección en especial en servicios cohortizados. sin embargo, su uso está asociado con una mayor dificultad para su colocación y retiro, lo que puede potencializar el contagio del usuario, la utilización debe hacerse bajo un protocolo supervisado y chequeado. las batas modificadas para ajustarse firmemente en el cuello pueden reducir la contaminación. en estudios ya descritos el uso de un respirador eléctrico purificador de aire con overol puede proteger contra el riesgo de contaminación mejor que una máscara n y una bata con un rr: . , intervalo de confianza (ic) del %: . a . pero fue más difícil su retiro con rr . , ic del % . a . . en un eca ( participantes), las personas con una bata larga tenían menos contaminación que aquellas con un overol. las batas pueden proteger mejor contra la contaminación que los delantales.( - ) los epp como batas y overoles deberían estar hechos de un material que cumpla con los requisitos mínimos de la asociación americana de químicos textiles:  tipo a: buena repelencia al agua, resistente a la penetración, pero mala permeabilidad al aire.  tipo b: buena repelencia al agua, buena permeabilidad al aire, pero poca resistencia a la penetración del agua.  tipo c: bata quirúrgica que tiene poca repelencia al agua y resistencia a la penetración de agua.  tipo d: hecho de fibras de polietileno de alta densidad, tela no tejida (tyvek®), tiene buena repelencia y resistencia al agua, mala permeabilidad al aire. no se puede emitir una recomendación a favor o en contra para el uso de respiradores elastoméricos como elementos de protección personal dentro de las unidades de cuidados amci ® intensivos. no hay evidencia que soporte la superioridad de los respiradores elastoméricos frente a los n , son más costosos, difíciles de utilizar y pueden implicar algún riesgo para el paciente. por tanto, su uso sólo debería considerarse frente a un desabastecimiento de los n y bajo la adopción de un protocolo institucional riguroso y bajo chequeo. los respiradores elastoméricos son respiradores ajustados a media cara o cara completa, esta última otorga protección ocular. su filtración está determinada por el filtro que se utilice, estos van desde partículas de nivel n a p . están hechos de material sintético o de goma que les permite desinfectarse, limpiarse y reutilizarse repetidamente. están equipados con cartuchos de filtro reemplazables. al igual que los respiradores n , los respiradores elastoméricos requiere entrenamiento adecuado para su correcta colocación y retiro. por eso es muy importante revisar el manual del usuario antes de su uso. los respiradores elastoméricos no deberían utilizarse en entornos quirúrgicos, debido al riesgo potencial de contaminación del campo quirúrgico, con el aire que sale de la válvula de exhalación. como recomendación de buena práctica, aprobado por la fda, debe colocarse una máscara quirúrgica encima de la válvula de exhalación para evitar este riesgo. solo se debe permitir el uso del respirador elastoméricos por clínica para evitar infecciones cruzadas, esto permitirá una protección esencial contra agentes infecciosos y la auto contaminación. aunque los cartuchos de filtro son finalmente desechables, están destinados a ser reutilizados hasta que ya no se puede respirar o se vuelven visiblemente sucio. generalmente se recomienda, en la mayoría de los casos, hacer recambios cada días. deben tener procedimientos de limpieza/desinfección actualizados y aprobados por su manufacturador.( , - ) recomendaciÓn se recomienda realizar la limpieza y desinfección de equipos biomédicos y de superficies las veces que sean necesarias y en el momento de egreso del paciente siguiendo los protocolos de cada institución. el desinfectante para este proceso debe ser de nivel intermedio o alto para superficies y equipos biomédicos y cumplir con las recomendaciones del fabricante según lo aprobado en el registro sanitario. fuerte a favor fundamento para la desinfección de las superficies ambientales hospitalarias y domiciliarias, la oms recomienda emplear un desinfectante que sea efectivo contra virus cubiertos (el coronavirus pertenece a esta categoría), específicamente, recomienda emplear alcohol etílico para la desinfección de algunos equipos biomédicos reusables (p. ej.: termómetros) y para las superficies, el hipoclorito de sodio o precursores de sodio como el dicloroisocianurato de sodio (nadcc) que tiene la ventaja de la estabilidad, la facilidad en la dilución y que no es corrosivo.( ) (tabla ). page se recomienda que el ingreso de un paciente a uci debe hacerse bajo un procedimiento estandarizado que incluya la coordinación y comunicación de los servicios vinculados, adecuación de la unidad de atención a las necesidades del paciente y la garantía de la bioseguridad del equipo multidisciplinario. se recomienda que cada institución establezca en sus procesos prioritarios el circuito del traslado que incluye el itinerario del traslado, el uso de ascensor, el número y la organización de los intervinientes sanitarios y no sanitarios (celadores, seguridad, limpieza), las medidas de protección empleadas por los mismos (epp, limpieza) y los recursos materiales necesarios durante el traslado. el traslado de pacientes con casos sospechosos o confirmados de covid- se puede presentar entre servicios a nivel hospitalario o entre instituciones con diferentes niveles de atención y deben tenerse las precauciones universales de un traslado seguro. una posición responsable es evitar el traslado de estos pacientes el máximo posible, a menos que sea imprescindible, teniendo en cuenta el riesgo/beneficio. considerar evitar traslados interinstitucionales solo por temas administrativos. el personal sanitario que realice el traslado debe contar con todos los epp, considerando este traslado como de alto riesgo de transmisión vírica. se debe utilizar mascarilla quirúrgica o n- , de acuerdo con el riesgo amci ® de aerosolización. hasta que la rt-pcr para sars-cov- este negativo se podrían retomar las prácticas habituales de traslado de los pacientes . ( ) ( ) ( ) capítulo . abordaje diagnóstico y covid- se recomienda en pacientes con diagnóstico o sospecha de infección por sars-cov- clasificar la enfermedad en leve, severo o paciente crítico, teniendo en cuenta los criterios de la clasificación por las fases y estadios de la enfermedad. se recomienda en pacientes con diagnóstico o sospecha de infección por sars-cov- , clasificados como críticos y que requieren de intubación orotraqueal realizar la clasificación por fenotipos ( o ), con el fin de proyectar una estrategia de ventilación mecánica. el covid- , es una enfermedad con una presentación clínica diversa, desde formas leves hasta presentaciones graves que incluyen el sdra, la mediana del período de incubación desde la exposición hasta el inicio de los síntomas es de aproximadamente a días, y el . % de los pacientes sintomáticos tendrán síntomas dentro de los . días después de la infección ( ) , que incluye fiebre, tos, disfagia, malestar general, mialgias, anorexia, náuseas, diarrea, anosmia y ageusia; la disnea se presentó entre los y días ( ) y puede representar progresión a covid- severo, que se manifiesta con hipoxemia, disfunción orgánica múltiple, documentación de arritmias cardíacas, rabdomiólisis, coagulopatía y choque ( ) . dentro del espectro de enfermedad, siddiki et al, proponen un enfoque estructurado por fases expresados en tres estadios (historia natural de la enfermedad), siendo el primero donde la patogenicidad viral es dominante, se incluye el periodo de incubación, síntomas leves, con multiplicación del sars-cov- centrándose principalmente en el sistema respiratorio gracias a la unión del virus con el receptor de la enzima convertidora de angiotensina (ace ), el hemograma puede revelar linfopenia y neutrofilia sin otras anormalidades significativas. el estadio es la enfermedad pulmonar establecida, neumonía viral, tos, fiebre con progresión en algunos casos a hipoxia con trastorno de los índices de oxigenación (pao /fio menor mmhg), hallazgos en imágenes de tórax (radiografía y/o tomografía) de infiltrados alveolares o vidrio esmerilado, mayor linfopenia y elevación de transaminasas ( a: sin hipoxemia, b: con hipoxemia). el estadio o fase de híper inflamación sistémica extrapulmonar se caracteriza por elevación de biomarcadores inflamatorios y estado protrombótico (il - , il- , il- , ftn -α, proteína c reactiva, ferritina y el dímero d), con presencia en las formas más graves de disfunción orgánica múltiple, lesión miocárdica (troponina y péptido natriurético de tipo b elevados), con fenómenos trombóticos, progresión a sdra y choque ( ) . amci ® la neumonía por sars-cov- , se característica por disociación entre la severidad de la hipoxemia y el mantenimiento relativamente bueno de la mecánica respiratoria, con compliance del sistema respiratorio en promedio de ml / cmh o; gattinoni, marini et al, proponen dos fenotipos de presentación de la insuficiencia respiratoria; el primero (tipo ) con una mecánica pulmonar adecuada, con baja probabilidad de reclutabilidad y con hipoxemia, relacionado al desbalance entre la perfusión y la ventilación; el segundo (tipo ) más acorde a las definiciones de sdra (csdra "covid- patient with sdra"), con una compliance pulmonar baja y reclutabilidad potencial( , ) ( ). se propone la siguiente clasificación clínica del covid- . ( , , y pacientes críticos de %, con tasa global de mortalidad de , %, siendo mayor entre los pacientes de a años con % y entre los mayores de años con . %, dentro del grupo de pacientes clasificado como crítico la mortalidad descrita fue del %( ). recomendaciÓn se recomienda en cuidado intensivo, realizar el diagnóstico de covid- del paciente sospechoso por medio de rt-pcr conociendo su alta especificidad, su variabilidad en relación con el tiempo y pérdida de rendimiento diagnóstico luego de la primera semana de inicio de los síntomas. se recomienda tomar la primera muestra para rt-pcr de hisopo nasofaríngeo o de cornete medio sobre hisopado oro faríngeo o de saliva, de ser negativo se puede repetir la prueba de a horas preferiblemente de tracto respiratorio inferior, esputo no inducido o en aspirado traqueal en paciente intubado. se recomienda el uso conjunto de rt-pcr e igm por elisa en pacientes con sospecha de covid- , primera rt-pcr negativa, que se encuentren entre la segunda y tercera semana desde el inicio de los síntomas, con el objetivo de mejorar la sensibilidad en la identificación de infección por sars-cov- . en cuidado intensivo, el diagnóstico de covid- se fundamenta con base en la presentación clínica compatible y factores epidemiológicos asociados con probabilidad de infección; el diagnóstico definitivo se realiza con pruebas de amplificación de ácido nucleico del virus (naat), la detección del genoma viral del sars-cov- se realiza por medio de reacción en cadena de la polimerasa por transcriptasa reversa (rt-pcr) dado a su especificidad del % ( , ) ; por lo cual todo paciente que cumple con la definición de caso sospechoso se le debe realizar rt-pcr, sars-cov- independientemente de si se encuentra otro patógeno respiratorio ( ) . las muestras para el diagnóstico por rt pcr se recolecta de las vías respiratorias superiores, nasofaringe, cornete medio u orofaringe; todos con alta especificidad. sin embargo, se sugiere recolectar los hisopos nasofaríngeos o de cornete medio por tener mayor sensibilidad ( ( / ) . en pacientes con neumonía severa a quienes se le realizó lavado broncoalveolar (bal) y rt-pcr entre los días y el % de las muestras fueron positivos, en pacientes no intubados con esputo no inducido el % de las muestras fueron positivas ( , ). wang et al, en un estudio de pacientes con covid- , las rt-pcr con tasas positivas más altas fue en muestras extraídas por bal ( %; de muestras) y esputo % ( de muestras) ( ) . para la detección del sars-cov- por rt-pcr en pacientes en cuidado intensivo, teniendo en cuenta la rigurosidad de aspectos de bioseguridad y aerosolización, se debe tomar la primera muestras en nasofaringe o cornete medio, si esta prueba es negativa se puede repetir en a horas, si este es el caso o existe más de días desde el inicio de los síntomas se prefiere una muestra del tracto respiratorio inferior, por esputo no inducido por personal de salud o por aspirado traqueal en pacientes intubados ( , ), aunque el rendimiento diagnóstico del bal es alto por lo general, se debe evitar la broncoscopia para minimizar la exposición de los trabajadores de la salud ( ) . la probabilidad de detección del arn de sars-cov- puede variar según la fase de la enfermedad, si bien una rt-pcr positiva confirma el diagnóstico de covid- , los reportes falsos negativos y la sensibilidad se ve influenciado por el tiempo desde la exposición e inicio de síntomas. kucirka et al, en un análisis de siete estudios evaluaron el rendimiento diagnóstico de la rt-pcr en relación con el tiempo desde el inicio de los síntomas o la exposición, con resultados expresados en tasa estimada de falsos negativos, siendo del % el día de la exposición, del % el día (estimado como primer día de síntomas, ic: % a %), % en el día (día desde el inicio de síntomas, ic: % a %) luego comenzó a aumentar nuevamente de % en el día (ic: a %) a % en el día (ic: a %) ( ) . la precisión y los valores predictivos de rt-pcr para sars-cov- no se han evaluado sistemáticamente, la sensibilidad de las pruebas moleculares está influenciada por múltiples factores como sitio y calidad de la muestra, técnica de procesamiento; probablemente las menores tasas de falsos negativos (sensibilidad entre y %) está entre el día y luego de inicio de los síntomas ( ) . a partir de aquí el rendimiento diagnóstico disminuye, por lo tanto, es importante que el intensivista valore estas consideraciones en el momento de tomar conductas, en cuanto tipo de aislamiento, tratamiento y pronóstico. amci ® las pruebas serológicas detectan anticuerpos contra el sars-cov- y ayudan a identificar pacientes que han tenido la enfermedad y algunos con la enfermedad activa, la seroconversión se ha descrito entre el día y , sin embargo, hay incertidumbre en la incidencia de la seroconversión ( ) . estas pruebas se usan principalmente en tamizaje poblacional y estudios de seroprevalencia; en cuidado intensivo el análisis de la igm por elisa contribuye a la detección de pacientes con infección reciente, además con el análisis conjunto con la igg se clasifica el estado de infección en agudo o convaleciente. las pruebas serológicas se realizan por diferentes técnicas como la inmunocromatográfica de flujo lateral, la inmunofluorescencia indirecta (ifi) y el ensayo de inmunoadsorción ligado a enzima (elisa) ( , ) . las pruebas serológicas no deben usarse como la única prueba para diagnosticar o excluir la infección activa por sars-cov- . la sensibilidad y la especificidad de muchas de estas pruebas serológicas son inciertas, así como su valor predictivo positivo. los anticuerpos detectables generalmente tardan varios días en desarrollarse. guo et al, documenta niveles de anticuerpos por elisa, con una mediana de detección de anticuerpos igm e iga de días (iqr, - ) y de igg de días (iqr, - ) después del inicio de los síntomas, con una probabilidad de resultados positivos de . %, . % y . % respectivamente; es probable que el rendimiento diagnóstico de igm por elisa sea mayor que la de rt-pcr después del quinto día luego de inicio de síntomas; cuando se combinan estas técnicas (elisa igm con rt-pcr) la tasa de detección positiva es del . % ( ) . zaho et al, en un estudio de pacientes con covid- , donde el , % estaba en condición crítica, la mediana del tiempo desde el inicio de los síntomas hasta la detección de anticuerpos (técnica elisa) fue de días para igm y días para igg; dentro de los primeros días desde el inicio de los síntomas solo el . % tenía anticuerpos detectables, entre los días a la sensibilidad de igm fue . % e igg de . %, luego de los días la sensibilidad igm e igg fue de . % y . % respectivamente; el uso combinado de rt-pcr y elisa igm presentó una sensibilidad del % entre los días a y del % entre los días a . ( ) la rt-pcr tiene especificidad del %, con adecuado rendimiento diagnóstico entre los días y luego del inicio de los síntomas con sensibilidad que varía entre el y %, con presencia ascendente de falsos negativos luego del día , por lo cual el diagnóstico debe tener consistencias epidemiológicas y clínicas (síntomas y hallazgos radiológicos compatibles con covid- ) donde una rt pcr negativa no excluye la enfermedad; la precisión y el tiempo para la detección de anticuerpos varían con la técnica utilizada, su uso es limitado en cuidado intensivo, sin embargo su identificación por técnica elisa en conjunto con rt-pcr mejora la sensibilidad y la probabilidad de falsos negativos, especialmente entre los días y desde el inicio de síntomas. faltan estudios que evalúen el rendimiento diagnóstico de las diferentes pruebas. amci ® se recomienda la medición de marcadores de severidad al ingreso a uci del paciente críticamente enfermo por covid- (hemograma, transaminasas, ldh, ferritina, troponina, dímero d y pcr) los cuales se han asociado con peor pronóstico en la enfermedad por covid- , logrando ofrecer intervenciones más tempranas. se recomienda no utilizar una periodicidad de rutina para la medición de seguimiento de biomarcadores de severidad en el paciente con sospecha o diagnóstico de covid- . en un estudio cohorte retrospectivo que evaluó pacientes diagnosticados con covid- desde el de enero de hasta el de marzo de , fang liu y colaboradores encontraron correlación en la elevación de il- y pcr con la gravedad clínica, lo que sugiere podrían usarse como factores independientes para predecir la severidad del cuadro, los pacientes con il- > . pg./ml o pcr> . mg/l tenían más probabilidades de tener complicaciones graves ( ) , así mismo en otro estudio multicéntrico retrospectivo de pacientes infectados se identificó resultados de laboratorio con diferencias significativas con elevación de glóbulos blancos, valores absolutos de linfocitos, plaquetas, albúmina, bilirrubinas, función renal, transaminasas, troponina, proteína c reactiva e interleucina (il ) en el grupo con desenlace de mortalidad contra los dados de alta ( ) . entre otros marcadores la troponina como lesión cardiaca (elevación de troponina por encima del percentil límite de referencia superior) se ha reportado en % a % de los pacientes con covid- en wuhan, china, en dos estudios retrospectivos por xiabo yang y colaboradores ( , ) . en una revisión sistemática de mayo , kermali m. y colaboradores exponen que existe evidencia a favor de los valores bajos de linfocitos y plaquetas y valores elevados de los biomarcadores il- , pcr, troponina, ldh, ferritina, proteína amiloidea a y dímero d, pueden relacionarse con la gravedad de la infección por covid- y su fuerte asociación con la mortalidad ( ) . estos resultados pueden usarse como un complemento en la práctica clínica para guiar a los médicos a identificar pacientes con mal pronóstico y la rápida implementación de medidas de soporte, monitorización y reanimación en la evolución de los pacientes críticos en la unidad de cuidados intensivos. solo en estudio, karmali et al, en , determinan en promedio entre a horas, la periodicidad en el seguimiento de estos, sin embargo, no se discrimina entre pacientes críticos y no críticos. consideramos que el seguimiento de estas pruebas debe estar ajustado al juicio clínico del médico intensivista tratante, según la evolución de los pacientes. se muestran el comportamiento de los biomarcadores mas frecuentes en la tabla . tabla . biomarcadores en pacientes críticos con sospecha o diagnóstico de covid- . tendencia de biomarcador en relación con la gravedad covid- pcr aumentada recomendaciÓn se recomienda la no medición de marcadores de inflamación o de severidad de forma rutinaria solo con el objetivo de iniciar un tratamiento específico o algoritmos terapéuticos en la enfermedad por covid- en pacientes críticos. el síndrome de liberación de citocinas o denominado "tormenta de citocinas" parece asociarse en pacientes con afecciones graves por covid- . la citocina proinflamatoria il- es la citocina mejor documentada en covid- correlacionada con la gravedad, el estado crítico del paciente, la carga viral y el pronóstico ( , , ) . se han descrito mayores niveles de citoquinas proinflamatorias (il- , il- , il- , factor estimulante de colonias de granulocitos, factor de necrosis tumoral e interferón gamma) asociadas a compromiso pulmonar severo en pacientes con infección por coronavirus, determinado por la rápida replicación del virus, infiltración masiva de células inflamatorias y trastorno severo de la inflamación ( , ) . igualmente, está asociada la presencia de linfopenia como biomarcador de mal pronóstico para covid- ( ) . hallazgos similares se encontraron en la pandemia de influenza a (h n ) de sin ser especificó su valor ( ) . las manifestaciones clínicas de la tormenta de citocinas incluyen síndrome de respuesta inflamatoria sistémica, hipotensión, síndrome de fuga capilar, insuficiencia renal, sdra, miocarditis, entre otras ( ) , algunos autores han determinado este cuadro como un síndrome de linfohistiocitosis hemofagocitica secundaria. es razonable pensar que, en pacientes con sospecha de tormenta de citocinas basado en los hallazgos de laboratorio, el manejo con inmunomoduladores puede resultar beneficioso, sin embargo, los resultados del manejo de la hiperinflación basado en pruebas diagnósticas han tenido resultados encontrados en pacientes con covid- . el uso de esteroides, inmunoglobulina endovenosa, inhibidores del receptor de citoquinas (tocilizumab) o inhibidores de janus kinasa, han disminuido los valores de los biomarcadores, días de hospitalización ( ) o necesidad de fracciones elevadas de oxígeno ( ) , sin embargo no han demostrado beneficio sobre la mortalidad y en algunos casos, si un aumento en la incidencia de infecciones bacterianas o fúngicas sobreagregadas ( ) . se recomienda realizar radiografía simple de tórax para todos los pacientes con sospecha o diagnóstico de covid- en uci. se recomienda realizar tac de tórax según disponibilidad de tecnología institucional, ante la incertidumbre diagnostica, teniendo en cuenta las condiciones clínicas, la tolerancia del paciente al traslado y los protocolos administrativos de seguridad. fuerte a favor fundamento se reconoce como el gold estándar diagnóstico de infección pulmonar por covid- a los estudios moleculares, sin embargo, estos presentan limitaciones: a) contaminación de las muestras b) errores en la técnica de la toma, c) muestra insuficiente para ampliación genética favoreciendo falsos negativos d) demora de reporte de los resultados. por lo anterior, se recomienda imágenes diagnósticas en la aproximación de paciente con sospechas de infección pulmonar por covid- ( , ) .  radiografía simple de tórax: ventajas: mayor accesibilidad que la tac de tórax y realización de la prueba a la cabecera del enfermo desventaja: baja sensibilidad en estadio temprano, después del día de inicio de los síntomas presenta aumento en el rendimiento diagnóstico  tac de tórax: ventaja: es ampliamente recomendada. alta sensibilidad en estadios tempranos. permite describir extensión, distribución, localización, densidades parenquimatosas, aplicables en clasificaciones y puntajes diagnósticos, pronósticos y de seguimiento en permanente evolución y mejoría. algunas asociaciones referentes como asociación china de radiología, en su recomendación de expertos propone clasificación tomografía en estadios temprana avanzada severo disipación desventaja: traslado del paciente hasta el tomógrafo (no todos los enfermos toleran el transporte) y tecnología no disponible en todos los niveles de atención page no se puede emitir una recomendación a favor o en contra para el uso de ecografía pulmonar a la cabecera del paciente crítico como herramienta diagnóstica o de pronóstico en con covid- . se puede considerar como una alternativa para la valoración imagenológica pulmonar en el paciente crítico con covid- cuando las condiciones del paciente no permitan su traslado. no se sugiere la utilización de la ecografía pulmonar para el seguimiento de lesiones pulmonares agudas en el enfoque del paciente crítico con covid- . puede utilizarse para determinar complicaciones asociadas a la enfermedad o en la inserción de dispositivos invasivos. en general los estudios de imágenes no representan un papel concluyente para el diagnóstico de covid- . la ultrasonografía en específico requiere estudios de validación, un programa de entrenamiento es operador dependiente, y se le atribuido limitaciones en la capacidad de discriminación en la cronicidad de las lesiones pulmonares. la ultrasonografía pulmonar puede servir como herramienta a la cabecera del paciente para mejorar la evaluación del compromiso pulmonar y reducir el uso de radiografías de tórax y tomografía computarizada ( ) , sin embargo no debe usarse para el diagnóstico inicial, pues éste se compone de criterios clínicos, radiográficos y microbiológicos que actualmente son el estándar de oro; la ecografía no los reemplaza debido a la baja especificidad en relación con el virus, se sugiere su uso como complemento en la valoración diaria del paciente, ojalá realizada por el mismo observador. la ecografía pulmonar es altamente sensible y puede revisar de forma rápida y precisa la condición pulmonar, creando un potencial para evaluar los cambios o la resolución con el tiempo, especialmente en la uci, escenario en el que cada vez se usa más para la detección de múltiples patologías pulmonares que se pueden demostrar junto con covid- , sin embargo, hasta la fecha no hay hallazgos específicos, ni patognomónicos que se relacionen con covid- en el examen ecográfico del paciente ( ) . la adopción de ultrasonido pulmonar puede reducir la necesidad de exposición a la radiación ionizante y, a su vez, reducir la cantidad de radiografías necesarias para la evaluación rutinaria del paciente, disminuyendo también la exposición de personal asistencial adicional como el uso de elementos de protección personal ( ) . es bien conocido el beneficio de la ecografía durante y después de la colocación de accesos venosos centrales para establecer la presencia o no de complicaciones inmediatas como neumotórax. a la fecha no hay publicaciones acerca de la utilidad del ultrasonido como herramienta para establecer pronóstico. se necesitan más estudios para evaluar la utilidad de la ecografía pulmonar en el diagnóstico y manejo de covid- ( ) . se recomienda no establecer un punto de corte en el valor de dímero d para el inicio rutinario de anticoagulación plena en el contexto de infección por covid- . se recomienda la administración de profilaxis antitrombótica según protocolo institucional independiente de niveles de dímero d en el paciente críticamente enfermo por covid- . los fenómenos inflamatorios inherentes a procesos infecciosos son considerados desde décadas previas, factores protrombóticos, no siendo una excepción la infección por covid- . en algunas publicaciones se hallan asociaciones con desenlaces cardiovasculares negativos ( ) y sugieren asociación entre niveles elevados de dímero d ( marcador de estado de trombosis ) y riesgo de embolismo pulmonar con or crecientes or de . a los días hasta . a los días de seguimiento ( ) . de igual manera, zhou et al, reportan asociación de dímero d mayor a mcg/ml y mortalidad ( ) sin embargo los estudios presentan limitaciones en su diseño, a pesar de ello algunos autores proponen anticoagulación como factor de protección en mortalidad sin precisar precisión en la dosis, tipo de heparina y selección de enfermos ( ) . finalmente la european heart journal en su entrega de farmacología cardiovascular desarrolla una propuesta en la cual combina un puntaje previo de riesgo de cid en uci a niveles de fibrinógeno; esta pudiera ser una herramienta para selección de pacientes a recibir anticoagulación sin embargo aún está en proceso de validación ( ) . por estos motivos, hasta el momento, no se tiene suficiente cuerpo de evidencia que permita hacer una recomendación basado en los niveles de dímero d como variable aislada para administración de anticoagulación terapéutica. se recomienda no utilizar de rutina la procalcitonina en un algoritmo diagnóstico, para diferenciar entre neumonía viral vs bacteriana o confirmar la presencia de una sobreinfección bacteriana en el paciente con sospecha o diagnóstico de covid- . se recomienda no medir de forma rutinaria la procalcitonina en pacientes con sospecha o diagnóstico de covid- como factor pronóstico. la procalcitonina es un biomarcador que ha sido incluido en algoritmos de diagnóstico y pronóstico durante los últimos años. schuetz et al, en , concluyen en una revisión sistemática de estudios, que la procalcitonina es segura dentro de un algoritmo, para guiar a los médicos tratantes entre iniciar o suspender antibióticos en neumonía adquirida en la comunidad; sin embargo en una revisión sistemática más reciente, kamat et al, reportaron una sensibilidad % [ic % %- %; i = . %], especificidad % [ % ic, %- %; i = , %]) para el inicio de tratamiento antibiótico para neumonía bacteriana, lo cual nos determina que la prueba es inespecífica para diferenciar entre infecciones virales vs bacterianas. en neumonía por sars-cov- , se han publicado algunos artículos evaluando el uso de la procalcitonina como prueba asociada al pronóstico de los pacientes. liu et al encontraron que la procalcitonina se asoció a mayor severidad de los cuadros de neumonía. hr, . ; % ci, . - . ; p= . . en este estudio también se tuvo un resultado similar con la proteína c reactiva y la il- . plebani et al, publican un metaanálisis, donde sugiere que los niveles elevados de procalcitonina se asocian a mayor severidad de la infección. (or, . ; % ci, . - . ). es importante mencionar, que en estos estudios se evaluaron otros biomarcadores de inflamación como interleucina , proteína c reactiva y ferritina; presentando todos ellos, aumento en sus valores y asociándose a severidad de la enfermedad; por tanto, se considera que, en particular, la procalcitonina elevada, no representa una diferencia en el pronóstico, comparado con otros biomarcadores de inflamación. hasta el momento no se han publicado estudios en infección por sars-cov- donde se evalué el papel de prueba diagnóstica para confirmar sobreinfección bacteriana o diferenciar entre neumonía viral vs bacteriana. ( , ( ) ( ) ( ) se recomienda no usar de forma rutinaria el uso de pruebas clínicas de laboratorios clínicos para determinar la resolución de la enfermedad crítica por covid- . se recomienda considerar la ausencia de dificultad respiratoria y fiebre por más de horas, requerimiento de oxígeno a baja concentración y bajo flujo, como indicadores clínicos de resolución de la fase crítica de la enfermedad por covid- . se recomienda no utilizar de forma rutinaria el uso de pruebas microbiológicas de erradicación viral, para determinar la resolución de la enfermedad en pacientes en uci con covid- . amci ® enfermedad está asociado tanto a la carga viral como a la respuesta hiperinflamatoria del huésped a la infección viral. en cuanto a la carga viral, en pacientes que tienen un curso leve de infección, el pico de la carga viral en muestras nasales y orofaríngeas ocurre durante los primeros - días tras el inicio de síntomas y prácticamente desaparece al día , mientras que en los que cursan con neumonía severa en uci, la carga viral es veces mayor y puede persistir la excreción viral hasta el día a ( ) . por esta razón, consideramos que en los pacientes en uci no es necesario confirmar la erradicación del virus o su negativización en muestra respiratorias, orina o heces, para determinar la mejoría clínica, curación o para el egreso del paciente crítico ( ) . en el contexto clínico, el pronóstico se ha asociado a la presencia de marcadores bioquímicos elevados, sin embargo, no existe evidencia que el seguimiento con estos marcadores iniciales de inflamación determine el momento exacto de la resolución de la enfermedad. varias organizaciones internacionales como el cdc de usa y el european centre for disease prevention and control ( mar ) ( , ), national centre for infectious diseases (ncid) singapore ( ), world health organization ( de marzo de )( ), han establecido criterios para resolución clínica y egreso hospitalario de los pacientes. estos criterios incluyen: ausencia de fiebre mayor a horas sin antipiréticos, mejoría de los síntomas respiratorios, ausencia de requerimiento de hospitalización por otras patologías, el resultado de dos ( ) rt-pcr para sars-cov- negativas, con intervalo de muestra mayor a horas. la utilidad de dichos criterios no ha sido evaluada en pacientes en cuidado intensivo. en uci, huang et al ( ) , describieron en pacientes a quienes se dio egreso, la ausencia de fiebre por días, mejoría radiológica y evidencia de erradicación viral, como criterios de alta. sin embargo, consideramos que deben primar los criterios clínicos sobre los paraclínicos, en el momento de definir el egreso de un paciente de cuidado intensivo, teniendo como principal indicador la ausencia de dificultad respiratoria y la mejoría en los índices de oxigenación, con requerimiento de oxígeno suplementario a bajos flujos y concentración ( ) . se recomienda la cánula de alto flujo, donde esté disponible, en pacientes con covid- a nivel del mar con hipoxemia leve (pao /fio < y > o sao /fio < y> ). en alturas superiores a los mts por encima del nivel del mar esta terapia se puede considerar en pacientes que no tengan hipoxemia severa (pafi< ). amci ® se recomienda en pacientes críticos por covid- el uso de la cánula de alto flujo en salas de presión negativa, donde estén disponibles, que garanticen la seguridad del recurso humano. si no se dispone de habitación con presión negativa se puede optar por habitación individual cerrada. se debe contar con todo el equipo de protección personal necesario para el personal sanitario y de apoyo. se recomienda colocar mascarilla quirúrgica por encima de la cánula nasal en el paciente con sospecha o diagnóstico de covid- y mantener una distancia mínima de metros con otros pacientes. se recomienda la intubación inmediata en pacientes críticamente enfermos con sospecha o diagnóstico covid- con índice de rox ([spo /fio ] / frecuencia respiratoria) < a las horas de iniciada la oxigenación con cánula de alto flujo teniendo en cuenta que el retraso en la intubación aumenta la mortalidad. se recomienda considerar la cánula de alto flujo en caso de agotamiento de ventiladores mecánicos. la cánula de alto flujo ofrece flujos de hasta litros/minuto, que aportan una fracción inspirada de oxígeno (fio ) constante que reduce el espacio muerto y produce una presión positiva que genera reclutamiento alveolar y puede redistribuir el líquido alveolar ( ) ( ) ( ) . se ha reportado que al generar aerosoles, aumenta el riesgo de contagio para el personal de salud ( , ( ) ( ) ( ) . hasta el punto de que se ha recomendado su uso en salas con presión negativa ( ) . recientes publicaciones han establecido que la cánula de alto flujo genera una corta distancia de dispersión de aerosoles con lo cual las medidas de distanciamiento, un adecuado equipo de protección personal y donde estén disponibles, realizarla en salas de presión negativa darían seguridad al uso de la cánula de alto flujo ( , ) . a pesar de que la experiencia en la actual pandemia ha sido escasa, basada en series de casos, estudios retrospectivos y de metodología limitada, ha resultado ser promisoria en cuanto a la mejoría en la oxigenación y la tolerancia por el paciente ( , , , , , ( ) ( ) ( ) ( ) ( ) . sin embargo, hasta el momento no se ha establecido que la cánula de alto flujo evite la intubación. la caf podría convertirse en un alto riesgo de mortalidad al prolongar la decisión de intubación y al favorecer la lesión pulmonar autoinducida (p-sili) por aumento del esfuerzo inspiratorio. por todo ello es necesario un estricto y estrecho monitoreo del paciente durante una o dos horas para definir si ha habido mejoría o no mientras se realizan estudios que demuestren que evita la intubación y genera desenlaces clínicos importantes como menor estancia en uci, menor estancia hospitalaria y menos días de ventilación mecánica. la terapia de oxígeno de caf podría ser considerada para pacientes atendidos en altitudes mayor a mts, que no tienen hipoxemia severa (pafi < ), la respuesta debe evaluar dentro de los a minutos posteriores a su inicio y los pacientes que no mejoran amci ® significativamente y progresa la dificultad respiratoria no deben mantenerse con esta terapia. el monitoreo del paciente con caf para la toma de decisión de éxito o fracaso de esta estrategia y considerar la posibilidad de intubación incluye la evaluación gasométrica, la oximetría de pulso, así como considerar los criterios para intubación: frecuencia respiratoria > por minuto, deterioro de conciencia, inestabilidad hemodinámica, pao /fio < (a nivel del mar), sao /fio < , índice de rox< , , spo < % ( , - , - , , ) . se recomienda crear o ajustar protocolos institucionales de sedoanalgesia basado en objetivos con escalas validadas. se recomienda el uso de analgesia multimodal que incluya analgésicos opioides, no opioides y bloqueos regionales en el paciente crítico por sospecha o diagnóstico de covid- . se recomienda sedación profunda con agentes como midazolam o propofol para mantener rass entre - y - en pacientes covid- con sdra severo, necesidad de uso de relajantes neuromusculares o posición prona. se puede considerar en planos de sedación superficial agentes como propofol o dexmedetomidina (coadyuvante) para mantener rass entre y - en pacientes seleccionados con sospecha o diagnóstico de covid- con sdra no severo. en la actualidad no se encuentra evidencia de alta calidad proveniente de ensayos clínicos, sino editoriales, series, reportes de casos y artículos de revisión de expertos ( ) ( ) ( ) . la creación y aplicación de protocolos de sedoanalgesia adaptados a cada institución ha mostrado disminución del tiempo en la uci y menores complicaciones ( , ) . es importante definir objetivos guiados por escalas, recomendándose sedación profunda o completa en situaciones especiales como ventilación mecánica invasiva por sdra severo, disincronía ventilatoria persistente, posición prona y bloqueo neuromuscular (bnm), como puede observarse en pacientes covid- con compromiso pulmonar severo. mientras que se debe procurar sedación ligera, cooperativa o no sedación en pacientes en ventilación mecánica invasiva en pacientes con sdra no severo, ventilación no invasiva y en el retiro de la ventilación ( , ) . los opioides han sido el pilar de la analgesia en dolor moderado a severo. el fentanilo es actualmente el más usado; el remifentanilo reduce el tiempo en ventilación en pacientes amci ® renales, hepáticos, ancianos y neurológicos; la hidromorfona se prefiere en el retiro de la ventilación y en pacientes extubados; y la metadona ha mostrado disminuir la tolerancia a opioides ( ) . se propone el uso de estrategias de analgesia multimodal asociando medicamentos no opioides como el paracetamol, ketamina a dosis analgésicas (< , mg/kg) en dolor somático, lidocaína en dolor visceral, y pregabalina en dolor neuropático ( ) . la sedación ligera o cooperativa son mejores con propofol en cuanto a tiempo de despertar y con dexmedetomidina para preservar funciones cognitivas y el impulso respiratorio, con menor desacondicionamiento ( ) . en sedación profunda, el propofol ha mostrado más fácil titulación y menos acumulación que el midazolam; sin embargo, su uso se ve limitado hasta horas y a dosis < , mg/kg/h, ante el riesgo de pris (síndrome relacionado con la infusión de propofol). el midazolam, sin dosis techo ni tiempo límite y de bajo costo, ha sido el más utilizado de los sedantes, disminuyendo su uso por su asociación con delirium y de retraso en los tiempos en ventilación; sin embargo, la pandemia covid- ha vuelto a aumentar su uso. la dexmedetomidina ha sido utilizada como adyuvante en sedación profunda, disminuyendo el consumo de sedantes, con menos efectos secundarios ( , ) . se muestran los medicamentos para sedoanalgesia y relajación neuromuscular que se pueden utilizar en los pacientes con covid- en la tabla . se recomienda iniciar una estrategia individualizada de ventilación mecánica ajustadas a las condiciones específicas de severidad en el paciente crítico con covid- . amci ® se recomienda la ventilación protectora en modos controlados por volumen o por presión que garanticen un volumen corriente < cc/kg de peso predicho teniendo como metas una presión meseta < cm h o y una presión de conducción < cm h o. se recomienda emplear fracciones inspiradas de oxígeno para lograr metas de saturación de acuerdo con la pao /fio entre y % en el paciente con sospecha o diagnóstico de covid- . en la paciente embarazada entre y %. se recomienda en el paciente crítico por covid- iniciar con peep individualizado a la severidad del compromiso pulmonar y ajustar el nivel de peep de acuerdo con la tabla de fio /peep. el estudio arma ( ) demostró que la ventilación con bajos volúmenes corrientes (vt) se asocia con reducción de: mortalidad (p= . ), en días libres de ventilación mecánica (p= . ) y días libres de falla orgánica (p= . ). una revisión sistemática posterior confirmó que el uso de bajos volúmenes se asocia con menor progresión a sdra ( ) . un metaanálisis que revisó estudios y meta-análisis en uci confirmó que la ventilación protectora era una de las tres intervenciones que mejora la sobrevida en pacientes con sdra ( ) . esto fue confirmado por landoni en un análisis de estudios multicéntricos con impacto en mortalidad en uci ( ) . recientes publicaciones han sugerido que en covid- puede haber dos fenotipos que se diferencian en la distensibilidad ( , ) . sin embargo, el mismo estudio arma demostró que "el beneficio de ventilación con vt más bajo fue independiente de la distensibilidad de las vías respiratorias, lo que sugiere que el vt más bajo fue ventajoso independientemente de la distensibilidad pulmonar". más aún, el uso de bajos vt se asoció con una reducción en las concentraciones de interleuquina lo cual explicaría el mayor número de días sin falla orgánica múltiple y sugeriría una reducida respuesta inflamatoria asociada a la ventilación protectora ( ) . con el tiempo la evidencia ha demostrado que la ventilación protectora, además de vt y presión meseta bajos, debe incluir presiones de conducción menores de cm h o ( ) ( ) ( ) . existe suficiente evidencia que demuestra que fio y pao altas se asocian con aumento en la morbimortalidad ( ) ( ) ( ) . en sdra el estudio arma y una más reciente publicación demostraron que el tener metas conservadoras de pao se asocia con mayor sobrevida ( , ) . hasta el momento, la literatura en ventilación mecánica ha demostrado que la mejor estrategia para ajustar el nivel de peep en sdra es la tabla de fio /peep ( , ) . amci ® se recomienda la ventilación mecánica protectora en sdra por covid- independiente del fenotipo de presentación. se recomienda la clasificación de fenotipos en sdra para pacientes con covid- para ajustar la toma de decisiones de manera individualizada en ventilación mecánica. el manejo ventilatorio en covid- tiene los mismos principios generales de los pacientes con sdra ( ) . sin embargo, la identificación de fenotipos podría impactar en la evolución y pronóstico ( ) . gattinoni ha postulado el desarrollo de un sdra típico (fenotipo h: con alta elastancia, alto cortocircuito, alto peso pulmonar) o una presentación atípica (fenotipo l: caracterizado por baja elastancia, bajo shunt, bajo peso pulmonar). ( ) . pelosi et al sugiere clasificar los pacientes con tres fenotipos similares ( ) . con base en tales fenotipos se han propuesto estrategias ventilatorias diferenciales para minimizar el daño inducido por el ventilador (vili) ( ): . el primer paso, en el fenotipo l, es revertir la hipoxemia aumentando la fio . . en el tipo l, hay varias opciones no invasivas: cánula nasal de flujo alto, presión positiva continua en la vía aérea (cpap) o ventilación no invasiva (niv). se debe evaluar el esfuerzo inspiratorio y, de estar disponible, medir la presión esofágica. la peep alta puede disminuir los cambios de presión pleural y detener el ciclo vicioso que exacerba la lesión pulmonar. sin embargo, la peep alta, en pacientes con distensibilidad normal, puede tener efectos hemodinámicos perjudiciales. en cualquier caso, las opciones no invasivas son cuestionables, ya que pueden asociarse con altas tasas de fracaso y retraso de la intubación. . la magnitud de las presiones pleurales inspiratorias puede determinar la transición del tipo l al h. la presión esofágica > cmh o aumenta el riesgo de vili y, por lo tanto, la intubación debe realizarse lo antes posible. los tipo l, si son hipercápnicos, pueden ser ventilados con volúmenes > ml / kg (hasta ml / kg). la posición prona debe ser usada solo en último caso, ya que las condiciones pulmonares son buenas. la peep debe reducirse a - cm h o, dado que la capacidad de reclutamiento es baja y el riesgo de falla hemodinámica aumenta. la intubación puede evitar la transición al fenotipo tipo h. . los pacientes tipo h deben ser tratados como sdra grave, incluyendo mayor peep, si es compatible con la hemodinamia, posición en pronación y soporte extracorpóreo. en el fenotipo se deben aplicar las estrategias de ventilación protectora convencional ( , ) . amci ® se recomienda en paciente con covid- considerar hipoxemia refractaria cuando no se obtienen las metas de oxígeno propuestas, a pesar de las maniobras ventilatorias recomendadas y cumple con los siguientes parámetros: pafi < , fio > , y peep apropiado, considerando la altitud. se recomienda considerar la utilización de ecmo, en sitios donde esté disponible y con alta experiencia para obtener resultados aceptables, en pacientes con hipoxemia refractaria luego de haber implementado ventilación protectora, relajación neuromuscular y posición prona. se recomienda administrar tromboprofilaxis en todos los pacientes con covid- con hipoxemia refractaria que no presenten contraindicaciones. la hipoxemia refractaria no es un concepto estático y absoluto, según la definición de berlÍn del sdra, se clasifica el sdra en leve, moderado y severo de acuerdo con la relación pao y fracción inspirada de oxígeno, con peep mayor de cms de h o. la hipoxemia severa es aquella que cuenta con una pafi menor de ( ) ( ) ( ) . la hipoxemia refractaria hace referencia a un estado de hipoxemia severa que a pesar de las diferentes estrategias ventilatorias no aumenta la pafi y tiene consecuencias en el estado ácido básico y metabolismo celular permitiendo una anaerobiosis ( , ) . para definir la hipoxemia refractaria deben coincidir varios escenarios , una pafi menor de , una fracción inspirada de oxígeno mayor de . , a pesar de un peep apropiado no se tiene en cuenta en la definición el ph ni la paco ni el tiempo transcurrido ( ) . algunas de las medidas terapéuticas no ventilatorias que se han empleado en sdra y covid- con hipoxemia refractarias son la oxigenación con membrana extracorpórea (ecmo) y la tromboprofilaxis o anticoagulación de rutina. la oxigenación extracorpórea a través de una membrana ha sido una estrategia controvertida en pacientes con hipoxemia refractaria de diferentes orígenes, en la epidemia de la influenza por el virus h n fue usada en pacientes con hipoxemia refractaria teniendo resultados aceptables ( ) . en el estudio eolia los resultados no mostraron mejoría en la supervivencia, aunque hay diferentes posiciones y estudios post hoc de este ensayo clínico con beneficios, su uso se limita a casos muy restringidos y en sitios de alta experiencia para obtener resultados aceptables ( ) . en sdra por covid- el ecmo se ha usado en hipoxemia refractaria entre un a . % en diferentes series con resultados variables ( , , ) . un tipo de pacientes hipoxémicos y con ventilación mecánica han presentado cuadros tromboembólicos pulmonares en estos casos la trombólisis de rescate con activador de plasminógeno tisular rtpa (alteplase) se ha recomendado con resultados alentadores en serie de casos, pero su evidencia es muy débil para ser recomendada( - ). dada la alta frecuencia de enfermedad tromboembólica reportada en covid- se ha reportado la utilidad de la tromboprofilaxis, especialmente en casos de dímero d o índice de sic elevado ( ) . amci ® se recomienda monitorizar sistemáticamente la oxigenación con los índices: pao /fio y sao /fio , y en donde esté disponible el monitoreo continuo con capnografía. se recomienda monitorizar de forma rutinaria la presión meseta y la presión de conducción como estrategia al pie de la cama para verificar la ventilación protectora. el sdra y covid- , es una condición dinámica que apenas se está caracterizando, hay varias presentaciones que no cumplen con todos los criterios de berlÍn ( , ) . gattinoni ha caracterizado en dos presentaciones el sdra en los pacientes con neumonía por coronavirus sars-cov- , una con alta compliance, mínima reclutabilidad; la otra con baja compliance, pulmones pesados y reclutabilidad, tal vez esta presentación sea el verdadero sdra ( , , ) . los pacientes que requieren ventilación mecánica por falla ventilatoria en covid- , son los que mayor mortalidad tienen al parecer por la lesión pulmonar inducida por la intubación tardía y el gran esfuerzo respiratorio con presiones transpulmonares oscilantes y muy negativas ( , , ) . la monitoria de estos pacientes soportados con ventilación mecánica tiene dos objetivos: el primero detectar el deterioro clínico para sugerir estrategias más avanzadas como el ecmo, y el segundo es evitar el daño pulmonar inducido por la ventilación mecánica. se debe tener presente la mayor posibilidad de contagio con el número de manipulaciones en el paciente, por esto nunca olvidar el perfecto uso de los elementos de protección personal y disminuir el número de contacto con el paciente. las metas que se buscan con la ventilación mecánica en el paciente con sdra por covid- son mantener una oxigenación adecuada teniendo en cuenta la altura sobre el nivel del mar con pao entre y mmh y metas de saturaciones reportadas entre y % a y % , mantener una ventilación adecuada evitando el espacio muerto , disminuir el trabajo respiratorio y protegiendo el pulmón del daño ocasionado por la ventilación mecánica y las repercusiones hemodinámicas ( ) .  el confort de los pacientes en ventilación mecánica es la principal señal de un uso adecuado del modo ventilatorio y los parámetros ventilatorios apropiados para la patología y demanda del paciente ( ) .  las curvas y bucles son herramientas indispensables para valorar la mecánica respiratoria del paciente soportado con ventilación mecánica, se puede diagnosticar amci ® las asincronías del paciente y el ventilador, el origen, tipo y frecuencia además de la respuesta al manejo. también se evalúa la resistencia de la vía aérea ( ) . variables fisiológicas:  es importante valorar la oxigenación del paciente, la literatura actual sugiere el monitoreo de la pafi es el más representativo y sencillo test para valorar la oxigenación y representa el shunt pulmonar, se debe hacer mínimo diariamente, o cuando se haga una intervención en el ventilador o paciente; en sdra por covid- la hipoxemia se relaciona directamente con mortalidad, debe mejorar con la ventilación mecánica ( , ) .  la medición de la paco indica la de la ventilación, la hipercapnia tiene relación directa con el espacio muerto en el paciente con sdra, y varios estudios la relacionan con la mortalidad. puede evaluarse directamente en los gases arteriales o relacionarla con el pco expirado por medios de la capnografía, gattinoni propone una forma de evaluarla al lado de la cama del paciente relacionando el etco /paco , cuando es < de sugiere un shunt elevado y mayor espacio muerto; áreas de pulmón ventiladas y no aireadas. otras tecnologías incluidas en el ventilador moderno como la capnografía volumétrica se está validando para evaluar el espacio muerto, la reclutabilidad y la titulación de peep ( , ) .  la saturación venosa mixta svo , refleja de manera subrogada la función ventricular, no todos los pacientes tienen catéter de arteria pulmonar para su medición por lo que se está reemplazando con el ultrasonido en la cabecera del paciente; recordar que el % de los pacientes con sdra cursan con falla ventricular derecha ( ) . monitoria de mecánica ventilatoria y protección pulmonar:  para evitar el daño pulmonar debe propender por un volumen corriente bajo ( - ml/kg de peso predicho) y presión plateau menor de cms h , para garantizar la ventilación con protección pulmonar ( , ) .  driving pressure ( presión cambiante de la vía aérea, presión diferencial o presión de conducción) es la presión plateau (presión pico en ventilación controlada por presión) menos peep, debe ser menor de cm h , está relacionado con aumento en la mortalidad en pacientes ventilados por que representa una medición indirecta del strain pulmonar porque relaciona el volumen corriente con la compliance del sistema respiratorio y este a su vez se relaciona con el volumen espiratorio pulmonar final ( ) .  la medición de la compliance del sistema respiratorio es necesaria y nos clasifica el paciente de acuerdo con su fenotipo para trazar el plan de manejo ventilatorio, cuando la compliance es baja, esto se puede hacer al lado de la cama del paciente con los ventiladores modernos ( , ) .  la construcción de la curva presión/volumen aún es una herramienta útil para ubicar el área de ventilación segura del paciente, evitando el atelectrauma y la sobredistensión pulmonar(estrés) , el peep se calcula dos puntos por encima del punto de inflexión inferior y el punto de inflexión superior nos indica hasta dónde podemos aumentar el volumen corriente este punto debe estar por debajo de cms h para evitar la sobre distensión, con los ventiladores modernos se puede construir esta curva ( , ) . amci ®  las curvas presión tiempo en pacientes ventilados con modos volumétricos pueden monitorizar la resistencia de la vía aérea, la compliance pulmonar, el trabajo respiratorio, las curvas de flujo puede también indicar si se presenta autopeep, resistencia aumentada de la vía aérea entre otras ( ) .  presión transpulmonar, en casos más complicados donde es más difícil obtener la meta de oxigenación a pesar del peep en aumento una opción es el catéter esofágico, para medir la presión transpulmonar en la inspiración y espiración y calcular así el stress pulmonar y evitar las presiones oscilatorias y sobre todo negativas para evitar el daño pulmonar. esta herramienta también ayuda a evaluar el trabajo respiratorio, y el diagnóstico de las asincronías que se presenten en el paciente ventilado ( , ) . se recomienda no utilizar de forma rutinaria la relajación neuromuscular en el paciente crítico con covid- con sdra. se recomienda utilizar la relajación neuromuscular en pacientes en posición supino o prono, que están fuera de parámetros de protección pulmonar (presión de conducción mayor y presión plateau mayor a ) con pafi menor y cuando ya no es posible limitar el volumen corriente. se debe considerar la utilización de protocolos estandarizados con el fin de disminuir la variabilidad, y según disponibilidad seleccionar el cisatracurio como primera opción, en caso de escasez se pueden utilizar otras opciones teniendo en cuenta su farmacodinamia y farmacocinética. el de marzo de , la organización mundial de la salud emite una serie de orientaciones para el manejo de la infección respiratoria aguda grave (irag) en pacientes con sospecha o diagnóstico de covid- . en el paciente críticamente enfermo con sdra moderado o grave (pao /fio < ) por covid- no está indicado de forma sistemática el bloqueo neuromuscular mediante infusión continua debido a que no se cuenta con evidencia suficiente que sustente mejoría en la supervivencia con respecto a una estrategia de sedación ligera sin bloqueo neuromuscular, se debe considerar su uso cuando se evidencia asincronía paciente-ventilador a pesar de la sedación, hasta el punto de que no se pueda limitar el volumen corriente de forma fiable, hipoxemia o hipercapnia que no mejoran con el tratamiento ( ) . recomendaciÓn se recomienda no utilizar de forma rutinaria oni en pacientes adultos que presenten sdra e infección por sars-cov- . fuerte en contra fundamento a la fecha (mayo de ) no contamos con estudios sobre el uso de óxido nítrico inhalado (oni) como tratamiento de pacientes con infección covid- . existe evidencia indirecta sobre el uso de oni en el síndrome de dificultad respiratoria aguda (sars-cov), y la infección por coronavirus en el síndrome respiratorio de oriente medio (mers-cov) ( ) . en cochrane realizó una revisión sistemática que incluyó ensayos de calidad moderada con pacientes adultos con sdra tratados con óxido nítrico inhalado. los resultados no mostraron ningún efecto estadísticamente significativo sobre la mortalidad (rr . , % ci . - . ). se mostró mejora transitoria en el índice de oxigenación a las horas (md (diferencia media) - , , ic del % - , a - , ) y mejoría en pao /fio a las horas (md , , ic del % , a , ). no se identificó diferencia significativa en los días libres de ventilación y finalmente se presentó aumento estadísticamente significativo en la incidencia de insuficiencia renal en pacientes con óxido nítrico inhalado (rr , , ic del % , )( ). amci ® en se realizó un estudio observacional que incluyó pacientes tratados de dos hospitales de beijing con oni como tratamiento para sars ( ) . en comparación con ningún tratamiento, oni mejoró la saturación arterial de oxígeno (spo ) de % a % (p< . ); se asoció a menor necesidad de oxígeno suplementario (p< . ) y menor necesidad y retiro de cpap y bial (p < . ). los cambios en radiografía de tórax mejoraron en de los pacientes que recibieron oni. sin embargo, debido a problemas graves de validez por pequeño tamaño de la muestra (n= , oni= , control= ), no aleatorización y no enmascaramiento en la asignación, se considera que este estudio cuenta con baja calidad metodológica, lo cual limita la interpretación de los resultados. en un estudio retrospectivo multicéntrico que incluyó pacientes con mers-cov en condición crítica en arabia saudita, se mostró que el manejo con ventilación no invasiva (niv) tenía mayor probabilidad de requerir óxido nítrico en comparación con los pacientes con ventilación mecánica invasiva ( , % vs , %, p a , ) ( ) . en una serie de casos en la que participaron pacientes con infección por mers-cov confirmada o probable, pacientes recibieron oni debido a hipoxemia refractaria. en el seguimiento a días, cinco de los pacientes continuaron vivos ( ) . los estudios sobre mers-cov se limitaron a una serie de casos y una cohorte retrospectiva con baja calidad de evidencia. en ambos estudios, los pacientes recibieron otras terapias de rescate (relajación neuromuscular, ventilación oscilatoria de alta frecuencia, ecmo y posición en prono), por lo tanto, se desconoce el efecto terapéutico clínico del oni en el tratamiento de la infección por mers-cov. a la fecha ( de mayo de ) tres ensayos clínicos evalúan el papel del óxido nítrico inhalado en pacientes con covid- y sdra leve/moderado, y como profilaxis para los trabajadores de la salud covid- (tabla ). tabla . comparación de ensayos clínicos que evalúan el papel del óxido nítrico inhalado en pacientes con covid- y sdra leve/moderado, y como profilaxis para los trabajadores de la salud covid- . no se cuenta con evidencia por el momento que respalde el uso de óxido nítrico inhalado en pacientes con covid- . los resultados de los ensayos en curso, así como ensayos clínicos de alta calidad son necesarios para apoyar su uso. sólo evidencia indirecta metodológicamente limitada de óxido nítrico en pacientes con sras mostró una mejor oxigenación, una menor necesidad de oxígeno suplementario y mejoría en la radiografía de tórax. en pacientes con sdra y mers-cov, no mostró un beneficio claro e incluso mostró un mayor riesgo de insuficiencia renal ( ) . otros estudios han evaluado el efecto tóxico asociado a su uso documentando metahemoglobinemia( ), inhibición de la agregación plaquetaria y formación de dióxido de nitrógeno ( ) . razones por la cuales guías recientemente publicada no recomienda su uso de forma rutinaria( ). recomendaciÓn se recomienda el uso temprano de la ventilación en posición prona, por al menos horas continuas, en pacientes con sdra por covid- con pao /fio < mmhg. la ventilación en posición prono como estrategia ventilatoria propuesta desde los años ( ), cuenta con evidencia que demuestra resultados positivos en cuanto a mejoría de mortalidad, mejoría en el trastorno de oxigenación y el reclutamiento alveolar en pacientes con sdra. los mecanismos por los cuales la posición prona conduce a la mejoría en el trastorno de oxigenación y del reclutamiento alveolar en los pacientes con sdra, incluyen ( ) ( ) ( ) ( ) : amci ®  mejoría de la relación ventilación/perfusión y mayor homogeneidad en la distribución de aire en los pulmones.  aumento del volumen de fin de espiración.  disminución del efecto compresivo del corazón en los pulmones.  mejoría del drenaje de las secreciones.  optimización del reclutamiento alveolar, con mejoría de la distribución del volumen corriente, a su vez, limita el desarrollo del daño alveolar pulmonar. los estudios coinciden en el efecto benéfico que esta terapia tiene en la mejoría de la oxigenación, el objetivo se centrará en evaluar las recomendaciones con respecto a:  beneficio de la terapia con respecto a la mortalidad al día , al día y a los meses.  beneficio de la terapia según el grado de severidad de sdra con respecto la relación pao /fio .  tiempo de terapia en posición prono con mayor beneficio.  número de sesiones de la terapia en posición prono.  recomendaciones según balance riesgo/beneficio en lo que respecta a los efectos adversos: retiro o desplazamiento no planeado de catéteres, obstrucción de tubo endotraqueal, neumonía asociada a ventilador, lesiones de presión. se eligieron artículos que con las características metodológicas consistentes ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . de estos ocho artículos se excluyó uno por corresponder a pacientes pediátricos ( ) . al evaluar los escritos de forma cronológica, se puede apreciar en los primeros artículos ( , ) , la dificultad para lograr enrolar (reclutar) el suficiente número de pacientes, de tal manera que algunos estudios fueron detenidos de forma prematura ( , , , ) . era entonces esperable que los resultados no fueran concluyentes, y que no lograran ser robustos, al no alcanzar el tamaño de muestra deseado, comprometiéndose la confiabilidad, el poder y corriendo el riesgo de obtener resultados falsamente negativos. sin embargo, se podía percibir en los diferentes estudios, una notoria mejoría en la oxigenación, sin repercusión en la mortalidad ( ) ( ) ( ) ( ) ( ) ( ) . por otro lado, es necesario tener en cuenta que los estudios iniciales ( ) ( ) ( ) ( ) no se realizaron con el uso de ventilación protectora asociada a la pronación. esto es un elemento pertinente, pues la ventilación protectora puede per se, brindar un efecto adicional en la mejoría de la oxigenación. otro efecto importante que debe analizarse es el bloqueo neuromuscular, cuya evidencia hoy sugiere su uso en criterios ya mencionados ( ) . con el tiempo y según la experiencia de cada centro, se fue utilizando con menor dificultad ( ) . sin embargo, no se tenía aún claro qué tipo de pacientes obtenían el mayor beneficio de la terapia, además que el tiempo de la terapia en posición prona, seguía siendo una incógnita. la evidencia reflejaba hasta el momento que los pacientes con sdra más severos y la terapia aplicada por más tiempo se asociaban con una tendencia a la reducción en mortalidad. para dirimir qué tipo de pacientes se beneficiarían más de la terapia, se tomó como base, la severidad del sdra según la relación pao /fio ( ) . a pesar de que los estudios iniciales( - ) enfrentaban el reducido tamaño de muestra, algunos análisis identifican beneficio de la mortalidad ( ) en pacientes con sdra más severo, constituyendo un probable umbral de beneficio cercano y por debajo de - mmhg ( ) . amci ® antes del estudio proseva ( ) en totalidad estudios reportan mortalidad al día ( , ) , un estudio mortalidad al día ( ) , uno estudio mortalidad al día ( )y uno mortalidad al día ( ) . sin embargo, al tomar todos los estudios, aún no se alcanzaba suficiente significancia estadística para reducir la mortalidad a los días y meses. con el estudio proseva -guerin et als -en el año ( ) , se alcanza más poder estadístico, demostrando un beneficio importante en pacientes con pao /fio menor de mmhg, ventilados con bloqueo neuromuscular y ventilación protectora en su totalidad, y logrando una reducción de la mortalidad al día y mantenida hasta el día con respecto al grupo control ( . % ( de participantes) versus . % ( de ) (p< . ). de tal manera que, según la evidencia disponible, en lo que al trastorno de oxigenación se refiere (según la relación pao /fio ), sugiere que el mejor candidato para esta estrategia ventilatoria es el paciente con sdra severo con una pao /fio menor de mmhg. es de primordial importancia detenerse a considerar por otra parte, el número de horas que se implementaría la terapia. el estudio inicial realizado por gattinoni et al. ( ) , llevó a los pacientes a un período corto de horas, sin encontrar resultados positivos en mortalidad. (resultados que no se pueden solamente atribuir a la calidad del estudio, sino también, al reducido número de horas de la ventilación en posición prono). mancebo et als (la abreviación latina para "otros "es et al.), y fernández et al. ( , ) por su parte, optan por ventilar un mayor número de horas ( - horas) obteniendo resultados (que sugieren una reducción en mortalidad) con tendencia a disminuir mortalidad. si bien diferentes metaanálisis sugieren no (la ausencia de beneficio) beneficio de la ventilación en prono ( , ) , los resultados son diferentes cuando se aborda la terapia con períodos mayores a horas ( ) . teniendo en cuenta, que el estudio con más poder estadístico ( ) postula horas de terapia en posición prono, la recomendación es la pronación por un tiempo mayor a horas, contemplando hasta las horas por sesión. otra pregunta que surge con frecuencia es el número de veces que se puede implementar la terapia. existen diferentes estudios en los cuales se indica el número de veces en promedio en que se llevó el paciente a posición prono ( , ) , otro protocolo en donde se estipula un número límite de días en los que se llevó a cabo la terapia( ), y otro estudios en los cuales no se precisó el número de sesiones de la terapia ( , ) . se podría entonces recomendar con respecto a la mayor evidencia disponible ( ) , pronar a los pacientes en varias sesiones ( en promedio) (depende de los criterios para continuar o suspender el prono que deben ser individualizados para cada paciente, en cada zona geográfica y en cada unidad de cuidados intensivos, es difícil saber si es el promedio para todos), y considerarla más veces si es necesario. en lo que respecta a los efectos adversos, tres estudios reportaron barotrauma y neumonía asociada a ventilador ( , , ) ; dos estudios desplazamiento de catéter central ( , ) y siete estudios reportaron extubación ( ) ( ) ( ) ( ) ( ) ( ) ( ) . sin embargo, los efectos adversos no alcanzaron significancia estadística para proscribir la terapia ( ) . recientemente fue publicado el consenso colombiano de sdra( ) en el cual se hace referencia a las recomendaciones para realización de ventilación prono. se presenta la lista de chequeo, y medidas que deben ser realizadas en la maniobra para lo cual se cuenta con la participación de terapia respiratoria, enfermería y médico. se recomienda implementar un protocolo de retiro de ventilación mecánica basado en la prueba de respiración espontánea y articulado con un protocolo de sedación y analgesia en el paciente críticamente enfermo por sospecha o diagnóstico de covid- . desde diciembre de , un número de casos de neumonía por síndrome respiratorio agudo severo sars-cov /covid- en wuhan china se identificaron, como causa de insuficiencia respiratoria aguda( ). el síndrome de dificultad respiratoria aguda (sdra) ocurre en el % de los pacientes hospitalizados y en el % de los pacientes admitidos a la unidad de cuidados intensivos (uci) en wuhan ( ) . mientras la ventilación mecánica es una intervención que potencialmente salva la vida, esta puede llevar a múltiples complicaciones y contribuir a la lesión pulmonar ( ) . es por todo esto que el retraso en el retiro de la ventilación mecánica puede aumentar el riesgo de amci ® infecciones, aumenta la sedación innecesaria, el trauma de la vía aérea y aumento en el costo de la atención de estos pacientes ( ) . el retiro de la ventilación mecánica es un proceso de tres pasos, el primero es conocido como preparación la cual depende de variables fisiológicas, criterios clínicos y predictores de weaning (destete), el segundo paso es el propio weaning, el cual consiste en la disminución del soporte ventilatorio entregado al paciente, con el objetivo de llevar al último paso que es la extubación( ), ilustración . por todo lo anterior, se recomienda realizar un proceso de retiro de ventilación mecánica invasiva adoptando un protocolo, seleccionando adecuadamente a los pacientes, ya que evidencia sugiere que la adecuada selección de los pacientes disminuye los días de ventilación mecánica, disminuye la estancia hospitalaria y la estancia en uci ( ) . se recomienda realizar el retiro de la ventilación mecánica siguiendo los pasos antes mencionados, iniciando con la preparación, la cual consiste en: preparación . asegurarse que la lesión pulmonar que llevó a la falla respiratoria esté resuelta. . adecuado intercambio de gas, definido como adecuados índices de oxigenación con peep (presión positiva al final de la expiración) a cmh o, y fio (fracción inspirada de oxígeno) < . . al igual que el proceso de intubación, es un proceso que genera aerosoles por lo cual se recomienda la aplicación de lidocaína dosis de , a , mg por kilogramo de peso, a minutos antes la extubación con el objetivo de disminuir el reflejo de tos ( ) y la exposición del personal de salud. se recomienda en el paciente con sospecha o diagnóstico de covid- a quien se considera realizar vmni, utilizarla en salas de presión negativa, donde estén disponibles, que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. se recomienda considerar la vmni en pacientes con covid- con hipoxemia leve (pao /fio < y > o sao /fio < y> ) en salas de presión negativas, donde estén disponibles que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. fuerte a favor se recomienda considerar la vmni en pacientes con covid- con hipoxemia leve (pao /fio < y > o sao /fio < y> ) y con historia de epoc o cuadro de edema pulmonar agudo asociado en salas de presión negativas, donde estén disponibles que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. se recomienda colocar doble filtro en el circuito del ventilador para reducir el riesgo de generación de aerosoles en vmni del paciente crítico con sospecha o diagnóstico de covid- . se recomienda la intubación inmediata en pacientes críticamente enfermos por covid- si se evidencia respiración toraco-abdominal, uso de músculos accesorios, frecuencia respiratoria > , hipoxemia (pao /fio < o sao /fio < ), fracaso ventilatorio (ph< . con paco > mmhg), hacor> . existe suficiente evidencia que demuestra que la vmni es una estrategia que reduce la mortalidad en pacientes críticos ( , , ) . además, reduce la necesidad de intubación. los números de casos necesarios a tratar (nnt) son: ocho para salvar una vida y para evitar una intubación ( ) . estos desenlaces son fundamentalmente en pacientes con epoc y edema pulmonar agudo ( ) ( ) ( ) ; también hay evidencia a favor, aunque menos fuerte, en pacientes inmunosuprimidos ( , ) . se ha planteado mayores tasas de éxito con interfaces faciales totales o con helmet ( , ( ) ( ) ( ) ( ) ( ) . por el contrario, en falla respiratoria hipoxémica hay evidencia en contra del uso de vmni ( ) . la experiencia previa con h n , sars y mers no apoya el uso de la vmni en falla respiratoria hipoxémica de origen viral ( ) ( ) ( ) ( ) ( ) ( ) . además, se ha cuestionado el uso de vmni en covid- por el riesgo de contagio al generar aerosoles ( ) . recientemente se demostró que la distancia de dispersión de aerosoles era menor de un metro ( ) . por otro lado, el fracaso de la vmni se ha asociado con alta morbimortalidad ( ) ( ) ( ) . ello obliga a evaluar estrictamente la posibilidad de éxito o fracaso. así, en falla respiratoria amci ® hipoxémica la escala hacor ha sido validada para este fin y un puntaje> contraindicaría la vmni ( ) . adicionalmente si se emplea la vmni el prolongar la decisión de intubación puede aumentar la mortalidad y es por ello necesario monitorizar estrictamente al paciente y evaluarlo para establecer, ojalá antes de dos horas, si el paciente responde a la vmni ( , , ) . las indicaciones para intubación en este caso son respiración toracoabdominal, uso de músculos accesorios, frecuencia respiratoria mayor de , hipoxemia (pao /fio < o sao /fio < ), fracaso ventilatorio (ph< . con paco > mmhg), hacor> o índice de rox (spo /fio )/frecuencia respiratoria) < ( , , , , , ) . recomendaciÓn se sugiere el uso de posición prono en pacientes no ventilados críticamente enfermos por covid- que no responden a la oxigenoterapia convencional de acuerdo con los protocolos institucionales, las condiciones de cada servicio y la tolerancia individual de cada paciente. débil a favor fundamento los pacientes con enfermedad por coronavirus (covid- ) están en riesgo de desarrollar un síndrome de dificultad respiratoria aguda (sdra) ( ) . en pacientes intubados con síndrome de dificultad respiratoria aguda grave, la posición prona (pp) temprana y prolongada (al menos horas diarias) mejora la oxigenación y disminuye la mortalidad ( , ) .debido a que las unidades de cuidados intensivos (uci) están sobrecargadas con pacientes con covid- , la pp en paciente despierto con respiración espontánea puede ser útil para mejorar la oxigenación y prevenir las transferencias hacia uci. un estudio describió la viabilidad del uso de la ventilación no invasiva y la cánula de alto flujo asociado a la pp estableciendo su tolerancia y su seguridad en pacientes con sdra moderado y severo( ); la pronación puede reclutar todas las regiones pulmonares y favorecer el drenaje de secreciones de la vía respiratoria, mejorando el intercambio gaseoso y la supervivencia en el síndrome de dificultad respiratoria (sdra) ( ) . en una comunicación corta proveniente de italia en el cual se incluyeron pacientes que son sometidos al pp asociada con el uso de ventilación no invasiva, se concluye que proporcionar niv en la posición prona a los pacientes con covid- y sdra en salas generales en un hospital en italia era factible. la frecuencia respiratoria disminuyó durante su implementación y la oxigenación mejoró durante una pronación posterior a su línea de base. si la intubación se evitó o se retrasó, queda por determinar ( ) . en otro reporte de caso, publicado recientemente por un grupo francés( ) de pacientes con covid- e insuficiencia respiratoria hipoxémica manejados fuera de la uci, el % amci ® fue capaz de tolerar pp durante más de horas. sin embargo, la oxigenación aumentó durante el pp solo un % y no se mantuvo en la mitad de los pacientes después del regreso a la posición supina. estos resultados son consistentes con los hallazgos de pequeños estudios previos de pp en pacientes no intubados ( ) . un ensayo clínico controlado que evalúe el uso del pp en pacientes no intubados puede ser un mecanismo para seleccionar pacientes que bien puedan beneficiarse de esta estrategia terapéutica. dada la evidencia débil que soporta el uso del pp en pacientes no intubados en términos de la disminución de la necesidad de entubación o ingreso a cuidados intensivos y la duda razonable de aumentar desenlaces deletéreos en aquellos pacientes en los cuales se retarde el tiempo de intubación, no se emite recomendación, a favor o en contra, del empleo de esta estrategia de manera rutinaria. en situaciones en las cuales hay limitación de recursos y de disponibilidad de camas en cuidados intensivos el uso de la pp asociada a vni o cánula de alto flujo podría ser una estrategia útil para mejorar la oxigenación en pacientes infectados con covid- e hipoxemia. se recomienda considerar la elevación de biomarcadores como la troponina i o t y el nt-pro-bnp en el paciente con covid- como indicadores de injuria miocárdica aguda, sin embargo, no reemplazan la ecocardiografía en el enfoque del paciente con sospecha de disfunción miocárdica. fuerte a favor fundamento la injuria miocárdica aguda asociada a covid- se reporta con frecuencia teniendo en cuenta los cambios en biomarcadores como la troponina y cambios electrocardiográficos, pero su impacto en la función cardíaca se desconoce y mucho menos su correlación con los cambios ecocardiográficos. los pacientes con covid- pueden desarrollar una serie de complicaciones cardiacas desde injuria miocárdica, arritmias, infarto, hasta miocarditis fulminante con falla cardiaca aguda y shock cardiogénico ( ) . la troponina i, se ha encontrado más elevada en pacientes con curso fatal por covid- ( ) . los niveles de nt-pro-bnp han sido reportados con elevación severa en pacientes con miocarditis y disfunción sistólica, con una disminución progresiva en relación a la mejoría de los pacientes, pero no parece tener un correlación significativa con el cambio de la fracción de eyección (fevi) ( ) . la evidencia clínica sugiere que la elevación de los biomarcadores es más relacionada al compromiso sistémico que el daño miocárdico directo, q. deng y colaboradores en un análisis retrospectivo de pacientes, reportaron niveles iniciales de troponinas normales casi en la mayoría, en el , % de los casos los niveles incrementaron significativamente, principalmente en los que fallecieron y solo pacientes tenían fevi menor al % y ninguno inferior al %, lo cual no sugiere una asociación entre las dos amci ® pruebas ( ) . en una publicación donde se compara el fenotipo de pacientes con covid- con un histórico de pacientes con sdra por influenza ( ) , se encontró que los primeros tenían mayor elevación de troponinas % vs %, pero en los parámetros ecocardiográficos contrario a lo que se esperaría los índices de rendimiento ventricular fueron mayores para el grupo de covid- : Índice cardiaco . vs . l/m/m ; fevi vs %; tapse vs mm, nuevamente aunque no fue uno de los objetivos del estudio, parece no encontrarse correlación entre los biomarcadores que sugieren injuria miocárdica y los parámetros ecocardiográficos, el cual constituye uno de los pilares de la exploración cardiaca. se recomienda considerar como marcadores iniciales de mal pronóstico en el paciente crítico con covid- con sospecha de disfunción miocárdica aguda la elevación persistente de troponina i, mioglobina o creatin kinasa; independiente de la fracción de eyección del ventrículo izquierdo evaluada mediante ecocardiografía. fundamento los pacientes con covid- admitidos a la unidad de cuidados intensivos presentan con frecuencia disfunción cardiaca primaria, que puede corresponder a cardiomiopatía por estrés o miocarditis viral, pero también pueden ser consecuencia del compromiso sistémico ( , ) . aunque parece que en los fenotipos cardiovasculares estudiados, el compromiso hemodinámico severo de la función sistólica izquierda y derecha es menor ( ) . en el estudio de deng y colaboradores con pacientes con covid- , la fiebre, la disnea, hipoxemia, la obesidad y niveles elevados de cpk, troponina y nt-pro-bnp se relacionaron significativamente con mayor severidad ( ) . en el subgrupo de pacientes con miocarditis frente a controles, el perfil clínico se describe con mayor edad, niveles de temperatura más elevados ( . ± . vs . ± . ; p: . ), mayor proporción de disnea ( , vs %), y de dolor torácico ( , vs , %). en los pacientes fallecidos el % tuvieron picos de elevación de troponina i y de nt-pro-bnp dentro la semana que precedió la muerte, el % presentaron alteraciones electrocardiográficas y solo el % presentaron fracción de eyección menor o igual al % ( ) . shi y colaboradores, enrolaron pacientes para describir el significado clínico del compromiso miocardio de pacientes con covid- en wuhan, pacientes fallecieron, de los cuales el % presentaron injuria miocárdica aguda ( ) . el área bajo la curva (auc) de la troponina i inicial para predecir muerte intrahospitalaria fue de , (ic %, de , - , ) con una sensibilidad y especificidad del %, el auc para mioglobina fue de . y para cpk-mb fue de , ( ). un punto de corte para el pico más alto de troponina i de . , tuvo un hazard ratio para mortalidad de . (ic %, . - . ; p= . ). en un análisis multivariado la edad avanzada, la respuesta inflamatoria y las enfermedades cardiovasculares subyacentes se asociaron con mayor riesgo de lesión miocárdica en pacientes con covid- . con la información disponible parece que los biomarcadores de lesión miocárdica aguda elevados amci ® al ingreso y de forma persistente pueden predecir el riesgo de mortalidad intrahospitalaria en los pacientes con sospecha o diagnóstico de covid- con afectación cardiovascular. se recomienda no realizar de forma rutinaria ecocardiografía en pacientes críticos con covid- . se debe practicar ecocardiografía en pacientes con sospecha o diagnóstico de covid- si presenta alguna de las siguientes condiciones: . síntomas y signos de insuficiencia cardíaca aguda de novo. . shock o deterioro súbito hemodinámico refractario a líquidos y/o vasoactivos con sospecha de origen cardiogénico. . sospecha de infarto agudo de miocardio o embolismo pulmonar para determinar intervenciones terapéuticas con un beneficio clínico. . cambios en el electrocardiograma, arritmias ventriculares o paro cardiorrespiratorio no explicados por otra causa. para nuestro conocimiento, en el momento no existe estudios clínicos que evalúen los criterios para realización de ecocardiograma en el paciente con covid- . las sociedades de ecocardiografía han recomendado realizar el ecocardiograma en el contexto clínico en el cual, la información obtenida proporcione un cambio en la conducta o se espere un beneficio clínico al realizar este procedimiento ( ) ( ) ( ) ( ) . igualmente, se recomienda realizar el examen a la cabecera del paciente y el escaneo debe ser dirigido a contestar preguntas específicas según el contexto clínico del paciente ( ) ( ) ( ) ( ) ( ) . ward et al, en su publicación describe cómo el uso del ecocardiograma limitado (dirigido) en la university of chicago medicine (ucm), aumentó significativamente durante la pandemia ( % frente a %, p < . ), posterior a la implementación de recomendaciones sobre el uso apropiado de la ecocardiografía en tiempos de pandemia ( ) . los pacientes con infección por sars -cov- pueden presentarse con comorbilidades cardiovasculares que potencialmente estén descompensadas y/o compromiso cardiovascular por covid- . en este último, podemos encontrar alguno de los siguientes fenotipos: falla cardiaca aguda en el marco de compromiso directo viral o secundario al estrés metabólico y liberación de citoquinas, síndrome coronario agudo, cor-pulmonar secundario a tep o por compromiso secundario al sdra ( , , , ) . las manifestaciones cardiovascular puede sospecharse en el marco de choque que no esté explicado por causas extracardiacas evidentes que no responde a líquidos, dolor torácico con clínica de síndrome coronario agudo, cambios electrocardiográficos y elevación de biomarcadores de lesión miocárdica, signos de falla cardiaca descompensada, deterioro súbito de la oxigenación, arritmias o paro cardiorrespiratorio ( , , ) . ante estas manifestaciones, el ecocardiograma podría ser útil para entender el origen de la descompensación aguda, al estar enfocado a amci ® responder preguntas acerca de la función ventricular global y segmentaria (en el abordaje de síndrome coronario agudo), compromiso del ventrículo derecho, alteraciones valvulares, derrame pericárdico, si existe una contribución cardiovascular al compromiso pulmonar, si en el marco del choque existe evidencia de componente cardiogénico y cómo podría guiarse/optimizarse el soporte hemodinámico de estos pacientes( - ). se sugiere en pacientes críticamente enfermos con covid- que cursan con shock y sdom, ajustar la monitoria a las condiciones clínicas del paciente y recursos disponibles. se puede considerar el cap para el monitoreo del gasto cardiaco, la valoración de la perfusión y orientar los elementos hemodinámicos del tipo de shock, el cap de gasto cardiaco continuo puede disminuir la exposición del personal de salud frente al catéter de medición convencional. se sugiere la utilización de la tdtp dependiendo de la disponibilidad del recurso para orientar el diagnóstico diferencial del sdra versus edema pulmonar cardiogénico en los pacientes con covid- . fundamento los pacientes con infección severa por sars-cov- , cursan con alto riesgo de falla renal y cardiovascular, con necesidad de un manejo restrictivo de líquidos, lo que justifica la monitoria estricta en uci ( , ) . el catéter venoso central es útil para la monitoria inicial de estos pacientes, sin embargo, su predicción a respuesta a volumen está limitada ( ) ( ) ( ) . la monitoría no invasiva tiene limitaciones en casos severos de inestabilidad hemodinámica, ventilación espontánea y en presencia de peep alto, lo que limita su uso para el cálculo de gasto cardiaco y la predicción de respuesta a líquidos ( , ) . la monitoria con cap puede ser considerada en pacientes con covid- que cursan con choque y doms, con el objetivo de realizar un diagnóstico definitivo de los componentes del choque, valorar la hipoperfusión, la función cardiaca y el estado de volemia ( , ) . igualmente, los pacientes con sospecha tep o compromiso del ventrículo derecho pueden beneficiarse de esta monitoria( ). richard et al, en su estudio determinaron los desenlaces asociados al uso de cap vs cvc en pacientes con shock, sdra, o ambos, sin evidenciar diferencias en mortalidad ( . % vs . % p =. ) o estancia hospitalaria. el uso de cap no garantiza la mejoría de desenlaces en pacientes con covid- , sin embargo, la presencia de una monitoria continua ayudaría a optimizar los recursos y disminuiría la interacción con el paciente, con menor exposición del equipo médico ( ) . la monitoria por tdtp puede utilizarse en pacientes con covid- que cursan con choque, buscando optimizar el manejo hídrico, valorar el agua extravascular pulmonar (evlw) y el índice de permeabilidad vascular pulmonar (pvpi) con el fin de establecer el diagnóstico amci ® definitivo del edema pulmonar: sdra vs cardiogénico ( ) ( ) ( ) . hu et al, en su estudio evaluaron los desenlaces del uso de evlw y la presión de cuña de la arteria pulmonar (pawp) como estrategias para el manejo de líquidos en pacientes sdra, no encontraron diferencias significativas en las tasas de supervivencia (p = , ). no obstante, en el grupo de evlw la duración de la ventilación mecánica y la estancia en la uci fueron significativamente menor (p < , ), al igual que el balance hídrico (p < . ), con mejoría significativa en los índices de oxigenación (p = . )( ). no se puede emitir una recomendación a favor o en contra para la utilización de un protocolo de ultrasonido rutinario a la cabecera del paciente (pocus). sin embargo, se podría considerar el uso en pacientes seleccionados, con los adecuados epp y desinfección de los equipos; donde el pocus pueda tener ventajas sobre otras modalidades de monitoria o en pacientes con limitaciones para monitoria invasiva que requieren evaluación del estado hemodinámico o determinación de severidad del compromiso pulmonar. las recomendaciones sobre la utilidad de pocus en pacientes con covid- están enfocadas principalmente en la evaluación de la severidad/progresión de la lesión pulmonar, diagnóstico de manifestaciones cardiovasculares, monitoria hemodinámica y en la guía de fluidoterapia ( , ( ) ( ) ( ) . en la valoración del compromiso pulmonar por covid- , el pocus ofrece una ventaja sobre otras modalidades de monitoreo, debido a la capacidad de enmarcar el compromiso pulmonar en una línea de tiempo según sus hallazgos: desde la aparición de un patrón de "líneas b", consolidaciones subpleurales con evolución a consolidaciones multilobares, irregularidades en el artefacto de la línea pleural y finalmente aparición de patrón de "líneas a" una vez inicie la recuperación, con adecuada correlación tomográfica ( ) ( ) ( ) ( ) . en la diferenciación del origen del choque, el pocus ha demostrado superioridad versus el concepto clínico al evaluar: función ventricular (incluyendo ventrículo derecho), vena cava inferior, líquido libre abdominal, lesiones aórticas, compromiso pulmonar y búsqueda de trombosis venosa profunda identificando tep ( , ) . en cuanto a la monitoria hemodinámica de pacientes con covid- , adicional a la función y gasto cardíacos, se recomienda variables dinámicas de respuesta a líquidos y ultrasonido pulmonar en el diagnóstico de sobrecarga hídrica ( ) . la variabilidad de gasto cardiaco calculado por pocus durante la elevación pasiva de miembros inferiores, identifica los respondedores a líquidos con sensibilidad del % y un lr (-): . [ %ci, . - . ]) ( ) . la variabilidad de la vena cava inferior muestra limitaciones en pacientes con aumento de presiones de cavidades derechas y respiración espontánea ( ) . en cuanto a otras modalidades de monitoría no invasiva, es importante conocer las limitaciones en el marco de compromiso hemodinámico severo, ventilación espontánea, alteraciones valvulares aórticas, entre otras ( ) . marik ( ) . el ultrasonido tiene limitaciones específicas como la necesidad de un operador experimentado y la adecuada calidad de las imágenes. adicionalmente, en los pacientes con covid- , esta modalidad de monitoreo requiere una mayor interacción con el paciente, mayor uso de epp en comparación con otras modalidades el pac( ). se recomienda perseguir al inicio de la reanimación del paciente críticamente enfermo con covid- , metas clínicas de fácil medición, como la presión arterial media (entre y mmhg) o el gasto urinario (mayor a , cc/k/h) y metas de perfusión como el lactato en sangre arterial (menor a mmol), la saturación venosa central de oxígeno (entre y %) y la diferencia veno arterial de co (menor a mmhg). se han reportado casos de disfunción ventricular como causa de choque asociado a covid- , sin embargo, no se ha descrito un manejo específico o cambios en las metas de reanimación para estos pacientes, las manifestaciones de hipoperfusión tisular son: alteración de la conciencia, oliguria, piel fría y moteada y pulso débil. a nivel de gases sanguíneos las metas de reanimación pueden ser globales como el lactato (en sangre arterial vn< mmol) y la diferencia veno arterial de co (pv-aco vn < mmhg) o regionales como la saturación venosa central de oxígeno medida en la sangre venosa tomada de un catéter central (vn: - %). se puede optimizar la perfusión, interviniendo los principales determinantes: fluidos para aumentar el volumen intravascular, inotrópicos para aumentar la fuerza de contractilidad, vasopresores para recuperar la presión de perfusión y transfusión de glóbulos rojos para aumentar la hemoglobina como transportador de oxígeno ( ) , las recomendaciones dadas en el documento anterior siguen siendo válidas e incluyen entre otras que todo paciente con covid- en estado de shock debe ser ingresado de forma inmediata a la unidad de cuidados intensivos, garantizando el aislamiento indicado, procurando recuperar la presión arterial media a valores > mmhg, para ello la utilización de un catéter venoso central en los pacientes que no responden al manejo inicial y el procedimiento debe ser realizado por el médico con mayor entrenamiento, idealmente guiado por ecografía si hay disponibilidad y las competencias, así mismo las estrategia de control estricto de fluidos para no generar efectos deletéreos relacionados a la sobrecarga de volumen, es lo más indicado. se recomienda en la reanimación inicial de pacientes en estado de shock con sospecha o amci ® diagnóstico de covid- , guiar la fluidoterapia con el uso de índices clínicos como el tiempo de llenado capilar, temperatura de la piel y depuración de lactato; en fases avanzadas donde el monitoreo clínico es insuficiente utilizar medidas dinámicas como la variabilidad de presión de pulso (vpp), la variabilidad de volumen sistólico (vvs), la respuesta a la maniobra de elevación pasiva de piernas o la prueba de oclusión teleespiratoria de acuerdo a los recursos disponibles y la experiencia. fuerte a favor fundamento sobre los objetivos de intervención y el tipo de agente vasoactivo a utilizar en pacientes con covid- y shock no existe una evidencia directa y las recomendaciones se basarán en evidencia indirecta de pacientes críticos con diversos tipos de shock en especial el séptico y vasopléjico. la falla circulatoria aguda asociado a sdra en covid- se presenta con una frecuencia del - % y en la admisión a urgencias la hipotensión y un lactato ≥ es infrecuente( , ). la baja sensibilidad del qsofa y crb- para predecir la severidad del covid- y la necesidad de intervenciones de terapia intensiva refleja lo infrecuente del shock en esta condición. factores como la vasoplejia, fuga capilar asociada al estado hiperinflamatorio, altos requerimientos de peep y disfunción cardiaca pueden ser generadores o contribuyentes del shock y deben ser considerados en el abordaje diagnóstico, en su interpretación para lograr tomas de decisiones adecuadas. en pacientes con sdra, una reanimación óptima de líquidos debe tomar en cuenta aspectos como tiempo (oportunidad), tipo (cristaloides balanceados y/o no balanceados o coloides) y volumen (ni mucho, ni poco) con el objetivo de disminuir la mortalidad, el tiempo de vm y de cuidados intensivos, sin que ello afecte los índices de oxigenación, de perfusión tisular y la morbilidad asociada con su uso inadecuado. la administración agresiva de líquidos puede empeorar la oxigenación y la disfunción ventricular, lo que potencializa un mayor tiempo de ventilación mecánica e incluso la mortalidad. la evidencia ha demostrado que una estrategia conservadora de fluidos (balance - +/- amci ® en una revisión sistemática y un metaanálisis de rct (n = . ) una terapia dirigida al aclaramiento temprano de lactato frente a una terapia guiada por la saturación venosa central de oxígeno (svo ), se asoció con una reducción significativa de la mortalidad (rr , ), menor estadía en la uci (dm , días), y menor duración de la ventilación mecánica (dm - , horas). pero se debe resaltar que un nivel alto de lactato no siempre indica hipovolemia; también puede ser causada por uso de adrenalina, agonistas beta o por disfunción mitocondrial, insuficiencia hepática e isquemia mesentérica ( ) . por otra parte, el llenado capilar (crt), una prueba técnicamente fácil y accesible, realizada cada minutos se asoció con una reducción no significativa de la mortalidad (hr , ) en comparación con la medición de lactato sérico cada horas. dado el potencial beneficio sobre mortalidad, duración de estancia en uci y la duración de la ventilación mecánica, así como su accesibilidad, sugerimos utilizar parámetros dinámicos de temperatura de la piel, tiempo de llenado capilar y / o medición de lactato sobre parámetros estáticos para evaluar la capacidad de respuesta a la fluidoterapia en pacientes con covid- y shock( ). se recomienda en pacientes adultos con covid- y estado de shock, escoger la norepinefrina como el vasopresor de primera línea y a la vasopresina el de segunda. si no se cuenta con norepinefrina el uso de vasopresina o epinefrina serían la primera elección; la dopamina no se recomienda por el mayor riesgo de arritmias. se recomienda iniciar dobutamina frente al aumento de la dosis de norepinefrina en pacientes en estado de shock con evidencia de disfunción cardíaca e hipoperfusión persistente a pesar de la reanimación inicial. fuerte a favor fundamento sobre los objetivos de intervención y el tipo de agente vasoactivo a utilizar en pacientes con covid- y shock no existe una evidencia directa y las recomendaciones solo pueden basarse en evidencia indirecta de pacientes críticos con sepsis y sdra. en pacientes en shock séptico los agentes vasoactivos para alcanzar una pam de - es un objetivo razonable. una presión media más alta puede incrementar . veces el riesgo de arritmias cardiacas y no está exento de riesgo de isquemia en las extremidades ( ) . para aproximarnos a la escogencia de los vasoactivos en shock séptico, basados en su perfil de riesgo/beneficio, la guía scandinavian society of anaesthesiology and intensive care medicine (ssai ) task force for acute circulatory failure( ), la revisión sistemática de cochrane database con rct con un n: ( ) y el ensayo clínico controlado de honarmand k et al con un n: , ( ) amci ®  la noradrenalina es el agente vasoactivo más ampliamente estudiado con el menor riesgo a priori de efectos no deseados, razón por la cual se sugiere usar como el agente vasoactivo de primera línea en pacientes con covid- y shock.  si la noradrenalina no está disponible la vasopresina o epinefrina se muestran como la mejor alternativa. los factores que determinan la elección entre vasopresina y epinefrina pueden incluir disponibilidad y el perfil de seguridad de estos agentes. con la vasopresina, la isquemia digital puede ser una preocupación y con epinefrina, la taquicardia, la isquemia miocárdica y el exceso de producción de lactato.  el uso de dopamina se ha asociado un . veces mayor de riesgo de aparición de arritmias frente a la norepinefrina y un posible aumento del riesgo de mortalidad. por ello la dopamina no debe utilizarse en pacientes con covid- y shock donde haya disponible de norepinefrina o las alternativas señaladas  en shock distributivo la adición de vasopresina a las catecolaminas evidenció baja certeza de reducción de la mortalidad (rr , ; ic del %: , a , ), alta certeza de una reducción de la fibrilación auricular (rr , ; ic del %: , a , ) y certeza moderada de un mayor riesgo de isquemia digital (rr , ; ic del %: , a , ). en vista de estos hallazgos se plantea la vasopresina como un agente vasoactivo de segunda línea a ser utilizado si la pam objetivo no se ha alcanzado con norepinefrina en pacientes con covid- y shock. no existe evidencia directa en pacientes con covid- y shock, para establecer una recomendación sobre cuál es el agente inotrópico óptimo. en una guía de práctica clínica de que evalúa el agente inotrópico óptimo en pacientes con insuficiencia circulatoria aguda (shock), no se identificaron rct que comparen dobutamina versus placebo o ningún tratamiento. con base en una justificación fisiopatológica, sugerimos agregar dobutamina, más que no suministrar ningún tratamiento, en pacientes con covid- y shock con evidencia de disfunción cardíaca e hipoperfusión persistente a pesar de la reanimación con líquidos y altas dosis de norepinefrina. el uso de dobutamina en estado de shock, incluso en pacientes con covid- con shock, debe ser investigado. recomendaciÓn se recomienda no suspender la medicación estándar para falla cardiaca en pacientes con sospecha o diagnóstico de covid- , especialmente los iecas, ara-ii, y b-bloqueadores, si la condición clínica permite continuar el uso de esta medicación, ya que no se ha podido confirmar una asociación nociva. amci ® previos alrededor de la relación independiente de la edad avanzada, enfermedad cardiovascular subyacente (enfermedad coronaria, insuficiencia cardíaca y arritmias), tabaquismo activo y epoc con muerte por covid- ( ) ( ) ( ) ; esos mismos reportes sugieren que las mujeres son proporcionalmente más propensas a sobrevivir a la infección por covid- que los hombre; existen además consideraciones especiales desde el punto de vista cardiovascular, que se deben tener en cuenta al decidir cualquier terapia en paciente afectados por covid- y existe la hipótesis de un efecto nocivo de la terapia estándar para falla cardiaca en estos pacientes ( ) . en un estudio observacional que incluye paciente con infección por covid- , se evaluó la relación entre la enfermedad cardiovascular subyacente, y la asociación entre la terapia farmacológica cardiovascular y la mortalidad ( ); respecto a los factores de riesgo cardiovascular, el . % de los pacientes tenían hiperlipidemia, el . % tenía hipertensión, el . % tenía diabetes mellitus, y en relación a los medicamentos, los sobrevivientes usaron más comúnmente ieca y las estatinas que los no sobrevivientes, mientras que no se encontró asociación entre la supervivencia y el uso de ara ii; respecto a los otros medicamentos incluidos b-bloqueadores, antiplaquetarios e insulina no se encontraron diferencias significativas; sin embargo, y teniendo en cuenta el impacto sobre mortalidad del uso de betabloqueadores en la falla cardiaca, consideremos que la decisión de continuar su uso debe basarse en el análisis clínico de cada paciente y su estabilidad clínica. se recomienda establecer un protocolo de reanimación ajustado al contexto del paciente con covid- con una organización administrativa ajustada a la pandemia que incluya las siguientes estrategias:  desarrollar una estrategia de prevención del paro cardiorrespiratorio en el paciente con sospecha o diagnóstico de covid- basada en la detección oportuna.  formar un equipo multidisciplinar formado en rcp con líderes médicos, de en enfermería y en terapia respiratoria, los cuales deben educar a todo el equipo de trabajo en la identificación de signos de alerta temprana, cambios abruptos de variables clínicas, técnicas de monitoreo, interpretación de paraclínicos y de alarmas de monitoreo.  entrenamiento del personal sanitario a través del uso de la simulación clínica en manejo de crisis, epp, y procesos de atención fundamentales en la atención del paro cardiaco en el paciente con sospecha o diagnóstico de covid-  promover la comunicación asertiva, planeación y retroalimentación de las intervenciones realizadas antes y después de un evento de paro cardiaco (briefing y debriefing), con el fin de establecer modificaciones que conllevarán a mejoras en la atención de futuros eventos. amci ® el pronóstico y sobrevida de un paciente con sospecha o diagnóstico de covid- que presenta paro cardiaco depende de la prevención a través del reconocimiento oportuno de las causas reversibles de éste, la no presencialidad y un ritmo de paro no desfibrilable lo hacen de mal pronóstico. la prevención va a depender del nivel de entrenamiento del equipo previamente, y en época de pico de pandemia cuando el talento humano especializado disminuya, se hace necesario que en los equipos de trabajo estén liderados por especialistas en la disciplina para que puedan guiar al equipo. los nuevos procesos de atención del paciente, la alta contagiosidad del virus, y el uso de nuevos medicamentos hacen que se requiera un entrenamiento del personal para estandarizar los procesos y disminuir el error médico. la crisis de covid- está ejerciendo una presión sin precedentes sobre las personas, los equipos y los sistemas organizacionales, conllevando a errores médicos que van desde la infección cruzada por el personal sanitario con la posibilidad de cometer errores en la atención. cada día trae nuevos desafíos: picos en volumen y gravedad, escasez de equipos y estrés en los médicos sobrecargados, que se manifiesta según la experiencia de wuhan en insomnio y depresión. se propone implementar una estrategia antes, durante y después del trabajo clínico, denominada circle up covid- desarrollada por center of medical simulation dirigida a convertir equipos de trabajo muchas veces insustituibles , en eficientes , seguros, fuertes y que se apoyan mutuamente incluyendo en mejora de la salud psicológica . impactando en el rendimiento del equipo , y promoviendo el bienestar y la resiliencia( - ) se recomienda establecer un protocolo de reanimación ajustado al contexto clínico del paciente con covid- que incluya las siguientes modificaciones:  implementar criterios de selección e inicio de maniobras de rcp en la atención del paro cardíaco basados en la bioética y en el pronóstico de supervivencia a corto y largo plazo de los pacientes.  promover la prevención del paro cardiaco mediante la detección oportuna del riesgo y definir intubaciones programadas.  asegurar la correcta protección con los epp necesarios al abordar el paro cardiaco y la intubación que son procedimientos generadores de aerosoles.  priorizar el manejo de la vía aérea antes del inicio de las compresiones torácicas, haciendo énfasis en la reducción de la exposición de aerosoles (código azul protegido).  utilizar filtro de alta eficiencia contra virus para todas las estrategias de ventilación (bolsa mascarilla con cierre hermético y en el circuito del ventilador).  promover la realización de la intubación por el operador de mayor experticia con uso de videolaringoscopio si está disponible y considerar el acceso supraglótico solo si el intento de intubación es fallido. amci ®  en caso de parada cardiaca en ventilación mecánica iniciar el masaje cardiaco evitando las desconexiones del circuito del respirador.  en caso de paro en posición prono si el paciente se encuentra vigil retornar rápidamente a la posición supino y si está en ventilación mecánica es razonable realizar compresiones en la espalda. fuerte a favor fundamento se hace necesario el entrenamiento en el manejo de los procedimientos generadores de aerosoles y se sugiere que el que realiza la intubación orotraqueal debe ser el más experto. el riesgo de aerosolización es de . en el proceso de intubación orotraqueal, el de compresiones torácicas es de , , ventilación mecánica no invasiva de , , ventilación manual pre-intubación de , , succión después de intubación , . se ha descrito que si no se ha capacitado previamente en el retiro de los epp existe más riesgo de auto contaminación y aumenta ésta sin un líder supervisor al retirarlo. según revisión de cochrane sobre ropa y equipo de protección para los trabajadores sanitarios para evitar que se contagien con el coronavirus y otras enfermedades altamente infecciosas da a conocer que la capacitación presencial, la simulación por ordenador y la capacitación por vídeo dieron lugar a menos errores a la hora de quitarse el epp que la capacitación impartida solo como material escrito o una conferencia tradicional ( ) ( ) ( ) ( ) ( ) ( ) . se recomienda no considerar la existencia de manifestaciones neurológicas específicas o típicas atribuidas a la infección por sars-cov- . se recomienda realizar la valoración neurológica integral del paciente con diagnóstico o sospecha de sars-cov- teniendo en cuenta manifestaciones frecuentes relacionadas a covid- : disgeusia, anosmia, cefalea, vértigo, confusión, delirium, alteración de estado de consciencia, eventos cerebrovasculares, ataxia, polineuropatías inflamatorias y convulsiones. una revisión sistemática realizada por asadi-pooya et al. entre diciembre y marzo de mostró que el % de los pacientes con covid- presentaron sintomatología neurológica. analizaron cinco artículos (n= ) donde eran retrospectivos, y era prospectivo, encontrando cefalea entre el y el %, vértigo entre el y el %, confusión en el %, alteración del estado de consciencia en el %, eventos cerebrovasculares en un %, ataxia en un , % y convulsiones en un , % ( ) . amci ® menor a participantes donde recolectarán información sobre el compromiso neurológico en pacientes con covid- con un seguimiento hasta febrero de ( ) . la hiposmia y la disgeusia de aparición súbita son manifestaciones clínicas muy prevalentes en pacientes con covid- evidentes aun en ausencia de sintomatología respiratoria alta. lechien et al publicaron un estudio multicéntrico que incluyeron hospitales europeos reclutando pacientes infectados con covid- levemoderado donde , % presentaron alteraciones relacionadas con el olfato y , % presentaron alteraciones relacionadas con el gusto, donde la anosmia se presentaba antes que cualquier otro síntoma en el , % de los casos y el , % de los casos no presentaba rinorrea u obstrucción nasal, la recuperación del olfato fue presente en el % de los pacientes y las mujeres fue el grupo poblacional más afectado (p= , ) ( ) . otras manifestaciones clínicas son las alteraciones de la agudeza visual, y dolor tipo neuralgia ( ) . respecto a las patologías psiquiátricas; la serie de mao comenta que el , % de los pacientes tiene clínica de alteración del estado de consciencia, concepto que se aproxima a la defunción de delirium. severance et al. encontraron que pacientes con sintomatología psicótica aguda presentaron niveles elevados de inmunoglobulina g para coronavirus del tipo hku , nl y oc con diferencias estadísticamente significativas respecto a los individuos controles (n= ) (p< , ). donde la respuesta inmune para nl fue asociado con el espectro-esquizofrenia (or: . , ci . - . , p= . ) pero no se correlaciona con desórdenes afectivos ( ) . aún no se ha descrito una correlación directa de este trastorno psicótico con covid- . los síntomas musculares se han observado en pacientes infectados por covid- incluyendo la miopatía del paciente en estado crítico (miopatía difusa no necrotizante con degeneración grasa de fibras musculares), la miopatía necrotizante (ligada a falla orgánica múltiple) y la miopatía de filamentos gruesos( ). se recomienda considerar como predictores clínicos neurológicos de alerta para sospechar covid- : anosmia, disgeusia, delirium y alteraciones neuromusculares inespecíficas sin otra causa aparente de explicación. se recomienda no establecer de rutina predictores neurológicos específicos de mal pronóstico en el paciente críticamente enfermo con covid- . se deben tener en cuenta los factores de riesgos generales de mal pronóstico para la población general como la edad avanzada, las comorbilidades cardiovasculares y el tabaquismo. page la proteína spike (s) del covid- es reconocida por la enzima convertidora de antígenos (eca ) de la célula huésped cuyo papel es el punto de entrada molecular a tejidos pulmonares, gastrointestinales y neuronales ( ) . la forma como el covid- ingresa al sistema nervioso central es desconocido, pero se especula que inicialmente invade terminales nerviosas periféricas y después llega al sistema nervioso central a través de una ruta guiada por sinapsis nerviosas con un patrón ascendente (ruta dada por el coronavirus hev y el oc- ) ( , ) . en modelos de roedores el covid- ingresaría al cerebro a través del nervio olfatorio, atravesando la lámina cribiforme propagándose por el tálamo y el tallo cerebral; explicándose por la expresión de los receptores de la enzima convertidora de antígenos (eca ) en la superficie de las mucosas nasales, las neuronas y la glía ( , , ) . una segunda forma de ingreso es a través de la vía hematológica mediante arterias cerebrales atravesando la barrera hemato-encefálica utilizando las células inflamatorias como un modelo similar al del caballo de troya ( , , ) logrando una ubicación definitiva en células neuronales y endoteliales del lóbulo frontal como lo demostraron en estudios post-mortem descritos por paniz -mondolfi et al, lo que explicaría los cambios comportamentales de paciente ( ) . la tercera forma de acceso al sistema nervioso central es mediante el drenaje del sistema linfático cerebral invadiendo ganglios linfáticos hiliares y mesentéricos con sintomatología gastrointestinal asociada ( ) . una vez ha logrado ingresar tiene la capacidad de infectar macrófagos, microglía, y astroglía los cuales secretan factores proinflamatorios como interleuquina , interleuquina , y factor de necrosis tumoral alfa ( ) . esta condición se exacerba con el desencadenamiento de la tormenta de citoquinas liderada por la interleuquina , interleuquina , interleuquina e interferón gamma ( ) . de esta forma los coronavirus siendo neurotrópicos ocasionan múltiples manifestaciones clínicas ya mencionadas, así como encefalitis, parálisis flácida, incluyendo la asociación con guillain-barré ( ) . los síntomas que harían sospechar la presencia de neurocovid- son la náusea, el vómito y la anorexia, estos síntomas pueden ser el reflejo del compromiso del virus en el área postrema del piso del cuarto ventrículo que hace parte del complejo vagal dorsal de la médula oblonga. sin embargo, estos síntomas pueden enmascararse como una respuesta inespecífica relacionada con un compromiso gastrointestinal ( ) . como se mencionó previamente la anosmia y la disgeusia son síntomas significativos para sospechar en covid- ( ) ; esto es debido a una lesión directa sobre el nervio olfatorio (i par craneal), y la lesión de alguno de los tres nervios encargados de registrar el sentido del gusto como lo son el vii, ix y x pares craneales, así como el compromiso del núcleo solitario y del tálamo como zona de relevo; de hecho el núcleo del tracto solitario es muy cercano al centro respiratorio que podría ocasionar disnea de origen central ( ) , otros núcleos como el núcleo dorsal motor del vago y el núcleo ambiguo están relacionados con funciones cardiovasculares a tener en cuenta ( ) . respecto a los factores de riesgo destaca el tabaquismo el cual aumenta la posibilidad de neuroinfección debido a interacciones funcionales entre el receptor nicotínico de acetilcolina y el receptor eca el cual está sobreexpresado en pacientes fumadores ( ) . amci ® en pacientes con infecciones severas vs infecciones no severas ( . % vs . %, p = . ), incluyendo eventos cerebrovasculares ( , % vs , % p= , ), alteración del estado de consciencia ( . % vs . %; p< . ) y lesiones musculoesqueléticas ( , % vs , % p< ) ( ) . la encefalopatía que se manifiesta como una alteración aguda o subaguda del estado de consciencia presentándose en pacientes con comorbilidades, factores de riesgo cardiovasculares, edad avanzada y deterioro cognitivo previo ( , , ) . así como aquellos individuos con hipoxemia la cual induce metabolitos anaerobios en el sistema nervioso central, edema celular, intersticial e isquemia ( ) . los eventos cerebrovasculares pueden ser desencadenados por cuadros de hipoxia, inmovilización, un incremento de la respuesta proinflamatoria o por predisposición a la hipercoagulabilidad ( ) . respecto a este último rubro tanto la edad (hazard ratio , /por año % ic , - , ) y la coagulopatía definida como como un tiempo de protrombina mayor a segundos, o tiempo de tromboplastina mayor a segundos (hr , % ic , - , ) fueron considerados predictores independientes de complicaciones trombóticas ( ) ; otros trabajos reportan incremento del conteo plaquetario y niveles elevados de dímero d ( ) . de hecho, un scoping review realizado por wilson y jack muestra que la presencia de eventos cerebro vasculares es un factor de riesgo de mal pronóstico para pacientes infectados por covid- ( ) . mao et al. reportaron eventos cerebro vasculares en pacientes con covid- , los factores de riesgo más relevantes fueron los clásicos factores de riesgo cardiovasculares (diabetes, hipertensión y edad avanzada), así como una presentación sistémica severa, teniendo como un denominador común el compromiso estructural de grandes vasos ( ) . la presencia de convulsiones (clínicas o subclínicas) puede ser una manifestación de eventos cerebro vasculares, meningoencefalitis o hipoxia cerebral. siendo los factores de riesgo más importantes en su exacerbación las alteraciones electrolíticas como hipocalcemia, las reacciones adversas a medicamentos y la epilepsia como comorbilidad de base ( , ) . otros tipos de coronavirus como lo son el e, , y oc se han aislado de líquido cefalorraquídeo (lcr) de pacientes con esclerosis múltiple sugiriendo posiblemente sean agentes etiológicos en la exacerbación de brotes de esta patología sin embargo aún no se ha documentado la asociación entre covid- y ésta condición ( ) . otras patologías en las cuales se ha asociado la presencia de esta familia de microorganismos es la enfermedad de parkinson, la esclerosis lateral amiotrofia, la neuritis óptica y la encefalitis aguda diseminada ( , ) . múltiples trabajos han documentado la asociación entre polineuropatía y coronavirus. la mayoría de ellos consideran que existe una estrecha relación entre una polineuropatía autoinmune exacerbada por la infección por coronavirus o bien un compromiso nervioso periférico inducido por bloqueo neuromuscular, alteraciones hidroelectrolíticas o disvitaminosis( ). amci ® se recomienda que todo paciente con acv isquémico se considere sospechoso de infección por covid- si presenta: sintomatología asociada sospechosa de infección por covid- , contacto cercano con individuos con sintomatología infecciosa, allegados con viajes recientes, si la historia clínica es atípica, si la información suministrada no es clara, si presenta deterioro del estado de alerta inexplicable, y si al examen físico presenta hallazgos compatibles con una infección por covid- . se recomienda que todo paciente con acv isquémico de quien no se pueda recibir información se considere sospechoso de covid- ya que el evento cerebro vascular es una complicación que se ha reportado en pacientes con infección por covid- . hay una gran evidencia que correlaciona la presencia de infección por covid- con factores de riesgo cardiovasculares. esto se demostró en un metaanálisis que incluyó estudios de china (n= ) donde las comorbilidades más frecuentes fueron hipertensión ( , %, % ic: - , %), diabetes ( , %, % ic , - , %), enfermedad cardiovascular ( . %, % ic: . - . ) y patologías respiratorias ( . %, % ic: . - . %). al comparar severidad vs no severidad la presencia de hipertensión tuvo un or de . ( % ci: . - . ), y la enfermedad cardiovascular un or de . ( % ci: . - . ) respectivamente ( ) . existieron pocos casos de hipercoagulabilidad en pacientes sin factores de riesgo cardiovasculares ( ) . la cohorte de wuhan (n= ) publicada por wang et al. mostró otros factores de riesgo que podrían eventualmente estar asociados a estado de embolia y trombosis como shock en , %, arritmias , % y miocarditis en un , %. situaciones que conllevarían a hipercoagulabilidad, lesión endotelial y eventualmente la aparición de acv. ( ) . en este orden de ideas existe una estrecha correlación entre la presencia de factores de riesgo cardiovasculares que ocasionarían acv y que podrían eventualmente estar relacionados con la patogenia de covid- . ante esta inquietud khosravani et al. publicaron en stroke un informe especial donde realizan ciertas recomendaciones para el abordaje de los pacientes con acv en el contexto de la pandemia por covid- de una forma rápida, eficaz y segura para los diferentes profesionales de la salud. surge así el código stroke protegido el cual consiste en:  usar elementos de protección personal y una mascarilla al paciente  ejecutar protocolo de aislamiento de contacto y gotas  ejecutar protocolo de aerosoles si el paciente está sometido a ventilación mecánica no invasiva, manejo de aspiración de secreciones o maniobras de reanimación cardiopulmonar básica y avanzada  si el paciente presenta deterioro del estado de alerta con necesidad de soporte ventilatorio alto con fracción inspirada de o mayor a % se recomienda intubar temprano y proceder con el transporte. se debe proceder con el código stroke protegido si el paciente presenta alguna de las siguientes condiciones:  si el paciente presenta sintomatología sospechosa de covid- (fiebre, tos, dolor torácico, disnea, cefalea, mialgias, emesis)  si existe algún contacto cercano con sintomatología infecciosa  si el paciente o alguno de sus allegados ha presentado viajes recientes amci ®  si el paciente es covid- positivo  si refiere al interrogatorio una historia clínica atípica o poco clara  si el paciente o alguno de sus acudientes es incapaz de suministrar información  si el paciente presenta deterioro del estado de alerta  si al examen físico se encuentran signos compatibles con patologías diferentes a covid- por último, estas son las recomendaciones en el momento de realizar el traslado a saber:  no apresurarse dentro de la sala de reanimación o dentro de la unidad de cuidados intensivos y mantener la calma  designar un líder para el traslado del paciente y para supervisar el uso adecuado de los elementos de protección personal  limitar el número de personas encargadas del transporte  evitar la contaminación con otras áreas del hospital( ). se recomienda no realizar neuroimagen de rutina en pacientes críticos por covid- con cefalea y anosmia, dado que no existe una evidencia concluyente que demuestre una estrecha correlación entre estos síntomas y hallazgos imagenológicos. fuerte en contra fundamento en la mayoría de los casos la cefalea es un síntoma no específico que no es característico de irritación meníngea la ocurrencia de cefaleas aisladas en ausencia de otros síntomas sugiere un mecanismo benigno más que un compromiso de sistema nervioso central ( ) . sin embargo, el covid- al tener una capacidad neuroinvasiva, se han reportado casos de encefalitis virales con o sin necrosis hemorrágicas de compromiso temporal mesial y talámico que ameritarían estudio de imagen diagnóstica ( ) . un estudio retrospectivo publicado por kandenmirli et al. evaluó pacientes en hospitales infectados con covid- de los cuales requirieron manejo en uci donde el % de ellos (n= ) presentaron sintomatología neurológica. la resonancia magnética cerebral fue realizada en % (n= ) de estos pacientes. el % tuvieron hallazgos agudos, el % tuvieron alteraciones corticales de la captación de señal en el modo flair, pacientes tuvieron anormalidades en la señal flair en la sustancia blanca profunda y subcortical, pacientes con lesiones en el lóbulo frontal, en el lóbulo parietal, en el lóbulo occipital, en el lóbulo temporal, en la corteza de la ínsula, y en el giro cingular ( ) . amci ® un paciente presentó trombosis de seno venoso y otro presentó un infarto en el tercio medio del territorio de la arteria cerebral media. en % de los pacientes no se encontraron hallazgos que sugieran compromiso intracraneal de covid- . una correspondencia escrita por helms et al. reportaron pacientes con sdra y covid- en dos unidades de cuidados intensivos en francia entre marzo y abril del , el % presentaron signos neurológicos como delirium evaluados mediante cam-icu ( %), agitación ( %), signos del tracto cortico espinal ( %), síndrome disejecutivo ( %). de ellos se realiza resonancia magnética en pacientes encontrando alteraciones en la perfusión en un %, un realce de leptomeninges en un %, y un acv isquémico en un %; dos pacientes asintomáticos presentaron áreas isquémicas con hiperintensidad focal ( ) . ante estos hallazgos los principales diagnósticos diferenciales son las patologías autoinmunes, encefalitis, convulsiones e hipoglucemia. los pacientes con compromiso frontal bilateral poseen hipoxemia que ocasionan hipoperfusión fronto temporal. las microhemorragias corticales (y no) son consecuentes de la ruptura de la membrana hematoencefálica resultando en este patrón mencionado. el estado postictal muestra un compromiso simétrico de la sustancia blanca. de esta forma hay que considerar otras condiciones como las comorbilidades cardiovasculares, las reacciones adversas a los medicamentos, e hipoxia inducida por sdra que ocasionarían patrones imagenológicos de confusión, que ponen en duda la estrecha relación entre covid- y hallazgos de resonancia magnética ( ) . en este orden de ideas necesitamos más datos para determinar cuáles son los hallazgos imagenológicos relacionados con neurotropismo y qué patrones pueden encontrarse directamente relacionados con la presencia de convulsiones, hipoxia o el desencadenamiento de una tormenta de citoquinas( , , ). se recomienda la valoración neurológica completa en los pacientes con sospecha o infección por covid- . se recomienda la monitorización electroencefalográfica continua por al menos h o según la consideración del especialista en neurociencias en el paciente en estado crítico con sospecha o infección por covid- en quien se sospeche crisis epilépticas o estatus no convulsivo. las manifestaciones neurológicas en pacientes con infección por covid- pueden estar presentes en el % de los casos ( , , ) . en el paciente en estado crítico con infección por covid- se han observado complicaciones vasculares que puede causar ataque cerebrovascular agudo isquémico o hemorrágico con tazas de entre el al % por lo que el examen neurológico debe realizarse para documentar la presencia de déficit amci ® neurológico focal que haga sospechar esta patología ( , , ) . es conocido que los pacientes en unidades de cuidado crítico tienen patologías que aumenta el riesgo de crisis o estado epiléptico, entre las cuales se encuentran ( ) ( ) ( ) ( ) :  pacientes sin patologías neurológicas hospitalizados en unidad de cuidado intensivo  hemorragia subaracnoidea  hemorragia intracraneal  trauma cráneo encefálico moderado a severo  infección del sistema nervioso central  tumor cerebral  encefalopatía hipóxico-isquémica algunas de estas patologías se presentan más frecuentemente en pacientes por covid- por lo que la monitorización electroencefalográfica continuar es requerida en este grupo de pacientes. el virus puede producir descompensación de paciente con epilepsia conocida, crisis por fiebre, crisis producidas por el estado crítico del paciente o las patologías subsecuentes que se han observado en esta infección ( ) . el tiempo de monitorización requerido debe ser entre a horas para lograr una sensibilidad de a %( , ). se recomienda realizar tomografía cerebral simple ante las manifestaciones neurológicas focales que nos hagan sospechar ataque cerebrovascular isquémico o hemorrágico. la resonancia cerebral puede ser necesaria como estudio complementario para determinar otros diagnósticos diferenciales en los pacientes con sospecha o infección por covid- . las manifestaciones neurológicas en pacientes con infección por covid- pueden estar presentes en el % de los casos ( , , ) . en el paciente en estado crítico con infección por covid- se han observado complicaciones vasculares que puede causar ataque cerebrovascular agudo isquémico o hemorrágico con tazas de entre el al % por lo que el examen neurológico debe realizarse para documentar la presencia de déficit neurológico focal que haga sospechar esta patología ( , , ) . la tomografía cerebral hace parte de la valoración inicial del ataque cerebrovascular isquémico y del estudio para determinar la presencia de otros diagnósticos diferenciales como ataque cerebro vascular hemorrágico en paciente con dicha patología puede ser necesario realizar angiotac con extensión a tórax en caso de sospecha de oclusión proximal o estudios endovasculares en pacientes que sea indicado ( ) . la realización de resonancia cerebral usualmente documenta alteraciones en los pacientes con covid- en el - % de los casos evaluados no relacionada con el acv. se recomienda la realización de punción lumbar en el paciente con sospecha o diagnóstico de covid- con base en los reportes de casos disponibles:  paciente con crisis epilépticas de novo.  paciente con alteración del estado de conciencia persistente a pesar de encontrarse metabólicamente compensado, descartado ataque cerebrovascular u otra causa de encefalopatía.  paciente con manifestaciones como mielitis, neuropatía craneal múltiple o sospecha de polineuropatía desmielinizante aguda. las capacidades neurotrópicas de los coronavirus en general han sido expuestas desde la infección por sarscov. en cuanto al sars-cov- , asadi-pooya y colaboradores( ), han descrito, la posibilidad de ingreso al sistema nervioso central tras el ingreso por la mucosa nasal o por una gran viremia en el torrente sanguíneo. se han descrito procesos inflamatorios asociados (encefalitis) y en previamente con el sars y el mers hasta lesiones desmielinizantes (encefalomielitis aguda diseminada). estas primeras manifestaciones en con el sars fueron presencia de crisis epilépticas de novo, en quienes excluyendo otras causas tanto por neuroimagen además de pruebas microbiológicas en lcr, les fue descubierto el sars cov mediante pcr rt. en la presente pandemia, takeshi moriguchi y colaboradores ( ) describieron el primer caso de encefalitis asociado a sars-cov- , en un hombre joven en japón, que, tras días de clínica respiratoria, desarrollo crisis epilépticas y alteración del estado de conciencia, sin antecedente conocido de epilepsia. se realizo imagen por resonancia cerebral, demostrando hiperintensidades a nivel temporal y lcr que mostró pleocitosis linfocitaria, se descartaron otros virus ( herpes, herpes zoster) y se le realizó pcr rt para sars-cov- siendo positiva. inclusive en este paciente los primeros hisopados faríngeos fueron negativos, pero los hallazgos en tomografía de tórax hicieron sospechar la infección por sars-cov- . otro caso descrito de encefalitis hemorrágica aguda, fue también descrito en la revisión de ahmad y colaboradores ( ) . donde una mujer joven presentó cuadro respiratorio de fiebre de días de evolución y compromiso del estado de conciencia. en esta paciente se aisló el sars-cov- en hisopado faríngeo y ante el compromiso severo del estado de conciencia, se realizó resonancia que mostró compromiso hemorrágico bitalámico, en regiones temporales e ínsula, apoyando que se tratara de una diseminación tras neuronal probablemente con puerta de entrada mucosa olfatoria y siguiendo la diseminación por el los tractos del primer nervio hasta la corteza entorrinal, que es la vía propuesta de infección descrita inclusive por grupos como el de montalvan ( ) y natoli ( ) . amci ® precisamente, en la revisión sistemática de montalvan ( ) se describen caso de mielitis en contexto de paciente con infección por sars-cov- , en quien se documentó la infección en lcr. se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov- en líquido cefalorraquídeo en los pacientes con sospecha o confirmación de infección por sars-cov- que realice crisis epilépticas de novo, en quien se descarte otras causas de estructuralidad (acv, tumores) o causas metabólicas (alteraciones hidroelectrolíticas, hipoglicemia, uremia etc.). se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov- en líquido cefalorraquídeo en pacientes con sospecha o confirmación de infección por sars-cov- con alteración franca del estado de conciencia, en quien se haya descartado como causante hipoxia, ataque cerebrovascular, alteración hidroelectrolítica o estatus no convulsivo mediante imágenes y electroencefalograma. se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov- en líquido cefalorraquídeo en pacientes con sospecha de encefalitis, mielitis o síndrome de guillain barré. en pacientes críticos, se ha recomendado búsqueda activa de compromiso del snc por sars-cov- , sobre todo en pacientes con crisis epilépticas que no sean sintomáticas a trastornos metabólicos, además de hallazgos imagenológicos y curso clínico. las descripciones hechas por asadi y takeshi ( , ) , muestran pacientes con cursos tórpidos, en los que el común denominador es una alteración del estado de conciencia persistente a pesar que otras variables (metabólicas, vasculares e infecciosas diferentes al sars-cov- ). de igual forma pacientes que realicen en contexto de la enfermedad, un cuadro de debilidad generalizada aguda, con arreflexia e incluso compromiso de nervios craneales como los descritos por zahra sedaghat( ). page amci ® se recomienda en los pacientes críticos con compromiso pulmonar por covid- , un manejo orientado de fluidos frente a una estrategia liberal, ajustando el balance de fluidos de acuerdo con la evaluación clínica y/o a la capacidad de respuesta a volumen para garantizar la perfusión renal. se recomienda ajustar la intensidad de la monitoria en el paciente crítico con covid- al grado de severidad de la enfermedad para alcanzar tempranamente metas de reanimación que se reflejen en menor riesgo de lesión renal aguda. se recomienda evitar el uso de medicamentos nefrotóxicos teniendo en cuenta la farmacocinética y farmacodinamia individual, así como las interacciones farmacológicas en el paciente crítico con covid- . el manejo de la volemia en los pacientes críticos ha cambiado considerablemente en las últimas décadas orientándose a un manejo titulado, evitando la administración empírica de altos volúmenes de líquidos durante la fase de reanimación de los pacientes siendo el flujo sanguíneo el determinante primordial del aporte tisular de oxígeno; los principales componentes de este deben optimizarse y balancearse para evitar la disoxia tisular. en estados de bajo flujo, los mecanismos compensadores neurohumorales producen una redistribución del flujo a lechos no esplácnicos y a nivel renal una redistribución corticomedular convirtiendo el tejido medular renal en una zona vulnerable a la lesión. de igual forma, el flujo sanguíneo renal se ve reducido de forma refleja en presencia de hipoxemia y/o hipercapnia. diversas fuentes de información nos han indicado cómo orientar adecuadamente el manejo del estado de perfusión tisular en los pacientes críticos con o sin una condición de shock, siendo la estimación aproximada del estado de volumen del paciente el pilar fundamental sobre el cual se basará toda la estrategia de restablecimiento o mantenimiento de la volemia del paciente con el fin de mantener la perfusión adecuada. aunque históricamente se prioriza la normalización del volumen en la reanimación de un paciente inestable, hemos comprendido la importancia que tiene el tiempo para lograr las metas en el pronóstico general del paciente y evidencias como el trabajo de ospina y cols. soportan el uso temprano de vasopresores para lograr de forma temprana metas de perfusión mientras buscamos la normovolemia. los pacientes con enfermedad pulmonar asociada a covid- deben mantenerse normovolémicos para preservar el flujo sanguíneo renal siendo la evaluación del estado de volumen un verdadero reto clínico. la fiebre, el aumento de pérdidas insensibles, la baja ingesta o las pérdidas por el tracto digestivo, pueden hacer que un paciente con covid- tenga hipovolemia, situación que puede desencadenar daño renal de no ser revertida apropiadamente y a tiempo. del otro lado del espectro, la administración liberal de líquidos además del potencial de empeorar la lesión pulmonar en presencia de una membrana alveolo capilar seguramente alterada, puede producir por sí misma un incremento en el riesgo de desarrollar lesión renal como lo demostró el estudio de grissom. desde el punto de vista de las metas amci ® hemodinámicas que se deben tener con un paciente con covid- , se recomienda seguir los lineamientos de la campaña sobreviviendo a la sepsis, orientadas a mantener un óptimo estado de volumen, unas presiones de perfusión en un rango que permita la regulación de los flujos regionales en los distintos órganos y un gasto cardíaco dentro de unos rangos establecidos para una perfusión sistémica óptima. el examen clínico sigue teniendo vigencia absoluta para una adecuada aproximación al paciente, por ello debemos buscar los indicadores clínicos tradicionales de hidratación (piel, mucosas, enoftalmos, edema, etc.), el llenado capilar como lo describe hernández y cols en el estudio andrómeda-shock, el estado de alerta, las funciones cognitivas y la rata urinaria son entre otros unos marcadores aceptables para hacernos una idea del estado de adecuación de la perfusión periférica. la oliguria como marcador de perfusión renal está presente en / parte de los pacientes al ingreso a uci sin tener en sí sola una implicación pronóstica, sin embargo, la persistencia de esta en el tiempo es un indicador de alerta y obliga a una evaluación más detallada de las diversas variables que pudieran ocasionarla. debemos recordar que la administración de cargas de líquidos solamente está justificada cuando hay una respuesta cardiovascular a dicha administración, situación que se puede prever con una prueba de elevación pasiva de las piernas o con métodos más invasivos como la variabilidad de la onda de pulso, del volumen sistólico y/o del gasto cardíaco, entre otros. el esfuerzo respiratorio del paciente, los volúmenes utilizados en las estrategias de ventilación protectora y las arritmias frecuentemente presentes en los pacientes con compromiso pulmonar limitan el rendimiento diagnóstico de diversos dispositivos utilizados para la evaluación del estado de volumen y el gasto cardíaco, situación que debemos conocer y manejar( , - ). se recomienda no utilizar de forma rutinaria la administración de tratamientos específicos antivirales para el paciente crítico con covid- , con lesión renal aguda o crónica se recomienda no utilizar de rutina remdesivir en los pacientes con falla renal crónica y debe suspenderse en los pacientes que desarrollan lesión renal aguda con tfg < ml/min. las intervenciones farmacológicas en los ensayos clínicos deben ajustarse a la farmacocinética y farmacodinamia específicas de cada molécula. la incidencia de falla renal reportada por criterios de kdigo, en los estudios chinos fue de . % de los pacientes críticamente enfermos y de . % en los pacientes con covid- y amci ® sdra. sin embargo, otros estudios han demostrado que hasta un %de pacientes con covid- que ingresan a la uci pueden presentar falla renal aguda. la falla renal en los pacientes con covid- es multifactorial como se ha descrito en preguntas anteriores por lo que se recomienda la toma diaria de creatinina sérica y el seguimiento continuo del gasto urinario y otros parámetros de la función renal como hematuria, proteinuria, tasa de filtrado glomerular, nitrógeno ureico en sangre, dímero d. los medicamentos que se emplean en el manejo de la infección covid- que actualmente incluyen oseltamivir, lopinavir/ritonavir, ribavirina, y la cloroquina o hidroxicloroquina son metabolizados principalmente en el hígado, aunque en la orina se encuentran metabolitos derivados de oseltamivir, ribavirina y de la hidroxicloroquina. por esto en ninguno de los estudios realizados en torno a la infección por sars-cov- se ha realizado ninguna recomendación en cuanto a la modificación de su dosis. la hidroxicloroquina por su parte se metaboliza a cloroquina, que a su vez se metaboliza a monodesetilcloroquina y a bisdesetilcloroquina. este medicamento no es dializable en las diálisis intermitentes y la única recomendación en los pacientes con falla renal, es hacer seguimiento electrocardiográfico estrecho para vigilar la prolongación del qtc. el favipiravir es un inhibidor de la polimerasa dependiente de rna que se encuentra en fase experimental para el tratamiento de la infección por sars-cov- . la eliminación de este medicamento se realiza por vía renal y en los pacientes con falla renal en estadios leves a moderados se ha encontrado una concentración dos veces mayor en el riñón que sus niveles en sangre; sin embargo, esto no se ha asociado con ningún evento adverso por lo que la recomendación actual es no disminuir la dosis en pacientes con falla renal. el remdesivir se elimina por vía renal por lo cual no se recomienda administrar en pacientes con falla renal y los pacientes que desarrollan falla renal con el medicamento durante los estudios han sido retirados de los ensayos clínicos. no se cuenta con estudios que evalúen el remdesivir en una tfg < ml/min. en la tabla se describen algunas intervenciones farmacológicas propuestas en medio de la pandemia para el manejo del covid- y su relación con la tasa de filtración según tfg. recientemente , izzedine et al publicaron una carta editorial en el kidney international may , donde alertan sobre el posible efecto nocivo de la hidroxicloroquina en la aparición de falla renal aguda al inhibir la autofagia celular que es un proceso importante en la remodelación de los túbulos renales, siendo estas células de alto recambio, pudiendo todo esto contribuir a la aparición de falla renal aguda( , , - ) se recomienda aplicar las indicaciones tradicionales de terapia de soporte renal en pacientes críticamente enfermos con covid- . se recomienda el inicio de terapia de soporte renal en pacientes críticos con covid- con hipercalemia severa o acidosis metabólica severa, refractarias a pesar del manejo médico óptimo o cuando el balance positivo de fluidos es deletéreo, con mayor requerimiento de oxígeno suplementario y que no responde a diuréticos. se recomienda el inicio temprano de terapia de soporte renal dentro de las primeras horas de una indicación absoluta, asegurando previamente una adecuada reanimación de la perfusión tisular. en ausencia de trastornos hidro-electrolíticos y severa sobrecarga de volumen, el tiempo de inicio de diálisis es controversial. amci ® se recomienda en pacientes críticos con covid- que requieren soporte renal, las modalidades de terapia continua o extendida si cursa con inestabilidad cardiovascular, de acuerdo con la disponibilidad institucional. se debe considerar en pacientes críticos con covid- , que requieren inicio de soporte renal preferir la vía femoral para inicio de la terapia para disminuir el riesgo de contaminación por proximidad, la siguiente vía se establecerá de acuerdo con la evolución y condiciones del paciente. en pacientes diagnosticados con covid- se puede presentar la insuficiencia renal aguda como parte de su enfermedad. en estudios observacionales de usa y china la ira se reportó entre un y % de los pacientes. la enfermedad renal en pacientes con covid- se puede manifestar como ira, hematuria o proteinuria, y conllevan un mayor riesgo de mortalidad. la ira se asocia con cambios hemodinámicos y liberación de citocinas, pero no se descarta citotoxicidad directa por el virus. en un estudio realizado en nueva york con pacientes covid- positivos se diagnosticó lesión renal aguda en % de ellos, siendo leve con aumento de creatinina dos veces por encima del nivel basal en % de los pacientes, moderada en % de los pacientes, y severa con más del triple de la creatinina basal en %. hubo hematuria en el % de los pacientes y proteinuria en el %. se requirió terapia dialítica en el % de todos los pacientes con diagnóstico de ira, y el % de los pacientes que requirieron diálisis estaban en ventilación mecánica. la ira fue notada dentro de las primeras horas de admisión a uci en el % de los pacientes y se relaciona con la severidad de la enfermedad. existen además predictores independientes: edad, raza negra, diabetes, hipertensión, enfermedad cardiovascular, ventilación mecánica, y uso de vasopresores. la terapia dialítica debe instaurarse precozmente una vez realizado el diagnóstico, idealmente dentro de las primeras horas, después de asegurado que se ha completado el proceso de reanimación correspondiente. las indicaciones te trr en pacientes críticos con ira en covid- no difieren del paciente crítico general y se debe considerar ante: manifestaciones severas de uremia, sobrecarga de volumen, trastornos ácidos básico, refractarios, hipercalemia severa con manifestaciones cardiovasculares. pero no hay datos clínicos que respalden el inicio temprano vs tardío en esta población particular. pero un planteamiento válido es que la sobrecarga de volumen en pacientes que desarrollan sdra es perjudicial, dificultando el soporte ventilatorio óptimo, por lo cual se puede considerar un umbral más bajo para el inicio de trr con esta indicación específica: sdra + sobrecarga de volumen + infección covid- ( ) . amci ® el paciente debe ser dializado en el cubículo de cuidado intensivo o en la habitación de aislamiento en los casos en que esté disponible y siempre evitar traslado a unidades con otros pacientes. la crrt es la modalidad preferida para este tipo de pacientes, pero debe quedar claro que esto depende de las facilidades de la institución que albergue al paciente y de la experticia de los profesionales. el acceso vascular en el paciente crítico general debe ser en su orden: vena yugular interna derecha, venas femorales comunes, vena yugular interna izquierda, y debe ser colocado por el médico encargado del paciente si está capacitado para ello, para evitar exposiciones innecesarias del personal de la salud. sin embargo, por precaución por el riesgo de contaminación, recomendaciones de expertos basadas en seguridad sugieren la utilización el catéter femoral. el tipo de catéter recomendado es un catéter doble lumen transitorio. sería ideal el monitoreo a través de cámaras del procedimiento para evitar el contacto prolongado del personal de enfermería durante el procedimiento de diálisis. en algunos casos específicos y de acuerdo con la disponibilidad, la diálisis peritoneal puede ser una alternativa. en los casos de crrt el líquido efluente no es contaminante para el personal de la salud. para terminar, es importante hacer énfasis en que en algunos hospitales ha habido escasez de insumos y esto puede llegar a convertirse en un serio problema. se recomienda para casos de fuerza mayor:  un litro de solución salina al . % con cloruro de potasio a necesidad  un litro de dextrosa al % en agua con meq de bicarbonato de sodio  un litro de solución salina al . % con gr de cloruro de magnesio  un litro de solución salina al . % con gr de cloruro de calcio esto nos da una solución de cuatro litros que contienen: meq/l de sodio, . meq/l de bicarbonato, . mmol/l de magnesio y . mmol/l de calcio, más una cantidad variable de potasio. esta solución se puede usar como líquido dializante en pacientes en terapias de reemplazo renal continuo. especial cuidado se debe tener en el proceso de anticoagulación, pudiéndose usar heparina no fraccionada, hbpm, y citrato en los centros donde se tenga experiencia ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . se sugiere no utilizar de rutina la trrc más hemoperfusión en el paciente crítico con covid- . débil en contra page amci ® se puede considerar en el paciente crítico con covid- con lesión renal aguda en quien se considere inicio de trrc, considerar la utilización de filtros de fibra hueca con propiedades adsortivas o asociado con cartuchos para hemoperfusión directa. el síndrome de liberación de citocinas (tormenta de citocinas) es un importante determinante en la transformación de infección por covid- de leve a moderado y progresión de la lesión de un órgano como el pulmón con neumonía y sdra a compromiso sistémico con inestabilidad hemodinámica, cid y fom. los pacientes afectados de tormenta de citocinas se encuentran con niveles altos de il- especialmente, además de il- , tnf, que se relacionan con pobres pronósticos y mayor mortalidad. la asociación entre la lesión alveolar y renal (eje pulmón-riñón) es evidenciada en estudio del por panitchote y cols con pacientes con sdra secundaria a neumonía y sin enfermedad renal preexistente que desarrollaron lesión renal aguda en el % con aki en el % de ellos. recientemente en estudio en china zhou, con pacientes afectados de covid- se encontraron como indicadores de mal pronóstico pacientes con altos niveles de dímero d, il- , troponina i, dhl, ferritina y choque séptico. las terapias de depuración extracorpórea han sido utilizadas como tratamiento en pacientes con lesión severa por covid- ; dentro de estas se cuentan la crrt, hemoperfusión aislada, intercambio plasmático (tpe), plasmafiltración y adsorción (cpfa) y crrt + hemoperfusión. dentro de los beneficios de la crrt se cuentan la estabilidad hemodinámica, estabilidad del medio interno, depuración de toxinas pequeñas y medianas, incluidos mediadores inflamatorios, cuando se utiliza terapia convectiva, además de permitir soporte nutricional. la asociación de este procedimiento con membranas especiales (an + metilsulfonato y polietilamina oxiris) permiten hacer adsorción de citocinas (il- ) y endotoxinas, por periodos de h por días consecutivos para manejo de tormenta de citocinas. la hemoperfusión aislada o asociada a crrt, también permite la remoción de il- , utilizando cartuchos ha , con procedimientos de a horas de duración por días consecutivos ( , ( ) ( ) ( ) ( ) ( ) ( ) . no se puede emitir una recomendación a favor o en contra sobre el uso rutinario de la plasmaféresis como opción terapéutica en la fase de inflamación del paciente con covid- . amci ® el coronavirus covid- puede inducir el síndrome respiratorio agudo severo (sars), que conduce a la disfunción inmune, la liberación excesiva de citoquinas inflamatorias, y a una serie de reacciones en cascada de activación de citoquinas, que resultan en lesiones alveolares difusas, formación de membrana hialina, exudación de fibrina y otras manifestaciones de lesión del pulmón. en casos severos, la tormenta de citoquinas sistémicas invade el sistema circulatorio, lo que lleva a una inestabilidad hemodinámica, shock y mods ( ) . los niveles de il- , il- , tnf-a y otras citoquinas inflamatorias en pacientes con covid- grave son significativamente más altos, lo que puede estar relacionado con un mal pronóstico ( ) . por lo tanto, la plasmaféresis se puede usar con seguridad y efectividad en pacientes con covid- grave, para eliminar mediadores inflamatorios de gran peso molecular. la seguridad depende como todas las terapias extracorpóreas de un personal de la uci entrenado, preparado y capacitado para aplicar las intervenciones en forma óptimas ( ) . las terapias extracorpóreas de soporte de órganos pueden representar una parte importante de la respuesta y los médicos y otros profesionales de la salud deben estar familiarizados con estas terapias sofisticadas. se debe hacer un llamado a la acción, para crear conciencia sobre las diferentes técnicas extracorpóreas, cada una con criterios específicos y modalidades de prescripción, entrega y monitoreo( , ). se recomienda no utilizar de forma rutinaria el uso de un tratamiento específico dirigido a pacientes con infección por sars-cov- /covid- comparado con el manejo estándar para mejorar desenlaces clínicos fuertes. actualmente no existe una terapia dirigida que se a efectiva para el manejo del virus; un número alto de estudios han surgido en los últimos dos meses, la mayoría sin el rigor metodológico suficiente para tomar decisiones adecuadas con respecto al manejo del amci ® paciente con infección por sars-cov- . el conocimiento en la estructura del virus y el mejor entendimiento en la fisiopatología de la enfermedad genera un sinnúmero de potenciales fármacos que han sido ensayados para el manejo de la enfermedad. en tiempos de pandemia, con una patología catastrófica en términos de vidas humanas y costos hospitalarios; es importante encontrar soluciones a desenlaces importantes como mortalidad, días de estancia en uci y en el hospital, aumento en los días libres del ventilador, disminución de complicaciones mayores debido a la enfermedad entre otros. hasta el momento no se ha documentado ninguna terapia específica que pueda impactar sobre estos desenlaces; pero la calidad de los trabajos, tampoco dejan claro sin él no usar ningún tratamiento específico mejora los desenlaces al menos al disminuir el número de complicaciones. este nuevo beta-coronavirus es similar al coronavirus del síndrome respiratorio agudo severa (sars-cov) y del síndrome respiratorio del medio este (mers-cov); por lo tanto, varias moléculas que habían sido evaluadas en este tipo de enfermedad rápidamente se abrieron paso a ensayos clínicos en paciente con covid- . estos ensayos principalmente observacionales, aleatorios pero abiertos con un número pequeño de pacientes no han permitido sacar adecuadas conclusiones y es frecuente como ver las diferentes guías de las principales sociedades del mundo cambiar de forma frecuente sus recomendaciones; no existes evidencia de estudios clínicos aleatorios y controlados que midan desenlaces fuertes, la premura de un tratamiento efectivo ha sacrificado el rigor metodológico que una investigación requiere. una estructura viral y replicación conocidas generan posibles dianas para que diferentes fármacos puedan ser investigados, antivirales tipo arbidol el cual inhibe la fusión de la membrana en la envoltura viral a algunos receptores; antimaláricos como la hidroxicloroquina y la cloroquina, las cuales inhiben la entrada viral y endocitosis por múltiples mecanismos, así como los efectos inmunomoduladores demostrados en el huésped; antivirales que impiden la replicación como el lopinavir o darunavir inhibiendo las proteasas o la ribavirina, el remdesivir o el favipiravir que actúan como análogos de nucleótidos o fármacos que actúan modulando la respuesta específica del huésped como el tocilizumab el cual se une al receptor de la il- inhibiendo el punto de acción de esta; los corticosteroides con múltiples efectos en la modulación del sistema inmunológico del paciente o los fármacos para evitar la respuesta secundaria a esta cascada inflamatoria como son los anticoagulantes. por último, se han buscado estrategias con el fin de mejorar la inmunización pasiva del huésped en el uso del plasma de pacientes convalecientes o el uso de inmunoglobulinas enriquecidas entre otros tratamientos propuestos para esta enfermedad. como vamos a ver más adelante, actualmente no existe un tratamiento específico con el nivel de evidencia suficiente para recomendar de manera generalizada; tampoco existe suficiente evidencia del manejo del soporte básico sin el uso de fármacos dirigidos, que demuestre que esta estrategia se deba implementar de manera sistemática en todos los pacientes; por lo tanto, a continuación trataremos de resolver las inquietudes con respecto a los diferentes medicamentos que han sido usados en la pandemia del sars-cov- /covid- . se recomienda no utilizar antimaláricos tipo hidroxicloroquina (hcq) o cloroquina (cq) para el manejo de pacientes con infección por sars-cov- /covid- . para la fecha no hay un adecuado sustento bibliográfico que soporte el uso de antimaláricos en la prevención o manejo de pacientes con infección por sars-cov- tanto leve, moderada como severa. los mayores estudios no muestran utilidad clínica y tendencia a mayores eventos cardiovasculares con el uso de antimaláricos en pacientes con infección por covid- comparado con no darlo. su utilidad se deriva principalmente de resultados en estudios preclínicos e in vitro; como los presentados por wang y cols donde evaluaron medicamentos de manera in vitro contra el covid- , siendo el remdesivir y la cq efectivos de manera in vitro contra el nuevo coronavirus( ); liu y cols, donde la hcq fue efectiva en inhibir la infección por sars-cov- in vitro que junto con su potencial antiinflamatorio tenía potencial para el uso clínico ( ) y yao y cols, donde la hcq fue más potente que la cq para inhibir el sars-cov- in vitro y fue recomendado para el uso en humanos en dosis de mg dos veces al día por el primer día, seguido de mg dos veces al día por días más mantendría la concentración efectiva del fármaco en el tejido pulmonar ( ) . los pocos estudios clínicos, son de baja calidad y no han mostrado mejoría ni eficacia en el uso de antimaláricos para el manejo de paciente adultos con covid- , algunos estudios iniciales con pocos pacientes con resultados favorables ( ) e incluso con recomendaciones para uso en las primeras versiones de guías internacionales para la amci ® hcq y cq, encontrando superioridad en estudios observacionales, de pocos pacientes, sin comparadores para inhibir la exacerbación de la neumonía, hallazgos de las imágenes pulmonares, promover una conversión negativa al virus y acortar el curso de la enfermedad; la cq tuvo un efecto notable tanto en términos de resultado clínico como de eliminación viral ( ) ; considerando la hcq y la cq como un tratamiento costo efectivo ( ) . estudios posteriores con un mayor número de pacientes no han logrado reproducir los estudios preclínicos iniciales; mahévas y cols, evaluaron la efectividad de la hcq en pacientes admitidos a cuatro hospitales en francia, con neumonía por covid- quienes requieren oxígeno, pero no se encontraban en uci, comparado con una población con manejo estándar; la hcq se usó a dosis de mg día en las primeras horas a la admisión, este estudio no soporte el uso de la hcq en pacientes admitidos al hospital con covid- que requieren oxígeno al no reducir de forma significativa la admisión a la uci, el sdra o muerte en el día después del ingreso ( ) ; por el contrario, se han reportado efectos secundarios frecuentes (prolongación del intervalo qt, hipoglucemia, cambios en el estado mental, alteraciones gastrointestinales y retinopatía); silvia borda y cols, evaluó la seguridad y eficacia de dos dosis de cq en pacientes con covid- severo en un estudio aleatorizado, doble ciego fase iib en pacientes adultos hospitalizados con infección por sars-cov- , los pacientes fueron expuestos a dosis altas de cq ( mg dos veces al día por días) o dosis bajas ( mg dos veces al día en el día y una vez al día por días), los hallazgos preliminares de este estudio sugieren que la dosis más alta de cq no debe recomendarse para pacientes críticos con covid- debido a sus posibles riesgos de seguridad, especialmente cuando se toman simultáneamente con azitromicina y oseltamivir; estos hallazgos no pueden extrapolarse a pacientes con covid- no severo ( ); tang y cols, evaluaron la eficacia y seguridad de la hcq con el manejo estándar en un estudio multicéntrico, abierto, aleatorio y controlado en china, pacientes con covid- positivo se incluyeron en el análisis de intención a tratar ( en el grupo de hcq y en el grupo estándar), la hcq fue administrada a dosis de mg día por tres días y mantenimiento con dosis de mg día (duración del tratamiento: dos a tres semanas en pacientes con enfermedad leve a moderada o enfermedad severa respectivamente); la administración de hcq no resultó en una significativa mayor probabilidad de conversión negativa comparado con el tratamiento estándar, los efectos adversos fueron mayores en el grupo de hcq ( ) . con todo esto la hcq y la cq, si se usan deberá ser bajo estudios experimentales aprobados con una estricta monitorización y vigilancia clínica de la frecuencia cardíaca y el intervalo qt, los niveles de glucosa, la función hepática y renal, y el cribado clínico de trastornos mentales y visuales en pacientes que reciben estos fármacos. debe evitarse hcq/cq en pacientes con enfermedades cardiovasculares subyacentes. nuevas evidencias con un mayor número de pacientes podrían sacar la hcq y la cq inclusive de estudios clínicos; barbosa y cols, evaluaron en un estudio cuasialeatorio comparativo el uso fuera de registro de la hcq en pacientes positivos por el sars-cov- , el pronóstico primario fue la necesidad de escalar el soporte ventilatorio, cambio en el conteo de linfocitos o cambio en el índice de neutrófilos/linfocitos, un total de pacientes fueron incluidos, en el brazo de hcq. la administración de hcq fue asociada con la necesidad de aumentar el nivel del soporte ventilatorio comparado con aquellos que no recibieron hcq al día del estudio, no hubo beneficios en la mortalidad, reconstitución inmunológica y riesgo de intubación ( ) . el estudio con un mayor número de pacientes proviene de la ciudad de new york; geleris y cols, examinaron la asociación entre el uso hcq y la intubación o muerte en un centro médico de ny, se analizaron . amci ® estaban más enfermos en términos de oxigenación, en este estudio observacional la administración de hcq no fue asociada con una disminución en el riesgo compuesto de intubación o muerte ( ) . se recomienda no utilizar antimaláricos tipo hidroxicloroquina (hcq) o cloroquina (cq) en combinación con azitromicina (az) para el manejo de pacientes con infección por sars -cov- /covid- . fundamento para la fecha la evidencia no favorece el uso combinado de los antimaláricos en combinación con la azitromicina; por el contrario, la combinación de estos dos medicamentos puede ser deletérea, inclusive con un aumento reportado en la mortalidad y la aparición de arritmias ventriculares de novo; estudios iniciales fueron promisorios, gautret y cols, evaluaron inicialmente el efecto de la hcq en la carga viral respiratoria en conjunto con el uso de azitromicina, la presencia del virus al día fue el pronóstico primario; pacientes con tratamiento mostraron una significativa reducción en la carga viral al día de la inclusión comparado con los controles; la azitromicina adicionada a la hcq fue significativamente más eficiente en la eliminación viral ( ) ; nuevamente gautret y cols, realizaron un estudio observacional, no controlado, no comparativo de pacientes tratados con la combinación de hcq más azitromicina, presentando una mejoría significativa en disminución de la carga nasofaríngea del virus y una menor tiempo de enfermedad ( ) ; luego million y cols, evaluaron la combinación de hcq y az en un estudio retrospectivo de . pacientes con sars-cov- tratados con hcq ( mg tres veces al día por días) + az ( mg en el día , seguido de mg al día por los próximos días), el pronóstico fue mortalidad, empeoramiento clínico (ingreso a uci) o persistencia viral; la administración de hcq+az en combinación antes de que aparecieran las complicaciones del covid- es segura y asociada a una baja mortalidad en los pacientes ( ) ; soportado además por estudios in vitro que demuestran que la combinación de hcq y az tienen efectos sinérgicos para el sars-cov- a concentraciones compatibles con las que se obtienen en pulmones humanos ( ) . otros estudios por el contrario no han encontrado resultados positivos, es así como, magagnoli y cols, en un análisis retrospectivo de pacientes confirmados con infección por sars-cov- en centros de veteranos de los eeuu, un total de pacientes fueron evaluados (hcq, n= ; hcq+az, n= ; no hcq, n= ), en este estudio no hubo evidencia que el uso de la hcq tanto sola o en combinación con la az, redujo el riesgo de ventilación mecánica en pacientes hospitalizados con covid- ; una asociación con un aumento en la mortalidad fue identificada en pacientes tratados con hcq sola ( ) . nuevamente los estudios con un mayor número de pacientes se encuentran en la ciudad de new york; rosenberg y col, describieron la asociación entre hcq, con o sin az en el pronóstico de pacientes hospitalizados con covid- ; un estudio de cohorte multicéntrico retrospectivo en pacientes hospitalizados con covid- en hospitales de ny, los pacientes recibieron ; en pacientes hospitalizados en el área metropolitana de ny con covid- , el tratamiento con hcq, az o ambos, comparado con ningún tratamiento, fue no significativamente asociado con diferencias en la mortalidad hospitalaria ( ) . recomendaciÓn se recomienda no utilizar de forma rutinaria el uso rutinario del lopinavir/ritonavir para el manejo de pacientes con infección por sars-cov- /covid- . en la actualidad no existe evidencia a favor o en contra en el uso del tratamiento con antirretrovirales con lopinavir/ritonavir en el manejo de pacientes adultos hospitalizados con covid- ; no se observó ningún beneficio con lopinavir/ritonavir más allá de la atención estándar. se está en espera de cierre de diferentes ensayos futuros que confirme o excluyan el uso de lopinavir/ritonavir en el paciente covid- . en diciembre de , un nuevo coronavirus, designado sars-cov- , ha causado una pandemia ( , , , ) ; cuando hablamos de enfermedad producida por covid- hablamos de enfermedades que van desde las enfermedades leves autolimitantes del tracto respiratorio hasta neumonía rápidamente progresiva, neumonía grave, falla multiorgánica y muerte. hasta este momento no existen agentes terapéuticos específicos para las infecciones por coronavirus. después de la aparición del síndrome respiratorio agudo grave (sars) en , entre los fármacos aprobados se identificó lopinavir, un inhibidor del aspartato proteasa tipo del virus de inmunodeficiencia humana (vih), que tiene actividad inhibitoria in vitro contra el sras-cov, el virus que causa el sars en los seres humanos y el ritonavir combinado con lopinavir para aumentar su vida media plasmática a través de la inhibición del citocromo p ( ) . se comenzaron estudios evaluando la respuesta antiviral in vitro de la combinación de lopinavir/ritonavir y ribavirina en pacientes con sars; comparados con pacientes tratados con ribavirina sola, que sirvieron como controles históricos; el pronóstico adverso (sdra o muerte) fue significativamente más bajo en el grupo de tratamiento comparado con los controles históricos ( . % vs . %, p = . ) al día del inicio de los síntomas; una reducción adicional en el uso de esteroides y de infecciones nosocomiales fue vista en el grupo de tratamiento con una disminución en la carga viral y aumento en el conteo de linfocitos ( ) ; del mismo modo, el lopinavir tiene actividad, tanto in vitro como en modelo animal, contra el coronavirus del síndrome respiratorio de oriente medio (mers-cov) ( ) . estos estudios previos son el soporte inicial para el uso del lopinavir/ritonavir en la epidemia del covid- ; cao y cols, en mayo de publicaron en china, un estudio controlado, aleatorizado en pacientes hospitalizados con prueba ; los efectos adversos gastrointestinales fueron más comunes con el lopinavir-ritonavir, pero los eventos adversos serios fueron más común con el grupo control; el tratamiento con lopinavir-ritonavir fue suspendido en pacientes ( . %) secundario a los eventos adversos ( ) . otro estudio hung y cols, en hong kong, evaluaron en un trabajo multicéntrico, prospectivo, aleatorizado, fase la eficacia y seguridad de la terapia combinada por días de lopinavir mg y ritonavir mg cada h, ribavirina mg cada h y tres dosis de millones de ui de interferón beta- b en días alternos en pacientes con covid- comparado con lopinavir/ritonavir cada h (grupo control); el resultado primario fue tiempo en la negativización de la pcr viral en el hisopado nasofaríngeo en paciente con covid- ; pacientes fueron ingresados, en el grupo de combinación y en el grupo control; en el grupo de intervención de forma significativa se negativizo la prueba de pcr de forma más rápida ( días [iqr - ]) que el grupo control ( días [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; hr . [ic % . - . ], p = . ); los eventos adversos fueron similares entre los grupos; ningún paciente murió durante el estudio ( ) . por último, un pequeño estudio de zhu y cols en china, con pacientes con sars-cov- ; evaluaron de forma retrospectiva los efectos antivirales y seguridad del lopinavir/ritonavir y el arbidol (antiviral aprobado en china y rusia para el sasr y la influenza), pacientes en el grupo de lopinavir/ritonavir y en el grupo de arbidol; los pacientes lopinavir/ritonavir presentaron un mayor tiempo para la negativización de la prueba de pcr viral (p < . )( ). se recomienda no utilizar de forma rutinaria remdesivir como antiviral para el manejo de pacientes con infección por sars-cov- /covid- . se debe considerar su uso en escenarios de estudios de investigación clínica aprobados. actualmente no hay disponibilidad del medicamento en el país (colombia) por lo cual no se incluye en los protocolos de manejo de paciente con covid- . en estados unidos el primer paciente con covid- mostró una mejoría significativa de sus síntomas con horas de tratamiento con remdesivir ( ) , lo que abrió la puerta a un nuevo tratamiento para el sars-cov- ; el remdesivir (gs- ) es un análogo de los nucleótidos que inhibe la rna polimerasa; con un amplio espectro antiviral, puede inhibir la replicación de múltiples coronavirus en las células epiteliales del sistema respiratorio ( ) ; estudiado ( ) . por último, un estudio publicado por antinori y cols, en milán, italia; de manera prospectiva (compasional) incluyó pacientes con neumonía por sars-cov- mayores a años bajo ventilación mecánica o con una saturación de oxígeno ≤ % al aire ambiente o un puntaje del national early warning score ≥ ; el pronóstico primario en cambio en el estado clínico en una escala ordinal de categorías ( = no hospitalizado de regreso a sus actividades diarias normales; = muerte); de los paciente ingresados, se encontraban en uci y en un piso de hospitalización de enfermedades infecciosas; un curso de días de remdesivir fue completado por pacientes ( %) y suspendido en , de os cuales ( . %) se descontinuo por eventos adversos; a los días, ( . %) pacientes de piso fueron egresados, permanecían hospitalizados y uno murió ( . %), en la icu ( . %) fueron egresados, ( . %) pacientes murieron, ( . %) aún se encontraban en ventilación mecánica y ( . %) estaba con mejoría pero aún hospitalizado; la hipertransaminasemia y la injuria renal aguda fueron los eventos adversos más frecuentes reportados ( . % y . %, respectivamente); los datos sugieren que el remdesivir puede beneficiar a pacientes con neumonía por sars-cov- hospitalizados por fuera de la unidad de cuidado intensivo ( ). page se recomienda no utilizar de rutina la ivermectina para el manejo de pacientes con infección por sars-cov- /covid- . para la fecha no se cuenta con la suficiente evidencia para emitir una recomendación para el uso de la ivermectina en pacientes con covid- , en estudios iniciales in vitro, caly y cols, demostró como la ivermectina, una droga autorizada por la fda como antiparasitario tiene un efecto antiviral de amplio espectro de manera in vitro, con una reducción significativa de la replicación viral en modelos experimentales ( ) , un estudio aún sin publicar, observacional multicéntrico de casos y controles (n: casos y n: controles), realizado entre el de enero y de marzo de , incluyó pacientes diagnosticados con covid- confirmados por laboratorio, la dosis fue de mcg/kg de ivermectina más la terapia médica de soporte en comparación con terapia médica sin ivermectina, el resultado principal fue la medición de supervivencia; en pacientes que requirieron ventilación mecánica, la mortalidad fue menor en el grupo de ivermectina ( , % y , % respectivamente) y las tasas de mortalidad global fueron más bajas con ivermectina ( no se puede emitir una recomendación a favor o en contra sobre el uso compasivo o rutinario de tocilizumab en pacientes con infección por sars-cov- /covid- . en pacientes individualizados se ha reportado desenlaces clínicos favorables. se puede considerar su uso en pacientes que cumplan con todos los siguientes criterios: ver tabla . el tocilizumab (tcz) un anticuerpo monoclonal humanizado igg k, el cual se puede unir de manera específica a los receptores solubles de membrana para la il- (sil- r and mil- r) y ha sido ampliamente usado en el tratamiento de enfermedades autoinmunes, tales como la artritis reumatoide, la enfermedad de still del adulto o vasculitis de grandes vasos ( ) . un primer estudio de xiaoling xu y cols, describió en china, pacientes tratados con tocilizumab, se documentó una mejoría en los síntomas, en los requerimientos de oxígeno y en los hallazgos imagenológicos de la tomografía de tórax; los niveles promedio de il- antes de la terapia fueron de . pg./ml; todos los pacientes recibieron lopinavir y metilprednisolona antes de la terapia. se trata de una serie con una muestra pequeña de pacientes en donde solo ( %) pacientes estaban en condición crítica ( ) . posteriormente luo y cols, en china, reportan el uso de tocilizumab en pacientes, con mejoría en el aumento de la pcr en todos los pacientes, excepto uno, y una disminución de la il . el nivel sérico de il- tendió a aumentar inicialmente y luego disminuyó en pacientes. los niveles medios de il- antes de la terapia fueron de . pg./ml; % de los pacientes recibieron metilprednisolona; el % fallecieron. es otra serie pequeña, con pacientes ( , %) en condición crítica ( ) . roumier y cols, en francia, estudiaron pacientes que recibieron tcz, observando que se redujo la necesidad de ventilación mecánica en comparación con los controles ( amci ® . ); en este estudio, no hubo diferencias en la reducción de la mortalidad y pacientes ( %) se encontraban en uci ( ) . klopfenstein y cols, también en francia, en un estudio retrospectivo de casos y controles, encontraron que pacientes que recibieron tcz (n= ), a pesar de tener más requerimiento de oxígeno, con resultados biológicos más pobres (mayor linfopenia y un nivel de pcr superior) al inicio del estudio que los pacientes sin tcz (n = ), presentaron el objetivo combinado (ingreso a uci y mortalidad) menor que los pacientes sin la terapia ( % vs %, p = . ); es otra serie, que disminuye la necesidad de ventilación mecánica ( % vs %, p = . ) de manera significativa ( ) . luego rimland ca y cols informan los primeros datos de pacientes con covid- tratados con tcz en los estados unidos, de ellos en ventilación mecánica; la pcr y el fibrinógeno mejoraron rápidamente, pero no hubo mejoría en otros marcadores o resultados clínicos. sólo a seis pacientes les tomaron niveles previos de il y de ellos dos tenían niveles bajos ( ) . en otro estudio mikulska y cols, próximo a salir en jama, en pacientes con sdra moderado a severo, hay mayor disminución de la mortalidad con el tratamiento combinado de tocilizumab y esteroides, en relación con cada una de estas terapias. por último, no todos los estudios han mostrado resultados positivos; kimmig y cols, de chicago (eeuu), en de los pacientes críticos con covid- , que recibieron tcz, se asoció con una mayor incidencia de infecciones bacterianas secundarias, incluida la neumonía asociada al ventilador ( . % vs. . % p = . ) ( ) . posterior a la recomendación hay nueva evidencia publicada y estudios aún sin publicar que puede soportar el uso de los inhibidores de la il- en pacientes con sars-cov- /covid- ; morena y col, en un estudio de tocilizumab como uso "off-label" en el tratamiento de neumonía por sars-cov- en milán, italia, este estudio abierto, prospectivo, describe las características clínicas y el pronóstico de pacientes con covid- confirmado y severo tratados con tcz iv, todos los pacientes, presentan niveles plasmáticos elevados de il- (> pg/ml) y saturación de oxígeno < % al aire ambiente, pacientes ( %) se encontraban con sistema de alto flujo de oxígeno y en ventilación invasiva, a los días luego del tratamiento se observa una caída dramática en la temperatura corporal y la pcr, con un incremento significativo en el conteo de linfocitos (p < . ); a los días del tratamiento, pacientes ( %) mostraron una mejoría en la severidad del cuadro; fueron dados de alta; ( %) mostraron empeoramiento de su cuadro clínico y de estos, murieron ( %). la mortalidad fue significativamente asociada con el uso de ventilación mecánica al inicio ( . % vs % de los pacientes en soporte de oxígeno no invasivo, p = . ), el efecto adverso más frecuente reportado fue la elevación de las enzimas hepáticas ( %), trombocitopenia ( %) e infecciones bacterianas serias e infecciones fúngicas en un ( %); los autores concluyen que el tcz ejerce un rápido beneficio sobre los marcadores inflamatorios y la fiebre, aunque no se consiguió un impacto clínico sobre el pronóstico, el riesgo aumentado de infecciones severas no es despreciable ( ) . capra y col, describieron pacientes en un hospital de italia con neumonía por covid- y falla respiratoria sin soporte ventilatorio y al menos uno de los siguientes: frecuencia respiratoria ≥ respiraciones/min, saturación ≤ % o pao /fio <= mmhg, los pacientes recibieron la terapia estándar para el momento (hidroxicloroquina, lopinavir y ritonavir) y fueron considerados el control; pacientes recibieron tzc con días de la admisión más el manejo estándar, los pacientes en el grupo de tratamiento mostraron de manera significativa una mayor sobrevida comparado con los pacientes control (hr para muerte, . ; % ic], . a . ; p = . ), ajustado para las características clínicas de base; de pacientes en el grupo de tcz y de en el grupo control murieron; % y . % de los pacientes que se dieron de alta en el grupo de tcz y en el control se recuperaron; la función respiratoria mejoró en el . % de los amci ® pacientes con tcz que aún se mantenía hospitalizados, donde el % de los controles empeoro y requirieron ventilación mecánica, dando al tcz un espectro positivo en términos de curso clínico y sobrevida en pacientes con covid ( ). guaraldi y col; evaluaron el papel del tcz en reducir el riesgo de ventilación mecánica invasiva en pacientes con neumonía severa por covid- quienes recibían tratamiento estándar para el momento (hidroxicloroquina, azitromicina, antirretrovirales y heparinas de bajo peso molecular) en un estudio retrospectivo, observacional en bologna, reggio emilia y módena, italia; el tcz fue dado a dosis de mg/kg de peso corporal de forma iv (con un máximo de mg) en dos infusiones separadas h o mg sc administradas en dos dosis simultáneas, una en cada muslo ( mg en total), cuando la formulación iv no se encontraba disponible; el pronóstico primario fue la combinación de ventilación mecánica invasiva o muerte; de pacientes ingresados, ( %) tenían neumonía severa por covid- y fueron incluidos, ( %) de pacientes en el grupo estándar requirieron ventilación mecánica, comparados con ( %) de pacientes tratados con tcz (p = · ; [ %] de pacientes tratados iv y [ %] de pacientes tratados sc); ( %) pacientes en el grupo estándar murieron, comparado con ( %; p < . ) pacientes con tcz ( [ %] del grupo iv y [ %] sc); luego de ajustar para sexo, edad, centro de reclutamiento, duración de los síntomas y puntaje de sofa, el tratamiento con tcz fue asociado con una reducción en el riesgo de ventilación mecánica invasiva o muerte (hr . , % ic . - . ; p = . ); ( %) de pacientes tratados con tcz fueron diagnosticados con nuevas infecciones en comparación con ( %) de pacientes en el grupo estándar (p < . ) ( ) . campochiaro y col, en un solo centro evaluó la eficacia y seguridad del tcz en pacientes con covid- severo, se diseñó un estudio retrospectivo en pacientes con características de hiper-inflamación (definida como una elevación tanto en la pcr, ≥ mg/l, normal < mg/l o ferritina ≥ ng/ml, normal < ng/ml en presencia de un incremento en la dhl > u/l), acompañado de un compromiso respiratorio severo, definido como hallazgos típicos en la radiografía y/o tomografía, la presencia de una saturación de oxígeno ≤ % al aire ambiente o una pao :fio ≤ mmhg ingresados a la uci, comparando pacientes con tcz iv al manejo estándar, pacientes fueron incluidos de los cuales fueron tratados con tcz; los pacientes se encontraban con alto flujo o ventilación mecánica no invasiva, a días de seguimiento, % de los pacientes con tcz experimentaron mejoría clínica comparado con un %del tratamiento estándar (p = . ); la mortalidad fue % en el grupo de tcz y % en el grupo estándar (p = . ); la incidencia de infección y trombosis pulmonar fue similar en ambos grupos ( ); somers y col, evaluaron un estudio observacional en pacientes con neumonía por covid- severo que se encontraban en ventilación mecánica, evaluando como pronóstico la probabilidad de sobrevida posterior a la extubación; pacientes fueron incluidos, recibieron tcz y no; en los modelos ajustados, el tcz, fue asociado con una reducción del % en el riesgo de muerte [hr . % ic . a . ]; aunque el tcz, fue asociado con un incremento en la proporción de pacientes con superinfecciones ( % vs. %; p < . ), no hubo diferencias en la mortalidad a días entre los pacientes tratados con tcz con o sin superinfecciones [ % vs. %; p= . ] ( ). price y col, también publicaron un estudio observacional de pacientes hospitalizados con covid- , los pacientes recibieron tcz si cumplían criterios del síndrome de liberación de citoquinas, se evaluaron pacientes; de los cuales ( %) recibieron tcz, estos pacientes que recibieron ventilación mecánica la sobrevida fue del % ( % ic, - ), luego del tcz pocos eventos adversos fueron reportados y tanto la oxigenación como los biomarcadores de inflamación mejoraron ( ) y por último, en un estudio preliminar con datos aún sin publicar perrone y col, evaluaron la eficacia del tcz en pacientes con neumonía por covid- , en un estudio amci ® multicéntrico fase en italia; se utilizó tcz, a dosis de mg/kg iv, una o dos administraciones con horas de diferencia; y casos fueron disponibles para un análisis de intención a tratar, pacientes murieron; las tasas de letalidad fueron de . % ( . % ic, . - . , p = . ) y . % ( . % ic, . - . , p < . ) a y días; el tcz redujo la tasa de letalidad a días pero no a días comparado con las esperadas sin presentar una toxicidad significativa; la eficacia fue más evidente en los paciente que no requerían ventilación mecánica ( ). recomendaciÓn se recomienda no utilizar de rutina bloqueadores de interleuquina- (anakinra) en pacientes con infección por sars-cov- /covid- . aunque su ventaja está en el perfil de seguridad, su vida media corta ( horas) y porque las infecciones oportunistas son raras ,no hay suficiente evidencia para emitir una recomendación sobre el uso de este medicamento; cavalli y cols, de milán, italia, realizaron un estudio de cohorte retrospectivo en pacientes con sdra moderado a severo con hiperinflamación (pcr ≥ mg/dl, ferritina ≥ ng/ml o ambos), manejados con ventilación mecánica no invasiva fuera de la uci y que recibieron tratamiento con hidroxicloroquina y lopinavir, los pacientes que recibieron anakinra, mg/kg dos veces al día intravenosa (n= , dosis alta) o mg dos veces al día subcutánea (n= pacientes, dosis baja) fueron comparados con una cohorte retrospectiva que no recibió anakinra (tratamiento estándar); la duración del tratamiento se prolongó hasta el beneficio clínico sostenido (reducción del % en la pcr, y una pafi > , durante al menos días consecutivos) o hasta la muerte, bacteriemia, o efectos secundarios (alt > veces valores de referencia); el tratamiento con anakinra a bajas dosis se interrumpió después de días debido a la escasez de efectos sobre la pcr y el estado clínico. a los días, el tratamiento con dosis altas de anakinra se asoció con una reducción en la pcr y mejoría en la función respiratoria en de pacientes ( %); en el grupo estándar, ocho de pacientes ( %) mostraron mejoría respiratoria a los días. en días de seguimiento, la sobrevida fue del % en el grupo de dosis altas de anakinra y del % en el grupo estándar (p = . ). se trata del primer estudio que demuestra seguridad y mejoría en los pacientes covid- , pero en el contexto fuera de la uci ( ) . huet y cols, de parís, francia, realizaron el estudio llamado ana-covid- en el que compararon pacientes tratados con anakinra subcutánea mg dos veces al día durante h, luego mg diarios durante días, con pacientes históricos; su criterio de inclusión fue tener una saturación de oxígeno del % o menos con un soporte de mínimo de l / min de oxígeno. la admisión a la uci por ventilación mecánica invasiva o muerte se produjo en ( %) pacientes en el grupo de anakinra y ( %) pacientes en el grupo histórico (hr . amci ® pacientes en el grupo histórico tuvieron un aumento en las aminotransferasas hepáticas ( ). recomendaciÓn se sugiere que la terapia con interferón sólo sea considerada en pacientes con formas graves de infección por covid- en el marco de un estudio clínico. estudios preliminares muestran que el virus del covid- induce una expresión muy débil de interferones en las células infectadas, lo que obstaculiza la respuesta inmune innata temprana a la infección y sugiere que el uso de interferón (ifn) exógeno para estimular la inmunidad antiviral ( ) . zhou q y col, en china, en un estudio observacional de pacientes con covid- con gravedad mixta proporcionó evidencia de muy baja calidad que la adición de interferón-α a la terapia con umifenovir no afecta el tiempo de eliminación viral o la duración en la estancia hospitalaria cuando se comparó con el umifenovir solo ( ) . hung if y cols, de hong kong, realizaron un ensayo multicéntrico, en adultos con covid- , en los que pacientes recibieron una combinación de lopinavir/ritonavir y tres dosis de millones de unidades internacionales ( · mg) de interferón beta- b en días alternos (grupo de combinación) y pacientes recibieron lopinavir/ritonavir (grupo control); la terapia de combinación fue segura y superior al control, para aliviar los síntomas y acortar la duración de la eliminación del virus y la estancia hospitalaria; se trata de un estudio fase , en pacientes con covid- leve a moderado (ningún paciente con ventilación en el grupo de combinación), en el que el ifn se administró en los primeros días de inicio de los síntomas y con el uso de un análogo de nucleósido oral (ribavirina), que no está en nuestras guías ( ) . se necesitan estudios de ifn solo o combinado en pacientes críticos con covid- . los efectos adversos de los ifn tipo i pueden limitar su uso para una intervención generalizada, como se propone en el brazo ifn-β con lopinavir/ritonavir del ensayo solidaridad de la oms. la administración por inhalación de vapor que se realiza actualmente en china ofrece la ventaja de acceso rápido al tracto respiratorio; sin embargo, la farmacodinamia y la farmacocinética de este modo de administración nunca se han evaluado. se sugiere no utilizar de rutina corticoides en el tratamiento de pacientes con sospecha o diagnóstico de covid- . no se puede considerar su uso profiláctico ni en pacientes con enfermedad leve sin requerimiento de oxígeno. débil en contra page en diciembre de , una serie de casos de neumonía de causa desconocida surgió en wuhan, hubei, china, con presentaciones clínicas muy parecidas a una neumonía viral; los análisis de secuenciación profunda de muestras del tracto respiratorio inferior indicaron un nuevo coronavirus, que se denominó novel coronavirus (covid- - ) ( ) . en la actualidad, en ausencia de terapia preventiva para sars-cov- , la piedra angular de atención para pacientes con covid- sigue siendo el manejo de apoyo, que va desde el tratamiento ambulatorio sintomático hasta el tratamiento intensivo completo con medidas de soporte en cuidados intensivos ( ) . dentro del manejo farmacológico se ha planteado la opción del uso de corticoides, la justificación estaría basada en la disminución de la respuesta inflamatoria del huésped a nivel pulmonar; es decir, un efecto inmunomodulador, ya que esta infección puede conducir a un síndrome de distrés respiratorio agudo (sdra); sin embargo, el beneficio puede verse superado por los efectos adversos, incluido el retraso en el aclaramiento viral y mayor riesgo de infección secundaria. a pesar de que la evidencia directa de corticoides en covid- es limitada, revisiones de los resultados en otras neumonías virales nos podrían orientar en principio en esta actual situación ( ) . teniendo en cuenta lo anterior; stockman y cols, en el . , realizaron una revisión sistemática sobre ensayos en pacientes con sars; quince ensayos examinan el uso de corticoides con diez o más pacientes en tratamiento; ensayos también recibían ribavirina; trece de estos estudios no fueron concluyentes; dos estudios describen un daño potencial con el uso de esteroides; en la literatura china estos autores encontraron catorce estudios con uso de esteroides en sars; doce fueron suspendidos por posible daño, la mayoría de estos ensayos se realizaron con muestras pequeñas de pacientes y de manera retrospectiva ( ) . arabi y cols, en noviembre de . , realizaron un estudio multicéntrico de cohorte retrospectivo en hospitales de atención terciaria de arabia saudita, donde se incluyeron pacientes; el uso de corticoides en pacientes con mers no se asoció con un cambio significativo a los días en la mortalidad y se documentó un retraso en la eliminación del arn de mers-cov ( ) . en cuanto hace referencia a la situación actual de pandemia por sars-cov- y compromiso pulmonar; wu y cols, en marzo de . realizaron un estudio retrospectivo de pacientes con covid- en china; para aquellos pacientes que desarrollaron sdra, el tratamiento con metilprednisolona estuvo asociado con una disminución del riesgo de muerte ( / [ %] con esteroides vs / [ %] sin esteroides; hr, . [ic %, . - . ]), con las limitaciones de los estudios retrospectivo, de un solo centro, con un limitado número de pacientes ( ). zha y cols, en marzo de . , describen el uso de corticosteroides en el tratamiento de pacientes con covid- ; no hallaron asociación entre la terapia con esteroides y el pronóstico de los pacientes sin sdra, siendo un estudio con una serie muy pequeña de pacientes ( ) . yang y cols, en marzo de . , en una revisión sistemática y meta-análisis que incluyó . pacientes de estudios, describen como el tratamiento con corticoides estuvo asociado con una mayor mortalidad (rr = . , ic % = . - . , p = , ), mayor estancia (wmd = . , ic % . - . , p = < , ) y una mayor tasa de infección bacteriana (rr = . , ic % . - . , p = < . ); con algunas limitaciones en este metaanálisis, la mayoría de los estudios incluidos son estudios de cohorte retrospectivos, controles históricos, con un bajo nivel de evidencia y una falta de ensayos controlados aleatorizados con buen diseño, sin un estándar uniforme para el tiempo y la dosis de los corticoides utilizado en los estudios; los efectos de los corticosteroides pueden ser influenciado también por otras opciones terapéuticas, como los medicamentos antivirales ( ) . por último, li y cols, en mayo de . , en otra revisión sistemática y metaanálisis, con respecto al uso de corticosteroides en sujetos con amci ® infecciones por sars-cov- , sars cov y mers-cov, se determinó que hubo retraso en la eliminación del virus sin mejoría en la supervivencia, reducción en la duración de la hospitalización o tasa de admisión en la uci y/o uso de ventilación mecánica; presentándose varios efectos adversos. debido a la preponderancia de los estudios observacionales en el conjunto de datos y los sesgos de selección y publicación, se concluye especialmente con respecto al sars-cov- , que se necesita mayor investigación con ensayos clínicos aleatorizados. internamente en este meta-análisis sugiere precaución al usar esteroides en pacientes con covid- ( ). horby y col en una rama del ensayo de evaluación aleatorizada de la terapia covid- (recovery), estudio aleatorizado, controlado, abierto que compara una gama de posibles tratamientos con la atención habitual en pacientes hospitalizados con covid- , compararon el uso de la dexametasona a dosis de mg día (oral o intravenosa) una vez al día por días o el alta según lo que ocurriera primero contra el manejo habitual; en pacientes aleatorizados que recibieron dexametasona se compararon con pacientes en manejo estándar; ( . %) pacientes en el grupo de dexametasona y ( . %) pacientes en el grupo control murieron a los días, con un riesgo relativo ajustado para la edad (rr . ; % ic . a . ; p < . ). la mortalidad relativa y absoluta variaron significativamente en relación al soporte ventilatorio al momento de la aleatorización; la dexametasona redujo las muertes en una tercera parte de los pacientes que recibieron ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p < . ), y una quinta parte en los pacientes que reciben oxígeno sin ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p = . ), pero sin reducir la mortalidad en paciente que no recibieron soporte respiratorio al momento de la aleatorización ( . % vs. . %, rr . , % ic . a . ]; p = . ) ( ). se recomienda no utilizar plasma convaleciente como tratamiento de rutina en paciente con sars-cov -covid- . se debe considerar su uso en el marco de un ensayo clínico y con alguno de los dos escenarios siguientes: escenario a (enfermedad severa), definida como uno o más de los siguientes: disnea, frecuencia respiratoria > /min, spo < %, pao /fio < o empeoramiento radiológico con aumento > % de los infiltrados pulmonares en - horas. escenario b (enfermedad que amenaza la vida), definida como uno o más de los siguientes: falla respiratoria, choque séptico, o disfunción multiorgánica. se recomienda no utilizar plasma convaleciente para profilaxis clínica de rutina contra la infección por sars-cov- , solo se debe considerar en el marco de un ensayo clínico. ( ) . una revisión sistemática y meta-análisis exploratorio realizado en identificó estudios de infección por coronavirus sars e influenza severa, el estudio reveló una reducción de la mortalidad, especialmente si el plasma convaleciente se emplea en la fase temprana de la enfermedad cuando se comparó con placebo o no tratamiento (or . ; ic del % . - . ); sin embargo, hay que tener presente que los estudios son de baja calidad, carecen de grupos control y puede tener riesgo moderado a alto de sesgo ( ) . se ha sugerido que el plasma convaleciente de pacientes que se han recuperado de covid- puede ser una terapia potencial, proporcionando inmunidad pasiva de los anticuerpos específicos contra sars-cov- y podría servir para prevenir y tratar la enfermedad ( ) . las personas que se han recuperado de la infección por sars-cov- pueden generar anticuerpos neutralizantes ( , ) que podrían tener aplicación en la prevención de infección en ciertos escenarios, como las personas con comorbilidades subyacentes que predisponen a enfermedad grave y aquellas con exposición de alto riesgo como los trabajadores de la salud y los expuestos a casos confirmados de covid- . existen algunos riesgos asociados con el uso de plasma convaleciente, unos conocidos y otros teóricos; los riesgos conocidos son aquellos asociados con la transfusión de hemocomponentes, incluida la transmisión de virus (ej. vih, vhb, vhc, entre otros) ( ); riesgo muy bajo, con los estándares de calidad actuales de los bancos de sangre; también se pueden presentar complicaciones no infecciosas, como las reacciones alérgicas, anafilaxia, reacción febril a la transfusión, lesión pulmonar aguda relacionada con la transfusión (trali), sobrecarga cardiaca asociada a transfusión (taco) y hemólisis si se administra plasma abo incompatible ( ) . los riesgos teóricos incluyen el empeoramiento de la infección dependiente de anticuerpos (antibody-dependent enhancement of infection -ade); el ade puede ocurrir en varias enfermedades virales e implica una respuesta inflamatoria exagerada ante la presencia de ciertos anticuerpos ( ) . otro riesgo teórico es que la administración de anticuerpos a las personas expuestas al sars-cov- puede evitar la enfermedad, pero modifica la respuesta inmune de tal manera que esos individuos monten respuestas inmunes atenuadas, lo que los haría vulnerables a la reinfección posterior, si se comprueba que este riesgo es real estos individuos podrían ser vacunados contra covid- cuando exista una vacuna disponible ( ) . durante el brote actual en china, se utilizó plasma convaleciente en algunos pacientes con covid- ( ), desde esta publicación se identificaron publicaciones relacionadas con el tema, entre todos los estudios fueron tratados con plasma convaleciente un total de pacientes( - ) (tabla ). shen y cols en marzo describieron en china el primer reporte en el cual el plasma convaleciente puede ser una opción de tratamiento en pacientes con covid- ; reportaron una serie de casos de pacientes críticamente enfermos con covid- y síndrome de dificultad respiratoria aguda (sdra), todos en ventilación mecánica, a quienes se les administró plasma convaleciente con anticuerpos neutralizantes [ a días después del inicio de la enfermedad (ddie)], todos ellos posteriormente mostraron mejoría clínica, la carga viral de los pacientes disminuyó y fueron negativas en los días posteriores a la intervención ( ) . de forma similar, duan y cols en mazo en china reportaron mejoría clínica en una serie prospectiva de casos de paciente severamente enfermos con covid- , que recibieron plasma convaleciente con un tiempo medio de . ddie ( a días) después del inicio de los síntomas, e hicieron amci ® una comparación con un grupo control histórico comparables en edad, género y severidad de la enfermedad ( ) . zhang y cols en mazo , en china reportaron una serie de casos de pacientes con covid- críticamente enfermos, en falla respiratoria con ventilación mecánica y dos de ellos con ecmo (membrana de oxigenación extracorpórea), a quienes se les dio tratamiento con plasma convaleciente en un tiempo medio . ddie, posteriormente todos tuvieron mejoría clínica ( ) . posteriormente ahn y cols en abril en corea, describieron una serie de dos casos de paciente con covid- severamente enfermos, en falla respiratoria y con ventilación mecánica, quienes además de recibir hidroxicloroquina, lopinavir/ritonavir y metilprednisolona, fueron tratados con plasma convaleciente entre - ddie, ambos pacientes se recuperaron y fueron liberados de la ventilación mecánica, uno fue dado de alta al momento del reporte ( ) . ye y cols en abril en china, describieron una serie de pacientes con covid- con anormalidades imagenológicas y deterioro clínico a pesar del tratamiento estándar y con pcr para sars -cov- persistentemente positiva, aunque no estuvieron en falla respiratoria o con ventilación mecánica, de hecho, una de las pacientes era portadora asintomática; todos recibieron plasma convaleciente entre a ddie, en todos los pacientes, excepto , hubo resolución de los cambios de vidrio esmerilado y consolidación, todos mejoraron y fueron dados de alta ( ) . zeng y cols en abril en china, reportaron una serie de casos con covid- en falla respiratoria y se compararon con controles que no recibieron plasma convaleciente por limitación en la disponibilidad y compatibilidad abo; a este grupo de paciente, se les administró plasma convaleciente en promedio . ddie, en todos los casos se negativizó la pcr para sars-cov- a los días después del tratamiento; sin embargo, contrario a los reportes de los estudios previos, en este grupo se murieron pacientes ( % vs %, p = . ), pero tuvieron mayor porcentaje de aclaramiento del virus (pcr sars-cov- negativa % vs . %, p = . ) antes de la muerte e incluso el tiempo de sobrevida fue mayor en el grupo de tratamiento (p = . ) ( ) . salazar y cols en mayo , en houston, texas, reportaron una serie de pacientes con covid- severa o amenazante para la vida, el desenlace primario fue seguridad y el secundario fue el estado clínico de los pacientes al día luego de la transfusión; al día posttransfusión, pacientes ( %) mejoraron con relación a su estado clínico basal, ( %) no tuvieron cambios y pacientes tuvieron deterioro clínico. siete de los nueve pacientes que mejoraron ( %) habían sido dados de alta; para el día post-transfusión, ( %) de los pacientes presentaron mejoría y pacientes más, habían sido dados de alta; tres pacientes permanecían sin cambios, pacientes se deterioraron y uno murió por una condición no relacionada con el plasma; el promedio de estancia hospitalaria fue de . días y la estancia hospitalaria luego de la transfusión fue en promedio días; hubo una disminución de los valores promedio de pcr desde . mg/dl el día , a , mg/dl y . mg/dl los días y respectivamente; al momento de la publicación del artículo, solo permanecían intubados pacientes; todos los pacientes que requirieron ecmo ya se habían liberado y ( %) fueron dados de alta ( ) . también en texas, estados unidos, ramachandruni y col en mayo , reportaron una serie de casos con covid- severa, falla respiratoria y en ventilación mecánica o pao /fio < , todos con comorbilidades; a los cuales les administraron metilprednisolona y posteriormente plasma convaleciente; compararon los valores basales de pao /fio y posterior a la intervención; encontrando, mejoría de la pao /fio en % luego del tratamiento con esteroides y en % luego de la administración de plasma convaleciente ( ) . finalmente, en respuesta al brote de covid- en los eeuu y las tasas de mortalidad reportadas, la fda en colaboración con la mayo clinic y la comunidad nacional de bancos de sangre desarrollaron un programa nacional de acceso ampliado para recolectar y distribuir plasma convaleciente donado por amci ® individuos que se han recuperado de covid- ; entre abril y mayo , ; fueron incluidos . pacientes con covid- severa o potencialmente mortal o con riesgo alto de progresión a covid- severa o potencialmente mortal en el programa nacional de acceso ampliado; en ese tiempo, un total de . pacientes inscritos recibieron transfusión de plasma convaleciente covid- . en una publicación reciente, joyner y cols en mayo , en estados unidos, hicieron un análisis de seguridad después de la transfusión de plasma convaleciente covid- humano con compatibilidad abo en . adultos hospitalizados con covid- grave o potencialmente mortal, % de los cuales se encontraban en uci; la incidencia de eventos adversos serios (eas) durante las horas siguientes a la transfusión de plasma convaleciente fue < %, incluyendo mortalidad ( . %); de los eas reportados, hubo incidentes reportados como eas relacionados, incluyendo muertes, eventos de taco, trali y reacciones alérgicas graves asociadas a la transfusión; sin embargo, solo (de ) eas fueron considerados definitivamente relacionados con la transfusión de plasma convaleciente por el médico tratante; en este grupo de pacientes, la tasa de mortalidad a los siete días luego de la administración del plasma convaleciente, fue del , % ( ) . a pesar que la tasa general de letalidad para la covid- parece ser aproximadamente . % ( ), la tasa de mortalidad reportada parece no ser excesiva si la comparamos con los informes de wuhan que sugieren tasas de letalidad del % para los pacientes hospitalizados ( ) y % entre los pacientes en unidad de cuidados intensivos ( ) . los nueve estudios mostraron mejoría en muchos aspectos, incluyendo el aclaramiento del virus, la disminución del suplemento de oxígeno y la ventilación mecánica, la normalización de los valores de laboratorio, y la recuperación en los hallazgos pulmonares radiológicos. todos los estudios, reportaron que no se presentaron eventos de seguridad o reacciones adversas serias relacionadas con la administración de plasma convaleciente en pacientes con covid- , excepto en casos relacionados, según criterio de los médicos tratantes, en el estudio publicado por joyner y cols ( ) . esta serie de estudios son alentadores; sin embargo, la mayoría de los reportes de casos tienen limitaciones significativas: carecen de los ajustes para factores de confusión críticos, incluidos los co-tratamientos, las características basales, la gravedad de la enfermedad y el momento de administración del plasma y deben ser seguidas de investigaciones adicionales. para establecer mejor el papel del plasma convaleciente es necesario realizar estudios dirigidos a los siguientes escenarios: . el uso como profilaxis post-exposición . evaluar si el plasma convaleciente es útil en paciente con enfermedad leve . el efecto del plasma convaleciente en pacientes con enfermedad moderada . el tratamiento de rescate con plasma convaleciente en pacientes que requieren ventilación mecánica debido a covid- . finalmente, trabajos que evalúen la seguridad y farmacocinética del plasma convaleciente en los pacientes pediátricos con alto riesgo. actualmente están en curso varios estudios para evaluar el tratamiento de pacientes infectados con sars-cov- con plasma convaleciente. una búsqueda realizada el de mayo de en clinicaltrials.gov con los términos "plasma convaleciente y covid- " mostró ensayos en curso sobre el uso de plasma convaleciente en pacientes con amci ® covid- , que nos ayudarán a resolver las inquietudes relacionadas con esta intervención ( ) . page se recomienda no utilizar de rutina las las inmunoglobulinas hiperinmunes en pacientes con infección por sars-cov- /covid- . la inmunoglobulina hiperinmune (h-igiv) se deriva de individuos con altos títulos de anticuerpos contra patógenos específicos y se ha utilizado con éxito en el tratamiento de infecciones, como el citomegalovirus y la gripe h n ( ) . se propone que la inmunoglobulina hiperinmune combinada con medicamentos antivirales puede ser efectiva en el tratamiento de pacientes con covid- , estos anticuerpos (ac) recogidos de los pacientes recuperados serán específicos contra covid- al aumentar la respuesta inmune en pacientes recién infectados ( ) . existe evidencia más sólida para el uso de h-igiv en el tratamiento de enfermedades virales. cheng y cols en enero de , realizaron una revisión retrospectiva en hong kong, que reveló que el plasma convaleciente de los sobrevivientes de sars-cov administrados a pacientes con sars-cov que tenían enfermedad progresiva resultó en tasas de alta significativamente más altas en el día y tasas de mortalidad más bajas, en comparación con los controles históricos ( ) . hung y cols en febrero de , realizaron un estudio de cohorte prospectivo sobre la efectividad del plasma convaleciente de los sobrevivientes de h n con un título de ≥ : ofrecido a pacientes de la uci con infección grave por h n ; los pacientes que rechazaron las infusiones de plasma convalecientes fueron controles; veinte de los pacientes recibieron sueros convalecientes, el tratamiento con plasma convaleciente condujo a una reducción significativa de la carga viral respiratoria, los niveles séricos de citocinas (il- , il- , tnfα) y la mortalidad ( ) . posteriormente hung y cols en agosto de , publicaron un estudio multicéntrico prospectivo, doble ciego, aleatorizado y controlado en el que compararon la efectividad de la inmunoglobulina hiperinmune (h-igiv) del plasma convaleciente de los sobrevivientes de h n versus la inmunoglobulina iv (igiv) normal, en pacientes con h n en uci con soporte respiratorio y recibiendo oseltamivir; este estudio mostró, una reducción de la carga viral y una mayor supervivencia en el grupo que recibió h-igiv dentro de los días posteriores al inicio de los síntomas, demostrando la superioridad de la inmunoglobulina hiperinmune sobre la igiv en el tratamiento de la infección grave por h n ( ). el uso de inmunoglobulina hiperinmune ha demostrado una clara efectividad en el tratamiento de la gripe y el sars-cov; sin embargo, el plasma se debe recolectar y procesar de pacientes convalecientes y verificar que tenga títulos adecuados. según la experiencia con el sars-cov, lo ideal es recolectar plasma de pacientes con un curso de enfermedad más leve ( ) . poco se sabe sobre la seguridad de la inmunoglobulina hiperinmune cuando se usa para el tratamiento de infecciones por coronavirus, los riesgos incluyen la exacerbación de la infección dependiente de anticuerpos (antibody-dependent enhancement of infection -ade)( ); el ade puede ocurrir en varias enfermedades virales, e implica una respuesta inflamatoria exagerada ante la presencia de ciertos anticuerpos; sin embargo, los estudios en sars y mers no proveen información suficiente para extrapolarse a la infección por sars-cov- . no se encontraron estudios con inmunoglobulina hiperinmune en el tratamiento de pacientes con covid- . no se puede emitir una recomendación a favor o en contra para el uso de la inmunoglobulina intravenosa como tratamiento adyuvante en pacientes con covid- severo. se debe considerar la inmunoglobulina intravenosa como tratamiento adyuvante en pacientes con covid- severo, en el contexto de estudios clínicos en los siguientes escenarios. escenario a (enfermedad severa), definida como uno o más de los siguientes: disnea, frecuencia respiratoria > /min, spo < %, pao /fio < o empeoramiento radiológico con aumento > % de los infiltrados pulmonares en - horas. escenario b (enfermedad que amenaza la vida), definida como uno o más de los siguientes: falla respiratoria, choque séptico, o disfunción multiorgánica. en las enfermedades virales, los anticuerpos ejercen su efecto por neutralización viral (bloqueo de la entrada de células virales y, por lo tanto, replicación), activación del complemento, opsonización y mediación de citotoxicidad celular dependiente de anticuerpos. la neutralización viral es específica de antígeno; otras actividades antivirales son antígeno-inespecíficas y se realizan en parte a través de interacciones fc: fc receptor. en la infección por sars-cov- , el principal antígeno objetivo asociado con la neutralización es la proteína spike, que es responsable de la unión del sars-cov- a las células epiteliales, incluidos los neumocitos; los anticuerpos en las inmunoterapias pasivas covid- son de naturaleza policlonal, con múltiples epítopos contra los paratopes de sars-cov- , incluido el dominio de unión al receptor en la proteína spike ( ) . la inmunoglobulina intravenosa (igiv) es un producto derivado del plasma de miles de donantes utilizados para el tratamiento de inmunodeficiencias primarias y secundarias, afecciones autoinmunes/inflamatorias, trastornos neuroinmunológicos y secuelas relacionadas con infecciones; la igiv proporciona protección inmune pasiva contra una amplia gama de patógenos; actualmente, la experiencia con el uso de igiv en el tratamiento de la infección por sars-cov- es muy limitada; sin embargo, la justificación del uso de ivig en la infección por sars-cov- es la modulación de la inflamación ( ) . la igiv se ha usado en el tratamiento de otros coronavirus, incluido el sars cov. stockam y cols, en septiembre de , en respuesta a una petición de "the world health organization -who", realizaron una revisión sistemática de los efectos del tratamiento en los pacientes con síndrome respiratorio agudo, incluida la igiv o el plasma convaleciente; se evaluaron cinco estudios sobre el uso de igiv o plasma convaleciente administrado además de corticosteroides y ribavirina, se consideró que estos estudios no fueron concluyentes ya que los efectos de la igiv o el plasma convaleciente no podían distinguirse de otros factores que incluían comorbilidades, estadio de la enfermedad o el efecto de otros tratamientos ( ) . wnag y cols, en mayo , hicieron un estudio prospectivo, en un solo centro, de infección por sars en taiwán, se administró igiv si el paciente tenía leucopenia amci ® o trombocitopenia, o si había progresión rápida de la enfermedad en la radiografía; un total de pacientes recibieron igiv, de los cuales tenían citopenias graves, uno de ellos tenía evidencia de síndrome hemofagocítico y paciente tuvieron progresión radiológica de la enfermedad; el estudio sugiere que la igiv condujo a una mejora significativa en el recuento de leucocitos y plaquetas, pero reconoce que no había un grupo de control para evaluar objetivamente las respuestas ( ) . lew y cols, en julio , reportaron un estudio retrospectivo de un solo centro en singapur, se encontró que los pacientes adultos con sars tratados con un régimen de pulso de metilprednisolona ( mg) e igiv ( . mg/kg) diariamente durante tres días consecutivos tuvieron una hazard ratio ajustada de . para mortalidad en comparación con el grupo no tratado, con una tendencia hacia una recuperación más temprana; sin embargo, este hallazgo no fue estadísticamente significativo (ic del %: . a . ; p = . ); además, este resultado tuvo como factor de confusión el uso concurrente de esteroides ( ) . aunque algunas de las preparaciones de igiv comercializadas actualmente (gamunex-c y flebogamma) contienen anticuerpos que reaccionan de forma cruzada contra el sars-cov- y otros antígenos de virus, in vitro( ), hasta la fecha, ningún ensayo clínico de alta calidad ha demostrado eficacia y seguridad convincentes de igiv en epidemias de coronavirus. a pesar de que los datos para el uso de igiv en la infección por sars y mers son débiles, la dosis alta de ivig puede ser útil en la infección grave por sars-cov- a través de la modulación inmune, saturando fcγr y reduciendo ade ( ) . en general, la inmunoglobulina intravenosa es bien tolerada y el perfil de seguridad es bien conocido. las reacciones adversas comunes son leves y autolimitadas, pero se sabe que en pacientes de alto riesgo se producen efectos adversos graves, como trombosis, disfunción renal y muerte. en cuanto la evidencia (tabla ); xie y cols en abril, en wuhan, china, realizaron un estudio retrospectivo, revisando casos de covid- severa (disnea, fr > /min, spo < % en reposo, pao /fio < , progresión imagenológica > % en - horas) o críticamente enfermos (falla respiratoria con ventilación mecánica, choque, disfunción orgánica múltiple) en el cual evaluaron la mortalidad a días como desenlace primario y como desenlace secundario evaluaron la mortalidad a días, días de estancia hospitalaria, de uci y la necesidad de ventilación mecánica; reportaron que el tratamiento con igiv dentro de las horas posteriores al ingreso no sólo redujo el uso de la ventilación mecánica comparado con el tratamiento luego de horas del ingreso ( . % vs . %, p = . ), sino que también redujo la duración de la estancia en el hospital ( . ± . vs . ± . días, p = . ) y la uci ( . ± . vs . ± . días, p = . ); mejorando en última instancia la mortalidad a los días (p = . ); concluyen, que el estudio demostró que el tratamiento con igiv en pacientes con covid- con neumonía grave puede mejorar los indicadores en poco tiempo y mejorar la eficiencia del tratamiento de los pacientes con alta efectividad ( ) . el tratamiento con dosis altas de igiv ( g/día durante días) al inicio del distrés respiratorio, sumado al tratamiento de soporte y en un caso combinados con antivirales (lopinavir/ritonavir) y metilprednisolona en covid- grave publicado por cao y cols en marzo, en wuhan, china ( ), demostró la elevación de los recuentos de linfocitos, disminución de los marcadores inflamatorios, recuperación de la oxigenación, resolución parcial/completa de las alteraciones radiológicas pulmonares y las pruebas de hisopos nasales y orofaríngeos negativos dentro de unos pocos días después del inicio tratamiento (< días). lanza y cols en mayo , reportaron en nápoles, italia, el caso de una mujer de años que tenía covid- severa y que venía con deterioro clínico a la cual se le venía dando tratamiento con hidroxicloroquina más azitromicina, no se le administraron amci ® esteroides por el riesgo de disminuir la depuración de la viremia, a quién se le administró igiv el día después de iniciados los síntomas con mejoría clínica rápida, normalización de los gases arteriales y disminución marcada de los infiltrados pulmonares al día y respectivamente; como evento adverso, reportaron hipotensión durante el inicio de la infusión que se mejoró al disminuir la velocidad de infusión. la paciente finalmente se recuperó, negativizó rt-pcr sars-cov- y fue dada de alta ( ) . se recomienda la tromboprofilaxis farmacológica en todos los pacientes confirmados o sospechosos de covid- severo, a menos que está contraindicada, en cuyo caso es razonable la implementación de profilaxis no farmacológica. en términos generales se reconoce que los pacientes hospitalizados con enfermedad médica aguda, incluidas infecciones como la neumonía, tienen un mayor riesgo de eventos tromboembólicos ( ) . tang y cols, en china describieron múltiples trastornos de la coagulación en pacientes con covid- , aquellos pacientes que no sobrevivieron tenían significativamente mayores niveles de dímero-d y productos de degradación de la fibrina y con tiempos de coagulación convencionales más alargados al ingreso (p < . ); . % de los no sobrevivientes y . % de los sobrevivientes cumplían criterios para coagulación intravascular diseminada( ); este mismo grupo realizó un estudio con pacientes con covid- severo, pacientes con coagulopatía asociada a covid- (cac), definida por un sic score ≥ la utilización de tromboprofilaxis redujo de manera significativa la mortalidad a días ( . % vs . %, p = . ) así como también en aquellos con un se recomienda no utilizar de rutina antiagregación en pacientes con covid- severo con el fin de prevenir desenlaces neurológicos adversos. no se encuentran estudios en la literatura para el uso de antiagregantes para el manejo específico del covid- ; el manejo de los eventos cardiovasculares en pacientes covid- no difiere de la población general sin la enfermedad. no se establecen diferencias en los estudios descritos, ni en las publicaciones hasta la fecha de la revisión ( ) . no se puede emitir una recomendación a favor o en contra sobre el uso de la anticoagulación terapéutica de rutina con heparinas de bajo peso molecular o heparina no fraccionada en pacientes con bajo riesgo de sangrado y con un curso clínico grave o crítico, que además tengan elevación del dímero d mayor a mcg/ml ( ng/ml) y/o fibrinógeno mayor a mg/dl. diferentes publicaciones describen como una estrategia de tratamiento basada en profilaxis con heparina de bajo peso molecular (hbpm) para tratar la coagulopatía grave por covid- podría no ser suficiente. especialmente porque estos pacientes tienen dentro de su coagulopatía, una predisposición mayor a la presencia de trombosis que al sangrado. además, los bajos niveles de antitrombina que se han descrito en estos pacientes, los hace más resistentes a la heparina, lo que sugiere que las dosis profilácticas ya sea de heparina no fraccionada o hbpm pueden ser inadecuadas( - ). asociaciÓn colombiana de medicina crÍtica y cuidados intensivos. amci ® no se puede emitir una recomendación a favor o en contra sobre la anticoagulación terapéutica de rutina con heparinas de bajo peso molecular en pacientes con covid- en estado crítico, que presenten elevación del dímero d mayor a mcg/ml ( ng/ml). tang y cols, en china con un estudio retrospectivo donde se incluyeron pacientes con covid- severo, se evaluó la presencia de trombosis como desenlace asociado. este estudio describe como niveles de dímero d por encima de ng/ml estaban asociados a una mayor probabilidad de muerte y en estos pacientes el tratamiento anticoagulante reduce de manera significativa la mortalidad ( . , ic % . - . ) ( , ) . no se puede emitir una recomendación a favor o en contra para la medición rutinaria de niveles de anti xa en pacientes con covid- en los que se decide hacer anticoagulación terapéutica con hbpm. se puede considerar la medición de niveles anti xa si se cuenta con la disponibilidad del recurso. harr y cols, en un estudio en donde se incluyeron pacientes con hiperfibrinogenemia relacionada a trauma, se evidenció como los niveles de fibrinógeno se correlacionaron significativamente con la consistencia del coágulo y adicionalmente como se genera una relación inversa entre los niveles de fibrinógeno y la actividad de las hbpm, lo que sugeriría una potencial resistencia a la heparina. basados en que los pacientes covid- presentan niveles de fibrinógeno en muchas ocasiones con niveles > mg/dl e incluso > mg/dl lo que hace razonable considerar que aquellos pacientes covid- que requieren dosis terapéuticas de hbpm y es posible evaluar los niveles de anti xa, hacer ajustes y monitoreo del nivel de anticoagulación sería una opción razonable ( ) . se recomienda el uso de hbpm o hnf para la anticoagulación terapéutica en pacientes con una indicación específica con diagnóstico de covid- . amci ® como previamente se describió, una revisión sistemática comparó las dosis fijas de hbpm subcutánea con dosis ajustadas de hnf intravenosa o subcutánea en personas con clínica sugestiva de tep, esta revisión demostró que la incidencia de tep recurrente fue menor con hbpm que en los participantes con hnf (or . , ic % . a , ), también se asoció con una reducción en el tamaño del trombo (or . , ic % . a . ), evidencia de baja calidad. sin embargo, no hubo diferencias en la mortalidad general entre los participantes tratados con hbpm y los tratados con ufh (or . , ic % . a . ). por otra parte, los protocolos de manejo en escenarios de coagulación intravascular diseminada (cid), proponen el uso de hnf por encima de la hbpm en pacientes en los que se indica la anticoagulación. más aún, la presencia de falla renal aguda es común en los pacientes con covid- , por lo que la opción de tratamiento con hnf tiene también escenarios en donde podrían ser de elección ( , ) . capítulo . procedimientos y covid- recomendaciÓn se recomienda realizar la preoxigenación en pacientes con sospecha o diagnóstico de covid- , cuando estén disponibles, en áreas de presión negativa con mínimo recambios de aire por hora o en instalaciones con ventilación natural o que tengan un recambio de aire de mínimo l/seg si están disponibles. se recomienda como complemento opcional durante la preoxigenación en el paciente crítico con diagnóstico o sospecha de covid- la caja de acrílico para protección durante la intubación, la caja no protege contra la generación de aerosoles fuera de esta y requiere para su uso, entrenamiento previo. si es difícil su uso retírela inmediatamente. se recomienda utilizar en la mascarilla quirúrgica sobre la mascarilla de oxigenación en el paciente crítico con sospecha o diagnóstico de covid- . se recomienda utilizar filtros hpfa entre la máscara y el dispositivo disponible para la preoxigenación en el paciente crítico con sospecha o diagnóstico de covid- . se recomienda en el paciente con sospecha o diagnóstico de covid- preoxigenación por a min, si el paciente luego de minutos no tiene incremento en la mejoría inicie la administración de medicamentos. se recomienda en caso de compromiso hemodinámico considerar ventilación a dos manos con cierre hermético de la máscara sobre la cara del paciente con frecuencias altas (> por minuto y baja presión). amci ® fundamento el manejo de la vía aérea es un procedimiento considerado generador de aerosoles, la enfermedad covid- tiene una alta tasa de transmisión y el personal de salud requiere el uso estricto del epp ( , ) , revise el enunciado para epp recomendado en esta guía. la posibilidad de permanencia del virus en algunos ambientes puede durar hasta horas, por esto una estrategia para proteger al equipo de salud y otros pacientes podrías ser estar en áreas con presión negativa, lo cual no es fácil de encontrar en nuestro contexto o que tenga un adecuado recambio de aire( ). como medidas complementarias se pueden utilizar opciones como las cajas acrílicas para intubación, esta disminuye el riesgo de contaminación por gotas, pero no elimina los aerosoles, es necesario previo entrenamiento. en caso de que la caja sea una limitante retírela inmediatamente ( ) . los pacientes covid- , clásicamente se presentan con tos, esto es un factor de riesgo para quienes manipulan la vía aérea, la utilización de mascarilla por parte del paciente debajo del dispositivo bolsa mascarilla , la cual también deberá tener un filtro de alta eficiencia para disminuir el número de partículas que pueden estar en el ambiente durante la preoxigenación y posible ventilación, ésta última la cual será evitada al máximo ( , , ) . la preoxigenación es una técnica que pretende barrer el nitrógeno y aumentar la disponibilidad de oxígeno para que cuando el paciente presente apnea por los medicamentos para la intubación o por su condición clínica, se disponga de un tiempo mayor sin desaturación crítica y riesgo de colapso cardio-respiratorio. considere que los pacientes con formas moderadas y severas de covid- , pueden tener más comprometida esta reserva respiratoria y puede no ser efectiva la preoxigenación y cuando inicia la apnea, la desaturación será más precoz. si después de minutos de preoxigenación no hay mejoría de la saturación arterial, considere fallida esta maniobra y considere mayor riesgo de hipoxemia severa con probabilidad de bradicardia extrema y paro cardiorrespiratorio. en caso de requerir ventilación por disminución rápida de la saturación de oxígeno arterial y considere necesario ventilación manual bolsa mascarilla a dos manos no debe ser vigorosa y debe utilizar filtros hpfa, estos reducirán los aerosoles en el ambiente( , ). se recomienda la utilización de cajas de acrílico para intubación del paciente con sospecha o diagnóstico de covid- como complemento durante la intubación para disminuir el riesgo de contaminación por gotas, sin embargo, no protege % la generación de aerosoles y si ésta hace más difícil la intubación retírela inmediatamente. amci ® se recomienda minimizar los intentos de intubación orotraqueal en el paciente con sospecha o diagnóstico de covid- , el primer intento debe procurar ser realizado por el más experimentado en el manejo de la vía aérea. se recomienda la intubación con videolaringoscopio en pacientes con covid- teniendo en cuenta disponibilidad y entrenamiento); esta alternativa puede ofrecer ventajas frente a la laringoscopia tradicional. se recomienda administrar medicamentos para asegurar la vía aérea en secuencia de inducción rápida, para obtener en el menor tiempo posible condiciones para la intubación ( a s). se recomienda en caso de intubación fallida por personal experto, considerar dispositivos supraglóticos como las máscaras laríngeas como medida de rescate con el riesgo de generación de aerosoles. se recomienda contar con disponibilidad de vasopresor y atropina en el sitio donde se realizará la intubación. en caso de contar con lidocaína se recomienda utilizar a dosis de mg/kg sin epinefrina. administrada minutos antes de la intubación. como se describió en el aparte de preoxigenación, se deben garantizar todas las medidas basadas en bioseguridad y protección adecuada para el personal de la salud, ubicación en áreas que cuenten con presión negativa o salas con un óptimo recambio de aire pueden ayudar a disminuir el riesgo de contagio. las medidas complementarias como la caja de taiwán o caja de intubación puede complementar de forma opcional estas medidas durante el proceso de aseguramiento definitivo de la vía aérea pero no garantiza % la eliminación de aerosoles, pudiendo ser un obstáculo para quien realiza la intubación, de ser así, se deberá retirar inmediatamente, por eso sólo es un complemento al epp, que es la verdadera protección en estos escenarios (se debe incluir máscaras n o ffp fpp ) ( , ) . el paciente críticamente enfermo puede tener comprometida de forma significativa su reserva respiratoria y hemodinámica por su cuadro clínico y puede empeorar por los medicamentos utilizados durante la intubación orotraqueal. las complicaciones en el manejo de la vía aérea se presentan cuando se realiza más de un intento dentro de los cuales están el traumatismo, desaturación e hipoxemia ( ) . es por esto por lo que se debe procurar que el primer intento sea realizado por el más experimentado en el manejo de la vía aérea e idealmente se logre la intubación en el primer intento con la menor hipoxemia secundaria ( , , ( ) ( ) ( ) . basado en el planteamiento anterior y considerando la ventaja de intubar pacientes críticamente enfermos en el primer intento dadas sus condiciones clínicas, la revisión de amci ® demandas asociadas a manejo de vía aérea en los estados unidos sigue siendo preocupante al considerar posibles causas la falta de entrenamiento y falta de aplicación de las guías y recomendaciones de manejo así como en la utilización de equipos adecuados para pacientes específicos ( ) ; se ha planteado especialmente en los pacientes covid- los cuales tienen un importante compromiso pulmonar y alto riesgo de desaturación, la posibilidad de encontrar una vía aérea difícil no predicha y dificultades en su manejo( , ). es por esto que se requiere del mejor dispositivo para manejo de vía aérea invasiva disponible, siempre y cuando se cuente con el adecuado entrenamiento previo en su uso, la ventaja de los videolaringoscopio se debe a la superioridad al compararla con la laringoscopia convencional, siendo en algunos grupos la primera opción para intubaciones electivas ( , ) . como se ha mencionado, el riesgo de una rápida desaturación en pacientes con enfermedad pulmonar, así como en pacientes con covid- , se debe utilizar la inducción de secuencia rápida la cual se utiliza para pacientes con estómago lleno en los cuales se quiere lograr condiciones de inconciencia y de intubación óptimas en el menor tiempo posible para disminuir el riesgo de broncoaspiración. en estos casos de falla respiratoria con tan mala reserva se quiere aprovechar la ventaja que ofrece esa técnica para tener en poco tiempo al paciente intubado con menor riesgo de desaturación. es así como los medicamentos en una inducción de secuencia rápida incluyen el opioide, hipnótico y relajante, estos dos últimos administrados simultáneamente y lavados con un bolo de cc. la opción del opioide en nuestro contexto suele ser fentanilo a dosis de a mcg / kg iv, dando a minutos de latencia para su efecto, luego el hipnótico que puede ser propofol entre y , mg / kg si la estabilidad hemodinámica lo permite o considera usar vasopresor simultáneo. en caso de preferir evitar la hipotensión la ketamina a dosis de a , mg / kg es una opción más estable hemodinámicamente. con relación al relajante neuromuscular la succinilcolina es la clásicamente utilizada dosis de a , mg/kg pero debido a sus efectos secundarios como hiperpotasemia, fasciculaciones, mialgias y un importante riesgo de hipertermia maligna algunos grupos no lo consideran, el rocuronio a dosis de , mg/kg ha demostrado lograr tiempos y condiciones de intubación similar a la succinilcolina sin los efectos secundarios de esta ( , ) algunos grupos han considerado no utilizarlos si la condición clínica del paciente es crítica pues éste período de latencia puede ser acompañado de una hipoxemia severa y paro cardíaco, por lo tanto sólo usan hipnótico y relajante neuromuscular. ante una vía aérea difícil no predicha en la cual no se logre la intubación, considere los dispositivos supraglóticos como las máscaras laríngeas los cuales son más fáciles de insertar en comparación con la técnica de intubación orotraqueal, y deben estar dentro del planeamiento y organización de elementos para manejo de la vía aérea invasiva. al lograr ventilar con este dispositivo se logrará una recuperación del paciente, pero se pueden generar aerosoles pues este mecanismo de cierre puede permitir escape de aire y macropartículas durante el ciclo respiratorio ya sea manual o mecánico ( , ) . el cuadro clínico de pacientes críticos y particularmente covid- , puede asociarse a inestabilidad hemodinámica y requerimiento de soporte vasopresor. la adición de medicamentos como los opioides o hipnóticos pueden asociarse a hipotensión la cual puede no responder a volumen, es necesario evitar episodios de hipotensión en especial en pacientes ancianos, con enfermedades cardiovasculares de base las cuales pueden tolerar menos estos cuadros de hipotensión, así como también pueden favorecer amci ® desbalance en la relación ventilación/perfusión a nivel pulmonar empeorando los cuadros de hipoxemia. la hipoxemia puede acompañarse de bradicardia y si no se corrige la ventilación o la bradicardia es muy probable que el paciente presente paro cardiorrespiratorio, por lo cual se recomienda utilizar una dosis de atropina para corregir la bradicardia, no mejorará la oxigenación, pero tendrá un tiempo adicional para recuperar la oxigenación y ventilación del paciente. en estos casos debe tener disponibles vasopresores y atropina desde el planeamiento de los medicamentos necesarios para el manejo de la vía aérea ( ) . se recomienda la intubación orotraqueal oportuna y no retrasar el inicio de la ventilación mecánica invasiva en los pacientes con sdra severo por covid- debido a mayor riesgo de desenlaces adversos. fundamento definir el momento de la intubación en esta población es un reto. la mayoría de los autores recomiendan el inicio "temprano" de la ventilación mecánica, sin embargo, la definición de cuando es temprano no es clara. este punto es motivo de análisis dado que a la luz de la evidencia actual la utilización de métodos no invasivos como la ventilación no invasiva y la cánula de alto flujo, para manejo inicial de pacientes con algún grado de hipoxemia es controvertido entre otras por el riesgo que supone al personal sanitario al ser un procedimiento generador de aerosoles. en el un estudio mostró que la intubación retrasada después de la falla al utilizar cánula de alto flujo o la ventilación no invasiva para pacientes con insuficiencia respiratoria moderada y grave se asoció con una mayor mortalidad. publicaciones recientes muestran que solo la quinta parte de los pacientes que murieron por covid- recibieron ventilación mecánica invasiva y soporte respiratorio más agresivo antes de la muerte, lo que indica que en muchos pacientes se habría retrasado la intubación. de los pacientes fallecidos solo el % recibieron tratamiento de oxígeno nasal o con mascarilla facial antes de su muerte. esta baja proporción puede tener varias explicaciones. primero, algunos pacientes con hipoxemia severa no tenían otros síntomas, como dificultad para respirar o disnea, es decir, desarrollaron una especie de hipoxemia silenciosa. en segundo lugar, la falta de suficientes ventiladores mecánicos invasivos es una razón importante que evitaría que los pacientes reciban intubación. tercero, el manejo de estos pacientes por un equipo de médicos no intensivistas; por lo tanto, pueden no estar seguros del momento en que un paciente requiere intubación. la serie de casos de la epidemia de covid- en wuhan mostró que la intubación tardía era común en la etapa inicial de la epidemia, mostrando que una de esas posibles razones del retraso incluía falta de ventiladores mecánicos invasivos y falta de capacitación clínica específica para el soporte respiratorio. recomendaciÓn se recomienda tomar la decisión de intubación orotraqueal en el paciente crítico con sospecha o diagnóstico de covid- utilizando una combinación de variables clínicas, gasométricas y hemodinámicas. tabla la intubación orotraqueal (iot) efectiva y segura, programada debe prevenir el colapso respiratorio y hemodinámico. siempre es necesario asegurar la escena del procedimiento de intubación con las consideraciones técnicas y de protección personal adecuadas. conocer los pasos para la realización del procedimiento de intubación orotraqueal (iot) contextualizados al paciente covid- , reduce los riesgos innecesarios. la iot es un procedimiento generador de aerosoles por lo tanto lo ideal es realizarlo en una habitación con presión negativa, sin embargo, la baja disponibilidad en el país obliga a utilizar otras alternativas de seguridad. una habitación de presión negativa es un cuarto que tiene una presión más baja que las áreas adyacentes, lo que mantiene el flujo de aire fuera de la habitación y hacía habitaciones o áreas contiguas. las puertas de la sala deben mantenerse cerradas, excepto al entrar o salir de la sala, y la entrada y la salida deben minimizarse. la intubación en el paciente crítico con covid- es de los procedimientos que mayor riesgo de aerosolización tiene, por lo tanto, se debe adoptar una posición de intervención oportuna, pero también segura, para evitar desenlaces desfavorables en el paciente y disminuir el riesgo de contaminación en el personal de salud, se recomienda individualizar cada caso mediante la combinación y análisis de los criterios clínicos, gasométricos y hemodinámicos de cada paciente. ( , ( ) ( ) ( ) se recomienda no realizar de rutina broncoscopia en los pacientes con sospecha o diagnóstico de covid- , debido al riesgo de generación de aerosoles. se puede considerar en atelectasias masivas con compromiso significativo de la oxigenación adicional a la lesión pulmonar per sé y la hemorragia alveolar para control local directo. en general la realización de broncoscopia en pacientes con sospecha o confirmación de covid- debe ser evitada y realizarse sólo con indicación de emergencia como cuerpo extraño en la vía aérea, hemoptisis masiva, obstrucción grave de la vía aérea central o atelectasia lobar o pulmonar completa( ). esto debido a la alta carga viral en la mucosa nasal y faríngea de los pacientes con infección por sars-cov- ( ) y la alta producción de aerosoles infecciosos que se generan durante este procedimiento. en caso de ser necesaria su realización, deberá ser llevada a cabo por el operador con mayor experiencia amci ® minimizando el tiempo de exploración y el personal expuesto en la sala. es mandatorio utilizar epp completo que incluya respirador fpp o fpp , bajo protocolo supervisado de donning y doffing ( ) . son de elección los broncoscopios desechables de un solo uso, pero de no ser posible se prefiere el uso de un broncoscopio flexible por encima de uno rígido por la más fácil manipulación de este. idealmente el procedimiento se llevará a cabo en el mismo cubículo del paciente que deberá contar con presión negativa y recambio de aire de a veces por hora. recomendaciÓn se recomienda no realizar de rutina la broncoscopia para la recolección de muestras para el diagnóstico de covid- en el paciente críticamente enfermo. la broncoscopia es una prueba de segunda elección para la toma de muestras respiratorias en los pacientes con sospecha o confirmación de covid- ( ) . la toma de muestras del tracto respiratorio superior por hisopado nasofaríngeo u orofaríngeo es el método primario y de elección para determinar la infección por sars-cov- . solo si resultaran dos pruebas negativas y persistiera una alta sospecha diagnóstica estaría indicado tomar muestras del tracto respiratorio inferior por broncoscopia, ya sea aspirado endotraqueal (bas) o lavado bronco alveolar (bal) ( ), prefiriendo la realización de minibal para la recolección de muestras ( ). las muestras deberán ser recogidas en un recipiente estéril, e introducidas en una bolsa con autocierre. deben manejarse con cuidado extremo evitando manipulaciones innecesarias y bajo protocolos de protección para el personal que las maneja, y trasladarse al laboratorio para su análisis. pueden almacenarse a - ºc las primeras h de su recolección; si se demorara más el análisis, precisa almacenarse a temperatura de - ºc ( ) . en una serie china la sensibilidad del bal fue de % frente a % en muestra de esputo (no recomendada) y % en hisopado nasofaríngeo ( ) por lo que el especialista que realiza el procedimiento deberá sopesar el riesgo de este procedimiento en cada caso, valorando que se beneficiarán aquellos pacientes que tengan una indicación adicional para su realización. se recomienda la realización de la traqueostomía cuando está indicada, en los pacientes covid- sospechosos y confirmados con pronóstico razonable de vida, después del o día de ventilación , previa valoración y consenso por el equipo quirúrgico y de cuidado intensivo, asegurando que las condiciones clínicas, ventilatorias y hemodinámicas se encuentran controlada. en el contexto de pacientes hospitalizados en cuidado intensivo, la traqueostomía se realiza para facilitar el destete de la ventilación mecánica, mejorar la limpieza de la vía aérea y el manejo de las secreciones, aumentar la comodidad de los pacientes y la movilización y disminuir la probabilidad de complicaciones como la estenosis traqueal; sin embargo no hay una clara disminución en la mortalidad ( ) ( ) ( ) . la infección actual por sars-cov , tiene diferentes estadios de gravedad, uno de ellos es el compromiso pulmonar el cual se caracteriza por un síndrome de dificultad respiratorio agudo (sdra). de acuerdo con el comportamiento de la covid- , entre un % a un % de los pacientes requieren ventilación mecánica ( , ) , este grupo de pacientes con manifestación grave del compromiso pulmonar requiere estrategias de protección pulmonar en la ventilación mecánica, sedación profunda y posiblemente parálisis muscular y puede tener una mortalidad entre el % y el % ( , ) . una de las principales características en éste grupo de pacientes es la mortalidad temprana, definida ésta como aquella que se produce en menos de días; de acuerdo a la experiencia en wuhan, china; leung ( ), reporta que la mortalidad se presenta en los primeros cinco días luego de la admisión hospitalaria y de acuerdo a lo referido por graselli et al ( ) , en la región de lombardía, italia, la mediana de mortalidad se presenta al día siete después del ingreso. con estas consideraciones, al principio de la pandemia y en las aproximaciones iniciales no se recomendaba realizar la traqueostomía en los primeros días posteriores a la intubación orotraqueal. sin embargo, con el conocimiento de la fisiopatología y las experiencias en otras series, como medida para facilitar la liberación de la ventilación mecánica, se ha podido realizar el procedimiento después de la primera semana de inicio de la ventilación. de acuerdo con el curso natural de la enfermedad, el paciente en promedio se intuba al día a de iniciado los síntomas, una semana posterior a la intubación para la traqueostomía, estaríamos alrededor del día de la enfermedad, donde los pacientes ya tendrán una disminución de la carga viral. esto sin embargo no evita la utilización de los epp necesarios. esta medida en específico fue discutida y consensuada entre la sociedad de medicina critica amci y la asociación colombiana de cirugía. no existe evidencia que permita evaluar el riesgo real de infección del personal asistencial de los pacientes con sospecha o diagnóstico de covid- en la realización de traqueostomías. se debe considerar en la traqueostomía y la realización de ésta como un procedimiento generador de aerosoles (organización mundial de la salud). se recomienda que los epp requeridos para la realización del procedimiento, deben incluir máscaras ffp o n , protección ocular, vestido antifluido idealmente desechable y amci ® guantes; este nivel de protección representa el mayor nivel de seguridad para realizar el procedimiento en el paciente con sospecha o diagnóstico de covid- . no existe actualmente artículos que permitan evaluar cual es el riesgo real de infección del personal asistencial en la realización de traqueostomías en pacientes con covid- ; quizás el ejemplo más cercano, es una serie de casos, reportada durante la epidemia del síndrome respiratorio agudo grave en el ( ), en el cual se realizaron traqueostomías sin ninguna infección del personal, en este reporte se aseguró un adecuado uso del equipo de protección personal (epp), el cual incluía las medidas de barrera, máscaras ffp y cuando existía la disponibilidad, respiradores con suministro de aire purificado. la traqueostomía y la realización de ésta es considerada por la organización mundial de la salud (oms) un procedimiento generador de aerosoles, bajo esta perspectiva, el epp requerido para la realización del procedimiento, debe incluir máscaras ffp o n , protección ocular, vestido antifluido idealmente desechable y guantes; este nivel de protección representa el mayor nivel de seguridad para realizar el procedimiento mencionado. es obligatorio, que todo el personal reciba el entrenamiento para la postura, el uso y el retiro de los epp, si estos pasos no se realizan de la forma adecuada, representan una fuente de contaminación( ). se recomiendan no esperar la negativización de la pcr para sars-cov para realizar la traqueostomía en el paciente con diagnóstico de covid- . fuerte en contra fundamento los estudios de zou et al y lescure et al ( , ) , muestran que la carga viral de los hisopados nasales y faríngeos es elevada en la primera fase de la enfermedad, con una disminución entre el día nueve al quince, pero esta puede permanecer detectable hasta por tres semanas ( ) . existen recomendaciones acerca de la necesidad de realizar la traqueostomía una vez la pcr para sars-cov sea negativa ( , ) ;aunque este esquema suena lógico, es importante tener presentes las siguientes consideraciones. la sensibilidad de una sola muestra para ruta-pcr puede ser sólo del %( ) y es posible que sea necesario realizar una segunda prueba para minimizar el riesgo para quien realiza el procedimiento, aunque esta aproximación no siempre puede ser viable desde el punto de vista clínico, epidemiológico y administrativo. amci ® recomendamos que la mejor estrategia es diferir la realización de la traqueostomía hasta días luego de la intubación cuando esta indicada, con el conocimiento acerca de la evolución natural de la enfermedad, en ese momento en la mayoría de los pacientes, lo más probable es que la condición ya se encuentre en la tercera semana desde el inicio de los síntomas, en cuyo caso lo más probable es que ya exista una disminución de la carga viral; este hecho no evita que se deba utilizar de la forma correcta los epp. recomendaciÓn se recomienda escoger la técnica teniendo en cuenta la experticia que tenga en el grupo tratante y la anatomía del paciente para la decisión de la técnica. se recomienda la guía ecográfica para disminuir la probabilidad de complicaciones del procedimiento si la anatomía es desfavorable para la realización de traqueostomía percutánea. se recomienda la traqueostomía quirúrgica en pacientes de riesgo elevado de complicaciones donde se requiere un control más rápido y seguro de la vía aérea. se recomienda no utilizar de forma rutinaria la utilización de broncoscopia para la realización de traqueostomía por vía percutánea. no hay evidencia directa hasta la fecha de publicación del consenso si existe superioridad entre las dos técnicas en el paciente con sospecha o diagnóstico covid- . en pacientes críticos no existen diferencia en los desenlaces cuando se evalúa el rendimiento de la técnica percutánea y la técnica quirúrgica, la elección de uno u otro método está dado por la anatomía del paciente, el entrenamiento de los profesionales y la disponibilidad de los diferentes insumos y técnicas. tampoco se ha logrado hasta la fecha evaluar durante la pandemia de covid- si existe una diferencia entre las dos técnicas y por lo tanto no es posible acercarse a una recomendación basada en la literatura. sin embargo, es importante que para la decisión de la técnica a utilizar se considere la anatomía del paciente y los siguientes aspectos:  no se recomienda la utilización de broncoscopia para la realización de traqueostomía por vía percutánea, ésta aumenta la generación de aerosoles y el número de personas expuestas a estos ( , ) . amci ®  si la anatomía es desfavorable para la realización de traqueostomía percutánea, la guía ecográfica puede disminuir la probabilidad de complicaciones del procedimiento ( ) .  la traqueostomía quirúrgica es una alternativa para la realización del procedimiento en las unidades de cuidado intensivo en momentos de sobrecarga laboral y adicionalmente puede tener un mejor y más rápido control de la vía aérea, especialmente en pacientes con riesgo elevado de complicaciones ( ) . recomendaciÓn se recomienda el uso de la terapia ecmo en sdra severo refractario por covid- (pao /fio < , posición prona, requerimiento de relajantes neuromusculares en algunos casos vasodilatadores pulmonares y maniobras de reclutamientos) sin respuesta clínica manifestado por:  pao /fio < mmhg por mas de horas  pao /fio < mmhg por mas de horas  ph < . + paco > mmhg por mas de horas además del criterio anterior, se recomienda tener en cuenta la edad, las comorbilidades y la expectativa de sobrevida del paciente con buena calidad de vida y en circunstancias donde no exista limitación de recursos. no hay estudios clínicos aleatorizados sobre el uso del ecmo en pacientes con covid- ( , ) . existe el estudio eolia ( ) , el cual fue detenido durante su realización, y de acuerdo a un análisis bayesiano posterior puede interpretarse como una disminución de la mortalidad en los pacientes en ecmo con sdra severo ( , , ( ) ( ) ( ) ( ) . de igual manera debe considerarse la racionalización de los recursos y el estado de prevalencia de la pandemia en un lugar determinado. el inicio de la terapia puede evaluarse en función de la cantidad de pacientes en falla respiratoria y la disponibilidad de personal y otros recursos; si el hospital debe comprometer todos los recursos en proveer medidas básicas de cuidado intensivo no debe utilizar el ecmo ( , ) . los pacientes jóvenes sin comorbilidades son considerados de alta prioridad al igual que los trabajadores de la salud ( , ) . amci ® se recomienda no desarrollar nuevos centros de ecmo en época de pandemia, sobre todo en situaciones con limitación de recursos. fuerte en contra fundamento actualmente se recomienda el uso del ecmo con las mismas indicaciones para sdra basado en la capacidad de las instituciones de salud para iniciar éste tipo de terapias ( ) . en épocas de crisis la capacidad de los hospitales está saturada y obliga a la reubicación y optimización de los recursos ( , ) . los centros que ofrecen la terapia en ecmo deben ser centros con resultados favorables y tiempos de soporte de pacientes relativamente cortos ( ) . cuando estamos en tiempos de capacidad hospitalaria convencional y existe disponibilidad de camas de cuidado intensivo se pueden ofrecer los servicios de ecmo vv, va, e-cpr inclusive a pacientes no covid- , cuando estamos en contingencia y capacidad nivel se debe hacer un triage respecto a pacientes jóvenes y ofrecer ecmo vv, va, escoger muy bien los casos para pacientes no covid- y no ofrecer e-cpr, cuando estamos en contingencia y capacidad nivel es porque ya se están usando sitios de expansión y están casi saturados se restringe el ecmo a todas las indicaciones y se prioriza a pacientes con indicaciones no covid- con mayor riesgo de sobrevida, el ecmo vv queda para pacientes jóvenes, con disfunción de órgano y covid- positivo, no se ofrecerá ecmo va o e-cpr y cuando estamos en capacidad de crisis es porque la capacidad total hospitalaria está sobresaturada y no es posible realizar ecmo tanto en pacientes covid- como en los no covid- ( , ) . se recomienda practicar e implementar medidas de capacitación y vigilancia continua para mejorar la higiene de manos, evaluando la adherencia a protocolos establecidos en los trabajadores de la salud mediante listas de chequeo y supervisión para evitar infecciones cruzadas en el entorno del paciente con sospecha o diagnóstico de covid- . se recomienda establecer protocolos específicos para reducir el riesgo de infecciones que se deriven de la interacción y el cuidado del paciente crítico con sospecha o diagnóstico de covid- . amci ® se recomienda implementar prácticas de cuidado para la prevención de contagio de covid- . se debe utilizar el equipo de protección personal (epp) para la prevención de enfermedades de componente infeccioso asociado a exposición con fluidos corporales derivados del paciente crítico con sospecha o diagnóstico de covid- . las infecciones relacionadas con la atención sanitaria (iras) son definidas por la organización mundial de la salud como aquellas "infecciones que se presentan en un paciente durante el proceso de atención en un hospital u otro centro sanitario que no estaban presentes o no se estaban incubando en el momento del ingreso; se incluyen las infecciones contraídas en el hospital pero que se manifiestan tras el alta hospitalaria y también las infecciones profesionales entre el personal del centro sanitario". las iras representan una importante carga de enfermedad que se asocia a un impacto negativo en la economía del paciente y del sistema sanitario. la organización mundial de la salud plantea la higiene de manos como la principal medida necesaria para reducir y prevenir las iras; por esta razón establece directrices sobre la higiene de manos en la atención sanitaria y basada en esta propone la estrategia multimodal para la mejora de higiene de manos. las estrategias mencionadas anteriormente han demostrado el incremento en el cumplimiento de higiene de manos y disminución en las infecciones relacionadas con la atención en salud. la estrategia multimodal se articula a través de cinco componentes: cambio del sistema, formación, evaluación/ retroalimentación, recordatorios en el lugar de trabajo clima institucional. a través de estos componentes, se garantiza que el centro sanitario cuenta con la infraestructura necesaria para practicar adecuadamente el lavado de manos incluyendo dentro de este el acceso a un suministro seguro continuo de agua, jabón, preparado alcohólico y toallas; a su vez se proporciona educación, evaluación y retroalimentación con regularidad a todos los profesionales sanitarios ( se recomienda realizar un plan de cuidados organizado y específico en paciente crítico con sospecha o diagnóstico de covid- , ofreciendo el uso óptimo de recursos e intervenciones. se recomienda evitar el uso de excesivo de papelería relacionada con los registros usados para gestión de insumos y atención de los pacientes. se recomienda realizar intervenciones educativas enfocadas a mejorar la adherencia y adecuado uso de los epp. se debe procurar el cuidado de los elementos de protección personal bajo un protocolo que conserve las condiciones de integridad de estos. la actual reserva de elementos de protección personal (epp) es insuficiente debido al aumento de la demanda global, por el incremento de casos de covid- y por la información errónea que ha conllevado a compras de pánico y almacenamiento. por esta razón la organización mundial de la salud a través de su guía: uso racional del equipo de protección personal para la enfermedad por covid- ha implementado las siguientes estrategias para optimizar la disponibilidad de (epp): usar los (epp) adecuadamente, minimizar la necesidad de (epp) y coordinar el suministro adecuado de (epp). ( ) la duración máxima del uso continuo de la n es de a horas, siguiendo las recomendaciones del manual de medidas básicas para control de infecciones en ips de minsalud. pero en lo cotidiano, ningún trabajador tolera a horas continuas con un respirador. por esto, su uso continuo en el sitio de trabajo dependerá de la necesidad de pausar para comer, para ir al baño, etc. en este caso, se guardará en una bolsa de papel para su nueva colocación, si tiene menos de horas, se desechará si está visiblemente contaminada o se torna húmeda. el reusó de la n dependerá de la casa del fabricante, de si contiene o no celulosa en su estructura del respirador. por ejemplo, la recomendación del consenso colombiano acin sobre la desinfección para los respiradores sin celulosa es con peróxido de hidrógeno vaporizado al % por minutos. los respiradores n de uso industrial tienen mayor contenido de celulosa que los de uso médico por lo tanto para procesos de esterilización, solo los n de uso médico podrán ser esterilizados mediante de peróxido de hidrógeno vaporizado (sterrad®)( - ). amci ® se recomienda elaborar el protocolo de pronación del paciente críticamente enfermo por covid- , garantizando el entrenamiento al personal de salud, organizando el recurso humano, dispositivos de apoyo y tiempo establecido para cambios de posición para prevenir las lesiones por presión en el paciente. fuerte a favor fundamento la estrategia de pronación es una alternativa eficiente en el manejo del síndrome de dificultad respiratoria en pacientes críticos y es fundamental la gestión del profesional de enfermería en la prevención de complicaciones y eventos adversos, lo cual aportará significativamente a la calidad del cuidado ofrecido favoreciendo las mejoras en la oxigenación. es importante optimizar los cuidados de enfermería en torno de los cuidados de piel en los pacientes en ventilación mecánica en decúbito prono, los estudios han demostrado como principal complicación las lesiones de presión con una incidencia hasta de . %, siendo las más frecuentes las grado y ( ). una lesión se puede producir si se supera una presión en el tejido capilar arterial de mmhg denominándose interfaz de presión. basándose en lo anteriormente mencionado, el cambio de posición es un componente integral de la prevención y el tratamiento de las upp, con una justificación sólida y de amplia recomendación en la práctica de enfermería ( ) . el uso de superficies especiales para el manejo de la presión (semp) a partir del estudio de defloor ( ), se determinó un antes y un después en el uso de las semp en conjunción de los cambios posturales. este realizo un importante aporte para reducir la incidencia de lesiones por presión comparado con los colchones de estándar. se recomienda promover actividades para controlar el nivel de estrés en el personal durante las jornadas de trabajo: identificar y reconocer los propios límites, buscar o proponer ayuda psicológica profesional cuando sea requerido, promover estilos de vida saludables, y organizar los turnos de trabajo asegurando periodos de descanso. se recomienda compartir las emociones con pares y superiores, analizar objetivamente las situaciones adversas, manejar fuentes de información objetivas y científicas, realizar pausas activas durante el turno y brindar espacios grupales para expresar emociones, miedos e incentivar al equipo de trabajo reconociendo su labor. amci ® se recomienda utilizar recursos de salud mental ocupacional, apoyo por enfermedad y licencia familiar, además de garantizar una adecuada dotación de personal. los estilos de superación personal y el crecimiento psicológico desempeñan un papel importante en el mantenimiento de la salud mental de las enfermeras. es razonable suponer que los niveles de ansiedad y estrés entre los profesionales de la salud son proporcionalmente más altos que los de la población general debido al contacto directo con pacientes infectados. esto puede explicar por qué las enfermeras de primera línea son excepcionalmente vulnerables a la fatiga y al agotamiento (wang, okoli, et al. ) , agotamiento mental, falta de moral del personal, control / autonomía de decisión, menor calidad de vida y baja satisfacción laboral (cheung y yip, ). ( ) . una investigación reciente realizada en china continental menciona el impacto negativo de la pandemia de covid- en los trabajadores de atención médica de primera línea, incluidos los mayores niveles de ansiedad (shanafelt, ripp y trockel, ), depresión (xiang et al. ), estrés postraumático síntomas, soledad e impotencia (xiang et al. ) ( , ) . los aspectos traumáticos y estresantes de la participación en una pandemia también ponen en riesgo el daño psicológico a los médicos ( ) . la experiencia psicológica de las enfermeras que atienden a pacientes con covid- se puede resumir en temas: primero, las emociones negativas presentes en la etapa inicial consisten en fatiga, incomodidad e impotencia que fue causado por el trabajo de alta intensidad, el miedo y la ansiedad, y la preocupación por los pacientes y sus familiares. segundo, los estilos de auto afrontamiento incluyeron ajustes psicológicos y de vida, actos altruistas, apoyo de equipo y coordinación racional. tercero, encontramos crecimiento bajo presión, que incluía un mayor afecto y agradecimiento, desarrollo de posición de responsabilidad profesional y autorreflexión. finalmente, encontraron que las emociones positivas ocurrieron simultáneamente con emociones negativas( ). se recomienda ofrecer mecanismos de apoyo para amortiguar el estrés relacionado con la pandemia por covid- . esto incluye intervenciones para pacientes y familias ofreciendo recursos de salud mental y educación al egreso, previo a este reforzar visitas virtuales. se recomienda anticipar las necesidades de salud mental de los pacientes, el personal y las familias para ofrecer una respuesta integral de salud pública. se debe incluir atención psicológica en la hospitalización para pacientes, familiares y personal afectado por covid- . se recomienda proporcionar atención de salud mental en las comunidades, mientras que se requiere distanciamiento social y los recursos del sistema de salud son limitados. amci ® se recomienda mantener una estrategia de comunicación asertiva con la familia, teniendo en cuenta la formación del personal sobre las estrategias para comunicar malas noticias. ser solidarios con el duelo de las familias y acompañar el proceso de afrontamiento aún en la distancia, identificando factores de riesgo para patología mental o duelo complicado, utilizando los recursos institucionales de salud mental para mejorar las intervenciones. la pandemia tiene el potencial de crear una crisis secundaria de angustia psicológica y desbordamiento del sistema de salud mental. los miembros de la familia pueden experimentar angustia, miedo o ansiedad por la hospitalización de un ser querido, particularmente cuando las medidas de control de infecciones restringen las visitas. la telesalud (incluida la cobertura de seguro para la telesalud), el suministro extendido de medicamentos, el aumento de la capacitación en salud mental del proveedor, el apoyo virtual de pares y los grupos virtuales de apoyo al uso de sustancias pueden ayudar a garantizar que se satisfagan las necesidades de salud mental de la comunidad ( ). el sistema de salud y los líderes de enfermería deben asegurarse de que su personal de enfermería clínica esté protegido y respaldado para que puedan proporcionar esta dimensión crucial de la atención de covid- . se recomienda crear grupos centralizados y definidos para atención de pacientes con sospecha o diagnóstico de covid- que se encarguen de elaboración, socialización e implementación de protocolos. estos deben incluir los aspectos de infraestructura, áreas delimitadas, utilización de epp, listas de chequeo, observadores, insumos y recursos que permitan atención integral. se recomienda organizar el plan de atención del paciente con sospecha o diagnóstico de covid- de enfermería con la asignación de actividades, número de personas según escalas que midan escalas de carga laboral para definir el número adecuado de los miembros del equipo de trabajo, tiempo de atención, gestión de recursos, gestión de riesgo y un líder por turno que garantice el cumplimiento fuerte a favor fundamento la implementación de estrategias de gestión en contingencias genera un trabajo organizado, enfocado en la prevención y tratamiento centralizado, elaboración y socialización de protocolos claros, áreas específicas, delimitadas y asignadas, con un uso racional del recurso humano que se despliega en fases, desde el inicio de la emergencia considerado como detección temprana hasta la atención directa de pacientes con sospecha amci ® o confirmación de sars-cov- . dentro de las fases tempranas, se busca la gestión de los recursos necesarios para la atención de estos pacientes, con una asignación de zonas o servicios y unas condiciones particulares, tratamientos específicos y actividades de atención especiales para las cuales se discriminan medicamentos, dispositivos e insumos necesarios para el cuidado de enfermería. los grupos de atención deben contar con capacitación, gestión y supervisión, apoyo logístico, apoyo psicológico y retroalimentación ( , ) . el plan de atención de enfermería debe tener presente la minimización de exposición, la prevención de infecciones en el personal y cuidados especiales derivados de la condición clínica de los pacientes con esta infección, altamente contagiosa y con síntomas o necesidades que rompe el modo operacional convencional y que requiere implementación basada en la práctica clínica. por lo tanto, el plan debe ser centralizado oportuno, ordenado, seguro y eficiente e incluye: relación enfermería/paciente de acuerdo a criticidad, capacitaciones y entrenamiento al personal de enfermería de línea de frente en el área crítica de aislamiento mediante videos, infografías y procesos prácticos (el contenido de capacitación incluye el uso de elementos de protección personal, higiene de manos, desinfección de áreas, manejo de residuos y esterilización de dispositivos de atención al paciente y manejo de exposición ocupacional), asignación de actividades clínicas (atención directa) y administrativas (supervisión, observador, líderes, gestión de recurso humano y medicamentos), soporte y contratación de personal adicional ante la contingencia con preparación académica o inducción, asignación de turnos razonables con períodos de descanso (alimentación, eliminación), coordinación con otros departamentos y optimización de flujos de trabajo, estrategias de control de infecciones y trabajo en equipo ( , ) . se recomienda que las muestras clínicas tomadas para el diagnóstico de covid- deben conservarse a temperatura entre - a °c, y luego de las horas deben permanecer congeladas a una temperatura de - °c. se recomienda que se realice el envío al laboratorio de salud pública de referencia dentro de las horas posteriores a la toma de la muestra del paciente. se recomienda que el transporte de las muestras debe realizarse con geles o pilas congeladas. se recomienda considerar que las muestras del tracto respiratorio bajo presentan la mejor certeza diagnóstica en pacientes con neumonía para adultos intubados y ventilados mecánicamente con sospecha de covid- . se recomienda contar con elementos de protección personal de acuerdo con las precauciones establecidas para el paciente con sospecha o diagnóstico por covid- para evitar la transmisión a profesionales de la salud. se debe evitar perder el circuito cerrado en los pacientes ventilados mecánicamente y valorar el riesgo de las acciones en pacientes con peep alta. se recomienda realizar la toma de muestra post mortem no invasiva por hisopado nasofaríngeo dentro de las primeras seis ( ) horas posteriores al fallecimiento, para que esta sea útil para su análisis. las muestras clínicas tomadas para el diagnóstico de coronavirus deben conservarse a temperatura entre - a °c, y luego de las horas deben permanecer congeladas a una temperatura de - °c. sin embargo, la muestra puede conservarse en un tiempo máximo de refrigeración por horas. no obstante, se sugiere que se realice el envío al laboratorio de salud pública de referencia dentro de las horas posteriores a la toma. si no se conserva la cadena de frío adecuada, la muestra puede ser inviable. el transporte de las muestras debe realizarse con geles o pilas congeladas ( , , ) .se debe tener en cuenta que no conservar la cadena de frío durante el transporte de la muestra, degradan la partícula viral, obteniéndose falsos negativos ( ) . las muestras del tracto respiratorio bajo presentan la mejor certeza diagnóstica en pacientes con neumonía. para adultos intubados y ventilados mecánicamente con sospecha de covid- en comparación al tracto respiratorio superior (nasofaríngeo u orofaríngeo). en el caso de aspirado traqueal, es importante considerar que para la obtención de las muestras para el diagnóstico de covid- se deben contar con elementos de protección personal de acuerdo a las precauciones estándar para evitar la transmisión a profesionales de la salud, circuito cerrado y valorar su realización en aquellos pacientes con peep alta ( ) . la toma de muestra post mortem no invasiva por hisopado nasofaríngeo se debe hacer antes de seis ( ) horas post mortem, para que esta sea útil para su análisis( , - ). se recomienda en los pacientes con diagnóstico covid- , monitorizar continuamente la oxigenación mediante saturación arterial de oxígeno con pulso oxímetro y la aparición temprana de signos clínicos de dificultad respiratoria durante la monitorización (aleteo nasal, cianosis, tirajes intercostales). se recomienda no suministrar de forma rutinaria suministrar oxígeno si la saturación de oxígeno (spo ) está por encima de %, y no se evidencian signos clínicos de dificultad respiratoria durante la monitorización continua del patrón respiratorio. se recomienda como parámetro importante para evaluar la oxigenación y guiar el suministro de oxígeno mediante los diferentes dispositivos la transferencia de oxígeno, medida por la pao / fio o sao /fio . se propone iniciar la oxigenoterapia por cánulas de bajo flujo y ajustar el flujo (máximo l) hasta alcanzar la spo objetivo ≥ %; si el paciente se encuentra en estado crítico iniciar con mascarilla con bolsa de reserva (a - l / min). una vez que el paciente esté estable, el objetivo de oxigenación es mantener niveles de spo entre y % en pacientes no embarazadas y entre - % en pacientes embarazadas. se recomienda no utilizar de forma rutinaria el uso de dispositivos que generan aerosoles durante la administración de oxígeno (dispositivos venturi o nebulizador de alto flujo o jet) en pacientes con sospecha o diagnóstico covid- . en las diferentes guías publicadas para manejo de pacientes positivos para covid- las metas de oxigenación durante la terapia de oxígeno en adultos recomiendan iniciar la oxigenoterapia a l / min y ajustar el flujo hasta alcanzar la spo objetivo ≥ % durante la reanimación; o use mascarilla con bolsa de reserva (a - l / min) si el paciente está en estado crítico. una vez que el paciente esté estable, el objetivo de oxigenación es > % de spo en pacientes, no embarazadas y ≥ - % en pacientes embarazadas( ). los dispositivos para la oxigenoterapia se pueden dividir en dos grupos, dependiendo de si cubren la totalidad o una parte de los requerimientos respiratorios del paciente. unos son de bajo flujo o para esfuerzos mínimos del paciente, estos dispositivos completan su ventilación con aire ambiente y los sistemas de alto flujo cubren la totalidad de los requerimientos inspiratorios del paciente. escalones terapéuticos: oxigenoterapia convencional a diferentes concentraciones de bajo flujo (son las cánulas nasales, las mascarillas simples y las mascarillas con reservorio), es el primer escalón terapéutico ante cualquier paciente que presente una situación de hipoxemia (spo ) < % respirando aire ambiente. el objetivo debe ser ajustar la fio (hasta . ) para mantener un nivel de oxigenación adecuado, considerado este como una spo > %. la administración de oxígeno se considera un procedimiento generador de aerosoles de riesgo bajo y por lo tanto es adecuado para pacientes covid- positivos( ). b. en adultos con signos de emergencia (respiración obstruida o ausente, dificultad respiratoria severa, cianosis central, shock, coma y / o convulsiones) deben recibir vía aérea amci ® de emergencia manejo y oxigenoterapia durante la reanimación para apuntar a spo ≥ %. una vez el paciente está estable, objetivo> % de spo en adultos no embarazadas y ≥ - % en mujeres embarazadas. c. para el manejo del paciente con covid- la máscara de no re inhalación se considera como la opción de preferencia para escalar el paciente antes de la intubación y considerar la transferencia a uci; esto se debe a que puede proporcionar altas fracciones inspiradas de oxígeno ( ) . d. los dispositivos que generan aerosoles durante la administración de oxígeno (dispositivos venturi o nebulizador de alto flujo o jet), no están indicados para manejo de covid- ( ). se recomienda aplicar las estrategias de retiro de la ventilación mecánica habituales para pacientes adultos críticos en general, hasta el momento no se ha construido una evidencia contundente para el destete en covid- . se recomienda en el paciente críticamente enfermo por covid- un descenso de la presión de soporte (psv) según tolerancia clínica, de esta forma el paciente podrá ser sometido a la realización de prueba de respiración espontánea con una presión de soporte de entre - cm h o. se recomienda que el destete automatizado puede ser considerado como una herramienta útil según disponibilidad de equipos para realizarlo. se recomienda no utilizar las maniobras que incrementan la aerosolización como la prueba de respiración espontánea en pieza en t o el cuff-leak test en el momento de realizar la medición de los predictores de éxito en el destete. fuerte en contra fundamento la realización de las pruebas de respiración espontánea sigue siendo un factor predictor importante en el éxito en el retiro del soporte ventilatorio mecánico y la indicación de tiempo de duración sigue siendo de a minutos debido a que las intubaciones realizadas en el mismo periodo de tiempo no han tenido diferencias significativas en el éxito del destete ( , ) . en los pacientes que han sido ventilados por más de horas y que el motivo por el cual fueron llevados a ventilación mecánica ya ha sido superado se debe establecer un protocolo de destete que debe incluir una prueba diaria de respiración espontánea y la minimización o retiro de la sedación (si no existe alguna contraindicación)( , ). la movilización temprana como factor coadyuvante en el éxito de la liberación mecánica ya se ha documentado en otros escenarios similares, razón por la cual la implementación amci ® temprana de este tipo de estrategias será un punto de vital importancia para recuperar la funcionalidad de los pacientes con covid- ( ) . se recomienda que la extubación de los pacientes críticamente enfermos por covid- se debe realizar con los elementos de protección personal requeridos para el riesgo de aerosoles. se recomienda no estimular la tos y el esputo inducido en los pacientes con sospecha o diagnóstico de covid- posterior a la extubación inmediata. se recomienda no utilizar de forma rutinaria la vmni en la falla respiratoria post extubación en pacientes críticos que no tengan una enfermedad concomitante que sea respondedora a la vmni como el epoc o edema pulmonar de origen cardiogénico en pacientes con sospecha o diagnóstico de covid- . fuerte en contra se recomienda mantener un umbral bajo para decidir intubación en caso de sospecha de fallo en la extubación en el paciente con sospecha o diagnóstico de covid- . la estricta monitoria y manejo del paciente posterior a la extubación surgen como un reto insoslayable para el personal de cuidado intensivo, enfocando todos sus esfuerzos en evitar la re-intubación, lo que se traducirá en un descenso significativo de la morbilidad y la mortalidad que supone una re-intubación ( ), la cual se puede definir como el no requerimiento de re intubación en las primeras horas post extubación ( , ) . en los últimos años la cánula nasal de alto flujo (caf) se ha convertido en una herramienta útil en el soporte de oxigenoterapia en los pacientes extubados que presenten riesgo de reintubación ( , ), y a la vez no presenten hipercapnia ( ) . la utilización de ventilación mecánica no invasiva de manera profiláctica en la falla respiratoria post extubación no ha demostrado tener éxito evitando la re-intubación en las primeras horas ( , ) excepto en las situaciones donde el paciente presente una enfermedad pulmonar o alteración cardiaca concomitante que sea respondedora a el manejo con vmni como lo son la enfermedad pulmonar obstructiva crónica (epoc) y el edema pulmonar de origen cardiogénico ( , ) . los pacientes extubados en los que se halla documentado epoc, se sugiere posterior a la extubación la implementación de una estrategia de niv de manera protocolaria ( , ), con una intensidad de hora cada horas durante un período mínimo de horas ( ). se recomienda utilizar en los pacientes con extubación reciente que no expresen predictores de riesgo de fracaso, sistemas de oxigenoterapia convencionales de bajo flujo que generen menos riesgo de aerosolización, fuerte a favor se podría considerar cánulas de alto flujo de oxígeno y/o la ventilación mecánica no invasiva (con una máscara facial adecuadamente ajustada y ramas inspiratorias y espiratorias separadas) como terapia de puente después de la extubación, pero se deben brindar las condiciones estructurales necesarias (habitaciones de presión negativa o habitaciones aisladas de puertas cerradas) y con epp estrictos para el personal sanitario. fuerte a favor fundamento las pautas de anzics establecen que la caf y/o la ventilación no invasiva (con una máscara facial bien ajustada y ramas inspiratorias y espiratorias separadas) pueden considerarse como terapia de puente después de la extubación, pero deben proporcionarse epp estricto en el aire. la terapia cpap o bipap (con alta presión espiratoria final) podría ser útil para prevenir la eliminación del reclutamiento en estos pacientes. en el momento de la extubación, los pacientes a menudo han estado enfermos durante más de una semana. es probable que su carga viral disminuya en ese punto, por lo que el riesgo de transmisión del virus puede ser menor (en comparación con la intubación inicial) ( ) . de no contar con predictores de que nos indiquen que podría fracasar la extubación se deben utilizar entonces sistemas de oxigenoterapia convencionales de bajo flujo que generen menos riesgo de aerosolización( ). se recomienda limpiar y desinfectar con frecuencia el área de retiro de epp, incluso después de que se haya completado cada procedimiento de eliminación. se debe limpiar esta zona, pasando de las áreas más limpias a las más sucias, antes de ingresar a la habitación del paciente y realizar el manejo y disposición final de residuos. se recomienda realizar la limpieza de superficies con un desinfectante adecuado o con una solución de hipoclorito sódico que contenga ppm de cloro activo (por ejemplo, un producto con hipoclorito en una concentración de - gr/litro, se hará una dilución : en el momento de su utilización). amci ® se recomienda que los recipientes que contengan los residuos deberán quedar en el lugar designado a tal efecto, que permanecerá cerrado hasta que, según el procedimiento de gestión de residuos de la institución sean retirados. los circuitos, filtros, succión cerrada y tot deben ser dispuestos en bolsas de color rojo las cuales deben ser de polietileno de alta densidad de . milésimas de pulgada y deben contar con un rótulo donde se indiquen: el nombre del generador, las palabras residuos biolÓgicos (covid- ) . una vez dispuesto, apretar y asegurar con nudo la bolsa de residuos y remover la bolsa de residuos del recipiente de residuos. posteriormente, desinfectar el exterior de la bolsa con solución desinfectante. luego colocar la bolsa de residuos en otra bolsa adicional de residuos y apretar y asegurar con nudo la bolsa de residuo. finalmente desinfectar la exterior bolsa de residuos con solución desinfectante. una vez terminada la disposición de los residuos de extubación, desinfectar los guantes con que manipuló los residuos con solución desinfectante y ubicar la bolsa de residuos dentro del vehículo de recolección interna de residuos. finalmente desinfectar el exterior de la bolsa de residuos con solución desinfectante. una vez terminada la disposición de los residuos de extubación, desinfectar los guantes con que manipuló los residuos con solución desinfectante y ubicar la bolsa dentro del vehículo de recolección interna. acogerse a la ruta sanitaria que asegure el menor riesgo de contaminación en el traslado interno de los residuos en la habitación del paciente (zona limpia) y zona sucia, se debe garantizar la ubicación de recipiente plástico de color rojo, liviano, resistente a los golpes, en material rígido impermeable, de fácil limpieza, y resistentes a la corrosión. los recipientes deberán ser lavados y desinfectados de acuerdo con los procedimientos establecidos por el prestador de servicios de salud( - ). se recomienda utilizar un ajuste de peep del paciente crítico por covid- , basado adicional a la tabla de peep, en las condiciones clínicas del paciente, en los índices de oxigenación, en la mecánica respiratoria del paciente y en los métodos de monitoreo disponibles. se recomienda titular la peep más alta que mantenga o mejore la relación safi y permita una presión plateau ≤ cmh o. se recomienda utilizar otras estrategias de titulación de peep probadas y con las cuales el equipo de trabajo esté familiarizado, dependiendo de la disponibilidad del recurso: ensayo peep decremental precedido por una maniobra de reclutamiento; titulación mediante la amci ® estimación de la presión transpulmonar con catéter esofágico o tomografía de impedancia eléctrica. fuerte a favor fundamento la titulación de la peep debe hacerse en función de la distensibilidad, oxigenación, espacio muerto y estado hemodinámico. puede titularse la peep mediante la estimación de la presión transpulmonar con catéter esofágico o tomografía de impedancia eléctrica. podría también titularse a partir de la fórmula (dp=plateau-peep) teniendo en cuenta que sea lógico el acoplamiento matemático fisiológico (lo que resultaría en una peep de cmh o si la presión plateau es de cmh o). la titulación de la peep requiere consideración de los beneficios (reducción de atelectrauma y mejora del reclutamiento alveolar) frente a los riesgos (sobre distensión inspiratoria final que conduce a lesión pulmonar y mayor resistencia vascular pulmonar)( , , ). se recomienda aplicar los protocolos de rehabilitación física como estrategia beneficiosa en el tratamiento respiratorio y físico de pacientes críticamente enfermos por covid- . se recomienda realizar la movilización precoz del paciente críticamente enfermo por covid- durante el curso de la enfermedad siempre que sea posible hacerlo de forma segura, asegurando la protección personal del personal sanitario. derivado del tratamiento médico intensivo para algunos pacientes con covid- , incluida la ventilación pulmonar protectora prolongada, la sedación y el uso de agentes bloqueantes neuromusculares, los pacientes con covid- que ingresan en la uci pueden presentar un elevado riesgo de desarrollar debilidad adquirida en la uci empeorando su morbilidad y mortalidad. por lo tanto, es esencial la rehabilitación temprana después de la fase aguda del síndrome de distrés respiratorio agudo (sdra) para limitar la gravedad de la debilidad adquirida en uci y promover la recuperación funcional. según la guía de la oms y la ops, enfatizan extremar el uso de los elementos de protección personal (epp) durante las intervenciones de rehabilitación física. la rehabilitación física proporciona intervenciones a través de movilizaciones, ejercicio terapéutico y programas individualizados a las personas que superan la enfermedad crítica asociada con covid- durante la ventilación mecánica y luego de esta, con el fin de permitir un retorno al hogar con funcionalidad. la prescripción de la movilización y ejercicio terapéutico debe de ser considera cuidadosamente en función del estado del paciente teniendo en cuenta, la estabilidad hemodinámica y clínica de la función respiratoria. cuando las movilizaciones, ejercicio terapéutico o programas de rehabilitación están indicados, debe realizarse una correcta planeación teniendo en cuenta amci ® la identificación/uso del personal mínimo necesario para realizar la actividad de manera segura. y el aseguramiento de todo el material que requerido esté a la mano y funcione correctamente y esté perfectamente limpio y desinfectado. si el material/equipo tiene que ser compartido con otros pacientes, límpielo y desinféctelo después de cada uso, entre paciente y paciente. se requiere personal entrenado específicamente para la limpieza y desinfección de los equipos, en una habitación aislada. y siempre que sea posible, evitar el traslado del material entre las áreas infectadas y no infectadas del hospital, manteniendo el equipamiento en las zonas aisladas ( ) ( ) ( ) ( ) ( ) . ( basados en un estudio preliminar aún sin publicar, se podría sugerir el uso de dexametasona a dosis de mg (oral o venosos) por días o hasta el alta si ocurre primero en pacientes hospitalizados con sospecha o diagnóstico de covid- que requieren suplencia de oxígeno, incluyendo aquellos con ventilación mecánica, que sean menores de años y con más de días de síntomas. amci ® actualmente no existe una terapia dirigida que se a efectiva para el manejo del virus; un número alto de estudios han surgido en los últimos dos meses, la mayoría sin el rigor metodológico suficiente para tomar decisiones adecuadas con respecto al manejo del paciente con infección por sars-cov- . el conocimiento en la estructura del virus y el mejor entendimiento en la fisiopatología de la enfermedad genera un sinnúmero de potenciales fármacos que han sido ensayados para el manejo de la enfermedad. en tiempos de pandemia, con una patología catastrófica en términos de vidas humanas y costos hospitalarios; es importante encontrar soluciones a desenlaces importantes como mortalidad, días de estancia en uci y en el hospital, aumento en los días libres del ventilador, disminución de complicaciones mayores debido a la enfermedad entre otros. hasta el momento no se ha documentado ninguna terapia específica que pueda impactar sobre estos desenlaces; pero la calidad de los trabajos, tampoco dejan claro sin él no usar ningún tratamiento específico mejora los desenlaces al menos al disminuir el número de complicaciones. este nuevo beta-coronavirus es similar al coronavirus del síndrome respiratorio agudo severa (sars-cov) y del síndrome respiratorio del medio este (mers-cov); por lo tanto, varias moléculas que habían sido evaluadas en este tipo de enfermedad rápidamente se abrieron paso a ensayos clínicos en paciente con covid- . estos ensayos principalmente observacionales, aleatorios pero abiertos con un número pequeño de pacientes no han permitido sacar adecuadas conclusiones y es frecuente como ver las diferentes guías de las principales sociedades del mundo cambiar de forma frecuente sus recomendaciones; no existes evidencia de estudios clínicos aleatorios y controlados que midan desenlaces fuertes, la premura de un tratamiento efectivo ha sacrificado el rigor metodológico que una investigación requiere. una estructura viral y replicación conocidas generan posibles dianas para que diferentes fármacos puedan ser investigados, antivirales tipo arbidol el cual inhibe la fusión de la membrana en la envoltura viral a algunos receptores; antimaláricos como la hidroxicloroquina y la cloroquina, las cuales inhiben la entrada viral y endocitosis por múltiples mecanismos, así como los efectos inmunomoduladores demostrados en el huésped; antivirales que impiden la replicación como el lopinavir o darunavir inhibiendo las proteasas o la ribavirina, el remdesivir o el favipiravir que actúan como análogos de nucleótidos o fármacos que actúan modulando la respuesta específica del huésped como el tocilizumab el cual se une al receptor de la il- inhibiendo el punto de acción de esta; los corticosteroides con múltiples efectos en la modulación del sistema inmunológico del paciente o los fármacos para evitar la respuesta secundaria a esta cascada inflamatoria como son los anticoagulantes. por último, se han buscado estrategias con el fin de mejorar la inmunización pasiva del huésped en el uso del plasma de pacientes convalecientes o el uso de inmunoglobulinas enriquecidas entre otros tratamientos propuestos para esta enfermedad. amci ® a los diferentes medicamentos que han sido usados en la pandemia del sars-cov- /covid- . de manera reciente en datos preliminares aún sin publicar horby y col en una rama del ensayo de evaluación aleatorizada de la terapia covid- (recovery), estudio aleatorizado, controlado, abierto que compara una gama de posibles tratamientos con la atención habitual en pacientes hospitalizados con covid- , compararon el uso de la dexametasona a dosis de mg día (oral o intravenosa) una vez al día por días o el alta según lo que ocurriera primero contra el manejo habitual; en pacientes aleatorizados que recibieron dexametasona se compararon con pacientes en manejo estándar; ( . %) pacientes en el grupo de dexametasona y ( . %) pacientes en el grupo control murieron a los días, con un riesgo relativo ajustado para la edad (rr . ; % ic . a . ; p < . ). la mortalidad relativa y absoluta variaron significativamente en relación al soporte ventilatorio al momento de la aleatorización; la dexametasona redujo las muertes en una tercera parte de los pacientes que recibieron ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p < . ), y una quinta parte en los pacientes que reciben oxígeno sin ventilación mecánica invasiva ( . % vs. . %, rr . , % ic . a . ; p = . ), pero sin reducir la mortalidad en paciente que no recibieron soporte respiratorio al momento de la aleatorización ( . % vs. . %, rr . , % ic . a . ]; p = . ) ( ) . no se emite recomendación a favor ni en contra para el inhibidor de la janus quinasa (baricitinib) en los pacientes con sospecha clínica o diagnóstico de covid- severo. uno de los reguladores conocidos de la endocitosis es la proteína quinasa asociada a ap (aak ); la interrupción de aak podría, a su vez, interrumpir el paso del virus a las células y también el ensamblaje intracelular de partículas del virus. uno de los seis fármacos de unión a aak de alta afinidad es el inhibidor de la janus quinasa (jak y jak ), llamado baricitinib, que también se une a la quinasa asociada a la ciclina g, otro regulador de la endocitosis ( ) . el baricitinib alcanza concentraciones plasmáticas suficientes para inhibir aak con mg o mg una vez al día; por su baja unión a proteínas plasmáticas y a su mínima interacción con las enzimas cyp, permite combinarlo con los antivirales. sin embargo, algunos piensan que el bloqueo de la señal jak-stat por baricitinib puede producir un deterioro de la respuesta antiviral mediada por interferón, con un posible efecto facilitador sobre la evolución de la infección por sars-cov- ; otras limitantes son la linfopenia (no dar si < cel./ mm ) y el aumento de la cpk. ( , ) . cantini y cols, en abril , en italia, administraron baricitinib a mg/día vía oral por semanas a pacientes con covid- moderado y los compararon con un grupo control; la terapia mejoró significativamente los parámetros clínicos, respiratorios y de laboratorio (pcr); ninguno de los pacientes requirió uci vs % del grupo control, sin eventos adversos. se amci ® trata de un estudio piloto de seguridad e impacto clínico en pacientes que no estaban en uci ( ) . ¿en pacientes hospitalizados con sospecha o diagnóstico de covid- el uso de n-acetil cisteína modifica el curso clínico de la enfermedad o genera beneficios en desenlaces clínicos de interés? basados en evidencia indirecta para el manejo del sdra y resultados observaciones en covid- , se podría utilizar el uso de n-acetil cisteina a dosis de mg/kg/día durante los primeros cinco días del sdra, aunque no se ha demostrado impacto en la mortalidad, su utilización parece relacionarse con una disminución significativa en la estancia en la unidad de cuidados intensivos y con disminución de los marcadores inflamatorios en pacientes con covid- . la severidad de la infección en covid- , en gran parte depende de la respuesta inmunológica de cada persona, sin embargo, se encuentran mecanismos fisiopatológicos de relevancia. sobreproducción de moco en vía aérea superior e inferior, que en parte explica la dificultad en la mecánica ventilatoria y los retos de ventilación en estos pacientes, la descarga desmedida de citoquinas proinflamatorias que se asocian a la falla multiorgánica y la coagulopatía asociada a la disfunción endotelial. esto mecanismos fisiopatológicos son comunes en el sdra, incluido los casos asociados a covid- ( ). amci ® enfermo con falla respiratoria aguda, la cual engloba falla respiratoria hipóxica (tipo ), falla respiratoria hipercápnica aguda (tipo ), sdra y lesión pulmonar aguda, se revisaron ensayos clínicos, más de pacientes. el análisis del grupo de n acetilcisteína intravenoso mostró una reducción de estancia en uci, de . días, con una heterogeneidad muy baja del %, con valoración de la evidencia calificada como de alta calidad y baja probabilidad de sesgo ( ) . en covid- , fue utilizada con recuperación completa en un caso severo de un paciente con déficit de glucosa fosfato deshidrogenasa (g pd), con control de la hemolisis y resolución del compromiso pulmonar. en pacientes sin déficit de g pd, también ha sido asociada a mejoría clínica y disminución significativa de los niveles de pcr y ferritina ( ) . en una revisión de costo efectividad nacional, se identificaron referencias, de ellos era revisiones sistemáticas de la literatura, dos de las cuales incluían metaanálisis (lu y zhang ), y fueron incluidos en la evaluación. estos estudios incluyeron información de ensayos clínicos que comparaban la aplicación de nac intravenosa frente a placebo o cuidado usual en pacientes con sdra. los tres estudios reportan como resultado de mortalidad rr de . con ic al % de . a . (lu ), rr de . con ic al % de . a . (lewis ) y rr de . con ic al % de . a . (zhang ) . para el tiempo de estancia en uci solo las revisiones con metaanálisis reportaron resultados, encontrando una diferencia de promedio de días de estancia de - . días con ic al % de - . a - . (lu ) y de - . días con ic al % de - . a - . (zhang ). una de las revisiones reportó que en ninguno de los estudios analizados se presentaron eventos adversos. no se encontraron resultados para los desenlaces de infección, sobreinfección, ni uso y tipo de antibiótico utilizado. en el análisis se encontró una reducción estadísticamente significativa de los días de estancia en uci de los pacientes que recibieron tratamiento con n-acetilcisteína intravenosa con dosis entre y mg/kg/día durante los primeros cinco días del sdra, en comparación con los pacientes que recibieron placebo o manejo usual. no se reportó diferencia estadísticamente significativa en la reducción de la mortalidad de los pacientes que recibieron nac. ( ) calidad de vida . ¿cómo podemos medir la calidad de vida, en los pacientes con covid- que egresan de la uci? se recomienda utilizar los marcadores disponibles de severidad y del riesgo de mortalidad por covid- en los pacientes internados en la uci. amci ® las secuelas inmediatas en los pacientes víctimas del devastador ataque sistémico del covid- durante su estancia en la uci son valorables, pero no se dispone de herramientas que permita medir el grado de afectación de la calidad de vida de estos pacientes posterior al egreso de la uci o de alta hospitalaria, por lo tanto, se sugiere realizar estudios de creación, validación y utilización de instrumentos de valoración de la calidad de vida en pacientes con covid- posteriores al alta hospitalaria. parte importante de los pacientes con diagnóstico de covid- que ingresan a la uci, evolucionan tórpidamente presentando deterioro progresivo de los diferentes órganos llegando en pocos días a una falla multiorgánica ( ) , estos pacientes presentan características clínicas y de laboratorio que se relacionan de manera significativa con mayor severidad y riesgo de mortalidad ( , ) . a pesar de conocer con alguna precisión el riesgo de severidad y mortalidad de los pacientes que ingresan a la uci, no disponemos de un score que nos permita evaluar y predecir el grado de afectación en la calidad de vida de los pacientes que logran sobrevivir. aproximadamente un , % del total de pacientes con enfermedad por covid- ingresan a uci, y de estos , % sometidos a ventilación mecánica ( ); lamentablemente los pacientes con enfermedad severa que logran sobrevivir y recuperarse han sido sometidos a una larga estancia en la uci y a ventilación mecánica invasiva con una intubación prolongada, que puede producir disfunción en la deglución impidiendo a la persona alimentarse de forma correcta y segura. es importante diagnosticar esta disfagia en los pacientes que se están recuperando del covid- y tratarla correctamente desde el principio para evitar complicaciones importantes como la malnutrición y la deshidratación, así como el riesgo de neumonía aspirativa. además de la disfagia, la fibrosis pulmonar y el riesgo de trombos son los problemas más frecuentes, pero no los únicos. una de las características de la enfermedad severa por covid- es que el virus provoca una enfermedad multiorgánica, con un amplio y heterogéneo abanico de secuelas cuyo alcance todavía se desconoce y aunque el órgano más afectado es el pulmón, puede afectar también otros órganos o sistemas incluido el snc, que en los casos más graves puede presentar encefalitis, delirios, desorientación y confusión, síntomas que pueden persistir tras el alta de la uci. otra secuela frecuente son las polineuropatías, esta afectación suele comenzar con una sensación de hormigueo en las extremidades y en los pacientes con covid- se presenta además con un cuadro de miositis que provoca debilidad y cansancio al caminar, a veces incluso en reposo; en algunos pacientes se presenta tal debilidad que dificulta llevar el alimento a la boca e incluso deglutirlo. sin embargo, la primera y más frecuente de las manifestaciones neurológicas del covid- es la pérdida del olfato, que a veces perdura como secuela tiempo después del alta. un estudio en pacientes ingresados en el hospital clínico san carlos de madrid revela que el % había sufrido anosmia en mayor o menor grado. la importancia de este síntoma radica en que las fosas nasales pueden ser la vía de acceso del virus al sistema nervioso central. amci ® otras posibles secuelas neurológicas asociadas a la infección por covid- son la ageusia, la cefalea y amnesia a corto y mediano plazo. también son importantes las secuelas que afectan al sistema cardiovascular. un estudio publicado en la journal o the american medical association advierte que un % de los pacientes presenta una elevación de las enzimas que indican daño en el miocardio. la inflamación que provoca el virus puede provocar directamente ese daño y también puede agravar el estado de pacientes que ya tengan una patología cardiovascular de base, muchas miocarditis son reversibles, pero hay una parte importante que deja como secuela una pérdida de la función contráctil. todavía se desconoce el alcance y es difícil medir el impacto de la enfermedad sobre el corazón porque en algunos casos, los síntomas de insuficiencia cardíaca se confunden con los de la neumonía. otra de las complicaciones más frecuentes, y potencialmente más grave, afecta al mecanismo de coagulación de la sangre. durante el ingreso hospitalario se han visto numerosos casos de ictus. la secuela más importante es el riesgo de que se formen trombos, que pueden ir al pulmón o al cerebro, y si se producen en las arterias, pueden dar lugar a un infarto, aunque este efecto es mucho menos frecuente. eso explica que algunos pacientes de covid- dados de alta hayan tenido que volver a ingresar por trombosis. finalmente es importante tener presente que a las afectaciones que haya podido producir el virus, hay que sumar las secuelas neurológicas propias de una estancia prolongada en una unidad de cuidados intensivos que también pueden ser graves y a veces no se distinguen bien unas de otras. debilidad muscular, desorientación, depresión y problemas psicológicos son secuelas muy habituales entre los pacientes que salen de la uci por enfermedades diferentes. por la anterior razón es difícil, por ahora, saber qué es efecto directo del virus y que puede derivarse del proceso de hospitalización. aún es difícil decir si los daños a largo plazo dependen del propio virus o de los efectos adversos del proceso tratante. sin embargo, este análisis de las posibles secuelas del covid- en el cuerpo, se presenta con más dudas que certezas. como es habitual en medicina, las causas pueden ser múltiples y muchas veces reflejan la participación de varias complicaciones que se han podido dar durante el proceso infeccioso directo o por la hospitalización, la información sobre los mecanismos de invasión del sars-cov- en todos los órganos sigue siendo, por ahora, escasa. y también lo es nuestro conocimiento sobre los efectos adversos de los medicamentos, muchos de ellos experimentales, que se han utilizado durante esta crisis. gran parte de lo que conocemos actualmente sobre los efectos de este virus proviene de la experiencia clínica de otros colegas y de las historias de pacientes que han sufrido la enfermedad, quedando todavía mucho por descubrir. dado el actual panorama, diferentes hospitales e instituciones de salud se preparan en torno a la rehabilitación, habilitando ya unidades multidisciplinares poscovid- para el seguimiento de estos pacientes y algunos centros están contactando con los pacientes dados de alta para evaluar su estado y hacer un seguimiento a su salud. también están en marcha varios estudios multicéntricos para evaluar el alcance de las secuelas, prácticamente todos los centros sanitarios deberán tener pautas de seguimiento y control para los pacientes ya dados de alta, creándose necesario la utilización simultánea de amci ® instrumentos de medición de calidad de vida, que en nuestro país colombia, ya se han utilizado previamente con este fin ( , ) . con este instrumento de medición de la calidad de vida se realizaron algunos estudios piloto tanto en pacientes crónicos como en la población general con el fin de determinar la comprensión del instrumento y factibilidad de aplicación del mismo en cuanto a la consistencia interna, la revisión realizada por vilagut y cols demostró que la aplicación de la escala arrojó en diversos estudios un alfa de cronbach igual o superior a . en todas las escalas excepto en función social ( ) . y aunque un número cada vez mayor de estudios mide los resultados físicos, cognitivos, de salud mental y de calidad de vida relacionada con la salud (cvrs) en los sobrevivientes adultos de la uci, los datos sobre las propiedades de medición de tales instrumentos son escasos y, en general, de calidad deficiente a justa. se necesitan análisis empíricos que evalúen el rendimiento de los instrumentos en adultos sobrevivientes de la uci para avanzar en la investigación en este campo ( ) . finalmente, el conocimiento de las secuelas y complicaciones dejadas por la infección del covid- , permitirá identificar importantes variables clínicas que acompañan a esta enfermedad y que afectan de manera importante la calidad de vida de los pacientes que padecen la enfermedad severa en la unidad de cuidados intensivos. en la actualidad no existen estudios para evaluar el riesgo de malnutrición aguda en pacientes hospitalizados por sars-cov- . experiencias con otras infecciones virales por influenza, se han identificado como factores asociados con mortalidad, la presencia de malnutrición, la adquisición de infección intrahospitalarias, la falla respiratoria y la presencia de infiltrados en la radiografía de tórax ( ) . las guías espen recomiendan utilizar el must o el nrs- ( ), para la tamización del riesgo nutricional, estos puntajes de tamización previamente han sido validados en múltiples patologías y contextos clínicos; sin embargo, existen otros puntajes útiles desde la perspectiva clínica como la valoración global subjetiva, mini-nutritional asessment( ), puntaje nutric ( ) y la global leadership initiative on malnutrition (glim) ( ) . el proceso de diagnóstico nutricional debe involucrar, dos componentes: la identificación del riesgo con la utilización de alguno de los puntajes previamente validados en otros contextos y posteriormente el diagnóstico de los pacientes con malnutrición y la valoración de la gravedad de ésta; en este último paso es importante la valoración del índice de masa corporal, los hábitos de consumo calórico y proteico, la presencia de inflamación, los trastornos gastrointestinales, las enfermedades de base y siempre que sea factible el cálculo de la masa muscular. tabla . en vista del riesgo de infección al personal de salud, no siempre será necesario la visita nutricional al paciente, ésta podría ayudarse con entrevista al familiar, interrogatorio vía amci ® telefónica y sólo en caso necesario el examen del paciente para lo cual se requiere el uso de equipo de protección personal completo. esta estrategia de interrogatorio al familiar o al paciente por vía remota o telefónica puede ayudar a identificar los patrones de consumo y los hábitos nutricionales de riesgo y en caso de ser necesario la valoración nutricional disminuye el tiempo de exposición a un ambiente contaminado. para la atención presencial de pacientes en el ámbito de cuidado intensivo, es necesario definir cuál es el riesgo que existe de infección para el personal de salud, para aclarar esta pregunta se debe definir si hay un riesgo de generación de aerosoles( ). aunque no existen pautas específicas para la nutrición en pacientes con covid- , las diferentes sociedades científicas han desarrollado guías de pauta clínica para la nutrición de pacientes con esta enfermedad ( , ) . idealmente la nutrición debe ser iniciada de forma temprana, esto se refiere al inicio en las primeras a horas del ingreso a cuidado intensivo o en las primeras horas luego de la intubación y el inicio de la ventilación mecánica( ) y se prefiere la vía enteral. aunque no existen estudios para evaluar el momento del inicio de la nutrición en pacientes con infección por sars-cov , el inicio temprano de la nutrición ha mostrado beneficios en términos de mortalidad y reducción de infecciones con dicha estrategia ( , ) . adicionalmente es importante, evaluar el riesgo de morbilidad y mortalidad asociado a la malnutrición aguda en el ámbito del paciente crítico, en los pacientes que no se alcance la meta de aporte calórico y proteico por vía enteral o que exista contraindicación para ésta, se debe considerar el inicio de nutrición por vía parenteral, especialmente cuando su riesgo nutricional agudo sea elevado (puntaje nutric ≥ , nrs ≥ ) ( ) ( ) . el choque no es una contraindicación para la utilización de nutrición enteral ( ) y no es una indicación para el uso de nutrición parenteral, quizás la mejor estrategia, es vigilar la presencia de disfunción gastrointestinal, en combinación con la presencia de intolerancia a la nutrición enteral, especialmente en pacientes con acidosis láctica en progreso y cuando sea necesario escalar la dosis de vasopresores o exista incapacidad para la reducción de éstos. no es necesario medir el residuo gástrico de rutina, es preferible iniciar procinéticos de forma rutinaria. la sonda debe colocarse con cuidado de evitar riesgo de contaminación, preferiblemente al entubar al paciente. algunos pacientes pueden presentar diarrea, ya que se ha descubierto la presencia de la proteína ace (receptor del virus sars-cov- ) en células del esófago, estómago, duodeno y recto. no existe evidencia que indique que la nutrición enteral durante la posición prono aumente el riesgo de complicaciones. sugerimos no suspender nutrición enteral al durante la pronación, se debe iniciar con dosis trófica de ml/h. amci ® se recomienda una estrecha monitorización de la tolerancia a la nutrición enteral para pacientes en posición prono. se recomienda para aumentar la tolerancia de la ne a los pacientes en posición prona, una elevación del tórax entre - º (posición de trendelenburg inversa) no realizar endoscopias digestivas para ubicación de sondas avanzadas recomendaciones de nutrición parenteral los pacientes con covid- pueden requerir niveles significativos de sedación y bloqueo neuromuscular, lo que puede aumentar la incidencia de intolerancia gastrointestinal. la nutrición parenteral (np) debe utilizarse donde la alimentación enteral no está disponible o no logra completar el % de los requerimientos. si existen limitaciones para la ruta enteral, se podría recomendar nutrición parenteral periférica (npp) en la población que no alcanza el objetivo proteico energético por nutrición oral o enteral. la np temprana debe 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of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition espen guideline on clinical nutrition in the intensive care unit safety and outcomes of early enteral nutrition in circulatory shock gastrointestinal function in intensive care patients: terminology, definitions and management. recommendations of the esicm working group on abdominal problems key: cord- -z twpb authors: mcaloose, denise; newton, alisa l. title: wildlife cancer: a conservation perspective date: journal: nat rev cancer doi: . /nrc sha: doc_id: cord_uid: z twpb until recently, cancer in wildlife was not considered to be a conservation concern. however, with the identification of tasmanian devil facial tumour disease, sea turtle fibropapillomatosis and sea lion genital carcinoma, it has become apparent that neoplasia can be highly prevalent and have considerable effects on some species. it is also clear that anthropogenic activities contribute to the development of neoplasia in wildlife species, such as beluga whales and bottom-dwelling fish, making them sensitive sentinels of disturbed environments. cancer is an important cause of morbidity and mortality in several wildlife species. virus-associated, carcinogen-related and novel transmissible tumours currently impact threatened and protected species. cancer can affect conservation outcomes by reducing reproductive success, altering population dynamics (see glossary) or directly or indirectly leading to population declines. in one species, the tasmanian devil (sarcophilus harisii), high cancer incidence threatens the species with extinction. the links between animal and human health and scientific discovery are long standing. viral oncogenesis is a familiar concept in both animal and human fields of medicine, and the study of animal viruses has led to important insights into the molecular basis of cancer. discovering the transmissible effects of the rous chicken sarcoma virus in (ref. ) led to the eventual identification of the src oncogene and its role in human cancer. identification of the ras oncogene originated from studies of murine leukaemia virus and our understanding of the crucial role wnt signalling pathways have in carcinogenesis resulted from studies of mouse mammary tumour virus . it is likely that animals, including wildlife, will continue to contribute to our understanding of cancer biology in the future. novel allograft tumours in tasmanian devils and dogs may influence the development of beneficial animal and human cancer immunotherapies, and wildlife that develop cancer from anthropogenic factors will continue to act as sentinels for animal and human health risks. what is learned about cancer through wildlife health monitoring will affect conservation of animals and wild places and affect human health, consistent with the one world, one health concept (one world, one health is a registered trademark of the wildlife conservation society) . cancer is a major cause of death in humans. the world health organization estimated that in , . million human deaths globally (approximately % of the total number of deaths) were due to cancer . however, despite its high incidence, cancer as a cause for human extinction is unlikely to warrant much consideration. until recently, the same could have been said for most wildlife species. in the few surveys that summarized wildlife mortality data, trauma and starvation were most frequently reported as causes of death , . however, over the past few decades wildlife health monitoring has increased and we are now gaining an improved -and occasionally alarming -perspective about the presence and impact of cancer in endangered species, such as the tasmanian devil, western barred bandicoot (perameles bougainville) and attwater's prairie chicken (tympanuchus cupido attwateri), and non-endangered species, such as the beluga whale (delphinapterus leucas). evaluating health and diagnosing disease in wild animal populations poses several challenges. as in human medicine, data and sample collection in wildlife is performed by networks of professionals and non-professionals across many disciplines, including veterinarians, veterinary pathologists, biologists, epidemiologists and citizens in field stations, universities, zoos and communities. access to live or deceased animals and sample collection can be complicated by environmental obstacles, such as thick jungle or wide dispersal of animals across oceans and vast savannahs, species-specific adaptations, such as flight, or tissue loss through environmental decomposition, predation or post-mortem scavenging. cancer in wildlife is most commonly detected during post-mortem examination with confirmation through histopathology. advanced cancer diagnostics, such as computed tomography and magnetic resonance imaging that are available for cancer detection and identification in humans are generally not accessible or are unavailable for wildlife. techniques such as immunohistochemical staining are of value but have limited applicability in wildlife owing to the species-specific nature of protein targets and consequently the antibodies required for detection. most animal cancer detection resources and the best developed networks exist in the companion animal and agriculture sectors. relatively limited resources, human or financial, are dedicated to consistent or widespread wildlife health monitoring and disease diagnostics. for these reasons and those listed above, cancer in wildlife goes largely undetected. establishing cancer prevalence is important if we are to understand the effect of cancer in wildlife species and its importance to conservation. however, determining valid cancer prevalence (or prevalence of any disease in wildlife) is not often achieved. most wild animals live and die in anonymity without being documented in census data, without baseline health information or without the causes of morbidity and mortality ever being determined or recorded. an exception to this general rule are small, geographically isolated populations, such as the critically endangered island fox (urocyon littoralis), for which population size and animal health can be feasibly monitored and valid disease prevalence can be established . it is more common to determine disease prevalence relative to a sampled group. for example, beluga whales inhabit arctic and subarctic waters along the coasts of alaska, canada, greenland and russia. the southernmost extent of the range includes an isolated population in the st lawrence river estuary (sle) where population and health monitoring have been ongoing for years . in this population, cancer was the second leading cause of death overall (accounting for % of mortalities) with tumours identified in % of adult animals found dead , a rate that is strikingly similar to that found in humans, in whom cancer is the second leading cause of mortality in the united states and accounts for . % of all deaths . in this case, cancer prevalence was determined for the local population rather than the species. as the sle beluga whales are a well-characterized group, accurate data for epidemiological modelling can be applied to the local population; similar disease prevalence and epidemiological investigations can be performed in captive collections of s c i e n c e a n d s o c i e t y abstract | until recently, cancer in wildlife was not considered to be a conservation concern. however, with the identification of tasmanian devil facial tumour disease, sea turtle fibropapillomatosis and sea lion genital carcinoma, it has become apparent that neoplasia can be highly prevalent and have considerable effects on some species. it is also clear that anthropogenic activities contribute to the development of neoplasia in wildlife species, such as beluga whales and bottom-dwelling fish, making them sensitive sentinels of disturbed environments. animals (box ) . in less well monitored or more dispersed populations, disease prevalence is often estimated from retrospective information in the absence of accurate population data, and in many cases small numbers of animals are assumed to be representative of the species as a whole, a situation that may or may not be accurate. in wildlife with cancer, the focus is generally not on treatment but rather is directed at understanding tumour biology, prevention and intervention strategies in spontaneous tumours, as well as efforts to affect conservation policy or introduce mandatory environmental abatement in anthropogenically induced tumours. research and resources for investigating wildlife cancer are extremely limited and successful outcomes for wildlife are more likely for tumours arising in association with anthropogenic activities and interventions that also affect humans. in one notable exception, intense activity is currently focused on tasmanian devils, in which a spontaneous transmissible tumour is spreading to epidemic proportions resulting in devastating effects on the population. in this case, efforts are underway to manage the remaining animals and develop conservation strategies without which species extinction is predicted. the tasmanian devil survives only on the island state of tasmania, australia, and is threatened by extinction owing to a contagious cancer, devil facial tumour disease (dftd) [ ] [ ] [ ] [ ] . it presents as a focal or multicentric neuroendocrine tumour , that generally affects the face and neck and progresses to cause considerable soft tissue disfigurement; metastasis, most commonly to regional lymph nodes or the lungs, occurs in up to % of the animals . complications associated with tumour growth or metastasis result in a mortality rate of % in affected animals. in the early s, tasmanian devils were considered to be common and the tasmanian devil population was estimated at , individuals ; however, since its first observation in (refs , ) , dftd has decimated the tasmanian devil population resulting in a population decline of % and the listing of the tasmanian devil as an endangered species by the international union for the conservation of nature and natural resources (iucn) in . current disease modelling predicts declines of % across % of territories with diseased tasmanian devils and a % reduction of the entire population over the next years , , . at these rates, extinction is a real possibility. evidence supports a novel mechanism of dftd persistence and spread within the population: allograft transmission. although initial transmission trials are not yet published, transplantation of cultured dftd cells and surgically implanted tumour tissue has produced tumours in unaffected tasmanian devils . further evidence supporting the allograft theory includes failure to identify pathogens (such as a virus) using routine light and electron microscopy . additionally, in all animals studied, karyotype analysis shows rather than chromosomes and consistent genetic aberrations, including loss of both sex chromosomes, no copies of chromosome , loss of copy of chromosome and the addition of unidentified marker chromosomes (m -m ) in tumour cells compared with host cells . furthermore, evaluation of multiple microsatellite and major histocompatibility complex (mhc) loci has confirmed genotypes that are identical in tasmanian devil tumours but differ from their hosts, consistent with an exogenous tumour source . despite having a competent immune system [ ] [ ] [ ] , ease of tumour spread is thought to be related to low mhc class i diversity and limited cell-mediated immunological reaction and activation (rather than the failure of immune cells to proliferate) to tumour cells in tasmanian devils , , . canine transmissible venereal sarcoma (ctvs) is the only other cancer in wildlife known to be naturally transmitted through an allograft ; the unintentional transfer of undiagnosed neoplastic cells through organ transplantation is the only comparison in humans [ ] [ ] [ ] . ctvs was first described and transmitted between dogs in by novinski (reviewed in ref. ). it is a histiocytic tumour that generally affects the external genital mucosa of sexually active dogs and is transmitted during breeding and the licking or sniffing of affected tissue , , . the tumour is locally extensive more often than metastatic and in many animals it regresses within several months; morbidity rather than mortality is therefore more common in ctvs. the tumour is present in free-ranging dogs worldwide and is most prevalent in tropical and subtropical countries . ctvs is thought to have developed in a wolf (canis lupus) or east asian breed of dog to , years ago , [ ] [ ] [ ] , and on the basis of genetic markers, murgia et al. suggest that ctvs arose from a single common ancestral neoplastic clone that subsequently diverged into the two current genetically distinct tumour subtypes that exist in dogs today . similarly to dftd, experimental studies have confirmed that ctvs is a clonal cell allograft. supporting evidence includes transmission trials , common specific chromosomal anomalies, such as tumour markers (in particular the long interspersed nuclear element insertion near myc , ), and genetic comparisons of the mhc, microsatellites and mitochondrial dna between tumour and host cells . impaired differentiation of dendritic cells, downregulation of classical mhc class i and absence of mhc class ii , , (rather than low mhc diversity as in dftd) are recognized as factors in host immune system evasion and successful transmission and progression in ctvs , , . circulating anti-tumour antibodies in affected dogs are implicated in natural tumour regression , , which if complete is associated with subsequent resistance , . recent scientific interest in characterizing immunological reactions between the tumour and host, including the disease monitoring of captive animals, primarily those in zoos and aquariums, reveals a wide range of spontaneous neoplasia across the vertebrate taxa. retrospective mortality studies in two zoos have shown similar rates of neoplasia in captive wildlife to those in domestic animals , . tumours, such as chromatophoromas in snakes , , hepatocellular or biliary carcinomas in bears , , and renal, biliary and apocrine tumours in black-footed ferrets , are known to wildlife pathologists through various descriptions in the literature. unusual tumours, such as kaposiform hemangioendothelioma in a tamarin (sanguinus oedipus) or multilobular osteoma and chondroma in an african wild dog (lycaon pictus) have also been observed (d.m., unpublished observations). in a few situations, management practices for captive animals have been associated with cancer development. for example, subcutaneously implanted microchips used for the permanent identification of individual animals have been associated with soft tissue sarcomas in laboratory mice , and rats , as well as in a few zoo animals -degus (octogon degus), feathertail glider (acrobates pygrnaeus) and egyptian fruit bat (rousettus aegyptiacus) . additionally, melengestrol acetate contraception in exotic zoo felids, such as tigers, lions and jaguars, is associated with a high incidence of mammary gland carcinomas ( % in one report ) and is therefore no longer a recommended method of contraception in these animals. role of tumour and host cytokine expression, such as transforming growth factor-β (tgf-β ), interleukin- and interferon-γ, in this unique canine tumour includes consideration of the potential applicability of ctvs as an in vivo model for developing cancer immunotherapies in humans , . genetic diversity is a common topic of discussion and scientific debate in the conservation community. low mhc diversity is implicated as a factor in disease susceptibility owing to its crucial role in immune system surveillance and resistance to infectious disease; it is of special concern in endangered species, such as the cheetah (acinonyx jubatus). low mhc diversity is thought to be important to the high susceptibility of cheetahs to infectious diseases, such as feline coronavirus and the unusually high acceptance of tissue allografts between cheetahs . low mhc diversity has been implicated in dftd transmission and may have been important in the early development of ctvs . current evidence and disease modelling suggest that transmission of dftd and ctvs occurs in a frequency-dependent , , , rather than a density-dependent manner. in frequency-dependent disease transmission models, transmission rate depends on the frequency of contact with an infected host and is independent of population size (for example, human sexually transmitted diseases are often modelled in this manner). if the modelling is correct, dftd could therefore lead to the extinction of the tasmanian devil despite its decreasing population density. in classical density-dependent disease transmission models (commonly applied to infectious diseases, such as measles or influenza) there is a population threshold below which disease is not maintained in the host , and in which disease disappears in the absence of alternative disease reservoirs. recent modelling by hilker et al. suggests that there are certain conditions under which density-dependent disease transmission could lead to extinction in the absence of reservoirs, such as when there is a strong allee effect as is proposed in the predatorprey relationship between golden eagles and the critically endangered island fox . if dftd transmission were density rather than frequency dependent, extinction could also potentially occur under certain densitydependent conditions. dftd is incurable and unpreventable. in an effort to avoid species extinction, scientists and conservationists are considering establishing isolated assurance colonies of tumour-free animals in geographically restricted areas in tasmania or zoos to guard against ongoing population declines while preventive therapies are developed and tested. it is a race against time to ensure the survival of the tasmanian devil. as the population loses ground to the disease, the tasmanian devil's crucial role as the top carnivorous marsupial in the tasmanian ecosystem and its survival will be challenged by native predators, such as the spottedtail quoll (dasyurus maculates) and eastern quoll (dasyurus viverrinus), as well as introduced species, such as the fox and domestic cat (felis catus) . if efforts fail and the tasmanian devil disappears, the tasmanian devil's role in the tasmanian ecosystem and its contribution to biodiversity will be lost forever and dftd will represent the first known instance of a contagious cancer causing the extinction of a species. the biology of dftd and its devastating and rapid effects on the tasmanian devil population are unique regardless of species. more commonly recognized mechanisms of tumorigenesis in both animals and humans include mutations of proto-oncogenes involved in cell cycle regulation, signal transduction and tumour suppression (such as ras, wnt or p ) or the effects of viral oncogenes, such as src. the effects and implications of oncogenic viruses and mechanisms of tumorigenesis in most wildlife species are poorly understood; however, in some species these issues and the consequences for conservation are in the process of being elucidated. attwater's prairie chickens and western barred bandicoots are endangered largely because of habitat destruction , and, in the case of the bandicoot, introduced predators such as foxes (vulpes vulpes) and domestic cats . wild attwater's prairie chicken populations totalled less than by the mid s and fewer than several thousand western barred bandicoots are thought to survive today . captive breeding programmes in both species were established in the s but have been hampered by cancer-causing oncogenic viruses. reticuloendotheliosis viruses (revs), a group of avian gammaretroviruses that are similar to mammalian type c retroviruses , , are associated with lymphomas in attwater's prairie chickens. revs have been isolated from attwater's prairie chickens at every captive breeding facility . natural infection most often causes runting, immuno suppression or a non-neoplastic syndrome associated with high mortality in young birds and b or t cell lymphomas in adults. experimental infection with rev apc- is oncogenic in japanese quail a decrease in population fitness, such as population decline, at low population density. in the lowest ecological regions (such as the sediment surface) of a body of water. an animal in the order cetacea; includes, whales, dolphins and porpoises. a benign tumour of the intrahepatic bile ducts. tumour of the pigment-producing, light-reflecting cells, xanthophores, erythrophores and iridiphores, in vertebrate and invertebrate species, such as amphibians, fish, reptiles, crustaceans and cepahalopods. an organochlorine insecticide that bioaccumulates in the environment and is carcinogenic. an animal in the orders carnivora or odobenidae (walrus), otariidae (fur seal and sea lion) or phocidae (true seals). marginal and long-term changes in birth, death, immigration, emigration and composition (such as, sex, age and class) of a population. an animal in the families trichechidae (manatees) or dugongidae (dugongs and sea cows). (coturnix coturnix japonica) and specific pathogen-free chickens and turkeys, causing cancer (primarily lymphoma) as early as weeks after hatching in inoculated quail embryos and days and weeks postinoculation in domestic chickens and turkeys, respectively . infected attwater's prairie chickens can be chronically and significantly infected and appear to be clinically healthy for months before disease expression, acting as reservoirs for virus replication and transmission to susceptible birds . western barred bandicoots infected with bandicoot papillomatosis carcinomatosis virus type (bpcv ), a novel oncogenic virus that contains genetic material from both papilloma and polyoma viruses, develop cutaneous and mucocutaneous hyperplasias, as well as papillomas, some of which undergo malignant transformation to squamous cell carcinoma , . in a review of western barred bandicoots with lesions, hyperplasias ( %), carcinomas in situ ( %) and squamous cell carcinoma ( %) were all common, as was the presence of concurrent benign and malignant lesions in individuals ( %) . histologically, positive indirect immunohistochemistry for conserved papillomavirus capsid antigens and identification of viral crystalline arrays in affected keratinocyte nuclei with transmission electron microscopy support a causal relationship between the virus and tumour development . virus-associated debilitation and tumour-associated death in rev-and bpcv -infected attwater's prairie chickens and western barred bandicoots, respectively, have led to limited population growth and are a risk to the survival of each species. the potential for disease transmission from captive to free-ranging remnant populations has implications for release and reintroduction efforts. unlike the situation for attwater's prairie chickens and western barred bandicoots, confirming the effect of virus-associated neoplasia in most wildlife species is often much more challenging. for example, in the marine environment, increases in spontaneous benign and malignant tumours have been identified over the past two decades , - . several viruses seem to have a role in the formation of these tumours but causal relationships remain to be confirmed, and understanding short-and long-term population effects will require ongoing and expanded monitoring. green turtles (chelonia mydas) around the globe are dying from herpes-associated fibropapillomatosis , - . these turtles are listed by the iucn as endangered owing to several factors, including habitat disturbance or destruction, over-harvesting of animals and eggs, boat strike and entanglement in fishing nets that lack turtle excluder devices. in some parts of their range fibropapillomatosis-associated death is now also considered to be a contributing factor to overall population decline. in well-monitored populations, such as those along the coasts of florida and the caribbean and hawaiian islands, fibropapillomatosis is thought to be an epidemic , and dramatic increases in prevalence of as much as % since the early s have been observed , . given its global distribution, some have suggested that the disease probably reflects a worldwide panzootic , , . fibropapillomatosis is most commonly observed in green turtles but has been described for all sea turtle species, including the critically endangered leatherback (dermochelys coriacea) , kemp's ridley (lepidochelys kempii) , and hawksbill (eretmochelys imbricata) turtles. fibropapillomatosis in sea turtles is characterized by benign nodular to papilliferous, fibropapillomas and fibromas rather than fibrosarcomas (which are observed but with less frequency) . tumours generally arise in the dermis but can be found in internal organs, such as the lung, liver, kidney and heart. tumours in non-cutaneous sites may reflect metastasis from primary cutaneous tumours or multicentric development that is secondary to systemic virus dissemination, as the virus has been detected with real-time pcr in tumours in cutaneous and non-cutaneous sites , . herpes viral inclusions are seen with variable frequency in tumour epithelium , , . debilitation and death from fibropapillomatosis occurs when tumour growth interferes with crucial functions such as feeding, sight and mobility. the cause, environmental persistence and mode of natural transmission, as well as the cofactors and mechanisms of tumorigenicity of marine turtle fibropapillomatosis, are under investigation. transmission studies and consensus in the scientific community favours a new chelonian alpha herpesvirus as its cause , . viral dna is consistently found in tumours and tissues of tumour-bearing animals and inoculation of fibroblasts from turtle fibropapillomas has produced fibromas in immunodeficient mice . however, to date this new turtle virus has not been cultured and koch's postulates have not yet been demonstrated. limited experiments on two cultured sea turtle herpesviruses, lung-eye-trachea disease virus and hv , have established that both can persist and retain their infectivity outside the host in the marine environment for up to hours . if fibropapilloma-associated turtle herpesvirus behaves similarly, environmental persistence could be important in natural disease transmission, especially in areas of high turtle density. other factors that could contribute to viral persistence and spread include mechanical vectors, such as leeches, which have been shown to carry sufficient viral loads (up to million copies) to be a potential vector . virus-associated papillomas and carcinomas are also described in several marine cetacean and sirenian species in freeranging and managed populations (table ) . in wildlife, tumours of the genital tract are important if they interfere with successful breeding, pregnancy or parturition. in one study, benign genital papillomas were present in . % of dusky dolphins and . % of burmeister's porpoises , and were considered important enough to interfere with copulation in % of burmeister's porpoises , . genital tract carcinoma is an emerging disease in california sea lions. before the early s, malignant tumours of any type were only rarely reported in pinnipeds , , . however, from to , % of sexually mature sea lions that were found stranded along the californian coast and died during rehabilitation had histologically aggressive, widely metastatic genital transitional cell carcinomas . in a subsequent report, . % of california sea lions that died during a series of unusual mortality events caused by harmful algal blooms from may to october, , had benign or malignant genital tumours . otarine herpesvirus- is the putative cause of genital carcinoma in california sea lions , . this virus is consistently found in tumours examined by electron microscopy or immunohistochemical staining and by pcr of the viral dna polymerase and terminase genes [ ] [ ] [ ] . otarine herpesvirus- is a gammaherpesvirus in the genus rhadinovirus and is closely related to human herpesvirus- (ref. ), the causative agent of kaposi's sarcoma , . interestingly, papillomaviruses cause cervical cancer in women, and human papillomaviruses and are considered to be high risk for the development of malignant cancer , . surprisingly, no papillomaviruses have been detected in sea lion or other marine mammal genital tract carcinomas , . the high prevalence of genital tract carcinomas in california sea lions is unprecedented in any pinniped species. despite apparently increasing cancer prevalence, tumours have not been associated with changes in population growth, which from to has been increasing at an annual rate of approximately . % per year . continued monitoring will be essential to determine long-term population effects, identify causes for high prevalence and establish potential environmental cofactors that initiate or promote tumour development. cancer is a multifactorial disease. in the domestic cow infection with bovine papillomavirus (the cause of oesophageal and rumenal papillomas) and exposure to ptaquiloside (a natural carcinogen in bracken fern) results in the malignant transformation of papillomas to squamous cell carcinoma . a similar interplay may occur between viruses and chemical cofactors in sea turtle fibropapillomatosis (that is, increased incidence of fibropapillomatosis in sea turtles in polluted bodies of water) , or sea lion genital cancer: an % higher level of polychlorinated biphenyls is found in the blubber of sea lions with genital carcinoma relative to those without genital carcinoma . systematic studies assessing the potential direct or indirect roles of these contaminants in tumour development have not been performed. however, the above examples suggest that in some wild populations, carcinogenesis reflects the combined effects of multiple factors, potentially including those from the local environment. high cancer incidence is reported in wildlife populations in environments that are heavily contaminated with anthropogenic chemicals. fish living in industrialized waterways suffer epizootics of liver and skin cancer - . in the population of beluga whales living in the sle, an environment that receives effluent from aluminium smelting facilities , cancer is the second leading cause of death . although several industrial and agricultural pollutants have been recovered from the estuary, polycyclic aromatic hydrocarbons (pahs) are a major concern, as they are recognized occupational hazards and human carcinogens [ ] [ ] [ ] [ ] . causal relationships between wildlife cancers and contaminant exposure are receiving increased attention owing to risks for both wildlife and humans. cancer epizootics have been recognized in many species of fresh water, marine and estuarine fish. the most intensively studied epizootics have occurred in industrialized areas of the united states and canada, such as the great lakes tributaries, including locations considered to be areas of concern (box ), puget sound harbours and bays of the east coast of the united states. these environments are all contaminated by pahs released from steel mills, creosote production plants and petroleum facilities , , , . epizootic tumours reported in these areas include hepatocellular adenomas, hepatocellular carcinomas, cholangiomas and cholangiocellular carcinomas in brown bullhead catfish (ictalurus nebulosus) and english sole (parophrys vetulus) , , epidermal and oral papillomas in brown bullheads and white sucker fish (catostomus commersoni) , and (rarely) squamous cell carcinomas and melanomas in brown bullhead catfish , . most affected species are bottom feeders, which suggests that a benthic lifestyle contributes to high cancer incidence through chronic exposure to contaminated sediment and consumption of contaminated invertebrates. pah profiles from species experiencing epizootic cancer support this theory, as they reflect the pah compounds found in the food items and compound profiles of the sediment in their habitats , , . support for causal relationships between environmental pollutant exposure and cancer in fish has been experimental and observational. fish laboratory models have demonstrated that dietary and intraperitoneal exposure to the pah benzo[a]pyrene (bap) produces hepatocellular or biliary tumours . additionally, skin and liver tumours have been induced by exposure to extracts prepared from pah-contaminated sediment. in one experiment, extracts from environmental sediment samples were painted onto the skin of brown bullhead catfish and this resulted in a % increased incidence of skin tumours over years . in a similar study with the same extract, dietary exposure resulted in both biliary and hepatocellular tumours . striking causal associations have also been made when environmental contamination decreased following closure of industrial facilities along affected waterways. for example, the prevalence of hepatocellular carcinoma in adult brown bullhead catfish living in the black river, ohio, united states, ranged from % to % in the early s . at that time, age-selective mortality owing to the high cancer prevalence nearly eliminated in canada and the united states signed the great lakes water quality agreement to address environmental issues in the great lakes region. in , the two countries designated areas of concern (aoc), locations selected owing to their proximity to urban and industrial centres, contaminated river sediment, potential sewer outflows and runoff from paved surfaces (see figure) . at each location, federal, state and provincial governments developed a remedial action plan (rap), a set of guidelines to abate anthropogenic environmental influences. success and eventual delisting are based on improvements in the chemical, physical or biological integrity of the site. many of the great lakes fish populations affected by high rates of cancer occur in areas of concern. there are many methods of measuring success, but decreases in fish deformity and tumour occurrence are one milestone included in most plans, a recognition that fish serve as sentinels of the health in these habitats. so far, of the areas have been delisted. two additional locations have been designated as areas of recovery. induction of small intestinal neoplasia fish older than years from the population . following a downturn in the steel industry, the coking facility located along the river closed in . pah levels in the sediment declined from , μg per g in to μg per g in . during that time, the incidence of liver cancer in brown bullhead catfish decreased by % and the percentage of -year-old fish in the population tripled . cancer is rarely reported in wild or captive cetaceans, and the literature consists predominantly of single cases of lymphoma, leukaemias and a wide range of other neoplasms (for example, granulosa cell tumours, seminomas, and cholangio, renal, squamous and anaplastic carcinomas) , , . however, beluga whales in the sle exhibit a high rate of cancer. small intestinal adenocarcinoma is the most frequent malignancy seen in sle belugas, in contrast to that observed in other cetaceans , , . additionally, among marine mammals, mammary carcinoma has been reported only in sle beluga whales , . industrial and agricultural environmental contaminants have been identified in the sle [ ] [ ] [ ] [ ] [ ] . high concentrations of bap have also been documented in sle tissue samples, and the concentrations of polychlorinated biphenyls, dichlorodiphenyl trichloroethane, mirex, mercury and lead are much higher in beluga whales from the sle than those living in the arctic , , . after a decline in the population owing to hunting pressures, the sle beluga whales received endangered species status from the canadian government in , but since that time there has been no evidence of population recovery , . comparative mortality data from the isolated sle beluga whales and a population of beluga whales in a less contaminated environment in northwest alaska indicate that beluga whales from the sle die at an earlier age, in part owing to the high rate of cancer , . high environmental levels of bap are proposed as an important factor in tumorigenesis in sle beluga whales; however, this idea is contentious and the role of these agents is under active investigation. bap is a potent procarcinogen, and the site of metabolism and carcinogenesis depends on the route of exposure [ ] [ ] [ ] . mice chronically exposed to oral bap develop small intestinal adenocarcinomas, gastric carcinoma and papillomas of the squamous portion of the stomach , . high environmental levels of bap in the sle, known human carcinogenicity and evidence from animal models has led to the theory that oral bap exposure in beluga whales has a role in their high incidence of intestinal cancer. the proposed source of exposure to bap in sle beluga whales is ingestion. beluga whales dredge the sediment, feeding on large numbers of invertebrates, animals known to bioaccumulate bap , . blue mussels in the saguenay river portion of the sle beluga whale habitat contain bap levels that are times higher than in blue mussels in adjacent habitats . pah exposure induces cytochrome p a (cyp a ) expression in hepatic and extrahepatic tissues and cyp a can serve as a biomarker for pah exposure. sle beluga whales show increased cyp a expression in multiple organs (including the liver, lung, urinary bladder and testis), which is consistent with systemic pah exposure and metabolism , . cyp a activates pahs into carcinogenic metabolites, and in animal models the proximal small intestine contains the highest concentrations of cyp a (ref. ). therefore, in sle beluga whales it has been suggested that ingested bap induces small intestinal enzyme activity, resulting in the high incidence of intestinal neoplasia observed (fig. ) . relationships between tumour development and environmental contamination are strongly suggested by scientific data and circumstantial evidence from wildlife studies. tumour epizootics in fish and the high cancer rate in the sle beluga whale population are important indicators of ecosystem health. care and caution must be applied to the selection of tumours used as environmental indicators. some epizootic cancers are solely viral (table ) or genetic in origin and therefore would not serve as appropriate indicators of environmental contamination. for example, spontaneous nephroblastomas of japanese eels (anguilla japonica) contain a mutant gene with a high level of homology to the human wilm's tumour suppressor (wt ) gene . therefore, only certain tumours can be indicators of environmental contamination and ecosystem health. similarities of high cancer incidence and tumour type between species support the conclusion of common risk factors in shared environments and show the value of wildlife populations as important indicators of environmental discord. wildlife cancer reveals itself as a series of challenges and opportunities for conservation. the above examples include cases in which cancer has limited population growth or has caused population declines through the novel mechanism of allograft transmission, viral oncogenesis and the effects of carcinogenic environmental contaminants. however, the list of examples is short and a more complete understanding of the role of tumours in wildlife population dynamics and the individual and interactive factors that drive tumorigenesis across a wide range of wildlife species is lacking but necessary. health monitoring, disease surveillance and scientific inquiry focused on understanding basic biology and interactive factors in wildlife cancer are crucial to our understanding of wildlife health, the role of cancer in wildlife populations and our ability to identify, assess and mitigate the risks for disease development in wildlife populations. key elements for improving environmental exposure to the procarcinogen benzo[a]pyrene (bap) is thought to have a role in the high incidence of small intestinal neoplasia in beluga whales in the st lawrence river estuary. the proposed mechanism involves intestinal exposure through ingestion of contaminated prey items and sediment followed by induction of cytochrome p a (cyp a ) in small intestinal epithelial cells. cyp a is responsible for oxidation and metabolism of bap to the carcinogen (bap , diol , epoxide). the activated carcinogen preferentially binds dna at the exocyclic nitrogen of the guanine residue, which is required for base pairing, resulting in the potential for g → t transversion during dna replication and disruption of tumour suppressors or proto-oncogenes. current limitations that exist in each of these key activities include identifying, coordinating and expanding existing surveillance networks; increasing and developing capacity for disease diagnostics and epidemiology; securing increased funding for multi-disciplinary scientific research and training aimed at identifying the complex mechanisms involved in wildlife tumorigenesis; integrating human and animal health surveillance systems; creating a unified animal and environmental health database; and improving current relationships, as well as establishing new collaborative relationships with stakeholders and policy makers. expanding the range and scope of each of these activities will have broad and direct benefits for wildlife and also potentially for the environment and humans. early recognition of cancer epizootics in wildlife has the potential to drive timely environmental mitigation and influence environmental policy. building capacity and leveraging expertise across disciplines will result in expanded opportunities for advancing our understanding of normal cellular processes and mechanisms of carcinogenesis as has occurred historically in investigations of wildlife cancer. as we look to the future, there is tremendous opportunity for achieving imagined advances in our understanding of wildlife 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to ebv associated membrane and viral capsid antigens in burkitt lymphoma patients the relation of infection with the hepatitis b agent to primary hepatic carcinoma a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks herpesvirus and the lucké tumor isolation of a new type c retrovirus (htlv) in primary uncultured cells of a patient with sézary t-cell leukaemia molecular characterization of a unique retrovirus associated with a fish tumor identification and characterization of an exogenous retrovirus from atlantic salmon swim bladder sarcomas the authors thank d. lyden, w. karesh and t. chang, whose thoughtful suggestions, especially early on, helped shape the content of the manuscript. they also acknowledge d. joly and the anonymous reviewers, whose critical reviews and comments markedly improved the final version. key: cord- -bhl up authors: rantsios, a.t. title: zoonoses date: - - journal: encyclopedia of food and health doi: . /b - - - - . - sha: doc_id: cord_uid: bhl up zoonoses are “those diseases and infections which are naturally transmitted between vertebrate animals and man.” relevant lists of zoonotic diseases are presented. factors, including environmental aspects that may influence the manifestation of zoonoses and their significance for public health, are discussed. foodborne, nonfoodborne, and vector-borne diseases are presented, followed by an article on preventative actions and control measures. specific reference is made to the ‘one health’ concept, since, for zoonoses containment, an integrated approach and cooperation between all responsible health professionals, at all levels, is a sine qua non precondition. according to the joint who/fao expert committee on zoonoses, second report, in the year , zoonoses (the expression zoonotic diseases is also used) are "those diseases and infections which are naturally transmitted between vertebrate animals and man." the transmission may take place directly or indirectly by means of vectors. the severity of zoonotic diseases in humans varies from mild symptoms to life-threatening conditions. zoonotic diseases are usually transmitted at the human-animal interface through exposure to animals (e.g., rabies), animal products (e.g., brucellosis and salmonellosis), or their contaminated environment (e.g., echinococcosis/ hydatidosis). transmission may occur by a diseased animal or by a clinically normal animal that, nevertheless, is able to transmit pathogens to humans (e.g., campylobacter and verotoxin producing escherichia coli (vtec)). a distinction is made between foodborne and nonfoodborne diseases. a concise list of zoonoses appears in table . emerging infectious diseases (eids) are diseases appearing in a geographic area or population for the first time. the term reemerging diseases is also used for ancient diseases that are 'forgotten' and thought to be controlled or extinct from a particular area or population and manifest a new appearance. the emergence of eids is thought to be driven by socioeconomic, environmental, and ecological factors. they are, mostly, related to free-living wild animals. the term neglected tropical diseases is used for a certain list of diseases recognized as such by the who, including a number of zoonotic diseases appearing in table . neglected tropical zoonotic diseases are ancient endemic zoonoses in europe and north america. they are largely controlled and tended to be overlooked, while the attention is shifted to newly emerged zoonoses. they can be, potentially, turned into a pandemic. although, at the time of the aforementioned who report, the number of zoonoses was at the level of , they are doubled by then and still counting. zoonoses may be bacterial, viral, parasitic, or due to unconventional agents (e.g., prions). it is estimated that out of the about human nosogenic factors, due to biological agents, % have multiple hosts and move between species. in addition, % of the eids, in the last $ - years, are zoonotic; again, % of which originate from wild animals. further to the significance of zoonoses for human health, one must not underestimate the impact they are having in preventing the efficiency in animal food production and enhancing problems in living animals and animal products international trade. as an indication of the significance of zoonotic diseases for human health, you can see, in table , the most important zoonoses in terms of human health impact, livestock impact, amenability to agricultural interventions, severity of diseases, and emergence. zoonoses present a major threat to human and animal health. these diseases, as already indicated, are multifactorial manifestations and therefore a reflection of the complexities of ecosystems in which animals and humans coexist. they are influenced by multiple interrelated global factors, including ecological evolution, human demographics, and behavior. emerging and reemerging diseases represent failures in understanding the socioecological systems we live in and respond to new conditions. what we learn from these failures will largely determine how successful we are in developing sustainable and healthy human communities. as can be seen in figure , humans, livestock, and wildlife can expose each other and spread, among them, potential pathogenic agents. any one of these players, therefore, can spill over pathogens to the others. more specifically, changes in human behavior, expressed as population increase and urbanization, and the demand for improved living conditions, requiring in turn proportional increase in food production and related economic activity, in particular agricultural development, are a decisive contributing factor. for example, water management, associated with still water collections, one way or another, increases mosquito breeding opportunities, which in turn may result in enhancing disease occurrence, like rift valley fever. such conditions are observed after the construction of dams and irrigation networks. similarly, intensive animal farming promotes disease transmission through untreated or poorly treated waste material spread in the environment or through ventilation system diffusion of contaminated material carrying pathogens. due to the problems related to high animal population density, maintained in intensive livestock production (e.g., pigs and poultry), which facilitated disease transmission, efforts were made to combat them with the use of antibiotics, either therapeutically or preventatively. however, these schemes resulted in widespread antibiotic-resistant pathogens, including those of zoonotic significance. these conditions, more specifically, can contaminate animal food products and potentially provide for the production of unsafe foods. it is important in this respect to mention that, according to eu food food production premises are considered as food business operations. consequently, they are obliged to certain responsibilities, related to putting together and effectively implementing and functioning a food safety management system (fsms), tailor-made for each particular activity. in table appears a list of zoonoses emergence linked to agricultural intensification and environmental changes. evidently, ecosystems are complicated, and the impact they are having on fauna and thereof on conditions potentially harboring zoonotic agents does not provide for a general interdisciplinary multipurpose approach, with patterns applicable in all cases. on the contrary, principal factors, among others, biological, ecological, economic, and social characteristics, and climatic conditions must be assessed, ad hoc, locally within each ecosystem. the implementation, therefore, of the concept of one medicine and one health, which, as an integrated approach, is very important for facing zoonotic diseases per se because not, only it contributes to animal and human health but also it significantly supports, apart from aspects of economic development, food safety and security. foodborne zoonotic pathogens are transmitted through consumption of contaminated food and drinking water. infectious agents present in foodstuffs include bacteria (e.g., salmonella and campylobacter), viruses (e.g., norovirus and hepatitis a virus), parasites (e.g., trichinella), and prions (infectious agents of bovine spongiform encephalopathy). challenges to food safety continue to increase in unpredictable ways, largely due to changes in food production, processing, distribution, and the environment, which may contaminate food; to emerging germs and toxins of food safety significance; and to new conditions, created by new food technology applications, with an impact on food safety (e.g., minimally processed foods and maintenance of cold chain). in table , a list of the most important biological hazards responsible for foodborne illnesses appears, along with the relevant most important foods involved in each case. nonfoodborne zoonotic diseases are transmitted through the following: • direct contact or close proximity with infected animals or through the environment. examples are the following: ○ avian influenza, a viral disease occurring mainly in poultry and other birds but transmissible to other animals or humans. ○ q fever, caused by the coxiella burnetii, affecting animals and humans. human infection mainly results from the inhalation of contaminated dust from the placenta and birth fluids or feces from infected animals. ○ salmonella infections, which can originate from contact with infected reptiles and amphibians such as pet snakes, iguanas, and frogs or their environment. ○ vtec, which can be acquired through contact with infected farm animals. for zoonoses prevention, the focus is on surveillance, rapid detection, and quick response. zoonotic infections in animals may produce a distinctive recognizable disease, such as rabies, or they may manifest themselves as a mild illness or the animal may be entirely asymptomatic. however, in this last case, if the pathogen is transmitted to humans, it may result in illness if they lack the specific immunity required. anyone, who has contact with animals, can get a zoonotic disease, but some people may be more at risk than others. risk table the most important zoonoses in terms of human health impact, livestock impact, amenability to agricultural interventions, severity of disease, and emergence data from the who and authoritative literature: when there are several authoritative estimates, the midpoint is given. note: high human mortality gets a double weight as the most important criterion for many stakeholders. total score ¼ (human death  ) þ (humans affected) þ (high livestock impacts) þ (farm intervention possible) þ (other concerns: severe or emerging disease). the maximum possible score is therefore and the minimum . factors for susceptibility to zoonoses, among others, are certain population groups, including professional and occupational groups working in close contact with animals (like livestock attendants, slaughterhouse workers, and veterinarians); homeless and poor people; and in general, people with a weakened immune system, children aged less than five, the elderly, and pregnant women. the factors promoting zoonotic disease outbreaks in humans, apart from frequent contact with animals, include intensive livestock production, poor animal and personal hygiene, and overlap with wildlife habitat. a common way for vector-borne diseases to spread is through the bite of a mosquito or tick. people can get diseases in most places, where they might have contact with infected animals and insects, including animal displays, farms, petting zoos, county or state fairs, pet stores, child care facilities or schools, nature parks, and wooded and bushy areas. in order to reduce the risks of transmission of zoonoses from pet animals to humans and also to production animals, the concept of responsible pet ownership (rpo) is advocated. it is recognized that education and awareness promotion of pet owners, for rpo, to prevent zoonoses related to companion animals, is of a paramount importance for eliminating these diseases. the who and efsa promote activities, of the general public's concern, for preventing zoonoses. they include the following: -promotion of awareness to understand the potential risk for human infection from zoonotic diseases, after contact with animals. -specific risk communication, as it may be required, through appropriate public actions. biosphere wildlife livestock humans peri-domestic wildlife figure pathogen flow at the wildlife-livestock-human interface. arrows indicate direct, indirect, or vector-borne candidate pathogen flow. in each host species, there are a vast array of constantly evolving microorganisms, some of which are pathogenic in the host. these are a source of new organisms for other host species, some of which may be pathogenic in the new host or may evolve in the new host to become pathogenic. if the pathogen is also transmissible in the new host species, then a new transmission cycle may be established. the rate and direction of candidate pathogen flow will depend on the nature and intensity of interaction between wildlife, livestock, and human compartments and the characteristics of the compartments ( table ) . adapted from proceedings of the national academy of science, may , , vol. , no , p. . -improvement in the level of personal hygiene by ○ acquiring the habit to wash hands thoroughly and frequently, after contact with animals. ○ closely supervising children to ensure they wash their hands properly and avoid hand-to-mouth activities (thumb sucking, eating, and use of pacifiers) after animal contact. -the use of registered insect repellents and products that contain repellents for use on clothing. accordingly, treat clothing and gear, such as boots, pants, socks, and tents. -inspection for and removal of ticks from the human body, with specific care for children. -limitation in the number of places around residential areas for mosquitoes to breed by eliminating places holding water. for health professionals, recommendations may include the following: -responsible services to systematically search for potential sources of human infection from animal sources and the environment -joint efforts and coordination among public health authorities and related professionals, both public and private -risk communication and information sharing among responsible health services and close coordination to manage risks related to the movement and trade of livestock -concerted actions for ○ good practices in the efficient implementation of biosecurity measures in farms and at border or territory crossings; ○ continuously reminding and training people, who work with livestock and in slaughterhouses, for the significant importance of personal hygiene practices; ○ the implementation of the one health concept zoonotic diseases are strongly influenced by social and economic practices. many of them are thus not only diseases related to climate and environment but also diseases of poverty. in epidemiological terms, conditions of poverty increase the probability of enhanced contact and hence increase the likelihood of epidemics. combating diseases like tuberculosis and malaria has much more to do with housing, nutrition, and water management than with any advances in biomedical science. animal migrations facilitate the global spread of pathogens and increase cross-species transmission. understanding, predicting, and controlling diseases at the human-animal interface are a huge challenge, for health professionals, in today's world, where international trade and travel globalized diseases. however, even when risks are identified, adequate underlying infrastructure and resources are required to take the measures needed, if outbreaks and emergencies are to be prevented or controlled. special consideration needs to be given to vector-borne diseases. vectors may move to long distances and therefore may introduce disease, in new geographic areas, by means of human traveling, international trade, animal movement (more specifically livestock and migratory birds), the wind, and changes in agricultural practices. health is a state of complete physical, mental, and social wellbeing and not merely the absence of disease or infirmity. it is the outcome of a complex of several interdependent medical, economic, sociocultural, environmental, and ecological factors. people's health and well-being and equally animal health and welfare are strongly interlinked. both also influence and are impacted by the health of the environment. health is a precondition for well-being and respectively welfare. wellbeing and welfare reinforce health. the advancement of the health and well-being of people and animals depends on effective and sustained cooperation between varied professions and disciplines, in both the public and private sectors (world veterinary association (wva) position). therefore, multisectoral horizontal links at all levels between human and animal health professionals, public and private, are needed to face zoonotic diseases. reducing relevant risks is impossible to be achieved alone by a particular sector, regardless of its importance. therefore, there is an increasing convergence to a one health approach, which incorporates, in an integrated manner, cross-sectoral, multidisciplinary cooperation. one cannot distinguish human, animal, and environmental health. the health of each one of them is the precondition for the health of the others (see again figure ). building sustainable national mechanisms for more effective cross-sectoral cooperation has greatly facilitated risk assessment and management of specific diseases, such as h n influenza and rift valley fever. at the international level, strong cooperation among the who, oie, and fao is improving the efficiency of surveillance, including data collection, risk assessment, and risk management options, allowing for consistent, sciencebased risk communication on global health threats at the human-animal-ecosystem interface. a strategic agreement outlines the sharing of responsibilities and enhanced coordination of complementary roles and activities between the fao, oie, and who at national, regional, and global levels. internally, cooperation takes place across departments, clusters, and regions of the aforementioned organizations. externally, further cooperation materializes with links and contacts to additional international partners, such as international agencies and networks, ngos, and academia, and to national agencies, such as institutions and administrative governmental units. according to statements made by eu officials in support of it, one health is linked to livelihood and equity and fits with eu objectives to promote global security, social justice, international cooperation, and multilateralism and fight poverty. further, there should be no resignation vis-à-vis the existence of different health standards across nations. table biological hazards responsible for most important foodborne illnesses and related foods campylobacter (poultry) e. coli o :h (ground beef, leafy greens, and raw milk) listeria (deli meats, unpasteurized soft cheeses, and produce) salmonella (eggs, poultry, meat, and produce) vibrio (raw oysters) norovirus in many foods (e.g., sandwiches and salads) toxoplasma (meats) for the eu, the one health movement has in many senses grown out of the response to influenza-related crises. the definition of one health chosen by the european commission in its external relations and actions reads as follows: the one health approach consists of (i) improving health and wellbeing through the prevention of risks and the mitigation of the effects of crises that originate at the interface between humans, animals and their various environments; (ii) for that purpose: (a) promoting a multi (cross) sectoral and collaborative approach; and (b) promoting a 'whole of society' approach to health hazards, as a systemic change of perspective in the management of risk. in may , a meeting was organized by the centers for disease control and prevention (cdc), atlanta, georgia, the united states, titled 'operationalizing "one health": a policy perspective.' 'critical enabling initiatives' such as 'training,' 'one health global network,' 'information clearing house,' 'needs assessment,' 'capacity building,' 'proof of concept,' and 'business plan' were identified as fundamental to moving forward one health. further, it seems reasonable to think that an improved coordination that includes intersectoral cooperation in surveillance, communications, outbreak response, and sample sharing community-based interventions for the prevention and control of zoonotic diseases is needed. there is further and more specific need for systematic cooperation between strong and autonomous public health services and strong and autonomous veterinary services, in the respect of their specific expertise. however, a culture of cross-sectoral cooperation does not yet exist all along the chain. fostering such a culture, stretching from the field level to that of international organizations, is the big challenge for successfully controlling zoonotic diseases in general and more particularly either emerging or neglected ones. there is a deficit in current university medical training, due to the fact that the whole training concept is geared to treat, rather than prevent, diseases and preserve and promote health. a cultural change putting emphasis on the prevention and appreciation of the importance of the connection, in terms of health and well-being, between humans, animals, and ecosystems, is required. combating zoonotic diseases is not how to clean up the disease mess after the fact, but on how to prevent the mess from occurring in the first place. it would almost appear that the ideological lenses through which diseases in general are being studied preclude acting on the evidence. this, if nothing else, should raise a warning flag that those who study disease are not necessarily well equipped to promote health. see also: escherichia coli and other enterobacteriaceae: food poisoning and health effects; milk: processing of milk. the european union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in emerging infectious diseases of wildlife -threats to biodiversity and human health mapping of poverty and likely zoonotic hotspots joint who/fao committee on zoonoses, second report global trends in emerging infectious diseases zoonosis emergence linked to agricultural intensification and environmental change one health agriculture-associated diseases: adapting agriculture to improve human health public health impact of zoonoses and international approaches for their detection and containment oxford textbook of zoonoses. biology, clinical practice and public health control (oxford textbooks in public health) paho/who zoonoses and communicable diseases common to man and animals: vol. i. bacterioses and mycoses chlamydioses, rickettsioses and viroses brucellosis: recent developments towards ''one health coordinating surveillance policies in animal health and food safety: "from farm to fork sharing responsibilities and coordinating global activities to address health risks at the animal -human-ecosystems interface. a tripartite concept note foodborne disease outbreaks: guidelines for investigation and control steps in a foodborne outbreak investigation html -who initiative to estimate the global burden of foodborne diseases key: cord- -j wbcr authors: prado-gascó, vicente; gómez-domínguez, maría t.; soto-rubio, ana; díaz-rodríguez, luis; navarro-mateu, diego title: stay at home and teach: a comparative study of psychosocial risks between spain and mexico during the pandemic date: - - journal: front psychol doi: . /fpsyg. . sha: doc_id: cord_uid: j wbcr context: the emergency situation caused by coronavirus disease (covid- ) has affected different facets of society. although much of the attention is focused on the health sector, other sectors such as education have also experienced profound transformations and impacts. this sector is usually highly affected by psychosocial risks, and this could be aggravated during the current health emergency. psychosocial risks may cause health problems, lack of motivation, and a decrease of effectiveness at work, which in turn affect the quality of teaching. despite their importance, there are hardly any studies that analyze psychosocial risks of non-university teachers during a health emergency such as that caused by covid- . objectives: the aim of this study was to analyze the perception of covid- and the psychosocial risks of non-university teachers comparing spain and mexico during the state of alarm caused by covid- . methods: data were collected from non-university teachers ( . % women; . % from mexico, . % from spain) aged – (m = . , sd = . ) via a self-completed questionnaire during the pandemic from march to april . results: data analysis suggests that inequity is the most important risk, followed by work overload. teachers appear to be moderately satisfied with the information on covid- and the measures taken, while their satisfaction with the available resources is lower. when comparing the two countries, significant differences can be observed in every risk considered except for social support, with lower levels in mexican teachers compared to spanish ones. in the case of the perception of covid- and its impact, the perception in general of levels of information, measures, and resources is better among mexican teachers than among spanish ones, who present higher scores of the impact of the health emergency. conclusion: the results underline the importance of the professional’s perception of resources during a health emergency, which could prevent to some extent burnout and possible alterations associated with it. the measures taken by the responsible entities and the provision of information do affect teachers not only directly but also indirectly by making them more vulnerable to psychosocial risks that could affect their health and professional performance, thus affecting students as well. on january , , the china center for disease control and prevention (china cdc) reported that a novel coronavirus had been detected as the causative agent for of the cases of pneumonia (european centre for disease prevention and control, c; holmes, ) . on january , , the world health organization (who) declared this first outbreak of novel coronavirus a "public health emergency of international concern" (world health organization, a). on march , , the director general of the who declared coronavirus disease (covid- ) a global pandemic (european centre for disease prevention and control, d; world health organization, b). as of march , , all european union (eu)/european economic area (eea) countries and more than countries worldwide are affected (world health organization, a ,world health organization, b . as of april , , many eu/eea countries started to adjust their response measures (i.e., gradual opening of school, small shops, and other businesses, etc.) (european centre for disease prevention and control, a) . data from the eu/eea show that around - % of diagnosed covid- cases are hospitalized, and % have severe illness. hospitalization rates are higher for those aged years and above and for those with other underlying health conditions (european centre for disease prevention and control, b) . since december , , and as of may , , , , cases of covid- have been reported, including , deaths (european centre for disease prevention and control, b) . as this is a new virus, no vaccine is currently available; it may be many months or even more than a year before a vaccine has been tested and is ready for use in humans (european centre for disease prevention and control, d) . main global data on cases and death are shown in table . as can be seen in table , spain and mexico are among the most affected countries, with spain ranking third in europe in terms of deaths reported by covid- , and mexico ranking third in terms of deaths reported by covid- in the americas. on one hand, in spain, the situation has been particularly complicated. as of may , , there have been , confirmed cases in spain, and , have died according to official data from the european centre for disease prevention and control (ecdc) (european centre for disease prevention and control, d). the first positive diagnosis was confirmed on january , (linde, ) , while the first death occurred on february in the city of valencia (caparrós, ) . in view of the rapid spread of the virus, since march , the spanish government has decreed a state of alarm (boletín oficial del estado, ), restricting the mobility of citizens to certain cases, such as the purchase of food and medicines or visits to medical centers or the workplace. all face-to-face teaching activities have been interrupted in spain since march , after the state of alarm was decreed (boletín oficial del estado, ), although some communities such as madrid or la rioja imposed this measure on their schools a few days earlier. in total, some million students from all educational stages are currently following their academic year at a distance (faro de vigo, ) . a large number of teachers took on the tasks of distance teaching without being previously trained for them, nor having specific resources for all this in many cases. this whole situation of stress caused by the pandemic, together with changes in the usual working conditions, could negatively affect not only the psychosocial risks of teachers but also its main consequences such as burnout. on the other hand, in the case of mexico, the first positive diagnosis was confirmed on february in mexico city (british broadcasting corporation, ) , almost a month after in spain. on march , a "health emergency due to force majeure" was declared as a result of the evolution of confirmed cases and deaths from the disease in the country, which led to the implementation of additional actions for its prevention and control (secretaría de salud, gobierno de méxico, ). currently, there have been , cases, of which , have been discharged and , have died, according to official data from the mexican government and ecdc (european centre for disease prevention and control, d; secretaría de salud, gobierno de méxico, ) . despite the fact that spain is one of the countries most affected by the pandemic in europe, and mexico in america, we could say that the phase in which both countries are in this sense is different, since at the moment, it seems that spain has reached its peak long before mexico, with the former being in a phase of decreasing new cases, while the latter seems to be in the middle of a phase of increasing new cases. of the total number of cases of ) , only , have been recorded in the last days, while of the total number of cases in mexico ( , ), , have been recorded in the last days, i.e., the figure has practically doubled in the last weeks (as of may , ) (european centre for disease prevention and control, d). the pandemic caused by covid- generates a series of impacts in all spheres of society, posing a challenge in practically all areas. during the pandemic, the population has had to adapt to a number of situations where uncertainty, fear, and, in many cases, pain have been present. these effects may translate into a range of emotional reactions (such as distress or psychiatric conditions), unhealthy behaviors (such as excessive substance use), and non-compliance with public health directives (such as home confinement and vaccination) in people who contract the disease and in the general population (pfefferbaum, ) . one of the many sectors that has had to adapt to this new situation and the demands that it entails is that of education. teachers at all levels of education have tried to maintain their fundamental role and continue to carry out their teaching duties, despite the uncertainty of the situation, the difficulties related to their own health and that of their loved ones, the lack of resources (material and knowledge), and the huge amount of extra work that adaptation to teach from home entails, including helping their students to cope to this situation. in particular, nonuniversity teachers (primary and secondary education), aware of the fundamental importance of learning for the development and future school performance of their students, have faced this situation by providing, in most cases, distance education, even if resources were often not available and uncertainty about the situation has always been present. in this context, the factors that are normally important for the good professional performance and well-being of teachers become even more relevant. among them are the so-called psychosocial risks. cox and griffiths ( ) define psychosocial risks at work as aspects regarding work design as well as the social, organizational, and management contexts of work that could potentially cause physical or psychological harm. psychosocial risks and work-related stress are among the most challenging issues in occupational safety and health, impacting significantly on the health of individuals, organizations, and national economies (bailey et al., ; bergh et al., ) . psychosocial risks arise from poor work design, organization, and management, as well as a poor social context of work, and they may result in negative psychological, physical, and social outcomes such as work-related stress, burnout, or depression (european centre for disease prevention and control, d). more specifically, psychosocial risks have been shown to be related to low job satisfaction (guadix et al., ) , health problems (bergh et al., ) , work accidents (fornell et al., ) , work-related stress (junne et al., ) , and burnout (maslach et al., ; elshaer et al., ) . psychosocial risks are closely related to work-related stress, which has been associated with a reduction in social interaction and the ability to concentrate at work, an increase in physiological pain and cardiovascular problems, and a higher incidence of mental illness such as depression and anxiety (nielsen et al., ) . in this same vein, the right management of psychosocial risk helps to prevent accidents and absenteeism (maslach, ) , to increase productivity (bakker and demerouti, ; bakker and wang, ) and to promote well-being at the workplace (hammer et al., ) . among the different theoretical models that exist to explain the appearance of occupational stress, karasek ( ) model is the one with the most theoretical and empirical support and it is the one that currently has the most influence and attention. it explains work-related stress according to the imbalance between psychological demands at work (e.g., workload, role conflicts, interpersonal conflicts, job insecurity,. . .) and the control level or resources that the employee has. according to this model, the employee health or well-being depends on the balance of the work demands and the resources that the employees have. when the demands are higher than the resources, it can create a feeling of work-related stress in the employee. in addition, the chronic work-related stress can cause burnout syndrome, being able to appear as several physical or psychosomatic symptomatology. thus, an excess of demands will produce a negative consequence in the employee, as higher burnout; however, having enough resources benefits the employee, decreasing the probability of having higher burnout (hatch et al., ) . among the different psychosocial risks, the following stand out because of their importance in relation to the crisis situation and the theoretical reference model: (a) role conflict: this is the situation in which a worker cannot simultaneously satisfy the contradictory role expectations in which he or she is involved. there is role conflict when a worker is being given work tasks without enough resources to complete them and receiving contradictory requests from different people. previous research has shown that problematic levels of distress were % more likely for workers reporting role conflict (johannessen et al., ) . having to teach from your own home often can bring some role conflict, since familiar conciliation might get more challenging for those teachers who also have to perform other roles, such as being parents, partners, and caregivers in general. (b) lack of organizational justice: lack of organizational justice refers to the extent to which employees perceive they are treated unfairly in their workplace and the perception of the absence of reciprocity in social exchanges (moorman, ; kobayashi and kondo, ) . low organizational justice is known to be a potential risk factor for poor physical and psychological health among employees (fujishiro and heaney, ; kobayashi and kondo, ) . (c) workload: it assesses quantitative and qualitative workload. quantitative workload refers to the amount of activities to be performed in a given period of time, while qualitative workload refers to the difficulty of the task and the volume of information to be processed in relation to the time available (gil-monte, ). a high workload has been associated with low levels of well-being and higher risks of health problems (pace et al., ) . in general, the adaptation to the current pandemic situation requires an extra load of work that teachers (and family and students) have to deal with. (d) interpersonal conflicts: it assesses the frequency with which workers perceive conflicts coming from the school management, colleagues, students, or relatives of the students. interpersonal conflicts have been associated to health problems, particularly to depression (kubik et al., ) . in the context of the covid- pandemic, uncertainty has often made it difficult to reach an agreement between school, families, students, and teachers about the best way to proceed, which new measures to take in order to adapt, and for how long this measures should be maintained. (e) emotional work: it refers to the effort, planning, and control necessary to express the organizationally desirable emotions during interpersonal transactions (morris and feldman, ; ortiz et al., ) . previous research has shown that problematic levels of distress were % more likely for workers reporting high emotional work (johannessen et al., ) . in the context of a pandemic, an extra burden of negative emotions in teachers (such as worry, uncertainty, and fear) would be expected. (f) job insecurity: the perceived threat of losing one's current job in the near future (heaney et al., ) , or that the employer did not comply with his or her obligations or promises (breach of psychological contract) (vander elst et al., ) , can have equally serious consequences as actual job loss (de witte, ) . particularly, job insecurity is considered a stressor that affects negatively the physical, psychological, and social health of the employee (cheng and chan, ; de witte et al., ; de witte et al., ; selenko et al., ) . on the other hand, as the karasek model points out, one of the most important resources to cope with psychosocial risk factors is the social support at work. social support at work is defined as the social climate in the work context involving the relationship between the supervisor and coworkers (karasek and theorell, ) . it involves both social-emotional and instrumental support. the former refers to the degree of social and emotional integration between coworkers and the supervisor, while instrumental support refers to the collaboration between coworkers and the supervisor to carry out work tasks (martín-arribas, ). an important potential source of social support is the emotional support of family, friends, and colleagues, which is particularly difficult to have on a confinement situation such as that originated by the covid- pandemic. social support and resilience protect individuals from threats to their mental and physical health by reducing or balancing the negative effects of the stressful events they experience in life (woodhead et al., ; sun et al., ) . as previously stated, a large number of demands and low resources produce a series of negative consequences for workers (karasek, ) , of which psychosomatic health problems and burnout syndrome are the most important due to their prevalence and associated consequences. the term psychosomatic refers to all those alterations in which mental processes influence areas of the organism (montiel et al., ) . among the most common are various types of symptoms affecting multiple organs and systems. examples of these are back pain, tension headaches, sleep problems, chronic fatigue, heartburn, tension diarrhea, or heart palpitations (jaradat et al., ) . burnout syndrome is defined as a prolonged response to chronic emotional and interpersonal stressors at work and is defined by the three dimensions of burnout, cynicism, and inefficiency (maslach et al., ) . the prevalence of burnout in education oscillates between and . % depending on the country and the study considered (ratto et al., ; villaverde et al., ) . a eurofound study (aumayr-pintar et al., ) showed that, in portugal, % of educational professionals had a moderate risk of burnout, and the employees with burnout syndrome increased from to % between and . in addition, their study (king et al., ) with school counselors in australia found that % of the sample experiences burnout. given this prevalence in recent years, the study of burnout in the education sector has become increasingly important (kim and burić, ; mclean et al., a; schonfeld et al., ) . most researches pointed out the importance of burnout on teachers (kaur and singh, ; yerdelen et al., ; schonfeld et al., ) , considering it as a risk for teachers that can negatively affect effective teaching (travers, ) , their interaction with their students (travers, ) , and their motivation for the job (mclean et al., b) , resulting in absenteeism (makhdoom et al., ) , depression (martínez-monteagudo et al., ), insomnia (gu et al., ) , or a decrease in the capacity to give support to the students (zapf et al., ; jennings and greenberg, ) . despite the impact of pandemics on the health and wellbeing of citizens, and more specifically of workers, and their clear influence on working conditions, or more specifically on their psychosocial risks, there are hardly any studies that have addressed the effect of a pandemic on psychosocial risks. there are even fewer studies comparing these types of factors during a pandemic in spanish-speaking countries. although there are studies carried out within the framework of different crises, allowing for contextualization of stress situations, these do not focus on the specific case of a pandemic like the one we are facing due to covid- . this situation is even more limited if we consider the impact on teachers. likewise, the few studies traditionally available have been carried out retrospectively, ignoring their perception of the pandemic, as well as the associated psychosocial risks during the times of greatest severity. after conducting a review of the literature, we were unable to observe any studies focused on teachers that analyzed the psychosocial risks of this group and their perception of the pandemic comparing two spanish-speaking countries at different phases or moments of the pandemic. therefore, the study presented here aims to fill this gap in the literature by offering a first approach to the perception of covid- by teachers and its relationship with psychosocial risks, comparing data from spain and mexico. the main aim of this study was to analyze the perception of non-university teachers regarding measures and resources implemented by institutions and governments and its impact on their daily work. also, to analyze the psychosocial risks of these professionals and its relation to the sanitary emergency caused by covid- comparing two spanish-speaking countries, spain and mexico, at a moment where the two countries were at different phases of the pandemic. data were collected from a sample of non-university teachers ( . % women and . % men; . % from mexico and . % from spain) aged - years (m = . , sd = . ) via a selfcompleted questionnaire during the covid- pandemic from march to april . from spain, participants were aged - (m = . , sd = . ), . % of whom were women and . % were men. from mexico, participants were aged - (m = . , sd = . ), . % of whom were women and . % were men. at the beginning of the study, the research team contacted different associations and institutions of education in order to reach non-university teachers and invite them via e-mail to participate in the study. in the online invitation, teachers were informed about the purpose of the study and also about how their anonymity and confidentiality were guaranteed. the time cost of completing the questionnaire was min. the eligibility criteria for participants were as follows. inclusion criteria: (a) to be a teacher in an institution other than university. (b) to be actively working during the moment of assessment. (c) to have signed the informed consent document and confidentiality agreement within the framework of the principles of the declaration of helsinki. the research included the variables and measurement instruments: different questionnaires were used to measure demands, resources, and consequences: the unipsico battery (gil-monte, ), the burnout assessment tool (bat) (schaufeli et al., ) , and the job insecurity scale (vander elst et al., ) . the demand factors include: taken from unipsico battery (gil-monte, ). role conflict is the situation in which a worker cannot simultaneously satisfy the contradictory role expectations in which he or she is involved. the scale is composed of five items (e.g., "i receive incompatible demands from two or more people"). participants are asked to score the frequency with which they have experienced the situation described in each statement on a likert-type scale from to ( = never; = very frequently: every day), with higher scores indicating higher levels of role conflict (scores above . are considered high, whereas scores equal to or below . are considered low). the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). extracted from unipsico battery (gil-monte, ). lack of organizational justice is defined as the perception of the absence of reciprocity in social exchanges. the scale is made up of five items (e.g., "i give up my skin at work compared to what i receive in return"). the response format is on a likert-type scale from to ( = never; = very frequently: every day), with higher scores indicating higher lack of organizational justice (scores above . are considered high, whereas scores equal to or below . are considered low). the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). taken from unipsico battery (gil-monte, ). it assesses quantitative and qualitative workload on a likert-type scale from to ( = never; = very frequently: every day). quantitative workload refers to the amount of activities to be performed in a given period of time, while qualitative workload refers to the difficulty of the task and the volume of information to be processed in relation to the time available. it consists of six items, three quantitative (e.g., "is it possible for you to work at a relaxed pace?") and three qualitative (e.g., "when you are working, do you encounter particularly hard situations?"), with higher scores indicating higher workload (scores above . are considered high, whereas scores equal to or below . are considered low). the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). extracted from unipsico battery (gil-monte, ). it assesses the frequency ( = never; = very frequently: every day) that workers perceive conflicts coming from the hospital management, colleagues, patients, and relatives of the patient. the scale consists of six items (e.g., "how often do you have conflicts with your colleagues?"), with higher scores indicating higher interpersonal conflicts (scores above are considered high, whereas scores equal to or below . are considered low). the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). it was measured using the job insecurity scale (vander elst et al., ) . it consists of five items (e.g., "i feel insecure about the future of my job") designed to measure quantitative job insecurity (i.e., insecurity to lose the job as such). respondents were asked to rate these items on a -point likert-type scale, ranging from ("strongly disagree") to ("strongly agree"), with higher scores indicating higher levels of job insecurity. the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). the resource factors include: extracted from unipsico battery (gil-monte, ). this is defined as the availability of help from other people. it evaluates the social support offered by your head of studies, the management of the center, and by your colleagues, in all cases in the form of emotional support and technical support. it consists of six items (e.g., "how often do your colleagues help you when problems arise at work?"). this was answered on a -pont likerttype scale ( = never; = very frequently: every day), with higher scores indicating higher social support at work (scores above . are considered high, whereas scores equal to or below are considered low). the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). the consequence factors include: included in the unipsico battery (gil-monte, ). it assesses the frequency of occurrence of psychosomatic problems related to the perception of sources of stress at work. it consists of nine items related to different systems of the organism (e.g., "have you been worried that, without making any effort, your breathing would be cut off?"). it was answered on a -pont likert-type scale ( = never; = very frequently: every day), with higher scores indicating higher psychosomatic problems (scores above . are considered high, whereas scores equal to or below . are considered low). the scale has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). it was assessed using the reduced version of the bat (schaufeli et al., ) . it consists of items that evaluate four scales: (european centre for disease prevention and control, c) exhaustion (e.g., "at work, i feel mentally exhausted"), mental distance (e.g., "at work, i have trouble staying focused"), emotional impairment (e.g., "i don't recognize myself in the way i react emotionally at work"), and cognitive impairment (e.g., "i make mistakes in my work because i have my mind on other things"). participants are asked to score the frequency that they have experienced the situation described in each statement on a likert-type scale from to ( = never; = very frequently: every day), with higher scores indicating higher levels of burnout. the instrument has obtained adequate psychometric properties in previous studies and in the present research (total sample: α = . ; mexico: α = . ; spain: α = . ). this is an ad hoc questionnaire of items constructed to measure different aspects related to the health emergency caused by the covid- . the aspects considered are: available resources (provided by the health center, regional government, and national government, e.g., "i feel that my center has put sufficient resources to deal with covid- in my daily work"), information {provided by the health center, regional government, and national government, e.g., "i consider that from the regional government [e.g., state of sinaloa (mexico)/or autonomous community (spain)] i have been given enough information to deal with covid- in my daily work"}, measures (taken by the health center, regional government, and national government, e.g., "i believe that sufficient measures have been taken by the national government to address covid- in my daily work"), and impact on work (workload, labor conflicts, work-related stress, and workrelated concerns and fears, e.g., "the covid- has increased my workload"). the subjects score on a likert-type scale his or her level of agreement or disagreement with the statements ( = totally disagree, = totally agree). scores range from to , with higher levels indicating greater satisfaction with the resources available, information, and measures taken, as well as higher levels of impact on work. the scale has obtained adequate psychometric properties ( a descriptive statistical analysis was performed for all study variables, as well as correlations and mean comparison analysis. all analyses were carried out using the statistical package spss (statistical package for the social sciences, version , armonk, ny, united states: ibm corp.). sociodemographic from the total sample of non-university teachers, were from mexico and from spain. the great majority worked in a public institute ( . %); % taught in kindergarten, as it can be seen in table , regarding psychosocial risks, that teachers in spain present medium levels on all of the psychosocial risks, whereas teachers in mexico present medium levels on lack of organizational justices and of social support and low levels on the rest of psychosocial risks. as it can be seen in table , during the pandemic, teachers in spain rated the resources, information available, and measures taken by the government and the hospital below the mean value of the answer scale, which points to a tendency to consider resources, information, and measures as insufficient. the highest scores from teachers in spain are regarding the impact of covid- on their jobs, although the scores in this case are also below the medium value of the answer scale. teachers in mexico, on the other hand, rated the resources and information available and the measures taken by government and the hospital above the mean value of the answer scale, which points to a tendency to consider resources, information, and measures as sufficient. the lowest score for teachers in mexico is the impact of covid- on their jobs. analysis of the mean comparison among the variables of the study was carried out between data from teachers in spain and mexico (table ). in general, it seems that the pandemic has a greater effect in the case of spain, since there are statistically significant differences in all dimensions except social support, with higher levels of risk and consequences in the spanish case. likewise, there is greater satisfaction with the available information, resources, and measures in the mexican case than in the spanish case, and finally, there seems to be a greater impact of the pandemic on the work and life of teachers in the spanish case in comparison with the mexican case. the results of the correlation analysis among the variables are shown in table . as it can be seen, almost all the variables are very strongly related. the only correlation that is not statistically significant is between the impact of covid- in the workplace and social support. when focusing on correlations among the variables in teachers from spain and mexico separately, the situation slightly changes ( table ). in the case of teachers in spain, many of the psychosocial risks correlate between them, except for job insecurity that does not appear related to any of the resting variables. also, from the covid- -related measures, information and measures are related with a higher number of psychosocial risks, whereas impact is less related to the rest of the variables (psychosocial risks as well as the resting covid- -related measures). on the other hand, in the case of teachers in mexico, variables are also very strongly related between them, although lack of organizational justice and job insecurity are less related to the rest of the variables. also, in contrast with the case of spain, impact appears related with the rest of the covid- -related measures. the current crisis caused by the coronavirus is a challenge not only in the health field but also in all spheres of society. in this context, professionals at all levels have had to adapt to new working conditions, in addition to dealing with the pandemic in their personal lives and as members of the community. among them, teachers of preschool and primary and secondary education have had to assume their important role in the best possible way, with limited means and resources and with the uncertainty of the moment and with the enormous responsibility that comes with educating and training children and adolescents, helping them to cope with the crisis and often providing relief as much as possible with homework and how to take school home (boletín oficial del estado, ; faro de vigo, ) . considering that teachers are vulnerable to burnout and job stress (zapf et al., ; jennings and greenberg, ; kaur and singh, ; yerdelen et al., ; travers, ; makhdoom et al., ; martínez-monteagudo et al., ; mclean et al., b; schonfeld et al., ; gu et al., ) , and therefore the negative consequences these can have on their health and professional performance (bergh et al., ; fornell et al., ; junne et al., ; european centre for disease prevention and control, d) , it is essential to study how psychosocial risks affect this group at a time of such vulnerability and general demand as the present. the literature on social risks to teachers in a pandemic context is extremely limited; however, it is critical to study the extent to which factors related to teachers' well-being may be affected during a health crisis such as the current one in order to ensure the well-being of teachers and, in turn, the children and adolescents in their care. this study has sought to explore the extent to which teachers are affected by psychosocial risks during the pandemic and how these risks relate to teachers' perceptions of the pandemic in terms of resources, measures, information, and impact. at the same time, it compares data of teachers in mexico with data of teachers in spain, two countries heavily affected by the pandemic and yet at very different stages of its development: spain in the midst of a drop in cases, mexico in the midst of a rise (european centre for disease prevention and control, d) . at the time of collecting the data, the coronavirus crisis was at its peak in spain, while in mexico, it was in a more initial phase. this facilitates the interpretation of some of the data found. the main results of the study show, on the one hand, that teachers in spain as well as teachers in mexico inform about perceiving lack of organizational justice during the pandemic but, at the same time, to perceive social support. teachers in spain, however, also inform about role conflict, workload, interpersonal conflict, psychosomatic problems, and burnout. these data go in line with previous literature about the social risks that teachers are exposed to (zapf et al., ; jennings and greenberg, ; kaur and singh, ; yerdelen et al., ; travers, ; makhdoom et al., ; martínez-monteagudo et al., ; mclean et al., b; schonfeld et al., ; gu et al., ) . regarding resources and information available about covid- , the impact of covid- on their jobs, as well as measures taken by responsible entities (national and regional government, as well as work center), data from teachers in spain point to a perception of insufficient resources, information, and measures and to a perception of a moderatehigh impact of covid- on their jobs. regarding teachers in mexico, data point to a perception of sufficient resources, information, and measures taken by responsible entities, as well as to a perception of a moderate impact of covid- on their jobs. when specifically comparing data from teachers in spain and mexico, the results highlight a difference between teachers in both countries: spanish teachers present more role conflict, lack of organizational justice, workload, interpersonal conflicts, job insecurity, psychosomatic problems, and burnout than teachers in mexico. at the same time, teachers in spain inform about less resources, information, and measures than teachers in mexico, but also about a bigger impact of covid- on their jobs, than teachers in mexico. the fact that teachers in spain are more affected by psychosocial risks during the pandemic and are more burned out by work could be due, on the one hand, to the phase of the pandemic at the time of data collection, as the pandemic situation was more severe in spain at the time that the study was conducted. however, it could also be due to the fact that teachers in spain perceive fewer resources, information, and measures taken by responsible institutions, which could in turn worsen some of the psychosocial risks and even be a direct risk factor for burnout. specifically, in terms of the relationship between psychosocial risk factors and covid- -related measures, these appear to be closely related, although it is true that in the case of teachers in spain, the relationship between covid- related measures and social risks is clearer than in the case of teachers in mexico. of the covid- -related measures, the least related to psychosocial risks is the impact of covid- on work, while of the psychosocial risks, the least related to the rest of the psychosocial risks and to covid- -related measures is job insecurity. these data indicate that teachers' perception of the measures taken by the responsible entities, as well as the perception of sufficient information and resources, could influence the psychosocial risks to which these professionals are exposed. as mentioned above, some of the differences are due, on the one hand, to the phase of the pandemic in which both countries were and, on the other hand, to the perception of resources by teachers to face the pandemic and the challenges it poses in their professional life. one of the main limitations of this study is that it presents an analysis of relationships between variables that does not allow for the establishment of causal relationships between them. furthermore, it is a cross-sectional study that does not allow for observing the evolution of the data as the pandemic caused by covid- progresses. future studies could make new measurements of the variables when the different phases of the pandemic have passed, which would allow the comparison of the variables taking into account the evolution of the health crisis, as well as the evolution of the psychosocial risks of teachers and the possible development of pathologies that, based on the scientific literature, have been related to the burnout and psychosocial risks described here. despite its limitations, this study shows data collected in a context never before seen, where data on psychosocial risks are not collected a posteriori but in the midst of a pandemic crisis. our data speak of a greater general attrition of teachers in spain, which indicates that the pandemic may indeed be related to greater sources of stress and psychosocial risks. at the same time, data from the present study underline the importance of the perception of resources by professionals, which could prevent to some extent the burnout and the possible alterations associated with it. it is difficult to carry out this type of study in these contexts for a number of reasons, but we believe that it is important to have data to support the fact that the measures taken by the responsible entities and the provision of information affect teachers not only directly but also indirectly by making them more vulnerable to psychosocial risks that could affect their health and professional performance, thus affecting students as well. if this is important in any context, it becomes even more important in a context where the emotional toll on society is more evident than ever. some of the main practical applications of this research would be to know the psychosocial risks during a pandemic in non-university teachers to discover the perception of resources, information, and measures adopted by the different public and private entities to deal with covid- , as well as to know the impact that this perception has had on the daily work of non-university teachers. these results can help make a difference between building resilience and developing burnout. any data that can clarify the relationships between the variables will be data that will benefit teachers, their students, and society in general. the results obtained in the present study allow to advance and consolidate the research on psychosocial risks during a pandemic while enabling the development of policies for action to improve teachers' coping with a pandemic and occupational health, which in turn will impact the outcomes of their work and society as a whole. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. the patients/participants provided their written informed consent to participate in this study. vp-g made a substantial contribution to the concept and design of the work, as well as on analysis and interpretation of data, drafted the article and revised it critically for important intellectual content, approved the version to be published, and participated sufficiently in the work to take public responsibility for appropriate portions of the content. mg-d made a substantial contribution to the concept of the work and acquisition of data, revised the article, approved the version to be published, and participated sufficiently in the work to take public responsibility for appropriate portions of the content. as-r made a substantial contribution to the concept or design of the work and on interpretation of data, drafted the article and revised it critically for important intellectual content, approved the version to be published, and participated sufficiently in the work to take public responsibility for appropriate portions of the content. ld-r made a substantial contribution to the design of the work and the acquisition of data, revised the article critically for important intellectual content, approved the version to be published, and participated sufficiently in the work to take public responsibility for appropriate portions of the content. dn-m made a substantial contribution to the concept and design of the work and acquisition of data, drafted the article and revised it critically for important intellectual content, approved the version to be published, and participated sufficiently in the work to take public responsibility for appropriate portions of the content. all authors contributed to the article and approved the submitted version. burnout in the workplace: a review of the data and policy responses in the eu. eurofound a national standard for psychosocial safety climate (psc): psc as the benchmark for low risk of job strain and depressive symptoms job demands-resources theory: taking stock and looking forward self-undermining behaviour at work: evidence of construct and predictive validity tailoring psychosocial risk assessment in the oil and gas industry by exploring specific and common psychosocial risks real decreto / , de de marzo, por el que se declara el estado de alarma para la gestión de la 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scale: a psychometric evaluation across five european countries personality traits that predict the burnout syndrome on mexican teachers stress, social support, and burnout among long-term care nursing staff emergency committee regarding the outbreak of novel coronavirus ( -ncov). geneva: who who director-general's opening remarks at the media briefing on covid- relationship between turkish elementary science teachers' occupational well-being and some contextual and demographic characteristics: a multivariate analysis emotion work as a source of stress: the concept and development of an instrument we would like to thank all the teachers who have voluntarily participated in this study even though they had a high workload and stress as a result of the health emergency caused by covid- . we would also like to thank the reviewers and the editor of the journal for their valuable contributions to this paper. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © prado-gascó, gómez-domínguez, soto-rubio, díaz-rodríguez and navarro-mateu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -x swoy d authors: kobayashi, nobumichi title: impact of emerging, re-emerging and zoonotic viral infectious diseases, in a virologist’s perspective date: - - journal: open virol j doi: . / sha: doc_id: cord_uid: x swoy d nan factors related to humans and society are the most responsible for emergence and spread of infectious diseases. at present, global population is estimated to be . billion and growing rapidly, described as "a population explosion". this increase is remarkable in sub-saharan africa and south asia. increase of population is a common issue in developing regions and facilitates poverty, and leads to urbanization associated with the growth of megacities ( cities as of ) with a population of more than million. high population and its density increase the risk for transmission of infectious pathogens via human-to-human contact, and low hygienic condition arising from undeveloped infrastructures (e.g., sewerage system). naturally, population explosion and urbanization facilitate the expansion of residence area of humans, which promote forest development associated with environmental destruction. during the process, humans may encounter unknown viruses which have been lurking in any animal but harmful to humans. although it has not yet been fully demonstrated, ebola virus is suggested to reside in some species of fruit bats as potential natural host, from which this virus might have transmitted to humans, causing ebola virus disease (evd). international tourists who traveled abroad reached over . billion in , and have been constantly increasing. the development of airline networks enables infectious pathogen to spread globally in a few days. such typical situation was evident for the outbreak of sars (severe acute respiratory syndrome) in , when the unknown pathogen, which was later revealed to be a novel coronavirus, was disseminated from china to at least countries within a week via air travel of infected patients. the recent outbreak of mers coronavirus in korea ( ) was also caused by a returnee from the middle east. spread of emerging viruses is indirectly related to socioeconomic problems including civil wars, an increase of refugees, and natural disasters. these human factors always compromise human health and increase the risk for emerging/re-emerging infectious diseases. more than virus species have been known to be able to infect humans. historically, the number of newly identified virus species/family has been constantly increasing since the th century, which was associated with the development of technology, from tissue culture and serological detection, to genetic identification represented by pcr and high throughput sequencing [ ] . among more than pathogens of humans, over % of them were considered to have originated in animal species (vertebrates). emerging viruses are considered more likely to be zoonotic. the number of infectious diseases outbreaks increased globally about times from the s to , associated with an evident increase of zoonosis as well as vector-borne disease, compared with human-specific infections [ ] . increase of zoonosis and vector-borne diseases is related to global changes in environment and ecosystem which may be caused by climate change associated with global warming. floods, fierce heat, and drought which arise as an influence of climate variation, may cause an increase of vectors, facilitating their move from an endemic area, and a decrease of natural enemies to vectors. phylogenetic analysis combined with chronological tracing indicated that recent global spread of chikungunya was caused synergistically by factors of humans, environment, vectors, and viruses [ ] . in the s and s, illegally dumped waste tires increased due to globalized trading and industrialization. these tires provided water pool for the proliferation of mosquitos, enhancing local endemicity. thereafter, viruses with vectors have disseminated via international airline network, associated with the occurrence of genetic diversity in viral genome. a mutation in the envelope protein conferred increased viral growth in mosquito, which facilitated spread of this vector-born disease. thus, spread of emerging viral diseases is considered to be caused by multifactorial mechanisms. currently, globally important viral infectious diseases may be classified into following categories with features such as ( ) high frequency worldwide (diarrhea, respiratory infections, etc.), ( ) pandemic concern (evd, etc.), ( ) directed to eradication/elimination (e.g., poliomyelitis), and ( ) neglected tropical diseases (e.g., rabies). these viral diseases are prone to be prevalent in developing countries, because of ) higher population (density), ) higher prevalence of vector/reservoir of virus pathogens, ) shortage of medical/preventive resources against viral diseases and ) low socioeconomic/hygienic status. furthermore, it is difficult to predict the emergence or spread of novel viral infections, and particularly zoonosis is impossible to eradicate because wild animals/vectors carry viral pathogens. despite such situations, scientists have a significant role to reduce the risks of emerging viral diseases. the first priority is to ensure adequate surveillance of viral diseases, i.e., to maintain epidemiological study on humans, animals, virus strains from various sources including environment, which is relevant to "one health" approach advocated recently. molecular epidemiology and population dynamics of viruses provide suggestions for the genetic, phenotypic, and epidemiologic trend of the viral diseases. outcomes from those efforts are definitely essential for developing diagnostic methods, therapeutic approaches, and vaccines. not applicable. the author declares no conflict of interest, financial or otherwise. the challenge of emerging and re-emerging infectious diseases emerging virus diseases: can we ever expect the unexpected? human viruses: discovery and emergence re-emerging and zoonotic virologist's perspective the open global rise in human infectious disease outbreaks virus evolution and transmission in an ever more connected world this license permits unrestricted use, distribution, and reproduction in any medium declared none. key: cord- - fh df authors: polyzos, stergios a.; mantzoros, christos s. title: making progress in nonalcoholic fatty liver disease (nafld) as we are transitioning from the era of nafld to dys-metabolism associated fatty liver disease (dafld) date: - - journal: metabolism doi: . /j.metabol. . sha: doc_id: cord_uid: fh df nan this special issue of "metabolism, clinical and experimental" is dedicated to nonalcoholic fatty liver disease (nafld), a disease closely linked with the insulin resistance (ir) syndrome or metabolic syndrome (mets) [ ] and its related comorbidities, including obesity [ ] , type diabetes mellitus (t dm) [ ] , dyslipidemia [ ] and cardiovascular disease [ ] . the prevalence of nafld has been estimated to be about %, being higher in selected populations with genetic predisposition (e.g. hispanics), or certain metabolic traits (e.g. obesity, t dm) [ ] ; the affected individuals have been projected to be millions in the usa and millions in the eu [ ] . nafld has been emerging as a major cause of advanced disease (i.e., cirrhosis and hepatocellular carcinoma) and liver transplantation [ ] , and is associated with higher hepatic and cardiovascular mortality [ ] . thus, nafld adds a heavy health and economic burden to societies all over the world in the st century [ ] . despite the high prevalence of the disease and the intensification of research efforts in the field, the noninvasive diagnosis [ ] and treatment [ , ] of nafld remain unmet medical needs. this is the second special issue of "metabolism, clinical and experimental", to be published approximately five years after the previous one [ ] , highlighting the importance of this largely metabolic disease, which continues to be a high priority for both researchers and clinicians alike. we are delighted to offer to scientists and practitioners all over the world outstanding pieces of work by leading researchers in the field who summarize herein recent scientific progress and their invaluable expert opinion on current status and future directions. the scope of this special issue is to provide a state-of-the-art update on the topic of nafld, which will prove to be useful for all clinicians who desire to have a brief, but still comprehensive overview of the disease to help guide them in their everyday clinical practice, as well as for researchers who desire to have an up-to-date summary of major milestones in the field, which could serve as a pivot for their research and could provide them future directions. j o u r n a l p r e -p r o o f in this special issue, first kechagias et al. reviews established and emerging factors contributing to the progression of nafld [ ] . they report that most nafld patients may remain asymptomatic, but - % of them are estimated to develop complications of cirrhosis with high risk of death. the presence of t dm may be the most important clinical predictor of liver-related morbidity and mortality in nafld. other risk factors that adversely affect the disease progression include, but are not limited to, nutrients (fructose, monounsaturated fatty acids and trans fatty acids), genetic polymorphisms and environmental factors, as well as the severity of mets [ ] . given the high priority of the presence of t dm in nafld, dewidar et al. summarize herein the bidirectional association between nafld and t dm [ ] . they start from associations at the cellular level and the interplay of their pathogenetic mechanisms, continue with the interpretation of clinical terms, and conclude with the translation of these associations into the potential future development of novel biomarkers and clinical trials in nafld. specifically for the disease management, the authors propose that lifestyle modification and certain drug classes used to treat t dm seem to be beneficial for the treatment of nafld and also recommend novel therapeutics that could be possibly beneficial for both nafld and t dm [ ] . another hot topic in the field of nafld is the need for a noninvasive diagnosis. liver biopsy remains the gold standard for grading and staging the disease. however, liver biopsy is invasive and has certain limitations, including sampling error and serious, albeit rare, complications [ ] . furthermore, performing serial liver biopsies to follow-up the progression of the disease, when there is no approved treatment, raises certain ethical considerations. thus, long et al. provide evidence towards noninvasive biomarkers proposed for nonalcoholic steatohepatitis (nash) and hepatic fibrosis [ ] . ideally, we need biomarkers for the diagnosis, risk stratification, prognosis and monitoring of the disease. these biomarkers would be useful not only to avoid unnecessary, and potentially dangerous j o u r n a l p r e -p r o o f in a small percentage of subjects, liver biopsies, but would also facilitate clinical trials to evaluate potential therapies and would simplify care in the future. omics are an emerging field in the noninvasive diagnosis of nafld and may also help us unravel the complicated pathogenesis of the disease [ ] . perakakis et al. highlight the amounting evidence of omics in the pathophysiology and noninvasive diagnosis of nafld, drawing not only from the literature, but also from their own contribution to the development of novel diagnostics and potential novel therapies [ ] . an important issue in the field of omics is the appropriate management and synthesis of the flow of information from genomics, epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and glycomics, so as to separate associations found possibly by chance from those opening new windows in our knowledge, that need to be validated and applied to practice, thus substantially helping in the diagnosis and treatment of the disease [ ] . handling of circadian clock-machinery as a potential therapeutic target of nafld [ ] . as mentioned above, nafld has emerged as a major cause of morbidity and mortality. mantovani et al. summarize the increasing body of clinical evidence, suggesting that nafld is linked with increased hepatic and extrahepatic morbidity and mortality [ ] . it seems that nafld has a bidirectional relationship with t dm, chronic kidney disease and j o u r n a l p r e -p r o o f cardiovascular disease, the latter representing the main cause of death in patients with nafld. importantly, apart from the higher risk of hepatocellular carcinoma (hcc), which may occur even in the absence of liver cirrhosis, nafld has been associated with extrahepatic malignancies, including colon, stomach, breast, uterus and possibly prostate cancer [ ] . the trends of morbidity and mortality are projected to follow the increasing trends of nafld prevalence, thus adding a considerable health and economic (direct and indirect) burden to the affected individuals and the societies worldwide. the rising trends of the prevalence of nafld and related liver cirrhosis make nafld the faster growing indication for liver transplantation. in this regard, majumdar and tsochatzis emphasize the multiple challenges needed to deal with during the pre-peri-and post-transplant period [ ] . the management of multiple metabolic co-morbidities, including obesity, t dm, dyslipidemia and cardiovascular disease, are considered important in the preand peri-transplant period. regarding the post-transplant period, the outcomes of liver transplantation in nafld patients seem to be similar to those observed after liver transplantation for other indications, including hcc. last but not least, the rising prevalence of nafld has resulted in increasing prevalence within the pool of potential liver graft donors, which in turn may adversely affect post-transplantation outcomes [ ] . given the shortage of graft donors globally, nafld seems to exacerbate the problem by affecting liver transplantation in two ways: it increases the need of liver transplantation whereas it also decreases the availability of appropriate graft donors. appropriate pharmacological management of nafld is expected to mitigate hepatic and extra-hepatic complications, as well the need of liver transplantation and possibly cardiovascular complications in the future. however, there is currently no licensed medication for nafld. pioglitazone and vitamin e are recommended by the latest guidelines as potential off-label treatment for selected patients with nash and hepatic fibrosis among diabetics [ , ] . statins should be also considered for nafld patients, especially those at high cardiovascular risk, such as patients with t d [ ] . polyzos and msdc- k (a ppar sparing modulator), are being evaluated in ongoing phase clinical trials. research efforts are also targeting selective ppar modulators, including chs- and pemafibrate, with the ultimate hope of launching drugs which will be exerting their beneficial effects without having the adverse effects of pioglitazone, a ppar-γ full agonist [ ] . nonetheless, while the approval of novel medications or their combinations are expected, physicians should also be focusing actively on recommending lifestyle modifications, i.e. exercise and a healthy dietary pattern (e.g. mediterranean diet), which remain the cornerstone of nafld prevention and management [ ] . while the compilation of this special issue was ongoing, two position articles on the nomenclature of the disease were published, proposing the change of the terminology from nafld to metabolic (dysfunction)-associated fatty liver disease (mafld) [ , ] . this proposed change was initially based on the ascertainment that the use of "nonalcoholic" overemphasizes the absence of alcohol, whereas at the same time it underemphasizes the significance of multiple metabolic factors contributing to the development and progression of nafld. alternative nomenclature could also be, and in our opinion more appropriately so, dysmetabolism-associated fatty liver disease (dafld), given that it is the metabolic dysfunction (dysmetabolism) and not metabolism that drives the fatty liver disease, which initiates the cascade leading to nash and complications. this novel terminology is expected to more accurately reflect the heterogeneous pathogenesis and multiple metabolic dysfunctions that are associated with the disease, and may have both research and clinical implications. it can also signify a change in mindset and focus that can hopefully drive more j o u r n a l p r e -p r o o f effectively the translation of current knowledge on the metabolic aberrations of the disease into differently designed clinical trials and, hopefully, new treatments [ , ] . more specifically, the evolving clinical presentation and the fact that the course of the disease is affected by multiple factors that may also change over time, add a dynamic dimension to the "multiple-hit" pathogenesis of nafld/mafld/dafld [ , ] . based on this concept, genetic and epigenetic factors interplay with age, sex, ethnicity and lifestyle habits, metabolic traits and gut microbiota, all adding to the heterogeneity of the disease and, importantly, to different responses to treatment [ , ] . accordingly, different phenotypes of nafld/mafld/dafld may be predominantly driven by genetic predisposition, environmental factors or metabolic aberrations (i.e. obesity, t dm) [ ] . this is apparently a seemingly useful concept when selecting the most appropriate management for each affected individual, but also when selecting patients for clinical trials. another important issue that also emerged recently is that there is a large group of patients, whose liver disease can be attributed to both metabolic aberrations and alcohol consumption [ ] . this group remains largely understudied, mainly owing to the previous classification into either alcoholic or nonalcoholic fatty liver disease and thus not considering the co-existence of both. the concept of a different nomenclature for nafld, i.e. one more oriented to the metabolic features of the disease, had been first proposed several years ago. several scientists had proposed a very similar terminology, i.e. mets-associated fatty liver disease (mafld) [ ] . it seems that a deeper knowledge of the pathogenesis of the disease, as well as the apparent failure of most clinical trials have now made the researchers and the clinicians more receptive to this, in our opinion, unavoidable change of the nomenclature. although mafld seems to reflect better the risk factors of the disease than nafld, and dafld may be even a more accurate descriptor, there is ongoing debate on whether this change could be premature, thus still leaving residual ambiguity in terms of the characterization of the disease and which will eventually necessitate an international consensus of all liver societies [ ] . although an international consensus does not appear to be imminent and may prove to be a j o u r n a l p r e -p r o o f slow process in terms of making progress towards a better characterization of the disease and its subtypes in the future, this does not eliminate the value of related proposals and international efforts towards eventual harmonization [ , ] . the prevalence of Νafld / mafld/ dafld continues to rise in parallel with the epidemics of obesity and t dm, which will continue to burden the affected individuals and societies with truly significant health and economic consequences [ ] . the association between Νafld / mafld/ dafld and t dm is so strong that has led to the suggestion to include Νafld / mafld/ dafld in the diagnosis and management plan of patients with t dm, thus handing Νafld / mafld/ dafld in a similar way to e.g. diabetic retinopathy or nephropathy in patients with t dm [ ] . being a highly prevalent disease without a standard noninvasive biomarker and an approved treatment renders Νafld / mafld/ dafld an attractive topic for researchers, clinicians, policymakers and industry [ ] ; it has been estimated that the drug market for nash will reach $us billion in the usa, japan, and european union- (england, france, germany, italy and spain) in [ ] . this has resulted in a race to develop the most appropriate medication(s) the soonest possible. thus, there are currently more than drugs in the development pipeline, most of which are under evaluation in early phase clinical trials, but also some are in phase clinical trials, as mentioned above [ ] . this explosion in trials raises the expectation that the first medication for nash will be hopefully approved in . nonetheless, owing to the heterogeneity of the disease, it is probably impossible for one medication to cover all nash patients. instead of waiting for the magic bullet, we recommend that we try to identify subgroups that would benefit from each medication and we believe that for many patients a combination treatment may be more efficacious in treating nash patients [ , ] . nonetheless, the efficacy and safety of combination therapies remain to be shown in future trials. deeper understanding of its pathophysiology is anticipated to result in better characterization of the disease and its subtypes. in this regard, any discussions on novel and j o u r n a l p r e -p r o o f possibly more appropriate terminology are towards the right direction [ , ] . in turn, better characterization may help the introduction of better noninvasive biomarkers for each subtype, thus entering an era of better characterization of the disease and of more individualized diagnosis and treatment. ideally, we would like to have a biomarker similar to glycated hemoglobin for diabetes, in other words a biomarker to be accurate, validated and specific, to help in the disease identification, staging, prognosis and follow-up, as well as, ideally, to guide the response to treatment. as for the treatment, the heterogeneity of the disease renders difficult to find an appropriate biomarker for all nafld/ mafld/ dafld patients. while waiting for a better characterization of nafld/ mafld/ dafld subtypes [ , ] , research on the best biomarker or on the optimum combination of two or more biomarkers could eventually increase the accuracy of diagnosis [ ] . serum biomarkers may be combined with each other, or with imaging (e.g. transient elastography, magnetic resonance elastography); a second biomarker or imaging may be utilized in a sequential mode, i.e. when the first one provides intermediate, promising but not fully desirable result [ ] . it is highlighted that nash and fibrosis are the main targets for discovering noninvasive biomarkers, with the latter regarded as the main histological prognostic factor of advanced disease [ ] . we also need biomarkers for the co-existence of nafld/ mafld/ dafld with and/or for future development of new-onset t dm [ ] , cardiovascular disease and most importantly biomarkers predicting not only morbidity but also mortality [ ] . apart from mafld/ dafld, the concept of "metabolic inflammation" has been recently proposed [ ] . metabolic inflammation starts on the basis of lipid accumulation in the hepatocytes (i.e. steatosis), which may progress to hepatic but also to systematic inflammation in some patients, thus resulting in multiorgan morbidity [ ] . thus, metabolic inflammation puts under one pathophysiological umbrella conditions that are well associated, including nafld, t dm, cardiovascular disease, but also neurocognitive impairment. several authors had previously referred to nafld as a systematic or multiorgan disease [ ] , a concept that may lead to a more holistic management in the future. it is also important to consider nafld in the setting of the recent coronovirus- pandemic. in this regard, liver injury in patients with covid- was frequent but mild, and followed a hepatocellular rather than cholostatic pattern [ ] . notably, the presence of nafld was independently associated with covid- progression; longer viral shedding time was also observed in nafld patients, in a retrospective study [ ] . another study showed that patients with nafld with high probability of hepatic fibrosis (as evaluated by the noninvasive indices fibrosis- or nafld fibrosis score) are at higher risk of severe covid- illness [ ] . although no definitive explanation exists, the authors hypothesized that nafld with significant fibrosis may amplify the covid- induced cytokine "storm" via the hepatic release of multiple cytokines, thus further complicating the disease progression [ ] . closely related to nafld conditions, obesity [ ] [ ] [ ] [ ] and t dm [ , ] , have been also associated with more severe illness from covid- . although ambiguity continues to exist, a position paper on the proposed care of patients with chronic liver disease in the era of covid- has been recently published, highlighting that patients with advanced liver disease and those subjected to liver transplantation are regarded as more vulnerable to covid- infection, i.e. having higher risk of infection and a more severe course [ ] . all the above represent challenges in the field of nafld/ mafld/ dafld, in which there are many questions to be answered and translated into clinical practice in a dynamically changing environment. it seems to be important to place nafld/ mafld/ dafld under the same umbrella with relevant comorbidities (e.g. obesity, t dm, dyslipidemia, cardiovascular disease) in an ever-changing environment with continuous and novel challenges (e.g. infections, chemicals, endocrine disruptors [ ] ). in this regard, approaching nafld/ mafld/ dafld in a holistic way may be more appropriate rather than facing nafld as a separate entity. of course, the holistic approach needs cooperation of different medical specialties (i.e., hepatologists, endocrinologists, cardiologists, internists, pathologists) and the will of policymakers to receive and integrate advice from teams of j o u r n a l p r e -p r o o f experts to design a path leading to the most effective diagnosis and therapy of this important and prevalent disease. no sources of financial support for this editorial. disclosure statement: sap has no competing interests. csm has been a shareholder of and reports grants through his institution and personal consulting fees from coherus inc and pangea inc, reports grants through his institution and personal consulting fees from esai and novo nordisk, reports personal consulting fees and in kind support with research reagents from ansh inc, reports personal consulting fees from genfit, p.e.s., intercept, astra zeneca, aegerion and regeneron, reports in kind support (educational activity meals at and through his institution) from amarin, jansen, boehringer ingelheim and travel support and fees from tmioa, the california walnut commission and the cardio metabolic health conference. highligths  this is a special issue of "metabolism" dedicated to nonalcoholic fatty liver disease.  experts in the field provide an overview of recent progress and developments in nafld and offer their expert opinion on future directions.  nafld remains a highly prevalent disease without an approved treatment. nonalcoholic fatty liver disease: updates on associations with the metabolic syndrome and lipid profile and effects of treatment with ppar-gamma agonists obesity and nonalcoholic fatty liver disease: from pathophysiology to therapeutics nonalcoholic fatty liver disease treatment in patients with type diabetes mellitus; new kids on the block non-alcoholic fatty liver disease and dyslipidemia: an update non-alcoholic fatty liver disease and risk of cardiovascular disease epidemiology and natural history of nonalcoholic fatty liver disease global burden of nafld and nash: trends, predictions, risk factors and prevention non-alcoholic fatty liver disease: a sign of systemic disease nonalcoholic fatty liver disease and alcoholic liver disease are major drivers of liver mortality in the united states economic and clinical burden of nonalcoholic steatohepatitis in patients with type diabetes in the necessity for timely noninvasive diagnosis of nonalcoholic fatty liver disease emerging and future therapies for nonalcoholic steatohepatitis in adults pharmacological management of nonalcoholic fatty liver disease nonalcoholic fatty future disease established and emerging factors affecting the progression of nonalcoholic fatty liver disease metabolic liver disease in diabetes -from mechanisms to clinical trials advances in non-invasive biomarkers for the diagnosis and monitoring of non-alcoholic fatty liver disease non-invasive diagnosis of non-alcoholic steatohepatitis and fibrosis with the use of omics and supervised learning: a proof of concept study the role of omics in the pathophysiology, diagnosis and treatment of non-alcoholic fatty liver disease perturbation of the circadian clock and pathogenesis of nafld complications, morbidity and mortality of nonalcoholic fatty liver disease changing trends of liver transplantation and mortality from non-alcoholic fatty liver disease clinical practice guidelines for the management of non-alcoholic fatty liver disease the diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the the use of statins alone, or in combination with pioglitazone and other drugs, for the treatment of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and related cardiovascular risk. an expert panel statement current and emerging pharmacological options for the treatment of nonalcoholic steatohepatitis adiponectin as a target for the treatment of nonalcoholic steatohepatitis with thiazolidinediones: a systematic review mafld: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease a new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement the multiple-hit pathogenesis of non-alcoholic fatty liver disease (nafld) the multi-hit process and the antagonistic roles of tumor necrosis factor-alpha and adiponectin in nonalcoholic fatty liver disease nonalcoholic fatty liver disease: multimodal treatment options for a pathogenetically multiple-hit disease non-alcoholic steatohepatitis -from nafld to mafld from nafld to mafld: implications of a premature change in terminology time to include nonalcoholic steatohepatitis in the management of patients with type diabetes phase drug pipelines in the treatment of nonalcoholic steatohepatitis nonalcoholic fatty liver disease: is it time for combination treatment and a diabetes-like approach? noninvasive tests in the assessment of nash and nafld fibrosis: now and into the future association between fibrosis stage and outcomes of patients with nonalcoholic fatty liver disease: a systematic review and meta-analysis significant fibrosis predicts new-onset diabetes mellitus and arterial hypertension in patients with nash metabolic inflammation-a role for hepatic inflammatory pathways as drivers of comorbidities in nonalcoholic fatty liver disease? non-alcoholic fatty liver diseases in patients with covid- : a retrospective study risk of severe illness from covid- in patients with metabolic dysfunction-associated fatty liver disease and increased fibrosis scores severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher in-hospital mortality obesity: the "achilles heel" for covid- ? letter to the editor: obesity as a risk factor for greater severity of covid- in patients with metabolic associated fatty liver disease letter to the editor: obesity as a risk factor for greater severity of covid- in patients with metabolic associated fatty liver disease letter to the editor: fasting plasma glucose associated with mortality rate in t dm patients with covid- infection commentary: covid- and diabetes mellitus: what we know, how our patients should be treated now, and what should happen next care of patients with liver disease during the covid- pandemic: easl-escmid position paper the emerging role of endocrine disruptors in pathogenesis of insulin resistance: a concept implicating nonalcoholic fatty liver disease key: cord- -a fynm authors: riggs, shannon m. title: chapter guinea pigs date: - - journal: manual of exotic pet practice doi: . /b - - . - sha: doc_id: cord_uid: a fynm publisher summary this chapter deals with the health and medical care issues of guinea pigs. guinea pigs have wide bodies with short limbs. they have a short, flat nose, laterally placed eyes, and hairless external pinnae. the dentition of the guinea pig is described as aradicular hypsodont. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. heavy food containers are recommended to make dumping of the receptacle more difficult. all food containers should be easy to disinfect and should be cleaned regularly, because guinea pigs have a habit of soiling their food bowls. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than °f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. c h a p t e r guinea pigs shannon m. riggs guinea pigs, cavia porcellus, are members of the caviidae family of the order rodentia. guinea pigs are native to mountainous regions of south america where they were domesticated as long as years ago. wild species of guinea pigs, or cavies, still inhabit columbia, peru, venezuela, argentina, brazil, and paraguay. domesticated cavies in these countries are used for food. in the wild, guinea pigs live in small groups and, therefore, are often more comfortable in the presence of other guinea pigs when maintained as companion animals. extensive breeding has resulted in numerous varieties of coat color and characteristics. the most common breeds are the american (or english) which has a short, smooth coat and the multicolored teddy (figure - ); abyssinian ( figure - ), which has a medium length coat in a whorled pattern; and the peruvian, which has a very long, smooth coat. guinea pigs have wide bodies with short limbs. a distinctive anatomic characteristic of species in the family caviidae is the number of digits on the front and rear feet ( digits front feet and digits rear). tails are usually very short or absent. the guinea pig has a short, fl at nose, laterally placed eyes, and hairless external pinnae. adult guinea pigs usually weigh between and g, with the males being slightly larger than females. the average life span of the companion guinea pig is approximately to years. the dentition of the guinea pig is described as aradicular hypsodont (e.g., all teeth have a relatively long crown and are "open rooted"). the maxilla is slightly wider than the mandi-ble, and the occlusal angle of the premolars and molars is marked compared to other rodent species. the dental formula of the guinea pig is (i / , c / , pm / , m / ) = . the maxillary incisors are much shorter than those set in the mandible. the molars and premolars are not easily visualized without special instrumentation because of the small size of the oral cavity and tendency for the involution of the buccal surface. females are sexually mature at weeks of age, whereas males on average reach puberty approximately weeks later. gestation is long, when compared to other rodents, at days. as a result of this long gestation period, young are precocial when born. juvenile pigs usually eat solid foods by or days of age. litter sizes range from to , with an average of to young. a female guinea pig should deliver her fi rst young before she is months of age. if birth has not occurred before months of age, the pubic symphysis becomes mineralized, with future pregnancies resulting in an inability of the sow to naturally deliver the babies. female guinea pigs that become pregnant after months of age invariably require cesarean section deliveries. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. wire-bottom cages may also be used; however, care must be taken to ensure that the mesh is small enough that a limb cannot become entrapped. an area of solid fl ooring should be provided, as uninterrupted time on wire mesh may predispose the guinea pig to pododermatitis. adequate space is needed in the enclosure for the guinea pig to move about unencumbered with enough space for a hide box. hide boxes or a secluded space is required for prey species (e.g., rodents) to reduce stress that may lead to disease problems. substrate products that contain aromatic oils (e.g., cedar and pine shavings) should not be used, as they can act as contact and respiratory irritants. appropriate bedding materials include recycled newspaper products, shredded paper, and aspen shavings. the enclosure should be cleaned thoroughly on a regular basis (e.g., times per week) because unsanitary conditions predispose the guinea pig to pododermatitis, respiratory, and other health problems. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. however, the sides of the enclosure should be high enough to prevent escape (approximately cm). heavy food containers are recommended to make dumping of the receptacle more diffi cult. all food containers should be easy to disinfect and cleaned regularly, as guinea pigs have a habit of soiling their food bowls. most guinea pigs readily accept drinking water from a sipper bottle, which will decrease spillage and will keep feces, urine, and bedding from contaminating the water. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than ° f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. all animals are very sensitive to environmental and/or nutritional changes. therefore, if changes have to be made, gradual exposure of the animal to the changes is recommended. an appropriate guinea pig diet includes a formulated, pelleted diet for that species, high-quality hay (e.g., timothy, orchard grass, oat) ad libitum, and ample fresh vegetables. as the animal's food intake is more dependent on volume consumed rather than calories consumed, a pet fed a predominantly pelleted diet (higher nutritional concentration) has a tendency to become obese. fruits and grains, if they are offered at all, should comprise a very small portion (< %) of the total diet and offered only as treats. because guinea pigs lack the enzyme l-gulonolactone oxidase, they are unable to synthesize ascorbic acid from glucose. therefore, guinea pigs require supplemental vitamin c in their diets. although commercial guinea pig pellets are manufactured with vitamin c, the supplement often degrades rapidly, especially if the pellets are subjected to high heat and humidity. vitamin c placed in drinking water also degrades rapidly and should be changed daily. to ensure that a guinea pig is receiving a proper amount of vitamin c, it is necessary to supplement a diet of pellets and hay with plenty of fresh foods or often a specifi cally manufactured vitamin c supplement tablet (oxbow, inc., murdock, ne). many green, leafy vegetables, such as kale, mustard greens, dandelion greens, parsley, and many others, are excellent sources of ascorbic acid (box - ). the vitamin c requirement of an adult, nonbreeding guinea pig is mg/kg/day. , in the united states, guinea pigs are not routinely vaccinated for infectious diseases. however, owners should be encouraged to have annual examinations that include an oral examination and a complete blood count. being prey species in the wild, guinea pigs are adept at hiding illness, and routine evaluations by a qualifi ed veterinarian may help in detecting abnormalities early. many health problems of guinea pigs are related to improper husbandry. during a routine veterinary visit, the owners should be asked to provide a detailed description of the animal's housing environment, including substrate, frequency of cleaning, ambient temperature, and exercise time. the diet history is also important. owners should be asked not only what the guinea pig is offered, but also in what proportions and of what foods the animal actually eats. most guinea pigs are quite docile and do not require aggressive restraint. often a hand on the animal's dorsum is adequate to restrain a guinea pig patient on the examination table ( figure - ). when transporting a guinea pig, support the body with one hand under the thorax and abdomen while placing the other hand on the back to prevent the patient from falling or jumping ( figure - ) . if chemical restraint is required, gas anesthesia with isofl urane or sevofl urane is generally well tolerated. anesthetic gases can be delivered via mask or induction chamber. mild sedation can be achieved with an intramuscular injection of a combination of midazolam ( . - . mg/kg) and butorphanol ( . - . mg/kg) intramuscularly. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. before beginning a physical examination, it is important to observe the animal before it has been stressed by restraint. a healthy guinea pig should be alert and aware of its surroundings. as guinea pigs are often shy animals, they may attempt to hide or escape. the examiner should use a thorough, systematic approach to focus on the respiratory character and rate, posture, and attitude of the animal. it is also important to have any instruments (e.g., transilluminator, stethoscope, thermometer, blood-collecting supplies) that may be necessary to decrease the amount of handling time for the patient. obtaining an accurate body temperature, heart rate, and respiratory rate is best accomplished at the beginning of the examination, as these parameters will invariably change with handling. an accurate weight should be obtained using an electronic gram scale. a "hands-on" physical examination should begin with the head; the veterinarian should assess the eyes for symmetry or discharge and check that the external pinnae of the guinea pig are hairless, as normal. the external ear canals often contain a small to moderate amount of dark, ceruminous debris. the nasal planum should be dry and fl at, whereas palpation of the ventral mandible may reveal deformities secondary to overgrowth of molar and premolar apices. the guinea pig coat varies somewhat with breed but, in general, should be smooth and shiny. guinea pigs often have a mild to moderate amount of dark sebaceous debris on the skin of the dorsum. older male guinea pigs may develop a focal accumulation of this debris at the base of the vertebral column, which may be referred to as the "grease gland." thoracic auscultation and abdominal palpation can be performed as in other patients. heart and respiratory rates will vary depending on the degree of stress a patient experiences. as pulse and respiratory rates can be very rapid, careful auscultation is necessary to detect subtle abnormalities (e.g., murmurs, crackles, wheezes). auscultation of gut sounds is also an important part of the guinea pig physical exam. a healthy guinea pig should have to borborygmi per minute. the practitioner should keep in mind that stress will decrease gastrointestinal (gi) motility; therefore, a stressed animal will often have a decrease in borborygmi. structures normally found on abdominal palpation include kidneys, urinary bladder, cecum, and intestines. fecal pellets are often palpable in the colon. careful examination may reveal the presence of abnormalities such as gi distention, masses, or, in females, ovarian cysts. limbs and joints should be carefully evaluated because thickened or painful joints may be indicative of a vitamin c defi ciency. a complete oral examination is an important part of the guinea pig exam. because of the potential stress associated with the oral exam, this evaluation should be reserved for the end. the oral cavity of the guinea pig is very narrow with a small opening, making visualization diffi cult ( figure - ) . instruments such as an otoscope with cone or a human nasal speculum will increase visualization of the caudal oral cavity ( figure - ). however, many dental lesions may be overlooked using these methods, and anesthesia is often required to adequately determine oral health. obtaining blood samples from guinea pigs for routine diagnostics is a challenging procedure. peripheral venipuncture sites available for most mammals (e.g., lateral saphenous vein and cephalic vein) are diffi cult to visualize in guinea pigs and only allow for the collection of small sample volumes. the jugular vein may be used; however, the short neck of the guinea pig and poor tolerance of the aggressive restraint by the patient restrict the availability of this site for blood collection. often to obtain an adequate blood sample, it is necessary to anesthetize the patient. once anesthetized, the large central vessels may be accessed with less stress on the patient and phlebotomist. acceptable venipuncture sites while the patient is anesthetized include the cranial vena cava ( figure baseline blood work is an important tool in the routine monitoring of health as well as in the diagnosis of disease. a complete blood count is essential for assessing red blood cell and white blood cell parameters (box - ). guinea pigs have heterophils rather than neutrophils as the predominant circulating granulocyte. heterophils lack myeloperoxidase, the enzyme that causes purulent, liquid exudates. therefore, the debris contained in guinea pig abscesses is often found to be very thick and caseous, a fact that must be understood when treating these conditions. a normal guinea pig white blood cell differential will consist of primarily heterophils and lymphocytes, usually with a greater proportion of lymphocytes. the remaining leukocyte types (e.g., monocytes, eosinophils, basophils) are normally present in very low numbers. early stages of a guinea pig's infl ammatory response are often characterized by a shift in the differential white cell ratio (e.g., increased heterophils, decreased lymphocytes) rather than an increase in total leukocyte count. this ratio shift makes evaluating the entire leukogram essential for health evaluation (box - ). the platelet count is also an important marker of infl ammation in guinea pigs and other small mammal species. large increases in the platelet count (> , , /μl) can be seen without an increase in the total white blood cell count. a cell type that is unique to guinea pigs is the kurloff cell. these large lymphocytes contain a cytoplasmic inclusion (e.g., a kurloff body). kurloff cells are noted most often in the peripheral blood of reproductive age females, although they are also identifi ed in male guinea pigs. the number of circulating kurloff cells will increase in response to exogenous estradiol and testosterone administration, although the effects were more dramatic with estradiol administration. , other studies have shown the disappearance of kurloff cells in spayed female and castrated male guinea pigs. the function of the kurloff cell is not completely understood, and these cells appear to lack lysosomes. at this time, there is no evidence that the kurloff cell has phagocytic activity. the activity of kurloff cells appears to most closely correlate with that of natural killer cells found in other species. plasma biochemical analysis is necessary for evaluation of organ function as well as plasma glucose and proteins. it is important to interpret the results of a chemistry panel in conjunction with history, physical exam fi ndings, and the results of other diagnostic tests. reference intervals for biochemical parameters are shown in box - . urinalysis is a useful diagnostic test in guinea pigs with signs of upper or lower urinary tract disease. urine samples may be obtained by free catch, fl oor catch, or cystocentesis. if urine is to be cultured, it is ideal to use the cystocentesis method of collection. ultrasound guidance and/or mild sedation of the patient will aid the veterinarian and/or veterinary technician in obtaining the urine sample. the urine of guinea pigs is typically yellow to amber in color, but it may be darker and more orange depending on the patient's diet. pigments in the urine can sometimes be mistaken for hematuria, so differentiation is important. because guinea pigs are herbivores, the ph of guinea pig urine is alkaline, usually . to . . crystalluria may be seen but is not a normal fi nding. if crystals are found in a urine sample, the guinea pig patient should be examined for urinary calculi. to minimize rotation, care should be taken to extend the limbs symmetrically when positioning the patient. because guinea pigs have stocky builds with short limbs, and because they resent aggressive restraint, sedation or anesthesia is helpful in obtaining diagnostic radiographs as well as in reducing the patient's stress ( figure - ). table - is a guideline for radiographic techniques used in common small mammal radiographic studies. dental malocclusion is a common disease problem in guinea pigs. radiographs of the skull are helpful in assessing the degree of malocclusion as well as potential bone involvement. in addition to lateral and dorsoventral views of the skull, right and lateral oblique views can help to localize lesions. magnifi ed views of the skull can be obtained by placing the patient on an elevated platform under the x-ray beam without changing the distance between the x-ray cassette and beam. ultrasound is another imaging modality that is very useful in the diagnosis of common guinea pig disease processes, such as ovarian cysts (figure - ) and urinary tract calculi. as with radiographs, sedation or anesthesia can assist in reducing patient stress and improve the quality of images. guinea pigs often have a large amount of gas accumulation in the gi tract, which obscures the ultrasound image, sometimes making this imaging technique of limited value. advanced imaging techniques, such as computed tomography (ct) and magnetic resonance imaging (mri), are useful tools in the diagnosis of disease in guinea pig patients. limitations of these modalities include limited availability, expense, and decreased image quality compared with that of larger patients. mri has the added limitation of requiring a significant amount of time (often > min) under anesthesia with limited monitoring ability. regardless of the present limitations, veterinarians should be aware that these techniques are available and that reference material exists that depicts normal anatomy. as guinea pig owners continue to demand high-quality care for their pets, these imaging techniques will likely become more commonplace in small mammal practice for these patients. microbiologic samples can be obtained for diagnosis of various guinea pig infections. exudates from nasal or ocular secretions can be examined for abnormal fl ora. some bacterial organisms (e.g., bordetella bronchiseptica) are diffi cult to culture. laboratories need to be advised as to the organisms in question so they can perform more specifi c diagnostic tests to obtain organism identifi cation. abscesses are another disease condition that may require culture for proper treatment. because guinea pigs form caseous abscesses, the purulent debris itself is typically not useful for bacterial isolation. for the best chance of identifying organisms within an abscess, a portion of the abscess capsule should be submitted to the laboratory. both aerobic and anaerobic bacterial cultures should be requested, especially if it is suspected that the abscess may have originated from a dental problem. roundworms and coccidia are seen in guinea pigs and can be identifi ed by fecal fl otation or fecal direct smear, as in other species. cryptosporidiosis has also been reported. identifi cation of cryptosporidium organisms usually requires acid-fast staining or immunofl uorescent antibody testing in addition to fecal fl otation. ectoparasites (e.g., mites, lice, fl eas) commonly infest guinea pigs. visualization of the parasites and/or their waste, as well as skin scrapings, microscopic examination of hair follicles, and tape preparations can be useful in the identifi cation of these parasites. the term enterotoxemia refers to the overgrowth of toxinproducing bacteria in the gi tract, particularly clostridium diffi cile. this can occur with stress, an abrupt change in diet, gi stasis, or inappropriate antibiotic administration. the gi fl ora of guinea pigs is predominantly gram positive, and administration of antibiotics with a primarily gram-positive spectrum (e.g., beta-lactam antibiotics, macrolides, lincosamides) can lead to the depletion of normal gut fl ora, allowing colonization by opportunistic bacteria (e.g., gram-negative organisms, clostridium spp.). the dentition of the guinea pig is described as aradicular hypsodont, meaning all teeth grow throughout the animal's life and are open-rooted. the dental formula of the guinea pig is i / , c / , p / , m / . the mandible of these animals is wider than the maxilla. the occlusal angle of the molars and premolars in the guinea pig is quite severe when compared with that of rabbits and other rodents (figure - ). guinea pigs lack the layer of yellow enamel present on the rostral surface of the incisors in most other rodents. dental malocclusion is a common disease process in guinea pigs. the development of malocclusion can be the result of multiple etiologies or a combination of factors (e.g., improper diet, genetics, trauma). incisor malocclusion alone is rare in guinea pigs, so a thorough oral examination is necessary to determine the presence of cheek teeth malocclusion. when the molars and premolars do not occlude properly, overgrowth of the crowns and reserve crowns takes place. with improper wear, sharp points can form on the buccal aspects of the maxillary cheek teeth and the lingual aspects of the mandibular cheek teeth. the mandibular cheek teeth can overgrow to such an extent that they entrap the tongue, making eating and drinking diffi cult (figure - ) . clinical signs can include inappetence or dysphagia, decreased fecal output, ptyalism, poor coat quality, and leth-argy. abscesses commonly occur at the apical aspects of overgrown molars and premolars. because the premolars and molars are elodont (open-rooted), it is possible that these teeth can overgrow and impinge on the nasolacrimal duct, causing ocular and nasal discharge. the reserve crowns can also overgrow into the nasal cavity, where oral bacteria may be seeded, causing rhinitis and sinusitis. diagnosis of dental disease is based on physical and oral examination fi ndings. careful palpation of the ventral mandible and maxilla may reveal bony protuberances corresponding to overgrowth of the apical surfaces of the cheek teeth. proper instrumentation is very important for adequate visualization of the oral cavity. dental speculums and pouch dilators are invaluable aids in obtaining a good view of the molars and premolars (figure - ) . however, many abnormalities can be overlooked in a conscious animal, so anesthesia is often required to obtain a thorough oral examination. visualization of the oral cavity and occlusal surfaces can be facilitated by the use of an endoscope (figure - ) . most endoscopes available in veterinary practice have a degree of angulation of the fi eld of view, allowing greater focal area. abnormal oral examination fi ndings may include an uneven occlusal surface or angle of the cheek teeth, formation of sharp points with or without associated ulceration of the oral mucosa, food impaction, and abnormal spaces (diastema) between teeth. imaging, including routine radiographs, magnifi ed skull radiographs, and computed tomography can be incorporated to better evaluate the extent and seriousness of the process (figure - ) . radiographic studies should include dorsoventral, lateral, and right and left oblique views. treatment is centered on restoring a normal occlusal plane to the teeth, a procedure that should be performed while the animal is under general anesthesia. unstable animals should be provided with supportive care before the anesthetic event. a high-speed surgical dental handpiece can be used to restore a normal occlusal plane and to reduce any sharp points. handheld trimmers are not acceptable, as they tend to crush teeth and can cause fractures and pulp exposure. dremel tools are also not considered acceptable because the small size of the guinea pig oral cavity will not allow for appropriate trimming. with any dental instruments, care should be observed to minimize oral soft tissue trauma. when a guinea pig is diagnosed with dental malocclusion, it is important to convey to the owners that this will be a lifelong problem for their pets. with many cases, routine occlusal adjustments will be necessary for the remainder of the animal's life. tyzzer's disease is the common term for bacterial enteritis caused by clostridium piliforme. infection with this organism is more commonly described in small rodents such as mice and hamsters, although it has also been described in guinea pigs as well as other mammalian groups. tyzzer's disease often presents acutely, with signs such as diarrhea, depression, and poor coat quality, often progressing rapidly to death. transmission is via the fecal-oral route. guinea pigs can be infected with c. piliforme without showing clinical signs. animals that are carrying the organism but showing no signs of illness have still been found to shed organisms. animals that carry the organism and then become immunocompromised (e.g., stress, immunosuppressive drug therapy) often develop clinical disease. clostridium piliforme is an intracellular bacterium that will not grow on routine culture media, so antemortem diagnosis is diffi cult. tyzzer's disease begins as an intestinal infection, but later spreads to the liver hematogenously, causing areas of necrosis. , positive identifi cation of the organism on necropsy requires examination of hematoxylin and eosin or silver staining of affected tissues. treatment should consist of fl uid and nutritional support as well as appropriate antibiotic therapy. unfortunately, the progression of the disease is rapid, making treatment unrewarding. prevention is key, focusing on owners' reducing housing stress, providing a proper diet, and maintaining a clean environment. cryptosporidiosis has been described as causing disease in guinea pigs in a laboratory setting. diagnosis was made histologically from affected animals that lost weight, had diarrhea, and suffered an acute death. cryptosporidium organisms were found in the brush border of the intestinal tract from the duodenum to cecum, with associated infl ammation. suspected coronavirus infection has been described in young guinea pigs. clinical signs in affected animals included anorexia, weight loss, and diarrhea. approximately half of the affected animals in the reported outbreak recovered. necropsies performed on animals that died or were euthanized showed lesions consistent with coronavirus infection resulting from an acute to subacute necrotizing enteritis involving primarily the distal ileum. coronavirus-like virions were identifi ed on transmission electron microscopy of feces collected from the lower gi tract at necropsy. outbreaks of salmonellosis have occurred in guinea pig colonies. some of the organisms that have been isolated include s. enteritidis, s. dublin, s. fl orida, s. poona, and s. bredeney. , salmonellosis has been described to affect guinea pigs in acute and chronic disease processes. with acute salmonellosis, guinea pigs often die after a brief illness characterized by nonspecifi c signs of illness and diarrhea. usually only a few pathogenic abnormalities are found at necropsy, and confi rmation of a diagnosis is dependent on culture of affected tissues. chronic salmonellosis is a wasting disease, lasting several weeks. splenomegaly, hepatomegaly, and mesenteric lymphadenopathy are common postmortem fi ndings in affected animals. guinea pigs that recover can remain chronic, intermittent shedders of salmonella organisms. yersinia pseudotuberculosis causes several disease syndromes in guinea pigs. , the most common presentation affects the mesenteric and colonic lymph nodes, infi ltrating these lymph nodes with caseous nodules. clinically, affected guinea pigs will lose weight, have diarrhea, and develop a generalized lymphadenopathy. transmission of the disease can be either vertical or horizontal. as stated previously, guinea pigs require dietary supplementation of ascorbic acid because they lack the enzyme l-gulonolactone oxidase necessary for synthesis of this compound. although diets formulated for guinea pigs are supplemented with ascorbic acid, it is important to remember that it breaks down very rapidly, usually within the fi rst days after production. if a guinea pig is not receiving vitamin c supplementation in its diet, the veterinarian can assume that the diet is defi cient in this important nutrient. ascorbic acid is a necessary component of collagen, and defi ciencies are often noted as manifestations of abnormal collagen synthesis. clinical signs of hypovitaminosis c can include lameness, hemorrhage, lethargy, anorexia, poor coat quality, and bruxism. diagnosis of hypovitaminosis c is based largely on clinical signs and history. radiographs will reveal changes to the costochondral junctions and widening of the epiphyses of long bones. recommended treatment includes fl uids and nutritional support, pain control, and parenteral vitamin c supplementation. attention should also be paid to improvement of the guinea pig's diet at home. in addition to causing skeletal and cartilage abnormalities, vitamin c defi ciency has been known to reduce immune function. in vitro, vitamin c defi ciency was demonstrated to cause a reduction in migration of macrophages in guinea pigs. urogenital dystocia dystocia most frequently occurs in primiparous sows that are bred after approximately months of age. at this time, the symphysis between the pubic bones becomes fused and will not expand to allow the passage of fetuses. cesarean section must be performed in these cases to save the sow and the young. factors other than age that can predispose a sow to dystocia include large fetuses in relation to sow size, uterine inertia, and obesity. urolithiasis occurs commonly in pet guinea pigs, and the common clinical signs associated with the disease include stranguria and pollakiuria, vocalizing when urinating, and hematuria. the underlying cause(s) of this condition is not completely understood but is likely associated with a genetic predisposition and/or the presence of a high-calcium diet. other less common underlying etiologies associated with urinary calculi formation include ureteral neoplasms (e.g., papilloma). calculi are primarily composed of calcium carbonate, although magnesium ammonium phosphate hexahydrate and calcium phosphate calculi will also occur. radiographic or ultrasonic imaging can be used to confi rm the location of the urinary calculi, which may be present in the renal pelvis, ureters, urinary bladder, or urethra (figure - ) . urinary tract calculi often require surgical removal. ideally, once removed, the calculus and/or a portion of the bladder wall should be cultured. it may also be helpful to analyze the composition of the stone to help determine ways of preventing recurrence. ovarian cysts are a common fi nding in middle-aged to older female guinea pigs. this disorder has a reported prevalence of % in female guinea pigs aged . to years. , multiple cysts can be present on one or both ovaries. clinical signs related to cystic ovaries can include abdominal distention, lethargy, and anorexia related to the space-occupying nature of the cysts. ovarian cysts that are actively producing hormones can produce bilaterally symmetrical alopecia of the fl anks. ovarian cysts can be quite large (up to several centimeters) and can often be identifi ed on physical examination. abdominal ultrasound is the most useful diagnostic tool for a defi nitive diagnosis. the most defi nitive treatment for cystic ovaries is ovariectomy or ovariohysterectomy, curing the current problem and preventing recurrence. in animals where surgery is not a viable option, ultrasound-guided percutaneous aspiration of ovarian cysts can be performed as a palliative treatment. however, without removal of the ovaries, the cysts will recur, requiring repeated procedures. pregnancy toxemia typically occurs in pregnant sows during the last weeks of gestation. as with other species, pregnancy toxemia is the result of a negative energy balance and the metabolism of fat. sows that experience pregnancy toxemia are typically overweight and become anorexic. clinical signs include lethargy, dyspnea, and anorexia, usually progressing to death within a few days. therapy of affected sows should center on providing nutritional support, correcting electrolyte imbalances, and preventing opportunistic infections. prognosis is generally considered poor, as many sows fail to respond to treatment. neoplasia of the reproductive tract is not commonly reported in guinea pigs, but several tumor types have been described. of the described reproductive neoplasms, the vast majority occur in female guinea pigs. uterine leiomyoma (often associated with ovarian cysts) and leiomyosarcoma, ovarian teratoma, and granulosa cell tumor are reported neoplasms of the reproductive tract. diagnosis, as in any other patient, should be based on the results of cytologic or histopathologic sampling. benign neoplasms may be resolved with ovariohysterectomy, but further diagnostics to determine the extent of local invasion should be performed before surgery takes place. a thorough diagnostic work-up, including imaging techniques (e.g., abdominal ultrasound, thoracic radiographs), should be performed to look for metastases of malignant tumor types. mycoplasma caviae has been isolated from the reproductive tracts of guinea pigs. these guinea pigs are often unaffected by the organism, but metritis has been suspected to be associated with infection. as with other species, mycoplasma spp. is suspected to cause reproductive problems (e.g., abortion and decreased fertility). respiratory disease is a common presenting complaint in guinea pig patients. clinical signs can vary from sneezing and upper respiratory signs to severe dyspnea and death. bordetella bronchiseptica is one of the most common respiratory bacterial agents associated with pneumonia in guinea pigs. many guinea pigs are carriers of the organism, which will cause clinical disease if the animal is stressed. a thorough history, obtained from the patient's owner, often reveals interactions with other species that are also subclinical carriers of b. bronchiseptica (e.g., rabbits, dogs). clinical signs noted in guinea pigs infected by the bordetella organism include nasal discharge, dehydration, tachypnea, and lethargy. diagnosis should be based on clinical and radiographic signs and history. confi rmation of the diagnosis can be determined with enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence diagnostic tests, or culture of exudates. treatment/preventive options for b. bronchiseptica infections in guinea pigs include an autogenous bacterin vaccine as well as three commercially available vaccines for bordetella spp. (porcine b. bronchiseptica and human b. pertussis), which were found to offer some protection against the development of bronchopneumonia in experimentally infected guinea pigs. streptococcus pneumoniae can cause pleuropneumonia, pleuritis, and peritonitis in guinea pigs. serologic detection of s. pneumoniae antibodies using elisa have been described in previously published papers. on postmortem examination, lesions found to be associated with s. pneumoniae infection included pleuritis, pleural effusion, lung abscessation, otitis media, pericarditis, and others. streptococcus pneumoniae was also identifi ed from septic arthritis lesions in a group of guinea pigs. five guinea pigs in a laboratory colony demonstrated multiple enlarged joints from which pure cultures of s. pneumoniae were isolated. several other individuals in this colony had previously died with typical s. pneumoniae lesions (e.g., pleuritis, pericarditis) and lesions consistent with hypovitaminosis c. the group with septic arthritis also had scorbutic changes on necropsy. streptobacillus moniliformis, the causative agent of rat-bite fever in humans, was isolated from a laboratory guinea pig with pneumonia. this organism is of particular importance because of its zoonotic potential. adenovirus has been shown to cause necrotizing bronchopneumonia in guinea pig populations, although it can also produce a transient, subclinical infection. , clinical signs include depression and dyspnea, but guinea pigs often die acutely without clinical signs. identifi cation of adenovirus dna from diseased lung tissue was achieved using a polymerase chain reaction technique in one study. development of this pcr assay for identifi cation of the virus has determined that the guinea pig adenovirus is distinct. histopathologic examination of affected tissues of animals infected with adenovirus will reveal the presence of characteristic intranuclear inclusion bodies. the most commonly reported neoplasm of the respiratory tract in guinea pigs is bronchogenic papillary adenoma. the prevalence of the tumor is as high as % in guinea pigs over the age of years. given the relatively more common occurrence of pneumonia in guinea pigs, bronchogenic papillary adenoma can often be misdiagnosed. for this reason, thoracic radiographs of guinea pigs with respiratory disease are highly recommended. yersinia pseudotuberculosis can cause septicemic pneumonia in guinea pigs. death usually occurs rapidly, after the development of coughing and dyspnea. at necropsy, severe congestion of the lungs is found grossly and through histologic evaluation of the tissues. trixacarus caviae are sarcoptoid mites that commonly affect guinea pigs (figure - ) . affected guinea pigs are intensely pruritic, sometimes to the extent of seizure development. as with other mite infestations, diagnosis is based on the identifi cation of the parasites on skin scrapings. once defi nitive diagnosis has been made, treatment with ivermectin and selamectin ( mg/kg q - wk) is usually effective. chirodiscoides caviae are fur mites diagnosed in guinea pigs. because it rarely causes clinical disease, treatment is usually unnecessary. , gyropus ovalis and gliricola porcelli are species of lice that are commonly identifi ed in guinea pigs (figure - ) . infested guinea pigs may be pruritic, but are usually unaffected. guinea pigs severely infested with these mites may demonstrate poor coat quality and alopecia. patchy hair loss without associated pruritus may be attributed to dermatophytosis, most commonly trichophyton mentagrophytes (figure - ) . lesions are circular and scaled and usually occur on the face and head. the diagnosis of dermatophytosis is made by a positive fungal culture. because of the zoonotic potential of these fungal organisms, care should be streptococcus zooepidemicus lancefi eld's group c is the causative agent of cervical lymphadenitis. this disease will cause severe swellings of the lymph nodes in the cervical region in guinea pigs. affected guinea pigs will frequently exhibit no other clinical signs but may become septicemic, with lesions affecting the heart, lungs, kidney, and skin. the most effective treatment for cervical lymphadenitis is complete surgical excision of the affected lymph nodes, followed by appropriate antibiotic therapy based on culture and sensitivity testing. lancing and draining the abscesses is often not curative, as the abscesses form thick capsules that harbor organisms, leading to recurrence. yersinia pseudotuberculosis has also been shown to cause cervical lymphadenitis in guinea pigs. although the affected guinea pigs are usually not ill, the concern is that rupture of the abscesses will release large amounts of this potentially zoonotic organism into the environment. another potential causal agent of cervical lymphadenitis is streptobacillus moniliformis. one report exists of a cervical mass, initially believed to be cervical lymphadenitis, which was histologically determined to be a thyroid papillary adenoma. the mass was apparently nonfunctional and the guinea pig appeared otherwise healthy, but surgical resection was curative. guinea pigs housed in cages with wire fl ooring are predisposed to developing ulcerated lesions on the plantar surfaces of their feet. mild lesions may appear as hyperemic, swollen areas of the weight-bearing surfaces. these lesions can progress to ulcerations with secondary infections (figure - ). vitamin c defi ciency has also been considered a predisposing factor for the development of pododermatitis, as affected animals may be in pain and reluctant to move, resulting in the development of pressure sores. ulcerated, infected lesions should be managed with appropriate antibiotics and antiinfl ammatory drugs, but the focus of treatment should be improving husbandry (e.g., providing appropriate fl ooring/bedding, vitamin c supplementation). trichofolliculomas are the most common cutaneous tumor seen in guinea pigs. these benign tumors often occur on the dorsum and are typically round and hairless (figure - ). another cutaneous abnormality that has been described in guinea pigs is cutaneous vascular malformation. the lesion described was a raised, ulcerated plaque on the animal's fl ank, which bled intermittently. ultimately, the cutaneous vascular malformation resulted in fatal hemorrhage. histologically, the lesion was described as an expansile mass extending into the skeletal muscle and consisting of multiple vascular spaces of varying sizes. these vascular spaces were lined with endothelial cells. compared to the incidence of neoplasia in other mammalian species, the incidence of neoplasia in guinea pigs appears low or is underreported. however, there have been several reported cases of neoplasia in guinea pigs. as more guinea pig owners seek quality veterinary care for their pets, reports of neoplasia will increase. lymphoma is the most commonly reported neoplasia in guinea pigs. clinical signs associated with guinea pig neoplasia include lymphadenopathy, splenomegaly, and hepatomegaly. leukemic and aleukemic forms of guinea pig lymphoma have been identifi ed. thyroid carcinoma has been reported in an adult guinea pig that demonstrated multiple masses in the ventral cervical region. as there was no evidence of neoplastic disease elsewhere on postmortem examination, this was considered a primary tumor. mesothelioma has been reported in the abdomen of an adult guinea pig. the guinea pig had died of complications associated with pneumonia, and the mesothelioma was diagnosed through a necropsy examination. the mass consisted of diffuse nodules on the serosal surfaces of numerous organs in the abdominal cavity. guinea pigs are native to cooler regions of south america and are therefore relatively intolerant of temperatures above ° f, lower if the environment is also humid. guinea pigs should be housed in well-ventilated enclosures at temperatures between ° and ° f to prevent heat stress. clinical signs of heat stress include rapid, shallow respirations, lethargy, poor peripheral perfusion, and ptyalism. treatment includes reducing the animal's core body temperature with cool water baths or applying alcohol to the feet and ears; in addition, fl uid therapy (either intravenous or subcutaneous) is recommended to improve perfusion. the prognosis for this condition is guarded. chlamydophila psittaci has been identifi ed as a disease-causing agent in guinea pigs; it usually causes a mild, self-limiting conjunctivitis. this intracellular bacterial disease usually occurs in young guinea pigs to weeks of age but has been reported in adults as well. in one outbreak, the typical conjunctival abnormalities were present with other, more signifi cant, clinical signs, such as rhinitis, abortion, and pneumonia. a defi nitive diagnosis can be achieved through giemsa staining, immunofl uorescent antibody testing of conjunctival scrapings, and serologic testing. guinea pigs have been reported to develop pathologic changes related to ingestion of forssk fern. , in these studies, guinea pigs fed fresh fern developed hematuria and hemorrhage of the bladder wall. one guinea pig fed dried fern developed proliferative urocystica and adenoma of the bladder, a fi nding sometimes considered precancerous in human patients. this guinea pig did not show clinical signs associated with the bladder abnormalities. rabies virus infection is uncommon in rodent species, but it has been described and should be considered a differential diagnosis for an ill guinea pig with suspect contact to wildlife, especially raccoons. a recent report of a rabies in a privately owned guinea pig described abnormal behavior (biting the owner) days after the guinea pig had possible interactions with a raccoon. in this guinea pig, rabies virus antigen was detected by immunofl uorescent antibody testing in the sublingual salivary gland, tongue, and buccal tissues, implying that the guinea pig could have transmitted the virus via a bite wound. sick guinea pigs are often anorexic and therefore dehydrated. restoration of normal hydration status is crucial for the successful treatment of many disease processes. replacement of fl uid defi cit and maintenance of normal hydration can be achieved by administering crystalloids substances through subcutaneous, intraperitoneal, intravenous, or intraosseous routes. because of the diffi culty in placing and maintaining catheters in peripheral veins and bones, the most common route for fl uid administration is subcutaneous. subcutaneous fl uid administration is generally well tolerated. fluids are administered under the skin of the cranial, dorsal thorax (figure - ) . butterfl y catheter needles are useful because they allow the patient to move around without pulling out the injection needle. maintenance fl uid rates for guinea pigs are - ml/kg/day. another important aspect of management for the sick guinea pig is nutritional support. anorexic guinea pigs can experience a change in their normal gi fl ora in as little as to hours. this change of gi fl ora can lead to ileus, colic, overgrowth of pathogenic bacteria, and enterotoxemia. commercial products are available that are palatable and high in fi ber. these products help to maintain gut motility (oxbow critical care for herbivores, oxbow hay company, murdock, ne). patients will often eat directly from a dish or -ml catheter tip syringe. for patients that are more resistant to eating, a technique that is useful, in my experience, is to remove the plungers from -ml or -ml syringes and fi ll them individually using a catheter tip syringe. although this method may seem tedious, it allows the delivery of small boluses of food to be incrementally dispensed. the gi tract of guinea pigs can be very sensitive to the effects of certain classes of antibiotics. the gi fl ora of guinea pigs is primarily gram positive, and administration of antibiotics with a primarily gram-positive spectrum can result in overgrowth of gram-negative and anaerobic organisms. enteral administration of penicillins (e.g., amoxicillin, ampicillin), macrolides (e.g., erythromycin, lincomycin), and fi rst-generation cephalosporins can result in overgrowth of opportunistic pathogens. ampicillin administered subcutaneously at doses of and mg/kg three times a day resulted in mortality rates of % and %, respectively, in a group of guinea pigs. at necropsy, clostridium diffi cile was cultured from the ceca of all fatalities. twenty-fi ve percent of guinea pigs administered mg/kg of cefazolin, a fi rst-generation cephalosporin, intramuscularly died of enterocolitis after several injections. all antibiotics should be used with caution in guinea pigs because of the possibility of disruption of the gi fl ora (table - ). guinea pigs can be induced using isofl urane or sevofl urane administered by mask or induction chamber (figure - ) . for lengthy or potentially painful procedures, preanesthetic medications may be used before induction of the inhalant anesthetic agent. as a preanesthetic, an injectable combination, midazolam ( . - . mg/kg) and butorphanol ( . - . mg/kg), has been used with success. the administration of premedications will also decrease the stress of induction. isofl urane and sevofl urane are both acceptable inhalant anesthetics for guinea pigs. the advantage of sevofl urane is that it does not appear to have as noxious a scent as isofl urane, thereby reducing breath holding during induction and producing a smoother anesthetic episode. after induction, the inhalant anesthetic may be switched to isofl urane if cost is an issue. guinea pigs should not be fasted more than to hours before anesthesia. because these animals are hindgut fermenters, withholding food for longer periods of time may disrupt gi fl ora. careful monitoring during anesthesia is essential. the number of respirations must be visually monitored for depth and character. heart rate and rhythm should also be monitored continuously using a pediatric stethoscope or doppler unit, which can be placed on a peripheral artery. changes in heart rate or respiratory rate can occur rapidly, so it is advantageous to have precalculated doses of emergency drugs (e.g., glycopyrrolate, epinephrine, atropine, dopram) drawn and available for use before anesthetic induction. intubation of guinea pigs is generally considered to be very diffi cult. the long, narrow oral cavity makes visualization of the glottis diffi cult. the soft tissues of the tongue and soft palate are continuous in the caudal oropharynx, leaving only a small aperture, the palatal ostium. intubation must be performed through this opening in the mucosal tissues. several techniques have been described for visualizing the glottis to assist with intubation. these usually involve alterations of laryngoscope blades so that they may be introduced into the oral cavity without traumatizing the delicate buccal mucosa. , another possible intubation technique utilizes a stethoscope altered so that an appropriately sized endotracheal tube can be affi xed to the end. the endotracheal tube is then inserted into the caudal oropharynx. the anesthetist will then listen through the ear pieces of the stethoscope for expiration. the tube is then gently introduced into the trachea. this technique requires practice to perfect but, once the procedure has been performed several times, is a reliable way to intubate guinea pigs. application of a small amount of lidocaine to the rima glottis can ease intubation. rigid endoscopes and otoscopes, when available, are also very helpful in visualizing the glottis for intubation. indications for an ovariohysterectomy procedure in guinea pigs include routine sterilization, dystocia, and reproductive tract disease (e.g., neoplasia, muco/hydro/pyometra, ovarian cysts). in general, the surgical approach and technique are similar to those used with domestic species. the patient is placed in dorsal recumbency, and a ventral midline incision is made through the linea alba. care must be taken upon entering the abdomen to avoid incising the large cecum, which is often situated along the ventral body wall. the body of the uterus can be located dorsal to the bladder. the ovaries lie caudally and laterally to the kidneys and are to mm in length. the abdomen of the guinea pig is deep, and the ovaries can be diffi cult to exteriorize. care must be taken to avoid tearing the short ovarian vessels. once the ovaries are removed, the uterus is ligated and transected in a manner similar to that used with other mammals. another method of sterilization that is reported in guinea pigs is ovariectomy. the incidence of uterine disease compared with ovarian disease in guinea pigs is quite low. the approach for removal of the ovaries is through small incisions of the dorsal-lateral body wall caudal to the last rib. the advantage of this approach is that the sensitive gi tract is not manipulated to reach the structures to be excised. orchiectomy is typically performed to prevent reproduction, to decrease undesirable sexual behavior, and to treat reproductive tract disease. the orchiectomy procedure in guinea pigs is performed by making an incision through the scrotum over each testicle. reactions to suture material are relatively common in guinea pigs after surgical procedures. these reactions can vary in severity from mild local irritation to abscessation. suture materials that cause a large amount of infl ammation, such as chromic gut, should never be used in guinea pigs. in general, monofi lament suture materials that are degraded by hydrolysis are preferred. advances in diagnosis and treatment of small exotic 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-fusfdkjw authors: lukiw, walter j.; vergallo, andrea; lista, simone; hampel, harald; zhao, yuhai title: biomarkers for alzheimer’s disease (ad) and the application of precision medicine date: - - journal: j pers med doi: . /jpm sha: doc_id: cord_uid: fusfdkjw an accurate diagnosis of alzheimer’s disease (ad) currently stands as one of the most difficult and challenging in all of clinical neurology. ad is typically diagnosed using an integrated knowledge and assessment of multiple biomarkers and interrelated factors. these include the patient’s age, gender and lifestyle, medical and genetic history (both clinical- and family-derived), cognitive, physical, behavioral and geriatric assessment, laboratory examination of multiple ad patient biofluids, especially within the systemic circulation (blood serum) and cerebrospinal fluid (csf), multiple neuroimaging-modalities of the brain’s limbic system and/or retina, followed up in many cases by post-mortem neuropathological examination to finally corroborate the diagnosis. more often than not, prospective ad cases are accompanied by other progressive, age-related dementing neuropathologies including, predominantly, a neurovascular and/or cardiovascular component, multiple-infarct dementia (mid), frontotemporal dementia (ftd) and/or strokes or ‘mini-strokes’ often integrated with other age-related neurological and non-neurological disorders including cardiovascular disease and cancer. especially over the last years, enormous research efforts have been undertaken to discover, characterize, and quantify more effectual and reliable biological markers for ad, especially during the pre-clinical or prodromal stages of ad so that pre-emptive therapeutic treatment strategies may be initiated. while a wealth of genetic, neurobiological, neurochemical, neuropathological, neuroimaging and other diagnostic information obtainable for a single ad patient can be immense: (i) it is currently challenging to integrate and formulate a definitive diagnosis for ad from this multifaceted and multidimensional information; and (ii) these data are unfortunately not directly comparable with the etiopathological patterns of other ad patients even when carefully matched for age, gender, familial genetics, and drug history. four decades of ad research have repeatedly indicated that diagnostic profiles for ad are reflective of an extremely heterogeneous neurological disorder. this commentary will illuminate the heterogeneity of biomarkers for ad, comment on emerging investigative approaches and discuss why ‘precision medicine’ is emerging as our best paradigm yet for the most accurate and definitive prediction, diagnosis, and prognosis of this insidious and lethal brain disorder. senile dementia is the progressive, age-related loss of memory and cognition sufficiently severe to irreversibly affect social, behavioral, perceptual, occupational, and functional capabilities. recent statistics indicate that globally, about~ million people live with dementia, now costing an estimated one trillion dollars in annual healthcare. by , the number of people with dementia is projected to increase to~ million. in the united states, alzheimer disease (ad), the leading cause of senile dementia in the elderly, currently affects about~ million people age and older; by , the number of people aged and older with ad will grow to a projected~ million if no medical breakthroughs occur to prevent, slow, or cure this incapacitating disorder of the human mind [ - ]. one hundred and fourteen years since its original description, despite immense research efforts and clinical trials employing multiple strategic therapeutic approaches, there is currently no adequate treatment or cure for this widely expanding socioeconomic and healthcare concern [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the discovery, characterization, and quantification of biomarkers as measurable substances or cognitive disruptions in the 'prospective ad patient', whose presence are indicative of disease, are urgently needed so that: (i) ad may be more accurately diagnosed, especially at an earlier 'prodromal' stage and with the goal of preventive and or targeted therapeutic strategies that may be implemented at the earliest signs of ad onset; and (ii) more effective and reliable integration of multi-modal biomarkers for ad that can streamline, support, and strengthen the diagnostic and therapeutic decision-making. remarkably, peer-reviewed publications on biomarkers for ad have yielded almost~ , original research reports and reviews since crossing the words 'alzheimer's disease' and 'biomarkers'; [ , , [ ] [ ] [ ] . these include observations on the classical and established ad biomarkers [ ] [ ] [ ] [ ] , including altered genetics (incorporating genome-wide association studies or gwas), genetic mutations and gene modifications (including methylation and post-transcriptional modifications), end-stage neurotoxic and pathogenic metabolic products that accumulate in ad brains, such as multiple forms of tau aggregates and amyloid-beta (aβ) aggregation species and plaques. ad biomarkers also include protein, lipid, proteolipid, inflammatory cytokine, chemokine, carbohydrate, microrna (mirna), and messenger rna (mrna) abundance, speciation, and complexity, as well as an evolving assortment of neuro-radiological and neuroimaging technologies (table ) . ad biomarkers are certainly useful in the detection of dementing illness during its progressive course, and their appearance and magnitude correlate with cognitive loss in a dynamic way, allowing clinicians to monitor responses to therapeutic intervention across a background of aging of the ad patient. table . multiple interrelated factors contribute to ad. the considerable heterogeneity of alzheimer's disease (ad) appears to be mediated in part by a highly interconnected network of biological factors, and each of these can be used as diagnostic biomarkers which appear to each have a variable potential to contribute to ad-type change. there is abundant evidence that all of these biomarkers and/or factors (listed alphabetically) contribute to ad initiation, onset, or propagation, and there may be other important biological factors and other elements that may contribute to this complex neurological disease that we have not yet recognized or even considered. data derived from each of these multiple biomarkers and factors combined is amenable to systems and network analysis, information integration, and the application of precision medicine that should ultimately yield a more accurate diagnosis of ad at any stage of the disease (see text for references; specific references to the biomarkers listed in table can be found in [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . abbreviations: bace = β-amyloid cleavage enzyme; crp = c-reactive protein (a blood-serum-based inflammatory biomarker); lncrna = long non-coding rna; psi, ps = presenilin , presenilin ; rrna = ribosomal rna; sncrna = sall non-coding rna. age and age-related effects; amyloid (aβ and aβ peptides); compartmentalization of biomarkers [brain tissue, extracellular fluid (ecf), csf, blood serum, urine]; cytokine storm (cytokines and chemokines); environment and environmental effects; epigenetics (methylation, mrna and mirna editing); exosomes and extracellular micro-vesicles (exs and emvs); gender and gender-related effects; genetics (mutations in bace, ps , ps , etc.,); gastrointestinal (gi) tract microbiome; innate immunity; neuro-inflammatory markers (crp); inter-current illness (cardiovascular disease); lifestyle (diet, smoking); messenger rna (mrna); microbial contribution (viral, bacterial, fungal, other); microbiome (oral, other); microrna (mirna); mirna-mrna linking patterns; misdiagnosis; oral microbiome and dental hygiene; other rna (sncrna, lncrna); overlapping neurological disorders: [downs syndrome (trisomy ), frontotemporal dementia (ftd), multi-infarct dementia (mid), neuro-vascular disease, prion disease (prd), etc.,]. as improvements in ad diagnostics are based on advances in both ad biomarker acquisition and the technologies used to gain these data, below we briefly discuss some of the most recent advances contributing in a major way to the more accurate and comprehensive accrual of important ad biomarker data. currently, the complex molecular cargos of exosomes and extracellular microvesicles (exs and emvs) have emerged as one of the most representative, significant, dependable, and trustworthy of all ad biomarkers. typically, ex and emv cargos consist of various mixtures of protein, lipid, proteolipid, cytokine, chemokine, carbohydrate, microrna (mirna) and messenger rna (mrna), and other constituents including end-stage neurotoxic and pathogenic metabolic products. these in part, consist of tau proteins, amyloid beta (aβ) peptides, alpha-synuclein, tar dna-binding protein (tdp- ) and others. exs and emvs (i) have been analyzed in the cerebrospinal fluid (csf), blood serum, and post-mortem tissues of ad patients; (ii) are derived from their cells of origin and typically contain hundreds of different signaling molecules, many of which are potentially pathogenic and may be involved in the horizontal spread of neurological disease from one brain region to another; (iii) may represent a defined class of plasma membrane-derived nanovesicles released by all cell lineages of the human central nervous system (cns); and (iv) as potential biomarkers, may contribute to an additional element of certainty into the diagnostic assessment of ad [ , , , [ ] [ ] [ ] . there is a wealth of data indicating that neurotropic microbes including both dna and rna viruses (such as herpes simplex or sars-cov- ) or bacterial phyla such as proteobacteria, verrucomicrobia, fusobacteria, cyanobacteria, actinobacteria, and spirochetes or microbe-derived viral, fungal, or prokaryotic cellular components or microbial neurotoxins have high affinity for neural cells of the human brain and cns [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . multiple independent laboratories continue to report the detection of viral, bacterial, fungal, protozoal, or other microbially-derived nucleic acid sequences or neurotoxins, such as highly inflammatory bacterial amyloid peptides, lipopolysaccharide (lps), and many microbe-derived endotoxins within ad affected brain tissues [ , , , , , ] . microbial biomarkers and systems biology approaches to understand host-microbiome interactions have been suggested by multiple ad researchers that both: (i) predict the risk of developing ad well before the onset of cognitive decline; and (ii) stimulate and/or accelerate the development of classical ad neuropathology [ , , , [ ] [ ] [ ] [ ] . whether these viral, bacterial, or other microbial dna-or rna-based nucleic acids or associated lipoproteins, liposaccharides, peptidoglycans, bacterial-derived amyloids, and/or neurotoxins originate from the gastrointestinal (gi) tract microbiome, a potential brain microbiome, or some dormant pathological microbiome is currently not well understood [ , , , , , ] . since , at least peer-reviewed research articles provide convincing evidence that multiple species of microbes, including viruses, bacteria (especially gram-negative bacteria), and other microorganisms or their secreted components are strongly associated with the onset and/or the development of ad-type change [ , , , , [ ] [ ] [ ] ] . if microbial presence in the brain is involved in the early initiation or propagation of ad, as currently suspected, then specialized rna-sequencing applications or nucleic acid-containing gene chips, electrochemical biosensors, or panels of microbial-derived s ribosomal rna (rrna) interrogated with nucleic acid probes derived from ad biofluids might be useful as novel ad biomarkers in the detection of microbial patterns of expression from human brain tissues at any stage or degree of ad neuropathology. dna microfluidic array technologies, quantitative reverse transcription pcr (rt-qpcr), rna sequencing, led-northern, western immunoassay, and electrochemical biosensors, integrated by advanced bioinformatics tools have uncovered families of up-regulated human brain enriched micrornas (mirnas) and their down-regulated messenger rna (mrna) targets. these have been found in short post-mortem interval (pmi) sporadic ad brain, in transgenic animal models of ad (tgad), in brain biopsies, and in biofluids from ad patients. genome-wide association studies (gwas), epigenetic evaluations, such as mirna-mrna linkage or association mapping for ad-relevant neurological pathways, should provide useful diagnostic approaches since it has recently become apparent that mirna-mediated mrna-targeted regulatory mechanisms involve a large number of pathogenic and highly integrated gene expression pathways in the cns [ , , [ ] [ ] [ ] [ ] . to cite one very recent example, the human-brain-resident, nuclear factor kappa b p /p (nf-κb p /p )-regulated microrna- a (mirna- a) is an inducible, -nucleotide, single-stranded non-coding rna (sncrna) easily detected in cns neurons and immunological cell types. an inducible mirna- a: (i) is significantly up-regulated in ad brain tissues, csf, and blood serum [ , ] ; (ii) is an important epigenetic modulator of inflammatory signaling and the innate-immune response in several neurological disorders; and (iii) is essential in the down-regulation of the innate-immune regulator complement factor h (cfh; [ , , , , , , ] ). lps-and nf-kb (p /p )-inducible micrornas, such as mirna- a and mirna- b, appear to contribute to neuropathological, neuro-inflammatory, and altered neuro-immunological aspects of both ad and prion disease (prd; [ , , , , [ ] [ ] [ ] ). interestingly, nf-κb-sensitive up-regulated mirnas and their down-regulated mrna targets appear to constitute an integrated nf-κb-mirna-mrna signaling network implicated in multiple ad pathophysiological processes [ , , , , , ] . hence, potential signaling pathways to the acquisition of the ad phenotype appear to occur in part via an integrated and highly complex system of multiple mirna-mrna interactions that define many key pathogenic and pro-inflammatory gene expression pathways. genetic and epigenetic signaling via mirna-mrna networks in the brain may be one of the most useful as potential biomarkers for early ad detection as they can detect subtle failure in multiple ad-relevant brain signaling systems and metabolic pathways [ , , , , ] . a number of neuro-radiological and neuroimaging technologies are currently being used to view physical atrophy and structural change in specific anatomical regions of the human brain, such as the hippocampus, neocortex (gray matter), white matter, ventricles, and other brain regions for the purpose of acquiring real time data for the diagnosis of ad [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these neuroimaging technologies and structural and functional imaging techniques include computerized tomography (ct; including dual-energy ct), positron emission tomography (pet), scintigraphic neuroimaging (pet-sn), diffuse optical imaging (dot), structural and functional magnetic resonance imaging (mri), including ultra-high field mri (uhf-mri), magnetoencephalography (meg), single-photon emission computed tomography (spect), cranial ultrasound, and functional ultrasound imaging (fus) in the search for anatomically-based diagnostic biomarkers for ad with high accuracy and sensitivity [ , , [ ] [ ] [ ] [ ] [ ] [ ] . neuroimaging techniques, hardware and software design, and imaging resolution are being constantly improved, updated, and refined [ , [ ] [ ] [ ] . for example, with high signal-to-noise (s/n) ratios, improved contrast and unparalleled spatial resolution, ultra-high field mri of ≥ tesla (t) has been highly successful in imaging the neuroanatomy of highly focused brain regions targeted by ad pathophysiology while providing additional information on morphological, quantitative, and subtle metabolic changes associated with early ad-type pathological alterations [ , ] . in vivo biomarkers for ad performed by recently implemented scintigraphic neuroimaging and employing amyloid binding pet agents along with non-scintigraphic biomarkers from blood (plasma/serum) and csf have provided unique and novel opportunities to investigate the pathogenesis, prodromal changes, and time course of the disease in living individuals across the ad continuum [ , , [ ] [ ] [ ] . imaging technologies have indicated that ad changes in brain tissues begin as much as~ years prior to the onset of clinical symptomatology [ , , , ] . the opportunities afforded by in vivo biomarkers of ad, whether by blood (plasma/serum) or csf examination or imaging technologies, are beginning to transform the strategic design of ad therapeutic trials by shifting the focus to the preclinical stages of the disease and massively integrating both molecular-based and neuroimaging data [ , , [ ] [ ] [ ] [ ] [ ] . classically, the diagnosis of ad was a clinic-pathological one and there was a considerable error rate in the clinical diagnosis, especially early in the course of the disease. the differential diagnosis for ad by exclusion was confounded by a great many clinically overlapping neurological disorders including, mainly, mid, ftd, prion disease, tumors, subdural hematomas, neurovascular disruption and disease, and others [ ] [ ] [ ] . early neurophysiological diagnostic observations of ad included a diffusely slow electroencephalogram (eeg) and reduced cerebral blood flow [ , , ] . early pet studies demonstrated that oxygen extraction in the ad brain was relatively normal, thus tentatively excluding ischemia as a potential pathogenic factor [ , , ] . morphological pathological changes including the appearance of amyloid-enriched senile plaques (sp) and neurofibrillary tangles (nft), widely distributed in neocortex but excluded from the basal ganglia, thalamus, and substantia nigra, and a severe loss of large neocortical neurons, were 'classical' diagnostic characteristics of the ad patient [ ] [ ] [ ] [ ] [ ] . usually at the family or care-giver's request, post-mortem neuropathological examinations of the deceased ad patient's brain were routinely performed by qualified ad-trained specialists and neuropathologists and brain tissues were often subsequently provided to ad researchers for further molecular-genetic and biomarker research including the examination for the presence of ad-relevant brain lesions. light microscopy, nft and sp amyloid dyes (such as congo red, thioflavin s, thioflavin t and methylene yellow), and antibody-based staining (such as e and g ), the evaluation, density, composition, and quantitation of nft and sp amyloid, and the examination of blood (plasma/serum) or csf amyloid were additional indicators of immune-or inflammatory-neuropathology in the individual ad patient, which often contributed to the confirmation of ad in the 'prospective ad patient'. currently, in many medical schools in the us and canada, the post-mortem examination of the ad brain still remains the classical exercise to certify and verify the existence of ad. it is generally appreciated that the application of 'precision medicine', involving massively integrated data sets of multi-faceted ad biomarkers, data-driven analytical methodologies, and the application of systems theory, will challenge and may eventually supersede the need for the classical post-mortem neuropathological examination of the brain in order to verify and confirm the diagnosis of ad [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , , , ] . there are inherent problems in current approaches to ad biomarker research: (i) as most early reports emphasized just one or at most a few ad molecular-genetic biomarkers, biophysical, and neuroimaging modalities without consideration of the other hundreds or thousands of proteins, peptides, carbohydrates, lipids, or dna and multiple species of rna that have been previously implicated as being 'probable' diagnostic markers for ad; (ii) very often the nature of the acquisition of ad biomarkers represented a 'snapshot in time' of one specific portion of the ad continuum that does not take into consideration the time course of changes observed in ad and/or the progressive nature of the disorder; (iii) because easily accessible and non-invasive ad biomarkers are often limited in their diagnostic applicability because of their overlap with other neurological diseases related to ad, such as down's syndrome, parkinson's and lewy body disease, prion disease, ftd, hippocampal sclerosis, and mid and stroke; and (iv) no single newly generated de novo biomarker has ever been found to be associated with ad; that is, fluctuations in the abundance of pre-existing ad biomarkers reflect significant and absolute differences in the quantity, speciation, and stoichiometric relationships of ad-related biomolecules, including indicators of pro-inflammatory and immune system dysfunction. put another way, no 'specific' ad biomarker 'suddenly appears' at the earliest onset, or propagation, or throughout any time-point during the course of ad, or at any stage of cognitive failure for that matter. rather, it is usually a quantifiable up-regulation or down-regulation of an already existing biomarker in a specific anatomical region or biofluid compartment that has been the most consistently observed in the progressively degenerating brain. to cite a recent example, over susceptibility genes and gene loci have been associated with late-onset ad and multiple models have been proposed [ ] , however, these associations are relatively rare and non-penetrant, occur in a few but not all ad cases, adding to the complexity and heterogeneity in the diagnosis of ad [ , , , , , , ] . to further confound the establishment of definitive ad biomarkers, ad is commonly associated with more than one single neuropathology, in the case of ad usually with cerebrovascular and/or neurovascular involvement, and every one of these ancillary neurological disorders can carry their own set of complex and often overlapping disease biomarkers [ , , , , ] . especially over the last years, the progressive and steadily increasing accumulation of molecular, genetic, epigenetic, neuroimaging, clinical, and geriatric data acquired from multiple ad cohorts has significantly increased our appreciation and understanding of the intrinsic variability and heterogeneity of ad biomarkers associated with the continuum of ad and other forms of progressive age-related neurodegenerative disease. the generation of massive datasets integrating multiple genetic-, epigenetic-, molecular-, and neuroimaging-derived biomarkers is enabling the application of clustering techniques and the identification and stratification of ad subtypes that may further categorize the multiple aspects of ad heterogeneity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these approaches hold great potential: (i) for improving both the diagnosis and prognosis of ad; (ii) for projecting the clinical and neurological evolution of ad for planning suitable directions in therapeutic mediation; (iii) in providing multiple opportunities for the more directed analysis of ad heterogeneity in a data driven manner; (iv) in providing strategic guidelines for more decisive therapeutic intervention and the more efficacious clinical management of ad; and (v) for advancing 'precision medicine' not only for the individual ad patient, but also for other cases of inflammatory neurodegeneration and neurological disease. multiple analytical molecular-genetic approaches, advances in geriatric psychiatry and clinical evaluation, advancements in neuro-radiological labelling techniques and neuroimaging technologies, integrated diagnostic and predictive strategies and methodological improvements, discoveries of the comprehensive pathophysiological profiles of complex multi-factorial neurodegenerative diseases: (i) are presently well within the capabilities and scope of contemporary clinical, medical, and diagnostic neurology; (ii) are currently yielding increasingly large volumes of biomarker data for both individual ad patients, large populations of ad cases and age-and gender-matched controls; and (iii) are providing a data-driven basis for the paradigm shift of using the 'precision medicine' approach in ad prevention, diagnostics, prognostics, and therapeutics [ , , , , , , ] . less common clinical presentations of ad are becoming increasingly recognized, adding to the increasing volume of ad biomarker data [ , , , , ] . since one of the pillars of 'precision medicine' is supported by biomarker-derived medical data, further improvements in the acquisition, integration, interpretation, and bioinformatics aspects of clinical data and the coordination and analysis of clinical, laboratory, molecular-genetic, and neuroimaging data, geriatric and psychological information and related healthcare resources should obtain significantly increased accuracy in the diagnostic synopsis for the "prospective ad patient". the significant heterogeneity of the ad condition: (i) will certainly benefit from an equally wide variety of ad biomarker-derived 'precision medicine'-oriented treatment approaches and/or data-driven pharmacological strategies; and (ii) whose biomarker-based therapeutic design will greatly improve the current situation regarding the healthcare, more effective and successful treatment, and the development of disease-modifying drugs for ad patients at any stage of the disease [ , , , , ] . the ongoing search for valid biomarkers for ad is being carried out globally in at least a dozen major geriatric, bioinformatic, neurobiological, neuro-genetic and neurological bioscience arenas: (i) those involving the age, gender, and geriatrics of the 'prospectivead patient'; (ii) in the genetics and epigenetics of the ad patient including messenger rna (mrna) and microrna (mirna) signaling patterns, complexity and genomic methylation research; (iii) in multiple biofluids from ad patients including the blood (plasma/serum) of the systemic circulation, the glymphatic system, the cerebrospinal fluid (csf) and/or urine; (iv); through the detailed analysis of molecular cargos from both biofluids and tissue-compartmentalized exosomes and extracellular microvesicles (exs and emvs); (v) throughout the peripheral nervous system (pns; typically using skin biopsies); (vi) via clinically-based geriatric, psychiatric, and neurological assessment and testing; (vii) via advances in neuro-radiological labeling techniques and neuroimaging technologies including cat, pet, pet-sn, mri, fmri; uhf-mri, dot, meg, spect, cranial ultrasound, functional ultrasound (fus) imaging, and immunohistochemistry involving confocal laser scanning microscopy and other advanced microscopic and neuroimaging techniques; (viii) from the quantitation and characterization of the load of microbial and microbial-derived components in the ad-affected brain; (ix) via the identification, quantitation, and characterization of ad-specific lesions including amyloid peptide-enriched sps and nfts; (x) after post-mortem examination and biopsies of ad cases, again matched up against those same biomarkers in age-and gender-matched neurologically normal controls to corroborate the prospective diagnosis of ad; (xi) via the comprehensive analysis of the potential contribution of overlapping progressive, age-related neurological disorders to ad-type change; and lastly (xii), through the assessment of the socioeconomic, environmental, and lifestyle factors of the 'prospectivead patient' ( table ). the recent application of highly integrated data sets of these multiple ad biomarkers and analytical techniques on large cohorts of ad patients and involving systems-biology and 'precision medicine' may well serve to unravel many of the more intricate aspects of ad heterogeneity and expand and build on current therapeutic strategies to more effectively address both the diagnosis and clinical management of this devastating neurological disease. acknowledgments: a.v. is an employee of eisai inc. this work has been performed during his previous position at sorbonne university, paris, france. h.h. is an employee of eisai inc. this work has been performed during his previous position at sorbonne university, paris, france. at sorbonne university he was supported by the axa research fund, the "fondation partenariale sorbonne université" and the "fondation pour la recherche sur alzheimer", paris, france. this work was presented in part at the society for neuroscience (sfn) annual meeting - october chicago il, usa. sincere thanks are extended to l. carver, e. head, w. poon, h. leblanc, f. culicchia, c. eicken and c. hebel for short post-mortem interval (pmi) human brain and/or retinal 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journal: disease-a-month doi: . /s - ( ) - sha: doc_id: cord_uid: kzy hdb abstract multiple sclerosis is a chronic disease that begins in late adolescence or adulthood. it is highly variable in its expression and severity. it is believed to be autoimmune in nature. the cause is unknown; both genetic and environmental factors have been implicated in the pathogenesis. ms generally presents with the acute or subacute onset of neurologic abnormalities that may wax and wane over many years. diagnosis is generally made by means of observation of the clinical course in conjunction with a neurologic examination and laboratory tests. these tests may include magnetic resonance imaging of the head and spine, lumbar puncture, and evoked potentials. treatment is based on general supportive care, the use of corticosteroids for relapses, and symptomatic management of ongoing problems. the frequency of relapses can be reduced with interferon-β (betaseron). copolymer and interferon-β la are being evaluated by the u.s. food and drug administration for approval for use for reduction in the frequency of relapses in relapsing-remitting ms. treatment of chronic progression is often attempted with immunosuppressive agents such as corticosteroids, azathioprine, and cyclophosphamide. use of other agents is being investigated. multiple sclerosis (ms) is a chronic disease of the central nervous system that typically begins in late adolescence or early adulthood. the cause is unknown, although the disease is believed to be autoimmune in nature. both genetic and environmental factors have been implicated in the pathogenesis of ms. a viral cause has been postulated, but no single virus has been confirmed to be associated with ms. the pathologic features of ms include the presence of demyelinating areas in the white matter of the brain with perivascular in-flammation and relative sparing of the axons. plaques are commonly found in the periventricular areas of the cerebral hemispheres, in the optic nerves, the brainstem, the cerebellum, and the spinal cord. the presence of inflammation in ms plaques and the presence of oligoclonal immunoglobulin bands suggest an autoimmune basis of the disease. characterization of the inflammatory cells in the plaques and in the cerebrospinal fluid has revealed a predominance of t cells. this finding has focused a great deal of attention on the trimolecular complex, which consists of the major histocompatibility complex, the t-cell receptor, and the antigen. consistent associations with dr , drbl , dqb , and the dw haplotypes have been identified in white persons. studies of restricted use of specific t-cell receptor regions in the immune process have not revealed a specific receptor in this disease. the antigen remains unknown, although many investigators are working with myelin basic protein and other proteins associated with myelin. two animal models, experimental allergic encephalomyelitis and theiler murine encephalomyelitis, are valuable in testing experimental immunotherapies and other aspects of autoimmune mediated demyelination. ms generally appears with the acute or subacute onset of neurologic abnormalities that may wax and wane over many years. common early symptoms include numbness, double vision, paraparesis, monoparesis, bladder control problems, optic neuritis, ataxia, or tremor. common ongoing symptoms include those just mentioned, vertigo, increasing spasticity, depression, emotional lability, gait abnormalities, fatigue, dysarthria, quadriparesis, constipation, incoordination, fatigue, and pain. diagnosis is made by means of observation of the clinical course in conjunction with the neurologic examination and laboratory tests. magnetic resonance imaging of the head and spine can be valuable in the evaluation of suspected ms. the presence of an elevated immunoglobulin g (igg) index or oligoclonal bands in the spinal fluid also can be helpful. evoked potentials can help confirm subclinical involvement of the eyes, vestibular function, or sensory tracts. the differential diagnosis of ms includes other demyelinating syndromes, particularly the monophasic syndromes, such as postinfectious encephalomyelitis, postinfectious transverse myelitis, and isolated optic neuritis. some infectious diseases, such as lyme disease, syphilis, and htlv- myelopathy, can be confused with ms. other autoimmune conditions, such as systemic lupus erythematosus, behcet's syndrome, sarcoidosis, and sjogren's syndrome, can cause symptoms similar to those of ms. some leukodystrophies and hereditary degenerative syndromes can be confused with ms. ms is often classified by its clinical course. benign ms is charac-dm, january terized by mild intermittent relapses with nearly complete resolution. relapsing-remitting ms is the most common form of the disease. it is characterized by episodes of acute or subacute neurologic dysfunction followed by periods of improvement and stabilization. secondary progressive ms begins with a relapsing-remitting course, but the disease gradually worsens, causing slow accumulation of neurologic signs and symptoms. ms that never has a relapsing-remitting course but begins with a slow progression of signs and symptoms is classified as primary progressive ms. treatment of ms is based on the progression of an individual case. general health measures include exercise, physical and occupational therapy, a balanced diet, and aggressive treatment of fever and overheating. treatment of relapses is recommended for moderate to severe relapses. corticosteroids are choice of treatment of relapses. steroids should be used with caution because of the large number of side effects associated with long-term use. the frequency of relapses can be reduced with interferon-i lb (betaseron). copolymer and interferon la are being evaluated by the u.s. food and drug administration for approval for use in reduction of the frequency of relapses in relapsing-remitting ms. these drugs soon may be available for clinical use. treatment of chronic progression is often attempted with immunosuppressive agents such as corticosteroids, azathioprine, methotrexate, and cyclophosphamide (cytoxan). other agents under investigation are cladribine and intravenous immunoglobulin. symptomatic treatment of the chronic symptoms of ms is important. treatment of symptoms can help patients remain functional and comfortable even with relatively severe chronic problems. fatigue can be treated with rest breaks during the day, exercise, and energy-conservation techniques. medications that may help are amantadine hydrochloride and pemoline. spasticity is a severe problem that causes contractures, pain, insomnia, and increased fatigue. it can be treated conservatively with physical therapy, particularly stretching exercises. baclofen and diazepam can also be useful and are often used alone or in combination. in patients with severe spasticity, baclofen can be administered with an intrathecal pump. urinary dysfunction is a common problem. a urologist usually is needed to define the type of dysfunction present. a hypertonic, spastic bladder can be treated with anticholinergic agents. a hypotonic bladder may require intermittent or long-term catheterization. detrusor-sphincter dyssynergia may require a combination of anticholinergic agents and intermittent catheterization. urinary retention, which causes frequent bladder infections, may require acidification of the urine or long-term administration of antibiotics. patients with severe retention may require urinary diversion. sexual dysfunction often requires a multidisciplinary approach, including counseling, modification of sexual techniques, medication, or prosthetic devices for men. tremor can be a severe, intractable problem. medications include clonazepam, propranolol, acetazolamide, or diazepam. emotional problems are common in patients with ms. emotional lability may respond to tricyclic antidepressant medications. depression is treated with antidepressant agents and counseling. pain is a prominent concern in many patients with ms. dysesthetic pain can often be managed with tricyclic antidepressants, carbamazepine, phenytoin (dilantin), or valproic acid. musculoskeletal pain is treated with antiinflammatory medications and physical therapy. cognitive dysfunction can be a disabling and distressing component of ms. documentation with neuropsychiatric testing may be helpful in managing these problems. current investigations of ms center on the concept of autoimmunity, possibly mediated by a viral illness. studies designed to define the role of the immune system in ms may be useful. medications designed to reduce a specific autoimmune response and medications that assist in stimulation of remyelination or improvements in quality of life are being developed. over the past few years, great strides have been made in understanding the role of the immune system, in improving diagnostic capabilities, and in managing the problems associated with ms. as this trend continues, we may have more diverse and effective therapies for the management of ms. table . jean martin charcot's description of the clinical and pathologic features of ms is the foundation of our knowledge of the disease.l the historical aspects of ms are reviewed in previous publications. s we are now entering a new phase of understanding brought about by careful clinical trials and the capability of monitoring the disorder with longitudinal magnetic resonance imaging (mri). in an individual patient, ms can be described by means of clinical observation. current concepts of the clinical courses, their relative frequencies, and mri characteristics of ms are portrayed in table . investigations with mri have changed the concept of ms by demonstrating more evidence of disease activity than is expected from clinical examination. disease activity, as measured with mri, is particularly high among patients with chronic progressive disease. the acute lesions of ms can now be demonstrated with gadolinium-enhanced mri. the initial event is associated with local disruption of the blood-brain barrier (fig. ) . as the abnormality evolves, increased signal intensity becomes evident on t -weighted images (figs. and ). the lesion may grow larger over a few days and then the areas of high signal intensity may begin to recede. over time, the lesions may completely resolve on tzweighted images. with each relapse, which is defined by new or newly enhancing lesions on mr images, the older areas of involvement may be reactivated. reactivation is associated with the development of permanent lesions on mr images. clinical correlation is frequently observed with areas of contrast enhancement or abnormal signal intensity in the cerebellum, brainstem, or spinal cord. abnormalities in the cerebral hemispheres are frequently periventricular in distribution and only occasionally correlate with specific symptoms or signs. , the accumulation of lesions in the frontal lobes is associated with a decline in memory. in addition, a change in the number of lesions on cranial mr images correlates with a change in overall clinical status as measured with standard scales.g observations made with mri are having a marked impact on both our basic knowledge of ms and on therapeutic trialsjo mri studies will provide considerable insight into the natural history of the disease and will be an excellent independent variable in future clinical trials. traditionally ms is thought to have a relatively high incidence in the northernmost latitudes of the northern hemisphere.l* this theory is based on the incidence of the disease in scandinavia and the northern united states. a similar association is documented in the southern hemisphere in australia and new zealand. these observations are supplemented by data from m igration studies, which demonstrate a relation between age at m igration and assumption of disease risk for the location. risk is conferred by exposure to an environ- certain populations are susceptible to ms, and certain populations are resistant to ms. for example, lapps in scandinavia have a very low incidence of ms, even though they reside predominantly in the far northern latitudes. in north america the disease is infrequent among hutterites and native americans. ms is uncommon in japan. the incidence of the disease in first-degree relatives of patients with ms is times that of the general population, suggesting that genetic factors influence disease expression. the results of populatidn-based studies of twins offer evidence that environmental and genetic factors contribute to the development of ms. these investigations show that the concordance in monozygotic twins is greater than %. it is less than % in dizy- gotic twinp suggesting that although genetic factors are important, environmental exposure also is important for disease expression. it is now commonly accepted that multiple genes influence autoimmune diseases in both animals and human beings. therefore polygenic inheritance is postulated for ms. like other autoimmune diseases, ms is more frequent in women, with a ratio of :l. the pathologic features of multiple sclerosis were first described by charcot,l who recognized plaques in.the white matter (scleroses en plaque) during pathologic examination of brain sections. these plaques were demonstrated to lack myelin and to contain perivascular inflammation. these features were established as the pathologic hallmarks of ms. the following discussion centers on the typical findings in ms. comparisons are made between ms and other forms of inflammatory demyelinating disease. the distribution of plaques within the white matter is restricted to the central nervous system (cns). plaques are found frequently in a periventricular distribution in the cerebral hemispheres. some of these plaques may be associated with the distribution of terminal veins. j plaques may occur anywhere within the white matter. when plaques are near the cortex, sparing of the subcortical myelinated fibers is often observed. plaques adjacent to gray matter may at times spread into the gray matter, including the cortex and deeper nuclei. plaques are frequently found in the white matter of the optic nerves, the brainstem, the cerebellum, and the spinal cord. plaques in these locations more frequently correlate with symptoms. within a plaque, axons are frequently preserved. the evolution of a plaque is not known. mri investigations show that the blood-brain barrier is locally disrupted at the onset of symptoms. pathologists disagree as to whether demyelination precedes inflammation or is secondary to inflammation. at present the latter view predominates. in acute plaques, the inflammatory response of lymphocytes, plasma cells, and macrophages is capable of producing or augmenting demyelination by direct and indirect mechanisms. the inflammatory response is predominantly perivenular, with a lesser response at the edges of or within plaques. the macrophages associated with acute plaques characteristically contain myelin fragments or myelin breakdown products.lg lymphocytes may contribute to the pathologic process by means of direct or indirect pathways. direct mechanisms include antibodyand cell-mediated immunity. t-cell-mediated reactivity is favored because most inflammatory cells are t cells. indirect mechanisms include the secretion of lymphokines and cytokines. the ability of molecules such as tumor necrosis factor to damage myelin or oligodendrocytes is the focus of ongoing research. cytokines may influence macrophage activation, stimulating the phagocytosis of myelin. in addition, the release of heat shock proteins may result in stimulation of ty cells, resulting in increased cytotoxicity. the cns lesions of ms can be classified as early active, active, inactive, early remyelinating, and late remyelinating, according to histologic criteria. the features of these lesions are detailed in table . studies of oligodendrocytes early in the course of ms have demonstrated relative preservation of these cells in some patients,z , and remyelination is possible in these patients. other patients have a striking loss of oligodendrocytes, making remyelination unlikely. these differences may reflect the severity of the injury at a specific site of demyelination, or they may indicate that the pathogenesis of demyelination varies among patients with ms. this may imply that an autoimmune basis for ms has long been suspected because of the inflammation in the cns and the presence of oligoclonal bands in the cerebrospinal fluid (csf). the inflammatory response is primarily lymphocytic and mononuclear. ~ the predominance of t cells among the lymphocytes has led investigators to evaluate the role of the t-cell receptor and its recognition of antigen combined with major histocompatibility antigens (mhc). this has been named the trimolecular complex. the t-cell receptor recognizes antigen in the context of the mhc molecule. in the case of mhc class ii molecules such as drz, the antigen fragments are bound in a cleft, which is presented to the tcell receptor for recognition. with regard to the components of the in the context of the trimolecular complex, it is important to note that ms has been associated with certain mhc or human leukocyte antigen (hla) markers. a consistent observation is the association of dr , drbl , dqb , and the du haplotype with ms. different hla associations are reported within ethnic groups. the mhc molecules may contribute to genetic susceptibility to the disorder, but they are only one of a number of factors that confer risk for the disease. j the presence of oligoclonal bands in the csf of patients with ms is frequently observed (fig. ) . these abnormal immunoglobulins are identified in a high percentage of patients with clinically definite ms, and they are present in approximately % of patients at the clinical onset of the disease. the oligoclonal bands in ms are of unknown specificity. small percentages may bind to known viral antigens in some patients. consistent binding of these antibodies to specific viral polypeptides or viral oligopeptides with homology to myelin components has yet to be demonstrated. the oligoclonal bands are not specific to ms and can be observed in patients with cns infections such as syphilis, subacute sclerosing panencephalitis, viral encephalitis, or meningitis. , if the infection is self-limited, the oligoclonal bands may be a transitory abnormality. in comparison, chronic infections of the cns are associated with persistence of the oligoclonal bands. in these settings, the antibodies that compose the oligoclonal bands have pathogen specificity. oligoclonal bands can be observed in patients with autoimmune diseases such as systemic lupus erythematosus. the probability that an environmental factor is involved in the pathogenesis of ms has stimulated interest in a viral cause. although viral isolates are reported from the cns of patients with ms, - g there are no consistent observations. attempts to detect viral nucleic acids by means of in situ hybridization and polymerase chain reaction (pcr) are in progress. these techniques are extremely sensitive and require rigorous controls. careful confirmation of any future viral isolates or viral nucleic acids by multiple laboratories is required. [ ] [ ] [ ] [ ] [ ] [ ] [ ] recent studies of tropical spastic paraparesis demonstrate that the retrovirus human t-cell lymphotropic virus type i (htlv-i) is involved in the pathogenesis of this disorder, which shares some clinical features with ms. ga it is clear, however, that htlv-i is not a pathogen in ms. there remains the possibility that a retrovirus or endogenous retrovirus could contribute to the pathogenesis of ms. there is considerable interest in the possibility that exposure to a virus may lead to an immunopathologic condition that results in ms. of particular note are investigations that demonstrate the potential of molecular mimicry to produce autoimmunity. the term molecular mimicry arises from the demonstration of shared homology between normal human myelin proteins and viral polypeptides. if an immune response is mounted to such a viral epitope, then it may be perpetuated by exposure of the shared region on the normal human protein. in ms, homology between myelin antigens and viral peptides is established. thus this mechanism could result in cns demyelination after viral infection. autoimmunity could also result from superantigenic stimulation of t cells by viral or bacterial proteins. superantigens are capable of binding to specific t-cell receptor proteins, producing nonspecific stimulation of relatively large numbers of t cells, which might cause clonal expansion of t cells reactive to myelin or oligodendrocyte antigens. s animal mqdels of demyelinating disease cns demyelination associated with inflammation is present in animal models of experimental allergic encephalomyelitis (eae) and theiler murine encephalomyelitis (tme). these models provide an opportunity for the investigation of autoimmune and virus-associated disease, respectively. eae is an autoimmune disease of the cns and a model for immunotherapy. a cd + t-cell population specific for a myelin antigen, either mbp or proteolipid protein, is required for initiation eae. eae and ms share characteristics that include cns demyelination, perivascular t cells, association with mhc class ii antigens, and possibly restricted tcr v-gene utilization. g the murine adoptive transfer model has another important feature of ms: the chronic relapsing clinical course. g this clinical course is useful for investigations of the immune response and immunotherapy not only during onset of the disease but also during relapse. the pathologic features of this murine transfer model are inflammation and prominent demyelination. g- eae is not associated with an environmental factor. the tme model of immune-mediated demyelination is of particular interest because it has important parallels with postinfectious encephalomyelitis and ms. in this model, antecedent mild or even subclinical viral encephalitis is followed by a period of quiescence and the eventual onset of demyelination. the virus is persistent during the demyelinating phase of the disease. this implies that either low-level expression of viral polypeptides or immunologic cross-reactivity between virus and myelin antigens is crucial for initiating demyelination. the demyelination in the tme virus model is mediated by t lymphocytes. these t cells may have viral specificity but produce demyelination. this mechanism would be relevant to ms if the suspected environmental factor were one or several viruses. as in ms and eae, t cells appear to initiate immunemediated demyelination in tme.!~~z~~ experimental immunotherapies are evaluated in these animal models and provide a basis for clinical trials in human beings. examples of these investigational treatments include cytokine transforming growth factor-i , (tgf , lymphokine-toxin, anti-t-cell receptor vb-specific monoclonal antibody, , t-cell vaccination, blocking peptides, anti-adhesion molecule specific monoclonal antibodies, g and nitric oxide synthetase inhibition. these experimental models provide an invaluable resource for the study of immunotherapy. although these experimental models are not likely to mirror the pathogenesis of ms, they are extremely useful in the study of cns inflammation and demyelination. ms is primarily a disease of young adults. most patients report their first symptoms between the ages of and years. the disorder rarely appears before the age of years or after the age of years, although it has been reported to occur in both children and the elderly. the symptoms of ms in children are essentially the same as those in adults; ataxia, numbness, and visual disturbance are the most common presenting symptoms. in elderly persons, a progressive onset is more common. ms is characterized by episodes of neurologic dysfunction, followed by periods of stabilization or remission. symptoms, once they appear, may partially or completely resolve or may be permanent. these episodes tend to develop over hours or days. sometimes the symptoms occur with almost strokelike suddenness, or they may develop slowly over a few weeks. once the symptoms have developed, resolution generally occurs over weeks or months. certain signs and symptoms are more common in the early stages of ms. these include numbness, double vision, monoparesis, paraparesis, bladder control problems, optic neuritis, ataxia, or tremor (table ) . dm, january numbness can be difficult to evaluate. numbness that suggests early ms includes an ascending numbness beginning at the feet. this may be a sign of transverse myelitis. hemiparesthesia, bilateral hand numbness, and dysesthesia in both hands, both feet, or on one side of the body, also are early symptoms of ms. the numbness is usually present for days, weeks, or months. many patients describe numbness or paresthesia with no objective abnormalities. if objective sensory abnormalities occur, they are more commonly reduction of vibration, proprioception, or stereognosis rather than pain or fever. the diplopia that occurs with ms is frequently partial or complete internuclear ophthalmoplegia, which is often bilateral. a small percentage of patients have sixth nerve palsy" or, more rarely, third or fourth nerve palsy. ww sometimes monocular diplopia is a symptom of optic neuritis. optic neuritis is usually characterized by monocular blurred vision, sometimes with scotomata and often with alteration of color vision. retroorbital pain or headache is common in patients with active optic neuritis. the pain may intensify with eye movement. motor weakness is usually accompanied by upper motor neuron signs, such as hyperreflexia or the babinski sign. paraparesis is the most common early symptom, but the weakness also can occur as hemiparesis or monoparesis. spas.ticity can be a later manifestation. signs and symptoms that commonly occur as ms progresses include vertigo, tremor, incoordination, increasing spasticity, depression, mood swings, cognitive abnormalities, impotence or other sexual dysfunction, weakness, lhermitte's sign, gait abnormalities, constipation, urinary incontinence, optic nerve pallor, fatigue, quadriparesis, dysarthria, loss of upper extremity coordination, and dysesthetic pain (table ) . uncommon but important problems include seizures, atypical facial pain or tic douloureux (trigeminal neuralgia), bowel incontinence, swallowing problems, hearing loss, and dystonia. bell's palsy is sometimes seen in patients with ms (table ) . the classic course of ms is one of intermittent neurologic signs and symptoms over many years. as time progresses, chronic problems accumulate. the amount of total disability varies from patient to patient. after a number of years, a patient's condition may stabilize permanently, but this does not always occur. d&f, january subtypes of disease ms can be divided into subtypes according to the course of the disease. there is a continuum among the various subtypes, and the disease in some patients does not fit into a pattern. benign ms accounts for % to % of cases and occurs more often in young women. in this type of ms, symptoms are mild and often sensory. resolution of neurologic problems is nearly complete. over the years, these patients rarely experience considerable disability. relapsing-remitting ms is the most common form of the disease. it is characterized by episodes of neurologic dysfunction [variably called exacerbations, relapses, or attacks) followed by periods of improvement and stabilization (called remissions). during a remission, not all symptoms resolve completely. the patient may be left with permanent disabilities, which may vary in severity. the condition of % to % of patients with an initial relapsingremitting course begins to worsen gradually over time, and neurologic signs and symptoms accumulate. this form of the disease is classified as secondary chronic progressive ms or relapsing-progressive ms. the latter term is also used to describe disease in patients who have sudden deteriorations in a stepwise manner without clinically significant recovery. primaryprogressive ms occurs in % to % of patients. disease in these patients begins with a slow progression of neurologic deficits with no history of relapse and may also have periods of stabilization or subacute worsening. common problems that appear and gradually worsen with time include spastic paraparesis, cerebellar ataxia, and urinary incontinence. clinical findings although no neurologic findings are pathognomonic for ms, certain abnormalities found during a physical examination can be helpful in providing a clue to the diagnosis of ms. these include internuclear ophthalmoplegia, which is rarely seen in other diseases and is especially rare in young adults. hyperreflexia and the babinski sign are common in early ms. optic nerve pallor can provide a clue to subclinical or resolved optic neuritis. altered color vision in one eye and a marcus-gunn pupil also are signs of optic neuritis. nystagmus is a common finding in patients with ms. many types of nystagmus are identified, including pendular nystagmus, small-amplitude nystagmus, or gaze-evoke nystagmus. a a absent abdominal reflexes in a slender patient who has not undergone an abdominal operation may be a helpful sign. a mild intention tremor with or without past-pointing is also an early sign, as is a positive romberg sign or difficulty with balance with divz,january tandem gait. subtle motor weakness or spasticity may also be found. loss of vibratory or proprioceptive sensation in the lower extremities is common early in the course of the disease. ms should be strongly suspected in young or middle-aged adults who describe symptoms consistent with the lhermitte sign in the absence of obvious cervical cord abnormalities. the lhermitte sign consists of paresthesia or an electric shock-like sensation that radiates up the head or down the spine on neck flexion or extension. other important abnormalities are gait disturbances, persistent binocular double vision when looking in a particular direction, or a history of optic neuritis or transverse myelitis. fatigue and depression are not criteria for the diagnosis of ms. no laboratory test is universally diagnostic for ms. certain studies can be helpful in confirming the presence of separation of lesions in space and time. examination of the csf can be valuable for two reasons. first, the pattern of csf findings can help confirm the presence of demyelinating disease. the protein level is often slightly elevated but is rarely greater than . g/l unless the patient is experiencing a severe exacerbation, particularly optic neuritis or transverse myelitis. a modest elevation in cell count, generally less than /mm , is seen in some patients. the cell pattern usually consists mostly of mononuclear cells. if more sophisticated testing is conducted, most cells can be identified as t lymphocytes. qualitative analysis of proteins can be helpful in suggesting the diagnosis of ms. at electrophoresis oligoclonal immunoglobulin bands can be identified in the csf but not in the serum of many patients with ms , (fig. ) . the igg index, a comparison between igg levels in the csf and igg levels in the serum, is elevated in many patients with ms. , g although these findings suggest ms, they also are found in other diseases, most commonly other inflammatory diseases of the cns. these diseases include lyme disease, systemic lupus erythematosus, progressive multifocal leukoencephalopathy, encephalitis, and subacute sclerosing panencephalitis. the ver is abnormal in approximately % of patients with ms, regardless of whether there is a history of optic neuritis. a slowed ploo in a patient without a history of optic neuritis can be paraclinical evidence of a second lesion and can be used to confirm a diagnosis of ms (fig. ) . g the baer is more difficult to interpret than the ver and is abnormal in approximately % of patients with ms. in the baer, five d&z, january consecutive waves are identified; these are numbered i-v the wave interval i-iii is considered the peripheral system. abnormalities in this wave suggest a lesion in the peripheral auditory nerve. the wave interval iii-v is generated from the central hearing areas in the brainstem. slowing in this area suggests a brainstem lesion. abnormalities in waves iii-v are seen in approximately % of patients with ms. the sser is a technically more difficult study than the other responses, but it is useful for identification of slowed central conduction in the sensory pathway in the spinal cord and brain. the sser is abnormal in approximately % of patients with definite ms. the sser also is useful in the identification of peripheral lesions, suggesting that peripheral neuropathy rather than a central lesion is the cause of numbness. the development of mri has been extremely important in both making the diagnosis of ms and helping researchers understand the dynamics of ms in patients with the disease. mri findings should be interpreted with caution, however. abnormal mri findings alone are not sufficient to confirm a diagnosis of ms without clinical evidence. , in patients with ms, patchy areas of abnormal white matter are seen on t%weighted and spin-echo images. these are most commonly found in the cerebral hemispheres in the periventricular areas. in some patients, however, lesions also are identified in the brainstem and cerebellum. mri also helps identify lesions in the cervical and thoracic spinal cord. gadolinium enhancement can be seen around some lesions, particularly if a patient is having an exacerbation or fairly rapid chronic progression. gadolinium enhancement is considered a sign of an active lesion. (table ). - g mr images should be interpreted with caution, particularly in patients with chronic illness of any kind or in patients older than years. fazekas et a . attempted to differentiate the mr images of healthy persons older than years from those of patients with ms. they identified the following three criteria for the diagnosis of ms: lesions abutting the lateral ventricles, lesion diameter greater than . cm, and lesions present in the posterior fossa. if two of the three criteria were met, the specificity for ms was % and the sensitivity was %. a follow-up study in which consecutive mris were examined yielded a sensitivity of % and a specificity of %" these criteria may be useful in the interpretation of mri findings in some patients, but they should be used with caution for patients with other diseases that can affect mri, such as hypertension and diabetes mellitus. patients with those diseases were excluded from the study by fazekas et al. the size and area of the lesions present on mr images correlate poorly with the patient's disability. , many patients with large lesions on mr images have minor clinical findings, whereas some patients with small lesions have severe disability. one area in which mri may indicate the severity of the problem is in the cognitive status of the patient. an increase in the area of the lesions in the cerebral hemispheres or thinning of the corpus callosum may correlate with poor cognitive function. the presence of lesions in the spinal cord does not correlate with disease severity. a recent study in which body coil imaging was used showed that % of patients with ms had lesions in the spinal cord that were identified by this technique. although the presence of lesions and the area and number of lesions did not correlate with a patient's level of disability, the presence of spinal cord atrophy did correlate with greater disability. patients with partial or complete transverse myelitis who subsequently are found to have ms often have lesions on mr images that correspond to the level indicated by symptoms and the level of neurologic findings (simnad v, rose jw, manuscript in preparation). the use of mri for the follow-up evaluation of ms has become an integral part of research into the course of the disease. however, because mri findings do not correlate with a patient's clinical condition, new abnormalities on mr images in the absence of clinical worsening should not be treated as an exacerbation of the disease. new abnormalities can, however, indicate that the disease remains active. mri should be repeated in patients in whom the diagnosis has not been confirmed or in patients who have new symptoms that suggest a second disease. as the choice of treatments of ms increases, monitoring of disease activity may become useful in determining the course of treatment. optic neuritis is often seen as a first demyelinating episode in patients with ms. the diagnosis of ms should be considered in patients with optic neuritis, and a careful history and examination should be performed to exclude other neurologic abnormalities. however, many patients who have a single episode of optic neuritis never have other demyelinating episodes. one study of patientsgo found that ms developed in % of women and % of men within years of an attack of optic neuritis. transverse myelitis, inflammation of an area of the spinal cord causing ascending weakness and numbness up to the level of the lesion, can also be seen as the initial demyelinating event in ms. other causes include infectious, postinfectious, and postvaccinal demyelination. sometimes the cause is never determined. when transverse myelitis occurs, an imflammatory lesion can be identified on mri images of the cervical or thoracic spinal cord. estimates of the risk of ms after an isolated episode of transverse myelitis range from % to %.g -g im'z, january the use of the cranial mr images in patients with optic neuritis or transverse myelitis may be helpful in predicting which patients are more likely to have additional problems. one prospective study identified patients with a single demyelinating episode such as optic neuritis or transverse myelitis. patients with abnormal mri findings at the time of the first episode had a % risk of a second episode within years. patients with normal mri findings at the time of the first episode had a % risk of development of a second lesion in years.g a syndrome in which optic neuritis and transverse myelitis develop with no other demyelinating events is called devic's neuromyelitis optica. in this disorder, cranial mri findings remain normal. this is considered a monophasic illness-both abnormalities occur within a year of each other, and patients may never have another demyelinating event. this is a rare syndrome.g the following characteristics are associated with a favorable prognosis: ( ) female sex, ( ) early age at onset, ( ) onset of symptoms referable to a single neurologic system, ( ) substantial recovery from relapses, ( ) early symptoms of numbness rather than corticospinal or cerebellar symptoms. unfavorable prognosis is associated with chronic progressive disease (either primary or secondary), older age at onset, and male sex.g -g dlagnostic criteria because of the difficulties involved in the diagnosis of ms, several criteria have been published to standardize the terms used to describe the certainty of the diagnosis. the two primary sets of criteria are those of poser et a . g and shumacher et a . the poser criteria are more recent and are summarized in table . it is important to remember that no abnormality should be used as a criterion if it can be explained by another medical problem. other conditions may commonly be confused with ms and should be considered in the differential diagnosis. the differential diagnosis depends in part on the clinical and laboratory findings in an individual patient. postinfectious encephalomyelitis is a subacute syndrome, possibly caused by an autoimmune response to a viral infection. patients with this illness experience the acute or subacute onset of confusion, disorientation, gait abnormalities, loss of bowel or bladder control, weakness, or other symptoms. abnormalities in the white mat- ter can be seen with mri, and evidence of inflammation frequently is seen in the cse the patient's condition may or may not return to normal; recovery may take months or even years. lyme disease is a prominent concern and appears to be a cause of intermittent neurologic events, the most common of which is bell's palsy. encephalomyelitis may develop, with vague symptoms of numbness, fatigue, and memory deficit. abnormalities in the white matter may be seen with mri, and csf findings may resemble those in ms, including mild leukocytosis and oligoclonal bands. patients may have a history of a tick bite, a rash, or recent arthralgia. lyme titers or a lyme pcr in the blood or csf may be helpful to these patients. systemic lupus erythematosus is a well-known syndrome that may cause transverse myelitis, strokes, encephalopathy, and optic abnormalities. clues to the differential diagnosis are systemic abnormalities such as hematuria or leukopenia, arthritis, or an elevated antinuclear antibody titer, erythrocyte sedimentation rate, or other blood measurement. sometimes both systemic lupus erythematosus and ms occur in the same patient. primary cns vasculitis can cause a syndrome similar to ms. differentiating features include prominent headaches, confusion, and sudden strokelike episodes. an elevated erythrocyte sedimentation rate may be present in some patients, as may an elevated csf protein level. patients may have an abnormal cerebral angiogram. bi-opsy of the temporal lobe or meninges may be helpful in the diagnosis of this syndrome. the htlv-i, a retrovirus, causes a syndrome known as tropical spastic paraparesis or htlv-i-associated myelopathy. it may cause progressive spastic paraparesis or generalized white matter disease. htlv-i is relatively rare in the united states but is present in some patients who have resided around the caribbean sea. behqet's syndrome can cause mri findings identical to those in ms. cardinal features of behqet's syndrome include oral ulcers, genital ulcers, and uveitis. variable features include involvement of the skin, eyes, joints, lungs, intestines, and heart and venous thrombosis. neuropsychiatric symptoms, including quadriparesis, pseudobulbar palsy, cranial neuropathy, cerebellar ataxia, peripheral neuropathic lesions, or cerebral venous thrombosis may be present. g, sarcoidosis and sjggren's syndrome are autoimmune diseases that may show lesions on mr images that resemble those of ms. meningeal enhancement is a clue to cns sarcoidosis. a chest radiograph may show granulomatous lesions suggestive of systemic sarcoidosis. although igg levels are raised in the csf of patients with cns sarcoidosis, oligoclonal bands are found in some patients. csf angiotensin-converting enzyme determination may be used to further differentiate cns sarcoidosis from ms. vitamin b deficiency and syphilis can cause posterior column abnormali& and dementia. tests for these problems should be performed when a patient with these symptoms is seen. certain leukodystrophies may appear in adulthood. these include adrenal leukodystrophy, krabbe's disease, and metachromatic leukodystrophy. mri findings in these diseases show large areas in which no normal white matter is present. female carriers of the adrenal leukodystrophy gene may have an ms-like syndrome. ~ g hereditary degenerative syndromes, such as familial spastic paraparesis, olivopontocerebellar degeneration, and spinocerebellar degeneration, may be confused with ms, particularly with primary progressive ms. in these diseases, mr images may be normal or may show atrophy of the brainstem, spinal cord, or cerebellum. the csf is normal in these patients. studies support the concept that exercise is beneficial for the patients with ms.ggjoo simple measures such as walking, using an exercise bicycle, and swimming may be of considerable value. exercise should be performed in a cool environment whenever possible to prevent heat-associated transient declines in neurologic function. swimming and water aerobics in pools that are not overly heated are particularly valuable, because the patient is cooled while exercising. physical and occupational therapy are often invaluable for maintenance or improvement of neurologic function. bracing disabled portions of limbs, particularly the ankle, provides considerable benefit. exercise regimens tailored to the patient may help to maintain or improve strength, range of motion, and mobility. devices that provide assistance with walking can be important in reducing the risk of falls, allowing for greater independence and increased activity. other assistive devices can be helpful in reducing fatigue and increasing independent activity. careful consultation with a specialist in rehabilitative medicine can assist the patient with management of work and daily activities.l"o it is advisable for persons with ms to maintain a balanced diet. weight control is a prominent concern. overweight patients with motor, sensory, or coordination deficits that impair ambulation are at particular risk of falls, which may result in serious injuries, including fractures. patients who are overweight and whose strength is decreased lose any reserve strength they may have because of their weight. some patients with ms lose weight and require dietary supplementation. patients with dysphagia may require feeding tubes to help prevent aspiration pneumonia. although various diets have been advocated for ms, there are no substantial data from controlled trials to support the assertions. as a general health measure, it is commonly suggested that patients with ms restrict cholesterol and fat in the diet. diets that meet the requirements of the american heart association are likely to be useful, because most patients with ms live into middle age and beyond. pregnancy is a concern among young women with ms. many studies of the effect of pregnancy on ms have been undertaken. an increased risk of exacerbations in the first months postpartum has been reported.lol-la however, the risk of exacerbations during pregnancy appears to be unchanged or slightly reduced.lo overall longterm disability does not appear to be altered by pregnancy.lo j the increased relapse rate seen during the postpartum period has been postulated to be caused by an increase in immune tolerance during pregnancy, followed by a return to normal in the postpartum dm, january period. it has also been postulated that the relapses are secondary to the decrease in the level of female hormones after parturition.lo*-lo in addition to the physical effects of pregnancy, another major concern is the care of an infant or child by a person with physical problems. persons with ms need to consider carefully whether they can handle the additional work of caring for a child. persons with chronic physical problems may need special provisions, such as extra assistance in the home or special equipment. the physician should discuss pregnancy, delivery, and child care with women of childbearing age. increased core temperature, whether due to heat exposure or to a febrile response, may lead to a transient increase in neurologic symptoms.lo if the event is due to heat exposure, the patient simply needs to rest in a cool environment and await recovery. if an infection is responsible, the source of the infection should be determined and treated. an antipyretic medication such as acetaminophen can then be administered. many patients with ms are susceptible to urinary tract infections and may not have clinical manifestations of the infection. in some patients this is due to impaired sensory capabilities, and some patients have chronic urinary symptoms that may not change substantially with an infection. one study of ms exacerbations pointed to an association with antecedent infection.lo if a patient has persistent worsening after an infection that has been appropriately treated and resolved, steroid therapy should be considered in the event the infection recurs. a relapse is considered to be the onset of new neurologic symptoms or marked worsening of old symptoms lasting longer than hours. certain conditions may mimic an exacerbation and should be ruled out or treated before steroid therapy is considered. these include fever, infection (commonly urinary tract infection or viral illness), overheating, fatigue, severe emotional stress, or the effects of medications such as baclofen, which can increase weakness. if these problems are appropriately treated, the patient's condition usually improves. mild relapses may be best treated without steroid therapy. the symptoms include a mild numbness, mild changes in bladder function, mild optic neuritis (visual acuity better than / ), slight increase in spasticity, or a dysesthetic pain syndrome. any new abnormality that does not change a person's ability to perform his or her usual daily activities may not require steroid therapy. in these patients, rest is sometimes helpful. patients with more severe worsen-ing may benefit from steroid therapy. the symptoms include gait disturbances, severe numbness or paresthesia, moderate to severe paresis, moderate or severe optic neuritis, severe vertigo, or marked impairment of eye movement. it is often appropriate for the physician to observe the patient for a few days before making a decision about the use of steroids. standard therapy for many years, immunosuppression with corticotropin (acth) or steroids has been used in the treatment of the exacerbations of ms. the primary effect of these agents is to shorten the duration of an attack, and no benefit has been proven in the overall outcome from an attack. steroids should not be given until an abnormality resolves because this may never occur. acth was the first immunosuppressant to be widely used in ms.lo although it is still given to some patients who respond well to the medication, acth has been largely supplanted by other steroids, most commonly prednisone and methylprednisolone. many different regimens have been used. a typical regimen is units by intravenous or intramuscular injection once a day for days. prednisone is commonly used for mild or moderate exacerbations of ms. although low doses do not appear to have any effect on an exacerbation, larger doses do appear to shorten the duration of an ms attack.log there is no standard treatment regimen; a dose of at least mg/kg per day is commonly recommended and should be continued for to days. our regimen is mg once a day by mouth for days, then tapered by mg every days. other regimens range from days to weeks or longer. methylprednisolone with sodium succinate (solu-medrol) is often used in the treatment of severe relapses, or when the patient's condition continues to worsen after several days of high-dose prednisone.'lo typical dosages range from to mg/day and last from to days. a typical dose is mg in ml of % dextrose in water over minutes every hours to a total of doses. another is mg in ml of % dextrose in water over minutes every hours for doses. an oral prednisone taper over about days to weeks may be used afterward. one study of optic neuritis suggested that high-dose methylprednisolone produces more favorable results than oral prednisone for patients with poor visual acuity. this study showed only a faster recovery time; follow-up examinations at year did not show any difference in final outcome."l the study involved patients who did not necessarily have a diagnosis of ms. however, a follow-up evaluation with patients in whom ms subsequently developed did suggest that the methylprednisolone-treated group had a longer time interval to the development of a second demyelinating event than dm,january . those who received prednisone or placebo.l for this reason, some neurologists believe that all attacks of ms should be treated with intravenous methylprednisolone. the side effects of steroids are well known. these include nonspecific immunosuppression leading to opportunistic infections, induction of hyperglycemia, fluid retention, hypertension, emotional abnormalities, hypokalemia, peptic ulcers, occasional aseptic necrosis of the femoral head or other bones, and demineralization of bone. chronic use may lead to cataracts, osteoporosis, muscle wasting, hypertension, diabetes, increased susceptibility to infections, and a cushingoid appearance. steroids should be used with caution. we have found the,following precautions helpful: administration of calcium and possibly vitamin d during the administration of steroids and restriction of foods with a high sugar or sodium content. we encourage our patients to eat foods rich in potassium, such as bananas, orange juice, and tomatoes. patients who experience indigestion may benefit from the use of histamine blockers such as ranitidine. some patients may need sedation with diazepam or other agents because of severe mood swings, anxiety, or sleeplessness. patients who receive high doses of methylprednisolone should be observed for hypertension, electrolyte imbalance, and hyperglycemia. these problems should be treated appropriately. occasional psychiatric symptoms, including depression, psychosis, and severe anxiety, may necessitate cessation of steroid therapy. betaseron, a recombinant interferon-& has been approved by the u.s. food and drug administration (fda) for use in ambulatory patients with relapsing-remitting ms. this approval followed a year, controlled, double-blind study that showed in patients treated with million units of betaseron administered subcutaneously every other day the relapse rate was reduced to . relapse per year compared with . relapses per year in patients given placebo.l an mri study performed with the same population revealed fewer new lesions in the treatment group than in the control group.lo the drug did not improve ongoing symptoms. the study was limited to patients with relapsing-remitting disease, and the findings should not be extrapolated to patients with chronic progressive disease. a study of the use of betaseron by patients with chronic progressive ms is planned. patients whose condition is stable would not benefit from the use of betaseron. there are problems with the use of betaseron. although the drug may be helpful in patients with frequent relapses, it does have seri-ous side effects. almost all patients experience local reactions at the site of injection, and some patients have had tissue necrosis at injection sites. the injection site must be changed regularly to reduce the likelihood of ulceration. many patients have a flulike reaction, which may include fever, chills, malaise, and myalgia. this reaction resolves with time and commonly lasts only a few months; however, it may last as long as a year. these symptoms can be partially controlled with acetaminophen or ibuprofen. liver function studies may show abnormalities, and leukopenia may be present. fatigue and emotional disturbances have been reported. our patients have experienced episodes of acute depression and anxiety, and one patient had an episode of uncontrollable rage. depression may necessitate temporary or permanent cessation of betaseron treatment. however, antidepressants, such as fluoxetine, sertraline, and paroxetine hydrochloride, may help counteract the depression. in a few cases, ms appears to worsen when the patient is taking betaseron. acute weakness develops in some patients with the first few injections. this is not always associated with fever and may resolve with time. menstrual irregularities have been reported, and betaseron cannot be used during pregnancy. some patients tolerate the medication better if the full dose is titrated up over approximately month. periodic blood tests to check for leukopenia and abnormal liver function are suggested. clinical trials of other preparations of interferon-a and interferon- are nearing completion. one clinical trial involved administration of a weekly intramuscular injection of interferon la. the results suggested that this drug reduces the likelihood of progression in patients with early disease. a phase iii clinical trial of another investigational agent, copolymer , has been completed. this drug appears promising in reducing relapses and has a good safety profile. j these agents will likely be available in the near future, pending fda approval. although most treatment aimed at chronic progression remains experimental, the use of intermittent intravenous methylprednisolone has become a common practice. most commonly, patients who experience subacute worsening may respond to a course of highdose solu-medrol similar to that given for a severe relapse. the condition of some patients appears to stabilize, at least temporarily, with this course of therapy. some patients with progressive disease may respond to a single dose of mg of solu-medrol in ml of % dextrose in water given over hour once a month for to months. subsequent treatments may be given every to weeks. azathioprine has been used for the treatment of chronic progres-sion with some success. studies have shown a modest benefit of azathioprine, primarily in stabilizing the condition of some patients.l j patients who take this drug should be examined for leukopenia or hepatotoxicity. about % of patients are unable to tolerate azathioprine because of fever, rash, or nausea. patients with continued progression during therapy with azathioprine or solu-medrol may benefit from combined therapy. cyclosporine was evaluated in a multicenter clinical trial and was found to have modest clinical benefit.llg the prolonged use of cyclosporine in patients with chronic progressive ms was complicated by side effects, principally nephrotoxicity and hypertension. the use of cyclophosphamide in the treatment of chronic progressive ms is controversia . - the results of clinical trials of this agent in chronic progressive ms are contradictory. the drug may have use in rapidly progressive ms that does not respond to steroid therapy. further investigation with mri and neuropsychological testing and careful clinical assessment should resolve the controversy. a number of promising phase iii clinical trials of therapeutic agents for relapsing-remitting ms are being conducted. for two of these agents, the -year placebo-controlled phase has been completed. these are an interferon-& given once a week by intramuscular injection, and copolymer . both drugs reduce the frequency of relapses and favorably influence disability. the interferon-l is identical to human interferon- and differs from betaseron in that it has the sequence of amino acids and glycosylation of human interferon.l the results of a review of the safety profile of this drug compared with that of betaseron will be of considerable interest. copolymer appears to have activity similar to that of betaseron with regard to reduction of relapses in ms.l , the side-effect profile appears to be favorable compared with that of betaseron. laboratory investigations demonstrate additive effects of copolymer and interferon-l in vitro. because the drugs theoretically act through different mechanisms, combined therapy might be possible. because of the results of a pilot study, oral myelin is being tested in a phase iii clinical trial. lz in the pilot trial, the efficacy of the drug was observed in only a subgroup of patients (dr -negative men). two pilot studies of the use of methotrexate for ms have been performed. j methotrexate in low doses is used for the treatment of rheumatoid arthritis, psoriasis, and crohn's disease. similar therapy may be of benefit to patients with advanced ms.lz a phase iii controlled trial and dose response testing will be of considerable interest. methotrexate should be used in clinical settings that allow careful neurologic and laboratory follow-up evaluation. dm, january cladribine by intravenous administration appears to alter the progression of ms. lz the drug has relatively selective toxicity for lymphocytes; however, the side effects can be substantial. additional studies to evaluate dose and route of administration are being initiated. the clinical effects of repeated dosage with this medication also require study. immunoglobulin therapy may be useful in ms; however, controlled trials of intravenous immunoglobulin (ivig) must be completed.lz this therapy may be useful in relapsing disease and can be considered for patients with both ms and diabetes. ivig therapy is not necessarily benign and can be responsible for the transmission of viral hepatitis. several clinical trials of monoclonal antibodies are in progress. a number of monoclonal antibodies with specificities for either lymphocytes or adhesion molecules are being subjected to initial trials in human beings. a monoclonal antibody that appears to lower lymphocytes and have an appreciable effect on the lesions of patients with ms as seen on mr images is being studied.lz ,%klptomtic therapy one of the most important aspects of the treatment of ms is helping patients manage their ongoing symptoms. because of the chronic nature of the problems associated with ms, medication and adjustments in lifestyle are used to help patients cope with their disabilities. table gives a summary of possible symptomatic treatments. fatigue can be disabling in patients with ms. it is described in different ways by different patients. the classic description of fatigue is increased weakness with exercise or as the day progresses. the patient may walk fairly well in the morning but need a cane or walker by afternoon. other descriptions include sudden attacks of sleepiness or excessive chronic sleepiness, even though the patient has had enough sleep at night.lzg patients who describe fatigue should be questioned closely about their sleep habits and other symptoms of depression. many patients with fatigue may have poor sleep habits or insomnia, which lead to daytime fatigue. depression is a common problem in patients with ms. if the fatigue is a product of depression, treatment of the depression should be helpful. fatigue is sometimes managed without medication. patients may respond to one or two brief ( to minutes) naps during the day. if this is not helpful or not possible, amantadine may be given to help control the problem. the mechanism of action of amantadine is not known, but it is helpful in approximately % of patients.lzg side effects, such as dizziness, headaches, nervousness, or edema, may limit the usefulness of the drug. pemoline is a cns stimulant that may be helpful in some patients. it should be used in low doses and should generally be given early in the day because it may cause insomnia. anxiety and anorexia are other problems that may occur with this drug. liver function studies should be performed periodically to monitor for hepatotoxicity. fluoxetine (prozac) may be helpful both to increase energy and to treat depression.* d&z, january vertigo vertigo can be an intractable and disabling problem. vertigo can occur in sudden spells that last a few minutes, or it can be chronic and last for hours. some physical therapy techniques involve habituation exercises to help with vertigo. medications that may be helpful include meclizine, promethazine hydrochloride, and low-dose diazepam. oscillopsia may occasionally respond to clonazepam or baclofen. vertigo with nausea and vomiting may respond to metoclopramide. spasticity can appear in many different ways. it may be seen at direct examination as a "catch" in the muscles with passive rapid movement of the limbs, or it may cause severe stiffness or rigidity. some patients may have severe spasms of the affected limb, which may be precipitated by movement or occur at night. these are most common in the lower limbs and may be either flexor or extensor spasms. the spasms can be quite painful. primary treatment of spasticity includes physical therapy with stretching exercises, combined with medication. baclofen is the most commonly used drug for spasticity, although its mechanism of action is not known. the dose of baclofen should be low when treatment begins and should be titrated slowly and carefully. patients who take an overdose of baclofen experience weakness. the dose of baclofen is extremely variable-some patients with only moderate spasticity tolerate high doses, whereas others with severe spasticity tolerate only low doses. other limiting side effects include drowsiness, confusion, and nausea. use of baclofen should not be discontinued abruptly but should be tapered over a few weeks. * diazepam in combination with baclofen may be helpful for patients with severe spasticity or those who cannot tolerate high doses of baclofen but need to control spasticity. diazepam can be used alone for spasticity, but it is not as effective as baclofen. diazepam can be particularly helpful for flexor or extensor spasms at night. dantrolene has limited value because of its hepatotoxicity and the weakness that accompanies the muscle-relaxant effect. it may be helpful in intractable cases of spasticity. the baclofen pump was developed for use in patients with intractable spasticity. this device is an intrathecal pump with a subcutaneous reservoir of baclofen that administers continuous doses of baclofen directly into the spinal canal. this method of administration can be effective. with the lower dose delivered directly to the spinal cord, patients seem to have fewer side effects than with other routes of administration. dose levels can be programmed to change throughout the day, so patients with problems that are worse during the night or another part of the day can take increased doses of the drug during those times. tizanidine is an agent used outside the united states for spasticity. it is being studied in the united states and may become available in the near future. other agents that may be useful in the treatment of spasticity include carbamazepine, phenytoin sodium, methocarbamol, and cyclobenzaprine hydrochloride. clonidine patches may be used for adjunctive therapy in patients with persisting spasms who are taking other drugs. spastic dysarthria is an uncommon symptom in ms. speech is hesitant and stuttering, and breath control is difficult. baclofen sometimes is helpful in this condition. bladder dysfunction is an extremely common problem in ms. examination of postvoid residual urine volume and urodynamic testing are extremely important in delineating the causes of bladder dysfunction. other urologic examinations, such as cystoscopy, may help eliminate mechanical problems as the cause of urinary dysfunction. consultation with a urologist skilled in the evaluation of neurologic dysfunction of the bladder is essential to the best therapeutic outcome. the most common problem is a spastic bladder. this is a small, hyperactive bladder. symptoms of this type of bladder dysfunction are urgency, increased frequency, and incontinence in which the bladder empties completely with brief warning. this condition can be treated with anticholinergic agents such as oxybutynin or propantheline . j sometimes baclofen or amitriptyline can be of use in the control of this problem (table ) . detrusor-external sphincter dyssynergia is a common problem. in this syndrome, the bladder attempts to empty, but the urethra remains closed. symptoms may be urgency and hesitancy, double voiding, and increased frequency with a feeling of incomplete emptying. anticholinergic or tricyclic agents alone may be of help with this syndrome, but more commonly a combination of anticholinergic drugs and intermittent catheterization is needed to control the problem. the patient performs self-catheterization two to four times a day. a flaccid bladder is less common than the other types of bladder dysfunction. this is an enlarged bladder that empties poorly. symptoms include hesitancy, double voiding, a feeling of incomplete emptying, and dribbling incontinence. untreated urinary retention can result in hydronephrosis. urecholine can be of use in a few patients. frequently, however, a schedule of intermittent selfcatheterization may be needed (table ) . patients with flaccid bladder or sphincter dyssynergia may have frequent urinary tract infections. acidifying agents such as hippuric acid or vitamin c may be useful in the prevention of infections. longterm administration of antibiotics should be avoided to reduce the risk z patients with severe bladder problems that are unresponsive to noninvasive therapy may require a chronic indwelling catheter or urinary diversion. these techniques may be required by patients who cannot perform intermittent self-catheterization. sexual dysfunction is common in both men and women with ms. women often report decreased sensation, lack of vaginal lubrication, difficulty achieving orgasm, or painful muscle spasms in the legs or pelvis during intercourse. men report diminished sensation and difficulty in achieving or maintaining an erection or experiencing orgasm. there is no simple answer to the sexual problems that occur with ms. a multidisciplinary approach is needed in which the physical and psychological aspects of sexual problems are considered. for women, treatment of muscle spasms with medications for spasticity may allow intercourse with less pain. techniques to increase vaginal and clitoral stimulation may help women experience orgasm. other methods of increasing arousal may be helpful. men are interviewed to determine whether there are other causes of erectile dysfunction. medications that may affect erectile function should be eliminated if possible. yohimbine, an a- -adrenergic receptor antagonist, can sometimes help restore function in a patient with borderline function . other methods, including papaverine or phentolamine injections, a vacuum erectile device, or a penile prosthesis, may be considered. inappropriate affect can be a problem in patients with ms. many patients have severe mood swings that can affect both their work and their social relationships. low-dose amitriptyline or another tricyclic antidepressant is frequently helpful in controlling mood swings. g depression is a common problem in ms. j , the suicide rate among persons with ms is estimated to be . times that of the healthy population. whether the depression is a primary symptom of ms or a situational problem is not known. physicians should be alert to the possibility of depression in their patients. full-dose antidepressant medications and psychological counseling may be beneficial. tremor can be a limiting factor in many patients with ms. treatment with medications is frequently unsuccessful. agents that may be useful include clonazepam, acetazolamide, propranolol, primidone, and diazepam. isoniazid has been reported to be helpful in some patients. we have found clonazepam to be the most helpful of these agents in our patients, but treatment may be limited by drowsiness. a common misconception is that pain is not a symptom in patients with ms. the truth is that pain is often a problem and may be a prominent concern for patients with ms. this can be a primary factor in the disease, or it can be a consequence of disability associated with the disease. much of the pain reported with ms is musculoskeletal and is related to abnormal use of muscles and joints. for example, patients who use a wheelchair may experience wrist, shoulder, or elbow pain from manipulating the wheelchair. patients with paraparesis or ataxia may experience back or leg pain from poor posture and balance when walking. these problems should be treated with antiinflammatory medications and physical therapy. primary ms pain is often dysesthetic. z the patient describes a burning sensation or perhaps even electric shock-like pain. this pain can be in any location, but it is most commonly in the lower extremities. some patients experience tic douloureux or atypical facial pain. this primary pain may be controlled with tricyclic antidepressants, phenytoin, or carbamazepine. in patients with refractory pain, valproic acid can be tried. z headaches can become a problem in patients with ms. it is not known whether these headaches are caused by ms or are a separate problem. both tension and migraine headaches are common, and treatment is similar to the treatment of headaches in patients who do not have ms. retro-orbital pain is frequently observed in patients with optic neuritis. these patients may require steroid therapy. spasticity and muscle spasms can cause severe pain. treatment of the spasticity helps the pain. many patients with ms experience cognitive abnormalities. unlike the dementia of alzheimer's disease, the cognitive deficits seem to be more scattered and tend to be retrieval deficits rather than memory loss. patients can have substantial cognitive difficulties but still have normal mini-mental state examination findings. neuropsychological studies have shown that as many as % of patients may have some cognitive difficulties. these difficulties can be important in terms of disability and ability to cope with illness. only a minority of patients have severe cognitive abnormalities. mr images in patients with cognitive problems tend to show a larger number and size of lesions in the white matter of the cerebral hemispheres. frontal lesions are more common in patients with cognitive difficulties.* the corpus callosum may be thinner than normal, as seen on sagittal images. patients with cognitive problems should undergo careful neuropsychiatric testing. sometimes depression or anxiety can be contributing factors in these symptoms. the minnesota multiphasic personality index or the beck depression scale in conjunction with cognitive testing may be helpful in differentiating emotional problems from structural cognitive deficits. proper treatment of the anxiety or depression may lead to improved cognitive function. recognition of the areas and degree of cognitive difficulty in patients with ms may be helpful in the care of the patients. patients may be able to learn ways of working around a problem. problems with a job may be related to cognitive problems, and ways of altering the job may be found. patients may become disabled from working because of these problems. this testing also may help the family understand the need for helping the patient deal with problems that have become too difficult to handle alone. cognitive rehabilitation techniques are being tested for patients with ms in some centers. further investigation is needed to evaluate the efficacy of these techniques. careful assessment of the patient's abilities and disabilities is crucial for proper management. in many patients, chronic symptoms cannot be prevented. symptomatic therapies are often effective for alleviating the afflictions produced by ms and for allowing the patients to live a productive and comfortable life. the cause of ms is unknown. theories revolve around the idea that the disease is either autoimmune or virus-mediated. it is still reasonable to question which pathologic feature is the inciting event. much research is focused on the t cell and potential mechanisms by which these cells could initiate ms. hla associations are found in many populations; however, hla markers are neither necessary nor sufficient to confer disease susceptibility, and other factors that confer disease susceptibility are being sought. at this time there is no confirmed evidence of a viral cause of ms. investigations with in situ hybridization and pcr technology are being conducted in an attempt to identify viral nucleic acids in the cns. perhaps these techniques will assist in unraveling the pathogenesis of ms. an intriguing possibility is that molecular mimicry may be re-sponsible for the initial generation of autoreactive lymphocytes. this mechanism involves exposure to viral or bacterial antigens, which generates an immunologic response that consists of reactive t-cell populations. because t cells cross-react with myelin peptides, a potential for demyelination exists. this theoretic mechanism is known to cause demyelination in rabbits. an interesting investigation of human mbp-reactive t cells demonstrates that mbp-specific t-cell clones can recognize multiple viral polypeptides presented by dr or dql mhc antigens. this would imply that ms could be generated by exposure to any one of a number of antigenic stimuli, such as influenza viruses or herpesviruses or even bacterial antigens. selected activated t-cell populations that enter the cns could then recognize a myelin epitope and initiate the autoimmune response, which would persist long after the inciting infection was cleared. recent investigation with mr spectroscopy demonstrates that white matter outside ms plaques may be abnorma . " these findings may signify that there is a fundamental abnormality in the white matter. whether these findings are secondary to genetic, biochemical, autoimmune, or viral factors remains to be determined. despite the deficiencies in our understanding of disease pathogenesis, therapy for ms has advanced. phase iii clinical trials with interferon- and copolymer have demonstrated modest but definite benefit. the mechanisms by which these drugs favorably influence the clinical course of ms remain to be elucidated. recent studies of chemotherapeutic agents suggest that control of chronic progressive disease may be a real possibility. future clinical trials will attempt to define the efficacy of and parameters for these therapies. another question that remains unanswered is whether the use of multiple-drug therapy might be beneficial in the treatment of ms. for example, combined therapy with interferon-i lb and copolymer may produce more benefit than either drug alone. in chronic progressive disease, the use of solu-medrol in combination with another immunosuppressant such as azathioprine or methotrexate also should be explored. remyelination is another topic of interest for future research. research is being conducted into the use of ivig as a remyelinating agent. in addition, oligodendrocyte transplant experiments are being conducted in canine modes and may eventually be used for human patients. research involving medications to improve the symptoms that limit the lives of many patients with ms is ongoing and should continue. -amino-pyridine and , -diamino-pyridine are being studied as agents that may improve conduction through poorly myelinated areas. these agents may reduce double vision, improve strength, and possibly reduce tremor. more research is needed to evaluate these dm,january and other compounds that may improve the quality of life of many patients with ms. although the cause of ms remains a mystery, important advances have been made in the 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vnz zzg authors: garcia, sònia title: pandemics and traditional plant-based remedies. a historical-botanical review in the era of covid date: - - journal: front plant sci doi: . /fpls. . sha: doc_id: cord_uid: vnz zzg pandemics are as old as humanity and since ancient times we have turned to plants to find solutions to health-related problems. traditional medicines based mostly on plants are still the only therapeutic possibility in many developing countries, but even in the richest ones, herbal formulation currently receives increased attention. plants are natural laboratories whose complex secondary metabolism produces a wealth of chemical compounds, leading to drug discovery – % of widespread use drugs are indeed of plant origin. their therapeutic potential is even bigger: although many plant-based compounds show inhibitory effects against a myriad of pathogens, few reach the stage of clinical trials. their mechanism of action is often unknown, yet traditional plant-based remedies have the advantage of a long-term experience in their use, usually of hundreds to thousands of years, and thus a precious experience on their safety and effects. here i am providing a non-systematic historical-botanical review of some of the most devastating pandemics that humanity has faced, with a focus on plant therapeutic uses. i will revisit the middle ages black death, in which a plant-based lotion (the four thieves vinegar) showed some effectiveness; the smallpox, a viral disease that lead to the discovery of vaccination but for which the native americans had a plant ally, an interesting carnivorous plant species; tuberculosis and the use of garlic; the spanish flu and the widespread recommendation of eating onions, among other plant-based treatments; and malaria, whose first effective treatment, quinine, came from the bark of a peruvian tree, properties already known by the quechua people. synthetic analogues of quinine such as chloroquine or hydroxychloroquine are now being revisited for the treatment of covid symptoms, as they are artemisinin and derivatives, other plant-based compounds effective against malaria. finally, i will give some hints on another facet of plants to aid us in the prevention of infectious diseases: the production of biotechnological plant-based vaccines. altogether, my aim is to stress the significant role of plants in global health (past, present and future) and the need of enhancing and protecting the botanical knowledge, from systematics to conservation, from ecology to ethnobotany. pandemics have shaped the history of mankind, and plants were usually the first available therapeutic choice. there is evidence of herbal preparations by egyptians around bc, later improved by greeks and romans, and widely documented in official drug books known as pharmacopoeias. still in our days, traditional medicines based mostly on plants are the only therapeutic possibility for many people in developing countries (akerele, ) . but, also in the first world, with wide access to the most modern drugs, the use of plant-based traditional medicine is experiencing a revival, as it is seen as safer and healthier than synthetic drugs. indeed, one advantage of traditional remedies over modern drugs is that their effects and margin of safety have been known for long. there is also a renewed scientific interest on plant-derived drug discovery, according to the current increasing publication trend on the topic (atanasov et al., ) . the rich secondary metabolism that characterises plants make them a source of compounds that may have a yet unknown therapeutic potential, only limited by the availability of resources to perform clinical trials. it is claimed that natural products (mostly from plant origin) will be the most important source of new drugs in the future (atanasov et al., ) . a recent editorial (nature plants, ) highlighted the need of funding and understanding botanical knowledge in the context of the current, and possibly future, pandemics. it is urgent to develop therapeutic tools to protect from high risk of infection (mitjà and clotet, ) and plant-based remedies with proven safety profiles could be one of the faster solutions. here i present a non-systematic review with a historical-botanical perspective on some of the most important pandemics that humanity has faced, and in some cases is still facing, and how certain plants or plantbased remedies have been used, and may continue being used, to treat these diseases, possibly including covid . the black death or black plague took place in the middle ages ( ) ( ) ( ) ( ) ( ) in eurasia, and still is the deadliest pandemic ever, with an estimated loss of million of human lives wiping out to % of european population in roughly four years (deleo and hinnebusch, ) . although this is the most know outbreak of the bacterium yersinia pestis, the much earlier -and longer-plague of justinian (from to ad) was also caused by the same pathogen (harbeck et al., ) , killing about - million people during two centuries. there have been other less spectacular, but still important plague outbreaks, arriving to the most recent ones in madagascar during the present decade. originated in china, the plague was usually spread by trade boats, whose rats carried fleas with the bacterium, which was transmitted to humans directly by the bite of the flea, and then between humans by contact or aerosol inhalation. there are several forms of the disease, the most common being the bubonic plague, which provokes the inflammation of the lymph nodes (buboes) as its most recognizable sign; a second form is the pneumonic plague which affects the respiratory system and is more deadly; the third form, the septicaemic plague, is the least common but has a mortality ca. % (byrne, ) . the antibiotic treatment, starting in early xxth century, reduced the death rate to about %- % which previously was between %- %; however, little is known on the remedies used before antibiotics were a reality and the major plague outbreaks occurred much earlier. in the middle ages, some preventive measures included, among others, carrying sweet smelling herbs to clear "the evil air" (which was believed to carry the pathogen) around the person (jones, ) , garlic for cleaning kidneys and liver, and lavender or chamomile teas to calm the stomach bile (khaytin, ) . a remedy named "the four thieves vinegar" was very popular: it consisted in several herbs, such as angelica (angelica archangelica), camphor (cinnamomum camphora), cloves (syzygium aromaticum), garlic (allium sativum), marjoram (origanum majorana), meadowsweet (filipendula ulmaria), wormwood (artemisia absinthium), and sage (salvia officinalis), brewed in vinegar (gattefosse, ) . before going out, people should apply it on hands and face for avoiding to contract the plague. some of these plants are well known flea repellents, so this may be one of the reasons for its efficacy. other herbs such as meadowsweet might have been included to release pain (as it contains salicylic acid, a precursor of aspirin) and to mask odours, a very helpful property considering that decomposing bodies were usually encounteredthe legend states that the name of the remedy might refer to thieves using it to rob the plague dead or sick (lucas, ) . another treatment coming from ancient greeks also gained popularity: the king mithridates antidote (totelin, ) , an extract of about fifty plants in a mixture with opium (papaver somniferum) paste, which if any, at least eased the pain or promoted a peaceful death. other prescriptions included lavender or rosewater baths, probably due to their antimicrobial and buboes healing properties. willow bark (another source of salicylates) was also given as a painkiller (khaytin, ) . a curious "prophylactic" plant-based remedy was recommended by the napolitan doctor angelerio during a plague outbreak in alguero in the xvi century ( - ) ( figure ): "any person going out from home must carry a cane (note: probably from the species arundo donax) six spans long, and as long as the cane is, one must not approach other people" (bianucci et al., ) . the origin of smallpox, a viral infectious disease caused by variola virus (two variants: v. major and v. minor) is unknown, but it dates back at least to the ancient egypt (third century bc), since some mummies showed smallpox-like eruption, the characteristic macules of the disease. as with the plague and other pandemics, the disease has occurred in several outbreaks around the world, the most recent in the late s. this virus has killed between and million people during the th century (koplow, ) until the global eradication campaign by the world health organisation (who) in . smallpox was the first infectious disease to have been eradicated ( ), the (only) second one being rinderpest, a viral illness of cattle. the smallpox vaccine (the first ever) was based on edward jenner's demonstration, by the end of the xviii th century, that inoculation with cowpox (a variant of the smallpox virus infecting cows) protected against the disease. actually, jenner's contribution popularized the practice of vaccination, a word coined by himself coming from the latin word vaccinus (i.e., or/from the cow) for the prevention of several other infectious diseases. however, before vaccination was discovered, how did people deal with the illness? particularly interesting was the approach of the native americans, which were deeply affected by the disease. by the end of xix th century several surgeons and practitioners related to the us army, as well as the prestigious botanist charles f. millspaugh ( ) , described the use of poultices and infusions from the indigenous medical flora based on the plant sarracenia purpurea (family sarraceniaceae) to be effective for treating smallpox, in a likely case of medical appropriation of the indigenous therapeutic knowledge (lawrence-mackey, ). known by native americans (mi'kmaq people) as mqo'oqewi'k, also named purple pitcher plant, it belongs to a genus of carnivorous species that use modified pitcher-shaped leaves to trap insects. possibly, the spotted appearance of the plant (figure ), resembling one of the main clinical signs of the disease (clarke, ) , inspired its use to the indigenous people. this may be another example of the doctrine of signatures, an ancient concept by which god somehow indicated to men what plants would be useful for, by certain signs (coles, ) , a pseudoscience which has caused more harm than good in general, although exceptions appear. compelling descriptions of their effectiveness were recorded, such as ''the greatest remedy known for the dreadful scourge'' or "'it seemed to arrest the development of the pustules, killing, as it were, the virus from within" (clarke, ) . the advent of vaccination put forward the botanical remedy, but the antiviral properties of sarracenia purpurea have been later demonstrated in vitro (arndt et al., ) . the authors showed that the plant extract was not only active against smallpox, but also against other poxviruses, papovirus sv- and various herpes viruses, including papillomavirus and epstein-barr virus-associated carcinomas, usually by inhibiting the virus replication at the level of early transcription (moore and langland, ) . another global and persistent pandemic is tuberculosis, caused by mycobacterium tuberculosis. together with smallpox, it is one of the oldest known diseases, since molecular data and archaeological evidence support that it coexisted with humans from the neolithic (gutierrez et al., ; nicklisch et al., ) . relevant figures in the history of medicine such as hippocrates or avicenna identified the disease, which involved coughing blood and fever and which was often lethal. actually, avicenna detected the infectious nature of the illness and based on tuberculosis, was probably the first to come up with the idea of quarantine to stop the spread of infectious diseases (shephard, ) . tuberculosis infected million people only in , of which . million died (unaids, ). it is found in every country, being the first infectious cause of death worldwide. from the hiv/aids outbreak, the combination of both is usually fatal where tuberculosis is endemic (mainly developing countries), as the immune weakening caused by hiv facilitates the onset of tuberculosis. this disease is indeed the final cause of death of many hiv infected people; paradoxically, while many could live now with aids they are dying from tuberculosis. the main reason is that the bacterium has developed resistance to most antibiotics, which usually have to be taken during long and tedious treatments. this is why researchers have turned to the search of effective alternatives also among medicinal plants, as some of them have already demonstrated anti-tuberculosis activity. besides, an effective plant-based treatment would be more affordable in poor countries which are those more affected by the disease. among the plants that are being investigated, garlic (allium sativum, family alliaceae), a former remedy already used to treat the plague (see above) stands out for its renowned properties, although it still far from being an alternative. the benefits of garlic (of which there are about varieties) are well-known already from the ayurvedic and unani medicine systems (raghunandana et al., ) as well as from the chinese traditional medicine, but also ancient greeks, romans and even egyptians used it to treat illnesses. it has been used as a food and folk medicine for centuries by many cultures. garlic has a variety of pharmacological virtues, including antimicrobial, anticancer, antioxidant (dini et al., ) , fungicidal and as a cure for heart diseases, among others (majewski, ) . the antitubercular and other antimicrobial activities of garlic, however, have been demonstrated in vitro but still, seldom in vivo. although garlic has more than biologically active compounds, allicin is the most relevant, albeit highly unstable; therefore, depending on the preparation of the garlic-based remedy the efficacy may not be as high as expected (majewski, ) . the wide antimicrobial and even antifungal spectrum of allicin is explained by its inhibitory effects on sulfhydryl metabolic enzymes. by interacting with these enzymes, allicin induces thiol stress in bacteria, which, among others, inhibits the growth of the microorganisms (müller et al., ) . malaria, caused by protozoan species of the plasmodium group, is an infectious disease coming from the bite of a mosquito, usually an infected anopheles sp. female. the current name of the illness was given by the italians around xix th century, as a contraction of the words "mal aria" (i.e., bad air) from the belief that the disease was transmitted by the "miasma" coming from marshes (macip, ) . the most typical symptom is fever, together with nausea and vomiting, tiredness, headache, occasionally yellow skin and in severe cases it can lead to seizures, coma and death. it is another ancient disease, spanning from the neolithic to our days, and currently found in all intertropical continents. recent studies detected the parasite in african monkeys, probably being the source of the disease, although it is still debated how it spread worldwide (molina-cruz and barillas-mury, ) . it is known that malaria arrived to europe by the first century ad, probably coming from the african rainforests and travelling by the nile to the mediterranean, where it spread to the middle east and from there to greece, italyhistorians hypothesize on the triggering role of malaria in the fall of the roman empire -and the rest of europe, even as far as england and denmark (karlen, ) . between the xvi th -xix th centuries the disease crossed the atlantic ocean probably on slave ships to reach the american continent (yalcindag et al., ) . it was suffered by presidents of north america such as g. washington or a. lincoln and it raged specially with native americans, taking thousands of their lives. the centers for disease control and prevention, the leading national public health institute of the united states, was founded because of malaria in . in the last century, probably - million people have died from the disease, accounting for %- % of deaths (carter and mendis, ) . at present malaria is mostworrying in sub-saharan africa, accounting for ca. % of current cases, although there is also a resurge in southern asia (arrow, ) . the most well-known and one of the most effective historical treatments against malaria is quinine, an alkaloid extracted from the bark of the cinchona tree (cinchona officinalis) belonging to family rubiaceae, the same of coffee. it is original from peru (where it is the national tree, although currently it is considered an endangered species) and the quechua traditionally used the ground bark of these trees to stop shivering because of cold, not for malaria treatment per se. most likely, spanish jesuits missionaries brought cinchona to europe for the first time, having observed how the quechuas used it to threat shivering, by the end of xvi th century -a second case of medical appropriation in this story. the tree was named (by c. linnaeus) after the spanish countess of chinchon, who was treated with its bark in peru back in the early xvi th -linneaus misspelled the name of the countess, omitting the first "h" in the name (meshnick, ) . it is also said that the countess may have introduced the curative bark to europe when she returned to spain, but it is currently considered that this a legend rather than what actually happened (haggis, ) . quinine is effective on the "cessation of febrile paroxysms" (stephens et al., ) , one of the main symptoms of malaria, and which has given its popular name to the species (fever tree). malaria outbreaks, however, continued to appear during centuries with no alternative to quinine. during world war i the german army was strongly affected by the parasite in the troops of east africa, as the allies controlled java, the main worldwide quinine producer. in an interesting historical moment that impelled science, the german government commissioned a search for a substitute to quinine, determined not to suffer again from its shortage. in chloroquine, a synthetic compound similar to quinine was synthesized at the bayer laboratories. much later ( ) another very similar derivative, hydroxychloroquine, was produced in the us. both chloroquine and hydroxychloroquine are used to prevent and treat malaria, being some of the antimalarial drugs of choice in areas where the disease is not resistant to them (a recurrent problem in this disease); they were preferred over quinine because of much less severe adverse effects, although at present there are many other even safer alternative drugs for the treatment of malaria. in recent years, these antimalarials have shown several immunomodulatory effects and they currently treat, mostly, diseases such as lupus erythematosus or rheumatoid arthritis. as with tuberculosis, the parasite tended to develop resistance to these treatments sooner or later, and researchers were urged to look for alternatives. a very popular one was found in the plant artemisia annua (sweet wormwood, from family asteraceae) (figure ) a remedy known in chinese traditional medicine as qing-hao for more than years (klayman, ) . chinese herbalists had been using it for treating haemorrhoids, chills, and fevers (trigg and kondrachine, ) . the species, as other members of genus artemisia such as absinthe or tarragon, is aromatic and bitter. one of the compounds responsible for its bitterness, a sesquiterpene lactone extracted from the glandular trichomes named artemisinin, is the active compound against malaria. the discovery of artemisinin is also very remarkable from the historical point of view. in , during the chinese cultural revolution under mao zedong's mandate, the secret "project " was a plant screening research program to find an alternative treatment for malaria, which was ravaging vietnamese army during vietnam war. in , dr. youyou tu a researcher of that program, isolated artemisinin, "rediscovering" the ancient remedy qing-hao. the drug started to be used in , a relatively short-period to establish a new medicine in the market, but it was also based on thousands of years of experience by the chinese traditional practitioners. nowadays artemisinin and its synthetic derivatives are one of the main defences against drug-resistant malaria in the asiatic southeast. however, who recommends it in combined therapy with other drugs, in part to avoid the development of resistance and in part to counteract the short half-life of artemisinin in plasma, leading to the artemisinin combination therapies (acts) which include companion drugs such as some cloroquine derivatives (e.g., mefloquine). in tu was awarded the nobel prize for the discovery of artemisinin, which represents an important contribution of china to the global health, as well as the first and awaited nobel prize in the sciences for china. it is considered the most significant milestone of tropical medicine of the last century, contributing to a better health and saving tens of thousands of lives every year in tropical developing countries of south asia, south america and africa. in the spring of started one of the deadliest pandemics in recent history, caused by one aggressive strain of the h n influenza virus (the same virus that caused the swine flu pandemic). it was popularly known as the spanish flu because in the context of world war i censorship minimized the effects of the pandemic to keep people's morale in the countries involved in the conflict, while in the neutral spain newspapers were free to report its effects, giving the impression that this country had been particularly devastated by the disease. during months, until summer , there were three waves of the pandemic, being the second the worst. a fourth, much fainter wave, took place in the spring of and after this one, the virus disappeared as it had arrived. it infected about half a billion people (ca. / of the world's population) and killed about million (with some estimates as high as million); for a reference world war i estimates range from to million deaths. the origin of the virus is unclear but it is thought that it started as a zoonosis from birds to humans which later was transmitted from humans to swine. the symptoms were an amplified version of those of normal flu, but typically deaths were caused by complications derived from a secondary pneumonia. contrary to other h n flu strains, this one was unusually lethal among young people, and almost one century later its high-virulence is only partly understood (tumpey et al., ) . since at that time there was neither vaccine, nor antibiotics to treat secondary pneumonia, the main prophylactic options were, as with the covid pandemic, to avoid contact through lockdown and quarantines, to increase personal hygiene and to use disinfectants widely. also, people turned to folk remedies and some recommendations got popular, such as the widespread advice "eat more onions!" (figure ) (arnold, ) . as with garlic, onion (allium cepa) has certain compounds (particularly a polyphenol named quercetin) which have demonstrated antiviral properties (lee et al., ; sharma, ) but still more research and clinical trials are needed. besides, in the usa a group of doctors known as "the eclectics" got positive results by treating the flu symptoms with plant remedies, together with other measures that included exercise. they reported a fatality rate ca. . % for their patients while the average in that pandemic was ca. % (abascal, ) . by selecting the herbs to match the symptoms, they used a wide variety of species. the most remarkable among them, and that have later proved therapeutic, were: gelsemium sempervirens (known as yellow yasmine, with antipyretic properties), eupatorium perfoliatum (boneset, already known by native americans to treat cold-related symptoms), actea racemosa (black cohosh, also used by native americans as a painkiller, probably due to the content in salicylic acids of its roots) and asclepias tuberosa (pleurisy root, used to treat respiratory problems and with expectorating properties) (abascal, ) . however, despite their long history of use, again there is little applied research on these plants. currently, flu is partly under control by the release of annual vaccination campaigns with newly synthesized vaccines that collect most of the virus' seasonal variability. in the latter most important flu pandemic ( ) besides the vaccine, oseltamivir (tamiflu ® ) a drug derived from the species ilicium verum (star anise, from family schisandraceae) was also crucial to treat most severe cases, although the production of this compound is limited by the low productivity of the tree, and synthetic derivatives are being developed (macip, ) . finding adequate treatments for flu is still and urgent task as the fear of a pandemic similar to the one in is a still a sword of damocles in the concerns of most epidemiologists. from the s, the science of "molecular farming" gives another potential role to plants on the prevention of infectious diseases, involving plants or plant cell cultures to produce recombinant proteins (rybicki, ) . the first steps of this approach were the "manufacturing" of the human growth hormone, monoclonal antibodies or human serum albumin in transgenic tobacco or sunflower plants ( barta et al., ; hiatt et al., ; sijmons et al., ) . other recombinant proteins more recently produced in plants -shifted from bacterial, mammalian or fungal cell to plants and plant cell cultures-and commercialized, include human type collagen i manufactured in tobacco, bovine trypsin in maize or human lysozyme and lactoferrin in rice (yang et al., ; hennegan et al., ; shoseyov et al., ; takeyama et al., ) . as with the mentioned proteins, the vaccine production would follow similar steps: isolation of a specific antigen protein, the one that triggers an immune response from the virus; the gene(s) encoding that protein is transferred to bacteria and these bacteria are used to infect plants, so the plant will in turn produce the antigen proteinthe vaccine. plants would provide a flexible, cheap and easily scalable method to manufacture vaccines. they would also be safer than traditional vaccines, because of the absence of pathogens of animal origin. plant-based vaccines for humans are not yet in the market, although some candidates have entered clinical trials (rosales-mendoza et al., ) . it is likely, however, that they will start being approved in the mid-term, at least in the cases of ebola or rabies, or in a longer term for seasonal influenza (rybicki, ) . the outbreak of covid caused by the coronavirus sars-cov- , originated in china (province of wuhan) in december and has caused , , infections and , deaths worldwide (updated th june ). even earlier than the pandemic status was declared by who ( th march ) researchers across the world engaged in hundreds of clinical trials, in an unprecedented quest for a cure to the disease, vaccine, drug or both. given the long time-frames that usually imply finding a good candidate, many research groups have turned to repurpose other drugs. the reasons are that the effects (including adverse or side effects) of these drugs are well known and have been used in broad population groups with different ages and idiosyncrasies, so the security margin is increased, allowing to save precious time in long trials. in this regard, antimalarials are potential candidates (schlagenhauf et al., ) and both chloroquine but particularly hydroxychloroquine (as explained above, synthetic derivatives of quinine, the antimalarial alkaloid coming from the bark of the fever tree) are being studied to fight covid , although it is perhaps too soon to draw conclusions on their efficacy. several studies have reported their utility for some patients and some national guidelines have recommended both drugs for treatment of covid (see colson et al., and singh et al., ) despite there has been certain controversy. the who halted studies on these drugs by the end of may prompted by an observational study reporting that hydroxychloroquine produced a higher mortality rate in hospitalised patients, but the study was soon retracted on the basis of questionable veracity of data and analysis (mehra et al., ) and trials on the drug have been resumed shortly after. the effectiveness of hydroxycloroquine taken at initial stages of the disease was recently tested in a multicentre randomised controlled trial based on previous experiences of post exposure prophylaxis (pep) drugs to prevent infections (mitjà and clotet, ) , but no benefit was observed beyond the usual care . other plant-based antimalarials, artemisinin and derivatives, are also being tested against sars-cov- , again not without controversy. in many african countries, an elixir based on artemisia annua extract, "covid-organics" is being distributed as a cure against covid . however, there is little scientific evidence of the effectiveness of such elixir and its extended consumption can have associated problems, the most important the development of resistance to the drug by the malaria parasites in a continent particularly sensitive to the disease. nevertheless, there is evidence that the extract of artemisia annua has antiviral properties, being active against sars-cov- (li et al., ) , herpes simplex (karamoddini et al., ) , hepatitis a (seo et al., ) , hepatitis b, bovine viral diarrhoea, and epstein-barr (haq et al., ) . this has stimulated the research of the potential use of artemisinin and derivatives (such as artesunate) to treat covid , which is now being conducted by several biotech companies (e.g., mateon therapeutics, artemilife) as well as by public research institutions (e.g., the liverpool school of tropical medicine, the max planck institute of colloids and interfaces). traditional chinese medicine has also had a say in the cure of covid : the national administration of traditional chinese medicine (natcm) organized a study in late january to identify potential treatments, and the lung cleansing and detoxifying decoction (lcdd) was widely used and studied through clinical trials; its apparently high effectiveness made that the natcm officially recommended lcdd as a treatment for covid (weng, ) . among the lcdd ingredients, there were species such as ephedra sinica (well known as decongestant and bronchodilator through the active compound ephedrine), atractylodes macrocephala (showing antiviral activity against influenza viruses in experimental assays) or scutellaria baicalensis (containing anti-inflammatory flavonoids), and the combination of these effects and others from the remaining ingredients likely counteracts covid by their synergistic activities. however, as weng ( ) points out, it is difficult to transfer the success of the lcdd treatment to other countries, both because the cultural acceptance of tcm is not present outsides china, and due to the lack of knowledge on the precise chemical composition and mechanism of action, which are required in modern therapy. once the first vaccine for covid is finally developed (with estimates ranging from september to several years) another plant may also play an important role in order to produce it in large amounts: nicotiana benthamiana, a close relative to tobacco. this species is the focus of the eu project newcotiana, coordinated by researcher dr. diego orzaéz at the csic, the leading public research body in spain. in this project, genome editing practises (e.g., crispr) will be used to transform the plant in a biofactory for the large-scale production of the vaccine once it is available. moreover, one company may be currently developing a covid vaccine based on the expression of a sars-cov- protein in tobacco -kentuchy bioprocessing, a biotechnological branch of british american tobacco (capell et al., ) . as rosales-mendoza et al. ( ) stated in a recent review, the production of a plant-based vaccine in the context of the current pandemic would have the advantages of low cost, fast and escalable production, easy administration, and safety. beyond plant-based vaccines, molecular farming through plants, usually by transient expression of target proteins, can also be deployed to produce diagnostic reagents, as well as antibodies and antiviral proteins for therapeutic use. the italian biotechnology company diamante is generating antigens, to use in elisa tests (serological), based on a sars-cov- protein also in tobacco plants (capell et al., ) . another eu consortium, pharma-factory is also developing plant-based platforms to produce medical, veterinary and diagnostic products, for dealing with covid and also other diseases. i hope that the reader finds this review useful to call for the important role that plants have played and still play in human health. as schlagenhauf et al. ( ) commented, plant-based remedies are, more than an "alternative medicine", the organisms to which we owe some of the most useful therapeutic tools. still in the era of wide implementation of (synthetic) drug treatments, we turn to plants in many cases when resistances appear, as shown. paradoxically, there is a human tendency to ignore plants, a form of cognitive bias known as "plant blindness" (allen, ) that should be opposed, perhaps by enhancing and implementing more widely the botanical education. in this context, it is also essential not only to maintain but to increase societal funding into basic sciences such as botany, as well as to foster collaboration between scientists from different disciplines, whose interaction may open new therapeutic possibilities. finally, i would like that this review serves as a little recognition to the usually ignored ethnobotanical traditional knowledge of many indigenous peoples across the world of which the so-called western culture has in most occasions, illegitimately appropriated. the author confirms being the sole contributor of this work and has approved it for publication. i am thankful to roi rodriguez (institut botànic de barcelona) who kindly revised this paper and contributed interesting observations, as well as to the reviewer. herbs and influenza: how herbs used in the flu pandemic can be effective in the next nature's medicinal bounty: don't throw it away plant blindness in vitro characterization of a nineteenth-century therapy for smallpox pandemic : the story of the deadliest influenza in history the locus of decisions about aids/hiv, malaria treatment: what does welfare economics say? a question discovery and resupply of pharmacologically active plantderived natural products: a review the expression of a nopaline synthase -human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue quinto tiberio angelerio and new measures for controlling plague in th-century alghero the black death potential applications of plant biotechnology against sars-cov- evolutionary and historical aspects of the burden of malaria dictionary of practical materia medica in three volumes adam in eden, or nature's paradise chloroquine and hydroxychloroquine as available weapons to fight covid- a plague upon the phagocytes the potential role of garlic (allium sativum) against the multi-drug resistant tuberculosis pandemic: a review aromatheŕapie -les huiles essentielles hormones veǵetales ancient origin and gene mosaicism of the progenitor of mycobacterium tuberculosis fundamental errors in the early history of cinchona artemisia annua: trials are needed for covid- yersinia pestis dna from skeletal remains from the th century ad reveals insights into justinianic plague improvement of human lysozyme expression in transgenic rice grain by combining wheat (triticum aestivum) puroindoline b and rice (oryza sativa) gt promoters and signal peptides production of antibodies in transgenic plants the new cambridge medieval history: c. -c. antiviral activities of aerial subsets of artemisia species against herpes simplex virus type (hsv ) in vitro man and microbes: disease and plagues in history and modern times arcgis storymaps. herbal medicine in the black plague qinghaosu (artemisinin): an antimalarial drug from china smallpox: the fight to eradicate a global scourge medical appropriation in the 'red'atlantic: translating a mi'kmaq smallpox cure in the mid-nineteenth century anti-influenza a virus effects of fructan from welsh onion (allium fistulosum l.) identification of natural compounds with antiviral activities against sarsassociated coronavirus nature's medicines; 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studies on allicin, the antibacterial principle of allium sativum (garlic) what does plant-based vaccine technology offer to the fight against covid- ? vaccines plant-based vaccines against viruses repurposing antimalarials and other drugs for covid- antiviral activity of herbal extracts against the hepatitis a virus efficacy of garlic and onion against virus the middle-ages: monasteries, medical schools and the dawn of state health care," in an illustrated history of health and fitness, from pre-history to our post-modern world human collagen produced in plants: more than just another molecule production of correctly processed human serum albumin in transgenic plants chloroquine or hydroxychloroquine for prevention and treatment of covid- syst. rev. , cd studies in the treatment of malaria: v. intramuscular injections of quinine alkaloid in simple tertian malaria plant-based vaccines for animals and humans: recent advances in technology and clinical trials mithradates' antidote-a pharmacological ghost commentary: malaria control in the s characterization of the reconstructed spanish influenza pandemic virus tuberculosis-good progress, but not enough plant solutions for the covid- pandemic and beyond: historical reflections and future perspectives multiple independent introductions of plasmodium falciparum in south america expression and localization of human lysozyme in the endosperm of transgenic rice key: cord- - ie c f authors: heimer, carol a. title: the uses of disorder in negotiated information orders: information leveraging and changing norms in global public health governance date: - - journal: br j sociol doi: . / - . sha: doc_id: cord_uid: ie c f the sars epidemic that broke out in late in china’s guangdong province highlighted the difficulties of reliance on state‐provided information when states have incentives to conceal discrediting information about public health threats. using sars and the international health regulations (ihr) as a starting point, this article examines negotiated information orders in global public health governance and the irregularities in the supply of data that underlie them. negotiated information orders within and among the organizations in a field (here, e.g., the world health organization, member states, government agencies, and international non‐governmental organizations) spell out relationships among different categories of knowledge and non‐knowledge – what is known, acknowledged to be known, and available for use in decision making versus what might be known but cannot be acknowledged or officially used. through information leveraging, technically sufficient information then becomes socially sufficient information. thus it is especially information initially categorized as non‐knowledge – including suppressed data, rumour, unverified evidence, and unofficial information – that creates pressure for the renegotiation of information orders. the argument and evidence of the article also address broader issues about how international law and global norms are realigned, how global norms change, and how social groups manage risk. to mean well-defined ignorance (gross and mcgoey : ) or a type of knowledge about the unknown (gross : ) . to be sure, influential research has considered such important matters as the distinctions between levels and kinds of ignorance, uncertainty, and risk, strategies for handling asymmetric information, and procedures for coping with dishonesty and duplicity (see, e.g., ackerlof ; arrow ; cook ; ericson and doyle ; goffman ; granovetter ; heimer b; knight knight [ ; shapiro ; williamson ) . what is missing, though, is a thorough incorporation of various forms of ignorance, such as non-knowledge, into existing theories of how people, groups, and organizations seek, assign meaning to, and use information (gross and mcgoey ; heimer ; mcgoey ) . researchers need to consider how exactly non-knowledge fits into the negotiated information orders that anchor organizational and interorganizational action. using sars as an example, this article examines negotiated information orders in global public health governance and the irregularities in the supply of the data that underlie them. information may be in short supply because it is suppressed, and it may also be of uncertain quality because it is incomplete or purposefully misleading. in effect, the sars case suggests, whether information is acquired from legitimate sources shapes not only the nature and quality of the information itself but also the uses to which it can be put. in addition to seeking information, then, actors strategically seek information from particular sources and deploy the information they have in hand to pressure others to augment or confirm existing information. through information leveraging, technically sufficient information becomes socially sufficient information. in this way, the article shows what a negotiated information order might look like when we more fully incorporate the social uses of non-knowledge and other forms of ignorance into our analysis. in particular, the article suggests that it is especially information categorized initially as non-knowledge -including suppressed data, rumour, unverified evidence, and unofficial information -that creates pressure for the renegotiation of information orders. although it is a truism that information is needed before rational decisions can be made, the importance of information for organizational decision making is often overestimated. since the pioneering work of herbert simon (march and simon ; simon ) , organization theorists have understood that the model of rational decision making was a poor description of reality and did not capture how information is actually used by organizations. because of limited cognitive and computational capacities, theorists suggest, organizations are only boundedly rational, accepting satisfactory solutions rather than continuing decision-making processes until they find optimal ones. besides using less information than might be expected, organizations also use it on a different timetable and for different purposes. for instance, information intensive solutions often are produced somewhat independently of the problems with which they are eventually matched (feldman ) . employing the metaphor of a garbage can, other scholars suggest that organizational decision making is not linear, but instead depends on how the semi-autonomous streams of choice points, problems, solutions, and participants come together (cohen et al. ; heimer and stinchcombe ) . moreover, information has symbolic as well as instrumental uses, often serving to legitimate decisions even when it plays little role in identifying problems and selecting or crafting solutions (feldman and march ) . decision making is a quintessentially social matter. decisions may depend less on whether decision makers have enough high-quality information than on whether they agree that the available information meets a variety of normatively or even legally established criteria. that is, whether or not information is technically sufficient, it must also be socially sufficient to be usable in decision making (heimer a) . information is technically sufficient if it can be used to answer key questions confronting an organization and if it can be used, perhaps with some modification, in an organization's decision-making algorithms. if decision makers cannot cite data of the sort conventionally used or recognized by their organizational field as sufficient for decision making, their decisions may be subject to challenge. a negotiated information order emerges when consensus is reached within or between organizations in a field regarding the criteria for socially sufficient information -about the type of information usable in decision making, the priority given to different types of information, and allocation of responsibility for gathering and interpreting that information (heimer a: ) . as these conceptual distinctions suggest, the symbolic nature of information penetrates even more deeply into organizational decision making than previous research might lead us to expect (feldman and march ; meyer and rowan ) . in particular, such symbolic considerations shape assessments of both decision-making processes and the information on which they are based. to work its symbolic magic, information must be seen as legitimate, and organizational actors will spar over whose data passes that test. but, crucially, such tests are layered. socially sufficient information is thus information that is widely agreed to be adequate to its intended purposes. technically sufficient information is more contested, with some actors touting its virtues and others casting doubt. technical sufficiency can therefore be a way-station along the path to social sufficiency or an intermediate category that permits some uses of information while prohibiting others. although participants experience these discussions as realist, social scientists would be quick to point out the deeply constructionist character of claims about the quality and veracity of information. the lines dividing categories of information are necessarily fluid, with discoveries shoring up some claims while undermining others and regularly adding to the stores of both knowledge and ignorance. as we will see, it is especially the boundary between knowledge and non-knowledge where contests are focused, because crossing that boundary makes otherwise prohibited actions possible. transposed into an organizational register, the dividing line between knowledge and non-knowledge takes the form of a distinction between technically and socially sufficient information. to say that the acceptability of information depends on a negotiated information order says only that the meaning of information is not given a priori but must be worked out collectively. norms about the sufficiency of information may be grounded in rules or laws. or they may reflect a broad, but informal consensus. what consensus is ultimately reached will depend on such factors as power differences, inter-organizational dependencies, and pre-existing loyalties. the preferences of powerful actors who have a vested interest in perpetuating practices associated with traditional types of information may have an outsized influence on the norms that emerge. previous agreements about the acceptability of various kinds and quantities of information provide important starting points, but will be less influential when decision makers face situations that seem unprecedented. thus negotiated information orders can be destabilized by modifications in technology, by the arrival of new problems or opportunities, or by changes in relationships among parties. three examples illustrate the importance of negotiated information orders in assigning meaning and determining how information is interpreted and used. clarke's mission improbable ( ) shows how information orders negotiated by powerful actors can exclude other voices that might challenge the meaning assigned to information. analysing organizations' plans to avert, control or cope with disasters, clarke considered the 'fantasy plans' created to clean up oil spills in open waters, evacuate long island in the event of a nuclear power plant accident, and protect the population during and after a nuclear war. in each case, rather than frankly acknowledging the impossibility of averting, controlling or mitigating disaster, key actors developed elaborate analogies and conducted careful simulations to convince themselves and others of the truth of essentially untenable propositions. the problem, of course, is that such analogies and simulations rarely worknuclear meltdown is not much like an ice storm, a major oil spill in open waters cannot be simulated by scooping up oranges from calm seas, and the evacuation of long island because of a nuclear accident cannot in good conscience be equated with the flow of people during rush hour. but when discussion is confined to a circle of experts, others may be unable to point out the obvious. here, the negotiated information order precluded consideration of information that other parties could have introduced by dismissing suppressed perspectives as unusable non-knowledge. with sars, as we will see, the first impulse of health workers, scientists, and policy makers was also to assume that they were seeing a variant of something they had encountered in the past -an atypical pneumonia or a disease caused by chlamydia (normile ; who a) . in sars, as in clarke's cases, suppressing information allowed actors at least temporarily to move forward with existing routines. even when divergent views are not completely suppressed, the context in which information is considered can shape conclusions. in last best gifts, healy ( ) asks how the major organizations supplying blood and blood products to american patients responded to early evidence that hiv could be transmitted through their products. in the american blood industry, a nonprofit whole-blood sector (the blood banks), reliant on donors, coexists with a for-profit plasma industry (the plasma fractionators) that purchases plasma from suppliers. between and , when no one was sure whether hiv could be transmitted through tainted blood and blood products, the us centers for disease control (cdc) presented their accumulating evidence and made recommendations about how to keep blood supplies safe (healy : - ) . representatives of blood banks and plasma fractionators received identical information, often at the same meetings. interestingly, though, these two sectors interpreted the information differently and adopted divergent strategies. blood banks, dependent on donors, saw blood borne transmission as 'still unproven' (healy : ) and were unwilling to ask intrusive questions about donor lifestyles and sexual practices. in contrast, plasma fractionators, working in a competitive market that made them more dependent on consumers than suppliers, adopted a policy of questioning potential donors and excluding putatively high-risk groups from their supplier pools. in short, the negotiated information order of the plasma fractionators led them to see the early information about hiv transmission as knowledge to be acted on, while blood banks' information order constructed the same information as non-knowledge to be ignored. with sars, people in different social contexts not only interpreted the data differently but also concluded that the data implied different things about their obligations under the ostensibly clear rules of the ihr. the final example shows how attempts to solve a new problem, not easily managed within the constraints imposed by an existing information order, can lead to modifications in that information order and changes in power relations in the field. contrasting the insurance of mobile rigs used for exploration and drilling with the insurance of fixed platforms used later for the production of oil in the norwegian north sea, heimer ( a) shows how norwegian insurers gradually altered the negotiated information order dominated by powerful british marine insurers. during the crucial early period of the exploration and development of the oil fields, insurers lacked the experience-based information needed for rating and underwriting, making them dependent on the reinsurance offered by british insurers. because multiple companies had to cooperate to assemble these insurance contracts with their uncertain risks and astronomical face values, insurers had to agree about what information was acceptable for rating and underwriting. british insurers stubbornly insisted on using conventional types of information. some types of information that, from a norwegian perspective, addressed key uncertainties thus could not be used simply because they had not been used in the past; social sufficiency dominated (alleged) technical sufficiency because of the requirement for consensus. because the total insurance capacity was insufficient to adequately insure the north sea oil fields, norwegian insurers were strongly motivated to create new routines for collecting and analysing data. they worked around and then modified conventions about what information could be used for ratemaking and underwriting. and gradually the situation changed. for mobile rigs, experiencebased data slowly became available and pooling risks over time and over similar units became increasingly feasible. this in turn further decreased dependence on the british reinsurance market and enabled norwegian insurers to be more flexible about what data to use and how to construct the policies. in contrast, little changed in the insurance arrangements for fixed installations, which were more expensive, less uniform, less numerous, and introduced later in the development of the oil fields. in the sars case as well, as we will see, pressure to change the rules became acute when new sources of helpful information became available but could not be efficiently exploited unless rules and norms were modified. as these examples demonstrate, organizations' information use is strongly shaped by social conventions. negotiated information orders spell out the relationships among different categories of knowledge and non-knowledge -what is known, acknowledged to be known, supplied by official sources, categorized as socially sufficient and therefore available for use versus what might be known, with varying degrees of uncertainty, but cannot be acknowledged or officially used. non-knowledge, which often comes from unofficial, back-channel sources, may be disregarded because it seems dangerous, threatening, harmful, or simply uncertain; ignored because decision makers are reluctant to bear the costs of retooling to collect, evaluate, and use new forms of information; or discarded because of the symbolic importance attached to information from official sources and the rights accorded to those who possess such high-value information. negotiated information orders thus introduce a modicum of stability in information use for some period of time until a new opportunity or danger arises. when that occurs, key actors pointing to the strategic value of certain information may successfully advocate for the reclassification of some non-knowledge as usable and technically sufficient and for the renegotiation of the information order. the arrival of a new disease -hiv, for the negotiated information order of the american blood industry; sars, for the ihr, the corresponding information order of global public health governance -can bring into sharp relief the irrationalities of established understandings about the reliability of information and the appropriate ways of using it. this article draws on a case study of sars to demonstrate how the deficiencies of the existing information order, institutionalized in the ihr, became painfully apparent in the wake of the epidemic. recognizing the substantial contributions that unofficial, previously illegitimate sources of knowledge could make in the fight against deadly infectious disease in turn helped to solidify the consensus around ihr reform efforts already underway in - when the sars epidemic occurred. in preparing this article, i have drawn especially on primary who documentation about the ihr and sars epidemic, supplemented by reports and commentaries from governmental bodies (e.g., the cdc and us congress) and non-governmental organizations and policy institutes (e.g., the national academy of medicine and chatham house). i have also drawn extensively on existing journalistic and scholarly accounts chronicling and analysing various features of the epidemic and scholarly articles and books investigating the epidemic's legal ramifications and the revision of the ihr. these documents were drawn from a larger body of primary and secondary materials collected primarily in - for a larger project examining the relationship between law and globalization in healthcare more generally. the article contends that in this case, a strong argument about technical sufficiency ultimately led to a new rule system that recategorized such non-knowledge as socially sufficient, legitimate, usable knowledge. international disease surveillance and global health governance have a long history before the sars epidemic. this history includes a century of international sanitary conferences to standardize quarantine regulations to prevent the spread of cholera, yellow fever and plague (and, previously, relapsing fever, typhus and smallpox); the crafting and revision of the international sanitary regulations (first written in ); and the formation of a series of international organizations to oversee disease surveillance and international public health, culminating with the creation of the world health organization, whose member states formally adopted the international sanitary regulations in . revised and renamed the international health regulations (ihr) in , these rules were in turn replaced by the revision, which went into effect in (fidler ; fidler and gostin ; gostin : - ; scales : - ) . a key issue in these agreements has been the collection and publication of information about disease outbreaks, with careful rules about who has to report and to whom, what they must report about, and what information they must transmit -in short, a negotiated information order that became more fully institutionalized over time. only with transparency, the argument went, was there any hope of protecting public health and curbing the spread of disease. yet, as the history of disease surveillance makes clear, because nations also worry about threats to trade, tourism and national reputation, they often strategize about what to reveal and on what timetable, hoping that diseases can be brought under control before discrediting information damages the economy or spoils the national reputation. the objective of the international conferences, conventions, and the ihr has been to induce more timely and more complete sharing of information, previously narrowly focused on reporting on a few infectious diseases and now more expansively redefined to include both infectious diseases and a wide variety of other threats to public health, by recognizing and working with this tradeoff. under the ihr (who ) , security against the spread of disease was to be achieved by requiring member states to notify the who of disease outbreaks within their borders (part ii, notifications and epidemiological information, articles - ) and maintain public health capabilities at ports and airports to monitor and reduce cross-border transmission of disease (part iii, health organization, articles - ). minimization of interference with trade and travel was to be achieved by specifying the range of responses states would be permitted or required to take in response to public health threats (part iv, health measures and procedures, articles - ). in effect, commitments to report outbreaks were traded for promises that responses to such information would be moderate, reasonable and scientifically grounded. even with this exchange in place, though, the record of compliance has been poor, and poor on both counts (carvalho and zacher ; fidler : ; kamradt-scott : - ; scales ; woodall ) . countries frequently failed to report disease outbreaks, but they also imposed overly restrictive protective measures, including quarantines and outdated vaccination requirements, that violated the spirit and the letter of the ihr rules on trade and travel. diseases continued to spread across borders whether or not travellers and goods were impounded, quarantined or otherwise delayed. although neither the who nor the member states seemed very committed to it, as the governing information order, the ihr continued to be consequential in shaping the circulation of information, categorizing information as actionable or not, and providing an excuse for states to shirk or evade pressures to report even as new transparency norms were emerging. the ihr's history suggests that this information order is primarily organized around concerns with trade and travel and has favoured the interests of rich countries (chorev b; fidler ; kamradt-scott ) . similar patterns of favoring the interests of rich countries in global health governance have been noted by other scholars (king ; erikson ; but see wenham on recent changes in emphasis). 'the rising commercial costs imposed by a system of uncoordinated, unregulated national quarantine practices meant that trade rather than health drove the development of international governance on infectious diseases', concludes fidler ( : ) . quite emblematically, the treaty and its predecessors focused only on diseases that seemed likely to be spread by trade and travel, and particularly those that might move from poor to rich countries. infectious diseases that plagued only poor countries, such as polio, were not listed, and south-south contagion was a secondary concern. adjustments were unidirectional: diseases were removed from the list, but re-emerging or new diseases were not added; no adjustments were made to take account of changes in modes and speed of transportation. the official rules of the ihr in some senses imagined a static information order in which states interacted with the who -what fidler ( ) describes as a westphalian system. yet the information order has evolved over time in important ways, with the official information order often out of step with informal practices. two key drivers of change have been innovations in information technologies, which vastly increased the amount of information available while simultaneously reducing state control of information, and the creation of new types of actors in the loosely organized global public health system. as reporting rules were first being developed, it was diplomats who certified that a ship's last port of call was disease free, allowing ships to avoid quarantine as they entered ports to offload cargo and passengers (fidler : ) . although diplomats no longer verify bills of health, the treaty's reliance on national reporters remains a core element of the reporting framework even though the new categories of actors (ngos, ingos, international health workers, laboratory workers, scientists, etc.) have access to much relevant information. thus an evolving information order peopled with these new actors co-existed with a static legal framework that only recently acknowledged and incorporated them. this meant that the who was unable to act even when it possessed information that it believed to be technically sufficient. because it was bound by a strictly formalized set of rules (few things are more rigid than a treaty with a long list of signatories), it could not adjust to evolving communication patterns. although analysts have often described disease surveillance as a collective action problem in which the global interest in transparency is pitted against national interests in episodic strategic concealment, characterizing the problem this way vastly understates the complexity of the interactions among actors. in particular, although it is nation states that have ihr treaty obligations, information about disease may be generated and controlled not only by non-state actors (as mentioned above) but also below the level of the nation-state, by agencies of the state, provincial health departments, individual public or private hospitals, and doctors and other medical personnel. as we will see, the norms and rules about how these lower level actors fit into the ihr negotiated information order have not always been entirely clear. until recently, the ihr made the who exceedingly dependent on official country reports by prohibiting the use of other sources of information. although promed-mail became an important unofficial source of information about threats to public health after its founding in , for many years the who was constrained from officially using it (woodall ). over time, the who's stance on these alternative sources of information evolved. a world epidemiological record piece suggested that 'public health authorities should give more attention to information from sources other than the public health sector, including ngos and the media. the capacity of public health authorities to rapidly respond to outbreak-related information from any source is essential for the efficiency and credibility of the entire surveillance effort' (who : ) . as the volume of information available from electronic sources and from health experts dispersed around the world increased, the pressure to use such information also increased. with increasingly sophisticated tracking systems, for instance, it became possible to demonstrate that deaths (even of particular named individuals) could have been prevented by earlier issuance of travelers' advisories (woodall ) . often, though, sub rosa information was less useful for issuing official warnings than for pressuring countries to report or for asking pointed questions about the adequacy or accuracy of reported information. 'they have accused us of spreading unfounded rumors and posting reports that have had no peer review. but we're just reporting what is being said or published. we tell health officials, you might as well report this, because you'll be reading it on promed tomorrow', commented charles calisher, an early moderator of promed (miller ) . some kinds of action required only that information be seen as technically sufficient (adequate in volume and coverage), but other kinds of action required that information also be socially sufficient (supplied by legitimate sources and arriving through specified routes). were the ihr ever an effective information order? undoubtedly the treaty was an improvement over earlier agreements, both in clarifying expectations and obligations and in institutionalizing a set of practices for reporting on disease outbreaks and keeping protective reactions in bounds. although it was an admirable attempt to create a worldwide consensus that balanced health interests against economic ones, it also had several clear deficiencies. an especially important deficiency was the limited coverage of the ihr, which cast doubt on the legitimacy of the treaty. beyond this severely limited coverage, the ihr were also compromised as an information order by a naïve conception of states as unitary actors and by rules that allowed the who to use only limited kinds of information supplied by specified, state-based actors. over time, informal norms supported fuller reporting on a broader range of threats and exploitation of information from unofficial as well as official sources. but in the medium term, although some nation-states adhered to the new norms, others hid behind the inadequate formal rules of the ihr, and still others continued to ignore even the limited formal requirements of the ihr. how well did this imperfect, outdated information order function when the ihr encountered sars, a new, deadly infectious disease that seemed poised to spread rapidly around the world? did the deficiencies of the information order in fact prevent the who from acting quickly and appropriately to contain the disease? many accounts of the - sars episode describe the chinese as concealing information or misrepresenting the situation in the first months, often suggesting that the country acted illicitly or illegitimately in doing so (altman ; the guardian ). yet closer examination of the record (see especially huang ) suggests that something considerably more complex occurred -there were multiple legitimate reasons for china to conceal early evidence of the outbreak. to begin, during the first days, there was nothing to report because no one understood that this was a new viral disease. because most apparently new diseases are in fact not new, physicians are reminded to think of horses not zebras when they hear hoof beats. as perhaps happened with sars, this advice sometimes leads people astray. with the benefit of hindsight, it is easy to conclude that chinese health workers should have been more diligent in forwarding reports about early cases of 'atypical pneumonia'. but we must be careful not to interpret actions taken in the confusion of the earliest days with knowledge acquired only later. still, local hospitals did call on provincial authorities for help. provincial authorities contacted the national ministry of health. a group of experts conducted an investigation. a report was prepared and circulated to all of the hospitals in the province. but here chinese law altered the disease's trajectory because the report became a state secret that could be shared only with specified people (such as the heads of hospitals). and then the trajectory was modified serendipitously when the report arrived in hospitals during the chinese new year celebrations. because no one read or acted on the report for a three-day period, precautionary measures were not implemented, creating an opportunity for the disease to spread. as noted, several months passed between the first appearance of sars and the first reports to the who. had the first suspicious cases been reported promptly, the disease likely would not have spread beyond guangdong province and hundreds of deaths could have been prevented. reports on the case suggest that in the earliest period, people 'knew' but 'didn't know' about the epidemic. and although healthcare workers, officials and other actors suspected a problem, at least in some instances they were either forbidden to share information or prohibited from acting on the information they received. in this case, the complex interplay of international, national and local rules and norms seems to have done as much to delay as to accelerate the spread of information about threats to public health. information about sars gradually leaked out, though, with a report from the chinese ministry of health finally reaching the who on february (who d). accounts of this period mention 'medical whistle-blowers' (see, e.g., eckholm ), promed-mail, the global health intelligence network (gphin, the 'rumor list'), the global outbreak alert and response network (goarn), and the move of the disease across borders into hong kong and then vietnam. although who personnel were investigating cases of what turned out to be sars in china as early as late february (enserink a (enserink : , the who issued its first alert about a severe form of atypical pneumonia only on march . according to david heymann (then executive director of who's communicable diseases cluster), vietnam was 'the trigger' for this announcement (enserink a (enserink : . a march report from carlo urbani, a who parasitologist consulting on a case in the french hospital in hanoi, provided the first indication that the new disease had spread beyond guangdong and hong kong. (urbani himself subsequently died from sars.) with the second who announcement, the world was informed that the atypical pneumonia, now named sars, was a new and very serious communicable disease. the secret was out. during this period, it could be hard to discern the signal in the noise. many things contributed to the noise -the irreducible uncertainties of the early days of a new disease, fear, mistakes, lack of preparation, incompetence, reputational concerns, and of course deliberate obfuscation. to be sure, there was ample evidence of outright concealment, foot-dragging, and obfuscation. the guangdong provincial government 'initially banned the press from writing about the disease and downplayed its significance' (enserink b (enserink : . although the who diplomatically reported cooperative efforts (see, e.g., who b), it carefully avoided comment on chinese silence or obfuscation between november and february, and even later. in fact, although chinese officials agreed to share information, their first promises were followed by more deceptions (fidler ; huang ; knobler et al. ). when the chinese government began to share information, who officials were still unable to get meetings with chinese health officials and were refused permission for travel to guangdong (enserink b (enserink : . when the early undercount of sars patients was attributed to the inadvertent exclusion of patients in military facilities, eckholm pointed out that the high proportion of beijing sars patients in military hospitals 'could [instead] indicate that patients were placed there to avoid their inclusion in civilian disease reports ' ( ) . what the nation-state does not know, it cannot report. but the ihr was little help in dampening the noise or strengthening the signal. although the ihr treaty was officially the governing document when the - sars outbreak occurred, it was an imperfect information order that did not authoritatively mandate a clear course of action. '[n]othing compelled china, or any other country, to tell the rest of the world what was happening within its borders early in ' (enserink b (enserink : . indeed, the shocking weakness of the international health governance system was surely a factor in china's failure to report the outbreak quickly. under the ihr, most disease outbreaks, including those of previously unknown diseases, were domestic business. but if china had no formal obligation to report, why was it so soundly condemned for its delay in transmitting information to the who? although the treaty -international law -did not require reporting, emerging norms around the management of global public health governance diverged from formal law. under these emerging norms, a failure to report a new disease, an environmental disaster, or some other occurrence that might affect global public health was a serious infraction (heymann ) . indeed, it was the conflict between these emerging norms and the existing treaty provisions, along with the emergence of new infectious diseases like ebola, that helped spur the ihr revision, first called for in a resolution, well ahead of the sars outbreak. an important difference between formal treaty obligations and norms, though, is that the first applies uniformly and the second does not. being a signatory to a treaty is a bright line. membership in a moral community is more ambiguous, with some treaty signatories more fully incorporated and others more peripheral. thus although the long silence of the chinese government was not technically a violation of the ihr, it nevertheless appeared dishonest and inappropriate to the international community, undermining rather than supporting emerging cooperative norms and in fact harming global public health by allowing the new disease to spread beyond china's borders. the institutional incoherence around global public health governance was in fact deeper than this; the treaty provisions were inconsistent with domestic law as well as with emerging norms. until treaty provision and domestic law are harmonized, health workers can be caught between local and global legal obligations, two distinct sets of rules laying out inconsistent requirements for partially overlapping groups of actors. although only state representatives were responsible for reporting to the who, domestic law compelled medical workers to preserve state secrets about the very matters that international norms -but not ihr treaty provisions -compelled them (or others in their chain of command) to report. many chinese actors were in a terrible bind, legally required to protect state secrets but morally obligated to share information so fellow citizens could protect themselves from a virulent emerging disease and so international bodies could study the disease and develop methods to combat it. individual and global interests both demanded transmission of information, yet the chinese state initially mandated secrecy instead. moreover, the ihr specified roles and obligations for only a few actors, thus offering no guidance about appropriate courses of action for many other actors who possessed relevant information. beyond legalistic matters about obligations to report or to conceal, the evidence from sars also suggests that fears about economic consequences of adverse publicity associated with disease outbreaks strongly shaped the thinking of chinese authorities (huang : ) . these economic concerns were in fact justified, though overstated, in hindsight. the economic effects of sars include much more than the cost of providing medical care for those affected, as analysts acknowledge. lee and mckibben ( ) estimated the short-term impact of sars to be about $ billion for alone if people expected the epidemic to be a one-time event and considerably higher if they behaved as if they anticipated recurrences. subsequent research suggests that the economic impacts were considerably smaller than anticipated and that recovery occurred quickly (keogh-brown and smith ) . although the economic impact was widely dispersed, the losses were greater in asian countries than in the rest of the world, with strong shocks to mainland china, which experienced a decline in foreign investment, and especially to hong kong whose service economy depends on travel and tourism. for government officials responsible for the overall welfare of a society, including both physical and economic health, worries about commercial impacts cannot be dismissed. as a negotiated information order, the ihr was thus ineffective, unstable, and ripe for change for a host of reasons. first, legal obligations were out of sync with the higher expectations of an evolving normative system. second, international law and domestic law often had not been harmonized and disagreed about whether threats to public health should be reported or kept secret, creating a serious conundrum for health workers. third, the ihr failed to take account of the social complexity of a system in which information was produced and controlled by a wide variety of actors, including not just official national representatives (e.g., ministries of health) and provincial or other substate actors (e.g., provincial departments of health), but also actors who were not state representatives but nevertheless had relevant roles and expertise (e.g., heads of hospitals, whether private, public or military), journalists, and private citizens all with varying relationships to the international treaty, emerging norms, and domestic law. fourth, although the ihr did not envision that the who would act on the basis of information other than that provided officially by nation-states, pressure to use such 'non-knowledge' had increased over time as information sources multiplied, tools to parse such information were created, and threats to public health came to seem increasingly urgent. one important effect of sars was to shift the boundary between official and unofficial knowledge, ultimately modifying the information order so that unofficial information of questionable quality could be used as leverage, forcing states to reveal what they might have preferred to conceal. the revision of the ihr was adopted by the world health assembly (wha), the governing body of the who, in and put into force in . just as revisions to the ihr were being crafted, the deficiencies of the existing legal framework were made glaringly apparent by the rapid spread of sars and the numerous -and avoidable -deaths it caused. although china had not in fact violated the existing treaty, it clearly violated emerging norms on the reporting of infectious diseases. the objective of the new treaty provisions was to induce earlier and fuller reporting by acknowledging the importance of non-state actors as suppliers of information and recrafting the information order so that previously unusable kinds of information -information that might have been seen as technically sufficient but was not socially sufficient -could now be used. the revision brought important changes in what has to be reportedany 'public health emergency of international concern'. along with this broader range of reportable threats, the ihr introduced a decision tool to replace the short, simple list and guide reporting; offered considerable guidance about who should report and how (e.g., mandates for designated reporters, now called 'national focal points'); and created tool kits for implementation including for harmonizing the ihr with domestic law (who ). in effect, these changes move the ihr from the realm of 'soft law' further into the domain of 'hard law' (abbott and snidal ) by making the rules more specific and more obligatory, by adding processes for interpretation of law and for dispute settlement, and by inserting rudimentary enforcement mechanisms. some of the work of hardening the ihr is delegated to individual member states as they bring domestic law into harmony with the ihr. as treaty provisions and domestic law are harmonized and gaps bridged, excuses for non-compliance are eliminated and domestic supports for compliance are added (see, e.g., the agreement between the australian federal government and its states and territories to ensure timely reporting [commonwealth of australia ; scales : ]). fidler ( ) argues that sars exposed the conflict between an outdated, unworkable, westphalian system of international governance and a world in which global diseases required a global governance system. states have lost their primacy, he suggests, in a world in which they can control neither the movement of disease nor the movement of information. believing it had the right to suppress information, the chinese government attempted to treat information about infectious disease as it always had: as a matter of state secrets. but in a world of cell phones and internet, text messages and email allowed both patients and physicians to circumvent the state. prohibiting news media from reporting the outbreak of the deadly disease did not keep individuals from communicating with one another inside china and sending information and questions to contacts outside the country. with the growth of new information technologies, state monopolies on information have decayed and the balance between socially and technically sufficient information has shifted. as the volume of information considered technically sufficient has increased and the who has developed more sophisticated techniques for extracting high-quality information, its capacity to pressure states to meet their treaty obligations has increased. something like an enforcement capacity, albeit one not formally recognized in the ihr, grew up in the midst of all this complexity. with the vote of the world health assembly (wha) and the subsequent revision of the ihr, this enforcement capacity has been recognized, endorsed and formalized, first with the wha's blessing of the who's use of unofficial information and then with the incorporation of this information use into the procedures outlined in the revised ihr. in this case, changes in practice preceded changes in the legal infrastructure as the who increasingly drew on information that did not come directly from the official reporters of member states. but in a pattern of 'punctuated globalization' (heimer ) , the legal framework seems now to have reclaimed the lead in moving forward global coordination around public health surveillance. as countries and agencies adjust to the ihr, we can expect the development of a host of new strategies for exploiting the opportunities created by this new framework. the revisions have required many countries to invest heavily in improving their systems for tracking and reporting threats to public health. this, in turn, has created an opening for many joint activities between rich and poor countries, including construction of new cdc facilities around the world (gootnick ) . do these changes then signal the end of the gap between actionable, socially sufficient information and technically sufficient information in global health governance? rather than an end to the gap, we should expect a shift of the gap's location. gaps arise because parties with imperfectly aligned interests have some incentive to game systems. such discrepancies between global, collective interests and regional, state or local interests will continue to exist and some evidence suggests both continued and fresh strategies for gaming and non-compliance (scales : - ) . the exact configuration of the gaps will change, of course, as the nature of the key actors changes (less emphasis on states, perhaps) and as technologies change (easier transmission of information by both official and lay actors). the gap itself will not vanish. states will remain relevant actorsindeed world politics suggests that national borders are as often reinforced as demolished and that states continue to have responsibilities and interests that might motivate them to conceal information. moreover, a clarification of treaty obligations and the introduction of a new lever for the who will not entirely resolve the problem. in the past, with no uncertainty about obligations to report, countries nevertheless failed to report outbreaks (carvalho and zacher ; fidler : ; kamradt-scott : - ; scales ) . although the who can more nimbly alert the world about an outbreak, it can do little beyond that: no sanctions, no fines, no cancellation of membership. and new incentives for non-compliance will continue to arise. until samples were used to create flu vaccines, countries had little reason to withhold samples of new influenza strains. but under a regime that protects intellectual property and gives those supplying samples no share of the income from the sale of resulting vaccines, countries now have an incentive not to offer their samples for the common good. when indonesia, responding to this incentive structure, began withholding flu samples, a new who working group developed a non-binding framework to encourage both virus and benefit sharing (fidler ; fidler and gostin ; scales ; smith ) . in the argument of this article, sars plays a central (albeit non-determinative) role. but is sars simply a useful case on which to hang the argument? or could the argument have been built around hiv/aids, h n , ebola, zika, or some other infectious disease? in fact, other diseases and sars are not interchangeable in this argument; sars is not 'merely' an example. because of historical timing, sars was the epidemic that brought the previously recognized failings of existing disease surveillance systems into the spotlight and stiffened the spines of those pushing for change. the features and timing of sars helped to bring the shortcomings of the ihr into sharp relief, undermining their legitimacy and making it essentially impossible for the who and public health specialists to continue working under the old rules. the legitimacy of the who increasingly depended on denying the legitimacy of the ihr. by the time sars appeared, the deficiencies of the ihr had become so glaringly apparent that the wha had endorsed the who's use of unofficial information even before the rules changed. but particular features of the disease, namely its brief incubation period and moderate transmissibility, meant that the adage that microbes do not respect national borders was all too applicable. local outbreaks of sars had global relevance in a way that local outbreaks of hiv/aids, with its long period of dormancy, did not. sars quickly became a global threat. but it also mattered that the disease arose in a country that wished to suppress information about the outbreak. in the age of the internet and cell phones, information, like microbes, neither respects borders nor governmental edicts on secrecy. thus sars brought to a head a long-standing clash between national governments' desires to keep secrets and new capacities to transmit information with or without governments' blessing. in fidler's view, 'china's behavior [at the start of the sars epidemic] put the final nail in the coffin of basing global surveillance for infectious diseases only on government information ' ( : ) as the rules required. sars was a 'historic moment in public health governance' (fidler : ) , the tipping point for new governance strategies (fidler : ) . in a limited sense, then, sars was a boon to the who because it provided an added inducement for the wha and member states to modify the rules in ways that benefited the entire group and gave the who and ihr new relevance. although the ihr's limitations had long been apparent, by making it impossible to deny that the treaty provisions were outmoded sars accelerated the process of reaching consensus on proposed changes. the revisions of the ihr attempted to deal with two kinds of ignorance: ignorance about outbreaks of known diseases and ignorance about newly emerging diseases and other threats to public health. before revision, the ihr had focused only on outbreaks of known diseases and therefore on ignorance that could in principle be reduced or even eliminated by full and honest disclosure. as it became clear that infectious diseases were not going to be eradicated, as new diseases continued to emerge, and as natural disasters, industrial accidents, air and water pollution, and so forth came to be understood as threats to public health, the ihr's focus shifted to these less tractable forms of ignorance and thinking changed about what should be reportable under the ihr. this expanded understanding of threats to public health brought both expanded obligations for states and expanded obligations for the who. the who's remit now included not just spreading the word and issuing advisories about a larger package of threats to public health, but also overseeing and orchestrating the scientific work of untangling the etiology, symptom patterns, modes of detection, and effective remedies for these threats. into this changed environment, the reworked information order introduced a more sophisticated understanding of the relationship between what was or could be known and what was unknown and perhaps even unknowable. the modified procedures of the ihr in some senses acknowledged the difference between technically sufficient information that was also socially sufficient -because it had been supplied by mandated state reporters -and technically sufficient information that was not socially sufficient because it travelled to the who by unconventional or even clandestine routes. but the loosening of constraints on the sourcing of information did more than simply make information usable by recategorizing previously unofficial, socially insufficient information. the modified procedures also opened the door to using information as leverage, with information of inferior quality or illegitimate provenance being used to pry loose information of better quality or from official sources. moreover, in casting a wider net and exhibiting its willingness to draw on an expanded network of informants and more variable kinds of information, the ihr seem to acknowledge the essential irreducibility of ignorance. when uncertainty cannot be eliminated, and when the transmission and withholding of information is at least in part a strategic game, an entity such as the who is in no position to sharply limit the information it will consider. the ihr, a renegotiated global public health information order, thus incorporate into their structure an acknowledgement of the complex relationship between knowledge and ignorance, socially sufficient information and technically sufficient information, and the socially constructed nature of these distinctions. although this article focuses on negotiated information orders in global public health governance, its argument and evidence address broader issues about how global norms change and how social groups manage risk. the story of the - sars epidemic, the core empirical component of the article, is about the possibility that a virulent new disease would become a devastating pandemic and about an emerging (but not yet formalized) obligation to inform the who about serious threats to public health. the comparison points -the threat of aids contamination in banked blood (healy ) ; threats from oil spills, nuclear power accidents, and nuclear war (clarke ) ; and threats from accidents on north sea oil rigs and platforms (heimer a ) -are also about how key actors assessed novel risks. in all of these cases, the assessment of the core risk was implicitly balanced against other risks -risks to trade and tourism for sars; risks to relationships with important constituencies for the blood banks (healy ) ; risks to desired investments in business and government enterprises (clarke ) ; and risks to vested interests in the insurance business (heimer a) . generally speaking, though, as discussions unfolded, only some of the risks were fully on the table, perhaps because people were not wholly aware of how other considerations were shaping their thinking, perhaps because of the questionable legitimacy of balancing other risks (trade and tourism, in the sars case) against threats to life and health. the result is often a pattern of minimizing assessments of danger and normalizing those (implicit) assessments. as noted earlier in the article, many disease outbreaks, even of the three reportable diseases, had not been reported to the who. somewhat like the normalization of deviance that diane vaughan ( ) so carefully describes in the challenger launch decision, the deviant non-reporting of disease outbreaks had been normalized. some countriesespecially poorer ones -were learning from one another that they would suffer no consequences from ignoring ihr treaty obligations. although the ihr were described as regulations to protect health in all countries, in fact they focused on stemming the spread of disease from poor countries to richer ones. as chorev ( a) suggests, international obligations perceived as coercive are more likely to be reinterpreted locally and perhaps ultimately transformed through processes of reactive diffusion. in the case of the ihr, reactive diffusion essentially made the already unenforceable ihr progressively less useful. but in the pre-sars period, the evidence in fact suggests a more complex process of normative change. two rather different norms were being institutionalized simultaneously in global public health governance. at the same time that ignoring ihr treaty obligations was becoming the norm in some circles, a different norm was spreading in other circles. some countries -especially the richer ones -were adopting a more cooperative stance, sharing information not only on ihr reportable diseases but also on other infectious diseases and threats to public health. it was this cooperative norm, not the norm of non-reporting, that ultimately diffused and, coupled with the sars epidemic, led to a reinvention of the ihr as a treaty with a few more teeth. how did this happen? here a comparison with the space shuttle launch decision is instructive. although nasa carried out rigorous, carefully scripted pre-launch reviews, contextual pressures to launch could subtly shift thinking about which risks could be dismissed and which warning signs ignored. over time, these modified assessments were institutionalized and the insularity of the process made it hard for alternative viewpoints to force a recalibration. the conflict between protecting against rare events and attending to business is utterly mundane (vaughan ) , so mundane that insurers have institutionalized methods for protecting key risk management tasks from production pressures (heimer b) . the job of the ihr, arguably, is to rebalance risk assessments so global public health interests are not regularly sacrificed when discrediting information about health threats is concealed to protect a country's trade and tourism. yet the ihr treaty gave the who few levers to induce such a rebalancing. unlike space shuttle launch decisions, though, global public health governance does not take place behind a single set of closed doors. thus, although a practice of non-reporting -normalized deviance -seemed to be developing in some sectors, changes in information technologies and communication patterns made secret keeping more difficult and shifted the balance in favour of the more cooperative norm. even with china's strict control over the internet and the press, text messages and emails spread news about 'atypical pneumonia', forcing public officials to acknowledge the outbreak. although any single medium might fail to pick up the news, the proliferation of methods for detecting signals makes suppression of information more difficult. a news blackout might make gphin, which scrapes information from news outlets, less effective, but have less effect on promed-mail, which relies on medical workers' postings. working together over some considerable period of time and in a series of discrete steps, the new information technologies and the emerging norm of information sharing reconfigured the rules about global public health governance and reshaped understandings about what information could be used and who could supply it. information technologies first reshaped some practices of the who. as the who began to use the unofficial information supplied by entities like gphin, it also initiated the process of redefining non-knowledge as technically sufficient, at least for some purposes. as the who rebuilt its routines to use unofficial information alongside official country reports, new relationships and resources (e.g., goarn) were created around those new information sources. both the suppliers of information and the who increasingly treated this new information as technically sufficient. with the endorsement of the wha, these new practices and new definitions of the adequacy of unofficial information were further institutionalized, moving one step further to a formal change in the treaty itself. with the adoption of the ihr, the process was complete -what had previously been categorized as unusable non-knowledge was first reconceptualized as technically sufficient, and ultimately accepted as socially sufficient for use in an expanded menu of actions. nevertheless, information categorized as unusable non-knowledge will always exist and will continue to be important precisely because it comes from different social locations than those tapped by official information. as mary douglas would remind us, we need the sentinels on society's margin to warn us of unexpected dangers every bit as much as we need people working in core institutions to protect us from more routine risks (douglas and wildavsky ) . although admittedly the uses of non-knowledge or clandestine knowledge are typically different than the uses of official knowledge, that should not lead us to underestimate either the vital strategic value of non-knowledge or the importance of using it efficiently in a smoothly functioning, adaptable information order. just ask kim philby or david john moor cornwell, aka john le carré. gphin in (who c , notes that '[m]ore than % of the initial outbreak reports come from unofficial informal sources, including sources other than the electronic media, which require verification' (who n.d.) . gphin is often credited with picking up news of a disease outbreak in china in late november (heymann and rodier : ) . set up in by the who and formally launched in , goarn is a collaboration of other networks, linking a wide variety of experts and combining both surveillance and response (fidler ; heymann ; heymann et al. ; who c: ) . as of , goarn includes as members over technical institutions and networks concerned in one way or another with public health (https://extranet.who.int/goarn/; last viewed march ). fidler ( ) , especially, credits goarn with a major role in containing sars. according to virologist malik peiris, 'if something untoward was happening across the border, it would come to hong kong pretty quickly' (enserink a (enserink : . vietnam was a reluctant trigger, though. as hospital staff fell ill, the vietnamese government had to be persuaded that this was not simply a 'private problem in a private hospital' but might instead be 'very important' (enserink a : . according to the who, the resurgence of cholera in south america and plague in india, as well as the emergence of new infectious agents such as the ebola virus, 'resulted in a resolution at the th world health assembly in calling for the revision of the regulations' (https://www.who.int/ihr/ about/faq/en/; last viewed march ). some observers (e.g., katz and fischer ; wenham ) contend that states have not lost their primacy. for similar assessments of the role of sars, see lazcano-ponce, allen and gonzález ( : ) and katz and fischer ( ) . to be sure, the new technologies were not free, and wenham ( ) notes that considerable human intervention was required to make sense of the volumes of information arriving through gphin and goarn. but these costs were disproportionately borne by richer countries, who also agreed to help build infrastructure and supply expertise for poorer countries. hard and soft law in international governance the market for "lemons": quality uncertainty and the market mechanism uncertainty and the welfare economics of medical care the international health regulations in historical perspective changing global norms through reactive diffusion: the case of intellectual property protection of aids drugs the world health organization between north and south a garbage can model of organizational choice risk and culture: an essay on the selection of technological and environmental dangers war stories sars: chronology of the 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'epidemic intelligence -systematic event detection international health regulations ( key: cord- -h qf oh authors: brown, keith d.; campbell, catherine; roberts, glenda v. title: precision medicine in kidney disease: the patient’s view date: - - journal: nat rev nephrol doi: . /s - - - sha: doc_id: cord_uid: h qf oh research in the field of nephrology continues to improve our understanding of the mechanisms that promote and drive kidney disease, including how human genetic variation might affect disease predisposition and progression. one of the goals of these research efforts is to inform and enable the implementation of precision medicine, whereby patient management is tailored to the individual according to the mechanisms underlying their disease to increase the chances of therapeutic success. to achieve this goal, we need a clearer understanding of the molecular pathways that underlie the many different causes of kidney failure. these research insights are being increasingly translated and implemented into clinical practice. in this viewpoint, we asked three individuals who have been affected by kidney failure for their views on the importance of understanding the drivers of kidney disease and, on a personal level, what they hope might be achieved with this information. limited clinical practice, is highly involved in kidney research. in the event that my focal segmental glomerulosclerosis should ever return, i'm hopeful that advances in our understanding of the molecular basis of kidney disease will mean that information will have a much larger role in the management of the disease than it might have had - years ago. catherine campbell. i am privileged to have had a kidney biopsy as this procedure is not always routinely available to patients with kidney disease. being aware of my biopsy findings, which included the presence patients with acute kidney injury (aki) and ckd is beginning to expand our understanding of the underlying causes of these conditions and, in some cases, to improve diagnostics. these discoveries are being shared within research institutions. clinical research discoveries are likely to be more rapidly adopted by physicians involved in clinical research, who are often exposed to the research findings during renal grand rounds or other academic meetings, often before those findings are published. often it takes years for clinical research findings to be adopted by physicians whose practice is outside of the university research environment. i think that patients who are managed by nephrologists who work at research institutions will probably be the most immediately affected because their physicians are exposed to the discoveries and insights that their research peers are developing. as researchers start to publish more of their discoveries, i expect to see more patients benefit from this enhanced molecular understanding. it is my hope that the inclusion of a 'lay abstract' in medical research articles will become a standard requirement by publishers. such abstracts would enable patients to understand the reported research findings and empower them to ask questions and discuss the research with their physicians, which would likely impact the management of their disease. we know that global kidney scarring is more pronounced in patients with late-stage disease than in those with early-stage disease. therefore, early-stage biopsies, coupled with an improved understanding of the molecular basis of kidney disease, might make it easier for pathologists and nephrologists to assess the potential causes of disease and its progression, which would likely influence how the patient's disease is managed. how important is it for you to understand the underlying mechanistic basis of your disease? k.d.b. the human body is a magnificent thing and i have always had an interest in human physiology. therefore, i have always been very interested in the pathophysiology and disease mechanism of kidney disease. when your own kidneys are failing, you of fibrotic scarring commonly found in chronic kidney disease (ckd), has helped me understand better the causes behind my kidney failure and has empowered me to manage my self-care. these findings have improved my desire to slow the disease by watching my diet and blood glucose levels, which helps me manage my diabetes, as well as monitoring my blood pressure, avoiding potential falls and resting to avoid fatigue. i have accomplished these goals successfully through medication compliance and regular lab draws and self-injections of an erythropoiesis-stimulating agent. knowing the molecular basis of my disease provides a foundational base for my care and the care of others. as a doctor of nursing practice, i believe it is within my personal and professional responsibilities to educate patients, families and the community of ways to prevent ckd. abstract | research in the field of nephrology continues to improve our understanding of the mechanisms that promote and drive kidney disease, including how human genetic variation might affect disease predisposition and progression. one of the goals of these research efforts is to inform and enable the implementation of precision medicine, whereby patient management is tailored to the individual according to the mechanisms underlying their disease to increase the chances of therapeutic success. to achieve this goal, we need a clearer understanding of the molecular pathways that underlie the many different causes of kidney failure. these research insights are being increasingly translated and implemented into clinical practice. in this viewpoint, we asked three individuals who have been affected by kidney failure for their views on the importance of understanding the drivers of kidney disease and, on a personal level, what they hope might be achieved with this information. viewpoint nature reviews | nephrology quickly learn all the various regulatory functions that the kidney performs. identifying the underlying mechanisms of my kidney disease has been a revolutionizing experience. i had always been somewhat focused on my blood sugars and blood pressure, but the results of my kidney biopsy relayed a surprise findingalthough i have diabetes, the main cause of my kidney disease is hypertension. also, as a child i had frequent urinary tract infections (utis), but a potential cause for their recurrence was never investigated. urinary incontinence has also been a lifetime challenge and embarrassment. in kindergarten i was punished when having to go to the bathroom during class. i was also punished by a family member for wetting on the way home from school, even though i had been to the bathroom before leaving. these growing pains were emotionally painful and humiliating. however, my biopsy results gave me some insight into these events, as the presence of fibrotic tissue is likely linked to complications from frequent utis that resulted in kidney infections . participating in the kidney precision medicine project (kpmp) consortium as a patient with ckd and being involved in community engagement and education have provided me with a basic understanding of the origins of my ckd -i have a family history of type diabetes and hypertension, kidney transplants in the short term. in the long term, i think the increased scientific knowledge we gain will lead to an eventual cure for kidney disease. advances are sure to come in genetic engineering and new medications that will correct whatever is causing an individual's kidney disease. c.c. the current limited treatment options can be devastating for patients with ckd. advances in precision medicine can only be viewed as positive for these patients. they are also vital to the families of patients with ckd to ensure early diagnosis and treatment in the future. for example, the presence of ckd risk factors with a strong genetic component, such as type diabetes, hypertension or heart disease in my family, might increase the likelihood that another family member might develop ckd. current research efforts include the kpmp, which aims to study kidney biopsy samples to develop a kidney atlas in which molecular information is catalogued to help identify the underlying causes of kidney disease. i hope that this project will lead to improved early disease diagnosis and specific treatments, as well as providing vital education for physicians, patients and their families. the community advisory board includes members from the community who have been selected to include a diverse population to facilitate community inclusion in education and research, for example, by recruiting individuals to provide biopsy tissue, and to enable health recommendations to reach the under-served and those with predisposition to kidney disease. biopsy samples are currently being collected from patients with aki or ckd and my long-term vision for maximizing the potential of these resources includes not only the continued procurement of biopsies but also the collection of biopsy samples from healthy individuals for comparison with those from individuals with kidney disease. i envision community-wide understanding and participation in rallying for the development of, and access to, appropriate treatments. today, medicare only provides years of anti-rejection drugs to transplant recipients, which disproportionally affects and limits the life expectancy of patients who cannot afford these medications independently. in the long term, patients and their families must be informed and empowered to speak up for their right to access appropriate treatment regimens. g.v.r. i hope that in the future, it will be standard of care to recommend a kidney biopsy to anyone who is diagnosed with kidney disease. considering the diagnostic which are heritable risk factors for ckd. although i cannot change the effects that these risk factors have had on my health, my improved understanding of the disease basics has educated and improved my self-care. it is very important to me to understand the underlying mechanistic basis for my disease because it enables me to take informed action. for instance, if i know that eating too much kale increases the likelihood of developing kidney stones and i have a propensity towards kidney stone development, then i can change my diet accordingly. what do you envisage precision medicine will mean for the diagnosis, prognosis and treatment of kidney disease in the future? what are your hopes for the short and long-term? k.d.b. i hope that new discoveries in precision medicine will, in the short-term, lead to treatments that will significantly delay the progression of kidney disease. once we find ways to better understand what cellular or molecular or genetic attributes are related to an individual's kidney disease, then treatments can be developed to modify or suppress these various causes of the disease. delaying the progression of kidney disease will hopefully reduce the need for dialysis and catherine campbell has been a registered nurse for more than years. her life purpose is committed to educating and servicing others in health concerns. she has made multiple contributions in volunteer service: as a patient participant in the kpmp, an american association for retired persons (aarp) public policy advocate, a board of directors member for the case management society of america, a life fellows advocate in the american college of healthcare executives, dallas/fort worth community advisory board for kidney patient support, an american association of kidney patient advocate and a governance committee member for the sigma nu upsilon chapter. in her continued commitment to the nursing profession, she mentors careerists in the american college of healthcare executives, as well as doctoral and master nursing students, and delivers online teaching to nursing students attending mount saint mary's university, los angeles. glenda v. roberts is the director of external relations & patient engagement at the university of washington center for dialysis innovation (cdi) and the kidney research institute (kri). she brings the patient voice to a number of nih/niddk government and industry research efforts, as well as the american society of nephrology (asn) covid- response team and the asn covid- transplant subcommittee. she is a member of the international society of nephrology (isn), the kidney health initiative (khi) patient and family partnership council (pfpc), can-solve ckd international research advisory committee and home dialyzors united advisory board, and has been an ambassador for the american association of kidney patients since . in addition to being a patient participant, glenda is a member of the steering committee, the collaborations committee, the return of results committee and the community engagement committee, as well as the director of communications, for the kpmp. www.nature.com/nrneph challenges resulting from the increase in scarring as kidney disease progresses, by identifying the cause of an individual's kidney disease earlier, we will hopefully be able to implement a treatment tailored for that individual to slow or reverse the progress of their kidney disease. most doctors do not recommend biopsies in the early stages -they tend to wait until a patient is stage or later -and one explanation is that a biopsy is surgery and thus discouraged. i hope that as doctors become exposed to the things that we're learning in the kpmp they'll appreciate the benefit of a biopsy. in the short-term, i hope that as many patients as possible will continue to participate in research projects, for example by signing up to get biopsies and contribute to the kpmp kidney atlas, so that researchers gain better insight into the cells and pathways that cause various diseases. this atlas will enable us to better divide the causes of kidney disease into subgroups and identify the appropriate treatment for each subgroup, rather than the one-size-fits-all approach that is used today. historically, diseases like cancer and kidney disease were treated with a one-size-fits-all approach. we see that the cancer research field is making good progress in terms of developing more personalized treatments, and the outcomes are encouraging. i am looking forward to the development of similar novel therapies and treatments for people with kidney disease. can you see any drawbacks to a precision medicine approach to kidney disease? is there likely to be an impact on family members? k.d.b. in my view, more scientific knowledge and evidence-based medicine are always better than less. however, one drawback when we're dealing with genetic testing is, of course, the concern about the privacy of an individual's genetic information. in a worst-case scenario, a person has to worry about things like insurability and employability if certain genetic information is revealed. even things like whether or not g.v.r. i do not see any drawback to precision medicine, per se. however, based on some historical practices by insurance companies, i think that some people will be reluctant to get a personalized diagnosis because it might be viewed as a pre-existing condition. i think that this is a legitimate concern. the question becomes whether the individual gets treatment now to slow or reverse the condition or whether they wait and let the disease put them at greater risk in order to get insurance coverage. hopefully, someday people will not have to worry about their family history or pre-existing conditions preventing them from getting insurance coverage. overview of the cellular and molecular basis of kidney fibrosis the kpmp is funded by the following grants from the niddk: u c dk , uh dk , uh dk , uh dk , uh dk , uh dk , uh dk , uh dk , uh dk , uh dk , uh dk and uh dk . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. g.v.r. is employed by the university of washington, has contributed to the national palliative care grant (patient advisor) and has participated in the following nih-funded projects: kpmp (director of communications), apollo (community advisory committee member) and apol (community advisory board member). k.d.b. and c.c. declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. to have children can be an issue if you are likely to pass on a gene that increases the risk of kidney disease in your offspring. as physicians and scientists learn more about the specific genetic and molecular characteristics of an individual that are causing or contributing to their kidney disease, this will likely have an impact on their family members. for example, family members who might have inherited these genetic predispositions to kidney disease will be faced with decisions about whether or not they want to undergo genetic testing. as with certain genetic variants that are related to certain cancers, some family members will want to get tested and begin whatever individualized treatment might be appropriate; others might prefer not to know whether they are predisposed to developing kidney disease.c.c. i do not foresee any drawbacks to precision medicine. genetics and personalized treatments are already recognized in other diseases. for example, regular mammography might be recommended for early detection of breast cancer in individuals known to have inherited genetic variants that research has identified as being associated with an increased likelihood of developing breast cancer. such hereditary risk variants might also be identified in the context of kidney disease. breast cancer research has also demonstrated, for example, associations between different lifestyles and health status, and the risk of developing breast cancer . it might be possible to obtain the same type of information about the development of ckd by advancing research through the study of kidney biopsy samples. recognizing the potential risks in a family might prevent children from developing ckd through education and information, which are vital to the future of our young and might enable them to improve their lifestyle and minimize the risks to their health. i might have benefited from this information at a younger age -there is no guarantee that this support would have decreased my kidney failure, but hopefully precision medicine will still help improve the lasting poor prognosis. key: cord- -z ifmr authors: mahy, b.w.j. title: emerging and reemerging virus diseases of vertebrates date: - - journal: encyclopedia of virology doi: . /b - - . - sha: doc_id: cord_uid: z ifmr in the last two decades, a large number of new viruses have been discovered, many of which are pathogenic in humans or other vertebrates. among the more important causes of virus emergence have been changes in human behavior, population, and increases in travel to distant countries. in addition, the application of new molecular technologies has led to the recognition of many viruses that hitherto went undetected. many of the new, emerging viruses have an rna genome, and many are zoonoses. the spread of human immunodeficiency virus, causing acquired immune deficiency syndrome, and the use of immunosuppressive drugs following transplant surgery, have increased the numbers of people in the population that are highly susceptible to emerging virus infections. the threat of a new pandemic of influenza virus in the human population stresses the need for development of better methods for detection, surveillance, and control of emerging virus diseases. it became apparent during the last two decades of the twentieth century that new infectious diseases were increasingly being recognized in the human and animal populations. this led to the establishment of a formal committee of the institute of medicine of the national academy of sciences, usa, who reported on their deliberations in , in a report edited by joshua lederberg and richard shope. this was followed years later by a second report, edited by mark smolinski, margaret hamburg, and joshua lederberg, which appeared in . among the factors they cited as contributing to emergence were microbial adaptation and change, human susceptibility to infection, climate and weather, changing ecosystems, economic development and land use, human demographics and behavior, technology and industry, international travel and commerce, breakdown of public health measures, poverty and social inequality, war and famine, lack of political will, and finally intent to harm. the advent of highly specific molecular techniques such as the polymerase chain reaction (pcr) in the early s permitted the detection and grouping of viruses on the basis of genome nucleotide sequence analysis, and in several respects these techniques have replaced serological analyses for the characterization of viruses. although it is still important to isolate viruses in cell culture for their complete characterization, it is now possible directly to detect viruses in diseased tissues by pcr, then, by sequencing the amplicon, to determine whether a new virus has emerged to cause the disease. in fact, many viruses which do not readily grow in cell culture can only be differentiated by sequence analysis. the papillomaviruses are an example. their study was very difficult until the advent of sequence analysis, which now has revealed more than types in humans, and many more in animals and birds. for differentiation, three virus genes (e , e , and l ) are sequenced, and if the combined sequence of these three genes differs by more than % from known papillomaviruses, the virus is considered to be a new type. other viruses which have not been grown in cell culture include many caliciviruses, and the ubiquitous anelloviruses, such as torqueteno (tt) virus, which can be detected and sequenced in the blood of most humans and many other vertebrate species. hepatitis c virus was originally described as non-a, non-b hepatitis virus because of the severe disease it caused but the virus would not grow in cell culture, and eventually was detected in blood known to be infected with the virus by reverse transcription of the rna present using random primers then expressing the resultant dna in the bacteriophage lambda gt . thousands of clones were screened using patient blood as a source of antibody before positive clones were detected, which then allowed the development of enzyme immunoassays that could detect the virus in blood and so were used to screen blood destined for transfusion, saving millions of lives worldwide. once the complete genome of hepatitis c virus was sequenced, it became apparent that there are many different genotypes circulating in the world, with different pathogenic properties. nucleotide sequence analysis has also been extremely useful in tracing the origins of viruses. for example, when hantavirus pulmonary syndrome, caused by a bunyavirus of rodents, sin nombre virus, was initially detected in in the four corners region of western usa, it was found that rodents inside a house where people had been infected carried a virus identical in sequence to virus isolated from human cases. however, rodents caught at various distances from the house had increasingly variable genome rna sequences, providing strong evidence that these rodents, deer mice (peromyscus maniculatus), were the source of the infection. subsequently more than other hantaviruses, some of which also cause hantavirus pulmonary syndrome in humans, were isolated from rodents throughout north and south america. each new virus seems to be associated with a different genetic variant of rodent host, and all rodents that carry the virus belong to the subfamily sigmodontinae, unique to the american continent. a particularly powerful tool for the initial recognition of an emerging virus is the application of immunohistochemistry to diseased tissues. provided a comprehensive collection of antibodies is available, the particular virus or related group of viruses can often be detected. for example, when hendra virus first appeared in in australia, causing the death of a horse trainer and of his horses, antibody against the virus was sent to the centers for disease control and prevention (cdc). then, in , cdc was asked to investigate a newly emerged epidemic that had appeared in malaysia, killing more the people and causing disease in many pigs. initially, it was suspected to be caused by a virus related to japanese encephalitis virus, but a virus was isolated from the pigs that replicated in vero cells, and reacted in an immunofluorescence test against the hendra virus antiserum. this could subsequently be used on patient tissues to study the pathogenesis of the disease, and after comparison of the genome sequences of hendra virus and nipah virus they were found to be closely related and are now classified in the genus henipavirus of the paramyxoviridae. finally, molecular methods can be used to detect new, emerging viruses in the absence of disease in the host. in , allander and colleagues searched for rna viruses in human respiratory secretions using random primer pcr, and discovered a hitherto unknown parvovirus with a sequence related to the bovine and canine parvoviruses, which are grouped together in the genus bocavirus. the new virus was called human bocavirus, and many research groups worldwide have now confirmed the presence of the virus, particularly in pediatric samples, although it is still not certain how important this virus is in causing morbidity and mortality. their method also amplified a human coronavirus from the respiratory samples, and when sequenced this turned out to be hku , a recently emerged coronavirus detected by scientists at hong kong university. it is possible that a systematic search of human samples using such molecular techniques might reveal more hitherto unknown human viruses. in some cases, the emerging viruses themselves have contributed to other viruses emerging and reemerging in the population. this is especially true of human immunodeficiency virus (hiv), the cause of acquired immune deficiency syndrome (aids), which rapidly spread following its emergence in the early s to infect more than million people worldwide by the end of the twentieth century. because of its severe effects on the immune system, the virus leads to numerous other infections in the hiv-infected population. for example, picobirnaviruses, that had been detected in fecal samples from chickens and rabbits, were difficult to detect in human fecal samples until a cohort of men with aids was examined, and in these humans picobirnavirus was detected for the first time. some rare diseases have become common in persons with aids. for example, the human polyomavirus known as jc virus can cause the rare brain disorder known as progressive multifocal leukoencephalopathy (pml). normally, the virus remains dormant in the kidney, but in hiv-infected individuals the hiv-encoded transactivator tat acts as a transactivator of jcv leading to pml which progresses to death within months after infection. other important virus infections which emerge in aids patients are human herpesviruses (cytomegalovirus, herpes simplex viruses and , varicella-zoster virus, and human herspesvirus , which causes kaposi's sarcoma). hiv is mainly spread through sexual activity between an infected and a noninfected person, and is most common in those who indulge in high-risk sexual behavior with multiple partners. it can also be transmitted by direct contact with infected blood, and is common in persons who indulge in intravenous drug use, particularly when needles, syringes, or equipment used to prepare drugs for injection are shared. it is therefore an example of a virus disease which is dependent on risky human behavior for its maintenance in the human population. the ability of such new infections to spread in the population has been greatly enhanced by population growth and ease of movement as a result of rapid air travel. a dramatic recent example of this was the appearance of the coronavirus causing severe acute respiratory syndrome (sars) in late which spread by air travel from a single infected chinese physician who infected persons in a hong kong hotel. these infected persons then traveled by air and spread the infection to more than individuals worldwide, % of whom died. the virus then apparently receded from the human population in july . only recently was it discovered that the sars coronavirus has a natural reservoir in chinese horseshoe bats (rhinolophus sinicus). some other species such as himalayan palm civets and raccoon dogs from which the virus has been isolated may serve as amplification hosts. following the recognition of the human coronavirus sars, research on coronaviruses intensified, and this led to the discovery in of two previously unrecognized human viruses, one found by hong kong university, called hku virus, and another reported almost simultaneously from the netherlands, called nl , and from yale university, called new haven coronavirus. the latter viruses probably represent two isolates of the same virus species. they are clearly associated with lower respiratory tract infection in children, but initially it was claimed that new haven coronavirus was also associated with kawasaki disease in children. this intriguing claim was rapidly investigated and refuted by several different groups in japan, taiwan, and elsewhere, and the cause of kawasaki disease, which has features resembling a virus infection, remains unknown. a majority of recent emerging virus diseases have been zoonoses (i.e., diseases transmitted from animals to humans under natural conditions). some of the more important of these include hiv- , which was transmitted to humans from chimpanzees in central africa around , and hiv- , transmitted from sooty mangabeys to humans in west africa around . other important recent examples are the viruses of the genus henipavirus. hendra virus was first recognized through a disease outbreak in some horse stables in hendra, queensland, australia, when horses and their trainer died from pulmonary disease with hemorrhagic manifestations in . the reservoir of the virus was found to be in large fruit-eating bats (pteropus spp.), and one year later a horse farmer miles away in mackay, queensland, died of encephalitis from the same virus. then, in , a related virus was discovered in malaysia following a major outbreak of respiratory disease in pigs and neurological disease in humans in their close contact. more than humans died, and in a successful effort to control the disease . million pigs were slaughtered. the causative virus was isolated from a fatal human case that had lived in nipah river village, and so was named nipah virus. hendra and nipah viruses are clearly members of the family paramyxoviridae, but have been placed in a separate genus as their rna genome is about kb in length, larger than that of any other paramyxovirus. nipah virus, like hendra virus, was found to have a reservoir in pteropus bats, and has since been identified in fatal human disease outbreaks in india in and bangladesh in . other new viruses which apparently have a reservoir in fruit bats include menangle virus, a new paramyxovirus which emerged in a commercial piggery near sydney, australia, to cause stillbirths and abortion in pigs. menangle virus also caused disease in two workers in the piggery. a new virus related to menangle virus emerged during an investigation of urine samples from pteropid bats collected on tioman island, off the coast of malaysia, in , and was named tioman virus. during the same investigation, a new orthoreovirus was isolated from pteropus hypomelanus in and called pulau virus, and more recently a related orthoreovirus called melaka virus was isolated from a human case of acute respiratory disease in melaka, malaysia. serological studies of sera collected from human volunteers on tioman island showed that % had antibodies against both pulau and malaka viruses. another important group of zoonotic diseases are rodentborne, and caused by members of the genus hantavirus of the family bunyaviridae. these viruses first emerged during the korean war of - , when thousands of un troops developed a mysterious disease with fever, headache, hemorrhage, and renal failure with a fatality rate of - %. it was more than a quarter of a century before the causative virus was isolated from field mice in korea, and named hantaan virus, the cause of hemorrhagic fever with renal syndrome (hfrs) in humans. then, in , a new hantavirus emerged in the four corners region of southwestern usa as the cause of a severe acute respiratory disease syndrome, with a fatality rate close to %, and named sin nombre virus. this virus was shown to be transmitted to humans by inhalation of virus present in the urine, feces, or saliva of deer mice (peromyscus maniculatus). it seems likely that this disease had existed for many years, and was only recognized in because of a clustering of human cases as a result of a regional upsurge in the rodent population resulting from climatic conditions causing increased availability of rodent food. fortunately, in most of these infections, humans appear to be a dead-end host, and transmission between humans does not occur except with the andes virus in south america. rodent-borne viruses of the family arenaviridae also cause a number of serious zoonotic diseases in humans. the 'old world' arenaviruses such as lassa fever virus have been known for some time, but still cause thousands of fatal hemorrhagic fever cases every year in west africa. however, 'new world' arenaviruses such as junin virus causing argentinian hemorrhagic fever and machupo virus causing bolivian hemorrhagic fever have long been recognized in south america. recently, new arenaviruses have emerged, probably as a result of deforestation, which results in rodents seeking shelter in human habitation, and brings them into closer contact with people. these viruses include guanarito virus that causes venezuelan hemorrhagic fever with % mortality rate from confirmed cases, and sabia virus isolated in that causes brazilian hemorrhagic fever with a high fatality rate, including two laboratory acquired cases. rabies is a zoonotic disease of great antiquity that has mainly been associated with carnivores, such as dogs. the virus is excreted in the saliva of infected animals, and following infection it moves through the nervous system to attack the brain, causing aggressive behavior which results in the animal biting humans and animals with which it comes into contact and thereby spreading the virus infection. fortunately, due to early work by louis pasteur, a vaccine was developed that protects humans or other animals from infection, and can also be given immediately post exposure, and the domestic dog population in the developed world is vaccinated and does not pose a risk to humans. however, in some developing countries, it is not uncommon for a rabid dog to bite and infect more than people before it can be put down, and worldwide there are still some human rabies deaths per year. using molecular sequencing techniques, it is now possible to distinguish the genotypes of rabies viruses associated with different species of host, as the virus has become adapted through frequent transmission between members of the same host species. in the usa, there are six recognized terrestrial animal genotypes, in raccoons in eastern states, skunks in north-central states, skunks in southcentral states, coyotes in southern texas, red foxes in alaska, gray foxes in arizona, and several genotypes associated with particular species of bat. in fact, most fatal cases of human rabies in the usa can now be traced to bats, which are often not detected when the person is bitten; so rabies is not suspected and vaccination is not undertaken until the disease has taken hold. many important virus diseases are spread by arthropods, and exposure to new arthropods and the viruses they carry is critical to the emergence of new virus diseases. dengue hemorrhagic fever is caused by dengue virus which is transmitted mainly by the asian mosquito (aedes albopictus), and dengue fever is one of the most rapidly emerging diseases in tropical regions of the world. there are four serotypes of dengue virus, and it seems that consecutive infections with two antigenic types can lead to the more serious disease of dengue hemorrhagic fever with shock syndrome, which, if untreated, can result in up to % mortality. unfortunately, through the importation of vehicle tires containing water from korea, the asian mosquito was introduced into the usa, and is now present in several regions of the southern states. it can act as a vector not only for dengue virus, but also for california encephalitis virus. in europe, the emergence of two important animal diseases has occurred through the movement of arthropod vectors into the iberian peninsula. african horse sickness virus causes a disease that can be fatal to horses, mules, and donkeys, and is transmitted by nocturnal biting flies of the genus culicoides. these were introduced inadvertently into spain, and the disease is now endemic around madrid and regions to the south. african swine fever virus is transmitted by ticks of the genus ornithodorus and it causes a fatal disease resembling classical swine fever in domestic pigs. it first emerged in portugal and spain in , france in , italy in , and cuba in . through slaughter of infected animals, the disease was eradicated from europe, except sardinia, by . the most recent dramatic example of the movement of a virus vector is provided by west nile virus, a flavivirus first isolated in uganda in . this virus uses birds as a reservoir host, and is transmitted from birds to humans and other vertebrates by mosquitoes. in , cases of encephalitis in new york were found to have been caused by a strain of west nile virus that was phylogenetically similar to a virus isolated from geese in israel. at the same time, many birds, especially corvids, began dying in new york state. since the introduction in , west nile virus has become well established throughout the usa and moved north into canada and south into the caribbean and into mexico. it is not known how the virus moved from israel to the usa, but the most reasonable explanation is that it was carried in an infected mosquito or possibly an infected bird in the hold of an aircraft. transmission by an infected human seems less likely since the titer of virus in human blood is usually too low for efficient mosquito transmission. it is clear, nevertheless, that once it arrived in north america, west nile virus found an extremely favorable environment with abundant avian and arthropod hosts that facilitated its spread throughout the american continent. the emergence of new viruses is likely to continue as viruses evolve and find new ecological niches in the human and animal population. it is noticeable that most newly recognized viruses have been rna viruses, perhaps since rna evolves at a faster rate than dna, for which host cells have developed efficient proofreading enzymes. it will be important in the future to detect new viruses before they can emerge to cause disease in the population. the sars epidemic provides an excellent example. before the epidemic, only two human coronaviruses were known, human coronaviruses e and oc . despite the fact that serious coronavirus diseases were well known in other vertebrates, such as feline infectious peritonitis and avian infectious bronchitis virus, it was not until the sars epidemic that research on human coronaviruses led to the discovery of three new human coronaviruses -sars, hku , and nl /new haven. there are other genera of viruses that cause serious disease in animals but have not been adequately investigated in humans. an example is the genus arterivirus, which has members causing serious disease in horses and pigs, but has not been reported at all in humans. this could be a worthwhile area for future investigation. another critical factor in the future control of emerging viruses is better vector control. when mosquito control was conducted using ddt, dengue fever virus was virtually eliminated from the americas in the s, but environmental concerns led to the widespread banning of the use of ddt, so that since the s there has been a considerable expansion of dengue fever in south america, with the appearance of dengue hemorrhagic fever there for the first time. there is a real need to improve mosquito control measures to control this disease. although there are prospects for a dengue virus vaccine, this is so far not available. finally, one of the most important viruses that continue to emerge in different antigenic forms is influenza virus. the main reservoir of influenza viruses is in birds, and over the past century several pandemics of influenza have emerged, the most serious of which was in . pandemic strains usually arise by a process of antigenic shift, where one of the genes encoding the hemagglutinin and/or the neuraminidase of influenza virus is replaced by one from birds. new pandemics occurred in (h n subtype), (h n subtype), and (h n subtype). since , there have been no new pandemics, but it is widely expected that another will occur. at the time of writing, there is worldwide concern that a highly pathogenic avian influenza virus (h n subtype), which has caused some human infections and deaths in persons in close contact with infected birds, might mutate or recombine to generate a virus which would be highly transmissible in the human population. plans are being developed in many countries and by the who to try to prepare for such an event by generating possible vaccines against such a virus and stockpiling antiviral drugs. emerging and reemerging virus diseases of plants identification of the major, parenteral non-a, non-b hepatitis agent (hepatitis c virus) using a recombinant cdna approach nipah virus: a recently emergent deadly paramyxovirus identification of new papillomavirus types critical review of the vector status of aedes albopictus west nile virus: epidemiology and clinical features of an emerging epidemic in the united states nipah virus encephalitis reemergence the widening scope of coronaviruses timing the ancestor of the hiv- pandemic strains a novel coronavirus associated with severe acute respiratory syndrome emerging zoonoses: crossing the species barrier the emergence and reemergence of viral diseases molecular and biophysical characterization of tt virus: evidence for a new virus family infecting humans genetic identification of a hantavirus associated with an outbreak of acute respiratory illness microbial threats to health, emergence, detection and response, pp. pp are we ready for pandemic influenza h n ? geminivirus a class of plant viruses with a genome composed of single-stranded dna encapsidated in geminate particles.pandemic an epidemic at the regional or worldwide level. satellite an infectious molecule that depends on a helper virus for crucial functions. key: cord- -qjfkvu n authors: tang, lu; zhou, yiwang; wang, lili; purkayastha, soumik; zhang, leyao; he, jie; wang, fei; song, peter x.‐k. title: a review of multi‐compartment infectious disease models date: - - journal: int stat rev doi: . /insr. sha: doc_id: cord_uid: qjfkvu n multi‐compartment models have been playing a central role in modelling infectious disease dynamics since the early th century. they are a class of mathematical models widely used for describing the mechanism of an evolving epidemic. integrated with certain sampling schemes, such mechanistic models can be applied to analyse public health surveillance data, such as assessing the effectiveness of preventive measures (e.g. social distancing and quarantine) and forecasting disease spread patterns. this review begins with a nationwide macromechanistic model and related statistical analyses, including model specification, estimation, inference and prediction. then, it presents a community‐level micromodel that enables high‐resolution analyses of regional surveillance data to provide current and future risk information useful for local government and residents to make decisions on reopenings of local business and personal travels. r software and scripts are provided whenever appropriate to illustrate the numerical detail of algorithms and calculations. the coronavirus disease pandemic surveillance data from the state of michigan are used for the illustration throughout this paper. coronavirus disease , an infectious disease caused by severe acute respiratory syndrome coronavirus (sars-cov- ) (world health organization, ), has become a global pandemic that has spread swiftly across the world since its original outbreak in hubei, china, in december . as of june , this pandemic has caused a total of confirmed cases and fatalities in more than countries. being one of the most lethal communicable infectious diseases in human history, it is expected that the covid- pandemic will continue spreading in the world population, causing even higher numbers of infections and deaths in the future. with no effective medical treatments or vaccines currently available, public health interventions such as social distancing have been implemented in most of the countries to mitigate the spread of the pandemic. one of the central tasks of statistical modelling is to provide a suitable risk prediction model that enables both government and public health workers to evaluate the effectiveness of public health policies and predict risk of covid- infection at the national and regional levels. such information is valuable for governments to assess the preparedness of medical resources (personal protective equipments and intensive care unit beds), to adjust various intervention policies and to enforce the operation of social distancing. modelling for infectious diseases has a profound role in informing public health policy across the world (heesterbeek et al., ; siettos & russo, ) . the outbreak of the covid- pandemic in december has led to a surge of interest in disease projection that ubiquitously relies on mathematical and statistical models. a crucial step in modelling disease evolution is to capture key dynamics of the underlying disease transmission mechanisms from available public health surveillance data, which enables reliable projection of disease infection into the future. a prediction model may help us foresee some possible future epidemic/pandemic scenarios and learn consequent impacts of current economic and personal sacrifices due to various control measures. because of both data quality and data limitations from public surveillance data systems, a statistical model should take the following features into account in its design and development. first, a statistical model should be able to make predictions and, more importantly, to quantify prediction uncertainties. forecasting is known to be a notoriously hard task, which depends heavily on the quality of data at hand and a certain model chosen to summarise the information from observed data and then to reproduce information beyond the observational time period. the chosen model is of critical importance to deliver prediction. this paper concerns a review of the family of classical compartment-based infectious disease models, which have been the most widely used mechanistic models to capture key features of infection dynamics. we begin with the most basic susceptible-infectious-removed (sir) model to build up the framework (section ), and this three-compartment model is then generalised to have more compartments to embrace additional features of infection dynamics (section ), such as the well-known fourcompartment model, susceptible-exposed-infectious-removed (seir) model, which takes the incubation period of contagion into account. given many types of factors potentially influencing the evolution of an epidemic, a single prediction value is insufficient to be trustworthy unless prediction uncertainty is reported as part of forecast analysis. quantification of prediction uncertainty is of critical importance, especially when a forecast is made at an early phase of an epidemic with limited data. building sampling variations in infectious disease models makes a statistical modelling approach different from a mathematical modelling approach. a clear advantage of a statistical model is that the model parameters, including those in the mechanistic model, can be estimated, rather than being specified by certain subjectively chosen prior information. second, the consideration of building sampling uncertainties in the modelling of infectious disease is a fundamental difference of a statistical modelling approach from a mechanistic modelling approach known in the mathematical literature of dynamic systems. a mechanistic model is typically governed by a system of ordinary differential equations, such as the existing three-compartment sir model consisting of three differential equations, which explicitly specifies the underlying mechanisms of an epidemic. this model is assumed to govern an operational system of disease contagion and recovery or death, which, in reality, cannot be directly observed. most of the time, public surveillance data are accessible, which represent only a few snapshots of the underlying latent mechanistic system of an epidemic. such gaps may be addressed by a statistical model that incorporates sampling schemes to explain how observed data are collected from the underlying infection dynamics. in turn, prediction uncertainty will reflect forms and procedures of the chosen sampling schemes specified in the statistical model. in this paper (section . ), we will introduce the state-space model as a natural and effective modelling framework to integrate the mechanistic model and sampling schemes seamlessly. third, given the scarcity of the available data in public health surveillance systems, the complexity of a model used for prediction should be aligned with the issue of parameter identifiability. for example, at the beginning of an outbreak, one should consider a simple model, which may be expanded over the course of an epidemic's evolution with increased data availability. to make the specified model useful to answer a certain question of practical importance, a relevant feature should be included in the model building. for example, in the study of control measures to mitigate the covid- spread, the model specification should incorporate a structure that is sensitive to the influence of a preventive policy. in section . , we will introduce an expansion of the basic sir model in that time-varying control measures are allowed to enter. the flexibility of permitting certain modifications is an important property of a model to be considered in an infectious disease model. in this field, all models need to be tailored with increased data and more knowledge from the literature as a disease evolves over time. from this point of view, compartment-based models are superior to other models because, for example, it is easy to add other compartments, such as an exposure compartment, a quarantine compartment or a self-immunisation compartment, to improve the mechanistic model, to answer specific question of practical importance and to capture distinctive data features for better prediction. fourth, as the epidemic evolves further, surveillance data become abundant and have higher resolution. for example, in the usa, the numbers of confirmed symptomatic covid- cases and case fatalities are recorded for each county. the average county population size in the usa is approximately , so a microinfectious model may be built upon county-level surveillance data to make high-resolution prediction and to assess the effectiveness of control measures at a community level. this paper (section ) will discuss this important extension of the classical sir model, essentially a temporal model, to a spatio-temporal model that enables borrowing of information from different spatially correlated counties in the improvement of risk prediction. this exemplary model generalisation sets up an illustration from a nation-level macromodel to a county-level micromodel. the latter is more relevant and useful for local governments to make decisions of business reopenings and for residents to be aware of local infection risk. last, to make research findings transparent and to place resulting toolboxes into the hands of practitioners, an open-source software package must be a deliverable. this is indeed a rather demanding task, as the ease of implementation and numerical stability impact the choice of statistical models and statistical methods for estimation and prediction. note that not every statistical model permits delivery of a user-friendly computing package that is general and flexible enough to handle various types of data. in this paper, we focus on the discussion of markov chain monte carlo (mcmc) methods that have been developed in the literature to perform estimation and prediction for state-space models (section . ). in this paper, we invite the readers on a journey of surveillance data, modelling, estimation and prediction, implementation and software development. after reading this paper, one should be able to use existing compartment-based models or to expand them in a study of an infectious disease epidemic, to improve estimation and/or prediction methods, or create one's own software. it is our hope that this paper may pave the path to learning, practising or developing new methodologies that are useful for a broader range of infectious disease modelling problems. multi-compartment models have been the workhorse for modelling infectious diseases since the early th century. they are a class of mathematical models used for describing the evolution of masses (in unit of proportions or counts) among the compartments of a varying system, with broad use cases in epidemiology, physics, engineering and information science. this is a dynamic system that is typically represented by a system of ordinary differential equations (odes) with respect to time, and, given a starting condition, the mass in each of the components is regulated by a function over time. an ode is a simple mathematical model to depict a trajectory of a functional trend. one of such examples used extensively in epidemiology is an exponential growth function, f.t/ d e t , which may be viewed as a solution to an ode of the form: df .t / dt d f .t/, or dy dt d y, where y is a function of time t, which obviously is y d f.t/ d e t with an initial condition f. / d . it is worth pointing out that this simple ode explicitly characterises the rate of change (speed or velocity) for function y d f.t/, rather than directly specifying a form for the function f.t/ itself. such rate-based characterisation is termed as 'dynamics' in the mathematical literature. clearly, this ode is not a statistical model as it does not provide a law of data generation; in other words, there is no randomness in this ode to reflect sampling uncertainties. a typical multi-compartment model consists of several odes for a vector of rates that are linked each other. this is referred to as a dynamic system. the forms of odes are specified according to relevant scientific knowledge about the understanding of the underlying dynamic mechanism related to an infectious disease. in the context of infectious disease modelling, the sir model is the most basic threecompartment dynamic system that describes an epidemiological mechanism of disease evolution over time (see figure ). in brief, the model describes the flow of infection states or conditions by (i) moving susceptible individuals to the infectious compartment through a transmission process (the first arrow) and (ii) moving infectious individuals to the removed compartment (either dead or recovered) through a removal process (the second arrow). at a given time, the total population n under a study is partitioned into the three compartments, denoted by s, i and r, and their sizes satisfying s c i c r d n. with a slight abuse of notation, this notation denotes either the type of compartment or the size of compartment, whichever is applicable in a given context. in other words, s, i and r are used to denote the sizes of the mutually exclusive subpopulations of susceptible, infectious and removed individuals, respectively. this compositional constraint, that is, s c i c r d n, may be interpreted in a term of probability (or proportion) as follows: at a given time, an individual in the population is either at risk (susceptible), or under infection by a virus (infectious), or removed from the infectious system due to recovery or death; that is,  s c  i c  r d , where  s ,  i and  r are, respectively, the probabilities of being susceptible, infectious and removed. this presents the primary constraint for a multi-compartment infectious disease model. more details of the sir model will be described in section . often times, the interest for such system lies in the function values over time, but the closedform analytical solution for such functions may not exist. for example, to answer the question of how many individuals will be infected with the covid- by the end of the year (or any future time) requires to know a calculator that computes the cumulative numbers of susceptible, infected and removed cases over time from the past to the future. unfortunately, in reality, functions relevant to this calculator are usually non-linear, and their exact forms are difficult to directly specify. in contrast, a set of odes helps better understand the disease transmission dynamics (i.e. traits of infectious diseases) and more conveniently captures their key features, where each ode may correspond to one mode of disease evolution. such odes for disease spread may be regarded as a model for the expected dynamic mechanism, serving as a systematic component in a statistical model. numerical methods such as the euler discretisation method or the runge-kutta approximation method (stoer & bulirsch, ; butcher, ) can be used to obtain approximate solutions of such odes with given boundary conditions. regardless of methods used, solutions to a dynamic system are deterministic functions. we illustrate a basic mechanistic model of disease spread in the succeeding text. additional review from deterministic and mathematical perspectives of multi-compartment models is given by anderson et al. ( ) and hethcote ( ) . example . consider the sir model for a hypothetical population with a constant population of n d residents and an initial condition of susceptible individuals, infectious individual and individual removed (either died or recovered). here subjects may be also regarded as % if the unit of proportion is used in the interpretation. the transitions between compartments, written in odes as in ( ), represent population movement from one compartment to another (see figure ). we consider an example withˇd : (a rate of moving from s to i) and d : (a rate of moving from i to r), leading to r dˇ= d : . here r is the socalled basic reproduction number that quantifies an average number of susceptible individuals contracting a virus from one contagious person in an environment of no preventive measures. this is a quite infectious scenario as we will see later. the r script in the succeeding text shows a scenario of obtaining the solution to the system of odes by standard ode solvers ( r package desolve) using the first-order euler method (not shown) or the runge-kutta fourthorder (rk ) approximation method (figure ). details about the rk method can be found in appendix a . . figure , on each of these days, the sum of the three values from the three curves is always equal to , presenting a time-varying redistribution of the individuals. with no control measures in this hypothetical infection dynamics, the susceptible compartment quickly drops and reaches an equilibrium state after days of the outbreak, and during the period of first days, the infectious compartment increases to a peak and then decreases to zero (no contagious individuals in the population) as all currently infected individuals move to the removed compartment, which is the exit of the system. despite relying on a valid infectious diseases mechanism, deterministic approaches have several drawbacks: (i) the actual population in each compartment at a given time is never accurately measured because we only obtain an observation around the mean; (ii) the nature of disease transmission and recovery is stochastic on the individual level and thus never certain; and (iii) without random component in the model, it is neither possible to learn model parameters (e.g. r ) from available data nor to assess prediction uncertainty. the latter is of critical importance given many unobserved and uncontrolled factors in surveillance data collection. in an early stage of the current covid- pandemic, the daily infection and death counts reported by health agencies are highly influenced by the availability of testing kits, reporting delays, reporting and attribution schemes, and under-ascertainment of mild cases in public health surveillance databases (see discussions in angelopoulos et al., ; banerjee et al., ) ; both disease transmission rate and time to recovery or death are also highly uncertain and vary by population density, demographic composition, regional contact network structure and non-uniform mitigation schemes (ray et al., ) . hence, statistical extensions are necessary to incorporate sampling uncertainty in estimation and inference for infectious disease models. the main focus of this paper will be given to a statistical modelling framework based on a class of state-space models, in which the systematic component is specified by multicompartment infectious disease models while the random component is governed by a certain sampling distribution of surveillance data. note that multi-compartment infectious disease models present a class of classical mechanistic models widely used in practice and that incorporating certain sampling distributions allows to make statistical estimation, inference and prediction with quantification of uncertainties. we organise the paper as follows. in the first part of the paper, we introduce a class of macromodels. we begin with the most basic sir mechanistic model in details, followed by some important extensions used to address representative scenarios of disease spread and infection evolution. examples include seir model with an additional compartment of exposure accounting for potential incubation period of infection and susceptible-antibody-infectious-removed (sair) model with an additional compartment of antibody accounting for potential self-immunisation after being infected. then, we formally introduce the framework of state-space models, a powerful statistical modelling approach that aims to model available surveillance data from public health databases with the utility of the underlying latent mechanistic model. in the second part of the paper, we introduce a class of micromodels. when an epidemic continues, data become abundant and of high resolution at community level. for example, the surveillance data of the covid- pandemic in the usa are collected from individual counties. this allows building county-level microinfectious models in addition to country-level or state-level macromodels. being a certain subgroup analysis, such micromodelling is appealing to address spatial heterogeneity across the more than counties in the usa and consequently improves the prediction accuracy. as far as the spatial modelling of infection dynamics concerns, we review the classical cellular automata (ca) that is extensively used to describe person-to-person interacting rules associated with epidemic spreading patterns in a population via relevant interlocation connectivity functions. this ca may vary spatially and temporally, which presents a principled way to extend a state-level macroinfectious disease model to a stratified microinfectious model. in addition to the case of geographical subgroups, other types of subgroups by, for example, age, race, income, political party and economy, are also of interest. our main objective of this paper is to introduce to readers the basics of infectious disease models, underlying modelling assumptions, statistical analyses and possible extensions. examples will be provided for demonstration purposes. this review targets readers who have had some statistical training but no prior experience in infectious disease modelling. the first infectious disease model (mckendrick, ; kermack & mckendrick, ) is widely known as the susceptible-infectious-removed model, or in short the sir model (see figure ). it is a three-compartment model for studying how infectious diseases evolve over time on the population level. it defines a mechanism of disease transmission and recovery for a population at risk by a dynamic system of three disjoint states: susceptible, infectious and removed. we note an important distinction between infectious and infected individuals. infectious individuals are those who are currently infected and not yet recovered or dead (currently infected individuals become infectious immediately in the sir model, although it may not be true in reality; see the seir model in section where currently infected individuals become infectious with a delay in time), whereas infected individuals could mean only currently infected or both currently and previously infected. for clarity, we will refer to currently infected as infectious so that the three states in the sir model are mutually exclusive. individuals in the susceptible state are not immunised and can become infected by coming into contact with infectious cases, so they are at risk at a given time. individuals in the infectious state contribute to the transmission of the disease until they ultimately recover or die, so they are contagious. individuals in the removed state include those who either recover or die (without distinction). this is an exit from the infection system, meaning that once an individual leaves this system (recovers or dies), he or she would never return to the system. this is true for people who die from the virus but may not be the case for recovered individuals. thus, in the sir model, there is a technical assumption that a recovered individual would become self-immunised to the virus and no longer impact the disease transmission. a possible way to relax this assumption is to create two separate compartments corresponding to recovery and death states, respectively, leading to a four-compartment infectious disease model. to make our presentation focused on the basic three-compartment model, we make this self-immunisation assumption in this section. given what we said earlier, the current version of sir is only applicable for diseases, where long-term immunity can be developed, and does not apply to recurring infectious diseases, such as the common cold. this is because the disease transmission rate is set as a constant in sir. in this section, we introduce the sir model in its basic deterministic form (section . ), define reproduction numbers (section . ), elaborate its assumptions (section . ) and properties (section . ) and present some technical extensions to the basic sir model. mechanistic extensions, such as modifications to the three-compartment sir model to account for additional components or disease mechanism, are discussed in section . we use s.t/, i.t/ and r.t/ to denote the time-course subpopulation sizes (i.e. the number of individuals) distributed into each of the three compartments at a given time t, where t is continuous. clearly, where n is the total population size, which is a fixed constant. the starting time is denoted as t d . the rates of change among these subpopulations are represented by a system of odes: ( ) ( ), these three odes define a dynamic system of three deterministic functional trajectories over time, including the susceptible trajectory s.t/, the infectious trajectory i.t/ and the recovered trajectory r.t/ for t . this sir dynamic system is well posed in the sense that non-negative initial conditions lead to non-negative solutions of the three functional trajectories. these trajectories collectively demonstrate the evolutionary mechanism of an infectious disease. the sir dynamic system in ( ) may be interpreted as follows. let us consider events occurring instantaneously at time t. in the first ode, the ratio i.t/=n represents the proportion of contagious individuals in the population, which may be thought of as a chance that a person in the at-risk population may run into a virus carrier. if each individual at risk has an independent chance to meet a contagious person, then, according to the binomial distribution, the expected number of susceptible individuals contracting the virus is s.t/i.t/=n. in reality, a person at risk may run intoˇ(say, ) contagious individuals, leading to a modified chanceˇi.t/=n. thus, instantaneously at time t, the system gains an additional number of infected cases equal toˇs.t/i.t/=n, and these cases will leave the susceptible compartment to enter the infectious compartment. such loss to s.t/ is attributed to the negative sign in the first equation. in the second ode, the first term is the number of new arrivals of contagious individuals and the second term is the loss of contagious individuals at a rate who either recover or die and then enter the removed compartment. the third ode is based on an absorbed compartment that always accumulates with new arrivals with no departure cases. in the literature, the transition rate represents the fraction of the infectious population that exits the infectious system per unit time. for example, d : means that the infection compartment will decay (or infectious individuals being recovered or dead) at an average rate %. in other words, = describes the expected duration ( days for d : ) over which an individual stays infectious under the exponential distribution of time for his or her sojourn. variations of the form in ( ) are often seen in the literature. among those, the most important sir specification is given as follows. because the total population n remains constant over the duration of infection, by dividing both sides of the ordinary differential equations by n, the rates of change in terms of population proportions can be derived, without changing the interpretation ofˇand . that is, ( ) where  s .t/,  i .t/ and  r .t/ are the probabilities (or proportions) of being susceptible, infectious and removed at time t, respectively. here the probability of being infectious  i .t/ is also known as the prevalence of disease in the epidemiology literature (see, e.g. osthus et al., ; wang et al., ) . a clear advantage of this alternative form of the sir model ( ) where the population size n is implicitly absorbed into the parameter of disease transmission rateˇ, which may be interpreted as a per capita effective contact in proportion to the population (see, e.g. johnson & mcquarrie, ). despite the differences in notations and presentations, they convey the same infection mechanism, but interpretations need to be given accordingly. although we use these model specifications exchangeably in this paper, the form given in ( ) is recommended to conduct practical studies. based on the two parametersˇand in an sir model, the ratio r dˇ= is termed as the basic reproduction number, which captures the expected number of new individuals who directly contract the virus from one contagious individual in an environment with no preventive measures. intuitively, it is a product of the infection rateˇand the infectious duration = . the basic reproduction number r does not depend on the distribution of people over the three compartments and presents a key appealing disease characteristic for describing and comparing across infectious diseases (see, e.g. chowell et al., ; ferguson et al., ; khan et al., ; liu et al., ). an epidemic is expected to occur when r > , or to disappear when r < . this is because in the sir model ( ), at the condition of s.t/=n , the former is equivalent toˇ> , leading to di.t/=dt .ˇ /i.t/ > , while the latter implies di.t/=dt < . the earlier interpretation of r relies on an implicit assumption that all contacts with a contagious individual are susceptible, which contrasts with the effective reproductive number. the effective reproductive number is defined as r e .t/ d r s.t/ n . it represents the expected number of newly infected individuals who contract the virus directly from a contagious individual at time t, given that each susceptible individual has a chance of s.t/=n to meet this contagious individual. this is not to be confused with the notation r.t/, the removed population. in the early outbreak of an infectious disease in a large population, r e .t/ r because s.t/=n . in contrast to r , which is only descriptive of the disease itself (or the progression of disease near time ), r e .t/ reflects the progression of the infectious disease in a population at any given time because it directs the sign of di.t/=dt corresponding to acceleration or deceleration of the infection dynamics. this may be seen by the second-order derivative d i.t/=dt ; a time, say t , at which d i.t /=dt d or the rate di.t /=dt reaches a peak, is referred to as a turning point (see the peak in the middle panel of figure ). hence, r is of most interest during the early phase of an epidemic, whereas r e .t/ is of most interest later on during the controlling phases of an epidemic. for example, the so-called herd immunity is the natural immunity developed when an epidemic reaches r e .t/ < . in other words, without interventions, it requires the proportion of susceptible individuals to be no more than =r , or the combined proportion of infectious and recovered to be at least =r in order to contain the spread. as another example, if an effective vaccine becomes available at time q t > , knowing r e . q t/ allows us to estimate the remaining proportion of population that needs to be vaccinated in order to control the epidemic (i.e. for achieving r e .t/ < ). figure shows that the effective reproductive number r e .t/ for example decreases as the group of susceptible individuals, s.t/, shrinks over time, eventually reaching below the threshold of at time . the value at time is r d r e . / d : , while r e . / d . the time of reaching this threshold also marks a special time of interest-when the number of active contagious individuals starts decreasing at time after reaching its maximum, as shown in the middle panel of figure . like every mathematical model, there are some assumptions and constraints such as boundary conditions that the sir model needs to satisfy. these restrictions define the circumstances where the sir model may be appropriate to use in practice. although some of them have been mentioned earlier, for the sake of self-contained summarisation, we list all key assumptions as follows. assumption : the population involved in the infection is closed with no additions or leakage of individuals, and the size of the population is fixed, say, n. this assumption may be satisfied by an epidemic that is rapid and short lived, during which disease evolution is not affected or is minimally affected by vital changes (e.g. natural births or deaths) and migration (i.e. immigration and emigration). technically speaking, the three compartments satisfy the condition of the form: assumption : individuals in the population meet each other randomly in that both probability and degree of interactions with one another remain constant over time, regardless of geographical and demographic factors. this is a strong assumption of homogeneity for the sir dynamic system that is governed by the same transmission and recovery param-etersˇand . in practice, such a homogeneity assumption may be easily violated. thus, modelling with heterogeneous dynamics of infection is an important and active research area in the literature on infectious diseases. assumption : one susceptible individual can only develop immunity (or selfimmunisation with antibody against the virus) through infection (i.e. no vaccination). in other words, as shown in figure , the infectious compartment is the only exit of the susceptible compartment, and there is no other state to which an at-risk individual would move next. once recovered from infection, one becomes immune to the virus for the remainder of the study period and would not return to be susceptible again. in effect, this is a rigorous definition of recovered case in the sir model. from a view of the graphic representation in figure , this implies that there is no connection from the removed compartment to the susceptible compartment, or in other words the removed compartment is the terminal state of the infection dynamics. it is worth pointing out that to date the validity of this assumption for the covid- pandemic remains unknown. in the literature, this condition is assumed for a certain period of time over which risk prediction is considered. assumption : the infection has zero latent period in that one becomes infectious once exposed. this is a key distinction of the sir model from the seir model. like many infectious diseases, the covid- has a reported average incubation period of between and days (li et al., ; pan et al., ) , which adds some additional complexity in the modelling of infectious disease dynamics. as a matter of fact, this latency of contagion is really the timing of being contagious and not that of being symptomatic. some studies have found that covid- carriers are most contagious in the early phase of illness prior to the occurrence of noticeable clinical symptoms (ip et al., ; he et al., ) . given these findings, it is tricky to see how the compartment of exposure for incubation would be added to extend the sir model for the covid- pandemic. assumption : because the sir model has constant transmission and recovery parameterš and , which are not time varying, the underlying infection is assumed to evolve in fully neutral environments with no mitigation efforts via external interventions such as a public health policy of social distancing, effective medication or fast testing kits for diagnosis. as far as the covid- pandemic is concerned, this is the biggest restriction of the sir model, which is not reflective of the reality-almost all countries with reported covid- cases have issued various non-pharmacological control measures. many researchers have proposed solutions to overcome this unrealistic assumption of the sir model in the analysis of covid- data (see, e.g. wang et al., ) . assumption : the population size n is large enough to have enough number of incidences, including the number of infections, the number of deaths and the number of recovered cases, so that the sir model parameters can be stably estimated with high precision. technically speaking, this is not a model assumption but a condition of sample size for statistical power. because this mechanistic model will ultimately be used for risk projection, a well-trained model with reliable data is necessary to not only produce an accurate prediction but also to adequately assess the prediction uncertainty. although these six assumptions specifically concern the sir model, most of these discussions or associated insights are useful to understand the restrictions of sir model extensions that will be presented in the remaining sections. knowing possible violations of a certain restriction on a multi-compartment model in data analyses gives rise to potential new research problems for further investigation. to further understand the mechanism of infection governed by the sir model, we now give a brief summary of its analytic properties that provide useful guidelines for us to build statistical models and methods to learn the sir model from available surveillance data from public health databases. ( ). more importantly, although the dynamic system defined by the sir model is continuous over time, available surveillance data are reported at discretised measurements over discretised time points. for example, most of the covid- public databases update data on a daily basis, in which 'a day' is the unit of time for measurement. knowing this discrepancy between the continuous time underlying mechanistic model and the sampling frequency at discrete times for available data is essential to create a statistical framework to link the sir model with the data at hand. property : the sir model is deterministic and does not contain any probabilistic components. it is noteworthy that dynamics and stochasticity are two different mathematical properties; a dynamic system (e.g. the sir model) is not necessarily stochastic, while a stochastic system is not necessarily dynamic. as shown in figure , the compartment sizes s.t/; i.t/ and r.t/ are time-varying functions with no random fluctuations, which are completely determined by the model parameters and the initial conditions of the sir model. obviously, this is a limitation of the sir model when it is applied for data analysis, where data collection is subject to profuse uncertainties and random errors. property : it is easy to show that the number of individuals at risk (in the entry of the system), s.t/, is monotonically non-increasing and that the number of removed cases (at the exit of the system), r.t/, is monotonically non-decreasing (see figure ). hence, the total number of individuals who have been exposed to a virus is equal to n s.t/ d i.t/ c r.t/, which is monotonically non-decreasing. i.t/, the number of active contagious cases, or the difference between the two groups of the exposed cases and the recovered cases, can be either increasing or decreasing. the middle panel of figure nicely conveys such directionality of movements, in which the time of i.t/ reaching the peak and the time of i.t/ reducing to zero are two important turning points of interest in epidemiology. the former indicates the turning point of disease mitigation, and the latter corresponds to the turning point of disease containment. property : it can be shown that i. / d (or equivalently,  i . / d ), meaning that the disease will eventually die out. this is because when t ! , the rate of prevalence  i .t/, given by .ˇÂ s .t/ / in ( ), will become negative at a certain time and then become more and more negative until converging to zero because  s .t/ is a decreasing function and  i .t/ is bounded in the succeeding text by zero. however, this property of decaying to zero is conditional on the assumptions listed earlier. violation of assumptions and are most likely to cause a disease to persist because the monotonicity of s.t/ used in the earlier argument is no longer valid. an example of such diseases includes seasonal influenza, where immunity does not last long. property : the sir model has a recursive property in that at any given time, disease progression (i.e. shapes of the three functions) is only dependent on their current values and not on other information from the past. this property of recursion should not be confused with the markov property that has exclusively used in the literature of stochastic processes under the conditional probability law. here there is no probability law involved in the recursive operation, which is indeed a fully deterministic recursion. such conceptual distinction may help understand the differences between dynamics and stochasticity. during an epidemic, various control measures are typically issued by governments to mitigate or contain the spread of the disease. a direct impact of these external interventions is that both the transmission and recovery rates are no longer constant over time. thus, an important generalisation of the sir model is to accommodate different degrees of mitigation policies, including social distancing, limiting transportation, mandatory mask wearing and city lockdown. as observed in the ongoing covid- pandemic, mitigation strategies are changing over time. limiting mobility of susceptible individuals and medically isolating contagious individuals in the population would reduce the rate of contracting virus, leading to a decreasing disease transmission rateˇ.t/. at the same time, gaining better knowledge on both treatment and self-management of symptoms and improving medical resources may increase the rate of recovery .t/ over the course of an epidemic. incorporating time-varying parameters into the sir model leads to an important extension of the basic sir model ( ): the form ofˇ.t/ can be specified mainly in two ways. one is to letˇ.t/ be either a parametric function (e.g. exponential decaying function) or a non-parametric function (smirnova et al., ; sun et al., ) , both of which may be estimated from available data. one useful feature for the use of a parametric function ofˇ.t/ is to incorporate seasonality in the transmission rate. it is well known that many infectious diseases spread most quickly in some of the winter months. especially, respiratory infectious diseases caused by some coronaviruses exhibit seasonal behaviours that are consistent with the trends of temperature and humidity (barreca & shimshack, ; sajadi et al., ) . accounting for such seasonal periodicity in the model would produce a better long-term prediction of an epidemic. as the public attention for covid- pandemic projection gradually shifts from the short term to the long term, it becomes increasingly important to take seasonality into account. following dietz ( ) , a simple way to introduce seasonality is to assume that the transmission rateˇfluctuates over the period of a year:ˇ. t à ; t d ; : : : ; ; whereˇ is the average contact rate, oe ; is the degree of seasonality with d reducing the model to the basic sir model, and oe ; / is the offset in time horizon so that peak transmission occurs at t d . other periodic functions or their combinations can also be used to model seasonality. as an alternative to a fully non-parametric function, wang et al. ( ) assume a form .t/ dˇ .t/, < .t/ Ä , where .t/ is a known function specified according to given control measures. this specification allows to assess the effectiveness of a target preventive measure, as well as to compare different preventive strategies. clearly, the model with .t/ Á represents disease progression in the absence of any mitigation effort, which sets up the baseline situation in the policy assessment and comparison. the flexibility in specifying .t/ allows easy incorporation of future business reopening events; for example, in the covid- pandemic, this function may be specified as a u-shaped curve in that control measures (e.g. social distancing) gradually relax after a certain time point (see more details from wang et al., , and some numerical results of the covid- data analysis). more discussions on the time-varying transmission rate are given in section . . the assumption of a fixed population size is restrictive, especially when an epidemic remains for a long period of time before it is contained. in this setting, inclusion of natural birth and death dynamics is needed to adequately characterise the time-varying size of each compartment in the sir model. first, let be the natural birth rate and let be the natural death rate. so, the population size will change according to the ode of the form in this case, there are three exits for natural deaths, each occurring at one compartment. an extension of the basic sir model is given as follows: ; as desired. note that when model ( ) is used, n.t/ will be automatically absorbed into the proportions and thus no longer appears in the model formulation. in this section, we review several four-compartment mechanistic models as extensions of the basic sir model introduced in section . being a simple version of a mechanistic model with three compartments, the sir model has some limitations in real-world applications. thus, extensions of this basic type to account for different disease mechanisms and assumptions have been widely considered in the literature. the commonly studied seir model takes into account an incubation period by adding an exposed compartment in between susceptible and infectious compartments (see figure ). the underlying assumption here is that individuals in this exposure subpopulation have contracted the virus but are not yet contagious and are bound to become contagious. in the current literature, most infectious diseases that are suitable for the sir model are believed to fit in the seir model. the exposed compartment may be regarded as a waiting room for virus carriers who are about to spread the virus in the population. let ı be the rate for an exposed individual becoming contagious. then, the basic sir model can be extended to a four-compartment model consisting of the following four odes: where e.t/ is the size of the exposed compartment at time t. in this case, the compositional constraint becomes this constraint is clearly satisfied by the seir dynamic system defined in ( ). let  e .t/ be the probability (or proportion) of being exposed to the virus. then, the rates based sir model ( ) can similarly be extended from the model ( ) earlier. technically, the seir model often suffers from the issue of parameter identifiability because determining a correct incubation period of an infectious disease and thus the parameter ı is a rather difficult task in practice. first, incubation period varies from one person to another; in the case of covid- , the incubation period ranges from to days, with a median of . days (lauer et al., ) . in another study of covid- patients in china, guan et al. ( ) have reported that the estimated incubation period is between to days with a median of days. it is clear that this quantity is very person dependent. second, ascertainment of contagion may be largely delayed because of shortage of virus testing sources. this length-biased sampling problem is notoriously challenging for the estimation of the incubation period (qin et al., ) . third, in the literature (e.g. he et al., ) researchers found that covid- carriers tend to be more contagious right after contracting the coronavirus than a week later because they are not self-quarantined in the absence of clinical symptoms. in other words, in the case of the covid- , the incubation period (or sojourn at exposed state) is too short to play a substantial role in the modelling of the pandemic. not all infectious diseases will develop long-term immunity. individuals may develop immunity after recovery only for some time and could lose immunity such that they become susceptible again. thus, recovered individuals rejoin the susceptible compartment after a certain duration of immunity. this disease evolution is intuitively called the susceptible-exposed-infectious-removed-susceptible (seirs) model. we assume no death in the removed compartment (see figure where the recovered branch in the removed compartment is connected to the susceptible compartment). an example of diseases studied using this model includes the common cold. this seirs model is defined as follows: where is the rate of losing immunity and becoming susceptible again after recovery. different from the seirs model, there are some infectious diseases where long-term immunity is yielded by individuals who survive from their infection. to build the self-immunisation into the infection dynamics, introduce an antibody (a) compartment to the sir paradigm, shown in the bottom thread of figure . because individuals who enter the antibody compartment will no longer be at risk of infection for a certain period of time, this compartment is indeed an exit compartment, at least over a certain time window within which immunity is active, in addition to the removed compartment. in some infectious diseases such as the covid- , the subpopulation of self-immunised individuals is not directly observed or clinically confirmed by the viral rt-pcr diagnostic tests because of mild or absent clinical symptoms. they are self-cured at home with no clinical visits. adding this compartment in the modelling can help to greatly mitigate the issue of under-reporting for the actual number of infected cases in the population. this dynamic system consists of four compartments, that is, susceptible, self-immunised, infectious and removed, with the following odes: where˛is the rate of self-immunisation, which is not identifiable because of the lack of observed data. an approach to estimating the rate parameter˛is to collect data of antibody serological surveys from the population. refer to for more discussions. this section mainly focuses on an introduction of statistical models to analyse surveillance data of an epidemic. each statistical model consists of two components: a systematic component and a random component. in the context of infectious disease data analysis, the former may be specified by a dynamic infectious disease model from sections and . the latter is built upon a random sampling scheme that enables a stochastic extension of the mechanistic model (e.g. sir model) given in the systematic component. essentially, the notions about disease transmission, recovery or other characteristics are used to define key population attributes or parameters in an infection dynamic system of interest, which will be estimated by available data via a statistical modelling framework, where some covariates may be incorporated to learn some subgroup-specific risk profiles. a clear advantage of statistical and stochastic extensions is the ability to quantify uncertainty in both estimation and prediction in connection to sampling variability. this added uncertainty is crucial to policymaking as models not only generate an average estimation or prediction but also present the best and worst possible scenarios for more robust and confident handling of epidemics, given that surveillance data are subject to various issues in the data collection. an example presented in britton ( ) vividly shows the uncertainty in the progression of an infectious disease. consider patient zero, who will go on and infect on average r number of other individuals, as defined by a certain disease mechanism. the number of individuals who contract the virus from this patient is in fact stochastic, varying around the expected number of infections r , which could be described by a distribution (e.g. poisson or negative binomial) with mean r on the support of non-negative integers. with a non-zero probability of taking the value zero due to the variability in human activities, there is a non-negligible chance that an epidemic is completely averted. the opposite could be an outbreak with a non-zero probability that infects tens of thousands of people. without modelling such uncertainty, we cannot see all these possibilities and associated likelihoods of their occurrences during the course of an epidemic (roberts et al., ) . infectious disease systems governed by the class of multi-compartment models, though describing the population average, are useful to describe individual-based stochastic processes if certain random components are introduced into the modelling framework. thus, the resulting statistical models present more natural approaches to the analysis of surveillance infectious disease data. before introducing statistical methodologies that are commonly used for parameter estimation, we distinguish model parameters into two categories. those that can be determined a priori with no need for estimation, which we term as hyperparameters. those that cannot be fully determined and need to be estimated using the data at hand, which we term as target parameters. the choices of which parameter should be a target parameter versus a hyperparameter vary widely across methods. intuitively, the more we know about the biological characteristics of a disease, the more parameters can be held fixed a priori in the analysis. it is however very difficult to determine most of the model parameters early in an outbreak because of the limited amount of knowledge and data about the disease. indeed, many model parameters are not identifiable because of the lack of relevant data availability. one such example is the rate parameter of immunity˛in the sair model ( ). as relevant knowledge accumulates, literature reveals increasingly precise characterisation of the disease, such as its latency period, recovery rate, death rate, immunity duration and antibody acquirement. such information is typically obtained from surveys of high-quality individual-level data, which may provide much better quantification of these hyperparameters than having to be re-estimated by epidemic models, which, on the other hand, are largely based on much coarser surveillance data. in the case of the covid- pandemic, this survey-based approach may be too costly to carry out in countries with large and heterogeneous populations. in general, target parameters are mostly those that are location specific, for example, transmission rate and fatality rate. they vary largely across regions because of non-uniform mitigation effort and hospital resources; hence, datadriven estimations are preferred. in section , we introduce an areal spatial modelling approach to account for spatial heterogeneity in the analysis of infectious disease data. because of the issue of parameter identifiability in some mechanistic models, specifying hyperparameters in the model fitting is inevitable. however, holding hyperparameters fixed at certain values according to some external data sources is indeed controversial, and the validity of consequent analyses is highly dependent on the appropriateness of these certain prior values. to relax this technical weakness, later in section , we introduce a bayesian framework in which such prior information (e.g. hyperparameters) enters the statistical model via certain prior distributions rather fixed values, so that the uncertainty on those hyperparameters is adaptively compensated with the amount and quality of observed surveillance data. such flexibility has a great advantage in synthesising prior evidence and observed data. to present this section at a reasonable technical level, most of the discussions in the succeeding text are given in the setting of the basic sir model, and generalisation to other compartment models should follow with slight modification. in closing, it is noteworthy that the frequentist statistical methods discussed in the succeeding text are based on a fundamental assumption of data collection; that is, the population-level compartment data s.t/, i.t/ and r.t/, and others if relevant, can be directly collected from the study population. in other words, at given time, every individual in the population can be observed directly for his or her current status of being susceptible, infectious, recovered or died. this is practically impossible. thus, the interpretation of the estimation results should be carried out with caution. in the sir model ( ), the transmission rateˇand recovery rate are two target parameters of interest. estimation ofˇand can be carried out through optimisation in search for a model that best fits to the data. a commonly used minimisation criterion is the least squares loss. giveň and , numerical approximations (e.g. runge-kutta methods) can be used to solve for the trajectories, s.t/; i.t/ and r.t/. these expected trajectories are then compared with the observed trajectories to compute a discrepancy score, such as the sum (over time) of the squared errors, represented as a loss function of target parameters. now, it remains to find the estimates of these parameters that give rise to the curve that best fits the data through standard optimisation tools. in this case, the optimisation pertains to a two-dimensional search, which should be computationally straightforward. even a greedy search is computationally cheap. we illustrate using both simulated data and real data in examples and , respectively. example . we first generate an observed sequence of cumulative infectious counts following example , namely, the sir model with the true parameter valuesˇd : and d : . for simplicity, we fix d : in this example. we then evaluate the sum of squared error (sse) loss between the expected cumulative infectious count i.t/ and its sample counterpart i obs .t/, and the value that minimises this loss gives an estimate ofˇ. figure plots the sse loss versusǔ sing the simulated data i obs .t/, t d ; : : : ; t, with t d ; ; , respectively. it is found that the sse loss is minimised at Ǒ d : as expected. the longer the observed sequence, the more curved around . the sse appears, so the better we can identify the minimum of the sse curve. the r script shows the example for the case of t d . note that the sequence we used to define the fit is i.t/, but s.t/ and r.t/ can also be used in the estimation. similarly, a two-dimensional grid search can be used for estimatingˇand jointly when is not fixed in which the data of r.t/ must be used in the estimation. here we present only one replicate for illustration. example . we apply the same approach as given in example for analysing the daily time series of the covid- cumulative infectious counts in michigan during march to may . details of the data are described in appendix a , including the i.t/ sequence. the already defined sir function from example is used as the dynamic model, and the already defined sse function from example is used as the loss function. by fixing d : (i.e. average contagious period of days) the following code computes the solution Ǒ d : using the first observations ( to march). we then increase the number of observations in the estimation; as shown in figure , the value of Ǒ decreases when more data are used. this is noticeably different from example where Ǒ remains constant regardless of the number of observations used. the gradual decrease in our estimate ofˇindicates a potential reduction in the transmission rate over time in michigan due to the enforcement of statewide social distancing. in other words, the assumption of a constant transmission rateˇis inappropriate for the michigan data. this result suggests a need for using a more proper modelling technique, which will be demonstrated in section . . being often used as a classic textbook example, this least squares approach is equivalent to the maximum likelihood estimation (mle) under the assumption that measurement errors are independent and normally distributed with a homogeneous variance. in general, this approach gives consistent estimation and does not require a distributional assumption for the data generation and thus can be applicable to non-normal data. however, the ordinary least squares loss used in the earlier example assumes that data are independently sampled over time, which is not true because the observations are time series and are thus temporally correlated. because of this, the least squares estimation is not efficient. cintrón-arias et al. ( ) have discussed the use of a generalised least squares approach to account for more complex error structure, including temporal autocorrelations. it is not always the best practice to directly use data of i.t/ and r.t/ in the estimation of the model parameters. the covid- projection by gu (https://covid -projections.com/) adopts a loss optimisation approach based on the seir model using only death counts due to quality concerns with infection counts (e.g. under-reporting issue). the model uses a discrete state machine with probabilistic transitions to minimise a mixture of loss functions, such as mean squared error, absolute error and ratio error. in the literature, there are many other estimation procedures (e.g. wallinga & teunis, ; cori et al., ; thompson et al., ) . some of these alternatives do not estimateˇand , but more directly target the effective reproductive number r e .t/ in estimation and inference. here we present the method of moments, another routine estimation approach in the statistical literature for estimating the model parameters in the sir model ( ). during the early phase of an epidemic, one may assume s.t/=n and set dt d (e.g. a time unit of day for discretisation), so that the second ode of ( ) leads to the approximate exponential function solution: ( ) at discrete times at which data are actually recorded. after estimate o is obtained, we obtain Ǒ immediately. however, the estimation of is only accurate during the early phase of disease outbreak because the approximation of s.t/=n is used. in the literature, other types of moments are also used to derive parameter estimates. for instance, using the approximation from the first ode of the sir model ( ) at discrete times, one can easily obtain the following expression: an estimate ofˇmay be obtained by averaging the quantities given in the right-hand side of the equation earlier. in the case whenˇ.t/ varies over time because of changes of a certain mitigation measure, the earlier method of moments estimator may still be applied locally with a possible utility of a kernel weighting function such as the nadaraya-watson estimator (nadaraya, ; watson, ) . a very similar approach leads to the following approximation: which may give rise to a non-parametric estimator of the effective reproductive number. although r e .t/ can be identified at each time point using data solely from t, for numerical stability, the same idea of a kernel weighting (e.g. running-bin method) smoother is applied to estimate r e .t/ at t (see, e.g. wallinga & teunis, ) . linear approximations are easy to implement; however, the variances produced from such linear fits are typically inadequate in describing the true randomness of an infectious disease to allow valid inference and prediction. alternatively, it is promising to investigate the local linear fitting method (cleveland & devlin, ) that produces non-parametric estimates of the time-varying model parameters to better reflect temporal dynamics of the infection. in both the least squares estimation and method of moments estimation, there are no explicit assumptions about probability laws for data sampling. implicitly, both methods are based on the sampling scheme on the entire population; that is, the current status of every individual in the study population is recorded. this is certainly not true in practice. to overcome this, some estimation methods are proposed to account for sampling variability under certain parametric distributions. distribution assumptions can be made for many quantities in an infectious disease model. some are fully specified based on given knowledge. for example, the distribution of incubation period of a disease can be represented as a probability mass function by days (lauer et al., ) . on the other hand, some distributions are only specified to be from a family of shapes, with the exact form to be estimated. we illustrate the latter using a stochastic sir model. stochastic sir models typically require the same assumptions as a deterministic sir model (section . ). to reflect the stochastic nature of disease transmission and recovery, stochastic processes such as a poisson process are used to model the accumulation of cases. following the earlier definitions ofˇand , the number of effective contacts in the population is a poisson process with rateˇn. of these contacts, only those between the contagious and susceptible will lead to new infections. hence, the counting process defined by the number of exposed (i.e. i.t/ c r.t/, or equivalently n s.t/) follows a poisson process with ratě s.t/i.t/=n. hence, the number of newly exposed in an instantaneous duration of dt follows a poisson distribution with meanˇs .t /i.t / n dt. on the other hand, the duration of time individuals staying infectious is assumed to be independent and identically distributed according to an exponential distribution with rate , and hence, the mean infectious duration is = . when we jointly consider all i.t/ infectious subjects at time t, exit events occur independently with a rate i.t/, and the gap times between two adjacent exits are exponentially distributed with mean =f i.t/g. in summary, the number of removed individuals is a counting process following a poisson process with rate i.t/. such stochastic formulation is commonly used, for example, in bailey ( ) and andersson and britton ( ) . through the earlier definitions, s.t/; i.t/ and r.t/ are now random variables that can be directly sampled. in fact, it suffices to assume only two of the three counting processes in order to define a stochastic sir model due to the constant sample size constraint. for demonstration, at time t, in an instantaneous time interval oet; t c dt/, we may specify a stochastic sir model as follows: where i.t/ d n s.t/ r.t/. as a result of this probabilistic formulation, the effective reproductive number is now defined as an expectation, that is, r e .t/ d efˇs.t/i.t/=ng. the stochastic sir model ( ) is specified in continuous time, and we would hope that dt is very small. in practice, approximation to ( ) is used by letting dt d or a unit of day, which is typically the smallest time unit used in public surveillance data. as a result, s.t/ and r.t/ at time t are used to approximate the average in the entire duration of oet; t c /. this approximation turns a continuous time stochastic model into a discrete time scholastic model to proceed with statistical analysis. other distributions, such as negative binomial or general dispersion family (song, ) , may be considered to handle the issue of overdispersion in the counting processes. with distributions in place, we turn the focus to estimation and inference by the maximum likelihood approach. maximum likelihood estimation is often preferred in a parametric model where the underlying probability distribution is properly chosen. for convenience, we take day as the time of unit. by discretising time based on observed sequences, that is, t d ; ; : : : ; t, observed daily increments of counts s.t/ d s.t/ s.t c / in the susceptible compartment and r.t/ d r.t c / r.t/ in the infectious compartment are conditionally independent, given historical accumulated counts s.t/ and i.t/, according to the definition of model ( ). the second model in ( ) contains only the removal parameter , so the log-likelihood function of with respect to the data of daily increments in the removed compartment, r.t/, and daily cumulative counts of infections, i.t/, can be written as where s. / d n and i. / d . however, one caveat in the simplistic likelihood formulations earlier is that the cumulative time series s.t/ and i.t/ are assumed to be directly measured without errors. in other words, the earlier likelihood accounts only for the sampling uncertainties in the increments not those in the cumulative counts, so the resulting statistical inference may suffer from underestimated standard errors. there are two types of statistical inference theory considered in this context, namely, the infill asymptotic theory and the outreach asymptotic theory. the former pertains to the situation where the sampling points increase within a fixed time window (i.e. fixed t), while the latter is a situation of practical relevance where the time window of the data collection tends to infinity (i.e. t ! ). britton et al. ( ) discuss the infill large-sample properties under the assumption that the complete epidemic data, that is, continuously observed counting processes .s.t/; i.t// t [ ,t] , are available. under such setting, the asymptotic distribution of the mle based on continuously observed trajectories is established. obviously, it is really rare in practice to collect infectious disease data via such infill sampling schemes. nevertheless, for the sake of theoretical interest, we refer readers to britton et al. ( ) and references therein. the outreach large-sample theory for the mle with discrete time series data provides a statistical inference relevant to most of infectious disease applications. as an epidemic evolves, the number of equally spaced time points (say, daily) for data collection increases. when sampling errors in both i.t/ and s.t/ are allowed, the likelihood earlier is indeed a kind of conditional composite likelihood (varin et al., ) . thus, the standard theory of composite likelihood estimation implies that the asymptotic covariance of the estimator is given by the inverse godambe information matrix (or a sandwich estimator). the sensitivity matrix in the godambe information is hard to obtain analytically because of the serial dependence in the time series. instead, one may take a non-parametric bootstrap approach similar to that considered by gao and song ( ) to evaluate the standard errors in order to conduct a valid statistical inference. conditional independence is a strong assumption for mathematical convenience in the mle. relaxing it has drawn some attention in the literature. for example, lekone and finkenstädt ( ) and allen ( ) construct likelihood-based approaches using discrete time markov chain seir models; becker ( ) and becker and britton ( ) consider the mle in the sir model using martingale methods when all transition events for each individual are observed. it is however unlikely that such individual-level details are observed in most surveillance data used for modelling of infectious disease mechanisms. estimators using less detailed data have been proposed (e.g. becker, ; rida, ) . as part of efforts on further relaxing strong conditions in the earlier stochastic sir model ( ), in section . , we review a state-space modelling approach that generalises the current likelihood model and estimation framework, where s.t/, i.t/ and r.t/ are not directly measured and rather treated as markov latent processes. also, hyperparameters are included via their prior distributions instead of fixed values, and a bayesian estimation similar to the mle is established through the mcmc approach. this class of state-space models is so far one of the most flexible statistical modelling frameworks to analyse infectious disease data. we highlight several software packages that are publicly available for estimation of parameters in the multi-compartment models. overall, additional efforts in this computational domain are needed. several packages focus on the estimation and inference for r and r e .t/. for example, obadia et al. ( ) , in their r package r , implements multiple methods, including a method of moments-type approach (dietz, ) , a bayesian method (bettencourt & ribeiro, ) and likelihood-based estimation procedures (forsberg white & pagano, ; wallinga & teunis, ; wallinga & lipsitch, ) . along this line, cori et al. ( ) and thompson et al. ( ) develop bayesian methods to estimate the effective reproductive number and are made available through the r package epiestim and microsoft excel (https://tools.epidemiology.net/epiestim.xls). their methods use a moving window approach, assuming that the reproduction number r t, in this window oet c ; t is constant. a gamma prior distribution is used to derive the posterior distribution of the r t, given new infectious counts. state-space models refer to a class of linear or non-linear hierarchical stochastic models with parametric error distributions. the conventional state-space model is not formulated as a bayesian model, but later, its bayesian formulation has gained great popularity because of the availability of mcmc methods for the estimation of the model parameters (carlin et al., ) . this class of models primarily attempts to explain the dynamic features of the state-space model framework is advantageous over the stochastic compartment models introduced in section . in the following aspects of statistical modelling: (i) state-space model does not assume that the compartment processes s.t/, i.t/ and r.t/ are directly observed, which are treated as latent processes to be estimated from observed data. (ii) state-space model allows an explicit sampling scheme to be part of the model specification, which enables the quantification of both estimation and prediction uncertainties in the statistical analysis. (iii) state-space model is built upon the compartment probabilities (or rates or proportions) that automatically adjust for potentially varying population sizes. this conveniently relaxes the condition of a constant population size in the basic sir model. (iv) state-space model provides a flexible statistical modelling framework that embraces time-varying model parameters and integrates prior knowledge of disease mechanisms (e.g. r value from other studies) via prior distributions of the model parameters. (v) implementation of mcmc methods in statespace modelling provides a powerful approach to parameter estimation and predictions using conditional distributions given the history. this is different from all estimation methods in section that are always formulated via marginal distributions under strong assumptions of sampling rules. a state-space model consists of two stochastic processes: a d-dimensional observation process fy t g and a q-dimensional state process f t g given as follows: the state process  ;  ; : : : is a markov chain with initial condition  p .Â/, and transition (conditional) distribution is given by y t j t f t .yj t /: the observation process fy t g is conditionally independent given the state process f t ; t g, and each y t is conditionally independent of  s ; s ¤ t; given  t , the conditional distribution is y t j t f t .yj t /. this model can be graphically presented by a comb structure shown in figure . according to cox et al. ( ) , the state-space model is a parameter-driven model in that the processes of the compartment proportions are unknown population parameters to be estimated, while the stochastic multi-compartment model such as the stochastic sir model in ( ) is a data-driven model where the compartment proportions are directly observed. as pointed out earlier, the validity of the latter is questionable in practice, especially in the analysis of the covid- pandemic data. let y s be the collection of all observations up to time s, namely, y s d .y ; : : : ; y s /. let be a generic notation for the set of model parameters. denote the conditional density of  t , given y s d y s , by f t|s .Âjy s ; /. then, the prediction, filter or smoother density is defined, respectively, according to whether t > s, t d s or t < s. this conditional density f t|s . jy s ; / is the key component of statistical inference in state-space models. to develop the maximum likelihood inference for model parameters in state-space models, the one-step prediction densities f t|t are the key components for the computation of the likelihood function (see chapter of song, ) . given a time series data fy t ; t d ; : : : ; ng, the likelihood of y n is where f .y i / is expressed as follows: where by convention, g . i / d f | . jy i /, conditional on an initial observation y at time . in the earlier likelihood evaluation, one-step prediction densities, f t|t , and filter densities, f t|t , can be respectively given by the recursions ( ) with the recursion starting with f | . / d p .Â/. in general, exact evaluation of the integrals in ( ) and ( ) is analytically unavailable, unless in some simple situations, such as both processes being linear and normally distributed. for the linear gaussian state-space model, all f t|s are gaussian, so the first two moments of ( ) and ( ) can be easily derived from the conventional kalman filtering procedure, as discussed in chapter of song ( ) . however, with some computational costs, all integrals in the earlier likelihood and the filter can be evaluated numerically by mcmc methods. recently, wang et al. ( ) have developed an extended sir (esir) model that is built upon a state-space model with two (d d ) observed time series of daily proportions of infectious and removed cases, denoted by y i t and y r t , which are generated from the q-dimensional underlying infection dynamics f t ; t g governed by a mechanistic sir model. in the case of the sir model, q d . as shown in figure , the latent process is a time series of the three-dimensional latent vector of population probabilities  t d  s t ;  i t ;  r t > that satisfies a three-dimensional markov process of the following form: where parameter Ä scales the variance. the function f. / is a three-dimensional vector as a solution to the sir model ( ), which determines the mean of the dirichlet distribution via the rk approximation. in comparison with the stochastic sir model in ( ), here the compartment proportions  t are unobserved and explicitly modelled by a markov process to account for temporal correlations, so the parameter estimation can be carried out with multivariate likelihood functions. because the serial dependence is accounted for in the statespace model, the resulting estimation and prediction are more powerful than those given in section . . two observed time series y i t ; y r t > that are emitted from the underlying latent dynamics of infection  t are assumed to follow the beta distributions at time t: where  i t and  r t are the respective probabilities of being infectious and removed at time t, and i and r are the parameters controlling the respective variances of the observed proportions. it is easy to see that y i t and y r t are conditionally independent given  t , and e y i t j t d  i t and e.y r t j t / d  r t , and d . i ; r ; Ä;ˇ; /. because y i t and y r t share a common latent variable  t , their marginal correlation is modelled. in fact, these two beta distributions define a sampling scheme of observed data, including daily empirical proportions of infectious cases and removed cases, which are a collection of daily signals from the underlying latent sir infection dynamic system. the earlier state-space model ( ), ( ) and ( ) is useful to assess the effectiveness of control measures (e.g. social distancing) via the projected epidemic evolution in the future time. to process, one can replace the constant transmission rateˇby a time-varying transmission rateˇ .t/, where .t/ is a given transmission rate modifier. it is specified as a function in time to reflect different forms and strengths of control measures. this results in an esir model proposed by wang et al. ( ) : where .t/ . obviously, the basic sir model is a special case with no intervention in place, .t/ Á . in general, the .t/ may be specified by a practitioner to reflect a particular control measure. for an example of the covid- in hubei province, china, a possible choice of .t/ given in the following is a step function that reflects government-initiated macroisolation measures: .t/ d figure (a)-(c), we obtain different types of transmission rate modifiers. alternatively, .t/ can be a continuous function, say, .t/ d exp. t/ or .t/ d expf . t/ ! g; > ; ! > , that reflects steadily increased community-level surveillance and personal protection (wearing face masks and washing hands) as shown in figure (d)-(f). note that this modifier function does not have to be a monotonic decreasing function and may take a u-shape to capture the relaxation of control measures. with such a modelling framework, one can carry out comparisons of different preventive protocols via the resulting projected infection risk  i .t/ or other epidemic features such as the time of the effective reproduction number r e .t/ < and the time of a disease recurrence associated with relaxed control measures. a clear advantage of the state-space model is that it enjoys the resilience of mcmc being a primary method for statistical estimation and prediction. in other words, the statistical analysis methods can be easily modified to accommodate changes made in the latent multi-compartment models and/or in the observed time series models. one example of the covid- pandemic modelling given in wang et al. ( ) is to extend the three-compartment esir model to a four-compartment model by incorporating stringent quarantine measures issued by the hubei government via a new addition of in-home quarantine compartment. this new model is termed as susceptible-quarantined-infectious-removed (sqir) model. this quarantine compartment collects in-home isolated individuals who would have no chance of meeting any infectious individuals in the infection system. so, it is another exit from the dynamic system in addition to the removed compartment. let .t/ be the chance of a susceptible person being willing to take in-home isolation at time t. the basic sir model in equation ( ) is then extended to include a four-dimensional latent process where  s t c  q t c  i t c  r t d . the quarantine rate .t/ may be specified as a dirac delta function with jumps at times when major quarantine policies are issued by the government. for example, one may specify the time-dependent quarantine rate function .t/ for hubei province as follows: .t/ d note that at each jump, the respective proportion of individuals would leave the susceptible compartment and enter the quarantine compartment. figure (g)-(i) shows three different types of in-home quarantine rates during the period of the covid- pandemic in hubei province. in a similar spirit to the sqir example of application ii earlier, consider an interesting extension of the basic sir model in the analysis of the us covid- data to include an antibody compartment to handle the subpopulation of self-immunised individuals. this four-compartment model is termed as sair model, which has been discussed in detail in section . . because the antibody compartment is also a second exit from the infection system, similar to the quarantine compartment, one can turn the sair model given in ( ) into a similar form of the sqir model in ( ), where .t/ is replaced by˛.t/, the rate of self-immunisation. it is known that the population immunity rate cannot be estimated from observed surveillance data, which needs to be figured out by using large-scale serological surveys in the population. thus,˛.t/ may be specified as a dirac delta function (e.g. figure (g)-(i)) with jumps at times when the surveys are conducted and function values based on the survey results. it is worth pointing out that although the sqir and sair models have very similar model structures, their interpretations are very different. the former is applicable to the case of very stringent self-isolation control measures in hubei, while the latter is reflective to the situation of selfimmunisation due to mild control measures in the usa, so that a substantial proportion of individuals who contracted the virus, recovered and became immunised. markov chain monte carlo has been extensively used for the estimation and prediction in the state-space model (see, e.g. carlin et al., ; chan & ledolter, ; czado & song, ; de jong & shephard, ; zhu et al., , for a vast literature on this topic). such popularity of mcmc in the state-space model is rooted in its power to handle the evaluation of high-dimensional integrals involved in the likelihood function ( ). the essential strategy for the calculation of each high-dimensional integral is to approximate it by a sample mean of the involved integrand. this sample average is obtained from many mcmc sample draws from posterior distributions of the model parameters, including the time series of the latent probability vector  t . let t be the current time up to which we have observed data . ( ) draw  .m/ t from the posterior h  t j .m/ t ; .m/ i of the q-dimensional latent process, at t d ; : : : ; t ; t c ; : : : ; t; ; .m/ i according to the observed process, at t d ; : : : ; t ; t c ; : : : ; t, respectively. prior distributions are specified for some of the hyperparameters; for example,  dirichlet y i y r ; y i ; y r , r dˇ= and follow some log-normal distributions, and i , r and Ä follow some gamma distributions or inverse gamma distributions, respectively. convergence diagnostics of the mcmc algorithm may use standard diagnostic tools such as the gelman-rubin statistic based on multiple chains with different initial values, monitoring trace plots of the model parameters and so forth. the r package coda provides a comprehensive toolbox of convergence diagnostics (brooks & gelman, ) . using the mcmc draws collected after the burn-in, various summary statistics may be obtained to estimate model parameters, conduct inference and make prediction. the summary statistics (e.g. posterior mean and posterior mode) from the in-sample draws of the model parameters can provide point estimates and % credible intervals with the left and right limits set respectively at the . th percentile and . th percentile, and those of the observed processes may be used to check the goodness of fit of a proposed model and to perform model selection via the deviance information criterion (spiegelhalter et al., ; gelman et al., ) . more importantly, the summary statistics from the out-sample draws of the latent process  t ; t > t provide point predictions and their % credible prediction intervals. it is interesting to note that the earlier mcmc implementation does not depend much on the form of the runge-kutta solution f. t ;ˇ; / in the latent process ( ). as long as a mechanistic infectious disease model has an approximate analytic solution f. /, the bayesian estimation and inference can be carried out using mcmc. such flexibility is appealing to develop software applicable for a broad range of practical studies. mcmc procedures are well suited for the estimation and inference in the setting of statespace models because of fast and reliable numerical performances. for the michigan data analysis example in section . , using an average personal computer, we spend . h completing all mcmc calculations of draws with thinning bin size of after the burn-in judged by four separate mcmc chains. this computing speed can be improved by using highperformance computing facilities and/or some recent posterior sampling methods. as suggested by zhou and ji ( ) for a state-space sir model, one may set a more efficient sampler over highly correlated posterior spaces by parallel-tempering mcmc algorithm (geyer, ) , which provides rapid mixing in mcmc chains. also, along the line of online learning, sequential monte carlo methods for posterior sampling (doucet et al., ; dukic et al., ) are promising, as they permit efficient updating of existing posteriors with sequentially arrived data, in the hope to avoid refitting the model by running mcmc from scratch using the updated complete data. wang et al. ( ) and have developed a series of extended sir models by introducing time-varying transmission rate, quarantine process and asymptomatic immunisation process (details in section . ). the proposed methods have been established in an open-source r package esir, available on github (https://github.com/lilywang /esir). this package calls rjags to generate mcmc chains and retains a few mcmc controllers from rjags. the package is also updated weekly with new summarised us state-level count data for the covid- pandemic. several robust methods that are developed specifically for the prediction of the covid- are cited by the centers for disease control and prevention (https://www.cdc.gov/coronavirus/ -ncov/covid-data/forecasting-us.html). to name a few, the bayesian approach (verity et al., ) developed by researchers at imperial college london (featured in adam, ) and the hybrid modelling approach (ihme covid- health service utilization forecasting team & murray, ) adopted by the university of washington institute for health metrics and evaluation (ihme) (discussed by jewell et al., ) have attracted great public and government attention. we refer to their original work for modelling details. it is difficult to appreciate the original work and followed comments without running real covid- data using their software, which is lacking for the ihme models, among some others. to increase research transparency, releasing software or computing code used in statistical methods to the public is strongly encouraged. we now illustrate the use of r package esir to analyse the covid- surveillance data during the period of march to june from michigan state, usa. the michigan data used in this analysis are listed in appendix a , including both i.t/ and r.t/. in the data analysis, we demonstrate the use of both the state-space model described in application i and the mcmc method, where the transmission rate modifier .t/ is set as exponential functions. from package esir, we can extract many useful statistics related to estimation and forecasting. for example, we can obtain both mean and median projections of the prevalence curve  i .t/; t > t as well as their % credible prediction intervals. in addition, this package provides the estimated first and second turning points of an epidemic. the former is the time when the daily number of new infectious cases stops increasing, while the latter is the date when the daily number of new infections becomes zero. mathematically, the first corresponds to the time t at which r  i t d or the gradient of p  i t is zero, and the second is the time t at which the rate of prevalence is zero p  i t .t/ d : the following is the r script to perform the data analysis: in the above program, we consider a time-dependent declining transmission rate with the modifier value .t/ d exp. t/ where the parameter is chosen so that the modifier equals to . on may. this value is determined based on the social distancing scoreboard posted by unacast, inc. (https://www.unacast.com/covid /social-distancing-scoreboard). one needs to set exponential d true, to activate such setting. alternatively, as shown in figure (a)-(c), one may use a step function by providing a vector of pi , values and the corresponding vector of changes dates in change_time. in the main function above, we let the starting date be march and conduct the estimation and projection of days ahead (t_fin d ) on june and after. we run four separate mcmc chains with different initial values, each with length of , kept from every draws (thn d ) (a thinning operation to reduce autocorrelations) after draws are dropped. thus, with a relatively squandering setting, we expect a better performance of convergence and reliable quantification of prediction uncertainty using sample quantiles. there are two different prior settings for sensitivity analysis. one follows the example code earlier, with the prior mean for the log-normal distribution of the basic reproduction number to be . , the removal rate . and thus the mean transmission rate . , and the other with all these values to be , . and . , respectively. the two distinct settings provide similar estimating and forecast results as can be seen in figure . their estimated reproduction numbers are . ( % credible interval [ . , . ]) and . ( % credible interval [ . , . ]), respectively, which are similar considering that their prior settings are quite different. the output gelman-rubin statistic developed by gelman and rubin ( ) are close to (data not shown). both pieces of evidence as well as stationary trace plots warrant the convergence of the the michigan covid- data have been preprocessed to smooth away some unnatural gaps caused by the clustered reporting issue as discussed in appendix a . figure shows an adequate model fitting, where all observed numbers of confirmed infections fall in the % insample credible intervals of prevalence  i t ; t Ä june. in contrast, the % out-sample credible intervals of the projected proportion (y i t ) are much wider, reflecting to the significant amount of uncertainty in the prediction. such uncertainty elevates as the time moves further away from the present time. despite the large uncertainty, the projected mean and median prevalence curves show a decreasing trend over time, which means that the social distancing works to mitigate the epidemic in michigan although the rate of improvement is moderate. also the fact that the two estimated turning points have occurred before june is another piece of evidence for the positive effects of the series of social distancing orders issued by the state governor since march . model diagnosis is an important part of a statistical analysis, which is typically conducted using various residual plots. as illustration, in this michigan data analysis, let n  t be the posterior means over the period of march to june. we consider residuals of the two observed processes, defined by figure shows that there are dominant lag- autocorrelation (the three coefficients are about . ) and no any additional significant autocorrelations beyond the lag- dependence. this confirms the assumption that the three latent processes are all the first-order markov processes. all mechanistic models discussed in the previous sections are useful to analyse the infection dynamics for a large population such as a country or a state in which most of model parameters may be assumed to be homogeneous and representing the entire population. this type of macromodelling approach is particularly valuable at the early phase of disease outbreak when the national public health administration aims to come up with nationwide macrointervention protocols with very limited amounts of relevant data available. once an epidemic evolves further into its middle phase, with more and more surveillance data collected from local communities, a macromodel is no longer suitable for an in-depth analysis of microinfection dynamics owing to the existence of substantial heterogeneity across local communities. this section concerns a review of significant extensions of infectious disease models by incorporating spatial heterogeneity across different geographical locations into modelling and analysis. the focus will be on the recent development of integrating the classical spatial cellular automata (ca) (von neumann & burks, ) with the previously discussed temporal multi-compartment models, leading to an important class of spatio-temporal multi-compartment models. this class of models is useful to predict local infection risk. technically speaking, the majority of existing macromechanistic models to study the spread of infectious disease are based on the assumption that the system is homogeneous in space. this means that the spatial characteristics that could potentially play a non-trivial role in the development and outcome of disease infection are not taken into consideration. this is a valid assumption if the population vulnerable to the infectious disease is mixed well and the human interventions (e.g. vaccination strategies) are homogeneous across different spatial locations. however, in reality, there exists substantial heterogeneity in the urbanisation, ethnic distribution, political views, governance and economic composition across different subgroups of individuals distributed over geographical locations, all of which will influence the spread of infectious disease and make the previous macromechanistic model not appropriate to address the dynamics spatially. one possible extension is to utilise partial differential equations (pdes) (murray et al., ) in spatial homogeneity, which is relaxed to allow area-specific spread patterns of epidemics. as noted in the literature, one limitation of pdes is that this approach ignores the fact that infectious disease is spread through person-to-person interactions, rather than by a continuous population. thus, pdes may lead to impractical results about the dynamics of an epidemic (mollison, ) . a natural strategy is to embrace a micromodel mimicking an interactive particle system, and ca is one of the well-studied systems with the strength of modelling spatially varying infection dynamics. originated in the works of von neumann and burks ( ) and ulam ( ) , the ca paradigm has been used in many applied fields, including the modelling of infectious diseases. when applied to model spatial variations of epidemic spread, ca has three distinctive features: (i) it treats individuals as discrete entities in order to study person-level movements in the infection dynamics. this high-resolution paradigm necessitates the incorporation of individual's heterogeneity such as residential address, age, race, pre-existing medical conditions and others in the modelling. in surveillance data, geographical information is publicly available (e.g. county that an individual lives), so it is feasible to utilise this variable in the extension of the macromechanistic model. (ii) ca allows to introduce local stochasticity; for example, the ca paradigm may be built upon a person-to-person infectious mechanism if individual-level information is available; otherwise, it may be based on a group-group infection process. (iii) ca is formulated in a network of particles (e.g. individuals, groups, villages and counties) with certain rules of connectivity and stochastic laws of disease transmission. this network topology is well suited for computations and simulations. because of these unique advantages, the ca paradigm has been employed by researches as an efficient method to study spread patterns of epidemics (beauchemin et al., ; ahmed & agiza, ; boccara et al., ; quan-xing & zhen, ; fuks & lawniczak, ; willox et al., ; rousseau et al., ; sirakoulis et al., ; fuentes & kuperman, ; liu et al., ; yakowitz et al., ; sun et al., ) . in the modelling of infectious diseases, the basic ca formulation involves three primary components: (i) a two-way array of cells (e.g. an age group or a county) that contain groups of individuals under study, and each individual belongs to one cell; (ii) a set of discrete states (e.g. susceptible, self-immunised, contagious, recovered and death) that describe different conditions of individuals during an epidemic; and (iii) some specific rules or updating functions that determine spatially how local interactions with a target cell from its neighbouring cells can influence and change the states of individuals in the target cell; all cells in a ca system achieve a global propagation of infection status updates instantaneously and continuously. in the application of the ca, determining neighbouring cells is tricky, and different types of neighbourhood topology have been proposed in the literature, including von neumann neighbourhood, moore neighbourhood, mvonn neighbourhood and extended neighbourhood (hasani & tavakkoli, ) (see figure for an example of these four neighbourhood types). in the modelling of influenza a viral infections, beauchemin et al. ( ) use a simple two-dimensional ca model to investigate the influence of spatial heterogeneity on viral kinetics. their study population consists of two types of cell species, the epithelial cells and the immune cells. the epithelial cells are the target of viral infection, and the immune cells are those fighting the infection. the ca model is built upon a two-dimensional square lattice with the moore neighbourhood (see figure (b)), where the condition of a certain cell will only be influenced by the eight closest cells around it. the set of states for the epithelial cells include healthy, infected, expressing, infectious or dead, while an immune cell can be in any of two states: virgin or mature. decision rules of updating the ca system are governed by parameters, such as infect_rate that models the probability of a healthy epithelial cell being infected by contacting each infectious nearest neighbour. detailed updating functions are discussed in beauchemin et al. ( ) . simulations show that the proposed ca model is sophisticated enough to reproduce the basic dynamic features of the cell-to-cell infection. different from the modelling of the influenza a viral infection earlier, fuks and lawniczak ( ) propose a lattice gas ca that is closely connected to an sir framework of an epidemic, where the interacting patterns of individuals are modelled. it is assumed that the status of individuals will change between three types, susceptible, infectious and recovered, denoted as fs; i; rg. the space where the epidemic takes place is set as a group of regular hexagonal cells, in which the individuals are located at the centre of each cell and can move through a channel that is created by connecting two centres of adjacent cells. the evolution of the ca occurs at discrete time steps under the operation of three basic functions, including contact c, randomisation r and propagation p. with the application of function c, an individual who is susceptible can become infected with probability . ˇ/ n i , whereˇis the transmission rate and n i is the number of infectious individuals within the same cell. meanwhile, an individual who is infectious can recover with probability , where is the recovery rate. the function r randomly assigns individuals in each cell to move through the channels, which contributes to modelling the mixing process of individuals. in the final propagation step, individuals simultaneously move to the cells that they are randomly assigned to by r. in addition to the basic epidemic dynamics modelled by the proposed lattice gas ca, fuks and lawniczak ( ) also study the effect of heterogeneous spatial distribution of individuals with states s, i and r and the influence of different types of barriers in controlling the spread of an epidemic. whereˇis the population macrotransmission rate and is the population macrorecovery rate. first, when the set v d ∅, that is, an empty set, the ca-sir model for cell .i; j/ reduces a celllevel sir model similar to that given in ( ). second, the numerator n i cp;j cq  i i cp;j cq .t / is the expected number of infectious cases yesterday (time t ) in a neighbouring cell .p; q/ v whose cell population is n i+p,j+q . so, the ratio is an empirical probability that a person in cell .i; j/ randomly runs in a contagious person from its neighbouring cell .p; q/. third, this random chance is weighted by a factor of intercell connectivity, denoted by ! .i;j / pq ; the stronger tie of cell .i; j/ with cell .p; q/, the higher likelihood of a person from cell .i; j/ running in contagious individuals in cell .p; q/. fourth, summing up all such likelihoods gives a total likelihood that an individual from cell .i; j/ would run in the virus carriers from all the neighbouring cells. a typical form of the intercell connectivity coefficient is given by ! .i;j / pq d c .i;j / pq m .i;j / pq , where c .i;j / pq and m .i;j / pq are broadly defined as a connection factor and a movement factor, respectively. they are used to characterise the intercell mobility or how easily individuals in the cells can move between the centre cell and its neighbouring cells. this ca-sir system, which is integrated with the sir model, can serve as a basis for the development of useful algorithms to emulate real-world epidemic infection spatially. . assume that there is a square array of cells that hold the population under the study of a certain epidemic. our target cell is the one at the centre (see figure ) we illustrate the predicted risk of infection with the covid- for all counties in michigan state using the state-space model with the mechanistic ca-esair latent process . in the first step, we apply the mcmc method to estimate the model parameters (ˇand ) and the vector of four probabilities  t of being susceptible, self-immunised, infectious and removed by fitting the esair model with the state-level surveillance data since march. this can be performed easily using the r package esir, which has been illustrated in section . . both the antibody rate function˛.t/ and the transmission rate modifier .t/ are pre-specified using other data sources with the detail given in the succeeding text. after getting the estimates of the model parameters, we use them as the initial values to make county-level risk prediction by the ca-esair model ( ). in this example, we consider only a -day ahead infection rate prediction (i.e. may ) for all the counties in michigan, namely,  i c .t c /. given that the covid- pandemic evolves fast in the state of michigan in early may , this kind of short-term forecast or nowcast is of great interest to the michigan government for timely decision making on either extending an existing governor's 'stay-at-home' order or relaxing this executive order. to perform the prediction, one important task is to specify the inter-county connectivity coefficient ! cc .t/. as discussed in , it is challenging to define ! cc .t/ objectively, as it involves many variables. in this illustration, we specify this coefficient as ! cc .t/ d cc expf Ár.c; c /g, where Á is a tuning parameter to be determined. briefly speaking, the first factor c,c is the inter-county mobility factor characterising the decrease of human encounters in terms of their potential movements between counties, which has been given online (https://www.unacast.com/covid /social-distancing-scoreboard). the second factor r.c; c / is a certain travel distance between two counties c and c in terms of both geodesic distance (karney, ) and 'air distance' based on the accessibility to nearby airports. in addition, the tuning parameter Á enables to adjust the scale of the travel distance by minimising the sum of (county-level) weighted absolute prediction error for the one-step ahead risk prediction of the infection rate. in addition to the specification of the connectivity coefficient ! cc .t/, the self-immunisation rate˛c.t/ is calculated based on the results of the new york statewide antibody test surveys released by the new york governor andrew cuomo on april (new york state report, ), and the transmission modifier function c .t/ is specified by the effectiveness score of state-specific social distancing using cell phone data in the usa from the transportation institute at the university of maryland (https://data.covid.umd.edu/). additional details of the determination of c,c , r.c; c /,˛c.t/ and c .t/ and the tuning of Á can be found in . figure (a) shows the -day ahead projected infectious rate for counties in michigan on may, and figure (b) plots the corresponding county-level weighted prediction errors (wpe), which is at the order of for the counties. the r package ca-esair is available on github (https://github.com/leyaozh/ca-esair). in this paper, we have presented the basics of multi-compartment infectious disease models from both deterministic and stochastic perspectives. we emphasise on the probabilistic extension of mechanistic models, which opens the door to a suite of statistical modelling techniques while still preserving the infectious disease dynamics in multi-compartment models. within the stochastic modelling framework, both the frequentist and the bayesian schools of modelling considerations and statistical methods are visited, along with high-level review and illustrative examples. epidemic models have played a key role in the past century to provide understanding of past and ongoing infectious diseases, and it is our belief that they will continually be valued and be improved to help us better understand the current covid- pandemic as well as future infectious diseases. we conclude with several remarks on future directions of stochastic infectious disease modelling. although publicly available surveillance data are useful to build preliminary models for the understanding of spreading patterns of infectious diseases, their data quality in terms of measurement biases and under-reporting has been known an outstanding issue that significantly impacts the validity of statistical analysis results (angelopoulos et al., ) . this is indeed an open problem to date with no appropriate solutions yet. with no insurance of reliable data, statistical methods, regardless of macromodels or micromodels, would fail to produce meaningful results. one potentially promising solution to such a fundamental concern is to build reliable and well-validated open-source benchmark databases that include not only traditional surveillance data but also personal clinical data from various sources such as hospital electronic health records, drug trials and vaccine trials. in addition, data from serological surveys and data from mobile devices or as such are also useful to increase information resolution and reliability, to remove major measurement biases and to calibrate data analytics. this task requires also efforts of data integration and international collaborations. research on the covid- pandemic certainly gives rise to a new opportunity of developing data integration methods to not only address challenges of data multi-modality but also overcome many data-sharing barriers and data confidentiality concerns. the population of self-immunised individuals is a significant source of bias in covid- surveillance data; they have never been captured by public health monitoring systems. according to survey results (new york state report, ), % of individuals in the city of new york have been tested antibody positive to the coronavirus. this simply means that a nationwide serological survey is a must in order to come up with an appropriate assessment for the underlying epidemiological features of the covid- pandemic in the usa. the design of this nationwide serological survey is a challenging statistical problem. solving it requires some innovative ideas and methods; for example, a cost-effective design of pooling several serum samples to perform a pooled test (e.g. gollier & gossner, ) , and an efficient design of hierarchical stratified survey sampling schemes. the sair model introduced in section . presents a basic framework for statistical models incorporating antibody serological surveys into the multi-compartment dynamics of infectious diseases. large-scale tracking data have played an important role in evaluating the effectiveness of social distancing in communities. the precision of intervention efficacy helps improve both estimation and prediction that directly impact government's decisions on tightening, extending or lifting control measures. one emerging data source pertains to the information of real-time cell phone locations, which allows better contact tracing so that individual data sequences can be recovered and used for modelling of personal risk and regional hotspots. a research group in the university of maryland (https://data.covid.umd.edu/) proposes several algorithms to process the cell phone data in the usa to extract key features of personal mobility, including location identification, trip identification, imputation of missing trip information, multilevel data weighting scheme, comprehensive trip data validation, and data integration and aggregation ghader et al., ) . however, these types of data are proprietary and subject to the issue of personal privacy (ienca & vayena, ) . integrating such data type or its summary statistics into infectious disease models should be encouraged, but in a cautious and responsible manner. in this field, statistical learning methods with differential privacy (dwork, ) are of great interest. statistical methodologies have been greatly challenged in the modelling and analysis of infectious diseases; almost every methodological troubling issue known the statistical literature surfaces, which presents new opportunities to statisticians and data scientists to develop innovative solutions. among many challenges, we emphasise a few of critical importance, which may be easily ignored in the new methodology development. we strongly advocate for the urgent need to build models that are transparent and reproducible (peng, ) . as most methods and models for the covid- pandemic are fairly recent and many have not yet been carefully peer reviewed, researchers should document the sources of data used, data preprocessing protocols, source computing code and sufficient modelling details to allow external validation from the public. such details are also necessary to allow others, who may have better quality data but without sufficient statistical expertise, to easily adopt new methodologies to obtain high-quality results. as mentioned in an original post by dr nilanjan chatterjee (https://link.medium.com/hquqilead ), transparency, reproducibility and validity are three criteria to assess and assure the quality of prediction models. his essay also mentioned the difficulty in reproducing the work given by the ihme to obtain accurate predictions and appropriate confidence intervals. similar to the ihme method that has no software available, gu's method for the covid- prediction (https://covid -projections. com/) that has recently received much attention does not provide software, either, unfortunately. without clear guidance and full reproducibility, even models that currently do well might fail in the future because predictions are relying on certain kinds of extrapolation assumptions that need to be unveiled to the scientific community with full transparency for validation and comparison. given that model projections for the covid- pandemic have been changing dramatically from day to day primarily because the underlying models are changing, the primary aim may be set at optimising prediction models for nowcasting or short-term projections and be aware of the probable worst case scenarios for longer-term trends. as shown in the data example in section . , the optimal tuning parameter is determined by the minimal short-term -day ahead prediction error. as pointed out by huppert and katriel ( ) , transmission models with different underlying mechanisms may lead to similar outcome in one context (e.g. short term) but fail to do so in another (e.g. long term). the further we project, the more we are uncertain about the validity of model assumptions. hence, extra caution is needed when reporting and interpreting long-term projection results. with the available surveillance data, making a nowcast of infection risk in next few hours is difficult; but it may become feasible when certain data sources of local information are accessible, such as electronic health records from local hospitals, viral testing results from local testing centres and mobile tracking data from individual cell phones. this requires a finer-resolution prediction machinery that may be established by generalising the ca to certain spatial point processes. despite being challenging, such prediction paradigm would be very useful and worth a serious exploration. because of the potential bias in surveillance data, either delayed reporting of infected case or inaccurate ascertainment of death caused by a virus, there are many measurement errors in data. this calls for statistical methods that can directly handle various data collection biases or are robust to such biases. there is little work performed in this important field of statistical modelling and analyses. in the current literature, model diagnostics for infectious disease models are largely lacking. given that most of the existing mechanistic models are based on certain parametric distributions (e.g. poisson processes), checking model assumptions is required. for example, for the proposed poisson process, the assumption of incremental independence and overdispersion should be checked. in addition, procedures of validating prediction accuracy are also important in which the choice of test data is tricky and needs to be guided by some objective criteria. a major weakness noted for the existing mechanistic models is the inflexibility of adding individual or subgroup covariates (e.g. age and race). the current strategy of handling these extra variables is via stratification, which would end up with strata with small sample sizes, so that subsequent statistical analyses lose power in both estimation and prediction of infection dynamics. an extension from the ca seems promising as the ca presents a system of particles distributed in different cells (or strata), where individual characterisations on particles may be added via covariates. the resulting model would assess and predict personal risk, as well as identify hotspots of new infection. this is worth serious exploration in the future with appropriate data available (e.g. electronic health records from hospitals). for a global pandemic such as the covid- that affects over countries in the world, an integrative analysis is appealing to understand common features of the pandemic so to learn different control measures. given the fact that a pandemic evolves typically in a certain time lag, experiences from countries with earlier outbreaks may be shared with countries with later outbreaks, where statistical methods may borrow relevant information to set up prior distributions in the model fitting. for example, the estimated reproduction number estimated from the european covid- data may be a hyperparameter in the statistical analysis of the us covid- data. there is a clear need of more comprehensive meta-analysis methods to better integrate data from different countries than using the data to create hyperparameters. along this line, one of the earliest attempts is to combine covid- forecasts from various research teams using ensemble learning (see, e.g. https://github.com/reichlab/covid -forecast-hub). most investigation efforts made by quantitative researchers have been relatively independent in an academic setting, and it is high time that policymakers and stakeholders are involved and play an active role in such modelling efforts. long-term projection of the covid- is most sensitive to and highly dependent on public health policy. a major source of uncertainty is due to the conflicting demands between public health (disease mitigation) and the need to sustain economic growth (livelihood), and the balance of the two is a moving target. one way to account for the modelling uncertainty is to factor in economic planning as a time-varying modifier of projection models. although some efforts have been made to incorporate economical data, most are retrospectively oriented, and we believe more efforts should be spent to prospectively incorporate expert inputs and economic forecasts. this is a research area of great importance worth serious exploration. we like to close this review paper by casting a few open questions of great interest to the public (at least to ourselves) that statisticians may help deliver answers with existing or new data to be collected by innovative study designs. we also hope that these questions motivate new methodological developments. question : how would researchers assess both timing and strength of the second wave of the covid- pandemic? is the second wave worse than the first one? answers to these questions need a relatively accurate long-term prediction of the infection dynamics. among so many different statistical models being able to predict future spreading patterns, we need to identify few ones or their combinations that are particularly useful to make long-term predictions. question : as many countries and regions started to reopen business, how would government monitor the likelihood of a recurring surge of covid- caused by business reopenings? does the social distancing measure help reduce a potentially rising risk? answers to these questions require adequate data that may not be easily collected by routine approaches. statisticians may work with practitioners to develop good sampling instruments and schemes for community risk surveillance. question : is face mask protective? if so, how to assess the compliance of face mask wearing? questions about the causal effect of face mask wearing on disease progression are very challenging. this is because there is no randomisation in the intervention allocation and many confounding factors are unobserved. question : is there evidence that the contagion of the coronavirus decays over time because of an increasing recovery rate of virus carriers and a decreasing rate of case fatality? statisticians ought to work out some thoughtful and convincing answers to the public. such surveillance data, there are data reporting gaps shown in figure a that are possibly caused by the so-called clustered reporting; that is, the recovered cases have not been released on the daily basis. to mitigate this data reporting artefact, we invoked a simple local polynomial regression procedure (loess) to smooth such unnatural jumps, resulting in a smooth fitted curve shown in figure a . the calibrated cumulative 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this research is partially supported by the national science foundation grant dms . although the two applications discussed earlier in section . give a framework of how ca models the dynamics of epidemic spread, white et al. ( ) provide a more direct incorporation of spatial ca with the temporal sir compartments at the population level, where each cell stands for a small population (e.g. a county) with different proportions of susceptible, infectious or recovered individuals. the resulting ca-sir given in white et al. ( ) is formulated by four parts (c, q, v and f). first, c d f.i; j/; Ä i Ä r; Ä j Ä cg defines the cellular space, or a collection of r c cells on a two-way array, where r c is referred to the dimension of the cells. second, q represents a finite set that contains all the possible states of a cellular space. in the case of the sir model, q d fs; i; rg corresponding to the susceptible, infectious and removed states. third, v d f.p k ; q k /; Ä k Ä ng is the finite set of indices defining the neighbourhood of each cell, and consequently, v ij d f.i c p ; j c q /; : : : ; .i c p n ; j c q n /g denotes the set of neighbouring cells for the central cell .i; j/. specifically, v d v f. ; /g represents all the neighbouring cells without the cell at the centre of consideration. fourth, function f stands for certain updating rules to govern the dynamics of interactions between cells in the a ca-sir system. for each cell at a discrete time t (say, today), its current status is described by three cell-specific compartments f s ij .t/,  i ij .t/ and  r ij .t/g, where  s ij .t/,  i ij .t/ and  r ij .t/ oe ; represent the cell-specific probabilities of being susceptible, infectious and recovered, respectively. clearly,  s ij .t/ c  i ij .t/ c  r ij .t/ d to form a microcell-level sir model. the ca-sir model is updated based on the following transition functions: for cell .i; j/ v, based on the basic ca-sir model proposed in white et al. ( ) , extensions can be easily applied to better model the dynamics of infectious diseases using real data. propose a spatio-temporal epidemiological forecast model that combines ca with an extended sair (esair) model to project the county-level covid- prevalence over counties in the continental united states. this model is termed as ca-esair model in which a county is treated as a cell. to carry out cell-level infection prevalence updates, the macroparametersǎ nd need to be estimated from the macromodel esair model. in comparison with the esir model discussed in section . , a new antibody compartment (a) is included in the esair model to account for the individuals who are self-immunised and have developed antibodies to the coronavirus. the inclusion of the antibody compartment can address the under-reporting issue known for available public databases and to build self-immunisation into the infection dynamics. in this way, better estimation of the macromodel parameters can be obtained. the esair model can be described using the following odes, which govern the law of interactive movements among four compartments or states of susceptible (s), self-immunised (a), infectious (i) and removed (r):where˛.t/ is the self-immunisation rate, .t/ is a time-varying transmission rate modifier,ˇis the basic disease transmission rate and is the rate of being removed from the system (either dead or recovered). the earlier esair model is an alternative expression of model ( ) based on the compartment probabilities. in order to apply the ca-esair system to model the epidemic spread in the usa, relax the classical ca-esair from spatial lattices (or cells) to areal locations of counties. let c be the collection of counties. here we consider the extended neighbourhood type (all counties are neighbouring ones given high mobility in the us population). for a county c c, n c denotes the county population size, and c c denotes the set of all the other counties except county c. for county c at time t, the county-specific probability vector is denoted by  c .t/ d . s c .t/;  a c .t/;  i c .t/;  r c .t// > . the ca-esair model at discrete times is expressed by the following form:where˛c.t/ is the county-specific self-immunisation rate and c .t/ is the county-specific transmission modifier. same as the parameter mentioned in the ca-sir model ( ) earlier, ! cc .t/ is a connectivity coefficient that quantifies the inter-county movements between counties c and c . by applying the proposed ca-esair model, have proposed a t-day ahead risk forecast of the covid- as well as a personal risk related to a travel route. the runge-kutta method is an efficient and widely used approach to solving ordinary differential equations when analytic closed-form solutions are unavailable. it is typically applied to derive a numerical functional system of high-order accuracy with no need of high-order derivatives of functions. the most well-known runge-kutta approximation is the runge-kutta fourth-order (rk ) method. for example, in the case of the mechanistic sir model ( ) where y is an unknown function in time t, which can be either a scalar or a vector. then for a preselected (small) step size h > , a fourth-order approximate solution of y satisfies at a sequence of equally spaced grid points y n ; n d ; ; : : : ; with jy n y n j d h, y nc d y n c h.k c k c k c k /; n d ; ; : : : ;where k d f .t n ; y n /;k d f .t n c h; y n c hk /:because four terms k , k , k and k are used in the approximation, the earlier method is termed as an rk method of the ode solution to function y. for a general rk approximation, refer to stoer and bulirsch ( ) . in the succeeding text, we list michigan data from march to june . the numbers of daily confirmed cases and deaths are obtained from the github repository jhu csse (https://github.com/cssegisanddata/covid- ), and the daily recovery data are collected from point acres (https://coronavirus. point acres.com). the daily cumulative numbers of deaths and recovered cases are then summed as the cumulative number of removed cases. in key: cord- - u cskii authors: nembaware, victoria; mazandu, gaston k.; hotchkiss, jade; safari serufuri, jean-michel; kent, jill; kengne, andre pascal; anie, kofi; munung, nchangwi syntia; bukini, daima; bitoungui, valentina josiane ngo; munube, deogratias; chirwa, uzima; chunda-liyoka, catherine; jonathan, agnes; flor-park, miriam v.; esoh, kevin kum; jonas, mario; mnika, khuthala; oosterwyk, chandré; masamu, upendo; morrice, jack; uwineza, annette; nguweneza, arthemon; banda, kambe; nyanor, isaac; adjei, david nana; siebu, nathan edward; nkanyemka, malula; kuona, patience; tayo, bamidele o.; campbell, andrew; oron, assaf p.; nnodu, obiageli e.; painstil, vivian; makani, julie; mulder, nicola; wonkam, ambroise title: the sickle cell disease ontology: enabling collaborative research and co-designing of new planetary health applications date: - - journal: omics doi: . /omi. . sha: doc_id: cord_uid: u cskii sickle cell disease (scd) is one of the most common blood disorders impacting planetary health. over , newborns are diagnosed with scd each year globally, with an increasing trend. the sickle cell disease ontology (scdo) is the most comprehensive multidisciplinary scd knowledge portal. the scdo was collaboratively developed by the scdo working group, which includes experts in scd and data standards from across the globe. this expert review presents highlights and lessons learned from the fourth scdo workshop that marked the beginning of applications toward planetary health impact, and with an eye to empower and cultivate multisite scd collaborative research. the workshop was organized by the sickle africa data coordinating center (sadacc) and attended by participants from countries, with participants connecting remotely. notably, from the standpoint of democratizing and innovating scientific meeting design, an scd patient advocate also presented at the workshop, giving a broader real-life perspective on patients' aspirations, needs, and challenges. a major component of the workshop was new approaches to harness scdo to harmonize data elements used by different studies. this was facilitated by a web-based platform onto which participants uploaded data elements from previous or ongoing scd-relevant research studies before the workshop, making multisite collaborative research studies based on existing scd data possible, including multisite cohort, scd global clinical trials, and scd community engagement approaches. trainees presented proposals for systematic literature reviews in key scd research areas. this expert review emphasizes potential and prospects of scdo-enabled data standards and harmonization to facilitate large-scale global scd collaborative initiatives. as the fields of public and global health continue to broaden toward planetary health, the scdo is well poised to play a prominent role to decipher scd pathophysiology further, and co-design diagnostics and therapeutics innovation in the field. s ickle cell disease (scd) is one of the most common blood disorders and is caused by a single point mutation, which promotes sickling of erythrocytes due to polymerization of hemoglobin s. each year, over , newborns are diagnosed with scd globally and the incidence rates are expected to increase (piel et al., ) . the increase in scd is compounded with its impact on morbidity and mortality rates. this has propelled scd from a neglected disease to a recognized planetary health challenge (ware, ) . as the fields of public and global health continue to broaden toward planetary health, new approaches toward knowledge coproduction and critical governance of medical innovations are urgently needed (haines, ; horton et al., ; Ö zdemir, ) . while traditional research efforts in scd are commendable, with a few exceptions, many are conducted in silos (makani et al., ) , focus on limited populations, and in some instances suffer from statistical underpowering. to promote collaborative research and translation of research into health care interventions, an scd ontology (scdo) was created by a multidisciplinary working group (group, ; mulder et al., ) . the working group is made up of members of the h abionet , the sickle pan africa network (span), and internationally recognized ontology experts. this working group currently operates under the auspice of the sickleinafrica consortium (makani et al., ) . the sickleinafrica consortium is made up of span and two national institutes of health (united states)-funded projects: the sickle pan-african consortium network (sparco) and the sickle africa data coordinating center (sadacc) (makani et al., ) . to our knowledge, the scdo is the most comprehensive resource of standardized hierarchical descriptions of knowledge in the scd domain and other related hemoglobinopathies (group, ) . work is already underway to translate this resource into french and portuguese and to create scdo-framed data standards for scd research. such scdodriven data standards are expected to support reliable and robust exchanges and translation of knowledge to planetary health action in the field, not to mention more efficient data integration and interoperability from different and diverse sites for retrospective and prospective research. currently, the scdo contains over standardized terms, which are categorized into upper classes, which include phenotypes, genetic modifiers, guidelines, and quality of life and care, among others. therefore, scdo data standards associated data elements and data collection tools encompass multiple disciplines. the scdo working group is continually updating the ontology, which is available on multiple platforms, including the project website (sickle cell disease ontology working group, c) , the european bioinformatics institute ontology look-up service (sickle cell disease ontology working group, b) , github (sickle cell disease ontology working group, a) , and bioportal (sickle cell disease ontology working group, ) . this expert review presents the highlights and the lessons learned from the fourth scdo workshop that marked the beginning of applications toward planetary health impact, and with an eye to empower and cultivate multisite scd collaborative research. the fourth scdo workshop took place in november in cape town, south africa and, as noted above, marked the beginning of the application phase of the ontology. the key aim of the workshop was to enable multisite scd cohort collaborative research and clinical trials by using the scdo to harmonize existing data elements from multiple sites. the structure of the scdo is now stable enough to support research and translational work (group, ) . in fact, the scdo framework has already been used to develop yet another disease-specific ontology, the hearing impairment ontology (hotchkiss et al., ) . the workshop objectives included collecting data elements from the scdo members who are actively engaged in data collection and research, in addition to demonstrating how data elements could be harmonized using the scdo. an online searchable platform, the global scd registry portal, (https://www.sickleinafrica.org/registries-list) was developed to facilitate uploading of existing research data elements preworkshop. other members of the scdo working group who could not attend the workshop are likely to upload their data elements in anticipation of a global cohort study. the process for using this platform is shown in figure . additional objectives included developing participants' knowledge and skills for establishing cohort studies and clinical trials. a total of participants attended the meeting from countries (brazil, cameroon, ghana, nigeria, rwanda, nembaware et al. the process map to register, view, and access the global scd registry portal. scd, sickle cell disease. senegal, south africa, tanzania, uganda, united kingdom, united states of america, malawi, zambia, and zimbabwe). participants included experienced scd researchers, junior researchers, and graduate students. the workshop provided an interdisciplinary global forum for participants to offer updates on scd activities at their sites and report on interesting research outputs from some of their studies. an update on the scdo was provided, followed by a session on data harmonization. brainstorming sessions were held to develop new research ideas that use the harmonized scd data or work across multiple sites, and discussions were held on how sites could get involved in clinical trials. the workshop included training activities in a variety of topics. an scd patient advocate also presented at the workshop, giving a patient perspective on their aspirations, needs, and challenges. the scdo was first released on bioportal in and published in a peer-reviewed publication in (group, ) ; however, the scdo is in constant flux as new terms are added and the structure refined. during this fourth scdo workshop, further additions and adjustments were made, particularly in the ''phenotype'' class, where concepts of abnormality versus complications were separated. in addition, the mapping of sickleinafrica data elements to the scdo (for data standards) was started. sparco aims to create a registry of , scd patients across three countries, tanzania, nigeria, and ghana, as a resource for future multisite cohort studies. all sites had achieved over % recruitment numbers of their targets. for example, ghana had registered patients representing . % of its target of patients. various stakeholder engagement activities are ongoing, of particular note are the public-private partnerships in ghana, aiming to make hydroxyurea widely accessible to scd patients. in addition, three sites are engaging and collaborating with government and advocacy organizations to ensure sustainability, delivering skills development activities for health care workers and researchers, developing standards of care for scd patients at different levels of the health care systems, and developing cohort and implementation research proposals. data from the three sites are collected with oversight from sadacc and the sparco hub. sadacc is also responsible for data archiving. the sicklegenafrica network is funded by the national institutes of health (nih) and is a collaborative project of genomics research coupled with capacity building (sickle-geneafrica, ). sicklegenafrica aims to recruit patients with scd (children year of age and older, and adults), who attend sickle cell clinics at six study sites in ghana, nigeria, and tanzania. the main objectives include identifying genetic markers of cytoprotection proteins that neutralize hemolysis danger-associated molecular pattern molecules and acute organ damage. in addition, the study aims to identify genome-wide determinants of malaria complications and echocardiographic abnormalities in patients with scd. ethics and community engagement are important components of the research. ethics and psychosocial research possibilities were presented by sicklegenafrica members during the workshop. a current survey being conducted within sicklegenafrica was shown as an exemplar of ethics research in autonomy and decision making, and return of individual findings. in addition, the european union arise network highlighted their work packages and ongoing work in scd research and training. cameroon: genetic modifiers of the severity of scd up to cameroonian scd patients were recruited in five regions of the country during two periods - and - . the aim of the project was to explore the frequency and influence of genetic variants known to modulate the severity of scd in cameroon. findings from the study were ( ) the known fetal hemoglobin (hbf)-promoting loci (bcl a and hbs l-myb) influence the expression of hbf in only . % (sickle cell disease ontology working group, ) of cameroonian patients, differing from the % to % observed in the african diaspora, ( ) % of cameroonian scd patients have a deletion on the alpha thalassemia gene (sickle cell disease ontology working group, a; sickle cell disease ontology working group, ) , and ( ) the predominant beta-globin gene haplotype is benin ( %), followed by cameroon ( %) (sickle cell disease ontology working group, b ). there was a high incidence of microalbuminuria ( %) and glomerular hyperfiltration ( %) in cameroonian children and adolescents living with scd. the study found that clinical indicators tend to contribute highly to kidney phenotypic variations in scd compared to the < % variation explained by six kidney dysfunctionrelated genetic variants. there is need for further genetic and environmental research in this subpopulation. given that scd and malaria have co-evolved over the years, this study investigated the evolutionary selection pressure exacted by malaria on the structure of the beta-hemoglobin (hbb) gene cluster in cameroonian individuals. briefly, differences in the genetic distance of various cameroonian ethnicities were observed, while longstanding knowledge of selection in the hbb gene cluster was confirmed. these findings can inform the design of future genetic association studies in specific african populations and guide the way inference is drawn from a few samples analyzed to the greater population. the recipient epidemiology and donor evaluation study-iii brazil scd cohort. this is a large multicenter cohort established to characterize health outcomes in the brazilian scd population. funded by the u.s. nih, the cohort was enrolled between and , with participants from four states (são paulo, rio de janeiro, minas gerais, and pernambuco). key publications to date are as follows: clinical and genetic ancestry profile of a large multicenter scd cohort in brazil. a description of patient recruitment and study procedures, and baseline data collected during enrollment visits ( - ) (carneiro-proietti et al., ) . the article also reports the sequencing of single nucleotide polymorphisms (snps) covering the entire genome. identification and characterization of hematopoietic stem cell transplantation candidates in an scd cohort. this study found that % of children and % of adults had at least one indication for hematopoietic stem cell transplantation according to ministry of health criteria (flor-park et al., ) . two groups were compared, those with at least one indication and those without indications. the first group showed more severe disease with respect to clinical characteristics. clinical and genetic predictors of priapism in scd (cintho ozahata et al., ) . this study describes a priapism prevalence of % in the cohort, with a higher occurrence in homozygous ss patients. priapism was associated with avascular necrosis and pulmonary hypertension. a genome-wide association study (gwas) also associated the risk of priapism with an snp in the transforming growth factor beta-receptor gene. there are other articles in preparation, including quality of life in children and adults, profile of s-beta and sb+ patients, pyrosequencing techniques for hb mutations, rhd and rhce variants, epidemiology of blood utilization and impact on chronic transfusion therapy and transfusion adverse events, risk and outcomes of hiv in scd, and transfusion-transmitted infections in the cohort. zambia: large-scale scd point-of-care screening in zambia, a multidisciplinary network in a rural area has demonstrated the feasibility of using dental services to facilitate large-scale scd point-of-care screening. scaling up of dental services and other public health approaches, including the expanded program for immunization, has great potential for scaling up screening for scd, including newborn screening in low-resource settings (chunda-liyoka et al., ) . to understand the burden of scd, the scd phenotype in zimbabwe, and gaps in the quality of care, a registry of children younger than years was launched in at a tertiary level hospital in harare. preliminary data from the small cohort have shown that hbss is the main genetic disease type. the scd registry pilot was successful and will be continued; however, there is a need for data harmonization with other centers in africa to allow collaborative work and data comparability. additional studies are required to further characterize the scd genotype and phenotype and establish newborn screening, and to provide data for policy makers and allow proper health care planning for patients with scd in zimbabwe. the sickleinafrica data elements are maintained on the redcap platform. following workshop sessions about redcap (basics, data elements, and data harmonization) using the scdo framework, participants highlighted what they considered the essential data elements in their scd registries. a consensus was reached and the sickleinafrica data elements adjusted accordingly. a live hands-on session on harmonization of new datasets was conducted for participants maintaining registries or collecting data for scd research. sadacc researchers had developed a spreadsheet that auto-identifies common data elements and python scripts that carry out the harmonization. participants provided their feedback on the experience and challenges faced during this harmonization process, which will be used to refine this process. data quality and assurance processes. participants received training on how redcap features can be utilized to improve data quality during patient recruitment. participants were introduced to r packages, which can calculate and summarize data quality-relevant information for a specified dataset. big data analytics. a video presentation was delivered by one of the co-leads of the big data analytics course, which was piloted to sickleinafrica researchers earlier in . background on the use of big data in scd was given and the different training modules were also described. ethics, legal and social implications. the ethics, legal and social implications (elsi) framework developed to support the sparco registry and sickleinafrica data governance issues among other items was finalized during this meeting by all participants. this included the data sharing and authorship policies and consent/assent forms. details of the processes followed in developing the elsi framework have been published in a separate article (munung et al., ) . the consortium agreed to develop elsi research questions, which could answer important questions for the implementation and application of the scdo for research purposes. it was agreed that patient engagement should be a core activity of the projects to maximize the benefits of the scdo to patient advocacy groups. the scdo is currently being translated into layperson language to allow patient groups to benefit from this resource. all sickleinafrica sites have extensive experience working with local scd patient groups; however, proper engagement models are lacking. it was agreed upon to work together with patient representatives across sites to design a patient engagement framework that is going to be used as a model within sickleinafrica. the scdo quality-of-life and care definitions were utilized to encourage workshop participants to interrogate their retrospective data, identifying possible quality-of-life indicators (e.g., pain, physical functioning, social functioning, education, and occupation) and possible calculation of disability-adjusted life year. for prospective studies, participants were presented with quality-of-life and care issues such as validation of measures, literacy, language differences, and consideration of appropriate pain, quality of life (e.g., eq- d-available in some african languages), health utility indices, and quality-adjusted life year assessments defined in the scdo. fifteen trainees from the pilot sickleinafrica big data analytics training were taught how to formulate research questions for literature reviews focusing on the scd. this training included an introductory lecture on scd. after the training, fellows presented their literature review concepts. to attend the next sickleinafrica consortium meeting, trainees would need to show substantial progress in the development of their literature reviews with plans to publish the article. the need for advanced training and expansion of this training to include proposal development and scientific writing, which is scheduled for the near future, was highlighted. challenges in the development of literature reviews included lack of time and limited access to journals. to support future collaborations among participants, training was provided in collaborative grant writing. the differences between writing collaborative grant proposals compared to single group proposals were highlighted. key skills identified were leadership, communication, and planning. the collaborative process involves orientation, conflict, and emergence and reinforcement phases (dopke and crawley, ) . the understanding of belbin team roles and their strengths and weaknesses were proposed to facilitate efficient teamwork. using scd as an example, information on the availability and accessibility of grant funding opportunities for groups in low-and middle-income countries was provided. this session described observational studies, distinguishing cross-sectional studies, case-control studies, and cohort studies. cohort studies were further distinguished as follows: concurrent prospective, nonconcurrent prospective, crosssectional prospective, truly retrospective, and cross-sectional retrospective. the session suggested that the scope of observational studies in sickleinafrica will mostly include cross-sectional studies, retrospective studies, and prospective nonconcurrent studies. the core of the session focused on potential challenges that could be encountered analyzing sickleinafrica data, which include reassessing the distribution of diseases and health statuses using available data, specifically: representativeness of the study populations the accuracy of diagnosis and definition of health statuses completeness of information on diseases and health statuses (missing data). due to variation across sites, analysis of sickleinafrica multicenter observational studies is likely to require extensive data quality checks. possible solutions to some of these challenges were discussed, including imputation of missing data and employing individual participant data meta-analysis for multicenter observational studies. examples were used to illustrate challenges and how they have been addressed in published studies such as the asia pacific cohort studies collaboration (kengne et al., ) studies collaboration et al., ) , the health surveys of england cohorts (kengne et al., ) , the pan-european epic-interact study , and the demographic health survey data (caleyachetty et al., ) . a representative from the african academy of sciences (aas) presented a prerecorded video talk on the current status, new priorities, and new opportunities for clinical trials in africa. aas aims to create a platform to support communities of clinical trials. subsequently, the main features and principles of clinical trials were discussed. a strong emphasis was placed on conducting research in an ethical manner. recent clinical trial examples (two from europe and one from africa) illustrated how quickly sidestepping or ignoring safeguards may lead to detrimental patient outcomes. the discussion was particularly timely, given the announcement a week earlier of a new major collaborative effort between the nih and the bill and melinda gates foundation to develop advanced therapies for scd (''nih launches new collaboration to develop gene-based cures for sickle cell disease and hiv on global scale j national institutes of health [nih],'' ), an effort that will require numerous clinical trials of novel therapies both in africa and overseas. in addition, the difference between early-phase and latephase trials was explained, with the double-blinded, placebocontrolled, randomized controlled trial showcased as the (often infeasible) gold standard for the latter. the challenges in generalizing trial results to entire patient populations, when the trials are usually carried out on convenience samples, were also discussed versus case studies. recent clinical trials with severe ethical transgressions leading to injury or death were highlighted, including ''abdullahi vs. pfizer,'' or ''tegenero,'' or ''bia - .'' the aas representative led a discussion on how a community of scd stakeholders could be created to support clinical trials across the continent (within the aas clinical trials community platform currently under development). therefore, in addition to the clinical trials proposed in table , the group also highlighted suggestions and their needs to the aas. integration of biomarker candidates in global clinical trials can help not only personalized medicine in the clinic but also inform broader generalization of trial results across world populations in the future (s xardas x and kendirci, ). basics of designing observational studies based on existing data were introduced. participants were encouraged to collaborate with a quantitative analyst starting in the conceptualization and design stages rather than only for the analysis stage. potential biases and limitations of observational studies, including confounding variables and other causal relationships, were demonstrated. the need to strike a balance between a priori analysis plans and the process of discovery was discussed, as well as the importance of high-quality descriptive work, besides embarking upon more sophisticated model-based analysis. some case studies were provided as examples for observational and clinical trial talks and are summarized in table . nembaware et al. in two sessions, participants developed detailed concepts for future studies. for the observational studies, two studies were proposed, a descriptive study and a genetic study. the descriptive study will focus on a meta-analysis across all the span countries and would include physical attributes, disease, and phenotypes of patients. the genetic study will be a gwas meta-analysis of hbf based on existing gwas data from tanzania, cameroon, brazil, and united states. in addition, research questions will be developed by the big data analytics trainees. groups were encouraged to align their clinical trials to the sparco research focus areas; a summary of the proposed clinical trial studies is in table . the scd research field has historically been trivialized for scientific and technological advancements due to a range of inherent biases that exist in common global frameworks. planetary health is a public health discipline established in , calls for critical governance and implementation frameworks that if adopted for the scd research field could provide effective planetary health solutions that consider and encompass natural systems upon which human health depends (haines, ; horton et al., ; whitmee et al., ) . a unique differentiating feature of planetary health is that not only is its scope broad but it is also critically informed, for intervention for poor growth in infants and young children with scd malaria and scd chemoprophylaxis regimens with or without insecticide-treated bednets example, by emphasizing and formally recognizing the importance of ecological and political determinants of health. ecological determinants include, for example, pandemics such as covid- , whereas political determinants include power asymmetries and historical and modern-day social injustices in science and quotidian life that are often overlooked and yet markedly impact the health of humans and planetary ecosystems. to these ends, this report demonstrates a shift in the scd field toward participatory research, highlighting the relevancy of a recently proposed planetary health knowledge critical governance framework to advocate for innovative and more participatory and inclusive approaches in global science and research (Ö zdemir, ) . in addition, the scdo has potential to enable tailoring of planetary biomedical and clinical solutions through promotion of human collaborative research across biological, social, ecological, and political determinants of health. for example, the scdo could enable implementation of panvigilance, a systems pharmacovigilance planetary approach, which holds great promise toward enhanced management of adverse drug reactions, drug safety, and efficacy. panvigilance integrates biomarkers from edge populations in clinical trial designs for improved detection of unintended drug effects, whether adverse, toxic, or therapeutic (s xardas x and kendirci, ). the scdo working group agreed on deliverables to foster collaborative research, strengthening research quality and scientific writing skills of the participants. most importantly, the workshop demonstrated the potential of scdo-enabled data standardization to enable large-scale global scd collaborative initiatives using existing disparate multisite datasets. while biomedical science and clinical applications broaden toward planetary health, the scdo is well poised to play a prominent role to decipher scd pathophysiology further, and co-design diagnostics and therapeutics innovation in the field. systolic blood pressure, diabetes and the risk of cardiovascular diseases in the asia-pacific region vitamin a supplementation every months with retinol in million pre-school children in north india: devta, a cluster-randomised trial prevalence of behavioural risk factors for cardiovascular disease in adolescents in low-income and middle-income countries: an individual participant data meta-analysis tobacco use in pregnant women: analysis of data from demographic and health surveys from low-income and middle-income countries clinical and genetic ancestry profile of a large multicentre sickle cell disease cohort in brazil application of a public health strategy to large-scale point-of-care screening for sickle cell disease in rural sub-saharan africa clinical and genetic predictors of priapism in sickle cell disease: results from the recipient epidemiology and donor evaluation study iii brazil cohort study characterization of inpatient cystic fibrosis pulmonary exacerbations. pediatrics strategies for increasing the efficacy of collaborative grant writing groups in preparing federal proposals identification and characterization of hematopoietic stem cell transplant candidates in a sickle cell disease cohort the sickle cell disease ontology: enabling universal sickle cell-based knowledge representation addressing challenges to human health in the anthropocene epoch-an overview of the findings of the rockefeller/lancet commission on planetary health from public to planetary health: a manifesto the hearing impairment ontology: a tool for unifying hearing impairment knowledge to enhance collaborative research azithromycin to reduce childhood mortality in sub-saharan africa association of c-reactive protein with cardiovascular disease mortality according to diabetes status: pooled analyses of , participants from four u.k. prospective cohort studies noninvasive risk scores for prediction of type diabetes (epic-interact): a validation of existing models smoking, diabetes and cardiovascular diseases in men in the asia pacific region sickle cell disease: tipping the balance of genomic research to catalyse discoveries in africa development of bioinformatics infrastructure for genomics research in h africa proceedings of a sickle cell disease ontology workshop-towards the first comprehensive ontology for sickle cell disease nih launches new collaboration to develop gene-based cures for sickle cell disease and hiv on global scale j national institutes of health (nih) innovating governance for planetary health with three critically informed frames global burden of sickle cell anaemia in children under five, - : modelling based on demographics, excess mortality, and interventions using dynamic treatment regimes to understand erythropoietinstimulating agent hyporesponsiveness comparison of aberrant behavior checklist profiles across prader-willi syndrome, down syndrome, and autism spectrum disorder panvigilance: integrating biomarkers in clinical trials for systems pharmacovigilance sickle cell disease ontology working group sickle cell disease ontology sickle cell disease ontology sickle cell disease ontology project-scdo is sickle cell anemia a neglected tropical disease? safeguarding human health in the anthropocene epoch: report of the rockefeller foundation-lancet commission on planetary health all authors contributed to the article and approved the final version before submission. we thank collin pillai from aas for his presentation. the authors declare they have no conflicting financial interests. this work was supported by the national heart, lung, and blood institute of the national institutes of health (award number u hl ) and (award number u hl ). key: cord- -xl fv qx authors: kahn, r. e.; morozov, i.; feldmann, h.; richt, j. a. title: th international conference on emerging zoonoses date: - - journal: zoonoses public health doi: . /j. - . . .x sha: doc_id: cord_uid: xl fv qx the th international conference on emerging zoonoses, held at cancun, mexico, – february , offered participants from countries, a snapshot of current research in numerous zoonoses caused by viruses, bacteria or prions. co‐chaired by professors heinz feldmann and jürgen richt, the conference explored topics: (i) the ecology of emerging zoonotic diseases; (ii) the role of wildlife in emerging zoonoses; (iii) cross‐species transmission of zoonotic pathogens; (iv) emerging and neglected influenza viruses; (v) haemorrhagic fever viruses; (vi) emerging bacterial diseases; (vii) outbreak responses to zoonotic diseases; (viii) food‐borne zoonotic diseases; (ix) prion diseases; and (x) modelling and prediction of emergence of zoonoses. human medicine, veterinary medicine and environmental challenges are viewed as a unity, which must be considered under the umbrella of ‘one health’. several presentations attempted to integrate the insights gained from field data with mathematical models in the search for effective control measures of specific zoonoses. the overriding objective of the research presentations was to create, improve and use the tools essential to address the risk of contagions in a globalized society. in seeking to fulfil this objective, a three‐step approach has often been applied: (i) use cultured cells, model and natural animal hosts and human clinical models to study infection; (ii) combine traditional histopathological and biochemical approaches with functional genomics, proteomics and computational biology; and (iii) obtain signatures of virulence and insights into mechanisms of host defense response, immune evasion and pathogenesis. this meeting review summarizes of the conference presentations and mentions briefly the articles in this special supplement, most of which were presented at the conference in earlier versions. the full affiliations of all presenters and many colleagues have been included to facilitate further inquiries from readers. addition to the summaries later of six presentations on this topic, this special supplement includes an article, monitoring of west nile virus infections in germany by dr. u. ziegler et al. which identified west nile virus (wnv) antibodies in migratory birds, but not in resident birds, in domestic poultry or in local horse populations throughout germany. the wnv antibody-positive species were found in birds that migrate to tropical africa or southern europe; however, wnv-specific rna could not be found in any of the samples. the conference opened with a presentation from professor m. a. diuk-wasser and her colleagues j. simpson and c. m. fosom-o'keefe (all yale school of public health, new haven, ct, usa) and g. molei, p. m. armstrong, and t. g. andreadis (center for vector biology and zoonotic species at the connecticut agricultural experiment station, new haven, ct, usa), ecology of west nile virus in the north-eastern united states. professor diuk-wasser began by noting that west nile virus (wnv) was introduced into new york city in by unknown means and was now considered endemic throughout the usa, with , human cases and , deaths in the usa since . it had been hypothesized that increased biodiversity leads to a decreased risk of exposure to zoonotic pathogens (keesing et al., ) . at issue is whether this 'dilution effect' or 'zooprophylaxis' for vector-borne pathogens applies only when vectors are generalist feeders, because the link between host diversity and pathogen transmission might break down when vectors exhibit host preferences. in the north-eastern united states, wnv perpetuates in an enzootic transmission cycle involving culex spp. mosquitoes and virus-competent avian hosts. previous studies had detected that a large proportion of c. pipiens and c. restuans bloodmeals were derived from american robins (turdus migratorius), suggesting a key role for this bird species in the wnv transmission cycle (kilpatrick et al., ; molaei et al., ) . the new haven-based research team tested for preferential feeding by conducting equal choice experiments (robins versus other bird species) (simpson et al., ) and by comparing the proportion of culex spp. bloodmeals acquired from robins to the proportion of robins in the local bird community. both methods indicated preferential feeding for robins. they were also able to identify robin communal roosts as amplification foci in greater new haven (diuk-wasser et al., ) . then, through field-informed mathematical modelling, they determined that host preferences were indeed key drivers of wnv transmission and that landscape attributes (such as urbanization) in combination with mosquito abundance and a measure of host community competence were the strongest predictors of pathogen prevalence (simpson et al., ) . thus, it was clear that pathogen prevalence and human risk of infection were best predicted by assessing the relative pathogen competence and attractiveness to vectors of all species in the host community, rather than using simple measures of biodiversity. in the next presentation, interactions among multiple tick-borne pathogens in a natural reservoir host, professor fish and his colleagues j. brown, m. fitzpatrick, s. usmani-brown, p. cislo and p. krause (yale school of public health, new haven, ct, usa) stressed that species interactions within a parasite community drive infection risk in a wildlife population (telfer et al., ) . at least five tick-borne pathogens are known to be transmitted by ixodes scapularis, the principal vector of lyme disease in the united states: (i) borrelia burgdorferi, an agent of lyme disease; (ii) anaplasma phagocytophilum, an agent of human anaplasmosis; (iii) babesia microti, an agent of human babesiosis; (iv) borrelia miyamotoi, an agent of relapsing fever; and (v) the powassan encephalitis virus. two or more of these pathogens can be transmitted either simultaneously by a single tick or sequentially by successive tick-bites, resulting in different permutations of mixed-infection studies. in the context of pathogen prevalence of ixodes scapularis nymphs, borrelia burgdorferi, has been found in . % of samples from the north-east and mid-western united states, while babesia miroti has been found in . % of samples from block island, rhode island. professor fish explained that several types of co-infections have been explored in an experimental system employing laboratory colonies of i. scapularis ticks and peromyscus leucopus white-footed mice, a natural reservoir host for these pathogens. outcomes of mixed infections in mice have been measured by r o , the fitness parameter and basic reproductive rate which indicates the number of secondary tick infections resulting from a primary infection (levin and fish, ) . the observed outcomes of dual mixed infections have been variable with both positive and negative effects on r o , while interactions have been mutual, unidirectional or null. these diverse pathogen interactions play an important role in determining the infection prevalence of host-seeking nymphs in nature, and consequently, in the risk of infection for humans. professor h. henttonen (finnish forest research institute, vantaa, finland) and his team h. leirs, e. r. kallio, k. tersago and l. voutilainen in collaboration with university of antwerp, belgium; university of liverpool, united kingdom; and the universities of helsinki and jyväskylä, finland, studied biome specific rodent dynamics and hanta epidemiologies in europe. their research sought to understand the main biomes and forest coverage in europe, the european hanta viruses and their carriers, and the biome specific dynamics of hanta virus carriers and the biome specific transmission dynamics and epidemiologies. within the bunyaviridae family of viruses, hantaviruses infect rodents (and insectivores) and cause haemorrhagic fever with renal syndrome (hfrs) in humans in the old world and hantavirus cardiopulmonary syndrome (hcps) in the new world. in a large european union project, eden (emerging diseases in a changing european environment, ), rodent-borne (robo) viral infections have been studied, along with tick-borne pathogens, leishmaniasis, west nile virus, malaria and rift valley fever. the most important aim of professor henttonen and his colleagues was to clarify the differences in boreal (northern) and temperate europe in the human epidemiology of nephropathia epidemica, by far the most common hantaviral disease in europe, caused by puumala hantavirus (puuv). the population dynamics of the host species, the bank vole, differ greatly in various parts of europe, driven by predation in the north and masting events in the temperate zone. consequently, the causes of rodent fluctuations are different. in addition, the role of landscape patterns (homogenous taiga vs. fragmented temperate forests) in rodent/virus dispersal is significant, as well as local environmental conditions (e.g. temperature and moisture), which affect virus survival outside the host. for example, in room temperature, puuv remains infectious for at least weeks outside the host, and possibly for much longer in cold temperatures and in moist conditions. these research findings are essential for human risk evaluation with regard to both long-term and seasonal occurrence of puuv in the environment. in spite of chronic infection of bank voles and the excretion of puuv in their faeces, urine and saliva, the shedding period is limited, which has significant implications for seasonal transmission dynamics in rodents. thus, within the same host/virus system, biomespecific puuv epidemiologies occur (kallio et al., ; tersago et al., ) , thereby highlighting the need for geographically comparative studies in europe (metla, ) . professor v. sambri and his team, p. gaibani, f. cavrini, a. m. pierro, m. p. landini and g. rossini (all regional centre for microbiological emergencies [crrem] , unit of clinical microbiology, st orsola-malpighi university hospital, bologna, italy) investigated usutu: a novel human pathogenic mosquito-borne flavivirus. this virus belongs to the japanese encephalitis serogroup within the mosquito-borne cluster of the genus flavivirus in the family flaviviridae. first isolated from mosquitoes of the genus culex in south africa in , the usutu virus (usuv) has since been isolated from mosquitoes, rodents and birds throughout sub-saharan africa and europe. the virus is thought to be maintained in nature in a mosquito-bird transmission cycle in areas with a minimum of at least ten hot days > °c, but no mammalian reservoir has yet been identified. professor sambri pointed out that it was not until september that usuv was found in the liver of a patient who underwent an orthotropic liver transplant (gaibani et al., ) . further study of the plasma and genome sequencing analysis confirmed the presence of usuv viremia. then usuv was detected in the livers of an additional four patients from the same area suffering from acute meningo-encephalitis during / . both serological assay and molecular assay have been used as new tools for the diagnosis of usuv infection. thus, it is now clear that usuv is a new emerging flavivirus pathogenic for humans. further studies are required to discover both the geographical distribution of this virus and the mechanisms by which humans acquire the virus. since this conference presentation, there has been increased awareness of the seriousness of usuv (vázquez et al., ) . according to the world health organisation (who) and unicef, . million children under the age of five die from diarrhoea annually (unicef/who, astroviruses cause infections within the small intestine and are associated with at least % of all sporadic cases and > % of all hospitalized cases. these rapidly evolving, nonenveloped, single-stranded rna viruses can be transmitted directly from infected individuals and animals, and indirectly through contaminated food and water. professor schultz-cherry's laboratory was the first to demonstrate that astroviruses induce diarrhoea by a novel mechanism: they possess an enterotoxin that disrupts intestinal epithelial barrier function independent of cellular damage or an inflammatory response (koci et al., ) . this occurs within h post-infection because of reorganization of the tight junction protein occludin and the actin cytoskeleton (moser et al., ) . in essence, within a complex pathogenic process, astroviruses cause diarrhoea by increasing intestinal barrier permeability. this is the first evidence showing that a viral coat protein is an enterotoxin. of great interest, the toxin can act independently of species barriers. given the increasing isolation of astroviruses from diverse species, there is increasing evidence that toxicogenic astroviruses could be associated with zoonotic disease. professor m. g. katze (department of microbiology and washington national primate research center, university of washington, seattle, wa, usa) set out a unifying approach to molecular biology in his presentation, systems and computational biology: emerging tools for exploring emerging viruses. he emphasized that modern day virologists and immunologists must do better in their search to understand how a virus kills and how effective vaccines can be developed, especially because traditional virology has yielded surprisingly little information about why some virus strains cause severe diseases while others remain innocuous. he pointed out that the case fatality rate for the influenza pandemic was about . % and that particular h n virus may have infected as much as one-third of the world's population. issues arise not only in understanding a virus, but also in understanding how hosts respond. for example, the virus infection resulted in very high expression of inflammatory, antiviral and immune cell genes very early in host infection (kash et al., ) . significant progress in overcoming existing and emerging viruses depends on biologists, mathematicians and computer specialists working together within a systems biology paradigm. such research begins with either in vitro studies of virus replication on cell lines or primary cell cultures, moving to nonhuman primate models of virus infection. then samples from the experiments are investigated at multiple time points and conditions; and high throughput data are then examined by data processing to prepare systematic evaluations of different host responses. data integration involving data analysis and modelling of key genes and pathways is then possible, followed by iterative processing of host perturbations and the use of viral mutants to discover specific applications to translational research. such a systems biology approach requires not only continuing experiments with virusinfected experimental systems but also significant efforts to maintain the hardware and software of an extensive laboratory computational infrastructure. it is this computing infrastructure, which permits the laboratory to go quickly from samples to pathway visualization, as the data analysis workflow moves from microarray images to gene expression data to pathway models. the mission of this virolab is to develop steadily over the years to come a virtual laboratory to confront the viruses involved in infectious diseases -influenza, ebola, marburg, hepatitis c, sars-cov, vaccinia, herpes simplex, west nile, hiv- , siv, measles, lassa, chikungunya and dengue fever. the three key characteristics of this integrated approach to so many infectious diseases are as follows: (i) to use cell culture, primary cells, nonhuman primate and human clinical models to study viral infection; (ii) to combine traditional histopathological, virological and biochemical approaches with functional genomics, proteomics and computational biology (haagmans et al., ); and (iii) to obtain signatures of virulence and insights into mechanisms of host defense response, viral evasion and pathogenesis (casadevaill et al., ) . for example, with the study of all respiratory viral diseases, a unifying hypothesis is that highly pathogenic respiratory viruses use both unique and common strategies to remodel the host cell to enhance virus replication, regulate disease severity and promote virus transmission (chang et al., ) . a highly significant new tool for studying these emerging viruses is next generation sequencing (ngs) which has already 'changed the way we think about scientific approaches in basic, applied and clinical research' to such an extent that 'the potential of ngs is akin to the early days of polymerase chain reaction (pcr), with one's imagination being the primary limitation to its use' (peng et al., ) . already, a good understanding of the 'timing' and extent of immune (innate)-mediated injury after virus infection has been achieved. furthermore, molecular 'disease' signatures associated with different pathogens in multiple animal species have been described at micro-rna, mrna, protein level, metabolite and lipid levels. such successful modelling of molecular events has made possible verifiable prediction about key nodes and bottlenecks, enabling the identification of novel host cell drug targets (diamond et al., ) . the translational impact of this research, in professor katze's view, will be immense, revealing a completely new and expanded host defense repertoire consisting of non-annotated noncoding rnas. despite all of these achievements, four crucial questions remain unanswered: (i) is systems biology too complicated and too expensive to become the pre-eminent approach in virology and immunology? (ii) are mathematicians and computer scientists up to the challenges? (iii) how will new technologies like next generation sequencing impact virus systems biology research, especially in the context of rna sequencing? (iv) how can new principal investigators best be identified and appointed? (virolab, ) . it has long been recognized that the emergence of any zoonoses is a complex process involving 'ecological interactions at the individual, species, community and global scale' (childs et al., , p. ) . this topic began with a presentation from professor a. a. aguirre that focused on the ecological framework in which any zoonotic disease should be considered. the role of bats as an important reservoir host for many dangerous zoonotic pathogens was then considered in some detail (cf. daniels et al., ; field et al., ; gonzalez et al., ; wang and eaton, ) . professor a. a. aguirre (department of environmental science and policy, george mason university and executive director, smithsonian-mason global conservation studies program, front royal, virginia, usa) presented emerging zoonotic diseases of wildlife: developing global capacity for prediction and prevention. he began by explaining that conservation medicine and more recently ecohealth have emphasized the need to bridge disciplines, thereby linking human health, animal health and ecosystem health under the paradigm that 'health connects all species in the planet' (aguirre et al., ) . in his view, the recent convergence of global problems such as climate change, biodiversity loss, habitat fragmentation, globalization, infectious disease emergence and ecological health demands integrative approaches breaching disciplinary boundaries. the international union for conservation of nature (iucn) maintains a red list of threatened species -an important initiative in view of the animal extinctions that have already occurred, of which . % were caused by disease (smith et al., ) . professor aguirre noted that the u.s. agency for international development (usaid) has been a major leader in the global response to the emergence and spread of highly pathogenic avian influenza (hpai). since mid- , it has programmed approximately $ million to build capacities in more than countries for monitoring the spread of hpai among wild bird populations, domestic poultry, and humans, and to mount a rapid and effective containment of the virus when it is found. recent analyses indicate that these efforts have contributed to significant downturns in reported poultry outbreaks and human infections and a dramatic reduction in the number of countries affected. furthermore, the usaid bureau for global health, office of health, infectious disease and nutrition (gh/hidn) recently funded two cooperative agreements, predict and respond, under its avian and pandemic influenza and zoonotic disease program to continue and expand this work. the goal of predict is to establish a global early warning system for zoonotic disease emergence that is capable of detecting, tracking and predicting the emergence of new infectious diseases in high-risk wildlife (e.g. bats, rodents and nonhuman primates) that could pose a major threat to human health. the goal of respond is to improve the capacity of countries in high-risk areas to respond to outbreaks of emergent zoonotic diseases that pose a serious threat to human health. the geographical scope of this expanded effort is directed to zoonotic 'hotspots' of wildlife and domestic animal origins (jones et al., ) . predict includes a program of smart (strategic, measurable, adaptive, responsive and targeted) surveillance that focuses on preventing the 'spilling over' from wildlife to humans or to halt these diseases rapidly after that spillover by understanding what factors induce emergence and rapidly identifying ways of prevention, control, and mitigation. the overall aim of smart is to promote an integrated, global approach to emerging zoonoses. this integration requires commitment from a broad coalition of partners and stakeholders including government agencies, universities and non-governmental organizations, collaborating for specific purposes and to generate in the future new international structures able to respond to these emerging zoonoses. with . billion animals being imported into the united states each year, as well as an extensive international trade in illegal animal exports ) and some % of emerging zoonoses worldwide having wildlife origins, professor aguirre stressed that ecohealth has become a necessity, not an optional policy goal. dr. g. a. marsh and his colleague dr. l.-f. wang (australian animal health laboratory [aahl], geelong, victoria, australia) began their presentation, bats: a mixed bag of new and emerging viruses, by pointing out that the ''old'' bat viruses were represented by many zoonotic pathogens, including rabies virus, yellow fever virus, st louis and japanese encephalitis viruses, and west nile virus. now bats have been identified as natural reservoirs for a number of new and emerging viruses -ebola virus, marburg virus, hendra virus and sars-like coronaviruses. there are some different bat species; and they often roost in high-density colonies of over one million flying mammals, which have, in a very real sense, been travelling for millions of years, exposing themselves to many pathogens; therefore, the resulting complexity is not surprising. key research questions include (i) why do bats seem to be able to co-exist with a great diversity of viruses without showing disease signs? (ii) what triggers the spillover of bat viruses into other animals? (iii) do bats control viral infection differently from other mammals? attempts to isolate viruses from bats have generally been unsuccessful. therefore, in an effort to improve the success rate for virus isolation, dr. marsh and his team have recently developed primary cell culture lines from numerous different species of bats (crameri et al., ) . the use of these bat cell lines, in combination with improved sampling techniques, has lead to recent isolation of hendra virus from a number of bat urine samples collected in several locations across queensland, australia, including those associated with human and horse virus spillover events (smith et al., ) . furthermore, this henipavirus surveillance program has led to the isolation of a number of novel viruses from two different virus families, whose zoonotic potential is not yet known. in an attempt to understand virus/host interactions, as well as to provide insight into the key factors involved in future spillover events, aahl has launched a number of international collaborative projects in south-east asia and ghana, west africa. c. kohl (sonntag et al., ) . the phylogenetic analysis of the genome sequence of bat adv- demonstrated a close relationship to canine adenovirus and (cadv- and cadv- ) (kohl et al., ) . the very similar genome organization supported the hypothesis of a shared ancient ancestor. interestingly, both cadvs are presenting untypical pathological features within the family adenoviridae. these adenoviruses were found to have an unusually broad host range and are causing a rather higher pathogenicity in a variety of carnivore hosts. the untypical pathological features might be understood as signs of a missing adaptation host and could provide a model to study ancient inter-species transmission events. this section of the conference addressed cross-species transmission of selected pathogens. in addition to the summaries below of three presentations on this topic, this special supplement includes an article, epidemiological survey of tryanosoma cruzi infection in domestic owned cats from the tropical southeast of mexico by dr. m. jiménz-coello et al. setting out how a significant public health problem in mexico has been caused by the crossspecies transmission of american trypanosomiasis (at) from triatomine bugs to domestic cats, representing a potential risk to humans. speaking on behalf of an extensive team of collaborators from a number of institutions -c. osborne, p. cryan, t. j. o'shea, l. m. oko, c. ndaluka, c. h. calisher, a. berglund, m. l. klavetter, r. a. bowen and k. v. homes -dr. s. r. dominguez (section on pediatric diseases, the children's hospital, university of colorado school of medicine, aurora, co, usa) began by noting that the first pandemic of the twenty-first century, the deadly sars virus, had its natural reservoir in bats. in his presentation, alphacoronaviruses in new world bats: prevalence, persistence, phylogeny and potential for interaction with humans, he suggested that bat coronaviruses (covs) may well be the ancestors of all group and covs. today bats had become a primary species encountered by humans in terms of potential exposure to significant disease agents. their research was tackling three important unanswered questions: (i) what is the prevalence and diversity of bat covs in new world bats? (ii) do bat covs persist in bat populations and/or individual bats? (iii) what are the potential interactions of infected bats with the human population? a -year study (osborne et al., ) had collected clinical and environmental samples from bats at rural sites and urban sites throughout colorado, as well as bat carcasses obtained from various counties throughout the state from the colorado department of public health and environment. of the , faecal or anal swab samples, , that is, %, were positive for cov rna. the highest prevalence of the virus was in juvenile bats; although rates of prevalence varied from year to year, late spring was the time when the virus peaked. although bat covs persisted within bat populations and their roosts, individually tagged cov-infected bats cleared their infections within weeks without apparent illness. new world bats of the same species in geographically distinct locations and over the course of several years harbour similar covs, and some new world bat covs may be able to infect bats of different genera. strikingly, bats, which had known or potential contact with humans, had a high prevalence of - % of cov infection. it is clear that significant opportunities exist for zoonotic transmission of coronaviruses from bats to humans and vice versa, especially as more than viruses have already been isolated from or detected in bat tissues. noting that many mammalian and avian species in addition to bats are susceptible to coronavirus infection, receptor proteins that include ace , apn and cea-cam . the recent emergence of sars coronaviruses from civets, bats and/or other reservoir species into humans depended upon a few amino acid substitutions in the receptor-binding domain (rbd) of s from the animal viruses that allowed them to recognize human ace instead of, or in addition to, receptors of their natural hosts (li, ) . alphacoronaviruses of pigs, cats, dogs and human coronovirus e use apn receptors of the host species, and all four viruses recognize feline apn (tusell et al., ) . in contrast, for human alphacoronavirus nl , the receptor-binding motif (rbm) with its three loops in the rbd binds specifically to human ace . in the rbds of the cat virus, fipv, professor holmes and her research team predicted three loops structurally similar to the nl rbms, and they constructed chimeric fipv rbds containing one, two or three rbms from nl . receptor-binding assays using enzyme-linked immunoassays (elisa), flow cytometry and co-immunoprecipitation identified three loops (rbms) in fipv rbd that are required for binding to feline apn. furthermore, substitution of only a few key amino acid residues within the rbms of fipv altered apn specificity and viral host range. thus, the emergence of alphacoronaviruses into new host species can occur when spontaneous mutations arise in the rbms that permit binding to variants of the apn receptor protein expressed by different host species. considering the interaction between human and swine h n viruses since , professor h. d. klenk (institute of virology, philipps university, marburg, germany) presented the mechanisms of pathogenicity and host adaptation of influenza viruses in the light of the new h n pandemic. he explained that there was now a clear scientific consensus that wild aquatic birds are the natural hosts for a large variety of influenza a viruses. occasionally these viruses are transmitted from this reservoir to other species, such as chickens, pigs and humans, leading to devastating outbreaks in domestic poultry and the possibility of human influenza pandemics. by the end of february , there had been , deaths, with the world health organization later confirming cases in countries and territories, with deaths in at least countries and territories before the spread of the h n virus diminished. however, professor klenk set out the evidence to support his view that the pathogenic and pandemic potential of this new h n virus is not yet exhausted. the host range and pathogenicity of any virus are polygenic traits depend on the interaction of different viral proteins with specific host factors. it has long been known that proteolytic activation and receptor specificity of the hemagglutinin (ha) are important determinants for pathogenicity and interspecies transmission, respectively. there is now considerable evidence that ha mutations altering receptor specificity and cell tropism of the pandemic influenza a virus (h n v) are linked to the d g amino acid substitution and are associated with a particularly severe outcome of infection (liu et al., ) . it should be remembered that the viral polymerase has to enter the nucleus of the infected cell to promote replication and transcription of the viral genome. adaptive mutations in polymerase subunits of avian viruses improve binding to importin alpha, a component of the nuclear pore complex in mammalian cells. as a result, nuclear transport of these proteins and efficiency of replication are enhanced. thus, the interaction of the viral polymerase with the nuclear import machinery is an important determinant of host range. some of the structural features typical for avian viruses have been preserved in the polymerase of the pandemic influenza a virus (h n v) suggesting that this virus has the potential to further adapt to humans. recent studies have shown that the ns protein, another important determinant of pathogenicity and host range, is sumoylated and that this modification enhances virus growth. interestingly, ns of h n v is not sumoylated (xu et al., ) . taken together, these observations support the view that the pathogenic and pandemic potential of the new virus is not yet exhausted. furthermore, because of the firm evidence of ha polymorphism in position , mutants and other mutations with altered receptor specificity will have to be closely monitored. in the subsequent discussion, it was noted that when a virus becomes highly pathogenic, this might block its spread if additional hosts are not readily available. furthermore, the role of co-infection with bacterial inflection was highly relevant in the - influenza pandemic and might well be relevant in a future pandemic. there have been at least three influenza pandemics every century since , with some evidence of earlier epidemics and pandemics after . in the cambridge world history of human disease, a. w. crosby ( ; p. ) has noted that although the black death and world wars i and ii killed higher percentages of the populations at risk, the - influenza pandemic was possibly 'in terms of absolute numbers, the greatest single demographic shock that the human species has ever received'. the summaries below of seven presentations on this topic highlight the diversity of influenza viruses in north america (cf. nelson et al., ) , while other relevant research has been published with respect to swine influenza viruses (sivs) in europe (kyriakis et al., ) . considerable research has now been carried out into how the highly pathogenic h n avian influenza virus spreads from wild birds and ducks to chickens and other species, including humans (rabinowitz et al., ; ma et al., ) . the studies of how influenza viruses can be genetically altered to become more transmissible have become a matter of much controversy palese and wang, ) . in addition, to the summaries below, this special supplement includes an article, lessons from emergence of a/goose/guangdong/ -like h n highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern china, in which dr. x.-f. wan has considered the emergence and ecology of influenza a viruses in southern china, especially the highly pathogenic h n virus. backed by an extensive team of collaborators, professor a. d. m. e. osterhaus (head, department of virology, erasmus medical centre, rotterdam, the netherlands) began his presentation, emerging and neglected influenza viruses, by explaining the complex aetiology of the influenza a, b and c viruses. while humans can serve as host species for all three viruses, influenza a can also be present in other mammals and avian species, influenza b in seals and influenza c in pigs. the severity of the disease is relatively high with influenza a, moderate with influenza b and low with influenza c, with the prevalence in humans high with both influenza a and b viruses, but lower with influenza c. furthermore, a clear distinction needs to be made between seasonal influenza, avian influenza and pandemic influenza. there are two different mechanisms of host adaptation -sequential mutations and genome reassortment. most recently, the new h n swine flu pandemic outbreak of drew attention to the speed with which an influenza virus could move around the world. however, the fact that this particular virus was not as virulent as first anticipated proved crucial in confronting the virus, even though it spreads rapidly among humans, unlike the much more virulent h n avian flu virus, from which more than people have died from more than verified cases from to (world health organization (who), ). although clinical evidence of h n avian influenza appears predominantly in diving ducks, a number of dabbling duck species -mallard, teal, wigeon and gadwall -appear to spread h n , generally acquired from wild birds, without showing major signs of disease. the likelihood of a major pandemic linked to h n has not decreased in the last years, even though publicity has certainly decreased. furthermore, professor osterhaus pointed out that the recent h n pandemic influenza outbreak indicated that the scientific community was wrong in its earlier belief that 'a pandemic strain could only arise from a subtype that had not previously been widely disseminated in humans [because] the h n virus has shown that human varieties characterized by different hemagglutinin (ha) molecules may follow separate lines of evolution and may generate potentially pandemic strains within an existing human ha subtype. hence, it is essential to develop methods for estimating how many antigenically different subtypes may reside within each ha type' (cf. rappuoli et al., ) . in the light of the continuing prevalence of many subtypes of influenza, there is a critical need for improved monitoring, especially in asia and africa, as part of a move from a reactive to a proactive approach, with greater research into the possibility of developing a universal vaccine. although there are increasing opportunities for virus infections to emerge and spread rapidly in our global society, new tools are being provided by research in molecular biology, epidemiology, genomics and bioinformatics. already early warning systems based on state of the art virus detection techniques, as well as targeted intervention strategies based on data about the mutual virus-host interaction have been instrumental in dealing with numerous viral threats, including sars and avian influenza. the extensive research of the department of virology at erasmus medical centre in rotterdam was highlighted by a further presentation, influenza pneumonia: the role of the alveolar macrophage, given by dr. d. van riel. highly pathogenic avian influenza (hpai) h n virus causes severe, often fatal, pneumonia in humans. the pathogenesis of hpai h n virus is not completely understood, although the alveolar macrophage (am) is thought to play an important role. the am resides in the pulmonary alveolus, the primary site of hpai h n virus replication in humans. it had been shown previously that hpai h n virus attaches abundantly to these am (van riel et al., ) . the aim of this study was to determine the response of primary human am to hpai h n virus, seasonal h n virus or pandemic h n virus, and to compare these responses with that of macrophages cultured from monocytes. hpaiv h n infection of am compared with that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to % versus up to %), lower virus production and lower tnf-alpha induction. infection of am with h n or h n virus resulted in even lower percentages of infected cells (up to %) than with hpai h n virus, while virus production and tnf-alpha induction were comparable. in conclusion, this study revealed that macrophages cultured from monocytes are not a good model to study the interaction between am and influenza viruses. furthermore, the interaction between hpai h n virus and am could contribute to the pathogenicity of this virus in humans, because of the relatively high percentage of infected cells rather than virus production or an excessive tnf-alpha induction (van riel et al., ). one virus of each pair was wild type, while the other carried the h y na mutation conferring resistance to na inhibitor oseltamivir. within each pair, the wild-type and oseltamivir-resistant virus caused disease of equal severity in ferrets and replicated to comparable virus titers in the upper respiratory tract. then, to assess the fitness of drug-resistant h n influenza viruses, the research team considered virus-virus interactions within the host by co-inoculating ferrets with mixtures of the oseltamivirsensitive and oseltamivir-resistant h n viruses in varying ratios (e.g. / ; / ; / ; / ; / ). using this novel approach, they demonstrated that the proportion of a/vietnam/ / -h y clones tended to increase, while the proportion of a/turkey/ / -h y clones tended to decrease. their findings suggest that the h y na mutation can affect the fitness of two h n viruses differently and is dependent on background na sequence. dr. govorkova pointed out that antigenic and genetic diversity, virulence, the degree of na functional loss of h n virus and differences in host immune response can also contribute to such differences. therefore, the risk of emergence of drug-resistant influenza viruses with uncompromised fitness should be monitored closely and considered carefully in pandemic planning. in a collaboration with c. corzo, k. juleen and m. gramer they initiated an active surveillance program in healthy pigs in multiple sites in , during a period coincident with the emergence of the h n pandemic in humans. their study, active surveillance for influenza viruses in north america, presented an analysis from months of data which indicated that similar viruses can be detected in both active and passive surveillance schemes and that there has been an explosion of diversity in swine influenza viruses (siv) in the united states. not only were a number of pandemic h n infections in swine detected, but a number of pandemic/endemic swine virus reassortants were found, albeit from healthy animals (ducatez et al., ) . virologically, the pattern of disease surveillance grounded in the activities of state diagnostic laboratories collecting information from diseased animals is representative; however, epidemiologically this data from diseased animals is not representative. reverse zoonoses have had a huge impact on siv in the united states (vincent et al., ) , and the pandemic virus is now endemic. however, in considering whether any particular reassortment causes alarm, it must be acknowledged that there is not yet a good model of risk, so h , like h , is going to be found in pigs for some time to come, but the consequences of this diversity in siv are not yet clear. the extensive collaboration now taking place in the study of swine influenza was evident in the presentation vessel pendulum began by explaining the three elements of how swine could be considered as a mixing vessel for influenza a viruses as formally proposed by scholtissek et al. ( ) : (i) swine are susceptible to infection with influenza a viruses from avian and human viruses; (ii) the avian viruses can adapt within the pig, producing novel reassortants; and (iii) these reassortants can then be shed and are infectious to man. the goal of this presentation was to test the first part of the mixing vessel hypothesis, concerned with the susceptibility of swine to avian and human influenza viruses, making use of both mixing vessel studies in pigs and genetic markers to investigate adaptation. dr. lager noted that the emergence of the h n highly pathogenic avian influenza virus that can transmit from avian species directly to man, and the presumption that the h n influenza jumped from birds to man has expanded our understanding of the swine mixing vessel hypothesis as a potential, but not exclusive, source of human pandemic viruses (taubenberger et al., ) . moreover, the emergence of the pandemic h n virus has re-emphasised swine as a potential source of pandemic virus. in this study, all of the challenge viruses (avian h , h , h ) induced a similar effect in pigs; challenge viruses did replicate in pigs; the infections were subclinical with mild pneumonias; most infections resulted in seroconversion; and none of them transmitted to contact controls. this series of studies suggests pigs could be easily infected with avian viruses; however, an adaptation step is needed to generate fit viruses that transmit among swine. parallel studies are currently underway testing the susceptibility of pigs to human seasonal influenza viruses. future studies using reverse genetics could investigate potential genetic markers for adaptation of avian viruses to swine which may provide insight into the interspecies transmission of influenza viruses. a in this study, an attempt was made to recreate the pandemic virus by co-infecting cells (in vitro) or a group of pigs (in vivo) with eurasian (sp ) and north american triple reassortant (ks ) sivs (ma et al., a) . infected pigs were co-housed with two groups of sentinel animals to investigate virus maintenance and transmission. the origin of each gene segment of viruses was determined, which were isolated from supernatants collected from co-infected cells or nasal swabs and bronchioalveolar fluid samples collected from infected and sentinel animals. different reassortant viruses were identified from co-infected cell lines; however, no virus with the genotype of ph n was found. less reassortant viruses were found in the lungs of co-infected pigs in contrast to those in co-infected cells. interestingly, only the intact ks was detected from nasal swabs from the second group of sentinel pigs. these results demonstrated that multiple reassortant events can occur within the lower respiratory tract of the pig; however, only a specific gene constellation is able to be shed from the upper respiratory tract. however, in this study, it was not possible to generate the ph n constellation using co-infection with the techniques described above and previously (ma et al., b) . in . she began by reflecting on the ability of swine to act as a reservoir for many influenza viruses, becoming infected with low mortality, regardless of influenza virus strain. the objective of the study was to further understand the porcine response to influenza and to compare this response to other animals infected with the same virus. to accomplish this objective, they used statistical and functional analysis of global gene expression to compare host transcriptional response during acute infection by a contemporary h n pandemic influenza virus (a/california/ / ) in swine, non-human primates and mice. using their data, they compared and contrasted the biological pathways most significantly associated with gene expression changes during acute infection across these species. their goal was to leverage data collected in their previous studies (ma et al., ; safronetz et al., ) to better understand influenza virus pathogenesis through a cross-species analysis that considered three crucial questions: (i) which genes change over the course of acute infection? (ii) what are the top functions altered during infection? (iii) how does functional response compare across the three species? despite challenges to data integration and interpretation, including the differences in transcript representation and annotation on the microarrays for the different species, the researchers found notable differences in response to influenza in the lungs of the three species. although similar functional groups of genes changed with infection in all three species, the nature of that response was species specific. swine exhibited an elevated transcriptional response that tapered by resolution of influenza. mice exhibited a decrease in many acute phase and immune response genes quickly followed by a steady increase in expression. host response in macaques was most pronounced and maintained over time. in considering the transcription of immune-related genes in swine, mice and nonhuman primates, they found that although the number of immune-related genes changing in each species was similar, the precise genes changing were very different, with only immune response genes commonly differentially expressed across all three species. this suggested that the nature of immune response within each species may be quite different. in response to the perennial question after any scientific experiment, ''where do we go from here?'' they offered four ideas: (i) time series analysis could reveal unique response kinetics across species, thereby leading to targeted analysis; (ii) data integration across multiple data types, including transciptomics, proteomics, mirna and ngs could generate a more complex, multidimensional view of response; (iii) as annotation of the different species-specific genomes improves, this information could be integrated into future analyses, making a better understanding of the biological responses to infection possible; and (iv) the gathering of this additional information could empower more precise analysis on what makes each species uniquely susceptible or resistant to influenza. in the firm view of these particular six researchers, studies such as this are necessary for a deeper understanding of influenza pathogenesis and demonstrate the utility of systems biology in the study of emerging viruses. three relevant articles on this topic have been published below, highlighting the global dimensions of both infection and treatment, no matter where the virus first emerges. the need for geographical comparative studies of the emerging hantavirus, puumala hantavirus (puuv), has already been indicated by professor henttonen and his team in their presentation summarized earlier in the opening topic of this meeting review. in a further investigation into the same hantavirus, dr. eckerle and her colleagues have presented an article within this special supplement entitled atypical severe puumala hantavirus infection and virus sequence analysis of the patient and regional reservoir host. in this article, they focus on the difficulties in the diagnosis of and treatment for a single patient and performed virus sequence analysis showing regional clustering in reservoir and host. in their more wide-ranging conference presentation, they investigated cytokine expression in a cohort of patients hospitalized with acute severe hantavirus infection during an epidemic in germany in (cf. faber et al., ) . elevated proinflammatory cytokines during the early phase of disease compared to healthy controls and increase in immunosuppressive tgf-b from early to later phase of disease supported the hypothesis of an immune-mediated pathogenesis of puumala hantavirus (sadeghi et al., ) . this finding indicates that the immune status of the host for old-world hantaviruses plays an important role, not only the virus itself. in a further article published in this special supplement, how ebola virus counters the interferon system, a. kühl and s. pöhlmann have reviewed which components of the innate immune system could be effective against the zoonotic transmission of ebola virus (ebov) to humans, which results in severe haemorrhagic fever and high case-fatality rates. their focus is on how the interferon (ifn) system, as a key innate defense against viral infections, is targeted by distinct ebov proteins, and on how specific effector molecules of the ifn system could form a potent barrier against the spread of ebov in humans. finally, in lassa fever in west africa: evidence for an expanded region of endemicity, dr. n. sogoba and his colleagues h. feldmann and d. safronetz have stressed the importance of increased surveillance for lassa virus across west africa. the seven presentations summarized below cover a number of haemorrhagic fever viruses. for example, an important example of a highly contagious and life-threatening haemorrhagic fever virus is crimean-congo haemorrhagic fever virus (cchfv), caused by a tick-borne virus of the bunyaviridae family (elliott, ) , first recognized in the crimea in , with an identical virus isolated in the congo in ; the incidence and geographical spread of this disease with its high human fatality rate have increased significantly in the past years. however, the causes of this increase are not yet clear (maltezou and papa, ) . in the light of the need to develop new therapies and effective, safe vaccines, the next seven research presentations could prove to be of considerable significance, not only for cchfv, but also for the hendra, nipah, lujo and ebola viruses. although these viruses have certain common features in their causes and consequences, each haemorrhagic fever virus needs to be carefully studied as a distinct entity. dr (peyrefitte et al., ) . moreover, it has already been shown that cchfv causes liver damage in infected patients and in the animal model (bereczky et al., ) . the research objectives were to consider: (i) how does cchfv affect hepatocarcinoma cell lines? (ii) is cchfv able to enter and replicate into these cell lines? (iii) does cchfv modulate the in vitro cellular response? to better understand the cchfv pathogenesis in liver cells, they analysed in vitro the host response induced after cchfv infection in huh (unable to produce ifn-beta) and hep-g (capable of producing ifn-beta) cell lines. they noticed that while in huh , cchfv infection elicited at day a cytopathogenic effect, no visible effect was seen in cchfv-infected hepg . this intriguing feature led them to analyse the viral parameters expecting a differential cellular response. both cell lines were shown to be permissive to cchfv and with a high viral yield as monitored by plaque titration assay, genomic and antigenomic strand quantification. these cchfv-infected hepatocarcinoma cell lines induced only il- secretion. in addition, a pro-apoptotic effect was observed in huh but not in hepg . interestingly, no type-i ifn was detected for hep-g during the kinetic study, suggesting a strong inhibition of ifn secretion. they concluded that cchfv does enter and replicate in hepatocytes and that hepatocytes could be involved in cchf pathogenesis associated with antigen presenting cells for cchfv dissemination. while cchfv did not induce ifn-beta secretion in hepatocyte cell lines, cchfv did induce the secretion of il- in hepatocyte cell lines. furthermore, cchfv induced a higher secretion of il- in the apoptotic huh cell line than in the nonapoptotic hep-g cell line. thus, this research indicated that il- production and apoptosis seemed to be markers of cchfv pathogenesis in hepatocyte cell lines. professor t. w. geisbert (university of texas, medical branch, galveston, tx, usa) presented an evaluation of countermeasures against hendra and nipah viruses in nonhuman primate models. he pointed out that the henipaviruses, hendra virus (hev) and nipah virus (niv) are enigmatic emerging pathogens that can cause severe and often fatal neurologic and/or respiratory disease in both animals and humans. guinea pigs, hamsters, ferrets and cats have been evaluated as animal models of human hev infection. a research team led by professor geisbert recently evaluated african green monkeys as a nonhuman primate model for henipavirus infection and discovered that they are the first consistent and highly susceptible nonhuman primate models of hev and niv infection rockx et al., ) . the severe respiratory pathology, neurological disease and generalized vasculitis manifested in both hev-and nivinfected african green monkeys provides an accurate reflection of what is observed in henipavirus-infected humans. these nonhuman primate models were then employed to evaluate several post-exposure treatments including ribavirin (which did not work) and a human anti-henipavirus monoclonal antibody (which was successful). dr the research was motivated by the awareness that neutralizing antibodies are probably the major effectors against this viral infection. the rationale of using rv vectors for the development of a niv vaccine was fourfold: (i) rv-vectored vaccines are not pathogenic regardless of the route of administration or the immune status of the host; (ii) rv-based vaccines are very efficacious even after a single immunization by the oral route; (iii) rv-based vaccines have the ability to target macrophages and dendritic cells, to induce th t-cell response and are capable of inducing long-lasting immunity; and (iv) postexposure prophylaxis using recombinant rv vaccines is very effective, even when the cns is already infected (faber et al., a,b) . the niv g gene was inserted into the non-pathogenic rv vectors spbaangas or spbaangas-gas, resulting in spbaangas-ng or the double gas variant spbaan-gas-ng-gas, respectively. further research led to four significant conclusions: (i) there are no detectable amounts of niv g present in recombinant nivg-rv particles; (ii) the presence of an niv g gene does not increase, but rather decreases the pathogenicity of the recombinant viruses; (iii) priming with nivg-rv triggers a strong niv g-specific memory response, which correlates inversely with vaccine concentration used for the priming; and (iv) a single immunization with nivg-rv is probably sufficient to protect against a niv challenge infection. arenaviruses are rodent-borne bisegmented ambisense rna viruses, which include lassa fever virus, lymphocytic choriomeningitis (lcm) and tacaribe the index case for this acute febrile illness virus was a travel agent living on a farm during in lusaka, zambia, who infected a local cleaner, as well as a paramedic and a nurse in johannesburg, south africa, all of whom died, with the paramedic infecting a further nurse who was treated with ribavirin and survived . the name of the virus originated from the first two letters of the two key cities, lusaka and johannesburg. four of the five infected persons died of haemorrhagic fever-like symptoms paweska et al., ). viral genome sequencing revealed that this virus differed from other arenaviruses by at least % and is highly pathogenic, with a case fatality rate (cfr) of % paweska et al., ) . in view of the uniqueness and high virulence of lujo virus (ljv), the research team developed a reverse genetics system to study the molecular characteristics of this novel arenavirus. this system will facilitate studies of ljv biology, development of antiviral screening assays and pathogenesis studies in animal models. t. cutts (national microbiology laboratory, public health agency of canada, canadian science centre for human and animal health, winnipeg, manitoba, canada) with his colleagues s. theriault (chief, applied biosafety research program, same centre) and g. kobinger (chief, special pathogens program, same centre) presented cytofixÔ inactivation of veroe cells infected with zaire ebola virus (zebov) both in vitro and in vivo. first, it was pointed out that removing infected tissues from high-containment laboratories requires implementation of a number of different decontamination techniques to render the organism inert and is subject to flexibility according to the laws of the country in which the laboratory is located. according to the canadian biosafety guidelines th edition, an organism may be removed from containment once it has been rendered inert, but no procedure is in place to validate these biosafety guidelines, and it is up to the individuals to implement the relevant guidelines (public health agency of canada, , p. . chap. . . ). methods such as gamma irradiation, formalin fixation, acetone and methanol permeation, plus the use of various other chemical agents, are common practices to preserve cellular tissue or blood components and to inactivate organisms (elliott et al., ; mitchell and mccormick, ; preuss et al., ; villinger et al., ; sanchez et al., ) . such methods still raise questions as to their effectiveness or their redundancy. furthermore, these inactivation steps can lead to the alteration of the target organism possibly affecting the qualitative and quantitative results. the focus of the applied biosafety research program was to evaluate and develop technologies and procedures relevant to biocontainment in the context of the laboratory, as well as to prevent unintentional and intentional release of dangerous organisms into the environment. using the commercial product, cytofix/cytoperm tm from bd biosciences, this research sought to inactivate vero e cells which had been infected with the deadly zaire ebola virus (zebov). the aim of the research was to determine the effectiveness and duration of cytofix/ cytoperm for fixing the cellular material infected with zebov. the veroe cells were infected with the wildtype zebov and a mouse adapted zebov(mazebov) and assayed after a -min and -min exposure to cyto-fixÔ followed by neutralization. samples of blood from a non-human primate infected with zebov were drawn at dpi and assayed for effectiveness in the same manner as the in vitro studies with cytofixÔ. in addition, vero e cells infected with mazebov were treated in the same manner and injected into balb/c mice to compose the in vivo studies. cytoxicity and neutralization assays were used to determine the effect (if any) the treatment had on both the virus and the health of host cells. results of the tissue culture tcid assay showed that a -min exposure to cytofixÔ inactivated a large portion of the cells containing infectious virions, while after a -min exposure, no detectable levels of virus were observed. blood samples from the non-human primates showed similar results to the cell culture assay having no detectable virus from infected cells after min of exposure. in vivo studies with mice showed that both a min and -min exposure time to cytofixÔ had a % survival rate after days post-infection, while the positive controls succumbed after to dpi. because laboratories differ in their preferences of technique, the time of inactivation also varies. what this research demonstrated was the effectiveness of a quick procedure of min for inactivating viruses within cells infected with zebov, thereby rendering organisms safe to remove from containment. has not yet been linked with disease in humans, the presence of antibodies against rebov in people working closely with infected macaques and swine indicates that humans can be infected with this virus (miller et al., ; miranda et al., ; barrette et al., ). however, research has been hampered by the fact that the only available disease model for rebov to date has been cynomolgus macaques. seeking new rebov disease models, the research team assessed various rodent models -the balb/c mouse, hartley guinea pig, syrian hamster and stat )/) mouse that lacked the signal transducer and activator of transcription (durbin et al., ) . although virus replication occurred in guinea pigs and hamsters, progression to disease was only observed upon inoculation of stat )/) mice. despite certain drawbacks set out in the journal article, the stat )/) mouse can be used to investigate the determinants of differences in pathogenicity in various rebov strains, as well as to assess vaccination and antiviral therapies (miller et al., ; miranda et al., ; durbin et al., ; barrette et al., ; de wit et al., ) . the unity of human, animal and ecosystem health outlined by professor aguirre, as well as the interactions among multiple tick-borne pathogens in a natural reservoir host set out by professor fish and his research team, both summarized in topic above, highlight the necessity of cross-disciplinary collaboration in studying zoonotic bacterial diseases (daszak et al., , pp. - ) . such collaboration is especially important in studying tick-borne infectious disease, which emerged so extensively in the united states during the last three decades of the twentieth century (paddock and yabsley, , p. ) . now, in an article published in this special supplement, beyond lyme: etiology of tick-borne human disease with emphasis on the southeastern united states, drs. stomdahl and hickling have explained that tick distributions are in flux, especially in the south-eastern united states, requiring health providers to think 'beyond lyme' to identify the specific tick species that bite humans and the different pathogens these ticks carry. in an international context, drs. wood and artsob have set out the increasing importance of travel-associated rickettsioses in their article, spotted fever group rickettsiae: a brief review and a canadian perspective. in a third article published in this special supplement, drs. verma and stevenson present an article on epidemiology of leptospirosis with its one million cases worldwide. in leptospiral uveitis -there's more to it than meets the eye! they hypothesize in detail about how the eye inflammation uveitis is triggered and stress the impact that 'understanding how this bacterium is able to induce this inflammatory process will be a key to the better management and prevention of the disease'. this continuum of basic research leading to understanding a disease and then to managing that disease and finally to preventing it offers a pattern of scientific discovery that is relevant to many other emerging zoonotic diseases. opening his presentation, the foodborne pathogen campylobacter jejuni exploits mammalian host cell receptors and signaling pathways, professor konkel noted that the per cent of c. jejuni isolates that are resistant to antibiotics is continuing to increase and that c. jejuni infections are frequently associated with serious sequelae, including guillain-barré syndrome. it is well understood that infection with c. jejuni is often a consequence of eating foods contaminated with undercooked poultry. however, c. jejuni pathogenesis is a highly complex process that is dependent on many factors including motility, adherence, cell invasion, protein secretion, intracellular survival and toxin production. acute illness, characterized by the presence of blood and leucocytes in stool samples, is specifically associated with c. jejuni invasion of intestinal epithelial cells. dissecting bacteria-host cell interactions are critical to understanding the infection caused by c. jejuni. previous work has shown that maximal invasion of host cells by c. jejuni is dependent on synthesis of the c. jejuni cadf and flpa fibronectin (fn) binding proteins and requires the secreted campylobacter invasion antigens [cia(s)] (larson et al., ) . to test the hypothesis that maximal cell invasion requires specific signalling events, binding and internalization assays were performed in the presence of numerous inhibitors of cell signaling pathways. the research team found that c. jejuni cell invasion utilizes components of focal complexes (fcs), as invasion is significantly inhibited by wortmannin (an inhibitor of pi- kinase) and pp (a c-src inhibitor). they further demonstrated that a wild-type strain of c. jejuni results in the activation of the rho gtpase rac . these observations are consistent with the proposal that c. jejuni binding to host cell-associated fn and secretion of the cia proteins trigger integrin receptor activation, which in turn promotes intracellular signalling and actin cytoskeletal rearrangement. on the basis of these data, they concluded that c. jejuni utilizes a novel mechanism to promote host cell invasion. the research findings professor konkel presented were recently published in cellular microbiology (eucker and konkel, ) . simple, fast and specific tests for pathogen identification are essential for epidemiological investigation of numerous diseases. within the field of immunodiagnostics, a quantitative determination of either antibody or antigen by antigen-antibody interaction can be made by lateral flow tests (also known as a dipstick or rapid tests). dr. e. baranova and her colleagues p. solov'ev, n. kolosova and s. biketov (all state research center for applied microbiology, obolensk, russia) began the presentation, development of lateral flow tests for the fast identification of zoonotic disease agents, by pointing out that lateral flow (lf) tests can be used in the field, as a diagnostic tool that produces results that can be read visually by the naked eye within min after sample application. the creation of an algorithm for the development of an appropriate lf test to identify biopathogens requires the development of a target antigen, obtaining specific antibodies (biketov et al., ) and then creating a lf-test formulation to be trial tested. the target antigens must have the ability to induce species-specific antibodies, as well as be characterized by surface localization with multiple epitope presentation on the surface. the antibodies need to have a specificity and sensitivity sufficient for application in the lf detection format, as well as the capacity to be preserved after labelling with gold particles and after immobilization on a surface. over a period of months, the research team developed and tested in the field lf tests for the detection of bacillus anthracis, which causes anthrax, yersinia pestis, which causes bubonic plague, and francisella tularensis, which is the causative agent of tularaemia (or rabbit fever). all three of these lf tests have now been made available as commercial products and are being used throughout russia for the rapid identification of these dangerous pathogens. drs. j. d. trujillo and p. l. nara (center for advanced host defences, immunobiotics and translational comparative medicine, iowa state university, ames, iowa, usa) have developed and validated a new approach to the diagnosis of infectious agents. dr. trujillo explained that they are employing novel polymerase chain reaction (pcr)-based methods for the detection and differentiation of current and emergent mycoplasma species relevant to human and animal medicine and biodefense. their presentation, titled novel sybr Ò real-time pcr assay for detection and differentiation of mycoplasma species in biological samples from various hosts, began by explaining the relevance of mycoplasma species, which are endemic, strict or opportunistic pathogens in human and animal medicine. moreover, mycoplasma species are important re-emerging pathogens and foreign animal diseases. importantly, mycoplasma species are difficult to culture or are un-culturable, and thus are difficult to impossible to detect by conventional diagnostic methods. moreover, current pcr methods have limited breath of species detection and differentiation, requiring the use of species-specific assays that are costly and time-consuming. their goal was to develop a pilot mycoplasma genus diagnostic assay to validate the novel application of high-resolution melt (hrm) methodology for rapid, sensitive and cost-effective detection and differentiation of various pathogenic mycoplasma species. dr. trujillo presented the validation and utilization of sybr Ò green dye in real-time pcr (qpcr) mycoplasma detection and differentiation assay (panmyco qpcr). this pcr assay utilizes primers specific for this genus (modified from s. c. baird et al., ) . this pcr assay results in the generation of small dna fragments of various base pair lengths called pcr amplicons. each amplicon has a melt temperature (tm) that is determined following qpcr. sequence of amplicon representative of the mycoplasma species present defines the melt temperature (tm) and allows for the use of amplicons tm in species identification with limited resolution and excellent sensitivity. the panmyco qpcr assay has similar sensitivity to a conventional nested pcr assay for mycoplasma bovis with a linear detection range of one colony forming unit (trujillo et al., ). additional work presented described increasing species resolution of this assay, by defining unique melt profiles for each mycoplasma species amplicon utilizing precision melt software from biorad, ca, usa to perform hrm analysis. greater than different species of mycoplasma found in bovine, caprine, ovine, avian and porcine hosts have been characterized with the panmyco qpcr and hrm analysis. occasionally, this testing has resulted in the detection of multiple species in a single sample or discovery of novel or emergent mycoplasma species. this data analysis method allows for the sensitive detection and rapid differentiation of numerous mycoplasma species in many different hosts. dr. trujillo concluded that this novel real-time pcr assay can detect and potentially differentiate all known mycoplasma species. moreover, this presentation demonstrated the novel use of genus-specific sybr green pcr and hrm analysis for the detection, differentiation and discovery of medically important pathogens. several additional translational research projects have been launched to demonstrate the importance and utility of the pan myco qpcr assay in the context of infectious disease surveillance. one translational research project focuses on validation of this novel molecular methodology for field detection assays. there is increasing awareness of the need for improved laboratory investigation, risk assessment, contingency planning and simulation exercises to respond effectively to zoonotic diseases (lipkin, ; westergaard, a and b; escorcia et al., ) . in view of the need to research into and respond to so many emerging zoonoses, it is relevant to note the fourfold classification of emerging zoonoses proposed earlier by silvio pitlik: type : from wild animals to humans (hanta); type +: from wild animals to humans, with further human-to-human transmission (aids); type : from wild animals to domestic animals to humans (avian flu); and type +: from wild animals to domestic animals to humans, with further human-to-human transmission (sars) (kahn et al., : p. ) . confronting outbreaks of these emerging zoonoses is often possible with an imaginative combination of laboratory investigation and extensive fieldwork (borchert et al., ; robinson, ) . three distinctive articles appear below on outbreak responses to zoonotic diseases, highlighting the importance of linking together basic research, practical action and an integrated one health-oriented approach. in a. grolla and nine co-authors from eight different institutions in five different countries have explained how two mobile laboratories were set up and capable of running within < h of arrival, providing safe, accurate, rapid and reliable diagnostic services as the ebola zaire outbreak began in the democratic republic of the congo. finally, in emerging and exotic zoonotic disease preparedness and response in the united states: coordination of the animal health component, dr. r. l. levings has set out the integrated approach of emergency management and diagnostics, veterinary services, animal and plant health inspection service, united states department of agriculture in the prevention of, the preparedness for, the response to and the recovery from a zoonotic disease outbreak. in all three of these areas -basic research, practical action and an integrated one health-oriented approach -much has been achieved in recent years, but much also remains to be achieved as soon as possible. even when those diseases are not transmitted to humans, there are substantive challenges, as highlighted in the next case study by woods on combating brucellosis in cattle in zimbabwe. in a practical, problem-oriented presentation, dr. p. s. a. woods (veterinary public health section, faculty of veterinary science, university of pretoria, onderstepoort, south africa and university of reading) with r. s. beardsley (pharmaceutical health services research, school of pharmacy, university of maryland, baltimore, maryland, usa) and n. m. taylor (veterinary epidemiology and economics unit, school of agriculture, policy and development, university of reading, reading, united kingdom) asked can we increase farmers' perception of their brucellosis susceptibility to improve adoption of preventive behaviors amongst small-scale dairy farmers in zimbabwe? she explained the background to the problem, presented a model that was used to develop a strategy to confront the disease and then set out the results and recommendations of the research team. brucellosis is an extremely infectious bacterium that causes abortion in cows, different syndromes in other animal species and malaria-like undulant fever, arthritis, depression and epididymitis in people. however, it had been controlled in zimbabwe until when financial constraints forced the government veterinary services to curtail disease surveillance and discontinue free vaccinations. small-scale farmers did not seek vaccination from other sources, partly because they were unaware of the necessity of vaccination, and also at that time brucellosis was absent from small-scale farming areas. however, uncontrolled cattle movements from to linked to invasions of large-scale farms resulted in dispersal of possibly brucella-positive cattle and movement of the disease into small-scale herds. the result was that brucellosis became a potential problem in these herds and now presents a serious zoonotic threat. preventing brucellosis requires movement control to stop brucella-positive cattle entering an area, as well as live vaccine for female calves. although there is no human-to-human spread of the disease, it is essential that people do not handle new-born calves or abortions from brucella-positive cows, nor drink unpasteurized milk from brucella-positive cows (arimi et al., ) . in essence, reducing the risk of brucellosis requires that farmers adopt appropriate preventive behaviours, with these control efforts and changes in behaviour being communitydirected in order to be sustainable. it was this stress upon community direction that formed the basis for funding by the wellcome trust to investigate the hypothesis that the level of a farmer's knowledge about brucellosis would influence subsequent preventive behaviour. the approach, based partly on the 'health belief model' (rosenstock et al., ) was grounded in the expectation that each small-scale farmer would make health behaviour choices according to individual perceptions about the disease and personal beliefs about their abilities and the costs required to change the risks of their cattle and families acquiring the disease. in this project, the independent variable was the level of an individual farmer's knowledge about brucellosis, while the dependent variables were two key preventive behaviours -decreasing cattle disease by calfhood vaccination and preventing zoonotic disease by milk pasteurization. the research was carried out in partnership with a national network of small-scale dairy cooperatives with all activities conducted with existing local personnel. the aim was to tailor the educational program to the initial knowledge or awareness of each community of farmers, recognizing the considerable difference in knowledge levels between-and within communities. local teams, not outsiders, developed appropriate educational materials, targeting those with the lowest levels of knowledge. completed survey questionnaires indicated a significant relationship between the initial level of farmers' knowledge about brucellosis and their calf brucellosis vaccination practices. the range of brucellosis knowledge among some small-scale farmers in southern zimbabwe was considerable, with % of farmers being unaware of the disease, % having limited knowledge and % having good knowledge. however, even amongst those farmers with a relatively high level of knowledge, % of farmers had not vaccinated their calves at the time of the survey. furthermore, there was a disappointingly low uptake of milk boiling despite a significant increase in knowledge about raw milk as a mode of infection for humans. although the information sessions did increase farmers' awareness of the dangers of zoonotic brucellosis, an exaggerated perception of the effectiveness of calf vaccination decreased the likelihood of safe milk practices. this outcome indicated the importance of reaching the women who were responsible for milk and food preparation. ongoing research is investigating whether increasing the role of nurses and environmental health technicians to emphasize human infection and to reach different family members, within a research paradigm which combined veterinary and human medicine, would increase the uptake of milk hygiene practices. there is increasing awareness of the need to balance transparency with carefully designed information disclosure strategies in the face of sudden outbreaks of foodborne diseases (national research council, ; taylor, ) . both consumers and producers must be rapidly informed of any significant dangers with specific food products; however, considerable misinformation can be spread if laboratory results are incomplete or inconclusive (palm et al., ) . recent experience with e. coli-infected sprouts in germany and listeria-infected cantaloupes the united states has highlighted the difficulties in identifying the original source of a disease outbreak, as well as the swiftness with which an unexpected food-borne disease can cause sickness and death (armour, ; blaser, ; buchholz et al., ; frank et al., ) . it should be noted that that there was no easily identified zoonotic link in either of these two food-borne diseases derived from bacteria, which killed people in the united states and throughout europe during ; however, as professor c. kastner points out later, a significant number of these food-borne diseases do have a zoonotic origin (parker et al., ) . two articles linked to this topic are published in this special supplement. first, there is emerging antimicrobial resistance in commensal e. coli with public health relevance by dr. a. käsbohrer and her colleagues. their aim was to assess the prevalence of and trends in antimicrobial resistance through active monitoring programs along the food production chains for poultry, pigs and cattle, as well as to collect isolates for resistance testing and then select certain isolates for further phenotypic and genotypic characterization. the research team found alarming rates of resistance to antimicrobials in zoonotic bacteria and commensals, as set out in their article, which could compromise the effective treatment for human infections. this work provides a basis on which to improve both risk assessment and risk mitigation strategies in the face of the increasing antimicrobial resistance to zoonotic bacteria and parasitic organisms within both humans and animals. second, in american trypanosomiasis infection in fattening pigs from the south-east of mexico, m. jiménz-coello and her colleagues have investigated the extent to which the protozoa trypanosoma cruzi (t. cruzi) is presenting in fattening pigs in yucatan, mexico, threatening parasitic infections in animals destined for human consumption. tackling the question of how to refine national and international strategies to combat food-borne zoonotic diseases, professor c. kastner (food science institute, kansas state university, manhattan, kansas, usa) considered the public health and economic impact of foodborne zoonotic diseases. he began by noting that each year in the united states, according to statistics from the centers for disease control, million people become sick from food-borne diseases, , are hospitalized and , die. a significant portion of these diseases have a zoonotic origin, with extensive product recalls and domestic as well as international trade disruptions (fung et al., ) . therefore, more than years ago, the us department of agriculture established a food safety consortium ( ) which focuses on food-borne zoonotic diseases involving beef in kansas, pork in iowa and poultry in arkansas. the continuing aim of that consortium is to develop long-term control strategies that identify the critical control points and control technologies, as well as short-term strategies to address incidental contamination, whether accidental or intentional. the us livestock industry in general and kansas in particular are vulnerable to food-borne zoonotic diseases. for example, in kansas, sources of contamination include feed, feedlots (which vary in size from , to , head per lot) and packing plants (which vary in size from , to more than , head per day per plant). beef processing points where mixing of different ingredients occurs are the most critical points for both incidental and intentional contamination. in the light of these challenges, a biosafety level research facility, the biosecurity research institute (bri) ( ) has been built on the kansas state university campus, to evaluate strategies to detect and control food-borne zoonotic diseases from production through processing. furthermore, in minneapolis, minnesota, ncfpd (national center for food production defense, ) has been operational since as a department of homeland security center of excellence. ncfpd has adopted a systems approach whose goals include to: (i) ensure significant improvements in supply chain security, preparedness and resiliency; (ii) develop rapid and accurate methods to detect incidents of contamination and to identify the specific agent(s) involved; (iii) apply strategies to reduce the risk of food-borne illness because of intentional contamination in the food supply chain and to develop the tools to facilitate recovery from contamination incidents; (iv) deliver appropriate and credible risk communication messages to the public; and (v) develop and deliver highquality education and training programs to develop a cadre of professionals equipped to deal with future threats to the food system. these research centers are essential to minimize the threat of food-borne zoonotic diseases. t. cutts (national microbiology laboratory, public health agency of canada, canadian science centre for human and animal health, winnipeg, manitoba, canada) presented comparative inactivation studies of listeria monocytogenes at room and refrigeration temperatures on behalf of a research team that included b. carruthers, c.-l. cross, s. theriault (chief, applied biosafety research program, same center) and himself. listeria monocytogenes, a non-sporulating, gram-positive bacillus, is found chiefly in ruminants, but can affect all species and causes listeriosis, an infrequent but serious illness that affects the central nervous system of humans and domestic animals (bortolussi, ; chan and weidmann, ). listeriosis can be acquired from the consumption of contaminated foods and has an incubation period ranging from to days (bortolussi, ; chan and weidmann, ). because of this variable incubation period and the fact that listeriosis leads to a mortality rate of - %, the applied biosafety research program at the national microbiology laboratory of the public health agency of canada considered the significance of proper decontamination of listeria in food processing environments (chan and weidmann, ). the importance of this work is indicated by the fact that somewhere from to % of ready-to-eat foods are thought to be contaminated with listeria (public health agency of canada, and . recently, listeria monocytogenes has gained notoriety because of its ability to grow at the low temperatures, high salt and low ph used in food processing plants (bortolussi, ) . therefore, a study was undertaken to determine the bactericidal efficacy of various liquid disinfectants and the effect that low temperatures have on the ability of these disinfectants to inactivate l. monocytogenes at conditions found in food processing plants. at both room and refrigeration temperature ( °), ethanol, javex, su and peracetic acid (paa) products outperformed all others. surprisingly, there was no significant variation in performance at room temperature compared with refrigeration temperature. however, as some organisms undergo changes during a temperature shift, it is crucial to test each disinfectant at the temperature at which it will be employed. bleach was found to be effective but is toxic, corrosive and residue forming, while the paa and ethanol compounds do not form residues and are not corrosive. as a result of these studies, major canadian food-processing plants have changed their decontamination procedures and are no longer using quaternary ammonium compounds (quats), which were previously used extensively. positive relations have been built up between companies and laboratories, leading to more relevant laboratory studies and industrial applications (public health agency of canada, ). a prion (proteinaceous infectious particle) has been defined as a 'malformed version of a normal cellular protein that apparently ''replicates'' by recruiting normal proteins to adopt its form, [thus becoming] capable of infecting other cells of the same, or a different organism' (prusiner, ; thain and hickman, , p. ) . although two nobel prizes in medicine have been awarded for prion research, to carleton gajusek in and to stanley prusiner in , the precise nature of the infectious agent remains unclear to such an extent that controversy continues about whether a prion is solely protein (brooks, , pp. - ) . whatever the cause, prion diseases are fatal chronic neurological diseases that affect the brains and nervous systems of many mammals, including humans (imran and mahmood, ) . prions can be detected in tissues by a number of research techniques, including infective bioassay, animal inoculation, western blot and immunochemistry. it is clear that prions can cause spongiform encephalopathies within both humans and animals (e.g. creutzfeldt-jakob disease, kuru, scrapie, transmissible mink encephalopathy, feline spongiform encephalopathy and bovine spongiform encephalopathy) (blood et al., (blood et al., , p. . summaries of the three presentations below offer further insights into the nature of prion diseases. in prion diseases, professor j. j. badiola and dr. c. akin (university of zaragoza, zaragoza, spain) focused on the outbreak of bovine spongiform encephalopathy (bse) ('mad cow disease') in the united kingdom, which led to a better understanding of the epidemiology and molecular characteristics of the disease. epidemic bse affected mainly the united kingdom, with a total of , positive animals compared to , in all other member states of the european union (oie, ). control and eradication of transmissible spongiform encephalopathies (tses) became a priority, not only in europe, but throughout the world. in , a reinforcement of the passive surveillance program and the establishment of an active one were established by the european commission for all the european union member states (european commission, ) . passive surveillance, focused on animals with clinical signs of the disease, and active surveillance was carried out in the following target groups: healthy slaughtered, fallen stock, emergency slaughtered and animals culled under bse eradication. apart from these measures, specific risk materials (e.g. tonsils, intestines, spleen, spinal cord and skull, including the brain and eyes) were defined and prohibited from being included in the human food chain. moreover, a banning of all meat and bone meal for animal feed was established (european commission, ) . the result of these powerful eradication measures has been a rapidly decreasing number of new bse cases, with less than cases detected worldwide in , of which were in the european union (oie, ) . the impressive containment of bse in the united kingdom from , reported cases in to in is testimony to the determination with which scientists, politicians, civil servants and farmers have worked together to bring the disease under control. professor c. i. lasmézas (dept of infectology, the scripps research institute, scripps, florida, usa) began her presentation, zoonotic potential of new animal prion diseases: assessment in non-human primates, by noting that the first demonstration of the transmissibility of a prion disease to non-human primates (nhps) was made in by carleton gajdusek when he transmitted kuru to chimpanzees. since then, animal and human prion diseases have been transmitted to a range of nhps. cynomolgus macaques have shown the highest selectivity with regard to the prion strain by which they can be infected and therefore seem to be the species of choice to assess the risk that any given animal prion strain can be pathogenic for humans (lasmézas et al., ) . prions were thought to be very difficult to transmit from one species to another; however, the experience of studying scrapie highlights the difficulties inherent in studying prion diseases in the laboratory. scrapie had been transmitted orally to other ruminants (goats) but only intracerebral inoculations had successfully transmitted scrapie to monkey, mouse or mink. however, the oral transmission of bovine spongiform encephalopathy (bse) to domestic cats in forced a revision of this earlier belief. transmissions of bse have now occurred orally to sheep, goat, monkey, mink, cheetah, puma, cat and mouse. intracerebral transmission of bse has also occurred to pig. furthermore, intraspecies oral transmission of bse has taken place within numerous speciesmonkey, mink, sheep, goat, cow, hamster and mouse. vcjd (variant creutzfeldt-jakob disease) is a new human disease, which was caused by eating ruminant-derived food products contaminated with bse. vcjd poses a public health problem because of the absence of preclinical diagnostic test, the long incubation periods of prion diseases in humans (possibly extending up to years) and the transmissibility of the disease by blood transfusion. the research team at the french commissariat a l' energie atomique (cea) demonstrated that bovine spongiform encephalopathy (bse) was transmissible to macaques within years with a % infection rate and caused a disease indistinguishable from the human variant of creutzfeldt-jakob disease (lasmézas et al., ) . this provided a model to study carefully the peripheral pathogenesis of vcjd, the oral infectious dose of bse and evaluate the risk of human-to-human transmission of vcjd by blood transfusion (herzog et al., ) . further, the research team used the macaque model to assess the zoonotic potential of emerging forms of bse called l-or h-type. the l-type bse presents with higher pathogenicity to macaques than classical bse (comoy et al., ) . therefore, continued precautionary measures remain necessary to protect the human food chain. experiments are ongoing at the national institute of allergy and infectious disease, hamilton, montana, to assess the risk linked to chronic wasting disease that is spreading throughout the usa. the closing acknowledgements of professor lasmézas to other researchers indicated both the complexity and importance of continuing work in prion diseases. furthermore, since the cancun meeting further important research has been published (hamir et al., ) . infectivity distribution studies of animals infected with bse prions animals are a matter of considerable importance in seeking to elucidate the route of infectious prions from the gut to the central nervous system (cns) open questions about this lethal journey from the gut to the brain, including where in the gut the disease begins, the initial steps of the neuronal bse pathogenesis, the ascension of bse prions to the brain, the haematogenous spread and the centrifugal contamination of the periphery (buschmann and groschup, ; hoffmann et al., ) . the scale of the research task was indicated by the fact that , samples were collected per animal autopsy, leading to some , frozen samples collected and archived at the friedrich-loeffler-institut. tissue samples were collected from the gut, the central and autonomous nervous system (ans) of the challenged bovines and then examined for the presence of pathological prion proteins (prp sc ). there was some variation among different animals. however, a distinct accumulation of prp sc was observed in the distal ileum, confined to follicles and/or the enteric nervous system, in almost all animals . bse prions were found in the sympathetic nervous system starting from months post-inoculation (mpi) on as well as in the parasympathetic nervous system from mpi on (kaatz et al., ) . a clear dissociation of prion infectivity and detectable prp sc deposition was obvious in tongue (balkema- . the earliest presence of infectivity in the brainstem was detected at mpi, while prp sc -accumulation was detected first after mpi. in summary, these results deciphered for the first time the centripetal spread of bse prions along the ans to the cns starting already half way during the incubation period. bse prions spread in cattle from the gut to the brain along the sympathetic, parasympathetic and spinal cord routes, possibly in that order of importance. spinal cord involvement may even not be necessary at all, but bse infectivity in the form of prp sc spills over into the periphery already in the pre-clinical phase. the modelling and prediction of emerging zoonoses is a fast-growing field of considerable complexity. of the five papers relevant to this topic, two have been published in full below in this special supplement. dr. g. zanella and her colleagues consider modelling transmission of bovine tuberculosis in red deer and wild boar in normandy, france. their mathematical model of the mycobacterium bovis infection within and between species takes into account the transmission of m. bovis through infected offal -the viscera of animals killed by hunters and left behind. when an animal was hunted in the brotonne forest in normandy prior to , it was eviscerated in situ and only the carcass taken away, with the raw viscera left behind. since , offal disposal has been required in brotonne forest; however, the regulation has not always been observed by hunters (unpublished correspondence with g. zanella, - december, ) an important benefit of mathematical modelling is that it permits consideration of all the elements involved in disease transmission within a population, thereby complementing field data, as well as testing the effects of control measures. thus, the direct transmission of the m. bovis infection within the red deer and wild boar populations can be distinguished from indirect transmission through contaminated offal. the model indicates that offal destruction is the key factor in infection control for both red deer and wild boar. the authors conclude that, in principle, the structure of this model is relevant to the situations where dead animals play an important role in disease transmission between two or more species. in a further article published in this special supplement, constructing ecological networks: a tool to infer risk of transmission and dispersal of leishmaniasis, dr. c. gonzález-salazar and professor c. stephens set out the role of ecological networks as a powerful tool for understanding and visualizing inter-species ecological and evolutionary interactions. taking the example of leishmaniasis in mexico, they show that such networks can be used not only to understand potential ecological interactions between species involved in the transmission of the disease, but also to identify the potential role of the environment in disease transmission and dispersal. strikingly, they show how potential interactions can be inferred from geographical data, rather than by direct observation. their findings have led to the prediction of additional reservoirs in mexico of many new species, including bats and squirrels. the resulting model can be used to understand and map potential transmission risk, as well as construct risk scenarios for the dispersal of leishmaniasis from one geographical region to another. such a risk assessment tool for leishmaniasis will be especially useful in the light of the bill and melinda gates foundation decision in january to join with major pharmaceutical companies and the world health organization in targeting leishmaniasis as one of the neglected tropical diseases to receive improved drugs, diagnostics, vector control strategies and vaccines (bill & melinda gates foundation, ; boseley, ) . however, the possibility of new reservoirs suggests it is hard to imagine that leishmaniasis can be completely eradicated. nevertheless, it is increasingly clear that leishmaniasis has a disturbing capacity to jump from species to species, so efforts to control the disease must be given a high priority (unpublished correspondence with c. stephens, february , ; cf. flanagan et al., ) . it is difficult to model and predict the distribution and impact of a new emerging virus. for example, the emergence in november in europe of a midge-borne virus member of the bunyaviridae family, named schmallenberg virus after the location in germany where it was first detected, has caused serious birth defects in lambs, goats and cattle (ecdc, ) . scientists, farmers, veterinarians, public health officials and consumers are all confronted with the uncertainty inherent in facing a new animal pathogen (farmers weekly, ) . appropriately, at the same time as this new virus has emerged, the animal health and veterinary laboratories agency (ahvla) has set up a new independent advisory group to evaluate veterinary surveillance in england and wales, although their original intent was in part to consider funding reductions (trickett, ) . modelling risk factors for zoonotic influenza infections is challenging because the infections are often rare; the laboratory assays are often difficult and imprecise, and the most definitive studies require intensive resources. this was the view of professor g. c. gray (emerging pathogens institute and college of public health and health professions, university of florida, gainesville, florida, usa) in his presentation, modeling risk factors for zoonotic influenza infections in man: challenges and strategies for success. in particular, serologic detections of these infections in humans may be confounded by crossreacting antibody, waning antibody from the infection of interest, inaccurate matching of the enzootic pathogen and the laboratory strain, laboratory errors and weakly powered statistical comparisons. the underlying question which professor gray and his research team is tackling is: which human, animal and environmental factors predict disease? these three factors can be viewed as a venn diagram with its intricate interactions. like understanding cardiovascular disease, how a person acquires a zoonotic influenza infection is a complex process, and predictive laboratory assays are imprecise. for example, with avian influenza viruses (especially h n , popularly known as 'bird flu'), poultry veterinarians, turkey workers, hunters and people without indoor plumbing may be at increased risk of aiv infection but infections are rare. subclinical or mild infections do occur; and occasionally aiv causes severe disease in persons exposed to sick birds. although aiv transmission from human-to-human seems rare, further cohort studies and more sensitive serological assays are needed. a basic scientist often tests hypotheses by: (i) carefully setting up an experimental setting; (ii) isolating confounding factors; and (iii) looking for statistically significant associations with an outcome. such a process is not possible for a number of emerging disease problems such as human infections with swine influenza virus (siv). experimental studies are not possible. hence, epidemiologists must perform observational studies of people most likely to be infected with siv and by looking at possible risk factor associations, infer causality. one must first determine settings where the prevalence of siv in expected to be high and then study those workers. for example, sivs are often endemic in large-scale modern production facilities. risk factors for sow-herd siv seropositivity involve pig density, whether there is an external source of breeding pigs, the total animals on the site and the closeness of barns. similarly, risks factors for finisherherd siv positivity must be considered -the number of siv-positive sows, size of herd, pig farm density and farrow-to-finish type of farm (poljak et al., ) . however, siv surveillance in pigs is largely passive and voluntary, so recognizing which pig workers to study is a challenge. detection of siv infections in man often requires a sentinel event (e.g. human illness with pig exposure or sick pigs). as pigs do not always have clinical signs of novel virus infection and often there is no compensation system to protect pig farmers, the pork industry is reluctant to permit the study of their workers for siv infection (gray and baker, ) . therefore, these observational studies are currently very difficult. professor gray concluded by pointing out that although there are numerous challenges in conducting epidemiological studies for zoonotic influenza, there are six substantive ways to control confounding variables: (i) design every study carefully; (ii) use non-animal-exposed controls; (iii) employ validated laboratory assay using zoonotic influenza strains; (iv) use multivariate modeling to examine cross-reacting serologic responses due to human viruses and vaccines; (v) consider proportional odds modeling; and (vi) consider employing a second unique serologic test (see gpl, ) . with the support of co-authors from different institutions, dr. k. j. linthicum (united states department of agriculture, agricultural research service, center for medical, agricultural & veterinary entomology, gainesville, fl, usa) presented two case studies about forecasting emerging vector-borne diseases. dr. linthicum began by pointing out that global climate variability, often linked to el niño conditions, can be used to forecast emerging vector-borne disease spread in local areas (linthicum et al., ) . these forecasts are possible because specific pathogens, their vectors and hosts are sensitive to temperature, moisture and other ambient environmental conditions. with consistent and reliable satellite observations, global sea temperatures, climate and vegetation can be observed. first, temperature plays a major role in its impact on aides aegypti mosquitoes transmitting dengue haemorrhagic fever virus in southeast asia (linthicum et al., ) and possibly also on how ae. aegypti transmits chikungunya virus in africa and asia , as well as on how anopheles species mosquitoes transmit p. vivax malaria in the republic of korea. vectorial competence is dependent upon the extrinsic incubation (ei) period in the mosquito vector. the ei represents the time from ingestion of the virus while feeding on a viremic host to the virus arriving in the salivary glands. the shorter the ei period, which occurs during higher ambient temperatures, the greater the vectorial competence (garrett- jones and shidrawi, ) . if data are available for a specific local area on the daily humanbiting rate (ha) of the mosquitoes, the daily rate of blood feeding (a) and the length of the ei cycle (n), it is possible to calculate vectorial capacity (rattanarithikul et al., ) . second, accurate measurements and understanding of how exceptionally heavy rainfall and flooding affects aides and culex mosquitoes and the introduction of virusinfected mosquitoes into susceptible vertebrate hosts enables forecasts to be made about when and where rift valley fever (rvf) will develop in sub-saharan africa and middle east (anyamba et al., ) . outbreaks of rift valley fever are known to follow periods of widespread and heavy rainfall associated with the development of a strong inter-tropical convergence zone over eastern africa (davies et al., ) . during periods of elevated transmission, there is a significantly increased risk of globalization of these and other arboviruses; however, the forecasting methods described provide . - months early warning before an outbreak and provide ample time for disease mitigation before the first cases appear (anyamba et al., ) . furthermore, the emergence and expansion of a number of disease vectors (e.g. mosquitoes, mice, locust) often follow the trajectory of the green flush of vegetation in semiarid lands. the ability to predict periods of elevated risk enables better prevention, containment or exclusion strategies to be drawn up to limit globalization of emerging pathogens. thus, it has been possible for the food & agricultural organization (fao) to create a system of alerts -the emergency prevention system for transboundary animal and plant pests and diseases (empress, ) . subsequent to dr lithicum's presentation, significant further work has been done to provide a genome-scale overview of gene expression in the malaria-transmitting mosquito anopheles gambiae (maccallum et al., ) , as well as to expand the vectorbase website with regularly updated genome information on two other mosquito species, aedes aegypti and culex quinquefasciatus, and numerous other organisms, including the tick species ixodes scapularis (lawson et al., ; niaid, ) . the ultimate aim of this research is to create a database that will facilitate a systems-level view of gene expression for many different organisms. reflecting on the numerous types of statistical analysis that are used to estimate confidence intervals for proportions in scientific studies, dr. s. guillossou and his colleagues professors h. m. scott and j. a. richt (dept. of diagnostic medicine and pathobiology, college of veterinary medicine, kansas state university, manhattan, kansas, usa) utilized the final presentation of the conference, estimates of low prevalences and diagnostic test estimates: what confidence do we really have? to illustrate the differences, limits and sometimes chaotic behaviour of different statistical approaches. dr. guillossou pointed out that there were more than different methods for determining a % confidence interval of a proportion. he stressed that it is always important to report the method of statistical analysis being utilized. in his view, the agresti-coull interval approach presents a satisfactory compromise between computational requirements and coverage probability (newcombe, ; brown et al., ) . ideally, the effects of coverage probability should be estimated and the most appropriate method chosen before reporting the findings or using proportions as inputs in any epidemiological study. what did this th international conference on emerging zoonoses achieve? there was the opportunity to meet old friends and make new friends, to share one's academic work and to reflect on what lies ahead with emerging zoonoses. it is now clear that human medicine, veterinary medicine and environmental challenges are a unity which must be considered under the umbrella of 'one health' (one health initiative, ) . viruses are continuing to jump from animals to people with unexpected consequences, because the evolution of any virus is impossible to predict. even the recent relatively mild swine flu virus infected % of the human population and killed some , people globally -far less than would have been the case if the virus had mutated to a more deadly form, as might easily have happened. the reality is, as professor nathan wolfe, professor in human biology at stanford university, has commented: 'as a species, we're not that focused on the things that have the most potential to be devastating to us as a global population, such as viruses. unless people take these things seriously, we're going to look back and say we had all the tools necessary to try to address these risks, and we basically ignored them because they weren't dramatic like a car accident or a hurricane' (geddes, ; kahn, ; wolfe, ) . this conference, many others and the th international conference on emerging zoonoses to be held in in berlin, are aimed at creating, improving and using the tools essential to address the risks of viral contagions in a global society. none. the organizing committee of the conference wishes to acknowledge the excellent services of the conference organizers, target conferences of tel aviv, israel, and the welcome financial contributions of medimmune, boehringer ingelheim vetmedica gmbh, prionics ag, center of excellence for emerging and zoonotic animal diseases (ceezad) and national center for foreign animal and zoonotic disease defense (fazd), as well as the poster prize donated by wiley-blackwell. we are also grateful to the wiley-blackwell staff who have contributed so significantly to this special supplement, especially rachel robinson and peter tubman, as well as to dr. klaus osterrieder for his helpful comments and to the presenters who have approved or improved every summary in this meeting review. this material is based upon work supported by the u.s. department of homeland security under grant award number -st-ag . the views and conclusions contained in this supplement are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the u.s. department of homeland security. additional funding has been provided by the kansas bioscience authority. conservation medicine: ecological health in practice prediction of a rift valley fever outbreak : prediction, assessment of the rift valley fever activity in east and southern africa - and possible vector control strategies climate teleconnections and recent patterns of human and animal disease outbreaks risk of infection with brucella abortus and escherichia coli o :h associated with marketing of unpasteurized milk in kenya fallout from listeria outbreak hits walmart: retail detection and identification of mycoplasma from bovine mastitis infections using a nested polymerase chain 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clinical aspects of human infection with avian influenza a (h n ) virus modification of non-structural protein of influenza a virus by sum key: cord- -t yioyl authors: rohr, jason r.; barrett, christopher b.; civitello, david j.; craft, meggan e.; delius, bryan; deleo, giulio a.; hudson, peter j.; jouanard, nicolas; nguyen, karena h.; ostfeld, richard s.; remais, justin v.; riveau, gilles; sokolow, susanne h.; tilman, david title: emerging human infectious diseases and the links to global food production date: - - journal: nat sustain doi: . /s - - - sha: doc_id: cord_uid: t yioyl infectious diseases are emerging globally at an unprecedented rate while global food demand is projected to increase sharply by . here, we synthesize the pathways by which projected agricultural expansion and intensification will influence human infectious diseases and how human infectious diseases might likewise affect food production and distribution. feeding billion people will require substantial increases in crop and animal production that will expand agricultural use of antibiotics, water, pesticides and fertilizer, and contact rates between humans and both wild and domestic animals, all with consequences for the emergence and spread of infectious agents. indeed, our synthesis of the literature suggests that, since , agricultural drivers were associated with > % of all — and > % of zoonotic — infectious diseases that emerged in humans, proportions that will likely increase as agriculture expands and intensifies. we identify agricultural and disease management and policy actions, and additional research, needed to address the public health challenge posed by feeding billion people. i nfectious diseases are emerging at an unprecedented rate with significant impacts on global economies and public health . the social and environmental conditions that give rise to disease emergence are thus of particular interest, as are management approaches that might reduce the risk of emergence or re-emergence . at the same time, undernutrition -the insufficient intake of one or more nutrients -remains a major source of global ill health [ ] [ ] [ ] [ ] . together, the unprecedented rate of infectious disease emergence and the need to sustainably feed the global population represent two of the most formidable ecological and public health challenges of the twentyfirst century [ ] [ ] [ ] [ ] [ ] (fig. ) , and they interact in complex ways (fig. ) . although modern agricultural technologies have reduced hunger, improved nutrition and spared some natural ecosystems from conversion to agriculture, current global agricultural production and distribution infrastructure still leaves billions of people's diets deficient in one or more crucial nutrients, with major consequences for global morbidity and mortality , and these deficiencies are expected to worsen with climate change [ ] [ ] [ ] [ ] . credible estimates are available for specific nutrient shortfalls -for example, . billion people suffering iron-or vitamin b -deficiency anemia , . billion people with insufficient dietary energy (that is, calorie) intake , % of pre-school age children and % of pregnant women at risk of vitamin a deficiency -but the world still lacks a rigorous, recent assessment of the population suffering shortfalls of any one or more nutrients, although the number is surely in the billions. by , the united nations projects that the global population will grow by nearly billion, to exceed billion people . meeting the united nations' sustainable development goal, to "eradicate hunger" (https://sustainabledevelopment.un.org/) for this expanding human population will necessitate a large increase in food supplies, with major changes to agricultural production and distribution systems, infrastructure and social protection programmes (fig. ) . despite large and, in many cases, growing inequalities, the global population is on average expected to become richer, and historically, with greater affluence has come greater consumption of food products in general and more animal-sourced foods in particular, both of which further increase food demand and thus the requirement for agricultural expansion and/or intensification . agriculture already occupies about half of the world's land and uses more than two-thirds of the world's fresh water , and recent studies have suggested that agricultural production might need to double or triple by to keep pace with projected population growth and food demand [ ] [ ] [ ] (fig. ) . meeting this demand using present agricultural production systems could require replacing > hectares of natural ecosystems with agricultural production, approximately % of the global land area, which is larger than the continental united states, although continuous efficiency improvements in agriculture will compel re-evaluation of these estimates. in turn, this agricultural expansion could result in an estimated ~ -fold increase in irrigation, ~ . -fold increase in fertilizer , , and -fold increase in pesticide use (fig. ) . the challenges of feeding > billion people and managing infectious diseases intersect in several ways ( figs. and ) . first, the expansion and intensification of agriculture are disproportionately occurring in tropical, developing countries , , , where % of deaths are attributable to infectious diseases , where the risk of disease emergence might be greatest, and where disease surveillance and access to health care, particularly for those infections that accompany extreme poverty, are most limited , . second, agricultural expansion and intensification have historically come with massive habitat conversion, contamination with animal waste and increasing use of agricultural inputs, such as pesticides and antibiotic growth promoters. beyond their direct adverse effects on human health , , agricultural biochemical inputs are known to have direct effects on emerging human infectious diseases, and can also serve as indirect drivers by contributing to the emergence of wildlife diseases that constitute important sources of emerging infections in humans , . however, the strengthening of agricultural production systems can also improve nutrition, which has pronounced benefits for combating many infectious diseases at the individual and population levels , . although concerns have been raised regarding the environmental impact of agricultural expansion and intensification , - , , , their effects on infectious disease risk have not been synthesized. here, we review both the beneficial and adverse effects of agricultural expansion and intensification on the transmission of human infectious diseases. we synthesize the pathways through which agricultural practices influence human infectious diseases, and vice versa, and identify opportunities to minimize the adverse consequences of agricultural growth while maximizing the human health benefits of agricultural development (fig. ). agricultural development can yield direct improvements to nutrition, and through several mechanisms, nutrition can be a critical determinant of infectious disease susceptibility and progression , (figs. and ). for example, immune responses are energetically costly and thus undernutrition often reduces the development and effectiveness of immune responses that can limit or clear infections. the relationship between infectious disease and nutritional agricultural production can improve human health by reducing food prices and enhancing nutrition, which can increase resistance to infectious diseases. however, freshwater habitats established for irrigation, as well as other agricultural inputs, often increase the risk of vector-borne diseases, such as malaria and schistosomiasis. in general, rural residents are most vulnerable to these increases in infectious disease, whereas consumers some distance away derive most of the benefits from increased food production. to maximize human health given the impending billion humans projected on the planet by , society must minimize the adverse consequences of agricultural growth while maximizing the health benefits. images by kate marx. status can function in reverse as well, because many parasitic infections place direct demands on host nutrition, causing undernutrition when food is limited . in fact, some parasites, such as helminths, can even cause eating disorders, such as geophagy (desire to eat soil), bulimia and anorexia . enduring infections often require rapid and effective repair of tissue damage caused by parasites, which is also costly. hence, certain infections can cause undernutrition, which itself can compromise both resistance and tolerance to infections , . by improving nutrition, agricultural development should facilitate combating many infectious diseases. for example, death rates from acute respiratory infections, diarrhoea, malaria and measles, diseases that on average kill more than a child every seconds ( million per year) , are much higher in children who suffer undernutrition than in those that do not , . in addition, poor maternal nutrition and associated impaired fetal growth are strongly associated with neonatal death from sepsis, pneumonia and diarrhoea ; undernourishment is a well-understood risk factor for . although the traditional green revolution approach to food production has succeeded in reducing undernourishment arising from insufficient calorie and protein intake, it has been very slow in reducing micronutrient deficiencies , which can be have significant effects on defence against disease. to make matters worse, where violence, unrest and terrorism impede access to food, such as in parts of africa and central asia, morbidity and mortality attributed to communicable diseases can be further exacerbated . although research suggests that improving nutrition will generally improve responses to infectious diseases, there may be important exceptions. for certain diseases, such as schistosomiasis and many respiratory infections, pathogenesis is a product of host immunity, often from hyperinflammation , . thus, undernutrition can actually decrease symptoms by reducing the strength and pathogenicity of immune responses . in addition, as nutrition improves, some parasites might proliferate faster than immunity can increase , resulting in more, rather than less, morbidity, with the classic example that dietary intake of iron can increase malariainduced mortality . in this section, we review the links between rural economy, infrastructure and infectious disease. , phosphorous (c), fertilizer and pesticide use (d), cropland area (e), and irrigated land area (f) and associated % prediction bands (light blue bands). 'nitrogen' refers to the normalized estimated global amount of nitrogen nutrients in fertilizers produced (originally reported in thousands of tonnes, now in metric tonnes). 'phosphorous' refers to the normalized estimated global amount of p o nutrients in fertilizers produced (originally reported in thousands of tonnes, now in metric tonnes). for a, data were collected from the world bank. for b-f, data were collected from the food and agriculture organization of the united nations and statistical models were developed based on the relationships between these variables and global human population density. after the best model was identified, year was substituted for human population density based on the fit in a. these projections should be evaluated with considerable caution because the models assume that human population size is the only factor that affects fertilizer and pesticide use and the amount of arable and irrigated cropland, when in reality, many factors affect these responses (for example, climate, diets, type of crops grown and so on). they merely illustrate that even an extremely parsimonious model seems to track past patterns tolerably well and thus could serve as a basis for coarse projections. see supplementary methods for details of the statistical models used to generate these figures and supplementary table for coefficients and statistics for the best-fitting models. economic development. economic development, especially agricultural development, has historically driven reductions in both infectious diseases and poverty across many settings. in fact, the poor financially benefit more from economic growth in the agricultural sector than in industrial or service sectors , . the early stages of economic development often involve the construction of infrastructure to facilitate food production and distribution, including roads, dams and irrigation networks , . more recently, the early stages of rural development often include rapid expansion of telecommunication, and to a lesser degree, electrification, both of which are promising but underutilized resources for disease monitoring and control in the developing world . other rural infrastructure that is more crucial to infectious disease prevention, such as safe water, sanitation and energy supplies, often follows or is developed concurrently , . in developing countries, rural infrastructure that provided access to sanitation and safe water explained % and % of the difference in the prevalence of malnutrition and child mortality rates between the poorest and richest quintiles, respectively . moreover, clean water, sanitation and electricity can also facilitate the construction of schools and health clinics that can help to further reduce disease through education, prevention and treatment. hence, if the history of the developed world repeats itself in the developing world, then it seems plausible that agricultural development necessary to feed billion people might help to reduce infectious diseases by promoting economic development and rural infrastructure. there are several important assumptions to this hypothesis, however, such as: ( ) it is possible to feed billion people; ( ) the rate of economic development will match or exceed the pace of human population growth; and ( ) developing countries are not in an infectious disease-driven 'poverty trap' , which is the notion that poverty increases the chances of acquiring and succumbing to disease, and chronic disease traps humans in poverty [ ] [ ] [ ] [ ] [ ] . if agricultural development lags behind population growth or if clean water supplies, sanitation and electricity do not immediately follow road, dam and irrigation construction, then the prevalence of many infectious diseases could remain unchanged or even increase as human populations grow . agricultural irrigation and freshwater redistribution. one major change in rural infrastructure that often accompanies agricultural development is the redistribution of fresh water, which has well-known consequences for the transmission of infectious diseases. for example, because % of crop production comes from only the % of agricultural land that is irrigated, and irrigated lands accounted for much of the increased yields experienced during the green revolution , the potential for increased irrigation infrastructure to exacerbate infectious diseases is a critical concern (fig. ) . importantly, agricultural development has caused both declines of certain types of fresh water, such as wetlands, and increases of others, such as dams, reservoirs and irrigation schemes. one of the largest drivers of global wetland losses has been conversion to agriculture , which has led to declines in diseases that rely on wetland habitats. for example, japan's successful eradication of schistosomiasis during the mid-twentieth century relied in part on conversion of wetlands to orchards in schistosomiasis-endemic areas , although replacing oxen with horses, which are less susceptible to schistosoma japonicum, as draft animals is also thought to have contributed substantially . similarly, some of the earliest successes in malaria control at the turn of the twentieth century occurred in the southern united states, where wetland drainageincluding for agricultural conversion -was one of a suite of antitransmission strategies used by the rockefeller foundation in its landmark malaria control efforts . although wetlands often decline with agriculture, dams, reservoirs and irrigation networks often increase, and this redistribution of fresh water has been widely associated with increases in some vectors and hosts of human pathogens . for example, construction of the aswan high dam in egypt and the accompanying irrigation network was associated with a rise in mosquito vectors and the disfiguring mosquito-borne disease lymphatic filariasis, commonly known as human elephantiasis . likewise, dam and irrigation construction resulted in increases in malaria in sri lanka and india , , and a sevenfold increase in malaria in ethiopia . a meta-analysis of studies revealed that humans living near irrigation schemes or in close proximity to large dam reservoirs had significantly higher risk of schistosome infections than humans that did not live near these water resources , at least partly because of increased freshwater habitat for intermediate snail hosts. similarly, a recent analysis of schistosomiasis case data from the past years across sub-saharan africa showed far-reaching effects of dams and irrigation schemes, with increases in schistosomiasis risk extending up to hundreds of kilometres upstream from the dams themselves . hence, dams, reservoirs and irrigation networks related to agricultural expansion are likely to increase vector-borne and waterborne diseases unless water resources are developed in deliberate and coordinated ways to mitigate these risks . urbanization, globalization and the movement of agricultural products. the world's population became majority urban in and urbanization continues to outpace population growth, especially in developing countries. urbanization necessarily extends food supply chains, as consumers reside further from farms and fisheries. although only % of the food produced globally for human consumption is traded across international borders , globalization is likewise elongating food supply chains. furthermore, as the costs of international travel have declined over time, the movement of people across borders has likewise increased. the expanded spatial scope and increased frequency, speed and volume of people and agricultural products moving within and among countries necessarily facilitates the spread of pathogens. for example, in ecuador, there is evidence that the construction of new roads has affected the epidemiology of diarrheal diseases by changing contact rates among people as well as between people and contaminated water sources . each year in the united states, there are already an estimated million people with illnesses, , hospitalizations and , deaths from food-borne infections , and imported foods -especially from developing countries with poor sanitation infrastructure and weak food safety enforcement -have been associated with a rise in food-borne illness . in addition, globalization is already thought to be a factor in the spread of human influenza viruses that spillover from poultry and swine , . although modernization of food supply chains, especially those linking farms in developing countries to high-income consumers in cities and high-income countries, has typically accelerated the diffusion of stricter food safety and quality standards , , as urbanization and globalization further extend food supply chains, enhanced monitoring for the spread of pathogens across transportation networks and strengthened food safety regulations will be needed. in this section, we review the effects of agricultural industrialization. anti-parasitic and antibiotic drug use. as the global human population increases, there will almost certainly be an increase in high density, industrialized livestock and aquaculture operations. currently, such livestock operations are vulnerable to devastating losses of animals to disease. for instance, in just the last years, an influenza a virus (h n ) and a foot-and-mouth outbreak led to the destruction of more than . million chickens and million livestock in china and great britain , respectively, and a 'mad cow disease' epizootic led to the slaughter of million cattle worldwide . similar scenarios have occurred in aquaculture. in the past three decades, there has been more than fourfold growth in industrial aquaculture worldwide , which should continue to increase as food demand grows and wild fish and shellfish captures push the limits of renewable production . bacterial outbreaks in aquaculture are common, especially in developing countries where there are sanitary shortcomings . in an effort to prevent these catastrophic disease-associated losses and improve animal growth, the agricultural industry uses a larger proportion of global antibiotic and anthelmintic production than human medicine, and most of the antibiotics are provided at non-therapeutic doses in the absence of any known disease , . in fact, although estimates are lacking for most countries in the world, in the united states, nearly nine times more antibiotics are given to animals than humans and, of the antibiotics given to animals, more than times as many are used nontherapeutically as therapeutically . this widespread use of antibiotics and anti-parasitic drugs (for example, anthelmintics) in industrialized agriculture and aquaculture could have important implications for human infectious diseases because it seems to be driving microbial resistance to these drugs, some of which are also used in human medicine , , . for example, livestock are a primary source of antibiotic-resistant salmonella, campylobacter and escherichia coli strains that are pathogenic to humans . there is evidence that antibiotic-resistance genes acquired from aquaculture are being transferred to human systems and these pathogens have subsequently caused outbreaks . anthelmintic resistance is also rife among parasitic worms of livestock and is strongly implied for parasitic worms of humans . as livestock and aquaculture production expand to address growing food demands, it is likely that current antibiotics and anthelmintics will become less effective because of evolved resistance, and thus infectious diseases of domesticated animals and humans will be more difficult to treat . pesticides, fertilizers and disease. pesticides, particularly insecticides, have shared value for suppression of agricultural pest populations and disease-carrying insect vectors, such as mosquitoes and other flies. because of the anticipated sharp increase in pesticide use by (fig. ) , insecticide resistance is also expected to increase, with important implications for the control of diseases carried by insect vectors. insecticides with widespread shared use for both crop protection and vector control include pyrethroid, organophosphate and organochlorine insecticides . several mosquito vectors of human and livestock pathogens have already evolved some resistance to these compounds . if agricultural expansion and intensification is accompanied by an increased use of insecticides, vector resistance may become more common and the control of vectorborne diseases more challenging. in addition to potentially driving vector resistance, increased pesticide use is likely to cause numerous other effects on host-parasite interactions. pesticides can alter disease risk by modifying host susceptibility to parasites [ ] [ ] [ ] . for example, many pesticides are immunomodulators that can increase infectious diseases of wildlife and humans , , or are endocrine disruptors of humans with potential downstream effects on immunity . even if pesticides do not directly affect immunity, detoxification of pesticides is energetically 'expensive' for the host, and thus pesticide exposure can reduce available energy resources for humans and zoonotic hosts to invest into parasite defences , . pesticides can also affect human disease risk by altering the densities of hosts or parasites or their natural enemies or mutualists , . several pesticides can alter host behaviours or be directly toxic to hosts and parasites, which in turn can modify contact rates between parasites and human hosts. in addition, pesticides can alter community composition, which can indirectly affect behaviours or densities of intermediate and zoonotic hosts and parasites. although chemical contaminants can be deadly to many free-living stages of parasites, both empirical trends and theoretical models suggest that stress associated with pesticide exposure can increase non-specific or generalist pathogens , , . in addition to the impacts that pesticides can have on infectious diseases, they can also contribute to non-infectious diseases, such as cancers, birth defects, miscarriages and impaired childhood development , which can further strengthen the poverty-infectious disease trap. indeed, among african agricultural households, there is evidence of an association between increased pesticide use and increased time lost from work due to sickness . in addition to an increase in pesticides, nitrogen-and phosphorous-based fertilizers are expected to increase threefold by to boost food production, and most of this increase will occur in tropical regions already rich with pathogens , , . although the effects of environmental nutrient enrichment on disease are indirect and complex, with some infections increasing and others decreasing, two reviews on the subject suggest that elevated nutrient levels more often than not exacerbate the impact of infectious disease , . for example, phosphorous enrichment can benefit mosquitoes that transmit malaria and west nile virus , . in addition, nitrogen-and phosphorous-based fertilizer use can increase the number of snails that transmit flatworms that cause human schistosomiasis , . conversion of natural habitat to agriculture can increase the abundance of ecotones (boundaries between ecological systems), change species composition and reduce native biodiversity , . ecotones play an important role in a number of important emerging infectious diseases , , and reduced biodiversity that accompanies agricultural intensification can increase zoonotic disease emergence and can worsen already endemic diseases [ ] [ ] [ ] . a recent global meta-analysis suggests that, based on the available literature, such biodiversity losses generally increase infections of wildlife and zoonotic infections of humans . however, studies of the relationship between biodiversity change and infectious disease risk tend to focus on a single parasite or disease, often of non-human animals, which limits the ability to determine more broadly the effects of agricultural intensification on the overall burden of infectious disease in human populations . recent studies have found that the local loss of dense forests, largely from agricultural expansion, affected diarrheal diseases, acute respiratory infections and general fever in cambodian children and infectious disease incidence in nigerian children over a decade , . proximate causes probably included reduced regulation of microbial contaminants in surface and ground waters, increased smoke from biomass burning, shifts in the ranges of insect vectors and decreased access to forest ecosystem services. a major concern is the potential for positive feedbacks between poverty, biodiversity loss, soil degradation and infectious disease , . several mechanisms can underpin these reinforcing relationships. the first arises as a result of poor rural peoples' reliance on limited biophysical assets for their livelihoods. as households clear forests, deplete soils or overharvest biota to meet near-term consumption needs, this resource degradation can compromise future economic productivity and increase disease risk. in addition, poverty, environmental degradation, biodiversity loss and disease can quickly become mutually reinforcing responses to natural shocks, such as droughts or floods, or to protracted conflict in a region. there are also more direct examples. for instance, global net losses of tropical forests remained unchanged during the s and s (at approximately million ha yr − or . % annually ; also see https://glad.umd.edu/projects/global-forest-watch and https:// www.globalforestwatch.org) and forest clearing can lead to transmission of zoonotic disease by increasing contact with wild animals. conversely, encroachment of people and domesticated animals into natural areas can introduce diseases to wildlife that can devastate wild populations and create reservoirs for the disease to be transmitted back to domesticated animals (see next section for further discussion and citations). especially as the agricultural frontier expands, considerable attention must be paid to monitoring and managing these biodiversity-poverty-disease feedbacks . a central tenet of epidemiology is that the incidence of many infectious diseases should increase proportionally with host density because of increased contact rates and thus transmission among hosts . hence, increasing human and livestock densities could cause increases in infectious diseases unless investments in disease prevention are sufficient to prevent these increases. given that most of the increase in human and livestock densities are expected to occur in developing countries where disease surveillance, pest control, sanitation, and medical and veterinary care are limited, there is little reason to expect that control efforts will keep up with the expected increases in infectious diseases associated with increasing densities of these hosts. as host densities and thus transmission increase, theory suggests that parasite virulence should also increase under some circumstances . when virulence of a pathogen is tied to its propagule generation within the body (such as occurs with many viral infections), the intermediate virulence hypothesis posits that an intermediate level of virulence maximizes parasite transmission because it balances producing many parasite offspring (increasing parasite fitness) with detriments to host survival due to pathology (decreasing parasite fitness). the balance in this trade-off determines parasite persistence. hence, as host densities increase and transmission becomes more frequent, the cost of increased virulence declines, shifting the optimum towards higher virulence . consequently, for pathogens that experience this virulence trade-off, increases in human, crop and livestock densities have the potential to augment both the incidence and severity of infectious diseases. feeding billion people -and the associated increase of land converted to agricultural production and livestock grazing -is expected to cause a surge in human-livestock, human-wild animal and livestock-wild animal contact rates, increasing the likelihood of 'spillover' events, which are defined as pathogen transmission from a reservoir host population to a novel host population , . as natural ecosystems are converted to crop land or range land, interactions among humans, and domesticated and wild animals, could increase . furthermore, if developing countries follow a trajectory similar to developed countries, then their demand for meat will increase, further increasing human-livestock, human-wild animal and livestock-wild animal contact rates . these interactions are crucial because % of livestock pathogens are capable of infecting multiple host species, including wildlife and humans , and based on published estimates from the s, over half of all recognized human pathogens are currently or originally zoonotic , , , as are - % of recent emerging infectious disease events , , (fig. ) . examples of recent zoonotic disease emergences with enormous impacts on either livestock, humans or both, many of which might have agricultural drivers, include avian influenza, salmonellosis (poultry and humans), newcastle disease (poultry), swine flu, nipah virus (pigs and humans), middle east respiratory syndrome (camels and humans), bovine tuberculosis, brucellosis (mostly cattle and humans), rabies (dogs and humans), west nile virus, severe acute respiratory syndrome (sars) and ebola (humans) , . to quantify the relationship between agricultural factors and disease emergence in humans through time, we used the human disease emergence database of jones et al. . we classified land-use change, food industry and agricultural industry as agricultural drivers of human disease emergence (see supplementary methods) . these analyses revealed that agricultural drivers were associated with % of all diseases and nearly % of zoonotic diseases that emerged in humans since (fig. ) . these values are even higher if we include the use of antimicrobial agents as an agricultural driver of human disease emergence, given that agricultural uses of antibiotics outpace medical uses in the developed world nearly nine to one , . several factors have materialized that facilitate spillover events associated with disease emergence . spillover appears to be a function of the frequency, duration and intimacy of interactions between a reservoir and novel host population . for example, influenza is believed to have jumped from horses to humans soon after domesticating horses and then made additional jumps to humans from other domesticated animals, such as poultry and swine . similarly, when free-range turkeys were prevented from interacting with wild birds and when interactions between domesticated pigs and wild fruit bats were reduced, influenza and nipah virus incidence , human pathogens that are currently or originally zoonotic , , and recent emerging pathogens that are zoonotic , . dropped, respectively, suggesting wild-animal sources of these infections , . in sub-saharan africa, the high frequency and duration of environmental interactions between different species of schistosoma worms infecting humans and cattle has undoubtedly facilitated their hybridization, and these hybrid schistosomes are more virulent to humans than their non-hybrid counterparts . these factors associated with spillover and disease emergence could be targeted to reduce transmission potential as human populations and agricultural productivity increase. given that most human population growth is expected to occur in developing, tropical countries, hunting, fishing and gathering pressures will almost certainly rise in subsistence economies , . the consequences of these pressures might initially increase contact rates between humans and wildlife. this could increase disease risk as bushmeat hunting is already thought to be responsible for several emerging human infectious diseases . to make matters worse, bushmeat hunting is the second biggest threat to biodiversity behind habitat loss, and biodiversity losses can contribute to disease emergence . however, if humans overexploit these natural resources, then human-wildlife interactions could eventually decline as species go extinct, which could reduce spillover events. changes in the composition of biodiversity associated with overhunting and overfishing can also have complex indirect effects on disease risk mediated by species interactions. for example, empirical evidence supports the notion that defaunation of large vertebrates in africa should increase zoonotic disease risk by reducing the predators and competitors (large herbivores) of rodents, a common reservoir of human pathogens . similarly, overfishing of snail-eating cichlids in lake malawi in africa seems to have been a causal factor in snail population and human schistosomiasis increases there . investigations of the effects of overexploitation on infectious diseases remain in their infancy, but the consequences for human populations could be profound. up to this point, we have focused on the effects of feeding billion people on infectious diseases, but the relationship is bidirectional. that is, human infectious diseases can also impact the agricultural and economic development necessary to feed the growing human population (fig. ) . for example, the overuse of antibiotics, anthelmintics and pesticides to prevent diseases is driving resistance to these chemicals that will compromise future crop, livestock and aquaculture production. in sub-saharan africa, areas with higher historical tsetse-fly abundance, the vector of the parasite (trypanosoma brucei) that causes african sleeping sickness in humans and cattle, experienced greater lags in the adoption of animal husbandry practices (for fertilizer and labour in agricultural enterprise) that hindered agricultural development and prosperity in africa long before and after europeans colonized . in rural subsistence communities, any source of ill health can significantly impact people's productivity, yields and agricultural output [ ] [ ] [ ] . for example, human immunodeficiency virus/aids has reduced average life expectancy in sub-saharan africa by years since , and a kenyan study found that crop production by rural subsistence-farming families dropped % after the death of a male head of household . many neglected tropical diseases (ntds) impose devastating productivity losses for affected people that can impede agricultural development from local to national to regional scales . some low-income communities appear mired in this poverty-disease trap, and thus might require substantial investment in health systems to promote the necessary agricultural and economic growth to pull them out of the povertydisease cycle [ ] [ ] [ ] [ ] [ ] . the goal of identifying potential changes to infectious disease risk associated with feeding the growing human population is not only to draw attention to this important current and future problem but to also encourage preparation for these changes by stimulating agricultural-and disease-related research, management and policy that could maximize the human health benefits of agricultural development . one urgent challenge is the problem of antibiotic, anthelmintic and pesticide resistance. analyses demonstrate that, in some cases, improvements in growth and feed consumption can be achieved by improved hygiene instead of antibiotics . indeed, all antibiotics as growth promoters were banned in the european union in . to curb antimicrobial consumption in food animal production, van boeckel et al. suggest: ( ) enforcing global regulations to cap antimicrobial use, ( ) adhering to nutritional guidelines leading to reduced meat consumption, and ( ) imposing a global user fee on veterinary antimicrobial use. in addition, as recommended by the world health organization, international organization for epizootics, and food and agriculture organization, national and international policies based on best management practices should be developed and implemented that document when and how antibiotics should be used, and agencies should be established to monitor their use, mandate reporting and enforce these policies . finally, given that host genetic variability can reduce disease risk , large-scale industrial livestock operations could add genetic variability into their artificially selected food animals in an effort to reduce epidemics and epizootics. these changes promise to secure the long-term viability of antibiotics and anthelmintics for curing diseases of humans and non-human animals. another challenge that seems surmountable is to enhance education and health literacy. not surprisingly, education has been documented as a major contributing factor to reducing infectious diseases, especially ntds, and reducing ntds can have reinforcing positive effects on the ability of humans to fight more deadly diseases, such as aids, malaria and tuberculosis . limiting ntds could also have knock-on effects for education and health literacy because ntds impede cognition, learning and school attendance . indeed, an investment of just us$ . per child for ntd control can result in the equivalent of an extra school year of education . this is likely an underestimate because of unaccounted for indirect effects of deworming on learning. a recent study revealed large cognitive gains among children who were not dewormed but had older siblings who were . thus, enhanced education and ntd control have the potential to synergistically fuel agricultural and economic development and facilitate escape from the poverty-disease trap. national and international shifts in investments could also potentially pay large dividends for nutrition, infectious disease control and poverty reduction. there is considerable evidence that the developing world will struggle to feed its growing human population because of the poverty trap of infectious disease . however, evidence also suggests that this trap might be broken through investments in health infrastructure and preventive chemotherapy [ ] [ ] [ ] . curing worm diseases has the potential added benefit of reducing nutritional needs of cured individuals by ceasing the feeding of their parasites. by reducing food demand, these drugs could also protect the environment by reducing the conversion rate of natural areas to agriculture. this, in turn, might help to curb climate change, which is one of the greatest threats to global food security and might also increase disease risk , . understanding the economic relationships among infectious disease treatment and prevention, food production, poverty and climate change in the developing world are important areas of future research. as human populations increase, there might be more pathogen spillover events that could result in new emerging human infectious diseases. historically, humans have combated emerging diseases through early detection followed by coordinated quarantine, as demonstrated by the sars outbreak in and the ebola outbreak in . we recommend continued and improved coordinated international disease surveillance, but this approach reacts to rather than prevents disease. we recommend a shift in both research and disease management efforts towards proactive management approaches. one proactive approach that has promise for preventing zoonotic disease emergence is biodiversity conservation , , but more research is needed to evaluate its effectiveness across various types of zoonotic diseases and its costs and benefits relative to more proven reactive public health interventions. ultimately, science needs a better understanding of pathogen spillover, the origins of disease emergence and post-spillover evolution so that human disease emergence can be better predicted and prevented , , . improved and diversified plant and animal genetic material could also help to simultaneously reduce hunger and disease. many efforts are already well underway; for example, introducing c traits into rice to enhance higher photosynthetic capacity in a warming world, and breeding drought-and flood-tolerant cereals, pulses and vegetables better suited to increased frequency of extreme weather events that can help sustainably enhance productivity. crops and livestock genetically adapted to resist more pests can reduce pesticide and antibiotic use. in addition, given that micronutrient deficiencies are now the most widespread source of undernutrition globally, commodity research must diversify beyond merely boosting productivity of staple cereals. far greater attention is needed on approaches that add mineral and vitamin content to foods . other, perhaps more challenging, interventions also merit attention. closing 'yield gaps' on underperforming lands, increasing cropping efficiency, eating less meat and excess per capita food consumption -which also have adverse health impacts on noninfectious diseases, such as high blood pressure, diabetes and heart disease -and reducing food waste and loss have the potential of doubling global food supplies , , , , while simultaneously allowing financial savings to be redirected towards health infrastructure to control disease. in addition, family planning, promoting female education and job market prospects, and enhancing early childhood survival have proved very effective at reducing human population growth . finally, producing food in more urban and suburban environments through vertical farming (the practice of growing produce in vertically stacked layers) also has potential to enhance food production locally, and might have reduced agrochemical and transportation costs and non-target effects relative to more traditional farming . investments into predictive models could also pay dividends. agricultural environments are complicated, multi-species systems that exhibit interactions at many levels. the complexity further increases from the tensions that arise in balancing expanding agricultural systems, social benefits and infectious disease risk. all of this complexity can overwhelm many analytical tools and experiments, which makes advances in mathematical modelling especially crucial to addressing these issues. box describes several examples of advances in mathematical modelling that illustrate their promise for analysing the links between agricultural practices and the dynamics of infectious diseases, projecting risks to future decades, influencing risks through either intentional (policies and interventions) or unintentional (continued habitat box | modelling tools for quantitative analysis of agriculture-infectious disease systems mathematical models facilitate the investigation of a complex web of interactions between agricultural systems, social dynamics and infectious diseases in the presence of the substantial nonlinearities inherent in these disparate processes. a notable example is a body of theory and analyses that have emerged to examine the links between agricultural systems and the dynamics of infectious diseases in the context of global development . the approach combines ecological and economic theory to model population subsistence and health, with the goal of examining the conditions under which subsistence and health needs of populations are met. the fundamental consumer-resource relationships that underlie the biological generation of capital via agricultural production can be formalized in ordinary differential equation models, which can be coupled with similarly structured models that estimate the gains and losses in human capital associated with population health and the increasing risk of acquiring and succumbing to infectious diseases associated with poverty. key insights emerge from such integrated models. high prevalence of infectious diseases among populations that rely heavily on subsistence, labour-intensive agriculture can reduce agricultural productivity, degrade human capital, undermine economic growth and generate conditions that systematically reinforce poverty . poverty, in turn, increases disease transmission as the lack of access to clean water and preventive care often associated with the lifestyle of the poor make them continuously exposed to parasites and pathogens embedded in the environment . malnutrition, a common consequence of poverty, may weaken immune response and increase susceptibility to disease. poor education and lack of resources lead to further reduction of health-seeking behaviour. modelling shows that the reinforcing feedback between poor health, labour productivity and capital formation triggers a cycle of increasing poverty and disease, known as disease-driven 'poverty trap' . it also shows that poverty traps are reinforced by the asymmetry between the slow pace of investment in disease protection associated with increasing capital due to agricultural development, and the relatively faster ecological changes in transformed landscape that foster disease transmission . modelling can also reveal the impact of temporary versus lasting structural interventions in changing the development trajectories of poor populations. these intrinsically quantitative and mechanistic investigations can indicate specific conditions for economic growth or resilience that can inform development strategies, via, for instance, targeted investments in agricultural development, human health or ecological conservation that may reinforce globally stable development equilibria . a final major contribution of such models is to encourage primary data collection in support of deeper understanding of system dynamics, and greater inference of underlying determinants of health and wealth. because there are strong traditions of primary data collection in population biology, epidemiology and other disciplines wherein mathematical modelling is being advanced, the expansion of model-based research on coupled agricultural-health systems is likely to yield new field data collection efforts and direct the design of experiments, which, in turn, will yield greater opportunities for model parameterization and validation. these new empirical field data are what are desperately needed to explore whether the predictions of highly stylized models are supported in settings far more complex than modelled. simulation modelling, in turn, can also permit exploration of the uncertainty intrinsic to field experiments that generate data in a specific place and time, advancing understanding of system performance under a range of feasible weather, market and other conditions not realized during the period of data collection. conversion and antibiotic use) actions, and incorporating economic and social costs of various public health or agricultural interventions. these models should help direct field experiments, which are desperately needed to parameterize and validate these models and to quantify key sources of uncertainty. ultimately, we believe 'win-win' scenarios for controlling infectious diseases, increasing agricultural productivity and improving nutrition are attainable. although this sort of integrative thinking has been historically rare because agriculture and public health have been perceived as disparate disciplines, it is beginning to slowly penetrate these distinct fields of study. for example, several agrochemicals seem to increase the risk of human schistosomiasis and agriculturally derived zoonotic pathogens, and thus researchers are actively attempting to identify agrochemicals that might 'kill two birds with one stone' , reducing crop pests and thus increasing food production while not increasing or even decreasing human pathogens [ ] [ ] [ ] . similarly, researchers are beginning to consider the introduction of prawns as biological control agents of schistosomiasis, which could simultaneously decrease disease and increase nutrition -because restored prawns can be fished, harvested and consumed without compromising their diseasecontrolling benefits , . we believe that more resourceful and creative thinking and interdisciplinary interactions (among medicine, public health, agriculture professionals, economists, anthropologists, sociologists, modellers, empiricists and wildlife biologists, in the developed and developing world) have great promise for sustainably and simultaneously improving human nutrition while controlling infectious diseases. in conclusion, we hope that by synthesizing the complex intersection of food production and human health, we have highlighted the value of increasing agricultural-and disease-related research, management and policy, maximizing the benefits of agricultural development while minimizing its adverse effects on human health and the environment, and preparing for the imminent changes driven by the billion people expected to inhabit earth by . global trends in emerging infectious diseases nigeria's invisible crisis hunger in africa: the link between unhealthy people and unhealthy soils maternal and child 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zoonotic pathogens and fecal indicator bacteria agrochemicals increase risk of human schistosomiasis by supporting higher densities of intermediate hosts modelled effects of prawn aquaculture on poverty alleviation and schistosomiasis control genetic dissimilarity between mates, but not male heterozygosity, influences divorce in schistosomes disease ecology, health and the environment: a framework to account for ecological and socio-economic drivers in the control of neglected tropical diseases the rise and fall of malaria under land-use change in frontier regions funds were provided by grants to j.r.r. from the national science foundation j.r.r. developed the idea and wrote most of the paper. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . / s - - - . correspondence should be addressed to j.r.r.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -e y f authors: kastritis, efstathios; kitas, george d.; vassilopoulos, dimitrios; giannopoulos, georgios; dimopoulos, meletios a.; sfikakis, petros p. title: systemic autoimmune diseases, anti-rheumatic therapies, covid- infection risk and patient outcomes date: - - journal: rheumatol int doi: . /s - - -x sha: doc_id: cord_uid: e y f as of june th about . million individuals have tested positive for, and more than , have died due to covid- . in this review we outline the pathophysiology that underpins the potential use of anti-rheumatic therapies for severe covid- infection and summarize the current evidence regarding the risk and outcome of covid- in patients with systemic autoimmune diseases. thus far there is no convincing evidence that any disease-modifying anti-rheumatic drug (conventional synthetic, biologic or targeted synthetic) including hydroxychloroquine, may protect against severe covid- infection; answers about their possible usefulness in the management of the cytokine storm associated with severe covid- infection will only arise from ongoing randomized controlled trials. evidence on covid- risk and outcome in patients with systemic autoimmune diseases is extremely limited; thus, any conclusions would be unsafe and should be seen with great caution. at present, the risk and severity (hospitalization, intensive care unit admission and death) of covid- infection in people with autoimmune diseases do not appear particularly dissimilar to the general population, with the possible exception of hospitalization in patients exposed to high glucocorticoid doses. at this stage it is impossible to draw any conclusions for differences in covid- risk and outcome between different autoimmune diseases and between the various immunomodulatory therapies used for them. more research in the field is obviously required, including as a minimum careful and systematic epidemiology and appropriately controlled clinical trials. autoimmune diseases are characterized by intrinsic immune alterations, which may lead to chronic inflammation in multiple organ systems. the diseases themselves and many of their treatments are associated with increased risk for severe infections [ ] [ ] [ ] [ ] . detailed research on the pathogenesis of these diseases has led to the development of targeted therapies, aimed mainly at cytokines and cells, which play key roles in the chronic inflammatory process. during the covid- pandemic, the risk and outcomes of patients with autoimmune diseases have become a cause of concern and require proper investigation. on the one hand, this population is, by virtue of the disease and its treatment, potentially vulnerable to suffer severely from covid- ; on the other hand, many of these patients have been and continue to be exposed to immune modifying therapies which may have the potential to treat some features and improve outcomes of covid- itself. the initial link came from observations indicating that severe covid- infection, manifested by acute respiratory distress syndrome (ards) and/or multi-organ failure, is associated in a proportion of patients with a "cytokine storm" [ ] [ ] [ ] [ ] . it follows then that certain cytokine-blocking agents may be beneficial in this scenario [ , ] and several of them are presently being tested in clinical trials internationally. in the meantime, data on the risks and outcomes of covid- infection in patients with autoimmune diseases who are already receiving such agents could be informative but remain scarce [ ] [ ] [ ] . in this narrative review, we outline the pathophysiologic mechanisms that underpin the potential utility of anti-rheumatic therapies in the context of severe covid- infection and summarize the current evidence regarding the risks and outcomes of covid- infection in patients with systemic autoimmune diseases. driven by data indicating that an immune overreaction, a "cytokine storm", may be a critical component of severe covid- infection [ ] [ ] [ ] , it has been hypothesized that the use of drugs blocking specific cytokines could be beneficial for some infected patients [ , ] . previous observations in patients infected with different viruses such as the influenza virus [ , ] and other coronaviruses like the sars-cov and mers-cov [ ] suggested that an exaggerated host immune response could participate in tissue injury leading to serious outcomes. data regarding the exact nature of host immune responses' against covid- are now starting to emerge [ ] . as is the case with other respiratory viruses, the initial innate antiviral immune responses in the respiratory tract are crucial for effective control of the infection. in the majority of cases (> %), these responses are adequate, leading to viral clearance and clinical improvement without significant associated tissue (mainly lung) injury. data regarding immune responses in recovered patients following mild or moderate illness are limited so far. on the other hand, in a proportion of patients (~ %), severe lung injury is observed which may lead in approximately % of patients to ards, respiratory and multi-organ failure or even death (~ %). in such cases, a dysregulated immune response has been described, usually during the nd week of infection, characterized more often by t cell lymphopenia (both cd and cd cells) [ ] , enhanced production and release of pro-inflammatory cytokines such as interleukin- (il- ), tumor necrosis factor-a (tnf-a), il- and g-csf [ ] [ ] [ ] ] , decreased production of antiviral type i and iii interferons [ ] and t cell exhaustion [ ] .this "hyperinflammatory state" which is being referred to as "cytokine storm" shares certain clinical (high fever), laboratory (increased c-reactive protein-crp, ferritin, serum il- and d-dimer levels) and immunological (macrophage and t cell activation) features with the macrophage activation syndrome or secondary hemophagocytic lymphohistiocytosis (hlh) seen in a subset of patients with infections (bacterial or viral), auto-immune or auto-inflammatory diseases (such as systemic onset juvenile idiopathic arthritis, adult still's disease and systemic lupus erythematosus) and after cart cell therapy in patients with leukemia or lymphoma [ ] [ ] [ ] . such observations underpinned the hypothesis that targeting pro-inflammatory cytokines with specific drugs used in autoimmune diseases may control the hyperinflammatory state in covid- infection; however, this approach also raises concerns for a blunted response to viral invasion [ ] . targeted anti-cytokine therapies neutralize individual mediators of inflammation rather than causing a complete shutdown of the innate and adaptive immune responses. hence, the risk from specific pathogens may differ between therapies. the most commonly used biologic disease modifying anti-rheumatic drugs (bdmards), i.e. the anti-tnf agents (infliximab, etanercept, adalimumab, golimumab and certolizumab) increase the risk of serious bacterial infections [ ] while there is some limited evidence that the may also increase the risk for influenza infection [ ] . humoral immunity may be affected more by some drugs (such as the anti-cd monoclonal antibody, rituximab) than other targeted anti-cytokine therapies, such as anti-il- ,-il- / il- or -il- a, which have not been associated with a significant increase in the risk of viral infections. so far there have been few studies assessing the role of cytokine-targeted (mainly il- and il- ) therapies in patients with covid- infection [ ] [ ] [ ] [ ] [ ] but none of them were randomized controlled studies. however, drugs with the potential to inhibit multiple cytokines may be riskier. these include the more recently developed targeted synthetic (ts)dmards, mainly the jak inhibitors. these drugs target jak and jak , affecting the function of several cytokines that are involved in antiviral responses (including type i interferons, il- , il- , il- and ifnγ) and could theoretically inhibit viral clearance [ ] . the flip side of this is that jak inhibition may block sars-cov- entry and pro-inflammatory cytokines that depend on jak signaling (including il- , il- and gm-csf) [ , ] . the majority of patients with autoimmune diseases, however, still receive the older conventional synthetic (cs) dmards and/or variable doses of glucocorticoids that may also have significant effects in the context of covid- infection. hydroxychloroquine has been suggested as a potential therapeutic agent for prophylaxis or therapy of covid- infection but the results so far have been conflicting [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there is concern regarding the use of moderate-to high-dose glucocorticoids in covid- infection [ , ] and so far these are only recommended for mechanically ventilated patients with ards [ ] . there are no data so far for other commonly used csdmards, such as methotrexate, azathioprine, cyclosporine and mycophenolate mofetil. in summary, whereas on the one hand b-or ts-dmards may theoretically increase the risk and severity of viral infections, there are, on the other hand, mechanistic and clinical observations suggesting that they may be useful in controlling specific aspects of the immune overreaction and thus improve the outcomes of severe covid- infection. indicative answers for both questions may arise from wellconducted observational studies in patients with autoimmune diseases receiving dmard therapy compared with the general population. a definitive answer for the latter question could only be provided as a result of appropriately conducted randomized controlled trials (rcts) in patients with severe covid- infection [ ] . a literature search in the electronic databases pubmed, scopus and web of science was performed until may th, using the terms "covid- " and "rheumatic diseases", "disease-modifying anti-rheumatic drugs", "biologics", "rheumatoid arthritis", "systemic lupus erythematosus", "spondyloarthropathies", "psoriatic arthritis", "ankylosing spondylitis", "vasculitis", "scleroderma", "jak inhibitors", glucocorticoids" or "corticosteroids" in order to identify relevant publications of patients with rheumatic diseases and covid- infection. articles were initially selected by their title and abstract and then the full text was searched for the appropriate relevant content. references from the selected articles were also manually searched for relevant articles. in table , we summarize data from all published studies and case series which described covid- infection in patients with autoimmune diseases. there is significant heterogeneity both in the general characteristics as well as the autoimmune disease characteristics and treatments of the patients included in these cohorts. the largest series with detailed data so far is from new york [ ] . it reports data on the outcome of patients with autoimmune/inflammatory diseases who had either confirmed ( patients) or suspected ( patients) covid- infection (table ) [ ] .the majority were females ( %) at a rather young age (mean years). most common diagnoses included spondyloarthritis (spa) and/or psoriasis (pso), inflammatory bowel diseases (ibd), rheumatoid arthritis (ra) or a combination of these diseases. compared to other patient cohorts, comorbidities were rather uncommon (hypertension: %, copd: %, diabetes: %). most patients were on biologics or jak inhibitors ( %) with few receiving glucocorticoids ( %). hospitalization was required for ( %), icu admission or mechanical ventilation in patient ( %) while there was only death at arrival in the er ( %). the rate for hospitalization was not different from that of the general population of new york ( %). an observational study from france monitored the clinical course of covid- infection in patients with systemic lupus erythematosus (sle) who were on long-term hydroxychloroquine therapy (median . years) ( table ) [ ] . comorbidities were common in this group including obesity ( %) and chronic kidney disease ( %). all but one patient had clinically quiescent sle, with a sledai score equal to . twelve ( %) patients were receiving glucocorticoids (usually at doses < mg/day) and seven ( %) were receiving additional immunomodulatory drugs. hydroxychloroquine and glucocorticoids were maintained at the same dose, while immunosuppressive drugs were discontinued or reduced. fourteen patients required hospitalization ( %), half of them (n = ) in the icu and finally patients ( %) died. although this study is also limited by small numbers, the authors concluded that hydroxychloroquine does not appear to prevent severe covid- infection. regarding the severity of covid- in sle patients, no safe conclusion can be drawn, but the high incidence of other comorbidities may confound these observations. in the relatively early phase of the outbreak in n. italy, monti et al. performed a survey in patients with chronic arthritis in their outpatient clinic to investigate potential infections with covid- or high-risk contacts (table ) [ ] . the authors gathered information from patients ( % with ra, % with spa, % treated with anti-tnfs, % with other bdmards and % with tsdmards). they identified confirmed and suspected covid- infections while another reported high-risk contacts but remained asymptomatic for the -week observation period. three patients (one with confirmed and two with suspected covid- ) were on hydroxychloroquine. all patients with symptoms of infection had their anti-rheumatic therapy temporarily withdrawn at the time of symptom onset. no significant relapses of the rheumatic disease occurred;' none of the patients with a confirmed or highly probable covid- developed severe respiratory complications or died and only one patient with confirmed infection, aged , required admission to hospital and received low-flow oxygen supplementation for a few days. all patients with confirmed covid- received at least one antibiotic course, and the hospitalized patient also received antiviral therapy and hydroxychloroquine. the authors also reported that among patients admitted for severe covid- during one month at their hospital, which was a referral center for covid- , none was receiving either bdmards or tsd-mards [ ] . an initiative driven by the global rheumatology alliance aims to continuously collect data internationally for patients with rheumatic diseases infected with covid- . initially data for patients were reported [ ] and more recently its updated form containing data for patients ( with confirmed and with presumptive diagnosis of covid- ) were published (table ) [ ] . the most common diseases were ra ( %), spa ( %), sle ( %) and other diseases ( %, including vasculitis, sjogren's syndrome etc.). medications included csdmards in %, bdmards in %, tsdmards in % and glucocorticoids in %. common comorbidities included hypertension in %, lung disease in %, diabetes in %, cardiovascular disease in % and chronic renal insufficiency in %. of the infected patients, % were admitted to hospital and % died. by multivariable logistic regression analysis, age > years (odds ratio-or . ), certain comorbidities (hypertension or cardiovascular diseases, lung disease, diabetes and chronic renal disease, ors . - . ) and high glucocorticoid dose (≥ mg/day, or . ) were associated with a higher risk for hospitalization, on the other hand, previous use of anti-tnf agents was associated with a lower hospitalization risk (or . ) [ ] . these results should be interpreted with caution since selection bias may have played a significant role here with inclusion of more severe cases. case reports of patients with autoimmune/rheumatic diseases who were infected with covid- are also table anti-inflammatory treatment and outcomes of patients with confirmed or suspected covid- infection n number, ra rheumatoid arthritis, spa spondyloarthritis, psa psoriatic arthritis, as ankylosing spondylitis, pso psoriasis, ibd inflammatory bowel disease, uc ulcerative colitis, cd crohn's disease, sle systemic lupus erythematosus, bdmards biologic disease modifying anti-rheumatic drugs, il- interleukin- , csdmards conventional synthetic dmards, mmf mycophenolate mofetil, nr not reported a some patients had more than disease haberman et al. [ ] mathian et al. [ ] monti et al. [ ] global rheumatology alliance [ ] total n (%) increasingly appearing in the literature. a brief report from italy described the incidence of covid-infection and outcome among patients with autoimmune/inflammatory diseases [ ] . most patients, ~ % were receiving csd-mards, % bdmards, while % were also receiving relatively low dose glucocorticoids (average . mg of prednisolone daily). only one registered patient ( . %) developed a confirmed covid- infection; this incidence was similar to that of covid- infection in the area ( . % at that time). an interesting case of a -year-old woman with granulomatosis with polyangiitis for years, who developed severe covid- infection while on therapy with rituximab and glucocorticoids has been reported [ ] . the patient had ear, nose and throat, orbital, lung, joint and skin involvement, anti-proteinase (pr ) antibodies and had been heavily pretreated (with cyclophosphamide-total dose of g, anti-tnf, mycophenolate mofetil, methotrexate, leflunomide, rituximab and steroids). few months before the pandemic she received four infusions of weekly rituximab ( mg/m ) and a maintenance dose of rituximab ( mg) was administered in march , while still under prednisone mg daily. she was diagnosed with covid- having presented with bilateral interstitial pneumonia days after her last rituximab dose. she received lopinavir/ritonavir for days but required mechanical ventilation, for ards, days after symptom onset. hydroxychloroquine was started following intubation and the authors report rapid clinical improvement with no need for mechanical ventilation after days and no need for oxygen supplementation after days, while nasopharyngeal rt-pcr became negative and the patient was discharged. the authors hypothesized that glucocorticoids and rituximab may have limited the cytokine storm and delayed the worsening of clinical status of the patient. a very brief discussion of a few cases of covid- infection in patients with autoimmune/inflammatory diseases from the uk has also been published, without any details reported [ ] . these cases include a -year-old male with crohn's disease on infliximab with mild disease who did not require hospitalization, a female with ulcerative colitis (of unspecified age) on tofacitinib and steroids who also did not require hospitalization and a -year-old female with crohn's disease on adalimumab who was hospitalized but did not require mechanical ventilation. as of june th about . million individuals have tested positive for, and more than , have died from covid- [ ] . despite these numbers, data regarding the risk of infection and the severity and outcome of the disease in patients with systemic autoimmune diseases remain extremely limited. the estimated prevalence of systemic autoimmune/inflammatory diseases in the general population ranges between and % [ ] . assuming an equal risk for covid- infection and death with the general population, one would have expected at least deaths of such patients so far. were data on them to have been collected systematically through international collaborations, these are useful numbers for detailed analyses of risk with potentially powerful clinical messages. one should take into account though that the percentages of patients with systemic autoimmune disease who receive immunosuppressives may be lower within the elderly population which is more vulnerable to severe outcomes. moreover, since males seem to have higher death rates than females [ ] , the female preponderance in systemic autoimmunity could be a protective factor. based on the available data at present, any conclusions would be unsafe and should be seen with great caution. analysis of the published data presented in this review suggest the following: (a) there is thus far no convincing evidence that any of the dmards (conventional synthetic, biologic or targeted synthetic) including hydroxychloroquine, used in the management of systemic autoimmune diseases may protect against severe covid- infection; (b) answers about the possible usefulness of dmards in the management of the cytokine storm and its sequelae during severe covid- infection will only arise from the currently ongoing rcts; (c) the risk of covid- infection in patients with systemic autoimmune diseases does not seem to be much higher than in the general population with similar comorbidities [ , ] ; (d) the severity of covid- infection (in terms of requirement for hospitalization, icu admission or death) does not appear to be particularly dissimilar in this population compared with the general population, although data from large series of patients are not available yet; (e) it is impossible at this stage to draw any conclusions for differences in covid- risk and outcome between different autoimmune diseases and between the various immunomodulatory therapies used for them. more research in the field is obviously required, including as a minimum careful and systematic epidemiology, basic laboratory research and well-designed and appropriately controlled clinical trials. author contributions all authors: ( ) contributed to the acquisition, analysis, or interpretation of data, ( ) drafted and critically revised the manuscript, ( ) approved the final version of the manuscript. 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coronavirus disease : open label, randomised controlled trial infectious diseases society of america guidelines on the treatment and management of patients with covid- clinical evidence does not support corticosteroid treatment for -ncov lung injury surviving sepsis campaign: guidelines on the management of critically ill adults american college of rheumatology guidance for the management of adult patients with rheumatic disease during the covid- pandemic covid- in immune-mediated inflammatory diseases-case series from new york clinical course of coronavirus disease (covid- ) in a series of patients with systemic lupus erythematosus under long-term treatment with hydroxychloroquine montecucco c ( ) clinical course of covid- in a series of patients with chronic arthritis treated with immunosuppressive targeted therapies rheumatic disease and covid- : initial data from the covid- global rheumatology alliance provider registries characteristics associated with hospitalisation for covid- in people with rheumatic disease: data from the covid- global rheumatology alliance physicianreported registry incidence and clinical course of covid- in patients with connective tissue diseases: a descriptive observational analysis rituximab for granulomatosis with polyangiitis in the pandemic of covid- : lessons from a case with severe pneumonia a ( ) identifying rheumatic disease patients at high risk and requiring shielding during the covid- pandemic who coronavirus disease geoepidemiology of autoimmune rheumatic diseases covid- pandemic: is a gender-defined dosage effect responsible for the high mortality rate among males conflict of interest none for this manuscript, for all authors. key: cord- - k g khw authors: tresker, steven title: a typology of clinical conditions date: - - journal: stud hist philos biol biomed sci doi: . /j.shpsc. . sha: doc_id: cord_uid: k g khw in the philosophy of medicine, great attention has been paid to defining disease, yet less attention has been paid to the classification of clinical conditions. these include conditions that look like diseases but are not; conditions that are diseases but that (currently) have no diagnostic criteria; and other types, including those relating to risk for disease. i present a typology of clinical conditions by examining factors important for characterizing clinical conditions. by attending to the types of clinical conditions possible on the basis of these key factors (symptomaticity, dysfunction, and the meeting of diagnostic criteria), i draw attention to how diseases and other clinical conditions as currently classified can be better categorized, highlighting the issues pertaining to certain typology categories. through detailed analysis of a wide variety of clinical examples, including alzheimer disease as a test case, i show how nosology, research, and decisions about diagnostic criteria should include normative as well as naturalistically describable factors. clinical medicine is replete with examples of diseases where there is a disparity between the diagnostic criteria used to ascertain the presence of the disease and the pathophysiological dysfunction of the disease. clinicians also study and treat a number of non-diseases, such as pregnancy, as well as conditions straddling the line between disease and risk for disease (such as the early stages of atherosclerosis). as christopher boorse wrote in ( - ) , "the distinction between normal variation and underlying disease is one of the most important features of medical theory, though in practice it is often hard to draw because so much clinical evidence is gross output." given that most clinical evidence is indeed gross output (blood glucose levels, for example), whereby laboratory tests at most only offer a proxy for or rough indication of the dysfunction present (e.g., defects in pancreatic beta cells, adipose tissue, the incretin system), ascertaining the presence of disease is not always as straightforward as it may seem. this is especially true for asymptomatic conditions, but also for conditions in which the dysfunction is mild or difficult to discern. although there are multiple nosologies of disease, there is less discussion in the philosophical or medical literature on the classification of clinical conditions (with some exceptions, such as kline, ; engelhardt, , ch. ; caplan et al., ; schwartz, schwartz, , a doust et al., ; and hofmann, , ) . clinical conditions include conditions that look like diseases but are not; conditions that are diseases but that (currently) have no diagnostic criteria; and other types, including those relating to risk for disease (-blinded for review-, , figure ). this paper is meant to fill that gap by examining factors important for characterizing clinical conditions, to arrive at a typology of clinical conditions. through analysis of a wide variety of detailed clinical examples, i show how diagnostic criteria (e.g., those promulgated by medical society guidelines for use in clinical practice). this highlights the fact that for many conditions the existence of pathophysiology is assumed on the basis of the presence of symptoms. yet recent calls for revisions in alzheimer disease (ad) and huntington disease diagnostic criteria advocate inclusion of ad and huntington disease biomarkers (jack et al., ; reilmann et al., ) . this underscores the notion that there is not always a close correspondence between ad and huntington disease clinical symptoms and their respective pathophysiological signs. indeed, pathological changes can be present in the brains of people with ad years before the manifestation of symptoms (jack et al., , ) , whereas people with huntington disease possess the mutant gene from birth and often undergo a long presymptomatic phase (reilmann et al., ) . how these and other conditions are defined (through diagnostic criteria) has profound implications for clinical practice, research, and society (regarding insurance, stigma, and other effects of being labeled with a clinical condition). a chief goal of this paper is to explain how normative considerations determine which typology categories a condition can be in and therefore whether biological dysfunction or symptoms are more important for defining a disease. the usefulness of the typology may lie in both identifying main distinguishing features of clinical conditions but also in using the distinctions to think through the implications of different classifications. dysfunction dysfunction as a necessary feature of disease has been extensively defended by boorse and others (e.g., schwartz, b; hausman, hausman, , , including those advocating so-called hybrid conceptions of disease (e.g., stegenga, ; wakefield, ) , so i will not repeat these arguments here. since diseases should be included in any typology of clinical conditions meant to reflect clinical practice and medical science, dysfunction accordingly must be a feature of that typology. a diagnosed condition does not automatically mean dysfunction is present, and undiagnosed disease does not necessarily mean there is no pathology present. improved diagnostic criteria could better capture the people who truly have the physiological dysfunction constitutive of a disease. however, as will be discussed later, normative considerations and not any intrinsic feature of the bst or the typology determine whether this should be done. the disparity between the diagnostic criteria used to ascertain the presence of disease and the underlying disease-defining dysfunction is known to practicing clinicians and medical researchers. for example, in acute kidney injury (aki) it is clear that the current diagnostic tools (such as serum creatinine concentrations) do not accurately reflect the underlying pathophysiology: indeed, the arbitrariness of the current cut-off of oliguria has been recognized in the field (md ralib et al., ; ostermann, ). yet because kidney biopsy is risky and impractical, more specific and sensitive diagnostic tools are needed than those commonly used. aki has multiple etiologies and proper diagnostic tools should distinguish these. as will be discussed below, similar situations exist for other diseases, including chronic ones. the typology can make sense of such situations, as well as nosological developments, and thus provides a conceptual framework for thinking about what may be readily apparent to medical professionals, but also what may be contested territory. on the bst asymptomatic disease is still disease because there is statistically subnormal physiological function. this dysfunction just does not ( on the typology they would still have a clinical condition, category e or g. another important factor for a typology of clinical conditions is the meeting of diagnostic criteria. this is because this factor determines whether a person is considered to have a disease (even if there is not always a correspondence between whether the person meets the bst's criteria for having a disease). diagnostic criteria comprise signs, symptoms, or both. they are how clinicians diagnose a condition, whether the criteria are implicit or explicitly stated in guidelines. in practice, diagnoses may proceed on the basis of criteria different from those listed in guidelines, such as a physician practicing when the american college of rheumatology's criteria for the diagnosis of fibromyalgia were followed who might have diagnosed a patient with fibromyalgia even in the absence of tender points, despite the requirement for their presence. however, this is not a problem for the typology because criteria are still being used and these criteria necessarily involve certain symptoms or signs. diagnostic methods (like a gold standard, say biopsy) are not the same as diagnostic criteria. the latter may encapsulate the former. a test itself is not a diagnostic criterion because it is a means by which a diagnostic criterion can be assessed as being met or not, typically by a cut-off value or the presence or absence of a feature (e.g., cancerous cells). there are many diseases for which the pathology is too difficult to assess, so the diagnostic criteria for the disease consist solely of symptoms or behavioral signs. meeting diagnostic criteria is how diseases are identified in clinical practice. in some cases this involves specific pathology, as identified through whatever tests are available that could indicate the key dysfunction is present. in other cases this might solely involve symptoms, which for some conditions, at least some of the time (e.g., ad), provide a strong indication the key biological dysfunction is present, whereas for other conditions (e.g., depression) sometimes only tenuously (if at all) link to pathology. the fact that most of the conditions listed in the diagnostic and statistical manual of mental disorders th edition (dsm- ; american psychiatric association, ) are based on behavioral criteria (i.e., polythetic symptom complexes) emphasizes the importance of symptoms to the diagnostic criteria of many conditions. it also shows how for some of the dsm- conditions biological dysfunction could be present, whereas for others absent, a point also made by olbert and gala ( ) on their way to arguing for the distinct role played by symptomatology in mental versus somatic disorders. the intriguing example they give involves the difficulty believing a person could have a mental disorder without that person experiencing that disorder's defining symptoms (e.g., depression diagnosed by biomarkers in a patient without any depression symptoms; olbert and gala, , ) . as will be discussed for ad, any incredulity this might engender depends on the condition; ad, like depression, is listed in the dsm- , but unlike depression has biomarkers available that can identify ad's neuropathological signature even in a patient not yet exhibiting dementia. this raises the possibility of asymptomatic diagnosis, something conceivable for a neurological condition but seemingly bizarre for a psychiatric one. many diseases go undiagnosed. the typology shows that there could be disease even if the most conclusive test is not performed; consider herpes sufferers before the tzanck smear was available. a person with painful pustules around their mouth could have an acne or herpes simplex outbreak. confirming herpes with the tzanck smear does not mean the disease was not present prior to performing the test. indeed, whether a test accurately identifies herpes simplex as the cause is a function of its sensitivity and specificity, which is why the tzanck smear is now rarely used in some locales given that polymerase chain reaction tests are available. in a clinic a person is diagnosed with a disease or not. if yes, that diagnosis might not really represent disease (at least based on the presence of dysfunction), or if no then the person still has a clinical condition, which is partly known through the presence of certain symptoms. some diagnostic criteria do a better job of identifying diseases than others. diagnostic criteria for diseases are not static. they change over time, as when an expert group revises diagnostic criteria. this can be of profound significance for the conceptualization of a condition. table illustrates this with respect to ad and how its typology category can change depending on the diagnostic criteria. diagnostic criteria influence a multitude of factors; in the united states a diagnosis is required for insurance reimbursement; diagnosis guides treatment; diagnosis labels a person, for better or worse; and diagnostic criteria provide the basis for identification of cases of a disease, enabling research on specific diseases (exemplified by the inclusion/exclusion criteria of clinical trials, which often list specific diagnoses). take the following factors as the basis for a typology of clinical conditions, not all of which (according to the bst) are diseases. because each factor is dichotomous, eight ( ) categories result: a. asymptomatic person without dysfunction and not meeting diagnostic criteria for a disease: this is a healthy person. this could be a risk-based condition (called a "risk-based disease" by schwartz [ ] when there is no dysfunction), such as osteoporosis, hypertension, hypercholesterolemia, or type diabetes. this is a person with disease, not exhibiting symptoms, who remains undiagnosed. examples include a person in the early stages or with a silent state of myasthenia gravis, type diabetes, ad, huntington disease, or any other disease wherein the pathophysiological processes begin years in advance of clinical presentation. for example, a person could have significant neurofibrillary tangles or aβ plaques without having symptoms of ad. compensated, controlled, or asymptomatic disease, whether as the result of treatment or the natural course of the disease. examples include multiple sclerosis or crohn disease in remission, or an infectious disease carrier. disease: symptoms being part of normal variation, such as in a cough, bereavement, fatigue after work, or some cases of enuresis. this is a person meeting diagnostic criteria that rely on the presence of symptoms, but who does not actually have a disease. many of the disorders listed in the dsm- could fall under this category. diabetes, whether or not it is evident to its bearer, consists of an unusual deficiency in insulin secretion and therefore in sugar metabolism. hepatic cirrhosis, nephritis, pancreatic cancer, and countless other pieces of local pathology can progress for a long time without depressing gross functions enough to be detected. they do, however, make standard tissue functions decline and fail in the affected part of the organ. (boorse, , ) similarly, there are diseases (categories c, d, g, and h) for which asymptomaticity represents pathophysiological variability, in the sense that some cases of the disease result in symptoms, whereas others result in asymptomatic disease, but disease nonetheless (as assessed by signs, such as results of laboratory tests). including infectious-disease carriers in category d reflects the fact that there is dysfunction even though a carrier may by asymptomatic. on the bst and by the lights of the typology such carriers would be diseased. however, wakefield argues that an asymptomatic infectious disease carrier is not diseased ( , ) . he offers the case of typhoid mary, acknowledging that dysfunction was present, but denying that she was diseased because there was no symptomatic harm to her (although there was harm to others). boorse recognizes that wakefield's view of disorder as a harmful dysfunction is similar to his own view of therapeutic abnormality ( , ) : "with dysfunction analyzing the "disease" part and harm analyzing the "clinical" part." however, not all symptoms cause harm. (caplan et al., ) is not a disease because it involves no dysfunction, yet the decision to "treat" an absconding slave would definitely involve a normative consideration-in this case, quite an evil one. an advantage of the bst is that it highlights the need for treatment for certain chronic, asymptomatic diseases well ahead of when they become clinically apparent. as boorse stated (writing in [ ] , but still relevant for today), "current disease classifications, now grounded in a century of scientific physiology and pathology, reveal a converse danger in the clinical approach: it favors acute symptomatic disorders over more serious chronic ones." by accounting for asymptomatic disease, the bst offers a conceptually robust basis for evaluating and guiding how diagnostic criteria are set for diseases. for example, it points to the presence of diseases for which some patients are diagnosed even if they do not have any dysfunction (category b, and possibly category f if the symptoms present are not reflective of any dysfunction). these include the risk-based conditions, discussed next. some conditions straddle the line between disease and risk for disease (doust et al., ; rogers and walker, ) . peter h. schwartz ( ) identifies a number of diseaseshypercholesterolemia, type diabetes, obesity, hypertension, osteoporosis-where a person could meet the diagnostic criteria for the disease yet not have an underlying dysfunction. instead, they could be at risk for more serious manifestations of the disease. a risk-based condition could thus be defined as a clinical condition for which a person could meet diagnostic criteria for a "disease" yet for which in some cases no dysfunction is present (it is not a disease by the bst). the diagnostic criteria are better seen as biomarkers of risk for more severe disease or other diseases. what distinguishes risk-based conditions from non-risk-based conditions is the lack of dysfunction present in risk-based conditions for some patients meeting the diagnostic criteria. typically, risk-based conditions are those for which overdiagnosis is a problem. this is because some cases of risk-based conditions should be "better understood as risk factors for developing disease in the future" (schwartz, , ) . although all the risk-based conditions schwartz identifies are continuous-variable "diseases" , not all continuous-variable "diseases" are risk-based conditions. hypothyroidism, for example, presents similar issues as with other continuous-variable "diseases", but because the range is so narrow for which dysfunction is present, there may be fewer cases of overdiagnosis than in type diabetes or hypertension. moreover, mild elevation of thyroid stimulating hormone might not always reflect dysfunction but could be a sign of normal aging (surks and hollowell, ) . schwartz ( a) equates pathology with dysfunction, which is different than suboptimal or below-average function. type diabetes, for example, becomes a disease when dysfunction is present but is still a disease for many hba c levels (i.e., the high ones), just not those that do not involve any underlying dysfunction. risk-based "diseases" as schwartz terms them is thus a misnomer on the typology (they should be called "risk-based conditions") because it is only on current expanded-boundary diagnostic criteria that they are (wrongly according to schwartz) considered diseases. risk-based conditions show how a condition can straddle the boundary between one and another typology category (b and d). when diagnostic criteria lower the threshold by which a person is considered to have a disease, more people who are not diseased may be classified as being diseased, resulting in overdiagnosis and the medicalization of normal variation. Élodie giroux ( , ) charges schwartz with not paying enough attention to the notion of functional efficiency with respect to risk-based conditions, and thinks he is unable to clarify whether a risk-based condition is normal or pathological. she thinks that medium or severe hypertension in itself is probably not a dysfunction. schwartz ( ) considers these levels dysfunctional, in contrast to mild hypertension which he considers to not involve dysfunction. giroux's ( , ) critique is motivated by the divergence between blood pressure as a quantitative variable representing the level of functional efficiency and as a direct measurement of dysfunction of the circulatory system. because she is correct that blood pressure is not a direct measurement of the dysfunction (i.e., functional efficiency) of the circulatory system (in most cases at least), she cannot find a basis for saying that any degree of hypertension represents dysfunction. indeed, hypertension, as well as blood glucose levels, could be indications of disease, although they are also potentially pathogenic themselves (such as with glucotoxicity or when blood pressure gets really high, as pointed out by boorse [ ] ). nonetheless, it is possible that there is dysfunction not only in medium or severe hypertension but even in mild hypertension. the dysfunction may just not rise to the level of clinically apparent dysfunction. there may still be dysfunction (such as at the level of the cell or molecule), that is undetectable with current clinical and diagnostic tools. but as boorse ( ) regarding ad, if ad were diagnosed solely on the basis of symptoms and not neuropathology, a person presenting with ad symptoms could not fall under category c. this is because category c involves asymptomatic people with dysfunction who do not meet diagnostic criteria for a disease. were ad's diagnostic criteria to include laboratory evidence of ad neuropathology, however, then an asymptomatic person with ad could fall under category c if they were to not meet all the diagnostic criteria. this could happen if the person were to possess ad pathology not detectable by current technology (i.e., they still have dysfunction) or if they were to lack some ad symptoms that might still be required for an ad diagnosis (i.e., alongside the neuropathological evidence). importantly, this clinically unapparent dysfunction is not the same as risk for frank dysfunction. it could increase such risk, however, in the same way as any disease could increase the risk for another disease. this clinically unapparent dysfunction is still disease though-disease that current diagnostic tools just have not caught up with yet. for example, years ago a person with the huntington disease expansion still had huntington disease even if unaware of their carrier status and not yet symptomatic. a similar situation exists for familial ad, because people with such genetic mutations are virtually guaranteed to develop dementia, the symptomatic hallmark of ad (dubois et al., , ). yet ad presents an even more intriguing test case for the typology than huntington disease. this is because most cases of ad are of the sporadic type (dubois et al., , ) and not all ad-biomarker-positive people develop cognitive impairment (jack et al., , ) . nonetheless, recently proposed research diagnostic criteria allow for an ad diagnosis based entirely on biomarker findings (dubois et al., ; jack et al., ) . although these new diagnostic criteria are intended for research, some clinicians around the world do use biomarkers as an aid to diagnosis (frisoni et al., ) . this means some addiagnosed people could fall under category b or d, depending upon whether biological dysfunction is present. the implications of this are discussed next. ad is a complex, multicausal condition clinically involving mild cognitive impairment (mci) or dementia, and neuropathologically involving the presence of neurofibrillary tangles and senile plaques composed of aβ (vinters, ) . ad offers an example of how the typology can help understand factors important for research, treatment, diagnosis, and nosology. apr , first, to know whether there is a theoretical disease present per the bst it is necessary to know the population distribution of the functional efficiency of a physiological part or process in a reference class (boorse, ) . taking the elderly as a reference class, the pathophysiological hallmarks of ad, whether at the symptomatic or presymptomatic stage, would only count as theoretical disease if they are not commonly found in the appropriate reference class. for the purposes of this discussion i will assume this to be true, although given the high prevalence of ad pathology in individuals aged greater than years (jack et al., , ) with only autopsy providing a definite confirmation. however, the existence of individuals with ad pathology but no ad symptoms, and ad symptoms but no ad pathology (jack et al., , ) , called into question this presumption. although autopsy can confirm an ad diagnosis, this is rarely done now given the availability of in vivo biomarkers previously validated against postmortem ad pathology. moreover, biomarker-based diagnostic criteria have recently been proposed for ad, enabling diagnosis independent of clinical symptoms, such as in the predementia phases. brain biopsy is rarely performed to diagnose ad because it has low diagnostic yield-not only because it is too difficult and risky (not least of which from general-anesthesia sequelae) but because it is not sensitive and specific enough (venneti et al., ; warren et al., ) . in the last few years, with the advent of more sensitive biomarkers ( however, a new preclinical stage was defined that could be diagnosed mostly or entirely on the basis of biomarkers (though in contrast to mci and dementia such diagnoses were to be used for research purposes only as biomarkers continued to develop [jack et al., ] ). with the recognition that early treatment, even before clinical onset, could result in a greater chance to arrest the disease than with later treatment (langbaum et al., , ; graham et al., , ; jack et al., , ; dubois et al., , ) , there have been intense efforts to use biomarkers to identify people, even if currently asymptomatic, who would likely develop dementia or prodromal ad. this has resulted in a tension within ad diagnostic criteria (or, alternately put, a lack of consensus in the ad field as to what the diagnostic criteria should be), between symptom/neuropsychological-testing-based criteria and biomarker-based criteria. this tension is borne out in different recommendations for research diagnostic criteria promulgated by ad research groups, such as the research framework of the nia-aa (jack et al., (jack et al., , and that of the international working group (iwg)/dubois ad diagnostic criteria (dubois et al., (dubois et al., , . the latest nia-aa framework defines ad as a biological entity, solely by its pathophysiology as evidenced by biomarkers, without consideration of symptoms (jack et al., ) . this allows for a biomarker-based diagnosis in asymptomatic people. by contrast, the iwg ad diagnostic criteria require both symptoms and biomarkers (dubois et al., ) . this could entail the existence of asymptomatic-at-risk-for-ad patients or presymptomatic patients, the latter carrying proven ad autosomal dominant mutations, which, because they have virtually complete penetrance, merit the "pre-" designation on the justified assumption that all such patients will develop dementia (dubois et al., , ) . a publication (dubois et al., ) allowed for a diagnosis of preclinical ad in the absence of symptoms, aligning the criteria with the nia-aa's view of ad as a biological, not syndromal, entity. these developments in diagnostic criteria do more than change how ad is identified-they go to the heart of how ad is defined. is ad a syndrome, a distinct type of neuropathology, or both? the distinction between a condition's diagnostic criteria and the definition of a condition is an important one, one the typology brings into sharp relief. this is by virtue of the symptomatology and dysfunction factors, because whereas a condition's definition may in some cases coincide with current diagnostic criteria, this is not always so. these cases of contested definitions (such as with ad) offer philosophically interesting and ethically important tests of whether biological dysfunction is more important than symptoms for defining a disease. the nia-aa research framework views dementia as a syndrome, not a disease, which is caused by ad (among other diseases): "the term "alzheimer's disease" refers to an aggregate of neuropathologic changes and thus is defined in vivo by biomarkers and by postmortem examination, not by clinical symptoms" (jack et al., , ) . yet this can be problematic because ad pathophysiological processes do not always lead to dementia. indeed, in cognitively unimpaired people aged greater than years, up to % have ad neuropathology (jack et al., , ) , and it is possible that these people do not develop dementia simply because they do not live long enough. in the terms of the typology, positive cases of ad (i.e., those meeting the diagnostic criteria) have gone from being categories f, g, or h to being categories a, b, c, or d when asymptomatic and e, f, g, or h when symptomatic. yet, as alluded to earlier, analyzed on the bst the asymptomatic or even prodromal stages may not be diseases at all, because the biological dysfunction may be too common, a point recognized by others (alexopoulos and kurz, , ; shermer and richard, ) . instead, they would be risk states. although there are some benefits to separating the pathological features from the clinical features (jack et al., ) , this has not been without its share of criticism, some authors going so far as to call the separation a messy divorce (mccleery et al., , ) . indeed, reconceptualizing ad as solely a biological entity privileges a disease approach instead of an illness approach. it could even result in a worry-type illness state characterized by anxiety over the putative certainty of developing dementia, compounded by the negative societal consequences of being labeled as having ad. in addition to challenging common notions of dementia as understood by lay people, cultural differences in how dementia is perceived around the world (alladi and hachinski, ) , as well as the unavailability of biomarkers in poorer regions, may make the reconceptualization of ad practical for only a minority of people. the ethically problematic nature and potential harm of asymptomatic or prodromal ad diagnoses include insurance, driving, and employment implications; additional costs for diagnostics and potentially ineffective treatments; needless worry and hypervigilance; and the stigma of being labeled with an ad diagnosis (le couteur et al., ; swallow, ; milne et al., ; shermer and richard, ) . turning people who may or may not have a disease into patients with a chronic illness (bedson et al., ) or patients-in-waiting (timmermans and buchbinder, ) similarly deserves careful deliberation, whatever the benefits the new research diagnostic criteria may hold. reconceptualizing ad as various states of risk may help avoid some of these problems, but still can result in a "diagnosis" of risk, which can be harmful given that interventions to reduce risk can also cause harm (accad and fred, ) . by contrast, there could be benefits to conceiving ad solely as a biological entity. as mentioned, category c allows for a biomarker-based diagnosis of ad. if an ad diagnosis depended on clinical symptoms, then a large number of asymptomatic people who have the pathophysiological defects of ad might not be considered for treatment because they would not be diagnosed. this is particularly important given that the window for affecting the pathogenesis of ad via treatment may exist years before symptoms become apparent. applied to the clinic, there are risks with this approach to diagnosis and treatment, however. this is especially true when the line between physiological and pathological changes is difficult to distinguish. for example, in cardiomyopathy, technological advances have resulted in identification of asymptomatic, nonhypertrophic, genotype-positive people. such individuals are at risk of being overdiagnosed because they are unlikely to become symptomatic or have deadly arrhythmias (quarta et al., ) . with respect to ad, diagnosis not dependent upon clinical symptoms is also subject to overdiagnosis if there are people with the pathophysiological defects who do not go on to develop ad symptoms. this could happen if the pathophysiological defects are normal signs of aging or if abnormal ad biomarkers are also present in old age, which in fact they are (vinters, ) . indeed, alexopoulous and kurz think there is practical validity in defining preclinical states of ad, such as by its effect on protective behaviors across the lifespan. however, they propose using the term "advanced brain aging" instead of "preclinical ad" on the grounds that they think a biomarker-based conceptualization of ad is conceptually invalid (alexopoulos and kurz, ) . although they are correct that ad biomarkers should be sufficiently validated and accurately distinguish between ad dementia and normal aging, ad neuropathology has been shown in a substantial number of brains of people aged less than years ; although, to be fair, not all such people will necessarily go on to develop ad symptoms in their old age). moreover, the pathology associated with ad has been shown to not be due to aging-related mechanisms (nelson et al., ) . these findings highlight that the pathophysiological deficits of ad can begin well ahead of clinical symptoms. while clinical dementia may sometimes be simply a product of normal aging, the dementia of ad is not. several studies have shown that there are significant ad brain lesions in people at early stages of clinical cognitive symptoms (reviewed in vinters, ) . the reason recent trials of ad therapies may have failed is because only symptomatic patients were enrolled, thus excluding the chance to arrest the disease by addressing the pathophysiological defects in their incipient stages when therapy might have been more effective (lyon, ; langbaum et al., , ; graham et al., , ; jack et al., , ; dubois et al., , ) , a fact underscored by the oftentimes sudden onset of severe symptoms, the varying rate of disease progression among patients, and the sometimes decades-long prodromal phase, which can complicate measurement of drug efficacy (graham et al., , ) . moreover, given ad's phenotypic, endotypic, and etiologic heterogeneity, characterizing the condition as a single disease is likely to lead to research and therapeutic dead-ends, such as with single-therapy solutions (huang and mucke, ) . how then should ad, or any other disease, be defined? defined according to the bst on the basis of biological dysfunction, only biomarker-assessed neuropathology is needed for an ad diagnosis. but this does not mean that all diseases need to be recognized by clinical medicine (blinded for review-, ). diagnostic criteria for ad that require symptoms in addition to biological dysfunction mitigates against the chance the person is merely a "carrier" and will never be affected in any significant way by the pathophysiology. however, each disease presents its own set of considerations, which militates against cookie-cutter solutions. although whether a disease involves readily identifiable pathological signs might help, on its own this is insufficient to guide the setting of diagnostic criteria (i.e., deciding what criteria should be used by which a person can be diagnosed as having a specific disease). what is also needed is the use of normative considerations to dictate what criteria are used (-blinded for review-, ). as shown with ad, understanding a condition's place in the typology may sensitize one to whether it may be possible to diagnose well before symptoms appear. for example, the beta-cell dysfunction and insulin resistance of type diabetes have been hypothesized to exist years before glucose levels become elevated (kahn, ) . type diabetes and other conditions can also stress the line between disease and risk. for example, vickers and colleagues ( ) propose unambiguous lesions as distinguishing disease from risk. they give the example of a torn aorta. however, this should not be taken to mean that type diabetes could not have unambiguous lesions. in the case of a torn aorta the lesion is at the level of the organ. with type diabetes unambiguous lesions could be at the level of the cell or lower. the idea of risk is complex when it comes to ad because it could refer to many things: the increased risk that apoe ε carriers, for example, have for developing ad (dubois et al., , ) but which is by no means large enough to guarantee that all or most carriers will develop ad (whether defined via symptoms or solely by biomarkers). other risk states, listed roughly in order of likelihood of resulting in dementia, include an asymptomatic person with some biomarker evidence of ad pathology, to such a person with more biomarker evidence, to a patient in the prodromal stage without biomarker evidence, to a prodromal patient with biomarker evidence, to a person with presymptomatic ad (i.e., familial ad), even if that person is not currently symptomatic (table ). the nature of the biomarker evidence results in different amounts of risk (jack et al., ) . a key difference between the nia-aa research framework and the iwg criteria is that whereas the former views asymptomatic but ad-biomarker-positive people as at risk for developing ad symptoms, the latter views these people as being at risk for developing ad (jack et al., , ) . this can conflict with lay intuitions about ad, a point recognized (but perhaps not adequately resolved) by the nia-aa: … we also recognize the deeply engrained historic use of the term "alzheimer" to denote particular syndromes. thus, we strongly recommend that a clinically ascertained syndrome consistent with what has historically been labeled "probable or possible ad" be referred to as alzheimer's clinical syndrome, but not as ad or some modified form of ad (e.g., "possible or probable ad"). (jack et al., , ) the key to differentiating when conditions such as ad should be viewed as risk instead of disease may be the confidence with which diagnostic methods can unequivocally identify if and when the asymptomatic cases will turn into symptomatic cases and the prodromal cases will turn into manifest cases (e.g., dementia). high confidence might call for viewing as disease whereas low confidence might call for viewing as risk. however, this is easier said than done and the typology cannot directly help in this regard. a direct consequence of the bst's conception of disease is its ability to distinguish cases where there is a disparity between the diagnostic criteria for a disease and the dysfunction putatively underlying the disease. recognition of this can contribute to robust research programs that lead to revised diagnostic criteria for diseases. it can also affect nosology, leading to the splitting of one disease into many, or the unification of previously unrelated diseases (e.g., as with tuberculosis) when a common underlying etiology is identified. the result can be earlier diagnosis and more timely, effective interventions. in the case of aki, for example, "[f]uture definitions [of aki] are likely to incorporate novel functional and damage biomarkers to characterize aki better" (ostermann and joannidis, , ) . as technology advances, the ability to probe into the pathophysiological defects of many diseases may improve, such that they can be treated, even if asymptomatic and representing just mild dysfunction. such proactive intervention well ahead of when a person becomes symptomatic may also make it possible to modify the disease process and hopefully avert the development of the disease, or mitigate certain aspects of its presentation. this is now true for rheumatoid arthritis, for which disease-modifying drugs are available. for other diseases, such as atherosclerosis and type diabetes, early lifestyle interventions (exercise, diet) have been shown to prevent their development or progression. for diseases in which there are neither cures nor effective treatments, such as huntington disease, the value of knowing in advance whether one has the disease may be less than for diseases in which therapeutic interventions are available. indeed, the rate of presymptomatic testing for huntington disease is very low (oster et al., ) . nonetheless, in huntington disease detectable pathophysiological changes are present one to two decades prior to clinical diagnosis (paulsen et al., ) . research trials aimed at preventive therapeutics can thus still benefit from this lag between the beginning of pathophysiology and the appearance of symptoms. the existence of risk-based conditions-category b-which in some cases medicalize individual variation, could lead to less diagnosis of disease (if recognized as not involving dysfunction), whereas for category c diseases it could lead to more diagnosis (if diagnostic criteria are changed to allow for laboratory evidence of pathology). understanding the nature of the categories could provide a conceptual toolkit for nosology and the setting of diagnostic criteria. this can lead to approaches that are directly relevant to patients. for example, patients falling within certain categories could be provided direct indicators of disease rather than treating risk factors for disease. measuring and using carotid artery total plaque area as a guide to therapy, for instance, has been shown to improve therapy in cardiovascular disease prevention clinics (spence and hackam, ) . similarly, measurement of intima-media thickness affected both physician and patient behaviors, leading to better treatment behaviors (korcarz et al., ) . making visible direct indicators of disease can in a way turn a sign into a symptom (i.e., the feeling engendered by witnessing the sign), with possible effects on an affected patient's motivation now that they can more palpably (or visually) experience a disease they may not yet suffer from. as a classificatory system, the typology has several beneficial features: it is simple, containing only eight categories; exhausts the conditions clinicians encounter; has mutually exclusive categories; is flexible, able to accommodate new conditions and diseases; and is compatible with and can be integrated with other classification systems, such as the international statistical classification of diseases and related health problems (icd). there are, however, some limitations. although the typology can be conceptualized as a framework to help medical researchers, philosophers, bioethicists, and others think through considerations surrounding risk, disease, and the classification of medical conditions, the typology might only make explicit approaches that are already utilized, such as direct application of the notion of risk to classify a condition as a disease or not. the typology has not been empirically tested for its usefulness. furthermore, the typology is silent on etiology, although it is fully compatible with classification systems that rely on etiology. ultimately, a key argument of this paper (and one for which a typology is not even needed), is that normative considerations-although absent on the conception of disease used here-do guide how disease is recognized in clinical practice. here the typology may be useful by showing how normative considerations determine whether biological dysfunction or symptoms are more important for a condition's diagnostic criteria. tensions between the relative importance of the typology's factors for a given condition can result in category reassignment, which can have enormous implications for how a condition is diagnosed, reimbursed by insurance, and viewed by patients. this is especially true when clinicians, patients, or researchers hold implicit biases against certain categories with respect to how conditions falling under those categories should be managed as a class. further work can clarify the degree to which this is true. in conclusion, by attending to the types of clinical conditions possible on the basis of three key features (i.e., symptomaticity, dysfunction, and the meeting of diagnostic criteria), clinical conditions as currently classified can be better categorized, highlighting the issues pertaining to certain typology categories. hopefully this can reduce overdiagnosis or treatment where none is merited and at the same time increase sensitivity to risk for those individuals for whom it is important. this way such individuals' conditions can be considered by medicine with just as much importance as disease in which dysfunction is readily apparent. risk-factor medicine: an industry out of control? the new conceptualization of alzheimer's disease under the microscope of influential definitions of disease world dementia diagnostic and statistical manual of mental disorders labelling chronic illness in primary care: a good or a bad thing? in: health 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iatrogenic disease medically unexplained symptoms in frequent attenders of secondary health care: retrospective cohort study diagnostic criteria for huntington's disease based on natural history the line-drawing problem in disease definition small tumors as risk factors not disease reframing the disease debate and defending the biostatistical theory progress in defining disease: improved approaches and increased impact on the reconceptualization of alzheimer's disease treating arteries instead of risk factors: a paradigm change in management of atherosclerosis effectiveness of medical interventions age-specific distribution of serum thyrotropin and antithyroid antibodies in the us population: implications for the prevalence of subclinical hypothyroidism expectant futures and an early diagnosis of alzheimer's disease: knowing and its consequences patients-in-waiting: living between sickness and health in the genomics era simulated brain biopsy for diagnosing neurodegeneration using autopsy-confirmed cases against diagnosis emerging concepts in alzheimer's disease the biostatistical theory versus the harmful dysfunction analysis, part : is part-dysfunction a sufficient condition for medical disorder? i thank chris boorse for helpful comments on this article. the generous support of the fonds voor wetenschappelijk onderzoek-vlaanderen (fwo) is acknowledged ( n). endnote i thank an anonymous reviewer for this journal for this point. i put "diseases" in scare quotes because while some continuous-variable conditions are diseases by the bst, some are not. key: cord- -yltc wpv authors: lessler, justin; azman, andrew s.; grabowski, m. kate; salje, henrik; rodriguez-barraquer, isabel title: trends in the mechanistic and dynamic modeling of infectious diseases date: - - journal: curr epidemiol rep doi: . /s - - - sha: doc_id: cord_uid: yltc wpv the dynamics of infectious disease epidemics are driven by interactions between individuals with differing disease status (e.g., susceptible, infected, immune). mechanistic models that capture the dynamics of such “dependent happenings” are a fundamental tool of infectious disease epidemiology. recent methodological advances combined with access to new data sources and computational power have resulted in an explosion in the use of dynamic models in the analysis of emerging and established infectious diseases. increasing use of models to inform practical public health decision making has challenged the field to develop new methods to exploit available data and appropriately characterize the uncertainty in the results. here, we discuss recent advances and areas of active research in the mechanistic and dynamic modeling of infectious disease. we highlight how a growing emphasis on data and inference, novel forecasting methods, and increasing access to “big data” are changing the field of infectious disease dynamics. we showcase the application of these methods in phylodynamic research, which combines mechanistic models with rich sources of molecular data to tie genetic data to population-level disease dynamics. as dynamics and mechanistic modeling methods mature and are increasingly tied to principled statistical approaches, the historic separation between the infectious disease dynamics and “traditional” epidemiologic methods is beginning to erode; this presents new opportunities for cross pollination between fields and novel applications. in , ronald ross coined the term bdependent happen-ings^to capture the fundamental difference between the study of infectious diseases in populations and other health phenomena [ ] . because infectious diseases are, for the most part, acquired from the people around us, our own future health status depends on that of our neighbors (e.g., the more people we know who are infected, the more likely we are to become infected ourselves). for acute infectious diseases, the health status of the population often changes quickly over time, with the number of people infectious, susceptible to being infected, and immune to the disease changing substantially over the course of an epidemic. further, the membership in each of these groups does not vary arbitrarily over time but is driven by often well-understood biological processes (box ). for instance, in the simple example of a permanently immunizing infection spread through person-to-person transmission such as measles, new susceptible individuals only enter the population through birth and immigration; these individuals can then only become infected by contact with existing infectious individuals, who, in turn, will eventually become immune or die and be removed forever removed from participation in the epidemic process. the epidemic dynamics of infectious diseases are driven by similar mechanistic relationships between the current and future health states of the population. the expected number of infections at some time t is illustrated for a directly transmitted disease in the above equation. dynamic and mechanistic models of disease spread, regardless of complexity, capture these relationships in order to improve inference or predict the disease dynamics. the study of infectious disease dynamics encompasses the study of any of the shared drivers of the mechanistic processes of disease spread with an eye towards better understanding disease transmission. as illustrated above, these include: the size of the susceptible population ( ): the number of people available to be infected. the dynamics of susceptibility is not shown here, but can itself can be complex, as new susceptibles enter the population through birth, immigration and loss of immunity. for many diseases (e.g., dengue, influenza), susceptibility is not a binary state, and complex models may be needed. the force of infection: the force of infection is the probability that any individual who is susceptible at a given time becomes infected (analogous to the hazard of infection). the size of the susceptible population times the force of infection is the reproductive number ( ). when this value is above , the epidemic will grow. when it falls below , it will recede. the infectious process ( ): the infectious process dictates the chances of becoming infected on a direct or indirect contact with an infectious individual. here represented as a per contact probability of infection, this itself can be a complex, multi-faceted process. the contact process ( ): the process by which infectious contacts are made, whether directly or mediated by a some vector or the environment, is one of the most complex parts of the infectious process. much modern research focuses on accounting the role of space and population structure in the contact process. previous infections ( ): fundamental to the nature of infectious diseases is the number of previous infections, however, these may not as directly lead to current infections as illustrated here if transmission is mediated by a vector or the environment. the natural history of disease ( ): how infectious people are at particular times after their infection determines their contribution to ongoing disease transmission, and fundamentally drives the speed at which epidemics move through the population. other aspects of disease natural history (e.g., the incubation period) may determine our ability to control a disease and its ultimate health impact. over the course of the twentieth century, the main body of epidemiologic research became increasingly reliant on models of statistical association, often with strong assumptions of independence between observations (hereafter referred to as associative models) [ ] . however, as a result of the need to deal with dependent happenings, there remained a strong subpopulation within infectious disease epidemiology that used models of an entirely different type. variously referred to as bmathematical,^bdynamic,^or bmechanistic^models, these models are characterized by having a mechanistic representation of the dynamic epidemic process that determines how the population's state at time t + depends on its state at time t (hereafter referred to as mechanistic models). historically, these models have more often been deterministic and built top-down from first principles rather than based on patterns in any particular dataset. however, as increasing computational power has caused an explosion in the types of models that can be subject to rigorous statistical analysis, there has been a shift toward more data-driven and statistical approaches and a greater focus on stochasticity and uncertainty. this confluence between principled statistical inference and mechanistic processes is paying huge dividends in the quality of the work being produced and the types of questions being answered across disciplines within infectious disease epidemiology. infectious disease models are being given a firmer empirical footing, while the use of generative mechanistic approaches allows us to use models as tools for forecasting, strategic planning, and other activities in ways that would not be possible with models that do not represent the underlying dynamic epidemiologic processes. in this manuscript, we review current research into dynamic and mechanistic models of infectious disease with a focus on how the confluence of mechanistic approaches, new statistical methods, and novel sources of data related to disease spread are opening up new avenues in infectious disease research and public health. for those interested in further pursuing the topic, we provide a list of key resources in box . recent work in infectious disease dynamics has been characterized by an increasing focus on data and principled approaches to inference. traditionally, deterministic models were a dominant tool for studying the theoretical and practical basis of disease transmission in humans and animals. this approach yielded important practical and theoretical results there exist a number of freely available resources that aid infectious disease modelling efforts. courses that form the basis of our understanding of disease dynamics [ •, ] but was limited in approach. deterministic models are usually parameterized through some combination of trajectory matching (i.e., minimizing the distance between observed and simulated data) and specifying parameters based on previous literature. this approach may be sufficient to describe the expected behavior of an infectious disease in a large population, but an increasing focus on how stochasticity and parameter uncertainty impact public health decision making, combined with the growing availability of computational power, has driven a move toward more statistically principled and data-driven likelihood-based approaches. illustrative of this evolution is the contrast between early descriptions of the key dynamic properties of hiv transmission with more recent dynamic characterizations of pandemic h n influenza (h n pdm), middle eastern respiratory syndrome (mers-cov), and ebola. in the late s, several papers were published laying out the essential properties of hiv transmission dynamics that would govern the course of the epidemic (at least in the near term) [ - ]. these papers presented deterministic epidemic models that captured the processes driving the epidemic and highlighted the key parameters, such as the speed of progression to aids, that needed to be investigated. uncertainty was largely addressed through scenario-based approaches (e.g., different future epidemic trajectories were presented for different plausible sets of parameters), and for the most part, different aspects of the transmission dynamics were derived from independent studies, with only the growth rate (i.e., doubling time) estimated from incidence data. while the parameters essential to characterizing epidemic dynamics remain largely unchanged for recently emerging pathogens, the approach to data and estimation is qualitatively different. integrated statistical frameworks built on markov chain monte carlo (mcmc) techniques are used to estimate all, or most, parameters from different datasets and to produce posterior distributions both for parameter estimates and forecasts of future incidence [ , •, ]. these methods allow innovative use of unconventional data sources, such as disease incidence among travelers [ , •], to estimate population incidence of the disease, and molecular data can supplement incidence data providing independent estimates of the same parameters (see discussion of phylodynamics below) [ , •]. these recent attempts to quickly characterize the properties of emerging diseases are emblematic of an increasing focus on developing statistical methods, grounded in dynamical models, to estimate key epidemic parameters based on diverse data sources. surveillance data is often used to estimate the reproductive number (r t , the number of secondary infections that a primary infection is expected to infect at any point, t, in an epidemic), incubation period, and serial interval (the expected time between symptom onset in a case and the people that case infects), as was done in recent outbreaks of mers-cov [ •, ] and ebola [ ] . surveillance data has also been paired with serological data to estimate force of infection (i.e., the hazard of infection) and basic reproductive number (r t , when the population is fully susceptible, designated r ) of several pathogens, including dengue and chikungunya [ ] [ ] [ ] . dynamic modeling approaches can also aid in the interpretation of surveillance data. state-space models (e.g., hidden markov models) have been used to pair our mechanistic understanding of disease transmission with a statistical inference framework by linking observed incidence and dynamics with underlying population disease burden and susceptibility (i.e., the population's state). notably, this approach has been used to estimate global reductions in mortality due to measles in the face of incomplete reporting [ •] . likewise, valle and colleagues used hybrid associative and mechanistic models to account for biases that treatment of detected malaria cases might have on estimates of key values such as the incidence rate [ ] . perhaps, the biggest limitation when attempting to characterize the parameters driving disease transmission remains data availability. data on disease transmission often comes from incomplete surveillance data or represents one aspect of a partially observed epidemic process. for example, epidemic curves are usually limited to symptomatic cases. similarly, key events in the transmission process, such as the exact time of infection, are generally not observable and have to be inferred from observed data. methods, such as the use of mcmc-based data augmentation and known transmission processes to infer the possible distribution of transmission trees, have been developed to deal with partially observed data and have been used to reconstruct outbreaks [ ] , characterize risk factors for transmission [ ] , and quantify the impact of interventions [ ] . a limitation of likelihood-based approaches, such as those mentioned above, is that it is often impossible or impractical to evaluate the data likelihood, in particular, for complex models and large datasets. to deal with this challenge, several blikelihood-free approaches^have been developed, including approximate bayesian computation (abc) and sequential monte carlo (smc) [ , ] . an advantage of these approaches is that they only require the ability to simulate from candidate models (i.e., if data can be simulated, calculation of the likelihood is unnecessary) and therefore can be more easily applied than methods that require iterative evaluation of the likelihood. abc has been used to integrate phylodynamic and epidemic models of influenza and other pathogens [ ] , and smc methods have been used to parametrize dengue transmission models using data from multi-centric vaccine clinical trials [ •] . despite important advances over the past decades in linking data and transmission models as tools of inference, many challenges remain and are the topic of continued research. inference for complex models and using large datasets remain challenging, in part, due to computational burden. mechanistic models offer promise as a way to simultaneously link data from diverse, heterogenous data sources (as in [ ] ), but this promise has yet to be realized, though some phylodynamics methods come close (see below). further, rapid inference on emergent epidemics remains a tool only used in high-profile epidemics [ •, ], and these inferential techniques remain inaccessible to field epidemiologists. scientists and physicians have tried to forecast the course of epidemics since the time of hippocrates. associations between incidence and extrinsic factors such as time of year, climate, and weather can and have been used to forecast infectious diseases [ ] [ ] [ ] . however, mechanistic models that capture the natural history of the disease (e.g., duration of immunity and cross protection) [ ] , mode of transmission [ ] , and movement patterns [ , ] can improve forecasts, particularly when associations with extrinsic drivers of incidence, such as climate, are weak or unknown (e.g., for emerging pathogens). in recent years, forecasts based on models that capture the underlying mechanistic processes of transmission and pathogenesis have become common. uses range from forecasting the peak timing and magnitude of an influenza season [ ] , to forecasting the spread and spatial extent of emerging pathogens such as zika, ebola, and chikungunya [ ] [ ] [ ] . the mechanistic underpinning of these models allows forecasts to take into account dynamic processes that may, otherwise, be impossible to capture, including changes in behavior and resource availability in response to an epidemic [ ] . approaches adopted from computer science, machine learning, and climate science have enhanced our ability to provide reliable forecasts with quantified uncertainty. particularly important are ensemble approaches [ ] , which integrate forecasts from multiple imperfect models or different parameterizations of the same model to calculate a distribution of potential courses of the epidemic [ , ] . ensemble approaches have been used for forecasting influenza in temperate regions [ ] , where influenza is highly seasonal, and more recently in subtropical areas such as hong kong, where the seasonal pattern is less distinct [ ] . similarly, ensemblebased climate models have been incorporated with infectious disease models to forecast climate-related disease including plague and malaria [ ] . these examples use multiple parameterizations of a single model. ensemble approaches can also be used to accommodate uncertainty in model structure by comparing estimates from parameterizations across different models, as is work by smith and colleagues where an ensemble of different individual-based models was used to estimate the impact of a future malaria vaccine [ ] . there has been an explosion in the number of forecasts being made to aid public health decision making, including a number of government-sponsored contests to forecast the progression epidemics of diseases ranging from influenza to chikungunya and dengue [ ] [ ] [ ] . as forecasts become more widely used, care must be given to ensure that the purposes of the model and uncertainty (both structural and statistical) are well communicated. in a recent outbreaks of emerging infectious diseases, like ebola, groups raced to make forecasts of the evolution and spatial spread of the outbreak [ , ] , with some predicting an epidemic size orders of magnitude greater than what was actually observed. while some of these extreme forecasts were made as worst-case planning scenarios, they were interpreted as likely scenarios, raising alarm and casting doubt on the validity of model-based forecasts, thus highlighting the importance of clear communication of a model's purpose and its limitations [ , ] . the quality of infectious disease forecasts and standards for their interpretation are far from the gold standard of methods and conventions used in the meteorology. improvement in both the methods used and their practical use remain critical areas of future research. the advent of bbig data^has opened up new avenues in how we parameterize and understand models of infectious disease spread. big data refers to massive datasets that are too large or complex to be processed using conventional approaches [ ] . however, advances in computing increasingly allow their use without large delays in processing time or unrealistic computing capacity requirements. one of the most successful attempts to use big data to understand disease dynamics has been the use of call data records (cdrs) to capture human mobility. for each call that is made or received, mobile phone operators capture the mobile phone tower through which the call is made. by tracking tower locations for a subscriber, we can capture where he or she is moving. in practice, to ensure confidentiality, cdrs are usually averaged over millions of subscribers to provide estimates of flux between different locations in a country. transmission models built upon cdr-based estimates of seasonal patterns of human movement have been used to explain patterns of rubella disease in kenya [ ] and dengue in pakistan [ ] . in both instances, models built on empirical human movements seen in cdrs outperformed alternative parametric models of population movement based on our theoretical understanding of human travel patterns (e.g., gravity models where movement is based on community size and distance [ ] ) and models where movement was not considered. cdr-based models have also been used to understand the dynamics of large-scale outbreaks such as ebola in west africa [ ] and challenges in malaria elimination [ ] . questions remain as to the generalizability of cdr-based analyses in settings where mobile phone ownership is low [ ] , and problems capturing flows between countries remain. however, the largescale penetration of mobile phones, even in resourcepoor settings, makes cdrs a hugely valuable data source for informing infectious disease models. another type of big data that has enormous potential for furthering our understanding of disease dynamics is satellite imagery. detailed satellite imagery can provide high-spatial-resolution estimates of key determinants of many infectious disease processes, including environmental factors (e.g., land cover), climatic conditions (e.g., precipitation, temperature), and population density throughout the globe [ , ] . in infectious disease epidemiology, such datasets have recently been used as the basis for statistical models that produce fine scale maps of disease incidence, prevalence, and derived transmission parameters (e.g., force of infection, basic reproductive number) for a large number of diseases. early efforts focused on mapping the global distribution of key drivers of malaria transmission [ , ] . these approaches have since been used to estimate the burden from a wide range of pathogens [ ] [ ] [ ] [ ] , vectors [ ] , and host reservoirs [ ] . these analyses have allowed disease burden and risk to be estimated in areas with limited surveillance capabilities, expanding our understanding of the global burden of many pathogens. high-resolution geographic data can gain additional power when paired with mechanistic models that capture changes in disease risk, as in recent analyses that accounted for the effect of birth, natural infection, and vaccine disruptions driving increases in measles susceptibility and epidemic risk in the wake of the ebola outbreak [ ] . finally, big data are increasingly being used with mechanistic models to more directly estimate disease burden in real time [ ] . for example, patterns in the usage of different google search terms have been shown to correlate well with incidence trends for diseases such as influenza [ , ] and dengue [ ] . it is worth noting that big data alone can typically only explain part of trends in incidence, and models that incorporate seasonal dynamics typically outperform models that rely solely on search terms. similar approaches have been used with wikipedia updates [ ] and social media sites such as twitter and facebook. electronic medication sales data and electronic medical records have also been proposed as novel data sources of disease trends [ ] . these approaches can provide estimates much faster than traditional surveillance systems, where it often takes weeks or months for results of cases to be aggregated and analyzed. mechanistic models can then be fit to this data to better understand seasonal or spatial parameters. for example, yang et al. used mechanistic models fit to google flu trend data to estimate epidemiological parameters such as the basic reproductive number and the attack rate for cities in the usa over a -year period [ , ] . phylodynamics, the study of how epidemiological, immunological, and evolutionary processes interact to shape pathogen genealogies, is among the newest and fastest-growing areas in infectious disease research [ ] . the term phylodynamics was coined in by grenfell et al., who observed that the structure of pathogen phylogenies reveals important features of epidemic dynamics in populations and within hosts [ ] . this relationship provides a theoretical framework for linking molecular data with population-level disease patterns using dynamic models. early methodological work in phylodynamics concentrated on the formal integration of the kingman's coalescent and birth death models from population genetics with standard deterministic epidemic models. the coalescent model provides a framework for estimating the probability of coalescent events (lineages converging at a common ancestor) as we move back in time given changes in population size [ ] . the branching patterns in a phylogenetic tree describe the ancestral history of sequenced pathogens, such that nodes closer to the root of the tree represent historical coalescent events while nodes near the tip represent recent events. the strong relationship between the genetic divergence of pathogens and time allows us to estimate the timing of coalescent events and estimate the rate of growth (or decline) of pathogen populations. these estimates are the critical link between genetic and epidemic models [ ] . the formal statistical integration of population genetic and epidemic models allows us to estimate the critical epidemiological parameters such as the basic reproductive number directly from pathogen sequence data [ ] [ ] [ ] . for example, magiorkinis et al. used sequence data from viruses collected over a -year period in greece to estimate subtype-specific reproductive numbers and generation times for hepatitis c [ ] . using data from the athena hiv cohort, which samples ∼ % of hiv-infected persons in the netherlands, bezemer et al. used viral sequence data to estimate reproductive numbers for hundreds of circulating transmission chains, showing that large chains persisted within the netherlands for years near the threshold for sustaining an epidemic (r = ) [ , ] . other phylodynamic applications have focused on elucidating the spatial dispersal pattern diseases such as influenza and hiv. in an analysis of nearly , influenza genomes, bedford et al. showed fundamental differences in the global circulation patterns of h n , h n , and influenza b viruses and that these were potentially driven by differences in the force of infection and rates of immune escape (i.e., antigenic drift) [ ] . likewise, faria et al. used hiv sequence data from central africa to reconstruct the early epidemic dynamics of hiv- using phylodynamic methods and showed that kinshasa in the democratic republic of congo likely served as the focal point for global hiv spread [ , ] . phylodynamics plays an important role in real-time infectious disease surveillance and targeted control [ ] . in recent epidemics of mers-cov and ebola, genomic data was used to assess transmission patterns, monitor viral evolution in populations, and inform epidemic control [ •, [ ] [ ] [ ] . analyses of hivepidemics among us and european men who have sex with men demonstrate that the amalgamation of epidemiologic and genomic data can be used to identify high-risk transmitters and optimal targeted intervention packages [ •, ] . however, the utility of real-time phylodynamic analysis in many settings remains hindered by inadequate infrastructure, few viral sequence data, and limited analytic capacity at local levels. initial phylodynamic models could only deal with simple epidemic patterns (e.g., exponential growth), and recent methodological work has focused on extending the phylodynamic framework to account for complex nonlinear population dynamics [ , ] . for instance, rasmussen and colleagues showed how phylodynamic models could be extended to integrate more complex stochastic and structured epidemic models using bayesian mcmc and particle filtering [ •]. others have focused on resolving transmission network structure from phylogenies [ , ] or integrating data across multiple scales by incorporating information on intra-host pathogen diversity and ecological processes directly into phylodynamic models [ ] . however, equally important recent work has shown that phylodynamic inferences can be highly sensiti ve to sam pl ing and unmeasured factors. simulation studies show that the relationship between phylogenetic trees and the underlying transmission networks is a complex function of the sampling fraction and underlying epidemic dynamics [ , ] and that failure to account for intra-host viral diversity may bias phylodynamic inference [ ] . here, we have focused on areas where we feel that there has been the most innovation in the use of dynamic epidemic models in recent years. this is not to imply that innovation has stopped in other areas where dynamic models play a key role. dynamic models have long been key to our understanding of epidemic theory. innovative models continue to be developed to deal with the challenges posed by pathogen evolution [ ] , complex immunological interactions [ ] , and host heterogeneity [ ] . there has been increasing emphasis on the use of dynamic models in informing public health policy since the early s when they played a key role in the response to the foot-and-mouth disease outbreak in the uk [ ] and assessment of the risk from a smallpox-based bioterrorist attack [ , ] . these uses have extended to endemic disease, such as a modeling analysis by granich and colleagues [ ] that highlighted the potential of btest-and-treat^strategies for hiv control. recently, dynamic models have played an important role in guiding the response to emerging disease threats, from pandemic influenza [ ] , to multi-drug resistant organisms [ , ] to mers-cov [ ] . many of the themes discussed throughout this manuscript have had a profound impact on these efforts, as does the need to report results and assumptions in a way accessible to policy makers. mechanistic models also crop up in other areas of epidemiology, often in less obvious ways. nearly all of the key methods of genetic epidemiology are based on a mechanistic understanding of the underlying processes inheritance, mutation, and selective pressure. social epidemiology at its core is based on the idea that our health depends on the behavior and health of those around us and, hence, has its own approaches to dependent happenings (though the terminology differs). recently, there has been increasing interest in using mechanistic modeling approaches similar to those used for infectious disease to understand health phenomena that are, in part, socially driven, such as obesity [ ] . physiological measurements are often founded on mechanistic models of processes within the body (e.g., use of serum creatinine to approximate the glomerular filtration rate, a key measure of kidney function [ ] ). infectious disease dynamics is, perhaps, unique in epidemiology in the number of researchers that it brings from non-health related disciplines, particularly physics, computer science, and ecology. this, combined with the unique aspects of infectious disease systems, has contributed to the use of models that are distinct from btradition-al^epidemiologic methods. however, the field is being transformed by the same forces that are transforming epidemiology in general: increasing access to technological tools and computational power; an explosion in the availability of data at the molecular, individual, and population levels; and a shift in what the important epidemiologic questions are as we eliminate old health threats and change our environment. increasing emphasis on principled 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coalescent under neutrality uses innovative methods to combine mechanistic models within phylodynamics to estimate transmission parameters modelling tree shape and structure in viral phylodynamics inferring epidemic contact structure from phylogenetic trees reconciling phylodynamics with epidemiology: the case of dengue virus in southern vietnam contact heterogeneity and phylodynamics: how contact networks shape parasite evolutionary trees how the dynamics and structure of sexual contact networks shape pathogen phylogenies within-host bacterial diversity hinders accurate reconstruction of transmission networks from genomic distance data core groups, antimicrobial resistance and rebound in gonorrhoea in north america age profile of immunity to influenza: effect of original antigenic sin insights from unifying modern approximations to infections on networks dynamics of the uk foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape containing bioterrorist smallpox emergency response to a smallpox attack: the case for mass vaccination universal voluntary hiv testing with immediate antiretroviral therapy as a strategy for elimination of hiv transmission: a mathematical model strategies for containing an emerging influenza pandemic in southeast asia improving control of antibiotic-resistant gonorrhea by integrating research agendas across disciplines: key questions arising from mathematical modeling modeling epidemics of multidrug-resistant m. tuberculosis of heterogeneous fitness estimating potential incidence of mers-cov associated with hajj pilgrims to saudi arabia social network analysis and agent-based modeling in social epidemiology estimating glomerular filtration rate from serum creatinine and cystatin c key: cord- -k b wqe authors: gerc, vjekoslav; masic, izet; salihefendic, nizama; zildzic, muharem title: cardiovascular diseases (cvds) in covid- pandemic era date: - - journal: mater sociomed doi: . /msm. . . - sha: doc_id: cord_uid: k b wqe introduction: covid- is the disease caused by an infection of the sars-cov- virus, previously known as novel coronavirus ( -ncov) respiratory disease. world health organization (who) declared the official name as covid- in february and in (th) march declared covid- as global pandemic. in june (th) , over million cases registered in the world, recovered . million and death over . . aim: the aim of this study is to retreive published papers about covid- infection deposited in pubmed data base and analyzed current results of investigations regarding morbidity and mortality rates as consequences of covid- infection and opinions of experts about treatment of afected patients with covid- who have cardiovascular diseases (cvds). methods: it’s used method of descriptive analysis of the published papers with described studies about corona virus connected with cvds. results: after searching current scientific literature (on pubmed till today is deposited more than . papers about covid- with consequences in almost every medical disciplines), we have acknowledged that till today not any evidence based medicine (ebm) study in the world. also, there are no unique proposed ways of treatments and drugs to protect patients, especially people over years old, who are very risk group to be affected with covid- , including patients with cvds. vaccine against covid- is already produced and being in phases of testing in praxis in treatment of covid- at affected patients, but the opinions of experts and common people whole over the world about vaccination are full of controversis. conclusion: frequent hand washing, avoiding crowds and contact with sick people, and cleaning and disinfecting frequently touched surfaces can help prevent coronavirus infections are the main proposal of who experts in current guidelines, artefacts stored on a web site. those preventive measures at least can help to everybody, including also the patients who have evidenced cvds in their histories of illness. authors analyzed most important dilemmas about all aspects of cvds, including etipathogenesis, treatment with current drugs and use of potential discovered vaccines against covid- infection, described in scientific papers deposited in pubmed data base. covid- is the disease caused by an infection of the sars-cov- virus, first identified in the city of wuhan, in china's hubei province in december . covid- was previously known as novel coronavirus ( -ncov), respiratory disease before the world health organization (who) declared the official name as covid- in february ( , ) . covid- spreads by contact with respiratory droplets that spread when an infected person coughs or sneezes. respiratory virus infection is a major source of global pandemics as a consequence of swift human-to-human respiratory tract transmission. within the past two decades, coronaviruses and influenza viruses have hit the world several times, causing significant mortality, economic loss and global panic. the sars outbreak in triggered deaths among more than . patients in countries, followed by the emergence of mers in , which resulted in deaths at least, among . patients in countries ( ) . besides coronaviruses, avian and swine influenza remain a concern for global public health in the h n pandemic alone. there were . laboratory-confirmed deaths and more than . deaths from respiratory disease worldwide ( ) . in late , a cohort of patients presenting with pneumonia of varying acuity and unknown aetiology in wuhan, china, heralded the outset of covid- . although covid- appears to have greater infectivity and lower mortality than sars and mers, many uncertainties (including route of infection, viral evolution, epidemic dynamics, appropriate antiviral treatment and strategies for disease control) remain. since there is no cure for this viral disease and a long wait is needed to find a vaccine, a rigorous epidemiological measure was first taken. the largest quarantine in history was made, between and million people in several chinese cities were quarantined; that included necessary hygiene measures such as: group meetings were canceled, schools closed and travel banned. some people believe that a later evaluation of all the more modern epidemiological measures will show whether all of them are justified, as experts point to procedures that curb a severe acute respiratory syndrome (sars) caused by another type of virus corona ( ) ( ) ( ) . cvds may become unstable in the setting of viral infection as a consequence of imbalance between infectioninduced increasing in metabolic demand and reduced cardiac reserve ( ) ( ) ( ) ( ) ( ) ( ) . till the may st, , . persons worldwide died as consequences of covid- infection and total number of infected people was , . citizens. web portal "worldometer" announced up-dated picture of global epidemiological facts since of the first registered patient infected with covid- in the world till today, and last number of patients who treated and survived was , . . from the same source on the may st , , . patients infected by covid- covid- infection is caused by a new beta-coronavirus, which the who has called (sars-cov- ) -severe acute respiratory syndrome. coronavirus is a disease that appeared in december in the chinese city wuhan and began to spread very quickly beyond the borders of china. in a short period of time, it took on all the characteristics of a pandemic and spread exponentially to almost all countries of the world. early covid- case reports suggest that patients with underlying conditions are at higher risk for complications or mortality -up to % of hospitalized patients have a chronic medical illness ( % cardiovascular or cerebrovascular disease). in the largest published clinical cohort of covid- to date, acute cardiac injury, shock and arrhythmia were presented in . %, . %, and . % of patients, respectively ( ), with higher prevalence amongst patients requiring intensive care. initially, the main complications of covid- were thought to be lung-related, then it was quickly observed that covid- is attacking many organs, including the heart muscle, vascular endothelium and the cardiovascular system in general, increasing morbidity and mortality, especially in patients with other cardiovascular risk factors presented (hypertension, diabetes, obesity, cerebrovascular and renal disease). in these patients, the morbidity is %. in a significant number of infected patients, severe myocardial damage, malignant arrhythmias and cardiac arrest occurred. it's interesting, sars-cov- can also lead to neurological symptoms, such as dysgeusia or ageusia, or to the appearance of hyposmia or anosmia ( ) . the virus damages the nerve endings in the olfactory epithelium and damages the olfactory bulb. therefore, the virus is neurotropic and can spread along the nerves and then damage them. it happens that the peripheral nerves are also affected, which manifests itself in the form of quadriplegia, and also causes cerebrovascular diseases. so, sars-cov- is neuroinvasive and can penetrate the brain and lead to neuronal death ( ) . it is interesting to mention that in % of patients, only gastrointestinal symptoms occur in the form of loss of appetite, abdominal pain, nausea and diarrhea. cardiovascular complications of influenza infection, including myocarditis, acute myocardial infarction and exacerbation of heart failure have been well-recognized during previous historical epidemics and make a significant contribution to mortality. also, previous coronavirus outbreaks have been associated with a significant burden of cardiovascular comorbidities and complications. the afore-mentioned clinical manifestations suggest that covid- is a systemic disease, that it is characterized by numerous tissues and organ damages. the presence of thrombi and microthrombi contributes to the manifestation of various clinical symptoms. at the same time, vascular endothelial dysfunction and coagulopathy occur ( ) . inflammation of the endothelium severely impairs its function. the virus not only leads to pneumonia, which in turn causes further complications ( ), but also to systemic endothelial inflammation. namely, the virus directly attacks the endothelial cells of blood vessels. induce endothelial damage in the small and smallest blood vessels of the lungs and leads to an enhanced inflammatory response by t cells. this is similar to an organ rejection reaction in case of transplantation. during the autopsy, a significant increase in new blood vessels was found in the lungs of the patient with covid- , through intussusception angiogenesis. therefore, there is a comprehensive inflammation of the endothelium of the heart, brain, lungs, kidneys and digestive tract. all of these ones have fatal consequences: severe microcirculation disorders occur that damage of the heart, lead to pulmonary embolism and clog blood vessels in the brain and digestive system. multiple organ failure occurs, ultimately resulting in death. systemic inflammation promotes thrombosis. massive inflammation stimulates the production of cytokines which in the liver leads to the formation of coagulation factors. for example, the level of fibrinogen in a severe patient with covid- is - g/l, while in a healthy person the value of fibrinogen is - g/l. endothelial cells located on the inside of blood vessels have ace- receptors on their surface, which serve as a gateway for sars-cov- . therefore, the wall of the blood vessel as well as the blood itself is affected at the same time. this explains why thromboembolic complications occur so frequently, including pulmonary embolism. many patients have increased d-dimer values as well as cutaneous changes in the limbs, suggesting thrombotic microangiopathy. diffuse intravascular coagulation and thrombosis of large blood vessels are associated with multisystem organ failure. renal blood vessels are often involved and ace- receptors are known to be found in the renal tubules. in severe forms of covid- the kidneys are affected in %, while in milder forms it's in %. therefore, sars-cov- not only attacks the lungs, but also affects the blood vessels of all organs. covid- is a systemic inflammation of the blood vessels, so we could see the whole clinical picture as covid- endothelism ( ) . from the point of view of cardiologists, covid- therapy is directed in two directions: it is necessary to stop the reproduction of the virus at the stage when it is most pronounced and at the same time to protect and stabilize the patient's blood vessels. this is especially true for patients who already have cardiovascular disease and some of the risk factors (hypertension, diabetes, increased cholesterol, obesity, smoking) with reduced endothelial function. it is important to mention that the endothelium of younger patients can more easily resist the attack of the virus. covid- is a primary respiratory disease, but many patients have cardiovascular disease, including the hypertension, acute heart damage and myocarditis. acute lung damage leads to increased heart rate, which then presents an additional problem, especially for patients with pre-existing heart insuficiency. cardiovascular disease can be primary if we take into account the activity of the renin-angiotensin system (ras) and angiotensin converting enzyme (ace- ), which is also present in the heart itself. sars-cov- , not only leads to damage of the respiratory system, but also to acute and chronic damage to the cardiovascular system ( ) . cardiovascular diseases that were present before a patient was infected with covid- significantly increased, both morbidity and mortality in these patients. this especially refers to the presence of hypertension, diabetes, coronary heart disease and kidney disease. patients with acute coronary syndrome, who are infected with sars-cov- virus, have a poor prognosis. in such patients, the functional cardiac reserve is reduced due to myocardial ischemia or necrosis. at the same time, the possibility that these patients will develop heart failure is quite high. respiratory failure and hypoxia can lead to myocardial damage as well as the reaction of the immune system itself. in wuhan, according to reports of chinese physicians, in patients infected with covid- and with acute coronary syndrome, the complete clinical picture was very severe and associated with high mortality ( ) . if patients already suffer from heart failure, infection with covid- leads to worsening their health condition and not seldomly to death. on the other hand, acute inflammatory responses can lead to ischemia in the presence of pre-existing cardiovascular disease. the inflammatory activity of atherosclerotic plaque in coronary blood vessels becomes even more pronounced during systemic inflammation, which makes atherosclerotic plaque more vulnerable and thus increases the possibility of its rupture. in such a situation, the vulnerable plaque should be stabilized with aspirin, statins, beta blockers, ace inhibitors, or angiotensin ii antagonists ( ) . inflammation can lead to endothelial dysfunction and to increased levels of procoagulant factors, which contributes to the forming of an occlusive thrombus and possibly to its rupture. if all these facts are considered, then an intense inflammatory response, along with the already present cardiovascular disease, can contribute to heart damage. vascular endothelial cell dysfunction in the presence of inflammation, and impaired myocardial function, can lead to cardiac decompensation or to its worsening, if it has been presented before. myocarditis, pericarditis and arrhythmias can also occur. rhythm disorders are manifested in the form of bradycardia, tachycardia and paroxysmal atrial fibrillation. the occurrence of myocarditis is the result of focal or global inflammation of the myocardium, necrosis and ventricular dysfunction. inflammation, by itself, plays an important role in the onset, development and prognosis of cardiovascular disease. the virus can penetrate directly into the myocardium and multiply and lead to its damage, necrosis and apoptosis (programmed cell death). focal myocarditis should be considered if precordial pain occurs during and after the acute phase of covid- , with clinical signs that would suggest that it is a coronary syndrome, but without coronary artery obstruction. c-reactive protein (crp), as well as other inflammatory cytokines, have become new risk factors. cardiovascular disease and cardiovascular risk factors represent an independent risk factor for cardiac death. troponin will be released in - % of patients infected with the sars-cov- virus, indicating that heart damage has occurred. increased values of troponin and its dynamic increase during hospitalization, shows that the risk of artificial ventilation is increased by times, and the risk of arrhythmias is increased, while mortality is increased by times. all of the above explains the connection between cardiovascular diseases and the occurrence of death in patients suffering from covid- . many patients infected with covid- die from cardiac arrest due to severe hypoxia, multiorgan dysfunction, or systemic inflammation. the occurrence of arrhythmias, such as atrial fibrillation, often occurs as part of covid- cardiomyopathy, among other things, because inflammation is a substrate for the occurrence of arrhythmias ( ) . ventricular arrhythmias occur, which can also be an introduction to cardiac arrest. qt prolongation may occur as part of myocarditis, as well as due to the application of chloroquine and hydroxychloroquine, especially if combined with azithromycin. this therapy is currently widely used in many clinics around the world, although the benefits of this therapy are highly debatable and the side effects can be very serious. the latest study, published in may nd, in the lancet, did not prove the benefit of hydroxychloroquine or chloroquine used as monotherapy or in combination with secondgeneration macrolides, azithromycin or clarithromycin. at the same time, there were ventricular arrhythmias and mortality increased. cytokine storm syndrome is a dysregulation of the immune system, and the appearance of severe symptoms that directly endanger the patient's life. when the sars-cov- virus enters our body, it first seeks protection, then it will reproduce and then spread further. our cells in the body provide the virus with extraordinary conditions to do all this. as sars-cov- infiltrates the cells of the respiratory system, it can thus hide better from our immune system and then multiply further. there are special cells in our body called t cells, and they fight infection. when t cells are activated they release cytokines, which further stimulate t cells to form, and then release even more cytokines. a type of t cells -called cytotoxic t cells -are formed. cytotoxic cells are such cells that kill infected cells. cytotoxic cells are chemically oriented to kill and they are ideally to stop the proliferation of viruses such as sars-cov . cytotoxic t cells only attack infected cells and kill them. the immune response has a chemical indicator that tells a strong immune response to stop in certain moment when the danger is neutralized. at that moment, the cytokine storm is very active and then the system is overloaded and functions no longer properly. the immune system is so irritated that it stops making a difference between an infected cell and a healthy cell and then attacks everything which found in its path. this is a very bad situation for the patient, because, not only does sars-cov- kill the cells in our body, but so our immune system ( ) . acute myocarditis, cardiac decompensation, or cardiogenic shock can occur during a cytokine storm. inflammatory cells in the presence of hypercytokinemia infiltrate the heart. there is a release of troponin and an increase in brain natriuretic peptide (bnp). inflammatory cytokine storm is accompanied by immunopathological changes in the lungs. patients infected with sars-cov- mostly die from acute respiratory distress syndrome (ards) ( ). this leads to acute pneumonia and to accumulation of fluid in the lungs, which requires intubation and ventilation. such a severe clinical picture develops six to seven days after the onset of infection. unfortunately, % of patients with acute respiratory distress syndrome do not survive. at the same time, patients die faster from cytokine storms than from infection. during a cytokine storm, death occurs due to the failure of several organs, which explains such a high mortality. proinflammatory cytokines such as interleukin , interleukin , interleukin , interleukin , interleukin , interleukin beta, and chemokinases such as ccl , ccl- and ccl , contribute to the development of ards. during the cytokine storm, there is an increase in d-dimer, ferritin, lactate dehydrogenase (ldh), crp, interleukin , interleukin , tumor necrosis factor alpha (tnf-alpha) and lymphopenia ( ) . a significant number of people with covid- do not develop a cytokine storm. however, some people have a tendency to develop a cytokine storm, if they possess a specific gene, which allows the immune system to react in a certain way. however, this has not been proven with certainty. this phenomenon of "immune storm" is not completely new. it has been observed in previous coronavirus-related epidemics, namely sars (severe acute respiratory syndrome) in and mers (middle east respiratory syndrome) in ( ). the phenomenon of "immune storm" can be observed in other viral infections, such as influenza. there are also non-infectious agents that can lead to an "immune storm", for example after an organ transplantation. the cytokine storm is unwanted and one of the most spectacular moments during treatment with car-t cells. this method consists of injecting t lymphocytes that have been genetically modified to fight tumors, leukemia or lymphoma. angiotensin converting enzyme- is especially present in the lungs, heart and other tissues. it serves, among other things, as a functional receptor for sars-cov- virus entry into cells. ace inhibitors (angiotensin converting enzyme inhibitors) and angiotensin ii antagonists (arbs) are first-line drugs used in the treatment of hypertension, cardiac decompensation, after a myocardial infarction and in chronic kidney disease. both ace inhibitors and arbs lead to increased ace- activity and at the same time, they could lead to increased virus activity. it has been hypothesized that these drugs could lead to worsening of sars-cov- infection. for now, there is no consensus that the use of ace inhibitors and arbs could reduce or increase the risk of developing covid- . however, it should be emphasized that so far there are no studies showing that drugs that block the renin-angiotensin system (ras), such as ace inhibitors: lisinopril, enalapril, trandolapril, or angiotensin ii antagonists such as losartan, candesartan, valsartan, increase ace - in human tissues ( ) . in addition, there are no animal or human studies to show that drugs that block ras increase ace- levels in the lungs. in contrast, studies in animals that have been infected with the coronavirus show that ace- plays an important role in protecting the lungs from more serious injuries caused by the virus. according to reports from china, the most common comorbidity in covid- was hypertension, and patients with hypertension required ventilatory support due to severe respiratory complications ( , ) . at the same time, these patients had increased mortality. the mechanism that connects hypertension and covid- is the immune system. immune system dysregulation occurred in patients with hypertension and covid- . poor blood pressure control in these patients leads to further dysregulation of the immune system in these patients. hypertension is associated with lymphocytes and cd + t cell dysfunction. immune aging of cd + t cells prevents them from effectively resisting viral infection. this contributes to pathological cytokine overproduction. probably all this could explain the connection between hypertension and covid- . it could be assumed that ace inhibitors and arbs, leading to better control of hypertension, could, at least in part, establish better function of the immune system, whose function is compromised ( ). in discussions about discontinuing therapy with ace inhibitors, the european society of cardiology, the international society of hypertension, the european society of hypertension, the european medicines agency (ema) and three american cardiac associations: american heart association, american college of cardiology and heart failure society of america, recommend not to stop taking ace inhibitors and arbs. because of these controversial views, a series of ongoing studies will give us an answer as to whether we should stop administering these drugs, or whether they should be given to patients infected with the sars-cov- virus. patients who have well controlled hypertension do not require frequent check-ups. many patients with hypertension due to covid- are in self-isolation in order to reduce the risk of covid- and therefore cannot go for follow-up examinations. these patients need to measure their blood pressure as often as possible at home and continue taking the therapy regularly. in case of an urgent consultation, for example due to an hourly increase in pressure, these patients are recommended a telephone consultation, until they can go to their regular check-ups with the doctor. in addition, hypertension often occurs in the elderly ( % of people over the age of develop hypertension) and at the same time the prevalence of hypertension increases sharply in very old people. the old age of the patient is a very important risk factor for complications in covid- and often with fatal outcome. what are the factors that correlate with a poor patient prognosis? it is evident that patients, younger than years, die significantly less from covid- than older patients. mortality is especially higher in men older than years ( ). mortality increases with each year of age by %. the risk does not increase linearly with age, but exponentially. men die more than women. this is explained by the fact that circulating ace- is higher in men than in women. at the same time, the values of ace- are higher in diabetics and people with cardiovascular disease. the presence of hypertension, diabetes and coronary heart disease contributes to the patient's poor prognosis. furthermore, increased ddimer values are a poor prognostic sign. according to one chinese study, d-dimer values in survivors were low and were below microgram/l. among those who died, d-dimer values increased significantly to micrograms/l ( ) . continuous increase in d-dimer is a poor prognostic sign. the appearance of lymphopenia and an increase of crp also do not indicate a good prognosis for the patient ( ). the therapy which is currently available in the world, can be divided into supportive, immunosuppressive, antiretroviral and possible new therapies ( ) . supportive therapy consists of oxygen administration as well as hemodynamic support. early intubation and invasive mechanical ventilation are crucial in patients with severe, progressive symptoms and where there is a great need for oxygenation. as heart damage has occurred, as it's mentioned earlier, it is necessary to include therapy given for cardiac decompensation as well as antiarrhythmics. due to thromboembolic complications, anticoagulant therapy must be included. antiviral therapy is also included ( ) ( ) ( ) . from the range of the drugs, anakinra (kineret) can be used, which blocks the activity of the specific cytokine interleukin (il- ). in addition, tocilizumab (actemra) is increasingly being used. tocilizumab blocks the activity of another cytokine, interleukin (il- ), which is thought to be a key of biological mediator responsible for the cytokine storm. during a cytokine storm, an increase in il- can have severe consequences for the cardiovascular system. tachycardia, atrial fibrillation, hypotension, and left ventricular dysfunction may occur with concomitant increase in gnp. in a recently published retrospective multicenter analysis in patients from wuhan, il- was a clinical predictor of mortality in covid- ( ) . in china, tocilizumab is approved for use in severe complications with covid- and where il- levels are elevated ( ) . in italy, too, tocilizumab has been used successfully in critically ill patients where complications of covid- have developed. similar studies with tocilizumab are underway in france, belgium and denmark. other therapeutic options include chloroquine, hydroxychloroquine, lopinavir/ritonavir (kaletra), ribavirin, favipiravir, intravenous immunoglobulin, and janus kinase (jak) inhibition. inhibition of jak can affect inflammation and virus entry into cells ( ) . the already mentioned therapy with corticosteroids and antibiotics is still possible. it should be emphasized that immunosuppressive therapy cannot be used in every patient. suppressing the immune system when the body fights infection can lead to serious side effects and increase the risk of secondary infection ( ) . in severe forms of covid- , chinese doctors in wuhan advocate determining laboratory parameters of the cytokine storm. this makes it possible to identify the patients who will benefit most from immunosuppressive drugs and significantly increase the chances of these patients surviving. effective treatment of a cytokine storm would be the fastest response to reduce mortality in some patients with covid- . currently, studies are underway examining various therapeutic options with the aim of determining which is the most suitable and most appropriate in the treatment of covid- ( ) . under the auspices of the world health organization (who), several large studies have begun, including the discovery study. the study will include patients. patients are divided into five groups and receive standard therapy given for sars-cov , antiviral therapy and, among others, the much-lauded hydroxychloroquine. a large solidarity study is also underway, also sponsored by the who. in solidarity study several therapeutic options are compared, namely remdesivir, lopinavir/ritonavir, lopinavir/ritonavir in combination with interferon beta- ; and chloroquine or hydroxychloroquine. remdesivir is the focus of clinical trials. a study done with remdevisir was published in the lancet ( ) . it is a randomized, multicenter, double-blind, placebo-controlled study showing that patients receiving remdesivir recovered more quickly if remdesivir therapy was started a maximum of ten days after the onset of symptoms. mortality was % in the remdesivir group and % in the placebo group. in short, it is important that remdesivir is superior to placebo and that it reduces recovery time in patients with covid- . at the present time, we need prospective, randomized, and placebo-controlled studies, which will provide us an answer -what is the real role and importance of remdesivir is in the treatment of covid- . there are currently other studies with remdesivir underway. the ema in its latest release recommends that remdesivir can be used as a compassionate use in very severe patients ( ) . the term compassionate use refers to the use of a drug that has not yet been approved and which is used outside clinical studies under very strictly controlled conditions. this allows the patient to access a drug that is still in the developmental stage. however, remdesivir is not yet registered in european union countries ( , , ) . even before the covid- vaccine is found, national leaders are faced with the dilemma of whether to immunize as much of the population as possible or whether compulsory vaccination carries the risk of movement, which is already prone to conspiracy theories about pharmaceutical companies and government authoritarian tendencies? preliminary results from a study conducted by the vaccine confidence project and orb international, in europe in early april, , while the infection was still on the rise, show that resistance to the vaccine is particularly strong in countries that have managed to avoid the worst pandemic ( , ( ) ( ) ( ) . in switzerland, where immunologists suggested mass vaccination as early as october last year, % of respondents said they did not want to be vaccinated. in austria, skepticism about the vaccine is at a similar level, as % of respondents said they would refuse the vaccination, according to world media ( ) . the number of germans who answered positively to the question about the vaccine against covid- from mid-april to mid-may, fell from % to %. in the uk, orb international conducted a survey on may th and th, and % of respondents said they did not want to be vaccinated. four european governments (of italy, germany, france and the netherlands) for their citizens has signed contract with british pharmaceuthical company "astrazeneca" to supply the region with its potential vaccine against the coronavirus -the british drugmaker's latest deal to pledge its drug to help combat the pandemic. the contract is for up to million doses of the vaccine, developed by the university of oxford, which will be produced until the end of the year , as announced minister of haelth of italy, roberto speranza. the deal is the first contract signed by europe's inclusive vaccines alliance (iva), a group formed by france, germany, italy and the netherlands to secure vaccine doses for all member states as soon as possible ( ) . but, virologists weigh evidence and often come up with somewhat contradictory advice, while public health authorities need weeks to formulate their messages ( , ) . patients with cvds must be observed by health professionals (specialists of cardiology, family practitioners, epidemiologists, etc) with special preventive measures regarding covid- infection ( ). the severity of the primary respiratory syndrome and risk of adverse outcomes is increased in patients infected with covid- and with pre-existing cardiovascular diseases. patients with coronary artery disease and heart failure may be at particular risk as a result of coronary plaque rupture, secondary to virally induced systemic inflammation, and rigorous use of plaque stabilizing agents (aspirin, statins, beta-blockers and angiotensin-converting enzyme inhibitors) has been suggested as a possible therapeutic strategy ( , , ) . electrocardiographic changes and troponin elevation may signalyzing the underlying myocarditis, and echocardiography frequently demonstrates subclinical left ventricular diastolic impairment (with a higher likelihood of the need for mechanical ventilation in those patients with systolic impairment and reduced ejection fraction) ( ). pro-coagulant effects of systemic inflammation may increase the likelihood of stent thrombosis and assessment of platelet function and intensified anti-platelet therapy should be considered in those patients with a history of previous coronary intervention. • author's contribution: all authors gave substantial contribution to the conception or design of the work and in the acquisition, analysis and interpretation of data for the work. each author had role in drafting the work and revising it critically for important intellectual content. each author gave final approval of the version to be published and they are agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. final proof reading was made by the first two authors. • conflict of interest: none declared. • financial support and sponsorship: nil. public health aspects of co-vid- infection with focus on 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covid- ? clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china pharmacologic treatments for coronavirus di-sease (covid- ). a review effective treatment of severe covid- patients with tocilizumab th responses in cytokine storm of co-vid- : an emerging target of jak inhibitor fedratinib covid- : consider cytokine storm syndromes and immunosuppression flooded by the torrent: the covid- drug pipeline remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial esc guidance for the diagnosis and management of cv disease during the covid- pandemic sleep and motion disorders of physicians and nurses working in hospitals facing the pandemic of covid- pharmacologist's view of the new corona virus key: cord- -t l zii authors: mayer, j.d. title: emerging diseases: overview date: - - journal: international encyclopedia of public health doi: . /b - - . - sha: doc_id: cord_uid: t l zii emerging infectious diseases are diseases that are either new, are newly recognized, or are increasing in prevalence in new areas. resurgent diseases are also usually grouped in this category, as is antimicrobial resistance. these diseases have been given formal recognition in the past two decades, although a historical outlook demonstrates that the phenomenon has probably been persistent, although largely undetected, through recorded history. emergence has accelerated recently, driven by factors such as demographic change, land use change, increased rapidity and frequency of intercontinental transportation, and other mostly social trends. continued infectious disease emergence poses, and will continue to pose, significant challenges for public health and for basic science. emerging and re-emerging infectious diseases have been major features of contemporary societies. indeed, there is evidence that history has been characterized by the constant interplay of humans and pathogens (mcneill, ) . however, it is impossible to say when the terms 'emerging infection' or 'emerging infectious diseases' were first used to describe new infectious diseases, or diseases that meet the criteria that are described in this article. the belief in the s that the threat of infectious diseases had been eliminated in developed countries was unfounded. a broader view of history would have demonstrated this. one possible reason for the optimism is that the s was a decade of optimism in general. in the united states, social programs were instituted to address inequities; humankind had not only orbited the earth, but landed on the moon; the gains of science and technology were impressive; economic expansion was equally impressive; poliomyelitis had been all but eliminated in the united states; and the sense of 'control' was widespread. beyond the borders of the united states, however, in africa, asia, latin america, and elsewhere, malaria proved to be a huge challenge to life, although its prevalence was decreasing, and diarrheal diseases continued to take their toll, particularly among the young. transportation links created the potential for transmission of infection between tropical regions and developed countries such as the united states. the potential for new diseases to emerge in the united states was there, and it took just a few years until this happened, catching the medical and public health communities by surprise. in discussions of emergence, both 'emerging infections' and 'emerging infectious diseases' are commonly found. while the two are closely related, they are not synonymous. an infection does not necessarily represent a state of disease. 'infection' suggests that an agent (usually a microbe) has become resident in the host. usually that agent is replicating in the host. however, the host need not show any sign of disease, in the sense that it can conduct its normal activities without hindrance. 'disease' is a state in which the normal functioning of the host is impaired, and both signs and symptoms are present -indeed, they are what limit normal function. an infectious disease is therefore a disease that is due to a pathogen. appeared de novo, or are being experienced in a region with greater intensity, or for the first time. some authors have used a more specific definition of emerging to diseases and have specified five types of emerging diseases: ( ) diseases that arise de novo, ( ) diseases that are newly recognized, ( ) diseases that have not previously existed in a specific area, ( ) diseases that had not yet made a species jump to humans until the present, and ( ) diseases that are increasing in prevalence. there are other definitions as well. the simplest definitions are frequently the most useful, and thus morse's definition will be used in this article. re-emerging infectious diseases are frequently thought of as being closely related phenomena to emerging infectious diseases. whereas emerging diseases denote diseases that are being experienced for the first time in a given location, re-emerging diseases are diseases that are reappearing in regions from which they have disappeared. usually eradication is due to deliberate efforts on the parts of government and public health agencies. for example, malaria control programs following the end of world war ii were instrumental in the elimination of malaria from some areas of the world, such as italy and spain. sometimes, malaria eradication was eliminated as part of multisector development programs. for example, the tennessee valley authority, created during the s primarily for flood control, hydroelectric power, and economic development, also had an explicit aim of malaria control. this resulted in the drainage of most swamps, and the elimination of malaria from this part of the united states. just as malaria was disappearing from many regions in the s, the next decade saw the resurgence of malaria, and the global prevalence of malaria has been increasing ever since. there are multiple reasons for this. these include anopheline spp. resistance to ddt, banning of ddt because of suspected environmental effects, and the development of resistance to chloroquine. malaria, then, is a re-emerging disease. another is tuberculosis. in many societies, tb had been nearly eliminated, but with the appearance of hiv/aids, immunocompromised individuals were much more susceptible to tb reactivation. tb, therefore, is also considered to be a re-emerging disease. the public and the medical and public health communities gradually came to realize that their complacency over the potential threat of infectious diseases was misplaced, and that new and emerging diseases constituted one foci of concern over health threats to the public. this change in attitude came gradually, and can be thought of as a series of historical 'moments,' each of which refocused attention on infectious diseases. while it is impossible to be exhaustive here, this section takes a roughly chronological approach in describing the events that led the public and professional communities to realize that infectious diseases had not been 'conquered.' the bicentennial of the united states was celebrated in , and there were many gala events around the nation in july. one was the meeting of the pennsylvania chapter of the american legion. the events surrounding this meeting were the first to bring the attention of both the population and the broad scientific and medical communities to the argument that infectious diseases in the united states had been 'conquered,' and both alarmed the public and aroused the curiosity of the scientific and medical communities because this appeared to be a new disease. indeed, before legionellosis was identified and antimicrobial treatment identified, legionellosis was called a 'monster disease. ' over members of the american legion who had attended the meeting developed an unusual respiratory illness, and it became clear that it was of bacterial etiology, although it was initially thought to be viral, due to its close clinical resemblance to influenza. approximately people died as a result of this outbreak. however, two things remained unclear. first, the pathogen could not be identified with conventional methods, and second, no common source of exposure could be identified initially, although the fact that the number of incident cases followed a typical epidemic curve suggested very strongly that there was some sort of common exposure to the pathogen. the news media seized upon this medical 'mystery,' and the public knew that they were dealing with an unknown infectious disease. this constituted a historical moment in contemporary american history, because it had been decades since something like this had happened. six months later, the bacterium was finally identified. legionella was not a new bacterium. stored samples from outbreaks as early as tested positive for legionella spp. however, the bacterium had not been identified in these outbreaks because it had not yet been described and characterized. in retrospect, most renowned is an outbreak that occurred in pontiac, michigan in , although the symptoms were milder than in the legionella outbreak in philadelphia. in fact, mild legionellosis with a nonpneumonic form is often called 'pontiac fever.' this is not the place to review the epidemiology, pathophysiology, and clinical aspects of legionellosis in depth. briefly, though, it usually has an acute onset, and is usually caused by legionella pneumophila, although other species are also pathogenic. in fact, there are species of the genus, and numerous serotypes. epidemiologically, l. pneumophila is by far the dominant species in human disease. the major reservoirs are bodies of freshwater, and the main mode of transmission is through small droplets that are inhaled from the environment. in the philadelphia outbreak, the source was finally traced to the air conditioning system in the hotel in which most attendees were lodged; the attendees were inhaling small particles in certain parts of the building. dozens of subsequent outbreaks have been traced to similar mechanisms. these have been not only air conditioners but also shower heads, aerosolizers in sinks, and whirlpools. virtually anything that aerosolizes fresh water is a potential mechanism by which legionellosis may be transmitted. symptoms of classic legionnaires disease are nonspecific and include fever, malaise, headaches, and myalgias. frequently, rigors will develop, as will a productive cough (in about half the cases). dyspnea (shortness of breath) is almost invariably present, and chest pain is common, as is a relative bradycardia for the elevated temperature. there are a number of abnormalities in laboratory tests, and chest films are markedly abnormal. a urine antigen test is available for one serotype, so laboratory diagnosis must frequently rely on more complex and time-consuming laboratory methods such as dfa. sputum cultures or cultures from bronchoalveolar lavage have been the mainstay of laboratory diagnosis. since laboratory methods do not show a definitive diagnosis until a minimum of days following onset, diagnosis is usually made on clinical grounds, and treatment is initiated based upon index of suspicion. erythromycin proved to be effective in , and other macrolides (azithromycin, clarithromycin) are highly effective. tetracycline and doxycycline are frequently used, as are the fluoroquinolones, such as levofloxacin. in hosts who are not immunocompromised, the prognosis is generally positive. there is no doubt that legionellosis was an emerging disease when it was first identified. its particular significance lies in its historical context -in the fact that this was the first occurrence that began shaking the optimism of the s and early s that infectious diseases had been conquered, and also in the fact that the etiology of an obviously infectious syndrome with a reasonably high case fatality ratio remained unknown for a number of months. chronologically, the next event to bring infectious disease to the attention of the public was another emerging infectious syndrome. in late and , a number of women in the united states became seriously ill with a syndrome characterized by high fever, shock, rash, hypotension, and capillary leak. this syndrome had been first described as such years earlier, although in retrospect it had been noted in the medical literature in the s. the paper identified toxic shock syndrome in males, females, and children -and the females were both menstruating and not menstruating. the outbreak was associated with menstruating women, many of whom were using superabsorbent tampons. although this was a major risk factor in the - outbreak, much of the public and many physicians were under the erroneous impression that toxic shock syndrome (tss) was necessarily associated with menstruating women who were using superabsorbent tampons. although tss is not necessarily associated with menstruating women, this does remain a risk factor in the epidemiology of tss. as with legionnaires disease, tss was a rare disease, yet the public's perception of it was out of proportion to its true prevalence -the risk was exaggerated. this is something that social scientists have called the 'social amplification of risk' in the context of new events that are potentially dangerous, but that nonetheless carry with them a low risk. amplification takes place as a result of media coverage, and as a result of intrapsychic processes that tend to amplify the threat of novel threats when the locus of control over the event is external to the individual. during the outbreak of toxic shock syndrome, newspapers were full of stories about tss and the sometimes deadly consequences of developing the syndrome. these were frequently on 'page above the fold' and necessarily caught the attention of the public. the same was true of television news. once this outbreak of tss appeared to be concentrated in one single group -menstruating women using superabsorbent tampons -the general public's fear of tss began to diminish, and the federal government mandated the withdrawal of those tampons from the market. the number of incident cases began a rapid decline, and was back to baseline of about cases per year by . some reports demonstrated that there was a decrease in the use of all tampons -not just superabsorbent tampons. it was already known in that toxic shock syndrome was caused by staphylococci (specifically, s. aureus). in these cases, treatment is threefold: removal of the tampon, indwelling tampon, or other hypothesized environmental cause; aggressive fluid resuscitation; and rapid use of antistaphylococcal antibiotics. other bacterial species can cause toxic shock syndrome. in rare cases, other staphylococcus species have been associated with toxic shock syndrome, and because they are coagulase-negative, they are difficult to treat. at this time, coagulase-negative staphylococci constitute the most common cause of hospital-acquired bacteremia. this sometimes results in endocarditis, and usually the only effective treatment is surgical valve replacement, particularly in the case of those who have had earlier valve replacement. aggressive antibiotic therapy is occasionally effective. should toxic shock syndrome be considered to be an emerging disease? it certainly was in , when the public was so concerned with its appearance. now, in , years after it was first described, this label is more questionable. what was most significant about toxic shock syndrome, however, was its historical significance. it followed the outbreak of legionnaires disease so closely that it turned the public's attention, once again, to infectious diseases, and to infectious diseases that had been unknown. it also reminded the biomedical community that infectious diseases had not been conquered. the issue at the time was whether legionnaires disease and toxic shock syndrome were anomalies, whether the assumption of the conquest of infectious diseases had clearly been erroneous, or whether these two outbreaks were harbingers of a new stage in 'epidemiologic history'a historical period during which emerging infections would become common and would catch the attention of the public, the public health community, the medical community, and government agencies. the public health and medical communities were divided on this. it would soon become clear, however, that the latter would hold true -that emerging infectious diseases would come to the forefront of public health, epidemiology, and the medical community. in the cases of legionnaires disease and tss, the social amplification of risk exaggerated perceived threats. nonetheless, the public became more attentive to infection. two other phenomena would solidify this attention. one was the appearance of hiv/aids in the united states, and the other was public attention that was drawn to hemorrhagic fevers, mostly in africa. the details of hiv/aids are covered elsewhere in this encyclopedia, and there will be no attempt here to duplicate this material. rather, this discussion concentrates on the significance of hiv/aids. when hiv/aids first appeared in several urban areas in the united states in , it appeared to be an anomalous syndrome. it was not called 'aids' until , when the centers for disease control (cdc) gave the syndrome that label. in the same year, researchers at cdc also linked one of the pathways of transmission to blood and blood products, causing a great deal of public concernif it was possible to contract aids through a frequently used medical practice, it had the potential of affecting millions of people. until then, aids was thought to be restricted to the gay community. in , blood banks were warned by the cdc that blood and blood products could definitely transmit aids, and surgeons and other medical personnel began rethinking the criteria necessary for transfusion. by , it was clear that the exponential increase in the number of incident cases was a definite trend. in and , two teams discovered that the pathogen causing aids was viral, and although it had a different nomenclature at first, there was a great deal of relief that the causal agent had been discovered. it is an interesting study in the sociology of science to analyze the competing claims by luc montagnier at the institut pasteur and robert gallo in the united states concerning their respective claims that they discovered hiv. it is now clear that montagnier discovered the virus. shortly after the virus was discovered and characterized, an antibody test was developed to detect hiv in vivo. this was quickly used to screen blood products as well as to detect hiv in individuals. whereas some people decried the slowness of the u.s. government's response to hiv, the time from the first presentation of a group of males with kaposi's sarcoma or oral thrush until the antibody test for a recently identified virus was only years. granted, the president of the united states, ronald reagan, had not even mentioned aids, and funding was less impressive than it could have been, but the time was quite short. the real challenge with hiv has been to find an effective vaccine, or to find a 'cure,' although antivirals have been effective in suppressing viral load in the majority of cases since - . the prevalence and mortality data are well-known. the best estimates are that globally, over million people are living with hiv/aids, and approximately million have died of hiv/aids. currently, about - million of those living with hiv/aids are women, and in developing countries, particularly in sub-saharan africa, hiv/aids is becoming, increasingly, a disease of women. currently, approximately two-thirds of those living with hiv/aids are in sub-saharan africa, but the increasing prevalence and incidence of hiv/aids in asia -and particularly, in india and china -are making east asia and south asia regions of tremendous concern. this is because each country has over billion people, and the prevalence rates do not have to be high to result in large numbers of infected people. the global significance of hiv/aids is that it, by itself, has altered demographic trends, and the political economy of nations and regions, not to mention the human suffering that this disease has exacted. in botswana and swaziland, for example, the gains in life expectancy during the th century have not only been completely reversed, but the life expectancy at birth is lower now than it was at the beginning of the th century. in the context of this article, hiv/aids is an emerging infectious disease par excellence. a generation ago, it was literally unheard of. now in all developed countries and in many developing countries, hiv/aids shapes many behaviors, is responsible for significant stigma, is feared, and causes a significant percentage of deaths. globally, hiv/aids is the fourth leading cause of death, although in many parts of africa, it is the leading cause of death. hiv/aids is an emerging infectious disease because of the historical rapidity with which it moved from an unknown localized zoonotic complex in west and central africa to the most prevalent infectious disease in the world. while the scientific evidence suggests that there were a number of species jumps of both hiv- and hiv- that occurred in africa, these were so localized and the societies isolated enough from the rest of the world that hiv went unnoticed. thus, it appeared as though the disease went from nonexistence to a major pandemic in a matter of a few years. and there is another major significant dimension. since hiv/aids appears to have originated in africa -'out there,' away from northern europe and north america -some have argued that hiv/aids acquired a certain nefariousness -a disease emerging from the dark, foreign, isolated jungle -the stereotypical cauldron of new diseases. viral hemorrhagic fevers have been in the public eye since , when there was a major outbreak of a hemorrhagic fever in the jos plain of nigeria. the disease came to be called lassa fever, caused by an arenavirus (lassa) that seemed particularly undesirable to the public. the virus is named after the town in which this outbreak occurred. like all hemorrhagic fevers, including dengue in some cases, one of the characteristics of lassa fever is that it can disturb the clotting/coagulation mechanism, resulting in disseminated intravascular coagulation (dic) and diffuse hemorrhage. the outbreak was publicized in the united states through the news media, perhaps because it was an 'exotic' or newsworthy event, and once again, the social amplification of risk was responsible for exaggerated fears of 'what if it spreads here?' that this outbreak occurred in sub-saharan africa, which, in the eyes of the north american public, may have been thought to be all 'jungle' (the jos plain is not rain forest) probably also contributed to the amplification of risk. serologic tests demonstrate that exposure to lassa virus is common in west africa. for example, in parts of nigeria, seroprevalence is positive in % of those tested; in sierra leone, the figure varies from - % depending on the region (richmond and baglole, ) . it is now known that humans are dead-end hosts, and that the rat species mastomys natalensi is the natural host. these rats are extremely common throughout sub-saharan africa. people become infected by inhaling aerosols from rat excreta, and risk is increased by eating them, which is a very common practice in west africa. modern modes of travel have allowed infected individuals who are either symptomatic or asymptomatic at time of entry to travel to other continents, where they require treatment for lassa fever. these cases have not been numerous, but cases have appeared in the united states and japan, as well as in several european countries. this has caught some clinicians unprepared, since they were not trained in tropical medicine and were unaware of how to diagnose or manage a viral hemorrhagic fever. the prevalence rate of lassa fever is much higher than was initially thought. in one series, lassa fever accounted for % of adult deaths in sierra leone, and as many as % of hospital admissions (richmond and baglole, ) . following the outbreak in , it took some time to investigate adequate treatment protocols, but now, aggressive fluid replacement and the use of antiviralsparticularly ribavarin -are the treatments of choice. ebola hemorrhagic fever and closely related marburg virus are both single-stranded rna viruses, as are other viruses that cause hemorrhagic fevers. ebola and marburg are filoviruses; ebola virus is actually a genus and there are four species. it was first described in the sudan in , and estimates are that mortality from this virus has now exceeded people. the case fatality ratio exceeds %, and may be as high as % in some cases. transmission is different than lassa fever. it is usually through direct contact with blood and bodily secretions from individuals who are ill with ebola fever, or from nonhuman primates who are also infected. evidence points to bats as the natural reservoir of ebola virus, but this is not certain. in several studies, however, bats have been shown to be infected by the virus (leroy et al., ) . this is highly suggestive, but it is not conclusive proof. like so many other viral hemorrhagic fevers, the symptomatology of ebola is very nonspecific and typical of viral syndromes in general. the clinician needs to have a high index of suspicion. at this point, the only certain treatment is supportive, and from a public health point of view, quarantine is of the utmost importance, since ebola fever is so contagious. this was well-documented by the news media in the outbreak in kikwit, democratic republic of the congo (drc, then zaire) in . this was so well-documented that once again it led to exaggerated perceptions of risk, with overtones of the 'exotic disease' from sub-saharan africa and its possible spread to the united states. recent advances in understanding the pathogenesis of ebola and the role of proinflammatory cytokines has led to the use of some recombinant products that block the progression of the inflammatory cascade to dic in some animal models. nonetheless, this approach has not been used in humans as of . there are three notable points that need to be mentioned concerning ebola. first is that it appears to be increasing in prevalence in africa. this may be because detection is better and the disease has been better described, both epidemiologically and pathophysiologically. second is that there is significant concern that ebola virus could be used as a biological weapon. it has thus been placed on the highest level (category a) of potential biological weapons by the cdc. finally, ebola, more than any other emerging infectious disease, typifies in the mind of the public the sort of dangerous, threatening disease risk that is associated with tropical areas, the 'jungle,' and the threats that are associated with a more interconnected world. bovine spongiform encephalopathy (bse), or 'mad cow disease' in nontechnical terms, is another infectious disease that focused public awareness on emerging infections. the pathogen in this case was unusual not only in the sense that it had not been described elsewhere, but also because the whole class of pathogens -prionshave been very rare. like another neurologic disease, kuru, bse turned out to be due to a prion. essentially, prions are very simple since they are just unusually folded and self-replicating proteins. they cannot even be described as organisms. the source of the prion is not known, although many speculate that it is somehow derived from sheep infected with scrapie. in , an unusual disease seemed to be affecting cattle in the united kingdom, and by the end of the year, over cattle had died because of spongiform encephalopathy. since it was apparent that the disease was contagious, over million cattle were intentionally slaughtered to limit contagion and ensuing effects on the cattle industry. by the mid- s, there was a clear epidemiologic association between bse and a variant of a neurodegenerative disease in humans that had been described in the middle of the th century: creutzfeldt-jakob disease (cjd). however, there were some notable differences between cjd and the disease that was affecting humans in the s. the median age of this new syndrome was much younger than in classical cjd; the median duration of survival from onset of symptoms was longer than in classical cjd; and pathological differences and differences on mri were apparent with this new variant. accordingly, the cjd associated with bse first was named 'new variant creutzfeldt-jakob disease' or 'nvcjd;' as time progressed, nvcjd was renamed 'variant cjd' or 'vcjd.' although there were very few cases of vcjd in the uk human population, the threat of this disease was great according to public perception. according to the world health organization (who), as of november , there had been cases of vcjd in the united kingdom, six in france, and one each in several other countries (who, ) . nearly all of those with vcjd died or would die within years. because of the realistic fear of contagion, several steps have been taken to limit the spread of vcjd. feeding practices for cattle have changed so that it is no longer legal to feed animal protein that might contain any tissues proximal to the central nervous system to other cattle. in the united kingdom, there was a ban on cattle over months old from entering the commercial food supply. in the united states, individuals who have lived in the united kingdom or who have spent more than months in the united kingdom are banned from being blood donors on the assumption that they might have consumed infected beef during their stay(s) in the united kingdom. a ban was instituted on importing cattle and cattle feed from the united kingdom, and, occasionally, from canada, in an attempt to prevent bse from spreading to the united states (kuzma and ahl, ) . while the number of incident cases of vcjd and bse have decreased in a typical epidemic curve pattern, the effects of the bse 'scare' have been tremendous. the very credibility of the uk government was threatened. the whole cattle and meat industries were severely hurt. on the other hand, surveillance techniques and understanding of cattle food chains were vastly improved. severe acute respiratory syndrome (sars) proved to be of great import in both the public awareness of emerging infectious diseases and in the testing and real-time construction of both domestic and international systems of public health surveillance and response. it was particularly important in terms of public awareness because it spread very rapidly on the international and intercontinental scales. sars apparently began as a few cases of a viral pneumonia in guangdong province in southeastern china in late . however, this was not immediately apparent to the global public health communities because it was not publicized by the chinese government. what catapulted sars to international attention in the media and in the public health community was the appearance and rapid increase of incident cases in guangdong in february (zhao, ) . sars spread rapidly to hong kong, where contact tracing eventually identified one night in a specific hotel where the index case stayed as being the epidemic focus. the index case infected at least others who were in the hotel at one time or another during that night. sars spread from hong kong to other areas of hong kong and to singapore, vietnam, and canada (toronto, ontario). the spread of all these cases has been traced to airplane travel, followed by localized spread by an index case. a case definition was developed based upon clinical presentation, which typically consisted of fever, initially, followed by lower respiratory signs and symptoms, sometimes resulting in acute respiratory distress syndrome and respiratory distress typical of acute lung injury as a response to the inflammatory cascade. just over cases were identified worldwide, and died, for a case fatality ratio just < %. a disproportionate degree of contagion occurred in intensive care units and areas of hospitals in which hospital personnel were exposed to respiratory excretions; close proximity -within m -to an infected patient who was undergoing endotracheal intubation was the single greatest risk factor for contracting sars. local measures to control the spread of sars consisted largely of quarantine and containment. in china, for example, separate quarters for sars patients were constructed very rapidly. in singapore, arriving and departing passengers were required to pass through automated temperature detectors, and anybody with a fever was required to undergo further medical evaluation. the same was true at most points of entry in most developed countries. since most cases were contracted in hospitals and health facilities, rigorous contact control procedures were instituted, and in some cases, hospitals were closed to visitors and new admissions. the identification of the pathogen causing sars constitutes a textbook example of how international cooperation in science and public health may occur when the willpower is there and the scientific capability exists. by mid-march , many leading laboratories with advanced virologic capabilities had agreed to cooperate in a network that was coordinated by the world health organization. within weeks, a pathogen was identified as a novel coronavirus, using a combination of methods: molecular polymerase chain reaction, culture, and electron microscopy, and shortly thereafter, the criteria of koch's postulates were met. thus, the evidence was quite clear that the new coronavirus was the pathogen. the virus was named the sars coronavirus, or, almost always, sars cov. the ecology of sars was not understood as quickly as the pathogen was identified. some features were identified within a number of months. first was the phenomenon of superspreaders, which is a concept that previously had received scant attention. in this case, it became apparent that a small number of individuals spread sars to a disproportionately large number of people. it is not clear whether this is because of behavioral factors, host-pathogen interaction, or environmental factors. what is fairly clear is that were it not for superspreaders, the epidemic would not have affected nearly as many people as it did. this is because the r , or number of people who one individual could infect, was inflated by superspreaders. thus there was a domino effect of contagion. in , bats were identified as the reservoir of sars cov. there had previously been some speculation about bats being the reservoir, but there was no solid evidence, and the reservoir had been a mystery. some had suggested that proximity of people to avian species could possibly be a factor in the pathogenesis of sars, because of the importance of this process in avian influenza. however, this turned out not to be the case with sars. sars is a prototype of an emerging infectious disease (berger et al., ) . there is no evidence that sars cov existed in the human population prior to the outbreak of late - . the specific syndrome surprised the public health and medical communities, yet its general features did not, and the emergence of new diseases had been a familiar concept since the u.s. institute of medicine report of . at the same time, the rapidity of the appearance of sars and its very rapid spread at every scale fueled public apprehension, and even hysteria in some cases. evidence exists that history has been punctuated by relatively regular influenza epidemics and pandemics. the rapidity of epidemic spread, leading to pandemics, is largely determined by the velocity of the prevailing transportation modes. severe epidemics and pandemics are caused by genetic shift, whereby the viral genome expressing surface antigens (hemagglutinin and neuraminidase) undergoes relatively major change. relatively minor epidemics occur because of genetic shift, in which the surface antigens undergo minimal yet detectable changes in their configuration. following genetic shift, people have minimal immunity to the virus, and are susceptible. in one sense, each year influenza constitutes an emerging infection, because the precise genome of the influenza viruses and the surface antigens undergo change. similarly, whenever a pandemic occurs, influenza represents a more significant emerging infection. on the other hand, influenza represents a disease entity that is not new to the population. thus, it is a matter of semantics whether to consider influenza to be an emerging infection. avian influenza may constitute the next serious pandemic threat. it has been known for decades that genetic reassortment occurs in southeastern china because of the proximity of humans, avian species, and swine. an unusual number of influenza epidemics appear to arise there. however, the concern over avian influenza arises from a slightly different situation. it has been known for some time that no less than influenza subtypes -different configurations of surface antigens -can infect aquatic bird species. it has been wellestablished that several of these subtypes can infect humans, although recent experience suggests that all subtypes that circulate in avian species may have the potential to infect humans. this is one of the reasons that has given rise to concern over the possibility of an avian influenza pandemic. this theoretical concern moved closer to reality in hong kong in , when one influenza strain (h n ) was transmitted directly from poultry to humans. this took place in 'wet markets' -markets in which live poultry are densely packed, and where people co-mingle with their intended purchases. the transmission in appears to have been limited: only cases were confirmed. however, the case fatality ratio was high. six of the people died. transmission also occurred with another strain - h n -in , and in and there was widespread transmission and mortality among chickens in hong kong. because of a concern over possible transmission to humans, and because of the devastating economic potential in the poultry industry, containment of this epidemic in poultry was partly obtained by the slaughter of millions of chickens and other poultry. avian influenza viruses have shown some propensity, since , for transmission to humans. so far, human cases of influenza that have been identified as avian strains have been limited to approximately , and these have all been in asia. human-to-human transmission has been implicated in only a few cases. if this is the case, what is the concern over avian influenza? because of the tendency for influenza viruses to mutate, many virologists and epidemiologists predict that there is a high likelihood that a mutation could occur that would facilitate human-to-human transmission of h n and other avian subtypes that have been transmitted to humans. if this occurs, then there is little doubt that this strain would spread rapidly among the human population, and would spread locally, nationally, and between continents in a manner similar to sars. other epidemiologists and virologists are more circumspect in their predictions, and argue that the probability of a mutation that would increase the propensity of avian influenza to spread from human to human is unknown. a minority of authorities argue that the probability is low. thus, in assessing the overall threat of avian influenza, the crucial question is whether the virus will spread readily from human to human. at this point (mid- ) , it is unknown whether this will occur. however, it is prudent public health policy to bolster surveillance systems, and governments are stockpiling neuraminidase inhibitors, which are medications that can moderate the course of influenza if taken early in the course of clinical disease, or sometimes prevent the onset of symptoms if taken prophylactically. similarly, there has been great emphasis on vaccine development and stockpiling. in response to growing public concern over emerging infectious diseases, both domestically and internationally, as well as to both interest and concern in the medical and public health communities, a major conference on emerging viruses was held at rockefeller university in . the conference was cosponsored by several government agencies. the conference participants reached many conclusions, but two of them were that emerging infections had become a major focus for scientific research and that emerging infectious diseases had become and would remain a major public health challenge for the united states. accordingly, the institute of medicine of the national research council of the united states took a proactive role and sought funding for a major study of emerging infections. the study was funded by a number of government units, and in early , a high-powered committee met in washington for the first time to: identify significant emergent infectious diseases, determine what might be done to deal with them, and recommend how similar future threats might be confronted to lessen their impact on public health. (institute of medicine, : vi) the committee issued a report in that quickly became a standard scientific and policy reference on emerging infectious disease. emerging infections: microbial threats to health was the first major comprehensive discussion of how emerging infections arise, and how they might be addressed by the public health community. the committee also identified the six 'factors' or causes of emergence. briefly, the factors that this committee identified were the following: human demographics and behavior; technology and industry; economic development and land use; international travel and commerce; microbial adaptation and change; and the breakdown of public health measures. it is notable that five of these six factors are social factors that are consequences of changes in society. even microbial adaptation and change, such as the development of antimicrobial resistance as a response to selective pressure, has a large behavioral dimension. this is partly a response to a technical innovation -the development of antimicrobials -and partly a response to a behaviorthe prescribing of those antimicrobials. of course, one dimension of this factor is the nonselective and improper prescribing of antimicrobials. this has several dimensions: the prescription of antibiotics when none are needed, the prescription of broad-spectrum antibiotics when narrowspectrum antibiotics are sufficient, the free availability of antibiotics in many developing countries on the street and in pharmacies where no prescription is needed, and the free use of late-generation antibiotics in the food industry to promote the growth of cattle, chickens, and other animals intended for human consumption. so, in fact, all of the six factors of emergence are social and behavioral in nature. it is ironic that despite the fact that both institute of medicine reports concluded that the major causes of emergence have been social, there have been very few social analyses of emerging infections. for example, emerging infectious diseases, a new journal founded in in response to the growing importance of emerging infections, has an explicit aim of including a social understanding of emerging infections in its contents, yet there have been very few articles written by social scientists in this journal, and very few articles with any social content have been published. the main point is that the overwhelming understanding of emerging infections has been 'biomedical.' this is not a criticism of either the journal or of any field in public health or medicine. in large part, this is the result of the sociology of knowledge and science. for whatever reason, few social scientists have become involved in research on emerging infections, whereas the same cannot be said about chronic diseases. some researchers have asked the question of why emerging infectious diseases are emerging now and in the societies where they are emerging, and have sought a more contextual understanding of emerging infections. david bradley asks a very penetrating question: [a]ttaching a microbiological label to an outbreak. . .does not answer either the micro-scale questions such as ''why is there an outbreak here, now, of this size, affecting these people?'' nor does it answer the macro-questions such as ''why are there more (or fewer) outbreaks this decade than last?'' nor does it answer the question ''what drives the overall worldwide trends in such problems?'' (bradley, : ) for example, a number of individuals have argued that emerging infections may represent another stage in the epidemiologic transition. our understanding of emerging infections has not been totally devoid of social analysis. inequality and poverty have become a major focus for the social analysis of health and disease. the argument is that through a complicated series of pathways that are yet to be fully understood, both poverty and inequality result in poor health status. this has not been applied extensively to emerging infectious diseases, although paul farmer's ( ) insightful work has been applied to emerging infections. in his critical analysis of emerging infection, farmer asks, ''emerging for whom?'' in other words, the diseases that westerners might label as emerging may have been present or endemic in poorer societies for a long time: if certain populations have long been afflicted by these disorders, why are the diseases considered ''new'' or ''emerging''? is it simply because they have come to afflict more visible -read more ''valuable'' persons? this would seem to be an obvious question from the perspective of the haitian or african poor. (farmer, : ) in other words, farmer argues, the concept of emerging infectious diseases is one of epistemology -the theory of knowledge. how do emerging diseases come to be categorized as 'emerging'? by implication, many of these diseases have been present in poorer societies for a long time. the evidence affirms this. hiv was probably present in small foci in central africa for decades to centuries; ebola was similarly endemic in west africa for an unknown period, as was lassa fever. what is novel about the past few decades is greater interconnection between places, allowing diseases, and news of diseases, to spread; better methods of detection; and changing settlement geographies that have brought people into different forms of contact with animal reservoirs. the root cause of the infectious disease emergence is human action, both intentional and unintentional. most of this action is the result of cumulative individual acts on a mass scale. for example, the mass urbanization of society in poorer countries is the sum of millions of individuals who move from rural to urban areas. this is largely the result of the perceived economic opportunities in urban areas, and the 'push' factor of lack of opportunity in rural areas. yet, taken together, millions of individual moves result in urbanization, and this urbanization facilitates the spread of diseases by the respiratory route, the fecal-oral route, and many other modes of transmission. the institute of medicine committee also developed a set of policy recommendations. these concentrated in two areas: the need for vastly increased resources for interdisciplinary training in infectious diseases because of the depleted workforce resources in this area; and the need to develop new surveillance and public health response systems, since the committee had determined that emerging infections did, indeed, constitute a major public health threat to the united states. this report was issued with a great deal of publicity. the u.s. public's attention was already focused on emerging infectious diseases as a result of legionnaires disease, viral hemorrhagic fevers, and toxic shock syndrome. now there was a major quasi-governmental report by a group of the nation's leading scientists who issued the sobering conclusion that: even with unlimited funds, no guarantee can be offered that an emerging microbe will not spread disease and cause devastation. (institute of medicine, : ) part of the institute's report identified specific microbes and diseases that could possibly threaten public health in the future. three of these were e. coli :h , cryptosporidiosis, and hantavirus. the report was prescient, because within a few years there were serious outbreaks of all of these. in , which was the year after the iom report was issued, there was a major outbreak of cryptosporidiosis on the south side of milwaukee, wisconsin. it caused diarrhea, ranging from mild to severe, in over people. cryptosporidium parvum is a protozoan parasite; evidence in animal models is that ingestion of even one oocyst can result in severe gastrointestinal symptoms. in humans, as few as oocysts can produce these effects (king and monis, ) . it is impervious to usual methods of water treatment, and only recently has an effective medication become available. the milwaukee outbreak was probably due to groundwater absorption of cattle feces, subsequent runoff due to both heavy rains and snow melting, transport of the oocysts to river tributaries, and movement of the oocysts into lake michigan, which serves as the water supply for the south side of milwaukee. the filtration plant for that water was ineffective in eliminating the oocysts. many of these events are putative, but together they constitute a logical chain. meanwhile, research is still proceeding on the ecology of cryptosporidiosis. understanding is progressing, but it is still incomplete. e. coli :h was also mentioned in the iom report as being an emerging disease. in january , the washington state department of health ascertained that an outbreak of :h was occurring in the state, and this outbreak was associated with having eaten at jack in the box fast-food restaurants. subsequently, it became apparent that the epidemic was not limited to washington, but also included idaho and nevada. the epidemiologic investigation of this outbreak was intricate, and implicated a chain of events. first, because meat inspection in the united states was inadequate, one theory is that e. coli :h from the bowels of cattle had gotten into meat that was sent to market when cattle were slaughtered, and the bowel was probably nicked or severed. another is that under stress, cattle defecate over one another, and fecal matter from one cow can contaminate the hides of other cattle. second, when this meat was ground into hamburger, it increased the surface area of the meat by several orders of magnitude, thereby allowing the pathogen a great deal of exposure. third, once this hamburger meat was shipped to jack in the box restaurants, it appears that hamburgers were being systematically undercooked, below industry standards. this allowed the e. coli to survive and enter the hosts' systems. the consequences of such infection can be severe, and were in , with those who were symptomatic frequently suffering from bloody diarrhea, fever, cramps, and, in the worst case, hemolytic uremic syndrome. the pathogenesis of this disease was only partially understood in , but understanding is more complete in . the third disease that was mentioned in the iom report that occurred shortly after its publication was hantavirus. in may , in the four corners area of arizona, new mexico, california, and utah, several males who were otherwise in good health developed a sudden serious respiratory disease that was thought to be a rapidly progressing acute respiratory distress syndrome, since this was the immediate cause of death. however, it was noted that these cases had formed a cluster, and investigators tried to find some sort of common source to explain a possible environmental exposure to explain this serious and sometimes fatal syndrome. though hantavirus had never been described in the united states, serologic tests in patients showed a surprising seropositivity to hantavirus. it was apparent that this was the pathogen that had caused the dozen deaths associated with the outbreak. the chain of events that led up to the outbreak is now fairly clear. winter was unusually warm in the four corners area as a result of el nino, and the spring was also unusually rainy. these two conditions led to the rapid and plentiful growth of pinon trees, which provided food for a number of rodents. there is consensus that the deer mouse (peromyscus maniculatus) population increased by an order of magnitude. testing demonstrated that about % of the mice that were trapped after this epidemic were infected with hantavirus, and studies demonstrate that households from which infected individuals came were far more likely to have heavy rodent infestations than were households of controls. more rigorous studies eventually showed that transmission occurred from rats to humans, and that many of the cases, in this instance, were associated with crawling under houses and other places in which rodent exposure was likely to occur. by , many of the predictions of the first institute of medicine report ( ) had been realized, and understanding of emerging infectious diseases had improved. there was greater focus on globalization as a process of disease spread, and the attacks on the world trade center and pentagon on september , focused attention on terrorism. a new institute of medicine committee was formed to consider the nature of microbial threats and emerging diseases, and the report of this committee was issued in (institute of medicine, ) . this report represented a rethinking of the factors of emergence, and presented a more nuanced understanding of the causes of emerging diseases, most of which were still social at one level or another. bioterrorism ('intent to harm') was specifically mentioned as a factor of emergence, as was lack of political will. policy recommendations for surveillance, response, and training were more detailed than in the report, and there was a more urgent tone to the need to respond to emerging threats. in this report, the emphasis on biological and social interaction was strong: genetic and biological factors allow microbes to adapt and change, and can make humans more or less susceptible to infections. changes in the physical environment can impact on the ecology of vectors and animal reservoirs, the transmissibility of microbes, and the activities of humans that expose them to certain threats. human behavior, both individual and collective, is perhaps the most complex factor in the emergence of disease. emergence is especially complicated by social, political, and economic factors. . .which ensure that infectious diseases will continue to plague us. (institute of medicine, : ) increasing resistance to antibacterials, antivirals, and other antimicrobials is frequently grouped under the heading of 'emerging infections.' resistance is certainly a constantly growing and very major public health problem, but this is of importance to emerging infections only in the sense that diseases that were once highly treatable with first-and second-generation antimicrobials are no longer treatable by them. the selective pressures exerted by antimicrobials have made numerous pathogens resistant to even the newest antimicrobials due to mechanisms that are now understood. for example, many respiratory pathogens are no longer treatable by b-lactam antibiotics since their b-lactam rings are cleaved by b-lactamases. there are fluoroquinolone-resistant strains of neisseria gonorrhoeae, resistant strains of staphylococcus aureus, and so on. the problem is most severe in hospitals, where severe infections once responsive to vancomycin are now resistant to this glycopeptide. several new antimicrobials have been developed, in part to address vancomycin resistance, but resistance to these medications developed within a few years of their introduction. thus, antimicrobial resistance is both a community problem and a hospital problem. there is great concern over multiple drug-resistant tuberculosis, which is defined as tuberculosis that is resistant to two first-line medications, and extensively resistant tuberculosis, which has a more complex definition specifying several medications. there is not space in this article to explore antimicrobial resistance in greater depth. the relationship between people and pathogens has been an integral part of history, and will continue to be. the progress in the diagnosis, detection, and clinical management of infectious diseases has been substantial. indeed, fauci ( ) has gone so far as to argue that: the successful diagnosis, prevention, and treatment of a wide array of infectious diseases has altered the very fabric of society, providing important social, economic, and political benefits. nonetheless, infectious diseases, aggregated together, constitute the second leading cause of death worldwide, and in many regions, they account for the dominant cause. moreover, emerging diseases will continue to emerge, because of constantly changing social and demographic conditions, as well as selective pressures. the prototypical emerging infectious disease, hiv/aids, has an uncertain future in the long run. perhaps a vaccine will be developed that will be inexpensive, and perhaps distribution systems will be developed that will transport the vaccine to points of demand. perhaps antiretrovirals will become extremely inexpensive, and perhaps the failure rate for antimicrobials of % will be overcome. however, it is unlikely under present conditions that all of these improvements will occur. thus, the future of hiv/aids is more sobering. the same is true of antimicrobial resistance. in an age of optimism when antimicrobials were developed and used successfully -perhaps the first years of antimicrobial use -concern over resistance was minimal. however, the fact that organisms adapt to changing environmental conditions and threats is something that has not been realized only recently. the inevitability of adaptation is undeniable, and the only way to meet the challenges of resistance is through a combination of appropriate antimicrobial use (including the use of narrow-spectrum antibiotics as soon as possible in the clinical course of an individual) and the development of new antimicrobials, as well as new understanding in the physiology and genetics of microorganisms, which might lead to the development of new technologies in addressing the pathogenic basis of disease. see also: aids, epidemiology and surveillance; antimicrobial resistance; severe acute respiratory syndrome (sars); transmissible spongiform encephalopathies; tuberculosis: overview; west nile disease. acromegaly a condition produced by overproduction of growth hormone, leading to excessive growth of the hands, feet, and jaw in postpubertal individuals and giantism in prepubertal children. adrenal glands two endocrine organs situated above the kidneys that make a series of hormones: cortisol (stress hormone), aldosterone (salt-retaining hormone), and catecholamines (stress hormones). autoimmunity a situation in which part of the body, often an endocrine organ, is recognized as 'foreign,' triggering an immune response that tends to lead to destruction of the endocrine gland. cushing syndrome excessive production of cortisol with loss of the normal circadian variation leading to weight gain, hypertension, and type diabetes mellitus. g protein proteins within the cell that transfer the hormone message from the receptor to specific parts of the cell. graves disease a combination of thyroid overactivity due to an autoimmune disorder and eye problems. hypothalamus part of the brain containing control centers for appetite, thirst, and pituitary hormone secretion. pituitary major regulator of hormone production. secretion of hormones regulated by the hypothalamus. severe acute respiratory syndrome (sars): paradigm of an emerging viral infection new and resurgent infectious: prediction, detection, and management of tomorrow's epidemics infections and inequalities: the modern plagues infectious diseases: considerations for the st century emerging infections: microbial threats to health in the united states microbial threats to health critical processes affecting cryptosporidium oocyst survival in the environment living with bse fruit bats as reservoirs of ebola virus plagues and peoples factors in the emergence of infectious diseases lassa fever: epidemiology, clinical features, and social consequences variant creutzfeldt-jakob disease sars molecular epidemiology: a chinese fairy tale of controlling an emerging zoonotic disease in the genomics era the coming plague: newly emerging diseases in a world out of balance new and resurgent infections: prediction, detection, and management of tomorrow's epidemics the changing face of disease: implications for society the challenge of emerging and re-emerging infectious diseases an emptying quiver: antimicrobial drugs and resistance the politics of emerging and resurgent infectious diseases disease in evolution: global changes and emergence of infectious diseases endocrine diseases: overview p c hindmarsh key: cord- -qun kyvw authors: pourbohloul, babak; meyers, lauren ancel; skowronski, danuta m.; krajden, mel; patrick, david m.; brunham, robert c. title: modeling control strategies of respiratory pathogens date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: qun kyvw effectively controlling infectious diseases requires quantitative comparisons of quarantine, infection control precautions, case identification and isolation, and immunization interventions. we used contact network epidemiology to predict the effect of various control policies for a mildly contagious disease, such as severe acute respiratory syndrome, and a moderately contagious disease, such as smallpox. the success of an intervention depends on the transmissibility of the disease and the contact pattern between persons within a community. the model predicts that use of face masks and general vaccination will only moderately affect the spread of mildly contagious diseases. in contrast, quarantine and ring vaccination can prevent the spread of a wide spectrum of diseases. contact network epidemiology can provide valuable quantitative input to public health decisionmaking, even before a pathogen is well characterized. p ublic concern regarding emerging infectious diseases is on the rise. the st century began with the emergence or reemergence of zoonotic diseases like severe acute respiratory syndrome (sars) ( ), avian influenza ( ) , monkeypox infection ( ), west nile virus disease ( ), mad cow disease ( ) , anthrax due to bioterrorist attacks ( ) , and unusual influenza epidemics ( ) . in addition to these new threats, public health officials face a large number of disease outbreaks every year in hospitals, schools, and other small communities. while development of vaccines and diagnostic tools proceeds at an unprecedented pace, development of tools for determining optimal intervention strategies lags behind. in response to this problem, we have found that mathematical models of disease transmission can be used to evaluate and optimize control strategies. such quantitative predictions can be empirically tested through randomized comparative trials, and mathematical models increasingly contribute to public health decisions regarding policy and intervention ( ) ( ) ( ) ( ) ( ) ( ) . we use contact network epidemiology to compare intervention strategies for airborne infectious diseases, including emerging diseases such as sars, for which epidemiologic data are limited. these methods are based on explicit mathematical models of the heterogeneous patterns of interpersonal contacts that underlie disease transmission in a community, be it a hospital, school, or city ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this approach differs from fully mixed compartmental models that assume that each person can infect every other person with equal probability ( ) . some compartmental models have been modified to include population heterogeneity and have provided insights into the long-term effects of intervention strategies ( ) ( ) ( ) ( ) . for communities with extensive heterogeneity in contact patterns, however, network models more explicitly capture patterns of disease transmission and thus enable more accurate and detailed predictions of the effect of control measures on the magnitude and distributions of outbreaks. vertex in the network, and each contact between people is represented as an edge connecting the vertexes. the number of edges emanating from a vertex is the degree of that vertex. this quantity indicates the number of contacts who potentially transmit disease to or acquire disease from a person. the variation of degree across the entire network, i.e., the degree distribution, is fundamental to determining the probability for spread of disease through a network of contacts. given the degree distribution of the contact network, one can analytically predict the fate of an outbreak. contact network epidemiology allows us to assess the vulnerability of a population to an infectious disease on the basis of the structure of the network (its degree distribution) and on the average transmissibility (t) of the disease ( , ) . t is the average probability that transmission will occur from an infected person (vertex) to an uninfected person. this parameter summarizes multiple aspects of transmissibility, including the contact intensity between persons, duration of infectiousness, and the host's susceptibility to the infectious pathogen ( , ) . we built an urban contact network model with , households with an average household size of . ( , persons) based on demographic information for the greater vancouver regional district, british columbia, canada. we used publicly available data from sources such as statistics canada to estimate the distribution of ages, household sizes, school and classroom sizes, hospital occupancy, workplaces, and public spaces ( ) ( ) ( ) ( ) ( ) . most of the edges in the network are undirected, meaning that transmission may occur in either direction (black edges in figure ). for example, persons living in the same household will have equal opportunities to infect each other. the remaining edges are directed, meaning that a person may infect another person but the converse is not true (gray edges in figure ). for example, suppose person a is healthy and has no reason to go to the hospital until he or she is infected with sars. at that point, person a will likely come into contact with and potentially spread sars to caregivers at the hospital. in contrast, if a caregiver at the hospital acquired sars while person a remained healthy in the community, then no opportunity would exist for transmission in the opposite direction. to model the directional flow of infected patients into a hospital, we include directed edges from persons in the population at large to caregivers in the hospital. in an urban setting, not all encounters are equally likely to lead to disease transmission. we capture this heterogeneity in ways. first, in the simulated urban network, the probability of a contact between persons depends on the location and nature of their overlapping daily activities. for example, persons in the same household are connected to each other with probability , while persons who encounter each other in a public space are connected to each other with a probability from . to . . second, after these connections are determined, we assign a distinct transmissibility, t ij , for each pair of connected persons i and j, that depends on the nature of their contact. for a given disease, the distribution of transmissibilities is based on empiric estimates for the diversity in infectious periods and the per day probability of transmission between persons who come into contact with each other. for more details, please refer to the online appendix, available from http://www.cdc.gov/ncidod/eid/vol no / - _ app.htm. in any given network exists a critical transmissibility value, t c , which indicates whether a large-scale epidemic is probable. any disease with average transmissibility <t c cannot cause sustained transmission within a population and will thus be limited to small outbreaks. such diseases die out because of the probabilistic nature of transmission before the disease has a chance to spread to the population at large. in this case, we can mathematically predict the expected size of small outbreaks, s. diseases with average transmissibility >t c will spark large-scale epidemics with probability s, which can also be estimated. the value of t c depends on the contact patterns within a community. roughly speaking, when abundant opportunities exist for transmission, disease will spread easily, and the epidemic threshold will be low. the equations for s, s, and t c , which are entirely in terms of the degree distribution and average transmissibility t, are presented in the online appendix. the epidemic potential of disease is commonly estimated by using the basic reproductive number r , the number of secondary infections arising from a single infection in a relatively naïve population ( , ) . this quantity is linearly related to the transmissibility of the disease, i.e., r = γt, where γ depends on the structure of the network (equations and in online appendix). when t is at the epidemic threshold (t = t c ), then r = . public health interventions aim to reduce the number of new infected cases, ideally decreasing the effective reproductive number of the disease below the epidemic threshold, r eff < . the difference between average transmissibility t and the basic reproductive ratio r is important. while both have threshold values that distinguish epidemic from nonepidemic scenarios (r = and t = t c ), t is determined by the transmission characteristics of the pathogen and the nature of human interactions, but not the numbers of contacts in a community, whereas r depends on all of these factors, particularly on the numbers of interactions within the community. for example, consider a single airborne pathogen spreading through a hospital, where abundant close contacts exist, and through a rural community, where close contacts are rare. the per contact probabilities of transmission (t ij ) may be similar in these settings because they are determined by the pathogenesis of the strain in the host, while the numbers of contacts are different. therefore, the average transmissibility t will be similar in the locations, while r will be substantially higher in the hospital than in the rural setting. the heterogeneous spread of sars worldwide suggested context-dependent patterns of transmission with relatively rapid spread through hospitals and relatively slow spread through communities ( ) . a notable exception to this pattern, the large cluster of sars cases outside a healthcare setting in the amoy gardens apartment complex in hong kong, seems to have spread through aerosolization of virus-laden sewage rather than direct person-to-person contact ( ) . when contact patterns within a community are extremely heterogeneous, explicitly modeling community structure and t makes more sense than assuming a universal r . we take this approach to evaluating disease control strategies in an urban setting ( ) . a primary public health goal is to bring disease from a value above an epidemic threshold to a value below the threshold, thereby eliminating the threat of a large-scale epidemic. this goal can be achieved through interventions that directly affect the transmissibility of the pathogen (t) or through interventions that modify patterns of interaction so that the epidemic threshold (t c ) is increased. we call these forms of intervention transmission-reduction and contact-reduction, respectively, and depict them graphically in figure . the solid curves represent the predicted size of an outbreak and the probability of an epidemic for an entire spectrum of t from to in an urban setting. all airborne pathogens have a transmissibility value within this range; = no transmission, and = every contact leads to transmission. thus, any disease can be mapped to a unique value on the curve. in our simulated urban contact network, the critical transmissibility threshold is t c = . . an outbreak of disease with t = . will almost certainly spark an epidemic in the absence of intervention (top circle in figure ). this value of t is equivalent to an r = for this contact network and thus corresponds to a moderately infectious disease like smallpox ( , ) . a successful intervention either reduces t so that it lies below t c (figure a ) or modifies the structure of the network so that t c rises above t ( figure b ). the first strategy can be achieved by interventions that reduce the probability of transmission per contact, such as face masks, gloves, gowns, handwashing, and other infection control precautions that prevent the exchange of respiratory droplets without eliminating contact. the second strategy involves modifying the contact network itself. interventions such as quarantine and closing schools and other public places effectively eliminate potential contacts (edges) between persons. interventions such as immunization and the prophylactic use of antibacterial or antiviral drugs are tantamount to removing persons (vertexes) from the contact network and therefore also alter the network structure. we mathematically assess the effect of such strategies by deleting edges and vertexes from the contact network and predicting the new probability of an epidemic and expected distribution of cases within the community. we evaluated a variety of commonly implemented public health interventions by changing the contact patterns within the network, transmissibility of the disease, or both. for each strategy, we calculated several epidemiologic quantities: ) the epidemic threshold, t c , which may be raised by contact-reduction interventions, ) the transmissibility of the disease, t, which may be reduced by transmission-reduction interventions, ) if t<t c , the expected size of a small outbreak, s, ) if t>t c , the probability of a large-scale epidemic, s prob , and ) if t>t c , the expected size of an epidemic, s, should one occur. based on calculations of these quantities, figures - report the effect of various interventions applied to a moderately contagious disease just above the epidemic threshold (left panel), where we believe sars to lie ( ) and a moderately infectious disease such as smallpox (right panel). gray entries correspond to unsuccessful interventions; white entries indicate strategies that are predicted to successfully move the pathogen below the epidemic threshold and thereby prevent a large-scale epidemic. although general use of face masks may have a moderate effect, its success hinges on correct use and level of compliance. for instance, face masks that are % effective will only prevent a large-scale epidemic of a sarslike disease if > % of the general population complies perfectly (figure ). if persons use face masks incorrectly or only partially, this intervention will be less likely to protect persons and the population as a whole. for moderately contagious diseases like smallpox, face masks alone will not protect large urban areas from an epidemic. figure also suggests that use of face masks by healthcare workers, while important for personal protection, offers limited protection to the population and does not predictably preclude an epidemic. one of the factors that influences the transmissibility t is the duration of infectiousness. the duration of effective infectiousness may be shortened, but not eliminated, by isolating persons immediately after diagnosis. although isolating an infected person will physically remove him from the network, the person may already have had a chance to infect others before being identified and isolated. for example, an infectious person who is isolated after the second day of a -day infectious period will have had days in which disease could be transmitted to close contacts. thus, isolation can be effective for diseases with low transmissibility but only if case identification occurs early in the infectious period. for such diseases, an isolation strategy that on average reduces the infectious period by % will prevent a large-scale epidemic (figure ). isolation will not preclude an epidemic for a highly trans- missible disease unless clinical and diagnostic tools can be applied early and confidently, which may not be the case for an emerging infectious disease. contacts between infected and susceptible persons can be eliminated during an outbreak through measures such as quarantine, closing public venues, and ring vaccination, or they can be eliminated preventatively through general vaccination strategies. figure predicts that simultaneous case-patient isolation and quarantine of close contacts substantially improves containment. for a mildly contagious disease, an outbreak can be controlled with a combination of isolation that reduces the infectious period by % and quarantine that successfully sequesters % of all casepatient contacts. much more rigorous isolation and quarantine are required for a highly contagious disease. such interventions require a strong surveillance infrastructure, reliable rapid diagnostic tests, and social acceptance. in figure , we show that such predictions can readily be translated into values of r eff . interventions that bring a population under the epidemic threshold are those that decrease r eff below . we emphasize that the predictions in figures and are specific to the underlying model of contact patterns in an urban setting and that, contrary to common interpretations, r eff (or r ) is not a universal constant but instead critically depends on structure of the host community. a general vaccination strategy is one in which a substantial proportion of the population is vaccinated at random. the success of this measure depends on proportion (coverage), vaccine efficacy, and disease transmissibility. the availability of a vaccine, therefore, does not guarantee prevention unless both delivery and vaccine-induced immunity are sufficient. for example, figure shows that a mildly contagious disease like sars may be thwarted by partial coverage (≈ %) with a moderately efficacious vaccine (≈ % vaccine efficacy). under this strategy, a moderately contagious disease can become epidemic unless a population receives % coverage with a % efficacious vaccine. ring vaccination of close contacts, on the other hand, is a very effective approach overall. this intervention, like quarantine, involves both transmission and contact reduction. identifying the index patient results in a reduced infectious period. subsequent identification and protection of his or her contacts through vaccination further limits the potential spread of the pathogen. figure considers the effect of ring vaccination on the population as a function of the effectiveness of patient isolation and the fraction of contacts that are successfully immunized. partial protection of contacts may stem from inadequate contact tracing or an ineffective vaccine. for example, vaccinating % of close contacts with a % efficacious vaccine is equivalent to vaccinating % of close contacts with a % efficacious vaccine. ring vaccination can be a successful strategy for a mildly contagious disease with even a moderate surveillance infrastructure or a partially efficacious vaccine. however, ring vaccination requires more successful case identification, contact tracing, and vaccination when implemented against a highly contagious disease. ring vaccination is only applicable to diseases with relatively long incubation periods that allow contacts to be identified, vaccinated, and develop a protective immune response. thus, this strategy is more appropriate for diseases like smallpox (incubation period days) than sars (incubation period - days). the white entries in figures - report the expected (average) size of small outbreaks for diseases below the epidemic threshold. any particular outbreak, however, may not be exactly equal to this average size. in the left panel of figure , we show the average and standard deviation of outbreak sizes over the range of transmissibility values below the epidemic threshold. for each value of t, we estimate the standard deviation by using , simulated epidemics on the original urban network (without intervention). for low t, outbreaks tend to be small and close to the average outbreak size s. as t increases toward the epidemic threshold, the distribution of outbreak sizes widens substantially, and s becomes less informative. given this variability, public health strategies should be based on bringing populations substantially under the epidemic threshold. our mathematical predictions are based on a single simulated urban network with , households with an average of . people per household. to address the sensitivity of the predictions to the particular pattern of contacts in the network, we stochastically generated urban networks of equal size and predicted the probability of an epidemic for the range of t above the epidemic threshold. since each of these networks has a unique degree distribution, the value of the epidemic threshold varies. in particular we find that the average epidemic threshold is . with a % confidence interval of . - . . recall that the network used in the analysis above has an epidemic threshold t c = . . the right panel of figure shows the mean probability of an epidemic across these networks with % confidence intervals. the probabilities for the particular network that we studied lie very close to the mean probabilities. the narrow confidence intervals suggest that our predictions are fairly robust to the particular architecture of the urban network. we further consider the effect of network size on these predictions in the online appendix. using contact network epidemiology, we evaluated various airborne infection control policies for a simulated urban setting like vancouver. this approach explicitly captures the heterogeneous patterns of interpersonal contacts we generated , epidemics for each of values of t from to the epidemic threshold. the solid curve represents the mean of outbreak size (µ), the dashed curve represents standard deviation above the mean (µ + σ), and the dotted line at the bottom shows the minimum size of an outbreak, which is always equal to , meaning that after the introduction of the first infected case the disease did not spread further. right panel: sensitivity of epidemic probability to network stochasticity. we generated different networks, each with , households. because of the stochastic nature of contact formation during network generation, these networks contain different numbers and configurations of edges and therefore have different degree distributions. the solid curve shows the mean probability of an epidemic across the networks for transmissibilities above the epidemic threshold, and the dashed curves are % confidence limits for the mean probability of an epidemic. that lead to disease transmission and allows rapid mathematical prediction of the probability and distribution of an epidemic. this analysis does not depend on computationally intensive simulations. furthermore, the approach allows one to quantitatively compare strategies that directly reduce the transmissibility of a pathogen or limit opportunities for a pathogen to spread. although each strategy has been considered on its own, these methods can easily predict the effect of combined interventions for an entire spectrum of airborne infectious diseases, including sars, smallpox, influenza, and meningococcal meningitis, among others. although the qualitative results of this analysis are applied to urban settings, the work is meant to be a proof of concept rather than to provide specific quantitative recommendations for urban control of communicable diseases such as sars and smallpox. until we have developed contact network models for a wide range of communities and assessed their generality, contact network epidemiology will need to be applied on a case-bycase basis. for example, hospitals can use these methods to improve control of nosocomial airborne infections. to start, each facility should model its particular network of patient-healthcare worker interactions, then calculate the effect of measures such as respiratory droplet precautions, grouping patients in cohorts, modifications to healthcare worker assignments, and vaccination ( ) . the success of contact network epidemiology depends not only on realistic models of contact patterns but also on reliable estimates of the average transmissibility of the pathogen, t. as a respiratory pathogen begins to spread through a population, epidemiologists can rapidly identify the mode and rate of disease transmission. these data can provide critical input for intervention strategies. historically, the rate of disease transmission has been measured and reported in terms of the basic reproductive number r , based on the doubling time of case counts in the early phase of an outbreak or epidemic. the value of r , however, may vary substantially, depending on the population in which it is measured. for example, recent estimates of r for sars ranged from . to . ( ) ( ) ( ) . in contrast, t is not subject to the particular patterns of interaction within a community and can be reliably estimated in diverse settings. measuring t is only slightly more involved than measuring r . for each case, one must measure not just the number of secondary cases, but also the total number of contacts of the case-patient during the infectious period and then divide the first value by the second. just as enormous molecular and technological resources are often mobilized to develop vaccines and diagnostic tools for emerging infectious diseases of public health importance, we should also harness the powerful quantitative mathematical tools that help assess disease interventions. when an airborne pathogen strikes, public health officials should be able to make scientifically grounded decisions about the competing medical, economic, and social implications following deployment of control measures. we illustrate that contact network epidemiology can provide detailed and valuable insight into the fate and control of an outbreak. integrating these tools into public health decision making should facilitate more rational strategies to manage emerging diseases, bioterrorist events, and pandemic influenza in situations in which empiric data are not yet available to guide decision making. identification of severe acute respiratory syndrome in canada who confirms human-to-human avian flu transmission the detection of monkeypox in humans in the western hemisphere the outbreak of west nile virus infection in the new york city area in infectious diseases. first u.s. case of mad cow sharpens debate over testing recognition and management of anthrax-an update widespread flu in united states exposes shortage of vaccine infectious diseases of humans: dynamics and control the foot-and-mouth epidemic in great britain: pattern of spread and impact of interventions impact of mass treatment on syphilis transmission: a mathematical modeling approach emergency response to a smallpox attack: the case of mass vaccination applying network theory to epidemics: control measures for mycoplasma pneumoniae outbreaks spread of epidemic disease on networks a mathematical model for predicting the geographic spread of new infectious agents containing bioterrorist smallpox the spread and persistence of infectious diseases in structured populations epidemic models: their structure and relation to data epidemics with two levels of mixing a deterministic epidemic model taking account of repeated contacts between the same individuals how viruses spread among computers and people modeling vaccination strategies against foot-and-mouth disease the structure and function of complex networks statistics canada bc statistics centre for health services and policy research at university of british columbia city of vancouver mathematical epidemiology of infectious diseases: model building, analysis and interpretation epidemiologic clues to sars origin in china evidence of airborne transmission of the severe acute respiratory syndrome virus network theory and sars: predicting outbreak diversity the mathematics of infectious diseases a model for a smallpox-vaccination policy sars outbreaks in ontario, hong kong, and singapore: the role of diagnosis and isolation as a control mechanism transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of severe acute respiratory syndrome we thank mark newman for technical guidance. key: cord- - qgzulgj authors: moni, mohammad ali; liò, pietro title: network-based analysis of comorbidities risk during an infection: sars and hiv case studies date: - - journal: bmc bioinformatics doi: . / - - - sha: doc_id: cord_uid: qgzulgj background: infections are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. sars is a threat which is similar to mers virus, but the comorbidity is the key aspect to underline their different impacts. one uk doctor says "i’d rather have hiv than diabetes" as life expectancy among diabetes patients is lower than that of hiv. however, hiv has a comorbidity impact on the diabetes. results: we present a quantitative framework to compare and explore comorbidity between diseases. by using neighbourhood based benchmark and topological methods, we have built comorbidity relationships network based on the omim and our identified significant genes. then based on the gene expression, ppi and signalling pathways data, we investigate the comorbidity association of these infective pathologies with other diseases (heart failure, kidney disorder, breast cancer, neurodegenerative disorders, bone diseases, type and type diabetes). phenotypic association is measured by calculating both the relative risk as the quantified measures of comorbidity tendency of two disease pairs and the ϕ-correlation to measure the robustness of the comorbidity associations. the differential gene expression profiling strongly suggests that the response of sars affected patients seems to be mainly an innate inflammatory response and statistically dysregulates a large number of genes, pathways and ppis subnetworks in different pathologies such as chronic heart failure ( genes), breast cancer ( genes) and bone diseases ( genes). hiv- induces comorbidities relationship with many other diseases, particularly strong correlation with the neurological, cancer, metabolic and immunological diseases. similar comorbidities risk is observed from the clinical information. moreover, sars and hiv infections dysregulate genes (anxa , gns, hist h c, rasa ) and genes (hba , tfrc, ghitm) respectively that affect the ageing process. it is notable that hiv and sars similarly dysregulated genes and pathways. only significantly dysregulated genes are common between sars-cov and mers-cov, including nfkbia that is a key regulator of immune responsiveness implicated in susceptibility to infectious and inflammatory diseases. conclusions: our method presents a ripe opportunity to use data-driven approaches for advancing our current knowledge on disease mechanism and predicting disease comorbidities in a quantitative way. electronic supplementary material: the online version of this article (doi: . / - - - ) contains supplementary material, which is available to authorized users. the term "comorbidity" refers to the coexistence of multiple diseases or disorders in relation to a primary disease or disorder in an individual [ ] . a comorbidity relationship between two diseases exists whenever they appear simultaneously in a patient more than chance alone [ ] . it represents the co-occurrence of diseases or presence http://www.biomedcentral.com/ - / / to chronic obstructive pulmonary disease (copd) [ , ] , obesity [ ] , mental disorders [ ] , immune-related diseases [ ] , cancer [ ] etc. comorbidity can be attributed to the disease connections on the molecular level, such as dysregulated genes, ppis (protein-protein interactions), and metabolic pathways as potential causes of comorbidity [ , , , ] . from a genetic perspective, a pair of diseases is connected because they have both been associated with the same dysregulated genes [ , ] , whereas from a proteomics perspective phenotypically similar diseases are related via biological modules such as ppis or molecular pathways [ , ] . population-based disease association is important in conjunction with molecular and genetic data to uncover the molecular origins of diseases and disease comorbidities. patient medical records contain important clarification regarding the co-occurrences of diseases affecting the same patient [ ] . during the last few years, several researchers have been conducted the disease comorbidity analysis to understand the origins of many diseases [ , , ] . goh, cusick, valle, childs, vidal, barabasi et al. and feldman, rzhetsky, vitkup et al. built networks of gene-disease associations by connecting diseases that have been associated with the same genes [ , ] , whereas lee, park, kay, christakis, oltvai and barabási et al. constructed a network in which two diseases are linked if metabolic reactions are associated between them [ ] . disease association studies from proteomic point of view have been studied by rual, venkatesan, hao, hirozane-kishikawa, dricot, li, berriz, gibbons, dreze, ayivi-guedehoussou et al. and stelzl, worm, lalowski, haenig, brembeck, goehler, stroedicke, zenkner, schoenherr, koeppen et al. [ , ] . rzhetsky, wajngurt, park and zheng et al. inferred the comorbidity links between disorders from the disease history of . million patients [ ] . however, all of these efforts have focused on the role of a single molecular or phenotypic measure to capture disease-disease relationships. in our work we have used disease-gene associations, ppis, molecular pathways and clinical information to obtain statistically significant associations and comorbidity risks among diseases. inflammation is a hallmark of many serious human infectious diseases associated to a wide variety of infections, such as hiv- [ ] . uk doctor max pemberton says "i'd rather have hiv than diabetes" as life expectancy among diabetes patients is lower than that of hiv [ ] . however, hiv has a comorbidity impact on the diabetes. also the flu can cause complications, including bacterial pneumonia, or the worsening of chronic health problems. asthma is the most common comorbidity in patients hospitalized for swine influenza (h n ) infection [ ] . dengue can cause myocardial impairment, arrhythmias and, occasionally, fulminant myocarditis [ ] . chronic medical conditions, such as heart disease, lung disease, diabetes, renal disease, rheumatologic disease, dementia, and stroke are risk factors for influenza complications [ ] . common chronic infections such as periodontitis or infection with helicobacter pylori may also increase stroke risk [ ] . moreover, the severity of pneumonia in patients coinfected with influenza virus and bacteria is significantly higher than in those infected with bacteria alone. the incidence of flu is higher in children and younger adults than in older individuals, but influenzaassociated morbidity and mortality increase with age, especially for individuals with underlying medical conditions such as chronic cardiovascular diseases [ ] . during the ageing process the immune system becomes compromised and it causes an increasing inflammation [ ] . in particular, chronic inflammation (inflammageing) and metabolic function are strongly affected by the ageing process [ ] . the ageing of populations is leading to an unprecedented increase different diseases like cancer and fatalities. it is reported that % of the elderly population has three or more chronic conditions [ ] . on the other hand, respiratory viruses are an emerging threat to global health security and have led to worldwide epidemics with substantial morbidity and mortality [ ] . coronaviruses (covs) cause respiratory and enteric diseases in human and other animals that induce fatal respiratory, gastrointestinal and neurological disease. severe acute respiratory syndrome (sars) is an epidemic human disease, is caused by a coronavirus (cov), called sarsassociated coronavirus (sars-cov) [ ] . sars patients may present with a spectrum of disease severity ranging from flu-like symptoms and viral pneumonia to acute respiratory distress syndrome and death [ ] . most of the deaths were attributed to complications related to sepsis, ards and multiorgan failure, which occurred commonly in the elderly for comorbidities [ ] . age and comorbidity (e.g. diabetes mellitus, heart disease) were consistently found to be significant independent predictors of various adverse outcomes in sars [ ] . children with sars have better prognosis than adults [ ] . advanced age and comorbidities were significantly associated with increased risk of sars-cov related death, due to acute respiratory distress syndrome [ ] . mild degree of anaemia is common in the sars infected patients and patients who have recovered from sars show symptoms of psychological trauma [ ] . another novel coronavirus mers-cov, which is a new threat for public health, has similar clinical characteristics to sars-cov, but the comorbidity is the key aspect to underline their different impacts [ , ] . mers-cov causes respiratory infections of varying severity and sometimes fatal infections in humans including kidney failure and severe acute pneumonia [ ] . despite sharing some clinical similarities with sars (eg, fever, http://www.biomedcentral.com/ - / / cough, incubation period), there are also some important differences such as the rapid progression to respiratory failure, which we have studied on comorbidities point of view. infection with the human immunodeficiency virus- (hiv) and the resulting acquired immune deficiency syndrome (aids) affects cellular immune regulation [ ] . hiv infection severely impacts on the immune system causing phenotypic changes in peripheral cells and dysregulates the innate immune system [ ] . significant number of hiv- infected patients exhibits osteopenia and osteoporosis, leading to higher incidence to develop weak and fragile bones during the course of disease [ ] . hiv has also been associated with an increased risk of developing both diabetes and cardiovascular disease [ ] . infection with hiv weakens the immune system and reduces the body's ability to fight infections that may lead to cancer [ , ] . people infected with human immunodeficiency virus (hiv) have a higher risk of some types of cancer (kaposi sarcoma, non-hodgkin lymphoma, cervical cancer, anal, liver, lung cancer, and hodgkin lymphoma) than uninfected people [ ] . many people infected with hiv are also infected with other viruses that cause certain cancers [ , ] . hiv infection even when controlled by highly active antiretroviral therapy (haart) is being linked to chronic inflammation [ ] . people with hiv- infection appear to have a markedly higher rate of chronic kidney disease than the general public [ ] . it is because some of the risk factors associated with hiv- acquisition are the same as those that lead to kidney disease because of the virus itself and some therapies (e.g. haart therapy). antiretroviral therapy for hiv may increase the risk of developing metabolic syndrome (abdominal obesity, hyperglycaemia, dyslipidaemia and hypertension) and thus predispose to type diabetes and cardiovascular disease. many of the biologic factors thought to be causally associated with inflammation in hiv disease are also thought to be causally associated with the inflammation of ageing [ ] . infections (acute and chronic conditions) are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. comorbidities related to flu have been recently investigated [ ] . comorbidities for tuberculosis have also been studied recently [ , ] . to understand the overall mechanism we have studied the comorbidity associations of sars and hiv infections. both hiv and sars are emerging infectious diseases in the modern world; each of these diseases has caused global societal and economic impact related to unexpected illnesses and deaths [ ] . sras is a significant public health threat and hiv is a long term chronic infection. since these two infections are associated with high mortality rates and there are no clinically approved antiviral treatments or vaccines available for either of these infections, we have selected these two infections for our study. centred on the sars and hiv- infections we have investigated highly heterogeneous disease comorbidity networks using the disease-gene associations, ppi subnetwork, molecular pathways and clinical information. we have presented a systematic and quantitative approach to discover human disease comorbidities using different sources of available mrna expression, protein-protein interactions, signalling pathways, disease-gene associations, disease-disease associations and disease-drug associations data. it has been shown that sars coronavirus infects and replicates in a wide variety of host cells in susceptible animals and human beings [ , ] . to understand the host response to this pathogen, we analysed the gene expression patterns of sars infected patients, compared to normal subjects using oligonucleotide microarrays from the ncbi geo (http://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse ) [ ] . we analysed the microarray gene expression data of over , genes from the pbmcs of sars patients, and compared with healthy control samples. we found that genes (p < . , > . fold change) were differentially expressed as compared to healthy controls in which genes were significantly up regulated and genes were significantly down regulated (see additional file : table s ). on the other hand, monocytes are the key immune responsive cells whose function is adversely impacted by hiv- . hiv- infection radically alters the monocyte phenotype, which is reflected in an hiv- induced gene expression analysis. monocyte gene expression microarray data were collected for control and hiv patients from the ncbi geo (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=gse ) [ ] . to find out the significant dysregulated genes during the hiv- infection, we have performed global gene expression analysis. we found that genes (p < . , > . fold change) were differentially expressed as compared to healthy controls in which genes were up regulated and genes were down regulated (see additional file : table s ). considering the significantly dysregulated genes of sars ( genes) and hiv- ( genes) infections, and gene-disease associations information, we have constructed two gene-disease associations networks (gdn), which are used to explore the shared genetic associations and disease comorbidity. starting from the bipartite graph we generated biologically relevant network projections and constructed multi-relational gene-disease network in which nodes are diseases or genes, and edges indicate association between gene and disease. this bipartite http://www.biomedcentral.com/ - / / graph consists of two disjoint sets of nodes, where one set corresponds to all known genetic disorders and the other set corresponds to all of our identified significant genes for sars and hiv- infections. the list of disorders, disease genes, and associations between them were obtained from the online mendelian inheritance in man (omim) [ ] , a compendium of human disease genes and phenotypes (see details in the methods section). we classified each disorder into one of disorder categories based on the physiological system affected as introduced in goh, cusick, valle, childs, vidal, barabasi et al. [ ] . in the gdn, nodes represent diseases class or genes, and two disorders are connected to each other if they share at least one gene in which mutations are associated with both diseases groups (figures and ). the number of interlinked genes between sars infection and other diseases indicates that immunological, hematological, neurological, metabolic and dermatological diseases categories are strongly associated with the sars infection (see figure and additional file : table s ). few genes are also shared between more than categories of diseases i.e those disease groups are also associated through at least that genes. for an instance, the gene atm shared among sars infection, cancer and immunological diseases. therefore, cancer and immunological diseases are also interrelated through the gene atm. among all these disease classes immunological diseases class is tightly correlated with the sars infection due to the highest number of genes ( genes) shared between them. on the other hand, the number of associated genes between hiv infection and other diseases indicates that neurological, metabolic, cancer and hematological diseases categories are strongly correlated with the hiv infection (see figure and additional file : figure the gene-disease association network centred on the sars infection is constructed based on the different categories of diseases that are connected and showed comorbidities with the sars infection through the different genes. red colour represents different categories of disorders and green colour represents different genes that are common with the other categories of disorders. the size of a disease node is proportional to the number of dysregulated genes shared between the infections/disorder groups. a link is placed between a disorder and a disease gene if mutations in that gene lead to the specific disorder. http://www.biomedcentral.com/ - / / figure the gene-disease association network centred on the hiv infection is constructed based on the different categories of diseases that are connected and showed comorbidities with the hiv- infection through the different genes. red colour represents different categories of disorders and green colour represents different genes that are common with the other categories of disorders. the size of a disease node is proportional to the number of dysregulated genes shared between the infections/disorder groups. a link is placed between a disorder and a disease gene if mutations in that gene lead to the specific disorder. table s ). few hiv dysregulated genes are also shared between more than categories of diseases such as the gene tgfb is shared among hiv infection, cancer and skeletal diseases. it is notable that significant genes ( upregulated and downregulated) are similarly dysregulated in the both sars and hiv infections. to observe the association of sars and hiv infections with other important diseases (chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, type and type diabetes), we have collected mrna microarray raw data associated with each disease from the gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) accession numbers are gse , gse , gse , gse , gse and gse [ ] . after several steps of statistical analysis we have selected the most significant over and under expressed genes for each infection and disease. we also performed cross compare analysis to find the common significant genes between each disease and sars/ hiv- infection. we observed that sars infection shares , , , , , , and genes corresponding to the chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, hiv- infection, type and type diabetes. on the other hand, hiv- infection shares , , , , , , and genes corresponding to the chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, sars infection, type and type diabetes. then we built disease-disease relationships network for sars and hiv- infection with other diseases (see figures (a) and (b) and additional file : table s and additional file : table s ). since genes do not function alone and they coordinate their activities in the form of complexes or molecular pathways. therefore two diseases are potentially inter-correlated to each other if they share at least one commonly associated pathway. for this http://www.biomedcentral.com/ - / / reason we have used reactome pathway database [ ] and selected the pathways related to these diseases as well as sars and hiv- infections. we have observed that diseases and infections shared pathways between them as shown in figures (a) and (b) and additional file : table s and additional file : table s . dysregulation in a protein subnetwork may yield the dysfunction of multiple protein subnetworks. therefore, multiple diseases may be caused by the malfunction of a protein complex. so, two diseases are potentially related to each other if they share one or more commonly associated protein subnetwork. to identify the association between diseases based on the ppi subnetwork, we used significantly associated disease protein pairs data from the hprd data base [ ] . to find statistically significant associations among diseases, we built disease networks centred on the sars and hiv infections in which two diseases are comorbid if there exists one or more protein subnetwork that are associated with both diseases. the disease similarity network and the protein-protein interaction network are integrated systematically and comprehensively in a simple and compact manner to formulate the disease comorbidity for the sars and hiv- infections as shown in figures and . we showed that sars and hiv infections shared ppi subnetworks with the other diseases or infections similar to the gene-disease and pathway-disease associations as shown in figures and . based on the gene expression, protein-protein interaction and molecular pathways data, we have found that both sars and hiv- infections have a strong association with other diseases or infections (chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, hiv/sars infection, type and type diabetes). these diseases and infections are also strongly correlated among them. we present the correlation strength and distance between a pair of these diseases and infections in figure . we show that some diseases (such as kidney disorders, breast cancer, osteoporosis and heart failure) are more associated with the sars infection (see figure ). kidney disorder is also tightly connected with the hiv- infection. the probability of occurring comorbidities between the more tightly connected diseases is more than that of others. it is notable that the patient medical records contain important evidence regarding the co-occurrences of diseases affecting the same patient. so, we constructed a phenotypic disease comorbidity network using million medical records of patients data from medpar and analysed its structural properties to better understand the connections among diseases and infections. nodes are unique diseases and edges indicate co-morbidity of the diseases. we included edges between disease pairs for which the co-occurrence is significantly greater than the random expectation based on population prevalence of the diseases. as pointed out in [ ] , the relative risk (rr ij ) overestimates relations involving rare infections and diseases, and underestimates relationships between very common disorders or infections. on the other hand, φ-correlation underestimates comorbidity between rare and frequent diseases, and discriminates associations between disorders of similar appearances. thus, we built a network by selecting only the statistically significant network edges having rr ij ≥ and φ ij ≥ . . figure summarises the set of all comorbidity associations among all diseases expressed in the study population by constructing a phenotypic disease network (pdn). in the pdn, nodes are disease phenotypes identified by unique icd- -cm (the international classification of diseases) disease codes, and links connect phenotypes that show significant comorbidity according to the relative risk rr ij ≥ and the correlation φ ij ≥ . . our phenotypic disease network consists of unique diseases nodes and co-morbidity relationships. sars-associated coronavirus icd- -cm diagnosis code is . , which is under the group of "viral and chlamydial infection in conditions classified elsewhere and of unspecified site" and icd- -cm diagnosis code . moreover, the icd- -cm code . is for the pneumonia due to sars associated coronavirus. so we have considered both icd- -cm codes . and . for our phenotypic sars comorbidity study. in our digit code data we have considered and for digit code data we have considered . . considering the relative risk rr ij ≥ between the disease group and other disorder categories, we have constructed the pdn as shown in figure (a), and considering the relative risk rr ij ≥ between the disease group . and other disorder categories, we have constructed the pdn as shown in figure (b). we presented only the most significant relative risk associations (see additional file : table s and additional file : table s ). the icd- -cm diagnosis code for the human immunodeficiency virus (hiv) infection is to , which is under the group of "infectious and parasitic diseases" and icd- -cm code ( - ). so we have considered both digit and digit icd- -cm codes for our phenotypic comorbidity studies related to hiv infection. considering the relative risk rr ij ≥ between the disease group and other disorder categories, we have constructed the pdn as shown in figure (a) and considering the relative risk rr ij ≥ and φ-correlation φ ij ≥ . between the disease groups under the sub categories of and other disorder categories, we have constructed the pdn as shown in figure (b). only the most significant relative risk association is represented (see additional file : table s and additional file : table s ). to observe the trend of phenotypic relative risk corresponding to the number of shared genes between http://www.biomedcentral.com/ - / / diseases, we have computed the number of shared genes between two diseases and their corresponding phenotypic relative risk of the occurrence of comorbidities as shown in figure . we observed that with increasing number of shared biomarker genes between diseases, the phenotypic relative risk is also increased. we may predict existing diseases of a patient and the prospective disease comorbidities through the identification of highly up and down dysregulated genes. so based on the available data we could predict the disease comorbidities and the level of the comorbidities using the regression model as figure . it is notable that ageing is also a "disease", not a natural process, for which age-related diseases increase exponentially with chronological time. so, to understand the impact of ageing on the disease comorbidities for sars and hiv infections we have considered the ageing data from the genage database (http://genomics.senescence. info/genes/human.html) [ , ] . after cross comparing http://www.biomedcentral.com/ - / / human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (sars-cov) [ ] . thus, a comprehensive evaluation of the complex epithelial signalling to sars-cov is crucial to better understand sars pathogenesis. since both of the sars-cov and mers-cov infections cause severe lung pathology we compare and contrast the genes expression level of sars-cov infection and mers-cov infection. to compare between sars-cov and mers-cov infections, and the affect on the disease comorbidities, we have performed the time series microarray data analysis for the both types of infections on lung compared to controls. we have considered gene expression microarray data from the ncbi geo (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=gse ) [ ] . from the analysis of sars-cov vs mock-infected controls (treated the same way except without the virus) we have found genes are highly significant and from the analysis of mers-cov vs mock we have found gens are highly significant (see details in the additional file : table s and additional file : table s ). interestingly, only genes (nfkbia, egr , ddit and ifit ) are common between these two infections (see figure ). however, only genes (nfkbia and egr ) play an important role and differentially expressed among the both infections in lung and also in sars infected pbmcs. then from the hierarchical cluster analysis of the differentially expressed genes of the lung infection by sars-cov and mers-cov, we observed distinct groups of genes that were significantly changed over time (see additional file : figure s and additional file : figure s , and additional file : table s and additional file : table s ). the log fold changes of the common genes (nfkbia, egr , ddit and ifit ) expression level for the infection of mers-cov and sars-cov are presented in the figures and . we observed that the log fold changes of nfkbia genes expression level is sharply upregulated in the both types of infections corresponding to time point. so nfkbia is an important bio-marker for the both mers-cov and sars-cov infections. it is also observed that the inflammatory genes nfkbia is a highly over expressed in the both pbmcs and lung cells for the infection of sars and also for the infection of mers in the lung cells (see figure ). indeed, the immune system plays a pivotal role in the outbreak of the inflammatory state. so in case of sars infection, the nfkbia gene plays an important role for the disease comorbidities. on the other hand, similar diseases share common genes and could be treated by the same drugs [ ] , which may allow us to make predictions for new uses of existing drugs. for an instance, the anti-diabetic drug metformin plays a major protective effect against cancer development and increases significantly higher survival rate of the cancer patients [ ] . the finding is that the earlier the metformin regimen was initiated, the greater the http://www.biomedcentral.com/ - / / preventive benefit for the cancer patient. there is an evidence that the antiviral medication, ribavirin, does not work in case of sars infection [ ] . to this end, we used connectivity map (cmap), which is a database of more than , drug transcriptional signatures in several cell lines [ ] . this database allows to identify of molecules that induce similar or opposite transcriptional changes relative to the query signature, based on their connectivity enrichment scores. as a query signature we used our highly dysregulated genes for the sars infection. we generated the connectivity score value ranges between + and - , where a highly positive score indicates that the drug induces changes similar to those induced by viral infection, while a highly negative score indicates that the drug reverses the expression of the sars signature. based on the connectivity score we have selected most potential positive and negative regulators of sars viral response (see details in the additional file : table s ). potential negative regulators indicate that drugs reverse the sars signature gene expression. among the negative potential regulator, the drug molecule tetracycline, zalcitabine, gibberellic acid, prestwick- and sulfaquinoxaline are more potential for the mcf cell line and vorinostat for the hl cell line. based on the data demonstrate the efficacy of different drug against sars virus can be predicted effective drug treatment for the emergent viruses. furthermore, immunomodulatory drugs that reduce the excessive host inflammatory response to respiratory viruses have therapeutic benefit to reduce the sars infection as well as disease comorbidities. we presented and analysed multi-relational disease comorbidity relationships of sars and hiv- infections with other diseases or infections based on the associations of genetics, proteomics, molecular signalling pathways and phenotypic disorders. the combination of molecular biology, genetics and clinical medicine has greatly facilitated understanding of how different diseases relate to each other. based on the combined genetics, ppis, pathways and clinical data, our disease networks can disclose potentially novel disease relationships that have not been captured by previous individual studies. the underlying hypothesis behind this line of research is that once we catalogue all disease-related genes, ppi complex and signalling pathways, if we do not consider environmental changes, we will be able to predict the susceptibility of each individual to future diseases using various molecular biomarkers and it will help us to enter an era of predictive medicine. our results indicate that such a combination of molecular and population-level data could help to build novel hypotheses about disease mechanisms. furthermore, if two or more diseases have associated comorbidity, the occurrence of one of them in a patient may increase the likelihood of developing the other diseases. we have also studied the differences between mers-cov and sars-cov in the host response. this enables rapid assessment of viral properties and the ability to anticipate possible differences in human clinical responses to mers-cov and sars-cov and their impact on comorbidities with respect to the general comorbidities conditions. we used this information to predict potential effective drugs against sars-cov, a method that could be more generally used to identify candidate therapeutics in future disease outbreaks. these investigation approach may also help to generate hypotheses and make rapid advancements in characterising the new viruses. we also found that patients' response of sars appears to be mainly an innate inflammatory response using nfk-bia, rather than any specific immune response against a viral infection such as hiv. however, hiv infection and highly active antiretroviral therapy (haart) also increase the immune reconstitution inflammatory syndrome (iris) and inflammation through the nf-κb pathways [ ] . moreover we have studied before about the impact of hiv infection on bone diseases and infection (e. g. osteoporosis and osteomyelitis). we observed that genes (e.g. rankl) and pathways (e.g. nf-κb) that are dysregulated by hiv infection also impact on the bone remodelling and bone related diseases. it is also recognised that inflammation plays a role in cancer aetiology, and various studies have found that inflammation may causes iris, obesity and tumour-promoting effects [ ] . moreover, inflammation is an important concomitant cause of many major age-associated pathologies such as cancer, neurodegeneration and diabetes [ ] . our study provides important evidence to associate diseases with the ageing process at the system level and helps to understand more about the comorbidities of the complex diseases. the ageing process itself is accompanied by a chronic low-grade inflammation, which is termed "inflammageing". the combination of metabolic-driven and age-driven inflammatory pathways plays a pivotal role in disease progression. this observation suggests that inflammageing and meta-inflammation can share stimuli and pathogenic mechanisms for comorbidities. we suppose that what is happening for the comorbidities we investigate is similar to what found for prions [ , ] . similar to most infectious agents, prion causes inflammatory responses by activating innate immunity through glial cells in the brain. the complete transcriptome of the prion brain at different time points is observed during the -week period [ , ] . at the beginning of the disease, both normal and diseased mouse networks were the same. although the disease started in the most unique network of prion accumulation and replication it is progressed to the other networks. based on this approach we may propose a pathway model for comorbidities how hubs genes dysregulate several other pathways to influence comorbidities. the number of dysregulated pathways could be proportional to the amount of dysregulation of hub genes. our pathway model may states that the hubs that are over turned on, may direct the signal to the different pathways creating comorbidities as shown in figure . for the infection, one of the pathways related to the inflammation starts dysregulation. with increasing time, both confidence level of inflammation and the number of dysregulated pathways are increased. moreover, with the increasing of inflammation the number of diseases for the comorbidities may increase. so initially infection dysregulates one signalling pathway of any cells and that causes other pathways may be dysrupted. in this way disrupting pathways increase more diseases in the same patient and make multimorbidity. disease genes play a central role in the human interactomes. overlapping component genes serve as bridges across the relatively independent functional modules or pathways. so perturbation in one pathway, such as the nf-κb signalling pathway, could be propagated throughout the other relevant pathways. we found sars and hiv- infections share significantly dysregulated genes as well as molecular pathways. both sars and hiv- viruses may infect and find an already existing comorbidity or generate a new comorbidity through the perturbation of the infected pathways. furthermore, it may provide us an opportunity to investigate the role of other http://www.biomedcentral.com/ - / / genes from the same pathway in the disease space. therefore, pathways could be used to represent the underlying biology of diseases and make prediction of disease comorbidities. in most of the cases, the correlativeness among genes, pathways and diseases are many-to-many, e.g. a disease is associated to many different genes and pathways; and a pathway is associated to many different diseases. this study suggests that a single pathway can be involved in many diseases whereas a disease may have dysregulation in many biological processes. hence, if a drug is already available to treat a disease through modulating the activity of a pathway, then it could potentially be used to treat other diseases that are strongly linked with the same pathway. on the other hand, when a disease shows dysregulation in multiple pathways, a pathwayguided combined drug may be employed in the treatment. moreover, the protein subnetwork-based approach to diseases may aid in drug discovery, in fact it can potentially be used to treat other diseases that are linked to the same protein complex. thus, our findings not only potentially help us to understand how different diseases are related based on their underlying molecular mechanisms but also provide insights into the design of novel, protein complex-guided therapeutic interventions for diseases. extending the concept of subclassifying patient cohorts to the single patient level refers to as personalised medicine. during the last few years, acceptance level of the personalised medicine is sharply increased as it has been apparent that standard treatment approaches are rarely efficient across the entire patient population. advances in highthroughput molecular assay technologies in the fields of genomics, proteomics and other "omics" is increasing the diagnostic and therapeutic strategies for personalised treatment. as a result, declining per-sample cost has given rise to numerous public repositories of biomolecular data. in particular, the availability of these data sets for many different diseases presents a ripe opportunity to use data-driven approaches to advance our current knowledge of disease relationships in a systematic way. the identified disease patterns can then be further investigated with regards to their diagnostic utility or help in predicting novel therapeutic targets. medicine will focus on each individual patient. it will become intrinsically proactive and will increasingly focus on wellness rather than disease. proactive and personalised medicine will bring fundamental changes to healthcare, taking carefully http://www.biomedcentral.com/ - / / targeted preventative or therapeutic action at the earliest indications of risk or disease comorbidities. we are entering into the genomic era of medicine, where a patient's genetic/genomic data is becoming important for clinical decision making, including disease risk assessment, disease diagnosis and subtyping, drug therapy and dose selection, risk assessment for adverse drug reaction, and family planning [ ] . today multi-scale and complex biomedical data are gathered and analysed to uncover combinations of predictive disease profiles. our genome, as well as multiple proteomes, multiple transcriptomes, multiple metabolomes, and other personalised data sets obtained at different points in our lives, will be readily available at affordable prices for each individual. in the near future, clinicians will have to consider genetic/genomic implications to patient care throughout their clinical workflow, including electronic prescribing of medications. therefore, for the implementation of the personalised medicine system, a model could be developed that will take individual genetic data. dysregulated biomarker genes will be identified from this genetic data and disease will be identified from the gene-disease association database. based on the information of the existing disease, the model will predict disease comorbidities using the disease-disease associations database. this will provide us to detect many diseases at the earliest detectable phase, even weeks, months, or maybe years before the symptoms appear and it will afford crucial insights into optimizing of our wellness. thus, personalised medicine will give fundamental new insights into disease mechanisms, and hence will open new opportunities for diagnosis, therapy and prevention from the disease comorbidities. in this study, we have considered all available categories of omics and phenotypic data to quantify the sars and hiv- infections centred comorbidity associations. we have shown that the phenotype disease network (pdn) has a heterogeneous structure where some diseases are highly connected while others are hardly connected at all. our findings showed that disease progression can be represented and studied using network methods, offering the potential to enhance our understanding of the origin and evolution of human diseases. detecting comorbidity in a large population is of clinical interest due to the fact that it may reveal new information useful for cause of diseases as well as for new treatment strategies. this study demonstrates the value of an integrated approach in revealing disease relationships and new opportunities for therapeutic applications. so we can say that this kind of approach will be helpful for making evidence-based recommendations about disease comorbidities. moreover, considering environmental factors (such as physiological stress, diet), ethnic group and gender discriminations are important factors in the comorbidity analysis. our network approach could be extended as a comorbidity map by integrating diet, exercise and other factors as in [ ] . the gene-disease associations data used in this study were collected from the online mendelian inheritance in man (omim) database (http://www.ncbi.nlm.nih.gov/ omim/). this omim database is the best-curated repository of all known disease genes and their associated http://www.biomedcentral.com/ - / / figure progressive temporal activation of pathways. a schematic view of networks becoming disease comorbidities increased for the perturbation of the pathways dysregulation advances with time. the red circles indicate increased levels of dysregulated gene expression relative to control and the red linked indicate dysregulated pathways that have been increased from infection as compared with normal control. the green indicated transcripts that are the same in control and infection condition. the four panels represent the network with time intervals of the infection progression. with time the inflammation confidence level is increased which is indicated by confidence interval. disorders [ , ] . genotype-phenotype relationships, as summarised in the omim database, contained more than human disease-genes associations involving diseases and disease associated genes. each entry of the omim is composed of four fields, the name of the disorder, the associated gene symbols, its corresponding omim id, and the chromosomal location. we selected the entries with the "( )" tag, for which there is strong evidence that at least one mutation is cause of the disorder. omim initially focused on monogenic disorders but in recent years has expanded to include complex traits and the associated genetic mutations that confer susceptibility to these common disorders [ ] . subsequently we classified each disorder into primary disorder classes based on the physiological system affected as introduced in goh, cusick, valle, childs, vidal and barabasi et al. [ ] . disorders having distinct multiple clinical features are assigned to the "multiple" class. this classification scheme reflects the phenotypic similarities among diseases in the same class and has been successfully used in the recent studies of systematic disease analysis [ ] . the gene expression data used in this study was obtained from the ncbi gene expression omnibus (geo) (http://www.ncbi.nlm.nih.gov/geo/) [ ] . we have considered different data sets for our analysis (accession numbers are gse , gse , gse , gse , gse , gse , gse , gse , gse and gse ) [ , , , , [ ] [ ] [ ] [ ] [ ] . these data sets contain data from the patients of different age and sex. after several rounds of filtering, normalization and statistical analysis, we had microarrays representing sars, mers, hiv- infections and other human diseases (heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, type and type diabetes). http://www.biomedcentral.com/ - / / the protein-protein interaction (ppi) data for human was obtained from the human protein reference database (hprd) [ ] . hprd contains the maximum number of ppi data among all publicly available literature-derived databases for human ppi [ ] . we have used the reactome knowledge base of human biological pathways database for our pathways association analysis [ ] . for the cross compare analysis between the sars and hiv infections, and ageing process we have download ageing data from the human ageing genomic resources (http://genomics.senescence.info/ download.html) [ , ] . they have collected human ageing genes after an extensive review of the literature. these genes are commonly dysregulated during the ageing process. to test the validity of the proposed disease associations, we examined the disease co-occurrence information at the population level. we obtained statistically significant pairwise comorbidity associations reconstructed from over million medical records in the us medicare claims database recorded in the icd- -cm format (http://www. icd data.com), which are frequently used for epidemiological and demographic studies and collected from [ ] . we used medpar records from to , where the dates and reasons for all hospitalizations were reported in icd- -cm format and it contains the diagnoses of elderly patients. each record consists of the date of visit, a primary diagnosis and up to secondary diagnosis. all diagnoses are specified by icd codes of up to digits. the first three digits specify the main disease category while the last two are used to give additional information about the disease. in total, the icd- -cm classification consists of different categories at the digit level and , categories at the digit level [ ] . to determine whether some existing drug compounds can reverse the sars infection signature, we used the publicly available connectivity map (cmap) database [ ] . cmap provides associations among genes, chemicals and disease or infection conditions. it is a collection of genome-wide transcriptional data from cultured human cells treated with , different compounds. the method of global gene expression analysis using oligonucleotide microarrays has proven to be a sensitive method to develop and refine the molecular determinants of human disorders [ ] . using this technology, we compared the gene expression profiles of sars, hiv and other diseases. to avoid the problems of comparing microarray data of different platforms and experimental systems, we normalized the gene expression data in each microarray sample (disease state or control) using the z-score transformation (z ij ) for each disease gene expression matrix using z ij = where sd is the standard deviation, g ij represents the expression value of gene i in sample j. this transformation allows for the direct comparison of gene expression values across various microarray samples and diseases. to combined more than one data series or experiments for a given disease, we employed a linear regression approach to obtain a combined t-test statistic between two conditions. data were log -transformed and we calculated expression level for each gene using a linear regression model : where y i is the gene expression value and x i is a disease state (disease or control). the coefficients β and β are the parameters of this model and were estimated by least squares. the t-test statistic, when estimating the value of β , is the same as the standard t-test statistic between disease and control states. time series microarray gene expression data analysis was divide into two steps: pre-processing and identification of statistically significant points by t-test, anova and regression analysis to find differently expressed gene profiles in different time points. in the first step, we preprocessed the experimental data using different statistical methods and finally followed by post less normalization, recommended by the golden spike project [ ] . in the second step, we have used a most suitable method "masigpro" (microarray significant profiles) to identify differentially expressed genes in time-course microarray experiments, which is a two step regression method successfully applied on more than one groups of time-series [ , ] . this two steps regression strategy is used to find genes with significant temporal expression changes and significant differences between experimental groups. this procedure first adjusts this global model by the leastsquared technique to identify differentially expressed genes and selects significant genes applying false discovery rate control procedures. then stepwise regression is applied as a variable selection strategy to study differences between experimental groups and statistically significant profiles. after finding differentially gene expression profiles among the group of experiments, the next step is to cluster them according to their profile similarities. the hierarchical clustering and the median gene expression profiles of clusters are performed according to the "masigpro" package in r [ ] . student's unpaired t-test was performed to identify genes that were differentially expressed in patients over normal samples and significant genes were selected. a threshold of at least . fold change and a p value for the t-tests of less than . were chosen. in addition, a two-way anova with bonferroni's post-hoc test was used to establish statistical significance between groups (< . ). pathways and functional categories were considered as over-represented when fisher's exact test p value was < . . for presenting the signalling and http://www.biomedcentral.com/ - / / interaction pathways of the different significant genes, we used cytoscape for data integration and network visualization [ , ] and reactome functional interaction (fi) cytoscape plugin for knowledge base of human biological pathways and network processes [ ] . for the gene disease association, we have considered the neighbourhood based benchmark and topological methods, which are better suited to our networks [ ] . in this case, topological refers to methods that rely only on the structure of the network to draw conclusions. we construct a gdn from gene-disease associations where the node in the network can be either a disease or a gene. this network can also be regarded as a bipartite graph. diseases are connected when the diseases share at least one significant dysregulated genes. let a particular set of human diseases d and a set of human genes g, genedisease associations attempt to find whether gene g ∈ g is associated with disease d ∈ d. if g i and g j , the sets of significant up and down dysregulated genes associated with diseases i and j respectively, then the number of shared dysregulated genes (n g ij ) associated with both diseases i and j is as follows: the co-occurrence refers to the number of shared genes in the gdn. the common neighbours is the based on the jaccard coefficient method, where the edge prediction score for the node pair is as: where e is the set of all edges. the number of shared pathways and protein subnetwork that links between diseases i and j are calculated using the equation and the link prediction score is measured using the equation . to estimate the correlation starting from disease cooccurrence, we need to quantify the strength of disease association for comorbidities by dipicting a distance between two diseases. for the analysis of the phenotypic data, we used the relative risk (rr ij ) as the quantified measures of comorbidity tendency of two disease pairs and checked φ-correlation (φ ij ) to measure the robustness of the comorbidity associations. the rr ij is observing in a pair of diseases i and j affecting the same patient. when two diseases co-occur more frequently than expected by chance, we will get rr ij > and φ ij > . however, rr ij and φ ij are not independent of each other and each carries unique biases that are complementary [ , ] . so, we used both measures of comorbidity to ensure the robustness of our investigations. the rr ij allows us to quantify the co-occurrence of disease pairs compared with the random expectation which is calculated as: ( ) where n is the total number of patients in the population, p i is the incidences/prevalences of disease i, p j is the incidence of disease j and c ij is the number of patients that have been diagnosed with both diseases i and j. for rr ij >= comorbidity is larger than expected by chance and for rr ij < comorbidity is smaller than expected by chance. to calculate the significance of the relative risk rr ij , we used the katz, baptista, azen and pike et al. method to estimate confidence intervals [ ] . according to their estimation, the % confidence interval for the rr ij between two diseases i and j is calculated by: lower bounds of the confidence interval (lb) = rr ij * exp(− . * σ ij ) and upper bounds of the confidence interval (ub) = rr ij * exp( . * σ ij ), where σ ij is given by: disease pairs within the % confidence interval are only considered if the lb value is larger than when rr ij is larger than , or if the ub value is smaller than when rr ij is smaller than . relative risk measure is intrinsically biased towards overestimation of relationships between rare diseases and underestimates the co-morbidity of more frequent diseases [ ] . this bias can be reduced by introduction of a φ-correlation measure. we can quantify the strength of comorbidities by calculating the correlation coefficient associated with a pair of diseases i and j as: where c ij is the number of patients affected by both diseases, n is the total number of patients in the studied population, and p i and p j are the morbidity or incidence of the i th and j th diseases respectively. the φ-correlation is the pearson's correlation for the variables which only take or values [ ] . for φ ij > comorbidity is larger than expected by chance and for φ ij < comorbidity is smaller than expected by chance. we can determine the significance of φ = by performing a t-test. this consists of calculating t according to the formula: t = φ √ n− √ −φ , where n is the number of observations used to calculate φ. to predict the comorbidities considering the primary or index disease we have calculated the conditional relative risk (conditional rr ij ) as follows: for all possible disease pairs i and j, for the cases that one index disease (i) is present (k = true) or absent (k = false). (p = . ). we have weighted the edges using a mutual information metric which quantifies how much greater the edge relationship is with respect to co-occurrence. the mutual information weight between two diseases i and j is defined as w ij = c ij p i + p j − c ij ( ) where c ij is the observed co-occurrence and p i and p j are the morbidity or prevalence of the i th and j th diseases respectively. to compare between sars-cov and mers-cov, a gene set enrichment analysis was undertaken using gsea [ ] . to find out the correlation (similarities) and distance (dissimilarities) among the diseases from the integrated analysis of multidimensional data (gene expression and protein protein interaction), we have applied euclidian distance measurement and metric multi-dimensional scaling (mds) using majorization [ ] . mds is a set of methods for discovering hidden structures in multidimensional data. based on a proximity matrix derived from variables measured on objects as input entity, these distances are mapped on a lower dimensional spatial representation. optimization problem is used to find mapping in target dimension of the data based on given pairwise proximity information while minimize the objective function. the particular objective function (or loss function) we used in this work is a sum of squares, commonly called stress. we used majorization to minimize stress and this mds solving strategy is known as smacof (scaling by majorizing a complicated function). stress majorization is an optimization strategy used in multidimensional scaling (mds) where, for a set of nm-dimensional data items, a configuration x of n points in r(<< m)-dimensional space is sought that minimizes the stress function σ (x). here r is that means the (r × n) matrix x lists points in -dimensional euclidean space. we have applied the cost function σ to measures the squared differences between ideal (m-dimensional) distances and actual distances in r-dimensional space as follows: x of dimension n × p as the individual's or judge's configuration, and x of dimension n × p as the object's configuration matrix. the least squares metric multidimensional scaling or mds problem is the minimization of σ and over all m × p configurations x. here w ij are given non-negative weights and d ij are given non-negative dissimilarities. the d ij (x) are the euclidean distances between rows i and j of x. thus d ij (x , x ) = p s= x is − x js ( ) where w ij ≥ is a weight for the measurement between a pair of points (i, j), d ij (x) is the euclidean distance between i and j, and δ ij is the ideal distance between the points (their separation) in the m-dimensional data space. note that w ij is used to specify a degree of confidence in the similarity between points (e.g. can be specified if there is no information for a particular pair). a configuration x which minimizes σ (x) gives a plot in which points that are close together correspond to points that are also close together in the original m-dimensional data space. programming scripts are freely available at www.cl.cam. ac.uk/~mam /comor/. http://www.biomedcentral.com/ - / / information regarding each of the clusters and genes is described in additional file : table s . additional file : table s . connectivity map results of predicted drugs per instance (for each drug and cells line) to reverse sars-cov for early and sustained signature (drugs with negative enrichment scores). the impact of cellular networks on disease comorbidity a dynamic network approach for the study of human phenotypes comor: a software for disease comorbidity risk assessment comorbidity of cardiovascular disease, diabetes and chronic kidney disease in australia. canberra: australian institute of health and welfare mortality after incident cancer in people with and without type diabetes impact of metformin on survival comorbidities in chronic obstructive pulmonary disease comorbidities of chronic obstructive pulmonary disease the incidence 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the risk ratio in cohort studies applied multiple regression/correlation analysis for the behavioral sciences. routledge gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles a majorization algorithm for solving mds submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank fp -health-f - for providing financial support, under grant agreement n (mimomics). additional file : table s . highly statistical significantly differential expressed genes between sars and control group in pbmcs. table s . highly statistical significantly differential expressed genes between hiv and control group. table s . phenotypic disease association for sars infection based on the icd codes at the -digit category level. only statistically significant links with high relative risk rr ij are considered. additional file : table s . phenotypic disease association for sars infection based on the icd codes at the -digit category level. only statistically significant links with high relative risk rr ij are considered.additional file : table s . phenotypic disease association for hiv infection based on the icd codes at the -digit category level. only statistically significant links with high relative risk rr ij are considered. additional file : table s . phenotypic disease association for hiv infection based on the icd codes at the -digit category level. only statistically significant links with high relative risk rr ij are considered.additional file : table s . highly statistical significant differentially expressed genes between sars-cov and reference group (mock) in lung epithelial cells.additional file : table s . highly statistical significant differentially expressed genes between mers-cov and reference group (mock) in lung epithelial cells.additional file : figure s . median expression profile of sars-cov vs mock using hierarchical clustering (ward method, pearson correlation) of statistical significantly differential expressed genes (p < . ). the information regarding each of the clusters and genes is described in additional file : table s .additional file : figure s . median expression profile of mers-cov vs mock using hierarchical clustering (ward method, pearson correlation) of statistical significantly differential expressed genes (p < . ). the the authors declare that they have no competing interests.authors' contributions mam and pl designed and mam implemented the analysis of the paper. mam and pl wrote the manuscript. both authors contributed to and approved the manuscript. key: cord- -le t l g authors: nan title: pathological society of great britain and ireland. rd meeting, – july date: - - journal: j pathol doi: . /path. sha: doc_id: cord_uid: le t l g nan claire m allen, d m hansell. mary n sheppard depanmenls of diagnosbc radrology and lung palhalogy royal bromplon nahonal hean and lung hosprlal, sydney slree:. london sw np percutaneous fine needle biopsy s an established diagnostic technique for lung lesion^ afirm diagnosisofbenign versus malignant is often achieved but histological interpretation of small fragments or groups of cells is difficult manual cutting (twcut) needles provide asuperior histological specimen but are associated with a high complication rate and have been mainly used for pleural lesions this s the first prospective study to assess the feasibility of obtaining histological samplesfrom lung lesionswing apoweredcuning needle (biopty gun) we have biopsied patients using biopty gun there were no major complications histological diagnoses were obtained in patients ( malignant, benign) the malignant lesions identified included nan-small cell carcinomas. adenocarcmamas. squamous cell carcinomas, bronchiolaalveolar cell carcinomas, -cell lymphomas, small cell carcinoma. atypical camnoid and metastatic breast carcmoma. the benign es ons included sarcoidosis. clyptogenic organising pneumonia. wegener's, resolving pneumonias with chronic inflammation the rad~ologistls assessmentaftheamauntoftissueobtainedconelated withgood histology. thethreefalsenegativespecimens obtained the radtdogist noted the inadequacy of the samples ~n two cases and the third was a geographic miss percutaneous biopsy using the biopty gun is a simple and effective means of obtaining good quality histological material from lung parenchyma with a high degree of diagnostic accuracy for bath benign and malignant lesions inorganic particulate matter in "normal" lung: a study using light microscopy (lm), scanning electron microscopy (sem), and energy dispersive x-ray analysis (edxa) london, onlano. l partmeni of palhology soulh sireel london onlano n a g canada h e possible association between inhaled inorganic matter and some cases of usual inter tltial flbrosis (uipi has been of longstanding interest to pathologists and cllnoians. unfortunately. most analytical techniques are impracticable ~n the context of a pathology sewice laboratory in an anempt to find a practicable s utm to this problem and to establish a baseline for inorganic particle load in a "normal" population, a study was undertaken using techniqueswhichwauld beavailable ~nmostlargepathologylaboratories ninecaseswereselectedfram the case(centra andperipheralparliansaf lower. middleand upperlobesjandexamined with thesemand edxaand compared with the lm appearance to determine the panicle type and distrlbutm m a "normal" population. a wide range of inorganic matter was ldentlfied canespanding to siiicb. aluminium and magnesium s cate , rutile and alumina "like" particles varying from < p to p in size in addition. trace elements including zinc. cadmium and increase in number of particles wasalso noted in areas of fibrosis which were present in two cases (old mflammatory disease) preseumably related to problems in particle clearance the findings of this pilot study suggest that although the sem and edxa will likely prove useful tools in the evaluation of lung biopsy soeclmens. the findinq of lnorqanic material in cases of uip must be interpreted with caution autopsy senicb whlch had no known expcsure to lnorganlc dust sectlons were taken from the nght lung each pulmonary adenomatosis has been described as adl~tinctive pathologlcal change seen in the lungsolexperimental area of replacement of normal alveolar lining cells by a taller more glandular type of eplthelwm. usually wlthwt significant cytological atypia. we describe cases i" whlch a smtlar change was seen as an mdental fcndlng ~n resection specimensfor primary pulmonary adenocaranoma. the lesions (usually multlpleand each mm orless m diameter) were identified in lung parenchymaat a distance from the tumour and consisted of thickened alveolar walls lined by prominent, distinctly atypical cells morphologically slmllar to type i pneumacytes and cytologically different to the associated turnour reactive changes " lung involved by obstrmtive pneumonitis were not included !n thts sews all of the associated tumwra were peripheral adenocarcinamas and all showed a pattern of alveolar wall spread at the tumour periphery clinically of the patients were female and all were smokers or ex-smokers the slgnlflcance of this lesion in the histogenesis of primary pulmonary ademcarcinoma is. as yet, unclear animals often i" assoclatlon wlth exposure to inhaled camnogens morphologically the lesion is a clrcumscrlbed departmen ofli~slopafhology sf richard's hosprlal. chrcheslei wesf sussex. po se a series of consecutive personally conducted autopsies in patlenfsdying suddenly outside hospnal and where the death was reported to h m coroner presented cot deaths were excluded. in cases the death had been reported because theattendingdoctarwas unwillingtoissueadeathcertlficate. in theother cases. deathwasnot duetonaturalcausesandfallowedsuicidearan accident oftheformetgroup, therewere t unsuspected malor findings cn patients ( . %). that s to say findings which were either the cause of death or would have led to admission to hospital far assessment and possible treatment they had been discovered in life the largest group were cardiovascular ( ) but there were cases of unsuspected malignant disease there were only five malor unsuspected findings in the group of unnatural deaths years). these results highlight the loss of teaching material in the coroner's system, at a time when hospnal postmortem rates are in universal decline. this matenal would be of value to medical students and to pathologists in traming. and largely comes from cases of lmle or no medico-legal significance. %) and these were in older sublects (mean age . a total of postwnortem specimens refened to the royal victoria ho~pdal electron microscopy unit dunng the years - havebeen reviewed thkscompnsed . vo of thetotalnumber of sewre-relatedcaaesreferred to theunit.oulof ocasesreferred. wereexaminedandrecorded indetail theremainderdidnotundergoelectron microscopic examination for various reasons, such as a concius~ve diagnosis being reached by light microscopy alone. and semi-thin sections showing severe tissue mtoiysis. the most common tssues referred for examination were lung, kidney. liver, brain and head the range of e m. studies carried out included transmission electron microscopy item). scanning electron microscopy (semi and x-ray microanalysis on sem. theelectron micrographs wererevlewedwlth respect totlssuepresewatlanand th wa correlatedwlth the timeintervalbetweendeathand autopsy. electron microscopy was considered, an review. to have been diagnostically useful in % of cases an which it was deployed c s herringtan, a k graham, k cooper, j mcgee it was shown prev usiy that the discrimination of human papillamavirus (hpv) types and by nlsh in archival biopsy matenal requires dlfferent conditions from those predicted by conventional solufion kinetic analysis the parameter tm' (tissue tm) was defined in order to describe these differences in this study. these pnnciples were extended to the discrimination of hpv . and in casesol cln the results of nlsh analysis werecompared with both immunohistochemistry for viral capsid protein and pcr typing these data demonstrate that crosshybndisatm of high nsk viral types occurs ~n clinical lesions under conventional hybridisation and stnngency washmgcondit ns thiscross hybridisation isnot due to thepresenceofviralcapsidproteinand sm relikelyto be areflectionoftheendpoint usedinn sh.i e thepresenceofvisiblesignal.practcally.multiplenlshsignalsduet closelyrelatedprobesinarchival materialarenot indicativeofmultiplehpvinfectionunlesstheyarepresent either ~n morphologically discrete areas of the biopsy or their presence has been confirmed by another molecular technique. motegenerally, thepresenceof asignal in nlsh usingaparticularprobedoes not implythattheidentity of the target nucleic acid is that of the probe medical students in the autopsy room shows that % thought faces should be covered during autopsy, and % thought that genitals should be covered. about half the group were indifferent to both proposals % thought the patient's identrty should be concealed from observers three students thought there should be no conversation at all. % thought that conversation should be limned to procedures and findings and % thought it should be limited to professional maners;conversely. % thought thereshwldbenolimitson thetopicsdiscwaed nearlyso% thoughtstudents should beencouragedtoassistbutnotpressuredintadoingsa. . % thoughtthatallshould becompelledtoassist, but % thought that students should onlv obsewe. and not be allowed to ass s.t. % of students thought that they should oni!iobsene autopsies on patients they had clerked. whereas % thought they should onlfbe on autopsies on patients they had not clerked. fmally. % thought that relations should give spmfic perm on before students observed autopsies. whereas . % thought not a quantitative study of the effects of fibroblast growth factor on wound strength and cellularity fibroblast growih factor (fgf) has potent angiogenic and fibrogenlc effects and is lmpllcated in the formatlon of granulation tissueand healing few attempts have been made toquantity theseeffectsmnvo. we havestudled arat skinwound modelusing red cellghostsasan fgfvehicle tensiometv ofthewoundsshowed amaxlmaleffectof the fgf after seven days when the wound strength was % above that m controls (p < ) this effect had disappeared by fourteen days. computerized image analys~ using a joyce lo& mini magiscan measured total nuclear content of areas i" the wounds. permitting a topographic analysis of cellulanty versus distance from the wound centre cellularlty effects showed adifferent time course from wound strength. a % increase at fouldays anda % decreaseat sevendays. relative tocontrols(bothp < . ) attwelvedaysthecellularityeffectwasstill sqnlflcant at a vo decrease but by twenw-one days it had dwappeared the results suggest that fgf causes an early transient increase in cellulardy and more rapid increase in wound strength: most of these cells are macrophages and fibroblasts suggesting a connection between thesetwo obsenat ns. the adhesion molecule$ integrin (cd ). integrin (cdl ). and intercellular adhesion molecule- (icam- , cd ) are essential to the intimate co-operation of antigen presenting cells (apcs), t cells and keratinocytes cyclosponn. which is an effective treatment for psonas~s, may cause immunosuppression by altering antigen presentation we have pertormed a quantitative immunohistochemical assessment of the effect of low dose ~yclosporin on the expression of p -integnn. p -integnn and icam-f m the epidermis in chronic plaque psonas s. staininglevels werecompared withclinicalresponseasassessed bythepsoriasisareaseverihllndex(pas score) pt-lntegnnand cam expressionon keratioocyteswerenotaltered bytherapy buttherewasasignificantdecrease inthemeanlevelsof p positivelargedendriticcells(apc )within theepidermis.bz-integnn wasnot expressed by keratinacytes there was a strong correlation between p expression and pas score after three months on cyclosporln and one month off therapy these results lndlcate that p stalnlng on large dendrltlc epidermal cells previous studies enumerating silver stained n~c l~l a r organiser regions in problematic cutaneous melanocytic lesions have yielded inconsistent. but generally favourable, resulis. it seems probable that such inconsistencies. arise largely from differences ~n fixation. staining and counting strategies. our group, having devised improved methods of agnor staining and counting. is now able to re-examine the potential role agnors in borderline lesions pilot work demonstrated potentially significant differences in agnor dispersal between benign and malignant lesions and i" this study bath agnor numbers and dispersal patiems have been evaluated a range of melanocytic lesions including banal naev~. dysplastic naevi. typical melanomas, spiu naevi, atypical spih le~ions and minimal deviation melanomas were collected virtualty all of the unusual lesions had been diagnosed by specialists in dermatopathology. using a combined assessment of agnor numbers and dispersal, it proved possible to discriminate borderlip lesions from banal naevl and typical melanomas. benign borderline lesions -such as spit naevi ~ possess numerous nor$ but display tight clustenng in contrast to malignant melanomas wherenordispersa isaprominentfeature discriminatinganetypeaf borderlinelesionfromanathercauld not be achieved however, in practice this distinction is probably less important than assigning a aoirary specimen to a benign or malignant group a larger prospective trial of agnors in melanocytic lesions currently in progress with a mean total exposure of pack-years. alihough p was expressed more commonly in adenocarcinoma ( rof lo)andsquamouscarc~noma( ~~of )than~nsmallcelltumours(l %of o),thiscouldbeaccountedfor by the smoking history. since patients with non-small cell carcinoma smoked more (a mean af pack-yeas) than those with small cell lesions (mean of pack-years). there was no relationship between p expression and sunlyal. s. a. sun. j. r. ~a s n e y depaitmenl ofpathology university of liverpol, p box , l!vemooi. l bx activation of thec-myconcqene with overexpression of itsancopratein product ocurs in bronchlal malignancles ofalltypes, but has beenmost extensivelystudied insmall celicarcmoma, wherensaverexpression~n culturedllnes has beenassociatedwithdevelopmentoffast-growingvanants whlchlosemuchofthelrendocrlnephenohlpeand aner their morphology. it has been suggested that these vanant lines might be the equlvalent of the large cell bronchial endocrine carcinomas sometimes seen i " yiyo, but this is not proven. we have used th myc - e monoclonal antibody and the avidin-biotin technique to study the panem of expression of the p oncoprotein product of the c-myc gene in turnour deposits of twelve subjects coming to necropsy with disseminated small cell carcinoma in an attempt to relate l o morphological vanablity and metastatic site. anhough expression bore no relationship to morphological vanation. it ohen differed markedly horn site to slte. whereas parts of the primary t m w r strongly overexpressed the protein ~n all but one wbfect, there was considerable vanability between secondary deposits. oossiblv mdicatina arelation~hi~ between c-mycex~resslon and propensltvfor metastasls to certain iocations. tumour growth rate is a key parameter of neoplastic aggression. and is determined by the balance of cell gain and loss.apoptos~sisama]ormadeoftumourcellloss, but linleis knownof itsregulation. dieremesin turnourgrowth conferred by hpvtypes and werestudied in aratfibroblast modelsystem. immortaiisedcells weretransfected with hpv and expression vectors. either alone or with activated c-ha-rasl. monoclonal cell lines were established. and their vector dna content was confirmed by pcr tumour cell ddfered in their growth properties m wvo and m vilro. hpv containing turnours were larger and showed less apoptosis than those containing hpv , although bothshowed moreapoptasls than thenodulestormed bytheparentfibroblastsalone. in all turnours the presence of ras greatly reduced apoplosis and increased the growth rate. very similar propetlies wereobswvedin culture, and apoptoticratesshowed astranglnveneconelatlonwlthratesofnet cellgrowth. hpvs appeared iostim"latetumourcei apoptasis, butthiswas suppressed byras. melowerapaploslsassoclatedwilh hpv compared with hpv may partly explain the more aggressive phenotype of cervical c a n m containing hpv . it hasbecomeapparent thatanumberof maleculesmaybeexpressed byrestingorqu~escentcellsandarelostwith transition into the cell cycle. in addition to being of biological interest, such molecules may prove useful as operatcanal mahers of quiescent cell populations in histological material and allow the further characterisation of cellular subpopulations. one such molecule is statin. a kd protein previously reported to be expressed only by cells ~n go. we have shown bylaserconfocalfluarescence microscopythat thestatin antigen is associated with the nuclear envelope with a dstnbution m l ar to that of nuclear lamins. using a monoclonal antibody ( - ) that recognises slatin we have defined the tissue distribution of statin immunoreactivlty in a range of continuously renewing, conditionaily renewing and non-renewing tissues. the distribution of immunoreactivlty s essentially as would be expected of a maher of quiescent ceils. in contrast. in established pancreatic carcinoma and other epithelial ceil lines. we have found $latin immunoreactivity ~n cycling cells using biotinylated k slatin double labelling techniques. in conciusion. statin lmmunoreactivitv in normal tissues correlates with auiescence but this realtionship is lost, at least in vifro. loss of cell-cell and cell-substratum adhesion are imponantfactors during turnourprogression. tumour promoten are compounds which although not carcinogenic themsews increase the frequency of turnour development in animals previously exposed to carcinogens. we have used the turnour promoter tpa on cultured human renal epithelia ~ells mimic nwplastlc transformation. following tpa treatment we haveexamined thedistribution of vlnculin, b integrin and actin within thetreatedcells byfluorescencemicroscopy.treatment of therenal epithelium by tpacauses arounding up of the cells and a loss of adhesion toeither laminin or fibronecttn substrata fiuorescent microscopic examination reveals a progressive loss of reactivity for vinculin and b inlegrin within local conlacts. these changes are accompanied by a redistribution of the actin microfilaments from orientated bundles of stress fibres to a circumferential arrangement these changesofareduction in focal contact componentsand disarganised actin cytoskeietan mimic the changes we have previously described in renal carcinoma. glutathione s-transferases are a diverse group of enzymes with an important role !n the metabolism faelgn compounds including some carcinogens and cancer chemotherapeutic agents. increased expression of gst pi is seen in manyanimal and humancancersandisassociated with resistancetocalboplatinandcisplatin in humanlung cancet cell iines. gsts are involved m steroid hormone transport and metabolism and have a role in the complex metabolic relationships between settoli ceils and germ cells. we have studied gst isoenzymne expression by an immunohistochemical method in stage i teratomata. stage i tetatomata, stage i seminoma, cases of intratubulargerm cell neaplasia( tgcn) and a groupof cryptorchid and normal testes. the stage i teratomata had beentreated with cisplatin based therapyand bothprimmaryandpost-therapymetaftatictumourtissuewerestudied. gstalphaexpression correlated with morphological evidence of epithelial differentiation in teratomata.therewas no difference in gst expression between stage i and stage i pnmary testicular iumours nor between primary testicularand post-therapy metastatic tumours. gstexpression did not correlate with survwal. gst pi was strongly expressed in the neoplastic germ cells of itgcn but was weak or negative in normal germ cells. this may be significant in view the potential for later contralateral turnour development tn patients treated by cisplatin based therapy. in summary, gstexpression in testicular germ cell turnours reflectedlheirdifferentiationandappsared to be unrelated l o therapy and subsequent survival. a case illustrating the usefulness of electron-microscopic examination of fine needle aspirates s described fine needleaspiration wasperlormedon asubcutanwusnoduleinthechsstwall ofaneldedyman whowassuspmted tobesuffering from bronchogeniccaronomaanclinicaland radiologicalfindings.mesmears werenotdiagnan~c. but ~n view of the history af asbestos exposure, the needle washings were submined for eiectron-microscopic examination, which showed mesathelial differentiation and a diagnosis of metastatic malignant mesotheiioma was suggested. an autopsy perlormed eight months later confirmed the diagnosis of malignant mesotheboma. t. dorman, a ti ismail, b cunan, m. leader pathology department hoyal college of surgeons m ireland, dublln many histologi~allymalignantfeaturessuch as hypercellularltyand high mitoticcounts, but clinically followa benign course. demoid tumours and fibromafoses can be densely cellular but usually quite bland histologically and are associated wdh infiltrative marginsand repeated local recurrences. in addtion irradiated tissue often contains many cellswithcytologicalfeaturesol malignancy thisstudyexaminesthepioidyofsuch lesionsbyboth olthecunently availabletechniques using dlsaggregated formalin fixed paraffin embedded tissue sixteen cases nf, dfsts. dts. fibromatoses( nci~ding palmar, plantar. retrcpentonealand soh tissue) and miscellaneouscases including leiomyoblastoma, inflammatory pseudosarcoma and plexiform histlocytama. one malignant fibrous histimytoma and t paragangliama were included as possible poslive controls. cases containing numerous irradiation fibroblasts were alm analysed all 'pseudosarcwnas' were euploid by both image analysis and flow cytometry the malignant fibrous histiocytoma was aneuploid by image analysis and flow cytometry and the paragangliomawasaneuplaid by imageanalysis and tetraploid by flow cytometv themndwonsof thisstudv are that pseudosarcomas are euploid and aneuploidy would appear to be confined to malignant tumours of mesenchymal ongm. other sites we collected cases to study their morphology and antigenic profile after staining for the epithelial markefficam . flow molecularweight keratins)andlp (high molecular wefghtkeratins), thegeneralmelanoma and neural markers s w. neurone specific enoias (nse). protein gene product . (pgp) and the melanoma s w i f l c marker hmb , the neuroepithelia markers leu and glial fibnllary acidic protein (gfap), and the intermediate ftlaments vimentln vim) and nsurofilament (nr the following turnour types emerged: i pure spindle celltype(n = ), ,purepolyganalcelltumaun(n = )comp sedofpleomorphiccells(n = ).undwmcellswith an alveolar in = ) or sheet arrangement (n = ). and mixed pleomorphic spindle and polygonal cell tumouffi (n = ). two tumouffi proved to be lymphomas an intraepithelial melanocytic component could be established in cases within adjacent respiratory or squamous melaplastic epithelium. in cases n e w trunks a~mclated with tumour contained increased numbers of atypical schwann cells. consistently expressed were s w ( and colon ovanan tumour antigen (cota). csais a heat stablemucin associated antigen present m normal colonic epithelial cells and is expressed m greater quantitites tn colon c adenmarcinomas cota is a heat stable antigen present n colanicneoplasiaand mumowwananturnours but not ~nnormalcalonicepithelium psmionsfrom to primary adenocarcinomas ( ovanan, colo-rectal, gastric. breast. oesophageal. prostatlc. pancreattc, endometnal and t gallb adder)and two adenmarcinomas metastatic tothe liverwere incubated with anti csa and anti cota antibodies using the p.a.p technique with positive and negative controls. while anti csa positivitywasseenin d ~ loni~aden carcin ma~( wsakand lo~tr~"g).~twasalsaseen n af ovanan ( weekand strong). of gastric ( weakand strong), of breast( weak and strong). of oesophageal ( weak and strong). of prostatic (all weak). of pancreatic ( weak and strong). of endometnal (all weak) and the gallbladder ademcarcinoma. both liver metastatic adenocarcinomas were negative in additim while anti cota staining was positive m of colonic adenocarcinomas ( weak and strong) and of ovarian adenocarcinomas.~twasalsapositive~n af gastnc( weakandzstrong). al lobreast( weakand stlmg), all esophageal(allweak) of pr ~tati~(allweak), pa"creatic(low akand t strong),all endometnal(all weak) and the case of gallbladder endocarcinoma (strons) both liver metastatic adenocarcinomas were negative. m e c o~c~u s~o~ of this study s that anti csa and anti cota are not adequately specific in the identification of a ~olonlc or ovarian origin af an adenocarcinoma and cannot reliably be applied to the identification of a metastatic adenocarcinoma of unknown primary site the early intfa-epithelial changes of adenocarcinoma of the nose and paranasal sinuses were sought in the histological sections from high grade adenocarcinomas and cyclindric cell carcinomas from this region none of which had had previous radiotherapy treatment. of the cases came from wwd-workers. i r m a polishw and from a plasterer attention was directed to the non-neoplastic epithellm of the surface and of the sera-muclnous glands. in casesofadenocarcinomaand t of cylindriccell carcinoma surfacechaogeswere detected these took the form of hyperplasm of goblet cells of the rsspiratq epithelwm accompanied by dysplastic changes of other epithelial cdls: m the latter these became enlarged and irregular m shape with large hyperchromatic nude and prominent nucleoli. lnthsmajorityof iesionsthesechangeswereisolatedin theepithelium. changesof thesetypes were never seen in sefomucinous gland epithelium. in addition similar changes were not noted m the coverlng respiratoryepithelium in casesofnasal polyps evenwhen severely inflamed thesemsmchangesso described haematoporphynn derivative sensitised tumwr tissue. s underwing evaluaton " widesptead and m i s t a n t papillary tumoursand ,n casesof rvldespreadseveredysplasra/carnnoma,nufu". arewewof spallents ( , m, ages years. pre-and post therapy) has revealed no change ~n histological grade and stage ~n patients, progression to invasion in t case and a redunion to mild urothellal atypla alone in case. local tissue changes following treatment were limnedto oedema and an acute lntlammatoryreactlon. no metaplastlc. stromalfibroblast or nene changes were seen variations ~n bladdw size. both increased and dlmtnlshed, wwe encountered the histological grading of tcc following pot shows little improvement. but studies are in progress to improve lhght delivery in the bladder, and hence improve treatment outcome. a armour. a. s. jack bpartment of pathology, umverslty of i d s . leeds. lsz jt follicular lymphoma is a disease characterised by widespread lymph node involvement usually at the time of presentation proper tie^ of lymphocyte homing and circulation appear to be mediated by a variety of cell adhesion molecules. the a m of this study was to compare mrmal nodal lymphocytes with the neoplastic populatlm m folllcularlymphoma. hall casesalloftheneaplasticfolliclesexpressed lcaml but thisyanedfromonlyafewcellsto % of cells within a follicle this dosltlvlty included dendmic retic~l~rn cells. germinal centre cells and neoplastic lymphocytes. leu (lymphocyte homing receptor) showed an inverse pattern of expression in all but cases the phenotype (icaml + l a is a feature of germinal centres and scanwed paracontcal blasts in reaclive nodes anhough there is uniformity of expression of lcaml in germinal centres which is not apparent in any folllcular lyumphoma case. this study showed a loss of lcamt and increased leu expression by neoplastic lymphocytes within follicles this may relate to the propensity of this disease to spread widely throughout the lymphatic system activation leads to an alteredfunction and aconcomitant aneration in the chemistry ofthe cell surface, which ~s a site nchincalbohydrate. rat lymph nodelymphacytes.eitherunactivatedoraclivatedfot days namixedlymphocyte reanlon. were treated with biotinylated iectins from a large panel. chosen to probe surface glycans lmlns were revealed with awdln-phycoerythnn and cell populations were analysed in a fluorescence activated cell-sorter double-stalnlng with fitc-monoclonal antlbodles defined the functional lymphocyte subsets. uea- and lta boundtonoce sof anytype. unactivatedb-cells boundall theremaininglectins.savempa. toagreaterextentthan did unactlvatedt-cellsand b-cell actlvatlon produced no change i" glycan sxpresslon. unactlvated and actlvatedtcells all expressed a . -linked sialyl residues, but o- . -linked sialyl expression was hetrogeneous, did not conespond toanysubsets,and wa~unchangedonanlvatian.alargegroupof lectinsshowedlow wno binding to unactlvatedt-cells, but boundonactivatianexactly in parallel to l- receptorexpressionctacantigen) thes~ngle structure gawgalnacal, galpl, glcnac-) galigalnacat , gal~ , glcnac-) r could account for their binding the presence of neoplastic (light chain restricted) b cell follicles in low grade b c d gastrolntestlnal (gi) mmphomaof although malt s not present in normal human gastric mucosa, lymphold t,ssue ,* acqulr& ,n response lo colonisationof themucosa by helrcob~cleipyioii. we have investigated the possibility that thisacquiied lymphoid t ssue is malt type whlch may povovlde the background i" which lymphoma can arl~e w e examined gast,,c b~ p ~e fr m casesafh~l~~~bacrerassaciatedgastntis.and biopsyandresectionspecimensfram casesaf t"mours has contr'buted to a peklstence of ihe more "iew arefofl'cle (fee) gastrlc cell lymphomaof malt in cases of hebcobaciergastritis prominent lymphoid follicles were idenfilled in aftheee b cell clusters were identified within the gastric epithelium. reminiscent oltheteatures seen the dome epithelium of sm intestinal peyer'z patch. thls b cell-eplthelh assoclatlon was not assaclated with the eplthelm cell changes or the glandular destructm seen ~n lymphoeplthellal i~s i o~s malt lymphomas. in / cases of gastric maltlymphoma helicobaclercould be identified oltheremaining cases. weiegastrecfamy specimens n which specimen washing may have contributed to the negativefindings. we suggest that gastrlc malt s acqulred in response to local immunological stimulation as a result of mucosal colonisation by hel~cobaclerpylon, and that the development of malt lymphoma is a subsequent event mucosa associated hmphold tlssue (malti has been explained on the bas of speclftc colonization at reactlve b lymphomasbe included in lhecatqoryof malt lymphoma but the frequent presenceotafoll~culapanem in these foltic es by ihe neaplastlc cantrocyte-like(ccl) it has been low grade cellthyro d we have ihe and 'nvesflgated the and genotype of' of primary low gradebcelllymphomasofthethyro'd alsodemonstratedfeatures ofmalt ymphama'ncludtng cclce and l y m p h~' t h~' a l l~' o n s . m e appearances and immunoh'sto'og~ofthefo'l'c'es werethoseofto'l'cularcalon'rat'on described 'ngimaltmmphomarather thanfccfoll'cular'~mphoma thepredomlnant iwg ~ane'naffoll~cularcolon~rat~~nconformed tothatdeslgnated cclcells show'ng astnk'ngly h'gh prol'feratton late no evidence of the '( ' ' transiocat'an was found in any on dna extracted from fresh (n = ) or paraffln embedded (n = ) t sue mese ilndlngs argue against a fcc lineage lor primary thyroid lymphomas and support their ncius on " the maltcategory we have used a panel of antibode$ to demonstrate stages of granulocyte maturation by immunoh~st chemistry i" decalcified. wax-embedded bane marrow trephine biopsies antibodies reactive with muramidase. u- -ant~tryps,n. neutraphil elaslase and cd react with early granulocyte precursors. cd and calgranulin identity later granulocytes. ihdiylduai antibodies dlfier rn the populations cells ldentlfled theantlbcdtes also react with cells of mwlocyte llneageand provide information concerning theorganlsatlon afmonopolesls about whlch liffle is known in normal marrow granulopoiesis is zonal wlth maturation occurring radially around trabeculae and blood vessels this pattern is exaggerate in reactive hyperplasia and chronic granulocytic leukaemia (cgl) there is marked disruption of this zonal organisation in myeloproliferative and myelodysplastic states, with considerable overlap patterns between these conditions in chronic mvelomonacvtic leukaemia (cmml) there is comdlete absence of zonal arrangement of granulopaiesis possibly due to monocytic proliferation obscuring the underlying marrow spaces and that by analogy with cgl cmml represents an exaggeration of this normal panern granulopoletlc panern we hypotheslse that monopoles normally occurs " a randomly dispersed fashion within as previously shown by us in animals untreated with cyclosparin a. the cell birth rate fell from an initial prelmmunisation valueof cellsit cellsihour to cells celldhouran day followed by a rise cellsit cellslhour on day however, in cyclosporin a treated animals the cell birth rate ( ce s/looo cells/hour) was signiticantlydepressed belowthecontrol pre-immunisation levelandremained suppressedupfaday fallowed by an abrupt rise these results are consisten with the hypothesis that t lymphocytes or their pmducts not only drwe the morphological appearancescompnsed lymphoepithelia lesions (in onecase). numerous lymphoid follicles and a diffuse infiltrate of monocytoid cells (centrocyte like cells) there was striking plasma cell differentiation and colonisation of lymphad folllcks by monacytoid cell?. and neoplastic plasma cells. lmmunahistochemistry convincingly demonstrated heavy and light chain restriction ~n all the biopsies from both the cases the bladder s developmentally related to the hind gut and this manifests " the variety of metaplastic epithelium seen at this site circulating cells. encauntenng the endothelial surface. make contacts an environment rich n glycan. a large panel of biotinylated lectins was used to probe far variations in the glycans expressed on endathelia of artenes. veins, arterioles, venules. capillanes, high endothelia vessels and lymphatics m a range of normal and pathological human tissues formalin-fixed, paraffln embedded specimens from the files of manchester royal infirmary and manchester royal eye hospital were used lectins were revealed with an avidin-peraxidasesystem no differences werefound between arterial. arteriolar, venular, veinous or lymphatic endothelia all expressed abundant complex-type n-linked glycans. of several subtypes. capillaries were highly variable and showed heterogeneity ~n their expression of ) outer chain sequences fn n-linked glycansand ) mucin-type sequences. both between different. normal organs and within an organ. implying that the surrounding tissue probably had a regulatory effect where endothelium was reactive, additional aneratiom wete seen and actively growmg endothelium in granulation tissue expressed hqhmannose siwctwes. high endolhelial ybss~is showed a much lower density and narrower range of glycan expression thandidad~acenlnormalcapillanes,despite theirknownvery highrateofglycansynthesisand secretion the iymphocyteiln-homing receplor(s) would be a component of this restricted porlfolio of glycan monocyte margination in atherosclerosis associated with immunological injury n. j. combs, p j gallagher. p. s bass clinical and experimental evidence indicates that immunological injury is associated with accelerated atherosclerosis. allograft recipients may develop accelerated atheroscleross and anlmalsgwen serum sickness and a high fat diet develop more extensive atherosclerosis than controls fed the diet alone we have tested the hypothesis that atherosclerosis ~n these animals is associated with increased adhesion of monocytes to the aortic endothelium. chronicswum sickness was induced in genetically hyperlipidaemic rabbits with nativeanionic bovine serum albumin (nbsa) or highly cationised protein (cbsa) rabbits given nbsa showed a spectrum of glomerular endocapillary prollferativechange thasegiven cbsa developed early membranous glomerulopathy in controls the numberofmanocytesadherent to theendothelium ranged from y isq mm and m animals given senm sickness - isq mm (nbsa)or ~ isqmm(cbsa) as therewerenosignificant intergroupdifferences theseiesultsdo not support the hypothesis that immunological injury increases monocyte adhesion to the aortic endothelium departmen! oipatholcgy, souihampfon universily hospdals soufhamplon. so xy amyloidcan beidentified nupta % of elderty hearts,especiallyin themyocardium oftheatrialappendages in a small prapanionofth%lecasesamylaid~salsapresent inthecardiacvalve~ butisolatedvalvulardepos,tsarerare. a year old male presented with bilateral leg weakness and an intradural turnour. he died days after spinal surgeryanddeposltsofaglloblaslomamunlforme wereldentlfledm both thecerebrumand thecord therewasno hlstory of cardiac disease but all four valves showed translucent verrucous thrkenlngs. these had a uniform eosnophilc hi~lologicalappearance. stainedwithcongo red and had ultrastnucturalfeatures of amyloid amyloid p proteinwas identified immunohistochemically ~n thesedeposits but negative multswere obtained withantibodies t o m . aland a proteins. mwewas no evidenceof cerebrovascular ormyocardial amyloidosis the involvementof all four cardiac valves. the stnking absence of amyloid i" other organs and the a~~o~l a t i o n with a widespread glioblastoma are unusuai but unexplained features of this case the ima s used as a bypass of narrowed coronary arteries it is said to be less prone than vein gratk to develop subsequent occlusivedisease. thisstudy of pairs of imasfrom subjects of vario~s ageswas to see if the histological structure ofthevessel might explain immunityto graft disease sixteen imas were fixed in distension byformalln at cm of water pressure and secllon~ taken at the level of each nb (first to stxth). all arteries undelwent similar changes along thelr length. wlth no slgniflcant difference between left and rlght lntimal changes were minor conssting of lbro-elastic muscular thickening in all age groups and in those who had died from vascular disease theinternalelastic lamellawaswelldefinedat all levels. buttheexternallamellawasclearlydefinedonlyatthelevel ofthefmhandslxthnbs(i.e. mthedetalartwyj. medral thrckne~~decreasedalongthelengthofthearter~esandinall cases changes from an elastic to a muscular structure, generally at the level of the fourth or fiflh rib the ratio of medial thickness to numbw of lmdlae in~reases along the artery. notably at its point of change muscle fibre orientation changes from inter-mixed cir~ular and longitudinal in the elastic part to predominantly circumferential in the muscular pan. the pronounced stnctural differences of the ima compared to the similar sired epicardia coronary arteries. which are muscular. may be of relevance in explaining their markedly different incidence of atheroma. naomi carter, s variend fatty change of the heart is a pwrly defined pathological entity which in the adult hean can be caused by severe hypoxia nutritional disorders. poisoning by selected drugs and catecholaminerelease it is most commonly seen ~n association with coronary artery disease little data exists with regard to the paediatric hean in pediatric deaths coming to post mortem over a year period. there were cases of myocardial fatty change of varying seventy and distribution detected ~n red -stained sections infection and congenital disordenwereimplicated n deaths infectionand cangenitaldisordenwereimplicated n deaths. fallcasesoffarmchangeand % of all deaths seven ofthe e~ases had acombined ~nfecti~u~andcangennalaetiology othercausesofdeath included turnour, traumaandcomplicationsofbinh % of casesofsudden infant dealhsyndrome(s ds) hada fatty heart only one case of the total cases showed deflnlte hlstaloglcal evldence of ischaemic myocardial damage. insome instances, thedegreeaffattychangemayberelatedtathedurationand seventyoftheundertyng condition someofthesechildren may havean occunnutritional orenlymedisarderwhich ~sexpressedatacellular level ~n the form o! fatty change and that contibutes to their early death. we havecampared thereparting oftemporalarterybiopsiesbetween - ( cases)and ( cases). the overall incidence of positive biopsies was . % and . % in each period the number of patients with clear clinical evidence of cranial arteritis was % but in - , % and in , % of these had positive temporal artery biopsies when the histology was reviewed approximately % of biopsies in each period had been erroneou~ly reported as healed or atypical artentis. in contrast. a true histological diagnosis of artetitis was missed in~nlya~inglepatient approximately % ofall patientswithaclinicaldiagnosisof giant cellarteritisdevelopedan additional symptom or pathological change associated with steroid treatment frequent final clinical diagnoses in patients with negative temporal artery biopsies were transient ischaemic attacks, cerebrovascular accidents. unexplained headache or migraine. polyarteritis or polymyalgia hwmatica these resuns confirm that one third of patients with deflnite clinical evidence of cranial arteritis will have negative biopsies pathologists continue to misinterpret normal arterial ageing changes as evidence of healed or atypical arteritis depanmenf hslopafhology and depanmen! resp raioiy medicne. sl banholomew s hospifal london ecla /be adhesion ~fepithelium to extracellularmatrices is mediated partly byafamily of heterodimeric molecules known as megrins we haveexamined theexpressionofthealpha- toalpha- integnn subunits in~unured human branchia epithelial cells. and in bronchial biops es from normal subjects and atopic asthamtics. we have also studied the expression of intercellular adhesion molecule-t (icam- , cd , rhinovirus receptor) br~nchialepithelia cells from surgical specimens were grown as explant cultures on glass covenlips. bronchial biopsies were taken from right upper and middle lobe carinas lmmunastaining was performed on acetone fixed cells and frozen tissue sections using alkaline phosphatase and immunoperoxidase techniques all biopsies showed strong positive staining of epithelium for alphe?, alpha~ and alpha- integnns. stainingforalpha- was weak or negativeand epithelium was negative foralpha- and alpha- except in twoasthmaticswhere itwas weakhi alpha- posittve. in contrast. cultured bronchial epithelial cellswere positive for all these mtegrinsexcept alpha- . epithelium was positive for cam- in asthmatics but negative all other biopsies. cultured cells were ~trongly positive for this molecule it concluded that expression of some adhesion molecules bv bronchial emhelium may vaw ~n relation to the cellular environment and that ths may be imponant tn disease l%panmen! of pafhology univeisrty of birmingham and departmenrs o! 'immunology and his!opalho!ogy eas! blood eosinophils are in a relatively inactive state with migration into tissues eosinophils became more activated these activated cells are hypodense compared to most blood eosnoph s low affinity receptors for both ige (fcerii. cd and igg (fcgriii. cd ) have been documented on activated. hypodense eosinophils this study assessed the expression of these proteins on ttssue eosmophk derived from nasal wlyps the blood and nasal polyps of seven patients undergoing nasal polypectomy were studied nornodense and hypodense eosinophils were isolated from venous blood by centrifugation an a discontinuous percoll gradient percoll wassimilarly used to obtainaneosinophilrichpreparationfromcellsearactedoutafthenasalpolyps cytospin preparationswetem?.de of these samples frozen sections of each polyp were also prepared lmmunostaining using an alkaline phosphataseianti-alkalln phosphatase detection system demonstrated that neither blood nor nasal polyp eosinophilsexpressed detectablecd orcdt l t i~p~~~i b l e t h a t e~~i n~p h l l s o f na~alp~lyp~aresimilarto blood eosinophils and are in a relatively inactive state lavage and biopsy studies. the relationships between mucosal inflammation. bronchospasm and bronchial hyperreactivity are unclear since bronchial smwth muscle has an essential rolein the pathophydogyof asthma. we haveexamined theextentto which it ~sin~ol~ed~nallergicinflammation bi p~ie~in~l~ding~moothmu clefr m asthmatics (age range who did not use steroids and controls (age range ) were embedded i" araldite and stainedformast cells ~monocional ant bodyaat)and eosinophils(monoclona antibody eg ). mast cell numben in the lamina propria and smooth muscle were similar for both asthmatic and control sub e m (mean values. astham lamina propna mm . smooth muscle timm , controls /mm and blmm'respectively) eosinophil numbers in the lamina propria were increased -fold in the asthmatics (p = ) but there was no significant increase in the number of eosinaphils rn the bronchial smooth muscle eosinophil numbers in the asthmatics correlated positively with fev, we conclude that the role of muco~al inflammation in the pathophysiology of asthma has yet to be determined we present cases ofextra-pulmonary pneumocystosis diagnosed on routinesurgical specimens (two biopsiesof liver. and one each of gastric mucosa. small intestine and a pen-anal mass). in each case. the histological features were similar to those seen in the lung. and as in other material from cases of aids, munlple pathology was often found extra-pulmonaly pneumocystasis s now being reported from a widerangeof clinical specialties. one reason forthismcreasemaybe that impraved patient suiyiy~i with"topicay inhalatiooalpentamidinetherapyallowsvisceral foci of infection to become clinically apparent this hypothesis is supported by the finding that ofour cases were taking nebulised pentamidme inthesehypersensltivitystatestothatobselved mbronchiectasis (a chronic suppurative lung condition not thought to in~olve a hypersensitivity aetiology) and in smokers and non-smokers with no evidence of active pulmonary inflammation ourresuns havesh wnthattheb:tlymphocyteratioisnodifferent ineaaandsarcoidfromthat seen ~n bronchiectasis and in normal lungs. we believe that thns is further evidence to suggest that bal is an unrepiesentativetechnlquetorlhestudyof interstitial lung diseasesand that morecansiderationshould begivento the possibility of humoral immune components in the pathogenesis of ea determine its effects upon the number of pulmonary neuroendocrine cells and their peptides. in one experiment. the concentration of noradrenaline in the lung was estimated by chromatography, and that of the peptides bombesm. neurotensin and caknonin gene-related peptide (cgrp) by radiaimmunoassay. there was significantly ies noradrenaline and bornbesin in the lungs of test rats than in controls but the levels of neurotensin and cgrp were unchanged. in a second experiment, pulmonary neuroendocrlne cells in histological sections were labelled with antiserato bombesin,calc,t~tonin,cgrp andproteingenepmduct [pgp s ) a~d =~" " t~d . t h~, e w =~ nochange in the number of labelled neuroendocrine cells expressed per unit area of lung or per unit length of airway between test and control rats for calcitonln. cgrp or pgp . . bornbesin-containing cells could not be identified in either group. an increase ~n pulmonary neuroendocrine cells could not be identified ~n either group. an increase in pulmonary neuroendacrine cells immunoreactive for bomtesin and calcitonin occu~s in the early stages of plexogenicpulmonaryartertopathy in man. theabsence ofsucha change in monocrotallnedulmonanhydertenslon in the rat suggests that this is a poor model for the human disease. this preliminary study was carried out to assess the quantitative expression of neuroendocrine and mast cells in adun humanlungsofcasesofasbestos-relateddisease.tencaseseachofasbestasis, pleuralplaques,carcinoma and mesothelioma were studied m comparison with ten normals wnh no history exposure to asbestos the lung sections were stained lor neuroendocrine markers neurone specific enolase (nso and chromagranln. and, chloroacstate esterase and toluidine blue for mast cells. there was a notable variation in the number of neuroendocrine and mast cells between the control and asbestos-related disease group the variation was also seen between the various asbestos-related diseases. though not statistically sbgnificant. the trend of the vanatton indicated that the individual diseases follow a particular pattern a n lhe expresston of these two cell popuipioiis. we have studied fibrous turnours of the pleura using morphology. immunohistwhemislry and eleclronmaroscopy. the findings were compared and contrasted with reactive pleural fibrosis and desmoplastlc mesothelioma. the fibroustumwn hadarangeof histologicalappearancesand % weremalignant ~nnature.theimmunophemtype was uniform and consistent with positive staining for vimentin and alpha m w t h muscle actin. this was ~n sharp contrast to findings ~n reactive pleural fibrosis and desmoplastic mesothelioma. uttrastructural appearances of the fibrous tumours of the pleura were supportive of a myofibroblastic ongin. we propose that fibrous tumours of the pleura arise from the submesothelial myofibroblast. the malignant fibrous turnours have a distinct immunohistochemical profile and electron microscopic features to differentiate l from themalignant mesenchymai mesothelioma. a study was undenaken to evaluate the use of immunaperoxidase stains on paraffin embedded tissue lo define the cell type in routine lung cancer preparations. and in particular to identdy a subgroup of turnours showing neuroendocrinedifferentiation. forty lour consecutivethoracotamycaseswere selected. following apilot study of cases. to assess digestion times and potentiaily useful antibodies, the remaining cases were processed using a battery of monoclonal antibodies: cytokeratr iaelfae , e ). neuron specitic enolase (nse), chromogranin. and beta microglobulm. in addition to the carcinoid turnours and oat cell carcinoma in the study large cell carcinomas and adenmarcinomas demonstrated positive neuroendocrine markers. uiirastnrcturally, dense core granules could be demonstrated in only / of the large cell carcinomas and in / adenocarcmomas. the discrepancy between the lmmunoperoxldase staining and electron microscopic features likely reflects the heterogeneityafthese turnours. in thisstudy noneof the turnours co-expressed neuroendocrine markersand beta microglabulm. however, the staining panern was inconsistent m the remaining cases high molecular weight cytokeratin ( pe ) was stronglyposltlve m all casesot squamouscell carcinoma and negativein everyihing else. in summary. monoclonal nse and chromogranin appeared to provide sufficient information to identity neuroendocrinedifterentiatian in thecasesexamined m this study. high molecularweight cytokeratinwasfound to bea usefuldiscriminaforforsquamouscelicarcinoma. beta microglobulin was negativemall the turnoursshowing neuroendocrine differentiation. butthe inconsistency of staining m non neuroendocrinetumaurs. made it less helpful for routine laboratory use although endocrine differentiation is the essence of small cell carcinoma of the bronchus, its occurrence m other morphological ("on-small cell) types of bronchial umour (large cell, squamous and adenmarcdnoma) is welldescribed however, its prevalence m such tumours is uncertam, estimates differing from study to rudy and accordingto how it is sought. we have examined, byimmunalabellmg, expression offiveendocrine markerproteins (neuron-specific enolase (nso, protein gene product [pgp) . , the bb isoenzyme of creatine kinase [ck-bb), synaptophysinands-t protein)m bronchoscopictissue biopsiesof "on-smallcellcarcinomaandasse~ns varlabiltty within and between tumour deposits in subjects coming to necropsy with disseminated disease exactly half of the tissue biopsy specimens immunolabelled for one or more marken; one for four, four for three, twenty lor two and five for one. possibly indicating an element of endocrine dtflerentiation inapparent from their morphology. expression was even more prevalent amongst the extensively-sampled turnours at necropsy, but since theinlraductionofthenewgeneralpractnianercontractmapril ,lhere hasbeenasignlicant increasein thenurnbeisof skin biopsiesreceivedmhistopathologydepa~ments in ourdepartment there has beenalhreefold increase in numbers of general practiioner skin biopsies. the aims of this study were to crnlcally appraise these biopsiesand comparethem tosimilarlysized skin biapsiesreceivedfrom hospital in-patientsviageneraland plastic surgeons for the six months prior to and aner st april . data collected included numbers received, ranae of pathological diagnoses, quality information supplied on the request card, accuracy clinical diagnoses. adequacy of excision, age. sex and sites of lesions the resuns showed a similar range of pathological diagnoses. the quality of clinical information supplied was comparable in the two groups as was the age and sex of patients. general practitioner biopsies were less common from the face. clinical recognition of lesions was somewhat less accurate amongst general practitioners than amongst hospital surgeons inadequate excision was more common m general practnioner cases. . % of general practitioner lesions were found unexpectedly to be premalignant or malignant (eight cases) and all ofthesewere inadequatelyexclsed. important implicationsemergingfromthisstudy are dlscussed an audit of skin biopsy specimens from general practitioners in grampian region: changes in requesting practice and specimen type assessmen ofresaction marginsofsurgicalspecimens~s becming more important m manyfieldsofpathology. we wwe interested in developing a means of assessing surgical margins dermatopatholqy in both conventionally removed skin ellipses and m skin ellipses removed during m o~s chemasurgery technique many of the conventionally used markers, such as indian ink, alcian blue and tipp-ex correction fluid are difficult to use i" that they are messytoapply. slow todry and show insufficient contrast wiih oneanother both lnthegrass specimen and microscopically weused"superman'paint,apaintusedforresinandplastermodels,wh,chcomesin awlderange of colours. the paint was easyto apply. did not run and dried qwckiy becauseof the variety of colours avaciablewe were able to apply contrasting c~l o u n to the vertical and horizontal axes of b x c~s i o~ af skm elhpses removed by mohschemosurgery.thepaint proyidedagaodmarkergrasslyand wasnot affected byfreezinqthetissue model paint provides another marker lor surgical bxcij~ii margins and s particularly useful for moh's chemasurgery where horizontal and vertical axes aremarked inorderto assess theadequacyoftheexcision.the model paint may also be usetul in other branches of surgical pathology where resection margins are important. in psoriasis there is altered bpidermal dmwentiattan and increased epidermal turnover both which involve changesin intercellularadhesion aquantnative imm~noh~st chernicaicompa~~sonof the expression of the integrin staining of integrin p subunlts - and e subunlts were disclosed usmg an andin-blatln peraxidase techn!que. large epidermal dendritic cells (antigen presenting cells) expressed p- the psoriatic skm.showed increased - a- . - . and p- expression. buta- and~- showednosignificantdifferencefromnormal thelncreasedlntegrin expression by keratinocytes seems to be a reflection of awered epidermal differentiation rather than increased keratinoc ?turnover themcrease in ~- pasitivedendriticceilscauidbeareflectianofalteredanfigen handkngin psonatic gin. interleukm- (il- ). used m the treatment of patients with metastabc disease fallmg to respond to conventional treatment, can induce regression oftumwrbuik in certain patients however, the systemlc administration ofil- is associated wlth a number of toxic effects, including dermatological compl#catlons. these have been poorly documented we have prospectivehl studied the dermatological reactions patlents treated wiih il- for metastats cdorectal ~arconoma. pre-and past-treatment biopsies were obtwied where possible, arid sect~ons stained with h e. giemsaand pas: hesh tissues weresubjected to immunophenotyplng.there were femaleand malepatients.with treatmentrour~sranginginnumbwfrom - onlyonepatlenthadapastmedlcal hlstoryof any skm complaint (eczema]. four patlenk suffered a dlffilse eryihematous reaction with mild desquamatm and dryness the other patient developed genwalised erythroderma and an additlonal photosensitivity type reaction. histology, anerthe initialcourse. revealed patchyspmgios s. exocylosis and basal layer epidermaldamagewnh a mild perivascular chronic inflammatory cell mfinrate. with suhsequent treatment there wasthlckenmg ofthe epiderrnls, plgrnent incontinence. dermal oedema and more marked chronic perivascular cell tnfiltrate. immunohistcchemistry revealed markedchanges inthe expression of cdt , hladr, cam- and cd m the derm . these changes were greatly heightsnedwith subsequent liealmsnts. with addnional changes tn other t cetl markers. clearly il- enhances the parenchymal expression of antibody-dependent and antigen-mdependent accessory molecules which are important in focusing the immune response. claire m. thornton. maureen y. waish weexaminedalfprimarycutaneaus malignant melanomas seen rn thmdepartmmtover a fweyear permd from to . the total number of tumoum was the number of cases of malignant melanoma both invasive and m m u , increased from cases ~n to cases in . superfmal spreading melanoma was the most common type of melanoma, accounting for % of the total cases this was followed by the nodular melanoma whch accountedfor %. wlthlentigamallgnamelanomaandacral lent~g~nousmelanomabe~ngtheleast commontypes. therewasanincreasmg numberoftumourspresentlngwlth abreslow'sdepthoflessthan t mm. thefigurermng from % of cases in lo % of cases m most cases were stdl of ciar*e kud ivanhough mcreasing numbersaftumour presented at clarke levels i and i wlth a corresponding reduction ~n cases presenting at clarke level v more lesions presented with a flat cross sectlonal profile ~n the later years the figures lncreaslng from % in % in i w o he numbor of i c s~s showlng surface ulceratan at predentatlon d-reased from % in t to % in themltotlcact~vlty. thedegreeofp~gmentalion. theintensiiyofthe~nnammatorycell infiltrate. the predominant cell type and the mcldence of vascular lnvaslon showed no change over the study penod classification of benign vascular tumours is notoriousiy difliwlt and ciinicopathological correlation is often imprecise. this almost certainly reflects the tendency of pathologists to lump together different lesions under the broad heading 'haemangioma', sometimes with capillarylcavernous subtyping. twelve cases of a distinctive subset of cavernous haemangiomas, to be known as sinusoidai haemangioma, are presented. these presented in adws ( female. male. mean age years. range & ). five arose in the upper limb and five an the trunk (of which two developed in mammary subcutis). all were solitary and presented as a bluish cutanmus swelling up to . cm in diameter of variable duration. one case was associated with ipsilateral gynaecomastia. average foiiow-up of . years has revealed no tendency far local recurrence or metastasis. histologically these were subcutaneous/deep dermal lesions with a lobular, sieve-like appearance and focally ill-defined margins. they were composed of dilated. thin-walled interwmmunicating vascular channels with a pseudopapillary architecture. thrombi were common and two cases showed central infarction. vascular spaces were lined by monolayered endothelium which was often plump and hyperchromatic but n d mitotic. distinction from wnventional cavernous haemangioma and angiosarcoma (particularly in breast lesions) is discussed. current methods forthe identification of herpes simplex virus (hsvj may fail to identity the presence ofthe virus in biopsy or autopsy material. we have investigated autopsy cases and neurosurgical biopsy cases of clinically suspected herpes simplex encephalitis by a nested polymerase chain reaction (pcr). dna was extracted from routineiyprocessedand paraffin embedded material byproteinase k incubation, phenol chloroform extractionand ethanol precipitation. nested pcr was performed using known oligonmlaotide primers designed from the hsvtype giywprotein d gene from an area with the lowest homology with hsv type . two of autopsy c a w and of neurosurgical biopsy cases ware pcr positive for hsv. the third neurosurgical biopsy case was not confirmed by pcr as being due to hsv. such primers lo hsv allow the rapid retraspeaive diagnosis of herpes simplex encephaliis and should be of value to neurapathologists. tissueembedded at lowtemperaturein lowicryl-k m resin hasbeenshownto besuitableforimmunogoldlabellng of cellular antigens, and to be capable of withstanding the processing required for hybridization of nucleic acid probas.theaimofthisstudywastoestablish basic wnditionssuitabieforhybridirationof digoxigenin labelled dna probes to lowicryl embedded material, both at the light and electron microscope levels. cultured haemopoietii cells wereembedded afler brief aldehyde fixation. digaxigenin labelled whole human dna or plasmid per (negative control) probeswereappliedtothinandsem-thinsectionsafterproteolytictilgestion and/or denaturation by heat or alkali. hybrids were detected in semi-thin sections by standard wlourimetric methods. and in thin sections by immunogold techniques. elaborate blocking procedures and prolonged washes were found to be unnecessary. specificnu~l~earsignaiwasseenatbdhthelightandemisv~iwiththewhalednaprobe,revealingdetailsof nuclear dna distribution not evident in paraffin sections or cytospun preparations. non-specific binding and background were minimal. signal was greatly reduced if the denaturing step was mined, and was slightly increased by protwlytic digestion, though at the expense of cytoplasmic morphological integrity. while the sensitivity of this system is limited by the fact that hybridization occurs only at the suriace of the section, it is a rapid and specific means of nudaic acid detection, and offers the dossibilitv of accurate iocalization of intracellular human and viral nucleic acid sequences at a fundamental level. of prostatic carcinoma, mouse liver, kidney and gut were used. acp was demonstrated using an kc-dye cwpling method. the acp was unaffected by the addition of mm tartrate. although the acp was known to be 'tartrate sensitive'. the addition of mm tartrate or mm sodium fluoride weakened but did not eliminate the reaction. mouse tissues, prostatic carcinoma and leiomyosarcoma tissues were processed using variausfixatives and embedding procedures. these were tested for acp and trap. the acp in the muilinucleated giant calk of the leiomyosarcoma survived standard formalin fixation and paraffin wax processing and was tartrate resistant. as expected mouse liver, kidney and gut acp and prostatic acp did nd survive most fixatives and embedding procedures. however acp could be demonstrated in those tissues processed bythe 'amex' method. i.e. fixed in acetoneat - o' c. processed throughmethyi benzoateand xyianeto paraffin wax. theacp.thus imalised in paraffin blocks was eliminated by the addition of mm tartrate to the incubating medium. if needed the amex pracedure prrsenes some tartrate sensitive acp in paraffin blocks. acp that survives standard fixation and embedding procedures is iikeiy to be tarfrate resistant. showed acp only in osteoclasts. various elements ot tissue processing procedures were examined to find the wnditionsnecessarytoachievemaximum acp localisation. tissue blockswere pr-ssbdintowaxusingstandard embeddingprocedures. threefixativeswereused. % neutral bufferedfmalin, formalcalcium and ethanol allat + 'cfor hours.twodecalcifyingfluids wereemployed, %ethylenediamine-tetraaceticacid(edta)ph . at + %, zo' c and 'c.andformicacidisodium citrateat+ %forw hats. formalinfixed edtatreatedtissuaat + % produced maximum acp activity. acp was shown in osteociasts, some ostwcytes, chandrocytes. cement lines., tide mark, periosteaicellsand large macrophage likecells inbona matraw. formalinfixed tissues decalcified in edtaatzo'cand 'c werenegativeforacpaswerealltissuesfixedinethana . lnformalcalciumfixedtissueand formic acidlsodium citrate decalcified tissue the acp reaction was weaker. with some elements, e.g. chondrocvtic acp missing. all the acp preserved through paraffin processing was tartrate resistant. it is well recognised that morphology and optical resoiution are vastly improved with resin as opposed to paraffin embedding of tissue. however, difficulties in producing consistent immunopetoxidase reactions on resin sections have caused many departments to abandon thetechnique. although antigenicity is preserved the low optical density of diaminobenzidine (dae) means that the reaction product is barely visible in thin resin sections. the aim of this study was to develop a method whereby antibodies wmmonly used on paraffin sections wuld be successfully applied to pm resin sections. tissues fixed for - hrs in % formol saline were partially dehydrated and infinratedwithlr whiteresinat °cfollowed bypolymerisationat cusingacatalyticmethod. pmsectionswere reacted with polyclonai and monocionai antibodms using a standard indirect immunoparoxidase technique. visualisationwas with a silveramplification systemfordae (amenham) appiiedasthetinalstage. excellent resuns have been obtained with a rangeof antibodies including s , von wiilibrand factor. immunoglobulins, uchll and l (dakoltd.). usingthistechniqueitisnowpossibletocombinehighresoiutioniightmicroscopywithaprecise immunocytochemicai reaction. the advantages are obvious and are particularly relevant in the field of lymph node pathology. however, at and gythe number in muscle dropped significantly . and hoursfoilowing treatment, but hadrecoverad to w n t m levels byfivedays. in the lamina propria, agnor numbersincreasedinitiailyaflerthe . and gy treatments but returned to control values by five days. with gy the agnor numbers showed a significant fall hours after irradiation: this decline continued up to five days. it is evident that &nor numbers within the small intestine are affected following irradiation. the variation in counts is dependent on dose, cell type and time since irradiation. karen m. britten, w. r. roche oepartment of pathology, southampton unwewty general hosp~lai, southampton, so xy in response to ever-increasing demands for immunophendyping in inflammatory disorders, we have developed anernatwefixation and embeddingtechniquesforsmall biopsy specimens. bronchial biopsieswerefixed in buttered formaiin and processed lor embedding in araidite or were fixed in acetone containing protease inhibitors and embedded in the water-soiubk resin glycol methacrylate (gma). gma allowed for the investigation of a full phenotypic profile akin to that which may be wfwmed in frozen section while yielding far superior morphology and greater numbers of d a n s from small biopsies. the phenotypic markers included those for t-ceiis (cd . cd , cd ,cd andlfal),macrophages(cdllc, cd ),mastcelis~b b andaat)andeosinophils(mbp, egl and eg ). wehaveaisodemonstratednautrophiieiastase,cytokinesandtheceiiadhesionmolecuiesicaml .elamand vcam. similar high qualty sections were obtained with araidne but the repertoire of antibodies was restricted to thoseantibodieswhichcannarmailybeappiiedin paraffin. wesuggestthatforsmaii biopsies whichrequiredetailed immunohistochemistry, such as in the areas of transplantation and mucosa immunology, fixation in acetone at - % with the inclusion of protease inhibrors and processing into glvcolmethacwiate with careful temderature control gives optimum results. these included % formaiin at vanous temperatures, microwave treatments. bouin's fluid and a wmmercialiy avaiiabieproduct "rapid fix".thetissueswereroutinelyprocessed. embedded in paranin wax andstained wilhthe haematoxylinandeosin method. an eval"ation~fthefi~alion methodswasaisocarriedoutforimmunocytachemicai stains. from microscopic examination of the results t was evident that for the haematoxyiin an+eosm stains. % formaiinat o'cwa~ wnsistentiytheoplimum methodof choice. farimmunocytochamistryali methodsrasuited in a poor performance using a standard trypsinisetion time. however, an acceptable result was achieved from % formalinat °canda microwave fixative method. byreducingthetrypsinisation time. this. however, required strict wntroi. thus it is possible to fix tissue within one hour. by a method which is cost effective and which can be used within limitations for immunacytochemistry. we set out to study the extent and time dependency of storage related artefacts in cytospin fluid (shandon). we assessedtheeffectof stwage in cytospin fluidon the nuciearmarphoiqlyof breast fnac. fnaspecimsns were coiiected on day and slides were made from each specimen on day . day and day . these were fixed with a spray fixative. between each sampk preparation, the cytospin colleaion fluid was kept at %. the slides were fauigen stained and nuclear morphological parameters (area, perimeter, form ar. farm pe. and convexity-concavity) weremeasured using aseescansoiitaire plus imageanalysis system. there wasasignlticantchange in ail the parametem betwwn day and day , and between day and day (p < . ). the measurements were repeatableon differentoccasionswithoutsignificant difference in theresuns. these results indicate thatstorage in cytospin fluid significantly aners nuclear morphology. the nuclei become progressively larger and more irregular in shape. il is therefore important to standardize the storage conditions of fna specimens. if accurate objective comparisons are required. the identification of macromolecular components of hydrated patholqlicai tissues revealed by low temperature scanningeiectron microscopy (ltsem) isan emergingfieldof enquiry. in orderthatthetechnicaidetaiisforltsem labeilingmaybeestablishedanexperimentaiprotocoi has beendrawninwhichiabeiparticiesofhighatomicnumber are visualized. the experiment comprises a mcdei system in which bovine serum albumen as a known amigen is dissolved in phosphatebutteredsalineand adsorbedontonitroceiiuiosemembtane. theantigen onthemembrane is subsequently reacted mth rabbt anti-cow antibody and then elther protein a-goid or goat anti-rabbn-gold. aner treatmentwithadeveiopertoaddaiayerofsiivertothegoidparticies,thepreparationsareanachedtostubs,rapidiy frozen in nitrogen slush at - ooc, coated with aluminium and observed on the ltsem stage at approximately - °c. backscaneredeiectronimagingathighacceleratingvoltages( kv)isusbdtodetectsilver-enhancedgoid partic es.these areciearlyvisuaiized. thefrozen hydrated preparationsarestabieunderthe described wndtions of ltsem operation. in contrast dry, wnventionai. sem preparations are beam sensitive; their initially observed delicate uitrastncture quickly dearades an exeosure to even the moderate i kw electron beams emdioved in secondary electron imaging. a new approach to systematic storage of pathological specimens for low temperature scanning electron microscopy as greater numbers of pathological specimens are stored for wnvenient future imaging by iow temperature scanning electron microscopy (ltsem). it is increasingly important to maximise expensive cryostare capacity. a cassenesystem hasthereforebeendeveiopedwhichincreasesthe hoidingcapacityof cryostoresbyafactord . over conventional bee storage. each c-he consists of an aluminium disc . mm in diameter wilh cylindrical wells mmindiametereveniyspacedand . mmapart. specimens. mountedan jeolstubs,areretainedinthe wells by set screws. five cassettes fit into each x mm glass storage jar, gwmg a capacny for a canister cryastore(w th @rs percanister)of oostub-mounted specimens an addtionai benefit ofthecassettesystem is that each specimen is afforded a mechanically and thermally protected environment. specimen collection for ltsem may take place in a distant operating theatre or laboratory. lmmobiiiration of specimens whilst they are in transit excludes any possibility of their being damaged during normal handling and improves the prospects for survivalof accidents.afurther benefitisthataparticuiar mountedspecimencannow bemorespeedily identlledand removed for ltsem from rs well. many pathological condtions are charactensed by the presence of cellular degeneration accompanied by cytoskeietai abnormalities. in many such diswders it is not clear whether cytoskeietai abnormalities are pftmary process or are part of a secondary response to cellular insun by other agents or mechanisms. we have used a fibroblast ceii culture mcdei to study the effects of physical distension on the cellular cyioskeielon. inert beads of and pm were introduced intoceils byallowingendocytosisor bymicroinjection.thecytoskeietai response tothese beads was studied using immunofiuorescence microscopy. beads introduced by endocytosis migrated to the prinuciear region and over hours became enmeshed in intermediate filament and mlcrotubuiar aggregates. actln microfilament organiselionwas not alfected. incontrast. microinjectionof beadsproduced an immediatecollapseof morofilaments,wiulvimenbnandtubuiin distribution being presened.the immediateresponseto microlnlectlon is similar to the collapse of actin filaments s w n upon thermal stress. this experimental model has shown !hat aggregates of cytoskeietai proteins may be produced within cells as a secondary responseto intraceiiuiar debris, and that microinjection may induce cytoskeietal abnormalnies similar to those seen in thermal stress. thedetection of numerical chromosome aberrations m interphasetumourceiis bynonisotopic insitu hybridisation has been previously described but the application d t h s technique to paraffin embedded material has been wmpiicatsd bythe requirementfottissuesectioning with the production of partial nuciei. inthisstudy, the analysis - pm thickparaffin sectionsolconventionally processed caski ceilsusing both human papliiomavlrus(hpv type l e a d achromosome specificalphoid probe wascompared with resunsomained usingintactceils.the use of sectioned material did not give signal distributions comparable to those obtained using whale cells. this is consistent wnh a mathematical model derived for the relationship between section thickness. nuclear size and nuclear retention in paranln sections. a method was therefore developed for the extraction and analysis of nuclei fromthick( pm)paraltinsectionsandappiiedtotheanaiysisof squamouscelicarcinomasofthecervix(n = ). the number of copies of chromosome varied from to and this variation was apparent both between lesions and between tumour ceiis within the same ieaon. this is comparable to resuns obtained wth cervical carcinoma derived csll lines. there was. however, no clear relationship between the presence of hpv sequences and chromosome number. these preliminary results suggest that the postulated loss of host suppression of hpv gene function by deletion of genes on chromosome does not occur through gross chromosomal abnormalities. membranous giomeruionephritls (mgn). an immune-mediated disease, is a frequent caused renal morbidity in man. cationic bovine serum albumin (cbsa), given to nzw rabbits in a chronic serum sickness-type protocol. is knowntoinduceglomerularchangessimilartothe humandisease. we~ssedtheenectofashortcourseofthe immunosuppressivedrug cyaon thedevelopmentof early stage.cbsa-induced mgn. fourteen male nzwrabbts received an iv immunismg dose of mg cbsa and p g e. coliendotoxin. one week later they commenced daily iy inlectionsaf mgcbsafor ~onsec~t~vedays. three rabbtsweresacftficed atthistime. sixofthe remaining rabbits were commenced on a short cou~sb of o m cya. whilst continuing to recewe daily doses of cbsa. the remainingrabbitswsregivencbsaonly.after consecutivedosesofcbsathesell anlmaisweresacnficed.ali rabbns given doses cbsa showed early stage mgn and those given doses of the cationic protein showed a more mature, established disease (thickened glomerular capillary wails wth diffuse. global. granular deposition of igg/c and subepithelial electmn dense deposits). four of the cya-treated cbsa rabbns showed a marked reduction in giomerular capillary wail c deposition. three of these rabbns had considerably less severe disease ultrastructuraiiy. these results suggest that cya may aker the course of cbsa-lnduced mgn. 'oroartment of pathology university of edmburgh m&~aischooi: late renal aliogran loss s due to arterial intimai proliferation and iumenai narrowing. there are few studies ofthe phenotypes of the intimai cells. we analysed these lesions by light microscopy and immunmyiochemistry using antisera against t-lymphocytes. b-iymphocytes. macmphages, smooth muscle cells, class ii hla or molecules and the proideration antigen pclo vessels were studied from gran nephrectomies resected between and months post-transplantation. we identified an arterial endolheliaiilis, r-rted in cardiac aliografts, but not emphaszed in renal graftrejection. panernsolarterial pathoiogywwerewgnisd: ( ) endotheiialitis in nnteriobuiar arteries without intimal proliferation, ( ) endothelialiis in larger arteries accompanied by intimal prdtferat!on of smooth muscle. ( ) '"inactive" lesions with thickened intima (i foam cells) but no endotheliaiitis, and ( ) 'natural" atherosclerosis of larger arteries. the endothellaidis tended to m u r in shmw sumving grafts. the pedominant cell was the macrophage. with fewer t-lymphocytes. pclo was expressed in mononuclear ceiis, smwth muscle cells and endothalid ceiis. pmicuiariy. but not exclusively in younger grafls. we proposb these ledonsevolve, variably. from an early endotheiiaiitis to late chronic vascular rejmlon or gralt athwmierosis. the predomlnance of the macrophage at ail stages, suggests it plays a significant roia in the evolution of these lesions. the rat is used to study the response of the renin-angiotensm system m diseases such as hypertension there are structural differences in the jga butthere are few comparisons of its response to stimulalion between species we used renin antisera and an lmmunoperoxidase technique to stain renin-containing cells (rcc) ~n rat (n = i t ) and human kldneys(l nephrectomy and autopsycases). westmulated therenin-angintensin system experimentally by clipping one renal artery ( rats) and inducing sodium depletion ( rats) and studied the analogous human diseases-renal artery stenosts ( cases) andaddison'sdissase( untreated and treated eases). wecounted the rcc and plotted their distribution on scatter diagrams. there were some dinerences in distribution between the species but in boththerewasagradient in distributionof rcc which predominated m the superficial renal cortex. in sodium depleted rats. recruitment of rcc in the iuxtamedullary jgas abolished this gradient. while in some of the animals with renal artery clip hypeitension the normal gradient was reversed, with most rcc in the deep cortex. in bdhu~nreatedaddison' diseaseand~nrenalanerystanosistherewasanoveralincrease(xs)m rcc butthenormal gradientoftheirdistnbution withintherenalcortexwasmainlained.theseresults haveimpl,cationsfortheroleolthe intrarenal renin-anglotensln system in the control of renal haemodynamcs. we have developed a model of adult human prostatic epdhelium that allows arch$tectuml and cytologioai features to be maintained. epithelial organolds produced by enzymic digestion of human benlgh prostatic hyperplasla tissue weresuspendedm type collagengelandsubcutaneouslyxenogranedintointact malenudemice.thexenogranis progressively invaded by mouse stromal cells thesesurround theepithelial organoids and supportthe reformation of epithelial structures with a lumen, lined by a mixed epithelial layer. as the lumen forms tall columnar epithelial cells begin to be seen, these progressively express the prostate specific epithelial markers. psa and psap further the xenograflsexpress appropriatecyiokeratin markers in boththeluminaland basaleplthellal cells. gelsplaced within a . pm millipore chamber, which do not undergo stromal invaston. lose all epithelial organisation with disorganised sheetsand ballsofcellsbeingfound.these cellsdonot express thesecretory markers. in the absence lanandr genic~tim~i~sep,th~l~~l structures wsth a lumen are formed but there are no tall columnar secretory cells and noexpression ofthesecretory markers. thismodel hasfurther been investigated todetermine theresponseof human prostatic cells growing in vivo to the antiandrogen flutamide and to a -aza-steroid e-reductase inhibitor these observations indicate the essential role of both stromal cells and androgens m dictating functional dlfferentlatnon this model will allow the dissection of the regulatory processes involved in prostatic differentiation. hepatocyte growth factor (hgf) is the most potent known mitogen for adult rat hepatocytes in pimary cuiture and isthoughtto haveanimportant rolelnlivergrowihandrepalr linle~sknownaboutthemechanismofactionof hgf on hepatocytes. since the adenylate cyclase system has been implicated in hepalocyie growth control. we examined the role of adenylate cyclase and cyclic amp wmp) in hgf-stimulated dna synthesis. human recombinant hgf (hrhgf. numl) had no elfect on baa or stimulated adenylate cyclase activity in membranes prepared from freshly isolated rat hepatocytes. similarly, hrhgf had noeffect on intracellular camplevels in cultured hepatocytes. furthermore, agents which increase camp inhibited hrhgf-stimulated dna synthesis m primary hepatocyte cui ures glucagon had an i . c. , of lo-'; fotskolin ( . ibmx ( "m). -broma camp ( pm) and dibutyryl camp ( pm) completely inhiblfed hrhgf-stimulated dna synthesvs. from thts. we conciude that adenylate cyclase and camp do not have a role m hgf-stimulated dna synthesis m pnmarycultures oladult rat hepatocytes. the receptor for hgf has recently been identified in tissues other than the liver, as c-met a protooncogenewith intrinsictyrosinekinaseactivitv. whether or notc-metactsasthsrecedtorfor hgf ~n heoatocvtes s currently under investigation a novel in wvomodel of intestinal differentiation is descnbed. fourteen-day, undifferentiatedfetal rat small intestine. stripped of the major part of s mesenchyme. then suspended in a type i collagen gel and renografted ~n a nude mouse. undergoessmall mntestbnal morphogenesis and cytod$ffeienbat$on. all tourmapr epithelial imeages. namely paneth. goblet. columnar and endocrine. are present. double labelling m sit" hybndlzatlon, employing biotinylated and digoxigenin labelled dna probes to whole rat dna and whole mouse dna, reveals an unusual ]uxtaposition of species specific stroma. the outer longitudinal smooth muscle layer, and the major part of the lamina propria. p isthemost commdnlyalteredgene~n humanturnours. mutati~nleadstothep~oducfionolanabnormalprotein which can be detected by immunohistology. such abnormalales are seen in a wide variety of turnours, including colon cancer. abnormal p protein levelshavebeen detewed in & % of sporadiccolorectaltumours. in order to determine i p mutations occur in carcinomas arising from dyspiasla. we have investigated the prevalence of such mutations m colorectal carcinomas from patients wr)h long-standing ulceratwe colitis %). lmmunocytochemicalstalnlng waspwformedon freshand paralflnembeddeduccarclnomasand sporadlc carcinomacontrols matched forsite, stageand grade six areas ofdysplasla, (fourassoclatedwlth uccancers)and sporadic adenomas were also stained. p protein was detected by immunohistochem~stry m the fresh uc cancers and / ( %) ofthe paraffin embedded uc cancers usmg the antibodies cmi, pab , pab and pab . slmllarresuitswere obtained in thesporadic carcinomasw,th fresh cancers positlvefor p and / ( %) of the paraffin embedded cancers. two areas of dysplasla which were associated with p postlve uc cancer~also showed positive p staining, alongwith an adenoma. our results indicatethatunlike k-ras mutations, p proteinabnormalities occurat a similarfrequencyln sporadic colorectal carcmomas, carcinomasar sing !n uc as well as being present in uc dysptasasla. this work suggests that p mutations play a mle m the dysplastacarcinoma sequence. threepanernsofstainingareloundin humancolonusingatechniquetodemonstrateo-acetylationofsialomucins (mpasj. % afindivdualsshowunifwmmpas-positivity. theremaindwareeitherent~~ympas-negativewmpasnegativewith occasional positive crypts. we suggestthatthisrepresentspalymorphismof an autosomal gene ( sa) controlling -acetylation of sballc acid. isolated crypt-restricted mpas-posltivky in otherwise negative indivtduais representing somatic mutation of the ma* gene in crypt stem cells of osa'/osa-individuals. to test this we have studied colons from patients with rectal carcinoma, half of whom had received cgy radiation days preoperataely. radialiond~dnotaffecttheprevalenceofthethreepnenotypesbutincreasedthefrequencyofmpasposltlvecryptsinanegativebackground(t ~ ~~~ . ~ to*, p < . ), largelyduetocryptsshowingseclorial mpas-positivlty ( . x lo* ys . x to , p < o.c€ql). consistent with incomplete crypt colonisation by a recently mutated phenotype. the prevalenceofthisosa*/osa~phenot~e(radiated %, non-radiated %) isveqcloseto the predicted heterorygosily rate ( . %. hardy-welnberg law). thew resuns suggest that human colonic crypts are monoclonal with a longer stem cell cycle than the mouse and that mpas staining provides a method for measuring human stem cell mutattonal load. pouchrris m leo-anal resarvoirs is a frequent complication of restorative practocolectamy which is associated wtih constderable morbidity. long term complications of pouchitis are unknown, however patient follow-up wlth sigmoidoscopic surveillanceis mandatory to assessdysplamc or neoplastic changes in resldual rectal mucosa. we examined epithelial cell proliferative activdy in the ileal mucosa of pouch biopsies using the antibody pclo which detects nuclear expression of the kd nuclear protein proliferating cell nuclear antigen (pcna). ten patienls with functtoning ,lea-anal pouch reservoirs of at least one year duration were biopsied and assessed histologically for evldence of pouchitis formalin fixed paraffin embedded biopsies from anterior and posterior pouch wall were examlned with pclo using the pap technique. ten terminal ileum sctions from right hemicokctomy specimens werenormalcontrols. cryptsin pouch biopsiesand normalcantralshadasimilarpciolabelling indexmeanof k and % (mean values - and w ) respectiveiy the sides and tips of villi had a significantly greater pclo labellmg indexinthepouch biopstes( mean. range - ). compared withnormaicontrols(t % mean, range - ) these resulk demmstiate an expanded proliferatwe epnhelial compartment in ileal pouch mucwhich in contrast to normal terminal ileum involves villous surfaces. considering the frequency and long term natllrs pouchitis. thesefindingssupportthe need for continued pathalogical and clinical assessmentof the ileal mucosa in the neo-rectum of these patients. we, cui, i c talbot, j. m. a. northover signiticant alterations in structure. function and gene expression of mltochondna have been reported in cobrectal turnours. but it $ not known rf these abnormalities are due to mitochondria genetic alteration. in this study, total cellular dna was isolated from l lo rectal carcinomas, adenomas and their adlacent histologically normal mucosa. these dna samples were digested separately with different restriction endonucleases. and then analysed by southern blotting using apurified mtdnaprobe. the restriction fragment pattern oftumourmtdnawas comparedta thatofeorrespondingnormalmucosalmtdna.theseresultsshowedthattherearenalargedeletions. insert~ons, or rearrangements $n turnour mtdna. and no single base changes m the delectable regions in spite of some polymorphic vanations. our results suggest that mtdna changes are unlikely to have a malor role in human colorectal tumoungenesis. hence, alterations in colorectal turnour mitochondria must be dependent upon other mechanisms crescentic colitis: the clinicopathological spectrum of a distinctive endoscopic feature in the sigmoid colon n. a. shepherd, s. gore, s. p. wilkmson oepariments ofnsfopafholcgyand gastreoterol~y, gloucestershre royal hospml, great western road, gloucesfw, crescentic coibs describes an endoscopic appearanc of the sigmoid colon charactwised by mucosal swellmg, eqhema and haemorrhagestrictly localised tothecrescentic mucosal folds. in alive yearpericd thisdiagnosiswas made m patrents, representvlg .a% of ail fbeopta endoscopies. there was a male predominance and most plients were middle-aged or elderly. dwerticulosis waspresent m most ( %) but theabnomallf~es wareconfined to the crescentic mucosal folds with sparing of the divenicular onfices. the malorlty of patients presented with a history of bleeding perano. histologically there wasaspectrum of changesvaryingfrommlnorvascularcongestian to florid active inflammatory disease with crypt architectural abnormalities mimicking ulcerative colitis. three patients presenting with crescentc colitis later developed the clinical, endoscopic and histopathological features of distal ulcerative coliitls: two other patients with a history of distal ulcerative colitis were found to have the charaderlstlc changes crescentic colitis only at endoscopy. three cases showed the histological features of mucosai prolapse the findings in thisstudy demonstratethat arelativelyspecificendoscopicfeaturemayexhibit a wide spectrum of pathological changes whilst luminal mucosal inflammation of the sigmoid colon, usually in a sociat on wiih diverticuioss. may mimic the pathology of chronic inflammatory bowel disease. asmall propartion of these cases may represenl a strictly locallsed form of chronic ulcerative colitis. of genome and antigen a n various anatomtcal sees the resuns have imolications lor the mechanism of e n t~ of mv into neurons and for mechanisms of transynaptic viral spread neuiopathology, fnstitute of pathology j chow, j tobias.' k co ston. t j chambers estrogen is generally considered to maintain bone mass through suppression of bone resorption we have previously demonstrated that administration of pharmacologic doses of estrogen increased bone formation in ovary-intact rats to assess theeffectsof physiological concentrationsol estrogen on boneformatian. estrogen was administered to ovariectamised rats fi n which bone resorption was suppressed by ahprbp. animals receiving exogenous p-estradtal (ed ( wglkg. t o wgikg and w pgkg daily for days) showed a dose-dependent increasemtrabecularbonevolumeaft %. . % and . % respectively, compared wnhthoseratstreated with ahprbp alone. the increase in bone volume was due to bane formation in e,-treated animals, in which bone resorption had been almost completely suppressed by ahprbp neither ovanectomy. ahprbp nor e,-treatment had asignificanteffecton thevolumeorrateoflormationofcortical bone thus. theiocreasedbonerasorptionwhichis aconsequence estrogen-dellclency entralns lncreased bone formation, whlch masks a slmunaneous reduction in estrogen-dependent bone formation. it thus appears that estrogen maintains bone volume not only through inhibition of bone resorption. but also through stimulation of bone formation. pgs m bone formation m vmo. which may represent a pathway common to bone anabolism that s observed !n response to many e""lio"mental t m"ll pgf , was wlthoul effect we found that nodule mductton by pgs occutred early the cunures, belore nodules lntegrln expression e n human bone was examkned by immunohistolqlical stalnmg of edta-decalcrfec and undecalcllned cryostat sections of fracture-and tumour-associated callus dbtalned at surgery and neonatal costochondral )unctions obtained at autopsy. cases were stained wlth a panel of well-charactsnsed monwlonai antibodies against p - . el- and uvp integrios using abc peraxidase and indirect immunofluorescence techniques. osteoclasts stained lor pl , p . u and ovp mtegnns. indicating that they express u p nlp- ) and uvp (classm vttranectin receptor) ostmblasts stained lor pl, and u , and osteocytes stained for p l and . indicating that ostmblasts express n pl nlp- ) and m gl nlp- . classical fibronectin receotorl and that vlp- expression s reduced or lost during differentiation to astwcytes j qulnn. n a. athanasau nufield depanment of paihology and bactenotogy, level , john radcoffe hospital, oxford, ox du osteoclasts are known to effect bone resorption in inflammation and malignancy but whether other cells of the mononuclear phagocyte system. particularly macrophages and macrophage palykaryons, are similarly capable of pathological bone destruction is uncertain macrophages derived from tumours (human lung camnomas. murine mmw-assmated mammary carcinomas) and inflammatory lesions (munne foreign body granubmas) were cuituredonboneslicesbothin thepresenceandabsenceaf stz slromalcells.theseneoplasticand inflammatory iesions contained a heavy macrophage infiltrate but no giant cells and no calcified tissue. there was superficial roughening ofthebonesurfacebymacrophagesboth inthepresenceandabsenceofst cellsand.aher t days. scattered areas of lacunar resorption n co-cultures of macrophages and st cells. normal pulmonary tissue macrophagesdid not produce resorption lacunaeunder these conditions. the results show that macrophages alone are capable of atype of low-grade boneresorption and that a subpopuwion of turnour inflammation-assaoated macrophages following specific interaction with slromal cells, can differentiate intocellscapable of the special sed function of high-grade lacunar bone resorpt on macrophages may thus directly contribute to the mteolysis associated with metastatic turnours and inflammatory lesms in bone differences in the grade of osteolysis may also account for clinical differences tn the degrees and rate at which pathological bane resorption occurs the george washmgton university medtcal center, washnqton, c , u s a for decades, new technological advances have been hailedor condemnedas representing the extinclion cla~s~cal diagnostic surgical pathology, but so far the reports of the death of our specialty have been grossly exaggerated. indeed, the newtechnologies of the sare seenas aids torathet than replacementsfor careful and intelligent gross and micmscopic examination and interpretation, which havealways remained the 'gold standard" against wnichnewtechniquesaremeasuted thedecade will bemarked by anexplosionof pos ibilitiesfort ueas well a non-tmue diagnosis, balanced by a shrinkage in both specimen sizes (already evident in breast pathology) and healthcare budgets.thus. thesurgical pathalogist will haveto becometoanevengreaterextentthecomplete physician. in order to beable to choose wiselyand economically from thediagnostic"menu"availab e. thesurgical pathologist w d also have a greater need than heretofore to be a competent cytopathologist as well, as many of his or her cases will also have fine needle aspiration material and a nuclear grading will assume greater significance o n tumor pathology. finally, the roles of the surgical pathologist ~n both research and patient care (including direct patteoi contact) will require emphasis m order to attract more medical students into our specialhl. womenorthosewithlowgradedisease.this hasledto theviewthathpv s~nielatedtocer~ c~l cancer asimple pcr protocol was developed using standards containing . - fg of hpv dna in ng of normal human placental ona toestimate levelsaf hpv dnain smearsor biopsies. the quantltativedistribution of hpv dna tn normal and abnormal ~ervlcal epithelium was mapped thmughout loop biopsy and hysterectomy specimens using micro-dissectionand histologyof alternate millimeter slices levels were measured in parallel c~n~c~i smears. preliminaryresuits show a low lwel of hpv dna(equiva ent to one copy per cells) was usual in women with normal smears or low grade abnormalities replication of hpv dna to a level of one copy or more per cell was limited to areas of cin ~ which may be very small and to atypical immature metaplasia. the level was reflected ~n the smear aswitch to a high level of hpv dna s a biological and potential diagnostic maherfor high grade precancer. cerv cal biopsies were stained by the cracker technique to demonstrate agnors. for the purposes of the study, b~opsiesweredividedlntaflvegroups-normal, kallocytosis.cini.cin iiandciniii.usinganiemps/ hastingan a m s photon framestore a customised image analysls system was created. the image s dllated and eroded to close any cavities an outlining procedure locates the agnors and records their mid-pants. from this st of midpants themeandistanceofeach agnor'slhreenearest neighboursiscalculated.thlsprocedurescanledoutfor several lelds of view. this study attempts to relate the shape of the associated "mean distance" histograms to the histologlca diagnoses in flry cases. primary adenocarcinoma of the cervix: a retrospective clinicopathological study of cases r ananoos, kamta nahar.' alison b,grigg.'s. roberts? sergin m. sma the patientfen healthy with a tendency togain weight being heronlycomplaint. gynaecological referral revealeda week gestation abdominal mass and bilateral varicose veins. laparotomy, lee oapharedomy, total abdominal hysterectomy and right salpingo-oophorectomy were carried out the len ovarian turnour was cyst~c, smoath surfaced. unilocular,cantainedturbid brownnon-greasycontentsand was cmln diameter.al cmsquarearea of lhe lining showed papillary tutmg. and the remainder was smooth. hiatology of these papillary areas revealed a papillomatow proliferative squamous neoplasm with keratinization and mild acute inflammatm. wthout evidence of cytologicalatypia.stromal invasionora benigncysticteratoma.theremainderofthecystshowednat swamous. cuboidal, or low-coiumnar type epithelium with an underlying vaguely endometrial type stroma in minute focl, with occasional small secondary cysts thls unusual squamous neoplasm was diagnosed as a proliferative epbdermoid meglomerularperipolarcell isan epithelial cell snuted atthe vascularpole ofthe glomerulus recently, theexistence of the penpolar cell has been doubted the aims of this study were first, to establish whether the peripolar cell is a m q v e cell type ~n the mammalian kidney. and second, to compare their numbers and morphology in different speces we used scanning electron microscopy to study - kidneys from each of mammalian species including man. we removed the glomeruli by microdissection and examined a minimum of vascular poles from each kidney penpolar cells were largest and most numerous m goat and sheep kidneys ( w% and % of glomerul~) meywerescant~estandsmallest humanand bovinekidneys( % and %).whileaspectsotsome per~polarcells resembled podocytesand otherperipolar cells had features reminiscent of parietal epithelialcells. in each ~pe~ie~wewereabletadistinguishperipolarcellsasaspeciflccellhlpe. weconcludefirst. that theglomerular peripolar cell s a unique cell type ~n the mammalian glomerulus, and second. that their morphology and number cystlc renal disease occurs in various forms characterised by dilatation of different pans of the nephron. the morphologoal, clinical and genetic features of these diseases is variable but some animal models have been developed m an attempt to understand the mechanism of cyst formation within the nephron. we describe a polycybtic . .ofthe kidneyinthscea-n mousew~hanx-linkedrecessive~mmunodetic,entsyndrome there is progressive cystlc dilatation affecting all parts of the nephron. the cyst lining is composed of a single l a y m c epithelium with focal nuclear crowding and the formation of micropapillary structures. the cystlc epithelial cells show subnuclear vacuolation focal basement membrane thickening is also a feature. there is no signdicant inflammatory infiltrate present within these ktdneys. election rni~roscoptc examination w e a l s that the subnuclear vacdation s due to loss of the membrane infoldings at the basal pole of the eplthelial cell wlth flud accumulatlon within the extracellular space. the basement membrane thickening is due to expansion of the lamina dense. the finding of a plycystic kidney lesion on these mice offers an opportunity to investigate the relationship beween the immune system and renal cyst formation. eqthropomtin. a circulating glywprotem, is the principal humoral regulator of elythropoiesis. it s produced in the kidney butthe precisecell oforigin iscontroversial. erythropoietin participates in a~ ~ssicaif dbackcontroi system whch attemots to restore oxvaen deiivew lo the tissues. normallv erdhrowiatin is resent in the sbnm at picomolar c mntrations bui revels may h e up to oid durilg ?&erehkpoxic ;tress. the mechanism linking renal oxygen sensing with erythropoietin synthesis is p o~l y understood but there s evidencethat cells of the innsr cortex respond to tissue hypoxia by producing elythropoietln. eryihropoietin geneexpression in the kidney can be detected by northern blot anaiysis within one hour of exposure to hypoxie stimulation. the cellular location of erythropaietin m-nger rnawasdetected inamurine model byinsdu hybridizationemploying radioiabeiled dna probes and autoradiography techniques. tubular cells of the inner renal cortex wbi b identifled as the main site of ervfhropoietin gene transcription. the location oterythropaetm production was confirmed by immunohistochemistry with anti-sera ra sed to pure recombinam dna-denvea human elythropoetin. the specific antibodies bound oniy lo tubular cell cytoplasm, confirming the tubular location of erythropoiet,n-producing cells. severe reversible acute renal failure and iga nephropathy r jackson, k l. c. mclay unrvemty diws~lon of pathology, giasgow ropl infirmary, castle sireel. glasgow, g osf whilst progressive, irreverable renel failure is well known in the evolution of a stgnificant proportion of cases of iga nephropathy, disease-related. reuerstbie, severe deteiioretion in renei function s a less well recognized phenomenon. relatively few cases aredocumented in the literatureand these have invariably been associated with m-iw haematuna. renal failure has been largely attributed to a combination of tubular obstruction by red - cast~andacutetubularn%crosisthaughttabetheresunof adirecttubulo-toxiceffectof released haemoglobin. in a review of cases of i@ nephropathy we have encountered two cases of this type. the clinical. histological. immunohistochemical and ultrastructural data relating to these cases are presented. on the basis of this evidence and an analysisof the relevant literature. isconcludedthat the accepted pathogenetic hypothesisdnot provide an entirelysatisfactaryexpianationof the phenomenon. itisfurlhersuggested that immunologically mediated injury dfrected at the glameruiar vascular pole and possibly the extraglamerular mesangium could compromise thetubular blood supply and might also lead to haemorrhage into the distal tubule at the macuia densa. scanning electron microscopy (semi when combined with electron microprobe analysis (mpa) s an expeditious and accurate method of identitying substances deposited in tissues. we report a case in which these techniques were used to study crystals in a transplanted kidney. the patient presented at age three with vitamin d resistant rickets and fanconi syndrome on the basis of cystlnosis. he gradually went into renal failure and received a kidney transplantfrom hismother. itfunctionedwsiifarafewmonths butthen deterioratedandwas removedone year after transpiantation.afewcrystalswereob~ed by light microscopy ma biopsytakenattwomonths. arepeat biopsy at four months was examined by sem and contained the typical hexagonal cryystais of cystine. the s o i i i sulphur peak obtained with mpa confirmed their elemental compojltion. the hydrophilic nature of the cystlne prevented their identificetion within routine sectionsfor transmission electron microscopy (tem). the hexagonal o~tline of aystaiswas confined to interstiitat macrophages rather than tubular arglomeruiar ceiis. pm frozen sections mounted on carbn pianchets diowed vlsualisation and analysis of the crystal deposits by sem. the use of appropriate processing methods may be crucial m identifying crystal dspasition within pathological specimens. during the year period - . new cases of dense deposit disease (m~~b,a"op,olif~=ti"~ giomerulonephritis type ill were diagnow in nonhern ireland. two further known patients developed recurrent disease in renal transplants. the mean age of onset was years (range eight- years) with five males and eight females affected. renal function was impaired in % patients at presentation which ranged from nephrotic syndrome ( %). nephrilidnephrotc syndrome ( %) and macroscopic haematura with mild protelnuria ( %) lo mild protemuria f microsopic haematuna ( %). serum c was universally low. haw of initial biopsies showed cr-entsusuailyaiatedwith rapidansefrenaifailure. % ofpatientsrequired diaiysisatorwithin twomonths of presentation. a further % d patmnts developed renal failure within three pars of disease onset of the six patientstranaplanted haw havedaveioped rmrrent diseasin their grafts six patients havenot required dialysisat ameanfoilaw upoffour yearr(ranse neloeightyears). mhaugh reialweiyuncommon, dense deposit disease isan important causeof renal failure in children and adolescents. we have confirmed that this disease has a poor outlook in terms of renal function with a tendency towards recurrence in grafts a review of renal biopsies from patients with diabetes mellitus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . showed with diabetic giomeruionephropathyalone, with other renal diseaseand with both diabetic giomerulonephropathyand other renal disease. eight with the histologiwl appearances of diabetic nephropathy without known diabetes were alsn identified. clinical details were available on ofthe patients. these showed that in type (insuiin dependent) diabetes ( ). most ( ) showed diabetic giomeruionephropathy, showed both diabetic glome~lon~phropathy and another diagoaas, and showed aniy another diagnosis in type (non insuiin dependent) diabetes ( ). showed diabetic glomerulonephropathy with showing another renal diagnosis without diabetic giombrulonephropathy, and with both diabetic glomeruionephropathy and another diagnosis. patients investigated with nodular glomerulosclerosis resembling that seen in diabetes but not known to be diabetic at the time of biopsy, showed abnormal glucose metabolism on investigation and t showed a paraprotein. this review showsthatinpatientswithciinicairenaldiseaseanddiabetesmeliitus,therenaldiseasecannotalways beassumed to be diabetic nephropathy especially ~n type diabetes where a wide range of other renal disease can occur histological findings of diabetic nephropathy may occur in patients before diabetes is diagnosed cllnlwiiy. a j. hawie, r. l. bryan in a biopsyof arenaltransplant. we notedan artmalabnormalitythat wasunrecorded in standard texts. in an arcuate artery. theiewastheappearanceafformation ofanewartery inside theoid, with layersof muscleandelastic fibres that in places resembled an internal elastic lamina. separated from the original internal elastic lamina by loose connective tissue. in a systematic study of consecutive transpiant biopsies and consecut~ve transplant nephrectomies. another six examples of this abnormality were found. these six and the index specimen showed changes cons~stent with chronic vascular rejection. in a systematic study of consecutive nan-transplant renal biopsies showing interstitial nephritis. another example ofthis lesion was found, in a kidney showing chronic interstitial nephntis. thischange isprobablyavmantfom of muscularisation ofthsarteriai intima, isseen in chronlc renal damage, and occurs in transplanted and native kidneys. localised myloidasis ofthe lower genito-unnarytractisararedisease. few studleshaveanempted tocharacterise theamyloidtypeusing immunohistochemicalstains. wereportaseriesofnin~casssinvoivingthebladder( ), lower ureter ( )and penile urethra( ). thesecomprised malesand pfemaies with an age ranged & years. nonsof the patients had evidence of systemic amyioidoss. three patients had a past history of lower genito-urinq tract infection and of these, one had repeated instrumentation. one patient had prostatic carcmma the cmmonast presenting wmpiaint washaematuriaand the mostfavouredsurgicaldiagnosiswasa neoplasm. fowpat#ents had repeat biapsiesfor persistent orrecurtentamyloidosiswith atimeintmal ofupto t yearsfram initial presentation. bght microscopy showed amyloid deposmon throughoutthebiopsies, in lamina propria. muscle. adiposetissueand vessel wails. with a variable giant cell reaction and lymphoplasmacytic infillrate. an abc-immunaperoxidase techniquewas usedwith antibodmsto pcamponent, serum amyioidaprote n prealbummandkappaand lambda light cham in an attempt to classify the amyloid type. this was found to b e d non-pa. non-prealbumm type. a negative or equivocal reaction was seen for kappa and lambda light chains however, such antibodies may not necessarily be immunoreactive with light chains or fragments of light chains m amyloid deposits. features of bile reflux type gastntis: glandular distmion (branching, "carkscrewmg"). nuclear regeneratwn, intramucosal smooth musclefibresandpaucityof inflammation. helicobacterpylanarganiams werenoted bileacid concentration #n gastrc aspirates collected by endoscopy at the time of biopsy was estimated using an optical densitymethod.gi casesshowingglandulardistorti~ hadraisedgasttic bileacidconcentration.m cases with mahednuclearregeneration hadraised bileacidsandof case~withinbamucosalsmoothmuxlefibres had raised bile acids nine of cases with raised bile acids showed a chronic inflammatory response the histological features of bile reflux type gastritis which correlate best with estimation of bile m gastric juice are glandular branching, "corkscrewing" and intramucosal smooth muscle fibres whereas pauclty of chronlc inflammation does not h pylon was identified in of cases with raised bile acid. aims to compare the bacterial flora of normal and inflamed appendices. and to conelate this with various histological features. to establish the incidence of yeninla infection ~n acute appendicihs !n southampton. methods resectedappendicesweresentheshta histopathologyandal cm portionwasremovedandcultured for aerobic and anaerobic organisma. the remainder was fixed ~n % buffered formalin and pocessed for rwtine histology results: appendices showed acute ap+endicitis, and were normal. histology there were statistically significant differences between the two groups for the p s e n c e o f faecollfhs (p < . ). fibrosis (p < . ) and prominent follicles (p < ) -all were more common ~n the normal appendices. microbiology no yer~inia was cultured in enher group. there were statistically significant differences in the number in each group which grew anaerobes and streptococci. anaerobes were more common in the normal group (p < . ). and streptococci morecommon in inflamedappendaes(p< ) conclusions. .thereisanaiiwed bactenalflora in acute appendicitis. yersinia does not contnbute to acute appendicitis in southampton. faecoliths. fibrosis and prominent follicles are significantly more common in normal appendces le antigen (using cat - ) a i d type h antigen using a specific moncbnal antiserum. we f w n d a widespread distribution oftype structures in both benign and malignant epnhelium oftheextrahepatic biliarytract and ampulla of vater. type h antigen expression was seen in benign epnhelium and in non-papillary tumoun and ampullary carinomas. lmmunohistochemical detection of tyw blood qrou antlqens does not adpear to be ofprwnostlc the majority of cases bearclose relationship to human ulcerativecolitisclinically, endoscopcallyarid i" re-nseto treatment one hundred postmortem iiyerswerestudiedfromconontoptamanns with totalseversul~ataecolitis who were pathogen free and had a histological picture resembling human uicerallve cdhs only iivwere normal in there was a mild penportal chronic inflammation. had extens~ve steatosts, had an appearance resembling chronic active hepatitis and ~n the histology resembled that of sclerosng cholangitja. other hepatic pathologies were s e n in smaller numbers. these changes parallel the itver disease seen in association wlfh "icbrativbcolitisinman. webelievetheconon toptamannprovidesthefirst modelof l i~e r d i~~~~e m u l~e r a t i v e~~l n to allow sturty of the pathogenesis of the enralntestinal manifestations of ulcerative colitis. r-rvolr and ilm-anal anastomosis. pre-and pan-surgical specimens were studied and compared using routine histological and histochemicaltechniqm themalontyofcasssexhibitedan lncreasem chronc lnflammatlon wlth n or noacute inflammation.the levelsof chronic innammation were found to bemost severe i" those cases which ~~fferedhompouchiti~. morphomstncalanalysisrevealedan~ncreaseincrypt depthtovillousheightration(cdvh) ~n % of cases. the cdvh in the pouchitis cases was greater than n the other specimens studied the resuns of m u m histochemical analysis did not show any charactenstic changes occumng. in the cases studied. lectm histochemistry demonstrated an increase an supranuclear staining of the pouches with dba. sea, wfa and wa. which is similar to that fwnd in poximal colon. staining with psa, lca. u f and lta revealed charactenstic changes in binding panem th-may mdjcale changes m tucosylation of certain cellular canponents. certain changes in lectin binding were found to be more sjgnificant in the pouchitis group of cases. the changes in the ra~ewoir mucosa are most irkely to be an adaptive response to the new mtra-lummal environment wlth the acqmdfon of cmain mime chaiacteristics. thereare alsosp~ciftc changes which occur to a greatsrdegree m the cases with wuchits, these may occur as a result of or lead to the occunence of puchms. these resuns may be cryostat sctionshomten patientswith ileal pouches lor extensive colitis, ten distal ulcerative ~olitics and five normal mall intestines were assessed immunohistochemically for macrophages (leu m antibody). rfdl gnterdigitating antigen presenting cells). and rfdt (resting macrophages)antigencantainingcellsin lminapropna. result~areexpressbdasape~=~~t=g~~f thetotal. normal small intestine contained a mean ? % and % (ranges - . - , and - ) leu m . rfdl and rfdt positive ~ell~re~pecl~~elywh~chcontrastswiththoseh~ghervaluesinpouchbiapsies- %, %and %(ranges - , - and )respectively. distal ulcerative coilticscontalned increased macrophagenumbers( . and % for the three antibodies) but have a predominance of the rfdi positive population in contrast to pouchitis patients in whomthe rfd positive cells predominate thesefindings demonswate remarkably high macrophagenumbersin pouchitis, howeverthe rfd positive sub-populations predominance suggests an aetiologv other than ulcerative colitis for muchitis. margaret balsltis. yah mahida the wide family of cell adhesion molecules (including the subgroups of the immunoglobulin supergene family. the integnn receptors andtheselectins) are involved in controlling interactions between endothella cells and leucacytes !n inflammatory states. this is partly by their influence on adhesion and migration of leucacytes to and through endothellurn. using monoclonal antibodies, we have compared expr~ss~on of the three cell adhesion molecules icam-t (interceiiular adhesion molecule- ). elam-t lendothelial leucocyte adhesion molecule-t and vcam- (vascular cell adhesion molecule-t) ~n normal colonic mucosa with muwsa from cases of inflammatory bowel disease. wehavefoundexpressianafthese threemoleculesto beincreased~ninflammatarybaweldiseaseandthis change involvesendothelial cells as well asleucocytes we will dixussthbrelationship between adhesion molecule expression and dlsease actlvlty and posslble therapeutic implications granulomatous enterocolitis associated with therapeutic irradiation . . mangham. k m newbold, s dover the universty of bmmgham department of palhology, sci ool of m&mi science the medrcal schwl, edggbaslon. chronic radiation-induced entero~olitis s a weli recognised complication of radiotherapy for mtra-abdominal or pelvbcmalignancy. the pathological features includepwforatlon. fistula formation. segmental necros~s. and stncture formation. the histologicalfeaturesareessentially lxhaamic in nature. consequent upon the characteristic vascular damage. two case of inadtation-induced granulomatous enterocolltis are described ~n which non-caseating epithelcoid granulomas were present ~n the bowel well. to our knowledge. granulomas have not been previously &="bed in radiation coldisor any other formof ixhaemic bowel disease thsgranulomaswere largely confined to the mmosz-associated lymphoid tissue and the draining lymph nodes. naked submucosal granulomas were, however atso present. this distribution is similar to that seen m crohn's disease and tuberculosis. no evidence of these, or any othersystem!c ~r a n " l~r n a t~~~~~n d i t l~n~ was present these cases suppon theview that granulomas in bowel disease are secondary to an tmpaired muwsal bamer to antigens and highlight the non-specifiaty of granulomas m the diaynoscs of inflammatozy bowel diseases between and % of crohn's disease patients have involvement of the upper gastrointestlna tract the identification of granulomata is normally required to make a specific diagnosts but this may be dinicult in small endoscopic biopsies. we present t o cases where there was a strong suggestion of upper gastrolntestinal crohn's disease with a definitive diagnosis established on more distal invoivbment. four of the biopsies contained granulomata. the remaindershowedeitherpatchy inflammation, local ulceration orvillo~s dtjtmtlon only in aneffort to establish whether crohn's disease biopsies contained specific macrophage or lymphoid populations we applied the~onoclonal antibodies. mt (cd ). uchll (cd ro)). muramidass. mac , alpha- antltwsin and kpt (cdg ) using immunohistochemistry. theresuns were compared with casesot "on-specific duodenitis orlelunltis crypt-restricted loss of g pd activity has been used to quantify carcinogen-induced somatic mutation of the x- samples of peripheral nerve (sciatic nerve) of mice from varylng age groups were examlned over the past years. development olage~related penpheral neivefibredegeneraflon was observed among mlce over months age. the spontaneous peripheral nauropathy was characterized by walierlan type degeneratlon. teased nerve flbres from mice of years old showed evidence of swelling of the myelin sheath. fragmentation d myelin into myelin balls or ovoids, with areas of segmental demyellnatlon. light microscopic examination of h e stained sections revealed axonal degeneration, and myelin fragmentation. with vawolation of nerve fibres. on ultrastructural studies. there was evidence of axa-myelin degeneration. axxoplasm showing dark stained bodles suggemng of degenerate mitochondria the myelin sheath a l~o showed disorganization and fragmentation myelin, sometimes with whorl lormatron rithcm the cyroplasm of schwann cetk, wtth evidence of autophagocytosis by schwann ceiis. in some inslancestherawere sgnsofproliferation at schwann cells. nervefibredegeneration affected both non-myelinated and myelmated fibres generally, therewereapparentlygreaternumbers fdegeneratenan-myelinated nerrefibres than myelmated nerve fibres. furthermore the numben of degenerate myelinat& nerve fibres were less than in seontaneous peripheral neuopathy in ageing rats (personal obsenat on) swntaneous peripheral neuropathy of sciat,cnenesmainlyappearedin miceaged manlhsormore, s eenonlvvelyr rely in mlceof months. younger mice did not show any evidence of this change age-related penpheral nelimpathy is not usually accornpan~~d by any clln,cal ,gns neur~. fhology. uhversrty ofiowa, iowa. u s a v~scuiarendothellalcclls(rn)conslitufet~elntertace betweenthe bloodstreamand thetcsske and pertoormsevetal key roles ~n the development immune and inflammatory responses endothellal cell from the braln display slgnlflcantly different momhoiogy from other en. having ilght ]unctlons between them and pauclty at micropinacytolic ves~cles dunng inflammatory conditions of the cns interaction between mlammataly cells and rbb en s an initial important event. it s known that expression of several adhesion molecules on b r m en is upregulated !n intlammatorycondtti~ns and cytokine-induction ofthese molecules has been investigated however an ~mparlanl mechanism of adheson molecule induction and hence modulation of tmflammatq cell/en binding might beviral infect onofbrainen wehaveexamined theabilityofmeaslesvirus(human )andherpes simplex virus to infect cerebral endothelial cells. adhesion of syngenelc splenocytes to both vlrally-infected and mock infected cells was determined using a chromiums assay. by h of infection with measles yins, cytopathic effect was eadent. and splenocyte adherence was increased to a mean of % of the control mock-infected aner h mock-infecled expression of the adhesion molecules meca- and mala-lon virally-infected cells was determined possible mechanisms for enhancement of adhesion will be discussed and subsequent implications of virus inlectiun of cerebral en ~n relation to homing of inflammatory cells into the brain. ultrastructulal dbsenamns were made on the mechanism and route of innanmatory cell diapedesis through cerebral vessel walls in an experimental model of can~ned~stempervlrusencephalamyel~t~s ~n the hamster migrating monocyies and lymphacyiesextended pseudopodiawhich contacted, indented and adhwedtoendothelwm. they lhen invaded the endnthel l cells. becoming enveloped by endothelial cytoplasmic processes which re-establishec the continuity of the vascular lining as the migrating cell passed through although mlgratlng cells were frequently seen close to intei endotreiml junctions. they were ever seen wlthln iprctlons. or between adlacent endothelial cellsand therewas noev!denceof opening of mterendothelial tlghtpnmons aherpasslng through the endothel~al layer. cells squeezed through small pores (migration porest in the t h r sub-endothellal basal lamina me present study confirms and extends previous observations on the operation u;#thr the cns of a trans-endothel~al. paraiunclianal route for d apedesis of mflammatary cells. ireland falbology royal viclona hospital, bcl/asl hi bl lrrlnrld a sewice for the blochemtcal dlagnosts of lysosamd torpge diseases has now been o n operalaon m betlast forslx y n a r~ it currently lids assays available for the measurement lysasamal acid hydrolases in plasma. serum. lkwcocvtes. cultured sk nfibroblasts. amnioticfluld and cultured amniatlccellstordlagnos~s of lysosomalstorage diaurders solar patients have been referred from throughout ireland wtth having a positive diagnosis of these. were diagnosed as being hamozygous for a specific lysosomal enzyme deficiency, were identified as having multiple enzyme acl~ciencies (mucolipidosis type -cell disease) and had heterozygote (carrier) enzyme ibvpis t t v latier. w~i e either parents (obligate hetemrygotes) or siblings of homozygotes and one was a heterozyquti' lor x~lirlhrll recessively inherited fabry's disease. in addttlon. prenatal diagnosis has been performed on motherswithafam,iy hi~toryofi-celldiseaseand/orhurler'ssyndmme.oneofthesepmvedto bepositivefor huller tht' resuiic the biochemical investigations of these cases are presented. high levels of circulating immunoglobulins are common ~n liver dlsbase. in alcohol c hver disease depos s of mmunaglobullna(lga) have been found in the liver and therenal mesanglum, and werepart acaseof a oyearoldfemale with a year history of cardiomyopathy and progressivemuscle weakness anerthe birth of her baby shewas subsequentlyflnedwith apacemaker. clin~cally, therewas sevweweaknessof neck flexors with proximodistal weakness tn both arms and mild weakness of htp flexors. the most stnking weaknesswasin herbreathmg muscles therewas no ptosisorfaaal weakness rellexeswere symmetrical and her plantarswere flexor herckwas normal. aquadriceps musclebiopsy revealed abnormal vanatcon o n fibre diameters affecting bothflbrelypes. occasional pink hyalme ~nclus~ons which stainedforacid phosphataseand wlth pas wwe seen i~i both fibre types electron microscopy showed these i n~l~s i o n~ to cwtsist of aggregates nm diameter filamentaenmeshedwithinacentral coreofdenseamorphousmatenal. inotherareastheamorphausmatsnal layas inegutar patches within !he sarcoplasrn mainly at the level of the "z" tine causing disintegration of the sacomere immunoelection microscopy wing collmdai gold showed that the denseamorphous material reacted slrongiyw<th desmiri antisera and calld therefore represent a phosphorylated form of the pratein. tubular aggrcgatesitas] hayebeendescr,bedinassociat,on witha widevarietyofdisorders wherethey most onen occurin type %fibres recentlytwolam~i~eshavebeenrepored with muscularweaknessandtasasthedom~nant biopsylinding wereporla thtrdfamilyol whatseemsla beadistinctcondition twopatients afalherandda-ohter presented with slowly progressive p.oxima weakness. limitation of eye movements and aciiilles t e o d a r.antractufes creatine kina-was s t times normal by light mcroscopy, basophllic accmulations were ~e e n in both fibre types clecson m!croscopy revealed that these conslsted tightly packed parallel tubular a m p (tas) these varied somewhat m theiiulliast,wct~ral appearance and were therefore clawfled accordingly thisreport lhrows furner bwdexe m support of a otstmct cllntcopathologicai enttry. the c-myconcaprotein (p ) is a dna-bmdmg protem. increased expression of which is assmated wth shartenmg of the gog, phaseof the cell cycle. quantitahon of p m breast cancers may have prognoshc and therapeutc implicatrons. flow cyiometnc quantitatton (fcmo) of p has been shown to correlate wxth certatn parameters m breast carcinoma. however, thsre are factors which we felt could potenttally confound this fcmq m foimalln flxed paraffinembeded(ffpe)tissues. bothnuclear(n)andcyt~plsmic(c)express~oncanoccur. dependlngonthetlssue fixative used. cytoplasmic stripping and exposure of nuclei to the digestwe enzyme, trypsin, occw dwng the preparation of the nuclear suspension from ffpe tissue. this study documents c-myc oncoprotein stainlog i" * tissue senion of p m a r y breast carcinomas: turnour cells, adlacent benign epithelium and stromal cells were assessed. the monccional antibody om- - (crb) was employed using the abc immunocytochemical technique thi~antibodyrecognisesp andacyiaplasm ~ kd protein the resultsoftrla trypsmsatm of tissue sbctionspriortostsiningwerealsonoted. positivbstainingwasseenin y~oftumaun. both nandcstainmg belng present in % benign epithelium, many endotheiial, fibroblast and lymphocyte nuclel showed posltlvlty trypsinisatwn ledto lossof staining ntensity.thusin ffpe breastcawnomas. ~, " g l e p~~~m = t =~f c m q~f om- - anti-c-mycantiobody isconfounded by facts((i) much extratumoural c-mycexpresslon cccurs, ( ) cytoplasmic stripping may iead to oncoprotrin loss and ( ) breast oancercells,iisinducedaver -fald by phorbolssten.amphiregulin bindstotheegfreceptotandcan be stimulatoryorinhibitorydependingan theceil line. ltal~oblndsto~tsown receptor, possiblyerbb- .weevaluated expression of amphiregulin in primary breast cancers using immunocyiochemistry. % of turnours showed staining using amonoclonal antibody toamphireguiin which wasdlffuseorfocal in tumourcells.amphiregulin was not expressed in stroma and much less often in normal breast ep!thellum than m tumours. immunohistochemistry waspositiveincases withthe highest mrnaexprsssion(concordance %)asmeasuredusingacdnaprobeand qualitativenortherblotsanddat blotsanabplatecounter all caseswereassayedforegfraod er. er'egfr-. er* egfr*. er-egfr-. er-egfr'. there was no association of amphtregulln expresston wlth other receptors. these results suggest amphiregulm may be an independant factor regulating growth of a substantla proportion of breast cancers. in h a s t cancer, overexpression of the c-erbb- oncogene is associated with poorer prognosis, and in a prwious study we found it to be associated with lack of response to endocrine therapy bolus doxorubicin with infmons of ifosfamiddmesna appears to be a very active chemotherapy combination for advanced breast cancer in young women. thirty-nine women (median age = yrs) with rapid recurrence of breast cancer (median disease free interval = mth) were treated with doxarubicin and ifosfamlde/mesna. and c-erbb- expression in their pnmary tumours (determined immunohistochsmically) related to treatment response. the tumours of patlents strongly expressed c-erbb- protein and of these patients had objective responses to chemotherapy in patlents with turnourseitherweakly expressingc-erbb- (n = )otshowingnostaining(n = )therewereobject,veresponses,n . these data indicate that c-erbb- expression ~n primary breast carclnoma, while associated with resistance to hormonetherapy. daesnot preclude responsetosystemic chemotherapy. and sugge~taposs~blerolefarc-erbb- status in directing treatment strategies for individual breast cancer patients. pmicularly adpvant therapies ~n early stage disease. transforming growth factor is a muitifuncyional regulatory protein which can either stimulate or inhibit cell proliferation. can inhibit immune responses and can enhance both angiogenesis and the formation of connective tlssue.tgfpmrnahas beendetectedin breastcancercelliines, andtheprotein hasbe~nfoundinthemediafrom several such lines. in view of the prominent stromal reaction seen in many breast cancen. tgfp may have a srgnificant role in tumour development and subsequent behawour. tgfb, protein expression has been examined ~mm~noh~stdchemically using a specific polyclonal antiserum in formalin fixed. paraffin embedded invaswe and in s,fu breast carcinomas occasional staining of normal acinar cells was seen. % of invawe cancers had prominent staining of cells for tgfb. often with associated stromal staining. % showed occasional cellular and stromal staining. and staining of stroma only wasobserved in %. theremainder werenegative staining of ins it^ carcinomas was less with only % having prominent staining and % being negative. there was no conelatkon with type or grade of tumour. or oestrogen receptor status but all carcinomas with prominent reactivity had metastasised to nodes. this along with the differences between lnvasive and m srtu carcinomas suggests that tgfp may be a determining factor for invasion and metastasis. depanment ofpathoiwy, univers,ty ofshemeld, poboxsq , shefiela s ul chromatin changes ~n neoplasticcells are well recagnised quantitation of nuclear chromatin distcibuhon is dcfficult becauseaf thevanetyof parameterswhich couldpossibly bemeasured in thisstmiyamethodof quantttatmg the distribution of dna was developed for a seescan solitaire plus image analysts system. the parameters homogeneity, condensation and clumping were calculated by segmenting the nucleus grey scale intensity into three levels, black, grey and white, byamethod of adaptivethresholding.theseleveis represent the darkwand lighter regions of chromatin distribution. the relative pr~portlons and positions of each of these regions were calculated by viewing the nucleus through a pixel wide computer generated "mesh" the system was used to compare benign and malignant feulgen stained breast fnac specimens. the homogeneily and clumping parameters were significantly different between the two groups (p < ). but complete discrimination was not possible in order to test the valtdity of the system. spicimens were graded subp wely ( to ) and compared with the image analysis results. there was % correlation for homogeneity and % lor clumping. indicating the difference in chromatin distribution s real and not a result of system error the role of nuclear dna analysis. oestrogen receptor and thymidine kinase (tk) activity as prognostic indicators in human breast cancer was assessed. these parameten were investigated in relation to traditional pmgnostlcators. theoveralloblectivewastodeveiopaprognosticindexthat canpredlctforsunlval.tumourmatenalfmm breast cancer patients was anaiysed the nuclear dna content and oestrogen receptor status were determined by static cylophotometry and a multipoint titration assay respectively thymldlne klnase actlvlty was assessed using atp or ctp as a phosphate donor. patients were followed-up for a median of months. the cox proprtlonal hazards model wasused todetermine which parameterscould predict survival.pathologicalgrade, tumours e. totaltkand ctpjatpratiowerefo~ndto beindependent predictonof survival.aprognosticscore wasderivedforeachpatlent and these were assigned to one of three prognostic groups. using these prognostic groups the rlsk of reduced sunwpi could be predicted in . % of patients. inconcluslon, the assesment of pathological grade, turnow s m and thymidine kinase activity provides a reliable means of predicting prognosis year periodarereviewed.the median follow-up was months. on the basiso margln contour, degreeofstmmal overgrowth and atypla, mitotic activity. nuclear pleomorphism and the presence of speclallsed stroma, benlgn and malignant phyllodestumounwereidentified turnoursoccurredin womenof msdlanage years whomost often presented with a palpable, occasionally painful mass with a medlan diameter of cm exclson ( ) or smple mastectomy(l ) werethemorefrequentformsof therapy. localrecurrenceswereexperlenced by gpatlentswhose pnmary tumours although classified as benign contained areas of stromal overgrowth and specnaltsed stroma: all had been primarily treated by a histologically inadequate local e~~i~i~n~r~~b c~t a n e o~~ mastectomy (l).these local recunenceswere successfully controlled by local surgery ( )and mastectomy( ) mf~vemal~gnant tumours treated by mastectomy, none has recurred locally or developed metastases. flve mcompletely emsed turnours whlch showed neither evidence of stromal overgrowth nor speciallsed stroma remaln disease free at years. it s concluded that histologically malignant phyllodes tumoun are unllkely to develop distant dlsease i treated appropriately andthatstromal overgrowth may indicatethe possibilityof recurrence ~n incompietelyexcisedbenign lesions. several recent studies have evaluated the incidence in primary breast carcinoma of "micrornetastases" ~n multiple sections of axillary lymph nodes. howbyb~, the significance of these findings ~n terms of prognosis and sunw remainsunclear. inanattempt toelaborateon this, wereviewedthecasesof patlentsdlagnosedas havlng'bodenegative'' breast carcinomaon routine histologyand in whom therewasafollow-upperladof at least fwe years. the anginal diagnostic h e slides were reviewed in conjunction with eptthellal immunohistochemical pr-aratlons usmgcea,ema. hmfg, andkeratin, andappropriateclinicalfollow-updatawasaccumulated.thestudycasesfell into two categories, those which retained a 'node-negatlve' status after revew, including immunohistachemical appraisal ( %). and those which demonstrated individual or grouped malignant epithelial cells ( %) ~n the peripheral or deeperainuses of the lymph node. of the former group. all pattents but one werealive with noevidence of recurrent breast carcinoma five yean after mastectomy. of the second cohort. posnttve far "micrometastases". % showed nodiseaserecurrence. while % had gross metatatic d i s e a s e o r h a d w c c~m b t o thecarcmoma within hefiveyearpenod.thefindingsdescnbed.aithough basedon low patient numbenln thisprelimmarysludy. indicate the appropriateness of a large multicentre sunw to evaluate completely the prognostic significance of these '"msrometastases". sadhna bhatnagar. j. mcclure. d a. kalbhen the injection of sodium iodoacetate ( . mg) into the avian knee joint on two consecutive days induces changes which macmscopically and radiologically resemble osteoarthrosis. knee-pnts from groups of six hens were studied atregularinterval~upt weeksalterinjection by histological and histochemlcel methods. characteristlcles~ons were identified and assessadon asemi-quantiative basisand indwdual ision profilesacrossthetime course of the expenment were constructed. during the first five weeksthe obvious changes wwe chondrocyte loss and depleted matrix staining. chondrone formation was also noted. fmm six weeks there was also tibravascular proliferation at the chondro-asseousiunction wim modelling activity and new bone fwmation. in addition near the insenion of ligaments there was chondrocyte differentiation and proliferation. the amount of cartilage fibrillation and flsswng increase during the expenment aithough there were so areas of apparent resurfacing. the decreased matrlx staining noted early in the expenmen stabilised and many lacunar spaces are occupied by chondmcytes towards the end. subchondral bone hyperplasia and osteaphyte formation are prominent persisting features. such sequentialcharacterisation may be usefulin a-ssinglhee(fectsofput=~~vetherapeuticcompounds onthe model. rodentachilles tendonsweiesubiected to midpointtenotomy and allowed to recover for various timesin sllu before the operaled tissue was mmved, placed into a millipore dinusion chamber and insened inhaprntoneally into syngeneic hosts. diffusion chambers were removed at weekly intervals such that the tom time post-operation :i.e. time sru plus time wnhin the diffusion chamber) was up to weeks. histological examination demonstrated that ectopiccartilage wasprodwed withinthediffusion chamber byatotal of weeks but only if thefirst weeks were recovery in situ. wnh increasing time. calcified cartilage, mtteoid and pnmary spongmsa bone was obseived evtdence suggests that the camlage forms via direct tendon metaplasia and that the bone may form as a result of dinerentialive changes within hypertrophic chondrocytes. tlbla and fibula appearto be directly exposed to tha surface of the medial femoral condyle and inner ridge of the lateral femoral condyle. the exposed load-bearing area of the femur and tibia contain demonstrable amyloid around chondrocytes and in the matrix below the surface. these areas are also covered with the densest hyalmd fibro-cartilage. amyloid p r e e u~r s from h e synovlum may penehatethe surface ofthe cartilage under pressure or precursm from cartilage matrix or chondrocytes may be converted into amyloid and are napped below the surface. the inpction of sodium todoacetate into avian knee iolnts is followed by the development of an osteoarthrotic pmess to studytheeftectal sodium lodoacetateon chondrocytemetabalism m wire, chickstmal chondrocytes grown i~th~ee-dimensionalcollagen gels wereexpmedtothe compound. sterna from chick embryos(l - days in om) were digested with collagenase releasing the chondrocytes which were then cultured in collagen gels. w i u m iodoacetate was added to -day old cultures at concentrations of . . and wg/rnl. ( , . and .o y lodm)tor hours. theeftectswere assessed lor up to days by: ) staining wnhfluorescein diacetate which pmducesfiuorescenceiniive. metabolisingceilsonly. )thelevelofproteoglycansinthemedium wasestimated by complexing with dimethylmethylene blue and measuring the increase in absorbance at nm. ) histologycultures were fixed in glutaraldehyde, embedded in paraffin, sectioned. and starned wm alcoholic toluidine blue. alcaan blueandpicmsinusred.treatmentwlthiodoacetateproducedadoserelateddecreaseincellnumbersand intheamount of proteoglycmsreleased into themedium (lessthan % at pg/ml).the histolagyshawed thatthe treated cells were smaller and produced less matrix. these results show that sadium iodoacetate reduces cell number and interferes wqth the manufacture of matrix components. instftute of pathology, ro i vcfona hosprtal, belfast. l. irelend theoblectivesof this study were tocorrelatecertain clmcal features of ependymoma. (i e. age, sex, anatomcai slte. turnour size. meningeal dissemination and raised intracranial pressure with certain pathological features. . e dna content, mnotic rate. immunocytochsmistry and ultrastwctural features) with prognoss a total cases was available for clinical pathological assessment univewty of liverpool, p box , liverpooi bx and 'sari andres unrvemty it is not surprising that wmal exposure to high altitude causes increased activity of the hypothalamic-pltultaryadrenocortical axis. but fflidence suggests that continued exposure to hypoxla leads it to perstst long after homeostas sis restored and acclimatization achieved their pltuatary glands were not sgnlflcantly different insizefromthoseof thesea-levelsubpcts, but c~ntainedlargerpopulat~onsofcort~cotrophs~n termsof the total population of their anterior lobes ( . vs . %: p < ) these fmdlngs are in keeping with those of previous studies of the hypothalamic-pituitary-=d~=~~cottical axis in man and animais exposed to prolonged hypoxia at natural high altitude or in the laboratory. they suggest that greater amounts of acth might be necessary to sus ain normal adrenocoltid function under such circumstances and that adrenocortical hyperplasla bone marrow wnsists of a haamopolstic system whch is nn intimate association with aftbrous stramal network.adun mammalian bonemanowstromais knownto have both chondmgenicand ostecgeniccapabilitiesinvlvoand stromal tissue has been shown to tom fibroblastic wlonies capable of giving nse to bone in vitro. however, the mqbility of manow stroma to form cartilage in who has not been addressed when grown in identical conditions to mammalian stroma. mechanically dissociated single cell suspensions of chick embryo bane manow hom > days in ovo and from day hatchlings readily lorm bath fibroblastic and cartilage colonies in vitro. cells within the cartilage colonies are polygonai in morphology, are separated by a relradile exhaceiiuiar matrix and svnthesise catilage specific pmeogiycans and collagens. as shown histochemicelly, biochemically and immunocytochemically. in contrast, adult chicken bone manow does not form cartilage. instead, the cells appear osteoblast-like and synthesise coilagens typical of bone. a. m. fianagan t. j. chambers &wtmment of h~slopatholosy. sl george's hosptal medlcel sch-i, london. sw ore osteaclastshavebeensuccessfullygeneratedinculturesofmurinehaemopoisticcells. itwouldbeuseful ifasmila, model were available to analyse the mechanisms of regulatlm of human mteaclast formaim ir normal and pathological states. although the osteoclast is present in reverai neoplastic conditions. it is not known whether it forms part of other haemowietic malignancies, lor example, polycythaemia rubra "era or chrmic lymphatic leuhemia. we used strategies based on our experience with murine osteoclastogenesis. however, we have been unabletogeneratefunctional human osteoclastscapableoiresorbingbone m vrtro. large numbersofmultinucleate cells developed in these cultures mese cells did not show a typical panem of reactivity with asteociast-specific manoclonal antibodies, nor did they bind ' sct but rath@r possessed an antigenic profile characteristic of macmphage polykaryons. it is pecullarthat human tissue fails to support osteoclast generation stnce cells of the other haemopoietic lineages were consistently generated in our cuitures. in murine cunuras it is known that a particular shomal cell type is requtred for osteoclastagen@sis; it is possible therefore that this cell population is spamin adult humantissue.miswouldreflecttheiownumberofosteoc asts presentinhuman adults comparedto mice elucidation of conditionssuitableforthe generation mthehuman osleoclastm wtrow~ll heldus understand the mechanisms by which it is regulated in health and disease spaceof one year. the other case s b middie-agedwoman who &=being affected bythe disease far sevaral years developed separate sarcomas in each lower limb dunng a nine month pwiod. each tumour was treated bv pamal amputation but she died from widespread metastases within one year of the first amputationthe distribution of proline- -hydroxylase in a range of human tissues shown by a monoclonal antibody, fib s. smlth, p. revell aepariment of mobd anatomy and bone and h m f research unrt, the london hospw medcal cdl€ge, london. e l fib . a commercially available monoclonal antibady to the b subunit at the enzyme proiine- -hydroxyla~ invalved~ntheproductionofcallagen, wasappl'edtaacetonebxedcryostat sect~onsofawiderangeoftissues. in hver, hspatocytesshowedvarying degrsesof labelling with the antibody, along with spindle shapedcells(sscs) in associated connective tissue in both tonsil and lymph node. sscs in the connective tissue were melled with the antibody along with a number of other cells (lymphocytes) within the germinal centres. in addition to this. perivascularcellsand cells intheconnectivetissue iayerimmediatelybelowtheepithelium also labeliedin sections of tonsil. in skin, very few cells in the dermis were positive for pm ne- -hydroxylase. as were also only a small numbetof epidermal cells. afew chondrocyteswere marked bythisantibody in artlcularcartilageand intervertebral disc,along withswneostwcytesin bone.manycellsinthepleuraandtheepimysiumof normal~keletalmusclew~e posotive. there was no staining at all in specimens of kidney. in addition to these 'normar tissues, examples of a seminoma, a breast carcinoma and a thymoma were examined. each of the turnours showed fib labeliiw of ssc~withinthestromalt~ssue.theseresultsshowthat fib b sele~tivelylabelssscs.whlcharapresumedto be f brobiasts. in a wide ranged both normal and pathological tissues. which produce and maintaln the collagen matnx. proime- -hydoxylase is an enzyme involved in the pmduction of collagen and demonshahle in the rer of fibroblasts. it may therefore be cmsldered as a possible matker for fibroblasts in tissue sections. we have applied the mouse moncclonal antlbdy fib , raised against the $ subunn of prol ne- -hydroxylase. to alcohol fixed paramn embeddedsections and acetonefixedcryostat sect~onsolnormal (n = ) and meumatoid (n = ) synovia. synoviocytes labelled very strongly, but the distribution vaned between samples. insome, mostsynoviocytes weremahed, whilein others ~nlythedeepiayerofcells wasmitive. it isnot known whether thts dillerence ~n prdine- -hydroxylase expression is due to the severity and duration of disease. drug treatment or other factors. it is of interest, however, that fib labelling was not wnflned to the type b (fibroblast-llke) synoviocytes and that the more supficlal type a synoviocytes also contained proline- hydroxylase. spindleshapsdeells i" thesublntlmaleonnectiva tissue were labelledma vanablemanner. inaddltlm to these findings for fibroblastic cells. a small cap of fib posnive cells was seen around lymphoidfollicles. sometimes polansed towards the synovial surface. this enzyme expression by lymphocytes is under further invsstlgatlo". vitronectin s an adhesive glycoprotein which shares several functional similarities with fibronectin. it is a major component of extracellular matnx and plays a role in cell-matrix mteradians, monocyte function. and the coagulation and wmplement systems. we have employed a monoclonal antibody to assess the distribution of vironectin in frozen synovial biopsies from patients with rheumatoid arthntis, ostwnhntis. ankylosing spmdyiitis andtraumatic non-inflammato~ control^ ( cases). lmmunoreactive vltronectin was identified in the synovium. similarto the panem of immunoreactiyefibronectin, itwas located in adensefibrillarpanem surrounding cellsof the synovial lining layer. vitronectin was also associated with fibres throughoutthe sub-intimal connective tissues. being most prolific in fibrotic areas. vltronectin was identified in the sub-endothellal layen of blood vessels. but unlike fibronecttn was not associated with basement membranes. similar distributlon patterns wme observed in all biopsies studied thelocalisation ofvitronectitin in synovial tissues suggestsapossibleroie inanachmmtofcellsto the extracellular matrix and that may be important in the pathophysiology of inflammation and repair manolayercultured articularchondrocytesare known to rapidly losethelrexpressionof collagen typeil.the purpose of this study was to compare the expression of various collagen types in twoand three-dimensional cultures articular chandracytes. in addition. the expression of s- w protein and its alpha and beta subunits was studied in monolayer cui ures. bavine articular chondrocytes were isolated by collagenase digestion from ankle joints and cuhured in monolayerandspheroid culturesin dmemwim % fcs lmmunocytochemical studieswere performed using the indirect peroxidase technique an monolayers affer methanollethanol ( vlv) fixation and on frozen sections of spheroids. the reaction was scored semi-quantitatively as negative (-), weakly (+) moderately (++) ontha basisofthenumbersafcasesrefsiredtoustottheidentificalionofjointcry~talsintissuesectians, webelieve that calcium pyraphosphatedihydrate(cppd) deposnion disease isbeing underdiagnosed byhistopathologists. a crltical light microscopic reviewof cases. all with thediagnosisconfimed bymicroanalysis(energydispersivexrayspectroscopyln thescanning electron microscope, infrared spectroscapywboth) revealed adistinctwefeathery or br"shlikeappearanceinal suchdeposits.thisfeaturewasapparentat lowpower, whileconvincingvisualisation of crystalswithinthedepositswasdiff~cuneven withanoil immersionobiective.thesignof birehingeneeof cppd crystals is more dificult to demonnrate i" tissue sections than synoviai fluids. due to iactois such as stain. fragmentation and heaping up of crystals, buf it can be more readily assessed in unstained sections or following microincineration of the section. in six of our c a m the deposits were exclusively within bone: demonstration depended on relatiyeunderdecalcification: deposits in this position have not previously been recognised. this study thus provides new information an the histological identdication of cppd deposns in tisues and implies heterogeneity in the pathogenesis of such deposits. in human knee osteoarthrosis (oa) overt unicornpartmental disease is frequently accompanied by a macroscopically normal second cornpartmsm. however. there are a number of light micmscopical changes within the latter which might amount to an "early osteoarthrosis". these are chondrocyte pmliferation and chondrone formation. decreased proteoglycan staining and disruption of the chandra-osseous interface with duplication of the tidemark and resorption ofthe cartilage by chondmclasts. tidemark duplication is also a feature of established oa re~resenti~gadistu~ancedfcabif~cat~oninrone ofarticularcartilage.slnceslwp~~t~i~~~~ knowncalciumion transponmoleculs.altsrationm~~sstainingpanernand~ntens~tym~grn beanlndexotearlyo~. sl~~protemstamng was studied in the tiblai plateau cartdage of cases: with unicornpartmental oa. with bicompartmental oa and controls. lntheianerall chondrocytesgavepositivemembrana and cytoplasmic staining (especially in zones e n d ) , d~u s e w e a k m a t~~c~l s t~~~~~g~~~~~e l , s t~o~g p a r i c e l l~i a~a~d~~t~~-t~~t o~~l s t~i~~~g~~~o~~s a~d . in overt and early oa the orderly malrlcal panem was disrupted with prominent patchy staining around chondrocyte clusters. the significance of the change in the matncal distribution ~n oa is unknown and unlikely lo be related to a disturbance of calcification in zone . road, london. wcl m e histogenesis at alveolar son part sarcoma (asps) is still unsenled: nonchromaffin paraganglioma. malignant granular cell myoblastoma, neuml tumour, myogenic tumour. habdomyosarcoma and renin-producing tumoui theories have been proposed lmmunohistochemical studies have yielded discrepant results we recentlyobsewed that the diastase resistant crystalline material in asps was strikingly reactive for vimentin by the immunoperoxidase method on paraffh sections one case. we then studied paraffin sections of seven other cases from our files between and ~ by routine light microscopy, immunohistochemistry and electron microscopy all eight cases were reactlve for vimentin. which highlighted the crystalline materlal as in the first case. vanabie numbers of immunoreactlve cells for desmin were found in three. and smooth muscle specific actin in four. all cases showed some reactivity for neuron specific enolase. and seven of the eight cases reacted for s- protein. the clinical and pathological lea lure^ of cases of synoviai sarcoma have been reviewed. these tumours are aggressive with a poor prognosis. typically affecting young aduns with a long history before presentation and detinltive diagnosis. a high index of suspicion is required if the diagnosis is not to be missed several features, including age, size. site. histological type (monophasicibiphasic), mitotic actlvity and necms~s have been investigated withaview toestablishing possible prognosticindicators. the bestguideto prognosisis assessment mitotic actlvity. size and histological type dld not affen the outcome. in this study the effecl of subcapsular orchidectomy on skeletal metastases from prostatic carcinoma was studied using bone histomorphomatnc paramsten. twenty-eight patients with bone metastases were studied immediately before and for seven months after orchidectomy. tetracycline labelled bone biopsies were taken from metastases and turnour free areas at the beginning and end of study. sixteen of the patients also underwent o~teocla~t inhibition tor six months using disodium pamidronate ( mg i.v. weekly for weeks then anemale weekly for five months) to enable differentiation of the skeletal response to castration. histamarphametnc anslysi~ tumour frse bonerevealed adrapin overall bonevolume. histological analysisofmetastasesshawedadecrease m osteoblastic activity but widespread osteoclast mediated ostealysis. tumour regression and manow recolonisation were present in most c~s b s but malignant foci remained in % of repeat metastatic biopsies, inducing a typical. lacalised disruption of bone metabolism. it is concluded that orchidectomy causes ostmblastic regression blrt induces increased ostboclast mediated bone destruction, which is most pronounced within metastases. although tumour regression and marrow recolonisation usually occur within metastases, active turnour foci anen perstsf. ( ) chairman s. g. silverberg, washington theeffectsafoncogeneson thedeveloping nelvoussystem havebeenstudiedinaneuraltransplant system ~ntats, taking advantage of the extraordinary capacity of fetal cns to differentiate in and fully integrate with, the adult host brain. gene transfer tnto tetal brain cells was mediated by in viiro infection with replication-defsctwe retroviral vectors. fetal rat brain suspensions were then stermtaxically injected into the caudoputamen of adult f rats.animals carrying transplants exposed in vim lo the polyoma medium tantigen developed endothellal hemangiomas in the graft which often led to fatal cerebral hemorrhage within - days alter transplantatm lntroductm of the viral sic gene caused astrocytic and mesenchymai tumors affer latency periods of months.following infection of fetal donor cells with a vector encoding the v-myc oncogene led to the development of only a single embryonal cns tumor whereas exposure to v-ha-ras and v-myc resulted in the rapbd induction of multiple malignant neoplasms. when injected intracerebrallyinto newborn ratsin vivo, complementation of theseongogenes led the development of malignant hemangioendotheliomas, undinerentiated neural tumors andlor leukmia.oncogene transferthus constitutes a challenging new model to assess the effects of translorm~ng genes on the developing nervous system. since the presentahon of molecular genetic evidence supporting the knudson hypothesis m the eariy s there hasbeen increasing interestinthe ideathatasimilartwo-hit mechanism mayaperateln sporadadlccancers therens now evidencbthatlassofonealleleandretentionottheother~nmutated/wildfarmaccuninsparad~ccalono. lung. renal and breast cancers in breast cancer lossof heterorygoslty(l h) has been demanstratedfarseveral alleleson chromosomes l p q and an the short arm of chromosome . loh has also been demonstrated on other chromosomes thegeneral viewisthat loh indicatesthatasuppressorgene may bepresentattheseloct. hawevet in interpreting lohthere sdifficulty indetermining when loh becomessignificant because itsincidencevariesfrom % / depending on the locus examined. in this presentation, knowledge about loh on chromosome p (and q where a new deletion has been recently discovered), and p will be presented and the pathqlenetic significanceand ciinicsl relevanceexamined. lnthe lattercontext anewrapid methodfordetemlnmg alleljc dosage will a l x be described which does not requlre rflp analysis. expelt systems - .e. computer software that can function as cansuhant, decision support system, or process contr~llerhas undergone a rapid development durlng the past decade. applications to histopathology have gnaw some ,ocqdiffuse maiignantmesotheliomas( mm) occurannuailymn. americaat thepresenttime.cumulat~~ely, substantial numbersot lesser-knownfarms of mesothelia turnourare also seen, including serous paptllary tumoun of the peritoneum, well differentiatedpapillarymesothellomas and benign cystic mesotheliomas, but incidencedata are not available.the relativeiyfrequent atypical reactive hyperplmasof w o s a membrane add further vanstytothe range of mesothellai lesions which may present diagnostic problems. the expenence of a u.s.-canadian mesotheliomapanel suggests that dmm vs. metastatic carcinomacontinues to bethe commonest mesatheliamarelateddiagnostieehaliengeandthatthedifficunyandalwayssolved bytheappiicationofspecialstains almostas common are le~ions in which the ditferential diagnosiscenters on dmm us. a reactive process (atypicai mesotheliai hyperplasia, fibrous pleurisy) within the laner group it is particulady difflcun lo obtain a strong consensus opinion, andfoilaw-uphasconfirmedthemaiorityopinioninonly %ofcases. sarcomatousdmmvs.sarcomanosisaiso a notable area of difficuny. most mesothelial lesions tn north amenca. including dmm. are reviewed within a short time of biopsy by a pathologist in a major teaching centre, only a small number ( - %) being subpct to further scrutiny by a panel or ~n the coune of epidemiological or clinical studies however, many cases resurface later ~n a legal sening when the material and clinl~al information s often available. the original diagnosis of the hosoital pathologist s usually endaned by the courts. sharon w. weiss llposarcoma is one of the most common forms of son tissue sarcoma and may be divided into several subwes well-differentiated. myxoid. round cali, phmwpha, and dedifferentiated. this presentation will discuss the ) diagnosticcnteriaof lipblasts ) d agnosisand behaviorof myxoidround cell iiposarcoma andthe ) behavior and incidence of "dedifferentiation" dwell-differentiated ihposarcoma. the diagnosis of lipsarcoma depends in part on the identdication of lipoblasts or pnmitive fat ceils. since iipoblast-l!ke cdls may be seen m a variety of conditions apart from iiposarcoma, strict diagnostic c!i tma must be applied in identlfving these ceiis. these cells contain a hyperchromatic. indented or scalloped, ecmtnc nucleus set in a cytoplasm containing one or more lipid-nch vacudes. these cells must also occur in an appropriate histaiqic background. vanous lesions which may contain ihpoblast-like cells and which may. therefore, mimic liposarcoma include fat necrosis, fat atrophy, silicone granuloma, signet nng carcinomas or melanomas, and a variety of malignant tumors with fixation artifact. myxold androundcell lipsarcomarepresentthemost commanfarmofsarcomaaccuningmearlytamidadunlifeandare commoniy located #n the region of the mlgh and popitteal fossa. although the designation of "myxold and "raund cely suggest two separateturnortypes. they represnt ends d a common h~stologlcspbctnm. myxaid liposeicoma represents the well differentiated end of the spenrum. whereas round cell represents the pcarly differentiated end.however, transitional or mixed forms ex~st. accounting for confusion as to how such tumors should be classlfled mymd liposarcomas are characterized by nodules of bland stellate or rounded cells set n hyaiuronic aod-rich strama. an intricate plexiform vasculature and numerous lipblasts are easily vdentaed. wlth progression to round celliiposarcomathecellsbecomelarger,moreatypicalandthestromalessmyxaid. lipoblastsaremoredlfticultto find and a plexiform vasculature s less apparent. recent work by evans suggests that evaluatlon of myxoidlfound cell ihpasarcomashould includethe percentage of round cell component wdhin atumorsincethisdirectclh/ mtluences prognosis. our policy is to carefully sampk such tmors, submining t section for each centimeter in greatest diameter of the tumor. a rough estimate of the percentage of a round cell component s made. we w a r d tumors havinglessthan faroundcellcomponentasgradei.thosehav,ngbetween & % roundcelicomdonent are considered grade . whereas those with more than % are considered grade . well dmerentiated liposarcoma is one of the most common sarcomas accuning m late adult life. characteristically affecting the deep musclesaftheextremities, theretropentonealspace. and the groin, this tumor ischaractenzed by varying amounts of mature fat interspersed with fibrous bands, atypical hyperchromaric spmdledceiis, and lipoblasts. these lesions are considered low grade sarcomas having a high rate of local recurrence but no abilny to metastaslze. recently evans and ammi eial suggested that well-differentiated liposarcomas occurring m the wbcutaneous tissue and muxles of the extremity cause so hnle morbidity that the use of the term "atypical lipma" should be used rather than ihposarcoma. in contrast. well-differentiated liposarcomas in the retropentoneum cause significant morbidihl and pose a significant risk of death from local disease. thus. nearly all pathologists agree that the term "weii differentiated lipasarcoma" should be retained for these tumors ~n the retroperitoneum. however. none of these recent studies have followed a large number of these tumors for a proianged period at time m order to assess the long term behavior and specihcalty risk that such es ons may progresswith time to a higher grade lesions ( . dedifferentiate). we have recently completed a follow-up study of cases of well ditferentiated iiposarcoma accurringmthemusc~oftheextremity,retropntoneum,andgroin. inalllmatlonsthenskof local recurrence was high. however,dedifferentiationoccurred~n % of casesandwasnotrestnnedtotumors~nanyparticularlacatlon. butcouldbe bestconelatedwiththedurationofthetumor. withmostcasesoccun~ngaflerl mmoreyean thus. dedifferentiation is not a site-dependent phenomenon, as has previously been suggested. but rather a time dependentphenomenon.anhough thesedatado notneee~~arily indicatethatwe should abandon theterm"atwca itpoma" they do indicate the need tor proianged foilow-up of patients wlth wei&-dlfferent,ated ddosarcoma and the small but deflnne risk of dedifferentiation as a long term complication of the disease. key: cord- -ji jbct authors: morens, david m.; folkers, gregory k.; fauci, anthony s. title: the challenge of emerging and re-emerging infectious diseases date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: ji jbct infectious diseases have for centuries ranked with wars and famine as major challenges to human progress and survival. they remain among the leading causes of death and disability worldwide. against a constant background of established infections, epidemics of new and old infectious diseases periodically emerge, greatly magnifying the global burden of infections. studies of these emerging infections reveal the evolutionary properties of pathogenic microorganisms and the dynamic relationships between microorganisms, their hosts and the environment. merging infections (eis) can be defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range" . eis have shaped the course of human history and have caused incalculable misery and death. in , a new disease -acquired immune deficiency syndrome (aids) -was first recognized. as a global killer, aids now threatens to surpass the black death of the fourteenth century and the - influenza pandemic, each of which killed at least million people , . of the 'newly emerging' and 're-emerging/resurging' diseases that have followed the appearance of aids (fig. ) , some have been minor curiosities, such as the cases of monkeypox imported into the united states , whereas others, such as severe acute respiratory syndrome (sars), which emerged in the same year , have had a worldwide impact. the anthrax bioterrorist attack in the united states falls into a third category: 'deliberately emerging' diseases. eis can be expected to remain a considerable challenge for the foreseeable future. here we examine the nature and scope of emerging and reemerging microbial threats, and consider methods for their control. we emphasize that emergence results from dynamic interactions between rapidly evolving infectious agents and changes in the environment and in host behaviour that provide such agents with favourable new ecological niches. about million (> %) of million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses (fig. ) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries , and particularly on infants and children (about three million children die each year from malaria and diarrhoeal diseases alone ). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities . eis have been familiar threats since ancient times. they were once identified by terms such as ȕșȓȖș ȝ (loimos) , and later as 'pestilences' , 'pestes' , 'pests' and 'plagues' . many examples can be cited in addition to the black death and the influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age . the age of discovery, starting in the fifteenth century, was a particularly disastrous period with regard to the spread of infectious diseases. importation of smallpox into mexico caused - million deaths in - , effectively ending aztec civilization , . other amerindian and pacific civilizations were destroyed by imported smallpox and measles [ ] [ ] [ ] [ ] [ ] . historians have referred to these events as apocalypses and even as genocide . for centuries, mankind seemed helpless against these sudden epidemics. but the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases [ ] [ ] [ ] led to enormous progress, notably the development of vaccines and ultimately of antimicrobials . in fact, the era of the identification of microbes had barely begun when optimists at the end of the nineteenth century predicted the eradication of infectious diseases . by the s, which witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in , and in , the us surgeon general stated that "the war against infectious diseases has been won" . some experts remained sceptical, aware of recurrent lessons from history. they were less persuaded by successes than alarmed by failures such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. richard krause, then the director of the us national institute of allergy and infectious diseases, warned in (ref. ) that microbial diversity and evolutionary vigour were still dynamic forces threatening mankind. as krause was completing his book the restless tide , aids -one of history's most devastating pandemics -was already insidiously emerging. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the past years, some of the factors that resulted in aids have also led to re-emergences of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalysed by wars, loss of social cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the challenge of emerging and re-emerging infectious diseases the classification of eis as 'newly emerging' , 're-emerging/resurging' or 'deliberately emerging' is useful because the underlying causes of emergence and the optimal prevention or control responses frequently differ between the groups. newly emerging infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (see box ); these include microbial genetic mutation and viral genetic recombination or reassortment, changes in populations of reservoir hosts or intermediate insect vectors, microbial switching from animal to human hosts, human behavioural changes (notably human movement and urbanization), and environmental factors. these numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. any discussion of recent eis must begin with the human immunodeficiency virus (hiv) that causes aids. hiv has so far infected more than million people worldwide . before jumping to humans an estimated - years ago , perhaps as a consequence of the consumption of 'bush meat' from non-human primates, hiv- and hiv- had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids might never have emerged had it not been for disruptions in the economic and social infrastructure in post-colonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure all promoted sexual practices, such as promiscuity and prostitution, that facilitate hiv transmission [ ] [ ] [ ] [ ] . such complex interactions between infectious agents, hosts and the environment are not unique to the epidemiology of hiv/aids. the examples cited below further illustrate how changes in population density, human movements and the environment interact to create ecological niches that facilitate microbial or viral adaptation. some infectious agents that have adapted to non-human hosts can jump to humans but, unlike hiv, are not generally transmitted from person to person, achieving only 'dead end' transmission. infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis , . examples include the arenavirus haemorrhagic fevers (argentine, bolivian, venezuelan and lassa haemorrhagic fevers) and hantavirus pulmonary syndrome (hps). viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioural factors. farming, keeping domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the first epidemic of hps, detected in the southwestern region of the united states in (ref. ) , resulted from population booms of the deer mouse peromyscus maniculatis, in turn caused by climate-related and recurrent proliferation of rodent food sources. increased rodent populations and eventual shortages of food drove expanded deer mouse populations into homes, exposing people to virus-containing droppings. the - malaysian nipah virus epidemic further illustrates the influence of human behaviours and environmental perturbations on newly emerging human infections. pigs crammed together in pens located in or near orchards attracted fruit bats whose normal habitats had been destroyed by deforestation and whose droppings contained the then-unknown paramyxovirus. virus aerosolization caused infection of pigs, with overcrowding leading to explosive transmission rates and ultimately to infections in pig handlers. variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. vcjd is caused by the humanadapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse) figure global examples of emerging and re-emerging infectious diseases, some of which are discussed in the main text. red represents newly emerging diseases; blue, re-emerging/resurging diseases; black, a 'deliberately emerging' disease. adapted, with permission, from ref. . epizootic/vcjd epidemic, primarily affecting great britain, probably resulted from the now-abandoned practice of supplementing cattle feed with the pulverized meat and bones of previously slaughtered cattle. bse itself is suspected to have emerged because of even earlier use of cattle feed containing the agent of sheep scrapie, a prion disease recognized by farmers more than years ago . alarmingly, the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans . the recent reports of variant strains of the bse prion suggest that the bse agent could be a more serious threat than other animal prions. infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses, as well as certain non-zoonotic diseases, which are discussed below. for example, legionnaires' disease, first identified in , is caused by legionella pneumophila, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems . campylobacter jejuni and shiga-toxin-producing escherichia coli (e. coli o :h and other agents of haemolytic-uraemic syndrome) infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios causing classical cholera (re-emerging; see below) and serogroup o cholera, and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis , seem to have come from environmental or animal organisms that have adapted to human-to-human 'faecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between and (ref. ) . scarlet fever, then the leading cause of death in children, is now rare, but has been largely supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever . when new microbes are discovered, their emergences as disease-causing pathogens may be delayed. for example, in , robert koch was unable to show that the newly discovered koch-weeks bacillus caused serious disease. more than a century later, a fatal ei dubbed brazilian purpuric fever was linked to virulent clonal variants of haemophilus influenzae biogroup aegyptius (the koch-weeks bacillus) . although the bases of emergences of new and more severe diseases caused by s. pyogenes and h. influenzae biogroup aegyptius are not fully known, in both cases complex microbial genetic events are suspected. the distinctive clonal variants associated with severe h. influenzae biogroup aegyptius disease have been shown by pcr (polymerase chain reaction)-based subtractive genome hybridization to contain not only a unique plasmid, but also unique chromosomal regions, some of which are encoded by bacteriophages . this research has narrowed the search for virulence determinants to unique proteins, some of which may have been acquired from other organisms by horizontal gene transfer. streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex than for h. influenzae biogroup aegyptius. many factors associated with streptococcal virulence have been identified in strains bearing the m surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection , . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus with kaposi sarcoma, and of helicobacter pylori with gastric ulcers and gastric cancer [ ] [ ] [ ] . some data even suggest infectious aetiologies for cardiovascular disease and diabetes mellitus , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range. re-emergence is caused by some of the factors that cause newly emerging infectious diseases, such as microbial evolutionary vigour, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related -for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria . the impact of both new and re-emerging infectious diseases on human populations is affected by the rate and degree to which they spread across geographical areas, depending on the movement of human hosts or of the vectors or reservoirs of infections. travel has an important role in bringing people into contact with infectious agents . an increase in travel-associated importations of diseases was anticipated as early as , when commercial air travel was still in its infancy . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute haemorrhagic conjunctivitis in (ref. ) , by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere (fig. ) . the persistent spread of hiv along air, trucking, drug-trafficking and troop-deployment routes is a deadly variation on this theme [ ] [ ] [ ] . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide (fig. ) . years of effective use of dichlorodiphenyltrichloroethane (ddt) led to the abandonment of other mosquito-control programmes, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine . research efforts focus on the development of vaccines and new drugs, and on re-establishing public health measures such as the use of bed nets. tuberculosis is one of the most deadly re-emerging diseases (fig. ) . the discovery of isoniazid and other drugs initially led to effective tuberculosis cures, empty sanitoria and the dismantling of public health control systems in developed nations. consequently, by the s, when tuberculosis had re-emerged in the era of hiv/aids, local and state health departments in the united states lacked field, laboratory and clinical staff and so had to reinvent tuberculosiscontrol programmes . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with aids, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug-resistant and multidrug-resistant strains , and a need for more expensive treatment strategies such as directly observed therapy. it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. . immune deficiency associated with aids, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than % of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than million people globally , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesviruses . breakthroughs in cancer therapy and in immunosuppressive therapies used to treat immune-mediated diseases and for transplantation , can also leave patients susceptible to opportunistic infections. human organ transplantation adds a further risk of infection with undetected pathogens in donor tissues, and transplantation of animal organs introduces the risk of transmission to humans of animal microbes . the emergence of zoonotic and vector-borne diseases can also be associated with human behaviours and environmental perturbation. although west nile virus is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally . a - epidemic in hawaii -fortunately without fatalities -is a reminder that dengue has also re-emerged in locations once considered to be dengue-free. usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopictus -a vector switch of potential significance with respect to dengue re-emergence . in the americas, including many us southern states, a. albopictus has been spreading into areas where a. aegypti mosquitoes are not found, and persisting for longer seasonal periods, putting tens of millions more people at risk of dengue infection. dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue haemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high insight review articles nature | vol | july | www.nature.com/nature population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes (for example, genes for the phage-encoded cholera toxin and the toxin-coregulated pilus (tcp) factor associated with attachment), and to undergo gene transfer events that lead to new strains containing further virulence gene combinations. these result in periodic cholera emergences that cause epidemics and pandemics . thus, although the disease we know as cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts' . these can lead to deadly pandemics, as occurred in , , and (refs , ) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts' , which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral haemagglutinin site that attaches to human cells. it remains something of a mystery how zoonotic influenza viruses mix with each other and with human strains to acquire the additional properties of human virulence and human-to-human transmissibility. before , mild cases of human disease associated with avian influenza viruses were occasionally reported . these events have become more frequent, sometimes resulting in severe cases of disease and death. avian influenza has recently made dead-end jumps to humans -for example, the hong kong outbreak of the newly emergent h n influenza, the h n epidemic in the netherlands, the - h n and h n epizootics in asia and elsewhere, and occasional cases of h n disease (fig. ) . meanwhile, back-switches of human h n viruses have emerged in pigs, from which both doubly mixed (pig-human) and triply mixed (pig-human-avian) viruses , have been isolated. such enzootic/zoonotic mixing is suspected to have occurred in the influenza pandemic of - , which was caused by an h n virus with an avian-like receptor-binding site . the predicted virulence genes of this virus are now being sought from -year-old pathology specimens and from frozen corpses . the implications of interspecies genetic mixing for future influenza pandemics are troubling. although much remains speculative about how influenza viruses emerge and spread, it seems clear that the process is driven by prolific and complex viral evolution (genetic reassortment and mutational 'drift'), interspecies mixing and adaptation, and ecological factors that bring humans into contact with animals and each other. by whatever means new influenza virus pandemic strains emerge, they eventually reach a critical threshold of human transmission beyond which epidemic and pandemic spread follows mathematically predictable patterns. the dynamics and determinants of such epidemic development have been studied since the nineteenth century for several infectious diseases. for influenza, both historical and prospective epidemics have been described or predicted using deterministic and stochastic mathematical models, often with surprising accuracy when compared with actual epidemic data. more complicated mathematical models that describe how diseases spread by means other than personto-person aerosol transmission have generally been less successful in describing and predicting epidemics, but have nonetheless been helpful in planning public health responses to epidemics caused by hiv , vcjd and other diseases. mathematical modelling is also used to determine the impact of emerging epidemics. for example, it has been difficult to estimate overall influenza mortality because fatal infections are often neither diagnosed nor accurately recorded in hospital records and death certificates, especially in the elderly. recent epidemiological attempts to obtain improved influenza mortality estimates from seasonal excess mortality data have indicated that influenza mortality may be insight review articles nature | vol | july | www.nature.com/nature greater than was previously suspected, because influenza deaths are frequently coded under seemingly unrelated categories such as cardiovascular diseases. the same approaches also show that other influenza-like deaths may actually be due to other agents, such as respiratory syncytial virus (rsv), a common childhood virus that in the past decade has emerged as a major cause of adult mortality . deliberately emerging microbes are those that have been developed by man, usually for nefarious use. the term 'deliberately emerging' refers to both naturally occurring microbial agents such as anthrax , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. deliberately emerging microbes include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity . as concepts, bioterrorism and biowarfare are probably not new. the alleged catapulting of plague-ridden corpses over enemy walls in the siege of caffa (the modern crimean port of feodosia, ukraine) and the dispatch of smallpox-impregnated blankets to indians by british officers in the seven years war ( - ) have frequently been cited as examples of bioterrorism or biowarfare , . two modern attacks have been well documented. in , an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election . a anthrax attack , in which a terrorist mailed anthrax-spore-filled letters to prominent figures, including two us senators, resulted in illness in at least people and the death of five of these individuals. public alarm was elevated by the knowledge that bacillus anthracis is a common and easily obtainable enzootic and soil organism found in laboratories worldwide, and that scientific technology had increased its lethality: the spores had been weaponized by being concentrated, finely milled and packed with a dispersal agent to increase their capacity to disseminate . the united states, the united kingdom, the soviet union and other nations once had sophisticated offensive bioweapons programmes that included the production of weaponized anthrax spores . soviet scientists continued to produce large quantities of organisms adapted for biowarfare and bioterror -among them the agents of smallpox, plague, tularaemia and marburg virus -for several years after their signing of the bioweapons and toxins treaty convention in , which forbade such activities . by , the soviet programme was annually producing , tonnes of weaponized anthrax spores, packing them into warheads and other delivery devices . before the anthrax attacks , the us scientific community had for several years been bolstering its biodefence research capacity. the anthrax attacks greatly accelerated this expansion as part of a national defence plan, which includes efforts to provide a knowledge base for the development of effective countermeasures against agents of bioterror, such as diagnostics, therapeutics and vaccines, and to translate this knowledge into the production and delivery of such measures . bioterror agents have been grouped into three categories of risk . the six category a agents (anthrax, smallpox, plague, tularaemia, viral haemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. infectious diseases will continue to emerge and re-emerge, leading to unpredictable epidemics and difficult challenges to public health and to microbiology and allied sciences. surveillance and response, the key elements in controlling eis, be they naturally occurring or deliberately engineered, depend on rapid clinical diagnosis and detection and containment in populations and , which along with state and local health departments and other agencies have been making significant strides in national surveillance-response capacity. the enormous influx of us government-funded research resources (largely through the national institutes of health) and public health resources (mainly through the cdc, and state and local public health agencies) in response to the increased threat of a bioterrorist attack will fortify the response capabilities related to all eis. however, it is clear that surveillance and other activities that traditionally fall within the domain of public health are not in themselves sufficient to adequately address the problem of eis. of critical importance are basic, translational and applied research efforts to develop advanced countermeasures such as surveillance tools, diagnostic tests, vaccines and therapeutics . genomics, proteomics and advances in nanotechnology are increasingly being exploited in diagnostic, therapeutic and microbial research applications, and in rational drug and vaccine design. direct and computational structural determination , prediction of protein-protein interactions between microorganisms and drugs, and sophisticated bioinformatics techniques support research in all of the above areas. these technologies have led to numerous advances in real-world utility against eis, most notably in the development of more than antiretroviral drugs that can effectively suppress hiv replication. where they are available and properly used in hiv-infected individuals, these medications have dramatically reduced hiv morbidity and mortality . gene-and protein-based microarrays can be used to detect pathogen signals, to monitor resistance to anti-infective agents, to characterize host gene responses to recent infections, and to facilitate the development of new drugs and vaccines . basic and applied research together have provided promising new vaccine platforms, such as recombinant proteins, immunogenic peptides, naked dna vaccines, viral vectors of extraneous genes encoding immunogenic proteins (including chimaeras), replicons and pseudovirions . many novel vaccine candidates are now being developed against eis such as hiv, ebola virus, west nile virus, dengue, the sars coronavirus, tuberculosis and malaria. of particular note are novel tuberculosis vaccines that recently entered clinical trials -the first time in more than years that new approaches to vaccination for tuberculosis have been assessed in humans . chimaeric flavivirus vaccines for west nile virus, dengue and japanese encephalitis virus are effective in animal models and are in various stages of clinical testing . our growing understanding of the human immune system is also helping to accelerate vaccine development. this is especially true in the case of innate immune responses, which are evolutionarily older, less specific and faster-acting than the adaptive responses that have been the traditional targets of vaccines . as we learn more about innate immunity and its relationship with the adaptive immune system, opportunities to create more effective vaccine adjuvants will emerge. for example, synthetic dna sequences that contain repeated cpg motifs mimic the stimulatory activity that bacterial dna fragments exert on the innate immune system. these sequences show promise as vaccine adjuvants that accelerate and augment immune responses . we can anticipate more progress of this kind as we continue to delineate the complex interactions between innate and adaptive immune responses. the sequencing of the human genome, the genomes of six other animals, including the mouse, and those of microbial vectors and microbes themselves (for example, p. falciparum and its mosquito vector, anopheles gambiae), have elevated microbiology to a wholegenome level. the ability to sequence microbial genomes in a few days or less, and to examine host-vector-microbe interactions at both the genome level and at the tertiary protein structural level, will help us to understand the molecular mechanisms that underlie the pathogenesis of infectious disease and host defences, including resistance and immune evasion. these advances will facilitate the development of new countermeasures. other fertile areas of research include the use of geographical information systems and satellite imaging to support field study and epidemic prevention (for example, predicting hps and rift valley fever epidemics in indigenous areas by satellite imagery of water and vegetation related to animal reservoir and vector prevalence). underlying disease emergence are evolutionary conflicts between rapidly evolving and adapting infectious agents and their slowly evolving hosts. these are fought out in the context of accelerating environmental and human behavioural alterations that provide new ecological niches into which evolving microbes can readily fit. it is essential that broadly based prevention strategies, as well as new and improved countermeasures (that is, surveillance tools, diagnostics, therapeutics and vaccines), be continually tested, refined and upgraded, requiring a strengthened relationship between public health and basic and clinical sciences. the challenge presented by the ongoing conflict between pathogenic microorganisms and man has been well summarized by a noted champion of the war on eis, joshua lederberg: "the future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled our wits versus their genes" . the global scientific and public health communities must confront this reality not only with wit, but also with vision and sustained commitment to meet a perpetual challenge. in figure of this review, the pie chart was drawn incorrectly, with the wedge sizes not in proportion to the total size. the correct figure is shown below. asthma and chronic obstructive pulmonary disease . million factors in the emergence of infectious diseases the wordsworth encyclopedia of plague and pestilence updating the accounts: global mortality of the - "spanish" influenza pandemic centers for disease control and prevention. multistate outbreak of monkeypox -illinois the severe acute respiratory syndrome investigation of bioterrorism-related anthrax, united states, : epidemiologic findings threats to global health and survival: the growing crises of tropical infectious diseases -an "unfinished" agenda emerging infectious diseases among indigenous peoples ǼșȓȖșȕșȍȓȊ -sive, pestis nuperae apud populum londinensem grassantis narratio historica epidemiology of the plague of athens the columbian exchange: biological and 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mitgetheilt und eingeleitet smallpox and the indians in the american colonies a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars biodefence on the research agenda: the world needs new and creative ways to counter bioterrorism threats in bioterrorism: i. cdc category a agents world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) preventing emerging infectious diseases: a strategy for the st century (department of health and human services protein structure prediction and structural genomics guidelines for using antiretroviral agents among hiv-infected adults and adolescents new technology platforms in the development of vaccines for the future the jordan report: th anniversary adjuvants of immunity: harnessing innate immunity to promote adaptive immunity recent advances in the discovery and delivery of vaccine adjuvants the value of complete microbial genome sequencing (you get what you pay for) the authors thank r. m. krause for helpful discussions, and j. weddle for graphic design. the authors declare that they have no competing financial interests.insight review articles key: cord- -wawui fd authors: tulchinsky, theodore h.; varavikova, elena a. title: communicable diseases date: - - journal: the new public health doi: . /b - - / - sha: doc_id: cord_uid: wawui fd publisher summary in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. public health has addressed the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. important for all health providers and public health personnel so as to be able to cope with the scale of these problems and to absorb new technologies as they emerge from scientific advances and experience, and their successful application. lived. it was to be observed, indeed, that it did not come straight on toward us; for the city, that is to say within the walls, was indifferently healthy still; nor was it got over the water into southwark; for though there died that week , of all distempers, whereof it might be supposed above died of the plague, yet there was but in southwark, lambeth parish included; whereas in the parishes of st. giles the agent-host-environment triad, discussed in chapter , is fundamental to the success of understanding transmission of infectious diseases and their control, including those well known, those changing their patterns, and those newly emerging or escaping current methods of control. infection occurs when the organism successfully invades the host body, where it multiplies and produces an illness. a host is a person or other living animal, including birds and arthropods, who provides a place for growth and sustenance to an infectious agent under natural, as opposed to experimental, conditions. some organisms, such as protozoa or helminths, may pass successive stages of their life cycle in different hosts, but the primary or definitive host is the one in which the organism passes its sexual stage. the secondary or intermediate host is where the parasite passes the larval or asexual stage. a transport host is a carrier in which the organism remains alive, but does not develop. an agent of an infectious disease is necessary, but not always sufficient to cause a disease or disorder. the infective dose is the quantity of the organism needed to cause clinical disease. a disease may have a single agent as a cause, or it may occur as a result of the agent in company with contributory factors, whose presence is also essential for the development of the disease. a disease may be present in an infected person in a dormant form such as tuberculosis, or a subclinical form, such as poliomyelitis or hiv. the virulence or pathogenicity of an infective agent is the capacity of an infectious agent to enter the host, replicate, damage tissue, and cause disease in an exposed and susceptible host. virulence is indicated by the severity of clinical disease and case fatality rates. the environment provides a reservoir for the organism, and the mode of transmission, by which the organism reaches a new host. the reservoir is the natural habitat where an infectious agent lives and multiplies, from which it can be transmitted directly or indirectly to a new host. the reservoir refers to the natural habitat of the organism, which may be in people, animals, arthropods, plants, soil, or substances in which an organism normally lives and multiplies, and on which it depends for survival or in which it survives in a dormant form. contacts are persons or animals who have been in association with an infected person, animal, or contaminated inanimate object, or environment that might provide an opportunity for acquiring the infective agent. persons or animals that harbor a specific infectious agent, often in the absence of discernible clinical disease, and who serve as a source of infection or contamination of food, water, or other materials, are carriers. a carrier may have an inapparent infection (a healthy cartier) or may be in the incubation or convalescent stage of the infection. communicable diseases may be classified by a variety of methods: by organism, by mode of transmission, by methods of prevention (e.g., vaccine preventable, vector controllable), or by major organism classification, that is, viral, bacterial, and parasitic disease. a virus is a nucleic acid molecule (rna or dna) encapsulated in a protein coat or capsid. the virus is not a complete cell and can only replicate inside a complete cell. the capsid may have a protective envelope of a lipid containing membrane. the capsid and membrane facilitate attachment and penetration of a host cell. inside the host cell, the nucleic molecule may cause the cell's chromosomes to be changed in its own genetic material or so that there is cellular manufacture and virus replication. viroids are smaller rna structures without capsids which can cause plant disease. prions are recently discovered (stanley prusiner, nobel prize, ) variants of viruses or viroids which are the infective agents cause of scrapie in sheep, and similar degenerative central nervous system diseases in cattle and in man (mad cow disease or creutzfeld-jakob disease in humans). bacteria are unicellular organisms that reproduce sexually or asexually, grow on cell-free media, and can exist in an environment with oxygen (aerobic) or in one lacking oxygen (anaerobic). some may enter a dormant state and form spores where they are protected from the environment and may remain viable for years. bacteria include a nucleus of chromosomal dna material within a membrane surrounded by cytoplasm, itself enclosed by the cellular membrane. bacteria are often characterized by their coloration under gram's stain, as gram-negative or gram-positive, as well as by their microscopic morphology, colony patterns on growth media, by the diseases they may cause, as well as by antibody and molecular (dna) marking techniques. bacteria include both indigenous flora (normal resident) bacteria and pathogenic (disease causing) bacteria. pathogenic bacteria cause disease by invading, overcoming natural or acquired resistance, and multiplying in the body. bacteria may produce a toxin or poison that can affect a body site distant from where the bacterial replication occurs, such as in tetanus. bacteria may also initiate an excessive immune response, producing damage to other body tissues away from the site of infection, e.g;, acute rheumatic fever and glomerulonephritis. parasitology studies protozoa, helminths, and arthropods that live within, on, or at the expense of a host. these include oxygen-producing, flagellate, unicellular organisms such as giardia and trichomonas, and amoebas such as entamoeba important in enteric and gynecologic disorders. sporozoa are parasites with complex life cycles in different hosts, such as cryptosporidium or malarial parasites. parasitic disease usually refers to infestation, with fungi, molds, and yeasts that can affect humans. helminths are worms that infest humans especially in poor sanitation and tropical areas. transmission of diseases is by the spread of an infectious agent from a source or reservoir to a person (table . ). direct transmission from one host to another occurs during touching, biting, kissing, sexual intercourse, and projection via droplets, as in sneezing, coughing, or spitting, or by entry through the skin. indirect transmission includes via aerosols of long-lasting suspended particles in air, fecal-oral transmission such as food and waterborne as well as by poor hygenic conditions with inanimate materials, such as soiled clothes, handkerchiefs, toys, or other objects. vector-borne diseases are transmitted via crawling or flying insects, in some cases with multiplication, and development of the organism in the vector, as in malaria. the subsequent transmission to humans is by injection of salivary gland fluid during biting, e.g., congenital syphilis, or by deposition of feces, urine or other material capable of penetrating the skin through a bite wound or other trauma. transmission may occur with insects as a transport mechanism, as in salmonella on the legs of a housefly. airborne transmission occurs inderectly via infective organisms in small aerosols that may remain suspended for long periods of time and which easily enter the respiratory tract. small particles of dust may spread organisms from soil, clothing, or bedding. vertical transmission occurs from one generation to another, or from one stage of the insect life cycle to another stage. maternal-infant transmission occurs during pregnancy (transplacental), delivery, as in gonorrhoea, breast-feeding, e.g., hiv, with transfer of infectious agents from mother to fetus or newborn. resistance to infectious diseases is related to many host and environmental factors, including age, sex, pregnancy, nutrition, trauma, fatigue, living and socioeconomic conditions, and emotional status. good nutritional status has a protective effect against the results of an infection. vitamin a supplements reduce complication rates of measles and enteric infections. tuberculosis may be present in an individual whose resistance is sufficient to prevent clinical disease, but the infected person is a cartier of an organism which can be transmitted to another or cause clinical disease if the person's susceptibility is reduced. immunity is resistance to infection resulting from presence of antibodies or cells that specifically act on the microorganism associated with a specific disease or toxin. immunity to a specific organism can be acquired by having the disease, that is, natural immunity, or by immunization, active or passive, or by protection box . vaccines and prevention "the greeks had two gods of health, aesculapius and hygeia, therapy and prevention, respectively. medicine in the twentieth century retains those two concepts, and vaccination is a powerful means of prevention. what follows is information on the vaccines that together with sanitation, make modem society possible, and that if wisely used will continue to bestow on mankind the gift of prevention, which according to proverb is worth far more than cure." source: plotkin, s. a., mortimer, e. a. . vaccines. second edition. philadelphia: wb saunders (with permission). infectious agent: a pathogenic organism (e.g., virus, bacteria, rickettsia, fungus, protozoa, or helminth) capable of producing infection or an infectious disease. infection: the process of entry, development, and proliferation of an infectious agent in the body tissue of a living organism (human, animal, or plant) overcoming body defense mechanisms, resulting in an inapparent or clinically manifest disease. antigen: a substance (e.g., protein, polysaccharide) capable of inducing specific response mechanisms in the body. an antigen may be introduced into the body by invasion of an infectious agent, by immunization, inhalation, ingestion, or through the skin, wounds, or via transplantation. antibody: a protein molecule formed by the body in response to a foreign substance (an antigen) or acquired by passive transfer. antibodies bind to the specific antigen that elicits its production, causing the infective agent to be susceptible to immune defense mechanisms against infections e.g., humoral and cellular. immunoglobulins: antibodies that meet different types of antigenic challenges. they are present in blood or other body fluids, and can cross from a mother to fetus in utero, providing protection during part of the first year of life. there are five major classes (igg, igm, iga, igd, and ige) and subclasses based on molecular weight. anfisera or antitoxin: materials prepared in animals for use in passive immunization against infection or toxins. source: jawetz, melrick, and adelberg, medical microbiology, . through elimination of circulation of the organism in the community. immunity may be by antibodies produced by the host body or transferred from externally produced antibodies. the body also reacts to infective antigens by cellular responses, including those that directly defend against invading organisms and other cells which produce antibodies. the immune response is the resistance of a body to specific infectious organisms or their toxins provided by a complex interaction of antibodies and cells including a. b cells (bone marrow and spleen) produce antibodies which circulate in the blood, i.e., humoral immunity; b. t cell-mediated immunity is provided by sensitization of lymphocytes of thymus origin to mature into cytotoxic cells capable of destroying virusinfected or foreign cells; c. complement, a humoral response which causes lysis of foreign cells; d. phagocytosis, a cellular mechanism which ingests foreign microorganisms (macrophages and leukocytes). surveillance of disease is the continuous scrutiny of all aspects of occurrence and spread of disease pertinent to effective control of that disease. maintaining ongoing surveillance is one of the basic duties of a public health system, and is vital to the control of communicable disease, providing the essential data for tracking of disease, planning interventions, and responding to future disease challenges. surveillance of infectious disease incidence relies on reports of notifiable diseases by physicians, supplemented by individual and summary reports of public health laboratories. such a system must concern itself with the completeness and quality of reporting and potential errors and artifacts. quality is maintained by seeking clinical and laboratory support to confirm first reports. completeness, rapidity, and quality of reporting by physicians and laboratories should be emphasized in undergraduate and postgraduate medical education. enforcement of legal sanctions may be needed where standards are not met. surveillance of infectious diseases includes the following: . morbidity reports from clinics to public health offices; . mortality reports from attending doctors to vital records; . reports from selected sentinel centers; . special field investigations of epidemics or individual cases; . laboratory monitoring of infectious agents in population samples; . data on supply, use, and side effects of vaccines, toxoids, immune globulins; . data on vector control activities such as insecticides use; . immunity levels in samples of the population at risk; . review of current literature on the disease; . epidemiologic and clinical reports from other jurisdictions. epidemiologic monitoring based on individual and aggregated reports of infectious diseases provide data vital to planning interventions at the community level or for the individually exposed patient and his contacts, along with other information sources such as hospital discharge data and monitoring of sentinel centers. these may be specific medical or community sites that are representative of the population and are able to provide good levels of reporting to monitor an area or population group. a sentinel center can be a pediatric practice site, a hospital emergency room, or other location which will provide a "finger on the pulse" to assess the degree and kind of morbidity occurring in the community. it can also include monitoring in a location previously known for disease transmission, such as hong kong in relation to influenza. epidemiologic analysis provided by government public health agencies should be published weekly, monthly, and annually and distributed to a wide audience of public health and health-related professionals throughout the country. feedback to those in the field on whose initial reports the data are based is vital in order to promote involvement and improved quality of data, as well as to allow evaluation of the local situation in comparison to other areas. in a federal system of government, national agencies report regularly on all state or provincial health patterns. state or provincial health authorities provide data to the counties and cities in their jurisdictions. such data should also be readily available to researchers in other government agencies, universities, and other academic settings for further research and analysis both on internet and hard-copy publications. notifiable diseases are those which a physician is legally required to report to state or local public health officials, by reason of their contagiousness, severity, frequency, or other public health importance (table . ). public health laboratory services provide validation of clinical and epidemiologic reports. they also pro- vide day-to-day supervision of public health conditions, and can monitor communicable disease and vaccine efficacy and coverage. in addition, they support standards of clinical laboratories in biochemistry, microbiology, and genetic screening. nosocomial or hospital-acquired infections constitute a major health hazard associated with care in institutions. in the united states, they occur in - % of hospital admissions and are the cause of lengthening of hospital stay and an estimated , deaths per year. in developing countries, nosocomial infection rates may occur in up to % of hospitalizations. this category of infectious disease most commonly includes infections of the urinary tract, surgical wounds, lower respiratory tract (pneumonias), and blood poisoning or septicemias. in the united states, up to % of hospital-acquired infections are caused by multidrug resistant organisms. staphylococcus infections resistant to many current antibiotics, for example, methicillin and vancomycin, are a notable cause of prolongation of hospitalization or even death. the increasing number of immunodeficient patients has increased the importance of prevention of nosocomial infections. where standards of infection control are lacking, in both developed and developing countries, hospital staff are vulnerable to serious infection. in developing countries, deadly new viruses, such as ebola and marburg viruses mainly affect nursing, medical, and other staff as secondary cases. surveillance and control measures are important elements of hospital management. hospital epidemiologists and infection control staff are part of modem hospital staffing. the cost to the health system of nosocomial infections is a major consideration in planning health budgets. reducing the risk of acquiring such infections in hospital justifies substantial expenditures for hospital epidemiology and infection control activities. with diagnostic related group payment for hospital care (by diagnosis rather than by days of stay) the good manager has a major incentive to ensure that the risk of nosocomial infections is minimized, since they can greatly prolong hospital stays, raising patient dissatisfaction and health care costs. an endemic disease is the constant usual presence of a disease or infectious agent in a given geographic area or population group. hyperendemic is a state of persistence of high levels of incidence of the disease. holoendemic means that the disease appears early in life and affects most of the population, as in malaria or hepatitis a and b in some regions. an epidemic is the occurrence in a community or region of a number of cases of an illness in excess of the usual or expected number of cases. the number of cases constituting an epidemic varies with the disease, and factors such as previous epidemiological patterns of the disease, time and place of the occurrence, and the population involved must be taken into account. a single case of a disease long absent from an area, such as polio, constitutes an epidemic, and therefore a public health emergency because a clinical case may represent a hundred carriers with nonparalytic or subclinical poliomyelitis. in the s, two to three or more cases of measles linked in time and place may be considered sufficient evidence of transmission and presumed to be an epidemic. a pandemic is occurrence of a disease over a very wide area, crossing international boundaries, affecting a large proportion of the population. each epidemic should be regarded as a unique natural experiment. the investigation of an epidemic requires preparation and field investigation in conjunction with local health and other relevant authorities. verification of cases and the scope of the epidemic will require case definition and laboratory confirmation. tabulation of known cases according to time, place, and person are important for immediate control measures and formulation of the hypothesis as to the nature of the epidemic. an epidemic curve is a graphic plotting of the distribution of cases by the time of onset or reporting, which gives a picture of the timing, spread, and extent of the disease from the time of the initial index cases and the secondary spread. epidemic investigation requires a series of steps. this starts with confirmation of the initial report and preliminary investigation, defining who is affected, determining the nature of the illness and confirming the clinical diagnosis, and recording when and where the first (index) and follow-up (secondary) cases occurred, and how the disease was transmitted. samples are taken from index case patients (e.g., blood, feces, throat swabs) as well as from possible vectors (e.g., food, water, sewage, environment). a working hypothesis is established based on the first findings, taking into account all plausible explanations. the epidemic pattern is studied, establishing common source or risk factors, such as food, water, contact, environment, and drawing a time line of cases to define the epidemic curve. how many are ill (the numerator) and what is the population at risk (the denominator) establish the attack rate, namely, the percentage of sick among those exposed to the common factor. what is a reasonable explanation of the occurrence; is there a previous pattern, with the present episode a recurrence or new event? consultation with colleagues and the literature helps to establish both a biological and epidemiologic plausibility. what steps are needed to prevent spread and recurrence of the disease? coordination with relevant health and other officials and providers is required to establish surveillance and control systems, document and distribute reports, and respond to the public's fight to know. the first reports of excess cases may come from a medical clinic or hospital. the initial (sentinel or index) cases provide the first clues that may point to a common source. investigation of an epidemic is designed to quickly elucidate the cause and points of potential intervention to stop its continuation. this requires skilled investigation and interpretation. epidemiologic investigations have defined many public health problems. rubella syndrome, legionnaire's disease, aids, and lyme and hantavirus diseases were first identified clinically when unusually large numbers of cases appeared with common features. the suspicions that were raised led to a search for causes and the identification of control methods. a working hypothesis of the nature of an epidemic is developed based on the initial assessment, the type of presentation, the condition involved, and previous local, regional, national, and international experience. the hypothesis provides the basis for further investigation, control measures, and planning additional clinical and laboratory studies. surveillance will then monitor the effectiveness of control measures. communication of findings to local, regional, national, and international health reporting systems is important for sharing the knowledge with other potential support groups or other areas where similar epidemics may occur. the centers for disease control and prevention (cdc), originally organized in as the office for malaria control in war areas, is part of the u.s. public health service. as of , the cdc had a budget of $ . billion, and employees include epidemiologists, microbiologists, and many other professionals. the cdc includes national centers for environmental health and injury control, chronic disease prevention and health promotion, infectious diseases, prevention services, health statistics, occupational safety and health, and international health. the epidemic intelligence service (eis) of the cdc in the united states is an excellent model for the organization of the national control of communicable diseases. young clinicians are trained to carry out epidemiologic investigations as part of training to become public health professionals. eis officers are assigned to state health departments, other public health units, and research centers as part of their training, carrying out epidemic investigation and special tasks in disease control. the cdc, in cooperation with the who, has developed and offers free of charge, a personal computer program to support field epidemiology, including epidemic investigations (epi-info), which can be accessed and down-loaded from the worldwide web. this program should be adopted widely in order to improve field investigations, to encourage reporting in real time, and to develop high standards in this discipline. cdc's morbidity and mortality weekly report (mmwr) is a weekly publication of the cdc's epidemiologic data, also available free on the internet. it includes special summaries of reportable infectious diseases as well as noncom- although an infectious disease is an event affecting an individual, it is communicable to others, and therefore its control requires both individual and community measures of protection. control of the disease is a reduction in its incidence, prevalence, morbidity, and mortality. elimination of a disease in a specified geographic area may be achieved as a result of intervention programs such as individual protection against tetanus; elimination of infections such as measles requires stoppage of circulation of the organism. eradication is success in reduction to zero of incidence of the disease and presence in nature of the organism, such as with smallpox. extinction means that a specific organism no longer exists in nature or in laboratories. public health applies a wide variety of tools for the prevention of infectious diseases and their transmission. it includes activities ranging from filtration and disinfection of community drinking water to environmental vector control, pasteurization of milk, and immunization programs (see table . ). no less important are organized programs to promote self protection, case finding, and effective treatment of infections to stop their spread to other susceptible persons (e.g., hiv, sexually transmitted diseases, tuberculosis, malaria). planning measures to control and eradicate specific communicable diseases is one of the principal activities of public health and remains so for the twenty-first century. treating an infection once it has occurred is vital to the control of a communicable disease. each person infected may become a vector and continue the chain of transmission. successful treatment of the infected person reduces the potential for an uninfected contact person to acquire the infection. bacteriostatic agents or drugs such as sulfonamides inhibit growth or stop replication of the organism, allowing normal body defenses to overcome the organism. bacteriocidal drugs such as penicillin act to kill pathogenic organisms. traditional medical emphasis on single antibiotics has changed to use of multiple drug combinations for tuberculosis and more recently for hospital-acquired infections. antibiotics have made enormous contributions to clinical medicine and public health. however, pathogenic organisms are able to adapt or mutate and develop resistance to antibiotics, resulting in drug resistance. wide-scale use of antibiotics has led to increasing incidence of resistant organisms. multidrug resistance constitutes one of the major public health challenges at the end of the twentieth century. antiviral agents (e.g., ribovarin) are important additions to medical treatment potential, as are "cocktails" of antiviral agents for management of hiv infection. antibiotic use is a health problem requiting attention of clinicians and their teachers as well as the public health community and health care managers, representing the interaction of health issues across the entire spectrum of services. organized public health services are responsible for advocating legislation and for regulating and monitoring programs to prevent infectious disease occurrence and/or spread. they function to educate the population in measures to reduce or prevent the spread of disease. health promotion is one of the most essential instruments of infectious disease control. it promotes compliance and community support of preventive measures. these include personal hygiene and safe handling of water, milk, and food supplies. in sexually transmitted diseases, health education is the major method of prevention. each of the infectious diseases or groups of infectious diseases have one or more preventive or control approaches (table . ). these may involve the coordinated intervention of different disciplines and modalities, including epidemiologic monitoring, laboratory confirmation, environmental measures, immunization, and health education. this requires teamwork and organized collaboration. very great progress has been made in infectious disease control by clinical, public health, and societal means since in the industrialized countries and since the s in the developing world. this is attributable to a variety of factors, including organized public health services; the rapid development and wide use of new and improved vaccines and antibiotics; better access to health care; and improved sanitation, living conditions, and nutrition. triumphs have been achieved in the eradication of smallpox and in the increasing control of other vaccine-preventable diseases. however, there remain serious problems with tb, stds, malaria, and new infections such as hiv, and an increase in multiple drug-resistant organisms. vaccines are one of the most important tools of public health in the control of infectious diseases, especially for child health. vaccine-preventable diseases ta b l e . annual incidence of selected vaccine-preventable infectious diseases in rates per , population selected years, united states, - disease the body responds to invasion of disease-causing organisms by antigenantibody reactions and cellular responses. together, these act to restrain or destroy the disease-causing potential. strengthening this defense mechanism through im-box . definitions of immunizing agents and processes vaccines: a suspension of live or killed microorganisms or antigenic portion of those agents presented to a potential host to induce immunity to prevent the specific disease caused by that organism. preparation of vaccines may be from: a. live attenuated organisms which have been passed repeatedly in tissue culture or chick embryos so that they have lost their capacity to cause disease but retain an ability to induce antibody response, such as polio-sabin, measles, rubella, mumps, yellow fever, bcg, typhoid, and plague. b. inactivated or killed organisms which have been killed by heat or chemicals but retain an ability to induce antibody response; they are generally safe but less efficacious than live vaccines and require multiple doses, such as polio-salk, influenza, rabies, and japanese encephalitis. c. cellular fractions usually of a polysaccharide fraction of the cell wall of a disease-causing organisms, such as pneumococcal pneumonia or meningococcal meningitis. d. recombinant vaccines produced by recombinant dna methods in which specific dna sequences are inserted by molecular engineering techniques, such as dna sequences spliced to vaccinia virus grown in cell culture to produce influenza and hepatitis b vaccines. toxoids or antisera: modified toxins are made nontoxic to stimulate formation of an antitoxin, such as tetanus, diphtheria, botulism, gas gangrene, and snake and scorpion venom. immune globulin: an antibody-containing solution derived from immunized animals or human blood plasma, used primarily for short-term passive immunization, e.g., rabies, for immunocompromised persons. antitoxin: an antibody derived from serum of animals after stimulation with specific antigens and used to provide passive immunity, e.g., tetanus. munization is one of the outstanding achievements of public health, as treatment of infectious diseases by antimicrobials is a major element of clinical medicine. immunization (vaccination) is a process used to increase host resistance to specific microorganisms to prevent them from causing disease. it induces primary and secondary responses in the human or animal body: a. primary response occurs on first exposure to an antigen. after a lag or latent period of - days (depending on the antigen) specific antibodies appear in the blood. antibody production ceases after several weeks but memory cells that can recognize the antigen and respond to it remain ready to respond to a further challenge by the same antigen. b. secondary (booster) response is the response to a second and subsequent exposure to an antigen. the lag period is shorter than the primary response, the peak is higher and lasts longer. the antibodies produced have a higher affinity for the antigen, and a much smaller dose of the antigen is required to initiate a response. c. immunologic memory exists even when circulating antibodies are insufficient to protect against the antigen. when the body is exposed to the same antigen again, it responds by rapidly producing high levels of antibody to destroy the antigen before it can replicate and cause disease. immunization protects susceptible individuals from communicable disease by administration of a living modified agent, or subunit of the agent, a suspension of killed organisms or an inactivated toxin (see table . ) to stimulate development of antibodies to that agent. in disease control, individual immunity may also protect another individual. herd immunity occurs when sufficient persons are protected (naturally or by immunization) against a specific infectious disease reducing circulation of the organism, thereby lowering the chance of an unprotected person to become infected. each pathogen has different characteristics of infectivity, and therefore different levels of herd immunity are required to protect the nonimmune individual. the critical proportion of a population that must be immunized in order to interrupt local circulation of the organism varies from disease to disease. eradication of smallpox was achieved with approximately % world coverage, followed by concentration on new case findings and immunization of contacts and surrounding communities. for highly infectious diseases, such as measles, immunization coverage of over % is needed to achieve local eradication. immunization coverage in a community must be monitored in order to gauge the extent of protection and need for program modification to achieve targets of disease control. immunization coverage is expressed as a proportion in which the numerator is the number of persons in the target group immunized at a specific age, and the denominator is the number of persons in the target cohort who should have been immunized according to the accepted standard: vaccine coverage = no. persons immunized in specific age group • no. persons in the age group during that year immunization coverage in the united states is regularly monitered by the national immunization survey by a household survey in all states, as well as selected urban areas considered to be at high risk for undervaccination. an initial telephone survey is followed by confirmation, where possible, from documentation from the parents or health care providers. the survey for july -june examined children born between august and november (i.e., aged - months, median age months). the results show improving coverage, with % having received three or more doses of dpt (diphtheria, pertussis, and tetanus), % with three or more doses of opv (oral polio vaccine), % with three or more doses of haemophilus influenzae, type b (hib), but only % with three or more doses of hepatitis b. however, only % had received all recommended vaccines at the recommended ages. eases that still cause millions of deaths globally each year. other important infectious diseases are still not subject to vaccine control because of difficulties in their development. in some cases, a microorganism can mutate with changes. viruses can undergo antigenic shifts in the molecular structure in the organism, producing completely new subtypes of the organism. hosts previously exposed to other strains may have little or no immunity to the new strains. antigenic drift refers to relatively minor antigenic changes which occur in viruses. this is responsible for frequent epidemics. antigenic shift is believed to explain the occurrence of new strains of influenza virus necessitating, for example, annual reformulation of the influenza vaccine associated with large scale epidemics and pandemics. new variants of poliovirus strains are similar enough to the three main types so that immunity to one strain is carded over to the new strain. molecular epidemiology is a powerful new technique used to specify the geographic origin of organisms such as poliomyelitis and measles viruses, permiting tracking of the source of the virus and epidemic. combinations of more than one vaccine is now common practice with a trend to enlarging the cocktail of vaccines in order to minimize the number of injections, and visits required. this reduces the number of visits to carry out routine immunization saving staff time and costs, as well as increasing compliance. there are virtually no contraindications to use of multiple antigens simultaneously. examples of vaccine cocktails include dpt (diphtheria, pertussis, and tetanus) in combination with haemophilus influenzae b, poliomyelitis, and varicella, or mmr (measles, mumps, and rubella) vaccines. interventions in the form of effective vaccines save millions of lives each year and contribute to improved health of countless children and adults throughout the world. vaccination is now accepted as one of the most cost-effective health interventions currently available. continuous policy review is needed regarding allocation of adequate resources, logistical organization, and continued scientific effort to seek effective, safe, and inexpensive vaccines for other important diseases such as malaria and hiv. new technology of recombinant vaccines, such as that of hepatitis b, holds promise for important vaccine breakthroughs in the decades ahead. internationally, much progress was made in the s in the control of vaccinepreventable diseases. at the end of the s, fewer than % of the world's children were being immunized. who, unicef, and other international organizations mobilized to promote an expanded programme on immunization (epi) with a target of reaching % coverage by . immunization coverage increased in the developing countries, preventing some million child deaths annually. bacillus calmette-gu rin (bcg) coverage rose from to %; poliomyelitis with opv (three doses) from to %, and tetanus toxoid for pregnant women from to %. since , there has been a decline in coverage in some parts of the world, mainly in sub-saharan africa. the challenge remains to achieve control or eradication of vaccine-preventable diseases, thus saving millions of more lives. part of the hfa stresses the epi approach, which includes immunization against diphtheria, pertussis, tetanus, po-liomyelitis, measles, and tuberculosis. an extended form of this is the epi plus program which combines epi with immunization against hepatitis b and yellow fever and, where appropriate, supplementation with vitamin a and iodine. the success in international eradication of smallpox is now being followed by a campaign to eradicate poliomyelitis and other important infectious diseases. diphtheria. diphtheria is an acute bacterial disease of the tonsils, nasopharynx, and larynx caused by the organism corynebacterium diphtheriae. it occurs in colder months in temperate climates where the organism is present in human hosts and is spread by contact with patients or carriers. it has an incubation period of - days. in the past, this was primarily an infection of children and was a major contributor to child mortality in the prevaccine and preantibiotic eras. diphtheria has been virtually eliminated in countries with well-established immunization programs. in the s, an outbreak of diphtheria occurred in the countries of the former soviet union among people over age . it reached epidemic proportions in the s, with , cases ( - ) with deaths in in russia alone. this indicates a failure of the vaccination program in several respects: it used only three doses of dpt in infancy; no boosters were given at school age or subsequently; the efficacy of diphtheria vaccine may have been low, and coverage was below %. efforts to control the present epidemic include mass vaccination campaigns for persons over years of age with a single dose of dt (diphtheria and tetanus) and increasing coverage of routine dpt vaccines to four doses by age years. the epidemic and its control measures have led to improved coverage with dt for those over years, and % coverage among children aged - months. who recommends three doses of dpt in the first year of life and a booster at school entry. this is considered by many to be insufficient to produce long-lasting immunity. the united states and other industrialized countries use a four-dose schedule and recommend periodic boosters for adults with dt. pertussis. pertussis is an acute bacterial disease of the respiratory tract caused by the bacillus bordetella pertussis. after an initial coldlike (catarrhal) stage, the patient develops a severe cough which comes in spasms (paroxysms). the disease can last - months. the paroxysms can become violent and may be followed by a characteristic crowing or high pitched inspiratory whooping sound, followed by expulsion of a tenacious clear sputum, often followed by vomiting. in poorly immunized populations and those with malnutrition, pneumonia often follows and death is common. pertussis declined dramatically in the industrialized countries as a result of widespread coverage with dpt. however, because the pertussis component of the vaccine caused some reactions, many physicians avoided its use, using dt alone. during the s in the united kingdom, many physicians recommended against vaccination with dpt. as a result, pertussis incidence increased with substantial mortality rates. this led to a reappraisal of the immunization program, with insti-tution of incentive payments to general practitioners for completion of vaccination schedules. as a result of these measures, vaccination coverage, with resulting pertussis control, improved dramatically in the united kingdom. pertussis continues to be a public health threat and recurs wherever there is inadequate immunization in infancy. a new acellular vaccine is ready for widespread use and will be safer with fewer and less severe reactions in infants, increasing the potential for improved confidence and support for routine vaccination. use of the new vaccine is spreading in the united states and forms part of the u.s. recommended vaccination schedule. tetanus. tetanus is an acute disease caused by an exotoxin of the tetanus bacillus (clostridium tetani) which grows anaerobically at the site of an injury. the bacillus is universally present in the environment and enters the human body via penetrating injuries. following an incubation period of - days, it causes an acute condition of painful muscular contractions. unless there is modem medical care available, patients are at risk of high case fatality rates of - % (highest in infants and the elderly). antitetanus serum (ats) was discovered in and during world war i, ats contributed to saving the lives of many thousands of wounded soldiers. tetanus toxoid was developed in . the organism, because of its universal presence in the environment, cannot be eradicated. however, the disease can be controlled by effective immunization of every child during infancy and school age. adults should receive routine boosters of tetanus toxoid once very decade. newborns are infected by tetanus spores (tetanus neonatorum) where unsanitary conditions or practices are present. it can occur when traditional birth attendants at home deliveries use unclean instruments to sever the umbilical cord, or dress the severed cord with contaminated material. tetanus neonatorum remains a serious public health problem in developing countries. immunization of pregnant women and women of childbearing age is reducing the problem by conferring passive immunity to the newborn. the training of traditional birth attendants in hygienic practice and the use of medically supervised birth centers for delivery also decreases the incidence of tetanus neonastorum. elimination of tetanus neonatorum by the year was made a health target by the world summit of children in . in that year, the number of deaths from neonatal tetanus was reported by unicef as , infants worldwide, declining to , in . immunization of pregnant women increased from under % in to % in - . despite progress, coverage is still too low to achieve the target of elimination. poliomyelitis. polio virus infection may be asymptomatic or cause an acute nonspecific febrile illness. it may reach more severe forms of aseptic meningitis and acute flaccid paralysis with long-term residual paralysis or death during the acute phase. poliomyelitis is transmitted mainly by direct person-to-person contact, but also via sewage contamination. large-scale epidemics of disease, with attendant paralysis and death, occurred in industrialized countries in the s and s, engendering widespread fear and panic and thousands of clinical cases of "infantile paralysis". growth of the poliovirus by john enders and colleagues in tissue culture in led to development of the first inactivated polio vaccine by jonas salk in the mid- s and gave hope and considerable success in the control of the disease. the development of the live attenuated oral poliomyelitis vaccine by albert sabin, licensed in , added a new dimension to its control because of the effectiveness, low cost, and ease of administration of the vaccine. the two vaccines in their more modern forms, enhanced strength inactivated polio vaccine (eipv), and triple oral polio vaccine (topv), have been used in different settings with great success. oral polio vaccine (opv) induces both humoral and cellular, including intestinal, immunity. the presence of opv in the environment by contact with immunized infants and via excreta of immunized persons in the sewage gives a booster effect in the community. immunization using opv, in both routine and national immunization days (nids) has proven effective in dramatically reducing poliomyelitis and circulation of the wild virus in many parts of the world. use of the enhanced strength ipv (eipv) produces early and high levels of circulating antibodies, as well as protecting against the vaccine-associated disease. in rare cases opv can cause vaccine-associated paralytic poliomyelitis (vapp), with a risk of case per , with initial doses, and case per over million with subsequent doses. approximately eight to ten cases of vapp occur annually in the united states, with clinical, ethical, and legal implications. use of ipv as initial protection eliminates this problem. experience in gaza and the west bank in the s and s, and later in israel, showed that a combination of ipv and opv is effective in overcoming endemic and imported poliovirus. opv requires multiple doses to achieve protective antibody levels. where there are many enteroviruses in the environment, as is the case in most developing countries, interference in the uptake of opv may result in cases of paralytic poliomeylitis among persons who have received or even doses of adequate opv. controversy as to the relative advantages of each vaccine continues. the opv program of mass repeated vaccination in control of poliomyelitis in the americas established the primacy of opv in practical public health, and the momentum to eradicate poliomyelitis is building. a combined schedule of ipv and opv would eliminate the wild virus and protect against vaccine-associated disease. the sequential use of ipv and opv was adopted as part of the routine infant immunization program in the united states in , but ipv alone was adopted in . there are concerns that exclusive use of either vaccine alone will not lead to the desired goal of eradication of polyomyelitis. progress in global eradication of polio has been impressive. global coverage of infants with three doses of opv reached % in as compared to % in . the african region of who had an increase in opv coverage from % in to % in . national immunization days (nids) were conducted in countries in and in , covering million children in . mopping up operations to reinforce coverage of children in still endemic areas is proceeding along with increased emphasis on acute flaccid paralysis (afp) monitoring. confirmed polio cases reported continued at - , per year in - . with continued national and international emphasis, and support of who, rotary international, unicef, donor countries, and others, there is a real prospect of a world without polio, if not by the year , then or shortly thereafter. measles is an acute disease caused by a virus of the paramyxovirus family. it is highly infectious with a very high ratio of clinical to subclinical case ratio ( / ). measles has a characteristic clinical presentation with fever, white spots (koplik spots) on the membranes of the mouth, and a red blotchy rash appearing on the rd- th day lasting - days. mortality rates are high in young children with compromised nutritional status, especially vitamin a deficiency. the measles virus evolved from a virus disease of cattle (rinderpest) some - years ago, becoming an important disease of humans with high mortality rates in debilitated, poorly nourished children, and significant mortality and morbidity even in industrialized countries. in the prevaccine era, measles was endemic worldwide, and even in the late s it remains one of the major childhood infectious diseases. it is one of the commonest causes of death for school age children worldwide. despite earlier predictions that measles deaths would be halved to , by , who reported . million measles deaths in that year and over million in . eradication in the first decade of the next century is a feasible goal, provided that there is an adequate international effort. measles immunization increased from under % worldwide in to % in - , but % in sub-saharan africa. single-dose immunization failed to meet control or eradication requirements even in the most developed parts of the world. a live vaccine, licensed in , was later replace by a more effective and heat stable vaccine, but still with a primary vaccination failure rate (i.e., fails to produce protective antibodies) of - %, and secondary failure rate (i.e., produces antibodies but protection is lost over time) of %. a two-dose policy incorporates a booster dose, usually at school-age, in addition to maximum feasible infant coverage of children in the - month period (timing varies in different countries). catch-up campaigns among schoolage children should be carried out until the routine two-dose policy has time to take full effect. nearly universal primary education in developing countries, offers an opportunity for mass coverage of school age children with a second dose of measles and a resulting increase of herd immunity to reduce the transmission of the virus. the two-dose policy adopted in many countries, should be supplemented with catch-up campaigns in schools to provide the booster effect for those previously immunized and to cover those previously unimmunized, especially in developing countries. the cdc considers that domestic transmission in the united states has been interrupted and that most localized outbreaks were traceable to imported cases. south america and the caribbean countries are now considered free of indigenous measles, based on their successful use of nids, although a large epidemic occurred in in brazil. it now appears that eradication has become a feasible target during the early part of the next century, with a strategy of levels of coverage in in-fancy with a two-dose policy, supplemented by catch-up campaigns to older children and young adults, and outbreak control. mumps. mumps is an acute viral disease characterized by fever, swelling, and tenderness usually of the parotid glands, but also other glands. the incubation period ranges between and days. orchitis, or inflammation of the testicles, occurs in - % of postpubertal males and oophoritis, or inflammation of the ovaries, in % of postpubertal females. sterility is an extremely rare result of mumps. central nervous system involvement can occur in the form of aseptic meningitis, almost always without sequelae. encephalitis is reported in - per , cases with an overall case fatality rate of . %. pancreatitis, neuritis, nerve deafness, mastiffs, nephritis, thyroiditis, and pericarditis, although rare, may occur. most persons born before are immune to the disease, because of the nearly universal exposure to the disease before that time. the live attenuated vaccine introduced in the united states in is available as a single vaccine or in combination with measles and rubella as the measlesmumps-rubella (mmr) vaccine. it provides long-lasting immunity in % of cases. mumps vaccine is now recommended in a two-dose policy with the first dose of mmr given between and months of age and a second dose given either at school entry or in early adolescence. mmr in two doses is now standard policy in the united sates, sweden, canada, israel, the united kingdom, and other countries. the incidence of mumps has consequently declined rapidly. local eradication of this disease is worthwhile and should be part of a basic international immunization program. rubella. rubella (german measles) is generally a mild viral disease with lymphadenopathy and a diffuse, raised red rash. low grade fever, malaise, coryza, and lymphadenopathy characterize the prodromal period. the incubation period is usually - days. differentiation from scarlet fever, measles, or other febrile diseases with rash may require laboratory testing and recovery of the virus from nasopharyngeal, blood, stool, and urine specimens. in , norman gregg, an australian ophthalmologist, noted an epidemic of cases of congenital cataract in newborns associated with a history of rubella in the mother during the first trimester. subsequent investigation demonstrated that intrauterine death, spontaneous abortion, and congenital anomalies occur commonly when rubella occurs early in pregnancy. congenital rubella syndrome (crs) occurs with single or multiple congenital anomalies including deafness, cataracts, microophthalmia, congenital glaucoma, microcephaly, meningoencephalitis, congenital heart defects, and others. moderate and severe cases are recognizable at birth, but mild cases may not be detected for months or years after birth. insulin-dependent diabetes is suspected as a late sequela of congenital rubella. each case of crs is estimated to cost some $ , in health care costs during the patient's lifetime. prior to availability of the attenuated live rubella vaccine in , the disease was universally endemic, with epidemics or peak incidence every - years. in unvaccinated populations, rubella is primarily a disease of childhood. in areas where children are well vaccinated, adolescent and young adult infection is more apparent, with epidemics in institutions, colleges, and among military personnel. a sharp reduction of rubella cases was seen in the united states following introduction of the vaccine in , but increased in , following rubella epidemics in - . a further reduction in cases was followed by a sharp upswing of rubella and crs in [ ] [ ] [ ] . an outbreak of rubella among the amish in the united states, who refuse immunization on religious grounds, resulted in cases of crs in . it is now thought that vaccination of sufficient numbers in the united states reduced circulation of the virus and protected most vulnerable groups in the population. in the past, immunization policy in some countries was to vaccinate school girls aged to protect them for the period of fertility. the current approach is to give a routine dose of mmr in early childhood, followed by a second dose in early school age to reduce the pool of susceptible persons. women of reproductive age should be tested to confirm immunity before pregnancy and immunized if not already immune. should a woman become infected during pregnancy, termination of pregnancy previously recommended is now managed with hyperimmune globulin. the infection of pregnant women during their first trimester of pregnancy is the primary public health implication of rubella. the emotional and financial burden of crs, including the cost of treatment of its congenital defects, makes this vaccination program cost-effective. its inclusion in a modem immunization program is fully justified. elimination of crs syndrome should be one of the primary goals of a program for prevention of vaccine-preventable disease in developed and developing countries. adoption of mmr and the two-dose policy will gradually lead to eradication of rubella and rubella syndrome. viral hepatitis. viral hepatitis is a group of diseases of increasing public health importance due to their large scale worldwide prevalence, their serious consequences, and our increasing ability to take preventive action. viral hepatic infectious diseases each have specific etiologic, clinical, epidemiologic, serologic, and pathologic characteristics. they have important short-and long-term sequelae. vaccine development is of high priority for control and ultimate eradication. hepatitis a. hepatitis a (hav) was previously known as infectious hepatitis or epidemic jaundice. hav is mainly transmitted by the fecal-oral route. clinical severity varies from a mild illness of - weeks to a debilitating illness lasting several months. the norm is complete recovery within weeks, but a fulminating or even fatal hepatitis can occur. severity of the disease worsens with increasing age. hav is sporadic/endemic worldwide. improving sanitation raises the age of exposure, with accompanying complications. it now occurs particularly in persons from industrialized countries when exposed to situations of poor hygiene, or among young adults when traveling to areas where the disease is en-demic. common source outbreaks occur in school-aged children and young adults from case contact, or from food contaminated by infected handlers. hepatitis a may be a serious public health problem in a disaster situation. prevention involves improving personal and community hygiene, with safe chlorinated water and proper food handling. hepatitis a vaccine has been recently licensed for use in the united states, and will probably soon be recommended for routine vaccination programs, as well as for persons traveling to endemic areas. hepatitis b. hepatitis b (hbv) once called serum jaundice, was thought to be transmitted only by injections of blood or blood products. it is now known to be present in all body fluids and easily transmissible by household and sexual contact, perinatal spread from mother to newborn, and between toddlers. however, it is not spread by the oral-fecal route. hepatitis b virus is endemic worldwide and is especially prevalent in developing countries. carrier status with persistent viremia varies from < % of adults in north america to % in some parts of the world. carders have detectable levels of hbsag, the surface antigen (i.e., australian antigen), in their blood. high risk groups in developed countries include intravenous drug users, homosexual men, persons with high numbers of sexual partners, those receiving tattoos, body piercing or acupuncture treatments, and residents or staff of institutions such as group homes and prisons. immunocompromised and hemodialysis patients are commonly carders of hbv. hbv may also be spread in a health system by use of inadequately sterilized reusable syringes, as in china and the former soviet union. transmission is reduced by screening blood and blood products for hbsag and strict technique for handling blood and body fluids in health settings. hbv is clinically recognizable in less than % of infected children but is apparent in - % of infected adults. clinically hbv has an insidious onset with anorexia, abdominal discomfort, nausea, vomiting, and jaundice. the disease can vary in severity from subclinical, very mild to fulminating liver necrosis, and death. it is a major cause of primary liver cancer, chronic liver disease, and liver failure, all devastating to health and expensive to treat. hepatitis b virus is considered to be the cause of % of primary cancer of the liver in the world and the most common carcinogen after cigarette smoking. the who estimates that more than billion people alive today have been infected with hbv. it is also estimated that million persons are chronic carriers of hbv, with an estimated - . million deaths per year from cirrhosis or primary liver cancer. this makes hepatitis b control a vital issue in the revision of health priorities in many countries. strict discipline in blood banks and testing of all blood donations for hbv, as well as hiv, and hepatitis c, is mandatory, with destruction of those with positive tests. contacts should be immunized following exposure with hbv immunoglobulin and hbv vaccine. the inexpensive recombinant hbv vaccine should be adopted by all countries and included in routine vaccination of infants. catch-up immunization for older children is also desirable. immunization programs should include those exposed at work, such as health, prison, or sex workers and adults in group settings. hbv immunization has been included in who's epi-plus expanded program of immunization. hepatitis c. first identified in , and previously known as non-a, non-b hepatitis, hepatitis c (hcv) has an insidious onset with jaundice, fatigue, abdominal pain, nausea, and vomiting. it may cause mild to moderate illness, but chronicity is common going on to cirrhosis and liver failure. the cdc estimates that million americans are chronically infected with hcv, with - , resulting deaths per annum, and the main cause of liver transplants. hcv is transmitted most commonly in blood products, but also among injecting drug users ( % of intravenous drug users were hcv positive in a vancouver study in ), and is also a risk for health workers. the disease may also occur in dialysis centers and other medical situations. person-to-person spread is unclear. prevention of transmission includes routine testing of blood donations, antiviral treatment of blood products, needle exchange programs, and hygiene. the who in has declared hepatitis prevention as a major public health crisis, with an estimated million persons infected worldwide ( ) , stressing that this "silent epidemic" is being neglected and that screening of blood products is vital to reduce transmission of this disease as for hiu hcv is a major cause of chronic cirrhosis and liver cancer. no vaccine is available at present, but an experimental vaccine is undergoing field trials. interferon and ribavirin treatment is reportedly effective in % of cases. hepatitis d. hepatitis d virus (hdv) also known as delta hepatitis, may be self-limiting or progress to chronic hepatitis. it is caused by a viruslike particle which infects cells along with hbv as a coinfection or in chronic carriers of hbv. hdv occurs worldwide in the same groups at risk for hbv. it also occurs in epidemics and is endemic in south america, africa, and among drug users. prevention is by measures similar to those for hbv. management for hdv is by passive immunity with immunoglobulin for contacts and high risk groups, and should include hbv vaccination as the diseases often coincide. there is currently no vaccine for hdv. hepatitis e. hepatitis e virus has an epidemiological and clinical course similar to that of hav. there is no evidence of a chronic form of hev. one striking characteristic of hev is its high mortality rate among pregnant women. the disease is caused by a viruslike particle with an incubation period of - days and is most common in young adults. sporadic cases as well as epidemics have been identified in india, pakistan, burma, china, russia, mexico, and north africa. hev results from waterborne epidemics or as sporadic cases in areas with poor hygiene, spread via the oral-fecal route. it is a hazard in disaster situations with crowding and poor sanitary conditions. prevention is by safe management of water supplies and sanitation. disease management is supportive care; passive immunization is not helpful and no vaccine is currently available. teria which causes meningitis and other serious infections in children under months of age. before the introduction of effective vaccines, as many as in children developed invasive hib infection. two-thirds of these had hib meningitis, with a case fatality rate of - %. long-term sequelae such as hearing impairment and neurological deficits occurred in - % of survivors. the first hib vaccine was licensed in , based on capsular material from the bacteria. extensive clinical trials in finland demonstrated a high degree of efficacy, but less impressive results were in seen in postmarketing efficacy studies. by , a conjugate vaccine based on an additional protein cell capsular factor capable of enhancing the immunologic response was introduced. several conjugate vaccines are now available. the conjugate vaccines are now combined with dpt as their schedule is simultaneous with that of the dpt. although the hib vaccine has been found to be cost-effective, despite initially being as costly as all the basic vaccines combined (i.e., dpt, opv, mmr, and hbv). for this reason, its use thus far has been limited to industrialized countries. the vaccine is a valuable addition to the immunologic armamentarium. it showed dramatic results in local eradication of this serious early childhood infection in a number of european countries and a sharp reduction in the united states. impressive field trials in the gambia showed a sharp reduction in mortality from invasive streptococcal diseases. the price of the vaccine has also fallen dramatically since the mid s. as a result, in , the world health organization recommended inclusion of hib vaccine in routine immunization programs in developing countries. influenza. influenza is an acute viral respiratory illness characterized by fever, headache, myalgia, prostration, and cough. transmission is rapid by close contact with infected individuals and by airborne particles with an incubation period of - days. it is generally mild and self-limited with recovery in - days. however, in certain population groups, such as the elderly and chronically ill, infection can lead to severe sequelae. gastrointestinal symptoms commonly occur in children. during epidemics, mortality rates from respiratory diseases increase because of the large numbers of persons affected, although the case fatality rates are generally low. over the past century, influenza pandemics have occurred in , , , and , while epidemics are annual events. the influenza pandemic of caused millions of deaths among young adults, by some estimates killing more than had died in world war i. it was the fear of recurrence of this pandemic which led the cdc to launch a massive immunization program in the united states in to prevent swine flu (the virus was a strain antigenically similar to that of the pandemic influenza) from spreading from an isolated outbreak in an army camp. the effort was stopped after millions of persons were immunized with an urgently produced vaccine when serious reactions occurred (guillain-barre syndrome, (i.e., a type of paralysis), and when no further cases of swine flu were seen. this demonstrated the difficulty of extrapolating scenarios from a historical experience. each year, epidemiologic services of the who and collaborating centers such as the cdc recommend which strains should be used in vaccine preparation for use among susceptible population groups. these vaccines are prepared with the current anticipated epidemic strains. the three main types of influenza (a, b, and c) have different epidemiological characteristics. type a and its subtypes, which are subject to antigenic shift, are associated with widespread epidemics and pandemics. type b undergoes antigenic drift and is associated with less widespread epidemics. influenza type c is even more localized. active immunization against the prevailing wild strain of influenza virus produces a - % level of protection in high risk groups. the benefits of annual immunization outweigh the costs, and it has proven to be effective in reducing cases of influenza and its secondary complications such as pneumonia and death from respiratory complications in high-risk groups. pneumococcal disease. pneumococcal diseases, which are caused by streptococcus pneumoniae, include pneumonia, meningitis, and otitis media. the capsular types of pneumococci selected out of known types of the organism for the vaccine are those responsible for % of pneumococcal pneumonia cases and - % of all pneumonia cases in the united states, and are responsible for some , deaths per year. this vaccine has been found to be cost-effective for high risk groups, including persons with chronic disease, hiv carriers, patients whose spleens were removed, the elderly, and those with immunosuppressive conditions. it should be included in preventive-oriented health programs, especially for long-term care of the chronically ill. because pneumococci cause bacterial meningitis, pneumococcal vaccine may be a future candidate for use in routine immunization programs for children (over age ). varicella is an acute, generalized virus disease caused by the varicella zoster virus (vzv). despite its reputation as an innocuous disease of childhood, varicella patients can be quite ill. a mild fever and characteristic generalized red rash lasts for a few hours, followed by vesicles occurring in successive crops over various areas of the body. affected areas may include the membranes of the eyes, mouth, and respiratory tract. the disease may be so mild as to escape observation or may be quite severe, especially in adults. death can occur from viral pneumonia in adults and sepsis or encephalitis in children. neonates whose mothers develop the disease within days of delivery are at increased risk with a case fatality rate of up to %. long-term sequelae include herpes zoster or shingles with a severely painful, vesicular rash along the distribution of sensory nerves, which can last for months. its occurrence increases with age and it is primarily seen in the elderly. it can, however, occur in immunocompromised children (especially those on cancer chemotherapy), aids patients, and others. some % of a population will experience herpes zoster during their lifetimes. reye's syndrome is an increasingly rare but serious complication from varicella or influenza b. it occurs in children and affects the liver and central nervous system. congenital varicella syndrome with birth defects similar to congenital rubella syndrome has been identified recently. varicella vaccine is now recommended for routine immunization at age - months in the united states, with catch-up for children up to age years and for occupationally exposed persons in health or child care settings. varicella vaccine is also recommended for nonpregnant women of child bearing years. cost-benefit studies indicate a : ratio if both direct and indirect costs are included (see chapter ). varicella vaccine is likely to be added to a "cocktail vaccine" containing dpt, polio (ipv), and hib. meningococcal meningitis. meningococcal meningitis, caused by the bacterium neisseria meningitides, is characterized by headache, fever, neck stiffness, delirium, coma, and/or convulsions. the incubation period is - days. it has a case fatality rate of - % if treated early and adequately, but rises up to % in the absence of treatment. there are several important strains (a, b, c, x, y, and z). serogroups a and c are the main causes of epidemics, with b causing sporadic cases and local outbreaks. transmission is by direct contact and droplet spread. meningitis (group a) is common in sub-saharan african countries, but epidemics have occurred worldwide. during epidemics, children, teenagers, and young adults are the most severely affected. in developed countries, outbreaks occur most frequently in military and student populations. in , meningococcal meningitis spread widely in the "meningitis belt" in central africa. epidemic control is achieved by mass chemoprophylaxis with antibiotics (e.g., rifampin or sulfa drugs) among case contacts, although the emergence of resistant strains is a concern. vaccines against serotypes a and c (bivalent) or a, c, w, and y are available. their use is effective in epidemic control and prevention institutions and military recruits, especially for a and c serogroups. vaccination is one of the key modalities of primary prevention. immunization is cost-effective and prevents wide-scale disease and death, with high levels of safety. despite the general consensus in public health regarding the central role of vaccination, there are many areas of controversy and unfulfilled expectations. a vaccination program should aim at % coverage at appropriate times, including infants, school children, and adults. immunization policy should be adapted from current international standards applying the best available program to national circumstances and financial capacities (table . ). public health personnel with expertise in vaccine-preventable disease control are needed to advise ministries of health and the practicing pediatric community on current issues in vaccination and to monitor implementation and evolution of control programs. controversies and changing views are common to immunization policy, so that discussions must be conducted on a continuing basis. policy should be under continuing review by a ministerially appointed national immunization advisory committee, including professionals from public health, academia, immunology, laboratory sciences, economics, and relevant clinical fields. bduring , the recommendation for polio virus was changed to doses of ipv in infancy. vaccine supply should be adequate and continuous. supplies should be ordered from known manufacturers meeting international standards of good manufacturing practice. all batches should be tested for safety and efficacy prior to release for use. there should be an adequate and continuously monitored cold chain to protect against high temperatures for heat labile vaccines, sera, and other active biological preparations. the cold chain should include all stages of storage, transport, and maintenance at the site of usage. only disposable syringes should be used in vaccination programs to prevent any possible transmission of blood-borne infection. a vaccination program depends on a readily available service with no barriers or unnecessary prerequisites, free to parents or with a minimum fee, to administer vaccines in disposable syringes by properly trained individuals using patientoriented and community-oriented approaches. ongoing education and training on current immunization practices are needed. incentive payments by insuring agency or managed care systems promote complete, on-time coverage. all clinical encounters should be used to screen, immunize, and educate parents/guardians. contraindications to vaccination are very few; vaccines may be given even during mild illness with or without fever, during antibiotic therapy, during convalescence from illness, following recent exposure to an infectious disease, and to persons having a history of mild/moderate local reactions, convulsions, or family history of sudden infant death syndrome (sids). simultaneous administration of vaccines and vaccine "cocktails" reduces the number of visits and thereby improves coverage; there are no known interferences between vaccine antigens. accurate and complete recording with computerization of records with automatic reminders helps promote compliance, as does co-scheduling of immunization appointments with other services. adverse events should be reported promptly, accurately, and completely. a tracking system should operate with reminders of upcoming or overdue immunizations; use mail, telephone, and home visits, especially for high risk families, with semiannual audits to assess coverage and review patient records in the population served to determine the percentage of children covered by second birthday. tracking should identify children needing completion of the immunization schedule and assess the quality of documentation. it is important to maintain up-to-date, easily retrievable medical protocols where vaccines are administered, noting vaccine dosage, contraindications, and management of adverse events. all health care providers and managers should be trained in education, promotion, and management of immunization policy. health education should target parents as well as the general public. monitoring of vaccines used and children immunized, individually and by category of vaccination can be facilitated by computerization of immunization records, or regular manual review of child care records. where immunization is done by physicians in private practice, as in the united states, determination of coverage is by periodic surveys. inspection of vaccines for safety, purity, potency, and standards is part of the regulatory function. vaccines are defined as biological products and are therefore subject to regulation by national health authorities. in the united states, this comes under the legislative authority of the public health service act, as well as the food, drug and cosmetics act, with applicable regulations in the code of federal regulations. the federal agency empowered to carry out this regulatory function is the center for drugs and biologics of the federal food and drug administration. litigation regarding adverse effects of vaccines led to inflation of legal costs and efforts to limit court settlements. the u.s. federal government enacted the child vaccine injury act of . this legislation requires providers to document vaccines given and to report on complications or reactions. it was intended to pay benefits to persons injured by vaccines faster and by means of a less expensive procedure than a civil suit for resolving claims. using this no-fault system, petitioners do not need to prove that manufacturers or vaccine givers were at fault. they must only prove that the vaccine is related to the injury in order to receive compensation. the vaccines covered by this legislation include dtp, mmr, opv, and ipv. development of vaccines from jenner in eighteenth century to the advent of recombinant hepatitis b vaccine in , and of vaccines for acellular pertussis, varicella, hepatitis a, and rotavirus in the s, has provided one of the pillars of public health and led to enormous savings of human life. vaccines for viral in-fections in humans for hiv, respiratory syncytial virus, papilloma, epstein-barr virus, dengue fever, and hantavirus are under intense research with genetic approaches using recombinant techniques. the potential for the future of vaccines will be greatly influenced by scientific advances in genetic engineering, with potential for development of vaccines attached to bacteria or protein in plants, which may be given in combination for an increasing range of organisms or their harmful products. recombinant dna technology has revolutionized basic and biomedical research since the s. the industry of biotechnology has produced important diagnostic tests, such as for hiv, with great potential for vaccine development. traditional whole organism vaccines, alive or killed, may contain toxic products that may cause mild to severe reactions. subunit vaccines are prepared from components of a whole organism. this avoids the use of live organisms that can cause the disease or create toxic products which cause reactions. subunit vaccines traditionally prepared by inactivation of partially purified toxins are costly, difficult to prepare, and weakly immunogenic. recombinant techniques are an important development for production of new whole cell or subunit vaccines that are safe, inexpensive, and more productive of antibodies than other approaches. their potential contribution to the future of immunology is enormous. molecular biology and genetic engineering have made it feasible to create new, improved, and less costly vaccines. new vaccines should be inexpensive, easily administered, capable of being stored and transported without refrigeration, and given orally. the search for inexpensive and effective vaccines for groups of viruses causing diarrheal diseases led to development of the rotavirus vaccine. some "edible" research focuses on the genetic programming of plants to produce vaccines and dna. vaccine manufacturers, who spend huge sums of money and years of research on new products, tend to work on those which will bring great financial rewards for the company and are critical to the local health care community. this has led to less effort being made in developing vaccines for diseases such as malaria. yet industry plays a crucial role for continued progress in the field. since the eradication of smallpox, much attention has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and some tropical diseases, such as malaria and dracunculiasis. eradication is defined as the achievement of a situation whereby no further cases of a disease occur anywhere and continued control measures are unnecessary. reducing epidemics of infectious diseases, through control and eradication in selected areas or target groups, can in certain instances achieve eradication of the disease. local eradication can be achieved where domestic circulation of an organism is interrupted with cases occurring from importation only. this requires a strong, sustained immunization program with adaptation to meet needs of importation of carriers and changing epidemiologic patterns. smallpox was one of the major pandemic diseases of the middle ages and its recorded history goes back to antiquity. prevention of smallpox was discussed in ancient china by ho kung (circe ao ), and inoculation against the disease was practiced there from the eleventh century ad. prevention was carried out by nasal inhalation of powdered dried smallpox scabs. exposure of children to smallpox when the mortality rate was lowest assumed a weakened form of the disease, and it was observed that a person could only have smallpox once in a lifetime. isolation and quarantine were widely practiced in europe during the sixteenth and seventeenth centuries. variolation was the practice of inoculating youngsters with material from scabs of pustules from mild cases of smallpox in the hope that they would develop a mild form of the disease. although this practice was associated with substantial mortality, it was widely adopted because mortality from variolation was well below that of smallpox acquired during epidemics. introduced into england in (see chapter ) it was commonly practiced as a lucrative medical specialty during the eighteenth century. in the s, variolation was also introduced into the american colonies, russia, and subsequently into sweden and denmark. despite all efforts, in the early eighteenth century smallpox was a leading cause of death in all age groups. toward the end of the eighteenth century an estimated , persons died annually from smallpox in europe. vaccination, or the use of cowpox vaccinia virus to protect against smallpox, was initiated late in the eighteenth century. in , a cattle breeder in yorkshire, england, inoculated his wife and two children with cowpox to protect them during a smallpox epidemic. in , edward jenner, an english country general practitioner, experimented with inoculation from a milkmaid's cowpox pustule to a healthy youngster, who subsequently proved resistant to smallpox by variolation (see chapter ). vaccination, the deliberate inoculation of cowpox material, was slow to be adopted universally, but by , over , persons in england were vaccinated. vaccination gathered support in the nineteenth century in military establishments, and in some countries which adopted it universally. opposition to vaccination remained strong for nearly a century based on religious grounds, observed failures of vaccination to give lifelong immunity, and because it was seen as an infringement of the state on the rights of the individual. often the protest was led by medical variolationists whose medical practice and large incomes were threatened by the mass movement to vaccination. resistance was also offered by "sanitarians" who opposed the germ theory and thought cleanliness was the best method of prevention. universal vaccination was increasingly adopted in europe and america in the early nineteenth century and eradication of smallpox in developed countries was achieved by the mid twentieth century. in , the soviet union proposed to the world health assembly a program to eradicate smallpox internationally and subsequently donated million doses of vaccine per year as part of the million needed to promote vaccination of at least % of the world population. in , who adopted a target for the eradication of smallpox. a program was developed which included a massive increase in coverage to reduce the circulation of the virus through person-to-person contact. where smallpox was endemic, with a substantial number of unvaccinated persons, the aim of the mass vaccination phase was % coverage. increasing vaccination coverage in developing countries reduced the disease to periodic and increasingly localized outbreaks. in , countries were considered endemic for smallpox, and another experienced importation of cases. by , the number of endemic countries was down to , and by only countries were still endemic, including india, pakistan, bangladesh, and nepal. in these countries, a new strategy was needed, based on a search for cases and vaccination of all contacts, working with a case incidence below per , . the program then moved into the consolidation phase, with emphasis on vaccination of newborns and new arrivals. surveillance and case detection were improved with case contact or risk group vaccination. the maintenance phase began when surveillance and reporting were switched to the national or regional health service with intensive follow-up of any suspect case. the mass epidemic era had been controlled by mass vaccination, reducing the total burden of the disease, but eradication required the isolation of individual cases with vaccination of potential contacts. technical innovations greatly eased the problems associated with mass vaccination worldwide. during the s, there was wide variation in sources of smallpox vaccine. in the s, efforts to standardize and further attenuate the strains used reduced complication rates from vaccinations. the development of lyophilization (freeze-drying) of the vaccine in england in the s made a heat-stable vaccine that could be effective in tropical field conditions in developing countries. the invention of the bifurcated needle (bernard rubin ) allowed for easier and more widespread vaccination by lesser trained personnel in remote areas. the net result of these innovations was increased world coverage and a reduction in the spread of the disease. smallpox became more and more confined by increasing herd immunity, thus allowing transition to the phase of monitoring and isolation of individual cases. in the last case of smallpox was identified in somalia, and in the who declared the disease eradicated. no subsequent cases have been found except for several associated with a laboratory accident in the united kingdom in . the who recommends that the last stores of smallpox virus should be destroyed in . the cost of the eradication program was $ million or $ million per year. worldwide savings are estimated at $ billion annually. this monumental public health achievement set the precedent for eradication of other infectious diseases. the world health assembly decided to destroy the last two remaining stocks of the smallpox virus in atlanta and moscow in . destruction of the remaining stock was delayed in to because of concern that illegal stocks may be held by some states or potential bioterrorists for potential use in weapons of mass destruction, concern regarding the appearance of monkeypox and a wish to use the virus for further research. in , the who established a target of eradication of poliomyelitis by the year . global immunization coverage with three doses of opv increased from some % in to over % in , with a slight decline in the period - . support from member countries and international agencies such as unicef and rotary international has led to widescale increases in immunization coverage throughout many parts of the world. the world health organization promotes use of opv only as part of routine infant immunization or national immunization days (nids). this strategy has been successful in the americas and in china, but india and the middle east remain problematic. eradication of wild poliomyelitis by the year will require flexibility in vaccination strategies and may require the combined approach, using opv and ipv, as adopted in the united states in to prevent vaccine-associated clinical cases. the combination of opv and ipv may be needed where enteric disease is common and leads to interference in opv uptake, especially in tropical areas where endemic poliovirus and diarrheal diseases are still found. the world bank estimated that achievement of global eradication would save $ million annually in the united states alone. since the eradication of smallpox, discussion has focused on the possibility of similarly eradicating other diseases, and a list of potential candidates has emerged. some of these have been abandoned because of practical difficulties with current technology. diseases that have been under discussion for eradication have included measles, tb, and tropical diseases such as malaria and dracunculiasis. eradication of malaria was thought to be possible in the s when major gains were seen in malaria control by aggressive case environmental control, case finding, and management. however, lack of sustained vector control and an effective vaccine has prevented global eradication. malaria control suffered serious setbacks because of failure in political resolve and capacity to continue support needed for necessary programs. in the s and s, control efforts were not sustained in many countries, and a dreadful comeback of the disease occurred in africa and asia in the s. the emergence of mosquitoes resistant to insecticides, and malarial strains resistant to antimalarial drugs, have made malaria control even more difficult and expensive. renewed effort in malaria control may require new approaches. use of community health workers (chws) in small villages in highly endemic regions of colombia resulted in a major drop in malaria mortality during the s. the chws investigate suspect cases by taking clinical histories and blood smears. . scientific feasibility a. epidemiologic vulnerability; lack of nonhuman reservoir, ease of spread, no natural immunity, relapse potential; b. effective practical intervention available; vaccine or other primary preventive or curative treatment, or vectoricide that is safe, inexpensive, long lasting, and easily used in the field; c. demonstrated feasibility of elimination in specific locations, such as an island or other geographic unit. . political will/popular support a. they examine smears for malaria parasites and a diagnosis is made. therapy is instituted and the patient is followed. quality control monitoring shows high levels of accuracy in reading of slides compared to professional laboratories. in the late s, there was widespread discussion in the literature of the potential for eradication of measles and tb. measles eradication was set back as breakthrough epidemics occurred in the united states, canada, and many other countries during the s and early s, but regional eradication was achieved combining the two-dose policy with catch-up campaigns for older children or in national immunization days, as in the caribbean countries. tuberculosis has also increased in the united states and several european countries for the first time in many decades. unrealistic expectations can lead to inappropriate assessments and policy when confounding factors alter the epidemiologic course of events. such is the case with tb, where control and eradication have receded from the picture. this deadly disease has returned to developed countries, partly in association with the hiv infection and multiple-drug-resistant strains, as well as homelessness, rising prison populations, poverty, and other deleterious social conditions. directly observed therapy is an important recent breakthrough, more effective in use of available technology and will play a major role in tb control in the twenty-first century. a decade after the eradication of smallpox was achieved, the international task force for disease eradication (itfde) was established to systematically evaluate the potential for global eradicability of candidate diseases. its goals were to identify specific barriers to the eradication of these diseases that might be surmountable and to promote eradication efforts. the subject of eradication versus control of infectious diseases if of central public health importance as technology expands the armamentarium of immunization and vector control into the twenty-first century. the control of epidemics, followed by interruption of transmission and ultimately eradication, will save countless lives and prevent serious damage to children throughout the world. the smallpox achievement, momentous in itself, points to the potential for the eradication of other deadly diseases. the skillful use of existing and new technology is an important priority in the new public health. flexibility and adaptability are as vital as resources and personnel. selecting diseases for eradication is not purely a professional issue of resources such as vaccines and manpower, organization and financing. it is also a matter of political will and perception of the burden of disease. there will be many controversies. the selection of polio for eradication while deferring measles when polio kills few and measles kills many may be questioned. the cdc published criteria for selection of disease for eradication are shown in box . . the who, in a review of health targets in the field of infectious disease control for the twenty-first century, selected the following targets: eradication of chagas' disease by ; eradication of neonatal tetanus by ; eradication of leprosy by ; eradication of measles by ; eradication of trachoma by ; reversing the current trend of increasing tuberculosis and hiv/aids. in , a conference in atlanta, georgia, reviewed the subject, which is still very much in a state of flux. table . summarizes the selection of diseases which are presently seen as controllable and those considered to be potentially eradicable. the subject will be under review in the years ahead. mycobacterium tuberculosis in humans and m. bovis in cattle. the disease is primarily found in humans, but it is also a disease of cattle and occasionally other primates in certain regions of the world. it is transmitted via airborne droplet nuclei from persons with pulmonary or laryngeal tb during coughing, sneezing, talking, or singing. the initial infection may go unnoticed, but tuberculin sensitivity appears within a few weeks. about % of those infected enter a latent phase with a lifelong risk of reactivation. approximately % go from initial infection to pulmonary tb. less commonly, the infection develops as extrapulmonary tb, involving meninges, lymph nodes, pleura, pericardium, bones, kidneys, or other organs. untreated, about half of the patients with active tb will die of the disease within years, but modern chemotherapy almost always results in a cure. pulmonary tb symptoms include cough and weight loss, with clinical findings on chest examination and confirmation by findings of tubercle bacilli in stained smears of sputum and, if possible, growth of the organism on culture media, and changes in the chest x-ray. tuberculosis affects people in their adult working years, with - % of cases in persons between the ages of and . its devastating effects on the work force and economic development contribute to a high cost-effectiveness for tb control. the tubercle bacillus infects approximately . billion people in the world today, causing over million cases and nearly million deaths in . during , new cases of tb included . million ( %) in southeast asia and the western pacific regions of who, with . million cases in india, and . million in indonesia. by , the incidence of tb may increase to . million new cases per year, a % increase over . between and , who estimates there were million new cases of tb, of which million cases were in association with hiv infection. during the s, an estimated million persons died of tb, including . million with hiv infection. a new and dangerous period for tb resurgence has resulted from parallel epidemiologic events: first, the advent of hiv infection and second, the occurrence of multiple drug resistant tb (mdrtb), that is, organisms resistant at least to both isoniazid (inh) and rifampicin, two mainstays of tb treatment. mdrtb can have a case fatality rate as high as %. hiv reduces cellular immunity so that people with latent tb have a high risk of activation of the disease. it is estimated that hiv negative persons have a - % lifetime risk of tb; hiv positive people have a risk of % per year of developing clinical tuberculosis. drug resistance, the long period of treatment, and the socioeconomic profile of most tb patients combine to require a new approach to therapy. directly observed treatment, short-course (dots), has shown itself to be highly effective with patients in poor self-care settings, such as the homeless, drug users, and those with aids. the strategy of dots uses community health workers to visit the patient and observes him or her taking the various medications, providing both incentive, support, and moral coercion to complete the needed to month therapy. dots has been shown to cure up to % of cases, at a cost of as little as $ per patient. it is one of the few hopes of containing the tb pandemic. in , who released a new strategy for control of tuberculosis over the next decade. the plan calls for new guidelines for control, new aid funds for developing countries, and enlistment of ngos to assist in the fight. the new guidelines stress short-term chemotherapy in well-managed programs of dots, stressing strict compliance with therapy for infectious cases with a goal of an % cure rate. even under adverse conditions, dots produces excellent results. it is one of the most cost-effective health interventions combining public health and clinical medical approaches. tuberculosis incidence in the united states decreased steadily until , increased in , and has declined again since. from to , there was an excess of , cases over the expected rate if the previous decline in case incidence had continued. this rise was largely due to the hiv/aids epidemic and the emergence of mdrtb, but also greater incidence among immigrants from areas of higher tb incidence, drug abusers, the homeless, and those with limited access to health care. this is particularly true in new york city, where mdrtb has appeared in outbreaks among prison inmates and hospital staff. from to , tb incidence in the united states declined by % and in some states, including new york, by % or more. this turnaround was due to stronger tb control programs that promptly identified persons with tb and initiated and ensured completion of appropriate therapy. aggressive staff training, outreach, and case management approaches were vital to this success. concern over rising rates among recent immigrants and the continued challenge of hiv/aids and coincidental transmission of hepatitis a, b, and c among drug users and marginal population groups show that continued support for tb control is needed. bacillus calmette-gurrin (bcg) is an attenuated strain of the tubercle bacillus used widely as a vaccination to prevent tb, especially in high incidence areas. it induces tuberculin sensitivity or an antigen-antibody reaction in which antibodies produced may be somewhat protective against the tubercle bacillus in % of vaccinees. although the support for its general use is contradictory, there is evidence from case-control and contact studies of positive protection against tb meningitis and disseminated tb in children under the age of . in some developed, low-incidence countries, it is not used routinely but selectively. it may also be used in asymptomatic hiv-positive persons or other high risk groups. the bcg vaccine for tuberculosis remains controversial. while used widely internationally, in the united states and other industrialized countries, it is thought to hinder rather than help in the fight against tb. this concern is based on the usefulness of tuberculin testing for diagnosis of the disease. where bcg has been administered, the diagnostic value of tuberculin testing is reduced, especially in the period soon after the bcg is used. studies showing equivocal benefit of bcg in preventing tuberculosis have added to the controversy. while those in the field in the united states continue to oppose the use of bcg, internationally it is still felt to be of benefit in preventing tb, primarily in children. a metaanalysis of the literature of bcg carried out by the technology assessment group at harvard school of public health concluded: on average, bcg vaccine significantly reduces the risk of tb by %. protection is observed across many populations, study designs, and forms of tb. age at vaccination did not enhance predictiveness of bcg efficacy. protection against tuberculous death, meningitis, and disseminated disease is higher than for total tb cases, although this result may reflect reduced error in disease classification rather than greater bcg efficacy. [colditz et al., jama, .] box . control of tuberculosis . identifying persons with clinically active tb; . diagnostic methods--clinical suspicion, sputum smear for bacteriologic examination, tuberculin skin testing, chest radiograph; . case finding and investigation programs in high risk groups; . contact investigation; . isolation techniques during initial therapy; . treatment, mainly ambulatory, of persons with clinically active tb; . treatment of contacts; . directly observed treatment, short-course (dots), where compliance suspect; . environmental control in treatment settings to reduce droplet infection; . educate health care providers on suspicion of tb and investigation of suspects. currently, the who recommends use of bcg as close to birth as possible as part of the expanded programme of immunization (epi). tuberculosis control remains feasible with current medical and public health methods. deterioration in its control should not lead to despair and passivity. the recent trend to successful control by dots despite the growing problem of mdrtb suggest that control and gradual reduction can be achieved by an activist, community outreach approach. the who in made tb control one of its major priorities, expressing grave concern that the mdr organism, now widely spread in countries of asia, eastern europe, and the former soviet union, may spread the disease much more widely. the disease constitutes one of the great challenges to public health at the start of the new century. acute infectious diseases caused by group a streptococci include streptococcal sore throat, scarlet fever, puerperal fever, septicemia, ersypelas, cellulitis, mastoiditis, otitis media, pneumonia, peritonsillitis (quinsy), wound infections, toxic shock syndrome, and fasciitis, the "flesh eating bacteria." streptococcus pyogenes group a include some serologically distinct types which vary in geographic location and clinical significance. transmission is by droplet, person-to-person direct contact, or by food infected by carriers. important complications from a public health point of view include acute rheumatic fever and acute glomerulonephritis, but also skin infections and pneumonia. acute rheumatic fever is a complication of strep a infection that has virtually disappeared from industrialized countries as a result of improved standards of living and antibiotic therapy. however, outbreaks were recorded in the united states in , and an increasing number of cases have been seen since . in developing countries, rheumatic fever remains a serious public health problem affecting school age children, particularly those in crowded living arrangements. longterm sequelae include disease of the mitral and aortic heart valves, which require cardiac care and surgery for repair or replacement with artificial valves. acute glomerulonephritis is a reaction to toxins of the streptococcal infection in the kidney tissue. this can result in long-term kidney failure and the need for dialysis or kidney transplantation. this disease has become far less common in the industrialized countries, but remains a public health problem in developing countries. the streptococcal diseases are controllable by early diagnosis and treatment with antibiotics. this is a major function of primary care systems. recent increases in rheumatic fever may herald a return of the problem, perhaps due to inadequate access to primary care in the united states for large sectors of the population, along with increased social hygiene problems. where access to primary care services is limited, infections with streptococci can result in a heavy burden of chronic heart and kidney disease with substantial health, emotional, and financial tolls. measures to improve access to care and pub-lic information are needed to assure rapid and effective care to prevent chronic and costly conditions. zoonoses are infectious diseases transmissible from vetebrate animals to humans. common examples of zoonoses of public health importance in nonindustrialized countries include brucellosis and rabies. in industrialized countries, salmonellosis, "mad cow disease" and influenza have reinforced the importance of relationships of animal and human health. strong cooperation between public health and veterinary public health authorities are required to monitor and to prevent such diseases. brucellosis is a disease occurring in cattle (brucella abortus), in dogs (br. cahis), in goats and sheep (br. melitensis), and in pigs (br. suis). humans are affected mainly through ingestion of contaminated milk products, by contact, or inhalation. brucellosis (also known as relapsing, undulant, malta, or mediterranean fever) is a systemic bacterial disease of acute or insidious onset characterized by fever, headache, weakness, sweating, chills, arthralgia, depression, weight loss, and generalized malaise. spread is by contact with tissues, blood, urine, vaginal discharges, but mainly by ingestion of raw milk and dairy products from infected animals. the disease may last from a few days to a year or more. complications include osteoarthritis and relapses. case fatality is under %, but disability is common and can be pronounced. the disease is primarily seen in mediterranean countries, the middle east, india, central asia, and in central and south america. brucellosis occurs primarily as an occupational disease of persons working with and in contact with tissues, blood, and urine of infected animals, especially goats and sheep. it is an occupational hazard for veterinarians, packinghouse workers, butchers, tanners, and laboratory workers. it is also transmitted to consumers of unpasteurized milk from infected animals. animal vectors include wild animals, so that eradication is virtually impossible. diagnosis is confirmed by laboratory findings of the organism in blood or other tissue samples, or with rising antibody titers in the blood, with confirmation by blood cultures. clinical cases are treated with antibiotics. epidemiologic investigation may help track down contaminated animal flocks. routine immunization of animals, monitoring of animals in high risk areas, quarantining sick animals, destroying infected animals, and pasteurizing milk and milk products prevents spread of the disease. control measures include educating farmers and the public not to use unpasteurized milk. individuals who work with animals (cattle, swine, goats, sheep, dogs, coyotes) should take special precautions when handling animal carcasses and materials. testing animals, destroying carriers, and enforcing mandatory pasteurization will restrict the spread of the disease. this is an economic as well as public health problem, requiring full cooperation between ministries of health and of agriculture. rabies is primarily a disease of animals, with a variety of wild animals serving as a reservoir for this disease, including foxes, wolves, bats, skunks, and raccoons, who may infect domestic animals such as dogs, cats, and farm animals. animal bites break the skin or mucous membrane, allowing entry of the virus from the infected saliva into the bloodstream. the incubation period of the virus is - weeks; it can be as long as several years or as short as days, so that postexposure preventive treatment is a public health emergency. the clinical disease often begins with a feeling of apprehension, headache, pyrexia, followed by muscle spasms, acute encephalitis, and death. fear of water ("hydrophobia") or fear of swallowing is a characteristic of the disease. rabies is almost always fatal within a week of onset of symptoms. the disease is estimated to cause , deaths annually, primarily in developing countries. it is uncommon in developed countries. rabies control focuses on prevention in humans, domestic animals, and wildlife. prevention in humans is based on preexposure prophylaxis for groups at risk (e.g., veterinarians, zoo workers) and postexposure immunization for persons bitten by potentially rabid animals. because reducing exposure of pets to wild animals is difficult, immunization of domestic animals is one of the most important preventive measures. prevention in domestic animals is by mandatory immunization of household pets. all domestic animals should be immunized at age months and revaccinated according to veterinary instructions. prevention in wild animals to reduce the reservoir is successful in achieving local eradication in settings where reentry from neighboring settings is limited. since , the use of oral rabies immunization has been successful in reducing the population of wild animals infected by the rabies virus. rabies eradication efforts, using aerial distribution of baits containing fox rabies vaccine in affected areas of belgium, france, germany, italy, and luxembourg, have been underway since . the number of rabies cases in these affected areas has declined by some %. switzerland is now virtually rabies-free because of this vaccination program. the potential exists for focal eradication, especially on islands or in partially restricted areas with limited possibilities of wild animal entry. livestock need not be routinely immunized against rabies, except in high risk areas. where bats are major reservoirs of the disease, as in the united states, eradication is not presently feasible. salmonella, discussed later in this chapter under diarrheal diseases, is one of the commonest of all infectious diseases among animals and is easily spread to humans via poultry, meat, eggs, and dairy products. specific antigenic types are associated with food-borne transmission to humans, causing generalized illness and gastroenteritis. severity of the disease varies widely, but the diseases can be devastating among vulnerable population groups, such as young children, the elderly, and the immunocompromised. epidemiologic investigation of common food source outbreaks may uncover hazardous food handling practices. laboratory confirmation or serotypes helps in monitoring the disease. prevention is by maintaining high standards of food hygiene in processing, inspection and regulation, food handling practices, and hygiene education. bacillus anthracis causes a bacterial infection in herbivore animals. its spores contaminate soil, worldwide. it affects humans exposed in occupational settings. transmission is cutaneous by contact, gastrointestinal by ingestion, or respiratory by inhalation. it has gained recent attention (iraq, ) as a highly potent agent for germ warfare or terrorism. limited supplies of vaccine are available. creutzfeld-jakob disease is a degenerative disease of the central nervous system linked to consumption of beef from cattle infected with bovine spongiform encephalopathy. it is transmitted by prions in animal feed prepared from contaminated animal material and in transplanted organs. this disease was identified in the united kingdom linked to infected cattle leading to a ban on british beef in many parts of the world and slaughter of large numbers of potentially contaminated animals. the tapeworm causing diphyllobothriasis (diphyllobothrium latum) is widespread in north american freshwater fish, passing from crustacean to fish to humans by eating raw freshwater fish. it is especially common among inuit peoples and may be asymptomatic or cause severe general and abdominal disorder. food hygiene (freezing and cooking of meat) is recommended; treatment is by anthelminthics. leptospiroses are a group of zoonotic bacterial diseases found worldwide in rats, raccoons, and domestic animals. it affects farmers, sewer workers, dairy and abattoir workers, veterinarians, military personnel, and miners with transmission by exposure to or ingestion of urine-contaminated water or tissues of infected animals. it is often asymptomatic or mild, but may cause generalized illness like influenza, meningitis, or encephalitis. prevention requires education of the public in self protection and immunization of workers in hazardous occupations, along with immunization and segregation of domestic animals and control of wild animals. vector-borne diseases are a group of diseases in which the infectious agent is transmitted to humans by crawling or flying insects. the vector is the intermediary between the reservoir and the host. both the vector and the host may be affected by climatic condition; mosquitoes thrive in warm, wet weather and are suppressed by cold weather; humans may wear less protective clothing in warm weather. the only important reservoir of malaria is humans. its mode of transmission is from person to person via the bite of an infected female anopheles mosquito (ronald ross, nobel prize, ) . the causative organism is a single cell parasite with four species: plasmodium vivax, p malariae, p falciparum, and p ovale. clinical symptoms are produced by the parasite invading and destroying red blood cells. the incubation period of approximately - days, depending on the specific plasmodium involved. some strains of p vivax may have a protracted incubation period of - months and even longer for p ovale. the disease can also be transmitted through infected blood transfusions. confirmation of diagnosis is by demonstrating malaria parasites on blood smears. falciparum malaria, the most serious form, presents with fever, chills, sweats, and headache. it may progress to jaundice, bleeding disorders, shock, renal or liver failure, encephalopathy, coma, and death. prompt treatment is essential. case fatality rates in untreated children and adults are above %. an untreated attack may last months. other forms of malaria may present as a nonspecific fever. relapse of the p ovale may occur up to years after initial infection; malaria may persist in chronic form for up to years. malaria control advanced during the s- s through improved chlovaquine treatment and use of ddt for vector control with optimism for eradication of the disease. however, control regressed in many developing countries as allocations for environmental control and case findings/treatment were reduced. there has also been an increase in drug resistance, so that this disease is now an extremely serious public health problem in many parts of the world. the need for a vaccine for malaria control is now more apparent than ever. the world health organization estimated that, in , sub-saharan africa (ssa) had million new malaria cases, with % of children up to age . over million deaths occur annually from malaria more than two-thirds of them in ssa. large areas, particularly in forest or savannah regions with high rainfall, are holoendemic. in higher altitudes, endemicity is lower, but epidemics do occur. chloroquine-resistant p. falciparum has spread throughout africa, accompanied by an increasing incidence of severe clinical forms of the disease. the world bank estimates that % of all disability-adjusted life years (dalys) lost per year in ssa are from malaria, which places a heavy economic burden on the health systems. in the americas, the number of cases detected has risen every year since , and the who estimates there to have been . - . million cases in . the nine most endemic countries in the americas achieved a % reduction in malaria mortality between and . southeast asian region reports some . million cases of malaria in and deaths from tb. this accounts for more than one-third of all non-african malaria cases. there is an increase in resistant strains to the major available drugs and of the mosquitoes to insecticides in use. vector control, case finding, and treatment remain the mainstay of control. use of insecticide-impregnated bed nets and curtains, and residual house spraying, and strengthened vector control activities are important, as are early diagnosis and carefully monitored treatment with monitoring for resistance. control of malaria will ultimately depend on a safe, effective, and inexpensive vaccine. attempts to develop a malaria vaccine have been unsuccessful to date due to the large number of genetic types of p. falciparum even in localized areas. a colombian-developed vaccine is being field-tested with partial effectiveness. research in vaccines for malaria has also been hampered by the fact that it is a relatively low priority for vaccine manufacturers because of the minimal potential for financial benefit. research on malaria concentrates on the pharmacological aspects of the disease because of increasing drug resistance. in , who has initiated a new campaign to "roll back malaria" and maintain the dream of eradication in the future. effective low technology interventions include community-based case finding, early treatment of good quality, insecticide use, and vector control. the use of community health workers in endemic areas, has shown promising results. local control and even eradication can be achieved with currently available technology. this requires an integration of public health and clinical approaches with strong political commitment. the rickettsia are obligate parasites, i.e., they can only replicate in living cells, but otherwise they have characteristics of bacteria. this is a group of clinically similar diseases, usually characterized by severe headache, fever, myalgia, rash, and capillary bleeding causing damage to brain, lungs, kidneys, and heart. identification is by serological testing for antibodies, but the organisms can also be cultured in laboratory animals, embryonic eggs, or in cell cultures. the organisms are transmitted by arthropod vectors such as lice, fleas, ticks, and mites. the diseases caused millions of deaths during war and famine periods prior to the advent of antibiotics. these diseases appear in nature in ways that make them impossible to eradicate, but clinical diagnosis, host protection, and vector control can help reduce the burden of disease and deal with outbreaks that may occur. public education regarding self-protection, appropriate clothing, tick removal, and localized control measures such as spraying and habitat modification are useful. epidemic typhus, first identified in , is due to rickettsia prowazekii. spread primarily by the body louse, typhus was the cause of an estimated million deaths, i.e., during war and famine, in poland and the soviet union from - . untreated, the fatality rate is - %. typhus responds well to antibiotics. it is currently largely confined to endemic foci in central africa, central asia, eastern europe, and south america. it is preventable by hygiene and pediculicides such as ddt and lindane. a vaccine is available for exposed laboratory personnel. murine typhus is a mild form of typhus due to rickettsia typhi, which is found worldwide and spread in rodent reservoirs. scrub typhus, also known as tsutsugamushi or japanese river fever, is located throughout the far east and the pacific islands, and was a serious health problem for u.s. armed forces in the pacific during world war ii. it is spread by the rickettsia tsutsugamushi and has a wide variation in case fatality according to region, organism, and age of patient. rocky mountain spotted fever is a well-known and severe form of tick-borne typhus due to rickettsia rickettsii, occurring in western north america, europe, and asia. q. fever is a tick-borne disease caused by coxiella burnetii and is worldwide in distribution, usually associated with farm workers, in both acute and chronic forms. regular anti-tick spraying of sheep, cows, and goats helps protect exposed workers. protective clothing and regular removal of body ticks help protect exposed persons. arthropod-borne viral diseases are caused by a diverse group of viruses which are transmitted between vertebrate animals (often farm animals or small rodents) and people by the bite of blood-feeding vectors such as mosquitoes, ticks, and sandflies and by direct contact with infected animal carcasses. usually the viruses have the capacity to multiply in the salivary glands of the vector, but some are carried mechanically in their mouthparts. these viruses cause acute central nervous system infections (meningoencephalitis), myocarditis, or undifferentiated viral illnesses with polyarthritis and rashes, or severe hemorrhagic febrile illnesses. arbovirus diseases are often asymptomatic in vertebrates but may be severe in humans. over antigenetically distinct arboviruses are associated with disease in humans, varying from benign fevers of short duration to severe hemmorhagic fevers. each has a specific geographic location, vector, clinical, and virologic characteristics. they are of international public health importance because of the potential for spread via natural phenomena and modem rapid transportation of vectors and persons incubating the disease or ill with it, with potential for further spreading at the point of destination. arboviruses are responsible for a large number of encephalitic diseases characterized by mode of transmission and geographic area. mosquito-borne arboviruses causing encephalitis include eastern and western equine, venezuelan, japanese, and murray hill encephalitides. japanese encephalitis is caused by a mosquito-borne arbovirus found in asia and is associated with rice-growing areas. it is characterized by headache, fever, convulsions, and paralysis, with fatality rates in severe cases as high as %. a currently available vaccine is used routinely in endemic areas (japan, korea, thailand, india, and taiwan) and for persons traveling to infected areas. tick-borne arboviruses causing encephalitis include the powassan virus, which occurs sporadically in the united states and canada. tickborne encephalitis is endemic in eastern europe, scandinavia, and the former soviet union. an epidemic of mosquito-borne encephalitis in new york city in included cases and deaths, due to the west nile fever virus, never before found in the united states. other insect vectors. it affects animals and humans who are in direct contact with the meat or blood of affected animals. the virus causes a generalized illness in humans with encephalitis, hemorrhages, retinitis and retinal hemorrhage leading to partial or total blindness, and death ( - %). it also causes universal abortion in ewes and a high percentage of death in lambs. the normal habitat is in the rift valley of eastern africa (the great syrian-african rift), often spreading to southern africa, depending on climactic conditions. the primary reservoir and vector is the aedes mosquito, and affected animals serve to multiply the virus which is transmitted by other vectors and direct contact with animal fluids to humans. an unusual spread of rvf northward to the sudan and along the aswan dam reservoir to egypt in - caused hundreds of thousands of animal deaths, with , human cases and deaths. rvf appeared again in egypt in . this disease is suspected to be one of the ten plagues of egypt leading to the exodus of the children of israel from egypt during pharaonic-biblical times. in , an outbreak of rvf in kenya, initially thought to be anthrax, with hundreds of cases and dozens of deaths, was related to abnormal rainy season and vector conditions. satellite monitoring of rainfall and vegetation is being used to predict epidemics in kenya and surrounding countries. animal immunization, monitoring, vector control, and reduced contact with infected animals can limit the spread of this disease. arboviruses can also cause hemorrhagic fevers. these are acute febrile illnesses, with extensive hemorrhagic phenomena (internal and external), liver damage, shock, and often high mortality rates. the potential for international transmission is high. yellow fever. yellow fever is an acute viral disease of short duration and varying severity with jaundice. it can progress to liver disease and severe intestinal bleeding. the case fatality rate is < % in endemic areas, but may be as high as % in nonendemic areas and in epidemics. it caused major epidemics in the americas in the past, but was controlled by elimination of the vector, aedes aegypti. a live attenuated vaccine is used in routine immunization endemic areas and recommended for travelers to infected areas. determining the mode of transmission and vector control of yellow fever played a major role in the development of public health (see chapter ). in , the who reported , cases and , deaths from yellow fever globally. dengue hemorrhagic fever. dengue hemorrhagic fever is an acute sudden onset viral disease, with - days of fever, intense headache, myalgia, arthralgia, box . dengue fever and dengue hemorrhagic fever, dengue fever, a severe influenza-like illness, and dengue hemorrhagic fever are closely related conditions caused by four distinct viruses transmitted by aedes aegypti mosquitos. dengue is the world's most important mosquito-borne virus disease. a total of , million people worldwide are at risk of infection. an estimated million cases occur each year, of whom , need to be hospitalized. this is a spreading problem, especially in cities in tropical and subtropical areas. major outbreaks were reported in colombia, cuba, and many other locations in . source: world health organization. . world health report gastrointestinal disturbance, and rash. hemorrhagic phenomena can cause case fatality rates of up to %. epidemics can be explosive, but adequate treatment can greatly reduce the number of deaths. dengue occurs in southeast asia, the pacific islands, australia, west africa, the caribbean, and central and south america. an epidemic in cuba in included more than , cases, and deaths. vector control of the a. aegypti mosquito resulted in control of the disease during the s- s, but reinfestation of mosquitoes led to incresased transmission and epidemics in the pacific islands, caribbean, central and south america in the s and s. outbreaks in vietnam included , cases in , another , cases in , and a similar sized outbreak in . indonesia had over , cases in with deaths, and in over , cases (january-may) with at least deaths. in , epidemics of dengue were reported in fiji, the cook islands, new caledonia, and northern australia. the who estimates , deaths and . million cases worldwide in . monkeys are the main reservoir, and the vector is the a. aegypti mosquito. no vaccine is currently available, and management is by vector control. lassa fever. lassa fever was first isolated in lassa, nigeria, in and is widely distributed in west africa, with , - , cases and deaths annually. it is spread by direct contact with blood, urine, or secretions of infected rodents and by direct person-to-person contact in hospital settings. the disease is characterized by a persistent or spiking fever for - weeks, and may include severe hypotension, shock, and hemorrhaging. the case fatality rate is %. marburg disease. marburg disease is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first seen in marburg, germany, in , following ex-posure to green monkeys. person-to-person spread occurs via blood, secretions, organs, and semen. case fatality rates can be over %. ebola fever. ebola fever is a viral disease with sudden onset of generalized illness, malaise, fever, myalgia, headache, diarrhea, vomiting, rash, and hemorrhages. it was first found in zaire and sudan in in outbreaks which killed more than persons. it is spread from person to person by the blood, vomitus, urine, stools, and other secretions of sick patients, with a short incubation period. the disease has case fatality rates of up to %. an outbreak of ebola among laboratory monkeys in a medical laboratory near washington, d.c., was contained with no human cases. the reservoir for the virus is thought to be rodents. an outbreak of ebola in may in the town of kikwit, zaire, killed persons out of cases ( % case fatality rate). this outbreak caused international concern that the disease could spread, but it remained localized. another outbreak of ebola virus occurred in gabon in early , with cases, of whom had direct exposure to an infected monkey, the remainder by human-to-human contact, or not established; of the cases died ( %). this disease is considered highly dangerous unless outbreaks are effectively controlled. in zaire, lack of basic sanitary supplies, such as surgical gloves for hospitals, almost ensures that this disease will spread when it recurs. lyme disease is characterized by the presence of a rash, musculoskeletal, neurologic, and cardiovascular symptoms. confirmation is by laboratory investigation. it is the most common vector-borne disease in the united states, with , cases reported between and . it primarily affects children in the - age group and adults aged - . lyme disease is preventable by avoiding contact with ticks, by applying insect repellant, wearing long pants and long sleeves in infected areas, and by the early removal of attached ticks. several u.s. manufacturers produced vaccines which are approved for animal and human use. in the mid s, a mother of two young boys who were recently diagnosed with arthritis in the town of lyme, connecticut, conducted a private investigation among other town residents. she mapped each of the six arthritis cases in the town, cases which had occurred in a short time span among boys living in close proximity. this suggested that this syndrome of "juvenile rheumatoid arthritis" was perhaps connected with the boys playing in the woods. she presented her data to the head of rheumatology at yale medical school in new haven, who investigated this "cluster of a new disease entity." some parents reported that their sons had experienced tick bites and a rash before onset of the arthritis. a tick-borne, spiral shaped bacterium, a spirochete, borrelia burgdorferi, was identified as the organism, and ticks shown to be the vector. cases repond well to antibiotic therapy. in over , cases ( . per , ) were reported from states, an increase from , in and , in . cases were mainly located in the northeast, north central, and mid-atlantic regions. the disease accounts for over % of vector-borne disease in the united states and was the ninth leading reported infection in . lyme disease has been identified in many parts of north america, europe, the former soviet union, china, and japan. a newly licensed vaccine is effective for people exposed to ticks but not general usage. personal hygiene for protection from ticks and environmental modification are important to limit spread of the disease. source: cdc, , mmwr, : - ; and cdc, , mmwr, , no. . lyme disease website http://www.cdc.gov/ncidad/disease/lyme/lyme.htm medically important parasites are animals that live, take nourishment, and thrive in the body of a host, which may or may not harm the host, but never brings benefit. they include those caused by unicellular organisms such as protozoa, which include amoebas (malaria, schistosomiasis, amebiasis, and cryptosporidium), and helminths (worms), which are categorized as nematodes, cestodes, and trematodes. public health continues to face the problems of parasitic diseases in the developing world. increasingly, parasitic diseases are being recognized in industrialized countries. giardiasis and cryptosporidium infections in waterborne and other outbreaks have occurred in the united states. parasitic diseases are among the most common causes of illness and death in the world, e.g., malaria. milder illnesses such as giardiasis and trichomoniasis cause widespread morbidity. intestinal infestations with worms may cause of severe complications, although they commonly cause chronic low-grade symptomatology and iron deficiency anemia. echinococcosis (hydatid cyst disease) is infection with echinococcus granulosus, a small dog tapeworm. the tapeworm forms unilocular (single, noncompartmental) cysts in the host, primarily in the liver and lungs, but they can also grow in the kidney, spleen, central nervous system, or in bones. cysts, which may grow up to cm in size, may be asymptomatic or, if untreated, may cause severe symptoms and even death. this parasite is common where dogs are used with herd grazing animals and also have intimate contact with humans. the middle east, greece, sardinia, north africa, and south america are endemic areas, as are a few areas in the united states and canada. the human dis-ease has been eliminated in cyprus and australia. while the dog is the major host, intermediate hosts include sheep, cattle, pigs, horses, moose, and wolves. preventive measures include education in food and animal contact hygiene, destroying wild and stray dogs, and keeping dogs from the viscera of slaughtered animals. a similar, but multilocular, cystic hydatid disease is widely found in wild animal hosts in areas of the northern hemisphere, including central europe, the former soviet union, japan, alaska, canada, and the north-central united states. another echinococcal disease (echinococcus vogeli) is found in south america, where its natural host is the bush dog and its intermediate host is the rat. the domestic dog also serves as a source of human infection. surgical resection is not always successful, and long-term medical treatment may be required. control is through awareness and hygiene as well as the control of wild animals that come in contact with humans and domestic animals. control may require cooperation between neighboring countries. tapeworm infestation (taeniasis) is common in tropical countries where hygienic standards are low. beef (taenia saginata) and pork (t. solium) tapeworms are common where animals are fed with water or food exposed to human feces. freezing or cooking meat will destroy the tapeworm. fish tapeworm (diphyllobothrium latum) is common in populations living primarily on uncooked fish, such as inuit people. these tapeworms are usually associated with northern climates. toddlers are especially susceptible to dog tapeworm (dipylidium caninum), which is present worldwide, and domestic pets are often the source of oral-fecal transmission of the eggs. the disease is usually asymptomatic. similarly, dwarf tapeworm (hymenolepis nana) is transmitted through oral-fecal contamination from person to person, or via contaminated food or water. rat tapeworm (hymenolepis diminuta) also mostly affects young children. onchocerciasis (fiver blindness) is a disease caused by a parasitic worm, which produces millions of larvae that move through the body causing intense itching, debilitation, and eventually blindness. the disease is spread by a blackfly that transmits the larva from infected to uninfected people. it is primarily located in sub-saharan africa and in latin america, with over million persons at risk. control is by a combination of activities including environmental control by larvicidal sprays to reduce the vector population, protection of potential hosts by protective clothing and insect repellents, and case treatment. a who-initiated program for onchocerciasis control started in is sponsored by four international agencies: the food and agriculture organization (fao), the united nations development program (undp), the world bank, and who. it covers countries in sub-saharan africa, focusing on control of the blackfly by destoying its larvae, mainly via insecticides sprayed from the air. prevalence in was reported by who as over million persons. the program has been successful in protecting some million persons and helping . million infected persons to recover from this disease. who estimates that the program will have prevented , cases of blindness by the year and has freed million hectares of land for resettlement and cultivation. the program cost $ million. this investment is considered by the world bank to have a return of - % in terms of large scale land reuse and improved output of the population. a who program, the african program for onchocerciasis control (apoc), started in , uses a new drug (ivermectin) and selective vector control efforts by spraying. this involves countries in africa, and in a similar program in south america. see website http://www/who.int/ocp and is financed by many donor countries, internation organizations, merck & company, and ngos. dracunculiasis (guinea worm disease) is a parasitic disease of great public health importance in india, pakistan, and central and west africa. it is an infection of the subcutaneous and deeper tissues caused by a large ( cm) nematode, usually affecting the lower extremities and causing pain and disability. the nematode causes a burning blister on the skin when it is ready to release its eggs. after the blister ruptures, the worm discharges larvae whenever the extremity is in water. the eggs are ingested in contaminated water and the larva released migrate through the viscera to locate as adults in the subcutaneous tissue of the leg. incubation is about months. the larva released in water are ingested by minute crustaceans and remain infective for as long as a month. prevention is based on improving the safety of water supplies and by preventing contamination by infected persons. education of persons in endemic areas to stay out of water sources and to filter drinking water reduces transmission. insecticides remove the crustaceans. chlorine also kills the larvae and the crustaceans which prologue larval infectivity. there is no vaccine. treatment is helpful, but not definitive. dracunculiasis was traditionally endemic in a belt from west africa through the middle east to india and central asia. it was successfully eliminated from central asia and iran and has disappeared from the middle east and from some african countries (gambia and guinea). the world health organization has promoted the eradication of dracunculiasis. major progress has been made in this direction. worldwide prevalence is reported to have been reduced from million cases in to million in , , in , and , cases in . eradication was anticipated for the year , and in the who established a commission to monitor and certify eradication in formerly endemic areas. india's reported cases fell from , in to in , and the country was free of transmission in . in , formerly high prevalence countries such as kenya reported no cases in , while chad, senegal, cameroons, yemen, and the central african republic less than cases each. eradication of this disease appears to be imminent. the who eradication program was developed successfully as an independent program with its own direction and field staff, but further progress will require the integration of this program with other basic primary care programs in order to be self-sustaining as an integral part of community health. community-based surveillance systems for this disease are being converted to work for monitoring of other health conditions in the community. schistosomiasis (snail fever or bilharziasis) is a parasitic infection caused by the trematode (blood fluke) and transmitted from person to person via an intermediate host, the snail. it is endemic in countries in africa, south america, the caribbean, and asia. there are an estimated million persons infected worldwide and more than million at risk for the disease. the clinical symptoms include fever, nausea, vomiting, abdominal pain, diarrhea, and hematuria. the organisms schistosoma mansoni and s. japonicum cause intestinal and hepatic symptoms, including diarrhea and abdominal pain. schistosoma haematobium affects the genitourinary tract, causing chronic cystitis, pyelonephritis, with high risk for bladder cancer the ninth most common cause of cancer deaths globally. infection is acquired by skin contact with freshwater containing contaminated snails. the cercariae of the organism penetrate the skin, and in the human host it matures into an adult worm that mates and produces eggs. the eggs are disseminated to other parts of the body from the worm's location in the veins surrounding the bladder or the intestines, and may result in neurological symptoms. eggs may be detected under microscopic examination of urine and stools. sensitive serologic tests are also available. treatment is effective against all three major species of schistosomiasis. eradication of the disease can be achieved with the use of irrigation canals, prevention of contamination of water sources by urine and feces of infected persons, treatment of infected persons, destruction of snails, and health education in affected areas. persons exposed to freshwater lakes, streams, and rivers in endemic areas should be warned of the danger of infection. mass chemotherapy in communities at risk and improved water and sanitation facilities are resulting in improved control of this disease. leishmaniasis causes both cutaneous and visceral disease. the cutaneous form is a chronic ulcer of the skin, called by various names, e.g., rose of jericho, oriental sore, and aleppo boil. it is caused by leishmania tropica, l. brasiliensis, l. mexicana, or the l. donovani complex. this chronic ulcer may last from weeks to more than a year. diagnosis is by biopsy, culture, and serologic tests. the organism multiplies in the gut of sandflies (phlebotomus and lutzomi) and is transmitted to humans, dogs, and rodents through bites. the parasites may remain in the untreated lesion for - months, and the lesion does not heal until the parasites are eliminated. prevention is through limiting exposure to the phlebotomines and reducing the sandfly population by environmental control measures. insecticide use near breeding places and homes has been successful in destroying the vector sandflies in their breeding places. case detection and treatment reduce the incidence of new cases. there is no vaccine, and treatment is with specific antimonials and antibiotics. visceral leishmaniasis (kala azar) is a chronic systemic disease in which the parasite multiplies in the cells of the host's visceral organs. the disease is characterized by fever, the enlargement of the liver and spleen, lymphadenopathy, anemia, leukopenia, and progressive weakness and emaciation. diagnosis is by culture of the organism from biopsy or aspirated material, or by demonstration of intracellular (leishman-donovan) bodies in stained smears from bone marrow, spleen, liver, or blood. kala azar is a rural disease occurring in the indian subcontinent, china, the southern republics of the former u.s.s.r., the middle east, latin america, and sub-saharan africa. it usually occurs as scattered cases among infants, children, and adolescents. transmission is by the bite of the infected sandfly with an incubation period of - months. there is no vaccine, but specific treatment is effective and environmental control measures reduce the disease prevalence. this includes the use of antimalarial insecticides. in localities where the dog population has been reduced, the disease is less prevalent. sleeping sickness. sleeping sickness a disease caused by trypanosoma brucei, transmitted but the tsetse fly, primarily in the african savannahs, affecting cattle and humans. some million persons are at risk in sub-saharan africa. who reported , new cases, a total prevalence of , cases, and , deaths from this disease in . prevention depends on vector control, and effective treatment of human cases. chagas disease is a chronic and incurable vector and blood transfusion borne parasitic disease (trypanosoma cruzi) which causes disability and death. it affects some million persons mainly in latin america, with some , new cases and , deaths occurring annually. about % of affected persons develop severe heart disease. brazil, which accounts for % of the cases prevalent in latin america, achieved elimination of transmission in , after uruguay ( ) and venezuela ( ) and followed by argentina ( ) . elimination of transmission is projected by who by the year . control is difficult, but control measures include reducing the animal host and vector insect population in its habitat by ecological and insectiside measures, education of the population in prevention by clothing, bednets, and repellents, and with chemotherapy for case management. amebiasis. amebiasis is an infection with a protozoan parasite (entamoeba histolytica) which exists as an infective cyst. infestation may be asymptomatic or cause acute, severe diarrhea with blood and mucus, alternating with constipation. amebic colitis can be confused with ulcerative colitis. diagnosis is by microscopic examination of fresh fecal specimens showing trophozoites or cysts. transmission is generally via ingestion of fecal-contaminated food or water containing cysts, or by oral-anal sexual practices. amebiasis is found worldwide. sand filtration of community water supplies removes nearly all cysts. suspect water should be boiled. education regarding hygienic practices with safe food and water handling and disposal of human feces are the basis for control. ascariasis. ascariasis is infestation of the small intestine with the roundworm ascaris lumbricoides, which may appear in the stool, occasionally the nose or mouth, or may be coughed up from lung infestation. the roundworm is very common in tropical countries, where infestation may reach or exceed % of the population. children aged - years are especially susceptible. infestation can cause pulmonary symptoms and frequently contributes to malnutrition, especially iron deficiency anemia. transmission is by ingestion of infective eggs, common among children playing in contaminated areas, or via the ingestion of uncooked products of infected soil. eggs may remain viable in the soil for years. vermox and other treatments are effective. prevention is through education, adequate sanitary facilities for excretion, and improved hygienic practices, especially with food. use of human feces for fertilizer, even after partial treatment, may spread the infestation. mass treatment is indicated in high prevalence communities. pinworm disease or enterobiasis. pinworm disease (oxyuriasis) is common worldwide in all socioeconomic classes; however, it is more widespread when crowded and unsanitary living conditions exist. the enterobius vermicularis infestation of the intestine may be symptomless or may cause severe perianal itching or vulvovaginitis. it primarily affects schoolchildren and preschoolers. more severe complications may occur. adult worms may be seen visually or identified by microscopic examination of stool specimens or perianal swabs. transmission is by the oral-fecal ingestion of eggs. the larvae grow in the small intestine and upper colon. prevention is by educating the public regarding hygiene and adequate sanitary facilities, as well as by treating cases and investigating contacts. treatment is the same as for ascariasis. mass treatment is indicated in high prevalence communities. ectoparasites. ectoparasites include scabies (sarcoptes scabiei), the common bed bug (cimex lectularius), fleas, and lice, including the body louse (pediculus humanis), pubic louse (phthirius pubis), and the head louse (pediculus humanus capitis). their severity ranges from nuisance value to serious public health hazard. head lice are common in schoolchildren worldwide and are mainly a distressing nuisance. the body louse serves as a vector for epidemic typhus, trench fever, and louse-borne relapsing fever. in disaster situations, disinfection and hygienic practices may be essential to prevent epidemic typhus. the flea plays an important role in the spread of the plague by transmitting the organism from the rat to humans. control of rats has reduced the flea population, but during war and disasters, rat and flea populations may thrive. scabies, which is caused by a mite, is common worldwide and is transmitted from person to person. the mite burrows under the skin and causes intense itching. all of these ectoparasites are preventable by proper hygiene and the treatment of cases. the spread of these diseases is rapid and therefore warrants attention in school health and public health policy. legionnelae, a gram-negative group of bacilli, with species and many serogroups. the first documented case was reported in the united states in , and the first disease outbreak was reported in the united states in among participants of a war veterans convention. general malaise, anorexia, myalgia, and headache are followed by fever, cough, abdominal pain, and diarrhea. pneumonia followed by respiratory failure may follow. the case fatality rate can be as high as % of hospitalized cases. a milder, nonpneumonic form of the disease (pontiac fever) is associated with virtually no mortality. the organism is found in water reservoirs and is transmitted through heating, cooling, and air conditioning systems, as well as from tap water, showers, saunas, and jaccuzzi baths. the disease has been reported in australia, canada, south america, europe, israel, and on cruise ships. prevention requires the cleaning of water towers and cooling systems, including whirlpool spas. hyperchlorination of water systems and the replacement of filters is required where cases and/or organisms have been identified. antibiotic treatment with erythromycin is effective. leprosy (hansen's disease) was widely prevalent in europe and mediterranean countries for many centuries, with some , leprosaria in the year . leprosy was largely wiped out during the black death in the fourteenth century, but continued in endemic form until the twentieth century. leprosy is a chronic bacterial infection of the skin, peripheral nerves, and upper airway. in the lepromatous form, there is diffuse infiltration of the skin nodules and macules, usually bilateral and extensive. the tuberculoid form of the disease is characterized by clearly demarcated skin lesions with peripheral nerve involvement. diagnosis is based on clinical examination of the skin and signs of peripheral nerve damage, skin scrapings, and skin biopsy. transmission of the mycobacterium leprae organism is by close contact from person to person, with incubation periods of between months and years (average of - years). rifampicin and other medications make the patient noninfectious in a short time, so that ambulatory treatment is possible. multidrug therapy (mdt) has been shown to be highly effective in combating the disease, with a very low relapse rate. treatment with mdt ensures that the bacillus does not develop drug resistance. mdt is covering % of known cases in , according to who reports, as compared to only % in . the increase has been associated with improved case finding. bcg may be useful in reducing tuberculoid leprosy among contacts. investigation of contacts over years is recommended. the disease is still highly endemic primarily in five countries, india, brazil, indonesia, myanmar, and bangladesh, and is still present in some countries in southeast asia, including the philippines and burma, sub-saharan africa, the middle east (sudan, egypt, iran), and in some parts of latin america (mexico, colombia) with isolated cases in the united states. world prevalence has declined from . million cases in , . million in , to less than million cases in . the world health organization expects to eliminate leprosy as a public health problem by the year , defined as prevalence of less than per , population, or less than , cases. trachoma is currently responsible for million blind persons or % of total blindness in the world. the causative organism, chlamydia trachomatis, is a bacteria which can survive only within a cell. it is spread through contact with eye discharges, usually by flies, or household items (e.g., handkerchiefs, washcloths). trachoma is common in poor rural areas of central america, brazil, africa, parts of asia, and some countries in the eastern mediterranean. the resulting infection leads to conjuncfival scarring and if untreated, to blindness. who estimates there are million cases of active disease in endemic countries. hygiene, vector control, and treatment with antibiotic eye ointments or simple surgery for scarring of eyelids and inturned eyelashes prevent the blindness. a new drug, azithromycin, is effective in curing the disease. the who is promoting a program for the global elimination of trachoma using azithromycin and hygiene education in endemic areas. chlamydia (chlamydia pneumonia) is suspected of playing a role in coronary artery disease by intraarterial infection, with plaque formation and occlusion of the artery by thrombi consisting mainly of platelets. if borne out, this will provide potential for low cost intervention to reduce the burden of the leading worldwide cause of death. sexually transmitted diseases (stds) are widespread internationally with an estimated million new cases per year, with . million new cases, over million total cases, and . million deaths ( ), aids has captured world attention over the past decade. the global burden of stds is enormous (table . ), and the public health and social consequences are devastating in many countries. sexually transmitted diseases, especially in women, may be asymptomatic, so that severe sequelae may occur before patients seek care. infection by one std increases risk of infection by other diseases in this group. syphilis is caused by the spirochete treponema pallidum. after an incubation period of - days (mean - ), primary syphilis develops as a painless ulcer or chancre on the penis, cervix, nose, mouth, or anus, lasting - weeks. the patient may first present with secondary syphilis - weeks (up to weeks) after infection with a general rash and malaise, fever, hair loss, arthritis, and jaundice. these symptoms spontaneously disappear within weeks or up to months later. tertiary syphilis may appear - years after initial infection. complications of tertiary syphilis include catastrophic cardiovascular and central nervous system conditions. early antibiotic treatment is highly effective when given in a large initial dose, but longer term therapy may be needed if treatment is delayed. gonorrhea (gc) is caused by the bacterium neisseria gonorrhoeae. the incubation period is - days. gonorrhea is often associated with concurrent chlamydia infection. in women, gc may be asymptomatic or it may cause vaginal discharge, pain on urination, bleeding on intercourse, or lower abdominal pain. untreated, it can lead to sterility. in men, gc causes urethral discharge and painful urination. treatment with antibiotics ends infectivity, but untreated cases can be infectious for months. drug resistance to penicillin and tetracycline has increased in many countries so that more expensive and often unavailable drugs are necessary for treatment. prevention of gonococcal eye infection in newborns is based on routine use of antibiotic ointments in the eyes of newborns. chancroid. chancroid is caused by haemophilus ducreyi. in women chancroids may cause a painful, irregular ulcer near the vagina, resulting in pain on in-tercourse, urination, and defection, but it may be asymptomatic. in men it causes a painful, irregular ulcer on the penis. the incubation period is usually - days, but may be up to days. an individual is infectious as long as there are ulcers, usually - months. treatment is by erythromycin or azithromycin. herpes simplex. herpes simplex is caused by herpes simplex virus types and and has an incubation period of - days. genital herpes causes painful blisters around the mouth, vagina, penis, or anus. the genital lesions are infectious for - days. herpes may lead to central nervous system meningoencephalitis infection. it can be transmitted to newborns during vaginal delivery, causing infection, encephalitis, and death. cesarian delivery is therefore necessary when a mother is infected. anti-viral drugs are used in treatment, orally, topically, or intravenously. chlamydia. chlamydia is caused by chlamydia trachomatis. in women, it is usually asymptomatic but may cause vaginal discharge, spotting, pain on urination, lower abdominal pain, and pelvic inflammatory disease (pid). in newborns, chlamydia may cause eye and respiratory infections. in men, chlamydia causes urethral discharge and pain on urination. the incubation period is - days and the infectious period is unknown. treatment for chlamydia is doxycycline, azithromycin, or erythromycin. chlamydia infection, not necessarily venereal in transmission, may be transmitted to newborns of infected mothers. chlamydia pneumoniae, presently under investigation as a possible cause or contributor to coronary heart disease, and is widespread in poor hygenic conditions. trichomoniasis. trichomoniasis is caused by trichomonas vaginalis. the incubation period is - days (mean = ). in women, trichomoniasis may be asymptomatic or may cause a frothy vaginal discharge with foul odor, and painful urination and intercourse. in men, the disease is usually mild, causing pain on urination. treatment is by metronidazole taken orally. without treatment, the disease may persist and remain infectious for years. (hpv). it is a sporadic disease which may be associated with cervical neoplasia and cancer of the cervix. hpv includes many types associated with a variety of conditons. the search for a hpv vaccine to prevent cancer of the cervix looks promising. in areas where a full range of diagnostic services is lacking, a "syndromic approach" is recommended for the control of stds. the diagnosis is based on a group of symptoms and treatment on a protocol addressing all the diseases that could possibly cause those symptoms, without expensive laboratory tests and repeated visits. early treatment without laboratory confirmation helps to cure persons who might not return for follow-up, or may place them in a noninfective stage so that even without follow-up they will not transmit the disease. std incidence between and is shown in table . , with decline overall except around , with subsequent further fall in incidence. screening in prenatal and family planning clinics, prison medical services, and selected years - disease syphilis ( [ ] [ ] [ ] [ ] [ ] [ ] and subsequent decline by more than % in reported cases includes all three stages of the disease as well as congential syphilis. rates are cases per , population, rounded. in clinics serving prostitutes, homosexuals, or other potential risk groups will detect subclinical cases of various stds. treatment can be carried out cheaply and immediately. for instance, the screening test for syphilis costs $ . and the treatment with benzathine penicillin injection costs about $ . in . partner notification is a controversial issue, but may be needed to identify contacts who may be the source of transmission to others. control of stds through a syndrome approaach based on primary care providers is being promoted by who. health education directed at high risk target groups is essential. providing easy and cost-free access to acceptable, nonthreatening treatment is vital in promoting the early treatment of cases and thereby reducing the risk of transmission. promoting prevention through the use of condoms and/or monogamy requires long-term educational efforts that are now fostered by the hiv/aids pandemic. increased use of condoms for hiv prevention is associated with reduced risk of other stds. training medical care providers in std awareness should be stressed in undergraduate and continuing educational efforts including personal protection as care givers. human immunodeficiency virus (hiv) is a retrovirus that infects various cells of the immune system, and also affects the central nervous system. two types have been identified: hiv , worldwide in distribution, and the less pathogenic hiv , found mainly in west africa. hiv is transmitted by sexual contact, exposure to blood and blood products, perinatally, and via breast milk. the period of communicability is unknown, but studies indicate that infectiousness is high, both during the initial period after infection and later in the disease. antibodies to hiv usually appear within - months. within several weeks to months of the infection, many persons develop an acute self-limited flulike syndrome. they may then be free of any signs or symptoms for months to more than years. onset of illness is usually insidious with nonspecific symptoms, including sweats, diarrhea, weight loss, and fatigue. aids represents the later clinical stage of hiv infection. according to the revised cdc case definition ( ), aids involves any one or more of the following: low cd count, severe systematic symptoms, opportunistic infections such as pneumocystis pneumonia or tb, aggressive cancers such as kaposi's sarcoma or lymphoma, and/or neurological manifestations, including dementia and neuropathy. the who case definition is more clinically oriented, relying less on often unavailable laboratory diagnoses for indicator diseases. aids was first recognized clinically in in los angeles and new york. by mid- it was considered an epidemic in those and other u.s. cities. it was primarily seen among homosexual men and recipients of blood products. after initial errors, testing of blood and blood products became standard and has subsequently closed off this method of transmission. transmission has changed markedly since the initial onslaught of the disease, with needle sharing among intravenous drug users, heterosexual, and maternal-fetal transmission becoming major factors. comorbidity with other stds apparently increases hiv infectivity and may have helped to convert the epidemiology to a greater degree of heterosexual transmission. the disease grew exponentially in the united states (table . ), but incidence of new cases nas declined since . aids has become a major public health problem in most developed and developing countries, reaching catastrophic proportions in some sub-saharan african countries affecting up to % of the population. hiv-related deaths were the eighth leading cause of all deaths in in the u.s., the leading cause among men aged - years of age, and the fourth leading cause for women in this age group. by , aids had been diagnosed in , persons and , had died. it is estimated that up to million persons are hiv infected in the united states. globally, deaths from aids totalled . million in , with an estimated . million person having died from this pandemic up to . in , an estimated . million person were hiv infected with . million new infection in . the declining incidence of new cases in the industrialized countries may be the result of greater awareness of the disease and methods of prevention of transmission. improving early diagnosis and access to care, especially the combined therapy programs that are very effective in delaying onset of symptoms, are important parts of public health management of the aids crisis. until an effective vaccine is available, preventive reliance will continue to be on behavior risk-reduction and other prevention strategies such as needle and condom distribution among high risk population groups. throughout the world, hiv continues to spread rapidly, especially in poor countries in africa, asia, and south and central america. the united nations reports that million persons are living with hiv/aids, % of them in developing countries, where transmission is % by heterosexual contact. every day, more than persons are infected, including children. in thailand, person in is now infected. in sub-saharan africa person in is infected, and in some cities as many as person in carries the virus. estimations of new infections per year in sub-saharan africa range from to million persons, while in asia the range is from . to . million new infected persons per year. lessons are still being learned from the aids pandemic. the explosive spread of this infection, from an estimated , people in to an anticipated million persons hiv infected, shows that the world is still vulnerable to pandemics of "new" infectious diseases. enormous movements of tourists, business people, truck drivers, migrants, soldiers, and refugees promote the spread of such diseases. widespread sexual exchange, traffic in blood products, and illicit drug use all promote the international potential for pandemics. war and massive refugee situations promote rape and prostitution, worsening the aids situation in some settings in africa. hiv has arrived in almost every country. however, there is the somewhat hopeful indication that the rate of increase, has slowed in the united states. this may be an indication either of higher levels of self-protective behavior, or that the most susceptible population groups have already been affected and the spread into the general population is at a slower rate. it is also possible that this may yet prove to be only a lull in the storm, as heterosexual contact becomes a more important mode of transmission. the eleventh international conference on aids, held in vancouver, canada, in july , reported signs that combinations of several drugs from among a number of antiretroviral medications are showing promise to suppress the aids virus in infected people. at a current annual price of $ , - , per patient, these sums well beyond the capacity of most developing countries. development of methods of measuring the hiv viral load have allowed for better evaluation of potential therapies and monitoring of patients receiving therapy. in developed countries, transmission by blood products has been largely controlled by screening tests; transmission among homosexuals has been reduced by safe sex practices; transmission to newborns has been reduced by recent therapeutic advances. safe sex practices and condom use may have helped in reducing heterosexual transmission. further advances in therapy and prevention with a vaccine are expected over the next decade. the hiv/aids pandemic is one of the great challenges to public health for the st century due to its complexity, its international spread, its sexual and other modes of transmission, its devastating and costly clinical effects, and its impact on parallel diseases such as tuberculosis, respiratory infections, and cancer. the cost of care for the aids patient can be very high. needed programs include home care and community health workers to improve nutrition and self-care, and mutual help among hiv carriers and aids patients. the ethical issues associated with aids are also complex regarding screening of pregnant women, newborns, partner notification, reporting, and contact tracing, as well as financing the cost of care. diarrheal diseases are caused by a wide variety of bacteria, parasites, and viruses (table . ) infecting the intestinal tract and causing secretion of fluids and dis- solved salts into the gut with mild to severe or fatal complications. in developing countries, diarrheal diseases account for half of all morbidity and a quarter of all mortality. diarrhea itself does not cause death, but the dehydration resulting from fluid and electrolyte loss is one of the most common causes of death in children worldwide. deaths from dehydration can be prevented by use of oral rehydration therapy (ort), an inexpensive and simple method of intervention easily used by a nonmedical primary care worker and by the mother of the child as a home intervention. in , diarrheal diseases were the cause of almost million child deaths, but by this had declined to . million, largely under the impact of increased use of ort. diarrheal diseases are transmitted by water, food, and directly from person to person via oral-fecal contamination. diarrheal diseases occur in epidemics in situations of food poisoning or contaminated water sources, but can also be present at high levels when common source contamination is not found. contamination of drinking water by sewage and poor management of water supplies are also major causes of diarrheal disease. the use of sewage for the irrigation of vegetables is a common cause of diarrheal disease in many areas. salmonella are a group of bacterial organisms causing acute gastroenteritis, associated with generalized illness including headache, fever, abdominal pains, and dehydration. there are over serotypes of salmonella, many of which are pathogenic in humans, the most common of which are salmonella typhimurium, s. enteritidis, and s. typhi. transmission is by ingestion of the organisms in food, derived from fecal material from animal or human contamination. common sources include raw or uncooked eggs, raw milk, meat, poultry and its products, as well as pet turtles or chicks. fecal-oral transmission from person to person is common. prevention is in safe animal and food handling, refrigeration, sanitary preparation and storage, protection against rodent and insect contamination, and the use of sterile techniques during patient care. antibiotics may not eliminate the carrier state and may produce resistant strains. shigella are a group of bacteria that are pathogenic in man, with four groups: type a = shigella dysenteriae, type b = s. flexneri, type c = s. boydii, and type d = s. sonnei. types a, b, and c are each further divided into a total of serotypes. shigella are transmitted by direct or indirect fecal-oral methods from a patient or carrier, and illness follows ingestion of even a few organisms. water and milk transmission occurs as a result of contamination. flies can transmit the organism, and in nonrefrigerated foods the organism may multiply to an infectious dose. control is in hygienic practices and in the safe handling of water and food. escheria eoli e. coli are common fecal contaminants of inadequately prepared and cooked food. particularly virulent strains such as o :h can cause explosive outbreaks of severe (enterohemmorhagic) diarrhoeal disease with a hemolytic-uremic syndrome and death, as occurred in japan in with cases and deaths due to a foodborne epidemic. other milder strains cause travellers diarrhoea and nursery infections. inadequately cooked hamburger, unpasturized milk, and other food vectors are discussed under food safety in chapter . cholera is an acute bacterial enteric disease caused by vibrio cholerae, with sudden onset, profuse painless watery stools, occasional vomiting, and, if untreated, rapid dehydration, and circulatory collapse, and death. asymptomatic infection or carrier status, and mild cases are common. in severe, untreated cases, mortality is over %, but with adequate treatment, mortality is under %. diagnosis is based on clinical signs, epidemiologic, serologic and bacteriologic confirmation by culture. the two types of cholera are the classic and el tor (with inaba and ogawa serotypes). in , a large scale epidemic of cholera spread through much of south america. it was imported via a chinese freighter, whose sewage contaminated shellfish in lima harbor in peru (box . ). the south american cholera epidemic has caused hundreds of thousands of cases and thousands of deaths since . prevention requires sanitation, particularly the chlorination of drinking water, prohibiting the use of raw sewage for the irrigation of vegetable crops, and high standards of community, food, and personal hygiene. treatment is prompt fluid therapy with electrolytes in large volume to replace all fluid loss. oral rehydration should be accomplished using standard ort. tetracycline shortens the duration of the disease, and chemoprophylaxis for contacts following stool samples may help in reducing its spread. a vaccine is available but is of no value in the prevention of outbreaks. viral gastroenteritis can occur in sporadic or epidemic forms, in infants, children, or adults. some viruses, such as the rotaviruses and enteric adenoviruses, af- in the s, peruvian officials stopped the chlorination of community water supplies because of concern over possible carcinogenic effects of trihalomethanes, a view encouraged by officials of the u.s. environmental protection agency (epa) and the u.s. public health service. in january , a chinese freighter arrived in lima, peru, and dumped bilge (sewage) in the harbor, apparently contaminating local shellfish. consumption of raw shellfish is a popular local delicacy (ceviche) and associated with cases of cholera seen in local hospitals. contamination of local water supplies from sewage resulted in the geometric increase in cases, and by the end of the pan american health organization (paho) reported an epidemic of , cases and deaths. the epidemic spread to countries, and in there were a further , cases and deaths spreading over much of south america, continuing in . in the united states, cases of cholera were reported in ; of these, cases and death were among passengers of an airplane flying from south america to los angeles in which contaminated seafood was served. in , cases of cholera were reported in the united states which were unrelated to international travel. these occurred mostly among persons consuming shellfish from the gulf coast with a strain of cholera similar to the south american strain, also possibly introduced in ship ballast. cholera organisms are reported in harbor waters in other parts of the united states (promed, , promed, . fect mainly infants and young children, and may be severe enough to cause hospitalization for dehydration. others such as norwalk and norwalk-like viruses affect older children and adults in self-limited acute gastroenteritis in family, institution, or community outbreaks. rotaviruses cause acute gastroenteritis in infants and young children, with fever and vomiting, followed by watery diarrhea and occasionally severe dehydration and death if not adequately treated. diagnosis is by examination of stool or rectal swabs with commercial immunologic kits. in both developed and developing countries, rotavirus is the cause of about one-third of all hospitalized cases for diarrheal diseases in infants and children up to age . most children in developing countries experience this disease by the age of years, with the majority of cases between and months. in developing countries, rotaviruses are estimated to cause over , deaths per year. the virus is found in temperate climates in the cooler months and in tropical countries throughout the year. breastfeeding does not prevent the disease but may reduce its severity. oral rehydration therapy is the key treatment. a live attenuated vaccine was approved by the fda in and adopted in the u.s. recommended routine vaccination programs for infants. adenoviruses. adenoviruses, norwalk, and a variety of other viruses (including astrovirus, calcivirus, and other groups) cause sporadic acute gastroenteritis worldwide, mostly in outbreaks. spread is by the oral-fecal route, often in hospital or other communal settings, with secondary spread among family contacts. food-borne and waterborne transmission are both likely. these can be a serious problem in disaster situations. no vaccines are available. management is with fluid replacement and hygienic measures to prevent secondary spread. giardiasis. giardiasis (caused by giardia lamblia) is a protozoan parasitic infection of the upper small intestine, usually asymptomatic, but sometimes associated with chronic diarrhea, abdominal cramps, bloating, frequent loose greasy stools, fatigue, and weight loss. malabsorption of fats and vitamins may lead to malnutrition. diagnosis is by the presence of cysts or other forms of the organism in stools, duodenal fluid, or in intestinal mucosa from a biopsy. this disease is prevalent worldwide and affects mostly children. it is spread in areas of poor sanitation and in preschool settings and swimming pools, and is of increasing importance as a secondary infection among immunocompromised patients, especially those with aids. waterborne giardia was recognized as a serious problem in the united states in the s and s, since the protozoa is not readily inactivated by chlorine, but requires adequate filtration before chlorination. person-to-person transmission in day-care centers is common, as is transmission by unfiltered stream or lake water where contamination by human or animal feces is to be expected. an asymptomatic carrier state is common. prevention relies on careful hygiene in settings such as day-care centers, filtration of public water supplies and the boiling of water in emergency situations. cryptosporidium. cryptosporidium parvum is a parasitic infection of the gastrointestinal tract in man, small and large mammals and vertebrates. infection may be asymptomatic or cause a profuse, watery diarrhea, abdominal cramps, general malaise, fever, anorexia, nausea, and vomiting. in immunosuppressed patients, such as persons with aids, it can be a serious problem. the disease is most common in children under years of age and those in close contact with them, as well as in homosexual men. diagnosis is by identification of the cryptosporidium or-ganism cysts in stools. the disease is present worldwide. in europe and the united states, the organism has been found in < to . % of individuals sampled. spread is common by person-to-person contact by fecal-oral contamination, especially in such settings as day-care centers. raw milk and waterborne outbreaks have also been identified in recent years. a large waterborne disease outbreak due to cryptosporidium occurred in milwaukee in described in chapter . management is by rehydration and prevention is by careful hygiene in food and water safety. helicobacter pylori. helicobacter pylori, first identified in , is a bacterium causally linked to duodenal ulcers and gastritis, contributing to high rates of gastric cancer (chapter ). it is an important example of the link between infection and chronic disease. this has enormous implications for prevention of cancer of the stomach, chronic peptic ulcers and large-scale use of hospitals and other medical resources (see chapter ). the control of diarrheal diseases requires a comprehensive program involving a wide range of activities, including good management of food and water supplies, education in hygiene, and, particularly where morbidity and mortality are high, education in the use of oral rehydration therapy (ort). oral rehydration therapy (ort) is considered by unicef and who to have resulted in the saving of million lives each year in the s. proper management of an episode of diarrhea by ort (table . ), along with continued feeding, not only saves the child from dehydration and immediate death, but also contributes to early restoration of nutritional adequacy, sparing the child the prolonged effects of malnutrition. the world summit for children (wsc) in called for a reduction in child deaths from diarrheal diseases by one-third and malnutrition by one-half, with em- phasis on the widest possible availability, education for, and use of ort. this requires a programmatic approach. public health leadership must train primary care doctors, pediatricians, pharmacists, drug manufacturers, and primary care health workers of all kinds in ort principles and usage. they must be backed by the widest possible publicity to raise awareness among parents. oral rehydration therapy is an important public health modality in developed countries as well as in developing countries. diarrhoeal disease may not cause death as frequently in developed countries, but it is still a significant factor in infant and child health and, even under the most optimal conditions, can cause setbacks in the nutritional state and physical development of a child. use of ort does not prevent the disease (i.e., it is not a primary prevention), but it is excellent in secondary prevention, by preventing complications from diarrhoea, and should be available in every home for symptomatic treatment of diarrheal diseases. an adaptation of ort has found its place in popular culture in the united states. a form of ort, marketed as "sports drinks," is used in sports where athletes lose large quantifies of water and salts in sweat and insensible loss from the respiratory tract. the wider application of the principles of ort for use in adults in dry hot climates and in adults under severe physical exertion with inadequate fluid/salt intake situations requires further exploration. management of diarrheal diseases should be part of a wider approach to child nutrition. the child who goes through an episode of diarrheal disease may have a faltering in growth and development. supportive measures may be needed following the episode as well as during it. this involves providing primary care services that are attuned to monitoring individual infant and child growth. growth monitoring surveillance is important to assess the health status of the individual child and the child population. supplementation of infant feeding with vitamins a and d, and iron to prevent anemia are important for routine infant and child care, and more so for conditions affecting total nutrition such as a diarrheal disease. in the developing world, respiratory infections account for over one-quarter of all deaths and illnesses in children. as diarrheal disease deaths are reduced, the major cause of death among infants in developing countries is becoming acute respiratory infections (aris). in industrialized countries, aris are important for their potentially devastating effects on the elderly and chronically ill. they are also the major cause of morbidity in infants in developed countries, causing much anxiety to parents even in areas with good living conditions. cigarette smoking, chronic bronchitis, poorly controlled diabetes or congestive heart failure, and chronic liver and kidney disease increase susceptibility to aris. aris place a heavy burden on health care systems and individual families. improved methods of management of such chronic diseases are needed to reduce the associated toll of morbidity, mortality, and the considerable expenses of health care. acute respiratory infections are due to a broad range of viral and, to a lesser extent, bacterial infections. it is the latter which can progress to pneumonia with mortality rates of - %. acute viral respiratory diseases include those affecting the upper respiratory tract, such as acute viral rhinitis, pharyngitis, and laryngitis, as well as those affecting the lower respiratory tract, tracheobronchitis, bronchitis, bronchiolitis, and pneumonia. aris are frequently associated with vaccine-preventable diseases, including measles, varicella, and influenza. they are caused by a large number of viruses, producing a wide spectrum of acute respiratory illness. some organisms affect any part of the respiratory tract, while others affect specific parts and all predispose to bacterial secondary infection. while children and the elderly are especially susceptible to morbidity and mortality from acute respiratory disease, the vast numbers of respiratory illnesses among adults cause large-scale economic loss from work absence. bacterial agents causing upper respiratory tract infection include group a streptococcus, mycoplasma pneumonia, pertussis, and parapertussis. pneumonia or acute bacterial infection of the lower respiratory tract and lung tissue may be due to pneumococcal infection with streptococcus pneumoniae. there are known types of this organism, distinguished by capsule characteristics; account for % of pneumococcal infections in the united states. an excellent polyvalent vaccine based on these types is available for high risk groups such as the elderly, immunodeficient patients, and persons with chronic heart, lung, liver, blood disorders, or diabetes. opportunistic infections attack the chronically ill, especially those with compromised immune suystems, often with life-threatening aris. mycoplasma (primary atypical pneumonia) is a lower respiratory tract infection which sometimes progresses to pneumonia. tb and pneumonocytis carynia are especially problematic for aids patients. other organisms causing pneumonias include chlamydia pneumoniae, h. influenza, klebsiella pneumonia, escherichia coli, staphylococcus, rickettsia (q fever), and legionella. parasitic infestation of lungs may occur with nematodes (e.g., ascariasis). fungal infections of the lung may be caused by aspergillosis, histoplasmosis, and coccidiomycosis, often as a complication of antibiotic therapy. access to primary care and early institution of treatment are vital to control excess mortality from aris. in developed countries, aris as contributors to infant deaths are largely a problem in minority and deprived population groups. because these groups contribute disproportionately to childhood mortality, infant mortality reduction has been slower in countries such as the united states and russia than in other industrialized countries. the continuing gap in mortality rates between white and black children in the united states can, to a large extent, be attributed to aris and less access to organized primary care. children are brought to emergency rooms for care when the disease process is already advanced and more dangerous than had it been attended to professionally earlier in the process. many field trials of ari prevention programs have been proved successful involving parent education and training of primary care workers in early assessment and, if necessary, initiation of treatment. this needs field testing in multiple settings. reliance on vaccines to prevent respiratory infectious diseases is not currently feasible. aris are caused by a very wide spectrum of viruses, and the development of vaccines in this field has been slow and limited. the vaccine for pneumococcal pneumonia has been an important breakthrough, but it is still inadequately utilized by the chronically ill because of its limitations, costs, and lack of sufficient awareness, and it is too expensive for developing countries. improvements in bacterial and viral vaccine development will potentially help to reduce the burden of aris. a programmatic approach with clinical guidelines and education of family and care givers is currently the only feasible way to reduce the still enormous morbidity and mortality from aris on the young and the elderly. the success of sanitation vaccines and antibiotics led many to assume that all infectious diseases would sooner or later succumb to public health and medical technology. unfortunately, this is a premature and even dangerous assumption. despite the longstanding availability of an effective and inexpensive vaccine, the persistence of measles as a major killer of million children per year represents a failure in effective use of both the vaccine and the health system. the resurgence of tb and malaria have led to new strategies, such as managed or directly observed care, with community health workers to assure compliance needed to render the patient noninfectious to others and to reduce the pool of carriers of the disease. current successes in reducing poliomyelitis, dracunculiasis, onchocerciasis, and other diseases to the point of eradication has raised hopes for similar success in other fields. but there are many infectious diseases of importance in developed and developing countries where existing technologies are not fully utilized. oral rehydration therapy (ort) is one of the most cost-effective methods of preventing excess mortality from ordinary diarrheal diseases, and yet is not used on sufficient scale. biases in the financing and management of medical insurance programs can result in underutilization of available effective vaccines. hospital-based infections cause large-scale increases in lengths of stay and expenditures, although application of epidemiologic investigation and improved quality in hospital practices could reduce this burden. control of the spread of aids using combined medical therapies is not financially or logistically possible in many countries, but education for "safe sex" is effective. community health worker programs can greatly enhance tuberculosis, malaria, and std control, or in aids care, promote prevention and appropriate treatment. in the industrialized and mid-level developing countries, epidemiologic and demographic shifts have created new challenges in infectious disease control. prevention and early treatment of infectious disease among the chronically ill and the elderly is not only a medical issue, it is also an economic one. patients with chronic obstructive lung disease (copd), chronic liver or kidney disease, or congestive heart failure are at high risk of developing an infectious disease followed by prolonged hospitalization. public health has addressed, and will continue to stress the issues of communicable disease as one of its key issues in protecting individual and population health. methods of intervention include classic public health through sanitation, immunization, and well beyond that into nutrition, education, case finding, and treatment, and changing human behavior. the knowledge, attitudes, beliefs, and practices of policy makers, health care providers, and parents is as important in the success of communicable disease control as are the technology available and methods of financing health systems. together, these encompass the broad programmatic approach of the new public health to control of communicable diseases. in a world of rapid international transport and contact between populations, systems are needed to monitor the potential explosive spread of pathogens that may be transferred from their normal habitat. the potential for the international spread of new or reinvigorated infectious diseases constitute threat to mankind akin to ecological and other man-made disasters. the eradication of smallpox paved the way for the eradication of poliomyelitis, and perhaps measles, in the foreseeable future. new vaccines are showing the capacity to reduce important morbidity from rubella syndrome, mumps, meningitis, and hepatitis. other new vaccines on the horizon will continue the immunologic revolution into the twenty-first century. as the triumphs of control or elimination of infectious diseases of children continue, the scourge of hiv infection continues with distressingly slow progess an effective vaccine or cure for the disease it engenders. partly as a result of the hiv/ aids, tb staged a comeback in many countries where it was thought to be merely a residual problem. at the same time an old/new method of intervention using directly observed short-term therapy has shown great success in controlling the tb epidemic. the resurgence of tb is more dangerous in that mdrtb has become a widespread problem. this issue highlights the difficulty of keeping ahead of drug resistance in the search for new generations of antibiotics, posing a difficult challenge for the pharmaceutical industry, basic scientists as well as public health workers. the burden of infectious diseases has receded as the predominant public health problem in the developed countries but remains large in the developing countries. with increases in longevity and increased importance of chronic disease in the health status of the industrial and mid-level developing nations, the effects of infectious disease on the care of the elderly and chronically ill is of great importance in the new public health. long-term management of chronic disease needs to address the care of vulnerable groups, promoting the use of existing vaccines and antibiotics. most important is the development of health systems that provide close monitoring of groups at special risk for infectious disease, especially patients with chronic diseases, the immunocompromised, and the elderly. the combination of traditional public health with direct medical care needed for effective control and eradication of communicable diseases is an essential element of the new public health. the challenge is to apply a comprehensive approach and management of resources to define and reach achievable targets in communicable disease control. access to e-mail and the internet are vital to current practice of public health and nowhere is this more important than in communicable diseases. there are many such information sites and these will undoubtedly expand in the coming years. several sites are given as examples. the internet has great practical implications for keeping up to date with rapidly occurring events in this field. outstanding encyclopedia database on infectious diseases (available via mdcassoc@ix.netcom.com at reduced price for promed users, and free to sub-saharan african sites) promed is an excellent, free report on current events in communicable diseases internationally; join via owner-promed @usa recommended readings centers for disease control. . update: international task force for disease eradication addressing emerging infectious disease threats: a prevention strategy for the united states. executive summary update: trends in aids incidence--united states one thousand days until the target date for global poliomyelitis eradication tuberculosis morbidity--united states measles--united states, . morbidity and mortality weekly report national adult immunization awareness week--october - , recommended readings ; and influenza and pneumococcal vaccination levels among adults aged --- years impact of the sequential ipv/opv schedule on vaccination cover-agemunited states advances in global measles control and elimination: summary of the international meeting recommended childhood immunization schedulemunited states impact of vaccines universally recommended for childrenmunited states progress toward global poliomyelitis eradication global disease elimination and eradication as public health strategies childhood immunizations rotavirus vaccines: who position paper. weekly epidemiologic record infectious diseases of humans: dynamic and control vaccines and world health: science, policy, and practice control of communicable diseases manual jawetz, melnick and adelberg's medical microbiology, twenty-first edition preventive medicine and public health, second edition efficacy of bcg vaccine in the prevention of tuberculosis. meta-analysis of the published literature manson's tropical diseases vaccination and world health principles and practice oflnfectious diseases immunization of adolescents: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the combination vaccines for childhood immunization: recommendations of the advisory committee on immunization practices, the american academy of pediatrics, the american academy of family physicians and the poliomyelitis prevention: revised recommendations for use of inactivated and live oral poliovirus vaccines diphtheria outbreakmrussian federation rubella and congenital rubella syndrome~united states compendium of animal rabies control, : national association of state public health veterinarians progress toward elimination of haemophilus influenzae type b disease among infants and children in the united states tetanus surveillance~united states, - recommendations and reports--vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of measles: recommendations of the advisory committee on immunization practices national, state and urban area vaccination coverage levels among children aged - months~united sates varicella related deaths among children--united states progress toward global poliomyelitis eradication ten great public health achievements--united states a ten-year experience in control of poliomyelitis through a combination of live and killed vaccines in two developing areas measles control in developing and developed countries: the case for a two-dose policy integration of vitamin a supplementation with immunization. weekly epidemiological record update cholera--western hemisphere, . morbidity and mortality weekly report isolation of vibrio cholerae o from oystersmmobile bay, - estimates of future global tuberculosis morbidity and mortality arbovirus disease--united states ~:~ other communicable diseases update: outbreak of legionnaire's disease associated with a cruise ship rift valley fever--egypt the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices update: trends in aids incidence--united states case definition for infectious conditions under public health surveillance guidelines for treatment of sexually transmitted diseases primary and secondary syphilis--united states global tuberculosis incidence and mortality during the th century pandemic: need for surveillance and research escherichia coli o :h diarrhoea in the united states: clinical and epidemiologic features the state of the world's children the rational use of drugs in the management of acute diarrhoea in children world health organization. . the malaria situation in aids: images of the epidemic. geneva: who. world health organization progress toward the elimination of leprosy as a public health problem the world health report : fighting disease, fostering development the world health report health for all in the twenty-first century. eb / . geneva: who. world health organization. . the world health report : life in the twenty-first century: a vision for all world health organization. . the world health report : making a difference key: cord- -iluqwwxs authors: lessler, justin; cummings, derek a. t. title: mechanistic models of infectious disease and their impact on public health date: - - journal: american journal of epidemiology doi: . /aje/kww sha: doc_id: cord_uid: iluqwwxs from the s through the s, lowell reed and wade hampton frost used mathematical models and mechanical epidemic simulators as research tools and to teach epidemic theory to students at the johns hopkins bloomberg school of public health (then the school of hygiene and public health). since that time, modeling has become an integral part of epidemiology and public health. models have been used for explanatory and inferential purposes, as well as in planning and implementing public health responses. in this article, we review a selection of developments in the history of modeling of infectious disease dynamics over the past years. we also identify trends in model development and use and speculate as to the future use of models in infectious disease dynamics. from the s through the s, lowell reed and wade hampton frost used mathematical models and mechanical epidemic simulators as research tools and to teach epidemic theory to students at the johns hopkins bloomberg school of public health (then the school of hygiene and public health) ( , ) . though never published by reed and frost (versions of the model were eventually published by their students ( , ) ), their model was one of the first mechanistic models of infectious disease transmission, and at a time long before digital computing, they may have been the first to use simulation methods to understand the epidemic process. reed and frost were pioneers in the study of infectious disease dynamics using mechanistic models, a field of epidemiology that has developed in parallel with the associative statistical models and methods of causal inference that dominate much of epidemiologic research. over the past century, mechanistic models have played an essential role in shaping public health policy, the way we study interventions aimed at controlling infectious diseases, and the theory on which disease control is based. mechanistic models differ from traditional statistical models such as regression models because their structure makes explicit hypotheses about the biological mechanisms that drive infection dynamics. such hypotheses range from simple representations of the time it takes to complete some part of the disease process (e.g., sartwell's lognormal representation of the incubation period ( )) to complex agent-based models that attempt to explicitly represent social interactions of people in an entire country ( , ) or even the world ( ) . regardless of scale, approach, and complexity, these models have more of the flavor of models in physics than the statistical models that are used in other branches of epidemiology, and in many cases they can be used to predict the effectiveness of hypothetical interventions in controlling disease spread. perhaps the first mechanistic model of infectious disease transmission used in assessing intervention strategies was a mathematical model of malaria transmission developed and refined by ronald ross in a series of papers published between and ( ) ( ) ( ) , pre-dating the work of reed and frost by decades. this model had a direct and powerful message for public health: malaria could be controlled and even eliminated through mosquito control, even if the vector could not be completely eliminated. ross used his theoretical framework to develop and advocate for multiple indices, including the prevalence rate and the entomological inoculation rate ( ) , that could effectively characterize the intensity of transmission in an area and identify goals for control. in the wake of the founding of the global malaria eradication program by the world health organization, george macdonald ( ) extended ross's work in order to justify the use of insecticide as a tool for global malaria eradication ( ) . in particular, he showed that increasing daily mosquito mortality from % to % would be adequate to eliminate malaria even in locations with the highest transmission intensities in africa. mechanistic models continue to play an important role in the fight against malaria. the work of ross and macdonald looms large to this day, with a recent review finding that the majority of models published since depart from central hypotheses of the ross-macdonald model in only a few key assumptions, if any ( ) . although there are numerous instances over the past century in which mechanistic models have contributed to the control of a single disease (see figure for some examples), their larger contribution may be in our general understanding of disease control. the prime example is the concept of herd immunity and the critical vaccination threshold. herd immunity is the indirect protection offered to members of the population susceptible to the disease (i.e., not immune and with the potential to be infected) by the immunity of surrounding individuals, and the critical vaccination threshold is the percentage of the population that must be vaccinated in order for the introduction of an infectious case to not spark an epidemic ( ) . to estimate the critical vaccination threshold, we must first understand one of the most critical concepts of infectious disease dynamics, the basic reproductive number, r . r is the number of cases that a single infectious individual is expected to cause in a fully susceptible population. this concept was first introduced in demography and underwent significant development by lotka while on a visit to the johns hopkins university school of hygiene and public health in (see heesterbeek ( ) for a full history of the development of r in infectious disease). although this value does vary by setting, for many pathogens it is remarkably consistent across contexts and serves as a rough quantification of pathogen transmissibility. based on dynamic models, it has been shown that if we vaccinate a proportion of the population equivalent to − /r , then the pathogen will fail to spread in that population. this is the critical vaccination threshold, and it has helped to set vaccination goals for a number of diseases, particularly when elimination is the goal. however, the dynamics of vaccines in real populations are complex, and mechanistic models have helped us to understand what to expect after changes in vaccination policy. for instance, immediately after the introduction of a vaccine or improvement in vaccination rates, a disease may appear to be eliminated from a population. however, this long honeymoon period may be followed by a large, resurgent, outbreak that bigger than the yearly epidemics seen before the introduction of vaccination (though the cumulative number of cases is still less than what would have been seen without vaccination) ( ) . these results have helped public health officials to understand that initial apparent vaccine successes may not last, as well as what to expect after introducing a new vaccine. mechanistic models have also been used to understand the optimal age range for vaccination campaigns ( , ) , how such campaigns should be timed ( ) , and how best to use vaccines when supplies are limited ( , ) . models have also been used to design active response strategies for vaccine use, including ring vaccination strategies such as those implemented in the smallpox eradication campaign ( ) . models were also used to assess strategies to respond to a bioterrorist release of smallpox in the early part of the st century and were influential in setting policy for response ( ) ( ) ( ) ( ) . one counterintuitive prediction of mechanistic models is that in rare cases, increased population immunity from vaccination can actually increase the incidence of severe disease. the poster child for the phenomenon is congenital rubella syndrome (crs). for most people, rubella infection causes a relatively minor infection characterized by fever and rash; however, when pregnant women are infected during the first trimester of pregnancy, it cause crs, which results in severe complications of pregnancy including congenital disorders and death of the fetus ( ) . because vaccination increases the average age of infection (by decreasing the hazard of infection), a vaccination program that does not achieve sufficient coverage can increase the number of pregnant women who are infected, thereby increasing incidence of crs ( ) . this is not simply a theoretical concept; although there have been no sustained increases in the incidence of crs (in part due to the public health response), both costa rica and greece experienced transient increases in crs burden after rubella vaccination ( , ) . in light of the threat of crs, mechanistic models have played an important role in setting world health organization recommendations for the introduction of a rubella vaccine. these recommendations encourage countries to wait to introduce the vaccine until measles vaccination rates (measles and rubella vaccines are usually given together) are high enough to guarantee a reduction in crs cases and to strongly consider vaccination campaigns in women of childbearing age before the vaccine is introduced ( ) . vaccination is only of a suite of control measures. another that is of particular importance in the control of macroparasite infections is mass drug administration. a key difference between microparasite and macroparasite dynamics is the huge variation in transmission potential of human hosts, with some individuals experiencing huge pathogen loads that contribute disproportionately to transmission within populations ( ) . here, strategies have taken an eye toward reducing overall population burdens of macroparasites, including targeting those with the highest burdens. theoretical explorations of the impact of heterogeneity in transmissibility have helped inform interventions and aided in the development of theory exploring the impact of heterogeneities in microparasites ( ) . (aids). ron brookmeyer ( ) used the incubation period distribution of hiv to "back calculate" the number of hiv infections that must have occurred over the previous course of the epidemic and predict the number of future hiv/aids cases in those already infected with hiv. he thereby linked an observable quantity (the number of aids cases) with an unobservable one (the number of people living with hiv). longini et al. ( ) then fit a more mechanistic model of disease progression to data from hiv-infected individuals in the united states army, achieving similar results by explicitly representing the biological process. when attempting to estimate global mortality from measles infection, simons et al. ( ) used a state-space model (i.e., a hidden markov model) which linked an underlying model of measles epidemic dynamics (the process model) with nationally reported measles incidence via an observation model ( ) . they thereby were able to estimate the extent to which national reports underestimated measles cases by reconciling these reports with what was likely given birth rates and a known epidemic process. planning for so called "black swans," which are unlikely but catastrophic events, is essential to ensuring security and population health. the prime example of an infectious disease black swan is the influenza pandemic, which is estimated to have killed - million people in years ( ) . governments and policy makers depend on simulations built on mechanistic models to decide the extent of these threats and what can be done to confront them. for the past decade and a half, there have been ongoing concerns that one of several strains of influenza a that have been known to infect humans from domestic poultry (h n , h n , etc.), might develop the ability to transmit efficiently in humans and cause a major pandemic. h n strains are seen as particularly concerning because of their high case fatality rate and the substantial increase in the number of human cases (particularly in southeast asia) that started in ( ) . independent teams of disease modeling experts developed sophisticated agent-based models of potential emergence events to determine whether effective antiviral agents could be used to contain an emerging influenza at the source ( , ) . these models showed that under reasonable expectations of the transmissibility of an emerging influenza (i.e., r in the . - . range), containment was possible, though perhaps not practical, as it would require the deployment of millions of courses of antiviral medication, very early detection of the disease, and rapid response. in parallel work, groups considered how the impact of a pandemic could be mitigated in the united states if the initial containment attempt was unsuccessful ( ) ( ) ( ) . the efforts of independent groups showed that something more than social-distancing measures (e.g., school closure, case isolation) would be needed to control a pandemic and that effective antivirals could help. in part on the basis of this work, the united states and other countries decided to stockpile antivirals to combat a future pandemic, a decision that has since been criticized by some ( ) . however, these criticisms have been focused on concerns about the efficacy of the stockpiled antiviral drugs ( ) rather than the results of the modeling work itself. the question of the probability of h n influenza evolving to become transmissible in humans has itself been the focus of mechanistic modeling ( ) . after research groups had identified different sets of mutations to the h n virus that would be sufficient to allow airborne transmission in a mammalian host ( , ) , russell et al. ( ) developed a mathematical model of the within host dynamics of influenza evolution. although the authors were unable to confidently estimate the probability of the emergence of a pandemic h n strain because of uncertainties about the underlying biological processes involved, they were able to identify the biological factors on which this probability would most strongly depend and recommend studies (e.g., deep sequencing of viral samples from h n -infected hosts) that might help to develop more precise predictions. there has been considerable debate surrounding the ethics of gain-of-function experiments for h n influenza ( ) , but if such experiments are to be justified, they must provide us a way to have advanced warning of a coming pandemic, a task that may only be possible through mechanistic models. however, to be successful, these models will require substantial additional theoretical work on how viral evolution interacts with the distribution of immunity in the population. in the event that an outbreak of an emerging disease does occur, mechanistic models are one of the first tools used to characterize the threat and plan a response. when a pandemic influenza strain emerged in , it was critical to quickly assess whether it had the potential to cause illness with high rates of fatality, like the virus that emerged in the pandemic of , or was a more mild disease, akin to what was seen in the pandemics of and . initial assessments relied heavily on dynamic models of a variety of types, including phylogenetic techniques paired with demographic models, models based on the probability of the observed number of introductions of pandemic h n into populations outside of mexico, analysis of epidemic curves, and the results of detailed investigations of early outbreaks ( , ) . analyses by a number of groups quickly showed that the emergent pandemic h n virus was behaving very much like alreadycirculating strains, and although it was still potentially a significant public health threat, it was unlikely to have a qualitatively different impact on mortality or morbidity than circulating influenza strains. in addition to its role in the response to the influenza pandemic, mechanistic modeling has played a role in the response to most of the emerging disease threats of this century, from foot and mouth disease in the united kingdom ( ), to severe acute respiratory syndrome coronavirus ( ) , to middle east respiratory syndrome coronavirus in saudi arabia ( ) , to ebola in west africa ( ) . the last of these shows both the power of mechanistic approaches and the dangers of its misuse. in the summer and fall of , the number of ebola cases in west africa was continuing to grow, and it was unclear how severe the epidemic would eventually become. to address this issue, as well as the threat of spread to other countries, a number of modeling exercises were conducted (e.g., gomes et al. ( ) ). of particular note was a model released by the centers for disease control and prevention that predicted that, without further intervention, . million cases of ebola would occur in liberia and sierra leone by mid-january ( ). this did not come to pass, and although the authors noted that such long-term projections were tenuous, the media and many in the public health community made much of this number. of course interventions and behavior change did occur, but the authors had also made tenuous assumptions about how the populations of liberia and sierra leone mix together, essentially treating each country as a homogenous entity. in contrast, the world health organization ebola response team, who also made projections based on an unconstrained epidemic, declined to forecast further than months into the future ( ), and though theirs was a moderate overestimate of total cases, they avoided publishing any panic-inducing overestimations (they projected approximately , cases by november , ; approximately , actually were reported by that point) ( ) . forecasting the course of disease spread is difficult to do well, particularly in the context of an active response. it also may be the least of what mechanistic approaches to disease epidemiology have to offer. the aforementioned work, particularly that of the world health organization ebola response team, also characterized important aspects of ebola's natural history and epidemiology, including its basic reproductive number (r ), the decline in r over the course of the epidemic, the incubation period, and the serial interval, properties of the disease that will be important to understand should it re-emerge. mechanistic and mathematical approaches aid not only in the response to particular diseases but also in illuminating basic epidemiologic principles and important parameters that dictate whether a novel (or existing) pathogen can be controlled. in a paper, fraser et al. ( ) confronted the question of why severe acute respiratory syndrome coronavirus was successfully contained, whereas influenza, hiv, and numerous others were not. they were particularly interested in the effectiveness of the tools available when first confronting a novel pathogen: contact tracing, isolation, and quarantine. they presented evidence that a critical determinant of the controllability of a pathogen is the amount of transmission that occurs before symptom onset, expressed by their parameter θ. pathogens that had a low proportion of all transmission occurring before symptom onset are easier to control because symptomatic individuals can be targeted with isolation or pharmaceuticals before they transmit to others. although forecasting is difficult, particularly in the response to an emerging disease threat, it remains a major goal of the disease-modeling community. because disease reporting is often delayed, forecasting includes not only projections into the future but also "now casting" of incidence based on more readily available information. this has led to a number of approaches in which models have been used to either process a data stream that is a proxy of the data of interest but available more quickly (e.g., google flutrends) ( ) or in analyses of ongoing outbreaks to assess (with available data) what might be the current situation given the limitations of the observation process and temporal lags in both reporting and outcomes being generated (e.g., calculating case fatality rates for the severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus outbreaks when many patients had yet to resolve) ( , ) . at a larger time horizon, several efforts have attempted to forecast the impact of interventions on future incidence. one of the most successful was a project that forecasted the impact of respiratory syncytial virus immunization campaigns on the temporal pattern of incidence in the united states. using mechanistic transmission models, pitzer et al. ( , ) made detailed predictions of the impact of vaccination on the multiannual dynamics of rotavirus, as well as the impact of the vaccine on genotype circulation. these forecasts of broad qualitative impacts of interventions are critical tests of models. detailed prospective predictions of changes that will occur with changes in health policy, which are then validated, will provide the best evidence of the utility of mechanistic models in the future. dependent happenings is the term coined by ronald ross ( ) to capture the fact that for infectious diseases, an individual's risk of infection depends on the disease status of those around them ( ) . this presents challenges for trial design and the interpretation of observational studies. cluster randomization and adjustment for intra-class correlation can be used to account for this effect in some cases ( ) , but mechanistic models are often useful in trial design or in interpretation of results when cluster randomization is imperfect or impossible. under these conditions, simulations studies have been used to help in study design settings, including vaccine studies ( , ) and combination approaches to hiv prevention ( ) . mechanistic models have been particularly revealing for studies of vaccine effectiveness. for example, a naïve approach would be to consider that all vaccines acted in the same way, providing complete protection for some fraction of the population. however, in reality vaccines may be leaky and provide protection only in some dimensions ( ) . vaccines may prevent infection all together (e.g., the measles vaccine) ( ), offer protection against pathogenic disease but still allow individuals to become infected and transmit the disease (e.g., acellular pertussis vaccines ( )), or only prevent onward transmission of the disease (e.g., transmissionblocking vaccines for malaria ( ) ). in order to anticipate and assess the impact of vaccines once scaled up to widespread use, the specific actions of the vaccine in reducing infection, onward transmission, and disease must be disentangled. these specific mechanisms will contribute differently to the direct, indirect, and total effects of a vaccine. these effects are increasingly targets of inference during trials ( ) , and developments in infectious disease theory have driven development of both inference tools and study design to measure specific impacts ( ) . in emerging outbreaks, simulation models have often been used as the framework to quickly quantitatively compare policy alternatives. the application of these models has yielded results ranging from broad information about the feasibility and potential impact of interventions to detailed recommendations about targeting of interventions. in the foot and mouth disease outbreak of , models were used to determine optimal culling strategies that specified operational details of those strategies, including the timing and spatial extent of culling. even outside of public health crises, infectious disease models play an important role in setting public health policy. cost-effectiveness analyses are often built on mechanistic models of disease spread ( , ) . models can help investigators choose between different intervention strategies, determine the potential of specific interventions, and compare investments across pathogens. infectious disease models play a critical role in incorporating indirect effects that can vary substantially across alternative programs. the design of immunization campaigns against human papillomavirus has to weigh the direct effects protecting women from human papillomavirus infection, as well as indirect protection resulting from immunization of both women and men. the tradeoffs of alternative programs in protecting individuals at risk of the most severe outcomes and those at little risk have been best evaluated in transmission models ( ) . increasingly important is the marrying of mechanistic disease models with operations research by explicitly modeling the logistical constraints on public health intervention. this approach can be key when preparing for outbreaks or bioterrorism, as speed of deployment, hospital capacity, and other logistical factors can severely impact the efficiency of disease containment and its subsequent spread ( , ) . likewise, a logistical analysis can assess the feasibility of novel diseasecontrol strategies, showing whether they are practical as well as efficacious; for instance, an analysis of the feasibility and potential effectiveness of passive immunotherapy in hong kong showed that this intervention could play an important role in controlling a mildly severe pandemic ( ) . as the price of computation drops and we enter the era of "big data," the role of mechanistic models will only increase. a powerful new synergy is the combination of mechanistic models of disease spread with phylogenetic techniques outlining the evolutionary relationship between infecting pathogens. genetic sequence data present samples of pathogens taken from a large population of pathogens both within a host and among all hosts. understanding the impact of different selective pressures on pathogens is inherently a task of population genetics. models of the population dynamics of pathogens have been incorporated into models in order to explain the phylogenetic structure of pathogens. sequence data have been used to infer basic reproductive numbers of pathogens ( , ) , harkening back to lotka's first use of the term to describe replication of organisms. in future work, we expect to see more direct integration of models with data at both population scales, as has been the tradition, and within models of infectious disease host scales. traversing these scales will be a key challenge to the field. targeted funding and the relatively new paradigm (at least for epidemiology) of sanctioning competitions to identify the best methods of disease forecasting continue to in invigorate the field. in the united states, the models of infectious disease agent study and the recently completed research and policy for infectious disease dynamics program have led to well over , publications and continue to invigorate research and training in the field ( , ) . similar initiatives in the united kingdom and other parts of europe, such as that from the medical research council's centre for outbreak analysis and modelling, have also been successful ( ) . competitions such as the national oceanic and atmospheric administration's dengue forecasting project ( ), the defense advanced research projects agency forecasting chikungunya challenge ( ) , and the us center for disease control and prevention's predict the influenza season challenge ( ) require researchers to assess and compare the performances of their models and stand by their predictions in the face of actual events. such initiative should serve to greatly improve the quality and number of models of infectious diseases, but this will only translate into improved public health if it is paired with greater engagement with policy and practice. in limited space, it is impossible to cover every important contribution that mechanistic models have made over the past century, and there is much important work that we have not covered. these contributions range from work showing the potential impact of test-and-treat strategies in hiv control ( ) , to analyses of how to best use a limited supply of cholera vaccines to control disease ( , ) , to fundamental work on the link between demographic characteristics and disease incidence ( ) . these omissions should not be seen as a reflection of the quality of the work, but rather merely as the result of our need to select only a few of many good options. the use of mechanistic models in infectious disease epidemiology has shifted over the course of years. the arc of their use spans beginnings as of a group of statistical and mathematical tools used by epidemiologists to understand a multitude of phenomena, to use and development by an increasingly specialized group of researchers over the course of the th century, to more general use by a broader group of researchers. this arc still bends. at their core, these methods provide frameworks of analysis that can be treated in the same way as other statistical tools of analysis. refinement of methods has led to a theoretical base and application toolkit that allows nonspecialists to analyze and understand infectious disease dynamics with mechanistic models. this broader ecosystem of modelers, which includes methods-focused researchers and public health practitioners, has led to encouraging progress in tying models increasingly to data and to the most salient infectious disease problems facing global health. memoir on the reed-frost epidemic theory a commentary on the mechanical analogue to the reed-frost epidemic model an examination of the reed-frost theory of epidemics some mathematical developments on the epidemic 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darpa challenge predict the influenza season challenge universal voluntary hiv testing with immediate antiretroviral therapy as a strategy for elimination of hiv transmission: a mathematical model the impact of a one-dose versus two-dose oral cholera vaccine regimen in outbreak settings: a modeling study a simple model for complex dynamical transitions in epidemics on the course of epidemics of some infectious diseases we thank c. jessica metcalf for suggestions and useful discussions.conflict of interest: none declared. key: cord- -jmetfa x authors: macdougall, heather title: toronto’s health department in action: influenza in and sars in date: - - journal: j hist med allied sci doi: . /jhmas/jrl sha: doc_id: cord_uid: jmetfa x this article compares the toronto health department’s role in controlling the influenza epidemic with its activities during the sars outbreak in and concludes that local health departments are the foundation for successful disease containment, provided that there is effective coordination, communication, and capacity. in , toronto’s moh charles hastings was the acknowledged leader of efforts to contain the disease, care for the sick, and develop an effective vaccine, because neither a federal health department nor an international body like who existed. during the sars outbreak, hastings’s successor, sheela basrur, discovered that nearly a decade of underfunding and new policy foci such as health promotion had left the department vulnerable when faced with a potential epidemic. lack of cooperation by provincial and federal authorities added further difficulties to the challenge of organizing contact tracing, quarantine, and isolation for suspected and probable cases and providing information and reassurance to the multi-ethnic population. with growing concern about a flu pandemic, the lessons of the past provide a foundation for future communicable disease control activities. (sars) in mount sinai hospital in april exemplifies the fear and concern that outbreaks of infectious disease provoke in the families of frontline workers. for historians, both the role of the media in highlighting the dangers of an epidemic outbreak and the response of health authorities recalled nineteenth-and twentieth-century reactions to cholera, typhus, yellow fever, smallpox, bubonic plague, and poliomyelitis rather than hiv/aids. but what part was toronto's health department to play in an international health crisis? as the sars outbreak once again demonstrated, local public health organizations are the foundation for concerted community efforts to manage disease and control public panic. by comparing and contrasting the way in which public health authorities in toronto managed the influenza pandemic and sars in , we can see how a century of medical advances had conditioned the public and health care professionals to expect prompt control of communicable diseases, speedy development of a prophylactic vaccine, and effective exchange of information at the provincial, national, and international levels. but both outbreaks also demonstrated the power of negative ethnic and class stereotyping, the impact of the media in both educating and frightening the public, and the high cost in terms of human lives and devastation of the local and national economies. in and , the worldwide influenza pandemic is estimated to have killed between and million people. for european and north american nations who were just coming to the end of world war i, with its toll of to million dead and wounded, the flu seemed to be the fourth horseman of the apocalypse. war, famine, pestilence, and death challenged canadians, americans, and their allies and foes both to respond to the immediate threat and to institute more formal national and international organizations to ensure that future pandemics were controlled before they could spread beyond their countries of origin. the great pandemic also gave further impetus to biomedical research that resulted in the discovery of the causative virus by british researchers in . as research continued, however, the complexity of influenza strains became apparent. but did public perceptions of the disease change? was it seen as a . in her study the gospel of germs: men, women, and the microbe in american life (cambridge, ma: harvard university press, ), nancy tomes presents a convincing argument about the impact of the "germ" theory on american attitudes to infectious disease and demonstrates how various groups adapted new behavior patterns and beliefs as a result. more recently, in "epidemic entertainments: disease and popular culture in early-twentieth-century america," am. lit. hist., , , - , she examines how contemporary problems such as the aids, ebola, and west nile viruses have been used by the media to create a climate of fear that prompts citizens to ignore significant public health threats by focusing on exotic and unlikely "risks." but her focus is on the way that advertising agencies used scientific discoveries in the mid-twentieth century to sell products by claiming to educate consumers in basic health principles. the use of radio and film for similar purposes is also analyzed to demonstrate the way that science becomes part of popular discourse and is, in turn, modified by popular perceptions. killer or simply as an annual nuisance that appeared in north america every fall and winter, after it had completed its attacks on the southern hemisphere and australasia? in , the question for many epidemiologists and health authorities was whether sars was the feared new version of the strain or another type of disease. lack of a readily available diagnostic test or specific symptomatology significantly hampered health authorities' response to the outbreak and prompted some officials to seek historical precedents for their containment efforts. by their very nature, epidemics reveal the strengths and weaknesses of the societies in which they occur. using toronto as a case study to examine the reaction of citizens and their health departments to influenza in and sars in provides an opportunity to probe into the changing role of local health departments and their staffs in two key crises. in , toronto was a bastion of white anglo-saxon protestantism, with less than % of its population of neither canadian nor british origin. the city had undergone a wave of physical expansion through the amalgamation of newly developed suburbs prior to and was the focal point for industry and commerce in ontario. as the provincial capital, it not only housed the legislature, the provincial board of health, the principal university, and the leading medical facilities, but also administered a budget equivalent to that of the provincial government. overshadowing these characteristics was toronto's fervent support of the war effort; it was the most imperialistic of canadian cities in , and for four long years, its , citizens provided volunteers for the canadian expeditionary force (cef), the canadian army medical corps (camc), and field hospitals in france, britain, and canada. civilians played their part and turned out munitions, food supplies, and clothing; bought war bonds; and planted victory gardens. the arrival of a virulent strain of influenza with the returning soldiers added further stress to the final days of the conflict and challenged existing public health staff to organize to combat disease with limited numbers, limited medico-scientific knowledge, and limited resources. by , the former city of toronto had been forcibly amalgamated with five surrounding municipalities to create a combined population totaling . million, nearly % of whom had not been born in canada. from on, the city had been a magnet for successive waves of refugees and immigrants seeking a better life for their children. by the s, toronto was the dominant economic engine for the nation. but as the federal and provincial governments adopted thatcherite and reaganite economic policies, the city lost much needed funding for its aging infrastructure and services. this did not bode well for toronto's health department, which relied on municipal taxes as well as provincial grants. furthermore, in the province updated the mandatory programs that local health units were expected to provide, and then changed the tax base to limit business taxes that toronto had used to fund innovative health and education programs. was toronto ready for a possible pandemic? the arrival of sars demonstrated the devastation that disease outbreaks impose as businesses and public facilities close in response to local, national, and international fears of disease transmission. indeed, one of the most striking differences between the two outbreaks was the administrative complexity created by the presence of competing provincial and federal authorities in . in , canada did not have a federal health department, provincial health departments were very small, and no international health agency . james lemon, toronto since : an illustrated history (toronto: james lorimer & company and national museums of canada, ), - , - , - . see also lila sarick, "visible minorities flock to city," globe and mail, february , a . sarick stated that census data indicated that % of the greater toronto area's population was visible minorities. the story noted that toronto's services and language classes were provided in many different languages and that these were under threat because of provincial plans to reorganize the education funding system. . gay abbate, "toronto board of health defies order to cut budget," globe and mail, july , a ; john spears, "budget blueprint holds line on taxes," toronto star, march , b . according to a tph budget fact sheet dated march , the department received . % of the $ . billion-dollar gross budget for the city. the $ . million allocated for tph services in was . % less than in and . % less than in . . in the british north america act, now known as the constitution act, , divided legislative powers between the federal and provincial governments. health, education, and social services were allocated to the provinces, while the federal government was responsible for national economic policy, the military, criminal law, agriculture, immigration, and only minor health duties such as immigrant inspection, quarantine, and the care of sick mariners and aboriginals. equivalent to the world health organization (who) existed. by comparing and contrasting the abilities of the two local medical health officers-drs. charles hastings and sheela basrur-to coordinate disease control efforts, develop and maintain sufficient capacity to respond to outbreaks, and communicate effectively with fellow citizens, the media, and external authorities, we will be able to gauge the impact of their activities during these crises. the parallels and differences in the two outbreaks demonstrate how the lessons of the past need to be deeply ingrained in both collective memory and public policy if present and future challenges are to be met with courage and effectiveness. - , - . had to be supported with economic arguments that demonstrated that spending on public health administration was an investment, not an expense. starting with a staff of three public health nurses in , hastings moved quickly to expand the health education component of his staff's work and in created a division of public health nurses. based in district offices shared with either the police force or social agencies, the public health nurses quickly became "guides, philosophers and friends" for the women and children in their areas. using a generalized system that stressed health education rather than curative services, toronto's department of public health (dph) devoted great attention to forging links with more than local voluntary groups through the neighbourhood workers' association (nwa). this reciprocal relationship intensified during world war i as many families received coordinated assistance from the dph and nwa as a greater emphasis on "scientific" social service developed. thus the concept of teamwork was well understood and widely shared when warnings about a flu epidemic began to arise in the spring and summer of . the influenza outbreak is thought to have begun at camp funston in kansas in march , and to have accompanied american troops to france, where it spread to the combatant armies. canadian soldiers began to fall ill during the spring, and the return of some troops during the summer of triggered the epidemic in canada. the federal government was responsible for military cases, but provincial medical officers and their municipal counterparts knew that they would be fighting the outbreak with limited resources since so many doctors, nurses, and inspectors were serving in the armed forces. on september, the toronto world reported cases in a military camp in ontario. for toronto's medical officer and its local board of health (lbh), this presented a challenge, because influenza was not a reportable disease under the ontario public health act, and most doctors were hoping that the outbreak would be similar to the one in - that had attacked primarily the elderly and apparently provided some immunity to those who survived. these hopes were soon dashed. military doctors were well aware that the flu was killing soldiers between the ages of twenty and thirty-nine with great rapidity. when the disease spread into the community, it devastated the workforce, made entire families ill, and left orphans and the elderly in its wake. but what could be done to stop it? communicable disease control was one of the main functions of municipal and provincial health departments in canada during the late nineteenth and early twentieth centuries, but in the past it had created opposition and imposed economic hardship on those who were quarantined in their homes or sent to municipal isolation hospitals. should these conventional tactics be used against the flu? as english canada's leading health department, toronto had a well-established division of communicable disease, a municipal laboratory for testing tb and diphtheria samples, an isolation hospital, and a division of vital statistics to provide the data needed for decision-making. but as hastings was well aware, the usual approach to controlling the spread of infectious disease was proving ineffective against influenza. articles in the october issue of the american journal of public health (ajph) and personal contact with health authorities in the united states made it clear to hastings, who was president of the american public health association (apha) in , and his provincial counterpart, dr. john w. s. mccullough, ontario's chief medical officer, that there was much disagreement about the benefits of these approaches. indeed, mccullough conducted a survey of provincial and state health officers on the merits of quarantine and isolation and found that the majority had concluded that "these measures are impracticable." but mayor thomas l. church, the press, and most of the public expected such actions, and in cities such as milwaukee, they were apparently effective. in toronto, however, quarantine and isolation were not implemented because the disease toll escalated so quickly as to render it ineffective on a case-by-case basis. in his capacity as president of the apha, hastings left toronto from to october to travel to boston, new york, and washington to see the ravages of the epidemic firsthand. since flu was not a reportable disease, the statistics for its spread and virulence are suspect, but each of the communities that experienced an outbreak quickly recognized its propensity to overwhelm standard disease control measures and facilities. when the disease first appeared in toronto, the moh and military authorities appealed for calm, provided a detailed description of the symptoms, strongly recommended resting in bed, and exhorted the sick to call for medical assistance. the first civilian casualty was a schoolgirl who died in toronto general hospital on september . in spite of growing public pressure for isolation and quarantine, hastings did not issue the order, because the bulk of cases were military men in the . the provincial board of health of ontario, "spanish influenza," pub. health j., , , . this item is followed on pages - by an article reprinted from chicago papers of october . chicago's health commissioner, john dill robertson, provided citizens with information from surgeon-general blue of the u.s. public health service that focused on the origin of the disease, its symptoms, and treatment. an editorial on page , entitled "influenza," reminded pub. health j. readers that there was ongoing controversy over pfeiffer's bacillus as the cause of influenza and noted that the connaught laboratories of the university of toronto were undertaking to study whether the causative agent was a filterable virus or b. influenzae and if a prophylactic vaccine were possible. base camp located in the city. but the child's death was a prelude to a typically rapid increase in cases and deaths; within a week, more than , students and staff out of the , students and , teachers were sick. the impact on the city's hospitals was immediate and overwhelming. by october, the toronto western hospital was full, and half the nurses at the grace hospital were ill. toronto general, the city's newest and largest facility, had almost % of its patients ill with flu by mid-october; eighty nurses fell ill, and three died. as a result, surgery was canceled except for emergency operations. similar problems beset the -bed st. michael's hospital, but the situation was further compounded by the absence of medical staff on duty overseas. the sisters of st. joseph used student nurses, their own teaching staff, and teaching sisters from loretto abbey to keep the hospital functioning during the epidemic. with a population of roughly , and the fear that % or more of the population would become ill if the european and american experience was repeated in toronto, the moh and his provincial counterpart moved swiftly to create additional hospital accommodation and train volunteers to care for the sick. two hotels were commandeered and turned into emergency hospitals. to staff them, the province issued a call for an ontario emergency volunteer health auxiliary that provided training to create a volunteer group known as the sisters of service. women's groups, teachers, and other women whose jobs were eliminated when their workplaces were closed attended the three-lecture course on the care of the sick and the sickroom. willing volunteers were then assigned to one of the six health department district offices or to the temporary hospitals. but as the staff at central neighborhood house, a settlement in one of toronto's slum areas, noted, few of the sisters of service were willing to serve in their part of the city. this was especially problematic for the poor and non-english-speaking immigrants because "the assistance of neighbours, usually freely rendered during illness, was negligible owing to the contagious nature of malady . . .," and this required settlement house workers to provide nursing, housekeeping, and child care during the epidemic. nevertheless, volunteer work was vital, as the public health nurses (phns) were working "to the point of exhaustion" dealing with the rapid increase in sick families. early in the outbreak, the moh informed the globe that the nurses were focusing their entire attention on assisting the sick, and that various inspectors had been put on twenty-four-hour duty to provide food, fuel, and other necessities to stricken families. according to the anonymous author of an in-house history of the public health nursing division: "as much hourly nursing care as could possibly be arranged was given, but it did not begin to cover the need. there were very few days that the nurses did not come into the district offices and relate some unbelievably harrowing stories." as the epidemic progressed, health department staff also caught the disease, and by october, of staff were ill, including twenty-two nurses and four doctors. to deal with the growing demand for nursing care and for food, fuel, and "bedding, night clothing, towels and even pneumonia jackets," the dph turned to the neighbourhood workers' association. using toronto's newspapers to publicize these needs, the nwa appealed to torontonians' patriotism and civic spirit by informing readers that any and all donations of soup, money, or volunteer time would be gratefully received and that the former would be delivered to stricken homes by boy scouts. depots to receive these items were set up throughout the city as torontonians rallied to care for the sick. the same issue of the papers reported that approximately fifty people a day were dying of flu or bronchopneumonia. the moh had already ordered schools to close, and various organizations such as the canadian and empire clubs as well as masonic lodges were canceling their meetings. the lbh and mayor church were in agreement that other places should also close to help prevent the disease from spreading, so on saturday, october, all theaters, moving-picture shows, pool and billiard rooms, and bowling alleys were closed for the duration. further precautions included prohibiting the circulation of public library books while allowing the libraries to remain open, and persuading toronto's churches to hold only a single service on sundays-mass for catholics in the morning, and evening services for protestants. the university was closed, and fifth-year medical students were assigned to assist busy general practitioners in making home visits and to work in the newly opened temporary hospitals. the health department also relied on the work of the victorian order of nurses and the st. elizabeth visiting nurses for bedside care of the sick. during the epidemic, the health department staff made , visits to stricken households, and its records indicate that there were approximately , deaths in , cases. the latter is probably an underestimate, given the extent to which the press of work prevented accurate reporting of cases and deaths. as well, the military was compiling its own statistics in the base hospital located in the . ibid. . "university classes cancelled," the toronto world, october , . the news story stated: "all students in the faculty of medicine are asked to volunteer their services to fight the epidemic." . "victorian order of nurses," pub. health j., , , . the von usually cared for maternity cases, but their small staff of eighteen volunteered to care for the sick during the flu epidemic. the st. elizabeth visiting nurses performed similar duties for catholic torontonians. . marion royce, eunice dyke: health care pioneer (toronto: dundurn press, ), - . can. j. med. surg., , , states that toronto suffered , deaths from influenza and , from pneumonia, for a total of , , which was , in excess of the normal october death rate of . . "the provincial board of health of ontario," pub. health j., , , noted that since influenza was not a reportable disease, "the only means we have of getting anywhere near the deaths caused by the epidemic is from returns made by undertakers . . . ." the result was an ongoing recalculation of the provincial morbidity and mortality rates as new information arrived. by , mccullough had concluded that ontario had experienced roughly , - , cases, with , deaths. eastern part of the city and at the base camp at the exhibition grounds. whether these were included in the city's tally is unclear. but the impact of the epidemic was profound. the newspapers contained short items noting the deaths of many specific individuals, advertisements apologizing for delays in delivering bread and milk, news stories describing board-of-health meetings and the actions that resulted from its deliberations, and hortatory calls for more volunteers. the world also printed an impassioned plea arguing the benefits of gauze masks and asking that "[e]verybody wear a mask to work on saturday morning." neither hastings nor mccullough felt that wearing masks in public was warranted, with the result that ontarians were not required to use them as were their counterparts in alberta and several u.s. states. the economic consequences of the epidemic were significant. munitions plants and other war industries slowed as workers became ill. the municipal firefighters and policemen took sick, as did trainmen and bell canada employees. the cold rainy weather added further stress to the epidemic when coal became difficult to obtain and fuel supplies for the sick and for industry diminished. in a society that lacked unemployment insurance, the task of responding to the needs of the sick and their families fell on a populace that had already donated its time, effort, and money to winning the war and buying victory bonds. nevertheless, the toronto board of trade created an influenza fund and worked with the nwa and other community groups to distribute the proceeds. by the beginning of november, the situation began to ease. the schools were supposed to open on november, but the fuel shortage postponed the reopening for a week. sporting events resumed, hospitals began to report empty beds, and on november the armistice was signed. the celebrations that this unleashed may have contributed to another wave of the flu, but for charles hastings, the epidemic revealed a crucial lesson: we require the centralization of authority. whether that be a public health service, a local government board, a department of health, a ministry of health or a secretary of health, it matters little, but all authority should be centralized under one department, if we are going to have efficient results. every human body may be a battlefield against these invisible foes. consequently, every individual must be trained a fighter, and though we march apart, we must fight together under one command. to his canadian counterparts, hastings was clearly calling for the creation of a federal health department, and in march , legislation to this effect was introduced. the ravages of the flu epidemic were cited as one of the factors justifying the extension of federal involvement in an area of exclusive provincial jurisdiction. but the real impact was at the provincial and municipal levels. in toronto, hastings and his staff had demonstrated the benefits of a well-organized department that had made links to other municipal services, local hospitals, and non-governmental organizations. their experience enabled them to move quickly to take command in a crisis situation. the role of provincial authorities was somewhat more complex. as chief medical officer (mo), lieutenant-colonel john w. s. mccullough had responsibility for all parts of the province that lacked permanent public health staff, but he was also deeply involved with his military duties. the solution was to allow hastings and his staff to demonstrate effective community engagement and then to use this model for the rest of the province. when standard disease control measures proved ineffective at stemming the rising numbers of cases, hastings turned to prevention. lessons from history," in which she reminds her readers that we still do not have an effective treatment for influenza, and that we too should use early twentieth-century techniques of providing information and immediate closure of all but essential services to ensure that "when our time comes, we will be able to match the intelligence, energy, coordination and cooperation of our forebears." see www.cmaj.ca/cgi/content/full/ / / / dc for the full text. he brought back a b. influenzae-based vaccine from his visit to the new york city laboratory to start flu vaccine production in toronto. most civilian and military health officers pinned their hopes for controlling the epidemic on either a preventive or a prophylactic vaccine. in the connaught laboratories had opened in toronto to produce diphtheria antitoxin, but it quickly became the main supplier of vaccines for the war effort. during the flu epidemic, dr. r. d. defries, the acting director, undertook the production and testing of flu vaccine using eighteen strains of the new york source and additional ones from canadian soldiers at the base hospital. although he was impressed by the impact of the vaccine on "desperate cases," he was alert to growing evidence that the vaccine was ineffective because researchers were unable to demonstrate that the pfeiffer bacillus was the cause of the disease and indeed had begun to argue that it was a filterable virus instead. defries later argued that "[t]he preparation and trial of vaccine was fully warranted by the existent knowledge of the disease and its etiology," while hastings commented in november that during the flu epidemic the medical profession was "severely censured for not having discovered a vaccine," indicating that the public too expected science to provide a preventive for the disease. but as many of the reports published in canadian and american medical journals indicated, there was little clinical evidence that preventive or prophylactic vaccination made a difference. and what about the citizens? one of the most striking features of the outbreak was the extent to which torontonians of all social classes suffered and yet sought to help each other. the middle class and well-to-do volunteered themselves and their cars to take food, medical and nursing supplies, and doctors and visiting nurses to their patients. workers tried to maintain essential services while their customers faced a final round of privation prior to the end of the war. teachers, homemakers, and nursing, medical, and dental students volunteered their services in hospitals and in the community. settlement workers noted that the poor were so severely affected that they were unable to provide assistance to their neighbors-a breach of customary practice. and various immigrant groups were presented with additional challenges, as the information provided in pamphlets and local newspapers had to be translated into languages they understood. as the anonymous scribe who wrote about public health nursing noted: "the epidemic lasted approximately two months and it was an unforgettable experience for us all." for the health authorities who had directed local and provincial or state efforts during the epidemic, the influenza outbreak provided a challenge to their authority and expertise that led figures such as sir george newman and victor c. vaughan to lament the inability of officials to either control or prevent the disease. at the rescheduled annual apha meeting in chicago in december , a committee was formed to prepare "a working program against influenza," which was published in the january issue of the apha journal. this comprehensive review of the strengths and weaknesses of the efforts to combat the disease justified its prescription for action by noting that health agencies "must act in light of present knowledge," even if that knowledge is limited or flawed. but it is clear that there were many variables that affected the progress of the disease, and that finding the cause and an effective vaccine was high on the medical community's agenda. for local health officers, however, the extent of public cooperation during ironically, the support that toronto's health department had received in proved limited. as the city returned to "normalcy" in , the mayor and board of control recommended budget cuts to municipal services, including the health department. the effective organizing and yeoman services that staff had performed during the flu epidemic were forgotten or ignored when a mild form of smallpox appeared in october . anti-vaccination groups organized rallies attended by some city council members who objected to hastings's dynamic leadership and his demand that mandatory vaccination be instituted. this well-established preventive measure was condemned as "german born compulsion" and rejected as antithetical to the principles of liberty and democracy for which the war had just been fought. were the anti-vaccinationists reflecting concern at the inability of the medical profession to prevent the flu epidemic through immunization, or was their opposition to compulsory vaccination a postwar rejection of the social and moral authority of progressive experts and their domination of the war effort? from to , municipal and provincial health departments continued to be legally responsible for control of communicable disease. but with the development of vaccines against childhood diseases, the eradication of smallpox, and the use of antibiotics to treat tuberculosis and sexually transmitted diseases, the war on disease appeared to be won. as attention and staff interest shifted to behavior modification and encouraging community development, the financial resources and personnel devoted to disease surveillance, infection control, and isolation/quarantine diminished. instead of tb sanatoriums, preventive measures, and mass chest screening and tuberculin testing, for example, the communicable disease control (cdc) unit in toronto was using directly observed therapy against a resurgence of tuberculosis in the late s. but would this client-specific approach prove effective against a future pandemic? what role would municipal health departments be expected to play in the event of such outbreaks? experts and pundits began to warn about the possibility of a worldwide pandemic of influenza during the s and s, in the wake of the and - of tuberculosis, followed by human deaths from avian flu, was coupled with growing concern about environmental degradation. in ontario, the pathogenic outbreak of e.coli as a result of water contamination in walkerton demonstrated the price that communities paid for failing to maintain basic services. a commission chaired by justice frank o'connor highlighted the effect of provincial government cuts to the ministry of the environment and noted that it had failed to share vital information with local and provincial health authorities. during the harris regime from to , the provincial government pursued tax cuts and reorganization of provincial services that focused on downloading duties to municipalities and regional governments. convinced that toronto and its surrounding cities-scarborough, north york, the borough of york, east york, and etobicoke-were duplicating services, the province compelled them to amalgamate in . this meant that the toronto health department had to incorporate staff from the other five municipalities, determine whether its programs and services were appropriate to the new city, and try to find economies that would assist the new city's budget committee in dealing with its declining revenues. the new moh, dr. sheela basrur, was a graduate of the university of toronto (m.d. , mhsc. ) who had been the moh of the east york health unit, which had approximately fifty employees. in she became the leader of over , staff, serving a population that was significantly different from its historical roots. in addition to expanding in terms of territory, the new city had a multi-ethnic population that included . % east and southeast asians, . % south asians, . % west asians, . % africans, % caribbeans, % north americans, . % british, and % aboriginals. fortunately, toronto health had been hiring community workers from the various ethnic groups since the s in recognition of the need to provide culturally sensitive approaches to health education and preventive services. but would toronto public health, as the new entity was known, be able to maintain its national and international reputation for innovative community-responsive public health services in the face of the province's mandatory programs and limited funding? the election of the progressive conservatives led by mike harris compounded the financial difficulties already facing toronto public health as a result of the recession of the early s. the harris regime was committed to cutting government spending and staff, dismantling publicly owned utilities, remaking the public education system, downloading as many social service and welfare activities as possible, and privatizing certain environmental and health services. for tph, staff cuts, program closures, and the pressure to reorganize and redefine future goals meant focusing on children, families, and specific "high-risk" groups such as hiv/aids victims and street people rather than expanding cdc activities. in addition, the city's many acute-care hospitals and long-term-care facilities were struggling to maintain service levels because of funding shortfalls and declining numbers of staff. a widespread flu outbreak in the winter of had resulted in the deaths of several citizens who had not received prompt assistance in overcrowded emergency wards. as a result, the province introduced mass flu vaccinations in the fall of . the immunization program was offered free to citizens through public clinics or their family physicians. but would this voluntary program be sufficient to protect torontonians from the feared pandemic? health canada had been attempting to develop a national flu pandemic program, but ontario was not supportive, preferring to develop its own approach, since the harris conservatives were engaged in an ongoing conflict with the chrétien liberals over which level of government had the authority to design health to alert them to this possible problem. by march, however, the tuberculosis test was negative, more people were sick, and infection-control experts at other toronto hospitals were working with tph to identify the new disease. on march, the world health organization had issued a global alert announcing outbreaks of atypical pneumonia in hong kong and hanoi, and this enabled tph and dr. allison mcgeer, an infectious-disease specialist at mount sinai hospital, to identify the mystery illness. "in consultation with provincial and federal health officials, tph held a press conference on march , activated its emergency response plan, established a public information hotline and assigned staff full-time to the outbreak investigation." this succinct statement fails to convey the sense of crisis that existed as all three levels of health authorities discovered the weakened state of communicable disease control measures. for more than fifty years, tph had not imposed quarantine on its citizens, and although the provincial health promotion and protection act contained provisions to do so, tph staff lacked recent experience. even more challenging was the lack of knowledge regarding the disease itself. what was its cause? how was it spread? where was it most likely to be contracted? what was the incubation period? how should it be treated? who should be responsible for informing the public, provincial and federal authorities, and the who about suspected and probable cases? the sars outbreak starkly revealed the lack of coordination between federal and provincial health officials, and this conflict added to the demands being placed on tph staff when they found themselves providing the same information to two different sets of officials. differences of opinion about the confidentiality of patient information further challenged tph containment efforts, since they needed names of contacts to determine who should undergo a tenday quarantine. in contrast to , when there had been a united front against influenza, the sars outbreak illustrated the gap between prevention at the community level and care in hospitals or other tertiary facilities. the situation was further complicated because of international air travel and the growing demand for preventive precautions at pearson international airport, located in mississauga, outside the bounds of tph's jurisdiction. with virtually no scientific information to guide them at the start, and confused lines of communication with senior governments, basrur and up to of her staff began to track cases, monitor contacts, provide infection-control advice to long-term-care facilities and hospitals with sars patients, and respond to growing public concern about the extent and nature of the outbreak. in addition to its printed materials, the tph website posted descriptions of the symptoms as well as guidelines on hand-washing and quarantine procedures in fourteen languages. more than staff did daily double shifts from a.m. to p.m. on the sars hotline, which received over , calls during the outbreak, , in a single day. although staff worked diligently, they were aware that the fragmentary information they provided early in the outbreak caused frustration for many callers. as justice campbell commented: "the problem was not lack of dedication and effort, but the fact that it was impossible in the middle of a rapidly expanding crisis to create the necessary infrastructure." nevertheless, in recognition of the ethnic diversity of the city and the origins of the outbreak, tph worked closely with the chinese community, which had created a community coalition concerned about sars. this group trained chinese-, mandarin-, and cantonese-speaking volunteers to staff a hotline (the numbers sound like the chinese word for "i'm willing to help you"), produced and distributed chinese-language sars material, did promotional activities for hard-hit chinese businesses, and raised research money for sars studies. during the course of the outbreak, tph's case management team was involved in , investigations that required consultation with infectious-disease specialists because the symptoms were atypical and no diagnostic test was available, even though the genetic sequence of the coronavirus was established by british columbia's michael smith genomic sciences centre on april. the lack of clear diagnostic criteria complicated control procedures because tph staff and their clinical colleagues were aware of the stigma attached to the disease and of the danger of missing a case. to compound their difficulties, technology failed at this critical moment. when the outbreak started, the only available diseasereporting system was a fourteen-year-old dos-based one known as rdis (reportable disease information system). it was quickly apparent that this disease-specific program would not work, and tph turned to paper files with post-it notes to keep track of cases and their contacts. within two weeks excel spreadsheets were also in use, but at no point was the technology sufficiently flexible to provide the type of information and analysis that would have enabled a clearer picture to emerge. the challenge of contact tracing and quarantine supervision was immense, as over , people were identified as contacts in each of the two waves of the disease and , spent ten days isolated in their homes. while they were in quarantine, staff from tph phoned once or twice a day to see if they had any symptoms and to find out if emergency food supplies from the salvation army or canadian red cross were required. in spite of frustration caused by having to review their situation with each tph staff member who contacted them, very few torontonians refused to comply with voluntary quarantine procedures. only twenty-seven isolation orders were issued during the outbreak. , , - , in which torontonians and health care personnel who had been quarantined reported that they had complied with quarantine requests "to reduce the risk of transmission to others," to protect community health, and because they saw it as their "civic duty." fear of legal consequences had little influence in the decision to undergo the hardship that ten days in isolation imposed. in a post-outbreak survey of health care workers and the general population who had been isolated, an american organization discovered that respondents cited "protecting others" as their main motivation for undergoing quarantine. this strong sense of personal and collective responsibility for community welfare mirrors the dedication of visiting nurses and volunteers during the flu epidemic. the good behavior by the general public may have stemmed in part from the growing recognition that sars was apparently a nosocomial infection. the outbreak was confined mainly to hospital staff, patients, visitors, and family members who had close contact with the index cases. but in response to growing concern about sars spreading more widely, the ontario government declared a state of emergency on march and ordered all of toronto's hospitals to move to code orange emergency procedures. as in , this resulted in the cancellation of all surgical procedures, limited emergency access, and the cancellation of appointments and elective procedures. all visitors were banned, including families seeking to care for dying relatives. four days later, this draconian measure was applied to the province in general to protect health care workers and to prevent the spread of sars into the general population. by the middle of april, the number of new cases was declining and health authorities began to think that the worst was over. . chapter of learning from sars describes the "quest for containment" between april and april, - , and notes that the media highlighted each story about possible community spread, leaving the impression that tph and provincial authorities were not doing their jobs effectively. provincial officials and hospital spokespeople had issued daily reports on the number of actual, probable, and suspected cases and provided the media with information to calm public anxieties over the easter and passover holidays. as in , religious groups were asked to use common sense and to avoid shaking hands, kissing, sharing common communion cups, and organizing large gatherings, including funerals. but as post-outbreak studies indicated, the mixed messages that the daily briefings provided did not convince external observers that the situation was under control. april the ban was lifted, but the international publicity and the continuing cancellation of conferences and conventions meant that toronto's economy was suffering greatly. the loss of jobs in the tourism and hospitality industries added to the stress, and the civic and provincial governments turned to marketing campaigns in an effort to reassure torontonians and visitors that the city was safe to visit. during late april and early may, staff from north york general hospital sought advice from tph regarding possible sars cases in the psychiatric ward and among elderly post-operative orthopedic patients. since none of these people could be linked epidemiologically to previous cases, the situation remained in flux until an icu nurse from north york general was admitted to the toronto western hospital with sars. in the interim, possible sars patients had been transferred to st. john's rehabilitation hospital and the baycrest centre for geriatric care. the province announced publicly that a second wave of the disease had appeared on may, and the criticisms of all the flaws and failures that external critics had been making about the city's inability to control the disease increased in volume. prime minister jean chrétien had already appointed a national commission led by dr. david naylor, then dean of medicine at the university of toronto, to investigate the outbreak, and now the ontario minister of health, tony clement, announced the creation of an expert panel on sars and infectious disease chaired by dr. david walker, dean of medicine at queen's university in kingston. and finally, on june, ontario's then-premier, ernie eves, named justice archie campbell to head a judicial commission to take testimony from patients, families, health care workers, and their representatives. these reviews made sars one of the most intensively studied disease outbreaks in canadian history, and the naylor, walker, and campbell reports all stressed the lack of coordination, capacity, and communication that bedeviled federal/provincial/municipal relations in ontario during the crisis. on june, the first nurse to die in the outbreak perished. her death was followed by that of a colleague on july, and by that of a family physician on august. out of the national total of cases, toronto had , with deaths. twenty-nine nurses, fourteen doctors, and thirty other health care workers, including respiratory therapists, radiology and ecg technicians, paramedics, registered assistants, housekeepers, clerical staff, and security personnel, suffered from sars, and many are still trying to recover. in comparison to the morbidity and mortality of the flu, these numbers may seem small, but a century of medical progress had conditioned the public and health care workers themselves to expect medical professionals to provide prompt diagnoses and effective cures. the apparent speed with which sars could spread and the . kylie taggart, "independent sars commission set up in ont.," med. post, , , . in this story, taggart notes that a ninety-six-year-old man who died at nygh was thought to be the index case for the second sars wave: a health care worker on the same floor may have contracted sars from her mother, who had been a patient in the scarborough grace hospital. . learning from sars, chapter , , . according to mark hume's article, "in search of a sars vaccine," china experienced , cases, with deaths, and was leading in the race to produce a vaccine against sars. worldwide, the disease infected , people in countries and killed , including the in toronto. . terry murray, "health-care staff have a 'duty' to treat," med. post, , , . lack of provincial laboratory support for diagnostic purposes left toronto health reliant on volunteers from other health units in ontario and medical researchers based in the city's hospitals for the information that it needed to determine whether individuals were at risk of contracting or spreading the disease. when experts like allison mcgeer became ill with sars, not only was there concern for her, but the experts with whom she had been consulting had to undertake ten days of quarantine during the height of phase one. the colleagues who cared for them, as well as the public health staff who supervised quarantine activities for their families, will never forget the stress that this outbreak brought. and authority. gradually, informal links with nearby health units emerged, as sars spread beyond toronto and york county into the peel and durham regions, but the shared sense of camaraderie that marked did not materialize, because there had not been the type of sustained contact and trust-building that had occurred in toronto from [ ] [ ] [ ] [ ] [ ] . both outbreaks demonstrated the logistical and political challenge that contagious diseases pose to local public health administrators. in , hastings and mccullough knew that their plans would be overset by lack of personnel. but they also knew that they could call on willing volunteers for support, and that the mayor and local board of health backed them. almost a century later, toronto public health had to people working in its communicable disease control section, but they were dealing with an unknown disease that quickly uncovered the gaps in existing procedures for infection control in public institutions. although tph staff had worked with the seventy-eight long-term-care facilities in the city to ensure that their infection control practices were effective, they had not provided the same level of service to acute care hospitals, because of budget cuts and because there were supposed to be infection control officers and committees in place. as a result, the trust that enabled hastings and mccullough to rely on their academic and hospitalbased colleagues for curative services did not exist, and tph moved to create effective relationships with toronto hospitals by establishing a communicable diseases hospital liaison unit. this was fully funded by the province from june to march , with a commitment for % funding thereafter. but as the toronto star reported, city bureaucrats think that unless the province pays the entire cost, the city should scrap the unit. not surprisingly, tph has argued that this unit is a critical part of future disease control efforts if a seamless transition from preventive to curative services is to be provided. in both outbreaks, communication was a vital part of the moh's role. in october , hastings responded promptly to press queries, relying on his well-established ties with various newspapers to ensure that a message of calmness and fortitude was presented. the extent of the epidemic meant that many reporters, typesetters, and delivery boys were among the ill, with the result that the official view was rarely questioned. as well, stories about the final days of the first world war occupied many readers' attention. in , the local press was initially very supportive and provided excellent summaries of existing knowledge regarding symptoms and where to seek help. the nightly news included the daily press conferences attended by senior provincial officials, local infectious-disease specialists, and dr. basrur. her calmness throughout the crisis had an impact, according to one toronto hospital's administrative assistant: "when the medical officer of health gets on tv and says everything is ok, we believe her." unfortunately, the information provided by hospital-based specialists and provincial authorities seemed to contradict the moh's steady confidence in her staff and their activities. as the naylor, walker, and campbell reports suggest, this approach was ultimately perceived as indicating a lack of leadership and a possible attempt at covering up the extent of the outbreak. in retrospect, a single spokesperson would have been advisable, but there was little that any of the officials could do to overcome the voracious appetite of the media for information. the information and misinformation that was broadcast internationally undoubtedly contributed to the who travel advisory and to the decline in tourism and convention business. as a result, politicians at the provincial and federal levels tried to demonstrate their faith in the disease control efforts by tph and its supporters by having widely publicized meals in chinese restaurants. gallant as these attempts to jump-start toronto's economy and promote solidarity with potential voters were, they did not mask the underlying tension between the two levels of government. tph was caught in the middle because it was the body that had to help people qualify for federal employment insurance, provide food and other necessities while they were in quarantine, and respond to all the calls for information that flooded the hotline. perhaps the most difficult ones to deal with were those asking for assistance in avoiding ethnic stigmatization. with its origins in china, sars provided critics of canadian immigration policy with a platform from which to vent their concerns. but the april outbreak among a charismatic filipino religious group meant that they too were treated with hostility and fear. as previously noted, nineteenth-and twentieth-century epidemics were replete with racist critiques directed against the presumed human vectors of diseases such as cholera, typhus, and plague. even aids prompted a similar response because of the high morbidity rate within the haitian community. but one of the striking features of the sars outbreak was the uniform condemnation of racist epithets by politicians, reporters, and concerned members of the public. and when it became clear that sars was predominantly hospital-based, health care workers also found themselves socially isolated. each of the official reports commented on the sense of "fear, anger, guilt and confusion" that health care professionals felt as they tried to protect themselves and their families from the disease and from the fear evinced by their fellow citizens. even more perturbing was the rift that appeared when provincial public health experts suggested that some of the in-hospital transmission occurred because of lack of hand washing, lack of proper use of n masks, and lack of common sense about staying home if symptomatic. a team from the centers for disease control and prevention in atlanta was invited to toronto to adjudicate this dispute, but well after the outbreak was over, it was revealed that very few of the n masks had been properly fitted. little wonder that hospital-based nurses who appeared before each of the commissions of inquiry were vehement in their criticism of the way the outbreak was handled. for these frontline workers, the sars outbreak demonstrated once again the gap between theory and practice in clinical settings and the continuing hierarchy that privileged medical rather than nursing and other staff. a century of evolution in professional identities and status expectations was laid bare by sars. in , flu was a known disease whose virulence seemed unaccountably to have mutated to the point that it became lethal. sars was an unknown virus whose incubation period, degree of virulence, symptomatology, treatment, and sequelae were determined through experience and monitoring events in hong kong, singapore, hanoi, and other stricken centers. in both instances, local public health agencies were the principal agents of the state because they provided the organization and staff to conduct casefinding home visits, arrange contact tracing and quarantine measures, and organize hospital accommodations for the seriously ill. these standard disease-control measures were overwhelmed by the magnitude of the epidemic, but the volunteer efforts of many citizens meant that the supportive care needed to prevent flu sufferers from succumbing to pneumonia and other sequelae was available. in , the unity of purpose that had linked toronto's health department, city hospitals, and the neighbourhood workers' association no longer existed. the hospital sector dominated much of the press coverage, and the cleavage between provincial officials and nurses' unions became widely known as a result. "name, blame, shame" replaced the deference to authority that had marked early twentieth-century news reports. nevertheless, the work of tph staff was recognized by international experts, and on july , mayor david miller presented dr. barbara yaffe, the acting moh, and frontline staff with the canadian public health association certificate of merit award for "their exceptional contribution in managing the sars crisis" by "controlling the outbreak and implementing one of the largest quarantines in modern history." such recognition from peers and colleagues across the country is welcome confirmation that in spite of all the flaws and failures, toronto public health fulfilled its obligations. and in his second interim report, justice campbell argued for the primacy of local and provincial medical officers, stating that they "must have the lead role in public health emergency mitigation, management, recovery, coordination and risk communication." when the sars outbreak began, reporters looked for parallels and historical models. the flu epidemic was cited by epidemiologists and historians as a possible parallel, largely, one suspects, because it has recently been the subject of renewed research and because it was worldwide. but was there perhaps another reason? were reporters and newscasters seeking reassurance that all would be well and that civilization would survive? in the western media, attention was divided between the war in iraq and the sars outbreak. in the twenty-first century, death in combat seems somehow more comprehensible than death from disease. but as environmental degradation proceeds and species-jumping viruses and bacteria multiply, the certainties that pervaded twentieth-century western medicine are beginning to fade. in their place is increasing respect for the ability of microorganisms to mutate and a determination to use all available scientific tools to combat threats to human health. to date, three vaccines have been developed against sars; the sino-european project on sars diagnostics and antivirals has reported that cinanserin, a drug for schizophrenia, is a useful therapy; and dr. josef penninger's research team has demonstrated that the protein ace can be used to combat the fluid buildup that killed sars patients. clarifying the clinical picture and finding effective medications may remove the fear that epidemic diseases create, but, as this review of disease control activities has demonstrated, age-old methods such as case identification, contact tracing, quarantine, and isolation are the first stage of containment and hopefully eradication. toronto's experiences in and demonstrate "the power of public health" as the bedrock of disease control efforts. but is it the historian's responsibility to point out the "lessons of the past"? if so, to whom should her observations be addressed? policymakers and public health administrators will be using the recommendations of the three reports as the foundation for change, and indeed, the federal government has already created a junior minister of state for public health, while ontario, under its new liberal government, has promised $ . million over the next three years to create the ontario health protection and promotion agency. dr. sheela basrur has been appointed the new chief medical officer of health, and the powers of the position have been expanded to enable future planning and better coordination. does this signal the senior governments' recognition of the crucial importance of prevention? has the balance of power within the biomedical world shifted, or will the sars outbreak fade from memory as quickly as the events of ? these questions will challenge future historians to explain the long-term impact of epidemic disease on society and to analyze the role of local health departments in the ever-expanding war on disease. the modern conception of public health administration causes of poverty the policy, spirit and programme of the neighborhood workers association america's forgotten pandemic some observations on the recent epidemic ´enlightening the public': the views and values of the association of executive health officers of ontario, - plague: a story of smallpox in montreal state medicine in transition: battling smallpox in ontario the value of a credit balance in public health administration doing good: the life of toronto's general hospital for the least of my brethren: a centenary history of st. michael's hospital (toronto and the control of influenza in ontario crossed wires put toronto on hit list disease is damaging ontario's economy, cabinet officials say first, tell the real story cutbacks fed sars calamity, critics say public-health spending cuts went too far, critics say fear factor: so just how big a risk is sars? what made the statement more surprising is that pat green's husband was a toronto firefighter and her son, derek, was a toronto transit commission bus driver, indicating that all three of them were in occupations that would be at risk if sars had been spreading in the community the learning from sars report estimated that sars would cost canada two billion dollars, while the former ontario auditor, erik peters, stated that sars-related spending by the provincial government would cost $ million, only $ million of which would come from federal coffers. see justice campbell's interim report-sars and public health in ontario, appendix e: the economic impact of sars. pestilence and restraint: haitians, guantánamo, and the logic of quarantine countless health care workers faced a fundamental conflict between self-preservation, and a professional obligation to serve the greater good nurses have long voiced concerns that their knowledge and experience is not taken seriously by senior decision makers. at north york general hospital, nurses alleged that administrators ignored their warnings of an impending second sars outbreak , , quotes dr. mark lipsitch of harvard university, who stated that "tph did a very good job under completely uncertain circumstances second interim report: sars and public health legislation killer viruses sars link to acute lung failure discovered in laboratory mice as a result of experience during the sars outbreak and growing concern about a future influenza pandemic, all three levels of canadian government have created pandemic influenza plans. see www.health.gov.on.ca for information on the ontario plan and its links to the federal plan key: cord- - bb authors: brownstein, john s; freifeld, clark c; reis, ben y; mandl, kenneth d title: surveillance sans frontières: internet-based emerging infectious disease intelligence and the healthmap project date: - - journal: plos med doi: . /journal.pmed. sha: doc_id: cord_uid: bb john brownstein and colleagues discuss healthmap, an automated real-time system that monitors and disseminates online information about emerging infectious diseases. the opportunity as developed nations continue to strengthen their electronic disease surveillance capacities [ ] , the parts of the world that are most vulnerable to emerging disease threats still lack essential public health information infrastructure [ , ] . the existing network of traditional surveillance efforts managed by health ministries, public health institutes, multinational agencies, and laboratory and institutional networks has wide gaps in geographic coverage and often suffers from poor and sometimes suppressed information flow across national borders [ ] . at the same time, an enormous amount of valuable information about infectious diseases is found in web-accessible information sources such as discussion sites, disease reporting networks, and news outlets [ , , ] . these resources can support situational awareness by providing current, highly local information about outbreaks, even from areas relatively invisible to traditional global public health efforts [ ] . these data are plagued by a number of potential hazards that must be studied in depth, including false reports (mis-or disinformation) and reporting bias. yet these data hold tremendous potential to initiate epidemiologic follow-up studies and provide complementary epidemic intelligence context to traditional surveillance sources. this potential is already being realized, as a majority of outbreak verifications currently conducted by the world health organization (who)'s global outbreak alert and response network are triggered by reports from these nontraditional sources [ , ] . in one of the most frequently cited examples [ ] , early indications of the severe acute respiratory syndrome (sars) outbreak in guangdong province, china, came in november from a chinese article that alluded to an unusual increase in emergency department visits with acute respiratory illness [ , ] . this was followed by media reports of a respiratory disease among health care workers in february , all captured by the public health agency of canada's global public health intelligence network (gphin) [ , , ] . in parallel, online discussions on the promed-mail system referred to an outbreak in guangzhou, well before official government reports were issued [ ] . these web-based data sources not only facilitate early outbreak detection, but also support increasing public awareness of disease outbreaks prior to their formal recognition. through lowcost and real-time internet data-mining, combined with openly available and summary points diseases is found in web-accessible information sources such as discussion forums, mailing lists, government web sites, and news outlets. sources can play an important role in early event detection and support situational awareness by providing current, highly local information about outbreaks, even from areas relatively invisible to traditional global public health efforts. useful, information overload and difficulties in distinguishing "signal from noise" pose substantial barriers to fully utilizing this information. automated real-time system that monitors, organizes, integrates, filters, visualizes, and disseminates online information about emerging diseases. real-time intelligence on a broad range of emerging infectious diseases for a diverse audience, from public health officials to international travelers. and other nontraditional sources of surveillance data can facilitate early outbreak detection, increase public awareness of disease outbreaks prior to their formal recognition, and provide an integrated and contextualized view of global health information. user-friendly technologies, both participation in and access to global disease surveillance are no longer limited to the public health community [ , ] . the availability of web-based news media provides an alternative public health information source in under-resourced areas. however, the myriad diverse sources of infectious disease information across the web are not structured or organized; public health officials, nongovernmental organizations, and concerned citizens must routinely search and synthesize a continually growing number of disparate sources in order to use this information. with the aim of creating an integrated global view of emerging infections based not only on traditional public health datasets but rather on all available information sources, we developed healthmap, a freely accessible, automated electronic information system for organizing data on outbreaks according to geography, time, and infectious disease agent [ ] ( figure ). operating since september , healthmap (http://www.healthmap. org/) is a multistream real-time surveillance platform that continually aggregates reports on new and ongoing infectious disease outbreaks [ ] . the system performs extraction, categorization, filtration, and integration of these reports, facilitating knowledge management and early detection ( figure ). through this approach, we seek a unified and comprehensive view of current infectious disease outbreaks in space and time worldwide. healthmap is designed to provide a starting point for real-time intelligence on a broad range of emerging infectious diseases for a diverse range of end users, from public health officials to international travelers [ , , ] . the system currently serves as a direct information source for approximately , unique visitors per month, as well as a resource for libraries, local health departments, governments (e.g., the us department health and human services and department of defense), and multinational agencies (e.g., the united nations), which use the healthmap data stream for day-to-day surveillance activities. many regular users come from the who, the us centers for disease control and prevention, and the european centre for disease prevention and control. knowledge sources. healthmap relies on a variety of electronic media sources, including online news sources through aggregators such as google news, expert-curated discussion such as promed-mail [ , , ] , and validated official reports from organizations such as the who. currently, the system collects reports from sources, which in turn represent information from over , web sites, every hour, hours a day. internet search criteria include disease names (scientific and common), symptoms, keywords, and phrases. the system collects an average of reports per day, with the majority acquired from news media sources ( . %). although most of the reports collected to date have been in english, healthmap also monitors information sources in chinese, spanish, russian, and french, with additional languages such as hindi, portuguese, and arabic under development. as healthmap reports are acquired solely from free news sources, operational costs are minimal. the web site is freely accessible on the internet without subscription fees. the use of international news media for public health surveillance has a number of potential biases that merit consideration. while local news sources may report on incidents involving a few cases that would not be picked up at the national level, such sources may be less reliable, lacking resources and training, and may report stories without adequate confirmation. furthermore, other biases may be intentionally introduced for political reasons through disinformation campaigns (false positives) or state censorship of information relating to outbreaks (false negatives). we have attempted to better understand some of these issues through ongoing analysis and evaluation research. we ran a -week evaluation of healthmap data, covering the period of october , through july . we found that pathogen diversity was substantial across news sources, with unique infectious disease categories reported through the google news feed alone (table ) . we found the frequency of reports about particular pathogens to be related not to their associated morbidity or mortality impact, but rather to the direct or potential economic and social disruption caused by the outbreak. for instance, we found substantial skew towards reporting on stories about avian influenza and food-borne illnesses. over the evaluation time period, countries had reports of infectious disease outbreaks, with the greatest reporting from the united states (n = ), the united kingdom (n = ), canada (n = ), and china (n = ) ( figure a ). there was a clear bias towards increased reporting from countries with higher numbers of media outlets, more developed public health resources, and greater availability of electronic communication infrastructure (approximated by number of internet hosts) ( figure b ). these trends are highly relevant for users of the system, and thus the individual impact of these factors on surveillance will form the basis of a detailed user guide currently under development. knowledge extraction. the system characterizes disease outbreak reports by means of a series of text mining algorithms. (a complete technical description of the system may be found elsewhere [ ] .) characterization stages include: (a) identifying disease and location; (b) determining relevance-namely, whether a given report refers to any current outbreak; and (c) grouping similar reports together while removing exact duplicates. once the reports are automatically processed, curators correct the misclassifications of the system where necessary (figure ) . currently, only one analyst reviews and corrects the posts. however, additional resources would enable more detailed multilingual curation and annotation of collected reports. extracting location and disease names from text reports presents the most formidable challenge. healthmap draws from a continually expanding dictionary of pathogens (human, plant, and animal diseases) and geographic names (country, province, state, and city) to classify outbreak alert information. however, disease and place names are often ambiguous, colloquial, and subject to change, and may have multiple spellings (e.g., diarrhea, common in the us, and diarrhoea, common in the uk). thus, the expansion and editing of the database requires extensive manual data entry. once location and disease have been identified, articles are automatically tagged according to their relevance. specifically, we identify whether a given report refers to a current outbreak ( "breaking news"), as opposed to reporting on other infectious disease-related news, such as vaccination campaigns, scientific research, or public health policy. in this case, healthmap makes use of a bayesian machine learning algorithm, trained on manually characterized existing reports, to automatically tag and separate breaking news. finally, duplicate reports are filtered, identified, and grouped based on the similarity of the article's headline, body text, and disease and location categories. using a similarity score threshold, the system groups related articles into clusters that provide the collective information on a given outbreak. knowledge integration and dissemination. healthmap is particularly focused on providing users with news of immediate interest and reducing information overload. overwhelming public health officials with information on outbreaks of low public health impact may distract them from investigating outbreaks of greater priority that might receive reduced media attention. thus, only articles classified as breaking news are posted to the site. although they are filtered from the initial display, other article types and duplicate articles are shown in a related information window, providing a situational report on an ongoing outbreak as well as recent reports concerning either the same disease or location, and links for further research (figure ) . healthmap also addresses the computational challenges of integrating multiple sources of unstructured information by generating meta-alerts of disease outbreaks. as false alarms can often be reduced by thorough aggregation and cross-validation of reported information, a composite activity score (or heat index) is calculated based on (a) the reliability of the data source (for instance, increased weight is given to who reports and reduced weight to local media reports); and (b) the number of unique data sources, with increased weight to multiple types of information (e.g., discussion sites and media reports on the same outbreak). this meta-alert derivation is based on the idea that multiple sources of information about an incident provide greater confidence in the reliability of the report than any one source alone. a wide range of further improvements are currently being developed across all components of the healthmap system. in particular, population and geography gaps in the coverage of the monitored sources need to be better understood and accounted for. for example, there are critical gaps in media reporting in tropical and lowerlatitude areas, including major parts of africa and south america-the very regions that have the greatest burden and risk of emerging infectious diseases (figure ) [ ] . monitoring other internet-based sources such as blogs, discussion sites, and listservs could complement news coverage. the use of click-stream data and individual search queries is also a promising new surveillance source [ ] . multilingual surveillance is critical for capturing greater geographic coverage and for providing earlier and more comprehensive reporting from local news media. potential future challenges include the possibility that news data sources that are freely available now will no longer be available if current business models change. in addition, the way news is reported online (content, format, communication standards, etc.) may change and develop in the coming years, which will require a re-tooling of the system in order to capture the appropriate information. potential future benefits of technological advances include better meta-data tagging if/when the semantic web becomes a reality. also, as location-based services become more widespread, including on portable devices, healthmap feeds can be tailored and targeted to specific users and their locations. future work must also focus on improving natural language processing capability to clearly identify the pathogen, filter nonpertinent reports and duplicates, and enhance the spatial resolution of location extraction. however, while improvements in machine learning techniques are undoubtedly critical, they cannot currently replace human analysis. the success of wikipedia has shown that leveraging collaborative human networks of trained public health professionals has the potential to support improved classification, severity assignment, conflict resolution, geocoding, and confirmation of reports on rare or unknown infectious diseases [ ] . a recently established collaboration between healthmap and promed-mail (http://www. healthmap.org/promed) is helping to pave the way for such a bidirectional system of classification and curation of information flow [ ] . continued system evaluation is also essential. the fundamental characteristics of different news source types need to be quantified, including sensitivity, specificity, and timeliness [ , , , ] . consideration should also be given to integrating unstructured online information sources with other health indicator data to provide a broader context for reports. pertinent data sets include mortality and morbidity estimates, laboratory data, field surveillance (e.g., vector and animal reservoir distribution), environmental predictors (e.g., climate and vegetation), population density and mobility, and pathogen seasonality and transmissibility. such integration could all articles related to a given outbreak are aggregated by text similarity matching in order to provide a situational awareness report. furthermore, other outbreaks occurring in the same geographic area or involving the same pathogen are provided. the window also provides links to further research on the subject. in this example, we show all alerts relating to a recent cholera outbreak in nigeria. yield a more precise relevance score for a given report, define populations at risk, and predict disease spread. healthmap is a member of a new generation of surveillance systems that mine media sources in near realtime for reports of infectious disease outbreaks, including gphin [ , ] , medisys, developed by the directorate general health and consumer affairs of the european commission [ ] , the us government-funded argus [ ] , and epispider [ ] . while internet-based online media sources are becoming a critical tool for global infectious disease surveillance, important challenges still need to be addressed. since regions with the least advanced communication infrastructure also tend to carry the greatest infectious disease burden and risk, system development must be aimed at closing the gaps in these critical areas. hence, achieving global coverage requires attention to creating and capturing locally feasible channels of communication. it also involves making the outputs of the system more accessible to users in these regions through user interfaces in additional languages and low-bandwidth display options, including mobile phone alerts. ultimately, the monitoring of diverse media-based sources will augment epidemic intelligence with information derived outside the traditional public health infrastructure, yielding a more comprehensive and timely global view of emerging infectious disease threats. a truly open and accessible system can also assist users in overcoming existing geographical, organizational, and societal barriers to information, a process that can lead to greater empowerment, involvement, and democratization across the increasingly interconnected global health sphere. implementing syndromic surveillance: a practical guide informed by the early experience disease surveillance needs a revolution global trends in emerging infectious diseases the new international health regulations: considerations for global public health surveillance rumors of disease in the global village: outbreak verification hot spots in a wired world: who surveillance of emerging and re-emerging infectious diseases use of the internet to enhance infectious disease surveillance and outbreak investigation official versus unofficial outbreak reporting through the internet global surveillance, national surveillance, and sars global public health intelligence network (gphin). th conference of the association for machine translation in the americas sars and population health technology the global public health intelligence network and early warning outbreak detection: a canadian contribution to global public health the internet and the global monitoring of emerging diseases: lessons from the first years of promed-mail internet and computer-based resources for travel medicine practitioners traveller's medicine on the internet healthmap: the development of automated real-time internet surveillance for epidemic intelligence infectious disease surveillance and detection: assessing the challenges-finding solutions netwatch: diseases on the move technology and public health: healthmap tracks global diseases get your daily plague forecast global awareness of disease outbreaks: the experience of promedmail promed-mail: background and purpose healthmap: global infectious disease monitoring through automated classification and visualization of internet media reports infodemiology: tracking flu-related searches on the web for syndromic surveillance internet encyclopaedias go head to head interactive map of promed reports available the emerging science of very early detection of disease outbreaks integrating syndromic surveillance data across multiple locations: effects on outbreak detection performance finding leading indicators for disease outbreaks: filtering, cross-correlation, and caveats identifying pediatric age groups for influenza vaccination using a real-time regional surveillance system health threats unit at directorate general health and consumer affairs of the european commission a heuristic indication and warning staging model for detection and assessment of biological events scanning the emerging infectious diseases horizon-visualizing promed emails using epispider blog: a regularly updated online journal containing news or commentary on a particular topic, generally produced by an individual or a small group of people. a sequential record of the actions performed by a user while browsing the internet, including web sites visited, searches performed, and hyperlinks followed.event-based surveillance: unstructured data gathered from sources of intelligence of any nature. structured data collected through routine public health surveillance systems.informal surveillance: information from individuals or news media sources, as opposed to official government or government-sponsored reports.listserv: an automated email forwarding system that allows any member of a group of people to easily send a message to all other members of the group. multistream surveillance: an approach that monitors multiple sources of information and may also integrate them into a unified analytical framework. key: cord- -icyut xa authors: pillaiyar, thanigaimalai; meenakshisundaram, sangeetha; manickam, manoj; sankaranarayanan, murugesan title: a medicinal chemistry perspective of drug repositioning: recent advances and challenges in drug discovery date: - - journal: eur j med chem doi: . /j.ejmech. . sha: doc_id: cord_uid: icyut xa drug repurposing is a strategy consisting of finding new indications for already known marketed drugs used in various clinical settings or highly characterized compounds despite they can be failed drugs. recently, it emerges as an alternative approach for the rapid identification and development of new pharmaceuticals for various rare and complex diseases for which lack the effective drug treatments. the success rate of drugs repurposing approach accounts for approximately % of new fda approved drugs and vaccines in recent years. this review focuses on the status of drugs repurposing approach for various diseases including skin diseases, infective, inflammatory, cancer, and neurodegenerative diseases. efforts have been made to provide structural features and mode of actions of drugs. sir james whyte black, winner of the nobel prize in medicine [ ] it is reported that the food and drug administration (fda) has approved agents against about human proteins [ ] , % of which comes under the classification of enzymes, transporters, g protein-coupled receptors (gpcrs), cluster of differentiation (cd) markers, voltage-gated ion channels, and nuclear receptors. it is a fact that the typical de novo drug discovery program takes to years [ , ] from the identification of lead molecule to market the drug and the probability of success rate is less than % [ ] . over five years, the number of new drugs approved by fda has been around per year, although billions of us dollars spent by various pharma industries on the research and development [ ] [ ] [ ] . the success rate is less than % of new drug discovery and development, which is far away from addressing an unmet clinical need for disease treatments. because, the effective therapeutics for complex diseases like alzheimer's disease (ad), parkinson's disease (pd), cardiovascular diseases, and neglected diseases are still lacking. this outcome strongly suggests that new strategies, approaches, and technologies are needed to accelerate drug discovery to advance the success rate of drug development. drug repurposing is a strategy consisting of finding new indications for already known marketed drugs used in various clinical settings or highly characterized compounds despite they can be failed drugs [ ] . it is a drug discovery program, which is faster and safer to develop medications against diseases/disorders for which no potential treatment is available. in recent years, the success rate of drug repurposing approach accounts for approximately % of the newly fda approved drugs, and vaccines. this is one of the main reasons for pharmaceutical companies to show their interest in drug repurposing approach. this approach does not require the initial six to years typically needed for the development of new drugs. additionally, many phases of de novo drug discovery and development can be by-passed, melanin protects human skin from the radiation, continuing irradiation can result in the risk of skin damage and malignant melanoma, a cancer of melanocytes. besides, the abnormal production of melanin leads to a serious of dermatological disorders including melasma [ ] [ ] [ ] [ ] , freckles, age spots, and post-inflammatory melanoderma. [ , ] . melanogenesis, a process of synthesis of melanin, is a complex enzymatic and biochemical catalyzed reactions, in which tyrosinase plays a rate-limiting step: hydroxylation of l-tyrosine to l- , -dihydroxyphenylalanine (l-dopa) followed by the oxidation of l-dopa to ldopaquinone, which serves as a substrate for the production of melanin [ ] . therefore, targeting tyrosinase has been recognized as a potential approach for controlling the abnormal production of melanin. tyrosinase is also an important target in the food industry as inhibition of tyrosinase can prevent the enzymatic browning of fruits and vegetables. besides, it essential for wound healing process and immune responses in many plants, sponges and some invertebrates. abnormal activities of tyrosinase have been linked to neurodegenerative disorders including parkinson's [ ] and huntington's diseases [ ] [ ] [ ] [ ] [ ] . thiourea and analogs. tyrosinase inhibitors can be broadly classified into two categories: (a) polyphenols, which are mostly natural products such as arbutin, hydroquinone and kojic acids, and b) thiourea and its derivatives. since the 's, phenylthiourea (ptu, ) has been well known as a tyrosinase inhibitor [ , ] . many research groups including ours have extensively studied the structure-activity relationships for ptu as tyrosinase inhibitors [ , ] . choi et al. screened the fda approved drug library that has closed structural similarity to ptu (see figure ) [ ] . for example, ethionamide ( ), a second-line antituberculosis drug used for the treatment of multi-drug resistant tuberculosis, shares a chemical similarity that led to the discovery of a new mushroom tyrosinase inhibitor with an ic value of . µm. other commercially available analogs of including, prothionamide ( ), thioisonicotinamide ( ), pyridine- -carbothiomide ( ), pyridine- -carbothiomide ( ) , and thiobenzamide ( ) were identified as potent tyrosinase inhibitors. in particular, compound had a strong inhibitory activity than other molecules, which suggests that the pyridine ring in compound can be replaced by other aromatic moieties, including a benzene ring. however, the poor inhibitory activity of isoniazid ( ), a first-line antituberculosis drug, suggests that carbothiomide group was crucial for tyrosinase inhibitory activity. moreover, comparing the inhibitory activities of drugs and implies that the additional aliphatic tail was not required for inhibiting the tyrosinase. inhibitory kinetic studies suggest that drug , and its analogs - were reversible and non-competitive. in cellular assay, drugs and markedly decreased the melanin content in b cells to the values of % and %, respectively, without inducing any cytotoxicity up to µm concentration. further studies suggest that the drug had strong inhibition of mammalian tyrosinase. however, the inhibition exhibited by drug , and its analogs were weaker than those obtained by ptu observed in enzyme, and melanin content assays. the same research group continued to search for molecules that are in clinical usage, and contain thiourea moiety [ ] . as a result, they could retrieve some thiourea containing drugs such as thioacetazone ( ) , ambazone ( ) , methimazole ( ) , carbimazole ( ) , thiouracil ( ) , methylthiouracil ( ) , and propylthiouracil ( ) . thioacetazone ( ) (also called as thiacetazone) is an anti-tuberculosis drug [ ] . ambazone ( ) is an oral antiseptic drug used in europe [ ] . the other five molecules ( ) ( ) ( ) ( ) ( ) are antithyroid drugs [ ] . these drugs, except , exhibited remarkable inhibitory activities against mushroom tyrosinase (see ic values of each in figure ), although they were comparatively weaker than ( µm) . kinetics studies of tyrosinase inhibition assigned these thiourea-containing drugs as non-competitive inhibitors. in cellular assay, using b cells, drug among other thioureas, significantly decreased the melanin content by % without inducing any cytotoxicity up to µm. further, enzymatic studies with cell extracts of b f cells confirmed that thioureacontaining drugs affected the function of mammalian tyrosinase. in an extended study, the same group repositioned thiopurine drugs - as tyrosinase inhibitors [ ] . thioguanine ( ) , a drug used for the treatment of leukemia, is one of the essential medicines required for a basic health system, recommended by the world health organization (who). it inhibited tyrosinase activity with a k i value of µm. in addition to that, two other thiopurine drugs such as mercaptopurine ( ) and azathioprine ( ) were discovered as tyrosinase inhibitors, and among them, drug showed stronger k i value (k i µm) than the drug . azathioprine ( ) exhibited a poor tyrosinase inhibition. these results suggest that the sulfur atom possibly plays an important role in the interaction of tyrosinase. interestingly, thioinosine ( ) , a metabolic product of mercaptopurine through the attachment of sugar moiety exhibited an excellent tyrosinase inhibitory activity with a k i value of . µm. further, kinetics studies classified the drugs , , and as competitive inhibitors. in the cellular assay, these drugs inhibited the melanin content without cytotoxicity up to µm. in particular, drug at -µm concentration, remarkably reduced the melanin content by %, without any apparent cytotoxicity. the thiopurine drugs were docked into four different crystal structures complexed with inhibitors tropolone, kojic acid, hydroquinone and ptu ( figure ) [ ] [ ] [ ] . in the molecular it also predicted that the intramolecular hydrophilic contacts with the residue of e- disappeared in mercaptopurine ( figure c ), whereas thioguanine ( figure b ) and thioinosine possess the contacts within . Å. thioinosine ( ) are represented. this figure was adopted from the publication of choi, j. et al [ ] . drugs repositioning for cancer therapy. as cancer is one of the leading cause of death worldwide [ ] , pharmaceutical companies invest billions of dollars in developing new anticancer drugs. the drug discovery and development process for the cancer treatment takes an average of years at a cost of approximately $ . billion [ ] . however, only % of the drugs that enter clinical trials are approved. this prolonged duration for the drug development and enormous cost of the (pre)clinical trials for their approval emphasizes the need for a drug repurposing approach (see figure for drugs repurposing in cancer therapy). aspirin ( ) , also known as acetylsalicylic acid, is one of the non-steroidal anti-inflammatory drugs (nsaids), which has been widely used as an analgesic and an antipyretic to prevent the heart attack and stroke. for the first time, gasic and co-workers reported the possible role of aspirin in cancer therapy. they discovered that the antiplatelet activity of in tumor-bearing mice was associated with a % reduction in lung metastasis [ ] . a recent study also indicated that the daily intake of the drug ( mg) produced a significant beneficial effect against gastrointestinal, esophageal, pancreatic, brain, prostate, and lung cancer [ ] . the mode of action aspirin is reported to modulate numerous molecules, which are associated with the tumorigenesis process [ ] . preclinical studies revealed that the anticancer activity of drug has been attributed by its inhibition of cyclooxygenase (cox) enzymes that promotes carcinogenesis through the synthesis of prostaglandins (pg), including pge [ ] . besides, drug was reported to inhibit the activation of transcription factor nf-κb, which is critical to regulating the expression of genes involved in apoptosis [ ] . in a study reported by li ling et al. compound not only inhibited proliferations and promoted apoptosis of cancer cells, but also delayed and overcame acquired resistance to targeted therapy. the underlying mechanism could be attributed to enhanced cancer stemness and activated nf-kb signaling in acquired resistant tumors were suppressed by aspirin and rendered resistant tumors more sensitive to aspirin than parental, sensitive cells in terms of proliferation, apoptosis and cancer stemness. on the contrary, aspirin has no effects on normal lung and mammary epithelial cell proliferation at concentrations used on lung and breast cancer cells. hence, aspirin could be a potential candidate for combination therapy for lung and breast cancers [ ] . other studies suggest that the anticancer property of aspirin ( ) has been linked to the phosphatidylinositol- -kinase (pi k) pathway, and ras-raf-mek-erk signaling cascade. while numerous studies suggest the potent anticancer activities of drug , the overall benefit is limited as it is associated with serious side effects including the gastrointestinal and renal toxicities. therefore, there is no clear recommendation to take for population-wide use. on the other hand, as a primary cancer prevention tool, many reports revealed using would have a greater benefit in the population at age - . thus, u.s. preventive services task force recommendation statement (uspstf), recommended for daily intake of for patients above years with increased risk of cardiovascular disease and colorectal cancer. celecoxib ( ) belongs to the family of nsaid, which has been used to treat pain and inflammation associated with rheumatoid arthritis (ra) and osteoarthritis (oa) [ ] . in , the fda approved celecoxib, which is a highly selective reversible inhibitor of cox- , a well-known inflammatory cancer target. because of its cox- inhibitory activity, the antitumor activities of drug have been extensively studied and shown to have chemopreventive activities against various cancer types. other than cox- , celecoxib targets glycogen synthase kinase (gsk) β, β-catenin, nf-κb, akt virus oncogene cellular homolog (akt), and b cell lymphoma (bcl)- families [ ] . in people with familial adenomatous polyposis (fap), a daily dosage of drug ( mg) significantly reduced the risk of colorectal adenomas. fda approved this compound to reduce colon and rectal polyps in people with familial adenomatous polyposis [ ] . however, it was associated with some drawbacks including gastrointestinal, renal, and cardiotoxic effects. ibuprofen ( ) is an nsaid, which has been primarily used to treat fever, pain, and inflammation. at the molecular level, ibuprofen inhibits cox, which converts arachidonic acid to prostaglandin. however, it is not selective towards any isoform of cox. the drug was marketed for the treatment of rheumatoid arthritis in the united kingdom ( ) , and in the united states ( ) as well. the anticancer activity of drug has been investigated in various cancer cell types. ibuprofen has been shown to inhibit the growth of prostate cancer cells [ ] . in adenocarcinoma gastric cells, drug showed antitumor effects, which have been mediated by the anti-angiogenesis, induction of apoptosis, and reduction of cell proliferation [ ] . the administration of drug induces apoptosis in metastatic melanoma cell lines [ ] . ibuprofen also reported to increases the chemosensitivity of cisplatin by decreasing the levels of heat shock protein s (hsp s) in lung cancer cells. hsp s are an important part of the cell's machinery for protein folding, and their function was associated with resistance to apoptosis [ ] . therefore, by blocking hsp s, ibuprofen increased the apoptosis by increasing the sensitivity of cisplatin. thalidomide ( ) is an immunomodulatory drug, which was originally developed as a sedative-hypnotic for the treatment of nausea during pregnancy. however, it was withdrawn due to its teratogenic effects. the drug was demonstrated whether it could be used for treating patients with refractory myeloma, because of its anti-angiogenic activity. after successful clinical evaluations, fda approved the drug for treating multiple myeloma. additionally, molecule also showed efficacy against several malignancies, including acute myeloid leukemia [ ] , myelodysplasia [ ] , and myelodysplastic syndrome [ ] . mechanistically, thalidomide binds to cereblon, which forms an e ubiquitin ligase complex, resulting in the rapid ubiquitination and proteasomal degradation of transcription factors, ikaros and aiolos [ ] . these two transcription factors are transcriptional regulators of b and t cell development [ ] . metformin ( ) is an orally available first-line drug and has been widely used for the treatment of type diabetes. the molecular mechanism of metformin involves the activation of adenosine monophosphate (amp)-induced protein kinase (ampk), a key enzyme regulating cellular metabolism. rapamycin (mtor), a gene that is involved in the survival of cancer cells is negatively regulated by ampk. metformin is also able to reduce the signals of mtor by inhibiting rag-mediated activation of mtor [ ] . in general, diabetic patients have an increased risk of several cancer types; especially diabetic women have a % risk of developing breast cancer. several studies suggested the anticancer property of drug . the drug at the dose of - mg/day has been shown to reduce the risk of cancer as well as its daily dose reduces the incidence of gastrological cancer in patients with diabetes [ , ] . several reviews and meta-analysis suggests that taking drug was associated with reduced risk of all cancer mortality in patients with diabetes [ ] . in a recent meta-analysis of several anti-diabetic drugs, it was found that the patients using drug had an overall reduced risk of cancer and decreased mortality rate by and %, respectively. whereas, the use of insulin was associated with an increased risk of cancer and mortality. the recent phase clinical trial studies using the occurrence of colorectal adenomas as a biomarker for cancer as a primary endpoint at year after intervention revealed that metformin reduced both occurrence and number of adenomas/polyps in the patients at low dosage level. methotrexate ( ) is a competitive inhibitor of dihydrofolate reductase (dhfr); a critical enzyme that involved in the synthesis of dna, rna, thymidylates, and proteins. the hydrofolate inhibitory activity of drug was responsible for its anti-leukemia activity. besides, drug was effective against a wide range of malignancies including breast, head and neck, leukemia, lymphoma, lung, osteosarcoma, bladder, and trophoblastic neoplasms [ ] . fda approved this compound in for the treatment of osteosarcoma, breast cancer, acute lymphoblastic leukemia, and hodgkin lymphoma. several reports suggest that the antitumor activity of methotrexate is also due to its ability to target inflammatory pathways. for instance, methotrexate was reported to suppress the nf-κb through the release of adenosine in cancer cells [ ] . rapamycin ( ) , also known as sirolimus, which was originally developed as an anti-fungal agent. however, drug was withdrawn due to its potent immunosuppressant and antitumor activities. mechanistically, the drug inhibits t cells and b cells by decreasing their sensitivity to il- through inhibition of mtor, which is highly upregulated in many tumor cells [ ] . in the year , fda, approved rapamycin for the prevention of allograft rejection. after that, this drug has been investigated for its anticancer properties. in recent studies, drug was reported to reduce the colony formation of leukemia progenitor cells in patients with acute myeloid leukemia [ ] . in addition to that, drug also showed efficacious in patients with imatinib-resistant chronic myelogenous leukemia through the suppression of vascular endothelial growth factor (vegf) mrna levels in leukemia cells with mild side effects [ ] . diclofenac ( ) is an acetic acid derivative of nsaid class, which has been used to treat pain and inflammatory diseases such as gout. its mode of action was believed to suppress the [ ] . in the phase ii clinical trial, it has been investigated in combination with calcitriol for recurrent prostate cancer. the results showed that the combination was well tolerated. statin family of drugs are lipid-lowering agents that inhibit the rate-limiting -hydroxy- methylglutaryl-coenzyme a (hmg-coa) reductase in the cholesterol biosynthesis pathway. statins are commonly prescribed to reduce cholesterol synthesis in patients with a high risk of cardiovascular disease. in addition to that, statins inhibit the mevalonate pathway that provides mevalonate, farnesyl, and geranyl pyrophosphate. these molecules are important for the cell cycle progression and cell proliferation, and therefore statins represent promising candidates in cancer therapeutics. in chronic myeloid leukemia cells, simvastatin ( ) [ ] . and other natural statins including mevastatin ( ) , lovastatin ( ) , and pravastatin ( ) displayed tnf-induced apoptosis through the downregulation of nf-κb mediated antiapoptotic gene products [ ] . the anti-cancer activity of statins was also investigated in animal studies, in which statins were effective in reducing the incidence and growth of tumors [ ] . several observational studies and meta-analysis supports the positive correlation of using statins with their chemopreventive effect in humans. meta-analyses revealed that the use of statins reduced the risk of patients with gastric cancer [ ] . as well as esophageal [ ] , and hepatocarcinoma cancer types [ ] . in a case-control study, drug with a dosage of mg/day for - years significantly reduced the incidence of colorectal cancer [ ] . depakine ( ) or valproic acid (vpa) is a short-chain free fatty acid mainly used to treat epilepsy, bipolar disorders, and migraine. its anticonvulsant activity has been attributed to the blockade of voltage-gated sodium channels and increased levels of gamma-aminobutyric acid (gaba) in the brain. anti-cancer activity of drug was first established in leukemia cells, in which the drug was shown to inhibit histone deacetylase (hdac) [ ] . depakine has also been found to suppress the production of cytokine and to modulate inflammatory signaling cascade. in human leukemia and human glioma cells, drug was able to suppress the production of il- and tnf-α [ ] . in prostate cancer cells, drug suppressed the il- through inhibition of nf-κb activity [ ] . some of the clinical trials of drug have advanced to phase ii for sarcomas, thyroid cancers, acute myelogenous leukemia, b cell lymphoma, breast cancer, melanoma, non-small, and small-cell lung cancers, prostate cancer, recurrent glioblastoma, and relapsed/refractory leukemia (www.clinicaltrials.gov.) recently, abdullah et al [ ] has shown that pitavastatin ( ) antagonizing the prc catalytic activity [ ] . treatment for acute myeloid leukemia (aml) has not significantly changed in the last decades and new therapeutic approaches are needed to achieve prolonged survival rates [ ] . bromocriptine ( ) is an ergoline derivative and dopamine agonist that is used in the treatment of parkinson's disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus [ , ] . repurposing strategy handled by lara-castillo, maría carmen, et al has shown bromocriptine as a potent anti-leukemia drug that mainly targets leukemia stem cells [ ] . [ ] [ ] [ ] . typically, the drug-discovery program to develop new potent anti-viral agents and to obtain approval for clinical use takes more than years. until now, no effective vaccines or drugs are approved treat these infections, although many are in (pre)clinical development. hence, the drugs that have been used for the treatment of other diseases or disorders that may inhibit the replication of coronaviruses (covs) might be useful in an attempt to save the life of several affected patients. in a search of potential anti-viral agents against mers-cov, de wilde et al identified four drugs such as chloroquine ( ) , chlorpromazine ( ) , loperamide ( ) , and lopinavir ( ) from the screening of fda approved drugs library ( figure ) [ ] . they all were able to inhibit the replication of mers-cov in the low micromolar range. in addition to that, all four drugs inhibited sars-cov as well as human coronavirus (hcov)- e, which suggests that they could be used for broad-spectral anti-viral activity. as a mode of action, compounds [ ] . this yielded a series of hit compounds, primarily categorized as, anticancer ( , ) , antipsychotics ( ) , antidepressent ( ) and antipsychotic ( ) pathways. interestingly, co-treatment of the drug with gemcitabine, a deoxycytidine analog that is commonly used for the treatment of cancers [ , ] , showed a synergistic anti-viral effect with a minimal cytotoxic effect. this supports the hypothesis of using them in a combination therapy to treat cov diseases. dengue fever is a life-threatening disease caused by four antigenically distinct dengue virus serotypes. it became a global burden, which causes approximately million infections each year, of which around , to , people die. even though a vaccine against dengue is available, its long-term protective action against each of the serotypes of dengue virus remains yet to be determined. besides, currently, no clinically approved antiviral therapy is available to combat this virus. among the many approaches applied to identify novel drugs for dengue fever, drug repurposing gained much attention to the scientific community. several antiviral, antimalarial, antidiabetic, antihistamine, anticancer, antipsychotic, antiparasite, and anticholesteremic drugs have been repurposed to combat dengue virus infection. a recent publication by botta et al. discussed each class of drugs and its repositioning in detail [ ] . to identify novel and potent drug candidates, approved drugs such as lovastatin ( ), chloroquine ( ) , prednisolone ( ), balapiravir ( ) , and celgosivir ( ) were investigated for the proof-off concept clinical trials for dengue viral infection ( figure ). although the results showed that they were safe in patients with acute dengue, drugs failed to meet prior-defined trial endpoints [ ] [ ] [ ] [ ] [ ] . besides, two clinical trials conducted in thailand and singapore involving ivermectin ( ) and ketotifen ( ), the preliminary result was quite promising as phase study of the drug suggested a reduction in serum ns levels and body temperature [ ] . recently, malakar et al. screened various classes of fda approved drugs including aminolevulinic acid ( ) , azelaic acid ( ), mitoxantrone hydrochloride ( ), quinine sulfate ( ) and tested their ability to inhibit dengue virus (denv) replication [ , ] . figure . representative example of drugs repurposed for dengue infections. aminolevulinic acid ( ), an endogenous non-proteinogenic amino acid, and azelaic acid ( ) are used to treat skin diseases [ , ] , while mitoxantrone hydrochloride ( ) , an anthracenedione antineoplastic agent used for the treatment of leukemia [ ] . quinine sulfate ( ) is a natural compound extracted from cinchona bark, which is commercially available in -mg tablets under the brand name qualaquin. it has been widely used to treat chloroquine-resistant plasmodium falciparum [ ] . its ability to reduce the viral replication was also demonstrated against herpes simplex virus (hsv) [ ] , and influenza virus [ ] . among these four drugs investigated, the drug was found to be very effective in inhibiting the replication of denv by about % compared to untreated controls, while the others showed only moderate reduction of about %. it was very impressive that drug was able to reduce virus replication of all four serotypes of denv in three different cell lines of human origin. at the molecular level, it inhibited denv replication by reducing viral protein and rna synthesis in a dose-dependent manner. moreover, drug enhanced the expression of genes related to innate immune response. these findings suggest that the efficacy of drug for stimulating antiviral genes, which led to reduce denv replication. from another set of prescribed drug candidates ( - ) were tested against blood-stage p. falciparum cultures ( figure ) and liver-stage p. berghei [ , ] . they all showed promising antimalarial activity with ic values ranged from . µm to . nm. drugs raloxifene hydrochloride ( ) salirasib ( , figure ) is a promising cancer drug candidate inhibits isoprenylcysteine carboxyl methyltransferase (icmt), a validated target for cancer drug development. recently, salirasib and its analogs with , , -triazole were repuposed for their pontential antimalarial activity [ ] . in general, triazole derivartives are known to have potent antimalarial activity [ ] . compound were investigated the in vitro toxicity to p. falciparum in the asexual stages and in vero cells. an antiplasmodial activity assay was performed using a simple, highsensitivity methodology based on nanoluciferase (nluc)-transfected p. falciparum parasites. the results showed that some of the analogs were active at low micromolar concentration, alzheimer's is the most common type of dementia. it affects around % of people over the age of , % over the age of , and more than a third of those over the age of [ ] . in the year , there were approximately . million people worldwide with ad of which dementia resulted in about . million deaths. it is estimated that there will be more than million people with dementia worldwide by . therefore, ad represents a major and rising public health concern, and there is an urgent need to develop more therapies that are effective. ad refers to a devastating condition leading to progressive cognitive decline, functional impairment, and loss of independence. accumulation of amyloid-β peptide (aβ)-enriched neuritic plaques, and neurofibrillary tangles, synaptic, and neuronal dysfunction, as well as loss in combination with the associated neurochemical changes in the brain, are the crucial pathological event for ad [ ] . recent work suggests that higher levels of total tau may potentiate the toxic effects of aβ [ ] . other factors such as inflammatory processes and mitochondrial function are also likely to have an important role [ ] . three acetylcholinesterase inhibitors including donepezil ( ), rivastigmine ( ) and are cost-effective [ ] . for example, acetylcholinesterase inhibitors improve cognition to above pre-treatment performance for ~ - months. the availability of these drugs has substantially advanced the treatment of patients with ad, but there is a persistent need to build on our increasing understanding of disease pathogenesis to develop more effective symptomatic treatments and disease-modifying therapies. drugs. the md simulation results exposed that these five drugs (risperidone, domperidone, verapamil, tamsulosin and cinitapride) showed better profiles with respect to their rmsd, rmsf, sasa and rg evaluations graphs and steady stable behavior in all docking complexes. in-vitro ache inhibition assay of the above best-screened drugs were performed by spectrophotometric method using acetylthiocholine iodide as substrate. the enzyme inhibition and kinetic mechanism of these drugs showed that cinitapride ( ) has good therapeutic potential with respect to standard and other drugs. it is well known that, cintapride ( ) is a gastroprokinetic agent and antiulcer agent of the benzamide class [ ] . it is an agonist of -ht and -ht receptors and as an antagonist of the -ht receptors [ ] . based on aforementioned results, it is justified that cinitapride has better repositioning profile which may be used in the treatment of ad after clinical assessment. efforts to develop more effective therapies have so far been unsuccessful with several highprofile clinical trial fails to demonstrate the benefit. the reasons for this are probably multifactorial. the majority of putative disease-modifying therapies that have been evaluated have targeted amyloid pathology. this lack of breadth in treatment approaches has been criticized, and some commentators have argued that a more sophisticated knowledge of disease pathways is needed before we can develop more effective candidate therapies. for example, phase ii trial of tarenflurbil ( , figure ) only provided a suggestion of benefit in a posthoc subgroup analysis [ , ] ; the putative mechanism of action via γ-secretase modulation and its related impact on amyloid pathology was never confirmed in patients with ad. subsequent phase iii trial had negative outcomes. although, the results of a phase ii trial of dimebon ( ) were much more favorable than of analog (figure ). it seems that the significant benefit seen in the treatment group was driven by a larger-than-expected deterioration in the group receiving placebo treatment, and the mechanism of action was not well characterized. in the central nervous system (cns), angiotensin ii mediates key processes including, the release of inflammatory mediators, vasoconstriction, mitochondrial dysfunction, and inhibition of acetylcholine release at central synapses. all of which are proposed to be relevant to ad and are potential targets for therapeutic intervention [ , ] . based on this background, it has been proposed that angiotensin receptor blockers (arbs) may confer symptomatic benefits on cognition. a large-scale screen of antihypertensive drugs by want et al. [ ] , identified the arb valsartan ( , figure ) as the only compound that was able to reduce aβ accumulation in cultured neurons and inhibit aβ aggregation in vitro. the same group was demonstrated that the reduced plaque burden as well as the improved learning and memory in cognitive tests (including the morris water maze task) following months of treatment with valsartan in month-old tg transgenic mice. as a result, the greatest benefits were seen at a dose of mg/ kg/day, which is equivalent to . times the maximum recommended dose for treating patients with hypertension. using another arb, olmesartan ( , figure ) by takeda et al. [ ] , demonstrated that daily treatment of young app mice for one month improved cerebral blood flow without affecting aβ - and aβ - levels. in the aβ - -injected mouse model, in which aβ fragments are injected intracranially to generate deficits, pre-treatment with telmisartan ( , figure ) increased cerebral blood flow and inhibited the plaque deposition [ ] . however, the physiological relevance of this model is unclear. perhaps, the most striking preclinical evidence comes from a study in which losartan ( , figure ) was administered intranasally to app/psen mice, at a dose much lower than that mediating hypotensive effects; drug led to a . -fold reduction in aβ plaques compared to vehicle-treated mice but also reduced the levels of pro-inflammatory mediators and increased the levels of anti-inflammatory mediator il- in the serum of these animals [ , ] . overall, there was significant reduction in the incidence of dementia in patients taking arbs compared to those taking the comparator cardiovascular drugs (hazard ratio: . ; % confidence interval: . - . ) and those taking angiotensin-converting enzyme (ace) inhibitor lisinopril (hazard ratio: . ; % confidence interval: . - . ). although the evidence for the potential of arbs in ad is conflicting, and difficult to interpret, in our view, there is a sufficient indication of potential benefit to merit further in vivo work to clarify the relative importance of different mechanisms, the optimal dose, and the optimal agent, which could lead to proof-of-concept study in patients with ad. the best evidence from in vitro and in vivo studies points to either drug or as a preferred arb, with some animal studies also highlighting drug as a potential candidate. however, there is an unfortunate disconnect between in vivo and clinical studies, as no formal studies to provide direct clinical evidence have so far been conducted on any of the most promising candidates. calcium channel blockers (ccbs) of the dihydropyridine class are widely used to treat hypertension and angina through their vasodilatory activity on smooth muscle vasculature. most of the drugs in this class have good blood-brain barrier penetration and induces cerebral vasodilatation, increased cerebral blood flow in animals and humans [ ] . in vitro studies have revealed that certain ccbs reduce aβ production, oligomerization, and accumulation, rescue aβ-induced neurotoxicity, and improve cell survival in the presence of aβ [ ] [ ] [ ] . ccbs have also been shown to reduce glutamate-induced cell death and levels of intracellular calcium [ ] . the ability of ccbs to prevent aβ - and aβ - production was investigated in chinese hamster ovary cells (cho) by paris et al. and iwasaki et al. in that study, amlodipine ( ) and nilvadipine ( ) (figure ) were identified as the only agents that inhibited aβ production [ ] . however, the concentrations studied were several-fold higher than can be achieved therapeutically. isradipine ( , figure ) was shown to have a neuroprotective effect against aβ-induced apoptosis in neuroblastoma mg cell lines. a protective effect in a drosophila melanogaster model of aβ-induced neurotoxicity and it's brain-penetrant in the xtg-ad mouse model of ad was also reported [ , ] . the differential effects of ccbs indicate that their potential benefits in ad are probably independent of their anti-hypertensive activity and may be specific to individual drugs within this class. dihydropyridines seem to be more effective than compounds with different chemical structures (such as verapamil or diltiazem), with evidence from preclinical studies that highlighted nilvadipine ( ) as the best therapeutic candidate. there was some clinical evidence regarding the potential benefit of the ccb nimodipine ( , figure ) in patients with clinically significant dementia. the evidence has been summarized in a cochrane review of nimodipine trials in more than , patients with dementia. the review reported that the treatment showed efficacy in improving cognition, but not activities of daily living, at doses of mg/day [ ] . however, the evidence was limited by small size and duration (mostly weeks) of trials as well as the lack of operational diagnostic criteria for ad or vascular dementia. tetracycline antibiotics are widely used to treat bacterial infections and are well tolerated in older people, but most of the treatment data pertain to short-term periods of exposure. studies related to ad have predominantly focused on minocycline ( , figure ) because it is the most lipophilic tetracycline, with greater blood-brain barrier penetration than other agents in this class. concerning preclinical studies, the drug has been shown to reduce aβ - aggregation, and promote disassembly of pre-formed fibrils in vitro studies. various groups have independently shown that drug reduces the levels of proinflammatory mediators and microglial activation in a range of mouse models of ad [ ] [ ] [ ] [ ] [ ] . besides, two of the three studies using transgenic mouse models of ad for days or more of treatment have shown a significant decrease in cerebral aβ accumulation and improvements in behavioral outcomes as well [ ] . one of these studies used -month-old xtg-ad mice and reported reductions in cortical amyloid levels following months of minocycline treatment, but no changes in tau pathology was observed. the third study reported that no benefit concerning amyloid accumulation or behavior over a treatment period of months. an additional study in a rat model of diabetes demonstrated a reduction in aβ - and aβ - levels and associated improvements in behavioral outcomes over weeks of treatment [ ] . doxycycline ( ) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies. compound is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in ad mice. balducci, claudia, et al. showed that -to month-old app/ps de (app/ps ) ad mice receiving under different treatment regimens recovered their memory without plaque reduction. an acute treatment was, also, sufficient to improve app/ps mouse memory, suggesting an action against soluble abos. this was confirmed in an abo-induced mouse model, where the abo-mediated memory impairment was abolished by a its pretreatment. although abos induce memory impairment through glial activation, assessing the anti-inflammatory action of , we found that in both the abotreated and app/ps mice, the memory recovery was associated with a lower neuroinflammation. our data promote as a hopeful repositioned drug counteracting crucial neuropathological ad targets [ ] . drugs that activate retinoic acid receptors (rars) are used to treat several skin-related conditions such as acne and psoriasis. retinoic acid is also vital for normal nerve function and repair. there is genomic and epidemiological evidence suggest that impaired retinoic acid signaling may contribute to the etiology of ad [ ] . chronic deprivation of retinoic acid in rats leads to deposition of aβ in the vasculature [ ] and dysregulation of amyloid processing in the cortex [ ] . it has been shown that treatment with retinoid x receptor (rxr) agonist bexarotene ( , figure ) , which is approved for the treatment of cutaneous t cell lymphoma, leads to pathological and behavioral improvements in transgenic mouse models of ad. acute treatment with drug (lasting less than days) caused a rapid reduction ( %) in aβ - and aβ - levels and aβ plaque burden in both young and old mice [ ] . chronic -day treatment resulted in a sustained reduction ( %) insoluble aβ levels. mechanistically, drug resulted in the upregulation of components of the high-density lipoprotein (hdl) pathway such as apolipoprotein e (apoe), which promotes the proteolytic degradation of aβ [ ] . further, potential mechanism of action of retinoids may include the upregulation of enzymes involved in amyloid clearance such as insulin-degrading enzyme [ ] and components of the apoe pathway [ ] . retinoids may also induce potentially beneficial changes related to insulin signaling and increased neurogenesis and promote neuronal differentiation of progenitor cells [ , ] . retinoids also act as antioxidants (by regulating sod and inhibiting glutathione depletion to reduce mitochondrial damage) as well as anti-inflammatory agents (by reducing the production of il- ) in vitro. both of these activities have potential importance in ad pathology [ , ] . therefore, there was a strong mechanistic rationale for the potential benefit of retinoid therapies beyond amyloid modulation, but there is a need to further clarify the impact of treatment on these pathways through in vivo studies. overall, studies in the literature indicate that retinoids have strong potential mechanistic plausibility as therapies for ad owing to their effects on app processing, aβ clearance, insulin signaling, and neurogenesis. out of the approved drugs, data for bexarotene have provided proof of concept as potential candidate for the treatment of alzheimer's disease as noted above, whereas acitretin ( , figure ) , which is known to penetrate tissues including brain may also be a promising candidate for ad [ ] . it is a long-term degenerative disorder of the cns, which mainly affects the motor system. as of , pd affected . million individuals, of which people died. it usually occurs people over the age of , of whom about one percent are affected. in addition to the classic motor symptoms caused by the death of dopaminergic neurons, parkinson's disease encompasses a wide range of nonmotor symptoms. although novel disease-modifying medications that slow or stop parkinson's disease progression are being developed, drug repurposing, which is the use of existing drugs that have passed numerous toxicity and clinical safety tests for new indications, can be used to identify treatment compounds. this strategy has revealed that tetracyclines ( ) are promising candidates for the treatment of parkinson's disease [ ] . tetracyclines, which are neuroprotective, inhibit proinflammatory molecule production, matrix metalloproteinase activity, mitochondrial dysfunction, protein misfolding/aggregation, and microglial activation. two commonly used semisynthetic second-generation tetracycline derivatives, minocycline ( ) and doxycycline ( ) , exhibit effective neuroprotective activity in experimental models of neurodegenerative/ neuropsychiatric diseases and no substantial toxicity. moreover, novel synthetic tetracyclines with different biological properties due to chemical tuning are now available. in this review, we discuss the multiple effects and clinical properties of tetracyclines and their potential use in parkinson's disease treatment. in addition, we examine the hypothesis that the anti-inflammatory activities of tetracyclines regulate inflammasome signaling. based on their excellent safety profiles in humans from their use for over years as antibiotics, we propose the repurposing of tetracyclines, a multitarget antibiotic, to treat parkinson's disease. isradipine ( ) is an l-type calcium channel blocker of the dihydropyridine class, which has been widely used for the treatment of high blood pressure to reduce the risk of heart attack and stroke. this drug came into medical use in the year . epidemiological data support that calcium channel blockers may have the potential to reduce the risk of developing pd. among dihydropyridines, drug has attracted very much as it inhibits the subtype cav ca + channels cav . and cav . , which are most likely mediate the risk in pd. moreover, the good brain bioavailability of drug has made it the most promising candidate for repurposing [ , ] . studies have shown that drug dose repentantly protect the dopaminergic neurons from -methyl- -phenyl- , , , -tetrahydropyridine mptp) and hydroxydopamine ( -ohda)-induced toxicity by reverting dopaminergic neurons to a latent juvenile pacemaking mechanism independent of calcium [ , ] . an open-label, doseescalation study assessing safety (steady-pd) of drug controlled release - mg/day in patients with early pd suggested that the acceptable tolerability at doses of ≤ mg per day; however at higher doses caused leg edema and dizziness [ ] . isradipine ( ) with mg being the highest dosage was again confirmed in another steady-pd-ii, a randomized, double-blinded trial, which was undertaken in subjects with early pd not requiring dopaminergic therapy [ ] . results suggest that the most common adverse effect was peripheral edema, which occurred in % of patients receiving drug ( mg). placebocontrolled phase iii clinical study to assess the efficacy of drug ( inosine ( , figure ) is a purine nucleoside, which has been used as a dietary supplement by athletes for improving aerobic performance. it has been shown to have neuroprotective roles by elevating the level of serum urate, a natural antioxidant and peroxynitrite scavenging property with potential benefits to patients with multiple sclerosis. many studies suggest that individuals with increased levels of urate in serum have a reduced risk of developing pd, as well as in patients with pd are associated with a reduced rate of disease progression. moreover, in toxin-based models of pd, increased urate levels have conferred protection against dopaminergic cell death stimulated by mptp, -ohda, and rotenone. akt-gsk- b signaling and nuclear factor (erythroid-derived- )-like (nrf ) were thought to involve in these effects. therefore, given the data supporting a neuroprotective role of urate, inosine has been repurposed in the pathogenesis of pd. the ability to raise urate level of drug in serum was demonstrated in sure-pd, a randomized, double blind, placebo-controlled in patients with early pd not yet requiring any medication. the results showed that inosine raised the mild urate level ( . - . mg/dl) or moderate urate elevation ( . - . mg/dl) after months and well-tolerated with favorable progression rate in updrs score, which amounted to ~ point per year on the total updrs scale. on the other hand, the elevated level of urate in serum have the risk of hypertension, coronary heart disease, and stroke over the long term. these side effects are potentially limiting its utility in older patients with pd. however, in patients of asian origin with pd, inosine elevated urate levels ( . - . mg/dl) without side effects after year of treatment was reported. the multicenter sure-pd trial, which involves a large number of patients ( patients), is currently underway intending to elevate the urate level to . - . mg/dl. the result of the study will be expected in the year . simvastatin ( ), since statins are also known to modulate various biological processes relevant to the pathogenesis of pd [ , ] . simvastatin has been repurposed for treating this disease. pretreatment with simvastatin preserved dopaminergic cells and motor behavior in rodents treated with -hydroxydopamine ( -ohda), promoting antioxidant protein expression or via modulation of nmda receptor and pro-inflammatory cytokine expression [ / ] . similarly, in -methyl- -phenyl- , , , -tetrahydropyridine (mptp) models, pretreatment with simvastatin suppressed activation of nf-κb, protected dopaminergic neurons, and improved the motor function [ , ] . although, stains, in general, have given encouraging preclinical studies, epidemiological data regarding the association between usage of statins and the risk of pd are unclear. moreover, the modest protective effect of statins that disappeared when adjusted for cholesterol level [ ] . however, due to the promising biochemical and pharmacological properties of simvastatin, it is currently being examined with patients having moderate-stage pd in phase ii double-blind, randomized, controlled, multicenter trial. nilotinib ( , figure ) is a selective c-abl tyrosine kinase inhibitor approved for the treatment of imatinib-resistant chronic myelogenous leukemia (cml). accumulating evidence suggests that c-abl activation has been linked in the pathogenesis of pd and other synucleinopathies. activated (phosphorylated) c-abl has been found in a high level in postmortem studies of patients with pd [ , ] . it was also reported that activation of c-abl in mice induces neurodegeneration in the hippocampal and striatal brain areas [ ] . continuous work has demonstrated that c-abl phosphorylation occurs as a result of mitochondrial dysfunction, and oxidative stress [ ] , which can promote the accumulation of α-synuclein in through effects on autophagy mechanisms [ ] and can further promote phosphorylation of parkin, causing inhibition of its ubiquitin e ligase activity, inducing mitochondrial dysfunction and dopaminergic neuronal death [ ] . all the above evidences propose that c-abl may be a promising therapeutic target in the management of pd. the cns penetration of nilotinib over other abl inhibitors has favored it to fetch more data in pd related studies. indeed, in preclinical models of pd, drug has been shown to cross the blood-brain barrier and reduces c-abl activity, ameliorating autophagic clearance of α-synuclein in transgenic and lentiviral gene-transfer models [ ] . more importantly, these effects were seen at doses far lower ( - mg/kg/day) than those used to treat cml, which is considered as the key characteristics for potential drug repurposing. furthermore, nilotinib prevented dopaminergic cell loss and motor impairments induced by mptp in mice, which were associated with inhibition of parkin phosphorylation and reduced accumulation of parkin substrate paris, thus hinting at another potential mechanism of action [ , ] . based on these preclinical data, a small, open-label proof-of-concept study was recently conducted to evaluate the safety and tolerability of nilotinib in patients with pd dementia or dementia with lewy bodies followed-up for weeks, followed by a final assessment weeks later [ ] . the necessary lowest choice of dose for the clinical study was taken as - mg. the authors reported that nilotinib was well tolerated, though one patient receiving mg was diagnosed with myocardial infarction, and two had transient qtc prolongations. there was also evidence of cns penetration with nilotinib csf plasma ratio of and % with and mg, respectively. due to numerous methodological limitations, these findings should be interpreted with caution [ ] . due to unwanted off-target nonselective tyrosine kinase inhibition; side effects of nilotinib at doses used to treat cml include cardiac conduction abnormalities. therefore, claims of tolerability should be interpreted with caution. indeed, the effects on efficacy was also highly impossible to be concluded. besides, it was also reported in the csf, none of the markers used were validated as biomarkers in pd; they can also vary greatly between patients and track poorly with disease stage and progression. this situation again raises questions about the optimum dose of nilotinib, its brain penetrance, assessments of cardiovascular effects in patients. parkinson's disease (pd) is a neurodegenerative disorder for which a greater prevalence and incidence is described in men. this suggests a protective effect of sex hormones in the brain. intellectual property related issues hinder the commercialization of repositioned molecule. in conclusion, repurposing of drugs has the potential to find and improve treatments for various diseases. the drugs listed above are only a few of plenty drugs that can be repurposed in various therapies. the drugs discussed in the perspective are summarized in table and compared to the previous uses of each drug with the repurposed use. the opportunities for drug repurposing are diverse, but a lot still has to be done for its exploration. drug repurposing or drug repositioning offers a new strategy to academic centers, research council programs, and not-for-profit organizations, as well as pharmaceutical and biotechnology companies for drug development. the main advantage of drug repurposing is the established safety of the known candidate compounds when compared to that of the development of novel therapeutic compounds. the time and cost required to advance a candidate into clinical trials can be substantially reduced because in vitro and in vivo screening, chemical optimization, toxicity studies, bulk manufacturing, and formulation development have already been completed in many cases, and can, therefore, be bypassed. moreover, these drugs have been on the market for many years, and consequently, the side effects are already known, and safety is therefore very high. for medicinal chemists, the repurposing of drugs on cancer is a rewarding job for the global healthcare system and possibly a step forward for people to get cheaper and safer medicines rather than afford for cancer therapy. however, time and minimum investment have to be spent to conduct further studies to improve the safety and success of the repurposed drugs. drug repurposing also offers the possibility to develop multi-target drugs that can interact with more than one pathway/protein at the same time. for complex diseases such as (neuro) inflammatory and degenerative disorders, the development of multi-target drugs will be an emerging area for the treatment having multiple advantages such as i) synergistic effect ii) reduced drug-resistance iii) better compliance iv) simplified pharmacokinetic and pharmacodynamic profile and a reduced risk of drug-drug interactions. on the other hand, the limitations of drug repurposing should also be considered. they involve a) technical challenges and legal requirements such as intellectual property rights which could hamper the whole process and are often hard to overcome b) serious problem on the development of resistant germs on account of consuming a drug for a variety of diseases c) owing to target selectivity, it is difficult to identify a drug that can cure or treat two different diseases on its own. nevertheless, drug repurposing is only a part of the solution for the sinking numbers of new diseases and its treatment. however, it is quite hard to replace the common way of identifying new drug candidates. in furuter, this approach could be improved in many differents ways: integrating data about repositioning drugs which are available in many public platforms, such as pubchem), the nobel chronicles a comprehensive map of molecular drug targets trends in development and approval times for new therapeutics in the united states drug repositioning identifying and developing new uses for existing drugs rebuilding big pharma's business model fda drug approvals genetic and rare diseases information centre drug repositioning: bringing new life to shelved assets and existing drugs drug repurposing: progress, challenges and recommendations a review of computational drug repurposing cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells melanins and melanosomes: biosynthesis, biogenesis, physiological, and pathological functions skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors melanogenesis-inhibitory and cytotoxic activities of triterpene glycoside constituents from the bark of albiziaprocera inhibitors of melanogenesis: a patent review recent updates in melanocyte function: the use of promising bioactive compounds for the treatment of hypopigmentary disorders inhibitors of melanogenesis: an updated review brain tyrosinase overexpression implicates age-dependent neuromelanin production in parkinson's disease pathogenesis novel tyrosinase inhibitory peptide with free radical scavenging ability tyrosinase-expressing neuronal cell line as in vitro model of parkinson's disease the reaction of alpha-synuclein with tyrosinase: possible implications for parkinson disease skaltsounis, design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors tyrosinase exacerbates dopamine toxicity but is not genetically associated with parkinson's disease degradation of tyrosinase induced by phenylthiourea occurs following golgi maturation inhibition of l-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase structural requirement(s) of n-phenylthioureas and benzaldehyde thiosemicarbazones as inhibitors of melanogenesis in melanoma b cells analogues of ethionamide, a drug used for multi drug resistant tuberculosis, exhibit potent inhibition of tyrosinase repositioning of thiourea-containing drugs as tyrosinase inhibitors pharmacovigilance and tuberculosis: applying the lessons of thioacetazone antithyroid drugs thiopurine drugs repositioned as tyrosinase inhibitors crystal structure of agaricusbisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone the unravelling of the complex pattern of tyrosinase inhibition exploring the interaction of n/s compounds with a dicopper center: tyrosinase inhibition and model studies united states cancer statistics: - incidence and mortality web based report cancer drug discovery by repurposing: teaching new tricks to old dogs systematic review update of observational studies further supports aspirin role in cancer treatment: time to share evidence and decision-making with patients? effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials aspirin, salicylates, and cancer the biology of prostaglandin synthesis and inhibition nonsteroidal antiinflammatory agents differ in their ability to suppress nf-kappa b activation, inhibition of expression of cyclooxygenase- and cyclin d and abrogation of tumor cell proliferation repositioning aspirin to treat lung and breast cancers and overcome acquired resistance to targeted therapy american society of health-system pharmacists targeting apoptosis pathways by celecoxib in cancer an international randomised trial of celecoxib versus celecoxib plus difluoromethylornithine in patients with familial adenomatous polyposis molecular mechanisms and bioavailability of polyphenols in prostate cancer inhibitory effect of ibuprofen on tumor survival and angiogenesis in gastric cancer cell ibuprofen and hydrogel-released ibuprofen in the reduction of inflammation induced migration in melanoma cells ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein single-agent lenalidomide induces complete remission of acute myeloid leukemia in patients with isolated trisomy thalidomide potentiates etoposide-induced apoptosis in murine neuroblastoma through suppression of nf-κb activation a combination of thalidomide and arsenic trioxideis effective and well tolerated in patients with myelodysplastic syndromes how thalidomide works against cancer suppression of aiolos and ikaros expression by lenalidomide reduces human ilc -ilc /nk cell transdifferentiation metformin, independent of ampk, inhibits mtorc in a rag gtpase-dependent manner type diabetes increases and metformin reducestotal, colorectal, liver and pancreatic cancer incidences in taiwanese: a representative population prospective cohort study of , individuals the potential effect of metformin on cancer: an umbrella review cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis mir- - p modulates resistance to methotrexate in human colorectal adenocarcinoma cells by downregulating hipk anti-inflammatory therapy in chronic disease: challenges and opportunities clinical activity of mammalian target of rapamycin inhibitors in solid tumors evaluation of combination of atorvastatin with sulindac or naproxen profoundly inhibits colonic adenocarcinomas by suppressing the p /beta-catenin/cyclin d signaling pathway in rats simvastatin potentiates tnf-alpha-induced apoptosis through the down-regulation of nf-kappab-dependent antiapoptotic gene products: role of ikappabalpha kinase and tgfbeta-activated kinase- role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements statin uses and mortality in colorectal cancer patients: an updated systematic review and meta-analysis statin use is associated with a reduced incidence of colorectal cancer: a colonoscopy-controlled case-control study statin use and the risk of colorectal cancer in a population-based electronic health records study the role of statins for primary prevention in non-elderly colorectal cancer patients statin use is associated with a reduced incidence of colorectal cancer: a colonoscopy-controlled case-control study sodium valproate and -aza- '-deoxycytidine differentially modulate dna demethylation in g phase-arrested and proliferative hela cells exploring the drug repurposing versatility of valproic acid as a multifunctional regulator of innate and adaptive immune cells therapeutic value of voltage-gated sodium channel inhibitors in breast, colorectal, and prostate cancer: a systematic review screening a library of approved drugs reveals that prednisolone synergizes with pitavastatin to induce ovarian cancer cell death towards the first targeted therapy for triplenegative breast cancer: repositioning of clofazimine as a chemotherapy-compatible selective wnt pathway inhibitor, cancer letters a drug repurposing screening reveals a novel epigenetic activity of hydroxychloroquine acute myeloid leukaemia in adults drug reformulations and repositioning in pharmaceutical industry and its impact on market access: reassessment of nomenclature differential actions of antiparkinson agents at multiple classes of mono-aminergic receptor i. a multivariate analysis of the binding profiles of drugs at native and cloned human receptor subtypes repositioning of bromocriptine for treatment of acute myeloid leukemia the genome sequence of the sars-associated coronavirus severe acute respiratory syndrome an overview of severe acute respiratory syndrome-coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy middle east respiratory syndrome-coronavirus (mers-cov): an updated overview and pharmacotherapeutics mers: south korea closes schools after third death severe respiratory disease associated with middle east respiratory syndrome coronavirus interaction of ethambutol with human organic cation transporters of the slc family indicates potential for drug-drug interactions during antituberculosis therapy screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome corona virus replication in cell culture effects of chloroquine on viral infections: an old drug against today's diseases? a systematic screen of fda-approved drugs for inhibitors of biological threat agents simulating henipa virus multi cycle replication in a screening assay leads to identification of a promising candidate for therapy pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents clathrin mediates infectious hepatitis c virus particle egress inhibitors of alpha virus entry and replication identified with as table chikungunya replicon cell line and virus-based assays mouse hepatitis virus type enters cells through a clathrin-mediated endocytic pathway independent of eps clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace with the cytoplasmic tail deleted small molecules targeting severe acute respiratory syndrome human coronavirus saracatinib inhibits middle east respiratory syndrome-coronavirus replication invitro gemcitabine: a critical nucleoside for cancer therapy synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses drug repurposing approaches to fight dengue virus infection and related diseases drug repositioning for dengue haemorrhagic fever by integrating multiple omics analyses effects of short-course oral corticosteroid therapy in early dengue infection in vietnamese patients: a randomized, placebo-controlled trial efficacy and safety of celgosivir in patients with dengue fever (celaden): a phase b, randomised, double-blind, placebo-controlled, proof-of-concept trial lovastatin for the treatment of adult patients with dengue: a randomized, double-blind, placebo-controlled trial ivermectin: a promising anti-dengue replication treatment pesented at th european congress of clinical microbiology and infectious diseases current status of dengue therapeutics research and development repurposing approved drugs on the pathway tonovel therapies. schein update on the management of rosacea: a status report on the current role and new horizons with topical azelaic acid cure of condylomaacuminata covering the glans penis using aminolevulinic acid/photodynamic therapy a phase study of azacitidine with high-dose cytarabine and mitoxantrone in high-risk acute myeloid leukemia quinine an old anti-malarial drug in a modern world: role in the treatment of malaria antiviral effects of quinine sulfate on hsv- hacat cells infected: analysis of the molecular mechanisms involved inhibition of influenza virus replication by targeting broad host cell pathways quinoline hybrids and their antiplasmodial and antimalarial activities evaluation of antiplasmodial potential of c and c modified quinolines: in vitro and in silico astemizole analogues with reduced herg inhibition as potent antimalarial compounds liver-stage malaria parasites vulnerable to diverse chemical scaffolds new leads for drug repurposing against malaria 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inhibition and kinetic mechanism approaches the prokinetic cinitapride has no clinically relevant pharmacokinetic interaction and effect on qt during coadministration with ketoconazole cinitapride protects against ethanol-induced gastric mucosal injury in rats: role of -hydroxytryptamine, prostaglandins and sulfhydryl compounds treating alzheimer's disease by targeting iron tarenflurbil phase study group. effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild alzheimer disease: a randomized controlled trial peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate alzheimer-related disease the renin-angiotensin system and antihypertensive drugs in alzheimer's disease: current standing of the angiotensin hypothesis? valsartan lowers brain β-amyloid protein levels and improves spatial learning in a mouse model of alzheimer disease angiotensin receptor blocker prevented β-amyloid-induced cognitive impairment associated with recovery of neurovascular coupling telmisartan prevented cognitive decline partly due to ppar-γ activation protective effects of intranasal losartan in the app/ps transgenic mouse model of alzheimer disease use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis favourable effects of nilvadipine on cognitive function and regional cerebral blood flow on spect in hypertensive patients with mild cognitive impairment identification of antihypertensive drugs, which inhibit amyloid-β protein oligomerization selective dihydropyiridine compounds facilitate the clearance of β-amyloid across the blood-brain barrier l-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for alzheimer's disease protective effects of ginsenoside rg against glutamate-induced neurotoxicity in pc cells selective antihypertensive dihydropyridines lower aβ accumulation by targeting both the production and the clearance of aβ across the blood-brain barrier a translational continuum of model systems for evaluating treatment strategies in alzheimer's disease: isradipine as a candidate drug nimodipine for primary degenerative, mixed and vascular dementia minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathyin vivo in transgenic mice minocycline as a potential therapeutic agent in neurodegenerative disorders characterised by protein misfolding reductions in amyloid-β-derived neuroinflammation, with minocycline, restore cognition but do not significantly affect tau hyperphosphorylation increases in β-amyloid protein in the hippocampus caused by diabetic metabolic disorder are blocked by minocycline through inhibition of nf-κb pathway activation doxycycline counteracts neuroinflammation restoring memory in alzheimer's disease mouse models evidence for defective retinoid transport and function in late onset alzheimer's disease disruption of the retinoid signalling pathway causes a deposition of amyloid β in the adult rat brain retinoic acid normalizes nuclear receptor mediated hypo-expression of proteins involved in β-amyloid deposits in the cerebral cortex of vitamin a deprived rats apoedirected therapeutics rapidly clear β-amyloid and reverse deficits in ad mouse models human apoe isoforms differentially regulate brain amyloid-β peptide clearance regulation by retinoic acid of insulin degrading enzyme and of a related endoprotease in human neuroblastoma cell lines interactions between retinoic acid, nerve growth factor and sonic hedgehog signalling pathways in neurite outgrowth sequential rarβ and α signallingin vivo can induce adult forebrain neural progenitor cells to differentiate into neurons through shh and fgf signalling pathways all-trans retinoic acid as a novel therapeutic strategy for alzheimer's disease towards retinoid therapy for alzheimer's disease kinetics of tissue distribution and elimination of retinoid drugs in the rat tetracycline repurposing in neurodegeneration: focus on parkinson's disease expression and , -dihydropyridine binding properties of brain l-type calcium channel isoforms rejuvenation protects neurons in mouse models of parkinson's disease the l-type channel antagonist isradipine is neuroprotective in a mouse model of parkinson'sdisease tolerability of isradipine in early parkinson's disease: a pilot dose escalation study phase ii safety, tolerability, and dose selection study of isradipine as a potential disease-modifying intervention in early parkinson's disease (steady-pd) simvastatin as a potential disease modifying therapy for patients with parkinson's disease: rationale for clinical trial, and current progress prospects of statins in parkinson disease simvastatin inhibits activation of nadph oxidase/p mapk pathway and enhances expression of antioxidant protein in parkinson disease models simvastatin prevents neuroinflammation by inhibiting n-methyl-d-aspartic acid receptor in -hydroxydopamine-treated pc cells neuroprotective potential of atorvastatin and simvastatin (hmg-coa reductase inhibitors) against -hydroxydopamine ( -ohda) induced parkinson-like symptoms simvastatin inhibits the activation of p ras and prevents the loss of dopaminergic neurons in a mouse model of parkinson's disease simvastatin prevents -methyl- -phenyl- , , , -tetrahydropyridineinduced striatal dopamine depletion and protein tyrosine nitration in mice confounding of the association between statins and parkinson disease: systematic review and meta-analysis novel regulation of parkin function through c-abl-mediated tyrosine phosphorylation: implications for parkinson's disease phosphorylation by the c-abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function nilotinib reverses loss of dopamine neurons and improves motor behavior via autophagic degradation of -synuclein in parkinson's disease models regulation of the c-abl and bcrabl tyrosine kinases a novel tyrosine kinase inhibitor amn (nilotinib) normalizes striatal motor behaviors in a mouse model of parkinson's disease, front the c-abl inhibitor, nilotinib, protects dopaminergic neurons in a preclinical animal model of parkinson's disease nilotinib effects in parkinson's disease and dementia with lewy bodies nilotinib-differentiating the hope from the hype repurposing sex steroids and related drugs as potential treatment for parkinson's disease drug repurposing: a promising tool to accelerate the drug discovery process the drug repurposing landscape from to : evolution, challenges, and possible solutions • repositioning is a promising approach for the rapid identification and development of new pharmaceuticals for rare and complex diseases key: cord- -se gdpna authors: jesus, isley; vanhee, valentin; deramaudt, therese b.; bonay, marcel title: promising effects of exercise on the cardiovascular, metabolic and immune system during covid- period date: - - journal: j hum hypertens doi: . /s - - - sha: doc_id: cord_uid: se gdpna with billion people in lockdown in the world, covid- outbreak may result in excessive sedentary time, especially in the population of vulnerable and disabled subjects. in many chronic disorders and diseases including type diabetes mellitus and hypertension, cardiovascular and immune beneficial effects of exercise interventions should be reminded. direct metabolic and endocrine link between type diabetes mellitus (t dm), hypertension, and coronavirus sars-cov- disease (covid- ) was recently reported [ ] . it is also important to note that with billion people in lockdown in the world, covid- outbreak may result in excessive sedentary time, especially in the population of vulnerable and disabled subjects. indeed, this population is very dependent on the caregivers in charge of their rehabilitation, since the trip to the patients' homes may be made more difficult during the outbreak. in many chronic disorders and diseases including t dm and hypertension, cardiovascular, metabolic and immune, beneficial effects of exercise interventions have been reported [ , ] . the intensity, volume, and mode of exercise may exert different activation of the hypothalamic-pituitary-adrenal axis, of the autonomous nervous system and of the resulting immunoregulatory hormones that influence immune response. exercise interventions may affect susceptibility to infection, as they were shown to modify monocytes and lymphocytes distribution, phenotype and cytokine production. as observed in many other chronic disorders and diseases including atherosclerosis and cardiovascular disease, chronic obstructive pulmonary disease, obesity, and insulin resistance, t dm is characterized by systemic inflammation and oxidative stress [ ] . animal models have shown that inflammatory cytokines expressed in adipose tissue were involved in obesity-linked insulin resistance. recently, macrophages were defined as key cells of innate immunity able to regulate metabolic homeostasis and inflammation [ ] . they express different anti or pro-inflammatory phenotypes modulating inflammation in metabolic tissues involved in glycemic homeostasis like adipose tissue, liver, pancreas and skeletal muscle. recently, growing evidences suggest the role of immune system in hypertension and the contribution of immune cells, cytokines, and innate and adaptative immunity in experimental models of hypertension [ ] . angiotensin ii, a key hormone of the renin-angiotensin system, is involved in the link between metabolic and immune response in hypertension through the activation of immune cells [ ] . although inflammation plays a central role in the development of t dm or atherosclerosis and hypertension, anti-inflammatory molecules are not recommended in current therapeutic strategies. potential candidates targeting tissue-specific inflammation, and minimizing the risks of systemic complications and/or comorbidities, are lacking. moderate exercise is known to exert beneficial health effects in patients with chronic disorders and diseases [ , ] . a randomized controlled trial was conducted to test whether an intensive lifestyle intervention (including - weekly aerobic training sessions with - min duration, of which to sessions were combined with resistance training) results in equivalent glycemic control compared with standard care in participants with t dm [ ] . in this -year follow-up study, . % of patient in lifestyle group and . % in the standard care group presented a reduction in glucoselowering drugs. moreover, . % of the participants in the lifestyle group have discontinued glucose lowering medication, suggesting that exercise may be an efficient treatment. interleukin- produced by skeletal muscle during exercise might improve glucose and lipid metabolism and exert direct anti-inflammatory effects by an inhibition of tumor necrosis factor alpha and by stimulating interleukin- receptor antagonist in patients with cardiometabolic diseases [ ] . in animal models, exercise was assessed to counteract deleterious cardiovascular effects of anti-inflammatory molecules like dexamethasone (i.e., capillary density decrease in skeletal muscles and arterial blood pressure increase). jesus et al. [ ] showed that weeks aerobic exercise training in rats prevented dexamethasone-induced hypertension and microvascular rarefaction by increasing antioxidant enzymes and improving the balance between apoptotic and angiogenic proteins. acute and chronic effects of exercise on immune response have been extensively studied in athletes [ ] . when done in excess, heavy exercise is associated with increased risk of illness, attributed to immune dysfunction. increased inflammatory biomarkers and increased risk of upper respiratory tract infections were observed after acute bouts of intense and prolonged exercise in athletes. during the post-race period, the increased susceptibility was correlated to suppressed salivary immunoglobulin a output, decreased natural killer cell activity and reduced t-and bcell function. at the opposite, moderate acute and chronic exercise-induced immune changes have been involved in the beneficial effects of physical activity to prevent cancer and cardiovascular diseases development [ , , ] . immune cells in anti-inflammatory responses are linked to mitochondrial fatty acid oxidation [ ] . recently, improvement of fatty acid oxidation-dependent respiration was reported in peripheral blood mononuclear cells of healthy volunteers with a sedentary lifestyle during low-intensity exercise [ ] . further studies are needed to assess the potential antiinflammatory effects of low-intensity exercise in patients with cardiometabolic diseases. concerning global health beneficial effects of low volume leisure-time physical activity, a large observational study showed that min/day for days a week of low-volume activity reduced all-cause mortality by %, cancer mortality by %, and mortality from cardiovascular disease by %, compared to individuals in the inactive group [ ] . in animal models of viral respiratory infection, chronic exercise reduced illness severity, viral load and resulted in greater anti-inflammatory effects than acute exercise during influenza infection. interestingly, epidemiologic data suggest that moderate exercise decreases mortality and incidence rate for influenza infection [ , ] . viral respiratory infections, including coronaviruses, induce severe inflammation, which is partially triggered by reactive oxygen species production and dysfunction of the host's antioxidant defense [ ] . interestingly, exercise interventions may stimulate antioxidant response, particularly via the nuclear factor erythroid -related factor (nrf ) transcription factor fig. schematic diagram representing known and hypothetical links between moderate exercise interventions and infectious diseases including covid- , inflammation, oxidative stress, and chronic diseases. inflammation and oxidative stress are increased by viral infection, type diabetes mellitus, cardiovascular diseases, and physical inactivity enacted by lockdown. increased inflammation and oxidative stress may contribute to the susceptibility of both chronic disease like type diabetes mellitus and infectious diseases. inflammation may be the link between chronic diseases development and comorbidity and increased susceptibility to coronavirus. moderate exercise interventions stimulate anti-inflammatory and antioxidant response, and prevent many chronic diseases development and comorbidity. that have been involved in antimicrobial defense and cardiovascular risk in metabolic diseases [ , ] . in this context, chronic moderate and adapted exercise may be doubly beneficial in t dm and cardiovascular diseases for preventing inflammation and viral respiratory infection, including coronavirus infection (fig. ). high incidence rates of overweight and obese patients are observed in covid- intensive care units and many data suggest that pre-existing comorbidities including hypertension, diabetes, and cardiovascular disease increase severity and mortality rate of covid- . whether exercise training programs, as secondary preventive interventions after healing, would confer immune protection to patients with cardiovascular and metabolic disease compared to sedentary controls, would deserve further investigations. the location of the exercise training programs, indoors or outdoors, should also be assessed. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. endocrine and metabolic link to coronavirus infection the compelling link between physical activity and the body's defense system exercise and sport science australia position stand update on exercise and hypertension nrf targeting: a promising therapeutic strategy in chronic obstructive pulmonary disease mechanisms of macrophage polarization in insulin signaling and sensitivity role of the immune system in hypertension angiotensin ii and leukocyte trafficking: new insights for an old vascular mediator. role of redox-signaling pathways effect of an intensive lifestyle intervention on glycemic control in patients with type diabetes: a randomized clinical trial anti-inflammatory effects of exercise: role in diabetes and cardiovascular disease training counteracts dex-induced microvascular rarefaction by improving the balance between apoptotic and angiogenic proteins mitochondria in the regulation of innate and adaptive immunity low-intensity exercise stimulates bioenergetics and increases fat oxidation in mitochondria of blood mononuclear cells from sedentary adults minimum amount of physical activity for reduced mortality and extended life expectancy: a prospective cohort study hygiene behaviors associated with influenza-like illness among adults in beijing, china: a large, population-based survey is exercise protective against influenza-associated mortality? redox biology of respiratory viral infections antioxidant and adaptative response mediated by nrf during physical exercise nrf : new insight in cell apoptosis key: cord- -qw qynqv authors: laskar, partha; yallapu, murali m.; chauhan, subhash c. title: “tomorrow never dies”: recent advances in diagnosis, treatment, and prevention modalities against coronavirus (covid- ) amid controversies date: - - journal: diseases doi: . /diseases sha: doc_id: cord_uid: qw qynqv the outbreak of novel coronavirus disease ( -ncov or covid- ) is responsible for severe health emergency throughout the world. the attack of severe acute respiratory syndrome coronavirus (sars-cov- ) is found to be responsible for covid- . the world health organization has declared the ongoing global public health emergency as a pandemic. the whole world fights against this invincible enemy in various capacities to restore economy, lifestyle, and safe life. enormous amount of scientific research work(s), administrative strategies, and economic measurements are in place to create a successful step against covid- . furthermore, differences in opinion, facts, and implementation methods laid additional layers of complexities in this battle against survival. thus, a timely overview of the recent, important, and overall inclusive developments against this pandemic is a pressing need for better understanding and dealing with covid- . in this review, we have systematically summarized the epidemiological studies, clinical features, biological properties, diagnostic methods, treatment modalities, and preventive measurements related to covid- . the emergence and spread of novel coronavirus ( -ncov) or the severe acute respiratory syndrome (sars) coronavirus (sars-cov- ) has threatened global public health. the coronavirus disease (covid- ), which is a transmission of sars-cov- to humans, was reported first in wuhan, hubei province, china in december . later, covid- rapidly spread worldwide creating a pandemic as there have been around , , reported cases and , reported deaths to date ( july ) affecting countries and territories around the world and two international conveyances (https://www.worldometers.info/coronavirus/). north america, south america, and asia are the most affected (maximum number of cases) continents by this pandemic so far (https:// www.worldometers.info/coronavirus/, data accessed on july ) ( figure a ). in spite of rapid development of knowledge, precautionary measurements, and clinical trials, researchers, regulatory bodies, and government administrations are facing a great challenge globally in various aspect to prevent covid- pandemic. the present situation is leading us to a still unknown future and conflicting paradox in the battle against sars-cov- . further, a plethora of documents have been published on "coronavirus" and "coronavirus disease" (year-wise keyword search for last years in pubmed on july ) research, where a maximum and a very large number of results were obtained in on both the keywords in comparison to previous years ( figure b) . nature index has also reported a hugely increasing global research publishing phenomenon on covid- pandemic as evidenced by , articles and , preprints (https://www.natureindex.com/news-blog/thetop-coronavirus-research-articles-by-metrics). a thorough and cumulative scientific knowledge about the progress against covid- amid various conflicts and controversies is the need of the time to set a clear directive in this battle. through this review article, we provide an overview of updated and rapidly evolving progress against covid- pandemic including various conflicts to the readers in a comprehensive manner. considering this, we have summarized diverse research areas covering the current known biological properties of sars-cov- , diagnostic tools for detection, therapeutic measurements for possible treatment, and prevention techniques to stop further spreading of this pandemic. still unknown future and conflicting paradox in the battle against sars-cov- . further, a plethora of documents have been published on "coronavirus" and "coronavirus disease" (year-wise keyword search for last years in pubmed on july ) research, where a maximum and a very large number of results were obtained in on both the keywords in comparison to previous years ( figure b) . nature index has also reported a hugely increasing global research publishing phenomenon on covid- pandemic as evidenced by , articles and , preprints (https://www.natureindex.com/news-blog/the-top-coronavirus-research-articles-by-metrics). a thorough and cumulative scientific knowledge about the progress against covid- amid various conflicts and controversies is the need of the time to set a clear directive in this battle. through this review article, we provide an overview of updated and rapidly evolving progress against covid- pandemic including various conflicts to the readers in a comprehensive manner. considering this, we have summarized diverse research areas covering the current known biological properties of sars-cov- , diagnostic tools for detection, therapeutic measurements for possible treatment, and prevention techniques to stop further spreading of this pandemic. coronaviruses (subfamily: coronavirinae, family: coronaviridae, order: nidovirales) are enveloped single stranded positive sense rna genomes that range in size from to kilobases [ , ] . coronavirus consists of four structural proteins: the nucleocapsid, envelope, membrane, and spike forming a core-shell morphology (figure ), whose diameter is in the range from nm to nm with spike like projections on its surface [ ] [ ] [ ] . the name, coronavirus (latin: corona = crown) came out due to the presence of a crown or the sun's corona-like spike (s) glycoprotein on viral surface forming club-shaped protrusions, which is also evidenced through the electron microscope [ ] [ ] [ ] . this transmembrane spike (s) glycoprotein on viral surface mediates the entry into host cells, forming homotrimers protruding from the viral surface [ ] . the receptor binding domain (rbd) in the spike (s) glycoprotein is the most mutable part of the coronavirus genome leading to generation of new properties and ability of virus to infect new cell types or even new species [ ] . based on phylogenetic relationships and genomic structures, the subfamily coronavirinae is divided into four genera (α-cov, β-cov, γ-cov, and δ-cov). α-cov and β-cov only infect mammals, whereas γ-cov and δ-cov infect generally birds and sometimes even infect mammals. β-cov and γ-cov are responsible for respiratory diseases in humans and gastroenteritis in animals [ , ] . presence of four coronaviruses (subfamily: coronavirinae, family: coronaviridae, order: nidovirales) are enveloped single stranded positive sense rna genomes that range in size from to kilobases [ , ] . coronavirus consists of four structural proteins: the nucleocapsid, envelope, membrane, and spike forming a core-shell morphology (figure ), whose diameter is in the range from nm to nm with spike like projections on its surface [ ] [ ] [ ] . the name, coronavirus (latin: corona = crown) came out due to the presence of a crown or the sun's corona-like spike (s) glycoprotein on viral surface forming club-shaped protrusions, which is also evidenced through the electron microscope [ ] [ ] [ ] . this transmembrane spike (s) glycoprotein on viral surface mediates the entry into host cells, forming homotrimers protruding from the viral surface [ ] . the receptor binding domain (rbd) in the spike (s) glycoprotein is the most mutable part of the coronavirus genome leading to generation of new properties and ability of virus to infect new cell types or even new species [ ] . based on phylogenetic relationships and genomic structures, the subfamily coronavirinae is divided into four genera (α-cov, β-cov, γ-cov, and δ-cov). α-cov and β-cov only infect mammals, whereas γ-cov and δ-cov infect generally birds and sometimes even infect mammals. β-cov and γ-cov are responsible for respiratory diseases in humans and gastroenteritis in animals [ , ] . presence of four corona viruses (hku , nl , e and oc ) have been found in human circulation which are generally cause mild respiratory disease [ ] . the -ncov is phylogenetically closely related to bat sars-like coronaviruses, hence name sars-cov- and belongs to β-cov genus lineage b [ ] . regarding pathogenicity and transmissibility, sars-cov- may differ from other known sars-cov due to a significant change in its spike glycoproteins (orf , and orf b) [ ] . doremalen et al. [ ] showed that the sars-cov- virus remained viable in aerosols (< µm) for at least up to h and was more stable on plastic and stainless steel than on copper and cardboard. sars-cov- was first identified from the samples (cultured human airway epithelial cells along with the virus from isolated bronchoalveolar lavage fluid) of adult covid- patients in wuhan, china. its morphology was analyzed by negatively stained sample under transmission electron microscopy (tem) [ ] . zhu et al. [ ] made a conclusion based on tem ultra-structural image of sars-cov- virus particles. this follows as (i) it is generally spherically shaped with a diameter ranges from to nm, (ii) it has an envelope with quite distinctive to nm protein spikes, and (iii) it has genetic material which matched to the genome from lineage b of the genus β-cov-showing more than % identity with a bat sars-like cov (bat-sl-covzc , mg . ). these observations are similar to the overall structure of coronaviridae family viruses. tem image of an isolate from the first united states case of covid- is also evidenced the spherically shaped viral particles (colorized blue) containing cross-sections through the viral genome (black dots) [ ] . the crystal structures of the unliganded sars-cov- main protease (m pro ) and its complex with an α-ketoamide inhibitor was used to provide some knowledge about the drug target for covid- [ ] . this m pro enzyme is essential one along with the papain-like protease(s) for processing the viral rna translated polyproteins. thus, inhibition of this enzyme may block the viral replication. the three-dimensional crystal structure, at . Å resolution, of the sars-cov- m pro is highly similar to the sars-cov m pro , due to the % sequence identity. dimerization (necessary for catalytic activity) of the m pro is regulated by domain iii (residues to ) mainly through a salt-bridge interaction between glu of one protomer and arg of the other. like sars-cov- , world also has seen an outbreak of worldwide pandemic and a large-scale fatal swine disease during the past two decades because of three other zoonotic (transmitted from animals to human) coronaviruses, such as sars in , middle east respiratory syndrome (mers) in , and swine acute diarrhea syndrome (sads) in . surprisingly, these viruses have been originated from bats, a natural source of various other highly lethal zoonotic viruses (such as hendra, nipah, ebola, and marburg viruses). sars and sads viruses were claimed to be originated in china [ , ] . based on these facts, scientists including chinese research group from wuhan (primary epicenter of also warned further possible coronavirus outbreaks from bats and with a high probability that outbreak will occur in china [ ] . both sars-cov and mers-cov bat β-coronaviruses crossed over to humans through an intermediary host, which was palm civet cats in the guangdong province of china (sars-cov, in - , mortality rate %) and dromedary camels in saudi arabia (mers-cov, in , fatality rate %), respectively [ ] . in the case of covid- , the virus was transmitted to human from bats, but the intermediary host animal(s) are not yet known. a study claimed that the intermediary host animal is pangolin due to the following findings on pangolin-cov, sars-cov- , and batcov ratg viruses: (i) at the whole-genome level, both pangolin-cov and sars-cov- share . % similarity among them, (ii) pangolin-cov and sars-cov- are reported to be the second closest relative to each other than to batcov ratg , (iii) in the receptor binding domain (rbd) of spike glycoprotein, five key amino acid residues involved in the interaction with human angiotensin converting enzyme (ace ) of pangolin-cov and sars-cov- are consistent, and (iv) only sars-cov- contains a potential cleavage site for furin proteases unlike both pangolin-cov and ratg [ ] . it was also concluded that the transmission of human sars-cov- virus from bat may include more than one intermediary host including pangolins [ ] . similar to sars-cov, the -ncov is reported to have the capability to transmit efficiently among humans due to familial cluster of pneumonia [ ] . several cases were reported person-to-person transmission of this virus not only through family settings, but were also in hospital and infected travelers [ , ] . person-to-person transmission of the sars-cov- infection is occurred via airborne droplets to the nasal mucosa in closed environments, close contact between people, unwashed hands, and touching contaminated surfaces with less possibilities. within the incubation period ranges from to days in general, sars-cov- may replicate locally in cells of the ciliated epithelium resulting cell damage and inflammation. primarily, respiratory secretions of any infected person are used to diagnose the presence of virus by special molecular tests including normal/low white cell counts with elevated c-reactive protein (crp) [ ] . additionally, abnormal computerized tomographic (ct) chest scan is also proved to be helpful to diagnose any infected person even for those with no symptoms or mild disease [ ] . the sars-cov- showed lower mortality but faster spreading than sars-cov and mers-cov. isolation of sars-cov- from oral swabs, bronchoalveolar lavage fluid, and stool proved them to be highly contagious [ , ] . sars-cov- infects human by interacting with a functional receptor, metallopeptidase named angiotensin converting enzyme (ace ), for its successful cellular entry ( figure ) [ , ] . crystal structure of the c-terminal domain of spike protein in complex with human ace (hace ) revealed an overall similar binding mode as that of sars-cov with hace [ ] . it was determined that -ncov uses ace as a cellular entry receptor in human, chinese horseshoe bats, civets, and pigs but not for mice and cells without ace protein expression capability [ ] . other coronavirus receptors, such as aminopeptidase n and dipeptidyl peptidase do not play any role for cellular entry of -ncov [ ] . previous studies revealed that almost all human organs are known to have ace mrna, though the protein expression of ace mrna was largely unknown. such ace receptor is found to be present in arterial and venous endothelial cells, arterial smooth muscle cells in the lungs, stomach, small intestine, liver bile ducts, colon, skin, kidney parietal epithelial cells, lymph nodes, and in the brain [ ] . the surface of lung alveolar epithelial cells and enterocytes of the small intestine also express ace protein allowing them to be infected by sars-cov- [ ] . the tissues of the upper respiratory tract are not the primary site of entrance for sars-cov, as oral and nasal mucosa and nasopharynx did not show ace expression on the surface of epithelial cells, rather upper respiratory tract might be susceptible to secondary infections from the infected lower respiratory tract. lower lungs may show higher opacity in the ct scans due to its more ace expression [ ] . higher viral loads have been recorded in the nose than the in throat, with similar viral loads seen in asymptomatic and symptomatic patients [ ] . coronaviruses generally are found to cause acute and chronic respiratory, enteric, and central nervous system diseases in humans as well as in other animals. the symptoms of a covid- patient are usually fever, cough, sore throat, breathlessness, fatigue, and feeling of discomfort. for most of the people, it is found mild. for elderly and the patient with comorbidities may develop pneumonia, acute respiratory distress syndrome (ards), and multi-organ dysfunction leading to death. many infected people are found to be asymptomatic causing a problem for early detection and controlling the spread of disease. mortality rate estimated by the world health organization (who) (as of march ) is . % (https://www.worldometers.info/coronavirus/coronavirus-death-rate/). further, speculation about the association of human coronaviruses with more serious human diseases (such as multiple sclerosis, hepatitis, or enteric disease in infants) are still under question due to no proper evidence [ ] . in absence of any approved therapeutics or vaccines for the treatment of covid- , who has promoted "test, isolate, and trace" method as a preventive measure. thus, early, rapid, and accurate diagnosis of covid- patients is becoming very crucial to control the sources of infection and to prevent further community spread. with a gradual understanding of biological properties of sars-cov- , various diagnostic methods and device strategy with point of care facilities have been developed for covid- detection worldwide. a summary of various diagnostic methods (table ) are presented for the covid- detection. various countries approved and implied different testing methods according to the regulation of their own health agencies based on situation and availabilities. below subsections are summarized the recent developments on diagnostic methods based on (i) nucleic acid, (ii) protein, (iii) chest scan and (iv) autopsy. nucleic acid-based detection strategy has been widely used against detection of various diseases, including coronavirus and recent covid- . in this section, we review some nucleic acid-based detection methods that are commonly being employed for the diagnosis of covid- . polymerase chain reaction (pcr) is an enzymatic method widely used in molecular biology to make millions to billions of copies of a specific dna sample [ ] . this method involves following steps in a series or cycles of temperature changes: (i) denaturation: separating the two strands of the dna containing the gene segment with the application of heat, (ii) annealing-marking gene segment of each strand of dna with a primer, (iii) primer extension: using a dna polymerase to assemble a copy alongside each segment, and (iv) repeat: continuously copy the copies [ ] . various pcr-based methods are an indispensable, common, and rapid techniques for scientist to amplify a minute nucleic acid sample to a large enough amount for a number of applications [ ] . due to high sensitivity and high sequence specificity, the pcr-based method has been used as a routine and reliable technique for detecting coronaviruses. coronavirus is a rna virus, so in general reverse transcriptase-pcr (rt-pcr) method is implied as follows: coronavirus rna is transcribed into cdna by reverse transcription, then the pcr is performed on cdna, and finally detection of pcr product through specific detection method(s) (gel visualization and sequencing) [ , ] . real-time: real-time reverse transcriptase-pcr (rt-pcr) detection method is evolved as a common platform for detection of all kinds of coronaviruses due to its low cost per test, less time-consuming process and more sensitive than the conventional rt-pcr assay [ , ] . the whole genome sequence of sars-cov- enabled to develop pcr-based kits to diagnose covid- in laboratory and clinical settings [ ] [ ] [ ] [ ] [ ] [ ] . corman et al. [ ] developed a robust diagnostic methodology considering the sars-related virus sequences available in genbank. a close genetic relatedness to the sars-cov and synthetic nucleic acid technology helped this process to design and validate such strategy without using any virus isolates and samples from infected person. such a technique can successfully discriminate -ncov from sars-cov. this approach provided the first version of the diagnostic protocol to the who from exclusivity testing on clinical samples ( january ). a real-time rt-pcr based test is found to be more sensitive than radiological test for pediatric patients [ ] . pediatric patients with milder symptoms, showed no clear clinical signs or chest x-ray findings but their real-time rt-pcr exhibited positive results. further, in this report, real-time rt-pcr showed positive results in rectal swab-testing but negative results in nasopharyngeal swab-testing for eight out of ten pediatric patients suggesting shedding of virus in the gastrointestinal tract and a possible fecal-oral transmission. wang's group [ ] reported that the rt-pcr based findings using different types of clinical specimens collected from infected individuals. in their study, it was found that viral loads were significantly correlated among pairs of throat swab and sputum samples. overall, real-time rt-pcr based method enables developing a high-throughput testing for rapid, on-demand, low-cost, reliable, quantitative detection technique against covid- in clinical settings [ ] . probe free: a team of indian institute of technology, delhi, india reported first probe-free real time pcr assay for covid- detection (http://www.iitd.ac.in/content/icmr-approves-probe-freecovid- -detection-assay-developed-iit-delhi- ). they have used comparative sequence analyses to identify unique regions (short stretches of rna sequences) in the sars cov- genome, which are not present in other human coronaviruses. in this highly sensitive assay, primers can specifically target unique regions (conserved in over fully sequenced) of covid- genomes, which was reported after extensive optimization using synthetic dna constructs followed by in vitro generated rna fragments. indian council of medical research has approved this technique as it does not require any fluorescent probes (thus low-price) but still useful for high throughput testing. isothermal amplification of nucleic acids is a rapid, efficient, and alternative amplification technique than pcr. this process can be applied at a constant temperature without any thermos-cycling apparatus, unlike in the case of pcr [ , ] . the isothermal amplification technique can be performed in water bath, on the cell surface, or even inside living cells, making it a superior technique over pcr [ , ] . based on reaction kinetics of isothermal nucleic acid amplification, it is divided to exponential amplification, linear amplification, and cascade amplification. these are further sub-divided into transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of rna technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of dna, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification, based on the developments in molecular biology of dna/rna synthesis [ , ] . furthermore, the use of microfluidic chips, capillary platforms, and test paper with isothermal amplification technique has been developed for single-cell or single-molecule analysis. among these, loop-mediated isothermal amplification (lamp) has been implied successfully for coronavirus detection [ ] [ ] [ ] [ ] . lamp technique can amplify target nucleic acid sequence using two or three sets of primers and a polymerase at a constant temperature (~ - • c) [ ] [ ] [ ] . in comparison to pcr-based technique, lamp can produce considerably higher amount of dna with high strand displacement and replication activity due to the use of additional pair of "loop primers". park et al. [ ] developed reverse transcription lamp (rt-lamp) assay(s) to detect genomic rna of sars-cov- . these rt-lamp assays (in combination with leuco crystal violet colorimetric detection method) can detect as low as copies of sars-cov- rna within min. these rt-lamp assays were highly specific towards sars-cov- compared to other human coronaviruses (hcov- e, hcovoc , mers-cov, and sars-cov). yu et al. [ ] also developed a rapid and sensitive isothermal lamp based method (ilaco) for the detection of covid- virus rna or cdna samples. in this method, ilaco was used to amplify a fragment of the orf ab gene using primers, which was proved to be specific for sars-cov- species (i.e., low chance for false positives) in comparison to the sequences of related viruses by the help of online tool primer-blast (including similar coronaviruses, influenza viruses and normal coronaviruses). ilaco can detect synthesized rna equivalent to copies of -ncov (performance is comparable to taqman based qpcr detection method), where reaction time varied from - min based on virus load in the collected samples. another lamp-based colorimetric detection method was reported to identify sars-cov- virus rna from purified rna or cell lysis (without an rna purification step) [ ] . the sensitivity of this portable method is equivalent to a commercial rt-qpcr test with only heating and visual inspection. zhu et al. [ ] demonstrated a successful and accurate diagnosis of covid- using one-step rt-lamp coupled with nanoparticles-based biosensor (nbs) assay (rt-lamp-nbs) within approximately h (from sample collection to result interpretation). they have employed two designed lamp primer sets (f ab-rt-lamp and np-rt-lamp), heating block (to maintain a constant temperature at • c), a real-time turbidity (la- c) and visual detection reagents (vdr) in addition to nbs interpretation to simultaneously amplify and detect genes of sars-cov- in a "one-step" and "single-tube" reaction. the sensitivity of sars-cov- rt-lamp-nbs was copies (each of detection target) per reaction, whereas no cross-reactivity was observed for all pathogens of non-sars-cov- (virus, bacteria, and fungi). the rt-lamp-nbs assay showed % the analytical sensitivity of sars-cov- for oropharynx swab samples of clinically diagnosed covid- patients and % specificity for clinical samples collected from non-covid- patients. crispr-cas (clustered regularly interspaced short palindromic repeats-crispr associated) is an adaptive immune system, which was discovered first in escherichia coli in and later also in other bacteria species. these are found predominantly in archaea ( % of genomes) than in bacteria ( % of genomes) [ , ] . being an immune system of archaea and bacteria, crispr and crispr-associated proteins deliver protection against invasive nucleic acids (such as dna, or rna from phages, plasmids, and other exogenous dna elements) [ , ] . scientists later exploited this immune responsive system by reengineering to target parts of genetic material for precise genetic alterations of any particular cellular type, which is the basis of crispr therapeutic and diagnostic platforms for human [ , ] . this adaptive immune system is also widely used as a tool for sars-cov- detection. crispr associated enzyme cas has already been utilized for rapid and portable sensing for successful rna-targeting [ ] . a specific high-sensitivity enzymatic reporter unlocking (sherlock) platform was developed by combining isothermal preamplification with cas to detect single molecules of rna or dna for dengue or zika virus [ ] . an updated sherlock protocol has been reported for multiplexable, portable, rapid, and quantitative covid- detection (https://broad.io/sherlockprotocol), which can target sequences in a range between and am ( - copies per microliter of input). another development of accurate crispr-cas -based lateral flow assay able to detect sars-cov- with % positive predictive agreement and % negative predictive agreement from respiratory swab rna extracts (less than min) [ ] . another newly developed method, sars-cov- dna endonuclease-targeted crispr trans reporter (detectr), was found to perform simultaneous reverse transcription and isothermal amplification by (i) rt-lamp for rna extracted (for nasopharyngeal or oropharyngeal swabs), (ii) cas detection of predefined coronavirus sequences, and (iii) cleavage of a reporter molecule confirms, which detects the virus [ ] . a fncas editor linked uniform detection assay (feluda) was developed for detecting nucleotide sequences, classifying nucleobase identity, and inferring zygosity [ ] . feluda is able to distinguish clear signatures of sars-cov- sequence in synthetic dna within one hour using a specific ribonucleoprotein (rnp) from non-specific rnp (such as h n or hbb). feluda can also clearly distinguish between two sars-cov- and sars-cov- sequences. this approach further can be developed as lateral flow assay on a paper strip to distinguish sars-cov- synthetic dna using sars-cov- specific rnp. protein-based testing has become as an alternative and additive detection strategy in addition to nucleic-acid based testing methods for coronavirus [ ] . in response to any infected viral protein antigens, antibodies (i.e., a blood protein produced in response to and counteracting a specific antigen) are generated in patient's body resulting a very specific antigen-antibody (ag-ab) serological interaction. detection of this specific antibody level(s) due to the sars-cov- infection can be useful for surveillance of covid- pandemic. this indirect serological test opens up wide range of possibilities, such as, (i) successful detection of asymptomatic patients, (ii) creating large a window of testing time even with a gradual decrease of viral load, (iii) protect community transmission due to false negative results by other methods, and (iv) proper guidance for individual quarantine period [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . kwok-yung yuen's group [ ] successfully detected antibodies generated in response to sars-cov- viral proteins at the time when the detection of the viral proteins become difficult due to gradual declining trend of viral load(s). serological test using enzyme-linked immunosorbent assay (elisa) for antibodies (immunoglobulin m, igm and immunoglobulin g, igg) is more confirmatory and unreliable results from oral swabs for -ncov detection [ ] . this test can be applied for respiratory, blood, or fecal samples. guo et al. [ ] conducted a covid- profiling study on early humoral response based on iga, igm, and igg response. this study found that igm and iga antibody were detected days, while igg was detected days after symptom onset, with a positive rate of . %, . %, and . %, respectively [ ] . the detection of igm by elisa was found more efficient than that of qpcr after . days of symptom onset [ ] . a successful immunological field-effect transistor (fet)-based biosensing device was developed for detecting sars-cov- in clinical samples, where the sensor was developed by conjugating a specific antibody against sars-cov- spike protein to graphene sheet coated fet [ ] . this rapid diagnostic device for sars-cov- antigen requires no sample pre-treatment or labelling. this is a highly-sensitive detection method for the sars-cov- spike protein at concentrations of fg/ml in phosphate-buffered saline and fg/ml in clinical transport medium, . × pfu/ml in culture medium and . × copies/ml in clinical samples. the false positive results in serological tests for covid- is a concern due to the presence antibodies generated against other coronaviruses (such as for common cold) irrespective of the presence of sars-cov- antibodies. recently, researchers have got a high frequency of cross-reactivity in plasma samples from covid- patients against the s protein of sars-cov- and sars-cov [ ] . however, more accurate antibody-based detection method can be made with additional features of infected patient in serological test, such as (i) combined (igm and igg) antibody assay rather than a single antibody test, (ii) more number of testing, (iii) report of elevated levels of c-reactive protein, d-dimer, lymphocytes, leukocytes, or blood platelets [ ] . there are growing concerns regarding the covid- testing despite of huge efforts in this direction. due to sudden outbreak and a huge increase of covid- cases, a sufficient number of covid- test kits are unavailable in hospitals and healthcare centers. further, an automatic detection system with a quick diagnostic capability could be an alternative or auxiliary method to prevent community spreading of covid- . in this situation, most countries have recommended the rt-pcr-based methods as the standard technique for covid- diagnosis. serological tests are also considered to be the primary technique for covid- detection. however, small hospitals, health centers in sub-urban and village areas, even private hospitals in sub-urban area may not have an approved rt-pcr testing center or pcr testing infrastructure facilities. on the other hand, chest-scan, a routine technique implied for prominent pneumonia pattern, has been evolved as useful non-invasive technique for covid- detection [ ] . both chest x-ray and computed tomography (ct)-scan were successful to distinguish the manifestations of typical pneumonia in the case of mers-cov and sars-cov infection [ , ] . these scans have helped to diagnoses suspected person to isolate and treat more quickly, even when the rt-pcr based test did not respond properly. such tests have proven lung histology (lung damage or holes/honeycomb-like appearance) of covid- patients [ ] . thus, chest-scan is useful for suspected covid- patients with negative rt-pcr result. . . . x-ray x-rays, a form of high-energy electromagnetic radiation, are shorter wavelengths than uv rays and longer wavelengths than gamma rays. x-ray machines are widely available sophisticated diagnostic imaging technique for body, bone and other dense objects that can block the radiation through a limited exposure to radiation. in addition, x-ray scans can be used for lung infections, pneumonia and tumors. an automatic prediction of covid- was successfully reported using chest x-ray images and a deep convolution neural network based pre-trained transfer models (resnet , inceptionv and inception-resnetv) [ ] . these pre-trained transfer models helped to obtain a higher prediction accuracy for small x-ray dataset. this model has end-to-end structure without manual feature extraction and selection methods, where the resnet is an effective one among all pre-trained models in the small dataset ( covid- vs. normal). computed tomography (ct) scan is a computer-assisted medical imaging device which combines cross-sectional (tomographic) scanned images of specific areas or virtual slices of any organ taken from different angles producing a d view of that particular organ. ct-scan is one of the methods used to diagnose various abnormalities of the chest (such as pneumonia, lung cancer etc.) [ , ] . thus, chest ct-scan is also being used as a fast, painless, non-invasive and accurate auxiliary diagnostic method in addition to the rt-pcr test for the suspected covid- patient [ ] [ ] [ ] . the national health commission of china included the chest ct findings as evidence of clinical diagnosis of covid- for patients in hubei province at the fifth edition of the diagnosis and treatment program of new coronavirus pneumonia due to the false-negative rate of rt-pcr test for covid- patient [ ] . several groups found chest ct scan more sensitive and better diagnostic tool in comparison to rt-pcr for covid- detection [ , ] . recent investigations demonstrated that the ct-scan of covid- patient(s) clearly showed bilateral pulmonary parenchymal ground-glass and consolidative pulmonary opacities, with a rounded morphology, crazy-paving pattern, linear opacities, and peripheral lung distribution [ , ] . in contrast, other study did not find any lung cavitation, discrete pulmonary nodules, pleural effusions, and lymphadenopathy [ ] . further, high-resolution ct (hrct)-scan for the chest is reported to be important tool to help clinicians to diagnose quickly and accurately the effected lung disease [ ] . artificial intelligence (ai) and deep learning-based automated ct image analysis of lung have been also developed to distinguish covid- pneumonia and influenza-a viral pneumonia [ ] [ ] [ ] . these automated deep learning based methods can produce graphical pattern of a particular covid- patient which is helpful for clinician to diagnose prior to pathogenic testing [ ] . in spite of such clinical diagnostic values, ct scan still fails to come at the forefront of covid- diagnosis due to the following reasons: (i) it is expensive, (ii) it requires technical expertise, and (iii) it is incapable of distinguishing sars-cov- pneumonia from other viral pneumonia and hysteresis. an autopsy report, through examination of a corpse by dissection, is an important source of information for research purposes to evaluate any disease causing death. autopsy has been proved to be hugely beneficial to diagnose emerging and reemerging infectious diseases, like covid- [ ] [ ] [ ] . in contrary, several groups raised their concern that the few autopsies have been performed on patients who died with suspected or confirmed covid- infection especially in the primary epicenters of pandemic (such as china and italy) [ , ] . despite the suggestion by who on performing post-mortem examinations for covid- deaths with following recommended safety procedures, many governments including italy discouraged the practice of autopsy during the period of increasing number of death and even some scientific report highlighted that the post-mortem examination does not have any primary diagnostic role, whereas autopsy may still have a clinical role in selected cases [ , ] . though based on autopsies, physicians can determine a profound change of the view of covid- disease not as a pneumonia but a systemic, vascular disease, putatively generated by autoimmunity [ ] . thus, a strong recommendation was urged to perform full autopsies on patients who died with suspected or confirmed covid- infection with recommended exceptional biosafety guidelines to reduce the further spread of potential infection from any corpse. there is currently no clinically proven therapeutic regimen to prevent and eradicate sars-cov- infection [ ] . covid- is being managed by the supportive treatment (oxygenation and ventilation, conservation fluid management). however, the use of broad-spectrum antibiotics [ ] . this section summarizes various treatment modalities for covid- ( figure ). important tool to help clinicians to diagnose quickly and accurately the effected lung disease [ ] . artificial intelligence (ai) and deep learning-based automated ct image analysis of lung have been also developed to distinguish covid- pneumonia and influenza-a viral pneumonia [ ] [ ] [ ] . these automated deep learning based methods can produce graphical pattern of a particular covid- patient which is helpful for clinician to diagnose prior to pathogenic testing [ ] . in spite of such clinical diagnostic values, ct scan still fails to come at the forefront of covid- diagnosis due to the following reasons: (i) it is expensive, (ii) it requires technical expertise, and (iii) it is incapable of distinguishing sars-cov- pneumonia from other viral pneumonia and hysteresis. an autopsy report, through examination of a corpse by dissection, is an important source of information for research purposes to evaluate any disease causing death. autopsy has been proved to be hugely beneficial to diagnose emerging and reemerging infectious diseases, like covid- [ ] [ ] [ ] . in contrary, several groups raised their concern that the few autopsies have been performed on patients who died with suspected or confirmed covid- infection especially in the primary epicenters of pandemic (such as china and italy) [ , ] . despite the suggestion by who on performing post-mortem examinations for covid- deaths with following recommended safety procedures, many governments including italy discouraged the practice of autopsy during the period of increasing number of death and even some scientific report highlighted that the postmortem examination does not have any primary diagnostic role, whereas autopsy may still have a clinical role in selected cases [ , ] . though based on autopsies, physicians can determine a profound change of the view of covid- disease not as a pneumonia but a systemic, vascular disease, putatively generated by autoimmunity [ ] . thus, a strong recommendation was urged to perform full autopsies on patients who died with suspected or confirmed covid- infection with recommended exceptional biosafety guidelines to reduce the further spread of potential infection from any corpse. there is currently no clinically proven therapeutic regimen to prevent and eradicate sars-cov- infection [ ] . covid- is being managed by the supportive treatment (oxygenation and ventilation, conservation fluid management). however, the use of broad-spectrum antibiotics [ ] . this section summarizes various treatment modalities for covid- ( figure ) . viral infection is always a major concern for morbidity and mortality in animals and humans worldwide. development of antiviral drugs have been always a pressing need to treat such viral infections. since the approval of first antiviral drug, idoxuridine in , drugs were clinically approved to treat nine human infectious diseases (human immunodeficiency virus, hiv; hepatitis b virus, hbv; hepatitis c virus, hcv; herpesvirus; influenza virus; human cytomegalovirus; varicella-zoster virus; respiratory syncytial virus; and human papillomavirus) [ ] . the antiviral drugs mostly inhibit the viral development rather than destroying the target pathogen unlike most antibiotics. a broad-spectrum antiviral is found to be effective against a wide range of viruses based on drug repurposing strategy [ ] . drug repurposing or drug repositioning is a cost-effective and time-efficient alternative strategy, which involves the recycle or re-use of clinically approved drugs for new disease instead of searching of new drugs [ , ] . in contrary to in vitro phenotypic screening of known drugs, in silico/computational drug repurposing strategy is a hypothesis-driven approach to identify the drugs for the treatment of any disease using big data analysis [ , ] . the drug-repurposing has been implied for several human diseases including antiviral drug development against coronavirus [ ] . various groups have proposed number of drug candidates through drug-repurposing (in vitro and in silico) for covid- treatment [ ] [ ] [ ] [ ] . who focused and initiated the "solidarity trial" (announced on march ) of four existing antiviral compounds/formulations ( figure ) to assess their clinical benefit against covid- [ , ] . viral infection is always a major concern for morbidity and mortality in animals and humans worldwide. development of antiviral drugs have been always a pressing need to treat such viral infections. since the approval of first antiviral drug, idoxuridine in , drugs were clinically approved to treat nine human infectious diseases (human immunodeficiency virus, hiv; hepatitis b virus, hbv; hepatitis c virus, hcv; herpesvirus; influenza virus; human cytomegalovirus; varicellazoster virus; respiratory syncytial virus; and human papillomavirus) [ ] . the antiviral drugs mostly inhibit the viral development rather than destroying the target pathogen unlike most antibiotics. a broad-spectrum antiviral is found to be effective against a wide range of viruses based on drug repurposing strategy [ ] . drug repurposing or drug repositioning is a cost-effective and time-efficient alternative strategy, which involves the recycle or re-use of clinically approved drugs for new disease instead of searching of new drugs [ , ] . in contrary to in vitro phenotypic screening of known drugs, in silico/computational drug repurposing strategy is a hypothesis-driven approach to identify the drugs for the treatment of any disease using big data analysis [ , ] . the drug-repurposing has been implied for several human diseases including antiviral drug development against coronavirus [ ] . remdesivir, an antiviral compound, which showed activity against multiple variants of ebola virus in cell-based assays and rhesus monkey model. chloroquine (cq) and its derivative hydroxychloroquine (hcq), antiviral compound(s) have been used to treat malaria and amebiasis. a combination of lopinavir and ritonavir, is co-formulated for hiv- treatment. another combination of lopinavir and ritonavir plus interferon-beta (lpv/rtv-ifnb) has been approved for the treatment of relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis. about clinical trials were identified by belhadi et.al., which includes open-label studies ( %), double-blind ( %), and single blind studies ( %) [ ] . they also classified the number of trials (n) and total numbers of planned inclusions (n) for lopinavir/ritonavir (n = , n = ), chloroquine (n = , n = ), hydroxychloroquine (n = , n = ), and remdesivir (n = , n = ). in contrary, several controversial reports including toxic side effects on these promising candidates under clinical trial(s) raised some challenging questions for researchers. those have been presented below: remdesivir, an antiviral compound, which showed activity against multiple variants of ebola virus in cell-based assays and rhesus monkey model. chloroquine (cq) and its derivative hydroxychloroquine (hcq), antiviral compound(s) have been used to treat malaria and amebiasis. a combination of lopinavir and ritonavir, is co-formulated for hiv- treatment. another combination of lopinavir and ritonavir plus interferon-beta (lpv/rtv-ifnb) has been approved for the treatment of relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis. about clinical trials were identified by belhadi et.al., which includes open-label studies ( %), double-blind ( %), and single blind studies ( %) [ ] . they also classified the number of trials (n) and total numbers of planned inclusions (n) for lopinavir/ritonavir (n = , n = ), chloroquine (n = , n = ), hydroxychloroquine (n = , n = ), and remdesivir (n = , n = ). in contrary, several controversial reports including toxic side effects on these promising candidates under clinical trial(s) raised some challenging questions for researchers. those have been presented below: a report presents that patients (with symptom duration of days or less) receiving remdesivir showed clinical improvement than those receiving placebo, but it did not make any statistically significant clinical benefits [ ] . an open-label non-randomized clinical trial study demonstrated significant decrease in viral load and carriage duration in covid- patients receiving hydroxychloroquine ( mg/day during ten days). this treatment showed enhanced effects in combination with azithromycin, but it identified serious methodological flaws [ , ] . another randomized clinical study did not make any difference in recovery rates upon hydroxychloroquine treatment in covid patients [ ] . however, a hype on cq and hcq has created drug shortages and affected other potential treatments (such as for patients with lupus). there was no significant benefit (clinical improvement) observed with lopinavir-ritonavir treatment [ ] . mortality and percentages of patients with detectable viral rna at various time points were similar in the lopinavir-ritonavir group and the standard-care group. it was also reported median time to clinical improvement was shorter by one day for lopinavir-ritonavir group than that observed with standard care. vaccination is one of the most effective and preventive medications against various diseases caused by pathogens (such as virus or bacteria). currently there are about approved vaccinations available against various life-threatening diseases, including measles, polio, tetanus, diphtheria, meningitis, influenza, typhoid, and cervical cancer (https://www.who.int/topics/vaccines/en/). a vaccine typically contains an agent (weakened or killed forms of any microbe, its toxins, or one of its surface proteins), which though resembles a disease-causing microorganism, but provides active acquired immunity to that particular infectious disease [ , ] . an antiviral vaccine helps to boost our natural immune response to an invading virus by priming it to recognize viral antigens. in general, antiviral vaccines can be classified as follows: (i) inactive or live-attenuated viruses, (ii) virus-like particle (vlp), (iii) viral vectors, (iv) protein-based, (v) dna-based, and (vi) mrna-based vaccines [ , ] . like many other diseases, the vaccine development is not successful and conclusive for coronavirus disease. until now, there is no proper vaccine is developed or approved for the treatment of human coronavirus diseases (such as sars-cov and mers-cov) [ ] [ ] [ ] . most big pharmaceutical companies also in the race to develop effective vaccines for cov infection [ , ] . the existing knowledge on previous strategies for cov vaccine developments can benefit the ongoing research as sequence analysis of the sars-cov- genome showed close relation to sars ( %) and to one bat ratg sars-like cov ( %) than to mers cov ( %) [ ] . liu et al. [ ] reported that patents (mentioned in cas content collection) are directly associated with anti-sars and anti-mers vaccines ( patents on inactive and live-attenuated virus vaccines, patents on dna vaccines, patents on viral vector vaccines, patents on vlp vaccines, and three patents on mrna vaccines) with a demonstrated immune response, which could be a huge boost for covid- vaccine developers. an accelerated evaluation of next-generation vaccine for covid- has been triggered as soon as the genetic sequence of sars-cov- is published on january [ ] [ ] [ ] [ ] . as of july , vaccine candidates have been identified (https://www.who.int/who-documents-detail/draft-landscape-of-covid- -candidatevaccines), of which candidate vaccines are in clinical evaluation ( table ) and candidate vaccines are in preclinical stages. associated problems and pitfalls are surely a major concern resulting huge roadblock for vaccine discovery against covid- . the pitfalls are many folded, which as follows: (i) antibody-dependent enhancement (ade) of viral replication for vaccinated people in future recurrence due to immune backfiring, (ii) rouge immunization resulting a huge damage on someone's own immune cells (such as neutrophil and basophil), (iii) in addition to immune response malfunctioning, three imperatives of vaccine effort: speed, manufacture and deployment at scale, and (iv) global access for a newly developed vaccine [ , ] . further, new findings and hypotheses stirred up the debate on covid- research making it difficult as well as relevant not only in therapy but also in producing a life-saving vaccine, without any risk of generating immunity-based complications. (i) molecular mimicry: two independent groups (lucchese and flöel; macario and cappello) proposed molecular mimicry as the culprit in determining multi-organ damages (including anosmia, leukopenia, and vascular damage) in covid- patients [ ] [ ] [ ] . indeed, there are confirmations that a number of human proteins share common epitopes with sars-cov- proteins and these epitopes are highly immunogenic. (ii) biphasic infection: due to (a) the ability of human coronaviruses to cause respiratory re-infections, regardless of pre-existing humoral immunity and (b) evidence on circulation of severe acute respiratory syndrome coronavirus type (sars-cov- ) in italy before the detection of first covid- case, an hypothesis was given on biphasic infection, the immunological result of a prior viral infections either by sars-cov- or different strains of coronaviruses, or potentially even other respiratory viruses resulting increased susceptibility to more severe forms of covid- , following a secondary infection with sars-cov- [ ] . this theory is sustained by anecdotal clinical reports of "biphasic infection" and "cytokine storm" through a possible ade immunological mechanism, which was already observed with infections sustained by other coronaviruses (such as sars-cov and mers-cov) or other viruses (such as west nile virus and dengue). convalescent plasma (cp) therapy involves the administration of antibodies to a susceptible person to prevent or treat an infectious disease providing an immediate immunity [ ] . in this therapy, donated blood from the infected person who've recovered from that infection have antibodies in their blood that can fight against the infection. the cp therapies have been tested since s as a possible alternative and/or auxiliary treatment including sars-cov, ebola, influenza a (h n ) etc. viruses [ , ] . additionally, cp therapy did not show any serious and immediate adverse effects in earlier cov treatment [ ] . the plasma with antibodies is prepared after separation of blood cells from the donated blood which can be extended to combat against covid- . cp therapy has been generally approved as experimental treatment for patients with critical conditions. duan et al. [ ] reported potentially improved clinical outcomes for severe covid- patients with a dose of ml of cp (derived from recently recovered donors with the neutralizing antibody titers above : ) with additional use of antiviral agents and maximal supportive care. this study confirmed a number of positive outcomes, including, (i) neutralizing antibody level increased rapidly up to : in five cases, while for other four cases maintained at a high level ( : ), (ii) increased oxyhemoglobin saturation within three days, (iii) increase in lymphocyte counts ( . × /l vs. . × /l), (iv) decreased c-reactive protein ( . mg/l vs. . mg/l), (v) varying degrees of absorption of lung lesions within seven days, (vi) undetectable viral load in seven patients who had previous viremia. additionally, treatment with human immunoglobulin is reported to increase same-day thrombotic event risk significantly ( . to . %) [ ] . transfer of blood substances may include inadvertent infection with another infectious disease agent and react to serum constituents resulting immunological reactions such as serum sickness [ ] . more high-quality studies, adequate selection of donors with high neutralizing antibody titers, central blood banking facilities to process the donated serum, efficient serological and virological assays, production and the use of cp according to precise ethical and controlled conditions are needed for implementing this therapy further. latest studies showed that ace , the key functional receptor of sars-cov- , is highly expressed in kidney (nearly times higher than in lung) resulting it to be a main target organ for sars-cov- attack [ ] . thus, novel coronavirus infections hugely damage the kidney of any severe covid- patient suffering from an immunological damage due to a cytokine storm, the imbalance of pro-inflammatory and anti-inflammatory factors. thus, auxiliary continuous blood purification could be an essential technology to take care of inflammatory factors, cytokine storm, electrolyte imbalance, and acid-base balance for any covid- patient resulting a reduced renal work-load and a possible recovery of renal function [ , ] . at present, extracorporeal blood purification technology is supplied for severely ill patient with novel coronavirus pneumonia as an auxiliary treatment to improve condition [ ] . there is an increasing awareness, tendency, and agenda to use traditional (t) medicine (indigenous health traditions) and complementary and alternative medicine (cam) (methods outside the biomedical mainstream, particularly in industrialized countries). globally, half the population uses t/cam as a preventive measure [ , ] . the national center for complementary and integrative health (nccih) of the national institute of health (nih), usa, has included various medical methods under the umbrella term cam, such as homeopathy, naturopathy, ayurveda, medicinal systems, and products originating from traditional medicine [ ] . further, herbalism, aromatherapy, acupuncture, massage, and reflexology are also various name and forms among the most popular branches of cam [ ] . the potential use of cam (sometimes along with conventional medicine) has been reported to be efficient therapeutic strategy against several virus associated diseases such as influenza, dengue, japanese encephalitis, hepatitis c, zika, and hiv [ ] [ ] [ ] . considering the efficacy of cam against coronaviruses with minimal reported adverse effects on host cells, ministry of ayush (ayurveda, yoga & naturopathy, unani, siddha and homoeopathy), government of india, recommended scientists, researchers and clinicians to pursue research and use of indian herbal drugs on covid- . through a memorandum, government of india is now trying to utilize the ayurveda, siddha, homeopathy, and unani system of medicine including prophylactic measures, intervention during quarantine, asymptomatic and symptomatic cases, public health research, survey, laboratory-based research etc. (https://www.ayush.gov.in/). indian traditional medicinal systems, one of the oldest treatments in human history (existed nearly years ago witnessed and scripted in ancient literature), played a significant role in encountering global health due to its antiviral, anti-inflammatory and antioxidant properties [ , ] . such a traditional branch of science could be a potential option against covid- because of its proved efficacy not only in treatment but also in preventive strategy for several diseases, including respiratory viral infections through immunity boosting, rejuvenating lifestyle, and dietary management [ , ] . ayush recommended selected traditional drugs for covid- as follows: http://ayush.mp.gov.in/sites/default/files/corona% advisory_ .pdf. [ ] . traditional chinese medicine (tcm) is also recently being included as one of the treatment modalities in china for covid- . previous record on efficacious tcm against respiratory tract infectious diseases, such as lianhua qingwen capsules (exerts independent antiviral effects) and shufeng jiedu capsules (synergistic antiviral effects with western medicine) on influenza viruses, has encouragd the practice of tcm against covid- [ , ] . wang et al. [ ] reported massive improvement for four covid- patients as a result of combination therapy including chinese and western antiviral medicine, where they used lopinavir/ritonavir (kaletra®), arbidol, and shufeng jiedu capsule (sfjdc) with necessary supportive care. however, lack of high-quality research, standard clinical trials, sufficient research articles, clear mechanism of action, rigorous population studies, prospective business model are also urgently required to establish the therapeutic effect and implementation of t/cam among mass population. in spite of some emphatic results through traditional, complementary, and integrative products, practices, and practitioners against covid- , north american and european governments are keeping their silence on these practices (https: //www.cdc.gov/coronavirus/ -ncov/prevent-getting-sick/prevention.html) and rather warned of possible harm and overselling (https://www.fda.gov/news-events/press-announcements/coronavirusupdate-fda-and-ftc-warn-seven-companies-selling-fraudulent-products-claim-treat-or). john weeks, editor-in-chief of the journal of alternative and complementary medicine, mentioned this double standard attitude as he pointed out that "no practices have definitive evidence for benefit against covid- , yet providers with other stripes are using experimental practices and off-label drugs every day in their desperate to ease patient suffering and elicit hope" [ ] . prevention is the utmost important strategy to fight against covid- in the present situation. several preventive measurements are taken to reduce the spread and transmission of the covid- infection. this is classified as follows (i) contact tracing, (ii) sharing or proper dissemination of information, (iii) precautionary measurements ( figure ). covid- [ , ] . wang et al. [ ] reported massive improvement for four covid- patients as a result of combination therapy including chinese and western antiviral medicine, where they used lopinavir/ritonavir (kaletra®), arbidol, and shufeng jiedu capsule (sfjdc) with necessary supportive care. however, lack of high-quality research, standard clinical trials, sufficient research articles, clear mechanism of action, rigorous population studies, prospective business model are also urgently required to establish the therapeutic effect and implementation of t/cam among mass population. in spite of some emphatic results through traditional, complementary, and integrative products, practices, and practitioners against covid- , north american and european governments are keeping their silence on these practices (https://www.cdc.gov/coronavirus/ ncov/prevent-getting-sick/prevention.html) and rather warned of possible harm and overselling (https://www.fda.gov/news-events/press-announcements/coronavirus-update-fda-and-ftc-warnseven-companies-selling-fraudulent-products-claim-treat-or). john weeks, editor-in-chief of the journal of alternative and complementary medicine, mentioned this double standard attitude as he pointed out that "no practices have definitive evidence for benefit against covid- , yet providers with other stripes are using experimental practices and off-label drugs every day in their desperate to ease patient suffering and elicit hope" [ ] . prevention is the utmost important strategy to fight against covid- in the present situation. several preventive measurements are taken to reduce the spread and transmission of the covid- infection. this is classified as follows (i) contact tracing, (ii) sharing or proper dissemination of information, (iii) precautionary measurements ( figure ). track, trace, and share of the information on covid- are major preventive steps to stop spreading of sars-cov- . a thorough, quick, and updated report should be always available covering various information (such as number of infected cases, casualties, regions affected in each country etc.) on easily accessible platforms. the promotion of "data science" or big data and data driven interdisciplinary research areas has helped a lot to control such global infectious disease or epidemics through extensive surveillance, sharing of epidemiological data, and patient monitoring [ ] . benefits of big data with technological advancement has facilitated the communication among regional, national and international healthcare agencies to monitor future host adaption, viral evolution, infectivity, transmissibility, morbidity, mortality, mental health impact and psychological effects due to covid- epidemic. here we have mentioned some authentic responsible agencies and tools along with several controversies aroused from the use of data science as follows: who: who is providing a daily "situation report" as an update on covid- pandemic (https://www.who.int/emergencies/diseases/novel-coronavirus- /situation-reports), where who receives the data from national authorities. worldometer: worldometer is a free reference website (managed by an international team of developers, researchers, and volunteers) that provides counters and real-time statistics on various topics such as world population, government, economics, society, media, environment, food, water, energy, and health (https://www.worldometers.info/). now the data in website is currently available in languages and will be available in more languages soon (https://www.worldometers.info/ languages/, accessed on may ). this reference website belongs to a united states of america (usa)-based digital company dadax (http://dadax.com/). presently, this website is being used quite popularly to provide various statistics (graphs, countries, death, symptoms, incubation, transmission, news) on covid- pandemic around the world (https://www.worldometers.info/coronavirus/) and the data is also trusted and used by several agencies including government organization of various countries. the speed and ease of communication is the heart of management to control the spread of any infectious diseases as it helps to build extensive surveillance, share of epidemiological data, and patient monitoring. mobile phone or especially smartphone with the help of various mobile apps (software application) can improve this purpose due to its speedy connectivity, computational power, remote access, electronic reporting, epidemiological data basing, real-time geospatial information, digitized process of contact tracing, more complete and shareable records, enhanced coordination among regional, national and global health agencies [ , ] . a past experience on using smartphones for geospatial tracking of infectious diseases such as hiv, ebola, and tuberculosis has helped to build these devices further to control covid- epidemics by implementing healthcare strategies, and improving general awareness among masses [ ] [ ] [ ] . further, smartphone embedded point-of-care testing and self-reporting facilities has helped to reduce risk of contracting covid- as these help actual and suspected patients with mild symptoms under self-quarantine to report remotely without going to any overcrowded hospital or healthcare centers [ , ] . who launched mobile app (https://github.com/worldhealthorganization/app) to accurately track and trace covid- cases. there are also several official contact tracing apps available for the citizen of various countries to inform a person's own health to help governments in strategic control (e.g., "aarogya setu" in india, "covid watch" in usa, "nhs covid- " in uk, "alipay health code" in china, etc.,). despite huge effort and help from data science, dueling in data caused also deviation, divisive and neglecting attitude regarding covid- . this further facilitates the spread of pandemic, shifting epicenters, more casualties, economic loss and so many other secondary problems created due to pandemic. here we have summarized some problems associated with data handling. reproductive number: the basic reproduction number (r ), an indication of the transmissibility of a virus in infectious disease epidemiology, has been used to represent the average number of new covid- infections generated by an infectious person in a totally naïve population [ ] . it is estimated that (i) the number of infections is likely to increase for r > , and (ii) transmission is likely to die out for r < . thus, a true estimation of r can be beneficial in terms of prevention of any infectious disease. liu et al. estimated higher reproductive number of covid- compared to sars coronavirus and also reported that the average reproductive number for covid- (r ~ . ) is considerably higher than that of who estimate (r = . ) [ ] . they mentioned that the current estimates of r for covid- are possibly biased due to insufficient data and short onset time. "risk of death" measurement: estimation of the case fatality risk or the risk of death among positive cases is a common epidemiological practice to measure the severity of infection for a given disease. kobayashi et al. [ ] mentioned several key epidemiological problems for assessment of the severity of covid- as follows-(i) "division of the cumulative number of deaths by that of cases tends to underestimate the actual risk because deaths that will occur have not yet observed, and so the delay in time from illness onset to death must be addressed"; (ii) "the observed dataset of reported cases represents only a proportion of all infected individuals and there can be a substantial number of asymptomatic and mildly infected individuals who are never diagnosed"; (iii) "ascertainment bias and risk of death among all those infected would be smaller when estimated using shorter virus detection windows and less sensitive diagnostic laboratory tests". they further suspected that the total number of reported covid- infections will be underestimated due to many undetected mild or asymptomatic cases that go. open data source: in this fight against covid- , an open data source is a big step forward in the age of big data. amaro et al. [ ] urged to make an effort through a "community letter" for sharing bimolecular simulation data on covid- to help and improve connection and communication among scientists and investigators working on simulation, experimental and clinical data. they committed to share methods, models, and results of their study openly and quickly to test findings, ensure reproducibility, test significance, eliminate dead-ends, and accelerate discovery on covid- applications. they committed to use preprint servers such as arxiv, biorxiv, and chemrxiv, open access data repositories such as zenodo, an open data sharing platforms for models and trajectories such as the molecular sciences software institute (molssi) sars-cov- biomolecular simulation data and algorithm store, the open science framework, the european open science cloud and several other agencies. they also make an appeal and encourage others for similar best practices on open data efforts in other research areas to prevent covid- . cyber-attack: in this grave situation due to covid- , the revolutionized advancement of information and communications technology is a crucial weapon to control the healthcare infrastructure in addition to businesses, deliveries of food and essential items to remote places, online grocery shopping etc. but there is a growing concern due to an increasing report of malicious attacks on information and communications technology during covid- as attackers find this an opportunity for financial gains and promoting evil intents. a times increase in spam email and % in malicious urls have been reported from february to march , in which the united states is found to be the epicenter for such targets [ ] . healthcare systems, government and media outlets, financial services are some of most important organization and industries, which are identified at the risk of cyber-attack [ ] . this team has identified the top ten such deadly cybersecurity threats amid covid- pandemic, which are as follows-(i) distributed denial of services (ddos) attack, (ii) malicious domains, (iii) malicious websites, (iv) malware, (v) ransomware, (vi) spam emails, (vii) malicious social media messaging, (viii) business email compromise, (ix) mobile threats, and (x) browsing apps. the worldometer website was reported under cyber-attack in march and was then hacked a few days later, resulting in incorrect information (dramatic rise on covid- statistics in vatican city) for approximately min (https://www.euroweeklynews.com/ / / /false-report-of- k-dead-in-vatican-city-lastnight-i-nearly-fell-off-my-chair-reading-it/). use of contact tracing covid- apps also raised some security and privacy concern as it keeps personal database of any individual [ , ] . thus there is a continuous debate on this issue and demand of more reliable apps among mass population, whereas governments are trying to make it a mandatory use as experts shown their confidence about these apps [ ] . social media activism: containment measures are primary guideline to tackle the novel coronavirus (covid- ) pandemic. due to moving out of physical public spaces, people are using online platforms as even more prominent and powerful tools to communicate social discussion and understand the unprecedented global crisis. however, ferrara reported after tracking and studying . m real-time english tweets about covid- (the large-scale data was collected since january , , the day the first covid- case was reported on us soil to the dataset up to march , the day before the united states government announced the state of national emergency due to the covid- pandemic) that the information on social media platforms are populated by bots, automated accounts [ ] . this study provided the evidence that high bot score accounts are used to amplify certain topics of discussion at the expense of others, such as (i) promotion of political conspiracies and divisive hashtags alongside with covid- content, and (ii) enabling participatory activism to shed light on issues that may otherwise be censored in china. thus, this study demands more nuanced and regulated discussion on social media platforms on covid- . behavioral changes and use of protective gears have been prescribed as the first and foremost precautionary step to stop the spread of sars-cov- . gradual understanding of the transmission of this virus and previous experience on successful behavioral imposition to control other epidemics such as aids (changes in sexual behavior, condom promotion, and government interventions) has helped a lot in that direction. though more efficient behavioral changes in daily lifestyle requires better understanding and proper implementation of the rules with time on covid- transmission. awareness on the risks from exposure level to respiratory droplets, airborne virus, contamination level from surfaces, concentration of transmission, incubation period, infectivity even before onset of symptoms in the incubation period, transmission from asymptomatic people will definitely be helpful to understand (i) behavioral changes and precautionary guidelines and (ii) use of protective gears. further use of telemedicine and robot can also play a crucial role as precautionary steps to combat against such infectious diseases. behavioral changes and precautionary guidelines: knowledge on covid- has helped to set precautionary guidelines in our day to day lifestyle. various health agencies such as who, the centers for disease control and prevention (cdc), national public health institute of the united states and many more recommended following general measures to prevent the spread of covid- -(i) complete washing of hands often using an alcohol-based hand sanitizer to kill the sars-cov- , (ii) avoid close contact, (iii) cover mouth and nose with a cloth or mask (e.g., n respirators) in public places, (iv) cover coughs and sneezes, (ii) avoid touching eyes, nose and mouth when outside, (iii) avoid travelling or gathering in crowded places, (iv) clean and disinfect frequently touched surfaces, (v) women with infants are encouraged to breastfeed their babies to enhance their immunity (https://www.cdc.gov/coronavirus/ -ncov/prevent-getting-sick/prevention.html, https://apps.who.int/iris/handle/ / ). in addition to hand hygiene practices, who also has given proper guidelines for . sanitation and plumbing of covid- hospitals, quarantine centers; . toilets and the handling of feces of covid- patients; . safe management of health care waste; . environmental cleaning and laundry at healthcare facilities; . safe disposal of greywater or water from washing personal protective gears, surfaces and floors; . safe management of dead bodies; . management of waste generated at home; . treatment and handling requirements for excreta (https://www.who.int/publications-detail/water-sanitation-hygiene-and-waste-managementfor-the-covid- -virus-interim-guidance). the guidance from national center for complementary and integrative health (nccih), national institutes of health prescribed a "healthy waiting" in life style, which includes social distancing (or physical distancing), mild exercise, stress reduction, restriction on smoking and alcohol (https://www.nccih.nih.gov/health/in-the-news-coronavirus-and-alternativetreatments). to prevent such health crisis and boost immunity with special reference to respiratory health, ministry of ayush, india recommended the following self-care guidelines in daily lifestyle (https://www.mohfw.gov.in/pdf/immunityboostingayushadvisory.pdf): further, home isolation or quarantine of suspected cases and those with mild illnesses have been entailed as prevention to reduce the burden on covid- hospitals for severe cases [ ] . as a preventive measurement, countries have imposed not only inter nation lockdown, but also total or partial lockdown inside their territory to minimize transmission from foreign nationals, social gathering and day to day activity [ ] . lockdown area are also classified based on infection progression and hotspot are also indicated to inform mass about the containment zone. thermal screening has become a common strategy to track the probable symptomatic cases at various juncture of transportation. it is obvious that such changes in daily life of general population and healthcare unit need more time to cope with. implementation of such changes is challenging in short time against rapid infectious nature of covid- . according to the psychiatrists and allied professionals, covid- pandemic and such forceful precautionary guidelines created subsyndromal mental health at multiple levels in the general population, among healthcare workers, and in vulnerable populations resulting the symptoms of anxiety and depression, self-reported stress, insufficient sleep [ ] . also, it is surely very conflicting to maintain balance between economy and public health during such long lockdown [ ] . every section of society specially those belonging to lower socio-economic state is being affected economically sooner or later due to such crisis, which create a desperation to neglect such imposed rules on mass population resulting further increase of covid- cases. protective gears: various protective gears are playing an important role to stop the spread pandemic. frontline healthcare professionals, in addition to healthy mass population are at a huge risk in covid- transmission from patients, suspected cases. as of april , more than healthcare workers including doctors have been infected (https://health.economictimes.indiatimes.com/news/industry/whosays-over- -healthcare-workers-across- -countries-infected-by-covid- / ). in during the sars outbreak, nearly % of those virus-affected were healthcare workers [ ] . this is an even more warning situation as it is important that the healthcare workers should be protected from infection to ensure uninterrupted medical facility and to prevent virus transmission to other patients. thus, use of protective gears such as personal protective equipment (ppe) kits, mask (tested n respirators), gloves and goggles are important accessories for healthcare professionals, whereas for mass population mask (more specifically n mask) is foremost important along with other protective guidelines. suddenly, very high demand of such protective gears resulted a huge shortage worldwide, which needs a huge workforce to support healthcare facilities in this crisis. additive manufacturing (i.e., d printing), an eminent technology for medical device preparation, came out as an innovative solution for covid- protective gears production with the help of other embedded technologies such as-(i) antimicrobial polymers and nanoparticles for making ppe, (ii) angiotensin-converting enzyme coated nanoparticles containing respiratory masks, chewing gums and nasal filters (iii) preparation of recyclable decontaminating nanofiber filtered face masks, etc. [ , ] . these ideas will surely be advantageous to prepare more effective protective gears for managing such pandemic, though further research is needed to implement such efforts to market. telemedicine: with the advancement of telecommunication technology, telemedicine has emerged as medical activity involving a doctor-patient distant interaction through telecommunication, which have been interchangeably known as telehealth (more politically correct term) or online health and e-health (fashionable term) [ ] . such telecommunication-based remote medical health facility is surely advantageous against any infectious disease outbreak and thus it is recommended by various health agencies for covid- as it (i) reduces person-to-person contact by obeying social distancing, (ii) maintain balance in facilities with limited workforce, (iii) evolves as an alternative cost effective health provider in comparison to traditional home visit [ ] [ ] [ ] . in spite of a promising future and willingness to use telehealth among patients, telemedicine suffers from various disadvantageous due to (i) less scope outside of emergency situations, (ii) clinician unwillingness and low acceptance among patients, (iii) problem with reimbursement, (iv) organization of the healthcare system. robotic gesture: robots already shown a huge potential as medical device for diagnosis to surgery including pediatric airway operation, oropharyngeal cancer operation etc. [ , ] . in similar fashion, to combat infectious epidemics like covid- , robots can play pivotal roles due to its abilities as follows-(i) large area cleaning and disinfection of contaminated surfaces (such as using uv light devices), (ii) diagnosis by automated or robot-assisted nasopharyngeal and oropharyngeal swab or blood testing, (iii) patient care by delivering medications and food to infected/suspected persons under quarantine, (iv) measuring vital signs, (v) assisting controls on transportation of mass population (such using thermal sensors and vision algorithms), (vi) help in telemedicine, (vii) facial tracking of any individual already having infection as a process of contact tracing, (viii) navigation in high-risk areas for sample transfer as well as delivery of medicines using autonomous drones or robot-assisted ground vehicles [ , ] . such robot-assisted strategy to control epidemic may speed up the process, reduce the risk of infection specially for frontline health care professionals, and increase workforce ( figure ). though despite such help in clinical care, logistics and reconnaissance, proper deployment of robots may face some problem due to privacy and security, incompatible diagnosis with the mutation of virus. tracing, (viii) navigation in high-risk areas for sample transfer as well as delivery of medicines using autonomous drones or robot-assisted ground vehicles [ , ] . such robot-assisted strategy to control epidemic may speed up the process, reduce the risk of infection specially for frontline health care professionals, and increase workforce ( figure ). though despite such help in clinical care, logistics and reconnaissance, proper deployment of robots may face some problem due to privacy and security, incompatible diagnosis with the mutation of virus. in conclusion, this article presented the current progress of four primary and important sphere of research in the battle against covid- pandemic, which are knowledge of the biological properties of virus, diagnostic modalities, treatment modalities, and prevention modalities. the huge development and deployment of advanced level of research and technology has helped to improve severely affected the global health condition due to covid- . a prior knowledge on previous viral outbreaks and pandemic has really helped researchers, health agencies and administrations a lot in response to covid- . in spite of such a plethora of research, the progress was hampered in many areas due to conflicting views and controversies about covid- . several such grey areas in transmission, best diagnostic methods with no false positive reports, possible treatment outcome, figure . possible preventive methodologies against infectious disease in future. in conclusion, this article presented the current progress of four primary and important sphere of research in the battle against covid- pandemic, which are knowledge of the biological properties of virus, diagnostic modalities, treatment modalities, and prevention modalities. the huge development and deployment of advanced level of research and technology has helped to improve severely affected the global health condition due to covid- . a prior knowledge on previous viral outbreaks and pandemic has really helped researchers, health agencies and administrations a lot in response to covid- . in spite of such a plethora of research, the progress was hampered in many areas due to conflicting views and controversies about covid- . several such grey areas in transmission, best diagnostic methods 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implications for coronavirus disease (covid- ) lessons for the future telemedicine in the era of covid- artificial intelligence: who is responsible for the diagnosis? robotic-assisted surgery for primary or recurrent oropharyngeal carcinoma evaluation of an ultraviolet room disinfection protocol to decrease nursing home microbial burden, infection and hospitalization rates handling, and testing clinical specimens from persons under investigation (puis) for coronavirus disease this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest.diseases , , key: cord- -hbhtbgzd authors: hui, zhang; jian-shi, huang; xiong, he; peng, lv; da-long, qiu title: an analysis of the current status of hospital emergency preparedness for infectious disease outbreaks in beijing, china date: - - journal: american journal of infection control doi: . /j.ajic. . . sha: doc_id: cord_uid: hbhtbgzd background in the event of a large-scale infectious disease outbreak, hospitals will play a critical role. the objective of our study is to understand the current status of hospitals preparedness for infectious disease outbreaks in beijing and to provide basic information for infectious disease prevention and control in hospitals. methods one hundred fifty-two secondary and tertiary care hospitals in beijing were surveyed by a standardized questionnaire. data related to hospital demographic information and their emergency plans, laboratory diagnosis capacity, medical treatment procedures for infectious diseases, stockpiles of drugs and personal protective equipment, and staff training were collected. results responses were received from ( . %) of the hospitals surveyed. overall, hospitals reported that the number of physicians and nurses in infectious disease accounted for only . % of the total physicians and . % of the total nurses, and surgery beds accounted for . % of all the fixed beds. approximately . % of the hospitals surveyed reported that they had an emergency plan, and none of those reported that their laboratories were able to isolate and identify all kinds of common pathogens of infectious diseases: . % of the hospitals had medical treatment procedures for all these infectious diseases, . % had stored specific drugs for treatment, . % had all personal protective equipment, and . % reported that their health care staff had been trained in hospital emergency preparedness for infectious diseases. in general, emergency preparedness for infectious diseases in tertiary care hospitals was better than that in secondary care hospitals; the preparedness at general hospitals was better than that at specialized hospitals; and that at teaching hospitals was better than that at nonteaching hospitals. conclusion emergency preparedness for infectious disease at hospitals in beijing was in an early stage of development during this survey. comprehensive measures should be developed and implemented to enhance their capacity for infectious disease emergency. infectious diseases are still a major public health problem worldwide in the st century. , prevention and treatment of infectious diseases not only relate to social development and people's health but also reflect development of health care in a region. , in the event of a large scale infectious disease outbreak, hospitals will be on the front line. , all hospitals should have emergency plans; prepare beds, drugs, and equipment; and educate and train staff in advance to respond to a large scale infectious disease outbreak or other public health emergency. severe acute respiratory syndrome (sars) crisis tested hospitals' capability in infectious disease emergency response in china and revealed its fragility, and the city of beijing was no exception. the sars crisis dramatically raised public awareness of health and led to national and local initiatives to improve emergency preparedness for infectious diseases. one year after these initiatives, are hospitals now well prepared for infectious disease emergencies in beijing? what are some important aspects of hospital preparedness that are likely ignored? to understand the current status of hospital preparedness for infectious diseases in beijing and to provide basic information for infectious disease prevention and control in hospitals, we surveyed all secondary and tertiary care hospitals in beijing between november and march . one hundred fifty-two secondary and tertiary care hospitals in beijing were evaluated, including general hospitals, hospitals of traditional chinese medicine, hospitals of integrated traditional and western medicine, specialized hospitals, medical emergency centers, and maternal and child health care centers. a complete and up-to-date list of hospitals was provided by the beijing health bureau. primary care hospitals were excluded from the study for reasons: ( ) they played a relatively less significant role in sars control during the crisis in beijing, and ( ) they were not designated for managing infectious diseases in beijing's health care system. a hospital preparedness for public health emergencies questionnaire was developed, based on authors' direct field experiences in china and literature and government documents of china and the united states, including the accreditation standards of comprehensive manual for hospital evaluation in beijing area, the hospital capability assessment for readiness of hawaii, the american hospital association chemical and bio-terrorism preparedness checklist, and the capability assessment for readiness (car). the questionnaire consisted of sections with items. in this article, only findings of the items covering the areas of hospital preparedness for infectious disease emergency were reported, including hospital demographic information and key components of hospital preparedness for infectious diseases: ( ) emergency plan (ep), ( ) laboratory diagnosis capacity (ldc), ( ) medical treatment procedures (mtp), ( ) specific drug stockpile (sds), ( ) personal protective equipment stockpile (ppes), and ( ) staff training (st). to ensure face and content validity, the draft questionnaire was evaluated by experts in hospital management and public health emergency preparedness and was revised based on their evaluation. feasibility of the questionnaire was tested by a pilot study in tertiary care hospitals and secondary care hospitals in beijing. the survey protocol, field study procedures, and quality control forms were revised based on the pilot study results. approval of this study was obtained from the institutional review board of the school of basic medicine, peking union medical college in beijing. the questionnaire was sent to the departments of medical administration of secondary and tertiary care hospitals in beijing, accompanied by an official document issued from the beijing health bureau, stating the importance of the survey to the construction of a public health preparedness system in the city. the directors of the departments of medical administration in hospitals were requested to be responsible for completing the questionnaire, and each of them organized a group of key persons involved in hospital emergency planning and/or infection control to answer the questions listed in the questionnaire. each returned questionnaire was carefully reviewed for completeness and consistency. for those with incomplete responses, a telephone call was made to ask the reason for the incomplete response. if no answer could be obtained, that questionnaire was considered as ''no'' response. a score for each of aspects of emergency preparedness was calculated by summing the number of items with ''yes'' answers to the questionnaire in each aspect. the higher the score, the better the hospital was prepared. all data were entered into a database with microsoft excel (microsoft corp, redmond, wa) software and cleaned, checked, and analyzed using statistical analysis system (sas) software version . (sas institute, cary, nc). descriptive statistics were used to present the data. comparisons of mean score for each of emergency preparedness aspects among different types of hospitals were performed by independentsample t test ( -tailed), with p # . as statistical significance. one hundred thirty-four of hospitals responded, with a response rate of . %. of the respondents, . % were tertiary care hospitals and . % secondary care; . % general hospitals and . % nongeneral; and . % teaching hospitals and . % nonteaching. there was a department of infectious diseases in . % of all the responding hospitals, with physicians and nurses specializing in infectious disease, accounting for . % and . % of the total, respectively. extra beds were provided in . % of responding hospitals in the event of public health emergency, accounting for . % of the fixed ones. there were isolation beds and intensive care unit (icu) beds in . % and . % of all the respondents, respectively. table shows the numbers of health care staff and beds. six aspects of hospital preparedness are described below. emergency plan. of hospitals responding, ( . %) had eps during the survey. among hospitals with ep, . % had a mechanism to initiate its ep, . % had a classification system of emergency level for different infectious disease outbreaks, . % had protocol for use of personal protective equipment, . % had a detailed plan for drug distribution, . % had measures to protect against secondary transmission, and . % reported that their eps were accessible to all medical staff. there were plans to enhance communication and cooperation between hospitals and hospitals and between hospitals and local centers for disease control and prevention in . % and . % of all the respondents, respectively. within the past months, . % of respondents had revised their ep at least once, and . % participated in response exercises for a largescale infectious disease outbreak. laboratory diagnosis capacity. we selected kinds of infectious diseases (plague, cholera, sars, anthrax, influenza, meningococcal meningitis, japanese encephalitis b, and brucellosis) to assess the laboratory diagnosis capacity, medical treatment procedures, and specific drug stockpile for infectious diseases control, based on the following criteria: ( ) class a infectious diseases, according to the law on communicable disease prevention and control of china (plague and cholera); ( ) infectious diseases caused by pathogen can potentially be used as a bioterrorist weapon (anthrax and brucellosis); ( ) infectious diseases with significant threat to life and health of the citizens of beijing based on surveillance data (sars, influenza, meningococcal meningitis, japanese encephalitis b). the results are listed in table . at the time of this survey, approximately half of the respondent hospitals reported that they could isolate and identify vibrio cholerae; approximately one fourth of the respondent hospitals could isolate and identify neisseria meningitidis, and less than % of surveyed hospitals reported that they could isolate and identify pathogen of plague, sars, anthrax, influenza, japanese encephalitis b, and brucellosis. none of the respondents' laboratories reported they could isolate and identify all kinds of infectious diseases pathogen. medical treatment procedures. our results revealed that . % of respondents had medical treatment procedures for all kinds of infectious diseases. as shown in table , . % of the respondent hospitals had special medical treatment procedures for treating sars or influenza patients; . % for cholera; . % for meningococcal meningitis; . % for japanese encephalitis b; and less than % for plague, anthrax, and brucellosis. special drug stockpile. special drug stockpile is defined as storage of certain drugs for treating patients with each of kinds of infectious diseases for at least days. of all respondents, . % had enough drugs stockpiled for all kinds of infectious diseases. a majority of respondent hospitals reported that they had an adequate stockpile of drugs for influenza and sars; more than half of respondents had an adequate stockpile of drugs for meningococcal meningitis and japanese encephalitis b; and less than half of respondents had an adequate stockpile of drugs for plague, cholera, anthrax, and brucellosis. personal protective equipment stockpile. of all respondent hospitals, . % had biohazard protective suits, . % had safety glasses, . % had ventilators, . % had n masks, and . % had powered airpurifying respirators available for health care personnel and other employees. however, only . % of the respondent hospitals reported that they had all of the above-mentioned types of personal protective equipment. staff training. among all the respondents, . % of respondent hospitals reported that they had a staff training program, with varied contents, including awareness of ep ( . %), medical treatment procedures ( . %), infectious diseases prevention and control ( . %), routes of transmission of infectious diseases ( . %), disinfection and sterilization ( . %), and principles of quarantine and isolation ( . %). overall, . % of respondents reported that their training curriculum included all above-mentioned aspects contents. mean scores for aspects of emergency preparedness were compared among different types of hospitals. as shown in table , the mean scores in all aspects of preparedness were higher in tertiary care hospitals than those in secondary care ones, but only the difference in score of specific drug stockpile between tertiary and secondary care hospitals had statistical significance. the mean scores in all aspects of preparedness were higher in general hospitals than those in nongeneral ones, but, only the difference in mean scores of aspects (medical treatment procedures, specific drug stockpile, and staff training) had statistical significance. the mean scores in all aspects of preparedness were higher in teaching hospitals than those in nonteaching ones, but only the difference in mean scores of aspects (ep, medical treatment procedures, specific drug stockpile, and staff training) had statistical significance. there are levels of government (central, provincial, and local) that play roles in funding health care services in china. after the sars crisis, the chinese government has invested . billion rmb to local governments, including million to beijing, to assist in developing regional response plans in . one of the major efforts is to enhance hospitals' infectious disease emergency response capabilities. the beijing government has developed a citywide response plan and invested . million rmb to implement and enhance the response capabilities. this study examined the current status of hospitals preparedness for infectious diseases in beijing. emergency preparedness means the planning and actions that ensure an organization's, or a community's, readiness to respond to an emergency in a coordinated, timely, and effective manner. lack of emergency response capability to infectious diseases is a worldwide question. even in america, hospitals may not have the capacity to deal with a sudden, large increase in the number of patients, as might be seen in a bioterrorist attack. because precautions avert perils, hospitals should formulate an ep; store staff, beds, and equipment; and educate and train their staff in advance to respond to a large-scale infectious disease outbreak or other public health emergency. sufficient emergency response personnel and equipment are necessary to respond effectively to a large-scale infectious disease incident. loutfy et al reported that hospitals' biggest challenge was insufficient personnel in toronto in the event of sars. our study indicates that, during the sars epidemic, hospitals were short of infectious disease physicians, nurses, and equipment in beijing. following public health emergencies, a substantial number of patients may present to hospitals within a relatively short period of time. higgins et al reported hospitals had the ability to surge beds or % of all licensed beds in kentucky. our study shows that different hospitals could provide different numbers of extra beds. most hospitals admitted that their extra beds were not enough to meet the demands. one hundred thirty-four respondent hospitals can provide extra beds, accounting for . % of all fixed beds in beijing. xiaotangshan hospital, beijing chest hospital, beijing ditan hospital, and beijing you'an hospital were the top hospitals reported that could provide surge beds; the total number was . these hospitals were designated hospitals for sars patients in beijing. therefore, it has been estimated that around extra beds at tertiary and secondary hospitals in beijing could be used as emergency. among all extra beds, at least beds could accept patients with infectious disease. during a sudden, large-scale infectious disease outbreak, hospitals will have to convert quickly from their current care capacity to surge capacity. therefore, the infectious disease emergency plan is needed before, during, and immediately after the infectious disease outbreak. in addition, reviewing and updating the emergency plan and have exercises periodically are important to enhance emergency response capability. at the time of this survey, virtually all respondents ( . %) had an ep. however, only . % of hospitals that had an ep reported that their facilities reviewed and updated their ep at least once, and only . % had participated in a disaster exercise for a large-scale infectious disease outbreak within the past months. no hospital or medical system can manage a public health emergency by itself. therefore, hospitals need to communication and cooperate with other hospitals and public health agencies. our survey reveals that . % respondents had a plan to enhance communication and cooperation with other hospitals, and . % of respondents had a plan to enhance communication and cooperation with local centers for disease control and preventions. the survey also shows that large a proportion of hospitals in beijing have specific drugs and medical treatment procedures for sars and influenza. only a small proportion of hospitals have specific drugs and medical treatment procedures for the other infectious diseases of public health significance. this may reflect the prioritization in infectious disease prevention and control strategies in beijing, partially based on the recent sars crisis and avian flu event. with the increasing trend of globalization, an infectious disease occurring in one region is prone to spreading to other regions. considering the coming summer olympics in beijing, the prioritization of beijing's infectious disease preparedness should be reconsidered. hospitals should prepare for responding to not only the infectious disease of immediate importance, but also those occurring rarely and emerging suddenly. detecting, isolating, and identifying an unknown pathogen accurately and timely are essential for selecting appropriate prevention and treatment measures. our results indicate that many hospitals do not have adequate laboratory diagnostic capability for the kinds of infectious disease of public health significance. however, the beijing health bureau has established the laboratory response networks to help hospitals that have inadequate laboratory diagnostic capabilities for infectious diseases. before the sars crisis, there had not been a large-scale infectious disease outbreak for a long period; therefore, hospitals' capability to identify and manage infectious diseases declined. education and training are the key measures to enhance medical staff's capability to respond to infectious diseases. our study shows that beijing hospitals realized the importance of training medical staff for infectious diseases, and that the majority of respondent hospitals reported that different types of training programs were offered to their staffs. however, the quality of training needs to be evaluated. the results from this study suggest that tertiary care hospitals emergency preparedness for infectious diseases is better than that of secondary care hospitals, general hospitals preparedness is better than that of nongeneral hospitals, and teaching hospitals preparedness is better than that of nonteaching hospitals. these results may reflect the fact that different types of hospitals have different functions and missions. it would be unreasonable or inappropriate to expect all hospitals in beijing to have the same level of preparedness for infectious diseases. however, infectious diseases do not respect our hospitals' classification system. therefore, a minimum preparedness requirement (eg, emergency plan, staff, beds, drugs, and equipment) for infectious diseases of public health significance should be applied to all types of hospitals. this study has several limitations. first, there may be a respondent reporting bias because we adopted a self-report method to evaluate the status of preparedness. however, the accompanied beijing health bureau's official document partially assures that a team approach at each hospital be applied to complete the survey as accurately as possible. the removal of a high-ranking official who hid or distorted information during the sars crisis has created an environment that encourages hospitals to report information accurately. with a pilot study, return questionnaire review, and follow-up investigation, we do not believe that the respondent reporting bias will have a significant impact on the survey results. second, our study only included secondary and tertiary care hospitals. primary care hospitals were not included in this study. although secondary and tertiary care hospitals are the mainstay for beijing's infectious diseases prevention and control, primary care hospitals roles in prevention and control infectious diseases should not be ignored. however, based on beijing's sars control experience, secondary and tertiary care hospitals' emergency response capacity reflects beijing's emergency response capacity for infectious diseases. third, only qualitative data were collected for certain questions such as the status of personal protective equipment and specific drugs stockpiles. the answers to these questions are ''yes,'' ''no,'' or ''don't know,'' which limited our ability to quantify hospitals personal protective equipment and specific drugs stockpiles preparedness. overall, our survey revealed that hospitals emergency preparedness efforts for infectious diseases were in an early stage of development at the time of this survey in beijing. to enhance preparation for dealing with infectious diseases of public health significance, hospitals should revise/update their ep timely, ie, planning is an ongoing process; hospitals should also have regular staff training and disaster exercises, enhance laboratory diagnostic capability, store appropriate antidotes and antibiotics, have sufficient personal protective equipments and related supplies, and pay close attention to communication and cooperation with other hospitals and public health agencies. in addition, more comprehensive instruments should be used periodically to assess the progress of hospital emergency preparedness. status and strategy of emerging infectious diseases controlling infections in the st century. a statement by the interacademy medical panel prevention of infectious diseases in medical institutions bioterrorism preparedness. i: the emergency department and hospital united states general accounting office. most urban hospitals have emergency plans but lack certain capacities for bioterrorism response atypical pneumonia crisis exposed five flaws of public health system evaluation of control measures implemented in the severe acute respiratory syndrome how to improve their capability of emergency response management in beijing beijing health bureau and chinese hospital association. the accreditation standards of the comprehensive manual for hospital evaluation in the beijing area public health practice program office. hospital capability assessment for readiness american hospital association. chemical and bioterrorism preparedness checklist federal emergency management agency. capability assessment for readiness car china's public health-care system: facing the challenges beijing commission of development and reform. national commission of development and reform distributed the national debt to strengthen beijing's health care system for public health emergency define: emergency preparedness prevention and control on public health emergency. wuhan: hubei science and technology press optimizing surge capacity: hospital assessment and planning assessing hospital preparedness using an instrument based on the mass casualty disaster plan checklist: results of a statewide survey the handbook of responding to public health emergencies. beijing: peking union medical college press institute of medicine. the future of the public's health in the st century strategy to cope with sars and public health emergency hospital management hospital bioterrorism preparedness linkages with the community: improvements over time key: cord- -wgdqu s authors: singh, meharban title: pediatrics in (st) century and beyond date: - - journal: indian j pediatr doi: . /s - - -z sha: doc_id: cord_uid: wgdqu s pediatrics is a dynamic discipline and there is awareness and hope for actualizing outstanding achievements in the field of child health in (st) century and beyond. improved lifestyle and quality of children’s health is likely to reduce the burden of adult diseases and enhance longevity because seeds of most adult diseases are sown in childhood. identification and decoding of human genome is expected to revolutionize the practice of pediatrics. the day is not far off when a patient will walk into doctor’s chamber with an electronic or digital health history on a cd or palmtop and a decoded genomic constitution. there will be reduced burden of genetic diseases because of selective abortions of “defective” fetuses and replacement of “bad” genes with “good” ones by genetic engineering. availability of totipotent stem cells and developments in transplant technology are likely to revolutionize the management of a variety of hematologic cancers and life-threatening genetic disorders. the possibility of producing flawless designer babies by advances in assisted reproductive technologies (arts) is likely to be mired by several ethical and legal issues. the availability of newer vaccines by recombinant technology for emerging infective and for non-infective lifestyle diseases is likely to improve survival and quality of life. there is going to be a greater focus on the “patient” having the disease rather than “disease” per se by practicing holistic pediatrics by effective utilization of alternative or complementary strategies for health care. due to advances in technology, pediatrics may get further dehumanized. a true healer cannot simply rely on technology; there must be a spiritual bond between the patient and the physician by exploiting the concept of psycho-neuro-immunology and body-mind interactions. in the years to come, physicians are likely to play “god” but medicine can’t achieve immortality because anything born must die in accordance with nature’s recycling blueprint. the medical science is likely to improve longevity but our goal should be to improve the quality of life. abstract pediatrics is a dynamic discipline and there is awareness and hope for actualizing outstanding achievements in the field of child health in st century and beyond. improved lifestyle and quality of children's health is likely to reduce the burden of adult diseases and enhance longevity because seeds of most adult diseases are sown in childhood. identification and decoding of human genome is expected to revolutionize the practice of pediatrics. the day is not far off when a patient will walk into doctor's chamber with an electronic or digital health history on a cd or palmtop and a decoded genomic constitution. there will be reduced burden of genetic diseases because of selective abortions of bdefective^fetuses and replacement of bbad^genes with bgood^ones by genetic engineering. availability of totipotent stem cells and developments in transplant technology are likely to revolutionize the management of a variety of hematologic cancers and life-threatening genetic disorders. the possibility of producing flawless designer babies by advances in assisted reproductive technologies (arts) is likely to be mired by several ethical and legal issues. the availability of newer vaccines by recombinant technology for emerging infective and for non-infective lifestyle diseases is likely to improve survival and quality of life. there is going to be a greater focus on the bpatient^having the disease rather than bdisease^per se by practicing holistic pediatrics by effective utilization of alternative or complementary strategies for health care. due to advances in technology, pediatrics may get further dehumanized. a true healer cannot simply rely on technology; there must be a spiritual bond between the patient introduction in order to improve human resource, children deserve high quality of healthcare at all social and ethnic levels without any discrimination [ ] . the futuristic model of pediatrics is likely to be greatly modified by technology boom with further erosion of doctor-parent/patient relationship [ ] . the day is not far off when a patient walks in the doctor's chamber with an electronic or digital health history on a cd or palmtop. he may be asked to walk through a screening device to decipher his genome and get baseline biochemical parameters. the child is likely to get a computer-based diagnosis with the help of apps-based algorithms and given a print out of the prescription. every disease and human behavior is genetically determined by human genome and epigenetics. the life events are predestined on the basis of genome, which is like a sophisticated horoscope or janampatri or bsubtle language of god^. identification and decoding of human genome is the greatest achievement of st century [ , ] . human genome project ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) has identified and sequenced , genes. around billion dna base pairs have been decoded by dna probes. the scientists have identified diseases, which are expressed through genes. on the basis of genome, we can plan personalized strategies for prevention, diagnosis and treatment of diseases with patient-specific or tailor-made drugs. human genome has highlighted one of the mysteries that % of human dna carries no instructions. nature can't be wasteful, it is unlikely to be bjunk dna^, and it seems to be the storehouse of untapped human mysteries and potential. it is possible to diagnose a large number of life-threatening or disabling genetic, chromosomal and developmental diseases in the fetus and offer bselective abortions^to ensure survival of bgenetically normal^human beings [ ] . it is likely to reduce the burden of disability so that resources are effectively utilized for improving quality of human life. when a genetically abnormal individual is born, it will be possible to replace bbad^genes by bgood^genes tagged to carrier viruses by state-of-the-art genetic engineering technology [ ] . the technology is already being used for correction of genetic abnormalities in children suffering from severe combined immunodeficiency syndrome (scid) due to adenosine deaminase deficiency (ada), duchenne muscular dystrophy (dmd), cystic fibrosis, hemophilia, familial hypercholesterolemia and some cancers. advances in genetic technologies have led to refinements of various assisted reproductive techniques (arts) and feasibility to produce bdesigner babies^. it is possible to produce clones of babies with identical genetic makeup by artificial twinning of an embryo or by a more complex technique of somatic cell nuclear transfer (scnt). the classical example of scnt is the production of dolly, the sheep, by scottish scientists. dolly was the exact replica of the bmother sheepŵ hose dna material of a somatic cell was transferred into the enucleated egg cell of another sheep in-vitro. the embryo was implanted into a surrogate mother and carried to term [ , ] . however, the newer arts have raised several ethical and legal issues. hematopoietic stem cell transplantation (hsct) by using multipotent hematopoietic stem cells derived from bone marrow, peripheral blood or umbilical cord blood shall be increasingly exploited for treatment of hematologic cancers, nonmalignant diseases like thalassemia, aplastic anemia, inborn errors of metabolism and autoimmune disorders [ , ] . the source of stem cells may be autologous (stored patient's own blood) or allogeneic, when stem cells are obtained from a compatible donor. in allogeneic stem cell therapy, risk of infection, mismatching, rejection and graft-versus-host disease (gvhd) is much higher. cord blood is a rich source of stem cells. a number of companies such as lifecell, babycell, cordlife, and stemade provide services for collection and cryopreservation of cord blood, which can be used for treatment of a number of life-threatening and lifestyle diseases in the donor, siblings and parents in later life. it is possible to repair a defective organ of the body by infusing totipotent natural or cloned stem cells [ ] . there will be increasing use of technology to assess, monitor and manage patients, with further depersonalization of pediatrics. the communication skills or bedside manners shall be increasingly replaced by technical interventions. the patients are likely to be increasingly fragmented into systems, organs, tissues, cells and dna. the age old symbol of physicians, the stethoscope, is likely to be replaced by digital stethoscope powered by iphone or android smart phone, hand-held ultrasound device, pulse oximeter and apps -based algorithms for making a diagnosis in the ambulatory clinic. dna and rrna probes are likely to be increasingly used for diagnosis of infective and genetic disorders [ , ] . it is envisaged that in due course of time, just a drop of blood will be enough to obtain values of most of the biochemical parameters. electronic devices in the form of a smart biometric bwrist watch^are in the pipeline to monitor vital signs, some biochemical parameters, hydration status and oxygen concentration. it is based on non-invasive pulse wave data collector using a modified applanation tonometry technique for recording real -time radial artery pulse waves. it can be used as a stand-alone device or paired with your iphone or android smart phone [ , ] . the work is in progress to develop sensors for assessing kidney functions and add-on displays for ekg and eeg. intelligent scales or cutting-edge wireless smart scales are being developed to monitor body weight, body mass index (bmi), body water, bone mass, and daily caloric intake. japan is in the forefront to develop smart loos or intelligent toilets to maintain effective bottom hygiene, assess certain body parameters and analyze body wastes. it is hoped that in the near future bgenometers^will be available to delineate the genomic characteristics of a person to predict personality and vulnerability to various diseases during the life span of a person, akin to an indian horoscope. raman spectroscopy is a quick, easy and non-invasive tool that can identify a large number of objects by virtue of their molecular size and dna characteristics. almost every material has its own raman pattern, based on how strongly its atoms are bonded [ ] . hand-held raman scanners are available which can be used for identification of drugs of abuse and explosives, diagnosis of cancer, identification of pathogens and allergens and estimation of blood components. three pillars, which are crucial for maintenance of sound health and good quality of life, include sound genetic constitution, safe environment, and intake of wholesome balanced food. food is indeed the breakthrough drug of the st century! almost y ago, hippocrates said, blet thy food be thy medicine and thy medicine be thy food^. there is a popular indian saying, bwhen diet is wrong, medicine is of no use. when diet is correct, there is no need for any medicine^. in order to tackle the widespread deficiencies of iodine, iron, zinc and vitamin a, food fortification or use of nutritional sprinklers are likely to become a reality in selected populations. protein hydro lysates and hypoallergenic foods shall be available for prevention and treatment of food allergies, which are emerging as public health problems in certain populations. the concept of functional foods is being increasingly exploited to prevent illness, promote health and improve quality of life. these foods have potentially positive effects on health beyond nutrition [ ] . they promote positive health and reduce the risk of diseases by virtue of phytonutrients, antioxidants, soluble and insoluble fiber, and probiotics [ ] . genetically modified (gm) foods are genetically engineered to produce changes in their dna for selective and mutation breeding. they are produced for better yield, resistance to pathogens and herbicides and for better nutrient profiles [ ] . most food modifications have primarily focused on cash crops like golden rice, bt cotton, and vegetable oils. but technology is not without travails. the safety of gm foods is controversial because their intake may be associated with greater risk of allergies, immune suppression, elaboration of toxins, emergence of antibiotic resistant super bugs and nutritional problems. the nutritional content of animal foods like milk, eggs, and meat can be improved by feeding the animal a diet rich in omega- fatty acids and docosahexaenoic acid (dha) [ ] . vaccines have accomplished near miracles in the fight against infections with virtual eradication of smallpox and polio from the world. however, the increasing number of vaccine shots is painful and frightening both to the children and their parents. a needleless pen-shaped device has been developed to deliver drugs and vaccines through painless supersonic waves. oral and mucosal vaccines are being developed against rotavirus, typhoid, flu, cholera, rsv, and measles. genes from bacteria and viruses are being inserted into the genetic makeup of fruits, vegetables and cereal grains to produce edible vaccines that are not destroyed by cooking or frying the food [ ] . there is hope that in the near future, antigen-primed or transgenic bananas, potatoes, tomatoes, lettuce, rice, wheat, soybeans and corn, shall be available as child-friendly vaccines [ , ] . each human being is unique by virtue of its dna. but in the modern system of medicine we prescribe the same medicine, in the same dose, through the same route and for the same duration for every patient, which is obviously too simplistic and naïve. x-ray crystallography can identify the atomic and molecular structure of a crystal and is being harnessed to discover and design new tailor-made or personalized drug molecules. pharmacogenomics is being used to produce specific drugs on the basis of genomic subgroups [ ] . it is possible to deliver the drug to the site of disease with the help of liposomes and carrier monoclonal antibodies. it is associated with decreased dosing, better efficacy, and reduced risk of adverse drug reactions. a number of natural biological response modifiers (brms) have been identified and are being exploited to control severe infections and certain malignant disorders [ ] . they include interferons, interleukins, tumor necrosis factor (tnf), colony stimulating factors (csf, g-csf, gm-csf), cytokines, imiquimod, and monoclonal antibodies. milstein and his co-workers have combined two types of immune cells to create hybrid clones of immune cells or hybridoma in order to produce specific antibodies for a wide range of targets [ ] . the availability of monoclonal antibodies has ignited the hope for prevention and treatment of life-threatening infections, for transport and delivery of drugs to the site of disease, destruction of cancer cells and identification of metastases with the help of radionuclide antibodies [ ] . efforts are being made to implant memory biochips, arrays of nano-polymer wires and develop other neurobionic interventions to take over the functions of damaged neurons. it has been shown that electrical stimulation of tongue with the help of a portable neuromodulator stimulator (pons) can facilitate the repair of damaged neurons. the chinese workers have produced progenitor cells from urine waste cells, which are useful for regeneration of neurons. it is feasible to produce cyborgs with superhuman capabilities like an iq of a genius, eyesight of an eagle and hearing of a bat. a large number of lasers are available for photodynamic and cosmetic dermatology for treatment of various disorders of pigmentation and birthmarks. they include carbon dioxide laser, q-switched lasers (ruby, nd:yag, and alexandrite), argon laser, pulse-dye and metal vapor lasers [ , ] . attempts are being made to produce artificial blood or blood substitute which is either hemoglobin-based or per fluorocarbon-based oxygen carriers [ ] . they are likely to serve the felt need of chronic shortage of blood and eliminate the risk of transmission of blood-borne diseases, immune suppression and other adverse effects of blood transfusion. the research workers at the massachusetts institute of technology (mit), cambridge, united states have identified doublestranded rna activated caspase oligomerisers (draco), which are credited with effective antiviral activity [ ] . they can serve as broad spectral antiviral agents for a variety of viral illnesses like dengue, flaviviruses, arenaviruses, h n influenza and rhinoviruses. imaging-guided interventions and keyhole or minimally invasive surgical procedures with fast recovery and minimal scarring have already become a reality [ ] . it is possible to replace each and every defective body organ by biological (human or animals like baboons and pigs) or synthetic spare parts like hearing aids, cochlear transplants, lenses, dentures, pace makers, heart valves, silicon implants, artificial joints and limbs. computer-aided surgical robots are being increasingly exploited to conduct routine and complex surgical procedures at a local site or a distant location. the robotic surgery is associated with advantages of smaller incision, greater precision, miniaturization, reduced blood loss, less pain and shorter duration of hospital stay. intelligent surgical knife (iknife) has been developed for bloodless incision and the vaporized smoke produced while cutting the tissues is analyzed by a mass spectrometer to diagnose malignancy real-time. a large number of electronically guided equipment like lasers, fiber optics, drills and staplers are being used to conduct surgical procedures more effectively and with greater safety. smart e-pants or electric underpants are available to prevent occurrence of bedsores in chronic and comatose patients. tele-or distant medicine has become a reality and canadian workers have made outstanding contributions in this venture [ , ] . it is possible to transfer the clinical case file through e-mail or clip stored in the cloud. imaging scans and electromagnetic waves from various body organs can be transmitted through a telephone line or an app. consultations can be sought globally at the touch of a button. teleconferences are being increasingly used for distant teaching. it is possible to provide global live coverage of complex surgical procedures through satellite [ ] . medicine is dynamic and pediatrics is far more dynamic with a rapid pace of developments to improve survival and quality of life. a large number of innovations have already become a reality or are likely to be introduced in the near future (table ) . lifestyle diseases and over nutrition among adolescents are emerging as public health problems because of intake of calorie-dense junk food, sedentary lifestyle and indulgence in excessive bscreen time^. obesity is associated with adverse health consequences such as syndrome x, adult-onset diabetes mellitus, hypertension and coronary artery disease. a large number of newer infective disorders like hiv, sars, bird flu, swine flu, zika, ebola, and multidrug resistant superbugs are causing serious health issues. following control of infective diseases by better public health interventions and immunizations, newer non-infective disorders like cancers, allergies, metabolic abnormalities, psychological and stress disorders, degenerative disorders like alzheimer's, and diseases due to pollutants, pesticides and toxins are assuming public health proportions. whenever man tries to improve survival and quality of life, nature tries to seek a balance by unleashing natural disasters. there is an ever-increasing scare of natural table list of future innovations and developments man has always strived to prolong life and cheat death but despite all the technological advances, medicine can never achieve immortality! nature is a huge recycling plant and birth-life-death-rebirth is in accordance with a pre-ordained celestial principle. nevertheless, scientists have made attempts to upload human neocortex through cloud with the help of brain-computer interfaces to achieve singularity and digital immortality. it is hoped that by , we will have new breed of computers who will have the ability to feel, perceive, interact and have artificial intelligence with the help of braincomputer interfaces [ ] . it sounds fictional but attempts are being made to use cryonics technology to preserve human organs or whole body for future resurrection by using cloning nanotechnology to bring it back to life when a cure is found for the disease that caused the death [ , ] . it appears man is trying to play god, but these attempts are most likely to prove futile. instead of prolonging human life, it is more important to improve the vitality of health and quality of life throughout the life span of a person. there is an increasing awareness that technology should not be allowed to further dehumanize medicine. there is a need to provide holistic care by focusing on the bpatient^having the disease and not on the bdisease^per se. the patients should be viewed in totality, and that too not in isolation but in context with the dynamics of ecology, family, friends, and society. instead of becoming passive recipients of drugs, patients and their parents, should become active participants in order to augment the process of healing. effective communication and showing due concern, compassion and empathy can energize the psycho-neuro-immunology axis of the patient. it is desirable that all approaches to healthcare should be exploited to provide healing. the alternative approaches to health care include ayurveda, unani, siddha, homeopathy (ayush), naturopathy, acupressure, acupuncture, reflexology, tai-chi, digong, reiki, yoga, meditation, visualization, magnetic therapy, gemology, aroma therapy, salt therapy, prayer and spiritual healing. in order to improve effective utilization of all complementary therapies, ministry of health, government of india has created a separate wing of indian system of medicine and homeopathy (ism and h) for effective utilization of herbal medicines and drugless therapies. our body is suffused with a cosmic life force energy field consisting of light, heat, sound, electric and magnetic waves emanating from various bchakras^. the bio field, which is called aura, halo or corona, extends beyond the skin and forms a protective sheath. there are seven chakras, which are located along the spine adjacent to various endocrine glands. they are linked with the body meridians, nadis, bhongan, and duct system. it is believed that life energy or prana flows into and out of our chakras. starting from base of the spine to the top of the head, the chakras include kundalini (coccyx), hara (sacral), solar plexus (navel), heart (midback), thyroid (base of throat), brow (third eye), and crown (sahasrara), which integrates all the chakras and is a source of astral energy (gold sun) or global consciousness or nonbeing. a healthy person, who has physical vitality, mental clarity, emotional, social and spiritual wellbeing, is likely to have bigger and brighter auras [ , ] . whenever, there is stress or dysfunction, whether physical, psychological, social or spiritual, the chakra energy field or halo is disturbed. human chakras can be scanned by various kirlian equipment like polycontrast interference photography (pip), digital aura scanning system (das), gas discharge visualization (gdv), medical thermal imaging (mti), resonant field imaging (rfi), and electro interstitial scanning (eis). when a chakra is found to be diseased or dysfunctional, it can be energized or activated with electric current, magnetic waves, healing touch, reiki and crystal healing. there is tremendous hope and scope for outstanding achievements in the field of medicine in st century. there will be a greater focus on public health interventions to improve social, community, and environmental factors to enhance survival and quality of life. healthy lifestyle and improvements in the health status and quality of life in children is associated with reduced burden of adult diseases because seeds of most adult diseases are sown in childhood. advances in clinical genetics are likely to revolutionize pediatrics. there will be no issue of survival of the fittest because everyone will survive. individuals born with defective genes will be managed by insertion of healthy cloned genes by further refinements in genetic engineering technology. molecular diagnostic tests are likely to be readily available with a possibility of having personalized or tailor-made medicines depending upon the genetic constitution of the patient. a number of newer vaccines for emerging infections are in the pipeline including realization of the revolutionary concept to produce vaccines for a large number of non-infective lifestyle diseases. the scientists are going to play bgod^to produce designer babies but it is unlikely to become a reality because of tremendous ethical and legal issues. future pediatrics will not merely focus on diseases but will pay attention to children and their parents to energize the psycho-neuro-immunology axis and provide holistic medicine by further refinements and exploitation of a variety of complementary and alternative approaches to promote health. medicine will become more patient-specific and less disease oriented. we shall be able to prolong life and ensure the quality of life worth living but we should not aim for immortality or resurrection of life. nature is a huge recycling plant and no body should try to arrest the divine process of birth-life-death-rebirth… however, there is a fond hope that in the next millennium, people are likely to have an iq of and live to an average age of y-but more importantly they are likely to have a good quality of life worth living. the next century of children's health care communication as a bridge to build a sound doctor-patient/parent relationship the human genome project implications of human genome project for pediatrics prenatal diagnosis and selective abortion: a challenge to practice and policy gene therapy: principles, practice, problems and prospects sheep cloned by nuclear transfer from a cultured cell line viable offspring derived from fetal and adult mammalian cells clinical applications of bloodderived and marrow-derived stem cells for non-malignant diseases hematopoietic stem cell transplantation: a global perspective multi-organ multi-lineage engraftment by a single bone marrow derived stem cell diagnostic applications of dna probes dna vaccines for non-infectious diseases: new treatments for tumour and allergy improved non-contact optimal sensor for detection of glucose concentration and indication of dehydration level wearable electronic devices monitor vital signs, activity level, and more single-pulse standoff raman detection of chemicals from m distance during daytime a new definition of functional foods by ffc: what makes the new definition unique? functional foods hlth dis functional foods: their role in disease prevention and health promotion european commission. a decade of eu-funded gmo research dietary enrichment of eggs with omega- fatty acids: a review new delhi: thieme medical and scientific publishers pvt ltd a spoonful of antigen edible vaccines genomics, personalized medicine, and pediatrics polysaccharide immunomodulators as therapeutic agents: structural aspects and biologic function continuous cultures of fused cells secreting antibody of predefined specificity strategies and challenges for the next generation of therapeutic antibodies photodynamic therapy in dermatology: recent developments lasers used in dermatology artificial blood broad-spectrum antiviral therapeutics robot-assisted surgery: the future is here telepediatrics in canada: an overview one hundred years of telemedicine: does this new technology have a place in pediatrics telemedicine and child health blessing or curse? nonpharmacological neurocognitive enhancement by bbrain engineering^ scientific justification of cryonics practice cryopreservation of organs by vitrification: perspectives and recent advances the human biofield and chakras: concepts and processes. moscow: xlibris corp the biofield hypothesis: its biophysical basis and role in medicine conflict of interest none.source of funding none. key: cord- -cw jrt authors: dennison himmelfarb, cheryl r.; baptiste, diana title: coronavirus disease (covid- ): implications for cardiovascular and socially at-risk populations date: - - journal: j cardiovasc nurs doi: . /jcn. sha: doc_id: cord_uid: cw jrt dennison himmelfarb and baptiste discuss the implications of coronavirus disease (covid- ) for cardiovascular and socially at-risk populations covid- is an infectious respiratory disease caused by the newly discovered pathogen, sars-cov- , a novel rna-dependent rna polymerase betacoronavirus that is thought to derive from bats at this time, there are no specific vaccines or treatments for covid- the best way to prevent and slow transmission is to be well informed about the covid- virus, the disease it causes, and how it spreads t he novel coronavirus disease emerged in december and, in less than months, evolved to a worldwide pandemic. [ ] [ ] [ ] this virus has spread rapidly, leading to an unprecedented global crisis that shows no signs of abating in the near future. as of wednesday, april , , there were more than cases and more than deaths reported globally. confirmed cases in the united states were at with more than deaths. statistical models predict that more than hospital beds will be used in the united states by the end of april . the impact of covid- on our daily lives, work, families, and overall operations is unprecedented in modern times, and the situation continues to change on a day-to-day basis. in addition to constantly evolving information, there are misconceptions among the public about the pathology, etiology, transmission, treatment, and risks associated with covid- . covid- is an infectious respiratory disease caused by the newly discovered pathogen, sars-cov- , a novel rna-dependent rna polymerase betacoronavirus that is thought to derive from bats. the incubation period for covid- is thought to be within days of exposure, and transmission occurs from humanto-human contact. the covid- virus spreads primarily through droplets of saliva or discharge from the nose when an infected person coughs or sneezes. our understanding of the pathobiology and clinical presentation of the virus, and risk factors for morbidity and mortality seen with covid- , although limited, is rapidly increasing (see table ). up to % of those infected are asymptomatic. this creates challenges to prevention efforts because these asymptomatic carriers are often unaware of their covid- status. most people infected with the covid- virus will experience mild to moderate respiratory illness and recover without requiring special treatment. older people and those with underlying medical problems such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop serious illness. , clinical presentation of severe cases of covid- is characterized by hypoxia, dyspnea, and greater than % of lung involvement on imaging. in critical cases, people infected with covid- will experience acute respiratory distress syndrome, multiple-organ failure, shock, and death. at this time, there are no specific vaccines or treatments for covid- . the best way to prevent and slow transmission is to be well informed about the covid- virus, the disease it causes, and how it spreads. the world health organization and centers for disease control and prevention (cdc) are reliable public information sources. early data suggest that those with covid- and hypertension or cardiovascular disease have a mortality rate of to times higher than the general covid- population, suggesting that they are highly susceptible to more severe effects of the disease. more than % of patients hospitalized with covid- in china had an underlying cardiovascular disease. furthermore, early reports of profound myocarditis and fatal dysrhythmias suggest a deleterious impact of covid- on the cardiovascular systems. [ ] [ ] [ ] acute and chronic cardiovascular complications of pneumonia, which is common with covid- , result from various mechanisms, including ischemia, systemic inflammation, and pathogenmediated damage. , chronic cardiovascular conditions may become exacerbated in the setting of viral infection as a consequence of imbalance between an infection-induced increase in metabolic demand and reduced cardiac reserve. patients with coronary artery disease and heart failure may be at a particular risk as a result of coronary plaque rupture secondary to virally induced systemic inflammation, and rigorous use of plaque stabilizing agents (aspirin, statins, b-blockers, and angiotensin-converting enzyme [ace] inhibitors) has been suggested as a possible therapeutic strategy. procoagulant effects of systemic inflammation may increase the likelihood of stent thrombosis, and assessment of platelet function and intensified antiplatelet therapy should be considered in those with a history of previous coronary intervention. it is not clear yet whether heightened systemic inflammatory and procoagulant activity persist after resolution of the covid- infection. in addition, there has been conjecture that ace inhibitors and angiotensin receptor blockers, ubiquitously used in cardiovascular patients, may increase a patient's susceptibility to the virus. however, currently, the american college of cardiology and american heart association have recommended against preemptively stopping or starting an ace inhibitor or angiotensin receptor blocker in the setting of covid- . , , with more than million americans having some form of cardiovascular disease, there is an urgency to increase awareness among healthcare providers of the potential impact of covid- in this high-risk population. the cdc released general preventive measures for covid- infection; however, socially at-risk persons and those with underlying cardiovascular and chronic conditions bear the burden of an elevated risk for developing severe complications and death. , by april , , general preventive measures including recommendations for frequent handwashing, social distancing, and curfews or stay-at-home orders have been sanctioned. hospitals and healthcare systems across the united states have suspended elective surgeries, procedures, and inpatient visits, changing the way people seek and receive healthcare. facilitated by new legislation, many healthcare providers now offer telemedicine and use mobile health technologies, in efforts to limit exposure to both patients and healthcare providers. these options provide protection and ongoing care for many high-risk individuals but are not feasible for all. the american heart association is advising intensified caution to those with underlying heart conditions including persons with diabetes and those with cardiovascular, chronic lung, and kidney conditions. additional recommendations from the heart failure society of america and american college of cardiology are noted (see table ). , , evidence to guide clinical decision making is being generated at an extraordinary pace. hypervigilance and close attention to guidelines are needed during this critical time. there is substantial concern that socially at-risk persons and those with cardiovascular conditions could experience delays in seeking healthcare as a result of self-isolation, low health and digital literacy, or lack of a primary care medical home. furthermore, persons who already have limited access to healthcare could be further compromised, specifically those who are ethnic minorities, have a low income, and experience food insecurity and housing instability, with lack of social support and transportation. the increase in self-isolation due to covid- , particularly among older persons, may also accelerate risks for cardiovascular, neurocognitive, and mental health problems. , in addition, there are concerns that this public health crisis may exacerbate discrimination, racism, and stigma because of widespread disinformation across social media and other outlets. assessment of health disparities after covid- is warranted to fully understand the burden this pandemic has on at-risk populations, including children who have been removed from the school environment as a preventive measure. all aspects of healthcare delivery are affected by this pandemic. the sudden and rapid advancement of covid- has created an unanticipated risk to healthcare providers. beyond transmission and contraction of the disease, frontline healthcare providers are at a higher risk how does covid- infection impact those with an underlying cardiovascular disease? • the covid- virus mainly targets the lungs and can affect the heart, making it work harder to carry oxygenated blood throughout the body, which increases the risk for heart attack and rapid-onset heart failure. • covid- infection could exacerbate symptoms of heart failure. • viral illnesses are known to destabilize plaques that can develop into blockages in arteries, putting one at an increased risk for a heart attack. • patients who are older in age and have comorbid cardiovascular risk factors and those with weakened immune systems who are infected with covid- are at the highest risk for developing serious complications, including death. • previous coronaviruses similar to covid- (sars and mers) have been linked to inflammation of the heart muscle, which increases the risk for heart attack, myocarditis, and rapid-onset heart failure. for experiencing anxiety, depression, and insomnia, due to burnout and compassion fatigue. the accelerating demands for hospital beds, personal protective equipment, and lifesaving apparatus such as ventilators and continuous renal replacement machines have introduced new financial burdens for otherwise high-resource health systems in the united states. although there is close daily monitoring of the ongoing global battle against covid- , examination of this pandemic's impact on healthcare workers and healthcare delivery systems is warranted. because of many states enforcing stay-at-home orders, healthcare providers are challenged with changing modes of practice for uncertain lengths of time. healthcare settings across the nation are expeditiously transitioning from in-person to telehealth visits to retain access to healthcare for those with chronic conditions. moreover, telehealth visits can be used to reduce visits to the emergency departments for nonurgent matters, a timely intervention to preserve emergency services for those with severe symptoms. if there is a silver lining, it may be the acceleration of the adoption of and expanded reimbursement for telehealth, broadening the reach and increasing the efficiency of chronic disease care. efforts to sustain these care improvements will be critical after the rapid spread phase of the pandemic. the covid- pandemic is expected to persist for months. we must become familiar with reliable sources of information such as the world health organization and cdc. the multitude of ongoing studies and clinical experience with individuals with covid- will provide us with much needed data to illuminate our understanding of the virus, its impact, and the potential effect of individual risk factors and medications. it is unclear at this time how the covid- will further impact the physical or mental health of individuals after recovery or overall health disparities among socially at-risk populations. as information about covid- is rapidly evolving, it is imperative that healthcare providers reinforce the general prevention guidelines in addition to recommendations for persons with cardiovascular disease by the american heart association, heart failure society of america, and american college of cardiology. world health organization. coronavirus centers for disease control and prevention. coronavirus disease cardiovascular disease and covid- coronavirus covid- global cases by the center for systems science and engineering (csse) at johns hopkins university (jhu). institute for health metrics and evaluation. covid- projections a pneumonia outbreak associated with a new coronavirus of probable bat origin the role of the american heart association in the global factors associated with mental health outcomes among health care workers exposed to coronavirus disease coronavirus precautions for patients, others facing higher risks table considerations for those with cardiovascular disease and other chronic conditions to maintain optimal cardiovascular health during the covid- pandemic: • frequent and consistent hand washing, avoid touching your face, and keeping home surfaces clean • stock up on medications, renew refills or obtain a -day supply (if health plan allows), and ensure sufficient supply for an extended time. • create a list of emergency contacts and keep this information in an easy-to-find place. • consider using delivery services for medication, groceries, and other essential services • take stock of food and beverages, following recommended diet as closely as possible and recognizing that many nonperishable canned foods are high in sodium • remain active at home, as much as possible, by continuing any at-home exercises one can physically tolerate such as walking • individuals who believe they have been exposed are advised to self-isolate (quarantine) for days to monitor symptoms. • avoid crowds and travel, and limit contact with others additional considerations specific to those with an underlying cardiovascular disease: • stay up-to-date with vaccinations such as flu and pneumonia manage stress by taking a few minutes each day to meditate, contact family and friends to stay socially connected, and improve sleep hygiene consider electronic consulting (telehealth) phone, videoconferencing, or messaging options for "visits continue taking angiotensin-converting enzyme inhibitors or angiotensin receptor blocker medications as prescribed for heart failure, hypertension, or ischemic heart disease • contact health provider immediately if experiencing fever or sore throat, and call if experiencing shortness of breath or warning signs of a heart attack or stroke • in the presence of covid- infection, consider need for intensified antiplatelet therapy in those with a history of previous coronary intervention coronavirus (covid- ) resources report of the who-china joint mission on coronavirus disease (covid- ) prevalence of comorbidities in the novel wuhan coronavirus (covid- ) infection: a systematic review and metaanalysis correlates of health literacy and its impact on illness beliefs for emergency department patients with acute heart failure prevalence and impact of cardiovascular metabolic diseases on covid- in china coronaviruses and the cardiovascular system: acute and long-term implications sars-cov : should inhibitors of the renin-angiotensin system be withdrawn in patients with covid- ? what heart patients should know about coronavirus life in the pandemic: some reflections on nursing in the context of covid- covid- and the consequences of isolating the elderly. the lancet public health virtually perfect? telemedicine for covid- key: cord- -ig j z authors: couetil, laurent; cardwell, jacqueline m.; leguillette, renaud; mazan, melissa; richard, eric; bienzle, dorothee; bullone, michela; gerber, vinzenz; ivester, kathleen; lavoie, jean-pierre; martin, james; moran, gabriel; niedźwiedź, artur; pusterla, nicola; swiderski, cyprianna title: equine asthma: current understanding and future directions date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: ig j z the havemeyer workshop brought together researchers and clinicians to discuss the latest information on equine asthma and provide future research directions. current clinical and molecular asthma phenotypes and endotypes in humans were discussed and compared to asthma phenotypes in horses. the role of infectious and non-infectious causes of equine asthma, genetic factors and proposed disease pathophysiology were reviewed. diagnostic limitations were evident by the limited number of tests and biomarkers available to field practitioners. the participants emphasized the need for more accessible, standardized diagnostics that would help identify specific phenotypes and endotypes in order to create more targeted treatments or management strategies. one important outcome of the workshop was the creation of the equine asthma group that will facilitate communication between veterinary practice and research communities through published and easily accessible guidelines and foster research collaboration. the effort to clarify the phenotype and terminology used to characterize horses with chronic inflammatory airway disease started in with a workshop in east lansing, michigan ( ) . several workshops were subsequently held with similar goals in mind with the latest hosted in cabourg, france in ( ) . in the last few years, the terminology has further evolved with the term equine asthma (ea) now being recommended to describe horses with chronic respiratory signs ranging in severity from mild to severe that were previously referred as inflammatory airway disease and recurrent airway obstruction, respectively ( ) . although strong evidence supports the role of exposure to environmental dust in the pathophysiology of both mild and severe ea, the potential role of infectious agents (bacterial and viral) has not been clearly established. the goal of the havemeyer workshop on equine asthma was to bring together researchers and clinicians from different disciplines who are actively investigating airway inflammation to discuss the latest information on this topic and provide some comparative perspective from human asthma. the workshop was designed to facilitate productive discussions that would inform potential future revisions of the american college of veterinary internal medicine (acvim) consensus statement on mild-moderate ea ( ) and provide future research directions. the present report follows the format of the workshop. the manuscript is organized thematically starting with the recent advancements in the understanding of the classification and diagnosis of human and equine asthma. the second part is centered on the etiology and pathophysiology of ea. the third and final section of the manuscript summarizes the extensive discussions conducted during the workshop with the goal of prioritizing future directions of ea research. clinical asthma phenotypes have been recognized for many decades but were collapsed into a unified hypothesis of asthma as an allergic disease in when age adjusted levels of immunoglobulin e were associated with asthma. it has taken more than years to consider the heterogeneity of asthma again with an emerging emphasis on endotypes, an intrinsically more interesting approach to understanding asthma pathobiology ( ) . the term "endotype" is used to describe a subtype of disease defined by a molecular mechanism, genetic variation or by treatment response ( , ) . cluster analyses of asthma cohorts have revealed groups with different ages of onset, lung function, concordance or lack thereof between measures of airway inflammation by sputum analysis and symptoms. a recent review of asthma by a panel of experts has focused on the need to recognize asthma in its diverse forms and to identify treatable traits. this extensive review has highlighted areas for future attention ( ) . the application of the analysis of gene expression to airway epithelial cells and sputum cells from well-characterized groups of asthmatics has led to the appreciation of asthma associated with t helper cytokines and non-t asthma ( ) . the former is the more allergic subset with higher ige and peripheral and sputum eosinophilia. non-t asthma has fewer of these features and is less responsive to inhaled corticosteroids. t cells that express interleukin- have been linked to severe neutrophilic asthma. these so-called th cells have been shown in animal models to be associated with steroid-unresponsiveness. the th cytokine interferon-γ likewise has been found to be expressed in the airways of severe asthmatics. in recent years there has emerged another lymphoid cell that participates in host responses to mucosal injury. these innate lymphoid cells are lineage negative, lacking the usual lymphocyte surface markers ( ) . they express similar panels of cytokines to the t helper subsets and are labeled innate lymphoid cell (ilc) , , and . they are rapidly activated by epithelial signals such as thymic stromal lymphopoietin (tslp), interleukins and , molecules termed alarmins. the secretion of il- and il- by ilc may lead to a pattern of inflammation previously interpreted as th . innate lymphoid cells are less steroid sensitive. additionally, alarmins prime cells such as dendritic cells and therefore may have a role in adaptive immunity as well as innate immune responses. the synthesis of amphiregulin, an epidermal growth factor receptor ligand, by ilc s but also th cells, is postulated to promote mucosal integrity. one could anticipate that viral infection of epithelial cells or damage by irritants giving rise to inflammation mediated by ilcs. however, their roles have yet to be fully explored. transcriptomic analysis of sputum has revealed three patterns of inflammation and gene signatures consistent with both th and ilc driven inflammation and oxidative stress ( ) . the descriptions of molecular mechanisms of inflammation may still be considered as a deeper form of phenotyping. however, the application of novel biologics to treat asthma is now implicating certain pathways in disease and therefore is providing us with true disease endotypes. most of the progress in the identification of treatable traits has related to the t phenotype. biologics targeting ige (omalizumab), il- and therefore, the eosinophil (mepolizumab, benrazilumab, rezlizumab) and the t cytokines (dupilumab) have all demonstrated efficacy in reducing exacerbations of asthma. recent results of studies targeting the alarmin tslp and therefore both t high and low asthma have confirmed efficacy against acute attacks of asthma. oxidative stress in asthma has not been specifically addressed. a problematic form of asthma is that associated with airway remodeling and fixed airway obstruction. the association with mucus plugging and eosinophilic inflammation has been recently identified as a potential factor in long term impaired airway function ( ) . severe equine asthma is typically a neutrophilic form of asthma although expression of t cytokines has been described ( ) . there is also evidence that il- is expressed in equine asthma and its effects on neutrophil survival are steroidinsensitive ( , ) . although neutrophilic human asthma is less steroid-sensitive than the eosinophilic phenotype, severe equine asthma is responsive to steroid treatment despite the presence of neutrophilic inflammation. severe equine asthma shares the structural remodeling of the airways with human asthma and a part of the remodeling change is reversible with steroid treatment as well as withdrawal from the inciting stimulus ( ) . studies of airway remodeling in human asthma with treatment have not addressed key components of remodeling such as increased airway smooth muscle mass. revised in and discussed the use of ea to describe these conditions ( ) . the revised consensus recognized that asthmatic horses of all severities have common clinical presentations (such as chronic cough, excess mucus, poor performance) but also a wide heterogeneity in terms of triggering factors, severity, and pathologic characteristics. a phenotype is the observable physical properties of an organism, including measurable laboratory findings, which is the result of the expression of the genes in response to the environment ( ) . identifying distinct phenotypes is of interest if they facilitate the diagnosis, the prognosis or allow the implementation of targeted therapy. while currently loosely defined, the ea phenotypes discussed in the consensus statements were based on clinical presentation (severe vs. mild/moderate), triggering factors (barn/hay or pasture), endoscopy findings (mucus) and bronchoalveolar cytology. from a clinical standpoint, further dividing ea as distinct "mild" and "moderate" phenotypes may promote recognition that asthma is an underdiagnosed cause of exercise intolerance in high performance horses. horses with a cough or increased respiratory rate at rest or following exercise will commonly undergo further diagnostic procedures to confirm asthma, or "anti-asthma" treatments will be implemented. however, this is generally not the case when no clinical signs suggestive of an airway disease are present. the term "mild ea" could describe the condition affecting these horses, while "moderate ea" would be used when clinical signs of airway disease (such as cough) are present, but without the periods of labored breathing at rest seen in "severe ea" ( ) . the inflammatory airway cell phenotypes (neutrophils, mast cells, eosinophils) were recognized in the and consensus statements ( , ) . future phenotypes may include the age (early or late) of appearance of clinical signs, or specific remodeling features affecting the airways, if these new features are shown to facilitate prognostication or the implementation of specific therapy. the future development of new portable and sensitive devices for measuring the lung function of horses (forced oscillation or flow interruption techniques), or the discovery of blood biomarkers for ea would help not only to facilitate the diagnosis of mild and moderate forms of ea in clinical practice, but also to possibly identify new phenotypes for these conditions. to date, different inflammatory pathways have been proposed as contributing to ea, which may eventually lead to novel therapies ( ) . the discrepancies between results of the different studies may be an indication of different endotypes in ea, although future studies on large cohorts of horses from multiple sites would be required before specific endotypes can be recognized. multicenter tissue banking could facilitate these studies. in summary, the acvim consensus statement recognized the currently known distinctive features of ea. further defining "mild" and "moderate" ea based on the presence or absence of easily identified clinical signs may promote the investigation of the subclinical (mild) phenotype. the identification of novel phenotypes and endotypes may lead to "precision medicine" where treatments most likely to help equine patients would be selected. this approach is now implemented in humans and may eventually be applicable to horses if supported by scientific research. severe equine pasture asthma (epa) is characterized by episodes of reversible airway obstruction in horses grazing pasture during the summer in hot humid climates ( ) . affected horses demonstrate neutrophilic airway inflammation, airway hyperresponsiveness extending throughout the season of remission, and airway remodeling ( , ) . the author's experience is restricted to epa as first described in horses residing in louisiana, and diagnosed in states with subtropical climates (mississippi, alabama, and florida) ( ) . veterinarians in regions of adjoining states and distant states (oregon) describe similar signs in horses grazing pastures during hot humid conditions. epa is described in the united kingdom where it differs in its association with hot dry weather or exposure to dust from harvest/burning of crops ( ) . epa demonstrates adult onset ( ± years; range - years) without sex predilection ( ) . asthma exacerbations generally begin in summer (july), persisting until temperature and humidity decrease (october/november) ( ) . fewer horses experience asthma in the spring. a history of prior seasonal cough and/or exercise intolerance may be identified. improvement within hours to days of isolation from pasture particulates in a stall environment is a key diagnostic feature of epa in the southeastern usa ( ) ; some severe cases necessitate isolation in a climate climate-controlled environment. in the author's experience, without adequate environmental management, disease severity is progressive and responsiveness to parenteral corticosteroids decreases. though specific agent(s) that elicit epa exacerbation are not identified, the response to stall housing implicates seasonal pasture-associated particulates. costa et al. reported increases in grass but not tree pollens were significantly associated with epa exacerbation using a pollen station ∼ miles from affected horses ( ) . in this regard, intact pollen is too large to reach the respirable zone of humans in order to elicit asthma, but moist conditions that are associated with epa exacerbations can shatter pollen and disseminate respirable particles ( ) . grass pollen sensitization is classically associated with th responses, ige-mediated hypersensitivity, and eosinophilic inflammatory infiltrates. however, chronic exposure to th sensitizing antigens and to complex antigen combinations that include th sensitizing antigens each generate th responses accompanied by neutrophilic airway inflammation that typifies epa ( , ) . subtopical grasses differ substantially from grasses in temperate and continental climates ( ) . pollen from subtropical grass subfamilies is important to rhinitis and human asthma in subtropical zones of australia, asia, india, africa, and america. pollination seasons for bahia and bermuda grass (spring through september/october) align to the season of epa exacerbation ( , ) . the pollen season for johnson grass is temperature dependent, flowering from may to july, with higher temperatures moving flowering later into autumm ( ) . a role for fungal triggering in epa exacerbation is suggested by the near identical clinical picture presented by horses with epa and barn dust-associated severe asthma, wherein a role for fungal triggering is substantiated in the latter ( ) . chronic neutrophilic airway inflammation characterizing both forms of severe equine asthma also aligns to th -mediated neutrophilic inflammation in fungal asthma models ( ) . of the more than species of fungi that exist in biotropic relationships with bermuda, bahia, and johnson grasses, curvularia, helminthosporium, alternaria, puccinia, epicoccum, and fusarium are implicated in eliciting human asthma ( ) . costa et al. identified fungal spores of the genus nigrospora, and curvularia, as well as basidiospores, as temporally associated with exacerbations of pasture asthma ( ) . these findings are in agreement with reported correlations between epa exacerbation and high dew point temperature ( ) . specifically, nigrospora conidia and basidiospore release increase with increasing relative humidity, resulting in a peak in spore counts during the early morning and aligning to the association of epa exacerbations with increased dew point temperature ( , ) . in contrast, conidia of cladosporium, alternaria, epicoccum, and dreschlera spp. are released during warm, dry, windy conditions, while precipitation is required for release of many ascospores. in this way, humidity influences fungi of relevance to asthma in different locales which could influence associations of pasture asthma in the uk with hot dry conditions, rather than hot humid conditions precipitating pasture asthma in the southeastern us. as a chronic and progressive disease of undetermined etiology, epa is most effectively managed by segregation from inciting grass pastures during warm seasons. the necessity to segregate horses from pasture, particularly at a time when they are typically extensively ridden and grazed, presents a conundrum that is ultimately detrimental for most affected horses. accordingly, there is a critical need to identify the agents that trigger epa in order to improve disease management. both veterinary practitioners and researchers muse about the diagnostic armamentarium available to physicians-if only we had the chest ct, the advanced lung function testing, the biomarkers-then we would be able to have a better diagnosis. a quick search of the literature, however, shows us that our counterparts face many of the same diagnostic dilemmas that we do, albeit often with higher bills! while pulmonologists have drawn up multiple guidelines to help in the diagnosis of asthma in humans with its multiple phenotypes and endotypes, physiciandiagnosed asthma criteria often fail to be consistent with the official guidelines rendering the results of large epidemiologic studies or clinical trials fraught with the perils of resting findings on nebulous datasets. various forms of spirometry or simple pulmonary function testing are readily available in human medicine, but few non-pulmonologists avail themselves of objective data, and instead rest on reported symptoms such as difficulty breathing on exertion, cough or positive response to bronchodilation ( ) . indeed, the gina toolbox identifies "lack of access to spirometry/bronchoprovocation tests" as a barrier to implementation of gina guidelines in human asthmatics ( ) . moreover, the heterogeneity in published algorithms for diagnosis of asthma-more than in the literature at last count-make even an algorithm-based diagnosis unsure ( ) . thus, the conclusion that symptom-based diagnosis is associated with a significant risk of over-diagnosis has been reached for asthma in humans ( ) . the current push in human medicine to refine both the phenotypes and endotypes for multiple different subtypes of asthmas aims to elucidate the underlying causes and thus treatments that may be very different. we are still searching for the criteria that will help us with this in equine medicine. if there are indeed mechanistically different groups of horses within the categories of mild, moderate, and severe ea that are associated with genetic differences or cellular or molecular biomarkers, then perhaps we will gain better understanding of treatment successes and failures and will be able more logically to choose clinical therapies and predict responses. the difficult case for the clinician and the researcher alike is not the horse with severe ea-because the history and clinical exam alone can often suffice to diagnose, and there is a visible relief in respiratory embarrassment with administration of bronchodilator (although it can take some time in horses with diaphragmatic exhaustion) ( ) . the difficult horse is the one with moderate/severe asthma in remission and the horse with mild-moderate ea. as was recently pointed out, the biggest difference that we note in the clinical diagnosis of horses with mild-moderate ea vs. severe ea is the presence of an increased respiratory effort at rest, which is due to the underlying pathophysiology of bronchoconstriction, increased mucus, and bronchiolar inflammation ( ) . the need, then, is to detect the mildly or subclinically affected horse. as veterinarians, we have at hand history, clinical signs, lung function testing, radiographs, endoscopy, analysis of airway secretions, blood biomarkers and clinical pathology which can be used in a minimum database in order to classify horses into clinically useful categories that have a pathophysiologic basis that can simultaneously allow us to diagnose, treat, and translate clinical cases into field research. a tentative diagnosis of ea in its most severe form can often be made on history alone, with the key component being the recognition of episodes of reversible respiratory embarrassment precipitated by exposure to specific triggers-namely, moldy hay in the northeast of the united states, and pasture allergens and particulates in the south. history in subclinical or mild cases is seldom of such definitive use; this does not mean that it is unimportant. such questions as parentage ( ) , type of feed and how it is fed ( ) , and heat and pollen counts at the time of diagnosis ( ) may be important risk factors for equine asthma. while it has been proposed that coughing and poor performance may serve to define a phenotype of moderate vs. mild ea ( ) , these signs are not sufficiently sensitive ( ) and would misclassify a subset of horses-they alert the clinician that moderate ea is likely, but the absence of these signs does not rule out disease. the connections between ea and viral or bacterial disease are not linear, but it is becoming increasingly clear that the connection exists ( , ) , thus a thorough history should include probing for past infectious respiratory disease. one of the best described questionnaire analysis tools for classification of horses based on history is the hoarsi index ( ), developed as a means of distinguishing among normal, mild-moderate ea or severe ea phenotypes. however, clinical signs and indices are insufficiently sensitive to distinguish horses with mildmoderate ea from normal horses or horses with severe ea in remission ( ) . proposed minimum database for both practitioners in the field and for research: a common history tool should be developed that addresses the main concerns of parentage if known, current and lifetime exposures to particulates and allergens including feeds and feeding practices, barn environment, vaccinations, travel history, and recent illnesses. multiple scoring systems have been shown to be useful for distinguishing healthy horses from horses with severe ea in exacerbation, but, similar to questionnaire indices, these scoring systems do not help in the more difficult problem of distinguishing horses with mild ea from healthy or severe ea in remission ( ) . indeed, years ago, robinson et al. found that even in horses with historical severe ea, clinical score failed to reflect low-grade airway obstruction, and suggested that without easily used, field-accessible testing equipment, lower airway disease would go underdiagnosed ( ) . recently, the adapted -point scoring system has been shown to be the most useful in discriminating mild from severe cases, but it is unlikely to distinguish normal from subclinical disease ( ) , and the ideass scoring system has recently been described as a useful scoring system for moderate-to-severe equine asthma ( ) . thus, while clinical scoring is essential to a good examination and careful research, and can potentially be useful in measuring response to treatment in the individual, it is insufficient in making the phenotypic distinction between mildly affected horses and healthy horses. proposed minimum database for both practitioners in the field and for research: the -point modified clinical score appears to best stratify horses with obstruction ranging from mild to severe. an application suitable for smart phone use would enhance the adoption of a common scoring tool. in human asthma, the gold standard is the detection of variability in pulmonary function using spirometry or other methods of lung function testing ( ) . unfortunately, lung function testing remains available only to a few specialized centers, as more recently developed portable lung function testing modalities are no longer on the market ( ) . initial reports from the author's laboratory of a simple field test of respiratory resistance using the interrupter technique hold promise for increased use of lung function testing in the future. while the classic esophageal balloon/pneumotachometer method is effective in demonstrating increased maximal pleural pressure and allows for calculation of pulmonary resistance and elastance as well as dynamic compliance in severe ea, it is not sufficient for demonstrating abnormal function in mildly affected horses in which baseline lung function is rarely abnormal and histamine or other bronchoprovocation or bronchodilation must be used in order in order to detect low-grade obstruction ( ) . unfortunately, in some studies, even histamine bronchoprovocation has not been sufficient to distinguish between normal horses and horses with mild asthma ( ) , and a lack of concordance between histamine bronchoprovocation and bronchoalveolar lavage (bal) cytology has been noted in several studies ( , ) . while lung function testing and histamine bronchoprovocation have shown moderate to strong correlations with bal cytology in some studies ( , , ) , others have not ( , ) . methods of performing histamine bronchoprovocation are equally important: studies in human asthmatics have shown that it is the total dose of histamine that is most important rather than the duration of exposure. a more precise method of dosing may be important to establish. in human athletes, indirect stimuli, such as cold air, hypertonic solutions such as mannitol, exercise, and amp are all considered more accurate and useful in predicting asthma than are direct stimuli such as methacholine or histamine; this is an area that requires exploration in equine pulmonology. while hay challenge is useful for research in severe ea, it is inappropriate in a clinical case, especially in a horse that is expected to do athletic work ( ) . in moderate to severe ea, variability in airflow should be demonstrated not through bronchoprovocation but through bronchodilation using either systemic (buscopan tm ) or inhaled (albuterol, ipratropium bromide) drugs to assess reversibility; it is possible that a h period of bronchodilation is necessary for maximum effect in horses with diaphragmatic fatigue ( ) . proposed minimum database for both practitioners in the field and for research: in research, lung function should be assessed and airflow variability/changes in airway caliber should be assessed with either bronchoprovocation or bronchodilation. more research is necessary to determine if field assessment of lung function after bronchoprovocation or bronchodilation is sufficient to determine change with the -point scoring system. it is essential that a robust, easily used system for testing lung function in the field be developed. unlike in human pulmonology, examination of airway secretions is a primary method of diagnosis in ea, be it mild, moderate or severe. although a standard volume of between and ml of saline using a m long endoscope or m bal tube is recommended ( ), this practice is not always followed, and cytology should be assessed keeping in mind that the amount of fluid infused will affect the cell percentages. the relationship between bal cytology and performance is still not clear. certainly, poor performance has been associated with what have been determined to be abnormal cell types or percentages ( ) . there has been much discussion as to what is normal on bal cytology; it likely depends on a combination of technique, environment and population. even the "stringent" definition proposed by couëtil et al. ( ) of < % neutrophils, % mast cells, % eosinophils, would be considered elevated in some high-performance populations ( , ) . although an earlier study found no evidence of a clear phenotype in mast cell vs. neutrophilic inflammation with respect to pulmonary gas exchange during exercise ( ) , recently, an increase in bal mast cells or neutrophils was shown to negatively affect performance ( ) . the way that cells are counted in bal cytology is also important, especially for rare cells. in our laboratory we count a minimum of cells at x for common cells such as macrophages and lymphocytes or neutrophils in mild ea, whereas for rare cells such as mast cells we count , cells. other techniques, such as using a -field differential for mast cells, are only useful if the cell density is high ( ) . the conundrum of whether to assess airway fluid from both lungs rather than blind sampling, or to pool samples, has also occupied attention from researchers. one group found that, depending on whether the "loose" or "stringent" categorization was used, - % of horses would have been categorized as control vs. mild-moderate ea if only one lung were used ( ) . as it is the rare practitioner who has a bronchoscope in the field, it is unlikely that even pooled samples ( ) , which may be a better representation of overall lung inflammation, will be taken other than in referral centers or practices. the problem is most important for rare cells. more attention will need to be paid in future to morphology and perhaps typing of cells. the existence of neutrophil extracellular traps (netosis) in horses with severe ea presents an additional method to determine response to treatment ( ) , and recently the presence of degenerate neutrophils has been shown to raise suspicion for bacterial infection ( ) . the question of macrophage morphology as an indicator of inflammation is also an area that will profit from further investigation ( ) . recently, as well, the paucigranulocytic phenotype has been described in which horses with clear signs of severe ea have low neutrophil percentages in the bal ( ) . this is thought to be due to mucus plugging of small airways that essentially sequesters neutrophils. although a recent publication showed a rather shocking % of highperforming european horses with mild-moderate ea had fungal elements in the bal ( ) , this remains to be confirmed in other populations. proposed minimum database for both practitioners in the field and for research: for the bal, at least -mls of saline should be used, and there is a preference for counting at least cells to adequately represent rarer cells. for research purposes where rare cells are of interest (e.g., mast cells or eosinophils), sampling of both lungs appears preferable. better categorization of cells through morphological descriptions including apparent neutrophil extracellular traps and notations of fungal or birefringent elements should be done. characterization of mucus on cytology may help to elucidate the paucigranulocytic phenotype. bal in the field will usually be done blindly with a specialty tube. the debate continues to swirl around the utility of tracheal wash vs. bronchoalveolar lavage, with malikides et al. ( ) finding a % disagreement in young racehorses, while derksen et al. ( ) determining that there was no correlation between bal and tw, and others finding no relationship between tracheal neutrophil counts and racing performance ( ); thus, tracheal cytology has been considered inappropriate for diagnosis of mild ea ( ). recently, however, a comparison of tw and bal in horses, along with evidence of mucus and endoscopy, found that only . % of horses would have been classified differently if they had had the other procedure, eventually concluding that there is no gold standard-except for mast cells, which are rare in the trachea, and thus, to be found, demand that a bal be performed ( ) . proposed minimum database for both practitioners in the field and for research: tracheal wash may be most practical for some practitioners in the field and has the added benefit of allowing for bacterial culture. the inability to assess mast cells adequately continues to limit this modality. in research settings, both tracheal aspirate and bal are preferable. many clinical diagnoses are made on the basis of endoscopic visualization of mucus, with strong support from the finding that tracheal mucus quite nicely correlated with racing performance or lack thereof ( ) . the recent consensus statement considers that the demonstration through tracheobronchial endoscopy of mucus grade / in racehorses or / for sport/pleasure horses is sufficient to diagnose mild-moderate ea and in support of this recommendation, rossi et al. ( ) found that visible mucus in the trachea is indeed likely to predict inflammation. there are varying degrees of certainty about mucus in the trachea predicting inflammation ( , , , ) . nonetheless, other studies have shown that mucus is insufficient to parse out mild vs. unaffected cases ( ) . endoscopy has also been shown to be useful in detecting an increase in upper airway abnormalities in horses with mild-moderate ea, with courouce-malblanc et al. ( ) raising the chicken-and-egg question of the relationship between mild-moderate ea and dorsal displacement of the soft palate, and more recently, wysocka and klucinski ( ) found that more horses with mild-moderate ea had dynamic pharyngeal abnormalities. it may be that the answer will rest in whether any of these modalities can help to define a phenotype rather than simply further describing an already understood phenotype. proposed minimum database for both practitioners in the field and for research: upper airway endoscopy should be performed to rule out upper airway cause of obstruction as a primary cause of signs or that might confound lung function testing. assessment of tracheal mucus should be performed. endobronchial biopsies offer an excellent method of sampling larger airways, although deeper layers cannot be accessed. the brass ring-being able to distinguish normal from remission or mild ea-remains elusive, however, as correlates were evident between histopathology and impulse oscillometry and showed a difference between horses in remission at pasture and those that remained stabled and treated with glucocorticoids, but did not show any difference between horses with severe ea in remission and controls ( ) . proposed minimum database for both practitioners in the field and for research: at this time, brushings/biopsies are not considered part of a minimum database. imaging is considered an important ancillary diagnostic in humans, but radiographs have not been shown to be sensitive or specific in horses with ea ( ) . chest ct is currently not feasible in large animals. while endobronchial ultrasound shows promise for the elucidation of airway smooth muscle thickening in severe ea, the ultimate goal of being able to detect low-grade disease in erstwhile healthy horses, or to distinguish normal from severe ea in remission remains elusive ( ) . proposed minimum database for both practitioners in the field and for research: at this time, imaging is not considered part of the minimum database. equine asthma encompasses mild to severe forms of chronic airway inflammation. severe ea affects ∼ - % of horses in countries with northern, cool climate ( , ) . mild-moderate ea affects - % of pleasure horses based on tracheal wash cytology (neutrophils > %) and up to % of racehorses based on bal cytology ( , ) . horses affected with severe ea experience exacerbation of clinical signs when exposed to organic dust originating from hay and bedding, in particular molds present in poor quality hay. as a result, clinical signs tend to be worse during the winter when horses are housed indoors for extended periods of time ( ) . some horses exhibit disease flare-ups while at pasture during summer months (epa) ( ) . these horses improve clinically during winter or after being housed indoor. a small percentage of horses appear to suffer from both classic severe ea and epa. horses with severe asthma tend to be mature (> years) to old animals and a genetic predisposition has been identified in some families ( , ) . the main clinical sign characteristic of severe ea is increased respiratory effort ("dyspnea") that can rapidly improve following bronchodilator administration. although the decrease in respiratory effort following bronchodilator administration can be detected within minutes of drug administration using lung function testing, clinical improvement may not be apparent to clinicians ( ) . acute exacerbation is associated with increased pulmonary artery and right-heart vascular pressures as well as increased pulmonary artery diameter on ultrasound ( ) . blood pressure return to baseline during clinical remission however, cardiac ultrasound abnormalities such as right ventricular wall thickness remained increased ( ) . surprisingly, severe ea is rarely fatal unless complications develop such as cor pulmonale ( ) . affected horses are more likely to be euthanized because owners get discouraged with the expense associated with chronic therapy and maintaining a low-dust environment ( ) . coughing and nasal discharge are non-specific signs of respiratory disease commonly reported in horse with severe ea ( ) . horses with a history of both coughing and mucoid nasal discharge are at increased risk of developing severe ea ( ) . thoracic auscultation may reveal increased breath sounds bilaterally, extended area of auscultation, and abnormal breath sounds (i.e., crackles, wheezes). however, the thick chest wall of horses makes auscultation an insensitive indicator of pulmonary disease, with abnormal findings obtained in < % of horses with severe ea ( ) . strict management changes or medical therapy will results in rapid improvement in clinical signs however, if exposure to triggering factors is not addressed improvement will be short lived or incomplete ( , ) . this form of mild respiratory disease is mainly subclinical with horses showing non-specific signs such as intermittent coughing and poor performance ( ) . however, mild asthma should not be ruled out in horses that do not cough because coughing is reported in only % of horses with mild asthma ( ) . coughing is associated with increased bal neutrophils ( ) . poor performance and reduced willingness to perform are associated with increased tracheal mucus scores in racehorses and show-horses, respectively ( , ) . in racehorses, poor performance has been associated with increased neutrophils and mast cells in bal fluid ( ) . there is an association between nasal discharge and increased tracheal mucus in racehorses ( ) . however, the association between tracheal mucus and bal cytology has not been reported yet. the term "remodeling" defines a process resulting in a tissue that is structurally and architecturally altered compared to its healthy counterpart. in asthma, structural alterations are represented by quantitative or qualitative changes of the bronchial wall components or their surrounding tissues, whilst architectural alterations refer to the skewed relationships among such structures. airway remodeling has been studied only in horses affected by severe ea. an increased expression of metalloproteinases and their tissue inhibitors has been recently reported in a group of horses with mild respiratory signs and bal cytology compatible with mild ea ( ) . however, the possibility that the horses studied were horses with severe ea in remission of the disease was not excluded. almost all airway components undergo remodeling in severe ea, both in peripheral (diameter < mm) and central airways. the airway smooth muscle mass as well as collagen and elastic fiber deposition are increased in the lamina propria of peripheral airways during severe ea remission compared to healthy airways ( , ) . mucostasis, mucus cell hyperplasia, peribronchiolar metaplasia, and interstitial fibrosis are more frequently detected in horses with severe ea in remission compared to controls ( ) . however, histomorphometric techniques revealed no differences in the number of mucus cells per mm of lamina reticularis or in the volume of stored mucosubstance in bronchial epithelial cells ( ) . central airway remodeling during disease remission is less pronounced compared to what is observed peripherally. whether airway submucosal structures are significantly altered during severe ea remission compared to control remain to be established ( , , ) . functionally, severe ea remission is associated with a normal lung function in spite of significant structural alterations of the airways. in these conditions, the respiratory resistance correlates with the amount of collagen within the lamina propria of peripheral airways ( ) , indicating that, in the absence of bronchospasm, peripheral airway stiffness is the major determinant of respiratory resistance in asthmatic horses. the functional implications of peripheral remodeling become more important during disease exacerbations, when most of the changes are further accentuated and the mechanics of breathing are altered ( , ) . there is no doubt that the major determinant of airway obstruction during severe ea exacerbations is smooth muscle contraction and that central airways play a major role ( ) . by definition, the force produced by a muscle is proportional to its cross-sectional area. given the increased smooth muscle mass (and cross-sectional area) during severe ea exacerbations ( ) , asthmatic muscle is "stronger" and able to contract the thickened lamina propria observed in severe ea, further reducing the airway lumen. increased mucus secretions into the airway lumen also contribute to airway occlusion ( ) . these same mechanisms operate in peripheral airways, where the effects on lung function are somewhat blunted by the fact that their overall contribution to pulmonary resistance is low, due to their large cumulative cross-sectional area ( ) . at this level, the more relevant functional effects of remodeling are the loss of lung elasticity and airway-parenchymal tethering. adequate small airway patency is guaranteed by their intimal connection to the lung parenchyma by elastic and connective fibers. when the lung inflates during inspiration, small airways are stretched and passively dilate. remodeling of elastic fibers and of the extracellular matrix within and around the airways and in the alveolar septa alters this mechanism, preventing the smallest airways from remaining open ( ) . the effect is even worse during expiration, when the lungs physiologically recoil and the airway diameter physiologically narrows. with a significantly impaired expiratory airflow, part of the air that reaches the alveoli remains trapped. this leads horses with severe ea in exacerbation to breath at increasing lung volumes [functional residual capacity ( ) ] in the attempt to maintain airway patency, which causes lung hyperinflation and enlarged fields of thoracic auscultation ( ) . anecdotal evidence to date has suggested that, although bal sampling is widely accepted elsewhere as the diagnostic tool of choice for cytological assessment of equine lower airways, tracheal endoscopy and tracheal wash-based diagnostics have remained the mainstay of routine clinical lower airway investigations in british thoroughbred racehorses in training. given the emphasis on bal in research, this would present a considerable challenge to furthering evidence-based respiratory medicine in this important equine population. in a recent study we investigated british racing veterinarians' rationales for current practices, and the challenges they face in relation to diagnosing and managing racehorse airway inflammation ( ) . qualitative data were gathered through semi-structured focus group discussions designed to capture current practices and opinions relating to the diagnosis and treatment of lower airway inflammation, as well as familiarity with and views on the most recent acvim consensus statement ( ), in which the term "mild-moderate equine asthma" was recommended. four british veterinary practices, two primarily serving the flat racing community and two primarily serving the national hunt (jump racing) community, in different geographical regions of england, were purposively selected to participate. focus group discussions were conducted at the practice premises, moderated by one of the authors (tk), an experienced qualitative researcher who is not a veterinarian. discussions were audio-recorded and transcribed verbatim, and transcripts were analyzed using an inductive, thematic analysis. in total, participants contributed to the focus group discussions (number per group ranged from to ). all were veterinarians (experience ranging from recent graduate to senior partner), with the exception of one laboratory team member and one veterinary student, and five were women. discussions lasted between and min. three key themes were developed through analysis of focus group data: (i) an over-arching theme of serving the racing industry within which two further themes (ii) disregarding of the consensus and (iii) the pragmatic clinician were nested. (i) serving the racing industry: this was a key driver of clinical approaches to racehorse respiratory health, which were strongly trainer-influenced in particular. the trainer selects horses for endoscopic respiratory assessment, often because of training and racing schedules rather than any clinical signs, and the approach to investigation and treatment is strongly influenced by trainer expectations. this varies with trainer personality, experience and training methods, as well as stage of the racing season, signalment of the affected animal and general health on the yard, and is in turn driven by commercial pressures of the racing industry. (ii) disregard of the consensus: the unanimous view across all four groups was that the condition defined as mildmoderate ea by current concensus ( ) is largely not seen in british racehorses which, in the participants' considerable collective experience, are affected predominantly with excess endoscopically-visible tracheal mucus largely attributed to bacterial infections. it was also considered unfeasible to fulfill two key aspects of the consensus case definition: waiting for chronicity of clinical signs (> weeks duration), and performing bal sampling. neither of these would be acceptable to trainers, according to participants, and participants themselves were not convinced of the extra value of bal sampling. the consensus statement was therefore seen as having been developed for outsiders, by outsiders without sufficient understanding of culture and practices on british racing yards. (iii) the pragmatic clinician: participants shared a strong professional identity as pragmatic clinicians often required to base clinical decision-making on direct personal or collective experience, rather than on research-based or laboratory evidence. cytological examinations of tracheal wash samples were defended as valuable when interpreted sequentially and combined with knowledge of the history and idiosyncracies of the individual horse and yard. although this approach was generally viewed positively as flexible and individualized, participants did also express some frustration with the sometimes unsatisfactory jigsaw of diagnostic information available to them, particularly in relation to discrepancies between clinical and laboratory findings. our work has highlighted a lack of alignment between clinical practice on british racing yards and international consensus on diagnosing lower airway inflammation, which constitutes a barrier to furthering development of a contextually-relevant evidence-base for this population. equine clinicians elsewhere may find themselves in disagreement with some of the opinions expressed, or practices described, by our study participants. however, these investigations were designed to understand the experiences and rationales of clinicians in the specific context of british racing practice. the strength and consistency of views expressed support the anecdotal evidence that, in this context, tracheal endoscopy and wash sampling are widely regarded as the best available means of providing the non-invasive monitoring of respiratory health expected by trainers and used to inform training-and racing-related decisions. it would be interesting to determine whether similar approaches are being taken elsewhere, particularly in populations of yearling and year old thoroughbred racehorses in training. given the considerable resistance to bal sampling in british racing, development of new tracheal-based or other minimally-invasive diagnostics, including appropriate biomarkers and suitably sensitive, portable lung function tests, would be valuable. furthermore, our participants' views that mild-moderate ea as defined by current consensus is largely not seen in british racehorses suggest that research furthering our understanding of the etiology and pathogenesis of airway inflammation in this equine population is still required. the respiratory system is an interface between the outer environment and the inner body. lower airways have historically been seen as a sterile milieu, thanks to the anatomical configuration, local surface immunity and mucus production and clearance systems ( ) . however, with the development of high sensitivity and high throughput technologies, the microbiota of the respiratory system has been described in healthy subjects in many species, including horses ( , ) . further investigation of the relationship between infectious agents, lower respiratory tract microbiota and the development of mild ea is warranted. we and others have reported descriptive results about the microbiota of horses with mild ea ( , ), but the causality between bacterial flora and the disease is far from being understood. studies on the microbiome use dna extraction followed by high throughput amplification and sequencing of the s amplicon ( ) . the sequences are then filtered and aligned against a taxonomy database to identify and organize operational taxonomic units (otus). descriptive analysis of the phyla, otus and bacterial species are then performed, followed by statistical analysis at the community level (within and between samples; alpha and beta diversity, respectively) and at the individual level (otu diversity analysis). statistical analysis can be used to compare between groups: healthy horses vs. those with mild asthma, upper vs. lower respiratory tract ( ) . the lower airways have a decreased richness (alpha diversity, corresponding to the number and proportion of each bacterial species) when compared to the upper airways in healthy horses ( ) . however, a very large majority of the same otus are present in both the upper and the lower airways, showing an overlap and some continuity in the bacterial population between the two anatomical environments in healthy horses. furthermore, treatment with corticosteroids did not affect the composition of the bacterial flora in the upper airways ( ) . the role of the upper airways microbiota in mild ea is unknown, but two studies did not find any difference in beta diversity of the upper airways between healthy horses and those with mild ea ( , ) . the relationship between bacteria and the lower respiratory tract of the equine host seems to be dynamic. as an example, a change in the environmental respirable particulates has an effect on the lower respiratory tract flora in horses. furthermore, treatment with systemic or nebulized dexamethasone induces some changes in the microbiota of the lower respiratory tract in both healthy and mild asthma horses ( ) . systemic dexamethasone administration decreased the evenness of the flora and increased the abundance of otus. there is an agreement between studies that the lower airways microbiota between healthy and mild ea horses are clearly different ( , ) . interestingly, streptococcus is one of the otus which differed with disease status, and was the otu with the greatest increase in relative abundance in mild ea. the effect of the environment on the composition of the lower airways' microbiota is also a common finding between studies ( , ). however, a study found that treatment with corticosteroids had more effect on the composition of the bacterial flora than changes in the environment ( ) . the microbiome studies are recent in equine medicine and are limited to being descriptive. the challenge for the scientific community will be to answer the causality dilemma of the chicken or the egg regarding the role of the airway microbiota in mild ea. asthma development in humans is most probably caused by the interaction of multiple factors, including genetics, allergen exposure, microbiome and invading pathogens. human rhinovirus, human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus, human enterovirus and human coronavirus are strongly associated with asthma exacerbations ( ) . the association between human rhinovirus-induced wheezing and the development of childhood asthma/wheezing has been confirmed in a recent meta-analysis ( ) . the risk for asthma by age years has been shown to increase (odds ratio . ) if children have been wheezing with rhinovirus during the first years of life ( ) . further, many prospective long-term follow-up studies have shown that human respiratory syncytial virus-induced bronchiolitis is associated with later development of asthma ( ) . however, the pathogenic role of respiratory viruses as triggers for the development and/or exacerbation in asthmatic human patients has not been fully characterized. changes in the immune response to viral infections in genetically predisposed individuals are very likely to be the main factor involved in the association between viral infection and asthma ( ) . the pathogenesis of ea remains incompletely defined. however, similar to human asthma, a multifactorial process is suspected. conditions associated with exercise, feeding and housing practices, location, seasonality, infection of the upper and lower airways and genetic influences have been linked to ea ( , , , ) and bacterial (streptococcus equi subspecies zooepidemicus, actinobacillus spp., pasteurella spp.) etiological agents have been linked to mild to moderate ea ( , ) . it remains to be determined if these agents are triggers for the development of ea or are secondary colonizers of already compromised airways. viral respiratory infections are one of the most common health problems in horses throughout the world ( table ). these infections are often self-limiting and a full recovery can be expected in most horses. young performance horses, such as racing horses, have an increased risk of respiratory viral infections. this relates to age susceptibility, commingling, stress and suboptimal biosecurity protocols ( , , ) . amongst respiratory viruses, only eiv and ervs have an affinity to the lower respiratory tract, leading to airway hyperresponsiveness. clinical signs associated with eiv are usually more severe than those seen with mild to moderate ea. further, no association has been determined between mild to moderate ea and infections with eiv, ehv- and ehv- ( , , ) . this is in sharp contrast to the detection of ervs (erav and erbv), known to cause subclinical or mild clinical disease ( , , ) . in a recent study, horses with mild to moderate ea were significantly more likely to have a positive titer as well as higher log-transformed titers to erav when compared to control horses ( ) . in another study, the detection of erbv by qpcr was significantly associated with coughing in standardbred racehorses in training ( ) . subclinical respiratory viral activity in horses with poor performance has been associated with ehv- and ehv- infection ( , ) . in a recent study, the detection of ehv- by qpcr in nasal secretions was significantly associated with mild to moderate ea ( ) . in another study, the detection of ehv- by qpcr was significantly associated with coughing and excessive tracheal mucus in standardbred racing horses ( ) . these results are in sharp contrast to two recent studies performed on swedish standardbred trotters, which were followed for months via qpcr analysis of nasal secretions and serology ( , ) . despite occurrence of poor performance and subclinical viral activity in the swedish standardbred trotters, the authors were unable to detect associations between ehv- /- and clinical respiratory disease and/or poor performance. these conflicting results reflect the ongoing challenges in establishing causality between mild to moderate ea and gamma herpesviruses, known to be ubiquitous in both healthy and clinically affected horses. in conclusion, associations between specific viruses detected via antigen or antibody detection and clinical signs of mild to moderate ea may suggest that viruses may play a role in triggering or exacerbating asthma. however, because some viruses are ubiquitous both in healthy and clinically affected horses or are often associated with subclinical disease, establishing causality is challenging and in need for further research. a growing body of research demonstrates the link between organic dust exposure and ea. introduction of horses to high dust environments not only induces profound bal fluid neutrophilia and airway obstruction in horses susceptible to severe asthma, but also significant neutrophilic airway inflammation in previously healthy horses ( , ) . outside of the experimental exposure setting, higher dust exposure has also been associated with increased risk of tracheal mucus accumulation in racing thoroughbreds ( ) . barn dust is a complex mixture, rich in potential sources of allergens as well as immunomodulators such as endotoxin and β-glucan ( , ) . in addition to individual horse factors such as age and susceptibility, this complexity may partially account for the heterogeneity of asthma phenotypes. respirable particulates, nominally < µm in diameter, have been linked to eosinophilic inflammation in young thoroughbreds entering race training ( ) and neutrophilic inflammation in actively racing thoroughbreds ( ) . increasing respirable endotoxin exposures have been shown to provide an apparent protective effect against neutrophilic inflammation at low doses ( ) , while high doses of endotoxin augment the inflammatory response to particulates ( ) , suggesting a non-linear response to inhaled endotoxin in the horse. mast cell inflammation has been found to be common in both young, untrained thoroughbreds ( ) and those that are actively racing ( ) , but unrelated to respirable dust or respirable endotoxin exposures. instead, bal mast cell proportions are related with respirable β-glucan exposures. conversely, inhalable dust exposures have not been found to affect bal inflammatory cell proportions. thus, inhalable particulates, those nominally < µm in diameter, appear to be less relevant than respirable particulates in equine respiratory health. setting exposure recommendations will require better understanding of the dose-response to inhaled non-infectious agents across wider ranges of age, breed, and discipline through study designs that include both exposure and respiratory health outcome measures and utilize appropriate statistical tools to relate them. advanced characterization of respiratory health, such as investigation of alveolar macrophage function and bal fluid cytokine profiles, coupled with extensive exposure assessment is likely to offer valuable insight into ea pathophysiology and identify new targets for intervention. miniaturization of optical particle counters has rendered realtime breathing zone exposure measurements on the horse both affordable and technically feasible. finally, the equine airway is arguably most susceptible to particle penetration during athletic exertion due to large tidal volumes and extension of the head and neck, yet the exposures that horses sustain during exercise are largely unexplored. such measures of exposure are complicated by the air speed and turbulence generated at the breathing zone during such activity and will require specialized sampling strategies. neutrophils are key actors in host defense, migrating toward sites of inflammation and infection, where they act as early responder cells toward external insults ( ). however, neutrophils can also mediate tissue damage in various noninfectious inflammatory processes. airway inflammation is one of the primary characteristics of an asthma-affected horse's response to aeroallergens with neutrophilic bronchiolitis being the main lesion ( ) . the mechanism by which airway inflammation develops in ea is a multifaceted and dynamic process. current knowledge suggests that the inflammatory component of this disease results from a combination of both the innate and adaptive immune responses ( ) . generally, airway inflammation involves activation of pathogen-specific inflammatory cells, modulation of gene transcription factors, and release of inflammatory mediators ( ) . within the airways, neutrophils likely contribute to bronchoconstriction, mucus hypersecretion, and pulmonary remodeling by release of proinflammatory mediators, including the cytokines interleukins and , neutrophil elastase, reactive oxygen species, and neutrophil extracellular traps (nets) ( ) ( ) ( ) ( ) . oxidative stress in horses with asthma is evidenced by the increase in elastase and decrease in ascorbic acid concentrations in balf associated with neutrophilia secondary to exposure to organic dust ( ) . the pathogenic role of nets has been described for many infectious and non-infectious human diseases, including respiratory cases with a massive influx of neutrophils into the airways ( ) . excessive net release is particularly deleterious in lung diseases because nets can expand easily in the pulmonary alveolar space and cause lung injury. furthermore, nets and their associated molecules can directly induce epithelial and endothelial cell death ( ) . the mechanisms that regulate neutrophil functions in tissues are complex and incompletely understood and must be regulated with exquisite precision and timing. timely apoptosis of neutrophils is central to the resolution of inflammation; dying neutrophils are known to stimulate their own efferocytosis, inducing macrophagic transition from a pro-inflammatory to an anti-inflammatory profile ( ) . thus, dysregulated apoptosis and mechanisms of inflammation may play an important role in the pathogenesis of ea. the persistence of apoptosis-resistant neutrophils in the airways of horses with asthma may also impede timely neutrophil clearance and delay the resolution of airway inflammation. the discovery and development of compounds that can help regulate ros, net formation, cytokine release and clearance of airway neutrophils would be highly beneficial in the design of therapies for ea ( ) . asthma is a highly heterogeneous condition of the lung. akin to the lining of the gastrointestinal tract, the lining of the airways is also in contact with external substances throughout life. ingested substances generally pass through the gastrointestinal tract unidirectionally, and a careful balance between processing of digested food materials, nutrient absorption and limiting immunoreactivity is maintained during homeostasis, with wellknown severe consequences of deviations in this balance. the airways function differently in that only gaseous substances normally pass into the distal alveoli and are exhaled in the reverse direction. inhaled particulates also have to be expelled in reverse direction toward the nasopharynx by largely mechanical means or taken up by alveolar macrophages for disposition with minimal inflammatory evocation ( ) . hence, a complex and selective epithelial barrier with differing functions characterizes both organs. the epithelium lining the airways has unique composition, morphology and function throughout the lung, and is intimately connected to subepithelial structures such as the basement membrane, mucous glands, smooth muscle, fibroblasts, endothelium and immune cells. the epithelium forms a barrier between inhaled components and the subepithelial constituents, and also has to balance efficient transfer of gases with controlled reactivity to non-gaseous components. while the lesions of severe ea manifest predominantly with inflammation, smooth muscle hyperplasia and fibrosis of the peripheral airways and surrounding tissues, the larger airways are exposed to the same inhaled substances and also have morphological, functional and molecular changes ( ) . research initially focused on the role of club cell secretory protein (ccsp), a member of the secretoglobin family produced by non-ciliated epithelial cells concentrated within the epithelium at the transition from bronchi to bronchioles. club cells are recognized as epithelial progenitor cells that can differentiate into ciliated and other specialized cells of the airway epithelium, participate in reduction of reactive oxygen toxicants through cytochrome enzymes, and their hydrophobic secreted protein inactivates a range of inflammatory mediators. horses with severe asthma have fewer club cells and lower concentration of ccsp in airway fluids, which may be a function of chronic inflammation resulting in reduced regenerative capacity of the airway epithelium ( ) . unique relative to other mammals, equids have two expressed ccsp genes that differ in of amino acids, and also in their interaction with hydrophobic molecules ( ) . recombinant eccsp increased neutrophil oxidative burst, phagocytosis and extracellular trap formation, lending support to the notion that loss of club cells has deleterious effects on lung health ( ) . whole transcriptomic changes in endobronchial epithelial biopsies from sites from th to th generation bronchi were investigated with next-generation sequencing. each horse served as its own control to identify changes in gene expression associated with an inhaled challenge since inter-individual variability exceeded changes attributable to the challenge. a bioinformatics pipeline including quality control measures to account for duplicates, variable sequencing depth and dispersion was implemented, results were mapped to the equine genome, and predicted proteins were procured with a combination of software and manual approaches to assign appropriate ensemble ids for analyzing interactions. an overall conservative analytic approach yielded genes differentially expressed in horses with severe asthma as a result of a challenge, with the majority up-regulated ( ) . not surprisingly, many up-regulated genes pertained to inflammatory mediators and effectors and were well-known members of protein interacting networks. however, somewhat more surprisingly, genes with altered expression also concerned more broadly epithelial cell formation and maintenance, and the circadian rhythm, suggesting that multiple cell properties are affected in exacerbated ea at the transcriptomic level. subsequent analysis of enriched gene sets in asthmatic horses further highlighted the importance of cell cycle regulation and repair pathways ( ) . transcriptomic studies of this nature yield a great deal of information, which requires subsequent confirmation regarding cell specificity, correlation with protein expression and function, and extension to a more robust number of affected and unaffected individuals. albeit, there is strong evidence to indicate that the bronchial epithelium is profoundly altered during exacerbation of severe ea, and this insight offers new venues for investigating the role of specific proteins and for potential therapeutic targets ( , ) . the entire spectrum of ea is influenced by interactions between the environment and genetics, but almost all research in this field has focused on the severe clinical phenotype. while no specific genetic risk factors have been reported for mild to moderate forms of ea, genetic susceptibility to certain bacterial lower airway infections could potentially be relevant ( ) . furthermore, mild but persistent respiratory signs such as occasional coughing and nasal discharge may represent early phenotypic indicators for an increased risk to later development of severe ea ( ) . this suggests that the genetics of milder forms of ea may be worth investigating in longitudinal studies. severe ea has been shown to be partly heritable in several breeds and has been the focus of genetic research involving family and epidemiological studies, whole-genome scans and investigation of candidate genes. reports of marked familial aggregation of severe ea date back years ( ) . parent, age, and stable environment have significant additive effects that increase the risk for developing severe ea as defined by a history of persistent frequent coughing and/or increased breathing effort ( , ) . offspring of affected sires have a more than -fold increased risk for developing severe ea ( ) . whole genome scans in high-prevalence families indicate two chromosome regions with a genome-wide significant association with severe ea ( ) . importantly, the associations differ between the families: region eca in one family and eca in another family. further association and gene expression studies indicate interleukin receptor as a candidate gene in a subset of ea-affected horses. molecular pathway analyses of genomic and proteomic data showed interactions between interleukin receptor and socs upstream of an important molecular cascade involving nuclear factor κb ( ) . so far, no causal genetic variant has been identified in interleukin . an allelic case-control genome-wide association study in the general warmblood population revealed another region on chromosome . the best-associated marker was located in the protein-coding gene txndc , which may be involved in regulating hydrogen peroxide production in the respiratory tract epithelium as well as in the expression of muc ac mucin ( ) . no genomic copy number variations were found to be associated with severe ea ( ) . integrative analyses combining gwas, differential expression (de), and expression quantitative trait loci (eqtls) were not able to uncover causative genetic variants that contribute to severe ea through gene expression regulation. however, results showed interesting similarities to human asthma with disease-associated genetic variants in clec a that also regulate gene expression of dexi ( ) . furthermore, global gene expression studies of mrna and mirna levels in these high-prevalence families have shown impaired cell cycle regulation and cd + t cell differentiation into th /th cells, respectively, in severe ea ( , ) . at present, none of these associations are useful genetic markers in the general population. most of the findings pertain to warmbloods only, or even only to certain lines and families. the fact that the chromosomal regions and the mode of inheritance do not agree between families indicates genetic heterogeneity for severe ea: depending on the genetic make-up of affected horses, different genes confer the susceptibility for the disease. it appears that the genetic basis of severe ea is robust, but remarkably complex. polygenic complexity, potentially with a larger number of genes that each may only contribute < % to the total genetic effects, may make it difficult to discover causative variants. nevertheless, the genetics of severe ea has revealed interesting links between severe ea, allergic skin diseases and susceptibility to intestinal parasites ( , ) . according to the national institutes of health, a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacologic responses to a therapeutic intervention ( ) . in practice, biomarkers include tools and technologies that can help in understanding the prediction, cause, diagnosis, progression, and outcome of treatment of a disease. although bal cytology has been recognized as the gold standard for diagnosing respiratory diseases such as ea, currently, sensitive and specific biomarker tests useful in routine laboratory diagnostics are being sought. a simple biomarker capable of distinguishing between animals with lower airway infections and those with non-infectious airway inflammation would be helpful. although the diagnosis of severe cases of ea is relatively easy, it is difficult to diagnose cases in remission or horses with a mild form of the disease. ideally, molecular biomarkers should reflect a feature of relevant pathological processes. in addition, biomarker assessment should be easy, low-cost, technically accurate, repeatable and have an acceptable risk. therefore, a measurement from easily obtainable body fluids or tissues is preferred, such as blood, urine, exhaled breath condensates, as opposed to bal, transbronchial biopsy or lung biopsy ( ) . several biomarkers are present or altered in the airways or circulation of horses with asthma. inflammatory markers such as acute phase proteins and cytokines have been studied as markers of systemic inflammation. however, the available literature on markers of systemic inflammation in horses with severe ea is not well-characterized and controversial ( , ( ) ( ) ( ) . apart from reports on differential expression of cytokines during the course of severe ea, only a few acute phase proteins have been investigated. haptoglobin is a suitable marker of both acute and chronic systemic inflammations, whereas high concentrations of serum amyloid a indicate acute inflammation. one study found no difference in the acute phase protein levels (serum amyloid a, c-reactive protein, haptoglobin) between horses with mild ea and those with other causes of exercise intolerance ( ) . another study found elevated haptoglobin concentration in horses with mild ea ( ) . surfactant protein d is a large multimeric collagenous glycoprotein produced mainly by type ii epithelial cells in the lungs and is also detectable in the serum. serum surfactant protein d has been identified as a potential systemic biomarker for some pulmonary diseases in humans, such as idiopathic interstitial fibrosis and acute respiratory distress syndrome. elevated serum levels of surfactant protein d have been detected in horses with mild ea ( , ) . circulating immune complexes are proteins that result from an immune response against an organism or antigens of various origin. in humans, circulating immune complexes are detectable in a variety of systemic disorders such as autoimmune diseases, allergies and infectious diseases ( ) . high levels of circulating immune complexes have been reported in horses with severe ea ( ) . another study found circulating immune complexes useful for differentiating healthy vs. severe ea, and monitoring corticosteroids therapy ( ) . the main group of enzymes responsible for collagen and other protein degradation in the extracellular matrix are matrix metalloproteinases (mmps), while tissue inhibitors of metalloproteinases (timps) lead to fibrosis formation. collagen is the main structural component of connective tissue and its degradation is a very important process in development, morphogenesis, tissue remodeling, and repair. in horses with severe ea, mmps, timps, and their ratios are useful in the evaluation of the severity of respiratory disease and in identifying subclinical cases ( ) . furthermore, mmp- , mmp- , timp- , and timp- are significantly decreased after therapy with inhaled glucocorticoid therapy ( ) . exhaled breath condensate is a promising source of biomarkers of lung disease in humans. exhaled breath condensate hydrogen peroxide concentration and ph were higher in horses with mild ea, vs. controls ( ) . additionally, both hydrogen peroxide and ph had a positive association with bal neutrophil percentage, while leukotriene b- demonstrated a positive association with bal eosinophil percentage. another study characterized the metabolomic profile of tracheal wash and exhaled breath condensate in healthy horses and those with severe ea ( ) . higher concentrations of histamine and oxidant agents, such as glutamate, valine, leucine, and isoleucine, as well as lower levels of ascorbate, methylamine, dimethylamine and o-phosphocholine, were found in the group of severe ea, compared to healthy controls. many biomarkers of ea have been studied-some are already being used in clinical settings, while others require further studies. however, history, clinical evaluation, and bal still constitute the basis for diagnosis of ea. immune response has mainly been investigated in the airways of horses with severe ea and more recently mild-moderate ea, while still representing one of the futures direction for research stated in the acvim consensus statement ( ). such characterization has mostly been performed through relative mrna expression of various cytokines in bal fluid, while several publications also reported protein concentration in bal fluid for few cytokines. various methodologies for cytokine mrna expressions have been published (e.g., sybr green or taqman technology, design of primers and probes, relative quantitation, etc.). variation in methodologies may ultimately prevent objective comparisons between reports, as well as the implementation of prospective, multicenter studies. such diversity should however not be considered as a scientific weakness, and methodological homogenization among the various research groups neither represents a prerequisite nor a final goal to be reached. however, evaluation of the methodological performances of different research laboratories might represent a relevant goal. in this manner, implementation of inter-laboratory comparisons based on international standards (e.g., iso/iec and iso ) warrants further consideration. let's consider for example mrna expression of two different cytokines by pcr in balf samples. as a first and informal procedure, a simple "blind test" could be performed among up to four different teams. in this procedure, the "reference lab" will provide the three other labs with aliquots of the same sample(s). each team will evaluate mrna expression for these two cytokines based on their own procedures, and comparisons of the results obtained and agreement among the teams can be evaluated. this "blind test" might then be repeated on a regular basis, systematically alternating the "reference lab" within the group. in the end, the procedure will provide an objective evaluation of the results diversity among the teams, but clearly will not determine whether several teams are more efficient than others for these specific analyses. a second and more structured procedure would require the specific synthesis of standards (mrna for two different cytokines in this case), and the development/validation of relevant conditioning and conservation procedures. a similar group of four different labs would first evaluate their ability to detect and quantify predetermined amounts of analytical standards (evaluation of the detection, not of the sample extraction, etc.). this step is a necessary preliminary, in the absence of reference methods. a panel of at least samples (previously calibrated with standards) would then be tested, including several identical ones (for repeatability) and submitted to the group (including a "self-shipment") for testing and further statistical analyses (agreement, etc.). once the methodological performance of the lab is considered acceptable for this panel, the procedure might then be repeated with another two cytokines and so on. in the end, the whole panel of standardized samples might allow the establishment of a labeling, accessible to any voluntary laboratory involved in equine asthma. mandatory considerations about such comparisons are that there is no trap, and this does not represent an overall examination of laboratories, but simple evaluations of procedures. all labs are expected to use their methodologies, whether or not the technologies are similar within the group. among others, samples conditioning, conservation, shipment and their associated costs will represent major issues to be considered, and this should be more broadly associated with virtuous initiatives such as the equine respiratory tissue biobank. several group discussions were conducted during the havemeyer equine asthma workshop to identify future research priorities. initial rotating small-group topic explorations (pathophysiology, risk-factors, diagnostic methods and phenotype definition) facilitated by members of the workshop organizing team, were followed by a final large group "roundtable" discussion of key directions for future ea research. the discussion was informed by data gathered directly from ∼ participants (i.e., all who attended the final roundtable), who were invited to propose up to three short-or long-term, focused or "big picture, " research topics or ideas that they considered to be key future research directions. these data were submitted anonymously, during the workshop, as free-text on paper and loosely arranged into broad categories for further open discussion. following the workshop, in order to present an accessible, systematic and non-selective summary of the ideas proposed by participants, the free-text data were collated in microsoft excel for content analysis using an approach based on recommended methods for quasi-qualitative data ( , ) . the text was transcribed verbatim and coded at two levels to categorize content into (i) broad topic areas (level ) and (ii) specific subsets of these topics (level ). all instances of each level topic code were then exported into online software (worditout) to create a word cloud (figure ) , in which the relative frequencies of occurrence of each topic are represented by font size. overall, responses were received, each proposing between and research ideas, resulting in a total of research ideas, which were organized into the broad topic codes presented in the word cloud. some research ideas encompassed more than one topic and were identified with multiple codes to reflect this. frequencies of occurrence of each code ranged from n= for "diagnostics" to n = for "genetics." specific proposed areas of interest in the dominant "diagnostics" category were the development of improved, non-invasive field diagnostics through the identification of suitable biomarkers, development of portable lung function tests, improved understanding of relative values of tracheal wash in comparison with bal cytology, or relationships between the two, and identification of gold standards for all of these diagnostic modalities. another key topic was phenotype distinction ( occurrences)-in particular to clarify any distinction between mild and moderate ea, and to determine whether or not such a distinction is valuable in terms of differing pathophysiology, diagnostic indicators, therapeutics or prognosis. as with many of these proposed topics, phenotype distinction rests on the back of the category "diagnostics"-pointing out a self-identified weakness on the part of ea researchers that the goal of identifying the horse with asthma so mild that it does not present as respiratory disease per se, continues in many cases to elude us and underscores a collective pragmatism that there is little benefit in understanding the fine points if we cannot definitively identify the case in the first place. ideas relating to therapeutics ( occurrences) included investigating the efficacy of different treatments including environmental management and any evidence for the value of antibiotics, as well as the development of optimal nebulized glucocorticoids, alternatives to corticosteroids, immunological treatments, respiratory probiotics, other novel therapeutics (e.g., marcks inhibitor peptide), and individualized treatments for different endotypes and phenotypes. suggestions relating to pathophysiology ( occurrences) included furthering our understanding of the role of environmental pollutants, of when a physiological response becomes a pathological response and of factors influencing progression from mild to severe equine asthma. standardization ( occurrences) referred in particular to the need to develop or agree on standardized diagnostic approaches, including in relation to bal collection techniques, laboratory processing and cytological methods and threshold values, context-specific reference ranges, development of a central repository of protocols and improved quality control protocols. a central repository of standard protocols was suggested. academic-clinical communication ( occurrences) was recognized as an area for general improvement. related research suggestions included improving our understanding of the views and practices of field clinicians, as well as their perceptions of disease progression and treatment efficacy, particularly in regions outside the uk (to build on the kinnison and cardwell uk study) ( ) . this would inform the enhancement of multidirectional communication between academia, referral and first opinion clinical practice, development of guidelines and apps for field practice and overall improved dialogue and engagement. better use of collaborative, epidemiological and longitudinal studies was suggested for many topics and included multicenter, cross-country collaborations, more use of the existing tissue bank and the initiation of a new equine asthma group. it is recognized that the ideas for research directions generated through this roundtable discussion at the end of a day workshop are subject to biases and influences relating to the interests, priorities and perceptions of workshop participants. however, by using and describing a systematic method of representing the ideas proposed, we have aimed at least to be transparent in our reporting of this. further, longer-term, international discussion and exchange of views will be facilitated by one of the key outcomes of this workshop, which was the development of the new equine asthma group. the aim of this group is to offer a platform of information for veterinary practitioners and horse owners as well as a resource for researchers to collaborate and exchange ideas on the understanding of ea. it was suggested that this group could lead some initiatives in line with the proposed areas of interest described above. there are plans for this group to develop some guidelines for the diagnosis and treatment of equine asthma, including for example the standardization of diagnostic methods, as mentioned above. development of an equine asthma group website and other communication tools are now underway as an internationally collaborative initiative. the havemeyer equine asthma workshop has paved the way for a better understanding of this many-faceted disease by bringing together researchers and clinicians to identify both the needs of the equine industry for effective treatments and at the same time focus researchers on the gaps in knowledge and understanding that will facilitate our ability to deliver on these needs. the participants made clear the requirement for more accessible, standardized diagnostics that will enable us to understand the underlying pathophysiology and identify specific phenotypes and endotypes and thus create more targeted treatments or management strategies. by creating an 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oxygen tension, mucus score and bronchoprovocation in horses with recurrent airway obstruction in a field setting physiological measurements and prevalence of lower airway diseases in trotters with dorsal displacement of the soft palate the occurrence of dynamic structural disorders in the pharynx and larynx, at rest and during exercise, in horses diagnosed with mild and moderate equine asthma (inflammatory airway disease) endobronchial ultrasound reliably quantifies airway smooth muscle remodeling in an equine asthma model radiographic scoring lacks predictive value in inflammatory airway disease a survey of horse owners in great britain regarding horses in their care. part : horse demographic characteristics and management a survey of horse owners in great britain regarding horses in their care. part : risk factors for recurrent airway obstruction analysis of risk factors for recurrent airway obstruction in north american horses: , cases ( - ) genetics of upper and lower airway diseases in the horse effects of n-butylscopolammonium bromide on lung function in horses with recurrent airway obstruction echocardiography in a horse with cor pulmonale from recurrent airway obstruction equine pulmonary disease: a case control study of referred cases. part : details of animals and of historical and clinical findings evaluation of coughing and nasal discharge as early indicators for an increased risk to develop equine recurrent airway obstruction (rao) coughing in thoroughbred racehorses: risk factors and tracheal endoscopic and cytological findings association of increased tracheal mucus accumulation with poor willingness to perform in show-jumpers and dressage horses metalloproteinases and their tissue inhibitors in comparison between different chronic pneumopathies in the horse effect of antigenic exposure on airway smooth muscle remodeling in an equine model of chronic asthma airway collagen and elastic fiber content correlates with lung function in equine heaves 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racing yards the microbiome and asthma evolution of the nasopharyngeal microbiota of beef cattle from weaning to days after arrival at a feedlot upper and lower respiratory tract microbiota in horses: bacterial communities associated with health and mild asthma (inflammatory airway disease) and effects of dexamethasone lung microbiome is influenced by the environment and asthmatic status in an equine model of asthma abcs of the lung microbiome role of viral infections in the development and exacerbation of asthma in children association between rhinovirus wheezing illness and the development of childhood asthma: a meta-analysis wheezing rhinovirus illnesses in early life predict asthma development in high-risk children respiratory syncytial virus in early life and risk of wheeze and allergy by age years immune mechanisms of respiratory viral infections in asthma a comparison of total, respirable, and real-time airborne particulate sampling in horse barns markers of respiratory inflammation in horses in relation to seasonal changes in air quality in a conventional racing stable herpesviruses in respiratory liquids of horses: putative implication in airway inflammation and association with cytological features multiple molecular detection of respiratory viruses and associated signs of airway inflammation in racehorses frequency of molecular detection of equine herpesvirus- in nasal secretions of horses with upper airway infection association between inflammatory airway disease of horses and exposure to respiratory viruses: a case control study longitudinal study of poor performance and subclinical respiratory viral activity in standardbred trotters surveillance programme for important equine infectious respiratory pathogens in the usa prevalence factors associated with equine herpesvirus type infection in equids with upper respiratory tract infection and/or acute onset of neurological signs from viral load of equine herpesviruses and in nasal swabs of actively racing standardbred trotters: temporal relationship of shedding to clinical findings and poor performance coughing, mucus accumulation, airway obstruction, and airway inflammation in control horses and horses affected with recurrent airway obstruction airway mucus in recurrent airway obstruction-short-term response to environmental challenge local airborne particulate concentration is associated with visible tracheal mucus in thoroughbred racehorses evaluation of nebulised hay dust suspensions (hds) for the diagnosis and investigation of heaves. : effects of inhaled hds on control and heaves horses exposure to inhalable dust, endotoxins, beta( -> )-glucans, and airborne microorganisms in horse stables environmental exposures and airway inflammation in young thoroughbred horses inhaled endotoxin and organic dust particulates have synergistic proinflammatory effects in equine heaves (organic dust-induced asthma) role of neutrophils in equine asthma recurrent airway obstruction in horsesan allergic inflammation: a review correlation between nuclear factor-κ b activity in bronchial brushing samples and lung dysfunction in an animal model of asthma antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone neutrophil extracellular traps in pulmonary diseases: too much of a good thing? front immunol neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones resolution of inflammation: an integrated view lung epithelium: barrier immunity to inhaled fungi and driver of fungal-associated allergic asthma development of a semiquantitative histological score for the diagnosis of heaves using endobronchial biopsy specimens in horses clara cell secretory protein is reduced in equine recurrent airway obstruction multiple secretoglobin a genes are differentially expressed in horses secretoglobin a and a a differentially regulate neutrophil reactive oxygen species production, phagocytosis and extracellular trap formation impaired response of the bronchial epithelium to inflammation characterizes severe equine asthma gene set enrichment analysis of the bronchial epithelium implicates contribution of cell cycle and tissue repair processes in equine asthma hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease bronchial epithelium as a target for innovative treatments in asthma evidence for transferrin allele as a host-level risk factor in naturally occurring equine respiratory disease: a preliminary study untersuchungen über die erblichkeit und das wesen des lungendampfes beim pferd [investigation into the nature and heritability of heaves in the horse sir frederick hobday memorial lecture. the genetic basis of some equine diseases a whole-genome scan for recurrent airway obstruction in warmblood sport horses indicates two positional candidate regions comparison of genomic and proteomic data in recurrent airway obstruction affected horses using ingenuity pathway analysis r a genome-wide association study for equine recurrent airway obstruction in european warmblood horses reveals a suggestive new quantitative trait locus on chromosome analysis of genomic copy number variation in equine recurrent airway obstruction (heaves) eqtl discovery and their association with severe equine asthma in european warmblood horses differential expression of serum micrornas supports cd + t cell differentiation into th /th cells in severe equine asthma impaired cell cycle regulation in a natural equine model of asthma multiple hypersensitivities including recurrent airway obstruction, insect bite hypersensitivity, and urticaria in warmblood horse populations insect bite hypersensitivity in horses is associated with airway hyperreactivity biomarkers and surrogate endpoints: preferred definitions and conceptual framework molecular biomarkers in idiopathic pulmonary fibrosis heaves, an asthmalike disease of horses markers of systemic inflammation in horses with heaves circulating immune complexes and markers of systemic inflammation in rao-affected horses acute phase proteins in racehorses with inflammatory airway disease serum surfactant protein d and haptoglobin as potential biomarkers for inflammatory airway disease in horses tests for circulating immune complexes circulating immune complexes in horses with severe equine asthma metalloproteinases and their inhibitors are influenced by inhalative glucocorticoid therapy in combination with environmental dust reduction in equine recurrent airway obstruction metabolomics of tracheal wash samples and exhaled breath condensates in healthy horses and horses affected by equine asthma is there anything else you would like to tell us" -methodological issues in the use of free-text comments from postal surveys working with words: exploring textual analysis in medical education research the authors are grateful to gene pranzo, president of the havemeyer foundation, for his support of the workshop on equine asthma. we are also thankful to boehringer ingelheim animal health, haygain, nortev, trudell medical international, and zoetis for their support of travel grants for participants and workshop activities. the workshop was made possible thanks to the generous support of the dorothy havemeyer foundation, boehringer ingelheim, haygain, nortev, trudell medical and zoetis. key: cord- -fk um nw authors: farver, carol f.; zander, dani s. title: molecular basis of pulmonary disease date: - - journal: molecular pathology doi: . /b - - - - . - sha: doc_id: cord_uid: fk um nw pulmonary pathology includes a large spectrum of both neoplastic and non-neoplastic diseases that affect the lung. many of these are a result of the unusual relationship of the lung with the outside world. every breath that a human takes brings the outside world into the body in the form of infectious agents, organic and inorganic particles, and noxious agents of all types. although the lung has many defense mechanisms to protect itself from these insults, these are not infallible; therefore, lung pathology arises. damage to the lung is particularly important given the role of the lung in the survival of the organism. any impairment of lung function has widespread effects throughout the body, since all organs depend on the lungs for the oxygen they need. pulmonary pathology catalogs the changes in the lung tissues and the mechanisms through which these occur. this chapter presents a review of lung pathology and the current state of knowledge about the pathogenesis of each disease. it suggests that a clear understanding of both morphology and mechanism is required for the development of new therapies and preventive measures. pulmonary pathology includes a large spectrum of both neoplastic and non-neoplastic diseases that affect the lung. many of these are a result of the unusual relationship of the lung with the outside world. every breath that a human takes brings the outside world into the body in the form of infectious agents, organic and inorganic particles, and noxious agents of all types. although the lung has many defense mechanisms to protect itself from these insults, these are not infallible and so lung pathology arises. damage to the lung is particularly important given the role of the lung in the survival of the organism. any impairment of lung function has widespread effects throughout the body, since all organs depend on the lungs for the oxygen they need. pulmonary pathology catalogs the changes in the lung tissues and the mechanisms through which these occur. what follows is a review of lung pathology and the current state of knowledge about the pathogenesis of each disease. we believe that a clear understanding of both morphology and mechanism is required for the development of new therapies and preventive measures. lung cancer is a major cause of morbidity and mortality throughout the world. the most recent estimates available from the surveillance, epidemiology, and end results (seer) program of the national cancer institute are that in over , people in the united states were diagnosed with cancer of the lung and bronchus, and over , will have died due to this disease [ ] . however, in the past decade incidence and mortality rates have begun to move in a more positive direction, particularly in men. overall, men show a decline in lung cancer incidence, while in women, although lung cancer rates grew from through , they stabilized from through [ ] . similarly, cancer death rates due to lung cancer have declined for men and have slowed for women. although, for women, lung cancer death rates have increased since , the rate of increase has slowed to . % annually from to [ ] . these trends parallel changes in the prevalence of tobacco smoking, the most important risk factor for development of lung cancer. given the tremendous societal and individual impacts of this disease, it is not surprising that the molecular biology of lung cancer is a major focus of investigation. elucidation of the molecular pathogenesis of these neoplasms has progressed significantly, offering insights into new, targeted therapies, and predictors of prognosis and therapeutic responsiveness. recognition of precursor lesions for some types of lung cancers has been facilitated by our expanded understanding of early molecular changes involved in carcinogenesis. the world health organization (who) classification scheme is the most widely used system for classification of these neoplasms (table . ) [ ] . although there are numerous histologic types and subtypes of lung cancers, most of the common malignant epithelial tumors can be grouped into the categories of nonsmall cell lung cancers (nsclcs) and small cell carcinomas (sclcs). nsclcs include adenocarcinomas (acs), squamous cell carcinomas (sqccs), large cell carcinomas, adenosquamous carcinomas, and sarcomatoid carcinomas. sclcs include cases of pure and combined small cell carcinoma. common pulmonary symptoms associated with these tumors include cough, shortness of breath, chest pain or tightness, and hemoptysis (coughing up blood). since some tumors cause airway obstruction, they predispose to pneumonia, which can be an important clue to the existence of a tumor in some patients. constitutional symptoms can include fever, weight loss, and malaise. some neoplasms will declare themselves with symptoms related to local invasion of adjacent structures such as chest wall, nerves, superior vena cava, esophagus, or heart. sclcs are known for early and widespread metastasis and are therefore particularly prone to being discovered through presentations as metastases in distant sites. some tumors are discovered due to pathophysiologic changes triggered by the release of soluble substances from tumor cells. endocrine syndromes due to elaboration of hormones are well recognized, and include cushing syndrome, syndrome of inappropriate antidiuretic hormone, hypercalcemia, carcinoid syndrome, gynecomastia, and others. hypercoagulability commonly occurs with lung cancers, leading to manifestations of venous thrombosis, nonbacterial thrombotic endocarditis, and disseminated intravascular coagulation. hematologic changes can include anemia, granulocytosis, eosinophilia, and other abnormalities. other paraneoplastic syndromes such as clubbing of the fingers, myasthenic syndromes, dermatomyositis/polymyositis, and transverse myelitis are noted in subsets of patients. when lung cancer is suspected, evaluation of the patient includes a thorough clinical, radiologic, and laboratory assessment, with collection of tissue or cytology samples to establish a pathologic diagnosis of malignancy and to classify the tumor type. fiberoptic bronchoscopy is often performed to collect samples for diagnosis. sample types can include transbronchial and endobronchial biopsies, bronchial brushings, bronchial washings, bronchoalveolar lavage samples, and transbronchial needle aspirates. submission of sputum samples for cytologic malignant epithelial tumors examination can provide a diagnosis in some cases, particularly for centrally located tumors such as sqcc and sclc. tumors arising in a peripheral location can also be sampled, in many cases, by fine needle aspiration or core needle biopsy performed under radiologic guidance. if a pleural effusion is present in combination with a lung parenchymal tumor, analysis of the pleural fluid cytology often allows one to establish a diagnosis. pleural biopsy, mediastinoscopy with biopsy, and wedge biopsy can also be performed, depending on the clinical and radiologic findings. for tumors with apparent distant metastasis, biopsy of the metastasis focus can both establish a pathologic diagnosis and determine the stage of the tumor. the prognosis of lung cancers is closely related to tumor stage. for nsclcs, the american joint commission on cancer tnm staging system is widely used (table . ) [ ] , and for sclcs, disease is classified as limited (restricted to one hemithorax) or extensive. overall, for lung cancers, the -year survival is . % for men and . % for women [ ] . an important factor leading to this relatively poor survival is the late stage at which many lung cancers are diagnosed. information from the seer database, from - , indicates that %, %, %, and % of patients were diagnosed with localized, regional, distant, or unstaged disease, respectively [ ] . the corresponding -year survival rates are . %, . %, . %, and . %, and year survival rates are . %, . %, . %, and . % [ ] . for patients with nsclcs, treatment depends on stage and comorbid conditions [ ] . surgical resection is the preferred approach to treatment of localized nsclcs, provided there is no medical contraindication to operative intervention. lobectomy or more extensive resection (depending on tumor extent) is usually recommended rather than lesser surgeries, unless other comorbid conditions preclude these procedures. tumor cm in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus t tumor with any of the following features of size or extent: > cm in greatest dimension, involves main bronchus ! cm distal to the carina, invades visceral pleura, associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung t tumor of any size that directly invades the chest wall, diaphragm, mediastinal pleura, parietal pericardium; or lies < cm distal to the carina but without involvement of the carina; or is associated with atelectasis or obstructive pneumonitis of the entire lung t tumor of any size that invades the mediastinum, heart, great vessels, trachea, esophagus, vertebral body, carina; or has separate tumor nodule(s) in same lobe; or is associated with a malignant pleural effusion. regional lymph nodes (n) nx regional lymph nodes cannot be assessed n no regional lymph node metastasis n metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, including intrapulmonary nodes involved by direct extension of the primary tumor n metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s) n metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene or supraclavicular lymph node(s). mx distant metastasis cannot be assessed m no distant metastasis m distant metastasis; includes separate tumor nodule(s) in a different lobe. occult t n m stage tis n m stage ia t n m stage ib t n m stage iia t n m stage iib t n m t n m stage iiia t n m t n m t n m t n m stage iiib any t n m t any intraoperative mediastinal lymph node sampling or dissection is also recommended for accurate pathologic staging and determination of therapy. subsets of patients also benefit from chemotherapy and radiotherapy. for more advanced nsclc and for sclc, chemotherapy and radiotherapy are the primary treatment modalities [ ] . rare patients with limited-stage sclcs can be considered for surgical resection with curative intent. development of lung cancer occurs with multiple, complex, stepwise genetic and epigenetic changes involving allelic losses, chromosomal instability and imbalance, mutations in tumor suppressor genes (tsgs) and dominant oncogenes, epigenetic gene silencing through promoter hypermethylation, and aberrant expression of genes participating in control of cell proliferation and apoptosis [ ] . there are similarities as well as type-specific differences in the molecular alterations between nsclcs and sclcs, and between sqccs and acs [ ] [ ] [ ] . oncogenes that play a part in the pathogenesis of lung cancer include myc, k-ras (predominantly acs), cyclin d , bcl , and erbb family genes such as egfr (epidermal growth factor receptor) (predominantly acs) and her /neu (predominantly acs) [ , ] . also, lung cancers often display abnormalities involving tsgs including tp , rb, p ink a , and new candidate tsgs on the short arm of chromosome (dutt , fhit, rasff a, fus- , bap- ) [ , ] . as research advances, these lists continue to grow, and as knowledge has expanded about the roles of these genes in carcinogenesis and tumor behavior, new targeted therapeutic agents have been designed to treat this disease ( figure . and table . ) [ ] . many other agents are under investigation. in cancers, chromosomal regions harboring tsgs and oncogenes are often deleted or amplified. allele loss involving loci in p - is a consistent feature of lung cancer pathogenesis [ , ] . wistuba et al. reported allelic losses of p, often multiple and discontinuous, in % of the lung cancers studied and in % of the precursor lesions [ ] . larger segments of allelic loss were noted in most sclcs ( %) and sqccs ( %) than in acs ( %) and preneoplastic/preinvasive lesions [ ] . there was allelic loss in the -kb p . deletion region in % of the lung cancers; % of the normal or reneoplastic/preinvasive lesions associated with lung cancers; and % of the normal, mildly abnormal, or preneoplastic/ preinvasive lesions found in smokers without lung cancer, but no loss was seen in the samples from people who had never smoked [ ] . p - deletions are also frequent and early events in the pathogenesis of lung carcinomas [ ] , and other common alterations include loh at q, q, q, and p [ ] . allelic losses that are more frequent in sqccs than acs include deletions at p (tp ), q (rb), p (p ink a ), p - , and several regions of p [ , , , ] . a recent study utilizing a bacterial artificial chromosome array to perform high-resolution whole genome profiling of sqcc and ac cell lines showed that regions of frequent amplification shared by both types of tumors included p; chromosome , q, q , q, and q; and common regions of deletion included p, q, p, p, q; chromosome ; and chromosome [ ] . however, acs appeared to have higher frequencies of deletion of chromosome ; p, q, q; and chromosome than sqccs, and possess small regions of amplification on chromosomes and not seen in sqccs. chromosome arms q and q were frequently deleted in ac but amplified in sqcc cell lines. both types of tumors showed deletion of chromosome arm p, but it was more frequent in the sqcc cell lines, while amplification of chromosome p was more frequent in acs. amplification of chromosome q was common to both types of tumors but showed frequent alteration at q - q in the sqcc lines and at q in the ac lines. inactivation of recessive oncogenes is believed to occur through a two-stage process. it has been suggested that the first allelic inactivation occurs, often via a point mutation, and the second allele is later inactivated by a chromosomal deletion, translocation or other alteration such as methylation of the gene promoter region [ ] . inactivating mutations in the tsg tp , which encodes the p protein, are the most frequent mutations in lung cancers. these mutations are found in up to % of nsclcs and over % of sclcs, and are largely attributable to direct dna damage from cigarette smoke carcinogens [ ] . tp mutational patterns show a prevalence of g to t transversions in % of smokers' lung cancers versus only % of lung cancers in nonsmokers [ ] . p protein is a transcription factor and a key regulator of cell cycle progression; cellular signals induced by dna damage, oncogene expression, or other stimuli trigger p dependent responses including initiating cell cycle arrest, apoptosis, differentiation, and dna repair [ ] . loss of p function in tumor cells can result in inappropriate progression through the dysregulated cell cycle checkpoints and permits the inappropriate survival of genetically damaged cells [ ] . the p ink a -cyclin d -cdk -rb pathway, which plays a central role in controlling the g to s phase transition of the cell cycle, is another important tumor suppressor pathway that is often disrupted in lung cancers. it interfaces with the p pathway through p arf and p waf/cip . thirty percent to % of nsclcs contain mutations of p ink a , including homozygous deletion or point mutations and epigenetic alterations, leading to p ink a inactivation [ ] . almost % of sclcs and smaller numbers of nsclcs, on the other hand, display loss of rb expression [ ] , and mutational mechanisms usually responsible include deletion, nonsense mutations, and splicing abnormalities that lead to truncated rb protein [ ] . p ink a leads to hypophosphorylation of the rb protein, which causes arrest of cells in the g phase. the active, hypophosphorylated form of rb regulates other cellular proteins including the transcription factors e f , e f , and e f , which are essential for progression through the g /s phase transition. loss of p ink a protein or increased complexes of cyclin d-cdk - or cyclin e-cdk lead to hyperphosphorylation of rb with resultant evasion of cell cycle arrest and progression into s phase [ , ] . cell cycle progression is inhibited by p waf/cip through its inhibition of the cyclin complexes. the %- % of nsclcs lacking detectable alterations in p ink a and rb may have abnormalities of cyclin d and cdk , which cause inactivation of the rb pathway [ ] . figure . provides an overview of the p and retinoblastoma (rb) pathways, showing the complex interactions between the components [ ] . epigenetic alterations (hypermethylation of the cpg island) of tsgs are also frequent occurrences during pulmonary carcinogenesis, and methylation profiles of nsclcs show relationships to smoke exposure, histologic type, and geography. methylation rates of p ink a and apc and the mean methylation index (mi) (a reflection of the overall methylation status) in current or former smokers were significantly higher than in never smokers; the mean mi of tumors was highest in current smokers; methylation rates of apc, cdh , and rarbeta were significantly higher in acs than in sqccs; methylation rates of mgmt and gstp in cases from the united states and australia significantly exceeded those from japanese and taiwanese cases; and no significant gender-related differences in methylation patterns were found [ ] . proto-oncogene activation and growth factor signaling are important in pulmonary carcinogenesis. the tyrosine kinase epidermal growth factor receptor (egfr) is frequently mutated in nsclcs, particularly in acs, and the mutational status is important in determining response to tyrosine kinase inhibitors. a related pathway, the phosphoinositide -kinase (pi k)/akt/mammalian target of rapamycin (mtor) pathway, is frequently deregulated in pulmonary carcinogenesis. as reviewed by marinov et al., this pathway has been reported to mediate the effects of several tyrosine kinase receptors, including egfr, c-met, c-kit, and igf-ir, on proliferation and survival in nsclc and sclc [ ] . clinical trials are ongoing, investigating the efficacy of the mtor inhibitor rapamycin and its analogues on lung cancer [ ] . her /neu is another related receptor tyrosine kinase that is upregulated in approximately %- % of nsclcs [ , ] , but unlike the situation with her /neu-positive breast cancers, treatment with anti-her /neu antibody (trastuzumab) does not seem to yield comparable benefits for nsclc when used alone or in combination with chemotherapy [ , ] . point mutations of ras family proto-oncogenes (most often at k-ras codons , , or ) are detected in %- % of lung acs and %- % of all nsclcs [ ] . although farnesyl transferase inhibitors prevent ras signaling, these agents have not shown significant activity as single-agent therapy in untreated nsclc or relapsed sclc [ ] . myc family genes (myc, mycn, and mycl), which play roles in cell cycle regulation, proliferation, and dna synthesis, are more frequently activated in sclcs than in nsclcs, either by gene amplification or by transcriptional dysregulation [ ] . vascular endothelial growth factor (vegf) is a homodimeric glycoprotein that is overexpressed in many lung cancers and directly stimulates endothelial cell proliferation, promotes endothelial cell survival in newly formed vessels, and induces proteases involved in the degradation of the extracellular matrix needed for endothelial cell migration [ ] . its angiogenic effects are mediated by three receptors: vegfr- , vegfr- , and vegfr- ; ligand binding leads to tyrosine kinase activation and activation of the signaling pathways required for angiogenesis [ ] . monoclonal antibodies to vegf (bevacizumab) and tyrosine kinase inhibitors to vegfrs have been developed and show promise for treatment of nsclc. a phase iii trial of bevacizumab showed significantly improved overall and progression-free survival when this agent was used in combination with standard first-line chemotherapy in patients with advanced nsclc, and several smallmolecule vegfr tyrosine kinase inhibitors have yielded favorable results in phase i and ii trials in nsclc [ ] . micrornas are a recently discovered class of nonprotein-coding, endogenous, small rnas which regulate gene expression by translational repression, mrna cleavage, and mrna decay initiated by mirna-guided rapid deadenylation [ ] . some micrornas such as let- have been suggested to play roles in carcinogenesis by functioning as oncogenes or tumor suppressors, negatively regulating tsgs and/or genes that control cell differentiation or apoptosis [ ] . investigations of the therapeutic potential of micrornas are also under way. in the version of the who classification scheme, ac is defined as "a malignant epithelial tumour with glandular differentiation or mucin production, showing acinar, papillary, bronchioloalveolar or solid with mucin growth patterns or a mixture of these patterns" [ ] . ac has become the most frequent histologic type of lung cancer in parts of the world. it occurs primarily in smokers, but represents the most common type of lung cancer in people who have never smoked and in women. a small subset of these tumors arise in patients with localized scars or diffuse fibrosing lung diseases such as asbestosis and interstitial pneumonia associated with scleroderma [ ] . these neoplasms usually arise in the periphery of the lung, and are more likely to invade the pleura and chest wall than other histologic types of lung cancers. radiologic studies can show one or more nodules, ground-glass opacities, or mixed solid and ground-glass lesions. on gross examination, the neoplasms are often solitary gray-white nodules or masses, sometimes with necrosis or cavitation, which pucker the overlying pleura. mucin-producing tumors can have a glistening, gelatinous appearance. other presentations include a pattern of consolidation resembling pneumonia (usually bronchioloalveolar carcinoma) ( figure . ), multiple nodules, diffuse interstitial widening due to lymphangitic spread, endobronchial lesions with submucosal infiltration, and diffuse visceral pleural infiltration and thickening resembling mesothelioma. common histologic patterns displayed by acs include acinar ( figure chapter molecular basis of pulmonary disease mixtures of these patterns are very frequent. less common histologic subtypes include fetal ac, mucinous (colloid) ac, mucinous cystadenocarcinoma, signet ring ac, and clear cell ac [ ] . acs usually exhibit differentiation toward clara cells or type ii pneumocytes or, less often, goblet cells. they manifest a range of differentiation extending from very well-differentiated tumors with extensive gland formation and little cytoatypia, to poorly differentiated, solid tumors that cannot be categorized as acs unless one orders a mucin stain (figure . ) . however, most examples include readily identifiable glands. invasiveness is reflected by the presence of neoplastic glands that infiltrate through stroma or pleura, stimulating a fibroblastic (desmoplastic) response ( figure . ) , or by cells in the lumens of blood vessels or lymphatics. in recent years, atypical adenomatous hyperplasia (aah) has been recognized as a precursor lesion for peripheral pulmonary acs. this lesion is defined as "a localized proliferation of mild to moderately atypical cells lining involved alveoli and, sometimes, respiratory bronchioles, resulting in focal lesions in peripheral part iv molecular pathology of human disease alveolated lung, usually less than mm in diameter and generally in the absence of underlying interstitial inflammation and fibrosis" (figure . ) [ ] . aah exists on a histologic continuum with bronchioloalveolar carcinoma (bac), which is defined as an in situ (noninvasive) form of ac, in which the neoplastic cells grow along alveolar septa (lepidic growth) without invasion of stroma or vasculature ( figure . , figure . ) [ ] . most bacs exceed cm in diameter and consist of cells with greater degrees of cytoatypia than aah. although aah is found in approximately % of patients without lung cancer at autopsy [ ] , it has been reported in %- % of lung resection specimens with all types of primary lung cancer and %- % of lung resection specimens with ac [ ] . the progenitor cell for bac and aah is believed to be an epithelial cell located at the junction between the terminal bronchiole and alveolus, termed the bronchioalveolar stem cell [ ] . a recently published large-scale study of primary lung acs, using dense single nucleotide polymorphism arrays, described significantly recurrent copy-number alterations in these tumors (table . ) [ ] . twenty-six of autosomal chromosome arms showed consistent large-scale copy-number gain or loss, and recurrent focal events, including amplifications and homozygous deletions, were found. although some of the alterations involved regions known to harbor a proto-oncogene or tsg, these genes remain to be identified in some of the other regions affected. amplification of chromosome q . was the most common event noted, found in % of samples. this region includes nkx - , which encodes a lineage-specific transcription factor (thyroid transcription factor- [ttf- ]) that activates transcription of target genes including the surfactant proteins, and may be an important proto-oncogene involved in a significant fraction of lung acs. immunohistochemical staining for ttf- can be performed to detect expression of this factor in most lung adenocarcinomas, aiding in the determination of the lung as the site of origin of the tumor (figure . ). additional work using small interfering rna (sirna)mediated knockdown of this gene in lung cancer cell lines with amplification led to reductions in tumor cell proliferation, through both decreased cell cycle progression and increased apoptosis, suggesting that gene amplification and overexpression contribute to lung cancer cell proliferation rates and survival [ ] . egfr and k-ras mutations are mutually exclusive mutational events in ac of the lung, which suggests the existence of two independent oncogenic pathways [ , ] . egfr is a receptor tyrosine kinase whose activation by ligand binding leads to activation of cell signaling pathways such as ras/mitogen-activated protein kinase (mapk) and phosphatidylinositol- -kinase, which in turn propagates signals for proliferation, blocking of apoptosis, differentiation, motility, invasion, and adhesion [ ] . tumor-acquired mutations in the tyrosine kinase domain of egfr, often associated with gene amplification, have been found in approximately %- % of nsclcs in the united states, and are associated with ac histology, never-smoker status, east asian ethnicity, and female gender [ , , ] . egfr mutations are frequently in-frame deletions in exon , single missense mutations in exon , or in-frame duplications/insertions in exon , and occasional missense mutations and double mutations can also be detected [ , ] . egfr mutation has an inverse correlation with methylation of the p ink a gene and sparc (secreted protein acidic and rich in cysteine), an extracellular ca þ-binding glycoprotein associated with the regulation of cell adhesion and growth [ ] . egfr status is an important predictor of response to egfr kinase inhibitors: patients with egfr mutations are most likely to have a significant response to egfr tyrosine kinase inhibitor therapy, and egfr amplification and protein overexpression have been reported to correlate with survival after egfr tyrosine kinase inhibitor therapy [ , ] . k-ras is a member of the ras family of proteins, which function as signal transducers between cell membrane-based growth factor signaling and the mapk pathways [ ] . k-ras mutations are associated with smoking, male gender, and poorly differentiated tumors [ ] . her (also known as egfr or erbb ), a member of the egfr family of receptor tyrosine kinases, is mutated in less than % of nsclc, and does not occur in tumors with egfr or k-ras mutation [ ] . the her mutations are in-frame insertions in exon and are significantly more frequent in acs ( . %), never smokers ( . %), asian ethnicity ( . %), and women ( . %), similar to egfr mutations [ ] . alterations in dna methylation appear to be important epigenetic changes in cancer, contributing to chromosomal instability through global hypomethylation, and aberrant gene expression through alterations in the methylation levels at promoter cpg islands [ ] . this lesion, which has been defined as a precursor lesion for peripheral pulmonary adenocarcinomas, consists of a wellcircumscribed nodule measuring several millimeters in diameter, in which alveolar septa are lined by mildly moderate atypical cells. epigenetic differences exist between egfr-mediated and k-ras-mediated tumorigenesis, and may interact with the genetic changes. a recent study showed that the probability of having egfr mutation was significantly lower among those with p ink a and cdh methylation than in those without, and the methylation index was significantly lower in egfr mutant cases than in wild-type. in contrast, k-ras mutation was significantly higher in p ink a methylated cases than in unmethylated cases, and the methylation index was higher in k-ras mutant cases than in wild-type [ ] . sqcc is defined as "a malignant epithelial tumour showing keratinization and/or intercellular bridges that arises from bronchial epithelium," in the who classification scheme [ ] . it is a common histologic type of nsclc that is closely linked to cigarette smoking. in most patients, this tumor arises in a mainstem, lobar, or segmental bronchus, producing a central mass on imaging known tumor suppressor genes and proto-oncogenes defined as found in either cosmic , cgp census , or other evidence; if there is more than one known proto-oncogene in the region, only one is listed (priority for listing is, in order: known lung adenocarcinoma mutation; known lung cancer mutation; other known mutation (by cosmic frequency); listing in cgp census). @myc is near, but not within, the peak region. ksingle gene deletions previously seen, this study provides new mutations as well. part iv molecular pathology of human disease studies. many of these tumors have an endobronchial component that can cause airway obstruction, leading to postobstructive pneumonia, atelectasis, or bronchiectasis. not infrequently, it is the pneumonia that prompts evaluation of the patient and leads to discovery of the tumor. less often, sqccs develop in the periphery of the lung. gross examination reveals a tan or gray mass that usually arises in a large bronchus and often includes an endobronchial component (figure . , figure . ). partial or complete airway obstruction can be associated with changes of pneumonia, bronchitis, abscess, bronchiectasis, or atelectasis. necrosis and cavitation are very common in these tumors. involvement of hilar lymph nodes by tan-gray tumor can be visible in some resected specimens. microscopically, the key features of this tumor are its keratinization, sometimes with formation of keratin pearls, and intercellular bridges ( figure . ). as is true of acs, the degree of differentiation of this tumor varies from very well differentiated cases, in which there are abundant keratinization and intercellular bridges and little cytoatypia, to very poorly differentiated cases, in which keratinization and intercellular bridges can be quite inconspicuous and the tumor consists of sheets of large atypical cells with marked cytoatypia and frequent mitoses. however, most cases fall more toward the middle of the spectrum. invasiveness is reflected by the presence of irregular nests and sheets of cells that infiltrate through tissues, stimulating a fibroblastic response, or by cells inside vascular or lymphatic spaces. invasive sqccs are often accompanied by sqcc in situ and dysplasia, their precursor lesions. these lesions arise in the bronchi and may be contiguous with the invasive tumor or exist as one or more separate foci. these precursor lesions can also be observed without coexisting invasive carcinoma. like sqcc, tobacco smoking is the main predisposing factor for sqcc in situ and dysplasia. unlike invasive sqcc, however, these lesions are not invasive-they do not extend through the basement membrane of the bronchial epithelium. grossly, they may be invisible or appear as flat, tan or red discolorations of the bronchial mucosa, or tan wart-like excrescences. microscopically, these lesions encompass a chapter molecular basis of pulmonary disease range of squamous changes that include alterations in the thickness of the bronchial epithelium, the maturational progress of squamous differentiation, cell size, and nuclear characteristics ( figure . , figure . ) [ , ] . as dysplasia increases from mild to moderate to severe, the epithelium thickens, and maturation is increasingly impaired. the basilar zone expands with epithelial cell crowding, the intermediate zone shrinks, and there is reduced flattening of the superficial squamous cells. cell size, pleomorphism, and anisocytosis usually increase, and there is coarsening of the chromatin and appearance of nucleoli, nuclear angulations, and folding. in carcinoma in situ, although the epithelium may or may not be thickened and the cell size may be small, medium, or large, there is minimal or no maturation from the base to the superficial aspect, and the atypical nuclear features are present throughout the entire thickness of the epithelium. mitoses appear in the lower third (mild or moderate dysplasia), lower two-thirds (severe dysplasia), or throughout the full thickness of the epithelium (carcinoma in situ). basal cells in the bronchial epithelium are believed to represent the progenitor cells for invasive sqcc, and the sequence of events leading to sqcc is believed to include basal cell hyperplasia, squamous metaplasia, squamous dysplasia, carcinoma in situ, and invasive sqcc (figure . ) [ , [ ] [ ] [ ] . regression of lesions preceding invasive sqcc can occur, particularly the earlier lesions [ ] . however, severe dysplasia and carcinoma in situ are associated with a significantly increased probability of developing invasive sqcc in patients followed over time with surveillance bronchoscopy [ ] . wistuba and colleagues evaluated sqccs and precursor lesions for loss of heterozygosity (loh) at chromosomal regions ( p , p . , p . - . , p , p - , p , q , p , q rb, and p tp ) part iv molecular pathology of human disease frequently deleted in lung cancer and found multiple, sequentially occurring allele-specific molecular changes in separate, apparently clonally independent foci, early in the pathogenesis of sqccs of the lung, suggesting a field cancerization effect [ , ] . they observed clones of cells with allelic loss at one or more regions in % percent of histologically normal epithelium and % of specimens with hyperplasia or metaplasia; increasing frequency of loh within clones with increasing histopathologic lesional severity; the most frequent and earliest regions of allelic loss at p , p - , p , and p ; increasing size of the p deletions with progressive histologic changes; and tp allelic loss in many histologically advanced lesions (dysplasia and cis) [ ] . an overview of the sequential molecular events leading to invasive sqcc is shown in figure . [ ] . large cell carcinoma is an undifferentiated nsclc without light microscopic evidence of squamous or glandular differentiation, although squamous or glandular features may be detectable by ultrastructural examination (figure . ) [ ] . histologic subtypes of large cell carcinoma include large cell neuroendocrine carcinoma (lcnec), combined lcnec, basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, and large cell carcinoma with rhabdoid phenotype [ ] . clinical signs and symptoms resemble those of other types of nsclc. most tumors develop as peripheral lung masses, except for basaloid carcinomas, which usually form centrally located masses. histologically, large cell carcinomas consist of sheets and nests of large cells with vesicular nuclei, prominent nucleoli, and moderate or abundant amounts of cytoplasm. lcnecs demonstrate neuroendocrine architectural features and immunohistochemical or ultrastructural evidence of neuroendocrine differentiation. basaloid carcinomas display nests of small, monomorphic, rounded or fusiform tumor cells with little cytoplasm, numerous mitoses, comedo-type necrosis, and hyaline or mucoid stromal degeneration. clear cell carcinoma consists of large tumor cells with clear cytoplasm. precursor lesions are not currently recognized for any of the subtypes of large cell carcinoma. however, basaloid carcinoma is associated with squamous dysplasia in about one-third of cases [ ] . large cell carcinomas are poorly differentiated carcinomas that can demonstrate features of ac (most frequent), sqcc, or neuroendocrine differentiation when examined by immunohistochemistry, electron microscopy, or molecular methods [ ] . these tumors often demonstrate losses of p, q, p, q, q, and p, and gains of q and p, more closely resembling acs than other histologic types of lung cancer [ ] . common molecular abnormalities include tp mutation, c-myc amplification, and p promoter hypermethylation, while k-ras mutation is less common [ ] . egfr tyrosine kinase domain mutation is not characteristic of large cell carcinomas, and egfrviii (deletion mutations in the extracellular domain of egfr) is uncommon [ , ] . the major categories of pulmonary neuroendocrine (ne) neoplasms include small cell carcinoma (sclc), large cell neuroendocrine carcinoma (lcnec), typical carcinoid, and atypical carcinoid. sclc and lcnec are high-grade carcinomas, typical carcinoid is a low-grade malignant neoplasm, and atypical carcinoid occupies an intermediate position in the spectrum of biologic aggressiveness. in one large series, the -year and -year survival rates for typical carcinoid were % and %, % and % for atypical carcinoid, % and % for lcnec, and % and % for sclc, respectively [ ] . by light microscopy, these tumors display ne architectural features including organoid nesting, a trabecular arrangement, rosette formation, and palisading. these patterns are more prominent in carcinoids than in lcnecs and may or may not be visible in individual sclcs. typical carcinoids contain fewer than mitoses per mm ( hpf) and lack necrosis ( figure . ), while atypical carcinoids show - mitoses per mm ( hpf) or necrosis, which is often punctate [ ] . sclc consists of small, undifferentiated tumor cells with scant cytoplasm and finely granular chromatin and absent or inconspicuous nucleoli ( figure . ). nuclear molding is characteristic, necrosis is common, and the mitotic rate is typically high, with a mean of over mitoses per mm [ ] . combined differences also exist in the characteristics of patients with carcinoids, as compared to patients with sclc and lcnec. patients with carcinoids are typically younger and less likely to smoke than those with sclcs and lcnecs, the vast majority of whom have a current or previous history of tobacco smoking [ , ] . rare patients with carcinoids have the multiple endocrine neoplasia (men ) syndrome, an association that is not seen with sclcs and lcnecs. in addition, an association with diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (dipnech) has been noted for carcinoids but not for sclcs and lcnecs, leading to classification of dipnech as a preinvasive lesion in the most recent version of the who classification scheme [ ] . dipnech is a diffuse proliferation of single cells, small nodules (ne bodies), and linear proliferations of pulmonary ne cells that may reside in the bronchial and/or bronchiolar epithelia ( figure . ) , and may be accompanied by extraluminal proliferations part iv molecular pathology of human disease (tumorlets and carcinoids) [ ] . however, morphologically identifiable precursor lesions for sclc and lcnec have not been established. molecular markers of pulmonary ne tumors include chromogranin a, synaptophysin (figure . ) , and n-cam (cd ). these markers are expressed by all categories of ne tumors, with higher frequencies observed in the carcinoids and atypical carcinoids than in small cell and large cell neuroendocrine carcinomas. gastrin-releasing peptide, calcitonin, other peptide hormones, the insulinoma-associated (insm ) promotor and the human achaete-scute homolog- (hash ) gene have also been reported as overexpressed by these tumors [ , ] . thyroid transcription factor- (ttf- ) is expressed by %- % of sclcs, %- % of lcnecs, and %- % of carcinoids [ ] [ ] [ ] [ ] . sclcs [ ] [ ] [ ] [ ] [ ] [ ] . more than % of sclcs and sqccs demonstrate large, often discontinuous segments of allelic loss on chromosome p, in areas encompassing multiple candidate tumor suppressor genes, including some of those listed previously [ , ] . atypical carcinoids show a higher frequency of loh at p, q, p , and p than typical carcinoids, but not as high as the high-grade ne tumors [ ] . some typical and atypical carcinoids possess mutations of the multiple endocrine neoplasia (men ) gene on chromosome q or loh at this locus [ ] , while these abnormalities occur with lower frequencies in sclcs and lcnecs, supporting separate pathways of tumorigenesis [ ] . men encodes for the nuclear protein menin, which is believed to play several roles in tumorigenesis by linking transcription factor function to histone-modification pathways, in part through interacting with the activator-protein- family transcription factor jund, modifying it from an oncoprotein into a tumor suppressor protein [ ] . oncogenes frequently amplified in sclcs include myc ( q ), mycn ( p ), and mycl ( p ), and additional amplified genes that represent candidate oncogenes include the antiapoptotic genes tnfrsf ( p ), dad ( q ), bcl l ( q ), and bcl l ( q ) [ ] . the myc proteins are transcription factors that are important in cell cycle regulation, proliferation, and dna synthesis, and can induce p arf , leading to apoptosis through p if cellular conditions do not favor proliferation [ ] . tsgs are inactivated in the majority of sclcs. eighty percent to % of sclcs demonstrate tp mutations, as compared to more than % of nsclcs, fewer atypical carcinoids, and virtually no typically carcinoids [ , ] . most of the tp mutations in sclcs are missense point mutations that result in a stabilized p mutant protein which can be easily detected by immunohistochemistry [ ] . p protein overexpression occurs frequently in high-grade ne carcinomas, but is unusual in typical carcinoids and intermediate in atypical carcinoids [ , ] . dysregulation of p produces downstream effects on bcl- and bax. antiapoptotic bcl- predominates over proapoptotic bax in the high-grade ne carcinomas, while the reverse is true for carcinoids [ ] . lcnecs resemble sclcs in their high rates of tp mutation and predominance of bcl- expression over bax expression [ ] . alterations compromising the p ink a /cyclin d /rb pathway of g arrest are consistent in high-grade pulmonary ne carcinomas ( %), primarily through loss of rb protein, but are less frequent in atypical carcinoids ( %) and are uncommon in typical carcinoids [ ] . mutations in the rb gene exist in many sclcs, with associated loss of function of the gene product [ , , ] . in another study, % of the ne carcinomas (excluding carcinoids) versus % of the non-ne carcinomas exhibited loh and loss of rb-protein expression [ ] . the hypophosphorylated form of rb protein functions as a cell cycle regulator for g arrest; cyclin d overexpression and p ink a loss produce persistent hyperphosphorylation of rb with consequent evasion of cell cycle arrest [ ] . recent data also suggest that in sclcs, overexpression of mdm (a transcriptional target of p ) or p arf loss leads to evasion of cell cycle arrest through the p and rb pathway ( figure . ) [ ] . the transcription factor e f- appears to play a role in cellular proliferation by activating genes required for s phase entry. e f- product is overexpressed in % of sclcs and % of lcnecs, and is significantly associated with a high ki index and bcl- :bax ratio > [ ] . a mediator of the proteasomal degradation of e f- , the s phase kinase-associated protein (skp ) f-box protein accumulates in high-grade ne carcinomas ( %), and its overexpression has been associated with advanced stage and nodal metastasis in pulmonary ne tumors [ ] . in the high-grade ne tumors, skp appears to interact with e f- and stimulate its transcriptional activity toward the cyclin e promoter [ , ] . telomeres play an important role in the protection of chromosomes against degradation. telomerases, the enzymes that synthesize telomeric dna strands, serve to counterbalance losses of dna during cell divisions. high telomerase activity has been noted in over % of sclcs and lcnecs [ ] [ ] [ ] versus % or fewer typical carcinoids [ , ] . expression of human telomerase mrna component (hterc) and human telomerase reverse transcriptase (htert) mrna were reported, respectively, in % and % of typical carcinoids; and in % and % of atypical carcinoids, lcnecs and sclcs, and telomere length alterations in lcnecs and sclcs were greater than in typical carcinoids [ ] . aberrant methylation of cytosine-guanine (cpg) islands in promoter regions of malignant cells is an important mechanism for silencing of tsgs (epigenetic inactivation). methylation of dna involves the transfer of a methyl group, by a dna methyltransferase, to the cytosine of a cpg dinucleotide [ ] . rassf a is a potential tsg that undergoes epigenetic inactivation in virtually all sclcs and a majority of nsclcs through hypermethylation of its promoter region [ , ] . ne tumors have lower frequencies of methylation of p , apc, and cdh (h-cadherin) than nsclcs [ ] . sclcs have higher frequencies of methylation of rassf a, cdh (e-cadherin), and rarb than carcinoids [ ] . promoter methylation of casp , which encodes the apoptosis-inducing cysteine protease caspase , was also found in % of sclcs, % of carcinoids, and no nsclcs, suggesting that casp may function as a tsg in ne lung tumors [ ] . although histologically defined precursors for sclc are lacking, a higher incidence of genetic abnormalities is found in the normal or hyperplasic airway epithelium of patients with sclc than nsclc [ ] . by extension, it has been suggested that sclc may arise directly from histologically normal or mildly abnormal epithelium, rather than evolving through a sequence of recognizable histologic intermediary changes [ ] . relatively little is known about molecular abnormalities in precursors of carcinoids. although carcinoids have been viewed as arising from tumorlets, q (int- ) allelic imbalance is significantly more common in carcinoids ( %) than in tumorlets ( %), and may represent an early event in carcinoid tumor formation [ ] . the int- gene lies in close proximity to men , a tumor suppressor gene frequently mutated in ne tumors [ ] . the molecular pathology of dipnech remains to be elucidated. mesenchymal neoplasms included in the who classification scheme (table . ) encompass a spectrum of malignant and benign proliferations that show differentiation along multiple lineages. overall, these tumors are much less common in the lung than are epithelial neoplasms. information about molecular pathogenesis has emerged for some of the mesenchymal neoplasms. pulmonary inflammatory myofibroblastic tumor (imt) is a lesion composed of myofibroblastic cells, collagen, and inflammatory cells that primarily occurs in individuals less than years of age, and is the most common endobronchial mesenchymal lesion in childhood ( figure . ) [ ] . synovial sarcoma is usually a soft tissue malignancy, but uncommonly arises in the pleura or the lung and often takes an aggressive course [ ] . pulmonary hamartomas are benign neoplasms consisting of mixtures of cartilage, fat, connective tissue, and smooth muscle, which present as coin lesions on chest radiographs and are excised in order to rule out a malignancy ( figure . ). many imts demonstrate clonal abnormalities with rearrangements of chromosome p and the anaplastic lymphoma kinase (alk) gene [ ] . the rearrangements involve fusion of tropomyosin (tpm) n-terminal coiled-coil domains to the alk c-terminal kinase domain, producing two alk fusion genes, tpm -alk and tpm -alk, which encode oncoproteins with constitutive kinase activity [ ] . like their soft tissue counterparts, more than % of pulmonary and pleural synovial sarcomas demonstrate a chromosomal translocation t(x; ) (syt-ssx) [ , ] . detection of this translocation can be very helpful for confirming the diagnosis of synovial sarcoma in this unusual location. most pulmonary hamartomas show abnormalities of chromosomal bands p , q - , or other regions [ ] , corresponding to mutations of high-mobility group (hmg) proteins, a family of nonhistone chromatin-associated proteins that serve an important role in regulating chromatin architecture and gene expression [ ] . malignant mesothelioma (mm) is an uncommon, aggressive tumor arising from mesothelial cells on serosal surfaces, primarily the pleura and peritoneum, and less often the pericardium or tunica vaginalis. the most important risk factor for mm is exposure to the subset of asbestos fibers known as amphiboles (crocidolite and amosite) [ ] . the incidence of this tumor in the united states peaked in the early to mid- s, and appears to be declining, likely related to decreases in the use of amphiboles since their peak period of importation in the s [ ] . these tumors are characterized by long latency periods between asbestos exposure and clinical presentation of the tumor, with a mean of - years [ ] . radiation, a nonasbestos fiber known as erionite, and potentially other processes associated with pleural scarring have also been implicated in the causation of smaller numbers of cases of malignant mesothelioma [ ] , and a role for simian virus (sv ) in the genesis of this tumor has been suggested by some, but remains controversial [ , ] . pleural mm most commonly arises in males over the age of . presenting features typically include a hemorrhagic pleural effusion associated with shortness of breath and chest wall pain. weight loss and malaise are common. by the time the tumor is discovered, patients usually have extensive involvement of the pleural surfaces. with progression, the tumor typically invades the lung, chest wall, and diaphragm. lymph node metastasis can cause superior vena caval obstruction, and cardiac tamponade, subcutaneous nodules, and contralateral lung involvement can also occur. from the time of diagnosis, the median survival is months [ ] . treatment may include surgery, chemotherapy, radiotherapy, immunotherapy, or other treatments, often in combination [ ] . the intent of surgery is usually palliative. whether extrapleural pneumonectomy with chemotherapy and radiotherapy can lead to cure is unclear [ ] . new agents are currently under investigation for their potential to improve the life expectancy and quality of life in patients with this aggressive malignancy. gross pathologic features of mm include pleural nodules which grow and coalesce to fill the pleural cavity and form a thick rind around the lung. a firm tan appearance is common, and occasionally the tumor can have a gelatinous consistency (figure . ). extension along the interlobar fissures and invasion into the adjacent lung, diaphragm, and chest wall are characteristic. further spread can occur into the pericardial cavity and around other mediastinal structures, and distant metastases can also develop. histologically, mm manifests a wide variety of histologic patterns. the major histologic categories include epithelioid mesothelioma, sarcomatoid mesothelioma, desmoplastic mesothelioma, and biphasic mesothelioma [ ] . epithelioid mesothelioma consists of round, ovoid, or polygonal cells with eosinophilic cytoplasm and nuclei that are usually round with little cytoatypia (figure . ). these cells most often form sheets, tubulopapillary structures, or gland-like arrangements, and some tumors can have a myxoid appearance due to production of large amounts of hyaluronate. sarcomatoid mesothelioma is composed of malignant-appearing spindle cells occasionally accompanied by mature sarcomatous components (osteosarcoma, chondrosarcoma, others). desmoplastic mesothelioma can be a diagnostic challenge due to its frequently bland appearance and resemblance to organizing pleuritis. it consists of variably atypical spindle cells in a dense collagenous matrix ( figure . ). helpful features for separating figure . malignant mesothelioma. the tan/white tumor involves the entire pleura surrounding and compressing the underlying parenchyma, which appears congested but relatively unremarkable. chapter molecular basis of pulmonary disease this tumor from organizing pleuritis include invasion of chest wall muscle or adipose tissue and necrosis. biphasic mesotheliomas include both epithelioid and sarcomatoid elements, each comprising at least % of the tumor [ ] . pathologic diagnosis of mm has been greatly assisted by the expanded availability of antibodies for use in immunohistochemistry [ ] . mesothelial differentiation can be supported by immunoreactivity with cytokeratin / , calretinin ( figure . ), hbme- , d - , and other antibodies. histologic distinction of epithelioid mesotheliomas from metastatic acs is a common need in practice, and a panel approach using calretinin and cytokeratin / , with other antibodies reactive with acs (cea, moc- , ber-ep , leu m , b . , and others) will usually be successful. electron microscopy can also be helpful in difficult cases by demonstrating long thin microvilli in many mms with an epithelioid component. pan-cytokeratin staining is helpful for supporting a diagnosis of sarcomatoid or desmoplastic mm as opposed to sarcoma, since most (but not all) sarcomas will not stain for pan-cytokeratin. other mesothelial and mesenchymal markers can also be useful for assisting in the differentiation of mm from histologically similar sarcomas. precursor lesions for mm have not been clearly defined from a histologic standpoint, although it is likely that an in situ stage exists [ ] . the term atypical mesothelial hyperplasia has been recommended for surface (noninvasive) proliferations of mesothelial cells of uncertain malignant potential [ ] . exposure to asbestos fibers is believed to trigger the pathobiological changes leading to the majority of mms. currently, it is believed that asbestos may act as an initiator (genetically) and promoter (epigenetically) in the development of mms [ ] . the degree to which tumorigenesis results from direct interactions of the fibers with the mesothelial cells, or through other mechanisms involving oxidative stress (or both), is unresolved [ , ] . multiple chromosomal alterations are often noted in mms, and inactivation of tsgs plays an important part in the pathogenesis of mm [ ] . a variety of genetic abnormalities have been reported including deletions of p - , p , p, q, q, p , q - , q, and proximal q, monosomy , and gains of q, p, p, q - , and q - part iv molecular pathology of human disease [ , ] . the most common genetic abnormality in mm is a deletion in p encompassing the cdkn a locus encoding the tumor suppressors p ink a and p arf , which participate in the p and rb pathways and inhibit cell cycle progression ( figure . ) [ , ] . recent studies have shown that sv large t antigen (present in some mms) inactivates the tsg products rb and p , raising the possibility that asbestos and sv could act as co-carcinogens in mm and suggesting that perturbations of rb-and p -dependent growth-regulatory pathways may be involved in the pathogenesis of mm [ ] . other common findings include inactivating mutations with allelic loss in the tsg neurofibromin (nf ), found at chromosome q [ ] , and inactivation of cdkn a/p arf and gpc (another tsg) by promoter methylation [ ] . loss of cdkn a/ p arf also results in mdm -mediated inactivation of p [ ] . however, in mms, unlike many other epithelial tumors, mutations in the tp , rb, and ras genes are rare [ ] . the wnt signal transduction pathway is also abnormally activated in mms and appears to play a role in pathogenesis [ ] . activation of the pathway leads to accumulation of b-catenin in the cytoplasm and its translocation to the nucleus. interactions with tcf/ lef transcription factors promote expression of multiple genes including c-myc and cyclin d. the mechanism of activation does not appear to involve mutations in the b-catenin gene, but may instead involve more upstream components of the pathway, such as the disheveled proteins [ ] . recent evidence also suggests that the phosphatidylinositol -kinase (pi -k/akt) pathway is frequently activated in mms, and that inhibition of this pathway can increase sensitivity to a chemotherapeutic agent [ ] . the wilms' tumor gene (wt ) is also expressed in most mms, but its role in the pathogenesis of mm is unclear [ ] . finally, egfr signaling in mms has recently become a focus of greater attention, and there are some data showing that the egfr is an early cell membrane target of asbestos fibers and is linked to activation of the mapk cascade [ ] . unfortunately, a phase ii clinical trial of gefitinib treatment in patients with mms did not show effectiveness, despite egfr overexpression in over % of cases [ ] . another study found that common egfr mutations conferring sensitivity to gefitinib are not prevalent in human malignant mesothelioma [ ] . further investigation continues into new, potentially efficacious agents for the treatment of mm. non-neoplastic pulmonary pathology comprises inflammatory and fibrosing diseases of the conducting airways, alveoli, vessels, and lymphoid tissue. this pathology may be localized or diffuse, may either have an obvious etiology or be idiopathic, and may cause injury that is reparable or irreparable. most importantly, an understanding of non-neoplastic lung pathology plays a vital role in the clinical management of these diseases. this section covers the major types of obstructive and interstitial diseases, the vascular lesions, the pneumonias, the occupational diseases, the major histiocytic conditions, and the most common developmental anomalies. this list does not include all of the non-neoplastic diseases that can affect the lung, but it represents those that are responsible for the majority of illness. also, the conditions highlighted within each of these categories are those about which we best understand the molecular biology of the disease mechanisms. obstructive lung diseases are characterized by a reduction in airflow due to airway narrowing. this airflow reduction occurs, in general, by two basic mechanisms: (i) inflammation and injury of the airway, resulting in obstruction by mucous and cellular debris within and around the airway lumen; and (ii) destruction of the elastin fibers of the alveolar walls, causing loss of elastic recoil and subsequent premature collapse of the airway during the expiratory phase of respiration. there are four major obstructive lung diseases: asthma, emphysema, chronic bronchitis, and bronchiectasis. asthma is a chronic inflammatory disease of the airways that affects more than million people worldwide. the prevalence of disabling asthma has increased over % since , ranging from as low as % in rural ethiopia to over % among children in parts of central and south america [ ] . in the united states, asthma affects approximately %- % of the population and is the leading cause of hospitalization among children less than years of age [ ] . clinically, the disease is defined as a generalized obstruction of airflow with a reversibility that can occur spontaneously or with therapy. it is characterized by recurrent wheezing, cough, or shortness of breath resulting from airway hyperactivity and mucus hypersecretion. the hyperresponsiveness is a result of acute bronchospasm and can be elicited for diagnostic purposes using histamine or methacholine challenges. the key feature of these symptoms is that they are variable-worse at night or in the early morning, and in some people worse after exercise. it has previously been assumed that these symptoms are separated by intervals of normal physiology. however, evidence is now accumulating that asthma can cause progressive lung impairment due to chronic morphologic changes in the airways. the treatment strategies for this complex disease are myriad. in atopic individuals, allergen avoidance should be the primary therapy. for example, in children, reducing exposure to house dust mites early in life decreases sensitization and the incidence of disease. for those who do develop the disease, avoidance of allergens later in life improves symptom control. established treatments for asthma flairs include inhaled corticosteroids, and short-acting and long-acting b -adrenoceptor agonists. phosphodiesterase (pde) inhibitors such as theophylline have been used for decades to treat asthmatic bronchoconstriction, but both cardiac and central nervous systems side effects have limited their use. newer pde inhibitors without side effects include non-xanthine drugs such as rofumilast. the pathologic changes to the airways in asthma are very similar to those seen in chronic bronchitis. they consist of a thickened basement membrane with epithelial desquamation, goblet cell hyperplasia, and subepithelial elastin deposition. in the wall of the airway, smooth muscle hypertrophy and submucosal gland hyperplasia are also present ( figure . ). in acute asthma exacerbations, a transmural chronic inflammatory infiltrate with variable amounts of eosinophilia may be present, resulting in epithelial injury and desquamation that can become quite pronounced. one sees clumps of degenerating epithelial cells mixed with mucin in the lumen airway. these aggregates of degenerating cells are referred to as creola bodies and can be seen in expectorated mucin from these patients. also present in these sputum samples are charcot-leyden crystals, rhomboid-shaped structures that represent breakdown products from eosinophil cytoplasmic granules ( figure . ). the changes seen in the walls of these airways represent long-term airway remodeling caused by prolonged inflammation. this remodeling may play a role in the pathophysiology of asthma. the amount of airway remodeling is highly variable from patient to patient, but remodeling has been found even in patients with mild asthma. currently, the effect of the treatment on this chronic pathology is unclear [ ] . the pathogenesis of asthma is complex, and most likely involves both genetic and environmental components. most experts now see it as a disease in which an insult initiates a series of events in a genetically susceptible host. no single gene accounts for the familial component of this disease. genetic analysis of these patients reveals a prevalence of specific hla alleles, polymorphisms of fc erib, il- , and cd [ , ] . asthma can be classified using a number of different schema. most commonly, asthma is divided into two categories: atopic (allergic) and nonatopic (nonallergic). atopic asthma results from an allergic sensitization usually early in life and has its onset in early childhood. nonatopic asthma is late-onset and, though the immunopathology has not been as well studied, probably has similar mechanisms to atopic asthma. although this nosology is convenient for purposes of understanding the mechanisms of the disease, most patients manifest a combination of these two categories with overlapping symptoms. th pathogenetic mechanisms of both types encompass a variety of cells and their products. these include airway epithelium, smooth muscle cells, fibroblasts, mast cells, eosinophils, and t-cells. the asthma response includes two phases: an early response comprising an acute bronchospastic event within - minutes after exposure, and a late response that peaks approximately - hours and that can have prolonged effects. if one wants to understand this complex response, it is best to divide it into three components: (i) a type hypersensitivity response, (ii) acute and chronic inflammation, and (iii) bronchial hyperactivity. type hypersensitivity in general, human asthma is associated with a predominance of type helper cells with a cd þ phenotype. these th -type cells result from the uptake and processing of viral, allergen, and environmental triggers that initiate the episode. the processing includes the presentation of these triggers by the airway dendritic cells to naive t-cells (th ), resulting in their differentiation into populations of th and th . the th differentiation is a result of il- release by the dendritic cells, and the th cells then part iv molecular pathology of human disease further propagate the inflammatory reaction in two ways. first, they release a variety of cytokines such as il- , il- , and il- that mediate a wide variety of responses. il- and il- stimulate b-cells and plasma cells to produce ige, which, in turn, stimulates mast cell maturation and the release of multiple mediators, including histamine and leukotrienes. second, these th cells secrete il- that, together with il- , also stimulates mast cells to secrete histamine, tryptase, chymase, and the cysteinyl leukotrienes causing the bronchoconstrictor response that occurs rapidly after the exposure to the allergen. il- from these lymphocytes also recruits eosinophils to the airways and stimulates the release of the contents of their granules, including eosinophil cationic protein (ecp), major basic protein (mbp), eosinophil peroxidase, and eosinophil-derived neurotoxin. these compounds not only induce the bronchial wall hyperactivity but are also responsible for the increased vascular permeability that produces the transmural edema in the airways. the cells can differentiate into th cells as a result of il- produced by dendritic cells. these th cells produce interferon-gamma (ifn-g), il- , and lymphotoxin, which play a role in macrophage activation in delayedtype hypersensitivity reactions as seen in diseases such as rheumatoid arthritis and tuberculosis [ ] . these th cells are predominantly responsible for defense against intracellular organisms and are more prominent in normal airways and in airways of patients with emphysema than in asthmatics. however, in severe forms of asthma, th cells are recruited and have the capacity to secrete tumor necrosis factor (tnf)-a and ifn-g, which may lead to the tissue-damaging immune response one sees in these airways (figure . ) [ , ] . acute and chronic inflammation the role of acute and chronic inflammatory cells, including eosinophils, mast cells, macrophages, and lymphocytes, in asthma is evident in the abundance of these cells in airways, sputum, and bronchoalveolar samples from patients with this disease. the number of eosinophils in the airways correlates with the severity of asthma and the amount of bronchial hyperresponsiveness. proteins released by these cells including ecp, mcp, and eosinophil-derived neurotoxin cause at least some of the epithelial damage seen in the active form of asthma. neutrophils are prominent in the more acute exacerbations of asthma and are probably recruited to these airways by il- , a potent neutrophil chemoattractant released by airway epithelial cells [ ] . these cells also release proteases, reactive oxygen species (ros), and other proinflammatory mediators that, in addition to the epithelial damage, also contribute to the airway destruction and remodeling that occurs in the more chronic forms of this disease. the susceptibility of the epithelium in asthma to this oxidant injury may be increased due to decreased antioxidants such as superoxide dismutase in these lungs [ ] . finally, mast cells are activated to release an abundance of mediators through the binding of ige to fceri, high-affinity receptors on their surface. allergens bind to ige molecules and induce a cross-linking of these molecules, leading to activation of the mast cell and release of a number of mediators, most notably histamine, tryptase, and various leukotrienes, including leukotriene d (ltd ), and interact with the smooth muscle to induce contraction and the acute bronchospastic response [ ] . allergen bronchial hyperactivity the cornerstone of asthma is the hyperactive response of the airway smooth muscle. the mechanism by which this occurs combines neural pathways and inflammatory pathways. as stated, the inflammatory component of this response comes predominantly from the mast cells. the major neural pathway involved is the nonadrenergic noncholinergic (nanc) system. although cholinergic pathways are responsible for maintaining the airway smooth muscle tone, it is the nanc system that releases bronchoactive tachykinins (substance p and neurokinin a) that bind to nk receptors on the smooth muscle and cause the constriction that characterizes the acute asthmatic response [ ] . in addition to these acute mechanisms, the airway also undergoes structural alterations to its formed elements. in the mucosa, these changes include goblet cell hyperplasia and basement membrane thickening. within the submucosa and airway wall, increased deposition of collagen and elastic fibers results in fibrosis and elastosis, and both the smooth muscle cells and the submucosal glands undergo hypertrophy and hyperplasia. these irreversible changes are a consequence of chronic inflammatory insults on the airways through mechanisms that include release of fibrosing mediators such tgfb and mitogenic mediators such as epidermal and fibroblast growth factors (egf, fgf). the exact mechanisms by which this occurs are not clearly defined, but the similarity of these factors with those involved in branching morphogenesis of the developing lung has led to a focus on the effect of inflammation on the interaction of the epithelium with the underlying mesenchymal cells [ ] . the term chronic obstructive pulmonary disease (copd) applies to emphysema, chronic bronchitis, and bronchiectasis, those diseases in which airflow limitation is usually progressive, but, unlike asthma, not fully reversible [ ] . the prevalence of copd worldwide is estimated at %- % in adults over the age of [ ] . though there are different forms of copd with different etiologies, the clinical manifestations of the most common forms of the disease are the same. these include a progressive decline in lung function, usually measured as decreased forced expiratory flow in second (fev ), a chronic cough, and dyspnea. emphysema and chronic bronchitis are the most common diseases of copd and are the result of cigarette smoking. as such, they usually exist together in most smokers. chronic bronchitis is defined clinically as a persistent cough with sputum production for at least months in at least consecutive years without any other identifiable cause. patients with chronic bronchitis typically have copious sputum with a prominent cough, more commonly get infections, and typically experience hypercapnia and severe hypoxemia, giving rise to the clinical moniker blue bloater. emphysema is the destruction and permanent enlargement of the air spaces distal to the terminal bronchioles without obvious fibrosis [ ] . these patients have only a slight cough, while the overinflation of the lungs is severe, inspiring the term pink puffers. the pathologic features of copd are best understood if one considers the whole of copd as a spectrum of pathology that consists of emphysematous tissue destruction, airway inflammation, remodeling, and obstruction [ ] . the lungs of patients with copd usually contain all of these features, but in varying proportions. the pathologic features of chronic bronchitis include mucosal pathology that consists of epithelial inflammation, injury, and regenerative epithelial changes of squamous and goblet cell metaplasia. in addition, the submucosa shows changes of remodeling with smooth muscle hypertrophy and submucosal gland hyperplasia. these changes are responsible for the copious secretions characteristic of this clinical disease, although studies have reported no consistent relationship between these pathologic features of the large airways and the airflow obstruction [ ] . the pathology definition of emphysema is an abnormal, permanent enlargement of the airspaces distal to the terminal bronchioles accompanied by destruction of the alveolar walls without fibrosis [ ] . the four major pathologic patterns of emphysema are defined by the location of this destruction. these include centriacinar, panacinar, paraseptal, and irregular emphysema. the first two of these are responsible for the overwhelming majority of the clinical disease. centriacinar emphysema (sometimes referred to as centrilobular) represents % of the cases and is a result of destruction of alveoli at the proximal and central areas of the pulmonary acinus, including the respiratory bronchioles ( figure . ). it predominantly affects the upper lobes the remaining two types of emphysema, paraseptal and irregular, are rarely associated with clinical disease. in paraseptal emphysema, the damage is to the distal acinus, the area that abuts the pleura at the margins of the lobules. damage in this area may cause spontaneous pneumothoraces, typically in young, thin men [ ] . irregular emphysema is tissue destruction and alveolar enlargement that occurs adjacent to scarring, secondary to the enhanced inflammation in the area. though this is a common finding in a scarred lung, it is of little if any clinical significance to the patient. though the emphysema in these lungs plays the dominant role in causing the obstruction, small airway pathology is also present. respiratory bronchiolitis refers to the inflammatory changes found in the distal airways of smokers. these consist of pigmented macrophages filling the lumen and the peribronchiolar airspaces and mild chronic inflammation and fibrosis around the bronchioles (figure . ). the pigment in these macrophages represents the inhaled particulate matter of the cigarette smoke that has been phagocytized by these cells. the macrophages in turn release proteases, which destroy the elastic fibers in the surrounding area, resulting in the loss of elastic recoil and the obstructive symptoms. in general, copd is a result of inflammation of the large airways that produces the airway remodeling characteristic of chronic bronchitis as well as inflammation of the smaller airways that results in the destruction of the adjacent tissue and consequent emphysema. the predominant inflammatory cells involved in this process are the alveolar macrophages, neutrophils, and lymphocytes. the main theories of the pathogenesis of copd support the interaction of airway inflammation with two main systems in the lung: the protease-antiprotease system and the oxidant-antioxidant system. these systems help to protect the lung from the many irritants that enter the lung via the large pulmonary surface area that interfaces with the environment. in the protease-antiprotease system, proteases are produced by a number of cells, including epithelial cells and inflammatory cells that degrade the underlying lung matrix. the most important proteases in the lung are the neutrophil elastases, part of the serine protease family, and the metalloproteinases (mmps) produced predominantly by macrophages. these proteases can be secreted in response to invasion by environmental irritants, most notably infectious agents such as bacteria. in this setting, their role is to enzymatically degrade the organism. however, proteases can also be secreted by both inflammatory and epithelial cells in a normal lung to repair and maintain the underlying lung matrix proteins [ ] . to protect the lung from unwanted destruction by these enzymes, the liver secretes antiproteases that circulate in the bloodstream to the lung and inhibit the action of the proteases. in addition, macrophages that secrete mmps also secrete tissue inhibitors of metalloproteinases (timps). a delicate balance of proteases and antiproteases is needed to maintain the integrity of the lung structure. an imbalance that results in a relative excess of proteases (either by overproduction of proteases or underproduction of their inhibitors) leads to tissue destruction and the formation of emphysema. this imbalance occurs in different ways in the two major types of emphysema: centriacinar and panacinar. in centriacinar emphysema, caused primarily by cigarette smoking, there is an overproduction of proteases primarily due to the stimulatory effect of chemicals within the smoke on the neutrophils and macrophages. though the exact mechanism is not completely understood, most studies support that nicotine from the cigarette smoke acts as a chemoattractant, and ros also contained in the smoke, stimulate an increased release of neutrophil elastases and mmps from activated macrophages, leading to the destruction of the elastin in the alveolar spaces [ ] . this inflammatory cell activation may come about through the activation of the transcription factor nfkb that leads to tnfa production [ ] . in addition, the elastin peptides themselves may attract additional inflammatory cells to further increase the protease secretion and exacerbate the matrix destruction [ ] . unlike centriacinar emphysema, panacinar emphysema is most commonly caused by a genetic deficiency of antiproteases, usually due to alpha- anti-trypsin (aat) deficiency, a condition that affects approximately , people in the united states [ ] . aat deficiency is due to a defect in the gene that encodes the protein aat, a glycoprotein produced by hepatocytes and the main inhibitor of neutrophil elastase. the affected gene is the serpina gene (formerly known as p ), located on the long arm of chromosome ( q - . ). the genetic mutations that occur have been categorized into four groups: base substitution, in-frame deletions, frame-shift mutations, and exon deletions. these mutations usually result in misfolding, polymerization, and retention of the aberrant protein within the hepatocytes, leading to decreased circulating levels. aat deficiency is an autosomal codominant disease with over allelic variants, of which the m alleles (m -m ) are the most common; these alleles produce normal serum levels of a lessactive protein [ ] . individuals who manifest the lung disease are usually homozygous for the alleles z or s (zz and ss phenotype) or heterozygous for the m alleles (mz, or sz phenotype) [ ] . an aat concentration in plasma of less than % of normal confers a risk for emphysema [ ] . in individuals with the zz genotype, the activity of aat is approximately one-fifth of normal [ ] . the second system in the lung involved in the pathogenesis of emphysema is the oxidant-antioxidant system. as in the protease system, the lung is protected from oxidative stress in the form of ros by antioxidants produced by cells in the lung. ros in the lung include oxygen ions, free radicals, and peroxides. the major antioxidants in the airways are enzymes including catalase, superoxide dismutase (sod), glutathione peroxidase, glutathione s-transferase, xanthine oxidase, and thioredoxin, as well as nonenzymatic antioxidants including glutathione, ascorbate, urate, and bilirubin [ ] . the balance of oxidants and antioxidants in the lung prevents damage by ros. however, cigarette smoke increases the production of ros by neutrophils, eosinophils, macrophages, and epithelial cells [ ] . evidence that damage to the lung epithelium and matrix is a direct result of ros includes the presence of exhaled h o and -isoprostane, decreased plasma antioxidants, and increased plasma and tissue levels of oxidized proteins, including various lipid peroxidation products. in addition to this direct effect, ros may also induce a proinflammatory response that recruits more inflammatory cells to the lung. in animal models, cigarette smoke induces the expression of proinflammatory cytokines such as il- , il- , tnfa, and il- from macrophages, epithelial cells, and fibroblasts, perhaps through activation of the transcription factor nfkb [ , ] (figure . ) . finally, there is some evidence that cigarette smoke further disturbs the oxidant-antioxidant balance in the lung by depleting antioxidants such as ascorbate and glutathione [ ] . bronchiectasis represents the permanent remodeling and dilatation of the large airways of the lung most commonly due to chronic inflammation and recurrent pneumonia. these infections usually occur because airway secretions and entrapped organisms cannot be effectively cleared. this pathology dictates the clinical features of the disease, which include chronic cough with copious secretions and a history of recurrent pneumonia. the five major causes of bronchiectasis are infection, obstruction, impaired mucociliary defenses, impaired systemic immune defenses, and congenital. these may produce either a localized or diffuse form of the disease. localized bronchiectasis is usually due to obstruction of airways by mass lesions or scars from previous injury or infection. diffuse bronchiectasis can result from defects in systemic immune defenses in which either innate or adaptive immunity may be impaired. diseases due to the former include chronic granulomatous disease (cgd), and diseases due to the latter include agammaglobulinemia/hypogammaglobulinemia and severe combined immune deficiencies. defects in the mucociliary defense mechanism that is responsible for physically clearing organisms from the lung may also cause diffuse bronchiectasis. these include ciliary dyskinesias that result in cilia with aberrant ultrastructure and cystic fibrosis (cf). congenital forms of bronchiectasis are rare but do exist. the most common include mounier-kuhn's syndrome and williams-campbell syndrome, the former causing enlargement of the trachea and major bronchi due to loss of bronchial cartilage, and the latter causing diffuse bronchiectasis of the major airways probably due to a genetic defect in the connective tissue [ , ] . the pathology of bronchiectasis is most dramatically seen at the gross level. one can see dilated airways containing copious amounts of infected secretions and mucous plugs localized either to a segment of the lung or diffusely involving the entire lung as in cystic fibrosis (figure . ) . microscopic features include chronic inflammatory changes similar to those of chronic bronchitis but with ulceration of the mucosa and submucosa leading to destruction of the smooth muscle, and elastic in the airway wall and the characteristic dilatation and fibrosis. these enlarged airways contain mucous plugs comprising mucin and abundant degenerating inflammatory cells, a result of infections that establish themselves in these airways following the loss of the mucociliary defense mechanism. bacteria may be found in these plugs, most notably p. aeruginosa. the pathogenetic mechanism of bronchiectasis is complex and depends on the underlying etiology. in general, the initial damage to the bronchial epithelium is due to aberrant mucin (cystic fibrosis), dysfunctional cilia (ciliary dyskinesias), and ineffective immune surveillance (defects in innate and antibody-mediated immunity), leading to a cycle of tissue injury, repair, and remodeling that ultimately destroys the normal airway. the initial event in this cycle usually involves dysfunction of the mucociliary mechanism that inhibits the expulsion from the lungs of organisms and other foreign substances that invade the airways. this may be due to defects in the cilia or the mucin. ciliary defects are found in primary ciliary dyskinesia, a genetically heterogeneous disorder, usually inherited as an autosomal recessive trait that produces immotile cilia with clinical manifestations in the lungs, sinuses, middle ear, male fertility, and organ lateralization [ ] . over proteins make up the axoneme of the cilia, but mutations in genes, dnai and dnah , which encode for proteins in the outer dynein arms, most frequently cause this disorder [ ] . in cf the main defect affects the mucin. in patients with this autosomal recessive condition, there is a low volume of airway surface liquid (asl) causing sticky mucin that inhibits normal ciliary motion and effective mucociliary clearance of organisms. this is due to a defect in the cystic fibrosis transmembrane conductance regulator (cftr) gene, located on chromosome that encodes a camp-activated channel which regulates the flow of chloride ions in and out of cells and intracellular vacuoles, helping to maintain the osmolality of the mucin. this protein is present predominantly on the apical membrane of the airway epithelial cells, though it is also involved in considerable subapical, intracellular trafficking and recycling during the course of its maturation within these cells. this genetic disease manifests in multiple other organs that depend on chloride ion transport to maintain normal secretions, including the pancreas, intestine, liver, reproductive organs, and sweat glands [ ] . the genetic mutations in cf influence the cftr trafficking in the distal compartments of the protein secretary pathway, and various genetic mutations produce different clinical phenotypes of the disease. over mutations of the cftr gene have been found. however, only four of these mutations occur at a frequency of greater than %. these mutations are grouped into five classes according to their functional deficit: group i, cftr is not synthesized; group ii, cftr is inadequately processed; group iii, cftr is not regulated; group iv, cftr shows abnormal conductance; group v, cftr has partially defective production or processing. approximately % of cf patients are in group ii and have the same mutation, f d cftr, a deletion of phenylalanine at codon [ ] . in these patients, most of the cftr protein is misfolded and undergoes premature degradation within the endoplasmic reticulum, though a small amount of the cftr protein is present on the apical membrane and does function normally. cf patients may have a combination of genetic mutations from any of the five groups. however, those patients with the most severe disease involving both the lungs and pancreas usually carry at least two mutations from group i, ii, or iii [ ] . systemic immune deficiencies cause bronchiectasis through the establishment of persistent infection and inflammation. there are four major categories of immune deficiencies. the first category consists of a number of genetic diseases that cause either agammaglobulinemia or hypogammaglobulinemia. these include xlinked agammaglobulinemia (xla) and common variable immunodeficiency (cvi). xla is caused by a mutation of the bruton's tyrosine kinase (btk) gene that results in the virtual absence of all immunoglobulin isotypes and of circulating b lymphocytes. in cvi there is a marked reduction in igg and iga and/or igm, associated with defective antibody response to protein and polysaccharide antigens. as expected, both of these diseases increase susceptibility to infections from encapsulated bacteria. the second category of immune deficiency is hyper-ige syndrome, a disease with markedly elevated serum ige levels that is characterized by recurrent staphylococcal infections. the third category is chronic granulomatous disease (cgd), a genetically heterogeneous group of disorders that have a defective phagocytic respiratory burst and superoxide production, inhibiting the ability to kill staphylococcus spp. and fungi such as aspergillus spp. finally, severe combined immune deficiency (scid) comprises a group of disorders with abnormal t-cell development and b-cell and/or natural killer cell maturation and function, predisposing these patients to pneumocystis jiroveci and viral infections [ ] . after the initial insult, the subsequent steps in the development of bronchiectasis include destruction of the epithelial cells and bronchial wall connective tissue matrix by the proteases and ros secreted by the neutrophils. this proinflammatory milieu is produced by multiple factors. first, infections can persist in these lungs due to defective host immune systems and mechanisms certain organisms have developed to evade these immune defenses. for example, pseudomonas aeruginosa, changes from a nonmucoid to a mucoid variant and also releases virulence factors to protect against phagocytosis [ ] . second, in the case of cystic fibrosis, neutrophils are directly recruited by proinflammatory cytokines, such as interleukin- (il- ), released from the bronchial epithelial cells as a result of the defective cgft protein [ ] . finally, the necrotic cellular debris and other breakdown products act as chemoattractants that recruit more inflammatory cells to the airway wall, further exacerbating the damage. the final phase of the repair and remodeling begins when macrophages invade and recruit fibroblasts that secrete collagen, leading to the fibrosis seen in the pathology. however, in the absence of effective airway clearance mechanisms, these ectatic airways remain a reservoir of infection that continues the cycle of inflammation and tissue destruction. the idiopathic interstitial pneumonias (iips) comprise a group of diffuse infiltrative pulmonary diseases with a similar clinical presentation characterized by dyspnea, restrictive physiology, and bilateral interstitial infiltrates on chest radiography [ ] . pathologically, these diseases have characteristic patterns of tissue injury with chronic inflammation and varying amounts of fibrosis. by recognizing these patterns, a pathologist can classify each of these entities and predict prognosis. however, the pathologist cannot establish the etiology, since these pathologic patterns can be seen in multiple clinical settings. the pathologic classification of these diseases, originally defined by liebow and carrington in [ ] , has undergone important revisions over the past years with the latest revision by the american thoracic society/european respiratory society in [ ] . the best known and most prevalent entity of the iips is idiopathic pulmonary fibrosis (ipf), which is known pathologically as usual interstitial pneumonia (uip). uip is a histologic pattern characterized by patchy areas of chronic lymphocytic inflammation with organizing and collagenous type fibrosis. these patients usually present with gradually increasing shortness of breath and a nonproductive cough after having had symptoms for many months or even years. imaging studies usually reveal bilateral, basilar disease with a reticular pattern [ ] . therapy begins with corticosteroids, advancing to more cytotoxic drugs such as methotrexate and cytoxan, but most current therapies are not effective in stopping the progression of the disease. the current estimates are that / , males and / , females have the disease, most of whom progress to respiratory failure and death within years [ ] . the pathology is characterized by a leading edge of chronic inflammation with fibroblastic foci that begin in different areas of the lung at different times. these processes produce a variegated pattern of fibrosis, usually referred to as a temporally heterogenous pattern of injury [ ] . because it occurs predominantly in the periphery of the lung involving the subpleura and interlobular septae, the gross picture is one of more advanced peripheral and basilar disease (figure . ) . the progression from inflammation to fibrosis includes interstitial widening, epithelial injury and sloughing, fibroblastic infiltration, and organizing fibrosis within the characteristic fibroblastic foci. deposition of collagen by fibroblasts occurs in the latter stages of repair. the presence of the abundant collagen produces stiff lungs that are unable to clear the airway secretions, leading to recurrent inflammation of the bronchiolar epithelium with eventual fibrosis and breakdown of the airway structure. this remodeling produces mucousfilled ectatic spaces giving rise to the gross picture of honeycomb spaces, which is seen in the advanced pathology ( figure . ) [ ] . theories of the pathogenesis of ipf have evolved over the past decade. early theories favored a primary inflammatory process, while current theories favor the concept that the fibrosis of the lung proceeds independently of inflammatory events and develops from aberrant epithelial and epithelial-mesenchymal responses to injury to the alveolar epithelial cells (aecs) [ ] . the aecs consist of two populations: the type pneumocytes and the type pneumocytes. in normal lungs, type pneumocytes line % of the alveolar wall, and type pneumocytes line the remaining %. however, in lung injury, the type cells, which are exquisitely fragile, undergo cell death, and the type pneumocytes serve as progenitor cells to regenerate the alveolar epithelium [ ] . though some studies have suggested that repopulation of the type cells depends on circulating stem cells, this concept remains to be fully proven. according to current concepts, the injury and/or apoptosis of the aecs initiates a cascade of cellular events that produce the scarring in these lungs. studies of aecs in lungs from patients with ipf have shown ultrastructural evidence of cell injury and apoptosis as well as expression of proapoptotic proteins. further, inhibition of this apoptosis by blocking a variety of proapoptotic mechanisms such the fas-fas ligand pathway, angiotensin, and tnfa production, and caspase activation can stop the progression of this fibrosis [ ] . the result of the aec injury is the migration, proliferation, and activation of the fibroblasts and myofibroblasts that leads to the formation of the characteristic fibroblastic foci of the uip pathology and the deposition and accumulation of collagen and elastic fibers in the alveoli (figure . ). this unique pathology may be a result of the increased production of profibrotic factors such as transforming growth factor-a (tgfa) and tgfb, fibroblastic growth factor- , insulin-like growth factor- , and platelet-derived growth factor. an alternative pathway might involve overproduction of inhibitors of matrix degradation such as timps (tissue inhibitors of matrix production) [ ] . in support of the former mechanism, fibroblasts isolated from the lungs of ipf patients exhibit a profibrotic secretory phenotype [ ] . multiple factors, such as environmental particulates, drug or chemical exposures, and viruses may trigger the initial epithelial injury, but genetic factors also play a role. approximately %- % of patients with ipf have a family history of the disease with an inheritance pattern of autosomal dominance with variable penetrance. two genetic mutations have been implicated in this familial form of ipf. one large kindred has been reported with a mutation in the gene encoding surfactant protein c, and six probands have been a b reported with heterozygous mutations in genes htert or htr, encoding telomerase reverse transcriptase and telomerase rna, respectively, resulting in mutant telomerase and short telomeres [ ] . adult respiratory distress syndrome (ards) represents a constellation of clinical, radiologic, and physiologic features in patients with acute respiratory failure that can occur after a variety of insults. ards is defined by clinical criteria that include a rapid onset of severe hypoxemia that is refractory to oxygen therapy, the presence of abnormal chest radiographs with evidence of bilateral alveolar filling and collapse, increased pulmonary artery occlusion pressure, and a resistance to improved oxygenation regardless of mechanical ventilation therapy [ ] . treatment of ards includes eliminating the underlying cause, protective ventilation strategies that improve oxygenation, and supportive treatment that may include administration of corticosteroids. the pathology of ards is diffuse alveolar damage (dad), whose histologic picture is one of inflammation and fibrosis that diffusely involves all of the structures of the alveolus and is similar throughout the affected areas of the lung [ ] . dad is divided into three major phases that follow each other chronologically after the original insult. these are exudative, proliferative, and fibrotic dad. the initial injury primarily involves the epithelium of the alveolar wall and the endothelium in the capillary, causing the destruction and sloughing of the type pneumocytes into the alveolar space and a breakdown of the tight junctions of the endothelium. in combination, these two events result in the loss of the epithelial-endothelial barrier of the alveolus and leakage of plasma from the capillary into the alveolar space. this flooding of the airspace with fluid markedly decreases oxygen exchange and causes the hypoxia that these patients experience. in addition, acute inflammatory changes of the endothelium also cause thrombi to form in vessels, adding to a decreased amount of blood circulating through the lung and further compromising gas exchange. as air is brought into the alveoli, the positive pressure within the alveolar space forces the plasma against the alveolar wall, producing a membranous morphology referred to as hyalin membranes characteristic of the first phase of dad, referred to as exudative dad (figure . ) . this initial injury is followed by a sequence of events that represent the lung's efforts to repair itself. first, type pneumocytes undergo hyperplasia and re-epithelialize the alveolar wall after the loss of the type cells. this re-establishes the epithelial barrier and, because these cells secrete surfactant, results in increased surfactant production, which lowers the surface tension of the alveolus and inhibits its collapse. because of the increased numbers of type pneumocytes, this is known as the proliferative phase of dad (figure . ) . in the final phase of dad, fibrotic dad, fibroblasts migrate in from the adjacent interstitium to the alveolar space and produce organizing and irreversible fibrosis within both the alveolar space and the interstitium. in addition to this mechanism, fibrosis may also occur in those areas where alveolar walls collapse when surfactant is decreased during the initial insult. the histopathologic picture during this fibrotic phase is one of thickened alveolar septa, intra-alveolar granulation tissue, microcyst formation, and areas of irregular alveolar scarring. in rare cases, these microcysts progress to large cysts, an adult equivalent of bronchopulmonary dysplasia. the cellular events of dad are complex and incompletely understood. in general, the disease can be broken down into two phases. in the first, a large influx of neutrophils and plasma enter the alveolar space. the role the neutrophils play in the initial cellular injury and death is unclear, but it is known that they are necessary for this injury to occur. in addition, clinical studies have shown that within the peripheral blood and bronchoalveolar lavages (bal) of these patients, neutrophils are present along with a myriad of proinflammatory cytokines, such as il- , il- , and tgfa, all of which are capable of recruiting them to the lung. also present in these fluids are mediators that recruit fibroblasts such as tgfb. all of these mediators are probably the result of upregulation of nfkb, a proinflammatory transcription factor, in alveolar macrophages. the adherence of neutrophils to the capillary endothelium in the lung occurs through adhesion molecules such as selectin, integrin, and immunoglobulins. neutrophil adherence and subsequent transmigration through the endothelium of the lung capillaries may cause some endothelial damage. however, most speculate that ros and reactive nitrogen chapter molecular basis of pulmonary disease species (rns) secreted by the neutrophils modulate the majority of this injury [ ] . this is supported by the finding that patients with ards have products of oxidative damage such as hydrogen peroxide (h o ) in the exhaled breath and myeloperoxidase and oxidized aat in the bal. the cell injury and death of the type pneumocytes most likely occurs via two mechanisms: lipopolysaccharide (lps)-induced caspase-dependent apoptosis and hyperoxia-induced cell death through apoptosis and nonapoptotic mechanisms [ ] . in the former, lps, an immunogenic component of the outer membrane of gram-negative bacteria, may trigger innate immune and inflammatory responses via toll-like receptors that bind fas-associated death domain protein and caspase- , leading to epithelial cell death. in hyperoxia-induced cell death, hyperoxia may induce the expression of angiopoietin (ang ) in lung epithelial cells. ang is an angiogenic growth factor that can activate caspase pathways and lead to apoptotic cell death [ ] . cell death in ards is not limited to these mechanisms, and further study of many of pathways by which this can occur is needed. lymphangioleiomyomatosis (lam) is a rare systemic disease of women, usually in their reproductive years (average age of years), that is characterized by a proliferation of abnormal smooth muscle cells giving rise to cysts in the lungs, abnormalities in the lymphatics, and abdominal tumors, most notably in the kidneys. in addition to sporadic cases (denoted as s-lam), lam also affects % of women with tuberous sclerosis (denoted as tsc-lam), a genetic disorder with variable penetrance associated with seizures, brain tumors, and cognitive impairment [ , ] . global estimates indicate that tsc-lam may be as much as -fold to -fold more prevalent than s-lam, though at least some suggest that tsc-lam may have a milder clinical course than s-lam [ ] . clinically, lam patients usually present with increasing shortness of breath on exertion, obstructive symptoms, spontaneous pneumothoraces, and chylous effusions or with abdominal masses consisting of either angiomyolipomas and/or lymphangiomyomas. chest imaging studies characteristically reveal hyperinflation with flattened diaphragms and thin-walled cystic changes. mortality at years from the onset of symptoms is %- % [ ] . lam appears as small, thin-walled cysts ( . - . cm) randomly throughout both lungs [ ] (figure . ) . microscopically, lam lungs contain a diffuse infiltration of smooth muscle cells, predominantly around lymphatics, veins, and venules. most notably, one finds smooth muscle cells in the subpleural with hemosiderin-laden macrophages in the adjacent field, and the macrophages are also seen on bronchoalveolar lavage specimens from these patients. the hemosiderin pigment in these lungs is thought to be secondary to microhemorrhages from the obstruction of the veins ( figure . ) [ ] . the smooth muscle cells in lam react to antibodies to hmb- , a premelanosomal protein. other melanosome-like structures are also found in lam cells, suggesting that these cells have characteristics of both smooth muscle and melanosomes [ ] . the lesional cells in lam are smooth muscle-like with both spindled and epithelioid morphology [ ] . these cells are the same in both s-lam and tsc-lam part iv molecular pathology of human disease and are a clonal population although they lack other features of malignancy [ ] . molecular studies reveal that the abnormal lam cell proliferation is caused by mutations in one of two genes linked to tuberous sclerosis: tuberous sclerosis complex or (tsc or tsc ). these two genes control cell growth and differentiation through the akt/mammalian target of rapamycin (mtor) signaling pathway [ ] . in this pathway, a growth factor receptor (such as insulin or pdgf receptors) becomes phosphorylated when an appropriate ligand binds, resulting in activation of downstream effectors and ultimately akt. the gene products of tsc and tsc are hamartin and tuberin, which act as dimers to maintain rheb (a member of the ras family) in a gdp-loaded state via statins, acting as a break to the akt/mtor pathway, thereby retarding protein synthesis and cell growth. in lam cells, loss-of-function mutations in these two genes remove this inhibition, leading to enhanced rheb activation, mtor activation (with raptor), and subsequent phosphorylation of downstream molecules which result in uncontrolled cell growth, angiogenesis, and damage to the lung tissue ( figure . ) [ ] . the abnormal proliferation of lam cells is thought to damage the lung through overproduction of matrix metalloproteinases (mmps), which degrade the connective tissue of the lung architecture, destroy the alveolar integrity, and result in cyst formation with air trapping [ ] . these destructive capabilities of the lam cells are enhanced by their secretion of the angiogenic factor vegf-c, which is thought to cause the proliferation of lymphatic channels throughout the lung [ ] . sarcoidosis is a multisystemic disease that involves the lung in over % of the cases [ ] . it is most common in the - -year age group and among females. in the united states, african americans are more commonly affected than caucasians [ ] . the clinical picture of sarcoidosis is variable, but most patients present with systemic symptoms including fatigue, weight loss, and fever. the most common finding on chest imaging studies is bilateral hilar lymph node enlargement and reticular, reticulonodular, and focal alveolar opacities within the lung parenchyma [ ] . pulmonary sarcoidosis is characterized by granulomas which consist of activated histiocytes, called epithelioid histiocytes that form nodules ranging in size from - microns (figure . ) [ ] . unlike infectious granulomas that usually contain areas of central necrosis, the granulomas in pulmonary sarcoidosis are predominantly non-necrotizing [ ] . also, the granulomas in sarcoidosis follow a distribution along the lymphatics, which includes the area in the subpleural, along the interlobular septae and around the bronchovascular area containing the bronchiole and branch of the pulmonary artery (figure . ). the granulomas occur much more commonly in the upper lobes, leading to the predominant upper lobe fibrosis and bronchiectasis that can be seen in longstanding sarcoidosis [ ] . despite over years of research on sarcoidosis, the etiology remains unknown. most agree that the disease is probably a result of environmental triggers acting on a genetically susceptible host [ , ] . a genetic basis of sarcoidosis has been suggested by studies that demonstrate familial clustering and racial variation [ , ] . further, complex inheritance patterns for the disease suggest that more than one gene may be involved [ ] . several genes of the major histocompatibility complex (mhc) region of the genome have been implicated. most are clustered on the short arm of chromosome that encompasses the human leukocyte antigen (hla) domain. the hla class i mhc molecules associated with sarcoidosis are the hla-b and hla-b class i alleles [ , ] . hla class ii molecules implicated in susceptibility include the hla-dr alleles [ , ] . genes other than mhc genes thought to regulate the susceptibility to sarcoidosis include those for chemokines such as macrophage inflammatory protein- a and rantes (ccr and ccr ) [ , ] . environmental factors that have been implicated are those that are aerosolized. therefore, these environmental agents have a mode of entry into the lungs and can cause granulomas in the lung, similar to sarcoidosis. these factors can be divided into two major categories, which include infectious and noninfectious agents. the mycobacteria have been the most extensively studied organisms. however, their role in this disease remains controversial due to the difficulty in identifying them by either culture or histochemical stains in sarcoid tissue. recently, molecular techniques have been able to demonstrate mycobacterial nucleic acid in sarcoid tissue [ , ] . however, even studies using this technology have not produced consistent results, and the role of these organisms in the disease requires further study. the immune response in sarcoidosis has two major features: (i) the initial event leading to granuloma formation and (ii) the progression of this granulomatous response to either resolution or fibrosis [ ] . the formation of the granulomas, triggered by activation of tcells and antigen-presenting dendritic histiocytes, results in a release of proinflammatory cytokines and chemokines, and recruitment, activation, and proliferation of mononuclear cells, predominantly t-cells. these activated t-cells are predominantly cd -expressing t-helper (th) cells, which release ifn-g and il- . alveolar macrophages at the site release tnfa, il- , il- , and other growth factors. this results in the granuloma formation and alveolitis, the characteristic morphologic features of the disease [ ] . the second phase of this immunologic response that leads to either resolution of the disease or persistence of the granulomas and fibrosis is less well characterized. ongoing granuloma formation and inflammation may be a result of the persistent presence of antigens, the excessive synthesis of chemotactic factors, or the part iv molecular pathology of human disease persistence of the mononuclear cells within the granulomas. importantly, the role of the t-cells in these granulomas is to secrete cytokines that attract, stimulate, and ultimately deactivate the fibroblasts that are responsible for the fibrosis that is seen in the chronic disease. the balance between the profibrotic mediators such as tgfb, insulin-like growth factor-i, platelet-derived growth factor (pdgf), and the antifibrotic mediators, such as ifn-g, probably dictates the natural history of sarcoidosis in the lung [ ] . genes involved in macrophage-derived cytokines, chemokines, and mediators of fibrosis are all possible candidates for the underlying genetic cause of this complicated disease. pulmonary alveolar proteinosis (pap) is a rare disease of the lungs characterized by accumulation of surfactant in the alveolar spaces. the names alveolar proteinosis, lipoproteinosis, or perhaps most accurately phospholipoproteinosis, apply equally to this entity. pap takes three forms clinically: (i) congenital ( %), (ii) secondary ( %- %), and (iii) idiopathic or primary ( %- %) [ ] [ ] [ ] . pap arises in previously healthy adults with the median age at diagnosis of approximately years and a male-to-female ratio of . : . the clinical presentation is variable and usually includes an insidious onset of slowly progressive dyspnea, a dry cough, and other symptoms of respiratory distress, including fatigue and clubbing. however, almost one-third of patients are asymptomatic and are found clinically by abnormal chest x-rays [ , ] . the secondary form of pap can be found in patients with environmental exposures, including fine silica, aluminum, titanium dioxide, and kaolin dust [ ] . also, secondary pap may be found in patients with malignancies, most commonly hematologic malignancies such as myelogenous leukemia [ , ] . chest imaging studies in both the idiopathic and secondary forms most commonly show fine, diffuse, feathery nodular infiltrates, centered in the hilar areas, sparing the peripheral regions [ ] . on chest computerized tomographs, the infiltrates may have a geometric-type shape, sometimes referred to as crazy paving [ ] . the most prominent microscopic feature of both idiopathic and secondary pap is the filling of the alveoli with finely granular period acid-schiff-positive diastaseresistant (pasd) acellular material (figure chapter molecular basis of pulmonary disease material consists of phospholipids ( %); surfactant proteins a, b, c, and d ( %); and carbohydrate (< %) [ ] . alveolar macrophages (ams) with prominent foamy cytoplasm are commonly seen, while alveolar septa are remarkably normal in appearance. in some alveolar spaces there are denser, more solid clumps of pas-d-positive material. definitive pathologic differences between the idiopathic and secondary forms of pap have not been well documented [ , ] . the etiologies of the two adult forms of pap have been well studied with the most known about the idiopathic variant. theories of the pathogenesis of this form have focused on the abnormal accumulation of the surfactant-like material within the alveolar spaces. since the regulation of surfactant levels in the alveoli depends on appropriate synthesis, recycling, and catabolism, the two opposing hypotheses have included overproduction versus decreased degradation of this material. in normal hosts, surfactant is essential to maintaining the low surface tension needed for proper alveolar inflation and gas exchange. the critical role of maintaining the proper composition and amount of surfactant in the alveoli is performed by two cell types: type pneumocytes and alveolar macrophages [ ] . the type pneumocytes synthesize surfactant in the endoplasmic reticulum and golgi, and store it as lamellar bodies [ ] , which are then delivered to and fuse with the apical plasma membrane, secreting the surfactant into the airways [ ] . catabolism of surfactant is carried out by type pneumocytes and ams. in pap, most evidence suggests that the clearance of surfactant by the am is decreased [ , ] . the first clue as to the underlying mechanism for this defect in am function came in when studies revealed that knockout mice deficient in granulocytemacrophage colony-stimulating factor (gm-csf) develop lung lesions similar to those in patients with pap [ ] . this rather serendipitous finding prompted explorations centered on the am and the effect diminished gm-csf might have on its cellular functions. subsequent studies from humans with pap revealed an autoimmune mechanism by which a circulating neutralizing antibody to gm-csf blocked its binding to the gm-csf receptor, depressing the effect of gm-csf on the ams [ ] [ ] [ ] . neutralizing antibodies to gm-csf have most often been identified in the idiopathic variant of pap. however, recently these antibodies have also been reported in patients with secondary pap [ ] . genes that control many functions in the am are controlled by signaling pathways initiated by gm-csf binding to the am. one pathway is mediated through a transcription factor pu. that controls genes involved in surfactant degradation, among other bactericidal functions [ , ] . another transcription factor, peroxisome-proliferator-activated receptor g (pparg), is also part of a pathway activated by gm-csf. pparg controls the expression of genes involved in intracellular lipid metabolism. ams from patients with pap have a deficiency of this transcription factor, which is correctable by gm-csf therapy [ ] . overall, the lack of gm-csf-initiated signaling in ams from patients with pap leads to inhibition of both pparg and pu. pathways. this results in decreased surfactant catabolism, intracellular lipid metabolism, and the accumulation of surfactant in the alveoli (figure . ). pulmonary hypertension consists of a group of distinct diseases whose pathology is characterized by abnormal destruction, repair, remodeling, and proliferation of all compartments of the pulmonary vascular tree, including arteries, arterioles, capillaries, and veins. the classification of these diseases has undergone a number of revisions. the most recent revision (in ) groups these diseases based on both their pathologic and clinical characteristics [ ] . there are five major disease categories in the current classification system: (i) pulmonary arterial hypertension (pah); (ii) pulmonary hypertension with left heart disease; (iii) pulmonary hypertension associated with lung disease and/or hypoxemia; (iv) pulmonary hypertension due to chronic thrombotic and/or embolic disease; and (v) miscellaneous causes, including sarcoidosis, histiocytosis x, and lymphangioleiomyomatosis. the clinical course of most patients with pulmonary hypertension begins with exertional dyspnea, and progresses through chest pain, syncope, increased mean pulmonary artery pressures and, eventually, right heart failure. the rate of this clinical progression varies among patients, from a few months to many years [ ] . treatment of these diseases focuses on blocking the mediators involved in the pathogenesis of the diseases. however, current therapies rarely prevent progression of the disease, and lung transplantation provides the only hope for long-term survival. the major group of this classification, pah, can be subdivided into familial pah, idiopathic pah, pah associated with other conditions (such as connective tissue diseases, hiv, congenital heart disease), and pah secondary to drugs and toxins (such as anorexigens, cocaine, and amphetamines). in these diseases, the primary pathology is localized predominantly in the small pulmonary arteries and arterioles. however, two other diseases in this group, pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis, involve predominantly other components of the pulmonary vasculature, the veins, and the capillaries, respectively. the pathologic changes seen in the pulmonary vessels of these patients primarily reflect injury to and repair of the endothelium. early pathologic changes include medial hypertrophy and intimal fibrosis that narrows and obliterates the vessel lumen. these are followed by remodeling and revascularization, producing a proliferation of abnormal endothelial-lined spaces. these structures are known as plexogenic lesions and are the pathognomonic feature of pah (figure . ) . in the most severe pathologic lesions, these abnormal vascular structures become dilated or angiomatoid-like and may develop features of a necrotizing vasculitis with transmural inflammation and fibrinoid necrosis. though the exact pathogenetic mechanism of pah remains unknown, research over the past years has begun to offer some clues. the familial form of pah, with a : female-to-male prevalence, has an autosomal dominance inheritance pattern with low penetrance. the genetic basis for this has been found to be germline mutations in the gene encoding the bone morphogenetic protein receptor type (bmpr ). these mutations account for approximately %- % of familial pah and %- % of patients with sporadic pah [ ] . approximately bmpr mutations have been identified in familial pah, each resulting in a loss of receptor function, either through alteration in transcription of the gene through missense, nonsense, or frameshift alterations in the codon or by rna spicing mistakes [ ] . the mechanism by which a single mutation to the bmpr gene induces vascular smooth muscle proliferation and decreased apoptosis that is not completely understood, but it most likely involves defects in the bmpr signaling pathway. bmpr is a receptor for a family cytokines (bmps) that are members of the tgfb superfamily of proteins that play a role in the growth and regulation of many cells, including those of the pulmonary vasculature. in the vascular smooth muscle cells of the lung, tgfb signaling causes a proliferation of smooth muscle in pulmonary arterioles, while bmpr signaling causes an inhibition of the proliferation of these cells, favoring an apoptotic environment. the bmpr signaling occurs through an activation of a receptor complex (bmpr and bmpr ) that leads to phosphorylation and activation of a number cytoplasmic mediators, most notably the smad proteins (mothers against decapentaplegic). these smad proteins, especially the smad , smad , and smad complex with smad , translocate to the nucleus where they target gene transcription that induces an antiproliferative effect in the cell. in familial pah, the bmrpr gene mutation may lead to insufficient protein product and subsequent decreased protein function, in this case decreased bmpr receptor function, decreased smad protein activation, and decreased antiproliferative effects in the vascular smooth muscle cells. the imbalance between the proproliferative effects of the tgfbs and the antiproliferative effects of the bmps results in the formation of the vascular lesions of pah (figure . ) [ , ] . despite these advances, questions regarding the pathogenesis of pah remain. most notably, why do only %- % of patients with the mutation develop clinical disease? some speculate that genes confer susceptibility but a second hit is required to develop the clinical disease, such as modifier genes or environmental triggers, perhaps drugs or viral infections [ , ] pulmonary vasculitides present as diffuse pulmonary hemorrhage and are usually caused by one of three major pulmonary vasculitis syndromes: wegener's granulomatosis, churg-strauss syndrome, and microscopic polyangiitis. all three diseases have similar clinical presentations and considerable overlap in their pathologic features as small vessel systemic vasculitides that affect the lung as well as other organs, most notably the kidney. wegener's granulomatosis (wg) is an unusual disease that affects the upper and lower respiratory tract and the kidneys. it usually presents between and years of age and is slightly more common in men than women. the clinical presentation depends on the affected organ, but when the lung is involved, hemoptysis is the major presenting symptom. chest imaging studies may show a variety of patterns, most commonly bilateral ground glass opacities with masses, usually in the lower lobes that may cavitate. immunologic testing of peripheral blood or end organ tissue can be helpful in revealing characteristic immunofluorescent staining patterns for antineutrophilic cytoplasmic antibody (anca), an antibody that targets two substances: proteinase (pr ) and myeloperoxidase (mpo). when present in either the blood or the tissue, the pattern of immunofluorescent staining can be cytoplasmic (canca) or perinuclear (panca). the former pattern is more commonly seen in wegener's granulomatosis, and the latter is more commonly seen in microscopic polyangiitis and churg-strauss syndrome (css). css is a systemic disorder defined by the presence of asthma, peripheral blood eosinophilia, and systemic vasculitis. similar to wg, it usually presents between and years of age, and a clinical diagnosis requires a history of asthma, a peripheral blood eosinophilia, neuropathy, an abnormal chest imaging study, and sinusitis. other organs involved include the heart, the central nervous system, kidneys (though less commonly than wg), gastrointestinal tract, and skin. chest imaging usually shows patchy, multifocal infiltrates; masses and cavitation are rare. laboratory tests reveal positive panca tests in % of patients. microscopic polyangiitis (mpa) is similar to both wg and css in that it is a systemic vasculitis that involves the lung and usually presents in the fourth or fifth decade of life. the clinical onset is usually sudden with fever, weight loss, myalgias, and arthralgias. the kidney is the main organ involved, and mpa is the most common cause of pulmonary-renal syndrome. lung involvement occurs in approximately % of the patients, and skin and upper respiratory tract are other common sites. similar to wg and css, anca testing is helpful with positive panca in % of patients. chest imaging usually shows bilateral infiltrates without masses, similar to css. treatment for all three diseases is immunosuppression with glucocorticoids or cyclophosphamide, and all three usually respond well, although wg has a greater relapse rate after treatment than either css or mpa [ ] . the pathology of wg, css, and mpa have overlapping features of an acute and chronic vasculitis that involves medium-and small-sized vessels in the lung. the inflammatory cell infiltrate that destroys the blood vessels is both lymphocytic and neutrophilic, and areas of fibrinoid necrosis are seen. however, in wg, there are characteristic areas of microabscesses that lead to masses of geographic necrosis with basophilia. scattered multinucleated giant cells are present, but no wellformed granulomas are seen. this helps to distinguish it from other vasculitides and infection (figure . ) . similarly, the pathology of css has distinguishing features, with the early pathology characterized by an eosinophilic pneumonia with areas of loosely formed granulomas with central necrosis containing degenerating eosinophils (figure . ). the infiltrate is predominantly eosinophils, but neutrophils, lymphocytes, and plasma cells are also present. capillaritis can be seen in wg, csg, and mpa, and all three have hemosiderin deposition present, both within alveolar macrophages and deposited in the connective tissue of the interstitium and the vessel walls. the pathogenesis of these three pulmonary hemorrhage syndromes is similar to the mechanisms of these diseases in the kidney. in general, these diseases in the lung and the kidney represent immune-mediated these lesions are thought to be the early form of the larger areas of geographic necrosis that produces the mass-like nodules found in these lungs. chapter molecular basis of pulmonary disease necrotizing vasculitides that have few or no immune deposits in the vessels but exhibit the presence of anca autoantibodies to myeloperoxidase (mpo) and proteinase (pr ), the components of primary granules of neutrophils. mpa and css are primarily diseases of mpo antibodies, and wg is primarily a disease of pr antibodies. the mechanism by which the ancas are induced is not known but may be part of an autoimmune response to environmental exposures early in life. these autoantibodies then inflict damage on the vessels through a mechanism that is not yet completely understood. one theory suggests that circulating ancas bind to pr and mpo on the surface of neutrophils and initiate a respiratory burst, degranulation, and apoptosis. ros and proteases are released and inflict endothelial and tissue damage on the adjacent vessel. the anca binding may also induce the release of proinflammatory cytokines and chemokines such as il- and tnfa that further contribute to the vascular inflammation. the second theory postulates that circulating immune complexes of excess anca antigen (mpo or pr ) and anca autoantibodies attach to the vascular endothelium and activate complement that results in the chemotaxis and adhesion of inflammatory cells, causing these cells to undergo a respiratory burst and, as in the first theory, release of ros and proteases that cause the vascular endothelial damage. in both theories, it is important to remember that mpo and pr are also present in monocytes and that anca autoantibodies may be involved with monocytes in similar ways to release inflammatory mediators [ ] . infectious diseases of the lung are a common cause of pulmonary disease given the constant exposure of the lungs to the environment. various organisms are capable of causing these infections, including common viruses and bacteria, as well as more uncommon fungi, parasites, and protozoa. the diagnosis of the specific etiologic agent can be challenging given that most have similar clinical features and many are difficult to identify in the lung tissue. this brief overview of the defense mechanisms the lung uses to protect itself will serve to introduce the pathology of these lung infections. the lung has multiple anatomic mechanisms by which it defends itself against invasion by various pathogens. first, the upper nasal cavities and respiratory tract serve as anatomic barriers to inhaled organisms. the ciliated epithelium and torturous cavities of the sinuses screen large organisms (typically larger than microns). for those particles that venture further down the respiratory tract, the cough reflex that the upper trachea elicits serves to expel them up and out. second, the mucociliary tree of the upper respiratory tract captures organisms that evade these two mechanisms. the bronchial epithelium contains cilia of up to microns in length that extend into the air surface liquid (asl). the asl is a bilayer of - microns in thickness consisting of a low-viscosity or watery lower layer that is covered by a high-viscosity or gel upper layer secreted by adjacent goblet cells. this sticky upper layer serves to trap organisms, and the coordinated beating of the cilia moves these entrapped invaders up this mucociliary escalator to the larynx, where they can be expectorated. present in the secretions of the large airways and within the surfactant lining the alveolar walls are soluble mediators secreted by various cells. these mediators include lysozyme and lactoferrin, which lyse bacteria and inhibit their growth; the defensins and cathelicidins, small peptides both with microbicidal properties; and surfactant proteins a and d at the alveolar level, which bind to microorganism and enhance phagocytosis and also have direct bactericidal activity [ ] . the major cells of the innate immune response of the lung are the alveolar macrophages (am) and the polymorphonuclear leukocytes (pmn). neutrophils phagocytize and destroy bacteria such as s. aureus, s. pneumoniae, and h. influenzae through a respiratory burst that generates nadph oxidase-dependent ros. in some instances, ams may ingest but not kill an organism. this occurs with such organisms as mycobacterium spp., nocardia spp., and legionella spp. because of the ability of these organisms to continue to replicate within part iv molecular pathology of human disease the am, cell-mediated immunity is required for their complete elimination. patients with defects in nadph oxidase are especially prone to respiratory infections by such organisms as s. aureus, nocardia spp. and aspergillus spp. bronchial epithelial cells are important in innate immunity through secretion of cytokines and molecules including il- , il- , il- , il- , and granulocytemacrophage colony-stimulating factor (gm-csf). these molecules attract macrophages as well as neutrophils and other inflammatory cells to the area to enhance the inflammatory response to the organism [ ] . bronchial epithelial cells also serve an important role in recognizing pathogens through patternrecognition receptors (prrs). natural killer (nk) cells are involved in the innate immune response with surface receptors that recognize cells infected with viruses such as rsv, influenza, parainfluenza, and rhinovirus. the nk cells release ifn-g, which recruit other immune cells to add to the antiviral response. dendritic cells are tissue histiocytes positioned around the airways and lymphatics in the lung that recognize pathogens and their antigens and trigger the proliferation and amplification of antigen-specific tcells. this immune response bridges the innate immune response to the adaptive immune responses and is especially important in fungal infections. this mechanism is mediated through toll-like receptors (tlrs) that are able to distinguish pathogens from self-components by triggering cytokine production through nfkb and ap- and expressing co-stimulatory molecules necessary for this t-cell activation [ ] . for those organisms that evade the basic, innate immunity of the lung, there are adaptive immune mechanisms that encompass both humoral and cellular immune mechanisms. humoral immunity is an important defense against encapsulated bacteria, most notably s. pneumoniae, and for other pyogenic bacteria such as h. influenzae, and staphylococci spp., and resolution of these infections requires the production of igg antibodies to the organisms. cellular immunity is especially important against such respiratory viral infections as influenza, rsv, cmv, varicella, and also against opportunistic infections. these viruses induce a cd þ and cd þ t-cell response that clears the lung of these viruses within - days post infection. granulomas are a common inflammatory response to both pathogens and foreign material. the most notable granulomatous infections in the lung are due to mycobacteria and fungal organisms. activation of cd þ t-cells by these organisms leads to proliferation and differentiation of these cd þ t-cells into t-helper- cells. the release of ifn-g by the th- cells activates lung macrophages to form epithelioid macrophages that have an increased ability to kill the microorganisms and express surface molecules that promote cell-to-cell fusion into giant cells. in addition, activation of these macrophages results in the release of numerous cytokines including ifn-g and tnfa. in patients who are deficient in cd þ t-cells or ifn-g, granuloma formation is very poor, altering the pathologic picture of these infections. this effect is most obvious in the nontuberculous mycobacterial infections, which have numerous patterns of injury depending on the immune status of their host. pneumonias can be broadly categorized into one of five major clinicopathologic categories, including (i) community-acquired pneumonias (acute and atypical), (ii) nosocomial pneumonias, (iii) aspiration pneumonias and lung abscess, (iv) chronic pneumonias, and (v) pneumonias in immunocompromised hosts. each type presents with a characteristic clinical pattern and may be caused by any of several pathogens so that treatment is many times empiric. the first category comprises community-acquired pneumonias (cap). these represent the majority of the lung infections that receive medical treatment, usually on an outpatient basis, with low (< %) mortality. patients hospitalized for these infections typically have other comorbidities. the responsible organisms include respiratory syncytial virus (rsv); rhinovirus, parainfluenza, and influenza virus; bacteria, including mycoplasma pneumoniae and rickettsia; and most notably chlamydia pneumonia. chlamydia causes what is termed atypical pneumonia with a clinical course characterized by a progressive onset of fever without chills, a dry cough, and chest imaging that reveals focal infiltrates. acute or typical cap presents abruptly with high fever, chills, productive cough, and radiographs with lobar or segmental consolidation. the most common pathogens are streptococcus pneumoniae, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. the second category, nosocomial pneumonias, consists of infections acquired within the hospital or from healthcare associated facilities. these infections are usually found in patients with predisposing risk factors and are a major source of morbidity and mortality, with some studies reporting a mortality range of %- %. the most common risk factors include respiratory ventilation, artificial airways, nasogastric tubes, supine positioning, and medications that alter gastric emptying. the responsible organisms include klebsiella spp., legionella spp., staphylococcus aureus, and pseudomonas aeruginosa. the third category includes aspiration pneumonias and lung abscesses. these infections occur in the setting of patients with aberrant swallow or gag reflexes that allow gastric or oral contents into the airways. the organisms where necrosis and cavity formation occurs include s. aureus, k. pneumoniae, the anaerobic oral flora, and mycobacteria. clinically, these infections may have an acute course with fever and dyspnea or a more insidious course, many times with patients first presenting with lung cavities, empyemas, or necrotizing pneumonias. the fourth category, chronic pneumonias, includes indolent infections that cause a localized mass-like lesion in an otherwise healthy host. nocardia and actinomyces spp. are the most common pathogens, but mycobacteria and fungi may also cause these pneumonias. the fifth category includes pneumonias that occur in the setting of an immunocompromised patient. these include a number of organisms that otherwise would not act as pathogens such as the viruses cmv and hsv, the fungi aspergillosis and pneumocystis pneumonia, and the bacterium mycobacterium avium complex. streptococcus pneumoniae streptococcus pneumoniae, a gram-positive diplococcus also known as pneumococcus or diplococcus pneumonia, is a common cause of bacterial pneumonia in infants and elderly patients, alcoholics, diabetics, and patients with immunosuppression. this pneumonia usually presents abruptly with chills, a cough with rust-colored sputum and pleuritis, with high fevers, tachycardia, and tachypnea. the characteristic gross pathology is a lobar pneumonia that progresses from a red acute phase to a gray organizing phase. a fibrinous pleuritis is common, which eventually organizes to entrap the lung parenchyma in a fibrous capsule [ ] . the microscopic examination reveals abundant fibrin, neutrophils, and extravasated red blood cells within the alveolar space and congested capillaries. hemophilus influenzae hemophilus influenzae is a gramnegative bacillus that inhabits the upper respiratory tract and can cause otitis media, epiglottitis, and meningitis, and usually enters the lung through aspiration or hematogenous spread. six serotypes are defined based on their capsular antigens, with type b the most common cause of pneumonias. this type of pneumonia is most commonly found in children or in the elderly with underlying chronic lung disease such as emphysema, cystic fibrosis, bronchiectasis, in patients with hiv infection, or in alcoholics. this bacterial pneumonia is usually preceded by a viral or mycoplasma infection that damages the mucociliary elements in the airways and allows for colonization by h. influenzae. the symptoms include fever; a productive, purulent cough; and myalgias. the incidence of this pneumonia as a common community-acquired pneumonia in children is quite low due to the advent of effective vaccines. however, it is increasing in incidence as a nosocomial infection [ ] . like pneumococcal pneumonia, the pathology of h. influenzae pneumonia is in a lobar distribution with a neutrophilic-rich infiltrate and a pleural effusion. necrosis and empyema may occur but are uncommon. staphylococcus aureus staphylococcal pneumonia is caused by staphylococcus aureus, gram-positive cocci that usually spread to the lung through the blood from other infected sites, most often the skin. though a common community pathogen, it is found twice as frequently in pneumonias in hospitalized patients. it often attacks the elderly and patients with cf and arises as a co-infection with influenza viral pneumonia. the clinical course is characterized by high fevers, chills, a cough with purulent bloody sputum, and rapidly progressing dyspnea. the gross pathology commonly reveals an acute bronchopneumonia pattern (figure . ) that may evolve into a necrotizing cavity with congested red/purple lungs and airways that contain a bloody fluid and thick mucoid secretions. the histologic pattern is characterized by a bronchopneumonia that spreads distally from the small airways into to the alveolar spaces (figure . ) to form abscesses that connect with the pleural surface and may result in empyemas. the treatment of this organism has become increasingly problematic due to antibioticresistant strains, most notably methicillin-resistant s. aureus. legionella pneumophila legionella are gram-negative bacilli found predominantly in aquatic habitats such as lakes, rivers, and ponds. standing pools of water from humidifiers and other water outlets may be other sources. approximately % of air conditioners contain these bacilli. though serogroups of legionella have been identified, cause the overwhelming majority of human pneumonia. the clinical disease takes two forms: (i) legionnaires' disease, named after the outbreak of pneumonia at the american legion convention in philadelphia; and (ii) pontiac fever, a self-limiting flu-like disease with nonspecific symptoms. legionella pneumonia presents as a severe infection of the lung with chills and rigors with a nonproductive cough. it can progress rapidly to systemic symptoms of nausea, vomiting, and diarrhea and can lead to renal failure and death without immediate antibiotic therapy. the infected lungs are remarkably red and congested and appear to be distended with fluid. the microscopic picture reveals fibrinopurulent exudates that fill the alveolar space mixed with a necrotic, cellular infiltrate of degenerating neutrophils and monocytes (figure . ). hyaline membranes may form in the periphery of the lesions, and pleural effusions consisting of fibrinoserous exudates are common. pseudomonas aeruginosa pseudomonas aeruginosa is a gram-negative bacillus that is found throughout the environment and in % of the airways of hospitalized patients. it usually enters the body through a disruption of the epithelial surface by cuts, burns, or therapeutic devices such as mechanical ventilators or intravascular catheters. pneumonias caused by this organism are usually found in intensive care units of hospitals and burn units, in patients with underlying chronic lung diseases including cystic fibrosis, emphysema, and in patients with prolonged hospitalization. the pathology is necrosis with a bronchopneumonia pattern that usually consists of an area of congestion and hemorrhage that is surrounded by a halo of tan/white consolidation (figure . ) . a necrotizing vasculitis with abundant organisms in vessel walls can be seen, and cavitation is common ( figure . ). in treated lungs, healed cavities or pneumatoceles may appear as smooth-walled fibrous cysts. other gram-negative bacilli gram-negative bacilli such as klebsiella pneumoniae, acinetobacter, and various enterobacteriaceae spp. are common nosocomial pathogens. similar to p. aeruginosa, these pathogens colonize the oropharynx and are usually introduced into the lung by inhalation or aspiration of oral contents. the most notable of these is friedlander's pneumonia caused by k. pneumoniae, the most common cause of gramnegative bacterial pneumonia. this typically occurs in men over years of age, usually in the setting of alcoholism, diabetes mellitus, or chronic lung disease. these patients produce large amounts of thick, bloody sputum, a product of the viscous mucopolysaccharide capsule of the organism, and present with severe systemic symptoms of hypotension and generalized weakness. the pathology of these pneumonias is similar to pseudomonas pneumonia with marked cavitation and abundant organisms on microscopic examination. nocardia spp. nocardiosis of the lung is caused by nocardia asteroides, a gram-positive rod found in the soil or organic matter. this infection is most common in immunocompromised adult patients and can be seen in the setting of pulmonary alveolar proteinosis, chronic lung diseases, and mycobacterial and other granulomatous diseases that affect the lung. its clinical course is indolent and usually begins - weeks before the patient presents for medical therapy. cough is common, often with thick, purulent sputum. in the immunocompromised setting, fever, chills, dyspnea, and hemoptysis are common, and weight loss may occur as the disease progresses. the pathology is remarkable for suppurative abscess formation with multiple cavities filled with green, thick pus. the inflammatory infiltrate consists of neutrophils, macrophages, and abundant necrotic debris with epithelioid histiocytes and giant cells within the wall of the cavity (figure . ). empyema and pleura involvement occur in the majority of cases. mycoplasma and rickettsia pneumonias mycoplasma pneumoniae pneumonia is among the most common infections of the lower respiratory tract and usually occurs in small epidemics in closed populations. it often presents with atypical features of a progressive onset, fever without chills, a dry cough, diffuse crackles on physical examination, and chest imaging studies that reveal patchy interstitial infiltrates. the pathologic features are a result of the attachment of the organisms to the bronchiolar epithelium where they cause epithelial injury and ulcerations through secretion of peroxide [ ] . in cases of severe infection, diffuse alveolar damage may be present. chlamydial pneumonia chlamydia spp. causes pneumonia in a variety of clinical settings. chlamydia trachomatis is an infection found predominantly in the postnatal period, chlamydia psittaci is the result of direct transmission from infected birds, including parakeets, parrots, and pigeons. chlamydia pneumoniae is the most common of the three and is a frequent cause of community-acquired pneumonia. it typically causes a very mild or asymptomatic infection with fever, sore throat, and nonproductive cough. the course of this infection may be severe in the elderly. chest imaging studies show alveolar infiltrates, and pleural effusions are present in the majority of cases. the pathology has not been well defined since the infection is usually self-limited. however, in experimental animal models there is a neutrophilic response in the early stages, and an interstitial, peribronchiolar, and perivascular infiltrate of lymphocytes, macrophages, and plasma cells in the latter stages of the infection. mycobacteria, a major cause of lung infections, are nonmotile, aerobic, catalase-producing, acid-fast bacilli. clinically significant lung infections can be caused by m. tuberculosis and by a group of nontuberculous mycobacteria (ntm). the latter group consists of over species, of which three cause the overwhelming majority of pulmonary disease. these are m. avium-intracellulare (m. avium complex), m. kansasii, and m. fortuitum-chelonae. throughout history, tuberculosis (infection with m. tuberculosis) was the major disease caused by these organisms and was responsible for worldwide morbidity and mortality. however, over the past two decades lung diseases caused by ntm have become much more common and now represent the majority of the pulmonary mycobacterial disease. mycobacterium tuberculosis pulmonary tuberculosis is spread by interpersonal contact through aerosolized droplets. once in the alveoli, the bacteria cause a cell-mediated inflammatory response that is capable of inducing granuloma formation and necrosis. as in all infections, the extent of the disease is a function of the host's immune response. the most susceptible part iv molecular pathology of human disease patients are those with certain conditions that include immunosuppression, diabetes, malignancy, renal failure, among others. clinically, an infected patient has a productive cough, fever, and weight loss, and may develop hemoptysis as the cavitation progresses and erodes into the pulmonary vessels. extensive involvement of the lung can produce significant dyspnea and pleuritic chest pain. the pathology of tuberculosis is primarily that of granuloma formation and acute pneumonia. the granulomas are predominantly necrotizing, and the pneumonia usually contains abundant fibrin and neutrophils that fill the alveolar spaces. the gross lesions are referred to as caseous or cheese-like, because of the amount of necrosis present. this caseous material can extend into airways and is commonly coughed up during the active disease. in chronic forms of the disease, the area can undergo fibrosis and involute into a firm, hard scar. there are three major clinicopathologic variants of the disease: (i) primary tuberculosis, (ii) postprimary or reactivation tuberculosis, and (iii) progressive fibrocavitary disease. primary tuberculosis. in this form of the disease, the initial site of infection can be anywhere in the lung, but is usually in the lower lobe or anterior segment of the upper lobe, the areas that receive the most ventilation. the lesion usually consists of a dense consolidation with acute pneumonia and necrotizing granulomas. cavitation may occur, especially in the setting of immunocompromised hosts. from these foci, the organisms may spread through the lymphatics to elsewhere in the lung, the hilar lymph nodes, and the bloodstream, and lay dormant for long periods of time. the combination of the primary site of infection and the involved hilar lymph nodes is known as a ghon complex [ ] . postprimary tuberculosis. this form of tuberculosis represents reactivation of old, scarred primary lesions long after the initial insult. the lesion can occur anywhere in the lung where the bacteria from the primary lesion have spread, but is usually apical. it consists of a focus or organizing pneumonia and fibrosis with central caseation. in an active lesion, the typical parenchymal pattern is an acute pneumonia with cavitation that expands to include the surrounding lung with aggregates of granulomas. the controversy surrounding this lesion arises as some evidence suggests that these lesions represent exogenous reinfection. the pathology of reactivation or reinfection may be indistinguishable, although reactivation tuberculosis may appear to arise out of a fibrotic, calcified chronic lesion [ ] . progressive fibrocavitary disease. this form of the disease may arise out of either primary or postprimary tuberculosis. however, the latter is the more common scenario. the cavities that develop in this form of the disease begin as a slowly progressive, necrotizing pneumonia with abundant granulomas (figure . ). the active disease may spread through the airways, causing ulceration, necrosis, and fibrosis of the surrounding bronchi and bronchioles. the extension of the disease in this way depends on the host, and patients with depressed immune systems can have large areas of the lung involved with massive pulmonary necrosis. usually, a fibrous capsule develops in the area of the cavitation, although inspissated necrotic material into the adjacent airways remains a continuous source of inflammation that can lead to reinfection and ongoing scarring [ ] . nontuberculous mycobacteria the nontuberculous mycobacteria (ntm) are ubiquitous inhabitants of our environment, isolated from soil, fresh and brackish water, house dust, birds, animals, and food, and are increasingly important in causing pulmonary disease. there are currently more than ntm species known. those organisms thought to be pathogenic to the lung include the clinical presentation of these lung infections can vary from minimally symptomatic small lesions discovered by routine radiography to sudden hemoptysis from advanced disease with severe cavitation (table . ). the two most characteristic lesions are those of diffuse infiltrates in an immunocompromised patient, seen most commonly in the hiv-positive population and an viruses most pulmonary infections are due to viruses from four major groups: influenza, parainfluenza, respiratory syncytial virus (rsv), and adenovirus (table . ) [ ] . the clinical presentations of these infections have some common features, including insidious onset, nonproductive cough, fever, and chest pain. chest imaging studies usually reveal bilateral, multifocal infiltrates, most without evidence of cavitation or pleural involvement. these infections are mild, self-limiting, and require no more than supportive therapy except in immunocompromised hosts, where the clinical course can be much more serious. also, immunocompromised patients are susceptible to other viruses such as herpesvirus and cytomegalovirus pneumonias, which are not common pathogens in normal hosts [ ] . since the s, a subset of pulmonary viral infections has emerged with a much more aggressive clinical course, most notably sars, coronavirus, and hantavirus. these viruses present with systemic symptoms of headache, myalgias, and weakness followed by a deteriorating clinical course with respiratory distress, shock and, in over % of the cases, death [ , ] . therapy for most respiratory viral infections is supportive, although antivirals are available for some viruses, mostly used in the setting of immunocompromised patients. ribavirin, a guanosine analogue, is the main antiviral used for rsv; m inhibitors or adamantanes (amantadine and rimantadine) are used against influenza a and neuraminidase inhibitors (oseltamivir and zanamivir) are used against both influenza a and b [ ] . cytomegalovirus is treated with ganciclovir, foscarnet, or cidofovir, while herpesvirus is treated with acyclovir [ ] . the pathologic patterns of injury for most viruses are similar, making morphologic distinctions among them difficult. however, some characteristic patterns emerge, most notably in those viruses that cause cytopathic changes. influenza, adenovirus, sars, coronavirus, and hantavirus all cause an acute lung injury pattern with diffuse alveolar damage, and in the case of the latter two viruses, evidence of hemorrhage and edema. influenza and adenovirus will also cause a necrotizing bronchiolitis due to their preferential infection of bronchial epithelial cells. finally, some viral infections can be distinguished by their characteristic cytopathic inclusions. adenovirus can be identified by characteristic smudge cells that present in advanced stages of the disease and represent adenovirus particles in the nucleus of an infected cell (figure . ). cytomegalovirus has both nuclear owl's eye inclusions, as well as cytoplasmic inclusions (figure . ). herpesvirus has glassy intranuclear inclusions and can also have multinucleation (figure . ). fungi are larger and more complex than bacteria, and their patterns of injury in the lung are different and in general more destructive. these pathogens are common in our environment and enter the lungs through inhalation. though many fungi are capable of causing pulmonary disease, most only inhabit the lung as colonizers. those of most concern for causing clinical disease include the endemic fungi of north america-histoplasma capsulatum, blastomyces dermatitidis, and coccidioides immitis-and two fungi that are commonly seen in immunocompromised hosts-aspergillus fumigatus and pneumocystis jiroveci. histoplasma capsulatum histoplasma capsulatum is a dimorphic fungus most prevalent in the middle portion of the united states from the great lakes to tennessee. the fungus is present in soil that has been contaminated with guano and other debris by nesting birds, most commonly blackbirds and chickens, and by bats. the organism lives in the environment as spores or conidia and germinates to form hyphae. these structures divide to create the yeast forms, which, when inhaled, induce granuloma formation in the lung. approximately % of people have skin tests that are positive for exposure to h. capsulatum, but most exposures do not cause clinical disease. disease typically occurs in people exposed to large amounts of organisms, such as construction workers who move large volumes of dirt or spelunkers who venture into bat-ridden caves. the acute disease has flu-like symptoms which are self-limiting. healed disease may leave behind calcified granulomas in the lung that appear as buckshot on chest imaging studies. the most chronic forms of this disease may slowly progress, giving rise to cavitating and fibrous lesions. in the immunocompromised host, disseminating histoplasmosis can be seen, although reactivation is uncommon [ ] . the pathology reveals characteristic necrotizing granulomas distributed around the airways (figure . ), which contain silver-positive yeast forms of - microns. these granulomas may resolve into scarred nodules, which can calcify and produce the characteristic chest images. cavities may form in the apices with progression of the disease, and the disseminated form of the disease has an abundance of organisms both within macrophages in the lung and throughout many organs in the body. blastomyces dermatitidis blastomyces dermatitidis is also endemic to the middle united states, including the ohio and mississippi river valleys. it is found in wooded terrain, usually during the wet seasons, putting campers and outdoorsmen at risk. the clinical disease takes two forms, cutaneous and systemic, the latter beginning in the lungs through inhalation. the acute pulmonary infection takes a nonspecific form with fever, malaise, and chest pain. imaging studies may show either infiltrates or a mass-like infiltrate. thus, blastomyces infection may mimic other diseases, and the diagnosis may be delayed. some patients go on to chronic disease with cavitation or progressive pulmonary blastomycosis, which manifests as acute respiratory distress syndrome, cavitary lesions, and a poor prognosis [ ] . the pathology of blastomyces infection is similar to histoplasmosis with necrotizing granulomas. however, the lesions are larger, showing more neutrophilic necrosis. the organisms are also larger ( - microns), with prominent broad-based budding, and are apparent on routine hematoxylin and eosin staining (figure . ). coccidioides immitis coccidioides immitis is found in the semi-arid desert climate of the southwestern united states. the organisms are inhaled as spores, causing an acute disease characterized by fever, chills, chest pain, dyspnea, and hemoptysis. chest imaging studies typically show consolidation and cavitation, and hilar lymphadenopathy is common. reactivation and dissemination are possible in patients with previous infection, whether or not they are immunocompromised patients [ ] . the pathology of pulmonary coccidioidomycosis is neutrophilic, suppurative, and granulomatous. the organisms appear as large spherules containing endospores, visible on silver stains. the spherules are - microns in diameter and the endospores that are released into the surrounding tissue proceed to mature into new spherules (figure . ) . as in histoplasmosis, cavitating lesions may have hyphal forms that begin to germinate. aspergillus fumigatus aspergilli are asexual mycelial fungi that are ubiquitous in the environment as airborne aspergillus spores. they are weak pathogens that produce invasive infections predominantly in immunocompromised hosts or in those with significant chronic lung diseases. in tissue, aspergilli form septate hyphae, - microns in diameter, with characteristic acute-angle, dichotomous branching (figure . ) . these organisms affect the lung in three major ways: (i) saprophytic growth in bronchi or pre-existent cavities; (ii) as an allergic or hypersensitivity reaction, predominantly in asthmatics; and (iii) invasive aspergillosis in immunocompromised hosts [ , ] . as a saprophyte, aspergillus produces surface growths or minute masses of hyphae, usually in bronchiectatic cavities, emphysematous bullae, or scars from previous lung diseases such as tuberculosis or sarcoidosis. the pathology is usually that of a fibrous-walled cavity containing degenerating hyphae (figure . ). in this setting, hyphae do not invade into the lung tissue, but surface erosion of a vascularized cavity may cause hemoptysis. aspergillus causes an immunologic response resulting in mucoid impaction or eosinophilic pneumonia in asthmatics, an entity known as allergic bronchopulmonary aspergillosis (abpa). pathologically, one sees mucoid plugs and superficial erosions of the airways with histiocytic inflammation, with only rare hyphal fragments present. the final form of the disease, invasive pulmonary aspergillosis, is found in severely immunocompromised, neutropenic patients. the hyphae, which disseminate through the blood, invade the blood vessels causing thrombosis, hemorrhage, and infarction to form typical targetoid lesions. this form of the disease has a poor prognosis despite aggressive antifungal therapy. pneumocystis jiroveci the taxonomy of pneumocystis jiroveci (formerly pneumocystis carinii) has changed over the past decade. previously thought to be a protozoan based on the histological characteristics of its trophozoite and cyst life forms, it has recently been placed in the fungal kingdom after ribosomal rna was found to have sequences compatible with the ascomycetous fungi [ ] . the inability to culture pneumocystis jiroveci has slowed the understanding of this organism. animal models have helped in defining the antigenic and genotypic differences among the various pneumocystis organisms, which has led to the proposal for species-specific strains, with p. jiroveci found in human infections [ ] . the molecular methods used for the typing these species examine a number of gene loci. most importantly, sequence analysis of the thymidylate synthase (ts) and superoxide dismutase (soda) gene loci, the epsp synthase domain of the multifunctional arom gene, and the mitochondrial small subunit ribosomal rna (mtssu rrna) locus have been used to distinguish the various pneumocystis species that infect different mammalian hosts [ ] . clinically, p. jiroveci causes disease predominantly in the immunocompromised setting. pneumocystis pneumonia (pcp) has been found during recent times most commonly in the aids population, but prior to this epidemic, it was found in malnourished infants and other severely immunocompromised hosts. because this organism has not been cultured, the diagnosis of pcp continues to be challenging. the clinical characteristics are nonspecific and vary with the patient's immune status. in the hiv population, patients typically develop a subacute onset of progressive dyspnea, a nonproductive cough, malaise, and a low-grade fever. in the non-hiv population, the presentation is more acute, with fulminant respiratory failure associated with cough and fever, and usually requiring mechanical ventilation [ ] . chest imaging studies typically show bilateral, symmetric, fine reticular interstitial infiltrates involving the perihilar area, which spread to involve the entire lung. figure . aspergillosis. aspergillus fumigatus grows within necrotizing cavities of the lung as branching septated fungal hyphae, as seen on this grocott methenamine silver stain. figure . aspergilloma. fungal hyphae from aspergillus fumigatus can colonize chronically inflamed lungs with cavities and may grow to form fungal balls with a dark, green color that are treated by surgical resection, as seen in this case of a lobectomy specimen. treatment is usually with trimethoprim/sulfamethoxazole and intravenous pentamidine. survival is %- % even in severely immunocompromised patients. the life cycle of p. jiroveci consists of three stages: trophozoite, cyst, and sporozoite. the trophozoite form, which adheres to the type epithelium, replicates and enlarges through three precyst stages before maturing into a cyst form that is found in the alveolar space. sporozoites develop within immature cysts through meiosis and mitosis. the mature cyst contains eight haploid sporozoites. the rupture of the cyst wall releases sporozoites into the surrounding environment where they mature into trophozoites. the pathology of the infection is predominantly due to the interaction of the organism with the epithelium. the attachment of the organism to the lung epithelium is via glycoprotein a present on the surface of the organism. the binding of the organism to the type cell occurs via surface receptors on the type cell that include macrophage mannose receptors. these interact with glycoprotein a and activate pathways in the organism that induce genes encoding for pathways that induce mating and proliferation responses, and for the formation of pheromone receptors, transcription factors, and heterotrimeric g-protein subunits [ ] . in addition to these genetic effects, the cyst wall contains chitins, polymers, and other substances, in particular, , -glucan, that maintain its integrity and induce the inflammatory response of the host. the , -glucan in the wall of the organism stimulates the release by the macrophages of reactive oxidant species and the generation of potent proinflammatory cytokines, such as tnfa, which bind to the organism and exert a toxic effect. once inside the macrophage, the organism is incorporated into the phagolysosome and degraded. tnfa also directly recruits other inflammatory cells including neutrophils, lymphocytes, and circulating monocytes, and induces the release of il- and ifn-g that recruit and activate inflammatory cells [ ] . in aggregate, the recruitment of these inflammatory cells and the mediators they release is responsible for the damage to the lung epithelium and endothelium that is seen in this disease [ ] . the pathology of pcp has typical and atypical variants. typically, the lung contains a dense interstitial plasma cell pneumonia that expands alveolar walls. the epithelium consists predominantly of type pneumocytes, and the alveolar spaces contain an eosinophilic, frothy exudate, which contains fine, hemoxylin-stained dots that represent a thickening in the cyst wall (figure . ) . in this form of the disease, the organisms are abundant and the diagnosis can usually be made by bronchoalveolar lavage. atypical pathologic variants include a necrotizing variant that has a pattern similar to the typical form with exudative alveolar infiltrates, but which undergoes necrosis and cavity formation. these cavities heal into fibrous-walled cysts, similar in gross appearance to those found in pseudomonas pneumonia. a third variant has wellformed granulomas involving the airways, a pattern common to histoplasmosis and tuberculosis. in this form, the organisms are rare and very difficult to find, even with tissue organismal stains. in general, the pathologic pattern of injury depends on the host's immune status, with the typical pathology found in severely immunocompromised hosts as the aids population and the atypical forms found in hosts with immune systems that are less compromised. pulmonary langerhans cell histiocytosis (plch) and erdheim-chester disease are histiocytic diseases that primarily affect the lung. other histiocytic diseases may affect the lung, such as niemann-pick disease, gaucher disease, hermansky-pudlak and rosai-dorfman disease, but these are not considered primarily lung histiocytic diseases. pulmonary langerhans' cell histiocytosis (plch) is a disease of the dendritic histiocytes of the lung referred to as langerhans' cells (lcs). this disease is part of a group of diseases that are characterized by a proliferation of langerhans cells in organs throughout the body that range from a malignant systemic disease as is seen in children [ ] to the pulmonary variant that is seen in adolescents and adults. plch is usually the result of inflammatory or neoplastic stimuli in lungs of smokers or in lungs involved by certain neoplasms [ ] . chest radiographs from patients with plch usually reveal bilateral nodules, predominantly in the upper lobes, which are worrisome for metastatic disease. treatment involves smoking cessation and steroid therapy. typically, the disease undergoes spontaneous regression. approximately %- % of patients will progress to irreversible end-stage fibrosis [ ] . the pathology of plch consists of airway-based lesions with a proliferation of lcs. the early cellular lesions contain a mixture of cells including langerhans' cells, lymphocytes, plasma cells, and eosinophils ( figure . ). though it was previously referred to as eosinophilic granuloma, eosinophils are not the major cell type present, and the lesion is, at best, a loosely formed granuloma. immunohistochemistry reveals the lcs to be diffusely, strongly immunoreactive to s- protein and cd a. ultrastructural analysis reveals intracytoplasmic organelles called birbeck granules, a normal constituent of langerhans' cells, in greater numbers in plch [ ] . the pathogenetic mechanisms of plch focus on defects in the homeostasis of dendritic cells (dcs) in the lungs of smokers and the role tobacco smoke may play in stimulating the proliferation of these cells [ ] . some studies suggest that stimulation of alveolar macrophages by chemicals in smoke results in secretion of such cytokines as gm-csf, tgfb, and tnfa [ ] . in transgenic mice, accumulation of dcs around airways may be a result of excess gm-csf [ ] . other theories suggest that cigarette smoke stimulates the secretion of bombesin-like peptide by the neuroendocrine cells in the bronchiolar epithelium and leads to a similar stimulation of alveolar macrophages and a cytokine milieu that promotes the proinflammatory proliferative changes [ ] . not all smokers get plch, leading to the suggestion that only smokers with an underlying genetic susceptibility will develop the disease. studies have established that in some cases the lcs in plch are clonal, suggesting that cellular abnormalities must play some part in the pathogenesis of the diseases [ ] . to support this, studies have shown genetic mutations and allelic loss of tumor suppressor genes in smokers with plch [ ] . the mechanisms by which this proliferation of lcs leads to the destruction of the bronchiolar epithelium and the other observed pathology are unclear. lcs in normal lungs have little ability to interact with t-cells or act as effective antigen-presenting cells, but the lcs of plch have a mature immunophenotype, expressing b - and b - , the co-stimulatory molecules needed for lymphostimulatory activity [ ] . whether this more mature immune phenotype leads to an unregulated immune response and destruction of the bronchial epithelial cells is not known. however, some studies have shown that bronchiolar epithelial cells may induce the expression of this mature phenotype by secreting cytokines in response to environmental stimulants such as cigarette smoke or viral infections, or by the development of hyperplastic or dysplastic lesions that express new foreign antigens [ ] . erdheim-chester disease (ecd) is a systemic non-langerhans' cell histiocytosis of adults that most commonly involves the long bones. involvement of other organs, including the lung, has been reported. lung involvement occurs in approximately %- % of the cases, and the patients usually present with cough, dyspnea, rhonchi, and pleuritic pain. radiographically, the lungs reveal infiltrates in a lymphatic distribution, predominantly upper lobe, with prominent interstitial septal markings that can mimic sarcoidosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . pulmonary involvement by ecd may have an unfavorable prognosis, and the fibrosis that ensues is one of the most frequently reported causes of death [ , ] . the treatment of ecd is variable with corticosteroids, chemotherapy, surgical resection, and radiation therapy reported [ ] . non-langerhans' cell histiocytes of dendritic cell phenotype are the main cells present in this disease. this infiltrate contains foamy histiocytes with scattered giant cells, a scant number of lymphocytes or plasma cells, and some fibroblasts. the histiocytes express cd (macrophage antigen) and factor xiiia (dendritic cell antigen), but express s- protein weakly or not at all, and do not express cd a. ultrastructural analysis reveals phagolysosomes, but no birbeck granules are present [ ] . this infiltrate that involves the lung is usually present in the pleura and subpleura, within the interlobular septa and around the bronchovascular structures. the remainder of the lung parenchyma is unremarkable, though fibrosis and paracicatricial emphysema can appear in the late stages of the disease [ ] . the etiology of ecd is not known, but this rare disease has been established as primarily a macrophage disorder [ ] . these histiocytes have abundant phagolysosomes and express the antigen cd a and are consistent with a phagocytic cell, most likely closely related to alveolar macrophages. the peripheral monocytosis and the proinflammatory cytokine profile that is found in these patients might suggest that the histiocytic infiltrate is a result of systemic monocytic activation and invasion of circulating monocytes into the tissues throughout the body [ ] . recently, an ecd patient was successfully treated by an agent toxic to monocytes, supporting the theory that these cells play a part in the disease [ ] . alternatively, end organ cytokine production by local inflammatory cells resulting in proliferation and differentiation of resident immature histiocyte populations may produce a similar picture. another interesting observation is that erdheim-chester has been reported to occur in patients with langerhans' cell histiocytosis [ ] , which may suggest that this is a disease where macrophages transition between two different phenotypes along the differentiation spectrum of tissue dendritic cells [ ] . whether this is a benign or malignant proliferation has not been established. of patients studied, clonality has been demonstrated in by polymerasechain reaction [ ] . environmental exposures are a major cause of lung disease and can cause a wide spectrum of both acute and chronic pathology. many organic and inorganic materials can cause lung damage, and because of their similar patterns of injury and long latent periods, it can be difficult to isolate the exact offending agent without a thorough clinical history. the two occupational lung diseases presented here-asbestosis and silicosis-represent pneumoconiosis, which are defined as diseases which result in diffuse parenchymal lung injury due to inhaled inorganic material. both have many pathologic patterns of injury that depend on the amount and length of time of exposure, and both can also cause neoplastic diseases of the lung. asbestos fibers are naturally occurring silicates that are commonly used in construction materials such as cement and insulation and in many textiles. they can be separated into two groups based on their mineralogic characteristics. serpentine fibers, named as such because they are long and curly, include chrysotile asbestos. amphibole fibers, more straight and rodlike, include predominantly amosite and crocidolite asbestos. in the united states most of the asbestos is chrysotile. the amphiboles are more pathogenic and are responsible for most of the neoplastic and non-neoplastic pulmonary diseases associated with asbestos exposure. by definition, asbestosis is bilateral diffuse interstitial fibrosis of the lungs that can be attributed to asbestos exposure. the disease, which mostly affects textile and construction workers, is usually the result of direct exposure over - years. the latency to clinical disease is inversely proportional to the level of exposure. the symptoms are a gradual onset of shortness of breath, a cough with dry rales at the bases on inspiration, and digital clubbing. in the early disease, the chest x-ray shows basilar disease that begins predominantly as thickening of the subpleural, but progresses as infiltrates and fibrosis that involve the middle zone, eventually leading to thickening of the airways and traction bronchiectasis. the apex of the lung is usually spared. the clinical findings are nonspecific and have considerable overlap with uip, so the diagnosis is usually made only when a history of significant exposure is discovered. the gross picture includes a bilateral lower lobe gray/tan fibrosis with honeycomb changes in late disease. microscopically, asbestosis can cause many patterns of injury in the lung, but the most common is collagenous deposition in the areas of the lymphatics where the fibers are in the highest concentration. these areas include the subpleural, interlobular septae, and around the bronchovascular areas that contain a bronchiole and a branch of the pulmonary artery. hyalinized pleural plaques are a common manifestation of asbestos exposure but are not specific for asbestos and can be found in the absence of pulmonary parenchymal disease. eventually, the fibrosis involves the alveoli beyond the bronchioles and causes distortion of the lung architecture to form remodeled, dilated airspaces similar to those seen in uip. distinguishing this fibrosis from other forms of fibrosing lung disease can be difficult, but the presence of ferruginous bodies, asbestos fibers coated by iron, proteins, and a mucopolysaccharide coat are indicative of significant asbestos exposures and support this diagnosis (figure . ) [ ] . figure . asbestosis. this cytopathologic preparation from a bronchoalveolar lavage specimen illustrates an asbestos fiber coated by an iron-protein-mucopolysaccharide substance and appears as a golden brown, beaded structure known as a ferruginous body. silicosis results from chronic, high-dose exposure to crystalline silica, which consists of silicon and oxygen with trace amounts of other elements, usually iron. the most common silica is quartz, which is present in large amounts in such rocks as granite, shale, and sandstone and is among the more fibrogenic of all silica types. thus, occupations most at risk for silicosis include sandblasting, quarrying, stone dressing, and foundry work where exposure to quartz is high. the disease takes three major clinical and pathologic forms that have different clinical characteristics. simple or nodule silicosis is marked by the presence of fine nodules cm, on chest imaging studies, usually in the upper lobes. patients with this condition are typically asymptomatic, with normal respiratory physiology. the pathology in these lungs reveals discrete, hard nodules that have a green/gray color, centered either on the small airways or in the subpleura. microscopically, these nodules have an early stellate shape that eventually transforms to a more whorled appearance with dustladen macrophages scattered throughout it. polarized light examination reveals weakly birefringent material. complicated pneumoconiosis represents similar pathologic findings only with larger and more circumscribed nodules, which coalesce into a large upper lobe mass, a condition known as progressive, massive fibrosis ( figure . ) . these patients are symptomatic with a productive cough and mixed pulmonary function tests with a reduced diffusing capacity as the fibrosis increases. diffuse interstitial fibrosis may occur; however, unlike asbestosis, this pattern is found in pneumoconiosis. when complicated pneumoconiosis is found with rheumatoid nodules in the setting of a patient with rheumatoid arthritis, this is known as caplan's syndrome. the pathogenesis of both asbestosis and silicosis depends upon inflammation and fibrosis caused by the inhaled fibers. in humans, the amount of fiber needed to cause fibrosis varies from person to person. this may be related to a difference in fiber deposition based on the size of the lungs or to the efficacy with which the lung clears these fibers [ ] . some studies have also suggested that fiber length determines the amount of pathology. however, this association has not been confirmed in humans for either asbestosis or silicosis. in both diseases, it is known that other factors increase the risk of developing disease. for example, smokers exhibit worse disease than nonsmokers with similar exposures to asbestosis. the mechanism for this effect is unclear, although speculation centers on the inhibition of fiber clearance in smokers. also, it is known that smoking enhances the uptake of fibers by pulmonary epithelial cells and in this way may increase the fibrogenic and inflammatory cytokine production by these cells. the cellular mechanisms by which both asbestos and silica fibers induce the inflammation and fibrosis are mediated predominantly through alveolar macrophages. in the case of silica, it is known that the uptake of these fibers into the alveolar macrophages is by way of a scavenger receptor expressed on the surface of the cell known as marco (macrophage receptor with a collagenous structure). once inside the cells, the fibers activate the release of ros that can lead to cellular and molecular damage through a number of pathways. first, ros can directly cause lipid peroxidation, membrane damage, and dna damage. second, silicainduced free radicals can trigger phosphorylation of cellular proliferation pathways through mitogen-activated protein kinases (mapks), extracellular signal regulated kinases (erks), and p . these pathways are also involved in the proliferation of fibroblasts in asbestosis and of mesothelial and epithelial cells in the neoplastic diseases associated with the inhalation of these fibers [ ] . in addition, these fibers can activate proinflammatory pathways controlled by such transcription factors as nuclear nfkb and activator protein (ap- ). these pathways result in the activation of the early response genes c-fos and c-jun and the release of proinflammatory cytokines such as il- as well as fibrogenic factors such tnfa [ ] . tnfa plays a prominent role in both diseases, and its regulation has been studied in animal models exposed to silica. it is now known that a transcription factor labeled nuclear factor of activated t-cells (nfat) plays a key role in the regulation of tnfa. figure . complicated pneumoconiosis/ progressive massive fibrosis. this sagittal cut section of lung reveals a large gray/black mass that extends from the apex to include the majority of the lung. the patient had a long history as a coal mine worker, and the microscopic sections revealed abundant anthracotic pigment and scarring in this area. binding sites for nfat have been found in the promoter region of the tnfa gene. the mediation of silica-induced tnfa transcription is probably via o -but not h o [ , ] . atresia of the lung represents a premature closure of the airway at any level of the bronchial tree including the lobar, segmental, or subsegmental airways. clinically, these children usually present between and years of age for symptoms of dyspnea, wheezing, recurrent pneumonias, or for incidental findings on a chest imaging study. these lesions are more common in the proximal segmental bronchi, right more often than left. when atresia is associated with anomalies of the vascular supply to the affected airway, the lesion represents a separate, aberrant segment of lung known as a sequestration, either intralobar or extralobar type. the pathology of bronchial atresias and sequestrations represents sequelae of chronic inflammation due to the accumulation of secretions in these blind-end airways. these features consist of cystically dilated airways with mucus and parenchymal fibrosis with honeycomb changes. in intralobar sequestrations (ils), the anomalous vessel is a muscular artery that enters through the pleura from an aortic source, usually from the thoracic area. ils are separate, isolated areas of lung invested with the normal visceral pleura without bronchial or arterial connections (figure . ). extralobar sequestrations (els) are pyramid-shaped accessory pieces of lung that have their own pleura with an artery from the lung but without airway connections. the category of congenital pulmonary cystic diseases represents the majority of congenital pulmonary disease and includes foregut cysts and cystic adenomatoid malformations. foregut cysts include bronchogenic, esophageal, and thymic cysts that form from defects in the foregut branching. clinically, these cysts are usually incidental findings on chest imaging studies, but they can present with complications due to infection or hemorrhage. pathologic features of these cysts include subtle differences that are usually only apparent after microscopic examination. grossly, these cysts usually arise proximally either within the mediastinum (over %) or in the proximal regions of the lungs, right more commonly than left, along the esophagus, and rarely within the lung parenchyma or below the diaphragm [ ] . microscopically, each cyst contains a simple cuboidal or columnar epithelium, ciliated or nonciliated, that may undergo squamous metaplasia. distinguishing among the three types of cysts requires the presence of other elements. bronchogenic cysts have submucosal glands and/or hyaline cartilage within their walls, and thymic cysts may contain residual thymus. congenital cystic adenomatoid malformations (ccam), now more commonly referred to as congenital pulmonary airway malformations (cpam), are segments of lung with immature airways and alveolar parenchyma. these are usually classified by their predominant cyst size into types - . type cysts, which contain a main large cyst of up to cm, are the most common. these cysts are distinguished from foregut cysts upon the recognition in the cpam of immature alveolar duct-like structures connecting to the surrounding lung parenchyma. this type of cpam is also notable, as it is known to undergo malignant transformation, usually to mucinous bronchioloalveolar cell adenocarcinomas. these anomalies arise due to defects during the various stages of development and are best considered within these developmental stages. the embryonic stage occurs within the first - weeks of life when the ventral wall of the foregut separates into the trachea and esophagus and branches to form the left and right lungs. the splanchnic mesenchyme that surrounds this foregut forms the vascular and connective tissues of the lungs. defects in this phase result in complete lack of lung development known as pulmonary agenesis and incomplete separation of the trachea and esophagus, causing tracheal-esophageal atresias and fistulas. the pseudoglandular stage, between weeks - of development, is a time of rapid development of the conducting airways including the bronchi and bronchioles and the expansion of the peripheral lung into the acinar buds. the mesenchymal tissue figure . intralobar sequestration. the tan and white mass involving this left lower lobectomy specimen represents chronic pneumonia and fibrosis in the sequestered area of the lung. the dilated airways are features of an endstage fibrosis that is commonly found in this entity. part iv molecular pathology of human disease that surrounds these buds begins to thin, becomes vascularized, and forms the cartilage that surrounds the more proximal branching airways. during the canalicular (week [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , saccular (weeks [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and alveolar (weeks to maturity) stages of development, the acinar buds continue to expand, and the mesenchyme surrounding this continues to thin. during the canalicular stage, the pulmonary vascular bed begins to organize, the distance between the blood in the vascular spaces and the air in the alveoli narrows, and the respiratory epithelium begins to form. the gas exchange unit of the alveolus becomes functional during the saccular stage with further differentiation of the respiratory epithelium to include clara cells, ciliated and nonciliated cells, and type cells with the first production of surfactant occurring during this period. this gas exchange unit continues to mature during the alveolar stage with the growth and septation of the alveoli. this process continues postnatally through - years of age. the different types of cpams arise at different stages of development. cpams , , and are a result of defects during the early embryonic and pseudoglandular stages of development, producing pathology with features of primitive alveolar buds and immature and abnormal airway cartilage structures. cpams and result from abnormal formation of the more distal airways and pulmonary parenchyma during the canalicular, saccular, and alveolar phases, causing pathology with immature alveolar, or alveolar simplification with enlarged alveoli [ ] . various genetic defects in the pathways that control lung morphogenesis have been associated with these congenital lung diseases. two major transcription factors are responsible for the normal branching morphogenesis. the first, thyroid transcription factor- (ttf- ), is a member of the nkx . family of hemeodomain-containing transcription factors. this factor plays a role in the lung epithelial-specific gene expression and proper lung bud development in the embryonic stage, as well as in the maturation of the respiratory epithelium. the second major factor is somatic hedgehog (shh)/gli, expressed by endodermally derived cells and required for branching morphogenesis. the development of the lung bud from the foregut endoderm depends on the appropriate expression of these lung-specific genes at the correct time in development. in the presence of genetic defects, aberrant lung development may occur. for example, mutations of various types in the shh/gli gene have been found to cause tracheoesophageal fistulas, anomalous pulmonary vasculature, and aberrant airway branching. also, deletions in the ttf- gene are associated with tracheoesophageal fistulas and a variety of forms of lung dysgenesis [ ] . finally, factors present in the surrounding mesenchyme play a role in inducing the proper development of the pulmonary endoderm. a prominent mesenchymal factor in this process is fibroblast growth factor (fgf), which modulates both the proximal and distal lung branching morphogenesis. deletions in this gene may cause lung agenesis and tracheal malformations [ ] . surfactant dysfunction disorders represent a heterogenous group of inherited disorders of surfactant metabolism, found predominantly in infants and children. pulmonary surfactant includes both phospholipids and surfactant proteins, designated surfactant proteins a, b, c, and d (sp-a, sp-b, sp-c, sp-d), synthesized and secreted by type cells beginning in the canalicular stage of lung development. damage to type cells during this time period can lead to acquired surfactant deficiencies. however, more commonly these deficiencies are the result of genetic defects of the surfactant proteins themselves. the major diseases are caused by genetic defects in the surfactant protein b (sftpb, chromosome p -p . ); surfactant protein c (sftpc, chromosome p ); and adenosine triphosphate (apt)-binding cassette transporter subfamily a member (abca , chromosome p . ). defects in sftpb and abca have an autosomal recessive inheritance pattern, and defects in sftpc have an autosomal dominant pattern. sp-b deficiency is the most common. it presents at birth with a rapidly progressive respiratory failure and chest imaging studies showing diffuse ground glass infiltrates. the gross pathology in these lungs consists of heavy, red, and congested parenchyma with microscopic features that range from a pap-like pattern to a chronic pneumonitis of infancy (cpi) pattern. in sp-b deficiency, the pap pattern predominates with a histologic picture of cuboidal alveolar epithelium and eosinophilic pas-positive material within the alveolar spaces that appears with disease progression. in the late stages of the disease, the alveolar wall thickens with a chronic inflammatory infiltrate and fibroblasts. this alveolar proteinosis-type pattern of injury can be confirmed with immunohistochemical studies that establish the absence of sp-b within this surfactant-like material. diseases due to abca or sftpc deficiency may present within a week of birth or years later; the former has a poor prognosis, but the latter has a more variable prognosis with some patients surviving into adulthood. indeed, sp-c mutations have also been recognized in some families as a cause of interstitial pneumonia and pulmonary fibrosis in adults [ ] . the pathology of sp-c deficiency has more cpi features and less proteinosis. in contrast, abca deficiency can have either pap or cpi features, with the former present early in the disease and the latter present in more chronically affected lungs [ ] . the sp-b gene (sftpb) is approximately kb in length and is located on chromosome . there are over recessive loss-of-function mutations associated with the sftpb gene. however, the most common mutation is a gaa substitution for c in codon , found in about % of the cases. the lack of sp-b leads to an abnormal proportion of phosphatidylglycerol and an accumulation of a pro-sp-c peptide, leading to the alveolar proteinosis-like pathology. sp-c protein deficiency is due to a defect in the sftpc gene localized to human chromosome . there are approximately dominantly expressed mutations in sftpc that result in acute and chronic lung disease. approximately % of them arise spontaneously, and the remainder are inherited. the most common mutation is a threonine substitution for isoleucine in codon (i t), found in % of the cases, including both sporadic and inherited disease [ ] . this mutation leads to a misfolding of the sp-c protein, which inhibits its progression through the intracellular secretory pathway, usually within the golgi apparatus or the endoplasmic reticulum [ ] . the absence of sp-c within the alveolar space causes severe lung disease in mouse models. infants with documented mutated prosp-c protein, the larger primary translation product from which sp-c is proteolytically cleaved, can have respiratory distress syndrome (rds) or cpi. in older individuals, pathologic patterns observed in the lungs with these mutations include nonspecific interstitial pneumonitis (nsip) and uip. in this affected adult population, the pathology and age of disease presentation vary even within familial cohorts, suggesting the involvement of a second hit, perhaps an environmental factor [ ] . the abca protein is a member of the family of atpdependent transporters, which includes the cftr, and is expressed in epithelial cells. mutation in this gene results in severe respiratory failure that is refractory to surfactant replacement. the cellular basis for the lack of surfactant in patients with this genetic mutation is not known. the presence of abnormal lamellar bodies within the type cells by ultrastructural analysis suggests a disruption in the normal surfactant synthesis and packaging in this disease. there is some evidence that this gene contains promoters that share elements consistent with their activation by the 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constitutes a large and often unknown reservoir. wildlife can also be a source for reemergence of previously controlled zoonoses. although the discovery of such zoonoses is often related to better diagnostic tools, the leading causes of their emergence are human behavior and modifications to natural habitats (expansion of human populations and their encroachment on wildlife habitat), changes in agricultural practices, and globalization of trade. however, other factors include wildlife trade and translocation, live animal and bushmeat markets, consumption of exotic foods, development of ecotourism, access to petting zoos, and ownership of exotic pets. to reduce risk for emerging zoonoses, the public should be educated about the risks associated with wildlife, bushmeat, and exotic pet trades; and proper surveillance systems should be implemented. deforestation, development of human habitat, and mining activities have been suggested as risk factors associated with the reemergence of vampire bat rabies in humans in the amazon basin. in , persons died of rabies transmitted by vampire bats, mainly in brazil ( cases) and colombia ( cases) ; only human cases of rabies were transmitted by dogs in all latin america ( ) . a similar trend was again observed for . when fi rst described in , kyasanur forest disease was restricted to a much smaller area ( square miles) in india than the actual , square miles of endemic zone ( ) . this tickborne disease occurs in evergreen rain forests interspersed with deciduous patches and clearings for rice cultivation and human habitations. forest workers are particularly at risk; their mortality rates may reach %. in , a major epidemic occurred during which several monkeys died, , humans were infected, and humans died. the outbreak occurred in previously undisturbed forest where some ha were clearcut to establish a cashew tree plantation. most of the human patients were immigrant laborers employed to clear the forest ( ) . as many as , human cases occur each year, and this number has increased in the past years. most cases occur during the dry season (january-may), when nymphal activity is maximal. such a zoonosis is a good example of deforestation and agricultural development leading to human habitat expansion into natural foci of a viral infection. because cleared areas were widely used for grazing of cattle, a major host for adult ticks, these areas favored the proliferation of the tick haemaphysalis spinigera. conversely, the reduction of traditional agricultural land and its replacement with forested areas, home to the main reservoirs and hosts of borrelia burgdorferi, in association with the settlement of persons in periurban areas, led to a considerable increase in human cases of lyme disease in the united states ( ) . an estimated . million wild ruminants, major amplifi ers for adult ixodes scapularis ticks, live in north america ( ) . from an estimated - million white-tailed deer inhabited north america before the arrival of europeans, the deer population was greatly reduced by habitat loss and unrestricted hunting. however, by the mid- th century, the population was restored throughout north america, and an estimated - million white-tailed deer are believed to inhabit the united states alone. in many areas of the eastern united states, populations have soared to previously unattained levels (www.aphis.usda.gov/ws/nwrc/is/living/deer.pdf). human activities may also be a source of wildlife infection, which could create new reservoirs of human pathogens. the recent outbreak of tuberculosis caused by mycobacterium tuberculosis in suricats and mongooses was one of the fi rst documented spillovers of a human disease within a wildlife population ( ) . banded mongooses were observed feeding regularly at garbage pits and were therefore exposed to human excretions and any infectious material from tuberculosis-infected humans. the emergence of argentine hemorrhagic fever in eastcentral argentina during the s, and its expansion to north-central argentina, has been directly linked to development of agricultural activities (mainly corn growing) that sustain the virus's main reservoir, the corn mouse (calomys musculinus). caused by the junin virus, argentine hemorrhagic fever affects primarily adult male agricultural workers, mainly during the harvest season ( ) . in the late s and early s, a rabies epidemic occurred in free-ranging greater kudus (tragelaphus strepsiceros) in namibia ( ) . the kudu population had increased considerably in response to favorable conditions and human-made environmental changes. suitable conditions for transmission in the kudu population after initial infection by rabid carnivores are provided by the social behavior of kudus, such as browsing on thorny acacia trees and resultant lesions in the kudus' oral cavity, and excretion of relatively high titers of virus in the saliva of infected animals ( ) . the outbreak of nipah virus infection in malaysia during - , which caused human cases of viral encephalitis and a % mortality rate, was also the result of several major ecologic and environmental changes associated with deforestation and expansion of nonindustrial pig farming in association with production of fruit-bearing trees ( ) . such combination led to infection of pigs, which developed respiratory and neurologic symptoms after indirect exposure to infected fruit bats that shed the virus. the sick pigs were a subsequent source of human infection ( ) . farming of wild animal species led to reemergence of zoonoses such as bovine tuberculosis in captive deer populations. deer at low population densities on natural range are less likely to be affected to any major extent by disease. however, disease becomes a factor in intensive management of deer ( ) . reemergence of zoonotic diseases that had been controlled from their domestic animal reservoirs is also of major concern. wildlife may become new reservoirs of infection and may recontaminate domestic animals; examples include bovine tuberculosis in the united kingdom associated with mycobacterium bovis infection in badgers (meles meles) ( ) and brucellosis in outdoor-reared swine in europe that resulted from spillover from the wild boar brucellosis (brucella suis biovar ) reservoir ( ) . wildlife trade provides mechanisms for disease transmission at levels that not only cause human disease out-breaks but also threaten livestock, international trade, rural livelihoods, native wildlife populations, and ecosystem health ( ) . worldwide, an estimated , primates, million birds, , reptiles, and million tropical fi sh are traded live each year ( ) . international wildlife trade is estimated to be a us $ -billion industry ( ) . translocation of wild animals is associated with the spread of several zoonoses. rabies was introduced in the mid-atlantic states in the s when hunting pens were repopulated with raccoons trapped in rabies-endemic zones of the southern united states ( ) . in eastern europe, raccoon dogs (nyctereutes procyonoides) are becoming a new reservoir for rabies, in addition to the established red fox reservoir, as raccoon dogs have spread into new habitats from accidental release of animals raised for fur trade ( ) . brush-tailed possums (trichosurus vulpecula) from tasmania were introduced into new zealand to establish a new species of fur-bearing animals. the translocated population proliferated and is now estimated to be > million, of which %- % are possibly infected by m. bovis, a permanent threat to the cattle-and deer-farming industries ( ) . translocation of hares from central and eastern europe for sporting purposes led to several outbreaks of tularemia, introduction of b. suis biovar to western europe, and subsequent encroachment of this brucellosis strain into the wild boar population of western europe ( ) . during - , b. suis biovar infections were reported in > outdoor-rearing pig farms in france ( ) . illegal trade can also be a possible source of human infection. in march , psittacosis developed in several customs offi cers in antwerp, belgium ( ) . a customs officer had been hospitalized with pneumonia days after exposure to parakeets illegally imported by an indian sailor. the risk of contracting psittacosis was . × higher for offi cers exposed to parakeets > hours than for those exposed only briefl y. similarly, a highly pathogenic avian infl uenza a h n virus from crested hawk eagles smuggled into europe by air travel has been isolated and characterized ( ); fortunately, however, screening of human and avian contacts indicated that no dissemination had occurred. another risk factor related to the emergence of zoonotic diseases from wildlife has been the considerable increase in consumption of bushmeat in many parts of the world, especially central africa and the amazon basin, where - . million tons and - million kilograms, respectively, are consumed each year ( ) . the simian foamy virus has been identifi ed as a zoonotic retrovirus that infects people who have direct contact with fresh nonhuman primate bushmeat; this fi nding indicates that such zoonoses are more frequent, widespread, and contemporary than previously appreciated. similarly, new retroviruses, human t-lymphotropic virus types and were found in persons who hunt, butcher, or keep monkeys or apes as pets in southern cameroon ( ) . the combination of urban demand for bushmeat (a multibillion-dollar business) and greater access to primate habitats provided by logging roads has increased the amount of hunting in africa, which has increased the frequency of human exposure to primate retroviruses and other disease-causing agents. similarly, several outbreaks of ebola virus in western africa have been associated with consumption of bushmeat, mainly chimpanzees that were found dead ( ) . traditional and local food markets in many parts of the world can be associated with emergence of new zoonotic diseases. live animal markets, also known as wet markets, have always been the principal mode of commercialization of poultry and many other animal species. such markets, quite uncommon in the united states and, until recently, in california, are emerging as a new mode of commercialization within specifi c ethnic groups for whom this type of trade assures freshness of the product but raises major public health concerns. the avian infl uenza epidemic, which began in southeast asia in and recently spread to other parts of the world, is directly related to infected birds sold live in traditional markets. live bird markets facilitate the spread of this avian h n virus by wild birds ( ) . similarly, the newly discovered severe acute respiratory syndrome-associated coronavirus was linked to trade of live, wild carnivores, especially civets, in the people's republic of china ( ). however, recent data suggest that civets may be only amplifi ers of a natural cycle involving trade and consumption of bats ( ) . trichinellosis has long been associated with consumption of undercooked meat from wild animals, such as bears, and now consumption of uncooked meat from deer and wild boar has recently been associated with emergence of severe cases of hepatitis e in hunters in japan ( ) . industrialized nations' new taste for exotic food has also been linked with various zoonotic pathogens or parasites, such as protozoa (toxoplasma), trematodes (fasciola sp., paragonimus spp.), cestodes (taenia spp., diphyllobothrium sp.), and nematodes (trichinella spp., anisakis sp., parastrongylus spp.). adventure travel is the largest growing segment of the leisure travel industry; growth rate has been % per year since (adventure travel society, pers. comm.). this type of travel increases the risk that tourists participating in activities such as safaris, tours, adventure sports, and extreme travel will contact pathogens uncommon in industrialized countries. the most commonly encountered rickettsial infection in travel medicine is african tick bite fever, caused by rickettsia africae and transmitted in rural sub-saharan africa by ungulate ticks of the amblyomma genus; > imported cases have been reported from several continents during the past few years ( ) . most patients are infected during wild game safaris and bush walks. moreover, because ecotourism is becoming increasingly popular with international travelers, more cases of imported rickettsioses are likely to occur in europe, north america, and elsewhere in years to come. cercopithecine herpesvirus (herpes b virus) is an alpha herpesvirus endemic to asian macaques, which mostly carry this virus without overt signs of disease. however, zoonotic infection with herpes b virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment ( ) . herpes b virus has been implicated as the cause of ≈ cases of meningoencephalitis in persons who had direct or indirect contact with laboratory macaques. a survey of workers at a balinese hindu temple, a major tourist attraction where macaques roam free, showed that contact suffi cient to transmit b virus occurred commonly between humans and macaques. furthermore, ( . %) of macaques at that location had antibodies to herpes b virus ( ) . petting zoos, where children are allowed to approach and feed captive wildlife and domestic animals, have been linked to several zoonotic outbreaks, including infections caused by escherichia coli o :h , salmonellae, and coxiella burnetii ( ) . more than outbreaks of human infectious diseases associated with visitors to animal exhibits were identifi ed during - ( ) . in an outbreak of salmonellosis at a colorado zoo, cases (most of them in children) were associated with touching a wooden barrier around the komodo dragon exhibit. salmonella organisms were isolated from case-patients, a komodo dragon, and the wooden barrier. children who did not become infected were more likely to have washed their hands after visiting the exhibit ( ) . exposure to captive wild animals at circuses or zoos can also be a source of zoonotic infection. twelve circus elephant handlers at an exotic animal farm in illinois were infected with m. tuberculosis, and had signs consistent with active disease after elephants died of tuberculosis. medical history and testing of the handlers indicated that the elephants had been a probable source of exposure for most of the infected persons ( ) . after an m. bovis outbreak in rhinoceroses and monkeys at a zoo in louisiana, animal handlers, previously negative for tuberculosis, had positive test results ( ) . exotic pets are also a source of several human infections that vary from severe monkeypox related to pet prairie dogs or lyssaviruses in pet bats to less severe but more common ringworm infections acquired from african pygmy hedgehogs or chinchillas. epidemiologic and animal traceback investigations confi rmed that the fi rst community-acquired cases of monkeypox in humans in the united states ( cases) resulted from contact with infected prairie dogs that had been housed or transported with african rodents imported from ghana ( ) . similarly, an outbreak caused by francisella tularensis type b occurred among wild-caught, commercially traded prairie dogs; f. tularensis antibodies in exposed person documented the fi rst evidence of tularemia transmission from prairie dog to human ( ) . african pygmy hedgehogs have been implicated in human salmonellosis cases in the united states and canada ( ) . in the united states, the number of commercialized reptiles, especially iguanas, imported per year has increased considerably to ≈ million. the number of human cases of salmonellosis, especially in very young children, increased dramatically in parallel with iguana pet ownership. the centers for disease control and prevention estimates that ≈ % of human infections with salmonellae in the united states are associated with having handled a reptile. most iguanas have a stable mixture of salmonella serotypes in their intestinal tract and intermittently or continuously shed salmonella organisms in their feces ( ) . eight cases of rabies caused by a new rabies virus variant were reported in the state of ceará, brazil, from through . marmosets (callithrix jacchus jacchus) were determined to be the source of exposure. these primates are common pets; most cases occurred in persons who had tried to capture them, and case was transmitted by a pet marmoset ( ) . in , encephalitis was diagnosed in an egyptian rousette bat (rousettus egyptiacus) that had been imported from belgium and sold in a pet shop in southwestern france. the pet bat was infected with a lagos bat lyssavirus and resulted in the treatment of exposed persons (y. rotivel, pers. comm.). emerging infectious diseases have a major effect on human health and can create tremendous economic losses. animals, particularly wild animals, are thought to be the source of > % of all emerging infections ( ) . leading factors for emergence of zoonoses are unbalanced and selective forest exploitation and aggressive agricultural development associated with an exponential increase in the bushmeat trade ( ) . similarly, the increase of ecotourism, often in primitive settings with limited hygiene, can be associated with the acquisition of zoonotic agents. therefore, development of appropriate programs for surveillance and for monitoring emerging diseases in their wildlife reservoirs is essential. most animal pathogens for which surveillance programs exist relate to farm animals, and few or no programs are specifi cally aimed at wildlife. two different but complementary approaches are ) to monitor the pres-ence of specifi cally identifi ed pathogens that have emerged as human pathogens and ) to investigate in a given wildlife species the presence of known or unknown infectious agents. furthermore, conservation of habitat biodiversity is critical for preventing emergence of new reservoirs or amplifi er species. key measures for reducing the dispersion of emerging zoonoses include sustainable agricultural development, proper education of tourists about the risks of outdoor activities, and better control of the live animal trade (exotic pets, wet markets, bushmeat). public health services and clinical practitioners (physicians, veterinarians) need to more actively educate the public about the risks of owning exotic pets and adopting wild animals. as suggested by kuiken et al. ( ) , it is time to form "a joint expert working group to design and implement a global animal surveillance system for zoonotic pathogens that gives early warning of pathogen emergence, is closely integrated to public health surveillance and provides opportunities to control such pathogens before they can affect human health, food supply, economics or biodiversity." major tasks that should be taken by the international community include better integration and coordination of national surveillance systems in industrialized and developing countries; improved reporting systems and international sharing of information; active surveillance at the interface of rural populations and wildlife habitats, especially where poverty and low income increase risks for pathogen transmission; training of professionals, such as veterinarians and biologists, in wildlife health management; and establishment of collaborative multidisciplinary teams ready to intervene when outbreaks occur. dr chomel is director of the world health organization/pan american health organization collaborating center on new and emerging zoonoses at the university of california, davis. his research focuses on zoonotic infections, especially those caused by bartonella spp., in domestic animals and wildlife and their effects on human health. risk factors for human disease emergence conservation medicine and a new agenda for emerging diseases update: multistate outbreak of monkeypox-illinois emerging or reemerging bacterial zoonoses: factors of emergence, surveillance and control diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the value of wildlife wildlife trade and global disease emergence bushmeat hunting, deforestation, and prediction of zoonoses emergence epidemiologic situation of human rabies in latin america in the encyclopedia of arthropod-transmitted infections emerging bacterial zoonotic and vector-borne diseases. ecological and epidemiological factors wildlife diseases and population medicine mycobacterium tuberculosis: an emerging disease of freeranging wildlife arenaviruses other than lassa virus rabies in the kudu antelope (tragelaphus strepsiceros) anthropogenic environmental change and the emergence of infectious diseases in wildlife advances in health and welfare of farmed deer in new zealand bovine tuberculosis and badger blame from the discovery of the malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis exotic-pet trade disease risks associated with wildlife translocation projects epidemiological surveillance of rabies in lithuania from to a psittacosis outbreak in customs offi cers in antwerp (belgium) highly pathogenic h n infl uenza virus in smuggled thai eagles emergence of unique primate t-lymphotropic viruses among central african bushmeat hunters isolation and phylogenetic characterization of ebola viruses causing different outbreaks in gabon transmission of avian infl uenza viruses to and between humans bats are natural reservoirs of sars-like coronaviruses complete or near-complete nucleotide sequences of hepatitis e virus genome recovered from a wild boar, a deer, and four patients who ate the deer rickettsioses and the international traveler b-virus (cercopithecine herpesvirus ) infection in humans and macaques: potential for zoonotic disease human exposure to herpesvirus b-seropositive macaques reports of zoonotic disease outbreaks associated with animal exhibits and availability of recommendations for preventing zoonotic disease transmission from animals to people in such settings an outbreak of salmonellosis among children attending a reptile exhibit at a zoo mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants epizootic of mycobacterium bovis in a zoologic park first reported prairie dog-to-human tularemia transmission hedgehog zoonoses prevalence of fecal shedding of salmonella organisms among captive green iguanas and potential public health implications alves araujo fa, de mattos ca public health: pathogen surveillance in animals key: cord- -al m lou authors: wolka, eskinder; zema, zewde; worku, melkamu; tafesse, kassahun; anjulo, antehun alemayehu; takiso, kassahun tekle; chare, hailu; kelbiso, lolemo title: awareness towards corona virus disease (covid- ) and its prevention methods in selected sites in wolaita zone, southern ethiopia: a quick, exploratory, operational assessment date: - - journal: risk manag healthc policy doi: . /rmhp.s sha: doc_id: cord_uid: al m lou background: the novel corona virus disease (covid- ) presents an important and urgent threat to global health and its effect is expected to get even worse in the middle- and low-income countries where the health system is weak and fragile. timely access to accurate information and public awareness on prevention methods is one of the feasible interventions in these countries. identifying level of public awareness on disease prevention is important to mitigate the pandemic. the aim of this study was to explore the level of awareness and prevention methods of covid- among residents in wolaita zone, southern ethiopia. methods: a qualitative study using a qualitative descriptive approach was conducted. community members engaged in different service sectors were selected purposively. a total of in-depth interviews were done. the transcripts were imported into opencode version . software packages. a qualitative thematic analysis approach was used to analyze the data. results: the findings revealed that . % of the participants had heard about the disease covid- and realized common modes of transmission. some participants linked the disease with resentment of god on people or anger of god towards human kind. importance of consuming hot drinks, ginger or garlic to prevent the disease was reported by participants. negative attitude towards quarantine and isolation centers and stigmatizing people with a cough were documented in this assessment. stigma and fear of isolation centers may prevent people from reporting the symptom of the disease and this can create favorable ground for the transmission. challenges like problem of consistent availability of water supply, affordability of materials used to keep hygiene by rural poor, and keeping physical distancing in different public gathering places were reported. conclusion: concerned bodies need to address gaps in public awareness by providing health education and continuous awareness creation. the novel corona virus disease (covid- ) is an infectious disease caused by a novel corona virus now called severe acute respiratory syndrome corona virus (sars-cov- ) and can be transmitted from person to person via contact with respiratory droplets. the novel corona virus disease (covid- ) presents an important and urgent threat to global health. since the outbreak in early december in the hubei province of the people's republic of china, the number of patients confirmed to have the disease and deaths from it increased at an alarming rate through time. it was initially reported to the world health organization (who) on december , . on january , , the who declared the covid- outbreak a global health emergency. on march , , the who declared covid- a global pandemic. [ ] [ ] [ ] despite public health responses aimed at containing the disease and delaying the spread, several countries have been confronted with a critical care crisis, and more countries will almost certainly follow. major prevention methods recommended by the who include; regularly and thoroughly clean hands with an alcohol-based hand rub or wash them with soap and water, maintaining social/physical distancing, avoid touching eyes, nose and mouth, and different protection measures based on the situations. it's very well known that the covid- outbreak has placed unprecedented demands on the health systems of many countries around the globe. this is expected to get even worse in the middle-and low-income countries where the health system is weak and fragile. in countries that are hardest hit, health facilities and workforce are currently swamped by activities related to controlling the pandemic. as a step for the preparation to respond to the covid- pandemic, african countries have established a taskforce for corona virus preparedness and response (aftcon) that works in tandem with the african union commission, africa centers for disease control and prevention. in line with this and as a member state of the african union, ethiopian's preparedness and the health system's capacity to respond to the covid- is no exception. ethiopia reported the first case of covid- on march , . the trend shows a dramatic increase in the number cases with more indications of transmission in the community especially in the capital, addis ababa. in ethiopia, four months after the announcement of the first case, covid- cases had been identified by july . while the number of cases that recovered from the disease was % ( ), the number of people who died due to covid- was (case fatality rate of . deaths per cases. the government of ethiopia has taken several measures such as shutting down of schools including universities, restricting the movement of people under a state of emergency (stay at home), introducing chains of training programs for health workers, identifying isolation areas and hospitalization sites, starting screening tests at bole international airport and different engagement in mobilizing resources including equipment, supplies, food, money and educating the people about the disease and preventive methods, and so on. in this covid- pandemic, in addition to other interventions, timely access to accurate information and public awareness on prevention methods can be the difference between life and death. the stakes are high in developing countries like ethiopia where millions of people have limited access to information because of low media access, insufficient internet penetration, illiteracy, and so on. in this pandemic, when everyone should be aware of the risks posed by covid- and, most importantly, be informed about how to protect themselves and their families and societies at large. currently, as part of a national effort, wolaita sodo university has been engaged in different intervention packages concerning the response to the covid- pandemic. one of the activities is public education and information dissemination by using local media and other methods. but little is known about level of public awareness of the disease and its prevention methods. so, this assessment generates important information regarding the level of public awareness of the disease which in turn contributes to designing better mitigation strategies. hence this study was conducted to explore the level of awareness and prevention methods of covid- among residents of wolaita zone, southern ethiopia. the assessment was conducted in selected districts (woredas) of wolaita zone, southern ethiopia. residents from damot gale, boloso sore and humbo areas were included in this study. a qualitative study with a descriptive qualitative approach was used. this method is a naturalistic inquiry and it is the method of choice for this assessment since straight description of an event or phenomenon is needed. community members engaged in the transport service, shopping, market places, hotels and cafeterias, governmental and non-governmental organizations and religious organizations were selected purposively. maximum variation sampling was used. maximum variation sampling is a type of purposive sampling by which the participants view on a specific topic amongst different types of groups will be explored. a total of study participants, who were engaged in different service sectors in selected assessment areas, were submit your manuscript | www.dovepress.com risk management and healthcare policy : included. the service areas addressed in the assessment were; civil servants ( ), traditional coffee (jebena buna) ( ), religious leaders ( ), motorbike drivers ( ), trade in market areas ( ), hotels ( ), barbers ( ) and shops/supermarkets ( ) . in-depth interview (idi) was chosen as the data collection method to capture awareness and practices related to covid- by using an interview guide. the interview guide mainly includes, brief socio-demographic characteristics, awareness and prevention methods of covid- and possible suggestions to tackle the pandemic. this method is useful to have as each participant has an opportunity to share feelings, perspectives, and experiences concerning the problem. a semi-structured interview guide which included socio-demographic components, awareness and prevention aspects of covid- was used to collect data. data collectors were health professionals with past experience and oriented on the study overview, objectives, participant selection, tool review and interview approach. the interview was audio-recorded by using a recorder. interview data were reviewed periodically during data collection until data saturation was reached, according to the judgment of the research team. audio records were transcribed and translated into english and imported in to opencode version . . a qualitative thematic approach was used to analyze the data. data coding was done in each category by using the software. once coding was complete, a code report was produced for each code, cleaned and prepared for synthesis. during synthesis and write up, meaning units were identified in relation to the aim of the study. emerging meaning units that were extracted from each topic of the analysis were coded and then combined together to form categories, depending on their differences and similarities. potentially eligible and interested participants received consent forms in their local language and were informed that their participation would be voluntary and there would be no personal consequences or benefits to participation. informed written consent was obtained from all participants. confidentiality and anonymity was maintained when quoting research participants and the participants confirmed their agreement on publishing their anonymised quotes. this study complied with the declaration of helsinki. written informed consent was obtained from the participants and a declaration of signed consent include the statement: i have read or had read to me the information sheet that explains the reasons for the study, and all about the group discussion that i am being asked to participate in, all the questions i had about the study have been answered. i clearly understand what is being asked of me if i agree to participate in this study. i also know that i have the right to leave the study at any time if i do not want to continue. i am aware that all the information that i give will be kept confidential. i agree and confirm my agreement on publishing anonymized quotes and take part in this study and i put my signature. this study was reviewed and approved by the research ethics committee of the college of health sciences and medicine, wolaita sodo university, ethiopia. letters of support were secured by the research team from all institutions and offices where data were collected. major thematic areas considered during analysis were awareness towards the disease, prevention methods and challenges and suggestions to mitigate the pandemic in local context and presented as follows. most of the participants, ( . %), included in this study were males and the mean age of the service providers is years old (table ) . participants of idis were asked, regarding the disease covid- , about its cause and common symptoms. accordingly, of participants indicated that covid- is a new disease which happened recently, affecting all people in all countries in the world, transmitted from person to person and caused by virus. participants obtained information about the disease from different sources; common sources of information mentioned by the participants include; radio, television, social media, health care workers, telecom messages during outgoing call, religious leaders and friends/neighbors. four of the participants linked the disease with resentment of god on people and some others had a confusion with the term zoonotic disease. one of the participants who is barber explained the disease as follows; as i heard from media, radio, tv, religious institutions and mobile health educators, it is a virus which is deadly and has no treatment that transmits from person to person through breathing or by air during coughing. it is affecting another participant from another area, who is engaged in hotel management service said; i heard the information from media and people talking about a disease called corona virus which i have not ever heard. i know that there is no detected case in our area but based on the information from media, cases are detected in other countries and addis ababa only in our country. since then, i am practicing the messages told by health professionals for prevention from the disease acquisition. (male participant, aged ) two of the participants noted that the disease is the result of the anger of god since people do not obey the rule of god and one of the participants who is a "bajaj" driver explained that; what i know is, it is the disease that came from god as result of our bad and evil acts, it has no drug to treat and the only option is pray to god to be protected from this type of disease. (male participant, aged ) another participant from another site similarly noted that the disease emerged by the order of god and he shared the above idea by saying; i believe that, corona virus disease came from god because of our sin and we should pray to alleviate the problem and we should try to fight it by using all possible methods. (male participant, aged ) a participant from a similar woreda emphasized to follow all the instructions from the government despite his strong belief in god for the reason of the emergence of the disease as well as solutions for it and noted the situation as follows; i recommend all people should kneel down for repentance. so far there are many evil acts we did, disobedience, homosexuality, genocide, ethnic targeted displacement, corruption, robbery and so many unethical acts. so, we have to ask forgiveness of god along with precautions from government. (male participant, aged ) one of the participants, who is civil servant in his occupation, said that "it is confusing for me whether this disease can be transmitted from animal to human through eating animal products like raw meat since there are recommendations that avoid consuming raw meat." this idea was shared by another participant from another woreda and he pinpointed the situation as follows; " . . . the disease can be transmitted via raw meat consumption so i think there is a possibility of acquiring it from animals" (male participant, aged ), participants of the assessment were asked to reflect their opinion on common symptoms and modes of transmission of covid- ; accordingly they forwarded their perspectives on symptoms of the disease and modes of transmission. cough, fever, sneezing, diarrhea, throat pain, difficulty breathing and head ache were common symptoms of the disease mentioned by several participants. based on the review of transcripts, participants indicated different modes of transmission of covid- , namely; hand shaking, hugging, sitting together, contact with droplets during coughing and sneezing, making contact with infected air and objects, skin penetration, and sharing clothes. concerning modes of transmission, one of the participants said; the virus can be transmitted through contact, sneezing droplets, when we are sharing clothes with others, hand shaking, when we talking there are small droplets they may transmit disease, when we are close each other or sit together. these are the ways of transmissions. (female participant, aged ) droplet inhalation and contact with the droplet was pinpointed by another participant and he said; corona virus is a disease that can be transmitted from person to person via contact and also individuals might contract this virus through droplet inhalation. in addition to this, if a person touch different materials that might have come in touch with the virus, it favors the virus transmission in the developed countries and now a days, has also been health problem in ethiopia. (male participant, aged ) based on the transcripts of interviews, some participants emphasized that the virus can be transmitted via skin penetration after hand shaking or making contact with an infected person. a bajaj driver from one of study woredas said; if we don't wash our hands, the virus can enter our body through penetration of skin of our hands. (male participant, aged ) the above concern was shared by another participant from the same town and he further elaborated the issue as follows; . . . at the time of hand shaking with the infected individual, uninfected individual can easily acquire the virus and at the same time it enters in to the body by penetrating hand, because it is very dangerous virus. (male participant, aged ) hand washing, avoiding hand shaking, wearing masks and gloves, using sanitizer, physical distancing, general personal hygiene, holy water, consuming hot drinks, chili papers, garlic and ginger were identified as prevention methods of the disease by participants of in-depth interview. according to the in-depth interview participants, hygiene was described in different ways namely; washing hands every minutes, taking showers five times per week, washing hands three times per day and using sanitizer, soap and water together. two participants assumed the fever screening as a final technique that declares their status and feel a sense of security. negative perception towards quarantine and isolation center was indicated by some of the participants. the following quotes describe different views as follows; as health professionals have been telling us washing hands every minutes, rubbing with alcohol, avoid hand shaking, making physical distance at least meter. (male participant, aged ) to prevent disease, keeping social distancing, we have been practicing social distancing in funeral areas yesterday as well as the day before yesterday. there were professionals coordinating this. another is minimizing social life. very important way is washing hand frequently. using masks, wearing gloves in the office is important since we are collecting revenues. avoiding hand shaking is another method. (female participant, aged ) a religious leader has briefed the importance of holy water to prevent the disease together with other precautions and he said; we make members to sit far apart, we order them to wash their hands and they use holy water without sharing drinking materials and i believe this is helpful to prevent the disease. (religious leader, male, aged ) risk management and healthcare policy : submit your manuscript | www.dovepress.com based on a participant response, hot drinks and homemade prevention options were key to prevent covid- . she described by her own word as follows; . . . this disease has come from foreign countries, we are ordered to pray god in churches, in addition we are using spices and herbs like garlic, ginger, chili papers and hot drinks which are important to prevent this infection. (female participant, aged ) based on the explanation of a participant, one time screening was considered as an adequate method to prevent the disease and he explained the issue as follows; . . . i don't know about it in detail but to prevent the disease as it has no drug, for myself i got tested for the disease while i was coming from shashemene, as i knew my status there is no problem but i want to teach others to get tested for the disease. (male participant, aged ) a participant described how they changed their usual office set up and trend in order to prevent the disease, she explained the situation as follows; in our office, our work has forces us to have frequent interaction with the people since we are collecting revenues. another thing that poses risk is money that we are collecting. we can't lock the job; because the revenue and tax that we collect is necessary for salary and other payments. with us there is finger print machine. since many people are touching it may transmit the disease. therefore we clean machine with alcohol after everyone is recording finger print and we are making the customers who use machine to rub their hands with alcohol. another is; we have hand washing corner at the gate. therefore everyone washes his/her hand before getting in to office. we serve the customer through window. we don't allow them to enter to the rooms. (female participant, aged ) participants noted challenges like consistent availability of water, affordability of materials used to keep hygiene for the rural poor, keeping physical distancing in funeral area and markets, lack of awareness (particularly in rural areas), difficulty of avoiding usual trend of greetings like hand shaking in rural areas, not obeying rules and instructions, misunderstanding on lifting of some of the restrictions like allowing motorbike movement, stigmatizing people with cough. in this study, the participants were also asked for any comments and suggestions on the ways of preventing the pandemic by overcoming the challenges and minimizing its burden. the major suggestions forwarded by the participants include; continuous awareness creation, law enforcement and providing support for poor people. the following quotes describe different views and suggestions as follows; still people are shaking hands each other in rural area, for example today it is market day, if someone caught by the disease, disease my spread to everyone. people salute each other while selling animals. they kiss each other's hand, to show their agreement for selling. therefore, it is good to teach the community continuously. (male participant, aged ) it is difficult to prevent this disease in our area. for example motorbikes carries two or more customers at a time, they do not respect the rule. at rural area; people have no awareness about the disease. they don't know even as the disease is killer. therefore, there should be intense teaching. still there is gathering, sitting together. there should be continuous teaching. (male participant, aged ) there is shortage of soap; food ingredients are expensive and not affordable for poor people. therefore, government should try to provide the supplies to the poor either as donation or by affordable price. (female participant, aged ) government should force the community. for instance; motor vehicle ordered to use by maximum of two persons at a time. but they not obey this. bajaj's do not obey too. therefore they should be forced to obey the rule. care should be taken in market areas and other gathering areas too. this is all that i can say. (male participant, aged ) . . . as i saw a great gap from both the government and the people. for example: majority of our people lives in the rural area but they have very little information about the disease so the government must address every kebele and each home. in addition to that as you know a lot of people are very poor and they obtain their expense from day to day activity so the government must support those in need. (male participant, aged ) sometimes peoples are not happy when you ignore hand shaking, they became emotional and respond aggressively. most of the time they said you didn't believe in god that is why you develop fear for the disease he protects us. so, this need attention. on the other hand, there is no enough water for hand washing in every part of the city it is limited to some special areas that may contribute for the spread of the disease. the most important thing that must need attention was people living in the rural area because there is no or little information about the virus because they lack mass media but majority of people are there, so the government must address this problem. in addition to this almost all the materials needed for protective purpose are in a very small amount or not at all. please as you said you are coming from university please try to solve this scarcity. (female participant, aged ) this quick assessment aimed to explore the level of awareness and prevention methods of covid- among residents of wolaita zone, southern ethiopia. the participants were civil servants, religious leaders, motorbike/bajaj drivers, merchants, hotel/café workers, barbers and people working in shops/supermarkets. the findings revealed that almost all of the participants heard about the disease covid- and based on their expression it is viral disease affecting everybody in all countries in the world and transmitted from person to person. some participants linked the disease with resentment of god on people and they strongly believe in praying to god to be protected from the disease. the implication of this finding is, it is still essential to provide necessary education for the community in order not to neglect all necessary prevention methods of the disease while praying to god which is well recognized by ethiopian government and currently it is included in the national television broadcasting program. some others had confusion with whether the disease is transmitted from animal to human because of advice like avoiding raw meat and other raw products. this shows the need for detail while educating people about the disease. findings from this assessment showed that most participants realized common modes of transmission of the disease, like contact with droplets during coughing and sneezing, making contact with infected air and objects via hands and so on, but some participants considered that the disease can be transmitted via skin penetration and this is also an important area that should be addressed during the education program. the assessment reveals that most participants got information from different sources and they had awareness of common symptoms of covid- . the findings showed several participants mentioned methods of prevention of the disease as per the recommendations by the world health organization (who) but some of the participants rely on consuming hot drinks, ginger or garlic to prevent the disease and giving them a sense of security, and this needs a precaution since these things are not scientifically proven prevention methods for covid- so far. in addition, issues like negative attitudes towards quarantine and isolation centers, stigmatizing people with a cough, considering screening by measuring temperature as a final result of their covid- status were documented in this assessment. so, it is helpful to consider these points during awareness creation and health education to mitigate the pandemic since issues like stigma and fear of isolation centers may prevent people from reporting the symptoms of the disease and this can create favorable grounds for the transmission. to ensure credibility of the findings in this study, data collectors who are very familiar with the study area were selected and trained. dependability refers to whether the findings of this study may be repeated in other places by other researchers and the study tools are available on request for others to do, consistently. transferability is the term used to refer to the external validity in qualitative studies, the study addressed different areas of the zone and service sectors and the finding is applicable in similar socio-cultural settings. this assessment has some limitations. although the study sites were drawn from different districts of the wolaita zone, the relative numbers of recruitment locations within these districts were few and not necessarily representative of the entire rural community. the overall number of respondents, though limited by available resources and time, was in line with similar qualitative quick assessments of this type. generally, most of the interviewed participants indicated that covid- is a newly emerged viral disease, affecting all people in all countries in the world, transmitted from person to person. some participants linked the disease with the anger of god on people and some others had confusion with zoonotic diseases. most participants mentioned common symptoms of the disease. participants indicated different modes of transmission of covid- , explicitly; hand shaking, hugging, sitting together, contact with droplets during coughing and sneezing, making contact with infected air and objects, skin penetration, and sharing clothes. hand washing, avoiding hand shaking, wearing masks and gloves, using sanitizer, physical distancing, general risk management and healthcare policy : submit your manuscript | www.dovepress.com dovepress personal hygiene, holy water, consuming hot drinks, chili papers, garlic and ginger were identified as prevention methods of the disease by participants of the in-depth interview. some participants assumed that screening by measuring temperature as a final technique that declares their status and felt a sense of security. negative perception towards quarantine and isolation centers was indicated by some of the participants. participants noted challenges like problems of consistent availability of water, affordability of materials used to keep hygienic by the rural poor, keeping physical distancing in funeral areas and markets, lack of awareness (particularly in rural areas), difficulty in avoiding the usual trend of greetings like hand shaking in rural areas, not obeying rules and instructions, misunderstanding on lifting of some of the restrictions like allowing motorbike movement and stigmatizing people with a cough. it is recommended to provide continuous awareness creation, community mobilization and strong law enforcement in some areas, like the transport sector. a local covid- prevention taskforce should regularly attend events like funerals and markets to facilitate physical/social distancing (this may include health care workers, religious leaders, community leaders, security and so on). novel coronavirus who declares public health emergency for novel coronavirus declares global emergency as wuhan coronavirus spreads:the new york times declares pandemic as number of infected countries grows. the new york times covid- ), who advice for the public lancet glob health novel coronavirus ( -ncove) reminiscent of spanish flu: a challenge to global public health systems (editorial) federal democratic republic of ethiopia, ministry of health the significance of saturation we would like to acknowledge the zonal health department and local administrations, without whose cooperation this assessment would have been impossible. study participants deserve special acknowledgment for all their cooperation in the data collection process and for providing genuine information that resulted in the accomplishment of this assessment. the authors report no conflicts of interest for this work. risk management and healthcare policy is an international, peerreviewed, open access journal focusing on all aspects of public health, policy, and preventative measures to promote good health and improve morbidity and mortality in the population. the journal welcomes submitted papers covering original research, basic science, clinical & epidemiological studies, reviews and evaluations, guidelines, expert opinion and commentary, case reports and extended reports. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- - ykvl u authors: binns, colin; low, wah yun; kyung, lee mi title: the covid- pandemic: public health and epidemiology date: - - journal: asia pac j public health doi: . / sha: doc_id: cord_uid: ykvl u nan in this issue of the journal, we publish a review of covid- infection by eminent virologists, mackenzie and smith (see in this issue). it is too early in the history of the covid- outbreak to write the full history, but their article provides a good outline of the emerging pandemic. the disease is causing widespread social disruption in many countries, and it has just been announced that the asia pacific academic consortium for public health (apacph) conference has been postponed indefinitely. the media are full of daily totals of new cases, hospital and intensive care admissions, and deaths. the actual numbers are dependent on the testing regimes in use at different locations. in the first months of , there were hundreds of papers and commentaries published on corona viruses, including a major clinical review in the lancet that has already had almost citations. corona viruses are a large family of viruses that can cause human diseases, but usually mild in nature, such as a common cold. there have been previous severe outbreaks of novel corona viruses: severe acute respiratory syndrome (sars) in and middle east respiratory syndrome coronavirus (mers-cov) in , which together caused more than cases. the case fatality rates (cfrs) were % for sars-cov and % for mers-cov. in this commentary, we will discuss additional public health issues that will assist in teaching at our public health institutions. since the first cases of covid- , the reported numbers have increased rapidly with more than . million cases and deaths to the end of february , and cases are now being reported from all of the more populous countries. the symptoms of covid- infection are nonspecific and include elevated temperature and cough. this, then, progresses to shortness of breath. a clinical report of the first patients from wuhan (n = ) with covid- infection gave the following details of clinical symptoms: fever %, cough %, myalgia or fatigue %, sputum %, and dyspnea %. the average time from the first onset of symptoms to the development of dyspnea was days. in the first report from outside of wuhan (from zhejiang province, n = ), the symptoms were fever %, cough %, sputum %, headache %, and myalgia or fatigue %. the disease is more likely to occur in people who have a chronic illness, are immunocompromised, or are older. those who contracted the virus and were older than years of age, and particularly older than years, were more likely to require admission to intensive care unit, and have an increased cfr (see table in mackenzie and smith, this issue). in wuhan, there were deaths from the confirmed cases, an overall cfr of . %. the initial clinical symptoms of covid- are the symptoms of a common cold and influenza. every adult gets to colds per year, and more frequently in children. with a world population of around . billion, this suggests that there are billion cases every year. screening all of these cases for possible covid- would obviously be impossible, and so testing for the virus is confined to those with other risk factors, including contact with confirmed cases and travel from outbreak epicenters. covid- is the latest in a continuing series of infectious disease epidemics in the history of the human race. as population numbers and population density increases, the likelihood of epidemics increases. probably, the greatest epidemic of all time was the influenza epidemic of , which caused an estimated million deaths worldwide and had a cfr of more than . % and which could have been as high as %. this was in the pre-antibiotic days, and before an influenza vaccine and antiviral therapy had been developed. infectious diseases remain a major challenge for public health. to assist in defining, containing, preventing, and ultimately in successfully treating those who become ill in an epidemic, it is important to understand the basic epidemiology of the outbreak. experts from many institutions have collaborated together in an effort to categorize the covid- outbreak. most infectious diseases have a spectrum of severity from clinically undetected disease through to death. figure is modified from the paper prepared by imperial college. it is likely that only the cases with severe symptoms or other risk factors (eg, contact with a known case) are being identified at present, but it varies between countries. for some diseases, the base of the triangle in figure would be much wider, and this may be the situation with covid- . the base of subclinical cases with minimal symptoms may be much greater than those with disease severe enough for hospital admission. the history of public health contains a number of examples of infectious diseases that were initially thought to have had a very high cfr only for it to be revised downward later. lassa fever was initially described as a very severe disease, with a cfr close to %. however, as more widespread epidemiological studies were undertaken in west africa, it was found that % or more of the population of sierra leone and guinea were seropositive to the disease without showing any symptoms. under these circumstances, the cfr for lassa virus is now thought to be low (< %), but the cfr for severe disease remains very high. when assessing the likely impact of an infectious disease, there are parameters that are considered: the likelihood of transmission of the disease (its capacity to spread), and the severity of the disease and its capacity to kill (or disable) those infected. these are assessed using the reproduction rate and the cfr. reproduction rate (r ): the base reproduction number (r ) for transmission indicates the number of secondary infections due to an initial case (r = virgin state, a population with no previous exposure to this infection ). in populations that have previously been exposed to the infection, and have some immunity, the reproduction rate may be lower. case fatality rate is defined as the proportion of reported cases of a specified disease that are fatal within a specified time. the cfr depends on the definition of disease, the accuracy of diagnosis (case detection), and the availability of treatment. transmissibility and severity are the most critical factors that determine the public health impact of an epidemic. a disease that has a high transmission rate and is very severe is the greatest public health risk. covid- has a high transmission rate, and the cfr appears to be greater than for influenza epidemics, and it is, therefore, potentially a major public health threat. infectivity is the tendency to spread the infection from host to host. the period of infectivity for some diseases commences before symptoms appear, making it far more difficult to control spread. the spectrum of clinical disease (see figure ) makes it difficult to define a case and even more difficult to calculate a cfr, as the denominator is not readily defined. in february, the cfr for covid- infection was estimated by the world health organization (who) to be %, much lower than for mers and sars, but estimates of the cfr have changed over time as the criteria for counting the number of cases in the denominator has changed to include very mild or even asymptomatic infections. changes in the denominator decrease the cfr and increase the reproducibility rate. in some situations, the reproducibility rate changes and the quarantine of passengers on a cruise ship in yokohama had a r times greater (as high as ) compared with the initial r in wuhan. the use of diagnostic tests has traditionally been reported using measures of sensitivity (proportion of true positives that are correctly identified by the test) and specificity (proportion of true negatives that are correctly identified by the test). at the present time, covid- disease is diagnosed by detecting the virus in throat and nasal swabs in patients who have symptoms of an upper respiratory tract infection who have been in a region with disease transmission. no other screening tests are yet available, and in the absence of "gold standard" for diagnosis, these parameters are not yet being calculated. incubation period is the time between exposure to an infectious agent and the appearance of clinical symptoms (or physiological evidence of disease). it is not known if transmission of the virus occurs during this period and before the presence of clinical symptoms. the who estimates of the incubation period for covid- range from to days, most commonly around days. modeling of the role of contact tracing and case isolation suggest that these are effective in the control of epidemics such as covid- . in australia, the home isolation (quarantine) of contacts or suspected cases is recommended for days. if transmission is occurring before symptoms appear, it makes it more difficult to control an infectious disease. the virus is contained in droplets from coughing and nasal secretions. it can survive up to hours on surfaces under favorable conditions. most transmission will occur through spread by hands and not from the direct inhalation of droplets. the who recommends the following to prevent transmission of the virus: • • regularly and thoroughly clean your hands with an alcohol-based hand rub or wash them with soap and water. • • maintain at least m distance between yourself and others, and in particular from anyone who is coughing or sneezing. • • avoid touching eyes, nose, and mouth. • • make sure you, and the people around you, follow good respiratory hygiene. this means covering your mouth and nose with your bent elbow or tissue when you cough or sneeze. then dispose of the used tissue immediately. • • stay at home if you feel unwell. if you have a fever, cough, and difficulty breathing, seek medical attention and call in advance. follow the directions of your local health authority. despite much research and success in some animal models, including primates, there is still no vaccine for sars. the first clinical cases of sars were noted in november , but it was not until months later that a causative agent was isolated. transmission and dispersion around the world occurred by respiratory aerosols and contact via hands, and in several cases, this was from touching door handles. there were reported cases of sars and deaths from many different countries. while there is no doubt that the virus has the potential to reemerge at present, it is not a clinical issue at present. sars has a lower transmissibility than covid- . the development, testing, and mass production of vaccines is always time-consuming before they can be deployed on a population-wide scale. the development, testing, and distribution of a vaccine will take years. despite the interest in sars, there is still no vaccine, and in other cases, important diseases have defied vaccine development, notably malaria and dengue. will covid- decline in the northern summer following the pattern of influenza? this may be because people are more likely to have closer contact with others because it is colder. it could also relate to relative humidity levels that are lower in winter. in western countries, when it is cooler in winter, respiratory infections increase (eg, influenza). however, this seasonality does not apply to influenza in india. a study of a new strain of influenza in vietnam shows that in the early years of this variant, there was significant transmission throughout the first year and the usual seasonal transmission pattern evolved a few years later. in australia, it is summer and hot, and the covid- is spreading here. this suggests that covid- transmission may not be related to climatic conditions and may not be seasonal in its early year(s) as the population is being exposed for the first time. the history of smallpox elimination provides an example of the role of epidemiology in defeating an infectious disease. the practice of variolation to prevent smallpox had been in existence for several centuries, particularly in the middle east, before edward jenner popularized the process and published his paper on the topic. vaccination against smallpox developed for almost centuries before the disease was certified as being eliminated in . what was needed was a thorough understanding of the epidemiology of the disease. by studying seasonal variation and transmission within an infection cluster, it was shown that with vaccination of perhaps % of the population, the immediate case contacts was just as effective as vaccinating % of the population. understanding the epidemiology of outbreaks proved to be the effective way of eliminating the disease. recording all of the details of the covid- outbreak is basic to understanding its epidemiology and the natural history of the disease and could provide the key to defeating the disease outbreak. there are several ethical issues that have already been raised in this outbreak. the importance of preserving the physical and mental health and availability of health workers in an epidemic situation is very important. the quality of health care depends almost entirely on them having a professional service ethic that motivates them to provide the highest quality care to all with the resources available to them. this often leads them to put their own health and safety at risk, especially in infectious disease outbreaks. after the sars outbreak of that caused illness and deaths among health workers, the ethics of exposing staff to the disease was still being debated in . the history of public health is full of the writings of the heroism of health workers providing care to those in need, despite endangering themselves. however, experience has shown that risks associated with outbreaks of life-threatening infections only receive attention after health workers have suffered serious adverse consequences. institutions need to prepare for outbreaks and provide the best available protective equipment to their workers and volunteers. a further ethical issue is the development of vaccines. if this proves possible, it will take several years to develop, test, and submit for approval. then issues of distribution and cost will need to be finalized. professor jeffrey sachs, health economist, public health advocate, and former advisor to the secretary general of the united nations for the development of the sustainable development goals, has written an editorial on the development of a vaccine for covid- . sachs states that in earlier public health emergencies, governments, nonprofit foundations, and international organizations took the lead in the development of preventive measures and made the vaccine or knowledge available freely. sachs discusses the example of jonas salk who did not patent the polio vaccine, making it affordable to programs all over the world. however, in the case of covid- , the present us administration is stating that commercial companies will develop the vaccine and its availability will depend on the market. will the profits of multinational companies come before the survival of the poor in lower income countries ? reducing the peak of new cases by slowing down the spread of new cases is very useful as it . reduces the load on diagnostic and treatment services . reduces the number of health workers who contact the disease and who can continue working . if the epidemic curve can be smoothed, it will result in a lower overall disease burden there are examples from the historical control of epidemics using public health measures that may be applied to covid- . an analysis of records from cities in the united states during the influenza pandemic shows that cities that implemented public health measures (isolation, banning meetings, etc) were successful in reducing epidemic peaks and overall mortality. this was in a naive population not previously exposed to this virus. hatchett et al documented the substantial differences between st. louis (which banned mass gatherings) and the higher death rates in philadelphia, which allowed the massed celebratory marches following world war i to proceed. useful public health interventions to reduce the peak of new cases include the following: . personal hygiene: no handshaking, no direct physical contact with others, keeping m distance from others, no coughing in public, do not touch your face, wash hands frequently with soap and water, and eat cooked food that is still hot. these measures require clean water supplies (also important for nutrition and food safety) . reducing person to person contact by banning public gatherings, closing schools, home quarantining, controlling public transport, and so on, has been effective, but disruptive, in some communities . isolation of cases and quarantine (usually self-quarantine) of contacts. there is more information on the who and centers for disease control and prevention websites. , in their modeling, hellewell et al suggest that isolation of cases and tracing of contacts may be successful in controlling an outbreak within months. in the current global situation, it appears that some countries have been able to slow the epidemic while others have been overwhelmed by peaks of cases. universities are presented with major challenges to manage the covid- epidemic. they are gathering places for thousands of young adults and academics who are usually older and are more vulnerable to complications. obviously, large gatherings should be avoided and wider use be made of online teaching and tutorials. on campus, the usual precautions of avoiding contact, keeping distance, and frequent handwashing should apply. within the memory of one of us (cb), these personal precautions were applied with success to polio, influenza, and hepatitis. we hope that these precautions will lead to a containment of the epidemic allowing time to continue development and clinical trials of a vaccine. the emergence of covid- is a serious global public health problem. the future direction of the epidemic is unknown. the size of the outbreak will depend on reducing transmission, which at the present time means using traditional public health measures. these include contact tracing and quarantine of cases, or sometimes the quarantine of localities. modeling suggests that these measures may be effective. health promotion programs should emphasize avoiding crowds, handwashing and hygiene, and extensive testing of at-risk persons. vaccine development is a slow process, and it will be a year(s) before it can become a component of public health interventions. schools of public health and research institute members of apacph are actively involved in basic research, epidemiology of outbreaks, and health promotion. this journal welcomes submissions that document the outbreak and contribute to the control of the disease. for the next few issues, we will publish a selection of letters and short papers that we are receiving on the covid- epidemic. in this day and age, there are more rapid means of communication, but the printed word remains unsurpassed as long-term record of what has happened. for our journal, the teaching of public health undergraduate and postgraduate students is important, and this will be a useful resource. the editors clinical features of patients infected with novel coronavirus in wuhan world health organization. who director-general's opening remarks at the media briefing on covid coronavirus disease (covid- ). symptoms of coronavirus the novel coronavirus pneumonia emergency response epidemiology team. vital surveillances: the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- )-china center for disease control and prevention. disease of the week. common cold. accessed march remembering gustav klimt and million others: the year anniversary of the greatest human epidemic lassa fever: epidemiology, clinical features, and social consequences covid- outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures world health organization. cholera case fatality rate preparation for possible sustained transmission of novel coronavirus: lessons from previous epidemics coronavirus: covid- has killed more people than sars and mers combined, despite lower case fatality rate diagnostic tests. : sensitivity and specificity feasibility of controlling covid- outbreaks by isolation of cases and contacts australian government department of health. coronavirus (covid- ) health alert world health organization. coronavirus disease (covid- ) advice for the public the severe acute respiratory syndrome severe acute respiratory syndrome: historical, epidemiologic, and clinical features differences in influenza seasonality by latitude, northern india chronological, geographical, and seasonal trends of human cases of avian influenza a (h n ) in vietnam, - : a spatial analysis jenner and the history of smallpox and vaccination infections and public health: who will win? epidemiology of smallpox in west-pakistan. ii. determinants of intravillage spread other than acquired immunity smallpox: ten years gone from cooperation to competition in national health systems-and back? impact on professional ethics and quality of care can healthcare workers reasonably question the duty to care whilst healthcare institutions take a reactive (rather than proactive) approach to infectious disease risks? public health ethics damien de veuster ( - ): a life devoted to lepers the trump administration's ludicrous approach to coronavirus vaccine public health interventions and epidemic intensity during the influenza pandemic colin binns, mbbs, phd , wah yun low, phd , , and lee mi kyung, phd key: cord- -hpnkou authors: pitlik, silvio daniel title: covid- compared to other pandemic diseases date: - - journal: rambam maimonides med j doi: . /rmmj. sha: doc_id: cord_uid: hpnkou in december , the first cases of a new contagious disease were diagnosed in the city of wuhan, the capital of hubei province in china. within a short period of time the outbreak developed exponentially into a pandemic that infected millions of people, with a global death toll of more than , during its first months. eventually, the novel disease was named coronavirus disease (covid- ), and the new virus was identified as severe acute respiratory syndrome coronavirus (sars-cov- ). similar to all known pandemics throughout history, covid- has been accompanied by a large degree of fear, anxiety, uncertainty, and economic disaster worldwide. despite multiple publications and increasing knowledge regarding the biological secrets of sars-cov- , as of the writing of this paper, there is neither an approved vaccine nor medication to prevent infection or cure for this highly infectious disease. past pandemics were caused by a wide range of microbes, primarily viruses, but also bacteria. characteristically, a significant proportion of them originated in different animal species (zoonoses). since an understanding of the microbial cause of these diseases was unveiled relatively late in human history, past pandemics were often attributed to strange causes including punishment from god, demonic activity, or volatile unspecified substances. although a high case fatality ratio was common to all pandemic diseases, some striking clinical characteristics of each disease allowed contemporaneous people to clinically diagnose the infection despite null microbiological information. in comparison to past pandemics, sars-cov- has tricky and complex mechanisms that have facilitated its rapid and catastrophic spread worldwide. on january , , the genome of a new coronavirus, now known as severe acute respiratory syndrome coronavirus (sars-cov- ), was posted on the internet. it had been isolated days before from patients developing varying degrees of pneumonia in wuhan, the capital of hubei province in china. immediately thereafter, a growing number of scientists worldwide became deeply involved in analyzing its molecular details. one of the major tasks that they focused on was synthetizing the proteins encoded by the viral rna and deciphering their structure and function. acutely aware of the pandemic potential of sars-cov- , some of these scientists immediately alerted selected vaccine producers, with the hope of triggering a swift process for vaccine design and development. , once the protein amino acid composition and the post folding structure of close to proteins in sars-cov- were defined, multiple computational searches were launched by a number of institutions, looking to repurpose extant drugs aimed against the newly discovered molecular targets. in parallel to this accelerated research, there was a marked and frightening spread of this new coronavirus throughout wuhan to a widening area in china, and it was subsequently exported to a rapidly growing list of countries worldwide. , this paper reviews the microbiological, clinical, and epidemiological characteristics of the coronavirus disease (covid- ) pandemic, as well as its socio-economic impact. in addition, covid- is compared to previous pandemics in human history. in the early days of the pandemic great effort was invested into understanding the life cycle of sars-cov- , so as to provide a basis for discovery of an effective vaccine to prevent covid- and/or a safe and efficacious drug to cure it, or at the least, to ameliorate its symptoms, shorten its duration, and/ or block its mechanism of transmission. being a virus, sars-cov- must invade host cells, hijack the cell's biologic machinery for reproduction, and, finally, release multiple daughter virions. research uncovered six steps in the life cycle of sars-cov- : ( ) attachment and entry; ( ) uncoating; ( ) guide ribonucleic acid (grna) replication; ( ) translation in the endoplasmic reticulum and golgi apparatus; ( ) assembly; and ( ) virion release. , the external surface of sars-cov- has multiple protruding elements called spike proteins which, after manipulation by host cell enzymes (furin and tmprss ), function as anchors for attachment to the host cells. , the cell surfaces of the upper and lower respiratory tract are covered with angiotensinconverting enzyme- (ace ) receptors, which are physiologically involved in blood pressure regulation. however, these receptors are also present in many other organs and tissues, helping to explain some of the extra-respiratory manifestations of covid- . once attached to the external membrane, sars-cov- covers itself with a portion of the host cell membrane and becomes an intracellular endosome. this structure undergoes partial uncoating allowing the release of grna into the cytoplasm of the host cell. the released strands of grna attach to host ribosomes, rna-dependent rna polymerase (rdrp), and together activate the grna replication mechanism. other released strands of grna undergo translation into structural, nonstructural, and coat proteins. the different basic blocks of the reproduced virus are finally assembled into multiple virions that are expelled to the extracellular space of the host. these released daughter virions are now ready to infect other cells or, even worse, other hosts. once these mechanisms had been clarified, multiple hypotheses relating to specific actions of different drugs were proposed. for example, chloroquine, hydroxychloroquine, and azithromycin all inhibit the uncoating of the invading endosomes. on the other hand, the antivirals remdesivir and favipiravir inhibit grna replication by rdrp. additional drugs, not only antivirals, have also been identified to tar-get the complex mechanisms of the intracellular viral cycle. the first human coronavirus was described by june dalziel almeida in . she had observed a viral structure seen under electron microscopy while being involved in a study investigating causes of the common cold. a paper submitted by almeida and her team described a crown-shaped structure supposed to be a new type of virus causing common colds. this paper was rejected as the editors claimed that "these microscopic observations resulted from distorted influenza viruses." , since this pioneering observation, around species of viruses in the subfamily coronaviridae have been described, the majority of which were found in animals, primarily though not only in bats. only some of the animal coronaviruses had been associated with a specific animal disease such as a severe type of bronchitis in poultry. on the other hand, many of these coronaviruses were isolated from healthy animals, again predominantly bats. based on current knowledge, there are seven species of human coronaviruses. four of them ( figure , table ) cause a mild upper respiratory tract infection manifested as a runny nose. occasionally, they involve the lower respiratory tract. the remaining three human coronaviruses are associated with a wider spectrum of disease severity. relatively frequently they cause severe pneumonia and other serious complications ( figure , table ). as indicated in figure , five of the seven human coronaviruses have well established intermediate hosts, based on both epidemiologic and genomic data. in the case of covid- , preliminary data suggest that several species of pangolins are the suspected intermediate host. pangolins are mammals covered by keratin scales. in china the pangolin is seen as an edible animal mainly, but traditional medicine in this country also attributes multiple curative properties to a powder obtained by mashing their scales. , although the transmissibility of both severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) is lower than that of covid- , the case fatality ratio is many times higher for mers and sars than for covid- (table ) . however, the number of covid- cases have markedly outnumbered the number of cases in both sars and mers. while clinical manifestations for the two last-mentioned infections were generally limited to the respiratory tract, although with higher degrees of disease severity, it is remarkable that there has been a wide spectrum of heterogeneous clinical manifestations in covid- cases. from a pathophysiological perspective, this phenomenon is explained by the ubiquitous presence of ace receptors throughout multiple organs and blood vessels. at a clinical level, some typical covid- manifestations described during the pandemic include "silent anoxia," a discrepancy between an extremely low oxygen level as measured by pulse oximeter and the simultaneous lack of dyspnea; signs of cardiac involvement including myocarditis, myocardial ischemia, and myocardial infarction ; hepatitis ; reddish discoloration of the toes mimicking frostbite or chilblains ; intravascular coagulation including pulmonary embolism ; encephalitis ; and acute renal failure (see also table ). recently a condition similar to kawasaki syndrome was described in a growing number of teenagers (as opposed to toddlers with classical kawasaki syndrome); it has been proposed to name the syndrome "pediatric multisystem inflammatory syndrome." the mechanism of this complication is an overreaction of the immune system. a comparison of the leading clinical identifiers of recognized pandemic diseases is provided in table . the simplest definition of a pandemic is a contagious infectious disease that has spread to multiple geographic areas or continents. the term "contagious" implies that the infection can be transmitted person-to-person, either directly or indirectly. various degrees of controversy emerge between members of the medical and scientific communities when defining a new disease as pandemic. according to the world health organization (who), "a pandemic is the worldwide spread of a new disease." however, even this condensed and crisp definition, not infrequently, leaves room for discussion. a convention, held at the beginning of the h n pandemic under the umbrella of the national institutes been published, mainly during this last decade. [ ] [ ] [ ] [ ] [ ] [ ] [ ] many web sites [ ] [ ] [ ] [ ] [ ] [ ] and review articles have also reviewed the history of pandemics. interestingly, there is a significant discordance in the inclusion or the exclusion of specific infectious diseases causing pandemics. table provides a chronological list of the major known pandemics reviewed in this article. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] obviously, written testimony on possible pandemics is lacking with regard to prehistoric times. however, it is important to recall that during the preagricultural era, the nomadic population of homo sapiens on earth was relatively small. consequently, we can assume that pandemics were relatively rare at that time. the known history of pandemics is based on the discoveries of documentation by ancient historians and other sources. table provides an overview of the known pandemics throughout history. [ ] [ ] [ ] [ ] [ ] [ ] [ ] due to the lack of consensus on the definition of a pandemic, there are some cases in which there is a discrepancy regarding the categorization of an epidemic as a pandemic. for example, the sars epidemic in - had all the characteristics of a potential dire pandemic, but fortunately its spread was interrupted by yet unexplained mechanisms. some pandemics such as aids have been widespread both in time and in vast geographic distribution. current knowledge marks the s as the beginning of the human immune deficiency virus (hiv) epidemic, when zoonoses from pan troglodytes troglodytes to humans occurred in africa. , similarly, the cholera and plague pandemics had a zoonotic origin. , figure provides some illustrative examples of the human (panel a) and zoonotic (panels b and c) factors contributing to the spread of pandemics, and devices developed to help avoid their spread (panels d, e, and f). intriguingly, and according to databases managed by both johns hopkins university and the european centre for disease prevention and control, plots of the daily global numbers of covid- infected persons during the second trimester of reveal an undulating and ascending pattern that is difficult to explain. however, they also show a progressive and steady decrease in mortality for that same period. this last observation merits a logical explanation, but there has not been enough research to date to provide one. the cumulative number and type of mutations detected during the first months of do not explain the reduced severity of sars-cov- . table also demonstrates the predominance of rna viruses as the cause of most pandemics, with the only exception being the variola virus, which is composed of dna. relatively few pandemics were caused by gram-negative bacteria. yellow fever epidemics occur mainly in africa and south america. the disease is transmitted by several species of mosquitoes, mainly by aedes aegypti. the disease life cycle has two possible scenarios: ( ) the sylvatic cycle, where the hosts are various types of animals, primarily monkeys; and ( ) the urban cycle, where the reservoir is human beings. outbreaks occur mainly during the rainy season. water accumulation, on land or in various objects, facilitates development of vector mosquitos. other viruses transmitted by aedes aegypti include dengue virus, zika virus, and chikungunya virus. for close to half a century, several ebola virus outbreaks have occurred in western and central africa. the long-term reservoir of this virus is a species of bats. due to massive deforestation, there have been episodes of viral spillover to humans and animals, including gorillas and chimpanzees. subsequently, human-to-human transmission occurred as the result of close contact with patients or bodies at burial ceremonies, and some convalescing patients continued to transmit the virus for some time after recovery. due to the high contagiousness of ebola, many health care workers were also infected. currently, there is an effective vaccine to prevent ebola (table ) as well as a drug composed of three types of antibodies (table ). in general, annual seasonal influenza in postpandemic years is caused by variants of the corresponding virus from the prior pandemics. influenza viruses involved in seasonal flu accumulate antigenic changes in a progressive fashion resulting in annual seasonal epidemics. an accepted parameter for the impact of annual influenza activity is the excess number of deaths. , interestingly, in , a reemergence of human h n viruses identical to those circulating before was attributed to an accidental "escape" of an old frozen laboratory specimen. epidemics and pandemics have had a very strong impact on human history. diseases like smallpox, measles, and plague decimated entire populations in several regions of europe, the middle east, and asia. at the end of the fifteenth century, the european conquistadores of the americas brought diseases with them that were unknown to the native population. due to the local lack of immunity to these newly imported viruses, smallpox and measles spread rapidly, causing fear and frustration among the natives due to the lack of natural resistance and a very high mortality rate; local communities were decimated and sometimes entire settlements were wiped out. this tragedy facilitated the conquest of the land and the massive conversion of indian tribes to christianity across the americas. beginning in the last decade of the twentieth century, the aids pandemic intensified its spread on all continents, inflicting the greatest damage in africa at the social, economic, and political levels. some of the manifestations of this megacatastrophe were the significant shortening of life expectancy, massive destruction of family units, and orphanhood. in addition, the profound immune suppression caused by hiv led to a rampant increase in the incidence and prevalence of tuberculosis and other infectious diseases. combined, these factors had a disastrous effect on the political structure of most african countries. , across history, pandemics have differentially infiltrated battling armies and in this way tipped the outcome of battles and war. during the last decades and as a valuable complement to the written documentation on ancient bacterial pandemics, paleomicrobiological studies relating primarily to the plague (yersinia pestis) have contributed greatly to the reshaping of our understanding of its epidemiology. there was much confusion and controversy regarding the epidemiology of plague in the multiple pandemics that occurred several centuries ago, until the studies of french investigators, drancourt and raoult and colleagues, which revolutionized several contentious concepts established by other investigators. their work centered on the skeletal remains of common graves from various epochs. [ ] [ ] [ ] [ ] by examining the bacterial dna in the pulp of relatively preserved teeth, they found genomic evidence for not only yersinia pestis but also for bartonella quintana, a bacterium transmitted by lice. , since yersinia pestis is also found in body lice, the investigators developed a laboratory rabbit model and exposed the animals to human lice infected with specific bacteria. additional epidemics investigated by the same scientists were found to be caused by bartonella quintana alone or combined with rickettsia prowazekii. these and other studies supported the theory that infected lice were key spreaders of yersinia pestis, sometimes found in polymicrobial infections. with regard to smallpox, several well preserved mummies have been found in egypt and other countries. some mummies, such as that of the egyptian pharaoh, ramses v, provided dermal evidence that they had terminally suffered from smallpox. however, genomic evidence for the actual virus has been found only rarely in mummies. this can be explained by the poor long-term preservation of viruses. other diseases documented by paleomicrobiological methodologies include tuberculosis, leprosy, and multiple parasitic infections. the past years have seen rapid development of genetic sequencing technologies for rna and dna. from the beginning, rna/dna research has been characterized by both exaggerated promises and inflated expectations regarding the importance of human genetic traits that predispose for both infectious and non-infectious diseases. unfortunately, to date, no human genetic markers predisposing to sars-cov- infection, nor the severity of covid- , have been found-although recent isolated exceptions to this statement can be found. for example, there may be a predisposition to covid- among humans with blood group a as opposed to other blood groups. in sharp contrast to human genomics, mapping viral rna mutations of sars-cov- has enabled a fairly accurate reconstruction of its transmission ( figure b ) and helped to determine its phylogenetic origins (figure and figure a ). , viral sequencing of samples obtained from covid- patients, and being able to mark its different mutations, has provided a much clearer and more accurate picture of viral transmission as opposed to results obtained by contact tracing. for example, a study of disease importation to continents, countries, or regions has surprisingly uncovered several shuffled patterns in the genomic data. when considering importation of the first covid- cases to the western united states, the initial investigation based on contact tracing indicated that those cases were epidemiologically unrelated. however, after sequencing multiple virus isolates, the researchers concluded that they were not only closely related, but probably also began with one patient of origin. a series of questions must be answered when attempting to determine the primary source of a pandemic virus like sars-cov- . as it is a coronavirus, the most probable source is a virus that originated in bats. to confirm this theory, genomic profiles of coronaviruses previously isolated from bats must be compared with coronavirus profiles isolated from patients. if these sequences are identical, then it can be assumed that the primary source is a bat. however, if the virus has been preserved in panel b, each circle represents a coronavirus isolated from a patient. different colors indicate a virus mutation. a genome sequencer device (machines in the figure) is used to determine the location and type of mutation: auuu, a segment of the viral genome adenine uracil uracil uracil; aucu, same segment of the genome after mutation cytosine replacing middle uracil; or gucu, same segment of the genome following another mutation: guanine replaced adenine. in the refrigerators of a virology laboratory, there exists a very real possibility of viral "escape" from the laboratory to the community. in this scenario, especially if the laboratory is located in the proximity of the epidemic epicenter, a laboratory origin of the pandemic virus would be a strong alternative. additional tests such as antibody testing of the personnel working in the suspected laboratory should provide further assistance in elucidating the true origin and pathway of the virus. other possible trajectories should also be investigated. for example, if early infections occurred in persons who had close contact to bats or other animals, such as animals raised on farms or held and sacrificed at wet markets, this would clearly point to a specific zoonotic link. another possibility is that the new virus was engineered for investigational purposes or with malicious criminal intentions. by looking at the structural details of the virus, molecular biologists have ruled out and published the arguments against this last-mentioned possibility. the covid- pandemic exemplifies the difficulties encountered when attempting to quantify the important numerical parameters of the disease. first of all, when trying to count the number of infected cases, this parameter is significantly underestimated, primarily because the proportion of people who are asymptomatic or suffering from mild disease are not yet known. secondly, the initial shortage of laboratory kits has limited the number of people who can be ideally tested. the same problem arises when attempting to calculate the basic reproduction number, i.e. the number of persons who were infected by a certain patient. in this case, it is not easy to draw a line to those who should be considered a contact. a similar bias can interfere with calculation of the case fatality ratio, i.e. the proportion of infected persons who have died of covid- . in addition to underdetection of mainly asymptomatic patients, some deaths due to covid- complications passed under the radar since the patients had not been tested for the virus. last but not least, the sensitivity and specificity of viral tests are not %, resulting in occasional false negative or false positive results. a complementary method to assess pandemic-related mortality is to measure excess deaths compared to previous years during the same season. in order to retrospectively estimate the number of infected persons, serologic tests are performed that measure the presence of specific igm and igg antibodies to sars-cov- . currently, there are many ongoing seroprevalence studies of anti-sars-cov- antibodies; however, the picture remains incomplete regarding the significance of these findings as markers of previous infections. in , a cholera outbreak erupted in the densely populated neighborhood of soho, london. john snow, a physician who lived in the vicinity, mapped the houses of the many affected persons with cholera. he discovered a water pump in the center of the mapped area that had been used by all infected persons for obtaining drinking water. on his advice, municipal authorities removed the pump handle, resulting in an immediate end to the epidemic, with no further cases. it was subsequently learned that human excrement pits had drained into and contaminated the water supply. this brilliant epidemiological investigation led to john snow being considered the "father of epidemiology." in the s a major epidemic of cholera occurred in peru. vibrio cholera contaminated the ballast water in ships that had arrived from india. massive contamination of large quantities of live fish occurred when the ballast water was discharged into the sea near the shore. fish were subsequently caught from this area, and thousands of peruvians developed cholera after consuming raw fish (ceviche), a popular traditional food. the epidemic was terminated abruptly when the health authorities recommended to discard the head and branchia (gills) when preparing the fish for consumption. following the spread of the covid- pandemic, the health authorities of most countries imposed a lockdown with various intervals of delay from the first detected case(s) so as to contain the local spread of the virus. subsequently, they also imposed the use of respiratory masks to reduce airborne transmission by infected persons and to prevent contagion among the uninfected population. data from several countries have shown that the earlier and more stringent the lockdown was applied, the better the efforts in containing the pandemic. vaccines are human-made molecular tricks aimed at cheating the immune system of the host, to make it "believe" that it has encountered a microorganism-causing disease (professor myron levine, personal communication). table lists the current availability of approved vaccines for pandemic diseases. paradoxically, some of the newer diseases such as aids and those caused by coronaviruses still lack an approved effective vaccine. on the other hand, smallpox, a very old but already eradicated disease, was (to the best of our knowledge) the first disease for which several types of "natural" empiric vaccines were used, initially by the chinese in ancient times, and subsequently by edward jenner in england in the eighteenth century. , interestingly, vaccination with smallpox vaccines was implemented several centuries before the discovery of viruses in general, and variola and vaccinia viruses in particular. as recently summarized by the who, there are currently at least different covid- vaccination research projects underway in the quest to prevent covid- . essentially, six groups of vaccines are being explored, three of which first require the isolation of viral particles. these virions are either: ( ) weakened (attenuated); ( ) killed by hot or chemical substances (inactivated); or ( ) fragmented viruses followed by isolation of small pieces of the virus (subunits). a second class of vaccine starts with genetically engineered pieces of either rna or dna, which subsequently are embedded in either: ( ) plasmids (dna); ( ) lipids (rna); or ( ) an adenovirus vector. in all cases, particles are diluted in a solution for initial animal testing (preclinical trials) and subsequent testing in humans (clinical trials). clinical trials are performed in three phases, each one having an increasing number of volunteers. phase i ( - volunteers) examines safety and immunogenicity. phase ii (> volunteers) examines safety, immunogenicity, and dose adjustments. phase iii (more than , volunteers) primarily examines infection prevention following exposure. if all three phases are successful, then the vaccine undergoes regulatory approval and subsequent mass production under strict quality control. the main problem with the need to develop a vaccine against a new pandemic microbe is that the process is lengthy and mined with multiple obstacles. , in the past, the elapsed time from vaccine development to approval and production could take up to years. however, in order to efficiently and rapidly cope with an emerging pandemic, ideally, the scientific world must be prepared with predefined "templates" to facilitate accelerated vaccine development. if a proposed vaccine causes serious side effects or alternatively is not sufficiently immunogenic, the development process should be restarted from the beginning. in some cases, when a clinical evaluation of a vaccine reaches phase iii, the number of newly infected cases drops rapidly, making it difficult to test the actual efficacy of the vaccine in preventing infection. in the quest for a covid- vaccine, there have been a few initiatives to recruit volunteers who would be challenged with the virus after being immunized. however, for this scheme to work, it is essential to have a very efficacious drug available for treating the infection, should the vaccine fail. in general, with regard to the efficacy of antiviral treatments, viral infections can be divided into three groups: ( ) infections lacking an effective antiviral therapy (e.g. sars, yellow fever, measles); ( ) infections in which antivirals do not cure the infection, but which do produce varying degrees of clinical improvement (e.g. influenza, aids, covid- ); and ( ) infections that can be cured by antiviral therapy (e.g. ebola, hepatitis c). table provides an overview of the treatments used for different pandemic diseases. despite an increase in antibiotic resistance, there remain multiple choices for treatment of bacterial pandemics. for example, the main pillar of medical treatment for cholera is the emergency replacement of large amounts of fluid lost as a result of diarrhea and vomiting, with antibiotics playing a secondary role in its treatment. the fatality ratio of untreated cholera is around %. however, prompt administration of appropriate amounts of fluids either orally or intravenously decreases the case fatality ratio to %. the overwhelming death toll from covid- has sparked a myriad of projects to identify drugs that can be repurposed on a fast track to for special treatment of patients with severe disease. in the meantime, the only drug that has shown some beneficial results in a double-blind randomized clinical trial compared to placebo is the antiviral remdesivir, which has been approved by the us food and drug administration and other regulatory institutions. the administration of convalescent plasma from recovering patients with covid- is now being examined at different sites, including new clinical trials, but conclusions regarding this therapy are still pending. the economic impact of past pandemics is hard to examine due to the lack of robust data. however, a retrospective look at the first months of the covid- epidemic reveals a catastrophic impact on the economies of most countries having to cope with significant numbers of cases. the harshest economic impact generally occurred in varying degrees in wealthier countries. one of the most important parameters for quantifying economic damage is the gross domestic product forecast, although different countries use other economic metrics. according to the majority of prognoses, the damage was expected to be greatest during the second quarter of . the primary reason for the severe economic impact of covid- has been the leading and widely justified slogan, "health before wealth." another parameter that has significantly contributed to the economic crisis worldwide is the swift increase in job losses. quantification of financial activities in selected populations may add important data to more accurate evaluation of the world economy as a result of the covid- pandemic. for example, listing the purpose of out-of-home visits (residential, parks, workplace, grocery stores, pharmacy) may contribute to a composite financial evaluation of representative family units. , despite the rapid and advanced progress in many medical disciplines since the end of the nineteenth century, the covid- pandemic has sadly demonstrated vast limitations worldwide in successfully coping with it. there is no doubt that the unexpected and yet fully unknown behavior of sars-cov- has strongly contributed to its pandemic status. for example, the high proportion of infected but totally asymptomatic persons has made containment challenging, to say the least. in some cases, the proportion of infected persons feeling absolutely well can approach almost %. for example, a covid- outbreak occurred on a cruise ship departing ushuaia in the province of tierra del fuego, argentina, and navigating to the antarctic peninsula. sampling the entire population on board revealed that a vast majority had contracted the infection, but % of them were asymptomatic patients. another tricky characteristic of the virus is that infected patients expelled virus particles through their respiratory tract, primarily during the early phases of the incubation period before they became symptomatic. selected examples from prior pandemics should illuminate our vision for the future. smallpox went from being a totally empiric vaccine to global eradication of the disease. however, aids, which is caused by a zoonotic retrovirus that translates its rna to dna and enters the human genome, presents an almost impossible challenge in approaching a total cure for the disease, although combinations of antivirals are able to halt or reverse the progression of the disease. this retrospective analysis and comparison of covid- with prior pandemic diseases can contribute to the improvement of a rationale and scientific approach to future epidemics or pandemics. the most important take-away point should be an understanding of the high degree of preparedness that is needed, including various protocols for social distancing that are adapted to the different transmission modalities of the involved microbes. in addition, multiple innovative protocols aimed at a robust accelerated vaccine development process are needed. disease-causing viruses, or colonizing species in the animal kingdom, should be evaluated for potential spillage to human beings. for those specific viruses, it is imperative to delineate seminal protocols that can be launched in emergency situations. recently, the new york times launched two very recent dynamic applications that allow tracking the daily status of therapies and vaccines for covid- . , readers can visit these sites to access updates regarding ongoing developments related to covid- vaccines and treatments. finally, the conclusion of this paper, at this point in time, is to stress the importance of ongoing refinement of interactions between government leaders, scientists, and economists, at both the national and international levels, so as to better grapple with the current (and any future) pandemic, as it unfolds. genomic epidemiology of novel coronavirus -global subsampling clinical characteristics of coronavirus disease china the trinity of covid- : immunity, inflammation and intervention moderna's work on a covid- vaccine candidate inovio urgently focused on developing covid- vaccine: because the world can't wait. inovio website. , inovio pharmaceuticals a sars-cov- protein interaction map reveals targets for drug repurposing european centre for disease prevention and control. covid- pandemic the architecture of sars-cov- transcriptome novel drugs targeting the sars-cov- /covid machinery covid- infection: origin, transmission, and characteristics of human coronaviruses structural variations in human ace may influence its binding with sars-cov- spike protein characterization of the receptor binding domain (rbd) of novel coronavirus: implications for development of rbd protein as a viral attachment inhibitor and vaccine physiological and pathological regulation of ace , the sars-cov- receptor network-based drug repurposing for novel coronavirus -ncov/sars-cov- she discovered coronaviruses decades agobut got little recognition studies on the structure of a coronavirus-avian infectious bronchitis virus mossman k. bats and coronaviruses zoonotic origins of human coronaviruses epidemiological analysis of covid- and practical experience from china probably pangolin origin of sars-cov- associated with the covid- outbreak crossref . tobin mj. basing respiratory management of covid- on physiological principles cardiovascular disease and covid- covid- and liver disease chilblain-like lesions on feet and hands during covid- epidemic covid- and its implications for thrombosis and anticoagulation neurological manifestations and complications of covid- : a literature review kidney disease is associated with in-hospital death of patients with covid- kawasaki-like disease: emerging complication during the covid- pandemic world health organization (who) pandemics: a very short introduction pandemics: a brief history plagues & history: past, present and future viruses: a scientific american library book a planet of viruses the evolution and emergence of rna viruses pandemic: a brief history of influenza, the plague, cholera, and other infectious diseases that have changed the world; how they started, how they ended, and the lessons learned by humanity of the worst epidemics and pandemics in history. live science website outbreak: of the worst pandemics in history how of history's worst pandemics finally ended. history website visualizing the history of pandemics. visual capitalist website the worst outbreaks in u.s. history a history of aids: looking back to see ahead crossref . sharp pm, hahn bh. the evolution of hiv- and the origin of aids plague: history and contemporary analysis attenuated sars-cov- variants with deletions at the s /s junction the influenza pandemic: years of questions answered and unanswered severe influenza: overview in critically ill patients paleomicrobiology of humans the history of epidemic typhus a personal view of how paleomicrobiology aids our understanding of the role of lice in plague epidemics human lice in paleoentomology and paleomicrobiology advancements in next-generation sequencing analysis of protein-coding genetic variation in , humans the effects of blood group types on the risk of covid- infection and its clinical outcome phylogenetic analysis of ncov- genomes where it went from there stunned the scientists a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterization and epidemiology of novel coronavirus: implications for virus origins and receptor binding the proximal origin of sars-cov- a systematic review of asymptomatic infections with covid- evaluation of covid- rt-q pcr test in multi-sample pools preliminary prediction of the basic reproduction number of the wuhan novel coronavirus -ncov diagnosis of the coronavirus disease (covid- ): rrt-pcr or ct? excess mortality estimation during the covid- pandemic: preliminary data from portugal diagnostic performance of covid- serology assays cholera and the water supply in the south districts of london ucla department of epidemiology, fielding school of public health new insights on the emergence of cholera in latin america during : the peruvian experience impact of complete lockdown on total infection and death rates: a hierarchical cluster analysis the jenner society and the edward jenner museum: tributes to a physicianscientist smallpox vaccines for biodefense draft landscape of covid- candidate vaccines the race for coronavirus vaccines: a graphical guide the covid- vaccine development landscape the sars-cov- vaccine pipeline: an overview remdesivir for the treatment of covid- -preliminary report deployment of convalescent plasma for the prevention and treatment of covid- economics in the age of covid- the economic impact of covid- covid- : in the footsteps of ernest shackleton coronavirus drug and treatment tracker coronavirus vaccine tracker. the new york times key: cord- - qcj uiy authors: langlois, isabelle title: viral diseases of ferrets date: - - journal: vet clin north am exot anim pract doi: . /j.cvex. . . sha: doc_id: cord_uid: qcj uiy distemper and rabies vaccination are highly recommended because of the almost invariable fatal outcome of these conditions. vaccination should constitute an important part of a ferret's preventative medicine program. with the current and anticipated development and licensing of new vaccines, practitioners are invited to gain awareness of the latest vaccine information. establishment of a practice vaccination protocol with regards to the site of administration of rabies and distemper vaccines is paramount to document any future abnormal tissue reactions. influenza is the most common zoonotic disease that is seen in ferrets. although it generally is benign in most ferrets, veterinarians must take this condition seriously. the characteristic continuous antigenic variation of this virus may lead to more virulent strains; the recent emergence of avian influenza virus outbreaks; and the increased susceptibility of elderly, young, and immunosuppressed individuals. shedding begins approximately days postinfection [ ] . the primary site of viral replication is the respiratory epithelium and lymphoid tissue of the nasopharynx [ , ] . the virus disseminates by way of peripheral blood leukocytes to the liver, kidneys, gastrointestinal tract, urinary bladder, and brain. viremia has been documented days postinoculation or infection and persists until the virus is neutralized by antibodies or the animal dies [ ] . gastric hypochlorhydria has been associated with cdv infection in ferrets [ ] . the lack of gastric acid was hypothesized to be due to direct action of the virus on gastric mucosa or was secondary to the viral effect on the central nervous system. infected ferrets become symptomatic after an incubation period of to days [ , , ] . in ferrets, natural cdv infection most often consists of a catarrhal phase followed by a fatal neurotropic phase [ ] . the initial phase is characterized by anorexia, pyrexia, conjunctivitis, and serous nasal discharge. an erythematous, pruritic rash appears on the chin (fig. ) and eventually spreads to the inguinal area [ , ] . hyperkeratosis of the footpads (fig. ) occurs inconsistently in ferrets [ ] . melena may be observed early in the course of the disease [ , ] . some ferrets die during the catarrhal phase secondary to bacterial infections, such as pneumonia. clinical signs that are seen in the neurotropic phase include hyperexcitability, muscle tremors, hypersalivation, seizures, and coma. ferrets die to days after being infected with ferret-adapted cdv [ , ] . the disease has a longer course when ferrets are infected with a wild canine strain; death occurs to days postinfection [ ] . sometimes a decrease in temperature is observed by the time the animals become moribund [ ] . a tentative diagnosis of canine distemper is based on the presence of typical clinical signs, severe leukopenia, a history of potential exposure to the virus, and questionable vaccination. a study demonstrated that virulent and attenuated cdv strains caused a severe leukopenia week after infection [ ] . leukocyte counts from animals that were infected with virulent strains remained low, whereas those of animals that received attenuated strains progressively increased to reach preinfection, or near preinfection, values days postinfection. the diagnosis of canine distemper is confirmed routinely by an immunofluorescence test on peripheral blood smear, buffy coat, or conjunctival scrapings in live animals. in experimentally-infected ferrets, a reverse transcriptase-polymerase chain reaction (rt-pcr) has been used to detect the virus in peripheral blood [ ] . recently, nested-pcr was found to be a sensitive and specific method for diagnosis of cdv infection [ ] . on postmortem examination, the gross lesions correspond to the aforementioned clinical signs. on hematoxylin and eosin-stained sections, round eosinophilic intracytoplasmic and occasional intranuclear inclusion bodies are present in epithelial cells of the trachea, bronchi, and urinary tract [ ] . other tissues, such as skin, gastrointestinal tract, salivary and adrenal glands, spleen, lymph nodes and brain, have been reported to contain inclusions [ ] . ferrets that have signs that are suggestive of canine distemper infection should be placed in isolation. supportive care, including fluid therapy, systemic and ophthalmic antibiotics, gavage feeding, and bathing with antipruritic shampoo, may be initiated. administration of immune anti-cdv serum also may be considered. thorough daily disinfection of the environment is indicated. in all cases, prognosis is grave. the virus is inactivated by heat, visible light, and commonly used disinfectants ( . % phenol, . % roccal, %- % sodium hydroxide, and . % formalin) [ , ] . some investigators reported that out of more than cases of distemper, not a single ferret survived the infection [ ] . blair et al [ ] treated distemper in a -month-old ferret while in the catarrhal phase. the ferret's life span and quality of life were improved with treatments, but the neurotropic phase could not be prevented and the animal was euthanized. when faced with an outbreak of canine distemper in a ferret colony, euthanasia and repopulation following disinfection is recommended. distemper is prevented best by vaccination. two vaccines are approved for use in ferrets in north america-fervac-d canine distemper vaccine (united vaccines inc., madison, wisconsin) and purevax ferret distemper vaccine (merial, athens, georgia and montre´al, quebec, canada). fervac-d is a modified-live virus vaccine of chick cell origin. a . % incidence of anaphylactic reaction has been reported with this vaccine [ ] . adverse events developed within minutes and were characterized by hyperemia, hypersalivation, and vomiting. purevax is a lyophilized vaccine of a recombinant canary pox vector that expresses the ha and f glycoproteins of cdv. the manufacturer uses the term ''ferret distemper'' vaccine on the product label to prevent confusion with their canine products; however, there is no such thing as a ''ferret distemper virus'' and ferrets are infected by cdv. the absence of adjuvant or entire distemper virus decreased postvaccination risks and the manufacturer reports an incidence of . % reversible anaphylactic reactions in its field safety trials. another modified-live canine distemper vaccine attenuated in primate cell line (galaxy d, schering plough animal health, omaha, nebraska) has been studied in ferrets. this vaccine was effective in preventing canine distemper in young ferrets that were challenged with virulent cdv after two vaccine inoculations [ ] ; however, the duration of immunity and the incidence of vaccine reactions are unknown. clinical use of this vaccine is extra label (not approved for use in ferrets by usda) and requires informed owner consent. postvaccinal distemper infections were reported in black-footed ferrets that were vaccinated with chicken embryotissue culture-origin cdv vaccine and in domestic ferrets that were vaccinated with canine cell products [ , ] . therefore, vaccination of ferrets with these products must not be performed. most ferrets that are obtained from pet stores at a young age received only one dose of cdv vaccine before leaving the breeding facility. repeated inoculations are required because maternal antibodies may interfere with proper immune response to cdv vaccine antigens. ferret kits should be vaccinated at weeks of age and every weeks for a total of vaccinations [ , ] . yearly booster vaccines are recommended [ ] . observation of the ferret for minutes following vaccination is advisable. in the event that a vaccine reaction occurs, administration of fluids, oxygen, antihistamine, and epinephrine may be indicated. parvovirus strains of varying virulence and immunogenicity cause aleutian disease. the disease was first reported in mink in the s and got its name because mink that were homozygous for the aleutian (blue) gene were affected most severely [ ] . the infection was documented in ferrets in the late s [ ] . at least three separate strains of aleutian disease virus (adv)-distinct from the mink strains-have been identified in ferrets [ , ] . the ferret strains are believed to be mutant strains of the mink parvovirus; the hypervariable capsid region of the ferret strains of adv is similar to that of the mink parvovirus [ ] . ferrets can be infected with the mink virus [ , ] and ferret adv can infect mink; however, the virulence is lower compared with mink that are infected with mink strains [ , , ] . transmission of the virus may occur by aerosolization; by direct contact with urine, feces, saliva, and blood; or by contact with fomites [ , ] . vertical transmission of adv occurs in mink [ , ] . the lesions that are caused by adv infection are immune mediated, but the mechanism by which adv interferes with the immune system is unknown. the severity of disease depends on the origin (mink or ferret) of the adv strain that is involved as well as the immune status and genotype of the infected individual [ ] . minks that are infected with mink adv strains deposit immune complexes in various organs that result in glomerulonephritis, bile duct proliferation, and arteritis [ ] . mink kits from antibody-free jills die from acute interstitial pneumonia when infected with virulent adv [ ] . affected individuals are immunosuppressed, and therefore, are more susceptible to influenza, viral enteritis, and distemper [ , ] . ferrets that are inoculated with ferret-adapted adv exhibit marked, persistent hyperglobulinemia and periportal lymphocytic infiltrates of the liver [ ] ; however, immunocompetent adult ferrets that are infected experimentally can develop a persistent infection without clinical disease [ ] . mink strains cause milder lesions and only a moderate elevation in gamma globulins in ferrets [ ] . most ferrets that show clinical signs are between and years old. ferrets can be infected for years before clinical symptoms are noted [ ] . any situation that leads to immunosuppression may play a role in the development of clinical disease. the clinical presentation of infected ferrets varies. some ferrets that are infected with adv die without clinical signs in good body condition [ ] . generally, ferrets show signs of a chronic wasting disease with progressive weight loss, malaise, and melena [ ] . acute dyspnea was described in one report [ ] . central nervous system signs, such as tremors, ataxia, paralysis, and convulsions, also have been reported [ , [ ] [ ] [ ] [ ] . affected animal also may present with fecal and urinary incontinence [ , ] . a presumptive diagnosis can be made based on a high gamma globulin concentration, the history, and clinical signs. although hypergammaglobulinemia is considered to be pathognomonic in mink, this feature is not always present in infected ferrets [ , ] . serum protein electrophoresis often shows that gamma globulins account for more than % of the total protein concentration [ , ] . other clinical pathologic findings of infected ferrets are variable. anemia, possibly attributable to hemolysis and decreased erythrocyte production, may be present [ ] . biochemical abnormalities, such as azotemia and elevated liver enzymes, may be seen according to damage that results from immune complex deposition. proteinuria and urinary casts that are secondary to kidney damage also may be observed [ ] . diagnosis of aleutian disease is confirmed antemortem with a positive serum titer coupled with hypergammaglobulinemia or lymphoplasmacytic inflammation in tissue biopsy samples. two serologic tests are available for adv testing-counterimmunoelectrophoresis (cep or ciep) (united vaccines, madison, wisconsin) and elisa (avecon diagnostics, bath, pennsylvania). the specificity and sensitivity of the elisa test has not been investigated in ferrets. therefore, results must be interpreted with caution. the cep test is used routinely in ferrets and is an effective method for identifying ferrets that have adv antibodies [ , [ ] [ ] [ ] [ ] . presence of antibodies without clinical disease for extended periods is possible [ ] . ferrets probably develop persistent and nonpersistent, nonprogressive forms of adv infection similar to mink [ , ] . detection of adv dna by in situ hybridization has been performed, but this method is not practical to screen for this condition [ ] . recently, polymerase chain reaction amplification of part of the capsid gene that is specific to adv and restriction fragment length polymorphism to distinguish the ferret types of adv from the mink types of adv are valuable, time-saving assets for diagnosis of this infection in ferrets [ ] . at necropsy, infected ferrets may have few or no gross lesions. hepatosplenomegaly, splenomegaly, and mesenteric lymphadenopathy have been reported [ , ] . the most consistent histologic findings of adv infection in ferrets are periportal infiltration of the liver by plasma cells, lymphocytes, and macrophages with stimulated lymphoid tissues [ , ] . bile duct hyperplasia, periportal fibrosis, and membranous glomerulonephrosis have been documented [ , , ] . in individuals that present with neurologic signs, perivascular lymphocytic cuffing in the brain and spinal cord (fig. ) and lymphoplasmacytic meningitis may be observed [ , , ] . there is no definitive treatment of aleutian disease in ferrets. symptomatic ferrets may benefit from use of anti-inflammatory medication or immunosuppressive drugs, such as prednisone and cyclophosphamide. in mink, treatment with cyclophosphamide has been used to control infections for up to weeks, but virus titers did not decrease [ ] . administration of gamma globulin-containing adv antibody may be considered because it contributed to decreased mortality rates in mink kits [ ] . control of aleutian disease is dependent on testing, cessation of breeding, and isolation of seropositive ferrets. all seropositive ferrets should be considered to be potential sources of adv and should be isolated from seronegative ferrets [ ] . in mink farms, testing by ciep and removing all individuals that have adv antibodies has been an efficient method to eradicate the disease [ ] . this approach, coupled with thorough disinfection, should be considered in any facility with a large number of ferrets. formalin, sodium hydroxide, and a phenolic disinfectant were efficacious against adv in the presence of organic material [ ] . there is no vaccine to prevent aleutian disease and vaccination probably is contraindicated because of the immune-mediated nature of this condition. in mink, vaccination exacerbated the severity of aleutian disease [ ] . in the late s, a novel diarrheal disease that affected domestic ferrets was reported by pet owners and ferret breeders in the mid-atlantic area of the united states. since that time, this condition has been diagnosed throughout north america and in several other countries. the disease was named epizootic catarrhal enteritis (ece) on the basis of similarities to the epizootic catarrhal gastroenteritis of mink [ ] . ece of mink is caused by a coronavirus that is related to transmissible gastroenteritis virus of pigs [ ] . research strongly implicates a coronavirus as the causative agent of ece because: ( ) microscopic lesions that were consistent with intestinal coronavirus infection were detected consistently in diseased ferrets; ( ) coronavirus particles were identified in the feces and enterocytes, but no other viruses could be identified; and ( ) immunohistochemical staining of jejunum showed coronavirus antigens in affected ferrets, but not in healthy individuals [ ] . the disease is characterized by high transmissibility, high morbidity, and a low mortality rate. ferrets show signs of lethargy and anorexia within to hours postinfection. vomiting is the first sign of gastrointestinal disease in most ferrets, but it may go unnoticed by some owners because it subsides within hours [ ] . subsequently, profuse green, bile-tinged diarrhea with a variable amount of mucus develops (fig. ) . the stool's appearance is responsible for the term ''green slime disease'' that also is used to describe this condition [ ] . the severity of clinical signs is highly variable; older ferrets that have concurrent diseases, such as insulinoma, long-standing infection with helicobacter mustelae, or adrenal neoplasia, are prone to develop severe clinical signs. ulcerations of the intestinal wall may occur which leads to the presence of blood in the feces. young ferrets tend to have mild or subclinical infection. the hypersecretory phase of uncomplicated ece often resolves within to days in healthy young animals. in some ferrets, this phase may be followed by a period of maldigestion or malabsorption of variable duration secondary to persistent lymphocytic inflammation of the intestinal wall. the feces become yellowish in color and contain grainy material that resembles bird seed. ece often can be diagnosed solely on the basis of characteristic historical findings and clinical signs [ ] . thorough collection of the history data often reveals exposure to an asymptomatic ferret to hours before the onset of clinical signs. generally, clinicopathologic findings are nonspecific. inanition may cause increased serum activity of alanine aminotransferase and alkaline phosphatase secondary to mobilization of peripheral fat stores to the liver, with resultant hepatocellular swelling [ , , ] . hypoalbuminemia may develop as a result of enteritis and malabsorption in chronically affected individuals. leukocytosis may be present in ferrets that have concurrent bacterial infection or gastric ulcers. definitive diagnosis of coronavirus infection often is difficult. coronavirus-like particles may be identified in the feces by electron microscopy during the acute phase of the disease process. characteristic histologic lesions that are seen in intestinal coronavirus infection, such as lymphocytic enteritis with villus atrophy, fusion, blunting and vacuolar degeneration or necrosis of the apical epithelium, may be identified on intestinal biopsy or necropsy specimens [ ] . ferrets may become dehydrated rapidly during the hypersecretory phase of infection. dehydration and electrolyte imbalances need to be addressed. fluids that are supplemented with dextrose and electrolytes may be administered orally, subcutaneously, or intravenously, according to the degree of dehydration. antibiotic may be indicated to prevent secondary bacterial infection, particularly in cases with suspected intestinal ulcerations. in these cases, administration of sucralfate and an h antagonist (eg, cimetidine) also are beneficial. sucralfate requires an acidic environment to be effective, so it should be given at least minutes before an h antagonist. syringe feeding with a highly digestible diet (science diet a/d, hill's prescription diet) may be indicated if anorexia persists. if clinical signs that are suggestive of maldigestion develop, oral administration of prednisone may be indicated. use of an anti-inflammatory dose of prednisone for to weeks, was followed by gradual tapering of the dose successfully. influenza viruses belong to the class orthomyxoviridae. human influenza types a and b are pathogenic to ferrets [ ] . ferrets also are susceptible to avian, seal, equine, and swine influenza a viruses, although only human, avian, and swine strains induce clinical signs [ ] [ ] [ ] [ ] [ ] . infection with influenza b virus less frequently results in illness and is associated with a milder clinical course [ ] . transmission of influenza virus from human to ferrets and from ferrets to humans was documented in the s [ , ] . ferrets are used extensively as an animal model for influenza virus pathogenesis and immunity studies because their biologic response to influenza infection is similar to that of humans [ , ] . influenza virus is transmitted by aerosol droplets from an infected individual, either to a human or a ferret. after intranasal inoculation, the virus localizes and replicates in great numbers within the nasal mucosa [ ] . following a short incubation period, the body temperature increases and then decreases approximately hours later [ ] . transmission of the virus begins at the height of pyrexia and continues for to days [ ] . as in humans, the disease is characterized by upper respiratory signs. clinical signs appear hours postinfection and include anorexia, malaise, fever, sneezing, and serous nasal discharge [ , ] . infection usually is mild in adult ferrets compared with neonates who can be severely ill [ ] . conjunctivitis, photosensitivity, and unilateral otitis also may be seen [ , ] . influenza infection may involve the lower respiratory tract in some susceptible animals [ , ] . usually, influenza virus is confined to the bronchial and bronchiolar tissues [ , ] . the disease may be fatal in -to -day-old ferret kits secondary to bronchiolitis, pneumonia, and aspiration of material from the upper respiratory tract [ , , ] . lancefield group c hemolytic streptococci have been involved in secondary bacterial pneumonia [ ] . influenza virus may infect the intestinal epithelium and cause limited enteritis [ , ] . hepatic dysfunction also has been reported in ferrets that were infected experimentally with influenza [ ] . neurologic symptoms, including ataxia, hind-limb paresis, and torticollis, were reported in ferrets that were infected experimentally with avian influenza a (h n ) viruses that were isolated from the outbreaks of disease in domestic poultry markets in hong kong [ , ] . generally, the diagnosis of influenza infection is based on the presence of compatible clinical signs, a history of exposure to infected individuals, and recovery from illness within to days. the differential diagnosis of any ferrets that has upper respiratory signs should include canine distemper. the usually mild and brief nature of influenza infections help to distinguish it from distemper. the use of virus isolation or hemagglutinin-inhibiting antibody titers on acute and convalescent serum samples rarely is needed for a diagnosis [ ] . an enzyme-linked immunosorbent assay has been used to detect antibodies against influenza a and may be used to obtain a diagnosis rapidly [ ] . clinical pathologic findings may present abnormalities. studies demonstrated an elevation in the neutrophil:lymphocyte ratio in the peripheral blood [ , ] . transient lymphopenia, with a loss of % to % of peripheral blood lymphocytes days postinfection was reported experimentally with avian influenza a (h n ) [ ] . plasma biochemical values generally are within reference ranges, but increases in concentrations of creatinine, blood urea nitrogen, potassium, albumin, and alanine aminotransferase were reported in some infected ferrets [ ] . in most cases, infected ferrets can be treated at home. owners should be instructed to let their ferret rest until fully recovered. offering affected animals their favorite diet, highly palatable food (chicken baby food, beef baby food), or a highly energetic diet (science diet a/d, hill's prescription diet) is indicated. force feeding and offering water by syringe can be performed as needed. treatments to relieve clinical symptoms should be done on a case by case basis. if coughing is persistent, a pediatric cough suppressant without alcohol (at the pediatric dosage on a per weight basis) has been used [ ] . to alleviate nasal congestion, the use of an antihistamine, such as diphenhydramine ( - mg/kg, by mouth, every to hours) [ , ] , or intranasal delivery of phenylephrine may help [ ] . antibiotics may be indicated to control secondary bacterial infections of the respiratory tract. neonates typically succumb to secondary bacterial infections. therefore, antibiotics may be useful in these patients to reduce mortality [ ] . the use of non steroidal anti-inflammatory drugs to control fever is of questionable benefit because fever seems to be important in restricting the severity of infection [ ] . experimentally, ferrets who received aspirin had cooler body temperature, but they shed more virus and their viral levels decreased less rapidly compared with ferrets that were not treated with an antipyretic. administration of antiviral medication has been studied in ferrets. amantadine hydrochloride ( mg/kg, by mouth, every hours) (symmetrel, bristol-myers squibb canada, montreal, quebec, canada) has been effective in treating ferrets that have influenza [ ] . zanamivir ( . mg/kg) (relenza, glaxo wellcome, mississauga, ontario, canada), given as a onetime intranasal dose was able to prevent influenza infection [ ] . administration of amantadine in ferrets rapidly produces antiviral resistance, but use of zanamivir does not [ ] . vaccination of ferrets against influenza virus generally is not recommended because it is usually a mild disease and the antigenic variation of the virus complicates vaccination [ , ] . experimentally, ferrets who recovered from influenza infection remained resistant to infection with the same strain for weeks following initial infection [ ] . ferret kits are protected from disease by milk-derived antibodies in immunized females [ ] . controlling influenza infection resides in avoiding exposure of susceptible ferrets to infected individuals, either ferret or human. owners should be advised to minimize contact with their ferrets if they have a respiratory infection and should be sure to wash their hands thoroughly before changing the animal's cage, food, and water. veterinarians who have respiratory infections may consider wearing a mask and gloves. rhabdovirus causes rabies, an acute and almost invariably fatal disease that affects many mammals and humans. over the past several years, there have been numerous reports of ferret bite injuries, including unprovoked attacks on infants and small children [ ] [ ] [ ] . these reports brought great controversy over the acceptability of keeping ferrets as pets with regard to the potential risk for rabies. this was because the period of viral shedding in the animal's saliva-before the onset of recognizable signs-was unknown at that time [ , , ] . to the author's knowledge, there is no reported case of human rabies secondary to a ferret bite. since , less than cases of rabies in domestic ferrets have been reported to the u.s. centers for disease control [ , ] . one of theses cases was attributed to vaccinating a ferret with modified-live virus rabies vaccine [ ] . rabies virus must contact nerve endings and enter nerve fibers before infection occurs. exposure to rabies virus does not always lead to productive infection [ ] [ ] [ ] . host response to rabies virus is influenced by the rabies virus variant, the viral dose, the route of transmission, the host species, and individual variations [ ] . infection occurs primarily by contact of nerve endings with infected saliva from a rabid animal as a result of a bite wound. contact with the conjunctiva or olfactory mucosa also can result in transmission [ ] . transmission of rabies through ingestion of an infected mouse was unsuccessful [ ] . the pathogenesis of rabies in ferrets has been studied using european red fox rabies variant, north central skunk rabies variant, and raccoon rabies variant [ ] [ ] [ ] . the mean incubation period is approximately month [ , ] . clinical signs reported include ascending paralysis, ataxia, tremors, paresthesia, hyperactivity, anorexia, cachexia, bladder atony, constipation, fever, and hypothermia [ ] [ ] [ ] . two of rabid ferrets that were inoculated with raccoon rabies variant showed aggressive behavior [ ] . signs were reported to be mild in ferrets that were inoculated with the european red fox rabies variant [ ] . mean morbidity period was to days [ , ] . mortality in ferrets that were inoculated with the european red fox variant was dose dependent [ ] . in the study that used skunk variant, ferret susceptibility was dose dependent and the incubation period was inversely proportional to dose [ ] . in contrast, ferrets that were inoculated with raccoon variant were only moderately susceptible-regardless of dose-and there was no correlation found between viral dose and incubation period [ ] . ferrets who survived experimental infection remained clinically normal except for one ferret that was given skunk rabies variant and had severe paralytic sequelae [ ] [ ] [ ] . the immunologic response of ferrets to rabies virus infection seems to vary depending on the rabies virus variant that was inoculated. virus neutralizing antibodies were demonstrated in of ( . %) rabid ferrets that were inoculated with skunk rabies variant compared with only of ( . %) rabid ferrets that were given raccoon rabies variant [ , ] . also, among ferrets that survived the infection, the proportion of seropositive cases was greater in ferrets that were given skunk rabies variant [ , ] . ferrets may or may not excrete rabies virus in their saliva depending on the virus variant to which they have been exposed. viral shedding was not documented in the saliva days postinoculation with european red fox variant [ ] . rabies virus was not detected in the saliva of any ferrets that were given skunk rabies variant, but it was isolated from the submaxillary salivary gland of one rabid ferret that was euthanized [ ] . shedding of rabies virus in the saliva was documented in ferrets that were inoculated with the raccoon rabies variant [ ] . rabies virus was isolated from the salivary glands of % of rabid ferrets and % shed rabies virus in their saliva. initial viral excretion ranged from days before the onset of clinical signs to days after the onset. viral shedding also was documented in of ferrets that were inoculated with a rodent strain of rabies virus [ ] . rabies should be included in the differential diagnoses of any ferret that has an unexpected onset of paralysis or acute personality change, especially if the ferret is unvaccinated, has access to outdoors, or lives in an area that is experiencing an epizootic of rabies. a ferret that is suspected of having rabies should be euthanized and its head should be submitted to the appropriate laboratory. generally, the diagnosis is based on direct immunofluorescent antibody testing of brain tissue [ , , ] . confirmation may be established by intracerebral inoculation of suckling mice or inoculation of tissue culture with homogenized brain tissue [ ] . the protective efficacy of killed rabies vaccine was demonstrated in ferrets that were vaccinated once subcutaneously with an inactivated rabies virus and were challenged year later with street virus of fox origin. vaccinated ferrets had a survival rate of % compared with less than % for unvaccinated controls [ ] . domestic ferrets that were vaccinated with a commercially inactivated rabies vaccine showed rapid induction of virusneutralizing antibody-a seroconversion that persisted at least months [ ] . based on these studies, killed rabies vaccines were approved for immunizing ferrets [ , ] (table ). ferrets should be vaccinated once at months of age or older and then annually thereafter [ ] . an animal is considered to be immunized if the primary vaccination was administered at least days previously [ ] . because a rapid anamnestic response is expected, an animal is considered to be vaccinated currently immediately after booster vaccination [ ] . local injection site reactions were reported to develop frequently with rabies vaccine [ ] . studies in cats showed that rabies vaccines more consistently produce granulomatous inflammation at vaccine sites, although leukemia virus vaccines are incriminated more often in the pathogenesis of vaccine-associated sarcomas [ ] [ ] [ ] . there is only one report of vaccineassociated sarcoma in a ferret [ ] . the ferret had been vaccinated for distemper and rabies on an annual basis in the dorsal area of the neck or interscapular area, so it is impossible to determine from which vaccine the tumor arose. practitioners should consider establishing a protocol in regard to the site of administration of rabies and distemper vaccines. this would allow the determination of which vaccine may be involved in the event that a tumor develops. a study demonstrated that the cellular response to the canary poxvectored rabies vaccine in ferrets was much milder than to the adjuvanted rabies vaccines [ ] . consequently, its future use may be associated with a decreasing number of local vaccine reactions. also, because persistence of lymphocytes and macrophages has been suggested to play a role in the pathogenesis of vaccine-associated sarcomas, the canary pox-vectored vaccine would be less likely to be involved in the oncogenesis of these tumors. although it shows great promise, practitioners should remember that canary pox-vectored rabies vaccine is not approved for use in ferrets in north america at this time. to the author's knowledge, there is only one report of anaphylactic reaction in a ferret following administration of an inactivated rabies vaccine [ , ] . this ferret previously had an anaphylactic reaction after receiving a distemper and rabies vaccine simultaneously. observation of ferrets for minutes following rabies vaccination is advisable. unvaccinated ferrets that are exposed to a rabid animal should be euthanized immediately [ , ] . if the owner refuses, the animal should be quarantined strictly for months and vaccinated month before being released [ , ] . ferrets that are vaccinated currently should be revaccinated immediately [ , ] . if a healthy ferret bites a person, it should be confined and observed for days [ , ] . the animal needs to be evaluated by a veterinarian at the first sign of illness during confinement. if signs that are suggestive of rabies develop, the animal should be euthanized and tested for rabies [ , ] . rotavirus belongs to the family reoviridae. all rotaviruses are unique because they possess double-stranded rna genome. generally, these viruses cause diarrhea in young animals and children, but they also can occur in older individuals [ ] . an atypical rotavirus was isolated from neonatal ferrets (mustela putorius furo) that had diarrhea at a large commercial farm in the united states. the disease was recognized at the ferret farm for several years and was referred as ''ferret kit disease'' [ ] . partial characterization identified this virus as an atypical rotavirus, based on the lack of rotavirus group a common antigen and on its distinct double-stranded rna electropherotype pattern in polyacrylamide gels. in finland, a rotavirus outbreak in ferret kits had a mortality rate that approached % [ ] . clinical signs of rotavirus infection occur in -to -week-old ferrets. soft yellow to green diarrhea is present with associated fecal staining or matted hair on their bodies. erythema of the anus and perineum also is reported [ ] . the disease was prevalent throughout the year at the american commercial farm, with a increased incidence in colder months. morbidity among primiparous jills was high (up to %); the morbidity rate decreased with each gestation to range between % to % in multiparous jills [ ] . the condition could be reproduced in -to -week-old ferret kits that were inoculated orally with viral preparations that were obtained from diarrheic ferrets; however, mortalities nor histologic lesions were observed in individuals that were infected experimentally [ , ] . antemortem diagnosis is difficult. viral particles may be detected by electron microscopy in clarified, ultracentrifuged fecal suspensions, following negative staining in symptomatic ferrets. in a study, % of diarrheic ferrets that were infected naturally were positive for rotavirus particles in their feces. the ferret atypical rotavirus does not react with the rotazyme test (abbott laboratory, chicago, illinois), a commercially available enzyme immunoassay [ , ] . the prevalence of this viral infection is unknown in ferrets because of the absence of reliable serologic tests. on postmortem examination, gross lesions are limited to the gastrointestinal tract. subtle histologic lesions that consist of mild blunting of the tips of the intestinal villi of the small intestine with enterocyte metaplasia to cuboidal cells may be observed. secondary bacterial infections may be a significant factor in the severity of the diarrhea. therefore, antibiotics are indicated and may contribute to accelerated recovery and decreased mortality rates. additional supportive care, including appropriate fluid therapy and force feeding, are indicated in most cases. in piglets, colostral antibodies play a key role in the protection against rotavirus [ ] . this also may be true for ferrets. ferret kits acquire most of their passive immune globulins from their mothers by intestinal transmucosal absorption from colostrum and milk; they acquire all of their iga from their mother's milk [ ] . the observed decrease in morbidity from ferrets of primiparous jills to ferrets of multiparous jills may be secondary to build-up of colony immunity with increasing age and exposure to the virus [ ] . oral vaccination of primiparous jills was attempted at a ferret breeding farm; however, this procedure was ineffective. atypical rotaviruses have not been cultivated successfully in cell culture [ ] . the ability to propagate the virus in cell culture in sufficient numbers will play a key role in the development of a vaccine. the infectious bovine rhinotracheitis virus (ibr) belongs to the family herpesviridae. there is only one published case report of spontaneous ibr infection in a ferret [ ] . the virus was isolated from the liver, the spleen, and the lungs of a clinically normal ferret. its diet consisted of % raw beef by-products; it was hypothesized that virus-laden raw beef was the source of infection. the pathogenesis by which the virus disseminated to the liver, spleen, and lung tissue has not been elucidated. in contrast to naturallyoccurring infection, experimental infection of ferrets with ibr virus by intranasal and intraperitoneal inoculations induced acute and chronic respiratory disease [ ] . considering that ibr virus can cause pathology in ferrets, these animals should not be fed raw meat or meat products [ ] . distemper and rabies vaccination are highly recommended because of the almost invariable fatal outcome of these conditions. vaccination should constitute an important part of a ferret's preventative medicine program. with the current and anticipated development and licensing of new vaccines, practitioners are invited to gain awareness of the latest vaccine information. establishment of a practice vaccination protocol with regards to the site of administration of rabies and distemper vaccines is paramount to document any future abnormal tissue reactions. influenza is the most common zoonotic disease that is seen in ferrets. although it generally is benign in most ferrets, veterinarians must take this condition seriously. the characteristic continuous antigenic variation of this virus may lead to more virulent strains; the recent emergence of avian influenza virus outbreaks; and the increased susceptibility of elderly, young, and immunosuppressed individuals. viral diseases canine distemper in black-footed ferrets (mustela nigripes) from wyoming pathogenicity of morbilliviruses for terrestrial carnivores a ferret model of canine distemper virus virulence and immunosuppression experimental distemper in mink and ferrets. i. pathogenesis gastric hypochlorhydria in ferret distemper a clinical guide to the pet ferret practical exotic animal medicine, the compendium collection canine distemper virus infection in the domestic ferret canine distemper virus (cdv) infection of ferrets as a model for testing morbillivirus vaccine 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inoculated with a raccoon rabies isolate the pathogenesis of rabies and other lyssaviral infections: recent studies susceptibility of carnivore to rabies virus administered orally zur frage der adaptationsfa¨higkeit von zwei in mitteleuropa isolierten tollwutvirusstaˆmmen an eine domestizierte und zwei wildlebende spezies. ein beitrag zur epidemiologic der tollwut . mitteilung: u¨bertragsversuche an frettchen mit einem nagerisolat. [the adaptability of two rabies virus strains isolated in central europe to one domesticated and two wild-living species: a contribution to epidemiology of rabies evaluation of an inactivated rabies virus vaccine in domestic ferrets serologic response of domestic ferrets (mustela putorius furo) to canine distemper and rabies virus vaccines national association of state public health veterinarians. compendium of animal rabies prevention and control canadian food inspection agency, animal health and production division, veterinary biologics section vaccine injection-site sarcoma in a ferret historical review and current knowledge of risk factors involved in feline vaccine-associated sarcomas feline vaccine-associated sarcomas focal necrotizing granulomatous panniculitis associated with subcutaneous injection of rabies vaccine in cats and dogs: cases ( - ) cats differ from mink and ferrets in their response to commercial vaccines: a histologic comparison of early vaccine reactions another interpretation of ferret's reaction to vaccination rabies postexposure prophylaxis isolation of an atypical rotavirus causing diarrhea in neonatal ferrets ontogeny of the ferret humoral response isolation of infectious bovine rhinotracheitis virus from mustelidae experimental infectious bovine rhinotracheitis virus infection of english ferrets (mustela putorius furo l) key: cord- -c o b j authors: matibag, gino c.; igarashi, manabu; la porte, ron e.; tamashiro, hiko title: advocacy, promotion and e-learning: supercourse for zoonosis date: - - journal: environmental health and preventive medicine doi: . /bf sha: doc_id: cord_uid: c o b j this paper discusses the history of emerging infectious diseases, risk communication and perception, and the supercourse lectures as means to strengthen the concepts and definition of risk management and global governance of zoonosis. the paper begins by outlining some of the key themes and issues in infectious diseases, highlighting the way which historical analysis challenges ideas of the ‘newness’ of some of these developments. it then discusses the role of risk communication to public accountability. the bulk of the paper presents an overview of developments of the internet-based learning system through the supercourse lectures that may prove to be a strong arm for the promotion of the latest medical information particularly to developing countries. we live in a dangerous world. yet it is also a world far safer in many ways than it has ever been. diseases that only recently were mass killers have been all but eradicated. advances in public health, medicine, environmental regulation, food safety, and worker protection have dramatically reduced many of the major risks we faced just a few decades ago. governance refers to how societies structure responses to the challenges they face. analyses of emerging and re-emerging infectious diseases (eids) have made it clear that national and international societies are confronting increased microbial threats ( ) ( ) ( ) . whether the focus is bioterrorism, hiv/aids, severe acute respiratory syndrome (sars), or avian influenza, germs increasingly pose dangers to human societies. germ governance concerns how societies, both within and beyond national borders, structure their responses to pathogenic challenges ( ) . the global nature of the microbial threat requires that governance address the borderless challenges presented by infectious diseases. the emergence of sars is a major global public health threat that requires a coordinated global response in terms of continued and improved surveillance and of research into a number of important public health issues. while much has been learnt about sars since it was brought to international attention in march , there remain many unanswered questions about where it came from, how it spreads, and the effectiveness of public health and other measures employed to control the disease. the overall goal of the "supercourse for zoonosis" is to show the most recent development in the knowledge of sars and other zoonotic diseases such as avian influenza and bovine spongiform encephalopathy (bse), inter alia, which have significant global impact not only on health but also on the economy. the specific objectives of "supercourse for zoonosis" are to develop a set of educational materials for the control of zoonotic diseases, to disseminate them effectively via the internet, to facilitate their use in the prevention and control of the diseases, and to promote human health while minimizing their economic impact. in the light of all these advances, it is most appropriate that all countries remain vigilant, not only with sars, but also to all zoonotic diseases that may toll the productivity of human, animal, ecological, and economical sectors of our daily lives. emerging infections (eis) can be defined as infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range ( ) . re-emerging and resurging infections are those that existed in the past but are now rapidly increasing either in incidence or in geographical or human host range ( ) . the term deliberately emerging refers to both naturally occurring microbial agents such as anthrax ( ) , and to bioengineered microorganisms such as those created by the insertion of genetic virulence factors that produce or exacerbate disease. about million (more than %) of million annual deaths worldwide are estimated to be related directly to infectious diseases; this figure does not include the additional millions of deaths that occur as a consequence of past infections (for example, streptococcal rheumatic heart disease), or because of complications associated with chronic infections, such as liver failure and hepatocellular carcinoma in people infected with hepatitis b or c viruses ( ) . the burden of morbidity (ill health) and mortality associated with infectious diseases falls most heavily on people in developing countries ( ), and particularly on infants and children (about three million children die each year from malaria and diarrheal diseases alone ( )). in developed nations, infectious disease mortality disproportionately affects indigenous and disadvantaged minorities ( ) . bacteria and viruses existed long before humans evolved, and bacterial diseases probably co-evolved with each species. many bacterial diseases that we see today have been around for as long as we have, others may have developed later. many examples can be cited in addition to the black death and the influenza pandemic, such as certain biblical pharaonic plagues and the unidentified plague of athens, which heralded the end of greece's golden age ( ) . importation of smallpox into mexico caused - million deaths in - , effectively ending aztec civilization ( , ) . with the beginning of microbiology, pathogens became apparent. the establishment of the germ theory and the identification of specific microbes as the causative agents of a wide variety of infectious diseases ( ) ( ) ( ) led to enormous progress, notably in the development of vaccines and ultimately of antimicrobials ( ) . by the s, which had witnessed the widespread use of penicillin, the development of polio vaccines and the discovery of drugs for tuberculosis, complacency had set in ( ) , and in , the us surgeon general stated that the war against infectious diseases has been won ( ) . some experts remained skeptical, aware of the current lessons from history. they were less persuaded by successes than alarmed by failures, such as the lack of progress against infections in the developing world and the global spread of antimicrobial resistance. the emergence of aids led to renewed appreciation of the inevitability and consequences of the emergence of infectious diseases ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in the past years, some of the factors that resulted in aids have also led to the re-emergence of historically important diseases such as cholera, diphtheria, trench fever and plague. many re-emergences have been catalyzed by wars, loss of cohesion, and natural disasters such as earthquakes and floods, indicating the importance not only of microbial and viral factors, but also of social and environmental determinants ( ) ( ) ( ) ( ) ( ) ( ) ( ) . these infections are those that have not previously been recognized in man. many diverse factors contribute to their emergences (table ) . numerous microbial, host and environmental factors interact to create opportunities for infectious agents to evolve into new ecological niches, reach and adapt to new hosts, and spread more easily between them. at the end of , an estimated . million (range . - . million) people around the world were living with hiv, including the . million (range . - . million) people who acquired hiv in . the epidemic claimed an estimated . million (range . - . million) lives in . sub-saharan africa remains the most affected region and is home to about % of the total number of people living with hiv worldwide ( ) . before jumping to humans an estimated - years ago ( ) , perhaps through consumption of 'bush meat' from nonhuman primates, hiv- and hiv- had ample opportunity to evolve in hosts that were genetically similar to man (the chimpanzee, pan troglodytes, and the sooty mangabey, cercocebus atys). but hiv/aids may never have emerged had it not been for disruptions in the economic and social infrastructure in postcolonial sub-saharan africa. increased travel, the movement of rural populations to large cities, urban poverty and a weakening of family structure, all these promoted sexual practices such as promiscuity and prostitution that facilitate hiv transmission ( ) ( ) ( ) ( ) . infections in animals that are transmitted to humans (zoonoses), and those transmitted from one vertebrate to another by an arthropod vector (vector-borne diseases), have repeatedly been identified as ranking among the most important eis ( , ) . viruses in these groups have co-evolved with specific rodent species whose contact with humans has increased as a result of modern environmental and human behavioral factors. farming, the keeping of domestic pets, hunting and camping, deforestation and other types of habitat destruction all create new opportunities for such infectious agents to invade human hosts ( ) ( ) ( ) ( ) ( ) ( ) ( ) . examples include the arenavirus hemorrhagic fevers (argentine, bolivian, venezuelan and lassa hemorrhagic fevers) and hantavirus pulmonary syndrome (hps). variant creutzfeldt-jakob disease (vcjd) is another example of a zoonotic disease emerging in humans. it is caused by the human-adapted form of the prion associated with the emerging epizootic (large-scale animal outbreak) of bovine spongiform encephalopathy (bse), commonly known as mad cow disease. the new bse prion has become uncharacteristically promiscuous: unlike most known prions, it readily infects multiple species in addition to humans. this suggests the possibility of further emerging diseases associated with prions with currently unknown transmissibility to humans ( ) . infectious agents indirectly transmitted to or between humans by way of human-modified environments account for other emerging zoonoses. legionnaire's disease, first identified in , is caused by legionella pneumophilia, whose emergence as a human pathogen might not have occurred were it not for the environmental niche provided by air-conditioning systems ( ) . campylobacter jejuni and escherichia coli infect agricultural animals, gaining access to humans through food, milk, water or direct animal contact. other enteric pathogens, such as the vibrios which cause classic cholera and the zoonotic protozoa cryptosporidium parvum and cyclospora cayetanensis ( ), seem to have come from environmental or animal organisms that have adapted to human-to-human 'fecal-oral' transmission through water. some eis come from microorganisms that once caused familiar diseases, but which now cause new or previously uncommon diseases. streptococcus pyogenes caused a fatal pandemic of scarlet and puerperal fevers between and ( ) . scarlet fever, then the leading cause of death in children, is now rare, but has largely been supplemented by other streptococcal complications such as streptococcal toxic shock syndrome, necrotizing fasciitis and re-emergent rheumatic fever ( ) . streptococcus pyogenes has been studied more extensively, but the basis of severe disease emergence seems to be more complex. many factors associated with streptococcal virulence have been identified in strains bearing the m surface protein as well as in other m protein strains, among them bacteriophage-encoded superantigen toxins and a protein known as sic (streptococcal inhibitor of complement), which seems to be strongly selected by human host mucosal factors. several lines of evidence suggest that changes in streptococcal virulence reflect genetic changes associated with phage integration, large-scale chromosomal rearrangements and possibly the shuffling of virulence cassettes (clusters of genes responsible for pathogenicity), followed by rapid human spread and immune selection ( , ) . infectious agents that are associated with chronic diseases are one of the most challenging categories of newly emerging (or at least newly appreciated) infections. examples include the associations of hepatitis b and c with chronic liver damage and hepatocellular carcinoma, of certain genotypes of human papillomaviruses with cancer of the uterine cervix, of epstein-barr virus with burkitt's lymphoma (largely in africa) and nasopharyngeal carcinoma (in china), of human herpesvirus with kaposi sarcoma, and helicobacter pylori with gastric ulcers and gastric cancer ( ) ( ) ( ) . some data even suggest infectious etiologies for cardiovascular disease and diabetes mellitus ( ) , major causes of death and disability worldwide. other associations between infectious agents and idiopathic chronic diseases will inevitably be found. re-emergence is caused by some of the factors that allow for newly emerging infectious diseases, factors such as microbial evolutionary vigor, zoonotic encounters and environmental encroachment. re-emergences or at least cyclical resurgences of some diseases may also be climate-related-for example, the el niño/southern oscillation (enso) phenomenon is associated with resurgences of cholera and malaria ( ) . travel has an important role in bringing people into contact with infectious agents ( ) . an increase in travelassociated importations of diseases was anticipated as early as , when commercial air travel was still in its infancy ( ) . this has since been demonstrated dramatically by an international airline hub-to-hub pandemic spread of acute hemorrhagic conjunctivitis in ( ), by epidemics of meningococcal meningitis associated with the hajj, and more recently by the exportation of epidemic sars (a newly emerging disease) from guangdong province, china, to hong kong, and from there to beijing, hanoi, singapore, toronto and elsewhere ( ) . plasmodium falciparum malaria was neglected for several decades, but is now among the most important re-emerging diseases worldwide. years of effective use of dichlorodiphenyltrichloroethane (ddt) had led to the abandonment of other mosquito-control programs, but the insecticide fell into disuse because of mosquito resistance and concerns about the insecticide's potentially harmful effects on humans and wildlife. consequently, malaria has re-emerged, and the situation has been worsened by the development of drug resistance to chloroquine and mefloquine ( ) . research efforts focus on the development of vaccines and new drugs, and on re-establishing public health measures such as the use of bed nets ( ) . the remarkable re-emergence of tuberculosis was fuelled by the immune deficiencies of people with hiv infection, which greatly increases the risk of latent mycobacterium tuberculosis infections progressing to active disease, and being transmitted to others. inadequate courses of anti-tuberculosis therapy compound the problem, leading to the emergence and spread of drug resistant and multidrug-resistant strains, and a need for more extensive treatment strategies such as directly observed therapy ( ) . it has been known for over a century that tuberculosis is a disease of poverty, associated with crowding and inadequate hygiene. the continuing expansion of global populations living in poverty makes tuberculosis more difficult to control. drug resistance, another factor causing microbial and viral re-emergence, may result from mutation or from bacterial acquisition of extraneous genes through transformation or infection with plasmids. sequential emergences of staphylococcus aureus that are resistant to sulpha drugs ( s), penicillin ( s), methicillin ( s) and to vancomycin in ( )-a last line of antibiotic defense for some multiply drug-resistant bacteria-are troubling. nosocomial enterococcus faecalis became fully resistant to vancomycin by , and then apparently transferred vana resistance genes to co-infecting staphylococci ( ) . methicillin-resistant staphylococci are now being isolated from livestock that have been fed with growthpromoting antibiotics ( ) , possibly contributing to resistance problems in humans. immune deficiency associated with hiv infection, and with chemotherapy for cancer, immune-mediated diseases and transplantation, has contributed to an enormous global increase in the numbers of immunosuppressed people over the past few decades (probably more than % of the world's population), setting the stage for the re-emergence of many opportunistic infections. hiv, which has infected more than million people globally ( ) , is the largest single cause of human immune deficiency and markedly increases vulnerability to a wide range of opportunistic pathogens, including pneumocystis carinii, various fungi, tuberculosis, protozoa and herpesvirus ( ) . the simultaneous emergences of encephalitis due to west nile virus (wnv) in the united states and russia ( , ) reflect abundances of eclectic vector mosquitoes and avian hosts in these locations. both were probably connected to endemic sites by virus carriage in migratory birds and travelers. the remarkable geographical spread of wnv in the five years since its introduction into the western hemisphere reflects an unfortunate confluence of viral promiscuity and ecological diversity ( ) . although humans are dead-end hosts for wnv, the risk of infection is greatly increased by marked zoonotic viral amplification and persistence in the environment. although wnv is now a major epidemiological concern in the developed world, dengue remains the most significant and widespread flavivirus disease to have emerged globally ( ) . usually transmitted by aedes aegypti mosquitoes, dengue has recently been transmitted by aedes albopticus-a vector switch of potential significance with respect to dengue reemergence ( ) . dengue re-emergence is further complicated by disturbing increases in a serious and formerly rare form of the disease, dengue hemorrhagic fever (dengue shock syndrome being its highly fatal form). these severe complications are thought to result from the evolution of dengue viruses to escape high population immunity, seen in increased viral virulence and human immunopathogenesis due to antibody-dependent enhancement of viral infection ( ) . cholera is also of interest, not only as an important cause of mortality, but also because of the complexity of factors that determine its re-emergence. both virulent and avirulent strains of these zoonotic bacteria are maintained in the environment and are rapidly evolving in association with phyto-and zooplankton, algae and crustaceans. such environmental strains seem to act as reservoirs for human virulence genes and to undergo gene transfer events that lead to new strains containing further virulence gene combinations ( ) . thus, although cholera has appeared to be clinically and epidemiologically stable at least since the third pandemic (in the s), modern evidence suggests that such apparent stability masks aggressive bacterial evolution in complex natural environments. influenza a viruses, which are endemic gastrointestinal viruses of wild waterfowl, have evolved elaborate mechanisms to jump species into domestic fowl, farm animals and humans. periodic gene segment reassortments between human and animal viruses produce important antigenic changes, referred to as 'shifts'. these can lead to deadly pandemics, as occurred in , , and ( , ) . in intervening years, shifted viruses undergo continual but less dramatic antigenic changes called 'drifts,' which allow them partially to escape human immunity raised by previously circulating influenza viruses. influenza drift is an evolutionary success story for the virus. influenza a has a seemingly inexhaustible repertoire of mutational possibilities at several critical epitopes surrounding the viral hemagglutinin site that attaches to human cells. deliberately emerging microbes are those that have been developed by humans, usually for nefarious use. they include microorganisms or toxins produced in a form that would cause maximal harm because of ease of dissemination, enhanced infectivity or heightened pathogenicity ( ) . two modern attacks have been well documented. in , an oregon religious cult spiked restaurant salad bars with salmonellae in an attempt to sway a local election ( ) . a anthrax attack ( ) , in which a terrorist mailed anthrax-sporefilled letters to prominent figures, including two us senators, resulted in illness in at least people and the death of five of these individuals. the united states, the united kingdom, the russian federation and other nations once had sophisticated offensive bioweapons programs that included the production of weaponized anthrax spores ( ) . in japan, the doomsday sect, aum supreme truth, carried out a nerve-gas attack on the tokyo subway in , and made a trial run on an anthrax weapon, using harmless vaccine bacteria as a test ( ) . bioterror agents have been grouped into three categories (a, b and c) according to risk ( ) . the six category a agents (anthrax, smallpox, plague, tularaemia, viral hemorrhagic fevers and clostridial botulinum toxin) are given top priority because they are highly lethal and readily deployed as weapons. category b and c agents include food-borne and water-borne organisms that incapacitate but usually do not kill. risk is the probability that exposure to a hazard will lead to a negative consequence ( ) . risk communication is an interactive exchange of information and opinion on risk among risk assessors, risk managers, and other interested parties ( ) . humans tend to fear similar things, for similar reasons. scientists studying human behavior have discovered psychological patterns in the subconscious ways we "decide" what to be afraid of and how afraid we should be. any given risk has a set of identifiable characteristics that help predict what emotional responses that risk will trigger. people's perceptions of the magnitude of risk are influenced by factors other than numerical data ( ) . the factors influencing risk perception are summarized in table . merely disseminating information without regard for communicating the complexities and uncertainties of risk does not necessarily ensure effective risk communication. wellmanaged efforts will help ensure that messages are constructively formulated, transmitted, and received and that they result in meaningful actions. the seven cardinal rules of risk communication are demonstrated in table . the fundamental goal of risk communication is to provide meaningful, relevant and accurate information, in clear and understandable terms, targeted to a specific audience ( ) . the goals of risk communication are summarized in table . it may not resolve all differences between interested parties, but may working in earthquake-prone areas hormone replacement therapy . risks perceived to be fairly distributed are more accepted than risks to be unfairly distributed. gun shots (in japan) traffic accidents . risks perceived to be natural are more accepted than risks perceived to be manmade. radiation from mobile phones radiation from the sun . risks perceived to be statistical are more accepted than risks perceived to be catastrophic. eaten by a shark (catastrophe) heart disease (statistics) . risks perceived to be generated by a well-known source are more accepted than risks perceived to be generated by a less known source. private industry government . risks perceived to be familiar are more accepted than risks perceived to be exotic. . risks perceived to affect adults are more accepted than risks perceived to affect children. asbestos exposure for children asbestos exposure in workplace . risks perceived with less uncertainty are more accepted than risks with high uncertainty. new technology conventional technology . risks perceived that could directly affect others are more accepted than risks that could affect oneself. table seven cardinal rules of risk communication* . accept and involve the public as a partner. the goal is to produce an informed public, not to defuse public concerns or replace actions. different goals, audiences, and media require different actions. . listen to the public's specific concerns. people often care more about trust, credibility, competence, fairness, and empathy than about statistics and details. . be honest, frank, and open. trust and credibility are difficult to obtain; once lost, they are almost impossible to regain. . work with other credible sources. conflicts and disagreements among organizations make communication with the public much more difficult. . meet the needs of the media. the media are usually more interested in politics than risk, simplicity than complexity, danger than safety. . speak clearly and with compassion. never let efforts prevent acknowledging the tragedy of an illness, injury, or death. people can understand risk information, but they may still not agree; some people will not be satisfied. * from: covello v and allen f. . (reference no. ) lead to a better understanding of those differences. it may also lead to more widely understood and accepted risk management decisions. effective risk communication should have goals that build and maintain trust and confidence. it should facilitate a higher degree of consensus and support by all interested parties for the risk management options being proposed. many considerations for effective risk communication, especially those involving the public, can be grouped in a sequence following the systematic approach of the risk communication process. this starts with gathering background and needed information, followed by the preparation and assembly of the message and its dissemination and distribution, with a follow-up review and evaluation of its impact ( ) . the general considerations for effective risk communication are demonstrated in table . risk communication efforts and programs need to be evaluated both regularly and systematically to determine their effectiveness and to provide for change when needed. communication aims and objectives need to be clearly stated if an evaluation is to be effective. this could include the proportion of at-risk population to be reached, adoption of appropriate risk reduction practices, and the extent of resolution of the crisis. it is important to learn from both positive and negative risk communication experiences, in order to adjust and improve ongoing communication activities. only through systematic evaluations, which are performed throughout the communication process, can that process be strengthened ( ) . globalization of disease prevention lectures, through the supercourse prevention project, is funded by the us national institutes of health (nih). the supercourse is an internet library of lectures on prevention, shared for free by , table goals of risk communication* . promote awareness and understanding of these specific issues under consideration during the risk analysis process, by all participants; . promote consistency and transparency in arriving at and implementing risk management decisions; . provide a sound basis for understanding the risk management decisions proposed or implemented; . improve the overall effectiveness and efficiency of the risk analysis process; . contribute to the development and delivery of effective information and education programs, when they are selected as risk management options; . foster public trust and confidence in the safety of the food supply; . strengthen the working relationships and mutual respect among all participants; . promote the appropriate involvement of all interested parties in the risk communication process; and, . exchange information on the knowledge, attitudes, values, practices and perceptions of interested parties concerning risks associated with food and related topics. table . japan has associated with the global health network through supercourse japan ( ) where a series of lectures in health, environment and sustainable development, epidemiology for decision-making, and zoonosis have so far been developed. the supercourse on health, environment and sustainable development was designed to provide an overview on health and environment in the context of sustainable development for public health students around the world, as well as decision makers, community leaders, scientists and professionals in government and non-governmental organizations, who are interested in health and environmental linkages in sustainable development ( ) . the supercourse on epidemiology for decision-making provides a learning resource for students of environmental and occupational epidemiology, and its main purpose is to promote the understanding and application of epidemiology in the prevention of environmental and occupational disease and the promotion of health ( ) . the supercourse on zoonosis aims to disseminate rapidly the latest information on animal diseases transmittable to humans. severe acute respiratory syndrome (sars) and bovine spongiform encephalopathy (bse) are some of the lectures discussed which will help in information sharing to developing countries ( ) . a joint action plan through the 'the pacific islands forum' was created in . its five priority policy targets are: ) enhancement of security in the pacific region, ) creation of a safer and more sustainable environment, ) improvement in education and human resources development, ) improvement in health, and ) promotion of more vigorous and continued trade and economic growth. the countries in the pacific region face a contradictory dilemma: some of the poorest countries have the most expensive telecommunications, yet information and telecommunication technology (ict) need to be extensively utilized. in these circumstances telemedicine, such as for remote clinical and pathological diagnosis, is too expensive and technically demanding to be widely used. since information in the future will be technology-and network-based, it is imperative for "supercourse asia" to fully exploit the potential of ict for health education and health service development in the region. the advantages of this approach are to use the most appropriate, inexpensive, opensource, and low-band ict, and to operate it through active national networking. the supercourse asia network (scan) is an offshoot of this initiative and aims that all children and adults will have equal access to health and education of sufficient quality to empower them to break the poverty cycle, to improve their quality of life (qol), and to participate effectively in national development. the mission is to alleviate poverty and improve health and qol of developing countries in the pacific region through advances in a cost-effective ict-based health educational system. the internet is the most inexpensive and speed efficient means to penetrate the remote places such as the pacific islands. japan, a leader of modern technology in the region, is in the best position to provide these technological and educational tools to contribute to the well-being of people in the region. the scan is a group of primarily academics, united by a common belief: the internet is the best way to disseminate knowledge, especially knowledge about health promotion and prevention. they are working towards facilitating the dissemination of health-related information over the internet and improving teaching in the field of education, social/preventive medicine, public health and epidemiology. the members are voluntary professionals in academia, healthcare, telecommunications, the government, ngos, and other public and private sector organizations who will work together towards developing "supercourse asia." they are not only active members of the scan but also the main contributors of "supercourse asia" lectures, reviewers, translators, and its major user group. the challenges for the scan are ) how to effectively utilize the national networks among the participating countries and within each participating country that may be geographically remote, ) how to evaluate the effects of the scan on the advancement of health and qol, and the alleviation of poverty though "supercourse asia" in the region, and ) how to sustain the network activities for many years to come. currently, the public has become concerned with information and safety, however, gaps between the understanding of safety and assurance are widening. health advocacy and promotion through e-learning are becoming more important in the framework of risk communication for the community. the supercourse lectures will serve as an international platform for sharing information, lectures, and ideas. understand the public perception of the risk through such means as risk surveys, interviews and focus groups expect different people to see the risk differently. preparation/assembly . avoid comparisons between familiar risks and new risks, as they may seem flippant and insincere unless presented properly. . recognize and respond to the emotional aspects of risk perceptions. speak with sympathy and never use logic alone to convince an audience characterized by emotion express risk in several different ways, making sure not to evade the risk question explain the uncertainty factors which are used in risk assessment and standard setting maintain an openness, flexibility, and recognition of public responsibilities in all communication activities accept and involve the public as a legitimate partner by describing risk/benefit information and control measures in an understandable way. . share the public's concern rather than deny it as not legitimate or as unimportant. be prepared to give people's concerns as much emphasis as the risk statistics be honest, frank, and open in discussing all issues if explaining statistics derived from risk assessment, explain the risk assessment process before presenting the numbers coordinate and collaborate with other credible sources evaluate the effectiveness of risk messages and communication channels. . emphasize action to monitor, manage, and reduce risk plan carefully and evaluate efforts it is based on the open source model, which allows free redistribution, shows the source code, and allows modifications and derived works this is especially useful for developing countries and minority populations with limited access to resources it aims to overcome the digital divide by improving access in places with low bandwidth internet connection it is a teaching support system -differing from a traditional distance education system it provides timely information for action -one of the greatest advantages of having a regional faculty base with varied areas of expertise which are very important in the field of public health (e.g., lectures on sars from the members in china and singapore, where it is most rampant) it is a "hyper-text comic book format hypertext links from text, images, tables, and pictures take the reader to other relevant supercourse lectures, images, or other websites on the internet oaks sc editors. emerging infections; microbial threats in the united states world health organization. removing obstacles to healthy development global defence against the infectious disease threat. world health organization globalisation of prevention education: a golden lecture factors in the emergence of infectious diseases threats to global health and survival; the growing crises of tropical infectious diseasesan unfinished agenda emerging infectious diseases among indigenous peoples epidemiology of the plague of athens the columbian exchange: biological and cultural consequences of . greenwood, westport: connecticut princes and peasants. smallpox in history. chicago: univ untersuchungen über bacterien. v. die aetiologie der milzbrand-krankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis spreading germs: diseases, theories, and medical practice in britain the greatest benefit to mankind: a medical history of humanity from antiquity to the present the evolution and eradication of infectious diseases infectious diseases: considerations for the st century oaks sc editors. emerg infect. microbial threats to health in the united states lederberg j editors. microbial threats to health in the united states: emergence, detection and response emerging and re-emerging infectious diseases: a multidisciplinary perspective emerging and re-emerging infectious diseases emerging infectious diseases in the st century emerging and re-emerging infectious diseases the origins of acquired immune deficiency viruses: where and when population migration and the spread of types and human immunodeficiency viruses the aids knowledge base slowing heterosexual hiv transmission variant creutzfeldt-jakob disease and the acquired and transmissible spongiform encephalopathies severe streptococcal infections in historical perspective emerging infections group a streptococcus: allelic variation, population genetics, and hostpathogen interactions genome sequence of a serotype m strain of group a streptococcus: phage-encoded toxins, the highvirulence phenotype, and clone emergence identification if herpesvirus-like dna sequences in aids-associated kaposi's sarcoma microbes and malignancy: infection as a cause of human cancers helicobacter pylori-associated diseases current clinical topics in infectious diseases el niño and health island epidemics epidemiology in relation to air travel acute hemorrhagic conjunctivitis: dealing with a newly emerging disease the severe acute respiratory syndrome two worlds of malaria research toward vaccines against malaria the global situation of mdr-tb methicillin (oxacillin)-resistant staphylococcus aureus strains isolated from major food animals and their potential transmission to humans centers for disease control prevention. staphylococcus aureus resistant to vancomycin-united states joint united nations programme on hiv/aids. aids epidemic update surveillance for aids-defining opportunistic illnesses, - . mmwr . cdc surveillance summary no. ss- the outbreak of west nile virus infection in the new york city area in outbreak of west nile virus infection west nile virus: epidemiology and ecology in north america dengue and dengue hemorrhagic fever antibody-dependent enhancement of infection and the pathogenesis of viral disease molecular ecology of toxigenic vibrio cholerae the next influenza pandemic: lessons from hong kong a molecular whodunit the chilling true story of the largest covert biological weapons program in the world-told from the inside by the man who ran it a large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars investigation of bioterrorism-related anthrax, united states, : epidemiologic findings japanese sect was close to bioterrorism journal says: live anthrax found at office threats in bioterrorism: i. cdc category a agents risk: a practical guide for deciding what's really safe and what's really dangerous in the world around you world health organization. the application of risk communication to food standards and safety matters, a joint fao/ who expert consultation supercourse: epidemiology, the internet, and global health seven cardinal rules of risk communication key: cord- -x oywbrj authors: wilson, brenda a. title: global biosecurity in a complex, dynamic world date: - - journal: complexity doi: . /cplx. sha: doc_id: cord_uid: x oywbrj biosecurity is emerging as a major global health priority for which innovative and unprecedented solutions are needed. biosecurity is a challenging biocomplexity problem involving multifaceted processes such as interactions between humans and nonhuman biota, anthropogenic environmental and ecological factors, and socioeconomic and political pressures. key to an effective biosecurity strategy will be fundamental understanding of evolutionary, anthropogenic and environmental driving forces at play in transmission and perpetuation of infectious diseases. biosecurity solutions will depend on increased support of basic biomedical research and public education, enhanced healthcare preparedness, alternative strategies for ensuringsafety, and improved interagency cooperation regarding global health policy. © wiley periodicals, inc. complexity, . i t is widely accepted by historians that there are certain dates upon which history seems to pivot, turning points that forever change the course of future events. the momentous months of september and october mark such a critical period in our recent history. our world as we knew it shifted, not just for the united states, but all nations. as a consequence, defense against bioterror agents came to the forefront as a major health priority in the u.s. and elsewhere. indeed, we were still reeling from the impact when sars swept across the globe in - , followed closely behind by the still ongoing worldwide spread of avian flu and the concomitant fear of it transforming into a human flu pandemic on the scale of that experienced in . but, these events are only at the pinnacle of a mounting number of impinging natural and imposed biohazards (table ) [ ] [ ] [ ] [ ] [ ] . importantly, these manmade and natural events have revealed a number of glaring gaps in our knowledge about infectious diseases, their transmission and perpetuation, and how to effectively combat them. existing and looming biological threats have now made biosecurity, which includes biodefense, the most pressing global health priority. in a rapidly changing world, biosecurity is at the intersection of every sphere of medical, biological, ecological, socioeconomic, and political system. although one might argue that the principal difference in the infectious disease threat today versus say , , or years ago is bioterrorism, the resources spend on preparing for a bioterror attack is viewed by most scientists as grossly exorbitant [ ] , particularly considering the small numbers of individuals who have been or could be affected by this type of attack and considering the relatively low medical relevance or prevalence of the diseases caused by the limited number of highpriority bioterror bioagents, the socalled ''category a select agents.'' and, while admittedly the preparedness and surveillance measures put in place for one has certainly helped to protect against the other (the improved global response to and curtailment of sars coming after the anthrax bioterrorist attacks is a prime example of this), most scientists feel that the limited resources available from an already overburdened system should instead be used for studying and preparing against the looming and potentially more devastating infectious disease threats from natural or accidental exposure [ ] , which could affect millions of people and animals and could have huge health and economic consequences. and thus, while the threat of bioterrorism must be considered, many scientists propound that the focus should be on the more urgent and dire problem of biosecurity, rather than just bioterrorism. it has been argued by many that there is no better creator of new highly potent biological threats than nature itself. however, there is also little doubt that anthropogenic environmental, socioeconomic, and ecological influences can have devastating impact on the extent and severity of the outcome of these natural biological hazards. risks to public health come from diverse scenarios ranging from epidemics to outbreaks during natural disasters to accidental exposures through poor food processing to deliberate releases or fear thereof ( table ) . finding solutions to these challenging biocomplexity problems will require integrated, multilevel, flexible, and interdisciplinary approaches that stretch traditional concepts. key to an effective global biosecurity strategy will be improved detection, prevention, treatment, and management of infectious diseases, but also better understanding of the intrinsic and extrinsic factors that contribute to their virulence and influence their transmission, prevalence, and perpetuation. thus to achieve this, we first need a better understanding of the critical evolutionary, anthropogenic, and environmental driving forces that contribute to natural and man-made biological threats. to gain a sense of the potential impact of biological threats on biosecurity, it is best to begin by considering the source and nature of the biological agents that pose biosecurity risks (table ) . manmade biological threats come in two flavors, deliberate and accidental. the concept of intentionally using biological agents as weapons is nothing new to warfare, and we are all aware of the american, russian, and other state-sponsored programs to develop biological agents as ''weapons of cases/ deaths total in from anthrax (perpetrator still at large) > , hospitalized/ deaths total in - from sarin gas (aum shinrikyo) cases/ deaths total in from salmonella (rajneeshee) death in from ricin (assassination of georgi markov) natural casualities: - m cases/ million deaths per year from malaria world-wide m cases/ , hospitalizations/ , deaths per year from foodborne illnesses in usa - m cases/ million deaths per year from tuberculosis world-wide > m cases/> m deaths total from hiv/aids world-wide, > . m deaths in usa m cases/ , deaths total from hepatitis c in usa mass destruction'' (wmd). these state-sponsored wmd programs have been largely dismantled [ ] , and instead the concept of using biological agents as bioweapons has now been usurped by individuals or small groups acting as terrorists engaged in a different form of warfare, where these agents are perhaps more accurately described as ''weapons of mass disruption'' (still wmd). we experienced a vivid and horrible display of this new brand of wmd with the anthrax attacks of , in which the u.s. postal system was utilized to dispense deadly disease, but even more notably, fear and turmoil. the cost of mounting a response to this new wmd has been enormous, not only in terms of billions of u.s. taxpayers' dollars, particularly directed toward biodefense (table ), but also in countless man-hours expended in ramping up other areas of security and healthcare preparedness and in the astounding disruption of lifestyle (e.g. inconveniences caused by intensified travel-related security measures). a growing number of scientists feel that the bioterror threat is exaggerated [ ] and that it is highly unlikely that any terrorist organization, foreign or domestic, could on their own develop from scratch a bioweapon capable of causing mass casualties. instead, it is more likely that the potential terrorists would steal or procure existing material and deploy it on a much smaller scale. in contrast, manmade threats resulting from inadvertent release, accidental contamination, or even from non-malicious intentional introduction of biological agents represent much more measurable concerns with known likelihood of risk. there have been a number of recent high-profile incidences that illustrate the havoc, alarm, and economic consequences that can result from widespread distribution of contaminated food because of accidental introduction of harmful microbes during food processing. what previously was seen only sporadically, such as at church socials, family gatherings or community picnics, moved abruptly into the public's eye with the largescale problem of undercooked fastfood hamburger meat contaminated with e. coli o :h , a toxin-producing bacterium that causes dysentery-like diarrhea and can cause kidney failure and death, especially in children and the elderly. this dangerous microbe has since been associated with over multistate or multination outbreaks of contaminated food, including meat, radish sprouts, apple juice, let-tuce, and most recently spinach. late last summer, e. coli o :h contamination of prepackaged fresh spinach led to cases of illness across states, with hospitalizations, kidney failures, and deaths [ ]. the outbreak, which was traced back to spinach obtained from a few fields in california [ ] , shook consumer confidence and cost the industry an estimated $ m in economic losses [ ] . there are many other examples of how the spread of natural threats can be greatly facilitated by our modern technologies, practices, and behaviors. viruses such as the marburg and ebola viruses, first discovered in the s and s, are examples of biological agents responsible for recently emerged diseases [ ] . ebola and marburg viruses are considered to be zoonotic diseases that are transmissible by close contact with animal species, but their spread has been facilitated by conditions in the country of outbreak, including political upheavals, reuse of needles, and cultural burial practices. zoonotic diseases are often perceived as only a problem of developing countries, where there is much closer contact with animals, both domestic and wild. however, living with animals is not limited to the third world. consider how many americans alone live with pets, sleeping with them, and even kissing them. it is interesting to consider that measles virus is closely related to canine distemper virus [ , ] , suggesting that at some point a dog-human transmission occurred or a common ancestral virus may have infected both. of particular and growing concern are high impact, foreign animal diseases, such as mad cow and foot-and-mouth diseases (table ). bovine spongiform encephalopathy (bse), commonly known as mad cow disease, is a fatal, progressive neurodegenerative disease of cattle caused by an infectious form of misfolded protein called a prion [ ] . transmission of bse occurs when healthy animals come in close contact, usually through ingestion, with prion-containing tissues from animals that have the disease. the first probable occurrence in cattle was in the early s, possibly as a result of feeding cattle meat and bone meal that contained scrapie-infected sheep products. scrapie is a prion disease of sheep and goats. industrial cattle-farming practices in europe prior to used rendered meat and bone meal, instead of the more common soybean meal used elsewhere, as a protein supplement in cattle feed. in the early s, a change in the rendering process in the u.k., in which a lower sterilization temperature was used for the steam boiling step in the process, is thought to be the major contributing factor to an increase in prions in the cattle feed that resulted in the bse epizoonotic outbreak. the uk epidemic peaked in with nearly , new cases per week, and by the end of there were over , cases of bse confirmed in the u.k. [ , ] . bse attracted particular attention because it now appears that it can also be transmitted to humans that consume tainted meat. since the first reported case in , at least people, in the u.k. and elsewhere, have died of a disease with similar neurological symptoms to bse, called variant creutzfeldt-jakob disease (vcjd) [ ] . for many of the vcjd cases, there is direct evidence that they had consumed tainted beef years before. the connection between bse and vcjd has a wider impact than just food safety--blood, tissues and organ donation programs are also affected and anyone having exposure to bse is a potential carrier [ ] . because of the long incubation period for prion diseases (years to decades), the full extent of the human vcjd outbreak is still not fully known, although the number of new cases appears to be declining. the long incubation period also makes testing for the disease in animals difficult because most livestock are slaughtered long before noticeable symptoms occur or even before plaques in the brain can be readily detected during inspection by necropsy. although many foreign animal diseases are not of serious concern to human health (i.e. humans may be affected only very rarely through direct contact with infected animals), they do pose considerable threat to our agricultural and food industries and could cost millions or even billions of dollars in economic and trade losses (hence their status as ''high impact'' diseases). foot-and-mouth disease (fmd) is a highly contagious, sometimes fatal viral disease primarily of cattle and pigs, but it has a wide host range. fmd occurs worldwide, but a number of areas, including north america, australia, new zealand, japan, most of europe, and parts of south america have been fmd-free for some time, mainly due to eradication through rigorous vaccination and culling programs. however, in , a major outbreak of fmd in the u.k. resulted in the slaughter of millions of animals, huge economic and trade losses estimated in the range of £ m, the temporary cancellation of sporting events and other outdoor events attended by farmers or those living in the country, and the implementation of strict policies on the sale and trade of livestock, as well as disinfection of all persons entering or leaving farming areas [ , ] . countries are recognized to be in one of three fmd categories: fmd present with or without vaccination, fmd free with vaccination, and fmd free without vaccination [ , ] . understandably, countries designated as fmd free without vaccination have the greatest export markets, so most developed countries have greatly enhanced their agricultural surveillance and trade policies to maintain their fmd-free status. innocuous introduction of foreign plants, animals, or insects can also have considerable ecological and economic impact on horticultural and agricultural industries. kudzu, a member of the pea family that is native to southeast asia, is an example of an invasive alien plant species that has caused considerable damage since its introduction to the southeastern regions of the u.s., where it is sometimes referred to as ''the plant that ate the south'' [ ] . kudzu was first intro-duced from japan into the u.s. in at the philadelphia centennial exposition, after which it gained some popularity as an ornamental shade vine. but, from to , the soil conservation service promoted its use as a means for controlling soil erosion. once established with a root system that can reach depths of up to feet, kudzu vines grow as much as a foot per day during a season with lengths up to feet. kudzu now covers over m hectares and poses a considerable threat to the otherwise high biodiversity of flora found in the south [ ] . a tremendous amount of money and effort is spent each growing season to prevent the highly prolific kudzu from overtaking roads, bridges, powerlines, local vegetation, and even homes, barns and other buildings. it costs an estimated $ m annually in lost cropland and management resources [ ] . for successful long-term control, the entire root system must be destroyed or the plant will grow back, and considerable effort has been made to find pesticides that can control this plant pest. key to an effective strategy to combat complex biological problems to ensure biosecurity will be a greater understanding of the driving forces that are important for transmission and perpetration of infectious disease and then the management and implementation of effective preventive or containment measures. the potential power of natural selection is obvious. time and again we have seen how rapidly microbes can evolve in response to selective pressure, such that certain behaviors or genetic traits tend to be eliminated from the gene pool, while others are maintained or changed. as a consequence, new biological threats are bound to emerge as we impose our influence on the environment. the goal of most pathogenesis research, of course, is to use our understanding of the ecology of host-microbe interactions and their role in pathogenesis to develop predictive models for disease progression and transmission. unfortunately, our current understanding is rudimentary at best, and we are just now beginning to tease out the intricacies of the co-evolution of pathogens with their hosts and environment and the role that these host-microbe interactions play in emergence of disease. human-induced evolution can be extraordinarily rapid and pervasive. the natural history of myxoma virus in american rabbits and its introduction into european rabbits as a means for controlling the rabbit populations in australia provides an interesting example of the co-evolution of a virus and its animal host [ ] . it also provides a glimpse into understanding the emergence of infectious disease. shortly after european rabbits were first brought to the americas in , they were found to succumb to a deadly and extremely infectious disease, which nearly half a century later was found to be caused by a vector-borne myxoma virus acquired through contact with the native, more resistant host, the common wild rabbit of south america. european rabbits were first introduced into australia in for hunting, but by the s they had become a major pest. to help in controlling the rabbit population, rabbits infected with this deadly virus were introduced in and the favorable weather conditions for mosquitoes that year helped to rapidly spread the disease, killing millions of rabbits. but, evolution and the power of natural selection then came into play. the myxoma virus that was first introduced killed over . % of infected rabbits, yet a few rabbits survived the exposure. within a year, new strains of the virus appeared that killed only % of infected rabbits, and in subsequent years even more attenuated viral strains appeared. under such strong selective pressure, the rabbits, too, evolved to gain increased resistance such that the original, highly lethal virus would no longer kill more than % of the rabbit offspring. clearly, genetic changes in both the viral and rabbit populations quickly altered the outcome of the disease in terms of severity and persistence. today, only % of infected rabbits succumb during a myxomatosis epidemic. one of the most devastating recently emerged diseases, whose initial and continuing spread can be attributed to human behavior, is that of hiv/aids. hiv is thought to have originated in nonhuman primates [ ] , but has become established in humans and is now transmitted human-tohuman through unprotected sexual practices, reuse of needles, and at first (although no longer) through contaminated blood supplies [ , ] . aids was first noticed in the early s as unusual occurrences of a rare cancer, kaposi's sarcoma, in young homosexual men. the social stigma associated with the disease gradually shifted with the realization that other populations were also at risk, including heterosexual and bisexual women, drug addicts, hemophiliacs, blood transfusion recipients, and babies born to hiv-positive mothers. the annual death toll rose linearly from with over , deaths to a height of nearly , in , before a noticeable decline was observed with the introduction in of a cocktail of three anti-hiv drugs. by the annual death toll was down to , and has since declined to around the , mark [ ] . however, although the death rate has declined, the number of cases reported annually in the u.s. still hovers around , [ , ] . host-microbe co-evolution over time appears to be in effect for hiv/ aids. there are now a considerable number of hiv-positive individuals who have survived for many years without acquiring aids. a large part of this is due to advances in anti-hiv medications and improved healthcare. however, even before the increased availability of hiv medications, there were a significant number of individuals engaging in high-risk behavior that appeared to be resistant to acquiring hiv. by examining these ''survivors,'' scientists found a genetic mutation (allele) in a surface receptor, called ccr- , which prevents the hiv virus from entering host cells [ ] . these individuals having the mutant receptor allele are mostly of european decent. in some parts of europe, up to % of the population carry at least one copy of the mutant receptor allele, while populations in the rest of the world do not carry the same allele [ ] . it is believed that this allele might have arisen through selective pressure from previous exposure of the population to another plague (perhaps bubonic plague, although this is not certain). but, importantly, the strong selective pressure that the new antiviral medications have placed on hiv has led to an accelerated deadly arms race. current antivirals, at an annual cost of over $ , per person, are targeted mainly against the viral outer coat proteins gp and gp , the processing protease, and the reverse transcriptase. although these viral protein targets are less variable than others in the viral genome, hiv has a high mutation rate and the strong selective pressure has caused the virus to rapidly evolve in response [ ] . the ever-evolving hiv makes developing new drugs a constant, and costly, challenge [ ] . other pathogens have been around for quite some time, but have recently acquired new properties making them increasingly more deadly. during the s, staphylococcus aureus emerged as one of the most common causes of hospital-acquired infections in the us. drug-resistant infections increase the risk of death, as well as the cost and duration of hospital stays. over a relatively short period of time s. aureus acquired genes that increased the bacterium's resistance to antibiotics [ ] . a timeline of the emergence of these new strains of s. aureus clearly demonstrates the evolution of a pathogen that is under strong selective pressure to survive (table ). once acquired, antibiotic genes can be spread from one microorganism to another through a process known as horizontal gene transfer, which involves uptake or transfer of dna encoding those resistance genes within or between different bacterial species. importantly, the antimicrobial resistance is maintained even after the selective pressure is removed. this exchange of genetic information is believed to contribute to the alarming rise in multidrug resistant bacteria [ ] . rapid development of antimicrobial resistance is forcing clinical and pharmaceutical researchers to devise alternative, innovative approaches to respond to this threat [ ] . hospitals are thought to be a major source of multidrug resistant bacteria, but agricultural practices involving usage of antibiotics as prophylactics and growth promoters in feed and crops have also played a role in its emergence [ ] . eye-opening evidence for just how prevalent genetic exchange occurs in natural environments is provided by the example of the substantial increase in antibiotic resistance among both community and clinical isolates of bacteria in the gut [ ] . tuberculosis (tb) is the leading cause of death in the world, and after a century of decline in the u.s., tb is once again on the rise, and alarmingly multiple drug-resistant strains have emerged. this increase in cases worldwide is attributable to a number of complex factors, including changes in the social structure and socioeconomic upheaval, the hiv epidemic, and a failure in some countries to improve public treatment programs. multidrug resistance in tb is a growing international health concern, because it has dramatically increased the difficulty in controlling the spread of tb and because of the high mortality rate associated with co-infection with hiv [ ] . co-infection of multidrug-resistant tb with hiv fuels the transmission of tb by accelerating the progression of latent tb into active disease because of the damage that hiv causes to the host immune system, which normally controls tb infection. individuals that test positive for both tb and hiv often die with - months. a major epidemic of tb-hiv infections has spread across the former soviet union due to socio-economic changes in the country that have led to overcrowded housing, and in particular overcrowded prisons [ ] . unfortunately, the pharmaceutical industry has largely abandoned tb drug development due to perceived nonprofitable consumer market--at risk populations are also the poorest [ ] . the spread of any disease that is transmitted from human to human is greatly facilitated under crowded conditions, which allow for efficient exposure to a higher initial inoculum of the infectious agent. sporting events, concerts, and other gatherings of large numbers of people in a confined area can promote human-to-human transmission, as well as exposure to new populations. legionnaire's disease, caused by the bacterium legionella pneumophila, was first recognized in when it struck a group of american legion conference attendees in philadelphia [ ] . this bacterial pathogen normally lives in fresh water as a parasite of amoeba, but unfortunately for us it can also live and thrive inside one type of our immune cells called a macrophage. the disease is acquired through aerosol exposure from contaminated water in ventilation systems, such as the air conditioning units in a hotel, or through aspiration during nasogastric tube feedings diluted with contaminated potable water in hospital or nursing-home settings [ ] . since the first widely publicized incident on the holland-america cruise line in , there have been numerous reports of cruise ship passengers succumbing to acute gastroenteritis caused by the norwalk virus [ ] . a cruise, where hundreds of passengers and crew mingle in close contact, can provide optimum conditions for a virus to spread through food, water, and direct contact. cruise ships are now required to report all gastrointestinal illnesses to the cdc before entering a u.s. port, especially if % or more of the passengers or crew are ill. what has been most economically troubling for the cruise line industry is the recalcitrant nature of the virus to decontamination efforts [ ] . overcrowding often leads to poor sanitation, resulting in accumulation of refuse, sewage, and vermin that thrive under such unsanitary conditions. natural disasters, civil disturbances, and war have caused large population displacements, with millions of people (and their animals) worldwide today living in refugee camps, which are overcrowded with poor sanitation and often without adequate food or clean water. these crowded camps provide ideal conditions for brewing and transmitting new infectious agents in malnourished or immunocompromised populations of humans and animals. the norwalk virus reared its ugly head once again during the katrina crisis in [ ] . of the estimated , evacuees sheltered temporarily at facilities in reliant park, a sports and convention complex in houston, texas, , persons reported symptoms of acute gastroenteritis during a -week period at the beginning of september. medical personnel, police, and volunteers having direct contact with patients also reported symptoms, suggesting secondary transmission. although the local public health officials and the cdc implemented extensive infection-control measures, including publicizing the need for enhanced hygiene techniques, the outbreak continued for an additional week before declining. habitat destruction (e.g. deforestation, slash-burn practices) and urban expansion can uncover natural reservoirs and expose humans and domestic animals to new disease-causing microbes. each year - zoonotic cases of the pneumonic disease tularemia, caused by the bacterium francisella tularenesis, are reported in the us, primarily in arkansas, missouri, and oklahoma [ ] . transmission usually occurs through arthropod bites, especially ticks or deerflies, but it can also occur through inhalation of contaiminated aerosols. in the late s, rabbits from arkansas and missouri were introduced to cape cod and martha's vineyard, and cases of tularemia in massachusetts were reported shortly thereafter. martha's vineyard experienced two larger outbreaks in and , which were linked to outdoor activities of mowing lawns and cutting brush [ ] . the humans were presumably infected by inhalation of microbe-contaminated animal remains mechanically aerosolized by the cutting action of the mowers or brush cutters. pollution and exposure to waste water or sewage can also lead to the emergence of new diseases. coral black-band disease is a globally distributed disease that has been causing the degradation of coral reef ecosystems. first reported in the s, the disease is observed as a pathogenic microbial consortium (mat) that migrates from the top to bottom of healthy coral, leaving behind dead exposed skeleton that disrupts the ecological and geological structures of coral reefs (see figure ) [ ] . a factor that appears to be contributing to the development and spread of coral black band disease is the pollution of seawater from industrial, municipal, and other terrestrial waste sites near the coral reefs [ ] . modern technologies have led to greater efficiency in production, marketing, and commerce of goods around the world. rapid transport of imported material and tourism related travel facilitate the spread of infectious diseases around the globe and are clearly contributing to the increased prevalence and severity of the diseases. exotic souvenirs, including wild animals and their associated microbes, have been imported illegally into the u.s. from various parts of the world. an outbreak of monkeypox in among residents of wisconsin, northern illinois, and northwestern indiana was the result of infection from prairie dogs bought at a pet shop in texas that became infected after contact with various exotic african rodents shipped from ghana and then distributed by other pet shop outlets in the midwest [ ] [ ] [ ] [ ] . rare zoonotic cases of monkeypox in humans had been reported previously only in remote villages of central and western africa near tropical rainforests where there is close contact with infected animals [ , ] . recent studies suggest that exposure to monkeypox in these areas has increased due to encroachment of humans into animal habitats. the cdc and fda subsequently embargoed all african rodents into the u.s. and banned the distribution or sale of african rodents and prairie dogs in the u.s. [ ] . trade routes and human practices have contributed to the spread of numerous diseases throughout history, but the speed with which they are spreading today have demanded the need for ever more rapid response and containment measures to be in place. an interesting example is that of cholera, caused by the cholera toxin-producing bacterium vibrio cholerae. in asia, cholera has been endemic for hundreds, maybe thousands of years, and cholera-like disease has been described in a number of ancient texts. the first well-documented epidemic in europe occurred in . since , seven major cholera pandemics have occurred [ ] . the first six were caused by the classical o biotype, whereas the seventh, which began in and persists today, is caused by the el tor o biotype. in , el tor reemerged in peru after a hiatus of over years, and rapidly spread throughout central and south america over the following couple of years, with more than . million cases and over , deaths [ ] . the spread of el tor in these countries could be traced along the major north-south coastal trucking route and is attributed to poor sanitation in these areas. the most recent cholera outbreaks have occurred in developing countries, such as angola, where civil strife has hindered water treatment and sanitation efforts [ , ] . the extent of the global cholera burden has been grossly underreported [ ] , in part due to limited resources, but also due to the detrimental effects such news can have on trade and travel to those regions. in some endemic areas, such as bangladesh, improved management strategies by the government and who, including aggressive rehydration therapy and antibiotics, have shortened the duration of illness and have reduced the fatality rates from natural cholera epidemics, which are largely seasonal in nature. in , a new strain of vibrio cholerae, designated o or ''bengal,'' caused a massive cholera epidemic in south asia [ ] . what was most disturbing about this new strain was its high prevalence in adults, suggesting that prior immunity gained during childhood through exposure to the classical or el tor o strains offered little or no protection against this new o strain. its subsequent spread to other asian countries lead some to worry that it may cause an eighth cholera pandemic, but luckily so far this has not materialized due to timely mobilization of effective control measures by researchers and healthcare officials. however, an emerging concern is the increased incidence of antibiotic resistant strains of vibrio cholerae in bangladesh. nearly all isolates are now resistant to the less expensive antibiotics, tetracycline, trimethoprinsulfamethoxazole, and erythromycin. although most are still sensitive to ciprofloxacin, the effective doses needed for treatment are increasing. seasonal changes in rainfall and sunlight can trigger periodic or transient emergence of some human pathogens such as cholera. an intriguing observation comes from the study of the annual epidemic profile of endemic cholera in the bengal region of bangladesh and india, where nearly all cases occur in a synchronized, explosive outbreak during major transitions of climate in the post-monsoon months of october and november [ ] . as the rains decline and sunlight increases there is a burst of algal and zooplankton bloom. it has been proposed that the increased concentrations of these particles (surfaces to which the bacteria adhere) in drinking water sources consequently increase the rates of ingestion [ , ] . during other times in the year, cholera cases occur only sporadically because the zooplankton sediment and there is less ingestion of bacteria-coated particles. recently, an additional factor has been credited toward the seasonal cholera epidemics, namely predation of the v. cholerae bacteria by bacteriophage (viruses that infect bacteria) due to amplification of the phage in the intestines of humans, followed by release into the environment [ ] . support for this model comes from the inverse correlation of the phage count with the abundance of toxigenic v. cholerae in water samples and with the incidence rates of cholera [ , ] . climate change can also dramatically alter the spread of arthropodborne diseases, which are most prevalent in a limited range of temperatures or environments preferred by these vectors. shifts in warming or cooling trends may extend or narrow the range of such vectors and the diseases they transmit. drought or flooding can also lead to spread of disease into new populations of animals or humans. the west nile virus is an example of a recently emerged vector-borne disease that has been introduced to a new geographic area. the virus was first isolated in uganda in and has since been known to cause disease in africa, west asia, europe, and the middle east [ ] . until when it caused a deadly outbreak in the new york metropolitan area, it had never been observed in the u.s., but now it has spread to every state, except alaska and hawaii, as well as canada and mexico. as of march , the cumulative number of human disease cases in the u.s. is , [ ] . the west nile virus is usually transmitted between birds by mosquitoes, but can be transmitted to humans and other hosts, particularly during favorable seasonal conditions with a hot dry summer followed by a wet fall, as what occurred in the new york area in . its introduction into the u.s. is thought to have occurred recently since the genetic profiles of the new york virus isolates suggest they came from a single source, which is related to a virus isolated in in israel [ ] . although not known for certain, it is possible that an infected bird could have been imported or an infected mosquito or tick may have hitched a ride on an international flight or on a ship carrying old imported tires infested with mosquito larvae. large holding and storage facilities for meat, grains, dairy and produce provide new habitats and breeding grounds for insects and vermin such as mice and rats. humans can be infected with the deadly hantavirus through inhalation of aerosolized virus present in dried rodent urine in grains or feedstuffs. in the southwestern u.s. experienced a mysterious outbreak of a new deadly respiratory illness in healthy people, which within a couple of months was identified by the cdc as a previously unknown type of hantavirus [ , ] . because the researchers knew that other hantaviruses were transmitted by rodents, they began trapping mice and rats in the area around the victims' homes and discovered that the deer mouse was the primary natural reservoir. further investigation revealed that there had been earlier unexplained deaths due to this hantavirus, but these cases were sporadic. the reason for the clustered outbreak in the could be connected to the unusually high numbers of mice in the area during that season [ , ] . for several years, the region had experienced drought, but in early , heavy snows melting and rainfall helped revive the flora and fauna in the region, such that the deer mice had plenty to eat. the mice increased dramatically in numbers, and consequently increased the likelihood of transmission to humans. intense cross and intraspecies interactions are conducive to transmission of a pathogen from one host to another. the rapidity with which microbes and viruses are able to evolve increases the likelihood of such close host-host contacts to cause the pathogen to ''jump'' across species barriers. like bse, chronic wasting disease (cwd) is a prion-mediated transmissible spongiform encephalopathy of cervids, such as mule deer, white-tailed deer, and rocky mountain elk [ ] . the potential for cwd to similarly cross the species barrier from cervids to humans is considered unlikely. but, because bse has been transmitted from cattle to humans (as vcjd), it is feared by some that cwd might also ''jump'' the species barrier. although cwd can be transmitted to cattle, sheep, and goats by direct inoculation into the brain [ ] , studies have not yet demonstrated that domestic livestock are susceptible via oral exposure, the presumed natural route of exposure to bse [ ] . it is feared that homology within critical amino acid sequences of the human and cervid proteins might facilitate cross-species transmission of cwd to humans, as what appears to have occurred for bse. thus, understanding how prions overcome resistance to transmission between species is crucial if we are to prevent future epidemics. although surveillance efforts for cwd in captive and free-ranging cervids are continu-ing, eradication of cwd from wild populations of cervids is unlikely with currently available management techniques. the potential emergence of a new disease-causing zoonotic agent that is transmissible between humans is a major concern. constant exposure and certain behaviors increase the likelihood that a virus will ''jump'' species. we experienced a frightening example of this with the rapid worldwide spread of the sars virus. alarmingly, we are currently at the brink of experiencing another such emergence, which could have devastating consequences on the human population. already the current spread of avian influenza a virus has resulted in the death from disease or culling of over m domestic poultry in asia, with an estimated $ b in economic losses to the asian poultry sector (table ) [ ] . a question on many people's mind today is whether another pandemic flu like the one in is inevitable [ ] . we already know that the circulating influenza h n virus can ''jump'' from birds to humans [ ] , but luckily we have not yet observed significant human-tohuman transmission other than a few cases through intimate unprotected contact with a critically ill index patient [ ] . will this fine dividing line be crossed soon? or, will this threat diminish before it evolves into a more human-specific virus? the truth is that we know very little about the specific factors that trigger a ''jump'' between species or a transition into a rapidly transmissible virus [ ] . we know even less about how to prevent these events from occurring or how to predict when they will occur. the current h n strain, first limited to poultry, quickly spread to migrating birds, but has now emerged in mammals and humans mostly through zoonotic contact. previously, it was widely accepted that avian viral strains could only readily infect humans after first having undergone genetic shuffling within swine, but now it appears that direct transmission from bird to human can occur [ ] . although its transmission from human-to-human is (luckily) still inefficient, the who, the cdc, and other organizations have already mobilized for just such an event [ ] . distinguishing a deliberately introduced infectious disease from a naturally occurring or emerging infectious disease is inherently more difficult due to their ''dual-use'' nature. the good news, though, is that effective medical treatment and prevention strategies for combating a naturally occurring infectious disease will most likely work just as well for one that is deliberately introduced. the exponential advances that have been made in the life sciences, medicine, and biotechnology have not only dramatically enabled our ability to respond to biological threats, but, sadly they have also increased the potential risks of malevolent exploitation and inadvertent misuse. indeed, a report from the u.s. national research council and the institute of medicine concluded that the breadth of potential biological threats is far wider than is commonly appreciated and will continue to expand in the future [ ] . the nih invests over $ b annually in medical research. since , nih has directed over $ b toward countering bioterrorism alone and currently spends over $ b of its annual budget on infectious diseases with over $ . b going toward emerging infectious diseases (table ) [ ] . considerable attention has been recently focused on developing better preparedness and surveillance (early warning) as strategies for more effective response to biological threats. this depends on having reliable, sensitive and rapid means for recognition of unusual events or unexpectedly high levels of common events. a number of animal and human health laboratory response networks have been established with the goal of maintaining an integrated national and international system for facilitating standardization and movement of information, for expansion of detection and diagnostics measures, for coordinating responses among federal, state, university and commercial clinical laboratories, and for identification of common source outbreaks. on the international front, the who and cdc have increased activities to build capacity for global disease detection and response, with immediate focus on and strengthening of influenza surveillance. the global alert and response network (goarn) was established in by who as a partnership of > institutions and networks to mobilize human and technical resources for the rapid identification and control of disease outbreaks that are of international importance [ ] . the global livestock early warning system (glews) has been formed by the food and agricultural organization (fao) of the united nations and the world health organization for animal health (oie) to strengthen epidemiological analysis and prediction of major animal diseases and zoonoses and to improve reporting from a variety of data sources that might impact disease transmission from animals to humans [ ] . the recent pandemics have also mobilized strengthening of the international health regulations, which were first established by who in to ensure maximum security against international spread of certain diseases (cholera, plague, yellow fever, and smallpox--although smallpox was removed from the list in ) with minimum interference of world commerce. in a revised set of regulations was adopted unanimously at the world health assembly to increase the roles and responsibilities of who and member states, including financing, developing, strengthening, maintaining and implementing core surveillance, and response capacities [ ] . biosecurity requires multipronged, flexible, and interdisciplinary approaches to combat the perpetuation and spread of infectious diseases. in all, simultaneous use of multiple strategies will be necessary for effective infection control and disease management. one such strategy is to use evolutionary engineering (combining predictive evolution and genetic engineering) to design vaccines and drugs based on predictive targets. for example, an innovative approach for control of e. coli o :h contamination of food and water was recently employed to eliminate the source for the organism. by vaccinating the animal reservoir--cattle--the researchers were able to prevent colonization of the cattle with the microbe, reducing the levels of bacteria shed in feces and thereby reducing the risk of human disease [ ] . this strategy was shown to significantly decrease the prevalence of e. coli o :h in a clinical trial conducted in a feedlot setting. improved sanitation and use of chlorinated drinking water has dramatically reduced the incidence of waterborne disease. evidence suggests that the cholera epidemic in south and central america was caused by a complex set of circumstances, including poor sanitation conditions, poor separation of drinking water and waste streams, and inadequate water treatment and distribution systems [ ] . indeed, outside of peru's capital lima, chlorination of drinking water supplies at the time of the epidemic was limited at best. improved water quality and sanitation have since reduced the incidence of cholera in south american countries. another simple, yet surprisingly effective strategy recently implemented has been the use of filtering water through multilayered cloth filters to remove the plankton and other particles to which the vibrio bacteria adhere [ ] . over the past years an unprecedented mobilization of resources have been directed at stopping the hiv pandemic, ranging from preventive strategies for persons at high risk for contracting hiv, such as educational counseling, testing, and referral services, to treatment with multiple drug regimens, to management measures aimed at improving the healthcare of persons living with hiv and preventing further transmission [ ] . although enormous success in prevention of hiv/aids in the u.s. has been achieved and we have learned a lot about how to approach rapidly evolving diseases from this experience, a number of prevention and treatment challenges remain. hiv prevalence remains high among homosexual men [ ] and racial/ethnic disparities have increased, especially among african-american men and women [ ] with prevalence among african-american men reported as high as % [ ] . new programs are needed to more effectively reach these populations [ ] . one approach toward improved treatment and reduction of drug resistance is the use of multidrug overkill (triple drug therapy). administration of just a single drug often leads to the development of resistance to that drug, but strong multidrug doses decrease the likelihood of multidrug resistance. for example, recent studies have shown that triple drug combination antiviral therapy in treating hivinfected persons offer superior viral suppression over other drug regimens [ ] . when two or more drugs are used simultaneously, each helps prevent the emergence of resistance to the other drug. effective regimens for the treatment of tb must contain four different drugs to which the organisms are susceptible. to illustrate how this might be so effective, consider that mutation rates in the tb-causing bacterium lead to a frequency of resistance to isonazid of in , to streptomycin of to , to ethambutol of to , and to rifampicin of to . bacterial mutants resistant to any single drug are naturally present in any large bacterial population. an inactive tb granuloma contains - bacteria, whereas an active tb lesion contains - bacteria. this means that the chance of gaining resistance to any one of the drugs is relatively high in an active lesion, but the chance of gaining resistance to multiple drugs is considerably less [ ] . the course of the four-drug treatment for tb usually lasts from to months. when adherence with the regimen is assured, the four-drug regimen is highly effective; however, a problem with tb treatment is that the drugs used are often counter-indicated, cause unpleasant side effects, and must be administered in series over a long period of time rather than simultaneously, which leads to problems with patient compliance [ ] . nearly half of individuals with tb do not complete their treatments. reduction of noncompliance can be achieved by direct observation of the patient to ensure full dosage. in developed countries, such as the u.s., this is relatively easy to achieve with the use of healthcare workers and family members. however, in developing countries, there are many obstacles to adhering to treatment regimens, and alternative strategies to improve compliance are needed. in addition to direct observation of treatment, other tactics include sending reminder cards or phone calls, monetary incentives, health education and counseling, and making access to clinic facilities easier [ ] . another strategy for reducing the prevalence of antimicrobial resistance is to remove the overall selection pressure by minimizing exposure to the drug, and especially withholding the most effective drugs, i.e. the ''drugs of last resort,'' until absolutely needed. this is becoming more and more difficult to accomplish with the accelerated rate of spread of antibiotic resistance through the overuse and over-prescription of antibiotics [ , , ] . indeed, many researchers were dismayed at the large distribution of ciprofloxacin (cipro tm ) to treat over , people after the anthrax attack in and warned that this widespread use could lead to resistance in other bacteria [ ] . the strain of bacillus anthracis strain used in the attack was equally sensitive to other less expensive and more commonly used drugs. ciprofloxacin is considered a ''drug of last resort'' because of its broad-spectrum and efficacy against many pathogenic bacteria, particularly those (such as staphylococcus) that are already resistant to other drugs. prescreening for sensitivity is another approach that allows for use of narrow-range rather than broad-spectrum antimicrobials, which further reduces the likelihood of resistance developing and spreading to other pathogens. management and implementation of effective preventive or containment measures in the event of natural or man-made biological threats will require increased infrastructure and diagnostic and surveillance capabilities. for example, the tremendous scale-up in food production, from the vast herds of cattle to the huge confined feedlots to the slaughterhouses to the many hundreds of distributors and supermarkets, has undoubtedly contributed to the emergence and prevalence of food-borne diseases such as that caused by e. coli o :h . the complexity of the modern food preparation and distribution process makes epidemiological tracking of the sources of contamination difficult, although there have been noticeable advances. in response to the need for improved agriculture and food biosecurity, congress passed the national agriculture and food defense act of , which will require enormous effort and financial commitment on the part of the government and agricultural and food industries. the logistics involved are daunting, particularly in coordinating efforts among many different agencies. one example is the food-borne diseases active surveillance network (foodnet), which is a collaborative project of the cdc, the usda, and the fda to monitor and q wiley periodicals, inc. study the epidemiology of food-borne diseases [ ] . in response to the growing need for better food surveillance, the fda implemented the hazard analysis and critical control point (haccp) program for prevention of food-borne diseases, which involves monitoring food distribution at critical control points where contamination is most likely to occur [ , ] . in the food safety and inspection service (fsis) of the usda established the food emergency response network (fern), which will work with the fda and its haccp program to integrate a laboratory network across the u.s. that can quickly respond to food-related emergencies [ ] . the fsis, as part of its task of protecting public health through food safety and defense, recently implemented a new assessment method, called carver shock [ ] , for identifying the most vulnerable target sites within a food processing system (table ). in considering the gaps in the u.s. agriculture and food defense capabilities, the main question that must be addressed is: exactly what level of security do the people of the usa want the agricultural industry to achieve, and our government to enforce, in terms of food safety and biosecurity? it is clear that we (as a nation and international community) are not happy with the current security status of our food and agricultural supplies. the outbreaks and ensuing deaths, economic losses and drop in consumer confidence resulting from contaminated spinach by e. coli o :h [ ] , mentioned above, amply demonstrate this point. but, if we take this episode as an example, one must question exactly what could have been done to prevent or further mitigate this outbreak than was already done. by all accounts, the surveillance, detection, evaluation, containment, and recovery measures were remarkably fast, accurate, and as good as one could possibly hope, considering the circumstances-far better than in previous incidents of a similar nature, thanks to improved measures newly in place. this was considered an accidental outbreak, but what if it had been a deliberate attack? undoubtedly, the response would have been little different. yet, what happened is still deemed by most as unacceptable. so, how could this be improved? most current measures are aimed at further improvements to detect or con-tain a future incident by increasing surveillance at the front-end, increasing diagnostics or enhancing epidemiological monitoring capabilities once an incident has occurred. but, just how much of an improvement would that be? clearly, what the public wants is a near certainty that such an event as occurred with the spinach will not happen again. part of the difficulty in adequately addressing this need is that current efforts are focused on too broad and vast a target (there are just too many steps in the food processing, where things can go wrong and something could slip through the cracks). instead, the focus for ensuring near complete biosecurity should be at the very end of the food processing chain, namely at the packaging and delivery stage. while all the proposed surveillance, detection, evaluation, containment and recovery measure will significantly reduce the possibility of future contamination, and thus should be done to the extent possible, they will not ensure the desired near-complete protection from an incident (which is what the consumer is demanding). so, how can this be achieved? many scientists see food irradiation as one viable solution. food irradiation of already processed and packaged food through promising new technology can eliminate disease-causing microbes from foods. the effects of ionizing radiation on food and on animals and people eating the irradiated food have been extensively studied and deemed safe [ ] [ ] [ ] , and indeed this technology has been implemented already for certain foodstuffs. yet, bringing this technology into use for most foods has been challenging, primarily due to misconceptions and fear about its safety on the part of the public and policymakers. closing this gap will require a ramping up of public engagement by the scientific community. somehow scientists must better communicate with the public and policymakers, convincing them of the benefit criticality-measure of public health or economic impacts of an attack to achieve terror goals accessibility-ability to physically access and egress from the target recuperability-ability of system to recover from an attack vulnerability-ease in accomplishing the attack effect-amount of actual direct economic loss from an attack as measured by loss in production recognizability-ease of identifying the target shock-the combined physical, health, economic and psychological effects of an attack the carver shock method rates each of seven attributes on a scale of - for identification of vulnerable sites in food processing and distribution system that might be targets for attack or points of contamination. of this technology and at the same time alleviating their fears of its use. to achieve global health biosecurity it is critical that the communication and educational barriers between nonscientists and scientists are overcome and that efforts by public healthcare, scientific and security policy communities are better integrated. enhanced coordination between multiple agencies is essential for implementing and maintaining global disease surveillance systems, public healthcare, and diagnostic and basic research laboratories. thus far, many redundancies in effort remain and interagency cooperation is still fragmentary. international cooperation has improved with regard to regulatory and containment (import/ export) policies, particularly in the area of travel restrictions during pandemics, but cooperation is still a bit shaky in other areas where commerce and political issues are concerned. and, much work remains in the areas of regulation of agricultural, societal and medical practices, pollution control, and prevention of habitat destruction. strengthening national and international capacities to prevent and control disease epidemics will require continued promotion of international cooperation and technical partnerships with institutions and networks around the globe to mobilize resources for control of disease outbreaks. regulation and oversight of research and use of potentially dangerous bioagents (''select agents'' or highrisk or high-impact bioagents) is well underway, but there remain disparities between that conducted in the u.s. and that done elsewhere in the world. biosafety and biosecurity of pathogens in laboratories and healthcare settings will require enhanced education for better preparedness and increased confidence of the public and policy makers in science. many scientists feel that policymakers have not fully understood the difference between biosafety and biosecurity and have consequently imposed a number of mandates and regulations that equate enhanced need for security with enhanced danger, i.e. greater biosafety risk. balancing biomedical and biotechnological advancement with biosecurity will always be at odds due to the ambiguous definition of what constitutes a potential biosecurity threat, but improved education will help with making those decisions. although warning and prevention are preferable to coping with the consequences of an attack, an emphasis must also be placed on improving public healthcare and basic research and education. it is critical that we develop a homeland and global biosecurity strategy that is applicable to both intentional and unintentional disease outbreaks. the best defense against any microbial threat is a robust public healthcare system in regard to its science understanding, capacity, and practice. there has been considerable advancement in the area of prevention, with improved surveillance and detection, and with new drugs becoming available at a remarkably rapid pace, but we are still only at the tip of the iceberg in our understanding of pathogen evolution, post-exposure treatment and control, prediction of epidemic versus pandemic spread, rates of transmission, impact of climate change, and preparation for handling a biothreat agent of unknown origin, whether it be from natural, accidental, or deliberate exposure. h n ) epidemic (h n ) england, turkey (h n ) (h n ) canada, turkey (h n ) ''spanish'' pandemic (h n ) australia, chicken (h n ) (h n ) germany, chicken (h n ) (h n ) england, chicken (h n ) ''asian'' - (h n ) pennsylvania ( m killed) (h n ) swine'' non-epidemic (h n ) hong kong (> m killed) (h n ) netherlands ( m killed) (h n ) canada ( m killed) -present (h n ) asia, pandemic report of the committee on emerging microbial threats to health in the st century on ''microbial threats to health: emergence, detection, and response global and regional burden of disease and risk factors measuring the global burden of disease and epidemiological transitions: - deaths: preliminary data for avian influenza: an omnipresent pandemic threat an open letter 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obsession with cipro foodborne diseases active surveillance network the integration of process analytical technologies, concurrent validation, and parametric release programs in aseptic processing use of microbial data for hazard analysis and critical control point verification-food and drug administration perspective food emergency response network -fern cooperative agreement food and drug administration center for food safety and applied nutrition, carver shock software tool for food defense and terrorism the role of irradiation in food safety irradiation of food-helping to ensure food safety food irradiation: a safe and useful technology key: cord- - tcikxl authors: paul, elisabeth; brown, garrett w; ridde, valery title: covid- : time for paradigm shift in the nexus between local, national and global health date: - - journal: bmj glob health doi: . /bmjgh- - sha: doc_id: cord_uid: tcikxl nan ► the covid- pandemic has triggered unprecedented measures worldwide, which have often been adopted in an 'emergency' mode and are largely reactionary ► alternatively, covid- needs to be appraised as part of a much bigger health picture, adopting a "systems approach" that enables interactions with other acknowledged and preventable health conditions, which often receive disproportionately low attention ► to do so requires a paradigm shift in global health governance, from a specific reactional paradigm to a systemic, coordinated and preventive paradigm ► it is necessary to adopt a holistic approach to health reflecting both a security approach and a health development approach, tackling upstream causes and determinants, aimed at helping populations reduce their individual risk factors and augment their natural immunity ► such preventive health policies must be tailored to local specificities and local environments, and health systems must be strengthened at the local level so as to be able to respond to population needs and expectations ► the current crisis calls for a paradigm shift in public and global health policies; and in the in the nexus between local, national and global health policies and systems the who declared the novel coronavirus disease (covid- ) an 'emergency of international concern' on january and a pandemic on march. according to who's situation report - , as of april , the epidemic has caused deaths worldwide. while it is seemingly nearing its end in china, where it was first reported, it is still on the rise in europe, in the usa and in other parts of the world, including in many low-income and middle-income countries (lmics). the pandemic has triggered unprecedented measures worldwide. many countries have installed travel bans, confinement and lockdown policies. these responses have been adopted in an 'emergency' mode, and are largely reactionary, aimed at mitigating the spread of the disease while waiting for a specific cure and/or vaccine to be developed. here we do not want to underestimate the risks caused by the pandemic, nor to question the measures taken by the who and governments. but we would like to express our concerns regarding four covid- -related issues, and advocate for a 'paradigm shift'that is, a scientific revolution encompassing changes in the basic concepts and experimental practices of a scientific discipline -to prepare for future crises. a shift in focus: covid- in the broader global health picture it is important to remember other acknowledged and preventable health conditions, when compared with the focus that covid- has triggered at global and national levels. respiratory diseases have been leading causes of death and disability in the world before covid- . it is estimated that, globally, four million people die prematurely from chronic respiratory disease each year; in particular, one million die annually from chronic obstructive pulmonary disease; pneumonia kills millions of people annually and is a leading cause of death among children under years old; each year . million die from tuberculosis; and lung cancer kills . million people a year and is the deadliest cancer. the who estimates that seasonal influenza kills up to people a year. in , an estimated people died of malaria. in , about women died during and following pregnancy and childbirth-that is approximately women each day. in the usa alone, a lower limit of bmj global health deaths per year was associated with preventable harm in hospitals. whereas infectious diseases seem to inspire the most terror among the public and policymakers, noncommunicable diseases are responsible for almost % of all deaths. depression affects million people globally and is the leading cause of disability worldwide, and nearly people die from suicide every year. the global boom in premature mortality and morbidity from non-communicable diseases has now reached a point where some have even suggested it to be a pandemic. moreover, climate change (through increased heat waves and disasters) and atmospheric and environmental pollution are expected to increase deaths and injuries, especially in lmics. in some debates, climate change has become more than a risk factor, with increasing calls for the who to declare it a public health emergency. from a public health perspective, covid- needs to be appraised as part of a much bigger health picture. for instance, beyond the lethality and direct mortality rates of covid- , attention should be paid to the interaction with other pathogens, as well as to the more indirect effects of its mitigation measures. indeed, the pandemic and its containment measures interact with, and impact on, other health conditions and will have system-wide effects, highlighting the importance of adopting a 'systems approach' to its resolution. a paradigm shift in global health governance the global health community, national security agencies and all governments have known that a pandemic like covid- was likely to come, yet global health policy has remained woefully unprepared nor fit-for-purpose. in , the g members proclaimed that ebola had been a 'wake-up call' for the need for better global cooperation. it was also recognised that antimicrobial resistance (amr) threatened to kill million people by , thus demanding urgent action. yet little has been done to address these existing global health governance shortcomings. for example, the lauded g and g response, the global health security agenda (https:// ghsagenda. org/), continues to speak in the terms of costly 'countermeasures' versus prevention and health system strengthening. moreover, the pandemic emergency financing facility (pef) (https://www. worldbank. org/ en/ topic/ pandemics/ brief/ pandemic-emergency-financingfacility), meant to deliver up to $ million in epidemic assistance to curb expansion into a pandemic, sits idle as a complicated 'loan mechanism' at the world bank, available to only a few countries (eg, china and india do not qualify for the money). there is also serious ambiguity about how the pef intersects and/or complements the who's contingency fund for emergencies (cfe) (https://www. who. int/ emergencies/ funding/ contingency-fund-for-emergencies). the cfe is available to more countries for more risks, and more quickly, but represents far less money than the pef (which, in theory, should come after the cfe, if you happen to prequalify for the loan). the 'one health' approach, which was meant to offer a more responsive research and policy agenda to combat zoonotic diseases, remains sluggish at best and underdeveloped in terms of including environmental factors, such as soil and water, which play a crucial part in amr and other threats. in terms of pathogen monitoring and response, the international health regulations, which are meant 'to help the international community and governments prevent and respond to acute public health risks that have the potential to cross borders and threaten people worldwide', are not fully implemented by many countries due to limited financial resources and political will, and have been violated in response to the covid- outbreak. what is more confounding is that many highincome countries like france have failed to fully implement the international health regulations, particularly in their overseas territories. in addition, other disease control mechanisms, like the who global influenza surveillance and response system (https://www. who. int/ influenza/ gisrs_ laboratory/ en/), remain inadequate and underfunded, with too few who laboratories and a market-based model where a global public good (pooled influenza knowledge) is turned into a private good (pharmaceutical profit), with historical inequities in terms of public health. moreover, many countries, like china, are incentivised not to raise the epidemic alarm too soon due to fears of diminished direct foreign investment (like with severe acute respiratory syndrome, h n and now covid- ) and fears that the government will be perceived as weak. these conditions of incapacity at the international level are exacerbated by a weakened who, whose budget has been radically reduced and ring-fenced. for example, the who used to receive three-quarters of its financing from assessed contributions levied on members. however, a change to a zero real growth policy for its regular budget in the s has meant it now only receives a quarter of its budget from member contributions. as a result, the who is dependent on extra-budgetary ring-fenced 'pet project' funding from donors to fill an increasingly shrinking budget. as the money flows to other multilateral health initiatives, the who's authority dissipates, with numerous organisations like the institute for health metrics and evaluation, the bill and melinda gates foundation and médecins sans frontières able to command greater epistemic authority, financial influence and response effectiveness. however, this expansion of initiatives creates a condition of policy fragmentation, which significantly weakens coordinated global public health. one real result of fragmentation of global health governance is an inefficient division of labour, where hundreds of actors such as the who, global fund, president's emergency plan for aids relief, united nations programme on hiv and aids, united states agency bmj global health for international development, world bank, the gates foundation and the clinton foundation (to name only a few) produce parallel programmes or bric-à-brac vertical health silos that have neither generated overall system strengthening in high burden countries nor allowed for effective global health policy. this creates two failures. first, contrary to sector-wide approaches, vertical 'pet-project' global initiatives often fail to promote sustainable long-term local health system strengthening, which is the best preventive defence for disease control (of all types, not just infectious diseases). second, the global level is woefully unprepared for epidemics, since global policy has remained reactionary, symptom-based and dependent on vaccine discoveries without full appreciation of other upstream determinants of disease and access to those vaccines. given the state of global health governance and inadequate investments in health system strengthening-as well as the failure, by many actors, to adopt a 'systems approach' to problem resolution -the spread and danger of covid- is not surprising. what is required, we argue, is to shift global health policymaking from a specific reactional paradigm to a systemic, holistic and preventive paradigm. there is no doubt that this approach will require serious resources, governance reform and political will. nevertheless, the global economic costs of covid- have already reached into at least a trillion dollars. thus, serious efforts to improve global and local health systems would be a small fraction of this cost, with a tried and true cost-saving philosophy that 'an ounce of prevention is worth a pound of cure'. beyond the 'pasteurian paradigm': a holistic view of health the emergency responses to covid- so far are based on the so-called 'pasteurian paradigm', which states that each disease is due to one pathogen; thus, for each disease there is one cure, targeting the responsible pathogen. in this case, laboratories are racing to find the cure or the vaccine against covid- -a vaccine which will come too late for the current epidemic, and will have limited efficacy if the virus mutates in the coming months or years. yet it is easy to see how the more pathogens there might be in the future (which there will be) the less this paradigm makes sense. moreover, the pasteurian paradigm has imposed its preferred research methodnamely, randomised control trials that try to isolate one variable from all possible variables-as the gold standard of science, relegating other approaches as near charlatanism. however, there is a multitude of evidence indicating that beyond a single pathogen, the development of a disease, as well as its outcome, is considerably affected by the physical and social parameters in which it operates, and that this is considerably affected by social, political, environmental and individual factors. this seems widely known by the public as far as chronic non-communicable diseases are concerned, but is also the case for infectious diseases, especially for emerging infections, in which the pathogenic role of social inequalities is recognised. moreover, the traditional frontiers between communicable and non-communicable diseases are being blurred by evidence of 'biosocial contagion'. in this light, the globalised world is now facing a 'syndemic'-that is, a synergy of epidemics that 'co occur in time and place, interact with each other to produce complex sequelae, and share common underlying societal drivers'. covid- is no exception, since its mortality rate varies significantly according to age, sex and comorbidities. as an alternative, we argue that it would be more effective, efficient and equitable to adopt a holistic approach to health. how to tackle the silent killers and how to prepare populations-including the most vulnerable -against future epidemics should be on the top of national and global health policy and research agendas. this should reflect both a security approach (fighting symptomatic issues) and a health development approach (tackling upstream causes and determinants). in doing so, the objectives should not be merely be the response mode, but a more concerted effort to limit environmental factors, protect biodiversity, reduce social health inequities, strengthen local health systems for preventive health, help populations reduce their individual risk factors and augment their natural immunity-notably through various 'healthy behaviours' and diets that are proven to strengthen the general immune system. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] like what recently took place in the field of evaluation of complex systems and policies, a 'realist' revolution of medical research is probably needed to help support this. it is ultimately important that the resulting policies are not copy-pasted from other countries, but adapted to each context, and backed by strong local health systems. by definition, preventive health policies must be tailored to local specificities, including local environments, and health systems must be strengthened at the local level so as to be able to respond to a population's needs and expectations. this is also the case for the response to covid- . viruses and epidemics have always existed, and will always exist, and should be anticipated. coronaviruses are a well-known family of viruses, and even if this one is particularly aggressive, its genome has been rapidly identified. the difference with this epidemic which is causing the semicollapse of health systems is that it has revealed a profound lack of national prevention and preparedness. in response to the epidemic, the most hit countries so far have faced a lack of equipment and critical care beds. in the uk and france, as just two examples, decades of austerity policies and an obsession with evaluating health facilities based on technical efficiency (ie, minimising inputs and increasing outputs) have considerably decreased the capacity of health systems to respond to above-average frequentation. the covid- emergency responses of many states have revealed important inconsistencies. in many european countries, the authorities have adopted a one-sizefits-all policy and imposed the same measures everywhere. more worryingly, some governments-notably in africahave not performed their own adapted risk assessment before copy-pasting strategies from abroad. this is problematic, since it makes little sense to use a predictive model developed from a country where the median age is and translate it to a country with a median age of , without adjusting the parameters. in addition, current policies fail to account for regional or transborder contextual parameters, where either more stringent or relaxed measures could be more suitable depending on geographical determinants. the universal lockdown of a whole country may not be necessary when there are only one or two epidemic outbreaks separated by hundreds of miles, especially if containment is quick and determined. what we suggest, in order to be effective, is that policies should fit each context and be adaptive at the territorial or ecosystem level, versus being unreflectively and uniformly bounded by national jurisdictions. this is the best way to not impose measures that are too coercive, which may face legal constraints and may be counterproductive, eroding public trust and cooperation. in the post-covid- recovery phase, we hope the lessons learnt from local, national and global responses to this pandemic will foster support, by policymakers and by the public, for tailored policy responses that support stronger and more integrated local health systems. in summary, the current crisis calls for a paradigm shift in public and global health policies. we will not be prepared for the next epidemic unless we take bold steps. first, global health policies should not be designed on a response mode to case-by-case threats, but should adopt a systems approach that can support a holistic picture of global disease burdens, risks and health conditions, as well as better consider the system-wide effects of adopted measures. second, countering current fragmentation in global health governance will require a substantial shift in global health policymaking from a reactional paradigm to a systemic and preventive paradigm, with meaningful commitments to human health security. third, there is a need to shift our focus from short-term curative policies based on the pasteurian paradigm, to long-term preventive and promotional policies based on a holistic view of people's health, which notably implies limiting environmental factors, reducing social health inequities, helping populations reduce their individual risk factors and augmenting their natural immunity. lastly, such holistic, preventive policies must be adapted to local contexts and implemented through strong local health systems able to have the 'cushion' capacity to respond to emergencies. twitter valery ridde @valeryridde acknowledgements we thank seye abimbola for inviting us to submit this commentary and for giving us critical suggestions for improvement, and eric muraille for advising on references on immunity. contributors ep and gb both had an initial idea for this paper and joined forces to arrive to this joint paper. they wrote the first draft and vr contributed to improving it. all authors contributed to the development of ideas, commenting on drafts and approved the final version. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests ep and vr have conducted consultations for various international and donor agencies. gb and vr have received funding from several research funding agencies. however, this article has been written in total independence of these contracts. patient consent for publication not required. provenance and peer review commissioned; internally peer reviewed. world health organization. coronavirus disease (covid- ) situation report - world health organization the structure of scientific revolutions forum of international respiratory societies. the global impact of respiratory disease -second edition up to people die of respiratory diseases linked to seasonal flu each year world bank group and the united nations population division a new, evidence-based estimate of patient harms associated with hospital care world health organization. noncommunicable diseases time to deliver -report of 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infection realistic evaluation. london: sage the coming plague: newly emerging diseases in a world out of balance. farrar, straus and giroux global preparedness monitoring board. a world at risk: annual report on global preparedness for health emergencies world health organization (acting as the host organization for the global preparedness monitoring board) healthcare resource statistics -beds why a one-size-fits-all approach to covid- could have lethal consequences [internet]. the conversation covid- -the law and limits of quarantine us emergency legal responses to novel coronavirus: balancing public health and civil liberties key: cord- -yf vynqe authors: agrawal, gaurav; aitken, john; hamblin, harrison; collins, michael; borody, thomas j. title: putting crohn’s on the map: five common questions on the contribution of mycobacterium avium subspecies paratuberculosis to the pathophysiology of crohn’s disease date: - - journal: dig dis sci doi: . /s - - - sha: doc_id: cord_uid: yf vynqe for decades, mycobacterium avium subspecies paratuberculosis (map) has been linked to the pathogenesis of crohn’s disease. despite many investigations and research efforts, there remains no clear unifying explanation of its pathogenicity to humans. proponents argue crohn’s disease shares many identical features with a granulomatous infection in ruminants termed johne’s disease and similarities with ileo-cecal tuberculosis. both are caused by species within the mycobacterium genus. sceptics assert that since map is found in individuals diagnosed with crohn’s disease as well as in healthy population controls, any association with cd is coincidental. this view is supported by the uncertain response of patients to antimicrobial therapy. this report aims to address the controversial aspects of this proposition with information and knowledge gathered from several disciplines, including microbiology and veterinary medicine. the authors hope that this discussion will stimulate further research aimed at confirming or refuting the contribution of map to the pathogenesis of crohn’s disease and ultimately lead to advanced targeted clinical therapies. crohn's disease is a chronic, inflammatory granulomatous disease that can affect the entire gastro-intestinal tract. involving the full thickness of the bowel, it is characterized by granulomas in approximately % of cases. a recent marked increase in the prevalence of the disease, particularly in developing nations and in the pediatric population, has generated further interest and research into this incurable condition. although no proven etiology exists as of yet, propositions such as autoimmune, t-cell-mediated immune responses to the resident gut flora, and numerous infective agents are among the most commonly debated and treated causes. given the apparent similarities to johne's disease, a chronic, granulomatous enteritis in ruminants caused by mycobacterium avium subspecies paratuberculosis (map), the suspicion for the past years is that map may be the causative agent of crohn's disease according to the observations of the scottish surgeon, tk dalziel [ ] , and likely illustrated earlier by the polish surgeon antoni leśniowski [ ] in . although dr burrill crohn commented initially upon its similarities to known mycobacterial infections of the gut, such as mycobacterium tuberculosis complex (mtb), particularly in that it was of a "granulomatous enteritis" nature [ ] , the organism was never found or isolated; consequently, over time several etiologies came to the fore [ ] . this conjecture is arguable due to the lack of a confirmatory diagnostic assay that implicates map in active disease. this review addresses common questions relating to the involvement of map in the pathogenesis of crohn's disease. members of the genus mycobacterium are microaerophilic gram-positive, rod-shaped bacteria that can form a mycolic (myco) bacterial cell wall that is lipid and cholesterol rich, which often retains acidic stains (acid-fast). members of this genus of actinobacteria can exist in a myriad of forms with three distinct groups recognized: mtb, m. leprae, and non-tuberculosis mycobacteria (ntm), which includes map. these range from behaving as obligate parasites to environmental saprophytes with varying rates of growth. when infecting humans, mycobacteria have a preference for residing within the lysosomes of the macrophage cell cytoplasm, mirroring the preference of mycobacteria in the environment where they sequester within amoebae in order to survive [ ] . map, like mtb, employs a variety of means to inhibit phago-lysosomal maturation that enables persistence within the macrophage [ ] . mycobacteria can exist in a number of adaptable forms such as extracellular/intracellular, cell wall deficient (cwd-also known as spheroplasts/l forms), acid-fast cell walled, and endospores. they have a relatively slow to the slowest growing rates of all bacterial species, which contributes to whether a particular mycobacterium is pathogenic or opportunistic (the host-microbe interaction is also an important factor [ ] ). slower growing organisms are more difficult to eradicate due to the majority of treatments targeting active respiration or cell walled forms. there is evidence to suggest that zoönotic pathogens may cause crohn's disease, from the emergence of johne's disease in animals, where the pathogenic features are well described and understood. a core principle is that an infection cannot be equated with pathogenicity. more recent discoveries, particularly in relation to the mycobacterial life cycle and of latency, strongly suggest the involvement of stealth cwd forms of mycobacteria. further factors include the pathogenicity of the bacteria and strain varieties, host immune-microbe interaction, macrophage functioning, the influence of the gut microbiome, optimal testing and protocols, treatment trials, and appropriate antibiotic combination chemotherapy. nevertheless, all of these observations require the gold standard of reliably and regularly culturing of map in human samples for confirmation and analysis. recent discoveries continue to implicate map as the initial promoter of crohn's disease through direct and indirect effects of inflammation. possible indirect consequences of infection in a predisposed host are a re-modulation of the gut microbiome from a gram-positive aerobic constituency to a gram-negative, anaerobic environment [ ] . the production of lipopolysaccharide and other signaling molecules from the latter groups of bacteria may also cause extraintestinal effects. the gut microbiome may be the common highway for a variety of inflammatory "autoimmune" diseases that cascade and overlap; it appears that map in crohn's disease is more than a simple direct "cause and effect" infective pathology. indeed, host-microbe interactions are essential to phenotypic expression of a disease. whether this is genetic, flora-related (dysbiosis), acquired defects (autophagy dysfunction), it is clear that an opportunity is presented for map to reproduce rather than colonize in the host, with persistence dependent on factors such as l forms and growth rates. it is estimated by the world health organization that one-third of the humanity has been exposed and infected with mtb [ ] , highlighting the widespread distribution and infectivity of mycobacteria in the environment. the observable characteristics of the genus mycobacterium are diverse, reflecting the environmental and pathogenic functions of certain members. nevertheless, there are some, such as the m. avium complex (mac), which bridge the divide between environmental members and the mycobacterial human pathogens mtb and m. leprae. this gradual scale of pathogenic potential, from commensal organism to opportunistic pathogen to an obligate pathogen, highlights the difficulty of assigning pathogenic functions to map, which is a member of the mac group. organisms in this cluster are characteristically opportunistically pathogenic. as an example, m. avium is seen in both healthy human controls although the organism is pathogenic in the presence of a declining or absent immune response. detection of mtb is considered to be proof of infection in the patient, whereas for mac members it is not so, as they have been associated with transient asymptomatic human carriage. after years, a consensus being considered is that map is not a human pathogen in its own right. characteristics of map are summarized in table . in light of the foregoing discussion, the authors have formulated five questions aimed at further understanding the involvement pf map in crohn's pathogenesis. cell wall-deficient mycobacterial (cwdm) species were discovered in the tissue of crohn's patients in [ ] , with subsequent identification of the isolates as a map after months of incubation [ ] . attempts were made to prove, by the use of koch's postulates (table ) , the pathogenicity of the organism in humans in an attempt to link map with crohn's disease. this was by using the technical methods available at that time. although further attempts to isolate map in crohn's disease tissue have had less success, the cardinal steps to proof of causation were described. these included the detection and isolation in culture of the putative pathogen and the subsequent introduction of the isolate into an animal model with induction of a "crohn's like" syndrome. to date, there have been few research attempts to assign causality that have duplicated that path likely due to the necessity of culturing the organisms until mycobacterial reversion of the cwdm occurs and the cwdm develop a cell wall. this necessity conflicted with the evidence that cell walled isolates of map in crohn's were quite rare, making it unlikely that cell walled map was a pathogen. on the contrary, the cell walled and the cwdm variety were rarely ever cultured from tissue. this inability to culture the target organisms led to the adoption of the surrogate detection method (polymerase chain reaction; pcr) in order to detect map in tissue. these molecular methods are preferred for detecting pathogens where culture is difficult with certain caveats. in favor of pcr for proof of pathogenicity are its ease to use, timely results, lower costs, and relatively high sensitivity and specificity of results. there are, however, two problems with pcr-false positives and false negatives, which in the instance of mtb infection, is a major reason why culture accompanies pcr. culture, the gold standard in mtb diagnosis, is also important for the detection of antimicrobial resistance, whereas pcr is confirmatory, providing a preliminary indication. the specific difficulties with reliance on pcr in the diagnosis of crohn's disease are numerous. it is an unreliable surrogate for culture. despite this, probes to detect genes and insertion sequences have been designed in order to identify specific sequences within the genome of map. one sequence, thought to be specific for map, is the is sequence though recent evidence suggests that other mycobacteria share this same gene sequence [ ] . although the f sequence appears to be more specific for map [ ] , in comparison with the - copies per organism observed for the is sequence, the f gene only has copy per organism. importantly, the numbers of these repetitive sequences influence the pcr detection rate, often requiring the more sensitive nested pcr methodology [ ] . many research trials comparing the detection rates of map using pcr in patients with crohn's disease versus healthy controls [ ] observed transient carriage of map in healthy controls [ ] , predictable given that members of mac are opportunistic pathogens. although one investigation was thought to have successfully isolated map in the majority of crohn's patients, subsequent investigations by other researchers demonstrated difficulties in reproducing the results using the same methods [ , ] , inferring that pcr for map is not reliable on its own due to a lack of disease correlation. on the basis of these observations, the utility of pcr for mtb diagnosis has been questioned. a study of samples of real-time (rt)-pcr for mtb dna as compared with mtb cultures showed that pcr was % sensitive and . % specific having a positive predictive value of . % and a negative predictive value of % [ ] . a systemic review and meta-analysis showed that mtb rt-pcr "may be better utilized as a rule out, add-on diagnostic test." [ ] . some consider that there is no association between map and crohn's disease, a conjecture supported by abundant table specific characteristics of map slowest growing mycobacteria known-takes weeks to reproduce and > h generation time multiple cellular forms, such as acid-fast bacilli with the ability to form spheroplasts or l forms strong tendency to form clumps. important for antibiotic chemotherapy treatment resistance to first-line anti-tuberculous drugs, in part due to genes/biofilm. low cure rates map has been cultured and grown in human blood but took months to do so can create dysbiosis of the local (gut) microbiome leading to inflammatory cascades disease is a result of host-microbe interaction and immune susceptibility of the host infection does not mean disease is expressed but may result in colonization/persistence/latency table koch's postulates the bacteria must be present in every case of the disease the bacteria must be isolated from the host with the disease and grown in pure culture the specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host the pathogen must be reisolated from the new host and shown to be the same as the originally inoculated pathogen circumstantial evidence. nevertheless, the american academy of microbiology concluded that people with crohn's disease have a : odds of having map in their blood or gut tissues compared with those who do not have crohn's disease [ ] . therefore, the current issue has progressed now to whether map causes cd or is an "innocent bystander" uninvolved in disease pathogenesis. growing and isolating an organism by culture of infected tissue is considered to be the gold standard in detecting bacteria [ ] . regarding map, the observed difficulties in the culture of cwdm forms has been a major obstacle for many researchers. the reasons for this are complex but can be distilled down to lack of experience with mycobacterial microbiological methods, and in developing media capable of sustaining growth of cwdm. the same culture methods used for johne's disease may not guarantee success in the cultivation of cwdm in humans. the task of cultivation of cwdm, though difficult, has been successfully carried out by some researchers albeit by time-consuming and complicated methods. lacking simple and straightforward methods and protocols for routine diagnoses, the proof of pathogenicity of map in crohn's disease remains obscure and arcane. in , the eminent german microbiologist robert koch formulated a set of principles termed "koch's postulates" designed to assign causality of a disease to bacteria using an animal model ( table ). the principles were first utilized for anthrax [ ] , a common disease of cattle caused by the bacterium bacillus anthracis and subsequently launched the field of medical bacteriology [ ] . problems exist though, since m. leprae and helicobacter pylori do not fulfill these criteria for causation, yet they are the recognized causes of leprosy and peptic ulcer disease, respectively. even during their early use, koch realized that his postulates could not explain the causative links between microorganisms and disease in all cases. an example was his acceptance of the causative bacteria of cholera (vibrio cholerae) despite its isolation from both sick and healthy people [ ] . although researchers have since attempted to apply koch's postulates to map and crohn's disease [ ] , their efforts have largely been thwarted due to the protean nature of map, which exists as a cwd form in humans but is present in ruminants as a cell walled form [ ] . specific genetic factors with unique host-microbe interactions are also thought to be involved in the pathogenesis of cd. these observations are summarized in table . crohn's disease patients do not deteriorate when treated with immunosuppressives, whereas in mtb infection, deterioration is common. the relative risk for mtb increases up to . - times with anti-tnf therapy depending on certain factors [ ] . hence, if crohn's disease is similarly the result of a mycobacterial infection, such as map, then there should be similar clinical deterioration instead of a positive response in a cohort of patients to anti-tnf therapy. nevertheless, mtb and map do not share the same ability to establish disease. there are for example, no cavitary lesions in crohn's disease that might suggest invariable pathogenicity, as is seen with pulmonary mtb. practically, the difference is that mtb is invasive, whereas map appears proinflammatory (in crohn's disease). both however share the same ability to trigger formation of granulomas and t-cell responses and as such are the main drivers of inflammation in crohn's disease and incidentally, are the main protective mechanism against intracellular pathogens in humans. importantly, drugs displaying several different mechanisms of action have historically been utilized for multiple purposes, e.g., hydroxychloroquine for malaria and rheumatoid arthritis treatment. similarly, the immunosuppressive drugs used in crohn's disease, such as thiopurines and biological therapies demonstrate anti-mycobacterial properties [ ] . though, infliximab reduces map titers in vitro [ ] , there is a suspicion that this may also lead to resistance of the organism through environmental pressures [ ] . current immunotherapies are targeted against the overexpression of cytokines in crohn's disease, such as interleukin (il)- , il- , and tnf-α, with underexpression of other cytokines, such as il- . these mirror the known immune responses to mycobacteria. tnf-α is essential for the clearance of intracellular pathogens and the control of mycobacteria via the augmentation of t-cell responses. this table barriers to the proof that mycobacteria are pathogenic there is no animal model for crohn's disease that supports an infectious trigger there is no readily available human isolate of a cwdm from infected crohn's tissue available to introduce into an animal there are no reliable published methods to re-isolate the cwdm from an artificially infected animal is through the promotion of macrophage activation and the differentiation and phagolysosome formation that optimizes cd + t-cell immunity; by promoting antigen presentation and apoptosis; and cross-priming cd + t-cells [ ] . the inability to clear mycobacteria from macrophages persistently raises tnf-α levels. furthermore, tnf-α is inhibited by induction of il- production, a key virulence factor produced by mycobacteria [ ] in order to create an intracellular sanctuary. this environment is created by down-regulating apoptosis and stimulating the release of soluble tnf receptor type from macrophages, which also inactivates tnf-α [ ] . il- is well studied by map investigators, since this factor is essential for communication between host innate and adaptive immune responses, in particular by macrophages hoping to attract t-cells. documentation of il- mediated suppression of interferon gamma (ifn-γ) secretion in peripheral blood of cattle and goats exposed to map infection enables persistence of map within the macrophage [ ] . the known excesses and deficiencies of cytokines and signaling molecules in crohn's disease has been compared with those observed in map infection, revealing similar patterns between the two [ ] . newer small molecule inhibitors currently being introduced as crohn's therapies such as pan and gut specific janus kinase (jak) inhibitors may also target the downstream effects of a pathological map infection, achieved by inhibiting jak and signal transducer and activator of transcription (stat)-the jak-stat pathway; a hallmark of ifn-γ induction, which is classically active in the clearance of intracellular pathogens. map subverts ifn-γ mediated activation of the infected macrophage with subsequent malfunctioning [ ] . there is an inability of the infected host to respond to, rather than produce, ifn-γ, hence this factor is raised unresponsively [ ] . this observation requires future confirmation in humans. there are no observable increase in rates among farmers and veterinarians, who are regularly exposed to map-infected cattle. therefore, environmental exposure appears to not be a risk factor for crohn's disease due to map. the prevalence of crohn's is estimated to be about . % in developed countries with an average of people affected per , population [ ] . comparatively, subgroups who exhibit greater exposure to johne's disease show only a mild increase of . % in the prevalence of crohn's disease, with studies reporting no association between the exposure to johne's disease and the development of crohn's disease [ ] . indeed, some studies have reported lower rates and a reduced mortality from inflammatory bowel diseases in farmers and in veterinarians when compared with the general population [ , ] . therefore, the hypothesis that map causes crohn's disease appears to be erroneous [ ] . from a microbiological viewpoint, the risk factor in cd for map involvement is a defective immune system. when coupled with an earlier exposure to the pathogen, the person is at a much higher risk of developing crohn's disease. with human immunodeficiency virus (hiv), patients are at a far higher risk of opportunistic infection with m. avium intracellulare (mac) due to their compromised immune system (reduced cd + count). it is also evident that crohn's, like hiv, is an emerging prevalent human disease, unlike other "autoimmune" diseases, that demonstrate a constant disease rate. this observation that the incidence of crohn's disease is increasing indirectly suggests that a significant environmental risk factor is present. observing that the rate of johne's disease is increasing in parallel with crohn's disease supports the hypothesis that crohn's is driven by a pre-existing genetic susceptibility married to an emerging animal pathogen, a hypothesis that can be further explored by knowledge of how the organism behaves when causing an inflammatory enteritis in cattle herds. indeed, in all animal species, map susceptibility is generally age-dependent-with exposure at an early age and during the pre-weaning phase essential for subsequent disease development [ ] . at a point later in life of the organism, typically during sexual maturation, a phenotypic change occurs, resulting in johne's disease [ ] . hence, according to this hypothesis, one's adult occupation should be irrelevant to the incidence of crohn's disease although exposure to cattle early in life should be contributory. furthermore, the majority of the aforementioned studies that compare the incidence of crohn's disease with livestock exposure are cross sectional since they do not take into account exposure to map-infected animals only, rather including exposure to any and all animals. evidence suggests that the younger this exposure occurs the greater the chance of controlling the organism [ ] with exposure to intracellular forms of map in early life incurring increased risk versus extracellular forms, which results in immune priming. although with the recent increase in incidence in the pediatric demographic, this would conflict, but it could suggest other environmental factors are at play, with one possibility being an increasing virulence of the organism from evolutionary pressures. another, is the host responses are influenced by the effect of dysbiosis, given the discoveries of the microbiome on mycobacterial susceptibility. research suggests that particular microbial signatures within the lung microbiome [ ] and even the gut microbiome [ ] are associated with the development of pathogenic tb infection. hence, one hypothesis is that the increasing prevalence of westernized diets and lifestyles reduces the diversity of resident microbiota [ , ] , including in children [ ] and thus contributes to immune susceptibility and the increasing incidence of crohn's disease in this population. since this hypothesis could explain the increasing incidence of crohn's disease in the developing world, further studies are required in order to link this relationship with the epidemiology of crohn's disease. map is detectable in rural environments with live map present in retail pasteurized milk, cheese, raw meat, and in domestic water supplies [ ] [ ] [ ] . hence, the prevalence of map in agriculture and in food and water supplies [ ] renders difficult the discrimination between neonatal exposure on the farm versus other modes of exposure. as a consequence, the identification of a suitable non-exposed group is challenging. perhaps most interesting apropos of the observation that breast feeding appears to be protective against crohn's disease, is the identification of map in retail powdered baby formula [ ] . this likely represents the most interesting epidemiological comparison since breastfed babies have a delayed exposure to map and most infant formula products are based on bovine milk. further research is required in this area for these observations. effective therapies against pathogens are designed on the back of sound knowledge of the target organism, which is currently lacking due to challenges in isolating and observing map. a contemporary example of this are the recent advancements in the in vitro propagation of hepatitis viruses and pcr-based methods for detecting hepatitis c infection, which led to extremely effective antiviral therapies [ ] . importantly, the difficulties in developing efficacious treatments against mycobacteria are not only present for map. despite a wealth of research into the lifecycle of mtb, current gold standard treatment is effective in % of cases and drops significantly in resistant strains: % in multi-drugresistant mtb (mdr-tb) and % in extensive-drug resistant tb (xdr-tb), respectively [ ] . therefore, applying this to map [ ] explains, in part, the lower-than-expected cure rates with antibiotic chemotherapy. although improved and evolved anti-mycobacterial therapy is required, the inability to culture the organism limits directly targeting organism-specific mechanisms, knowledge of which are needed to develop improved treatments. alongside resistant strains, eradication of cwdm is especially problematic. currently, amat is non-curative and the optimum length of treatment is unknown. there are legitimate concerns of arrhythmias from qt interval changes/or prolongation, due to the involvement of a macrolide (clarithromycin) and a riminophenazine (clofazimine). nevertheless, an underlying abnormal cardiac repolarization is a prerequisite for arrhythmia induction [ ] and that qt prolongation is unique to the individual and is a poor predictor of ventricular arrhythmias [ ] . electrocardiographic monitoring has proven useful to optimize pharmacovigilance, identify those at risk from the interaction, and implement effective dosage adjustments. guidance from m. leprae, m. avium and mdr-tb treatment suggest long-term regimens of > months are required [ ] and are safe [ , ] . the who's international drug monitoring programme reports that ventricular arrhythmia is not a common side effect of clofazimine [ ] and even more safety data will be provided from the important stream phase and global tb alliance phase trials [ ] . as mentioned, guidance from m. leprae and m. avium treatment suggests that long-term regimens of > months are required [ ] for treatment of map. yet, even the regimens themselves can vary substantially in their composition and efficacy, e.g., multi-drug therapy (mdt) and fixed duration (fd) therapy in leprosy treatment [ ] . despite this, several advancements in map treatment have occurred over time; rifabutin is now preferred to rifampicin due to its higher intracellular concentrations and tissue distribution that are essential for targeting intracellular map. furthermore, rifabutin has a significantly longer half-life than rifampicin ( h compared with . h). the higher lipophilicity of rifabutin ( -fold higher oil/water partition coefficient than rifampicin) could account for this difference alongside having a lower minimum inhibitory concentration (mic) for most pathogens. compared with rifampicin, rifabutin has a narrower induction spectrum and %- % weaker cyp a induction properties, thus altering its bioavailability and interactions with other medications. furthermore, the previously utilized antibiotics isoniazid and pyrazinamide are now thought to be ineffective for cwd ntms such as map, due to natural resistance [ ] . accumulating evidence suggests that a spheroplastic phase of map (a cwd form) is able to avoid antigenic immune recognition by the host, thereby helping explain the persistent relapsing nature of crohn's disease, if the map hypothesis is indeed correct. therefore, in order to target the known forms of map, it would be advisable to use at minimum a -drug combination, especially for the minimally metabolically active spheroplastic forms. a significant argument against the contribution of map to crohn's pathogenesis has been the lack of cure demonstrated with anti-mycobacterial therapy along with the lack of a significant p value in one large randomized control trial (rct) of anti-mycobacterial therapy [ ] . this study is often cited as a basis of evidence for ineffective treatment effects of map-active antibiotics as the patients with crohn's disease were not cured: a primary endpoint of the study. nevertheless, care must be taken on the basis of a limited understanding of the target organism and unreliable detection methods. it may be more appropriate to conclude that rather than a lack of causation, there was a lack of efficacy in the treatment. in addition to the admissions made by the authors, there are several follow-up articles to the study that highlight flaws and inaccurate conclusions [ ] [ ] [ ] [ ] [ ] . for a rct, there was no placebo arm since it is considered to be unethical to have such a group for crohn's patients, thus highlighting the flaws of rcts in real-world practice. prospective observational controlled studies are likely to be more clinically useful in this situation. furthermore, the design of the study removed without replacement subjects who did not achieve clinical remission on prednisolone, rendering it underpowered. the "per protocol" analysis used fluctuating denominators and inverted criteria from "remission" to "relapse," confusing interpretation while rendering it statistically invalid. importantly, an "intention-to-treat" reanalysis of the raw data demonstrated that amat was statistically of benefit for inducing and maintaining remission, as published in the lancet [ ] . these two studies are summarized in table . a second large-phase iii rct trial of amap, awaiting peer review, appears to contain promising data. although there are still limitations with this study, preliminary results have shown a statistically significant response in achieving remission using amat; (p < . ) as the primary endpoint [ ] . the map us rhb- study examined the safety and efficacy of amat in moderate-to-severe crohn's disease cases. importantly, the amat doses used in this study were higher than those used previously [ ] . the placebo arm was given standard immunosuppressive therapy due to the ethical considerations governing the control arm. at week , there were more subjects achieving the primary endpoint of remission (cdai < ) in the treatment arm than in the placebo arm ( % vs. %, p = . ). furthermore, the late induction of remission from week to week was also significantly greater than placebo ( % vs. %, p = . ). the week assessment does not represent preservation or durability of remission but rather captures the later induction of remission. the treatment arm showed a statistically significant reduction in biochemical markers of disease (crp or fecal calprotectin) at week ( . % vs. . %, p = . ), ( . % vs. . %, p = . ), and ( . % vs. . %, p = . ) alongside an endoscopic response in a subgroup of patients. a follow-on rct has a primary outcome measure of remission (cdai < ) at week . importantly, key secondary outcomes include measurement of map by pcr of blood at baseline and at several timepoints during treatment, measurements not available in the first map us study [ ] . this trial of amat was based on previous effectiveness in several case series and clinical trials reports [ ] [ ] [ ] [ ] [ ] [ ] [ ] and would be an important contributor to the accumulating higher quality evidence required to determine the effectiveness of amat. a summary of recent published trials of amat in the therapy of crohn's disease is presented in table . this review has attempted to acknowledge difficult questions regarding the contribution of map to crohn's disease by involving other disciplines and knowledge. zoönotic diseases are infections that originate in animals and jump species barriers to infect other species, including humans, and often occur unknowingly. transmission and infectivity differ with the species reservoirs present. the bubonic plague is caused by yersinia pestis from rats and more recently sars-covid- is thought to have originated from bats or pangolins [ ] . current management of crohn's disease relies on immunosuppression, with amat rarely, if ever, offered as a treatment option. nevertheless, this could be regarded as a lost opportunity from a lack of specific mycobacterial knowledge and stems from core issues for the lack of progression in the treatment and cure of this condition. specialization and reliance on current molecular technology entails a danger of neglecting and ignoring organisms for what they are and indeed, recognizing that assessment of bacterial behaviors could be beyond the current level of knowledge, awareness, and technology. the input of infectious diseases, microbiologists, and veterinarians are paramount to explain, present different viewpoints, and complement the knowledge base of the gastroenterologist. the future involves the essential and urgently required discovery of an accurate diagnostic test to truly assess the distribution, infectivity, and pathogenicity of map in crohn's patients. moreover, evolved forms of therapy are needed such as optimal amat variations, vaccines, and phage therapy. though great strides have been made, the true behavior of mycobacterium avium paratuberculosis in humans cannot yet be fully understood or elucidated, though the resolution of this -year-old question could be close to being answered. chronic interstitial enteritis granulomatous colitis crohn's disease of the colon the many faces of crohn's disease: latest concepts in etiology free-living amoebae, a training field for macrophage resistance of mycobacteria from mouth to macrophage: mechanisms of innate immune subversion by mycobacterium avium subsp. paratuberculosis the microbiome and tuberculosis: early evidence for cross talk. mbio role of commensal gut bacteria in inflammatory bowel diseases world health organisation. latent tb infection: updated and consolidated guidelines for programmatic management spheroplastic phase of mycobacteria isolated from patients with crohn's disease possible role of mycobacteria in inflammatory bowel disease an is -like sequence found in a mycobacterium sp. other than mycobacterium avium subsp. paratuberculosis detection of mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using is and f -based qpcr assays new pcr systems to confirm real-time pcr detection of mycobacterium avium subsp mycobacterium avium subspecies paratuberculosis causes: crohn's disease in some inflammatory bowel disease patients culture of mycobacterium avium subspecies paratuberculosis from the blood of patients with crohn's disease validation and standardization of is and f realtime quantitative pcr assays for the specific detection and quantification of mycobacterium avium subsp. paratuberculosis culture of mycobacterium avium subspecies paratuberculosis (map) from the blood of patients with crohn's disease: a follow-up blind multi center investigation clinical evaluation of real time pcr assay for detection of mycobacterium tuberculosis dna in respiratory specimens effectiveness of real-time polymerase chain reaction assay for the detection of mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis mycobacterium avium paratuberculosis: infrequent human pathogen or public health threat die Ätiologie der milzbrandkrankheit, begründet auf die entwicklungsgeschichte des bacillus anthracis robert koch and the 'golden age' of bacteriology limitations of the henle-koch postulates is crohn's disease caused by a mycobacterium? comparisons with leprosy, tuberculosis, and johne's disease. lancet unraveling the transcriptional regulatory networks associated to mycobacterial cell wall defective form induction by glycine and lysozyme treatment the risk of tuberculosis related to tumour necrosis factor antagonist therapies: a tbnet consensus statement treatment of severe refractory erythema nodosum leprosum with tumor necrosis factor inhibitor etanercept treatment of crohn's disease patients with infliximab is detrimental for the survival of mycobacterium avium ssp. paratuberculosis within macrophages and shows a remarkable decrease in the immunogenicity of mycobacterial proteins targeted combination antibiotic therapy induces remission in treatment-naïve crohn's disease: a case series targeting mycobacterium tuberculosis tumor necrosis factor alpha-downregulating genes for the development of antituberculous vaccines interleukin produced by macrophages inoculated with mycobacterium avium attenuates mycobacteria-induced apoptosis by reduction of tnf-α activity why interleukin- supplementation does not work in crohn's disease patients the role of il- in mycobacterium avium subsp paratuberculosis infection involvement of mycobacterium avium subspecies paratuberculosis in tnf-α production from macrophage: possible link between map and immune response in crohn's disease mycobacterium avium subsp. paratuberculosis inhibits gamma interferon-induced signaling in bovine monocytes: insights into the cellular mechanisms of johne's disease worldwide incidence and prevalence of inflammatory bowel disease in the st century: a systematic review of population-based studies lack of association between the occurrence of crohn's disease and occupational exposure to dairy and beef cattle herds infected with mycobacterium avium subspecies paratuberculosis mycobacterium avium subspecies paratuberculosis, crohn's disease and the doomsday scenario occupational mortality from inflammatory bowel disease in the united states - crohn's disease in people exposed to clinical cases of bovine paratuberculosis us) committee on diagnosis and control of johne's disease. diagnosis and control of johne's disease a synthesis of the patho-physiology of mycobacterium avium subspecies paratuberculosis infection in sheep to inform mathematical modelling of ovine paratuberculosis chronische autoimmunerkrankungen und kontakt zu tieren (chronic autoimmune disease and animal contact) study group: contact with farm animals in early life and juvenile inflammatory bowel disease: a case-control study meta-analysis of the lung microbiota in pulmonary tuberculosis the gut microbiome signatures discriminate healthy from pulmonary tuberculosis patients the western diet-microbiomehost interaction and its role in metabolic disease the impact of western diet and nutrients on the microbiota and immune response at mucosal interfaces the impact of diet and lifestyle on gut microbiota and human health are we closer to understanding why viable cells of mycobacterium avium subsp. paratuberculosis are still being reported in pasteurised milk? survival of mycobacterium avium subspecies paratuberculosis in retail pasteurised milk persistence of mycobacterium paratuberculosis during manufacture and ripening of cheddar cheese food safety: emerging pathogens detection of viable mycobacterium avium subspecies paratuberculosis in powdered infant formula by phage-pcr and confirmed by culture the polymerase chain reaction: essential for the development of curative therapy for hepatitis c world health organisation correlation between rpob gene mutation in mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of crohn's disease antibiotic-induced cardiac arrhythmias qt prolongation is an unreliable predictor of ventricular arrhythmia a trial of a shorter regimen for rifampin-resistant tuberculosis clarithromycin, rifabutin and clofazimine for treatment of disseminated mycobacterium avium complex disease in aids patients safety and availability of clofazimine in the treatment of multidrug and extensively drugresistant tuberculosis: analysis of published guidance and metaanalysis of cohort studies a practical handbook on the pharmacovigilance of medicines used in the treatment of tuberculosis: enhancing the safety of the tb patient tuberculosis treatment and drug regimens fixed-duration therapy in leprosy: limitations and opportunities mechanisms of pyrazinamide action and resistance two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for crohn's disease flawed australian cd study does not end map controversy importance of the australian crohn's disease antibiotic study australian crohn's antibiotic study opens new horizons the australian antibiotic trial in crohn's disease: alternative conclusions from the same study antimycobacterial therapy for crohn's disease: a reanalysis placebo-controlled, multicenter, parallel group study to assess the efficacy and safety of add-on fixed-dose anti-mycobacterial therapy in moderately to severely active crohn's disease (map us). united abstract : oral presentation. rhb- , a fixeddose, oral antibiotic combination against mycobacterium avium paratuberculosis (map) infection, is effective in moderately to severely active crohn's disease. american college of gastroenterology open label efficacy and safety of anti-map (mycobacterium avium ssp. paratuberculosis) therapy in adult crohn's disease: full text view: clinicaltrials.gov [internet two-year-outcomes analysis of crohn's disease treated with rifabutin and macrolide antibiotics open clinical trial of rifabutin and clarithromycin therapy in crohn's disease treatment of severe crohn's disease using antimycobacterial triple therapy-approaching a cure? dig liver dis antimycobacterial therapy in crohn's disease heals mucosa with longitudinal scars primary treatment of crohn's disease: combined antibiotics taking center stage a meta-analysis of anti-mycobacterial therapy for crohn's disease long-term antibiotic treatment for crohn's disease: systematic review and meta-analysis of placebo-controlled trials anti-mycobacterium paratuberculosis (map) therapy for crohn's disease: an overview and update profound remission in crohn's disease requiring no further treatment for - years: a case series anti-mycobacterial antibiotic therapy induces remission in active paediatric crohn's disease. microorganisms are pangolins the intermediate host of the novel coronavirus (sars-cov- )? key: cord- -biwnqa f authors: luke, anthony; d'hemecourt, pierre title: prevention of infectious diseases in athletes date: - - journal: clinics in sports medicine doi: . /j.csm. . . sha: doc_id: cord_uid: biwnqa f the sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. one must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. this article reviews preventive strategies for infectious disease in athletes, including immunization recommendations and prophylaxis guidelines, improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, insect-borne disease precautions, and prevention of sexually transmitted diseases. a special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. i nfections in sports can be serious medical problems. they can affect individual athletes, resulting in morbidity and decreased performance [ ] . they can also be spread to other athletes, putting them at risk for similar disease and complications. the sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. one must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. the authors aim to translate recommendations in public health to practice in a sports medicine setting. sports physicians have an opportunity to see athletes during adolescence and adulthood who might not come in for routine health maintenance except when related to their sports. primary prevention of infectious disease, the ideal goal, deals with avoiding the development of the disease before infection occurs. immunizations are an example of primary prevention and have been the most successful public health programs for disease prophylaxis. secondary prevention for infection control involves prevention of spread to others. athletes are often exposed to many different people, travel to compete in various environments locally and internationally, and engage in higher-risk activities, often in close contact with others [ ] . the authors discuss the preventive strategies for infectious disease in sport, including ( ) a review of immunization recommendations and prophylaxis guidelines, ( ) improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, ( ) insect-borne disease precautions, and ( ) prevention of sexually transmitted diseases (stds). a special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. athletes may share personal items (eg, towels, water bottles, and soap) and equipment (eg, weights). they may live in dormitories or in hotel rooms while traveling, which leads to close contact and high exposure to teammates. they participate in higher-risk activities [ , ] . fewer athletes practice safe sex, which can lead to stds in homosexual and in heterosexual individuals. athletes also use more illicit drugs and alcohol, which can place them at risk of intravenous needle exposure [ , ] . steroids, hormones, and vitamins are other substances that some athletes are injecting [ , ] . tattoos are popular among athletes, which are another source of needle infection risk. active immunization involves the administration of all or part of a microorganism or a modified product of that microorganism, such as an antigen or protein. this administration stimulates an immune response to develop protection against future exposure to the infection [ ] . although most vaccines are over % effective, they are not guaranteed to promote immune protection [ ] . immunizations typically involve inactivated vaccines or live, attenuated viruses. inactivated vaccines include killed virus or bacterial proteins to stimulate one's immune system to develop antibodies to any similar virus or bacteria. more side effects are associated with the live, attenuated viruses-typically local pain and, rarely, hypersensitivity reactions to vaccine constituents. a mild febrile illness, a recent exposure to an infectious disease, pregnancy, breastfeeding, nonspecific allergies, and family history of an adverse event after immunization, including seizures, are not contraindications for immunization [ ] . sports medicine physicians need to consider the following indications for immunizations (tables and ) : ( ) routine health maintenance; ( ) catch-up immunizations for failed or missed immunizations; ( ) immunizations of high risk groups (ie, splenectomy, chronic disease, immunocompromised); ( ) travel to an endemic area; ( ) close contact with an infected individual, or ( ) recent potential exposure to an infectious agent. when doing preparticipation physical examinations, it is sometimes assumed that athletes have received all their immunizations. proof of immunizations is required by schools and colleges, although exceptions can be given to individuals refusing to receive immunizations. in a study surveying , minnesota children who entered kindergarten in , by months of age, % of students had received the measles, mumps, and rubella (mmr) vaccine, and only % had received their fourth dose of diphtheria, tetanus, and pertussis vaccine (dtap) [ ] . vaccination rates can vary substantially by age, race/ethnicity, and neighborhood [ ] . white, non-hispanic students usually have higher vaccination rates than children of other racial/ethnic groups. it has been estimated that % of whites, % of hispanics, and % of african americans are fully immunized [ ] . although mmr vaccines have been administered for many decades and incidences of disease are presently low [ ] , the diseases can still occur in the adult population. between and , % of the measles cases occurred in college students [ ] . enzyme-linked immunosorbent assay tests for antibodies to mmr are available to detect for immunization status [ ] . in a series of students, ( %) were found to be seronegative to measles alone, ( %) were seronegative to rubella alone, and ( %) were seronegative to measles and rubella. eighty-six percent of the individuals seronegative to measles had previously received a dose of measles vaccine. following a second injection, conversion to seropositive status rose to % and % for measles and rubella, respectively. these data support the need for a two-dose vaccine schedule [ ] . pneumococcal vaccine is administered to prevent streptococcus pneumoniae infections. a conjugate heptavalent is given to during the first years of life. a polysaccharide vaccine is provided to high-risk individuals older than years against types of streptococcus pneumonia that account for % of invasive disease [ ] . high-risk groups include patients who have asplenia, sickle cell disease, diabetes mellitus, cirrhosis, immunocompromised states, chronic cardiac or pulmonary disease, or age years or older. immunity following vaccination is successful for periods of to years, requiring booster injections [ ] . hepatitis b is a blood-borne virus transmitted through sexual contact and parenteral exposure to blood and blood components [ ] . hepatitis b has a greater risk for transmission in sports than hiv. the risk of hiv transmission is estimated to between in million games and in million games [ , ] . the risk arises if bleeding and skin exudates from an infected individual come into contact with open wounds in another athlete, particularly during contact and collision sports. there are no confirmed cases of spread of hiv through sports [ ] ; however, out of high school sumo wrestlers at one club developed hepatitis b [ ] . another case series reported on of american football players who developed hepatitis b over a period of months [ ] . contact through open wounds, cuts, and abrasions were the suspected routes of transmission. although hepatitis a is a considered immunization in athletes who are traveling to endemic areas, routine vaccination for hepatitis b is recommended for all individuals after birth using single or combination vaccines [ ] . a three-dose immunization schedule is typically used after years of age, with injections at months, month, and months, although there is an optional four-dose schedule [ ] . the licensed vaccines have had % to % efficacy of preventing hepatitis b, with immunity lasting years or longer [ ] . immunizations for hepatitis b should be checked during the preparticipation physical examination, and catch-up immunizations recommended to the individual (see table ). if individuals are uncertain about their immunization status, serologic testing for antibody to hepatitis b surface antigen can determine immunity. when athletes are known to be infected with hepatitis b, secondary prevention includes education on personal hygiene, appropriate management of open wounds, proper use of protective equipment, safe sex practices using a condom, and avoidance of intravenous blood transmission (eg, through needle sharing and illicit drug use). bordetella pertussis, which is responsible for whooping cough, is a gram-negative coccobacillus transmitted by way of airborne droplets [ ] . although tetanus and polio have been controlled well with the use of vaccines [ ] , the rate of pertussis cases has been increasing in adolescents and adults despite routine immunizations [ ] . most cases occur in patients years or older [ ] . the infection is most concerning for infants because immunity is not complete until older ages. the spread to infants is typically from adults. pertussis usually presents with nonspecific upper respiratory tract infection symptoms for to weeks (catarrhal stage), after which the paroxysmal and sometimes uncontrollable cough develops [ ] . the cough is not necessarily always followed by the classic ''whooping'' sound, and pertussis should be considered with any persistent, prolonged cough. the whole-cell pertussis vaccine is estimated to be approximately % effective [ ] . this vaccine is still recommended for use in the routine immunization of young children; however, the immunity provided begins to decline at to years following vaccination, which makes adolescents and adults susceptible [ ] . rare adverse reactions from the vaccine include hypotonic, hyporesponsive episodes, high fever, seizures, and anaphylaxis [ ] . two acellular vaccines have been introduced that are as effective as whole-cell vaccines and have fewer adverse reactions [ ] . these vaccines are combined with tetanus toxoid and reduced diphtheria toxoid (dtap). the centers for disease control and prevention (cdc) recommends use of these dtap boosters rather than the tetanus-diptheria (td) booster starting after to years of age [ ] . for pertussis, individuals are most contagious during the first to weeks during the catarrhal stage but should be considered contagious until weeks after the paroxysmal stage ends or after taking antibiotics for days [ ] . diagnosis of pertussis infection is best performed through polymerase chain reaction assay (sensitivity, %; specificity, %) or through direct fluorescent antibody testing (sensitivity, %; specificity, %). nasal swab cultures (sensitivity, %; specificity, %) are routinely performed; however, they have high false negative rates and take to days to yield results [ ] . physicians in the united states are legally required to report cases of pertussis to state public health departments [ ] . it is estimated that % of susceptible household contacts will be infected after close contact [ ] . antibiotic prophylaxis is recommended for close contacts of persons who have pertussis to prevent outbreaks [ ] . preferred drugs are azithromycin for days, clarithromycin for days, or trimethoprim-sulfamethoxazole or erythromycin for days, which are similar for prophylaxis and treatment [ ] . influenza presents with constitutional symptoms of fever, chills, malaise, fatigue, and myalgia in addition to upper respiratory tract symptoms of a sore throat, cough, and rhinitis. rarely, more serious conditions can occur, including encephalopathy, transverse myelitis, myocarditis, and pericarditis [ ] . immunogenicity is determined by hemaglutinins and neuraminidases on the virus surface. antigenic drift can occur that can mutate the virus into different strains. transmission occurs by way of respiratory droplets. the virus has an incubation period of usually days (range, - days), and adults are infectious from the day before symptoms begin to approximately days after the illness starts [ ] . symptoms usually last a week, although less likely, symptoms can last longer than weeks. these symptoms can be very disruptive for treatment and challenging for the athlete to keep training and competing. a case series of students, mostly healthy adolescents at a ski school in austria, reported a severe outbreak of influenza a, leading to an attack rate of %, with % becoming ill within days of the outbreak. two students were hospitalized with pneumonia and died [ ] . influenza vaccines contain strains of antigenically equivalent strains of influenza similar to those annually recommended: influenza a (h n ), influenza a (h n ), and a b virus. depending on the emergence and spread of new strains, other virus strains can be added to update the vaccine [ ] . the efficacy of influenza vaccine is approximately % to % for individuals under age years [ ] . vaccination for influenza should occur in the fall (october or november), at the beginning of the flu season (box ) [ ] . antibodies develop approximately weeks after vaccination [ , ] . inactivated influenza vaccine is generally appropriate for all populations requiring influenza vaccine. three influenza vaccines were available in the united states for the to season: fluzone (manufactured by sanofi-pasteur); fluvirin (manufactured by novartis); and fluarix (manufactured by glaxosmithkline). the typical dose is . ml administered intramuscularly, usually in the deltoid muscle. live, attenuated influenza vaccine (laiv) is approved for use in healthy, nonpregnant individuals aged to years. the laiv is administered by way of a nasal spray once in each nostril (flumist, manufactured by medimmune). individuals who have a hypersensitivity or anaphylactic reaction to components of the flu vaccine or to eggs should not be vaccinated [ ] . adults reported having a % reduction in severe febrile illnesses after laiv compared with placebo [ ] . side effects from laiv increased in adults within days of immunization compared with placebo and consisted mainly of nasal congestion ( . % versus . %) and sore throat ( . % versus . %), which lasted, on average, days. less common complaints were tiredness, cough, and chills. there was no significant difference in the number of mild febrile illnesses between immunization and placebo groups [ ] . injections can be scheduled to occur at the optimum time during the athlete's competitive schedule to minimize concern about side effects. when inactivated influenza vaccine shortages occurred in previous years, the vaccine was recommended for high-risk groups as priority; however, the general recommendation now is to offer the immunization annually to anyone who wishes to reduce the likelihood of being ill with influenza or transmitting the virus if they should become infected [ ] . although this policy cannot be directly translated into a benefit for the athlete, depending on the level of athlete, the use of the laiv may also be beneficial to prevent lost time from sport. influenza vaccine has been suggested for competitive athletes and essential personnel, especially before international events occurring during the influenza season [ , ] . treatment with antiviral medications can reduce the duration of uncomplicated influenza a and b illness by approximately day when administered within days of illness onset [ , ] . recommended antiviral treatment should be given for days [ ] . four antiviral agents are currently available: amantadine, rimantadine, zanamivir, and oseltamivir [ ] . the influenza a virus, however, has become resistant to amantadine and rimantadine, which are presently not recommended to be used as first-line drugs [ ] . zanamivir (ralenza, dry powder taken by orally inhaled route) and oseltamivir (tamiflu, capsule or oral suspension) are neuraminidase inhibitors and can be used to treat patients and to control influenza outbreaks in closed settings. although typically used in nursing homes, an outbreak in a dormitory may require chemoprophylaxis [ ] . there are limited data to suggest that serious complications from influenza, such as lower respiratory tract infections, may be reduced [ ] . the use of antiviral medications for prophylaxis of influenza is unclear and is not yet recommended for routine seasonal control [ ] . the use of oseltamivir, however, has been recommended in specific cases, especially if there is high risk of spread such as household contacts and if individuals have not been immunized [ ] . oseltamivir was used to treat of patients, including athletes during the salt lake city winter olympics, with medications given to close contacts, which was believed to limit the spread of influenza [ ] . clinical history and physical examination are still the mainstays for diagnosing influenza. rapid swab tests are available and take approximately minutes to detect the influenza virus. the tests are less sensitive ( %- %) and specific ( %- %) than the traditional viral cultures [ ] . they have moderate sensitivities for influenza antigens and are more likely to produce false negative rather than false positive results [ , ] . direct and indirect fluorescent antibody staining tests are also available, but they are ordered more in hospitals because they take to hours to obtain results [ ] . viral cultures are still the ''gold standard'' for confirming the presence of influenza and identifying the strains and subtypes [ ] . neisseria meningitides is a serious concern, especially for the adolescent and college populations. an alarming trend during the s demonstrated an increase in meningitis deaths in college students, with living in dormitories being a risk factor. the disease can be spread by asymptomatic carriers. students living in dormitories were to times more likely of getting infected than those living in other types of accommodations [ ] . freshmen who lived in dormitories had an elevated risk of meningococcal disease (odds ratio, . ; % confidence interval, . - . ; p ¼ . ) compared with other college students [ ] . aside from the risk of death, % to % of survivors of meningitis have serious sequelae such as neurologic disability, limb loss, and hearing loss [ ] . routine vaccination with meningococcal vaccine is recommended for college freshmen living in dormitories and for other populations at increased risk. the cdc advisory committee on immunization practices recommends routine vaccination of young adolescents ( - years old) with meningococcal vaccine (mcv ) at the preadolescent health care visit [ ] . therefore, sports medicine physicians may be faced with higher frequency of checking for meningococcal immunization status for high school and college athletes. a tetravalent conjugate vaccine (menactra, sanofi pasteur) is available against neisseria meningitidis isolates a, c, y, and w- in a . -ml single-dose vial. over the age of years, % of the meningococcal infections are caused by strains c, y, or w- (cdc, unpublished data, ) [ ] . another vaccine, menomune (aventis pasteur limited), has been licensed since and has a similar immunogenicity profile to menactra and is delivered subcutaneously as a . -ml dose. menactra and menomune have serum bactericidal protection ranging from . % and . % for strain w- and . % and . % for strain y, respectively [ ] . revaccination may be necessary for individuals at high risk after years [ , ] . common side effects with mcv were local pain in just over % of patients, followed by swelling, induration, and redness in approximately . % to . %. fever was reported in . % of children years old or younger and in . % of adults [ ] . close contacts are at high risk and should be treated with chemoprophylaxis ideally within hours of identifying the index patient [ ] . the goal of treatment is to reduce nasopharyngeal carriage of n meningitidis. after more than days after the onset of illness in the index patient, chemoprophylaxis is not necessary [ ] . a single dose of ciprofloxacin ( mg orally) or ceftriaxone ( mg by intramuscular injection), or rifampin ( mg twice a day for days) is recommended for adults. children between month and years old may take rifampin ( mg/kg every hours for days), or ceftriaxone ( mg intramuscularly) if younger than years [ ] . one dose of azithromycin ( mg) was also shown to eradicate n meningitidis and may represent another treatment option [ ] . human papillomavirus (hpv) is associated with % of cervical cancers and anogenital, head and neck, and nonmelanoma skin cancers. it is an std and can be diagnosed by abnormal cervical cell changes seen on pap smear [ ] . this is a common infection, especially in sexually active adolescents and university students [ ] . primary prevention is now possible with two new vaccines: a bivalent vaccine against hpv types and and a quadravalent vaccine against types , , , and . the vaccines have a three-dose schedule: , , and months (bivalent vaccine) and , , and months (quadravalent vaccine). at . years, the bivalent vaccine was effective for producing a persistent antibody response against hpv and , with more than % seropositivity and . % effectiveness ( % confidence interval, . - . ) in reducing the number of reported abnormalities on pap smear, colposcopy, or both [ ] . routine vaccination with three doses of quadrivalent hpv vaccine is recommended for girls to years old but can be started in girls as young as years. girls and women aged to years who have not been vaccinated previously or who have not completed the full vaccine series are recommended to receive a catch-up series. the vaccine is intended to be administered before potential exposure to hpv through sexual contact [ ] . secondary prevention involves checking the affected individual's partners for signs of genital warts and other stds. regular cervical screen is recommended. use of condoms and education on spread is important. hpv infection persists for life; however, the degree and duration of contagiousness is yet unknown [ ] . athletes traveling need to consider the endemic diseases in the geographic location where they are competing. they should be aware of risks of acquiring common diseases, their accommodations (urban versus rural), local foods, and customs. immunizations should ideally be planned or more months in advance to allow for adequate time to administer vaccines (table ). there are many resources for information about prevention of infectious diseases for travelers (table ) . mosquito-borne disease a number of arthropods, such as mosquitoes and ticks, can transmit diseases. mosquito-vector diseases include west nile virus, yellow fever virus, and dengue virus. west nile virus, a flavivirus, has demonstrated a seasonally endemic epidemiology with geographic variation in the united states, especially in california, arizona, and colorado [ , ] . this disease typically presents between july and october, although cases have presented between april and december. the prevention of mosquito bites is the cornerstone of prevention. an athlete in an endemic area should wear an insect repellant such as deet (n,n-diethyl-mtoluamide), picaridin (kbr- ), or oil of lemon eucalyptus (p-menthane- , diol). deet and permethin may be applied to the clothing [ ] . if a sunscreen is used concomitantly, the insect repellant should be applied on top of this and removed at the end of the day. long-sleeved shirts that are tucked into long pants are also useful. tick-borne diseases include rickettsial diseases, lyme disease, babesiosis, tickborne relapsing fever, and occasionally, tularemia and q fever (table ) . certain athletes who participate in rural outdoor activities are more susceptible to tick bites. these sports include cross-country running, training in multiple sports in rural areas, and recreational outdoor sports such as fishing and hiking. children are more at risk to tick bites. three more common rickettsial illnesses are rocky mountain spotted fever, human monocytotropic ehrlichiosis, and human granulocytotropic anaplasmosis [ ] . the infectious organisms responsible for these illnesses maintain their lifecycles in mammals and ticks. their prevalence reflects the geographic locations and the seasonality of the tick abundance. their season is usually from april to september, but they can present throughout the year. newer rickettsial diseases are emerging. these potentially lethal diseases are difficult to diagnose because they often mimic viral syndromes. as many as % to % of patients are initially misdiagnosed [ , ] . with rocky mountain spotted fever, more than % of cases are reported in the five states of north carolina, south carolina, tennessee, oklahoma, and arkansas [ ] . the presentation most often manifests as a sudden febrile illness with headache, myalgia, and a maculopapular rash that spreads in a centripetal pattern. rickettsia rickettsii has a predilection for endothelial cells and can cause a diffuse vasculitis and an untreated mortality of %. the diagnosis is based on clinical presentation, with epidemiologic, geographic, and seasonal considerations. laboratory testing may be supportive with thrombocytopenia and mild liver enzyme elevation. serologic testing is supportive only on a delayed basis with acute and convalescent titers. human monocytotropic ehrlichiosis and human granulocytotropic anaplasmosis can also present with acute headache, fever, and myalgia. laboratory evaluation often demonstrates leukopenia, thrombocytopenia, and transaminase elevation. common tick-borne illnesses that have been reported in the northeast united states are lyme disease and babesiosis, which are transmitted by the tick ixodes scapularis [ ] . babesiosis can cause a febrile illness and possibly life-threatening anemia and thrombocytopenia. lyme disease is a rickettsial disease caused by borrelia burgdorferi. as such, concurrent disease may be caused by the same tick bite (see table ). there are no proven vaccines for these tick-borne illnesses, but all are preventable by careful vigilance and protection. the key to prevention is to understand the regional epidemiology and seasonality of the diseases. vaccination for lyme disease (lymerix) was originally approved; however, the manufacturer took the vaccine off the market due to declining sales. there was a % efficacy after two doses and a % efficacy after three doses [ ] ; however, the protection diminished after years. ticks thrive in a wooded environment and at the edge of woods with surrounding high vegetation. ticks are uncommon in well-mowed lawns. relative tick-free zones can be created by placing wood chips or gravel around recreational areas to separate the woods [ ] . other landscape management tips include removing clippings and leaves, keeping stone walls clean of leaves, and restricting the use of groundcover, such as pachysandra, where pets and children may play. widening woodland trails andkeeping in the center of the trail while walking may be helpful. when traveling in wooded areas, light-colored clothing is helpful to identify the tick. long pants tucked into tightly woven socks and closed shoes minimize exposure. deet at % to % should be applied to the skin. permethrin may be applied only to the clothing. clothes should be removed and cleaned and dried after exposure. the clothes dryer is effective in killing ticks. one should carefully check for ticks in the nymphal phase that may be the size of a pin head. careful inspection should be done of the hair, ears, axilla, belly button, and legs. children and pets should also be checked. it is also important to monitor pets that may travel in the woods and return indoors. the technique of tick removal is critical. tweezers with fine tips should be used close to the skin and pulled directly away. squeezing the body may allow contamination of the disease into the host [ ] . lyme disease is not contracted until at least hours of tick adherence [ ] ; however, ehrlichiosis may transmit in less than hours. preventive antibiotics are generally not indicated because less than % of bites are lyme infected, especially with a flat tick. after a high-risk exposure (when the tick has been engaged for more than hours and is engorged), a single dose of mg of doxycycline is believed to be effective [ ] . most infectious diseases are spread from contact with the microorganism directly or indirectly from the infected individual. athletes frequently interact with teammates, coaches, athletic trainers, and physicians and share equipment, water bottles, towels, and supplies. this interaction is particularly a concern, with the recent outbreaks of methicillin-resistent staphylococcus aureus (mrsa) infections among sports teams [ , ] . three categories of potential risk factors for spreading infection have been suggested: ''sharing'' (sharing soap/towels/water bottles with teammates), ''skin injury'' (cuts, abrasions), and ''close contact'' (locker adjacent to infected teammate, living on-campus) [ ] . good personal hygiene can help reduce colonization of bacteria. bacterial counts can range from to million colony-forming units per square centimeter on the hands [ ] . universal body fluid precautions-for example, using disposable gloves when examining the oral cavity or wounds and frequent hand washing-can reduce the risk of infection. mrsa is transmitted from an infected patient to the gloves of a health care worker in approximately % ( %- %). physicians, in particular, have a low compliance for using gloves and washing their hands [ ] . proper surgical hand washing is recommended to be to seconds with soap, a -second rinse with water, followed by complete drying with a towel. the use of rinses and gels with concentrations of % to % alcohol take seconds to use and are effective at killing organisms [ ] . the use of chlorhexidine soap has been useful for reducing mrsa infections. viruses and bacteria can exist on equipment. mrsa was found in the taping gel and whirlpool in the training facilities of a professional football team [ ] . using diluted bleach ( part bleach in parts water) to cleanse training areas and equipment is recommended [ ] . routine cleaning schedules for shared equipment should be established and recorded. for upper respiratory tract infections, isolation of those who have had close contact with someone who has a confirmed or suspected infection, especially those who have active symptoms such as persistent fever and cough, is an effective and practical method of avoiding contact [ ] . any athlete who has a scratch, abrasion, or laceration or who has potentially infectious skin lesions such as vesicular or weeping skin lesions should be removed from play until the area can be securely covered with occlusive bandages or dressings to prevent leakage of blood or serous fluid [ ] . uniforms with fresh blood should be removed and replaced immediately after stopping any bleeding. bleach diluted with tap water in a : ratio can be used to wash equipment that has had contact with blood or body fluid. body substance precautions should be taken by health care professionals at all times when treating open wounds. one type of bacteria that has become more common in the hospital and a community-acquired infection is mrsa. although contact sports such as wrestling and football have been commonly associated with mrsa spread, this infection has also been discovered in minimal-contact sports such as fencing [ ] . three factors are associated with mrsa spread in sports. first, even with sports that have minimal contact, there are often abrasions and chaffing from clothing and hot environments. second, equipment is often shared and there is potential for transmission of bacteria. third, many sports have sufficient skin-to-skin contact to transmit organisms. subsequently, health care providers should strongly encourage good overall and hand hygiene in addition to covering all wounds and limiting shared equipment. it is crucial to have an ample supply of soap and water and alcohol-based hand cleansers. athletes, staff, and coaches should be educated in proper first aid for wounds, in recognition of wounds that are potentially infected, and in seeking medical attention for lesions that have concerning signs, especially large pustules or boils. athlete's foot, tinea pedis, is a common ailment not only during the hot summer months but also during the winter months with indoor sports. a number of prevention items include washing feet daily; drying between the toes; wearing cotton, nonsynthetic socks; wearing bathing shoes in public showers; and wearing sandals in warmer weather. jock itch, tinea cruris, is best prevented by showering immediately after athletic endeavors and wearing cotton briefs. a good talc powder may be used for prevention of athlete's foot and jock itch. ring worm, tinea corporis, is best prevented by avoiding contact. contact athletes such as wrestlers should not participate until any lesions have cleared or can be safely and effectively covered. athletes may manifest risk-taking behavior and subsequently be at increased risk for stds [ ] . the preparticipation examination affords the opportunity for the clinician to address these concerns. the cdc has emphasized the five intervention strategies [ ] , which include education on sexual behavior, identification of asymptomatic individuals, diagnosis and treatment of infected individuals, counseling of sexual partners of persons who have an std, and pre-exposure vaccination when applicable. individuals at risk should be questioned about partners regarding number and same or opposite sex. information about the type of sexual activity, the use of protection, and history of previous stds should also be identified. preventive measures for an std include abstinence if an individual or partner is actively infected and undergoing treatment. pre-exposure prophylaxis is relevant in several situations. hepatitis b vaccine is recommended in all individuals potentially exposed to stds. hepatitis a vaccine is recommended for all men who have sex with men or users of illicit drugs (injectable and noninjectable). for girls and women aged to years, the new quadrivalent vaccine for hpv types , , , and is recommended due to the higher associated risk of cervical cancer. most condoms are made of latex and are quite effective in std prevention. in one study of partners of hiv-infected individuals, partners were % less likely to seroconvert than those who did not use condoms [ ] . the male condom can also reduce the transmission of gonorrhea, chlamydiosis, and trichomiasis [ ] . there may be some added protection against herpes simplex virus and a % reduction of hpv transmission [ , ] . when an individual is allergic to latex, certain polyurethane condoms are likely just as effective, although they may break more readily. conversely, natural-membrane condoms such as lambskin are too porous to be used for std prevention. only water-based lubricants should be used with latex condoms because oil bases will weaken the latex. the female condom is a double-ringed polyurethane sheath that is used vaginally and during anal receptive intercourse that is effective in limited trials in preventing hiv/stds [ , ] . spermicides and nonbarrier contraception have no role in std prevention. finally, providers should encourage patients who have stds to notify their partners. often, this notification is pursued by the public health department. in the event of exposure to hiv by sexual exposure or needle stick, hiv prophylaxis is often undertaken and should be immediate. prophylactic treatment usually involves the hospital infectious disease division to determine the best combination therapy. education is paramount in public health and in the prevention of infectious diseases. athletes are a high-risk population often due to their increased exposure to different people and environments and, sometimes, their outgoing lifestyle behaviors. primary prevention can be promoted through accurate immunizations; appropriate, planned health maintenance; good hygiene practices; and behavior modification to minimize high-risk activities. secondary prevention can be achieved through vigilant surveillance for reportable illnesses, proper education and containment for reducing spread if an illness occurs, and timely prophylaxis with medications and immunizations in certain cases. virus infections and sports performance-a prospective study lifestyles and health risks of collegiate athletes: a multi-center study an outbreak of measles at an international sporting event with airborne transmission in a domed stadium influenza vaccination for athletes? pneumococcal vaccine for olympic athletes and visitors to spain. barcelona olympic organizing committee medical and public health services at the atlanta olympic games: an overview summary of notifiable diseases-united states severe acute respiratory syndrome and sport: facts and fallacies prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) sports participation and health-related behaviors among us youth aids in a bodybuilder using anabolic steroids hiv infection associated with injections of anabolic steroids blood borne infections in sport: risks of transmission, methods of prevention, and recommendations for hepatitis b vaccination the infectious complications of anabolic-androgenic steroid injection active and passive immunization a statewide survey of immunization rates in minnesota school age children: implications for targeted assessment and prevention strategies swanson's family practice review: a problem-oriented approach evaluation of a two-dose measles, mumps, and rubella vaccination schedule in a cohort of college athletes antibody response of pneumococcal vaccine: need for booster dosing? bleeding injuries in professional football: estimating the risk for hiv transmission an outbreak of hepatitis b in members of a high school sumo wrestling club horizontal transmission of hepatitis b virus among players of an american football team a comprehensive immunization strategy to eliminate transmission of hepatitis b virus infection in the united states: recommendations of the advisory committee on immunization practices (acip) part : immunization of infants, children, and adolescents red book: report of the committee of infectious diseases pertussis: a disease affecting all ages duration of immunity against pertussis after natural infection or vaccination summary of notifiable diseases-united states randomised controlled trial of two-component, three-component, and five-component acellular pertussis vaccines compared with wholecell pertussis vaccine. ad hoc group for the study of pertussis vaccines systematic review of the effects of pertussis vaccines in children preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee on immunization practices red book: report of the committee of infectious diseases comparison of pcr, culture, and direct fluorescent-antibody testing for detection of bordetella pertussis recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: cdc guidelines influenza a outbreak among adolescents in a ski hostel effectiveness of influenza vaccine in health care professionals: a randomized trial parenteral influenza vaccination induces a rapid systemic and local immune response safety, efficacy, and effectiveness of live, attenuated, cold-adapted influenza vaccine in an indicated population aged - years effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial study indicates influenza vaccine beneficial for college athletes safety and efficacy of the neuraminidase inhibitor zanamivir in treating influenza virus infection in adults: results from japan. gg group effectiveness of neuraminidase inhibitors in treatment and prevention of influenza a and b: systematic review and meta-analyses of randomised controlled trials adamantane resistance among influenza a viruses isolated early during the - influenza season in the united states impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations antivirals for influenza in healthy adults: systematic review treatment and prevention of influenza: swedish recommendations evaluation of diagnostic tests for influenza in a pediatric practice influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza prevention and control of meningococcal disease. recommendations of the advisory committee on immunization practices (acip) risk factors for meningococcal disease in college students control and prevention of meningococcal disease: recommendations of the advisory committee on immunization practices (acip) effectiveness of meningococcal serogroup c conjugate vaccine years after introduction azithromycin compared with rifampin for eradication of nasopharyngeal colonization by neisseria meningitidis risks for incident human papillomavirus infection and low-grade squamous intraepithelial lesion development in young females genital human papillomavirus infection: incidence and risk factors in a cohort of female university students sustained efficacy up to . years of a bivalent l virus-like particle vaccine against human papillomavirus types and : follow-up from a randomised control trial acip provisional recommendations for the use of quadrivalent hpv vaccine red book: report of the committee of infectious diseases the incidence of ehrlichial and rickettsial infection in patients with unexplained fever and recent history of tick bite in central north carolina the epidemic of west nile virus in the united states laboratory evaluation of mosquito repellents against aedes albopictus, culex nigripalpus, and ochierotatus triseriatus (diptera: culicidae) physician knowledge of the diagnosis and management of rocky mountain spotted fever: mississippi rocky mountain spotted fever: clinical, laboratory, and epidemiological features of cases rocky mountain spotted fever in the united states, - endemic babesiosis in another eastern state: new jersey vaccination against lyme disease with recombinant borrelia burgdorferi outer-surface lipoprotein a with adjuvant. lyme disease vaccine study group prevention of lyme disease effect of tick removal on transmission of borrelia burgdorferi and ehrlichia phagocytophila by ixodes scapularis nymphs duration of tick bites in a lyme disease-endemic area prophylaxis with single-dose doxycycline for the prevention of lyme disease after an ixodes scapularis tick bite a clone of methicillin-resistant staphylococcus aureus among professional football players methicillin-resistant staphylococcus aureus in a high school wrestling team and the surrounding community recurring methicillin-resistant staphylococcus aureus infections in a football team on washing hands an investigation of contact transmission of methicillin-resistant staphylococcus aureus transmission of blood-borne pathogens during sports: risk and prevention methicillin-resistant staphylococcus aureus infections among competitive sports participants-colorado sexually transmitted diseases treatment guidelines effectiveness of condoms in preventing sexually transmitted infections a randomized controlled trial to reduce hiv transmission risk behaviors and sexually transmitted diseases among women living with hiv: the willow program effect of condoms on reducing the transmission of herpes simplex virus type from men to women condom use and the risk of genital human papillomavirus infection in young women use-effectiveness of the female versus male condom in preventing sexually transmitted disease in women use of reality ''female condoms'' for anal sex by us men who have sex with men. hivnet vaccine preparedness study protocol team key: cord- -apgdzgfz authors: lewis, thomas j; huang, jason h; trempe, clement title: reduction in chronic disease risk and burden in a -individual cohort through modification of health behaviors date: - - journal: cureus doi: . /cureus. sha: doc_id: cord_uid: apgdzgfz introduction health risk factors, including lifestyle risks and health literacy, are known to contribute to the chronic disease epidemic. according to the centers for disease control and prevention (cdc), chronic diseases account for % of healthcare costs, morbidity, and mortality. in the united states, healthcare providers attempt to modulate a limited set of risks. however, chronic diseases continue to proliferate despite expansion of wellness programs and drugs to manage and prevent chronic conditions. pandemics, exemplified by severe acute respiratory syndrome coronavirus (sars-cov- ), show that people in good health suffer mortality rates at % the rate compared to those with pre-existing chronic conditions. healthcare costs and morbidity rates often parallel mortality rates. new root-cause risk and health tools that accommodate low health literacy and are linked to personalized health improvement care plans are needed to reverse the chronic disease epidemic. reported here is a study on manufacturing employees in the midwest us using a personalized and group approach to chronic disease reversal and prevention which may also find utility in pandemic severity and policy decisions. methods health, lifestyle, behavior, and motivation data were collected on individuals at the beginning of a nine-month disease reversal and prevention program. the data were updated every two to six months over the period. inputs included information from a novel health risk assessment, serum biomarkers specific for chronic disease, and traditional medical information. using all these data we generated robust, personalized, and modifiable care plans that were implemented by the participant and guided by a care team including health coaches and medical providers. periodic renewal of profile data and biomarkers facilitated adjustment of care plans to optimize the path toward health goals set mutually by the participant and the care team. results ninety percent of participants experienced a favorable reduction in chronic disease biomarkers. the reduction in serum biomarkers coincided with a reduction in disease and risk attributes based on medical chart data and before and after interviews. hemoglobin a c, for example, lowered in all but one participant concomitant with reported improved energy and reduced need for medications in the majority of participants. markers of inflammation lowered across the population. most importantly each individual reported improvement in their overall health. conclusions this simple, inexpensive, root-cause based risk and health approach generates a “do no harm” action plan that guides a care team, including the participant, on a path to improved health. the data demonstrate that changes in a novel risk calculator score coincide with changes in sensitive biomarkers for chronic disease. when the risks of an individual are reduced, the biomarkers reflect that change with self-reported wellbeing also improved. this program and process may be of value to society plagued with escalating levels of chronic disease and merits further study and implementation. developed nations, and particularly the united states, continue to confront a chronic disease crisis. the world health organization (who) reported that in , non-communicable chronic diseases including: cardiovascular diseases, diabetes, cancers, and chronic respiratory diseases, accounted for / of deaths worldwide [ ] . the institute of medicine reported that america is less healthy compared to high income nations in obesity, diabetes mellitus, heart disease, chronic lung disease, and disability [ ] . the organization of economic cooperation and development (oecd) tracks the health of developed nations. the u.s. scores in the lower half among these nations on all major indicators of health, and longevity. when considering that the per person per year cost of healthcare in the united states is more than two and a half times that of the oecd nation average, yet our residents live . years less, a health paradox exists in the united states. this u.s. paradox is the worst cost-to-value benefit for chronic disease outcomes compared to the average of other developed nations. the chronic disease management system is failing people at both ends of the health spectrum. a root of the problem is that health and prevention recommendations currently supported by the major medical societies have proven ineffective at reversing or preventing chronic diseases. laboratory tests in common use remain of limited scope and provide little insight into chronic health status. pharmaceuticals prescribed based on test results have poor absolute statistical success at preventing or reversing disease. these assertions are borne out since % of the nation's nearly $ trillion in annual health care expenditures are for chronic conditions per the cdc. and the situation is not improving, for example, cardiovascular disease mortality, managed with statin drugs, blood pressure medications, and other usual care approaches across broad members of the u.s. population, increased nationally by . % between and [ ] . on average, residents of the united states with five or more chronic conditions spend times more on health services than people with no chronic conditions [ ] . as of , % of u.s. adults had at least one chronic condition, and % had more than one chronic condition. five percent of the population accounts for an estimated %- % of total health care expenses [ ] . the most expensive health conditions account for % of total health care expenses. the financial and productivity costs impact our corporations, who fund over half of the national healthcare at a price of roughly % of their gross revenues. and much of this cost is segmented in high-cost beneficiaries where, for example, the top % of claimants cost $ , /y compared to the population mean of $ /y. in a report compiled by the health care cost institute, there is a surprising large turnover from year to year among the highest cost healthcare spenders. three out of five top spenders in any given year were low or moderate spenders in the prior year. in , only % of the top % of spenders were in the top % of spenders in . moreover, this trend was consistent in each year from to . there is a need for better predictive analytics to determine who is and, more importantly, will be in this significantly high-cost segment of any population as a current tool, claims data, lacks predictive power. in pandemics, standard tests provide little information on projected outcomes, rather they simply indicate exposure and potential immunity. healthy people are much less likely to die compared to unhealthy or older people. physiological health, the main concern of practicing clinicians, is not well characterized through these tests. further, the main cause of death appears to be cytokine storm syndrome which is driven by innate, not adaptive immunity [ ] . thus, antibody testing does not adequately describe disease risk or severity. validated data on severe respiratory viral diseases and the correlation between mortality, immunocompromised status and existing chronic conditions in infected individuals indicate that a broad set of blood-based biomarkers may best serve to stratify risk and to set policy on containment strategies in populations [ ] . currently, the policy is being established with an incomplete set of evidence. in vivo blood biomarker analysis offers considerable opportunities for individual and population risk measurement. these tests afford fast analytical turn-around time, quantitative measurement, accessibility, serial monitoring and ready availability. in some instances, rapid and continuous monitoring is available. the measurement of and changes to a broad range of modifiable risk factors, and biomarkers connected to immune system activity through cytokine surrogates, offers the most important opportunity for the prediction of disease and improvement in global chronic and pandemic disease status. most industries recognize the value of early problem intervention. in the waste management industry, for example, there is a clear hierarchy of: . source reduction, . recycling, . treatment, and . land disposal. in healthcare, there is also a potential for a four-tiered approach to health maintenance: . prevention, . mitigation of asymptomatic disease in people with elevated predictive biomarkers, . mitigation upon the earliest detectable signs of early disease (dry macular degeneration is an example), and . advanced root-cause mitigation approaches within disease management approaches. most of the efforts in today's healthcare is on disease management with usual care which is only a small part of this suggested four-tiered approach. the who addressed major causes of chronic diseases with modifiable risk factors being: unhealthy diet; physical inactivity; and tobacco use. in addition, the who stated "these causes are expressed through the intermediate risk factors of raised blood pressure, raised glucose levels, abnormal blood lipids, overweight and obesity. the major modifiable risk factors, in conjunction with the non-modifiable risk factors of age and heredity, explain the majority of new events of heart disease, stroke, chronic respiratory diseases and some important cancers. the relationship between the major modifiable risk factors and the main chronic diseases is similar in all regions of the world." studies show that the u.s. experiences the same risks as exist globally. dietary factors, alone, are associated with nearly half of all cardiometabolic deaths. the highest proportions of cardiometabolic deaths were estimated to be related to excess sodium intake, insufficient intake of nuts/seeds, high intake of processed meats, and low intake of seafood omega- fats. dramatic changes in disease rates among migrating populations indicate that the primary determinants of these diseases are not genetic but environmental factors, including diet and lifestyle [ ] . studies on twins separated at a young age corroborate that chronic disease is much more related to environmental factors. expansion of the depth and breadth of risk assessment and concomitant prevention and disease amelioration programs represent an unmet healthcare need. a well-studied disease prevention arena is corporate wellness programs. most of these programs rely on "usual care" that includes: basic dietary recommendations, weight loss, smoking, alcohol consumption and metabolic and lipid index targets. a broad-based team of wellness professionals and academics evaluated workplace wellness programs. they unanimously concluded that few wellness programs meet expectations and most are abysmal failures. what separates bad, good, and great programs is "a combination of good design built on behavior change theory, effective implementation using evidence-based practices, and credible measurement and evaluation." to further support the need for more thorough risk assessment, in a global study of risks, the authors concluded "increasingly detailed understanding of the trends in risk exposure and the relative risks for each risk-outcome pair provide insights into both the magnitude of health loss attributable to risks and how modification of risk exposure has contributed to health trends [ ] . these types of data clearly illustrate a path to improved health outcomes through broader and deeper precision and personalized assessment. the risk evaluation tool used in this study, the chronic disease assessment™ (cda), is an on-line health risk assessment and mitigation tool and involves answering approximately questions that probe deeply into lifestyle and environmental sources of risks, behaviors, health attitudes, readiness to change, current and past complaints, problems and diagnoses. the output of the cda is an overall risk score and then sub-sets of scores by risk categories, and a score for each question/answer combination. the overall raw risk score is converted into a letter "grade" reflecting the extent of the individual's risk "portfolio." the letter grade is provided to participants as an easily understood value for their risks, to overcome a lack of health literacy that especially impacts high risk populations. in addition, the cda output generates a series of actions that provide personalized education and actionable solutions to each risk in a participant's risk portfolio. finally, a health revival care plan™ is generated from the risk portfolio, and adjusted by the health coach and the participant, to create a simplified, personalized roadmap to overcome risks and improve health. a major impediment to health improvement is low health literacy. the de facto intervention perpetuating this problem is a prescription for symptom management that requires little knowledge by the patient. deficits in health literacy are associated with poorer health outcomes and higher health-related costs for both individuals and systems. improved health literacy has been associated with reductions in risk behaviors for chronic disease, and decreased rates of hospitalization [ ] . health literacy is a critical and under-examined component of health disparities. according to the national assessment of adult literacy, over a third of u.s. adults have basic or below basic health literacy and have difficulty managing common health-related tasks. limited health literacy poses a significant economic burden to our society, with national estimates indicating that low health literacy costs the u.s. healthcare system from $ to $ billion each year in healthcare dollars [ ] . the nexus of this program, including the cda risk portfolio, actions, care plans, and health coaching, is designed to meet and exceed the united states department of health and human services national action plan to improve health literacy's three goals: ensuring equitable access to health information; creating 'person-centered health information and skills' and supporting the development of the skills needed to attain and maintain good health. a final important aspect of this process, not articulated by the action plan, is illumination of the connection of risks to problems and complaints. the cda collects and reports risks, problems, and complaints together. thus, participants are able to "connect the dots" between risks and problems, like oral health and joint pain or carbohydrate intake and fatigue, as examples. these upstreamdownstream connection realizations improve health literacy and stimulate more sustainable change which manifests in the adoption of actions and plans to eliminate the risks as a solution to their problems as opposed to the usual care option of a drug to control symptoms. this process empowers individuals to be a participant in their own health improvement through recognition of their control over causes and outcomes. within this study, health coaches interacted face-to-face and electronically with participants and groups of participants to implement care plans. coaching activates patients to change through collaborative learning and social support. patient engagement and p medicine, defined as predictive, preventive, personalized and participatory, is an increasingly important component of strategies to prevent and reverse chronic disease, at least within the functional and integrative medical communities. interventions that tailor support to the individual's level of activation, and that build skills and confidence, are effective in increasing patient activation. more highly activated people are more likely to engage in healthy behavior such as eating a healthy diet and getting regular exercise while avoiding health-damaging behavior such as smoking and illegal drug use. these behavioral changes have led to lower rates of hospitalizations and emergency department visits, compared to less activated patients [ ] . a bridge between risk factors and modification in certain intermediate factors like blood pressure and obesity are changes to blood-based biomarkers, which are more objective measures of health. the most routinely performed tests in usual care are for the assessment of kidney and liver health, blood chemistry, lipid markers and metabolic markers. heart disease continues to be the number one cause of morbidity and mortality in the u.s. and globally despite the broad use of cardiovascular disease medications for both prevention and intervention. a study of , patients hospitalized with a heart attack between and showed that almost % had ldl cholesterol levels within guidelines [ ] . these data imply there is room for testing to augment evaluation of cardiovascular risk and cause. in older populations, "concentrations of homocysteine alone can accurately identify those at high risk of cardiovascular mortality, whereas classic risk factors included in the framingham risk score do not" [ ] . in healthy men, adding creactive protein levels to traditional risk factors, the reynolds risk score, improved cardiovascular risk prediction. the intermountain risk score uses common blood measures and assesses risk from the group of markers to develop a risk score. although limited in application, this scoring system has been reported to be predictive of increased mortality risk and provides patients with a more definable goal, the improvement of the score. more comprehensive assessments for risk and disease are emerging including the "allostatic load" and "inflammaging" concepts. each of these approaches considers a broader molecular view, rather than an organ system view of disease. according to mcewen, "when these (our body's) adaptive systems are turned on and turned off again efficiently and not too frequently, the body is able to cope effectively with challenges that it might not otherwise survive. however, there are a number of circumstances in which allostatic systems may either be over-stimulated or not perform normally, and this condition has been termed "allostatic load" or the price of adaptation" [ ] . diabetes is a relevant example, where insulin production is frequently elevated in response to regular highly absorbable carbohydrate intake. claudio franceschi coined the term "inflammaging" in to describe the concept of low-grade chronic inflammation and its impact on health. inflammaging was described as an extension of the "network theory of aging" [ ] . similar to the allostatic load, a global reduction in the capacity to cope with a variety of stressors and a concomitant progressive increase in proinflammatory status are considered the major characteristics of the inflammation aging process and susceptibility to premature disease and mortality. biomarkers for inflammaging are readily available and inexpensive but seldom obtain in usual care, especially in the implementation disease prevention strategies. according to gay et al., the allostatic load leads to dysregulation of the neuroendocrine system and subsequent elevation in inflammatory markers, leading to metabolic syndrome and chronic diseases such as cardiovascular disease [ ] . thus, the allostatic load and inflammaging are both measured, at least in part, with inflammatory markers like c-reactive protein, cortisol levels, glycosylated hemoglobin, white blood cell counts, and fibrinogen as examples. independent of inflammatory markers, multiple biomarkers, in general, improve the predictive power of a panel. in a study of people assessed with biomarkers, persons with multi-marker scores in the highest quintile as compared with those with scores in the lowest two quintiles had elevated risks of death and major cardiovascular events of . and . (adjusted hazard ratios), respectively [ ] . this far exceeds the predictive hazard ratio for cholesterol which varies from . to . depending upon the study [ ] . a hazard ratio of < means cholesterol levels were determined to be protective and stave off early mortality. numerous studies and reviews consistently show the value of multiple markers in real-world prediction of disease events and premature mortality. the chronic disease temperature™ (cdt) risk scale used in this study combines emerging concepts for improving the evaluation of disease risk and measurement of active disease. the significant attributes of the cdt scale are: . consideration of multiple biomarkers, . selection of markers based on traditional and new predictive markers based on inflammaging and the allostatic load, . harmonizing each marker to a standard endpoint -increase in early mortality risk, . consideration of risk contribution based on log-linear deciles of marker levels and individual marker hazard ratios for mortality, and . combination of the risk values from each marker into a single number score to accommodate limited health literacy and to set an understandable objective target for health improvement. the aggregate cdt score is an indicator of early mortality and associated total morbidity, while the values for each marker reflect both mortality risk and disease risk based on the association of a given marker to disease. this single number may be an important bridge to better health literacy as most patients do not understand the meaning of their current lab values. the cdt does not constitute a medical diagnosis of disease any more than does any individual marker, like homocysteine, but does statistically afford better predictive capability and measurement of disease progression or regression. the cdt output promotes the concept that health and disease lie on a log-linear continuum rather than being an on/off switch. in this study, the implementation of risk assessment, health and disease measurement, care plans, and frequent measurement leading to continuous improvement represents a needed response to challenges society faces from chronic diseases. this "systems approach" is designed to better connect across fragmented divisions in healthcare without bias of discipline. that is, fundamentally, most chronic diseases are connected at root-cause physiological processes. the ultimate goal is to create new risk/plan/action/outcome connections that facilitate learning opportunities and iterative advancement in treatment and preventative methods for chronic disease. another consideration is the order applied to the interventions including "in series" or "in parallel". an example is diabetes that needs to be managed for the prevention of heart disease, yet these diseases lie in different medical silos. the final objective ensures that workup of any individual patient, regardless of the presumed scope of the illness, embraces all possible causal factors. the purpose of this study was to assess the effectiveness and safety of this novel care model for the prevention and reversal of a broad spectrum of chronic diseases and complaints over a nine-month period. primary endpoints to assess the effectiveness of the intervention were changes in health risk assessment scoring, changes in documented health complaints, changes in medication usage, changes in vital signs, changes to individual blood-based biomarkers designed to measure chronic health, and changes to the multiple marker risk score, the cdt. we conducted an open-label, randomized, controlled, before-and-after nine-month study of a high intensity remote and on-site care intervention named a health revival process (hrp). participants included a group of individuals who, at the time, were employed by a mid-west fortune , manufacturing company with approximately , employees at that site. no formal control group was established but non-participant health status over the period was tracked using claims data for diagnoses, complaints, medication use, and healthcare spending. participation was voluntary and recruitment started in november of , implemented by our company and the employer, focused on higher claims and more chronically sick individuals who were motivated to overcome unresolved chronic health issues. no rigid participation inclusion criteria were used other than each individual had at least one diagnosed chronic condition, was formerly or currently on a medication for a chronic disease, and was a high healthcare claimant (> $ /year currently or within the past three years) if that data was available. not all participants had claims data from previous years mainly due to their health plan and choice or employment history with the company. from those interested in the program and met the criteria, retrospective health data (medical claims) were reconciled to finalize the -person cohort without consideration for a specific type of condition. although not a formal clinical study, all procedures performed in the program involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. ethical oversight was provided by the existing primary care clinic management organization, but not under any formal written agreement other than to monitor for patient safety. informed consent, medical releases, and participation contracts were obtained from all participants included in the program. these documents were completed after each participant was provided detailed information about the program. all data were acquired in strict conformance with health data privacy laws by medical personnel and all stored data were contained on a health insurance portability and accountability act (hipaa) compliant cloud. the health revival process: participants in the health revival process (hrp) underwent medical history review, completed a +/-question online health risk assessment (chronic disease assessment™ (cda)) and laboratory testing for serum-based biomarkers (chronic disease temperature™ (cdt)). participants without a medical exam within the past months had one perform by our doctor, to determine their baseline health and risk status. all data obtained on participants, including problems, diagnoses, health complaints, reconciled medications, vitals, food journal, and other measurements were entered into our proprietary health revival software. upon qualifying, hrp participants began one-on-one health coaching encounters. the initial coaching session included active listening by the coach and reconciliation between the output of the cda risk assessment and health concerns, problems, and information articulated by the participant at that first encounter. any discrepancy between the responses to the cda questions and information presented to the health coach were verified and the cda was appropriate updated and annotated. the health coach, using the recommendations promoted by the up-to-date hrp software record, developed a personalized health roadmap referred to as a person's health revival care plan™ (hrcp). our medical doctor reviewed and finalized that document and then conferred with the coach on next steps appropriate for the participant. our doctor and the non-hrp medical doctors ensured that the recommendations and suggestions made by the health coach did not violate the health coaches license to provide advice. there was reasonable fidelity to the hrp delivery as everyone saw the same coach and doctor over the same time period. however, participants experienced different levels of coaching and doctor intensity based on the extent of their risk portfolio and medical needs. participants had continuous access to their health information, suggestions, and progress within the hrp software which maintained much of their personal health information. specifically, a participant could follow, track, and monitor changes in health measures and interact with specific, personalized content (written, audio, and video) curated automatically by the hrp system based on their personalized health information inputs including, their cda risk assessment, cdt markers, and vital signs recorded in the system. in addition, the participant portal included their personalized hrcp that created the structure for their hrp. participants were able to work with their health coach on the plan, follow the plan through a selfguided process, or some hybrid between the two paths. the health coach was able to monitor the self-guided process through system feedback that included reports on system logins and completed "actions" and "goals." completed actions occurred after a participant accessed content relevant to a health risk determined by the hrcp and choose a selection after reviewing the information like "completed" or "deferred." most participants relied on the health coach for structure, direction, and motivation. figure shows features of the patient portal health revival dashboard. participants in the hrp retained their existing providers for acute disease management and routine medical checkups. care coordination between existing providers and hrp care team occurred as needed. in particular, the hrp doctor and pcp discussed any possible interactions between supplements, lifestyle changes, and current medications. of frequent concern was a need to change the dose or use of insulin, other diabetic medications, blood pressure, cholesterol, and corticosteroid medications as the participant's health improved through lifestyle modification. frequency and type of biomarker tracking, beyond the before-and-after cdt labs, were individualized by the hrp doctor on the basis of care needs and progress as recorded by participants in updated cdas and health coaches in updated hrcps and coaching notes. participants on insulin were contacted at least weekly to assess any potential for hypoglycemia. either the pcp or the hrp doctor made medication modifications. chronic disease assessment: the cda was available electronically through a web browser and included +/-questions through a series of shorter surveys, with the participant able to stop and restart the survey at any point where they left off. each question included fixed answer choices from a single option to as many as options depending upon the scope of the question. in some instances, a question allowed for multiple answer selections while in others, only a single response could be recorded. each question/answer combination was given a risk score and assigned one or more attributes of risk based on our study of the medical literature for potential outcomes associated with the specific behavior attribute. the cda included consideration of common risks and disease root causes determined from our own clinical experience and published clinical case studies, population and randomized clinical trials. for example, the cda gave considered to oral health, gut health, eye health status, macro-and micronutrient imbalances, and indications of chronic and occult infection. the output of the cda was a raw score that converted to a letter "grade" reflecting the extent of the risk "portfolio" determined by the assessment. each letter grade, a-f, spanned a range of raw scores. the purpose of the letter grade was to convert a numeric value into a more meaningful and recognizable risk level. in addition, the software interface outputted an action or a series of actions for many of the question/answer pairs, through if/then/else logic. the actions are those known or believed to lead to a reduction in that risk based on peer-reviewed published studies or our own experience. the actions are bundles of content that explain the risk or potential risks and offer suggestions to ameliorate the risk. all this content was made available to participants through their health revival dashboard (hrd). example questions from the cda are provided in figure . chronic disease temperature: routine labs and the cdt biomarker panel were drawn at the on-site clinic by non-hrp technicians working for the primary care provider company. the labs drawn on each individual and the thresholds for chronic health considerations are described elsewhere [ ] . in summary, reference intervals were not used to determine health status and risk. instead, our team of biostatisticians reviewed the medical literature to determine the threshold level or levels for each biomarker, they indicated a statistically significant increase in early mortality associated with that marker. each marker was assigned escalating risk based on log-linear curve fitting to published information on mortality risk and biomarker raw value. many of the cdt biomarkers are common biomarkers with some being less commonly obtained in usual care. the ordering doctor of record was held responsible for ensuring any participant with an abnormal lab value, based on usual care reference intervals, notified the participant and arranged for appropriate care to correct the abnormal value. the "tighter" thresholds used as part of the cdt labs were used only by the hrp doctor and not used for making a medical diagnosis. instead, this more sensitive scale of normal vs abnormal lab values was used to measure changes in participant's physiology concomitant with lifestyle changes. this more sensitive scale for each biomarker and the overall cdt value, helped assess participant's health trends by recognizing that disease is not an on/off switch, but rather a continuum. for example, and hba c level of . %, although not a diagnosis for type diabetes, is a strong indicator of future type diabetes. a goal value of < . % was established for all participants to optimize insulin sensitivity and avoid future diabetes risk, assuming that value was obtained through lifestyle improvement and not pharmaceutical intervention. this logic was applied to all cdt markers. the interventions affected by the hrp through the hrcp were individualized to each participant and included a consideration of the participant's readiness to change and the likely sustainability of any given change as determined by their responses to chronic disease assessment questions and discussions with their health coach. the intensity of coaching was predetermined, but not fixed, by the cda grade. the coaching time allotted to each participant based on the cda grade is provided in table . doctor time allocation was approximately / th coaching time. the main risk considerations were a reduction in inflammation through: more movement, increased nutrient density of foods, improvement in digestion/absorption by improving gut balance and activity, increasing probiotic and prebiotics foods, elimination of high glycemic foods, better oral health maintenance, increased intake of healthy fats and omega- fatty acids, increased micronutrients to support hormone production, stress reduction, brain health through reducing whole-body inflammation, and consumption of greater amounts of fat-soluble vitamins, as examples. no specific nutrition program was recommended to the participants as a whole. instead a simple process of substitution of one food, considered of low nutrient value for another of higher value, was made as recommended by the health coach. recommendations were made on an individualized basis based on participant preferences, to affect a gradual and sustainable change from the standard american diet (sad) that was prolific throughout the cohort, to a new food consumption pattern with increased micronutrient density, fiber, and fat with less carbohydrate and sugar consumption. short term (one to three months) nutritional ketosis was suggested for a few highly insulin resistant and diabetic patients but no participants fully achieved nutritional ketosis during the nine-month period. however, these participants achieved a significant reduction in total carbohydrate consumption with a shift to low glycemic index food and those containing higher concentrations of marine, monounsaturated, and saturated fats. supplements were provided as part of the program and compliance with supplementation was near % based on self-reporting and resupply orders. at the outset of the program, after evaluation of cdt labs, cda reported dietary information, and food journals, the participants were provided any or all of the following supplements based on individually assessed deficiencies: multivitamin/mineral ( qd); vitamin d ( - , iu, qd); cod liver oil ( - g, qd); magnesium glycinate ( - mg, qd); vitamin k ( mcg, qd); probiotic ( - billion organisms, qd). supplements were provided only when deemed appropriate by the hrp doctor and were phased in then phased out over the nine-month period as the doctor determine that nutritional deficiencies or insufficiencies were being mitigated by the program. the purpose of the supplements was to quickly overcome apparent nutritional deficiencies. as part of the hrcp, foods that contained nutrients provided by the supplements were recommended and, when adopted, enabled the gradual elimination of supplementation without compromising nutritional needs. the main behavioral change strategy, executed by the doctor and coach care team, was to slowly and gradually ease a participant into change. the frequency of coaching sessions, dictated by the need of the participant (cda letter grade), including their burden or disease and risk, and their motivation, was adjusted to improve compliance. most participants suffered from some health ailment that impacted their daily wellbeing. improvement in general wellbeing, which started to be noticed by participants by the end of month , provided the motivation to continue to adopt gradual modifications to lifestyle. to help participants understand that the hrp is not a quick fix, our coaches explained that we have determined a general "rule of thumb" for the time required to improve health significantly. if a disease, like diabetes, has been slowly developing over five years, it will take at least five months of effort to reverse the disease, assuming the interventions are appropriate. outcome measures: in-clinic vital signs, health risk assessment (cda) risk score and list, and biomarkers were obtained at baseline and at the end of the -month program. problems, complaints and medications were reconciled at each health coaching and doctor encounter. fasted and non-fasted blood draws were obtained by clinic pcp staff using routine procedures. samples were provided to and analyzed by quest diagnostics using standard operating procedures. primary outcomes were: changes in biomarker values; risk scores; reported diagnoses; vital signs; weight; and medication use. secondary outcomes included reported complaints, for example, lack of energy, chronic pain, sleeplessness, mood issues, and general lack of wellbeing. the baseline demographics of the final hrp participants are presented in table . all participants were caucasian of european heritage. at baseline, % of hrp participants were actively taking pharmaceuticals for a medical problem and % were diagnosed with at least chronic condition. this reflects a substantially higher percentage of chronically ill individuals compared to the u.s. national average of % of u.s. adults having at least one chronic condition. on average, the group was taking . prescriptions per person. the major class of medications included: diabetes medications, injectable insulin, statins, blood pressure lowering, pain, mood (ssris), bisphosphonates, steroids, thyroid hormone, and proton pump inhibitors. the final participant number of was established after left the program. two were dismissed from the program for compliance reasons, four left early to join a weight loss program, two left because of the time commitment, and two left due to potential interactions between current medications and supplements as encouraged by their pcp, for a total of . %. there was no clear demographic trend between those who remained in the program versus those who dropped out. chronic disease assessment™ (cda): on average, the -participant cohort lowered their cda score by points ( %) from a raw value of to a new value of over six months. each point lowered reflected a reduction in a disease risk or resolution of a health problem or complaint. risks were scored on a - scale, with representing a minor risk or problem and representing a major risk or problem. the initial population cda grade was d+, assigned based on a range of raw numeric scores calculated from the survey, and the final grade after months of the hrp was c+. ninety-four percent of the group experienced an improvement in their risks while % of the cohort experienced a worsening of their cda grade ( figure ) . participants did not have their initial cda answers to refer to when they retook the assessment six months into the program. chronic disease temperature™ (cdt): on average, the -participant cohort lowered their cdt score from . to . . the cdt is based on a "degree" scale calculated by adding the risk contribution from each biomarker to . to arrive at the participant's cdt. the average chronic disease risk reduction in cdt was % (figure ). all % of participants whose cda grade worsened also experienced an adverse change in their cdt score. three participants improved their reported cda grade but witnessed an adverse change in their cdt. the cdt included several markers that are classified as "acute phase" reactants. in "acute-phase proteins and other systemic responses to inflammation," the authors explain that markers of chronic systemic inflammation are also subject to change acutely [ ] . for example, c-reactive protein elevates during the acute phase of pneumococcal pneumonia. c-reactive protein has an acute phase relaxation half-life of approximately one day upon removal of the insult whereas the half-life of fibrinogen is about one week. two of the three participants with an improved cda but worsened cdt experienced adverse physiological changes due to documented acute circumstances. retest was not available during the nine-month program to confirm our suspicion about the cause of the elevation. one recently underwent surgery and was recovering slowly. another participant was receiving ongoing treatment for a complex acute condition managed by the patient's pcp. other participants whose cdt worsened were in the hrp and a calorie-restricting weight loss program administered by third parties. the weight loss program was calorie counting-based with no guidance provided on macro and micronutrients. these individuals, although in the hcp, were less flexible to our coach's dietary suggestions because of the calorie restriction. for example, a participant refused to take cod liver oil because each capsule contributed approximately calories to their daily calorie allotment. our results demonstrated, in this small subgroup, that people in poor health and with a highly elevated cdt, confirming their health status objectively, may be contraindicated for a sustained calorie restriction program without nutritional guidance. although the literature is rich in studies suggesting that calorie restriction improves lifespan and reduces inflammatory markers, emerging studies emphasize that calorie restriction must be implemented without malnutrition that comes from low nutrient-dense foods processed foods. macro-and micronutrient intake of all participants was monitored with a food journal. worsening in cdt markers in people on a calorie-restricted diet correlated to micronutrient malnutrition exacerbated by reduced total calorie intake. malnutrition status was established in these individuals by determining their daily nutrient recommendation from the dri calculator for healthcare professionals provided by the usda and comparing the results to nutrients consumed based on available nutritional labels for foods consumed. there are numerous studies on the association between lifestyle behaviors and chronic disease risk. in large prospective studies, like the nurses' health study, vague conclusions are made about the association of smoking, regular physical activity, maintaining normal body mass index, eating a healthy diet and chronic disease proliferation [ ] . the individualized cda risk values potentially increase the precision, personalization, and accuracy of risk-to-disease relationship measurement. figure provides a view of the change in the cda risk score and its relationship to the cdt value for the biomarker panel at the beginning and end of the -month hrp program for the entire population. the same data are presented in figure as bubble chart with the before and after data superimposed on the same scale. temperature score) before and after six months of the hrp program notable is the reasonably smooth relationship between the two risks scores, the subjective cda and the more objective cdt. we conclude, from these data, reducing the most basic health risks, over time, may lead to a reduction in cytokine burden with often concomitant change in diagnosed chronic diseases. increasing the "n" in our database and making appropriate adjustments to assigned subjective risk values within the algorithm offers the potential to improve the correlation between determinants of health risks and physiological health status. the individualized statistics for the cda, cdt, and biomarkers comprising the cdt to evaluate participants health and risk are provided in table . score mean before mean after mean difference standard deviation t test value cdt is the chronic disease temperature biomarker score as a relative value with . considered optimal and elevated values indicating chronic risk; hba c is expressed as a %; glucose is expressed as mg/dl; nlr is the neutrophil to lymphocyte ratio; hs-crp is high sensitivity c-reactive protein or c-reactive protein, cardiac expressed as mg/l; insulin is expressed as uiu/ml; hdl is expressed as mg/dl; triglycerides are expressed as mg/dl; vitamin d is expressed as ng/ml; uric acid is expressed as mg/dl; wbc is the white blood cell count expressed as cells/ul; rdw is the red blood cell distribution width expresses as a %; ab neutrophils are neutrophils (absolute) expressed as cells/ul; esr is the sedimentation rate-westergren expressed in mm/hr; fibrinogen is fibrinogen activity expressed as mg/dl; homocysteine is expressed as umol/l; and aip is the atherogenic index of plasma expressed as the log(triglycerides/hdl) glycosylated hemoglobin (a c): a c, a -day retrospective average of blood glucose, contributes to an assessment of metabolic risk along with fasting glucose and insulin. a current therapeutic goal in usual care is to lower the a c value of diabetics, those with a c values above . %, with pharmaceuticals. the accord study shows that tight pharmaceutical control of blood sugar in those with severe insulin resistance suffer a significant increase in adverse cardiovascular events and mortality compared to those with less tight control [ ] . lifestyle interventions offer another approach to glycemic control and does so without risk of hypoglycemia and other side effects of the pharmaceutical approach. in the cohort of , none had optimal a c levels, defined as . %- . %. even a . % increase in a c above % increases the year risk for diabetes (odds ratio > . ) and the risk of diabetes increases exponentially with a c. in the participant cohort, at the end of the hrp, % (all but ) lowered their a c value ( insulin is the most sensitive marker for early metabolic risk because it increases first as an individual becomes insulin resistant. even values slightly above normal, and well below a diagnosis of diabetes, contribute to serious chronic diseases in the future, including alzheimer's and cardiovascular disease. type diabetes is associated with increased risk of cancer. hyperinsulinemia (elevated insulin levels) and insulin resistance are apparently the link. in a -year mortality study, individuals in the highest quintile of serum insulin had a % higher risk of cancer mortality and a % higher risk of gastrointestinal cancer mortality [ ] . the authors of this study concluded that hyperinsulinemia/insulin resistance is associated with cancer mortality independently of diabetes, obesity/visceral obesity and metabolic syndrome. in the -person cohort, participants ( %) were at elevated metabolic and associated chronic risk. six of the ( %) experienced a double digit drop in fasting insulin, of ( %) dropped from the high-risk category to a lower risk level, ( %) lowered insulin levels sufficiently to reduce their cancer risk severity category, ( %) changed little and stayed in the same risk category and move up one risk category ( [ ] . in the -person cohort, % of participants who were at high risk for cardiovascular disease, based on hs-crp level > , lowered that risk ( table ) . [ ] . the aip average value before the hrp was . and lowered to . at the end of the hrp. conventionally the rdw test, which is a part of a complete blood count, is used to help determine anemia status. however, it is also a marker of inflammation and often tracks with crp. red blood cells elongate and deform when flowing through capillaries, which may explain the association between red blood cell widths, vascular inflammation, and increased cardiovascular morbidity and mortality. in the -person cohort, % of participants at elevated risk for cardiovascular disease based on rdw levels lowered their risk ( table ) . white blood cell counts (wbc) is a predictor of strokes, heart attacks, and fatal heart disease. in the women's health initiative involving , women from - years of age, those with approximately , white cells per ml had more than double the risk of fatal heart disease than women with cells per ml [ ] . white blood cell counts in the normal range for acute indications are now more widely recognized as a predictor of adverse chronic outcomes. in the -person cohort, three individuals had high cardiovascular disease risk based on wbc levels, and % lowered that risk through lifestyle modifications. in addition, % of those with moderate risk moved to either low or very low risk as assessed by risk quartiles for wbc. in general, % of participants moved from a high to a lower risk status ( table ) . multiple studies show a significant inverse relationship between -hydroxy vitamin d (d ) status and cancer mortality. in a fifteen-year study of nearly , participants, an increment increase of ng/ml was associated with a % reduction in total cancer incidence, % reduction in total cancer mortality and statistically significant reductions in colorectal, pancreatic, esophageal, oral, and pharyngeal cancer mortality [ ] . for cancer, optimal d levels are above ng/ml. at the start of the program, eight participants had optimal levels and that number increased to by the end of the program. insufficient vitamin d, as defined for bone health are values below ng/ml. initially there were participants insufficient for blood d and none were insufficient at the end of the program. the population d levels went from to ng/ml, on average, by the end of the hrp. these data indicate a high degree of compliance with the program recommendations as the increase in d status was largely attributable to consistent supplementation. in general, the increase observed required daily supplementation of iu d daily ( table ). the neutrophil-to-lymphocyte ratio (nlr) is reported to be a robust outcome prognosticator in existing solid tumor cancers. in a study on breast cancer, patients with an nlr > . had substantially higher oneyear and five-year mortality rates compared to those with an nlr < . . the nlr value has similar predictive ability for cardiovascular mortality [ ] . in the cohort of , had nlr above the threshold for adverse cancer outcomes. sixteen of ( %) saw their nlr ratios return to very low risk (normal values) by the end of the program ( and associated changes to cancer and cardiovascular mortality prognosis medication prescription reduction was achieved as part of the outcome measurement. the cohort experienced a % reduction in medication usage, reduction in dose in %, and an avoidance of two costly medications. the prices included in table , below, where the actual pharmacy costs realized by the health plan and did not include any co-pay. the cohort experienced a reduction in chronic disease burden. chronic disease reduction was determined by changes in any of the following: actual change to a medical diagnosis, elimination of a medication associated with an existing diagnosis, changes in a vital sign that indicated a migration out of a diagnosis that was affected without the use of a medication, or change in a biomarker value or values that were initially used to make the diagnosis into a "normal" range without the use of medications (table ) . this participant presented with major risk factors and complaints including lack or exercise, fast food diet, high carbohydrate diet, daily high fructose corn syrup containing beverage consumption, use of omega- containing oils in cooking, statin drug daily prescription for primary cardiac event prevention, severe arthritis, psoriasis, and cataract. the severity of the psoriasis and her job function put her at risk of imminently going on disability. she had seen multiple specialists, was placed on antihistamines and topical steroids but her psoriasis condition continued to worsen. the next treatment option for her was to be etanercept which she declined pending the outcome from the hrp. she indicated that she had not washed her hands without pain in over a year. the hrp included -minute semi-monthly health revival coaching following the participant and care team agreed upon care plan. cholesterol-lowering drug usage was eliminated in the first month as directed by our medical director. health coaching focused mainly on food substitutions, increasing activity, value and use of supplements, a limited set of supplements, and additional care to her oral hygiene. after six months of intensive health revival coaching, many risk factors and complaints, revealed on her cda report, were either removed or reduced including nagging chronic pain. her main complaint, debilitating psoriasis slowly, but completed resolved in five months ( figure ) . however, the first signs of improvement in her psoriasis condition did not appear until month of the program. normally, in the case of autoimmune diseases like psoriasis, food sensitivities or allergies must be addressed. this participant was unwilling to eliminate some of the common allergens like gluten and dairy. she was placed on a modest supplement regiment based on nutritional deficiencies determined from food journaling, including: cod liver oil ( g/day); vitamin d ( , iu/day) and a multivitamin/mineral supplement (taken per label instruction), and the other general supplements included in the "methods" section. positive changes in lab values included: -hydroxy vitamin d status ( to ng/ml); white blood cell counts ( , to , ) ; rdw ( . % to . %); and fibrinogen ( to mg/dl). case : rheumatoid arthritis and type diabetes - -year-old male factory worker with a high school education (table ) . over the first five months, the participant lost pounds through a reduction in carbohydrate consumption, but with no significant change in daily calorie intake. the participant embarked on a substitution diet where, over five months, gluten-containing foods were removed from his diet and replaced with vegetables and marine-and animal-based fats. he was also put on a modest supplementation program including cod liver oil ( g/day); vitamin d ( iu/day); magnesium glycinate ( mg/day); vitamin k ( mcg/day) and a multivitamin/mineral (per label instruction). his type diabetes was reversed as illustrated by his a c dropping from . % to . % and his fasting glucose dropping from to < mg/dl. his pain was substantially eliminated, based on a subjective pain score of / initially, to / . his ra improved to enable him to be able to bend his fingers into a full fist for the first time in over five years (figure ). this participant reported with a severe autoimmune disease, polychondritis, that produced monthly painful flares in cartilage above her shoulders including her ears and eyes. long-term use of steroidal antiinflammatories were implicated in the cataracts and a breast lump that was removed surgically. the cataracts had progressed sufficiently to cause her to be on disability and be unable to drive a car. cataract surgery was not an option due to the severity and unpredictability of eye flares that could cause extremely adverse outcomes if they coincided with surgery. she had seen several specialists including local rheumatologists, natural doctors, and doctors from cleveland clinic with no relief to her condition. she had researched polychondritis on her own, prior to joining this program and eliminated gluten and dairy from her diet but this change did not alter the disease severity or frequency. this participant had made significant changes in her lifestyle prior to this program as reflected in her cda grade, but these changes were insufficient to improve her blood biomarkers indicated by the high cdt value of . , indicative of serious health risk and poor prognosis. our health revival process guided her to continued better choices and involved semi-monthly -minute lifestyle coaching. the main changes made over a six-month period included: increasing healthy fats, reducing carbohydrate intake, increasing micronutrient density, stopping nicotine dependence, improving digestive health with optimizing food choices including increasing stomach acid status, and repopulating gut microflora. at month in the program, her eye and ear flares had subsided sufficiently to allow for a meaningful reduction in eye and oral steroids, ( mg/day to mg/day prednisone). in addition, she was able to have successful cataract surgery which enable her to start driving again, and return to work, both of which were curtailed over one year. the polychondritis may never be cured, however, with appropriate lifestyle management, it is no longer impacting her quality of life. prevention and reversal of chronic and non-communicable diseases continue to be a largely unmet need. a fresh approach is clearly warranted to curb this global scourge. one impediment is the lack of precision and personalization of risk with "poor diet" as an example. and there is a lack of measurement of a broad array of minor, yet important, risks that can easily be overcome. the same suite of risks is continually presented to individuals who historically have not been able to modify or overcome them, with smoking or alcohol consumption as examples. according to khullar in, "we're bad at evaluating risk, how doctors can help," a broader approach involves helping patients systematically identify what's important to them, and based on these goals and preferences, suggesting to them how to think about their options [ ] . this logic is best applied across the entire time-line that defines the slow and insidious development of chronic disease. it starts with lifestyle decisions and habit development early in life that perpetuate into mid-life and then into old age. measurement and a proper medical "workup" regardless of presumed health status is a key strategy and potential motivating factor that is currently lacking. changes in chronic disease biomarkers in asymptomatic people may afford an early warning sign of stealth changes to which many may respond. pathology changes, identified with advanced diagnostics, which generally develop after a long incubation period detectable with proper biomarker evaluation, may facilitate change in the more recalcitrant. each individual has their own motivations. thus, providing patients with an array of choices and recommendations along the health/disease continuum has a higher probability of inciting action and improving outcomes or preventative actions. this study evaluated a new population risk and health assessment and mitigation system where measurements of risk and disease were made across the disease continuum by using finely tuned biomarkers and risk assessment. the output was a broad-reaching care plan assembled through integration of current health survey results, biomarkers, problems, complaints, medications, vital signs, verbal input from the participant to the health coach, and contributions from the care team. the remediation path to improved health developed as a consensus between the participant and care team, of agreed upon steps and actions, that were malleable as the process moved forward. adjustments were made based on participant preferences, success and failures, a solid health data. according to khullar, "patients need to understand their values but also their possible futures. the idea is not to reduce uncertainty, but to help patients clearly envision what life would look like in one outcome versus another, and to better prepare them for the various futures that might unfold." this program was designed to give participants options beyond the management of disease once it has struck. and it included regular monitoring and concomitant course adjustments to help participants attain their goals. this study prospectively observed adults with chronic conditions and unresolved health complaints that remained unresolved under usual care management and treatment. following six months of hrp, participants achieved subjective and objective improvement in health status with % seeing a reduction in multiple blood-based biomarkers and % achieving a reduction is a broad measure of determinants of health risk factors. concurrently participants reported weight loss ( % of the total and % of those with a reported weight loss goal), reduction in reported pain, sleeplessness, memory issues, heartburn, skin rashes, migraines, and daily fatigue. the diabetics in the program had all progressively worsen over the previous two years, as measured by fasting glucose, hba c, and medication usage and all improved under the hrp program. hrp meaningfully improved hba c, fasting insulin, neutrophil-to-lymphocyte ratio, hs-crp, vitamin d, white blood cell counts, red blood cell distribution width, absolute neutrophils -all part of the cdt panel. in addition, hdl, fasting glucose, triglycerides, gfr, atherogenic index of plasma (aip) liver enzymes, and blood pressure improved is most participants with initial abnormal values. aip is emerging as a valuable representation of increased mortality risk. improvement in this lab value ratio was consistent with previous studies using carbohydrate-restricted interventions. however, although the hrp included some level of carbohydrate restriction, this was not a mandate and carbohydrate consumption goals were not set. instead, participants were afforded broader options that met each at their level of readiness to change and did not overwhelm anyone with unachievable objectives. in general, small swap-out suggestions were agreed upon at each encounter. the "pure" study reports are a set of studies that describe components of the nutritional approach used in the hrp. in "fruit, vegetable, and legume intake, and cardiovascular disease and deaths in countries (pure): a prospective cohort study," fruit, vegetable and legume consumption recommendations were - g/day to achieve maximum benefit at reducing non-cardiovascular and total mortality [ ] . the hrp coaches encouraged consumption of three to four servings of these foods per day, focusing on lowest glycemic index choices. in "associations of fats and carbohydrate intake with cardiovascular disease and mortality in countries from five continents (pure): a prospective cohort study," healthy fats were found to be indicated for a reduction of total mortality risk and saturated fats were shown to be inversely associated to stroke risk. the hrp coaches guided participants to swap out carbs, sugars, and some protein in favor of healthy fats in foods and cooking oils with emphasis on increasing saturated, mono-saturated, and marine fats. obtaining nutritional ketosis for a two to three months window was suggested for all the diagnosed type diabetics; however, none achieved sustained ketosis but their metabolic markers indicated improvement of their diabetic conditions during the -month hrp. this suggested that the broader, more personalized risk reduction approach of this hrp, compared to strict carbohydrate restriction, affords metabolic profile results without the potential risks associated with carbohydrate starvation in insulinresistant subjects. reducing whole body inflammation was the primary objective of each encounter, not just reducing the glycemic value of food. examples included switching out proinflammatory for anti-inflammatory cooking oils, lowering glycemic value and load of substituted foods, reducing frequency of fast food consumption, improving oral hygiene, managing stress, establishing better sleep and rest patterns, enhancing hydration, improving micronutrient density of foods consumed, establishing more frequent movement routines, and consuming more gut-supporting foods. the regular health coach encounters that included reviewing risk factors, vitals, and medication usage, with doctor supervision, may have provided behavior reinforcement. further, it is plausible that this multi-risk amelioration care model allowed from both broader and greater adoption and improvements compared to interventions focused on fewer factors. in this hrp we effectively leveraged credible measurement and evaluation, linked these findings to participant's unresolved and nagging health complaints, and facilitated behavioral change leading to health improvement in most participants. the program did not rely only on usual care measures of health. participants were not confronted with high hurdles to health improvement that often discourage engagement. instead, the program centered around meeting a person at their level of readiness and capitalizing on small triumphs that eventually led to measurable health improvements recognized by the individual that led to a cycle of improvement rather than deterioration. no episodes of adverse events were attributable to the hrp. one insulin-dependent type diabetic participant showed a sudden increase in fasting insulin, from . to μu/ml, which was reported to his pcp for medication adjustment. several participants reported dizziness and either the hrp or pcp lowered their blood pressure medication dose that, in all cases, resolved the complaint. prior studies have demonstrated favorable cost reductions in broad-based wellness and disease management programs. most of the cost-saving and health maintenance were attributed to the management of existing disease rather than prevention and required a strong evidence-based approach. a strength of this hrp was an emphasis on root-causes of and reversal of disease rather than just case management. additionally, this study reflected a real-world workplace environment with a distribution of both white-and blue-collar workers participating and with a range of diseases and aliments. weaknesses included a lack of a representative control group, single location and participants were mostly caucasian. the study was not of sufficient size and duration to measure hard endpoints including mortality and adverse health events. future studies of this nature could include multisite randomized controlled trials with greater racial and ethnic diversity, and longer duration. this highly personalized and scalable health revival study protocol demonstrated that a broad array of chronic health complaints and problems can be controlled and reversed by methodically eliminating seemingly small lifestyle-induced health risks. it also demonstrated that the lifestyle risk tool, the chronic disease assessment™, and the biomarker panel, the chronic disease temperature™, that were used to develop care plans, changed in correspondence with participant-and medical staff-reported health improvements. therefore, these tools may be valuable for the measurement and mitigation of chronic disease risk and chronic diseases generally. importantly, the implementation of this program is low cost, using inexpensive on-line survey tools, biomarkers, and health coaching. additionally, this program is well suited to be implemented in large populations through surveying, obtaining labs through national networks, and performing group coaching sessions based on common risks identified through the risk assessment tool. more studies using this overall hrp approach are required to validate the measurement methods, processes, and outcomes. this approach offers a potentially important health delivery modality in a world with escalating chronic disease morbidity and mortality. acquired in strict conformance with health data privacy laws by medical personnel and stored and managed in a hipaa compliant emr. animal subjects: all authors have confirmed that this study did not involve animal subjects or tissue. conflicts of interest: in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. global status report on noncommunicable diseases. world health organization (ed): world health organization the us health disadvantage relative to other high-income countries: findings from a national research council/ institute of medicine report heart disease strikes back across the u.s even in healthy places multiple chronic conditions in the united states consistently high turnover in the group of top health care spenders the cytokine storm and pre-cytokine storm status in covid- -a model for managing population risk for pandemics and chronic diseases association between platelet parameters and mortality in coronavirus disease : retrospective cohort study. platelets. disease control priorities in developing countries. nd edition. the international bank for reconstruction and development/the world bank global, regional, and national comparative risk assessment of behavioural, environmental and occupational, and metabolic risks or clusters of risks, - : a systematic analysis for the global burden of disease study effects of health literacy on health status and health service utilization amongst the elderly is patient activation associated with future health outcomes and healthcare utilization among patients with diabetes lipid levels in patients hospitalized with coronary artery disease: an analysis of , hospitalizations in get with the guidelines use of framingham risk score and new biomarkers to predict cardiovascular mortality in older people: population based observational cohort study stress and the individual: mechanisms leading to disease . arch internal med network theory of aging meeting physical activity guidelines is associated with lower allostatic load and inflammation in mexican americans multiple biomarkers for the prediction of first major cardiovascular events and death atherogenic index of plasma and triglyceride/high-density lipoprotein cholesterol ratio predict mortality risk better than individual cholesterol risk factors, among an older adult population quarterback your own health -how to take and lower your chronic disease temperature acute-phase proteins and other systemic responses to inflammation diet, lifestyle, biomarkers, genetic factors, and risk of cardiovascular disease in the nurses' health studies the association between symptomatic, severe hypoglycaemia and mortality in type diabetes: retrospective epidemiological analysis of the accord study insulin resistance/hyperinsulinemia and cancer mortality: the cremona study at the th year of follow-up rosuvastatin in the primary prevention of cardiovascular disease among patients with low levels of low-density lipoprotein cholesterol and elevated high-sensitivity c-reactive protein: rationale and design of the jupiter trial leukocyte count as a predictor of cardiovascular events and mortality in postmenopausal women: the women's health initiative observational study prospective study of predictors of vitamin d status and cancer incidence and mortality in men the predictive value of elevated neutrophil to lymphocyte ratio for long-term cardiovascular mortality in peripheral arterial occlusive disease we're bad at evaluating risk. how doctors can help . the new york times fruit, vegetable, and legume intake, and cardiovascular disease and deaths in countries (pure): a prospective cohort study the authors thank the invaluable assistance of dr. michael l. carter, dr. trent austin, and jasmin lewis human subjects: consent was obtained by all participants in this study. neco irb issued approval t.the neco irb has approved this study as it was conducted in conjunction with routine clinical practice. all procedures performed in the program involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. ethical oversight was provided by the existing primary care doctors who were not part of the program. informed consent, medical releases, and participation contracts were obtained from all participants included in the program. these documents were completed after each participant was provided detailed information about the program. all data was key: cord- - wt z r authors: porcelli, brunetta; verdino, valeria; bossini, letizia; terzuoli, lucia; fagiolini, andrea title: celiac and non-celiac gluten sensitivity: a review on the association with schizophrenia and mood disorders date: - - journal: auto immun highlights doi: . /s - - - sha: doc_id: cord_uid: wt z r an association between many psychiatric and gluten-related disorders has been known for some time. in the case of schizophrenia and mood disorders, the major psychiatric disorders, there is much evidence, not without contradictions, of a possible association between schizophrenia and celiac disease. the association between mood disorders and gluten-related disorders, especially celiac disease, has only been studied for depression, often coupled with anxiety, and very recently for bipolar disorder. since non-celiac gluten sensitivity is now known to be different from celiac disease, many studies have shown that gluten sensitivity is also associated with major psychiatric disorders. here we review the literature on the association between schizophrenia/mood disorders and celiac disease/gluten sensitivity, pointing out the differences between these associations. an increased rate of autoimmune diseases has been demonstrated in individuals with psychiatric disorders such as schizophrenia (scz) [ , ] and mood disorders (md) [ , ] . individuals diagnosed as having an autoimmune disease are also likely to be at increased risk of psychiatric disorders. thus, the involvement of autoimmune mechanisms is increasingly held responsible for the pathogenesis of psychiatric disorders. among autoimmune diseases, celiac disease (cd) has been described for years in association with scz [ ] [ ] [ ] , as celiac patients are felt to be at increased risk of schizophrenia. however, it has recently been discovered that the immune response to gluten in schizophrenic patients diverges from that in cd [ , ] . as far as mood disorders are concerned, since they have not been systematically studied as disorders, their association with cd is neither conclusive nor systematic and is limited to depression, often coupled with anxiety. moreover, when a positive statistical association between depression and celiac disease has been found, the percentage frequencies varied from to % [ ] . there is still a lack of consensus about the nature of this association, with two opposing schools of thought: the first posits that specific mechanisms are implied in a common pathogenesis, while the second considers depression not specifically linked to cd but rather a chance event [ ] . regarding bipolar disorder (bd), to date only a few studies have investigated its association with cd. the first report showed increased risk of developing depression but not bipolar disorder in patients diagnosed with cd, while prior depression and bd were positively associated with subsequent onset of cd [ ] . in a recent study, celiac disease was not demonstrated to determine a statistically significant risk of bipolar disorder [ ] . however, like for schizophrenia, recent research showed that immunological reactivity to gluten, different from cd, can also be present in bipolar disorder [ ] [ ] [ ] . this reactivity towards gluten is known as non-celiac gluten sensitivity, or more simply gluten sensitivity (gs), recently found to be different from cd [ ] . here we review the major literature on the association between schizophrenia/mood disorders and cd/gluten sensitivity, highlighting the differences between these associations. a new nomenclature and classification for gluten-related disorders was recently proposed [ ] to sort out the confusion created by new data on different reactions to gluten. based on pathogenesis, gluten-related disorders can be divided into three types: ( ) celiac disease, gluten ataxia and dermatitis herpetiformis, all with autoimmune pathogenesis; ( ) wheat allergy, characterized by allergic mechanisms; ( ) gluten sensitivity in which neither allergic nor autoimmune mechanisms are involved. though gs and cd cannot be distinguished clinically, the two conditions usually show several differences: even in the absence of epidemiological studies, gs is estimated to have prevalence six times higher than that of cd. gluten sensitivity is also characterized by a lack of intestinal damage, negative cd serology (anti-tissue transglutaminase antibodies and anti-endomysial antibodies) and a possible presence of biomarkers of native gluten immune reaction (anti-gliadin antibodies) [ ] . moreover, while cd is strongly associated with specific human leukocyte antigen (hla) class ii genes, known as hla-dq and hla-dq , these genes are present in only about % of patients with gs [ ] . a recent study also demonstrated that gut permeability does not occur in gs, unlike in cd [ ] . thus, gs only seems to be characterized by activation of innate immune response, rather than adaptive response, which is responsible for detectable mucosal damage in cd. finally, analysis of intestinal mucosal specimens from cd patients has demonstrated increased expression of il- a, lacking in gs patients [ ] . on top of that, it has been observed that the prime manifestations of gs are often extra-intestinal, such as behavioral changes, bone or joint pain, muscle cramps, leg numbness, weight loss and chronic fatigue. this is why gluten sensitivity may easily go unrecognized and untreated, representing a risk factor for neurologic and psychiatric complications even greater than cd [ ] . a connection between schizophrenia and gluten began to be noticed in the s, as indicated by clinical cases like that reported by bender [ ] . during world war ii, dohan [ ] sustained the hypothesis of this link by recording fewer first hospital admissions for scz in five countries. he correlated this phenomenon with decreased consumption of wheat and rye in wartime. the reduced supply of cereals seemed to determine a lower incidence of schizophrenia, suggesting that they were related. another observation was that a great number of admissions for scz occurred in countries like ireland with higher availability of wheat and rye than in england and wales [ ] . studies on the effect of elimination of gluten from the diet of scz patients further strengthened the existence of an association between gluten and schizophrenia. in fact, scz patients whose symptoms improved after introduction of a cereal-and milk-free diet showed an interruption or reversal of clinical improvement during wheat challenge [ ] . at that time, the evidence pointed to a link between schizophrenia and gluten, and the authors suggested that celiac disease and scz had the same pathogenesis, namely the harmful effect of wheat. they hypothesized that the common clinical manifestations might signal a genetic link. this called for further studies to establish whether gluten consumption was a triggered genetic susceptibility, as suggested by dohan [ ] . subsequent observations often provided contrasting results. epidemiological studies, such as that of the national danish register [ ] , found that celiac disease occurred before onset of schizophrenia and that antibody-based diagnosis was a risk factor for scz. a higher than expected prevalence of certain autoimmune disorders (including cd) was also found [ ] before onset of schizophrenia in patients diagnosed with this mental illness and their relatives. almost at the same time, ludvigsson [ ] demonstrated that individuals with cd may be at increased risk of non-affective psychosis but not scz. however, analysis of data collected by the uk general practice research database indicated that the risk of scz in patients diagnosed with autoimmune gastrointestinal disease (cd, ulcerative colitis and crohn's disease) was no higher than in the general population [ ] . studies on the effect of eliminating gluten from the diet of schizophrenic patients, reviewed by kalaydjian et al. [ ] , showed that psychotic symptoms only declined in a subset of patients. these results are therefore contradictory: on one hand they suggest a common autoimmune pathway for celiac disease and schizophrenia, but on the other they refute it. until a few years ago, cd and gluten sensitivity were considered synonymous for celiac disease. uncertainty in disease classification and the absence of reliable methods, especially in older studies, may therefore explain these conflicting results. some studies may also have been biased by the fact that that certain scz patients were not tested for the antibodies. however, this bias was overcome by studies in which antibody levels characterizing celiac disease were determined in schizophrenic patients. a study conducted in israel to detect anti-endomysial antibodies (ema) showed no difference between a group with chronic scz and a group of healthy controls [ ] . since ema are very specific for celiac disease, this result could be interpreted as pointing to certain negativity for cd. however, since antitissue transglutaminase antibodies (a-ttg) and anti-gliadin antibodies (aga) were not investigated, this result cannot exclude gluten sensitivity in this group of patients, as suggested by increasing evidence that gs is frequent in schizophrenia [ , ] . given the lack of consensus on the prevalence of true cd in schizophrenia, samaroo et al. [ ] tried to characterize the immune response to gluten in a subset of schizophrenic patients with elevated aga titres. in this group, the elevation of aga was not associated with increased prevalence of a-ttg, deamidated gliadin peptide (dgp) antibodies (another serological diagnostic marker of cd), or with celiac-related hla genes. the authors concluded that these patients did not have celiac disease, but that scz patients probably have different mechanisms evoking an immune response against gluten, independent of transglutaminase and hladq and/or dq molecules. a longitudinal prospective study on antibody reactivity to gliadin, deamidated gliadin and tissue transglutaminase led to the same conclusions [ ] . patients with recent-onset psychosis showed higher levels of igg and iga aga than individuals with multi-episode schizophrenia and individuals without a psychiatric history. these results confirmed those of samaroo et al. [ ] : the immunological pattern of schizophrenia diverged from that of celiac disease, since increased iga a-ttg and igg dgp antibodies were absent except for a marginal elevation in iga a-ttg titre measured in the recent-onset psychosis group. again, disease-related differences in the prevalence of dq /dq alleles associated with cd were not found. cascella et al. [ ] designed a study to evaluate the immunological pattern of a group of schizophrenic patients taking part in the clinical antipsychotic trials of intervention effectiveness. from the serologic characterization of this patient sample, two groups emerged: one with elevated iga aga indicative of gluten sensitivity and the other with ema and/or a-ttg plus iga aga, markers indicative of celiac disease. thus the great majority of schizophrenic patients showed a dramatically higher prevalence of aga antibodies than the comparison group, while reactivity to a-ttg was only mildly increased and reactivity to ema was even lower. the discrepancy between the levels of a-ttg and ema was recognized to be crucial, since they target the same antigen, and this indicated the need for further research. the authors suggested that the results may have been biased by the different assay techniques used: indirect immunofluorescence for ema and elisa for a-ttg. another theory was that the low level of ema determined the milder intestinal damage observed in gs. to explain the discrepancy between the levels of a-ttg and ema, cascella et al. [ ] screened the same population of schizophrenic patients for transglutaminase antibodies (a-ttg- ) [ ] . as gluten causes an immune response to ttg, it was reasonable to suppose that ttg isoforms other than ttg could be involved, for instance ttg- , which would not cause intestinal but psychiatric manifestations. the authors looked for a-ttg- antibodies, which are primarily expressed in the brain and are reported to be elevated in gluten ataxia; they observed increased prevalence in scz patients. a reasonable explanation is that gluten may reach the brain through a breach in the blood-brain barrier and stimulate an immune response similar to that observed towards intestinal mucosa, leading neurons to expose ttg- . as well, gluten seems to trigger release of zonulin, a modulator of the blood-brain barrier and the precursor for haptoglobin- , an inflammatory marker found elevated in a subgroup of schizophrenic patients. this could explain increased blood-brain barrier permeability, allowing passage of gluten peptides and activated inflammatory cells. it also suggests the potential implications of a gluten-free diet (gfd) for the treatment of schizophrenia. a recent study [ ] analyzed a group of schizophrenic patients before they started a gfd, distinguishing cd from gs patients on the basis of antibodies (positivity for a-ttg or ema and for aga, respectively). their response to the diet was tested by evaluating psychotic symptoms. the diet seemed to improve symptoms and extrapyramidal effects, without interfering with the therapy or the behavior of these patients. to date, this is the first study concerning elimination of gluten from the diet of patients previously screened for celiac disease and gluten sensitivity by serological markers. the results are encouraging, though the sample and duration of the trial were not sufficient for definitive proof, especially in case of gs, where the data were insufficient to discern how long it takes to see an improvement. there is still lack of conclusive and systematic results about association between mood disorders and celiac disease. many authors found a positive statistical association with celiac disease [ , [ ] [ ] [ ] [ ] while others did not [ ] [ ] [ ] [ ] . however, these results were limited to depressive symptoms, which were frequently investigated in association with anxiety, and bipolar disorder has seldom been studied [ ] . regarding a possible association between mood disorders and gluten sensitivity, there have been no systematic studies. the few studies are limited to the association bipolar disorders/gluten sensitivity [ ] [ ] [ ] . in , thaysen observed peculiar fatigue in patients with intestinal complaints and in , dicke posited that gluten-free diet improved the mood of adult celiacs. in , dayne described improved behavior in celiac children on gfd and noticed that adults not compliant with the dietdeveloped problems like headache, insomnia and depression [ ] . in , goldberg studied a group of patients with diseases of the small intestine by standardized psychiatric assessment, finding that % had minor affective disorders [ ] . the main risk factors he identified for comorbidity of idiopathic steatorrhoea and psychiatric illness were a positive family or personal psychiatric history and depressive personality traits prior to onset of small intestinal pathology. the author suggested a genetic link between psychiatric illness and the enzyme defect suspected to be responsible for steatorrhoea. later evidence supported the central role of malabsorption, believed to cause reduced central metabolism in the monoamine pathways [ , ] . hernanz and polanco [ ] sustained this theory, observing impaired levels of tryptophan in blood samples of untreated children with celiac disease. nevertheless, malabsorption seemed unable by itself to determine sufficient depletion of tryptophan for psychiatric disorders, such as lifetime depressive and disruptive behaviour, observed in association with cd in a group of adolescents [ ] . thus, pro-inflammatory cytokines, such as ifn-c, produced by activated t-cells in response to ingestion of gluten, were considered to contribute massively to serotonin dysfunction, affecting the kynurenine-niacin pathway and enhancing the hypothalamus-pituitary-adrenal axis. carta et al. [ ] were the first to evaluate a possible role of autoimmunity in the pathogenesis of mood disorders, suggested by the finding of anti-thyroid peroxidase (anti-tpo) antibodies in celiac patients where major depressive disorder, dysthymic disorder and panic disorder had significantly high frequency. subclinical thyroid disease was suspected to be a risk factor for these psychiatric disorders. the authors suggested that cytokines produced in immune reactions may exert an effect on the hypothalamic-pituitary-adrenal axis, also affecting other brain circuits involved in mood control. they supposed that common neuroendocrine dysregulation partook in the pathogenesis of affective disorders and autoimmune diseases. in another study they showed that recurrent brief depression, diagnosed according to icd- criteria, had even higher prevalence than major depression in celiac patients [ ] . in celiac patients undergoing single-photon emission computed tomography, addolorato et al. [ ] observed that regional cerebral blood flow was more abnormal in untreated celiacs than in healthy controls, while there was no difference in cerebral perfusion between treated celiac patients and healthy controls. ciacci et al. [ ] evaluated a sample of celiac patients, normal controls and with chronic persistent hepatitis by means of a modified version of the zung self-rating depression scale and concluded that depression was a feature of cd unaffected by age at diagnosis, duration of illness or compliance/non-compliance with diet, whereas personal psychological resources appeared to be fundamental in developing or maintaining depression. in a general population-based cohort study in sweden, ludviggson et al. [ ] also found increased risk of subsequent depression in individuals suffering from celiac disease. siniscalchi et al. [ ] observed that fatigue was common in celiac patients and was not related to gfd, though it seemed less intense in treated patients. they found that celiac patients on gfd showed stronger depression symptoms than celiacs on a normal diet. data gathered by hauser [ ] showed that the duration of the gfd was not related to depressed mood, and this was the same for anxiety. nonetheless, while cd patients on gfd were not more depressed than controls, female cd patients on gfd seemed at increased risk of developing anxiety disorder. if on one hand there are encouraging results, like those just cited, supporting the hypothesis that depression and anxiety are related to celiac disease, on the other hand different studies found no significant association. for instance cicarelli et al. [ ] found that, although depression appeared to be associated with cd, as found in previous studies, the mood symptoms of celiac patients were the result of dysthymic disorder, not depression, diagnosed by dsm iv. according to fera et al. [ ] , affective disorders in celiacs may be ascribed to difficulties in adjusting to the chronic nature of the disease rather than directly to the disease itself. accomando et al. [ ] administered two depression assessment questionnaires to cd patients on gfd for more than years to determine the existence of a relationship between gfd and depression. the results failed to show any difference between celiac patients and healthy controls. roos et al. [ ] used the psychological general well-being index to assess celiac patients on gfd for many years. long-treated adult patients showed no difference in psychological well-being with respect to controls, suggesting that signs of depressed mood are not a feature of well-treated celiac disease. the state of current understanding of this topic is extremely confusing. a meta-analysis [ ] comprising results from reports on anxiety and depression in adult celiac disease failed to show a higher frequency of depression in celiac patients than in patients with other chronic illnesses. in , the association of bipolar disorder with celiac disease and gluten sensitivity was investigated for the first time by dickerson et al. [ ] . antibody assessment of bd patients showed significantly higher levels of igg aga than in controls. there was also an eloquent increase in igg dgp antibodies in the bd group, whereas iga aga and iga/igg a-ttg levels did not differ between the two groups. dickerson et al. [ ] continued to study the longitudinal trend of these antibodies in patients admitted during a manic episode and at -month follow-up. only igg aga was significantly elevated in the acutely manic group. however, these levels were not significantly different from those of controls at -month follow-up. among manic individuals, elevated levels at follow-up were significantly associated with re-hospitalization in the -month follow-up period, suggesting a correlation between gs and the clinical course of manic symptoms. another clinical study screened a sample of psychiatric inpatients [ ] for a wide spectrum of autoantibodies, finding significantly higher igg aga levels in inpatients than controls. bipolar disorder appeared to be the disorder most associated with autoantibodies. generally, the association of gluten sensitivity with schizophrenia has been more systematically investigated than its association with mood disorders. in bipolar disorder, a-ttg has never been found significantly different from controls [ ] , while in schizophrenia iga a-ttg [ ] and igg a-ttg [ ] have been found slightly higher than in controls. however, the increase in iga a-ttg [ ] was not accompanied by an increase in levels of anti-endomysial antibodies (ema), excluding the possibility of celiac disease. regarding schizophrenia, antibodies to gliadin did not show significant affinity for deamidated gliadin peptides, again excluding the possibility of celiac disease but suggesting a response to gluten with different antigen specificity in a majority of gluten-sensitive individuals with schizophrenia [ ] . on the contrary, dickerson et al. [ ] found evidence of increased levels of igg dgp in bd patients, though this result was not replicated in the later study [ ] . there have also been few studies on bipolar disorder and gluten sensitivity, however, they all yielded results consistent with an increase in igg aga levels [ ] [ ] [ ] , whereas some studies on schizophrenia and gluten sensitivity only found an increase in iga aga [ , ] . the effect of elimination of gluten from the diet has never been studied in bipolar disorder, whereas for schizophrenic patients it is described throughout the literature [ ] . a recent study [ ] tested response to a glutenfree diet in a group of scz patients who were previously considered to have celiac disease or gluten sensitivity on the basis of antibody assessment. the recent characterisation of non-celiac gluten sensitivity shows that like celiac disease, this gluten-related disorder is also associated with major psychiatric disorders. in this review of the literature on the association between scz/ md and cd/gs, we found that for many years, studies on the association between schizophrenia and celiac disease provided contrasting results [ - , , ] . celiac patients were thought to be at higher risk of developing scz than the general population [ ] [ ] [ ] . studies on the association between mood disorders and celiac disease did not use a systematic approach and provided contrasting results [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, these results were always limited to depressive symptoms, frequently investigated in association with anxiety, while bipolar disorder was studied to a lesser extent [ ] . with regard to the association between these major psychiatric disorders and gluten sensitivity, the results are not always concordant, essentially due to the fact that despite a body of data on gs, this disorder is not yet completely characterized and there are still no serological markers for its diagnosis. regarding the association between schizophrenia and gluten sensitivity, most recent studies agree on the involvement of gluten sensitivity rather than celiac disease in a subgroup of scz patients [ , , , , ] . as far as the association between mood disorders and gluten sensitivity is concerned, no systematic studies have been conducted. only bipolar disorder has been studied, assessing antibodies in a sample previously diagnosed with psychiatric disease [ ] [ ] [ ] . to our knowledge there have been no studies in which gs was evaluated in patients with confirmed unipolar depression. a deeper understanding of the immune alterations documented in bipolar disorder and schizophrenia is currently impossible. many theories have been put forward. focusing on bipolar disorder, immune activation is not likely to directly affect the brain [ ] . altered mechanisms of absorption of food protein and therefore processing of food antigen are possibly involved, in line with documented heightened immune activation towards milk caseins [ ] in this disorder. it is also worth noting the increasing evidence in support of the role played by infections, more than autoimmune diseases, in the pathogenesis of mood disorders [ ] . infection by toxoplasma gondii [ ] and other neurotropic pathogens, such as influenza and coronavirus, is associated with md [ ] . in schizophrenia, toxoplasma is suspected to directly affect the brain and the action of cytokines. its passage may occur by alterations in intestinal permeability due to mimicry of enterocyte epitopes by gliadin [ ] . a recent study has demonstrated that toxoplasma gondii [ ] infections raise gluten antibody levels in mice, suggesting that it may alter transcellular passage of gluten peptides. alterations in commensal gut microbiota have also been suggested to dysregulate the immune system, leading to production of antibodies [ ] . another recent study found that levels of scd , a marker of translocation of commensal microbiota, were significantly correlated with anti-tissue transglutaminase igg in bipolar disorder [ ] . we currently do not know whether alterations mainly involve intestinal breakdown, absorption, immune response to gliadin, autoantibody cross-reactivity, production of neuroactive peptides in altered digestion processes [ ] or activation of t cell bystander and consequently b cells with antibody production [ , ] . altered permeability, gut microbiota alterations and infections could therefore all be putative pathogenetic mechanisms involved in scz and md. further studies on the association between schizophrenia/ mood disorders and immune reaction to gluten are warranted to establish the threshold for therapeutic intervention, as only one study has systematically studied the effect of a gluten-free diet in schizophrenic gs and cd patients [ ] . new insights into gluten sensitivity have helped to understand recent results showing immune responses to gluten proteins in certain psychiatric disorders but the different findings are 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following toxoplasma gondii infection in mice discordant patterns of bacterial translocation markers and implications for innate immune imbalances in schizophrenia key: cord- -zjw fbfd authors: bhaskar, sonu; bradley, sian; chattu, vijay kumar; adisesh, anil; nurtazina, alma; kyrykbayeva, saltanat; sakhamuri, sateesh; moguilner, sebastian; pandya, shawna; schroeder, starr; banach, maciej; ray, daniel title: telemedicine as the new outpatient clinic gone digital: position paper from the pandemic health system resilience program (reprogram) international consortium (part ) date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: zjw fbfd technology has acted as a great enabler of patient continuity through remote consultation, ongoing monitoring, and patient education using telephone and videoconferencing in the coronavirus disease (covid- ) era. the devastating impact of covid- is bound to prevail beyond its current reign. the vulnerable sections of our community, including the elderly, those from lower socioeconomic backgrounds, those with multiple comorbidities, and immunocompromised patients, endure a relatively higher burden of a pandemic such as covid- . the rapid adoption of different technologies across countries, driven by the need to provide continued medical care in the era of social distancing, has catalyzed the penetration of telemedicine. limiting the exposure of patients, healthcare workers, and systems is critical in controlling the viral spread. telemedicine offers an opportunity to improve health systems delivery, access, and efficiency. this article critically examines the current telemedicine landscape and challenges in its adoption, toward remote/tele-delivery of care, across various medical specialties. the current consortium provides a roadmap and/or framework, along with recommendations, for telemedicine uptake and implementation in clinical practice during and beyond covid- . coronavirus disease has challenged the status quo of how we approach, deliver, and receive modern medicine ( ) ( ) ( ) ( ) . according to the american telemedicine association, telemedicine is defined as "the remote delivery of healthcare services and clinical information using telecommunications technology" ( ) . it allows for patient care while minimizing the need for physical interaction, thus reducing infection transmission and healthcare facility burden. it can be utilized for ongoing management of chronic conditions, medication compliance, physician-topatient consultation, and other remote services ( , ) . this can be leveraged to benefit broader populations through telehealth platforms and assisted technologies such as the internet of things (iot). telemedicine and digital technologies demonstrate exceptional potential in improving access and delivery in remote settings. there is also an opportunity to exploit the power of artificial intelligence (ai) algorithms to design a better pandemic preparedness and response plan ( ) . health systems have had to adapt to address emerging needs quickly, and many medical subspecialties have transitioned from in-person outpatient care to remote tele-or e-health. broadly, telehealth technologies can be deployed for targeted purposes relevant to a pandemic ( ) . remote assessment of patients could be undertaken, circumventing visits to outpatient clinics or primary care providers. patient continuity for those with chronic diseases is essential during a pandemic ( , ) . such patients are also at high risk of infection and poor outcomes, including mortality, among covid- -positive patients ( ) . notably, telemedicine also limits infection exposure to healthcare staff, can provide rapid access to subspecialists who are not immediately available in person, and allows for multidisciplinary team discussions. this is crucial in pandemic settings, as the safety of healthcare professionals is essential to ensure the sustainability of health systems to cater to emergent cases and maintain ongoing care. patients with flu-like symptoms can be triaged, and telemonitoring using video surveillance could be considered for patients who are homebound such as the elderly or frail. telemedicine can increase access for certain populations who are challenged during limited healthcare facility visitation, stay-home orders, and quarantine, such as single parents, immunocompromised patients, and patients who rely on the assistance of others for transportation. monitoring of patients along with remote delivery of home-based exercise, physiotherapy, psychological counseling, social work consultations, and speech and language interventions could be undertaken through telemedicine. our previous work analyzed the status and deployment of telemedicine during covid- across the geographical divide (bhaskar et al., under review) . in this article, we analyze the uptake of telemedicine across various medical subspecialties and organizational settings with a focus on the current covid- pandemic and propose an operational roadmap for further integration of telemedicine or tele-technologies across health organizations. as hospital systems become strained by the surge of covid- patients, methods to improve the efficiency of emergency departments (eds) are required, while maintaining standards of patient care. telemedicine supplies a potential avenue for triage of critical cases. remote and ambulatory monitoring of patients can allow for remote triage and assessment of emergencies such as acute myocardial infarction (mi), allowing patients to bypass the ed ( ) . automated forward triage systems that use algorithms to categorize patients into risk groups could also be utilized, as ed physicians experience considerable time pressure. current examples include the multi sources healthcare architecture (mhsa) algorithm and the electronic modified early warning scorecard ( ) . telemedicine has also been used to triage, expedite, and streamline the local covid- screening process, thereby reducing the strain on healthcare facilities and practitioner exposure. the new york presbyterian hospital, a world leader in digital health innovation, has demonstrated an effective method to reduce the burden of milder presentations ( ) . they established an ed-based telehealth express care service, in which after presentation and triage at the ed, patients with milder cases are taken into a private room for a teleconsultation with a physician. prescriptions and patient instructions are then printed to the room, and the patient is discharged. this dramatically reduces ed waiting times and allows the hospital to deal with everincreasing ed presentation numbers ( ) . as patients become anxious about ed infection risk, systems such as these are required, and patients need to be able to effortlessly contact eds to query whether their symptoms require a presentation. cardiology is one of the first specialties in which comprehensive telemedicine systems have been implemented. monitoring of heart rhythm in patients with implanted or real-time wearable devices has allowed ecg with holter monitoring, echocardiography records, and virtual auscultation. an emerging body of evidence suggesting cardiac involvement in covid- patients has concerned cardiologists ( , ) . this includes cardiovascular complications such as cardiac injury, heart failure, myocarditis, pericarditis, vasculitis, and arrhythmias ( ) ( ) ( ) . patients with pre-existing cardiovascular conditions who contract covid- also experience inordinately poor outcomes, including a -to -fold rise in mortality ( ) . due to the covid- pandemic, the american college of cardiology urgently updated its guidance on "telehealth: rapid implementation for your cardiology clinic, " in which it encouraged remote monitoring and virtual visits of patients with cardiac problems ( ) . the development of prognostic models based on the recently launched new european register capacity-covid will help to understand the role of underlying cardiovascular disease (cvd) in patients with covid- ( ) . virtual options can significantly increase efficiency compared to in-person doctor appointments ( ) . notably, non-invasive telemonitoring in patients with heart failure reduces allcause mortality and number of hospitalizations, as well as improves the quality of life ( ) . in february , the italian society of cardiology published data on the implementation of telemedicine in cvd patients and reported crucial involvement of telemedicine in the prehospital triage for st-elevated myocardial infarction (stemi) cases and remote monitoring by primary care physicians ( ) . an american heart association (aha) statement emphasized the role of telemedicine in pediatric cardiology through advanced video technologies like tele-echocardiography, fetal echocardiography in prenatal diagnosis, screening for congenital heart diseases, and confirmatory echo tests, external rhythm monitoring, catheterization laboratory, and personal tele-electrophysiology ( ) . due to their comorbidity risk, efforts to prevent covid- infection in cvd patients should be undertaken seriously by reducing hospital admission and outpatient visits ( ) . treatment adherence is one of the significant issues in the long-term management of cvds ( ) . the utilization of mobile phones through mobile health (mhealth) can be one of the reliable potential solutions in this area through measures such as electronic pillboxes and text reminders ( ) . the unique advantage of portable devices and smartphones is the ability to reach most patients and caregivers. the widespread use of mobile technologies makes medical support more effective, faster, safer, and less expensive in both outpatient and inpatient settings ( ) . mhealth can play an increasingly important role in cardiac care, extensively applied in triage, interventions, management, patient education, and rehabilitation. telehealth solutions are critical now, as we aim to minimize patients at high and very high cardiovascular risk being hospitalized and provide ongoing support to cvd patients during the covid- pandemic. in poland, some other systems have been tested in heart failure patients ( , ) , including e-oximeter, allowing for monitoring of heart rhythm and blood saturation, which might help to decide whether those quarantined should be hospitalized during covid- . telemedicine allows for prompt assessment of potential emergent neurological cases and can aid those with hospital access issues and those requiring fast acute assessment ( , ) . acute stroke outcomes are vastly impacted by the speed at which treatment is given, whether it be through tissue plasminogen activator (tpa), endovascular clot retrieval (evt), or antihypertensives. during times of physician shortages, as doctors become re-purposed for covid- purposes, rapid approaches to acute stroke management are needed ( ) . reperfusion treatment viability through computed tomography (ct) can be assessed remotely, allowing reperfusion treatment using tpa and/or evt to be efficiently undertaken. furthermore, telemedicine can be utilized to determine which patients require an urgent transfer from non-evt-capable hospitals to evtcapable hospitals ( ) . a program developed in germany known as transit-stroke, in which rural hospitals established a telemedicine network, saw an improvement in patient outcomes as neurological assessment was made faster, treatments were issued within the required timeframe, and h neurologist access was enabled ( ) . similarly, successful programs have been undertaken worldwide, such as telestroke programs in hawaii and south california ( ) . there is also evidence to suggest that patients who receive acute stroke assessment through telemedicine do not perceive decreased physician empathy compared to those who receive physical consultation ( ) . this somewhat relieves concerns about impaired patientphysician connection through telemedicine. while telemedicine decreases the time it takes to analyze head cts, more work is needed to ensure that this benefit applies equally across different telestroke programs ( ). mobile stroke units (msus) go beyond this to provide ct scanners and stroke personnel within an ambulance vehicle. such programs exist in locations such as melbourne (australia), various states in the us, and hamburg and berlin (germany), among others ( ). msus improve acute ischemic stroke outcomes by reducing the time to reperfusion; however, further development is needed in the treatment of hemorrhagic stroke. telemedicine could also allow ct assessment of mild traumatic brain injuries (such as concussions). this can help to determine if the patient requires transfer to a major hospital or can be treated locally and will also allow for post-concussion checkups ( ) . vulnerable patients who require respiratory management and/or critical care are at increased covid- risk due to their impaired state and require effective management with the aid of technology ( ) . in , the society of critical care medicine (sccm) tele-icu committee in the united states published an update on developments in telehealth critical care (tcc) ( ) . they described three emerging trends in tcc: hub-andspoke structure in which a central hub provides remote technical support, administrative support, and integration to a network of hospitals; decentralized structures in which consultations and patient reviews will be made on a case-by-case and request basis between two sites; and a hybrid structure in which a centralized structure exists but direct contact between spokes can be made for, e.g., specialist consultations. barriers to tcc included cost and reimbursement issues, lack of responsibility for individual hospitals, and legislative issues ( ) . a systematic review and meta-analysis of telemedicine in the us intensive care unit (icu) setting demonstrated decreased mortality and length of hospital stay with telemedicine incorporation ( ) . however, a statistical difference between an active model or high-intensity passive model, in which continuous patient telemonitoring is conducted, and a lowintensity passive model, in which only teleconsultation with an intensivist is conducted, was not ascertained and is an area for further research ( ) . patients with respiratory issues are at higher risk of covid- severe infections due to issues such as ventilator reliance and decreased cough function ( ) . this includes patients with chronic respiratory conditions such as chronic obstructive pulmonary disease (copd), bronchial asthma, interstitial lung diseases, as well as chronic neurological conditions such as neuromuscular diseases ( , ) . telemedicine aids respiratory patients through data collection, such as monitoring of vitals and ventilator status, and by transmitting these data for constant monitoring. in the case of under-resourced or under-developed critical care units in low and middle-income countries (lmics) (bhaskar et al., under review) , frequent international tele-education can serve to upskill doctors and spread critical care knowledge, such as ventilator management ( ) . patients with non-acute diseases require ongoing support and cannot be neglected during covid- times ( , , , ) . studies have shown that telemedicine can lead to similar outcomes as face-to-face delivery of care in the management of patients with heart failure, hypertension, and diabetes ( , ) . ongoing monitoring of these patients is required to prevent acute manifestations, hospitalization, or disease progression ( , ) . the differences within medical subspecialties and individual patients need to be considered, rather than broadly implementing uniform telemedicine approaches across all departments. for example, infectious disease cases can be complicated and require careful consideration of patient history and investigation findings. in these cases, asynchronous consultations, in which the physician reviews data before supplying patient recommendations, will be helpful ( ) . in other fields such as neurology, cardiology, and endocrinology, realtime, interactive consultations might be more applicable ( , ) . patients with neuromuscular issues are particularly at risk due to covid- ( ). patients with motor neuron disease (mnd)/amyotrophic lateral sclerosis (als) are among those who experience considerable disability and will require multidisciplinary telehealth ( ). types of telehealth include teleadvice, teleconsultation, tele-prescription, videoconferencing, home-based self-monitoring, and remote non-invasiveventilation (niv) monitoring. videoconferencing involves consultation with a health professional, home-based selfmonitoring involves taking one's own measurements and submitting them to a physician, and remote niv monitoring involves remote monitoring of the patient's niv data ( ) . the use of telehealth with als patients has been shown to be associated with positive benefits such as reasonable adoption rates, personalized data, and efficient consultations ( ) . other movement disorders such as parkinson's disease (pd) also require ongoing multidisciplinary care ( ) . established programs such as the ontario telemedicine network, the parkinsonnet infrastructure in the netherlands, and that of kaiser permanente in the us all display the ability to integrate telehealth into pd patient care ( ) . areas for growth include the reimbursement of nursing homes that utilize telemedicine, acceptance by patients and physicians, and reimbursement of at-home telemedicine programs ( ) . furthermore, global partnerships can increase international telehealth integration. for example, the international parkinson and movement disorders society africa section, established in the usa, launched a -year program to deliver specialist care to disadvantaged areas in africa using whatsapp tm . diagnosis of pd could also be aided by telehealth, with the unified parkinson's disease rating scale (updrs) and montreal cognitive assessment (moca) for pd both being able to be performed remotely ( ) . such tele-tools have also been recently proposed in the times of covid- for familial hypercholesterolemia patients, who require continuous monitoring of their health due to lifelong high levels of cholesterol and increased cvd risk ( ) . in migraine and headache patients, telemedicine could be used to assess new headache profiles for possible covid- symptomology or standard outpatient consultations ( , ) . cancer patients are another group at risk of covid- infection due to their immunosuppressed states, which could have fatal outcomes subsequent to infection ( ) ( ) ( ) ( ) ( ) . oncologists would use telemedicine for ongoing monitoring and compliance with cancer patients ( , ). this could be useful in monitoring adverse reactions to ongoing chemo-or radiotherapy, as well as to identify patients who might be at high risk of emergent medical attention, such as those at risk of venous thromboembolism. cancer patients could also be offered multidisciplinary care, including psychological interventions, physiotherapy, and specialized interventions such as mindfulness training, to improve the overall quality of life ( ). overall, telemedicine offers opportunities for cancer patients to access specialist care in the comfort of their homes. approaches to the use of telemedicine and mobile technologies in increasing access to novel drugs or interventions through clinical trials should be expeditiously pursued. telemedicine could also be used in palliative care and end-of-life planning involving patients' carers, family, and multidisciplinary care team ( ) . teledermatology is another promising perspective in the diagnosis and monitoring of skin lesions, including cancer ( ) . non-acute ophthalmological telemedicine has been implemented for retinal scans relating to diabetic retinopathy, retinopathy of prematurity, and other non-acute retinal monitoring ( ) . fundus scanning and optical coherence tomography imaging are being sent to remote trained healthcare practitioners (hcps) for evaluation and additionally are being evaluated by ai analysis using deep learning. these non-acute services are also being utilized locally by emergency and urgent care services to a certain extent ( ) . chronic patients must adhere to medications during this time and should not stop treatment regimens without consulting their physician ( , ) . patients taking immunosuppressants, steroids, or pain medications may be concerned about their covid- risk, and contact with their physicians needs to be ensured. adherence to medications can be monitored through mhealth and telehealth means ( ) . such examples include digital adherence technologies (dats) or electronic directly observed therapy (edot) for patients with tuberculosis ( ) . measures include ingestible sensors, video observation, digital pillboxes, and smartphone applications and have been trialed in china, india, belarus, and the us ( , ). the european respiratory society (ers) task force has described the implementation of remote home mechanical ventilation and physical therapy for patients with chronic respiratory disorders ( ) . the emphasis is on promoting common standards of clinical criteria as well as analyzing the cost/benefit ratio and evaluating reimbursing rules to implement in different countries ( ) . tele diagnosis uses patient data to aid remote diagnosis and can be utilized to identify those with bulbar and respiratory weakness. telemedicine strategies such as electronic inhalers, chipped nebulizers, self-monitoring through apps, and text reminders increase medicine compliance in patients with asthma, copd, and cystic fibrosis (cf) ( ) . furthermore, the diagnosis of copd through telemedicine means such as spirometry tracing and teleconsultation provides an opportunity to utilize technology to increase patient care. further studies are needed to stratify which patients, in terms of severity, will be best suited to a telemedicine management approach. another area of potential growth is in using ai algorithms to determine developing copd exacerbations ( ) . telemedicine for asthmatics tends to be more focused on treatment compliance and self-monitoring and can be useful in helping patients learn more about their disease, such as recognizing patterns of asthma triggers ( ) . other barriers to care include the risk that patient data may be manipulated, networks potentially becoming compromised, and inconclusive data on the benefit of telehealth on specific diseases such as copd ( ) . obstructive sleep apnea (osa) is one such disease in which remote monitoring can be utilized to prevent patients from having to spend time in a sleep clinic or respiratory clinic ( ) . home polysomnography devices can be used to track patients' breathing and oxygen levels; however, further work is needed to lower the rate of false negatives to the level of in-person sleep clinics ( ) . a prospective study of patients used a portable spirometer, with bluetooth capabilities and connected to a mobile phone application, to trace results and connect the patient to a physician for analysis ( ) . this allowed the patient's breathing difficulties to be assessed and categorized as asthma, copd, or normal breathing function ( ) . this study shows promising results for remote diagnosis of chronic breathing conditions; however, it does not preclude the need for future testing in some more complicated cases. other smartphone applications have utilized microphones and questionnaires to analyze and detect breathing difficulties associated with other pulmonary conditions such as coughs and lung cancer ( ) . covid- could impose severe stress on sleep clinics and may limit in-laboratory polysomnography sleep studies for osa assessments and diagnosis. home-based telepolysomnography for osa assessment could be explored so that the delayed diagnosis and the associated impact on patients could be minimized. patients with osa often require continuous positive airway pressure (cpap) while sleeping to improve symptoms and achieve proper rest ( ) . in order to see sustained results, patients need to use cpap for at least h at night, combined with lifestyle changes such as weight reduction and smoking cessation ( ) . low adherence to cpap remains a continuous problem for osa patients due to lack of motivation, discomfort, loud noise, and claustrophobia ( ) . telehealth provides an opportunity to increase cpap adherence by monitoring device output data and patient self-tracking of lifestyle factors. when usage falls, the patient can be contacted to discuss their reasons for low adherence and to motivate them to continue use ( ) . telemedicine could be used to monitor bulbar function in patients with a compromised bulbar function such as als ( , ) . the rapid decline in bulbar function could be captured using technologies that are useful in delivering specialist multidisciplinary care ( ) . other diseases in which bulbar function may be impaired include myasthenia gravis, spinalbulbar muscular dystrophy, and riboflavin transporter deficiency ( , ( ) ( ) ( ) . telemedicine can aid with rehabilitation following acute incidents such as stroke and traumatic brain injury (tbi) ( , ), as well as chronic conditions that require ongoing rehabilitation efforts such as copd, cvd, diabetes, and obesity ( ). stroke telerehabilitation programs involving consultations, exercises, games, and therapy aspects have shown positive outcomes such as improving patients' functional abilities and mental health ( ) . other benefits include increasing patient motivation and ease due to being in a home setting ( ) . it is important that patients receive enough support in areas such as technical setup and troubleshooting. the telerehabilitation in heart failure patients (telereh-hf) trial in poland demonstrated that a -week hybrid comprehensive telerehabilitation (hctr) program consisting of remote monitoring of training at patients' homes was well-tolerated ( , ) . however, the positive effects of the intervention didn't translate into improvement in clinical outcomes over a follow-up period of - months in comparison to standard care ( ) . a systematic review similarly found that telerehabilitation allowed for equal or more significant patient outcomes than center-based rehabilitation programs in stroke ( ) . furthermore, wearable devices can be used in the rehabilitation of various neurological diseases such as stroke, pd, multiple sclerosis, and tbi. inactivity is associated with various comorbidities and is often a result of chronic neurological disease or acute accident recovery. remote monitoring through wearable devices can track activity, gait, and any falls throughout rehabilitation ( ) . tbi can result in cognitive issues such as sleep disturbance, photophobia, memory, and behavioral changes ( ) . it is crucial that patients are not discharged without a follow-up plan. a neuropsychological test battery in the few years following moderate-to-severe brain injury and inpatient rehabilitation is vital to assess any cognitive decline and plateau. during covid- times, it is necessary to move outpatient testing of this sort to remote delivery, wherever feasible and while maintaining efficacy. the brief test of adult cognition (btact) has been shown to be effective over the telephone in patients with tbi to assess cognitive state ( ) . remote monitoring of physical activity by physiotherapists and patient consultation with neurologists can also be achieved through telemedicine. however, clear guidelines for rehabilitation management and evidence of efficacy through different delivery systems are lacking ( ) . pulmonary rehabilitation is essential for patients with chronic respiratory issues such as copd and can be achieved through telehealth measures such as monitoring, consultation, and education ( ) . this is important in copd, as potential exacerbations need to be monitored, and lower levels of rehabilitation access are associated with increased rates of hospitalization ( ) . additionally, personal movement tracking devices involving accelerometers are helpful in tracking patient exercise, which is an essential area of pulmonary rehabilitation ( ) . telehealth rehabilitation still faces major hurdles, however, such as cost-effectiveness, patient training, and the lack of regulatory frameworks surrounding personal health devices ( ) . according to the who, about million people annually need palliative care, and only % of them receive it ( ) . the importance of primary healthcare in palliative care was highlighted by the first who global resolution on palliative care in . the project echo (extension for community healthcare outcomes), as one of the examples, shows the potential of telemedicine in the training of patients, their family members, and medical workers in palliative care ( , ) . the training of palliative care via telemedicine/telehealth for outpatients in primary care will increase the coverage and quality of both care and life for these patients. telehealth, including mobile applications, plays a role in making patients more adherent to both pharmacological and non-pharmacological therapies; in remote monitoring of clinical parameters such as cardiovascular and respiratory system function; as well as in monitoring of diet and physical activity. given the overload of respiratory diseases and the flu-like presentations in routine practice, telemedicine offers an alternative that is particularly relevant in the covid- era. mental health support to frontline health workers, patients, and carers will be crucial, as long isolation, lack of social interaction, as well as anxiety over one's own and others' health will take a toll on well-being ( ) ( ) ( ) ) . psychotherapy, psychiatry, and counseling are easily converted to a teleconference format through platforms (such as-but not limited to-zoom tm and skype tm ) and should be utilized by frontline health workers, patients, and carers where necessary ( ) . anecdotal evidence also suggests that patients experiencing paranoid, anxiety, or post-traumatic stress disorders, who may be particularly affected by the covid- climate ( ), may feel more comfortable undergoing telepsychiatry over in-person psychiatry. online delivery will further help to resolve issues such as lack of access to practitioners in rural settings and cultural and linguistic barriers ( ) . furthermore, psychoeducation and mental well-being advice can be leveraged through smartphone apps and digital outreach programs ( ) . these services will become increasingly crucial in the pandemic setting, as physical isolation and frontline work pose both access issues and mental health stressors. the ethics of such teleservices needs to be ensured, with patient confidentiality, referral and billing practices, and physician eligibility being upheld ( ) . psychiatrists, psychotherapists, and psychologists need to ensure that they are maintaining their own mental health during this time, with programs such as professional supervision being of help ( ). in , nearly one-fifth of the european population was aged over years old ( ) . an aging population has put significant pressure on public spending; therefore, telemedicine can improve the scale and efficiency of delivery and ongoing management of elderly patients. elderly patients with mild cognitive impairment or dementia who might be at high risk of an acute condition should be identified using mobile technologies and telemedicine, and telemedicine solutions for the elderly should be easy to use and possibly automatic ( ). this would avoid unnecessary burdens to public health facilities. telemedicine can also be used to act as an interface of the local nursing care staff, carers, and patients with medical specialists. elderly patients will benefit from remote allied health delivery. patients who have had a recent surgery could be monitored at home or in nursing care facilities, preventing extended hospital stays. elderly patients with diagnosed mental health conditions could also benefit from telemedicine. however, self-efficacy and digital literacy presumably have a significant impact on the uptake of telehealth among the elderly ( ) . recent data from the us confirm that the most vulnerable age group for covid- is people over years old, and the highest mortality is observed in those aged and older ( ) . in ontario, canada (as well as in italy and the us), % of deaths related to covid- occurred in retirement homes and long-term care ( , ) . strict zero-visitation policies have had debilitating effects for some elderly patients, particularly those with dementia ( ). telemedicine has been utilized to connect family members with these patients to prevent further decline in mental status and provide comfort. this is useful, as family members have voiced concerns that physically distanced visits such as through windows may further confuse their loved ones. telehealth allows continual monitoring of vitals, physical examination, ongoing clinical management, and communication with patients. in elderly patients with limited accessibility, telemedicine could provide an alternative, easy-to-access service. elderly patients often suffer from social isolation, and telehealth can bring a sense of community. furthermore, by using ai, falls can be detected among elderly patients ( ) . ai can provide personalized medicine solutions to help identify patients at risk of harm. primary healthcare physicians and nursing homes should watch for signs of depression in the elderly, particularly as it has been shown that telemedicine is competent in managing depressive symptoms in the elderly ( ) . telemedicine can be useful in delivering interventions in congregate settings ( , ) . challenges in congregate settings include high population density, limited mobility, built environment issues, and limited access to health. this can make the prevention and management of covid- onerous while preserving human rights and ethical issues. some of the potential target populations include refugees and migrants ( ) , those living in incarceration, orphanages, old-age homes, or childcare centers; and schools. these populations are especially vulnerable to infection such as covid- , where an outbreak can have facility-wide implications and adverse health consequences and fatality. a simulation study on the possible impact of covid- outbreak in a bangladeshi refugee camp found a dire need for dramatic increases in healthcare capacity and infrastructure ( ) . existing approaches to control an outbreak, should it occur, would not be practically feasible, necessitating innovative solutions as well as novel and untested strategies in humanitarian settings ( ) . telepsychiatry to monitor and deliver interventions in congregate settings, especially among refugee populations living in resource-constrained areas ( , ) , could be an alternative when traditional therapy is not possible. telepsychiatry programs for congregate settings should be developed, and further studies are needed to evaluate their long-term impact on patient monitoring and care ( , ) . telemedicine systems are not novel concepts and have been used to good effect for programs such as forward triage in eds, critical care monitoring, and physician communication. existing systems will need to be reallocated, and innovations will be pushed through in order to provide care across all medical fields and to reduce hospital burden. this needs to be achieved within the constraints of funding, legislation, and supply-chain barriers. temporary government funding will be necessary to roll out telemedicine to both rural and urban settings, as well as relaxations to legislation that allow practitioner reimbursement of telemedicine services ( ) . a study by sayani et al., addressing the cost and time barriers in chronic disease management through telemedicine in lmics, found telemedicine to be economically beneficial not only by reducing the socioeconomic barriers to cost and access but also by increasing the uptake of services ( ) . another systematic review of studies conducted on costs of home-based telemedicine programs from to found that home telemedicine programs reduced care costs, although detailed cost data were either incomplete or not presented in detail ( ) . the data on the cost-effectiveness of telemedicine solutions in different medical areas remains inconsistent and confounded by many variables, including the type of disease and "digital maturity" of healthcare systems. however, in critical situations such as the covid- pandemic, telemedicine is proven necessary, and costing, billing, and reimbursement solutions are needed. there are variations in reimbursement policies across regions and healthcare systems. one of the major barriers has been harmonizing a standard reimbursement policy that is acceptable to all stakeholders and sustainable. we recommend that an integrated framework involving public and private parties could help develop a less complicated and streamlined reimbursement structure. notably, the adoption of a "flip the switch" health insurance strategy in north carolina to reimburse telehealth visits "at parity" with conventional office visits for all healthcare providers and specialists is timely and essential. in the long term, the impact of these strategies on healthcare quality and healthcare costs needs further study. healthcare providers must lead the way here in the covid- crisis to explore innovative approaches such as b b monitoring. certain limitations may act as roadblocks in the uptake, implementation, and scale-up of telemedicine and supporting technologies. considerable training is required to ensure patients can familiarize themselves with video teleconsultations and the use of supportive technologies. physicians also need targeted technical, clinical, and communication training based on their subspecialty needs. issues of limited access to broadband and internet facilities are an area that particularly limits the deployment of telemedicine in remote areas and under-resourced settings. telehealth requires reliable broadband access, which is not always acceptable both for clinics in rural areas and for patients living in such areas. when using telemedicine technology, legal restrictions and a lack of clarity as to what is permitted are possible, and these restrictions force telemedicine providers to proceed with caution. some conditions are not considered in the legislation of health systems. it is still not entirely clear whether virtual consultations and video surveillance will be fully paid in hospitals or will be evaluated as shorter visits so that the rates will be lowered. physician licensing and stability of the telemedicine infrastructures are issues of relevance in under-resourced settings. several critical medical procedures cannot be replaced by telemedicine, nor can it be offered to everyone, and there are many excluded groups of patients, including those with deficiencies (e.g., deaf and blind patients) and elderly patients. the effectiveness of telemedicine relies on the possibilities of the implementation of these tools in the given hospital/healthcare system, preparations/training of physicians/nurses, and awareness of the patients. figure | text, audio, or video means. effective telemedicine has several requirements, including culturally appropriate and available infrastructure; regulatory oversight and privacy compliance such as through the health insurance portability and accountability act of (hipaa); integration of technologies with existing data such as electronic health records (ehrs), apps, and monitoring devices; and insurance coverage such as medicare or private-payer schemes. credentialing on both sides is essential. the consultation should start with verification of the patient's identity through name, age, phone number, date of birth, and address. the physician should then clearly specify that this is a telemedicine consult and that no audio or video of the communication will be recorded. it is imperative that health record information is protected. the physician should then clearly and explicitly ask for consent, whether that be verbal, text, or video. at the start of the consultation, the physician should assess if acute care is required and make a cursory determination if telemedicine consultation is sufficient. if necessary, the physician should supply an immediate referral or advise the patient to seek immediate medical attention. during a typical consultation, the patient will be evaluated; and specific diagnostics and treatment would be recommended based on the assessment of the healthcare provider; and follow-up could be scheduled either in person or virtually. the physician should go through records, clinical history, and investigations including pathology and diagnostic reports, and obtain any additional information that the patient can provide. a general, non-specialist examination should be obtained, and any vital signs that the patient has the means to measure should be gathered. beyond this, when introducing technologies and measures to overcome gaps in the healthcare system, it is essential not to simply ask, "where are the gaps, " but also to define the standards and ideals of care and continually iterate toward these ideals. as mentioned before, telemedical consultations do not approach the same level of fidelity that an in-person physical exam yields, between physical exams, body language, vocal intonations, and odors. as such, the fidelity of the technology involved with telemedical consults must continually iterate to reach the same level of fidelity and information that an inperson visit might yield. in this vein, virtual and augmented reality technologies, while evolving, hold promise for the future of telemedicine, particularly in envisioning a future in which high-fidelity physician and patient "avatars" may meet in a virtual space for a telemedical consult, replicating aspects of an inperson visit through immersive technologies. covid- has expedited the uptake of telemedicine across various specialties. the rapid move by various bodies, associations, and providers to use telemedicine in maintaining patient continuity while limiting covid- risks of exposure to patients and healthcare workers will have a long-term impact well-beyond the current pandemic. teleconsultation needs are varied across specialties, and therefore, specialty-specific guidelines and recommendations need to be developed. a scoping list of various telemedicine studies across medical subspecialties (telemedicine vs. standard care) has been provided in table . a comprehensive workflow that critically profiles various telemedicine enablers has been proposed in figure , and recommendations to improve various factors are listed in table . the proposed workflow (figure ) provides a practical telemedicine framework cognizant of relevant requirements and considerations, and a step-by-step pathway to streamlined telemedicine delivery. this could be used as a template (for further customization or adaptation) by individual medical subspecialties. current challenges and recommendations to improve telemedicine include ( ) : (i) infrastructure capacity [formation and expansion of dedicated telemedicine units and workforce; cloud-based infrastructure to support telemedicine associated bandwidth traffic; liability, maintenance, and safety of telemedicine platforms; ongoing and regular maintenance and servicing of telemedicine hardware and software; awareness, education, and training to build confidence about telemedicine use among providers and consumers; compulsory telemedicine modules for medical students and continued professional development (cpd) workshops/courses for healthcare providers and medical informaticians/technologists; targeted courses aimed at re-skilling clinicians]; (ii) integration with existing data (standardized patient-specific information and consent form with telemedicine opt-in/out option); (iii) regulatory oversight issues (setup of telemedicine regulatory authority; accreditation/licensing of providers using telemedicine; guidelines for telemedicine use in inter-state and -nation settings; standardization of telemedicine related technologies and services with regulatory oversight, audit, and reporting; appropriate measures and oversight to protect privacy, security, and confidentiality of patient data; legal frameworks for telemedicine-specific information storage, sharing, and access); and (iv) insurance/payers (guidelines for telemedicine insurance; streamlined payment facilities for making and receiving payments; bundled services payments and insurance coverage). another important and emerging area is the use of text messaging [short message service (sms) or multimedia message service (mms)] as a model for service delivery ( ) ( ) ( ) ( ) ( ) ( ) . text messaging has proven efficacious in diabetes self-management, smoking cessation, weight loss, physical activity, and adherence to medication regimens [such as in human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids) patients who are on antiretroviral therapy] ( ) . a systematic review on text messaging interventions identified the following issues: identification of intervention characteristics, ensuring intervention effects last over a longer duration of time, and cost-effectiveness of these interventions ( ) . issues of privacy and security are also poignant in this context. nevertheless, text messaging offers potential benefit as a public health intervention toward chronic disease management ( ) ( ) ( ) ( ) , medication adherence, and secondary prevention ( ) . perceptions and experiences/satisfaction, regarding telemedicine services, of the patients and providers is important in improving telemedicine implementation, delivery, and impact ( ) ( ) ( ) ( ) ( ) . a systematic review on patient satisfaction with telemedicine highlighted methodological deficiencies in published studies ( ) . a study on patient and clinician experience with telemedicine found that virtual video visits may provide effective follow-up and increased convenience in comparison to routine in-person visits ( ) . another study found a perception of patients with type diabetes that telemedicine can improve their access to care ( ) . further studies focusing on communication issues and the quality of interpersonal relationships during telemedicine consultations and how these factors affect healthcare delivery using this medium are required ( , ) . some specialist examinations, including neurologist consultation, can also be conducted. the american academy of neurology has issued guidelines for telemedicine consultation ( ) . physicians can assess mental status; any visual, auditory, or cognitive deficits; comprehensive speech; cranial nerves; apparent tremors; and gait. motor examinations can also be conducted with the aid of a caregiver in order to help ascertain strength, tone, reflexes, dermatome sensation, and cerebellar function. in such a case, consent must be gained from both the patient and the assistor. special considerations may apply for pediatric patients or adults with intellectual disabilities. based on the severity of symptoms, the patient may require a management plan, including specific treatment, health education, and counseling if necessary. patients can be prescribed ongoing prescriptions, specific medications, or add-on medication to optimize regimes, given that there is no ambiguity about diagnosis and the medications are not dangerous. if there is any ambiguity about diagnosis, this must be recognized as a limitation of this mode of telemedicine, and documentation must be made. further tests should be done or referred for in-person consultation if necessary. it should be noted that detailed examination of tone, strength, and reflexes; comprehensive eye examinations; and examinations that require specific maneuvers such as vestibular examinations should be avoided, as examination findings won't be accurate. these recommendations will also need to be adjusted according to individual state or federal legislation. the future of telemedicine beyond the current covid- pandemic will depend on how we address existing challenges, building resilient health systems ( ) ( ) ( ) . further randomized controlled trials to evaluate the long-term effects of telemedicine-based interventions in various patient populations should be planned. telemedicine will play a major role as a "safety net" during the pandemic. the covid- pandemic is causing an unprecedented public health crisis impacting healthcare systems, healthcare workers, and communities. the covid- pandemic health system resilience program (reprogram) consortium is formed to champion the safety of healthcare workers, policy development, and advocacy for global pandemic preparedness and action. sbh devised the project, the main conceptual ideas, including the proposal for a new telemedicine workflow, the proof outline, and coordinated the writing and editing of the manuscript. sbh and sbr wrote the first draft of the manuscript. sbh encouraged sbr to investigate and supervised the findings of this work. all authors discussed the results and recommendations and contributed to the final manuscript. we would like to acknowledge the reprogram consortium members, who have worked tirelessly over the last days in contributing to various guidelines, recommendations, policy briefs, and ongoing discussions during these unprecedented and challenging times despite the incredibly short timeframe. we would like to dedicate this work to our healthcare workers who have 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training. the opinions expressed in this article are those of the authors and do not necessarily represent the decisions, official policy, or opinions of the affiliated institutions.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © bhaskar, bradley, chattu, adisesh, nurtazina, kyrykbayeva, sakhamuri, moguilner, pandya, schroeder, banach and ray. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -okw la b authors: nan title: chapter health effects date: - - journal: interface science and technology doi: . /s - ( ) - sha: doc_id: cord_uid: okw la b abstract there are beneficial as well as harmful aerosols. according to their nature, harmful particles can be classified into three categories: chemically toxic, infectious, and radioactive. in general, there is a relationship between the response and the dose received. a biochemically active particle may contain only a small amount of active agents. in this respect, an inhaled particle simply acts as a carrier that facilitates delivery of deleterious or beneficial components to specific surface areas of lung airways. in view of the tortuous narrow passageways and sharp turns they have to go through, aerosol particles are an effective carrier. as an indication of their effectiveness, about one half of all -m particles inhaled through the mouth deposit in the alveolar region. there are beneficial as well as harmful aerosols. according to their nature, harmful particles can be classified into three categories: chemically toxic, infectious, and radioactive. in general, there is a relationship between the response and the dose received. a biochemically active particle may contain only a small amount of active agents. in this respect, an inhaled particle simply acts as a carrier that facilitates delivery of deleterious or beneficial components to specific surface areas of lung airways. in view of the tortuous narrow passageways and sharp turns they have to go through, aerosol particles are an effective carrier. as an indication of their effectiveness, about one half of all -~tm particles inhaled through the mouth deposit in the alveolar region. in addition to respiratory disease, some types of deposited particles can cause systemic diseases following their dissolution and absorption. the period between first exposure to an agent and the appearance of symptoms varies depending on the characteristics of the agent and disease. some symptoms appear within hours from an acute exposure, whereas others may have latency periods as long as years. synergistic or antagonistic effects play an important role. synergism represents the interaction of two or more agents that produces an adverse effect greater than the sum of the effects resulting from exposure to each agent separately. antagonism is an interaction between two or more agents that results in a reduction in adverse effect of each agent. host factors have a strong influence on biological responses to harmful particles. the main host factors include state of health, genetic trait, immunological status, and psychological state such as anxiety and stress. inhalation of particulate toxicants can lead to various types of responses including fibrotic, systemic, irritant, allergic, mutagenic, teratogenic, and carcinogenic responses. injury of tissues may result from the inhaled compound or its metabolic products. effects may differ considerably between direct exposure of epithelial cells to toxicants and exposure to the toxicants after phagocytosis. toxicity also depends on particle size and physicochemical properties of the agent. concentration of the agent and duration of exposure are two important factors influencing response. acute and chronic exposure may result in different adverse effects. pneumoconioses, characterized by scars due to increase in interstitial fibrous tissue, are diseases resulting from retention of certain types of particles in the alveolar region. different types of dusts may produce different patterns of fibrotic lesions. for example, crystalline free silica found in mines, foundries, blasting operations, and glass manufacturing can give rise to a nodular type of fibrosis (silicosis). on the other hand, asbestos fibers can cause a diffuse fibrosis (asbestosis) if they exceed ~tm in length and . ~tm in width (lippmann, ) . coal workers' pneumoconiosis has a complicated form. most pneumoconioses result in shortness of breath and chronic cough, but pneumoconioses from certain types of dust, such as iron oxide, may have no evidence of functional impairment. systemic response arises from chemical toxicants that affect certain organs or tissues. target sites include respiratory tract, gastrointestinal tract, hematopoietic system, liver, kidney, bones, endocrine system, and nervous system. herbicides, pesticides and heavy elements such as mercury, lead, cadmium, arsenic, molybdenum, and selenium are known systemic toxicants. an example of systemic response is metal fume fever, an acute flu-like illness with symptoms of throat irritation and dry cough. the symptoms appear within hours following heavy exposure to fumes of metal oxides. irritant response generally results from inhalation of particulate sulfates, pesticides, and droplets of sulfuric acid or other strong acids. they cause inflammation in affected regions such as rhinitis, pharyngitis, laryngitis, bronchitis, pulmonary edema, and pneumonia. allergic response involves sensitization by initial exposure and reaction to subsequent exposure. such response can lead to bronchial asthma. examples of allergens include nickel, cobalt, arsenic, chromium, organic dusts, herbicides, and insecticides. inhalation is a route of entry for airborne mutagens (agents that produce changes in dna), teratogens (agents that cause developmental malformations), and carcinogens. among known or suspected chemical mutagens are ddt, sodium arsenate, cadmium sulfate, and some lead salts. examples of known or suspected chemical teratogens include dioxin, organic mercury, cadmium sulfate, and sodium arsenate. there is a broad variety of chemical carcinogens including cigarette smoke, soot, asbestos, arsenic, chromates, and certain petroleum products. changes in lung tissues can begin when an individual is exposed to a cancer-causing substance. a few abnormal cells may appear in the lining of bronchi. if exposure to the carcinogen continues, more abnormal cells can appear and lead to formation of a tumor. in addition to asbestosis, asbestos fibers can cause bronchial cancer and mesothelioma. the latency period is years or longer. to have chronic toxicity, inhaled asbestos fibers must exceed the following critical length limits" ~tm for mesothelioma and ~tm for lung cancer. in addition, fiber width must be less than . ~tm to induce mesothelioma and larger than this limit to cause lung cancer (lippmann, ) . cigarette smoke, containing particles and vapors, has been identified to be the cause of a plethora of diseases. in addition to cancers of the lungs, larynx, esophagus, bladder, cervix, kidney, pancreas, and stomach, smoking can give rise to emphysema, chronic bronchitis, pneumonia, chronic heart and cardiovascular diseases, abdominal aortic aneurysms, acute myeloid leukemia, cataracts, and gum disease. central bronchial airways are the most common sites for lung cancer in cigarette smokers (schlesinger and lippmann, ) . the diseases resulting from exposure to ambient aerosols include pulmonary emphysema, bronchitis, and, perhaps, lung cancer. asthmatics are particularly susceptible to the adverse effects of aerosol acidity in ambient air. emphysema is a condition of over-inflation of structures in the alveolar region. the over-inflation arises from a breakdown of alveolar walls. early symptoms of emphysema include shortness of breath and cough. the primary cause of emphysema is cigarette smoking. there is also an inherited form of emphysema due to deficiency of a protein called alpha -antitrypsin. bronchitis is an inflammation of the lining of bronchial airways. when bronchial airways are inflamed, less air is able to flow to and from the alveolar region and a heavy mucus or phlegm is coughed up. cigarette smoking is the number one cause of chronic bronchitis. dusts and fumes in ambient air and workplaces are other common causes of this disease. higher rates of chronic bronchitis are found among workers exposed to particles of high concentrations, such as coal miners, grain handlers, and metal molders. emphysema and chronic bronchitis in combination comprise chronic obstructive pulmonary disease (copd). a copd patient has difficulty breathing because ( ) the airways lose their elasticity and therefore cannot keep open properly; ( ) some alveolar walls are destroyed; ( ) airway walls are inflamed; and ( ) cells in airways make more mucus than usual, which tends to clog the airways. ambient aerosol is a mixture containing numerous chemical species of natural and anthropogenic origins. these particles are either directly emitted into the atmosphere from many different types of sources or formed in air by gas-to-particle conversion processes. harmful components identified in highly polluted air include acid sulfate species, heavy metals, reactive organic compounds, and peroxides. at sufficiently high doses these components can produce local respiratory diseases or systemic disorders. however, none of the harmful components mentioned above exists in ambient particles at sufficiently high concentration levels to cause a specific disease. ambient particles are harmful when their concentration exceeds a certain limit; epidemiological studies have indicated a strong association of increases in human morbidity and mortality rates with increased concentrations of ambient particles in a certain size fraction (wilson and spengler, ; vedal, ) . complexity in chemical characteristics of ambient particles has led to considerable difficulty in identifying the components responsible for adverse health effects. thus, it remains largely unclear which specific components are responsible. concentration alone cannot explain the causal relationship. according to the threshold limit values recommended by american conference of governmental industrial hygienists, a worker can be exposed to nuisance or inert dust at the concentration of mg/m in the respirable fraction without measurable injury. questions have been raised whether ambient particles are a carrier of shortlived, difficult-to-quantify, but harmful compounds. friedlander and yeh ( ) have provided supporting evidence for the involvement of peroxides in the adverse health effects of particulate pollutants. reactive oxygen species (ros) in particles also have been shown to play an important role. highly reactive oxygen-containing species, such as hydroxyl radical and hydrogen peroxide, are collectively described as ros. in ambient air, photochemical reactions involving reactive organic gases can produce ros, which are distributed in the gas and particulate phases. combustion aerosols, such as vehicular exhaust, also contain relatively high concentrations of ros. hung and wang ( ) have reported that the concentrations of particulate ros in ambient air are strongly associated with photochemical activities. there is a good correlation between the concentrations of ambient ozone and ros in submicron particles, especially in the ultrafine fraction that are freshly produced by either photochemical reactions in ambient air or combustion processes in vehicular engines. aerosol particles can serve as an effective carrier for ambient peroxides and reactive oxygen species to reach the alveolar region. in the absence of particles, inhaled peroxides and ros mainly deposit in tracheobronchial airways because of their high solubility and diffusivity. when these reactive species are adsorbed on particle surfaces, they can easily reach the alveolar region and thereby lead to an adverse effect greater than in tracheobronchial airways. because of their small sizes and surface characteristics, ultrafine particles appear to be more toxic than larger particles. respiratory effects are shown to be associated with the number of ultrafine particles (peters et al., ) . a recent study indicates that ultrafine particulate pollutants are capable of inducing oxidative stress and mitochondrial damage (li et al., ) . pulmonary effects of ultrafine particles have been reviewed by oberd rster ( ) . studies on rodents indicate that ultrafine particles administered to the lungs cause a greater inflammatory response than larger particles of the same total particle mass do. surface chemistry appears to be an important contributing factor of their high toxicity. furthermore, it appears that deposited ultrafine particles largely escape capture by alveolar macrophage and therefore have higher probabilities to enter the pulmonary interstitium. these results lend support to the hypothesis that ultrafine particles are causally involved in adverse responses seen in susceptible groups of the population. the need for further studies on ultrafine particles has been stressed in a couple of recent reports (national research council, ; friedlander and pui, ) . inhalation is an important route of entry for bacterial and viral pathogens. single bacterial particles can remain airborne for a long time and therefore have high probabilities to be transmitted through air. viruses in droplets generated by sneezing and coughing of a patient can survive for hours and gain access to another host while they are airborne. droplet size plays an important role in determining the atmospheric spread of infectious diseases. sneezing and coughing can produce droplets of various sizes. the distance to which a droplet can be transported depends on the rate at which the droplet evaporates. large droplets settle out of air quickly and, even if they are inhaled, may not reach the pulmonary region. for many pulmonary diseases, the susceptible part is the alveolar region of the respiratory tract. however, smaller droplets can evaporate rapidly to less than ~m in diameter. it is mainly these residual particles, named droplet nuclei (wells, ) , that can spread the diseases. tuberculosis and bacterial pneumonia are two common respiratory diseases caused by bacteria. symptoms of tuberculosis include feeling tired, loss of appetite, fever, coughing up blood, and night sweats. pneumonia is a serious infection of the lungs. when the lungs are infected, the alveoli fill with pus and other liquid, thereby greatly reducing the ability to transfer oxygen to the bloodstream. legionnaires' disease is a form of bacterial pneumonia. it has acquired its name, because the first known outbreak occurred in the bellevue stratford hotel in philadelphia where a convention of the pennsylvania department of the american legion was held. in that outbreak, over people contracted this previously unknown type of pneumonia as a result of exposure to mist from comaminated water that was used to cool the air in the hotel's air conditioning system. the disease can also be contracted by inhaling mist from water sources such as whirlpool baths and showers that are contaminated with legionella bacteria. anthrax is a disease caused by bacillus anthracis, a spore-forming bacterium. its symptoms are similar to a common cold initially, but progress to severe breathing problems and shock in a few hours to a few days. to contract the pulmonary form of anthrax, it requires inhalation of to thousand spores into the alveolar region of the lungs. droplets that deposit in the nose are not likely to cause the disease. examples of diseases due to viruses include influenza, common cold, viral pneumonia, and severe acute respiratory syndrome (sars). when a person is infected by influenza virus, the tissues of respiratory tract become swollen and inflamed. prominent symptoms of flu are high fever, muscle aches, severe headache, dry cough, and sore throat. patients with common colds have milder fever, stuffy nose, sneezing, coughing, and also sore throat. flu and common colds mostly spread through transfer of virus-containing droplets by inhalation or hand-to-hand contact. sars is a respiratory disease due to a type of coronavirus. first reported in asia in february , its symptoms include high fever (temperature higher than . ~ shortness of breath, headache, coughing, diarrhea, and malaise. most sars patients develop pneumonia. according to the world health organization, a total of , people worldwide were infected during the outbreak. of these, died. a probable transmission route of the sars virus is the spread of droplets produced by the cough or sneeze of a patient. infection can occur if these droplets deposit on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. the most common radioactive particles in ambient air are radon daughters. after release from soil, radon gas continues to decay and generate a series of radioactive products. these radon daughters easily attach to ambient particles and become a source of radiation exposure for the general population. occupational exposure to radioactive particles mainly occurs in uranium, tin, and hematite mines. the primary health effect from inhalation of radioactive particles is cancer. damage caused by ionizing radiation at the cellular or molecular level can give rise to uncontrolled growth of cells. this is a stochastic effect re:~ulting from long-term, low-level exposure to radiation. increased doses lead to increase in probability of occurrence. all regions of the respiratory tract are susceptible, but the frequency of incidence differs from region to region. the types of radiogenic tumors and target tissues have been reviewed by icrp ( ) . soluble radionuclides in deposited particles can be absorbed into bloodstreams. depending on their metabolic behavior, these radionuclides can be retained in various organs for different periods of time. a broad variety of pharmaceutical agents can be effectively delivered through inhalation to treat respiratory and systemic diseases. examples of aerosolized agents for treating respiratory disorders include bronchodilators and mucolytics. antibacterial and antiviral agents have been used in aerosol form for treating pulmonary infections. for aerosol therapy of systemic illnesses, various degrees of success have been achieved with hormones, vaccines, antibiotics, and cardiovascular drugs. among the agents tested for systemic medication, aerosolized insulin and heparin appear to be very promising. two proven classes of aerosolized drugs for treatment of asthma and chronic obstructive pulmonary disease are -adrenergic agonists and glucocorticoids. inhalation of -adrenergic agonists can relax bronchial smooth muscle, stimulate skeletal muscle, and inhibit release of inflammatory mediators. short-acting -adrenergic agonists can provide bronchodilation within minutes of inhalation. glucocorticoids are useful for treatment of inflammation. anticholinergics are among other common agents for treatment of asthma and chronic obstructive pulmonary disease. as bronchodilators, anticholinergics agent have a slower onset but a comparable duration of action as -adrenergic agonists. aerosolized formulations of corticosteroids have become commonly used agents in treating respiratory diseases such as asthma, cystic fibrosis, and obstructive lung disorders. these formulations are able to influence existing inflammation. mucolytics such as n-acetylcysteine can give rise to rapid liquefaction of viscid mucus. when administered in aerosol form, they can help maintain adequate mucus viscosity in patients with chronic obstructive airway disease. . a review of the sars epidemic in indicates that, in some countries, a majority of cases were caused by nosocomial infection. discuss possible transmission routes of the sars coronavirus in hospitals. . a potential terrorist weapon is the release of plant and animal pathogens in aerosol form. these pathogens can have devastating effects on agriculture if they are released at points where they can spread out rapidly. what strategies can be used to protect agriculture from such terrorist attacks? the submicron atmospheric aerosol as a carrier of reactive chemical species: case of peroxides emerging issues in nanoparticle aerosol science and technology (nast) experimental determination of reactive oxygen species in taipei aerosols human respiratory tract model for radiological protection ultrafine particulate pollutants induce oxidative stress and mitochondrial damage effects of fiber characteristics on lung deposition, retention, and disease research priorities for airborne particulate matter. i. immediate priorities and a long-range research portfolio pulmonary effects of inhaled ultrafine particles respiratory effects are associated with the number of ultrafine particles selective particle deposition and bronchogenic carcinoma ambient particles and health: lines that divide on air-borne infection; ii. droplets and droplet nuclei particles in our air: concentrations and health effects key: cord- - lbopaso authors: hansildaar, romy; vedder, daisy; baniaamam, milad; tausche, anne-kathrin; gerritsen, martijn; nurmohamed, michael t title: cardiovascular risk in inflammatory arthritis: rheumatoid arthritis and gout date: - - journal: lancet rheumatol doi: . /s - ( ) - sha: doc_id: cord_uid: lbopaso the increased risk of cardiovascular morbidity and mortality in rheumatoid arthritis and gout has been increasingly acknowledged in past decades, with accumulating evidence that gout, just as with rheumatoid arthritis, is an independent cardiovascular risk factor. although both diseases have a completely different pathogenesis, the underlying pathophysiological mechanisms in systemic inflammation overlap to some extent. following the recognition that systemic inflammation has an important causative role in cardiovascular disease, anti-inflammatory therapy in both conditions and urate-lowering therapies in gout are expected to lower the cardiovascular burden of patients. unfortunately, much of the existing data showing that urate-lowering therapy has consistent beneficial effects on cardiovascular outcomes in patients with gout are of low quality and contradictory. we will discuss the latest evidence in this respect. cardiovascular disease risk management for patients with rheumatoid arthritis and gout is essential. clinical guidelines and implementation of cardiovascular risk management in daily clinical practice, as well as unmet needs and areas for further investigation, will be discussed. cardiovascular disease is the most frequent cause of death worldwide. the global burden of disease study showed that · million people died of cardiovascular disease globally, accounting for % of all deaths. well established, traditional risk factors for cardiovascular dis ease comprise age, sex, race, hypertension, diabetes, smok ing, and hyperlipidaemia, all of which are included in various prediction models. however, over the past years several nontraditional risk factors, such as chronic inflam mation, have emerged as amplifiers of cardiovascular disease risk. rheumatoid arthritis is the most common auto immune arthritis, with a prevalence of up to %, and is char acter ised by a symmetrical polyarthritis with pos sible sys temic manifestations. rheumatoid arthritis is an accepted independent risk factor for cardiovascular disease, driven by the underlying chronic inflammatory pro cess. however, traditional cardiovascular risk factors remain important. gout is the most common crystalinduced, auto inflammatory joint disease with a prevalence of between · % and · %. gout occurs when monosodium urate crystals are deposited in joints and soft tissues. hyperuricaemia-defined by a serum urate concentra tion above the saturation point (ie, ≥ · mmol/l [≥ · mg/dl])-results predominantly from reduced renal excretion of uric acid, which is a consequence of genetics, comorbidities, and therapies. a continuum has been sug gested, from asymptomatic hyperuricaemia to asympto matic subclinical crystal deposition detectable only by ultrasound or dualenergy ct, to the clinical inflammatory state of gout flares, to chronic gouty arthritis with tophi and gouty bone erosions. if not treated adequately, gout is a debilitating disease with systemic manifestations, such as monosodium urate crystal deposition in organs and worsening of cardiorenal function. , in addition to gout flares, patients with gout frequently have a high burden of cardiovascular comorbidities, which might explain, in part, the high cardiovascular mortality when compared with the general population. during the past years, gout has been shown to be an independent cardiovascular risk factor, with higher cardiovascular mortality than in the general population. in this review, we will discuss epidemiological data on cardiovascular disease in rheumatoid arthritis and gout, not only for atherosclerotic disease but also for venous thrombotic disease and heart failure, as clinical and subclinical prevalence of the two diseases is higher than previously thought. the underlying pathophysiology of increased cardiovascular risk relevant to inflammatory arthritis, as well as the observed effect of antiinflammatory and disease modifying treatments such as uratelowering therapies in gout, will be reviewed and discussed. increased cardiovascular risk in patients with inflam matory arthritis necessitates cardiovascular risk assess ment and current management guidelines and their practical implications will be discussed. finally, we con sider topics that need further research with the aim to decrease the cardiovascular burden of patients. patients with rheumatoid arthritis have up to a twotimes higher risk of developing atherosclerotic cardiovascular disease than the general population, similar to patients with diabetes. the risk of ischaemic heart disease is increased in patients with early rheumatoid arthritis and symptom duration of less than year, and probably even in the subclinical stage. the risk of cerebrovascular incidents is increased by about % (relative risk · , % ci · - · ), whereas the risk of myocardial infarction is doubled (relative risk · , · - · ). moreover, patients with rheu matoid arthritis have almost twice the risk of developing congestive heart failure (rate ratio · , % ci · - · ), including both heart failure with preserved ejection fraction and heart failure with reduced ejection fraction. several factors contribute to increased cardiovascular risk, including comorbidities such as diabetes, dyslipidae mia, and hypertension; albeit the data for hypertension are somewhat conflicting. lipids seem to have paradoxi cal associations with cardiovascular risk in rheumatoid arthritis. during active disease, low total cholesterol and ldl cholesterol are associated with increased cardio vascular risk (the socalled lipid paradox). effective anti rheumatic therapies resulting in reduced disease activity of rheumatoid arthritis reverse the cholesterol reduction, thus leading to increased lipid concentra tions, and lipid concentrations in well controlled rheumatoid arthritis are generally stable and similar to those in the general population. , , furthermore, patients with rheu ma toid arthritis also have more than a two times increased risk of venous thrombotic disease compared with the general population (cumulative inci dence of · % [se · ] vs · % [ · ], p= · ). studies show that allcause mortality among patients with rheumatoid arthritis was % higher than in the general population, primarily because of cardiovascular disease ( %), with a median shortened life expectancy of - years. in one study, the estimated standardised cardiovascularmortality ratio was · ( % ci · - · ), which is substantially less than reported in earlier studies. the improvement in cardiovascular mortality in patients with rheumatoid arthritis over the past years could be attributed to the early initiation of more effective anti rheumatic treatments (conventional synthetic and biologi cal diseasemodifying antirheumatic drugs [dmards]). decreased disease activity following effec tive therapy is associated with a lower cardiovascular risk, and vice versa, whereas cardiovascular risk remains unchanged in patients who do not respond to biological dmards. however, it should be noted that about % of increased cardiovascular disease risk in patients with rheuma toid arthritis is associated with traditional cardiovascular risk factors. a large retrospective database study in the uk ( patients with gout vs without gout) showed that the prevalence of hypertension in patients with gout at baseline was twice as high compared with the control group ( % vs %). patients were also more often obese ( % vs %) and hyperlipidaemic ( % vs %) with more prevalent use of statins ( % vs %). a high prevalence of these traditional risk factors was also seen in the large national health and nutrition examination survey - , including · million us patients with gout. moreover, the national health and nutrition examination survey showed that % of patients with gout had diabetes compared with almost % in the nongout population (or · , % ci · - · ). in a large retrospective dutch cohort of primary care patients with gout, ( %) of patients already had established cardiovascular disease at cohort entry compared with ( %) of in the nongout control group. after years of followup, ( %) of patients with gout had developed cardiovascular disease, compared with ( %) of in the nongout control group. there was an even higher incidence of cardiovascular disease ( %) in patients managed by rheumatologists, possibly explained by a higher portion of severe gout seen in this setting. gout is an independent risk factor for cardiovascular disease; however, the strength of the association of hyper uricaemia and gout with other traditional cardio vascular risk factors makes distinguishing the isolated influence of gout on that risk difficult. although atherosclerotic disease, hypertension, and chronic kidney disease are associated with elevated serum urate concentrations, the causal relationship and direction of association between urate and these disorders is still under debate. , in the past decade, several observational studies have suggested an association between hyperuricaemia, gout, and congestive heart failure. a crosssectional study of patients in the usa found the prevalence of congestive heart failure to be % in patients with gout versus % in patients without gout. another study in patients with coronary artery disease showed that patients with gout had a higher prevalence of congestive heart failure ( [ %] of ) than those without gout ( [ %] of ). furthermore, individuals with asymptomatic hyperuricaemia already have an increased risk of con gestive heart failure, , and there appears to be a linear relationship with serum urate concentrations. gout increases the risk of mortality from cardiovascular disease (hazard ratio [hr] · , % ci · - · ). during a mean followup of years among patients with gout, ( %) of deaths had a cardio vascular attribution. looking for trends and possible improvement of cardiovascular mortality in gout, analysis of a medical record database representative of the uk compared cumulative mortality rates of individuals with gout versus nongout between - and - . the investi gators found that the high mortality in patients with gout remained unchanged, whereas mortality rates for rheumatoid arthritis have improved over the same time period. this mortality gap might be related to suboptimal gout care (insufficient allocation of medication, insufficient treat ment, and low drug adherence), as well as insufficient management of cardiovascular comorbidities. gout speci fic character istics that are associated with high cardio vascular risk are elevated serum urate concentrations (> · mmol/l [> · mg/dl]), longer disease duration (≥ years), oligo articular or polyarticular disease, and review joint damage and tophi, which all reflect a more severe disease state. although rheumatoid arthritis and gout have different pathogenic stimuli (not known for rheumatoid arthritis; monosodium urate crystal deposition for gout), different immune pathways (eg, antibodymediated in rheumatoid arthritis; interleukin [il] driven in gout flares), and in most cases different clinical presentation (more chronic symmetric synovitis in rheumatoid arthritis; mostly mono arthritic gout flares with asymptomatic periods), imaging studies have shown that in gout, subclinical inflamma tion and synovitis are present, even during asymptomatic periods. importantly, persistent inflammation is known to be a key mechanism in the development of cardiovascular disease. , cardiovascular disease in patients with gout and rheumatoid arthritis can be divided into three main categories: atherosclerosis, thromboembolism, and cardiac dysfunction (figure ). the pathophysiological mechanisms of these diseases are different. however, the inflammatory processes of gout and rheumatoid arthritis in the develop ment of cardiovascular diseases partly overlap. postulated shared mechanisms of rheumatoid arthritis and gout are systemic inflammation, reactive oxygen species (ros)induced oxidative stress, and endothelial dysfunction, all of which lead to atherosclerosis (figure ). the pathways of inflammation in rheumatoid arthritis and gout share some components of the innate and adaptive immune system, including activated neutrophils. exaggerated neutrophil activation has been linked to auto immunity and autoinflammation in rheumatoid arthritis and gout. one of the supposed links is the formation of neutrophil extracellular traps (nets). netosis involves a neutrophil cell death process in which dna is extruded, together with cytoplasmic and granular contents, to trap and eliminate extracellular pathogens and neutralise inflammatory cytokines. this process, which was first described in gout by schauer and col leagues, could explain the selflimiting character of gout flares. extra cellular dna exerts cytotoxic and pro thrombotic effects and might be a causal link between inflammation and coagulation. moreover, complexes of presumably net borne dna stimulate vascular plasma cytoid dendritic cells, with a strong interferon type response, promoting atherogenesis. nets might also directly cause endothelial dysfunction by activation and induction of damage to endothelial cells, illustrated by the establishment of nets in atherosclerotic lesions and arterial thrombi in humans. in addition to neutrophils, different proinflammatory cytokines play a central role in the pathogenesis of rheumatoid arthritis and gout. in rheumatoid arthritis, the reason for immune system activation has not been completely elucidated. however, proinflammatory cyto kines, such as tumour necrosis factor (tnf), il , and il , are important in the inflammatory process. in gout, the central inflammatory pathogenic process is assumed to be hyperuricaemia, leading to monosodium urate crystal deposition. these crystals provoke a host response, result ing in recurrent gout flares by acting as a danger signal for the innate immune system through activation of the nlrp inflammasome with the immedi ate release of mature il β by resident macrophages, as well the release of other cytokines. the nlrp inflammasome has an important role in the initiation of a gout flare. inflam masome activity causes oxidative stress, mito chondrial dysfunction, endoplasmic reticulum stress, and lysosome rupture, which are all important events in atherogenesis and could lead to atherosclerotic plaque instability and ultimately rupture. ros are a group of small reactive species that play crucial roles in the regulation of biocellular processes. the balance between ros and antioxidant species is essential to maintain cellular homoeostasis. hence, an imbalance in oxidant and antioxidant mechanisms leads to a state of oxidative stress. in several chronic diseases, including rheumatoid arthritis, there is abundant oxidative stress. although essential for vascular homoeostasis, an excess of ros might lead to vascular injury. vascular injury being the result of a complex cascade, including oxidative modification of lipoproteins, endothelial activation, and leucocyte migra tion and differentiation resulting in accelera tion of atherogenesis. oxidative stress might also link gout with cardiovascular disease. although soluble urate has antioxidant properties, during urate generation the catalytic enzyme xanthine oxidase produces substantial amounts of ros that affect biocellular mechanisms. there is some experimental evidence that in patients with gout, higher activity of xanthine oxidoreductase (xo) is associ ated with larger amounts of ros. indirectly, it was shown that inhibiting endotheliumbound xo resulted in reduced ros with favourable effects on vascular function. , endothelial dysfunction is a maladaptive state with disruption of vascular homoeostasis resulting from a proinflammatory state. by expressing adhesion molecules and inflammatory cytokines, endothelial cells amplify an inflammatory cascade facilitating monocyte infiltration in the subendothelial layer. ldl cholesterol accumulates in the subendothelial space and is oxidised, which is essential for the development of atherosclerotic plaques. endo thelial dysfunction is the earliest phenomenon in the development of atherosclerosis and was first described in rheumatoid arthritis in ; it has been observed in patients with early rheumatoid arthritis without traditional risk factors and in those with longstanding disease. in rheumatoid arthritis, systemic inflammation with pro inflammatory mediators, such as il β and tnf, is assumed to play a role in the development of endothelial dysfunction, as shown in rodent models. impaired endotheliumdependent arterial responsive ness has been observed in patients with untreated gout, compared with individuals without gout, with the degree of impairment related to both serum urate and high sensitive creactive protein concentrations. there are published case series and case reports of urate crystal deposition in the spine and solid organs in histological samples fixed with alcohol (no dissolution of crystals). a current observational study investi gated alcoholfixed specimens of coronary arteries from explanted hearts with polarisation microscopy and detected strongly birefringent urate crystals in about % of coronary arteries. whether or not crystal deposition in the vessels with subsequent local inflammation con tributes to a higher cardiovascular risk in these patients is not known. the immune system and coagulation system are linked to increased activity of the fibrinolytic system in patients with inflammatory joint diseases. tissue factor initiates the extrinsic coagulation cascade and is found on extravascular cells, such as monocytes and neutrophils. creactive protein, tnf, il , and com ple ment activation might amplify tissue factor synthesis in monocytes and endo thelial cells, and high concen trations of tissue factor have been found in patients with rheumatoid arthritis, particularly in those with a high disease activity. conse quently, increased concentrations of coagulation factors were shown in patients with rheumatoid arthritis. the role for tnf seems plausible, as in the general population this cytokine might induce an imbalance between clotting and fibrinolysis, resulting in a hypercoagu lable state. further more, a role for il was suggested by a randomised trial in patients with rheumatoid arthritis that showed that il receptor blockade reduced coagulation activation parameters by more than % compared with placebo. to date, data on the role of cytokines in the develop ment of thromboembolism in gout are not available and observational data suggests that gout is an independent risk factor for the development of deep vein thrombosis and pulmonary embolism. in addition to extensive neutrophil activation in gout, increased platelet reactivity has been detected. this finding might serve as one explanation for a prothrombotic state and association with thromboembolism in gout. initially, cardiac dysfunction in patients with rheumatoid arthritis was assumed to be secondary to ischaemic heart disease. however, the incidence of congestive heart failure (heart failure with preserved ejection fraction and heart failure with reduced ejection fraction) cannot be explained by atherosclerotic disease alone. a casecontrol study showed that patients with rheumatoid arthritis had a rapidly increasing risk of developing nonischaemic conges tive heart failure after clinical onset. congestive heart failure was seen more frequently in patients with rheuma toid arthritis compared with patients with osteo arthritis (adjusted risk · % [ % ci · - · ] for rheumatoid arthritis vs · % [ · - · ] for osteoarthritis). patients with rheumatoid arthritis treated with tnf blocking agents had significantly less congestive heart failure than those who were not receiving antitnf treatment. another study found an increased prevalence of heart failure with preserved ejection fraction in patients with rheumatoid arthritis, which correlated primarily with disease activity and with antiinflammatory treat ments (ie, conventional synthetic dmards and biological dmards). in addi tion, patients with rheumatoid arth ritis are susceptible to more rapid subclinical changes in diastolic func tion than are the general population. altogether, these studies suggest that systemic inflam matory activity and cardiac dys function cannot be attrib uted to athero sclerosis alone. chronic systemic inflammation has two distinct path ways in the development of cardiac dysfunction ( figure ) . first, chronic systemic inflammation leads to myo cardial review remodelling, and, specifically, to diastolic dysfunc tion. this process starts with inflammationinduced microvascular dysfunction, leading to deposition of collagen with subsequent stiffness and hypertrophy of cardio myocytes and a decreased ability of the myocardium to contract and relax, which might evolve into heart failure with preserved ejection fraction. second, systemic inflam mation and traditional cardiovascular risk factorinduced coronary microvascular dysfunction and activation lead to athero sclerosis with ischaemic heart disease. this ischaemia leads to autophagy, apoptosis and necrosis of cardio myocytes, and collagen deposition in the interstitial space giving rise to systolic ventricular dysfunction. in severe cases, heart failure with reduced ejection fraction might develop. several studies have assessed the effect of anti inflammatory therapy on cardiac function in rheumatoid arthritis, and a systematic review revealed that biological dmard therapy probably has favourable effects on cardiac dysfunction in rheuma toid arthritis. remarkably, the pathogenesis of cardiac dysfunction in hyperuricaemia and gout has not been adequately investigated to date. previous studies showed an associ ation between serum urate concentration and inflamma tion, thus suggesting a comparable pathogenesis with rheumatoid arthritis. furthermore, increased serum urate concentration and higher activity of rosgenerating xo in gout might lead to endothelial dysfunction with decreased nitric oxide production. hypertension might also have a causal role because gout and hyperuricaemia are independent predictors of developing hyperten sion. one interventional study assessing uratelowering therapy in patients with heart failure did not find signifi cant beneficial effects of allopurinol, probably because there was already advanced structural myocardial damage and multimor bidity in the patient population. studies with early urate lowering inter ventions are needed to answer the ques tion of whether, and to what extent, urate concentration and the associated inflam ma tion in gout con stitutes an independent risk factor for cardiac dysfunction. most nonsteroidal antiinflammatory drugs (nsaids), including the cyclooxygenase inhibitors, are associated with about a twotimes increased cardiovascular risk. the excep tion is probably naproxen, although the pub lished literature is conflicting in this respect. cardio vascular risk is associated with cox selectivity, and the lower cox selectivity of naproxen than of other nsaids (eg, cyclooxygenase inhibitors) is assumed to translate into a lower cardiovascular risk. in clinical practice, weighing the benefits of these drugs against cardiovascular risk is often difficult. the first risk model that could aid in clinical decision making regarding the use of these drugs was published in . this model was based on the precision trial, a large randomised controlled trial in which patients with osteoarthritis or rheumatoid arthritis were random ised to naproxen, ibuprofen, or celecoxib. a major toxicity risk calculator was developed from easily accessible cardiovas cular risk factors (ie, age, gender, diabetes, cardio vascular disease, hypertension, current smoking, statin use, serum creatinine, rheumatoid arth ritis, and haemato crit) to calculate a year risk score yielding three risk categories: low (< %), inter medi ate ( - %), or high risk (> %). however, external validation is necessary before such a calculator can be implemented in daily clinical practice. there has been a longstanding debate on whether steroids (eg, low dose of prednisolone, < mg daily) have unfavourable cardiovascular effects in an inflammatory situation. this effect is suggested by observational studies, but these data are confounded by indication because of high disease activity and should therefore be inter preted with caution. hopefully this debate can be settled by the gloria trial (nct ), a multicentre, year trial assessing the safety and effectiveness of a daily mg dose of prednisolone versus a matching placebo added to standard of care in older patients (aged ≥ years) with rheumatoid arthritis. systemic inflammation with elevated concentrations of circulating cytokines, such as il- and tnf, induce oxidative stress and endothelial activation. consequently, presentation of adhesion molecules (vcam- and e-selectin) by endothelial cells leads to monocyte infiltration in the myocardium. these monocytes produce tgf-β, resulting in the differentiation of fibroblasts into myofibroblasts, with subsequent deposition of collagen in the interstitial space. in addition, intracellular oxidative stress results in disrupted crosstalk between endothelial cells and cardiomyocytes, leading to stiffness and hypertrophy of cardiomyocytes with a subsequent decreased ability to contract and relax. these processes ultimately lead to preclinical diastolic ventricular dysfunction, which might evolve into heart failure with preserved ejection fraction. ischaemia, mostly secondary to atherosclerosis, leads to autophagy, apoptosis, and necrosis of cardiomyocytes, and deposition of collagen in the interstitial space. this condition can give rise to systolic ventricular dysfunction, which in severe cases can lead to heart failure with reduced ejection fraction. adapted from paulus and tschöpe, one of the first studies suggesting that cardiovascular mortality risk in rheumatoid arthritis could be reduced by methotrexate was published in . of patients with rheumatoid arthritis with a mean followup of years, ( %) patients had died, ( %) of which were cardiovascular deaths. methotrexate use was associ ated with a % reduction in cardiovascular mortality (hr · , % ci · - · ). methotrexate also has favourable effects on nonfatal cardiovascular events, with metaanalysis showing a risk reduction of % in comparison with treatment without methotrexate. other conventional synthetic dmards, such as sulfasalazine, might also have favourable cardio vascular effects. in addition to beneficial effects on the lipid profile, hydroxychloroquine has been associated with a reduc tion in cardiovascular events in patients with rheumatoid arthritis. the recent association of highdose hydroxy chloroquine with qt prolongation in patients with covid has raised questions about the use of hydroxy chloroquine in patients with rheumatoid arthritis. as such, it is important to note that disease activity of rheumatoid arthritis itself is associated with qt prolonga tion. furthermore, there have been no published reports indicating that the use of hydroxychloquine in patients with rheumatoid arthritis is associated with an increased risk for qt prolongation. hence, there is no indication to restrict the use of hydroxychloroquine in people with rheumatoid arthritis to require baseline electrocardiograms. the first largescale investigation that compared the effect of antitnf agents versus conventional synthetic dmards on cardiovascular mortality in patients with rheu matoid arthritis found an adjusted hr for death of · ( % ci · - · ). the risk of acute coro nary syndrome in rheumatoid arthritis was studied in patients from a swedish registry ( patientyears) compared with the general population; the hr for acute coronary syndrome was · ( % ci · - · ) for patients who were previously untreated with biolo gics and · ( · - · ) for patients using tnf inhibitors. a metaanalysis of studies with patients with rheumatoid arthritis investigated the reduction in cardio vascular events with methotrexate and tnf inhibitors in comparison with patients previously untreated with these agents. cardiovascular events were observed and the relative risk was · ( % ci · - · ) for metho trexate and · ( · - · ) for tnf inhib itors. hence, favourable cardiovascular effects might be mediated through an overall reduction in inflammation and are not drug specific. in , janus kinase (jak) inhibitors were suggested to be associated with an increased risk of thromboembolic disease and concerns have been reported by the us food and drug administration (fda) about the safety of tofacitinib. these concerns emerged from a postmarketing trial evalu ating the safety of tofacitinib at two doses ( mg twice daily and mg dose twice daily) versus a control group given a tnf inhibitor. there was an increased inci dence of pulmonary embolism and an increased overall mortality in patients taking mg tofacitinib twice daily. subse quently, a warning was issued indicating that tofacitinib mg twice daily is contraindicated in patients who have an increased thromboembolic risk. in view of these events, it is important that the prescribed dose for the treatment of rheumatoid arthritis is restricted to mg twice daily. the trial programme for baricitinib, the other jak inhibitor currently licensed for rheumatoid arth ritis, identified more thromboembolic events in the mg group in comparison with the placebo group, and as a result the mg dose was not approved in the usa. by contrast, a warning was issued in europe, indicating that the mg dose should not be used in patients with rheumatoid arthritis with an increased risk of venous thrombo embolism. further studies are needed to investi gate and to differentiate between the potential adverse effect of jak inhibitors and the hypercoagulable character of rheumatoid arthritis. firstline treatment of gout flares is usually with an nsaid or cyclooxygenase inhibitor. the duration of treatment, in general, is short, as gout flares in the early stages of disease are often limited to a few days. whether or not this treatment increases cardiovascular risk is not known. furthermore, there are no data on the cumulative yearly dose of nsaid or cyclooxygenase inhibitor intake for patients with recurrent gout flares, as these drugs are often available without prescription. in advanced gout in particular, chronic use of these drugs might be relevant with respect to adverse cardiovascular effects. colchicine is frequently used for treatment of acute gout flares and is also given as a lowdose regimen to prevent gout flares during initiation of uratelowering therapy. colchicine was first used as an antiinflammatory treatment in cardiovascular disease. a narrative review showed that colchicine has beneficial effects on athero sclerosis with plaque stabilisation, reduction of cardiovascular damage, and reduction in recurrence of acute coronary syndromes. colchicine might also have protective effects in stable coronary artery disease. two retrospective cohort studies in patients with gout showed a lower incidence of combined cardiovascular out comes in patients treated with colchicine. , by contrast, a trial showed that shortterm lowdose colchicine does not improve endothelial function in patients with coronary artery disease. however, an exploratory analysis indicated that endothelial function was significantly improved in the subgroup of patients with leucocyte activation, further indicating the important role of inflammation in athero sclerosis. ongoing trials, such as the colcot trial (nct ), will hopefully settle the debate about the efficacy of lowdose colchicine for secondary preven tion in patients with coronary disease. in patients with gout, more research is needed to establish the effects of colchicine on cardiovascular risk. gout flares that do not respond to nsaids, cyclo oxygenase inhibitors, or colchicine are frequently treated with steroids, either systemically or by intraarticular injec tion. as data on patients with gout are scarce, one can only speculate as to whether the typically short treatment duration has any unfavourable cardiovascular effects. in , the antiil β antibody canakinumab was approved by the fda and the european medicines agency for gout inflammation refractory to conven tional antiinflam matory treatment. a trial of the il receptor antagonist, anakinra, showed efficacy in controlling inflammation during gout flare without cardio vascular safety issues. the cantos trial, which investi gated antiinflammatory therapy with canakinumab for secon dary cardiovascular prevention in patients with myocar dial infarction, showed only a modest decrease in recurrent cardiovascular events (about %) compared with placebo, and this result was independent of lipid lowering effects. whether patients with gout had a cardio vascular bene fit from il βblocking therapy was not answered by this trial. in patients with diagnosed gout, uratelowering therapy with a treattotarget strategy to reduce serum urate below · mmol/l ( mg/dl) is an essential therapeutic intervention recom mended by current gout guidelines. the efficacy of a treattotarget approach on regression of tophi, frequency of gout flares, and mridetected synovitis has been shown. to reach the serum urate target, patients could be treated with uratelowering drugs such as xo inhibitors (eg, allopurinol), uricosurics (and combination with xo inhibitors-eg, benzbromaron), or a recombinant uricase (eg, rasburicase). uratelowering therapy should, in theory, reduce the risk of cardiovascular disease in gout. this therapy could decrease the risk by directly lowering urate concentration or indirectly through xo inhibition, with a subsequent reduc tion in oxidative stress and improvements in endothelial function. a systematic review and metaanalysis of randomised controlled trials revealed that uratelowering therapy with allopurinol, a purine xo inhibitor, lowered the incidence of major cardiovascular events (or · , % ci · - · ), total cardiovascular events (or · , · - · ), myocardial infarction or the need of urgent revas cularisation (or · , · - · ), and new or worsening hyper tension (or · , · - · ) in patients with various cardiovascular conditions versus the control group, but did not affect overall or cardiovascular mortality. these effects were not observed with nonpurinelike xo inhib itors, such as febuxostat. however, febuxostat lowered the composite event rate (cerebral, cardiovascular, and renal events, as well as all deaths) in patients with hyper uricaemia who were at risk of cerebral, cardio vascular, or renal disease compared with a nonfebuxostat group (hr · , % ci · - · ). in a systematic review and metaanalysis in patients with gout uratelowering therapy with an xo inhibitor was not shown to reduce cardiovascular events compared with placebo. in addition, another systematic review and metaanalysis showed that there was no significant association between allcause mortality and allopurinol use in patients with gout, albeit the number of studies was small. finally, a trial of nurseled gout care in a primary care setting consisting of extensive patient education and involvement, as well as a treattotarget strategy, was shown to be superior in reach ing a serum urate con centration of less than · mmol/l ( mg/dl) compared with standard care ( % vs %, risk ratio · , % ci · - · , p< · ). fewer deaths were observed in the nurseled care group, but numbers were too small to draw firm con clusions. to date, data regarding the effect of uricosurics or uricase treatment on cardiovascular risk in gout are absent. in general, available evidence is not sufficient to draw definite conclusions and further studies are needed. in , the fda issued a public safety alert with a warning of increased risk of death with febuxostat compared with allopurinol. this warning was based on results from the cares trial. although febuxostat was shown to be noninferior to allopurinol for the primary outcome (a composite of death and major cardiovascular events; hr · , upper limit of the onesided · % ci · , p= · for noninferiority), the incidence of secondary outcome measures, including death from any cause (hr · , % ci · - · ) and cardiovascular death (hr · , · - · ), was significantly higher with febuxostat than with allopurinol. definite conclusions from this trial are difficult to draw: first, no significant difference was observed in the primary outcome of the trial, second, there was a very high rate of discontinuation of therapy, with the majority of deaths occurring after stopping the drug, and finally, a control group without a xo inhibitor was absent. the fda therefore first mandated febuxostat not to be used in patients with cardio vascular disease but thereafter restricted the approved use to patients who could not be treated effectively or had severe sideeffects with allopurinol. important data will come from the fast trial, comparing the cardiovascular safety of allopurinol and febuxostat in the management of symptomatic hyperuricaemia (eudract number: - - , isrctn ). the recognition of an increased cardiovascular disease risk in arthritis prompted the european league against rheumatism (eular) to set out evidencebased recom mendations for the management of cardiovascular disease risk in inflammatory arthritis, and these guidelines were updated in . rheumatoid arthritis is also now accepted as an independent cardiovascular disease risk factor by the european society of cardiology guidelines. to reduce the risk of cardiovascular disease in patients with rheumatoid arthritis, optimal disease control is neces sary, and cardio vascular disease management should be the responsibility of the rheumatologist. risk assessment review should be done at least once every years, and should be reassessed following major changes in dmard therapy. cardiovascular risk management is also important for patients with gout, since gout is associated with a high rate of traditional cardiovascular risk factors and is also considered an independent risk factor for cardiovascular disease and associated mortality. in the update of eular's evidencebased recommendations for the management of gout, two overarching principles were that every patient with gout should receive advice regarding lifestyle and that they should be screened for cardiovascular comorbidities and cardiovascular risk factors. how ever, no specific recommenda tions for cardiovascular risk management were given. this omission was one of the main reasons for the assembly of a eular taskforce to develop recommenda tions for the management of athero sclerotic cardiovascular risk in rheu matic and musculo skeletal diseases, including sys temic lupus erythematosus, antiphospholipid syn drome, vasculitis, and gout. in europe, the systematic coronary risk evaluation calculator is used for cardiovascular disease risk pre diction. this model estimates the year risk of cardio vascular mortality and includes age, gender, smoking status, total cholesterol to hdl cholesterol ratio, and systolic blood pressure. however, systematic coronary risk evaluation was developed solely for the general popula tion and therefore underestimates the cardio vascular risk in patients with rheumatoid arthritis. to correct for this underestimation, eular recommends use of a multi plication factor of · . importantly, eular advises screening for cardiovascular disease risk in all patients with inflammatory arthritis and states that the rheumatol ogist is responsible for its initiation. subse quently, several prediction models for cardiovascular disease risk, such as qrisk , have incorporated rheu matoid arthritis in their risk model. the expanded risk score in rheumatoid arthritis is derived from the corrona registry and includes rheumatoid arthritis specific features (laboratory data are not needed). this risk score appears to perform as well as qrisk . the the table shows the characteristics of these algorithms. thus far, no goutspecific cardiovascular risk scores have been developed, probably because of an absence of con sistent data on cardiovascular outcomes. statins reduce cholesterol concentrations and cardio vascular disease in patients with rheumatoid arthritis to a similar extent as in the general population. furthermore, statins are not associated with more adverse events in comparison to the general population. the trace ra study compared the effect of mg atorvastatin with placebo in patients with rheumatoid arthritis and found a significant reduction in ldl cholesterol in the atorvastatin group. in this group, ldl concentrations were similar to those observed in the general population. a risk reduction of % in major cardiovascular events compared with placebo was observed, although this difference did not reach statistical significance possibly due to a lower than expected event rate. furthermore, statins have anti inflammatory properties that might translate into further reductions in cardiovascular disease risk as well as a modest reduction in rheumatic disease. a metaanalysis has shown the pleiotropic effects of statins, with a decline in inflammation markers (oestrogen receptor, creactive protein), proinflammatory cytokines (tnf, il , and il ), and fewer tender and swollen joints. the eular recommendations for gout advise syste matic screening and care in terms of cardiovascular diseases and risk factors. this strategy can be complicated because many drugs targeting comorbidities can aggravate hyperuricaemia and gout by lowering renal excretion of uric acid-eg, antihypertensive drugs such as β blockers, nonlosartan angiotensin receptor blockers, thiazides, and loop diuretics. this is also true for lowdose aspirin, but as the effect is very modest, use of lowdose aspirin for cardioprotective reasons should not be precluded in highrisk patients with gout. statins (atorvastatin and simvastatin), and also fenofibrate, have uratelowering effects by increasing renalurate excre tion. the effects of dietary measures in rheumatoid arthritis are still uncertain because of an absence of adequately powered studies. observational studies show that diet, exercise, and stress are associated with outcomes such as inflammation or disease activity. therefore, eular recom mends a general healthy lifestyle for all patients with rheumatoid arthritis, defined as a healthy diet (mediterranean), regular exercise, and smoking cessation. exercise improves cardiorespiratory fitness and decreases cardiovascular risk in patients with rheumatoid arthritis. there is lowtomoderate quality evidence for beneficial effects of weight loss with respect to lowering serum uric acid, achieving serum uric acid targets, and preventing flares in patients with gout. the eular guideline recom mends that patients with gout receive advice regarding lifestyle that includes avoidance of alcohol and sugar sweetened drinks, and discourages excessive intake of meat and seafood (purinelow diet). in addition to purines, the intake of fructose is also associated with an increase in uric acid production and should be avoided. guidance on smoking cessation is similar to the general population as smoking has multiple adverse effects, such as increasing the risk of cardiovascular disease, respir atory disease, and cancer. smoking also seems to have a pathogenic role in rheumatoid arthritis, promoting disease activity and reducing response to antirheumatic therapy. in contrast to rheumatoid arthritis, a relationship between smoking and the development of gout has not been found. many studies have indicated poor adherence to cardio vascular disease risk, management in rheumatoid arth ritis. for example, in a crosssectional study ( %) of patients with rheumatoid arthritis had hypertension, of which ( %) were treated with antihypertensive medications. moreover, despite treatment, many of these patients had suboptimal blood pressure. also, hyper cholesterolaemia is under diagnosed and undertreated in patients with rheumatoid arthritis. notwithstanding guidelines for the implementation of cardiovascular risk, management in clinical practice is often difficult. nevertheless, successful implementation of cardiovascular disease risk management was shown in a dutch programme for cardiovascular disease care. this programme included a collaboration between primary care and secondary care professionals with a shared laboratory system for primary care physicians and rheumatologists, in which primary care physicians received reimbursement for additional costs from health care insurance companies. lipid screening (as a proxy for cardiovascular disease risk management) was done in ( %) of patients with rheumatoid arthritis and increased to almost % after primary care physicians were sent reminder letters. implementation of cardiovas cular risk management for patients with gout has not been syste matically studied and to what extent it has been applied in either primary or secondary care is unknown. unfortunately, despite several guidelines for cardiovas cular disease risk management, implementation in daily clinical practice is still poor. a study in the uk confirmed this in a group of patients with gout in primary care. monitoring of lipids ( [ %]), blood pressure ( [ %]), and glucose ( [ %]) within month after their first gout consultation was infrequently recorded. this finding indicates the urgent unmet need for optimisation of cardiovascular risk management in these patients. at the same time, we should not forget that patients often also have other comorbid conditions. optimal preven tive treatment requires attention to these comorbidities, which are commonly seen in inflammatory arthritis and include osteoporosis, fatigue, and depression. the rationale is that the underlying mechanisms of these common comor bidities, particularly in rheumatoid arth ritis, overlap. in our view, such a multi morbidity, holistic approach is the optimal way to achieve substantial improvement in the quality of life of these patients. lastly, further randomised controlled trials with cardiovascular end points are needed, especially in gout, to ascertain optimal serum urate concentrations to improve the cardiovascularrisk profile. another relevant question is whether early uratelowering therapy-in case of asympto matic hyper uricaemia and evidence of crystal deposi tion by ultrasound or dual energy ct-has favourable cardio vascular effects, as shown for early treat ment intervention in patients with rheumatoid arthritis. rheumatoid arthritis and gout-two inflammatory joint diseases with different underlying causes-are associated with about a - % increased risk of cardiovascular disease compared with the general population. these patients not only have inflammation of joints but also experience systemic effects, including cardiovascular and haematological manifestations. different underlying pathophysiological mechanisms, such as systemic inflam mation, elevated oxidative stress, endothelial dysfunction, and changes in lipid profiles, might contribute to a substantially higher cardiovascular risk in these patients. the increased cardiovascular risk includes not only a higher rate of ischaemic cardiovascular disease but also subclinical heart failure, which seems far more prevalent than previously thought. early therapeutic intervention with current antirheumatic treatment in rheumatoid arthritis has shown favourable effects on cardiovascu lar disease risk. unfortunately, in gout, evidence that uratelowering therapy has consistent beneficial effects review on cardiovascular outcomes is still scarce. following the recognition that inflammation has an important causative role for cardiovascular risk in gout, anti inflammatory therapy and uratelowering therapies are expected to reduce the cardiovascular burden of these patients. therefore, optimal antiinflammatory control of patients with rheumatoid arthritis and effective urate lowering therapy in patients with gout are the main treatment goals to date. in addition to adequate control of disease activity, attention of the treating physician should be given to the treatment of concomitant cardio vascular diseases and management of risk factors-eg, adequate control of hypertension and dislipidaemia. for individual cardio vascular risk estimation in patients with rheumatoid arthritis and gout, the use of estab lished cardiovascular risk scores (eg, systematic coronary risk evaluation) could be implemented in daily assess ments. the inclu sion of a multiplication factor for gout in cardiovascular risk scores, as already accepted for rheumatoid arthritis, should be considered. all authors searched and screened references. the figures were constructed by mb and dv. the drafts were cowritten by rh, dv, mb, akt, mg, and mtn. all authors contributed to the final writing and editing of the submitted manuscript. dv has received speaker fees from novartis. akt has received speaker fees from berlinchemie menarini, novartis, and pfizer; personal fees and nonfinancial support from astrazeneca; and personal fees from grünenthal, outside the submitted work. mg has received speaker fees from sobi and roche, and research support from berlinchemie menarini and grünenthal. mtn received consulting fees from abbvie, celgene, celltrion, eli lilly, janssen, and sanofi; speakers fees from abbvie, bristolmyers squibb, eli lilly, roche, and sanofi; and research funding from abbvie, bristolmyers squibb, celgene, eli lilly, janssen, msd, mundipharma, novartis, pfizer, roche, and sanofi. all other authors declare no competing interests. we followed the main steps in writing a narrative review, and mtn formulated cardiovascular research questions addressing epidemiological and pathophysiological aspects of rheumatoid arthritis and gout, cardiovascular effects of drug treatment, and management of cardiovascular risk. references for this review were identified through searches of pubmed with the terms "rheumatoid arthritis", "gout", "hyperuricemia", "gouty", "arthritis, "gouty", "atherosclerosis", "cardiovascular disease", "cardiovascular event", "myocardial ischemia", "cerebrovascular disease", "stroke", "angina pectoris", "coronary disease", "coronary artery disease", "coronary arterial disease", "peripheral artery disease", "peripheral arterial disease", "congestive heart failure", "cardiac dysfunction", and "cardiovascular risk management" from jan , , to april , . articles were also identified through searches of the authors' own files. only papers published in english were reviewed. the final reference list was 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rheumatoid arthritis patients despite an explicit cardiovascular risk screening programme implementation of the eular cardiovascular risk management guideline in patients with rheumatoid arthritis: results of a successful collaboration between primary and secondary care prescription and comorbidity screening following consultation for acute gout in primary care key: cord- -qv ycdhe authors: miller, maureen; hagan, emily title: integrated biological–behavioural surveillance in pandemic-threat warning systems date: - - journal: bull world health organ doi: . /blt. . sha: doc_id: cord_uid: qv ycdhe economically and politically disruptive disease outbreaks are a hallmark of the st century. although pandemics are driven by human behaviours, current surveillance systems for identifying pandemic threats are largely reliant on the monitoring of disease outcomes in clinical settings. standardized integrated biological–behavioural surveillance could, and should, be used in community settings to complement such clinical monitoring. the usefulness of such an approach has already been demonstrated in studies on human immunodeficiency virus, where integrated surveillance contributed to a biologically based and quantifiable understanding of the behavioural risk factors associated with the transmission dynamics of the virus. when designed according to strengthening the reporting of observational studies in epidemiology criteria, integrated surveillance requires that both behavioural risk factors – i.e. exposure variables – and disease-indicator outcome variables be measured in behavioural surveys. in the field of pandemic threats, biological outcome data could address the weaknesses of self-reported data collected in behavioural surveys. data from serosurveys of viruses with pandemic potential, collected under non-outbreak conditions, indicate that serosurveillance could be used to predict future outbreaks. when conducted together, behavioural surveys and serosurveys could warn of future pandemics, potentially before the disease appears in clinical settings. traditional disease-outcome surveillance must be frequent and ongoing to remain useful but behavioural surveillance remains informative even if conducted much less often, since behaviour change occurs slowly over time. only through knowledge of specific behavioural risk factors can interventions and policies that can prevent the next pandemic be developed. no other modern epidemic or pandemic mobilized the global health community to action like the - ebola virus disease outbreak in western africa. following the outbreak, calls for pandemic-threat warning systems came from both traditional public health policy-makers , and national governments. as currently conceptualized, the first step in the identification of a pandemic threat requires an outbreak of sufficient size to come to the attention of medical personnel who are sufficiently influential and persistent to ensure action. once an outbreak is verified, well-established protocols for disease investigation and control can be swiftly put in place -although it may be many months before the main risk factors and most effective control measures are identified. the ebola outbreak in western africa probably began in december but it took another year before traditional burial practices were found to be a leading cause of the rapid spread of the causative virus. although human behaviours often increase the risk of acquiring an infectious disease, the systematic investigation of human risk behaviours is seldom included in disease surveillance strategies. however, behavioural surveillance to improve the understanding of human immunodeficiency virus (hiv) and acquired immunodeficiency syndrome has been ongoing for decades. behavioural assessment was key to the identification of injecting drug users as a high-risk group for hiv infection in the early s. it was also crucial in documenting the risks posed by hiv to women. subsequently, an innovative, practical method, which combines biological outcome data with behavioural risk factor data -i.e. exposure variables -was developed to document hiv transmission dynamics. such integrated biological-behavioural surveillance has since become well established and standardized and been frequently implemented globally. , it has contributed extensively to a biologically-based and quantifiable understanding of the behavioural risk factors associated with the acquisition and transmission of hiv and the early identification of subgroups of the population that may be more vulnerable to hiv infection. more recently, data from integrated surveillance have been used to evaluate the impact of evidence-based interventions to prevent hiv infection and to monitor treatment uptake. similar surveillance could help identify behavioural risk factors and high-risk subgroups for zoonotic infections such as ebola -potentially before diseases of pandemic potential are identified in clinical settings or major outbreaks occur in communities. approximately half of the emerging pandemic threats are zoonotic in origin. , at the time of writing, the most lethal and costly pandemics of the st century, i.e. avian influenza, ebola, middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars), have all been caused by zoonotic viruses. [ ] [ ] [ ] [ ] little is known about either the risk factors that lead to the initial spillover of a zoonotic disease into human populations or the dynamics of any subsequent humanto-human transmission. much more is known about (i) the locations of so-called hotspots where, many scientists believe, new pandemics of zoonotic disease are likely to emerge; (ii) the kinds of ecological and environmental activities that have been associated with spillover and outbreaks of zoonotic disease in the past; and (iii) the distinct spatial groupings of specific infectious diseases on a global scale, and the associ-abstract economically and politically disruptive disease outbreaks are a hallmark of the st century. although pandemics are driven by human behaviours, current surveillance systems for identifying pandemic threats are largely reliant on the monitoring of disease outcomes in clinical settings. standardized integrated biological-behavioural surveillance could, and should, be used in community settings to complement such clinical monitoring. the usefulness of such an approach has already been demonstrated in studies on human immunodeficiency virus, where integrated surveillance contributed to a biologically based and quantifiable understanding of the behavioural risk factors associated with the transmission dynamics of the virus. when designed according to strengthening the reporting of observational studies in epidemiology criteria, integrated surveillance requires that both behavioural risk factors -i.e. exposure variables -and disease-indicator outcome variables be measured in behavioural surveys. in the field of pandemic threats, biological outcome data could address the weaknesses of self-reported data collected in behavioural surveys. data from serosurveys of viruses with pandemic potential, collected under non-outbreak conditions, indicate that serosurveillance could be used to predict future outbreaks. when conducted together, behavioural surveys and serosurveys could warn of future pandemics, potentially before the disease appears in clinical settings. traditional disease-outcome surveillance must be frequent and ongoing to remain useful but behavioural surveillance remains informative even if conducted much less often, since behaviour change occurs slowly over time. only through knowledge of specific behavioural risk factors can interventions and policies that can prevent the next pandemic be developed. integrated surveillance for pandemic threats maureen miller & emily hagan ated ecological and virological barriers to the dispersal and establishment of those diseases. in the development of pandemic-threat warning systems, integrated biological-behavioural surveillance can be tightly focused on specific viral families in the high-risk population subgroups that live in identified hotspots and are environmentally or occupationally exposed to animals. the remainder of this article presents an overview of issues relevant to the design of rigorous behavioural surveys to assess the spillover of emerging zoonotic disease and the associated transmission risk factors, which is the first step in designing effective integrated surveillance. we identified community-based serological surveys of viruses with pandemic potential as a possible source of useful biological outcome data. we summarize the results of such serosurveys, conducted in nonoutbreak settings in africa, and evaluate their usefulness -especially when used in combination with behavioural surveillance -in the prediction of future outbreaks. when designed according to strengthening the reporting of observational studies in epidemiology criteria, integrated surveillance requires that both disease-indicator outcome variables and behavioural risk factors be measured in behavioural surveys. behavioural risk factors, i.e. exposure variables, simply represent the population prevalence of behaviours that may or may not increase the risk of disease. without the outcome variables, the exposure variables are of little use in elucidating the mechanisms of the spillover of zoonotic disease to humans or of subsequent human-to-human transmission. effective surveillance requires questions that assess a range of animal exposures, document experiences of unusual illness and measure contextual factors that can lead to an increase or decrease in the probabilities of behavioural risk factors and disease. in studies on zoonoses, the assessment of behavioural risk factors is complicated because different zoonotic diseases may be associated with different kinds of animal exposure. the spillover of zoonotic viruses from wildlife, the source of most emerging zoonoses, has been difficult to document. behavioural risk may be either direct or indirect. direct contact with primate blood or bodily fluids has been associated with several zoonotic viruses found in humans, such as human t-lymphocyte virus, simian foamy virus , and, possibly, ebola. indirect contact was responsible for the transmission of nipah virus to humans, which was mediated through date-palm sap contaminated with the urine of infected bats, and hantavirus transmission is most frequently associated with inhalation of aerosolized virus from the excreta of infected rodents. both general exposure to animals, e.g. when buying live animals at market, and more intimate exposure, e.g. during the slaughter of animals or as the result of animal bites, must therefore be assessed. syndromic surveillance is widely used to monitor illnesses of unknown etiology in clinical settings and can provide a useful referent in the identification of the outcome variables to be measured in behavioural surveys. several zoonotic diseases, such as avian influenza, mers and sars, were identified via syndromic surveillance networks, from localized increases in the incidence of influenza-like illness or severe acute respiratory infection. by using standardized definitions, it should be easy to develop questions or symptom checklists for syndromic surveillance based on self-reported data. such definitions already exist for influenza-like illness, severe acute respiratory infection and other syndromes consistent with zoonotic infection, such as encephalitis of unknown origin, fever of unknown origin and haemorrhagic fever of unknown origin. the risks posed to humans by exposure to animals may be modified by various biological, ecological, economic, political and sociocultural factors. for example, poverty can place individuals and communities at the forefront of zoonotic disease risk through several mechanisms. exposure to dense or diverse rodent populations in urban or rural environments , and the displacement of wildlife populations as land is cleared for crops are some mechanisms. understanding the context within which spillover to humans can occur is an important component in the prevention of zoonotic outbreaks. the same behavioural risk factors may be risky in one context but not in another. for instance, the sharing of a water source with animals displaced by a change in land use may only have an adverse effect on human health if there is faecal-oral transmission of the zoonotic pathogen to humans. if such transmission requires contact with the animal blood, then the sharing of the water source should not increase the risks of either spillover or transmission. once human-to-human transmission of a zoonotic pathogen occurs, additional risks come into play, often as a consequence of the human infection, and these should also be measured pre-emptively. burial practices and health-care-seeking practices were associated with explosive increases in, respectively, the incidence of human infection with ebola virus in western africa and mers in the republic of korea. even in the absence of detailed biological outcome data, behavioural surveillance may be used to assess relationships between behavioural risk factors and self-reported experiences of unusual illness that are consistent with the symptoms of zoonotic disease. this could be done both rapidly and at a scale that facilitates epidemiologically relevant analyses. although not as conclusive as biological data, self-reported data could provide substantially more information than is currently available. in the field of pandemic threats, biological outcome data could address the weaknesses of any self-reported data collected in behavioural surveys. the ideal source of biological outcome data, i.e. data that provide the strongest evidence of zoonotic disease spillover, would be community-based screening for acute infection with zoonotic viruses. however, as such infection is rare under nonoutbreak conditions, many thousands of individuals would usually have to be screened for a meaningful analysis of the behavioural risk factors. serological assays, in which previous exposure to a virus is demonstrated by a positive result, can provide alternative biological outcome data. since many more individuals may have been exposed to a virus than are currently ill with the virus, serology can provide the larger number of individuals, with known viral exposure, required for powerful analyses of behavioural risk factors. we therefore investigated the results of serosurveys for their potential usefulness in the prediction of future outbreaks. we focused on studies conducted in communities under non-outbreak settings in africa. we collated results presented in peer-reviewed publications -in english, french or spanish -that we identified via google scholar and web of science searches that ended on december (available from the corresponding author). we used "africa", "antibody", "serology", "serosurvey", "zoonoses" and "zoonotic disease" as search terms. we identified additional relevant results through the citations in the articles identified in the searches. serosurveys of zoonotic viruses have been conducted since the discovery of the ebola virus in the s, mostly during or shortly after an outbreak of zoonotic disease. our searches revealed serosurveys of zoonotic viruses in africa that had been conducted during non-outbreak conditions. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] to identify any associations between population subgroup risk and seroprevalence, we divided the subjects of the serosurveys into three risk categories, based on the limited data from previous research on zoonotic disease spillover. for example, hunters have been consistently found to be a high-risk population subgroup, , , , , followed by rural populations, who have been categorized as medium-risk because of their close and regular proximity to wildlife. , , , , , , randomized or representative samples of general populations , have been considered to be low-risk. serological assays for several different zoonotic pathogens were conducted as part of serosurveillance in each of the studies included in our analysis. use of these assays led to the recording of a total of unique zoonotic pathogen seroprevalences that ranged from % to %. of these seroprevalences, nine were recorded for high-risk population subgroups, for medium-risk and for low-risk. evidence of previous exposure to a zoonotic pathogen, i.e. a seroprevalence of more than zero, was detected in eight ( %) of the high-risk population subgroups, ( %) of the medium-risk and seven ( %) of the low-risk (available from the corresponding author). high seroprevalences, i.e. seroprevalences of at least %, represented the results for all eight of the high-risk subgroups with evidence of previous exposure to a zoonotic pathogen, ( %) of the medium-risk subgroups with such evidence and three ( %) of the low-risk subgroups with such evidence (available from the corresponding author). exposure to wildlife therefore appeared to be associated both with any evidence of viral exposure and with high seroprevalence. since the first known outbreak in , the united states centers for disease control and prevention have recorded documented ebola outbreaks in africa. more than % of the subjects included in serosurveys in gabon in and liberia in showed evidence of exposure to ebola virus, that is decades before an ebola outbreak was first reported in either of these countries. between . % and . % of the subjects included in ebola serosurveys in three countries that have never reported an ebola outbreak, the central african republic, , , madagascar and zimbabwe, were also found to be seropositive. although serological assays exist for the coronaviruses that cause mers and sars, hantaan viruses and paramyxoviruses, most serosurveys of zoonotic viruses in africa have focused on haemorrhagic fevers. most of the serosurveys we reviewed had also been done before the widespread availability and use of viral detection tests. recently, population-based serosurveys have been increasingly adopted, in recognition of their utility in preparing health authorities for potential outbreaks or epidemics. , , current serological assay methods tend to be labour-intensive and to suffer from cross-reactivity that prevents distinction between several antigenically related viruses. however, the last few years have witnessed major advances in the development of economically feasible methods for comprehensive serological profiling , and these should facilitate the investigation of zoonotic spillover into human populations. we review the potential contributions that integrated biological-behavioural surveillance could make to pandemicthreat prediction, prevention and risk mitigation. if we are to mitigate the risk of a zoonotic disease outbreak, we need a better understanding of the mechanisms behind the spillover of zoonotic disease into human populations. by making such mechanisms the focus of integrated surveillance, we should be able to: (i) monitor the presence and prevalence of behavioural risk factors and the seroprevalence of specific zoonotic pathogens within particular population subgroups; (ii) deploy targeted control and mitigation strategies rapidly; and (iii) evaluate the efficacy of prevention policies and interventions. although traditional disease surveillance must be frequent and ongoing to remain useful, behavioural surveillance remains informative even if conducted much less often, since behaviour change occurs slowly over time. to be effective as a prevention tool, integrated biological-behavioural surveillance should be implemented as a baseline measure -to identify behavioural risk factors, determine the prevalence of those risk factors, especially in any population subgroups that are considered at higher risk of zoonotic spillover, and establish seroprevalence. should an outbreak occur, a database that documents local behaviours and practices, as well as the context within which such behaviours occur, can contribute to the development of appropriate and feasible strategies for disease control and mitigation. finally, data from integrated surveillance will be invaluable in both informing realistic and effective interventions and policies for the prevention of zoonotic spillover and transmission, and evaluating the impact of such interventions and policies efficiently. relative to the economic, social and political costs of epidemics, prevention will always be less expensive , if the targets of prevention activities are well understood and acted upon. the fact that success can feel more like the status quo is a challenge unique to prevention. the political commitment for prevention activities will often be less than that for a reactive response elicited by the emergence of a terrifying new infectious disease. however, political support may be improved if surveillance is made to be, and appear, more cost-effective, by focusing on specific diseases in population subgroups who live in ecologically fragile hotspots. , there is substantial overlap between areas considered to be hotspots for zoonotic disease spillover and those considered hotspots for hiv, perhaps the best known zoonotic virus. , this overlap opens a real possibility of merging attempts to detect zoonotic disease spillover with pre-existing population-based systems that have been used to investigate the hiv epidemic for several decades. for example, the demographic and health surveys program is implemented globally in settings without high-quality civil registration and has extensive experience in collecting integrated biological-behavioural surveillance data in community settings. integrated surveillance will never be a viable alternative to traditional clinical disease surveillance for assessing active viral infections. rather, it can serve to complement virus detection efforts, by potentially identifying pandemic threats before the need for large-scale clinical intervention. as current behaviours may not reflect the behaviours that originally exposed the individuals who are found seropositive to the virus of interest, both current and lifetime behaviours need to be investigated. this is the strategy that has proved successful in identifying subtle exposure risks in the field of hiv, such as backloading of syringes with drug solution by injecting drug users. in identifying specific behavioural risk factors, integrated biological-behavioural surveillance will be most effective when the reported syndromic symptoms are recent, e.g. occurring in the previous months, and their probable association with a zoonotic virus can be confirmed by a positive serological test result. current pandemic-threat warning systems rely almost exclusively on disease surveillance in clinical settings. standardized biological-behavioural surveillance, in which both disease outcome data -self-reported and biological -and behavioural risk factors are measured, would complement traditional surveillance and greatly advance the understanding of behaviours and practices that could be targeted for risk mitigation and, ultimately, for prevention. the implementation of integrated biological-behavioural surveillance need not be frequent to be informative and useful in preventing the spillover of zoonotic agents with pandemic potential. ■ Отличительной чертой -го века являются вспышки заболеваний, оказывающие разрушительный эффект на экономику и политику. Хотя причины пандемий кроются в поведении людей, современные системы наблюдения, предназначенные для выявления угроз пандемии, в значительной мере полагаются на мониторинг исходов заболевания в клинических условиях. Стандартизованное, комплексное, биологически поведенческое наблюдение можно и следует применять в условиях общин как дополнение к подобному клиническому мониторингу. Целесообразность такого подхода уже была продемонстрирована в исследованиях вируса иммунодефицита человека, в рамках которых комплексное наблюдение способствовало биологически обоснованному и поддающемуся количественной оценке пониманию факторов поведенческого риска, связанных с динамикой передачи вируса. Для комплексного наблюдения, разработанного в соответствии с критериями устранения недостатков предоставления информации в наблюдательных исследованиях по эпидемиологии, необходимо измерение фак торов поведенческого риска, т. е. переменных подверженности воздействию, и выходных переменных индикаторов заболеваемости в рамках исследований поведения. В сфере угроз пандемии с помощью информации о биологических исходах можно было бы устранить недостатки предоставленных респондентами данных, полученных в рамках поведенческих исследований. Судя по данным серологических исследований вирусов, имеющих пандемический потенциал, которые были получены в условиях отсутствия вспышки, эпидемиологический надзор может быть использован для прогнозирования будущих вспышек. Проведение поведенческих и серологических исследований вместе позволило бы спрогнозировать будущие пандемии теоретически до того, как заболевание возникнет в клинических условиях. Польза традиционного наблюдения исходов заболеваний зависит от частоты и непрерывности его проведения, в то время как поведенческое наблюдение позволяет получить ценную информацию, даже если осуществляется гораздо реже, поскольку поведение со временем изменяется медленно. Только зная конкретные факторы поведенческого риска, можно разработать мероприятия и политики, способные предотвращать будущие пандемии. los brotes de enfermedades perjudiciales para la economía y la política son una característica del siglo xxi. a pesar de que las pandemias se ven impulsadas por el comportamiento humano, los sistemas de vigilancia actuales para identificar amenazas de pandemia dependen enormemente del seguimiento de los resultados de las enfermedades en entornos clínicos. la vigilancia integrada y normalizada de datos biológicos y del comportamiento podría y debería utilizarse en comunidades como complemento de dicho seguimiento clínico. su utilidad ya se ha demostrado en varios estudios sobre el virus de la inmunodeficiencia humana, en los que la vigilancia integrada contribuyó a la comprensión cuantificable y biológica de los factores de riesgo del comportamiento asociados con la dinámica de la transmisión del virus. al estar diseñada según los criterios del fortalecimiento de la notificación de los estudios observacionales en epidemiología, la vigilancia integrada requiere tanto los factores de riesgo del comportamiento (es decir, las variables de exposición) como las variables de resultados del indicador de enfermedades se midan en encuestas sobre el comportamiento. en el campo de las amenazas de pandemia, los datos de los resultados biológicos podrían abordar la debilidad de los datos autodeclarados recopilados en las encuestas sobre el comportamiento. la información de las encuestas serológicas sobre virus con potencial pandémico, recopilada en condiciones en las que no se había reportado un brote, indica que podría utilizarse la vigilancia serológica para predecir futuros brotes. al realizarse juntas, integrated surveillance for pandemic threats maureen miller & emily hagan las encuestas serológicas y sobre el comportamiento podrían advertir sobre futuras pandemias, probablemente antes de que la enfermedad aparezca en entornos clínicos. la vigilancia tradicional de los resultados de las enfermedades debe ser constante y frecuente para que sea útil, aunque la vigilancia sobre el comportamiento sigue siendo meramente informativa, incluso si se realiza con menos asiduidad, dado que los cambios de comportamiento se producen lentamente con el paso del tiempo. 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données sérologiques actualisées chez l'homme evidence for henipavirus spillover into human populations in africa outbreaks chronology: ebola virus disease comprehensive serological profiling of human populations using a synthetic human virome determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics geneva: joint united nations programme on hiv/aids innovations in health and demographic surveillance systems to establish the causal impacts of hiv policies syringe-mediated drug-sharing (backloading): a new risk factor for hiv among injecting drug users key: cord- -a jmc lq authors: wu, tong; perrings, charles; kinzig, ann; collins, james p.; minteer, ben a.; daszak, peter title: economic growth, urbanization, globalization, and the risks of emerging infectious diseases in china: a review date: - - journal: ambio doi: . /s - - - sha: doc_id: cord_uid: a jmc lq three interrelated world trends may be exacerbating emerging zoonotic risks: income growth, urbanization, and globalization. income growth is associated with rising animal protein consumption in developing countries, which increases the conversion of wild lands to livestock production, and hence the probability of zoonotic emergence. urbanization implies the greater concentration and connectedness of people, which increases the speed at which new infections are spread. globalization—the closer integration of the world economy—has facilitated pathogen spread among countries through the growth of trade and travel. high-risk areas for the emergence and spread of infectious disease are where these three trends intersect with predisposing socioecological conditions including the presence of wild disease reservoirs, agricultural practices that increase contact between wildlife and livestock, and cultural practices that increase contact between humans, wildlife, and livestock. such an intersection occurs in china, which has been a “cradle” of zoonoses from the black death to avian influenza and sars. disease management in china is thus critical to the mitigation of global zoonotic risks. today, an increasingly urban and interconnected world faces growing threats from emerging infectious diseases (mcmichael ; kapan et al. ; bradley and altizer ) . this is of particular concern in the developing world, where managing fast-spreading epidemics in the growing number of megacities is a pressing challenge (rees ) . recent epidemics have underscored the importance of linkages between host habitats and the global network of cities. the ebola virus, for example, has long survived among wildlife reservoirs in the hinterlands of africa, ''breaking out'' in towns and cities in conspicuous but otherwise local epidemics. as in earlier outbreaks, the epidemic is thought to have origins in the consumption of wild animal protein, while its spread occurred in densely populated african cities. the international threat it posed stemmed from the increasing air travel connections between these and other cities around the world. in the case of arboviruses like zika, dengue, chikungunya, west nile, and malaria, whose vectors have found ready habitat in urban areas, the primary mechanism for the spread of disease from one city to the next is international trade and travel (hay et al. ; tatem et al. ; alirol et al. ; weaver ; kraemer et al. ) . the same is true of coronaviruses such as severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome (mers). the latter emerged in saudi arabia in , having been transmitted between animal reservoirs such as camels and their human handlers. it has since spread throughout the surrounding region, and travel-related human infections have been recorded in europe, north america, and east and southeast asia (parlak ; zumla et al. ) . urbanization and globalization have made outbreaks of these diverse zoonoses difficult to control, even with unprecedented levels of international cooperation (khan et al. ; weaver ; chan ; kraemer et al. ) . for most emerging infectious diseases, prevention is better than cure-ex ante mitigation of disease risk is more economically efficient than ex post adaptation to an outbreak (murphy ; graham et al. ; voyles et al. ; langwig et al. ) . among mitigation strategies, vaccination has been a widespread and long-established practice for many dna viruses such as chicken pox or small pox. however, vaccination remains problematic for most rna viruses, including ebola, sars, and avian influenza, due to their higher mutation rate; vaccination is simply not a feasible way to prevent the emergence of many novel zoonoses, which will inevitably encounter immunologically naïve populations. therefore, mitigating the risks from emerging and reemerging zoonoses requires preemptive measures against their socioecological drivers (pike et al. ) . identifying areas where the convergence of risk factors is occurring with greatest intensity, and at the largest scales, is a logical first step in the development of a mitigation strategy. in this regard, china may be an important outlier among countries. assessment of the risks posed by zoonotic diseases requires an understanding of how socioeconomic, and ecological conditions affect two phenomena: emergence (the irruption of a pathogen originating in wildlife or livestock into human populations) and spread (the transmission of disease among both animals and people). in this article, we review the evidence for changes in zoonotic risks in china. more particularly, we show how income growth, urbanization, and globalization affect the likelihood of emergence and spread, using sars and avian influenza as topical and representative examples, but also referring to other diseases when relevant. we discuss the policy implications of changes in the epidemiological environment in china, and consider how the mitigation of zoonotic risk in china could benefit the global risk environment. china's rate of economic growth over the last years has been exceptional. real per-capita gdp (in purchasing power parity terms) rose from usd in to usd in , an average annual growth rate of over %. while this has generated the resources necessary to improve biosecurity and healthcare, it has also increased the likelihood of disease emergence and transmission. the presence of major migratory bird pathways (conduits for the transmission of influenzas), habitats that encourage mixing between wild and domesticated birds, and a dramatic increase in demand for fresh meat have increased the likelihood of disease emergence. at the same time, urbanization and the growth of international trade and travel have increased the likelihood of disease spread (wang et al. ; alirol et al. ; zhu et al. ; gong et al. ; li et al. a, b) . in , only one quarter of china's population lived in cities; today, over % are urban residents, and by there will be at least one billion city-dwellers, or % of the population (peng, ) . concurrently, china's integration into international networks of trade and travel has occurred rapidly. between and , china's exports to the rest of the world grew annually by around %, and although exports in this period were dominated by manufactures, exports of food and live animals grew at an only slightly lower rate. more importantly for china's exposure to global disease risk, imports of food and live animals were - times larger than exports in , and have since grown by around % year (world bank ). the epidemiological boundary separating humans from wildlife-and livestock-borne pathogens has been breached repeatedly throughout history. at the turn of the twentieth century, it was estimated that % of all known human pathogens and % of all emerging diseases were zoonotic (taylor et al. ). zoonotic ''spillover'' into human populations can occur in numerous ways. direct contact between people and pathogen-carrying animals through, for example, the consumption of infected wildlife or livestock is a common pathway of emergence (patz et al. ; murray and daszak ) . increasing per-capita income has led to increasing percapita meat consumption, and this has occurred more rapidly in china than in any other major economy. tracking rapid rates of gdp growth and rural-to-urban migration, china's meat consumption has risen by around one-third since the turn of the century (fig. ) . the pattern of consumption is also changing: while pork remains the main source of animal protein, chicken consumption and production are increasing more rapidly. between and , the growth in poultry numbers was around ten times the growth in pig numbers (wang et al. ) . nor has the growth rate of these stocks slowed. in , china had the world's largest stocks of poultry and swine, at . billion and million individuals, respectively. by comparison, u.s. stocks stood at . billion and . million individuals (fao ) . what makes the changing pattern of meat consumption significant for infectious disease transmission in china is a persisting social preference for live and freshly slaughtered meat (the primary interface for animal-to-human transmission of many zoonoses) (pi et al. ) . as the consumption of meat grows in the coming decades, so will contact between consumers and live or freshly slaughtered animals. over the next decade, per-capita consumption of chicken is expected to grow at an annual rate of . %, compared to . % for pork. while meat imports will likely increase, most of the growth in supply is expected to be from domestic sources. annual production of poultry, pork, and beef is projected to rise from million metric tons (mmt) today to mmt by (westcott and trostle ) . the resulting increase in the levels and densities of pigs and poultry enlarges the potential pathogen reservoirs for zoonoses, especially influenzas. a central mechanism that brings human, livestock, and wild animal populations together in china is the extensive network of wet markets-markets that sell live and freshly slaughtered domesticated and wild animals. although found in many parts of the world, wet markets are particularly common in east and southeast asia. in china, they are the primary source of meat purchases, particularly of poultry. in shanghai, for example, where highly pathogenic avian influenza (hpai) h n first emerged in , million of the approximately million chickens consumed annually were purchased at wet markets (pi et al. ) . the spillover of h n and h n into human populations in china has been closely linked to these markets (yu et al. (yu et al. , . wet markets are frequently underregulated, have unhygienic environments with inadequate sanitation, and are subject to poor surveillance and little biosecurity (woo et al. ) . although the role of wet markets is expected to diminish in the coming decades, they will still account for a significant, perhaps even the majority, share of poultry sales (pi et al. ) . income growth among urban consumers will increase the demand for meat, much of it bought in wet markets. indeed, dietary preference for freshly slaughtered meat and the enduring popularity of wet markets will remain important drivers of zoonotic risk for the foreseeable future. the growth of cities and changing agricultural conditions have shaped infectious disease ecology in china since at least the tang dynasty (seventh to tenth century ce), creating and connecting reservoirs of pathogens and vectors (jannetta ) . however, the speed and scope of urbanization over the past three decades have been significantly greater than at any other time in the past, creating a primarily urban population for the first time in china's history. the rate of urbanization has also been markedly higher than that of other industrialized and industrializing countries. for instance, in , at the start of its own economic liberalization program, india had an urbanization level of . % compared to china's . %. today, china's urbanization has reached . % compared to india's . % (un ). in china, this has expanded the interface of contact between humans, wildlife, and livestock. urbanization and associated land-use changes, in conjunction with rising meat consumption, have brought reservoirs of wildlife diseases into closer contact with livestock and people (wang et al. ; daszak ; daszak et al. ; myers et al. ) . in particular, the emergence of hpai strains has become more likely in southern china, where the growth of an increasingly affluent urban population has driven an increase in poultry production and land-use changes that brings humans, domesticated animals, and wildlife into closer contact (davis ; wallace et al. ) (fig. ) . changes in the configuration of natural, agricultural, and built-up land cover, as well as in the biotic and abiotic fluxes among them, also affect disease risks to people (reisen ; meentemeyer et al. ) . large and growing populations of livestock-particularly poultrydistributed across china are ideal sites of viral mutation and interspecies influenza transmission, most notably between wild and domesticated birds. china is also crossed by multiple migratory flyways, which allow numerous waterfowl and other bird species to carry avian influenza into and out of the country (chen et al. ; kilpatrick et al. ; takekawa et al. ; prosser et al. ) . across east asia, intensively cultivated rice fields are populated by poultry and ducks, but are also ready habitats for hpai-carrying waterfowl (gilbert et al. ; paul et al. ; martin et al. ; gilbert et al. ) (fig. ) . the growing number of species infected by recent outbreaks of avian influenza suggests that epidemic risks are growing as a result (webby and webstter ) . livestock populations are also staging posts for pathogens to enter human populations. for instance, poultry farming in china's urban and peri-urban areas increases the likelihood of h n spread (kapan et al. ; fasina et al. ). the risk of pathogen spread from diseases contracted in wet markets is exacerbated by the concentration and interconnectedness of human populations associated with urbanization (fang et al. ; hogerwerf et al. ; paul et al. ) . wet markets in urban areas are now recognized to be the primary locus of infection for h n . the high density of people makes cities, particularly the large cities that have appeared rapidly in china, force multipliers of pathogen transmission (alirol et al. ). patel and burke ( ) argue that the outbreak of sars in hong kong in and ''demonstrated how dense urban living could ignite a global health crisis.'' the first phase of the sars epidemic involved its spread through the interconnected metropolises of the pearl river delta (prd) (wang et al. ; zhu et al. ; li et al. b ). the prd was also an epicenter for hpai h n (see box ) and remains a potential hotspot for other influenzas (fig. ) , though this has yet to be reflected in public health policies in the area (fabre and rodwin ) . one reason for the effectiveness of cities as force multipliers, relative to rural areas, is the higher prevalence of a range of communicable and noncommunicable diseases, including sexually transmitted diseases and cancers. such conditions increase vulnerability to infection (alirol et al. ; gong et al. ; li et al. b) , while comorbidity can magnify the potential virulence of zoonotic pathogens and thus their spread (weiss and mcmichael ) . in many chinese cities, public health management has not kept pace with demographic and economic changes. despite progress in recent decades, immunization coverage for even common infections such as tuberculosis, measles, and tetanus remains inadequate (gong et al. ). infection risk is also related to the social inequality and dislocations caused by urbanization. in the pearl river delta in , for instance, % of migrants did not have access to medical insurance (fabre and rodwin ) . this deprivation may deter people from seeking preventative care, or even immediate care after possible infection. the epidemiological implications of disease comorbidityincluding the risks of ''super-spreaders''-are perhaps even more significant at the international level. for instance, had the first sars carrier reached the dense precincts of durban, with its high incidence of aids, rather than the more ordered and hygienic environment of toronto, the outcome may have been much worse (weiss and mcmichael ) . of course, the probability that an infection is transmitted abroad to a given city depends on the volume of trade and travel involved, but since trade between china fig. the intersection of dense chicken and duck populations, human populations (concentrated by urbanization), and migratory birds increases the likelihood of interspecies transmission and the emergence of new influenza strains. sources generalized bird migration routes adapted from fang et al. ( ) ; poultry distribution data from robinson et al. ( ) and south africa has been growing more rapidly than trade with china's main international markets, this is not a trivial observation. china is now the world's largest trading nation, and in recent years its trade to nearly every region of the world has increased significantly (fig. ) . the global infectious disease risks created by china's trade growth stem from the fact that international markets facilitate the movement of pathogens around the world as freely as commodities and people (perrings et al. ; knobler et al. ; tatem et al. ; hulme ; perrings ; kilpatrick ) . in history, there have been several notable moments when trade and travel have bridged the natural epidemiological discontinuities created by geography. the most famous of these is the black death of the thirteenth and fourteenth centuries, during which the plague bacillus yersinia pestis spread from china to europe along trade routes maintained by the mongol empire. the same disease had also earlier spread across eurasia from china in the sixth century (wagner et al. ) . that outbreak, known as the plague of justinian, had killed tens of millions across the mediterranean region and critically damaged the economic and geopolitical fortunes of the byzantine empire (mcneill ) . not all epidemics have chinese origins, though. several centuries later, overseas expansion by european powers led not only to the political and economic subjugation of foreign peoples, but also to the introduction of many new species-pests and pathogens among them (crosby ). the growth of maritime trade facilitated massive movements of people, plants, and animals, as well as the pathogens that these passengers and cargo carried, across the world's oceans. the so-called columbian exchange had particularly severe effects on human health on both sides of the atlantic. old world diseases such as smallpox, typhoid, typhus, and measles were introduced to the western hemisphere by colonizers, resulting in significant depopulation and a decisive shift in the balance of power (mcneill ; diamond ; crosby ) . nevertheless, china has remained a persistent and important source of infectious zoonotic disease. for instance, a plague outbreak in southwestern china engulfed the country and then spread to the rest of the world in the late s. the spillover was likely mediated by rat-borne fleas, brought into contact with people due to ecological encroachments from settlement expansion (benedict ) . this plague first spread to the port cities of the chinese coast and thence to southeast asia, the united states, and europe. in the western u.s., the bacillus remained epizootic among rodent species well into the twentieth century (mcneill ) . the archetypal modern pandemic-the one that remains a touchstone for thinking about global infectious disease risk-is the - spanish flu. the severity of this pandemic was in large part a result of the integration forced by global conflict. propagated by the movements of millions of servicemen during and after world war i, this strain of h n influenza may have infected as many as million people, or a quarter of the world's population, and killed as many as - million (taubenberger and box hpai and china's urbanization hpai h n first emerged in southern china in the late s. after several outbreaks, contained with varying degrees of efficacy, it has now spread across the world, infecting people, poultry, wild birds, and other wild and domesticated animals. income growth has driven an increase in china's protein consumption, which has resulted in a nearly -fold increase in domestic poultry stocks since (fao ) . urban wet markets are still the primary sources of poultry purchases-in shanghai, for instance, million of the million chickens sold in were from wet markets (pi et al. ) . that year, shanghai, china's largest city at million people, was the site of hpai h n 's emergence. the most likely areas of future h n spread in china are urban areas with a high density of wet markets . china is also traversed by several migratory bird pathways, bringing growing and ever-denser populations of poultry and people into contact with influenza-bearing wild birds (chen et al. ; takekawa et al. ). the heavily urbanized areas of southern china-such as the metropolitan pearl river delta, home to over million people and a high concentration of poultry production-is at particular risk for the emergence of hpai, and may be an important outlet for its spread within china and internationally (davis ; wallace et al. ) morens ). recent forensic studies tracking mortality rates and other contemporary evidence suggest that, contrary to its name, this pandemic actually originated in china (langford ; humphries ). indeed, china has been the epicenter of influenza both before and after the ''spanish flu.'' at least two of four historically documented pandemics originated in china before , as did both of the subsequent pandemics (potter ) (fig. ) . the mechanism behind the global spread of diseases after has been the ever-closer integration of the world economy. habitat suitability and transport distance determine the potential dispersal patterns of infectious disease vectors (tatem et al. ) , while the relative costs and benefits of trade and infectious disease determine the likelihood that pathogens will be spread this way (perrings ) . the potentially high cost of the sars epidemic led Ó royal swedish academy of sciences www.kva.se/en to prompt preventive action, but the volume of travel meant that the pathogen still managed to reach every continent. in east asia alone, sars resulted in at least a % decline of gdp (brahmbhatt ) . it has been estimated that the economic losses from a major influenza pandemic could be as high as $ . trillion ( . % of global gdp)-a downturn on par with the great depression-and cause over million deaths (mckibbin and sidorenko ) . given its role as the ''cradle of influenza'' (davis ) , and many other zoonoses, china should be a focus of international efforts to mitigate future infectious disease risk. it is likely that the factors that facilitated the global spread of the plague and the - , , and influenza pandemics that originated in china are even more forceful today. the lesson of recent decades is that zoonoses such as hpai, sars, and ebola cannot be reliably contained at the local, national, or even continental level. it follows that infectious disease risk mitigation is a product not only of the probability of emergence, but also of the probability that an outbreak will be propagated to other parts of the world. design and implementation of risk mitigation strategies require an understanding of the factors affecting the probability that zoonoses will emerge, and the likely pattern of their spread (mcmichael ; daszak et al. ; mcmichael ; daszak ; castillo-chavez et al. ) . with regard to china, in particular, this involves understanding the way that income growth, urbanization, and globalization interact with predisposing socioecological conditions (including changes in the interface between wild and domestic species, and cultural practices surrounding the consumption of those species) to alter the likelihood of emergence. we also need an improved understanding of the role of more widespread processes in changing epidemiological environments. climate change is expected to alter ecosystem processes and functioning in ways that will influence the emergence and reemergence of infectious diseases worldwide (morens et al. ; piao et al. ) , particularly for vector-borne pathogens (hales et al. ; chretien et al. ) . in china, climate change, including changes in the el nino-southern oscillation (enso), is expected to increase human vulnerability to a spectrum of infectious diseases such malaria, dengue, and japanese encephalitis (bi et al. ; bai et al. ) . mitigating the infectious disease risks of climate change-both for vectorborne and directly transmissible zoonoses-requires a deeper understanding of how it interacts with urbanization and globalization to alter the vulnerability of human populations (tong et al. ) . encouragingly, a recent survey of provincial public health workers in china found that the large majority had ''accurate'' (i.e., in line with existing scientific consensus) perceptions and knowledge about climate change and its likely impacts on infectious diseases (wei et al. ) . a second requirement for successful policy is to look beyond standard epidemiological measures for risk indicators. for avian influenza, outbreaks among wild birds, poultry, and people reveal patterns that suggest new indicators (fig. ) . the urban areas bestriding the pearl river and yangtze river deltas were the emergence areas for h n and h n , respectively. empirical studies have shown how both outbreaks were facilitated by similar socioecological changes (as discussed in preceding sections). because of this, it has been argued that wet markets could be used as an early-warning system to detect emerging zoonoses (webster ) , and that control measures could focus on the norms and incentives underlying human-to-animal interactions in the marketplace (goldman et al. ; woo et al. ; gao ; pi et al. ) . a third requirement is to enable public health infrastructures to respond to a range of threat indicators. sars and hpai, and the experiences of dealing with other zoonotic risks in recent years, have motivated chinese policymakers to improve their capacity to respond to emerging infectious diseases. responsiveness, information dissemination, and infectious disease surveillance have all improved since the initial sars and h n outbreaks (wang et al. ) . the ministry of health has created the world's largest online, real-time, case-based reporting system, called the china information system for disease control and prevention, with coverage from the national down to the county level (wang et al. ; gong et al. ; li et al. a, b) . this system is connected to a network of center for disease control and prevention (cdc) institutes (http://www.chinacdc.cn/en), which collaborates with government-funded labs and other academic organizations focused on zoonotic diseases (wang et al. ; zhang et al. ) . as of , there were cdc institutes across china (nbs ) . nonetheless, surveillance and the overall public health infrastructure still have several weaknesses, namely undercoverage of rural areas (where zoonoses, particularly those borne by livestock, may originate), lack of training for health professionals in poorer areas, and a low per-capita level of funding (tong et al. ) . indeed, these weaknesses may have been reflected in the fact that management of zoonotic diseases has largely been ad hoc. the reactions to hpai outbreaks included widespread wet market closures and trade restrictions. while this limited the spread of h n after its initial occurrence (webster ; he et al. ; yu et al. ) , it was also very costly to authorities, vendors, and consumers, and is unsustainable as a policy framework for the long run (gao ) . additionally, given the significant traditional values attached to wet markets and the live animal trade, policies of that kind may abrade cultural sensibilities. for instance, abrupt and prolonged closures of live animal markets may deprive people of a traditional venue for social interaction (goldman et al. ; woo et al. ; gao ) . improving public awareness and knowledge has been one form of intervention, but more generally management has tended to take the form of response rather than prevention. but given the changing zoonotic risks, mitigation (e.g., management at the human-animal-wildlife interface in anticipation of mutation and spillover) is likely to be more cost effective than adaptation (e.g., reducing contact rates through social distancing and trade and travel restrictions after an outbreak) (pike et al. ) . a fourth requirement is to build the collective capacity to mitigate international risk. evidence that this has received higher priority in recent times is china's greater involvement in world health organization (who) initiatives (wang et al. ; zhang et al. ). the u.s. agency for international development also has active programs in china assessing the risks of emerging zoonoses (e.g., the emerging pandemic threats program: http://www.usaid.gov/ept /). such ventures may provide an important medium for ''science diplomacy''-i.e., using research collaboration and the exchange of ideas as a platform to improve geopolitical relationships-between the two largest economies, and trading nations, in the world (hoetz ) . additionally, chinese health workers have, since , received training from the who and the u.s. center for disease control, although as of only had graduated from the program (tong et al. ) . as with domestic disease surveillance and management efforts, this has implications for the allocation of resources needed to support initiatives and institutions. finally, the underlying research efforts to model risk at different spatial scales and inform policy need to include factors that affect not only the abundance of susceptible, latent, infectious, and recovered individuals, but also the likelihood of contact and transmission. there would be value in exploiting a class of models in economic epidemiology that addresses the decisions made by people and policymakers that affect the likelihood of both host contact and infectious disease transmission . income growth, rising trade in goods and services, and the demographic and land-use changes caused by urbanization all affect private infectious disease risk management, and so should inform the public response. the development of infectious disease models for china that capture such risk factors would have the potential both to enhance management domestically, and to comprehend the risks from trade and travel links with the rest of the world. in certain respects, the nature of infectious disease risk mitigation is similar to the nature of climate change mitigation. in both cases, there is a closing window for timely action. in both cases, too, the mitigation of global risk depends heavily on the efforts of a small number of countries, each of which has a disproportionate impact on global risk (pike et al. ) . to that end, improving the management of infectious disease risk in china is a necessary, though not sufficient, condition for managing such risks globally. it has been argued that the world has been undergoing an epidemiological transition, in which rising incomes and the dissemination of improved technologies and good practices has shifted the burden of disease away from communicable toward noncommunicable diseases (mckeown ; sepulveda and murray ) . in part, this is because development generates greater resources for biosecurity and the prevention and treatment of infectious disease. there is ample evidence that an epidemiological transition is underway in china. rising affluence has lessened the burden of infections that once were socially devastating, such as malaria and tuberculosis, but has increased noncommunicable diseases, such as cancer, heart disease, and obesity . at the same time, globalization has increased the potential for domestic infections to be exported to countries where infectious diseases are still the greater part of the disease burden (bygbjerg ) . indeed, emerging infectious diseases have been identified as one of a few ''catastrophic risks'' facing humanity in the twentyfirst century, especially for developing countries (rees ) . in china-which, despite its remarkable development in recent decades, remains an ''emerging'' economy-novel zoonotic risks have accompanied the classic health trends of the epidemiological transition (cook and drummer ) . large developing countries such as india, indonesia, and nigeria have a similar set of predisposing socioecological risk factors to china-e.g., large and growing human and livestock populations, high levels of interaction between species, and large-scale ecological change. as the forces of economic modernization accelerate, so could the zoonotic risks that such countries face. unlike china, many of these countries are still in the early stages of the epidemiological transition. while they are becoming more exposed to disease risk through the growth of trade and travel, they still experience many of the public health conditions that increase vulnerability to infections. for instance, ''silent epidemics''-i.e., those caused by high-risk pathogens that have not received international attention, and that are only pervasive at a local scale-may yet flare into epidemics of global impact. a salient example is buffalopox, an emerging and reemerging zoonosis that has recorded many animal outbreaks and human cases in south asia. the pathogen dynamics indicate a reasonably high level of transmissibility between livestock and people, and the forces of income growth, urbanization, and globalization could contribute to its further spread (singh et al. ; venkatesan et al. ) . the degree to which china's public health authorities and researchers, along with their international collaborators, keep pace with income growth, urbanization, and globalization-and how these changes interact with china's predisposing socioecological conditions-will be a major force shaping global epidemiology. china is not the only emerging infectious disease hotspot, but it is among the most important. as the world continues to navigate a potentially new era for infectious diseases, the management of risk in china will be critical to the management of risk everywhere. tong wu (&) is a doctoral candidate in the school of life sciences at arizona state university, as well as a visiting scholar in the school of public policy and management at tsinghua university. his primary research interests include the economics of infectious diseases, ecosystem management, 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acute respiratory syndrome and influenza? the impact of climate change on infectious disease transmission: perceptions of cdc health professionals in shanxi province social and environmental factors in the emergence of infectious diseases usda agricultural projections to infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections world development indicators emergence of chronic non-communicable diseases in china human influenza a (h n ) cases, urban areas of the people's republic of china effect of closure of live poultry markets on poultry-to-person transmission of avian influenza a h n virus: an ecological study ecohealth and the black death in the year of the rat understanding and harnessing the health effects of rapid urbanization in china middle east respiratory syndrome ann kinzig is a professor in the school of life sciences and the school of sustainability at arizona state university. she is also the chief research strategist for the global institute of sustainability at asu. her research interests include ecosystem services, biodiversity, and the spread of pests and pathogens. peter daszak is the president of ecohealth alliance, an international nonprofit organization focused on research and public education based in new york city. his primary research interests include disease ecology and conservation medicine, and he has studied a wide variety of zoonoses, including sars, avian influenzas, and ebola. address: ecohealth alliance, west th street - th floor, new york, ny , usa. e-mail: daszak@ecohealthalliance.org key: cord- -o nab authors: dahlhausen, bob title: future veterinary diagnostics date: - - journal: j exot pet med doi: . /j.jepm. . . sha: doc_id: cord_uid: o nab the development of rapid, accurate, and sensitive diagnostic methods for detecting pathogens is the basis for treating, controlling, and eradicating infectious diseases of veterinary importance. scientific and technological advancements have revolutionized the field of veterinary diagnostics. genome sequencing has allowed efficient, sensitive, and specific diagnostic assays to be developed based on the detection of nucleic acids. the integration of advances in biochemistry, proteomics, engineering, and medicine offers enormous potential for the rapid and accurate diagnosis of viral, microbial, genetic, and metabolic disease. in the future, polymerase chain reaction assays, microarray testing, genomic analysis, and metabolic profiling will be accomplished in a rapid, portable, sensitive, and cost-efficient manner. s ince the discovery in by james d. watson and francis crick that dna and rna provide the code to life, there has been a global effort to understand how the genome sequences of numerous organisms are related to health and disease. the rapidly advancing field of diagnostics, tests used to detect a medical condition or causative pathogen responsible for an infection, will drastically change the practice of veterinary medicine in the near future. the ability to detect nucleic acids has had and still has a major impact on diagnostic processes in clinical medicine. classical pathogen detection and identification are based on culture methodology, biochemical tests, and microscopy. the culture method is still a core technology in many clinical laboratories because it can provide important information about the viability of the pathogen and its susceptibility to therapeutic agents. likewise, the historic gold standard for diagnosing viral infection has been selective cultivation followed by electron microscopy. recovery of the live virus remains a very slow, technically demanding, and expensive diagnostic tool. the development of rapid, accurate, and sensitive diagnostic methods for detecting disease pathogens is fundamental for treating, controlling, and eradicating infectious disease. rapid scientific and technological developments have revolutionized the possibilities of diagnosing and treating disease. researchers now have tools for observation at the level of the atom, to sequence entire genomes, and to understand the molecular basis of disease. recent biotechnological developments in microtechnologies and nanotechnologies have led to the proliferation of new, rapid diagnostic tests that hold promise for the improved management and control of infectious disease in animals. emerging technologies are rapidly being adapted to improve our ability to de-tect new etiological agents, to provide rapid, accurate diagnostic tests for previously difficult infections, and to develop new response-based paradigms for diagnostic testing. molecular tools in particular are advancing toward laboratory-on-chip and fully integrated technologies that are capable of same-day detection and pathotyping of agents in clinical specimens. future diagnostic technologies that are rapid, portable, low cost, and of greater sensitivity/specificity will aid the veterinarian in the early disease detection in a point-of-care setting. scientific advancements over the previous decades have enabled the sensitive detection and characterization of bacterial and viral nucleic acids. amplification technologies (e.g., polymerase chain reaction [pcr] , nucleic acid sequence-based amplification [nasba] ) allow for specific detection of genetic targets. furthermore, technological improvements in sequence determination systems allow for organisms to be fully characterized as to genotype, subtype, variant, mutants, and resistance patterns. for the veterinary clinical laboratory, commercially standardized assays are not readily available. most laboratories offering nucleic amplification tests (nat)-based diagnostics use methods developed inhouse or those adapted from research publications. there is no standardization among these in-house assays or an official regulatory body to monitor quality assurance. differences in laboratory experience and technique, primer and probe combinations, methods of nucleic acid isolation, and control standards contribute to the reproducibility and variability observed among laboratories. assays must be precise and reproducible and have positive and negative controls in place to prevent invalid test results. it is also recommended that these assays have an internal control to validate nucleic acid isolation and to test for amplification inhibitors. in a blind study, veterinary laboratories offering nat diagnostics for psittacine circovirus were evaluated. the findings of this investigation revealed that accuracy was % for laboratories, % for laboratories, and % for laboratory, whereas laboratory had a test specificity of %. the authors concluded that although results from most laboratories were accurate, both false-positive and false-negative results were reported by at least laboratories. they also concluded that laboratory may be generating large numbers of falsely reported results. veterinarians must realize that the accuracy of re-sults depends completely on the accuracy of the operating standards of the individual laboratory. laboratories should always strive for the highest standards of assay precision. differences in assay precision make it difficult to compare data from different laboratories without defined standards. another consideration in nat-based diagnostics concerns sample quality. preservation of sample integrity is essential for accurate test results, especially if detection or quantification of rna is desired. unlike dna, rna is unstable and is readily degraded by naturally occurring endonucleases (rnases) that are present in the environment and tissues. samples for rna testing should be handled with gloves to prevent contamination with rnases present on the skin. samples for rna testing should be promptly submitted to the laboratory or stored in a freezer at Ϫ °c until forwarded for testing. samples may also be treated with buffers designed to minimize rna degradation, with several commercial additives being available for this purpose, but their use varies among laboratories. it is best to consult the specific laboratory for their protocol for sample submission. although dna tends to be more stable than rna, naturally occurring endonucleases that degrade dna do exist. test samples should be aseptically collected and refrigerated or frozen until submission. the ability of pcr to detect extremely low levels of dna in a submitted patient sample results in the very real potential of falsepositive test results due to dna contamination. pcr is a technique that started as a research tool and was eventually integrated into medical diagnostic applications. in , kleppe demonstrated that dna could be artificially replicated in vitro with a dna polymerase enzyme. combined with methods allowing for the synthesis of dna oligonucleotides, kary mullis developed the technique of pcr in and won the nobel prize in chemistry for his work. pcr has become one of the most widely used technologies in veterinary research and molecular diagnostics of infectious disease. the wide range of applications include the detection of dna or rna derived from infectious bacterial, viral, mycoplasmal, parasitic, protozoal, or fungal organisms; evaluation of specific genetic defects; and quantization of cytokines and cellular growth factors. pcr testing can be used on a wide range of samples, including tissue from biopsies or necropsies, blood, feces, tissue, soil, cerebrospinal fluid, and swabs of mucosal surfaces. pcr uses the highly specific molecular recognition ability of watson-crick base pairing to provide the selectivity needed for a nucleic acid probe to bind to a targeted dna sequence and allow for its exponential amplification. specific fragments of dna are replicated by cyclical heating and cooling through repeated temperature stages in a thermal cycler unit (fig ) . each cycle can double the amount of existing target dna. repeatedly amplifying the target sequence over to cycles theoretically allows for millions ( ) of dna copies to be produced. pcr assays are thus extremely sensitive. they are capable of detecting femtograms ( Ϫ g) of target dna sequence in a large mixture, or up to a microgram ( Ϫ g) of nontarget dna. the dna obtained at the end of the reaction can be used for a variety of applications, including sequencing, cloning, or as a probe for in situ hybridization. the nested pcr consists of sequential pcr amplifications. the product from the first-round pcr is used as the template for a second pcr amplification. a second set of primers, internal to the first set, amplify a smaller fragment of dna within the first-round product. in regular pcr, primers may bind to incorrect regions of the dna, giving unexpected amplification products. the benefit of nested pcr is that increased specificity is achieved through confirmation of the correct primer binding during the first pcr amplification process. the nested pcr is more sensitive than conventional single-round qualitative pcr. the implementation of nucleic acid technology into routine diagnostics has greatly advanced with the introduction of real-time pcr. real-time pcr amplification uses the detection and quantification of a fluorescent reporter molecule whose signal increases in proportion to the amount of target amplification product generated. basic methods involve the detection of a dna-binding dye, such as sybr green (molecular probes, eugene, or usa). the dye intercalates between double-stranded dna formed in the pcr reaction and, when exposed to an excitation source, fluoresces. a laser scanner detects the fluorescence emitted by dye-impregnated dna strands formed through the pcr amplification. software traces this fluorescence as a running graph as the amplification reaction in real time (fig ) . a more specific strategy for real-time assays includes the use of a confirmatory probe sequence that matches part of the amplification product. these real-time amplicon detection technologies include taqman (applied biosystems, inc., foster city, ca usa) or hydrolysis probes, molecular beacon technology, and hybridization probes. fluorescence is only detected if the probe is bound to the amplified target, thereby enhancing specificity to an already highly specific assay. these probes do not react with primer-dimers or other nonspecific products that may be generated during the pcr reaction as may occur with sybr green reactions. however, a welldesigned and optimized sybr green amplification is a very effective testing technology. unlike taqman probes, the sybr green reporter remains intact so a melt curve analysis can be performed on the amplification product. melt curve analysis measures the dissociation characteristics of doublestranded dna during heating. melt curve analysis is sensitive enough to detect single-nucleotide polymorphisms (snp) and can distinguish between homozygous and heterozygous gene alleles by the dissociation patterns produced (fig ) . the advancement of this technology to produce highresolution melt analysis will provide higher sensitivity for snp detection within entire dye-stained amplicons for genomic analysis. the main advantage of real-time detection is that it accurately quantifies the number of rna or dna copies present in the test sample. real-time technologies can measure dna presence over a large dynamic range, providing important information regarding the magnitude of viral involvement within a test sample. this information is helpful in distinguishing clinically active infections from transient environmental exposures. quantification also aids in the treatment of patients with chronic infectious disease and monitoring disease progression and resolution. standard pcr techniques cannot be used to detect the presence of rna-based viruses in clinical samples. the direct amplification of rna in pcr systems is not possible because of the polymerase enzyme's inability to synthesize dna from an rna template. the inability to synthesize dna from an rna template is overcome by the use of a reverse-transcriptase (rt) enzyme before the pcr reaction. in rt-pcr, rna is converted into a complementary dna (cdna) strand by an rna-dependent dna polymer-ase (reverse transcriptase). the cdna produced is then used as a template in a conventional pcr reaction. it is important that all genomic dna within the test sample be destroyed before the rt-pcr to prevent masking the presence of cdna derived from the rt step. in addition to diagnosing rna viral infections in an individual patient, rt-pcr has been applied to monitoring disease in flocks and large groups of animals. environmental air-sampling, referred to as "sniffing," was paired with real-time rt-pcr to detect exotic newcastle disease virus in commercial poultry operations in . the same approach was successfully used to detect h n influenza in commercial quail. this methodology offers the advantage of detecting virus in contaminated environments by collecting airborne particulates, including virus comingled with dust and debris, without having to handle individual birds. pcr currently is still the main target amplification technique used in both research and diagnostic laboratories. a second target amplification technique, known as nasba, was specifically developed for the quantitative amplification of rna targets such as retroviruses and other rna viruses. , nasba's main advantage is that it works at isothermic conditions and can give quicker and more sensitive results than pcr. it has been used to develop rapid diagnostic tests for several pathogenic viruses with singlestranded rna genomes, including influenza a, footand-mouth disease virus, and severe acute respiratory syndrome (sars)-associated coronavirus. loop-mediated isothermal pcr (lamp) is a -step amplification reaction that amplifies a target dna sequence with high sensitivity and specificity under isothermal conditions. lamp uses a dna polymerase with inner and outer primers that recognizes separate regions within a target dna sequence. the assay has high specificity because the amplification reaction only occurs when all regions within the target dna are correctly recognized by the primers. lamp exhibits less sensitivity to inhibitory substances present in biological samples than regular pcr. lamp has been applied to pathogens that cause food-borne diseases. testing kits that use this technology are commercially available for detecting salmonella spp., legionella spp., listeria spp., verotoxinproducing escherichia coli, and campylobacter spp. many amplification methods, including pcr, ligase chain reaction, nasba, and strand displacement amplification, are used in commercially available diagnostic test kits. these diagnostic test kits are promoted as providing a rapid, sensitive, and specific detection of important pathogens, especially those that are not readily identified by more conventional methods. conventional detection and identification of noroviruses are performed by electron micros-copy, which is less sensitive and more time consuming than nat diagnostics. molecular-based testing for multiple pathogens in a large number of animals is expensive. the development of new variations of pcr have allowed for simultaneously performing multiple tests on a single diagnostic sample, thereby affording some savings on a per-test basis. in multiplex pcr, multiple primer pairs are used to amplify more than one gene or pathogen in a single pcr reaction. multiplex assays must be carefully designed for specific primer selection. primers selected must be to regions that are uniquely different and characterized by low affinity for primer-to-primer interactions. nucleic amplification technologies will continue to be a benchmark for pathogen detection in veterinary diagnostics. currently, there are reference sequences for viral genomes and reference sequences for viroids in genbank, an open-access public database of nucleotide reference sequences (http://www.ncbi.nlm.nih.gov/genomes/viruses/ viruses.html). the exponential replication of dna afforded by the pcr process allows for excellent diagnostic sensitivity and specificity. however, the instability and variability inherent in molecular enzymatic processes limit the application of this technology outside an institutional setting to a research facility or dedicated molecular laboratory. new technologies such as the lab-on-a-chip have been developed and have potential in veterinary point-of-care diagnostics. defined as a miniaturized analytical laboratory constructed on a small chip, this technology has promising applications when combined with nat diagnostic processes. both pcr and lamp assays have been used to facilitate the development of labon-a-chip-based nat devices. such devices can offer molecular-based testing in a rapid, effective, low-cost, and disposable automatic format as a pointof-care diagnostic tool. the concept of the dna or oligonucleotide array began in the mid- s as a derivative of the southern blot technique in which dna fragments are linked to a substrate and probed with a known gene or target dna sequence. the "probe" refers to the dna that is immobilized on a solid support such as a glass slide or a silicon wafer, whereas "target" refers future veterinary diagnostics to the pool of nucleic acids whose identity or abundance is being detected. both high-density microarrays for high-throughput screening applications and lower-density microarrays have been developed for diagnostic applications. microarray technology represents an important tool for the investigation of various areas of biology, mainly because of its ability to simultaneously detect and analyze thousands of different genes in a single trial. a dna microarray is a miniaturized form of dot blot in a predefined, ordered fashion at a high density. it consists of an arrayed series of thousands of microscopic spots of dna oligonucleotides, each containing picomoles ( Ϫ moles) of a specific dna sequence. the dna spots (gene chips) are attached in an equidistant, ordered arrangement to a solid surface, such as a glass, plastic, or silicon chip. alternative microarray platforms include polyacrylamide gel pads, microsphere beads, -dimensional films, and solid-state electronics. microarrays may have from a hundred to thousands of test sites that can range in size from to m. dna arrays are different from other types of microarrays only because they either measure dna or use dna as part of the detection system. hybridization is performed with corresponding probes attached to the solid support. these can be cdnas, oligonucleotides of varying length, or genomic sequences that are either radioactively or fluorescently labeled. artificial single-stranded dna or rna ligands, short oligonucleotides or peptide molecules that can be generated against amino acids, drugs, proteins, and other molecules are called aptamers. aptamers bind to a specific target molecule with high affinity and selectivity ranging from the micromolar to the nanomolar level, and they can discriminate between closely related targets. , a high-density array can be generated by applying aptamers to the substrate with robotic pins or inkjet technology, or by an in situ photolithographic synthesis process. these techniques allow for the production of a high-density chip containing thousands of short probes targeted at different loci within a single gene or multiple genomes. high-density microarrays may have up to test sites in a ϫ cm area. the core principle of dna microarrays is based on hybridization probing, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between nucleotide base pairs. hybridization occurs between cdna sequences such as the nucleic acid probes immobilized on the solid surface of a chip and the mobile dna, cdna, or messenger rna (mrna) sample. a high number of complementary base pairs in a nucleotide sequence allows for tighter noncovalent bonding between the strands to occur. probetarget hybridization is usually quantified through the detection of a fluorophore, silver, or chemiluminescence-labeled target. after the hybridization step is complete, the microarray is scanned. the fluorescent tags are excited by a laser, read by a microscopic camera, and computer integrated into a digital image of the array. software analyzes each microarray spot calculating the red-to-green fluorescence ratio and subtracting out background data. the nature and relative abundance of nucleic acid sequences in the target are determined. dna microarrays have been used for the detection of infectious and genetic disease, cancer diagnostics, and forensic and genetic identification purposes. microarray systems are very definitive and highly scalable because hundreds to tens of thousands of possible dna elements can be interrogated in a single experiment. examination of animal and zoonotic pathogens typically focuses on objectives: ( ) determining the presence or absence of a pathogen; ( ) determining pathogen viability; and ( ) enumerating the pathogenic agents of a specific disease. dna microarrays are well suited for determining the presence or absence of specific pathogens. dna microarrays also permit relatively rapid interrogation of a clinical sample against thousands of genetic targets, allowing for simultaneous detection and discrimination among hundreds of pathogenic agents of veterinary interest. however, dna microarrays are not very useful in inferring pathogen viability. the first important step in microarray-based pathogen detection is probe selection and design. many issues associated with probe design for dna microarrays can impact the overall fidelity of the assay and particularly the levels of specificity and sensitivity. one of the first applications of a bacterial high-density dna probe array was for the diagnostic capability of identifying mycobacteria spp. using rt-pcr and a dna microarray based on the mycobacterial heat shock protein, this technology was used for the rapid identification of different mycobacterial species in mixed infections and array-based genotyping of mycobacterial organisms. other applications to bacterial infections have included the diagnosis of salmonella spp., escherichia coli o :h , mycoplasma hyopneumoniae, gene profiling of pasteurella multocida, mycobacterium avium subsp, paratuberculosis spp., rhodococcus equi, borrelia burgdorferi, staphylococcus aureus enterotoxin genes and clostridium perfringens toxin genes, bacillus anthracis, c. perfringens, brucella spp., bordetella pertussis and b. parapertussis, and actinobacillus pleuropneumoniae. microarray technology has also been used to generate a "lipopolysaccharide array" against the outer membrane of gram-negative bacteria. monoclonal antibodies specific for escherichia coli o , e. coli o , francisella tularensis, and salmonella enteric serovar typhimurium o antigens were used to evaluate the assay. the detection limit of antibodies was reported to be ng/ml, which is -fold more sensitive than conventional immunofluorescence assays. the diagnosis of viral diseases requires the identification of the viral pathogen in the clinical sample and a subsequent correlation between the identified virus and the clinical disease. conventional methods of viral detection include isolation and in vitro culture, and immunological methods such as direct fluorescence antibody or enzyme immunoassay. with some viral disease agents, identification is readily accomplished. other viruses (e.g., norovirus), however, are refractory to in vitro culture. immunological methods, dependent on the quality and availability of antibodies, may also be ineffective for identifying viruses with constantly evolving serotypes. although pcr has revolutionized viral diagnostics by increasing detection sensitivity and specificity, the maximum number of viruses detectable in a single assay is relatively small. in diseases where different viruses are present or where no etiologic agent has been identified, the value of these conventional methods may be limited. unlike pcr technology where the target agent must be known to use specific test primers, microarrays can allow for the rapid diagnosis of multiple pathogenic agents in disease outbreaks and epidemics of unknown etiology. one of the first studies in multiple pathogen detection used a microarray chip to discern viral pathogens, porcine reproductive and respiratory syndrome virus and foot and mouth disease virus. the microarray detection device demonstrated rapid and accurate detection of these specific viruses. in another study, a low-density microarray, using capture-oligonucleotides targeting the conserved influenza matrix gene, accu-rately subtyped h n , h n , and h n influenza a viruses in less than hours. , researchers led by joseph derisi of the university of california at san francisco have combined genome databases of sequenced viruses with dna microarray technology. using available sequence data from viral genomes, they designed a long oligonucleotide viral microarray. the microarray was capable of simultaneously detecting hundreds of viruses in a single clinical sample. the approach used randomized primers in an rt-pcr to amplify any viral rna that was present in the clinical sample. the pcr product was then hybridized to the microarray comprising probes, representing nearly respiratory virus genomes. viruses that were represented on the microarray were readily detected and identified by specific hybridization to the appropriate oligonucleotides. this methodology enabled the simultaneous detection of hundreds of viruses, including essentially all respiratory tract viruses. this microarray system greatly expanded the spectrum of detectable viruses in a single assay while simultaneously providing the capability to discriminate among viral subtypes. similarly, sengupta and colleagues developed microarrays with probes to distinguish among various influenza viruses. although initial efforts were focused on only a few hundred viruses, derisi and colleagues devised a microarray representing all known human, animal, aquatic, and plant viruses called the virochip. the most highly conserved genomic sequences from within viral families were chosen for representation on the microarray. viruses represented included double-and single-stranded dna viruses, retroviruses, and both positive-and negative-stranded rna viruses. dna fragments of virtually every virus ever discovered, about , different viral sequences, were represented on the virochip. the virochip greatly accelerates the ability of researchers to diagnose viral infections and identify new viral epidemics within a very short time. this technology can help to rapidly identify known viruses and classify new ones based on their genetic makeup. the virochip has been credited with the rapid recognition of a novel coronavirus in , the etiologic agent responsible for sars. although the virochip represents sequences from known referenced viruses, equally important is its potential to facilitate viral discovery in diseases of unknown etiology. as new viruses evolve, they still maintain certain characteristic-conserved regions in the viral genome. by using highly conserved sequences from all known viral taxonomy, the viro-chip can identify similar sequences in previously future veterinary diagnostics unknown agents and newly evolved viruses within recognized families. proventricular dilation disease (pdd) has been diagnosed in psittacine birds for the past years, but the underlying etiologic cause has not been identified. although the disease exhibits characteristics of a viral infection and numerous viral etiologies have been proposed, confirmation of a definitive agent remained elusive. in , kistler and coworkers used the virochip to interrogate samples from pdd case/control series collected on different continents. tissues from birds with confirmed pdd displayed a novel bornavirus signature in . % of the pdd cases and none of the control tissues. the bornavirus-positive samples were further confirmed by virus-specific pcr testing and the complete genome sequenced. currently, genotypes of the avian bornavirus have been identified. microarray-based viral detection offers a powerful alternative for determination of viral subtypes. classic serotyping of viruses is tedious and limited by availability of antisera. conserved array elements are capable of broadly detecting many, if not all, viral serotypes. unique hybridization patterns are often observed for different viral serotypes, which enables rapid identification. because of the high resolution of microarray hybridization, it will see future application in the study of viral pathogenesis and diagnosis of veterinary diseases. microarray hybridization is a viable approach for detecting unsequenced or uncharacterized viruses and novel virus discovery. it can be used to differentiate among viral subtypes and provide important information on how a new virus relates to those that have been classified. application of this type of information can provide clues about viral origin and possible treatment strategies. this comprehensive and unbiased analysis of viral prevalence in a given biological sample also increases the feasibility of introducing molecular testing for viruses that are easily detected using classical diagnostic testing methods. when target nucleic acids are abundant, then direct microarray interrogation of pathogen targets is achievable in the absence of amplification. direct hybridization strategies involve extracting rna or dna from the sample and applying the material directly to a microarray without additional amplification. direct hybridization provides the least bias in gene detection, but also has the lowest level of ana-lytical sensitivity. another strategy bypasses these limitations and yields an extremely broad-reaching and unbiased detection strategy. it uses microarrays composed of carefully selected viral sequences, coupled to a random pcr amplification. one approach is to amplify one or more universal genes (e.g., s rrna, s rrna, s rrna genes) and to screen for pathogen-specific polymorphisms. greisen and coworkers were one of the first groups to amplify fragments of the s rrna gene using group-specific pcr primers and, with southern blots that had pathogenspecific probes, identified pathogenic bacteria in cerebrospinal fluid. microarrays can be used in a similar fashion to interrogate pcr products to distinguish between multiple pathogens. a second pcr strategy is to use multiplex pcr to amplify a number of discreet, pathogen-specific genetic markers that are subsequently detected on a dna microarray. simultaneous detection of multiple pathogens should be viewed in the context of an entire analytical process that spans sample collection to final testing. physical differences between target organisms often require very different strategies to extract nucleic acids for analysis after which the dna microarray can then serve as the actual detection device. the reader is referred to the review by wang for an encompassing review of microarray technology. in the area of microarrays for microbiological applications, several general reviews are available. specific reviews are available on dna microarray analyses of host-pathogen interactions and the use of microarrays for the molecular diagnosis of mycobacteria spp. generalized reviews in the medical-related areas include microarrays for disease gene discovery, microarrays in medicine, and microarrays for molecular pathology. early methods of dna sequencing, determining the nucleotide order of a dna segment, gene, or genome, were costly and slow. although the first sequence of a human gene locus was published in , it took years to report the sequence of the entire human genome in . the recent development of cost-effective, high-throughput sequencing now allows for efficient, full sequencing of an organism's genome. as dna sequence data have become more available, the field of comparative genomics has rapidly progressed. attention has turned to generating whole genome assemblies and genomics resources for veterinary species. in july , a publicly accessible annotated genome assem-bly of the domestic dog, and subsequently a draft sequence of the domestic cat, , were released. with complete genome sequences now available for the dog, cat, and horse, the tools are now available to research very specific questions about animal genomes and cancer. dna microarrays have technologically advanced genomics research. dna microarrays are used to detect dna or rna (most commonly as cdna after rt) and are referred to as gene expression analysis or expression profiling. gene expression analysis is based on the assumption that cells react to changes in their environment by increasing or decreasing transcription of appropriate genes. these changes in expression levels can be detected or quantified by using specific scientific methodology such as realtime rt-pcr and microarray technology. the use of a distinct dna array for gene expression profiling was first described in to identify genes whose expression was modulated by interferon. determining the level, or volume, at which a certain gene is expressed is called microarray expression analysis. miniaturized microarrays were first applied to expression profiling in . in veterinary medicine, genome libraries have allowed for the development of new, high-density microarrays to facilitate whole genome and gene-targeted profiling at high resolution and throughput. new, high-density microarrays have rapidly advanced our knowledge and understanding of pet and food animal genomes and have fostered the development of a rapidly growing class of molecular diagnostics focused on diagnostic testing for genetic traits encoded by genomic dna. scientists can use a microarray to examine the expression of hundreds or thousands of genes within a single sample, in a single assay. through computer analysis, the amount of mrna bound to each site on the array is precisely measured, generating a profile of global gene expression within the cell. once researchers have characterized the expression patterns of various genes involved in many diseases, the expression pattern of cdna from an individual can be hybridized to determine if it matches the expression pattern of a known disease. if a gene is over expressed in a certain disease state, then more sample cdna, compared with control cdna, is detected. in microarray comparative genomic hybridization, the increased or decreased expression of a particular gene(s) is determined. a change in the number of copies of a particular gene may correlate to a certain disease state. microarrays may also be used in veterinary diagnostics by comparing gene expression of infected cells or tissues with that of uninfected cells or tissues. expression profiling can also identify genes whose expression is changed in response to disease-causing agents. revealing how multiple genes work together to produce physical and chemical responses and analysis of patterns of coordinated gene expression can help identify genes involved in the development of various diseases. a canine-specific microarray using mrna and expressed sequence tag sequences has been developed. the elucidation of these biological networks (e.g., disease, development, nutrition) and identification of associated molecular mechanisms will allow for the development of more precise diagnostics for these processes. dna microarrays can be designed specifically for genotyping, thereby enabling them to measure single base pair changes at many thousands of points throughout a genome. dna microarrays are useful for genotyping point mutations, single nucleotide polymorphisms (snps), and short tandem repeats (strs) among alleles within or between populations. snp technology has been diversely applied in areas such as analyzing forensics, measuring disease predisposition, evaluating germ line mutations in individuals and somatic mutations in neoplasias, diagnosing infectious and genetic disease, and diagnosing cancer. once an snp pattern is associated with a particular disease, snp microarrays can be used to test an individual for that disease expression pattern. a determination can be made if the individual is susceptible or at risk of developing that specific disease condition. in sheep, diagnostic microarrays are used to determine specific snps that predispose animals to scrapie, a prion protein disease. individuals with these snps accumulate the prion protein disease cellular protein within their central nervous system. initial microarray platforms available for veterinary oncology were genomic and cdna microarrays generated by binding dna fragments onto a glass or silicon surface. more recently, high-density oligonucleotide arrays, where short dna sequences are synthesized directly on the surface of a "chip," have allowed for thousands of features to be represented. dna copy number variation, gene expression levels, and genetic alterations can be determined at a substantially increased resolution. high-density microarrays represent a powerful tool for comparative genomic studies. microarraybased comparative genomic hybridization merges molecular diagnostics with traditional chromosome analysis. , applications for high-density microarrays have been extended to many areas of medicine and medical genetics. new syndromes have been discovered, and the phenotypes of existing disease conditions have been expanded. a comparative study of canine chronic myelogenous leukemia showed that dogs diagnosed with chronic myelogenous leukemia also present with a functionally active genetic translocation previously identified in humans. this study resulted in the first molecular cytogenetic test for the presence of a clinically significant genomic alteration in a veterinary cancer. several tests are now available for canine patients to identify oncogenes and clonal lymphocyte populations. these tests can be placed into broad categories: ( ) detection of individual mutations in oncogenes, ( ) detection of chromosomal translocations, deletions, and duplications, and ( ) detection of clonality in lymphoma and leukemia through unique antigen receptor genes of b-and t-cells. each of these testing methods has been used in veterinary medicine on an experimental basis and is now being offered by several laboratories. the original emphasis on microarrays was on dna technologies. researchers have exploited the diversity of this technology to further extend applications to the development of protein, antibody, and carbohydrate microarrays. macbeath and schreiber developed the first small-molecule microarray in and the first protein microarray in . soon afterward, other array types were developed, such as cell arrays, carbohydrate arrays, and proteome arrays. newer microarray formats that developed at the turn of the st century provide a host of other biomolecules that can be presented on a chip, including proteins (e.g., whole proteomes, enzymes, antibodies), , small molecules (e.g., drug-like molecules, peptides, carbohydrates), , and even whole cells and tissues for simultaneous, multiplexed experimentation. within the last decade, protein microarrays have entered the field of proteomic research. , pro-teomics is the term used to describe the study of proteins expressed by a genome. technologies that had previously been established for dna microarrays were adapted to the generation of protein arrays with glass slides being initially used as a solid support in planar microarrays. bead arrays or liquid array systems rely on the use of different bead types that can be distinguished either by color, size, or shape. currently, protein microarrays are highly miniaturized and parallelized solid-phase assay systems that use a large number of different capture molecules immobilized in microspots. protein microarray technology has enormous potential for in vitro diagnostics and disease monitoring. protein microarray technology has enormous potential for application within the veterinary diagnostic testing arena. antibodies in an immunoassay format have been widely used and are well established as highly sensitive tools for disease detection. with monoclonal antibody production, it is possible to produce pure and highly specific antibodies against almost any type of antigen. as a next-generation tool, protein arrays in their role as miniaturized, multiplexed immunoassays are perfectly suited for generating a maximum of diagnostically relevant information from very minute samples. protein microarrays offer unparalleled throughput, minimal reagent consumption, and sensitive simultaneous detection of multiple targets. currently, there is rapid advancement of antibody microarrays that have been developed for clinical, biothreat, and point-of-care applications. early applications included the development of a protein microarray with antibodies, which was used to subtype the most common salmonella serovars, and a similar microarray was developed for e. coli. lipopolysaccharide, carbohydrate-based, and whole-cell microarrays have also been used for antibody-based detection of pathogens such as francisella tularensis. , liquid array technology has been adapted to the detection of avian influenza (ai) virus antibody in commercial poultry. the assay uses recombinant ai virus nucleoprotein conjugated to microspheres to detect the influenza-specific antibody. the ai virus liquid array has a . % sensitivity and a . % specificity. this liquid array format can theoretically detect different analytes in a single assay. therefore, this technology has the potential to simultaneously detect and subtype all influenza hemagglutinin and neuraminidase proteins, as well as antibodies specific to the other relevant avian diseases. protein microarrays with high pathogen proteome content offer a valuable platform for highthroughput serology. these antigen microarrays have been used to identify seropositive individuals by using the presence of serum antibodies to detect exposure to a specific pathogen. zhu and coworkers monitored the antibody profiles of patients with sars using protein microarrays containing purified coronavirus proteins. immunoreactivity against the coronavirus nucleocapsid proteins remained high for to days postinfection, which provided a means to check for exposure long after infection has occurred. the integration of dna and protein-based microarray methods may extend the range of rapid clinical diagnostic testing. in protein expression analysis, proteins or biomarkers from complex biological samples are identified and correlated with diagnostic patterns that are unique to specific cancer and disease conditions. the main challenge in the development of biomarker diagnostics is the biological variability among patient samples as well as the large dynamic range of biomarker concentrations. both disease and cancer biomarker discovery (oncoproteomics) has been studied extensively using proteomics. biomarkers have been identified that correlate with bladder cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal stromal tumors, glioma, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, and urothelial carcinoma. protein microarrays designed with hundreds of copies of protein-coding oligonucleotides or "bio-barcodes" can serve to amplify the protein target and are capable of detecting proteins down to attomolar concentrations. besides protein expression analysis, protein microarrays can also be used to determine the functional analysis of proteins, including protein interaction involving immobilized proteins or peptides, low molecular weight compounds, dna, oligosaccharides, tissues, or cells. protein microarrays have been developed for the detection of allergen-specific immunoglobulin e reactivity and detection of specific auto antibodies associated with autoimmune diseases. in the field of autoimmune disease diagnostics, multiplexed assays are currently entering the diagnostic market, and in the future, sets of tumor marker panels may also be applied to monitor treatment therapy for specific disease processes. immunoassay panels have been developed for fertility, cardiac disease, tumors, cytokines and growth factors, cell adhesion molecules, thyroid function, and drug residues. virtually every biological component from diverse small molecules, macromolecules (e.g., dna, proteins), and entire living cells have been placed on microarrays. a biomarker is "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to therapeutic intervention." research regarding biomarkers associated with disease has greatly advanced in both the human and veterinary medical fields. the challenge of this information is to determine which marker or combination of markers is optimal for the condition to which they are applied. evidence from research studies suggests that using multiple markers may be superior to using single markers alone. multiple marker combinations provide information of greater diagnostic and prognostic value than any single marker alone. the most promising use of veterinary biomarkers are those used to evaluate cardiovascular disease in dogs and cats. [ ] [ ] [ ] currently, circulating natriuretic peptides are regarded as the most promising markers used in the evaluation of such patients. other markers include those of myocyte injury typified by troponins, markers of myocyte stress (adrenomedullin), matrix metalloproteinases, markers of endothelial function, and markers of inflammation (c-reactive protein). concentrations of some of these substances are known to change in response to heart failure and the treatment of cardiac disease in veterinary patients. , clinical application of biomarkers in the future includes the detection of subclinical diseases, diagnosis of acute or chronic syndromes, risk determination, therapy selection, and monitoring of disease progression or response to treatment. presently, most biomarker testing takes place at dedicated laboratories, thereby increasing the expense of the test and the time needed to perform the test. the development of biosensor technology will enable biomarker testing to be accomplished in a smaller, faster, cheaper, and portable format. a biosensor can be generally defined as a device that consists of a biological recognition system and a transducer. a biosensor is a compact analytical device that integrates a biological element on a solid-state surface, enabling a reversible biospecific interaction with an analyte. the interaction of the analyte with the bioreceptor produces an effect measured by the transducer, a device used to convert the information into a measurable signal. the biological component of a biosensor can include any molecule used for biomolecular recognition. biosensors have been de-signed using specific binding proteins, lectins, nucleic acids, membranes, whole cells, antibodies, or antibody fragments. biosensors that detect the immunological interaction between a specific antibody and antigen are referred to as immunoreaction-based biosensors or immunosensors. some biosensors use aptamers, single-stranded dna or rna oligonucleotide sequences that recognize various target molecules with high affinity and specificity. these ligand-binding oligonucleotides mimic properties of antibodies in a variety of diagnostic formats. there are main types of biosensors: biocatalytic and bioaffinity-based biosensors, and they are primarily categorized on the detection principle applied. biocatalytic sensors use enzymes to catalyze a signaling biochemical reaction, and bioaffinity-based sensors monitor the binding event itself. transducers convert the biological interaction to a detectable signal and can be based on electrochemical (e.g., potentiometric, amperometric, conductometric), optical, microgravimetric, or thermometric (calorimetric) principles. biosensors have been designed using carbon nanotubes and nanoparticles for signal amplification. combining the molecular specificity of biological recognition with an operationally simple transducer, extend these small units' diagnostic capability toward portability and ease of use. recent developments in immunosensors have produced solid state systems that allow for rapid and continuous monitoring of antibody-antigen reactions in real time. the specificity of the biosensor system is determined by the immobilized detection molecule. innovative strategies using biomarkers represent alternative strategies for reliable cancer testing and for detecting clinical markers predictive of cardiovascular and other diseases. biosensors using antibodies or dna/rna strands can detect infectious disease agents and be used for hormone and drug assays. , currently, most biomarker testing takes place at a dedicated, centralized laboratory, but in the future this technology will provide a means of rapid, convenient, and economical testing at the "point of patient care." in cancer, there is a strong connection between early detection and positive patient outcome, thereby improving patient survival and disease prognosis in many cases. in addition to changes within the host genome, complex molecular alterations such as over or under expression of a protein can occur during the course of tumorigenesis. biosensors can detect these changes and can analyze molecular biomarkers for tumor diagnosis and classification, monitor response to treatment, and detect recurrence of the disease. cancer biosensors have been developed in a panel format for the simultaneous detection of different cancer biomarkers. , because most cancer biomarkers are not specific to a particular tumor, the use of an array to detect multiple markers increases the diagnostic value of their definitive diagnosis. recently, biosensors have been developed to detect breast cancer, ovarian cancer, gastrointestinal tract carcinoma, chronic myelogenous leukemia, and prostate cancer. prostatespecific antigen is the most reliable tumor marker to detect prostate cancer in its early stages. the recently developed biosensors for prostate-specific antigen detection are sensitive to a limit of pg/ml. the importance of inflammatory markers in the early detection of cardiovascular disease has been demonstrated. biosensors are being developed to detect c-reactive protein, cardiac troponin, myoglobin, and natriuretic peptide in a sensitive and efficient manner. based on a competitive immunoassay in a portable biosensor, good correlation between salivary cortisol and levels of "free" cortisol in serum has been demonstrated. a nucleic acid biosensor is an analytical device that integrates an oligonucleotide with a signal transducer. the nucleic acid probe is immobilized on the transducer and acts as the biorecognition molecule to detect dna/rna fragments. several dnabased biosensors have recently been developed for the detection of virus-related sequences and other infectious agents. dna or rna ligands can also be synthesized to be used to detect amino acids, drugs, proteins, and other molecules. dna or rna ligands can bind to their targets with affinity ranging from the micromolar to the nanomolar level and can discriminate between closely related targets. there is a growing need for small, fast, efficient and portable biosensors. the analysis of thousands of small molecules (metabolites) (e.g., sugars, organic acids, amino acids, nucleotides) by means of a global approach is referred to as metabolomics. combined with pattern recognition processes, metabolomics defines a metabolic phenotype (metabotype) in the study species. this approach combines high-throughput sample analysis with computer-assisted pattern recognition techniques whereby the full metabolite profile (metabolome) of a cell, tissue, or organism is determined. metabolomics is a powerful investigative tool for studying the biochemical effects of disease and screening for potential pharmacologic agents. alterations in the metabolic profile often present much earlier in the course of disease than induced histopathological changes. metabolomics can be a sensitive, early indicator of a disease process, and because metabolites are conserved across species lines, screening patterns can be applied to a wide range of species. the application of metabolomics in veterinary medicine is in its infancy. whitfield and colleagues used metabolomics to distinguish canine congenital portosystemic vascular anomalies from acquired hepatopathies. other early applications include the analysis of cadmium toxicity in rodents, assessment of sublethal stress in aquatic organisms, and neurotransmitter deficits in cerebral tissue from a mouse model of human batten disease. although current applications to the field of veterinary medicine are rare, metabolomics use in early preclinical safety assessment make it a rapidly developing tool in the field of drug investigations. metabolomics enables a noninvasive systems assessment of a broad spectrum of biologic responses by an individual to the effects induced by therapeutic compounds, which could impact all stages of veterinary drug research and development. molecular theranostics is an emerging area in which molecular diagnostic tools are used to provide rapid (less than hour), accurate, and informative diagnostic microbiology assays. conventional microbial culture and sensitivity testing methods require at least days' time because of the reliance on growth and isolation of microorganisms. veterinarians must frequently treat patients empirically with broad-spectrum antimicrobial agents, which may not be needed or may not be effective. the common use of broadspectrum antimicrobials is associated with an increasing rate of microorganism resistance, which may complicate patient treatment. early diagnosis and treatment of diseases reduce the risk of the patient developing long-term disease complications. for some diseases, a prompt treatment will also reduce further transmission of the disease to other animals or humans. assays based on the detection of nucleic acids offer enormous potential for the rapid and accurate diagnosis of microbial infections. this assay-based testing can be extended to include the detection and characterization of genes or mutations associated with antimicrobial resistance and virulence. advances in genomics and proteomics will provide the essential nucleotide or amino-acid sequence data required to design accurate assays. efficiency will also depend on the development of rapid, simple, and efficient methods for microbial nucleic acid or protein extraction from a variety of clinical samples. finally, rapid and specific assays, and the ability to detect all or most targeted microorganisms in multiplex target amplification systems, or signal amplification technologies, are needed. this will help future technologies reach a level of analytical sensitivity appropriate for testing directly from clinical samples without previous enrichment, thereby leading to more judicious antimicrobial usage and a concurrent reduction in antimicrobial-resistant organisms. to be useful, diagnostic methods must be accurate, simple, and affordable. clinical virology in real time drivers of biodiagnostic development laboratory reporting accuracy of polymerase chain reaction testing for psittacine beak and feather disease virus repair replications of short synthetic dna's as catalyzed by dna polymerases the nobel prize foundation. kary b. mullis nobel lecture on december rapid west nile virus molecular beacons: probes that fluoresce upon hybridization the light cycler: a microvolume multisamplefluorimeter with rapid temperature control loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases using molecular beacons to detect single-nucleotide polymorphisms with real-time pcr nucleic acid amplification and related techniques in microbiological diagnostics and epidemiology nucleic acid sequence based amplification (nasba) future veterinary diagnostics detection of highly pathogenic and low pathogenic avian influenza subtype h 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of an individual human genome sequence, comparative analysis and haplotype structure of the domestic dog initial sequence and comparative analysis of the cat genome state of cat genomics venkatasubbarao s: microarrays-status and prospects identification of interferon-modulated proliferation-related cdna sequences quantitative monitoring of gene expression patterns with a complementary dna microarray tools of the trade: diagnostics and research in domestic animal cytogenetics comparative analysis of transcript abundance in pinus sylvestris after challenge with a saprotrophic, pathogenic or mutualistic fungus the development of a high-density canine microarray determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays united states department of agriculture, veterinary services. the national scrapie surveillance plan genomewide analysis of dna copy-number changes using cdna microarrays comparative genomics using candida albicans dna microarrays reveals absence and divergence of virulence-associated genes in candida dubliniensis medical applications of array cgh and the transformation of clinical cytogenetics evolutionarily conserved cytogenetic changes in hematological malignancies of dogs and humans-man and his best friend share more than companionship molecular diagnostics of hematologic malignancies printing proteins as microarrays for high-throughput function determination progress in protein and antibody microarray technology advances in functional protein microarray technology small molecule microarrays: recent advances and applications peptide microarrays: next generation biochips for detection, diagnostics and high-throughput screening microarrays in infection and immunity protein microarrays and proteomics protein microarrays: promising tools for proteomic research protein microarrays for diagnostic assays development of a novel protein microarray method for serotyping salmonella enterica strains use of miniaturized protein arrays for escherichia coli o serotyping lipopolysaccharide microarrays for the detection of antibodies bacterial cell microarrays for the detection and characterization of antibodies against surface antigens a microsphere immunoassay for detection of antibodies to avian influenza virus severe acute respiratory syndrome diagnostics using a coronavirus protein microarray contribution of oncoproteomics to cancer biomarker discovery new analytical tools for studying autoimmune diseases biomarkers and surrogate endpoints: preferred definitions and conceptual framework the effect of heart disease, heart failure and diuresis on selected laboratory and electrocardiographic parameters in dogs lymphocyte subpopulations and hematologic variables in dogs with congestive heart failure benchmarks for the assessment of novel cardiovascular biomarkers biomarkers in cardiovascular disease: beyond natriuretic peptides immunosensorsprinciples and applications to clinical chemistry new clinical markers predictive of cardiovascular disease: the role of inflammatory mediators point-of-care biosensor systems for cancer diagnostics/prognostics multiplex measurement of seven tumor markers using an electrochemical protein chip a disposable multianalyte electrochemical immunosensor array for automated simultaneous determination of tumor markers biomedical and clinical applications of immunoassays and immunosensors for tumor markers a nanoparticle label/ immunocromatographic electrochemical biosensor for rapid and sensitive detection of prostatespecific antigen detection of cortisol in saliva with a flow-filtered portable surface plasmon resonance biosensor system nucleic acid biosensors for environmental pollution monitoring an introduction to metabolomics and its potential application in veterinary science metabolomics as a diagnostic tool for hepatology: validation in a future veterinary diagnostics naturally occurring canine model the initial pathogenesis of cadmium induced renal toxicity utilizing in vivo nuclear magnetic resonance spectroscopy to study sublethal stress in aquatic organisms high resolution h nmr based metabolomics indicates a neurotransmitter cycling deficit in cerebral tissue from a mouse model of batten disease molecular testing in laboratory medicine key: cord- -vubszdp authors: li, lucy m; grassly, nicholas c; fraser, christophe title: genomic analysis of emerging pathogens: methods, application and future trends date: - - journal: genome biol doi: . /s - - - sha: doc_id: cord_uid: vubszdp the number of emerging infectious diseases is increasing. characterizing novel or re-emerging infections is aided by the availability of pathogen genomes. in this review, we evaluate methods that exploit pathogen sequences and the contribution of genomic analysis to understand the epidemiology of recently emerged infectious diseases. when a pathogen crosses over from animals to humans, or an existing human disease suddenly increases in incidence, the infectious disease is said to be 'emerging'. the number of emerging infectious diseases (eids) has increased over the last few decades, driven by both anthropogenic and environmental factors [ ] . these include the expansion of agricultural land, which increases the exposure of livestock and humans to infections in wildlife [ ] ; a greater volume of air traffic, enabling eids to rapidly spread across the world [ , ] ; and climate change, which alters the ecology and density of animal vectors, thereby introducing diseases to new geographic locations [ ] . novel strains of existing pathogens also have the potential to cause large epidemics. the over-and misuse of antimicrobial drugs have contributed to the growing number of drug-resistant pathogen strains [ , ] . detecting, characterizing and responding to an eid requires co-ordination and collaboration between multiple sectors and disciplines. laboratory-based research helps to characterize the pathogen and its interactions with host cells, but is less useful for quantitative understanding of population-level disease dynamics. modeling approaches enable a large number of hypotheses to be tested, which might not be logistically or ethically feasible in laboratory and field experiments. in addition to characterizing past disease dynamics, modeling future trends informs decisions regarding outbreak response and resource allocation [ ] . modeling plays an especially important role in epidemiological studies of infectious disease spread, because the transmission of infectious disease between individuals is not directly observable. at the individual level, transmission times and who infected whom are typically unknown. and at the population level, disease burden needs to be inferred from observable data. important public health questions such as how quickly an epidemic spreads and how many people will be infected are hard to quantify without a mechanistic understanding of underlying factors driving disease transmission. by expressing disease spread in mathematical terms, statistical properties of epidemics can be estimated to help address specific questions regarding disease spread and control efforts [ ] . another discipline contributing to the study of eids is pathogen genomics. as sequencing technology has become more accessible and affordable, genetic analysis has played an increasingly important role in infectious disease research. sequencing pathogens can confirm suspected cases of an infectious disease, discriminate between different strains, and classify novel pathogens. in addition to examining individual pathogen sequences, multiple sequences can be analyzed together using phylogenetic methods to elucidate evolutionary [ ] and transmission [ ] history. just as mathematical models of disease transmission help to capture the epidemiological properties of an infectious disease, modeling the molecular evolution of pathogen genomes is important for phylogenetic methods. besides characterizing the genetics and evolution of a pathogen, mathematical models used in population genetics link demographic and evolutionary processes to temporal changes in population-level genetic diversity. the coalescent population genetics framework was developed so that demographic history could be inferred from the shape of the genealogy linking sampled individuals [ , ] . more recently, the birth-death model has been applied to infectious diseases to infer epidemiological history from a genealogy [ , ] . given the link between pathogen evolution and disease transmission, there is a trend towards integrating both epidemiologic and genetic data in the same analytical framework [ ] [ ] [ ] . in this review, we provide an overview of recent developments in genomic methods in the context of infectious diseases, evaluate integrative methods that incorporate genetic data in epidemiological analysis, and discuss the application of these methods to eids. over the last two decades, sequence data have increased in quality, length and volume due to improvements in the underlying technology and decreasing costs. as a result, pathogen sequences are regularly collected during routine surveillance and clinical studies. just as mathematical modeling can be used to analyze surveillance data to reveal details of disease transmission (box ), analysis of pathogen genomes employs mathematical frameworks to elucidate pathogen biology, evolution and ecology (figure ). at the most basic level, mathematical models are used to find the optimal alignment of pathogen sequences. multiple sequence alignment is useful for finding highly conserved or variable regions, shedding light on the molecular biology of the pathogen. furthermore, coupling sequences with clinical information can help identify the contribution of polymorphic sites to disease. revealing the evolutionary history of a pathogen requires a quantitative description of relatedness. based on polymorphic sites in the sequence alignment, a model of sequence evolution is then used to reconstruct the phylogeny [ ] . often, there is insufficient genetic diversity in the sample to fully infer the phylogeny without ambiguity. in such a case, it is useful to consider a tree as an unknown set of parameters and obtain its posterior probability distribution using a bayesian framework, such as the markov chain monte carlo (mcmc) approaches [ , ] . biological samples from which pathogen genetic material is sequenced are usually associated with geographic or temporal information (figure b ). when this additional information is available, phylogenetic methods can reveal the spatiotemporal spread of the pathogen in the population. if an outbreak is densely sampled, then the pathogen phylogeny provides information about the underlying transmission network and helps to uncover who infected whom [ , ] , though phylogenetic clustering alone is usually not sufficient to prove direct transmission or direction of infection ( figure b) . incorporating sampling times helps to convert a phylogeny specified in units of nucleotide substitutions to a phylogeny specified in units of time [ ] . the conversion is straightforward if sequence evolution follows a strict molecular clock, whereby the rate of substitution remains constant over time. however, selection pressure and population bottlenecks can lead to changes in the rate of substitution [ ] . more flexible models have been developed to incorporate time-varying rates of evolution [ , ] . with branch lengths in units of real time, the start date of an epidemic can be estimated. whereas phylogenetics aims to delineate the relationship between individuals, population genetics aims to link population processes to observed patterns of genetic diversity. inferences regarding pathogen population history are based on the genealogy, or ancestry, of sequences from sampled individuals, and often carried out in a retrospective population genetics framework known as the coalescent [ ] (box ). a genealogy describes the ancestry of sampled individuals. going backwards in time, pairs of lineages coalesce when they share a common ancestor, until the last two lineages coalesce at the time of the most recent common ancestor (tmrca) for the entire sample. since the turn of the century, the coalescent has been increasingly applied to infectious disease research to infer epidemic history from pathogen sequences, thereby linking pathogen evolutionary history to disease epidemiology ( figure c ). the method is especially useful for analyzing infectious diseases with mild or asymptomatic infections, for which case-based surveillance data severely underestimate prevalence, because the coalescent assumes a small sample compared to the population size [ ] [ ] [ ] . other approaches have been developed to make epidemiological inferences from genetic data. of particular note is the birth-death model [ ] , which describes the rates of transmissions, recoveries and deaths, and sampling events in terms of the sample genealogy [ ] . just as there are coalescent methods incorporating population structure [ ] [ ] [ ] and compartmental models [ ] [ ] [ ] , similar methods exist in the birth-death framework [ , ] . unlike the coalescent framework, the birthdeath model is still valid for densely sampled populations, which makes it more useful for studying small outbreaks. however, accurately inferring epidemiological parameters depends on correctly specified sampling proportions [ ] . although the two approaches are methodologically different, both aim to reconstruct pathogen population history and produce estimates of epidemiological parameters, such as the reproductive number (r ). the focus on the coalescent framework in this review is due to its more pervasive use in the literature and its greater versatility when integrated with epidemiological models compared to birth-death models. because of the simplistic assumptions of population genetics models, the population size inferred using coalescentbased methods cannot be directly interpreted as pathogen population size (prevalence of infection). it is rather the effective population size, n e (box ), which refers to the size of a wright-fisher population that would produce the same level of genetic diversity as observed in the sample. in real populations, the variance of the offspring distribution (box ) is higher than expected in a wright-fisher population due to heterogeneity in host infectiousness, non-random mixing of the population, and migration events. the consequence of a large variance is that there is a greater discrepancy between the effective and census population sizes [ ] . accounting for the dispersion of the offspring distribution is especially important when analyzing infectious disease data because of the widespread occurrence of transmission heterogeneity [ ] . another statistical property of epidemics affecting the results of modeling studies is the generation time distribution, which describes the time between infection of the primary case and of secondary cases. obtaining an estimate of the generation time is important for two reasons. first, estimates of r from the initial growth rate of an epidemic depend on the generation time distribution [ ] . as r is the mean of the offspring distribution, its value affects the relationship between the effective population size, n e , and the census population size, n. second, the coalescent model was originally specified in units of generations, and so estimates in this framework need to be converted to natural units using the generation time, t g . because transmission events are rarely observed, the generation time distribution is often approximated by the distribution of the serial interval, which is the time between onset of symptoms in the primary and secondary cases. the two distributions generally share the same mean but might have different variances [ ] . furthermore, the observed generation time decreases as the epidemic grows but increases again after the epidemic peak due to right censoring [ ] . as both sequence and surveillance data contain information regarding the transmission process, simultaneously analyzing both datasets should yield more accurate estimates of epidemiological parameters than separate analyses [ ] . the recently established discipline of phylodynamics takes an interdisciplinary approach to understand the pathogen phylogenetics and epidemiology in terms of disease transmission. most efforts thus far have focused on enhancing phylogenetic and population genetic analyses by incorporating spatial and temporal information about the sequences. the molecular clock model assumes a constant rate of evolution and thus helps to estimate the time of the most recent common ancestor of the sample, which approximates the start date of an epidemic. molecular clock analysis has been used to date the emergence of a range of emerging pathogens from hiv [ ] to multidrug-resistant streptococcus pneumoniae [ ] . linking geographic information with sequences can reveal the spatial spread of infectious disease. phylogenetic reconstruction of seasonal influenza (h n ) sequences has revealed the contribution of viral circulation in temperate regions to the global genetic diversity of influenza, and determined that not all epidemics in temperate regions are seeded by strains from south east asia [ , ] . also using global sequences, hepatitis c virus (hcv) subtypes were shown to spread from developed to developing countries [ ] . finally, phylogeographic analysis of methicillin-resistant staphylococcus aureus samples identified england as the source of the emrsa- lineage [ ] . by contrast, there have been relatively few studies incorporating genetic data into epidemiological frameworks. although genetic analysis plays an important role in elucidating transmission links in disease outbreaks [ , , ] , its integration with epidemiological models to understand population-level disease dynamics has been more limited. in one of the first papers to link coalescent inference to mathematical models in epidemiology, the effective population sizes of hiv- subtypes a and b were estimated from the maximum likelihood trees of viral sequences [ ] . in addition to revealing population sizes, pybus et al. [ ] estimated the r values of hcv subtypes ( a, b, and ) by inferring the epidemic growth rate from viral genealogy. taking integration a step further, the coalescent process has been described for compartmental epidemiological models such as the susceptible-infected-recovered (sir) model, thereby enabling epidemiological parameters to be inferred from the genealogy [ ] . to infer demographic history from both pathogen genomes and epidemiological data, rasmussen et al. [ ] developed a markovian framework in which the population size at each time step was estimated by taking into account both the surveillance data and the genealogy. the epidemic history reconstructed using both datasets was more accurate than when analyzing each type of data separately. in all the above methods, the genealogy of the sampled sequences was fixed. however, there might be great uncertainty regarding the order and the timing of coalescence, especially if the sequences are sampled within a short time period. while genealogical reconstruction using bayesian mcmc approaches allows phylogenetic uncertainty to be incorporated into estimates of population size [ , ] , an integrative model is lacking in which uncertainties arising from both genetic and epidemiological data are incorporated during demographic reconstruction. models of pathogen evolution and mechanistic models of disease spread have increased in complexity. there is also greater computational power to test these models with data. however, these sophisticated models have mostly been applied to infectious diseases for which abundant data are available. for example, new methods are most often tested on the hiv- pandemic [ , , , ] , for which data have been extensively collected from various settings and sources since the virus was first characterized three decades ago. it is worthwhile to evaluate how genomic methods have been applied to other diseases that have emerged more recently. in this section, we will present three case studies of recently emerged infectious diseases to illustrate the power and shortcomings of genomic methods discussed in this review. since emerging in guinea in march , ebola virus (ebov) has spread to other countries in western africa, resulting in the largest outbreak of ebola since it was first identified in . the first viral genomes were made available just a month after alarm was raised about a new ebola outbreak in guinea [ ] , with further sequences collected in sierra leone [ ] . by aligning all the genomes, a number of polymorphic sites were identified, including eight in highly conserved regions of the genome. further association studies are needed to clarify the role of these genetic variants in determining disease outcome. using the sampling dates of the sequences and a molecular clock model, phylogenetic analysis of ebov sequences revealed a start date of february in guinea, spreading to sierra leone by april [ ] . uncovering the relationship between the ebov lineage and previous ebov outbreaks has proved trickier than understanding the disease dynamics during the outbreak. initial phylogenetic analysis suggested that lineages causing the present outbreak did not cluster with ebov strains that caused earlier outbreaks in central africa [ ] . however, dudas and rambaut [ ] noted that the divergence of guinea sequences from those of previous outbreaks was because they were sequenced most recently and had accumulated the highest number of substitutions. assuming that the ebov genome followed a molecular clock model, the authors re-rooted the tree to a lineage that caused an outbreak in [ ] . instead of silently circulating in west africa, the ebov lineage causing the current outbreak likely descended from a lineage that previously caused outbreaks in the democratic republic of congo. these studies highlight two issues. first, correct rooting of a phylogeny is important for accurate inference of past epidemic history. correct rooting can be achieved by using an out-group, but one was not available in the case of this ebov strain. this leads onto the second issue. without sequences from animal hosts, the mechanism by which ebov was sustained between outbreaks remains unknown. middle east respiratory syndrome coronavirus (mers-cov) first appeared in saudi arabia in , and has since been reported in several neighboring countries in the arabian peninsula and on other continents [ ] . despite the dearth of sequence data, coalescent-based analysis of genomic sequences produced estimates of the tmrca (march ; % confidence interval (ci): november to june ), r ( . ; % ci: . , . ), and doubling time ( days; % ci: , days) [ ] . without further sequencing of the animal reservoirs, the authors could not infer whether these estimates applied to the animal reservoir or the human epidemic, because the methods are agnostic as to where transmission and evolution occur. the credible intervals around the estimates were unsurprisingly large given the small sample size. unlike the ebov outbreak, which is sustained by human-to-human transmission [ ] , there appears to have been multiple introductions of mers-cov into the human population. identification of the animal reservoir is therefore crucial for establishing risk factors of infection and planning appropriate interventions to control the disease. since bats are reservoirs for other coronaviruses, their being a reservoir host is possible. a nucleotide-long region of the rna-dependent rna polymerase gene was found to be % identical between a viral sample from a patient in saudi arabia and from a bat nearby, though the region is known to be highly conserved [ ] . however, antibodies against human mers-cov have been detected in dromedary camels [ ] , the camel mers-cov genome is similar to human mers-cov [ ] , and there are reports of close contact between patients and camels [ ] . phylogenetic analysis of coronavirus sequences from bats, dromedaries and humans indicate a bat origin, with dromedary camel as an intermediate host [ ] . it is possible that there are other animal reservoirs not yet sampled, which highlights the need to carry out extensive animal surveillance to characterize the emergence of an infection in humans. unraveling the complex evolutionary history of pandemic h n influenza with sequences collected over three decades from humans, pigs and birds, the origin of the pandemic h n influenza a strain (pdmh n or 'swine flu') was elucidated soon after emergence. within two months of the first reported case of swine flu in humans, genomic analysis of the novel influenza strain had been carried out. a phylogeny was constructed for each of the eight genomic segments with sequences from humans, swine and birds. comparison of these eight phylogenies revealed a complex history of reassortment with a mixture of gene segments from all three groups. the start of the pandemic was estimated to be the end of or early , and the dates of the reassortment events leading to pdmh n were also obtained [ ] . without good surveillance of influenza in the animal reservoir, the origin of the novel strain would have been difficult to uncover. by analyzing hemagglutinin sequences collected over a one-month period, the start date of the epidemic was estimated to be in late january [ ] . repeating the phylogenetic and molecular clock analyses with a further sequences shifted the estimated start date two weeks earlier. fitting an exponential growth model to the sequence data, r was estimated to be . , slightly lower than inferred from epidemiological data but with overlapping confidence intervals. to determine at which point during the pandemic coalescent analysis would have provided accurate and precise estimates of evolutionary rate, r and tmrca, real-time estimates of these parameters were obtained for genomic sequences collected in north america [ ] . accurate estimates could have been obtained as early as may, when viral genomes had been sequenced. more precise estimates could have been obtained by the end of june, when had been sequenced. however, inclusion of more sequences of longer length only slightly improved the accuracy of initial estimates [ ] . most statistical models in population genetics have focused on the application of such methods to viruses, although this bias is perhaps unsurprising given the large proportion of eids caused by viruses [ ] . whole-genome sequencing of bacterial isolates is becoming more widespread, and can help to uncover genetic determinants of clinical severity, elucidate pathogen-host interactions, and quantify evolutionary rates at within-and between-host levels [ ] . epidemiological investigations using bacterial genomes have also been possible. even though bacteria acquire point mutations at a lower rate per base than viruses, longer bacterial genomes have provided sufficient genetic resolution for phylogenetic analysis. for example, whole-genome sequencing has been used to refine the tuberculosis transmission network built using contact information [ ] , and to investigate an outbreak of methicillin-resistant staphylococcus aureus in a hospital and surrounding community in near real-time [ ] . the need for longer sequences when conducting epidemiological studies of bacterial infections adds to the per-sample cost of sequencing, and more computational resources are required for coalescent-based inference of pathogen history. however, this latter limitation may be overcome by only analyzing polymorphic sites if samples are similar. demographic reconstruction of emerging bacterial pathogens using coalescent-based approaches has been limited compared to work on viral pathogens. in one such study, the temporal changes in genetic diversity of streptococcus pneumoniae in iceland were estimated based on the coalescent model [ ] . this study was limited to a single multidrug-resistant lineage in a single location, with data collected over decades. over longer evolutionary time-scales, the accumulation of diversity through recombination can obscure phylogenetic relationships. more complex evolutionary models would be required to taken into account these genomic changes, increasing the uncertainty surrounding demographic estimates from genomic data. in addition to performing analyses with longer sequences, there is also a need to develop methods that exploit as many sequences in the sample as possible. for population studies, available sequences are often subsampled to remove individuals from the same household or in the same close contact network to have a representative sample of the population. furthermore, sequences from the same individuals are often discarded, though these may be informative for within-host evolution. although some effort has been made to link within-host to between-host evolution [ , ] , the effect of within-host evolution on population genetic inference is still not well studied. combining analyses across different scales could improve the accuracy of epidemiological predictions and provide better mechanistic explanations of observed trends. genomic studies have contributed to better understanding of eids and their spatiotemporal spread. sophisticated statistical methods have been developed to uncover the epidemiological features of infectious diseases based on the genealogy of their sequences. there is also growing when the distribution cannot be computed analytically [ ] . obtaining estimates of r and t g is not always sufficient to predict epidemic trajectory if there is significant heterogeneity between individuals. the offspring distribution with mean r and variance σ describes the probability distribution of the number of secondary infections caused by each infected individual. in compartmental models, the offspring distribution is not explicitly specified but follows from the specification of the model -in the case of the sir model it follows a geometric distribution. for certain diseases, the offspring distribution is more dispersed than captured by the geometric distribution [ ] . in other words, most individuals cause no further infections whereas a few individuals are super-spreaders who cause the majority of infections. accurate estimate of σ is important for predicting epidemic outcome and assessing control measures. effort to integrate genomic analysis with analysis of epidemiological data. in recent cases of eids, genomic data have helped to classify and characterize the pathogen, uncover the population history of the disease, and produce estimates of epidemiological parameters. just as compartmental models can be fitted to surveillance data to infer the epidemiological dynamics of an infectious disease (box ), the coalescent framework allows inference of population history from pathogen sequences. the coalescent model describes the statistical properties of the genealogy underlying a small sample of individuals from a large population. in the simplest case, the forward-time dynamics of the population is assumed to follow the wright-fisher model, in which the haploid population has discrete, non-overlapping generations, undergoes neutral evolution, and remains the same size [ , ] . extensions to the coalescent have assumed more complex population dynamics described by deterministic population equations [ ] , compartmental disease models [ ] , or non-parametric approaches [ , , , ] . within this framework, going backwards in time, individuals in the current generation are randomly assigned to parents in the previous generation. if two individuals have the same parent, then a coalescent event has occurred. eventually, all lineages in the sample coalesce to a single individual known as the most recent common ancestor of the sample. the rate of coalescence is inversely related to population size. if the population follows the wright-fisher model, evolutionary changes are selectively neutral, so the shape of the genealogy reflects only demographic changes. global trends in emerging infectious diseases agricultural intensification, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis geographic expansion of dengue: the impact of international travel spread of a novel influenza a (h n ) virus via global airline transportation hantavirus epidemic in europe emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally 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epidemiological models phylodynamics of infectious disease epidemics simple epidemiological dynamics explain phylogenetic clustering of hiv from patients with recent infection rates of coalescence for common epidemiological models at equilibrium uncovering epidemiological dynamics in heterogeneous host populations using phylogenetic methods dating phylogenies with sequentially sampled tips birth-death skyline plot reveals temporal changes of epidemic spread in hiv and hepatitis c virus (hcv) hatzakis a: integrating phylodynamics and epidemiology to estimate transmission diversity in viral epidemics superspreading and the effect of individual variation on disease emergence how generation intervals shape the relationship between growth rates and reproductive numbers a note on generation times in epidemic models generation interval contraction and epidemic data analysis lemey p: hiv epidemiology. the early spread and epidemic ignition of hiv- in human populations variable recombination 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center for disease control and prevention: middle east respiratory virus (mers) middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat who rapid pandemic assessment collaboration: pandemic potential of a strain of influenza a (h n ): early findings real-time characterization of the molecular epidemiology of an influenza pandemic insights from genomics into bacterial pathogen populations whole-genome sequencing for analysis of an outbreak of methicillin-resistant staphylococcus aureus: a descriptive study the genealogical population dynamics of hiv- in a large transmission chain: bridging within and among host evolutionary rates teller e: equation of state calculations by fast computing machines the genetical theory of natural selection evolution in mendelian populations sampling theory for neutral alleles in a varying environment an integrated framework for the inference of viral population history from reconstructed genealogies smooth skyride through a rough skyline: bayesian coalescent-based inference of population dynamics genomic analysis of emerging pathogens: methods, application and future trends we would like to thank nick croucher for discussions on bacterial genomics. ll is funded by a medical research council doctoral training partnership studentship. the authors declare that they have no competing interests. key: cord- - e ijp authors: zhang, ke; hou, yuansi; li, gang title: threat of infectious disease during an outbreak: influence on tourists' emotional responses to disadvantaged price inequality() date: - - journal: ann tour res doi: . /j.annals. . sha: doc_id: cord_uid: e ijp the ongoing covid- pandemic has negatively influenced the global tourism industry. despite the documented negative impacts of diseases on tourism demand and people's perceived health risk, researchers have seldom examined the psychological responses of tourists travelling during an infectious disease outbreak. we therefore conducted three studies to examine this key aspect, and our findings indicate that tourists have a strong negative emotional reaction towards disadvantaged tourism-related prices in response to a high (vs low) infectious disease threat. furthermore, risk aversion acts as an underlying mechanism driving this effect: tourists are more risk aversive under the threat of an infectious disease, which consequently magnifies their negative emotional reaction. at last, theoretical and practical implications of these findings for tourism are discussed. the outbreak of infectious diseases imposes a major effect on tourism demand and the economies of destinations (page, song, & wu, ) . infectious diseases documented in the tourism literature include avian flu, swine flu (h n ), severe acute respiratory syndrome (sars), lyme disease, and foot-and-mouth disease (donohoe, pennington-gray, & omodior, ; lee & chen, ; lee, song, bendle, kim, & han, ; miller & ritchie, ) . a new and ongoing infectious disease outbreak, covid- , was declared a pandemic by the world health organization on march , (who, . as a result, many countries and regions have introduced travel restrictions, which are exerting a severe negative economic influence on the tourism sector worldwide (yang, zang, & chen, ) . based on the estimations of the united nations world tourism organization (unwto) in april , global tourism could experience a - % decline in global international tourist arrivals this year, leading to a us$ - billion decrease in tourist spending. this underscores the fact that tourism is unfortunately one of the industry sectors hardest hit by the covid- pandemic (unwto, ) . the negative effects on the tourism sector and its destinations are due not only to travel restrictions but also to potential tourists' reduced willingness to travel during epidemics or pandemics. understandably, the danger of catching an infectious disease has become a major health concern, which influences tourists' willingness to travel and ultimately decreases their intention to visit places that may be hazardous to their health (jonas, mansfeld, paz, & potasman, ; page, ). thus, tourists tend to avoid visiting destinations that may be regarded as dangerous and risky (carter, ) . for example, wen, huimin, and kavanaugh ( ) prior research has tested how disease threats can influence macro-level variables in the tourism industry. for example, page et al. ( ) showed that the swine flu pandemic significantly decreased inbound tourism demand in the uk. blake, sinclair, and sugiyarto ( ) revealed that the foot-and-mouth disease outbreak significantly reduced tourism expenditure in the uk, which greatly affected its gdp. yang et al. ( ) recently provided the first empirical evidence of covid- 's impact on the tourism economy and provided some policy implications for welfare. another growing stream of research is the examination of tourist perceptions of health risks, which are a major determinant of tourists' intentions to visit a destination (chien et al., ) . rittichainuwat and chakraborty ( ) demonstrated the negative impact of infectious diseases (i.e., sars and bird flu) on thailand's destination image and tourists' travel intentions, which was driven by the health risk concerns of potential tourists. some destinations that are greatly affected by infectious diseases (e.g., africa affected by hiv) become perceived as high-risk destinations by tourists (carter, ) , who then show protective behaviour prior to departure (jonas et al., ) . page ( ) divided travel behaviour associated with health and disease into three phases: pre-travel, travel and post-travel. most investigations into the impact of disease on tourist perspectives at the individual level have focused on the pre-travel phase. however, it is important to determine how the threat of disease impacts tourists during the travel phase, as tourists are exposed to unfamiliar environments while travelling, which may involve health challenges, thereby increasing their susceptibility and vulnerability to disease compared to when they remain at home (chien et al., ) . the sudden outbreak and rapid spread of infectious diseases (e.g., covid- ) further contributes to the psychological challenges to under-prepared tourists in disease-affected areas and even in neighbouring unaffected areas. psychology researchers have begun to investigate the influence of disease on individuals' feelings and behaviours. it has been shown that a disease threat can lead people to dehumanise out-group members (navarrete & fessler, ) , conform to majority views (wu & chang, ) , become less extroverted and make avoidant movements (mortensen, becker, ackerman, neuberg, & kenrick, ) . however, other consequences of a disease threat in the tourism context require exploration. in the current research, we extend the literature by focusing on how disease impacts tourists' price perceptions, specifically their emotional reactions to disadvantaged price inequality. price inequality is defined as a situation in which consumers find they have paid more or less than comparable others for specific products or services (gelbrich, ; xia et al., ) . price inequality may occur in many consumption scenarios. for example, as some firms adopt discriminative or dynamic pricing strategies as a revenue management practice, customers may be charged different prices for the same product at various occasions (kopalle et al., ) . also, competing firms rely on price promotions to attract consumers, which enables consumers to pay different prices at different stores (bolton, warlop, & alba, ) . there are two reasons for the focus of current research on tourists' emotional responses to disadvantaged price inequality (i.e., consumers paying more than a reference group pays). first, literature findings suggest that disadvantaged price inequality, relative to advantageous price inequality, can do greater harm to firms (jin, he, & zhang, ; xia et al., ) . therefore, investigating this type of price inequality has direct managerial implications for service providers in the travel destinations. second, it has been posited in prior work that the emotional responses of customers are direct and key psychological consequences when they face disadvantaged price inequality (finkel, ; jin et al., ; xia et al., ) . specifically, researchers have recently revealed that individuals' reactions to disadvantaged inequality are primarily based on an emotional process, whereas their reactions towards advantaged inequality involve more complex cognitive functions (gao et al., ) . in addition, tourists' emotional reactions drive many downstream consequences such as their perceptions of the destination, overall enjoyment, and whether they recommend the destination to others (prayag, hosany, muskat, & del chiappa, ) . combining all these points, it is theoretically and practically meaningful to directly examine tourists' emotional responses to disadvantaged price inequality. it has been found that a series of antecedents may influence consumer responses to price inequality, such as the characteristics of transactions (xia et al., ) , customer attribution of inequality (gelbrich, ) , the social status of customers (jin et al., ) , and so forth. however, scarce literature has paid attention to the impact of the ambient environment on customer responses to price inequality. marketing researchers have investigated the environmental influence (e.g., type of store) on consumer responses to price information (büyükkurt, ; dodds, monroe, & grewal, ) , while few studies have been performed to examine the role of environmental factors associated with public health. in this study we fill this research gap by examining how the threat of an infectious disease can shape tourist reactions to disadvantaged price inequality. the mediating role of risk aversion neuberg et al. ( ) documented that humans have threat-management systems to avoid risks when facing disease threats, which we refer to as risk aversion. prior studies showed that the risk aversion induced by health threats can manifest in humans' feelings and emotions (e.g., anxiety, fear, and disgust), which enable people to detect and avoid potential health threats. for example, individuals tend to feel disgusted by contaminated food, which acts as a risk-avoidance response (rozin, millman, & nemeroff, ) . also, the risk-aversion tendency can lead individuals to be aversive to others with cues of disease threats, including physical disability and obesity (crandall & moriarty, ; park, schaller, & crandall, ) . more importantly, these emotional responses associated with risk aversion are found to be able to spill over to influence people's mental states and behaviours in non-health domains (schaller & park, ) . for example, schaller and park ( ) suggested that a disease threat may induce aversion towards social interaction with others, because more frequent interaction signals a higher risk of being infected. in addition, researchers have posited that a disease threat can induce more aversive attitudes towards foreign outgroup members because of the inference that they are associated with a higher infection risk (faulkner, schaller, park, & duncan, ) . disease threats can even facilitate individuals' endorsement of conservative political attitudes, which are linked with riskaversion and uncertainty avoidance (helzer & pizarro, ; jost, glaser, kruglanski, & sulloway, ; murray & schaller, ) . all of these findings resonate with the proposition of lopes ( ) that people facing a threat to their own security generally become more risk averse. it has been demonstrated that individuals with a higher risk-aversion tendency are more sensitive to possible threats and losses, and thus more motivated to minimise the possibility of these occurring (consiglio & van osselaer, ; molden, lee, & higgins, ; neuberg et al., ) . specifically, risk-averse (vs risk-seeking) individuals have been shown to care more about whether they fall below a certain reference point (higgins, ; scholer, zou, fujita, stroessner, & higgins, ) . in the context of disadvantaged price inequality, tourists engage in upward comparisons with peer tourists and consider the prices paid by peers as a reference point (jin et al., ; lee & fay, ) . therefore, disadvantaged price inequality can be perceived as a type of financial loss for tourists who have paid a higher price (oh, ; rondan-cataluña & martin-ruiz, ) . as tourists with higher risk aversion are more sensitive to losses, we propose that these tourists would show a stronger negative emotional response when facing such price inequality. in sum, previous findings suggest possible links between disease threat, risk aversion, and negative emotional reactions to disadvantaged price inequality. in summary, we present the following two hypotheses. hypothesis . a high (vs low) disease threat strengthens tourists' negative emotional reactions to disadvantaged price inequality. hypothesis . the effect proposed is driven by tourists' heightened risk aversion. three studies were conducted to investigate whether the threat of an infectious disease magnified the negative emotional reaction to a disadvantaged price inequality. similar to previous literature on disease threat and human behaviour (murray & schaller, ; wu & chang, ) , we combine survey (study ) and experimentation (studies a and b) methods. all of the studies were completed during february on amazon mechanical turk using american samples, before the who declared the covid- outbreak to be a pandemic. during this period, there were only confirmed covid- cases in the united states and no related deaths had been reported. therefore, these studies did not suffer from a possible confounding effect due to the actual pandemic (i.e., all participants might perceive a high disease threat, which would possibly lead to ceiling effect). in study , we found that tourists' perceived threat of infection were positively associated with their negative emotional reaction to a disadvantaged price inequality. in study a, we manipulated the disease threat, and conducted a content analysis that revealed that a higher threat increased tourist risk aversion. in study b, we manipulated the disease threat and showed that a higher disease threat led tourists to be more risk averse, which in turn magnified their negative emotional reaction to a disadvantaged price inequality. the participant profiles for three studies are presented in table . study served as an initial test of the relationship between perceived disease threat and negative emotional reaction to disadvantaged price inequality. specifically, we proposed that the higher the disease threat perceived by tourists, the more negative would be their emotional reaction when they faced a disadvantaged price inequality. we recruited u.s. participants during the second week of february , on amazon mechanical turk (mturk). mturk is an online crowdsourcing platform with participants from various backgrounds, which has been commonly used in social science research (hwang & mattila, ; paolacci & chandler, ; zhang, hou, li, & huang, ) . we used a correlational study design and excluded seven people due to their extreme response timing in completing the survey. one hundred and forty-six participants ( . % female, m age = . years, sd = . ) remained. the sample size was regarded as appropriate, as a power analysis using g*power software (faul, erdfelder, buchner, & lang, ; kim, zhang, & park, ; lu, lee, gino, & galinsky, ) showed that approximately participants would provide % power to detect a medium-sized effect in a correlation analysis (i.e., . ) with a false positive rate of %. all of the participants completed two surveys. the first was the perceived vulnerability to disease (pvd) scale (duncan, schaller, & park, ) , which consists of items to assess individual differences in perceived threat from an infectious disease (wu & chang, ) . the scale consisted of two subscales, a -item perceived infectability subscale to assess participants' perceived susceptibility to infectious diseases (e.g., 'i am more likely than the people around me to catch an infectious disease.'), and an -item germ-aversion subscale measuring participants' discomfort when faced with the threat of pathogen transmission (e.g., 'it really bothers me when people sneeze without covering their mouths.'). participants responded to all items on a -point likert scale ( = strongly disagree, = strongly agree). in the second task, participants indicated their opinions regarding a hypothetical travel scenario. thus, participants imagined that they were on a flight to another city, and that they found by talking with a nearby passenger that the passenger had paid us$ for their flight ticket, while the participants themselves had paid us$ for his/her ticket almost on the same day. the participants' negative emotional reaction to this disadvantaged price inequality was measured on a -item scale (i.e., how sad/ angry would you feel in this situation, = not sad/angry at all, = very sad/angry; adapted from jin et al., ) . finally, the participants provided their demographic information, such as their sex ( = male, = female), age, and a -point scale measuring annual household income ( = less than $ , , = $ , or more). we averaged the scale items measuring negative emotional reactions (r = . ) as the dependent variable. as previous researchers using the pvd scale have either treated all items as a single scale or examined the two subscales independently (duncan et al., ; faulkner et al., ; wu & chang, ) , we performed both of these analyses. first, we examined the association between the total pvd score and tourists' negative emotional reactions. we averaged the items in the pvd scale (α = . ) as the independent variable and regressed the negative emotional reaction on the pvd score. the results revealed that there was a positive association between pvd score and negative emotional reaction: β = . , t[ ] = . , p < . . second, we separately averaged the items in the perceived infectability subscale (α = . ) and in the germ-aversion subscale (α = . ). the results of these two regression analyses showed that tourists' negative emotional reactions were positively associated with both perceived infectability (β = . , t[ ] = . , p < . ) and germ aversion (β = . , t[ ] = . , p < . ). all of these effects (i.e., the effects of scores in the pvd scale, the perceived infectability subscale and the germ aversion subscale on emotional reactions) remained robust (all ps < . ) after including sex, age and income as covariates. therefore, the results supported our hypothesis. the findings from study provided preliminary support of the association between a disease threat and tourists' emotional reactions to disadvantaged price inequality. in the next studies, we manipulated disease threats so as to examine their causal impacts on tourists' emotional reactions, and then shed light on the underlying mechanism. study a had two objectives. first, it served as a pilot test to check the effectiveness of our manipulation of the disease threat. second, it was used to investigate the effect of a disease threat on tourists' risk aversion, which may act as the potential mechanism underlying tourists' emotional reaction towards disadvantaged price unfairness. we predicted that a high disease threat would lead tourists to be more risk averse. we recruited u.s. participants on mturk during the third week of february . we used a one-factor (disease threat: high vs. low) between-subjects design. seven people were excluded due to their extreme response timing in completing the survey and a total of participants ( . % female, m age = . years, sd = . ) remained. this sample size was regarded as appropriate because a power analysis using g*power software (faul et al., ; kim et al., ; lu et al., ) showed that approximately participants in each condition would provide % power to detect a medium-sized effect (i.e., . ) at a false positive rate of %. participants were randomly assigned to a high-threat or low-threat condition. they first engaged in a mental visualisation task and imagined themselves were in a one-week trip to a new city. they then read a piece of local news describing a fictitious influenzalike infectious disease, through which we manipulated disease threat. the manipulation was based on previous literature (agrawal & wan, ; menon, block, & ramanathan, ) . specifically, the content of this piece of news varied depending on conditions. in the high-threat (vs low-threat) condition, participants learned that the new influenza-like disease, x- , occurred in the city they were visiting (vs. some cities km away), that its transmission rate was . (vs. . ), similar to (vs. much lower than) the normal influenza, and that it was often contracted through droplets when infected people coughed, sneezed or talked (vs. through very close, prolonged personal contact with infected people), making it easy (vs. difficult) to contract. to ensure that the participants fully understood the news, they had to provide correct answers to three multiple choice questions before they could proceed to the next page. participants first responded to the manipulation check question (i.e., 'how likely will you catch x- when travelling in this city?' = not at all likely, = very likely). a higher score indicated a higher disease threat (menon et al., ) . next, they were asked to write down their feelings when travelling in this travel destination after reading the news, through which we coded the risk aversion level of each participant. finally, the participants provided their demographic information, such as their sex ( = male, = female), age, and a -point scale measuring annual household income ( = less than $ , , = $ , or more). the perceived disease threat was higher in the high-threat condition (m = . , sd = . ) than in the low-threat condition (m = . , sd = . ): t[ ] = . , p < . , d = . . thus, the manipulation of the disease threat was successful. following the content analysis method suggested by lu et al. ( ) , we invited two coders who were blind to the study hypotheses and manipulations to read each participant's writing, and then responded to a -point scale (i.e., "risk aversion means individuals are inclined to protect themselves against the risks around. please read the writing of this respondent, and then rate the level of risk aversion expressed in this respondent's writing". = very low, = very high, icc( ) = . ). we then averaged the two ratings as the risk aversion score. an independent t-test showed that risk aversion was significantly higher in the high-threat condition (m = . , sd = . ) than in the low-threat condition (m = . , sd = . ): t( ) = . , p < . , d = . , supporting our hypotheses. the regression analyses including sex, age, and income as covariates showed that the effect of disease threats ( = high, = low) on risk aversion was robust, β = . , t[ ] = . , p < . . the results of study a confirmed that the manipulation of the disease threat was effective. more importantly, the content analysis showed that higher disease threat led tourists to become more risk averse. next, in study b, we investigated the causal effect of the disease threat on the participants' emotional reactions to disadvantaged price inequality while directly measuring risk aversion as the underlying mechanism. k. zhang, et al. annals of tourism research ( ) study b had two objectives: first, we replicated the proposed effect of the disease threat on tourists' emotional reactions to a disadvantaged price inequality by manipulating rather than measuring the disease threat; second, we directly examined tourist risk aversion as the mechanism underlying the proposed effect. specifically, we predicted a mediation model in which a higher disease threat leads participants to be more risk averse at the travel destination, which in turn magnifies their negative emotional reaction to a disadvantaged price inequality (i.e., disease threat → risk aversion → negative emotional reaction). the study employed a one-factor (disease threat: high vs low) between-subjects design. we recruited u.s. participants on mturk during the fourth week of february . we excluded people due to their extreme response timing in completing the study. a total of participants ( . % female, m age = . years, sd = . ) remained. participants were first engaged in the same mental visualisation task (i.e., imagining they were in a one-week trip to a new city, and reading a piece of local news) as in study a, in which we manipulated the disease threat. next, participants were presented with a scenario regarding their travel experience in the city. specifically, they imagined that they were taking a -day package tour at the destination, during which a conversation with another tourist on the tour bus alerted them to the fact that they had paid a higher price (i.e., $ ) than the tourist paid (i.e., $ ) for the tour package. next, all of the participants responded to the same items as in study to indicate their negative emotional responses (i.e., how sad/angry would you feel in this situation? = not sad/angry at all, = very sad/angry). participants then completed a scale measuring their risk aversion after reading the news about x- . the scale measuring riskaversion was adapted from the literature (consiglio & van osselaer, ; sharma, ) to suit our research context. specifically, the scale has three items (i.e., 'at that moment, i was inclined to be cautious of the risks around me', 'at that moment, i thought a lot about how to stay safe from the risks around me', and 'at that moment, i was motivated to protect myself against the risks of being exposed to the disease'; = strongly disagree, = strongly agree). at last, the participants provided their demographic information, such as their sex ( = male, = female), age, and a -point scale measuring annual household income ( = less than $ , , = $ , or more). the perceived disease threat was higher in the high-threat condition (m = . , sd = . ) than in the low-threat condition (m = . , sd = . ): t[ ] = . , p < . , d = . , confirming that our manipulation of disease threat was effective. we averaged the two items measuring participants' negative emotional reaction (r = . ). an independent t-test revealed that the negative emotional reaction to the disadvantaged price inequality was higher in the high-threat condition (m = . , sd = . ) than in the low-threat condition (m = . , sd = . ): t[ ] = . , p = . , d = . . the regression analyses including sex, age, and income as covariates showed that the effect of disease threats ( = high, = low) on negative emotional reaction was robust, β = . , t[ ] = . , p = . . therefore, these results support our hypothesis. to test the underlying mechanism, we examined the mediating role of risk aversion. first, we coded the disease threat condition ( = high, = low) and averaged the items of risk aversion (α = . ). then, we examined the mediation model through both path analysis and a bootstrap analysis (kim, chen, & zhang, ; preacher & hayes, ) . the path analysis results revealed that the disease threat was positively associated with both the negative emotional reaction (β = . , t[ ] = . , p = . ) and the risk aversion (β = . , t[ ] = . , p < . ). after including disease threat and risk aversion as predictors and negative emotional reaction as the outcome, the effect of risk aversion was significant (β = . , t[ ] = . , p = . ), while the effect of disease threat became non-significant (β = . , t[ ] = . , p > . ; see fig. ). this showed that risk aversion mediated the effect of the disease threat on the tourists' negative emotional reactions to the disadvantaged price inequality. in addition, the results of a bootstrap analysis with bootstrapping samples (process macro model ; preacher & hayes, ) further confirmed that the influence of the disease threat on the negative emotional reaction to the disadvantaged price inequality was significantly mediated by risk aversion (indirect effect = . , se = . , % ci [ . , . ], excluding zero). the mediation model was robust after incorporating sex, age, and income as covariates (indirect effect = . , se = . , % ci [ . , . ], excluding zero). these findings thus supported our initial hypothesis. the results of study b supported both hypotheses and . first, study b replicated the finding of study by showing that a k. zhang, et al. annals of tourism research ( ) higher disease threat led to a stronger negative emotional reaction to a disadvantaged price inequality. more importantly, study b offered evidence of the mediating role of risk aversion. specifically, a high disease threat made the tourists more risk averse. in this case, they tended to show stronger negative emotions when facing a disadvantaged price inequality. in other words, the tourists were more sensitive to inequality when facing a higher disease threat. these findings discovered the underlying mechanism of the proposed effect and also have important practical implications for tourism management under an epidemic or pandemic threat. it is particularly important that tourism-service providers have knowledge about tourists' psychological needs and experiences, as this enables providers to select effective business strategies to ensure that their services meet customer expectations and offer satisfactory experiences. this becomes particularly crucial in crisis situations such as the threat of infectious diseases, as tourists' emotional responses to service encounters are likely to be magnified by this threat. given the increasingly frequent outbreaks of infectious diseases such as covid- , and their dramatic impact on the tourism industry, research in this area is urgently needed. we responded to this need by conducting the present study, which makes theoretical, methodological, and practical contributions. to the best of our knowledge, this research represents the first attempt in the tourism literature to investigate the impact of infectious diseases on tourists' psychological states. the findings of this study provide initial evidence for the significant influence of infectious diseases on tourists' emotional responses to price information. our carefully designed studies revealed that the perceived threat of an infectious disease tended to magnify tourists' negative emotional reactions to disadvantaged price inequality. notably, the impact of diseases on consumer psychology is an emerging field of research. thus, by focusing our study on a new consequence of this impact, from a tourism perspective, we have made a novel addition to this emerging line of research. our determination of the mechanism of the above impact represents another useful theoretical contribution. that is, we have found that risk aversion is the underlying process by which infectious diseases can lead to more negative emotional responses to disadvantaged price inequality. this means that when faced with an infectious disease threat, tourists are more likely to demonstrate risk aversion. in this context, tourists perceive disadvantaged price inequality as financial loss, and more risk-averse tourists tend to develop more negative emotional reactions. our findings therefore shed light on the mediating role of risk aversion in the effect of the threat of infectious diseases on negative emotional responses to disadvantaged price inequality. from a methodological perspective, we used a two-stage, between-subjects experimental design, and conducted both content analysis and statistical hypothesis testing to ensure the robustness of the findings. to the best of our knowledge, our research described herein was among the first wave of researchers applying an experimental design to analyse the effect of infectious diseases on tourism. the findings offer useful practical implications. because this study is set in the general context of infectious diseases, its implications are not restricted to the present covid- outbreak, but are general to a broader context. when there is a destinationspecific or global outbreak of an infectious disease, such as covid- , there is threat to tourists and the tourism industry, the magnitude of which depends on the scale and severity of the epidemic. if tourism businesses continue to operate during such an outbreak, including before and after travel restrictions and destination lockdown, tourists will encounter various tourism services during their trip, and may potentially experience disadvantaged price inequality and perceived price unfairness, as described in the experiments of this study. from the service provider's perspective, there can be multiple reasons for price inequality, including the service provider's own revenue management practices (e.g., price increases due to cost variations, discounts due to competition pressure and declining demand, or price discrimination in different market segments), as well as multiple channels of distribution that allow travel intermediaries to exercise their own pricing strategies. pricing strategies are commonly exercised in a competitive market, especially when there is over-supply against insufficient or reduced demand, such as during a pandemic. thus, the findings of this study have important implications for the pricing decisions of tourism businesses during an infectious disease outbreak. specifically, when a destination faces the threat of an infectious disease such as covid- , tourism businesses operating at the destination need to be cautious in offering discounts or promotions, implementing dynamic pricing or price discrimination strategies, and must carefully manage their multi-channel distribution strategies. tourism businesses must also consider the balance between short-term profitability and long-term business sustainability, and taking a cautious decision on increasing prices during an infectious disease outbreak, even though the operating cost may increase due to the infectious disease outbreak (mckinsey & company, ) . a lesson can be learned from the haidilao case, as introduced earlier in this article. price inequality may be inevitable in tourism markets during an outbreak of an infectious disease, as many tourism businesses need to react to the decline in demand or increased production costs. where a new pricing strategy has to be implemented, these tourism businesses must pay particular attention to communicating these price variations to customers clearly and effectively. full information on different pricing options should be made available to customers, such as making clear any restrictions that apply to lower-priced services (e.g., advance booking or inflexible cancellation), and all of the benefits that accompany higher-priced services. in sum, the key to avoiding negative emotional reactions from customers is to make them feel that the price inequality between services is acceptable. therefore, in addition to effective communication, tourism businesses need to understand what pricing strategies their customers perceive as acceptable and fair. where multi-channel distribution is used, the principal service provider needs to work closely with travel intermediaries to understand each other's latest pricing strategies and ensure that full information of their respective offers, such as restrictions of price discounts, is made available to potential customers. these aspects are relevant to the present covid- pandemic, because tourists are still likely to feel threatened by the pandemic during the recovery period after travel restrictions are lifted, and will continue to do until the pandemic has completely finished. therefore, tourism businesses need to bear in mind that tourists during this period may exhibit heightened emotional reactions to new revenue management practices and pricing strategies. in light of the propensity of risk aversion as the underlining mechanism of the effect revealed in this study, tourism businesses need to work closely with destination marketing organisations to manage tourist risk perceptions, such as releasing timely updates about heath and safely issues at the destination through multiple channels, including the businesses' own social media. in particular, in the recovery phase of covid- and similar pandemics, marketing should be focused on domestic markets and nearby regions where there is better knowledge of the destination and less-biased risk perception. the empirical findings of this study contribute to the initial wave of tourism research on the impact of disease threat. however, there are several limitations of this study which suggest avenues for further research. first, the current research provides empirical support of tourists' increased propensity for risk aversion and stronger negative emotional reactions to disadvantaged price inequality when under the threat of an infectious disease. subsequent research could investigate other psychological states and perceptions of tourists under the threat of infectious diseases, together with the mechanisms underlying these. for example, in response to infectious disease threat, might tourists prefer tourism products of national (vs. international) brand because of in-group favouritism. second, while our study focused on tourists' emotional reactions to a disadvantaged price inequality, scholars should also consider the effects of infectious diseases on other price perceptions in the tourism context, such as a price-quality inference, and further study the impact on tourists' behaviours. for instance, would infectious disease threat influence tourists' price-quality inference of tourism products and could such an effect further lead to refusal to repurchasing the tourism service? finally, while we used controlled experiments with high internal validity, future work could use other methods (e.g., big data or field studies) that are based on larger samples and could offer 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system (and why it matters) when risk seeking becomes a motivational necessity measuring personal cultural orientations: scale development and validation can you accept that haidilao's prices rise approximately % after reopening? online at april saw the sharpest increase in grocery store prices in nearly years the impacts of sars on the consumer behaviour of chinese domestic tourists the social impact of pathogen threat: how disease salience influences conformity the price is unfair! a conceptual framework of price fairness perceptions coronavirus pandemic and tourism: dynamic stochastic general equilibrium modeling of infectious disease outbreak tourists and air pollution: how and why air pollution magnifies tourists' suspicion of service providers his research interests include consumer psychology and behavioral economics senior lecturer in marketing at school of business and management professor of tourism economics at the university of surrey, uk. his research interests include quantitative methods in tourism demand analysis the authors acknowledge the research grants awarded by the national natural science foundation of china [grant number: , ]. key: cord- -berrojmq authors: burri, christian title: sleeping sickness at the crossroads date: - - journal: trop med infect dis doi: . /tropicalmed sha: doc_id: cord_uid: berrojmq human african trypanosomiasis (hat; sleeping sickness) is a disease with truly historic dimensions [...]. these activities marked the start of incredible efforts of the colonial powers to control the disease, but particularly also of the vertical approach towards disease control, with special programs run in parallel with the public health system-which is now one of the challenges in the elimination "end game" with very few patients remaining requiring an integrated public health approach. the detection of the causative agent, the mode of transmission and the first documented major epidemics coincided with the advent of modern pharmacology. it is, therefore, no surprise that there was an interest to find drugs against this disease. in , the colonial powers, in a joint conference, decided to give drug development a high priority and in course, several molecules were developed. the history of the oldest drug still in use against hat, suramin, is described in the context of the history in the article of madeja et al., not only is the drug still manufactured, but also the continued support of bayer since the year is a major contributing factor allowing us to write about the elimination of hat today. the activity of inorganic arsenic-based compounds had already been recognized in the mid- th century and the treatment of "nagana" (animal trypanosomiasis) was described by david livingstone in and david bruce in . this knowledge led to the development of the first organo-arsenic compound atoxyl ® in by paul ehrlich, who used trypanosomes as a model to screen molecules, but was mainly searching for drugs against syphilis. wolferstan thomas in liverpool subsequently showed that atoxyl ® was effective against t. b. gambiense. however, atoxyl ® , meaning 'non-toxic', caused severe adverse drug reactions particularly affecting the optic nerve; it was only active against early stage hat and was followed by tryparsamide in . tryparsamide was developed in the usa and was the first drug to be active against the late stage, although not very active and also prone to extensive resistance development. tryparsamide was also very toxic, with a dose dependent risk damaging the optic nerve. this is documented in a horrifying report from when a lieutenant of the french army in cameroon doubled the prescribed dose of tryparsamide to speed up the recovery of patients. two days later, all these patients were blind. this event prompted the swiss chemist and physician dr. friedheim to investigate alternative drugs. the introduction of a triazine ring lead to the development of melarsen (disodium p-melaminyl-phenyl arsonate) in , which proved to be a very efficient drug against t. b. gambiense, but was still very toxic. in , friedheim first described the trivalent arsenoxide form of melarsan, known as melarsenoxide. he reported its use in treatment of human sleeping sickness in . the main achievement was the reduction of the duration of therapy to six weeks, with two weeks of seven daily injections of . mg/kg each, spaced by an interval of one month. in a further step, capping the arsenic in melarsen oxide with british anti-lewisite, an antidote to the arsenical warfare agent lewisite, reduced the toxicity by a factor of the order of , but the trypanocidal activity only by a factor . . the new drug was called melarsoprol (mel b; arsobal ® ) [ ] . it remained the mainstay of second stage hat treatment until the early s despite the long treatment duration of days, the known related adverse drug reactions, particularly the encephalopathic syndrome that occurs in about % of the patients treated and leads to their death in about % of cases, and the potential for drug resistance. another line of research was the molecules with a diamidine structure discovered in the s. these molecules were detected by serendipity in the search for hypoglycaemic compounds, with the idea in mind that this effect might compromise the very prominent and particular glucose metabolism of trypanosomes. several compounds, however, proved to have a direct trypanocidal effect with the three compounds stilbamidine, pentamidine, and propamidine identified to have the highest activity [ ] . pentamidine does not penetrate the blood brain barrier so its use is limited to first stage hat; despite this drawback it is still is in use today, for treatment of children below six kilograms. the distinctly better safety profile of the drugs only active against first stage disease (pentamidine and suramin) versus melarsoprol was also the advent of the consistent performance of lumbar puncture to determine the disease stage and make a treatment decision. the need for a lumbar puncture was, for over years, a characteristic of hat treatment, a source of patient distress, stigma and technical limitation of treatment. some years after the discovery of pentamidine, the diamidines again became the focus of drug development, although not successful until today. the development of the organo-arsenicals, diamidines, and also drugs developed much later and still in use like eflornithine and nifurtimox, including their drawbacks related to the potential for drug resistance are described in detail in the contribution of de koning. the "scramble for africa" was an investment with very high political and economic stakes, and sleeping sickness was not just a disease; it had become the colonial disease. besides drug development, the responses of the anglophone and francophone colonial powers to trypanosomiasis differed significantly. francophone countries chose to concentrate directly on the medical problems presented by the disease in humans. this included the introduction of "mobile teams" actively searching and screening population for hat cases. this method of systematic case detection and treatment with the aim of elimination of the parasite reservoir was suggested by the french military surgeon eugène jamot and such activities started in in cameroon ("atoxylisation") [ ] . subsequently, the prevalence of hat declined from as high as % in to . - . % in , leading to an expansion of the methodology to other countries. after the second world war, atoxyl ® was replaced by pentamidine and a regular application of the drug every six months to the population at risk was introduced ("pentamidinisation"). in the s in the belgian congo alone, some two million people were subjected to this preventive mass drug administration [ ] . due to the partial presence of t. b. rhodesiense in their territories, the anglophone countries were confronted with the more widespread problem of disease in domestic livestock, which also presented a reservoir for human disease. their approach included vector control (traps, spraying), bush clearing, and game destruction [ , ] , and later the chemopreventive use of veterinary drugs (diminazene, isometamidium, and homidium). the control measures were overall very successful and progressively controlled the disease, reaching a very low, generalized transmission by the mid- s, with a minimum of cases declared in africa in [ ] . however, the measures taken were very costly, but above all very unpopular in the communities. this led to concealment where possible, and made the longterm goal of elimination by chemotherapy difficult, if not impossible [ ] . we could observe similar tendencies when conducting clinical trials in the s; potential patients were mostly hiding away or fleeing the villages at the beginning of mobile team campaigns for reasons of fear of stigmatization, lumbar puncture, pain, and the possibility of being treated with the dangerous drug melarsoprol. the in-depth understanding of the communities' beliefs, needs, and approaches is therefore key in a successful elimination attempt; insight on these topics are presented in the papers by and falisse et al., lee et al., and palmer et al. a factor mentioned by winslow in , which may still be under-researched today, is the relationship between the disease and poverty, particularly inadequate food supply, as the disease leads to unused land, which creates malnutrition [ ] . such a view would require an even more integrated approach towards disease control and elimination. when the colonial powers withdrew from africa between and , a new era began. the young nations created their own institutions with the goal of continuing research towards the elimination of hat (e.g., kenya trypanosomiasis research institute (ketri), nigerian institute for trypanosomiasis research (nitr), uganda trypanosomiasis research organisation (utro), and programme sur la recherche sur la trypanosomiase in côte d'ivoire (prct)). these institutions were reinforced since the s by internationally funded institutes dedicated wholly or partly to trypanosomiasis research (e.g., international laboratory for research on animal diseases (ilrad) in kenya, international centre of insect physiology and ecology (icipe) in kenya, international trypanotolerance centre (itc) in the gambia). however, in the course of monetary adjustments in the s, the decreasing funds available, and the emergence or increase of other health priorities, institutions devoted to a single disease were no longer sustainable and they were continuously integrated or transformed into multilateral institutions. overall, in the years after the independence process, the expenditures for hat were reduced, and awareness and surveillance of the disease decreased. the number of mobile teams was decreased, and it was attempted to transfer activities to the public health system-without having the respective tools, approaches, and knowledge [ ] . this, together with social instability, conflicts, and insecurity constraining disease control interventions led to a significant resurge of gambiense hat in the s and s [ ] , mainly affecting angola, congo, southern sudan, and the west nile district of uganda [ ] . at the end of the s, the situation was comparable to the one in the and ; the number of reported cases was almost , with , cases suspected [ ] . in , we published a special issue in the journal tropical medicine and international health [ ] , asking ourselves whether there were new approaches to roll back hat. not only was this the time of , hat cases suspected in countries of sub-saharan africa, it was also the time when the organo-arsenic drug melarsoprol was still the only treatment available for second stage hat. treatment with melarsoprol required around days of hospitalization with numerous and very painful injections, very severe adverse drug reactions like an encephalopathic syndrome were common and the mortality rate under treatment was as high as - %. in those days, an oncologist tried to comfort us, saying that a % treatment success rate for a disease with an inevitably fatal course was fantastic. we did not share this view, and rather expressed a dream: to make hat "an ordinary" disease, which follows the usual pattern "test, treat, track"-without the need of a lumbar puncture to make treatment decisions, without a high mortality rate under treatment, without the pain, without the stigma. elimination was a very far-fetched goal at this time, but there were some first positive signals that the dimensions of the problem and its impact on society and development were being recognized. the conclusions adopted by the international scientific committee of trypanosomiasis research and control (isctrc) in reflected a new awareness of the disease. the african union member states were urged to give highest priority ranking to african trypanosomiasis in their development programs, and it was recommended that urgent and particular attention should be given to surveillance and intervention in epidemic areas, to drug availability and resistance, and to the implementation of operational research to respond to the needs of control programs. at their meeting in lomé in july the oau heads of state and governments signed a declaration of intent to eradicate tsetse flies on the african continent-something that will likely not happen, but it was the turning point towards manifold activities which make us today work towards elimination of hat as a public health problem of and the interruption of transmission by . at the same time, médecins sans frontières' access to medicines campaign were able to make a compelling case that society needed to rethink drug discovery paradigms for neglected diseases. aventis (now sanofi) was persuaded to repurpose and develop the failed anti-cancer drug eflornithine for use against hat and to donate it at no cost to the who for distribution in africa. millions of dollars were also provided by aventis/sanofi to the who, who could now develop new screening and intervention programmes [ ] . bayer signed a similar contract with the who, a success story and joint effort which has been renewed by both companies until today, and which is one of the strong drivers in control and now elimination. in , the bill and melinda gates foundation selected hat to be one of the first diseases they targeted through the consortium of parasitic drug development (cpdd) and shortly thereafter, the drugs for neglected diseases initiative (dndi) was founded. the beginning of the century was truly an exciting time for neglected diseases and for hat in particular. the changes and significant impact on funding were later summarized in the landmark publication "the new landscape of neglected disease drug development" [ ] . the changed situation immediately led to the initiation of several large scale activities in drug development and the term "elimination" in the context of hat was mentioned for the first time by dr. jannin, leading the anti-hat efforts at the who in [ ] . drug development had been virtually dormant for about years. although the cultivation and test methods for drug screening for anti-trypanosomal drugs had been developed in the s and s [ ] , the money for pursing lead compounds in preclinical work, translational studies and large-scale trials was too scarce in these days. during the s, some initial limited drug activities were carried out on shoestring budgets: eflornithine, which had initially been developed in the s as a potential anti-cancer drug, was found to be active against second stage gambiense hat. this discovery in the s was a scientific breakthrough [ ] and eflornithine was shown to be much safer compared to melarsoprol. the drug received orphan drug status by the us food and drug administration in ; however, production was stopped after a few years and only resumed after significant public and political pressure. eflornithine, however, was only introduced for treatment in a limited number of centres by msf in , but not until by the national sleeping sickness control programs because of its limited availability, the initial high costs, and particularly the logistical challenge to transport the drug and its associated bottles of sterile water per treatment. the turning point was when who launched a kit format and coordinated training of staff from national sleeping sickness control programs [ ] . furthermore, in the s nifurtimox, developed against chagas disease was used in experimental settings mainly to treat melarsoprol refractory cases [ ] . in the mid- s, the pharmacokinetics of melarsoprol was elucidated. the assessment of a subsequently proposed abridged days regimen in a large scale trial with patients in angola (impamel i) allowed the replacement of the empirically derived complex schemes lasting from - days in [ ] [ ] [ ] . whereas the new regimen had major socio-economic advantages, the disappointment was that the frequency of the worst adverse drug reaction, the encephalopathic syndrome, remained at levels of - % of patients treated, still resulting in death in - % of those in whom encephalopathy developed. the metabolism of melarsoprol was elucidated somewhat later [ ] . the finding that the major metabolite melarsenoxide covalently bound to a midsize protein triggered another large-scale clinical trial, which led to the elucidation of the nature of the encephalopathic syndrome. the early s were dominated by the development of the oral prodrug pafuramidine against first stage hat; the program failed at a late point of development, but it contributed much to the understanding of hat chemotherapy and the conduct of clinical trials against hat, which it is described in detail by the paper of dickie et al. in parallel, several trials assessing combinations of eflornithine, melarsoprol, and nifurtimox were conducted. in all trials, the efficacy was better in the combination arms compared to the monotherapies. however, combinations containing melarsoprol resulted in very high frequencies of severe adverse drug reactions and were rapidly abandoned [ ] . a multiple-centre trial, conducted in the republic of congo and the democratic republic of the congo (drc) compared nifurtimox-eflornithine combination therapy (nect) with the standard eflornithine therapy. nect reduces the number of eflornithine infusions from to , the total amount of eflornithine by half and the hospitalization time by one-third [ ] . based on the favourable results of the trials conducted, nect was included for treatment of second stage gambiense hat into the who's essential medicines list in [ ] , and for children in [ ] . nect can be considered a milestone improvement: under optimal conditions, fatality during treatment is . % compared to - % under melarsoprol [ ] . the complexity of its application still restricts the use to the second stage disease, meaning that the lumbar puncture for diagnostic staging is still required [ ] , continuing until today. to identify better alternatives, the drugs for neglected diseases initiative initiated a major compound mining effort in to explore new and old nitroimidazoles as drug leads against human african trypanosomiasis. one of the compounds screened, fexinidazole, proved to be orally active against t. b. gambiense and t. b. rhodesiense in animal studies and had an excellent safety profile. the development of this orally active compound is described in detail in the papers of neau et al., and dickie et al., fexinidazole received a positive scientific opinion from the european medicines agency for treatment of gambiense hat in late , it was approved by the drug regulatory authority of the drc and added to the who list of essential medicines in , and the first official application in the drc happened at the end of january on world ntd day in a public ceremony. this deliberate coincidence of the date depicts the new integrated thinking of hat control and elimination in the framework of ntds clearly. fexinidazole will be an essential component towards hat elimination. however, it has some limitations, which will hamper its widespread use in the field: its absorption is dependent on simultaneous food intake, or else only subtherapeutic drug levels are reached; based on the observation of a lowered efficacy in patients with advanced disease, a lumbar puncture for staging still is necessary in such patients; and the drug has not been tested yet for children below six years [ ] . hence, the search for "the magic bullet" [ ] continues-with an excellent starting position compared to years ago: for the first time in history, we can speak of a modest pipeline of anti-hat drugs. one most promising candidate is in late clinical development, several compounds are well advanced in pre-clinical stages, and medicinal chemistry and lead selection work is continued as currently, the leading novel class of molecules are the boron-containing benzoxaboroles. one candidate, scyx- , acoziborole, entered phase ii/iii assessment in [ ] . the compound is described in the publication of dickie et al. should the development program be successful, acoziborole would further revolutionize the efforts to eliminate and sustain elimination of hat. due to its long half-life of h, it can be potentially used as a single-dose treatment and should it be well tolerated this would provide further options for decentralized use, and maybe even for "ring-treatment" of patient contacts following the example of ring-vaccinations used, e.g., in the control of the ebola virus. with fexinidazole, and potentially even more with acoziborole, the focus will turn away from the discovery and development of better tools, to the understanding of the implementation, optimal use, including the needs and perception of patients. the clinical research programs have contributed to the reduction of cases: new strong partnerships were formed as described by taylor et al. and the conduct of clinical trials in a number of endemic areas per se has had an impact through staff training, attention to disease, and intensified active case search and treatment of a large number of patients as described by mbo et al. besides the improvements of the renewed interest of governments and improved drug treatment, there are several other reasons for the decrease of hat prevalence: the advances in diagnostics are one of the major factors. the serological card agglutination test for trypanosomes (catt) first published in [ ] had a paramount impact on how patients could be screened by mobile teams. the test was adapted and improved several times, and despite its disadvantages (insufficient specificity to confirm diagnosis, only available in larger batches, cold chain necessary), it has kept its place in hat diagnosis. the mini-anion exchange chromatography for trypanosomes (maect) which increases the sensitivity to detect the parasite in the blood significantly was already published in [ , ] , however, only the increased funding available allowed its more consistent use and therefore detection of cases with low parasitemia. the introduction of rapid diagnostic tests is a true advancement, but also lacks the specificity needed to make a final treatment decision [ ] . additional tools were recently developed but so far only introduced to a limited extent into routine use (e.g., loop-mediated isothermal amplification (lamp) [ , ] ; immune trypanolysis test [ ] ), which will both play a role in the "end-game". the question, however, is how such tests will be used in the future, and in what settings. the currently ongoing research program ditect-hat is set up do exactly that: it seeks to validate the performance of diagnostic tools and algorithms for early and rapid diagnosis of gambiense hat for passive case detection, post-elimination monitoring, and for assessing the therapeutic response [ ] . in addition to the optimization of the technical aspects, however, it is of paramount interest to know about local settings, preferences, and the loss of skills in areas with decreasing patient numbers. the paper by palmer et al. reports on such investigations carried out in uganda. benhamou et al., through a case report on a repeatedly misdiagnosed patient, gives us an insight on future challenges for rapid diagnosis if knowledge and interest in the public health system is not maintained and broadened. another unresolved caveat of diagnosis is that a number of patients are determined to be seropositive, but thereafter hat cannot be confirmed. it will be one of the leading discussions when defining future strategies, and what to do in such cases. nkieri et al. investigated the extent of this phenomenon in the still affected regions of drc. on one hand, relapses have always been a major challenge in the treatment of hat and have made follow-up periods of up months after treatment necessary [ ] . on the other hand, until a few years ago, the dogma was "infected, but not treated inevitably leads to the death of the patient". reports on patients surviving for longer periods despite infection with trypanosomes emerged in the past few years [ ] . one of the compartments where trypanosome may survive seems to be the skin [ , ] . this might also explain how hat can re-emerge in so-called silent foci as illustrated by a nine-year-old child, who was diagnosed with gambiense-hat in ghana in , years after the last detected case [ ] . in this light, the findings of mudji et al. also have importance: over ten years after treatment in the framework of clinical trials, a number of patients revisited presented continued signs and symptoms seen in hat (lymphadenopathy, severe headaches, sleep disturbances); since no trypanosomes could be identified by any means, the implication of these findings remain open at this point. in any case, the existence of such long-term cases has a sudden and dramatic impact on the view of hat epidemiology and hat elimination [ ] . besides further epidemiological, parasitological, and molecular research, mathematical modelling may help to improve our epidemiological knowledge and inform about elimination strategies [ ] and their related costs [ ] . this field has significantly developed against all odds in the past years: trypanosomiasis with its extremely focal distribution and the many external factors influencing its transmission has been a true headache over two decades for all modellers and predictive mappers. studies of existing gambiense-hat models in a few foci (i.e., drc, guinea, and chad) suggest that some type of additional infection reservoir is needed to match the observed dynamics of reported hat cases [ , ] . this could arise from another human reservoir (including undiagnosed and latent infections), an animal reservoir, and/or heterogeneities in human risk exposure and surveillance coverage [ ] . the french colonial forces had completely dismissed the value of vector control due the successes of the treatment strategy proposed by laveran. however, vector control may play a larger role in gambiense hat elimination than anticipated. historical investigations, practical intervention studies, and modelling demonstrate the significant role that vector control can play in the control of gambiense hat. recent models suggest vector control will be essential if we are to reach the set target of elimination of the diseases as a public health problem by and beyond [ , ] . the fact that neither modelling nor vector control are represented in this edition does not represent a valuation of these topics. this special issue comes in a very timely moment, because it is now important to secure what has been achieved, to understand missing pieces, and to finish the work. however, several challenges have to be overcome to not to end up, again, in disaster. in , the world health organization, which has played an instrumental role in the control, set the goal for the elimination of human african trypanosomiasis (hat), caused by trypanosoma brucei gambiense (ghat), as a public health problem for and for the total interruption of transmission to humans for . the efforts to maximise output and optimize innovation by the who has intensified since, and several stakeholders and expert groups have been created and convened [ ] . since , the spectacular decrease of the case number has continued: some , cases were reported in , far fewer than the targeted milestone of cases, and in [ , ] . first of all, "donor fatigue" must be avoided. the elimination to "no transmission" in the drc where over % of the cases are nowadays occurring is a herculean task, which will not happen without considerable and continued funding. the conventional measurements of success (e.g., us$ spent per daly prevented) inevitably fail in an elimination scenario. naturally, the amount of money spent per patient identified and treated will soar, so the question to the health economists is, rather how much money do we lose in case efforts would not be continued, factoring in the needed future efforts to re-start and control the disease again. decisions on priorities will be necessary, too: whereas the total number of patients has massively decreased, the area in the drc they are coming from has not. therefore, a vast area still has to be kept under surveillance; this area has to be gradually reduced by safely "closing" focus by focus in order to not jeopardize the efforts. the elimination of hat, malaria, and guinea worm were all believed at a certain point to only be a matter of time-before new reservoirs became known (guinea worm), pestidicide, and drug resistance set in (malaria), and interest was decreased in a premature belief in success (malaria and hat). secondly, the human factor will start to play a key role: in theory, fexinidazole could be applied in , fixed health facilities ( ) an increase up by % from [ ] , and it will be many more, should acoziborole make it to application in a few years. however, hat is a massively stigmatized disease, linked to many beliefs and bad spirits. traditionally, patients after treatment were excluded from working and sexual intercourse for six months [ ] . therefore, the questions are: "will the disease be recognized by the younger physicians and nurses who have never seen a case of hat?" "is the medical staff willing to recognize a suspected case, given this will create a massive workload including trouble with the relatives of the patient and village, paperwork, an invasion of specialists for diagnosis and follow up activities?" "is the medical staff that was told for over years that hat belongs into the specialized hands of the vertical programs willing to assume this task and challenge?" "are the patients willing to accept hat as a diagnosis anymore?" "can we overcome wrong dogma and information?" and "will patients falling into the respective category accept a lumbar puncture?" these questions can only be addressed through the thorough understanding of beliefs, perceptions, preferences, and decision-making processes. therefore, the social and anthropological science, as well as health economics, will start to play a key role in the "end game". the articles presented in this issue by falisse et al., and lee et al. are contributing to this area. thirdly, peace, stability, and a minimal standard of living for the people in the remote regions most affected are necessary to achieve disease elimination-this condition has not yet been met. there is little the scientific community can directly contribute to this-however, knowing that disease and poverty are inextricably linked [ ] , our efforts have to continue. finally, rhodesiense hat (the east african form of the disease) was reduced to as little as cases in [ ] , goals have been reached and it may be seen as a quantité négligable. however, the disease with a zoonotic reservoir has the potential for spectacular returns, and this real danger still exists as described in the contribution of matovu et al. the surveillance and the knowledge of local medical staff has dwindled, innovation was absent for t. b. rhodesiense for decades, and serological instead of microscopic tests are used for diagnosis of other diseases making accidental diagnosis impossible. from january to october , a total of - cases were reported per month from all treatment centres in malawi; in november to january , this number surged to and higher. the cases were reported from populations around the two geographically separate wildlife reserve areas, vwaza and nkhotakota; the reason for this increase is, so far, unknown (personal communication, world health organization, control of neglected tropical diseases, geneva). this outbreak causes major concern and should be a serious warning to everyone who is of the opinion that sleeping sickness has been conquered. similarly, other unexpected priorities, such as the current sars-cov- epidemic, may at all times derail a fragile health system. as soon as the mental and financial attention and priority is on another disease, signals of a hat resurgence may well be overlooked-we should now be well aware about the consequences and impact of late reactions and exponential transmission. compared to when i wrote the conclusion of the hat special issue in [ ] , we are at a completely different point today. we celebrated several marvellous scientific successes in the meantime, tools were improved, patients numbers down-but to reach the set goals and to get completely rid of this horrible disease, the conclusion is the same again: "the goal must now be to maintain the momentum" and "even the biggest efforts of the research scientists, field workers, development agencies, and companies will fail if they are not paralleled by achievements in the political field bringing peace, stability, and a minimal standard of living to the people in the remote regions most affected". the author declares no conflict of interest. the history of african trypanosomiasis. parasites vectors the history of sleeping sickness african trypanosomiasis-centennial review the elusive trypanosome pharmacological aspects of the trypanocidal drug melarsoprol chemotherapy of protozoal infections: amebiasis, giardiasis, trichomoniasis, trypanosomiasis, leishmania and other protozoal infectious the development of drugs for treatment of sleeping sickness: a historical review epidemiology of human african trypanosomiasis control of human african trypanosomiasis: back to square one sleeping sickness-a growing problem? are there new approaches to roll back trypanosomiasis (editorial) the new landscape of neglected disease drug development commentary: sleeping sickness-a growing problem? new developments in human african trypanosomiasis effects of the ornithine decarboxylase inhibitors dl-α-difluoromethylornithine and α-monofluoromethyldehydroornithine methyl ester alone and in combination with suramin against trypanosoma brucei brucei central nervous system models treatment options for second-stage gambiense human african trypanosomiasis. expert rev. anti-infective ther nifurtimox in late-stage arsenical refractory gambiense sleeping sickness efficacy of new, concise schedule for melarsoprol in treatment of sleeping sickness caused by trypanosoma brucei gambiense: a randomised trial efficacy of -day melarsoprol schedule years after treatment for late-stage gambiense sleeping sickness effectiveness of a -day melarsoprol schedule for the treatment of late-stage human african trypanosomiasis: confirmation from a multinational study investigations of the metabolites of the trypanocidal drug melarsoprol nifurtimox-eflornithine combination therapy for second-stage african trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase iii, non-inferiority trial control and surveillance of human african trypanosomiasis: report of a who expert committee; world health organisation update on field use of the available drugs for the chemotherapy of human african trypanosomiasis new who guidelines for treatment of gambiense human african trypanosomiasis including fexinidazole: substantial changes for clinical practice a 'magic bullet' for african sleeping sickness ) for the serological diagnosis of t. b. gambiense trypanosomiasis the separation of trypanosomes from blood by anion exchange chromatography: from sheila lanham's discovery years ago to a gold standard for sleeping sickness diagnosis miniature anion exchange centrifugation for detection of low parasitemias: adaptation for field use sensitivity and specificity of hat sero-k-set, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by trypanosoma brucei gambiense: a case-control study loop-mediated isothermal amplification for detection of african trypanosomes using detergent-enhanced lamp for african trypanosome detection in human cerebrospinal fluid and implications for disease staging immune trypanolysis test as a promising bioassay to monitor the elimination of gambiense human african trypanosomiasis diagnostic tools for human african trypanosomiasis elimination and clinical trials (ditect-hat). . available online untreated human infections by trypanosoma brucei gambiense are not % fatal the skin is a significant but overlooked anatomical reservoir for vector-borne african trypanosomes the dermis as a delivery site of trypanosoma brucei for tsetse flies do cryptic reservoirs threaten gambienses-leeping sickness elimination? mathematical models of human african trypanosomiasis epidemiology seeing beyond : an economic evaluation of contemporary and emerging strategies for elimination of trypanosoma brucei gambiense quantitative evaluation of the strategy to eliminate human african trypanosomiasis in the democratic republic of congo evaluating long-term effectiveness of sleeping sickness control measures in guinea the impact of vector migration on the effectiveness of strategies to control gambiense human african trypanosomiasis human african trypanosomiasis control: achievements and challenges all past events/information related to human african trypanosomiasis monitoring the elimination of human african trypanosomiasis: update to the flipside of eradicating a disease; human african trypanosomiasis in a woman in rural democratic republic of congo: a case report whose elimination? frontline workers' perspectives on the elimination of the human african trypanosomiasis and its anticipated consequences this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -mq x nw authors: blagosklonny, mikhail v. title: from causes of aging to death from covid- date: - - journal: aging (albany ny) doi: . /aging. sha: doc_id: cord_uid: mq x nw covid- is not deadly early in life, but mortality increases exponentially with age, which is the strongest predictor of mortality. mortality is higher in men than in women, because men age faster, and it is especially high in patients with age-related diseases, such as diabetes and hypertension, because these diseases are manifestations of aging and a measure of biological age. at its deepest level, aging (a program-like continuation of developmental growth) is driven by inappropriately high cellular functioning. the hyperfunction theory of quasi-programmed aging explains why covid- vulnerability (lethality) is an age-dependent syndrome, linking it to other age-related diseases. it also explains inflammaging and immunosenescence, hyperinflammation, hyperthrombosis, and cytokine storms, all of which are associated with covid- vulnerability. anti-aging interventions, such as rapamycin, may slow aging and age-related diseases, potentially decreasing covid- vulnerability. humans and other animals (including the worm [ ] and the fly [ ] ) do not die from aging itself but from agerelated diseases such as ischemic heart disease (ihd), hypertension, diabetes, cancer, alzheimer's and parkinson's diseases, age-related macular degeneration, osteoporosis and sarcopenia (as we will discuss, even seemingly non-deadly diseases such as osteoporosis can lead to deadly complications). the incidence of these diseases increases exponentially with age. some diseases such as obesity, hypertension and diabetes develop earlier in the course of aging. other diseases, such as alzheimer's disease and macular degeneration, are usually diagnosed later [ , ] . age-related diseases may also occur in younger people with genetic predisposition and environmental exposure hazards. but even without these factors, diseases develop because they are quasi-programmed (see "quasi-programmed aging section"). these diseases are not diseases of civilization, as it may seem. humans simply now live long enough to develop them. of course, "hazards of civilization" can accelerate them at a younger age. aging and its diseases cannot be separated. healthy aging, or aging without diseases, is merely a slow aging, when biological age is less than chronological age. during a period of seemingly healthy aging, pre-pre-diseases and pre-diseases are progressing until they eventually reach clinical manifestations. thus, healthy aging progress to unhealthy and pre-diseases become diseases [ ] . age-related diseases and covid- vulnerability are highly intertwined. patients, who die from covid- , otherwise would die from age-related diseases such as heart disease, cancer, diabetes, hypertension, just a year later. covid- approximately doubles a patient's aging-dependent risk of dying during one year. for example, (numbers are very approximate), a sixty year old woman has % chance to die from aging before her st birthday. at that age, if infected, the death rate from covid- is around % for females. if infected, a patient has approximately doubled chances to die compared with usual age-related mortality during one year. as david spiegelhalter put it: "getting covid- is like packing a year's worth of risk into a week or two". https://medium.com/wintoncentre/how-much-normalrisk-does-covid-represent- e . children and young adults have a very low risk of death from aging-related diseases, so that risk remains extremely low even when doubled. although natural mortality is relatively high in the youngest age group, especially in infants, they do not die from age-related diseases of course. instead, infants are vulnerable to bacterial infections and candida infections due to underdeveloped immune system [ ] . low covid- mortality in the pediatric age group [ ] is consistent with the notion that covid- vulnerability is not due to a "weak" immune system. in contrast, as we will discuss in the next section, it is hyper-functional immune response that leads to death from covid- in the elderly by causing cytokine storm. aging severe covid- is characterized by hyperinflammation, cytokine storm, acute respiratory distress syndrome (ards), damage to the lung, heart and kidneys [ ] [ ] [ ] [ ] . in response to viral replication, hyperfunctional monocytes and macrophages infiltrate the lung, causing hyper-inflammation and hyper-secretion of cytokines such as interleukin (il)- , il- , il- , il- ra, interferon-γ inducible protein (ip)- , tumor necrosis factor (tnf)-α, ferritin, monocyte chemoattractant protein (mcp)- , macrophage inflammatory protein (mip) -α, granulocyte-colony stimulating factor (g-csf), c-reactive protein (crp) and procalcitonin. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this leads to leukocyte recruitment, vascular permeability, edema and further pulmonary damage in vicious cycle [ , , , , ] . hyper-inflammation becomes systemic, in turn causing hyper-coagulation and thrombosis, disseminated intravascular coagulation [ ] . this causes injury of distant organs such as the kidneys. pre-existing organ damage (late stages of agerelated diseases) exacerbates organ damage caused by cytokine storm [ , , ] . in addition, cellular hyperfunctions and systemic hyper-inflammation may lead to cellular exhaustion, such as exhaustion of lymphocytes (lymphopenia) [ ] [ ] [ ] . hypercoagulation is associated with hyperactive fibrinolysis and increased d-dimer blood levels [ ] . cytokine storm is a systemic hyperfunctional response ( figure ). of course, age-related hyperfunctional response, such as cytokine storm, is not caused by lifelong accumulation of molecular damage. aging is not caused by molecular damage after all. instead it's a continuation of developmental/growth programs that lead to hyperfunctions and in turn eventually to dysfunctions. hyperfunction theory of quasi-programmed aging "quasi" means "resembling" or "seemingly, but not really." quasi-program of aging is not a program but a continuation of developmental programs that were not switched off upon their completion [ , ] . they purposelessly unfold, leading to age-related diseases, secondary organ failure and death. quasi-programmed (program-like) aging is associated with higher than optimal cellular and systemic functions, which eventually, via cellular exhaustion and organ damage, lead to functional decline ( figure ). for example, starting from birth, blood pressure increases and continues to increase after organismal growth is completed. therefore, hypertension is the most prevalent age-related disease. in turn, hypertension can cause organ damage: stroke, infarction and renal failure. similarly, obesity develops in post-development as a continuation of growth (yet, it can be prevented by low caloric diets, illustrating that quasi-program of aging can be decelerated). hyperfunction is an excessive normal cellular function: contraction by smooth muscle cells (smc), adhesion and aggregation by blood platelets, insulin secretion by beta-cells, lipid accumulation by adipocytes, secretion by stromal and immune cells, oxidative burst by leukocytes, just to name a few. when higher than optimal, they cause vasoconstriction and hypertension, thrombosis, hyperinsulinemia, hypertrophy, hyperplasia, obesity, hyper-secretory phenotype or senescence-associated secretory phenotype (sasp), hyper-inflammation and so on. hyper-function is not necessarily an absolutely increased function. it may be also insufficiently decreased function (relative hyperfunction). levels of igf- and growth hormone decrease during lifespan. despite this decrease, igf- levels are still higher than optimal (relative hyper-function) because further genetic decrease in igf- levels (by genetic means) extends health span and lifespan in mammals [ ] [ ] [ ] . cellular hyperfunctions may eventually switch to cellular exhaustion and loss of functions at late stages. during the course of type ii diabetes, mtor overactivation and hyperinsulinemia eventually lead to beta-cell exhaustion and insulin insufficiency, from prediabetes to diabetes [ , ] . as another example, after puberty, hyperstimulation of the ovary eventually leads to oocyte exhaustion and menopause (see figure in ref. [ ] ). depletion of naïve lymphocytes is another example, as reviewed here later. age-related alterations are mostly noticed when they switch to functional decline, which is a late event. in some cases, functional decline can be primary and programmed. for example, thymus involution (replacement of t cells by adipocytes) starts early in life, accelerates at puberty and continues later. still loss of thymocytes and their niches may be in part due to adipocyte hyperplasia and hypertrophy [ ] . in fact, obesity accelerates involution, whereas calorie restriction decelerates it [ , ] . furthermore, the oblation of sex hormones decelerates or even reverses thymus involution [ ] . thus, involution is triggered by adipocyte hyperplasia and increased production of sex hormones during puberty [ ] . quasi-programmed aging is not driven by molecular damage. it is driven by nutrient/hormone/cytokinesensing and growth-promoting signaling pathways such as target of rapamycin (tor; mtor), which are involved in developmental growth and later cause hyperfunctional aging and its diseases [ , ] . what is the cause-effect relationship between age-related diseases and covid- lethality? do patients die from age-related diseases, complicated by covid- ? or, in contrast, do these various diseases make covid- infection lethal? both scenarios take place to some extent. however, the relationship is mostly indirect. both age-related diseases and covid-vulnerability result from the same underlying cause (figure ). this is why they are highly correlated. the cause is aging itself. aging is manifested by a sum of deadly -and not so deadly -diseases and conditions ranging from cancer to grey hair. although not all diseases seem to be deadly, they can cause complications such as stroke, ventricular fibrillation, renal failure, lung edema. even sarcopenia and osteoporosis lead to falls and broken bones culminating in a deadly sequence of events. cosmetic manifestations such as aging spots and wrinkles, while not deadly by themselves, can be manifestations of other diseases. for example, baldness correlates with prostate enlargement [ ] , and the later can lead to urinary obstruction and renal failure. diseases occur together. for example, chronic obstructive pulmonary disease (copd) is associated with diabetes, cardiovascular disease and hypertension [ ] . if a person has one disease (e.g., diabetes), this patient has higher chances of having other diseases (e.g., hypertension, ihd, cancer) or conditions, including covid- vulnerability, which is revealed only during infection but can be predicted by pre-existing diseases. aging is initially driven by an increase in cellular and systemic functions (hyperfunction), leading to age-related conditions. for example, hypertension is a systemic hyperfunction due to hyperfunction of multiple cell types such as arterial smooth muscle cells (asmc). similarly, covid- -vulnerability is associated with hyperfunction of inflammatory cells that, in response to covid- infection, causes cytokine storm, hypercoagulation and damage of the lung and distant organs. the covid- vulnerability syndrome is an agingrelated disease, strictly dependent on biological age, associated with other age-related diseases, and exemplified by hyper-functional response to infection. with hundreds of cell types acting in concert, the immune system is so complex that we cannot discuss age-related alterations without oversimplification. the most noticeable alteration is that memory t and b cells replace naive t and b cells [ ] . (this seems natural since life-long exposure to pathogens replaces naïve cells by memory cells). replacement of naïve immune cells decreases adaptive responses to novel antigens such as sars-cov- . in contrast, immune protection by memory t cells from viral re-infection with known pathogens is usually increased with age [ ] . immune responses are roughly divided into (a) innate responses, carried mostly by neutrophils, macrophages and nk cells, which react to pathogen rapidly and nonspecifically, and (b) adaptive responses, carried by t and b lymphocytes, which are delayed, slower and specific (e.g., antigen-specific clonal expansion of t and b lymphocytes and antibody production by b lymphocytes) [ ] [ ] [ ] . in the elderly, immune responses to sars-cov- / are "stuck in innate immunity," with insufficient progression to adaptive immunity [ ]. however, decline in adaptive response, such as antibody production, plays little role in covid- mortality. it is hyper-functional innate immunity, hyper-inflammation, cytokine storm and hyper-coagulation that lead to organ failure and death. in agreement, hyper inflammatory response rather than high virus numbers leads to death of sars-cov-infected old nonhuman primates [ ] . aging is associated with diseases of immune hyperfunction such as autoimmune disorders with paradoxical increase in certain signaling pathways and cytokine levels [ ] [ ] [ ] . in the elderly, innate immune cells are in a state of sustained activation, producing pro-inflammatory cytokines [ , [ ] [ ] [ ] . increased pro-inflammatory activity by the innate immune system, especially by monocytes/ macrophages, is a state of alertness and hyper-reactivity on the cost of potential age-related inflammatory diseases [ , [ ] [ ] [ ] . whereas some functions are decreased, others are increased. according to the inflamm-aging concept, innate immune system overtakes adaptive immune system in aging. cause-effect relationships are bi-directional: immunosenescence (namely, a decrease in adaptive response) is a cause and consequence of inflamm-aging [ , [ ] [ ] [ ] . we can consider inflamm-aging as an example of hyper-function. while some functions are decreased, others are increased. hyper-function is damaging. (in analogy, increased electric power, without an adaptor, would damage a laptop). damaging hyper-functions can lead to loss of function and cellular exhaustion. and vice versa, loss of function may cause compensatory hyper-functions of another components. cellular senescence is a continuation of cellular growth, when actual growth is completed [ , ] . in proliferating cells, cellular mass growth is balanced by cell division. cells grow in size and then divide. when the cell cycle is blocked (e.g., p and p ), then growth-promoting pathways such as mtor and mapk drive conversion to senescence (geroconversion) [ , , ] . during geroconversion, cells become hypertrophic and "fat". cellular functions increase: hyper-secretion and lysosomal hyper-function are manifested by sasp and beta-gal staining. hyper-activated growthpromoting pathways cause compensatory resistance to growth factors/insulin, permanent loss of re-proliferative potential [ ] . rapamycin, everolimus, pan-mtor and mapk inhibitors slows down geroconversion, maintaining reversible quiescence instead of senescence [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . geroconversion is a continuation of cellular growth [ , ] . similarly, aging is a continuation of developmental growth (see figure in ref. [ ] ). when the developmental program is completed, it becomes a quasi-program of aging. as discussed in detail, chronically activated nutrient-sensing and growth-promoting pathways drive age-related diseases, culminating in organismal death [ , ] . age-related diseases are quasi-programmed. aging is a common cause of age-related diseases, a sum of all agerelated diseases. they are diseases of hyper-function, secondary hypo-function and compensation reactions [ ] ; they are deadly manifestations of aging. from activation of cellular functions to systemic hyperfunctions, from diseases to organ damage and death, hyperfunction theory of quasi-programmed aging describes the sequence of events [ ] . and as discussed in , suppression of aging by gero-suppressants, such as rapamycin, will prevent and treat all age-related diseases [ ] . this point of view is becoming widely accepted and, in recent literature, quasi-programmed model of diseases ( ) is called "geroscience hypothesis" [ , ] . if aging were functional decline due to accumulation of molecular damage, then it would be near to impossible to restore functions and rejuvenate the immune system. in contrast, if functional decline is secondary to hyperfunctions (see figure in ref. [ ] ), these hyperfunctions can be suppressed pharmacologically to restore lost functions. typical drugs are inhibitors of their targets, rather than activators, so they decrease functions of their targets. by decreasing hyper-functions, which otherwise lead to secondary loss of functions, rapamycin may restore "lost" functions ( figure ). rapamycin improves vaccination against viruses such as influenza in old mice, monkeys and humans [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . importantly, rapamycin increases pathogenspecific but not graft-reactive cd + t cell responses [ , ] . therefore, rapamycin and everolimus can both be used to prevent donor organ rejection and improve adaptive immunity against new pathogens [ ] . differentiation is an increase of tissue-specific cellular functions. terminally differentiated b, t, and nk cells can rapidly react to already known pathogens [ ] . decrease in naïve t and b lymphocytes (and thus diminished response to novel antigens) results in part from cellular hyper-differentiation in the immune system [ , ] . hyper-functional differentiation can be counteracted by rapamycin [ ] . as another example, age-related exhaustion of stem cells is partially due to loss of quiescence caused by growth over-stimulation [ , [ ] [ ] [ ] . in general, senescent cells characterized by hyper-proliferative drive coupled with cell cycle arrest [ ] . in young mice, mtor hyperactivation causes senescence of hematopoietic stem cells (hsc) and decreases lymphopoiesis [ ] . in old mice, rapamycin rejuvenates hematopoiesis, and improves vaccination against influenza virus [ ] . third, production of lymphoid cells may be decreased because of disruption of hypoxic niches due to adipocytes hyperplasia [ , ] . hypoxic niches can preserve hsc [ , ] probably because hypoxia inhibits mtor and cellular senescence [ ] . in agreement, rapamycin preserves hscs [ , , , ] reduces the proportion of memory cells and maintains a pool of naïve t cells [ , ] . aging fourth, growth factor (gf)-and insulin-resistance is loss of function because cells cannot respond to gf/insulin. but it may be caused by over-activated mtor, which via s k/irs feedback loop blocks insulin and gf signaling. rapamycin abrogates the loop restoring signaling [ ] [ ] [ ] [ ] [ ] [ ] . a high prevalence of age-related diseases, often called "diseases of civilization," is a success story of modern medicine. in the past, most people did not live long enough to develop age-related diseases and those who developed them died soon after. due to medical advances, people survive to on average, despite suffering from age-related diseases. standard medicine preferentially extends life span, without necessarily affecting health span (see figure in ref. [ ] ). for example, defibrillation and coronary stenting can save life but not cure heart disease. it is anti-aging interventions that extend health span, delaying diseases, thus extending lifespan. aging is a common cause of all age-related diseases. by suppressing aging, anti-aging interventions may delay all age-related diseases [ ] . as a well-known example, low calorie diets such as calorie restriction, intermittent fasting, and low carbohydrate diets extend both health and lifespan. figuratively, low calorie diets prolong life by improving health. nutrients and obesity activate growth-promoting pathways (e.g., mtor), thus accelerating development of quasi-programmed (age-related) diseases. obesity is associated with all age-related diseases from cancer to alzheimer's and from diabetes to sarcopenia. covid- vulnerability is also associated with obesity [ , , , ] . according to hyperfunction theory, obesity accelerates aging and all age-related conditions including covid- vulnerability. diabetes is one of main risk factors of death in covid- [ , , , , , ] . can type diabetes, an agerelated disease, be reversed? in remarkable studies, it was shown that a brief course ( - weeks) of very low calorie diets (vlcds) can reverse type ii diabetes. in one study, vlcd reversed diabetes in % of patients with up to a -year history of diabetes [ ] . vlcd is most effective for its prevention and at early stages of diabetes [ ] . this anti-aging modality is so simple that remission can be achieved at home by healthmotivated individuals [ ] . simultaneously, it treats other age-related diseases such hypertension [ ] . obesity is associated with other diseases of hyperfunction from diabetes and sarcopenia to cancer and alzheimer's' disease. since age-related diseases are predictors of covid- mortality, vlcd in theory may decrease covid- vulnerability. in the soil of easter island, a complex bacteria produces anti-fungal antibiotic rapamycin to suppress yeast growth but, as a by-product, it also suppresses yeast aging (quasi-programed aging is a continuation of growth). approved for human use in , rapamycin (sirolimus) and its close analog everolimus are widely used in several diseases including cancer and organ transplantation. hundreds of clinical trials (and twenty years of clinical practice) have ensured their safety and good tolerability especially in healthy older adults [ ] . currently, several anti-aging clinics prescribe rapamycin out of label to prevent age-related diseases and slow aging. hundreds of recent reviews discussed rapamycin and everolimus in detail, so i will just emphasize a few points: . crucial prediction of hyper-function theory of quasi-programmed aging in was that rapamycin will slow aging, extend healthspan and lifespan and decrease all age-related [ ] . it has been confirmed: it extends lifespan in animals from worm to mammals. in some strains of short-lived mutant mice, it extends life span two fold [ , ] . . rapamycin slows geroconversion to cellular senescence in cell culture [ ] . . mtor is a potential therapeutic target in chronic obstructive pulmonary disease copd [ ] , [ ] . rapamycin (sirolimus) is already approved and successfully used in lymphangioleiomyomatosis (lam), a progressive, cystic lung disease, associated with inappropriate activation of mtor [ ] . longterm daily use of rapamycin improves lung function without causing serious side effects (and of course no even minor side effects in the lung, given that rapamycin improves lung function) [ ] . . despite widespread misunderstanding, rapamycin and everolimus do not cause diabetes. in contrast, they prevent diabetic complications in animals with diabetes (see for references [ ] ). in rodents, in some conditions they may cause symptoms of starvation pseudo-diabetes similar to prolong fasting and ketogenic diet [ ] . although, the johnson study found a slight but significant correlation between medicare billing for insulin and the use of rapamycin in renal transplant patients, this correlation was mechanistically explained by interaction of rapamycin with two other drugs used in the same patients [ , ] . in cancer patients, everolimus may cause reversible hyperglycemia as a mild, infrequent and reversible side effect after several weeks of daily high doses of everolimus and rapamycin [ ] . mechanistically, everolimus decrease insulin production, not causing insulin resistance [ ] . if anything, everolimus and rapamycin can be considered to treat complications of type ii diabetes and prevent hyperinsulinemia and obesity ( [ ] and references within). what actually contributes to type diabetes is excess of nutrients (and especially carbohydrates), which activate mtor and cause hyperinsulinemia and insulin resistance. as soon as covid- epidemic started, it become clear that covid- vulnerability is an aging-dependent condition and the use of rapamycin (sirolimus) was immediately suggested by independent researchers [ , , [ ] [ ] [ ] [ ] [ ] . these proposals were based on a mixture of several rationales, which need to be clearly distinguished. in theory, there are at least three independent applications of rapamycin and everolimus for covid- . currently, they all are still hypothetical. . anti-aging effect ( figure ). by decreasing biological age and preventing age-related diseases, a long-term rapamycin therapy may in theory decrease covid- mortality rate in the elderly. anti-aging application is especially important because it is beneficial regardless of covid- . after all, mortality rate from aging and its diseases is %, causing more than million deaths in the usa annually. continuous use of rapamycin is expected to improve health, decrease age-related diseases and extend healthy lifespan, rendering individuals less vulnerable, when infected with the virus. vulnerability (log scale) increases exponentially with age (blue line). the line ends at age , a maximum recorded age for humans. in theory, a continuous rapamycin treatment would slow down an increase of the vulnerability with age (red line). the increase is still logarithmic but at a different slope, because rapamycin slows the aging process. the maximum lifespan, in the absence of covid- , is extended because the % natural death threshold is achieved later. . rejuvenating immunity. as we discussed in section "figuratively, rapamycin rejuvenates immunity" [ ] , mtor inhibitors can improve immunity to viral infections, improve immunization and vaccination to some viruses such as flu [ - , , , ] . in addition, viruses such as flu [ ] and coronavirus (mers-cov) [ ] depend on mtor activity for replication. currently, however, there are no data regarding covid- . although aimed to evaluate safety, phase clinical trial "sirolimus in covid- phase (sirco- )" may reveal anti-viral effects too https://clinicaltrials.gov/ct /show/nct . . potential suppression of cytokine storm and hyperinflammation ( figure ). as we discussed in the section "cytokine storm is a hyperfunction", cytokine storm and hyper-inflammation is a main cause of death in covid- pneumonia [ - , , , , - ] rapamycin, an antiinflammatory agent, inhibits hyper-functions, cellular senescence and decrease secretion of cytokines ( [ , , ] . rapamycin inhibits the jak /stat signaling pathway [ ] and reduces ifγ and tnf-α levels [ ] . rapamycin (sirolimus) treatment improves outcomes in patients with severe h n pneumonia and acute respiratory failure and was associated with improvement in virus clearance, and shortened ventilator days [ ] . clinical trial "sirolimus treatment in hospitalized patients with covid- pneumonia (scope)" has been started https://clinicaltrials.gov/ct /show/nct . this review is intended for a professional audience, to stimulate new ideas and to aid the global efforts to develop effective treatments for covid- disease. this article does not represent medical advice or recommendations to patients. the media should exercise 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(sasp) mechanistic insights into impaired dendritic cell function by rapamycin: inhibition of jak /stat signaling pathway adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h n pneumonia and acute respiratory failure supplementary key: cord- -guciupc authors: hajj hussein, inaya; chams, nour; chams, sana; el sayegh, skye; badran, reina; raad, mohamad; gerges-geagea, alice; leone, angelo; jurjus, abdo title: vaccines through centuries: major cornerstones of global health date: - - journal: front public health doi: . /fpubh. . sha: doc_id: cord_uid: guciupc multiple cornerstones have shaped the history of vaccines, which may contain live-attenuated viruses, inactivated organisms/viruses, inactivated toxins, or merely segments of the pathogen that could elicit an immune response. the story began with hippocrates b.c. with his description of mumps and diphtheria. no further discoveries were recorded until a.d. when the smallpox vaccine was described. during the eighteenth century, vaccines for cholera and yellow fever were reported and edward jenner, the father of vaccination and immunology, published his work on smallpox. the nineteenth century was a major landmark, with the “germ theory of disease” of louis pasteur, the discovery of the germ tubercle bacillus for tuberculosis by robert koch, and the isolation of pneumococcus organism by george miller sternberg. another landmark was the discovery of diphtheria toxin by emile roux and its serological treatment by emil von behring and paul ehrlih. in addition, pasteur was able to generate the first live-attenuated viral vaccine against rabies. typhoid vaccines were then developed, followed by the plague vaccine of yersin. at the beginning of world war i, the tetanus toxoid was introduced, followed in by the pertussis vaccine. in , the expanded program of immunization was established within the who for bacille calmette–guerin, polio, dtp, measles, yellow fever, and hepatitis b. the year witnessed the launching of the international aids vaccine initiative. in , the who passed a resolution to eradicate polio by the year and in ; the first vaccine to prevent cervical cancer was developed. in , “the decade of vaccines” was launched, and on april st , the united nations launched the “shot@life” campaign. in brief, the armamentarium of vaccines continues to grow with more emphasis on safety, availability, and accessibility. this mini review highlights the major historical events and pioneers in the course of development of vaccines, which have eradicated so many life-threatening diseases, despite the vaccination attitudes and waves appearing through history. vaccines constitute one of the greatest success stories within the health sector. they form part of a multifaceted public health response to the emergence of pandemics. this review is general in nature. it highlights the major historical cornerstones in the development and progress of various types of vaccines since the beginning and through the ages until today. it recognizes the major pioneers whose work has made a difference in the advancement of this vital health field, despite all the anti-vaccination movements that appeared through the ages. multiple reviews were encountered during our literature search; however, each of those reviews dealt with a specific aspect of vaccination like effectiveness of a particular vaccine, or side effects of another or even attitudes toward vaccines. consequently, this work tried to put together the major achievements through history stressing the importance, continuous vital role, and the need for immunization for health prevention and protection as well as its impact on human experience. the physiological mechanisms behind vaccination are well established. vaccination activates the immune system and induces both innate and adaptive immune responses thus leading to the production of antibodies, in the case of a humoral response, or to the generation of memory cells that will recognize the same antigen, if there is a later exposure. periodic repeat injections can improve the efficacy and effectiveness of inoculations ( ) . the approval of a vaccine abides by a set of well-established international rules and regulations. prior to their approval by the respective health authorities, scientists test vaccines extensively in order to ensure their efficacy, safety, and effectiveness. next to antibiotics, vaccines are the best defense that we have to date against infectious diseases; however, no vaccine is actually % safe or effective for everyone. this is attributed to the fact that each body reacts to vaccines differently ( ) ( ) ( ) . significant progress has been made over the years to monitor side effects and conduct research relevant to vaccine safety. in addition, vaccine licensing is a lengthy process that may take years or longer. the food and drug administration (fda) and the national institute of health (nih) require that vaccines undergo the required phases of clinical trials on human subjects prior to any use in the general public. this process is becoming more complex as more caution and care is being allocated to the quality of the market product. furthermore, vaccines can be divided into different categories depending on the way that they are prepared including liveattenuated vaccines, inactivated vaccines, subunit vaccines, conjugate vaccines, and toxoids. live-attenuated vaccines are used more frequently for viruses rather than bacteria, since the former contain a lesser amount of genes and can be controlled more easily ( ) . the most common method in formulating live-attenuated vaccines involves passing the virus through successions of cell cultures to weaken it. this will produce a form of the virus that is no longer able to replicate in human cells. however, it will still be recognized by the human immune system, hence protecting the body from future invasions. examples of such vaccines are measles, rubella, mumps, varicella (more commonly known as chickenpox), and influenza. the disadvantage of using this technique is that the virus may transform into a more virulent form due to a certain mutation and cause illness once injected into the body. although this rarely occurs, it must always be taken into consideration ( ) . by using heat, radiation, or certain chemicals, one can inactivate a microbe. the microbe will no longer cause illness but can still be recognized by the immune system. poliovirus and hepatitis a are common examples of inactivated vaccines. this type of vaccine has the disadvantage of being effective for a shorter period of time than live-attenuated vaccines. multiple boosters of the vaccine are sometimes required to improve effectiveness and sustainability ( ) . a subunit vaccine contains only portions of the microbe that can be presented as antigens to the human immune system instead of the microbe as a whole. the antigens or the microbe portions that best activate the immune response are usually selected. an influenza vaccine in the form of shots is an example. in addition, a recombinant subunit vaccine has been made for the hepatitis b virus. hepatitis b genes are injected into maker cells in culture. once these cells reproduce, the desired antigens of the virus are produced as well, and these can be purified for use in vaccines ( ) . conjugate vaccines are designed from parts of the bacterial coat. however, these parts may not produce an effective immune response when presented alone. hence, they are combined with a carrier protein. these carrier proteins are chemically linked to the bacterial coat derivatives. together, they generate a more potent response and can protect the body against future infections. vaccines against pneumococcal bacteria used in children are an example of conjugate vaccines ( ) . some bacteria release harmful toxins that cause illness in infected individuals. vaccinations against such types of bacteria are prepared by inactivating or weakening the toxin using heat or certain chemicals. this will help prepare the immune system against future invasion. the vaccine against tetanus caused by the neurotoxin of clostridium tetani is a good example of a toxoid ( ) . the generation of vaccine-mediated protection is a complex challenge. effective early protection is conferred primarily by the induction of antigen-specific antibodies. the quality of such antibody responses has been identified as a determining factor of efficacy. efficacy requires long-term protection, namely, the persistence of vaccine antibodies and/or the generation of immune memory cells capable of rapid and effective reactivation upon subsequent microbial exposure ( ) . the exponential development of new vaccines raises many questions about their impact on the immune system. such questions related to immunological safety of vaccines as well as triggering conditions such as allergy, autoimmunity, or even premature death ( ) . such issues were always looked for and monitored and some vaccines were even stopped because of these issues. recent vaccine models rely on both a cell-mediated response and a humoral immune response with highly specific antibodies and have shown an adequate amount of success. this, however, has not been the case for a few diseases such as tuberculosis where the humoral immunity mounted by the bacille calmette-guerin (bcg), the only currently used human vaccine, is inefficient in conferring proper immunization ( ) . however, t cells do take part indirectly in the production of antibodies and of secreted biological molecules (e.g., interferon) for protection. it seems that a proper mounted immunity is better achieved by vaccineinduced antibodies, whereas a t cell immune response is needed for disease attenuation. hence, a robust understanding of b and t cell function is needed for proper immunization ( ) . multiple determinants modulate the primary vaccine antibody response in healthy individuals; they include the vaccine type, live versus inactivated, protein versus polysaccharide, and use of adjuvants ( ) . they also include the nature of the antigen and its intrinsic immunogenicity ( ) , the dose of the antigen, the route of administration, the vaccine schedule, and the age at administration ( ) . in addition, genes play a direct role in the body's response to vaccination even in healthy individuals ( , ) . for each of the above determinants, there might be a particular mechanism involved and is further influenced by other factors including extremes of life, acute or chronic diseases, immunosuppression, and nutrition status ( ) . early life immune responses are limited by ( ) limited magnitude of antibody responses to polysaccharides and proteins, ( ) short persistence of antibody responses to protein, ( ) influence of maternal antibodies, and ( ) limited cd + t cell and interferongamma responses. such factors are difficult to study in human infants due to neonatal immune immaturity and the inhibitory influence of maternal antibodies, which increase with gestational age and wane a few months post-natal ( ) . on the other hand, in elderly persons, the immune system undergoes characteristic changes, termed immunosenescence, which leads to increased incidence and severity of infectious diseases and to insufficient protection following vaccination ( ) . vaccines induce both innate (non-specific) and adaptive (specific) immune responses, which decline substantially with age thus leading to the decreased efficacy of vaccines in elderly persons. in the elderly, the innate immune response will witness a reduced phagocytic capacity of neutrophils and macrophages, a decrease in their oxidative burst, and impairment in the up-regulation of mhc class ii expression among other parameters ( ) . in addition, persistent inflammatory processes occur with increasing age and may reduce the capacity to recognize stimuli induced by pathogens or vaccines. for the elderly, improved special antigen delivery systems are needed to overcome these limitations ( ) . furthermore, the adaptive immune response is functionally defective in the elderly. the involution of the thymus with aging leads to a decrease in content and in output of mature naïve t cells into the periphery, which hampers the induction of adaptive immune responses to neoantigens. in the context of primary vaccination, this causes reduced response rate ( ) ( ) ( ) ( ) ( ) ( ) . b cells also undergo age-related changes that aggravate the functionality of b cells colonies. as effector b cells accumulate, naïve b cells decrease in number and this leads to a reduction in the diversity of antibody responses. in brief, vaccines tailored to the needs of the elderly will have to be developed, taking into consideration these limitations in order to improve protection in this population. in , weinberg and szilagyl eloquently approached the issues of efficacy and effectiveness clarifying the road to correctly answer the relevant but complex question: "how well does the candidate vaccine prevent the disease for which it was developed?" they highlighted clearly the distinction between efficacy (individual level) and effectiveness (population level), which are often confused terms that fit well into the new paradigm of translational research ( ) . at about the same time, curns et al. elaborated on the distinction between the epidemiologic concepts of vaccine efficacy and effectiveness within the context of translational research ( ) . such concepts were also addressed earlier, but slightly differently, by clemens and co-workers in two separate publications in and , and also by orenstein et al. in ( - ) . accordingly, vaccine efficacy is measured as the proportionate reduction in disease attack rate when comparing vaccinated and unvaccinated populations. vaccine efficacy studies always have rigorous control for biases through randomized prospective studies and vigilant monitoring for attack rates ( ) . in addition to proportionate reduction in attack rates, these studies can furthermore assess outcomes through hospitalization rates, medical visits, and costs. despite the complexity and expenses that arise from the initial trials, they are needed to establish vaccine efficacy ( ) . on the other hand, the related but distinct concept of vaccine effectiveness has always been compared to a "real world" view of how a vaccine reduces disease in a population. as such, it can evaluate risks versus benefits behind a vaccination program under more natural field conditions rather than in a controlled clinical trial. vaccination program efficiency is proportional to vaccine potency or efficacy in addition to the degree and success of immunization of the target groups in the population. in brief, it is influenced by other non-vaccine-related factors that could influence the outcome. the "real world" picture provided by vaccine effectiveness data is desirable in planning public health initiatives, an advantage that makes these studies attractive. translating research data into real public health application are a process that has been reengineered by the nih as part of a road map for future research. consequently, a new expanded definition of translational research, consisting of four steps was proposed, which fits nicely within the continuum of vaccine research ( ) . in this new process of phase i to phase iv clinical trials, safety, immunogenicity, efficacy, and post-licensure effectiveness of a particular vaccine are assessed ending up in phase iv with the burden of the disease ( ) . vaccines stood the test of time and many techniques have been introduced into the world of vaccination. practitioners used to write articles about their vaccinating instruments and techniques. according to john kirkup, vaccinators and physicians used various instruments and techniques to inject the vaccinating material into the human body. more than different vaccinating instruments have been recorded in british, american, german, and french catalogs between the years and ; most of them are out of use nowadays ( ) . there are multiple major landmarks in the history of vaccines. it was reported that the origin goes as far back as hippocrates, the father of modern medicine, b.c. he described mumps, diphtheria, and epidemic jaundice among other conditions ( ) . the earliest methods of immunization and protection against smallpox date back to about a.d., and are attributed to the chinese. it has been said that the son of a chinese statesman was inoculated against smallpox by blowing powdered smallpox sores into his nostrils ( ) . another method used for inoculation was the removal of fluid from the pustules of an infected individual and subsequently rubbing it into a skin scratch of a healthy individual. this procedure was later introduced into turkey around , long before reaching europe ( ) . it took six centuries for variolation to be introduced to great britain, in ( ) . the eighteenth century was marked by several major events that started with the spread of variolation from turkey and china to england and america, followed, in the late eighteenth century, by edward jenner's breakthrough of vaccination. variolation, derived from the latin word varus, meaning "mark on the skin, " or inoculation, derived from the latin word inoculare, meaning "to graft, " are two words that were used interchangeably in describing the aforementioned immunization process. by , variolation was introduced to england after the pursuit of an english aristocrat, lady mary wortley montague, who had been personally inflicted with an episode of smallpox. after being informed of the method of variolation, she made the embassy surgeon, charles maitland, perform the procedure on her year-old son in in turkey. in , dr. charles maitland performed the first english variolation on lady montague's year-old daughter after their return to london ( ) . lady montague became a great proponent of the procedure and worked thoroughly on advocating this process for its ability to protect against the spread of smallpox. data from the u.s. national library of medicine and the nih showed that - % of those variolated died as compared to % of those who contracted the disease naturally. correspondingly, rev. cotton mather and dr. zabdiel boylston introduced variolation in america and were also great advocates of this procedure especially since, in the same year, there was a smallpox epidemic in boston that killed hundreds ( ) . however, lady montague, rev. mather, and dr. boylston faced great opposition regarding their promotion of variolation even with the presentation of the comparative analysis of fatality rates, which reached % for those variolated compared to % for the naturally occurring disease ( ) . despite some variolation-related deaths, the word of inoculation kept spreading along with data suggesting that variolation was still the safeguard against the spread of smallpox. in addition, benjamin franklin, who lost his son in , wrote: "i long regretted that i had not given it to him by inoculation, which i mention for the sake of parents who omit that operation on the supposition that they should never forgive themselves if a child died under it; my example showing that the regret may be the same either way, and that therefore the safer should be chosen" ( ) . in , dr. william heberden, at his own expense and with the support of benjamin franklin, wrote a pamphlet entitled "some account of the success of inoculation for the small-pox in england and america: together with plain instructions by which any person may be enabled to perform the operation and conduct the patient through the distemper" ( ) . toward the late eighteenth century came jenner's breakthrough in finding a safer immunizing technique than variolation, which is vaccination. the method of variolation had low yet significant death rates; therefore, physicians were on the quest of finding a new and more secure method of immunization with minimal or no death rates. on this basis, an english physician named edward jenner ( - ) searched for a cure for smallpox, a debilitating disease that rendered the world helpless. jenner became interested in certain individuals who were immune to smallpox because they had contracted cowpox in the past. he personally witnessed this when he learned of a dairymaid that was immune to smallpox due to her previous infection with the cowpox virus, usually transmitted from infected cattle. during that time, an english farmer named benjamin jesty personally took charge of inoculating his wife and children with fresh matter from a cowpox lesion in one of his cows out of fear of having his wife and children become victims of the smallpox epidemic. he applied this method after having contracted cowpox himself and believing he was immune to smallpox. he never published his results even though his wife and children did not show symptoms after being exposed to smallpox ( ) . during these years, there were still outbreaks of smallpox. george washington, after surviving smallpox, ordered mandatory inoculation for his troops in ( ) . after many speculations on the role of cowpox and its immunizing effect against smallpox, jenner, in , inoculated an -year-old boy named james phipps using matter from a fresh cowpox lesion on the hands of a dairymaid named sarah nelms who caught them from her infected cattle. after several days, jenner inoculated the boy again but this time with fresh matter from a smallpox lesion and noted that the boy did not acquire the disease proving that he was completely protected ( ) . a few years later, word of his success circulated among the public, and jenner wrote "an inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of england, particularly gloucestershire and known by the name of cowpox, " after adding several cases to his initial achievement with the boy phipps. at first, his publication and achievement did not stir any interest in his community, but with time, word of jenner's breakthrough began spreading ( ) . the late eighteenth century was characterized by the implementation of the new process of immunization, vaccination, which required the inoculation of fresh matter from cowpox lesions into the skin of healthy individuals. the nineteenth century was a major landmark in the history of vaccines since it witnessed discoveries made by louis pasteur, the father of microbiology, and robert koch, the scientist who discovered the germ responsible for tuberculosis ( ) . in the beginning of the nineteenth century, the term "vaccination" was introduced by richard dunning from the latin word for cow "vacca. " after becoming aware of the fact that vaccination was more secure than variolation, several physicians initiated movements against the use of variolation and advocated for its eradication. dr. jean de carro, for example, aided in the elimination of variolation and its substitution with vaccination. some of the major efforts implemented in america were initiated by dr. benjamin waterhouse, who received the vaccine from edward jenner and vaccinated his own family. he later proved that they acquired immunity when they remained asymptomatic after he infected them with smallpox. waterhouse worked effectively on making vaccination universal in the u.s. unfortunately, like any other medical breakthrough, problems arose both because waterhouse aimed at making profit and the public was not ready to implement these procedures. however, after breaking his initial monopoly, waterhouse accepted to share his vaccines and made the supplies available to other physicians ( ) . despite all these efforts, smallpox epidemics continued to occur and jenner stated in a pamphlet that he wrote, "the annihilation of the small pox, the most dreadful scourge of the human species, must be the final result of this practice. " eradication was finally achieved years later. the time it took could be attributed to the fact that jenner did not think of the necessity of revaccination nor of the instability of vaccines, which made them unable to handle different environmental conditions, including countries other than england ( ) . the late nineteenth century was distinguished by pasteur's achievements that made him the father of vaccines after creating the first laboratory vaccine. louis pasteur ( - ), a french chemist and microbiologist, was the first to propose the "germ theory" of disease in addition to discovering the foundations of vaccination ( ) . he studied chicken cholera and received strains of bacteria causing anthrax and septic vibrio. pasteur started his experiments by intentionally infecting chickens by feeding them cholera-polluted meals and then recording the fatal progression of the illness. at first, pasteur was using fresh cultures of the bacteria to inoculate the chickens, most of which did not survive. during that time, pasteur had to go on a holiday, so he placed his assistant in charge of injecting the chickens with fresh cultures. however, his assistant accidentally forgot to perform the injections, and the bacterial cultures were left in a medium that was exposed to room air for about a month. later, the attendant injected the chickens with the now "attenuated" strain of bacteria resulting in mild, nonfatal symptoms. pasteur later re-injected these chickens, but this time with fresh bacteria. to his surprise, they did not get ill. ultimately, pasteur reasoned that what made the bacteria less deadly was exposure to air, mainly oxygen. pasteur used the french verb "vacciner" during the years and to describe how he was able to provide total body immunity through vaccination by inoculation of an attenuated virulence which was the first vaccine made by a human in the laboratory ( ) . pasteur also developed the anthrax vaccine in his laboratory, not long after performing his studies on chicken cholera. in , pasteur used his own anthrax vaccine, which contained attenuated live bacterial cultures in addition to carbolic acid, and demonstrated that all vaccinated animals survived while the control group died ( ) . during the same year, louis pasteur in france and george miller sternberg in the u.s. almost simultaneously and independently isolated and grew the pneumococcus organism. later in , pasteur successfully fought rabies that was endangering the european livestock by using his attenuated rabies vaccine obtained from desiccated brain tissue inactivated with formaldehyde, which provided immunity to dogs against rabies in his experiments ( ) . he reported his success to the academy of sciences in france, and a year later, he applied his original vaccine h after a -year-old boy was bitten several times by a rabid dog. the boy survived after being first inoculated with the most attenuated organisms, then subsequently with less attenuated organisms each day for days ( ) . in , the pasteur institute was established as a rabies treatment center as well as an infectious diseases research and training institute. after pasteur's successful live vaccines, a new type of vaccine was introduced in the last few years of the nineteenth century. these were killed vaccines, which were directed against three chief bacterial causes of human morbidity: cholera, typhoid, and the plague. the first cholera vaccine used to immunize humans was actually a live vaccine developed by jaime ferran ( - ), which provided a high level of protection during the epidemic in spain. however, the first killed vaccine for cholera was developed in by wilhelm kolle ( - ) and was used in japan in with over % efficiency. the credit for developing the killed typhoid vaccine during the s goes to both richard pfeiffer and almroth wright who made great contributions. wright was later credited for carrying out the "first large-scale vaccination using a killed typhoid vaccine" ( ) . finally, the killed vaccine for plague was first developed in by haffkine, who was one of pasteur's followers, when an epidemic struck bombay. during this period, vaccine production was taken over by factorytype laboratories, which formed the precursors of the biological products supply houses. many types were produced. paul ehrlich ( - ), a german physician and scientist who worked under a contractual collaboration with behring, noted the existence of toxoids in the late s. he also promoted enrichment and standardization protocols. these protocols enabled the exact determination of quality of the diphtheria antitoxins. in , it was demonstrated that toxoids could be used to durably immunize guinea pigs. it is crucial to briefly address the historical background of the bacterial infections that led to some of the earliest and most successful use of toxoids, inactivated forms of bacterial toxins, for the purpose of immunization. until the twentieth century, diphtheria, tetanus, and pertussis proved to be significant causes of illness and death with no effective treatments or prevention in sight. fortunately, advances in improved the prognosis of numerous future patients ( ) . at the end of the nineteenth century, especially in and , the cholera and typhoid vaccines were developed, followed by the introduction of the plague vaccine. the latter was preceded by the preparation of anti plague horse serum at the pasteur institute by alexandre yersin. yersin demonstrated disease protection in animals. later, he went to china to try his vaccine on humans during a plague epidemic ( ) . diphtheria is a potentially fatal disease that primarily involves tissues of the upper respiratory tract and kills its victims slowly by suffocation. in , a german physician, edwin klebs ( - ), was able to successfully isolate the bacteria that proved to be the etiological agent of the disease. it was later proved that toxin production is initiated only after the bacteria are themselves infected by a specific virus or a bacteriophage carrying the toxin's genetic instructions ( ) . in france, during the year , emile roux discovered the diphtheria toxin. his discovery led to the development of passive serum therapies through the scientific contributions of many, including emil von behring and paul ehrlich ( ) . similarly, the etiological agent of pertussis, commonly known as the "whooping cough, " was found to be a bacterium isolated from infected patient tissues in ( ) . tetanus was similarly a significant cause of mortality usually resulting from dysfunction of the autonomic nervous system or the respiratory muscles ( ) . in , another german scientist, arthur nicolaier ( - ), correlated tetanus with an anaerobic soil bacterium found in wounds. a few years later, the japanese investigator shibasaburo kitasato ( - ) was able to isolate this bacterium ( ) . at the beginning of world war i in , the tetanus toxoid was introduced following the development of an effective therapeutic serum against tetanus by emil von behring and shibasaburo kitasato. the rabies and typhoid vaccines were then licensed in the u.s. as the etiology of these destructive diseases was slowly being uncovered, by shibasaburo kitasato along with emil von behring ( ) . they discovered that the serum of animals that had been exposed to sub-lethal doses of the bacteria involved in tetanus and diphtheria was protective against the lethal effects associated with these pathogens by having an antitoxin effect when injected into another animal. additionally, this discovery, which earned behring the inaugural nobel prize for physiology and medicine in , was the concept of passive transfer in addition to serum therapy. he proved that serum could be acquired from immune animals and transferred to others as protection ( ) . once this concept made its way to clinical practice in , technical problems were faced while developing the right antitoxin concentration and potency. as a result, in the early twentieth century, the u.s. congress enacted the biologics control act legislation "to regulate the sale of viruses, serums, toxins, and similar products" to ensure medication quality control. nevertheless, with the increasing use and popularity of antitoxins derived from animal serum, scientists began to observe a syndrome now called serum sickness, or a reaction to immune-complexes formed from combining high concentrations of antigens with antibodies. this eventually led to the use of human rather than animal serum in order to decrease the frequency of adverse events; still, serum therapy was not perfect in preventing disease due to the frequency of adverse events and its brief duration of action. later on, combining diphtheria toxin and antitoxin in the same syringe proved much more effective in decreasing mortality rate. this combination became commercially available in . this was the first step in the shift from passive to active immunization ( ) . in , gaston ramon ( - ), a french veterinarian working at the pasteur institute, used a diphtheria toxoid produced by formalin and heat inactivation without the use of antitoxin to safely induce active immunity in humans. this product, termed anatoxine, was the basis for the novel and clinically effective toxoid vaccine against diphtheria. experiments followed to improve the durability of the protective response of the vaccine, and in , the importance of aluminum salts as an adjuvant added to the vaccine to augment the immune response to the antigen, became apparent ( ) . this was discovered by alexander thomas glenny ( - ) who proved that toxoid alone produced a lower level of antibody and immunity than desired, whereas better immunity was achieved when an inflammatory reaction was triggered. with these significant improvements, tetanus and diphtheria toxoids became routinely used across america and europe in the s and s ( ) . since then, refinements have been made to these vaccines to yield higher purity and reduce the number of booster doses. nowadays, widespread childhood vaccination is reducing the burden of these diseases. while this is a huge advantage, vaccines may potentially produce adverse effects that can discourage their acceptance by some populations. this has led to numerous safety movements which culminated in the congressionally legislated national childhood vaccine injury act in the s created to compensate families for selected adverse events potentially related to mandatory childhood vaccinations ( ) . nevertheless, global recommendations continue to call for routine immunization of children against diphtheria, tetanus, and pertussis with the combined dtp vaccine to sustain immunity in childhood and adolescence. dtp has, therefore, become one of the most widely used vaccines to achieve widespread immunity across age groups ( ) . tuberculosis, otherwise known as the "great white plague, " is another disease that started spreading as an epidemic once industrialization began. this disease caused approximately % of deaths in the eighteenth and nineteenth centuries across all socioeconomic groups ( ) . a french physician named jean antoine villemin ( - ) demonstrated that the mode of transmission of disease is through the respiratory system. robert koch ( - ) , known as the founder of modern bacteriology, revealed in that the causative agent of the disease is mycobacterium tuberculosis, which later became known as koch's bacillus ( ) . following this discovery, koch created what later came to be known as koch's postulates, which listed the criteria necessary for proof of bacterial causality: "the organism must be present in diseased tissues; it must be isolated and grown in pure culture; and the cultured organisms must induce the disease when inoculated into healthy experimental animals" ( ) . in , two bacteriologists working in the pasteur institute in lille, albert calmette ( - ) and camile guerin ( - ), announced their discovery of mycobacterium bovis, which is a strain of tubercle bacilli that could be used to create a vaccine against tuberculosis. this occurred after it became evident that different forms of the bacterium were required to prevent or treat tuberculosis, including non-pathogenic, attenuated, or killed tubercle bacilli from different sources, including human, bovine, and equine. this strain had an attenuated virulence while maintaining its antigenicity and became known as bcg ( ) . bacille calmette-guerin vaccinations proved to be successful in animal studies in and were soon used as an oral vaccine to immunize humans against tuberculosis. in , the bcg vaccine, constituted by the live-attenuated m. bovis, was first used in newborns. it has become the most widely administered of all vaccines in the who expanded program for immunization, but has been estimated to prevent only % of all potentially vaccinepreventable deaths due to tuberculosis ( ) . despite its imperfections, bcg remains the only effective vaccination for protection against human tuberculosis ( ) . yellow fever is a highly fatal infection caused by a small, enveloped, single-stranded rna virus and results in renal, hepatic, and myocardial injury, along with hemorrhage and shock ( ) . unlike previously mentioned diseases, the history of yellow fever is highly uncertain and filled with misconceptions. early work on immunization against the disease began with carlos finlay in the s and s when koch's postulates were becoming increasingly accepted. finlay proposed that mosquitos carried the yellow fever "germ. " he attempted to prove it by feeding mosquitos that had fed on yellow fever patients. however, it was later revealed that his process failed due to the lack of an incubation period within the mosquito, which is a transmission requirement that finlay was unaware of ( ). since , significant advances have been made in creating a vaccine by the yellow fever commission, which was originally led by walter reed ( - ) along with jesse lazear, aristedes agramonte, and james carroll. reed's experiments took finlay's discovery one step further by adding an incubation period of approximately weeks and achieved the same positive results. when mosquitos bite non-immune individuals after feeding on individuals who had yellow fever, none of the non-immune subjects died and very few suffered disease. this led the commission of investigators to a major discovery, namely, the identification of the asibi strain, which is the parent strain of the present d vaccine, obtained via continuous indirect passage through the aegypti mosquitos and direct passage through monkeys. in addition to identifying the etiological agent of the disease, the commission also identified rhesus monkeys as susceptible hosts, hence providing a means for testing future vaccine attempts. this paved the way for max theiler and other rockefeller foundation scientists to develop a successful live-attenuated vaccine for yellow fever in . "the most important experimental passage seriesdesignated d -used a virus that had been subcultured eighteen times in whole mouse embryos, followed by passages in wholeminced chick embryo cultures, after which the virus was passed in minced chick embryo depleted of nervous tissue. " theiler himself was actually one of the first individuals to be successfully vaccinated. the vaccine was quickly implemented, and alternative vaccines shown to be more dangerous were discontinued ( ) . influenza has proved to be very difficult to trace back in history due to its non-specific symptoms and features. it was not until the early twentieth century that influenza outbreaks began to be systematically studied due to well-documented clinical descriptions and epidemiological data. in , the "spanish flu" influenza pandemic was responsible for - million deaths worldwide and more than one-half million in the u.s. this virus was unusual because it spread so quickly, was so deadly ( ) . richard e. shope ( - ) , a physician who conducted his research in the department of animal pathology at the rockefeller institute in princeton, was the first to isolate influenza virus; a member of the orthomyxovirus family, from a mammalian host in ( ) . he was able to induce the syndrome of swine influenza in pigs by applying respiratory secretions intranasally. he also isolated a bacterium from the respiratory tract of infected pigs called haemophilus influenzae suis. when this bacterium was combined with a filterable agent and inoculated, the pigs developed the clinical manifestations of swine influenza. these two agents seemed to act synergistically with the virus to damage the respiratory tract hence creating the suitable environment needed for the virus to exercise its pathological effects. in , scientists from the british national institute for medical research including christopher andrews, wilson smith, and patrick laidlaw successfully isolated and transmitted the influenza virus from humans. throughout this year, "burnet has successfully cultivated the organism in chick embryos; other influenza types had been recognized; neutralizing antibodies had been identified and quantitated; and viral surface glycoproteins, h and n had been described" ( ) . these discoveries led scientists to introduce the inactivated vaccine in the mid- s that is still used to this day ( ) . the influenza a/b vaccine was initially presented to the armed forces epidemiological board in . it was licensed following the war and used for civilians in in the u.s. starting , a series of vaccines were licensed for haemophilus influenza type b (hib) polysaccharide vaccines. these vaccines are recommended routinely for children at and months of age. the vaccine was, however, not consistently immunogenic in children < months of age. in , the protein-conjugated hib vaccine was licensed and in the next years, it became available. during , a combined vaccine hib conjugate and hepatitis b was licensed. later on, in , the first nasally administered influenza vaccine was licensed. this live influenza a and b virus vaccine was indicated for healthy, non-pregnant persons ages - years. the contracts to develop vaccine against the h n avian influenza virus were awarded to aventis pasteur and to chiron in . during the following year, an inactivated, injectable influenza vaccine was licensed. it was indicated for adults years of age and older. during the same year, the fda approved afluria, a new inactivated influenza vaccine, for use in people aged years and older. two years later in , the department of health and human services, supported the building of a facility to manufacture cellbased influenza vaccine. it also directed toward development of a vaccine for novel influenza a (h n ). during the same year, the fda approved four vaccines against the h n influenza virus high-dose inactivated influenza vaccine (fluzone high-dose) for people aged years and older. in , the fda approved several vaccines: hibmency a new combination of meningococcal and hib vaccine for infants; flucelvax, which is the first seasonal influenza vaccine, manufactured using cell culture technology and a quadrivalent formulation of fluarix ( ) . unfortunately, one of the difficulties in dealing with influenza is the continuous mutability of the viral genome necessitating annual reassessments and reformulations of the vaccine. this has led to a suboptimal effectiveness of influenza vaccines, which are only successful against strains included in the vaccine formulation or strains of homogenous subtype. several pandemics were caused by the influenza virus: during the years - , the "asian" influenza pandemic caused by h n influenza virus resulted in an estimated , deaths in the u.s. alone and in the years - , the "hong kong" influenza pandemic caused by an h n influenza virus induced roughly , deaths in the u.s. ( ) . future studies should focus on producing vaccines protective against variant strains and creating surveillance systems to detect novel strains in time to formulate the proper vaccines. poliomyelitis, or polio, is an intestinal infection spread between humans through the fecal-oral route. it is a disease of the developed nations striking younger individuals most frequently in warmer weather. one of the most famous polio victims, president franklin d. roosevelt, founded the national foundation for infantile paralysis in , later known as the march of dimes ( ) . it is well established that better hygiene decreases childhood exposure to the disease, when infection would usually be milder since protective maternal antibodies are present ( ) . in , the nobel prize in medicine was awarded to john enders, thomas weller, and fredrick robbins for their discovery of the ability of poliomyelitis viruses to grow in tissue cultures ( ) . two major lifelong competitors were involved in the race for the polio vaccine, jonas salk and albert sabin . salk took a more traditional route using a killed-virus approach, which did not involve natural infection in acquiring immunity. instead, his approach involved a fully inactivated virus that still had the ability to induce protective antibodies. sabin, on the other hand, set out to create a live-virus vaccine based on the belief that this would trigger natural immunity and provide a lasting protection. salk had speed, simplicity, and safety on his side since a killedvirus did not have the ability to revert to virulence, whereas the live-virus vaccine could be given orally, establish longer lasting immunity, and offer passive vaccination through the excreted weakened virus potentially immunizing a large portion of nonvaccinated communities ( ) . not surprisingly, salk's vaccine was the first to make it to the population. following successful clinical trials in , six companies began mass production of the vaccine. unfortunately, salk's vaccines were soon suspended and recalled when contaminated samples were found in the market due to poor monitoring and control in some laboratories leading to serious health consequences and national panic. the first cutter polio vaccine incident was reported on april , with more cases reported just a day later with the number eventually rising to of those vaccinated and in of their close contacts. on april , the laboratory of biologics control requested that cutter laboratories recall all vaccines and the company did so immediately. on may , the surgeon general recommended that all polio vaccinations be suspended pending inspection of each manufacturing facility and thorough review of the procedures for testing vaccine safety. the investigation found that live polio virus had survived in two batches of vaccines produced by cutter laboratories. large-scale polio vaccinations resumed in the fall of ( ) . at the same time, sabin had been making great advances with his live-virus vaccine since . after successful clinical trials conducted in the soviet union that left polio virtually wiped out with no safety issues, it soon became the vaccine of choice in the west. the polio vaccination assistance act was enacted by congress and was the first federal involvement in immunization activities. it allowed congress to appropriate funds to the communicable diseases center [later the centers for disease control and prevention (cdc)] to help states and local communities acquire and administer vaccines. at the beginning of the s, the oral polio vaccine types , , and as well as the trivalent product were licensed in the u.s. the first were developed by sabin and grown in monkey kidney cell culture, while the trivalent oral polio was developed to improve upon the killed salk vaccine ( ) . as a result, in the late s, the cdc recommended switching back to salk's killedvirus polio vaccine, while the who also advocated the switch for polio-free nations and the continued use of the favored live-virus vaccine for routine immunization ( ) . the last two cases of wild type polio were reported in an unvaccinated amish in and in a -year-old boy from peru in ( ) . in , the enhanced-potency inactivated poliovirus vaccine was licensed. following successful developments in the polio vaccine, attention soon shifted to three other common viral diseases of childhood: measles, mumps, and rubella. the measles virus is an rna virus from the genus morbillivirus belonging to the paramyxooviridae family. it causes an acute illness that includes fever, cough, malaise, coryza, and conjunctivitis, in addition to a maculopapular rash. in general, measles is a mild disease but, like many others, has the potential to cause serious complications. in addition, measles is known to be one of the most contagious human diseases causing major outbreaks to occur very often. until the year , measles was still the leading cause of vaccine-preventable childhood deaths worldwide ( ) . john enders ( enders ( - , known as the "father of modern vaccines" had a particular interest in revealing the virus responsible for measles. he isolated the edmonston strain of the virus in , which was named after the child from whom it was isolated. a formalin-inactivated measles virus vaccine derived from this strain was subsequently licensed in the u.s. in . however, following the discontinuation of this vaccine in due to short-lived and incomplete immunity, over further attenuated vaccines were developed and used throughout the world, most of which were also derived from the edmonston strain ( ) . the first live-virus measles vaccine, rubeovax, was licensed in . other live-attenuated virus measles vaccines were eventually licensed in the u.s. in . the recommended age for routine administration was changed from to months of age. the first national measles vaccine campaign was launched in . the world recorded a % decreased incidence compared to the pre-vaccination years. in , a second live, further attenuated measles virus vaccine was also licensed. in , both the advisory committee on immunization practices (acip) and the american academy of pediatrics (aap) issued recommendations for a routine second dose of the measles vaccine. during the midto-late s, a high proportion of reported measles cases were in school-aged children ( - years) who had been appropriately vaccinated. these vaccine failures led to new national recommendations of a second dose of measles-containing vaccine ( ) . mumps is another acute viral illness. it is the only virus known to cause epidemic parotiditis in humans accompanied by fever, anorexia, headache, and malaise. k. habel and john enders isolated the virus in ( ) , and trials of formalin-inactivated mumps vaccine in humans began the same year by joseph stokes and colleagues and by enders. this approach was abandoned in the s due to short-lived immunity, and work began to develop live-attenuated mumps vaccines in by the vaccinologist maurice hilleman ( hilleman ( - and colleagues ( ) . hilleman isolated the wild type virus from his daughter, jeryl lynn, who contracted the virus at the age of and was recovering from it. it became known as the jeryl lynn strain of mumps virus. the mumps livevirus vaccine was licensed in december , ( ) . trials with this attenuated virus resulted in % protective efficacy and the vaccine was licensed in the u.s. in . this strain is still used to produce mumps vaccines until this day. it is given as part of the measles, mumps, and rubella (mmr) vaccine ( ) . rubella is a rash disease in children and adolescents caused by a filterable virus. it poses a severe threat to pregnant women and their children by potentially causing congenital deafness and cataracts. in , a rubella epidemic swept the u.s. resulting in . million cases of rubella infection, with an estimated , newborns having congenital rubella syndrome (crs), along with fetal and neonatal deaths in the thousands ( ) . the rubella virus was detected and isolated by two groups of scientists, thomas weller and franklin neva at harvard medical school, in addition to paul parkman and colleagues at the walter reed army institute of research (wrair). similar to measles and mumps, inactivated whole virus vaccines proved ineffective, so efforts turned to discovering a live-attenuated vaccine ( ) . in , paul parkman left wrair and joined harry meyer jr. at the nih division of biological standards, and the pair developed the first live-attenuated rubella vaccine in , hpv- , which was subsequently included in the initial mmr vaccine used in the u.s. in the s ( ) . maurice hilleman discovered the superior ra / vaccine that became the only vaccine used outside of japan starting in the late s. this vaccine maintained its preference due to many factors including increased durability and harmlessness to fetuses of inadvertently vaccinated pregnant women ( ) . in , three rubella virus strains were licensed in the u.s.: hpv- strain grown in dog-kidney culture, hpv- grown in duck-embryo culture, and cendehill strain grown in rabbit-kidney culture. a decade later, in , the ra / (human diploid fibroblast) strain of rubella vaccine (meruvax ii) by merck was licensed. all other strains were discontinued. merck's combined trivalent mmr as well as the combined measles and rubella vaccine (m-rvax) developed by maurice hilleman and colleagues, was licensed by the u.s. government in ( ) , and is still in use today. moreover, the age for routine vaccination with mmr vaccine was changed from to months in the year of . the next vaccine that combined measles, mumps, rubella, and varicella antigens (proquad) was licensed in . it was indicated for use in children months to years. in response to the association of this vaccine with autism, in , the eighth and final report of the immunization safety review committee was issued by the institute of medicine concluded that the body of epidemiological evidence favors rejection of a causal relationship between the mmr vaccine and autism ( ) . combination vaccines hold many advantages including reduced need for several injections, therefore, reducing the incidence of vaccination site reaction ( ) . the etiological agent of clinical hepatitis, identified by its distinguishing yellow jaundice, was found to be infectious in the early s. the different hepatitis strains, a and b, were first differentiated in ( ) . in the mid- s, blumberg and coworkers and prince discovered hepatitis b surface antigen in the circulating blood of carriers of the infection. deinhardt et al. soon followed this discovery with that of the hepatitis a virus ( ) . provost et al. successfully prepared a killed hepatitis a vaccine in , which proved to be safe and highly effective in extensive clinical trials. the first inactivated hepatitis a vaccine (havrix) was licensed in . the following year, a second inactivated vaccine (vaqta) also became available ( ) . hepatitis b, on the other hand, rarely causes any severe risk as a primary infection. however, those who develop a chronic persistent infection may continue to have severe disease for the rest of their lives. this may even lead to cirrhotic destruction of the liver due to host immune response to the virus. the discovery of the surface antigen particles of the hepatitis b virus by blumberg and colleagues in the plasma of human carriers was followed by attempts to create a vaccine. in , a killed hepatitis b vaccine was developed and clinical trials began in proving the safety and efficacy of the vaccine. merck and pasteur institute subsequently independently licensed the plasma-derived vaccine in ( ). on july rd , the recombinant hepatitis b vaccine (recombivax hb) was licensed. using recombinant dna technology, merck scientists developed a hepatitis b surface antigen subunit vaccine. three years later, on august th , the recombinant hepatitis b vaccine (engerix-b) was licensed. a decade later in , the fda approved a two-dose schedule of hepatitis b vaccination for adolescents - years of age using recombivax hb (by merck) with the -μg (adult) dose at and - months later. at the beginning of the new millennium, in , a combined hepatitis a inactivated and hepatitis b (recombinant) vaccine, twinrix was licensed. the following year, a vaccine combining diphtheria, tetanus, acellular pertussis, inactivated polio, and hepatitis b antigens (pediarix) was licensed ( ) . in conclusion, fortunately, both hepatitis a and b are now preventable due to the discovery of these highly effective vaccines that proved to maintain long-term immunity in vaccinated individuals ( ) . in , the world health assembly called for global smallpox eradication, which was launched the following year. during the first year of the program , cases of polio were reported in countries that were endemic to smallpox. four years later, the cdc recommended discontinuation of routine vaccination for smallpox in the u.s. following a greatly reduced risk of disease ( ) . during the s, especially in , the expanded program on immunization was created within who, in response to poor immunization levels in developing countries (< % of children in ). the following vaccines were used by the expanded program on immunization: bcg, polio, dtp, measles (often mmr vaccine), yellow fever (in endemic countries), and hepatitis b. three years later, in october , the last case of naturally acquired smallpox occurred in the merca district of somalia. in the same year, the first pneumococcal vaccine was licensed, containing serotypes (of the known serological groups) that composed % of all bacteremic pneumococcal infections in the u.s. ( ) . on may , the world health assembly declared the world free of naturally occurring smallpox. on the other hand, in july , two enhanced pneumococcal polysaccharide vaccines (pneumovax and pnu-imune ) were certified. these vaccines included purified capsular polysaccharide antigens of streptococcus pneumoniae and replaced the -valent polysaccharide vaccine licensed in . a few years later, in , the world health assembly passed a resolution to eradicate polio by the year ( ) . later on, in , the japanese encephalitis (je) inactivated virus vaccine (je-vax) was licensed. during the year , the expanded program for vaccine development and the vaccine supply and quality program were merged creating the global program for vaccines and immunization. during the same year, the western hemisphere was finally labeled as "polio-free" by the international commission for the certification of polio-eradication. the was another monumental year with the launching of the international aids vaccine initiative (iavi) that called for the speedy development of a human immunodeficiency virus (hiv) vaccine for use worldwide. this in turn led to the introduction of the scientific blueprint for aids vaccine development. iavi was funded by several ngos and foundations. it is a collaborating center of the joint united nations program on hiv/aids (unaids) whose efforts led finally lead to the first possible vaccine against hiv (aidsvax) which reached phase iii trials, the largest recorded human hiv vaccine trial at that time. the trial involved volunteers from the u.s., canada, and the netherlands, the majority of whom were men who have sex with men ( ) . preliminary results from the trial of aids vax (vaxgen) vaccine were reported in early . while the vaccine was shown to be protective amongst non-caucasian populations, especially african-americans, the same effect was not reproducible in caucasians ( ) . during the same year, the children's vaccine program was established at who's program for appropriate technology in health (path). the program's goal was to provide vaccines to children in the developing world and to accelerate research and development of new vaccines. the first vaccines purchased were hib, hepatitis b, rotavirus, and pneumococcal, which were not commonly used in the developing world ( ) . at the beginning of the new millennium, the western pacific region of the world was certified as polio-free. during the next years, the european region also became certified as polio-free. in , the fda licensed the first vaccine developed to prevent cervical cancer (gardesil), precancerous genital lesions and genital warts due to human papillomavirus (hpv) types , , , and . the first smallpox vaccine for certain immune-compromised populations was delivered under project bioshield on july th . the following year , the who declared the "decade of vaccines" and in , the united nations foundation launched shot@life campaign ( ) . varicella ("chickenpox") is caused by the varicella zoster virus (vzv). michiaki takahashi, professor of virology at the research institute for microbial diseases at osaka university, successfully produced the oka vaccine strain of live, attenuated varicella vaccine in the s. takahashi was able to make this remarkable advance at a time when very few viruses had been attenuated to produce efficacious live-virus vaccines including yellow fever, polio, measles, mumps, and rubella as previously mentioned. the vzv vaccine is the first and only licensed live, attenuated herpesvirus vaccine in the world. numerous trials in the early s continued to prove the safety and efficacy of the vaccine in both healthy and immunocompromised, high-risk individuals. as a result of these successful trials, the live varicella virus vaccine (varivax) was licensed in for the active immunization of persons months of age and older ( ) . about years later, in , varizig, a new immune globulin product for post-exposure prophylaxis of varicella, became available under an investigational new drug application expanded access protocol ( ) . as a herpesvirus, vzv possesses the unique ability to establish latent infection subsequent to primary infection. zoster results from reactivation of latent vzv that spreads through nerves to the skin. therefore, one fear associated with this vaccination was the possibility that it could increase the incidence and/or severity of zoster when compared to natural disease. conversely, it was actually shown that following vaccination, zoster is less common than after natural infection ( ) . in , the fda licensed a new vaccine to reduce the risk of shingles in the elderly. the vaccine, zostavax was approved for use in people aged years of age and older ( ) . rotavirus is the leading cause of severe diarrhea and vomiting (severe acute gastroenteritis) among young infants and children worldwide. no significant difference was found in the incidence of rotavirus in industrialized and developing countries, suggesting that vaccination may be the only way to control the impact of this severe disease. dr. ruth bishop and colleagues were the first to describe rotavirus in humans in . it was clear, early on, that a naturally acquired first infection, whether symptomatic or asymptomatic, was the most effective defense against severe reinfection, and subsequent infections progressively created greater protection. therefore, the goal was to create a vaccination that mimicked the effectiveness of naturally acquired immunity following infection. the development of live, attenuated, oral, safe, and effective rotavirus vaccines was then attempted starting in the mid- s. dr. albert kapikian and colleagues, at the nih, developed the rrv strain that was subsequently used to develop the rrv-tv, or the rotashield, live oral, and tetravalent vaccine licensed in to be used in infants at , , and months of age ( ) . however, due to several reported cases of vaccine-associated intestinal intussusception, rotashield was pulled off the market in the u.s. months after its introduction on the th of october, . in , the national institute of allergy and infectious diseases (niaid), part of the nih, awarded a new license agreement for rotashield to biovirx, inc. of minneapolis, mn, usa, which planned its global commercialization. in , history of intussusception was added as a contraindication for rotavirus vaccination ( ) . clark, offit, and plotkin then produced the rotateq vaccine by merck based on their bovine strain wc in , which was licensed in by the u.s. fda. this vaccine, live oral and pentavalent, is destined for use in infants ages - weeks ( ) . another vaccine, rotarix, was also licensed in . it is a liquid given in a two-dose series to infants from to weeks of age. before being licensed, both vaccines were shown to be safe and effective in rigorous clinical trials ( ) . during the past two decades, improvements in environmental health have contributed tremendously to disease vector control. however, substantial challenges remain in dealing with the newly emerging diseases such as severe acute respiratory syndrome (sars), h n , h n , and h n influenza, middle east respiratory syndrome (mers-cov), rotavirus, ebola virus, and a variety of other viral, bacterial, and protozoal diseases ( ) . the role of vaccines in the control and protection from the above mentioned emerging diseases cannot be overemphasized. actually, the importance of inducing protective immunity through vaccination came out to be the most powerful tool and effective strategy to prevent the spread of emerging viruses among populations, in particular, among people that are immunologically naïve and susceptible hosts. such emerging diseases represent a major public health concern; they affect livestock and humans thus threatening the world's economy and public health. vaccine strategies for emerging pathologies are limited by sudden appearance of the pathogen and the delayed time consuming traditional vaccine development process. novel methods to rapidly develop vaccine are being experimented, whereby investigators are working to achieve a better understanding of the nature of the interactions between the immune system and a panel of novel harmful microbes. on this basis several novel strategies have been developed and applied. such strategies included the use of ( ) recombinant proteins, or nanoparticles like in sars-cov and mers-cov, ( ) synthetic peptides like the case of influenza virus, in managing or even in preventing the emergence of new infectious diseases, a plan should be developed to strengthen surveillance and promote a multi-partners response within local, national, and global programs. with the high burden of emerging infectious diseases (eid) it becomes an essential part to find an effective method of either preventing or controlling their spread, that is where the role of vaccines prevails. it is significant to mention that the average case fatality rate for ebola is around % and outbreaks are affecting both developed and developing countries. another emerging disease, mers-cov, has caused the death of around % of people reported to have contracted the disease. another disease with high health and economic burden would be rotavirus which was estimated to have annual direct and indirect costs of around $ billion with "more than , physician visits, more than , emergency department (ed) visits, , to , hospitalizations, and to deaths each year in children younger than years" (cdc, ) . these are few of the facts regarding the affliction of eid most of which have no approved vaccine yet. on the other hand, the influenza virus which was estimated to cause an average of , deaths annually with a $ billion cost of an epidemic, showed that with the introduction of its vaccine, studies proved it to be % effective in preventing death ( ) . these figures have managed to influence many governmental and non-profit organizations to intervene either through governmental funding of vaccines where the congress provides yearly international eid funding to several u.s. governmental agencies or through international non-profit organizations which are the leaders in global health innovation ( ). vaccines remain among the most reliable and effective medical interventions in providing the means to fight debilitating and preventable diseases thus ensuring the continuity of mankind and saving lives. through reviewing the factsheets provided by the world health organization, which provide statistical data on the mortality and morbidity percentages before and after the introduction of the vaccines, one can comprehend the vital role vaccines have played up till this day. some of the figures that depict the impact of vaccines in decreasing mortality and morbidity include more than % decrease in polio cases since , with cases reaching , from over endemic countries down to cases as reported in with only endemic countries, measles vaccine has prevented the death of around . million children during - , in general vaccines prevent around million deaths annually worldwide ( ) . this success in providing better public health does not negate the economic burden of vaccination. vaccination programs require excessive funding to ensure proper handling and maintenance of vaccines, adequate staffing and ongoing provision over efficacy and safety of vaccines and the development of newer vaccines ( ) . nevertheless, the economic and social burden related to the expenses in hospitalizing affected unvaccinated people still outweighs the aforementioned burden. moreover, better health in the society would promote economic growth and productivity. consequently, public awareness and public efforts agree on the importance of vaccination and the implementation of policies regarding mandatory vaccinations as a way to decrease outbreaks of preventable diseases and improve global health and prosperity. as early as the introduction of vaccines, campaigns against vaccination were raging. as with any new medical intervention there are safety concerns that arise which might be deleterious to the public health. concerns regarding vaccines often follow a path that starts with the hypothesis of a potential adverse event that is impulsively announced to the public without having reproducible studies to confirm this hypothesis, and thus it would take the public several years to regain trust in the vaccine. a notable example in the recent history would be of the paper published by andrew wakefield in the british medical journal the lancet in , linking the mmr vaccine to autism. however, his research was discredited and the paper was retracted from the journal after it was proven that actually there is no link between mmr vaccine and autism as per the systemic review by the cochrane library ( ) . the battle against vaccines did not reach a halt, and there are still ongoing campaigns that come from religious, political, community-based, and even individual-based grounds raising even ethical issues regarding the mandatory vaccinations proposed by the government. according to the cdc, this year % of the children were vaccinated in the u.s., leaving % unvaccinated due to religious and philosophical exemptions or even parental refusal due to the fear of vaccine's side effects and concerns regarding autism from vaccines ( ); this is still a critical number since the unvaccinated would pose risk of outbreaks even among the immunized, which necessitates the need for additional awareness campaigns regarding the importance of vaccination since vaccines remain the only plausible measure of protection against preventable diseases. actually, a trend was reported in the health news lately, in the u.s., that pediatricians refuse to offer medical care for children whose parents declined their vaccination. vaccination has been of great importance throughout centuries (tables and ). people started with inoculation techniques dating back to a.d. with the chinese, turks, and asians. with every century and with every curious physician, inoculation techniques improved gradually giving rise to newer vaccination techniques with edward jenner and later on, louis pasteur and others. however, there is still plenty of room for improvement with the presence of ongoing epidemics and the spread of newly emerging diseases. one important goal is to strengthen the science base for vaccine development and for public health action and disease prevention. despite the common belief that infectious diseases were virtually eliminated by the middle of the twentieth century, new and reemerging infections are appearing along with drug resistant infections in the past two decades in the various parts of the world and whose incidence threatens to increase in the near future, due to changes in human demographics and behavior, immigration, and speed of international travel among other things ( ) ( ) ( ) . the importance of vaccine safety continued to grow throughout the twenty-first century, with the development and licensure of new vaccines added to the already robust immunization armamentarium. scientists also perfected new ways of administering immunizations including edible vaccines and needleless injections. however, formulated or delivered, vaccines will remain the most effective tool we possess for preventing disease and improving public health in the future. despite the antivaccination campaign and the association of vaccines with some side effects, vaccines continue to remain a cornerstone in global health. the distinctions between national and international health problems are losing ground and could be misleading, the "world is a village. " clinicians and public health workers need to interact on regular basis with veterinarians and veterinary public health. actually, good examples of the necessity of such collaboration is the emergence of sars-cov and mers-cov, it shows clearly how coronaviruses can spillover from animals into humans at any time, causing lethal diseases. foodborne diseases could lead to regional and international outbreaks which might constitute a threat to national and global security. centers for disease control and prevention. vaccines and immunizations: the basics epidemiology of vpds, vaccine safety the complicated task of monitoring vaccine safety vaccine safety: current and future challenges niaid. topics: vaccines the college of physicians of philadelphia. articles -different types of vaccines general aspects of vaccination the immune response to bcg vaccination of newborns the methodology for determining the efficacy of antibodymediated immunity aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen presenting cell type twin studies of immunogenicity-determining the genetic contribution to vaccine failure biology of immune responses to vaccines in elderly persons the influence of genetic factors on the immune response as judged by pneumococcal vaccination of mono-and dizygotic caucasian twins genetic regulation of immune responses to vaccines in early life vaccine epidemiology: efficacy, effectiveness, and the translational research roadmap the aging innate immune system reduction in acute gastroenteritis hospitalizations among us children after introduction of rotavirus vaccine: analysis of hospital discharge data from us states resolving the pneumococcal vaccine controversy: are there alternatives to randomized clinical trials? evaluating new vaccines for developing countries. efficacy or effectiveness? assessing vaccine efficacy in the field. further observations translational research and pediatrics the evolution of surgical instruments the history of pediatric infectious diseases the college of physicians of philadelphia. history of vaccines timelines. the college of physicians of philadelphia ( ) surgeons, smallpox, and the poor: a history of medicine and social conditions in nova scotia the immunization action coalition. vaccine timeline. the immunization action coalition jenner and the history of smallpox and vaccination experience in massachusetts and a few other places with smallpox and vaccination the medical side of benjamin franklin history of vaccine development (smallpox eradication: the vindication of jenner's prophesy chapter history of vaccine development. (pasteur and the birth of vaccines made in the laboratory chapter the history of anthrax vaccines: a biography (rabies) vaccines: a biography (killed vaccines: cholera, typhoid, and plague) vaccines: a biography (toxoid vaccines) historical review of pertussis and the classical vaccine preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccines recommendations of the advisory committee on immunization practices (acip) a brief history of vaccines and vaccination vaccines: a biography (tuberculosis and bcg chapter the history of tuberculosis yellow fever: a disease that has yet to be conquered vaccines: a biography (yellow fever chapter a history of influenza vaccines: a biography (influenza chapter vaccines: a biography (polio chapter a brief history of polio vaccines vaccines: a biography (measles, mumps, and rubella chapter the history of vaccines and immunization: familiar patterns, new challenges vaccines in historic evolution and perspective: a narrative of vaccine discoveries history of vaccine development (three decades of hepatitis vaccinology in historic perspective. a paradigm of successful pursuits chapter vaccines: a biography (varicella and zoster chapter vaccines: a biography (rotavirus chapter pandemic preparedness and response -lessons from the h n influenza of emerging infectious diseases: a cdc perspective vaccination greatly reduces disease, disability, death and inequity worldwide historical comparisons of morbidity and mortality for vaccinepreventable diseases in the united states vaccines for measles, mumps and rubella in children vaccination coverage among children in kindergarten -united states historic dates and events related to vaccines and immunization ih is the first author. she provided the idea and followed along with aj the execution of the work and final editing. nc and sc did the literature search for the eighteenth and nineteenth century and the respective preliminary writing about this period. ss and rb did the literature search for the twenty-first century, and about general aspects of vaccination and the respective preliminary writing about this period.mr did the literature search regarding vaccine efficacy and effectiveness in the context of the transnational research map and regarding vaccination instruments and inoculating techniques. ag did the literature search and the preliminary writing for the early history of vaccination and wrote a draft of a manuscript about this period. al edited thoroughly and commented on the final manuscript. aj supervised the whole process from inception to the final submission and edited the whole manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -azkamnpa authors: ecker, david j; sampath, rangarajan; willett, paul; wyatt, jacqueline r; samant, vivek; massire, christian; hall, thomas a; hari, kumar; mcneil, john a; büchen-osmond, cornelia; budowle, bruce title: the microbial rosetta stone database: a compilation of global and emerging infectious microorganisms and bioterrorist threat agents date: - - journal: bmc microbiol doi: . / - - - sha: doc_id: cord_uid: azkamnpa background: thousands of different microorganisms affect the health, safety, and economic stability of populations. many different medical and governmental organizations have created lists of the pathogenic microorganisms relevant to their missions; however, the nomenclature for biological agents on these lists and pathogens described in the literature is inexact. this ambiguity can be a significant block to effective communication among the diverse communities that must deal with epidemics or bioterrorist attacks. results: we have developed a database known as the microbial rosetta stone. the database relates microorganism names, taxonomic classifications, diseases, specific detection and treatment protocols, and relevant literature. the database structure facilitates linkage to public genomic databases. this paper focuses on the information in the database for pathogens that impact global public health, emerging infectious organisms, and bioterrorist threat agents. conclusion: the microbial rosetta stone is available at . the database provides public access to up-to-date taxonomic classifications of organisms that cause human diseases, improves the consistency of nomenclature in disease reporting, and provides useful links between different public genomic and public health databases. thousands of different microorganisms affect the health and safety of the world's populations of humans, animals, and plants. infectious microorganisms include species of bacteria, viruses, fungi, and protozoa. many different medical and governmental organizations have created lists of the pathogenic microorganisms most relevant to their missions. for example, the centers for disease control and prevention (cdc) maintains an ever-changing list of notifiable diseases, the national institute of allergy and infectious disease (niaid) lists agents with potential for use in bioterrorist attacks, and the department of health and human services (hhs) maintains a list of critical human pathogens. unfortunately, the nomenclature for biological agents on these lists and pathogens described in the literature is imprecise. organisms are often referred to using common names, alternative spellings, or out-dated or alternative names. sometimes a disease rather than a particular organism is mentioned, and often there may be multiple organisms or co-infections capable of causing a particular disease. not surprisingly, this ambiguity poses a significant hurdle to communication among the diverse communities that must deal with epidemics or bioterrorist attacks. to facilitate comprehensive access to information on disease-causing organisms and toxins, we have developed a database known as "the microbial rosetta stone" that uses a new data model and novel computational tools to manage microbiological data [ , ] . this article focuses on the information in the database for pathogens that impact global public health, emerging infectious organisms, and bioterrorist threat agents. it provides a compilation of lists, taken from the database, of important and/or regulated biological agents from a number of agencies including hhs, the united states department of agriculture (usda), the cdc, the world health organization (who), the niaid, and other sources. we curated these lists to include organism names that are consistent with the national center for biotechnology information (ncbi) nomenclature and to provide sequence accession numbers for genomic sequencing projects (if available). important synonyms or previously used names that identify the organisms are also shown. we have organized the lists according to phylogenetic structure. this paper provides graphic representations of the phylogenetic relatedness of important pathogenic organisms. the goal of the database is to provide an informative, readily accessible, single location for basic information on a broad range of important disease causing agents. the database will help users to avoid the pitfalls of confusing nomenclature and taxonomic relationships and allow access to literature on in-depth studies. the database can be accessed at http://www.microbialrosettastone.com/. the microbial rosetta stone database was designed to link genomic sequence data, taxonomic classification, and epidemiological information in a way that facilitates searches and data updates. the intent is not to replace currently available tools, like those available from ncbi, but instead serve to resolve ambiguities and data conflicts. one of the main goals of building this informatics infrastructure was to support the rapid determination of the causative agent in the event of a bioterrorist action or emergence of a previously unknown infectious disease. the organisms listed in the additional files are represented according to phylogenetic hierarchy in the figures. the symbols used in the figures connect the organism names back to one or more of the additional files, which allows visualization of the fact that organisms are often represented in multiple additional files. figure is the symbols key. figures and show the phylogenetic relationships of cellular life forms including bacteria, fungi, protozoa, and the plants and animals that produce toxins, figure shows the phylogenetic relationships of dna viruses, and figures , , and show the phylogenetic relationships of rna viruses. for optimal utility, the genomic sequence data, taxonomic, and epidemiological information were linked in a way that facilitates searches, data updates, and data curation. the microbial rosetta stone system will serve as a translator to resolve ambiguities and data conflicts between other public databases and literature and, when unable to resolve ambiguities, provide enough context for users understand the nature of the ambiguities. the database provides links to literature on experimental protocols for pathogen identification and to the genomic and biochemical information specific to a species, strain, or isolate. the data model and query tools are described in detail in hari et al. [ ] . the following sections describe the information and literature sources used to populate in the database for globally important and emerging pathogens and potential bioterrorist agents. in the developing world, nearly % of infectious disease deaths are due to six diseases or disease processes: acute respiratory infections, diarrhea, tuberculosis, hiv, measles, and malaria [see additional file ] [ ] . in both developing and developed nations, the leading cause of death by a wide margin is acute respiratory disease [ ] [ ] [ ] . in the developing world, acute respiratory infections are attributed primarily to six bacteria: bordetella pertussis, streptococcus pneumonia, haemophilus influenzae, staphylococcus aureus, mycoplasma pneumonia, chlamydophila pneumonia, and chlamydia trachomatis [ ] . these bacteria belong to four different taxonomic classes and illustrate how similar parasitic lifestyles can evolve in parallel within unrelated bacterial species (figure ) . major viral causes of respiratory infections include respiratory syncytial virus ( figure ), human parainfluenza viruses and ( figure ), influenza viruses a and b ( figure ), as well as some adenoviruses ( figure ) [ , ] . the major causes of diarrhoeal disease in the developing and developed world have significant differences due to the great disparity of availability of pure food and water and the general nutritional and health status of the populations. important causes of diarrhoeal disease in the developing world are those that tend to be epidemic, especially vibrio cholera, shigella dysenteriae, and salmonella typhi [ ] . these organisms are gammaproteobacteria (figure ) that use many different metabolic pathways to ensure their survival in a wide range of environments. in the united states there is a much lower incidence of diarrhoeal disease overall, and a relatively greater impact of direct human-to-human infectious transmission. the most important causes of diarrhoeal disease in the united states are bacteria such as escherichia coli, campylobacter species, clostridium difficile, listeria monocytogenes, salmonella enteritidis, and shigella species ( figure ) ; viruses, such as norwalk virus ( figure ) and rotaviruses ( figure ) ; and parasites such as cryptosporidium parvum, cyclospora cayetanensis, entamoeba histolytica, giardia lamblia, while microsporidia are responsible for a smaller number of cases ( figure ). infectious disease agents important to the public health in the u.s. are monitored by the cdc and listed in additional file [see additional file ] . there are no set criteria for inclusion on the notifiable disease list; rather, the list is created by the cdc in cooperation with state health departments. as diseases occur less frequently and new diseases emerge, the notifiable disease list changes. the list provides links to case definitions of each disease, including the etiological agent(s) responsible. in cases where the etiological agent was not listed or was symbol key figure symbol key. these symbols are used in the figures through to link pathogen names to their originating additional files. figure bacterial pathogens. bacterial phylogeny is based on work by hugenholtz et al. [ ] . bacterial phyla are shown on the leaves, with the firmicutes and the proteobacteria subdivided to the class level (taxa are shown in italics). their phylogeny is further broken down to the order level with the use of smaller branches. these two phyla account for roughly three quarters of the described infectious bacterial species (with the noticeable exception of deltaproteobacteria pathogens). proteobacteria contain both important plant and animal pathogens. proteobacterial plant pathogens are not clustered, but are observed in alpha, beta, and gamma subdivisions. some plant and animal pathogen species share the same genus classification (e.g. ralstonia and pseudomonas), and for at least one species, burkholderia cepacia, different subspecies are plant and human pathogens. of the bacterial phyla currently recognized in the ncbi taxonomy, thirteen are not present in figure due to the absence of noteworthy pathogens. some of these missing phyla are relatively important, like the cyanobacteria, but most of the remaining phyla are restricted to a handful of species and/or environmental niches. also of interest is the relative weight of the infectious agents within their respective phylum: while virtually all spirochaetes and chlamydiae constitute potential infectious agents due to their parasitic lifestyle, the phylum actinobacteria has few pathogens relative to the overall diversity of the phylum. it should be cautioned that our current view of bacterial diversity is biased towards cultivatable organisms, which represent a small fraction of bacterial diversity [ ] . therefore, this compilation represents the well-recognized infectious bacterial agents, but should in no way be considered exhaustive or complete. unspecific (i.e. brucella spp.), further research was done to determine an etiological agent and this information is in additional file [see additional file ] . each year in the united states, there are approximately million cases of food-borne illness, including , hospitalizations and , deaths [ , ] . in an estimated to % of these cases, chronic sequelae develop. these sequelae include renal disease, cardiovascular diseases, gastrointestinal disorders, neural disorders, and autoim-mune disease [ ] . the estimated cost of food-borne illness in the united states is $ billion annually [ ] . mishandling of food is believed to be responsible for % of all outbreaks of food-borne disease in developed nations, primarily due to a lack of education [ , [ ] [ ] [ ] . food-borne pathogens [see additional file ] are also important because they represent one of the largest sources of emerging antibiotic-resistant pathogens. this is due in part to the administration of sub-therapeutic doses of antibiotics to food-producing animals to enhance growth. for example, certain strains of salmonella show eukaryotic pathogens figure eukaryotic pathogens. eukaryotic pathogen life forms are clearly dominated by the fungi and protists. within the fungi, the phylum ascomycota has many human, animal, and plant pathogens and is a major source of toxins. protist species are responsible for globally important diseases such as the malaria-causing plasmodium species and the leishmania and trypanosoma species that cause significant mortality in the developing world. the eukaryotic phylogeny tree is based on ribosomal and housekeeping gene sequence analysis [ ] , but different taxonomy levels are simultaneously represented as the topology has not yet been reliably determined. resistance to eight or more antibiotics [ ] . studies have shown that antibiotic resistance in salmonella cannot be traced to antibiotic use in humans, suggesting that antibiotic use in animals is the primary cause of resistance [ ] . while much is known about the major microbes responsible for diseases, there are still many undiagnosed cases of infectious disease. it has been estimated that as many as three-fifths of the deaths from acute gastroenteritis per year in the united states are caused by an infectious organism of unknown etiology [ ] . four of the major causes of food-borne infections (campylobacter jejuni, escherichia coli o :h , listeria monocytogenes, and cyclospora cayetanensis, figure ) were only recently recognized as causes of food-borne illness [ ] . zoonoses are a special class of pathogens as they co-evolve with the reservoir host, not with humans. although not always the case, when zoonotic pathogens infect humans they have a tendency to cause severe disease, much like a commensal microorganism that infects unusual areas of the body [ ] . zoonoses can be broken down into two basic groups: those spread by direct contact with the infected animal and those spread via an intermediate vector [ ] . zoonoses can infect humans through many vectors. members of the genus hantavirus are spread by rats [ ] , west nile virus by mosquitoes [ ] , and campylobacter species by family pets [ ] . in , there was an outbreak of monkeypox (figure ) in the midwestern united states, which was attributed to the importation of pet animals [ ] . viruses, protozoa, and bacteria can all be transmitted either directly or via a vector. however, these groups are clearly differentiated on a phylogenetic level. [ ] . of all the agents infectious to humans, a majority are zoonotic in origin [ ] . several viruses responsible for human epidemics have made a transition from animal host to human host and are now transmitted from human to human. human immunodeficiency virus (figure ) , responsible for the aids epidemic, is one example [ ] . although it has yet to be proven, it is suspected that severe acute respiratory syndrome (sars), caused by the sars coronavirus ( figure ) , also resulted from a species jump [ ] . for many years, robert g. webster has studied the importance of influenza viruses in wild birds as a major reservoir of influenza viruses and has clarified their role in the evolution of pandemic strains that infect humans and lower animals [ ] . intriguingly, it appears that whenever a virus (or any other pathogen or pest) is eradicated another appears to fill the environmental niche. for example, in regions where the wild-type poliovirus ( figure ) has been eliminated, nonpolio enteroviruses have been associated with outbreaks single-stranded positive strand rna viruses figure single-stranded positive strand rna viruses. the togaviridae and flaviviridae, and to a lesser extent the coronaviridae and picornaviridae, families are the most prominent human pathogens. important plant viruses affect major cereal grains causing severe crop damages worldwide and affecting the nutrition of entire populations. of paralytic disease that clinically exhibit symptoms of poliomyelitis. the most common non-polio enteroviral causes of acute flaccid paralysis (afp) are human coxsackieviruses a and a and human enterovirus (figure ) . the poliovirus epidemics that emerged in the mid th century are now believed to have arisen not due to a change in the properties of the virus, but due to an improvement of public sanitation and personal hygiene, which delayed the acquisition of enteric virus infection from infancy to childhood. since maternal antibodies protected infants, they usually underwent silent immunizing infections, whereas when older children were infected they more often suffered paralytic poliomyelitis [ ] . proteobacteria are important plant and animal pathogens, and it is interesting that proteobacterial plant pathogens are not clustered on the phylogenetic tree, but are observed in alpha, beta, and gamma subdivisions ( figure ). some plant and animal pathogen species share the same genus classification (e.g. ralstonia and pseudomonas), and for at least one species, burkholderia cepacia, different subspecies are plant and human pathogens. in viruses, plant and animal pathogens are generally found in distinct families, with the notable exception of the families rhabdoviridae ( figure ) and reoviridae (figure ) that harbor both. most plant-specific virus families are not shown in figures , , , , but for rice alone at least ten unrelated virus species have been reported to infect cultures worldwide. the overall economical effect of plant pathogens has been estimated to reduce crop production by one fifth [ ] . foot-and-mouth disease almost destroyed the beef industry in the united kingdom and losses were very high. bird flu in south east asia affected the poultry industry severely. influenza a is a bird virus that jumped from birds via pigs to humans and as a human pathogen has a large impact on the economy [ ] . bioterrorism can be defined as an attack or threat of an attack using bioweapons on humans and/or their assets to create fear, to intimidate, to inflict harm, and/or affect economic well being. such acts may be motivated by political, religious, ecological, or other ideological objectives. any microorganism or toxin capable of causing disease or harm in humans, plants, or animals has the potential for illicit use and thus the list of potential agents could be vast. however, not all organisms or toxins make useful biological weapons. displaying biological weapons from a phylogenetic perspective provides insight into how organisms that have been historically used as biowarfare agents are related to important human and agricultural pathogens and also provides insight into other similar organisms that might be considered as weapons in the future. the properties that make organisms amenable for use as biological weapons have been discussed extensively [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the most important features include: ) accessibility; ) consistent ability to cause death or disability; ) culturability; ) possibility for large scale production; ) stability and ability to retain potency during transport and storage; ) delivery potential; ) stability and retention of potency after dissemination; and ) infectivity and toxicity [ ] . the infectious agents considered by the niaid to have high potential for bioterror use are listed in additional file [see additional file ] . validated and potential bioweapons agents are listed in additional file [see additional file ] . agents that have been used to commit biocrimes [ ] are in additional file [see additional file ]. the pathogens regulated by the hhs and the usda are shown in additional file [see additional file ] and additional file [see additional file ] , respectively. in addition to infectious microbes, toxins derived from biological sources are contained on these lists. we identified the biological source of each toxin, and listed the source organism in the additional file containing the toxin. all regulated toxins can be found on the hhs select agent list [see additional file ] and five are also regulated by the usda [see additional file ] . the regulated toxins (four small molecules and seven peptides or proteins) are produced by a wide variety of organisms: five are pro-duced by bacteria [ ] [ ] [ ] , two by fungi [ , ] , two by plants [ , ] , one by an animal [ ] , and one by a protist [ ] . the bacteria that produce toxins are indicated in figure and the eukaryotic toxin producers are shown in figure . the conotoxins are a collection of five families of related peptide toxins produced by the various species of snails in the genus conus [ ] . the two fungal toxins, diacetoxyscirpenol and t- toxin, are produced by multiple species of the genus fusarium [ , ] . of the five regulated bacterial toxins, four are peptides or proteins and tetrodotoxin [ ] is a small molecule. tetrodotoxin is produced by a variety of gammaproteobacteria that colonize the puffer fish [ ] . these toxins are relevant both due to concern that they may cause outbreaks of disease through accidental food contamination and because they are potential bioweapons. according to the cdc [ ] , an average of cases of botulism, caused by a bacterial toxin (figure ), are reported each year. interestingly, numbers of cases of food-borne and infant botulism have not changed significantly in recent years, but instances of wound-botulism have increased primarily due to the use of black-tar heroin, especially in california. ricin is a toxin produced by the plant ricinus communis (figure ) [ ] . when prepared as a weapon, there are three methods of delivery: ingestion, injection, or inhalation [ ] . one of the most famous documented uses of ricin was in the assassination of georgi markov, a bulgarian defector who died three days after being shot with a ricin pellet [ , ] . pathogens that can be genetically manipulated [see additional file ] represent a unique bioterrorist threat, particularly as technology to genetically alter microbes becomes more commonplace. for the purposes of this work, viruses considered as having a high potential danger for bioterrorist engineering were derived from the literature reports and from the authors' judgment balancing the considerations of terror potential with the technical difficulty of use. dna viruses (figure ) with large genomes can be manipulated in culture, and are susceptible to engineering events such as insertion of genes not normally present in viral genomes. for example, the entire orthopoxvirus genus of the poxvirus family is considered to have high potential for bioengineering because of a much-publicized paper that describes the engineering of an orthopox member, ectromelia virus, to express mouse interleukin- , resulting in a virus with extraordinary virulence [ ] . another member of the orthopoxvirus genus, vaccinia virus, is commonly used in molecular biology research and is extensively used in vaccines and vaccine research [ ] [ ] [ ] [ ] . thus, because they are highly infectious, will tolerate inserted genes, and can be manipulated easily in culture, poxviruses must be considered high-risk agents for acts of bioengineering. mutations to create vaccine-and drug-resistant strains are also possible in other largegenome dna viruses. adeno-associated viruses are small dna viruses that are members of the family parvoviridae. they are satellite viruses that depend upon the presence of their helper dna viruses, including members of the families adenoviridae and herpesviridae, for replication. like vaccinia virus, adeno-associated viruses have been used extensively as vectors for gene therapy, and therefore represent a risk for genetic modification and subsequent terrorist use. rna-genome viruses are naturally more resistant to insertion of genes, but can be made resistant to drugs and potentially even to vaccines by introduction of mutations. most critically, rna viruses may be created in the laboratory by total chemical synthesis to generate active virus particles or reconstituted from cloned dna. total chemical synthesis was demonstrated recently for poliovirus ( figure ) [ ] . since this publication in , the technology to chemically synthesize viruses has advanced substantially. chemical synthesis of rna viruses would eliminate the need for the bioterrorist to obtain stock cultures and would allow creation of any wild-type or mutant strain of virtually any virus. other positive-strand rna viruses ( figure ) that have been reconstituted from cloned dna samples include kunjin virus [ ] , human rhinovirus [ ] , foot-and-mouth disease virus [ ] , transmissible gastroenteritis virus, a coronavirus [ , ] , all three genera of the family flaviviridae [ ] , and a variety of plant and insect viruses [ , ] . thus, the positivestranded viruses must be considered a high-risk area for bioengineering, where the relative risk for each virus must be considered roughly equivalent to the terrorist potential inherent to the virus. negative-strand rna viruses ( figure ) can also be reconstituted by total chemical synthesis or from cloned genomic dna by a reverse genetics approach [ ] . many negative-strand rna viruses, including ebolaviruses, lake victoria marburgvirus, hantaan virus and lassa virus, are important human pathogens and potential bioterrorist agents. influenza virus a is a particularly noteworthy negative-strand rna virus due to its potential to cause pandemic disease, the availability of stock cultures of many strains, and the potential for total chemical synthesis by reverse genetics techniques [ ] . for bacteria, if virulence factors have previously been modified successfully in a species, that species was considered to have a high potential for bioengineering and was included on the chart. however, numerous other methods for engineering bacterial genomes exist. bacterial genomes consist of one or more chromosomes and additional genetic information may exist in the form of plas-mids. plasmids replicate independently of the chromosomal dna and can be transferred between bacteria through transformation. genes encoding virulence factors may be found in either chromosomes or plasmids. in one example of an early bioengineering event, the selective growth of an antibiotic-sensitive species with an antibiotic-resistant species in the presence of the antibiotic resulted in transference of resistance. current techniques allow for the splicing of genes encoding virulence factors from one species followed by insertion into another. however, the complexity of the bacterial genome makes it difficult to successfully transfer a gene with a high rate of expression. this manuscript provides a visualization of the results obtained from use of the microbial rosetta stone database, a database that uses a new data model and novel computational tools to synthesize information on important human, animal, and plant pathogens [ ] . information on critical pathogens and diseases has been collected from many sources. organism names from all lists were converted to ncbi species names and diseases were linked to particular pathogens, which facilitated computational analysis and linkage to public genomic databases. this database should facilitate access to information on disease-causing organisms and toxins. the database can be accessed at http://www.microbialrosettastone.com/. agent lists were collected from various sources as described in more detail for each additional file below. government agency lists (additional files , , , , and ) were taken directly from the specified agencies, while additional tables were compiled using cited literature. organism names from all lists were converted to ncbi species names. this included changes from species synonyms to ncbi-accepted nomenclature and expansion from genus to genus species (e.g. shigella to shigella dysenteriae or shigella sonnei) to represent the most important species (when known). where possible, disease names were associated with the name of the most predominant organism that causes the disease. the additional files contain taxonomic information, the ncbi name of the agent and any well-known synonyms, and accession numbers for the complete genome of each agent (if available). names of the infectious agents listed in the additional files were placed in tree-structure drawings shown in the figures that display the biological relatedness of the organisms. cellular life forms were organized according to currently accepted phylogenetic relationships. bacterial phylogeny (figure ) is based on work by hugenholtz et al. [ ] . the eukaryotic phylogeny tree (figure ) is based on ribosomal and housekeeping gene sequence analysis [ ] . the branching order presented in figure should be considered tentative as the on-going recognition of additional protist lineages is likely to alter the topology of the eukaryotic tree in the near future [ ] . the phylogeny of dna viruses (figure ) was derived primarily from the work of iyer, aravind & koonin [ ] . the common origin of rna viruses and their tentative relationships ( figures , , ) is based on an extensive analysis of their rnadependent rna-or dna-polymerase (manuscript in preparation). the rna virus phylogenetic trees were created using maximum parsimony on protein sequences. the branching of double-stranded rna viruses is for now left unresolved in light of their apparent polyphyly [ ] . the symbol key is shown in figure and symbols are used in the other figures to link the pathogens in the figures to additional files. the agents listed in additional file [see additional file ] were taken from a list maintained by the who. the who tracks the occurrence of morbidity and mortality of global infectious diseases. because of the great disparity in infectious disease prevalence between developed and developing nations, the who divides the world into demographically developed nations (including the united states, europe, japan, australia, and nations of the former soviet union) and demographically developing nations (including india, china, other asian nations, pacific island nations, africa, latin america, and the middle east) [ ] . the who lists leading causes of death worldwide by disease categories. the disease can be caused by multiple diverse organisms or co-infections of different organisms, as is the case for acute respiratory disease, or by specific organisms, such as acquired immune deficiency syndrome (aids) or tuberculosis. in the former case, we identified the most prominent organisms responsible for the disease for display in additional file . the cdc-notifiable-disease list represents those diseases that offer the greatest threat to public health in the united states. additional file [see additional file ] lists the agents on this list. there are no set criteria for inclusion on the notifiable disease list; rather, the list is created by the cdc in cooperation with state health departments. as diseases occur less frequently and new diseases emerge, the notifiable disease list changes. an up-to-date version of the list can be found online on the cdc website [ ] . the online list provides links to case definitions of each disease, including the etiological agent(s) responsible. in cases where the etiological agent was not listed or was unspecific (i.e. brucella species), further research was done to determine an etiological agent and literature used is cited in legend for additional file . the most common causative agents of food-and waterborne illness were taken from a publication by tauxe [ ] and are listed in additional file [see additional file ] . hubálek [ ] published a list that constitutes a representative subset of emerging infectious diseases, which was used as the basis for additional file [see additional file ] . the complete list of all species classified as emerging is substantial [ , ] . the number of pathogens listed in the emerging disease table at http://www.microbialrosettas tone.com/ is significantly larger than that found in additional file . the niaid strategic plan for biodefense research, which lists agents with potential for "high morbidity and mortality; potential for person-to-person transmission, directly or by vector; low infective dose and high infectivity by aerosol, with commensurate ability to cause large outbreaks; ability to contaminate food and water supplies; lack of a specific diagnostic test and/or effective treatment; lack of a safe and effective vaccine; potential to cause anxiety in the public and in health care workers; and the potential to be weaponized [ ] ." the properties that make organisms amenable for use as biological weapons have been discussed extensively [ , ] . for the purposes of the database, we have categorized microorganisms or toxins as validated bioweapons if they have been documented as used, or prepared for use, as biological weapons [ ] . potential bioweapons are defined as agents that have some properties that are considered useful as bioweapons or agents for terrorism, or that have been the subject of serious bioweapons research and development [ , ] . greenwood et al. used a hierarchical ranking of infectious agents and toxins of biological origin [ ] to classify agents as potentially useful biological weapons. greenwood et al.'s highest scoring agents overlapped substantially with our list of validated bioweapons and these are shown in additional file [see additional file ] . the agents listed in additional file and additional agents listed in additional file [see additional file ] could be used in commission of a biocrime. biocrimes are similar to traditional crimes that harm specific individuals except that the weapon is biological in nature. a number of biological agents have been used to commit biocrimes [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and the potential list of agents that can be used is enormous. indeed, it is impossible to anticipate the next organism that will be used for illicit purposes. documented cases and accessibility were the criteria for choice of agents in additional file . the high potential for bioengineering category [see additional file ] includes both organisms that have been actually modified in biological weapons research programs in the past, and organisms with properties that make them amenable to future engineering. placement in the latter category was based upon published reports of bioengineering for peaceful purposes [ ] [ ] [ ] [ ] [ ] [ ] and the author's (dje) analysis. the agents on the hhs list are shown in additional file . hhs regulates the possession of biological agents and toxins that have the potential to pose a severe threat to public health and safety. hhs's select agent program [ ] regularly reviews and updates the list of select agents and critical human pathogens [ ] . the usda is required by federal law to protect animal and plant health. high consequence livestock pathogens and toxins are agents that the usda considers to have the potential to pose a severe threat to animal or plant health, or to animal or plant products [ ] . these organisms are listed in additional file [see additional file ] . the pathogens on the hhs [see additional file ] and usda lists include agents that span all domains of life, including bacteria, viruses, four fungi, two protists, one prion, and toxins. a significant number of these agents appear on both the hhs and usda lists. dje conceived of the study, organized the structure and contributed to the research and writing, and was overall responsible for the development of the research and the manuscript. rs, vs, tah, kh, jam, and jrw contributed to the literature analysis, organization of written sections, and writing of the manuscript. pw conducted extensive research, analysis, and constructed and linked the tables. bb contributed the sections on biocrimes and bioterrorism. cb-o contributed the viral taxonomy components of the manuscript. cm contributed the phylogenetic organization sections, plant pathogen research, and generated the figures. the microbial rosetta stone project ii: a database system for tracking infectious microorganisms the microbial rosetta stone database: a common structure for microbial biosecurity threat agents the global burden of disease: a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in and projected to (global burden of disease and injury epidemiology of acute respiratory infections in children in developing countries epidemiology of acute 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bacterial gastrointestinal infections: registry based study lyme disease surveillance in england and wales niaid biodefense research agenda for cdc category a agents. national institute of allergy and infectious diseases niaid biodefense research agenda for cdc category b and c priority pathogens. national institute of allergy and infectious diseases antifungal activity of a virally encoded gene in transgenic wheat acute hemorrhagic conjunctivitis caused by coxsackievirus a variant evaluation of the duopath verotoxin test for detection of shiga toxins in cultures of human stools cfr part -possession, use, and transfer of select agents and toxins; interim final rule botulinum and tetanus neurotoxins: structure, function and therapeutic utility distribution and characterization of tick-borne encephalitis viruses from siberia and far-eastern asia possession, use and transfer of biological agents and toxins. department of agriculture globally important human pathogens. pathogens are indicated on the phylogenetic charts when they cause at least . organisms responsible for emerging infectious diseases. literature used in conversion of disease names to pathogen responsible and in population of the table included: [ , , , , [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . key: cord- - y fedcr authors: chang, christopher title: unmet needs in respiratory diseases: “you can’t know where you are going until you know where you have been”—anonymous date: - - journal: clin rev allergy immunol doi: . /s - - - sha: doc_id: cord_uid: y fedcr the care of patients with respiratory diseases has improved vastly in the past years. in spite of that, there are still massive challenges that have not been resolved. although the incidence of tuberculosis has decreased in the developed world, it is still a significant public health problem in the rest of the world. there are still over million deaths annually from tuberculosis, with most of these occurring in the developing world. even with the development of new pharmaceuticals to treat tuberculosis, there is no indication that the disease will be eradicated. respiratory syncytial virus, severe acute respiratory syndrome, and pertussis are other respiratory infectious diseases with special problems of their own, from vaccine development to vaccine coverage. asthma, one of the most common chronic diseases in children, still accounts for significant mortality and morbidity, as well as high health care costs worldwide. even in developed countries such as the usa, there are over , deaths per year. severe asthma presents a special problem, but the question is whether there can be one treatment pathway for all patients with severe asthma. severe asthma is a heterogeneous disease with many phenotypes and endotypes. the gene for cystic fibrosis was discovered over years ago. the promise of gene therapy as a cure for the disease has fizzled out, and while new antimicrobials and other pharmaceuticals promise improved longevity and better quality of life, the average life span of a patient with cystic fibrosis is still at about years. what are the prospects for gene therapy in the twenty-first century? autoimmune diseases of the lung pose a different set of challenges, including the development of biomarkers to diagnose and monitor the disease and biological modulators to treat the disease. the study and management of lung diseases is of concern to allergist/immunologists and pulmonologists. the most common chronic lung disease is asthma, but there is an immunological basis for many other lung diseases, which span a vast clinical spectrum of lung disorders ranging from infectious lung disease to cancer. several significant challenge areas include the diagnosis and treatment of certain specific infectious lung diseases, including viral lower respiratory infections caused by respiratory syncytial virus, rhinovirus, metapneumovirus, coronovirus, and enterovirus. severe acute respiratory syndrome (sars) coronavirus (sars-coa) was responsible for the outbreak of severe acute respiratory syndrome in the early s that originated in asia and led to significant mortality in those afflicted. other viruses such as bird flu and swine flu have the ability to cause respiratory tract disease as well. tuberculosis is another primarily respiratory infection that has been resistant to eradication. new strains of multidrug-resistant tuberculosis have emerged. other challenges involve the genetic diseases cystic fibrosis and alpha- -antitrypsin deficiency, autoimmune lung diseases, lung diseases that are part of a systemic autoimmune disease, and chronic obstructive pulmonary disease (copd). the diagnosis and treatment of hypersensitivity pneumonitis still poses problems to clinicians. biomarkers are continually being developed in lung and other cancers, but more research is needed [ ] . the genetics of cystic fibrosis are now well elucidated, but the development of a successful gene therapy has been unexpectedly slow (table ) . for many of these conditions, it was and still is expected that the ongoing development of new waves of molecular biology techniques, coupled with computerized automated analysis that can provide transcription signatures, will help identify differences between patients with these diseases and spur on the development of relevant and effective treatments. the subsequent discussion takes on some of the more interesting and challenging issues in respiratory disease over the past two decades, with a focus on what to expect in the future as well. asthma is one of the most common chronic diseases worldwide. the who estimates that there are over million asthmatics worldwide and , deaths per year. in the usa, there are million asthmatics, . million of whom are children. until recently, the mortality rates from asthma had been steadily increasing. it is only in the past - years that the mortality rates have begun to stabilize and are perhaps even on a decline. the increasing incidence of asthma has been attributed to various theories, perhaps the most popular of which is the "hygiene hypothesis" [ ] . this attributes the rise in incidence to the reduced rate of infectious diseases, cleaner living environments, and, in general, those features that accompany a higher standard of living. but, conversely, the annual death rates per , asthmatics tend to be higher in the economically poorer countries. this emphasizes the most significant global concern regarding asthma care, which is access to care and medications. in developed countries, this problem is less prevalent, and in countries such as the usa, we are primarily focused on asthma education and compliance issues. in both situations, severe asthma is still a problem in that it impacts quality of life and still contributes to considerable morbidity and mortality. one of the problems of severe asthma is related to defining the disease. asthma is inherently very heterogeneous, and many phenotypes of severe asthma have been described. it is increasingly clear that there is no single medication that works for all patients with severe asthma. while patient compliance is an issue, we frequently fall into the trap of believing the problem is always patient compliance, when in fact, it may be that patients are not taking their medications because they perceive that they do not work. nevertheless, the explosive growth in the availability of new pharmaceutical interventions provides us with an arsenal of controller medications, at least in the developed world. more medications are being developed every day, including the newer class of biological modulators against cytokines and chemokines [ ] , such as anti-il [ , ] and anti-il rα [ , ] . the next challenge will involve the identification of suitable patients for these new and existing treatments. but how will this be done? researchers over the years have attempted to define asthma phenotypes, trying to categorize asthma patients based on demographic and clinical characteristics in an attempt to define certain patient groups for certain preferential treatments [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in general, this has led to more confusion as the different research groups do not necessarily come up with the same classifications. moreover, asthma in children is also significantly different from that in adults, and the response to medications varies based on multiple and not a single factor. the choice and exclusion of confounding variables has been a thorn in the side of those attempting to define a specific asthma phenotype. it has also not been found that specific childhood asthma phenotype will develop into a specific adult asthma phenotype. the concept of asthma endotypes has now been the subject of much research because endotypes address the underlying mechanism of a disease [ ] [ ] [ ] [ ] . however, this is also confusing because of the highly redundant inflammatory pathways that can lead to disease. the use of biomarkers to identify those patients who are at high risk for severe, lifethreatening exacerbations has not been entirely fruitful. the recent commercial introduction of fractional exhaled nitric oxide (feno) to identify patients with eosinophilic inflammation is a potentially useful tool, but more is needed to guide treatment. the identification of a gene for asthma has revealed that this is more complex than one might have expected. there are now over genes that have been attributed to asthma. some of the more likely candidates include adam , ormdl , dennd b, filaggrin, chi l , and il- . moreover, the existence of shared genes between asthma and other comorbid conditions such as obesity has also been demonstrated [ ] . ultimately, asthma, like many other diseases, is a heterogeneous disease with many phenotypes and endotypes [ , ] . not one single treatment will fit all, and the art of medicine may be to find the right peg for the corresponding hole [ ] . the twenty-first century has ushered the concept of personalized or genomic medicine, which utilizes advancements in molecular biology, such as proteomics, metabolomics, and genomics, to identify the optimal therapy for each individual patient. cystic fibrosis-what happened to gene therapy? it has now been years since the identification of the gene responsible for cystic fibrosis (cf) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the initial study describing the gene named "cystic fibrosis transmembrane conductance receptor (cftr)" was first published in the late s by tsui et al. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this discovery led to a localization of the gene to chromosome , leading to widespread belief that a cure for cf utilizing gene therapy was right around the corner. the landmark development of a molecular biology technique which became known as genetic targeting in mice, pioneered by capecchi [ ] [ ] [ ] [ ] , evans [ , ] , and smithies [ ] [ ] [ ] [ ] [ ] , promised gene replacement in humans in a similar way to that performed in mice. twentyfour years later, there are new advancements in the management of cystic fibrosis with regard to pharmacological and supportive respiratory treatment, but still no cure. these advancements have prolonged the longevity of patients with cf, but their life expectancy still only averages to be about years. so, what happened to gene therapy? what happened to all that promise that buoyed the cystic fibrosis community back in the s? the idea behind gene therapy is to introduce a missing of malfunctioning gene into the cells of a patient using a "harmless" virus that can be manufactured to carry a normal copy of the diseased gene. this technique can either target cells globally or be restricted to a certain group or location of cells. but gene therapy has not progressed as smoothly or as quickly as anticipated. previously unrecognized barriers became apparent [ ] . these included the fact that studies were initially focused on molecular or biochemical outcomes and not on clinical efficacy. moreover, the administration of the gene would need to be repeated due to epithelial turnover. repeated administration leads to host recognition, which may inhibit gene expression. questions on which cells to target in order to achieve efficacy has slowed research. the use of viral material, including viral dna and liposomes, could potentially lead to inflammatory responses. the point is that unexpected consequences were discovered as research proceeded, and this has had an impact on the progress of gene research. in , a group of british investigators began large-scale trials of gene therapy delivered by encompassing the gene in fat globules and delivering the gene by nebulization. future methodologies will utilize a viral delivery strategy, but this is still several years into the future. gene therapy for cystic fibrosis is not dead, but certainly moving at a far slower pace than originally expected. current treatments are not curative for cystic fibrosis. gene therapy and stem cell transplants are two techniques that are still under investigation, and it may turn out that only certain mutations may be candidates for gene therapy. mutations in the cf gene have in fact been subclassified into six classes [ ] , each with its own pathogenic or physiologic characteristics. for example, the common f del mutation results in reduced amounts of cftr channel expression, which leads to exacerbation of the disease [ ] . respiratory syncytial virus-how to develop an effective vaccine? respiratory syncytial virus is an infection of the lower respiratory tract that causes significant morbidity and mortality in infants and young children [ ] . globally, there are over million new lower respiratory tract infections per year and approximately , deaths per year in children fewer than years of age. the majority of these deaths occur in lowincome countries where access to care may be limited. the search for a vaccine for respiratory syncytial virus (rsv) has been ongoing for many years, but like the previous case of gene therapy in cystic fibrosis, this also has been a challenge to achieve. in the absence of a vaccine, researchers have developed passive immunization to the virus in the form of a monoclonal antibody to rsv, named palivizumab. the current global strategies for the development of an rsv vaccine now target four areas: infants < months of age; infants > months of age and young children; pregnant women for whom passive immunization can be implemented; and the elderly, in whom rsv can also have significant morbidity [ ] [ ] [ ] . the main challenges that have prevented the development of an effective vaccine so far revolve around the fact that even natural infection to rsv does not provide long-term protection from reinfection. an earlier study actually found that the vaccine may actually accentuate the disease, especially in young infants who may not have a fully mature immune system and may be unable to effectively engage in somatic mutation, leading to a suboptimal b cell repertoire [ , ] . this may also apply to older infants or young children who may still be rsv-naive. the issues in adults are just the opposite as most adults have been exposed to rsv and thus have rsv antibodies. the effectiveness of passively immunizing pregnant women in order to deliver igg across the placenta to the fetus must be weighed against possible adverse effects of such a "vaccine" on the fetus. in the elderly, the challenge has been the ability to boost immunity through active immunization in an individual who is becoming immunosenescent. there are now several vaccine development programs that pursue a variety of vaccine strategies. a live virus vaccine trial has shown that a particular vaccine candidate, medi- , is safe, although effectiveness has not been proven [ ] . other strategies include subunit rsv vaccines, the use of dnaconjugate vaccines to boost antigen presentation, and further development of passive antibody prophylaxis that is focused on f or g proteins [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . an additional challenge with all of these strategies is that effective vaccine studies in animals have not been translated into successful human trials. tuberculosis is a global health problem. tuberculosis is an infection caused by the bacterium mycobacterium tuberculosis, which primarily affects the lung, but can also affect other tissues, including bone and the nervous system. tuberculosis is believed to be one of the oldest infections to reportedly affect mankind, with archeological and anthropological studies showing evidence of infection in humans over , years ago. it continues to top the charts of mortality and morbidity in developing nations. it is estimated that in , there were . million new cases of tb, as well as a prevalence of . million. the . million deaths in from tb in patients without hiv and , deaths in patients with hiv is a shocking statistic that shows how far we have come, yet how far we still are from effectively eradicating the disease [ ] . two very effective drugs have been developed to treat tuberculosis, isoniazid (inh) and rifampin. however, tuberculosis continues to be a global health problem. tuberculosis is second to hiv/aids as the infection with the highest mortality globally. more recently, an increase in multiple drug-resistant tuberculosis (mdr-tb) has been observed, with most cases from india, china, and russia. there were , reported cases of mdr-tb in the world in . of these, it is estimated that . % fall into the category of xdr-tb, which is an even more resistant form. thus, drug resistance is a significant challenge in the treatment of tb in the twenty-first century. another challenge stems from the fact that about one third of patients with hiv/aids are infected with tb, although many may not yet be symptomatic. the who defines six core functions and six strategic approaches to combat tb (table ) . overall, % of tb cases occur in just countries, and % of cases occur in china. however, as overall healthcare accessibility and technology improves, there have been significant declines in the rate of tb in some of the asian countries, including cambodia and china. there is no dispute that the dots and "stop tb strategy," as outlined in table , have contributed to significant progress in the control of tb, but significant challenges remain. one of the areas that are being focused on now has to do with the identification of biomarkers. biomarkers can play several roles in the improvement of treatment of tb in the world. the main areas of focus for research in biomarkers are to identify those that can perform the following functions: ( ) prediction of a curative state, ( ) prediction of reactivation of tb, and ( ) prediction of immunity to tb. in the past, biomarkers for tb would normally involve culturing for the organism, but the development of molecular biology techniques has afforded us the use of non-culture biomarkers. the types of approaches, as described by wallis et al. in [ ] , categorize biomarker development into functional categories ( table ) . non-culture biomarkers include cytokine levels, quantifiable genetic measures of the presence of tb, other serological tests, imaging techniques, gene transcription profiles, and micro-rna profiles. the development of a vaccine has been an ongoing challenge in the quest for a cure for tb, and limitations in these studies include the lack of valid biomarkers to assess protection in clinical trials. the cytokines interferon-γ and il- have been studied as biomarkers for latent tb infection. a tuberculosis-stimulated interferon-γ release assay can detect sensitization to tb antigens, but, unfortunately, cannot differentiate between resolved and persistent latent infection. interleukin- is a marker of innate immunity. elevated levels of plasma interleukin levels seen in tb patients indicate increased activation of innate immunity, which was not observed in controls. a chemokine that was observed to be elevated in tb were ccr [ ] , while the levels of the apoptosis inhibitor bcl expression were reduced [ , ] . il- levels actually correlated with the radiographic extent of the disease [ , ] . hiv appears also to modify cytokine production in patients with tb [ ] . other cytokines and chemokines have been profiled as potential biomarkers for tb disease activity [ ] [ ] [ ] [ ] [ ] [ ] , although to this date, none has been shown to be useful for widespread clinical application. fifty years ago, it was the introduction of pyrazinamide and rifampicin that reduced the duration of therapy from months to months. the value of biomarkers may be reflected in the implications of being able to predict cure and latency of infection, as well as active infection. from a therapeutic standpoint, this may mean the difference between the current regimen of months of treatment versus a possible reduction in the duration of therapy for certain patient groups. but this depends on the biomarker being significantly accurate as to not lead to inadequate treatment of affected individuals. though a half century has passed since the development of effective treatment for drug-susceptible tb, the period of treatment is still at least months of standard therapy, i.e., rifampicin and isoniazid for months and pyrazinamde and ethambutol during the initial months of treatment. efforts to reduce the length of treatment are dependent on the discovery of more effective treatment as well as being able to monitor the treatment. at the same time, more and more patients with tb are demonstrating multiple drug resistance forms including mdr or xdr tb. the addition of second-line drugs for the treatment of these more difficult to treat patients is plagued by longer periods of treatment, higher cost, and reduced efficacy. the drugs often used for second-line treatment include aminoglycosides, terizidone, protionamide, capreomycin, fluroquinolones, cycloserine, and ethionamide. the mortality of mdr and xdr tb in hiv patients is particularly high, and researchers have focused on novel approaches to combat this global health issue, including revamping of efforts to develop a vaccine for tb [ ] [ ] [ ] [ ] [ ] [ ] , treatment of latent infection to reduce reservoir pools of infection, and the development of predictive biomarkers. one should not forget that for years we have had a vaccine for tb, the bcg vaccine named after albert calmette and camille guerin. while this vaccine has been used primarily in underdeveloped countries and has been administered to over billion people worldwide, the limitations include an inability to protect against adult tb and adverse effects when used in hiv-positive tb patients. further research is ongoing to develop an improved replacement or enhancement vaccine for bcg [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, as we develop new biomarkers and new treatment strategies, one should remember that the infective landscape of tb is not a static target either. the emergence of multidrug-resistant strains as mentioned above means that the disease may become more difficult to treat and that the development of biomarkers must take new medication use into consideration. moreover, the development of biomarkers requires the availability of large patient samples, such as those that can be found in biobanks of large high-volume treatment centers. the willingness of patients to contribute to research, and the regulatory landscape for the collection of samples for research, could potentially negatively impact the ability to conduct large-scale studies on biomarkers. for a comprehensive review of biomarkers, diagnostics, drug treatment, and vaccine development, the reader is referred to an excellent series of articles published in lancet in [ , , ] . the incidence of pertussis appears to be increasing over the past years. this trend flies in the face of an availability of, first, the killed whole organism pertussis vaccine dtwp and, subsequently, the acellular vaccine (dpap). the acellular vaccine was less reactogenic because it incorporated a step during manufacturing that removed endotoxin, which was a major reason for the adverse side effect profile. it was clear that vaccination against pertussis afforded much improved prevention of the disease, as illustrated by the -fold decreased rate for pertussis between the s and s. dtwp vaccines were used until the s, when technological development in the s and s led to the introduction of commercially available acellular pertussis vaccines [ ] . the continuing increase in pertussis cases leads to an obvious question. is this a failure of an immunization program? earlier, we discussed several examples that illustrate the difficulty of bringing an effective vaccine to market (tb and rsv). the problems associated with pertussis are clearly different. there are several reasons that may explain why we have not eradicated the disease. it is well documented that the dtap vaccine is not as immunogenic as the whole organism dtwp vaccines. studies have indicated that the overall [ ] efficacy of dtap vaccines is below %. we do not normally test for pertussis titers following vaccination, except in studies. but there are those who suggest that the increase in pertussis cases is mainly due to the increased ability to diagnose the disease and to an increased awareness. yet another challenge may be that pertussis has mutated over the years, rendering the vaccine less effective. because our current vaccine is < % effective, the ability of mutant strains to evade destruction would lead to their preferential survival. it has been shown that pertussis vaccine which contains more antigens, such as the five-component vaccine that contains pertussis toxin (pt), filamentous hemagglutinin (fha), pertactin (prn), and fimbriae (fim / ), is superior to those containing fewer antigens. it has also become apparent that the balance of antigens in the vaccine may play a role in determining efficacy. for example, a whole-cell pertussis vaccine with antigenic components pt, fha, and fim was compared to an acellular pertussis vaccine in children. the whole-cell vaccine produced high levels of antibodies to prn and fim, but low levels to pt and fha, in contrast to the acellular pertussis vaccine which produced high levels of antibody to pt and fha. more than one study has suggested that the vaccine components may antagonize each other. it has been found that the antibody levels to fha do not correlate with the efficacy of the vaccine; furthermore, it has been suggested that adding fha to a vaccine may actually suppress efficacy. linked epitope suppression is another concept to explain the failure of pertussis vaccines. while one would expect that the presence of antigens that do not deliver a brisk vaccine response should not affect the response to other vaccine antigens, this may not be the case. if there were a linkage suppression between two epitopes on the pertussis vaccine, then this could be a significant consideration in vaccine design as one would then need to exclude certain antigens from the vaccine component mix. suffice to say that vaccine development is a very complex topic, and it involves various techniques, including the use of classical adjuvants such as aluminum salts, emulsions or liposomes, or novel adjuvants such as toll-like-receptor agonists, saponins, or immune stimulating complexes [ ] . one cannot discuss challenges in pertussis without commenting on the fear of immunizations held by many families, even in developed countries such as the usa. the refusal of many parents to immunize their children, for whatever reason they may have, plays a significant role in our inability to eradicate several diseases, including pertussis, measles, rubella, and so on. this phenomenon is not limited to the uneducated population as many highly educated individuals appear to choose not to subject their children to the relatively miniscule real or imagined risks of immunizations. certainly, one has an astronomically higher risk of being killed in a car accident than dying from a vaccination. and yet, people who refuse to vaccinate their children do not stop driving or riding in cars. the problem with all this is that achieving "herd" immunity is important in total eradication. this means that if only a very small portion of the population do not immunize their children, there is still a possibility of eradicating a disease, but as more people choose to remain unimmunized, the likelihood of successful eradication becomes less and less. unfortunately, people who choose to remain unimmunized fail to understand that in order that everyone not have to get immunized in the future (as in the case of smallpox), everyone must be immunized in the present [ ] . severe acute respiratory syndrome, or sars, was an infection that reached epidemic proportions during the early part of the s. it originated in china and rapidly spread to neighboring countries, and eventually, it was reported in many other distant nations, including europe and the usa. although the number of cases was far fewer, by several orders of magnitude, than the common flu, it caught the attention of healthcare professionals and public health officers because of its high mortality, with , cases and deaths reported between and , for a mortality rate of . %. sars was later identified to be caused by a coronavirus and was named sars-coa. travel histories of infected people were instrumental in tracing the origin of the disease [ ] . but where did this virus come from and how did it suddenly cause so much havoc? initially, in may of , the virus was traced to civets, a cat-like mammal that is occasionally consumed as food by the chinese. however, these creatures were eventually dismissed as the original source because of the lack of appearance of further infected civets. in , reservoirs of sars-like viruses were found in chinese horseshoe bats. these were often brought to market, and it was thought that the virus was passed to humans at that time. phylogenetic studies further provided evidence that genetically, it was likely that the sars coronavirus evolved from viruses that infected the horseshoe bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . so can sars happen again? in fact, smaller outbreaks that do not attract as much attention may already be occurring in a manner that mimics sars. a middle east respiratory syndrome or mers has been reported to also be caused by a coronavirus. the mortality in this case is high as well, of cases, or > % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . mers is also thought to have originated in bats. interestingly, many other viruses are thought to be transmitted by bats, including ebola, hendra, and rabies. the cellular receptor for mers is cd , or dipeptidyl peptidase [ ] . lessons learned from the sars epidemic included the fact that transmission can often be facilitated by the very medical workers who are trying to save patients [ , ] . strict infection control measures are critical in limiting the spread of the disease, including the use of negative pressure rooms, n masks, and gowns. the optimal dosing of antiviral medications and the timing of supportive measures of respiratory care are still unknown, but previous experience should guide us to be more vigilant if this ever occurs in a large-scale fashion in the future. autoimmune lung diseases-biomarkers and biologics-sparing the steroids? anti-neutrophil cytoplasmic antibody (anca) was discovered in [ , ] . the antigenic targets of anca include myeloperoxidase or proteinase- , and the antibodies against these antigens were for a long time referred to as p-anca and c-anca, respectively, the p and c indicating their cellular staining pattern (perinuclear or cytoplasmic). this latter nomenclature has since been discouraged, and the antibodies should be referred to in the context of their specific antigenic target [ , ] . an understanding of the pathogenesis of anca-associated vasculitis (aav) would help in the development of more effective drugs to treat this group of diseases [ ] , which include granulomatosis with polyangiitis (formerly wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (egpa or churg-strauss disease), and microscopic polyangiitis [ ] . for toxin-mediated aav, a meta-analysis has shown a link between crystalline silica and aav [ ] . silica is an inflammasome activator and is believed to trigger the activation of pro-inflammatory cytokines leading to the disease. other triggers include drugs, most commonly hydralazine, propylthiouracil, and penicillamine (all of which are known to stimulate b lymphocyte activation), and infectious trigger such as staphylococcus aureus. avoidance of exposure may prove to be a useful methodology to prevent disease, but as of this time, the triggers of aav are too diverse and too controversial to recommend any particular avoidance strategy. another disease state that affects millions, perhaps billions, of people worldwide is allergies. allergies that affect the respiratory tract can take the form of allergic asthma or simply allergic rhinitis and conjunctivitis. the hygiene hypothesis would indicate that because of our cleaner living environments, we are developing higher rates of sensitivities to common allergens, both indoor and outdoor, as well as higher rates of autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one of the main avenues of treatment has always been avoidance, but we have not really been able to define what exactly constitutes effective avoidance. many of the studies done on dust mite and animal dander avoidance have not been able to identify a single avoidance measure that can make a significant impact on outcome, although perhaps a comprehensive avoidance plan may provide some relief. studies are needed to ( ) determine whether indeed effective avoidance is possible in the face of indoor and outdoor exposures to allergens and ( ) the extent to which avoidance of allergens, if possible, provides a significant benefit to patients. the diagnosis and management of respiratory diseases is plagued by numerous unmet needs, affecting both common diseases such as asthma to uncommon occurrences such as sars. some of the problems are common and ultimately involve improving our understanding of the mechanisms of disease. only then can we develop tools to help us manage these patients. the advent of molecular biology has allowed us to develop genetic analyses of patients, and this has led to a realization that these diseases are frequently not one disease, but many [ ] . thus, the genomic and personalized medicine acts of and are important legislatures that will hopefully divert increased funding into studies that will help us identify which treatment should optimally be used on which patient [ , ] . understanding the pathogenesis of these diseases is another significant unmet need which requires commitment of funding and resources [ ] . the respective contributory roles of genes, epigenetics, and the environment in the pathogenesis of lung diseases must be better elucidated [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . with better understanding of disease comes better diagnostic markers and better treatment. other unmet needs circle around the availability of biomarkers to both diagnose disease and vaccines and other novel treatments to manage disease. new techniques for vaccination which will hopefully alleviate concerns of vaccines among vaccine-apprehensive patients may help lead to the eradication of infectious respiratory disease such as tb, pertussis, and rsv. there is much work yet to be done, and it is important to remember that diseases are not static either, especially in the context of pathogen-related diseases as pathogens are also very adept at evading our efforts to destroy them. the challenges outlined in this article are far from comprehensive, and specific unmet needs exist for a variety of other respiratory diseases, including alpha- -antitrypsin deficiency, hypersensitivity pneumonitis, chronic obstructive pulmonary disease, lung cancer, pulmonary hypertension in the newborn, and others. while each disease state may have its own unique set of challenges, in general, one can categorize these into three major areas of need, namely, diagnosis, treatment, and prevention (fig. ). more specifically, these 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disease do infections facilitate the emergence of systemic sclerosis? air pollution in autoimmune rheumatic diseases: a review the role of environmental estrogens and autoimmunity systemic lupus erythematosus one disease or many? the genomics of autoimmune disease in the era of genome-wide association studies and beyond the genomics and personalized medicine act of genes, epigenetic regulation and environmental factors: which is the most relevant in developing autoimmune diseases? the influence of sex and gender on the immune response genes, tolerance and systemic autoimmunity the x chromosome and immune associated genes twin studies in autoimmune disease: genetics, gender and environment genetics of asthma susceptibility and severity the genetic and environmental causes of pulmonary fibrosis epigenomics of idiopathic pulmonary fibrosis who's new stop tb strategy key: cord- -y z sa y authors: baumgärtner, wolfgang; löscher, wolfgang title: re-emergence of neuroinfectiology date: - - journal: acta neuropathol doi: . /s - - - sha: doc_id: cord_uid: y z sa y nan infections of the central nervous system (cns) represented an area of major concern in the pre-antibiotic and pre-vaccination era. following the wide-spead introduction of antibiotic therapy and the implementation of national vaccination programs, many infectious diseases appeared to be successfully contained, and the health threat posed by infectious pathogens seemed to belong to the past. however, in recent years the number of reported cases of infectious agents causing cns infections in the form of emerging and re-emerging diseases has been increasing [ ] . among the most devastating infectious diseases of the cns that plague today's world are cerebral malaria, rabies, toxoplasmosis, bacterial meningitis, arbovirus encephalitis and human immunodeficiency virus-associated neurological diseases [ , , , [ ] [ ] [ ] . viral and bacterial cns diseases represent an important but relatively neglected area of medicine in developing countries [ ] . similarly, parasiteinflicted diseases of the cns represent a major threat to public health indeveloping countries; this threat is also present in the western world but the disease burden is less [ , ] . overall, the burden of infectious cns diseases is reinforced by the fact that survivors may suffer from life-long etiology, discover the underlying mechanism and design prevention and intervention strategies. the cause of an inflammatory cns disease frequently remains undetermined. approximately % of patients with suspected cns infection lack an etiological diagnosis and one-third of these patients ultimately die as a result of the illness [ ] . however, present-day researchers have acquired a new diagnostic tool, namely, metagenome sequencing, and this tool is increasingly being applied to the identification of pathogens [ ] . intensified search with this tool and with new technologies have resulted in the identification of various pathogen vectors, including bats, domestic pets and wildlife animals, for different agents, such as the hendra and borna viruses, japanese encephalitis infection and middle east respiratory syndrome (mers) coronavirus [ , , , ] . this issue of acta neuropathologica includes a cluster of three review articles on neuroinfectiology with special emphasis on viral and bacterial diseases as well as the role of these diseases in epilepsy [ , , ] . these reviews reflect the plethora and diversity of causes, consequences and reaction patterns in neuroinfectiology. the review by ludlow et al. [ ] depicts emerging and re-emerging neurotropic and non-neurotropic viruses that cause cns diseases and sheds some light on the mechanisms underlying both the direct and indirect as well as the immediate and delayed consequences of these diseases. viral diseases of the cns, many arising from zoonotic pathogens, can be caused by a variety of viruses, including the bunyavirus, nipah virus, hendra virus and rabies, polio, tick-borne encephalitis, herpes and measles viruses [ , , , ] . viral cns infections are commonly caused by mosquito-borne viruses, such as the west nile, chikungunya and japanese encephalitis viruses, which have in recent times expanded their geographic range. in addition, some viruses, including the bat henipaviruses nipah virus and hendra virus, the borna virus as well as the japanese encephalitis virus, have crossed the human species barrier (spill-over infection) [ , , ] . various bacteria, including streptococcus pneumoniae, neisseria meningitidis, haemophilus influenzae, enterohemorrhagic escherichia coli (ehec) and listeria monocytogenes, are among the most common bacterial causes of cns diseases in humans. others, such as streptococcus suis, are zoonotic pathogens with great regional differences as formidably described by the review of doran et al. [ ] . in africa's so-called "meningitis belt", which stretches from senegal to ethiopia, outbreaks of meningitis due to meningococcal disease caused by neisseria meningitidis occur regularly, killing thousands and infecting tens of thousands each year [ ] . complications and long-term sequelae after the initial bacterial infection include epileptic seizures, hydrocephalus, infarction, herniations and persistent defects after healing. noteworthy, septic patients may develop septic encephalopathy, a potentially irreversible acute cerebral dysfunction which is clinically characterized a slowing of mental processes, impaired attention, memory dysfunction, delirium and/or coma [ ] . epilepsy with recurrent unprovoked (spontaneous) seizures may be a serious consequence of cns infections [ ] . the current terminology for the concept(s) and underlying cause(s) of epilepsy refers to three categories, i.e. genetic, structural/metabolic and unknown. this categorization replaces the previously used terms of idiopathic, symptomatic and cryptogenic. epilepsy resulting from various processes, including traumatic brain injury, neoplasms, ischemic or hemorrhagic stroke and cns infection, belong to the structural/metabolic category. congenital and developmental issues and genetic conditions are mostly associated with the development of epilepsy in younger patients, whereas infection, head trauma and tumors leading to epilepsy may occur at any age. in survivors of cns infections, the risk of unprovoked seizures is approximately % in developed countries, but much higher in resourcepoor countries. in their review, vezzani et al. [ ] comprehensively describe the infectious diseases and sterile (non-infectious) inflammatory responses, as well as the associated underlying complex pathogenetic mechanisms, which result in the development of epilepsy. these authors also describe those factors which play a critical role during epileptogenesis and which should be considered in prevention strategies. the causes of degenerative, inflammatory and behavioral disorders, including alzheimer disease, parkinson disease, multiple sclerosis, guillain-barré syndrome, encephalitis lethargica, congenital malformations, schizophrenia and bipolar disorder, are largely unknown. however, recent studies have been (re)-focusing on the role of neuroinflammation and infections as driving forces. in addition, new concepts in which infections early in life are considered to be predisposing factors with clinical manifestation(s) decades later are now being studied [ , [ ] [ ] [ ] . factors contributing to the emergence of new pathogens include changes in human demographics and behavior, intensification of international travel (tourism), commerce (global trade), increased economic development and land use, increasing importation of infected animals and exotic pet trade and the altered migration of vectors (such as birds and arthropods) and their adaption in new environments, in part facilitated by global warming [ , ] . faced with complex patterns of global changes, the researcher analyzing the interconnections among humans, companion animals, livestock and wildlife requires integrated approaches. these complex interactions and their conceptual interpretation depend on a multidisciplinary and cross-sectoral approach involving experts in both human and veterinary medicine as well as those in ecology, as envisioned by the one health-one medicine concept [ , ] . in addition to opportunistic infections, activation of silent-primarily non-pathogenetic-agents may represent a future threat as potent drugs are available to treat autoimmune disease, organ transplants and cancer. in addition, more patients with congenital immunodeficiency live longer and may be exposed to these pathogens. similarly, the gut microflora has been shown experimentally to play an important role as a trigger of cns inflammation [ ] . furthermore, t cells become licensed in the lung to enter the cns [ ] . these different modes of pathogen-host or immune system interaction may represent future avenues by which to study the pathogenesis of inflammatory and degenerative lesions in the nervous system, as outlined in the recent review by bauer et al. [ ] which focuses on progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome. neuroinfectiology represents an emerging multidisciplinary field which centers on the complex interactions between cns and pathogen-associated cellular and molecular processes, inflammation, immune responses, degeneration, stem cell homeostasis as well as tissue repair and regeneration. in order to combat this challenge extensive cross-fertilization between scientists from various fields is needed. new pathogens associated with cns involvement have emerged in recent years. these represent a major threat to public health, are of great economic relevance and represent a medical challenge due to the lack of appropriate diagnostic and treatment strategies [ ] . global trading, tourism and ecological and demographic changes will contribute to outbreaks of diseases due to these emergent agents. globalization of human travel and industrial exchange facilitates the spread of infections worldwide within a short time period. in addition, developments of immunomodulatory drugs to treat immune-mediated disease might cause opportunistic infections or activation of latent agents. new molecular detection systems will improve our ability to rapidly diagnose and recognize emerging and re-emerging pathogens and the host genetic factors involved in disease susceptibility, but the development of new strategies for diagnosis, prevention and treatment of neurological disorders will only be efficiently addressed by an interdisciplinary approach bridging the fields of neuroscience and infection medicine. moreover, little is known about the role of pathogen-related predisposing factors, mechanisms causing acute disease in individuals and factors resulting in long-term cns disturbances. overall, a more conceptual understanding of neuroinfectiology is pivotal for the development of successful prevention and treatment strategies. future studies in neuroinfectiology will address questions relating to the mechanisms of direct and indirect as well as acute, delayed and long-term damage, the role of misdirected immune responses in lesion initiation and the progression as well as prevention of cns infection by developing appropriate intervention strategies and potential beneficial approaches for tissue regeneration. infectious agents associated with schizophrenia: a meta-analysis progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome (iris) commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination kristensson k ( ) infectious diseases of the nervous system and their impact in developing countries parasitic diseases of the central nervous system host-pathogen interactions in bacterial meningitis emergence and re-emergence of viral diseases of the central nervous system an orthopoxvirus-based vaccine reduces excretion after mers-cov infection in dromedary camels neuroinfectiology. subspecialty with a future a variegated squirrel bornavirus associated with fatal human encephalitis gobal research priorities for infections that affect the nervous system teaching "one medicine, one health neurotropic virus infections as the cause of immediate and delayed neuropathology emerging roles of pathogens in alzheimer disease neuropsychiatric sequelae of nipah virus encephalitis t cells become licensed in the lung to enter the central nervous system the ecology of emerging neurotropic viruses assembly of viral genomes from metagenomes burden of neuroinfectious diseases on the neurology service in a tertiary care center tackling meningitis in africa emerging viral infections of the central nervous system: part emerging viral infections of the central nervous system: part infections, inflammation and epilepsy progress in clinical neurosciences: sepsis-associated encephalopathy: evolving concepts guillain-barré syndrome from "one medicine" to "one health" and systemic approaches to health and well-being acknowledgments the research underlying this editorial was in part supported by niedersachsen-research network on neuroinfectiology (n-rennt) of the ministry of science and culture of lower saxony, germany. key: cord- -brhvfsgy authors: miller, ryan s.; farnsworth, matthew l.; malmberg, jennifer l. title: diseases at the livestock–wildlife interface: status, challenges, and opportunities in the united states date: - - journal: preventive veterinary medicine doi: . /j.prevetmed. . . sha: doc_id: cord_uid: brhvfsgy abstract in the last half century, significant attention has been given to animal diseases; however, our understanding of disease processes and how to manage them at the livestock–wildlife interface remains limited. in this study, we conduct a systematic review of the scientific literature to evaluate the status of diseases at the livestock–wildlife interface in the united states. specifically, the goals of the literature review were three fold: first to evaluate domestic animal diseases currently found in the united states where wildlife may play a role; second to identify critical issues faced in managing these diseases at the livestock–wildlife interface; and third to identify potential technical and policy strategies for addressing these issues. we found that of the avian, ruminant, swine, poultry, and lagomorph diseases that are reportable to the world organization for animal health (oie), are present in the united states; ( %) of these have a putative wildlife component associated with the transmission, maintenance, or life cycle of the pathogen; and ( %) are known to be zoonotic. at least six of these reportable diseases—bovine tuberculosis, paratuberculosis, brucellosis, avian influenza, rabies, and cattle fever tick (vector control)—have a wildlife reservoir that is a recognized impediment to eradication in domestic populations. the complex nature of these systems highlights the need to understand the role of wildlife in the epidemiology, transmission, and maintenance of infectious diseases of livestock. successful management or eradication of these diseases will require the development of cross-discipline and institutional collaborations. despite social and policy challenges, there remain opportunities to develop new collaborations and new technologies to mitigate the risks posed at the livestock–wildlife interface. despite significant attention given to animal diseases in the last half-century, our understanding of disease processes, and how to manage them at the livestock-wildlife interface, remains limited (rhyan and spraker, ) . the increasing role of wildlife in the emergence of livestock fig. . the number of publications in english language journals identified in scopus database with the words "wildlife" and "parasite" or "disease" in the title, abstract, or key words. decker et al., ; rhyan and spraker, ) , potentially exacerbating pathogen transmission processes between them. globally, the role of wildlife in livestock diseases is expected to increase (siembieda et al., ) in conjunction with human population growth, which is expected to reach billion by . this will create increased demand for animal protein thereby increasing livestock populations (anonymous, ) . the demand will further increase potentially infectious contacts between livestock and wildlife leading to an increased potential for new zoonotic diseases to emerge. all of these challenges will require an improved understanding of the ecology of pathogens at the livestock-wildlife interface along with development of tools and mitigations to manage these pathogens. historically, managing diseases affecting both livestock and wildlife as a single, linked system in north america, has presented several obstacles. conflicting agency and institutional missions, program goals, and cultural differences that limit the potential for developing comprehensive mitigation of pathogen transmission contribute to hampering efforts in this area. nevertheless, research and policy at the livestock-wildlife interface has received increased attention in recent years with the number of scientific publications in english journals addressing this topic rising dramatically (fig. ) . this is driven, though not exclusively, by a rapid increase in the number of zoonotic disease events associated with wildlife in the latter part of the th century (dobson and foufopoulos, ; ostfeld and holt, ; decker et al., ) . three-fourths of all emerging infectious diseases (eids) of humans are zoonotic with most originating in wildlife (taylor and latham, ; jones et al., ) . a large proportion ( %) of livestock pathogens-and an even higher proportion ( %) of carnivore pathogens-infect multiple hosts including wildlife (cleaveland et al., ) . therefore, diseases that arise from the livestock-wildlife interface are of paramount importance and must be an area of focus for animal health authorities (siembieda et al., ) . one example, nipah virus-classified as an emerging infectious pathogen-recently moved from its natural host (fruit bats) to domestic swine, causing disease and mortality in both swine and local agricultural workers and resulting in economic losses (epstein et al., a) . the nipah virus outbreak in malaysia destroyed the malaysian swine industry while the associated human fatalities simultaneously created massive public panic (epstein et al., a) . this newly recognized virus was carried by fruit bats for decades and emerged as a result of newly occurring habitat destruction, climatic changes, and the encroachment of food-animal production into wildlife domains (epstein et al., b) . although little discussed, pathogen transmission at the livestock-wildlife interface is frequently bi-directional (bengis et al., ) . in contrast to conventional thinking, livestock have introduced several pathogens, such as bovine brucellosis and tuberculosis bacterium, to naïve wildlife populations in north america. these two pathogens are found in at least five wildlife populations (tessaro, ; sweeney and miller, ) and create significant challenges for disease control at the livestock-wildlife interface. in some instances, spillover events from livestock into wildlife impact conservation of species of concern (dobson and foufopoulos, ; nishi et al., ; joly and messier, ; cross et al., ) . an example is the transmission and introduction of bovine brucellosis and tuberculosis from livestock into native wood bison (bison bisonathabascae) populations in canada, which has created a conservation challenge for the species (tessaro et al., ; nishi et al., ) . another well-publicized example is the introduction of brucellosis into native bison and elk populations of the yellowstone ecosystem in (meagher and meyer, ; meyer and meagher, ) . this resulted in a wildlife management challenge due to conflicts between livestock and bison preservation. the presence of brucellosis poses continued risk for transmission back into livestock creating biological, social, and policy challenges (cross et al., (cross et al., , . obstacles faced by wildlife managers and livestock authorities for mitigating contact between wildlife and livestock has resulted in significant efforts to develop technology that reduces contact and is economically feasible. however, development of effective tools that can be readily deployed has been met with a host of challenges. many devices prove to be ineffective or only effective for a short duration (vercauteren et al., (vercauteren et al., , a . the most successful tools have involved fencing technology (e.g. high fence, wire mesh, electrified high-tensile steel wire, or polytape) that reduces contact between wildlife and livestock feed (vercauteren et al., b (vercauteren et al., , . however, fencing suffers from limitations such as the need for relatively frequent maintenance. more recently, research has focused on the use of historic tools such as livestock protection dogs to prevent contact between livestock and wildlife. in some cases these traditional tools have proven to be the most effective (vercauteren et al., (vercauteren et al., , . in addition to the challenges faced in developing effective mitigation tools is gaining social acceptance of their use by farmers, which is fundamental in successfully using these tools (brook and mclachlan, ) . however, there remains a need for identifying new economically feasible tools that wildlife and livestock managers can deploy to reduce contact at the livestock-wildlife interface. improving our understanding of the biological and anthropogenic processes that promote contact between wildlife and livestock is critical for limiting pathogen transmission at this interface. given the frequently bidirectional nature of pathogen transmission, cooperation is required between livestock owners, animal health officials, and wildlife managers if control efforts are to be successful. conflicts will undoubtedly continue to challenge wildlife managers and livestock authorities seeking solutions, which can only be found through the creation of new partnerships and the strengthening of existing ones that bridge the gap between wildlife and livestock agencies at all levels. here we conduct a systematic review of the english scientific literature to evaluate the status of diseases and pathogens at the livestock-wildlife interface in the united states. specifically, the goals of the literature review were three fold: first, to evaluate domestic animal diseases currently found in the united states where wildlife may play a role; second, to identify critical issues faced in managing these diseases at the livestock-wildlife interface; and third, to identify potential technical and policy strategies for addressing these issues. we highlight two examples of emerging diseases at the livestock-wildlife interface in north america, which pose management challenges and offer an opportunity for comprehensive disease management by facilitating cross-agency and state-federal partnering. in the united states, there are currently avian, ruminant, swine, and lagomorph diseases reportable to the oie. of those, are listed as present in the united states (anonymous, b) . our review of these pathogens identified ( %) which have a potential wildlife component associated with the transmission, maintenance, or life cycle of the pathogen, and ( %) are known to be zoonotic (tables and ). of these pathogens, ( %) have an arthropod vector involved in the transmission while the remaining % involve direct or indirect transmission. sixteen ( %) of the diseases present in the united states affect multiple species of livestock, all of these have a wildlife component, and % are zoonotic. of the oie reportable diseases affecting cattle, out of are present in the united states and have a wildlife component; have zoonotic potential. a wildlife component has been identified for out of ( %) oie reportable avian diseases with of these recognized as zoonotic. of the avian, ruminant, and swine diseases, are currently actively managed in the united states with of these having a federal eradication or control program (table ) . thirteen ( %) of these actively managed diseases have a wildlife component and at least (bovine tuberculosis, paratuberculosis, brucellosis, avian influenza, rabies, and cattle fever tick [vector control]) have a wildlife reservoir that is a recognized impediment to eradication due to continued spillover to domestic populations. of these diseases, (bovine tuberculosis and brucellosis) have foci of infection in wildlife as a result of spillover from livestock-further complicating eradication programs. specific estimates of direct and indirect costs to livestock and recreational hunting industries, and to governmental agencies resulting from pathogen transmission at the livestock-wildlife interface, are elusive; however, some estimates are available for specific diseases. reestablishment of bovine babesia sp. to its historic range in north america via adaptation of babesia sp. vectors to whitetailed deer would cost approximately $ . billion to the cattle industry (anderson et al., ) . in michigan, the loss of bovine tuberculosis accredited-free status is estimated to result in total agriculture and livestock losses of approximately $ million per year (horan and wolf, ) . furthermore, the michigan department of natural resources spent an estimated $ million on defining the extent of the disease in wildlife and initial management steps alone (o'brien et al., ) and to date has spent an estimated $ million on control, surveillance, and management of the disease. rabies-an important zoonotic disease with significant public health, agricultural, and ecological impacts-is known to impose a financial burden on countries around the world. the centers for disease control and prevention estimates that the united states spends in excess of $ million annually on rabies prevention, detection, and control (anonymous, a) with more than $ million spent on wildlife vaccination alone (sterner et al., ) . avian influenza, which has a well-documented wild waterfowl reservoir, continues to plague the domestic poultry (hahn et al., ; corn et al., ; kirkpatrick et al., ; spickler et al., ) direct, indirect avian chlamydiosis ducks c , turkeys c , chickens o gulls r , ducks r , herons r , egrets r , pigeons r , blackbirds r , grackles r , house sparrows r , killdeer r , raptors s , shorebirds s , migratory birds s (vanrompay et al., ; thomas et al., ; spickler et al., ) direct house finches a , american goldfinches a , purple finches a , eastern tufted titmice a , pine grosbeaks a , evening grosbeaks a , others a (thomas et al., ; luttrell et al., ; ley et al., ; spickler et al., ) direct, indirect bluetongue sheep c , goats c , cattle sc wild ovine species a , cervids a , water buffalo a , pronghorn a , (williams and barker, ; stallknecht et al., ; robinson et al., ; spickler et al., ; hoff and trainer, ) arthropod-borne bovine anaplasmosis cattle c cervids r (woldehiwet, ; kuttler, ) arthropod-borne (anonymous, ) arthropod-borne (reisen et al., ; emord and morris, ; komar et al., ; tate et al., ; schmitt et al., ; spickler et al., ) arthropod-borne equine influenza equids c wild birds r , numerous other species s (munster et al., ) direct, indirect equine piroplasmosis equids c uncertain (kellogg et al., ; spickler et al., ) arthropod-borne equine rhinopneumonitis equids c numerous species u (kinyili and thorsen, ) direct, indirect fowl cholera poultry c wild birds r (thomas et al., ; petersen et al., ; botzler, ; blanchong et al., ) direct, indirect infectious bovine rhinotracheitis/infectious pustularvulvovaginitis cattle c several implicated u (zarnke, ; kinyili and thorsen, ) (thomas et al., ; brugh and beard, ; seal et al., ; clubb and hinsch, ; spickler et al., ) direct, indirect (daszak et al., ; thomas et al., ; spickler et al., ) arthropod-borne (thomas et al., ) direct, indirect * bovine babesiosis is not present in cattle in the united states however the causative agent has been reported in wildlife and a vector eradication program exists. ** b. ovis has been found to cause poor semen quality in red deer but abortions have not been reported. the role potential role of red deer is still in doubt. *** the united states is considered free from new castle disease in poultry however new castle disease is present in free ranging species and is included here for completeness. c = clinical sc = subclinical c-sc = may be clinical or subclinical o = occasional reports r = reservoir s = spillover a = affected species (not a true reservoir, nor a spillover host) u = uncertain industry in the united states with estimated outbreak associated losses ranging from $ to $ million (capua and alexander, ; saif and barnes, ) . estimated impacts to the united states in the event of an epizootic avian influenza pandemic are at least $ billion (meltzer et al., ; arnold et al., ) . other livestock diseases with wildlife reservoirs including brucellosis, bovine viral diarrhea, and several poultry diseases are associated with significant losses in livestock production. concepts for integrated and adaptive management systems for eids at the livestock-wildlife interface are proposed by multiple authors (thirgood, ; wasserberg et al., ( s) and sweden ( s) however surveillance systems are established in norway, finland, france, united kingdom, italy, spain, switzerland, and the united states (morner et al., ; pedersen et al., ) . the united kingdom (sainsbury et al., ; lysons et al., ; hartley and gill, ; hartley and lysons, ) has developed a program to implement integrated risk management and eid monitoring systems for wildlife. these nascent emerging systems have common themes, which may be adaptable to the united states. in the existing literature, five interdependent aspects of disease management are suggested as being necessary for successfully addressing disease issues at the livestock-wildlife interface: ( ) horizon scanning (issue identification); ( ) risk analysis and assessment; ( ) risk mitigation; ( ) surveillance and monitoring; and ( ) disease control and management. these components, described as integrating sequentially with feedback loops, incorporate learning about the system. as information about the disease agent is improved, management is adapted thereby improving actions performed in the other components (fig. ) . this process of adaptive management has been well described in the ecological and wildlife management literature (kendall, ; mccarthy and possingham, ) , but concepts related to adaptive management have only recently been proposed as a method for managing disease systems (thirgood, ; wasserberg et al., ). rapid identification of new and emerging infectious diseases (horizon scanning) in wildlife is critical to protecting animal agriculture and human health. there is mounting concern over the zoonotic potential, and subsequent wide-ranging socioeconomic impacts, associated with wildlife-borne eids (jones et al., ) . recent examples of eids emerging from wildlife include nipah virus in swine (chua et al., ) , severe acute respiratory syndrome (sars) in humans (riley et al., ) , and h n hpai in domestic poultry, wild birds, and humans (ferguson et al., ) . in addition, new issues continue to emerge with well-documented disease systems, such as bovine tuberculosis and brucellosis influencing agricultural systems and wildlife management in north america (olsen, a; o'brien et al., ) . other eid's of concern to agriculture are certain to emerge in the future, some of which may disperse rapidly across broad geographic scales (cleaveland et al., ; siembieda et al., ) . the risks of existing and new eid's to disperse rapidly highlight the need for robust systems for early identification of pathogens, which may have important health, social, economic, or other management consequences. risk analysis is an often broadly used term referring to risk characterization, risk communication, and risk management, which provides support for decision making and policy in the face of uncertainty (suter, ) . in the case of animal disease, risk analysis is an important tool used to identify and characterize the potential risks posed by implementation of policy or by specific events such as importation of livestock. risk analyses form the foundation from which animal health policy is established. however, for diseases at the livestock-wildlife interface, quantitative risk assessments are often difficult. challenges for conducting quantitative risk assessments often result from incomplete information related to the disease status of wildlife or limited understanding of the potential contact between wildlife and livestock leading to pathogen transmission. in addition accurate quantitative data describing the spatial distribution, movement, population structure, and population density are typically unavailable limiting inference to the population or understanding population level risk factors. data quantifying important epidemiologic parameters necessary for describing disease risk such as contact rates, disease status of wildlife, wildlife population size, or biological process of the pathogen in wildlife are often unstudied or poorly understood. risk assessments often assess the risk of pathogen transmission from wildlife to livestock (daniels et al., ) . however, for many diseases of livestock in north america (e.g. bovine tuberculosis, brucellosis) the initial transmission event is from livestock to wildlife, which in some cases results in the establishment of a wildlife reservoir for the pathogen posing continued risks to livestock (tessaro, ; sweeney and miller, ) . for these reasons the most successful and useful risk analyses consider the bi-directional nature of transmission and address questions of risk using statistical methods to explicitly incorporate uncertainty. in addition, studies that estimate contact between livestock and wildlife to understand potential for pathogen transmission are needed. mitigating transmission risk between livestock and wildlife has received considerable attention (vercauteren et al., b (vercauteren et al., , wasserberg et al., ) . the ability to eliminate livestock pathogens from north american wildlife populations has been rare and when successful required extensive culling of wildlife. an example is the eradication of foot-and-mouth disease from the united states in which required the culling of , deer from the stanislaus national forest in california (williams and barker, ) . wildlife removal strategies can have unintended consequence, which was exemplified in the united kingdom where wildlife behavior was changed as a result of culling increasing the risk of bovine tuberculosis transmission to cattle (woodroffe et al., ). in addition protected wildlife can complicate control or eradication efforts to control disease (meyer and meagher, ) . eradication efforts requiring the culling of large numbers of wildlife are likely untenable in the united states today, thus preventing establishment of livestock diseases in wildlife populations is a central pillar of long-term risk mitigation strategies. implementing risk mitigations may offer the greatest potential for reducing economic and social impacts resulting from shared diseases. this often involves modifying animal husbandry practices to reduce contact between livestock and wildlife-including modified livestock housing, which reduces contact with peri-domestic wildlife or altered feeding practices, which reduces available forage for wildlife. other risk mitigations include tools that prevent direct contact between wildlife such as frightening devices, fencing, or livestock protection dogs (vercauteren et al., (vercauteren et al., , b (vercauteren et al., , . however, the development and implementation of these tools comes with their own set of challenges. successful implementation often includes changing social behaviors of livestock producers and developing new tools to manage risk mitigation which are cost effective and efficacious over the long term. other risk mitigations may include identifying and reducing or eliminating risky management practices-such as allowing contact between livestock and wildlife which may foster the emergence of new pathogens in the united states. these may include translocation of wildlife or domestic and international wildlife trade. the need to develop comprehensive surveillance systems that integrate livestock, wildlife, and human components has been suggested (mörner et al., ) . robust surveillance systems in wildlife and at the livestock-wildlife interface to provide early detection of newly emerging eids or spillover and spillback of pathogens between livestock and wildlife is essential. developing a comprehensive national monitoring system for eids in wildlife that is logistically and fiscally sustainable could yield economic benefits for livestock health management as a whole by reducing indemnity costs associated with spillover of disease from wildlife to livestock or by helping prevent spillover from livestock to wildlife through early detection. the objectives of such a system could be enhanced by close integration with existing livestock and wildlife health programs to guide "when", "where", and "how" surveillance is conducted. in addition, existing programs would benefit from closer working relationships between wildlife biologists, ecologists, epidemiologists, and veterinarians to improve efforts focused on reducing pathogen transmission (boadella et al., ) . one obstacle to developing long-term, comprehensive surveillance efforts at the livestock-wildlife interface is inconsistent funding for these activities (leighton et al., ; stitt et al., ) . funding has typically been in response to emergency directives (e.g. hpai h n surveillance) and focused for a short period until the threat is perceived to no longer exist. this has, predictably, generated problems for developing a comprehensive national infrastructure that can be maintained over the long-term. in addition, there are disease systems that have plagued agriculture for decades, such as bovine tuberculosis, that do not obtain sufficient levels of funding to fully address risks for introduction into new wildlife hosts such as feral swine (sweeney and miller, ) . another challenge faced by wildlife surveillance systems is that they often rely on hunter observations and reports, which are focused on game species. this increases the difficulty of identifying emergence of disease in non-game species. finally, due to challenges associated with working across agency departmental boundaries, such as reduced communication, differing priorities, perceived competition in missions, and cultural differences wildlife surveillance efforts often remain less than fully coordinated which reduces their overall benefit. once a pathogen is identified at the livestock-wildlife interface, active management and control of the disease agent is often the only method for reducing impacts to human health, agriculture, and recreational hunting industries (boadella et al., ) . integrated strategies that bring wildlife, human, and agricultural agencies together offer the greatest opportunity for success. management of diseases at the livestock-wildlife interface often requires long-term engagement using a combination of altered livestock husbandry practices, active disease suppression in wildlife, and prevention of transmission using mitigation techniques. if surveillance and risk management activities at the livestock-wildlife interface are to be successful, we must recognize the complex nature of current and emerging diseases. these diseases can involve different health jurisdictions, socio-economic dimensions, and a wide range of stakeholders (i.e. livestock industry, conservation organizations, recreational hunters, etc.). we must promote strategic collaboration and partnerships across various disciplines, sectors, departments, ministries, institutions, and organizations at country, regional, and international levels (binder et al., ; fao et al., ) . with the recent focus on "one health", which recognizes that human, animal (both domestic and wild) and ecosystems are tightly linked, successful management of disease requires an integrated approach where efforts are focused in concert across these domains (king et al., ; welburn, ) . in response to the one health focus several countries have developed specific plans to address wildlife health as it relates to human and domestic animal health (sainsbury et al., ; hartley and lysons, ) . however, obstacles still remain in developing robust systems which integrate across the domestic, wild animal, and human domains. in most countries, sector-specific institutions have clear roles, responsibilities, and budgets-but mechanisms for cross-sector collaboration typically do not exist. developing collaborations often proves difficult even mandated from the highest levels of government, as exemplified by continued outbreaks of highly pathogenic avian influenza in several countries (fao et al., ) . the united states suffers from similar limitations, due in part to the bicameral regulatory and legal authority for oversight of livestock and wildlife. states have clear ownership of wildlife; federal regulatory authorities do not always extend to control disease in livestock to manage the disease in wildlife. thus, the effective control of disease incursions from wildlife to livestock requires state and federal livestock management agencies to foster positive working relationships with wildlife agencies. unfortunately, such relationships frequently have not been developed resulting in a decision making process on livestock disease management in which wildlife appear as an afterthought, when often they are integral to disease maintenance and spread. involving all relevant stakeholders (i.e. livestock industry, wildlife conservation groups, wildlife health authorities, livestock health authorities, etc.) in the development of regional or ecosystem-level livestock disease management planning, from the beginning of the process, increases the likelihood of success (loomis, ) . one example of ongoing challenges animal health authorities face is found in the management of brucellosis in the yellowstone ecosystem. controversy has surrounded the management of brucellosis in the bison and elk within the ecosystem. these issues have often pitted federal, state, agricultural, and wildlife agencies against one another. several have noted that one of the most important constraints to managing brucellosis in yellowstone is jurisdictional inertia, or the unwillingness of agencies to relinquish their existing domains of territorial control (lavigne, ; mcbeth and shanahan, ) . this example underscores the need for wildlife, human, and agricultural agencies to develop strong working relationships prior to emergence of disease. integrated approaches to prevention before observing outbreaks in both wildlife and livestock may offer an opportunity for agencies to foster working relationships prior to a crisis. development of clear mechanisms and agreements will enhance collaboration and interaction at all levels and should include incorporation of the roles and mandates of the various institutions and agencies involved. often the agreements and working relationships that are established occur only at the highest levels of the organization resulting in little benefit to those working to implement program objectives. opportunities for professional interactions and working relationships needs to be created and supported at the field level in addition to the administrative level (fao et al., ) . historically, integrated cross-disciplinary collaboration between livestock and wildlife agencies has been a challenge. however, many programs managing animal health diseases could benefit significantly from increased communication and collaborations that combine program objectives and activities across agency jurisdictions. while challenging from a political and cultural perspective, the outcome could be beneficial and would enhance the ability to quickly identify and respond to new and emerging disease issues. integrating state and federal livestock and wildlife agencies into the disease program planning process could reap future rewards. below we illustrate the potential for crosssector collaboration using two disease eradication programs-cattle fever tick eradication and bovine tuberculosis eradication-facing challenges presented by the livestock-wildlife interface. other disease eradication and management programs that address issues associated with the diseases and pathogens listed in table would also likely benefit from increased collaboration across livestock, wildlife, and human agencies at both state and federal levels. bovine babesiosies, caused by hematoprotozoan parasites of the genus babesia, is globally among the most significant tick-borne disease of cattle (white et al., ; martinez et al., ) . in north america, the most important vectors of bovine babesiosis are rhipicephalus microplus and r. annulatus-collectively known as cattle fever ticks. cattle fever ticks were extirpated from the united states in after a nearly -year eradication campaign (graham and hourrigan, ; bram et al., ) . the eradication campaign exploited the perceived narrow host range of cattle fever ticks in combination with highly effective and now banned acaricides, which allowed the program to focus almost exclusively on the treatment of cattle (bram et al., ) . reestablishment of cattle fever ticks and bovine babesia to their historic range in north america is estimated to cost $ . billion in control efforts and cattle production losses (anderson et al., ) . as a result, animal health authorities and livestock producers consider mitigating this risk a priority. in recent years, there have been increasing infestations of cattle fever ticks on cattle along the texas-mexico border ( de león adalberto et al., ) . historically considered to be highly host specific for cattle, there is increasing evidence that white-tailed deer and other ungulates are suitable hosts for cattle fever ticks with infested deer found in locations absent of cattle (cantu et al., ) . in texas, cattle fever ticks have been recovered from free-ranging and captive-exotic ungulates including axis deer, fallow deer, elk, red deer, aoudad sheep, and nilgai antelope (mertins et al., ) . due to the potential ineffectiveness of treating tick infestations in cattle with currently approved methods, such as mandatory removal of cattle from affected pastures for a period of time (i.e. pasture vacation) and treatment of cattle with acaricides the treatment of white-tailed deer and other wildlife has become necessary. a recent study indicates that cattle fever ticks have a high degree of genetic fluidity, which may allow them to adapt to new host species and therefore provide a potential pathway for reestablishment in the united states via wildlife hosts (de meeus et al., ) . white-tailed deer are also increasingly being recognized as a potential reservoir for the babesia species (b. bigemina, b. divergens, and b. bovis) which cause clinical disease in cattle ( de león adalberto et al., ) . surveys for babesia in northern mexico and texas have identified molecular and serological evidence for the presence of b. bigemina and b. bovis in white-tailed deer and in nilgai antelope populations (cantu et al., ; cardenas-canales et al., ) . these changes in the host-pathogen system, and gaps in the understanding of cattle fever tick ecology and the host range of babesia, require the formulation of more effective control strategies that include both wildlife and livestock. to effectively address these challenges, state and federal agencies representing both livestock and wildlife authorities need to partner to develop policy that integrates surveillance and risk mitigations across both cattle and wildlife populations. an historic limitation of the program has been the nearly exclusive focus on controlling cattle fever ticks on cattle . recently the program has begun to deploy mitigations to control ticks on wildlife; however, the program is limited by a lack of operational tools to mitigate infestations on wildlife and a regulatory framework that would integrate management of the disease across wildlife and livestock authorities. while challenging, this offers an exciting opportunity to develop effective strategies and methods to address surveillance at the livestock-wildlife interface and to develop new mitigations that reduce the risk of infestation. bovine tuberculosis (btb), identified in nine geographically distinct wildlife populations in north america and hawaii, is endemic in at least four populations, including members of the bovidae, cervidae, and suidaefamilies (sweeney and miller, ) . the emergence of btb in north american wildlife poses a serious and growing risk for livestock and human health and for the recreational hunting industry. experience in many countries, including the united states and canada, has shown that while btb can be controlled when restricted to livestock species, it is almost impossible to eradicate this disease once it has spread into ecosystems with free-ranging maintenance hosts. recent epidemiological models suggest that once btb is introduced, the probability of becoming established in a wildlife population once introduced is at least % (ramsey et al., ) . spillover into wildlife-and establishment of new foci of infection in wildlife-would be costly to the cattle industry and animal health authorities. in addition, new foci of wildlife infection would complicate eradication efforts. therefore, preventing spillover of mycobacterium bovis into wildlife may be the most effective way to mitigate economic costs of btb. historically, wildlife control efforts for btb have focused solely on potential spillover into wild cervid species. however, m. bovis has been isolated from free-ranging swine (i.e. wild boar and feral swine) in at least countries (letts, ; corner et al., ; essey et al., ; o'reilly and daborn, ; aranaz et al., ; serraino et al., ; palmer, ) . new evidence from mediterranean ecosystems supports the role of wild swine as maintenance hosts of btb-sustaining infection and transmitting the pathogen to other species (aranaz et al., ; naranjo et al., ) . circumstances favoring btb transmission between wildlife and livestock in the mediterranean include artificial increases in wild game populations stimulated by a robust hunting industry, feeding and baiting of wildlife, and intensive cattle grazing in proximity to wild swine (hermoso de mendoza et al., ) . all of these characteristics likely apply to conditions in north america. particularly worrisome is the recent appearance of feral swine in the state of michigan where the potential exists for interaction with btb-infected white-tailed deer and cattle. regions of the southern united states also pose a risk where high densities of feral swine, an established hunting industry, significant baiting and feeding of wildlife, and introductions of btb infected cattle from mexico continue to occur (sweeney and miller, ) . furthermore recent evidence indicates that m. bovis may be present in free ranging white-tailed deer in northern mexico. one study report the presence of m. tuberculosis complex identified using amplification of dna from a tissue by pcr . the authors also report histopathology consistent with m. bovis infection observed in white-tailed deer. another study reported the frequent detection of antibodies against mycobacterium antigens in a cross-sectional survey of white-tailed deer in northern mexico (medrano et al., ) . while the risks posed by wildlife have been recognized, current investigations and response to potential spillover events from cattle to wildlife (cervid or swine), where disease is exceedingly more difficult to control or eradicate is inconsistently managed. few standards are in existence which establish best practices for investigating potential spillover into wildlife hosts. developing national policies and working relationships across agencies responsible for domestic and wildlife health at the state and federal level would have long-term benefits for preventing the risk of introduction of btb into new wildlife host populations. nearly % of the pathogens present in the united states have a potential wildlife component. to successfully manage and control these pathogens at the livestock-wildlife interface will require the development of cross-discipline collaborations and establishing common goals between agencies and organizations that in some cases have rarely worked together. we believe the principles of adaptive management offer the greatest opportunities to formulate a framework from which collaborations can be developed to manage diseases at the livestock-wildlife interface. eid monitoring systems for wildlife that incorporate and implement integrated risk management in an adaptive management framework offer the best opportunity for success. in addition, new and creative funding mechanisms that bring livestock and wildlife animal health authorities along with livestock industry and wildlife stakeholders together will need to be created. despite these social and policy challenges, there remain opportunities to develop new collaborations-along with the development of new technologies-to mitigate disease risks at the livestock-wildlife interface. we believe that two diseases eradication programs-bovine tuberculosis and cattle fever 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impacts of the cattle tick (boophilus microplus) under climate change chronic wasting disease infectious diseases of wild mammals chronic wasting disease of deer and elk: a review with recommendations for management the natural history of anaplasma phagocytophilum bovine tuberculosis in cattle and badgers in localized culling areas serologic survey for selected microbial pathogens in alaskan wildlife we would like to acknowledge the insightful comments and critical review of early versions of this manuscript by dr. steve sweeney, dr. tom deliberto, dr. reginald johnson, dr. kathe bjork, dr. tracey lynn, and mr. allan nelson. we also would like to recognize the diligent efforts of ms. mary foley for supporting our continued literature and library science inquiries and ms. carol losapio for her editing contribution. we also thank two anonymous reviewers for their critical and insightful comments on this manuscript. key: cord- - z cm authors: zou, kelly h.; li, jim z.; salem, lobna a.; imperato, joseph; edwards, jon; ray, amrit title: harnessing real-world evidence to reduce the burden of noncommunicable disease: health information technology and innovation to generate insights date: - - journal: health serv outcomes res methodol doi: . /s - - - sha: doc_id: cord_uid: z cm noncommunicable diseases (ncds) are the leading causes of mortality and morbidity across the world and factors influencing global poverty and slowing economic development. we summarize how the potential power of real-world data (rwd) and real-world evidence (rwe) can be harnessed to help address the disease burden of ncds at global, national, regional and local levels. rwe is essential to understand the epidemiology of ncds, quantify ncd burdens, assist with the early detection of vulnerable populations at high risk of ncds by identifying the most influential risk factors, and evaluate the effectiveness and cost-benefits of treatments, programs, and public policies for ncds. to realize the potential power of rwd and rwe, challenges related to data integration, access, interoperability, standardization of analytical methods, quality control, security, privacy protection, and ethical standards for data use must be addressed. finally, partnerships between academic centers, governments, pharmaceutical companies, and other stakeholders aimed at improving the utilization of rwe can have a substantial beneficial impact in preventing and managing ncds. noncommunicable diseases (ncds) are the leading causes of mortality globally and a major global health challenge that affects people in all countries, regardless of their socioeconomic status (world health organization ; gbd disease and injury incidence and prevalence collaborators ) . approximately % of ncd-related deaths are considered premature, occurring before the age of years, and approximately % of premature deaths occur in low-and middleincome countries (world health organization ) . however, the prevalence of many ncds increases with age (gbd disease and injury incidence and prevalence collaborators ; global burden of disease study collaborators ). the continued growth of the aging population, which is often pronounced in developed economies with relatively advanced healthcare systems, will increase the prevalence of ncds and accentuate disease burden. the world health organization (who) has identified main types of ncds that contribute the greatest burden. they are cardiovascular diseases (cvds), cancers, chronic respiratory diseases, and diabetes ( fig. ). in , ncds accounted for % ( million) of deaths worldwide, with % of these deaths attributable to cvd, % to cancer, % to chronic respiratory disease, and % to diabetes (world health organization ). in the us, cvd accounted for approximately of every deaths that year (benjamin et al. ). in addition, neurological conditions and mental health disorders, such as anxiety disorders, migraine, major depressive disorder, bipolar disorder, and alzheimer's disease, have emerged as major causes of disability (global burden of disease study collaborators ). indeed, mental health and well-being have been highlighted by the united nations as important components of the goal to reduce premature mortality from ncds by % over the next years (united nations ). ncds are characterized by their long duration or continual recurrence (i.e., chronic) and slow progression, and many ncds have high prevalence. for example, hypertension alone is diagnosed in over % of adults in a vast majority of countries across north america, latin america, europe, asia, africa, and oceania (clarivate analytics ). ncds are also major causes of morbidity and disability, including anxiety, depression, pain, and mobility impairment (lisy et al. ) . data from the global burden of disease study showed that the most common chronic sequelae, as consequences of disease, are largely attributable to ncds (global burden of disease study collaborators ). those who are most socioeconomically disadvantaged are often at a high risk of developing ncds (murray et al. ; marmot and bell ; nulu ) . furthermore, in the era of covid- , ncds pose an even greater threat. according to the centers for disease control and prevention, "people of any age with the following conditions are at increased risk for covid- : cancer; chronic kidney disease; chronic obstructive pulmonary disease (copd); immunocompromised state from solid organ transplant; obesity (body mass index [bmi] of or higher); cvd; sickle cell disease; and type diabetes mellitus" (centers for disease control and prevention ). the economic costs of ncds are burdensome to countries around the world (bloom et al. ) . besides ncd treatment and control, the negative impact also includes the reduced productivity at work, more days absent from work (absenteeism), and early retirement, both for individuals with ncds and due to premature death. these costs can lower family economic status, as well as national economic output. furthermore, national funds that need to be deployed toward treatment of ncds take away the funds that might otherwise be invested in infrastructure, research, and education (chen et al. ) . studies also suggest that the medical and economic burdens due to ncds, which are already high, continues to increase, especially in less-developed economies, as well as in middle-and high-income countries (global burden of disease risk factors collaborators ; timmis et al. ). therefore, how the social determinants of health (sdoh) affect ncds is an increasingly important area of focus (marmot and bell ) . governments around the world and international organizations, such as the world bank and who, are increasingly committed to ncd prevention and control (world health organization ). efforts to reduce the burden of ncds are increasingly shifted from treatment to prevention. this is because the majority of ncds occur as a result of modifiable risk factors. therefore, reducing and controlling these risk factors is an effective means of reducing the burden of ncds. the who has called for ncd prevention efforts to focus on the following modifiable behavioral risk factors (tobacco use, physical inactivity, the harmful use of alcohol and unhealthy diets; fig. ) and metabolic risk factors (raised blood pressure, overweight/obesity, hyperglycemia, and hyperlipidemia). prevention not only reduces ncd-related suffering to patients but is also more cost-effective to societies. for example, programs targeted at preventing or treating ncds can have a significantly beneficial effect, with recent estimates suggesting that every us $ spent on tackling ncds will have a return of at least us $ in the following years (world health organization ). a global comprehensive approach is needed to reduce the burden of ncds, which requires the collaboration across various sectors, including academia, industry and governments (upjohn ). this approach should reduce the risk factors for ncd and promote interventions to prevent and control ncds and is especially important during the covid- pandemic (hassan et al. ) . organizations such as the who have realized the importance of innovative data visualization to help educate on both the importance of reducing the global burden of ncds and the stopping the spread of the covid- during . real-world data (rwd) and real-world evidence (rwe) play an important role for international organizations, governments, and societies, helping them to make informed decisions regarding ncd prevention and control. according to the us food and drug administration (fda), rwd means data collected outside of the framework of randomized, controlled trials (rcts) (us food and drug administration ). furthermore, rwe is generated through the analysis of rwd and is used to answer specific clinical and research questions. the lack of rcts designed to assess the burden and degree of the comorbid conditions occurring with ncds means that rwe is the greatest resource available to study this important area. global advances in information technologies and telecommunication infrastructures have enabled a massive amount of rwd to be generated from diverse data sources. the wide variety of data sources include pharmacovigilance databases, electronic medical records (emrs) including medical images/imaging data and free-text notes from healthcare providers, electronic health records (ehrs), administrative insurance claims, patient registries, population health surveys, medical researches including genomics studies, data collected from digital apps and digital recording devices including wearable devices, and various other sources (fig. ) . rwd can be claims and transactions for healthcare resource utilization, electronic health records, surveys, linked datasets, and other digital data collected outside a traditional clinical trial. because rwd can be generated at a low cost (relative to rcts) and rapidly (e.g., streamed from wearable devices), it is often stored and processed in considerable quantity. thus, it can be so-called big data due to its "volume" (there's a lot of it), "variety" (the data takes many different forms), "velocity" (the data changes or are updated frequently) and "veracity" (the data may be of poor/unknown quality) (ibm ; seth ). the immense potential value of data in the modern world has led to it being described as the "the new oil" (the economist ). pharmaceutical companies are now investing in their rwe programs to increase their capabilities in this arena, across all aspects of the drug development and approval process (deloitte ; davis et al. ; morgan et al. ). rwe is the foundation for the understanding of disease epidemiology, including rates of disease incidence and prevalence, awareness, diagnosis, treatment, and control. rwe plays a critical role in quantifying disease burden, which can be measured according to the following aspects: patients' lives saved/lost, gain/loss of daily function, work productivity, and income; quality of life; healthcare resource usage. rwe can also be used to detect vulnerable populations (e.g., elderly and persons at high risk of ncds) and identify the most influential risk factors, which may lead to new and non-traditional solutions to clinical problems (batra and cheung ) . recently, rwe has been increasingly used in the regulatory arena for gaining label expansion, as well as accelerating drug approvals due to regulatory authorities now being more receptive to reviewing rwe (katkade et al. ; zou et al. ) . there is an opportunity to use rwe to study diverse populations that are frequently underrepresented in both clinical and observational studies. for example, patients who richardson et al. ; onder et al. ; hassan et al. ). these comorbid ncds such as cvd, chronic pulmonary disease and diabetes have also been demonstrated to worsen the clinical outcomes and increase the risk of death in those infected with covid- (docherty et al. ; bergman et al. ). this understanding can help deploy preventive strategies to identify people at most risk of contracting severe covid- . this will in turn avoid overburdening the healthcare system. finally, rwe can be used to measure and evaluate the effectiveness and cost-effectiveness of treatments, as well as programs targeting sdohs , and public policies for ncds. global and societal efforts to reduce the populations' risk of ncds (world health organization , ) can be supported by using rwe to monitor incidence and prevalence trends of ncds and risk factors, and to target prevention measures at populations that are vulnerable to ncds. on a larger scale, risk reduction and increased access to interventions can be attained through collaboration among public health policymakers, payors, healthcare providers, and patient groups. disease management can improve, as primary care providers adjust their treatment approaches, access to screening, detection, and treatment services, and more people have access to and can benefit from palliative care. the rationales for these changes will rely heavily on the availability and intelligent use of rwe. only when stakeholders clearly understand the context and implications of ncds can they begin to effect and direct changes. another important aspect to consider is regional and racial differences and variations of the prevalence of ncds and the various risk factors for these diseases (world health organization . this aspect can provide key information as to the effectiveness of local policy initiatives targeted at ncds as well as the influence of various factors and behaviors that could influence ncds, e.g., smoking and obesity (kontis et al. ; office of disease prevention and health promotion ). although rcts remain critical in determining treatment safety, efficacy, and mechanisms of action (collins et al. ) , their focus of patient selection and controlled clinical trial setting make it difficult to generalize the findings of rcts to real-world clinical practice, which often has many confounding factors affecting the effectiveness of the treatment (sherman et al. ). in addition, rcts often focus on the effects of a single disease on outcomes, rather than multiple related conditions, which given the high levels of cooccurrence of ncds, can be a limitation. furthermore, rcts are time-consuming and are becoming increasingly costly to conduct (baumfeld andre et al. ). this is especially true due to the chronic nature of the diseases being discussed. for example, patient followup in rcts is often insufficient to have a clear understanding of patient safety. in contrast, rwe allows a treatment's effectiveness and tolerability to be evaluated in real-world practice, and therefore, rwe is important to assess the long-term safety of medications and can help identify rare adverse events (collins et al. ) . furthermore, rwe provides tremendous opportunities to develop a more holistic understanding of patients and more effective approaches for comprehensive disease management. this is key to modifying patients' behaviors such as improving adherence with treatment, which in turn helps optimize outcomes (see below). while it is premature to suggest that rwe will replace rcts, rwe provides an important complementary mechanism to rcts for healthcare professionals seeking to find novel solutions to address the burden of ncds. for example, rwe has been used for many years to study adherence with medications to treat ncds and evaluate methods of improving adherence with these medications. poor adherence to medications to treat ncds is a major global issue. low adherence with treatment increases the morbidity and mortality burden of ncds, even in high-income countries (khan and socha-dietrich ; brown and bussell ; cutler et al. ) where effective therapies are available, disease burden can only be reduced if patients adhere to the treatment for the prescribed duration (world health organization ; shau et al. ) . improving patient adherence to existing interventions increases treatment effectiveness, resulting in significant overall cost-savings associated with disease burden. therefore, an additional key use of rwe in ncd prevention and control is to better understand how we can improve the complex issues surrounding treatment adherence and persistence (cramer et al. ) . to address this challenge, rwe can be used to estimate adherence and persistence rates and assess the factors associated with these rates . such information can be translated into realistic plans based on authentic insights to improve the proportion of patents adherent with their therapeutic regimens. the availability of rwd may depend on technology, digitization, data capture systems, and data flow regulations (us food and drug administration ). rwd is usually not collected for research purposes and thus can be messy and in many different formats or forms. data accuracy, reliability, and quality must be taken into account when using rwd for research (sherman et al. ) . to make the most of these research opportunities, innovative digital/analytic capabilities and technologies must be enabled and elevated globally for use by medical researchers serving in industry, government, and academia. given the variety of data sources for rwd, important issues arise around data integrity, integration, access, interoperability, standardization, quality control, security, privacy protection, and ethical standards for data use. informed consent is a key consideration in rwd. the us food and drug administration in its guidance for institutional review boards and clinical investigators states that "no investigator may involve a human being as a subject in research covered by these regulations unless the investigator has obtained the legally effective informed consent of the subject or the subject's legally authorized representative. an investigator seeks such consent only under circumstances that provide the prospective subject or the representative sufficient opportunity to consider whether or not to participate and that minimize the possibility of coercion or undue influence. the information that is given to the subject or the representative shall be in language understandable to the subject or the representative. no informed consent, whether oral or written, may include any exculpatory language through which the subject or the representative is made to waive or appear to waive any of the subject's rights, or releases or appears to release the investigator, the sponsor, the institution, or its agents from liability for negligence" (us food and drug administration ). however, informed consent needs to be carefully examined and discussed. nevertheless, informed consent can limit the access to certain types of data or information from some regions. advances in the blockchain technology can also enable dynamic informed consent (mamo et al. ) . when data are available and in aggregate, cutting-edge innovations might be introduced to address such challenges. this is because, in order to be of any use, structured data (i.e., encoded in a standardized data format) and unstructured data (e.g., free-text physician notes) from various sources may be combined and curated into databases using interoperable systems, which can be complicated. this is further complicated when databases obtained from various divergent sources are housed in separate repositories and data warehouses, which creates a major barrier to accessing and collating the relevant data. language differences between countries also present a common challenge in this regard. furthermore, in many countries, data are collected and stored in siloed patient registries in non-standardized ways. these registries are often not connected with each other and contain diverse sets of information, which complicates analysis further. even within the same country, such as the united states, linking patient-level data across various sources can be problematic. sometimes this can be accomplished using tokens that are generated from certain subsets of patient protected health information under the health insurance portability and accountability act of (hipaa) rule (us department of health & human services , ; centers for disease control and prevention ). other times, linkage can occur according to dates or locations of the treatment (curtis et al. ) . in many instances, some level of probabilistic matching, such as propensity score matching, may be required across multiple data sources or datasets (desai et al. ). sensitivity analyses based on different probabilistic matches can be considered to examine the robustness of the matching algorithm. databases can also be difficult to standardize; different diagnostic and treatment codes need to be harmonized (observational health data sciences and informatics ). similarly, it is important to establish and utilize standard and specific analytical methods and algorithms. for example, the observational health data sciences and informatics (ohdsi) group is at the forefront of harmonizing and standardizing the analysis of observational data and has created a common-data model (hripcsak et al. ) . this, along with similar initiatives, will improve data reproducibility and the potential for groups to collaborate while working towards a common aim. an additional complication in the analysis of rwd can be data and privacy protection laws, such as the california consumer privacy act of (ccpa) (state of california department of justice ) or the european union general data protection regulation (gdpr) (european commission ), which will need to be considered carefully when utilizing data from the european union, even without containing personal identifiable information. moreover, when utilizing data from digital devices, it is necessary to consider how best to accurately combine data. for example, glucose monitors may not calculate summary measures in exactly the same way, which necessitates getting the raw data and using software package such as "cgmanalysis" in r to compute values in a consistent manner (vigers ) . once these initial challenges are addressed, robust data curation and analytic algorithms must be derived that are tailored toward specific patient populations. these algorithms will aid the identification of persons with, or at risk of, ncds and help to address the challenge of ncds throughout a patient's ncd journey, including detection, screening, diagnosis, treatment, and monitoring. to achieve this, the current shortage of data translators (who act as shepherds with subject-matter expert knowledge) and data scientists will need to be addressed. the medical and scientific insights gained through rwe-generation can potentially impact global policies to reduce the burden of ncds. in addition, rwe can help provide opportunities to analyze both cross-sectional and longitudinal data to assess and monitor the effectiveness of various interventions designed to target ncds and risk factors for ncds. there is a critical need to address the substantial burden associated with ncds. this is even more urgent in the covid- era given the increased risk of poor clinical outcomes in patients with ncds who become infected with covid- . some of the limitations associated with rcts, notably their often narrow focus, can be addressed by using rwd. therefore, over the coming years, we anticipate seeing more of the power of rwd and rwe being harnessed to address the immense healthcare burden associated with ncds. innovative techniques for both capturing and analyzing data will be utilized. for example, a distributed research network is useful for generating rwe. to enhance the effectiveness and efficiency of healthcare delivery, it is important to understand the risk factors for disease progression, treatment patterns, and utilization. educational initiatives regarding the potential for rwd and rwe to deliver patient-centric, value-base healthcare will lead to further improvements in the management of ncds. however, it is also important to strike the right balance of understanding the challenges and limitations of rwd and rwe to ensure that they are utilized correctly. finally, partnerships can play a key role in improving the utilization of rwe. fruitful collaborative research opportunities exist across different healthcare stakeholders, including academia, industry and government, based on health information technology and innovation for gaining valuable insights. this will, in turn, have a substantial beneficial impact on the prevention and management of ncds. role of real-world evidence in informing cancer care: lessons from colorectal cancer trial designs using real-world data: the changing landscape of the regulatory approval process american heart association council on epidemiology and prevention statistics committee and stroke statistics subcommittee: heart disease and 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technologies real-world evidence-what is it and what can it tell us? most valuable resource is no longer oil of cardiology: cardiovascular disease statistics united nations: sustainable development goal : ensure healthy lives and promote well-being for all at all ages ( ). https ://susta inabl edeve lopme nt.un.org/conte nt/docum ents/ % age nda% for % sus taina ble% dev elopm ent% web upjohn: noncommunicable diseases worldwide: an evidence-based review of key research and strategies to develop sustainable solutions -a white paper us food and drug administration: a guide to informed consent, guidance for institutional review boards and clinical investigators us food and drug administration: guidance for industry electronic source data in clinical investigations cgmanalysis: clean and analyze continuous glucose monitor data health statistics and information systems: disease burden and mortality estimates noncommunicable diseases (ncds) and mental health: challenges and solutions adherence to long-term therapies world health organization: global action plan for the prevention and control of noncommunicable diseases noncommunicable diseases: key facts an acute respiratory infection runs into the most common noncommunicable epidemic-covid- and cardiovascular diseases harnessing real-world data for regulatory use and applying innovative applications key: cord- -dl vjwfn authors: sattar, yasar; ullah, waqas; rauf, hiba; ul hassan virk, hafeez; yadav, sunita; chowdhury, medhat; connerney, michael; mamtani, sahil; pahuja, mohit; patel, raj d.; mir, tanveer; almas, talal; moussa pacha, homam; chadi alraies, m title: covid- cardiovascular epidemiology, cellular pathogenesis, clinical manifestations and management date: - - journal: int j cardiol heart vasc doi: . /j.ijcha. . sha: doc_id: cord_uid: dl vjwfn abstract coronavirus disease (covid- ) is a rapidly progressing global pandemic that may present with a variety of cardiac manifestations including, but not limited to, myocardial injury, myocardial infarction, arrhythmias, heart failure, cardiomyopathy, shock, thromboembolism, and cardiac arrest. these cardiovascular effects are worse in patients who have pre-existing cardiac conditions such as coronary artery disease, hypertension, diabetes mellitus, and coagulation abnormalities. other predisposing risk factors include advanced age, immunocompromised state, and underlying systemic inflammatory conditions. here we review the cellular pathophysiology, clinical manifestations and treatment modalities of the cardiac manifestations seen in patients with covid- . coronavirus disease (covid- ) is a rapidly progressing global pandemic that may present with a variety of cardiac manifestations including, but not limited to, myocardial injury, myocardial infarction, arrhythmias, heart failure, cardiomyopathy, shock, thromboembolism, and cardiac arrest. these cardiovascular effects are worse in patients who have pre-existing cardiac conditions such as coronary artery disease, hypertension, diabetes mellitus, and coagulation abnormalities. other predisposing risk factors include advanced age, immunocompromised state, and underlying systemic inflammatory conditions. here we review the cellular pathophysiology, clinical manifestations and treatment modalities of the cardiac manifestations seen in patients with covid- . severe acute respiratory syndrome coronavirus- (sars-cov- ), the virus causing the coronavirus disease (covid- ) pandemic, has involved more than million cases worldwide. the us has the highest number of infected patients with more than million cases and , deaths by the second week of june, [ , ] . the respiratory symptoms including acute respiratory distress syndrome (ards) are well discussed in the literature. however, the extrapulmonary manifestations with likely cellular cytotoxicity is not well studied [ ] . the cardiovascular sequela of covid- can cause contractility disorders, arrhythmias, pericardial disease, vascular insufficiency, and sudden cardiac arrest. we sought to review cellular cytotoxicity, clinical symptoms, diagnosis and management of cardiovascular complications in covid- . shortly after the outbreak of covid- pneumonia in wuhan, china covid- , its causative agent of sars-cov- was first reported in january, [ ] . this outbreak has rapidly spread across china and globally through person to person transmission. the mean incubation period of this virus ranges between - days, therefore the travelers and suspected contacts are advised to quarantine for days. the basic reproduction number ranges from . to . and can be as high as . in intensive social contacts [ ] . the most common symptoms at the disease onset include fever, sore throat, cough and myalgia. the infected patients may also present with cardiovascular disease (cvd) like acute coronary syndrome(acs) and congestive cardiac failure(chf) [ ] . a study of patients have reported hypertension ( . %), coronary artery disease ( . %) and congestive cardiac failure( . %) as common underlying co-morbidities in confirmed covid- cases [ ] . another study comprising , cases reported five-fold increase in case fatality rates in patients with underlying cvd as compared to patients without cvd ( . % vs . %) [ ] . the impact of covid- on the cardiovascular system is evidenced through multiple studies which report myocarditis in - %, heart failure in %, arrhythmias in % and thrombotic complications in % of hospitalised covid- cases [ , ] . the covid- infection is initiated through binding of s-protein of sars-cov- with the host receptor angiotensin-converting enzyme (ace ) which mediates its entry into the cells. ace- is highly expressed on the pulmonary epithelial cells, cardiac myocytes and vascular endothelial cells which is responsible for extensive cardiopulmonary symptoms [ ] . upon binding with ace- , s-protein cleaves at dibasic arginine site by host protease tmprss to generate s and s subunits. the s subunit induces membrane fusion and viral endocytosis in the cell. after viral entry into the cell, the viral rna is released in the cytoplasm where it replicates and processed into virion-containing particles which fuses with the cell membrane to be released for widespread infection. sars-cov- also internalizes and downregulates the expression of ace- on the cell surface [ ] . since ace- primarily converts angiotensin i and ii to cardioprotective peptides, angiotensin - and angiotensin - ; its loss on cell surface may potentiate cardiac damage. additionally, the loss of ace- on vascular endothelium may exacerbate endothelial dysfunction, inflammation and thrombosis [ , ] . the ace- expression in vascular endothelial cells is linked to underlying pathological state, age and gender. it's activity is reduced in vessels with established atherosclerotic plaques and diabetes whereas it is increased in women and young adults due to a potential role of estrogen [ , ] . since the ace- levels are downregulated in covid- , any underlying factor that diminishes ace- expression compromises the cardioprotective action of ang - /ang - , further promoting the vascular damage. the reduced ace- also induces cytokine release through dysregulating renin-angiotensinaldosterone system, depressing mas receptor (ace /masr axis) and activating ace /bradykinin b r/dabk axis [ ] . these cellular effects are translated into exacerbation of underlying cardiovascular disease or new onset of cardiac symptoms. the cardiac complications of covid- can be divided into electrical and mechanical dysfunction. the electrical aberrance is seen in arrhythmias whereas pericardial, myocardial, valvular and vascular complications arise due to mechanical dysfunction. arrhythmia in covid- can be secondary to electrolyte imbalance, pulmonary disease, medication side effects, activated protein kinase c (pkc), or direct oxidized ca + /calmodulindependent protein kinase ii (camkii) [ , ] . in particular, hypokalemia and hypomagnesemia are commonly seen in covid- due to angiotensin ii-mediated urinary excretion and gastrointestinal losses [ ] . the hypoxia in ards can increase the risk of arrhythmias as evidenced in an experimental study by clark et al. which reported arrhythmias in % of the hypoxic rats [ , ] . the widely used drugs chloroquine, hydroxychloroquine, azithromycin, and lopinavir/ritonavir can also trigger arrhythmias by prolonging qt [ ] . the upregulated angiotensin ii and angiotensin ii receptor type axis (angii-at r) activates the pkc which interferes with initiation and propagation of action potentials leading to arrhythmias [ , ] . another probable mechanism is that ang ii activates nadph oxidase via phosphorylation and translocation of p phox [ ] . the activated nadph oxidase increases the reactive oxygen species (ros) which oxidizes the camkii into ox-camkii. the ox-camkii in turn phosphorylates ryanodine receptor (ryr ) leading to increased diastolic sarcoplasmic ca + leak which triggers delayed after depolarizations and induces atrial and ventricular arrhythmias [ , ] . the systemic inflammation in covid- may also dysregulate the post-translational modification of cardiac ion channels resulting in arrhythmia [ , ] it is also noteworthy that viral proteins of sars-cov- , orf and orf , activate nlrp inflammasomes which inturn promotes atrial fibrillation [ , ] . elevated angiotensin ii levels facilitate an inflammatory cascade via nf-kb, elevated il- , tgf-β and vascular endothelial growth factor (vegf). these cytokines promote serosal inflammation and fibrosis that may present as pericarditis [ ] [ ] [ ] [ ] . sars-cov- can cause pericarditis that may progress to pericardial effusion. pericarditis associated with pericardial effusion creates physical stress and can further complicate takotsubo cardiomyopathy [ ] . the combined cases of myocarditis and pericarditis cases due to covid- have also been reported [ , ] . the clinical presentation may overlap with other covid- symptoms like fever, chest pain, and severe hypotension. the diagnosis is made through a combined diagnostic approach including electrocardiography, echocardiography, and chest radiographs. myocardial dysfunction in the setting of covid- can present as acs, myocarditis, heart failure, stress cardiomyopathy, and shock. several mechanisms have been suggested for the myocardial injury associated with covid- . sars-cov- can induce systemic inflammatory response syndrome (sirs) through a combination of cytokine release syndrome (crs) and dysregulated immune activity that leads to myocardial dysfunction [ , ] . the immune system recognizes sars-cov- via recognition of intrinsic viral rna patterns, or the pathogen-associated molecular patterns (pamps). this identified pathogen attaches to the pattern recognition receptor (prrs), which initiate host-immune defense against the virus. the interaction of pamps with prrs in combination with a dysregulated inflammatory response in the setting of sepsis leads to cytokine storm. cytokines contribute to myocardial damage by facilitating the release of reactive oxygen species (ros), endogenous nitric oxide, and superoxide anion ( figure ). eventually, damage-associated molecular proteins (damps) like heat-shock proteins, highmobility group box (hmgb ), histones, and oxidized lipoproteins are released from the damaged myocardium which further activate the inflammation and injure myocytes. these excessive damps create an ongoing vicious inflammatory cycle resulting in septic/covid- cardiomyopathy [ , ] . the notch signaling pathway also promotes inflammatory cytokines, facilitates the sars-cov- infection, and plays a major role in structural cardiac remodeling therefore a therapy directed at modulation of notch pathway can limit the covid- infection and limit the inadvertent remodeling [ ] . the sars-cov- may cause direct myocardial cytotoxicity via c proteinase-mediated apoptosis, disruption of host protein translational mechanisms, loss of cellular homeostasis, and dysregulated host-immune response [ , ] . after viral infection of cardiomyocytes, the immune system triggers the infiltration of natural killer cells, macrophages, and lymphocytes. these cells initially confer protective effects, but hyper-activation can worsen the severity of myocarditis and lead to cardiomyopathy [ ] . biopsy specimens in myocarditis have demonstrated infiltration of mononuclear cells in the cardiac myocytes causing focal or diffuse damage [ ] . in addition, the hypoxemia in covid- can induce increased intracellular calcium levels which can result in cardiomyocyte damage and eventual cardiomyopathy [ ] . covid- can also cause stress cardiomyopathy due to emotional or physical stress on cardiac myocardium in the setting of anxiety and widespread inflammation [ ] .the myocardial stunning in the setting of catecholamine surge and coronary microvascular dysfunction may be exacerbated by covid- from ras dysregulation and plaque disruption [ ] . increased catecholamines further worsens the microvascular dysfunction through increasing cytokine production of il- via alpha- adrenergic receptor signaling in immune cells [ ] . the excessive catecholamines may also worsen the transient hypokinesia through beta -coupling from gs to gi [ ] . the combined effects of catecholamine surge and microvascular dysfunction in covid- may result in hypercontraction of the left ventricular apex and outflow tract resulting in stress cardiomyopathy. the increased cardiometabolic demand in systemic infection coupled with hypoxia secondary to pulmonary dysfunction can create myocardial oxygen supply/demand mismatch. these events when accompanied by inflammation induced hypercoagulation, microthrombi and plaque rupture can lead to cardiac ischemia and acute coronary syndrome [ ] . it is unclear whether the sars-cov- -mediated myocardial damage will have long-term implications on cardiac function and survival therefore follow up data on survivors is required to better assess the long-term outcomes. the symptoms of acute coronary syndrome (acs) include chest pain, dyspnea, and sinus dysfunction which can overlap with covid- symptoms [ ] . a case series of covid- patients with stemi reported that % of cases manifested chest pain at the time of stelevation. among these patients, % died of myocardial infarction whereas % died of non-coronary myocardial injury [ ] . the diagnosis of acs requires elevated troponin along with either echocardiographic or electrocardiographic abnormalities [ ] . the ventricular dysfunction in covid- may present with features of heart failure, cardiogenic shock, and myocarditis [ ] . their clinical presentation will overlap with the symptoms of covid- including respiratory distress and hypotension. the diagnosis can only be ascertained by utilizing various diagnostic modalities such as cardiac markers, echocardiography, mri, and biopsy. a study reports that heart failure is a common manifestation of covid- patients, seen in % of affected patients [ ] . cardiogenic shock was reported in a few cases with depressed left ventricular ejection fraction, left ventricular hypokinesis, and elevated cardiac markers [ , ] . a case of myocarditis showed similar diagnostic features but presented without the typical respiratory features of covid- [ ] . cardiovascular complications may present in isolation without respiratory symptoms in covid- . the ace- is widely expressed on the stromal fibroblasts of cardiac valves, particularly aortic valves. it is evidenced from the rt-pcr that the ace- expression is downregulated in stenotic valves. the depressed ace /angiotensin-( - )/mas receptor axis may potentiate inflammation, fibrosis and valvular sclerosis [ ] . simultaneously, upregulated angii-at r axis induces inflammatory cytokines like tnf-α and il- amplifier (il- amp) which further induces pro-inflammatory cytokines and recruits macrophages to create a positive feedback loop of inflammation [ ] . these downstream effects of sars-cov- infection result in valvular damage which may not present acutely due to the slowly progressive nature of cardiac valvular disease. however, periodic follow-up of cardiac function is essential in covid- survivors to timely identify this pathology. hypertension is frequently seen in covid- patients however it is unclear whether it is a risk factor for acquiring covid- . the unregulated angiotensin ii stimulates vasoconstriction and systemic inflammation which may worsen the underlying hypertension through oxidative stress and endothelial dysfunction. the reactive oxygen species (ros) like superoxide, interacts with nitric oxide to form peroxynitrite which reduces the availability of no and inhibits the enos activity [ ] . the elevated levels of inflammatory markers like crp and tnf also destabilize enos and limit no production. since no has vasodilatory and anti-inflammatory effects, its reduced bioavailability can exacerbate hypertension. the ros can also impair the vascular structure and function by directly inducing cell damage [ ] . hypercoagulation is another commonly seen manifestation of the disease [ ] [ ] [ ] . diffuse cytokine storm, elevated angiotensin ii, endothelin- , plasminogen activator inhibitor (pai- ), tissue factor, ros, and extrinsic clotting cascade are all interrelated pathophysiological components of hypercoagulation [ ] (figure ). the endothelial cells line the intima of the vasculature and are closely connected by adherents, tight and gap junctions which maintain the endothelial integrity [ ] . in sars-cov- , excessive cytokines, ros and proteases from the activated neutrophils may damage these junctional proteins, resulting in trans-endothelial migration of neutrophils [ ] . these neutrophils degranulate and form neutrophil extracellular traps which release damps (dna and neutrophil elastase) that further damage the junctional proteins and endothelial layer [ ] . this leads to greater serum contact with subendothelial tissue, which activates von-willebrand factor, platelet adhesion, and the coagulation cascade. inflammation leads to increased pai- , which increases endothelial fibrin deposition and reduced thrombolysis, ultimately contributing to the hypercoagulable and prothrombotic state of covid- ( figure ). the coagulation profile in covid- patients may show thrombocytopenia, elevated ddimer, prolonged prothrombin time, international normalized ratio (inr), and short activated partial thromboplastin time (aptt) [ ] . this coagulation disarray predisposes patients to thromboembolic complications. a case study reports an incidence of acute pulmonary embolism with right ventricular failure in a covid- patient which demonstrated elevated d-dimers at ng/ml [ ] . the increased risk of thrombosis further predisposes the risk of disseminated intravascular coagulation (dic), venous thromboembolism (vte) and multi-organ failure. there are multiple risk stratification tools to ascertain the risk of vte in covid- [ ] . another study has shown that . % of the non-survivors of covid- had dic with elevated d-dimer [ ] . given the high mortality in patients who develop dic, there is an expert consensus as reviewed by song et al. regarding the management of coagulopathy in these patients. current recommendations emphasize the importance of routine monitoring of coagulation parameters and the implementation of scores to diagnose covid- related coagulopathy. a variety of different drug treatments are being investigated in covid- infection despite the lack of definitive evidence about their efficacy. the proposed drug regimens include hydroxychloroquine, azithromycin, lopinavir/ritonavir, tocilizumab, remdesivir and ribavirin [ ] . the role of hydroxychloroquine in treating covid- patients with cvd is controversial. it inhibits binding of sars-cov- with ace- receptor by blocking tmprss- , and inhibits viral entry by altering endosomal ph [ ] . this drug has shown positive outcomes in terms of viral clearance however its safety in patients with cardiovascular disease is not clearly established due to potential side effects of polymorphic ventricular tachycardia, long qt-syndrome and increased risk of sudden death [ ] . a clinical trial of patients reported high dose of chloroquine is associated with severe arrhythmias and deaths and was therefore halted [ ] . since different drugs used in covid- have a certain risk of pro-arrhythmic side effects, it should be balanced with the anticipated benefit of therapy and administered under close monitoring. arrhythmias in covid- are managed based on etiology including repletion of electrolytes, discontinuation of medications causing arrhythmia, managing volume status, or suppressing catecholamine surge in covid- [ , [ ] [ ] [ ] . brugada syndrome can be treated by alleviating the underlying triggers including fever, or pneumonia, or icd placement in cases of brugada- [ ] [ ] [ ] . furthermore, in the setting of shock in covid- , the use of vasopressor such as dobutamine, epinephrine or pde-inhibitors such as milrinone should be avoided due to synergistic proarrhythmic activity of these medications. in hemodynamically stable arrhythmias, beta-blockers should be used and epinephrine should be avoided especially during the catecholaminergic surge in covid- [ ] . the pericarditis in covid- is treated with steroids and supportive care however pericardiocentesis may be required in pericardial effusion. the collected fluid should be checked for cytology, autoimmune reactions, cultures and tested for sars-cov- [ ] . the choice of treatment in covid- patients with stemi depends on the availability of a pci facility, or fibrinolytic and institutional policy of covid- stemi cases. anticoagulant therapies, such as unfractionated heparin or enoxaparin, may also be considered in covid- with stemi and nstemi [ ] . in covid- with nstemi, the decision to perform pci depends on the global registry of acute clinical events (grace) score > , or thrombolysis in myocardial infarction (timi) > or hemodynamic instability [ ] . as per the protocol by one chinese medical center, for stable patients who tested positive for covid- with stemi, it is reasonable to administer thrombolytics if onset of symptoms is < hours, pending no contraindications. elective pci can be considered once the patients are treated and have recovered from covid- . however, in unstable patients and those who do not have severe pneumonia, the recommendation assessed the risks and benefits of early pci [ ] . since dual antiplatelet therapy (dapt) after pci in covid- patients has increased bleeding risk, the new strategy is to reduce the duration of aspirin to - months and allow prolonged use of p y inhibitors. the early antiplatelet therapy with p y antagonists like ticagrelor, is beneficial after pci in covid- due to the inhibitory effects on platelet activation and intravascular thrombus formation [ ] . furthermore, the management of cardiovascular cardiomyopathy is based on type of manifestation. the treatment of hemodynamically stable heart failure involved optimal medical regimen including aldosterone antagonists, diuretics, and beta-blockers [ , ] . in patients with cardiogenic shock, iatrogenic agents such as milrinone, a phosphodiesterase -inhibitor (pde- i) can be used under close observation [ ] . there is widespread controversy about the use of acei, arbs, and neprilysin inhibitors (sacubitril) in covid- exacerbated heart failure. acei/arbs can worsen covid- by increasing ace expression which may amplify the chances of viral invasion [ , ] (figure ). on the contrary, ace inhibitors can be beneficial by increasing ace- and further opposing the action of angiotensin ii receptor type (at r) through increasing ang - and, ang - downstream effects [ ] (figure ) . a case-control study by mancia et al. showed no association of risk of covid- with the use of acei in overall case patients (odds ratio, . [ % ci, . to . ]) and those with fatal outcomes (odds ratio, . [ % ci, . to . ) [ ] . however, we still need evidence from different randomized clinical trials to support the use of acei/arbs in these patients. the summary of balance between at r and ang - , ang - is shown in figure . in patients with refractory cardiogenic shock in the setting of covid- , veno-arterial (vav) extracorporeal membrane oxygenation (ecmo) can be helpful [ , ] . many covid- patients present with acute respiratory distress syndrome and pulmonary embolism which can increase the right ventricular afterload resulting in impaired right ventricular function. echocardiogram and other advanced imaging modalities can play a significant role in determining the right ventricular dysfunction and ascertain the prognosis [ ] . covid myocarditis can be treated with immunosuppressants including steroids, cyclosporine, azathioprine, and immunoglobulins [ , ] . il- antagonists can be efficacious as they inhibit cytokine storms in covid- [ ] . the steroid use is controversial as chen et al. suggested that steroids might not be helpful in viral myocarditis however few reports have shown favorable outcomes in fulminant covid- cases [ , ] . in addition, a placebocontrolled trial has shown safety of intravenous immunoglobulin (ivig) in idiopathic dilated cardiomyopathy or biopsy-proven viral myocarditis with no added benefit in improvement of lv function [ ] . ivig can be considered a salvage therapy in fulminant myocarditis with hemodynamic instability [ ] . preliminary studies about the use of convalescent plasma have also demonstrated efficacy in critical covid- infection [ ] . statins also have a potential benefit in limiting cardiovascular complications in covid- through inhibiting toll-like receptors (tlr)-nf-κb pathway. it's use may also counter the hyperlipidemic sideeffects of other antiviral drugs used in covid- [ ] . multiple studies have supported the use of acei and arbs in hypertension due to their potential to improve the clinical symptoms and reduce mortality. a study reported a lower rate of severe disease and il- levels in covid- patients with hypertension who were being treated with acei /arbs. hence, these drugs may prevent the worsening of blood pressure due to inhibition of inflammatory response [ ] . the international society on thrombosis and haemostasis (isth) recommends vte prophylaxis in hospitalised covid- patients. in non-icu hospitalised cases, standard or intermediate dose of low molecular weight heparin (lmwh) is preferred for thromboprophylaxis however in icu patients, a combined approach of prophylactic or intermediate-dose lmwh with mechanical compression device is advised after assessing the bleeding risk. the duration of postdischarge thromboprophylaxis in covid- patients ranges between to days [ ] . a retrospective study has shown reduced -day mortality in covid- patients with elevated d-dimers who used heparin as compared to non-heparin users. (p= . ). hence, prophylaxis with low molecular weight heparin may have a role in preventing covid- associated thrombotic complications and mortality [ ] . in cases of diagnosed vte, parenteral anticoagulation with lmwh is preferred in an in-patient setting whereas direct oral anticoagulants (doac) are given after hospital discharge. the total duration of treatment in diagnosed vte is at least months [ ] . there is no evidence that antiplatelets increase the risk of severe covid- hence it should be continued for conditions like recent mi or pci within the last months. the decision for antiplatelet therapy is individualised on a case to case basis depending on the platelet count, bleeding risk and drug-drug interaction [ ] . covid- infection has been linked to myocardial injury, leading to severe disease progression. although incredible efforts are being done to accurately comprehend the mechanism of myocardial injury, it is prudent to expand the randomized controlled data. cardiologists 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critically ill extracellular potassium modulation of drug block of ikr. implications for torsade de pointes and reverse use-dependence covid- infection unmasking brugada syndrome. heartrhythm case rep the known into the unknown: brugada syndrome and covid- . jacc case rep sars-cov- , covid- , and inherited arrhythmia syndromes. heart rhythm catecholaminergic polymorphic ventricular tachycardia: the cardiac arrest where epinephrine is contraindicated st-elevation myocardial infarction in patients with covid- : clinical and angiographic outcomes. circulation. management of acute myocardial infarction during the covid- pandemic how to balance acute myocardial infarction and covid- : the protocols from sichuan provincial people's hospital antiplatelet therapy after percutaneous coronary intervention in patients with covid- : implications from clinical features to pathologic findings management of the hospitalized covid- patient with acute cardiomyopathy or heart failure renin-angiotensin-aldosterone system inhibitors in patients with covid- effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme statement addresses concerns re: using raas antagonists in covid- renin-angiotensin-aldosterone system blockers and the risk of covid- myocardial localization of coronavirus in covid- cardiogenic shock first case of covid- complicated with fulminant myocarditis: a case report and insights. infection prognostic value of right ventricular longitudinal strain in patients with covid- coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin a clinical trial of immunosuppressive therapy for myocarditis. the myocarditis treatment trial investigators the imperfect cytokine storm: severe covid- with ards in patient on durable lvad support corticosteroids for viral myocarditis controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy treatment of critically ill patients with covid- with convalescent plasma statin therapy in covid- infection covid- , hypertension and cardiovascular diseases: should we change the therapy? scientific and standardization committee communication: clinical guidance on the diagnosis, prevention and treatment of venous thromboembolism in hospitalized patients with covid- anticoagulation in covid- covid- and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review increased ros can also cause camk-ii activation causing early depolarization and arrhythmias. increased et also leads to increased thrombosis by increased endothelial dysfunction by oxidizing low density lipoprotein and pai- . lipopolysaccharide binds to receptor tlr- increasing nfkbeta ang - have a positive role in our body, and can fight against covid but their levels, and downstream pathways are decreased in covid- patients due to low ace- r levels. enos, npr a/b r, at r, masr normally counteracts the negative effects of at r abbreviations: sars-cov- : severe acute respiratory syndrome coronavirus- il- : interleukin ; ang ii: angiotensin ii ang - : angiotensin - ; ace: angiotensin converting enzyme; at r: angiotensin ii receptor type ; at r: angiotensin ii receptor type natriuretic peptide receptor a/b guanylate cyclase; tnfα: tumor necrosis factor alpha; inos: inducible nitric oxide synthase; enos: endothelial nitric oxide synthase; tgf-β: transforming growth factor beta; ros: reactive oxygen species; camk-ii: calmodulin dependent protein kinase ii; cam: calmodulin; cgmp: cyclic guanosine monophosphate atrial/brain natriuretic peptide tlr : transmembrane lipopolysaccharide receptor ; icam: intercellular adhesion molecule; vcam: vascular cell adhesion molecule; nf-κb: nuclear factor kappa-light-chain-enhancer of activated b cells; pge : prostaglandin e; masr: mitochondrial assembly g protein coupled receptor; nepa: neprilysin a receptor; nepb: neprilysin b receptor; npr a/b: neprilysin receptor a/b; hdac: histone deacetylase; gata: globin transcription factor; nfat: nuclear factor of activated t-cells; ppar-γ: peroxisome proliferatoractivated receptor gamma sodium ion; ncx: sodium-calcium exchanger stemi: st-segment elevation myocardial infarction color coding: green frames resemble the protective and anti-inflammatory mechanisms, which are imbalanced by the pro-inflammatory covid- state depicted in red causing overt inflammation and cell destruction; brown color depicts the potential therapeutic targets that can help balance this inflammatory state; yellow color shows all enzymes involved in raas or covid key: cord- - w zepx authors: bassareo, pier paolo; melis, maria rosaria; marras, silvia; calcaterra, giuseppe title: learning from the past in the covid- era: rediscovery of quarantine, previous pandemics, origin of hospitals and national healthcare systems, and ethics in medicine date: - - journal: postgrad med j doi: . /postgradmedj- - sha: doc_id: cord_uid: w zepx after the dramatic coronavirus outbreak at the end of in wuhan, hubei province, china, on march , a pandemic was declared by the who. most countries worldwide imposed a quarantine or lockdown to their citizens, in an attempt to prevent uncontrolled infection from spreading. historically, quarantine is the -day period of forced isolation to prevent the spread of an infectious disease. in this educational paper, a historical overview from the sacred temples of ancient greece—the cradle of medicine—to modern hospitals, along with the conceive of healthcare systems, is provided. a few foods for thought as to the conflict between ethics in medicine and shortage of personnel and financial resources in the coronavirus disease era are offered as well. in december , a series of pneumonia cases of unknown origin were reported in wuhan, hubei province, china. on january , after ascertaining the viral origin of the disease, the who recognised a coronavirus responsible for the disease and named it as the novel coronavirus ( -ncov). again, based on its genetic similarity to already known coronaviruses, the international committee on taxonomy of viruses renamed the previously termed -ncov as severe acute respiratory syndrome coronavirus (sars-cov- ). conversely, the first coronavirus or coronavirus was responsible for sars epidemic in . on january , the who released a public health emergency of international concern. on february , the who formally named the disease as covid- . on march , the who formally recognised the global spread of covid- as a pandemic: the second in this century and the first to be caused by a coronavirus. the first description of human coronavirus was published by the scottish virologist june hart almeida in the british medical journal in , while working in st thomas's hospital medical school (london). since the spokes around the viral edge reminded researchers of a crown, the virus was named corona (crown in latin) in a subsequent paper published in nature ( ). since march , sars-cov- has changed our daily life, behaviour and maybe the whole human history. sars-cov- has spread throughout our nightmares with the same strength as it has had in breaking into our lives. quarantine, spread, isolation, lockdown, cocooning, masking, social distancing, hand hygiene, blood tests, vaccine, immunity: all these words are now part of our daily speeches, pervading the most subconscious of our thoughts. our healthcare system is now facing huge challenges with the spread this virus, and so it is getting a favourable occasion to review the history of the previous infections which the human being had to face in the past, with the ultimate aim of educating young doctors to learn from those events. in daily conversations, people often use the term 'quarantine' to refer to separating sick people with a contagious disease from the healthy ones in various ways. quarantine or lockdown is a forced segregation to prevent a communicable disease from spreading. the term 'quarantine' comes from the italian word 'quarantena', which means a period of days (in italian, is 'quaranta'; the latter derives from the latin word quadrāgintā). during the th century, days was the length of strict isolation required for ships suspected of carrying an infectious or contagious illness before their passengers and crew were allowed to land. this practice was usual in venice in the s, in an effort to stave off plague ('black death'). it entered english language in the early s. an early mention of 'isolation' dates back to the th century b.c. or maybe earlier in the leviticus, the third book of the old testament in the bible. in that book, the procedure of separating infected from healthy people to prevent leprosy from spreading, according to mosaic law was described, (if the shiny spot on the skin is white but does not appear to be more than skin deep and the hair in it has not turned white, the priest is to isolate the affected person for seven days. on the seventh day the priest is to examine him, and if he sees that the sore is unchanged and has not spread in the skin, he is to isolate him for another seven days). however, nowhere in these forerunner reports was the term 'quarantine' used. after arriving in southern europe in , plague spread rapidly and reached england, germany and russia by . over these years, one-third of the european population passed away. the dramatic impact of the epidemic led governments to put in place extreme infection control measures. for instance, in , viscount bernabo of reggio, italy, declared that every person with plague was to be taken out of that city into the fields, where to die or to recover. the date, july , is one of the highest achievements in the history of medicine. originally, the period of isolation was limited to days. as reported in the first available documents, before entering the seaside city-state of ragusa in dalmatia (now dubrovnik in croatia), newly arrived people had to spend days (a 'trentine') in a restricted place in the islands in front of the city (see box ), [ ] [ ] [ ] waiting to see whether the symptoms of black death would develop. the city chief doctor jacob of padua was responsible for the decision. at that time, ragusa was an important adriatic harbour belonging to the maritime republic of venice. a specific decree (veniens de locis pestiferis non intret ragusium nel districtum) was published in dubrovnik's book of laws, the so-called green book (latin: liber viridis). the -day period outlined in the 'quarantine' edict was known in italian as a 'trentino' ('thirty' in english). four were the outlined point in the edicts, namely: ( ) those coming from black death-endemic regions would not be admitted into ragusa until they had waited in isolation for days; ( ) none from ragusa was allowed to access the isolation area, under penalty of being compelled to stay there for days; ( ) subjects not indicated by the great council as responsible of those being isolated were not allowed to bring them food, under penalty of remaining with them for days; and ( ) whoever did not accomplish with the above stated regulations would be fined and subjected to isolation for . in , the great council of ragusa released a new decree (de ordinibus contra eos qui veniunt de locis pestiferis anno factis), which specified again the -day duration of quarantine and determined the place. penalties were imposed for those breaking rules, and three healthcare officers (called kacamorti) were appointed to supervise the compliance with quarantine provisions. the penalties for not complying with the provided regulations were represented by a money fine or prison sentence or severe corporal punishment. the penalties were applied only to common people but not to aristocracy. in addition, the importation of goods from the countryside for the entire duration of the epidemic was forbidden. in , the venetian senate, the main deliberative and legislative body of the republic of venice, prolonged the waiting period to days, thus giving birth to the term 'quarantine'. over the next years, similar edicts were published in marseilles, venice, pisa and genoa. since disease was considered as a divine punishment for sinners, the biblical -day period of purification had crossed over into health practices and the term 'quarantine' had great symbolic and religious significance to medieval christians. in the sacred scripture, when god flooded the earth, it rained over days and nights; moses stayed on mt. sinai for days; jesus fasted and was tempted by the devil in the desert for days; after childbirth, a new mother was expected to rest for days. on the basis of a th-century tale by the genoese gabriele de' mussi, the devasting black death came over italy carried by genoese sailors who sailed from caffa/kaffa (now feodosija, ukraine) to their home city ( / ). in fact, in , the tartar forces captained by jani beg khan had laid a military operation in which his forces surrounded caffa, in an attempt to cut off essential supplies, with the aim of compelling the genoese to surrender and removing them from one of the cornerstones of europe's defence against eastern attack. after several unsuccessful assaults due to the outbreak of the black death, jani beg khan's army catapulted infected bodies over the city walls into caffa, using the plague to weaken the defenders. that is the first known example of biological warfare in the human history. a caffa street (via caffa in italian) is still present in the city centre of genoa to remember that event (figure ). however, the number of crimean harbours under mongol army control suggest that caffa is unlikely to have been the only source of plague-infested ships heading to europe. in addition, the overland caravan routes from mongol territories might be considered responsible for infecting europe as well. this plague was well described by one of the most famous italian authors of the th century, giovanni boccaccio ( - ), in his book the decameron written in tuscan vernacular (italian). the book is a collection of short stories told by a group of seven young women and three young men sheltering in a villa just outside florence to escape the black death that afflicted that city. boccaccio probably conceived his masterpiece of classical italian renaissance prose after the plague epidemic of , which came to a standstill by . the black death in the th century resulted in more than million deaths and is considered one of the greatest public health disasters in recorded history as well as one of the most dramatic examples ever of a periodically re-emerging disease. between and , the black death box origin of the term 'lazaret' ► all merchants, sailors and goods coming from 'suspicious lands' could not enter ragusa, unless they have spent a month on quarantine on uninhabited islands of mrkan, bobara and supetar in front of the city. a few wooden shelters (wooden so that they could be burned if needed) were built on them because of the unfavourable weather. this sort of simple dwelling was called lazaret (italian: lazzaretto). the term derives from the biblical story of beggar lazarus, who was raised from the dead out of the tomb by jesus christ. following that, another lazaret was built on a small island in the lagoon in front of venice in . in , the maritime republic of genoa followed the example of venice, and in , the old leper hospital in marseille was turned into a black death hospital. the great lazaret of marseille, perhaps the most complete of its kind, was built in on the island of pomègues. the procedures in the lazarets in the mediterranean sea did not differ a lot from those of the british empire in the levantine and north african trade. as soon as cholera arrived ( ), a few new lazarets were built, especially near the harbour of bordeaux in the human history, similar isolation centres were built for those with leprosy (leprosarium) or tuberculosis (tubercolosarium). killed about one-third of the european population, and a significant percentage of that in asia. only a few centuries later, it was discovered that plague or black death is a bacterial disease caused by the gram-negative pathogen yersinia pestis, discovered in by the swiss/ french physician alexandre yersin and later ( ) renamed 'yersinia' in his honour. it is mainly spread by infected fleas from rats. according to current knowledge, the bubonic plague has an averaged -day period from infection to death; therefore, the european quarantines would be highly successful in determining the health of crews from potential trading and supply ships. ('when leaving his surgery on the morning of april , dr. bernard rieux felt something soft under his foot. it was a dead rat lying in the middle of the landing.' albert camus , french writer, in his book the plague). following that, in the human history, quarantine was declared many times when an infectious disease threated the population of a town, region or country. the most dangerous pandemics in the human history are summarised in box . as a general rule, the more the human beings became civilised -leaving agrarian life, building towns, forging trade routes to connect remote regions one another and fighting wars for supremacy-the more pandemics showed up. in the usa, the process of developing national quarantine policy required many years. originally, as to many other matters, the single federal states were responsible for handling the influx of infectious illnesses. however, repeated outbreaks of yellow fever induced the congress to release the national quarantine act in , thus creating the premises for federal involvement. in , a cholera outbreak led officials giving the federal government more authority to impose the requirements. by , the quarantine system was totally nationalised. according to the us centres for disease control and prevention, there is a marked distinction between quarantine and isolation. as to the first term, people are put in 'quarantine' when they have neither signs nor symptoms, as they are not infected, but have been or may have been exposed to a communicable disease. this can help preventing the disease from spreading. conversely, people are put in 'isolation' when they are symptomatic or are reasonably believed to be infected with a communicable disease. this is to totally separate them from those who are not infected. isolation may be voluntary or imposed by a federal, state or local public health order. the history of quarantine in the usa is summarised in box . covid- widespread is putting healthcare systems under significant pressure worldwide. a large number of patients are needing to be admitted to hospitals at the same time for intensive care. available beds, ventilators and personnel are often not enough to face this ongoing emergency. the first facilities for sick people were not hospitals but temples dedicated to 'healing gods', such as imhotep for ancient egyptian, asclepius for the greek and aesculapius in ancient rome. at that time, prayers, sacrifices and dream interpretations played a crucial role in the healing pathway, but the ancient doctors also stitched cuts, set broken bones and administered opium for pain. the foundations of medicine are in the island of delos, which belongs to the cyclades archipelago in the greek sea. according to local mythology, delos was the birthplace of apollo, who is acknowledged as the original source of health and healing. he taught the art of surgery to chiron, the centaur, who in turn taught the same to asclepius, the greek healing god: his daughters were hygieia ('hygiene', the goddess of cleanliness), iaso (the goddess of recovery from illness), aceso (the goddess of the healing process), aegle (the goddess of good health) and panacea (the goddess of universal remedy). religious worship of asclepius was in the island of kos, which was also the birthplace of hippocrates, who is recognised as the father of 'rational' medicine. in athens, the temple dedicated to asclepius ( th century b.c.) had a large room for inpatient to be healed (pure in the body and spirit). greek physicians treated patients on the basis of house calls as well, which was a practice that continued for hundreds of years. the temples became medical practice schools in greece in the th century b.c. the word 'hospital' derives from the latin word 'hospes' for host or 'hospitium' for a place to entertain. the roman military hospitals were often used as healthcare facilities. each room was provided with three beds, thus representing a kind of ancient ward. similar hospitals were likely to be available also for gladiators and slaves, owing to their financial value. however, public hospitals were not at patients' disposal, and physicians examined them at home after being called. claudius galenus, who was born in greece and then moved to rome, was initially a surgeon for gladiators (coerusicus) and after that a physician and a philosopher. he became the emperor marcus aurelius' personal doctor and wrote about the plague, which afflicted rome during his reign. since marcus aurelius' family name was antonine, the pandemic disease was called 'antonine plague'. galenus contributed widely to the progress in medicine and his books were considered fundamental up the middle ages. as the roman empire got converted to christianity, the church's role in providing for the sick persons became firmly established. many monasteries were built, and generally they included rooms for pilgrims, the poor and the sick. the emperors from the th century onward, including charlemagne, decided that a hospital should be built beside every cathedral and provided with large, open wards. in the meantime, doctors kept on making house calls to the upper class. the strong religious influence in early healthcare is testified by the duties of the warden of st mary's hospital in england in . not only he was required to guarantee the quality of the care but also to hear the confession of the patient before admission ('pure in the body and spirit'). the wards, housing multiple patients, kept on being expanded and became the standard of care for public hospitals over centuries. the cross-shaped plan, which is thought to have had origin in florence (italy) during the italian renaissance, was conceived with an altar in the middle and multiple wards radiating from it. florence was well recognised for the high level of its hospitals and doctors, as testified by martin luther, during his visit. as the wards became larger, they often became the repository of infections. by the mid- s, one of the largest hospitals in paris had deteriorated to awful conditions, with wards which had over beds with multiple patients per bed. the wards were dark, poorly ventilated and dirty. a significant improvement was reached, thanks to florence nightingale, the founder of modern nursing, after seeing a mortality rate of over % at a military hospital in turkey during the crimean war. a new type of hospital, made up of pavilions rather than wards, was conceived. pavilions provided patients with fresh air and daylight, thus improving their recovery and reducing infections. this approach is sometimes called 'the nightingale ward' and was used first in st thomas' hospital in london. even at the old religious hospitals, nobles could have better housing bed by making donations. this approach was expanding in the late s, so that in , the first 'pay' hospital was opened in london with eight private single-bed rooms. as time passed, each country set up its arrangements for meeting and keeping people healthy, treating the sick and protecting from infectious diseases. healthcare innovation is a challenging topic for legislators and governments worldwide. only the most developed, industrialised countries (about of the in the world) have established healthcare systems, while the others are too poor for providing their citizens with any kind of mass medical care. healthcare system models are usually summarised in terms of three main models (or their combination), with expected different opinions in relation to which is the best of them. the first model is called bismarck model. germany has the world's oldest national social health insurance system, whose origin dates back to with the otto von bismarck's sickness insurance law. it uses an insurance system-the insurers are called 'sickness funds'-which are usually financed jointly by employers and employees by means of payroll deduction ('mutualistic' system). the bismarck model-based system is not aimed at making profits. doctors and hospitals tend to be private in bismarck countries. for example, japan has way more private box summary of the history of quarantine in the usa ► : in the harbour of philadelphia, the first quarantine station was built after a previous yellow fever outbreak in . ► : release of the national quarantine act, which shifted quarantine power from single states to federal government. ► : the federal government quarantine authority was set up. ► : the national communicable disease centre replaced the federal government quarantine authority. ► s: the number of quarantine stations was reduced from to . hospitals than the usa. the bismarck model does not provide with universal health coverage, since it requires employment for health insurance and stores its resources only for those who contribute financially. the second is the beveridge model. it was developed by sir william beveridge in in the uk. it is a centralised system working through the establishment of a national healthcare service (in the uk, it is the national health service). in summary, the government acts as the single-payer, because of taking off all competition in the market to maintain costs low and standardise benefits. the national healthcare service being the single-payer controls what doctors can do and what they cannot. this model is funded only by taxes, and there are no out-of-pocket fees for patients or any cost-sharing. everyone who is a tax-paying citizen is guaranteed the same access to care, so that this model is also defined as 'universal'. one criticism of the beveridge model is its potential risk of overuse. owing to the lack of restrictions, free access can potentially allow patients to demand healthcare services that are unneeded or wasteful. the result leads to increasing costs and taxes. the third model is the free market model. health is considered as a consumer good, in a free market for insurance companies aimed at making profits. it is the model adopted in the usa, where healthcare is not provided and financed by the government. those who have no health insurance have to pay the bill out-of-pocket at the time of a treatment is provided. as told, especially in europe, a combination of the abovestated models is possible. what is sure is the fact that, due to covid- rapid widespread, all these systems are facing an enormous funding crisis so far, whose consequences are also ethical. the word 'deontology' comes from the greek word 'deon', which means 'duty'. thus, deontology is also named as duty-based ethics. in deontology, reason is used to reach an agreement as to ethical standards to follow, so one has the duty to act according to those ethical principles. deontology is also called non-consequentialist ethics, because the rightness or wrongness (morality) of an action is judged on the basis of how it fits with ethical principles, not on its practical result. in this respect, the influence of the philosopher emmanuel kant is evident. according to kant, the moral law or 'categorical imperative' is an ethical principle, a universal law for all people. the essence of morality is in these principles. one of the oldest ethical code in the human history is the hippocratic oath. it was written by the ancient greek physician hippocrates ( b.c.- b.c.). the traditional hippocratic medical principle is 'primum non nocere' (first of all, avoid any harm) and doing good. its modern version, released in by lasagna, dean of the school of medicine at tufts university (usa), is still considered as a guide to conduct by the medical profession in modern ethics of deontology. since the introduction of the current bioethical paradigm by beauchamp and childress in , there has been much controversy about whether its four principles are universally and effectively applicable. these principles are ( ) respect for autonomy, ( ) justice, ( ) beneficence and ( ) nonmaleficence. the reported issues as to applicability are related to many factors, including the continuous changing of social structures and conditions, and the ongoing advances in technology. as to ethics, doctors have often to face other subjects who may have had no medical training, such as bioethicists, moral philosophers, theologians, clergy, hospital administrators, lawyers, attorneys, judges. living in a country with limited economic resources is another factor sometimes hampering the practical application of these ethical obligations. persad et al suggested four ethical principles to guide the distribution (allocation) of the available scarce medical resources, namely ( ) treating people equally, ( ) giving priority to the worst off, ( ) maximising benefits and ( ) promoting and rewarding social usefulness. within each of these four principles, there are two further contrasting ethical sub-principles (for a total of eight sub-principles). for example, regarding 'maximising benefits', two sub-principles are provided, that is, 'saving the most lives' and 'saving the most life years'. during covid- pandemic, this is one of the new challenges faced by doctors on the front line. since our medical systems have often only a limited amount of available resources (in terms of nurses, physicians, beds, treatments, equipment, money), doctors may have to choose whether treating a patient or another, based on his/her life expectancy. in addition to clinical responsibilities, the covid- outbreak is posing a very real ethical dilemma: what is a doctors' responsibility to serve patients despite personal risk? is this risk eventually to extend to medical trainees? the american medical association code of ethics states that a physician's responsibility to provide urgent care during disaster situations holds 'even in the face of greater than usual risk to the physicians' own safety, health or life'. there are also other new and still unanswered questions. how can a doctor maintain a patient's humanity during an overwhelming pandemic with limited resources and time? is the principle of delivering 'empathetic care' to be redefined under the current circumstances, and, if so, what does that look like? is sacrificing personal freedom with lockdown to protect the safety of communities justified? is sacrificing the individual healing and dignity of patients to protect the health and welfare of the general population allowed? should individual rights be limited in favour of supporting more vulnerable people? we are social creatures and the principle of 'solidarity' implies that we act supporting the most vulnerable members of our society and that we must not left them alone in scary times. in a more personal way, all of us have a friend or beloved one who is at risk in case of covid- . what would we want others to do to protect them? an interesting ethical perspective is represented by the immunity-based licences recently suggested, similarly to those for drivers or aeroplane pilots. however, one may argue that the latter are a certification of ability to work, not of health or personal state. unlicenced people should be subject to social or economic exclusion as well as personal liberty limitations. never the current restrictions were applied before in western societies. in ancient greece, crisis had also the meaning of 'change'. the covid- outbreak found most of countries unprepared, with scarce resources stored to afford the sars-cov- emergency. coronavirus has been dismantling our daily life in all its features (health, behaviour, economy). in a catastrophic perspective, since a second outbreak cannot be excluded, we should be ready to cope with it by building new specific covid- hospitals and preparing nurses and general practitioners to face the emergency at the very early beginning, thus preventing patients from the need to be admitted to hospitals. all the above have strong ethical consequences and imply a new way to allocate resources. there are two possible long-term strategies: 'long-term mitigation' and 'suppression'. the first implies sustained or intermittent social distancing until herd immunity is reached or a vaccine discovered. conversely, the second is aimed at identifying and isolating cases until a vaccine is available. suppression requires dropping case numbers quickly to be effective, by using excellent testing and contact tracing infrastructure. nowadays, a new pandemic is threatening our hypertechnological, but even so defenceless world. due to its sudden and too recent appearance, evidence-medicine on this issue is still poor to provide clinicians with reliable data. a new viral disease (the great invader) has surprised an unarmed world. it causes pneumonia, extrapulmonary complications and often death. the first case was reported in china on november but was not recognised soon. eight more cases appeared in early december , with researchers starting to think about a still unknown virus as their possible cause, but only on december, they were reported to the who country office. on february , the infection was officially named covid- . following that, the virus spread beyond chinese borders and, by mid-march, reached more than countries. on march , the who announced that covid- was officially a pandemic. the spread is anywhere near finished. without a vaccine available and due to the lack of a reliable therapy, a vast majority of the countries identified persistent lockdown as the only way to limit contagion from spreading without control. overall, the -century-old 'quarantine' word has back in fashion. doctors are now facing new challenges, which involve ethics and may lead to make difficult and painful choices for their patients and themselves, owing to the paucity of the available resources. the current healthcare systems are unlikely to be prepared to cope with this sudden and unexpected emergency and should be reformed or updated (from lazareth/leprosarium/ tuberucolosarium to 'covid-sarium', ie, a specialised centre with personnel for swabs and immunology tests). we should be careful that the 'cure' for covid- is not worse than the disease itself. the universal and unprecedented public health preventive measures, including lockdown and social distancing, are damaging our economy and increasing unemployment worldwide. lastly, as doctors, the current covid- age is emphasising some of the moral problems of modern medicine, which is more than ever split between ethics and financial rationalisation. epidemiology of sars-cov- : numbers matter! a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china covid- : first coronavirus was described in the bmj in the story of quarantine some historical comments on quarantine: part one some historical comments on quarantine: part two the holy bible. old testament historical sketches of quarantine the origin of quarantine small oversights that led to the great plague of marseille ( - ): lessons from the past people with leprosy (hansen's disease) during the middle ages a state of deference: ragusa/dubrovnik in the medieval centuries the origin of the word "quarantine a short history of quarantine (victor c. vaughan) biological warfare at the siege of kaffa the medieval plague: the black death of the middle ages boccaccio as therapist: plague literature and the soul of the city alexandre yersin: discoverer of the plague bacillus biology of plagues: evidence from historical populations encyclopedia of pestilence, pandemics, and plagues disease and history quarantine in times of emergency: the scope of s (ix) of the constitution the rod and the serpent: history's ultimate healing symbol a review of the principle mythical gods in ancient greek medicine the hospitalized legionnaire at the rhine front at the outset of the roman principate the evolution of the hospital from antiquity to the end of the middle ages the mediaeval hospitals of england (illustrations) hospitals' evolution through the ages infection control, from hippocrates to protocols nightingale and the war the history of hospitals and wards a view of health care around the world the birth of medical deontology the hippocratic oath and the ethics of medicine principles of biomedical ethics principles for allocation of scarce medical interventions fair allocation of scarce medical resources in the time of covid- medical ethics: four principles plus attention to scope the code of medical ethics of the american medical association the ethics of covid- immunity-based licenses ten reasons why immunity passports are a bad idea clinical and health policy challenges in responding to the covid- pandemic importance of suppression and mitigation measures in managing covid- outbreaks making evidential claims in epidemiology: three strategies for the study of the exposome new coronavirus outbreak: framing questions for pandemic prevention coronavirus disease infection and the cardiovascular system burnout in the age of covid- contributors ppb and gc conceived and wrote the paper; mrm and sm contributed to reference research and final approval for the paper to be published.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.competing interests none declared.patient consent for publication not required.provenance and peer review not commissioned; internally peer reviewed.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. pier paolo bassareo http://orcid.org/ - - - key: cord- - ko n authors: cunningham, andrew a.; daszak, peter; wood, james l. n. title: one health, emerging infectious diseases and wildlife: two decades of progress? date: - - journal: philos trans r soc lond b biol sci doi: . /rstb. . sha: doc_id: cord_uid: ko n infectious diseases affect people, domestic animals and wildlife alike, with many pathogens being able to infect multiple species. fifty years ago, following the wide-scale manufacture and use of antibiotics and vaccines, it seemed that the battle against infections was being won for the human population. since then, however, and in addition to increasing antimicrobial resistance among bacterial pathogens, there has been an increase in the emergence of, mostly viral, zoonotic diseases from wildlife, sometimes causing fatal outbreaks of epidemic proportions. concurrently, infectious disease has been identified as an increasing threat to wildlife conservation. a synthesis published in showed common anthropogenic drivers of disease threats to biodiversity and human health, including encroachment and destruction of wildlife habitat and the human-assisted spread of pathogens. almost two decades later, the situation has not changed and, despite improved knowledge of the underlying causes, little has been done at the policy level to address these threats. for the sake of public health and wellbeing, human-kind needs to work better to conserve nature and preserve the ecosystem services, including disease regulation, that biodiversity provides while also understanding and mitigating activities which lead to disease emergence. we consider that holistic, one health approaches to the management and mitigation of the risks of emerging infectious diseases have the greatest chance of success. this article is part of the themed issue ‘one health for a changing world: zoonoses, ecosystems and human well-being’. by the s, the human burden of infectious diseases in the developed world was substantially diminished from historical levels, largely due to improved sanitation and the development of effective vaccines and antimicrobial drugs [ ] . the emergence of a series of novel diseases in the s and s (e.g. toxic shock syndrome, legionnaire's disease), culminating with the global spread of hiv/aids, however, led to infectious disease rising back up the health policy and political agendas [ ] . public concern about emerging infectious diseases (eids) has been heightened because of the perception that infectious diseases were previously under control, because of their often rapid spread (e.g. severe acute respiratory syndrome; sars), because they often have high case fatality rates (e.g. ebola virus disease) and because the development of drugs and vaccines to combat some of these (e.g. hiv/aids) has been slow and costly. by the s, authors had begun to review similarities among these diseases and identify patterns in their origins and emergence [ , ] . similarities included a skew to zoonotic pathogens originating in wildlife in tropical regions (e.g. ebola virus), and that emergence was associated with environmental or human behavioural change and human interaction with wildlife (e.g. hiv/aids) or with domestic animals which had interactions with wildlife (e.g. nipah virus) [ - ] . emergence was found to be exacerbated by increasing volumes and rates of human travel and globalized trade [ ] . by the end of the s, the study of eids was a staple of most schools of public health, a key focus of national health agencies, a book topic and the title of a scientific journal [ ] . novel diseases continued to emerge, often from unexpected reservoirs and via new pathways. for example, between and , three new zoonotic viruses (hendra, menangle and nipah viruses) emerged from pteropodid bats in australia and southeast asia [ ] . each of these was transmitted via livestock (horses or pigs), and each belonged to the paramyxoviridae. around this time, emerging diseases were identified in a series of well-reported die-offs in wildlife, including canine distemper in african lions (panthera leo) in the serengeti, chytridiomycosis in amphibians globally, pilchard herpesvirus disease in australasia and west nile virus in corvids and other birds in new york [ - ] . pathogens were also implicated for the first time in species extinctions, or near-extinctions, e.g. canine distemper in the black-footed ferret (mustela nigripes), chytridiomycosis in the sharpsnouted day frog (taudactylus acutirostris) and steinhausiosis in the polynesian tree snail, partula turgida [ - ] . novel diseases and their emergence in people and wildlife were reviewed, and commonalities in the underlying causes of emergence discussed, in a paper published at the end of the decade [ ] . here, we re-examine some of the key conclusions of that paper, review how the field has progressed years on and identify some of the remaining challenges to understanding and mitigating the impacts of disease emergence in and from wildlife. prior to , wildlife diseases were mostly studied to improve zoo animal survival and welfare, with little published on the diseases of free-living wildlife unless they affected heavily hunted species (e.g. deer in north america) or were considered a threat to livestock health (e.g. tuberculosis, rinderpest). while non-infectious diseases had been widely recognized as important drivers of species declines (e.g. ddt poisoning of raptors [ , ] ), only a small number of researchers investigated infectious disease as a factor in, often covert, wildlife population regulation [ , ] . the role of infectious diseases in mass mortality events or population declines was often considered controversial or secondary to other factors [ ] , and their role in species extinctions often disputed [ , ] . the first definitive identification of disease as a cause of species extinction was published in following the demise of the last population of the polynesian tree snail p. turgida due to a microsporidian infection [ ] . this added to evidence that infectious agents had caused the extinction in the wild of the black-footed ferret, the extinction of around one-third of hawaiian honeycreepers and the slime mouldinduced decline of eelgrass (zostera marina) beds in the usa, leading to extinction of the eelgrass limpet (lottia alveus) [ , - ] . during the s, wildlife mortality events caused by infectious diseases were reported in zoos, in wildlife translocation programmes and in other conservation programmes [ - ] . perhaps the most important of these was the discovery of amphibian chytridiomycosis, caused by the chytrid fungal pathogen batrachochytrium dendrobatidis, which was first recognized in the s and has since been implicated in the decline or extinction of over species of amphibian [ , , , ] . this disease continues to threaten amphibians globally and has been described as 'the worst infectious disease ever recorded among vertebrates in terms of the number of species impacted, and its propensity to drive them to extinction' [ ] . amphibian chytridiomycosis appears to have emerged contemporaneously in australia and central america, associated with large-scale die-offs and extinction events, although in retrospect it might have been causing amphibian mortalities and declines in north america prior to this [ ] . proving that a disease is a cause of population declines in wildlife requires longitudinal population and pathogen data, which are often very difficult to collect. thus, a series of papers disputing the role of chytridiomycosis in amphibian declines ensued, with most suggesting that this disease either emerged secondarily to other factors, or that it was not the cause of declines/extinctions [ - ] . long-term datasets have since been published which provide convincing evidence that amphibian chytridiomycosis alone can cause mass mortalities leading to population declines [ ] . policy measures to control amphibian chytridiomycosis, however, have been slow to be enacted, with the first international policy measure (listing of chytridiomycosis by the world organisation for animal health) occurring in [ ] and with the implementation of measures recognized to mitigate the spread of this disease still not being enacted by the international community [ ] . public and political reaction to the more-recent emergence of white nose syndrome (wns) in north american bats provides evidence that the conservation implications of wildlife eids are becoming more widely accepted. the causative agent of wns is the fungus pseudogymnoascus destructans which colonizes the skin of a range of temperate-zone bats, often causing death during hibernation [ ] . only year after the initial discovery of the disease in the usa in january , visitors to bat caves across the country were being advised to reduce visits and to implement biosecurity measures, and by , caves in over states were closed to the public. the disease has been the focus of a series of grants, formation of multi-disciplinary research partnerships and significant efforts to identify pathogenesis, transmission pathways and potential control measures [ , ] . although there is a growing recognition of the impact of pathogens on wildlife, the significance of infectious disease as a cause of historical extinctions is likely underestimated due to a previous relative lack of infectious disease focus and diagnostic capability [ ] . collaboration among ecologists, conservation biologists and veterinary pathologists is relatively recent and increased pathological and epidemiological involvement in studies of the causes of wildlife declines are critically needed to identify and understand disease threats to wildlife and how to mitigate them. in addition to identifying an apparently growing trend of disease threats to wildlife, daszak et al. [ ] highlighted wildlife as the source of a series of high-impact, recently emerging pathogens affecting people. these authors reiterated the widely proposed hypothesis that most emerging pathogens rstb.royalsocietypublishing.org phil. trans. r. soc. b : originate in wildlife and spillover into human hosts due to a range of ecological, demographic and socio-economic changes [ , , ] . prior to , these wildlife-origin pathogens were known to include ebola and marburg virus, hiv- and hiv- , sin nombre virus, nipah, hendra and menangle virus, west nile virus, borrelia burgdorferi and others. since then, other human diseases have emerged from wildlife, including middle east respiratory syndrome (mers) and different subtypes of avian influenza, and further advances have been made in our understanding of patterns of zoonotic disease emergence. a series of papers analysed a database of all known human eids and confirmed that the majority are of animal origin, with viruses being a particularly important group [ - ] . further analysis of an updated version of this database identified that eids had increased in frequency (even accounting for increased numbers of researchers), with the proportion of those emerging from wildlife hosts increasing substantially over the last four decades of the twentieth century [ ] . the emergence of bat-origin viral eids of people during the s was highlighted by daszak et al. [ ] . since then, it has been shown that bats are reservoir hosts of a striking number of zoonotic viruses, including high-profile pathogens with high case fatality rates, such as nipah and hendra paramyxoviruses, filoviruses, sars-like coronaviruses and possibly also mers coronavirus [ , ] . this led some authors to propose that bats harbour a disproportionate number of emerging zoonoses compared with other mammalian groups [ - ] : a hypothesis that has been supported by two separate analyses of mammal virus datasets [ , ] . understanding why bats host so many zoonotic pathogens that cause lethal diseases in humans and how spillover from bats to humans occurs is important in order to control these, and possibly as-yet-undiscovered, diseases [ , - ] . there are likely to be multiple causes of novel disease emergence, but the human-mediated transport of pathogens (often in infected hosts) or vectors across geographical or ecological boundaries, a process termed 'pathogen pollution', has been identified as a major driver of this in wildlife [ ] and also in plants [ ] . the anthropogenic spread of pathogens has been responsible for the emergence of a series of high-profile wildlife eids, including the two known agents of amphibian chytridiomycosis, b. dendrobatidis and b. salamandrivorans [ , ] . subsequent research indicates that this is only part of the story, as it appears that the global pandemic lineage of b. dendrobatidis arose from a single hybrid origin via an ancestral meiosis, possibly via the anthropogenic mixing of allopatric lineages [ , ] . there is a substantial volume of research that shows how, once evolved, this virulent lineage has been introduced globally via the international trade in amphibians and via the human-assisted introduction of invasive species [ , - ] . in recent years, a body of literature has developed the concept of the ecosystem service of disease regulation. while still controversial, and probably not universal [ ] , this proposes that natural biodiversity limits the exposure and impact of many pathogens, including those that are zoonotic, through a dilution or buffering effect, thus limiting opportunities for pathogen spillover from wildlife to people [ ] . when biodiversity is depleted (usually by human activities), this ecosystem service is impaired and zoonotic pathogens are more likely to emerge, as has been shown for hantavirus [ ] and for b. burgdorferi, the causative agent of lyme disease [ , ] . also, alteration of species complements (again, usually due to anthropogenic impacts), rather than loss of biodiversity per se, can alter infection dynamics and lead to increased zoonotic disease risk [ ] . our understanding of the interactions between ecosystem change, disease regulation and human well-being, however, is in its infancy. almost years since the threats to conservation and human health that wildlife eids represent was first highlighted, there has been little effort to put in place policies to reduce risk. detecting and preventing the importation of infected hosts is widely used to prevent importation of many domestic animal diseases of economic or public health importance. some countries even enact this principle for the movement of people, whereby they conduct (often cursory) surveillance for infected persons arriving at their international borders, particularly during human pandemics [ , ] . the world health organisation provides guidance and training on this through its international health regulations (http:// www.who.int/ihr/en/). rules and regulations for international trade, including of animals and their products, are created and enforced by the world trade organisation (wto), which has the remit of ensuring 'that trade flows as smoothly, predictably and freely as possible' (www.wto. org). the wto agreement on sanitary and phytosanitary measures was enacted on january with the aim of protecting human, animal and plant life from disease-causing agents. while countries have discretion in what should be included, they are guided by the world organisation for animal health (oie) list of diseases of international importance. although the oie has a remit of protecting biodiversity, only two pathogens are listed for this purpose: b. dendrobatidis and ranavirus [ ] . most countries, therefore, use import controls to only protect against domestic animal diseases of obvious public health or economic importance, such as rabies and foot and mouth disease; diseases restricted to wildlife are not included even when oie-listed. in addition, trade agreements often prohibit barriers to international animal movements for the purposes of infectious disease control. for example, countries within the european union have little ability to prevent the spread of pathogens via within-eu trade unless as part of a specific eu disease control programme. even where technically legal under wto rules, there appears to be reluctance by countries to unilaterally impose restrictions on non-listed diseases in case they create an economic disadvantage or are subsequently found to be in breach of international trade regulations. it is possible that the international spread of amphibian chytridiomycosis would have been reduced if such measures had been implemented for this disease [ ] . perhaps learning from this, in january , the usa banned the importation of salamanders following the emergence of b. salamandrivorans in order to protect native wildlife from this novel pathogen [ ] . such protective action was enacted relatively rapidly following the discovery of b. salamandrivorans as a novel lethal fungus infecting and killing captive and wild salamanders in europe [ , , ] challenges remain to understanding the wildlife origins of zoonotic eids. it is often difficult, time-consuming, logistically challenging and very expensive to identify the origins of newly emerged pathogens of humans. for example, viruses similar to hiv/aids were discovered in non-human primates in the early s, but identification of the true progenitor viruses in chimpanzees took almost a decade of additional research [ ] . similarly, the origins of ebola and marburg viruses have been investigated for over years. to date, however, despite indications that bats are the natural reservoir hosts of these viruses, clear evidence has only been found for marburg virus infection in bats in limited locations [ - ] . identifying putative reservoir host(s) is just the beginning. in order to identify actions to prevent or mitigate future zoonotic spillover, both an understanding of the ecology of the pathogen in its natural host(s) and of human -host interactions are required [ ] . for example, substantial efforts have been conducted to understand immunological, behavioural and ecological characteristics of bats as part of a strategy to control zoonotic spillover from bats [ ] [ ] [ ] . long-term, multi-disciplinary studies that systematically investigate the ecology of zoonotic pathogens in their wildlife hosts along with the risk characteristics for spillover are critical to better predict and prevent future pandemics [ ] . such a study, which included years of field data collection on fruit tree distribution, pig farm management, viral dynamics and satellite telemetry of fruit bats, analysis of climate trends, experimental infection of bats under biosafety level- conditions and mathematical modelling of virus infection dynamics, identified the intensification of the pig industry as the driver of the zoonotic emergence of nipah virus in malaysia [ ] . these results informed government policies to separate pigs from bats via the removal of fruit trees from pig farms and the relocation of farms away from forested areas [ ] , since when no further nipah virus disease outbreaks have occurred in malaysia. eid events have been the focus of intense research over the past two decades, even though the numbers of people diagnosed with them are often relatively small. this disproportionate focus on eids probably relates to the dislike of human society for uncertainty, or put more simply, fear of the unknown. this may lead to perverse scenarios in which fear of disease can have a greater impact than the direct impact of the outbreak itself. for example, during a recent ebola virus epidemic in west africa, more people are estimated to have died from malaria due to their avoidance of healthcare facilities, where they feared they might catch ebola, than the thousands that died from the virus itself [ ] . indeed, when one considers the overall impact of zoonotic diseases on the human population, the largest (diagnosed) burden is associated with well known and fully recognized (in the industrial north), but neglected, diseases such as brucellosis, rickettsioses and rift valley fever [ ] . this predictable burden falls heavily on the global poor-poverty being the major risk factor for most zoonoses, which in turn causes some communities to suffer disproportionately from the burden of zoonotic disease [ ] . the neglect of such diseases includes diagnostic neglect (and confusion with other conditions such as malaria [ ] ) and historic and current research neglect; all of which feeds into therapeutic neglect. the delivery of the united nations sustainable development goals, which should result in much reduced poverty and improved health, will in themselves reduce the substantial burden of zoonotic disease. one health is the term used when approaches to tackling disease ( particularly zoonoses) consider all components that might lead to, or increase, the threat of disease. these include environmental and ecological/wildlife components as well as domestic animal and human factors. the last encompasses behavioural as well as medical issues, including cultural, political and other socio-economic drivers that might result in disease occurrence or spread. the review by daszak et al. [ ] was perhaps the first 'one health' review of emerging diseases, in that it brought together veterinary, ecological, conservation and human medical perspectives on disease emergence. the field of one health has expanded substantially since , diversifying to produce new journals, such as one health, ecohealth and the lancet planetary health, the one health platform, the international association of ecology and health, the planetary health alliance and a series of one health institutions in the usa, europe, australia and increasingly also in developing countries. the success of this multi-disciplinary approach has been driven largely by the synergistic impact of combining detailed and logistically challenging field sciences (e.g. ecology, field biology) with analytical approaches (e.g. epidemiological modelling, pathogen phylogenetic analysis) and laboratory science (e.g. serology, pathogen diagnostics, immunology). challenges remain, however. importantly, while the conservation, ecological and veterinary professions are increasingly engaged with one health, substantial elements of the medical profession are not aware of, or involved in, this approach. despite their neglect, a number of zoonotic diseases are eminently controllable or manageable by one health approaches, including infectious causes of abortion in livestock, which frequently result in febrile human disease, and human rabies transmitted via dog bites. control or prevention is best achieved through integrated public health, veterinary medicine, animal management and ecological approaches. one particular challenge for this is in the case of some zoonotic infections that do not cause clinical signs in their animal hosts, one of the most common examples of which is campylobacter spp. infection of poultry, which globally is the most frequent cause of food poisoning in humans [ ] . is it, then, the responsibility of farmers and vets to ensure that people do not become infected, or of public health practitioners or the general public through improved kitchen hygiene and behaviours? here, this would involve reduced infection of poultry (the role of farmers and veterinarians), reduced contamination of meat (the responsibility of veterinary public health workers) and preventive measures in the kitchen (hygiene and proper cooking), which are the domain of public health workers and the public [ ] . one health approaches are required at the policy and governance levels, too. responsibility for preventing and treating zoonotic disease, in both a developing and rstb.royalsocietypublishing.org phil. trans. r. soc. b : developed world setting, for example, often falls in between government ministries of health and agriculture (and for wildlife, ministries of environment and forestry) and this can structurally prevent the simplest of solutions from being implemented. an important example is rabies in humans transmitted through dog bites which kills around people annually [ ] and causes fear in many more in rabies endemic regions. the disease is easily preventable (and arguably open to eradication) through repeated annual or biannual mass vaccination of dogs [ ] . in many countries with a high burden of rabies in dogs, considerable sums are spent by the public and ministries of health annually on post-exposure prophylaxis (pep-often given after dog bites whether or not the animal was known to be rabid). the expense of this repeated treatment usually dictates that far more is spent on treatment than would be required to vaccinate all dogs in the same region. however, in many countries, the dog is regarded as a pest and not an agricultural animal for which ministries of agriculture have responsibility. in others, the dog does fall under the agricultural ministry, but these ministries are typically far less well resourced compared with ministries of health, thus rabies, which does not relate to food animals, is not prioritized. the obvious solution is for a synergized one health approach with the ministries of health supporting prophylactic vaccination programmes for dogs delivered by their typically far less well-resourced ministries of agriculture. this, however, rarely seems to happen and continued expenditure on bite management and pep continues. one health programmes addressing rabies have been extremely successful when appropriately resourced [ , ] ; however, they often fail to influence national government policy and are rarely adopted long term [ ] . in addition to the high costs of dealing with endemic zoonoses, such as rabies, emerging and re-emerging zoonoses can have substantial economic impacts. the cost implications of zoonotic eids were highlighted by daszak et al. [ ] as a rationale for policy measures, but methods for calculating the economic consequences of disease emergence have not advanced in the interim. despite clearly high financial impacts associated with some eids, few detailed economic analyses of their impact have been undertaken. estimates of the cost of the sars outbreak, for example, range from $ to $ billion, while the true costs of most eids have never been estimated [ ] . pike et al. [ ] approached the problem of disease emergence in the same way as the climate change phenomenon. they used the increasing frequency of emerging disease events reported by jones et al. [ ] to analyse two strategies to deal with the rising costs of eids over time: adaptation, whereby we adopt a business-as-usual approach and continue to cause increased eid events, then target control programmes after emergence; and mitigation, whereby we deal with the underlying drivers (e.g. wildlife trade, deforestation) and reduce the frequency of eid events. pike et al. [ ] show that mitigation strategies are more cost effective in the long term, with a -fold return on investment, and that these need to be enacted on a global scale within the current generation or the cost of eids becomes unaffordable. what would these global strategies entail? we highlight three approaches. first, a series of emerging diseases have been linked to the wildlife trade, or consumption of wildlife (e.g. sars, ebola). the health implications of the trade in wildlife have not been widely used to implement controls, or advocate for reduction in consumption, and may be a more effective message than its conservation impacts. this needs to be done judiciously, however, as disease spillover is a rare event and both bushmeat hunters and consumers will be wary of public health messages that do not fit with their experiences [ , ] . second, a revision of an earlier analysis of global drivers of disease emergence [ ] shows that land-use change correlates strongly with the emergence of zoonoses from wildlife (p daszak , unpublished observation). in malaysia, analyses of the economic cost of diseases that emerge due to land conversion for palm oil production (e.g. malaria, leptospirosis) are currently being used to advise industry where to reduce long-term impact. identifying land-use changes that lead to disease emergence informs policies for mitigation strategies. this could be done, for example, via the incorporation of wildlife and zoonotic disease threats in environmental impact studies, an approach for the prevention of disease emergence suggested by daszak et al. [ ] . third, targeted global surveillance programmes to identify novel pathogens of zoonotic potential before they emerge may increase our capacity to reduce their risk of emergence. for example, a series of laboratories now specialize in identifying novel viruses from wildlife hosts, e.g. bats [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the usaid emerging pandemic threats programme specifically targets emerging disease hotspots to identify novel viruses from bats, rodents and primates, to characterize high-risk behaviours in people and to identify potential mitigation strategies [ ] . while these programmes have already identified over new viruses from viral families with known zoonoses in the last few years, challenges remain in how to identify those with the highest (or any) risk of zoonotic emergence. this indicates that a change in approach is required, building on rapidly expanding databases of pathogen sequences, phenotypic characteristics and host-pathogen interactions. for example, the rapid incorporation of novel viral sequences into diagnostic tests may lead to more rapid identification of related, previously unknown, pathogens that emerge in outbreaks. using this approach, combined with a one health perspective that targets the underlying drivers of emergence, could result in the identification of pathogens that already are spilling over from wildlife hosts sporadically at low levels, enabling measures to be taken to reduce pandemic risk. since the synthesis paper by daszak et al. [ ] highlighted emerging disease threats of, and from, wildlife and the main drivers underlying these, further advances have been made in our understanding of the origin, size and potential scope of these threats. endemic zoonoses, however, continue to be relatively neglected, often with a lack of local and international realization of the extent to which they impact human health and well-being. this is partly due to issues surrounding local capacity and knowledge and partly because, unlike eids, they are not seen as a threat to people in the developed world. both eids and endemic zoonoses, however, can be tackled using a one health approach, including the rstb.royalsocietypublishing.org phil. trans. r. soc. b : identification and mitigation of human activities that lead to disease emergence and spread. one health approaches to dealing with disease threats from and to wildlife are still relatively young and untried, but all evidence points to them being most successful and 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endemic canine rabies the changing landscape of rabies epidemiology and control achieving population-level immunity to rabies in free-roaming dogs in africa and asia effective vaccination against rabies in puppies in rabies endemic regions rabies control and elimination: a test case for one health economic optimization of a global strategy to reduce the pandemic threat ebola and bushmeat: myth and reality henipavirus rna in african bats hantavirus in bat bats host major mammalian paramyxoviruses receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus isolation and characterization of a bat sars-like coronavirus that uses the ace receptor a sars-like cluster of circulating bat coronaviruses shows potential for human emergence authors' contributions. a.a.c. conceived the idea for the article. all key: cord- -k dqe lk authors: hoelzer, karin; bielke, lisa; blake, damer p.; cox, eric; cutting, simon m.; devriendt, bert; erlacher-vindel, elisabeth; goossens, evy; karaca, kemal; lemiere, stephane; metzner, martin; raicek, margot; collell suriñach, miquel; wong, nora m.; gay, cyril; van immerseel, filip title: vaccines as alternatives to antibiotics for food producing animals. part : new approaches and potential solutions date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: k dqe lk vaccines and other alternative products are central to the future success of animal agriculture because they can help minimize the need for antibiotics by preventing and controlling infectious diseases in animal populations. to assess scientific advancements related to alternatives to antibiotics and provide actionable strategies to support their development, the united states department of agriculture, with support from the world organisation for animal health, organized the second international symposium on alternatives to antibiotics. it focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the development and licensure of alternatives to antibiotics. this article, the second part in a two-part series, highlights new approaches and potential solutions for the development of vaccines as alternatives to antibiotics in food producing animals; opportunities, challenges and needs for the development of such vaccines are discussed in the first part of this series. as discussed in part of this manuscript, many current vaccines fall short of ideal vaccines in one or more respects. promising breakthroughs to overcome these limitations include new biotechnology techniques, new oral vaccine approaches, novel adjuvants, new delivery strategies based on bacterial spores, and live recombinant vectors; they also include new vaccination strategies in-ovo, and strategies that simultaneously protect against multiple pathogens. however, translating this research into commercial vaccines that effectively reduce the need for antibiotics will require close collaboration among stakeholders, for instance through public–private partnerships. targeted research and development investments and concerted efforts by all affected are needed to realize the potential of vaccines to improve animal health, safeguard agricultural productivity, and reduce antibiotic consumption and resulting resistance risks. alternatives to antibiotics can help minimize the need for antibiotics by helping to prevent and control infectious diseases in animal populations. as such, safe and effective alternatives are crucially important to the future success of animal health and production. to assess scientific advancements in the research and development of alternatives to antibiotics, highlight promising research results and novel technologies, assess challenges associated with their commercialization and use, and provide actionable strategies to support their development, the united states department of agriculture (usda), with support from the world organisation for animal health (oie), organized the second international symposium on alternatives to antibiotics. the symposium focused on six key areas: vaccines; microbial-derived products; non-nutritive phytochemicals; immune-related products; chemicals, enzymes, and innovative drugs; and regulatory pathways to enable the licensure and development of alternatives to antibiotics. this two-part manuscript synthesizes and expands on the scientific presentations and expert panel discussions from the symposium regarding the use of vaccines as alternatives to antibiotics that can reduce the need for antibiotic use in animals. part synthesizes and expands on the expert panel discussions regarding the opportunities, challenges and needs related to vaccines that may reduce the requirement for use of antibiotics in animals, while part two focuses on highlighting new approaches and potential solutions. a general discussion of the importance of antibiotic resistance and the opportunities, challenges and needs related to vaccines as alternatives that may reduce the need for use of antibiotics in animals is provided in part of this review, including a discussion of the properties of ideal vaccines, how current vaccines compare to these ideal vaccines, and how investment decisions around research and development of vaccines are made. this second part of the manuscript will highlight specific research advancements in the area of veterinary vaccines. as mentioned in part one of this manuscript, most pathogens invade the host at the mucosal surfaces, such as the gastro-intestinal (gi) tract. the gi tract constitutes the largest surface area of the body and is exposed daily to vast numbers of foreign antigens derived from feed, the microbiota and pathogens [ ] . within the intestine a complex cellular network has evolved to prevent unwanted immune responses to innocuous antigens, for instance feed or microbiota, while allowing swift protective responses against agents that cause infectious disease. key to keeping enteric pathogens at bay is the presence of protective pathogen-specific secretory iga (siga) at the site of entry, which prevents the adhesion of micro-organisms to the intestinal surfaces and neutralizes their enterotoxins. triggering robust and protective intestinal siga responses usually requires the local administration of vaccines [ ] . although live attenuated oral vaccines have had tremendous success, resulting for instance in the near global eradication of poliovirus [ ] , concerns on the dissemination of vaccine strains into the environment and rare cases of reversion to virulence, leading to vaccine-induced disease, have driven oral vaccine development to nonliving or vectored vaccines [ ] . however, oral vaccination is challenging due to several hurdles imposed by the cellular and molecular architecture of the gut: (i) the harsh environment of the stomach and small intestine, including the low ph, digestive enzymes, and bile salts, required to digest feed also easily destroys vaccines, (ii) a poor uptake of vaccine antigens by the intestinal epithelial barrier and (iii) the tolerogenic mechanisms that pervade the intestinal tissues, leading to peripheral and oral immune tolerance upon oral administration of antigens via the induction of foxp + regulatory t cells. this often results in a low immunogenicity of oral vaccines and requires innovative strategies to deliver the vaccine antigens to the intestinal immune system as well as the inclusion of adjuvants that promote innate and adaptive immunity [ ] . the mucosal immune system in the gut can be divided in inductive sites, where sampled antigens stimulate naive t and b cells, and effector sites, where effector cells perform their functions, e.g. assisting in the production of siga. in the small intestine, the inductive sites comprise the gut-associated lymphoid tissues (galt) and the mesenteric lymph nodes, while the effector sites constitute the lamina propria and the surface epithelium [ ] . the galt itself is composed of peyer's patches (pp), appendix and isolated lymphoid follicles. the presence of other galt-like structures, such as lymphocyte-filled villi (rat, human) and cryptopatches (mouse) is dependent on the species. interestingly, while in birds and most mammals pp or their equivalent are scattered throughout the small intestine, in pigs, ruminants and dogs the pp in the distal small intestine (ileum) are continuous. fish and reptiles on the other hand lack pp and the intestinal immune system in these species is composed of epithelial leukocytes . . next generation anticoccidial vaccines autogenous vaccines to reduce the need for antibiotic use conclusions references and rare, small non-organized lymphoid aggregates. it remains largely unknown how these species-specific differences might affect the efficacy of oral vaccines. from their entry point, which is typically the oral cavity, to their delivery site, most commonly the small intestine, the integrity of delivery systems and the stability of vaccine components are at risk. lysozyme in saliva, the low gastric ph together with pepsin and intestinal proteases can degrade oral vaccines. enteric coating of vaccine components with ph-responsive polymers with a dissolution threshold of ph might protect against gastric degradation and results in the release of their contents in the small intestine [ ] . in this context, ruminants pose an additional problem to vaccine stability as their polygastric gastro-intestinal tract effectively degrades substances including vaccines. site-specific delivery of oral vaccines to the small intestine is favorable as the mucus layer covering the small intestinal epithelium consists of only one layer, which is loosely adherent, less thick and patchy as compared to the colonic mucus layers and might promote their access to the intestinal epithelium. in addition, the small intestine is less densely populated by the microbiota, which might further disrupt the integrity of the delivery systems and the stability of vaccine components. underneath the mucus layer, a single layer of intestinal epithelial cells prevents uncontrolled access of the luminal content to the underlying intestinal tissues, further restricting uptake of oral vaccine antigens. crossing of the epithelial barrier by vaccines could be enhanced by exploiting antigen sampling routes in the small intestine or by adopting strategies used by enteric pathogens to colonize or invade the host [ ] . the best-known sampling route in the gut is associated with microfold (m) cells. these specialized intestinal epithelial cells reside within the follicle-associated epithelium covering the peyer's patches and take up macromolecules, particulate matter and microorganisms [ ] . many enteric pathogens hijack m cells to invade the host by binding to apical receptors. for instance, the invasin protein of yersinia species interacts with β integrin on m cells, leading to infection [ ] . likewise, gp marks m cells in many species and binds to fimh, a subunit of type i pili on escherichia coli and salmonella enterica. this interaction results in uptake of fimh + bacteria and initiates mucosal immunity [ ] . although many groups have focused on improving antigen uptake by targeting oral vaccines to m cell-specific receptors, these cells represent only a small, species-specific percentage of the total intestinal epithelial cell population. although m cell numbers increase from the cranial to caudal small intestine and m cell targeting strategies work quite well in rodent models, they mostly fail in larger animals due to the long passage time needed to reach the distal small intestine, where the gut-associated immune system is most pronounced. besides m cells, sampling of luminal antigens also occurs by intestinal mononuclear phagocytes via transepithelial dendrites. this sampling mainly occurs by cd c + cx cr + macrophages, which transfer the antigens to cd + dendritic cells (dcs). these dcs then drive the differentiation of regulatory t cells (tregs), which subsequently induce tolerance to these proteins [ ] . in the steady state, goblet cells can also transport small soluble proteins (< kda) across the epithelium to tolerogenic dcs via so-called goblet cell-associated antigen passages [ ] . absorptive intestinal epithelial cells or enterocytes, constituting > % of the small intestinal epithelium, may also sample the luminal content through receptor-mediated transcytosis. for instance, the neonatal fc receptor (fcrn), an mhc class i-like fcγ receptor, is expressed on the apical surface of enterocytes and transcytoses igg, immune complexes or fc-coated nanoparticles from the lumen to the basolateral surface of the epithelium [ ] . similar to m cells, it might be worthwhile to target apical receptors exploited by enteropathogens on small intestinal enterocytes to promote uptake of antigens by the epithelial barrier. a potential candidate would be aminopeptidase n (anpep), a zinc-dependent peptidase present in the brush border of small intestinal enterocytes, which serves as an entry receptor for several coronaviruses and also binds f fimbriae, a colonisation factor produced by porcine-specific enterotoxigenic e. coli. anpep also transports f fimbriae as well as microparticles functionalised with anpep-specific monoclonal antibodies across the intestinal epithelial barrier, resulting in robust intestinal siga responses, at least in piglets [ , ] . although the selective targeting of vaccine antigens to apical receptors might promote their uptake by the epithelium via transcytosis, this process is in itself insufficient to trigger protective intestinal immunity upon oral vaccination and explains the need to include adjuvants. these adjuvants should act on antigen presenting cells as well as intestinal epithelial cells to promote the induction of protective siga and cell-mediated immune responses. indeed, enterocytes not only provide a physical barrier separating the intestinal lumen from the host tissues, but also relay information on the luminal content to the underlying immune cells through the secretion of inflammatory or tolerogenic mediators. for instance, during the steady state, enterocytes produce thymic stromal lymphopoëtin (tslp) and transforming growth factor (tgfβ), which imprint a tolerogenic phenotype on intestinal dendritic cells [ ] . in contrast, upon infection enterocytes secrete il- and il- [ ] . this probably facilitates a switch from a tolerogenic to an immune-inductive environment, allowing activation of intestinal antigen presenting cells. as yet the most effective adjuvants for oral application are the enterotoxins from vibrio cholera (ct) and enterotoxigenic e. coli (etec) (lt). due to inherent toxicity, dmlt was developed, a nontoxic lt mutant retaining its adjuvanticity. this dmlt triggered intestinal memory responses upon oral vaccination with a nonliving etec vaccine and seems a promising candidate to be included as adjuvant in oral vaccines [ , ] . similarly promising strategies have been reported for eimeria [ ] . recent studies have shown that eimeria-induced il- production is critical in the initiation of early innate immune response in coccidiosis and blocking of il- production by exogenous il- -neutralizing antibody reduced both the intracellular development of eimeria and the severity of intestinal lesion [ ] [ ] [ ] . in summarizing this part, future design of oral vaccines should be tailored to the needs of the target species, focus on the selective delivery of vaccines to epithelial receptors to promote their transport across the epithelial barrier, induce protective immune response in the target tissues, and should include a mucosal adjuvant able to trigger memory siga responses. endospores, or spores, are produced by many bacteria as a response to nutrient deprivation. the spore is a dormant entity about μm in size that can germinate, allowing a nascent cell to emerge and enter vegetative cell growth [ ] . the spore carries remarkable resistance properties, being typically resistant to high temperatures (typically - °c), desiccation, irradiation, and exposure to noxious chemicals [ ] . the two principal sporeforming bacterial genera are bacillus and clostridia with the latter being exclusively anaerobic. members of the bacillus genus are being used as probiotics, that is, microorganisms that are added to the diet to improve the balance of microbial communities in the gi-tract and are therefore beneficial to human or animal health [ , ] . typical species include bacillus clausii, bacillus coagulans and bacillus subtilis. for a long time, it has been assumed that bacillus spores are soil organisms yet the evidence supporting this is actually rather sparse. instead, spores are found in the soil in abundance but live, vegetative cells, are rarely if ever found other than in association with plants or in the animal gut. mounting evidence shows that spores, although found in the soil, are mostly dormant and are shed in the feces of animals, which are their natural hosts [ ] . the consumption of spores associated with soil-contaminated plant matter enables spores to enter the gi-tract, transit the gastric barrier unscathed and then germinate and proliferate in the intestine before excretion as dormant spores [ ] . evidence suggests that spore forming bacteria comprise as much as % of the gut microbiota, indicating that the ability to form spores enables bacteria to survive in the environment as well as entering and transiting the gastric barrier of animals [ ] . the extraordinary resistance properties of bacillus spores coupled with their ease of genetic manipulation, and their successful use as probiotics, makes them attractive candidates for the delivery of heterologous antigens for vaccination. spores have been used as vaccine vehicles in a number of ways, differing principally in whether spores are genetically modified or not. in all cases b. subtilis has been utilized due to the excellent genetics available. using genetic modification, a chimeric gene consisting of a fusion between a b. subtilis anchor gene and an open reading frame encoding a putative protective antigen is first constructed. the next step is introduction of the chimera into the b. subtilis chromosome using a gene transfer technique, typically dna-mediated transformation, a process in b. subtilis that is straightforward. typically, the anchor is the ′-end of a gene encoding a spore coat protein such that the chimera is displayed on the spore coat. surprisingly, heterologous antigens displayed on b. subtilis spores are mostly stable and do not appear to suffer extensive degradation. using this approach a number of candidate antigens have been displayed and then evaluated in animal models. for example, spores displaying a tetanus antigen ttfc conferred protection to a lethal dose of tetanus toxin when administered orally [ , ] . mice dosed orally with spores expressing part of the alpha toxin of clostridium perfringens were protected to challenge with alpha toxin [ ] . a more recent example is that of clostridium difficile where a c-terminal fragment of the toxin a (tcda) could be stably expressed and when administered orally to hamsters conferred protection to c. difficile infection [ , ] . this particular vaccine has now entered clinical evaluation in humans [ ] . using a non-genetically modified organism (gmo) approach it has been shown that spores can adsorb antigens efficiently onto their surface and surprisingly this is both strong and stable, and reflects the unique biophysical properties of the spore [ ] . for the adsorption approach, it has been shown that the gastric barrier is particularly corrosive and adsorbed antigens are labile, but for intranasal delivery this method appears satisfactory. using this approach inactive (killed) spores can be used and success has included studies showing protection to influenza (h n ) [ ] and significant reduction in lung counts of animals challenged with mycobacterium tuberculosis [ ] . a unique feature of spores is their ability to enhance immune responses and this adjuvant effect has been characterized in depth [ - ]. however, the use of spores as mass-delivery vehicles for vaccines has several limitations. oral delivery clearly is the preferred approach but appears to work effectively only for the gmo approach. oral delivery also raises issues of tolerance and may prove to be a limiting factor. sublingual delivery has also been explored; this approach appears to provide levels of protection that are equivalent to oral delivery, but requires more doses [ , ] . nasal delivery is suitable but raises potential safety issues. for animal vaccines, spores are attractive since they are currently used as feed probiotics but also because they can survive the high temperatures used for feed production and may offer long-term utility. as mentioned already, spores have been manipulated for protection against c. perfringens but there now exists the opportunity to develop spores for protective vaccination to necrotic enteritis, an important poultry disease caused by c. perfringens that has been identified as a high vaccine research priority by the oie ad hoc group (see additional file in http://doi.org/ . /s - - - ). one application that is particularly promising is the use of spore vaccines in aquaculture. with intensive fish farming, bacillus spores are being used as probiotic feed supplements. for shrimp farming, viral diseases have devastated the industry and one of the most important shrimp pathogens is white spot syndrome virus (wssv) that causes seasonal outbreaks of disease [ ] . a number of groups have developed b. subtilis spores that display the vp capsid protein of wssv and when administered in feed appears to protect against white spot disease [ - ]. the mechanism for protection is intriguing; even though shrimp are not thought to produce antibodies, it is clear that presentation of the viral antigens does produce some level of specific immunity. despite the progress being made with spore vaccines one key issue remains: the containment of gmos. because spores are dormant with the potential to survive indefinitely in the environment, the use of recombinant spores in spore vaccines is likely to raise environmental concerns and successful regulatory approvals may be slow or impossible to secure. for human use, it is likely that a case can be made that the recombinant spore vaccines addresses an unmet clinical need, but for animal use devising a method for biological containment will be crucial. technological advancements now make it possible to genetically engineer bacteria and other microorganisms that deliver heterologous antigens in a way that can stimulate mucosal as well as humoral and cellular systemic in veterinary medicine, oral vectored vaccines have been instrumental in the eradication or control of rabies in wildlife reservoirs [ , ] . oral vectored vaccines have also been developed for several other veterinary applications, including some economically important diseases of food-producing animals that are associated with considerable antibiotic use such as porcine circovirus type- (pcv- ); in some cases, the vaccine vector is a chimera containing parts of multiple microorganisms-for instance, an attenuated live vaccine may be used as the vector-and the resulting vaccine simultaneously confers protection against multiple diseases, for instance marek's disease and infectious bursal disease or newcastle disease and avian influenza [ , ] . the development of some vaccine vector systems has been very successful and numerous veterinary vaccines have been developed based on them; the canarypox virus vector system alvac, for instance, has been used for the development of a range of veterinary vaccines including against rabies, influenza, and west nile virus [ ] . similarly, adenovirus vectors have also been widely used in veterinary medicine, both in companion and food-producing animals [ ] . vaccine platforms such as these are particularly valuable as they can allow for the rapid development of vaccine candidates in response to emerging vaccine needs, but the possibility of anti-vector immunity can restrict their usefulness [ ] . research and development of additional vaccine vector platforms is therefore needed. salmonella strains that express foreign antigens, either chromosomally or plasmid-based, have yielded promising results in several species including mice, humans, pigs and chicken [ ] [ ] [ ] [ ] [ ] [ ] . diseases for which these salmonella vectored vaccines were investigated include influenza, brucella abortus, post-weaning diarrhea and heterologous strains of salmonella [ ] [ ] [ ] [ ] . the use of pasteurellaceae as vectors for modified live vaccines against shipping fever in calves is currently under investigation, with promising preliminary findings [ ] . use of this vector system for other diseases including pinkeye has been suggested [ ] . in-ovo vaccination is a mass-vaccination strategy that is mainly used in broiler chickens albeit occasionally also in broiler-breeder and layer chickens [ ] . eggs are injected in the hatchery, typically during the third week of embryonic development around day or . to vaccinate, a small hole is made in the shell at the blunt end of the egg and the vaccine is injected below the chorion-allantoic membrane into the amniotic cavity or directly into the embryo. commercial in-ovo vaccination systems that automatically inject the eggs have been available since the early s. more than % of broiler chickens in the us are vaccinated in ovo, and in brazil that fraction equals % [ ] . the most common use of in-ovo vaccination is for marek's disease, potentially combined with vaccines against other diseases such as gumboro or newcastle disease. the ability to deliver a clearly defined vaccine dosage to every single chick and to invoke early protection in the chicks is among the main benefits of this technology, but it is labor-intensive, causes stress for the chicks, and high sanitary standards need to be followed during vaccine preparation and injection to manage infection risks [ , ] . in addition, the location of the vaccine injection is critical for efficacy. it has been shown, for instance, that if marek's disease vaccine is accidentally deposited into the air cell or allantoic fluid, adequate protection is not achieved [ ] . the stage of embryonic development can have profound effects on vaccine safety and efficacy [ ] . one study, reported that vaccination of - dayold embryos with herpes virus of turkeys (hvt) led to pronounced lesions and embryonic deaths, while vaccination on days did not cause detectable lesions [ ] . embryonic age at vaccination has also been shown to be correlated with antibody titers [ ] . maternal antibody titers actually increase after the typical age for in-ovo vaccinations and peak just after hatch [ ] . this can interfere with proper vaccine responses. however, evidence suggests that some vaccine strains are more affected by maternal antibodies than others [ ] . deliberate vaccine development may therefore limit the often disruptive effects that can be caused by maternal antibodies [ ] . other factors that need to be considered in the development of a successful in-ovo vaccination program include the characteristics of the vaccine or vaccines to be used, the type of incubator in which the eggs are housed in the hatchery, and the breed and age of the parent flock [ ] . in-ovo vaccination strategies are promising means of reducing antibiotic use in poultry production and have been the subject of intense research. importantly, they can provide robust and early protection against immune suppressive diseases such as infectious bursal disease [ , ] and vaccines against multiple diseases have been successfully combined. for instance, studies have shown that in-ovo vaccination strategies can simultaneously confer protective immunity against marek's disease, infectious bursal disease, newcastle disease, fowl poxvirus, coccidiosis, and necrotic enteritis [ , ] . other combination vaccines under investigation include vectored vaccines that simultaneously provide protection against newcastle disease and infectious bursal disease [ ] . in-ovo vaccination strategies have also been explored for other poultry diseases with promising results. this included an avian influenza vaccine based on a non-replicating human adenovirus vector [ ] , a recombinant viral vector vaccine against infectious laryngotracheitis [ ] , recombinant protein eimeria vaccines [ , , ] and a fowl adenovirus vectored vaccine against inclusion body hepatitis [ ] , among many others. a mycoplasma gallisepticum vaccine for in-ovo vaccination of layer chickens has also recently been evaluated, even though high chick losses at hatch were reported for the medium and high doses of the vaccine that were investigated [ ] . therefore, in-ovo vaccination strategies are capable of controlling several economically important poultry diseases. many of these diseases are viral, but can predispose animals to secondary bacterial infections. therefore, in many cases, in-ovo vaccines are promising alternative approaches to the use of antibiotics. clostridium perfringens is widespread in the environment and in the gastrointestinal tract of most mammals and birds. however, this bacterium is also one of the most common pathogens of food-producing animals, causing disease only under circumstances that create an environment which favors growth and toxin production, such as stress, injury, or dietary changes [ ] . the bacterium itself is not invasive, but causes disease through the production of a wide array of toxins and enzymes. however, no single strain produces this entire toxin repertoire, resulting in considerable variation in the toxin profiles and disease syndromes produced by different toxinotypes of this bacterium [ ] . while some of these toxins act only locally, other toxins which are produced in the gut exert their action in other internal organs or can act both locally and systemically [ ] [ ] [ ] . to date, efficacious vaccines are only available for the diseases caused by systemic action of the toxins and vaccination against enteric diseases still remains a challenge. however, some of these enteric diseases caused by c. perfringens are of major economic importance and lead to considerable use of antibiotics. amongst them are necrotic enteritis in broilers and necro-haemorrhagic enteritis in calves. despite the fact that much research is being directed to the development of novel vaccines against these c. perfringens-induced enteric diseases, several key barriers still have to be overcome. in general, clostridial vaccines require multiple doses to achieve full immunity. unfortunately, parenteral booster immunizations are impossible in the broiler industry, where mass parenteral vaccination is only feasible at the hatchery, either in ovo or on day-old chicks. because single parenteral vaccination at day of hatch offers no protection, other delivery methods need to be developed [ ] . oral vaccines can more easily be administered to birds, without the need of individual handling of the chicks and are therefore recommended. however, some questions arise when developing an oral vaccine as compared to the parenteral administration route. in addition to the fact that maternal antibodies can block the immune response in young chicks, also the induction of oral tolerance has to be circumvented and an efficient way to present the antigens to the mucosal immune system has to be developed. oral tolerance is a common problem in mammals and fish when developing oral vaccines. this is in contrast to chickens, where oral tolerance is age-dependent, and only an issue in -to -day-old chicks. after that age, protein antigens have been shown to induce a robust immune response and oral vaccination schemes are thought to be feasible [ ] . one appealing strategy for the delivery of vaccine candidates to the mucosal immune system is the use of attenuated or avirulent bacteria as antigen vehicles [ ] . attenuated recombinant salmonella strains which express c. perfringens antigens have been tested in several studies as oral vaccine vectors, leading to some promising results. however, the amount of protection afforded by these vaccines is not as high as compared to multiple doses of parenteral vaccination, and seems to depend on the colonization level and persistence of the vaccine strain [ ] [ ] [ ] [ ] . this indicates that the use of live vectors to express antigens derived from c. perfringens strains in the gut of broilers is a promising approach, but the vaccine delivery strategy still needs to be optimized to achieve optimal antigen presentation to the mucosal immune system and provide improved protection. alternatives to attenuated salmonella strains can be bacillus subtilis spores or lactobacillus casei, which both have a gras status and have the potential to be used as vaccine carriers for clostridium antigens [ , ] . b. subtilis has the advantage that the heat-stable spores can easily be incorporated in the feed and l. casei has known probiotic effects that facilitate the development of mucosal immunity. however, these types of vectors still have to be tested for their capacity to induce a good immune response, in particular against heterologous antigens, in broilers and whether they are able to provide protection against necrotic enteritis. another issue to be addressed when developing a vaccine against c. perfringens-induced enteric diseases is the choice of the antigens to be included in the vaccine. c. perfringens-induced diseases are the result of the toxins and enzymes that are produced and vaccination of chicks with c. perfringens supernatants provides protection against experimental necrotic enteritis [ , ] . however, the protective capacity of the supernatants depends on the strain used for supernatant preparation, indicating that full protection might be determined by an effective combination of different bacterial immunogens [ ] . in order to elucidate the optimal mixture of antigens to protect against necrotic enteritis, challenge trials are being performed mostly using parenteral vaccination schemes. once the ideal combination of antigens is known, this will have to be adapted to oral delivery strategies. several c. perfringens antigens have been evaluated as potential vaccine candidates. the tested antigens include both c. perfringens toxins (e.g. alpha toxin and the netb toxin) and highly immunodominant proteins identified in postinfection serum from birds immune to necrotic enteritis [ ] . in general, immunization studies of broilers with a single antigen all resulted in some level of protection against experimental necrotic enteritis. remarkably, immunization with netb toxin, which is essential to cause disease in broilers, does not afford higher levels of protection than vaccination with other toxins or proteins. however, when birds were vaccinated either via the parenteral or the oral route, with a combination of both netb toxin and alpha toxin, higher levels of protection were obtained [ , ] . in order to obtain full protection against c. perfringens-induced enteric diseases, not only antibodies that inhibit toxin activity might be needed; a combination of antigens targeting also bacterial proliferation, colonization and/or nutrient acquisition could be more efficient than either one of the individual approaches. indeed, in a recent study disruption of the putative adhesin-encoding gene cnaa resulted in a reduced ability to colonize the chicken intestinal mucosa and to cause necrotic enteritis [ ] . this strengthens the idea that vaccine antigens that target bacterial colonization might be indispensable to obtain a working vaccine against c. perfringens-induced enteric diseases. additional vaccine targets might be enzymes that aid in breakdown of the host tissue and nutrient acquisition, such as, amongst others, mucinases, collagenases and hyaluronidases. in contrast to the extensive efforts to develop a vaccine against necrotic enteritis in chickens, considerably less research has been directed to vaccination against necro-haemorrhagic enteritis in calves. the recent demonstration of the essential role of alpha toxin in necro-haemorrhagic enteritis and the proposition of a pathogenesis model will allow for the more targeted development of a vaccine [ , ] . in calves as in chickens, protection against c. perfringens-induced necrosis can be obtained by antibodies against a mixture of toxins, at least in an experimental model for bovine necro-haemorrhagic enteritis [ ] . furthermore, antibodies against alpha toxin alone, which is essential to cause intestinal disease in calves, are not sufficient to provide the same level of protection as antibodies directed against a mixture of c. perfringens proteins, indicating that a mixture of different antigens will be needed to provide full protection [ ] . in order to fully protect calves against c. perfringens-induced enteric diseases, antigens that target bacterial colonization and proliferation might be of equal importance as antigens targeting the toxin activities. next, it has to be explored whether parenteral vaccination is sufficient to induce a protective immune response or if a combination of systemic and mucosal immunity is needed when not only the bacterial toxins but also bacterial colonization is targeted. as administration of multiple parenteral doses of a vaccine to calves is more feasible than for chicken, it may be assumed that the development of a vaccine against necro-haemorrhagic enteritis is more straightforward and that c. perfringens supernatants can be used as a vaccine preparation. however, native toxins cannot be used as vaccine antigens due to safety issues. inactivation of clostridial toxins is generally achieved by formaldehyde treatment, which risks residual formaldehyde in the vaccine preparation, incomplete inactivation of the toxins, and batch-to-batch variation. moreover, formaldehyde inactivation can induce changes in the tertiary protein structures of relevant antigens and influence the immunogenicity of the vaccines. indeed, vaccination of both chickens and calves with formaldehyde inactivated c. perfringens supernatants or toxins have resulted in a good antibody response, but these are unable to protect against intestinal disease [ , ] . to overcome the need of chemically inactivating the c. perfringens toxins, current research focusses on the use of recombinant toxoids to develop a vaccine against c. perfringens-induced diseases. while this may be a good strategy to obtain a safe and protective vaccine on a laboratory scale, the production process is more laborious and time-consuming than production of conventional toxoids, especially because of the required purification steps [ ] . therefore, recent studies have explored the use of efficient low-cost alternatives, such as non-purified recombinant clostridial toxins and even recombinant bacterins, with success [ ] [ ] [ ] . in summary of this section, considerable progress has recently been made in the development of efficacious vaccines against c. perfringens-induced enteric diseases. the main issue that hampers a breakthrough in this field is the identification of a defined combination of antigens that is able to provide full protection against disease. these antigens will most likely target both the bacterial toxins and the bacterial colonization and proliferation. for the broiler industry, once the ideal vaccine antigens have been identified, development of an oral vaccine is needed. coccidiosis, an enteric disease cause by protozoan parasites of the genus eimeria, remains a major economic and welfare concern for the poultry industry globally. seven species (eimeria acervulina, e. brunetti, e. maxima, e. mitis, e. necatrix, e. praecox and e. tenella) are known to infect chickens, and at least six others infect turkeys [ , ] . the costs associated with coccidial disease are difficult to calculate, but have been estimated to exceed billion us dollars for the chicken industry alone, worldwide [ ] . because coccidiosis is a predisposing factor for the occurrence of necrotic enteritis, the true economic burden is likely even higher. all eimeria species can cause disease but the severity and clinical symptoms vary among species, and there is little or no cross-protection across species or some strains [ , ]. modern poultry production systems require effective control of coccidian parasites, typically through the routine use of anticoccidial drugs in feed or water. in the european union, eleven different anticoccidial drugs are currently licensed and between and tonnes are sold for use in animals for markets such as the uk every year [ ] . anticoccidial drugs can be divided into two groups, synthetic or chemical anticoccidials and ionophores, which are products of fermentation [ ] . in some countries such as the us, ionophores are classified as antibiotics, albeit with low human medical importance. the ionophores currently dominate the anticoccidial drug market, largely because they provide incomplete protection, even against naïve field strains without any drug resistance. low levels of parasites survive and induce protective immunity against the prevailing local parasite strains, without causing clinical disease [ ] . anticoccidial drugs provide an efficient means of controlling coccidial parasites and are highly cost-effective. however, drug resistance is widespread and increasing consumer concerns related to drug use in livestock production and residues in the food chain encourage the use of alternatives such as vaccination. notably, because coccidiosis is a predisposing factor for necrotic enteritis and other secondary bacterial infections, efficient control of this parasite is important to minimize the use of medically important antibiotics, including those deemed critically important for human health, in poultry production. the first anticoccidial vaccine was marketed in [ ] . it is a live parasite vaccine which includes multiple wild-type (i.e., non-attenuated) eimeria species. exposure to limited levels of such non-attenuated parasites permits the induction of a natural immune response in the chicken, resulting in protection against subsequent coccidial challenge. however, because protective immune responses against eimeria are fully species specific, the inclusion of each individual target species is necessary if comprehensive protection is to be achieved, which results in relatively complex vaccine formulations. such vaccines commonly include between three and eight parasite species or strains. the approach has been highly successful, although the lack of attenuation has been associated with reduced flock performance following vaccination and occasional clinical disease (reviewed elsewhere [ ]). in response to this limitation, a second generation of live eimeria vaccines has been developed using attenuated parasite lines. for most of these vaccines, attenuation was achieved by selecting for so-called precocious strains, which typically exhibit reduced pathogenicity with fewer and/or smaller rounds of asexual replication. these attenuated strains retained their ability to immunize. the first live attenuated anticoccidial vaccine was launched in , and several similar vaccines have been developed since using the same approach [ ]. nonattenuated and attenuated anticoccidial vaccines have become popular in the breeder and layer sectors, but are less widely used in the much larger broiler sector due to their relatively high cost compared to anticoccidial drugs and their limited availability. because eimeria cannot replicate effectively in vitro, the production of these live vaccines can only be achieved in eimeria-free chickens and separate chickens have to be used for each species or strain to be included in a vaccine. despite these production concerns billions of anticoccidial vaccine doses are sold every year, but more would be required to fully meet the growing demand. efforts to improve on first and second generation live anticoccidial vaccines have included extensive attempts to identify antigens that are appropriate for use in subunit or recombinant vaccines. in addition, progress has been made on the preparation of novel adjuvants and some promising results have been obtained, although data on their use in poultry has so far remained fairly limited [ ] . as an example, one vaccine is formulated from a crude mix of affinity purified e. maxima gametocyte antigens [ ], although the levels of protection achieved have remained controversial and production of the vaccine still requires parasite amplification in chickens. numerous studies have suggested that defined antigens such as apical membrane antigen , immune mapped protein , lactate dehydrogenase and so are highly promising vaccine candidates (reviewed elsewhere [ ] ). studies of eimeria field populations have reported limited diversity in many of these antigens, indicating that recombinant vaccines for eimeria may succeed even though antigenic diversity has undermined equivalent vaccines for related parasites such as plasmodium [ , ] . however, at present no recombinant anticoccidial vaccine is close to reaching the market. one of the biggest remaining challenges is how to deliver the antigens in an affordable, effective, and, most importantly, scalable manner. a range of vectored expression/delivery systems have been suggested including fowlpox virus (fwpv), hvt, salmonella typhimurium, yeasts such as saccharomyces cerevisiae and the tobacco plant nicotiana tabacum, with several showing promise [ ] . most recently, it has been suggested that eimeria itself might function as an expression/delivery vector for vaccine antigens [ ] [ ] [ ] . the ability to express and deliver anticoccidial vaccine antigens from multiple parasite species in a single transgenic line could provide an opportunity to streamline anticoccidial vaccine production from as many as eight lines to just one or two. using an attenuated vector species such as e. acervulina can improve productive capacity enormously and reduce vaccine cost. the parasite vector may also provide some ability as an adjuvant and methods for on-farm delivery are well established [ ] . in summary of this section on new coccidiosis vaccines, as pressure to reduce antibiotic drug use in livestock production increases it is clear that the demand for coccidial vaccines is stronger than ever. in the us, approximately - % of broiler companies use programs that include vaccination to control coccidiosis [ ] . this trend is primarily driven by demands to produce "no antibiotics ever" poultry products. however, it has also been shown that some coccidial vaccines provide an opportunity to replace drug-resistant field parasites in a poultry house with susceptible vaccine strains. while current european attenuated vaccines are limited by their lower reproductive potential, live vaccines do retain considerable unexplored potential. a better understanding of the underlying immune mechanisms through which these nontraditional approaches operate is needed to allow further progress. ultimately, it is clear that novel vaccines must be cost-effective, compatible with high standards of animal welfare, scalable and easy to deliver. autogenous vaccines (av) are also known as emergency, herd-specific or custom made vaccines. although the legal basis and exact definition differs from country to country, avs are used worldwide (e.g. eu, usa, canada, brazil, china, indonesia, australia, egypt) and have a long history of use. the use of avs for the control of fowl cholera has been well-documented [ , ] . as a common definition, all avs are made from inactivated bacterial or viral strains which were isolated from the same flock in which the vaccine is to be used. the use of avs is only allowed if no licensed vaccine is available, or it is respectively ineffective or does not cover the current pathogen strains in the flock. the definition of a flock varies and may include integrated concepts of production chains in different places; to address the issue, the concept of an epidemiological link has recently been proposed by the co-ordination group for mutual recognition and decentralised procedures [ ] . licensed vaccines have advantages compared to avs, including obligatory good manufacturing practice (gmp) production. licensed vaccines are also produced in bigger batches with defined strains and a high level of quality which makes their efficacy and safety predictable. however, licensed vaccines are not available in all cases. to generate avs, selected bacterial or viral strains are usually combined with a proper adjuvant. several viral or bacterial species can be used in a combination vaccine and different serotypes can also be combined in a polyvalent vaccine. the combination of inactivated viruses and bacteria is also an option. bacterial avs are accepted in all countries of the economic european area, whereas viral avs are not allowed in european countries including france, denmark and spain [ ] . a critical role in the successful production and use of an av falls to the isolation of vaccine strains. therefore diagnostic samples must be carefully obtained, based on appropriate choices regarding which sick and untreated animals to select for sample collection, which necropsy material to select, and which cultivation conditions and strains to use after results from sero-, toxo-or virulencetyping. for that purpose several methods like pcr, maldi-tof ms, slide agglutination or dna sequencing are available. because of the fundamental importance of the strain choice for the production of an adequate av, close collaboration between diagnostic laboratory and vaccine production is critical. each production is custom-made and numerous adjuvants, viral and bacterial isolates, including serotypes, toxins and species, provide countless combinations. this underlines the importance of experience as the basis in the production of high quality avs. the veterinarian also has obligations regarding diagnosis, ordering and responsibility for the administration of the vaccine. a variety of bacterial components are often used in avs. these include for poultry: depending on the animal species and age at vaccination different adjuvants can be used. as a standard adjuvant with good safety and efficacy, aluminium hydroxide is often used for production. polymer and other gel-like adjuvants are also available for production in aqueous mixtures. oily adjuvants, especially for water-in-oil emulsions, require a more sophisticated mixing procedure because of the need of a stable emulsion. furthermore oily vaccines might pose safety concerns. however, these induce a promising long lasting immune response because of a depot effect. in the case of organic animal production use of plant oil might be an option in order to avoid unwanted hydrocarbons. the risk of adverse effects, which depend on the adjuvant-antigen combination, can be decreased by standardization of the protocols. more data regarding the efficacy and safety of avs in field studies should be collected because clinical safety and efficacy is not regulated. . further steps in quality control include the inactivation test, endotoxin content or stability tests. some producers offer gmp production, and gmp production is required in some countries such as finland or sweden [ ] . in most countries gmp is only recommended. this example shows the vast differences in national legislation regarding the definition and interpretation of avs. because of worldwide circulation of animals and their pathogens a harmonization of manufacture, control and use of immunological veterinary medicinal products like av is important, and the aim at the economic european area [ ] . in summary, avs are a valuable option in certain situations where commercial vaccines are either not available or expected to lack efficacy because of a mismatch between circulating and vaccine strains. the selection of adequate clinical isolates and vaccine formulations requires considerable expertise and the effective use of avs depends on adequate manufacturing and appropriate veterinary oversight. regulatory differences among countries create a highly fragmented legal landscape that would benefit from further harmonization. vaccines are proven strategies for the prevention or control of infectious diseases in animal populations. therefore, they are promising alternatives that can reduce the need to use antibiotics in food-producing animals and their direct mitigating impact on antibiotic consumption has been demonstrated in a number of studies, even though the relationship between antibiotic use and vaccination is not in all cases clear-cut. the ideal vaccine is safe, effective against a broad range of pathogens, and easily adapted to mass-application. at the same time, it is cheap to produce and use, easy to register across key jurisdictions, and generates durable protection, ideally after a single administration. existing vaccines still fall short of these ideals. in fact, many current vaccines have a number of shortcomings with regard to safety, efficacy and/or user-friendliness that limit their ability to replace antibiotic use. overcoming these challenges will take close collaboration and innovative new approaches. public-private partnerships represent one promising governing structure for assuring such close collaboration across public and private sectors. investments in basic and applied research are equally needed to overcome these challenges, and research needs will have to be prioritized to ensure scarce resources will be preferentially dedicated to areas of greatest potential impact. research to characterize and quantify the impact of vaccination on antibiotic use is equally needed. yet, some data demonstrating the ability of vaccines to reduce antibiotic consumption are already available. similarly, key research breakthroughs and a number of highly promising vaccination approaches are already in development. these include new oral vaccines based on bacterial spores, live vectors, or new delivery strategies for inactivated oral vaccines; they also include new vaccination strategies in-ovo, combination vaccines that protect against multiple pathogens, the use of recent biotechnological advances, and comprehensive approaches to manage diseases 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immerseel f ( ) toxin-neutralizing antibodies protect against clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis recombinant alpha, beta, and epsilon toxins of clostridium perfringens: production strategies and applications as veterinary vaccines potency against enterotoxemia of a recombinant clostridium perfringens type d epsilon toxoid in ruminants potential protective immunogenicity of recombinant clostridium perfringens alpha-beta -beta fusion toxin in mice, sows and cows protective potential of recombinant non-purified botulinum neurotoxin serotypes c and d host specificity of turkey and chicken eimeria: controlled cross-transmission studies and a phylogenetic view we would like to thank the organizers, sponsors, and participants of the nd international symposium on alternatives to antibiotics on which this manuscript is based, in particular the united states department of agriculture (usda) and the world organisation for animal health (oie). the authors declare that they have no competing interests.authors' contributions kh, fvi, and cg planned the manuscript. kh led the drafting of the manuscript. lb, dpb, ec, smc, bd, eev, eg, kk, sl, mm, mr, mcs, nmw, cg, and fvi provided additional information and contributed to writing the manuscript including drafting selected sections and reviewing the manuscript. fvi and cg revised the manuscript. all authors read and approved the final manuscript. key: cord- - yi tz authors: poon, l. . c.; yang, h.; dumont, s.; lee, j. c. s.; copel, j. a.; danneels, l.; wright, a.; costa, f. da silva; leung, t. y.; zhang, y.; chen, d.; prefumo, f. title: isuog interim guidance on coronavirus disease (covid‐ ) during pregnancy and puerperium: information for healthcare professionals – an update date: - - journal: ultrasound obstet gynecol doi: . /uog. sha: doc_id: cord_uid: yi tz nan in response to the world health organization (who) statements and international concerns regarding the coronavirus disease (covid- ) outbreak, the international society of ultrasound in obstetrics and gynecology (isuog) is issuing the following guidance for management during pregnancy and puerperium. given the uncertainty regarding many aspects of the clinical course of covid- in pregnancy, frequently updated information may help obstetricians and ultrasound practitioners in counseling pregnant women and further improve our understanding of the pathophysiology of covid- in pregnancy. this statement, which is an update on our previous interim guidance (appendix s ), is not intended to replace other previously published interim guidance on evaluation and management of covid- -exposed pregnant women and should be considered in conjunction with relevant advice from organizations such as: american college of obstetricians and gynecologists (acog): https://www.acog.org/clinical-information/phys ician-faqs/covid- -faqs-for-ob-gyns-obstetrics centers for disease control , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), is a global public health emergency. since the first case of covid- pneumonia was reported in wuhan, hubei province, china, in december , the infection has spread rapidly to the rest of china and beyond , . coronaviruses are enveloped, non-segmented, positivesense ribonucleic acid (rna) viruses belonging to the family coronaviridae, order nidovirales . the epidemics of the two β-coronaviruses, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), have caused more than cumulative cases in the past two decades, with mortality rates of % for sars-cov and % for mers-cov [ ] [ ] [ ] [ ] . sars-cov- belongs to the same β-coronavirus subgroup and it has genome similarity of about % and % with sars-cov and mers-cov, respectively . sars-cov- is spread by respiratory droplets and direct contact (when body fluids of an infected person touch another person's eyes, nose or mouth, or an open cut, wound or abrasion). it should be noted that sars-cov- has been found in a laboratory environment to be viable on plastic and stainless-steel surfaces for up to h, whereas on copper and cardboard it is viable for up to h . sars-cov- also remains viable and infectious in aerosols for hours, raising the possibility of airborne transmission. the report of the world health organization (who)-china joint mission on coronavirus disease (covid- ) estimated a high r (reproduction number) of - . . the latest report from who , on april th , estimated the global mortality rate of covid- to be . %. however, other reports, which utilized appropriate adjustment for the case ascertainment rate and the time lag between onset of symptoms and death, suggested the mortality rate to be lower, at . % . huang et al. first reported on a cohort of patients with laboratory-confirmed covid- pneumonia. they described the epidemiological, clinical, laboratory and radiological characteristics, as well as treatment and clinical outcome of the patients. subsequent studies with larger sample sizes have shown similar findings , . the most common symptoms reported are fever ( . %) and cough ( . %) . myalgia or fatigue ( . %), expectoration ( . %) and dyspnea ( . %) are also reported . diarrhea ( . %) and nausea and vomiting ( . %) are less common . breslin et al. observed similar covid- severity in pregnant patients ( . % mild disease, . % severe disease and . % critical disease) to that reported in non-pregnant patients . on admission, ground-glass opacity is the most common radiologic finding on computed tomography (ct) of the chest ( . %) . no radiographic or ct abnormality was found in of ( . %) patients with non-severe disease and in five of ( . %) patients with severe disease. lymphocytopenia was reported to be present in . % of patients on admission . elevated c-reactive protein and lactic dehydrogenase were observed in . % and . % of patients, respectively. breslin et al. screened asymptomatic pregnant patients admitted to the labor ward, and found that . % of them tested positive; however, . % of these patients developed symptoms during admission or early postpartum . universal testing for covid- remains a topic of debate and its need is determined mainly by local protocol and prevalence of the disease. a recent study from new york, usa, reported that a relatively large proportion ( . %) of patients without any symptoms admitted for delivery tested positive for sars-cov- . of these patients, % developed fever before discharge from the hospital. this indicates the potential problem with triaging patients based merely on symptoms in areas with widespread community infection. pregnancy is a physiological state that predisposes women to respiratory complications of viral infection. due to the physiological changes in their immune and cardiopulmonary systems, pregnant women are more likely to develop severe illness after infection with respiratory viruses . in , pregnant women accounted for % of patients infected with influenza a subtype h n virus, but they accounted for % of h n -related deaths . in addition, sars-cov and mers-cov are both known to be responsible for severe complications during pregnancy, including the need for endotracheal intubation, admission to an intensive care unit (icu), renal failure and death , . the case fatality rate of sars-cov infection among pregnant women is up to % . currently, however, there is no evidence that pregnant women are more susceptible to sars-cov- or that those with covid- are more prone to developing severe pneumonia , - . over and above the impact of covid- on a pregnant woman, there are concerns relating to the potential effect on fetal and neonatal outcome; therefore, pregnant women require special attention in relation to prevention, diagnosis and management. based on the limited information available as yet and our knowledge of other similar viral pulmonary infections, the following expert opinions are offered to guide clinical management. case definitions are those included in the who's interim guidance, 'global surveillance for covid- caused by human infection with covid- virus' . • a patient with acute respiratory illness (fever and at least one sign/symptom of respiratory disease (e.g. cough, shortness of breath)) and a history of travel to or residence in a location reporting community transmission of covid- during the days prior to symptom onset; or • a patient with any acute respiratory illness and who has been in contact (see definition of contact below) with a confirmed or probable covid- case in the last days prior to symptom onset; or • a patient with severe acute respiratory illness (fever and at least one sign/symptom of respiratory disease (e.g. cough, shortness of breath)) and who requires hospitalization and in the absence of an alternative diagnosis that fully explains the clinical presentation. a contact is defined as a person who experienced any one of the following exposures during the days before and the days after the onset of symptoms of a probable or confirmed covid- case: note: for confirmed asymptomatic cases, the period of contact is measured from days before to days after the date on which the sample that led to confirmation was taken. • a suspected case for which laboratory testing for covid- is inconclusive; or • a suspected case for which testing could not be performed, for any reason. a person with laboratory confirmation of covid- , irrespective of clinical signs and symptoms. evidence suggests that a proportion of transmissions occur from cases with no or mild symptoms that do not provoke healthcare-seeking behavior . under these circumstances, in areas in which local transmission occurs, an increasing number of cases without a defined chain of transmission is observed and a lower threshold for suspicion in patients with severe acute respiratory infection may be recommended by health authorities . any suspected case should be tested for sars-cov- using available molecular tests, such as quantitative reverse transcription polymerase chain reaction (qrt-pcr). lower-respiratory-tract specimens likely have a higher diagnostic value compared with upperrespiratory-tract specimens for detecting sars-cov- . the who recommends that, if possible, lowerrespiratory-tract specimens, such as sputum, endotracheal aspirate or bronchoalveolar lavage, be collected for sars-cov- testing . if patients do not have signs or symptoms of lower-respiratory-tract disease or specimen collection for lower-respiratory-tract disease is clinically indicated but collection is not possible, upper-respiratory-tract specimens of combined nasopharyngeal and oropharyngeal swabs should be collected. if initial testing is negative in a patient who is strongly suspected of having covid- , the patient should be resampled, with a sampling time interval of at least day, and specimens collected from multiple respiratory-tract sites (nose, sputum, endotracheal aspirate). additional specimens, such as blood, urine and stool, may be collected to monitor the presence of virus and the shedding of virus from different body compartments. when qrt-pcr analysis is negative for two consecutive tests, covid- can be ruled out. the who has provided guidance on the rational use of ppe for covid- . when conducting aerosol-generating procedures (e.g. tracheal intubation, non-invasive ventilation, cardiopulmonary resuscitation, manual ventilation before intubation), healthcare workers are advised to use respirators (e.g. fit-tested n , ffp or equivalent standard) with their ppe , . the centers for disease control and prevention (cdc) additionally considers procedures that are likely to induce coughing (e.g. sputum induction, collection of nasopharyngeal swabs and suctioning) as aerosol-generating procedures and cdc guidance includes the option of using a powered air-purifying respirator , . chest imaging, especially ct scan, is essential for evaluation of the clinical condition of a pregnant woman with covid- [ ] [ ] [ ] . fetal growth restriction (fgr), microcephaly and intellectual disability are the most common adverse effects from high-dose (> mgy) radiation exposure , . according to the american college of radiology and american college of obstetricians and gynecologists, when a pregnant woman undergoes a single chest x-ray examination, the radiation dose to the fetus is . - . mgy, which is negligible, while the radiation dose to the fetus is . - . mgy from a single chest ct scan or ct pulmonary angiogram [ ] [ ] [ ] . chest ct scanning has high sensitivity ( %) for diagnosis of covid- . in a pregnant woman with suspected covid- , a chest ct scan may be considered as a primary tool for the detection of covid- in epidemic areas . informed consent should be acquired (shared decision-making) and a radiation shield applied over the gravid uterus. because of the logistics involved in performing a ct scan on critically ill patients, and the need for thorough cleaning of the ct unit after imaging a covid- patient, a portable chest x-ray is an acceptable alternative to a ct scan. a ct pulmonary angiogram is generally used in preference to a ventilation/perfusion scan on clinical suspicion of pulmonary embolus and should not be withheld during pregnancy. during the covid- pandemic, it has been proposed that ultrasound examination of the lungs of a pregnant woman with suspected covid- could be carried out at the same time as the obstetric scan, in order to minimize the risk of radiation as well as streamline the clinical assessment of these patients. this mode of lung imaging could also be considered when chest x-ray and ct scan are not available. however, management should be determined by the clinical features and severity of the disease, and not be based merely on diagnostic imaging. a practical guide on how to perform lung ultrasound examination in pregnant women with suspected covid- was published recently . in brief, the lung ultrasound scan can be performed using any type of machine and any type of transducer (including linear, convex and microconvex). detailed guidance regarding cleaning of ultrasound equipment and transducers in the context of covid- has been provided in the article 'isuog safety committee position statement on safe performance of obstetric and gynecological scans and equipment cleaning in context of covid- ' . on ultrasound, horizontal 'a-lines' are the hallmark of the normal lung. when the lung loses normal aeration, but is not completely consolidated, it generates different shapes and lengths of vertical artifacts, usually called 'b-lines'. when the density of the peripheral lung parenchyma is increased, ultrasound examination shows a white area (the so-called 'ultrasonographic white lung'), in which neither a-lines nor separated b-lines are visible. consolidation appears as an irregular hypoechoic area, and pleural effusion appears anechoic. a videoclip and images demonstrating these findings can be found in the original article . following the obstetric abdominal ultrasound examination, with the patient in a supine position, the examiner can simply move the probe from the abdomen to the chest, scanning the anterior and lateral areas of the thorax. the examination should cover the whole pulmonary area, from basal to upper areas of the thorax. four vertical lines (right mid-axillary line, right parasternal line, left parasternal line and left mid-axillary line) can be followed in order to perform a systematic examination. with the patient in a sitting or lateral position, the posterior paravertebral surface of the thorax should then be scanned, from basal to upper areas or along posterior-axillary lines according to the patient's position. refer to appendix . triage of pregnant patients who potentially have covid- is of great importance in order to reduce the risk of exposure for patients and healthcare workers alike . setting up a triage station outside the obstetric ward and outpatient clinic is essential, allowing for systematic and thorough screening (appendix ) for symptoms (e.g. fever, cough, sore throat) and risk factors (based on travel history, occupation, contact and cluster (tocc)). temperature should be checked. when concern is raised about a potential covid- patient, i.e. due to symptoms and/or tocc risk factors, a surgical facemask should be put on the patient and she should be separated from other patients, preferably in an isolated covid- unit. healthcare workers should don appropriate ppe for the management of pregnant women with suspected/probable/confirmed covid- , , . refer to appendix . suspected, probable and confirmed cases of covid- should ideally be managed by designated tertiary hospitals with effective isolation facilities and protection equipment . suspected/probable cases should be treated in isolation and confirmed cases should be managed in a negative-pressure isolation room, when available; otherwise, designated covid- units can help reduce spread by cohorting affected patients with dedicated staffing. designated hospitals should set up a dedicated operating room and a neonatal isolation ward. ideally, the operating room and neonatal isolation ward should have negative-pressure ventilation. when it is not possible to set up negative-pressure ventilation for operating rooms, it is advisable to discuss with the hospital engineer whether it is appropriate to switch off their positive-pressure ventilation. all attending medical staff should don ppe (fit-tested n , ffp or equivalent-standard respirator, eye protection (goggles and/or face shield), disposable fluid-resistant and impermeable protective gown and double gloves) when providing care for confirmed cases of covid- , , . however, in areas with widespread local transmission of the disease, health services may be unable to provide such levels of care to all suspected, probable or confirmed covid- cases. pregnant women with a mild clinical presentation may not initially require hospital admission, and home confinement can be considered, provided that this is possible logistically and that monitoring of the woman's condition can be ensured . if negative-pressure isolation rooms are not available, patients should be isolated in single rooms, or grouped together once covid- has been confirmed. for transfer of confirmed cases, the attending medical team should don ppe and keep themselves and their patient a minimum distance of m, or feet, from any individuals without ppe. when an inpatient has confirmed covid- , vigilant maternal monitoring (including oxygen saturation monitoring) is of paramount importance, allowing for rapid initiation of supportive care . although not yet validated in covid- pregnant patients, a modified early obstetric warning score (meows) can be used to enable early recognition of critical illness , . an adapted meows chart is provided in appendix . when the maternal condition requires additional care, this should not be withheld due to pregnancy. respiratory indications for transfer to an isolated negative-pressure room in the icu include pulmonary edema, need for airway protection and necessity of mechanical ventilation . patients with hypoxemic respiratory failure should be admitted to the icu as soon as possible. multidisciplinary care (obstetricians, maternal-fetal-medicine subspecialists, intensivists, obstetric anesthetists, internal-medicine or respiratory physicians, midwives, virologists, microbiologists, neonatologists, infectious-disease specialists) is essential, particularly because some pregnancy-related diseases can cause findings similar to those of severe covid- (e.g. pulmonary embolism) and because the physiologic changes of pregnancy may affect management (e.g. optimal maternal positioning, changes in respiratory physiology affecting appropriate ventilator settings). particular attention should be paid to fluid and electrolyte balance. symptoms should be treated, for example with antipyretic medicines. maternal vital signs (preferably using meows) and oxygen saturation level should be monitored vigilantly to minimize maternal hypoxia. arterial blood-gas analysis should be conducted. repeat chest imaging (when indicated) should be performed. complete blood count should be evaluated regularly, with renal-and liver-function testing and coagulation testing. ( ) the approach to symptomatic treatment and surveillance is the same as for suspected/probable cases. conservative fluid administration is advised , . fluid balance should be evaluated regularly to minimize the risk of fluid overload . isotonic crystalloid fluid is the first choice of fluid to be administered. ( ) currently, there is no proven antiviral treatment for covid- patients, although a number of drugs are being trialed therapeutically in patients with severe symptoms. decisions regarding antiviral, antimalarial and antibiotic treatment should be undertaken in conjunction with local infectious-disease experts, and with the obstetrician providing advice on potential maternal or fetal effects of any treatment regimen. a summary of potential treatments, including hydroxychloroquine, lopinavir/ritonavir, interferon β- b, tocilizumab, azithromycin and remdesivir, is provided in appendix s . ( ) in non-pregnant covid- patients, comorbidities such as hypertension or diabetes seem to increase the risk for progression to severe disease, with poorer clinical outcome . therefore, it is advisable to monitor closely pregnant patients with these comorbidities and to be aware of this increased risk. ( ) the degree of severity of covid- pneumonia is defined by the infectious diseases society of america/american thoracic society guidelines for community-acquired pneumonia (appendix s ) , . ( ) severe pneumonia is associated with a high maternal and perinatal mortality rate; there is, therefore, a requirement for aggressive treatment, including supporting measures with hydration, oxygen therapy and chest physiotherapy. the case should be managed in a negative-pressure isolation room in the icu, with the woman in a semi-recumbent or prone position, if feasible. support should be provided by a multidisciplinary team . ( ) antibacterial treatment: appropriate antibiotic treatment in combination with antiviral treatment should be used promptly when there is suspected or confirmed secondary bacterial infection, following discussion with microbiologists. ( ) blood-pressure monitoring and fluid-balance management: in patients without septic shock, conservative fluid-management measures should be undertaken . excessive fluid can worsen hypoxemia in severe disease without shock , . in patients with septic shock, fluid resuscitation and inotropes are required to maintain an average arterial pressure ≥ mmhg and a lactate level < mmol/l , . the hour- surviving sepsis campaign bundle of care is a concise and practical approach to initial care for (suspected) sepsis . this bundle of care is provided in appendix s . the who advises administration of - ml crystalloid intravenous fluid in the first - min, as a bolus . ( ) oxygen therapy: supplemental oxygen should be used to maintain oxygen saturation > % , ; oxygen should be given promptly to patients with hypoxemia and/or shock, and the method of ventilation should be according to the patient's condition and following guidance from the intensivists and obstetric anesthetists . accelerated hypoxemia in pregnancy is possible, due to increased oxygen consumption and reduced functional residual capacity . the intensivist should be aware of a higher likelihood of difficult intubation and greater risk of aspiration during pregnancy. ( ) medically indicated preterm delivery should be considered by the multidisciplinary team on a case-by-case basis. early delivery may aid ventilation, allowing for prone ventilation if required. ( ) even in confirmed covid- patients, other causes for maternal collapse should be examined . it has been reported that viral pneumonia in pregnant women is associated with an increased risk of preterm birth, fgr and perinatal mortality . based on nationwide population-based data from taiwan, it was demonstrated that pregnant women with viral pneumonia (n = ) had an increased risk of preterm birth, fgr and having a newborn with low birth weight and apgar score < at min, compared with those without pneumonia (n = ) . a case series of pregnant women with sars-cov in hong kong, china, reported three maternal deaths, that four of seven patients who presented in the first trimester had spontaneous miscarriage, four of five patients who presented after weeks had preterm birth and two mothers recovered without delivery but their ongoing pregnancies were complicated by fgr . currently, there are limited data regarding the impact on the fetus of maternal sars-cov- infection. there is an apparent increase of iatrogenic preterm birth but not of spontaneous preterm birth; therefore, cervical-length screening is not recommended. fever is common in covid- patients. previous data have demonstrated that maternal fever in early pregnancy can cause congenital structural abnormalities involving the neural tube, heart, kidney and other organs [ ] [ ] [ ] . however, a study of pregnant women reported that the rate of fever in early pregnancy was %, while the incidence of fetal malformation in this group was . % . among the viable pregnancies with data collected at - weeks of gestation, in the pregnant women with a reported temperature > • c lasting - days in early pregnancy, compared to those without a fever in early pregnancy, the overall risk of fetal malformation was not increased (odds ratio = . ( % ci, . - . )) . previous studies have reported no evidence of congenital infection with sars-cov, and currently there are no data on the risk of congenital malformation when sars-cov- infection is acquired during the first or early second trimester of pregnancy . if appropriate, giving advice via telephone or videoconferencing to patients with suspected/probable/confirmed covid- can be considered . when the patient has to be seen in hospital, early triage and isolation measures should be applied; all healthcare workers attending these patients should wear appropriate ppe , . management in a negative-pressure room is advised for confirmed cases. however, if this is not available, a dedicated single consultation room, from which any unnecessary equipment has been removed, is advised. thorough disinfection between patients, according to local protocols, is essential, especially of high-touch surfaces , . hospital admission might be required for pregnant patients with suspected/probable/confirmed covid- , either because of the disease itself or for obstetric reasons. a separate (part of the) obstetric ward should be reserved for these patients, preferably with negative-pressure rooms for confirmed cases. this ward should use dedicated equipment, such as cardiotocography (ctg) or ultrasound machines . this equipment should not be removed from the room/ward without appropriate disinfection. prompt review by senior team members and, if necessary, multidisciplinary review, is advised for these patients . even if a patient is managed in an isolation ward, woman-centered and skilled care with psychosocial support remains important . additionally, thromboprophylaxis must be considered for all pregnant women who are managed as inpatients, especially in those with severe disease, unless delivery is imminent (within h) , , . the royal college of obstetricians and gynaecologists advises prophylactic low-molecular-weight heparin for all pregnant women admitted with covid- , and this should also be considered in outpatient self-isolating patients on a case-by-case basis, according to risk factors . however, if the patient decompensates rapidly, a thorough risk-benefit analysis should be made regarding the administration of thromboprophylaxis, due to safety concerns regarding its use in conjunction with neuraxial analgesia. following maternal assessment, ctg for fetal heart-rate (fhr) monitoring, at an appropriate gestational age according to local practice, should be undertaken, as well as ultrasound assessment of fetal growth and amniotic fluid volume, with umbilical artery doppler if necessary. in severe covid- cases, the fetal scan can be performed once the patient is stabilized. all sonographers/sonologists should don appropriate ppe when undertaking the ultrasound scan . adequate cleaning of ultrasound equipment and transducers should be performed before further use . pregnant women with confirmed sars-cov- infection who are asymptomatic, or recovering from mild illness, should be monitored with -weekly ultrasound assessments of fetal growth and amniotic fluid volume, with umbilical artery doppler if necessary , . when the infection is acquired in the first or early second trimester of pregnancy, a detailed morphology scan at - weeks of gestation is indicated, and these pregnancies should be monitored carefully after recovery. the pregnancy should be managed according to the clinical findings, regardless of the timing of infection during pregnancy. all clinical visits for obstetric emergencies should be carried out in agreement with current local guidelines. all routine follow-up appointments should be postponed by days or until positive test results (or two consecutive negative test results) are obtained. recommendations on how to prioritize obstetric ultrasound services are provided in separate documents , . refer to appendix . covid- itself is not an indication for delivery, unless there is a need to improve maternal oxygenation. for suspected, probable and confirmed cases of covid- , delivery should be conducted in a negative-pressure isolation room whenever possible. the timing and mode of delivery should be individualized, dependent mainly on the clinical status of the patient, gestational age and fetal condition . in the event that an infected woman has spontaneous onset of labor with optimal progress, she can be allowed to deliver vaginally . continuous fetal and frequent maternal monitoring is essential in these patients. therefore, for optimum care as well as for the protection of the medical team, given evidence of presence of the virus in feces and the inability of healthcare workers to use adequate ppe during the delivery, water birth should be avoided , . shortening the second stage by operative vaginal delivery can be considered, as active pushing while wearing a surgical mask may be difficult for the woman to achieve . with respect to a pregnant woman without a diagnosis of covid- , but who might be a silent carrier of the virus, we urge caution regarding the practice of active pushing while wearing a surgical mask, as it is unclear if there is an increased risk of exposure to any healthcare professional attending the delivery without ppe, because forceful exhalation may significantly reduce the effectiveness of a mask in preventing the spread of the virus by respiratory droplets . during labor, excessive intravenous fluid should be avoided, especially when administering oxytocin, since this could worsen fluid overload due to its antidiuretic effect . oxytocin should be administered in an isotonic crystalloid such as . % nacl solution. induction of labor can be considered when the cervix is favorable, but there should be a low threshold to expedite the delivery when there is fetal distress, poor progress in labor and/or deterioration in maternal condition. septic shock, acute organ failure or fetal distress should prompt emergency cesarean delivery (or termination, if legal, before fetal viability) . donning ppe is time-consuming, and this may prolong the decision-to-delivery interval, but it is imperative. women and their families should be informed about this possible delay, which may be of particular importance in category- cesarean delivery. both regional anesthesia and general anesthesia can be considered, depending on the clinical condition of the patient and after consultation with the obstetric anesthetist. as general anesthesia is considered an aerosol-generating procedure, regional anesthesia is preferred. when appropriate, an early epidural should be considered for a laboring woman with confirmed covid- . concerning the presence of a birth-partner during childbirth, we advise adherence to local protocols. it is advisable to give the asymptomatic birth-partner a surgical facemask and they must follow strict hand hygiene . however, when the partner is symptomatic, they must remain in isolation and therefore cannot attend the delivery. for preterm cases requiring delivery between and + weeks of gestation, we urge caution regarding the use of antenatal steroids (dexamethasone or betamethasone) for fetal lung maturation in a critically ill patient, because this can potentially worsen her clinical condition and the administration of antenatal steroids might delay the delivery that is necessary for management of the patient, especially when there is a need to improve maternal oxygenation . the use of antenatal steroids should be considered according to risk-benefit analysis and in discussion with infectious-disease specialists, maternal-fetal-medicine subspecialists and neonatologists , . we advise against the use of steroids in women at risk for late preterm delivery (between and weeks of gestation). such risk-benefit analysis and discussion should likewise be applied to the use of tocolysis in women presenting with spontaneous preterm labor. miscarried embryos/fetuses and placentae of covid- pregnant women should be treated as infectious tissues and should be disposed of appropriately. at present, it is uncertain whether there is a risk of vertical mother-to-baby transmission of sars-cov- . in two studies, with a combined total of pregnant women with covid- in the third trimester, amniotic fluid, cord blood and neonatal throat swab samples tested negative for sars-cov- , suggesting there was no evidence of vertical transmission in women who developed covid- pneumonia in late pregnancy , . furthermore, in the study by qiu et al., vaginal secretion samples tested negative for sars-cov- rna . notably, a neonate born to a pregnant woman with covid- tested positive for sars-cov- rna in the pharyngeal swab sample h after birth, but it was subsequently confirmed that qrt-pcr testing of the placenta and cord blood was negative for sars-cov- , suggesting that intrauterine vertical transmission might not have occurred , . two studies recently explored the possibility of vertical transmission of sars-cov- in a combined total of seven affected pregnancies by testing for sars-cov- -specific antibodies (immunoglobulins g and m (igg and igm)) in neonatal serum samples , . their conclusion, that sars-cov- could be transmitted in utero, was based on the presence of igm antibodies, detected by recently developed automated chemiluminescence immunoassays, in blood drawn from three neonates following birth. however, for all three cases, the neonatal respiratory samples tested negative for sars-cov- rna. in the study of dong et al., the observed rapid decline (within days) of anti-sars-cov- igg levels in the infant, along with a decline in igm antibodies, strongly suggests that neonatal anti-sars-cov- igg antibodies were derived transplacentally from the mother, and not actively induced by the presumed neonatal infection . in order to further investigate the possibility of vertical transmission of sars-cov- , appropriately matched biological samples, including cord blood, placental tissue, amniotic fluid and amnion-chorion-interface swab, should be collected immediately after delivery, using aseptic technique, from women with covid- . a neonatal pharyngeal swab can also be collected. if possible, testing for sars-cov- of the miscarried fetus/placenta of covid- pregnant women should be undertaken. in addition to testing for sars-cov- rna by qrt-pcr, serological testing could be an important supplement in order to clarify the issue of vertical transmission of the virus. longitudinal follow-up for - months of infants born to covid- women should be undertaken . regarding neonatal management of suspected, probable and confirmed cases of maternal covid- , the umbilical cord should be clamped promptly and the neonate should be transferred to the resuscitation area for assessment by the attending pediatric team. there should be different healthcare workers taking care of the mother and the baby in order to minimize the risk of cross-contamination. there is insufficient evidence regarding whether delayed cord clamping increases the risk of infection to the newborn via direct contact . in units in which delayed cord clamping is recommended, clinicians should consider carefully whether this practice should be continued. there is also currently insufficient evidence regarding the safety of breastfeeding and the need for mother-baby separation , . if the mother is severely or critically ill, separation appears to be the best option, with attempts to express breastmilk in order to maintain milk production. for this, there should be a dedicated breast pump and the machine should be washed thoroughly, according to the manufacturer's recommendations, after each use . if the patient is asymptomatic or mildly affected, breastfeeding and colocation (also called rooming-in) can be considered by the mother in coordination with healthcare providers, or may be necessary if facility limitations prevent mother-baby separation. since the main concern is that the virus may be transmitted by respiratory droplets rather than breastmilk, breastfeeding mothers should wash their hands and wear a three-ply surgical mask before touching the baby. in case of rooming-in, the baby's cot should be kept at least m from the mother's bed, and a physical barrier, such as a curtain or glass, may be used , . the need to separate mothers with covid- from their newborns, with the consequence that they are unable to breastfeed directly, may impede early bonding as well as establishment of lactation . these factors will inevitably cause additional stress for mothers in the postpartum period. as well as caring for their physical wellbeing, medical teams should consider the mental wellbeing of these mothers, showing appropriate concern and providing support when needed , . at the time of writing, there are no effective drugs or vaccines to prevent covid- . therefore, personal protection should be considered in order to minimize the risk of contracting the virus. a. good personal hygiene should be maintained: during the covid- pandemic period, close contact with others should be consciously avoided, participation in any gathering in which a distance of at least m between individuals cannot be maintained should be avoided, attention should be paid to hand washing and hand sanitizer (with % alcohol concentration) used frequently , . b. a three-ply surgical mask should be worn when visiting a hospital or other high-risk area. c. medical assistance should be sought promptly for timely diagnosis and treatment when symptoms such as fever and cough are experienced. common symptoms at onset of illness include fever, dry cough, myalgia, fatigue, dyspnea and anorexia. ideally, medical staff assigned to care for suspected, probable or confirmed cases of covid- should minimize contact with other patients and colleagues, with the aim of reducing the risk of exposure and potential transmission. i. medical staff who have been exposed unexpectedly, while without ppe, to a covid- pregnant patient, should be quarantined or self-isolate for days. j. pregnant healthcare professionals should follow risk-assessment and infection-control guidelines following exposure to patients with suspected, probable or confirmed covid- . . pregnant women with confirmed covid- should ideally be managed by designated tertiary hospitals, and they should be informed of the risk of adverse pregnancy outcome. . negative-pressure isolation rooms should be set up for safe labor, delivery (including cesarean section) and postpartum (including neonatal) care. . during the covid- pandemic period, a detailed history regarding tocc and clinical manifestations should be acquired routinely from all pregnant women attending for routine care. . chest imaging, especially ct scan, should be included in the work-up of pregnant women with suspected, probable or confirmed covid- . . suspected/probable cases should be treated in isolation and confirmed cases should be managed in a negative-pressure isolation room. a woman with confirmed infection who is critically ill should be admitted to a negative-pressure isolation room in the icu. . antenatal examination and delivery of pregnant women with covid- should be carried out in a negative-pressure isolation room on the labor ward. human traffic around this room should be limited when it is occupied by an infected patient. . all medical staff involved in management of infected women should don appropriate ppe (fit-tested n , ffp or equivalent-standard respirator, eye protection (goggles and/or face shield), disposable fluid-resistant and impermeable protective gown and double gloves). . management of covid- pregnant women should be undertaken by a multidisciplinary team (obstetricians, maternal-fetal-medicine subspecialists, intensivists, obstetric anesthetists, midwives, internal-medicine or respiratory physicians, virologists, microbiologists, neonatologists, infectious-disease specialists). . vigilant maternal monitoring (including oxygensaturation monitoring) of all covid- patients is paramount, allowing for rapid commencement of supportive care. the use of a meows chart is recommended. . timing and mode of delivery should be individualized, dependent mainly on the clinical status of the patient, gestational age and fetal condition. . both regional anesthesia and general anesthesia can be considered, depending on the clinical condition of the patient and after consultation with the obstetric anesthetist. if possible, regional anesthesia is preferable due to a lower risk of transmission in comparison with general anesthesia. . at present, limited data suggest that there is no evidence of vertical mother-to-baby transmission in women who develop covid- in late pregnancy, although case reports of potential vertical transmission have emerged. . there is currently insufficient evidence regarding the safety of breastfeeding and the need for mother-baby separation. if the mother 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african mothers prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection a randomized, controlled trial of ebola virus disease therapeutics first newborn baby to receive experimental therapies survives ebola virus disease appendix flowchart for assessment of coronavirus disease (covid- ) risk in obstetric-unit attendees. ct, computed tomography; ppe, personal protective equipment; qrt-pcr, quantitative reverse transcription polymerase chain reaction; sars-cov- , severe acute respiratory syndrome coronavirus ; tocc, travel, occupation, contact and cluster. the following supporting information may be found in the online version of this article:appendix s summary of changes from previous interim guidance appendix s potential drugs for treatment of covid- in pregnant women appendix s infectious diseases society of america/american thoracic society criteria for defining severe community-acquired pneumonia , appendix s hour- surviving sepsis campaign bundle of care key: cord- - dcp rd authors: bonfá, eloisa; gossec, laure; isenberg, david a.; li, zhanguo; raychaudhuri, soumya title: how covid- is changing rheumatology clinical practice date: - - journal: nat rev rheumatol doi: . /s - - - sha: doc_id: cord_uid: dcp rd the emergence of covid- in early led to unprecedented changes to rheumatology clinical practice worldwide, including the closure of research laboratories, the restructuring of hospitals and the rapid transition to virtual care. as governments sought to slow and contain the spread of the disease, rheumatologists were presented with the difficult task of managing risks, to their patients as well as to themselves, while learning and implementing new systems for remote health care. consequently, the covid- pandemic led to a transformation in health infrastructures and telemedicine that could become powerful tools for rheumatologists, despite having some limitations. in this viewpoint, five experts from different regions discuss their experiences of the pandemic, including the most challenging aspects of this unexpected transition, the advantages and limitations of virtual visits, and potential opportunities going forward. by the largest tertiary public hospital in latin america, consisting of , beds and eight specialized institutes (heart and lung, orthopaedic, psychiatry, children, cancer, central, rehabilitation and radiology institutes). as the clinical director of the hospital and one of the coordinators of the covid- crisis committee, i was involved in the decision to isolate the central institute (containing beds) solely for patients with covid- (ref. ). this decision meant that the other seven institutes remained at low exposure for covid- . all non-covid patients from our general tertiary emergency unit and from more than specialized ward units allocated in the central institute, including the rheumatology unit, were transferred to these covid-cold institutes. patients from the rheumatology unit were transferred to the orthopaedic institute, along with patients from almost all specialized clinical wards. each specialized ward was allocated to one unit that had approximately % as many beds as were previously allocated to that ward. overall, the pandemic resulted in delays in non-emergency hospitalizations. one main challenge during this period was to divide the team between those who would work in the non-covid- area and those who were recruited to exclusively care for patients with covid- in the isolated covid- institute. a safe hospitalization flow for inpatients and employee safety was quickly established and upon suspicion of covid- , the patient was rapidly transferred to the transition area of the isolated covid- institute. another challenge was to increase the number of intensive care unit (icu) beds available in this central institute from to in months. to achieve this goal, we had to convert surgery rooms into icu beds. during the first months of the pandemic (april-july) in são paulo, > , patients with severe covid- were hospitalized in the isolated institute, and icu beds accounted for more than half of these patients. in terms of patients with rheumatic diseases, the number of hospitalizations decreased by ~ % compared with the same period in the previous year and the number of patients in our rheumatology outpatient clinics decreased by ~ %, to travel into health-care facilities, including for diagnostic tests and clinical laboratory monitoring or even infusions. the result was that with the covid- pandemic, many of the tools that we commonly wielded became unwieldy. this issue was particularly problematic for patients seeing us for a first visit, for patients who were failing to respond to therapies, for patients who needed to be seen urgently for concerning new symptoms or for patients who needed a referral to another specialist for evaluation and work-up of related independent diagnoses. in many instances, we used inadequate temporizing measures rather than a durable solution. for example, some patients with newly diagnosed inflammatory arthritic diseases were prescribed courses of prednisone until an in-person visit became possible. covid- has also taken an emotional toll on our patients, as, like many, they struggled to balance their personal lives as our society shifted towards a lockdown and with the anxiety of a pandemic. abstract | the emergence of covid- in early led to unprecedented changes to rheumatology clinical practice worldwide, including the closure of research laboratories, the restructuring of hospitals and the rapid transition to virtual care. as governments sought to slow and contain the spread of the disease, rheumatologists were presented with the difficult task of managing risks, to their patients as well as to themselves, while learning and implementing new systems for remote health care. consequently, the covid- pandemic led to a transformation in health infrastructures and telemedicine that could become powerful tools for rheumatologists, despite having some limitations. in this viewpoint, five experts from different regions discuss their experiences of the pandemic, including the most challenging aspects of this unexpected transition, the advantages and limitations of virtual visits, and potential opportunities going forward. nature reviews | rheumatology reducing from a mean of ~ , patients per month to ~ , patients per month. the rheumatology biological center, a separate unit dedicated exclusively to patients under biologic therapy, remained opened during the pandemic, and the number of appointments reduced by only ~ % compared with the same period in the previous year. zhanguo li. as a rheumatologist practicing at peking university people's hospital, beijing, the biggest challenge during the covid- pandemic has been how to manage patients with rheumatic diseases remotely using online systems, social media platforms (such as wechat) or telephone calls, because the patients simply could not physically attend the hospital. this alternative access to care was unprecedented and was previously even prohibited by our medical systems and insurance policies. the situation was extremely challenging for rheumatologists and patients for quite a few months, as rheumatologists had no existing online, regulated system for prescribing treatments. consequently, the ceasing of medication or inappropriate self-management occurred in many patients across the country, resulting in flares of disease in some patients. was scarce or lacking, and i feared bringing covid- back home. this situation challenged my conviction that my job as a rheumatologist is the best in the world! for me, the second biggest challenge to managing my (non-covid) patients over the past months has been my fear of putting them at risk through my prescriptions. i mainly see patients with inflammatory arthritis, most of whom are treated with biologics or other targeted therapies. initially, we had no information as to the potential risk associated with such treatments, in terms of increasing the risk of or severity of covid- . thus, whereas i have always prescribed such treatments with the conviction of helping my patients, the challenge here is a profound rethinking of the benefit-to-risk balance of my prescriptions. eloisa bonfá. for the first time, the rheumatology outpatient clinics of our hospital provided virtual care over the phone to define which patients could have their visit postponed, which patients needed a change in prescription or which patients had to come to the clinic for an appointment. postponing all previously scheduled rheumatology outpatient appointments was a challenging task owing to the large number of patient appointments per week (approximately ), and it required a team of staff fully dedicated to this assignment. those health-care workers who were at a high risk of severe illness from covid- were selected for this job. this procedure required several adaptations for the medical staff and patients due to the lack of previous experience with virtual care, as telehealth was only endorsed by the federal council of medicine during the pandemic . several measures of care and risk assessment were established for patients who were required to come into the clinic for an appointment, such as screening for covid- symptoms at entry and at the reception as part of the routine clinical assessment. patients were recommended not to attend a face-to-face appointment if they had any symptoms of covid- . other adaptations included reviewing appointment scheduling, physical distancing in waiting rooms, hand hygiene care and appropriate personal protective equipment. mask wearing is still mandatory in brazil for any outside activity during the pandemic and is also compulsory for patients during appointments. david isenberg. managing patients with serious autoimmune rheumatic diseases (who are often on steroids, immunosuppressives and/or biologics) who you cannot see and examine and do blood tests on has been a huge challenge. it is clear that many patients who have been carefully shielding have not wanted to come to hospital (at the university college hospital, situated in the centre of london) and some have clearly tried hard to deny (to themselves as well as to their physicians) the fact that their underlying disease was getting worse. we had a particularly troubling time months into the pandemic when, in a period of about week in april, we had to admit six patients with systemic lupus erythematosus (sle) who were experiencing acute flares -three of whom went straight into the icu and two of whom died. gossec. an overall and overarching challenge to my practice as an academic full-time rheumatologist at sorbonne université and pitié-salpêtrière hospital, paris, france, was my inner turmoil. when i was young, i spent a few months doing volunteer medical work in a developing country, but for me, this role led to less personal risk than the current pandemic, especially as personal protection equipment eloisa bonfá is a full professor of rheumatology and the clinical director of the largest tertiary public hospital of latin america. her main clinical and research interests are systemic lupus erythematosus and autoimmunity, with relevant contributions in the fields of autoantibodies, vaccines and drug monitoring in autoimmune diseases. she graduated at the university of são paulo medical school, brazil, and undertook specialist training in rheumatology in the same university followed by a -year rheumatology research fellowship at the hospital for special surgery, new york. laure gossec is a professor of rheumatology at sorbonne université and pitié-salpêtrière hospital, paris, france. she has a half-time clinical position where she mainly sees patients with inflammatory arthritis, and a half-time teaching and research position. her main research interests are patientreported outcomes and quality of life, as well as e-health and big data in psoriatic arthritis, spondyloarthritis and rheumatoid arthritis and she has authored more than papers. she is a past-chair of the epidemiology standing committee of eular. david isenberg is the academic director of rheumatology at university college london, uk. he has run both general and autoimmune rheumatic disease clinics for over years. his major research interests are in the structure, function and origin of autoantibodies and improving the assessment of patients with autoimmune rheumatic diseases. zhanguo li is a professor and head of the department of rheumatology and immunology at the peking university people's hospital, china. he is the past president of aplar, and the president of the clinical immunology committee at the chinese society for immunology. he is editor-in-chief of the chinese journal of rheumatology. his research interests are the mechanisms and immune therapy of rheumatic diseases, including rheumatoid arthritis and systemic lupus erythematosus. soumya raychaudhuri is a professor at harvard medical school, and a practicing rheumatologist at the brigham and women's hospital arthritis center. he is also appointed at the broad institute, and the university of manchester. he spends most of his time running a lab that is focused on defining mechanisms of disease in rheumatoid arthritis, and other immune-mediated diseases, using computational biology, genetics and functional genomics. www.nature.com/nrrheum zhanguo li. to adapt to the totally unexpected changes to clinical practice, one option in my department of the people's hospital was to set up a consultant team consisting of rheumatologists to provide medical service free to patients with rheumatic diseases, supported technically by an internet company. it was the first rheumatologist team to provide such support to patients in the country. many patients nationwide were helped by this group over a -month period, from early february to late march . in addition, we used a previously developed smartphone application (smart system of disease management (ssdm)) as a patient self-care instrument to evaluate disease activity and remind patients to contact rheumatologists. the ssdm system was designed for a research project , and the clinical value was also clearly shown in the patients who used this ssdm system during the initial months of the covid- pandemic. david isenberg. my practice has changed completely. during the first months of the pandemic, no routine appointments were offered (although an emergency clinic once a week was available) so that all outpatient consultations took place over the phone or occasionally by video conferencing. for patients with longstanding, well-established disease and on low or moderate doses of steroids and immunosuppressives, i was reasonably content to miss seeing the patients at routine follow-up appointments, but increasingly i have become concerned about the inadequacies of what can be done when not seeing patients face-to-face. among the pleasures and responsibilities of running clinics in an academically inclined institution are doing research and educating both undergraduate and postgraduate students. the introduction of more remote patient assessment has had, and will always have, a detrimental effect on both. it will be harder to recruit patients to trials. we cannot, for example, perform acr , acr or acr assessments of our patients with rheumatoid arthritis or british isles lupus activity group (bilag) assessments of our patients with sle, to help determine their eligibility for a clinical trial. the patient cannot agree to have their blood taken remotely for a project. likewise, teaching opportunities are restricted if we cannot, for example, demonstrate the use of the cross fluctuation test to show fluid in the knee of a patient, identify an enlarged liver or spleen or identify an extensor plantar response. these problems will obviously be detrimental for patient care too. to encourage my patients to come in and see me, especially when so many of them are worried about covid- and the personal risk to themselves. although these changes are essential to our ability to see patients in person, they do make the experience of being a doctor somewhat less personal. implementing social distancing has meant that i see fewer colleagues and staff. it also means that many of the spouses and family members that often accompanied my patients are no longer present. i no longer greet my patients in a crowded waiting room, rather they are brought in from an empty waiting room. masks are essential to protect our patients, especially those on immunomodulatory therapies, but they do make non-verbal cues harder to glean. overall, in-person visits continue to be essential, but they do not feel quite as warm or friendly. on the other hand, virtual visits have been much more effective than i might have anticipated. our clinical infrastructure has enabled video visits, which have proven to be far more productive than a simple phone call. the video visits are very practical and effective for my longstanding patients who are doing well on established therapeutics. previously, some patients who live further away might have taken a day off to drive into boston -in some instances from out of state -for a physical visit. for some of these patients, the ability to do a visit virtually has saved them valuable time. the virtual visit is often more efficient as visits can be easily started and ended, and the next visit can be started immediately. but the virtual visit has definite limitations. most obviously, the inability to do an in-person physical examination and joint examinations cannot be reproduced via video. the exam is essential for assessing our patient's disease activities or making diagnoses, and taking care of new patients or patients with active disease can hence be really challenging. video visits expose the digital divide of our society, and some of our patients are unable to fully take advantage of our infrastructure, especially those who are of fewer means, have poorer internet access or are older and less comfortable with technology. david isenberg. although apocryphal, there is a story that the then chinese premier zhou enlai, when asked by henry kissinger, richard nixon's secretary of state, laure gossec. my professional life has profoundly changed since february. my research activities usually involve very frequent travels to other countries, which have completely stopped since february. my academic work as a professor of rheumatology involves face-to-face interactions with students, which likewise have disappeared completely and have been replaced (partly) by online courses, which are by essence much less interactive. as regards my clinical work, my practice has changed because the hospital has become a place of dread and doom. my patients with inflammatory arthritis do not want to come to the hospital anymore, and i myself feel reluctant to ask them to come. for this reason, for months, all of my patient clinics were switched to teleconsultation, where no physical examination is possible and where the quality of care is lower. in the hospital, instead of accommodating patients with severe rheumatic diseases, our beds were taken over for patients with non-rheumatic diseases, for whom my added value and competency is much lower. one of the fun and interesting parts of my work is interactions within the medical and non-medical team as well as with colleagues outside of rheumatology (such as through staff meetings). most of this social interaction has now disappeared, replaced somewhat by e-mail exchanges. soumya raychaudhuri. i spend most of my time running a research lab in an academic setting. that part of my life has completely changed. like many workplaces, we have moved almost entirely to virtual work environments. hence, research and education has become much less interactive and we have had to shift our culture to accommodate this major change. my clinical practice is within the bwh arthritis center, which is a large clinic that hosts , patient visits per year. my practice specifically has shifted to include more virtual visits and fewer in-person visits. from march to july, my practice was almost entirely virtual. for in-person visits, to reduce the risk of infection for our staff and our patients, the bwh arthritis center has made dramatic changes in the way we interact with each other and with our patients, the flow of patients in and out of the clinic and the clinic rooms, how clinic rooms are turned over and many other components. the changes have been well executed and have affected every aspect of our clinical experience. the result is that i feel confident i do think the situation will mostly go back to normal, as my clinics require the use of physical examinations and ultrasonography. i am planning to keep around % of consultations online for patients in the long term. soumya raychaudhuri. i think that some of the changes will be here to stay. boston is a challenging city for many of our patients to get in and out of, particularly those who are coming from far away, or for those for whom driving or navigating public transportation is hard. for these patients, especially for routine follow-up visits, a virtual visit can offer real advantages. there are patients all over new england who would benefit from access to a referral centre. i can imagine if our institution or others are able to build a great virtual care infrastructure, we could be in a position to expand the scope of patients who our physicians are connecting with and caring for. laure gossec. barriers to online consultations include poor access to the internet for some patients, low-quality internet connection on either side, a lack of user-friendly medical files and also a psychological reluctance from patients regarding online consultations (most patients prefer to see me face-to-face). the wearing of masks is also a barrier to my clinical practice. it hinders the interactions with my patients, which makes shared decision-making (probably the most rewarding part of my clinics) more difficult. will it be that masks will push us back in time, to paternalistic prescriptions? who can say? zhanguo li. current barriers are the lack of a 'telehealth' and medical support system for patient care, which can facilitate patients and doctors in terms of consultations, efficient follow-up and clinical studies. if a second wave of covid- comes, we will face the same difficulty as we had a few months ago. eloisa bonfá. the most important adaptation is consolidation of the regulatory framework for telemedicine in brazil, including reimbursement for this activity. another notable barrier that is expected is the serious economic crisis resulting from the covid- pandemic that will limit investment resources in all areas including health. this limitation of resources will hinder the development and implementation of innovations. hopefully, increased solidarity, a hallmark of this crisis, and regional cooperation will help to overcome the challenges we will have during reconstruction. soumya raychaudhuri. i think telemedicine and virtual medical care could become really powerful tools for the right patient with the right infrastructure. i think that we need to make sure that our patients have access to a proper it infrastructure to mitigate access issues. if language is a barrier, we need to have a means of enabling translation services during our virtual visits. to realize the full potential of virtual care, we need to be able to arrange services and testing for our patients within their communities. after the visit, having an integrated health-care system that enables seamless data transfer is essential. with such an integrated health-care system, arranging imaging, lab work, therapeutic infusions and other services near to home becomes possible without cumbersome administrative barriers. currently, for my more distant patients, i often need to bring them into boston for tests and services. in many cases, they have alternative facilities near to their home, but those facilities are not connected to our system, and arranging local testing and services is challenging without extensive administrative effort. david isenberg. i anticipate that it will be even more important to stress to patients, if their disease is worsening and they have not been seen by a physician (or nurse), that they must contact the hospital and arrange a face-to-face appointment as soon as possible. from the administrative point of view, there will need to be greater flexibility about determining whether patients are to be seen face-to-face or via a telephone consultation. closer links with general practices will also be necessary as, in my experience, some general practitioners have been reluctant to take on routine monitoring of patients on immunosuppressive medication. zhanguo li. many opportunities lie ahead, as long as we focus on the needs of patients and rheumatologists. undoubtedly, more patient-associated and doctor-associated activities will be held online, providing opportunities for patient education and for his opinion on the effects of the french revolution, replied "too early to say". i think the same is true for assessing the long-term effects of covid- . the pandemic has highlighted the value (at least in the short term) of fully electronic record systems, which makes it possible to see patient records, including letters, imaging and blood test results, remotely. i can certainly envisage that some routine follow-up appointments can be undertaken remotely and safely (provided local blood tests can be done), which may well reduce the numbers of patients attending specialist clinics. eloisa bonfá. engaging back to 'normal' activities will take time and it will probably have to wait for a vaccine. until then, all adaptations and risk assessments will remain. but one of the major gains the covid- pandemic will bring is the consolidation of telemedicine and televisits in the care of patients. taking into account that many patients with rheumatic diseases have mobility difficulties, telehealth will provide an alternative approach to the care of these patients, when possible. furthermore, in a large city such as são paulo, with chaotic traffic and long distances, the possibility of avoiding public transportation, not only to prevent the spread of covid- but also to avoid other issues beyond the pandemic, will be more convenient for the patient. zhanguo li. covid- has certainly changed rheumatology practice. although the patient volume has now returned to normal in china, the demographics of patients attending outpatient clinics have altered in terms of disease severity and distance of travel. patients with mild diseases who live in remote areas now tend to see their local doctors, rather than come to rheumatology centres. laure gossec. at this stage, i do not really foresee the situation ever fully getting back to normal. it seems to me that social distances will be increased for a long time. in france, we usually hug and kiss a lot, which i do not think will go back to normal anytime soon. as for my professional life, i do not foresee going back to my previous rate of travel related to my research activities. i also think that medical teaching will be profoundly modified now with much more online resource use and much less face-to-face teaching. from that point of view, we were quite late in france in adopting these teaching methods, and this pandemic might well be an opportune moment for this change. as for my patients, www.nature.com/nrrheum virtual conferences, although patients with severe or difficult-to-treat disease will still need face-to-face appointments with their rheumatologist. laure gossec. improving access to best care, through online consultations but also by improving the patient trail (that is, the way in which patients first see their general practitioner before being referred to a rheumatologist) and decreasing the delay before a consultation, is a priority. better use of online resources and maybe of rheumatology nurses, if they are allowed to play a bigger role in france, are options to move forward, which may be facilitated by the covid- pandemic. soumya raychaudhuri. the implementation of effective virtual visits will be really powerful for rheumatology. the need for an in-person visit will always be there, especially for patients with very active disease or for new patients with uncertain diagnoses. but for patients who we know well, managing them to some extent virtually will have great value. i think in practice these are the patients we talk to on the phone informally and e-mail with. so, having a formal mechanism to take care of them will be beneficial to them and to us. david isenberg. by doing more telephone consultations and reducing the numbers of patients attending clinics face-to-face, it should be possible to reduce the waiting times for patients referred to rheumatologists. i am, though, becoming increasingly concerned about the 'downsides' of what has happened in the past months, notably the missed occurrences of increased disease activity in patients, the loss of educational opportunities for physicians and the difficulties in undertaking translational research. eloisa bonfá. innovations associated with self-care, including smartphone apps and wearable technologies, consolidated during the pandemic, are interesting alternatives for the management of several chronic conditions and will certainly also be useful for patients with rheumatic diseases. above all, there is no way back and the acceleration of digital transformation and the improvements in internet speed that occurred during the pandemic will continue and will transform our lives. this change will provide new opportunities for physicians to update their knowledge on the field and for continuing medical education online, without the need for physical travel. in addition, for organizations, a new way of dealing with administrative work took place with changes in workflows, including replacement of meetings with e-mails, increased working from home and accelerated automation that will forever change the way we work. how a premier u.s. drug company became a virus 'super spreader transmission of -ncov infection from an asymptomatic contact in germany overcoming barriers to providing comprehensive inpatient care during the covid- pandemic letter no. / , federal council of medicine, brasilia (mauro luiz de britto ribeiro to luiz henrique mandetta decree no. . , legislative assembly of the state of são paulo covid- in patients with rheumatic disease in hubei province, china: a multicentre retrospective observational study published online xx xx xxxx l.g. receives research grants from amgen, galapagos, janssen, lilly, pfizer, sandoz and sanofi, and receives consulting fees from abbvie, amgen, bms, celgene, gilead, janssen, lilly, novartis, pfizer, samsung bioepis, sanofi-aventis and ucb, all unrelated to the present paper. s.r. is an employee of brigham and women's hospital, and has recently served as a consultant for abbvie, biogen, gilead, merck and pfizer. he is a founder of mestag. he currently receives research funding from biogen. e.b., d.a.i. and z.l. declare no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -vq ei do authors: johnston, marjorie c.; porteous, terry; crilly, michael a.; burton, christopher d.; elliott, alison; iversen, lisa; mcardle, karen; murray, alison; phillips, louise h.; black, corri title: physical disease and resilient outcomes: a systematic review of resilience definitions and study methods date: - - journal: psychosomatics doi: . /j.psym. . . sha: doc_id: cord_uid: vq ei do background findings from physical disease resilience research may be used to develop approaches to reduce the burden of disease. however, there is no consensus on the definition and measurement of resilience in the context of physical disease. objective the aim was to summarize the range of definitions of physical disease resilience and the approaches taken to study it in studies examining physical disease and its relationship to resilient outcomes. methods electronic databases were searched from inception to march for studies in which physical disease was assessed for its association with resilient outcomes. article screening, data extraction, and quality assessment were carried out independently by reviewers, with disagreements being resolved by a third reviewer. the results were combined using a narrative technique. results of articles, met the inclusion criteria. of these studies, was of high quality, were of moderate quality, and were low quality. the common findings were that resilience involves maintaining healthy levels of functioning following adversity and that it is a dynamic process not a personality trait. studies either assessed resilience based on observed outcomes or via resilience measurement scales. they either considered physical disease as an adversity leading to resilience or as a variable modifying the relationship between adversity and resilience. conclusion this work begins building consensus as to the approach to take when defining and measuring physical disease resilience. resilience should be considered as a dynamic process that varies across the life-course and across different domains, therefore the choice of a resilience measure should reflect this. globally, the prevalence of disease is expected to rise as populations live longer, bringing greater costs to individuals and to health services. , consequently, there is a need to develop interventions and activities to reduce this burden of disease. promoting resilience to physical disease may be one such approach. resilience was defined in a recent review by windle: …the process of effectively negotiating, adapting to, or managing significant sources of stress or trauma. assets and resources within the individual, their life and environment facilitate this capacity for adaptation and "bouncing back" in the face of adversity. across the life course, the experience of resilience will vary. however, despite windle's and others attempts to produce an agreed definition, there is no universally accepted definition of resilience. [ ] [ ] [ ] the variability in the definition of resilience used by studies of physical disease resilience was highlighted in a recent systematic review by stewart and yuen. this examined the factors associated with predicting or promoting resilience in the physically ill and found that a range of variables including social support, psychologic factors, and coping strategies were associated with resilience. resilience research stems from the developmental psychology field and much of it focuses on children. although early life experiences affect resilient outcomes in later life, it is also apparent that research specifically focusing on physical disease and resilience is a more recent development in the resilience field. , the wide variations in the way resilience is defined and operationalized in the physical disease literature are a barrier for those wishing to study resilience and may explain the lack of robust evidence-based resilience interventions for health. better consensus regarding the definition and operationalization of resilience in relation to physical disease is an important step toward the development of interventions aiming to reduce the burden of physical disease. the aim of this systematic review was to summarize the definitions of physical disease resilience and the approaches taken to study resilience in studies examining physical disease and resilient outcomes. the preferred reporting items for systematic reviews and meta-analyses checklist was used to guide the method development and reporting of findings. medline, embase, psycinfo, pubmed, and the cochrane database of systematic reviews were searched from inception to march , studies in which physical disease was assessed for its association with resilient outcomes were included. peerreviewed quantitative studies with or more participants were eligible. the sample size restriction was applied for pragmatic reasons, as our early work demonstrated that studies of physical disease and resilient outcomes often involved complex analyses with multiple variables and that analyses with smaller populations were often underpowered. studies examining only psychiatric disorders and resilience at a family or community level were excluded, as were studies not in the english language. the authors were contacted for studies that were not accessible. titles, abstracts, and full texts were screened independently by reviewers (m. c. j. and t. p.) and any disagreement was resolved by a third reviewer (c. b.). the references of included articles were screened for relevance. primary data extraction was carried out by m. c. j. and t. p., with others acting as independent second reviewers (c. d. b., a. e., and l. i.). the data extraction form was prepared by m. c. j. and finalized after discussion with the other reviewers. data extraction included the study characteristics, the theoretical definition of resilience given by the authors, and the measures of resilience used. any disagreement between the reviewers following this process was resolved by the third reviewer (c. b.). we used the scottish intercollegiate guidelines network critical appraisal checklists to assess the quality of included studies. the quality of the study was not used as an exclusion criterion but as a guide to inform interpretation of the findings. owing to the heterogeneity of the studies and their findings, a meta-analysis was not carried out and the results were combined using a narrative technique. of articles, met the study criteria ( figure) . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the study characteristics are reported in table . of johnston et al. these studies, were cross-sectional and were longitudinal; were from north america, were from europe, and were from asia. the sample size at analysis ranged from - . the average age was years or older for of the studies. [ ] [ ] [ ] , of the studies including both sexes, had a greater proportion of women compared with men. [ ] [ ] [ ] , , , , of the studies reporting ethnicity, had predominantly white ethnicity. [ ] [ ] [ ] [ ] in addition, study was of high quality, were of moderate quality, [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and were low quality. , there was complete agreement between the reviewers for data regarding the theoretical definition of resilience and the resilience measurement. arbitration by the third reviewer was required for the quality assessment in studies. hardy does not succinctly state aim. "we hypothesize that stroke survivors with more extensive social ties and greater emotional and instrumental social support immediately after stroke will experience greater improvements in cognitive function over months of follow-up and achieve a higher level of cognitive functioning months after stroke." longitudinal cohort based on unsuccessful randomised controlled trial the goals of the current study were to identify the life events that older persons experience as most stressful, to determine how often each type of event is identified as most stressful (particularly among those with a recent serious illness), to evaluate the perceived consequences of these events for the lives of older persons, and to evaluate the relationship between demographic factors and measures of health and functional status and these perceived consequences. this study aims to investigate the association between resilience and health behaviours (such as physical activity and consumption of fruit and vegetables) in elderly individuals. we explore the physical, psychological, and social factors associated with reporting a good qol in the context of poor seizure control and socioeconomic disadvantage ("resilient" outcome) and the factors associated with reporting a poor qol in the context of good seizure control and socioeconomic advantage ("vulnerable" outcome). a theoretical definition of resilience was provided by of studies (table ) . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the following component parts of the definition were common to most studies: ( ) an adversity must be experienced to demonstrate resilience (all studies). - , - ( ) resilience is the ability to maintain healthy levels of function over time despite adversity or to return to normal function after adversity ( of studies). [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( ) resilience is a dynamic concept as opposed to a fixed personality trait ( of studies). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] all studies described adversity in their theoretical definition. this is variously described as "adverse events" or "adversity," , , , , , "a potentially highly disruptive event," , "potential trauma," "stressful situations," and "traumatic events." lundman et al. and perna et al. did not identify an adversity in their study design, although they discussed it in their theoretical definitions of resilience. , many studies referred to a previous work by bonanno et al. and defined resilience as maintaining healthy levels of function over time despite adversity. , , , , others similarly defined resilience as returning to normal function after adversity, retaining normal function, or adapting successfully to stressful situations. in contrast to this, studies included the concept of thriving under adversity, , and the highquality study by taylor et al. defined resilience as unexpected positive outcomes in adverse conditions. the study by perna et al. was the only one that stated it was studying resilience as a protective personality factor. this was their justification for not including an adversity in their study design. the remaining definitions treat resilience as a dynamic process, e.g., in studies, bonanno et al. described the ability to maintain stable functioning following adversity as evidencing resilience. , resilience measures there were approaches taken when measuring or identifying resilience in the included studies (table ) : ( ) measuring it using a new or established "resilience scale." , , [ ] [ ] [ ] ( ) identifying it based on outcomes observed in study participants. [ ] [ ] [ ] [ ] , furthermore, studies identified resilience using new , or established resilience scales. , , , in these studies, participants completed questionnaires from which scores indicating levels of resilience were derived. most studies then defined categories of resilience on the basis of scores. , , , for example, scali et al. used an abridged form of the connor-davidson resilience scale, which was split into low, intermediate, and high resilience groups based on tertiles of scores. the remaining studies identified resilience based on outcomes observed in study participants. [ ] [ ] [ ] [ ] , for example, outcome categories for bonanno et al. were based on the severity of the outcome, with the resilient group comprising those with the absence of or only one posttraumatic stress disorder symptom. costanzo et al. defined the resilient group as those in which there was no change in functioning before and after the adverse event. furthermore, studies examined how individuals responded psychologically to an adversity (severe acute respiratory syndrome and breast cancer ) and the way in which these responses changed over time. in these studies, the resilient group was defined as those who demonstrated high psychological functioning following the adversity that changed little over time. , the high-quality study by taylor et al. identified resilience on the basis of better-than-expected outcomes following adversity rather than the maintenance of healthy levels of function over time. the study identified resilience in those who had experienced the adverse event (epilepsy) together with additional disadvantage (poor seizure control and socioeconomic disadvantage) while still maintaining a good outcome (good quality of life). the authors distinguished this from an "expected good" outcome (those who experienced epilepsy but no additional disadvantage and demonstrated good quality of life). there were general approaches taken in studying physical disease resilience when resilience was the johnston et al. bonanno ( ) defined adult resilience as "the ability of adults in otherwise normal circumstances who are exposed to an isolated and potentially highly disruptive event such as the death of a close relation or a violent or lifethreatening situation to maintain relatively stable, healthy levels of psychological and physical functioning …as well as the capacity for generative experiences and positive emotions. (pp. [ ] [ ] ." resilience based on outcomes. outcome categories defined based on ptsd symptoms (assessed using the national women's study ptsd module) at mo following september terrorist attack. ( ) resilient group: high mean score for psychologic functioning on the sf- mcs that changed little over time (i.e., slope that was essentially zero). ( ) chronic dysfunction group: low mean sf- mcs score that changed little over time (i.e., slope that was essentially zero). ( ) recovered group: initial low mean sf- mcs score followed by steep positive increase/slope. ( ) delayed dysfunction group: initial high mean sf- mcs score followed by steep negative increase/slope. in defining these, they drew on previous work by bonanno et al. costanzo et al. o'leary and ickovics have proposed a model to describe three potential responses to adversity (o'leary & ickovics, ), which has been further elaborated by carver (carver, ) . following initial decline in functioning after adverse experience, carver described survival with impairment as continuing compromised functioning, but he distinguished this pattern from resilience, defined as a return to normal or baseline functioning, which is then further distinguished from thriving, described as exceeding one's original level of functioning. resilience based on outcomes. range of measures of "functioning" were compared before and after diagnosis: mental health, mood, psychologic well-being, social well-being, and spirituality/religiosity. results were interpreted as follows: "impairment indicates a decline, resilience indicates no change, and thriving indicates improvement in functioning from wave (prediagnosis) to wave (postdiagnosis)." glymour et al. nr resilience based on outcomes. implied definition of cognitive resilience is maintenance of cognitive function and absence of dementia. nr explicitly. resilience appears to be based on improvement in cognitive function between day ("baseline") and mo after stroke. hardy et al. resilience has been viewed as the process by which individuals survive or even thrive under adversity, incorporating both the internal traits, such as hardiness or high self-efficacy, and the external factors, such as social support, that promote coping. resilience measured using a resilience scale/questionnaire. authors did not set out to measure resilience and instead interpret their findings using resilience theory in their discussion. questions assessing the consequences of stressful life events were adapted from the resilience module of the asset and health dynamics among the oldest old study. authors interpreted individuals who had positive responses to the negative event as responding "resiliently." "resilience is considered to be the most common outcome following exposure to potential trauma. bonanno ( ) proposed four distinct patterns of adjustment in response to potential trauma: ( ) chronic disruption of normal functioning, ( ) recovery with a relatively mild and short-lived disruption of functioning, ( ) delayed disruption of functioning, and ( ) resilience with little or no disruption of functioning." references the same work as and therefore has a similar operationalized definition and the same resilience groups. resilience based on outcomes. psychologic distress measured using chinese health questionnaire (chq- ) at time points ( d, mo, mo, and mo) after surgery for breast cancer. authors defined their trajectories of psychologic distress identified by latent class growth curve analyses as: ( ) resilient: relatively stable levels of low distress across assessment points. ( ) chronic distress: stable high levels of distress at each measurement. ( ) recovered: initial elevated distress that gradually declined. ( ) delayed recovery: initially relatively low distress that elevated before reducing again. lundman et al. resilience has been referred to as a kind of plasticity that influences the ability to recover and achieve psychosocial balance after adverse experiences and as the ability to bounce back in the face of adversity. resilience in older people has been described as the ability to achieve, retain, or regain physical or emotional health after illnesses or losses. resilience measured using a resilience scale/questionnaire. this study treated resilience as a component part of "inner strength." inner strength was a sum score created from factor analysis of assessment scales -the resilience scale (wagnild and young), the sense of coherence scale, purpose in life scale, and the self-transcendence scale. perna et al. resilience is generally understood as the ability to adapt successfully to stressful situations (luthar et al., ; schumacher et al., ) . resilience measured using a resilience scale/questionnaire. in our study, resilience is conceptualized as protective personality factor, referring to the ability to adapt successfully to stressful experiences. used a short version of the resilience scale developed by wagnild and young. groups defined based on the resilience score: resilient/high resilience ¼ scores in upper third of scores; nonresilient/low resilience scores ¼ scores in middle or lower third of scores. scali et al. resilience has been defined as the capacity of individuals to cope with traumatic events, namely the capacity to "maintain relatively stable, healthy levels of psychological and physical functioning as well as the capacity for generative experiences and positive emotions" (bonanno, ) . resilience measured using a resilience scale/questionnaire. used the connor-davidson resilience scale cd-risc , an abridgment of cd-risc (a -item scale). three groups examined: low-, intermediate-, and high-level resilience groups based on tertiles of scores. taylor et al. resilience can be conceptualized as the process of achieving unexpected positive outcomes in adverse conditions, as opposed to an individual trait. resilience based on outcomes. four groups identified based on seizure control and socioeconomic status: outcome: physical disease may influence resilience either by being an adversity leading to resilience or a variable modifying the relationship between adversity and resilience (table ) . in studies, the disease of interest was considered to be the "adverse event." [ ] [ ] [ ] , , in of these, all study participants were selected on the basis of having the physical disease being studied, and so these studies did not examine whether physical disease led to increased or decreased resilience. , , , , in the remaining study, costanzo et al. compared cancer survivors with those without a cancer history. the study found cancer survivors had impairment relative to the comparison group in mental health, mood, and some aspects of psychologic well-being, but they showed resilient social well-being, spirituality, and personal growth. in the remaining studies, physical disease was studied for its association with resilient outcomes in populations exposed to adverse events such as bereavement or illness. the nature of the physical diseases that was considered varied. more than physical disease was included in studies. , , , , hardy et al. found no association between physical disease and resilience. , the remaining studies did find a statistically significant association, although the direction of this association varied. , [ ] [ ] [ ] for example, scali et al. found women scoring at an intermediary level of resilience were significantly more likely than those with low resilience to have been exposed to a recent breast cancer, whereas perna et al. found a greater prevalence of "high resilience" in those without disease when compared to those with disease. we systematically reviewed studies examining physical disease and its relationship to resilient outcomes to summarize the range of definitions of physical disease resilience and the approaches taken to study it. the component parts of the resilience definition shared by most studies were as follows: an adversity must be experienced to demonstrate resilience, resilience is the ability to maintain healthy levels of function over time despite adversity or to return to normal function after adversity, and resilience is a dynamic concept as opposed to a fixed personality trait. this is in agreement with flourished across many disciplines of psychology and health because of the rising popularity of positive psychology in these areas. although sparsely studied in the diabetes population, in other areas of chronic illness and stress such as hivþ men, survivors of violent trauma or battered women in shelters, resilience has been found to be associated with better emotional and physical health. resilience measured using a resilience scale/questionnaire. a resilience factor score was derived using scales, used to measure: optimism, self-esteem, self-efficacy, and self-mastery. three groups identified: low, moderate, and high resilience based on the lower, middle, and upper tertiles of the resilience factor score. windle's definition and also with others including those in the developmental, the psychobiology, and the neurobiology literature. , , , this provides support for the fact that the definition of resilience in the context of physical disease is no different from when it is used in other contexts. it is noteworthy that many studies of physical disease refer to resilience in terms of returning to or maintaining normal function. , , , this implies a resilient outcome need not necessarily be an exceptional outcome, particularly if function before the adverse event was not at a high level. indeed, for those whose baseline functioning was low before the adverse event, the measurement of good function after the event may be evidence for thriving not resilience. thriving is commonly defined as not solely returning to a normal level of function (as for resilience) but achieving a higher level of functioning. , therefore, the concept of stroke all participants had disease. * lam et al. surgery for breast cancer all participants had disease. * taylor et al. epilepsy all participants had disease. * yi-frazier et al. diabetes all participants had disease. * adverse event population exposed to (if described) personal illness or injury, death of a family member or friend, illness or injury of a family member or friend, and nonmedical events. self-reported physiciandiagnosed chronic conditions, dichotomised into z or o . not associated with resilience. hardy et al. lundman et al. did not include. range of diseases considered. resilience included in inner strength score. conditions significantly associated with inner strength: copd, heart failure, and osteoporosis. conditions not significantly associated: cerebrovascular disease and cataract. perna et al. none. disease (present/absent): diabetes mellitus, myocardial infarction, and stroke. higher prevalence of high resilience in those without disease compared with those with disease. scali et al. recent breast cancer, lifetime psychiatric diagnoses or lifetime traumatic event (included cancer disease, death of close relative and other life-threatening illness). breast cancer women scoring at an intermediary level of resilience were significantly more likely than those with low resilience to have been exposed to a recent breast cancer. copd ¼ chronic obstructive pulmonary disease; sars ¼ severe acute respiratory syndrome. n all selected on basis of having the disease, so no assessment of whether having it led to increased resilience. johnston et al. thriving is different and its place in the resilience spectrum should be considered further and clarified. there were approaches to measuring or identifying resilience in the included studies: measuring it using a new or established "resilience scale" or identifying it based on outcomes observed in study participants. in all but studies, resilience was measured at a single time point following the adversity. , given the consensus that resilience is a dynamic concept that varies across the life-course, it may be more appropriate to measure resilience at greater than one time point. [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] it would also seem reasonable that measured outcomes either encompass more than one life-course domain or that authors are clear that by measuring resilience using a single outcome only one domain of resilience is therefore being assessed. of the studies examining outcomes, used composite health indicators such as the short-form questionnaire , , and did not. , , composite outcome measures may capture a wider spectrum of the resilient outcome, as may selfrated health measures, which are viewed as being reliable and valid measures of individual and population level health and have been found to be powerful predictors of future morbidity and mortality. , the characteristics of the resilience measurement scales were not well described by studies but all included a range of psychometric properties. a recent methodologic review of resilience measurement scales concluded that many were lacking conceptual adequacy by focusing on psychometric properties and failing to examine resilience across multiple levels. this involved scales used by studies in this review, including the wagnild and young resilience scale , and the connor-davidson resilience scale. , , , we found that physical disease may be considered as an adversity after which an individual may or may not demonstrate a resilient outcome. our work also found that physical disease may act as a variable that promotes or reduces resilience following an adverse event. the promotion of resilience may be an example of a "steeling effect," whereby a negative experience strengthens resilience to another stress. in addition to this, there was a large degree of variability between studies. the nature of the adversity experienced, the characteristics of the disease, the timing and the nature of the measurement of resilience, and the individual's sociocontextual and personality risk and protective factors are all likely to affect the resilient outcome. [ ] [ ] [ ] there are few systematic reviews in the resilience literature and no previous reviews have summarized the literature surrounding the definition and measurement of resilience in studies of physical disease and resilient outcomes. therefore, our review addresses an important research gap regarding how resilience is defined and measured in physical disease. in the literature, the term "resilience" has been used interchangeably with psychological terms such as hardiness, mastery, and thriving. , , however, there is debate as to whether these terms represent component parts of resilience rather than being synonymous with resilience. therefore, we did not include these concepts as search terms. however, we acknowledge that as many in the literature treat these terms as synonymous with resilience, we may have missed some potentially informative studies regarding the relationship between physical disease and resilience. we also only concentrated on those studies in which resilience was an outcome in those with physical disease and have not included studies in which physical disease is an outcome in those assessed to be resilient. we applied fairly strict exclusion criteria to ensure that our results could be reported in an informative and useful manner. despite our restrictions, there were still large differences across studies in study design and quality. as a result, the reviewers encountered challenges in reaching agreement in these areas. of particular note, the inclusion of the study by glymour et al. was subject to some debate owing to its focus on "cognitive resilience," which is generally treated as a component part of cognitive reserve and thus could be seen as a more specialized form of resilience than our review in relation to physical disease aimed to cover. the sample size restriction was applied for pragmatic reasons as our early work demonstrated that studies of physical disease and resilient outcomes often involved complex analyses with multiple variables. therefore, analyses of populations with fewer than participants were often underpowered. this was further validated by the classification of the studies with the smallest sample sizes in this review as being "low quality." the limitation to adults only is reasonable, given that the burden of physical disease is known to increase with age. owing to the differences across included studies, we were unable to carry out a meta-analysis; however, we have presented our results in a structured manner to assist interpretation. the definitions of resilience used in these studies of physical disease and resilience broadly reflect the definition proposed by windle, which supports its use by future researchers of physical disease resilience. our results strongly suggest that resilience is a dynamic process rather than a personality trait, and therefore, it may be more appropriate to measure resilience longitudinally and using more than one outcome measure. it is unlikely to be feasible for researchers to examine the entire range of potential resilience outcomes across an individual's lifecourse in a single study. but there is at the very least, a need to be transparent about the form of resilience being measured in a particular study and a need to acknowledge the limitations of only measuring resilience at a single time point. disease prevalence is rising as our population ages and findings from physical disease resilience research may be used to develop evidence-based approaches to promote resilience to reduce the burden of disease. therefore, improving consensus regarding the definition and measurement of physical disease resilience is important, and this work begins to build this consensus by summarizing approaches taken by current researchers in the field. disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study epidemiology of multimorbidity and implications for health care, research and medical education: a cross-sectional study what is resilience? a review and concept analysis the construct of resilience: a critical evaluation and guidelines for future work what is resilience? a systematic review of resilience in the physically ill the human capacity to thrive in the face of potential trauma implications of resilience concepts for scientific understanding preferred reporting items for systematic reviews and meta-analyses: the prisma statement scottish intercollegiate guidelines network what predicts psychological resilience after disaster? the role of demographics, resources, and life stress psychological resilience and dysfunction among hospitalized survivors of the sars epidemic in hong kong: a latent class approach psychosocial adjustment among cancer survivors: findings from a national survey of health and well-being social ties and cognitive recovery after stroke: does social integration promote cognitive resilience? stressful life events among community-living older persons resilience of communitydwelling older persons trajectories of psychological distress among chinese women diagnosed with breast cancer inner strength in relation to functional status, disease, living arrangements, and social relationships among people aged years and older socioeconomic position, resilience, and health behaviour among elderly people measuring resilience in adult women using the -items connor-davidson resilience scale (cd-risc). role of trauma exposure and anxiety disorders factors predictive of resilience and vulnerability in new-onset epilepsy a personfocused analysis of resilience resources and coping in patients with diabetes psychobiology and molecular genetics of resilience the psychobiology of depression and resilience to stress: implications for prevention and treatment resilience and thriving: issues, models, and linkages the choice of self-rated health measures matter when predicting mortality: evidence from years follow-up of the australian longitudinal study of ageing self-rated health. comparisons between three different measures. results from a population study a methodological review of resilience measurement scales dr. marjorie c. johnston is funded by a clinical academic fellowship from the chief scientist office, scotland, uk (caf/ / ) and is also supported by the farr institute @ scotland. the protocol development and screening of titles and abstracts occurred while dr. johnston was an employee of nhs grampian. dr. terry porteous was funded by a grant from the university of aberdeen, uk, pathways to a healthy life theme.disclosure: the authors disclosed no proprietary or commercial interest in any product mentioned or concept discussed in this article. key: cord- -crhv gnt authors: sun, ying; dong, yanli; wang, lifeng; xie, huan; li, baosen; chang, christopher; wang, fu-sheng title: characteristics and prognostic factors of disease severity in patients with covid- : the beijing experience date: - - journal: j autoimmun doi: . /j.jaut. . sha: doc_id: cord_uid: crhv gnt covid- has become one of the worst infectious disease outbreaks of recent times, with over . million cases and , deaths so far. our study investigated the demographic, clinical, laboratory and imaging features of patients with covid- in beijing. patients were classified into four groups, mild, moderate, severe and critically ill. the mean age of our patients was years of age (range – ) and there was a slight male predominance ( . %). thirty percent of our patients had severe or critically ill disease, but only % of severe and % of critically ill patients had been to wuhan. fever was the most common presentation ( . %), but cough was present in only slightly over half of the patients. we found that lymphocyte and eosinophils count were significantly decreased in patients with severe disease (p = . and p = . , respectively). eosinopenia was a feature of higher levels of severity. peripheral cd (+), cd (+) t lymphocytes, and b lymphocytes were significantly decreased in severe and critically ill patients, but there was only a non-statistically significant downward trend in nk cell numbers with severity. of note is that liver function test including ast, alt, ggt and ldh were elevated, and albumin was decreased. the inflammatory markers crp, esr and ferritin were elevated in patients with severe disease or worse. il- levels were also higher, indicating that the presence of a hyperimmune inflammatory state portends higher morbidity and mortality. in a binary logistic regression model, c-reactive protein level (or . , [ci, . – . ]; p = . ), cd t lymphocyte counts (or . , [ci, . – . ]; p = . ), and d-dimer (or . , [ci, . – . ]; p = . ) were independent predictors of disease severity. in december , several patients with a pneumonia of unknown etiology were treated in wuhan, china. by late december , a novel coronavirus originally named -ncov was found to be the etiology of these illness. the terminology was later updated by the who whereby the disease is named covid- and it is caused by the virus sars-cov- [ , ] of those infected with covid- had severe disease, with a case fatality rate of . %. as of april , , the case fatality rate of covid- in china is approximately . % [ ] . at this time, the epidemic situation across china has been effectively controlled, and most of the new cases in china are currently imported from abroad. this study aims to evaluate the characteristics and prognostic factors of disease severity in patients with beijing. this study evaluated the characteristics and prognostic factors of disease severity in confirmed non-imported covid- patients in beijing. diagnostic criteria for covid - pneumonia was based on the new coronavirus pneumonia prevention and control program ( th edition) published by the national health commission of china [ ] . various specimens including respiratory samples from throat swab or sputum, urine, blood or stool samples were collected and tested for sars-cov- following who guidelines for quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) [ , ] . positive results of covid- infection were defined as any positive test from the above specimens. routine serial hematologic and biochemical tests were performed after admission, and the serum samples were screened for common respiratory pathogens, including antibodies to adenovirus, chlamydia pneumoniae, mycoplasma pneumoniae, respiratory syncytial virus, coxsackie virus a , legionella pneumophila and mycobacterium tuberculosis and all were negative. chest x-ray and chest ct scans were obtained for all patients and monitored serially. data were analyzed with spss v. . (spss inc., chicago, il, usa). continuous variables are expressed as the mean ± standard deviation (sd) or as the median (range). the analysis of variance or kruskal-wallis rank sum test was used for comparison between multiple groups. categorical variables were expressed as a number (%). intergroup comparison was performed using chi-square test. spearman correlation coefficient was used to describe the association between different variables and adverse events. the logistic regression model was used to identify factors associated with adverse events. odds ratios and their associated % confidence intervals (cis) were used as measures of effect size. p value less than . (two-tailed) was considered to be statistically significant. patients were categorized into mild, moderate, severe and critically ill levels of severity based on the criteria shown in table . eight patients were identified to have mild disease ( . %), had moderate disease ( . %), had severe diseases ( . %) and nine were critically ill ( . %). patients with severe or critical disease accounted for . %. table shows the demographic, epidemiologic, and clinical features of patients at admission. the median patient age was years, with a range of to years of age. figure shows the age and demographic characteristics of patients with covid- . covid- preferentially affects males ( of , . %). the sex distribution for the various levels of severity showed that among the mild cases, ( . %) were male, among moderate cases, ( . %) were male, among severe cases, ( . %) were male and among critically ill patients, ( . %) were male (figure ). there was a statistically significant difference among the four groups (p< . ). the mean bmi of the patients was . ± . , and there was no statistically significant difference among the four groups. thirty-five patients ( . %) had a travel history to wuhan in the days before the onset of symptoms, and twenty-six ( . %) patients without a travel history to wuhan had a history of contact with a person from or near wuhan in the days before the onset of illness. thirty-four patients ( . %) of patients had close contact with patients with covid- and ( . %) had a history of family clustering. patients with coexisting conditions were more susceptible to severe disease and of our patients had or more coexisting diseases, including hypertension in cases, diabetes mellitus in , cerebral infarction in , cardiac arrhythmia in , prostate cancer in , bronchial asthma in , pulmonary tuberculosis in , claustrophobia in , thyroid disease in , chronic hepatitis in , and chicken pox in . this is shown in table . the most common symptoms were fever ( . %) and cough ( . %), but diarrhea was not common. laboratory tests on admission showed leukopenia in patients ( . %), lymphopenia in patients ( . %), thrombocytopenia in patients ( . %), anemia in patients ( . %), and eosinopenia in patients ( . %) ( table ). the leukocyte, neutrophil, lymphocyte, eosinophil counts and hemoglobin level differed significantly among the four severity groups. eosinopenia was not found in patients with mild disease but was found in patients ( . %) with moderate disease, patients ( . %) with severe disease and critically ill patients ( . %). lymphocyte subset analysis (table ) showed that cd +, cd + t lymphocytes and b lymphocytes were significantly decreased in severe and critically ill patients. natural killer (nk) cells, a key component of innate immunity against infection [ ] , trended lower with increasing severity, but there was no statistically significant difference among the four groups. prothrombin time (pt) and prothrombin activity (pta) did not significantly differ among the four groups, but fibrinogen (fib) and d-dimer levels showed a statistically significant difference, the levels were much lower in the mild and moderate disease group compared with the severe disease groups. alanine aminotransferase (alt) levels were elevated in patients ( . %), aspartate transaminase (ast) levels were elevated in patients ( . %), alkaline phosphatase levels (alp) were elevated in patients ( . %), and gamma-glutamyl transferase (ggt) was elevated in patients ( . %), but only ast level had statistically significant difference among the four groups (p= . ). albumin level was decreased in patients ( . %), lactate dehydrogenase (ldh) levels were increased in patients ( . %), and both of albumin and ldh levels showed a statistically significant difference among the four groups. both creatinine and urea levels were increased in patients with severe diseases, and there were significant differences between the four groups. creatine phosphokinase, which is mainly found in cardiac myocytes, was elevated in patients ( . %). though there was no statistically significant difference in these four different clinical classifications, the levels of creatine phosphokinase in the severe and critically ill groups were much higher than in the mild and moderate patients. c-reactive protein level (crp), erythrocyte sedimentation rate (esr), serum ferritin and interleukin- (il- ) levels all had a significant elevation in the severe and critically ill groups. on admission, all patients in the mild group had normal imaging. ground-glass opacities were found in the remaining cases. seven moderately ill patients had single lobe focal ground-glass opacities and moderately ill patients had multiple unilateral ground-glass opacities. twenty-four moderately ill patients, severely ill patients and critically ill patients had diffuse multiple bilateral ground-glass opacities. pleural effusions were found in case with moderate illness, with severe illness and patients who were critically ill (table ) . patients were categorized into two groups according to clinical outcome, defined by those with or without an adverse event. adverse events were defined as any of the following: respiratory rates ≥ breaths per minute, pao /fio ≤ mmhg, peripheral oxygen saturation (spo ) ≤ %, or admission to the intensive care unit. the group with adverse events includes patients with severe disease or who are critically ill. figure shows the relationships between clinical characteristics and disease severity, using the spearman correlation coefficient. the following variables showed significant positive correlation to the disease severity (p< . ): advanced age, sputum production, shortness of breath, and higher neutrophil count, ast level (p< . ), ldh level, ggt level, crp level, esr level, serum ferritin level, interleukin- . of these, d-dimer, and serum ferritin level had a particularly strong correlation. there was also a positive correlation between ferritin levels and liver function tests (table ) . patients with higher total lymphocyte count (p< . ), t lymphocyte count (p< . ), cd + t lymphocyte count (p< . ), cd + t lymphocyte count (p< . ), b lymphocyte count (p< . ), albumin level (p< . ), and sodium level (p< . ), were negatively correlated with disease severity, and a travel history to wuhan in the days prior to the onset of disease was also negatively correlated with disease severity (p< . ). the variables which showed a significant correlation with disease severity were included in a binary logistic regression model to identify that c-reactive protein level, cd + t lymphocyte count, and d-dimer were independent predictors of disease severity (table ). the identification of predictive variables may assist physicians in the evidence-based treatment of covid- . we analyzed the demographic, clinical, laboratory and imaging features of patients with covid- in beijing to determine potential biomarkers that may affect the prognosis and management of these patients. covid- can occur in any age group. our data showed that mild illness appears predominately in those under years of age. older individuals develop higher levels of severity of disease. we showed that there was a slight male predominance overall. laboratory analysis at the time of admission in our cohort of patients demonstrated that lymphocyte and eosinophil counts were significantly decreased in patients with severe disease (p= . and p= . , respectively). eosinopenia was not found in mild disease, but was present in . %, % and . % of patients with moderate, severe and critical illness. evaluation of lymphocytes subsets demonstrated that cd +, cd + t lymphocytes and b lymphocytes were both reduced in severe disease and critically ill patients. natural killer (nk) cell counts were on a downward trend with increasing severity, but there was no statistical difference among the four groups. adam et al reported that in patients with acute exacerbations of chronic obstructive pulmonary disease, eosinopenia increases the risk of treatment failure and in-hospital mortality [ ] . other studies have also demonstrated eosinopenia in late stage bacterial pneumonias, as well as covid- [ ] . this is consistent with our results of eosinopenia in more severely affected patients. it has been shown that both sars-cov- and sars-cov- use the same angiotensinconverting enzyme (ace ) receptor to enter host cells [ , ] . the pathophysiology following viral entry is under intense study, and our data showed the total t lymphocytes, cd +, cd + t lymphocytes and b lymphocyte counts were all significant decreased in severe and critically ill patients on admission. these immunological characteristics were also seen in sars-cov- infection [ ] . these results indicate that sars-cov- , like its predecessor has a negative impact on t-cell mediated immunity [ ] . in other viral infections, such as hiv, cd + lymphocytes have been found to migrate to various tissues such as lymph nodes and bone marrow, supporting the theory that homing of cd + or cd + lymphocytes may be responsible for depletion of these cells in the peripheral blood [ ] . in dengue virus, t cells have been capable of skin homing [ ] . so the possibility exists that the reason for the lymphopenia in covid- could be the result of homing of lymphocyte subsets to the lung, although the actual clinical evidence for this has yet to be uncovered. liver injury in patients with sars-cov- infection is not rare [ ] . we found that alt levels were elevated in . % of patients, ast elevated in . %, alp elevated in . %, and ggt elevated in . % of patients, although only ast level was much higher in the critically ill patients and showed significance difference among the four groups. however, albumin level was much lower and ldh levels were much higher in the severity patients, and there was a statistically significant difference among the four groups (p= . and p= . ). of these patients, only two had a history of liver disease with normal liver function, one with hepatitis b undergoing antivirus treatment, the other with nafld. we found that the inflammatory markers crp level, esr, serum ferritin and interleukin- levels were elevated in severe and critically ill groups, and that serum ferritin level was positively associated with alt, ast and ldh levels, but not with alp and ggt. serum ferritin levels can be affected by iron status and may indicate a hyperimmune state. it is a marker for hemophagocytic lymphohistiocytosis [ , ] , which is a known complication of viral infections. it is often accompanied by an increase in certain cytokines such as il- rα [ ] . serum ferritin has been found to correlate with the presence of or severity of disease in other diseases states, including inflammatory conditions such as macrophage activation syndrome [ ] . severe disease, characterized by massive alveolar damage and progressive respiratory failure, may be related to a cytokine storm event [ ] . in cytokine storm, increases in interleukin (il)- , il- , granulocyte colony stimulating factor (g-csf), interferon-γ inducible protein , monocyte chemoattractant protein , macrophage inflammatory protein -α, and tumor necrosis factor-α may be observed. in addition to the above cytokines, the proinflammatory cytokines such as il- β and il- are often significant contributors to the host response to infections and increased morbidity and mortality [ ] . il- is an example of a multifunctional cytokine involved in the mediation of the immune response and inflammation [ , ] . il- is produced mainly by monocytes, but also be produced by interstitial fibroblasts, alveolar macrophages in the lung [ , ] . il- has also been shown to be involved in the pathogenesis of acute lung injury [ ] . histological characteristics in the lung of covid- patients showed diffuse alveolar damage and interstitial lymphocytes infiltrates [ ] . il- may act as a functional mediator to recruit lymphocytes into the lung lesions, which can further secrete il- in a vicious cycle, thus aggravating the lung injury and hastening death. covid- primarily attacks the lungs, but other organ systems can sustain injury as well, including the heart and liver. disease severity increased with advancing age. our findings indicated that c-reactive protein level, cd t lymphocyte count, and d-dimer were independent predictors of disease severity in beijing covid- patients. group between mild disease and critically ill, group between moderate disease and severe disease, group between moderate disease and critically ill, group between severe disease and critically ill. group between mild disease and critically ill, group between moderate disease and severe disease, group between moderate disease and critically ill, group between severe disease and critically ill. the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak the deadly coronaviruses: the sars pandemic and 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cd lymphocytes in the blood of hiv(+) individuals migrate rapidly to lymph nodes and bone marrow: support for homing theory of cd cell depletion understanding the human t cell response to dengue virus liver injury in covid- : management and challenges the use of ferritin to identify critically ill patients with secondary hemophagocytic lymphohistiocytosis rate of decline of ferritin in patients with hemophagocytic lymphohistiocytosis as a prognostic variable for mortality. pediatr blood cancer ifn-gamma and cd drive distinct pathologic features during hemophagocytic lymphohistiocytosis iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases clinical features of patients infected with novel coronavirus in wuhan, china role of the inflammasome, il- beta, and il- in bacterial infections biology of multifunctional cytokines: il and related molecules (il and tnf) il , and il b polymorphisms are associated with severe influenza a (h n ) virus infection in the mexican population il- and tumor necrosis factor synergistically stimulate fibroblast il- production and stabilize il- messenger rna human alveolar macrophage and blood monocyte interleukin- production acute lung injury: a clinical and molecular review china was the earliest country to experience rapid spread of the virus. while most of the , plus infected cases and , deaths occurred in hubei province we studied patients with a mean age of years in beijing, of whom % had severe or critical disease only % of severe and % of critically ill patients had been to wuhan lymphocytes and eosinophils were significantly decreased in patients with severe disease lymphocyte subsets cd +, cd +, b lymphocytes were significantly decreased in severely ill patients patients showed elevated liver enzymes, and inflammatory markers c-reactive protein, erythrocyte sedimentation rate, ferritin, and il- levels were elevated in severe disease t lymphocyte counts and d-dimer were independent predictors of disease severity manuscript title: characteristics and prognostic factors of disease severity in patients with covid- : the beijing experience.all the authors have made their substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; and drafted the work or revised it critically for important intellectual content,and so on.ying sun: design of the work, methology, analysisis, interpretaion of data, preparetion, wrote-original draft and revised it for imporatant intellectual content; yanli dong and lifeng wang: acquisition, data curation, analysisis, software, drafted the work and revised it for imporatant intellectual content; huan xie: collect data, investigation, analysisis, software; fu-sheng wang, christopher chang and baosen li: design of the work, supervision, writing, reviewing and editing.as the first author, i have approved the final version to be published; and i agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.author signature ying sun apr , key: cord- -naz vct authors: rostal, melinda k.; olival, kevin j.; loh, elizabeth h.; karesh, william b. title: wildlife: the need to better understand the linkages date: - - journal: one health: the human-animal-environment interfaces in emerging infectious diseases doi: . / _ _ sha: doc_id: cord_uid: naz vct wildlife are frequently a neglected component of one health; however, the linkages between the health of wildlife and human, domestic animal, and environmental health are clear. the majority of emerging zoonotic diseases are linked to wildlife, primarily driven by anthropogenic land changes. despite this risk, wildlife have important links to people as environmental indicators, food security and safety, and through human livelihoods. this chapter will describe these linkages and demonstrate the need to understand these linkages through targeted surveillance and understanding the ecology of wildlife diseases. while the management of wildlife diseases presents a significant challenge, such practices will greatly improve the health of people, domestic animals, wildlife and the environment. one health is the all-encompassing concept that recognizes the inextricable links between the health of people, animals (wild and domestic), and the environment. while the link between people and domestic animals is well recognized, as they have been used for food, work, and products for millennia, the link between the health of people and wildlife is often neglected. this may be due to a perceived distance between wildlife and people and a lack of understanding of the important linkages that unite human health with the health of all animals. in an ever more urbanized and globalized world, the distance between people and wildlife is shrinking and these linkages are becoming ever more evident. in this chapter, we will discuss the linkages between human health and wildlife from multiple perspectives. this includes direct linkages with human health, such as emerging and nonemerging zoonotic diseases, as well as linkages between wildlife health and the environment, food security and the health of domestic animals, and sustainable human livelihoods. wildlife are an important component of one health, but is often neglected due to difficulty in conducting health studies and limited data and funding. we will further discuss the importance of understanding these linkages with wildlife through targeted surveillance, understanding the ecology of wildlife diseases, and the management of wildlife diseases. when wildlife are discussed in the context of one health, it is frequently in terms of their role as hosts to emerging infectious diseases (eids). with the number of eids significantly increasing during the past years, recent work has demonstrated that the majority are zoonotic ( %) and approximately % of those are of wildlife origin (jones et al. ). an eid is frequently defined as a disease that has recently been jumped into a new host, has evolved a new pathogenicity, is increasing in incidence, or has expanded into a new geographic range (lederberg et al. ; jones et al. ) . while outbreaks of eids may seem like rare events, as a group they cause hundreds of thousands of deaths annually (bogich et al. ) and a single event may cost us$ - billion to the global economy (newcomb ) . the process of disease emergence is complex and often multifactorial, but it can be better understood using broadscale, ecological approaches in identifying these factors, or drivers. in the first attempt to classify the underlying drivers of disease emergence, the institute of medicine (iom) identified six factors including: human demographics and behavior; technology and industry; economic development and land use; international travel and commerce; microbial adaptation and change; and breakdown of public health measures (lederberg et al. ) . in , seven additional drivers were added to the iom report including: human susceptibility to infection; climate and weather; changing ecosystems; poverty and social inequity; war and famine; lack of political will; and intent to harm (smolinski et al. ). iom's classification of the ''factors in emergence'' largely paved the way for current research investigating the underlying drivers of infectious disease emergence. it is important to note that these drivers are not mutually exclusive, and that factors may act in concert and will vary at different stages of the emergence process. for example, anthropogenic land use change has resulted in cross-species transmission of disease or initial emergence, from animal hosts to humans both directly by increasing human contact with animal populations, and in other cases indirectly by changing vector populations (daszak et al. ; patz et al. ) . climate and weather can augment these affects and modulate outbreak size; particularly with vector or waterborne diseases, and global trade and travel has facilitated the spread of those diseases (hufnagel et al. ). these eid drivers function on a different scales, vary geographically (keesing et al. ) , and can be attributed to a combination of environmental, ecological, political, and social forces. despite ongoing research investigating the role of eid drivers, additional studies and modeling approaches are needed to more fully understand the complex mechanisms of emergence (bogich et al. ). one important driver of zoonotic disease emergence that has often been overlooked by the one health community is the wildlife trade. trade in wildlife, both legal and illegal, can lead to the introduction of zoonoses and/or foreign animal diseases that may impact domestic animals or native wildlife species (karesh et al. ) . the illegal wildlife trade is estimated to be approximately us$ billion, the second largest black market after narcotics (karesh et al. ) . it is likely that this estimate is overly conservative as a recent study found that within one tropical country, venezuela, - million animals are traded annually at an estimated cost of us$ million (asmüssen et al. ). an estimated . billion live wild animals were legally imported into united states between and . nearly, % of those animals were destined for the pet trade (smith et al. ). with the magnitude of stressed, and possibly immune compromised, wild animals being transported on a global scale, it is not surprising that this has resulted in the spread and transmission of diseases that affect native wildlife, domestic animals, and people. previous outbreaks of important zoonotic diseases have already been attributed to the wildlife trade; notable, among these in the twenty-first century are severe acute respiratory syndrome (sars) and monkeypox. sars is a novel coronavirus that shifted from bats into civets and humans causing severe morbidity and mortality (case fatality rate of . - . %) (donnelly et al. ) . globalization and airline networks expidited the spread of sars from its point of origin in southern china to infect people in countries, making it the first pandemic of the twenty-first century (zhou and yan ) . the ''wet market'' where sars emerged in guangzhou, china had stalls where wild mammals, domestic animals, reptiles, and birds were sold in conditions of poor hygiene and in close proximity. after the sars outbreak was traced back to these markets, the chinese government reportedly confiscated , wild animals (karesh et al. ) . the most likely scenario of initial spillover and emergence was that rhinolophid bats harboring a sars or sars-like coronavirus were kept in cages in close proximity to civets that contracted and amplified the virus in the markets (li et al. ) . however, the evidence demonstrating bats as the original natural reservoir of sars-like coronaviruses was not discovered until years after the initial outbreak (li et al. ; field ). ongoing research has now revealed large numbers of novel coronaviruses from bats, and the hypothesis of sars originating from these hosts has been further validated (woo et al. ; yuan et al. ) . monkeypox emerged under similar conditions through the legal pet trade in the us in . it is hypothesized that prairie dogs (cynomys spp.) were in contact with a shipment containing gambian rats (cricetomys spp.) and african dormice (graphiurus spp.) at a wholesale pet store. the prairie dogs became ill, as did people in contact with them (guarner et al. ) . despite the serious threat of eids from wildlife in the legal and illegal trade, few international programs exist to screen imported wildlife for pathogens of concern. a recent study analyzed wildlife products confiscated by the us customs and border protection at john f. kennedy airport in queens, ny as well as seizures from airports in philadelphia, washington, dc, houston, and atlanta . smith et al. ( ) detected simian foamy virus (sfv) and several herpesviruses from bushmeat samples, including nonhuman primates, e.g., chimpanzees (pan troglodytes), mangabeys (cercocebus spp.), and guenons (cercopithecus spp.), that were imported from guinea, liberia, and nigeria. while sfv has not yet been shown to be pathogenic in people, approximately % of bushmeat hunters in close contact with dead primates were found to be infected with sfvs (wolfe et al. ) . wolfe et al. ( ) demonstrated sporadic transmission of sfvs to humans, and offered a better understanding of how human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids) may have emerged from primates. we know that simian immunodeficiency virus (siv) jumped several times into people hunting and consuming nonhuman primates before mutating into the pandemic hiv strains that are now circulating (heeney et al. ). this recent work by smith et al. ( ) demonstrates that zoonotic pathogens can be transported in bushmeat and potentially could cause an outbreak in a location far from its endemic region, underscoring the need for better port surveillance and regulation of this trade. left unregulated, legal and illegal wildlife trade can potentially have a large impact on human health, as well as direct impacts on wildlife and domestic animal health (discussed below). the health of wildlife is closely linked to the health of the environment and can be extremely sensitive to anthropogenic changes. this includes direct physiological and behavioral responses to chemicals and pollution as well as competition and other effects from the introduction of nonnative wildlife and/or new pathogens. animals have long been used as indicators of a toxic environment. the proverbial ''canary in a coal mine'', as later memorialized by sting in a song, stems from the use of canaries to detect trace amounts of methane and carbon monoxide in mines since the early s. their death indicated to miners that they needed to evacuate the mine to prevent asphyxiation. dichlorodiphenyltrichloroethane (ddt) was one of the first global acknowledgments of chemicals affecting nontarget animals. ddt was found to reduce the eggshell thickness of multiple bird species (porter and wiemeyer ) . it was later discovered that bioaccumulation of the chemical through the trophic levels had devastating effects on the populations of certain top predators and insectivorous birds (e.g., raptors such as peregrine falcons (falco peregrinus) and bald eagles (haliaeetus leucocephalus) (grier ) . these effects became widely distributed by rachel carson's ''silent spring'' (carson ) and wildlife and plants were increasingly recognized as important indicators of man-made environmental health threats. during the s, the us environmental protection agency (epa) began using ecological risk assessment (in addition to human risk assessment) to evaluate the risk of agrochemicals or other manufactured chemicals, superfund sites, as well as air and water pollution (epa ) . ecological risk assessment depends on scientific assessment of the risk a chemical poses to a wide variety of plants and animals, including invertebrates, fish, birds, and small mammals (epa ). while the system continues to be improved, risk assessments have been important in identifying the detrimental effect of acid rain (beamish ), perfluorinated chemicals ( van de vijver et al. ) , and most recently endocrine disruptors (kloas and lutz ) . these ecological risk assessments have contributed to several us national environmental laws that were created to protect the health of people. in addition to top predators, small mammals are frequently used in ecological risk assessments and have been proposed as sentinels for heavy metal contamination. talmage ( ) suggests that small mammals make good indicators of environmental pollution because of their abundance, widespread distribution, short dispersal distance, generalized food habits, short life span, high reproductive rate, and relative ease of capture. in particular, they can be used to assess the environmental contamination of landfills and mine areas (torres et al. ) . insectivorous mammals appear to be the best indicators as they are exposed more directly through invertebrates that may consume soil (e.g., earthworms) (hamers et al. ) . in particular, small mammals have been used successfully to assess cadmium, fluoride, lead, and mercury exposures (talmage ) . one relatively recent study found that both rodents and children living around a mining site in mexico had nearly twice the levels of lead and arsenic as the respective controls from the reference site (jasso-pineda et al. ) . despite the many studies that have successfully demonstrated that various rodent species can be used as environmental indicators for heavy metals and chronic pollution and the frequent use of rodents in risk assessments for new chemicals on the market, rodents are rarely used for regulatory purposes (e.g., for long-term monitoring of mining sites) (handy et al. ) . when used appropriately, environmental indicators could be a very valuable tool in long-term monitoring of the risk of pollution and contamination of both terrestrial and aquatic habitats (lam and gray ; jasso-pineda et al. ) and should be utilized more frequently. as with zoonotic eids, emerging diseases of wildlife have increased during recent decades (daszak et al. ) and have frequently been linked to anthropogenic ecological changes. specifically, it is likely that trade, travel, invasive species, and poor biosecurity measures are driving many of these diseases. we highlight this with two emerging fungal pathogens that have caused devastating impact on two vertebrate groups, chytrid fungus in amphibians and white-nose syndrome (wns) in bats, with indirect one health consequences for human health and food security. during the late twentieth century, amphibian populations began to decline on a global scale (heyer et al. ; young et al. ) . chytridiomycosis (batrachochytrium dendrobatidis; chytrid fungus) was determined to be the causative agent in many of the declines (daszak et al. ) . this fungus caused multiple species declines (even local extirpation of multiple amphibian populations in some areas) in pristine habitats in the americas and australia (daszak et al. ) . it is believed that the chytrid fungus was spread through the trade of african clawed frogs (xenopus laevis), the original host, and the north american bullfrog (rana catesbeiana) (weldon et al. ; schloegel et al. ). r. catesbeiana is frequently raised and traded for food, with greater than , kg of this species sold within brazil every year (schloegel et al. ). it has been suggested that out of more than species of amphibians noted to be rapidly declining due to enigmatic causes (stuart et al. ), a significant portion of them may have been due to chytrid (skerratt et al. ). this fungus demonstrates how seemingly unrelated anthropogenic actions (wildlife trade) can have far-reaching effects on the environment and wildlife. additionally, the loss of amphibians may disrupt ecological processes, such as a reduction in predation on mosquito larvae, resulting in consequent indirect impacts on human or animal health. wns is an emerging disease of hibernating bats caused by the fungus geomyces destructans (blehert et al. ; lorch et al. ) . it was first documented in the us in , and was most likely introduced accidentally by people traveling to and from europe, where it is ubiquitous and causes no bat mortality (puechmaille et al. ). this cold-loving fungus thrives in the winter environment of bat hibernacula and disrupts the hibernation and physiology of over-wintering bats. bat mortality is frequently attributed to starvation and dehydration as the infection causes arousal during the winter leading to the depletion of the fat reserves of the hibernating animals (cryan et al. ; reeder et al. ). the fungus grows on the muzzle and wing membranes of susceptible bats. mortality rates at many hibernacula are extremely high, commonly in the range of - % (turner et al. ). wns has rapidly spread south and west across the us, being confirmed in us states and four canadian provinces; and by early , the us fish and wildlife service estimated that over . million bats have died from the disease. wns is predicted to cause the local extirpation or possibly the extinction of the little brown bat (myotis lucifugus), which was once the most populous bat in the us (frick et al. ) . once a wildlife disease such as wns is established, control becomes very difficult. researchers are currently working to understand the environmental and life-history variables that allow the fungus to persist and spread, with hopes that areas or microclimates can be set aside for management intervention ahead of the epidemic wave (boyles and willis ; wilder et al. ; langwig et al. ) . other unproven solutions being developed include antifungal treatment, maintaining rescue captive breeding colonies, and artificially heating caves. wns, like chytrid, is an introduced fungal pathogen and arguably they are two of the most significant wildlife diseases-threatening a wide range of species with possible ecological and global extinction (fisher et al. ) . the value of bats in control of agricultural pests in north america alone has been estimated to us$ . billion annually, with a loss of bats translating into increased production costs (pesticides and other pest control methods) and smaller crop yields (boyles et al. ). as introduced above, food safety and security is an important component of one health, especially as the fao estimates that . billion people are undernourished worldwide (fao ) . wildlife is linked with food security as wildlife can contaminate foodstuff with zoonotic diseases, bushmeat is a major protein source for people living in many tropical countries, and wildlife and domestic animals can share significant pathogens. foodborne illnesses pose a serious threat to public health with growing economic and international trade ramifications. standard epidemiological public health methods are frequently used to investigate foodborne outbreaks among people. however, foodborne diseases are good examples of the intricate link between human and animal populations, and the surrounding environment. in , a virulent strain of e. coli o :h was linked to spinach and affected approximately people in states (cdc ) . a typical epidemiological investigation of this outbreak would have extended only to human morbidity, mortality, assessments of risk and probable source, laboratory diagnosis, and clinical treatment. however, when domestic and wild animal health and ecology were considered, warnert ( ) found the same strain of e. coli o :h isolates that caused the human outbreak in wild pig feces, the feces of several cows, and in a stream on one of the four spinach farms in the area. thus, a one health perspective integrating our knowledge of epidemiology, clinical diagnostics, the environment and ecology, was required to fully investigate and understand this outbreak and has great utility in understanding the foodborne illness outbreaks. foodborne pathogens from wildlife span the taxonomic spectrum from helminthes to viruses. emerging foodborne diseases represent the majority of foodborne illnesses in the us and an even larger percentage of the foodborne illnesses are likely due to yet undescribed pathogens (tauxe ) . many of the known foodborne pathogens and up to % of foodborne eid events are zoonotic and many may be linked to wildlife. commonly, both wild and domestic animals are implicated as sources of food contamination (beuchat and ryu ; doyle and erickson ; newell et al. ; gorski et al. ; cima ) . despite this, definitive identification of a specific source animal or species is rare, particularly as epidemiological investigations often occur long after the index case, as it can take time for an outbreak across various states or counties to be detected. however, in some cases, detailed ecological studies can determine the exact route of food contamination from wildlife. for example, nipah virus, a bat-borne emerging encephalitic paramyxovirus, is primarily transmitted through contaminated date palm sap in bangladesh. with annual outbreaks in people and very high fatality rates ([ %), this is a pathogen of special concern (luby et al. (luby et al. , . recent serological and pathogen discovery studies have shown that pteropus giganteus, a large fruit bat, is likely the primary reservoir for this virus (epstein et al. ) . further, using techniques of wildlife surveillance and infrared camera traps, the exact mechanism of transmission was determined. bats were observed feeding from date palm sap collecting pots at night and currently specific interventions that do not entail bat eradication are being developed to prevent this transmission (nahar et al. ; khan et al. ). wild animals provide a substantial portion of our food globally, with nearly half of all seafood coming from wild sources. in some regions of the world, wild meat from terrestrial animals represents a primary source of protein on which people are dependent. the volume of wild meat (''bushmeat'') harvested from central africa alone totals more than billion kg per year (wilkie and carpenter ) . this volume of meat, almost all of which is processed and distributed to consumers with few if any modern hygiene practices, provides a constant opportunity for human exposure to both rare and common foodborne pathogens (karesh et al. ; smith et al. ). modeling has been used to indicate direct linkages between health and bushmeat consumption. golden et al. ( ) used generalized linear mixed-model regression to suggest that if bushmeat were removed from the diet of children in madagascar, hemoglobin concentration would decrease by . g/dl leading to a likely increase in anemic children of nearly %. the overconsumption of wildlife resources may soon lead to the loss of bushmeat protein to diets. fa et al. ( ) predict that the percentage of dietary protein consumed through bushmeat is unsustainable and will decrease from an estimated % in to % by in the congo basin. this prediction is based on increasing wildlife extraction to production ratios that will ultimately lead to a decline in wildlife abundance. without the availability of bushmeat or the redistribution of global food sources, food insecurity in this region is likely to increase. this link between bushmeat hunting and food insecurity has been supported by the results of a study, which found the alleviation of food security concerns of villagers in zambia by improving domestic animal production over years led hunters to turn in , snares and firearms, saving an estimated , wild animals (lewis and jackson ). wildlife can be reservoirs of important diseases of domestic animals, some of which were originally diseases of livestock and are now maintained in wildlife populations despite eradication of the disease in domestic animals. diseaseinduced morbidity and mortality in domestic animals as well as economic sanctions associated with the presence of certain diseases can adversely affect the supply of food animals. certain wildlife diseases can cause morbidity and mortality in domestic animals that are accidental or dead-end hosts. one such example is malignant catarrhal fever, caused by alcelaphine herpesvirus of wildebeest (connochaetes taurinus), which can cause acute mortality in cattle (russell et al. ). while its significance is limited to regions endemic for wildebeest (or zoos housing them), outbreaks of the virus can cause significant losses and hardships for local herders in africa. some diseases have larger economic ramifications, such as foot-and-mouth disease (fmd). though the virus affects all species with cloven hooves (order: artiodactyla), there are specific strains that are more likely to circulate in different geographic locations and possibly primary reservoir species (e.g., cattle or african buffalo, syncerus caffer) (klein ). while the virus is not highly fatal to the animals, trade restrictions to prevent the introduction of the virus in fmd-free regions has led to it becoming an economically important virus. it is estimated that during the fmd outbreak in the uk, losses to agricultural industry and the food supply chain amounted to £ . billion (thompson et al. ) . outbreak response led to the destruction of million animals (thompson et al. ) . it is likely that the virus was imported into the country from cattle in virus endemic regions (samuel and knowles ) . some diseases of concern have their origins in domestic animals, but have now been established into wildlife populations where they can then be retransmitted back to domestic animals (daszak et al. ). in the us, brucella abortus is generally believed to be introduced to the continent by cattle (meagher and meyer ) . after a successful eradication campaign, all states were declared free of brucellosis in domestic cattle herds in ; however, the maintenance of the bacteria in herds of elk and bison in the greater yellowstone area has allowed the pathogen to persist and continue to cause outbreaks. the debate over how to eradicate brucellosis from the country is ongoing. management methods that have been discussed include: test and cull, depopulation, and prohibiting winter feeding sites for elk as well as vaccination (olsen ) . a similar story follows the introduction of bovine tuberculosis (mycobacterium bovis) into wildlife hosts (see box ). one health includes considering pathogens originating from domestic animals that can severely affect wildlife. rinderpest virus was introduced into africa during the early s. the virus swept across the continent killing susceptible cattle and wild artiodactyls en masse. it has been reported that over . million cattle died in southern africa and up to % of the african buffalo population was decimated (plowright ) . fortunately, rinderpest was not able to be maintained in large wildlife populations without the presence of cattle (plowright ) . it is believed that the massive loss of multiple species of grazers (buffalo, wildebeest etc.) actually led to a change in the ecosystem of the region that is still in effect today, although as these species are recovering, the ecosystem is likely reverting to that which was believed to have been prior to the outbreak (holdo et al. ). rinderpest is now the second virus that has been eradicated through the use of vaccinations, after smallpox virus (yamanouchi ) . box . bovine tuberculosis: a persistent linkage between wildlife and domestic animals wildlife reservoirs of domestic animal diseases can make it exceedingly difficult or even impossible to eradicate economically important pathogens. often, diseases that originated in domestic animals persist in wildlife at such low prevalences that they are not detected in the wildlife until the disease is controlled or eliminated in domestic animals. in the us, a program to eliminate bovine tuberculosis (mycobacterium bovis; btb) was initiated in . by , every state had been declared to be free of btb (knust et al. ) , when a case of btb was reported in a white-tailed deer (odocoileus virginianus; wtd) in michigan. the outbreak was confirmed after of hunter-killed deer tested positive for btb (schmitt et al. ) . genetic analyses have confirmed that this strain of btb has been circulating in the wtd population at a low prevalence (o'brien et al. ) . efforts by the michigan department of agriculture and michigan department of natural resources (mdnr) initially focused on depopulation of wtd in the -county affected area and a ban on feeding or baiting deer during winter. while they have been successful in decreasing the prevalence by %, low levels of btb ( . % prevalence) continue to circulate in the wtd and occasionally spillover into cattle, leading to depopulation of the cattle herd (o'brien et al. ) . the circulation of btb in wildlife has a high economic cost to agriculture in the region as michigan now has a splitstate tuberculosis accreditation (the upper peninsula of michigan is still considered tuberculosis free, while the rest of the state is divided between accredited free, modified accredited advanced, and modified accredited, depending on the distance from the nidus of btb. it is estimated that btb and the accreditation change cost michigan's agriculture industry us$ million from to and us$ million from to (thiel ) . eradication of btb in the wtd population has been prevented partially due to the lack of public support for continued lethal population control. hunters contribute us$ million to michigan's economy and with other sympathetic electorate, who desire to view deer in their yards, have significant political clout (o'brien et al. ) . political pressure to decrease the level of lethal control has lead the mdnr to look to developing new vaccine-related technologies. while no such vaccine has yet been developed, pressure continues to mount as wtd in minnesota were found to have btb (most likely a cattle strain) following outbreaks in cattle in (knust et al. ) . several other wildlife species are known to be reservoirs of bovine tb in countries beyond the us, making it very difficult to eradicate globally. (o'brien et al. ) . bovine tuberculosis demonstrates the difficulty of sustainable control of a zoonotic and economically costly disease. the challenges of either eradication or control in wildlife also highlight the cost-effectiveness of prevention, i.e., investment in efforts to prevent domestic animal diseases from becoming established in wildlife populations would be more cost-effective and more less effort than trying to remove a pathogen from wildlife populations. many of the most biologically diverse regions coincide with human populations living at the highest poverty levels. as people raise themselves out of poverty, they can provide their families with better medical care, leading to better health. one proposed method of community development for improving livelihoods and health is the sustainable use and conservation of wildlife. an important component of ensuring sustainable use of wildlife is engaging all of the relevant stakeholders, especially the people living around the conservation areas whose livelihoods can be directly impacted by wildlife. people living in areas with free-ranging wildlife are frequently in conflict with wildlife, e.g., crop raiding. the sustainable use of wildlife can lead the community to accept the risks of coexisting with wildlife to support conservation and the health of the natural ecosystem. ecotourism has been defined by many different groups, here we will use the same definition as stronza and pêgas ( ) -nature tourism that intentionally seeks to deliver net positive contributions to environmental conservation and sustainable development for local communities. this definition links conservation directly with the health and development of the local communities. this concept aims to harness resources from the ever-growing tourism industry (walpole and thouless ) . for example, in kenya wildlife tourism grossed nearly us$ . billion in tourism-related industries in , with . million visitors to parks and game reserves (knbs ) . that amount of earnings contributes significantly to the national economy. however, despite the significant earnings from wildlife-based tourism and safari hunting, the equitable distribution of the funds is important in achieving the sustainability of wildlife resources. the success of ecotourism can be evaluated by measuring local economic benefits and participation as well as conservation indicators. there are many factors that may contribute to the success or failure of ecotourism, including: the presence of a flagship species, the biodiversity index and ease of viewing wildlife, the popularity of a particular location, the attitudes and current livelihood of the local communities, the perceived risk of wildlife to the community (e.g., crop raiding, disease, safety, or competition with, or consumption of domestic animals), and the perceived cost of living near the protected area (e.g., loss of access to cultivable or grazing land, watering holes, and inability to hunt) (walpole and thouless ) . a recent analysis of perceived cost from villages around kibale national park, uganda suggested a distinct geographical variation in households with perceived loss compared to those with perceived benefit. households within . km of the park boundary perceived the highest losses, while benefits were perceived up to km from the boundary (mackenzie ) . salafsky et al. ( ) worked with local communities to establish and support business operations and evaluate them with financial, social, and conservation indicators. they found that community participation in an operation was significantly linked to conservation success, even if the focus of the operation itself was not involved in conservation, such as the example from zambia discussed earlier where snares were turned in. interestingly, few operations were able to cover their costs after years and those that did required strong management systems to remain financially viable (salafsky et al. ) . in particular, creating successful community-based ecotourism programs can be very difficult given the competition and the high cost associated with start-ups in resource poor areas. it can take several years before such operations are able to cover their costs and it is not always clear that benefits for human health will trickle down from these operations (kiss ; walpole and thouless ) . few projects have successfully linked wildlife conservation directly to health care, although there are a few examples of large-scale initiatives seeking to do so. in the qomolangma national nature preserve in tibet, a collaboration among the villages, government, and various ngos led to the training of local villagers to protect the nature preserve with the benefits being improved access to basic health care. several individuals from local villages were educated in: preventative health care, distribution of medicines, environmental protection, ecotourism, poverty reduction, and income generation. these trainees provide services and education to the villagers. the success of the program was measured by a doubling of the estimated wildlife populations in the preserve, a decrease in logging by two-thirds, a decrease in the incidence of diarrheal diseases, and a reduction of infant mortality by % (melnyk ; taylor-ide and taylor ) . rarely mentioned in one health publications, discussions, or meetings is the topic of anthropozoonoses-diseases transmitted from humans to animals. much of the original literature on this subject area comes from studies with nonhuman primates, especially the great apes. butynski ( ) provides an extensive review of anthropozoonotic risks to great apes, including measles, herpesviruses, poliovirus, mycobacterium tuberculosis, sarcoptes scabeii, and a number of intestinal parasites. one survey found that % of tourists visiting sepilok orangutan rehabilitation centre in sabah, malaysia reported having one or more symptoms of an infectious disease while they were visiting the center (muehlenbein et al. ) . it has long been recognized that human tuberculosis (m. tuberculosis) can infect nonhuman primates. standard practices for captive nonhuman primates include routine testing of both the nonhuman primates and people who have contact with them (e.g., zoo keepers). interestingly, there is genetic evidence that suggests m. tuberculosis is significantly older than strains found in domestic livestock (m. bovis). it has been hypothesized that m. bovis evolved from an existing human pathogen or a common ancestor (brosch et al. ) . this is consistent with evidence of human tuberculosis preceding the to , -year-old domestication of animals (gutierrez et al. ) . excluding anthropozoonotic diseases from one health discussions alters the discourse to a narrow anthropocentric view of the world. the prevention of transmission of human diseases and improved human health can provide simple and cost-effective methods to protect wildlife from anthropozoonotic diseases. the linkages discussed above clearly indicate that wildlife health is intricately tied to the health of people, domestic animals, and the environment. despite these linkages and their key role as reservoirs of human eids, global efforts for wildlife health surveillance are lacking and underfunded. wildlife health surveillance can be used to better understand the pool of pathogens that may spillover into people or domestic animals; it can also be used to track the spread of wildlife diseases through populations. this surveillance can be used to investigate the ecology of the pathogen and hosts, which in turn can facilitate the prevention and control of important diseases. frequently, responses to emerging disease outbreaks are reactive and costly (childs and gordon ) . surveillance based on disease-specific control programs has successfully mobilized financial resources and delivered short-term results against disease-specific objectives (oliveira-cruz et al. ) . however, some have criticized this approach for focusing exclusively on a single disease and failing to reduce the risk of most pandemics (oliveira-cruz et al. ; travis et al. ). further, pathogen-specific surveillance often lacks sustainability and cannot be scaled-up, as benefits and outcomes are generally limited to the target area and funding cycle (oliveira-cruz et al. ) . to better target wildlife surveillance, there are several factors that need to be considered including geographic risk of emergence, the host species of the greatest concern for zoonotic spillover, and transmission pathways. the initial work to identify geographic ''eid hotspots'' found that countries with high biodiversity and human density are at the greatest risk for outbreaks (jones et al. ). most of these hotspots are located within developing countries, which often lack the infrastructure to conduct wildlife surveillance, either active or passive, and the ability to conduct diagnostic assays for rare or new diseases. one example of a multinational effort is the usaid predict project (see box ), which is based on initiating wildlife surveillance and investigating viral diversity in wildlife in these geographic hotspots. as zoonotic disease surveillance in wildlife clearly represents a great challenge (i.e., there are , + mammal species globally), predictive modeling and known patterns in host range can be used to focus the effort on the species and pathogens that pose the greatest risk of zoonotic emergence. new tools may make it possible to predict general patterns of host range in unsampled hosts, given known patterns from the past years of the published literature, knowledge of a species ecological and life-history traits, and some measure of surveillance or sampling bias from both a host and disease perspective. initial reviews of the literature were largely descriptive and grouped host species at higher taxonomic levels (e.g., ungulates, carnivores, rodents, and nonmammals) (cleaveland et al. ; woolhouse and gowtage-sequeria ; woolhouse and gaunt ) . more recent studies have tested patterns of pathogen-host range in a more mechanistic way, by explicitly including information on phylogenetic relatedness, although these studies are usually limited to a single host group or pathogen, e.g., bats and rabies (streicker et al. ) ; primates (davies and pedersen ) ; and fish ectoparasites (krasnov et al. ; poulin ) . similar approaches are currently being used to look at patterns of zoonotic disease emergence for all known mammal viruses and to test mechanistic drivers of cross-species viral emergence (bogich et al. ) . another useful way to focus surveillance efforts, speed up early detection, and reduce the risk of cross-species transmission is to target transmission pathways at specific human-animal interfaces. a key advantage of this approach is that surveillance efforts and control measures for one route of transmission should also mitigate a number of infectious diseases sharing the same transmission pathway. a multipathogen approach targeting disease transmission routes would be a useful way to target pathogen surveillance and control. it may also effectively focus prevention efforts, achieve early detection, and reduce additional risk of transmission. box . the usaid predict project: establishing a global wildlife surveillance network the predict project is part of the us agency for international development's emerging pandemic threats program. this project is developing a global wildlife virus surveillance system in countries that are in geographic hotspots (jones et al. ) in the amazon basin, mexico, southeast asia, and china, the gangetic plain, and the congo basin. this active surveillance system is aimed at understanding the importance of various human-wildlife interfaces. based on phylogenetic modeling, rodents, bats, and nonhuman primates were selected as target taxa due to their higher likelihood of harboring zoonotic pathogens (olival et al., unpublished data) . specifically, the project is targeting bushmeat sold in markets, wildlife that is collected by hunters, and wildlife living in proximity and/or conflict with people. predict is also investigating how changes in land use (using a landscape development index) can affect biodiversity and viral diversity of wildlife across a gradient of urban areas, rural areas with forest fragmentation, and in areas of pristine forest. this project works within each country's infrastructure to build surveillance and diagnostic capacity. viral discovery is conducted using degenerative primers to target viral families of zoonotic importance, which is then confirmed through genetic sequencing. in addition, deep sequencing methods are being used to discover new viruses. this diagnostic method maximizes the likelihood of discovering viruses, instead of targeting specific pathogens that may not be present. the predict project is standardizing surveillance methods across the globe to target potentially zoonotic viruses before they spillover. it is an example of combining high-level modeling with on-the-ground field data to target a surveillance system to efficiently detect potential pandemic viral threats. understanding wildlife diseases necessitates a multidisciplinary team, including epidemiologists, ecologists, and medical professionals. the ecology of the reservoir hosts as well as other competent species can be used to target disease management and mitigation (see box ). the importance of this is clear when considering pathogens such as ebola virus, where we have only recently discovered the probable reservoir (leroy et al. ), yet often cannot trace the transmission events from bats to nonhuman primates and/or people. further, new surveillance is finding evidence for ebola-like viruses in natural mammal reservoirs (e.g., bats and primates) well outside of their previously known range in africa (nidom et al. ; olival et al. unpublished data) . these recent findings point to a more urgent need to implement general, not pathogen-host specific, strategies to prevent zoonotic disease spillover from wildlife, i.e., by targeting transmission pathways or specific groups of hosts as were mentioned above. highly pathogenic avian influenza a/h n is a good example of the importance of understanding the ecology and epidemiology of a zoonotic disease in wildlife. this strain of avian influenza was first diagnosed in people in and in subsequent outbreaks has had an observed case fatality rate up to % (kandun et al. ) , although seroprevalence data indicate that it may be as low as - % (li et al. ) . when a/h n spread into europe and africa in there was an immediate reaction and assumption that it was transported by migrating birds, despite the lack of data at that time. the role of wild birds in the transmission and maintenance of a/h n remains controversial. outbreaks of a/h n in wild bird populations have occurred in isolation of poultry outbreaks and caused severe morbidity and mortality in some species. more than , birds at qinghai lake in china died during an outbreak, % of which were bar-headed geese (ansar indicus) (chen et al. ) . similar outbreaks occurred in mongolia and europe, indicating that there is occasional long-distance transmission by migratory birds (alexander ) . the effectiveness of longdistance transmission varies by species, as some species are severely affected by a/h n and others may be nonclinical shedders (based on studies in domestic mallards) (sturm-ramirez et al. ) . modeling by kilpatrick et al. ( ) suggests that the spread of a/h n may be a combination of the trade of poultry, the commercial trade in wild birds and transmission through migratory birds. their model suggests that the spread throughout asia was primarily due to poultry trade, and the spread in africa was partly due to poultry trade and partly due to migrating birds and the spread in europe was most likely through migrating birds. however, transmission by migratory birds appears to be rare, as proposed by gilbert et al. ( ) , who suggest that wild birds stopping over in areas at high risk for poultry strains of a/h n may occasionally transmit the virus via migration. a lack of sufficient information on the frequency or likelihood of spread through migratory birds indicates that further surveillance of a/h n should be conducted to better understand the transmission dynamics. without further ecological and epidemiological studies on the dynamics of a/h n , wild birds may have continued to be blamed for the maintenance of a/h n . it is known that waterfowl can be a mixing vessel for various subtypes of avian influenza (hatchette et al. ) , and it is rational to assume that they are the reservoir for a/h n . however, the seroprevalence of the h subtype is low in apparently healthy wild water bird populations (kang et al. ) . analyses of epidemics of a/h n in thailand found a strong association with the presence of free-grazing domestic ducks (gilbert et al. ) . additional analyses by gilbert et al. ( ) suggest that the presence of domestic ducks is the main factor associated with risk of a/h n in south asia, while human population and chicken density were also associated. recent modeling suggests that moderately sized flocks of poultry could maintain transmission of a/h n , whereas isolated small flocks or large commercial flocks are unlikely to maintain the virus (hosseini et al., unpublished data). thus, ecological studies of a/h n in both wild and domestic birds will continue to be important in understanding the maintenance of this virus. box . vulpine rabies: the importance of understanding ecology for control of rabies in europe vulpine rabies was first introduced to poland in and has since radiated out through eastern and western europe (anderson et al. ) . passive surveillance of wildlife throughout europe found that red foxes (vulpes vulpes) represented % of all wildlife diagnosed with rabies (who ) . attempts to control the fox population through culling did not succeed at preventing the spread of rabies, which expanded at an annual rate of - km. several papers in the early s illustrate the need for approaches and models that combined research on the viral pathogenesis, fox ecology, mapping of the development of epidemics, and importantly, the contact rate between foxes (anderson et al. ; macdonald and bacon ) . the social structure and density of foxes in some urban areas (five foxes per kilometer) would require a culling rate of - % to have a high probability of eliminating rabies (smith and wilkinson ) . culling adult foxes leaves empty territories that are quickly filled by dispersing yearlings and other young foxes. additionally, cub production is density dependent; thus, if culling were to decrease the population, more susceptibles would be introduced into the system as the reproductive rate of foxes would increase (macdonald and bacon ) . vaccination has been demonstrated to be effective in parts of europe and has two major advantages over culling: ( ) immunity decreases the likelihood of contact between two susceptible foxes and ( ) the reproductive rate remains stable, preventing the surge of susceptibles that follows a decrease in fox density (macdonald and bacon ) . for point-source infections, such as what would happen if rabies was introduced into britain, culling is more likely to be successful than in areas where rabies is endemic, especially as control efficacy may vary according to season (smith and wilkinson ) . the current control method for britain is to cull foxes within km of the point source followed by a ring of vaccine bait to prevent escape (smith ) . the ecological and epidemiological theory behind the development of control strategies for rabies in foxes can be transferred to other similar systems. in developing countries, the domestic dog remains the primary rabies reservoir. research in india has shown that trap, vaccinate (with or without sterilization), and release programs have led to a decreased number of rabies cases in humans, and may have led to a decrease in the stray dog population as well (reece and chawla ) . understanding the ecology of rabies in foxes and other carnivores has lead to a significant decrease in human rabies cases. wildlife are elusive, and have different ownership and custodian status among states and countries, and are often perceived in a variety of ways (emotional, religious, cultural, or utilitarian, etc.) by the general public; all of which necessitates new methods of disease control that consider the whole ecosystem including human interactions. artois et al. ( ) recently wrote a review of methods for controlling disease in wildlife and the risks associated with these methods. the primary goals of control are to limit the number of susceptibles or to treat/eliminate infected individuals to limit infectious period. lethal control and vaccination are the primary methods available to limit the number of susceptibles. lethal control, or culling, has frequently been shown to be very difficult to maintain in large populations of wildlife with high reproductive or immigration rates and is increasingly considered socially unacceptable (caughley and sinclair ) . further, culling is generally not a viable option when dealing with outbreaks in rare or endangered species, and the act of culling itself actually increases the human-wildlife contact interface and potential transmission of zoonoses. vaccination is increasingly being considered as a control option and is predicted to be the most efficient method to control hosts with relatively low reproductive rates. however, the production of a vaccine that is efficacious, stable in the environment, and easily deliverable (frequently orally) makes vaccination a difficult control method (artois et al. ). in addition, most effective wildlife vaccines (e.g. for rabies) are modified live vaccines that have the potential to harm nontarget species. another possible control method is fencing or other physical barriers to prevent direct contact. this is being recommended to farmers in michigan and minnesota as a method to prevent btb transmission from wtd sharing feed or entering cattle lots (palmer et al. ). low-tech bamboo skirts are also being used to keep bats out of date palm sap harvest areas to prevent the transmission of nipah virus (nahar et al. ). however, fences and barriers can sometimes interrupt nontarget species as well as the local ecosystem, e.g., in southern africa where fences prevent the migration of large herbivores, such as elephants (loarie et al. ). innovative 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in asia acknowledgments we would like to acknowledge the generous support from our funders for this work. including the us agency for international development emerging pandemic threats predict project, a niaid nonbiodefense emerging infectious disease research opportunities award r ai - , an nih/nsf ''ecology of infectious diseases'' award from the john e. fogarty international center ( r -tw ). key: cord- - afuen k authors: ensari, a. title: the malabsorption syndrome and its causes and consequences date: - - journal: pathobiology of human disease doi: . /b - - - - . - sha: doc_id: cord_uid: afuen k the indication for a small intestinal biopsy is usually the work-up of malabsorption, a clinicopathologic picture caused by a number of infectious and noninfectious inflammatory conditions. the biopsy is generally taken through an endoscope, by either forceps or suction, from the duodenum or proximal jejunum. depending upon the underlying condition, morphological abnormalities are seen in malabsorption range from normal mucosa with increased intraepithelial lymphocytes (gluten-sensitive enteropathy, viral gastroenteritis, food allergies, etc.), villous shortening with crypt hyperplasia (celiac disease (cd), treated cd, tropical sprue, and bacterial overgrowth), to completely flat mucosa (cd, refractory sprue, enteropathy-induced t-cell lymphoma, and autoimmune enteropathy). infectious agents that affect gastrointestinal tract can be grouped as food-borne and water-borne bacteria, opportunistic infections (bacterial, fungal, and viral), viral infections (extremely rarely biopsied), and parasitic and helminthic infections. the majority of these infections are, however, self-limited. although biopsy is more invasive, the use of this procedure allows detection of other causes, including whipple's disease, other protozoan forms of diarrhea (e.g., cryptosporidiosis, isosporiasis, or cyclosporiasis), crohn's disease, or lymphoma that may also present as diarrhea and malabsorption. the malabsorption syndrome and its causes and consequences a ensari, ankara university medical school, ankara, turkey elsevier inc. all rights reserved. autoimmune enteropathy this is a rare condition caused by autoantibodies to intestinal mucosa primarily affecting children who suffer from severe chronic diarrhea within the first months of life. celiac disease celiac disease (cd), also known as celiac sprue, nontropical sprue, gluten-induced enteropathy, or gluten-sensitive enteropathy (gse), is a chronic inflammatory disorder of the small intestine induced by a t-cell-mediated immune response and characterized by malabsorption after ingestion of wheat gluten or related proteins in rye (secalins) and barley (hordeins) in individuals with a certain genetic background. common variable immunodeficiency (cvid) cvid is primarily a dysfunction of b-cell differentiation resulting in hypogammaglobulinemia and diminished ability to produce antibodies. deamidation detachment of an amino group by tissue transglutaminase from gliadin that becomes negatively charged. enteroendocrine cell dysgenesis this disorder is associated with neurogenin mutation that is a protein involved in gut and pancreatic endocrine cell development, located on chromosome q . . enteropathy-induced t-cell lymphoma (eitcl) this highgrade t-cell lymphoma is the most fearsome complication of cd in patients with long-standing malabsorption. eosinophilic gastroenteritis this is a spectrum of diseases characterized by eosinophilic infiltration of various segments of the gi tract together with peripheral eosinophilia and coexisting allergies. graft-versus-host disease (gvhd) donor t cells of cytotoxic subtype incite an immunologic reaction to certain host cells resulting in injury to gi mucosa. gastrointestinal complications are common in bone marrow transplant recipients rather than solid organ transplantation. intestinal lymphangiectasia intestinal lymphangiectasia is defined as the presence of dilated lymphatics within the intestinal mucosa. it can be either primary due to a structural abnormality of the lymphatic system or secondary due to a local neoplastic or inflammatory condition causing lymphatic obstruction. intraepithelial lymphocyte t cell residing in the surface epithelium bearing t-cell receptors. the majority of iels are t cells, which are mostly cytotoxic t cells expressing ab tcr on their surface. the population specifically expanded in gse is the cd þ/cd À/cd À, gd tcr-bearing iels, which is only % of the total in normal mucosa. intraepithelial lymphocytosis less than intraepithelial lymphocytes/ enterocytes. characteristic finding in celiac disease but may also be found in a variety of intestinal disorders causing malabsorption. malabsorption malabsorption syndrome refers to the clinical picture comprising diarrhea, steatorrhea, malnutrition, weight loss, abdominal pain, and anemia due to maldigestion, mucosal/mural problems, or infections. microvillus inclusion disease (mvid) mvid is an uncommon congenital enteropathy characterized by severe, intractable diarrhea within the first week of life due to defective myo b gene located on q encoding myosin vb that regulates distribution of endosomes. it is characterized by loss of microvilli and numerous microvillus inclusions in the apical cytoplasm of the enterocytes. neonatal enteropathy severe intractable chronic (> - weeks) diarrhea starting immediately after birth and resulting in failure to thrive of the infant. peptic duodenal disease this represents a continuum of the same disease process of acute and chronic inflammation of the duodenal mucosa resulting from the toxic effects of excess gastric acid. chronic h. pylori infection is highly associated with peptic disease of the duodenum in more than % of the cases. refractory sprue refractory sprue is defined as incomplete or no response to gfd for at least months. it can be either primary, as lack of initial response to diet, or secondary, as unresponsiveness to diet in the form of a relapse. primary refractory sprue can include many different pathological conditions mimicking cd, comprising collagenous sprue, ulcerative jejunitis, lymphocytic colitis, collagenous colitis, and eitcl. small intestinal bacterial overgrowth (sibo) stasis caused by motor/neural disorders such as diabetes and scleroderma, diverticula, and surgical anastomoses results in abnormal proliferation of facultative aerobe bacteria normally colonized in the intestinal lumen. tissue transglutaminase the enzyme found in the lamina propria beneath the surface epithelium of small intestinal mucosa that removes an amino group from gliadin (deamidation) and adds the remainder of the peptide to the existing protein. tropical (postinfectious) sprue this is a rare disorder of unknown etiology, mainly occurring among residents or visitors to the tropical countries. malabsorption is usually initiated by an infection followed by colonization of the small bowel by enterotoxigenic bacteria. tufting enteropathy (te) te causes severe diarrhea in the first week of life together with various dysmorphic features including choanal atresia and esophageal/rectal atresia in some of the affected infants. epcam mutations on chromosome p have recently been suggested as the responsible genetic abnormalities. it is characterized by epithelial tufts or buds along the luminal aspect of the enterocytes. small intestine is the part of the gastrointestinal tract where much of the absorption takes place due to its large surface area provided by the numerous microvilli covering intestinal villi and the digestive enzymes on its surface actively secreted to optimize uptake of dietary substances. adequate absorption takes place when there are mechanically intact digestive organs to produce the necessary enzymes as well as an adequate blood supply, motility, and microflora. malabsorption develops when malfunction in any of these components leads to failure of absorption of nutrients resulting from a wide variety of causes ( figure and table ), the mechanisms of which can be classified into three groups: (i) maldigestion, which is related to mixing and digestive mediators; (ii) mucosal or mural causes including celiac disease (cd); and (iii) microbial causes (table ) . physiologically, there are three phases of absorption, luminal digestion, mucosal digestion, and mucosal absorption. the luminal phase consists of the breakdown of proteins, carbohydrates, and fats by digestive enzymes and bile, while mucosal phase involves hydrolysis of carbohydrates and peptides by enzymes of enterocyte membrane (disaccharidases and peptidases), and absorption phase comprises transepithelial transport of nutrients, fluid, and electrolytes to the blood vessels through enterocytes. malabsorption can, therefore, result from defects in either of these phases. in the succeeding text, the mechanisms of malabsorption are further discussed for each of these phases: . the luminal phase is where dietary fats, proteins, and carbohydrates are hydrolyzed and stabilized by digestive enzymes and bile. diseases often associated with this phase include À enterokinase and trypsinogen deficiencies that can lead to protein malabsorption, À impaired micelle formation that can cause problems in fat stabilization and the resulting fat malabsorption due to deconjugation of bile salts, À stasis of intestinal content due to a variety of factors (motor and anatomical abnormalities and small bowel contamination from enterocolonic fistulas) that can cause bacterial overgrowth. . bacterial overgrowth can cause decreased luminal availability of substrates (carbohydrates, protein, and vitamins). À the mucosal phase relies on the integrity of the brush border membrane of intestinal epithelial cells to transport digested products from the lumen into the cells. À impaired brush border enzyme activity may lead to lactose intolerance and sucrase-isomaltase deficiency. infectious chemical/toxic hc hc figure malabsorption develops when components of the mucosa are disrupted or damaged by a wide variety of injuries or stimuli including infectious, immunologic and inflammatory, and toxic or chemical causes. À impaired nutrient absorption can be inherited or acquired deficits. inherited defects include glucosegalactose malabsorption and abetalipoproteinemia. À acquired defects that are more common may be caused by decreased absorptive surface area (intestinal resection) or damaged mucosa (celiac sprue, tropical sprue, giardiasis, and crohn's disease). . transport abnormalities may be caused by lymphatic obstruction or vascular insufficiency. these include conditions like defective chylomicron synthesis in abetalipoproteinemia, submucosal infiltration in the intestinal wall due to lymphoma or amyloidosis, and lymphatic obstruction in intestinal lymphangiectasia. figure summarizes the phases of malabsorption. regardless of the cause, malabsorption syndrome refers to the clinical picture comprising diarrhea, steatorrhea, malnutrition, weight loss, abdominal pain, and anemia. undigested food produces diarrhea, due to its voluminous effect in the bowel lumen. although all three major nutrients (fat, carbohydrate, and protein) may be malabsorbed, clinical symptoms usually only develop with carbohydrate and fat malabsorption. small intestinal biopsy is an indispensable component of the diagnostic work-up of patients with malabsorption and/or chronic diarrhea secondary to mucosal damage. though complete villous flattening usually indicates cd when coexisting crypt hyperplasia is present, there are entities that may cause villous flattening and crypt hyperplasia other than cd. the majority of disorders causing malabsorption, on the other hand, produce mild to moderate villus blunting and crypt hyperplasia without any specific diagnostic feature. pathologists can also be faced with a patient with malabsorption or chronic diarrhea and a biopsy that appears normal or near-normal architecturally on microscopy. table highlights mucosal pathology of malabsorption. digestion begins in the mouth with mechanical disruption of food by chewing and mixing it with enzymes in the saliva. further mechanical digestion continues in the stomach until the semiliquid food or chyme is formed. digestion is completed when chyme is delivered into the duodenum and treated with pancreatic enzymes and bile salts. when normal gastric function is lost due to surgical procedures such as partial or total gastrectomy, the partially digested food rapidly passes into the duodenum causing osmotic pressure and diarrhea. enzymatic activity is equally crucial for absorption of nutrients. pancreatic enzymes and intestinal brush border hydrolases act in harmony for chemical modification of complex carbohydrates, proteins, and lipids. therefore, any disorder causing defective nutrient hydrolysis as in zollinger-ellison syndrome (zes) where excess hcl (acid) production may inactivate gastric lipase and proteolytic pancreatic exocrine enzymes, pancreatic enzyme deficiency due to chronic pancreatitis, or a malignancy obstructing the outflow of pancreatic juices into the duodenum may all lead to malabsorption. there are reduced bile salt synthesis due to severe liver disease (cirrhosis) and impaired bile salt secretion due to chronic cholestasis (intrinsic liver disease that causes bile duct damage or extrahepatic bile duct obstruction): bile salts are thus not available to form micelles to aid in fat solubilization for absorption. bile salts may be inactivated in the intestinal lumen by overgrowth of bacteria in small intestinal bacterial overgrowth syndrome (sibo). in patients with conditions that predispose to intestinal stasis, either by anatomical or motility abnormalities bacteria may overgrow in the proximal small intestine ( figure ). these excess bacteria deconjugate bile salts leaving them unconjugated and unable to participate in micelle formation. this leads to malabsorption of fat and fatsoluble vitamins a, d, e, and k. disaccharidase deficiency including lactase, sucrase, and maltase deficiencies may be either primary or secondary that may be differentiated by histology. low levels with normal histology may suggest primary deficiency, while the preceding disorder such as celiac disease may also be diagnosed by histology in secondary deficiency. lactase deficiency: among the disaccharidase deficiencies, lactase deficiency is by far the most common form worldwide. lactase is a disaccharidase located on the surface of intestinal microvilli, which cleaves lactose to glucose and galactose. lactase deficiency or hypolactasia in adulthood causes fermentation of lactose by intestinal microflora resulting in abdominal discomfort and diarrhea. the diarrhea is caused by the osmotic effects of lactose, glucose, and galactose in the colonic lumen, while short-chain fatty acids produced by fermentation of lactose cause flatulence ( figure ) . rare primary congenital deficiency may occur in infants, while secondary deficiency may result from disorders causing mucosal injury including celiac disease, tropical sprue, and inflammatory bowel disease (ibd). intermittent distribution of lactase on the brush border is revealed by immunohistochemistry, hydrogen breath tests and response to an exclusion diet are the diagnostic procedures for lactase deficiency. conditions leading to a decrease in surface area of the small intestine are among the leading causes of malabsorption as they also cause shortened transit time and defective exposure to digestive enzymes. this may be in the form of either decreased intestinal mucosal mass as in surgically resected small bowel or short bowel syndrome or may result from functional loss of the mucosa as in cd, tropical sprue, autoimmune enteropathy, and intestinal lymphangiectasia. celiac disease: cd, also known as celiac sprue, nontropical sprue, gluten-induced enteropathy, or gluten-sensitive enteropathy (gse), is a chronic inflammatory disorder of the small intestine characterized by malabsorption after ingestion of wheat gluten or related proteins in rye (secalins) and barley (hordeins) in individuals with a certain genetic background. the pathogenesis involves a t-cell-mediated immune response and autoreactive b lymphocytes that produce autoantibodies directed against gliadin, endomysium, or tissue transglutaminase (ttg) in individuals with a genetic susceptibility related to human leukocyte antigen (hla)-dq and hla-dq . the mechanism(s) by which gluten injures the epithelium has not been fully established. however, immunologic, genetic, and environmental factors all seem to be important. figure mechanisms of malabsorption with respect to luminal, mucosal, and mural/transport phases. a. immune mechanisms. multiple observations favor the conclusion that celiac disease is an autoimmune disorder occurring in the context of a combination of genetic and environmental factors, including the following: i. serum antibodies to gluten and its gliadin fraction are virtually always present in active celiac disease. a recent study showed subepithelial deposition of activated complement igg and igm in proportion to circulating antigliadin levels, suggesting humoral epithelial injury. antireticulin antibodies, antiendomysium antibodies, and anti-ttg antibodies also correlate with disease activity and have diagnostic specificity. ii. there is presence of numerous intraepithelial lymphocytes (iels), chiefly in the injured surface epithelium (these are mostly t cells, but with a high proportion gamma/delta types). this is highly consistent with cellmediated injury that is somehow facilitated by gluten. iii. steroids can cause patient improvement comparable to that with a gluten-free diet (gfd). iv. patients with celiac diseases show a highly significant excess of other autoimmune diseases (e.g., insulindependent diabetes mellitus). b. genetic factors. genetic factors include the following: i. there are a strong tendency for celiac disease to run in families ( - % occurrence in first degree relatives) and also a high ( %) concordance for celiac diseases in identical twin pairs. ii. there is a high correlation between celiac disease and presence of hla-b , dr (dr ), dr , and dq histocompatibility loci. the strongest association ( %) is with a specific hla dq molecule that is seen in only - % of the general population. the genetics seem to be 'complex' and a multigenic hla-associated susceptibility is favored. iii. there are overlap with the blistering skin condition dermatitis herpetiformis, including similar mucosal alterations, and overlap in hla markers and improvement in the skin lesions on a gfd. c. environmental factor(s). it seems likely that an environmental factor is a necessary precondition, along with appropriate genetic makeup and gluten ingestion, for an individual to develop celiac disease. support for this conclusion comes from the following: i. the fact that only one member of an identical twin pair is sometimes affected by celiac disease. ii. there is evidence of a possible role for viral infection: in one study, serological findings of prior exposure to an adenovirus (type ) were unusually common. in addition, a type adenovirus antigen shares an amino acid sequence with gliadin. although more recent studies have not confirmed a direct correlation between celiac diseases and adenovirus , the concept remains an attractive paradigm. mucosal pathology in cd may be summarized as follows: following uptake of gluten by the enterocytes, ttg deamidates gluten molecules that facilitate their binding to hla-dq and dq -bearing antigen-presenting cells. when gluten is presented to lamina propria cd þ helper t cells, they become activated to secrete inf gamma and induce upregulation of il that in turn boosts iel-mediated cytotoxicity. iels, under the influence of il , gain nk-cell-like properties and cause enterocyte damage ( figure ). tissue damage leads to ttg secretion and deamidation of more gluten peptides. because of the immunopathologic basis of the disease, there is a complex and heterogeneous population of inflammatory cells in the lamina propria comprising plasma cells that locally produce antigliadin antibodies and antiendomysial antibodies, t cells that include predominantly helper t cells as well as few cytotoxic cells. also, neutrophils, eosinophils, and mast cells may be found in varying numbers, though grading this inflammatory reaction is difficult and impractical. since none of these changes is specific to cd, intraepithelial lymphocytosis (ielosis) and architectural changes effecting villous to crypt ratio remain as the main diagnostic parameters of pathological evaluation. gluten sensitivity has also been known to affect other parts of the gastrointestinal tract, ( ) normal absorption of carbohydrates with intact brush border hydrolase activity. lactase acts upon luminal lactose and cleaves it to glucose and galactose that can then be absorbed by the intestines. ( ) in lactase deficiency, this cleavage cannot take place leaving lactose intact until it reaches the colon where fermentation by intestinal microflora occurs resulting in gas formation. the intact lactose causes osmotic diarrhea due to its voluminous effect in the lumen. in case of hypolactasemia, little cleavage of glucose and galactose occurs. scfa, short chain fatty acids. pathogenesis of celiac disease (cd). following uptake of gluten by the enterocytes, tissue transglutaminase deamidates gluten molecules, which facilitates their binding to hla-dq and dq -bearing antigen presenting cells. when gluten is presented to lamina propria cd þ helper t cells, they become activated to secrete certain cytokines including inf gamma and il . the latter induces cd þ t-cell influx to the surface epithelium, thus causing intraepithelial lymphocytosis (ielosis). intraepithelial lymphocytes (iels) under the influence of il gain nk-cell-like properties and cause enterocyte damage. including esophagus, stomach, and large intestine, and interestingly enough, iels are also increased in these areas in the form of lymphocytic esophagitis, gastritis, and colitis. clinical presentation varies from full-blown malabsorption with weight loss, diarrhea, and steatorrhea to more subtle symptoms such as folate or iron deficiency anemia, flatulence, episodic diarrhea, loose stools, neurological problems, osteoporosis, and vitamins k and d deficiencies in as many as % of patients. in children, usually within a few months of introducing the child to wheat-based foods, the classical syndrome of chronic diarrhea, steatorrhea, abdominal distension, and failure to thrive appears between months and years of age. both weight ( % below th centile), though more often, and growth ( % below th centile) are affected in these children. current estimates show that the incidence is at : in wheat-eating populations such as western europe and north america, while the incidence continues to rise in eastern societies, possibly, as a result of 'western style' eating habits. the diagnosis of cd currently relies on clinicopathologic studies including hla subtyping, serological tests, mucosal biopsy, and the effects of a diet free of gluten on the symptoms. based on a substantial amount of clinical research including a sequence of dynamic studies, the morphological continuum of gluten sensitivity was first introduced by marsh. marsh classification is comprised of three consecutive states of mucosal damage (types - ), including infiltrative lesion (marsh type ) characterized by increased iels in the villus epithelium in an otherwise normal mucosa with normal villous-crypt ratio, hyperplastic lesion (marsh type ) characterized by crypt hyperplasia with normal villi showing increased iels, and destructive lesion (marsh type ) characterized by flat mucosa with crypt hyperplasia and increased iel ( figure ). in its classical form, cd results in shortened, widened villi or even totally flat mucosa with hyperplastic crypts (figure (a) and (b)). these architectural changes are preceded by an increase in the number of iels over the normal numbers (figure (a) and (b)), corresponding to the cell-mediated immune nature of the disorder, and it is this group of cases that cause difficulty in the differential diagnosis. iels have been considered to be responsible for the epithelial damage observed in cd, although the exact mechanism is still not known. the vast majority of iels are t cells, which are mostly cytotoxic t cells expressing ab tcr on their surface. the population specifically expanded in gse is the cd þ/cd À/ cd À, gd tcr-bearing iels, which is only % of the total in normal mucosa (figure (a) and (b)). currently, the normal upper limit of iels is accepted as lymphocytes/ enterocytes (a ratio of iel per enterocytes) in h&e sections, whereas iels/ enterocytes (or a ratio of : ) in cd immunostained slides are considered as the upper limit of normal. iels are active components of the mucosal immune system that is under ongoing threat from luminal antigens such as gluten, microorganisms, drugs, and other toxic molecules, all of which can cause an increase in iels. though ielosis in a normal mucosa is an increasingly reported pathological feature in cd, it is by no means diagnostic, since overlap in the iel counts occurs between cd and non-cd patients. various pathological processes, including food allergies other than cd, helicobacter pylori-associated duodenitis, giardiasis, graft-versus-host disease (gvhd), tropical sprue, viral enteritis, nonsteroidal anti-inflammatory drug (nsaid) injury, chemoradiotherapy-induced enteritis, autoimmune enteropathy, immunodeficiencies, and crohn's disease, can induce ielosis with or without associated architectural changes. entities causing ielosis and flat mucosa are listed in table . refractory sprue comprises patients with incomplete or no response to gfd for at least months. histopathologically, most cases with refractory sprue show flat or near flat mucosa with a dense mononuclear infiltrate of mainly plasma cells and lymphocytes in the lamina propria and a massive increase in iels. the lymphocytes in the villous, crypt epithelium, and lamina propria are normal to medium in size with a normal cytological appearance. however, refractory sprue can be divided into two subtypes by means of t-cell clonality: type refers to cases with no clonality on tcr gene rearrangements and cd þ cd þ phenotype, while type comprises cases with cd þ cd À phenotype and showing clonal expansions proved by monoclonal rearrangements of the tcrgd gene. these features have led to the idea that these cases represent a form of in situ or 'cryptic' t-cell lymphoma. collagenous sprue refers to a variant entity having a thick collagen table beneath the surface epithelium of a mucosa that also has other typical features of cd, including ielosis and flat mucosa. enteropathy-induced t-cell lymphoma (eitcl) is the most fearsome complication of cd in patients with long-standing malabsorption. they usually suffer from severe malabsorption refractory to gfd, causing weight loss and complications such as perforations and bleeding due to ulcerative lesions. histopathologically, the mucosa is flat with ulcerations and crypt hypoplasia together with an atypical population of neoplastic lymphocytes infiltrates the mucosa. lymphoma cells are pleomorphic large cells that are double-negative (cd À and cd À) with cell surface markers but are almost always positive with cd that is also associated with poor prognosis. this is a rare disorder of unknown etiology, mainly occurring among the individuals living in or visiting the tropical countries. tropical sprue results from a nutritional deficiency or a transmissible microorganism and/or a toxin. malabsorption is usually initiated by an infection followed by colonization of the small bowel by enterotoxigenic bacteria. despite the fact that a single etiologic agent has not been identified, there is much evidence that an infection initiates and sustains tropical sprue: (a) it occurs in certain specific geographic areas (e.g., west indies and indian subcontinent) and enteric infections are common in these locations. (b) in some areas, it is epidemic. (c) aerobic enterobacteria colonize the patient's small intestine and these may be toxin-producing (note that this differs from the stasis syndrome in which anaerobic bacterial overgrowth is central (see in the succeeding text)). (d) recovery after treating tropical sprue with broad-spectrum antibiotics is usually rapid and dramatic. (e) some have postulated that a protozoan infection such as cyclospora may play a role. the entire small intestine including the ileum is involved with equal severity in tropical sprue, causing vitamin b and folate deficiencies resulting in megaloblastic change in the crypt epithelial cells. the mucosal lesion is similar to cd with varying degree of villous blunting, though completely flat mucosa is rare. increase in lamina propria lymphocytes, plasma cells, neutrophils, and eosinophils as well as iels that are more prominent in the crypts than the surface epithelium is usually present. upper gi involvement in ibd has been increasingly reported in the past years. both crohn's disease and ulcerative colitis may indeed affect the upper gi, including the duodenum. though the exact frequency of upper gastrointestinal involvement in crohn's disease is not known, it can reach up to - % of cases in some centers. most patients with involvement of the duodenal bulb have concomitant gastric antral involvement in the form of focal active inflammation. however, distal duodenal disease is usually associated with ileal and colonic involvement. symptoms of upper gi involvement are epigastric pain, nausea and vomiting, and rarely diarrhea. endoscopically, aphthous ulcers and granularity of the duodenal mucosa are seen. mild to moderate villous blunting can be found in the duodenum together with patchy or segmental chronic inflammation in the lamina propria and gastric mucin cell metaplasia (figure (a) and (b) ). granulomas are uncommon, but when found, they are usually more often in the stomach than the duodenum and can be helpful in the differential diagnosis. there is a group of patients with ulcerative colitis that presents with diffuse duodenal involvement. this has been reported particularly in patients with pancolitic presentation of ulcerative colitis who do not respond to steroids. histologically, duodenal mucosa may show active duodenitis with distortion of the crypts, cryptitis, and even crypt abscesses. regardless of the type of ibd, mucosal involvement of the small bowel may lead to a decrease in mucosal surface area and table conditions that are included in the differential diagnosis of gse there are conditions causing malabsorption through mechanisms involving not only the mucosa but also the intestinal wall such as scleroderma, amyloidosis, eosinophilic gastroenteritis, and neuromuscular disorders like intestinal familial neuro/myopathies. the pathology common to all the aforementioned conditions is the resultant defective peristaltism leading to luminal stasis and bacterial overgrowth that cause malabsorption. gi involvement is very common in systemic amyloidosis. gi symptoms comprise malabsorption, hemorrhage, nausea, and vomiting. endoscopic findings are nonspecific and biopsies from the rectum and stomach with sufficient submucosal tissue aid a correct diagnosis as deposition of amyloid protein may be present in vessel walls, muscularis mucosae, and nerves and rarely as extracellular globules in the lamina propria. appropriate histochemical stains such as congo red or sirius red are necessary for diagnosis, while immunohistochemistry can be used to classify the type of amyloid fibril protein. deposition of amyloid in the vessels causes them to become leaky and fragile leading to hemorrhage. deposits in the wall, in either the muscle or nerve bundles, cause impaired motility, while mucosal deposits lead to malabsorption and diarrhea due to diminished mucosal surface area by obliteration of the villi. is a spectrum of diseases characterized by eosinophilic infiltration of various segments of the gi tract together with peripheral eosinophilia and coexisting allergies. disorders causing gastrointestinal mucosal eosinophilia are presented in table . the disease may be patchy and may involve either only the mucosa or mural and serosal involvement may also be found. the mechanism is believed to be mediated through activation of th lymphocytes leading to an increased production of proallergenic interleukins, especially, il- , il- , and il . while il- promotes maturation of eosinophils and migration from the bone marrow into the circulating bloodstream, il- and il- upregulate the production of eotaxin- by the epithelium, a chemokine responsible for attracting the eosinophils into the mucosa ( figure ) . as a result, mature eosinophils accumulate in the intestine, are activated, and degranulate releasing multiple cytotoxic agents. though the mechanism of fibrogenesis is still unclear, il- , by inducing fibroblast-myofibroblast transdifferentiation, may be the critical molecule for tissue fibrosis as well as smooth muscle hyperplasia that leads to defective peristaltism and malabsorption in this condition. histological abnormalities consist of mild to severe eosinophilic infiltrates in the lamina propria and intraepithelial eosinophils both in the surface and crypt epithelium, forming eosinophilic crypt abscesses, and in the muscularis mucosae together with mildly increased iels (figure (a) and (b) ). due to the frequent use of nsaids, particularly in the elderly, they are among the common causes of small intestinal mucosal injury. nsaids cause nonspecific erosions and ulcerations in the mucosa that may eventually lead to formation of strictures in as high as - % in patients using these drugs. a rare but special form of strictures is known as the 'diaphragm disease.' it involves numerous weblike mucosal septa projecting into the lumen causing obstruction and mucosal stasis. the diaphragms are commonly located in the terminal ileum, leading to protein-losing enteropathy. histology of these diaphragms comprises reactive epithelium and prominent submucosal fibrosis lying perpendicular to the surface. this is the most common enteropathy of childhood. it is a rare condition caused by autoantibodies to intestinal mucosa primarily affecting children who suffer from severe chronic diarrhea within the first months of life. the patients may have a variety of autoantibodies to self-antigens in different organs including the pancreas, thyroid, and small intestine and may suffer from other autoimmune disorders such as rheumatoid arthritis, myasthenia gravis, psoriatic arthritis, hypoparathyroidism, idiopathic thrombocytopenic purpura, and atrophic gastritis. the hallmark of the disease is the presence of autoantibodies to goblet cells, enterocytes, parietal cells, or islet cells. there are two variants of the disease, namely, ipex (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) and apeced (autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy) syndromes. ipex is an x-linked fatal condition causing prolonged diarrhea. apeced or autoimmune polyglandular syndrome (aps- ) is an autosomal recessive disease characterized by severe enteropathy. histological findings of villous flattening and crypt hyperplasia are similar to gse, but the resultant secretory diarrhea is unresponsive to gfd or total parenteral nutrition (tpn). there are lymphoplasmacytic infiltration in the lamina propria and ielosis, though not as intense as in cd, and they are mainly bone marrow figure mechanism of eosinophilic enteritis is mediated through activation of th lymphocytes leading to an increased production of proallergenic interleukins, especially, il- , il- , and il . while il- promotes maturation of eosinophils and migration from the bone marrow into the circulating blood stream, il- and il- upregulate the production of eotaxin- by the epithelium, a chemokine responsible for attracting the eosinophils into the mucosa. located in the deeper crypts together with crypt epithelial cell apoptosis. there may be absence of goblet cells, paneth cells, or endocrine cells depending on the autoantibodies. primary enteropathies of infancy are composed of epithelial defects including microvillus inclusion disease (mvid), tufting enteropathy (te), and enteroendocrine cell dysgenesis. mvid ('microvillus atrophy') is an uncommon congenital enteropathy characterized by severe, intractable diarrhea within the first week of life. the disease was first described by davidson et al. in as severe secretory diarrhea occurring during the first week of life with villous atrophy in the intestinal biopsy. it has been demonstrated that myo b gene located on q encoding myosin vb that regulates the organization of intracellular transport and cell surface polarity in epithelial cells. myosin vb-deficient enterocytes display disruption of cell polarity as reflected by mislocalized apical and basolateral transporter proteins including cd , altered distribution of certain endosomal/lysosomal constituents including rab gtpases, and disorganization in the distribution of actin myofilaments ( figure ) . although described as a disease of the small intestine, there are few reports of colonic involvement. successful outcomes of small intestinal transplantation have been reported, and evidence suggests that an early transplant might be beneficial. diagnosis rests on light and electron microscopic examination of small intestinal biopsy. duodenal biopsies show moderate villous blunting, with no active inflammation in the lamina propria or ielosis. changes in the enterocytes are typically found at the villous tips rather than villous bases and crypts. light microscopy shows an irregular vacuolated appearance in the apical cytoplasm of the enterocytes with extensive or patchy absence of the brush border (figure (a) and (b) ). the surface epithelial brush border shows an abnormal pattern of staining by periodic acid-schiff (pas) and cd instead of its normal linear staining pattern. ultrastructural examination revealing intracytoplasmic inclusions that are lined by intact microvilli is the diagnostic hallmark of the disease (figure (c) ). these inclusions are present in the absorptive surface epithelial cells of the small intestine and are associated with poorly developed surface brush border microvilli. also known as epithelial dysplasia, congenital te also causes severe diarrhea in the first week of life together with various dysmorphic features including choanal atresia and esophageal/ rectal atresia in some of the affected infants. though the exact genetic abnormality is not yet known, epcam mutations on chromosome p have recently been suggested as the responsible genetic abnormalities. it has been speculated that there is abnormal epithelial basement membrane adhesion in the intestinal mucosa. in general, infants with te develop watery diarrhea within the first days after birth. stool volumes may be as high as - ml kg À body weight per day, with electrolyte concentrations similar to those seen in small intestinal fluid. the growth is impaired. histology is in harmony with the term 'tufting' as the surface enterocytes display focal crowding resembling tufts, buds, or small papillae, some of which seem to drop off into the lumen on sections (figure (a)- (c) ). colonic mucosa may also be involved. deposition of laminin and heparin sulfate in the basement membrane, increased desmoglein expression, abnormalities in the desmosomes, and abnormal distribution of a b integrin molecules are the other features that have been reported. this disorder is associated with neurogenin mutation, which is a protein involved in gut and pancreatic endocrine cell development, located on chromosome q . . patients present with congenital diarrhea and eventually develop type i diabetes. they are tpn-dependent and may require bowel transplantation. the intestinal mucosa demonstrates nonspecific features of villous abnormalities, whereas chromogranine immunostaining reveals lack of enteroendocrine cells in the mucosa. immunodeficiency states are commonly associated with malabsorption often resulting from opportunistic infections. they may be either primary such as selective iga deficiency and common variable immunodeficiency (cvid) or secondary including iatrogenic, immunoglobulin loss due to proteinlosing enteropathy and acquired immunodeficiency syndrome (aids). selective iga deficiency is the most common immunodeficiency in the general population. it is characterized by lack of iga production, major mucosal immunoglobulin. patients have no or very low levels of iga or selective iga in their serum but igm and igg secretion are present. the disease may be congenital or induced by infection or drugs. due to the absence of iga, b-cell maturation is defective, while some patients may also have t-cell abnormalities. accordingly, iga-containing plasma cells, but not others, are absent in the mucosa. most common clinical features are diarrhea, malabsorption, and bacterial infections. mucosa is usually normal, though completely flat mucosa can be found in some patients. cvid is the second most common immunodeficiency syndrome after selective iga deficiency. this is not a single entity but a group of conditions. with various underlying abnormalities, many of them are immune regulatory, including hyperactive t-cell suppression or reduced t-helper function. cvi appears after infancy and often is first discovered in adulthood. there is variable deficiency of iga, lgm, and igg, but iga production and igm production are typically reduced or absent. it is primarily a dysfunction of b-cell differentiation resulting in hypogammaglobulinemia and diminished ability to produce antibodies. patients present with diarrhea and recurrent chronic infections. it is characterized by a paucity or absence of plasma cells in the lamina propria despite its normal lymphocytic content usually in the form of lymphoid aggregates and ielosis in the surface epithelium. increased apoptosis is also a common feature, while in a small percentage of the cases, granulomas may be present in the lamina propria. the mucosa may also harbor infectious agents such as giardia, cryptosporidium, and microsporidia resulting from the immunodeficient state. gastrointestinal complications are common in bone marrow transplant recipients rather than solid organ transplantation. the main cause of these complications comprises gvhd, infections, toxicity of medication, and preexisting gi disease. patients suffering from acute gvhd are at higher risk for gastrointestinal infections. donor t cells of cytotoxic subtype incite an immunologic reaction to certain host cells resulting in injury to gi mucosa. patients with gvhd present with secretory diarrhea and abdominal pain and at times hemorrhage. upper gi involvement is more common in children, while colonic involvement is usually more severe than small intestinal disease. malabsorption may develop when small intestinal mucosa is severely damaged. microscopic findings vary from damage to individual epithelial cell in the form of apoptosis to total mucosal ulceration and are graded according to snover's criteria ( table ) . deficiencies associated with carbohydrates, amino acids, electrolytes, and vitamins are rarely biopsied as the intestinal mucosa is either normal or only slightly abnormal, while deficiencies of lipid transport cause abnormalities in the intestinal mucosa. among the latter, abeta/hypobetalipoproteinemia and chylomicron retention disease are the most commonly encountered disorders in infants with malabsorption. the patients suffer from diarrhea and fat malabsorption with low serum cholesterol and triglycerides and peripheral acanthocytosis. duodenal mucosa shows lipid vacuolization of surface enterocytes with preserved villous architecture (figure (a)- (c)). it is an autosomal recessive disease characterized by the absence of apolipoprotein b (apob), the transport protein of dietary fat from the small intestinal mucosa into the bloodstream. mutations in microsomal triglyceride transfer protein (mtp) located in q -q result in defective lipid transfer from endoplasmic reticulum to apob ( figure ). sar b gene encoding protein carrier gtpase located in q is mutated in chylomicron retention disease. lipid assembly is disorganized in both of these disorders resulting in low levels of serum lipids accompanied by various table grading of mucosal changes in gvhd (snover) grade grade grade grade crypt epithelial cell apoptosis apoptosis with crypt abscess individual crypt loss total mucosal denudation neurological manifestations due to malabsorption of fatsoluble vitamins, particularly vitamins e and a. intestinal lymphangiectasia is defined as the presence of dilated lymphatics within the intestinal mucosa. the form, known as secondary intestinal lymphangiectasia, is associated with a local neoplastic or inflammatory condition causing lymphatic obstruction. conditions associated with secondary intestinal lymphangiectasia include lymphoma, carcinoma, crohn's disease, systemic lupus erythematosus, behçet's disease, trauma, cardiac disorders, and complications after liver transplantation and radiation therapy. secondary form affects one side of the body resulting in unilateral edema but not protein-losing enteropathy. primary intestinal lymphangiectasia, on the other hand, is a rare congenital disorder characterized by severe protein-losing enteropathy, peripheral edema, steatorrhea, chylous effusion and lymphocytopenia, growth retardation, and severe hypoalbuminemia. there is a major structural abnormality of the lymphatic system consisting of dilatation and tortuosity of lymphatic vessels resulting in lymphatic stasis in the intestinal wall. the resultant lymph leakage causes loss of proteins, vitamins, and other nutrients. protein loss also causes immunoglobulin deficiency resulting in an immunodeficient state. primary form is usually diagnosed before the age of years. though entire small intestine can be involved, duodenum is the most commonly affected part of small intestine. the mucosa shows features of lymphangiectasia on endoscopy as numerous white spots or nodules that correspond to the dilated lymphatics in superficial mucosa involving the villus tips with formation of 'lacteals' (figure (a) and (b) ). these are dilated lymphatic vessels with endothelial lining and pale basophilic material of lymph fluid in their lumen. infectious agents that affect the gastrointestinal tract can be grouped as food-borne and water-borne bacteria, opportunistic infections (bacterial, fungal, and viral), viral infections (extremely rarely biopsied), and parasitic and helminthic infections. the majority of these infections are, however, selflimited. those patients that undergo endoscopic biopsy often have chronic or debilitating diarrhea, or malabsorption leading to systemic symptoms, or a history of immunocompromise or other significant clinical scenarios. clinicopathologic information regarding exact symptomatology and colonoscopic findings, as well as facts including travel history, food intake (such as sushi or poorly cooked beef), sexual practices, and immune status, can greatly aid in the diagnosis. ancillary techniques including histochemistry, immunohistochemistry, and molecular methods like in situ hybridization and pcr can increase diagnostic accuracy. gastrointestinal infections can cause mucosal inflammation that represents various patterns of tissue response. histological patterns of gastrointestinal infections are summarized in table . though these patterns can be observed in any part of the gastrointestinal system, there are some organ-/sitespecific findings of infectious diseases. a wide variety of infectious agents including, viruses, parasites, and bacteria can affect small intestinal mucosa. endoscopic picture may vary between erythema and granularity to erosions and ulcerations. the majority of infections cause only mild and nonspecific alterations in the intestinal mucosa including, ielosis, lamina propria inflammation, and rarely architectural changes, while some can be diagnosed by identifying the microorganism in biopsy specimens such as giardiasis, whipple's disease (wd), and opportunistic infections including microsporidiosis, cryptosporidiosis, and cytomegalovirus (cmv) infection. the general pathogenetic mechanism shared by many small intestinal infections depends on the interaction of the infectious agent with the mucosal components including the enterocytes and the immune modulators and effector cells in the lamina propria. this interaction requires an intact mucosa with intact enterocytes as well as an intact innate and adaptive intestinal immune system. the way that intestinal immune system recognizes pathogens from nutrients and microflora residents is via the recognition of pathogen-associated molecular patterns (pamps). these pattern recognition receptors include toll-like receptors and nod-like receptors that sense extracellular and intracellular pamps, respectively. they are specifically expressed in response to certain microbial stimuli, especially to invasive microorganisms. the immune effectors or modulators, on the other hand, are products of distinct genes, which may be either constitutively expressed (i.e., alpha defensins) or inducible (i.e., beta defensins) molecules by infectious stimuli. bacterial enteropathogens have a variety of virulence properties that determine the pattern of interaction with the intestinal mucosa and the clinical presentation. bacterial adherence and bacterial translocation are the two major mechanisms by which intestinal damage occurs. enterocyte adherence results from interaction between bacteria and its receptor on the epithelial cell surface. bacterial translocation, on the other hand, refers to the transportation of bacteria from the lumen through the mucosa to distant sites such as the mesenteric lymph nodes, spleen, and liver. bacterial overgrowth, impaired host defenses, and disruption of mucosal barrier are the main causes of bacterial translocation ( figure ). though numerous bacteria may cause small intestinal infection, they usually involve the colonic mucosa as well, in the form of enterocolitis. therefore, those that mainly affect the small intestine will be discussed here. all causes of bacterial overgrowth including motor/neural disorders such as diabetes and scleroderma, diverticula, and surgical anastomoses are related to stasis. bacterial overgrowth syndrome is more prevalent in elderly populations because of diminished gastric acid secretion and consumption of a large number of medications that can cause hypomotility. in addition, many patients with irritable bowel syndrome may suffer from this condition. normally, small intestinal lumen is colonized by facultative anaerobe or obligate bacteria, particularly bacteroides species, which are most abundant in the distal ileum. any disbalance of this complex intestinal microbiome, both qualitative and quantitative, might have serious health consequences resulting in sibo. the diagnosis of bacterial overgrowth relies on the presence of increased intestinal volume and bacterial concentrations, hydrogen breath tests, and response to antibiotics. small bowel aspirate and quantitative cultures with more than cfu ml À considered the cutoff are the diagnostic procedures. the resultant picture involves deconjugation of bile acids by bacterial enzymes causing loss of deconjugated bile acids in stool and decreased bile acid pool that is not enough for lipid digestion/absorption. moreover, intraluminal consumption of nutrients such as carbohydrates and amino acids, vitamin b , and iron by bacteria via competition and damage to small bowel enterocytes by bacteria causes a sprue-like histological and clinical picture. proposed mechanisms for bacterial overgrowth are presented in table . the mucosal biopsy may be normal or histopathologic picture may vary between mild to moderate villous shortening and blunting with increased chronic inflammatory cells in the lamina propria and iels and/or neutrophils. as these features are usually patchy in nature, multiple biopsies should be taken. the diagnosis is difficult for the pathologist without clinical correlation. whipple's disease wd is a rare, relapsing chronic, and systemic bacterial infection caused by an actinomycete, tropheryma whipplei. there is evidence of a host-related and possibly genetically determined increased susceptibility to whipple's disease related to innate bacterial translocation figure mechanisms of intestinal damage in bacterial infections. bacterial adhesion results from the interaction between bacteria and its receptor on the epithelial cell surface, whereas bacterial translocation refers to the transportation of bacteria from the lumen through the mucosa to distant sites like mesenteric lymph nodes. defects in t cells and mononuclear cells. persistent macrophage dysfunction may predispose to the infection since macrophages are unable to degrade the phagocytosed bacteria. patients present with malabsorptive diarrhea, arthralgia, lymphadenopathy, and cardiac and central nervous system symptoms. malabsorption in whipple's disease is due predominantly to blockage of nutrient movement in the lamina propria and in the mesenteric lymphatic drainage system by pas-positive macrophages. lipid accumulation in the mucosa and mesenteric lymph nodes is a prominent feature in the small intestine and led to whipple's name for the disorder -'intestinal lipodystrophy.' the characteristic feature in the small intestinal biopsy consists of blunted villi with distended lamina propria resulting from the accumulation of foamy histiocytes containing pas-positive rod-shaped, rounded, or sickle-shaped bacilli. mycobacterium avium-intracellulare complex (mai) is a significant cause of opportunistic infection in immunocompromised individuals, particularly those with aids, particularly in the pre-haart (highly active antiretroviral treatment) era. the organism is acquired through exposure to aerosols, food, water, and soil. the gi tract is affected twice as frequently as the lungs, the small intestine being the preferred site. healthy people do not develop active disease after exposure, while those with a low cd þ t-cell count are at risk. patients suffer from malabsorption, abdominal pain, fever, and weight loss. small intestinal biopsy reveals villous blunting and distension of the lamina propria by histiocytes loaded with acid-fast bacilli without well-formed granulomas that may be present in immunocompetent hosts. fungal infections of the gi tract have become more common over the years as numbers of patients with organ transplants, aids, and other immunodeficiencies have increased. gi fungal pathogens predominantly affect the immunocompromised individuals as opportunistic infections. almost all fungi may involve the small intestine during the disseminated gi involvement but histoplasma and cryptococcus have particular predilection for the small intestine and colon. histoplasma capsulatum commonly involves the ileum causing ulcers and obstructive masses in the lumen. histologically, lymphohistiocytic infiltration usually involving the mucosa and submucosa with ulceration, resembling wd, is observed. granulomas are only present in a small percentage of cases. histoplasma organisms are present in the cytoplasm of histiocytes as ovoid yeast forms. cryptococcus neoformans may involve the gi tract in patients with disseminated disease. histological features include round to oval yeast forms with budding and occasional hyphae or pseudohyphae. there may be necrotizing, suppurative inflammation and granuloma formation in many cases. giardiasis is the leading gi protozoan disease worldwide with an increase in summer and autumn. infection occurs by fecaloral transmission, contaminated food and water, and personto-person transmission. a number of studies have found that children and patients with hypogammaglobulinemia, agammaglobulinemia, iga deficiency, and/or achlorhydria have a higher incidence of infection with giardia. however, a humoral immunodeficiency state is not necessary for infection. immunosuppressed patients are more likely to have long-term infections with chronic diarrhea and malabsorption, while immunocompetent patients are more likely to have an acute self-limited diarrheal illness or become asymptomatic carriers. in its classical picture, diarrhea, abdominal pain, boating, nausea and vomiting, malabsorption, and weight loss are the main presenting symptoms. giardia exists in two forms: the motile trophozoite and the infective cyst. following ingestion, the cyst forms two trophozoites in the duodenum. these are pearshaped with two nuclei or sickle-shaped in sagittal plane appearing faintly basophilic on h&e sections. trophozoites are typically found in the lumen near the surface or as attached to the enterocytes (figure (a) and (b)). giardiasis is a typical example of enteric infection that presents with normal or near-normal mucosa, although more severe change in the villous morphology resembling cd may rarely be seen. in children, giardiasis can cause lymphoid hyperplasia in the small intestinal mucosa. coccidial infections are commonly observed in immunocompromised patients, particularly those with aids. however, all except microsporidia may also infect healthy individual and cause diarrhea accompanied by fever, weight loss, and abdominal pain. the immunocompromised patients may suffer from malabsorption. among the most common coccidial organisms infecting the small bowel are cryptosporidium parvum, cyclospora cayetanensis, isospora belli, microsporidium, cryptosporidia, microsporidia, and i. belli are often missed, because of their small size, intracellular location, and poor staining with usual tissue stains thereby require special stains. cryptosporidiosis is usually associated with normal duodenal histology in mild infections, whereas severe inflammation and villous atrophy may complicate serious infections. the diagnosis relies on the detection of tiny acid-fast oocytes in fresh stools. its characteristic appearance is a tiny spherical or ovoid basophilic organism attached to the apical aspect of the enterocyte both in the surface and crypt epithelium ( figure ) . they are best seen with modified kinyoun stain. cyclospora is similar to cryptosporidium but it is larger and is usually found in the enterocyte cytoplasm. they stain with modified acid-fast stains. isospora infection may lead to more morphological abnormalities in the form of villous blunting, crypt hyperplasia, and a mixed inflammatory infiltrate in the lamina propria, predominantly rich in eosinophils. the parasite is banana- shaped and can be found in the cytoplasm of the enterocyte (figure ) highlighted by a giemsa stain. although helminthic infections affect the entire gi tract, those that predominantly infect the small bowel will be discussed here. infection with ancylostoma duodenale, a hookworm, causes chronic blood loss and malabsorption and bloody diarrhea leading to iron deficiency anemia. patients often have peripheral eosinophilia with mucosal eosinophilic infiltrates. mucosal ulcerations are common when there is heavy parasitic infection. strongyloides stercoralis also causes malabsorption, diarrhea, and eosinophilia. the intestinal surface is hemorrhagic and eroded covered with a pseudomembrane of exudate and fibrin. larvae, adult forms, and eggs may all be present within the crypts (figure ). fasciolopsis buski inhabits the upper small intestine. the infection is usually asymptomatic unless there is heavy parasitic infection. symptoms include nausea, diarrhea, and gi hemorrhage. large adult parasites attach to the intestinal mucosa inciting an inflammatory reaction. biopsies from patients with enteric viral infections seldom if ever cross the stage of the surgical pathologist, as they are detected in stool samples rather than biopsy specimens. some common enteric viruses known to cause diarrhea include, adenovirus, rotavirus, coronavirus, echovirus, enterovirus, astrovirus, and norwalk virus. rotavirus is the most common cause of severe childhood diarrhea and diarrheal mortality worldwide, particularly in children under the age of years. patients develop fever, severe watery diarrhea, and vomiting leading to dehydration and acidosis. biopsy changes are very nonspecific and include increased inflammatory cells in the lamina propria, degenerative epithelial changes, and widening of villous tips. the diagnosis of rotavirus is generally made by stool immunoassay and/or culture, and the disease is rarely biopsied. adenovirus infection is also frequent as a cause of childhood diarrhea. however, it may also cause diarrhea in immunocompromised patients, especially those with human immunodeficiency virus (hiv) and aids. histological features of adenovirus infection include epithelial cellular changes such as loss of maturation, disorganization, and degeneration. characteristic inclusions usually crescent-shaped may be seen, especially in immunocompromised patients, within the nuclei of surface epithelium (particularly goblet cells). useful aids to diagnosis of adenovirus infection include immunohistochemistry, stool examination by electron microscopy, and viral culture. pathologists usually get a specimen from adenovirusinfected patients when the patient develops intussusception. cmv infection may be diagnosed in patients with immunosuppression or ibd even when the patients have not been treated with steroids. it is particularly important to consider this diagnosis in patients with steroid-resistant disease as treating the cmv may prevent the need for other medical therapy or surgery. symptoms may vary with the immune status of the patients and the site of the infection. most commonly abdominal pain, diarrhea, fever, and weight loss are observed, while isolated cmv infection of the duodenum may lead to severe gi bleeding. it usually causes ulceration of the mucosa and the viral inclusions are frequently found at the ulcer base. the characteristic owl's eye inclusions are preferentially found in endothelial cells and stromal cells but only rarely in enterocytes (figure (a)- (c) ). though h&e sections are diagnostic, immunohistochemistry may be applied when in doubt. cmv infection may mimic ibd both clinically and histologically. diarrhea in the setting of hiv infection may be related either to the infection itself, to the treatment, or to the opportunistic infections. gastrointestinal complications of hiv infection have dramatically decreased after the introduction of haart protocols. however, the effects of antiviral drugs on the gi tract together with superimposed infections are still commonly seen in these patients, especially in those with sustained low levels of cd þ t-cell counts. also, these patients may have atypical presentations of opportunistic infections that occur during or after therapy. hence, a cd count < cells mm À is the marker for those patients who should be searched for opportunistic infections for their gi symptoms. histological features of 'hiv enteropathy' include crypt hypertrophy, increased apoptosis in the enterocytes, and villous atrophy that are reminiscent of gvhd. hiv-associated pathology of the small intestinal mucosa is summarized in table . table causes of hiv-associated mucosal pathology malabsorption work-up: utility of small bowel biopsy pathophysiological approach to chronic diarrhoea the nonneoplastic small intestine diarrhoea due to small bowel diseases the pathology of malabsorption: current concepts an approach to duodenal biopsies small bowel review: disease of the small intestine clinical value of duodenal biopsies -beyond the diagnosis of coeliac disease gastric and oesophageal intraepithelial lymphocytes in paediatric coeliac disease intraepithelial lymphocytes in the villous tip: do they indicate potential coeliac disease? intraepithelial lymphocytosis in architecturally preserved proximal small intestinal mucosa patchy villous atrophy of the duodenum in childhood coeliac disease coeliac disease refractory sprue syndrome with clonal intraepithelial lymphocytes evolving into overt enteropathy-type intestinal t-cell lymphoma refractory coeliac disease coeliac disease: an update for pathologists gluten-sensitive enteropathy (celiac disease): controversies in diagnosis and classification morphometric analysis of intestinal mucosa v. quantitative histological and immunohistochemical studies of rectal mucosae in gluten sensitivity morphologic features suggestive of gluten sensitivity in architecturally normal duodenal biopsy specimens proximal small bowel mucosal villous intraepithelial lymphocytes prospective study of the role of duodenal bulb biopsies in the diagnosis of celiac disease gluten, major histocompatibility complex and the small intestine: a molecular and immunobiologic approach to the spectrum of gluten sensitivity ("coeliac sprue") morphology of the mucosal lesion in gluten sensitivity small intestinal biopsies in celiac disease: duodenal or jejunal? virchows arch celiac disease without villous atrophy in children: a prospective study the histopathology of coeliac disease: time for a standardized report scheme for pathologists how many duodenal biopsy specimens are required to make a diagnosis of celiac disease? variability of histologic lesions in relation to biopsy site in gluten sensitive enteropathy immunoglobulin a autoantibodies against transglutaminase in the small intestinal mucosa predict forthcoming coeliac disease collagenous spruean unrecognized type of malabsorption the role of esophagogastroduodenoscopy in the initial evaluation of childhood inflammatory bowel disease the face of tropical sprue in the significance of duodenal mucosal atrophy in patients with common variable immunodeficiency: a clinical and histopathologic study diagnosis and management of small intestinal bacterial overgrowth amyloidosis of the gastrointestinal tract: a -year, single-center, referral experience autoimmune enteropathy: a review and update of clinical management post-infectious irritable bowel syndrome: the past, the present and the future diagnosis of gastrointestinal graft-versus-host disease update in gastrointestinal allergic diseases loss of both phospholipid and triglyceride transfer activities of microsomal triglyceride transfer protein in abetalipoproteinemia microvillous inclusion disease-an ultrastructural diagnosis: with a review of the literature intractable diarrhea with tufting enteropathy: a favorable outcome is possible common variable immune deficiency in children-clinical characteristics varies depending on defect in peripheral b cell maturation endoscopic and histologic diagnosis of intestinal graft-versus-host disease after marrow transplantation tufting enteropathy: a newly recognized clinicopathological entity associated with refractory diarrhea in infants eosinophilic gastrointestinal disorders (egid) small intestinal bacterial overgrowth (sibo) in irritable bowel syndrome: frequency and predictors absence of cell-surface epcam in congenital tufting enteropathy microvillous inclusion diseasean ultrastructural diagnosis: with a review of the literature graft-versus-host disease of the gastrointestinal tract functional characterization of mutations in the myosin vb gene associated with microvillus inclusion disease upper gastrointestinal mucosal disease in pediatric crohn's disease and ulcerative colitis: a blinded, controlled study diffuse duodenitis associated with ulcerative colitis primary intestinal lymphangiectasia (waldmann's disease) gastric metaplasia and chronic inflammation at the duodenal bulb mucosa gastric metaplasia: its role in duodenal ulceration chronic diarrhea as the presenting feature of primary systemic al amyloidosis: serendipity or delayed diagnosis? histology of stomach and duodenum in crohn's disease gastric epithelium in the duodenum: its association with h. pylori and inflammation primary intestinal lymphangiectasia: is it always bad? two cases with different outcome human immunodeficiency virus-associated gastrointestinal disease: common endoscopic biopsy diagnoses whipple disease. clinical review of cases gastrointestinal infections in the immunocompromised host neurologic presentation of whipple disease: report of cases and review of the literature cytomegalovirus infection in steroid-refractory ulcerative colitis: a case-control study pathology of food-borne infectious diseases of the gastrointestinal tract: an update infective disorders of the gastrointestinal tract giardiasis: a histologic analysis of cases giardia: overview and update chorioretinitis and vitreitis due to tropheryma whipplei after transplantation: case report and review role of upper endoscopy in diagnosing opportunistic infections in human immunodeficiency virus-infected patients a rare cause of an ileocecal fistula in an aids patient. gastrointestinal infection by histoplasma capsulatum infection identified with internal transcribed spacer primer sets key: cord- -mey e gd authors: albers, heidi j.; lee, katherine d.; rushlow, jennifer r.; zambrana-torrselio, carlos title: disease risk from human–environment interactions: environment and development economics for joint conservation-health policy date: - - journal: environ resour econ (dordr) doi: . /s - - - sha: doc_id: cord_uid: mey e gd emergence of covid- joins a collection of evidence that local and global health are influenced by human interactions with the natural environment. frameworks that simultaneously model decisions to interact with natural systems and environmental mechanisms of zoonotic disease spread allow for identification of policy levers to mitigate disease risk and promote conservation. here, we highlight opportunities to broaden existing conservation economics frameworks that represent human behavior to include disease transmission in order to inform conservation-disease risk policy. using examples from wildlife markets and forest extraction, we call for environment, resource, and development economists to develop and analyze empirically-grounded models of people’s decisions about interacting with the environment, with particular attention to lmic settings and ecological-epidemiological risk factors. integrating the decisions that drive human–environment interactions with ecological and epidemiological research in an interdisciplinary approach to understanding pathogen transmission will inform policy needed to improve both conservation and disease spread outcomes. soon after the discovery of sars- -cov, covid- became a pandemic event that spread worldwide, infected millions of people, and created unprecedented economic impact (who a; guan et al. ) . current evidence suggests that covid- resulted from a zoonotic pathogen "spillover" event-transmission of a pathogen from wildlife to humans (andersen et al. ) . the majority (~ %) of the emerging infectious diseases (eids) since had an animal origin with most of these (> %) caused by pathogens associated with wildlife (jones et al. ) . although pandemics are rare, zoonotic disease incidence and novel spillover events cause frequent and serious health, social, and economic losses in tropical low and middle income countries (lmics) (morse et al. ) . as covid- spread around the globe, several countries in latin america experienced an extreme outbreak of dengue and other vectorborne diseases (lorenz et al. ) and the democratic republic of the congo endured their eleventh ebola outbreak since (who b). because zoonotic spillover events occur in human-animal interfaces, policy to address such disease risks requires interdisciplinary analysis of the epidemiological, environmental, and human processes occurring on the landscape. data analyses find that disease emergence and biodiversity loss share common anthropogenic drivers: land use change, habitat fragmentation, expansion of the agriculture frontier, and wildlife harvest and trade (patz et al. ; wolfe et al. ; loh et al. ; huong et al. ) . given that people's decisions are the root of these drivers, the choices people make about interactions with the environment influence both conservation outcomes and infectious disease risk (murray and daszak ) . in many lmics, human-environment interactions occur during activities that are integral to daily life and are a function of specific socio-economic and institutional settings (albers and robinson ; nielsen et al. ) . establishing policy to increase conservation and reduce disease risk requires understanding both people's decisionsincluding land use, resource extraction, and market activities-within their context, and how environment-human interactions lead to disease risk. environment, resource, and development economists explore models and data to determine why and how humans interact with the environment in lmics, which informs conservation policy. here, we review how these economic frameworks capture-or do not capture-drivers and characteristics of the human-environment interaction, while reflecting the natural and socio-institutional settings of lmics. we then propose how modeling frameworks can be expanded to incorporate the disease risk posed by that interaction to inform needed socio-enviro-epidemiological research and policy analysis, using an iterative process of data collection and modelling in an interdisciplinary setting. finally, we discuss how expanded models can provide insight into behavioral drivers of risk and inform design of policy for two general types of pathogen transmission pathway from wildlife to humans: direct-contacting a pathogen through hunting, marketing, or consumption of wildlife, with an example from indonesian wildlife markets; and indirect-exposure to pathogens while undertaking livelihood activities, with an example of malaria transmission. filling this research gap will address calls from many conservation and health actors to incorporate human dimensions of disease risk into joint conservation-health policy (romanelli et al. ; díaz et al. ; whitmee et al. ; who ) . the effectiveness of conservation policy is dependent on its ability to influence human-environment interactions. conservation economics in lmics provides both empirical and theoretical models of human behavior that inform conservation policy to address anthropogenic causes of biodiversity loss, as called for by conservation scientists (reddy et al. ; dobson et al. ) . these frameworks recognize that patterns of land use are informed by, and inform, decisions of people in lmics that cause biodiversity loss (e.g. pfaff ; andam et al. ; pfaff et al. ; albers et al. ) . both environmental and socio-economic characteristics of lmics inform people's decisions and interactions with their environment and are often critical considerations in conservation policy. governments, large scale land actors, and individual people make decisions that combine to determine the pattern of landscapes and land uses. in turn, tropical lmic people's land use and environmental interaction decisions consider patterns of government investments in roads and protected areas, larger scale land users, and agricultural policies (albers et al. ) . here, we discuss the empirical analyses that identifies drivers of conservation outcomes, critique their ability to identify policy tools to influence individual decisions, and describe existing lmic models of human-environmental interaction with conservation policy levers to form the basis for the next section's discussion of incorporating disease risk in these frameworks. investigating deforestation, land use change, fragmentation, and park effectiveness, econometric analysis of spatial imagery and gis datasets identifies correlations between physical and socioeconomic characteristics and conservation outcomes (e.g. pfaff ; andam et al. ; pfaff et al. ; busch et al. ; leblois et al. ) . roads both create fragmentation and encourage follow-on land conversion and fragmentation (saunders et al. ; freitas et al. ) , and protected areas and community management alter fragmentation (sánchez-azofeifa et al. ; southworth et al. ; nagendra et al. ; sims ) . although such empirical analysis is important for identifying big-picture issues and correlations, this style of empirical analysis presents three challenges for policy to influence people's behavior. first, such analysis obscures the incentives driving behavior and considers the actions of people only implicitly, which complicates identification of behavioral policy levers. second, remotely sensed datasets often do not contain information about socio-economic and institutional settings that influence behavior, such as which forests are effectively managed by community organizations or which villages face costly market access ghate et al. ). third, heterogeneity of cultures, landscapes, and other characteristics of individual lmics make extrapolating empirical results from one location to another difficult (leblois et al. ) , such as across settings with different acceptability of eating primate meat between muslims and christians (nyanganji et al. ) . conservation economics modeling work in lmics emphasizes the role of the socioinstitutional and ecological settings in which people make decisions on human-environment interactions (e.g. gunatileke and chakravorty ; albers and robinson ) . in lmics, many production and labor allocation decisions are influenced by natural environments, such as agricultural activities and non-timber forest product (ntfp) extraction, including hunting (robinson ; nielsen et al. ) . costly or limited market access influences decisions about how much to interact directly with natural environments for extraction and farming when facing a subsistence need or cash requirement (robinson et al. ; sills and abt ; muller and albers ; ghate et al. ). decisions to consume or trade wildlife depend on market access, prices, and opportunity costs (damania et al. ; wilkie et al. ) , in addition to other aspects of the household's production function including protecting crops from wildlife (johannesen and skonhoft ; bulte and rondeau ) . lack of clearly defined or enforced property rights create incentives that result in de facto open access use and habitat degradation (bulte and engel ; lópez-feldman and wilen ) . models that incorporate spatial decisions-about extraction and land use-provide a more explicit representation of the human-environment interactions that drive conservation outcomes and reflect the impact of property rights, community management, resource density, and market access on those interactions (robinson et al. albers et al. ). these economic decision models provide a lens through which to interpret data correlations and highlight policy levers for steering human interactions with the environment. developing general models of people's decisions within their social and ecological context, augmented with appropriate observation and data, can help form context-specific policy that focuses on changing people's behavior in pro-conservation ways that data analysis identifies as policy priorities. economists, health scientists, and conservation actors alike call for more research and policy that simultaneously addresses conservation and disease risk (salkeld et al. ; civitello et al. ; pattanayak et al. ) . often, conservation policy mitigates disease risk simply because conservation policy reduces contact between people and natural environments and wildlife. deforestation, land use change, and fragmentation all correlate with both biodiversity loss and zoonotic disease risk (patz et al. ; suzán et al. ; morand et al. ) . ecological models predict the geographic locations where new diseases may emerge or from which wildlife species they may emerge, but, despite including demographic variables, these models do not explicitly consider the interactions of people with their environment, which is a factor in the risk of zoonotic disease transmission (allen et al. ; olival et al. ). that transmission occurs through a combination of environmental, epidemiological, and human behavioral factors: first, human decisions determine the characteristics of the ecological landscape; second, the resulting environmental conditions determine the distribution of, and intensity of infection in, reservoir hosts; and third, the choice of activity and amount of time allocated to specific activities (hunting, farming, harvesting ntfps) drive the risk of exposure to pathogens (plowright et al. ) . as an example, the likelihood of mosquito-borne pathogen transmission depends on the abundance of mosquito vectors on a landscape, the pathogen load within the mosquito population, the chance that a pathogen-carrying mosquito bites a person, and the probability that the pathogen successfully infects the human (baeza et al. ). to depict risks on the landscape, disease ecologists create global emerging infectious disease "hotspot" maps that correlate past emerging infectious disease events, demographic data, and environmental variables to estimate the spatial relative risk of zoonotic spillovers worldwide. these models were developed at a coarse spatial resolution due to the sparsity of the data and multi-scale variables (allen et al. ) . human decisions around resource use and environmental interactions occur at a finer scale, making those interactions difficult to represent in this framework despite their critical role in determining the risk of disease. to address how humans influence zoonotic disease risk borne from environmental interactions, these hotspot maps can be combined with economic decision models at fine resolution that specify markets and institutions, landscape patterns, and resource use in lmics, and thereby illustrate the decisions behind where and how people overlap with pathogen hosts, which influences their pathogen exposure. using conservation economics models as a basis for developing policy that addresses both conservation and disease risk requires modeling extensions and new information. first, because both the amount of time and the locations of human-environment interactions contribute to disease risk within the ecological setting, expanding models of decisions about agricultural expansion, wildlife hunting and marketing, and resource extraction to incorporate space and time as decision variables provides a mechanism for characterizing the disease risk of such actions and for policies to internalize the disease risk into decisions. second, because both the ecological landscape of pathogens and human activities vary seasonally, economic models at the sub-annual level could identify seasonally targeted policies to mitigate disease risk. third, although empirical analysis signals the role of fragmentation in both biodiversity loss and zoonotic disease incidence, little ecological, economic, or interdisciplinary research depicts the process of fragmentation in a manner that helps define policy tools that influence people's actions in ways that could limit conservation losses and disease risk from fragmentation. in addition to such extension of modeling frameworks to address disease risk in the lmic context, we propose an interdisciplinary and iterative process of on-the-ground observation; economic, behavioral, and epi-ecological modeling to guide further field work; and data collection in a standardized manner to inform further research and policy efforts. that iterative process might begin with observation, both finding correlations in data and through stakeholder input; followed by economic analysis of models of human decisions parameterized with empirically-relevant values to identify the critical mechanisms and drivers of the human-environment interaction and potential for policy; followed by prioritization of ecological or economic data collection to form policy in specific settings; and an iteration back to broader data analysis to discern policy impact. such interdisciplinary analysis incorporating economic modeling will improve our ability to define factors that influence people's decisions involving interaction with the environment and the related disease risk. in the next two sub-sections, we discuss how to extend economic models of environmental interactions in lmics to incorporate disease risk in settings in which the risk is directly related to the actions taken by people, such as wildlife marketing, and in settings in which the disease risk derives indirectly or incidentally from the environment, such as siting livestock operations at the forest margins. the trade in legal and illegal wildlife is worth billions of dollars, with hundreds of millions of animals and plants traded globally (fukushima et al. ) . wildlife trade creates a direct human-wildlife interaction through intentional contact with the live or dead wildlife species at any point along the supply chain-from hunting to trading and selling to consumption (daszak et al. ; kruse et al. ; breed et al. ) . for example, sars-cov and sars- -cov are linked to wildlife trade in china, hiv to primate bushmeat hunting, monkeypox virus to the exotic pet trade, and h n and h n to domestic poultry (karesh et al. ; gilbert et al. ; andersen et al. ) . capturing, raising, and transporting live animals brings many individuals into close proximity, creating opportunities for inter-and intra-species pathogen transmission and potentially spillover to humans. for example, sars originated in wet markets where the pathogen spilled over from bats to civets (li et al. ) , and sars- -cov may have originated with pangolins as an intermediary host (andersen et al. ) . in these cases, the lack of regulations and poor biosafety at nodes on the supply chain increased the risk for vendors and purchasers further downstream. as the starting point of the wildlife supply chain, hunting activity that supplies markets poses a risk of disease spread as hunters interact with live animals (wolfe et al. ) . given that interaction, the drivers of hunting decisions can be targeted by policy to reduce disease risk and promote conservation. households face tradeoffs in their decisions to allocate time or land between agriculture and wildlife harvesting (bulte and horan ) . when these households also consume harvested wildlife, the income elasticity of demand for bushmeat drives the response to an alternative livelihood policy, identifying this elasticity as priority data (damania et al. ). response to policy may also differ when the hunted species are considered a nuisance to agriculture and are taken to defend crops as well as for consumption or sale. this dual purpose of hunting affects the tradeoffs hunters face when reacting to policy (johannesen and skonhoft ; bulte and rondeau ) . empirical analyses find variation in the response of hunters to the relative costs of hunting in an environment, such as park proximity; labor tradeoffs with other activities, particularly livestock; and household characteristics (e.g. brashares et al. ; foerster et al. ; van velden et al. ) . hunters may respond to the increased opportunity cost of hunting by switching to more efficient hunting techniques and more valuable species, which does not address disease risk and presents a conservation risk when those valuable species are protected species (damania et al. ) . when hunters cannot discriminate in the species they hunt, enforcement policies may have unintended consequences around consumption of protected species (robinson ) . hunters' decisions rarely include disease risk mitigating activities, perhaps due to a lack of knowledge of the risk the animal presents (harrison et al. ) . policies to alter incentives for bushmeat hunting and consumption include enforcement of illegal hunting, community-based conservation, and alternative or additional livelihoods and protein sources (foerster et al. ; van velden et al. ) . models predict the outcomes of these policies, showing in what circumstances alternative livelihoods, enforcement, and expanding protected areas lead to negative conservation outcomes (damania et al. ; johannesen ; robinson ) . policies that reduce hunting itself create conservation and disease risk mitigation but can impose economic burdens on hunters and demand for wildlife/bushmeat continues to create incentives for hunting. policies that promote safer hunting practices, information about disease risk, and incentives to hunt lower risk species directly alter the disease risk within the context of ongoing hunting. between the hunters and the final markets, traders, middlemen, and sellers in the wildlife/bushmeat supply chain serve a crucial role in lmics by providing access to markets, and also face disease risk. yet, few economic models and empirical analyses include the role of these other actors, which misses additional points of human-disease interaction that could respond to policy (bowen- jones et al. ; cowlishaw et al. ; kamins et al. ; nielsen et al. ; bachmann et al. ; van vliet et al. ; latinne et al. ) . middlemen with sufficient market power can pay lower prices to hunters than middlemen operating competitively and prevent the system from reaching the open access outcome (tháy et al. ). this role of middlemen is relevant in modeling wildlife supply chains and the resulting disease externality because they drive two elements of the disease risk: the amount of wildlife being harvested and the number of people directly contacting the wildlife. all points of human-wildlife contact along the supply chain represent areas of zoonoses spillover risk and possible opportunities for changing human behavior with policy levers. both local and global consumers contribute to the demand for wildlife and bushmeat (mcnamara et al. ; fukushima et al. ) . local demand in lmics is largely driven by wealth, nutritional needs, and prices (wilkie et al. ; fa et al. ; godoy et al. ) . analysis of demand across countries finds a range of income elasticities and price elasticities for bushmeat (wilkie and godoy ; east et al. ; wilkie et al. ; mcnamara et al. ) . policy can be aimed at manipulating the prices of bushmeat to reduce demand, and thereby reduce disease risk to consumers and the supply chain by reducing supply, but variation across settings in demand response imply a need for contextspecific information. here we demonstrate the role of balancing observation and data collection with modeling of people's decisions around their interaction with wildlife to inform conservation and disease policy. the wildlife markets in north sulawesi, indonesia sell a variety of wildlife and domestic animals, including bats like large flying foxes, which could potentially lead to disease spillover events. bat sales in these markets have been increasing for decades (clayton and milner-gulland ; lee et al. ; latinne et al. ) and concerns about declining bat populations and potential pathogen spillovers led to calls for policy to reduce bat harvesting (sheherazade and tsang ; latinne et al. ) . latinne et al. ( ) conducted stakeholder interviews with hunters, local collectors, middlemen, and vendors, and collected market data during - to describe the supply chain and estimate quantities of marketed bats. an important feature of these markets is that regional resource use and cultural differences between geographic regions create the current structure of the supply chain. demand for bat protein stems from christians in northern sulawesi, where hunting has historically occurred and reduced bat populations. current supply is transported from provinces in southern sulawesi, which are predominantly muslim and do not consume bats, and therefore have larger populations to hunt (clayton and milner-gulland ; lee et al. ; sheherazade and tsang ) . hunters sell the bats to either to local collectors or directly to middlemen, who transport the bats to the markets in the north (latinne et al. ) . by identifying the actors and collecting data on revenues and costs, latinne et al. ( ) identifies two salient characteristics of this market for policy development-hunters operate under open access conditions and middlemen transport animals to markets, factoring transportation costs into prices. combining observations, stakeholder interviews, and data collection, followed by modeling the entire supply chain, highlights specific incentives to target in order to conserve bats and mitigate pathogen spillovers from wildlife. the construction of the trans sulawesi highway in allowed vendors to travel further to collect wildlife and expanded the region over which bat harvest creates conservation losses and disease risk (clayton and milner-gulland ) . policies such as highway tolls that increase transportation costs to middlemen could reduce the volume of bats traded because hunters cannot readily access markets. in this case, data collection on the elasticities of demand for bat meat would be a critical piece of information to collect to determine whether the middlemen may pass on the price increase to consumers. second, policies aimed at providing alternative income sources to hunters could increase middlemen costs and reduce both harvest and disease risk. the policy could also increase the practice of middlemen transporting hired hunters to harvest the area, and disease risk and conservation concerns would continue (latinne et al. ) . to counter coordination between middlemen and hunters, policies that provide incentives for people in southern sulawesi to both forego hunting and protect bats from hunting could prevent these migrant hunters from accessing bat habitat. despite the valuable ecosystem services provided by bats, including fruit pollination, bats are generally considered a nuisance species by fruit farmers on sulawesi, which limits the farmers' interest in providing such incentives to bat hunters (banack ; latinne et al. ) . closer collaboration between conservation and health organizations could support payments for ecosystem services to potential bat hunters to discourage bat harvest and disease risk while promoting bat conservation for bat pollination and seed dispersal services. the iterative process of field work and modeling the supply chain, informed by stakeholder interviews, establishes a fuller picture of the disease externality risk at each contact point and the decisions of all actors and forms the basis for policy analysis aimed at reducing human-wildlife contact's disease risk and promotes conservation. some risk of disease arises through frequent human interactions with the environment in which contact with wildlife hosting a pathogen is incidental or an unintended consequence. these types of environmental interactions tend to drive persistence of endemic infectious diseases. for example, while harvesting fuelwood or other ntfps, individuals increase their exposure to arthropods or non-target wildlife species that are carriers of infectious pathogens like malaria and yellow fever (barros and honório ) . in southeast asia, pathogen transmitting mosquito species are found in greater abundance near deforested land and oil palm plantations (young et al. ) . the expansion of agricultural land, in particular oil palm and rubber in liberia and sugar-cane in bolivia, has increased the frequency of contact between plantation workers and rodents that are attracted to these environments, increasing thus the risk of hemorrhagic fevers (e.g. lassa fever in africa or ordog fever in south america) (olugasa et al. ; patterson et al. ; gibb et al. ) . the decision to site livestock operations near forest habitat increases the likelihood of interactions and conflicts between livestock and potential wildlife reservoirs for pathogens. for example, the emergence of nipah virus in malaysia was directly linked to agricultural intensification. pig farms were established in close proximity to forest; farmers planted fruit trees for additional income with the unintended result of attracting bats, which are natural reservoirs of nipah virus . in each of these examples, the risk of disease is endogenous in the decision to interact with the environment, but the risk is incidentally related to that decision. in these settings, the context of when, where, and how individuals interact with the environment plays an important role in the risk of disease. indirect interactions with disease risk are driven by choices individuals make about how and where they spend their time with respect to activities such as subsistence farming, collecting ntfps, mining, or working in the agricultural sector. these choices affect the land covers, habitat degradation, and wildlife with which individuals interact. as an example of a modeling opportunity, the ecological literature indicates that disease risk is greatest in altered or degraded natural habitats (murray and daszak ) , which could link to people's decisions as a function of resource quality. modeling the choice of labor allocation within a particular socio-institutional setting explicitly allows for representing trade-offs of allocating time to specific activities across land covers. labor allocation results can be combined with ecological models or data on vector or pathogen abundance across landscapes to determine individual pathogen exposure risk. the setting's institutions can also influence disease risk. for example, introducing markets for fuelwood substitutes alters the labor allocation to that environmental interaction and exposure to pathogens. similarly, establishing community property rights in forests that have been degraded by overextraction in de facto open access mitigates disease risk by reducing pathogen prevalence and reducing extraction within the forest. with more contextual information about activity choices from socio-economic field work, the daily and seasonal timing of activities can further inform infection risk. for example, if a wildlife species has crepuscular activity and agricultural work is conducted in the cool early morning, high vector activity and human presence on the same landscape may create a high-risk setting for pathogen transmission (vittor et al. ) . policy to reduce the risk of disease transmission through indirect interactions can take one of two approaches, either limiting resource extraction or limiting exposure to pathogens in risky environments. limiting the extent of extractive industries can simultaneously address conservation and health policy goals, but limiting opportunities for economic activities can result in illegal resource extraction, and disease incidence is often greater around illegal resource extraction operations (castellanos et al. ) . to limit extractive industries would therefore require creating alternative (legal) economic opportunities or payment for ecosystem service programs (cárdenas ) and monitoring and enforcing regulations. alternatively, policies can be developed using information about which specific actions increase human contact with the pathogen, and therefore create the greatest risk of pathogen transmission. for example, if oil palm plantation workers live near fields, increasing their risk of exposure to rats and lassa fever, housing can be sited in locations farther away from the source of risk. finally, disseminating information about how specific activities increase disease risks may be a viable policy to mitigate pathogen transmission. here, we explore an example of how leveraging empirical findings and expanding existing models, in combination with socio-economic and environmental observations, can inform policy to reduce malaria risk. a majority of the empirical disease research examines disease incidence from incidental interaction between humans and disease vectors stemming from economic activities such as land use change. ecological research suggests that deforestation opens the forest canopy, which modifies moisture and sunlight that reaches the ground and creates ideal breeding conditions for mosquitoes, which can transmit malaria (de castro et al. ; vittor et al. vittor et al. , . simultaneously, deforestation reduces the abundance of species that feed on mosquitoes, amplifying both the density of the vector and pathogen (baeza et al. ). in the amazon region of south america, the malaria vector species anopheles sp. occurs in greater abundance at forest edges and agricultural landscapes and in lower densities in forests and protected areas; anopheles abundance and diversity varies across land cover types (space); and anopheles feeding activity varies by time of day and season (stefani et al. ; bauch et al. ) . these findings imply that where and when people choose to perform activities is important in exposure to pathogens and disease risk. combining evidence from the natural sciences with economic models that explicitly represent when and where individuals spend time between, for example, collecting ntfps in a protected area and working in agricultural fields can provide insight into how incentives shape risk of infectious disease. existing econometric studies relating deforestation to malaria incidence have been conducted for south america, africa, and asia, and rely on geospatial datasets in combination with either regional epidemiological data (valle and clark ; hahn et al. ; terrazas et al. ; garg ) or individual health survey data (berazneva and byker ; bauhoff and busch ) . while the majority of studies find either positive correlation or causation between deforestation and disease incidence, these results are not consistent across all published works (e.g. valle and clark ; bauhoff and busch ) . although the increasing availability of geospatial and large datasets provides insight into global trends and hotspots for diseases, how people's decisions contribute to the risk of disease spread cannot be discerned from data at the parcel or pixel unit of analysis. as a result, the policy implications and ability to generalize results from these empirical studies are limited to identifying specific demographics that can be targeted for malaria prevention and control programs and broad statements about curbing rates of deforestation. as one possible starting point, albers et al. ( ) 's model of labor time allocation between wage labor and ntfp extraction incorporates both an individual spatial extraction path decision and an aggregate spatial equilibrium of ntfp extraction in a forest. that framework could be expanded to replace wage labor with agriculture, to further refine the timing and seasonality of decisions, and to reflect a range of socio-institutional and ecological settings. this modeling framework can be validated and parameterized using stakeholder interviews and observational data. then, overlaying this economic spatial land use model with ecological models that determine mosquito bite rate probabilities in agricultural land, fringe forest, deeper forests, and protected areas allows the model's solution to define the malaria transmission risk facing each individual based on their where and when they decide to perform specific activities. analyzing the spatial pattern of resource extraction decisions as a function of ecological, socio-institutional, and market settings can reveal the characteristics of settings and individuals that drive malaria risk and provide insight for policy development. simultaneously considering the daily and seasonal timing of human and vector activity (mosquitoes are most active at dawn and early evening, and most abundant around rainy seasons (de castro et al. ) ) can avoid creating policies that unintentionally increase pathogen transmission risk. for example, creating alternative fuel sources may decrease the total time spent in forests extracting fuelwood but focus that reduced time in ntfp extraction in fringe forests that have higher mosquito abundance, thereby increasing disease risk. joint consideration of the impact of policies on conservation and health outcomes can avoid perverse incentives and unintended outcomes. supplementing sensitivity analysis with stakeholder observations and data to describe particular settings produces stronger understanding of the drivers of decisions that create the disease risk and identifies the information and data necessary to provide the right policies for the ecological and institutional setting, across seasons. environment, resource, and development economists are particularly well-suited to developing frameworks of people's decisions to interact with their environments; and such models can identify policy levers that alter people's actions in ways that promote both conservation and disease risk mitigation. we suggest three guides for policy-relevant research: data analysis to identify correlations between land/resource characteristics and disease risk is necessary and important but not sufficient to guide joint conservation and disease-risk mitigating policy. empirical analysis that defines correlations but is not specific to people's decisions does not provide information about how human-environment interactions affect conservation or disease spread, which implies that policy levers are difficult to identify below generalities, such as "slow deforestation," "limit fragmentation," and "close wildlife markets." modeling people's decisions to interact with natural resources in a lmic setting, while using an iterative process that incorporates context and data, enables policy design based on people's decisions and disease spread mechanisms that can be modified for specific settings. because empirically-informed models characterize decisions about interactions with the environment, they can be used to identify how people react to policies that drive both conservation and disease risk outcomes. in the absence of detailed data at every site of conservation and disease policy interest, general models can prioritize particularly important data collection to enable the model's application in other eco-institutional settings. although economic models of people's decisions to interact with the environment are important for defining conservation-disease policy, interdisciplinary research with ecologists and epidemiologists is necessary to accurately reflect the mechanisms of disease spread that matter for policy. the risk of disease spread is a function of people's choices and of how those choices lead to pathogen exposure, which implies that economic models must be paired with data and models of those disease risks. economic models of humanresource interaction that differentiate across choices of locations and time spent, and across seasons, may be necessary to characterize human infection risk. in turn, economic policy analysis can prioritize further research into ecological mechanisms that drive risk in specific environments. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were 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prediction of zoonotic disease emergence world health organization ( a) coronavirus disease (covid- ) situation world health organization ( b) new ebola outbreak detected in northwest democratic republic of the congo: who surge team supporting the response world health organization ( ) connecting global priorities: biodiversity and human health identification of mosquito bloodmeals collected in diverse habitats in malaysian borneo using coi barcoding publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -yz buegp authors: gushulak, bd; weekers, j; macpherson, dw title: migrants and emerging public health issues in a globalized world: threats, risks and challenges, an evidence-based framework date: - - journal: emerg health threats j doi: . /ehtj. . sha: doc_id: cord_uid: yz buegp international population mobility is an underlying factor in the emergence of public health threats and risks that must be managed globally. these risks are often related, but not limited, to transmissible pathogens. mobile populations can link zones of disease emergence to lowprevalence or nonendemic areas through rapid or high-volume international movements, or both. against this background of human movement, other global processes such as economics, trade, transportation, environment and climate change, as well as civil security influence the health impacts of disease emergence. concurrently, global information systems, together with regulatory frameworks for disease surveillance and reporting, affect organizational and public awareness of events of potential public health significance. international regulations directed at disease mitigation and control have not kept pace with the growing challenges associated with the volume, speed, diversity, and disparity of modern patterns of human movement. the thesis that human population mobility is itself a major determinant of global public health is supported in this article by review of the published literature from the perspective of determinants of health (such as genetics/biology, behavior, environment, and socioeconomics), population-based disease prevalence differences, existing national and international health policies and regulations, as well as inter-regional shifts in population demographics and health outcomes. this paper highlights some of the emerging threats and risks to public health, identifies gaps in existing frameworks to manage health issues associated with migration, and suggests changes in approach to population mobility, globalization, and public health. the proposed integrated approach includes a broad spectrum of stakeholders ranging from individual health-care providers to policy makers and international organizations that are primarily involved in global health management, or are influenced by global health events. several current emerging threats and risks exposing public health vulnerabilities are linked to global processes, such as economics, trade, transportation, environment and climate change, and civil security. many of these processes are influenced or affected by the migration and mobility of human populations. , international migration, which is a supporting component and a consequence of globalization, increasingly affects health in migrant source, transit, and recipient nations. the flow of populations between locations with widely different health determinants and outcomes creates situations in which locally defined public health threats and risks assume international or global relevance. this fact is illustrated by the rapid awareness, detection, and response to the emergence of a novel influenza a/h n virus in the spring of . , global demographic predictions indicate that the forces promoting and supporting international migration will continue to do so, and may become stronger in all regions of the world as populations attempt to move up gradients of opportunity (such as economic, educational, security, health). both the diversity (the term 'diversity' describes the dissimilarities between host and migrant populations relevant to the determinants of health (such as genetics and biology, environment, behavior, and socioeconomics); 'disparity' reflects the burden of inequalities present at both individual and societal levels, which affects the determinants of health.) and the nature of modern mobility and migration frequently exceed the capacities of and thereby challenge existing policies and programs designed to address migration and health. the goals of this paper are to promote a change in thinking and approach to global health issues that reflects emerging evidence and importance of population-based factors, as opposed to disease or pathogen-based regulatory frameworks that have been traditionally used. this approach focuses on factors of human population mobility and shifting demographics that affect both health determinants and disease outcomes. attention in the public health community is traditionally drawn toward the potential and real effects of infectious diseases associated with migration. common examples observed in nations that receive migrant populations include tuberculosis [ ] [ ] [ ] [ ] and hepatitis b. [ ] [ ] [ ] however, sustained migration between disparate health environments also affects the longer-term epidemiology of chronic noninfectious diseases; this has a downstream impact on health promotion, health services for disease prevention and management, and occupational health outcomes at migrant destinations. as a result of these impacts, migration exerts increasing influence on public health policy, education of health-service providers, health system design, and service delivery. examples of these influences include the need to regulate or manage the use of alternative medicines and pharmaceuticals imported by migrants, or the introduction of alternative medical procedures. [ ] [ ] [ ] in terms of public health, migration has implications for recognition of threats, as well as for surveillance and response capacity. migration also influences broader aspects of the 'health of the public,' including the background burden of chronic or latent diseases (both infectious and noninfectious) and patterns of preexisting immunity; it also influences the use and uptake of disease prevention and health promotion interventions, and health-care service utilization in general. , ensuring that necessary information is both available and understood by diverse communities is an increasingly important aspect of public health planning and preparedness [ ] [ ] [ ] in nations with large mobile populations. this was recently shown by responses to the threat of influenza a/h n importation, which included quarantine, isolation, or preventive interventions. [ ] [ ] [ ] traditional regulatory approaches to public health risks in the context of migration are commonly disease oriented or event based. frequently, these concepts assume or are based on the view that a degree of homogeneity or similarity characterizes migrant populations; they also relate to the administrative or legal status of the population. this article puts forth the observation that many of the important public health aspects of migration originate from or are based on the diversity and disparity of the populations themselves and extend beyond the legal and temporal processes involved in changing one's residence. addressing migration-associated health threats and risks will be more effectively accomplished if approached from this population-based framework, rather than from traditional disease-or immigration status-based views. it is proposed that the development of standardized, programmatic approaches to health and migration that are based on collective international evidence would be an effective strategic and operational approach to global health. failure or further delays in addressing emerging health issues associated with migration and population mobility will continue to impair the effectiveness of policies and programs designed to tackle modern global health challenges. , methods the peer-reviewed literature was accessed through electronic searchable sites (pubmed, promed, and others) using standard search strategies for literature related to migrants, mobile populations, and specific disease outcomes. in addition, publicly available reports from international and national organizations and agencies were accessed for information on migrants, mobile populations, and health. organizations and agencies included the world health organization, international organization for migration, international labour organization, other united nations (un) agencies, world bank, centers for disease control and prevention (atlanta, usa), european centres for disease prevention and control, the health protection agency (uk), and others. in all the literature or reports, population demographics, source country, reporting region, and health outcome measurements were sought. as the study involved no contact with patients or individuals or personal medical information, research ethics approval was not sought. several factors associated with human population mobility make it a significant determinant of future health threats and risks for all regions of the world. these include the number of people on the move, as well as the diversity and disparity of population characteristics between source, transit, and recipient destinations. taken together, these factors exert a significant influence on the globalization of disease threats and risks. a third factor that continues to challenge objective measurement of health outcomes in migrants is the lack of standardization in terminology that applies to mobile populations. doing so would allow correlation and differentiation of populations on the basis of health-risk characteristics rather than administrative categories. part of the growth in migration is simply due to the increase in human population (the rate of growth is no longer increasing). the global population in was estimated at b . billion people. the global population estimate in was . billion. in , it was estimated that there were million international migrants. current estimates of international migrants place their numbers at b million. if this population were considered a national population, international migrants would be the world's fifth largest nation. a report from the un showed that % of all migrants resided in nations; migrants constitute at least % of the total population of nations; % of international migrants live in high-income economies; % of migrants have moved from a low-to-middle income country to a similar economically designated nation; and % of migrants have moved from a low-to-middle income nation to a high-income nation. ('south-to-south' migrants are about as numerous as 'south-to-north' migrants.) of the b million international migrants reported by the un in , b % arrived in the united states alone. these trends can be associated with profound demographic impacts on migrant source and destination countries. these global population figures, although important in their magnitude, also reflect significant differences in the demographic and health determinants of the migrants themselves. there are millions of migrant workers who leave their families behind to be supported by their financial remittances. the migrant worker population is increasingly composed of women, particularly within asia. these population figures also comprise refugees and internally displaced individuals who seek a safe haven and security after fleeing conflict and disaster (see table ). in addition, there are those populations for whom the numbers and statistics are less definitive. this refers to population movements of migrants without legal permission or authority to migrate, who enter and reside unofficially in a host country. the clandestine nature of these unauthorized migrant ('unauthorized migrants' is emerging as the preferred terminology for international migrants who arrive without the necessary approvals, permissions, or documents (such as identification, citizenship cards, visas, passports, and other travel documents). irregular migrants, undocumented migrants, and those who are smuggled or trafficked outside their own country are included in this terminology.) flows, including smuggling and trafficking, makes the determination of their numbers difficult and estimates inexact. the quality of data for unauthorized migrants decreases as the migration process becomes less formally organized. nonstandardized use of terminology describing migrants a major challenge in assessing health impacts of migration is the lack of agreed definitions and consistency in the use of terminology to describe migrant populations over time. some migratory movements may involve international travel across formal boundaries, whereas others, such as rural-urban migration and the internal displacement of populations through conflict or disaster may remain internal national processes. each of the processes and movements can be associated with several descriptive or definitive terms that can vary over time, use, location, and legal or administrative standards. the terminology used to describe a group of migrants in one location, for example, immigrants, may refer to a different migrant group in another setting. in some settings, the term 'immigrant' refers only to those accepted as residents in the destination country and granted legal status to remain there. in other jurisdictions, the term 'immigrant' may be used to refer to any foreign national including those without a formal legal status. although many health risks are associated with movement between locations with different health determinants and outcomes, many definitions of migrant populations currently are based on administrative measures, such as the duration of stay. the un distinguishes migrants according to the duration of stay, classifying them into long-term migrants (residence in a country other than the usual place of residence for months) and short-term migrants (a period of residence of months but o year). this definition does not apply to those individuals traveling for business, to visit friends or relatives, to seek medical treatment, or to undertake pilgrimage. this variability in the use of terminology and lack of direct descriptions related to health creates challenges in the analysis and interpretation of health outcomes associated with migration. international organizations and agencies are attempting to standardize the terminology of migration, but these activities are largely based on administrative and residency principles and may not adequately reflect the health characteristics and determinants of the person or population. furthermore, many modern migration flows are temporary or reciprocal, reflecting the global integration of economic, educational, and labor markets. such movements include populations composed of temporary and seasonal workers, international students, or medical tourists, as well as the growing numbers of those with dual or multiple citizenship and right of residence. these populations, although experiencing and reflecting the health and public health influences of migration, are not systematically captured in traditional national or international immigration statistics. it has been proposed that the health aspects of migration can be better addressed through a standardized, population health-based functional approach rather than by the administrative consideration of processes of modern migration (see table ; modified from gushulak and macpherson ). emerging health threats, risks, and challenges resulting from migration identifying threats related to migrant populations has been driven by historical outbreaks of transmissible infectious diseases of public health significance, such as plague and cholera. as seen in recent years with the severe acute respiratory syndrome (sars, ) and influenza a/h n ( pandemic), many national responses with regard to migrant populations tend to be traditional, on the basis of the principles of border inspection, isolation or quarantine, and exclusion. in migration health, threat and risk identification, assessment and management rarely occur 'pre-event.' examples of poorly studied health threats of potential societal and public health importance include domestic violence against migrant women in destination locations, , long-term impact of dietary changes , on the incidence of cardiovascular disease, diabetes, and certain forms of cancer in foreignborn migrants and their locally born offspring, or the importation of health services or pharmaceutical products from less-regulated environments, representing traditional but often unregulated or unmonitored patterns of self-care. efforts aimed at addressing these challenges can begin through the identification of vulnerabilities within different migrant populations. once identified, demographic and population-based risk analysis can reveal the extent to which mobility globalizes risks for national health systems. some health factors associated with migration are simply a function of the size of the populations on the move, and can be considered as affecting all migrants. there are specific factors associated with vulnerability, risk of illness, and adverse health outcomes that are not equally distributed across migrant groups. they may be relatively more prevalent in some migrant cohorts reflecting uneven influences of behavioral, environmental, genetic, biological, and socioeconomic determinants of health. migrants originating from areas of poverty, those who are forcibly displaced by conflict or environmental calamity, those with limited educational and linguistic skills, and those who are dependent on their communities for protection (such as people with preexisting health conditions, unaccompanied minors, the elderly, the young ,and single-parent families) are at greater risk of adverse health outcomes. at the same time, new arrivals who are subjected to legal, economic, and/or social exclusion can be very vulnerable to contracting disease resulting from poor living environments and exploitative working conditions, including lack of access to health care and preventive services. through a combination of genetic or biological, behavioral, environmental, and socioeconomic determinants of health, many global populations display major differences in the frequency and expression of poor health. the introduction of population movement into an area characterized by sustained differences in measures of population health allows for the transfer or elaboration of these characteristics between locations. this is a concept with far-reaching implications for health maintenance and promotion, disease prevention, intervention and health-services management, and education and training programs. in the sphere of infectious diseases, population mobility is one of the underlying factors in the emergence and reemergence of diseases of international public health importance as shown by communicable disease outbreaks. prevalence disparities between migrant source and destination countries can also exist for noninfectious diseases and conditions, although the direction of disparity from high to low prevalence and vice versa may vary depending on the condition being studied. as a consequence, migrant-receiving nations are sometimes required to respond to adverse health outcomes that originate beyond their jurisdiction and normal planning considerations. in addition, migrants who are subjected to legal, social, or economic isolation and deprivation may develop levels of ill health much different from those seen in the local or host population. taken together, evidence clearly suggests that health interventions and attempts to mitigate adverse health outcomes in migrant communities may require approaches that differ from those required by the locally born community. , the impact of migration on national health and disease epidemiology many economically advantaged nations have gained the benefits of long-standing and effective public health and disease-control programs. the effective control of tuberculosis in much of the high-income world means that the major remaining public health challenges presented by the disease in developed countries are related to migration. in terms of noninfectious conditions and chronic diseases, programs directed at detection and treatment for malignancies (cervical, breast, and colon), interventions to manage anemia and some endocrine disorders, and the significance of maternal-child health issues have resulted in the moderation of the impact of these illnesses locally. migration from less economically advantaged areas will affect the epidemiology of diseases in 'low-incidence' host environments. this is particularly true for diseases that occur at very low incidence levels, or which have been virtually eliminated locally. the relationship between migration and disease epidemiology at the destination of the migrant is not always negative. differential risk and variability in adverse health outcomes may exist between migrant and host populations for a number of diseases. variations in prevalence may go in several directions or vary over time and across generations. health impacts of population mobility may occur in an epidemiologically neutral context. large populations of labor migrants moving within asia or western europe represent situations in which there may be no significant health disparities between mobile and host populations, and any significant migrant effects on health instead are related to the scale of demand for services. finally, population mobility may result in situations in which new arrivals exhibit more positive health characteristics than those observed in the host population. examples of the latter type are observed in the context of some noninfectious or chronic diseases, such as cardiovascular diseases and eating disorders, and are described as representing the 'healthy immigrant effect.' , even this 'healthy immigrant effect' concept varies depending on time, social situation, and clinical condition. although preexisting health conditions and illnesses can be associated with changes in disease patterns, the act of migration can also pose new threats and health risks. postmigration public health impacts include the consequences of ill health when newly arriving migrants experience isolation, social exclusion, and/or poverty. in situations in which migrant communities' access to health or social services is limited, post-arrival susceptibilities may increase, manifested by the expression of a more severe or advanced disease. programs and strategies designed to promote and improve the health of migrants and mobile populations vary between nations. they often reflect national health priorities and local migration dynamics. examples include developing migrant-friendly clinics and hospitals, ensuring that some categories of unauthorized migrants, such as those without documents, can receive care without being reported to immigration authorities, providing medical care or screening guidelines indicating the role had by mobile populations in the epidemiology of a given disease or illness, and in terms of public health risk, considering migrant and mobile populations in emergency and contingency plans for disease risk mitigation. health system challenges migration-associated adverse health outcomes present two sets of challenges to health systems in migrant-receiving nations. the first challenge is the early appreciation and recognition of the diversity and disparity components of the population, which could result in significant migrant health issues. early recognition can be achieved through effective engagement of the health-services system by a health professional or the migrant. the size of the migrant population and the diversity within migrant communities and populations can mask significantly different groups with disease vulnerabilities. health practices may differ significantly by group, particularly in health-promotion strategies and approaches to disease screening between source and host nations (for example, hypertension, diabetes in pregnancy, colon, uterine or breast cancer). these differences may also extend to infectious disease prevention and control measures, such as vaccination or outbreak response. if health indicators are collected or recorded in terms of broad administrative classifications, such as 'migrant' or 'place of birth,' health risks in populations composed of internally displaced, refugees, or trafficked individuals may be obscured. the second set of challenges is related to access to care for migrant populations. even with the recognition of adverse health outcomes in migrant populations, providing secure access to equitable health services for populations of migrants can be difficult. these difficulties can exist even in nations with long-standing immigration programs. for nations that have only recently begun to deal with the growing dynamics of international migration, the difficulties can be much greater. professional and population education, training, and orientation to societal values in health promotion and maintenance, disease prevention, and health services utilization also take time and commitment to achieve. new and evolving populations of migrants and mobile populations can result in rapid arrival and growth of large communities with diverse characteristics, which include social, linguistic, cultural, and economic status; these can be associated with disparate health outcomes. access to and utilization of health and medical services by some foreignborn communities may have a different pattern than those of the host population. , specialized services encompassing linguistically and culturally competent providers, designed for the problems of migrants, will be required to ensure adequate health-care programs and service delivery models. similar features may need to be integrated into public health and disease-control programs designed to mitigate health threats or risks. this is reflected in the need to have educational and instructional information prepared in the language of migrants at an appropriate level for comprehension, and the need for translation or visual tools to deliver messages in a culturally appropriate manner. depending on the location and health sector capacities, these forces can affect the design and function of the health program. migrant-specific programs may be more effective for the migrant community, but they may engender additional costs and resource demands. additional challenges occur as a result of migrant diversity itself and can be seen in many locations where demands or needs for culturally competent health services can extend across several nationalities and ethnicities. strategies to deal with these situations include ( ) support for acculturalization and integration to allow migrants to better use domestic medical and health services and ( ) the provision of migrant-specific or migrant-friendly health services. national, regional, and municipal differences in migrant history and demography make it unlikely that a single approach will be applicable to all venues. however, modern information technology and networking does provide opportunities for the sharing and exchange of best practices and information across cultural environments. , the legal or administrative status of the migrant does affect access to services and care. migrants in an unauthorized situation and some foreign-born women have been shown to have a lower utilization of health services than the local population. for example, health services may be too costly for migrants who do not have health insurance coverage. although linguistically appropriate health services are available and affordable, they may not be used because of migrants' lack of information about their rights and entitlements or out of fear for deportation. limitations to traditional responses to the health challenges of migration traditional approaches to health and migration frequently deal with specific diseases, primarily communicable diseases of public health significance that may be associated with the arrival of migrants. , coordinated attempts at the international level to manage infectious disease transmission were organized and consolidated into the international health regulations (ihr), which was revised in . some nations with integrated and long-standing immigration programs have systematically screened applicants for permanent residency status (immigrants) and some other classes of mobile populations (such as temporary resident applicants, including foreign students or migrant workers ), for various health conditions and illnesses. immigration medical screening, quarantine, and isolation have been used in attempts to address the possible introduction of health threats by exclusion. major immigration-receiving nations continue to use these processes to reduce the impact of health disparities in arriving mobile populations. important as they are from a legal and administrative perspective, programs and policies that continue to embrace responses of inspection and exclusion will be increasingly costly and ineffective in the context of modern migration and population mobility. furthermore, attempting to manage or mitigate health risks in arriving travelers, when many of the health risks may be latent or subclinical, without affecting international travel and commerce is operationally and logistically impractical. modern migration is an integral component of the broader process of globalization and is intimately linked to other nonhealth aspects of globalization, such as global trade and economics, safety and security, and environmental and climatic changes. population mobility underpins the labor and economic demands for human capital. it also helps mitigate the social, demographic, and economic impacts of aging populations in many economically advanced nations where increased migration is required to sustain labor markets and population growth. in addition, modern migration is greater in magnitude and more diverse in health demographics than the traditional immigration process. at present, many people migrate temporarily or travel back and forth between their community of origin and their destination. as international migration will be an increasingly important aspect of human activity, improving the health of migrants and reducing adverse health outcomes related to migration can be expected to be a growing concern globally. some migrant-receiving nations are beginning to appreciate the impact of chronic illnesses in migrants. this includes the demand on and the use of health and medical services by foreign-born populations, and the impact of lifestyle and behavioral factors on health and the health sector. [ ] [ ] [ ] [ ] [ ] standardized approaches to manage health implications associated with population mobility have been proposed, which extend beyond traditional immigration administrative processes to encompass an integrated health framework for modern population mobility (see table ). managing health threats, risks, and challenges in a global context the need for modern and global approaches to population mobility and health is not an abstract goal. considerable attention in the field of migrant health is devoted to the investigation and improved documentation of health indicators among migrant populations in receiving nations. however, many of the health threats, risks, and challenges related to health outcomes due to migration result from factors and influences present outside the jurisdiction and hence the direct influence, of the migrant-receiving nations. , even in nations where long-standing immigration programs are a component of national policies, the health aspects of migration may not be addressed systematically and much of the attention toward migrant health issues is only national in scope. some regional strategies have been proposed, but analysis suggests that they may not be evenly applied or supported. , as a result, there is a paucity of systematic programs and policies to support the health of migrants. to improve global health management and preventive health practices, there is a need for coordinated international actions and partnerships between governments and organizations in nations of origin, transit, and destination. studies have suggested that primary health prevention endeavors such as tuberculosis control in countries of migrant origin are more economical over the longer term than traditional immigration screening programs and policies. they address better universal access in support of equity and the right to health, and have secondary preventive benefits manifested through the improvement of health indicators in migrant source countries. there is growing interest in and appreciation of efforts to address the importance of health and migration at the global level. in , the who world health assembly resolved to take on the issue through its member states by adopting a resolution on migrant health. approaching the topic through coordinated, international action will require considerable changes in many current national and regional health strategies and disease-control policies. national programs based on immigration status, nationality, historical roles of national borders, and traditional travel patterns will need to be redesigned to allow for equitable access to health services for migrants, and greater exchange of information and data to improve research into migrants' health. threats, risks, and challenges will need to be conceptualized in terms of mobility and population dynamics, and to consider migrant origin and transit practices. developing multisectoral approaches the health of migrants is intrinsically linked to all determinants of health but particularly to the unequal distribution of socioeconomic determinants, including income status, housing and accommodation, education, nutrition, sanitation, and employment. , as a consequence, societal responses will be most effective if they are multidisciplinary and involve stakeholders from all relevant sectors working together to reduce adverse outcomes and improve the health of migrants. international dialogue and activities in the field of migration are centered around the principles and policies of more effectively managing migration for the benefit of origin and recipient nations. sustaining and improving the health of migrants is a lateral issue that must be integrated into all aspects of migration management. this implies integrating migration into health policies and strategies that are directly related to desired health outcomes. it implies an increasing awareness among health-care providers and educators, as well as migration policy makers on how to address health threats, risks, and challenges associated with or resulting from population mobility and disparities in health services between geographical locations. a systematic approach to managing adverse migration health outcomes must reflect and integrate the several patchwork policies that have been evolving in many nations for more than a century to deal with situational aspects of migration. at present, various policies exist that address issues related to the health of trafficked and smuggled migrants, labor migrants, those traveling for medical and religious reasons, and for those applying for formal immigration. other broader policy instruments deal with aspects of health for bona fide refugees and asylum seekers or refugee claimants, detained and incarcerated migrants, and for those being returned or deported. integrated health policies that respect the rights of migrants will greatly facilitate coordinated approaches. these must be based on standardized international terminologies and principles reflecting the tools of the un, international organizations, and national programs. systematic actions that support migrant health improvement access to health services, and those that address the specific vulnerabilities of certain migrant populations, will assist nations in developing programs to meet current and future demands. these measures are in the global and national public health interest of sending and receiving communities from a social equity and equality perspective. prioritized programs to reduce disparities responsible for the greatest health risks several of the adverse health outcomes related to migration, particularly those associated with infectious diseases, are already the subject of international and in some cases global attention. many of these diseases are being addressed through initiatives that involve international and regional programs dedicated to improving global health. they include international efforts to expand immunization (gavi), reduce the impact of high-burden diseases (such as tuberculosis, hiv/aids, and malaria), manage the implications of pandemic influenza, and improve public health responses in general (ihrs). although migration and population mobility may feature in some aspects of these endeavors, there is a paucity of integrated collective action on migration-associated components. integrating a migration component into these activities can facilitate the global approach to disease control and demonstrate immediate benefit for both source and recipient nations. mobile populations are one of the means by which locally arising risks can become global challenges. mitigation programs and control strategies must encompass migration components in terms of both threat-to-risk assessment and intervention planning. the importance of these issues has been noted during responses to global health events such as sars ( ) and the more recent pandemic influenza ( ) event, wherein travel-related control measures included screening, inspection, isolation, quarantine, and exclusion. the scale of migration and population mobility has required many of these responses to have cultural and linguistically appropriate services. to be effective, such programs need to reflect the ongoing health impacts of migration that extend well beyond the 'immigration' process. a relatively new phenomenon in international population mobility is the number of migrants who, greatly facilitated by modern travel industry, return to their place of origin to visit relatives or friends; they are known as vfr (visiting friends and relatives) travelers. vfr travelers typically take longer trips, stay in local homes or accommodations, eat locally prepared meals, and take fewer pretravel precautions. many vfr travelers return to their country of origin with children who were born at the new place of residence and lack the immunity their parents acquired before migration. these migrant-related populations of vfr travelers have been identified as being associated with increased adverse health outcomes. , education and training in health and migration processes of migration and population mobility have complex ethical, legal, administrative, and social components that relate to the health of the migrant and host communities. studies have shown that the lack of familiarity with migrant health conditions or the nature of health determinants in migrant communities can negatively affect the effectiveness of care. better understanding of the nature of the health aspects of migration can prevent some adverse health outcomes in international migrants through activities that support the early detection and treatment of health problems in these populations. this is accomplished through early access to and availability and affordability of health care for newly arriving migrants. minimum standards for the provision of linguistically and culturally appropriate tools that assist in health-care delivery , are emerging health-system requirements in some nations. as the world becomes increasingly mobile, multicultural health-care providers in many locations will need to acquire greater capacity to understand, study, and address health needs of migrant communities. migration and international population mobility are facts of global life. the volume of mobile populations within and beyond national boundaries is bringing increasingly high numbers of diverse and disparate populations together. this may occur in areas where traditional administrative approaches to managing migration processes cannot address the health-care needs of migrant populations nor the impacts that these populations may have on transit, recipient, and home communities. addressing the health needs of migrants improves migrant health, avoids stigmatization and marginalization, may reduce long-term health and social costs, protects global public health, facilitates integration, and contributes to social and economic cohesion and development. this paper posits that many of the health challenges associated with modern migration have their origins in the international and global extension of inter-regional disparities in health indicators and determinants. mobile populations provide population bridges across these gaps in health indicators, and effectively globalize risks and negative health outcomes. national and international policies and programs reflecting existing geo-political boundaries represent traditional approaches to dealing with health and migration but are becoming progressively ineffective in the face of migration that is growing in volume and diversity, while extending across locations with disparate health determinants and outcomes. related to specific migrant populations or individual diseases and varying between nations, these traditional administrative approaches may not represent the most effective methods and means of meeting the current or future needs of an increasingly diverse and globalized population. the recent resolution by the who world health assembly reflects the growing international recognition of the importance of the health effects of modern population mobility. addressing migration health needs through a global lens will be best achieved with regional and global strategies that consider public health aspects previously considered to be limited to national needs. policies, programs, and health services for migrants may still be prepared and delivered at a local and national level, but they should be based on and reflect international and global principles and standards. systematic and integrated approaches to migration health, based on global processes and migrants' rights, represent an effective method of managing these issues. globalization: people, perspectives, and progress modernity at large: cultural dimensions of globalization globalization and health: a critical appraisal the signature features of 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edn the organization: geneva health disparities among travelers visiting friends and relatives abroad geosentinel surveillance network. illness in travelers visiting friends and relatives: a review of the geosentinel surveillance network health care provision for illegal migrants: may health policy make a difference? language barriers between nurses and asylum seekers: their impact on symptom reporting and referral culturally competent healthcare systems. a systematic review office of minority health. us department of health and human services cultural barriers to health care for refugees and immigrants. providers' perceptions tuberculosis control in vulnerable groups strengthening health systems in poor countries: a code of conduct for nongovernmental organizations trends: refugees, asylum-seekers, returnees, internally displaced and stateless persons . the organization press release world tourism exceeds expectations in -arrivals grow from million to million in two years. the organization department of economic and social affairs, population division. international migration vision -forecasting international student mobility a uk perspective towards a fair deal for migrant workers in the global economy office to monitor and combat trafficking in persons trafficking in persons report the opinions expressed in this paper are entirely those of the authors and do not necessarily reflect or represent the positions of any organization to which the authors have been or are currently associated. key: cord- -zpzncgiv authors: galimberti, andrea; cena, hellas; campone, luca; ferri, emanuele; dell'agli, mario; sangiovanni, enrico; belingheri, michael; riva, michele augusto; casiraghi, maurizio; labra, massimo title: rethinking urban and food policies to improve citizens safety after covid- pandemic date: - - journal: front nutr doi: . /fnut. . sha: doc_id: cord_uid: zpzncgiv the ongoing pandemic caused by the coronavirus disease (covid- ) is literally changing the world. from december to date, more than million cases have been reported worldwide and global health institutions are acting to slow down the virus transmission and are looking for possible prevention strategies in case of a new outbreak. as in other endemic or pandemic phenomena, the issues mostly covered by scientific and media attention are related to the diagnostic and therapeutic approach of covid- . however, a still neglected issue regards the adoption of a more systemic approach considering the close connection among the infection, the environment, and human behaviors, including the role of diet and urban management. to shed light on this issue, we brought together a faculty group involving experts in environment and biodiversity, food safety, human nutrition, and behavior, bioprospecting, as well as medical doctors having a deep knowledge of the complex historical relationship between humanity and vector-borne infections. two main aspects emerged from the integrative overview of the current covid- pandemic: (i) the scientific community should start sharing social actions and policy advocacy based on the assumption that human health strongly depends upon a sustainable exploitation of natural resources in populated areas; (ii) the specific strategic role of the cities in developing sustainable food systems and promoting healthy dietary patterns. definitely, some priority issues should be addressed to achieve these goals, such as global efforts to increase food safety and security, which would benefit from urban and peri-urban agriculture enhancement, smallholder food producers support, and ecosystem services and local biodiversity maintenance. the ongoing pandemic caused by the coronavirus disease (covid- ) is literally changing the world. from december to date, more than million cases have been reported worldwide and global health institutions are acting to slow down the virus transmission and are looking for possible prevention strategies in case of a new outbreak. as in other endemic or pandemic phenomena, the issues mostly covered by scientific and media attention are related to the diagnostic and therapeutic approach of covid- . however, a still neglected issue regards the adoption of a more systemic approach considering the close connection among the infection, the environment, and human behaviors, including the role of diet and urban management. to shed light on this issue, we brought together a faculty group involving experts in environment and biodiversity, food safety, human nutrition, and behavior, bioprospecting, as well as medical doctors having a deep knowledge of the complex historical relationship between humanity and vector-borne infections. two main aspects emerged from the integrative overview of the current covid- pandemic: (i) the scientific community should start sharing social actions and policy advocacy based on the assumption that human health strongly depends upon a sustainable exploitation of natural resources in populated areas; (ii) the specific strategic role of the cities in developing sustainable food systems and promoting healthy dietary patterns. definitely, some priority issues should be addressed to achieve these goals, such as global efforts to increase food safety and security, which would benefit from urban and peri-urban agriculture enhancement, smallholder food producers support, and ecosystem services and local biodiversity maintenance. the ongoing pandemic caused by the coronavirus disease (covid- ) is literally changing the world ( , ) . from the first documented human patient in wuhan (hubei, people's republic of china) in december , on august , more than million cases have been reported worldwide, of which more than six million still active ( % in serious or critical conditions) and almost k deaths. who and other authorities soon realized that it was no longer possible to contain the virus spread, but only to slow down its transmission and try, at least, to reduce "pressure" on national health systems. as in other endemic or pandemic outbreaks, the issues mostly covered by scientific and media attention are related to the diagnostic and therapeutic approach of covid- contagion. however, greater consideration should be given to a systemic approach considering the close connection between this disease, the environment and human behaviors, in a framework of building a safer, more sustainable and healthier world ( ) . how is it possible that a virus from a chinese market has spread to other continents so quickly, penetrating the heart of cities and killing the weakest citizens? to shed light on this issue, we brought together a faculty group involving experts in environment and biodiversity, food safety, human nutrition, and behavior, biological activity of natural products as well as medical doctors having a deep knowledge of the complex historical relationship between humanity and vector-borne infections. we believe that unlike the pandemics of the past, the factors triggering the current spread of covid- outbreak, should be analyzed not only by scientists and politicians but also by societal stakeholders. many european countries most afflicted with covid- have started thinking ahead are now facing with the "phase two" of the covid- situation by operating the recovery of industrial and social activities keeping, at the same time, the infection spread as low as possible. in such a context, the linear science, which analyzes those biological variables dealing with the pathogen and its infectivity to find possible solutions [e.g., a vaccine or a therapy ( )], clashes with the "post-normal science" (psn) approach, for which, societal values (e.g., the right to freedom, economic needs, and relational aspects) claim their importance ( ) . pns is designed to deal with situations of uncertain facts, values in dispute, high stake and urgent decisions [( ); figure ]. pns should be operated together with responsible research and innovation (rri) tools (https://www.rri-tools.eu/aboutrri) that offer a broad set of strategies to address global challenges throughout the analysis of "real-world complexities' considering new scientific knowledge and technologies, but also the needs of different stakeholder categories. the contrast between these approaches to science is exerting some pressure on governments that even proposed solving the problem of covid- with autonomous strategies or hypothesized to reach a sort of herd immunity by sacrificing an entire generation of older people ( , ) . this situation demonstrates the lack of organization by the modern society to address complex global issues. our contribution aims at supporting the adoption of a pns response to covid- pandemic, also considering the geopolitical and social aspects which caused the dramatic susceptibility to such infection. for this reason, we aimed at better addressing two main concepts regarding covid- "effect" on cities and citizen-led community responses, to prevent future pandemic events. the first point to discuss is the unexpected permeability of cities to this virus and hypothesize how the spillover from wildlife of such a kind of pathogens can reach urban centers. this evaluation is of primary concern to better define suitable prevention strategies for limiting or blocking the current one and other future pandemic spreads. what characterizes covid- contagion, is that it fully took advantage of globalization facilities, enabling rapid spread of the virus across the world. generally, accurate controls are performed on goods transported worldwide but in case of novel pathogens (e.g., the covid- ) diagnostic options are very limited. moreover, when human mobility is a major factor in the spread of infectious diseases, we should acknowledge that screening measures adopted at airports or customs should be implemented ( ) . temperature screening alone may not be very effective as it may miss travelers incubating the disease or concealing fever during travel, or it may yield false positives (e.g., having fever of a different cause), therefore it should be accompanied by other health messages, questionnaires and data collection ( ) . a different scenario occurs for the trading of food commodities for which strict regulations about quality and safety impose the adoption of analytical tools and innovation technologies to prevent the spread of foodborne pathogens or other contaminants ( , ) . in most cases, food quality evaluation is based on bioindicators, both at chemical and microbiological level ( , ) . most diagnostic techniques allow to characterize the internal microbiome and virome ( ) ( ) ( ) ( ) . considering covid- , what can be said with certainty is that such controls did not occur in the wuhan market where the virus started its incredible global spread. this situation further remarks the concept that pandemics are strictly linked to insufficient or absent food safety assessment and disease prevention protocols (e.g., as happening every time ebola viruses outbreak in central africa). it is necessary to remark that the food regulations across countries vary and the quality standards of the same food items produced in different countries are not the same ( , ). the current covid- pandemic, perhaps more than others, highlights that it is necessary to align food security protocols on a global scale since country specific inadequacies may cause serious global consequences. therefore, we believe that to prevent future pandemic outbreaks it is more and more important to consider issues arising from the establishment of supranational risk-governance systems. these typically operate in a framework of compromise between the local governance and local producers needs and the global safety for human health. safety is a priority for all the stakeholders associated with the entire food supply-chain. moreover, after the covid- emergency, it is desirable that citizens enhance its awareness toward the topic of food control and the concept of food safety will acquire a stronger social meaning based on "shared values, beliefs and norms that affect human mindset and behavior" ( ) . consumers' behavior and choices will help modify food supply-chains safety to prevent zoonoses and reduce other risks for humans. ( )] provides a framework to map systems uncertainty against decision stakes. global citizenship is based on rights to be actively involved in debates, responsibility, shared decisions (following careful risk evaluation), and actions to control and implement shared strategies. with regard to covid- , we know that a bat species and/or the malayan pangolin have been found to be likely reservoir hosts for the virus; however, the definitive identity of any intermediate host that might have facilitated spillover to humans is still unknown ( , ) . overall, the identification of the vector has a relatively important value. the central point is that the unceasing exploitation of wildlife and habitat has dramatically increased the risk of exposure to zoonotic diseases, as already and sadly demonstrated for example by hiv, ebola and h n ( ) . but how calculating the risk of these phenomena? are stakeholders and citizens aware of the risks? these elements are fundamental for a pns discussion that is necessary to drive the path of food safety. it is time to realize that food safety cannot rely only on the production chain but potential risks to human health and the environment should be considered as well. the increasing advances in scientific and technological tools have now been adopted to assess such risks, thus opening a new era of "prediction" rather than "reaction" to reduce pathogen contamination and foodborne outbreaks ( ) . for example, the current trends in food safety research rely on the application of (i) genomic analyses for foodborne pathogen identification and traceability, (ii) geographic information systems (gis) to prevent and predict the spatial spread of pathogens outbreak, (iii) tools adapted from landscape ecology (species distribution and niche modeling) and social network analysis for predicting patterns of disease outbreaks, as well as guidance for interventions, and (iv) meta-analysis tools to confer an overall summary of available study findings, providing generalizable estimates and generating strategic highlights to be used by policymakers and decision makers ( ) . overall, risk prevention remains the key factor. the history of pandemics teaches us that almost all recent human pandemics and most of the emerging infectious diseases originated from animals (mainly in wildlife). it is known that species more resistant to human pressure are likely to become the new competent hosts of vector-borne diseases and then to become the most probable spillover agents toward human hosts ( , ) . furthermore, we must remember that biodiversity perturbation and its trivialization is the main trigger of virus spillover events ( ) , as probably happened for covid- (figure ) . given these assumptions, the international food policies concerning food safety should consider biodiversity and ecological interventions to prevent zoonotic spillover events. this would be especially urgent in rural areas, where farming and livestocking often overlap with wildlife species ranges and it has been documented that livestock species usually act as figure | list of the principal pandemic and emerging zoonotic viruses showing human-to-human transmission after the spillover is occurred. summarized host and spillover interface data are provided as in ( ) (mouse: directly from wildlife; swine: directly from domestic animals; mosquito: transmission by vector involving wildlife or domestic host respectively; u: unknown). detailed references for each listed virus are provided in ( ) . frontiers in nutrition | www.frontiersin.org intermediate hosts of spillover events (e.g., influenza a and sars coronavirus, figure ). for this reason, it is also time to rethink urban areas by projecting proximity buffer zones to prevent direct contact between agricultural/zootechnic activities and natural habitats. finally, the conservation of natural biodiversity and its related species interactions are essential conditions to reduce the risk of spillover events ( ) . on the whole, a cooperative work (rri-driven) involving human-health agencies, agricultural authorities, farmers, and natural resource managing institutions, could be essential to promote the global ecological management to avoid the spread of a new putative pandemic "covid- " or other risky vector-borne pathogens that may adversely affect human health, the environment and economy. our second consideration regards the fragility of citizens, especially the weakest ones such as the elderly, and their sensitivity to diseases. it is now clear that these social categories are the most susceptible to severe covid- outcomes, particularly if they already suffer from multiple pathologies. diabetes is the most common comorbidity observed in infected deceased patients in italy, after hypertension ( ) . furthermore, recent data showed a high prevalence of obesity ( %) and overweight ( %) in italian patients, median age years, from different italian icus, confirming evidence available so far in the literature supporting impaired immune response to viral infections ( ) . therefore, beyond the infectious capacity of this virus, it is important to focus on those elements of modern society which could increase citizens' vulnerability, including diet, lifestyle and environmental factors, strictly linked to morbidity and mortality for all non-communicable diseases (ncds) ( , ) . although this concept is well-established, today, the global average consumption of healthy foods is substantially lower than the reference dietary intake, whereas overconsumption of highly processed, energy dense, and nutrient-poor foods is increasing ( ) . the mediterranean diet is considered by unesco as one of the "intangible cultural heritage of humanity" with multiple health benefits, including fortification of immune defenses. however, epidemiological data on covid- would seem to contradict this belief since mediterranean countries (e.g., italy and spain) have the highest number of confirmed covid- cases in the world. five years after the expo , dedicated to the theme "feeding the planet, energy for life, " the city of milan, which hosted the event, is under siege by covid- pandemic. recent dietary changes within the mediterranean basin, with a decreased consumption of plant foods, increased consumption of fast meals and junk food, and negative health consequences such as rise in obesity rates and in ncds incidence (e.g., diabetes, cardiovascular diseases, and cancer) are partially responsible of this burden ( ) . the modern-day change in food choices is the results of lifestyle standardization, enhanced technologies in food production and processing and limited time for culinary activities. this caused for example the progressive erosion of mediterranean food cultures ( , ) . moreover, environmental emergence, such as water scarcity in most mediterranean countries and land wasting also has deleterious consequences on mediterranean food production. global climate changes have also produced the failure of several crops, fisheries, and livestock productions, and the declining of mediterranean biodiversity and agrobiodiversity does not allow the selection of new varieties and resistant breeds. so, once again the erosion of the environment and biodiversity is closely connected to health risks ( ) . how to react to these social and environmental changes showing serious health consequences? the sustainability of the food supply chain is certainly essential ( ) . kinnunen et al. showed that less than one-third of the world's population can meet their food demand within a -km radius ( ) . we should also change our view of food and diet which should no longer, or rather not only, considered an energy source but a reservoir of bioactive molecules beneficial to human health. greater consumption of health-promoting foods and limited intake of unhealthier options are intrinsic to the eating habits of certain regional diets such as the mediterranean diet ( ) . healthy dietary patterns positively influence health and promotes the prevention of common non-communicable diseases (ncds), strengthening host community defenses ( , ) . this concept assumes a particular importance since the over years old citizens could be more at risk of being infected by covid- , not only for intrinsic conditions due to natural aging processes and comorbidities development, but also for inadequate nutritional status and related inadequate intake of macronutrients (e.g., proteins and healthy fatty acids, like omega- ), micronutrients (e.g., vitamins a, b , b , c, d, e, and folate), trace elements (e.g., zinc, iron, selenium, magnesium, and copper) and phytochemicals which are pillars in preventing many chronic degenerative diseases and supporting the immune system ( ) . this would seem to be true even for younger patients with metabolic and cardiovascular diseases, showing severe acute respiratory distress syndrome (ards) caused by covid- ( ) . these considerations are also well-known for past pandemics. a recent retrospective data analysis from the pandemic flu, showed that nutrition played a consistent role in the severity of the disease and was related to mortality also in younger age groups ( ) . more recently, chronic malnutrition has been correlated to high morbidity and mortality during the influenza pandemic ( ) . similarly, malnourished children appear to be at increased risk for viral pneumonia ( ) . in dietary recommendation, fat quality has to be addressed ( ) , since evidence shows the need to achieve a balance between dietary intake of omega- and omega- for optimal nutrition ( ) , especially in those subjects more vulnerable to malnutrition and "silent inflammation" which disposes to a greater propensity to viral infections ( , ) . certainly, the presence of an unbalance between pro-and anti-inflammatory lipid mediators has been reported in literature ( ) and it is important to remark that science has already given solutions ( ) which are not adopted in new diagnostic policies for primary and secondary prevention. moreover, food supplements such as vitamins c and d might also be considered to help both innate and adaptive immune cells ( ) ( ) ( ) ( ) . studies on human coronaviruses (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov), have highlighted that secondary metabolites of some plant species seem to inhibit virus proteins, cellular infection, and intracellular replication ( ) . extracts of spontaneous plants such as root tubers from rheum officinale baill. (rhubarb), root tubers or vines from polygonum multiflorum thunb., (polygonaceae) showed an inhibitory activity against the interaction of sars-cov s protein with ace ( ). procyanidins and other secondary metabolites extracted from cinnamomi cortex (cinnamomum cassia j. presl) can reduce the virus infection by interfering with endocytosis ( ) . the extract of cimicifuga rhizoma, meliae cortex, coptidis rhizoma, phellodendron cortex, and sophora subprostrata radix showed an ability to inhibit of rna-dependent rna polymerase and/or proteases crucial for coronavirus rna replication ( ) . finally, the antiviral activity of licorice (glycyrrhiza glabra l.), containing glycyrrhizin, inhibits replication, absorption and penetration of the sars-cov acting on the early steps of the replicative cycle ( ) . in this field, traditional chinese medicine is working actively to identify dedicated compounds to specifically contrast covid- infection ( ) . this practice relying on biodiversity and known as "bioprospecting, " may be a good strategy to find compounds having a positive effect on human health as well as to find new raw materials to produce novel and fortified foods for modern citizens. recently, di marco et al. ( ) suggested that the risk of emerging infectious diseases (eids) is a key aspect for developing suitable policy strategies at the global scale. within this framework, the conservation of biodiversity and food production are two additional pillars that should be considered to prevent drastic environmental changes and the risks of zoonoses, and virus spread. in figure we aimed at further remarking this concept including additional factors strictly related to covid- pandemic. from a nutritional point of view, addressing subclinical micronutrient deficiencies is one of the first steps that must be considered to improve resistance to infectious diseases like covid- (and other pathogens) ( ) . it is therefore recommended that micronutrient testing, such as vitamin d measurement, should be applied in the annual check up of selected individuals at high risk of deficiency ( ) . a nutritional approach, ensuring a long-term sustainability, is essential to improve micronutrient status by increasing the availability and consumption of micronutrient-rich foods ( ) . besides lifestyle changes, including diet has been shown to positively affect metabolic and cardiovascular diseases, which are the most frequent comorbidities associated to severe covid- disease. the spread of inappropriate eating habits and inactivity in western societies, particularly among the younger, "comfortably off " generations, has led to the development of chronic degenerative diseases defined as "comfort" diseases ( ) early in life, leading to an increase in premature deaths for ncds. food is readily available in developed countries but there is an evident split-up between scientific evidence, food choices, and dietary patterns of consumers. this, over time has favored the spread of the obesity epidemic and other diet-related diseases. it is time to acknowledge that environmental factors exert a major influence on dietary behavior, primarily by facilitating meals consumption away from home and by minimizing time dedicated to meal preparation and consumption and secondly, making food of poor nutritional quality available on the market and appealing for appearance, taste and price. this burden is exerted by market rules that affect behavior and food choices with scarce public awareness of the potential negative impact on health. it is necessary that science, technology, education, legislation, and community policies combine to create the urban structures and environment required to encourage healthy lifestyle including dietary choices, not just for few, but for everyone ( ) . more efforts must be addressed to reduce exposure to ambient air pollution, strongly associated with population density ( ), promoting chronic inflammatory state and affecting resilience to infectious diseases not last covid- ( ) . finally, western medicine generally tends to identify pharmacological molecules that act on specific disease mechanisms; however, human body complexity and individual answers are sometimes underestimated. thus, it could happen that infected patients die more due to comorbidities associated to infection with covid- than for covid- per se. the time has come to apply a systems biology approach where drugs, foods and lifestyle work in synergy to promote patient healing and prevent further infections, gaining a holistic approach to community health ( ) to support the further personalization of health and social care ( ) . for these reasons, in our scheme, we stressed the impact of environment and food system of therapeutic approaches. this integrative framework demands both an increased attitude of sharing by the scientific and technical community as well as a social participation since we believe that, as previously anticipated, food safety and human health should be regarded more as a social issue. our final suggestion is to start a frank and open scientific discussion on covid- issues and future risks for new pandemic outbreaks, continuing the legacy of expo , declared in the milan urban food policy pact, signed by more than cities in the world ( ) . in this document, the strategic role of the cities in developing sustainable food systems and promoting healthy diets is stated, yet acknowledging all the differences in their natural and policy endowments, including economic background and cultural innovation, managing vast public resources, infrastructure, investments, and expertise. these issues will be fundamental especially for those countries that are coming out of covid- lockdown restrictions and where it is more expected that political and social contrast will emerge if different stakeholders needs and opinions will not be analyzed and considered for planning the recovery after the pandemic event. we would like to undermine and integrate this overview with few take-home messages that arise from the teachings of this dramatic situation we are experiencing firsthand: (i) food safety is a global issue. the unsafety of local food markets, like the wuhan's one, can exert severe and global impact. (ii) smallholder food producers play a key role in feeding cities, by helping to maintain resilient, equitable, culturally appropriate food systems, and promote sustainable diets. in cities with a high percentage of elderly, local food production should be tailored for specific targets to maintain adequate nutritional status, including fortification of immune system. similarly, in developing countries, smallholder farms should improve the production of local crops rich in macro and micronutrients to improve food security and health of local populations ( ) . (iii) acknowledgment that urban and peri-urban agriculture may offer opportunities to protect and integrate biodiversity into urban landscapes and food systems, thereby contributing to synergies across food security, ecosystem services, and human wellbeing. this is very important to prevent the spillover of viruses but also to offer better efficacy of new drugs synthesized to fight future diseases. a unified approach to nutritional screening and assessment is recommended guiding toward integrated panels of biomarkers to investigate the nutritional status and predict future health outcomes of the individual and moving from stratified to personalized to precision nutrition ( ) . the whole scientific community should start sharing directions, social actions and policy advocacy recognizing that the health of people is closely connected to the health of biodiversity and ecosystems where they live. in this context, the "one health" approach (https://www. cdc.gov/onehealth/basics/index.html) represent a good starting point for successful public health interventions by fostering collaboration across all sectors, taking the right steps to promote health not for the few but for the whole planet. the original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s. this opinion paper was conceived by ml, ag, and hc. all authors contributed to its execution and write up with ml leading on the manuscript structure, biodiversity-related aspects were contributed ag and mc, nutrition aspects by hc, botanic and phytochemistry aspects by md, es, ef, and lc, and medical aspects by mr and mb. covid- : what is next for public health? the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak the junction of health, environment and the bioeconomy: foresight and implications for european research and innovation policies the early landscape of coronavirus disease vaccine 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patients with covid- disease vitamin d testing and treatment: a narrative review of current evidence improving diets and nutrition: food-based approaches. rome: cabi dietary attitudes and diseases of comfort available online at air pollution and population health: a global challenge is there an association between exposure to air pollution and severity of covid- infection? oxford covid- evidence service team centre for evidence-based medicine ( ) putting the patient back together-social medicine, network medicine, and the limits of reductionism developing the power of strong, inclusive communities. a framework for health and wellbeing boards office for public management potential role of neglected and underutilized plant species in improving women's empowerment and nutrition in areas of sub-saharan africa the authors are grateful to davide magnani for artworks support. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer ml declared a past co-authorship with one of the author mr to the handling editor.copyright © galimberti, cena, campone, ferri, dell'agli, sangiovanni, belingheri, riva, casiraghi and labra. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -iu flbcl authors: chiotos, kathleen; hayes, molly; kimberlin, david w; jones, sarah b; james, scott h; pinninti, swetha g; yarbrough, april; abzug, mark j; macbrayne, christine e; soma, vijaya l; dulek, daniel e; vora, surabhi b; waghmare, alpana; wolf, joshua; olivero, rosemary; grapentine, steven; wattier, rachel l; bio, laura; cross, shane j; dillman, nicholas o; downes, kevin j; oliveira, carlos r; timberlake, kathryn; young, jennifer; orscheln, rachel c; tamma, pranita d; schwenk, hayden t; zachariah, philip; aldrich, margaret l; goldman, david l; groves, helen e; rajapakse, nipunie s; lamb, gabriella s; tribble, alison c; hersh, adam l; thorell, emily a; denison, mark r; ratner, adam j; newland, jason g; nakamura, mari m title: multicenter interim guidance on use of antivirals for children with covid- /sars-cov- date: - - journal: j pediatric infect dis soc doi: . /jpids/piaa sha: doc_id: cord_uid: iu flbcl background: although coronavirus disease (covid- ) is a mild infection in most children, a small proportion develop severe or critical illness. data evaluating agents with potential antiviral activity continue to expand, such that updated guidance is needed regarding use of these agents in children. methods: a panel of pediatric infectious diseases physicians and pharmacists from geographically diverse north american institutions was convened. through a series of teleconferences and web-based surveys, a set of guidance statements was developed and refined based on review of the best available evidence and expert opinion. results: given the typically mild course of covid- in children, supportive care alone is suggested for most cases. for children with severe illness, defined as a supplemental oxygen requirement without need for non-invasive or invasive mechanical ventilation or extra-corporeal membrane oxygenation (ecmo), remdesivir is suggested, preferably as part of a clinical trial if available. remdesivir should also be considered for critically ill children requiring invasive or non-invasive mechanical ventilation or ecmo. a duration of days is appropriate for most patients. the panel recommends against the use of hydroxychloroquine or lopinavir-ritonavir (or other protease inhibitors) for covid- in children. conclusions: antiviral therapy for covid- is not necessary for the great majority of pediatric patients. for children with severe or critical disease, this guidance offers an approach for decision-making regarding use of remdesivir. in december , the novel coronavirus severe acute respiratory syndrome coronavirus (sars-cov- ) emerged in wuhan, hubei province, china, as the cause of a severe respiratory disease, coronavirus disease (covid- ) . as of august , , over million people worldwide have been infected, including over . million in the united states (us) alone, with over , deaths reported globally ( ) . in light of this public health crisis, there has been significant interest in identifying potentially efficacious antiviral therapies, including novel and "repurposed" medications. to guide pediatric clinicians in the use of these agents, and leveraging the sharing antimicrobial reports for pediatric stewardship (sharps) collaborative, we developed an initial antiviral guidance document, published in april , based on best available evidence and expert consensus ( , ) . in the few months since this initial publication, new evidence has emerged demonstrating the efficacy of the antiviral medication remdesivir in shortening time to clinical recovery in adults with covid- , while several other studies have shown ineffectiveness of hydroxychloroquine and lopinavir-ritonavir ( ) ( ) ( ) ( ) ( ) . based on these data, the us food and drug administration (fda) issued an emergency use authorization (eua) for remdesivir, and the previously issued eua for hydroxychloroquine was revoked ( , ) . further, additional observational studies have provided insight into the clinical epidemiology of covid- in children, demonstrating that while most young patients experience mild illness, a small proportion develop severe illness associated with adverse clinical outcomes, including need for pediatric intensive care unit (picu) admission and mortality ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . finally, in april , a newly recognized hyperinflammatory syndrome seemingly associated with covid- emerged, referred to by the royal college of paediatrics and child health as pediatric inflammatory multisystem syndrome temporally associated with sars-cov- (pims-ts) and by the world health organization and us centers for disease control and prevention as multisystem inflammatory syndrome in children (mis-c) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . affected children present with evidence of multisystem inflammation, with variable manifestations that may include fever, cardiovascular shock, gastrointestinal symptoms, or dermatologic or mucocutaneous changes ( ) ( ) ( ) ( ) ( ) . the syndrome, while fortunately rare with an estimated incidence of in , persons < years of age often necessitates picu admission and has resulted in rare mortalities ( ) . considering the rapidly expanding evidence base regarding optimal antiviral therapy for covid- , yet an ongoing paucity of pediatric-specific data, we reconvened the expert panel to update our initial guidance document. we remind the reader that this document is not a guideline, and we emphasize the ongoing importance of critical review of emerging literature to inform treatment decisions. we additionally refer the reader to guidelines published by the infectious diseases society of america and the national institutes of health ( , ) . a panel of pediatric infectious diseases physicians and pharmacists from geographically diverse north american institutions developed and refined a set of consensus guidance statements through a series of teleconferences and web-based surveys. the panel considered three major questions: ) what criteria define the pediatric population in whom remdesivir should be prescribed? ) does the presence of any underlying medical condition or characteristic warrant different criteria for remdesivir use based on an increased risk of covid- -related morbidity or mortality? ) should any other agents with potential antiviral activity be used to treat covid- ? following each consensus statement, we summarize our rationale and the relevant available evidence, prioritizing human studies. given the overall limited nature of pediatric data, a systematic review was not performed, nor was the available evidence formally evaluated using grading of recommendations assessment, development, and evaluation (grade) or other methodology. of note, this panel considered only antiviral use, with use of corticosteroids and other immunomodulatory therapies reviewed elsewhere ( ) . this guidance document has been reviewed and endorsed by the pediatric infectious diseases society. a statement of "recommend" reflects the panel's view that the evidence base for or against a therapy is sufficiently strong that departures from these recommendations could be viewed as outside the range of usual practice. a statement of "suggest" reflects the panel's view that there is weighting towards risk or benefit from the therapy. a statement of "consider" reflects the panel's uncertainty about the risk or benefit from the therapy. guidance statement: the panel recommends remdesivir be used only in children with positive sars-cov- viral testing. rationale: the clinical presentation of covid- in children is heterogeneous and overlaps significantly with other infections. administration of remdesivir without confirmation of sars-cov- infection poses a significant risk of exposing patients to unnecessary harms without the possibility of benefit and may deplete scarce remdesivir supplies. a rare exception might be made for critically ill patients with a high suspicion for covid- (based on a highly consistent clinical presentation combined with high local prevalence or contact with a confirmed case) for whom a significant delay in sars-cov- test results is anticipated. in such a scenario, empiric initiation of remdesivir could be considered while awaiting test results. guidance statement: the panel recommends that clinical criteria, particularly respiratory support requirements, be used to define scenarios in which treatment with remdesivir is considered. rationale: respiratory support requirement has been used to define illness severity categories in the published clinical trials evaluating remdesivir efficacy (table ) . because the potential benefit of remdesivir across illness severities may differ, we suggest respiratory support requirement be the primary determinant of whether remdesivir is used. the panel additionally recognizes that the spectrum of clinical presentations varies in children, and that rapid deterioration in clinical status and/or cardiovascular compromise may be additional considerations. finally, the panel also considered radiographic criteria, but because radiographic infiltrates are common, even among well-appearing, clinically stable children, respiratory support requirement was favored as the more objective and therefore relevant measure ( ) . guidance statement: remdesivir is suggested for children with severe covid- . rationale: we regard covid- cases as "severe" if there is a new requirement for supplemental oxygen (or an increased requirement from baseline) but without the need for new or increased non-invasive or invasive mechanical ventilation or ecmo (table ) . remdesivir is suggested in this population based on a randomized trial demonstrating a shorter time to clinical recovery in hospitalized adults treated with remdesivir, with the greatest benefit in the subgroup requiring supplemental oxygen without need for mechanical ventilation. however, the clinical course of severe covid- may be milder in children, and therefore the benefit of remdesivir is less certain, necessitating continued case-by-case assessments of benefit and risk in children. this assessment should be informed by illness severity, illness trajectory, hypothesized risk factors for poor clinical outcomes as detailed in section ii, and remdesivir availability. when available, patients should be enrolled in clinical trials. guidance statement: remdesivir should be considered for all children with critical covid- , unless there are contraindications. rationale: we regard covid- cases as "critical" if there is a new or increased need for non-invasive or invasive mechanical ventilation, hemodynamic instability requiring vasoactive agents, multisystem organ failure, or a rapidly worsening clinical trajectory ( table ) . the aforementioned randomized trial demonstrated no difference in time to clinical recovery among the subgroup of adults requiring mechanical ventilation or ecmo. the benefit of remdesivir therapy is therefore uncertain in this population. however, given extreme illness severity and lack of pediatric-specific data evaluating efficacy, remdesivir should be considered on a case-by-case basis in all critically ill children. this assessment should be informed by illness severity, illness trajectory, duration of ventilation (with initiation earlier in the intubation course favored), and remdesivir availability. when available, patients should be enrolled in clinical trials. rationale: based on the duration recommended in the fda eua and no difference in outcomes in a randomized trial comparing versus days of therapy in adults, we suggest a duration of up to days for most children with covid- who are treated with remdesivir. for children requiring mechanical ventilation or ecmo, a duration of up to days is recommended in the fda eua and was the duration studied in the placebo-controlled trial establishing remdesivir's efficacy in hospitalized adults. however, given data suggesting no difference in outcomes between versus days of therapy, and uncertainty as to whether remdesivir provides any clinical benefit at all for critically ill patients, we suggest a duration of - days, with up to days considered on a case-by-case basis for those patients not improving after days of therapy (table ) . remdesivir is a nucleoside analog prodrug which, when activated, binds to viral rna polymerase, resulting in premature rna chain termination ( , ) . the fda issued an eua for remdesivir on may , for adults and children with severe or critical covid- , which was subsequently expanded to include all hospitalized patients on august , ( ) . prior to the eua, remdesivir could be obtained through single patient expanded access ("compassionate use") requests through the manufacturer, gilead scientific. in vitro data while other nucleoside analogs (e.g., ribavirin) are ineffective against coronaviruses due to the proofreading capability of a unique '-to- ' exoribonuclease and resultant high-fidelity viral replication, remdesivir maintains activity despite the existence of this exoribonuclease ( , ) . in vitro studies also demonstrate a low likelihood of developing resistance, further supporting use of remdesivir ( ) . halfmaximal effective concentration (ec ) for sars-cov- was low in vero e cells ( . m), while cytotoxic concentration was high, suggesting remdesivir specificity for viral rna polymerase and a wide therapeutic index ( ) . the results of four randomized trials evaluating the efficacy of remdesivir have been published at the time of this guidance document. the adaptive covid- treatment trial (actt) (nct ) was a national institutes of health (nih)-funded, multicenter, double-blind, placebo-controlled trial evaluating the efficacy of a -day course of remdesivir in hospitalized adults with covid- . the primary outcome was time to recovery, defined as either hospital discharge or hospitalized without need for ongoing medical care, and was measured on an eight-point ordinal scale. preliminary results demonstrated that patients treated with remdesivir had a median time to recovery of days as compared to days in the placebo group (rate ratio . , % confidence interval [ci] . - . ). in a subgroup analysis stratified by respiratory support requirement, time to recovery was reduced in the group requiring supplemental oxygen only, with no difference in patients not requiring supplemental oxygen (i.e., patients with moderate disease) or in patients requiring mechanical ventilation (invasive or noninvasive) or ecmo (i.e., patients with critical disease). no statistical difference in mortality was detected ( . % in the remdesivir arm versus . % in the placebo arm). there were no differences in key safety outcomes between treatment groups, including anemia, acute kidney injury, or hepatic transaminase elevations ( ) . additional analysis of these data is ongoing. an industry-sponsored trial compared versus days of remdesivir therapy in a cohort of patients aged ≥ years with severe covid- , defined as pulmonary infiltrates on imaging and an oxygen saturation < % on room air or need for supplemental oxygen at randomization (nct ). the primary outcome was clinical status on day using a -point ordinal scale, similar to the previous study. after adjustment for baseline clinical status, which was more severe in the -day group, no differences in the primary outcome were detected in the two treatment arms ( ) . a second industrysponsored, open-label, non-placebo-controlled randomized trial compared standard care versus a -or up to -day course of remdesivir in hospitalized patients with moderate covid- , defined as pulmonary infiltrates on imaging and an oxygen saturation > % at randomization (nct ). the primary outcome of this study was clinical status score on day following randomization on a -point ordinal scale, with differences in the distribution of scores across treatment groups reported as odds of a better clinical status. relative to patients randomized to standard care, patients randomized to days of remdesivir had a greater odds of a higher clinical status (odds ratio . , % ci . - . ) at day , whereas no statistical difference in clinical status was detected in the -day group. significant limitations to this study include ) the unblinded nature of the trial and ) uncertainty as to how to translate the summary odds ratio presented in the primary analysis into a quantifiable and clinically meaningful difference in outcome ( ) . finally, a double-blind, placebo-controlled randomized trial compared remdesivir to placebo in hospitalized adults with severe covid- , defined as radiographically confirmed pneumonia and oxygen saturation of < % on room air or an arterial partial pressure of oxygen to fractional inspired oxygen of ≤ mmhg. similar to the previous two trials, an ordinal outcome scale was used, with the primary outcome of time to clinical improvement defined as the first day within days of randomization that patients experienced improvement of ≥ ordinal levels. this trial was halted prior to target enrollment due to the decline in covid- cases in china, but there was no statistical difference in the primary outcome between treatment groups. complicating the interpretation of these results, use of additional therapies, including antivirals, immunomodulators and corticosteroids, was permitted ( ) . additional published data describing use of remdesivir for covid- include case reports and case series ( ) ( ) ( ) . there are no comparative clinical data evaluating the efficacy or safety of remdesivir for covid- in pediatric patients. most covid- in children is asymptomatic or of mild or moderate severity ( , , , ( ) ( ) ( ) ( ) ( ) . in a large case series of , confirmed and suspected pediatric covid- cases in china, > % had asymptomatic, mild, or moderate infections ( ). data from the us is consistent with these findings, with the majority of children managed as outpatients ( , ) . children receiving remdesivir have been included in several pediatric case series, though data related to clinical outcomes and adverse events are not specifically reported ( , ( ) ( ) ( ) ) . a multinational european cohort of children demonstrated that ( %) had mild or moderate disease, with patients requiring picu admission and just four mortalities. remdesivir was used in patients, but the impact on clinical outcomes was not reported ( ) . similarly, among a cohort of hospitalized children in the us, . % required icu admission and only one mortality was reported. among the children with data on antiviral medication use, just nine received remdesivir ( ) . finally, among children aged < years enrolled in a randomized trial evaluating the efficacy of remdesivir for ebola, there were no serious adverse events attributed to remdesivir in children ( ). the fda-recommended dosing of remdesivir for children is summarized in table ( ) . these recommendations are based on adult physiologically based pharmacokinetic (pbpk) modeling and reflect those used in the aforementioned ebola trial, as well as those recommended for use under the single-patient expanded access ("compassionate use") program. while these doses are expected to provide similar drug exposure to those observed in healthy adults, there are no published pharmacokinetic studies that validate this approach. pediatric providers should be aware that remdesivir is available as an injectable solution and a lyophilized powder, which differ in their concentration of sulfobutylether-β-cyclodextrin sodium salt (sbecd), a renally cleared excipient. the injection solution contains g sbecd per mg vial, whereas the lyophilized powder formulation contains g sbecd per mg vial. for pediatric patients < kg, remdesivir lyophilized powder is used to limit cyclodextrin exposure to less than mg/kg ( ) . finally, while remdesivir is a substrate for cyp c , cyp d , and cp a in vitro, it has a low potential for drug-drug interactions as its metabolism is likely mediated by hydrolase activity. that said, providers are nevertheless encouraged to check drug interactions prior to use ( ) . the fda has warned of possible antagonism between remdesivir and hydroxychloroquine based on in vitro data, so concomitant use of these drugs is not recommended ( ) . several ongoing clinical trials in the us are evaluating the efficacy of remdesivir alone or in combination with various immunomodulatory agents, including baricitinib (nct , nct ), tocilizumab (nct ), and merimepodib (nct ). additionally, remdesivir is being studied in the who-sponsored solidarity trial (nct ). finally, an industry-sponsored study evaluating the safety, tolerability, pharmacokinetics, and efficacy of remdesivir from birth to years of age is underway to inform optimal pediatric dosing (nct ). evidence summary: the pathogenesis of mis-c remains unknown, but the syndrome is presumed to be post-infectious based on three main observations. first, a region's incidence of mis-c has been reported to peak about month after its peak in acute covid- cases ( , ) . second, some affected children have preceding symptoms consistent with acute covid- ( ) ( ) ( ) . finally, a substantial proportion of affected children have positive sars-cov- serology but negative rt-pcr assays, suggesting that their hyperinflammatory state may reflect a post-infectious phenomenon with an aberrant immune response ( , , ) . however, the sars-cov- testing profile is variable in patients meeting criteria for mis-c, with some patients having positive rt-pcr results, sometimes without sars-cov- antibodies ( ) ( ) ( ) . it is unknown whether rt-pcr positivity in mis-c represents replication-competent virus that may act as an ongoing inflammatory trigger. in addition, the overlap of clinical and laboratory features between acute covid- -associated hyperinflammatory syndrome and mis-c can result in diagnostic uncertainty. until the pathogenesis of mis-c is better understood, we suggest limiting remdesivir use to select patients with mis-c and a positive viral testing, including those with an ambiguous clinical presentation, severe illness, or an rt-pcr cycle threshold result suggestive of a high viral load. another potential consideration for remdesivir use is concurrent use of immunosuppressive therapy -particularly corticosteroids -that may impair virologic control. guidance statement: there are no definitive data to support any specific risk factor for severe in children. rationale: the majority of pediatric data related to covid- remains descriptive in nature, including population-level epidemiologic studies and single-and multi-center case series describing primarily hospitalized and/or critically ill patients. the reported prevalence of any comorbidity in these series varies widely, ranging from % to %. nevertheless, the panel recognizes that pediatric clinicians are likely to consider comorbidities when weighing the risks and benefits of antiviral therapy on a case-bycase basis, and in making these decisions may consider: ) the available, albeit limited, pediatric covid- literature; ) risk factors associated with severe covid- in adults; and ) pre-existing medical conditions in children associated with worse clinical outcomes for other viral infections. we have therefore summarized relevant data and highlighted hypothesized risk factors ( table ) that clinicians may consider in determining whether to administer remdesivir. evidence summary: although there is no standard definition for children with medical complexity (cmc), this term generally refers to children who have multiple chronic health conditions, may be dependent on medical technology, and may have functional limitations (e.g., due to neurologic impairment, developmental delays, or genetic syndromes) ( ) . this group's risk of decompensation with pulmonary infections is likely driven by a combination of factors, including abnormalities in mucociliary clearance, muscle tone, and craniofacial structures, as well as potentially delayed recognition of illness due to impairments in communication ( ) . it would therefore not be unexpected if cmc experienced a more severe course following sars-cov- infections, though empiric data confirming this assumption are lacking, and interpretation of published studies is confounded by inconsistent definitions of cmc. in a cross-sectional study of children admitted to north american picus, ( %) were classified as medically complex, whereas only an estimated . % of children in the us are cmc ( , ) . in a large european study of children, both underlying pulmonary disease and neurologic disease were associated with increased risk of picu admission, though whether children in these categories would have met the definition for cmc based on the severity of the underlying condition was not reported ( ) . similarly, two smaller us case series highlight a numerically higher prevalence of children with underlying genetic and neurologic conditions among patients requiring picu admission, though these differences did not achieve statistical significance ( , ) . collectively, these data support the possibility that cmc may be at risk for severe covid- , and medical complexity therefore could be considered in making antiviral treatment decisions. the pediatric cohort described by dong and colleagues remains the largest to date and included children in the < -year category ( ). of these, % had mild to moderate symptoms or were asymptomatic. a more recent case series of children from italy found that infants < months old were at increased risk of critical disease compared to older children, though no deaths occurred. in this series, % of the children who received icu-level care also had comorbidities, and out of the infants in the icu did not require respiratory support ( ) . similarly, a multinational european study including children identified an association between age < month and icu admission, as compared to outpatient management or management on the general ward. however, the degree of respiratory or inotropic support was not reported, so illness severity cannot be assessed, and as in the prior report, there were no mortalities in this age group ( ) . in the report of covid- in us children described above, among infants < year of age, % required icu-level care, compared with % of the children > year of age, suggesting that young age is not associated with increased risk of severe disease ( ) . likewise, the covid- -associated hospitalization surveillance network (covid-net) recently reported surveillance of children hospitalized in the us with covid- , among whom infants < months of age accounted for . % ( / ) of the pediatric hospitalizations. further analysis of clinical severity in of these hospitalized patients did not separate out infants < year but found that children aged - years did not have increased rates of icu-level care compared to older children ( . % vs %). no children - years of age required invasive mechanical ventilation ( ) . several additional reports have also described infants experiencing only mild infection who frequently improved without any intervention ( ) ( ) ( ) . this includes a case series of hospitalized infants < days, none of whom required supplemental oxygen or intensive care ( ) . overall, data are insufficient to suggest that young age alone is a risk factor for severe covid- . initial reports of covid- in the us suggested that adolescents were not at risk of severe disease ( ) . however, as the pandemic has unfolded, single-center reports on the us experience among hospitalized children have suggested an increased proportion of severe respiratory disease in adolescents compared with younger children and infants ( , , ) . a large multicenter report of north american picu hospitalizations describes that the majority of picu admissions occurred in the -to -year-old age group ( ) . as risk factors among adolescents similar to those identified in adults (e.g., obesity) could contribute to icu admissions, older age could be considered a risk factor for increased covid- severity and therefore could be considered in guiding antiviral treatment decisions. in a cohort of children admitted to picus in new york city with acute covid- , % had hematologic malignancy/immunosuppression listed as a comorbidity ( ) . in a separate cohort from the north american picu collaborative study group, % of patients had malignancies or were otherwise receiving immunosuppression ( ) . outcomes were not stratified by co-morbidity in either study, and it remains unclear whether immunocompromised status is a risk factor for severe disease requiring icu-level care. in a large multinational european study, children with malignancy accounted for % of the cohort requiring picu admission ( ) . cohorts of pediatric cancer patients from new york, madrid, and italy had both low rates of infection and low morbidity when infected ( ) ( ) ( ) ( ) . covid-net reported . % of all hospitalized pediatric patients with underlying disease were immunocompromised ( ) . no cohort studies of pediatric solid organ transplant patients with covid- have been published, though case reports and anecdotal information suggest that severe covid- in this population is rare ( ) ( ) ( ) ( ) . finally, in two electronic registries of pediatric covid- cases, severe disease or death appeared uncommon in children who were immunocompromised or in those receiving treatment for cancer ( , ) . these data suggest that children who are mildly to moderately immunocompromised are not at higher risk of severe covid- ; however, the limited number of studies and lack of comparative data preclude an assessment of risk in severely immunocompromised children (table ) considering the limited available data in sars-cov- -infected children, and extrapolating from other viruses, children with severe t-cell deficiency or dysfunction may be at risk of more severe disease and may exhibit longer viral shedding than non-immunocompromised children. these factors could be considered in deciding whether to prescribe remdesivir (table ). we remind clinicians to consider the potential for drug toxicity and drug-drug-interactions given the numerous medications that immunocompromised patients receive, particularly for patients receiving other experimental agents. adult data suggest that, in addition to older age, presence of underlying cardiovascular disease, including coronary artery disease, cardiomyopathy, and hypertension, and chronic respiratory disease are associated with covid- -related morbidity and mortality ( ) ( ) ( ) ( ) ( ) . however, differing etiologies of cardiopulmonary disease in children make direct application adult data challenging, with congenital heart disease as well as bronchopulmonary dysplasia being additional pediatric considerations (table ) . patients with congenital heart disease have not consistently been reported in series describing severely or critically ill cohorts, though one large european series reported that % of non-picu versus % of picu patients had congenital heart disease ( , ) . limited experience suggests a high prevalence of underlying pulmonary disease among children with severe covid- , including children with chronic respiratory insufficiency or failure resulting in technology dependence (i.e., chronic invasive or noninvasive mechanical ventilation) ( , , , ) . a significant prevalence of asthma among children with sars cov- infection has been described in some reports, although data are insufficient to demonstrate an association between underlying asthma and increased risk of severe covid- ( , ) . however, there is evidence to support more severe outcomes from other respiratory viral infections, such as influenza ( , ) , parainfluenza ( ), rsv ( ) ( ) ( ) ( ) , and non-covid- coronaviruses ( , ) , in children with chronic cardiac and pulmonary conditions. presence of severe underlying cardiac or pulmonary disease could therefore be considered when weighing risks and benefits of potential antiviral therapy ( ) . data from retrospective studies suggest that being overweight (bmi > th - th percentile for age and sex) or obese (bmi ≥ th percentile for age and sex) is an independent risk factor for hospitalization and severe manifestations of covid- in adults ( ) ( ) ( ) ( ) ( ) ( ) . being overweight or obese is common in children ( ) , but unlike adults, comorbid cardiovascular disease (e.g., hypertension, diabetes, or renal disease) less often complicates these conditions. initial reports of hospitalized children with covid- are mixed with regard to disease severity in obese children. for example, a case series of children hospitalized with covid- in new york city indicated that obesity was the most prevalent comorbidity, with a significant association with mechanical ventilation in children ≥ years of age ( ) , whereas other series have not demonstrated this association ( , ) . recognizing the limitations of these small series reporting unadjusted analyses, and considering the growing body of evidence supporting an association between overweight and obesity and covid- severity in younger adults, an elevated bmi could be considered when determining whether to administer remdesivir, particularly when associated with cardiovascular comorbidities ( , ) . based on observational data, adults with diabetes mellitus appear to be at elevated risk for several complications of covid- , including progression to severe disease, development of ards, and death ( , , ( ) ( ) ( ) . however, diabetes mellitus has not emerged thus far as a clear independent risk factor for complications of covid- in children ( , ( ) ( ) ( ) . this may be due in part to limited pediatric data, but may also be due to a higher prevalence of type versus type diabetes in children relative to adults, as well as a higher prevalence of associated comorbidities in adults, including obesity ( ) . based on emerging data that support obesity as a risk factor for severe covid- , obesity may be an important comorbidity modifying risk for complications of covid- in children with diabetes mellitus ( , ) . a related issue is that while use of concomitant medications acting on the renin-angiotensin-aldosterone system has been hypothesized to influence risk for covid- -associated complications, current evidence does not support a detrimental effect of these medications ( ) . when considering remdesivir in a pediatric patient with covid- , diabetes mellitus and associated comorbidities such as obesity could be considered in the decision-making process, but diabetes mellitus should not be the sole rationale for choosing to administer antiviral therapy. exposure to angiotensin-converting inhibitor or angiotensin receptor blocker therapy should not influence risk assessment in decisions to administer remdesivir. the initial human data that led to enthusiasm surrounding use of hydroxychloroquine, with or without azithromycin, included small observational studies and randomized controlled trials early in the pandemic ( ) ( ) ( ) ( ) ( ) this study was terminated prior to target enrollment due to waning of the pandemic in china, but no difference in the primary outcome of virologic clearance at days ( % versus %) or time to virologic clearance ( versus days) was detected ( ) . finally, a brazilian trial compared the impact of hydroxychloroquine plus azithromycin versus hydroxychloroquine alone versus standard therapy on day clinical status using a -point ordinal scale in just over hospitalized patients with mild to moderate covid- . no difference was detected in any treatment arm ( ) . also supporting the lack of efficacy of hydroxychloroquine are the results of a randomized trial comparing hydroxychloroquine to placebo for post-exposure prophylaxis following a moderate-or high-risk exposure to sars-cov- . no difference in sars-cov- rt-pcr positivity or sars-cov- -compatible illness within days was detected ( ) . most observational studies have also demonstrated a lack of benefit of hydroxychloroquine. mahavas and colleagues performed a multicenter study evaluating the impact of hydroxychloroquine compared to standard care on icu-free survival at days in a cohort of hospitalized adults with severe covid- . following inverse probability of treatment weighting (iptw), no difference in icu-free survival was detected ( % versus %; hr . , % confidence interval . - . ) ( ) . similar findings were demonstrated by geleris and colleagues, who found no difference in a composite outcome of intubation or death with hydroxychloroquine versus standard care following iptw in a single-center cohort of patients (hr . , % confidence interval . - . ) ( ) . three large observational studies additionally considered the combination of hydroxychloroquine and azithromycin. the first was a multicenter study comparing hydroxychloroquine alone (n= ), hydroxychloroquine plus azithromycin (n= ), or no hydroxychloroquine (n= ) in a cohort of men admitted to us veterans affairs hospitals. unadjusted mortality was highest in the hydroxychloroquine group ( . % versus . % in the combination therapy group versus . % in the untreated group). following propensity score adjustment, hydroxychloroquine therapy remained associated with mortality (hr . , % confidence interval . - . ), while hydroxychloroquine plus azithromycin was not (hr . , % confidence interval . - . ). cause of death was not reported in this study, and given its observational design, it is possible that the association between hydroxychloroquine use and death is the result of residual confounding ( ) . a second study utilized data from the new york state department of health to compare in-hospital mortality in a cohort of patients, including children. no difference in mortality was detected among patients treated with hydroxychloroquine alone (n= ), azithromycin alone (n= ), hydroxychloroquine plus azithromycin (n= ), or neither drug (n= ). cardiac arrest was more common in the group receiving hydroxychloroquine and azithromycin relative to no treatment, but not in patients receiving either drug alone ( ) . finally, a retrospective study including , hospitalized adults suggested reduced in-hospital mortality among patients treated with hydroxychloroquine (hr . ; % ci . - . ) or the combination of hydroxychloroquine and azithromycin (hr . ; % ci . - . ) relative to neither drug ( ). however, a notable limitation was that the hydroxychloroquine-treated patients more often received steroids, a therapy that has been shown to reduce covid- related mortality. this raises the possibility that the mortality benefit seen in the hydroxychloroquine-treated groups was, in fact, driven by the receipt of steroids, especially when considered in the context of multiple randomized trials demonstrating no benefit with hydroxychloroquine therapy. in addition to the lack of efficacy, several reports have highlighted the potential for qtc prolongation with concomitant hydroxychloroquine and azithromycin therapy, occurring in up to % of treated patients ( , ) . the high-dose arm of a randomized trial comparing high-dose ( mg twice daily for days) to low-dose ( mg twice daily on day , followed by mg daily for a total of days) chloroquine was terminated after detection of higher mortality in the high-dose arm ( ) . there are no comparative observational studies or randomized trials evaluating safety or efficacy of hydroxychloroquine or chloroquine in children. frequency of hydroxychloroquine use has been reported in several pediatric case series and was as high as % in a us cohort of critically ill children ( , ( ) ( ) ( ) ) . several authors have evaluated dosing strategies for hydroxychloroquine and chloroquine in children using pharmacokinetic modeling; however, detailed discussion of these studies is beyond the scope of this review ( ) . lopinavir-ritonavir is a protease inhibitor approved by the fda for treatment of pediatric hiv. the ritonavir component inhibits the cyp a metabolism of lopinavir, increasing plasma levels of lopinavir. it is a preferred therapy for children weeks to years of age who require antiretroviral therapy and is an alternative antiretroviral agent for children > years of age ( ) . it is not fda approved or authorized for use in the treatment of sars-cov- infection. its hypothesized mechanism of action for sars-cov- is inhibition of the viral proteinases papain-like proteinase and c-like proteinase, which are key enzymes in coronavirus polyprotein processing. an in vitro study of the antiviral activity of lopinavir in vero e cells demonstrated an ec of . m, which is well above the trough lopinavir serum concentration with dosing used for hiv and doses used in studies of sars-cov- ( , ) . no animal data exist evaluating lopinavir-ritonavir for sars-cov- . a randomized controlled trial compared lopinavir-ritonavir to usual care in hospitalized adults with severe covid- . there was no difference between the groups in time to clinical improvement, defined as a two-point improvement on a seven-point clinical severity scale between the groups ( days versus days), -day mortality ( . % versus %), or virologic clearance. the lopinavir-ritonavir group did experience shorter icu length of stay ( versus days). concerns about the generalizability of these findings include: ) a relatively small sample size, such that only a large difference in outcome was detectable; ) lopinavir-ritonavir was started late in the disease course (median of days after symptom onset), perhaps beyond the time of peak viral replication; and ) a high mortality rate in this cohort, perhaps limiting ability to extrapolate these data to other, less sick patients ( ) ( ) . two smaller trials have reported consistent findings, with no differences in virologic clearance in hospitalized adults treated with lopinavir-ritonavir or another protease inhibitor, darunavir/cobicistat ( , ) . published observational studies largely do not support use of lopinavir-ritonavir for treatment of covid- and highlight a high prevalence of adverse effects, particularly gastrointestinal effects, as well as potential drug-drug interactions from prolonged cytochrome p a inhibition ( ) ( ) ( ) . there are no comparative observational studies or randomized trials evaluating safety or efficacy of lopinavir-ritonavir or other hiv protease inhibitors for treatment of sars-cov- infection in children. reports of use are sparse and limited to case series, the largest of which included children treated with lopinavir-ritonavir, all of whom recovered ( , ) . fund. md has nih-supported collaboration with gilead sciences (u ai ) but does not receive monetary support from gilead. ar is a prior consultant for pfizer outside the scope of this work. there is insufficient evidence to support young age alone as a risk factor for severe covid- . there is insufficient evidence to definitively support older age (i.e., the adolescent age group) as a risk factor for severe covid- . however, based on the higher prevalence of adolescents in published pediatric cohorts relative to younger children, older age could be considered in making antiviral treatment decisions. there is insufficient evidence to definitively support severe immunocompromise as a risk factor for severe covid- in children. however, given the limited evidence base, and based on adult studies of covid- and extrapolation from other viral infections, severe immunocompromise could be considered in making antiviral treatment decisions. evidence to date suggests that mild/moderate immunocompromise should not be considered a risk factor for severe covid- in children. there is insufficient evidence to definitively support underlying cardiac disease as a risk factor for severe covid- in children. however, based on adult studies of covid- , extrapolation from other viral infections, and limited data in children with covid- , presence of underlying cardiac disease could be considered in making antiviral treatment decisions. there is insufficient evidence to definitively support underlying pulmonary disease as a risk factor for severe covid- in children. 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early-stage covid- patients in south korea clinical and epidemiological features of children with coronavirus disease (covid- ) in zhejiang, china: an observational cohort study key: cord- -gbmnoo authors: callender, lauren a.; curran, michelle; bates, stephanie m.; mairesse, maelle; weigandt, julia; betts, catherine j. title: the impact of pre-existing comorbidities and therapeutic interventions on covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gbmnoo evidence from the global outbreak of sars-cov- has clearly demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from covid- . this is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. not all comorbidities confer the same risk, however, many affect the function of the immune system, which in turn directly impacts the response to covid- . furthermore, the myriad of drugs prescribed for these comorbidities can also influence the progression of covid- and limit additional treatment options available for covid- . here, we review immune dysfunction in response to sars-cov- infection and the impact of pre-existing comorbidities on the development of covid- . we explore how underlying disease etiologies and common therapies used to treat these conditions exacerbate covid- progression. moreover, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of covid- in patients with pre-existing comorbidities. the novel severe acute respiratory syndrome coronavirus (sars-cov- ), and subsequent sars-cov- induced coronavirus disease has spread on an unprecedented scale. according to the world health organization (who), as of the th july , there have been , , cases and , covid- related deaths worldwide. evidence from the global outbreak has clearly demonstrated that individuals with pre-existing comorbidities such as hypertension, cardiovascular disease, and diabetes are at a much greater risk of dying from covid- ( , ) . this is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. given that comorbidities are associated with high mortality among covid- patients, a better understanding of the biological mechanisms that underpin this risk are needed to enable development of appropriate preventative and therapeutic strategies. the immune system plays a vital role during covid- , and the degree of immune dysfunction correlates with disease severity ( , ) . severe covid- cases are associated with significant lymphopenia and an overactivated innate immune response resulting in hyperinflammation ( ) . many covid- associated comorbidities affect the function of the immune system, which in turn directly impacts the response to covid- . furthermore, the myriad of drugs prescribed for these comorbidities will also influence the progression of covid- and limit additional treatment options available for covid- . here, we review the current sars-cov- literature and explore how preexisting comorbidities adversely affect covid- outcome. furthermore, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of covid- in patients with pre-existing comorbidities. in december , several cases of an infectious pneumonia with an unknown etiology emerged in wuhan province, china. by january , a novel coronavirus termed sars-cov- was identified as the cause. the virus spread rapidly and was classified as a pandemic by the who on the th march . however, this is not the first pathogenic human coronavirus to emerge in the last decade. in , a severe acute respiratory syndrome (sars) coronavirus (sars-cov) with animal to human transmission was reported in guangdong province, china ( , ) . prior to sars-cov, four human coronaviruses belonging to the alpha and beta genera of the coronaviridae family had been identified: hcov- e, hcov-oc , hcov-nl , and hcov-hku ( ) . however, unlike the previously identified coronaviruses, sars-cov was phylogenetically distinct ( ) . furthermore, rather than causing upper respiratory tract infections with mild common cold symptoms, sars-cov caused severe lower respiratory tract infections, resulting in viral pneumonia and risk of developing acute respiratory distress syndrome (ards). the sars-cov outbreak lasted months, and infected , individuals across different countries, with a mortality rate of ∼ % ( ) . in , a second novel human coronavirus emerged in saudi-arabia and was termed middle east respiratory syndrome (mers) coronavirus (mers-cov). similar to sars-cov, infection with mers-cov can cause fatal pneumonia. to date, the who has reported , mers-cov cases, with a mortality rate of ∼ % ( ) . while the previous two coronavirus outbreaks were relatively well-contained, the unprecedented spread of the current pandemic has demonstrated increased infectivity of sars-cov- . early genome sequencing from china revealed that the k base-pair viral genome of sars-cov- shared . % sequence identity with sars-cov, whereas a bat coronavirus previously detected in rhinolophus affinis shared % sequence identity ( , ) . whilst mers-cov utilizes dipeptidyl peptidase- (ddp ) for cell entry, sars-cov, and sars-cov- share the same cell entry receptor; angiotensin converting enzyme ii (ace ) ( , ) . ace is recognized by the s -subunit of the spike protein and is ubiquitously expressed in the epithelia of the nasal cavity, airway tract and the alveolar space. notably, reports have shown that the receptor binding domain of sars-cov- s has a higher affinity for ace , which may be one contributing factor to the increased viral pathogenesis of sars-cov- ( , ) . the scale of the ongoing pandemic demonstrates the need for a more comprehensive understanding of the disease, and of the contributing factors such as pre-existing comorbidities, which are proving detrimental for disease severity and outcome. clinical presentation of covid- varies greatly. a metaanalysis of studies from countries ( , patients) reported . % of cases as mild, . % as severe and . % as critical ( ) . most healthy individuals are asymptomatic or present with mild/moderate respiratory illness ( ) . the majority of critical cases occur in older (≥ years) or comorbid individuals ( , , ) . covid- symptoms are typical of pathogenic human coronaviruses (figure ) ( ) , with fever and cough reported most commonly, however other common symptoms include dyspnoea, sore throat, sputum production, fatiguem and headache ( , , ) . more recently, olfactory dysfunction such as anosmia has been described in covid- patients ( , ( ) ( ) ( ) ( ) . furthermore, rare gastrointestinal symptoms such as nausea, diarrhea and vomiting have also been described ( , , ) . patients with severe covid- can develop serious and potentially fatal complications such as ards, thromboembolic events, septic shock and multiple organ failure (figure ) . due to the severity of these complications, many are associated with critical covid- patients who require intensive care ( , ) . due to underreporting and large inter-country variation, the fatality rate remains unclear. for instance, in italy the overall case-fatality rate has been reported as . % compared to . % in china ( ) . however, despite the variation, the evidence clearly demonstrates a higher fatality rate among those who develop severe covid- ( ) . the immune system plays a vital role during covid- , and the degree of immune dysfunction correlates with disease severity (figure ) ( , ) . during sars-cov- infection the immune system becomes activated, resulting in local inflammation, the recruitment of monocytes, dendritic cells (dcs), natural killer (nk), t and b cells. this response may manifest as mild/moderate disease resulting in a fever, cough and fatigue, however this will be followed by resolution of both the infection and inflammation. in severe covid- cases, severe lymphopenia and the accumulation of functionally exhausted t and nk cells result in an inability to mount an effective antiviral immune response to clear sars-cov- ( , ) . furthermore, interleukin (il- ) levels remain elevated over time, and are accompanied by high levels of il- , il- , il- , tumor necrosis factor-α (tnf-α), c-x-c motif chemokine (cxcl- ), monocyte chemoattractant protein- (mcp- ), and macrophage inflammatory protein- α (mip- α) resulting in systemic cytokine storm ( ) . this uncontrolled systemic hyperinflammation can cause the development of critical and potentially life-threatening complications such as severe pneumonia, ards, septic shock and multiple organ failure ( , , ) . both lymphopenia and hyperinflammation figure | prevalence of covid- symptoms and complications. data obtained from a quantitative meta-analysis of studies ( ) . it's important to note that % of all cases analyzed in the meta-analysis were hospitalized cases. therefore, the proportion of symptoms and complications are representative of this and the overall proportions with regards to all covid- cases will be much lower. the prevalence of anosmia ( ) and thromboembolic events ( ) were obtained independently from smaller cohorts and may therefore change as more data is published. figure | immune response in mild/moderate and severe covid- cases. mild/moderate covid- is characterized by local inflammation, the recruitment of monocytes, dcs, nk cells, t and b cells, followed by resolution of the infection and inflammation. severe covid- is characterized by severe lymphopenia, t cell exhaustion, and systemic hyperinflammation that can cause the development of critical and potentially life-threatening complication such as severe pneumonia, ards, septic shock, and multiple organ failure ( , , ) . are being reported in the majority of covid- cases admitted to hospital and is associated with a poor prognosis ( , , ) . more detailed examination has demonstrated negative effects on all lymphocyte subpopulations including cd + and cd + t cells, b cells, and nk cells ( ) . high-dimensional analysis of circulatory immune profiles in mild, moderate and severe covid- patients by mass cytometry revealed that proportions of naïve cd + t cells, tgfβ + cd − naïve cd + t cells, dcs, and macrophages are associated with mild cases, whereas a sharp decline in the proportion of cd + t cells and nk cells was observed in severe cases ( ) . interestingly, single-cell rna sequencing of pbmcs isolated from hospitalized covid- patients revealed a novel population of developing neutrophils, which appeared to be closely related to plasmablasts, in patients that had developed ards ( ) . as this was a small cohort of patients, further studies are needed to assess whether this novel subset of neutrophils plays a role in the development of ards and other covid- complications. functionally, cd + t cells and nk cells in severe covid- patients exhibited more signs of exhaustion than mild/moderate patients ( , ) . for example, elevated programmed cell death protein- (pd- ), cytotoxic t-lymphocyte-associated protein- (ctla- ) and t cell ig and itm domain (tigit) on cd + t cells and increased nkg a on nk cells ( , ) . as exhausted t and nk cells are less able to mount an effective antiviral immune response, it is unsurprising that these subsets are unable to eradicate sars-cov- and correlate with severe covid- cases. in addition to t cell changes, humoral immunity against sars-cov- is starting to come to light. evidence of sars-cov- specific antibodies was demonstrated in a study of hospitalized patients. igg and igm sars-cov antibodies were present in % of patients within week of onset and % by day ( ) . in another study, most patients developed robust antibody responses between and days, and although delayed, a stronger antibody response was observed in critical patients ( ) . these findings were also mirrored in a study of covid- patients, who all showed a positive igg response by day , followed by seroconversion to igm ( ) . interestingly, antibody titers were found to be higher among severe covid- patients ( ) , however the authors acknowledge that interpreting an association between antibody response and disease severity is difficult due to the small sample size of severe and critical patients in their study. a recent study reported low variable plasma antibody titers in convalescent individuals, however they found binding domain specific antibodies with potent anti-viral activity in all individuals ( ) . older individuals (≥ years) are more prone to severe covid- and have a higher mortality rate ( , , , ) . clinically, older patients have more pronounced immune dysfunction compared to younger patients, as lymphocyte counts are lower and pro-inflammatory cytokine levels higher ( ) . this is not surprising as aged immune systems are associated with immunosenescence and chronic low-grade inflammation, termed inflammaging ( ) . although immunosenescence affects all aspects of the immune system, much of the deterioration in protective viral immunity can be attributed to defective t cell immunity ( ) . the decline of naïve t cell output due to thymic involution ( ) and the accumulation of senescent t cells leads to reduced viral host immunity ( ) . in mice, cd + t cells were shown to be crucial against sars due to their important role in sars-cov clearance. this protection was lost in aged mice as senescent cd + t cells responded poorly to antigen ( , ) . moreover, in addition to inflammaging, the accumulation of senescent cd + t cells and b cells with distinct senescenceassociated secretory phenotypes ( , ) in older individuals results in elevated baseline inflammation, further increasing susceptibility to hyperinflammation and cytokine storm upon sars-cov- infection. in addition to age, biological sex and ethnicity have also been implicated in covid- outcomes. although no major sex differences exist when examining absolute number of covid- cases, disease incidence is higher in males when comparing older individuals (≥ years). furthermore, initial reports from china suggested a male bias in mortality ( , ) , which has now been reported in out of countries that have reported sex-disaggregated data, revealing a global male case fatality rate of . % compared to . % in females ( , ) . this finding is consistent with data obtained from the previous sars and mers epidemic ( ) ( ) ( ) . the predominant hypothesis to explain these biological sex differences is that estrogen plays a protective role against covid- . following the sars epidemic, studies in mice demonstrated that ovariectomy or pharmaceutical blocking of estrogen in female mice resulted in elevated immune cell infiltration in the lung and consequently a more severe disease outcome ( ) . in support of this, researchers in china reported that lower levels of estrogen were associated with more severe covid- cases in women ( ) . although the exact molecular mechanisms underpinning how estrogen protects against covid- are yet to be confirmed, the influence of estrogen on aging and immunity, ace levels, and sex-related risk factors for comorbidities have all been suggested ( , , ) . due to these benefits, researchers in the uk have commenced investigations into the effects of hormonal therapies such as the contraceptive pill and hormone replacement therapy, however no data has been published yet. more recently, data has emerged suggesting that black, asian and minority ethnic (bame) individuals are at a greater risk of acquiring sars-cov- and have worse clinical outcomes ( ) . for instance, in the uk two thirds of covid- fatalities among healthcare workers were bame individuals ( , ) . underlying comorbidities, which are more prevalent in bame individuals, in addition to cultural, behavioral and socio-economic differences have been proposed as possible causes ( ) . however, more data is needed in order to truly establish whether a relationship between covid- and ethnicity exists. evidence from the global outbreak has demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from covid- ( , ) . however, a greater understanding of the biological mechanisms that underpin this risk is needed to develop appropriate preventative and therapeutic strategies. here we review the major comorbidities identified in a number of meta-analyses ( figure ) ( , ). after identification, independent searches were conducted in order to comprehensively assess the impact of each comorbidity and its associated therapies on sars-cov- risk, covid- progression and outcome, and future covid- therapy options. hypertension has been repeatedly reported as the highest pre-existing comorbidity in covid- patients ( , , , - ). retrospective analysis revealed that patients with hypertension have an increased risk for severe infection and mortality ( , ) . however, whether hypertension itself or the use of hypertensive therapies are responsible for these statistics is currently unknown. hypertensive patients are commonly treated with renin angiotensin system inhibitors, such as ace inhibitors (acei) and angiotensin-receptor blockers (arb). as acei and arb can significantly increase ace expression ( ), many speculate they are responsible for the increased risk to hypertensive patients ( ) . conversely, a retrospective review of hospitalized covid- patients, of which . % had underlying hypertensions, indicated that acei and arb may be protective effect against covid- , as the percentage of severe cases were lower in patients treated with acei/arb ( . %) when compared to those treated with other anti-hypertensive treatments such as calcium channel blockers, β-blockers, and diuretics ( %) ( ) . however, patients treated with non-aci/arb were also found to have a higher incidence of additional comorbidities ( ), which may have been responsible for the development of severe disease. due to the conflicting evidence and opinions among the scientific community, it remains unclear whether treatment with acei/arb has a positive or negative impact on covid- progression, however many are continuing to examine this. ( ). hypertension ( %), cardiovascular disease ( . %), and diabetes ( . %) were the most prevalent pre-existing co-morbidities. as this data is representative of hospitalized patients, the prevalence of these among mild/moderate cases may be different, and as more data is analyzed these may change. frontiers in immunology | www.frontiersin.org cardiovascular disease has also been highly reported among covid- patients and is associated with an increased mortality rate ( , , ) . furthermore, cardiovascular complications such as thromboembolic events, myocarditis, acute coronary syndrome, arrythmia, cardiogenic shock and heat failure, have been documented in covid- patients without prior cardiovascular disease ( ), demonstrating a significant impact of sars-cov- infection on the heart. in a case series of covid- patients, those with underlying cardiovascular disease had a mortality rate of . %, which was further increased to . % in a subset of patients who had both underlying cardiovascular disease and elevated troponin t levels, indicative of myocardial injury ( ) . two possible explanations for the increased prevalence and mortality among patients with comorbid cardiovascular disease have been proposed. firstly, cardiovascular disease is commonly treated with renin angiotensin system inhibitors as described above ( , ) , and secondly, ace is highly expressed in the heart ( ) . a recent study analyzing the cellular distribution of ace in human heart tissue obtained from covid- patients identified that ace was highly expressed in pericytes, cardiomyocytes and fibroblasts ( ) . furthermore, cd , an additional binding receptor for sars-cov, was specifically expressed in macrophages ( ) . due to the increased presence of macrophages in cardiovascular disease, the authors speculate that cd + macrophages may enhance viral entry into the human heart. collectively, this study indicated an intrinsic susceptibility to sars-cov- infection in the heart, which could explain the high susceptibility to covid- among cardiovascular disease patients and the higher incidence of acute cardiac injury in non-cardiovascular disease patients. according to numerous reports severe covid- patients are at heightened risk of thromboembolic events, with - % of critically ill covid- patients reported to have developed thromboembolic complications ( , ( ) ( ) ( ) ( ) . systemic inflammation and subsequent activation of coagulation are both contributing factors to this increased risk ( , ) . coagulation abnormalities have been reported throughout the covid- pandemic and the term covid- -associated coagulopathy (cac) has been used to describe patients displaying coagulation changes ( ) . elevated levels of prothrombin, fibrinogen and ddimer, in addition to elevated inflammatory markers such as c-reactive protein (crp) and il- , are markers of cac ( ) . in particular, increased d-dimer levels highly correlate with disease severity, as elevated d-dimer presenting at admission or over time are associated with increase mortality in covid- patients ( ) . due to the high incidence of thromboembolic events, the use of thromboprophylaxis for patients admitted to hospital with severe covid- has been suggested ( , ) , and cac should be monitored carefully in all hospitalized covid- patient, particularly those with pre-existing risk of thromboembolic events. diabetes is the third most prevalent underlying comorbidity in covid- patients ( , , , , ) . type diabetes is a multifactorial disease characterized by chronic inflammation and impaired metabolism and has become an increasing risk to human health. diabetic individuals have an increased susceptibility to infection ( ) , and are at a high risk of developing multiple comorbidities such as cardiovascular disease ( ) . one study on covid- found that diabetic patients were more likely to develop pneumonia and were responsible for . % of severe cases, but only % of mild/moderate cases ( ) . there are a number of reasons as to why people with diabetes are more likely to develop severe covid- . firstly, chronic inflammation in diabetic patients increases their susceptibility to hyperinflammation and the development of cytokine storm. this has been already reported in covid- patients, as il- and crp levels were found to be significantly higher in diabetic patients ( ) . secondly, it is well-documented that hyperglycaemia can impair the immune response, increase oxidative stress and is associated with the onset of premature senescence ( , ) . consequently, diabetic patients that are unable to control their blood glucose levels may have an even greater vulnerability to severe disease. finally, in addition to disease etiology, the treatment of diabetes may also impact covid- development. as previously discussed for hypertension and cardiovascular disease, the use of renin angiotensin system inhibitors may increase susceptibility to sars-cov- infection ( , ) . furthermore, dpp inhibitors commonly used to treat diabetes have an anti-inflammatory effect, resulting in reduced macrophage infiltration, which could impair the innate immune response during covid- ( ) . obesity is associated with most of the common covid- comorbidities such as hypertension, cardiovascular disease and diabetes ( , ) . the global prevalence of obesity varies greatly, for example obesity is more common in the united states and europe than it is in asian countries ( ) . consequently, covid- severity and mortality rates may also vary as a result of this. one study reported a higher bmi in patients with severe infection, and when comparing survivors vs. nonsurvivors, it was reported that . % of non-survivors had a bmi above kg/m , which was a significantly higher proportion than survivors ( ) . however, the cohort for this study was small (n = ) and further retrospective analysis of existing studies is needed to clarify the impact of obesity on covid- . following the previous h n pandemic, retrospective analyses reported that obesity was associated with increased risk of severe infection and mortality ( ) , which is in line with the increased risk of infection in obese individuals ( ) . in addition to increased risk of comorbidities, obesity is also linked to an impaired immune response ( ) , with evidence of impaired antibody ( ) and t cell ( ) responses. furthermore, expression of ace is upregulated in adipocytes of obese individuals and therefore may act as a potential target for sars-cov- ( ). the incidence of cancer as a covid- comorbidity has been low. in an extensive meta-analysis of , patients, malignancy accounted for just . % of comorbidities reported ( ). however, a nationwide analysis in china reported that . % of covid- cases had active cancer, and these patients had a higher proportion of serious events in comparison to individuals without cancer ( ) . at this current time, it is unclear whether cancer patients are at high risk of covid- due to an immunocompromised state linked to certain cancer therapies. in one particular study, patients that underwent recent chemotherapy or surgery had a higher risk of clinically severe events than those who did not. however, a limited number of just patients were included in this analysis ( ) , demonstrating the need for more thorough research. furthermore, not all cancer patients should be considered equally immunocompromised. cancer patients treated with immuno-oncology therapies such as immune checkpoint inhibitors (ici) could be more immunocompetent than patients undergoing chemotherapy ( ) . nonetheless, there are two major concern associated with the use of immuno-oncology therapies during covid- . firstly, there is potential overlap between covid- interstitial pneumonia and possible pneumological toxicity from anti-pd- /pdl- agents, which can be fatal. although this is a rare immune-related adverse event, it has been reported in . - % of patients treated with anti-pd- /pdl- monotherapy, and - % of patients treated with anti-pd- /anti-ctla- combination therapy ( , ) . secondly, there is a risk of cytokine release syndrome associated with t cell-engaging immunotherapy, such as chimeric antigen receptor (car) t cells. given that cytokine storm has been linked to a negative outcome in covid- due to the development of ards and multiple organ failure, ici and car-t cell therapies may exacerbate this hyperinflammatory state and increase mortality in these patients ( ) . overall, despite the lower incidence of cancer among covid- patients, those being treated with immunocompromising therapies such as chemotherapy, and those susceptible to immune-related adverse events in response to immuno-oncology therapies should be monitored carefully. as infection with sars-cov- results in an acute respiratory disease that can progress to ards, respiratory failure and potentially even death, it is reasonable to speculate that patients with pre-existing respiratory disease would be at increased risk of severe covid- . surprisingly, this risk is not as striking as one might anticipate, as the prevalence of asthma was just . % in a study of covid- patients in china ( ) . furthermore, the incidence of chronic obstructive pulmonary disease (copd) among hospitalized covid- patients was reported to be just . % in a meta-analysis of , patients ( ). conversely, the center for disease control and prevention (cdc) recently released data of us hospitalizations indicating that copd is present in . % of patients ( ) . a possible explanation for this global variation could be due to differences in therapeutics agents and disease management. for instance, a global survey assessing the severity and control of , asthmatic adults worldwide published that individuals in japan and asia-pacific regions reported less severe disease than those in europe and the united states ( ). the prevalence of chronic liver disease in covid- patients is estimated to be % ( , ) . however, liver damage has been repeatedly reported as a common complication in response to covid- ( , , ) . in a study of , patients in china, elevated levels of the liver enzyme aspartate aminotransferase (ast) were reported in % of mild/moderate covid- patients, and % of severe covid- patients ( ) . the same study also reported elevated levels of alanine aminotransferase (alt) in % of mild/moderate covid- patients and % of severe covid- patients ( ) . consequently, it has been proposed that liver damage associated with severe covid- patients is due to dysregulated innate immunity against sars-cov- , or hepatoxicity in response to treatments, rather than pre-existing liver disease. chronic kidney disease is associated with an increased risk of pneumonia, and elevated mortality from infectious diseases has been reported in patients with end stage renal disease ( ) . in an extensive meta-analysis, chronic kidney disease accounted for . % of comorbidities in covid- patients ( ). while the prevalence of chronic kidney disease among covid- patients is low, those with pre-existing kidney disease have been associated with severe disease and increased mortalities ( ) . ace expression in the kidney is elevated in chronic kidney disease ( ) . however, elevated ace expression in the kidney does not appear to correlate with increased susceptibility to sars-cov- in the same way as it does in the heart. nonetheless, chronic kidney disease is associated with persistent, low-grade inflammation which could exacerbate covid- symptoms. several factors contribute to this inflammation such as elevated cytokines including il- and crp, oxidative stress and impaired metabolism ( ) . therefore, the underlying pathogenesis of chronic kidney disease may increase vulnerability to hyperinflammation and cytokine storm upon sars-cov- infection, resulting in severe covid- . autoimmune diseases are conditions characterized by inappropriate immune activation and destruction of healthy cells. lymphopenia is common among autoimmune diseases such as type diabetes, rheumatoid arthritis and systemic erythematosus lupus ( ) , and since lymphopenia is regarded as a major risk factor for developing severe covid- , individuals living with an autoimmune condition may be perceived as high risk. however, unlike other comorbidities mentioned above, autoimmune diseases have not been reported as a risk factor in the current meta-analyses. one possible explanation could be that autoimmune diseases are often treated with drugs designed to restrict immune activation, many of which are now being repurposed for covid- and will be discussed below. furthermore, potential alterations in patient behaviors in order to shield themselves from infection could influence reporting of autoimmunity as a risk factor. as the pandemic continues to accelerate globally, so does the race to develop an effective therapy to protect against and treat covid- (figure ) . however, robust efficacy and safety assessments are needed, particularly with regards to their use in comorbid patients, as reviewed below. the sars-cov- genome and spike protein structure was discovered very rapidly ( ) , enabling focussed development of both rna-and protein-based vaccines. however, vaccine development is a challenging and time-consuming process. following the sars-cov epidemic, several vaccines were developed and assessed using animal models. vaccination with live virus was shown to cause complications in mice such as lung damage ( , ) , and although recombinant spike protein based vaccines were able to protect animals from sars-cov challenge, they were ineffective at inducing sterilizing immunity ( ) . other vaccines with inactivated sars-cov and mers-cov have demonstrated reduced viral titers, reduced morbidity and greater survival in animal models ( , ) . however, the rapid eradication of sars-cov reduced demand for development. some mers-cov vaccines are currently in pre-clinical and clinical development ( ) , however as mers-cov is less closely related, it is unlikely that these will cross-protect against sars-cov- . learnings from previous coronavirus outbreaks indicate that antibody responses are not particularly long-lived, with sars-cov antibodies lasting on average just years ( ) . this apparent lack of long-term protection exacerbates the need for an effective vaccine or vaccine programme to protect against potential recurrent seasonal sars-cov- infections. on the th july , the who reported candidate sars-cov- vaccines under development worldwide ( ) . some of the most promising include an mrna vaccine; mrna- ( ), a dna plasmid vaccine ( ) , and an adenovirus vaccine; chadox ncov- ( ) . although clinical evaluation is underway, outcomes are not available yet. furthermore, frontiers in immunology | www.frontiersin.org vaccine efficacy depends upon an individual's ability to mount a strong immune response against it. consequently, vaccine regimens that grant protection in healthy individuals may not be adequate for older individuals and those with comorbid conditions who have increased immunosenescence ( ) . for influenza, the use of adjuvant ( ) and high dose vaccines ( , ) have been developed and implemented to increase vaccine immunogenicity for older and high-risk comorbid adults. therefore, similar measures are likely to be needed to fine tune a sars-cov- vaccine once it becomes available. in the interim, rolling out a sars-cov- vaccine with proven efficacy in healthy individuals could result in herd immunity, preventing transmission to those more vulnerable and offer indirect protection. another therapeutic approach intended to prevent covid- is convalescent plasma therapy. previous use during the sars ( ) and mers ( ) epidemics, and h n pandemic ( ), demonstrated reasonable efficacy and safety. evidence from those outbreaks revealed that convalescent plasma contained neutralizing antibodies ( ) , and a meta-analysis from sars-cov and influenza studies, reported a significant reduction in mortality following convalescent plasma therapy ( ) . several convalescent plasma studies for sars-cov- have been reported. in china, a small pilot study was conducted to test convalescent plasma collected from recently recovered patients on severe covid- patients, four of which had underlying conditions including hypertension and cardiovascular disease ( ) . from the patients, had high neutralizing antibody titers of ≥ : . no serious adverse events were reported following transfusion, and all patients experienced improved symptoms within to days post-transfusion. however, there are many caveats to this study, the very small sample size and the use of concomitant treatments mean it is not possible to ascertain if clinical improvements were due to convalescent plasma. consequently, additional large scale, controlled, randomized trials are needed and are currently underway. despite no adverse events being reported in the small sample sizes currently being tested for covid- , plasma transfusions are not without risk. as with any transfusion, there is a risk of transfusion transmitted infections, albeit small ( ) . perhaps of more concern are the non-infectious risks such as allergic reactions, transfusion related acute lung injury characterized by acute hypoxemia and pulmonary oedema, and transfusion associated circulatory overload, characterized by hypertension, tachycardia, tachypnoea and dyspnea ( ). these are of particular concern for severe covid- patients with extensive lung damage, and for those with pre-existing hypertension, cardiovascular disease or renal failure ( , ). due to the increased prevalence of comorbidities among covid- patients, those predisposed to transfusion-related adverse events will need to be carefully evaluated. as transfusion volume and rate have both been identified as risk factors for transfusion associated circulatory overload ( ) , covid- trials should examine these parameters to ensure safety in comorbid patients. while many scientists attempt to develop novel therapies to prevent covid- , others are focusing their attention on repurposing drugs that are already on the market (figure ) . at present, the most contested repurposed drug is chloroquine and its analog hydroxychloroquine. following the sars-cov epidemic, researchers found that in vitro treatment of chloroquine could increase endosomal ph and impair terminal glycosylation of ace receptors on the cell surface, therefore inhibiting sars-cov-ace interactions and preventing virus entry ( , ) . similarly, after the mers-cov epidemic, researchers demonstrated that chloroquine could inhibit in vitro replication of mers-cov in well-established cell lines ( ) and primary mature antigen presenting cells ( ) . however, as sars-cov and mers-cov resolved relatively quickly, continued assessment of chloroquine remained limited. furthermore, although in vitro treatment can inhibit sars-cov- ( ), the use of chloroquine and hydroxychloroquine for covid- in the clinic is highly controversial. a series of early clinical trials conducted in china reported apparent efficacy of chloroquine phosphate in treating covid- ( ) . this was followed by a small, non-randomized study of just patients from france, which demonstrated a clinical benefit from the use of hydroxychloroquine, but lacked an appropriate control group and a long-term follow-up ( ) . however, an observational study of , patients treated with hydroxychloroquine report no sign of efficacy for covid- ( ) . this was further confirmed by results from the large randomized evaluation of covid- therapy (recovery) trial that examined , hospitalized covid- patients treated with hydroxychloroquine and reported no significant difference in mortality when compared to , standard of care patients ( , ) . lopinavir is a protease inhibitor often formulated with lowdose ritonavir and traditionally used to treat hiv patients ( ) . lopinavir/ritonavir was previously assessed in a small, nonrandomized study conducted in hong kong during the sars-cov epidemic. this study demonstrated that lopinavir/ritonavir treatment resulted in reduced viral load and milder disease, with less recurrence of fever, diarrhea, and improved chest radiographs when compared with those treated with ribavirin, an alternative antiviral ( ) . during the current outbreak, two independent case studies of covid- patients hospitalized in south korea, reported a reduction in viral load and subsequent recovery of patients following the administration of lopinavir/ritonavir ( , ) . however, a randomized control study in china of severe covid- patients demonstrated no difference in mortality, or in the amount of viral rna detected ( ) . furthermore, recent findings from the recovery trial comparing , patients randomized to lopinavir/ritonavir to , standard of care patients concluded no clinical benefit from the use of lopinavir/ritonavir ( , ) . moreover, protease inhibitors such as lopinavir/ritonavir have been associated with hepatotoxicity during hiv treatment ( , ) , and drugto-drug interaction (ddi) with certain statins, for example rosuvastatin, can increase the risk of myopathy ( , ) . therefore, administration to covid- patients with preexisting liver disease and those being treated with statins could prove detrimental. like other members of the protease inhibitor class, lopinavir/ritonavir has also been associated with metabolic changes that can result in hyperglycaemia ( ) , hyperlipidaemia ( ) , and insulin resistance ( ) . due to the lack of efficacy and increased risk of toxicity to certain covid- patients lopinavir/ritonavir could be prove more harmful to patients and should therefore be avoided. oseltamivir and favipiravir, two antiviral treatments traditionally used to treat influenza, have also been examined for use in covid- . oseltamivir is a neuraminidase inhibitor, which has been extensively used as a prophylactic against influenza. in a small, single-center study of patients with sars-cov- pneumonia in china, . % of patients received oseltamivir alongside anti-bacterial drugs such as moxifloxacin, ceftriaxone, azithromycin, and glucocorticoid therapy ( ) . the study concluded no effective outcomes based on oseltamivir ( ) . however, the small study size, lack of appropriate control and the fact that many patients remained hospitalized at the time of publications limits full interpretations. favipiravir is an antiviral with potent inhibitory activity against viral rna-dependent rna polymerase. experimentally, favipiravir demonstrated effective sars-cov- inhibition in vero e cells ( ) . furthermore, a small, open-label, non-randomized comparative study of patients in china, compared clinical outcomes of patients treated with favipiravir and lopinavir/ritonavir ( ) . the median time until viral clearance was days with favipiravir compared to days with lopinavir/ritonavir. at day , ct scans of the chest from patients treated with favipiravir demonstrated significant improvements. adverse events occurred in % of favipiravir treated patients compared to % of lopinavir/ritonavir treated patients ( ) . however, despite initial promise more robust clinical data is needed in order to establish the efficacy and safety of favipiravir as a treatment of covid- . another antiviral agent being examined for use in covid- is remdesivir. when metabolized into its active form, remdesivir inhibits viral rna polymerases, causing a decrease in viral rna production. remdesivir has been shown to inhibit sars-cov and mers-cov in human airway epithelial in vitro models ( , ) , and in combination with interferon beta, remdesivir has been shown to be superior to lopinavir/ritonavir in a mers-cov mouse model ( ) . despite some initial controversial findings ( ) , results from more robust clinical trials have demonstrated reasonable efficacy. for instance, preliminary results from the national institute of allergy and infectious diseases adaptive covid- treatment trial involving , patients, demonstrated that remdesivir accelerated recovered by % compared to placebo ( ) . furthermore, recent results from the phase simple trial investigating the use of remdesivir in patients with moderate covid- showed that patients receiving remdesivir treatment were % more likely to have improved clinically by day than standard of care patients ( , ). due to the success of these two trials, the use of remdesivir has been approved by the fda, ema, uk, and japan as a treatment for covid- . despite proven efficacy, adverse events in response to remdesivir have been reported in % of patients of which % were severe ( ) . these included septic shock, multiple organ dysfunction syndrome, acute kidney injury and hypotension. therefore, the use of remdesivir in comorbid patients with increased susceptible to these adverse events requires further evaluation. as severe covid- cases are characterized by hyperinflammation, the use of immune modulating and anti-inflammatory treatments to prevent severe lung injury and disease progression are being explored. due to their immunomodulatory properties, mscs are being clinically assessed to treat inflammatory conditions such as systemic lupus erythematosus ( ) and graft vs. host disease following allogeneic haemopoietic stem-cell transplantation ( ) . consequently, a pilot study was initiated to investigate the potential therapeutic benefit of mscs for covid- infected patients in china. the study involved just covid- patients who were monitored for days post-msc injection ( ) . msc treatment was well-tolerated and no adverse events occurred during treatment. furthermore, virtually all clinical symptoms subsided, with patients being discharged days post-msc injection ( ) . mass cytometry of pbmcs revealed that peripheral lymphocytes, regulatory cd + cd c + cd b mid dcs and il- increased. whereas, crp, tnf-α and overactivated cxcr + cd + /cd + /nk cells decreased to days following injection compared to placebo group. mscs were shown to be ace negative, meaning they were immune to sars-cov- infection ( ) . although this pilot study shows promise, more robust clinical data is required to validate therapeutic benefit and safety. moreover, the use of mscs would require clinical grade msc production and may not be a plausible solution for many healthcare systems. il- is considered the key cytokine responsible for the induction of cytokine storm during sars-cov, mers-cov and sars-cov- infections ( , , ) . consequently, the recombinant humanized anti-human il- receptor monoclonal antibody tocilizumab, currently used to treat rheumatoid arthritis (ra), has been examined for use during covid- . tocilizumab first demonstrated effectiveness in a small retrospective study of patients with severe covid- pneumonia in china ( ) . oxygen intake was reduced, and improved symptoms occurred in % of patients. lymphocyte levels returned to normal in % of patients and crp levels reduced significantly in . % of patients ( ) . several case studies have also demonstrated rapid clinical improvements following tocilizumab ( ) ( ) ( ) . although these initial studies are promising, the full clinical trial data is still unavailable. furthermore, although safety profiles for tocilizumab are well-established for intermittent use in ra, the safety of tocilizumab when used in combination with antiviral agents and other comorbid therapies has not been established. other anti-inflammatory treatments now being tested clinically for their use against covid- are janus kinase (jak) and bruton's tyrosine kinase (btk) inhibitors. the jak-signal transducer and activator of transcription (jak/stat) pathway mediates the signal transduction of numerous cytokines in a number of immune cells such as t cell, nk cells, and dcs ( ) . consequently, jak inhibitors have emerged as effective treatments for many autoimmune and immunemediated disease. at present, a number of jak inhibitors such as ruxolitinib ( ), baricitinib ( ) , and fedratinib ( ), are being assessed as a potential treatment for covid- . preliminary results from a pilot study evaluating hospitalized patients, of which were treated with baricitinib, demonstrated significant reductions in serum il- , il- β, and tnf-α, as well as a rapid recovery of circulatory t and b cell frequencies following baricitinib treatment ( ) . consequently, a phase adaptive covid- treatment trial (attc- ) has now been established in order to evaluation the use of baricitinib in combination with remdesivir compared to remdesivir alone ( ) . however, despite promising initial findings, as jak inhibitors block a wide range of cytokines including ifn-α, which is crucial during early innate immunity in response to viral infections, the impact of this on viral clearance needs to be evaluated. furthermore, ruxolitinib and baricitinib have both been associated with increased weight gain, cholesterol and albumin levels ( , ) . although no causal association has been reported yet, the issue should not be dismissed and covid- patients with metabolic and cardiovascular comorbidities should be carefully considered before use. bruton's tyrosine kinase (btk) is a key regulator of cell surface receptors expressed primarily in b cells, but also in monocytes/macrophages and neutrophils ( ) . currently, btk inhibitors are used to treat various b cell malignancies and chronic graft vs. host diseases ( ) . as btk can regulate il- , tnf-α, and mcp- , btk inhibitors are being tested in combination with car-t cells ( ) , to alleviate cytokine release syndrome. furthermore, in chronic lymphocytic leukemia, btk inhibitors have been shown to increase cd + and cd + t cell, and significantly downregulate pd- and ctla- ( ) , highlighting a potential reversal of t cell exhaustion, which could be beneficial in covid- . consequently, clinical trials to assess the use of btk inhibitors against covid- are underway. the use of corticosteroids to treat covid- remained largely uncertain until recently. although individuals being treated with long term corticosteroid were instructed to continue with their medication, the use of corticosteroids specifically to treat covid- was not recommended ( ) . this was largely due to the unknown impact of immune suppression on viral clearance and potential adverse outcomes. however, preliminary data from the recovery trial evaluating , patients randomly allocated to receive dexamethasone has proven significant clinical improvements ( ) . dexamethasone is a steroid used to reduce inflammation in a myriad of inflammatory conditions and now reported to reduce covid- related deaths among patients receiving respiratory support by one-third ( ) . in response to these findings the demand for dexamethasone to treat the most critical covid- patients has surged globally ( ) . whilst these findings are exciting, safety data detailing potential adverse events and the impact of co-medications, such as nonsteroidal anti-inflammatory drugs, has not yet been reported and thus dexamethasone should still be considered carefully prior to administration. patients with pre-existing comorbidities are at a greater risk of dying from covid- . however, not all comorbidities confer the same risk. by exploring the underlying disease etiologies and common therapies used to treat these conditions, we have discussed their impact on covid- . comorbidities closely associated with age, chronic inflammation and dysregulated metabolism such as hypertension, cardiovascular disease, and diabetes are the most prevalent comorbidities. however, many of these comorbidities are strongly associated with each other. consequently, many patients will have multiple comorbidities and therefore while we have discussed these individually, the reality is that a combination of factors will be at play. furthermore, as multiple drug use is inevitable for patients with pre-existing comorbidities, the impact of overlaying 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doc_id: cord_uid: ozkly infectious diseases are diagnosed by detection of a bacterium, virus, fungus, protozoan, or helminth in a patient with a compatible clinical illness. the methods of detection include cultivation of bacteria and fungi on growth medium, isolation of viruses in cell culture, and identification of the agent biochemically, antigenically, or genetically. infectious diseases can also be identified by detection of a specific immune response, usually antibodies, that develop during the course of illness. visualization of an agent in infected tissue can provide a diagnosis based on specific morphological characteristics or identify the category of organism, for example, gram-positive or gram-negative bacterium or virus (e.g., eosinophilic cytoplasmic inclusion bodies in neurons in rabies virus infection). methods that detect and allow visualization of antigens (immunohistochemistry) or nucleic acid sequences (in situ hybridization) provide more specific diagnoses. detection of specific nucleic acid sequences amplified by polymerase chain reaction is a powerful molecular diagnostic tool. since the elucidation of the etiology of the first infectious disease, anthrax, more than years ago, koch's postulates have been applied and modified as novel technologies and agents have emerged. during the last years, more than previously unknown agents of infections have been identified in emerging infectious diseases, a phenomenon that is likely to continue. dh walker, university of texas medical branch, galveston, tx, usa elsevier inc. all rights reserved. contagion the ability of a disease to spread by exposure of a person to an infected patient. cytopathic effect the microscopic observation of damage to a cell in culture after infection by a pathogenic agent, usually a virus. differential diagnoses a list of potential diagnoses in a patient who develops a particular set of clinical manifestations. immunohistochemistry a method by which antigens such as those of a particular agent are detected in a tissue section by microscopy by reaction with an antibody and subsequent colorimetric or fluorescent signal-generating methods. in situ hybridization a method for the detection of a genetic sequence in tissue by annealing with a complementary nucleic acid probe coupled with colorimetric, fluorescent, or radiographic detection. opportunistic infection infection with a normally nonpathogenic organism that occurs in an immunocompromised host. prior to the work of koch and pasteur demonstrating in that anthrax is caused by the microscopic bacterium bacillus anthracis, the concept of infectious diseases did not exist. indeed, at that point, there was only one curiosity that could be called an infectious disease, namely, anthrax. robert koch's teacher, jacob henle, had published in a theoretical treatise, 'on miasmata and contagia,' in which he put forth the criteria that one would have to meet to establish that a microorganism caused disease. these criteria now known as koch's postulates are considered the fundamental steps by which an agent is proven to cause a particular infectious disease (table ) . three centuries earlier, hieronymus fracastoro had described clearly the principles of contagion, that is, that exposure of a person to a specific disease such as measles could result in the individual's development of measles, not smallpox, and exposure to smallpox resulted in cases of smallpox, not measles. the scientific identification of viruses, bacteria, fungi, algae, protozoa, and helminths as etiologic pathogens of specific diseases then exploded after elucidation of the etiology of anthrax from the late nineteenth century to today. progress employed available tools such as microscopy, experimental animals, and microbial cultivation techniques and advanced more rapidly with the knowledge of the nature of viruses, immunologic methods, electron microscopy, and molecular methods. when a patient seeks medical attention because of illness, the physician searches for evidence to establish a diagnosis that leads to treatment that will cure or ameliorate the illness. prior to the medical encounter, the diagnostic possibilities are enormous ranging from medical and surgical diseases to the field of psychiatry. infectious diseases in theory offer the greatest opportunities for specific nonsurgical treatment of illness. the key to a favorable outcome is an astute clinician who seeks appropriate clinical, occupational, social, and travel history; considers wisely the potential epidemiological exposures; and gathers incisive data from the physical examination, radiological studies, and laboratory testing. the intermediate result is a prioritized list of differential diagnoses with focus on identifying diseases with the highest clinical consequences and those that are amenable to treatment. specific diagnoses require isolation of the agent in culture, microscopic visualization of the pathogen in tissue lesions, and/or detection of a specific host immune response to the organism. these tasks are accomplished by clinical microbiology and immunology laboratories and pathologists. bacteria comprise the group of agents of the greatest volume and therapeutic consequence that are cultivated in the microbiology laboratory. the culture results must be interpreted with knowledge of the pathogenic potential of the bacterial species isolated and the normal microbial flora at the anatomical site that was the source of the cultured sample. the recognition of contaminants and opportunistic pathogens requires specific knowledge and judgment. escherichia coli could be the cause of septic shock or enterocolitis or merely normal flora or a contaminant. on the other hand, mycobacterium tuberculosis is always a pathologically significant isolate. there are numerous approaches to the isolation of bacteria that are designed to recover particular pathogens that involve the appropriate media containing particular nutrients, salts, inhibitors of other organisms, and growth conditions (e.g., temperature, ph, and anaerobic atmosphere). selection of culture media (nonselective, selective, and differential) for a particular specimen is mostly based on the clinical history and the specimen source (blood, feces, csf, and sputum). automation of blood culture systems has greatly expedited and improved the isolation of infectious agents from blood. it is necessary to specify whether the culture requested is routine, anaerobic, or other specific microbial groups (e.g., mycobacteria) or a particular agent (e.g., brucella). some bacteria grow slowly requiring that cultures be requested to be held longer if certain organisms are suspected. some bacteria have yet to be cultivated (e.g., treponema pallidum). although a few fungi grow on some standard bacterial media, most fungi are recovered in the mycology laboratory on media designed especially for their propagation. fungi that are widely disseminated as spores in the environment and may contaminate cultures or normal yeast flora of some body sites require judgment to initiate or withhold antifungal treatment. identification of fungi has been accelerated greatly in microbiology laboratories by performing either hybridization tests or polymerase chain reaction (pcr) on media growing a fungus that is not identifiable by conventional morphological techniques such as blood culture bottles that contain yeast growth. identification of mycobacteria has also been greatly improved by the availability of dna-based tests on specimens inoculated into liquid medium. once mycobacterial growth is flagged by the automated system, molecular identification is possible, shortening the time until appropriate treatment is initiated. viral infections can be diagnosed by propagation of viruses in cell culture. the presence of viral replication in monolayers of cells in the virology laboratory is classically recognized by injury to the cells caused by the viral infection, known as cytopathic effect. many viruses do not cause microscopically detectable injury to the cell monolayer, and there is no cell type that is universally susceptible to all families of viruses. moreover, there are viruses that have yet to be recovered in cell culture. it is wise to provide the name of the patient's condition (e.g., meningoencephalitis or pneumonia) to the virology laboratory and to indicate the particular viruses that are suspected in order that appropriate cells and procedures will be employed. viruses that are recovered in cell culture can be identified by immunologic and molecular methods. direct detection by such methods has replaced a large portion of viral isolation efforts. some viruses (e.g., the herpes viruses including herpes simplex virus types and , cytomegalovirus, varicella-zoster virus, and epstein-barr virus) cause infections that persist long after the acute illness. recovery of any of these viruses other than in the classic acute infection may reflect asymptomatic viral shedding or an opportunistic reactivation causing clinical illness. the viral quantities are greater in clinically significant conditions. the immune system, which evolved to control infectious diseases, also provides an approach to determine the diagnosis of an infectious disease. the adaptive immune response recognizes antigens of the infecting agent by production of antibodies and generation of antigen-specific t lymphocytemediated cellular immunity. the antigens that are recognized range from epitopes that are present on only one species, subspecies, or strain to antigens that are shared by agents in a genus, family, or larger group. the patient's antibodies to particular antigens that are detected and measured in the serology laboratory frequently identify the agent of the infection. various serological tests employ the methods of immunofluorescence, enzyme immunoassay, agglutination, western immunoblot, or others. the drawbacks and pitfalls of the serological diagnostic approach include the frequent absence of antibodies in serum at the time of the patient's presentation for medical diagnosis early in the course of illness prior to their production, the occurrence of cross-reactive antibodies stimulated at an earlier time by other organisms that may be misinterpreted as diagnostic of the current illness, the occurrence of persistent specific antibodies stimulated by a previously resolved infection, and misinterpretation of the reported results owing to lack of knowledge of the levels of antibodies that are generally considered diagnostic. although it is sometimes possible to observe infectious organisms by direct examination of a clinical specimen macroscopically (e.g., worms passed in feces) or microscopically (e.g., t. pallidum by dark-field microscopy of a genital lesion of suspected syphilis), diagnostic evaluation usually begins with a stained smear of sputum, cerebrospinal fluid, urine, wound exudate, or other specimen. the pathologist examines microscopic sections stained routinely with hematoxylin and eosin (h&e). large organisms such as helminths are readily observed and can be identified by their specific morphological characteristics by one with particular expertise. routinely stained sections reveal brown-pigmented fungi, which stand out from the pink and blue of eosin and hematoxylin. table henle-koch postulates for the demonstration that a microorganism causes a disease . the organism occurs in every case of the disease in question and under circumstances that can account for the pathological changes and clinical course of the disease . it occurs in no other disease as a fortuitous and nonpathogenic parasite . after being fully isolated from the body and repeatedly grown in pure culture, it can induce the disease anew some eukaryotic protozoa and fungi can be detected and identified by the presence of pigment (e.g., some malarial parasites), subcellular structures (e.g., kinetoplasts of leishmania), or particular size, reproductive process, and cell wall structure (e.g., broad-based budding and refractile cell wall of blastomyces dermatitidis and large sporangiophores containing endospores of coccidioides immitis). some viruses cause pathological changes in the infected cells such as the formation of syncytial multinucleated giant cells and accumulations of proteins often of viral origin that are stained with eosin or hematoxylin (viral inclusions) in the nucleus or cytoplasm. recognition of the cytopathic effects and eosinophilic or basophilic cytoplasmic or intranuclear inclusions can lead to a specific diagnosis such as rabies or cytomegalovirus infection or point to a group of viruses that cause similar pathological changes such as herpes simplex viruses and and varicella-zoster virus. more often, infectious organisms are detected in smears of clinical samples or histological sections that have been stained by methods such as the gram stain that color gram-positive bacteria dark blue and gram-negative bacteria pink. the morphology of the bacteria as cocci or bacilli further characterizes the agent. the acid-fast stain is particularly useful for detecting mycobacteria and diagnosing tuberculosis. bacteria that stain poorly or not at all by the gram method can be visualized after silver impregnation staining (e.g., the warthin-starry method), which can detect treponema, legionella, leptospira, and indeed nearly all bacteria. a separate silver-staining technique (e.g., gomori's method) is very useful for detecting fungal yeast and hyphae. the only pathogenic fungus with a mucin capsule, cryptococcus, is identified by a stain such as mucicarmine. the most powerful tool for specific microscopic diagnosis of infectious diseases is immunohistochemistry. antibodies to antigens of particular bacteria, viruses, fungi, and protozoa have been produced, standardized, and made commercially available to react with the agents after appropriate processing of the tissue sections. after incubation of the antibody with the appropriately prepared microscopic section, the unbound antibody is washed away. only antibody bound to the infectious agent's antigens remains bound to the tissue section. the bound antibody is reacted with a series of reagents that deposit a colored precipitate on the antigen, allowing the detection, anatomical localization, and morphological analysis that has the ability to identify the agent and establish the diagnosis. the location of the agent is often very particular, and the expected size and shape of an organism add weight to confidence in the diagnosis. each infectious species contains a unique genome, dna for bacteria, fungi, protozoa, and some viruses and rna for the other viruses. transcription of the genetic dna into messenger rna results in many copies of some rna sequences. in situ hybridization is the method of detecting specific dna or rna sequences by annealing complementary dna or rna containing a label that is used to generate a visible product at the location of the target nucleic acid sequence in infected tissues. those powerful techniques have not been exploited for the diagnosis of infectious diseases as extensively as immunohistochemistry. however, they are very effective and can be used to distinguish among even closely related organisms. detection of dna or rna with nucleic acid sequence of a specific infectious agent powerful methods for the amplification of the nucleic acid sequences of few genomes of a bacterium, virus, or other agent to produce and detect millions of copies of the targeted sequence have provided a sensitive approach to the diagnosis of infections. the key to the technique is polymerase chain reaction (pcr), a heat-stable dna polymerase from organisms that reside in thermal springs. thirty or more thermal cycles of dna synthesis initiated by primers with specific dna sequences yield logarithmic increase in the particular dna fragment. for rna genomes such as those of rna viruses, a step of enzymatic conversion of rna to dna is performed using reverse transcriptase. identification of a novel, causative agent of a previously unknown infectious disease is a special challenge. in , the us surgeon general stated, "it's time to close the book on infectious diseases. the war on infectious diseases is over, and we have won." it was believed for the next quarter of a century that vaccines and antibiotics had controlled infectious diseases. in reality, approximately previously unknown agents of infectious diseases have been identified beginning in with marburg virus, the original agent of an african viral hemorrhagic fever. because it is clear that the emergence of infectious diseases will be a continuing occurrence, medical personnel should understand how these mysteries are solved. there are four steps in the discovery of emerging pathogens: ( ) awareness of the presence of an unknown disease, ( ) detection of an infectious agent in association with the disease, ( ) determination that the agent causes the disease (koch's postulates revisited), and ( ) determination that the etiologic agent is novel. awareness of an unknown disease may occur because of the abrupt onset of a cluster of cases of a severe illness or recognition of distinctive gross or microscopic pathological lesions. severe acute respiratory syndrome caused by a novel coronavirus is a recent example. in contrast, a syndrome of unknown etiology may have been well defined clinically for many years prior to discovery of an infectious etiology. gastric and duodenal ulcers caused by helicobacter pylori and cat scratch disease caused by bartonella henselae are examples of such diseases for which a microbial agent was unsuspected or long elusive, respectively. the methods for the initial detection of a previously unknown agent have included microscopy, bacterial culture, cell culture, animal inoculation, electron microscopy, cross-reaction of antigens in serological tests, immunohistochemistry, and pcr amplification followed by dna sequencing of the products. these are the same methods used to diagnose infection experimentally and characterize the agents. the methods used to support the etiologic role of the pathogen have included experimental animal infection, self-experimentation, laboratory accident, and demonstration of the development of a specific immune response to the agent. demonstration that the pathogen is novel includes classical biochemical phenotyping, antigenic analyses, dna or rna gene sequencing, unique ultrastructural morphology, and identification of a novel toxin. the field of emerging infectious diseases is where diagnostic infectious diseases and basic science meet to solve current biomedical infectious challenges. the roles of the astute clinician, pathologist, and laboratory technologist have been important. knowledge of microbiology, virology, immunology, molecular biology, and both cutting-edge and classic technologies has been critical to effective application to the discovery of novel pathogens. often, identification of the genus of the agent allows the selection of effective antimicrobial treatment that would have been previously unknown. the payoff of knowledge is noteworthy. pathology of infectious diseases infectious diseases: atlas, cases, text. the mcgraw-hill companies emerging pathogens: challenges and success of molecular diagnostics immunohistochemistry of infectious diseases diagnosing emerging and re-emerging infectious diseases: the pivotal role of the pathologist koneman's color atlas and textbook of diagnostic microbiology infectious disease pathology: clinical cases detection of infection or infectious agents by use of cytologic and histologic stains key: cord- - cckppk authors: klimek, ludger; pfaar, oliver; worm, margitta; eiwegger, thomas; hagemann, jan; ollert, markus; untersmayr, eva; hoffmann-sommergruber, karin; vultaggio, alessandra; agache, ioana; bavbek, sevim; bossios, apostolos; casper, ingrid; chan, susan; chatzipetrou, alexia; vogelberg, christian; firinu, davide; kauppi, paula; kolios, antonios; kothari, akash; matucci, andrea; palomares, oscar; szépfalusi, zsolt; pohl, wolfgang; hötzenecker, wolfram; rosenkranz, alexander r.; bergmann, karl-christian; bieber, thomas; buhl, roland; buters, jeroen; darsow, ulf; keil, thomas; kleine-tebbe, jörg; lau, susanne; maurer, marcus; merk, hans; mösges, ralph; saloga, joachim; staubach, petra; jappe, uta; rabe, klaus f.; rabe, uta; vogelmeier, claus; biedermann, tilo; jung, kirsten; schlenter, wolfgang; ring, johannes; chaker, adam; wehrmann, wolfgang; becker, sven; freudelsperger, laura; mülleneisen, norbert; nemat, katja; czech, wolfgang; wrede, holger; brehler, randolf; fuchs, thomas; tomazic, peter-valentin; aberer, werner; fink-wagner, antje-henriette; horak, fritz; wöhrl, stefan; niederberger-leppin, verena; pali-schöll, isabella; pohl, wolfgang; roller-wirnsberger, regina; spranger, otto; valenta, rudolf; akdis, mübecell; matricardi, paolo m.; spertini, françois; khaltaev, nicolai; michel, jean-pierre; nicod, larent; schmid-grendelmeier, peter; idzko, marco; hamelmann, eckard; jakob, thilo; werfel, thomas; wagenmann, martin; taube, christian; jensen-jarolim, erika; korn, stephanie; hentges, francois; schwarze, jürgen; o´mahony, liam; knol, edward f.; del giacco, stefano; chivato pérez, tomás; bousquet, jean; bedbrook, anna; zuberbier, torsten; akdis, cezmi; jutel, marek title: use of biologicals in allergic and type- inflammatory diseases during the current covid- pandemic: position paper of Ärzteverband deutscher allergologen (aeda)(a), deutsche gesellschaft für allergologie und klinische immunologie (dgaki)(b), gesellschaft für pädiatrische allergologie und umweltmedizin (gpa)(c), Österreichische gesellschaft für allergologie und immunologie (Ögai)(d), luxemburgische gesellschaft für allergologie und immunologie (lgai)(e), Österreichische gesellschaft für pneumologie (Ögp)(f) in co-operation with the german, austrian, and swiss aria groups(g), and the european academy of allergy and clinical immunology (eaaci)(h) date: - - journal: allergol select doi: . /alx e sha: doc_id: cord_uid: cckppk background: since the beginning of the covid- pandemic, the treatment of patients with allergic and atopy-associated diseases has faced major challenges. recommendations for “social distancing” and the fear of patients becoming infected during a visit to a medical facility have led to a drastic decrease in personal doctor-patient contacts. this affects both acute care and treatment of the chronically ill. the immune response after sars-cov- infection is so far only insufficiently understood and could be altered in a favorable or unfavorable way by therapy with monoclonal antibodies. there is currently no evidence for an increased risk of a severe covid- course in allergic patients. many patients are under ongoing therapy with biologicals that inhibit type immune responses via various mechanisms. there is uncertainty about possible immunological interactions and potential risks of these biologicals in the case of an infection with sars-cov- . materials and methods: a selective literature search was carried out in pubmed, livivo, and the internet to cover the past years (may – april ). additionally, the current german-language publications were analyzed. based on these data, the present position paper provides recommendations for the biological treatment of patients with allergic and atopy-associated diseases during the covid- pandemic. results: in order to maintain in-office consultation services, a safe treatment environment must be created that is adapted to the pandemic situation. to date, there is a lack of reliable study data on the care for patients with complex respiratory, atopic, and allergic diseases in times of an imminent infection risk from sars-cov- . type- -dominant immune reactions, as they are frequently seen in allergic patients, could influence various phases of covid- , e.g., by slowing down the immune reactions. theoretically, this could have an unfavorable effect in the early phase of a sars-cov- infection, but also a positive effect during a cytokine storm in the later phase of severe courses. however, since there is currently no evidence for this, all data from patients treated with a biological directed against type immune reactions who develop covid- should be collected in registries, and their disease courses documented in order to be able to provide experience-based instructions in the future. conclusion: the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyps, and spontaneous urticaria should be continued as usual in patients without suspected infection or proven sars-cov- infection. if available, it is recommended to prefer a formulation for self-application and to offer telemedical monitoring. treatment should aim at the best possible control of difficult-to-control allergic and atopic diseases using adequate rescue and add-on therapy and should avoid the need for systemic glucocorticosteroids. if sars-cov- infection is proven or reasonably suspected, the therapy should be determined by weighing the benefits and risks individually for the patient in question, and the patient should be involved in the decision-making. it should be kept in mind that the potential effects of biologicals on the immune response in covid- are currently not known. telemedical offers are particularly desirable for the acute consultation needs of suitable patients. course in allergic patients. many patients are under ongoing therapy with biologicals that inhibit type immune responses via various mechanisms. there is uncertainty about possible immunological interactions and potential risks of these biologicals in the case of an infection with sars-cov- . materials and methods: a selective literature search was carried out in pubmed, livivo, and the internet to cover the past years (may -april ). additionally, the current german-language publications were analyzed. based on these data, the present position paper provides recommendations for the biological treatment of patients with allergic and atopy-associated diseases during the covid- pandemic. results: in order to maintain in-office consultation services, a safe treatment environment must be created that is adapted to the pandemic situation. to date, there is a lack of reliable study data on the care for patients with complex respiratory, atopic, and allergic diseases in times of an imminent infection risk from sars-cov- . type- -dominant immune reactions, as they are frequently seen in allergic patients, could influence various phases of covid- , e.g., by slowing down the immune reactions. theoretically, this could have an unfavorable effect in the early phase of a sars-cov- infection, but also a positive effect during a cytokine storm in the later phase of severe courses. however, since there is currently no evidence for this, all data from patients treated with a biological directed against type immune reactions who develop co-vid- should be collected in registries, and their disease courses documented in order to be able to provide experience-based instructions in the future. conclusion: the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyps, and spontane-ous urticaria should be continued as usual in patients without suspected infection or proven sars-cov- infection. if available, it is recommended to prefer a formulation for self-application and to offer telemedical monitoring. treatment should aim at the best possible control of difficult-to-control allergic and atopic diseases using adequate rescue and add-on therapy and should avoid the need for systemic glucocorticosteroids. if sars-cov- infection is proven or reasonably suspected, the therapy should be determined by weighing the benefits and risks individually for the patient in question, and the patient should be involved in the decision-making. it should be kept in mind that the potential effects of biologicals on the immune response in covid- are currently not known. telemedical offers are particularly desirable for the acute consultation needs of suitable patients. the clinical symptoms of infection with the novel coronavirus (severe acute respiratory coronavirus ; sars-cov- ) became known as the "coronavirus disease (covid- )" on february , [ ] . the international committee on taxonomy of viruses (ictv) called this novel human pathogenic virus sars-cov- [ ] . the global spread of the sars-cov- pandemic and patients with severe covid- courses pose a major challenge to healthcare systems worldwide. the coronavirus that caused the severe acute respiratory syndrome (sars-cov) in / has approximately an % nucleotide sequence identity with sars-cov- [ ] . sars-cov- is a betacoronavirus of the subgenus sarbecovirus, subfamily orthocoronavirinae, and the th member of the coronaviridae family that can infect humans. it can be isolated from human samples obtained from respiratory secretions, nasal and pharyngeal swabs and isolated on cell cultures [ , , ] . it is covered by a lipid membrane that can be disrupted by detergents and is different from the middle east respiratory syndrome-related coronavirus (mers-cov), from sars-cov, and from the coronaviruses responsible for the common cold ( e, oc , nl , and hku ) [ ] . abbreviations. angiotensin-converting enzyme the incubation period after an infection with sars-cov- can be of up to days, during which the infected person can be asymptomatic but nevertheless transmit the virus. in a high number of patients, the infection leads to symptoms of the upper and lower airways, and, less frequently, also of other organ systems (nervous system, gastrointestinal tract, kidneys, blood vessels). in the worst case scenario, multi-organ failure and respiratory failure can result, as has been described for other coronavirus infections (sars-cov- , mers-cov) [ , , ] . in more severe cases, infection with sars-cov- can lead to pneumonia, severe acute respiratory syndrome, renal failure, and death [ , , , , ] . higher age and comorbidities such as chronic airway diseases (particularly copd), diabetes mellitus, cardiovascular diseases, obesity, and immune deficiency of various origins have been described as risk factors of a severe course [ , , , ] . the need for intensive care treatment and invasive ventilation is associated with high mortality. we will present clinical and immunological aspects that have to be considered with regard to the covid- pandemic in patients treated with biological therapy against ige and mediators of type inflammation (table ) . the characteristics of the immune response after infection with sars-cov- are still insufficiently understood. while various forms of the course of covid- and the infection with the virus have been described, it is still unclear which immunological background influences the course of the disease. this is also true for the role of the innate table . recommendations on treatment with biologicals during the covid- pandemic in patients with asthma, atopic dermatitis, urticaria, or crswnp. recommendations for biological treatment in non-infected patients during the covid- pandemic or in patients who have recovered from covid- infection termination of biological treatment is not generally necessary; and biologicals should be continued as scheduled, particularly in severe cases, based on an individual risk-benefit analysis. in mild-to-moderate covid- courses, or when sars-cov- infection is suspected, biologicals can be continued in the indications discussed here if a patient-based risk-benefit analysis supports the decision and the patient consents after having been informed about the limited availability of data. prolongation of the injection interval can be considered (as indicated in the summary of product characteristics) to limit the necessary physician-patient contacts to a minimum. in severe covid- courses, prolongation of the injection interval (as indicated in the summary of product characteristics) or treatment interruption should be considered in the indications discussed here. the risk of the possible requirement of systemic glucocorticosteroids must be considered. biological treatment can be continued during the current covid- pandemic in asymptomatic patients with negative pcr tests, in patients without known exposure or contact with sars-cov- -positive people, and in patients who have completed an adequate quarantine period. in a quarantine situation, telemedical support might be feasible, in particular with the aim of continuing the basic therapy with topical steroids, inhaled bronchodilators, antihistamines, etc. in accordance with the relevant guideline recommendations or with the aim to expand those therapies according to the patient's needs. biological therapy in patients without evidence of sars-cov- infection can be started for approved indications during the current covid- pandemic, self-administration of biologicals should generally be preferred; this is made easier if user-friendly pen systems for self-application are available. adequate patient training is required. practices and allergy centers must be prepared for the current covid- pandemic by following the recommendations of the who and of national and regional authorities. these recommendations should be continuously updated and adapted to new scientific findings and recommendations made by authorities. crswnp = chronic rhinosinusitis with nasal polyps. and adaptive immune system with regard to sars-cov- infection. while natural killer (nk) cells traditionally play an important role in the early phase of viral infections, cd + t helper cells come into action in the subsequent phase [ ] . early antibody secretion and production in the mucosa-associated lymphatic tissue initially include antigenspecific igm, iga, and, later, igg antibodies, and are essential for immune response [ , , ] . macrophages are activated and secrete inflammatory cytokines, with type interferons (type ifn) playing the most prominent role. in infections with other coronaviruses (e.g., sars-cov- ), type ifn is responsible for the adequate initiation of the immune reaction, and patients with delayed or insufficient ifn production have a more severe disease course [ ] . the activation of apoptosis or pyroptosis in epithelial cells serves as an antiviral defense, but excessive immune reactions can also contribute to local tissue damage through synergistic effects [ ] . an excessive production of pro-inflammatory cytokines has already been observed in sars-cov- , mers-cov, and recently also in sars-cov- infections, and has been described as a cytokine storm [ , ] . natural igm, and probably also mannose-binding lectin (mbl), are believed to be the first line of defense against sars-cov- [ ] . these antibodies and mbl recognize glycans and are abundant in children and young adults. however, they decrease dramatically with age and are over times lower at the age of ≥ than at the age of - years [ ] . as they are part of the innate immunity, they are the only antibodies able to recognize sars-cov- before the adaptive immune response is initiated [ ] . if the virus enters the lungs early enough, it can replicate in an unhindered manner, as no or only little resistance exists. the resulting inflammation with a massive activation of local mediators (complement and coagulation cascades interleukin- (il- ), cytokine storm) can cause damages that lead to complications or, in some cases, even to death [ ] . furthermore, the ability of sars-cov- viruses to penetrate the cell via receptors such as ace and tmprrss could explain the different severities of covid- in different patient groups [ ] . extensive damage to the lungs leads to rapid clinical deterioration and usually to the need for intensive care treatment, which can be observed typically - days after infection. the risk of kidney, liver, and/or other organ damage as well as of consumption coagulopathy is significantly increased. affected patients usually have highly increased interleukin (il)- β, il- , il- , and tnf-α levels ( figure ) [ ] . the therapeutic blockade of one or more of these cytokines has been discussed as a potential future therapy option for severely affected patients in whom il- can be massively increased [ ] . il- plays a central role in the cytokine storm, and tocilizumab has already been used as a biological with anti-il- effects in covid- [ , ] . approved indications for anti-il- or anti-il- r antibodies (such as tocilizumab, sarilumab) currently include, for example, rheumatoid arthritis, juvenile rheumatoid arthritis, and castleman disease. the immune reactions of type and type described here are contained by other cytokines, such as il- and tgf-β, and type inflammation could possibly counteract the cytokine storm. increased levels of eosinophilic granulocytes, as one of the key cells of type inflammation, have been ascribed a protective effect in severe viral infections, although the mechanism of action has not yet been identified [ ] . on the other hand, low blood eosinophil counts could simply reflect the severity of the infection. the interaction of sars-cov- with its receptor on the cells of the respiratory system, the membrane-bound angiotensin-converting enzyme (ace ), which is responsible for the entry of the virus into the host cell, is far better investigated [ ] . therefore, the reduced expression of ace in the airway epithelial cells of patients with allergic asthma is being discussed as a potentially protective factor against sars-cov- infection [ ] . it can be assumed that only the interaction of the individual cytokine responses leads to an adequate and effective immune response in coronavirus infections. however, imbalances between type , type , and type reactions might have a significant negative or positive impact on the course of the viral infection ( figure ). figure . sequence of immunological events in sars-cov- infection: if sars-cov- infects cells via the surface receptors ace and tmprss , this leads to active replication and release of the virus, the cells decay through pyroptosis. damps are released that are recognized by neighboring epithelia, endothelia, and alveolar macrophages and trigger the release of pro-inflammatory cytokines such as il- , ip- , mip α, mip β, and mcp . this attracts monocytes, macrophages, and t cells, which, when ifn-γ is added, activate another inflammatory, self-reinforcing cascade. in defective immune responses (left), this can lead to accumulation of immune cells and overproduction of pro-inflammatory cytokines, which then damage the lung and may lead to a cytokine storm with multi-organ failure. additionally, non-neutralizing antibodies produced by b cells may enhance the infection and lead to further organ damage. in a healthy immune response (right), the initial inflammation attracts virus-specific t cells to the infection site where they can eliminate the infected cells before the virus spreads further. neutralizing antibodies selectively block the virus, alveolar macrophages recognize and phagocytize affected cells and neutralized viruses. altogether, these processes clear the viral infection with minimal damage of respiratory tissue and lead to recovery. reproduced from tay et al. [ ] . ace = angiotensin-converting enzyme ; ade = antibodydependent enhancement; damp = damage-associated molecular pattern; g-csf = granulocyte colonystimulating factor; ifn = interferon; il = interleukin; mip α = macrophage inflammatory protein α; sars-cov- = severe acute respiratory syndrome coronavirus ; tnf = tumor necrosis factor. so far, there is insufficient evidence to indicate which risk factors cause a severe course of covid- . a history of lung disease has been considered a potential risk factor for developing covid- and possibly also for a severe course. bronchial asthma, which is the most important allergic indication for biologicals targeting ige and type inflammation, is possibly one of these diseases. however, since many patients with pulmonary disease also have other comorbidities, some of the suspected factors could turn out to be confounders once further studies are carried out. thus, it remains unclear whether patients with type- -associated bronchial asthma without any other possible risk factors should be considered high risk for a severe covid- course. the currently available data rather contradict this. there is only limited data on covid- in connection with a type- -associated disease, and its prognostic value is very limited. the currently available studies do not indicate an increased risk for patients with allergies, asthma, or other atopy-associated diseases ( table ) [ , , , , , , ] . for example, in wuhan and italy, the percentage of seriously ill or deceased covid- patients with known bronchial asthma was far below the prevalence of asthma in these places [ ] . it also remains unclear why in many patients not only lymphopenia but also eosinopenia was detected at the time of admission [ ] . neither decreased nor increased eosinophil levels have so far been clearly associated with certain clinical courses of sars-cov- infection. in recent years, several biologicals have been approved in europe that block ige antibodies or the interleukins il- , il- , and il- , which are relevant in type inflammation, or their receptors [ , , , , , , , , , ] . omalizumab has been approved for the treatment of severe allergic bronchial asthma in adults and in children older than years with proven sensitization against a perennial airborne allergen and reduced lung function. another indication is antihistamine-resistant chronic spontaneous urticaria in adults and in adolescents older than years. mepolizumab, benralizumab, and reslizumab are report of patients taking oral glucocorticosteroids because of breathing difficulties due to covid- and known asthma who were hospitalized week later with acute respiratory insufficiency wang et al. [ ] (wuhan, china) of studied patients had asthma zhang et al. [ ] (wuhan, china) study of patients of whom had chronic urticaria, had asthma, and had unclear adverse drug reactions grasselli et al. [ ] (lombardy, italy) study including , , of whom had a history of: bronchial asthma, anemia, inflammatory bowel disease, chronic respiratory insufficiency, endocrine disorders, chronic pancreatitis, diseases of the connective and supporting tissue, organ transplantation, epilepsy, neurological disease (reported as "other" in the study) dreher et al. [ ] (aachen, germany) result: covid- patients with a history of respiratory disease develop ards more frequently ( vs. %; vs. patients; of these, vs. patients with asthma; n = ) ards = acute respiratory distress syndrome. il- or il- receptor blockers, available for adults and, partially, also for children. dupilumab has been approved for the treatment of: (i) atopic dermatitis in adolescents older than years, (ii) severe type- -dominant asthma in adults and (iii) chronic rhinosinusitis with nasal polyps (crswnp) in adults. table shows the situation of approval in germany, austria, luxembourg, and switzerland. self-administration by the patient is listed separately as it significantly facilitates care for suitable patients during the sars-cov- pandemic. viral infections of the upper and lower airways have been associated with the development and exacerbation of allergic disease [ , , ] . infection and the persistence of virus particles in the mucosa could inhibit the efficacy of the local innate immune system and promote type inflammation. the blocking of type inflammation by therapeutic antibodies against ige, il- , or the il- or il- /- receptors has so far not been suspected to increase the risk of viral infections. however, il- has a dual role in viral infections due to two different haplotypes in the il- gene. it can promote infections with the herpes virus and the norovirus [ ] as well as with the ebola virus, which is related to the coronavirus [ ] . on the other hand, il- can also inhibit viral infections by promoting innate immunity [ , , ] . thus, more evidence from clinical observations is necessary to be able to provide clear recommendations with regard to covid- . table gives an overview of the frequency of viral infections occurring as adverse events in trials on these monoclonal antibodies. there have been reports on the lower incidence of viral infections under anti-ige treatment with omalizumab, since this therapy may increase the functionality and the production of ifn-α by plasmacytoid dendritic cells (pdc). this leads to an enhanced antiviral defense and to a reduction of virus-induced asthma exacerbations [ , ] . also, for type blockade with anti-il- antibodies (mepolizumab, reslizumab) or anti-il- receptor antibodies (benralizumab), the risk of respiratory viral infections in the active-agent study groups was equal to or lower than the risk in the placebo groups (table ). it has not yet been clarified whether the blockade of type inflammation or of ige influences the risk of developing covid- or its course. in the case of a cytokine storm, possible negative effects induced by blocking the type immune response situation are conceivable; but these effects require further investigation. the first reports show that the disease course is not worse in covid- patients with eosinophilic diseases under biological therapy [ , ] . however, further study results should be awaited, especially considering the fact that sars-cov- changes rapidly due to mutations [ ] . meta-analyses by agache et al. [ , , ] have shown a slightly increased rate (low to medium risk of association) of adverse events when anti-il- / r, anti-il- / r, and anti-ige are used in severe asthma, in- dependently of covid- . thus, there is no clear recommendation regarding the decision-making to continue or temporarily interrupt a biological therapy during an infection with sars-cov- . treatment interruption could entail the risk of suboptimal control of the allergic disease or, in the case of exacerbations, the need for systemic glucocorticosteroids, for which an increased risk of a possibly more severe covid- course has been described [ ] . recommendations for the management of allergic/atopyassociated diseases under anti-type- therapy during the covid- pandemic (table ) to ensure an appropriate, high-quality, and accurate care for patients on anti-type- treatment with underlying atopic-eosinophilic or allergic disease, antibody therapy should be continued and remain unchanged during the ongoing pandemic when there is no evidence of sars-cov- infection. to cope with the current shortfalls in hospitals and the more difficult hygiene conditions, telephone or telemedical follow-up should be considered in suitable patients when technical and medical requirements allow for it. for this purpose, comprehensive patient training with regard to documentation of the disease activity and, where applicable, to self-administration of the medication is desirable. this is facilitated by the partial availability of user-friendly pen systems for self-application. in general, in countries with low infection numbers and a consequent relaxation of covid- -associated restrictions, there is no contraindication for starting biological therapy in patients without evidence of a current sars-cov- infection. according to the current state of knowledge, biological therapy for the indications discussed here can be continued in mild to moderate cases of sars-cov infection/ covid- disease, if an individual consideration of risks and benefits supports this decision. the risks and benefits have to be assessed by a specialist, and it is recommended to in-form the patient about the fact that only limited data are available. in severe courses of covid- , prolongation of the dosing interval or treatment interruption should be considered. when doing so, the risk of the potential requirement of treatment with systemic glucocorticoids should also be taken into account. in a quarantine situation, a telemedical approach might be feasible, in particular with the aim of continuing or expanding the basic therapy with topical steroids, inhaled bronchodilators, antihistamines, etc. in accordance with the relevant guideline recommendations [ , , , , , , ] . if hospitalization due to the exacerbation of asthma-or type- -associated diseases becomes necessary, current guidelines on diagnosis and treatment must be followed. sinus surgery for crswnp should, if possible, be delayed in patients with suspected or confirmed covid- disease. in the case of urgently indicated systemic therapy for severe atopic dermatitis, consideration should be given to therapy with either biologicals, classic immunosuppressants, or systemic glucocorticosteroids, although systemic glucocorticosteroids are not recommended due to their broad immunosuppressive effect (see above). for cyclosporin a (cya) as an approved therapeutic option for atopic dermatitis, in vitro studies have suggested antiviral effects [ ] . t-celldirected immunosuppression performed after organ transplantation (cya, tacrolimus) is being discussed as a possible protective factor against serious clinical complications of sas-cov- infection [ ] , as well as the use of cya in covid- [ , ] . however, reliable clinical data have not yet been published. possible metabolic interactions between cya and lopinavir/ritonavir (cyp inhibitors) have to be taken into account. severe covid- courses have been reported in two patients with atopic dermatitis treated with dupilumab [ ] . the currently available data suggest that the risk of developing a severe course of covid- is probably not increased in patients with allergies and atopy-associated diseases. however, there is a lack of study results including subgroup analyses on seriously ill atopy patients and their treatment. the effects of ige or type inflammation blocking on sars-cov- infection have not yet been clarified. in cases of a mild to moderate covid- course, we advise to continue biological therapy for the indications mentioned here, if the patient-based assessment of the benefits and risks supports this approach and if the patient agrees after adequate information about the limited availability of data. in severe courses of covid- , prolongation of the dosing interval or treatment interruption should be considered for the indications discussed here. this assessment should be patient-based and should consider the risk of the possible requirement of systemic glucocorticosteroids. in all other patients, in whom neither a suspected nor a proven sars-cov- infection is present, the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, crswnp, and spontaneous urticaria can be continued unchanged or can be re-started in the current sars-cov- pandemic. the use of telemedicine for treatment support and patient education is recommended and can facilitate the continuation of biological administration by self-injection. s. korn received speaker and personal adviser's fees from astrazeneca, gsk, novartis, and sanofi outside the submitted work. u. darsow was a lecturer, principal investigator, and consultant for alk abello, bencard, and novartis pharma outside the submitted work. o. palomares received research grants and/ or personal fees from allergy therapeutics, amgen, astrazeneca, diater, glaxosmithkline, s.a., inmunotek s.l., novartis, sanofi-genzyme, and stallergenes outside the submitted work; he was a member of advisory boards of novartis and sanofi-genzyme. t. biedermann was a consultant to or received personal lecture fees or research grants from alk-abelló, celgene-bms, lilly deutschland gmbh, mylan, novartis, phadia-thermo fisher, sanofi-genzyme, regeneron. r. valenta received research grants from viravaxx, vienna, austria, and from hvd life sciences, vienna, austria, and acts as a consultant for viravaxx. r. buhl received personal lecture and/or consultant fees from astrazeneca, boehringer ingelheim, chiesi, cipla, novartis, roche, sanofi, and teva as well as research support for universitätsmedizin mainz from boehringer ingelheim, glaxosmithkline, novartis, and roche, outside the submitted work. r. brehler received personal fees for lectures and/or consultancy and/or clinical studies from alk, allergopharma, almirall, astra zeneca, bencard, gesellschaft zur förderung der dermatologischen forschung und fortbildung e.v., gesellschaft für information und organisation mbh, gsk, dr. pfleger, hal, leti, merck, novartis, oto-rhino-laryngologischer verein, pierre fabre, pohl boskamp, stallergenes, thermo-fischer, biotech tools, genentech, circassia. a. bossios reports personal consultant and/or lecture fees from novartis, astrazeneca, gsk, and teva outside the submitted work. j. bousquet reports personal fees from chiesi, cipla, hikma, menarini, mundipharma, mylan, novartis, sanofi-aventis, takeda, teva, uriach, kyomed-innov, and purina outside the submitted work. j. schwarze received personal fees from mylan, f f events; support from industry for educational acivities of the scottish allergy and respiratory academy as well as the children and young people's allergy network scotland outside the submitted work; support from industry for eaaci; j. schwarze is eaaci secretary general - . j. hagemann received speaker fees from novartis pharma. m. wagenmann received personal consultant and/or speaker fees from alk-abelló, allergopharma, astrazeneca, bencard-allergie, genzyme, hal-allergie, infectopharm, leti pharma, meda pharma, novartis, sanofi aventis, stallergenes, teva. l. klimek reports grants and/or personal fees from allergopharma, meda/mylan, hal allergie, alk abelló, leti pharma, stallergenes, quintiles, sanofi, asit biotech, lofarma, allergy therapeut., astra-zeneca, gsk, inmunotk outside the submitted work; he is a member of the following organizations: aeda, dghno, deutsche akademie für allergologie und klinische immunologie, hno-bv gpa, eaaci. coronaviridae study group of the international committee on taxonomy of viruses. the species ssevere acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china clinical features of patients infected with novel coronavirus in wuhan pathogenic human coronavirus infections: causes and 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or an inhaled corticosteroid boost to prevent fall asthma exacerbations no evidence of increased risk for covid- infection in patients treated with dupilumab for atopic dermatitis in a high-epidemic area -bergamo sars-cov- immunogenicity at the crossroads. allergy. . epub ahead of print efficacy and safety of treatment with dupilumab for severe asthma: a systematic review of the eaaci guidelines-recommendations on the use of biologicals in severe asthma efficacy and safety of treatment with biologicals (benralizumab, dupilumab, mepolizumab, omalizumab and reslizumab) for severe eosinophilic asthma. a systematic review for the eaaci guidelines -recommendations on the use of biologicals in severe asthma stellungnahme zur anwendung von glukokortikosteroiden bei entzündlichen erkrankungen der oberen atemwege (u. a. allergische rhinitis/chronische rhinosinusitis) während der aktuellen covid- -pandemie -empfehlungen des Ärzteverbandes deutscher allergologen (aeda), des deutschen berufsverbandes der hno-Ärzte (bvhno) und der ags klinische immunologie eaaci guidelines on allergen immunotherapy: house dust mite-driven allergic asthma guideline for the diagnosis and treatment of asthma -guideline of the german respiratory society and the german atemwegsliga in cooperation with the paediatric eaaci guidelines on allergen immunotherapy: hymenoptera venom allergy cyclophilins and cyclophilin inhibitors in nidovirus replication immunosuppression drug-related and clinical manifestation of coronavirus disease : a therapeutical hypothesis epub ahead of print cyclosporine therapy during the covid- pandemic is not a reason for concern role of cyclophilin a during coronavirus replication and the antiviral activities of its inhibitors safety of dupilumab in severe atopic dermatitis and infection of covid- : two case reports the trinity of covid- : immunity, inflammation and intervention considerations on biologicals for patients with allergic disease in times of the covid- pandemic: an eaaci statement praxis für kinderpenumologie/allergologie am kinderzentrum dresden (kid) praxis und klinik für dermatologie/allergologie am schwarzwald-baar klinikum germany klinische abteilung für allgemeine hno division of allergy and immunology klinik für dermatologie, allergologie und venerologie medizinische hochschule hannover, hno-klinik key: cord- -fbl jjb authors: cassini, alessandro; colzani, edoardo; pini, alessandro; mangen, marie-josee j; plass, dietrich; mcdonald, scott a; maringhini, guido; van lier, alies; haagsma, juanita a; havelaar, arie h; kramarz, piotr; kretzschmar, mirjam e title: impact of infectious diseases on population health using incidence-based disability-adjusted life years (dalys): results from the burden of communicable diseases in europe study, european union and european economic area countries, to date: - - journal: euro surveill doi: . / - .es. . . . - sha: doc_id: cord_uid: fbl jjb the burden of communicable diseases in europe (bcode) study aimed to calculate disability-adjusted life years (dalys) for selected diseases in the european union (eu) and european economic area (eea). methods: dalys were estimated using an incidence-based and pathogen-based approach. incidence was estimated through assessment of data availability and quality, and a correction was applied for under-estimation. calculation of dalys was performed with the bcode software toolkit without applying time discounting and age-weighting. results: we estimated that one in inhabitants experienced an infectious disease episode for a total burden of . million dalys ( % uncertainty interval (ui): . – . ) between and ; % of which was related to the acute phase of the infection and its short-term complications. influenza had the highest burden ( % of the total burden), followed by tuberculosis, human immunodeficiency virus (hiv) infection/aids and invasive pneumococcal disease (ipd). men had the highest burden measured in dalys ( % of the total), adults years of age and over had % and children less than years of age had %. age group-specific burden showed that infants (less than year of age) and elderly people ( years of age and over) experienced the highest burden. conclusions: these results provide baseline estimates for evaluating infectious disease prevention and control strategies. the study promotes an evidence-based approach to describing population health and assessing surveillance data availability and quality, and provides information for the planning and prioritisation of limited resources in infectious disease prevention and control. countries of the european union (eu) and european economic area (eea) increasingly face the challenge of how best to allocate limited resources for infectious disease prevention and control. evidence to determine priorities is often limited and epidemiological data may be unavailable, of uncertain quality or difficult to communicate to decision makers. burden of disease estimates, using composite health measures, provide clear and comprehensive information for transparent and accountable decision making and have the potential to play an important role in health policy formulation [ ] . numerous studies have addressed the challenge of estimating disease burden regionally, nationally and globally [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in high-income countries, the incidence of infectious diseases has decreased over the last century, but recent outbreaks of emerging and re-emerging diseases worldwide, such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), measles, avian and pandemic influenza, chikungunya virus, ebola virus disease (evd) and zika virus disease, have resulted in a renewed focus on infectious diseases [ ] [ ] [ ] [ ] [ ] [ ] . in addition, the traditional boundaries between non-infectious diseases and infectious diseases have become blurred as increasing evidence of the aetiological role of the latter in triggering noninfectious conditions is available [ , ] . in , the european centre for disease prevention and control (ecdc) commissioned a pilot disease burden study using seven selected infectious diseases in order to propose a methodology for a burden of disease study tailored towards infectious diseases and assess the feasibility of, and interest in, such an approach [ ] . based on this pilot, the burden of communicable diseases in europe (bcode) project was launched [ ] , funded by ecdc and implemented in collaboration with a european consortium led by the dutch national institute for public health and the environment (rivm) and consisting of academic and national health institutes from eu countries. the main objective of the bcode project was to develop a methodology to assess the impact of infectious diseases on population health in eu/eea countries. it also intended to promote an evidence-based approach to assess population health, foster analysis of surveillance data quality and availability, facilitate the communication of complex health information to decision makers, and provide a tool for the planning and prioritisation of infectious disease prevention, preparedness and control measures. to achieve these objectives, a methodology was developed [ , ] that uses a composite health measure, the disability-adjusted life year (daly) [ ] , to express the disease burden of an infectious disease in a single metric and is therefore suitable for comparing their relative burden. in line with the overall objectives of the bcode project, the specific aim of the bcode - study described in this paper was to provide a baseline the error bars indicate the % uncertainty intervals. average annual estimate of the eu/eea burden of selected infectious diseases surveyed by ecdc and measured in dalys. the methodological framework of the bcode - study was based on the bcode project [ , ] . this methodology uses an incidence-based approach with a disease progression pathway to estimate dalys, an outcome measure that describes the impact of years lived with disability (yld) following the onset of a disease and of years of life lost due to premature mortality (yll) compared with a standardised life expectancy [ ] . the incidence-based approach acknowledges current and future sequelae of infections, and sets the baseline for estimating the impact of prevention and control interventions. the disease progression model (i.e. outcome tree) links possible sequelae to an initial infection and allocates that future burden to the time of infection. to calculate dalys, the incidence of acute, symptomatic disease is a key input variable. besides the number of symptomatic infections, computation of dalys requires several additional age group and sexspecific variables. these variables include the risk of developing short-and long-term complications (health outcomes), their duration, and weights reflecting their severity. these variables are described through disease models or outcome trees, which represent the progression of a disease over time by ordering relevant health outcomes following infection and illustrating their conditional dependency [ , ] . to determine the life expectancy at age of death, we used the same standard reference life table as the global burden of disease study (gbd ) [ ] . disability weights were selected from the set developed by the european disability weight project [ ] . outcome trees, their parameters and literature reviews for each disease included in this study are described in the bcode toolkit, version . [ ] and are available in supplement . no age-weighting and time-discounting was applied. diseases for inclusion in the present bcode - study were selected from those listed in decision / /ec with amendments, which fall under the mandate of ecdc as part of its responsibilities for epidemiological surveillance in support of the identification, assessment and communication of threats to health due to communicable diseases in the eu/eea countries [ ] . the selection criteria were data availability, incidence, outbreak potential and whether the disease is preventable with widely used vaccines (supplement ). final disease selections were made by an ad hoc working group of the ecdc advisory forum, a board of experts from eu/eea countries advising the ecdc director [ ] . results represent the burden of infectious diseases in all of the eu/eea countries, except for croatia, which joined the eu in . however, due to the wide variability of data availability and/or quality across countries and in order to balance data quality and representativeness, for some diseases the estimation was based on a subset of countries. details are available in supplement . reasons for excluding countries included data availability (e.g. countries not reporting surveillance data to ecdc) and data completeness (e.g. countries reporting only aggregate or sentinel-based surveillance data but with the denominator population being unreported or unknown). age group and sex-specific demographic data were obtained from the eurostat database, [ ] . cases of diseases notified to ecdc through the european surveillance system (tessy), a database of communicable diseases cases in eu/eea countries, were used as the main data source for estimating incidence of acute infections. in order to remove the effect of large fluctuations in incidence data, for example that because of seasonality of disease or outbreaks, notified cases during five years, to , were averaged to obtain an annual notified number of incident cases. the annual number of cases was estimated in a stepwise approach, generally by multiplying the age group and sex-specific number of cases notified to ecdc by a multiplication factor adjusting for underestimation [ ] . for full details see supplement and table . in order to determine the most suitable multiplication factors, we reviewed the available tessy data. the first step involved determining the availability of notification data: which countries reported and for which years. countries not reporting or reporting limited information on sex and age of cases data were excluded from the study. the second step involved reviewing annual notification rates separately for each country, and the third step involved comparing the average rates across different countries. during these steps, together with ecdc surveillance experts, we considered surveillance systems' characteristics, including case definition, case-based vs aggregate reporting, compulsory vs voluntary reporting, comprehensive vs sentinel surveillance and whether or not the surveillance system had national coverage. notification rates were also reviewed in relation to epidemiological circumstances (e.g. outbreaks and disease exposure), reporting practices, healthcare providers' awareness, and healthcare system characteristics. for a number of diseases, i.e. campylobacteriosis, chlamydia infection, congenital toxoplasmosis, influenza, pertussis and salmonellosis, it was concluded that it was not possible to estimate the incidence from notified data and alternative methods were applied (see supplement ). in particular, no published large community study was found for influenza except for the results of the flu watch cohort study in the united kingdom [ ] , which we chose as the main data source to model the incidence of influenza in eu/eea countries. all the approaches above were explored in order to estimate the incidence of acute hepatitis c virus (hcv) infection in the general population. however, only published serological studies based on limited populations at risk were found, which would have introduced an unmeasurable bias and uncertainty and would not have allowed to estimate the incidence in the total population. therefore, we excluded hcv infection from our disease burden estimation as no reliable data on the annual incidence of acute hcv was identified. for each disease, a model was generated using the bcode toolkit. within each model, the age groupspecific and sex-specific annual number of cases, multiplication factors adjusting for underestimation and population were inserted in the software. uncertainty intervals (ui) were expressed as uniform ( values) or project evaluation and review techniques (pert) ( values) distributions; we ran the models at , iterations of the monte carlo simulations, without time discounting and age-weighting. for each disease, results included dalys per case and the following per , population: incidence, deaths, yll, yld and dalys. for all the outputs, we showed the median and the % ui. the bcode - study used a combination of aggregate health information (i.e. without personal identifiers) notified to ecdc through tessy and information stemming from the scientific literature; therefore, informed consent was not required. other information included in the study was drawn from published literature. we estimated that between and , the selected infectious diseases accounted for , cases per , population per year ( % ui: , - , ) and there were . deaths per , population annually ( % ui: . - . ) ( table ). figure ). these four top-ranking infections accounted for % of the total burden of communicable diseases in eu/eea countries. legionnaires' disease, campylobacteriosis and hepatitis b had a significantly lower burden compared to the the error bars indicate the % uncertainty intervals. four diseases discussed above. invasive haemophilus influenzae disease, invasive meningococcal disease, chlamydia, salmonellosis, pertussis and shiga toxin/ verocytotoxin-producing escherichia coli (stec/vtec) infection had an even lower burden. the remaining diseases were ranked with a significantly lower burden. yll accounted for % of the total burden ( figure ). diseases with higher incidence and mortality as compared with other diseases were found to be influenza, campylobacteriosis and salmonellosis ( figure ), although only the former has a high burden in dalys. pertussis and chlamydia have high incidence and low mortality, whereas tb, hiv/aids, ipd, legionnaires' disease, hepatitis b virus (hbv) infection and invasive haemophilus influenzae disease (ihid) had low incidence and high mortality. in terms of burden of congenital infections in the newborns, almost all the burden ( %) was attributable to toxoplasmosis, listeriosis and rubella infections ( table ). the diseases with the highest number of dalys per case, which represents the individual burden and to a certain extent the severity of the disease, were rabies and variant creutzfeldt-jakob disease, which are ultimately fatal conditions. hiv/aids, invasive meningococcal disease, listeriosis, tb, ihid, legionnaires' disease, hbv infection, ipd, congenital toxoplasmosis, tetanus and diphtheria followed, with dalys per case ranging from . to . . diseases determined to have a high individual and population burden were legionnaires' disease, ipd, hiv/aids and tb, while influenza was determined to have a low individual but high population burden (figure ). most dalys, around %, were due to infections occurring in males. considering more detailed results presented in supplement , diseases such as tb, hiv/ aids, legionnaires' disease, were found to impact mostly men while chlamydia and gonorrhoea had a higher burden in women. when considering dalys over the total population, % occurred in children less than years of age, % in individuals less than years of age and % in individuals aged years and over (see supplement ) ; most dalys were found in age groups between and year of age ( figure ). however, when considering the age group-specific dalys per , population of the age group, those with the highest overall burden were infants under one year of age and individuals years of age and over ( figure ). compared with the age groups of between and years of age (adults) and years of age and over (elderly population), the total burden of disease in the population under years of age is lower (figure ). the diseases with the highest burden in the under years age group are hbv infection, influenza, ihid, ipd and invasive meningococcal disease (imd). hiv/aids, tb and influenza are the diseases with the highest burden in the adult population, whereas influenza, ipd and tb have the highest impact in the elderly population. the acute phase of diseases had the highest impact on the total burden ( %) (see supplement ) . this was the result of the outcome trees that modelled case fatality proportions (cfp) as a direct risk to the acute infection. the high share of ylls ( % of total dalys, see table ) compared with ylds was due to the limited amount of time lived with a disability, which is typical for infectious diseases. the final ranking of the burden of disease gives a new picture of the impact of infectious diseases when compared with notification data (figure ). this study presents the estimation of the burden of selected infectious diseases in the eu/eea in dalys, adopting an incidence-and pathogen-based methodology and a consistent approach to surveillance and outcome data assessment. the results allow ranking of infectious diseases taking morbidity, disability and premature mortality resulting from acute infections and their sequelae into account. the incidence-based approach chosen for this study allows for the effect of future long-term complications of a disease to be included in the calculation of dalys, resulting in a more comprehensive estimate of the effect of prevention and control interventions [ ] . compared to a prevalence-based approach, in the incidence-based dalys, the potential future burden avoided, for example, by vaccination as a possible intervention measure, is included [ , , ] . we did not apply time discounting, which is generally applied in economic studies, because we did not consider there to be reasons justifying the decline of healthy life years over time. similarly, age-weighting was also not applied because it was considered that a healthy life year should be valued equally, irrespective of the age at which it is lived or lost. both choices are consistent with current methodologies used by the world health organization's (who) foodborne disease burden epidemiology reference group (ferg) and the global burden of disease studies [ , ] . access to healthcare varies across countries but is largely universal. although healthcare and surveillance systems vary, incidence data included in this study is mainly based on cases of disease notified to national surveillance systems and reported to tessy during years when the reporting procedures were considered to be consolidated. surveillance in the eu/eea differs in terms of purpose and systems for collecting data. this study enabled a thorough review of surveillance data availability and quality for each disease and each country. as a result, this study increases our knowledge and indicates areas for improving european infectious disease surveillance. the averaging of annual number of cases over years removed the effects of large fluctuations in incidence, i.e. flattened the effects of outbreaks. however, it could still be valuable to show the effect of an outbreak given that such can cause rankings to substantially change from the baseline. for example, the burden of disease was . dalys per , bulgarian population per year considering the measles outbreak of just under , cases [ ] . this burden would have led to this outbreak ranking fourth in our results, between hiv/aids and ipd. our study ranked influenza as the infectious disease with the highest impact on population health in the eu/ eea. although the cfp chosen for the influenza disease model was low, the incidence was significantly higher than that of any other disease included in our study ( table ). the main driver of the high burden of influenza is the contribution of premature mortality associated with the infection (yll). our study estimated a mortality of . per , population, slightly lower than the ecdc-estimated annual average influenza deaths in eu/eaa countries of . per , population (range: . - . ) within the same period based on the published figures of , deaths (range: , to , ) [ ] . similar mortality rates were published in national studies in the netherlands: . to . however, it is important to note the limitations of our estimation of dalys for influenza, namely the single incidence data source, the flu watch cohort study in the united kingdom, representing a limited geographical region [ ] which may have a different epidemiological profile and vaccination coverage from other eu/eea countries. however, the southern hemisphere influenza and vaccine effectiveness research and surveillance project (shivers) in new zealand found a very similar incidence of symptomatic influenza (personal communication, sue huang and don bandaranayake, july ). moreover, the netherlands national burden of disease study [ ] our results for influenza support the recommendations of the council of the european union [ ] , reiterated by the ecdc influenza vaccination report [ ] , to develop a national seasonal influenza vaccination action plan and to achieve target vaccination coverage for older population groups, people with chronic medical conditions, pregnant women and healthcare workers. our estimation of tb mortality rate was in line with national notified deaths of tb. for example, the eurostat mortality for tb was . per , population in the eu countries in [ ] , very much in line with our estimated rate of . per , population. our findings reinforce the need for increasing efforts in eu/eea countries to eliminate tb. hiv/aids has a high burden of disease in europe despite the low mortality risk compared with the preantiretroviral treatment era. this is reflected in the overwhelming contribution of yld to the total dalys (ca %). as significant hiv transmission continues in europe [ ] and the high associated burden found in our study highlights the need to strengthen prevention and testing efforts. this study estimated that . deaths per , population were due to hiv/aids. given our incidence-based approach, one must consider that this estimation is a projection of future mortality rates for people being infected in the time-period analysed, i.e. to . the eurostat (eu countries) notified standardised death rate from hiv/aids went from . per , population in to . per , population in [ ] . in our model, we projected a lower fatality assuming further decrease in the future due to improved treatment options, increased testing/ early ascertainment of cases and increased treatment compliance. published data on observed number of deaths of ipd are comparable to those in our study: in our study cfp was % (see supplement ) while in a european study in countries, death was reported in . % to . % of cases and changed according to age [ ] , in line with the models used for our study. similarly, published ipd incidence and mortality estimates in the netherlands based on sentinel surveillance and statistical estimation methods [ ] reported incidence of . per , population and deaths of . per , population, which are very similar to those presented in our paper. based on our study, most of the burden of ipd is experienced by adults over years of age, although children aged under years also significantly contribute to the total dalys (see supplement ) . these findings are relevant to discussions about vaccination strategies since, according to ecdc's report on invasive bacterial diseases in , 'the majority of infections were caused by serotypes covered by the -valent pneumococcal conjugate vaccine pcv ' [ ] . ranking of diseases can be also tailored to specific age groups as illustrated in figure . it is interesting to note that all five top ranking diseases among the less than years of age group are preventable through vaccination. within the adult population, further research on the main risk groups affected by hiv/aids and tb, which by far are the two infections with the highest impact, would be advantageous in order to better inform intervention strategies. the elderly population is mostly affected by respiratory diseases (influenza, ipd, tb and legionnaires' disease) and gastro-intestinal diseases (campylobacteriosis and salmonellosis). age-specific vaccination campaigns could help prevent the burden of influenza and ipd in particular. results from this study must be placed in a broader perspective. recently, the burden of a six selected healthcare-associated infections (hais) was estimated in dalys based on the bcode methodology [ ] . their cumulative burden of dalys per , population in that study was almost twice the one found in this one. these results imply that, among those surveyed by ecdc, hais represent the infections with the highest burden on european population. however, the methodological differences relating to the syndromic approach chosen for the burden of hais vs the pathogen-based approach of this study may limit comparing the results of the two studies. in particular, a number of diseases included in the current study may have been healthcare-associated (e.g. legionnaires' disease, diseases attributable to streptococcus pneumoniae), leading to some degree of double-counting [ , ] . however, this would likely be limited given that other diseases included in the present study may be uncommon causes of hais (e.g. infections due to neisseria meningitidis, tb, hepatitis a and b and invasive meningococcal disease) [ , ] . the global burden of disease study (gbd ) estimated dalys for a large number of diseases [ ] . by downloading gbd country-specific estimates from the global health data exchange (ghdx) website and totalling the dalys for , we were able to estimate the eu/eea burden of several infectious diseases included in our project. hiv/aids and tb overwhelmingly ranked higher than other infectious diseases in both studies. however, the gbd calculated prevalent dalys, assuming a steady state and not taking into account the projected future burden, so comparisons must be made with caution. the ontario burden of infectious disease study (onboids) [ ] used a comparable incidence-based daly estimation approach as applied in our study. similarly to our findings, the onboids found that infections caused by streptococcus pneumoniae, hiv and influenza virus had high burden. differences in ranking of diseases might be explained by differences in, for example, case definitions and disability weights. epidemiological differences should also be considered, as for example, the incidence of tb is higher in eu/eea countries than in ontario. another burden of disease study based on incidence is the world health organization (who) food-borne disease burden epidemiology reference group (ferg) [ , ] . considering the overlapping diseases, the diseases with the highest burden based on the published results for who european region eur a of that study and the results of this study were campylobacteriosis, salmonellosis and listeriosis. the only difference, the burden of stec/vtec, might be due to the higher risk of developing haemolytic uraemic syndrome (hus) and end-stage renal disease (esrd) in the bcode toolkit disease progression model. one strength of our study is that it is based on a rigorous assessment of national surveillance systems, which provided important information on sensitivity; where possible, notified data for a disease was adjusted specifically for each country. for example, country multiplication factors based on a self-reported survey of the national sensitivity towards ipd surveillance [ ] were applied to the notification data and dalys were estimated using the resulting cumulative number of cases. in other instances, such as for legionnaires' disease, countries were grouped according to ecdc disease programme expert opinion into higher, intermediate and lower surveillance system sensitivity and different multiplication factors were then applied for each sensitivity group. the sensitivity of surveillance systems does not only depend on their intrinsic characteristics; systems are also prone to temporary changes, for example, during outbreaks, when increased awareness might also increase willingness and capacity to detect and report cases. for the estimation of the incidence of measles, for example, notification of cases from countries and years experiencing an outbreak were adjusted with a multiplication factor of . [ ] , as opposed to . for other countries and years [ , ] . another strength of this study is that it captures the different risks of developing sequelae or death according to age group. examples include the age group-specific risk of developing hus after stec/vtec infection, a crucial step towards the estimation of its burden, and the redistribution of the cfp of salmonellosis, campylobacteriosis and influenza according to observed agespecific mortality data in order not to overestimate the number of deaths in younger age groups [ ] . disability weights included in the disease models are derived from a study performed in europe and thus have the potential to better reflect the preferences and values of the eu/eea population [ ] . most infectious diseases cause temporary mild disabilities; it is important to note that according to the methodology used to estimate disability weights, these may differ substantially for similar health states [ ] . a further strength is that the freely accessible and transparent methodology, parameters and variables of this study allow for reproducing the estimates and making comparisons with results from similar studies. for example, the burden of infectious diseases in the netherlands is a national study that was based on the bcode project methodology with national adaptations [ ] . a number of limitations need to be taken into account when interpreting the results of this study. first, the selection of diseases was limited to those included in decision / /ec with amendments. this list does not include other infectious diseases with a potentially significant burden in eu/eea countries, such as infections with human papillomavirus (hpv), helicobacter pylori, rotavirus, norovirus and human respiratory syncytial virus (hrsv). second, multiplication factors adjusting for underestimation of notified data were selected from information found in the literature. few country-specific multiplication factors were available and ranges based on the limited number of published studies were applied consistently across eu/eea countries. moreover, multiplication factors were not adjusted for different age groups, although some diseases causing diarrhoea, such as salmonellosis for instance, had high notification rates in children. this may be due to a testing bias, i.e. children may be tested more often, or to their reduced immunity or to higher exposure. regardless of the reason, this means that there is a risk that the results may be underestimated or overestimated. third, the disease models (outcome trees) in the bcode toolkit are based on several assumptions [ ] . variables for each model parameter represent the available information in the literature and the age-specific risk of developing a certain sequelae or death was often not available. outcome trees were developed considering the incidence of disease and the risks of developing sequelae as currently observed in eu/eea countries. therefore, treatment and preventive measures were implicitly considered and this should be taken into account when interpreting the results. for example, vaccine-preventable diseases (vpds) with high coverage had a lower burden of disease, but they had the potential to substantially increase their burden and their resulting position in the final ranking during outbreaks. in addition, the disease models included in this study are static and do not consider future infection transmissions. dynamic models, such as sir compartmental models for infection transmission, should be developed when assessing the impact of prevention and control interventions. fourth, the probability of developing sequelae or death were estimated based on the limited information in the literature, except in some cases where information was derived from surveillance data (supplement ), and considered the competing risks of dying or developing complications to the extent possible. at older ages, for example, co-morbidities may worsen the severity of a given infectious disease, suggesting modification of disability weights or the need to consider the attributable fraction due to the infections as opposed to the other underlying condition. fifth, the burden of hbv was based on the average annual number of acute infections but like other longterm disease progression pathways, other subsequent stages of the disease have an impact on population health. it would be beneficial to complement the incidence-based hbv results with prevalence studies given that our burden of hbv estimates do not consider prevalent long-term complicated cases. the methodology and results in this study are based on a fully transparent and reproducible approach. we believe that the burden of disease methodology described in this study provides a clear and comprehensive view on the impact of infectious diseases on population health. calculation of dalys through incidence-based disease progression models represents a comprehensive approach suitable for infectious diseases and provides useful information for prioritisation and planning in public health, among other purposes. for example, a recent scientific opinion by the european food safety authority recommended using the bcode approach for ranking risks [ ] . another example is the slovenian national estimation of the burden of tick-borne encephalitis that identified age groups with the highest dalys in order to inform vaccination strategies [ ] . however, as quantitative results alone might not fully encompass all unknowns, uncertainties and variability [ ] other dimensions of health should be considered. burden of disease measured in dalys could be integrated with risk-ranking methodologies such as multicriteria decision analysis (mcda). that being said, this study provides useful information for planning and prioritising surveillance strategies and intervention options aimed at preventing and controlling infectious diseases as the estimates provide a useful picture of the impact of 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control (ecdc) burden of six healthcare-associated infections on european population health: estimating incidence-based disability-adjusted life years through a population prevalence-based modelling study hospital epidemiology and infection control bennett & brachman's hospital infections world health organization estimates of the global and regional disease burden a data synthesis surveillance of invasive pneumococcal disease in eu countries: towards a european system? vaccine large measles outbreak at a german public school the global burden of measles in the year --a model that uses country-specific indicators has the measles mortality reduction goal been achieved? a natural history modelling study review of disability weight studies: comparison of methodological choices and values a software tool for estimation of burden of infectious diseases in europe using incidence-based disability adjusted life years scientific opinion on the development of a risk ranking toolbox for the efsa biohaz panel estimating the annual burden of tick-borne encephalitis to inform vaccination policy impact of food and water-borne diseases on european population health. current opinion in food science surveillance atlas of infectious diseases. stockholm: ecdc estimation of incidence of symptomatic salmonella and campylobacter infections based on sero european centre for disease prevention and control (ecdc) european centre for disease prevention and control (ecdc)/ world health organization regional office for europe (who/ europe) we are grateful to prof. johan giesecke for his invaluable help in supporting and steering the bcode project throughout the years. we would also like to thank the following ecdc colleagues for their valuable scientific input: zero akyol, andrew amato, sabrina bacci, julien beauté, ida czumbel, niklas danielsson, birgitta de jong, erika duffell, karin haar, janusz janiec, csaba ködmön, otilia mardh, gordon nichols, taina niskanen, lucia pastore celentano, pasi pentinen, anastasia pharris, emmanuel robesyn, ettore severi, gianfranco spiteri, johanna takkinen, lara tavoschi, wim van bortel, marieke van der werf, eva warns-petit, therese westrell, robert whittaker, herve zeller. we also acknowledge mike catchpole, chief scientist at ecdc, for his help in revising the manuscript.we would also like to thank other members of the bcode consortium: cheryl l. gibbons none declared. conceived the project: ac, ec, pk, ah, mek. analysed the data: ac. wrote the first draft of the manuscript: ac. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indicate if changes were made. key: cord- -eskefcwk authors: herrington, cs; coates, pj; duprex, wp title: viruses and disease: emerging concepts for prevention, diagnosis and treatment date: - - journal: j pathol doi: . /path. sha: doc_id: cord_uid: eskefcwk viruses cause a wide range of human diseases, ranging from acute self‐resolving conditions to acute fatal diseases. effects that arise long after the primary infection can also increase the propensity for chronic conditions or lead to the development of cancer. recent advances in the fields of virology and pathology have been fundamental in improving our understanding of viral pathogenesis, in providing improved vaccination strategies and in developing newer, more effective treatments for patients worldwide. the reviews assembled here focus on the interface between virology and pathology and encompass aspects of both the clinical pathology of viral disease and the underlying disease mechanisms. articles on emerging diseases caused by ebola virus, marburg virus, coronaviruses such as sars and mers, nipah virus and noroviruses are followed by reviews of enteroviruses, hiv infection, measles, mumps, human respiratory syncytial virus (rsv), influenza, cytomegalovirus (cmv) and varicella zoster virus (vzv). the issue concludes with a series of articles reviewing the relationship between viruses and cancer, including the role played by epstein–barr virus (ebv) in the pathogenesis of lymphoma and carcinoma; how human papillomaviruses (hpvs) are involved in the development of skin cancer; the involvement of hepatitis b virus infection in hepatocellular carcinoma; and the mechanisms by which kaposi's sarcoma‐associated herpesvirus (kshv) leads to kaposi's sarcoma. we hope that this collection of articles will be of interest to a wide range of scientists and clinicians at a time when there is a renaissance in the appreciation of the power of pathology as virologists dissect the processes of disease. copyright © pathological society of great britain and ireland. published by john wiley & sons, ltd. in this, the seventeenth annual review issue of the journal of pathology, we have collected together a series of review articles that each deal with an aspect of 'viruses and disease'. study of the relationships between a viral infection and the host are critical, not only for enhancing our knowledge of known viral diseases but also for understanding how viruses may be involved in diseases of unknown aetiology [ ] , and for attempting to predict how viruses emerge from animals to infect humans [ ] . as in many other arenas, pathology plays a vital part in improving our understanding of the mechanisms by which viruses cause disease. pathological assessment sheds light on the ways viruses interact with cells and tissues, spread within and transmit between hosts. the eventual pathological outcomes are a complex interplay between the host response to, and the direct effects of, the virus infection. in keeping with these considerations, this issue contains articles that address the roles that cell and tissue biology and pathology are playing in the elucidation of the mechanisms of virus-associated diseases. in , the subject area of viruses and disease is particularly pertinent, since we live at a time when viruses are regularly in the news. this is exemplified by the ongoing outbreak of ebola virus in west africa and, at the time of writing, the importation of infections into europe and the usa. such events act as timely reminders of the potential for viruses to cause global disease and it is fitting that our first article deals with filoviruses. zaki and colleagues have produced a tour de force, describing what ebola and marburg viruses are and how and why they are so highly pathogenic in humans. as two of introductory article and the complex way in which these viruses induce disease through multiple effects on the immune system and endothelium [ ] . in the first of two reviews on coronaviruses, grolinski and baric discuss the importance of the innate immune response in the development of lung pathology following human coronavirus infection, whilst van den brand, smits and haagmans review the available clinical and pathological data on middle east respiratory syndrome (mers) in comparison to severe acute respiratory syndrome (sars), and how these inform our understanding of both the different pathological effects of these viruses and their responses to interferon-based therapies [ , ] . staying with the theme of emerging infectious diseases and viral zoonoses, de wit and munster review the pathogenesis of encephalitis and vasculitis caused by the outbreaks of nipah virus. they focus on dissemination of the virus through its host, current knowledge of the determinants of pathogenicity and the routes of zoonotic and human-to-human transmission, emphasizing the key role played by animal models in advancing our understanding of this virus and the diseases it causes [ ] . karst et al review the molecular pathology of norovirus (the cause of 'winter vomiting disease'), one of the main causes of acute gastroenteritis, but also now appreciated as causing chronic effects and extra-intestinal spread [ ] . muehlenbachs et al provide a comprehensive review of the enteroviruses, including their diverse pathological effects and how histopathology, immunohistochemistry and molecular studies have provided insight into tissue tropism and viral pathogenesis [ ] . similarly, lucas and nelson combine to provide an equally comprehensive review of the diversity of diseases associated with human immunodeficiency virus (hiv)- infection, including discussing the additional problems that are likely to be encountered as successfully treated hiv-infected individuals age [ ] . mumps and measles are two of the oldest-recognized infectious diseases and have been the subjects of intensive worldwide vaccination programmes. it has been suggested that it may be possible to eradicate measles virus (mv), and even though this is probably not feasible for mumps virus (muv), huge advances in reducing the disease burden have been achieved for both. despite these successes, these viruses continue to cause human disease in both the developing and the developed world. rubin et al have combined their expertise to review the renewed interest in mumps pathogenesis arising from the resurgence of this disease in the highly vaccinated populations in europe and the usa. they emphasize the need to develop clinically-relevant animal models of disease, which are necessary to ensure that unsafe vaccines are not licensed [ ] . in addition, ludlow et al provide an up-to-date review of measles pathogenesis. they highlight how recent advances in understanding virus-receptor usage explains why the virus infects both epithelial and immune cells and how the inter-related, and opposing, effects of these two distinct tissue tropisms modulate disease [ ] . these reviews are followed by articles that provide an in-depth analysis of human respiratory syncytial virus (rsv) and influenza. in the first of these, pickles and devincenzo review the pathogenesis of bronchiolitis caused by rsv, with a particular emphasis on the cytopathological effects of viral infection on columnar cells, leading to the pathological sequelae of bronchiolar obstruction and inflammation [ ] . the review on influenza by kuiken et al discusses how the olfactory system acts as an important conduit for virus spread to the central nervous system. using data from human and animal infections, they provide compelling evidence that many viruses use this route and discuss current understanding of the molecular mechanisms involved [ ] . the next two articles are dedicated to the diseases associated with the human herpes viruses cytomegalovirus (cmv) and varicella zoster virus (vzv), each of which infects the majority of humans in the world and establish lifelong, latent infections. in their review of cmv, griffiths et al discuss how epidemiology, and molecular and cellular pathology, account for the variable clinical aspects of cmv-associated disease and how the host immune responses designed to control the virus contribute to immune impairment [ ] . in their review of varicelloviruses, ouwendijk and verjans include an up-to-date analysis of both human and animal infections. these studies, using an excellent animal model of disease and targeted pathology approaches driven by macroscopic imaging, have been highly informative in progressing our understanding of the pathogenesis of varicella and herpes zoster in terms of primary infection, latency and reactivation [ ] . the idea that viruses can cause cancer is more than a century old [ ] and it is now well recognized that a number are responsible for human cancer [ ] . in this annual review issue, we have six articles that provide updates on the mechanisms involved in virus-induced cancers and the potential for targeting these viruses therapeutically. there are three reviews on epstein-barr virus (ebv). the first article, from vockerodt et al, reviews recent insights into the mechanisms of ebv-associated lymphoma development [ ] , whilst lo and colleagues re-evaluate the mechanisms introductory article exploited as targeted therapy for ebv-associated malignancies [ ] . feltkamp et al provide a comprehensive review of the role played by β-human papillomaviruses (hpvs) in human basal and squamous carcinomas of the skin, and their synergy with immunosuppression and exposure to uv radiation [ ] , contrasting with the more well-defined relationship between α-hpv types and cervical and other squamous cancers. ringelhan et al provide a timely update on the role of chronic hepatitis b virus infection in the pathogenesis of hepatocellular carcinoma (hcc), which involves both direct effects of viral gene expression and indirect effects of infection [ ] . finally, but by no means least, gramolelli and schultz discuss recent advances in identifying and understanding the mechanisms by which kaposi's sarcoma-associated virus causes the unusual vascular tumour after which it is named, with an in-depth discussion of the viral proteins that are responsible [ ] . these articles not only provide a comprehensive view of how viruses cause disease but also demonstrate the critical role that pathology plays in this field [ ] . recently there has been a true renaissance in appreciating the power of sound observations, where pathology is not 'just descriptive' but rather is the bedrock for meaningful hypothesis-driven research. this illustrates the unique opportunities for virologists and pathologists interested in dissecting the molecular mechanisms of viral disease to work together, develop partnerships and drive the field forward. such studies will help extend our understanding of virus transmission from animals to humans and from humans to humans. the need to understand transmission fully is exemplified by the current ebola virus outbreak in west africa. moreover, the development of animal models that can be used to test novel vaccines and antivirals is vital. only by understanding how these interventions modulate the resulting disease, through the involvement of pathologists, can basic scientists know their translational goals are closer. we hope that these contributions demonstrate that this is the case not only for the cancer-associated viruses, where the interface with pathology is more widely appreciated, but also for the more classical infectious diseases, for which tissue-based studies can also provide essential information in the context of 'real' disease. going forward, the increasing availability and use of 'omics' technologies in virology [ ] [ ] [ ] has tremendous potential for rapidly identifying new viruses and their host interactions, including immune responses. nevertheless, in this new era, pathology will continue to be a vital component of identifying the true relationships between viruses and human disease, and we hope that this annual review issue will serve as a blueprint for future studies in the diagnosis, treatment and prevention of virus-related conditions through an improved understanding of the processes involved. orphan viruses, orphan diseases: still the raw material for virus discovery prediction and prevention of the next pandemic zoonosis tissue and cellular tropism, pathology and pathogenesis of ebola and marburg viruses pathogenesis of middle east respiratory syndrome coronavirus molecular pathology of emerging coronavirus infections animal models of disease shed light on nipah virus pathogenesis and transmission the molecular pathology of noroviruses tissue tropism, pathology and pathogenesis of enterovirus infection hiv and the spectrum of human disease molecular biology, pathogenesis and pathology of mumps virus pathological consequences of systemic measles virus infection rsv and its propensity for causing bronchiolitis the olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system the pathogenesis of human cytomegalovirus pathogenesis of varicelloviruses in primates the history of tumor virology why do viruses cause cancer? highlights of the first century of human tumour virology the epstein-barr virus and the pathogenesis of lymphoma the role of epstein-barr virus in epithelial malignancies epstein-barr virus-encoded ebna and zebra: targets for therapeutic strategies against ebv-carrying cancers human β-papillomavirus infection and keratinocyte carcinomas the direct and indirect roles of hbv in liver cancer: prospective markers for hcc screening and potential therapeutic targets the role of kaposi's sarcoma-associated herpesvirus in the pathogenesis of kaposi's sarcoma cell culture and electron microscopy for identifying viruses in diseases of unknown cause application of next-generation sequencing technologies in virology genomic approaches to studying the human microbiota all authors contributed to writing the manuscript. key: cord- -zq ms w authors: louvardi, maya; pelekasis, panagiotis; chrousos, george p.; darviri, christina title: mental health in chronic disease patients during the covid- quarantine in greece date: - - journal: palliative & supportive care doi: . /s sha: doc_id: cord_uid: zq ms w objective: to investigate the effect of the time spent on quarantine on distress, anxiety, depression, and somatization of chronic disease patients during the covid- quarantine in greece and the differences in these parameters between healthy individuals and chronic disease patients. method: the sample consisted of healthy individuals and patients (respiratory, autoimmune, cardiovascular, endocrine, patients with other diseases, and patients with more than one disease) completing sociodemographic assessments as well as the -dimensional symptom questionnaire ( dsq) during march , to may , . pearson's correlation was used to search for the association between time spent on quarantine and the dsq subscales (distress, anxiety, depression, and somatization). independent sample t-test and glass's Δ were used for differences between healthy individuals and chronic disease patients in these subscales, an analysis also carried out between healthy individuals and all patient subgroups. results: no statistically significant correlations were noted between the dsq subscales and the quarantine duration, both for the patient and the healthy individuals’ group. chronic disease patients had significantly higher levels of distress (p = . , Δ = . ) and somatization (p = . , Δ = . ), but not there were no significant differences in anxiety (p = . , Δ = . ) and depression (p = . , Δ = . ). concerning head-to-head comparisons between the healthy individuals’ group and each patient group, significantly higher scores in distress were found only for patients with respiratory diseases (p = . , Δ = . ). regarding somatization, significantly higher scores were noted for the healthy individuals’ group compared with patients with autoimmune diseases (p = . , Δ = . ), respiratory diseases (p = . , Δ = . ), other diseases (p = . , Δ = . ), and more than one disease (p = . , Δ = . ). no statistically significant differences were found in anxiety and depression. significance of results: the results of this study indicate that interventional programs for chronic disease patients during quarantine should focus on distress and somatization, not on anxiety and depression. respiratory patients might have more supportive care needs compared with patients with other diseases. undoubtedly, covid- is the biggest threat of global public health for , since despite its low case fatality ratio (cfr), the easiness in transmission leads to a worldwide pandemic (heymann and shindo, ) . the value of r of the disease during the early phase of the pandemic was estimated at . - . (zhao et al., ) , making the essential use of strict public health measures to set the transmission under control (guo et al., ) . several studies have investigated the impact of covid- on patients with chronic diseases. through an analysis of , cases in china, it was found that the cfr was . % for cardiovascular disease patients, . % for diabetes, . % for chronic respiratory syndrome, and % for hypertension, while the cfr of the general population was . % (wu and mcgoogan, ) . in addition, even though the original data have not been published so far, it is widely accepted that patients receiving immunosuppressant or immunomodulatory therapy (e.g. those affected by multiple sclerosis) have an increased risk of complications and mortality from covid- (willis and robertson, ) . furthermore, the disease outbreak has lead to treatment delays in patients with chronic diseases, such as cancer, which might be disturbing and affect the treatment outcome. for example, delaying therapies for gastrointestinal and lung cancer patients was considered necessary in china during the disease outbreak to prevent the risk of infection (moujaess et al., ) . hence, patients with chronic diseases consist a highly vulnerable group during the covid- outbreak, which has to be further studied. quarantine is a must-use measure during a disease outbreak to protect public health (tognotti, ; parmet and sinha, ) . in the current pandemic, this approached has been firstly used in china to set covid- under control and to protect the health of vulnerable populations, such as patients with chronic illness (guo et al., ) . nevertheless, it is necessary to investigate the economic, societal, and psychological impact of this policy in order to improve the way it is implemented and to design strategies minimizing the negative effects, since the implementation of quarantine raises several ethical dilemmas (upshur, ; cetron and landwirth, ) . to date, limited studies have investigated the mental health of people with chronic diseases while implementing quarantine policies. more specifically, in a relevant study in south korea during the mers outbreak in , it was found that those with history of physical disease had increased odds for clinically significant anxiety - months after the quarantine (jeong et al., ). an additional study in the same country on quarantined hospitalized hemodialysis patients during the mers outbreak found that % and . % met the criteria for clinically significant anxiety and depression, respectively (lee et al., ) . concerning the outbreak of covid- and related quarantine measures, a recent study in spain during march - , found that chronic disease patients had higher levels of stress, anxiety, and depression compared with healthy individuals (ozamiz-etxebarria et al., ) . thus, chronic disease patients might have increased supportive care needs while placed in quarantine. to date, no other relevant studies have been published, leading to several literature gaps, especially since the previous studies have not investigated patients with different types of chronic diseases while implementing quarantine policies. in that context, this study investigated mental health parameters of patients with chronic diseases during the covid- outbreak in greece. the aims of the study were the following: (a) to search for an effect of quarantine duration on anxiety, depression, somatization, and distress of chronic disease patients and (b) to investigate differences between healthy individuals and patients with chronic diseases in anxiety, depression, somatization, and distress during the covid- quarantine. this study was part of a wider ongoing research to investigate mental health parameters in the greek population, which started at december . the research has gained approval by the appropriate committee of the medical school of the national and kapodistrian university of athens and complies fully with the declaration of helsinki. at the present study, all the eligible participants from the dataset completing the assessments between march , and may , were extracted to be further analyzed. the inclusion criteria in the present study were the following: (a) age over years old and (b) being able to communicate in greek. the inclusion and exclusion criteria have not changed at any stage of the data collection process. the sociodemographic data of the participants included gender (male/female), age ( - / - / - /over ), family status (married/unmarried living with a partner/unmarried living alone/divorced or widowed), having children (yes/no), educational level (primary/secondary/tertiary), smoking status (current smoker/occasional smoker/nonsmoker), and occupational status (unemployed/student/private sector worker/public sector worker/ freelancer or businessman woman/pensioner/house worker). the -dimensional symptom questionnaire ( dsq) consists a self-reported instrument including items scored on a likert-type scale ( = no to = very much or always). this instrument includes four different subscales, measuring distress, anxiety, depression, and somatization (terluin et al., ) . the dsq has been previously validated in greek (tsourela et al., ) . in this study, the a level was . for depression, . for anxiety, . for distress, and . for somatization. as mentioned above, this study is part of a wider research investigating mental health parameters in the greek population. several posts on social networks containing a link leading to the assessments, also explaining the study purpose, the inclusion criteria, and the anonymity of participation, were used for participant recruitment. the posts were placed on groups with general content to increase the representativeness of the study sample. the participants pressing the link were guided to the assessments, which were completed instantly. the time needed to complete the assessments was estimated at approximately min. the participants analyzed in the present study were those that have completed the assessments from march , till may , . the beginning of this time interval was one week after the start of the quarantine, since the recall period of the dsq is one week. thus, since the quarantine started at march , , using participants who completed the assessments prior to march , would lead to recalling symptoms placed before the beginning of the quarantine. the end of this interval refers to the last day of the quarantine in greece. in the present study, the participants analyzed were those with no chronic disease (n = ) and those with the following diseases: cardiovascular (n = ), autoimmune (Ν = ), chronic respiratory diseases (n = ), and endocrine disorders (n = ). these disease groups were included since at least participants were found in the dataset for each of these subgroups. patients with other diseases (e.g. schizophrenia) and patients with multiple chronic diseases (e.g. asthma and cancer) were analyzed in separate groups (n = and , respectively). thus, the first group consisted of healthy individuals and the other six patients with different types of chronic diseases. the statistical analysis of the present study was carried out by the use of spss statistical software version (spss, inc., chicago, il). absolute values and proportions were used for descriptive analysis regarding the demographic characteristics of the study. since according to the kolmogorov-smirnov test the normality of the dsq subscales was not violated, independent sample t-test was applied to investigate differences between healthy individuals and each subgroup with chronic disease. the effect size was calculated by the use of glass's Δ. pearson's correlation was used to investigate the relationship between the duration of quarantine and the dsq subscales for the chronic disease and the healthy individuals' group. the p-value was set at . for all the analyses. the sociodemographic data of the participants are presented in table . as indicated in the table, the sample consisted mostly be females ( . %). a considerable proportion of participants was between - years old ( . %) and unmarried who lived alone ( . %). most of the participants did not have children ( . %), were nonsmokers ( . %) and had received tertiary education ( . %). private sector workers ( . %) and public sector workers ( . %) were the most frequent types of occupational status. further information is presented in table . the correlation between quarantine duration and the dsq subscales score is presented in table . as indicated in the table, there was no correlation between quarantine duration and the subscales' scores both in the healthy individuals and the chronic disease patients group. with regard to differences in dsq subscales between healthy individuals and chronic disease patients, the relevant analysis is presented in table . as indicated in the table, chronic disease patients had statistically significant higher scores in somatization ( p = . ) and distress ( p = . ). the differences in anxiety and depression were not statistically significant ( p = . and . , respectively). the mean value of the dsq distress subscale was . for healthy individuals (sd . ). statistically significant differences were noted only when compared with patients with respiratory diseases ( p = . ), as shown in table . the mean value in the dsq anxiety subscale was . for healthy individuals (sd . ). as indicated in table , there were no statistically significant differences with any of the patient groups. the mean value of dsq depression was . for the healthy individuals group (sd . ). the comparisons with each of the patient groups of the study did not lead to statistically significant differences in depression, as shown in table . the mean value of the dsq somatization subscale was . for the healthy individuals group (sd . ). concerning the differences in somatization between healthy individuals and each patient group, the relevant analysis is presented in table . as indicated in the table, most patient subgroups had statistically significant higher scores of somatization. this study aimed to investigate the correlation between time spent on quarantine and the dsq subscales in chronic disease patients in contrast to the relevant correlation in healthy individuals, as well as to search for differences in the scores of these subscales based on the disease status of the participants. the first main finding of the study is that there is no correlation between the dsq subscales and time spent on quarantine neither for the healthy individuals nor for the chronic disease group. the second finding regards the significantly higher levels of distress and somatization in chronic disease patients, while anxiety and depression have no difference compared with healthy individuals. the analyses per disease indicate that endocrine and cardiovascular disease patients are not significantly affected, since they did not differ from healthy individuals in any of the dsq subscales. autoimmune patients, those affected by more than one disease and patients with other diseases, differ from healthy individuals only in somatization. patients affected by respiratory diseases have additional supportive care needs, since they differ from healthy individuals in more than one parameter (somatization and distress). of note, all the analyses indicated a nonsignificant effect on patient depression and anxiety. the absence of significant associations between the dsq subscales and quarantine duration is in line with previous studies concerning the association between quarantine duration and psychopathological manifestations in the general population. for example, during the covid- massive quarantine in china, it was found that time was unrelated to the intensity of anxiety levels (hu et al., ) , while, in a previous study in toronto, canada during the sars outbreak, it was found that quarantine duration was not significantly related to depressive symptoms, although there was a trend toward a positive association (hawryluck et al., ) . the results of the present study confirm and expand the current knowledge, since, both in the healthy individuals and in the chronic disease patient group, there was no association between the dsq subscales and time spent in quarantine. a paradoxical finding of the present study has to do with the absence of higher depressive levels for chronic disease patients during the lockdown, since, according to the previous study in greece, chronic disease patients have higher levels of depressive symptoms compared with healthy individuals (gerontoukou et al., ) . the absence of differences could be attributed to mechanisms activated during societal threat in collective-oriented societies, such as greece. more specifically, during the capital controls, a study in breast cancer patients found that depressive symptoms were queerly lower during that period of economic and political destabilization, a finding that could be attributed to an increase of social support toward the vulnerable during the general societal threat (pelekasis et al., ) . similar mechanisms could have been activated during the current crisis, leading to higher support toward patients with chronic diseases, and positive effects on depressive levels, leading to the absence of differences from healthy individuals. nonetheless, since this study found higher levels of distress and somatization for chronic disease patients, developing and implementing appropriate mental health interventions during quarantine policies is essential. in addition, as indicated from a study during the sars outbreak, psychopathological manifestations could lead to extra secretion of catecholamine, which causes instability of myocardial electricity and adverse outcomes on cardiovascular disease patients (pan et al., ) . as for patients with autoimmune diseases, such as multiple sclerosis, it is supported that covid- -related anxiety could lead to disease exacerbations (ahadi et al., ) . thus, developing and applying appropriate interventions for chronic disease patients during a pandemic is essential. as for the content of those interventions, aiming only at mental health parameters might be somehow restricting, since chronic disease patients might have additional supportive care needs while placed in quarantine. interestingly, it has been supported that an underrated threat for chronic disease patients, such as cardiovascular, regards the absence of physical activity during quarantine, which could lead to a further risk for adverse outcomes (mattioli et al., ) . for that reason, it is essential to design appropriate interventions targeting both the improvement of mental health and the adoption of a healthy lifestyle during the quarantine. in greece, recent studies testing the effectiveness of a relevant culturally oriented intervention entitled "pythagorean maya louvardi et al. self-awareness" indicate a wide range of benefits for mental health and health behaviors of patients with chronic diseases, such as cancer and multiple sclerosis (darviri et al., ; anagnostouli et al., ; charalampopoulou et al., ) . thus, developing and applying an internet-based intervention using this content could lead to improvements in mental health and health behaviors of patients with chronic disease during a future covid- quarantine or generally in future pandemics. a few limitations for this study have to be reported. at first, internet data collection is prone to selection bias, since potential participants with no or low access to the internet have a smaller chance to be included (bethlehem, ) . even though carrying out an internet-based data collection process was unavoidable due to the lockdown, this leads to a selection bias that cannot be ignored. the young age of the study participants and the high proportion of those with no children reflect this type of bias. in addition, there could be a sample size bias, since not using a relevant formula to determine the optimal sample size indicates that the results of a study are prone to this type of error (campbell and machin, ) . finally, it is unclear if the differences recorded between patients and healthy individuals are similar to those under normal circumstances, since patients with chronic diseases, especially those with advanced illness, have a generally higher risk of psychopathology compared with healthy individuals (turner and kelly, ; tremblay and breitbart, ; gerontoukou et al., ; rabiee et al., ) . relevant studies have to be carried out in other countries to understand if the differences reported at the present study account only for greece or are general. more specifically, greece is considered as a successful case in covid- outbreak management (tsiotas and magafas, ) . hence, studies in other countries more significantly affected by the outbreak (e.g. italy, spain, and france) could allow us understand if the effects are similar across different countries or are influenced by the degree that each country was affected by the pandemic. this study investigated the effect of massive quarantine during covid- in greece on distress, anxiety, depression, and somatization of patients with chronic diseases. distress and somatization were increased in chronic disease patients, while anxiety and depression were not. endocrine and cardiovascular disease patients were not significantly affected, since they did not differ from healthy individuals in any of the dsq subscales. patients affected by respiratory diseases had additional supportive care needs, since they scored significantly higher from healthy individuals in more than one parameter (somatization and distress). autoimmune patients, those affected by more than one disease and patients with other diseases differed from healthy individuals only in somatization. since this study indicates that chronic disease patients have additional supportive care needs, developing and delivering relevant interventional programs during pandemics is essential. funding. this research received no specific grants from any funding agency in either the public, commercial, or not-for-profit sector. conflict of interest. the authors hereby state that they have no potential conflicts of interest to declare. palliative and supportive care psychiatric advice during covid- pandemic for patients with multiple sclerosis a novel cognitivebehavioral stress management method for multiple sclerosis. a brief report of an observational study selection bias in web surveys medical statistics: a commonsense approach public health and ethical considerations in planning for quarantine the effects of pythagorean self-awareness intervention on breast cancer patients undergoing adjuvant therapy: a pilot randomized controlled trial pythagorean self-awareness serves successfully as a new cognitive behavioral-based technique in multiple sclerosis physical and psychosocial well-being and quality of 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china: summary of a report of cases from the chinese center for disease control and prevention preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak key: cord- - op d t authors: wang, xingyuan; zhao, tianfang; qin, xiaomeng title: model of epidemic control based on quarantine and message delivery date: - - journal: physica a doi: . /j.physa. . . sha: doc_id: cord_uid: op d t the model provides two novel strategies for the preventive control of epidemic diseases. one approach is related to the different isolating rates in latent period and invasion period. experiments show that the increasing of isolating rates in invasion period, as long as over . , contributes little to the preventing of epidemic; the improvement of isolation rate in latent period is key to control the disease spreading. another is a specific mechanism of message delivering and forwarding. information quality and information accumulating process are also considered there. macroscopically, diseases are easy to control as long as the immune messages reach a certain quality. individually, the accumulating messages bring people with certain immunity to the disease. also, the model is performed on the classic complex networks like scale-free network and small-world network, and location-based social networks. results show that the proposed measures demonstrate superior performance and significantly reduce the negative impact of epidemic disease. though the medical conditions are improved significantly, epidemics have never been away from human world, especially in developing countries. of growing concerns are adverse synergistic interactions between the emerging diseases and other infectious. poor sanitation and lack of medical knowledge lead to the wide spreading of disease in developing countries, such as ebola and mers (middle east respiratory syndrome coronavirus). besides, some of traditional contagions are resistant to drug treatments now, such as malaria, tuberculosis, and bacterial pneumonia. these kinds of diseases are defined as new emerging infectious disease (eid), which have increased in the past years and will keep growing in the near future [ ] . there are several remarkable characteristics in the eid: unpredictable, not preventable, irremediable, high mortality, rapid transmission and wide scope of influence. the expansion of disease spreading can lead to social panic at some level. it is hence imperative to study effective control strategies to prevent the disease diffusion. many of researchers have tried to explore the prevention measures of eid. as a practical method, the information diffusion on epidemic dynamic has attracted much attention in recent years. a path-breaking work in this field was taken by funk et al., who proposed an epidemiological model that considers the spread of awareness about the disease [ , ] . lima et al. proposed that the propagation of disease can be reduced by the spreading of immune information, which make individuals resistant to disease and then work against the epidemic propagating [ ] . a model of competing epidemic spreading over completely overlapping networks was proposed by karrer and newman, revealing a coexistence regime in which both types of spreading can infect a substantial fraction of the network [ ] . wang and tang distinguish two types of disease spreading and proposed the dynamic model of asymmetrically interacting and disease spreading. their research focuses on three problems: the different network structures and information spreading dynamics; the asymmetric effects of one type of spreading dynamics on another; the timing of the two types of spreading [ ] . moreover, several researches are also meaningful to us [ ] [ ] [ ] [ ] [ ] [ ] . trpevski et al. explored the rumors propagation in multiple networks [ ] . zhang et al. proposed a model considering time delay and stochastic fluctuations [ ] . ababou et al. investigated the spreading of periodic diseases and synchronization phenomena on exponential networks [ ] . rakowski et al. put forward an individual based model to study the effects of influenza epidemic in poland. a simple transportation rule is established to mimic individuals' travels in dynamic route-changing schemes, allowing for the infection spread during a journey [ ] . all the above efforts are worth approving but the specific ways of message spreading are ignored. this paper enriches the research in this area, and the proposed methods are shown to have a significant effect in epidemic prevention. the paper proceeds as follows. in section , we first describe the characteristics of epidemic, then the siqm models are developed, which contain the disease prevention measures based on quarantine and message delivery. section gives the sensitivity analysis of the model on classical complex networks. in section , the common regularity of human mobility and experiment results on location-based social network are given. the paper is concluded in section . normally, the process of infectious disease can be divided into three stages: susceptible period, infection period, recovery period [ ] . the infection period is further divided into latent period and invasion period. the former is characterized by the tiny indisposition; the latter is featured by onset of clinical signs and symptoms. moreover, according to the research of lessler et al., the latent period is essential to the investigation and control of infectious disease [ ] . in this period, the infectious individuals have obtained the ability to spread diseases, yet without obvious symptom to arise attention, e.g., the latent period of influenza remains only one to three days, yet the epidemic can sweep through a city in less than six weeks. furthermore, according to the health experts sartwell's research, the period of latent period varies between individuals in the same regular fashion as do other biological characteristics [ ] . the distribution of days seems to follow the ''normal'' curve. based on their theory, we investigate several common infectious diseases and collect their incubation period in table and then get the average length of latent period which approximates to days. to sum up, two assumptions are proposed here as the basic precondition of model: ( ) the infection period of epidemic is divided into latent stage and invasion stage in epidemiology. because the disease in former stage is hard to observe and diagnose, we preset a relative low value to its isolating rate which ranges from . to . . the rate in invasion period ranges from . to . ( ) to simplify the model, the average length of latent period is uniformly set to days, which is important to the experiments in section . the value is deduced by results shown in table . individual status in the model is divided into four types: susceptible, infected, in quarantine and in messaging. the susceptible means the people who are vulnerable to the disease. the infected denotes people who have already been infected consciously or unconsciously. when in quarantine, it means the infected ones have been isolated and will not spread disease anymore. when in ''messaging'' status, the specific persons, denoting the ones who have just been isolated or their directly connected neighbors, would deliver specific messages to their neighbors. timely isolation and message-delivery are the main prevention measures in our model, especially the latter. just imagine that a new infectious disease outbreaks at a certain area and the messages about disease are open to public a few months later, it would be a large cost to control its scale, duration and damage. consider the epidemic sir (susceptible-infectious-recovery) model, which is firstly proposed by kermack and mckendrick [ ] . now we improve the model by modifying the ''recovery'' status to ''quarantine'' status then constructing an enclosed and one-way evolution system. infection rate is defined as u and isolation rate as v. finally, the modified partial differential equations are given by: with nodes (termed as v ) representing individuals and edges (termed as e) representing connections between individuals. each node i can be in one of three statuses: susceptible, infectious and isolated. the status is described as a status vector, containing a single in the position corresponding to the current state, and everywhere else. let the probability function of each status is set to the state transition processes is shown in fig. . status is the initial state, and status is the final state. when connected to the node in status , nodes in status may turn into status at a certain rate. nodes in status could keep their status or convert into status on either latent period or invasion period. evolution of the model is given by the formulas where multi realize can be interpreted as a realization of random realization for the probability distribution prob i (t), or a mapping from probability vector to status vector. for each node i, u i (t) and v i (t) represent its infecting rate and isolating rate respectively. in the study in section . , the isolating rate of disease is defined as {v |v ∈ ( , . )} when in latent period. when in invasion period, the isolating rate is defined as {v |v ∈ [ . , )}. the infecting rate u i (t) is given by each node in status tries to infect its neighbor node at time t. each try may be successful with a rate β. if j ∈ l i , set l ij = , and l ij = otherwise. in formula parameter θ could take any of values in the set { , , , , , , , , , }, which stand for the quality of information. it could be measured by the characteristics of messages like authority, accuracy, availability, etc. the higher value corresponds to the higher quality. moreover, the information quantity is defined by lima et al. have proposed that the public-facing information distribution may cause a considerable amount of neglect [ ] . therefore, the directional message delivery is essential to warn the crowds who are under high risk of being infected. simultaneously, as the receiving and accumulating of messages, the newly received messages perform a gradually decreasing effect. accordingly, geometric series is adopted here to simulate the process. owing to its convergence properties, m i (t) will never be over . n i and n i , representing the total amount of direct and indirect messages respectively, are given by the messages, sent from one node (in status ) to its direct-neighbors, are defined as direct messages, while the messages sent to the direct-neighbors' neighboring nodes are defined as indirect messages. a and b are the influence rates of two types of messages. for node i, n i (t) represents the total number of its direct-neighbors who are isolated, and n i (t) represents the total number of its neighbors's neighboring nodes who are isolated. represent the ratio of nodes in status , and respectively; let for the average value of infectious rate and average information quantity respectively. to facilitate understanding, a simple example is given in fig. . initialize the global parameters with the length of latent period is defined as one time unit. each node in status could spread disease to its neighbors and get isolated at a random probability of v (in latent period) or v (in invasive period). each node in status sends messages to its neighbors and neighbors' neighboring nodes. those received messages will improve the nodes immunity and decrease infection rate. concretely, ''remark '' is calculated by all the infectors are finally isolated, and several lucky nodes become immune to the disease like node . this section presents the results of model behavior on complex networks. ba scale-free networks and ws small-world networks are contained, generated by algorithms. all the experiments are performed initially with two nodes in status and others in status . if there are n nodes sorted in number in network, then the (n/ )th, ( n/ )th nodes are picked up as the initial nodes in status . first consider the scale-free network, which is generated by ba algorithm [ ] . the network begins with m nodes. every time adding a new node, we connect it to m (m < m ) nodes which are already in the network. finally, the average degree of network ⟨k⟩ is equal to m, and degree distribution p k is approximate to k . in fig. , we present the results of model behavior under different conditions and situations. if without message diffusion ( fig. (a) and (b) ), most of nodes become infected in the epidemic. under quarantine measures, there is a red and unimodal curve shown in fig. (b) , which represents the number of nodes in status . statistical results show that the speed of disease spreading is delayed fivefold, and the peak value is reduced by %. in fig. (c) , we present a more acceptable result, where over a third of population is preserved from the disease only if received several messages in a medium-degree quality, and the peak of infection is decreased by around %. let then we get three dash lines which are nearly overlapping the solid lines. it could be equivalently represented for s (t) = p (t) , i (t) = p (t) , q (t) = p (t). moreover, a clear comparison can be seen in fig. (d) , where u (t), which denotes the average infection rate, is investigated under three different situations as mentioned above. another finding is shown in fig. (e) and (f). as the value of v , which denoted the isolation rate in latent period, grows from . to . , the peak value of i (t) reduces from . to . (fig. (e) ); with the same parameters, we change the value of v from . to . , then obtain the peak value i max (t) ≈ . (fig. (f) ). there is a particular conclusion: the increasing of isolation rate in invasion period, as long as over . , contributes little to the preventing of epidemic; the improvement of isolation rate in latent period is the key to control the spreading of disease. next in fig. (g) , the influences of message quality and isolation rate are measured. for the same parameters as before, the higher value of θ corresponds to the lower value of i (t). it can be predicted by i ∼ − /θ . that is another conclusion is obtained here: even though a higher value of θ corresponds to the lower infection rate, once θ > the effect is changed slightly. it implies that the disease is easy to control as long as the messages achieve a certain quality. in fig. (h) and (i), we display the steady-status behavior of the model on ba networks with different value of m. similar results are obtained by the experiments on -nodes network and , -nodes network. the clustering coefficient in scale-free network [ ] is get by it is obvious that there is not obvious characteristic of clustering in scale-free network. m (t ) represents the average information quantity on the stabilizing time t . q (t ) =  t t= i(t) denotes the ultimate scale of population in quarantine. they are used to measure the population immunity and magnitude of disease. as m changes from to , the average information quantity presents exponential distribution, which illustrates that the flow of information is highly improved as the increasing of average degree ⟨k⟩. the proportion of isolators presents a fluctuating distribution. for m = , both m (t ) and q (t ) are sustained at around . . at this point, despite the weak immunity of population, the epidemic will not expand for the low connectivity of network. in this part, we consider the model behavior on small-world networks. the networks are generated by ws algorithm, which are firstly distributed in an irregular circular shape and contain n nodes. every node connects to k nodes nearby, with k / in the clockwise and k / in the anticlockwise and n >> k >> ln (n) >> . later rewire each edge at probability p. change the parameter p from to then the generating network is translated from regular network into random network. self-loops or multiple edges between nodes are not allowed. clustering coefficient in ws small-world network [ ] is given by the steady-status in ws network achieves the same level (q (t ) ≈ . ) as in ba network by setting p = . and k = , which can be studied from fig. (a) and (b) . by increasing the value of p, the randomness of network is improved. the networks with high regularity and low degree are hard to spread disease, e.g. the networks in fig. (a) with p = . , k = ; the increasing k contributes to the disease spreading theoretically but finally is balanced by the increasing value of m (t ); in fig. (c) , we find that in a limited range, the more irregular the network is, the more easy the disease spreads; finally in fig. (d) , the small-world network is transformed into a completely random network by setting p = . there seems to be no remarkable differences between results in fig. (b) and (d). it illustrates that the network topologies contribute little to disease spreading, as the generating parameter p is over . . fig. (e) and (f) give a comparison about the model behavior on scale-free networks and small-world networks. results show that the infectious disease spreads more rapid in scale-free network. epidemics in small-world networks appear three characteristics corresponding to the number of infectors: slower growth, slower convergence and lower peak. in most studies, the meta-population networks are fixed. yet in actual world, the positions of individuals change every time. thus it is meaningful to consider the regularity of human mobility when analyzing the disease spread in real world. cho, myers and leskovec have made a research about this issue [ ] . as they put forward, ''short-ranged traveling is periodic both spatially and temporally and not effected by the social network structure, while long-distance travel is more influenced by social network ties''. they have proved that social relationships can cover about %- % of all human movements, while periodic behavior covers %- %. we conclude their achievement systematically below as the experimental basis: • human activity presents the periodic prosperity and recession. the period is on a weekly basis. from monday to friday, human activity is highly consistent, i.e., moving from home to work station then going back home. on weekends, the activities are scattered and inconclusive. • km is the typical human radius of ''reach'' as it takes about - h to drive such distance. because of the geographically non-uniform distribution over the earth, human tend to cluster in cities and the homes of friends at around km present a similar kink. • user home locations are not explicitly given yet can be inferred by defining the home location as the average position of check-ins within the scope of km. this method to infer home locations is demonstrated with % accuracy. likewise, we say that user a ''visits'' her friend b if a checks-in within radius r of b's home (set r = km). • long-distance travel of people is usually influence by friends. the relative influence of a friend who lives km away is times greater than the influence of a friend who lives km away. • there is a % possibility that a user who travels more than km from her home will pay a visit to an existing friend's home. moreover, the probability remains constant after the km mark though the number of friends decreases with the distance. • ''check-in'' data is with a spatial accuracy of about km. above all, individuals can be geographically divided into: the intimates (with a largest probability to be infected) within km; the fellows (with a larger probability to be infected) within the range of - km; the potential contacts (with a medium probability to be infected) beyond km but within km; the distant contacts (with small probability to be infected) beyond km. four types could transform into each other as distance changes. table . in fig. we show the geographic position (left panel) and relational network (right panel), where nodes are positioned using the geographic locations of mar. . initially, all the individuals are distributed around the world, mostly in north america, europe, japan and new zealand. it seems like that people who lived in america, europe and japan tend to keep a close contact with each other. especially, there are most lines scattered from america to other districts. it is supposed that americans tend to travel around the world and live in foreign countries, meanwhile keeping a close touch with their internal friends. the mechanism of disease spreading and preventing is as follows. for node i, which has been infected, the first week is defined as latent period, when it is hard to observe the obvious symptoms of disease. the infected node would keep its periodic activities in this period, and most of neighbors within scope of km would be infected unconsciously. until the next week (invasion period), the node would be isolated at a high probability of v . if isolated, the node would deliver immune messages to its direct and indirect neighboring nodes. if not, it would continue to spread disease within the scope of km. the process is derived from eqs. ( . ) and ( . ). without any prevention measures, the disease would spread all over the world within three months (fig. ). it can be attributed to the convenient and fast movement of human beings. in fig. (a) , the red line describes the diseases which are infectious in both latent period and invasion period, while the orange line depicts diseases which are infectious only in latent period. making a comparison, the former is over . % than the latter. after analyzing, we summarize two reasons to express the weak distinction: firstly, most neighboring nodes have been infected and the space of infection becomes smaller in invasion period; secondly, the high isolation rate reduces the number of infectors. the green line in fig. (b) represents the number of infectors while taking preventive measures. compare to the previous red line, number of infectors decreases over . %. we display the details of disease spreading process in fig. . green points represent the susceptible people; red points stand for the infected people; blue points denote both the isolators and immune people (who collect enough messages). source of infection is located in north america. to simulate the free spreading process of disease, there is no prevention measure in the beginning. as we can see from the first four panels, the uncontrolled disease spreads from america to the europe and asia successively. in fact, the prevention measures are started at the fourth week but remain with a weak and limited power. we attribute it to the latent period of disease, which is set to a week basis. later in the fifth and sixth weeks, the regional infectious disease evolves into a global outbreak. most of patients are experiencing the invasion period clinically and may be isolated at a very high rate. the immune measures show their great effects right now. the covering square of blue points is expanding. after nine weeks, the epidemic is under control completely. in this paper we provide a novel model about the control of infectious diseases and perform it on different networks. the main conclusions are as follows. first, in the model, two kinds of disease prevention measures are proposed: quarantine and message delivery. under the premise of medical research, we assume the rates of patients who are isolated in latent period and invasion period are different. besides, the specific messages sent from the isolators to their direct and indirect neighbors are originally proposed. information quality and information accumulating effect are also considered in the model. second, several valuable results are obtained by simulating the model in scale-free and small-world networks: (i) the increasing of isolation rate in invasion period, as long as over . , contributes little to the prevention of epidemic; the improvement of isolation rate in latent period is key to control the spread of disease. (ii) infectious diseases are easy to control as long as the propagating message achieves a certain quality. (iii) in the scale-free network with a very low degree (⟨k⟩ = ), the epidemic will not diffuse on the network despite the weak immunity in population. (iv) compared to scale-free network, epidemics in small-world network appear three characteristics corresponding to the number of infectors: slow growth, slow convergence and lower peak. finally, in general, the mobility and communication of population can be time-varying. based on the specific research about human periodic activity, we constitute a social network which has a certain similarity with the real world, in which the activity of human presents periodic trend and certain randomness. experimental results show that the proposed strategies have great effect on the control of disease. risk factors for human disease emergence the spread of awareness and its impact on epidemic outbreaks endemic disease, awareness, and local behavioural response disease containment strategies based on mobility and information dissemination modeling the dynamical interaction between epidemics on overlay networks asymmetrically interacting spreading dynamics on complex layered networks model for rumor spreading over networks complex dynamics in a singular leslie-gower predator-prey bioeconomic model with time delay and stochastic fluctuations spreading of periodic diseases and synchronization phenomena on networks influenza epidemic spread simulation for poland-a large scale, individual based model study effects of epidemic threshold definition on disease spread statistics analysis of the impact of education rate on the rumor spreading mechanism the mathematical theory of infectious diseases and its applications incubation periods of acute respiratory viral infections: a systematic review the distribution of incubation periods of infectious diseases extending the sir epidemic model design, synthesis and biological evaluation of functionalized phthalimides: a new class of antimalarials and inhibitors of falcipain- , a major hemoglobinase of malaria parasite fruit bats as reservoirs of ebola virus marburg hemorrhagic fever measles virus for cancer therapy effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) are we there yet? the smallpox research agenda using variola virus hospital outbreak of middle east respiratory syndrome coronavirus a contribution to the mathematical theory of epidemics emergence of scaling in random networks mean-field theory for clustering coefficients in barabási-albert networks on the properties of small-world network models friendship and mobility: user movement in location-based social networks this research is supported by the national natural science foundation of china (nos: , and ), and program for liaoning excellent talents in university (no: lr ). key: cord- -aqh zxgh authors: el homsi, maria; chung, michael; bernheim, adam; jacobi, adam; king, michael j.; lewis, sara; taouli, bachir title: review of chest ct manifestations of covid- infection date: - - journal: eur j radiol open doi: . /j.ejro. . sha: doc_id: cord_uid: aqh zxgh coronavirus disease- (covid- ) is a viral pandemic that started in china and has rapidly expanded worldwide. typical clinical manifestations include fever, cough and dyspnea after an incubation period of - days. the diagnosis is based on rt-pcr test through a nasopharyngeal swab. because of the pulmonary tropism of the virus, pneumonia is often encountered in symptomatic patients. here, we review the pertinent clinical findings and the current published data describing chest ct findings in covid- pneumonia, the diagnostic performance of ct for diagnosis, including differential diagnosis, as well the evolving role of imaging in this disease. serum immunoglobulins (igm and igg) can be used to diagnose recent infection to covid- [ ] . for example, a study demonstrated that the positive detection rate is increased ( . %) when combining igm assay with rt-pcr compared to rt-pcr alone ( . %) [ ] . a total of original papers published in english totaling over patients were reviewed [ , . reviews and case reports were excluded. most reviewed papers originate from china [ , - , - , - , - ] and the remainder from japan [ , ] , korea [ ] and italy [ , ] . three papers were in collaboration between chinese and us institutions [ , , ] . chest radiography (cxr) findings in patients with covid- have been described in small case series [ , ] . cxr is negative in %- . % of cases despite positive findings on chest ct ( fig. ) [ , ] . the most common radiographic finding is opacification, often with a peripheral and lower lung distribution [ , ] (fig. ) . thus, cxr is not recommended for routine diagnosis of covid- , but may be helpful for follow-up. a low dose, chest ct without intravenous contrast is generally used for evaluation [ ] . typically, covid- pneumonia presents with bilateral peripheral patchy ground-glass opacities (ggo) with or without consolidation [ , , ] (fig. , ) . ggos may have a rounded morphology [ , , , , ] in up to % of the cases (fig. ) . superimposed interlobular septal thickening can also be present, resulting in a crazy-paving pattern (fig. ) . vascular enlargement, air bronchograms, and a halo sign have also been described (fig. ) . the ggos and consolidative lesions are larger than cm in % of cases [ ] . the lower lobes and posterior portions are more frequently involved [ ] . mediastinal and hilar lymphadenopathy, pleural effusions or thickening, discrete pulmonary nodules, and pulmonary cavitation have been rarely described in the chinese series [ , , ]. an italian study described lymphadenopathy in up to % of cases [ ] . table shows the prevalence of various imaging findings on chest ct at initial presentation. a japanese study described that asymptomatic patients tend to have more ggo ( %) and less consolidation ( %) compared with symptomatic patients ( % and %) [ ] . cxr and chest ct can be negative in up to . % of patients, especially in early and/or non severe disease [ ] . in mild cases, ct findings consist more frequently of ggo alone ( %), followed by ggo with consolidation ( %) [ ] . severe cases with a clinical acute respiratory distress syndrome (ards) picture present with widespread dense consolidative opacification on ct (see below). several studies categorized covid- ct findings into several stages based on time since the onset of symptoms [ , , , , ] . these were classified into main successive stages: early, intermediate and late [ ] and a fourth resorptive stage [ ] .  early stage (≤ days): more than half of the patients have negative chest ct ( %). the remaining patients have predominantly ggo ( %) and consolidation ( %). imaging findings when present were often unilateral [ ] . similar findings were described in another study [ ] , which demonstrated that in early disease . % of cases present with a solitary lesion seen in % of cases in the right lower lobe.  intermediate stage ( - days): as the disease progresses, more ggo ( %) and consolidation ( %) are noted bilaterally ( %) and with a peripheral lung distribution ( %). only % of patients have negative chest ct [ ] . % and % of patients have ggo and consolidation, respectively. the imaging findings are bilateral in % and peripheral-predominant in % of cases [ ] .  absorption stage/fourth stage (> days): % and % of patients have ggo and consolidation, respectively. the imaging findings are bilateral in % and peripheralpredominant in % of cases [ ] . when patients improve, the lung disease evolves and organizes, and fibrous bands may appear [ , , , ] . a "reverse-halo sign" is occasionally observed and can be an indication of organizing pneumonia [ , ] . in severe cases, the consolidation and ggo increase and involve all five lung lobes, resulting in a dense consolidative appearance and characterized by diffuse alveolar damage, usually with a poor prognosis [ ] (fig. . ) it has been suggested that imaging can help predict severity of covid- disease. for example, a study showed that a severity score based on cxr at initial presentation in nonelderly patients ( - y) predicts outcome (hospital admission and intubation) [ ] . another study suggested that severe/fatal cases were more likely to have more extensive and diffuse disease with more frequent architectural distortion and bronchiectasis compared to mild/common cases [ ] . however, more data is needed particularly in comparison with blood tests, such as cytokine levels. oncology patients: a retrospective study in oncologic patients showed similar findings of ggo ( %) and patchy consolidation ( . %). the presence of the latter at admission was associated with worse outcome [ ] . pediatric and pregnant patients: few studies have described chest ct findings in children [ ] [ ] [ ] [ ] who tend to have milder findings. the largest study included patients [ ] , % of whom had normal chest ct, % had ggo, and % had consolidation with a surrounding halo (which was considered a typical finding in this population). bilateral disease was observed in % of cases. co-infection with another pathogen was identified in %- % of the cases [ , ] . another study [ ] found that pregnant women have more consolidation (up to %) as compared with the non-pregnant population ( %). pregnant woman also manifested with bilateral and peripheral-predominant ggo. pleural effusions were more commonly encountered in pregnant than in non-pregnant women, up to % versus %. elderly population: older patients (> y) have similar (classic) chest ct findings [ ] . however, they tend to have more areas of lung involvement with more lobes affected and more pleural thickening [ ] . similar findings were obtained in another study [ ] , where patients older than years had more extensive disease with more consolidation as compared with patients younger than . they tended to have more architectural distortion, bronchiectasis, mediastinal and hilar lymphadenopathy and pleural effusions and worse outcomes than younger patients [ ] . covid- and pulmonary embolism: an increased incidence of acute pulmonary embolism in hospitalized covid- patients has been reported [ ] . out of patients, % had an acute pulmonary embolism and it was associated with elevated d-dimer (up to . ug/ml). covid- can be associated with pulmonary embolism especially when d-dimer is elevated (fig. ) , and preventive anticoagulation has been recommended in hospitalized patients [ ] . [ , ] . a study performed by bai et al found that of radiologists had high specificity ( - %) and moderate sensitivity ( - %) in distinguishing covid- pneumonia from other viral pneumonias, although there was variability in radiologist performance [ ] . compared to viral pneumonia, covid- tends to have a more peripheral distribution, greater ggo, increased vascular enlargement and a "reverse-halo sign". zhao et al also demonstrated that covid- had more frequent bilateral ggos compared to non-covid- viral infections [ ] . key findings less frequent in covid- pneumonia compared to non-covid pneumonia included air bronchograms, centrilobular nodules, tree-in-bud opacities, bronchial wall thickening and a reticular pattern [ , ] . other coronavirus organisms with genetic resemblance to covid- can produce similarly devastating pulmonary syndromes, including sars ( . % mortality rate) and mers ( . % mortality rate) [ , ] . while there have been no documented cases of sars since , mers cases are still reported sporadically. all three of these infections cause peripheral multifocal airspace opacities (ggo and/or consolidation), without pneumothorax, cavitation or lymphadenopathy. differences in the imaging appearances have been reported, as sars tends to be unilateral and focal in distribution ( %) and mers patients can develop pleural effusion ( %) [ , ] . both sars and mers are associated with constriction of the pulmonary vasculature, whereas enlargement of the vasculature has been reported in covid- [ ] . pulmonary fibrosis was reported as a late manifestation in mers, but not in sars. the longterm sequelae of covid- remain to be determined; however, early publications indicate a fibrotic phase characterized by reticulation, interlobular septal thickening and traction bronchiectasis [ , ] . finally, a variety of other noninfectious pulmonary diseases can produce imaging findings on ct that may overlap with covid- pneumonia, including pulmonary edema, ards, organizing pneumonia, prior treatment (i.e. drug toxicity or radiation pneumonitis/fibrosis), pulmonary infarction, alveolar hemorrhage, and interstitial lung diseases [ ] . both ards and bacterial superinfection can complicate covid- pneumonia. while there is an abundance of literature being published at a rapid rate, results of these studies must be scrutinized and interpreted with caution as a variety of sources of bias may produce artificially high performance characteristics of ct and certain imaging findings for covid- pneumonia [ ] . ultimately, given the variable and non-specific nature of the imaging findings in covid- , integration of clinical history, laboratory tests, in addition to quantitative approaches including machine learning or artificial intelligence (ai, see below), are needed for accurate assessment. several studies compared the accuracy of chest ct against rt-pcr [ , , , , ] . abnormal chest ct findings can be present despite a negative rt-pct with a sensitivity ranging between %- % [ , , , ] . however, according to raptis et al this high sensitivity can be explained by a selection bias of the studied population and low threshold for positive disease on chest ct [ ] . using rt-pcr as a reference, specificity of chest ct ranges between %- % and accuracy between %- % [ , ] . the positive predictive value and accuracy of chest ct were higher in patients older than years. % of cases had improvement of imaging findings before negative conversion of rt-pcr [ ] . the positive rate of rt-pcr was %- % [ , , ] . this can be explained by the fact that rt-pcr was obtained through throat swabs which have low positive rates [ , ] as viral pneumonia does not usually produce purulent sputum [ ] . with the rapid evolution of this outbreak, the global community of radiologists has convened to construct guidelines and position statements regarding the appropriate use of imaging during this pandemic. early in the outbreak, hospitals in china were using chest ct in many suspected patients, as well as to follow-up the progression of lung disease. in the us, the centers for disease control and prevention (cdc), the american college of radiology (acr), the society of thoracic radiology (str), and the american society of emergency radiology (aser) issued their position statements recommending against the use of ct for widespread screening and diagnosis of covid- , instead reserving ct for those cases with clinical suspicion for complications like abscess or empyema [ ] [ ] [ ] . this was largely spurred by the early research on chinese patients which showed that imaging, specifically ct, has a wide range in sensitivity for detecting covid- pneumonia [ , ] . in addition, the ct imaging features of covid- j o u r n a l p r e -p r o o f page of are sometimes nonspecific and can overlap with other infections as discussed above. the positive predictive value of ct is low in locations where covid- prevalence is low [ ] . the multinational consensus statement of the fleischner society [ ] recommended chest ct for patients with moderate to severe symptoms or with mild disease with associated comorbidities or with worsening respiratory symptoms. the european society of radiology (esr) and the european society of thoracic imaging (esti) outlined their recommendations in a consensus document, stating that chest ct should not be performed as a screening tool in patients with "mild or no symptoms" [ ] . however, they add on that chest ct may play a useful role in patients with milder symptoms who have co-morbidities (e.g. diabetes, obesity, chronic respiratory disease, etc.). in their statement, they also note that with further research, information gained from a baseline ct in covid- patients may play an important role in predicting who will have poor outcomes or need ventilation. with the spread of covid- globally and the increasing number of cts performed in patients under investigation (puis), there have been frequent questions in the radiology community regarding the optimal way to report ct findings potentially attributable to covid- pneumonia. a national expert panel of thoracic radiologists gathered to produce a consensus statement on this topic, subsequently endorsed by the radiological society of north america (rsna), the str, and the acr [ ] . chest ct findings were classified for covid- pneumonia into four groups: typical appearance, indeterminate appearance, atypical appearance, and negative for pneumonia. standardized language templates are provided in [ ] with the goal of decreasing reporting variability and increasing clarity by using consistent language. the question of whether to include terminology such as "coronavirus" or "covid- " in reports remains an area of debate. the authors acknowledge that for patients with unexpected findings that could be attributable to covid- , the matter is complex and consultation with local clinical colleagues is needed to establish an approach. the term "viral pneumonia" is considered a reasonable alternative. given the variable sensitivity in rt-pcr for the diagnosis of early covid- and the overlap in the imaging appearances with other pulmonary diseases, ai approaches have been applied in an effort to improve the diagnostic accuracy of ct. in a recent retrospective multicenter study of , chest cts from , patients, a deep learning model detection neural network (covnet) in an independent test set demonstrated excellent per-exam auc, sensitivity and specificity of . , % and %, respectively for detecting covid- compared to cap and other nonpneumonias [ ] . other studies using deep learning approaches have found similar results with reported aucs up to . [ , ] and accuracy up to . % [ ] for the classification of covid- from non-covid- pulmonary infection. a recently published algorithm integrating chest ct and clinical history in patients presenting early in the course of the disease reached an auc of . and had higher sensitivity ( . %) when compared to a senior thoracic radiologist ( . %) and a thoracic radiology fellow ( %). the algorithm was able to identify % of cases presenting with positive rt-pcr but normal chest ct. this method may have a role for a quick diagnosis of covid- pneumonia with ct [ ] . more work in terms of performance and clinical implementation/acceptance is needed. page of covid- is a new rapidly spreading pandemic. it has typical ct findings with ggos and consolidation often with a peripheral and lower lung distribution. in early disease, imaging findings can be absent; therefore, ct chest cannot be used as a screening method, and rt-pcr remains the reference diagnostic test. ai may play a role in the rapid diagnosis of covid- , but more data is needed to assess its added value. there is no conflict of interest associated with this publication and there has been no financial support for this work posterior chest x-ray shows ill-defined opacities in the lateral aspect of the right lung (arrows). (b) axial noncontrast chest ct image shows ground-glass opacities with a peripheral distribution in both upper lobes (arrows). there is superimposed interlobular septal thickening with visible intralobular lines in the ground-glass opacity (crazy paving pattern). 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covid- patients and the radiology department -advice from the european society of radiology (esr) and the european society of thoracic imaging (esti) radiological society of north america expert consensus statement on reporting chest ct findings related to covid- . endorsed by the society of thoracic radiology, the american college of radiology, and rsna rapid ai development cycle for the coronavirus (covid- ) pandemic: initial results for automated detection & patient monitoring using deep learning ct image analysis deep learning enables accurate diagnosis of novel coronavirus (covid- ) with ct images deep learning system to screen coronavirus disease artificial intelligence-enabled rapid diagnosis of patients with covid- more than lobes affected uncommon findings centrilobular nodules tree-in-bud nodularity key: cord- -rvwxysvc authors: panfili, f. m.; roversi, m.; d’argenio, p.; rossi, p.; cappa, m.; fintini, d. title: possible role of vitamin d in covid- infection in pediatric population date: - - journal: j endocrinol invest doi: . /s - - - sha: doc_id: cord_uid: rvwxysvc purpose: covid- is a pandemic of unprecedented proportion, whose understanding and management is still under way. in the emergency setting new or available therapies to contrast the spread of covid- are urgently needed. elderly males, especially those affected by previous diseases or with comorbidities, are more prone to develop interstitial pneumonia that can deteriorate evolving to ards (acute respiratory distress syndrome) that require hospitalization in intensive care units (icus). even children and young patients are not spared by sars-cov infection, yet they seem to develop a milder form of disease. in this setting the immunomodulatory role of vitamin d, should be further investigated. methods: we reviewed the literature about the immunomodulatory role of vitamin d collecting data from the databases medline and embase. results: vitamin d proved to interact both with the innate immune system, by activating toll-like receptors (tlrs) or increasing the levels of cathelicidins and β-defensins, and adaptive immune system, by reducing immunoglobulin secretion by plasma cells and pro-inflammatory cytokines production, thus modulating t cells function. promising results have been extensively described as regards the supplementation of vitamin d in respiratory tract infections, autoimmune diseases and even pulmonary fibrosis. conclusions: in this review, we suggest that vitamin d supplementation might play a role in the prevention and/or treatment to sars-cov- infection disease, by modulating the immune response to the virus both in the adult and pediatric population. the recent epidemic of covid- has rapidly become an emergency threat for the health systems of all infected countries by overloading the medical facilities and intensive care units and causing thousands of deaths. in order to allow the national health systems to organize and set more beds, devices and medical personnel, the local governments have taken extraordinary measures to restrain the spread of the epidemic, including limitation of individual movements, shutdown of all less necessary economic activities and isolation of areas and cities where the epidemic has clustered. given the dire circumstances, the efforts of the researchers have been focused on the prevention and treatment of this disease. nonetheless, while the development of new drugs or vaccines would take a long time before human testing and commercialization, clinical trials on available pharmaceuticals with a potential effect against covid- could more easily yield useful results and, as a matter of fact, are already being performed. vitamin d is usually known for its role in the maintenance of bone health and calcium-phosphorus metabolism, yet many other roles of this hormone have been recently discovered, such as modulation of the immune response in both infectious and autoimmune diseases. just recently, it is being hypothesized that vitamin d supplementation could be used as a preventive or event therapeutic option in covid- , but more studies are needed to validate this association. in the following, we describe vitamin d immunomodulatory role and effect on some infectious filippo m. panfili and marco roversi have contributed equally to the manuscript. and autoimmune diseases that might share similarities with covid- , based on the current and future understanding of the pathogenesis of this infection. in december , an epidemic of pneumonia caused by a novel virus later identified as severe acute respiratory syndrome coronavirus (sars-cov- ) started in the city of wuhan, hubei province, china. recently, the world health organization (who) has declared coronavirus disease , a pandemic of global health concern. most people infected by sars-cov- develop interstitial pneumonia with ground-glass opacities at the ct scan, while a minority of patients develop acute respiratory distress syndrome (ards), and need hospitalization in intensive care units (icus). as of april , , a total of , , laboratoryconfirmed cases and , deaths have been documented [ ] . in italy, the first european country to deal with a rapid increase in the number of covid- cases in the last month, the epidemic is cornering the national health system (ssn), which usually provides high-level and free-for-all health care. despite the total shutdown of the country to limit the spread of the epidemic and the increasing of icu personnel, devices, and beds are timely met, the recent estimates predict a gloomy development of the epidemic, with clinical facilities at their maximum load and intensive care specialists forced to allocate the life-saving care to the youngest and fittest [ ] . that is, making the uncomfortable choice between who lives and who dies. the demographics of the covid- outbreak proves that elderly males, with or without comorbidities, are the most severely affected across all populations. in a large report of , cases from the chinese center for disease control and prevention [ ] , % of cases were aged - years and % were aged years or more, with a case-fatality rate of % and . %, respectively. critical disease requiring admission in icu and mechanical ventilation was observed in % of all cases, with a case-fatality rate of %. in a sample of laboratory-confirmed cases of covid- [ ] . % of patients were females and . % of patients were years old or younger. although children appear to be the least affected across all ages, they might still be responsible of the transmission of the virus. in a cohort of pediatric cases of covid- [ ] up to . % of patients did not have any symptoms of infection or radiologic features of pneumonia and % of these patients had radiologic features of pneumonia without presenting any clinical sign. in a report on . pediatric cases of covid- more than % of patients with clinically or laboratory diagnosed infection had asymptomatic, mild, or moderate disease. half of the % critical cases of covid- in this study were less than year of age [ ] . most people depend on sunlight exposure to produce the required amount of vitamin d [ ] . differences in sunlight-dependent production of vitamin d is greatly influenced by season, latitude, time of day, skin pigmentation, sunscreen use, and age, with the elderly generating % of the vitamin d produced by younger ones in the same amount of time. as very few nutrients naturally contain vitamin d, dietary intake of vitamin d is generally insufficient. thus, fortification of food and/or oral supplementation is often necessary. in adults, vitamin d supplementation is recommended for all ages ( ui/day from to years of age; ui/day > years of age; up to / ui/day to maintain a blood level of vitamin d above ng/ml), with a higher dosage required for pregnant or lactating women and subjects at risk of osteoporosis [ ] . in the pediatric population, vitamin d supplementation ( ui/day from birth to months of age and ui/day beyond months of age) is usually recommended for the prevention of rickets and osteomalacia [ ] . the ultraviolet b radiation is absorbed by -dehydrocholesterol in the skin, leading to its conversion to previtamin d , which is rapidly transformed into vitamin d (cholecalciferol). the molecule then undergoes further processing in the liver [ -hydroxycholecalciferolorcalcifediol] by cytochrome cyp r and in the kidney ( , -dihydroxy cholecalciferol or calcitriol) by cytochrome cyp b , before reaching its cellular targets. here, the activated vitamin d binds to the nuclear vitamin d receptor (vdr) and forms an heterodimeric complex with the retinoic acid x receptor that recognizes specific dna sequences, known as vitamin d responsive elements (vdre), resulting in the expression of the vitamin d responsive genes via a variety of transcriptional factors (fig. ). although vitamin d is usually acknowledged for the maintenance of bone health and calcium-phosphorus metabolism, many other roles of this hormone have been recently discovered, such as stimulation of insulin production, effects on myocardial contractility, prevention of inflammatory bowel disease (ibd), and promotion of thyroid-stimulating hormone (tsh) secretion. furthermore, the immunomodulatory role of vitamin d has been the subject of several studies [ ] . to date, it is known that the vdr activation can regulate the expression of more than genes, many of which are involved in innate and adaptive immunity. vdr is expressed in almost all immune cells, including activated cd + and cd + t cells, b cells, and antigen-presenting cells, such as macrophages and dendritic cells. the receptor acts as a modulator of innate and adaptive immunity [ ] . it is also known that vitamin d enhances the expression of two antimicrobial peptides called cathelicidin and β-defensin, and that play a key role in innate immunity [ , ] . these peptides are involved in direct microbicidal effects and have also shown pleiotropic effects in inducing immunomodulatory responses to pathogen stimuli. in particular, human cathelicidin peptide ll exhibits a variety of effects, through interacting with formyl peptide receptor-like (fprl ), recruiting neutrophils, monocytes, and t cells to infectious sites. it also promotes apoptosis of infected cells and showed a potent antiviral effects on a variety of viruses, such as hiv- , influenza viruses, hsv - , rhinovirus, and hcv [ ] . a number of studies reported a high prevalence of vitamin d deficiency among hiv-infected individuals. more specifically, faster hiv progression and severity, lower cd + counts, increased risk of mortality, and increased vulnerability to mycobacterium tuberculosis were reported [ ] . although the effect of normal to high levels of vitamin d on increasing cd + count is still unclear, a recent review proved that vitamin d plays an important role in reducing the immune activation of hiv-infected patients. in particular, the paper reports that supplementation with a daily dose of vitamin d between - iu for at least weeks can reduce the expression of cd and ki in cd + t lymphocytes, inflammatory monocytes (cd + cd +), as well as the expression of pd + (an exhaustion marker) in cd + t cells, with an increase in cd +/cd + t cell ratio. this leads to the important assumption that brisk supplementation in vitamin d-deficient hiv-infected patients could help reduce chronic inflammation and comorbidities [ ] that are still frequent among these patients despite the constant amelioration of combination antiretroviral therapy (cart) [ ] . human cathelicidin peptide ll also modulates the recognition of viral dsrna by toll-like receptor (tlr ). it is known that activation of toll-like receptors (tlrs) generate antimicrobial activity against intracellular pathogens and it has been demonstrated that tlr activation expressed by human macrophages can upregulate the expression of vitamin d receptor and vitamin d- -hydroxylase genes, leading to the production of antimicrobial peptide (cathelicidin) and stimulate the intracellular killing of mycobacterium tuberculosis [ ] . vitamin d also promotes selftolerance by shifting the cytokine patterns from a th- to a th- environment. this results in a reduction in th -and in this picture, we resumed on the left side the principal functions of vitamin d. in particular, we enlightened the anti-proliferative and anti-inflammatory function on adipose tissue, the anti-fibrotic effect on lung tissue, the inhibition of the aromatase enzyme with subsequently reduction in estradiol, the preventing role in developing autoimmune diseases and its immunomodulatory role on the innate and adaptive immune systems. on the right side, it is shown the "cytokine storm" caused by sars-cov- . we highlighted also the possible interaction role of dpp- /cd -r, reduced by supplementation of vitamin d with sars-cov- . ace- angiotensin-converting enzyme- , rxr retinoid x receptor, tlrs toll-like receptors, vdr vitamin d receptor, vdre vitamin d response element, wbc white blood cell th -stimulating cytokines with depletion of th- cells (which are known to be linked to tissue damage and inflammation) and upregulation of regulatory type- (t reg) cells [ ] . it has also been demonstrated that vitamin d is capable of inducing autophagy and apoptosis in infected cells, throughout several mechanisms [ ] . finally, -hydroxyvitamin d and , (oh) d also modulate t-cell immunity, reducing pro-inflammatory type cytokines (such as, il- , ifn-γ, il- , il- , tnf-α, and il- ) and increasing antiinflammatory type cytokines (such as, il- , il- , and il- ) [ ] . more specifically, , (oh) d inhibits proliferation of plasma cell and immunoglobulin secretion, and it induces b cell apoptosis [ ] . in summary, many studies have demonstrated the immunomodulatory influence of vitamin d both in adult and pediatric patients, by enhancing and coordinating the innate and adaptive response in different pathologic conditions (table ). several studies have demonstrated that higher levels of vitamin d are associated with better prognosis and outcome in infectious diseases [ ] . indeed, vitamin d has been extensively studied as a putative preventive and therapeutic agent for acute respiratory tract infections (artis) in both adults and children, especially in developing and low-income countries, owing to its safety and low cost. as a matter of fact, pneumonia is the leading cause of death in children in the world [ ] . a great number of studies have hypothesized a positive correlation between vitamin d deficiency and the risk of developing artis [ ] . however, the link between vitamin d deficiency and acute respiratory infections occurring during the winter season, despite being frequently brought up in the literature, has not been unequivocally confirmed [ ] . moreover, discrepancies between different age groups have been observed. vitamin d supplementation, as already described, seems to decrease proinflammatory cytokines in the lung via modulation of both macrophages and t lymphocytes activity [ ] . in a recent meta-analysis, including randomised controlled studies (rcts), it has been demonstrated that vitamin d supplementation reduced the risk of developing acute respiratory tract infections, with a higher protective effect in those who received weekly vitamin d supplementation or in those with low levels of vitamin d at baseline ( -hydroxyvitamin d levels < nmol/l) [ ] . while it is supposed that serum levels of vitamin d between ng/ml and ng/ml should be adequate to provide an immunomodulatory effect, there has been great uncertainty on the vitamin d supplementation regimen to adopt [ ] . a recent systematic review studied the role of vitamin d as an adjunctive therapy to antibiotics in acute childhood pneumonia. it included seven rcts conducted in low-income countries that involved children. nonetheless, owing to the different supplementation regimens adopted for each study and the lack of reporting on the etiology of pneumonia, only low-to very low-quality evidence was made available [ ] . in fact, its role as possible adjuvant to antibiotics treatment of acute childhood pneumonia has already been undetermined in a previous cochrane review [ ] . a more recent study also showed how high-dose vs standard-dose wintertime vitamin d supplementation did not reduce viral upper respiratory tract infections in young healthy children [ ] . nonetheless, the ineffectiveness of vitamin d in the younger might be due both to the lower prevalence of vitamin d deficiency, when compared with the elderly, and to a putative threshold effect of vitamin d in preventing arti. it has been, indeed, demonstrated that the negative correlation between levels of vitamin d and respiratory tract infections, should be attributed to its active form ( , -oh -vit d); therefore, restoring the levels of inactive vitamin d might not be sufficient in some patients, like those affected by chronic kidney or liver disease [ ] . interestingly, it has also been demonstrated that vitamin d supplementation in patients with ventilator-associated pneumonia (vap) can significantly reduce il- , that can be considered a prognostic marker, and the mortality rate in patients treated with vitamin d was significantly lower than that of the placebo group. the authors concluded suggesting the administration of vitamin d at a high intramuscular dose ( . ui) as an adjunct to the standard treatment of vap patients [ ] . in a variety of animal models pretreatment with , -oh -d proved effective in mitigating or preventing the onset of type diabetes mellitus (dm ), multiple sclerosis (ms), rheumatoid arthritis (ra), systemic lupus erythematosus (sle), crohn's disease, thyroiditis, psoriasis, polymyalgia rheumatica, and autoimmune gastritis [ , ] . an interesting study conducted on . subjects showed a statistically significant correlation between vitamin d status and development of autoimmune disease in danish population [ ] . interestingly, in another study specific allelic variants of vdr (especially bsmi, apai, taqi, and foki polymorphism genotypes) have been associated with higher susceptibility to develop an autoimmune disease (i.e., bsmi and foki polymorphism for sle or apai, bsmi, and taqi polymorphisms for ra) [ , ] . this is of particular interest and it could explain the variability of responses to vitamin d supplementation in autoimmune and infectious diseases across the different regions of the world [ ] . a significant inverse correlation was reported between disease activity and serum vitamin d concentration in sle [ ] . a preventive role of vitamin d has also been demonstrated in dm . in this autoimmune disease using calcitriol supplementation reduces serum levels of antibodies and slows the progression of β cell destruction down in the early stages of the disease [ ] , interestingly, it has also been demonstrated that in systemic sclerosis (ssc) [ ] the vdr could act as a negative regulator of tgf-β/ hydroxyproline, col a , col a and alfa-sma mrnas ↓ prevention of bleomycin-induced lung fibrosis in a murine model [ ] smad signaling, thus making vitamin d a putative antifibrotic treatment in the early stages of the disease. although the immunoregulatory function of activated vitamin d has been widely demonstrated, its role in modulating disease activity in patients with autoimmune diseases, such as rheumatoid arthritis was only recently showed [ , ] . in a german study on a selected cohort of patients with juvenile idiopathic arthritis (jia), vitamin d levels not only proved to be lower than those of their peers from the general population, but were also found to be inversely correlated with disease activity and the risk of progression to an extended disease course and/or jia-associated anterior uveitis [ ] . however, many other studies could not demonstrate a significant inverse correlation between vitamin d levels and jia disease activity, probably owing to the diverse geographical origins of the patients and the lack of an established single measure that could serve as an accurate indicator of childhood disease activity [ ] . although a sudden increase in the incidence of kawasaki disease (kd) was noted in the province of bergamo, italy, which was profoundly affected by the covid- epidemics [ ] , another study reported a significant severe vitamin d deficiency in a cohort of children with kd as compared to healthy controls, and low levels of vitamin d seems to correlate to the risk in developing coronary artery aneurysms (p = . ) and non-aneurysmatic cardiovascular lesions (p < . ) [ ] . moreover, vitamin d deficiency has been associated with resistance to intravenous immunoglobulin in kd (defined as persistent or recrudescent fever ≥ h after the completion of the initial infusion), suggesting a potent immunomodulatory role of this vitamin [ ] . of note, most of the aforementioned studies could not define the optimal concentration of (oh) d and the corresponding dietary requirements or treatment regimen suitable to the given disease. recently, it has been demonstrated that high levels of vitamin d (directly activated by respiratory tract cells through cyp b ) could reduce pulmonary fibrosis by decreasing the levels of pro-inflammatory cytokines (il- beta) produced by pulmonary fibroblast cell lines in a mouse model of bleomycin-induced lung fibrosis [ ] . in another study vitamin d administration prevented bleomycin-induced lung fibrosis in mice, by decreasing the levels of hydroxyproline and col a , col a , and alfa-sma mrnas [ ] . in the same study, pretreatment with vitamin d reduced profibrotic stimuli and restored tgfb -induced downregulation of vdr mrna levels. elsewhere, vitamin d deficiency proved to activate the ras pathway with induction of tgf b- [ ] . another pathway inhibited by , (oh ) vitamin d [ ] is the wnt/beta-catenin signaling, which has also been reported as involved in pulmonary fibrosis. the demographics of the covid- outbreak proves that elderly males, with or without comorbidities, are the most affected across all populations. the available data on the epidemic are also showing a lesser involvement of vast areas lying in the tropics. although this could easily relate to the lower median age of the population of developing countries, it is harder to make such an inference when looking at the markedly slow march of the covid- epidemic in countries of the southern hemisphere, such as australia [ ] . just recently [ ] , it has been directly hypothesized that vitamin d supplementation could be used as a therapeutic combination in covid- , based on the epidemiology of the disease, and on the decreased vitamin d status observed in calves infected with bovine coronavirus [ ] . in the emergency setting that followed the spread of the covid- pandemic new therapeutics have been empirically administered on the basis of former experience in the management of diseases sharing a few similarities with covid- -associated ards, such as inflammatory autoimmune diseases. a growing interest in the role of tocilizumab, a monoclonal antibody directed against interleukin- (il- ), which is commonly used in the treatment of rheumatoid arthritis, ensued the publication of works regarding its potential use in the prevention of severe cytokine release syndromes, such as in car-t cell treated pediatric oncologic patients [ , ] . this so-called cytokine storm has been postulated and confirmed [ ] as the main responsible for the lethal pulmonary involvement that is being observed in covid- and was thoroughly studied in former sars epidemic [ ] . in order to confirm tocilizumab therapeutic potential in ventilator-assisted covid- patients, a clinical trial is currently ongoing in china [ ] and in europe [ ] . just recently, normal to high blood levels of vitamin d proved to act synergistically with tocilizumab in patients with rheumatoid arthritis by suppressing il- enhanced osteocyte-mediated osteoclastogenesis and reducing disease activity [ ] . although the data on covid- survivors are still lacking, a further downside of the pandemic might be the development of pulmonary fibrosis, which has been widely described as a common complication of ards [ ] . here, vitamin d supplementation before and after the infection could play an antifibrotic role that yet need to be delved into. finally, we would highlight some points that should be further investigated. there is, in fact, a vast literature that shows how obesity in children is closely related with low levels of vitamin d, reaching the prevalence of % in the united states. interestingly, in a recent review, it was also showed that increased adipose tissue, altered adipocyte function and development of adipocyte hypertrophy is linked to an altered adipokine secretion profile, with increase in tnf-alpha, il- , and il- b levels. even more interestingly, the study proved that patients receiving long-term vitamin d supplementation had a reduction in adipose tissue inflammation by inhibition of tnf-alpha activity [ ] . prepubertal children have generally lower androgen levels, with an elevated estrogen to androgen ratio. it has been demonstrated that low estrogen levels are related with an increased il- beta, il- , and tnf, increased activity of th cells and high androgen levels are related with an increase in il- beta, il- , and a reduction in tnf, ifn-gamma, il- , il- , gata . it is also clear that sex hormones can differentially influence, along with other genetic polymorphisms and environmental factors, development of innate and adaptive immune responses. in a murine model, the hormonal changes of puberty upregulated the expression of genes associated with innate and adaptive immune responses in males and females, respectively [ ] . it has also been demonstrated that high levels of vitamin d seem to reduce aromatase activity (which is in turn increased by high levels of pro-inflammatory cytokines levels), thus containing the effects related to increased peripheral estrogen metabolism, such as b cell overactivity. that means that low levels of vitamin d can increase the risk of developing autoimmune diseases in young women [ ] . till date, only one recent paper addressed the relationship between vitamin d levels and the clinical outcomes of patients with covid- [ ] . the author conducted a multinomial logistic regression to explore the association between serum (oh)d level and clinical outcomes of cases with laboratory-confirmed infection of sars-cov- . interestingly, serum (oh)d proved to be a predictor of severe (or . , p < . ) and critical (or . , p < . ) covid- . a recent review proposes the supplementation of vitamin d in covid- patients based on the promising findings of rcts conducted in other viral infections [ ] . according to the emerging relationship between vitamin d status and alleged covid- infection, vitamin d supplementation has already been proposed elsewhere [ ] . although we do not assume that vitamin d plays a role in the pathogenesis of covid- , we do believe that its putative role in preventing or even treating the disease urgently needs to be further addressed. at the moment of writing, an interventional randomized clinical trial has been proposed at the university of granada, with enrollment of participants, proposing vitamin d supplementations (a single dose of , ui of vitamin d) in preventing and treating mild forms of suspected covid- [ ] . in a recent paper, it is assumed that vitamin d prophylaxis (without overdosing) could reduce, especially in patients with hypovitaminosis d, the severity of illness caused by sars-cov- [ ] . the importance of treating the hypovitaminosis d along with an early nutritional supplementation has been highlighted for the potential preventing role of malnutrition sequelae in these patients [ ] . on the basis of the possible direct and indirect effect of vitamin d on immune system and cytokines production, we speculate a possible influence of this vitamin on the immunologic response to the virus and/or a modulating effect on the drugs being administered, namely hydroxychloroquine and anti-il and anti-il agents. author contributions dr fmp conceptualized and designed the article, collected data, drafted the initial manuscript, and reviewed and revised the final manuscript. dr mr conceptualized and designed the article, drafted and wrote the manuscript, and reviewed and revised the final manuscript. dr df conceptualized and designed the article, coordinated and supervised data and reviewed and revised the final manuscript. dr mc critically reviewed the manuscript for important intellectual content. dr pd reviewed the manuscript for important intellectual content. prof pr reviewed the manuscript for important intellectual content. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. funding no funding was secured for this study. conflict of interest the authors have no financial relationships relevant to this article and no conflict of interest to disclose. cledoes not contain any studies with human participants or animals performedby the sole author d. fintini. informed consent for this type of study, informed consent is notrequired. world health organization. coronavirus disease (covid- ) outbreak covid- and italy: what next? lancet characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention [published online ahead of print clinical characteristics of coronavirus disease in china sars-cov- infection in children epidemiology of covid- among children in china s) sunlight and vitamin d for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease evaluation, treatment, 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patients hospitalized for the novel coronavirus disease (covid- ): rationale and feasibility of a shared pragmatic protocol key: cord- -aoz jbf authors: bartlett, john g. title: why infectious diseases date: - - journal: clin infect dis doi: . /cid/ciu sha: doc_id: cord_uid: aoz jbf infectious diseases is a broad discipline that is almost unique in contemporary medicine with its ability to cure and prevent disease, to identify specific disease causes (microbes), and to deal with diverse, sometimes massive outbreaks. the value of the infectious disease practitioner is now magnified by the crisis of antibiotic resistance, the expanding consequences of international travel, the introduction of completely new pathogen diagnostics, and healthcare reform with emphasis on infection prevention and cost in dollars and lives. infectious disease careers have great personal rewards to the practitioner based on these observations. it is unfortunate that we have been so effective in our work, but relatively ineffective in convincing the healthcare system of this value. students of medicine have multiple career options with various attractions and concerns. so it is with the discipline of infectious diseases. as with all medical specialties, infectious diseases has unique features that are important to highlight: among medical specialties, this one is consistently changing, often unpredictable, usually exciting, and incredibly rewarding for health impact. it is also often challenging and seemingly underappreciated, at least until needed. these facts appear to be relatively idiosyncratic to this discipline with a menu of priority pathogens that is in constant flux and weaponry that will change in unpredictable ways. the extraordinary kinetics and ability to intervene successfully using public health, preventive vaccines, and disease-limiting antimicrobials are its great strengths. the following are some of the highlights and unique features of a career in the science and practice of infectious diseases. the menu includes the litany of epidemics, heroic efforts to conquer disease, our expectations with antimicrobials, vaccines and public health, and challenges that may lead to transformative interventions. the field of infectious diseases is kinetic, unpredictable, and layered with surprises that sometime require heroic efforts from a diverse field of scientists and practitioners. • on october , a patient with fever and confusion was seen at a florida medical center by dr larry bush, an infectious disease physician. he examined the cerebrospinal fluid, saw boxcar gram-positive rods, diagnosed anthrax, and predicted bioterrorism [ ] . this was strong stuff at a time no one had thought much of bioterrorism anywhere in decades and especially in an obscure, small town in florida. the ensuing epidemiologic investigation showed anthrax spores in this patient's workplace, the local postal service, and a letter received by the patient. this was the index case of the anthrax bioterrorism epidemic that shook the country in .the result was a major national preparedness response to not only bioterrorism, but also preparedness for natural disasters, epidemics, and other major public health threats. • in , dr alan steere, a rheumatologist from yale school of medicine, led an investigation of an outbreak of arthritis involving children and adults in connecticut. most of the patients had asymmetric swelling and pain of large joints, especially knees, and some also had an erythematous, annular rash [ ] . it was initially called "lyme arthritis," but most physicians thought it was simply juvenile rheumatoid arthritis. dr steere was convinced it was an infection and moreover, that it was arthopod-born based on epidemiology and clinical features. his relentless pursuit of the pathogen was finally rewarded with the discovery of the newly recognized spirochete in the blood and in typical skin lesions [ ] . the specific agent was subsequently defined in a another extraordinary effort, this time by dr willie bergdorfer, who had spent much of his career studying the microbiology of the hindgut of ticks; he successfully isolated the pathogen that he considered his last and most important scientific project [ , ] . that agent is named in his honor: borrelia burgdorferi [ ] . • dr robin warren, a pathologist in australia, made the historic discovery that gastric biopsies from patients with gastritis showed a large burden of curved bacteria. no one paid attention until a young gastroenterologist, dr barry marshall, agreed to study the association. this pairing was considered an "odd couple"; dr warren was described as quiet, thoughtful, and persistent whereas dr marshall was self-described as brash and determined [ ] . subsequent studies consistently showed the association between this curved microbe with gastritis and peptic ulcer disease, but there was almost uniform opposition from both gastroenterologists and infectious disease physicians. larry altman, noted medical editor for the new york times, wrote that never in his experience had he witnessed such fierce opposition from the medical community to the possibility that peptic ulcer disease was caused by a microbe (l. altman, personal communication, may ) [ ] . this prompted proponents, drs barry marshall and alan morris, to perform the ultimate experiment-they swallowed a flask of helicobacter pylori (and suffered from the experience; l. altman, personal communication, may ) [ ] . the long-term result of this unrelenting battle is now well known: h. pylori is accepted as the cause of peptic ulcer disease and its sequela, guidelines for diagnostic testing and treatment are based on h. pylori as the pathogen, this agent is listed as a class carcinogen, and nobel prizes were awarded to drs warren and marshall [ ] . • in early september , dr april pettit, an infectious disease physician in tennessee, saw a patient with aspergillus meningitis following an epidural steroid injection [ ] . this prompted her to notify dr marion kainer at the tennessee health department, who then set up shop with a sleeping cot in the health department to facilitate a nonstop investigation [ ] . this was the beginning of the infamous national epidemic of exserohilum rostratum meningitis associated with the contaminated steroids that led to cases and deaths in states. credit here is to dr pettit for recognition and prompt notification, to dr kainer for her aggressive response on behalf of the victims, and to the centers for disease control and prevention (cdc) for the hasty intervention. (somewhat disappointing is the fact that compounding pharmacies are still unregulated.) • in , dr anthony fauci read the july edition of morbidity and mortality weekly report [ ] describing gay men with kaposi sarcoma or pneumocystis carinii pneumonia in california. for the first time in his life, dr fauci had what he called "chill pimples" ("chill bumps"), and this led to a career change to find the cause, treatment, and prevention of aids. this must be now viewed as possibly the most remarkably successful attack on an important infectious disease since fleming discovered penicillin. • in february , severe acute respiratory syndrome (sars) was a newly described, severe disease in humans that was often fatal and appeared to travel by air routes, but had no established pathogen or treatment. dr klaus stohr at the world health organization (who) identified the finest virology laboratories in the world and asked them to collaborate to define the pathogen with the condition that all information would be shared on the internet and there was no ownership of the data. the participating labs with varying skills were spread throughout the world, so the daily conference calls started with "good morning, good afternoon, and good evening." the etiologic agent was described in an unauthored "global alert" on april and in the lancet with "multicentre collaborative network" as the authors (see [ ] ). this unselfish collaboration under strong leadership is credited with the rapid solving of a global crisis that eventually showed cases with deaths in countries. • these anecdotal experiences (bioterrorism, lyme disease, peptic ulcer disease, iatrogenic fungal meningitis, human immunodeficiency virus [hiv]/aids, and sars) illustrate the unpredictable challenges and some of the unique responses that have left a major imprint on medicine. it is noteworthy that all started with strong leadership and came to closure with either elimination or successful management. epidemics of infections are predictable to occur, but largely unpredictable in time, place, microbe, and consequences. the following highlights some of the recent epidemic records and surprises in this category: • west nile virus was first reported in new york city in and reached a -year zenith for reported cases in with cases, including % with the dreaded neuroinvasive form of the disease [ ] . • coccidioidomycosis: the total number of reported cases increased -fold in years, from in to in [ ] . • malaria reported in us travelers reached a record high of cases in [ ] . • chikungunya virus reached a record number of cases in the caribbean with > reported cases, including in us travelers to st martin [ ] . this pathogen is highlighted because global warming is expected to make it endemic in the southern united states and because of its substantial morbidity with possible long-standing arthritic complications [ ] . • measles: the largest number of annual reported cases in the united states in years was noted during - . it now appears that will be worse [ ] . measles was declared eradicated in but has now become a problem, primarily in those refusing vaccination, but also in some with documented vaccination [ ] . measles continues to be an important infectious disease challenge due, in part, to the extraordinary public health issues that cost one health system $ to deal with the potential epidemiologic consequences of a single case [ ] . • pertussis: this infection is resurgent in the united states and europe, with increased cases including epidemics in children and adults and involving both vaccinated and unvaccinated individuals [ ] . this is thought to reflect waning immunity to the acellular vaccine and the need for a new vaccine [ ] . • meningitis: - reporting showed outbreaks of neisseria meningitidis meningitis, on college campuses and among gay men in new york city and los angeles [ ] . • influenza: this is a continual concern based on the everpresent threat of pandemics with devastating consequences ( - , - , - , - , - ) that seem difficult to predict or control [ , ] . limitations of current expertise were illustrated with influenza a(h n ) swine flu, as the standard concept based on historic precedent is that new influenza epidemics come from asia in the wintertime, but this one came in the eastern hemisphere in the summertime [ ] . the more recent threats that could pose serious consequences are influenza a(h n ) and influenza a(h n ) [ ] [ ] [ ] . both show high mortality rates, but little evidence so far of that single critical mutation permitting attachment to the hemagglutinin antigen to permit sustained person-to-person transmission [ ] . • middle east respiratory syndrome (mers) coronavirus: this coronavirus is a major global concern with analogies to the sars coronavirus in terms of its perceived potential to become a global epidemic with high mortality and no apparent treatment [ , ] . of immediate importance in the united states is recognition of risk with appropriate diagnostic testing, isolation, and management of persons with severe, unexplained pneumonia associated with recent travel to the arabian peninsula (mers) [ ]. • foodborne disease: widespread foodborne epidemics are now a common consequence of the massive food distribution system that permits contaminated beef or lettuce from mexico to reach stomachs in distant multistate areas, with medical consequences involving hundreds or thousands of people. this includes the more recent emergence of the gii. sydney strain of norovirus. these outbreaks seem likely to continue, with unpredictable pathogens in unpredictable places [ ] [ ] [ ] . • heartland virus: a recently encountered tick-borne disease in tennessee and missouri with cases and deaths [ ] . • polio-like virus infection with extremity paralysis has been recently reported in and possibly children in california [ ] . • ebola virus: who has reported an outbreak in guinea involving a new clade of this usually fatal infection [ ] . this listing could continue almost indefinitely. the point is that epidemics are the domain of infectious diseases and public health, with the expectation for management or prevention of outbreaks with requirements for detection, reporting, isolation, and case management. the listing here includes diverse pathogens, some life-threatening diseases, infections with important public health implications, an upsurge of pediatric infections in adults, many travel-related infections, multiple public health threats, and the continuous concerns for influenza and foodborne disease. the major weaponry of the infectious disease catalog includes antibiotics, vaccines, and public health. these categories are remedial reading, but some facets are worthy of emphasis. the value of antibiotics seems obvious. the first patient to receive penicillin was a young woman with β-hemolytic streptococcal bacteremia with fever of . °c- . °c daily for weeks. she received penicillin intravenously starting march , promptly recovered, and survived to age years [ ] . this would appear to be "evidence-based medicine" with an n = . the following statement from dr walsh mcdermott in summarizes this breakthrough especially well: "penicillin gave more curative power to a barefoot, itinerant care provider in the deepest reaches of africa than the collective powers of all physicians in new york city" [ ] . the more recent experience with bacterial resistance and sparse pipeline threatens this miracle, but antiviral development is quite different, primarily for hiv and hepatitis c virus (hcv). it now appears that patients with hiv can achieve near-normal longevity [ ] . hcv infection is even more impressive in terms of speed of progress and ability to cure. the hcv treatment story reflects the efficiency of basic science to define targets, pharmaceutical skills of industry, well-organized trial networks, and a regulatory agency (us food and drug administration [fda]) that facilitated product development [ ] . the impact of vaccines is also impressive. a comparison of annual incidence of vaccine-preventable diseases in the united states reported for the period prior to availability of the designated vaccine compared with its incidence in shows the decrease in polio as %; diphtheria, %; rubella, . %; mumps, . %; invasive type b haemophilus influenzae, . %; and pertussis, a disappointing %. a recent report concluded that the global total for lives saved by vaccines exceeds million [ ] . the impact could be substantially greater with more global access, fewer refusals, and a better pertussis vaccine. a recent cdc analysis of annual costs associated with major nosocomial infections totaled $ . billion per year in the united states with the following rank order by median cost/case: central line bacteremia, $ ; ventilator-associated pneumonia, $ ; surgical site infection, $ ; clostridium difficile infection, $ ; and catheter-associated urinary tract infection, $ . this illustrates the challenge and the priorities [ ] . another challenge is epidemics involving nosocomial pathogens, as shown with the klebsiella pneumoniae carbapenemaseproducing bacteria (kpc) in the national institutes of health (nih) clinical center. this began with a patient transferred from a new york city hospital with a kpc infection and became the source of an institutional outbreak that required extraordinary efforts to control, including a wall constructed to isolate cases, removal of plumbing (as a possible source), use of matrix-assisted laser desorption/ionization time-of-flight (maldi-tof) molecular diagnostics to detect cases and carriers, hydrogen peroxide room aerosols, and "whole house" surveillance cultures. the epidemic was finally halted, but the toll was cases and fatalities over months [ , ] . another kpc epidemiologic investigation showed widespread distribution of this microbe from a long-term acute-care facility in the chicago area [ ] , and others have demonstrated distribution of kpc by air travel from india to europe [ ] . these epidemics require extensive resources and specialized skills; they will be expected to increase substantially in the era of "bad bugs." there is no specialty field in medicine that demonstrates shifting priorities like infectious diseases. to illustrate this point, i have summarized the "hot topics" discussed in the "what's hot in infectious diseases" presentation to the annual meeting of the american college of physicians in , compared with the presentation in , to illustrate the nearly complete change of priorities in a relatively short time. avian influenza, rabies (first survival without vaccine), west nile virus, bioterrorism, transfusion-associated jacob-creutzfeldt disease, usa strain of methicillin-resistant staphylococcus aureus (mrsa), sars, and chlamydia pneumoniae and its role in coronary artery disease and influenza. carbapenemase-producing gram-negative bacilli, colistin, constant infusion of β-lactam antibiotics, molecular diagnostics, a litany of epidemics, new pathogens (mimivirus, borrelia miyamotoi, emmonsia species, and bradyrhizobium enterica), c. difficile gene sequencing, the microbiome, and hcv. note that the -year interval resulted in a completely new agenda for what was considered timely and important in the field based on rapid changes in topical microbes, new epidemics, and new diagnostics, (but not new antimicrobials). it is impossible to predict the menu for . it is now known that genes for resistance to antimicrobial agents were well established in bacteria at least million years before evidence of human life [ ] . the use of antibiotics has selected for these genes by mendelian laws, making it increasingly difficult to control previously treatable infections. this problem was predicted by the father of antibiotics, alexander fleming, who, in , wrote that ". . . the public will demand the drug and . . . then will begin an era . . . of abuses. the microbes are educated to resist penicillin and a host of penicillin-fast organisms is bred out which can be passed to another individual and perhaps from there to others until they reach someone who gets a septicemia or a pneumonia which penicillin cannot save. in such a case, the thoughtless person playing with penicillin treatment is morally responsible for the death of the man who finally succumbs to an infection with penicillin-resistant organisms. i hope this evil can be averted" [ ] . also of note is the prediction by nobel laureate joshua lederberg: "the future of humanity and microbes will likely evolve as . . . episodes of our wits vs their genes" [ ] . it now appears that fleming's prediction is a harsh reality and evolutionary microbial resistance genes are gaining the upper hand, reflecting the combination of massive antibiotic use and lack of new pharmacologic agents. the result is the alarming escalation of antibiotic resistance that is global and applies to nearly all categories of treatable pathogens, leading some to predict "the postantibiotic era." this resistance has been declared a "crisis" by the infectious diseases society of america, the cdc, who, the us congress, and the us president. a disturbing observation in the united states is the conspicuous absence of a national plan to deal with resistance, including the lack of a living record of antibiotic consumption and resistance correlated by location and trajectory. this is in sharp contrast to the european union, which includes countries with official languages and diverse cultures, but has systematically collected data on antibiotic consumption and microbial resistance patterns for years [ , ] . this has resulted in multiple publications with data reviews, studies of interventions, messages to consumers such as an ebug internet program for students, a european antibiotic awareness day, standardized methods to collect data [ ] and a recent -point plan with budget to address the issue [ ] . their data are striking in showing the dramatic association between per capita antibiotic use and national resistance patterns. for example, antibiotic consumption in greece is nearly times that of the netherlands, so we expect more resistance problems in greece, but the magnitude of this difference is alarming: bacteremic carbapenemase strains among all bacteremic k. pneumoniae isolates appear to be about times more common in greece, and mrsa as a percentage of all s aureus isolates is about times higher [ ] . the european union appears to have a mature and substantive model to learn from, with the important caveat that it functions well because there is no claimed ownership, as there are equal partners. there are also some good national programs that have successfully addressed specific problems to learn from: • eu data for showed that france had embarrassingly high antibiotic use rates, accompanied by increasing resistance by s. pneumoniae. this prompted a national campaign targeting prescribers and consumers on antibiotic abuse and its consequences. the goal was a % reduction in antibiotic prescriptions for the entire country; they achieved a % reduced resistance [ ] and also achieved the largest decrease in per capita antibiotic consumption for any nation in the history of the global antibiotic fund [ ] . • a recent report from israel showed a national campaign to reduce the incidence of kpc. analysis of their results with a prevention bundle showed a reduction from per patient-days to . per patient-days [ ] . • the united kingdom addressed the issue of the epidemic nap- strain of c. difficile through gene sequencing and aggressive antibiotic control. the result was a national % reduction in c. difficile infection rates [ ] . the examples given are based on national data addressing major challenges with impressive results. in the united states, this remains a unanswered challenge, but is also an opportunity for the skills of the infectious disease discipline in terms of data collection, evaluation, interventional trials, and policy implementation into practice, primarily in the form of antibiotic stewardship. recent reports using gene sequencing suggest that conventional methods of infection control could substantially improve this effort. examples: ( ) results from the united kingdom have largely disproven conventional teaching regarding the epidemiology of c. difficile infection [ ] ; ( ) this technology also appears to contradict some contemporary concepts about transmission patterns of s. aureus [ ] ; and ( ) it has proven to be a valuable tool in outbreak investigation of kpc infections in a hospital [ ] and in a large community outbreak of kpc involving multiple facilities [ ] . it seems clear that as this technology gets faster and cheaper, it will be embraced as an infection control standard [ ] , although there needs to be caution and skill in interpreting results [ ] . this work in developing countries is another attractive career option based on need, probability of impact, and unique special programs such as the president's emergency plan for aids relief, the bill & melinda gates foundation, and others. some of this is direct patient care, but possibly very attractive targets for impact are the development and implementation of innovative programs that deal with the vast need combined with minimal resources [ ] . the new healthcare system should value infectious disease expertise based on its important role in addressing resistance and costs associated with nosocomial infections. nevertheless, it is feared that the current structure and payment system are not constructed as a good fit to prioritize infectious disease skills. specifically, there is no code for preventing infections, conserving antibiotic use, or preventing resistant pathogens. this might be an erroneous conclusion, or the situation may change as the system matures and becomes serious about addressing the crisis. "bundles" to deal with healthcare efficiencies are in vogue and could be a strength of infectious diseases. an example is the -step central line bacteremia prevention bundle that proved effective in trials [ ] . generalized adoption of this bundle was predicted to save lives and $ . billion per year in us hospitals [ ] , and subsequent actions by clinicians, regulatory agencies, and stakeholders have resulted in an estimated % decline in central line bacteremia rates [ ] . • stewardship: solving or reducing the problem of antibiotic resistance largely depends on antibiotic development and reducing antibiotic abuse. the major on-site forces for improving smart antibiotic use at the point of care are antibiotic stewards-preferably infectious disease or pharmacy personnel trained in this skill to improve the speed of detecting resistant or epidemic pathogens. the tools are obvious to infectious diseases-trained clinicians, but often require methods that are not well inculcated into hospital or clinic practice. methodology with proven value for antibiotic conservation include shortcourse regimens (virtually always wins or ties in trials), use of procalcitonin to facilitate decisions on when to start or stop antibiotics, use of molecular diagnostics to improve pathogentargeted antibiotic decisions, outpatient infusion therapy to reduce inpatient risk (and cost), optimal use of the agents we have, waiting room with notices that the doctor will prescribe antibiotics only according to guidelines, acknowledgement of possible microbiome harm, and possible use of social network media [ , ] . nevertheless, there must be caution: the new us healthcare system represents socialized medicine largely managed by capitalists, which invites both quality and chicanery. for example, the centers for medicare and medicaid services' " -hour rule" for treating community-acquired pneumonia had improved outcome advantages, but also led to overprescribing, declined use of diagnostics, perceived antibiotic abuse, and increases in c. difficile infections. given the priority of cost containment and its relevance to infectious diseases, infectious disease training should probably include attention to the business of medicine. • molecular microbial diagnostics: these are rapidly being developed and introduced into clinical use for detection of epidemic pathogens or resistance genes with advantages of speed, precision, and sensitivity. most polymerase chain reaction (pcr)-based tests define a specific pathogen with extraordinary sensitivity within minutes. the fda has approved these pcr tests to detect at least viruses and bacteria [ ] . these tests may also be useful for early detection of epidemic pathogen or resistance genes [ ] . it seems clear that the introduction of molecular tests for general use may be difficult to interpret in the context of clinical care, so these new tests will require a substantial stewardship from the infectious disease community. this was illustrated in a trial to guide antibiotic decisions based on results of a pcr-based diagnostic to detect mrsa in purulent soft tissue infections that had no significant impact on antibiotic selection [ ] . gene sequencing will be a new and important role for the infectious diseases-trained clinician as it becomes more readily available for defining transmission patterns to inform infection control practice. • microbiome: study of the microbiome at various anatomical sites represents a major nih-sponsored initiative that could possibly translate into important opportunities to treat or prevent multiple conditions [ , ] . this work is at the dawn of development, but the early reads suggest a potential role in obesity, allergies, autoimmune disease, cancer, diabetes, heart disease, and other conditions [ , ] . it is also apparent that antibiotics have a profound and long-lasting impact on the microbiome [ ] . this field requires a transformation in our conventional understanding of infectious diseases, as the "pathogens" are communities of microbes that communicate in contrast to the koch postulate of "one microbe, one infection." an example is a recent report showing that volunteers fed steak and eggs (lecithin) have conversion by gut flora to trimethylamine-n-oxide, which is a marker of atherosclerosis [ ] . this microbial interaction could be altered with antibiotics. the long-term goal is to define associations and intervene possibly with antibiotics and probiotics; this work may also illustrate potential harm to redefine risk-benefit ratios for antibiotics. • bundles: another potentially important role for infectious diseases-trained clinicians is the development of bundles that prevent infectious disease complications. an example is central line bacteremia, as described above [ ] [ ] [ ] . that experience can now be applied to multiple iatrogenic infection risks associated with specific patients or procedures, possibly prioritizing those with the greatest healthcare consequences as described above. the role of infectious diseases is to define the bundle, design the study, and then implement them when results are convincing or even mandated. specialized skills in the management and study of infectious diseases are an increasingly important specialty in contemporary medicine. the roles of practitioners in the discipline are diverse, usually important, and sometimes critical, but commonly undervalued by contemporary priorities in healthcare systems and healthcare reform. it would be difficult to find another discipline in medicine that has such extraordinary diversity, surprises, value in patient care, and clinical relevance for both domestic and international applications. for many trained in medicine, joining the field of infectious diseases is simply the right thing to do. supplement sponsorship. this article was published as part of a supplement titled "the john bartlett festschrift: celebrating a career in medicine," sponsored solely by the department of medicine of the johns hopkins school of medicine in recognition of john bartlett's contributions to medicine. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. index case of fatal inhalation anthrax due to bioterrorism in the united states lyme arthritis: and epidemic of oligoarticular articular arthritis in children and adults in three connecticut communities the spirochetal etiology of lyme disease interview with willie bergdorfer, ph.d. interview by vicki glaser lyme disease-a tick-borne spirochetosis? how the discovery of borrelia bergdorferi came about nobel prize winners robin warren and barry marshall two win nobel prize for discovering bacterium tied to stomach ailments long-term follow-up of voluntary ingestion of helicobacter pylori 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intestinal microbial metabolism of phosphaditylcholine and cardiovascular risk key: cord- -vak p ac authors: rocha, francisco airton castro; duarte-monteiro, ana margarida; da mota, licia maria henrique; pinto, ana carolina matias dinelly; fonseca, joão eurico title: microbes, helminths and rheumatic diseases date: - - journal: best pract res clin rheumatol doi: . /j.berh. . sha: doc_id: cord_uid: vak p ac there has been a progressive interest on the modifications of the human defense system following insults occurring in the interface between our body and the external environment, as they may provoke or worsen disease states. studies suggest the billions of germs that compose the gut microbiota influence one’s innate and adaptive immune responses at the intestinal level, but these microorganisms may also impact rheumatic diseases. the microbiota of the skin, respiratory and urinary tracts may also be relevant in rheumatology. evidence indicates that changes in the gut microbiome alter the pathogenesis of immune-mediated diseases such as rheumatoid arthritis and ankylosing spondylitis but also of other disorders like atherosclerosis and osteoarthritis. therapeutic strategies to modify the microbiota, including probiotics and fecal microbiota transplantation, have been received with skepticism, which, in turn, has drawn attention back to previously developed interventions such as antibiotics. helminths adapted to humans over the evolution process, but their role in disease modulation, particularly immune-mediated diseases, remains to be understood. the present review focuses on data concerning modifications of the immune system induced by interactions with microbes and pluricellular organisms, namely helminths, and their impact on rheumatic diseases. practical aspects, including specific microbiota-targeted therapies, are also discussed. the coevolution between germs and humans has been marked by mankind development-related modifications. migratory flows and industrialization have affected farming, food intake, housing, and clothing, causing a profound impact on the environment [ ] . the most compelling evidence of genetics' relevance to the pathophysiology of rheumatic diseases is the association of the human histocompatibility antigen hla-b with inflammatory spondyloarthropathies (spa). hla-b prevalence varies across populations and some hla-b variants have been associated with a decreased prevalence of ankylosing spondylitis, highlighting the complexity of this association [ ] . of interest, concordance rates for identical twins in rheumatoid arthritis (ra) vary from to %, which is similar to the - % concordance rate in type i diabetes, a disease considered to have a clear and strong genetic influence [ ] . since most prevalent rheumatic diseases are not directly associated with specific genes, it is likely that various genes, together with environmental agents, influence the natural course of such disorders. the possibility that germs (microbes) present in the human body modulate the immune response is a classical concept. however, direct causality relying on koch's postulates has been difficult to prove, thereby limiting the association of a specific germ with a disease. fastidious germs and asymptomatic carrier states represent challenges when attempting to implicate a specific insulting agent as the cause of a disease. germs populating "open" body areas such as the skin, oral cavity, respiratory, and gastrointestinal systems, as well as "extracellular" and secretory components are collectively called the microbiome, a term that does not encompass multicellular agents that may also be relevant in this context like worms and fungi. modern biotechnology is based on complex dna sequencing rather than culture and staining procedures. although this has greatly improved the identification of germs, it has also added to the complexity of when attempting to understand parasite-host interactions [ , ] . microbiome issues have attracted the interest of various researchers who have produced excellent articles and reviews on the subject of germs and human interactions. nonetheless, not much attention has been granted to helminths, which might potentially have a role in this scenario. a decrease in the prevalence of infections and infestations was previously associated with an increased prevalence of allergic and autoimmune diseases among populations living in western developed countries. moreover, water purification, massive vaccination and other sanitary measures provided health benefits to people living in industrialized countries [ ] . still, these apparently healthful "cleaning" strategies could also reduce human microbiota diversity, which could in some way negatively impact one's health. likewise, improvement in health care practices can also render adverse events. recent outbreaks, such as the arboviruses epidemics in brazil in the last years [ ] and the ongoing coronavirus pandemic, lead to the immediate incorporation of individual practices aiming at the protection against contamination. attitudes like avoiding contact with other people and the use of handrails in public transportation, or wearing gloves and masks are deliberately taken in an attempt to prevent infection [ ] . while this course of action is understandable during a pandemic, excessive handwashing or showers, avoidance of child contact with unprotected soil, grass, and animals, may compromise the stimulation of the human immune system, thereby jeopardizing the body's response to infections [ , ] . although it is still debatable, reduced breastfeeding and increase in caesarean delivery as well as massive vaccination were also shown to have a negative impact on immune development, being associated with increased prevalence of allergic and autoimmune diseases [ ] . following this line of thought, researchers have previously compared the immune system with the central nervous system based on the idea that establishing connections and plasticity are probably vital to the functioning of both systems. based on this assumption, avoiding contact with germs may result in more vulnerability [ ] . one possible mechanism for the breastfeeding benefit is the presence of oligosaccharides in human milk, which could modulate the intestinal microbiota. it was recently shown that mice exposed to these oligosaccharides did not develop a type experimental diabetes. that effect was linked to modifications of the intestinal microbiota, leading to an increase in short-chain fatty acid content in the gut. xiao et al. ( ) used their results to justify the benefits of exclusive breastfeeding in humans. we should consider that oligosaccharides isolated from human milk rather than mouse milk were offered to the animals. applying that premise to mammalian milk it might well be that cow's milk could be beneficial to humans, provided that both contain the same type and amount of oligosaccharides [ ] . examples of a possible link between robust sanitary measures and increased incidence of immune-mediated diseases include the augmented prevalence of asthma in developed countries [ ] , at the same time that autoimmune diseases appear less frequent or less severe in nigeria, where proper sanitation is still to be implemented [ ] . inflammatory bowel diseases, e.g. crohn's disease, are considered perfect models of the association between changes in dietary habits and less exposure to germs with increased incidence of diseases considered to be immune-mediated. crohn's disease is currently more prevalent in wealthy populations living in the northern hemisphere, but numbers appear to be increasing in developing countries, possibly due to dietary modification and sanitary improvements [ ] . other immune-mediated diseases including type diabetes, systemic lupus erythematosus (sle), ra, and multiple sclerosis have also been reported to be less prevalent in developing countries [ ] . "everything is autoimmune until proven otherwise" was once said, claiming for a ubiquitous role of autoimmunity in any disease state. thus, splitting diseases into immune or non-immune-mediated would merely serve didactic purposes [ ] . innate immune mechanisms operate in close association with the adaptive immune responses, modulating the body's defense against tissue damage caused by a germ, trauma or cancer. this also applies to housekeeping processes such as digestion, breathing, and senescence, which generate by-products that may cause harm to the host. for instance, uric acid released in the event of cell death alerts the immune system, unleashing apoptosis to avoid systemic and potentially harmful inflammation [ ] . atherosclerosis and cardiovascular damage have been associated to changes in the intestinal microbiota. of interest, studies have linked the processing of phosphatidylcholine in the intestine to increased atherogenesis by producing trimethylamine-n-oxide (tmao) [ ] . accordingly, modification of the intestinal microbiota by dietary changes, repopulation of the gut flora or administration of probiotics could lead to a marked reduction in plasma and urine tmao levels, which was associated with a decreased risk of major cardiovascular events [ , ] . although we may not consider atherosclerosis a bona fide immunemediated disease, the above-described results indicate that microbiota changes could alter disease course. limitations of these studies include assessment of tmao levels systemically as well as evaluation of the microbiota only at the intestinal level. in fact, antibiotics might have altered the microbiota in other sites such as the skin, oral cavity, and respiratory tract, which were not directly evaluated. although causality is hard to prove, the data reinforces the need to pursue the concept of an association between atherosclerosis and microbiota. it is beyond our scope to fully discuss all aspects of the microbiome. we will focus in bacteria and their products, and helminths as triggers or modulators of disease processes in rheumatic diseases. the microbiota has gained more and more relevance as a driver in the pathogenesis of various rheumatic diseases including ra, sle and, more recently, osteoarthritis (oa) [ , ] . the gastrointestinal tract appears as a natural target, partially because it harbors a great and diverse number of bacteria. nutritional habits, antibiotic use, constipation as well as gastrointestinal infections are some factors that may alter the bowel's microbiome. considering the huge number of cells linked to immune mediation in the gut, e.g. cell population in peyer's patches, modifications of the gut flora definitely impact one's immune response [ ] . animal studies showed that the modification of the intestinal bacterial flora influenced the severity of antigen-induced arthritis (aia) [ ] and experimental allergic encephalomyelitis -i.e. the most commonly used animal model for human multiple sclerosis [ ] . changes in the cytokine profile of those animals, with predominance of type t helper (th) cell responses and increase in the number of t regulatory (treg) cells, were associated with improvement following restoration of a "normal" flora in germfree animals that received faecal transplantation [ ] . issues regarding innate immune mechanisms are receiving more attention, given their importance to the physiology of the gastrointestinal tract. integrity of the mucosa with a protective layer of mucus, acid ph in the stomach, biliary fluid, maintenance of junctions between epithelial cells as well as presence of antimicrobial secreted peptides, e.g. defensins and cathelicidins, and immunoglobulin a constitutively present in the lumen represent first-line defenses against invaders. distinguishing "normal flora" from invading organisms can be made by pattern recognition receptors (prr) exposed in intestinal epithelial cells, which include toll-like receptors (tlr) and nucleotide-binding oligomerization domain (nod)-like receptors. sensing of pathogens or endogenous stimuli released from dying or damaged cells by prr leads to downstream activation of intracellular signal transduction inflammatory pathways [ , ] . a concerted effort between both innate and adaptive immune responses is then constructed to face damage. it has been proposed that the cost to the immune system progressively increases in a stepwise process resulting from engagement or disruption of successive protective barriers [ ] . simple as it may seem, normal peristalsis contributes to dispatch pathogenic material. during constipation, delayed bowel emptying favors proliferation of germs, and disturbance of vesical bladder clearance leads to recurrent urinary tract infections, particularly in women [ , ] . in developing countries with poor sanitation, recurrent episodes of diarrhea compromise maintenance of a normal flora due to disruption of the gut epithelia, as well as to altered restoration of the normal flora secondary to speedy bowel movements [ ] . on the other hand, seniors are more susceptible to constipation. this may further compromise gut flora and favor the development of diverticula, which are potential sites of inflammation [ ] . tissue-resident lymphocytes, eosinophils, macrophages, and treg cells are present in the intestinal barrier acting to contain the inflammatory insult. breaches in the gastrointestinal barrier expose immunocompetent cells located in the lamina propria and increase systemic migration of germs or their constituents (antigens) allowing their encounter with professional dendritic antigen-presenting cells in mesenteric lymph nodes, amplifying an inflammatory response that should have been kept locally. in the appropriate unlucky host, bearing a susceptible genotype, the triggering of a complex immune response spreads systemically [ , , ] . although unproven, one may speculate that the highly irrigated synovial tissue with fenestrated capillaries and easily accessible immune competent cells, such as type a synoviocytes, are a preferable harbor to those germs or their parts (antigens), where they can foster a sustained immune response. medications used to treat rheumatic diseases also modify our microbiome. sulphasalazine, which has antibiotic properties, is still an option to treat inflammatory bowel disease and chronic inflammatory arthropathies [ , ] . minocycline, marketed primarily as an antibiotic, was until recently used as a disease-modifier drug in ra [ ] . methotrexate, a cornerstone in the treatment of ra, as well as leflunomide may have part of their therapeutic benefit linked to changes in the host microbiome [ ] . more recently, fexofenadine, a compound to treat chronic allergic diseases, was shown to inhibit tumor necrosis factor activity, revealing an immunomodulatory role to fexofenadine not hitherto described [ ] . chloroquine also has antiviral activity and has just been shown to inhibit in vitro coronavirus replication in concentrations that could be achieved in clinical practice [ ] . of interest, the recent worldwide arbovirus chikungunya outbreak has been associated with joint manifestations, which could derive from a direct viral activity or the triggering of existing subclinical inflammation in the joints [ ] . in addition, other viruses like epstein-barr and parvovirus b have long been associated with the pathogenesis of immunemediated diseases. hence, the recent proposed antiviral chloroquine activity could well be an additional mechanism to explain its effective response to treat various rheumatic diseases, including the ability to ameliorate musculoskeletal manifestations following chikungunya infection [ ] . vulnerability to infections particularly in the immunocompromised host occurs in "open systems" such as the skin, respiratory, urogenital, and gastrointestinal tracts. periodontitis has been shown to have a major role in the pathogenesis of ra. however, recent epidemiological data may question periodontitis relevance in ra pathogenesis. using modelling to extrapolate collected data, both the prevalence and burden of ra were reported to be higher in high-income populations where oral hygiene is presumably better [ ] . edentulation is highly prevalent among low-income brazilians and chronic periodontitis is the major cause of tooth loss during adulthood [ ] . yet, ra burden was shown to be milder in brazil and other low-income areas of the globe, as compared to wealthy regions. also, we have reported very low childhood health assessment questionnaire (chaq) scoring, meaning a favorable outcome, among low-income juvenile idiopathic arthritis (jia) patients living in one of the less developed areas in brazil. indeed, it is surprising that the chaq scores were lower than those collected among jia patients evaluated in the pediatric rheumatology international trials organization (printo) and childhood arthritis and rheumatology research alliance (carra) registries that gather data from high-income populations, with presumably better sanitation and oral hygiene. although it may just be coincidental, there is a high prevalence of helminthiasis in our low-income population leading us to speculate whether helminths or their products down-modulate inflammation in autoimmune diseases, thus decreasing severity [ ] ( figure ). in developing regions, despite low-income and low-literacy that negatively impact disease outcome, helminths' infestations may down-modulate inflammation, resulting in less damage. maintaining skin integrity is probably as relevant as avoiding gut damage for adequate immune homeostasis. ultraviolet radiation, trauma, and tick bites are common causes of lesions inflicted to the skin, paving a way to stimulate immune competent cells located in the dermis. stratification of skin layers and the presence of continuous capillaries and fat tissue keep inflammatory stimuli away from direct contact to "hidden" professional antigen presenting cells of the skin, such as the dendritic langerhans cells. in addition, innate immune mechanisms, including tight junctions between cells, pigmentation, hair follicles, sweat and sebaceous material prevent access to cells located in the dermis. as an analogy to the gut barriers, it seems that every effort is made in order to avoid access of germs, or their parts (antigens), to satellite lymph nodes where antigen presentation and systemic widespread inflammation ensues. it was suggested that a homeostatic response of intestinal cells to commensals is under tight regulation, including the production of antibodies to those germs. that tolerance could be broken during infections, triggering the proliferation of cd + t cells both to the invading pathogen as well as to commensals, developing memory cells to the new infecting agent. in a specific host that equilibrium could be lost leading to unresolving inflammation like that occurring in crohn´s disease [ ] . similar mechanisms could be the reason for the occurrence of gastrointestinal manifestations in spa. a recent report has shown that skin-resident t lymphocytes constitutively expressing interleukin (il)- a coexist with commensals, contributing to tissue integrity. harmful stimuli represented by trauma, infection or insect bite brake barriers, promoting cell plasticity so that the cytokine repertoire is now enriched with il- and il- , leading to prolonged inflammation. therefore, skin barriers help maintain homeostasis by avoiding contact of stimuli to professional cells of the immune system [ ] . once a certain stimulus, in a susceptible host, surpasses that initial defense, systemic inflammation is unleashed, provoking non-resolving autoinflammatory or autoimmune diseases, whether affecting mainly innate or adaptive immune responses, respectively. as mentioned above, worldwide epidemiological data show that the burden of ra varies across the globe, with numbers appearing to be higher in developed countries [ ] . previous estimates of ra have suggested a . - . % prevalence in wealthy regions, with lower prevalence in underdeveloped countries, varying from . to . % [ ] . however, we have to bear in mind that more accurate data were collected in developing regions. thus, despite some discussion, research suggests ra is less prevalent and/or less severe among low-income populations. paradoxically, delay in diagnosis, less access to specialists, failure to comply with treatment and unavailability of disease-modifying compounds presumably carry a negative impact to the treatment of chronic inflammatory arthropathies [ ] . regarding sle, it was shown that low-income patients living in the northeast of brazil had similar long-term damage evaluated by the systemic lupus collaborating clinics' (slicc) criteria, compared to patients from developed countries [ ] . helminthiasis are still highly prevalent in the northeast of brazil and, according to the recommendations of the world health organization, it is current practice to prescribe prophylactic anti-helminthics to decrease the burden of parasite infections and helminths' infestations (https://extranet.who.int/rhl/topics/preconception-pregnancy-childbirthand-postpartum-care/antenatal-care/who-recommendation-preventiveanthelminthic-treatment; downloaded in feb , ). helminths have for a long time been considered relevant modulators of the immune response [ ] . there are reports on the benefit of the administration of thichuris suis eggs to treat patients with crohn's disease [ ] . our group has shown that animals subjected to collagen-induced arthritis and treated with a crude ascaris suum extract have decreased inflammation with reduction of cytokine release into the inflamed joints [ ] . we then identified the gene sequence of a peptide from the a. suum extract present in the celomic fluid of that worm, which shows antiinflammatory activity in mice subjected to aia. intramuscular administration of a plasmid with the correspondent gene sequence decreased inflammation in aia mice, apparently through a shift of the macrophage population into an m phenotype (unpublished data). it is worth mentioning that oral administration of a. suum extract, rather than viable worms as with t. suis experiments, was effective, providing a proof-of-concept to support the idea that helminthic products released into the bowel modulate the inflammatory response [ ] . it has also been previously shown that an excretory-secretory product of acantocheilonema vitae has an immunomodulatory role, being able to decrease osteoclast-mediated bone resorption via modification of the gut microbiota [ ] . helminths or their products might have beneficial and harmful effects to the host. difficulties in establishing a net result should not prevent efforts to dissect those mechanisms, both to understand the pathophysiology of immune-mediated diseases as well as to develop therapeutic alternatives (figure ) [ , ] . possible mechanism where secretory products from a. suum drive macrophage differentiation into an m phenotype, leading to the decreased release of inflammatory mediators. in , inman wrote "perhaps a word of caution is needed for the critics and a word of encouragement for those hunting microbes in the joints", when referring to the possible role of infection in the development of rheumatic diseases, specifically spa [ ] . he compared this concept to the relationship between helicobacter pylori infection and gastritis, which was at first met with skepticism, but is nowadays accepted as a fact [ , ] . when talking about "microbes in the joints", one can discuss the topic from the perspective of infection and how it can contribute to the development of certain rheumatic diseases, but also in a broader sense, considering the human microbiome (more specifically, the gut microbiome) and how it can be related to these disorders, specifically inflammatory arthropathies [ , ] . the understanding of the depth of this relationship could, in the future, yield new forms of treatment [ ] . spa, as a group, are characterized by findings of asymmetric oligoarthritis, axial involvement, particularly of the sacroiliac joints, and inflammatory back pain, enthesitis, and characteristic extra-articular features, including acute anterior uveitis and psoriasis [ , ] . ankylosing spondylitis (as), psoriatic arthritis (psa), reactive arthritis (rea), and inflammatory bowel disease (ibd)-related arthritis [ ] all share an association with hla-b [ ] . the hla-b subtype is the one that's most commonly associated with spa [ ] . in fact, the presence of this allele is the greatest risk factor for the development of spa [ ] . parallel to the genetic predisposition, there has been mounting evidence for the role of infection in the pathogenesis of spa [ , ] . the clearest example of this association is illustrated by rea [ ] . for a long time, there has been evidence that chlamydia trachomatis (an obligate intracellular parasite of eukaryotic cells [ ] ) plays a role in the development of inflammatory arthropathies [ ] . the pathogenesis of chlamydia-related arthritis can be considered distinct from that associated with enteric bacteria since it involves metabolically active organisms residing long-term within monocytic cells in synovial tissues, after resolution of the primary genital infection and migration of the cells to the joint, a process that is known as persistence [ , [ ] [ ] [ ] . there is also evidence that differences in the expression of heat shock protein- genes may contribute to organism persistence [ ] . another interesting aspect to consider is that only a minority of patients with chlamydia genital infection develop rea. this could be explained by the findings of gérard ( ) that showed only ocular serovar group chlamydiae in synovial biopsies from arthritis patients, who were pcr-positive for c. trachomatis dna in that tissue and not genital serovar. this could mean that the composition of the initial inoculum influences the probability of arthritis development [ ] . these data, if confirmed, could be relevant when thinking about a way to monitor which patients will go on to develop or have a greater risk of developing arthritis. nevertheless, it is important to note that these findings were based on the evaluation of only four patients [ ] . similarly, c. pneumoniae has also been accepted as an etiologic agent for inflammatory arthritis [ , ] . earlier, it was believed that antibiotic treatment of patients with chlamydia-induced arthritis was ineffective [ , , , ] , but recent studies indicate antibiotic therapy might be beneficial [ , ] . however, this efficacy is not complete as the proposed treatment ( months of oral antibiotic therapy with either doxycycline or azithromycin, both combined with rifampin) improves joint symptoms in % of patients and leads to remission in % [ ] , indicating other mechanisms might be involved in the development of arthritis which, if better understood, could lead to the development of novel therapeutic targets [ , ] . besides chlamydia, enteric pathogens such as campylobacter jejuni, clostridium difficile, escherichia coli, salmonella, shigella, and yersinia, and respiratory pathogens like chlamydia pneumoniae or mycoplasma pneumoniae have also been shown to give rise to reactive joint processes. evidence for the benefit of antibiotic therapy directed at those bacteria is sparse, since it doesn't appear to alter the course of the arthritis [ ] . the actual concept of rea makes history of infection a required criterion for its diagnosis, but, unlike in septic arthritis, inflammatory arthritis is not directly caused by infection of joint tissue (positive culture), instead it follows an extra-articular infection [ , ] . as for the other spas, which comprise the majority of the diseases in this group, with rea accounting for less than % of the overall disease burden [ ] , the role of infection (whether there is a role or what that role is) is not as well established. nonetheless, there has also been some evidence regarding this matter [ , , , ] -various authors have discussed the role of chlamydia, not only in rea but also in undifferentiated spa [ ] . rashid et al. ( ) reported a link between elevated levels of klebsiella antibodies and as in various geographical locations [ ] , and the development of this disease appears to be related to the presence of k. pneumoniae aerogenes in the gastrointestinal tract [ ] . the role of hla-b has been a focus of research when trying to understand spa. in rea, hla-b is present in to % of patients [ ] . there are several theories that attempt to define how exactly hla-b predisposes to spa and how it is implicated in its pathogenesis [ , ] . this is of particular relevance in the context of the arthritogenic peptide hypothesis, according to which microbial peptides mimic certain self-peptides. specifically, hla-b may have structural features that are shared with bacteria, causing reactivity of hla-b -specific, cd + bearing cytotoxic t lymphocytes, leading to autoimmunity and tissue damage and inflammation [ , ] . zhang et al. ( ) stated that as seems to be related to k. pneumoniae infection in hla-b positive patients, based on consistent findings of raised anti-k. pneumoniae antibodies and the presence of molecular mimicry between the hla-b molecule and bacterial antigens [ ] . because some patients who are hla-b negative also develop rea, other investigations have focused on the role of specific microbial factors in the pathogenesis of the disease. for example, in patients with rea and documented s. typhimurium infection it has been suggested that salmonella outer membrane protein is able to stimulate interleukin (il)- /il- production in synovial immune cells, possibly contributing to arthropathy [ ] , considering the well-established role of this pathway in spa [ , ] . there has also been some focus on the role played by the microbes that inhabit the human body and do not represent a state of infection -in this case, the focus has been centered around the gastrointestinal microbiota (oral and gut microbiota) -and its possible implication in the pathogenesis of spa [ ] [ ] [ ] . changes in the homeostasis and balance of the microbiota can lead to subtle but perhaps profound alterations in the balance with the host's immune system, leading to a state of inflammation that may contribute to disease. the correlation between hla-b and as has been known since the 's [ , ] . this relationship is also true for the other spa, as stated above. studies indicate inflammatory bowel disease, or, at least, intestinal inflammation, is more prevalent in spa patients (as or others) and some genes associated with as are also associated with ibd [ , ] , including genes related to gut physiology and immunology. based on these associations, it has been postulated that imbalances and defects in gut microbiota can play a role in the pathogenesis of spa [ , ] , since there's a significant clinical, genetic, immunological, and microbiological overlap between ibd and spa [ ] . not surprisingly, there has also been evidence that hla-b affects the gut microbiome and its metabolites, and perhaps the handling of bacteria during infection [ , ] . the gut microbiota is acquired post-partum and its composition can be influenced by various factors during its development until around years of age, when it seems to stabilize. during a person's lifetime, the gut microbiota can be affected by diet and nutrition and also antibiotic use -disturbances in its balance can be a factor for disease [ ] . a study that analyzed biopsies from the terminal ileum of as patients showed the microbiome in as patients was different from healthy subjects, with greater abundance of bacteroidaceae, lachnospiraceae, porphyromonadaceae, rikenellaceae, and ruminococcaceae and lower abundance of prevotellaceae and veillonellaceae [ ] . data has shown that hla-b can determine an increase in the intestinal permeability, which results in continuous antigenic stimulation with activation of effector t cells [ , , ] . hla-b can also induce an endoplasmic reticulum stress response and promote autophagy/unfolded protein responses (upr), resulting in the release of tumor necrosis factor (tnf), interleukin- (il- ), overexpression of il- by paneth cells, and interferon-γ and increase in th cells [ ] . il- can regulate the maturation of autoreactive th cells and induce chronic inflammation by stimulating il- , il- , il- , and tnf production in neutrophils and macrophages [ ] . there has also been evidence that hla-b may interact directly with gram negative bacteria, either by provoking an immune response through antigen mimicry (as previously discussed) or by altering genes expressed by bacteria, thus affecting dendritic cell function and influencing the immune response [ ] . despite the relationship between spa, hla-b and the microbiome, the best example of the influence of gut dysbiosis in the pathogeneses of rheumatic diseases is still ra and its relationship with citrullinated proteins [ ] [ ] [ ] . proteins are ubiquitous in the human body, assuming different forms and specific functions. after syntheses in ribosomes, they are further altered and specialized through posttranslational modification (ptm), which includes various processes. one of such processes, involving the conversion of arginine amino acid into citrulline amino acid, is called citrullination and occurs physiologically during apoptosis. this "biological operation" is catalyzed by peptidylarginine deiminase enzymes (pads) and produces an amino acid that is not encoded by the genome, being solely produced by this process [ ] . there has been extensive research and data that demonstrate the role of pads in the development of immune-mediated diseases [ ] , and in the field of rheumatology, this is particularly relevant in respect to ra. in humans, there are five pad isoenzymes ( to ), expressed in various tissues and organs that contribute to many of the body's physiological functions. they are actively expressed in the intestinal epithelium, making it a source of citrullinated peptides [ ] . changes in oral microbiota can also influence progression and outcomes in ra [ ] and porphyromonas gingivalis (pg) has been ascribed a major role. it is present in the oral cavity and proliferates in patients affected by periodontitis. the pad expressed by pg can lead to citrullination and may explain the close association between ra and periodontitis, an inflammatory disease of the oral mucosa [ ] . there are actually reports of a direct correlation between the serum level of antibodies and pg plus acpas in ra patients [ ] . however, as we mentioned above, discrepancies concerning increased ra burden in regions with better oral hygiene may question the relevance of periodontitis in ra pathogenesis [ ] . when considering the role of gastrointestinal microbiota and infection in rheumatic diseases, the best characterization was achieved in ra, through the understanding of the process of citrullination and its relationship with oral microbiome, mainly pg. in spa, altered intestinal permeability and hla-b 's role in the induction of that disruption may trigger antigenic stimulation with t cell activation [ ] . it is also relevant to refer, in respect to citrullination, that it was thought that citrullinated proteins were specific to the synovium of ra patients, but recent reports revealed citrullinated proteins are also present in the synovium of non-ra inflammatory conditions [ ] , which may account for its probable role in other inflammatory arthropathies. antibiotic use may be considered a mixed blessing in in the context of therapeutics. while it is undisputable that antibiotics are lifesaving in many cases, widespread and unnecessary use of such compounds, particularly those with a broad-spectrum profile, leads to the development of bacterial resistance as well as alterations to the microbiome. modifications of the immune response occur secondarily to the eradication of commensal microorganisms, which are important stimuli to intestinal cells. probiotics are commonly regarded as a useful and harmless strategy to reconstitute the gastrointestinal flora following antibiotic use. probiotics can be routinely prescribed by physicians and other health care professionals, and may also be found as over-the-counter products, on the premise of restoring homeostasis. however, there is a lack of adequate studies documenting those benefits, not to mention that probiotics are marketed without complying with the strict rules used for drugs' approval. by not being appropriately regulated, neither the safety nor the efficacy of probiotics have been specifically documented. it was recently shown that supplementation of probiotics to humans or mice that received broad-spectrum antibiotics led to delayed restoration of the previous normal flora. on the other hand, autologous fecal microbiota transplantation (fmt) shortened the time for reconstitution of the gut flora [ ] . although suez et al. ( ) have correctly discussed limitations and extrapolations from their single study in healthy volunteers, the results justify a closer look into the advertised benefits of probiotic use, regardless of indication, highlighting the need for data to adequately document harms and benefits. antibiotics are also used as modulators of the immune response. sulphasalazine is indicated in spa and ra as a disease-modifying compound due to its immunomodulatory activity [ , ] . until recently, tetracyclines (minocycline) were used as disease-modifying antirheumatic drugs because of their anti-inflammatory effects involving the inhibition of proinflammatory cytokines and matrix metalloproteinase activity [ ] . gold salts had their efficacy at least partially linked to antimicrobial activity and were formerly in use for ra treatment. regarding rea, and despite the difficulties in pointing to a specific germ as the causative agent, prolonged antibiotic use has been associated with decreased symptoms and disease duration [ , ] . other medications already in use may have unsuspected antibiotic activity as the recently described antiviral chloroquine response. however, the most common and effective treatment for chronic inflammatory arthropathies relies in down-modulating inflammatory response-related targets with immune suppressors, aiming for symptom relief and halting structural tissue damage. following the boom in microbiome research, other microbiota directed therapeutic interventions appeared. for instance, changing dietary habits as a means to increase the supply of short-chain fatty acids by ingesting fiber is also an appealing strategy to improve immune function, because it is perceived as a non-harmful alternative in addition to the low-calorie profile benefit. a high-fiber diet favors the production of short-chain fatty acids, particularly propionate and butyrate, which have antiinflammatory and immunomodulating activities, and were associated to bone-sparing effects in ra patients. mice subjected to short-chain fatty acids' administration had decreased bone resorption partially mediated by down-regulation of tnf receptor associated factor (traf)- and nuclear factor of activated t-cells, cytoplasmic (nfatc ) genes [ ] . as a therapeutic alternative to treat chronic inflammation, fmt attracts both interest and skepticism. the successful use of fmt in patients affected by clostridium difficile infections refractory to antibiotic treatment came as a proof-of-concept promising study [ ] . despite its recognized benefits, the procedure has its risks. harmful contaminants can be supplied together with the "good guys" that would restore the intestinal flora after fmt. indeed, the u.s. food and drug administration (fda) issued a safety alert on june , about the serious adverse reactions due to the transmission of multi-drug resistant organisms following fmt (www.fda.gov/media/ , downloaded on feb , ). the logistics behind the necessary standardization of appropriate fmt "components" include storage, "quality" control regarding donors, recipients, expiration date, to mention a few issues. given the difficulty in translating fmt into clinical practice and the inconsistent data on probiotic use, microbial active pharmaceutical ingredients have also been developed as treatment strategies for rheumatic diseases. biological therapies involving the delivery of live organisms are a major challenge and regulations are being developed, but it was not without great care that the industry introduced biologicals into the therapeutic armamentarium of immune-mediated diseases. a recent review addressed issues about the development of microbial therapeutics, which is still in its early stages. uncontrolled proliferation of a living organism, toxicity and hypersensitivity reactions as well as long-term stability are some of the challenges that need to be overcome. pharmacokinetics, pharmacodynamics and the supply chain are matters of concern, particularly if one considers worldwide distribution. gut barriers, including acidic ph, bowel movement, mucus layer, bile salts, to name a few, also affect the bioavailability of oral vaccines or stability of microbe-derived orally active compounds [ ] . challenges represent opportunities for improvement. considering the interest of big pharma companies on live biotherapeutics, the marketing approval of a specific microbial therapy will likely happen in a near future. * identify causal relationships between microbiome perturbations and human rheumatic disease * define roles, for skin, respiratory, and urogenital tract microbiota in the pathogenesis of rheumatic diseases; * conduct high-quality clinical trials with pharmaceutical grade probiotics in rheumatic diseases; * identify mechanisms by which antibiotics modulate f rheumatic diseases; * for patients with rheumatic disease, determine "healthy" and "harmful" dietary components that are simple,affordable, and readily available. 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autoimmune disorders peptidylarginine deiminase from porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis association of periodontitis with rheumatoid arthritis: a pilot study specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: relevance to antifilaggrin autoantibodies post-antibiotic gut mucosal microbiome reconstitution is impaired by probiotics and improved by autologous fmt short-chain fatty acids regulate systemic bone mass and protect from pathological bone loss fecal microbiota transplantation--an old therapy comes of age microbial therapeutics: new opportunities for drug delivery item group: ig manuscript title: microbes, helminths and rheumatic diseases chapter andar rodolfo teófilo - - -fortaleza -ce -brazil phone e-mail address: arocha@ufc.br the authors declare no conflict of interest practice points * antibiotic use should be made within specific indications, thereby trying to limit changes to the microbiota; * attention should be given to innate immune mechanisms as modifiers of disease states; * proven dietary recommendations for the treatment of rheumatic diseases are not yet available * general recommendations, such as avoiding excess salt and fat, and consuming fresh vegetables, are current best practice * benefits of probiotic use, at least in rheumatic diseases, demand bona fide data key: cord- -mejd blb authors: lewnard, joseph a; reingold, arthur l title: emerging challenges and opportunities in infectious disease epidemiology date: - - journal: am j epidemiol doi: . /aje/kwy sha: doc_id: cord_uid: mejd blb much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. initially submitted september , ; accepted for publication november , . much of the intellectual tradition of modern epidemiology stems from efforts to understand and combat chronic diseases persisting through the th century epidemiologic transition of countries such as the united states and united kingdom. after decades of relative obscurity, infectious disease epidemiology has undergone an intellectual rebirth in recent years amid increasing recognition of the threat posed by both new and familiar pathogens. here, we review the emerging coalescence of infectious disease epidemiology around a core set of study designs and statistical methods bearing little resemblance to the chronic disease epidemiology toolkit. we offer our outlook on challenges and opportunities facing the field, including the integration of novel molecular and digital information sources into disease surveillance, the assimilation of such data into models of pathogen spread, and the increasing contribution of models to public health practice. we next consider emerging paradigms in causal inference for infectious diseases, ranging from approaches to evaluating vaccines and antimicrobial therapies to the task of ascribing clinical syndromes to etiologic microorganisms, an age-old problem transformed by our increasing ability to characterize human-associated microbiota. these areas represent an increasingly important component of epidemiology training programs for future generations of researchers and practitioners. infectious diseases; methods; modeling; surveillance abbreviation: ebov, ebola virus. the priority afforded to infectious diseases within epidemiologic research has been fluid over the past years or longer. despite the lasting prominence of early investigations into measles, cholera, plague, typhoid fever, malaria, and yellow fever ( ) ( ) ( ) ( ) ( ) ( ) , the intellectual tradition of modern epidemiology stems largely from studies of chronic diseases dating to the post-world war ii era, when such conditions came to surpass infectious diseases in morbidity and mortality in highincome countries amid improvements in living conditions and the introduction of numerous antibiotics and vaccines. this epidemiologic transition co-occurred with a shift in focus for epidemiologic research (and training programs) toward the multifactorial etiology of chronic conditions ( ) . in parallel, early th-century work on the "dependent happenings" of communicable diseases ( ) ( ) ( ) ( ) yielded to the development of today's core biostatistical methods for chronic diseases, premised on the independence of outcomes among subjects ( ) ( ) ( ) . since the late th century, the emergence of human immunodeficiency virus and acquired immunodeficiency syndrome and other infections has renewed interest in infectious diseases and their health, economic, and security implications ( ) . outbreaks of severe acute respiratory syndrome, pandemic influenza a h n , ebola virus (ebov), and zika virus have prompted international responses, whereas influenza a h n and h n , lassa fever virus, nipah virus, and middle east respiratory syndrome coronavirus, among other agents, have been a source of regional concern. the incidence and endemic range of "neglected" infections, including dengue and cholera ( , ) , have expanded, and antimicrobial resistance has threatened to derail the control of tuberculosis, typhoid, malaria, gonorrhea, yaws, and invasive bacterial infections ( ) ( ) ( ) ( ) ( ) ( ) . although highly effective vaccines are available, measles and yellow fever have resurged on multiple continents, due to gaps in vaccine coverage ( , ) , while short-lived vaccine-induced protection has facilitated unexpected resurgences in diseases once on the path to elimination, such as pertussis and mumps ( , ) . after decades of relative obscurity in the mid- th century, infectious disease epidemiology has experienced an intellectual rebirth in response to disease emergence. repopulation of this field by scientists trained not only in clinical medicine but ecology, demography, and quantitative sciences has led to the adoption of methods scarcely addressed in traditional public health training programs. cluster-randomized trial designs, for example, have become commonplace for evaluating infectious disease interventions, quantifying indirect effects resulting from contagion ( ) . classical models of ecological dynamics have been adapted to address the transmission and control of infectious agents, while our expanding ability to integrate such models with epidemiologic data through bayesian statistics has enhanced their relevance to policymaking ( ) ( ) ( ) . most recently, sequencing and phylogenetic analysis have afforded an unprecedented view into the population structure and dynamics of pathogens ( , ) . here, we review developments in infectious disease epidemiology together with their implications for research and practice, and for the training of future epidemiologists. we first consider the role of epidemiologic surveillance in the context of pathogen emergence and the integration of surveillance data into quantitative studies of transmission. next, we discuss challenges in causal inference, including the evaluation of public health interventions against emerging pathogens and the difficulties of attributing clinical syndromes to microbial agents. surveillance justifiably has been seen as a core public health function, with its important role articulated by langmuir in ( ) . in the united states and elsewhere, surveillance for diverse infectious (and noninfectious) diseases has typically relied on an essentially passive system of reporting by healthcare providers or laboratories, often mandated by public health laws. although such passive systems of disease reporting have proven invaluable, their limitations, such as incomplete, often inconsistent detection of cases and delayed detection of outbreaks, are well documented ( ) . as a result, active surveillance systems that do not depend on providers and laboratories to report have been developed and promoted. the centers for disease control and prevention-funded emerging infections program and its components (e.g., foodnet, abcs), initiated in , is notable example of such a system, as are international versions promoted through the centers for disease control and prevention's global health security initiative ( ) . such systems, while expensive to develop and maintain, are of great value, especially when they collect biological specimens (e.g., isolates of bacterial and viral pathogens) for typing and support analytic epidemiologic studies (e.g., casecontrol studies of vaccine effectiveness ( ) ). nevertheless, these systems, too, may have limitations, particularly in their ability to detect in a timely fashion outbreaks caused by novel microbial agents, prompting interest in alternative methods. in this article, we consider proposed alternatives, highlighting their benefits and challenges. beyond efforts to quantify the incidence of infectious diseases of known etiology, disease surveillance has been advocated as a means of mitigating the threat posed by novel pathogens. large-scale efforts to identify pathogens with the potential to spill over from animals to humans have received notable investment, for instance from the us agency for international development emerging pandemic threats program ( ) . more recently, metagenomic sequencing has enabled the number of known viruses to be multiplied in such studies ( ) . the aim of those working on the global virome project, launched in , to characterize within years all . million viruses thought to exist ( ) . however, the pathway for translating data sets produced by such activities into actionable threat-reduction programs remains unclear. that only approximately viruses are known to infect humans (and an even smaller handful to cause major epidemics) may constrain the value of large-scale virus discovery for identifying high-risk pathogens, as well as viral determinants of pathogenic potential ( ) . spillover events causing recent epidemics-including h n in mexico, middle east respiratory syndrome in saudi arabia, and ebov in west africa-have been poorly predicted by factors long believed to drive disease emergence ( ) . accordingly, interest in expanding our catalogue of potential pathogens should be weighed against our persisting need to enhance the detection and control of outbreaks of known pathogens ( ) . for instance, ebov epidemics in the democratic republic of the congo in took weeks to be identified, with dozens of suspected cases having already accumulated ( , ) . serological studies have received increasing enthusiasm for monitoring emerging pathogens of significance to humans ( , ) . the prevalence of antibodies indicating previous exposure may provide valuable information about the frequency of animal-human spillover events and the potential for person-toperson spread, overcoming reporting biases that favor detection of large outbreaks under traditional surveillance. moreover, the low cost of multiplex assays makes integrated surveillance of multiple pathogens plausible. although serosurveys have bolstered recent efforts to understand the geographic range and clinical spectrum of ebov and zika virus infections ( , ) , the enhancement of dengue hemorrhagic fever risk by prior exposure ( ) , and the role of immunologic history in influenza susceptibility and vaccine response ( ) , there remain few examples of public health programs undertaking serological studies for routine surveillance, at least in civilian populations ( ) . outside of laboratory-based surveillance, the increasing availability of passively collected "big data" on the health and behaviors of individuals has prompted enthusiasm about enhancing disease surveillance through alternative data streams. initiatives such as the promed-mail network and healthmap ( , ) compile and disseminate news about outbreaks from media and other sources, aiming to trigger investigation by public health organizations. data such as emergency department visits, medication sales, online search queries, and social media postings have also been suggested as real-time indicators of outbreak activity, although their integration into public health responses remains a subject of debate ( ) ( ) ( ) . the need to overcome reporting biases is a central challenge, because observations may be too nonspecific to distinguish between meaningful and spurious signals in settings with high technological capacity while also being insensitive to even high-risk events in resource-poor settings ( , ) . although nontraditional data sources have, in some applications, supported inferences about epidemic dynamics ( ) , limited information about cases from such sources remains a barrier. for instance, models fitted from news reports of recent measles and mumps outbreaks have yielded considerable underestimates of vaccine coverage ( ) ( ) ( ) , underscoring the importance of field investigations. forecasting the incidence of diseases has been a more successful application of these emerging data streams and dataanalytic approaches. although the acknowledged failure of google flu trends-a prediction approach based on internet search behavior-yielded important lessons about nonmechanistic forecasts, approaches based on machine learning and crowdsourced human judgment have provided the most accurate within-season predictions of us influenza activity in recent comparisons ( ) ( ) ( ) . given expanding interest in forecasting among researchers, funding agencies, and other stakeholders, there is a clear and compelling need to evaluate whether such forecasts can enhance the success and efficiency of public health response efforts. mathematical modeling as a means to understanding infectious disease spread dates to studies by sir ronald ross ( ) . although the use of models to connect data such as age of infection to transmission dynamics of endemic infections has longstanding precedent ( , ) , assimilation of outbreak data for near-term assessments of control priorities is a comparatively recent phenomenon. integration of modeling with the public health response to epidemics of bovine spongiform encephalopathy and foot-and-mouth disease in the united kingdom and the severe acute respiratory syndrome epidemic ( - ) has led to expectations for near real-time modeling studies during major outbreaks. in recent experience, models of the spread of pandemic influenza a h n ( ), cholera ( ), middle east respiratory syndrome ( ), ebov ( , ) , chikungunya virus ( ), zika virus ( , ) , yellow fever ( ) , and plague ( ) have all been published within weeks of the respective outbreak notifications. although the circumstances of particular epidemics dictate what data may be available and pertinent, methods for fitting models to data have generally focused on exponential growth rates in cases ( ) or the distribution of the serial interval ( ) . methods based on the latter class of data offer the advantage of illustrating real-time changes in reproductive numbers ( ) ; however, the requisite information from patient line lists is seldom available. reliance instead on ecological data exposes models to numerous vulnerabilities, notably the inability to discern individual risk factors (and thus the population meaningfully at risk). these shortcomings may prevent models from predicting reductions in transmission before depletion of the susceptible population. a challenge thus lies ahead in determining the role of models in outbreak response and the best practices for communicating modeling results. although the ability of models to evaluate prophylactic strategies may be considered a benefit, recommendations to act against remote future risks have sometimes triggered resistance among stakeholders ( ) . during the west african ebov epidemic, for example, attention to worst-case model-based projections prompted some to question the reliability of the models ( ), reflecting an important discrepancy between public understanding of modeling as a forecasting tool and the intended uses of models for scenario-based comparisons ( , ) . this use of modeling has been better understood in attempts to communicate the impact of interventions after the fact ( ). the ease of sequencing pathogen genomes has afforded a new view into transmission during outbreaks. use of sequence data to identify transmission clusters in the presence of unsure epidemiologic links dates to the early years of the human immunodeficiency virus and acquired immunodeficiency syndrome epidemic ( ) . in recent years, sequencing has aided efforts to track the sources of unexplained epidemics of cholera in haiti ( ) and ebov in west africa ( ) , and has shown increasing utility for reconstructing the geographic spread of pathogens ( , ) . a particular advantage of phylogenetic analysis is the possibility of estimating unobserved epidemiologic quantities, such as the reporting fraction ( ) and reproductive numbers for subcritical transmission ( ) , which remain difficult to assess from traditional case-notification data. beyond reconstructing the demographic history of pathogen lineages, recent years have seen progress toward joint analysis of epidemiologic and sequencing data ( ) . such "phylodynamic" approaches have shown particular relevance for emerging infections, including distinguishing the role of repeated introductions and subsequent local transmission ( ) ( ) ( ) ( ) . whereas most applications have been tailored to specific data sets and assumptions, the development of generalized methods for joint inference of epidemiologic and phylogenetic parameters remains a priority ( ) to support real-time analysis. the ability to rapidly develop and deploy countermeasures to mitigate the threat posed by emerging infections has received increasing recognition as a component of public health preparedness. however, outbreaks are difficult environments in which to evaluate interventions. during the west african ebov epidemic, the feasibility of a new paradigm for development and evaluation of interventions in emergencies was demonstrated by accelerated vaccine safety, immunogenicity, and efficacy studies ( ) . the coalition for epidemic preparedness innovations was established in , with an initial focus on vaccines against nipah virus, middle east respiratory syndrome coronavirus, and lassa fever virus, in addition to adaptable vaccine platforms for novel threats ( ) . lessons learned in ebov vaccine trials will have an influential bearing on evaluations during future emergencies. despite efforts to accelerate evaluation of candidate vaccines, incidence had reached low levels by the time phase iii efficacy trials were ready to begin, posing a threat to their statistical power: a planned trial in liberia was canceled due to declining transmission ( ) , and no cases of disease occurred in a second trial in sierra leone ( ), preventing efficacy assessments. in a stepped-wedge trial in guinea, clusters of primary and secondary contacts of ebov disease cases were randomly assigned to immediate or delayed vaccination; no cases were reported among vaccine recipients during the trial ( ) or in subsequent field deployments of the vaccine, supporting a conclusion of near % vaccine efficacy. debates surrounding design of these trials highlight methodological questions requiring additional attention. in the guinean trial, a "ring" vaccination scheme helped maximize power by enrolling contacts of known cases ( ) . however, the choice of individual-or cluster-level randomization within rings was debated. because members of a vaccinated cluster are exposed to direct protection through vaccination and indirect protection due to reduced transmission within their clusters ( ), cluster-randomized trials have weaker statistical power than individually randomized trials of the same size ( ) . moreover, the direct effect measured in individually randomized studies may be a preferred, transportable efficacy measure ( ) . uses of simulation helped in planning vaccine trials tailored to the real-world circumstances of the ebov outbreak ( ) and enabled trialists to compare alternative designs in terms of ethical mandates ( ) . simulation-guided design further presents the opportunity for applying adaptive trial methods ( ) in the context of infectious disease outbreaks, where dynamic trends in incidence may highlight the benefits of such approaches. in addition to efficacy trials for new interventions, observational studies are needed to assess licensed interventions against evolving and re-emerging pathogens. most commonly applied in evaluations of influenza vaccines, test-negative designs have become popular in routine ( ) and exploratory ( ) studies of vaccine effectiveness. by measuring vaccine effectiveness from the exposure odds ratio of vaccination among individuals seeking care who test positive or negative for a pathogen of interest, this design seeks to overcome associations of health-care seeking with vaccination status ( ) . however, it is uncertain whether health-care seeking and other sources of confounding are appropriately controlled for, and whether measures accurately capture vaccine direct effects ( , ) . uncertainty about the validity of estimates that routinely inform vaccine policy-making demonstrates the need for formal evaluations of such studies and strategies to reduce bias. time series analyses of public health surveillance data provide another approach to measuring the real-world impact of vaccination on disease incidence, with the advantage of identifying the overall effect of a vaccination program resulting from direct and indirect protection ( ) . although the ecological nature of such designs permits the introduction of biases from changes in diagnostic practices or health-care seeking, such studies nonetheless have offered important insights where other approaches failed. limited reductions in influenza-related deaths among elderly persons amid increases in influenza vaccine coverage during the s provided an important indication that the "healthy vaccinee" effect accounted for astonishing and implausible protection against all-cause mortality among elderly influenza vaccine recipients in cohort and case-control studies ( ) ( ) ( ) . newer methods continue to improve public health inferences obtained from time-series data. in a recent evaluation of invasive pneumococcal disease incidence, trends expected under continued use of -valent pneumococcal conjugate vaccine provided a counterfactual condition for measuring the impact of the switch to a -valent vaccine targeting emerging serotypes ( ) . bayesian averaging of models encoding differing pre-and postvaccination trends and change points provides a generalized strategy for defining such counterfactual comparisons ( ) . other signals of transmission intensity in surveillance data, such as age of infection and sub-or multiannual periodicity, may provide additional insights while reducing sensitivity to fluctuations in reporting effort ( , ) . the re-emergence of pathogens against which vaccines are widely deployed, such as varicella, pertussis, and mumps in the united states, poses additional challenges for conducting vaccine effectiveness studies. situational factors may undermine researchers' ability to establish the extent to which cases owe to primary or secondary vaccine failure in all or certain vaccine recipients, and whether emerging pathogen lineages are escaping vaccine-driven immune pressure. for instance, high compliance with vaccine schedules may limit variation in individuals' vaccination status and exposures, necessitating large samples to detect factors influencing vaccine performance ( ) . because re-emergence most likely reflects the expansion of or several pathogen clades, limited pathogen diversity may hinder the application of conventional approaches to identifying microbial determinants of vaccine escape ( , ) . novel methods to distinguish null from vaccine-driven mutations in antigencoding regions ( , ) may streamline efforts to identify vaccine escape, while mathematical modeling provides a basis for comparing candidate hypotheses with observations ( , ) . because vaccine studies are typically powered for primary clinical endpoints, long-term observational studies are needed to monitor for rare vaccine-attributable adverse events. such studies have been crucial to identifying safety concerns such as intussusception after rotavirus vaccination ( ) and to refuting spurious links, such as autism onset after measles-mumpsrubella vaccination ( ) . however, unique challenges arise in vaccine safety studies; at the individual level, vaccination and adverse-event detection may be confounded due to health-careseeking behavior, whereas at the population level, age-related confounding may occur when vaccine recommendations are based on the individual's age. ecological designs taking advantage of natural experiments have proven useful in numerous studies of vaccine safety ( , ) but inconclusive for certain classes of rare events, including those that also result from the vaccine-targeted infection ( , ) . recent years have seen growing interest in case-only methods offering the ability to reduce or rule out individual-level sources of confounding. case-crossover methods are common among such approaches ( , ) and resemble matched case-control studies by sampling "control" periods from the person-time contributed by case individuals before an adverse event. self-controlled case-series methods similarly benefit from the use of cases as their own controls, following a cohort logic in estimating the relative incidence of adverse events after vaccination within specified risk periods ( ) ; with adequate sample size, researchers may be able to use such analyses to eliminate or greatly reduce potential time-or age-related confounding. in response to the growing threat posed by antimicrobial resistance, the world health organization and national governments have prioritized bringing novel antimicrobial drugs to market ( ) . these plans will necessitate phase iii trials in which the efficacy of new therapeutic agents is addressed and possibly phase iv studies, in which the optimal use of new and existing drugs, either singly or in combination, can be determined. whereas patients traditionally have been enrolled in antimicrobial treatment studies on the basis of target bacterial species infections or clinical syndromes, it is uncertain within what strata such trials may yield transportable inferences. rather than merely the infecting pathogen's baseline resistance or susceptibility phenotype, strata may be defined by factors such as pathogen lineage, mutational barriers to resistance development ( ) , and presence of horizontally transferable resistance elements in cocolonizing agents or environmental sources ( ) . stratification based on interpatient and even intrapatient tumor heterogeneity is an emerging feature of cancer therapy trials and may provide a template for such designs ( ) . in addition to clinical endpoints, carriage of susceptible and resistant bacteria, including commensal agents not purposefully targeted by treatment, can inform the impact of treatment on resistance selection in targeted and bystander species ( ) . whereas between-group differences in the absolute prevalence of colonization with resistant organisms ( ) are tested for routinely, stratified measurements (see the reports of shrag et al. ( ) and feikin et al. ( ) , for example) of the effect of treatment on acquisition and clearance of susceptible and resistant pathogens are more informative of the underlying biology ( ) and may detect signals of selection masked by simpler betweengroup comparisons ( , ) . studies are also needed to address optimal deployment of new and existing antimicrobial drugs in clinical practice. the tradeoff between maximizing a drug's impact and minimizing resistance selection has led policymakers to ration certain new drugs as last-resort treatments. however, in recent experience, such decisions have ignited ethical debates ( ) . coupling mathematical modeling with field-based studies has proven useful for understanding the effectiveness of antimicrobial use policies, as highlighted in recent evaluations of the risk of resistance under population-wide access of the tuberculosis drug bedaquiline ( , ) and antimicrobial cycling to limit resistance selection in hospital settings ( , ) . whereas certain efforts we have discussed have been made to identify novel microorganisms able to cause human infection ( , ) , most epidemics caused by emerging pathogens have been recognized first by clusters of anomalous syndromes-such as cardiopulmonary syndrome caused by new world hantaviruses, severe acute respiratory syndrome caused by a coronavirus, and congenital abnormalities caused by zika virus -before the role or even existence of the etiologic microorganism had been characterized. the problem of ascribing a clinical syndrome to an etiologic agent is among the oldest in epidemiology, dating at least to the th century, when robert koch laid out criteria for such inference (i.e., koch's, or more correctly, henle-koch's, postulates ( ) ). however, these postulates have long been recognized as inadequate, particularly for illnesses caused by viruses, and thus of largely historical interest ( ) ; for instance, the notion that a pathogen should be absent from healthy individuals is incompatible with the prominence of carriage and asymptomatic infection in the natural history of numerous pathogens. a growing appreciation of the complexity of the human microbiome, and the likelihood that intricate mixtures of microorganisms at diverse body sites may be either the cause or consequence of both detrimental and beneficial physiological states, has further highlighted the difficulty of linking a given health outcome to infection by a single microorganism. the now-recognized critically important role of persistent infection and the inflammation it can produce in diverse cancers and possibly other chronic diseases has further diminished the relevance of koch's postulates and the age-old distinction between infectious and chronic diseases, as illustrated in the well-known example of human papillomavirus causing cervical, anal, and oral cancers ( ) . the best causal understanding of such relationships has come from randomized trials demonstrating that infection-preventing interventions are efficacious against downstream chronic illness, such as peptic ulcers due to helicobacter pylori and chronic wheeze due to respiratory syncytial virus ( , ) . natural experiments following the same intuition have provided additional evidence of such relationships, such as measles-induced immunosuppression ( ) , malnutrition and stunting due to enteric infection ( ) , and complex or chronic otitis media due to tissue damage from acute early-life disease ( ) . such relationships have proven difficult to probe in the absence of a randomized or natural experiment, because of the likelihood that confounding factors influence individuals' risk for initial infection as well as chronic sequelae. new paradigms for ascribing an etiologic role to microorganisms and resulting host responses are clearly needed and may prove important in efforts to quantify the health impacts of infectious disease interventions. the recognition in the s and s that infectious diseases were not, in fact, disappearing 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years of age in resourcepoor environments prevention of early episodes of otitis media by pneumococcal vaccines might reduce progression to complex disease the authors received no specific funding for this article. conflict of interest: none declared. key: cord- -ca avyjf authors: zhang, lei; wilson, david p. title: trends in notifiable infectious diseases in china: implications for surveillance and population health policy date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ca avyjf this study aimed to analyse trends in notifiable infectious diseases in china, in their historical context. both english and chinese literature was searched and diseases were categorised according to the type of disease or transmission route. temporal trends of morbidity and mortality rates were calculated for eight major infectious diseases types. strong government commitment to public health responses and improvements in quality of life has led to the eradication or containment of a wide range of infectious diseases in china. the overall infectious diseases burden experienced a dramatic drop during – , but since then, it reverted and maintained a gradual upward trend to date. most notifiable diseases are contained at a low endemic level; however, local small-scale outbreaks remain common. tuberculosis, as a bacterial infection, has re-emerged since the s and has become prevalent in the country. sexually transmitted infections are in a rapid, exponential growth phase, spreading from core groups to the general population. together human immunodeficiency virus (hiv), they account for % of all death cases due to infectious diseases in china in . zoonotic infections, such as severe acute respiratory syndrome (sars), rabies and influenza, pose constant threats to chinese residents and remain the most deadly disease type among the infected individuals. therefore, second-generation surveillance of behavioural risks or vectors associated with pathogen transmission should be scaled up. it is necessary to implement public health interventions that target hiv and relevant coinfections, address transmission associated with highly mobile populations, and reduce the risk of cross-species transmission of zoonotic pathogens. china has experienced a large decline in the spread and burden of infectious diseases since the early s, associated with effective and large-scale public health interventions and large populationbased vaccination programmes. china successfully eliminated infectious diseases -including smallpox from the general chinese population in early s [ ] , years before its global eradication -and another infectious diseases, including poliomyelitis, have more recently been eliminated [ ] . a further diseases, including measles, are thought to be contained well at low endemic levels [ ] . surveillance systems for infectious diseases in china are mainly hospital based. the latest available statistics (from ) indicate that china has , county hospitals, , township hospitals and , medical clinics [ , ] . hospitals at the prefecture level or above are usually equipped with reference laboratories that are capable of carrying out molecular surveillance for cases. together with laboratories from academic institutions, they form the front line of surveillance for outbreak detection and notification of infectious diseases in china. all hospitals and clinics are obliged to report both suspected and confirmed cases of notifiable infectious disease to their nominated county centre for disease control (cdc). after recording the details of the reported cases (including their geographical location, demographicl information and infection status), county cdcs send the information to the country's central cdc, through the national infectious diseases monitoring information system database, which was established in [ ] . this reporting mechanism bypasses the previous stepwise hierarchical reporting framework, thus allowing information to flow directly from grassroots cdcs to china's central disease database. however, regional, provincial and national cdcs do not have equal access to all of the data in the database: their access rights are limited to their own administrative regions, and only national cdcs has the full access to all data [ ] . in addition to their responsibility for verifying the disease information from their administrative regions, municipal and provincial cdcs are required to report to the appropriate level of the bureaus of health or department of health and to form networks with local research bodies, universities and other health organisations. at the top of the hierarchy, china's central cdc is the overseeing organisation with responsibility for assembling and analysing diagnostic data for all diseases and then presenting a final report to the ministry of health, which is the main public health policymaker in china. the central cdc is also the only legitimate office for disseminating infectious disease information to the public, but provincial and municipal cdcs are also authorised to publicise the information to people in their jurisdiction, under the supervision of the ministry of health [ ] . in contrast to disease surveillance systems in europe, the chinese surveillance system uses a multilayer administrative mechanism that enables rapid and efficient upward flow of epidemic information. china is a populous country of . billion people [ ] . over the last years of economic reform in the country, there have been environmental, demographic, social and behavioural changes in the population. as china's ties with the rest of world become increasingly strong, infectious diseases in china no longer remain a domestic issue. a thorough review of infectious disease surveillance in china is timely and important for the country's disease prevention and control strategies. currently, infectious diseases are classified as notifiable in china. this study reviews trends in notifiable infectious diseases in china, in their historical context, discusses the current epidemiological state of these infections and their implications for disease surveillance and public health interventions. the review is structured by category of disease or transmission route. currently, infectious diseases are notifiable in china, classified as a, b or c according to their epidemic levels and potential population threats. groups a and b (total diseases) represent categories of diseases with high risk of outbreaks or that are likely to result in rapid spread once an outbreak occurs. mortality and morbidity related to group a and b diseases are reported and published by the chinese ministry of health on a monthly basis. group c diseases are less infectious and, when outbreaks occur, are epidemiologically less severe. they are required to be reported only when outbreaks occur. in this review, we searched published peer-reviewed research articles as well as online reports and grey literature from to relevant to disease surveillance in china in the following databases: pubmed, chinese scientific journals fulltext database (cqvip), china national knowledge infrastructure (cnki) and wanfang data. keywords used in the database search included ['chinese' or 'china'] and ['infectious diseases surveillance' or 'surveillance system' or 'infectious diseases monitoring' or 'infectious diseases information' or the type of infectious disease or the name of individual infectious diseases]. we also searched governmental reports, reports of non-governmental organisations and other grey literature from online sources. we then collated data on notified cases and mortality related to these diseases, using the latest available information from the ministry of health [ ] . notifiable infectious disease data, including morbidity and mortality rates, was summarised by chinese ministry of health and published annually in chinese health yearbook and online accessible through cnki database. notably, this dataset does not contain data on sars and influenza a(h n ) virus infection. hence we did not include them in our statistical analysis, but describe them in the text. the selected diseases were categorised into eight major types according to their diseases characteristics and origins. the morbidity and mortality for each disease type were calculated as the sum of the corresponding rates of individual diseases. the total number of diagnosed and death cases were estimated by multiplying morbidity and mortality rates by the overall chinese population in the study years. case fatality rate was defined as the percentage of persons diagnosed with the disease who die as a result of the illness during the calendar year, and was estimated by dividing mortality rate by morbidity rate of the diseases. further, for each individual disease, we calculate the disease-specific mortality rates among the chinese population in five-year intervals ( - and - ) . for each disease, the mean annual increase or decrease during - , with % confidence intervals, is estimated, based on linear regression of logarithmic values of the number of annual death cases. trends in infectious diseases in china, china, - morbidity of notifiable infectious diseases in china, represented by estimated numbers of new cases, declined substantially (. %) from , cases per million in to a nadir level of , cases per million in (figure a ). since then, the rate of new infectious disease cases gradually reverted and maintained an upward trend. in , the estimated rate of infectious diseases among the general chinese population reached over cases per million population. the composition of diagnosed diseases cases also changed substantially, in , the three most reported diseases were gastrointestinal diseases ( . %), vector-borne diseases ( . %) and vaccine-preventable diseases ( . %), corresponding to a total of . % of all diagnosed cases ( figure a ). additionally, these three types of diseases account for . , . and . million reported cases respectively during the period - (table ). in contrast, in , although gastrointestinal diseases remained the dominating disease type ( . %), the proportion of all cases that were due to viral hepatitis quickly rose to . %. sexually transmitted diseases re-emerged, and together with hiv, consisted of a substantial . % of all reported cases. in , the three most frequently reported disease types included viral hepatitis ( . %), bacterial infections ( . %) and stis and hiv ( . %), which account for . , . and . million diagnosed cases respectively during the period - (table ) . rapid declines in infectious diseases mortality and its similar saddle pattern were also observed in the past years. the overall mortality rate in china decreased from cases per million in to cases per million in , then it gradually reverted to cases per million in ( figure b ). vaccine-preventable diseases, bacterial infections and gastrointestinal diseases were the greatest causes of death, accounting for . %, . % and . % of reported infectious diseases death cases among chinese population in ( figure b) . however, the rank of composition shifted to zoonoses ( %), viral hepatitis ( . %) and quarantinable diseases ( . %) in . since then, the proportion of deaths caused by stis and hiv, and bacterial infections rapidly increased. by , stis and hiv ( . %) has become the mostly deadly infectious disease, followed by bacterial infections ( . %) and zoonoses ( . %). during the period - , these three types of diseases have led to approximately , , , and , deaths in china, respectively (table ) . case fatality rate measures the percentage of deaths among people who contracted a disease. during - , the disease type with the highest fatality rate is zoonoses, consistently causing - % of deaths among the infected population. following that, quarantinable diseases killed . - . % of its infected population during the same period. in comparison, fatality rates in vaccinepreventable ( . (table ) . plague, cholera and epidemic haemorrhagic fever (which is caused by hantaviruses and mainly transmitted by rodents) have had diverse impacts on the chinese population at different stages of recent history. during to , there were notified outbreaks of plague across china, which resulted in an estimated . million deaths. from to , the mean number of annual notifications of plague infection was , cases per year [ , ] . this was then substantially reduced to cases per year over the following to years. since , the number of annual reported plague cases has increased to approximately , which is equivalent to . per , population per year [ , ] . there is no observed trend in the table . estimated number of diagnosed cases, death cases and case fatality rate of eight major infectious disease types in china in -year intervals. period period - period period - period period - period period - period period - period period - period period - quarantinable diseases mean annual mortality due to plague during - (table ) . cholera has caused two major outbreaks in china in recent decades: the first started in , leading to a large peak in the number of cases in , with an annual notification rate of four cases per , population ( figure a ). the rate then gradually subsided but there was a second major outbreak in the early s. during to , cholera cases were reported at relatively low incidence levels: the disease is well controlled, with a mean annual decline in mortality due to the disease of % ( % ci, - %, table ). despite a -fold decrease in the epidemic haemorrhagic fever notification rate since and continuous decline in the diseasespecific mortality rate, china is still the country most severely affected by this disease, with % of the global cases of haemorrhagic fever with renal syndrome (hfrs) occurring in china [ , ] . hfrs remains an important public health issue in china, as an estimated , - , new cases are reported each year [ ] . it is important to establish effective strategies to reduce the incidence of hfrs. the mean annual decline in mortality due to this disease is % ( % ci, - %, table ). persistent public health campaigns have been effective in reducing the number of rodents, which has been largely responsible for the decreasing trend in plague and epidemic haemorrhagic fever notifications. improved hygiene and sanitary conditions have also led to the decline in incidence and mortality due to cholera. overall, these quarantinable diseases persist at low endemic levels, but still pose potential threats. in the early s, a nationwide vaccination programme was implemented to eradicate smallpox, measles, poliomyelitis, tuberculosis (tb), pertussis, diphtheria and tetanus. measles vaccine (one-dose schedule) was introduced in the country in , and from it has been administered routinely to all infants through the country's expanded programme on immunisation [ ] . measles mortality and morbidity has declined continuously and substantially since . during to , the incidence was less than one per , population (figure b), with fewer than deaths due to measles reported annually [ ] . however, limited small-scale outbreaks continue to occur among susceptible children in rural areas with low routine immunisation coverage and among susceptible children of 'floating immigrants' (the relatively large population of internal chinese immigrants who leave their rural hometown and move to urban areas to seek better employment opportunities) [ ] . in addition, although a two-dose measles vaccination schedule has been recommended in china since , the second dose is widely perceived as a nonmandatory booster. coverage of the second dose is not reported, but is thought to be low [ ] . a global effort to eradicate polio began in [ ] ), wild-type poliovirus was eradicated in china in [ , ] ; extensive surveillance for acute flaccid paralysis is thought to have strongly contributed to polio eradication in the country [ ] . vaccination against diphtheria, pertussis and tetanus was also made mandatory for all infants under the country's expanded immunisation programme since s. in , vaccination coverage of the vaccine against the three diseases reached . %, comparable with polio vaccination ( . %) and measles vaccination ( . %) [ ] . since , the total annual incidence of reported cases of diphtheria, pertussis and tetanus decreased to below . cases per , population ( figure b ). widespread vaccination against these diseases is one of the important contributors to improvements in child health in china: infant mortality rate dropped from % in to . % in [ ] . mandatory vaccination of children throughout the past decades has been demonstrated to be successful in containing the spread of these diseases in china. it is estimated that polio vaccination alone has saved a total of , , children in china from disabling disease and suffering [ ] . mortality due to pertussis and tetanus experienced significant annual decays at rates of % ( % ci, - %) and % ( ci%, - %) ( table ) . trends in tb are discussed in the section on bacterial infections. the number of reported cases of bacillary and amoebic dysentery has declined rapidly since in china (figure c ). an initial large decline occurred in the mid- s, largely due to substantial improvements in sanitary conditions and quality of drinking water. however, approximately million diarrhoeal episodes were still reported in china each year at the end of the s: % of the episodes were in children under five years of age [ ] . shigella bacilli and entamoeba histolytica have been found to be the main aetiological organisms [ ] . a live oral shigella vaccine was then developed in in china, which provided - % protection against s. flexneri serotype a and s. sonnei infection [ ] . between and , reported deaths due to dysentery decreased by a mean of % ( ci%, - %) per year ( table ). the number of notified cases of typhoid and paratyphoid fevers is very low in china, with an annual rate of . notifications per , population in (figure c ). the number of reported deaths due to these infections has decreased at a mean rate of % ( % ci, - %) per year (table ) . however, these infections remain endemic in some rural areas of southern china, where there is a lack of adequate sewage disposal and safe water supplies [ ] . a large, localised outbreak occurred during to in xing-an county in the southern province of guangxi, which resulted in an annual incidence rate of to cases per , population [ ] . subsequently , doses of vi polysaccharide vaccine were administered in and the epidemic quickly subsided. no further typhoid fever outbreaks were reported in china. national public health campaigns since in china effectively improved hygiene and sanitary conditions, which substantially reduced most of vector-borne infectious diseases. in , a total of , , malaria cases were reported nationally, accounting for % of the total number of reported infectious disease cases and an incidence of , per , population. malaria disproportionately affected some regions, with incidence rates as high as , per , population in hainan province [ ] . in and , there were major outbreaks of the disease, leading to a national incidence of , and , , respectively, per , population [ ] . it took considerable time for the incidence to fall: the number of cases did not decrease to levels until , but it has since continued to decrease. by , the rate of notifications decreased to . per , population, corresponding to more than a % reduction and accounting for only . % of the total number of reported cases of notifiable conditions [ ] . data for to indicate that the mean annual mortality rate due to malaria was reduced to . per , population (table ) , corresponding to only reported deaths per year countrywide. malaria no longer represents a major threat to the chinese population. the ongoing governmentmobilised mass movement for vector control is probably an effective method for eliminating mosquitoes and their habitats and consequently reducing the disease burden due to malaria in the country. the universal healthcare system provided an effective base for the implementation of countrywide interventions [ ] . as a result of the national public health campaigns to eliminate infectious disease vectors, forest encephalitis (also named russian spring-summer encephalitis), tick-borne relapsing fever, tsutsuga-mushi disease (also known as scrub typhus) and epidemic typhus were reduced to very low levels by the late s and were then no longer notifiable. the number of visceral leishmaniasis (also known as kala-azar), japanese encephalitis, malaria and dengue fever epidemics has also decreased, with few cases per year (figure d) . however, there are signs that climate change may have led to local outbreaks of some these vector-borne diseases in the past decade in china [ ] . rabies is one of the zoonotic diseases on the rise in china. during a -year period between and , a total of , human rabies cases were reported, corresponding to an incidence rate of . cases per , population. three major epidemics were observed [ , ] : the first occurred in the mid- s when the annual number of reported cases reached a peak of about , . the epidemic subsided in the s and then started to surge again in the early s. after reaching a sustained peak from until the end of the decade, at a level of , to , cases per year [ ] , in the number of cases was at its lowest, with reported cases [ ] . as the disease in humans is closely associated with transmission of rabies virus by infected dogs, in the past epidemics, strict policies for vaccination of owned dogs and elimination of stray dogs were put in place, resulting in effective control of the epidemic. however, since , due to a new wave of rapid expansion of the pet dog population in urban china, there has been a third outbreak, in which the number of cases has increased rapidly: in , the number of notified cases climbed to , [ , ] . the rabies-related mortality rate increased during to by a mean of % ( % ci, - %) per year ( table ). this should serve as a call for action to prevent the further spread of rabies in china. severe acute respiratory syndrome (sars), caused by a coronavirus that was transmitted to humans through close contact with civet cats, spread from guangdong, china, quickly leading to a worldwide epidemic in . china reported , ( %) of the , sars cases globally and sars-related deaths during the epidemic, which lasted for approximately a year [ ] . the first human case of highly pathogenic avian influenza a (h n ) virus infection was reported in hong kong in . by , mainland china had avian influenza outbreaks among birds in provinces and a total of human cases and deaths had been reported [ ] . in , the epidemic had spread to countries and resulted in deaths, out of cases [ ] . in comparison, the influenza a(h n ) pandemic caused more than , human cases and deaths in china alone [ ] , despite the virus being much less virulent than influenza a(h n ) virus. china, with concentrated human activity in its fast-growing major cities that may lead to effective transmission of human-tohuman transmission of h n , is very vulnerable to the transmission of respiratory infectious diseases of zoonotic origin. during the mid-to-late stages of the sars outbreak and, more apparently throughout the course of the influenza pandemic, the chinese government responded swiftly with openness and transparency to control both diseases and prevent further episodes. in both cases, the epidemics caused widespread social panic. instead of the ministry of health, the state councilsrepresenting the highest administrative authority of the chinese government -assumed direct leadership in combating the epidemics. case notifications for leptospirosis, anthrax and brucellosis have remained at low levels since . there was an increase in the number of notified case of brucellosis from . per , population in to . per , population in , but this was probably due to improved surveillance and is believed not to reflect an increase in incidence [ ] . the annual number of notified cases of leptospriosis and anthrax dropped below . per , population in . the mortality rate due to brucellosis remains very low and stable, whereas the rates due to leptospriosis and anthrax have dropped significantly, by % and %, respectively, during to (table ) . china has the second largest tb epidemic in the world (after india) [ ] . in , some , , patients were living with pulmonary tb in china, corresponding to an incidence of per , population, and resulting in , deaths from tb in the same year [ ] . since , an estimated , additional tb reactivation cases have arisen per annum in china due to human immunodeficiency (hiv) coinfection [ ] . the tb epidemic has spread unevenly in china: in , china reported a national tb prevalence of . % [ ] , whereas the southern province of guangxi, which borders vietnam, reported a much higher prevalence of . % [ ] . in , in adopting dots -the internationally recommended tb control strategy -china launched a -year infectious and endemic disease control project in an effort to curb its expanding tb epidemic in of its provinces [ , ] . it is estimated that the number of chinese people infected with tb between and decreased by % and that , tb deaths were averted. with a success rate greater than % [ ] , dots has led to the avoidance of an estimated . million tb cases [ , ] . however, since , multidrug-resistant (mdr) tb and extensively drug-resistant (xdr) tb have emerged as a severe public health issue in the country. in , china reported approximately , mdr tb cases, accounting for about one third of the estimated global burden of mdr tb [ ] . in some provinces, the proportion of mdr tb among newly notified tb cases and previously treated cases was found to exceed % and %, respectively [ ] . the latest study indicates that the prevalence of mdr tb among tb patients is as high as . %, and . % of mdr tb cases were xdr during to [ ] . the mortality due to tb increased at a rate of % ( % ci, - %) per year. hydroelectric projects that led to the formation of a large number of dams in china, as well as climate change, have substantially increased the risk of schistosomiasis occurring and its spread to non-endemic areas of the country, leading to small-scale outbreaks and increases in rates of case notifications near the affected areas [ , ] . the incidence of schistosomiasis has increased from . cases per , population in to . per , population in (figure f ). since the founding of china in , laws have been enacted to make commercial sex illegal. brothels were closed down and sex workers were sent to camps to be 're-educated'. special institutions for treatment of sexually transmitted infections (stis), at the time mostly syphilis, gonorrhoea and chlamydia, were established for the training of medical personnel, who were then sent to areas previously associated with large sex industries to treat stis at beginning of s. during s, a universal healthcare system was established that provides treatment for sti patients free of charge. designated laboratories for stis were built for diagnosis and research [ ] . more importantly, population-based health education campaigns about stis were carried out, and detection and treatment of stis were portrayed as patriotic actions [ ] . as a result, the number of cases of notifiable stis decreased from million in to reportedly eliminated from [ , , ] . however, notifiable stis have re-emerged, leading to a fastspreading epidemic in the country. in , the incidence for stis was . per , population and in , the rate increased to . per , population, which corresponded to . million new sti cases [ ] . the annual growth rate of the number of sti cases in china exceeds % [ ] . the rise of sti incidence is mainly due to unprotected sexual acts and other high-risk behaviours [ , ] . since early s, underground prostitution has again become rampant in major cities in china [ ] . the number of chinese men, increased purchasing power of urban residents and trends away from traditional chinese family values of fidelity all led to an increased number of male clients for commercial sexual services [ ] . in addition, lack of sexual health knowledge among commercial sex workers and their inability to effectively negotiate condom use during sexual acts substantially increased the risk of sti transmission [ , ] . the previously hidden population of men who have sex men (msm) is becoming more overt due to increasing acceptance in society. high-risk sexual behaviours, including a low percentage of condom use, in this population is also contributing to the fast transmission of stis [ ] . for example, in , the incidence of syphilis in china was . per , population: it increased -fold to . cases per , population in and further to . cases per , population in [ , ] (figure g ). in particular, msm, injecting drug users (idus) and female sex workers have the highest syphilis prevalence in china, of - % [ , ] , . % [ ] and - % [ ] , respectively. the first case of acquired immunodeficiency syndrome (aids) in china was reported in , but a local hiv epidemic was not detected until when a cluster of infections was diagnosed among idus in yunnan province [ ] . before , china had fewer than , reported hiv/aids cases, but in , the number rose to , . the latest estimates are that , people were living with hiv/aids in china at the end of , with , new infections added every year since then [ ] and , - , annual aids-related deaths [ ] . although the hiv/aids epidemic first began and predominated among idus, it rapidly spread to other population groups through both heterosexual and homosexual transmission. the latest figures demonstrate that heterosexual transmission has accounted for approximately - % of new hiv infections since [ ] , which may level at or exceed the percentage ( - %) caused by transmission due to the sharing of injection equipment among idus [ , ] . notably, hiv infection has become more prevalent among msm, as male homosexual activities account for approximately % of new infections [ ] . aids mortality rate increased from . in to . cases per million in , then to . cases per million in , corresponding to an annual growth rate of % ( % ci, - %) the rate is expected to increase sharply if the epidemic remains uncontrolled and there is no large procurement of antiretroviral drugs. cumulatively, over infants were infected through mother-to-child transmission by and it was estimated that about million chinese were carriers of mycobacterium tuberculosis in [ ] : coinfection of hiv and m. tuberculosis could therefore have a large epidemiological impact, in the absence of appropriate public health interventions. viral hepatitis is prevalent in china, but the prevalence differs according to the type of virus. before the s, hepatitis a virus (hav) was the most prevalent. in the early s, the annual number of reported hav diagnoses was more than per , population. this dropped by % to per , population in to [ ] (figure h ). the mean annual death rate due to hav decreased at a rate of % ( % ci, - %) during to ( table ). the decrease is probably a result of mass vaccination efforts in introducing the hav vaccine (h strain) in the s and improvement of hygiene and sanitary conditions that broke the cycle of food and water contamination [ ] . according to data [ ] , hav persists at low, endemic levels ( figure h ). in comparison, infections of hepatitis b virus (hbv), which is transmitted mainly through body fluid from mother to child, and hepatitis c virus (hcv), transmitted predominantly by intravenous injections or blood transfusion, are becoming alarmingly widespread in china. the annual number of new hbv cases increased rapidly from per , population in and reached per , population in , despite an increasing vaccination coverage of newborns from % in to . % in [ ] . hcv infections followed a similar trend and in , the annual notification rate was about . per , population. the hcv and hbv prevalence among the general population was . % and %, respectively [ ] . mortality due to hbv and hcv increased at annual rates % ( % ci, - %) and % ( - %) respectively. in contrast to hav infection, which always causes acute hepatitis but never develops into a chronic condition, hcv and hbv infections often cause chronic hepatitis and may develop into cirrhosis and hepatocellular carcinoma [ ] . coinfection of hbv or hcv with other pathogens causing chronic infections, such as hiv, results in substantial increases in the disease burden on individuals, accelerates disease progression and complicates treatment [ , ] . since hbv and hcv can both be transmitted through exchange of body fluids, coinfection of these hepatitis viruses with hiv is common in at-risk groups in china. approximately - % of the , registered idus in china are infected with hcv [ , , ] and - % of them are infected with hbv [ , ] . the prevalence of hiv/hcv coinfection is . % in idus [ ] . among blood donors, the prevalence of hiv/hcv coinfection is . - . % [ , ] , whereas the figure for hiv/hbv coinfection is . % [ ] . in comparison, coinfection of hiv and hcv among the general population remains unknown as there is currently no reports on its prevalence. however, coinfection is expected to be increasingly noticeable as the hiv epidemic transforms into a generalised epidemic in china [ ] . overall, this study investigated the temporal trends of major types of notifiable infectious diseases in china. our analysis indicated that while the morbidity and mortality of most infectious diseases reduced substantially during - , there is an increasing trend of re-emergence of previously prevalent diseases and emergence of new infectious diseases since , in particular, stis and hiv, viral hepatitis and zoonoses diseases. these diseases accounted for the majority of deaths associated with infectious diseases in china since . consistent with this, analysis of case fatality rates indicated a higher percentage of deaths among zoonotically infected people ( - %) and an elevated proportion ( . %) among stis and hiv-infected individuals. our study has important implications for the surveillance and control of infectious diseases in china. first, the persistent small outbreaks of particular infectious diseases across the country indicate that any decrease in prevention efforts will probably trigger re-emergence of the diseases. surveillance activities are therefore essential to inform and prioritise public health responses. although efficient and well-developed disease surveillance systems have been implemented in many urban areas, hygiene conditions, health services and monitoring of patterns of spread and disease burden are largely lacking in rural china [ ] . second, the rapid rise in the number of notified cases of stis, especially hiv infection, and viral hepatitis in china is associated with growth of the sex industry, increasingly frequent risky sexual behaviours and an increasing number of sexual partners in the general chinese population. the newly implemented internetbased national infectious diseases monitoring information system database not only provides a platform for integrating epidemiological data on hiv infection and other stis, but also initiated second-generation behavioural surveillance of the at-risk populations [ ] . our analysis indicates that both epidemiological and behavioural surveillance of hiv infection and other stis is essential to understand and forecast the trends in these epidemics. extending surveillance efforts into population groups that were previously considered to be less at risk may further improve the quality and reliability of surveillance data. third, coinfections, especially those involving hiv, are likely to become major public health issues in the future. we demonstrate that tb/hiv, hbv/hiv and hcv/hiv coinfections have become increasingly prevalent in china and the trend is likely to continue in the absence of strong public health interventions. thus, inclusion of testing for tb and viral hepatitis coinfections as part of hiv voluntary counselling and testing could be useful among at-risk populations. scaling up of free-of-charge antiretroviral therapy should also include treatment for these coinfections. fourth, surveillance of zoonotic infections becomes increasingly important due to the interactions between humans and animals in china. china has established an efficient surveillance system for human infectious diseases [ , ] : a parallel national centralised system for surveillance of disease in animals may be useful for reducing the risk of animal-to-human transmission. one of the potential limitations in our investigation may be poor data quality in the passive notification-based surveillance system. it is widely perceived that underreporting exists in the chinese infectious diseases surveillance system, but little is known about the extent of underreporting as systematic evaluations have never been conducted [ ] . this issue requires further consideration in the future. an overview of prevalence studies would be useful for comparison with numbers of notified cases in order to estimate numbers of undiagnosed cases. a comprehensive evaluation would also involve assessments of the efficiency and effectiveness of the surveillance system, both quantitative and qualitative. understanding changes in testing rates in various population groups will also be useful for interpreting trends in notification data. but such investigations are beyond the scope of this review. infectious disease surveillance and interventions in china face several major challenges. the country's major economic reform in has created a large economic disparity between rural and urban areas, resulting in a large population of floating immigrants (also known as 'peasant workers'). this accounts for % of the total chinese population in [ ] . the vast majority of these immigrants are from rural environments but work in urban areas, away from their families, and hence regularly travel between the two sites. in addition to the natural population growth of major cities, the inflow of internal immigrants substantially increases the population size and density in urban areas. these immigrants often live in overcrowded residential areas with compromised hygiene conditions. female immigrants often become targets of those seeking to recruit them for commercial sex work [ , ] . these floating immigrants are often more disadvantageous than the local residents. they tend to be less educated compared with others in urban areas who have permanent resident status and less likely to seek medical treatment when sick due to financial constraints or lack of health insurance [ ] . not surprisingly, the population of floating immigrants has been found to be more vulnerable to infectious diseases, especially stis, hiv and viral hepatitis [ ] . additionally, their high-risk behaviours and mobility promotes transmission of infectious disease agents and creates a major challenge for disease detection and control. given the characteristics, behaviours and the increasing population size of floating immigrants, it becomes apparent that the capacity of current chinese healthcare system cannot meet the needs of both the surveillance activities and health problems of these migrant workers. the infectious disease surveillance system needs to be tailored for its high mobility, and a dramatic expansion of the private health sector is required to address the health needs of this population. transmission of zoonotic infectious agents relies on close interaction between infected animals and humans. as a result of the increasing social trend of keeping house pet dogs, it is estimated that the number of pet dogs in china reached - million in [ ] . however, only % of dog owners register their animals with government authorities and only - % of dogs are vaccinated against rabies [ , ] -coverage of greater than % is needed to sufficiently control rabies, as determined by the world health organization (who) [ ] . the high rabies prevalence ( . %) among dogs and the expanding number of pet dogs pose a continual and increasing threat of transmission of rabies virus from dogs to humans in china [ ] . in addition, the consumption of game meat, especially in southern china, is one of the important sources of zoonotic diseases [ ] . markets in guangdong provinces sell live poultry, fish, reptiles and mammals, including dogs and civet cats, for food [ ] : the housing of these live animals, often in packed conditions, together with human activities, makes such markets the ideal hub for cross-species transmission of zoonotic agents [ ] . since there is no systematic surveillance of these live markets, it is difficult to estimate the number of such markets and the amount of game meat consumed by residents in southern china. the sars epidemic in demonstrates that transmission of pathogens originally associated with animals can cause worldwide outbreaks in human populations. the current absence of both epidemiological and behavioural data, including their frequency of contacts and means of animal handling, on animal infectious diseases is a strong barrier to effective surveillance and control. environmental changes facilitate the emergence and transmission of bacterial infections. the formation of a large number of dams in china, as a result of the increasing implementation of hydroelectric projects, have substantially increased the risk of schistosomiasis emergence and its spread to non-endemic areas of the country. further, the dense population conditions in urban china and the high mobility of its floating migrants substantially promote the rapid transmission of tuberculosis [ ] . strengthening infectious diseases 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of avian influenza severe acute respiratory syndrome analysis of factors affecting the epidemiology of tuberculosis in china key: cord- -yoy hj j authors: sansonetti, philippe j title: covid‐ , chronicle of an expected pandemic date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: yoy hj j what is covid‐ ? what are the causes, parameters, and effects of this disease? what are the short‐ and long‐term prospects? philippe sansonetti, infectious disease specialist and chief editor of embo molecular medicine, explains why the fate of the epidemic is in our hands.[image: see text] w hat is covid- ? what are the causes, parameters, and effects of this disease? what are the shortand long-term prospects? philippe sansonetti, infectious disease specialist and chief editor of embo molecular medicine, explains why the fate of the epidemic is in our hands. it is difficult to believe in plagues when they fall on your head (albert camus, the plague ( , ed. gallimard) ). the covid- pandemic is a plague as described by camus; it is urgent and vital for our society to realize this. it is not too late, but time is running out. it is the third coronavirus-associated epidemic globally in < years after sars in and mers in . during these two epidemics, we were first worried, then reassured after it was over, and we did not much afterwards in terms of preparedness, therapy, and vaccine. today, in the absence of treatment and vaccine, the evolution of the covid- epidemic is nonetheless still in our hands. charles nicolle ( nicolle ( - , professor at the collège de france and director of the institut pasteur in tunis, wrote in destin des maladies infectieuses ( ): so, there will be new diseases. it's a fatal fact. another fact, also fatal, is that we will never be able to detect them from their origin. knowledge of infectious diseases teaches men that they are brothers and united. we are brothers because the same danger threatens us, united because contagion comes to us most often from our fellow men. we also, from this point of view and whatever our feelings are towards them, stand together with animals, especially domestic animals. he was already predicting the emerging pathogens of the late th century and st century. coronaviruses are a huge family of singlestranded positive rna viruses. alpha-coronaviruses are endemic and infect mammals including humans and cause, for example, mild respiratory and intestinal diseases in children. beta-coronaviruses like sars-cov- (the official name of covid- virus) on the other hand are well adapted to their reservoir, the bat, but not to humans, which explains why human infections are so damaging. other members of this family, the gamma-and delta-coronaviruses infect birds and fish and, at least for the time being, have not been associated with human disease. one thing that this epidemic has taught us is that we, scientists now, possess the technology and capabilities to quickly react: as soon as the virus emerged in the city of wuhan, china, deep sequencing and bioinformatics allowed for the diagnostic of this novel virus in a few days. the viral genomic sequence was then quickly shared and used to develop a detection test based on realtime pcr for rapid diagnosis and for initiating epidemiological studies all over the planet. now compare this with the years it took to identify hiv thirty years ago through conventional growing of the virus in in vitro culture. molecular diagnosis has revolutionized this field, and despite the initial delays in communicating about this epidemic, chinese doctors and biologists quickly reported the first evidence for sars-cov- , and provided the first sequences, clearing the way for the global scientific community to further develop diagnostic tools and engage in a race to discover dedicated drugs and vaccines. the name coronavirus comes from the protein s (s for spike) and its crown-like shape. the s protein plays a central role for host specificity and pathogenicity as it binds to the ace receptor on the target cell surface and mediates entry of the viral rna. the phylogenetic tree shows that sars-cov- is very close to sars and mers , and the diseases they cause are very similar. the same is true for their origin: bats are the main reservoir of coronaviruses. the phylogeny is therefore relatively well known, and one can apply the lessons learned during previous outbreaks, even if no vaccine or therapy is available so far. sars-cov- is a textbook case of an emergent pathogen after a species jump ("zoonosis"). we have observed these zoonoses for decades, particularly in tropical regions (ebola is one such example; marí saéz et al, ) . generally, an emergent pathogen can give rise to two scenarios. the virus is ill-equipped and has little capacity to mutate and therefore to adapt and stabilize in the new species. human infection is abortive as the virus is not able to establish human-tohuman transmission. still, under this scenario, the disease can be very severe as was the avian influenza h n caused by direct bird-to-human transmission, reaching~ % mortality, but for which limited, if any, human-tohuman transmission was reported (wang et al, ) . the virus is better adapted to its new host, thus better able to cross the species barrier, and its adaptation is facilitated by further mutations due to poor fidelity of the enzyme that replicates the positive strand of viral rna. this is what happened with sars-cov- , which jumped relatively easily from bats to humans via an intermediate mammal reservoir. the disease caused by sars and mers was more serious, and the mortality rates were higher ( % and %, respectively). sars-cov- however is often associated with milder pathologies. this is a typical trade-off: less virulent, but more transmissible versus more virulent but less transmissible. this balance is extremely important and defines the disease profile. the natural reservoir is a certain bat species; it is impressive to see how far these animals are capable of carrying these emerging viruses, such as corona. this was the case with ebola in africa, and with the nipah virus in malaysia, which appeared in the late s. as human behavior changes ecological conditions, these bats increasingly come into contact with animals which are susceptible for a zoonotic species jump and which further replicate the virus. this generates a risk zone around humans, since any contact with these intermediate animals can cause another species jump into humans. in the case of sars , it is assumed that the intermediate animal was the webbed civet, a feline particularly frequent in asia; for mers, it was the camel. for ebola, it probably was great apes (malvy et al, ) while direct bat-to-human transmission was also considered (marí saéz et al, ) . for sars-cov- , some claim that the intermediate reservoir is the pangolin. indeed, numerous studies have shown that the virus circulating in pangolins is very close to the one observed in humans, suggesting that the current pandemic results from human behavior and animal trafficking. we are constantly threatened by these emerging diseases so-called anthropocene diseases, which are essentially or even exclusively linked to the omnipresent imprint that humans leave on the planet. what is true for the climate and the environment is also true for infectious diseases, in particular emerging ones, and the three are linked. this is a story in three acts: (i) stochastic species jumps, (ii) possible transmission to and between human, and (iii) pandemic explosion. it is interesting to note that the distribution map of covid- clusters and hotspots overlaps with the map of intercontinental air flights (which carried billion passengers in ). we can clearly see the role transport plays in the transmission and global spread of these diseases, turning them into pandemics more readily than even before in human history. however, as we speak, more and more cases are being reported in the countries of the intertropical zones, reaching southern america and africa where containment will be difficult to achieve. there are also important environmental aspects linked to the anthropocene era, such as intensive agriculture and husbandry that profoundly modify ecosystems and cause unanticipated encounters between animal and human habitats, rising temperatures in equatorial regions that drive animal population migration to cooler regions and political instability that causes mass displacement of refugees. also, different national healthcare systems and a lack of global information exchange and coordination can lead to a delay in diagnosis in some countries, but the comparison of thefts and foci of infection is striking. in how far these effects had an influence on the covid- pandemic requires further study once the pandemic has passed its apex. parameters of the epidemic r (basic reproduction rate) is the average number of secondary infections produced when an infected individual is introduced into a population where all individuals are susceptible. if the r is less than , there is no epidemic situation; as soon as it is greater than , there is an epidemic. in the case of covid- , this number is between and . this is therefore a typical epidemic situation. for the spanish flu of - , the r was . ; tuberculosis is and therefore extremely contagious; and for measles, r ranges between and . the incubation period is - days, and all evidences converge to indicate that the patients are contagious from the onset, when they are yet asymptomatic, unlike sars in where the contagion only appeared with the peak of viremia after several days after infection. the sars-cov- virus is highly contagious: people transmit while they are still asymptomatic, or just begin to experience mild symptoms. the attack rate, the number of newly infected patients compared to the general naive population, is high, much higher than the seasonal flu. severe cases are around - % and require hospitalizations that last on average between and days, which is what threatens our health systems. covid- is therefore a disease with high epidemic potential that puts a major strain on the healthcare system. this is why governments decided to put in place different strategies to attenuate the progression of the disease. the mortality rate is relatively low. when we will be able to get a full picture of this pandemic, we will most likely notice that it was between and %. it seems higher during periods of exponential spread as is currently the case in the usa, france, spain, or italy. not necessarily because the disease is more severe during this period, but likely because death count is indisputable, while it is difficult to assess the number of infections, which is always higher than observed. undoubtedly, the majority of infected people develop a mild form of this disease, which makes accurate calculation of the mortality rate difficult at present. at the time of publication, mass serological testing is being rolled out in countries such as germany, particularly in hotspot areas, which will give a much more accurate estimate of mortality rates. but the mortality rate increases due to the stress on the healthcare system and the availability of hospital beds. % mortality, % of severe cases, those are not large numbers statistically speaking, but compared to the number of infections, and taking into account the transmissibility and infectivity of the virus, we are reaching a situation that endangers our healthcare system. this is what legitimizes the flatten-the-curve policy. yet, one good news: children younger than years-old are mostly not ill even though they can get infected and transmit the virus. the receptor for sars-cov- and sars-cov- , angiotensin ii converting enzyme (ace ), is an enzyme attached to the surface of cells, including lungs, pneumocytes and endothelium, endocardium, kidney, liver, and intestine. it came as a surprise that the virus binds to an important enzyme for the regulation of blood pressure. this could explain the severity of the disease in the respiratory tract with instances of pneumonia and ultimately, acute respiratory distress syndrome (ards) observed in elderly subjects or individuals with chronic comorbidities (i.e., diabetes, hypertension, cardio-respiratory insufficiency, chronic immunosuppression). the virus targets the blood-lung barrier, affecting the oxygen exchange zone. acute respiratory distress syndrome has classic inflammatory signs, a so-called "cytokine storm" characterized by a significant increase in cytokines and proinflammatory chemokines. what is less typical for corona infections is the destruction of the alveolocapillary barrier, which seldom happens also in covid- , but when it does, requires fast intervention. ards can also occur in younger individuals during the healing phase, which, in this case though, may be linked to the immune response. the adaptive immune response specific to the virus is still poorly understood. we know that this virus affects ifn-driven immunity, but we do not know why, or which effectors blunt this immune response. it is possible that the virus has acquired this strategy in bats that express constitutively low level of ifn. further work is urgently needed to learn to control the disease efficiently and sustainably. the future of the covid- pandemic is in our hands. so far, we can only rely on prevention and symptomatic treatment of severe forms. regarding prevention, health authorities have three main options. the first-which may seem cynical-considers that the more people become infected, the quicker the population will become immune until the epidemic peters out as the virus finds no more immunologically naive individuals. this is the principle of group/herd immunity. if % of the population were infected by sars-cov- , the epidemic will stop. but this would come at the cost of a very brutal epidemic peak with a high number of severe cases and deaths. we saw it during the epidemic of the asian flu in the united kingdom in . for a week to ten days, the health system imploded, because the health personnel were sick, the equipment insufficient, and the number of seriously ill patients exploded. totally opposite is the "chinese approach": to isolate cities and individuals, which undeniably appears effective in controlling the epidemic. the risk is that because only few individuals have been infected, the attack rate was reduced due to strict confinement, hence many individuals are still immunologically naive and vulnerable to a second viral wave and risk an epidemic rebound. the intermediate option, which has been taken in most of european countries, is to flatten the curve over time with different levels of isolation and social distancing, hoping that even if less than % of the population will be infected, above all, the healthcare system will hopefully be preserved. the approach is based on a concept of social distancing (two meters/ feet apart) and individual hygiene. it seems that in addition to airborne contamination, contaminated hands are also a major vector, either by direct contact with an infected patient, or indirectly by contact with a contaminated surface on which the virus seems to be able to survive for many hours. so, no kiss, no hug, no handshake and absolute hand hygiene. avoid touching face unless hands have been thoroughly washed with soap or decontaminated with a hydro-alcoholic gel. the importance of wearing masks for the general population is still debated in some countries that quickly ran short of these masks. in agreement with attitudes in asian countries, wearing masks is now strongly advised to complement hygiene measures. common sense starts to prevail. unfortunately, the measures that have been implemented so far have been clearly insufficient, as we have seen in italy, spain, france, uk and as we now see in the usa. confinement and isolation at home have therefore been decided for an extended period at the cost of a huge economic risk, given the intrinsic dynamics of the epidemic. failure of these measures in these countries will have to be compared with their success in asian countries like south-korea, taiwan, singapore, and to a certain extent in germany. large implementation of diagnostic combined with isolation of sars-cov- positive individuals may be part of the answer. no matter the strategy taken by health authorities, we need to convince ourselves that our fate is in our hands. we stepped into another world in only a few weeks. what we valued yesterday-our daily activities, our hobbies, our workmust be weighted in relation to the gravity of the situation. finally, it is essential to find antiviral treatments for severely affected patients and to block transmissibility from individual to individual. a few options are as follows: (i) "repositioning" of drugs already tested for other viruses, such as hiv, or drugs that are already commercially available; (ii) better understanding of the pathophysiology of ards in order to develop a dedicated pharmacology using repositioned and novel molecules; and (iii) developing an effective vaccine. the risk of rebounds justifies the absolute necessity of a vaccine that is the only way to achieve durable elimination of covid- and even eradication of sars-cov- , including in low-income countries with fragile health systems. design, development, validation, clinical studies, and registrations with regulatory agencies takes between and years on average for a standard vaccine. this is incompatible with the urgency of an emerging epidemic like covid- . fortunately, novel paradigms of vaccine discovery and development are on their way to considerably shorten the delays. nonetheless, we know that we would not have a vaccine available until at least next year. in the meantime, in spite of enormous difficulties, we need a global mobilization for more basic and applied research from academia and the pharmaceutical and vaccine industry. as much as we have been able to massively reduce the time to identify novel pathogens, we need to reduce the time to develop the tools of their control. again, our future is in our hands. adapted from an article published in french in laviedesidees.fr, march . ebola virus disease investigating the zoonotic origin of the west african ebola epidemic probable limited person-to-person transmission of highly pathogenic avian influenza a (h n ) virus in china key: cord- -rqlkwoya authors: rajewsky, nikolaus; almouzni, geneviève; gorski, stanislaw a.; aerts, stein; amit, ido; bertero, michela g.; bock, christoph; bredenoord, annelien l.; cavalli, giacomo; chiocca, susanna; clevers, hans; de strooper, bart; eggert, angelika; ellenberg, jan; fernández, xosé m.; figlerowicz, marek; gasser, susan m.; hubner, norbert; kjems, jørgen; knoblich, jürgen a.; krabbe, grietje; lichter, peter; linnarsson, sten; marine, jean-christophe; marioni, john c.; marti-renom, marc a.; netea, mihai g.; nickel, dörthe; nollmann, marcelo; novak, halina r.; parkinson, helen; piccolo, stefano; pinheiro, inês; pombo, ana; popp, christian; reik, wolf; roman-roman, sergio; rosenstiel, philip; schultze, joachim l.; stegle, oliver; tanay, amos; testa, giuseppe; thanos, dimitris; theis, fabian j.; torres-padilla, maria-elena; valencia, alfonso; vallot, céline; van oudenaarden, alexander; vidal, marie; voet, thierry title: lifetime and improving european healthcare through cell-based interceptive medicine date: - - journal: nature doi: . /s - - - sha: doc_id: cord_uid: rqlkwoya here we describe the lifetime initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. this mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. the analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. the timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. the application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in europe over the next decade. here we describe the lifetime initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. this mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. the analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. the timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. the application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in europe over the next decade. although advances in medicine have led to remarkable progress in certain disease areas, most chronic disorders still cannot be completely cured. this is mainly because most such diseases are detected only late in their progression, once gross physiological symptoms manifest themselves, at which point tissues and organs have often undergone extensive or irreversible damage. at this stage, the choice of interventions is typically quite limited. it is difficult to predict whether a patient will respond to a particular treatment (often invasive or aggressive therapies that can be of modest benefit), or whether therapy resistance will emerge and lead to a relapse. despite technology-driven revolutions that enable a patient's physiology to be investigated at the level of molecules , and placed in the context of tissues , , in most cases our ability to detect and predict diseases at an early stage is limited by our incomplete mechanistic understanding of disease at the cellular level. cells develop and differentiate along specific lineage trajectories to form functionally distinct cell types and states , which, together with their neighbouring cells, underlie and control normal physiology ( fig. ) . however, we have not been able to systematically detect and understand the molecular changes that propel an individual cell along these trajectories during normal development or ageing, or the molecular causes that trigger deviations from healthy trajectories and drive cells and tissues towards disease (fig. ) . timely detection and successful treatment of disease will depend crucially on our ability to understand and identify when, why, and how cells deviate from their normal trajectories. more accurate cellular and molecular diagnostics will enable us to intercept disease sufficiently early to prevent irreparable damage. to achieve this interceptive medicine (fig. ) , we need to invest in approaches that provide a detailed molecular understanding of the basis of disease-related heterogeneity in tissues, with sufficient molecular, cellular and temporal resolution. several challenges need to be overcome in order to understand complex disease landscapes, which comprise of vast numbers of potential cellular states (fig. ) . first, we need to resolve normal cellular heterogeneity across space and time to begin to define the cell types, states and cell-cell interactions that normally exist in the body. this is a main goal of the human cell atlas consortium . however, to discover the cellular bases of diseases requires that we track cellular heterogeneity and the molecular composition of cell trajectories in health and during disease progression longitudinally-throughout an individual's lifetime. second, we need to understand the molecular mechanisms and complex networks that define a cell's state, and control its function, fate and trajectory over time, to be able to reconstruct a cell's history and predict its future. this is essential for selecting the optimal intervention for an individual patient. thus, systematic and longitudinal profiling of samples from many individuals is required. third, we have yet to develop the computational frameworks required for integrating temporal data and patient profiles, with large cohorts to identify regulatory changes and to dissect the causes and manifestations of disease. current attempts to model human disease have not succeeded in integrating the thousands of molecular phenotypes that are acquired from patients. finally, we are limited by our lack of knowledge of the underlying causes of disease. to predict any given patient's response to a specific therapy may require testing or modifying cells from the patient in an experimental system, a challenge that has yet to be routinely implemented. to address these challenges experts from different disciplines came together in to form the lifetime initiative (https:// lifetime-initiative.eu). it has since grown to be a pan-european community consisting of more than research institutions with support from companies and several funding agencies and national science academies. in the initiative was awarded a coordination and support action by the european commission to develop a strategic research agenda (sra) for a large-scale, long-term initiative with a roadmap for implementing cell-based interceptive medicine in europe in the next decade. the ambitious goal is the early detection and interception of complex diseases, as well as the ability to select the most effective therapeutic strategy for a patient. between march and june the initiative established several multi-disciplinary working groups (listed in the supplementary information), organized numerous workshops, meetings and surveys (and thereby engaged the wider community) and commissioned stakeholder interviews and an impact study. the european commission will use lifetime's sra during the planning of the next research and innovation framework programme: horizon europe. here, we outline lifetime's vision and key aspects of the sra towards establishing cell-based interceptive medicine. central to lifetime's vision and approach is the development and integration of new technologies, such as single-cell multi-omics, high-content imaging, artificial intelligence (ai) and patient-derived experimental disease models. the application of these integrated approaches to medical challenges and their incorporation into both experimental and clinical workflows are expected to directly benefit patients. for example, appropriate single-cell based biomarkers will give physicians early warning that a cell or tissue is entering a disease trajectory. understanding disease heterogeneity at the cellular level and knowing the molecular aetiology of a disease will allow researchers to systematically identify drug targets and resistance mechanisms and to define therapeutic approaches, based on a given disease's molecular or cellular vulnerability. this strategy differs markedly from classical approaches to drug discovery . the stratification of patients on the basis of underlying disease mechanisms, assessed in situ within single cells, will help physicians to select the most appropriate treatment(s) or to use combination therapies that are tailored to the individual. these will be used first to identify cells that are deviating from the healthy trajectory, to steer them away from disease, and later to reduce the threat of relapse (fig. ). this transformative single-cell data-driven approach has the potential to increase the success rates of clinical trials and the efficacy of novel therapeutic interventions in clinics over the next decade. overall, the lifetime strategy is likely to affect both diagnosis and treatment, to greatly improve health and quality of life, and to reduce the societal burden of diseases such as cancer, neurological and neuropsychiatric disorders, infectious diseases, and chronic inflammatory and cardiovascular diseases. below, we outline the development and implementation of technology at the heart of lifetime's approach, describe lifetime's mechanism for identifying medical priorities, discuss the required infrastructures in europe, interactions with industry and innovation, ethical and legal issues, describe lifetime's education and training vision, and estimate the expected impact of the lifetime approach on medicine and healthcare. lifetime builds on and will collaborate with related international initiatives that are paving the way by producing reference maps of healthy tissues in the body, such as the human cell atlas (hca) and the nih human biomolecular atlas program (hubmap) . single-cell technologies-particularly transcriptomics-are generating the first reference cell atlases of healthy tissues and organs, and are revealing a previously hidden diversity of cell subtypes and functionally distinct cell states . single-cell analyses of patient samples are beginning to provide snap shots of changes in cell composition and pathways that are associated with diseases such as cancer [ ] [ ] [ ] [ ] [ ] [ ] , chronic inflammatory diseases , , alzheimer's disease [ ] [ ] [ ] , heart failure , and sepsis . because pathophysiological processes within individual cells involve different molecular levels, understanding the underlying mechanisms requires the integration of current single-cell approaches. lifetime proposes the integration of several approaches . this includes combining transcriptomics ( fig. ) with methodologies that provide additional information on chromatin accessibility, dna methylation, histone modifications, d genome organization, and genomic mutations [ ] [ ] [ ] . future developments will enable the incorporation of single-cell proteomes, lipidomes, and metabolomes, which will add key insights into different cellular states and their roles in health and disease. in addition to specific cell subtypes and the role of cellular heterogeneity, it is crucial to investigate the surrounding tissue context and organ environment. new spatial '-omic' approaches, particularly spatial transcriptomics, include information on the locations of diseased cells, their molecular makeup and aberrant cell-cell communication within the tissue [ ] [ ] [ ] [ ] [ ] [ ] [ ] . advanced imaging approaches also now enable the systematic spatial mapping of molecular components, in situ, within cells and of cells within tissues , [ ] [ ] [ ] [ ] [ ] . the cellular context, with respect to different immune and stromal cell types, extracellular components and signalling molecules that contribute to disease progression, will help to identify the roles of specific cell types and interactions in diseases , [ ] [ ] [ ] . the implementation of cell lineage tracing approaches , which link cellular genealogies with phenotypic information about the same cells, may help us to understand how populations of cells develop dynamically to form the specific architecture of a healthy or a diseased tissue. lifetime proposes to develop the necessary single-cell methodologies and end-to-end pipelines (fig. ), which will be integrated into robust, standardized multi-omics and imaging approaches, and scaled to profile hundreds of thousands of patients' cells . this will require an in-depth analysis of longitudinal human samples obtained from patients and cohorts, including european and national clinical trial groups as well as initiatives collecting longitudinal biological material connected to well-annotated clinical information (fig. ). linking these data to clinical outcomes will identify the cellular parameters that are permissive to a therapeutic response, for example, during checkpoint blockade immunotherapy , , or treatment of multiple myeloma . by detecting rare drug-resistant cells that are present before , or that emerge during treatment , therapeutic regimens and combinatorial treatments can be adapted to improve outcomes. handling these large molecular datasets will require sophisticated and distributed computational and bioinformatics infrastructures (see 'implementation and infrastructure'), as well as the development of tools to integrate and ensure the interoperability of different data types, including single-cell multi-omics, medical information and electronic health records. lifetime will work with ongoing european and national efforts to integrate molecular data into electronic health records and to establish standards and interoperable formats to address specific disease challenges. this process will promote the development of advanced personalized models of disease. to be able to implement routine longitudinal sampling of patients, we need to develop approaches for sampling small biopsies, including liquid biopsies, that will detect individual cells or cell-free dna released from pathological cells before and during therapy . multi-dimensional descriptors of cell states from patients taken from different stages of disease or therapy will be used to derive new biomarker sets or to enhance current panels. collaboration with ongoing atlas projects, industrial partners and regulatory authorities will be key for benchmarking and deriving the new standards that will enable us to deploy these new methods in the clinic. we hope that this will achieve earlier disease detection and guide the appropriate selection of drug targets and therapies (fig. ) . unlocking the potential of unprecedented amounts of integrated digital information (including molecular data describing how individual cells make decisions) requires ai, in particular machine learning approaches that can identify meaningful molecular patterns and dependencies in the datasets , . although such approaches have proven very useful when applied to medical imaging data and have enabled the identification of subtle disease-associated changes , medical imaging cannot capture the full complexity of human physiology nor the status of a disease at the single-cell level. high-content imaging, together with information about gene expression, chromatin states, and protein and metabolic parameters, will contribute to the stratification of disease phenotypes. machine learning and advanced modelling approaches will be used to integrate and analyse the different layers of cellular activity, and can generate multi-scale and potentially even causal models that will allow us to infer regulatory networks and to predict present and future disease phenotypes at the cellular level , - (fig. ) . the deep integration of machine learning technologies with spatial multi-omics and imaging technologies and data has the potential to usher in a new age of digital pathology to aid in decision-making by physicians (fig. ) . by considering not only anatomical, physiological and morphological aspects, but also multidimensional molecular and cellular data, it will be possible to provide a more granular representation of a patient's disease state to complement the pathologist's slides and bulk measurements in tissues (for example, of mrna or metabolites). we envision as the final goal the incorporation of new ai-based decision-aiding systems that will integrate and interpret available molecular, cellular, individual disease trajectory and imaging information. interpretable and accountable ai systems will also provide the basis for clinical recommendations. the integration of cellular information should lead to a more precise description of a patient's molecular and physiological history, and will guide early detection, allow predictive prognosis, and guide recommendations for therapeutic interventions to deliver more precise and effective treatments (fig. ) . targeting cellular trajectories through time. a, cells are programmed to develop and differentiate along many different specific lineage trajectories (blue trajectories) to reach their functional state. when these normal lineage processes go awry, it can cause a cell to deviate from a healthy state and move towards a complex disease space (coloured manifolds defined by multi-dimensional molecular space-including gene expression, protein modifications and metabolism), as shown by red trajectories. b, many diseases are detected only at a relatively late stage with the onset of symptoms (red trajectory) and when pathophysiological changes can be at an advanced stage (red cells). at this point, cells, tissues and organs have undergone extensive and often irreversible molecular and physiological changes since the initial events that caused them to deviate from a healthy state. hence, the choice of interventions may be limited and often involves harsh or invasive procedures. c, understanding the early molecular mechanisms that cause cells to deviate from a healthy to a disease trajectory will provide biomarkers for the early detection of disease, and new drug targets and innovative therapies to intercept diseases before the onset of pathophysiology and the manifestation of symptoms. understanding the cellular origin and aetiology of disease from a patient-centred perspective requires systems that faithfully recapitulate key aspects of a patient's pathophysiology, and render them experimentally tractable to test mechanistic hypotheses and predictions. organoids are an emerging experimental system that allow aspects of organ development, regeneration and pathophysiology to be modelled , , (fig. ) . derived from adult or pluripotent human stem cells, organoids can capture individual features that are unique to each patient and can be interrogated molecularly in space and time. importantly, by comparing organoid models from diseased and healthy individuals, unique disease features can be extracted even if the specific genetic cause of a disease is unknown. therefore, organoid models offer a valuable tool for achieving some of the main goals of lifetime, especially in cases in which repeated access to patient tissues is limited or impossible (for example, in neurological and neuropsychiatric disorders). despite their promise, organoids still require substantial development to harness their full potential for disease modelling (fig. ) . lifetime proposes to advance the models to capture the full degree of cellular heterogeneity and tissue-specific structural and metabolic conditions , and to incorporate key physiological aspects, such as immune responses, vascularization or innervation. because complex interactions between multiple tissues and organs are involved in many diseases, it will be necessary to develop tissue engineering principles that combine multiple organoids in pathophysiologically relevant crosstalk ('organoids-on-a-chip'). to optimize translational potential, lifetime will engage in standardizing, automating and scaling organoid approaches, to allow systematic derivation, propagation and banking of organoids. such industrialization is also needed for large-scale chemical or genetic perturbations (for example, crispr-cas screens), and for elucidating the genetic bases of disease variability and drug response at population-relevant scales, in both the preclinical and clinical contexts (fig. ) . the resulting mechanistic dissection, enabled by large-scale perturbations, will be used to validate corresponding ai models of disease interception and progression. in addition to organoids, in vivo model systems are necessary to translate the science from the bench to humans. a complex biological system is required to study the myriad of host-disease and host-pathogen interactions associated with complex diseases, such as infectious diseases, cancer or alzheimer's disease. the use of animal models is single-cell multi-omics and imaging technologies will be developed for high-throughput applications. different modalities will be combined to provide insight into underlying mechanisms, based on coordinated changes between different regulatory molecular layers. insight into cellular genealogies and cellular dynamics will require the integration of lineage tracing tools. technologies will also need to be scaled for clinical deployment. the integration and analysis of large, longitudinal multi-omics and imaging datasets will require the development of new pipelines and machine learning tools. these include the development of causal inference and interpretative machine learning approaches to create molecular networks for predictive and multiscale disease models. patient-derived disease models such as organoids will be further developed to improve tissue architecture and the incorporation of physiological processes such as vasculature, nerve innervation and the immune system, to provide models that more faithfully recapitulate disease processes. improved knowledge of disease mechanisms will require the application of large-scale perturbation tools to organoids. tissue-tissue and organ-organ interactions will be recreated using microfluidics and organ-on-a-chip technologies to study key systemic interactions in diseases. single-cell multi-omics analysis of patient-derived samples (such as blood or tissue) or personalized disease models (for example, organoids and experimental disease models) will be profiled longitudinally to cover the different disease stages. large-scale multidimensional datasets will provide quantitative, digitalized information that will provide information about the decision-making processes of cells. these will be analysed using ai and machine learning to arrive at predictive models for disease trajectories, providing cellular and molecular mechanisms of disease onset and progression. models will be validated using large-scale perturbation analysis and targeted functional studies in disease models, which will be used in an iterative process to improve both computational and disease models. important for understanding the complex temporal relationships that occur in diseases, such as those involving the vasculature, immune system and pathogens as well as neuronal networks in the brain. life-time will therefore improve the clinical relevance of animal models and make use of approaches in which patient-derived tissues can be integrated into in vivo models [ ] [ ] [ ] [ ] [ ] to study the dynamics of cellular heterogeneity in space and time. lifetime, as a community, has the capacity to develop and integrate these technologies, which often require expertise and specialized instrumentation that are located in distinct laboratories. a coordinated effort can achieve the required benchmarking and standardization of technologies, workflows and pipelines. this will also ensure that the data, software and models generated adhere to fair (findable, accessible, interoperable, and reusable) principles (see 'implementation and infrastructure'), are available across national borders, and are in full compliance with international legislations such as the european general data protection regulation. moreover, lifetime will ensure that technologies, including ai and organoids, will be developed in an ethically responsible way in collaboration with patients, putting the patient at the centre (see 'ethical and legal issues'). lifetime has initiated a mechanism, called launchpad, to systematically identify medical challenges that can be addressed through lifetime's approach and have a direct effect on patient care. initially, the focus has been on five disease areas that are a substantial burden to society: cancer, neurological and neuropsychiatric disorders, infectious diseases, chronic inflammatory diseases and cardiovascular diseases. other disease areas will be continuously monitored (for example, rare mendelian diseases and metabolic diseases), and research programmes initiated as technologies and infrastructures develop. the lifetime launchpad has defined several criteria to identify the medical challenges. these include: societal impact (including incidence and prevalence, disease severity, economic impact and the pressing need for new and more efficient clinical treatments and early detection), evidence for cellular heterogeneity that limits current clinical avenues, availability of samples from biobanks, relevant preclinical models, existence of patient cohorts including those enabling longitudinal studies, clinical feasibility and ethical considerations, as well as alignment with national and eu funding priorities. subsequently, multidisciplinary working groups, including clinicians, in each disease area have used these criteria to define the following disease challenges and to develop ten-year roadmaps to address them in the lifetime sra . despite cancer broadly covering hundreds of individual tumour types, there are critical knowledge gaps that are common to all cancer entities, including the mechanisms of early dissemination and therapy resistance. metastatic dissemination of a subpopulation of cancer cells is a leading cause of death in almost all cancer types. successful treatment of advanced and metastasized forms of cancer remains difficult, despite the development of targeted therapies and immunotherapies, owing to the emergence of drug or therapy resistance. to address these medical priorities, lifetime recommends focusing on understanding the cell types and states-malignant cells and their microenvironment-that are involved in early stages of cancer dissemination, and the reprogramming of cellular states during disease and their effect on resistance to therapies. for neurological disorders, a major challenge is a lack of understanding of the early events in disease onset to enable the development of disease-modifying therapies. the lack of access to longitudinal samples from patients necessitates the establishment of cohorts of patient-derived disease models to understand the cellular heterogeneity associated with disease. the discovery of pathways and biomarkers that will allow the stratification of patients on the basis of the cellular mechanisms that drive a disease will make it possible to design new clinical trials to reevaluate drugs that were previously tested without such stratification, and to broaden the drug target portfolio. as seen during the coronavirus disease (covid- ) pandemic, it is important to be able to understand infection mechanisms and the host response in order to rapidly identify the most likely effective treatment for an infection. at the same time, the continuous rise of antimicrobial resistance requires the discovery of new therapeutic strategies. a key medical challenge for infectious diseases is to understand the cellular response to infections and to develop precision, immune-based therapeutic strategies to combat infections. chronic inflammatory diseases impose a high burden owing to their long-term debilitating consequences, which result from the structural destruction of affected organs or tissues. current therapies treat the symptoms but do not cure or fully control the chronic inflammatory pathophysiology. while different targeted therapies exist, they are expensive and their success is limited by high rates of non-response to treatment. consequently, there is an urgent need to explore and understand how cellular heterogeneity contributes to the pathology of inflammatory diseases and how this relates to the predicted course of disease and the response of a patient to one of the numerous available therapies. many cardiovascular and metabolic diseases lack effective therapies owing to a lack of knowledge of their underlying causes and the link between abnormal cardiac cell structure or function and pathophysiology. the identified medical priority is to understand the cellular and molecular mechanisms involved, in order to enable early diagnosis and the design of new mechanism-based therapies for precise clinical treatment. the lifetime disease roadmaps can be divided broadly into three phases : first, immediate research into the identified medical challenges using established, scaled single-cell technologies, computational tools and disease models; second, the development of new technologies that are required to address specific medical challenges, including the development of spatial multi-omics and imaging approaches and advanced patient-derived model systems for longitudinal analyses; and finally, the application of these next-generation technologies to the longitudinal analyses of patient samples, or patient-derived models, combined with machine learning to generate patient trajectories and predictive models of disease. the resulting predictions and biomarkers will be validated in prospectively collected patient cohorts within clinical trials that will also include longitudinal liquid biopsies. the routine clinical use of predictors and biomarkers for risk stratification of patients and resulting interventions-where feasible-is the pre-final step. the final step is the extension of predictors and biomarkers to the analysis of large longitudinal patient cohorts, such as national cohorts, for developing secondary and tertiary prevention approaches based on the new biomarkers. during the implementation of these roadmaps, the initiative will establish an experimental design working group to develop systematic procedures to ensure that research samples are acquired from diverse cohorts (including age, sex, and ethnicity). this will require the development of strict criteria for the inclusion of samples and to ensure appropriate coverage of critical metadata. they will also define standardized procedures for the acquisition and processing of samples from different pathology sites (depending on the disease area). it is envisaged that during disease challenge pilot projects, an experimental design oversight body will determine, using early data, the number of diseases that should be studied as the initiative develops, with recommendations on the sample sizes required to obtain sufficient statistical power. the scale of the data that will be generated and analysed, the cross-disciplinary and international structure, and the ambition of lifetime to pioneer novel analytics using ai, place lifetime in an excellent position to shape the next generation of computational infrastructure for medical and biological data in europe. this will require close interaction with and evolution of the established european infrastructure (fig. ) , such as the european open science cloud (eosc) and high-performance computing infrastructures through the european high-performance computing (eurohpc) initiative. lifetime will also interact with related european life sciences research infrastructures to create added value and to avoid duplication of effort in strategies and tools for sharing and accessing data and the development and application of standards. as medicine is inherently decentralized, lifetime will also help to connect eu medical systems and develop large federated european data infrastructures. fragmentation of research across borders, disciplines and timeframes needs to be overcome. the generation of data and development of technology by lifetime will be harmonized across expert groups and centres, allowing the results to be quickly applied in clinics. thus, a coordinated approach is required that integrates the multidisciplinary expertise of single-cell technologies, data science, and organoids as well as in vivo models across europe. it must also engage clinicians and patients to achieve medical impact. to address these challenges, lifetime proposes a multidisciplinary network of lifetime centres (fig. ) with different complementary thematic clusters across europe, each working in close association with hospitals. these connected, flexible innovation nodes will share resources, gather the necessary critical mass for global competitiveness, and be open for collaboration with the entire scientific community. lifetime centres should deliver a number of key functions: • serve as platforms for the development and advancement of breakthrough technologies for single-cell research in -omics and imaging, ai (in particular machine learning), and experimental and computational disease models. • closely and actively collaborate with patients, clinicians, hospitals and healthcare systems, in some cases with a specific disease focus. • set standards in data generation, standardization and management, implementing fair principles. • set standards in ethical, legal and societal issues (elsi) programmes by working together in multidisciplinary teams aimed at responsible research and innovation. • offer opportunities to collaborate, test and benchmark new methodologies and analysis methods; for example, in adaptive experimental design. • offer unique opportunities to industry to translate recent knowledge and novel technologies from the laboratory to the market. • provide an early access programme to new technologies developed by companies. • function as open, interconnected education hubs, delivering training in the new technologies to researchers, scientific personnel and clinicians, as well as providing engagement activities for patients and the public. lifetime aims to analyse data that are inherently distributed across different clinical centres in different countries, which is a substantial challenge. these data are usually not accessible outside a national, regional clinical care system or specified data 'safe havens'; when they are accessible, accredited systems are often required for storing the data and information governance may be at the hospital, federal or international level. this means that a federated approach is the only way to access and integrate information from various european healthcare systems. thus, the lifetime data and computational network, building on cloud technologies, will provide the necessary capacities to enable federated analytics across the lifetime centres and will provide a technical and legal framework for integrating core information structures, multi-omics assays, imaging, ai and machine learning technologies, and health records (fig. ) . a joint data coordination centre, following a multi-level approach, will ensure transparent data access control, compatibility and standardization. within this framework, lifetime will also coordinate and pioneer open data sharing and reuse and collaboration, including models of access before publication of data. to start this cooperative lifetime centre network, the initiative can build on initial developments and programmes by lifetime members in a number of european countries; for example, the vib single-cell accelerator programme in belgium, the berlin cell hospital/clinical single-cell focus in germany, the uk's sanger/ebi/babraham single cell genomics centre, and the lifetime single-cell centre in poland. to avoid duplication and lack of standardization, the lifetime cell centre network should be coordinated through an entity or framework that optimizes coordination and support to achieve the lifetime vision. funding for specific research projects that involve one or more lifetime centres could come from a portfolio of private and public funding opportunities, on both the national and pan-european levels. the network will interact closely with key european efforts and will contribute to eu strategies and programmes. collaborations with the private sector will be key for the rapid translation and delivery of technologies, instrumentation, diagnostics and therapies (fig. ) . currently, more than companies support life-time's vision. these span multiple sectors as well as industrial associations and networks such as the european federation of pharmaceutical industries (efpia) and the euro-bioimaging industry board (ebib). the transformation of breakthrough discoveries into solutions to improve the health of european citizens will involve several crucial steps. these include the creation of a unifying framework that fosters and streamlines pre-competitive interactions between academia and industry at the interfaces of computer science, single-cell biology, -omics, imaging, patient-derived disease modelling and precision medicine. a large-scale collaboration platform across europe should be developed that provides umbrella agreements, regular meetings, dual training of early-career scientists in academia and industry, and exchange programmes. this will enable joint projects between public and private sectors that span the entire biomedical innovation cycle from discovery research and technology development to implementation in hospitals and the healthcare industry. cross-sectoral collaborations between small, medium-size and large companies with different development timelines and distinct business models is crucial to stimulate innovation. to expedite the identification of, and investment in, emerging technologies developed in academic and industrial laboratories, successful local initiatives such as tech watch and accelerator programmes (for example, the vib single-cell accelerator) should be scaled and coordinated at the eu level. lifetime aims to create a networking and match-making platform for individuals and academic and industry organizations that share the goal of developing and integrating breakthrough technologies and applying them in the clinic to benefit patients. further measures could foster innovation and entrepreneurship. for example, a pre-seed, pre-incubator funding scheme based on competitive calls to support start-up or technology transfer ideas. the creation of a dedicated european ecosystem is also essential. this will require additional key measures, such as the development of enabling digital environments and the promotion of early disease interception with all necessary stakeholders (for example, patients, regulators, payers, and others), as described in the lifetime call for action launched in december (https://lifetime-initiative.eu/ make-eu-health-research-count/). the implementation of lifetime's vision triggers relevant ethical questions from all societal groups that are directly affected by the project (patients, clinicians and scientists), and from society in general. lifetime aims to pioneer a real-time or parallel elsi programme that will predict, identify, monitor and manage the ethical impact of the biomedical innovations that arise from research and technology development, ensuring that implementation follows ethical guidelines. lifetime's elsi programme can be used as a testing ground for other international interdisciplinary initiatives (fig. ) . ethical issues will be identified and managed as early as possible, and the programme will ensure that ethical and research integrity guidance is implemented throughout the entire research process to stimulate positive effects and mitigate negative ones . specialists in bioethics, public engagement, ethics of technology and lawyers have identified lifetime's ethical and societal priority areas. these include questions related to the derivation, storage and use of organoids, the use of ai, data ownership and management, anonymization of data, equity of access to such revolutionary medical care, the definition of health and illness, and transparent science communication to society . to initiate a relationship of trust with the public, we will include diverse modes of communication and engagement, for example through art, citizen science and public dialogue, contributing to scientific literacy, and promoting individual critical thinking and public participation in decision-making processes. the introduction of interceptive medicine into clinical practice in parallel with a multidisciplinary research programme will require capacity building in health and research systems, and substantial deployment of technology in clinics. this will lead to a collaborative, fast-developing and interdisciplinary environment in research and in hospitals, which will require new training inputs. to respond to these needs, lifetime will create an education and training programme, ensuring the sustainable application of new technologies and the implementation of new medical and scientific approaches (fig. ) . importantly, this will be done in an integrative scheme that intersects the multiple lifetime disciplines and areas of action: disruptive technologies applied to medical challenges, technology transfer and innovation, research integrity, data management and stewardship, ethical and societal issues, communication and emotional skills, or management of medico-scientific and collaborative projects. each lifetime training activity will be based on multi-lateral education: basic researchers will teach other researchers and clinicians about the potential of the technological solutions, while clinicians will teach researchers about the clinical needs and biological challenges of the diseases in focus. this will strictly follow the idea of bench to bedside and back. the programme will have an inclusive philosophy to ensure that it can provide training to the wider community, including researchers, clinicians, technical operators, managers and staff of technology platforms, as well as administrators, patients and the lay public. lifetime envisions the organization of cycles of colloquia and outreach activities to inform the public, the formulation of short-term courses compatible with a culture of lifelong learning and adaptability, and interdisciplinary masters and phd programmes. through education and training, lifetime will engage and inform society, will develop community interactions new professional curricula and will train a new generation of highly skilled medical scientists and support staff, in order to foster scientific and medical excellence in an ethical, responsible and inclusive framework. medicine and healthcare are rapidly expanding pillars of our economy. eu countries collectively spend more than € , billion per year on healthcare for their million citizens. given the dimensions and spiralling healthcare costs associated with an ageing population, these numbers will continue to increase unless we can mitigate the damaging effects of ageing. we expect that coupling current health monitoring with early detection and disease interception will have a major economic impact. in europe, % of the population will soon be over years old, with an age distribution that will continue to change until % are over years old in . given the prevalence and cost of caring for people with degenerative conditions and the increase in chronic lifestyle-induced diseases, the knowledge and technologies developed by lifetime are urgently needed to detect these diseases earlier, and to avoid their worst manifestations. lifetime would also have an impact in the era of unexpected pandemics such as covid- by rapidly determining the cellular and molecular basis of the disease. this would identify potential therapeutic strategies for patient subgroups as well as representing a starting point for the development of effective new therapies. one of healthcare's largest outstanding issues is that many patients do not respond to commonly prescribed treatments. whereas well-controlled randomized clinical trials provide evidence for the statistical utility of a given therapy, in practice often many patients must be treated before a single patient will show a measurable benefit. other patients may not benefit at all or even be harmed , leading to an economic loss that is estimated to be in the hundreds of billions of euros per year. the variable therapeutic responses that originate from the cellular and genetic heterogeneity that exists in cancer and other complex diseases, contributes not only to the failure of treatments, but also to the rising cost of drug development, which is currently estimated at around € - billion per drug. in silico models for disease trajectories generated by lifetime will enable the integration of personal genetic and lifestyle information into predictive models of disease course. this will allow physicians to determine and implement optimal therapeutic strategies that are tailored to the individual (precision medicine) with sophisticated timing of disease interception. the knowledge gained will also contribute to more appropriate selection of patients for clinical trials. recent advances in key single-cell technologies, ai and patient-based experimental systems, such as induced pluripotent cells and organoids, have set the stage for their integration and deployment to improve mechanistic molecular understanding, prediction, and treatment of disease onset and progression. patients will benefit from cell-based medicine through the earlier detection of diseases at a stage where they can be effectively intercepted. the integrated technologies will enable the selection, monitoring and, if necessary, modification of therapeutic strategies for an individual to improve clinical outcomes based on high-resolution cellular information. within the next decade, the obtained molecular mechanistic information has the potential to revolutionize drug discovery processes and clinical trial design, and eventually to be incorporated into clinicians' daily decision-making processes. as the lifetime community continues to grow, new individuals, institutions and companies are encouraged to join and contribute to establishing a european platform to implement single-cell and data-driven medicine to address the growing burden of complex and chronic diseases. a brief history of human disease genetics integrative omics for health and disease modeling development and disease with organoids organogenesis in a dish: modeling development and disease using organoid technologies scaling single-cell genomics from phenomenology to mechanism the human cell atlas the lifetime initiative. lifetime strategic research agenda single-cell genomic approaches for developing the next generation of immunotherapies the human body at cellular resolution: the nih human biomolecular atlas program global characterization of t cells in non-small-cell lung cancer by single-cell sequencing single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma dysfunctional cd t cells form a proliferative, dynamically regulated compartment within human melanoma single-cell transcriptomic analysis of primary and metastatic tumor ecosystems in head and neck cancer dissecting the multicellular ecosystem of metastatic melanoma by single-cell rna-seq single-cell rna-seq reveals aml hierarchies relevant to disease progression and immunity tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type i ifn and fibrosis relevant pathways defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry a single-cell atlas of entorhinal cortex from individuals with alzheimer's disease reveals cell-type-specific gene expression regulation a unique microglia type associated with restricting development of alzheimer's disease single-cell transcriptomic analysis of alzheimer's disease single-cell reconstruction of the adult human heart during heart failure and recovery reveals the cellular landscape underlying cardiac function an immune-cell signature of bacterial sepsis multi-omics profiling of mouse gastrulation at single-cell resolution scnmt-seq enables joint profiling of chromatin accessibility dna methylation and transcription in single cells simultaneous quantification of protein-dna contacts and transcriptomes in single cells spatial transcriptomics and in situ sequencing to study alzheimer's disease dissecting cellular crosstalk by sequencing physically interacting cells molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region gene expression cartography visualization and analysis of gene expression in tissue sections by spatial transcriptomics single-cell and spatial transcriptomics reveal somitogenesis in gastruloids high-definition spatial transcriptomics for in situ tissue profiling super-resolution chromatin tracing reveals domains and cooperative interactions in single cells microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms rna imaging. spatially resolved, highly multiplexed rna profiling in single cells visualizing dna folding and rna in embryos at single-cell resolution spatial reconstruction of immune niches by combining photoactivatable reporters and scrna-seq the single-cell pathology landscape of breast cancer a structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis unravelling cellular relationships during development and regeneration using genetic lineage tracing b cells and tertiary lymphoid structures promote immunotherapy response high-dimensional single-cell analysis predicts response to anti-pd- immunotherapy chemoresistance evolution in triple-negative breast cancer delineated by single-cell sequencing toward minimal residual disease-directed therapy in melanoma application of cell-free dna analysis to cancer treatment deep learning: new computational modelling techniques for genomics eleven grand challenges in single-cell data science high-performance medicine: the convergence of human and artificial intelligence multi-omics factor analysis-a framework for unsupervised integration of multi-omics data sets computational methods for single-cell omics across modalities the book of why: the new science of cause and effect building models of brain disorders with three-dimensional organoids lab-built brains xenograft and organoid model systems in cancer research interrogating open issues in cancer precision medicine with patient-derived xenografts hallmarks of alzheimer's disease in stem-cell-derived human neurons transplanted into mouse brain development of a chimeric model to study and manipulate human microglia in vivo stem-cell-derived human microglia transplanted in mouse brain to study human disease the fair guiding principles for scientific data management and stewardship the syscid consortium & rosenstiel, p. systems medicine in chronic inflammatory diseases real-time ethics engagement in biomedical research: ethics from bench to bedside thinking 'ethical' when designing a new biomedical research consortium people in the eu: who are we and how do we live? what happened to personalized medicine? lifetime community working groups thomas bourgeron , sarion bowers , dries braeken , catherine brooksbank , nils brose silvie van den hoecke , ibo van de poel , andre van der ven , julie van der zee department of medical biochemistry and biophysics, karolinska institutet human genetics and cognitive functions unit flanders institute for biotechnology (vib), ghent, belgium. department of medicine & surgery, università degli studi di milano-bicocca communication networks, content & technology, european commission, brussels, belgium. institute of pharmacology and toxicology centre for gene regulation and expression department of brain sciences a. i. virtanen institute for molecular sciences alzheimer's disease and other cognitive disorders unit, fundació clínic per a la recerca biomèdica alzheimer center we acknowledge all participants that have attended and contributed to lifetime meetings and workshops through many presentations and discussions. we thank j. richers for artwork and a. sonsala, a. tschernycheff and c. lozach for administrative support. lifetime has received funding from the european union's horizon research and innovation framework programme under grant agreement . author contributions all authors contributed to the writing of the article and provided comments and feedback. they all approved submission of the article for publication. the individuals listed at the end of the paper are members of working groups that contributed to the writing of the lifetime strategic research agenda (listed in full in the supplementary information). please note that the complete lifetime community is much broader and includes many associates and supporters that are actively contributing to and advocating for lifetime (further information can be found at https://lifetime-initiative.eu). supplementary information is available for this paper at https://doi.org/ . /s - - - . correspondence and requests for materials should be addressed to n.r., g.a. or s.a.g. peer review information nature thanks michael snyder, ali torkamani and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. reprints and permissions information is available at http://www.nature.com/reprints. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -w catjj authors: degeling, chris; johnson, jane; kerridge, ian; wilson, andrew; ward, michael; stewart, cameron; gilbert, gwendolyn title: implementing a one health approach to emerging infectious disease: reflections on the socio-political, ethical and legal dimensions date: - - journal: bmc public health doi: . /s - - - sha: doc_id: cord_uid: w catjj background: ‘one health’ represents a call for health researchers and practitioners at the human, animal and environmental interfaces to work together to mitigate the risks of emerging and re-emerging infectious diseases (eids). a one health approach emphasizing inter-disciplinary co-operation is increasingly seen as necessary for effective eid control and prevention. there are, however, socio-political, ethical and legal challenges, which must be met by such a one health approach. discussion: based on the philosophical review and critical analysis of scholarship around the theory and practice of one health it is clear that eid events are not simply about pathogens jumping species barriers; they are comprised of complex and contingent sets of relations that involve socioeconomic and socio-political drivers and consequences with the latter extending beyond the impact of the disease. therefore, the effectiveness of policies based on one health depends on their implementation and alignment with or modification of public values. summary: despite its strong motivating rationale, implementing a one health approach in an integrated and considered manner can be challenging, especially in the face of a perceived crisis. the effective control and prevention of eids therefore requires: (i) social science research to improve understanding of how eid threats and responses play out; (ii) the development of an analytic framework that catalogues case experiences with eids, reflects their dynamic nature and promotes inter-sectoral collaboration and knowledge synthesis; (iii) genuine public engagement processes that promote transparency, education and capture people’s preferences; (iv) a set of practical principles and values that integrate ethics into decision-making procedures, against which policies and public health responses can be assessed; (v) integration of the analytic framework and the statement of principles and values outlined above; and (vi) a focus on genuine reform rather than rhetoric. the recent ebolavirus (ebov) outbreak in west africa and continuing human infections with a novel h n influenza a virus in mainland china are salient reminders of how human and nonhuman health are inextricably linked. nonhuman animals are the source of % of emerging and re-emerging infectious disease (eid) threats to human health [ ] , and more than half of all established human pathogens [ ] . the threats posed by eids are dynamic. eids are caused by pathogens that can change their behaviour over timeeither through genetic modification or through changes in the patterns and pathways of transmission [ ] . social, economic and political systems can either promote or inhibit pathogen transfer, and the incidence and pathogenicity of the disease [ ] . while a lack of data makes quantitation difficult, eids and zoonoses account for a significant proportion of the global disease burden [ ] . eids and emergence of zoonotic pathogens, including human immunodeficiency virus (hiv), are direct causes of an estimated million deaths worldwide each year [ ] . a one health approach is increasingly considered to be the most effective way of managing eid threats [ , ] because it represents an acknowledgement of certain facts about the nature of disease, which are then deployed to structure the response. one health is grounded in a recognition that human, animal and environmental health are interdependent [ ] , that animal species provide a shared reservoir for pathogen exchange and spread, and that many eids are driven by varied and dynamic human-animal interactions [ , ] . the response one health offers is to deconstruct the disciplinary silos [ ] which have separated biomedical and social sciences devoted to the study of human disease from those devoted to nonhuman disease and ecological concerns [ , ] . inter-disciplinary research is called for and required, as is interventionist practice at local, national and international levels involving: policymakers, planners, regulators, physicians, veterinarians, ecologists, public and animal health officials, environmental health officers, microbiologists, and other allied natural and social scientists [ , ] . although principally associated with eid prevention and control, one health is also relevant to prevention and control of endemic and zoonotic animal diseases, as well as securing food safety [ , ] . considering the magnitude and complexity of global issues surrounding infectious disease and food security, the one health approach has the potential to provide the creative, effective and sustainable solutions required. despite its strong motivating rationale, implementing a one health approach can be challenging. dealing with eids in an integrated and considered manner can be highly problematic, especially in the face of a perceived crisis. in this paper we examine the socio-political, ethical and legal considerations implied by a one health approach to eids. first we describe how a one health approach could galvanise and enhance current capacity in eid prevention and control. making reference to case examples, we then identify and characterise sociopolitical, ethical and legal concerns that have the potential to limit the effectiveness of one health interventions. finally, we draw on this data to provide guidance as to how these concerns and issues might be addressed, and point to remaining challenges to the likely success of the one health approach to eid control and prevention. in order to explore the broader implications of a one health approach we employed philosophical and qualitative methods to map existing and potential scientific, ethical and political responses to eids in australia and our region. the overarching philosophical approach is that of developing sustained arguments that critically analyse the existing literature and reconceptualise or refine key concepts. this conceptual information is often observed in exemplars and paradigm cases. in particular we focused on materials pertaining to the social, political and ethical consequences of responses to the risks posed to human health and wellbeing by hendra virus [hev], nipah virus [niv] and rabies virus [rbv] in australasia, and compared them with international responses to canonical examples of pandemic and food borne zoonoses severe acute respiratory syndrome (sars) [ ] and bovine spongiform encephalitis/variant creutzfeldt jacob disease (bse/vcjd), respectively. a synopsis of the characteristics and burdens of these diseases and the pathogens that cause them are outlined in boxes and . because our aim was to generate inductive insights and develop a robust set of argumentsrather than a comprehensive catalogue of every case example or publicationthe sample evolved iteratively from searches of textual sources such as publicly available international (e.g. who) and government reports; academic databases (e.g. pubmed); online/print news services (factiva); organizational newsfeeds (centers for disease control); and the websites of major one health collaborations [ ] . materials in the sample were read and qualitatively reviewed through an iterative process of testing, revising and refining our definitions, principles and theoretical generalisations [ , ] against the emerging conceptual map and feedback from the research team. led by the first author, this cycle of searching, mapping and critical analysis continued until a period where new textual materials were not providing substantive new insights and the team was confident that a position of conceptual saturation had been achieved. in what follows we draw on these analyses and reflections to describe the content, context and nature of the challenges that need to be faced for the effective implementation of a one health approach to eid control and prevention. findings eid prevention and control strategies require a one health approach one health is a holistic approach that emphasizes, but is not restricted to, the need to understand and regulate the environmental context (human-animalecosystem interface) of disease emergence and expression [ ] . eids are characterized by their complexity and uncertainty as to their causes, consequences and likely solutions [ ] . in broad terms, the occurrence and cross-species transmissibility of many emerging pathogens, like ebolavirus (ebov) and h n , arise from human activities such as changes in land use, growth in global trade and travel and intensification of animal husbandry practices [ ] [ ] [ ] . the speed with which our understanding of the biology and epidemiology of h n has developed demonstrates how much our ability to respond to new eid threats has improved over the last few decades. yet despite advances in immunobiology and genomics that have contributed to diagnostics, therapeutics, and vaccine development, the threat of eids to human health and community wellbeing persists. part of the reason why eid threats remain in spite of scientific advances, are that eid events are not simply about pathogens jumping species barriers. the threats posed by eids are comprised of complex and contingent sets of relations that involve socioeconomic and sociopolitical drivers and consequences, with the latter extending beyond the impact of the disease. the social, cultural and economic impacts of zoonoses are significant. the examples contained in tables and demonstrate the difficult balance between the human health risks and socioeconomic and cultural costs of eid control [ , ] . policy decisions should be based on sound evidencebut it is often the case in dealing with eids that the evidence required is absent or fluid. eid events are often dynamic situations that are characterised by uncertainty. as events unfold new evidence is created. consequently decisions made on the basis of present data can be seen as wrong in the future, as more evidence and a better understanding emerges. official reviews of canonical eid events such sars [ ] and bse/vcjd [ ] share two key findings: (i) that actions to reduce risk should not be predicated on scientific certainty; and (ii) that policies to deal with the risks and effects of an eid need to be founded on widely held values, so that people understand, in advance, the kinds of choices that will have to be made. this suggests that the one health approach needs more than inter-sectoral collaboration and robust health legislation, as the unique nature of eids critically limits the effectiveness of scientific, top-down and technocratic approaches to governance [ ] . hendra virus infection is endemic among at least two species of flying fox in australia and causes rare, but catastrophic, human infection [ ] . loss of habitat has led to increasingly intense incursions of flying foxes into populated rural and peri-urban areas and promoted the 'spill-over' of hendra virus into horses and then to people [ ] . hundreds of people have been directly exposed to hendra virus, with seven confirmed human infections and four deaths since . with over one hundred dead horses and persistent risk, the emergence of hendra has had significant impact on equine and tourist industries in north eastern australia, diverted major research resources and caused significant distress and controversy in the broader community [ , ] . nipah virus, a close relative of hendra, is endemic in east asian flying fox populations. in , after a program of deforestation and agricultural development in eastern malaysia it spread to pigs then humans and other animals, causing respiratory disease and severe encephalitis [ ] . it subsequently was reported in india and bangladesh. humans can be infected directly from bats, by ingestion of contaminated food and from other humans. among confirmed human cases, the overall mortality was greater than % [ ] . nipah control programs devastated malaysia's pig industry and caused high unemployment and dislocation of rural populations, at a cost of more than us$ billion to the national economy [ ] . nipah virus has been identified by who as a likely cause of future pandemics. rabies virus infects the central nervous systems of people, wildlife and domestic mammals. the disease is transmitted by bites from infected animals and once it becomes symptomatic, it is virtually always fatal. , people die and . million receive post exposure prophylaxis annually, costing $ billion [ ] . rabies is endemic in much of south east asia but its range is expanding. focusing on australia, the continent is free from rabies, but the current expansion of the disease in indonesia [ ] is a genuine threat to northern regions. although likely controllable in domestic dog populations [ ] , if rabies were to become endemic amongst wild or feral animals in this setting, current modelling indicates it would be almost impossible to eradicate [ ] . table significant historical (i.e. effectively eradicated) eids severe acute respiratory syndrome (sars) is a human respiratory infection, caused by a coronavirus isolated from chinese horseshoe bats [ ] . it was first reported in asia in and, within a few months, spread to thirty seven countries in the americas, europe and asia. it affected more than people and caused deaths, before being successfully eliminated by concerted international efforts. the outbreak and fear that another pandemic could occur are estimated to have cost canadian and east asian economies us$ billion [ ] . bovine spongiform encephalitis/variant creutzfeldt jacob disease (bse/ vcjd) is a rare but fatal human neurodegenerative condition, caused by consumption of bovine products contaminated with the prions that cause bse. since vcjd was first identified in , cases have been reported in the uk and forty nine elsewhere. the world bank estimates that the direct costs of vcjd/bse to date exceed more than us $ billion. infected herds and the control measure imposed to prevent further infections devastated agricultural communities. the impacts of the emergence of a new zoonotic disease amongst the british public were far broader than agriculture, including the cessation of uk plasma production because of potential iatrogenic infection. with an estimated one in uk residents carrying vcjd, the burdens will continue well into this century [ ] . the success of one health depends on more than scientific knowledge and technical achievement because some of the issues that arise in addressing eid risks are socalled 'wicked problems' [ ] . when a new eid threat emerges there are rarely ready-made solutions and health policymakers and practitioners are often forced to make tragic choices that may contravene widely held values. considerations must include the need to protect public health and the wider social, economic and environmental impacts of proposed interventions. economic and political interests can complicate the decisionmakers' motives and decision-maker uncertainty is compounded by policy decisions becoming entangled in political, ethical and legal considerations [ ] [ ] [ ] . as events surrounding the ebov outbreak in west africa illustrate, the importance placed on a specific eid threat at any one time also depends on who is setting the agenda [ ] . therefore to be successfully implemented, the one health approach must address a range of socio-political, ethical and legal challenges that arise as a consequence of the spread of infection within and between species. most of these challenges are not unique to one health, but are shared by any approach to addressing eids. however these challenges frequently go unrecognized. in the following section we will clarify the nature of these issues so they can be addressed later in the paper. ( )socio-political challenges a focus on individualism, perceptions, short term solutions, populism and avoiding controversy are features of political life, which can prove challenging for eid policy and work against developing effective strategies for addressing eids. policy responses to eid events such as nipah and hendra virus infections (outlined in box ) tend to focus on necessary and proximal causes (what individuals do to put themselves at direct risk from an infectious pathogen) because the science about other aspects of eids is often complex, uncertain and lacking a clear narrative. compounding this, our moral psychologies have evolved to respond to direct harmsnot indirect distal causal stories. many people in liberal democracies believe that they are entitled to rights and freedoms that cannot be sacrificed merely for the marginal gains of others. as the discourse surrounding climate change and other wicked problems illustrates, this promotes technological solutions because they do not require substantive changes in human behaviours and underlying values systems. [ ] the net result is that the policy focus for eid prevention and control tends to remain on individual behaviours rather than the structural drivers of emergence and transmissiona case example being the focus on vaccine development and the husbandry practices of horse owners in response to the zoonotic risks of hendra virus [ , ] .. the political impetus for action in response to many eids is not necessarily scientific evidence but societal perceptions. indeed, in the face of scientific uncertainty and ethical ambiguity, ideological perspectives and short-term political considerations often supplant efforts to devise effective long-term interventions [ , ] . political imperatives to avoid, or at least minimise, public concern whilst dealing with eids can also prove challenging. in the case of bse, powerful interests dominated early government responses, leading policymakers to make decisions that avoided public controversy, but had major economic consequences. as the crisis unfolded, expertise became politicized leading to conflict between agencies and policy inconsistency between health communication strategies and the measures being taken to minimize the risks to human health [ ] . even when the link between bse and vcjd became clear, existing feed bans were poorly enforced and risk communication was dominated by fear of public panic [ ] ; even as the decision was made to remove all potential sources of human infection from the uk food supply, messages were confused and policy implementation impeded by poor co-ordination between agencies [ ] . a common but problematic response to eid threats has been to invoke the precautionary principle. roughly speaking, the precautionary principle can be applied in situations where human activities create a scientifically plausible, but uncertain, risk of significant harm. in response the principle advocates that actions ought to be taken to avoid or reduce the harm, and that these actions need to be proportionate to the seriousness of the potential harm. in other word, in the absence of evidence take a conservative approach. however applying the precautionary principle to eids in an attempt to protect the public can result in what, in retrospect, amounts to an excessive response. this occurred with attempts to control nipah infection, where significant damage was inflicted on industry, livelihoods and the economy. similarly, experience with highly pathogenic avian influenza (hpai) h n in china and se-asia showed that overzealous policy responses can destroy livelihoods and threaten food supplies [ , ] . in vietnam alone, almost million birds were culled in in an attempt to eradicate hpai. although many birds were owned by large commercial operations, others were kept by 'backyard' farmers and villagers. mass culling of poultry appears decisive, but places excessive burdens on vulnerable populations, is ineffective in the context of extensive 'backyard' poultry farming and can, in fact, promote the spread of disease [ ] . a similar scenario is currently playing out with rabies control in bali. unfortunately, the precautionary principle and analytic tools and concepts appealed to in this domain, fail to deliver what is required at times of eid outbreaks since they do not advance public engagement or help resolve disagreements in times of uncertainty [ , ] . philosophical critiques of the precautionary principle applied to eids have also shown its limitations, including that defining criteria by which to judge a threat as plausible and a response proportionate, often will only substitute one uncertainty for two others [ ] . ( )ethical challenges the effectiveness of an eid control policy will depend on the context of its implementation and particularly its alignment with stakeholder and public values [ , ] . in modern liberal democracies at least some consensus over what is in the public interest and an understanding of the values which support it, is therefore required for the successful implementation of eid responses. yet this is precisely what has been lacking in outbreaks where fracture lines, differences and value conflicts have become apparent. when the stakes are high, evidence and the implications of actions are uncertain, the situation is complex and resources may be limited but where decisions need to be made, differences are exposed. such differences could be around beliefs about how to deal with ecological and environmental issues, which may conflict with the importance people attach to public goods, protection of individual autonomy and animal welfare [ ]. these conditions of crisis and division are conducive to undesirable consequences including public fear, mistrust, misinformation and non-compliance with public health directives. for example in canada during the sars crisis, leaders were unprepared for the range of ethical conflicts that arose, including those over: individual freedom versus the common good; healthcare workers' safety versus their duty to care for the sick; and economic costs versus the need for containment [ ] . as indicated in box , both the outbreak itself and fear that another outbreak could occur had significant economic consequences. any approach which hopes to successfully respond to eid threats, including a one health approach, needs to address the ethical concerns articulated above. to this end, potentially conflicting values and logic must be negotiated to realise effective, sustainable and just solutions. prioritisation and resource allocation require political processes based on fundamental ethical questions about what is valuable, what is to be protected and, ultimately, what is dispensable. to be effective, public policy must be consistent with the values of citizens to whom it is applied, otherwise it can become mired in controversy about whose values should prevail [ , , ] . therefore, one of the first and most important tasks of policy work is to establish how the public interest is best defined. ( )legal challenges the legal environment in which eid policy is made and in which responses to outbreaks occur, presents its own set of challenges. the law surrounding eid responses in most jurisdictions is diffuse, complicated and often subject to re-interpretation on the basis of whose interests are given primacy at the time decisions are made. moreover, in many countries different approaches by state/provincial and local authorities, overlaid by federal/national powers, complicate regulation so much that 'hard law' is often replaced by resort to 'soft law' of executive and administrative powers and international instruments, such as the international health regulations (ihr) [ ] . this may add complexity and confusion to the eid regulatory structures, rather than facilitating public health responses to a new threat. such confusion provides a salient reminder that even in 'global law' approaches to eids, the sovereign state remains the institution responsible for regulation and control [ ] . public health law responses to eids tend to be oriented towards controlling cross-border pathogen transfer and community outbreaks rather than the underlying deficiencies and structural conditions from which the threats emerge. other laws, such as environmental law, may be more useful in addressing structural conditions for emergence. changes in land use and agricultural intensification in developing societies are major drivers of eid. however, the cost of laws that restrict development may be greater global health inequities, with consequential effects for health outcomes. in order to clarify eidrelated legal tensions between economic development and health security, a more explicit recognition is needed of who are the primary beneficiaries and who bears the costs of a one health approach to eids [ ] . legal clarity around the frameworks designed to protect populations from eids is critical to providing an enabling infrastructure to co-ordinate and support the one health-based work of policymakers, development planners, human and animal health-workers and biosecurity agencies. the health of humans, animals, and ecosystems are interconnected. a one health approach promises a better understanding of how to prevent and control eids at the human-animal-ecosystem interface. however the socio-political, ethical and legal challenges of eids illustrated above highlight how responses to infectious disease threats are intrinsically value laden. when a new infectious pathogen such as hendra or nipah virus first appears, or a known threat such as rabies or ebola encroaches on a new setting, there is limited scientific evidence or past experience to guide decisions or determine whether a planned response will be proportionate. vastly different interpretations of eid events and their likely outcomes might be supported by the available data. policymakers and practitioners therefore have little guidance as to what they should do when faced with a nascent infectious disease threat, only what they can do. as others [ ] [ ] [ ] have cogently argued, they must therefore ask themselves: whose health is being prioritized; which public and which good are we seeking to protect? notwithstanding recognition of a need for complementary work on values-based questions that inevitably surround eid risks and eid control, the adoption of the one health approach, so far, has not included development of a comprehensive, ethically-informed policy and implementation framework; this has limited its practical utility [ , ] . despite rhetorical and some financial support for one health as the guiding ethos by which to address interconnected human, animal and environmental health issues, its impact will be minimal unless implications of uncertainty on, and potential conflicts between, human values and political processes are recognised and articulated. any attempt to address these ethical and normative dimensions must take into account the dynamic nature of eid risk management. a policy that seems reasonable today may be inappropriate tomorrow, in light of new evidence. and when situations are uncertain, decision-makers inevitably fall back on their values. therefore, a solid framework based on shared values is needed to support decision-making surrounding eids when "evidence" isor may beunreliable, and rapidly changing or fluid. what is needed to guide a one health approach to eids? to successfully meet the challenges described above, particularly the necessity to align eid policy with public values, a one health approach needs to engage in the following. (i) social science and economic research to help catalogue and describe the drivers, mechanisms and social and political configurations through which eids become threats to human, animal and ecological health [ , ] . the complex connections between individual social needs and the local socioeconomic context of affected or at-risk communities, need to be understood and addressed by policymaking processes. this should ensure that manifest injustice, livelihood-based decisions and other social and cultural factors do not undermine the effectiveness of favoured control measures. without adequate knowledge of specific local arrangements, there is a danger that insufficiently nuanced or unified approaches to eids will actually undermine the heterogeneous relationships and contingent practices that make health possible in circumstances of structural disadvantage [ , ] . the social sciences are analytically broader and more policy focussed than the natural sciences. whereas the natural sciences tend to frame infectious disease threats narrowly as matters of biological integrity and security, such that barrier technologies and hygiene practices dominate the logic of interventions [ ] , social science approaches go beyond this. building social scientific evidence for use in conjunction with natural scientific evidence about eids aligns with the growing realization that eid emergence is as much about the social and economic configuration of capital flow as it is about the biological features of host-pathogen interactions. current approaches to the economic and structural drivers of eid emergence still presume that state and market neoliberalism is part of the natural order, even as evidence is mounting that these systems of development are central to the problem [ , ] . moreover, the current emphasis on microbiology and focus on newer molecular techniques to characterise pathogens, is drawing attention away from developing better understandings of the environmental, economic and social drivers of eids. while this is understandable given the desire for vaccines and drugs to solve eids, if one health researchers and practitioners broaden their approach to causality to include upstream, social and economic systemic causes, questions and issues that have been traditionally bracketed or thought best avoided will become central to the cross-sectoral collaboration implied by one health. framework (ohaf) needs to be pursued. such a framework would catalogue case-based experiences and reflect the particular dynamics of specific eids, and promote inter-sectoral collaboration and knowledge synthesis, including integration of information about social, cultural and economic impacts, control measures and uncertainty [ , ] . the framework would serve as a prompt to ensure that minority perspectives are represented and all relevant concerns are considered. an ohaf could provide a rubric for comparisons between outbreaks. this would allow the inherent complexities of economic and societal responses to eids to be compared, to inform policy processes. it is vital for discussions about eid prevention and control to have this kind of sound empirical foundation, because uncertainty and media coverage have the potential to drive bad policy. development of an ohaf could be facilitated by adopting well established and methodically rigorous processes such as framework analysis, produced by the national centre for social research (uk) [ ] , or multi-criteria decision analysis [mcda] developed within the field of decision science [ ] . in the first instance framework analysis would allow for systematic incorporation of the perspectives and contributions of different scholarly disciplines and expert stakeholders. framework analysis facilitates movement between different datasets, thematic areas, theoretical resources, and levels of abstraction without loss of conceptual clarity [ ] . the framework method is used to organize and manage research and interpretation through the process of summarization, which is codified into a robust and flexible matrix that allows the policymaker/researcher to analyze data both by case and theme. it is commonly used in areas such as health research, policy development and program evaluation. equally, mcda methods offer an alternative and potentially complementary approach to ohaf development. comprised of a suite of analytic strategies, mcda have been shown to be valuable tools for prioritization and decision-making in animal and human health [ ] . mcda provides a framework to compare policy alternatives with diverse and often intangible impacts, which can be particularly useful in determining and justifying the prioritization and mobilization of limited research and public health resources [ , ] . (iii)genuine processes of public engagement across the developed and developing world are also essential to a successful one health approach. these processes are not so much about engaging in deliberative democracy for policy decision-making, as about defining the principles and values that should guide decision-making. this means procedural inclusiveness alone is not enough to ensure transparency and reflexivity, to capture people's preferences and to effectively communicate with the public [ ] . the successful implementation of the one health approach to eids will depend on public trust and cooperation. public support for unpalatable measures is more likely if citizens understand the issues, and policy implementation reflects community values and preferences. to this end, citizens' juries have been employed in the uk, australia, the us and elsewhere [ ] [ ] [ ] [ ] to explore similar issues and identify citizens' preferences. they represent informed public opinion better than other social research methods (e.g. surveys or focus groups) because they give participants factual information, bring them into a structured and constructive dialogue with experts, provide them with time to reflect and deliberate, and allow them to represent their views directly to policymakers. to be successful, one health needs to be about more than disease prevention and control. the dynamic, unpredictable effects and risks to peoples' lives of eids necessitate a public health and biosecurity infrastructure equipped to address the ethical problems that arise. eid management must therefore be based on normative principles as well as local knowledge, operational experience and disease-specific scientific and economic evidence. this means that governments and policy-makers need to explain and justify the values that underlie decision-making and engage the public in discussions about ethical choices, so that when difficult decisions arise in the face of uncertainty, they will be accepted as fair and essential for the public good [ ] . this necessitates that the guiding values and likely ethical choices need to be articulated in a formal statement in advance, as in the heat of emerging health threat, decision makers will be under pressure from many sources to 'do something quickly'. (v)integration of an ohaf and spv with the ihr and relevant national health and biosecurity legislation is essential so that policymakers and practitioners can dynamically test their decision-making. our response should of course be based on the best scientific evidence, but eids are not just scientific issues, they also have significant social, ethical and animal rights dimensions. experiences of infectious disease threats such as bse/vcjd and sars indicate that there have been problems combining evidence and human values at both local and policy levels [ , ] . the communicability of diseases between species raises social, ethical and legal issues that have not been clearly elucidated or adequately addressed. our response to nonhuman animal disease is not determined solely by bio-scientific knowledge; the way people and animals live with and amongst each other is also shaped by social norms, economic imperatives and human values. in matters of public health it is no longer sufficient to ask what works and what is the strength of the evidence; we also need to ask ethical questions about how we should seek to live, and what is the right thing to do [ ] [ ] [ ] . consensus about the best approaches to eid control and prevention are not always possible, however an agreed set of guiding principles and values can be a means to ensure dialogue, if not always agreement. the development of an ohaf and spv will also promote clearer communication about public risk. significant eid threats have major implications for distribution of scarce resources, access to and regulation of health services and maintenance of social order. as described above it is also clear that policy and legal responses to eid threats are often highly politicised and compromised by failure to communicate clearly with the public. policymakers responsible for responding to disasters such as eids typically find that there is a dissonance between transparency that may appear alarmist versus withholding information to avoid panic. regardless of advice, people will make their own decisions based on their interpretation of available information, from formal and informal channels. so public communication, before and during a public health emergency, is frequently as important as political decisions and regulatory changes [ , ] . this means that, to be effective, a one health approachlike any eid policymust deal with scientific uncertainty, whilst addressing the socio-political, ethical and legal dimensions of effective health communication and intervention strategies [ ] . by exposing decision-making processes to reveal the scientific and normative uncertainties and ethical complexities, the introduction of an ohaf and a spv into one health theory and practice may incorporate iterative deliberation and learning into eid policy processes. (vi)finally, one health must be about genuine reform rather than merely rhetoric. a one health approach rests on the assumption that the cross-sectoral integration of expertise, research methodologies and public health infrastructure will inevitably improve capacity for disease-risk prediction and effective intervention. however, calls for increased intersectoral co-operation by public health practitioners, clinicians, scientists and policy-makers are not a new phenomenon. for example in the s advocates of "new public health" called for health authorities to turn their attention to the social, economic and environmental factors that affect healthrequiring the realignment and policy integration of health departments with other government agencies [ , ] . unfortunately in this case as others, attempts at promoting inter-sectoral approaches rarely move beyond rhetoriceven when driven by the best intentions and supported by substantial resources [ ] [ ] [ ] . the problem is that arguments that promote the need for greater co-operation between sectors tend to focus on the likely benefits of collaboration rather than what reform would entailthat is, what needs to be done organisationally and politically to achieve the desired outcomes [ ] . established 'sectors'whether orientated towards human or animal health, agriculture or the environmenthave genealogies, traditions and rationalities of "what we are here for" that have been shaped by social, political and administrative processes [ ] . as institutions, they are philosophically and structurally resistant to change that diverts resources and re-orients practices away from their own sectoral priorities [ ] . in essence, they have their own constituencies to serve. as a consequence, establishment and implementation of mechanisms that enhance information-sharing, collaboration and inter-sectoral co-operation, such as working groups and interdepartmental committees, have rarely delivered the outcomes promised in the past. responses to bse/vcjd in the uk [ ] , hpai in south east asia [ ] , and recent case studies of one health programs in uganda [ ] , suggest that more work is needed to coordinate implementation and overcome sectoral interests. the complexity of the problems posed by eids mean that organising effective control and prevention programs will require genuine cross-sectoral integration and, potentially, re-sectoring of some institutional and professional responsibilities [ ] . and as the recent ebolavirus disease outbreak illustrates, there must also be sustained social and political willingness to achieve control. if one health is genuinely the way forward, as we believe it is, then we should do more than talk about its potential benefits. without genuine cross-sectoral reform and a radical broadening of the scope of its inquiry into how specific social, cultural and spatial configurations promote the risks of eid emergence, one health is in danger of becoming merely a rhetorical strategy to avoid conflict between its core disciplines, whereby practitioners, researchers and policymakers will espouse the methodological and moral case for interdisciplinary collaboration yet remain in their silos [ ] . even if these barriers are overcome the one health approach will only succeed if it explicitly acknowledges local contingent and contextual dimensions of disease risk and disease expression and the political impacts of scientific uncertainty, while also seeking to accommodate the values and preferences of 'at risk' and affected individuals. further, we suggest that decision making around eids requires an ethical framework that reflects the values of affected and 'at risk' communities, privileges justice, takes account of human flourishing, protects animal health and welfare and is developed in consultation with relevant stakeholders and the public. eid risk management is a major global public health issue to which one health represents a promising approach, but its potential benefits have not been fully realised [ , ] . despite recognition that the social and cultural dimensions are critical to the success of one health, social scientists are yet to play a central or substantive role in shaping research programs and interventions [ , ] . at the same time as the literature on the ethics of pandemic responses and preparedness continues to grow, the one health approach to eids has received little formal ethical consideration. even the most ethically attuned existing frameworks for biosecurity and infection prevention and control provide only general operational principles that do not guide actions in times of uncertainty. if one health is to be meaningful − let alone successful − more attention must be paid to how these different types of knowledge are brought together and brought to public attention. effective responses to eids are likely to be delayed or precluded unless all the socio-political, ethical and legal implications are articulated, publicly 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a framework for health promotion does health promotion need a code of ethics? early response to the emergence of influenza a (h n ) virus in humans in china: the central role of prompt information sharing and public communication the new public health what is the "new public health joining up or pulling apart? the use of appraisal to coordinate policy making for sustainable development what's new about the "new public health"? learning lessons from past mistakes: how can health in all policies fulfil its promises? the significance of 'sectors' in calls for urban public health intersectroralism: an australian perspective one world-one health and neglected zoonotic disease: elimination, emergence and emergency in uganda ecological aspects of hendra virus cross-species virus transmission and the emergence of new epidemic diseases the hendra virus report: an investigation into agency responses to hendra virus incidents between new directions in conservation medicine: applied cases of ecological health henipaviruses: unanswered questions of lethal zoonoses the nipah virus outbreak and the effect on the pig industry in malaysia the cost of canine rabies on four continents review of rabies epidemiology and control in south, south east and east asia: past, present and prospects for elimination development of a novel rabies simulation model for application in a non-endemic environment australian veterinary emergency plan (ausvetplan) review of bats and sars an update on the assessment and management of the risk of transmission of variant creutzfeldt-jakob disease by blood and plasma products the work was funded by nhmrc grant # and seed funding from the marie bashir institute for infectious disease and biosecurity and the school of public health at the university of sydney. the funding source has had no involvement in how the paper was interpreted or written. the authors declare that they have no competing interests.authors' contributions cd led the conceptualization, review, critical analysis and drafting of the article. jj, gg, ik, aw, mw, and cs all made significant contributions to the critical analysis and drafting of the paper. jj and cd led the final preparation of the paper for submission. all authors read and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -burm nxm authors: eckmanns, tim; füller, henning; roberts, stephen l. title: digital epidemiology and global health security; an interdisciplinary conversation date: - - journal: life sci soc policy doi: . /s - - - sha: doc_id: cord_uid: burm nxm contemporary infectious disease surveillance systems aim to employ the speed and scope of big data in an attempt to provide global health security. both shifts - the perception of health problems through the framework of global health security and the corresponding technological approaches – imply epistemological changes, methodological ambivalences as well as manifold societal effects. bringing current findings from social sciences and public health praxis into a dialogue, this conversation style contribution points out several broader implications of changing disease surveillance. the conversation covers epidemiological issues such as the shift from expert knowledge to algorithmic knowledge, the securitization of global health, and the construction of new kinds of threats. those developments are detailed and discussed in their impacts for health provision in a broader sense. the term digital epidemiology is in this special compilation defined by marcel salathe as epidemiology that uses data that was generated outside the public health system, i.e. with data that was not generated with the primary purpose of doing epidemiology (salathe ) . arguably a narrow definition, we will use this conceptualization as the starting point for our conversation. the so defined digital epidemiology promises faster detection of disease outbreaks and improved surveillance as well as reduction in administrative and financial burden, among other things. at hand in the following conversation is less the question if those promises are kept. instead we are interested to reflect epistemological/methodological, ethical/legal, social/political, and organizational aspects and implications corresponding to the promise of digital epidemiology. what will be the relationship of traditional and digital epidemiology? will a possible change influence the scope of public health and global health? tim eckmanns, henning füller and stephen roberts discuss political implications of digital epidemiology. digital infectious disease early detection systems such as the promed-mail, global public health intelligence network (gphin), healthmap, the now closed google flu trends or the syndromic surveillance system essence are central elements of global public health surveillance. however, with increasingly digitalized (algorithmic) global public health surveillance systems and related data-driven epidemiological analyses (e.g., digital epidemiology and other research methodologies), there seem to emerge epistemological shifts, as well as methodological ambivalences and diverse social and political effects. you, henning and stephen, both work from a social (or rather political) science perspective on the societal implications of digital epidemiology, which is shaped by multiple imperatives, e.g., of 'global health security' as well the potentials of big data. over the past two decades, i would argue, we have seen an unleashing of the algorithm across practices of health security and surveillance. algorithmically-guided infectious disease surveillance systems have proliferated across global health geographies, seemingly in response to a series of interconnected and complex transformations within global health governance (ghg), as well as the practice of international relations and international security. we have seen the rise of a seeming 'epidemic of epidemics' from the late twentieth century onward, including the emergence of hiv-aids, novel strains of avian and swine influenza, sars, ebola, mers, the zika virus, and the re-emergence of cholera, polio and multi-drug resistant tuberculosis across low and middle income countries (lmics) clinical and public health surveillance practices with their routinised processes of data collection, analysis, and dissemination from national health institutes have increasingly fallen out of pace with the capacity to timely identify the globalised spread of novel and re-emergent pathogens. correspondingly, the rise of the digital era, resultant from technological interconnectivity and innovation, has generated infinite, voluminous and diverse data at a rate never feasible in history. between and , . quintillion bytes of data have been produced continually, every day in the mere span months (ibm ). celebrated for the capacity to connect the operational 'dots' between these seemingly unintelligible and largely unstructured streams of data in the surveillance and identification of infectious disease outbreaks, the algorithm has emerged as salient and novel technology of security in the pre-emption of pandemic threats in the twenty-first century. to firstly illustrate this shift, in late november , the global public health intelligence network (gphin), a semi-automated online health surveillance system, which piloted the use of retrieval algorithms to filter international media sources, identified the early reporting of a form of atypical pneumonia circulating in guangdong province, china. the 'algorithmic gaze' of gphin identified the origins of the severe acute respiratory syndrome (sars) in advance of months of traditional public health and governance authorities. more than a decade following the rapid spread of sars, healthmap, an online health surveillance system, identified again, via algorithmic processing of digital data streams, the emergence of a mysterious hemorrhagic fever occurring in macenta, guinea. healthmap critically captured and presented strategic epidemic intelligence detailing the emergence of the ebola virus disease (evd) on march , days in advance of the official notification of outbreak by the guinean health authorities. digital epidemiology -from expert knowledge to 'knowledge without truth' the increasing integration of algorithmically-driven infectious disease surveillance systems contemporary logics of health security are critical and significant for a number of reasons (roberts and elbe ) . first, reflective of a growing recourse to the harnessing of novel information sources to contain pandemic illness, the who, via the revision of the international health regulations ( ) , has clearly authorised the collection, assessment and utilisation of non-governmental sources of epidemic intelligence and data (article . ), without prior clearance of member-states. (world health organization ) in this regard, the algorithm emerges a new purveyor of varied, voluminous and expedited data sources to be leveraged in the risk assessment of future infectious disease threats. epistemically, what we can see is how the centralisation of the algorithm within security technologies such as digital disease surveillance systems re-contour previous relations and understandings of knowledge production, the practice of surveillance and the regulation of pandemic risk. the cultivation of knowledge to address the contingent within past 'regimes of truth' were largely sustained, as illustrated by foucault by the 'avalanche of statistical numbers' (hacking ) . increasingly however, within these contemporary security technologies, the vs of big data (volume, variety, and velocity) are now being mined, scanned, and reassembled via algorithmic processing of data to produce findings and alerts on the next pandemic. information and 'truths' about the physical world and the contingent threat of infectious disease are increasingly extracted in the forms of signals and signs of the realm of the digital, and no longer solely generated from statistical processes via human analysis. furthermore, as the conceptual work of antoinette rouvroy ( rouvroy ( , rouvroy ( , , has demonstrated, algorithms have emerged within health surveillance technologies as purely 'rational' or 'objective' instruments of forecasting, indifferent to the causes of phenomena and seeking only to accrue maximal reservoirs of data to address that which constitutes the contingent or the uncertain. what this means therefore is information and knowledge generated by these algorithmic techniques now appear to bypass the traditions of human assessment, analysis, hypothesis, testing and trial which were essential to the statistical calculation of the contingent. rouvroy has referred to this dissemination of this new form of understanding future-situated uncertainty as "knowledge without truth", represented in the context of this discussion by disease tracking systems including gphin and healthmap, which have, with upward intensity sought to apprehend infinitely expanding data sources through an intensified recourse to algorithmic-suffused disease surveillance. what is absolutely vital to emphasise here is that amid the widespread deployment of big data analytics and increasingly sophisticated algorithms for tracing the next outbreak, little critical assessment has been formulated by global health security theorists and practitioners on the ramifications 'digital' turn of health surveillance and the implications of big data and algorithmic surveillance practices on individuals, populations and states. thus, these continued shifts towards employing advanced algorithms to make sense of unprecedented amounts of information (leese ) , across practices of contemporary disease surveillance must be continually matched with equally robust interrogations of the unforeseen or unprecedented implications of securitization by algorithms in the realms of ethics, law, politics and society. thanks, stephen for underlining the function of algorithms in current approaches of disease surveillance in global health policies. in addition, i pose that the rationale of an 'emerging diseases world view' (king ) is similarly influential for public health surveillance on a domestic scale. especially in the us, systems of syndromic surveillance have been explicitly employed to answer the challenges of the 'next pandemic' with a new algorithmic form of public health monitoring. going a bit into the details of one specific example of syndromic surveillance, i want to illustrate the problem of "knowledge without truth" stephen mentioned above. the argument is that those systems 'call back' in several ways, influencing both truth claims and practices of public health provision. my empirical example is a study on the use of the "electronic surveillance system for the early notification of community-based epidemics" (essence) in the u.s. national capitol region, an application of syndromic surveillance that received considerable attention as a pilot project (füller ) . technically the essence system provides the server infrastructure to draw together diverse data-sources that are considered indicative for public health. its 'syndromic' approach consists in the integration of several so called surrogate data, signals of diseases or public health problems generated before a confirmed medical diagnosis (velasco et al. ). in the case of essence, such surrogates are for example emergency department chief complaints, daily over-the-counter sales of the two big pharmacy chains cvs and rite aid, reports on absenteeism data gathered from public schools and others. essence claims to provide an unmatched situational awareness partly due to the near real-time nature of those data (collected and reported at least daily). given the amount and unstructured nature of this data, the system employs algorithms to continually search the gathered data-stream for unusual patterns and a gui to visualize and map resulting alerts. if there is an unusual coocurrence of for example the sale of headache pills and school absenteeism in a region, the system will flag out a warning. importantly, the base for this pattern recognition are at no times diagnosed health problems but assumptions generated through the association of different data sets. eventually the system promises to automatically provide an early notification of any unusual public health event before it has been medically diagnosed (fearnley ) . the turn towards infection control and surveillance in public health and the introduction of syndromic surveillance systems have both been contested early on and from several vantage points (reingold ) . the focus here is to point out the performative character of technologies and their related practices in altering the goals and modes of public health provision. my argument centers on the fact that the system is constantly producing health related truth claims. whether it is just quietly monitoringas it does most of the timeor in the rare cases that it is flagging out a public health emergency, the system claims a certain truth about the health of the monitored population. in both cases, the algorithmically produced knowledge becomes performative in different ways. both forms of truth claims illustrate the 'knowledge without truth' problematic stephen already mentioned. on the one hand, those systems introduce a new expectation and a demand to constantly assure the normal state of affairs. new technologies of surveillance are employed to be able to illustrate an absence, to be able to constantly assure that there is nothing to worry about, as kezia barker argues (barker ) . in order to be aware of unusual events, resources, work and infrastructure are invested to extensively monitor the routine state of public health. but this additionally generated knowledge does not provide a qualified, actionable truth about the state of public health. trying to see short-term events, those systems measure against the baseline of the 'normal'. in its usually quiet mode of monitoring, the systems make the implicit claim about a 'normal' , 'well functioning' , 'unproblematic' state of public health, ignoring any long-term and structural health issues. on the other hand, in the case of actually flagging out an incidence, automated monitoring systems such as essence are problematic in their rendering of disassociated facts into medical truths. the threat of an emerging public health event is especially burdening for the executive branch of the local state. decision-makers are pressured to act early, at best before the expected cascading of an infection gets out of control. this expectation makes it tempting to base a decision on the syndromic signals as they are readily available and -through the included mapping tool -often clearly localized. while those signals are explicitly handled as an additional but clearly undiagnosed source of information among epidemiologists and public health experts, for the executive branch they have a tempting appeal of providing a near real-time situational awareness and as such an actionable grasp on the emerging public health event. importantly, using essence as a base for decision-making approaches the signal as if it was an authoritative medical fact instead of just an indicator for the clustering of certain syndromes. the danger of misinterpretation as the algorithmically generated knowledge travels contexts may result in wrongly employed public health interventions with negative social effects. besides the problem of false positives prevalent to those systems (fearnley ) the system always suggests a spatialized source of the problem that may or may not be medically justified. employing public health interventions based on those seemingly objective and localized realities can easily mean the wrong allocation of scarce resources and attention or effect an unjustified stigmatization of a 'problematic' area. from my perspective, as a medical infectious epidemiologist and public health expert who advises on the development of new surveillance systems and who constantly needs to be aware of their effectiveness as well as the consequences of their use, stephen and henning's analyses offer extremely important contributions on how to think about and evaluate increasingly digitized health-and infectious disease control. to add to this, i would, in the following, like to make a few further comments about the epistemic and political aspects of the digitization of infection control. in particular, i am able to speak to activities and experiences at germany's national public health institute, the robert koch institute (rki), and to those at the world health organization (who), where i was within the framework of the west african ebola outbreak ( to ) (owada et al. ) . first of all, i agree with stephen's analysis that there is the risk, as a result of the successive propagation of algorithmic approaches and technologies for infectious disease control, an epidemiology traditionally based on diagnostic findings and controlled statistical processes is becoming increasingly marginalized and, in parallel, the necessary verification loops are being replaced in favour of 'big data' ideologies and trends of dataification. in this context, it seems to me that widespread assumptions that advance the idea that a digital, unofficial infectious disease surveillance and monitoring is quicker than traditional, official information and reporting systems need to be modified. it is true in retrospect that existing digital systems and their associated early warnings could have been faster if their first signals had been correctly named or interpreted at an early stage. at the same time, however, it is mostly ignored that even official state authorities often have knowledge about specific events at relatively early stagesonly that they either initially withhold such information or distribute it in other ways according to the official information/notification systems, e.g. the example of stephen, the authorities of guinea were aware that there was something going on, but they waited with the reporting. so have i experienced it at the who: few countries directly provided all available information to the organization. it can also be observed again and again that official information either minimizes or plays to the media or other entities in a targeted way. in this context, non-state surveillance platforms such as promed or healthmap, for example, should be commended especially for their dimension of political transparency, as they put pressure on governments not to keep information from the public as much as possible. at the same time, however, the increasingly digitized identification, analysis and distribution of epidemiological indications of infectious disease these platforms enable not only leads to increasing likelihood of false positives, but also to specific problems of an immediate, uncontrollable communication of risk. the danger of panic and the great effort required to avoid panic are to be feared. henning provides very important information in this regards. he describes that specific public health actors (here: local health authorities) may be compelled to equate technologically-generated signals with epidemiologically certified public health events, and, on the basis of these unproven indications, initiate public health measures. further, this is also a problematic development from the perspective of resource retention in an already thinly-resourced public health service. early responses and over-reactions from political decision-makers or the media are to be feared in equal measure. in the broader context of the focus and framework of a 'global health security' , such potentially exaggerated perceptions and reactions are tied to perceptions of elevated threats of infectionwhether from (quasi) natural or man-made infection (e.g., in the context of war or incidents of terror)and, consequently, to urgent demands for comprehensive and constant attention, outbreak detection, and further crisis/disaster preparedness measures. as a result of this, infectious disease epidemiology is increasingly being, in my humble opinion, in an irritating way integrated into the national and international security architectures. so it was during the west african ebola outbreak in sierra leone and liberia, two of the three hardest hit countries, that the military was constantly present in the planning of public health measures. e.g. in one situation in sierra leone i remember this resulted in prioritizing quarantining over other public health measurements like community engagement. quarantining is not per se negative but in this particular case turned out very ambivalent as the measure evoked strong resistance among the population and potential new infected individuals increasingly were actively hidden as a consequence. also in non-outbreak times, the cooperation between security forces and public health entities is becoming increasingly narrow. this can be seen, for example, in the global health security initiative, which addresses both the biological threats of pandemic flu and possible threats from chemical or radio-nuclear terrorism. these perspectives as well as the social aspects and subsequent costs of a digitized infection control should be discussed. they are closely linked to the imperatives of constant monitoring and early detection, as well as the similar focus of a 'global health security'. as such, they should be considered with the view not only to the (not new) anticipated restrictions or marginalizations associated with classical, structural and also socially-reformed, areas of public health, as well as to further possible negative costs resulting from of a 'securitized' public health. i would like to hear from you -stephen and henning -especially with regard to these broader health and social policy debates, from your social science perspectives, what is your understanding of 'global health security' in general and of the 'preemptive security logic' , which is often discussed in this context, especially? henning i would suggest to understand "global health security" as a set of preferences and truth claims that are currently framing our understanding of health issues of international relevance. this understanding results from a perspective, that interrogates threat discourses and related policies as a structured but contingent formation of problem descriptions. problems do not exist 'naturally' but they have to be articulated and put on the agenda in a process of social interaction. this approach draws back to michel foucault and his proposal to acknowledge a power/knowledge nexus in general and specifically the power effects of truth claims. according to this, articulating and framing an issue are powerful ways to predetermine the range of thinkable approaches and solutions. by using the term "global health", policy-makers, non-governmental actors and academic observers are drawing together several health problems into a common frame, but also marking this frame as a field of intervention and claiming its relevance. the contours of this frame are still blurry and there exist numerous approaches to define "global health" (brown et al. ; farmer et al. ; fassin ) . there is no accepted definition and "global health […] is more a bunch of problems than a discipline". (kleinman ) the ongoing emergence of a problem field "global health" is an interesting moment then, where new truth claims are put forward and a new understanding of related issues such as 'health' and 'the global' are formed. those newly related ideas are powerful as they are confining the agenda setting and plausible goals and methods of intervention. approaching "global health" from this angle, what is striking from the outset is a strong undercurrent of security. the recent surge of "global health" can be attributed to a confluence of two separate discourses. on the one hand, globalization is increasingly narrated as a health risk. an "emerging diseases" discourse paints the picture of a global spread of infectious diseases due to unparalleled levels of global connectivity and frequency of global travel (barrett et al. ). on the other hand, the concept of national security is being reimagined, facing a new multi-polar and complex world order. today, in order to achieve national security, one has to look beyond military dominance and to take societal issues such as health, poverty but also climate change as security threats into account (redclift and grasso ) . for example in the us, facing the threat of bioterrorism, public health has become a concern for the department of homeland security and international infection control resurfaced as a security issue. both the fear induced by 'globalisation of disease' and the rethinking of national security are underlining a new relevance of global health issues. the resulting tremendous development in global health policies and programs accordingly are often following a security rationale (genest ) . one example is the newly installed global health surveillance mechanisms and the revised international health regulations (ihr) (fidler ) . the recent conception presents global health as part of a security problem rather than as a humanitarian issue. this securitization of 'global health' has already been described in some detail (cook ; king ; pereira ) . here i want to underline the corresponding shift in the perception of threats and its implications. current problems of global health security are often depicted as essentially incalculable. emerging diseases, acts of intentional bioterrorism, food security in an increasingly global connected distribution system, antimicrobial resistant agents, − more than ever we now seem to be confronted with "unknown unknowns". we not only do not know when those events will happen, but we even do not know what the threat is exactly. the reformulated international health regulations (ihr) tellingly have shifted from monitoring a fixed catalogue of diseases to the obligation to warn about anything unexpected. according to the ihr, the national health agencies have now to signal any unspecific "public health emergencies of international concern" (world health organization ) to the who. this specific perception of "global health security problems" as incalculable threats calls for a certain pre-emptive and outbreak-oriented intervention. the implications of the employed "preemptive security" logic have been detailed in critical security studies (de goede and randalls ; lakoff and collier ; massumi ; caduff ) . as those studies have shown, preemption often demands the extension of (technological) surveillance and orients efforts towards the event and away from structural conditions. comparable tendencies have been shown for current "global health" policies, for example an orientation towards containment of an event rather than the search for a broader structural prevention (rushton ) . to sum it up, i would argue that global health is currently presented as a problem and has been put on the political agenda in a way that calls for a very specific answer in the form of a "preemptive security logic". firstly, the underlying truth claims about the problems to solve frame the emerging field of global health partly as security issue. secondly, the incalculability problem evoked in many threat discourses of current global health thinking demands a certain security rationale. the problem of an unknown unknown has to be dealt with preemptively. this way of presenting the problem of global health then implicitly constraints plausible interventions. approaching health as a security issue does often not tackle the actual problems of health on the ground. for example, this approach inclines to invest scarce resources into monitoring and surveillance rather than education and local health infrastructure. in order to reach the goal of more substantial health policies it is important to be aware of this securitization bias in the current problematization of global health. building further on excellent points articulated by henning, this epistemic shift in government and politics towards 'global health security' has been resultant, as i argue, from significant larger geopolitical transformations, and new reconsiderations of security perspective, in a post-cold war era of rapidly proliferating non-traditional security challenges, which extend beyond traditional security correlations of the state/military, are transnational or global in scope, and again, to underscore the centrality of henning's earlier points, which cannot be prevented entirely, only addressed through coping mechanisms and the development of techniques of preemption and forecasting (caballero-anthony ). the rise of global health security and its securitizing processes have transformed the ways in which international relations and global politics are understood, orientated and practiced. in the united nations security council (unsc) adopted resolution (unsc ) which emphasised that the current hiv/aids pandemic, if unchecked, posed a risk to international security and stability, marking the first time in which a health threat was discussed before the un body mandated to maintain international peace and security (fidler ) . years following the seminal security council resolution on hiv/aids, the united nations launched its first and only to date, military mission to combat the spread of an infectious disease outbreak. known as the united nations mission for ebola emergency response (unmeer), the first ever un emergency health mission sought to contain the spiraling west african ebola outbreak following the un security council resolution , which determined that the ongoing outbreak in west africa 'constituted a threat to international peace and security' (unsc ), and we can understand these grand transformations within global politics and international relations as permeated by emergent logics to preempt both occurring public health emergencies and also probable future pandemics. contrastingly, for critical theorists, global health security has emerged as a concept which denotes a novel biopolitical project, or rather, the appearance of a new governmental problem in public health: how to effectively manage 'emerging infectious diseases' at a global scale (lakoff ) . contemporary global health systems are therefore problematized not only by the rapid emergence of pathogens on a global scale, but the risk posed by these circulating pathogens are no longer calculable using tools of risk assessment, which are based on patterns of historical incidence (ibid). in this regard, global health security rationalities, i assert, galvanise and accelerate the facilitation and development of novel techniques and practices of anticipatory or preemptive security, which emphasise the real-time, continuous and cost-effective surveillance of potential disease outbreak and public health emergencies. increasingly, in an era of innumerable digital data sources, the preemption of health risks are managed and analysed via an assemblage of innovative and evolving surveillance practices which combine multiple data sources and disease-tracking techniques, enacted at local, regional and global levels. syndromic surveillance platforms, and digital epidemic intelligence systems including promed-mail, gphin, healthmap, bio-caster, epispider, and the now-defunct google flu trends can thus be conceptualised as new governmental technologies of overarching global health security practices, developed and installed around yet unforeseen events in order to halt or preempt the 'sudden, circular bolting' of pandemic phenomena (foucault ) . collectively then, in my view and building upon the expert points provided by henning, processes of securitization of global health and the rise of preemptive security logics have advanced calls for the deployment of novel security technologies and surveillance apparatuses over the past two decades. these calls have been met with the re-drawing of disease surveillance operations and the launching of new technologies which now seemingly patrol digital datascapes in the surveillance of potential public health emergencies. such novel technologies constitute critical components of an evolving ensemble of new governing practices, knowledges, techniques and rationalities of health security, increasingly influenced by digitised, automated and computerized algorithms. . as components in an emergent socio-technological apparatus of security for the strengthening of global health governmentalities, it is also crucial to consider the ways in which these expanding digital syndromic surveillance systems re-contour previous understandings of the temporalities, form and practice of preemption in the identification of forthcoming pandemics. firstly, the rise of syndromic surveillance technologies for the forecasting of probable disease outbreaks, departs significantly from previous methodologies to identify and further preempt pathogenic threats. as seen with the steady integration of algorithmic programming over the past two decades from promed-mail, to gphin, and to healthmap, syndromic surveillance technologies increasingly draw upon and aggregate open-source data pulled via algorithmic processing from the realm of the digital to inform contemporary practices of health security in the non-digital/physical world. within the politics of preemption, this marks a novel transition towards the harnessing of infinite online data sources, afforded by increasingly sophisticated algorithms to identify unusual data correlations or patterns indicative of a potential disease outbreak. in turn, this represents a process that is distinct and divergent from previous methodologies of health surveillance which utilised clinical and laboratory testing, analysis, observation, and the collation of statistics in order to render visible and intelligible, occurring or emergent infectious disease outbreaks. in the new era of digital disease surveillance, the data warehouse emerges alongside the traditional clinic as a new critical site of surveillance and zone of security praxis in the preemption and surveying of disease risk. further to this, novel techniques to preempt looming pandemic threats via these digital syndromic surveillance systems now also correspond with new problematizations of data and knowledge forms in the securitization of uncertain [pathogenic] futures. unlike previous systems of infectious disease surveillance which were routinely marked by an incompleteness of data in which to understand forthcoming pandemic risks, the deluge of 'big data' of the early twenty-first century has now reversed this problematization of data.. contemporary digital disease surveillance systems and the practice of health security are no longer hindered by a scarcity of data but rather burdened by an excess of infinitely generating, unstructured and diffuse streams of digital data. in order then to preempt and track the emergence of disease outbreaks in a present world that is submerged in data sources, digital disease practices must navigate, as matteo pasquinelli ( ) writes, 'vast data oceans' to detect that which constitutes the anomaly, be it common patterns of behaviours in social media, buying or selling tendencies in stock markets, the oscillation of temperatures in a specific region, or suspicious keywords in disease surveillance networks (ibid). again, in this new practice of 'navigating vast data oceans' , the digital algorithm emerges once more as a strategic, pragmatic and celebrated technology of government with the capacity to apprehend, process and project new insights of disease patterns from troves of digital data which manifest beyond human cognitive and analytic capacities. thus, the politics of preemption in the present era of elevated pandemic threat are intimately intertwined with expanding recourses to apprehending big data sources and employing algorithmic processing techniques to produce advanced alerts, indications and insights of potential pathogenic uncertainties. indeed, during several critical public health emergencies over the past two decades, a combination of big data sources and algorithmic techniques produced meaningful and advanced insights into emergent public health emergencies, including during the early and critical stages of the emergence of severe acute respiratory syndrome (sars) in china and ebola in guinea. however, the success and rise of the algorithm in these health histories should not distract from the imperative for continued meaningful-and indeed critical investigations and interrogations of emergent digital disease surveillance practices which utilize diffuse big data sources and processing of such data streams via algorithm. algorithms are not only famously opaque, but have also been shown to be cantankerous, if not delicate technologies, illustrated famously by a false reporting of a cholera outbreak in the united states by google in , as a result of oprah winfrey picking love in the time of cholera as book of the month in her book club (simonsen et al. ). however, as technology and innovation advance, algorithms are getting smarter, more insightful and more precise, but the growing commonplace of these knowledge producing machines with intensifying technical complexities makes the monitoring and regulation of these data-processing technologies ever the more urgent and vital. the ascendancy of the era of big data and the rise of digital disease surveillance systems have afforded unprecedented new opportunities towards the enhancement and bolstering of disease detection capacities in an era increasingly preoccupied with the emergence of future security challenges-among them pandemic illness. the objective of this discussion has been to provide an overview and highlight the potential gains and benefits yielded by these new data sources and processing techniques, while also emphasising that key ethical, legal, political and societal concerns abound and must not be sidelined in contemporary efforts to accrue maximal data reserves and to effectively track and detect the next pandemic before it occurs. tim dear stephen, dear henning, thank you very much for this inspiring conversation. again, it made clear the necessity of an interdisciplinary and social sciences inspired debate about contemporary epidemiology and public health. for me three insights emerge. first of all, the gains in timeliness and scope of digital epidemiology come at the cost of providing a different type of knowledge. the information provided through such systems is not the same as the traditional expert knowledge based on human assessment, analysis, hypothesis, statistical testing and trials but an algorithmic 'knowledge without truth'. the status of this knowledge may not be totally clear in all the different contexts where it is used. this may result in ill-informed decision making. a driving force for the demand of digital epidemiology is a reformulated conception of global health. a common thread running through the diverse debates about global health policies today is the issue of security. this securitization of global health does frame current policies. specifically, threats to global health are increasingly identified as incalculable emergencies (unknown unknowns). this results in a demand for preemptive ways to act on those emergencies before they have evolved. this preemptive security logic also fosters an unlimited big data surveillance as a practice of 'navigating vast data oceans'. for sure these points need further critical examination. thus i am looking forward to future interdisciplinary exchange and discussion. biosecurity: securing circulations from the microbe to the macrocosm emerging and re-emerging infectious diseases: the third epidemiologic transition the world health organization and the transition from "international" to "global" public health non-traditional security challenges, regional governance, and the asean political-security community (apsc) cook ah. securitization of disease in the united states: globalization, public policy, and pandemics. risk, hazards crisis public policy precaution, preemption: arts and 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infectious disease surveillance in the big data era: towards faster and locally relevant systems security council resolution on the responsibility of the security council in the maintenance of international peace and security: hiv/aids and international peace-keeping operations unsc ( ) security council resolution social media and internet-based data in global systems for public health surveillance: a systematic review. the milbank quarterly world health organisation. fifty-eighth world health assembly resolution wha . : revision of the international health regulations. geneva: world health organization tim eckmanns thanks klaus scheuermann for intellectual input. german federal ministry for education and research (förderkennzeichen: gp ). authors' contributions all authors contributed equal to the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - wtyk r authors: sabroe, ian; dockrell, david h.; vogel, stefanie n.; renshaw, stephen a.; whyte, moira k. b.; dower, steven k. title: identifying and hurdling obstacles to translational research date: journal: nat rev immunol doi: . /nri sha: doc_id: cord_uid: wtyk r although there is overwhelming pressure from funding agencies and the general public for scientists to bridge basic and translational studies, the fact remains that there are significant hurdles to overcome in order to achieve this goal. the purpose of this opinion article is to examine the nature of these hurdles and to provide food for thought on the main obstacles that impede this process. it has achieved much, including the sequencing of the entire human genome, but it is already under serious attack for its failure to deliver effective therapies in many areas. this opinion article will provide a subjective view of our understanding of translational research, identify obstacles to its successful development, and propose a series of initiatives to improve the effectiveness of translational research strategies. originating from latin, translation means to 'carry across' . in biomedical research, the goal of translational science is to develop a thorough operational understanding of the human organism in health and disease, with the goal of 'carrying across' this knowledge to alleviate disease and suffering and to improve the quality of human existence. to be translational in medicine we must acquire knowledge from the broad arena of molecular and cellular biology and then apply this knowledge to human disease. the quality of our scientific output (perceived as a change in disease incidence and/or the development of a therapy) is largely dependent on the quality of the input data and the methods for their processing and interpretation, although the process of generating effective translational science is not as linear (that is, from molecules to models to humans) as is often thought. failure to ask the appropriate questions of optimized systems leads to the acquisition of knowledge that might be less relevant than anticipated. further corruption of the process comes as a result of limitations in our models, which are often not fully appreciated (or are simply ignored) at the time of the study. additionally, grant agencies must be sufficiently flexible to allow researchers to follow up on novel observations because many of the most exciting developments arise from unexpected findings. determining what research is intrinsically translational, or has been translated effectively, is therefore surprisingly difficult, and in a healthy global biomedical research environment, translation will continue to mean very different things to different groups. few scientists will see a process through from the conception of a hypothesis to the development of a specific medical therapy. as scientists, we are nonetheless required to measure our performance in terms of our 'translational potential' , particularly when it comes to justifying and generating funding and publications. therefore, in the absence of being able to measure contributions to health directly, we often quantify individual success using surrogate markers (such as publications and their impact, prestige, and funding), which depend on the prevailing concepts of what constitutes importance. however, these markers may be flawed for this purpose. a more global assessment of the output of translational science might consider whether the scientific community has improved specific disease outcomes, quality of life or life expectancy. specific examples of global success might not be as common as we would wish, but increased successes in the areas of organ transplantation or the ability to eradicate diseases such as measles virus highlight our ability to be successful. the improved treatment of many cancers through the combination of good science and high-quality clinical trials of new therapies or combinations of therapies has been strikingly impressive and provides many examples of highly effective translational science , . there remains, however, a lack of available mechanisms by which to relate our individual contributions to the global progress of translational science, and many factors conspire to impede our progress. the translation of basic scientific research faces a myriad of hurdles, both obvious and occult. these revolve around our understanding of the nature of the translational process, the integration of the outputs of different technological approaches to disease, the use of models, access to tissues and appropriate materials, and the need for support in increasingly complex areas such as ethics and bioinformatics. in addition, owing to technological advances, well-meaning safeguards of personal privacy have been imposed in relation to carrying out research in humans, and these have, in fact, greatly impeded progress in translational studies. problems with the models. entirely appropriate restrictions on what research can be done in humans have contributed to the status quo in which the mouse has become an indispensable model for translational biology; however, it is often not possible to predict biological responses in humans accurately based solely on results obtained from animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . within immunity, exposure of mice to their own species-specific commensals and pathogenic organisms might contribute to a species-specific immunological phenotype , that affects the translational relevance. for example, studies of the effects of irak (interleukin- receptor-associated kinase ) deficiency have revealed increased identifying and hurdling obstacles to translational research susceptibility to a greater range of pathogens in mice than is observed in humans who are deficient for irak . where good potential translational concepts are generated from models, determining how to move to the human can be challenging, as highlighted by difficulties in calculating tolerogenic doses of insulin in the prevention of diabetes . in addition, technical limitations, or the dominance of prevailing models, can sometimes limit the scope of in vivo work, as illustrated by studies of airway disease, where studies in mouse models are focused predominantly on the important t helper (t h ) component of asthma but are poor models for the contribution of other mediators. mouse models are also complicated by our limited understanding of the phenotypes of human diseases , . designing perfect models of diseases that we do not fully understand is a tall order, and chronic diseases that involve life-long interactions between the host and the environment present a particularly difficult challenge [ ] [ ] [ ] [ ] . developing truly chronic models of disease is, however, heavily militated against owing to ethical concerns about prolonged suffering. moreover, the typically short-term nature of research funding, where outputs must be deliverable at a reasonable cost and within the time frame of a project grant, hinders the development of chronic models of disease. it is equally clear that work on a single cell line is often poorly predictive of the behaviour of the whole organism. although the use of primary cells from normal tissues can overcome some of these problems, the phenotype of these cells might be altered by their removal from the microenvironmental influences in vivo, and they often show markedly different responses to those of primary cells that are obtained from diseased tissue. therefore, rodent models remain essential and cannot be replaced by in vitro approaches at present, but are an imperfect translational conduit for both biological and practical reasons (such as the difficulty in establishing chronic disease models). other models such as flies and zebrafish (danio rerio) have many advantages for forward genetics and other related studies, but have substantially greater differences to humans than mammalian models. primates are the most compatible mammalian models for immunological research, and have provided invaluable insights into diseases such as hiv, but their broader use is not feasible because of major ethical concerns, as well as other practical and cost-related issues. even these models can have limitations. recently, differential expression of siglecs (sialicacid-binding immunoglobulin-like lectins), which are inhibitory receptors thought to downregulate immune-cell activation, has been noted between apes and humans , and such differences in immune responses between species require careful consideration, as highlighted by the development of a 'cytokine storm' when individuals received an agonistic cd -specific antibody that did not show the same effects in monkeys , . research is being increasingly hindered by bureaucracy. evident at many levels in research management, nowhere is this more of a problem than in the areas of access to human samples and tissues, and in the establishment of clinical collaborations. liaison with industry also provides additional challenges . furthermore, some of the most exciting areas of current research, such as work with embryonic stem cells, are subject to special ethical concerns. even the simplest research involving humans, or archived specimens from humans, is often encumbered by a multistaged, intimidating application process that might dissuade individuals from carrying out the proposed study. overcautious interpretation by ethics committees and regulatory authorities of what is ethical results in restrictive practices that can, for example, prevent re-screening of dna banks for relevant markers and mutations, or apply over-elaborate protection of clinical phenotype data. although it is absolutely clear that robust systems are needed to prevent exploitation of patients or clinical data, it is often unclear how the ethical standards that are applied to the review of proposed studies are developed. apparently arbitrary standards that might be perceived to be more ethical simply because they are more restrictive seem to be ever more on the increase. it is also unethical to excessively hinder research, but this imperative sometimes seems to come second to apparently minor scruples over details such as the methods of recruitment. although the introduction of national standards to ensure timely review of proposals has, in the united kingdom, begun to address these issues, increasingly complex review processes are frustrating and sometimes of questionable ethical value. the area of personal privacy is particularly complex. the majority public view is supportive of the use of some personal data in confidential research . however, although many individuals are less concerned that well-regulated data usage is an invasion of privacy than some ethics committees seem to be, due consideration is needed of the views of the minorities who are more concerned with their privacy. the future potential for apparently confidential genetic research resulting in the identification of individuals through the cross-referencing of dna databases is clearly of concern , . in addition, governments tend to respond to highly public episodes of research offences or clinical misconduct with legislation. this runs the risk of creating additional administrative burdens for the majority of individual scientists who practise ethically and well, without ensuring the prevention of future wilful misdeeds by a rare minority. increasingly, clinical practice is becoming more defensive and less willing to engage in research as a response to a society that is becoming more risk-adverse, litigious and blame-centred when adverse events occur. this generates further barriers to research in humans and requires a frank reappraisal of what experimentation is acceptable in humans or on samples that are derived from humans. additional problems arise in setting research priorities. without effective public input, we run the risk that research priorities become the whim of changing government focuses, which lack the stable, long-term input necessary for meaningful scientific advancement. although charities and private foundations that are centred on specific diseases provide important contributions, their focus is often too narrow to tackle the wider research agenda. this has led to a culture of increasing financial pressures that are governed by short-term policies, which require rapid outputs, and piecemeal funding, stifling longer term, more open-ended research that might produce real translational progress . for example, political pressures to study agents of bioterrorism or emerging infectious diseases such as sars (severe acute respiratory syndrome) can be welcomed and are clearly important; however, more long-term commitment to studying infections of historical global importance is also essential. with respect to developing long-term goals, the national institutes of health translational biomedicine must be seen by scientists and the wider public in the context of a broad-based body of science that has the potential to contribute, in the near or long term, to the advancement of human health. increased financial and political pressures on scientists to maximize directly measurable patient benefit from research carried out over short time frames runs the risk of destroying a healthy science base and should be actively resisted. a continued and robust engagement of the public is mandatory for scientists, because unless the public values the broad sweep of translational biomedical science, funding will inevitably be diverted away from science or applied only to those areas of research that immediately influence patient care. as scientists, we must learn to articulate not only the promise of science, but also the difficulties that are associated with moving an idea along to the product stage, so that unrealistic expectations of perfect medications for every disease in short time frames are not raised. we must also wrestle the debate on the usefulness of in vivo models away from the more extreme ends of the anti-vivisection movement so that a constructive, rather than confrontational, discussion can evolve. equally, we must foster a culture of research in the curricula of medical students, and promote the goals of translational research to basic science undergraduate and graduate students. we must seek to marry the skills of basic scientists with those of clinicians to convey the idea that strong translational research underlies improved health and is inseparable from the provision of good standards of clinical care. improving our use of the models? in many biomedical fields, a series of debates have highlighted deficiencies in our current in vivo models. strain differences and variance in experimental conditions between research groups pose major challenges in comparing the results derived from different studies. a collaborative investment in phenotyping human disease by clinicians and basic scientists is required for developing robust models. there is clearly an opportunity to define the characteristics, behaviour and relevance of model systems, particularly with a view to standardizing optimal strains and protocols. for example, multiple models exist for common diseases such as asthma and rheumatoid arthritis , although harmonizing protocols and strains for particular features of a disease must avoid the loss of sufficient diversity in order to allow the finding of the unexpected. this challenge could be met by interest groups or individuals working in a particular area, by large national funding agencies and/or international funding initiatives, or alternatively by specialist societies. such debates might facilitate the comparison of data between laboratories and between species, and might highlight the components of specific diseases that are ripe for the development of new in vivo models and protocols (for example, there remains a great need to more effectively model the role of the innate immune system in acute and chronic asthma), broadening the number of disease processes or phenotypes that are modelled in pathology. although much work will continue to focus on the mouse, for some diseases comparative or independent studies in other species will continue to be important. beyond the scope of this article, there are issues ahead with respect to subject selection for early clinical trials and the development of individualized treatment regimes that are based on pharmacogenetic and individual disease phenotypes , . figure | developing interactive models. a | in vivo experimentation, perhaps in particular the generation and characterization of knockout mouse strains in experimental models of disease, is often viewed as the gatekeeper between in vitro science and the generation of drug targets that are appropriate for human disease. although central to an effective understanding of immune biology and the role of new candidate drug targets, the predictive value of in vivo experimentation is less than desired, particularly in the context of studies of single knockouts in specific disease models and mouse strains. b | an example of a more holistic network in which multiple lines of evidence allow the refinement of objectives and target relevance in order to increase the chance of successful drug discovery. such approaches reflect the approach of many researchers, but (acknowledging that no branch of science is 'easy') the main difficulties associated with undertaking human-based research run the risk of degrading the quality of data that arise from an integrated scientific approach. the tendency to view translational research as a linear process in which mice are the gateway between basic science and humans does rodent models a great disservice. in particular, the classical route of identifying genes in vitro, followed by generating knockout animals in vivo, has, in general, been poorly predictive of the consequences of targeting specific molecules in humans. advances in medical sciences that have emerged in this manner are vastly outnumbered by those that arise by serendipity or through less predictable routes. a more holistic integration of in vivo models with in vitro science and studies in the human is needed when summarizing the translational relevance of a system, in which in vivo models contribute strongly to an iterative strategy, but are not themselves the final arbiters (fig. ) . the potential to identify medicines for use in human disease by screening less complex biological systems has long been recognized in the pharmaceutical industry, and there is now considerable interest in the use of model organisms such as caenorhabditis elegans and zebrafish in high-throughput screens for new drugs , . it is becoming increasingly evident that these systems can be used to identify therapeutic targets in the immune system . in this way, understanding the biology of pathways and gene products is deferred, and the ability of a compound to intervene in complex biological processes is directly tested, as highlighted in recent studies of calcium-channel antagonists in c. elegans . combining the use of mice and humans in effective strategies. it seems self-evident that research which aspires to influence pathogenesis in humans needs to be carried out on the human system. at the same time, we must also anticipate the potential for doing harm that might accompany any new approaches to treating diseases, which militates against a rapid progression to phase i human trials. nonetheless, the extraordinary difficulties that are associated with carrying out human studies in parallel with animal models has facilitated a culture in which such studies rarely occur, and in which prioritizing research is not driven by the inclusion of such processes. indeed, almost quixotically, work in human tissues and cell lines is sometimes not deemed to be of importance until verified in a mouse with a targeted mutation. bridging this divide requires the scientific community to value more highly the studies that seek to bring mouse and human studies together, and to appreciate that in human studies a lack of phenotype or subtle modifications of processes might be as important as models that generate dramatic phenotypes. from simple co-culture models of normal human tissues and ultimately to the generation of whole organs or representations of whole organs in the laboratory, we are now beginning to produce in vitro human systems that can complement essential work that is currently only achievable in vivo. increasing success with new gene-delivery systems, combined with new technologies such as gene knockdown by rna interference, might allow us to overcome the inability to study humans with targeted gene deficiencies. such models are in their infancy, and cell-culture-based approaches are, in many scientists' views, farther from human biology than techniques that investigate biology in mice. it would seem that both are required, and therefore, a major thrust to develop standardized co-culture models that are based on primary human cells from healthy and diseased subjects would provide a complementary scientific base to our strong expertise in the mouse. our commitment to this development is essential, not only to boost the translation of science to the human, but also to ensure that we honour the principles of reduction, refinement and replacement (minimizing the number of animals that are used and finding alternatives wherever feasible) that are central to all animal experimentation. we are faced with many difficulties in generating appropriate human tissue models, including defining differences in similar cell types that are pathologically relevant (for example, comparing the biology of endothelial cells isolated from the umbilical cord with those isolated from different microvascular beds). we also need to determine the optimal representative culture conditions for primary cells. for example, when should epithelial cells be studied at an air-liquid interface, or when should leukocyte-endothelial interactions be studied under flow? importantly, diseased tissues are often modified by inflammation, genetic traits and epigenetic processes that require further consideration when translating from the biology of health to the biology of disease. nevertheless, we have recently shown that simple co-cultures of primary human cells can in some circumstances replicate inflammatory systems that are observed in vivo in mice , ; such co-culture models are becoming increasingly common. the future ability to study systems that resemble organs or complete tissues, and verifying the work of simple co-culture experiments in such systems, offers a future we should not only embrace, but also be actively working towards at every opportunity. it is routine in clinical practice to combine drugs with similar or differing modes of action when treating disease. there is also increasing appreciation of the roles of cellular and molecular networks in the aetiology of disease , a fact that is implicitly recognized by the need for in vivo models in which complex interactions between tissues and cells can be studied without a need to identify the full panoply of the systems involved (box ). in the context of an inflammatory disease, we have recently proposed that these networks are best described by the terminology of 'contiguous immunity' , whereby, in disease, temporally and spatially contiguous networks comprising multiple processes of innate and adaptive immunity are in continual dialogue and evolution . it is curious, then, that so many studies aim to land a single 'killer blow' on pathological processes, rather than considering the impact of therapeutic combinations. our increasingly deeper understanding of the complexity of the inflammatory process often results in the targeting of downstream components for which there might be redundancy or very specific functionality. the targeting of such systems in combination, although complicated to achieve and less satisfying with respect to the identification of single clear targets, is nonetheless likely to have relevance for successful translation. allowing access to tissues and overcoming ethical anxieties. the diversion of research funding into bureaucracy and the delays in productivity that result from meeting statutory requirements for ethical practice contribute to the erosion of research capacity. research cannot do without governance, but it is disappointing that increasing regulation has not been met by increased support for ethical human-based research, and www.nature.com/reviews/immunol the future ability to study systems that resemble organs or complete tissues … offers a future we should not only embrace, but also be actively working towards at every opportunity. although many processes conspire to make research harder, few exist to make it easier. a simplification of the regulations and national-level funding that provides templates for ethical applications, together with training and support for investigators, would do much to make this easier. because many projects involve international collaborations, simplified standard international regulations would also greatly facilitate progress. many research projects have conceptually similar goals (such as the tackling of inflammation in a tissue), and generic pre-approved protocols requiring modest local adaptation would obviate many problems. in the united kingdom, the proposed establishment of a single health research board to coordinate government-funded health research provides an opportunity to develop programmes that examine the effects of government regulation on research and mechanisms for overcoming the issues discussed earlier. research on human tissues could be enhanced by improved access to normal and diseased specimens. the establishment of central and devolved services whose function would be to source human tissues in an ethical manner, characterize the donors and tissues anonymously, and make these resources freely available to investigators in research-active countries would transform biological research (indeed, some progress is being made here, but there is still much to be done). an emphasis on dialogue with scientists and flexibility with respect to supporting science would be required to underpin access to these resources by researchers. where materials are scarce, such systems could be supported by expertise in cell-line immortalization. the development of the lung tissue research consortium by the national heart, lung and blood institute is an exemplar of a clinical data and tissue bank that offers enormous potential for research in lung disease; in addition, the development of the uk biobank shows it is feasible for human resources to be sourced ethically in ways that allow relatively broad use in medical research according to need. the proposed tissue banks would promote a positive view of human science working in parallel with mouse biology, which also requires commitment and support from the public to make it work. 'omics'. one area in which large-scale biology is excelling is the use of public-access databases (such as those pertaining to gene expression (genomics) or the production of metabolites (metabolomics)) , but we are missing opportunities to further expand these databases. local and national guidelines define the optimal management of many diseases, but it can be argued that the providers of health care are not given appropriate opportunities to engage with the scientists whose work is developing the future treatments for these diseases. scientific initiatives that are associated with national care plans could drive disease phenotyping and tissue collection for tissue, dna, rna, protein and metabolite databanks with good the difficulty in designing in vitro and in vivo experiments to model human disease has inevitably required and generated simplifications of pathology, often leading to relatively linear models of disease (for example, tissue damage, followed by antigen presentation, the generation of immunological memory and autoantibodies, and a resulting autoimmune disease). in reality, pathology is generated by networks that can exhibit substantial plasticity over the course of a disease. these principles are illustrated in the figure. a disease process -pathology -is represented at the centre of a series of simple conceptual networks, components of which are left intentionally blank to avoid attempting to define specific diseases. each component (or node) within the network might have different roles at different times in a disease, or if active at more than one tissue site, might even have different roles simultaneously in a disease process. therefore, the depicted connections are plastic over time and in individual microenvironments. in a cellular network. pathology can be considered in the context of networks of cells that are recruited or resident at inflammatory sites, whose communication through cytokines and other molecules regulates inflammation. in a cytokine network. pathology is driven by the interrelated actions of cytokines, again forming a dynamic plastic network. in a process network. process behaviour (for example, angiogenesis, scarring and leukocyte recruitment) will contribute differently to pathology at specific tissue locations and different times in the disease. as an illustration, wound-healing responses might contribute to the resolution of normal tissue architecture, the development of fibrosis, or the regulation of inflammatory cell recruitment and survival. depending on the nature and duration of the stimulus driving the woundhealing process, and the location in which it is set, multiple resulting pathological phenotypes are feasible. it is the nature of tissues to contain multiple cells and supporting structures that are physically associated, with many more cells that can transit through or become resident. we have proposed that the immunity seen in pathology rarely falls clearly into categories of innate and adaptive immune, or t helper (t h ) and t h responses. rather, pathology is generated by a networked interaction that changes over time. the networked relationships of immune pathology might be better described as 'contiguous immunity', in which multiple processes or networks can be operational and in dialogue in the same space (physically contiguous); equally, processes might be linked together in evolving patterns (temporally contiguous). understanding these networks, and, where necessary, developing new models to elucidate and target them, is essential to effective translational biology. ifn, interferon; il, interleukin. public access. good clinical practice not only should focus on ensuring that existing best practice is reliably reproduced, but also should put equal weight to research and development of practice through translational research. it is not just the priority of scientists to engage with clinicians: the imperative is equally strong that clinicians should engage more with basic scientists. in both cases it is important that such engagement is facilitated and supported by national policy. another major challenge is to take the input of a large amount of descriptive data generated by an 'omics' approach and both interpret and reapply it to a translational problem in a hypothesis-driven manner. as we learn to integrate complex data sets with in vitro cell biology and in vivo models, we might begin to generate virtual phenotypes, allowing conclusions to be drawn on the basis of the relatedness of a series of data sets to the questions asked. in essence, all researchers do this when they read published data, but the process is inherently subjective and formalizing such integrated biology could be more objective and so better inform future experiments. we are now experiencing an unprecedented blossoming in available technologies, unparalleled through history, which makes biomedical science extraordinarily exciting. with this comes an ever greater financial burden and increasingly complicated ethical issues. an increasing focus on the need for effective translation from the use of these resources highlights the many obstacles that impede such progress. we have highlighted these difficulties, and have suggested strategies to rejuvenate and maximize our translational potential. tyrosine kinases as targets for cancer therapy adjuvant chemotherapy for breast cancer - years later pro: mice are a good model of human airway disease modelling the human immune response: can mice be trusted? modeling allergic asthma in mice: pitfalls and opportunities con: mice are not a good model of human airway disease the mouse trap satisfaction (not) guaranteed: re-evaluating the use of animal models of type diabetes modelling infectious disease -time to think outside the box? lost in translation: barriers to implementing clinical immunotherapeutics for autoimmunity distinct mutations in irak- confer hyporesponsiveness to lipopolysaccharide and interleukin- in a patient with recurrent bacterial infections pyogenic bacterial infections in humans with irak- deficiency severe impairment of interleukin- and toll-like receptor signalling in mice lacking irak- phenotypes in asthma: useful guides for therapy, distinct biological processes, or both? evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics loss of siglec expression on t lymphocytes during human evolution differences in immune cell 'brakes' may explain chimp-human split on aids cytokine storm in a phase trial of the anti-cd monoclonal antibody tgn national survey of british public's views on use of identifiable medical data by the national cancer registry confidentiality in genome research no longer de-identified mouse model of airway remodeling: strain differences murine models of asthma rodent models of rheumatoid arthritis concordance among gene-expressionbased predictors for breast cancer a genomic strategy to refine prognosis in early-stage non-small-cell lung cancer a small-molecule screen in c. elegans yields a new calcium channel antagonist in vivo drug discovery in the zebrafish a transgenic zebrafish model of neutrophilic inflammation capturing complex d tissue physiology in vitro cooperative molecular and cellular networks regulate toll-like receptor-dependent inflammatory responses agonists of toll-like receptors and activate airway smooth muscle via mononuclear leukocytes pulmonary perspective: targeting the networks that underpin contiguous immunity in asthma and copd a bioinformatician's view of the metabolome we thank the many scientists whose stimulating conversations have in some measure been represented in these pages. i. the authors declare no competing financial interests. ian sabroe's homepage: http://www.shef.ac.uk/medicine/staff/sabroe.html lung tissue research consortium: http://www.ltrcpublic.com uk biobank: http://www.ukbiobank.ac.uk access to this links box is available online. key: cord- -rb ggonx authors: chaber, anne-lise title: the era of human-induced diseases date: - - journal: ecohealth doi: . /s - - - sha: doc_id: cord_uid: rb ggonx nan a global political and research infrastructure has grown around the fight against ''zoonoses,'' or diseases transmitted between vertebrates and humans. the one health or ecohealth approach and its integration of human, animal, and environmental health and the concomitant critique of the epistemological fragmentation of the western scientific tradition can be applauded, but scientific and political focus needs to shift from zoonotic disease to human-induced disease. human-induced disease includes both infectious-i.e., zoonoses and disease from animal origin-and non-infectious anthropogenically driven diseases. human-induced disease as the label for diseases-both infectious and non-infectious-caused by human activities and their environmental impact emphasizes the role of the human in disease transmission and could serve reshaping our approach to disease management and prevention. zoonotic diseases have been at the cornerstone of the one health/ecohealth approach. zoonoses incurred an estimated $ billion in direct costs and over $ in indirect losses in the last years (world bank ). these events generate moral panic, legitimate new spheres of legal activity, and spur new arguments for the funding of research. the arsenal of disease-combatting weapons grows constantly, evidenced by mass immunization or eradication of animal reservoirs. this dynamic derives from an anthropocentric perception and the psychologically useful imagination of a species barrier. most viruses and bacteria are not pathogenic but symbiotic, with humans hosting some , billion bacteria compared to their own billion cells. bacteria have a vital role in human bodily function and are integral elements of human cells (mitochondrion), and viruses constitute at least % of human dna. humans share most of the viruses, bacteria, and fungus with the rest of the animal kingdom, and thus it should come as no surprise that zoonotic pathogens were the cause of more than % of emergent infectious disease events in the last years, with % of these originating in wild fauna (keusch et al. ). the biologically relevant question is what allows or prevents infection by microorganisms, whether or not the infected species is human. the direct link between zoonosis and human activities and demographic growth is established. land use modification for urbanization, food production, and agricultural change accounts for around % of all zoonotic emerging infectious diseases (eids) (keesing et al. ). demographic, societal, and behavioral change gave rise to human immunodeficiency virus (hiv/aids) (de sousa et al. ) and outbreaks of syphilis (d' angelo-scott et al. ) . anthropogenic environmental change leads to the emergence of infectious diseases in wildlife (daszak et al. ) the advent of mass travel exacerbates the historically established dissemination of infectious disease along pathways of migration. examples are bubonic plague (yersinia pestis), cholera (vibrio cholerae), seasonal influenza, severe acute respiratory syndrome (sars), and malaria (plas-modium falciparum) (tatem et al. ) . the trade in goods and animals is directly linked to outbreaks such as human monkeypox virus in north america (karesh et al. ) or h n in the united arab emirates (naguib et al. ) . it is nevertheless common that cause is attributed to the animal and not to human behavior as evidenced, for example, by the mass culling of badgers and the ibex to control tuberculosis or prevent brucellosis in europe. this also evidences the diminished value to humans of wild animals and comes despite the universal declaration of animal rights (unesco and revised, ) which states that ''action endangering the survival of a wild species, and every decision leading to such act form a genocide, i.e. a crime against the species'' (article ). when human health is concerned, even hypothetically, such principles are quickly abandoned in the urge to displace consequence as cause. is reducing the risk of transmission by tackling human activities amplifying disease transmission really out of our hands? human-induced diseases do not only encompass infectious disease such as zoonoses whose spread is enhanced by human activities, but it also includes chronic disease linked to environmental perturbation caused by human activities. environmental degradation is a sensitive topic since environmental health is a concept that is still lacking definition, consensus and true global liability and accountability. the world health organization (who) estimates that ambient air pollution caused . million deaths throughout the world in (who and others ). the organization for economic co-operation and development (oecd) claims this death rate increased by % in china, % in india (oecd ), and % worldwide between and . it estimated that the annual economic cost of illness and premature mortality linked to air pollution is $ billion (oecd )-a figure that is % of the world's annual public budget for human health. the international monetary fund reported the use of fossil energies costs $ billion a year (parry et al. ) in disease, premature death, and environmental damage. this figure is . times more than the combined public health budgets of countries and yet industries that exploit fossil energies are subsidized directly and indirectly by more than $ billion annually (imfdirect-the imf blog ). environmental degradation is estimated to be the cause of % of world mortality (pimentel et al. ). human activity gives rise to multiple forms of toxic pollution affecting both our health and the environment. for example, production of garments for international markets takes place mainly in developing countries and involves unregulated tannery operations that generate chromium pollutants known to have carcinogenic effects on both animals and humans. in industrial plants with poor waste treatment and disposal infrastructure were responsible for the lead pollution linked directly to the death from chronic illness of , people living mainly in low and middle income countries (harris and mccartor ) . the institute for health metrics and evaluation also estimated that lead exposure accounted for . % of the global burden of idiopathic intellectual disability, % of the global burden of ischemic heart disease, and . % of the global burden of stroke. unesco's world water assessment programme estimates that industry is responsible for the annual accumulation of - million tons of sludge, heavy metals, and other toxic wastes, and that % of untreated industrial waste in developing countries is dumped directly into water systems (united nations educational, scientific and cultural organization ). [for people of] poor countries, resource degradation is the most tragic, forced as they are to overuse natural resources from which depends their survival. they are driven to sacrifice their future to ensure precarious everyday life. that is why in many countries, acting against poverty includes protecting the environment.-un secretary general, th june , rio da janeiro. a plethora of examples is already linking human diseases-both infectious and non-infectious-to environment perturbation in developed and developing countries. concerns are also raised regarding the impact of climate change on human disease burden. climate change is shifting ecological dynamics leading to potential increase in vector-borne disease such as lyme disease (brownstein et al. ) or malaria (martens et al. ) , zoonotic diseases like hantavirus infection (tian et al. ) , and non-infectious disease such as ciguatera fish poisoning (gingold et al. ) . even though environmental health and human health are deeply connected, environmental health is difficult to protect without consensus on definitions, common objectives between beneficiaries, and long-term planning. con-ventions of international environmental law introduced the concept of legal protection for species, sites, habitats and ecosystems. three concepts of nature coexist: it is a law project (unesco ) in which the common heritage dimension is underlined; it is a legal subject (united nations ); or it is a law purpose (unep )? the international criminal court announced in september that it will now prioritize crimes that result in the ''destruction of the environment'', ''exploitation of natural resources'', and the ''illegal dispossession'' of land (international criminal court ) . yet legality and illegality are often controversial. policies intending the liberalization of international trade do not take into account resource consumption, pollution, or negative societal impact in the producing and exporting countries (pearson ) . traded goods are not priced to incorporate such costs, and as such benefits of trade are distorted. a multitude of supranational environmental agencies, commissions, programs, and secretariats exist, but there is no global authority able to levy an appropriate pollution tax or fines on national governments or economic agents. it is becoming increasingly apparent to many that solutions and actions cannot be left to government or supranational government agencies. consumers are boycotting products with negative social, environmental, and health impacts such as slavery in clothing factories, harvesting of exotic timber, or methylmercury contamination in seafood. environmental citizenship is a recent and theoretically complex concept (bell ) . it is defined by the united nations environmental programme as an ''attempt to make environmental conservation and sustainability an important duty of citizenship that citizens all over the world should be aware of'' (unep). many multinational enterprises are now engaged in corporate citizenship programs to promote sustainable development, including the simultaneous consideration of economic growth, environmental protection, and social equity in business planning and decision-making. yet the willingness and ability of governments to reflect environmental values of their citizens vary greatly among countries (pearson ) . the individual remains the fundamental element of society. how powerful can one individual be? the internet and its powerful networking effect is beyond control of established institutions. it creates opportunities for horizontal communication, develops new forms of democracy and social participation, and could bring people closer to having impact on issues of common concern such as health, social justice, and the environment (barcena ) . could billions of individuals, scattered through the world but connected via the internet, become the strongest and most active pillar of environmental protection and thus enhance health and improve social justice? the term human-induced diseases might have the potential to put the human back into perspective and unify concerns and efforts. human-induced disease as the label for diseases (infectious and non-infectious) caused by human activities emphasizes the role of human and could serve bringing together scientists, politicians, industrials and laymen in common pursuit. human-induced diseases need to be named in order to be collectively claimed. effect of climate change on lyme disease risk in north america anthropogenic environmental change and the emergence of infectious diseases in wildlife high gud incidence in the early th century created a particularly permissive time window for the origin and initial spread of epidemic hiv strains ciguatera fish poisoning and climate change: analysis of national poison center data in the united states global energy subsidies are big-about us$ trillion big, imfdirect-the imf blog the world's worst toxic pollution problems report international criminal court, the office of the prosecutor ( ) policy paper on case selection and prioritisation wildlife trade and global disease emergence impacts of biodiversity on the emergence and transmission of infectious diseases sustaining global surveillance and response to emerging zoonotic diseases potential impact of global climate change on malaria risk outbreaks of highly pathogenic avian influenza h n clade . . . c in hunting falcons and kept wild birds in dubai implicate intercontinental virus spread the cost of air pollution: health impacts of road transport ecology of increasing diseases: population growth and environmental degradation global transport networks and infectious disease spread anthropogenically driven environmental changes shift the ecological dynamics of hemorrhagic fever with renal syndrome who, others (who) ( ) burden of disease from ambient air pollution for towards a one health approach for controlling zoonotic diseases unesco world heritage centre-convention concerning the protection of the world cultural and natural heritage i would like to acknowledge dr. françois moutou and prof. claude saegerman for their advices and inspiring comments. my sincere thanks also go to fleur toulat, anya stafford, and rachel johnson for editing and polishing the english language. key: cord- -xqphom x authors: papanikolaou, ilias c; sharma, om p title: tropical lung diseases date: - - journal: hunter's tropical medicine and emerging infectious disease doi: . /b - - - - . - sha: doc_id: cord_uid: xqphom x nan the term "tropics" refers to the region of the earth lying between the tropic of cancer and the tropic of capricorn. in the tropics, warm climate, poverty, lack of education, and poor sanitation provide an ideal environment for pathogens, vectors and intermediate hosts to flourish [ ] . in this vast landmass, respiratory infections are a major cause of morbidity and mortality in children and adults [ ] . in a typical tropical clinic, - % of outpatients have respiratory complaints, and - % of inpatients have lung disease (table - ) [ ] . many tropical patients suffer from lung diseases that are found worldwide, e.g. asthma, bronchiectasis, chronic obstructive lung disease, hiv infection-related lung disease, and lung cancer. numerous dust diseases, e.g. silicosis, asbestosis, byssinosis, hypersensitivity pneumonitis, and diseases due to microbial contamination of agricultural products, remain under-recognized. diseases associated with pulmonary symptoms and infection that are concentrated in the tropics include malaria, pulmonary schistosomiasis, melioidosis, paragonimiasis, echinococcal cysts, tropical eosinophilia, and diseases related to nutritional deficiencies [ ] . in addition, individuals who come in contact with birds or animals may develop zoonoses such as tularemia, psittacosis, q fever and leptospirosis [ ] . in the tropics, indoor air pollution caused by biomass fuels used for cooking and heating of the homes and huts is an important cause of obstructive lung disease and chronic lung infections [ ] . the following are the common tropical pulmonary conditions: l pneumonia: typical and atypical l eosinophilic pneumonias and tropical pulmonary eosinophilia l bronchiectasis, asthma and chronic obstructive pulmonary disease (copd) l pleural effusion l nontuberculous granulomatous lung disease l occupational lung diseases. a reasonable approach to the patient with lung disease in the tropic starts with age, occupational exposure, physical examination, hiv status, chest x-ray and blood tests. in children, bacterial pneumonia is the most common and life-threatening disorder. known immunodeficiency suggests tuberculosis, fungi and opportunistic pathogens. peripheral blood eosinophilia with either a pleural effusion or diffuse parenchymal consolidation may suggest a parasitic infection, or, when combined with wheezing, tropical pulmonary eosinophilia. worldwide diseases like copd may affect nonsmoking individuals due to indoor pollutants. streptococcus pneumoniae is the most common bacterial cause of pneumonia. upper respiratory involvement often precedes the onset of pneumococcal pneumonia, which is characterized by fever, chills, malaise and sweating. the patient is flushed and febrile with a rapid pulse and respiratory rate. dyspnea is associated with a nonproductive cough, and sputum, if present, may be thick, tenacious or "rusty". severe pleuritic chest pain causing tachypnea and grunting respiration is often present. such symptoms are abrupt in young, immunocompetent patients ( fig. . ) [ ] . in elderly patients, symptoms may be few and can be dominated by confusion, delirium and prostration [ ] . physical examination of the affected lung, usually the lower lobe, reveals diminished lung expansion, impaired percussion note, decreased breath sounds, crepitations (crackles/rales) and bronchial breath sounds. cyanosis is common and a herpes simplex eruption may be seen on the lips. with proper treatment, most patients with pneumococcal pneumonia improve clinically and radiographically within - weeks. when resolution occurs, fever subsides within a week as the temperature decreases following a crisis pattern ( fig. . a) . delayed resolution is seen in smokers, the elderly, and in those with poor nutrition, diabetes or other comorbid illnesses. staphylococcal pneumonia (staphylococcus aureus), accounts for - % of acute community-acquired pneumonias. it is an important cause of pneumonia in children, the elderly, patients recovering from influenza, people with diabetes mellitus, and those who are immunocompromised. methicillin-resistant staphylococcus aureus (mrsa) causes illness in % of cases of upper or lower respiratory tract infection in the community and in % of patients who are hospitalized. patients with staphylococcal pneumonia are usually ill with high fever, shaking chills, chest pain, cough and purulent sputum. chest x-ray films show patchy consolidation and cavities. sputum examination is an important aid in the diagnosis of pneumonia. color, amount, consistency and odor are helpful: mucopurulent sputum is commonly found in bacterial pneumonia or bronchitis; scanty watery sputum is often noted in atypical pneumonia; "rusty" sputum is seen in pneumococcal pneumonia; and currant-jelly or dark-red sputum suggests klebsiella pneumoniae. foul-smelling expectoration is associated with anaerobic infections due to aspiration, ilias c papanikolaou, om p sharma give an inhaled bronchodilator for days* • soothe the throat and relieve the cough with a safe remedy • if coughing for more than weeks or if having recurrent wheezing, refer for assessment for tb or asthma • advise the mother when to return immediately • follow-up in days • if wheezing (even if it disappeared after rapidly acting bronchodilator) give an inhaled bronchodilator for days* • soothe the throat and relieve the cough with a safe remedy • if coughing for more than weeks or if having recurrent wheezing, refer for assessment for tb or asthma • advise the mother when to return immediately • follow-up in days if not improving a blood count usually reveals leukocytosis in bacterial pneumonia, leukopenia in viral infection, and eosinophilia in parasitic infestation. when available, chest x-ray is extremely helpful ( table - ). tuberculosis is omnipresent in the tropics; upper lobe lesions with or without cavities strongly suggest tuberculosis. in children, the integrated management of childhood illness (imci) guidelines for treating pneumonia are recommended (see fig. . ) [ ] . nevertheless, a patient's illness has to be assessed based on geography, prevalence of potential etiologies, virulence of the organism, and the drug sensitivity pattern (box . ). in some areas, particularly papua new guinea, south africa and spain, resistance of the pneumococcus to penicillin is common. for children with non-severe pneumonia, the world health organization (who) recommends oral trimethoprim-sulfamethoxazole (tmp-smx) or oral amoxicillin for days [ ] . in severe pneumonia in hospitalized children, the policy in low-income countries is to first give benzylpenicillin injections, changing the therapy to oral amoxicillin when the child responds. in very severe pneumonia, in children in low-income settings, chloramphenicol may be given first with benzylpenicillin and gentamicin in combination as an alternative [ , ] . atypical pneumonia is caused by mycoplasma pneumoniae, chlamydia pneumoniae, legionella spp., viruses, tuberculosis, fungi and parasites. this syndrome is not extensively studied in the tropics because of the expense involved in culturing and isolating various organisms and obtaining serologic and immunologic tests. mycoplasma pneumoniae infections occur worldwide, affecting mostly school-aged children and young adults. a typical patient with mycoplasma pneumonia is an older child or young adult with an insidious onset of fever, malaise, tightness of the chest, and dry brassy cough. constitutional symptoms are out of proportion to the respiratory symptoms. hemoptysis, pleural pain and gastrointestinal symptoms are uncommon. the tropical physician should be aware of the non-respiratory manifestations of mycoplasma infection, including anemia, myringitis, stevens-johnson syndrome, hepatitis and neuritis [ ] (see table - ). leptospirosis is common in tropical areas where sanitation is poor and water supply primitive. epidemics of leptospirosis occur after high rainfall in monsoon seasons when the water supply is contaminated by sewage or animal urine. about half of the patients with leptospirosis have fever, cough, hemoptysis and pneumonitis [ ] . other features are jaundice, conjunctivitis and impaired renal function. melioidosis, caused by burkholderia pseudomallei, is endemic in southeast asia (vietnam, cambodia, myanmar), northern australia and west africa. melioidosis is hyperendemic in northern australia, and in parts of northeastern thailand it is an important cause of fatal community-acquired pneumonia [ ] . patients become infected while wading through fields, paddies, and flooded roads. clinical presentation is protean and nonspecific. the radiologic picture of upper lobe infiltration and cavity formation can be indistinguishable from tuberculosis [ ] . diagnosis requires isolation of the organism. the mortality rate ranges from % to % but is higher in hivinfected and immunocompromised hosts. respiratory symptoms of cough and chest pain in typhoid are present in up to % of cases at the onset of the disease. pulmonary infiltrates may be associated with positive sputum cultures for salmonella typhi. a fever chart showing continuous fever is highly suggestive of enteric fever. diagnosis may be difficult without blood and stool culture facilities. in brucellosis, the lungs are involved in about % to % of cases, usually following inhalation of organisms. abnormalities include bronchopneumonia, solitary or multiple lung nodes, miliary interstitial lung disease, lung abscess and pleural effusion. organisms can be identified on stains or sputum cultures. tularemia is a generalized infection caused by francisella tularensis and occurs after skin or mucous membrane contact with infected mammals or through the bite of an arthropod, usually a tick or biting fly. diagnosis should be considered in the presence of a skin ulcer associated with fever, generalized lymphadenopathy, cough and signs of pneumonia. pneumonia, either primary from inhalation of an infected aerosol or secondary to systemic infection, occurs in about % of cases. children with malnutrition and edema should be admitted to hospital pneumonic plague is less common than either bubonic or septicemic disease. nevertheless, fatal bronchopneumonia can occur without lymphadenopathy and is characterized by watery, bloody sputum. a sputum gram stain can show bipolar stunted rods. pneumonic plague and tularemic pneumonia should be considered when a severe, rapidly progressive bronchopneumonia is reported in an endemic area, and "typical" bacterial pneumonias have been ruled out. in slaughterhouses, meat-processing plants, and areas with sheep and goat husbandry, q fever (coxiella burnetii) can cause epidemics of atypical pneumonia. inhalation of dried infected material is the chief source, and fever, headache and dry cough are the main symptoms. occasionally, the sputum is blood-streaked. bornholm disease (caused by coxsackieviruses and occasionally other enteroviruses), also known as epidemic pleurodynia or devil's grip, causes chest discomfort and cough. widespread epidemics of bornholm disease occur in the pacific islands and south africa. in - , an unusual coronavirus was responsible for more than cases of a severe acute respiratory syndrome (sars) that spread via international travel across continents from its origin in guandong province, china. the sars coronavirus was previously unknown in humans; a possible reservoir was identified in civet cats and raccoons. after droplet inhalation of the virus, there was an incubation period of - days, then fever, cough, malaise and headache occurred. pulmonary inflammation was characterized by desquamation of pneumocytes, hyaline membrane formation and acute respiratory distress syndrome (ards). the chest x-ray showed diffuse opacities or consolidation, especially in the lower lung fields. recovery could be slow and some patients developed fibrosis. mortality was - %, with the elderly and those with cardiovascular problems being especially at risk. kawasaki disease occurs in children under years of age. this acute multisystem disease of unknown cause is characterized by fever of days duration and four of five clinical features: non-purulent conjunctivitis; injected (or fissured) lips or pharynx or strawberry tongue; cervical adenopathy; a maculopapular rash; and changes in the extremities (erythema and edema of the palms and soles, associated with desquamation). pneumonitis occurs in % of the children and coronary artery dilatation and aneurysms in - % of untreated cases. in brazil there has been a seasonal rise of the condition at the beginning and end of the monsoon season [ ] . cryptococcus neoformans and c. gatti are saprophytic fungi distributed worldwide and are particularly abundant in soil contaminated by pigeon droppings in the tropics as well as in temperate countries. pulmonary infection results from inhalation of the organisms from environmental sources [ ] . systemic helminth infection usually elicits eosinophilia and increased ige. although eosinophilia can be a clue to a pulmonary helminth infestation, the definitive diagnosis requires demonstration of ova or larvae in sputum, bronchial alveolar lavage fluid, pleural fluid or lung biopsy [ ] . loeffler's syndrome refers to "simple" pulmonary eosinophilia with no or minimal systemic and pulmonary symptoms. in many helminth infestations (ascaris, strongyloidiasis, hookworm), the larvae migrate through the lung and can cause fever, cough, dyspnea, wheezing, hemoptysis and lung infiltrate. schistosomes cause two clinical syndromes. in acute disease, immature schistosomula pass through the lung, and can lead to fever, eosinophilia and pulmonary infiltrate. in chronic schistosomiasis, especially when portal hypertension has led to venous shunts, eggs can bypass the liver and plug pulmonary capillaries and arterioles, producing granuloma and pulmonary hypertension. radiographs may show dilated pulmonary arteries ( fig. . ). in paragonimiasis, the lung is the predominantly involved organ. the diagnosis must be considered in a patient from southeast asia with cough, hemoptysis (which is recurrent in > % of cases), a pulmonary cavity and pleural effusion. tropical pulmonary eosinophilia, typically in india and other south asian countries, causes immunologic hyperresponsiveness to wuchereria bancrofti, brugia malayi or other microfilariae. clinical presentation consists of nocturnal cough, wheezing, fever and weight loss. chest radiographs show diffuse interstitial miliary infiltrates ( fig. . ) ; there is a high eosinophil count. in developed countries, serum ige and antifilarial antibodies can be used to confirm the diagnosis (table - ) . bronchiectasis is a chronic, debilitating condition. dilatation and distortion of the airways leads to impaired mucociliary clearance, which encourages bacterial colonization and bronchial inflammation. patients have fever, chronic cough, mucopurulent sputum, hemoptysis (table - ) , wheezing, dyspnea and malaise (box . ) . the primary cause of copd is smoking l copd affects men and women equally l copd is not curable but can be prevented the diagnosis of bronchiectasis in developed countries is confirmed by computed tomography of the chest (fig. . ) ; whereas, in the tropics, the diagnosis is mainly clinical and depends upon a compatible history, presence of finger clubbing, sputum that settles into three layers (mucoid or frothy, mucopurulent, and purulent) and a chest x-ray, if available. treatment includes regular chest percussion, broadspectrum antibiotics for exacerbations, and influenza and pneumococcal vaccinations. the incidence of asthma in the tropics is low for unclear reasons; however, the disease remains underdiagnosed and untreated. "all that wheezes is not asthma" is a dictum that is true in the tropics, as there are many entities that cause wheezing and difficulty in breathing, including tropical eosinophilia and mitral stenosis. asthma monitoring in the tropics can be achieved by using an inexpensive peak flow meter. treatment should fit the frequency and severity of attacks. betaagonists and cromolyn sodium (sodium cromoglycate) are usually available. oral corticosteroids in short courses can be used to control severe episodes; however, long-term use of systemic corticosteroids, without adequate monitoring, is not safe. aerosol inhalers are of great value but they are expensive, difficult to use, and require painstaking teaching. chronic obstructive lung disease is a progressive disease which is characterized by airway obstruction that is only partially reversible by bronchodilator therapy. the term copd encompasses chronic bronchitis and emphysema. once a common disease of men, copd is now as frequent in women because of increased tobacco use and the widespread use of dung and biomass for indoor cooking and heating in low-income countries (box . ) . the most common symptoms are dyspnea and chronic cough. it is mild and occurs only on heavy exertion. with progression of airway obstruction, patients become more short of breath and eventually cannot breathe at rest. physical examination in the early stage is normal, but in advanced disease, prolonged expiration and expiratory wheezes are audible. in severe cases, the thoracic cage becomes barrelshaped with increased anterior-posterior diameter; percussion note is hyperresonant. when chest x-ray and pulmonary function testing are not available, a peak-flow meter is an inexpensive device to assess severity of airway obstruction and monitor the response to treatment. cessation of smoking is essential. oral theophylline and beta-agonist drugs control symptoms. antibiotics (ampicillin, tetracycline and sulfa drugs) are available to treat copd exacerbations in the tropics. pleural effusion is a frequent condition with variable clinical signs and symptoms. small effusions can remain silent and are often detected only on chest radiography. large effusions are associated with dyspnea and diminished chest movements on the affected side. vocal fremitus is reduced; percussion note is stony dull; and auscultation reveals diminished breath sounds and decreased vocal resonance. sometimes, bronchial breathing is heard at the upper level of dullness. in addition there may be a pleural friction sound. exudative effusions typically have cell counts, protein and biochemical markers opposite to those of transudates. exudates can be further classified into neutrophilic, lymphocytic and eosinophilic. neutrophilic exudates may be due to bacterial infection, gastrointestinal diseases, pulmonary embolism, collagen-vascular diseases (cvd) and asbestos-related benign effusion. pleural effusion occurs in about % cases of pneumonia, and can progress to a complicated effusion (pleural fluid ph< . , positive gram stain) or to an empyema, both necessitating pleural fluid drainage with a chest tube thoracostomy in addition to antibiotic treatment. empyema can occur in pneumococcal, staphylococcal (most often) and klebsiella infections. a right-sided pleural effusion may be associated with amebic liver abscess. the disease presenting with the highest pleural fluid lymphocytosis is tuberculous pleuritis; however, early in the course, there can be a neutrophilic exudate. a large volume of pleural fluid should be obtained for examination for acid-fast bacilli. in about one-third of cases, the tuberculin skin test is negative initially and converts to positive after - weeks. knowledge of the hiv status of a patient with pleural effusion, if positive, significantly inclines to a tuberculosis. an eosinophilic exudate is more common in the tropics. endemic parasitic and fungal infections are major causes of such an effusion. ascariasis, echinococcosis and paragonimiasis are some of the causative parasitic infections. paragonimiasis is associated with low pleural fluid glucose and low ph. fungal diseases responsible for such an effusion are histoplasmosis, cryptococcosis and coccidioidomycosis. in the absence of chest x-ray or biopsy evidence, it is not possible to diagnose pulmonary involvement due to sarcoidosis and other granulomatous diseases. consequently, in the tropics, these disorders remain undiagnosed. the possibility of sarcoidosis should be considered in a patient with dyspnea, uveitis, hepatosplenomegaly, peripheral lymphadenopathy, chronic skin lesions, and a chest x-ray film showing bilateral hilar adenopathy [ ] . the occupational disorders result from human social activity, and as such are preventable. the dusts that provoke occupational disorders can be classified into: those that induce granulomatous reaction (e.g. beryllium, talc and organic antigens); those that cause fibrosis (e.g. silica, asbestos and coal); and those that cause neither inflammation nor fibrosis, thus remaining inert (e.g. iron, barium and tin) ( podoconiosis is an endemic nonfilarial elephantiasis occurring in individuals exposed to red clay soil derived from alkaline rock. a chronic and debilitating disease, it exerts a large economic burden. the silica particles are found in the skin, lymph nodes and lymphatics of affected and unaffected individuals. these individuals have reduced lung function as compared with adults living in areas of low silica concentration [ ] . immunological aspects of tropical lung disease pneumonia: the forgotten killer of children. geneva: unicef/ who parasitic lung infections tropical infections and the lung pulmonary disease world health organization, family and community health cluster, department of child and adolescent health and development. consultative meeting to review evidence and research priorities in the management of acute respiratory infections (ari) influence of age on symptoms and presentation in patients with community acquired pneumonia integrated management of childhood illness (imci) for high hiv settings clinical efficacy of co-trimoxazole versus amoxicillin twice daily for treatment of pneumonia: a randomised controlled clinical trial in pakistan chloramphenicol alone versus chloramphenicol plus penicillin for severe pneumonia in children chloramphenicol versus benzylpenicillin and gentamicin for the treatment of severe pneumonia in children in papua new guinea: a randomised trial approach to the patient with tropical pulmonary disease pulmonary complications of leptospirosis the epidemiology of melioidosis in australia and papua new guinea melioidosis after brief exposure: a serological survey in us marines kawasaki disease: a clinical and epidemiological study of children in brazil lung biology in health and disease: tropical lung disease lung biology in health and disease: tropical lung disease assessment of respiratory function in patients with podoconiosis key: cord- -a pu rlu authors: perakakis, nikolaos; stefanakis, konstantinos; mantzoros, christos s. title: the role of omics in the pathophysiology, diagnosis and treatment of non-alcoholic fatty liver disease date: - - journal: metabolism doi: . /j.metabol. . sha: doc_id: cord_uid: a pu rlu non-alcoholic fatty liver disease (nafld) is a multifaceted metabolic disorder, whose spectrum covers clinical, histological and pathophysiological developments ranging from simple steatosis to non-alcoholic steatohepatitis (nash) and liver fibrosis, potentially evolving into cirrhosis, hepatocellular carcinoma and liver failure. liver biopsy remains the gold standard for diagnosing nafld, while there are no specific treatments. an ever-increasing number of high-throughput omics investigations on the molecular pathobiology of nafld at the cellular, tissue and system levels produce comprehensive biochemical patient snapshots. in the clinical setting, these applications are considerably enhancing our efforts toward obtaining a holistic insight on nafld pathophysiology. omics are also generating non-invasive diagnostic modalities for the distinct stages of nafld, that remain though to be validated in multiple, large, heterogenous and independent cohorts, both cross-sectionally as well as prospectively. finally, they aid in developing novel therapies. by tracing the flow of information from genomics to epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and glycomics, the chief contributions of these techniques in understanding, diagnosing and treating nafld are summarized herein. nafld is recognized as the most common liver disease in developed countries with its incidence continuously rising in parallel to the increasing incidences of obesity and type diabetes (t dm) [ ] . significant efforts are currently underway to delve deeper into the pathophysiology of the disease, to create non-invasive tools for its diagnosis and staging, as well as to develop highly effective and specific treatments. the disease is characterized initially by hepatic lipid accumulation (nonalcoholic fatty liver; nafl), that can often progress to non-alcoholic steatohepatitis (nash), liver fibrosis or cirrhosis, as outlined in detail elsewhere in this special issue [ ] . several triggers and series of events stimulating necroinflammatory processes in the liver have been recognized and described as "the multiple hit" pathogenetic mechanism leading to advanced nash [ ] . additionally, multiple factors (such as lack of physical activity, unhealthy nutrition, concomitant alcohol consumption and presence of other metabolic diseases) have been associated with increased risk of disease progression [ ] . still, we are not able to predict with great accuracy which patient and when will develop hepatic inflammation or fibrosis. j o u r n a l p r e -p r o o f assessed [ ] , and histologically-proven gwas [ ] of more than total patients each, re-affirmed the robust clinical and histological genome-wide associations of pnpla , tm sf , gckr, and hsd b . novel fibroinflammatory loci in slc a and slc a were also studied [ ] . however, a bench-to-bedside conversion of current results through tangible diagnostic and therapeutic applications has yet to take place. pnpla rs constitutes the chief nafld predictor, adjusted for known risk factors (or= . ) [ ] . yet its addition in an early predictive model of nafld, using routine variables, contributes trivially to score accuracy (area under the receiver operator characteristics (auroc) = . and . respectively) [ ] . pnpla is included in the nash score (aspartate transaminase (ast, insulin, pnpla genotype) and the clinlipmet score (nash clin score, metabolomic and lipidomic variables) with aurocs of . and . respectively, yet its contribution to nash prediction is not underlined [ ] . in a more recent phenome-genome wide study of patients, clinical data (age, sex, bmi, blood counts) comprise a fitter nafld predictor than rs genotyping (auroc = . and . respectively) and have % higher sensitivity compared to genotyping alone. even though rs carriage was associated with . years earlier nafld detection, its addition does not markedly amplify the forecasting model (auroc = . ) [ ] . cumulative genetic risk scores (grs) are frequently discussed (table ) . a grs of previously mentioned snps in pnpla , tm sf , gckr and mboat indicates a -fold increase in nafld risk starting from its second tertile [ ] . when coupled with a multivariate insulin resistance score, a grs j o u r n a l p r e -p r o o f of pnpla and tm sf snps detects nas ≥ (auroc= . ) and fibrosis ≥ when age included (auroc = . ), yet grs alone have aurocs of ≤ . [ ] . pnpla and tm sf polymorphisms are also included in a novel nash score alongside diabetes, insulin resistance, ast and c-reactive protein levels, which predicts nash more effectively compared to the older nash score (aurocs of . and . respectively) and has enhanced accuracy in diabetic cohorts (auroc = . ) [ ] . lastly, a recent investigation of individuals formulates a grs comprising pnpla , tm sf and hsd b snps. a maximum risk score of designates a % increase in alanine aminotransferase (alt) levels and a pronounced risk of cirrhosis and hcc (or= and respectively) [ ] . genetic testing in nafld is currently not recommended by the american association for the study of liver diseases [ ] . thus far, genomics have not identified a consistent marker that reflects the accurate histologic features of nafld, partially owing to limited population sizes, inconsistent nafld validation methods and scarcity of biopsy-confirmed models. most importantly, genomics are not dynamic processes, thus they cannot reflect the impact of environmental factors (high caloric intake, presence of obesity, t dm etc.) in the pathogenesis and progression of nafld. multi-gene models are promising tools of patient risk stratification but require additional validation. pnpla has also been investigated through early genetic interventions. preand post-translational silencing of pnpla in p.i m knock-in mice j o u r n a l p r e -p r o o f challenged with a sucrose-rich diet drastically improves intrahepatic fat profiles [ , ] . additionally, pnpla silencing ameliorates steatosis and nas independent of genotype, and improves inflammation, fibrosis and levels of acute phase, chemo-attractant and pro-fibrotic proteins, in p.i m knockin mice fed a nash-inducing diet [ ] . however, pnpla protects from hypercholesterolemia, gallstones, gout and acne in humans [ ] , therefore future pilot studies must be carefully considered. tm sf is another possible target, yet its substantial cardioprotective effects [ ] indicate the complexity of such an approach, since the chief cause of death in nafld patients is cardiovascular disease [ ] . patient genotyping prior to therapeutic scheme initiation implies compelling curative applications. for instance, pnpla p.i m carriers would not respond to statins and inhibitors of de novo lipogenesis, in contrast to tm sf and gckr carriers [ ] . moreover, hsd b has already been implied as a therapeutic target [ ] , while it was recently established that carriage of hsd b rs diminishes the fibrotic effects of pnpla rs , thus indicating a novel objective for pathway-targeting therapies [ ] . further research could uncover powerful hierarchical and therapeutic tools in nafld genomics, jumpstarting a new era of clinicogenomics. j o u r n a l p r e -p r o o f progression of nafld. given the continuous evolvement of personalized medicine, we expect the information from genomics to be used in the near future for identifying the patients at higher risk for presenting a more aggressive course of disease and/or for potentially predicting the response to specific treatments. these may justify more intensive follow-ups or guide treatment decisions in high-risk populations. sfince genomics are not dynamic processes and do not reflect the effects of environmental factors on nafld, we see less potentials for them as diagnostic markers and we expect for them to have only a small, if any, contribution in non-invasive diagnostic tests. finally, due to the technical and clinical challenges related to geneengineering as well as due to the complex pathophysiology of the disease, we recognize at the moment limited perspectives in the development of gene therapies for nafld. epigenomics investigates the epigenetic modifications on cellular genetical material. several studies have assessed the impact of epigenetic modifications in the development and progress of nafld ( figure ) as well as in the association of nafld with other metabolic diseases by focusing on dna methylation, histone modifications and mirna expression profiles that can significantly affect transcriptional activity. significant alterations in dna methylation are observed when moving from nafl to nash and liver fibrosis [ ] [ ] [ ] . these changes include alterations j o u r n a l p r e -p r o o f in dna methylation that affect the expression of genes involved in glucose, lipid or acetyl-coenzyme a (coa) metabolism (pc, plcg , acly) [ ] , insulin-like signaling (e.g. igf- , igfbp ) and mitochondrial function (nadh dehydrogenase ) [ ] . additionally, given that a specific dna methylation signature develops with increasing age in humans, nash seems to accelerate epigenetic age by promoting changes in methylation that are associated with hepatic collagen content [ ] . an untargeted evaluation of dna methylation in liver tissues of patients with nafld identified almost , cpg sites that were differentially methylated in patients with advanced liver fibrosis (f -f ) compared to those with no or mild fibrosis (f -f ). % of these sites were hypomethylated and % were hypermethylated in advanced liver fibrosis in nafld, whereas % of the reported methylations correlated with gene expression levels. the hypomethylation was associated with higher expression of genes related to tissue repair, liver inflammation, fibrosis and carcinogenesis (e.g. fgfr , col a , casp , ccr , ccl ) whereas the hypermethylation with lower expression of genes involved in lipid and aminoacid metabolism [ , ] . these findings were also replicated in other studies [ ] . dna methylation seems to be particularly involved in the activation of hepatic stellate cells and their differentiation to myofibroblast that are crucial procedures for hepatic fibrogenesis [ , ] . changes in methylation of specific genes have been linked with these processes. genes promoting fibrogenesis such as tgfβ and pdgfα are hypomethylated and thus highly expressed whereas genes inhibiting hepatic stellate cells activation such as the pparα, pparδ and pparγ are hypermethylated and j o u r n a l p r e -p r o o f thus lower expressed in the liver of patients with advanced fibrosis compared to the ones with mild disease [ , ] . apart from their involvement in nafld development and progress, hepatic alterations in dna methylation may be associated with systemic metabolic outcomes. specifically, a case-control study in nafld patients has shown that increased methylation in specific sites of the promoter of the ppargc a, which encodes the major regulator of mitochondrial biogenesis, is associated with lower mrna expression of ppargc a in the liver, lower ratio of mitochondrial to nuclear dna and increased insulin resistance, thus linking hepatic mitochondrial dysfunction of nafld with peripheral insulin resistance [ ] . in agreement with the above finding, a study following untargeted procedures has identified methylations that are affecting mrna expression of hepatic genes involved in insulin signaling that are also highly correlated with fasting insulin independently of the presence of t dm, thus providing further evidence for the relation of hepatic dna methylation with insulin sensitivity [ ] . finally, a recent study focused on the identification of differentially methylated regions that form networks which are associated with the progression of nafld. they identified two important networks, one that included genes that affect cytoskeleton organization, transcriptional activity and cell proliferation and another that was associated with metabolic pathways. the cpg methylation levels in both networks were affected by age and fasting plasma glucose levels and for the second network the changes in methylation levels were partially corrected by controlling weight and blood glucose levels [ ] . j o u r n a l p r e -p r o o f histone modifications are also important epigenetic changes that affect transcriptional activity and refer to several posttranslational procedures such as acetylation, phosphorylation, methylation and ubiquitination. among them, acetylation status has been most vigorously studied and is considered the net result of histone acetylation by histone acetyltransferases (hats) and histone deacetylation by histone deacetylases (hdacs) [ ] . acetylation neutralizes lysin's positive charge, thus leading to a looser chromatin structure that facilitates transcription. the transcriptional coactivator p belongs to hat proteins, whereas sirtuins belong to nad-dependent class iii hdacs. glucose-induced activation of p increases the transcription of carbohydrate-responsive element-binding protein (chrebp) resulting in the stimulation of lipogenic genes through histone acetylation and thus promoting the development of nafld [ ] . tannic acid attenuated the effects of p leading to a decrease in the lipogenesis-related genes and to an improvement of nafld in mice [ ] . similarly, inhibition of cdk protein reduces the formation of c/ebpα -p complexes reducing liver steatosis and correcting age-associated liver changes [ ] . p may also be involved in the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts [ ] . among the sirtuins, especially sirtuin (sirt ) has been shown to participate in the regulation of hepatic metabolism and insulin sensitivity, with hepatic deletion of sirt resulting in steatosis and inflammation, whereas overexpression of sirt protecting from nafld [ ] [ ] [ ] . similarly, sirt deficiency leads to insulin resistance, hyperlipidemia and steatohepatitis in mice [ ] whereas a polymorphism of sirt is associated with metabolic syndrome and nafld in humans [ ] . hdac , a member of j o u r n a l p r e -p r o o f human class i hdacs has been also implicated with circadian metabolic rhythm and its deletion leads to hepatic steatosis in mouse liver [ ] . altogether, there is increasing evidence that both dna methylation as well as histone modifications play a crucial role in the development and progress of nafld. an important question is whether the numerous epigenetic changes observed in nafld in combination with the highly dynamic nature of epigenome can serve as diagnostic or prognostic markers of nafld or even as markers of treatment response. very few studies have addressed this point so far. one of them that included a small number of patients with nafld has shown that alterations in hepatic dna methylation may be sufficiently reflected in plasma. in this study, % of plasma dna methylation of pparγ was used to identify subjects with severe (kleiner - ) vs mild fibrosis (kleiner - ). the threshold of methylation was % and demonstrated % sensitivity and % specificity to differentiate between the two above categories [ ] . although validation of the findings in largest and more heterogenous cohorts is needed, it is one of the first studies to suggest that changes in dna methylation can have beyond their pathophysiological significance also value as potential noninvasive biomarkers for the diagnosis and staging of nafld. a second study has also investigated differences in methylation of dna from peripheral blood leukocytes that may be able to discriminate between nash vs nafl. the study identified six genes that their methylation correlates with lobular inflammation and can be potentially used as diagnostic biomarkers (with the j o u r n a l p r e -p r o o f best one showing % specificity and % sensitivity) for differentiating nash from nafld [ ] . finally, in a large epigenome-wide association study consisting of , participants of european ancenstry, cpgs were found in peripheral blood to be associated with hepatic fat as assessed by computed tomography or ultrasound imaging and among them one was also associated with risk for new-onset t dm [ ] . nevertheless, significantly more and larger studies are needed in order to decide whether epigenetic changes in the blood can have any diagnostic or prognostic value. targeting epigenomic changes for the treatment of diseases seems to be theoretically a very attractive option for a number of reasons. first, epigenomic alterations are involved in fundamental pathophysiological mechanisms of a disease through their impact on gene expression. second, epigenetic changes are also highly dynamic and reversible, thus correction of them holds promise for reversal to "normal status". for example, after bariatric surgery, robust changes in methylation have been reported. hypermethylation and consequently reduction of transcriptional activity of ptpre (encoding protein-tyrosine phosphatase epsilon) may improve hepatic insulin sensitivity [ ] . exercise may also reduce methylation and improve mrna levels of mitochondrial genes, thus improving mitochondrial function [ ] . third, it may be possible to combine treatments targeting epigenome with other drugs, having in that case a complementary role. such treatments are currently being developed mainly against cancer diseases and consist of pharmacological dna methylation inhibitors (dnmtis), hdac inhibitors (hdacis) or activators. in nafld very few of them have been tested so far. j o u r n a l p r e -p r o o f among them, the sirt activator, resveratrol, improved dyslipidemia and steatohepatitis in a mouse model of atherogenic nafld [ ] . several ongoing human clinical trials (two of them in phase iii) are currently investigating the efficacy of resveratrol in human nafld but results from the first studies have shown no significant effect on hepatic fat content [ ] [ ] [ ] . other medications are currently also being developed and have shown promising results in animal studies, such as the -deazaneplanocin a (dznep) [ ] or the hdac inhibitor saha but none of them is in a more advanced stage of clinical development [ ] . epigenomics are one of the least investigated "omics" in nafld. the few studies performed so far have shown that the extensive hepatic epigenetic modifications observed in nafld are causally related to the disease. it is still unclear though how accurately the hepatic epigenome is reflected in peripheral blood and thus it is questionable whether epigenetic changes studied at the level of peripheral blood cells may have any significant diagnostic value. given also the limited amount of information from pathophysiological studies investigating the dynamic of epigenetic changes with nafld development and progression, it is probably premature to expect any drugs to be developed in order to target exclusively epigenome of nafld in the near future. genetic predisposition determines nafld transcriptomic signatures, as shown by recent investigations on the carriage of pnpla i m on transcriptome variability [ ] . as described above, transcriptomics are also linked to epigenetic modifications in nafld [ , ] . through global microarray transcriptional snapshots, nafld and especially nash are characterized by overexpression of genes associated with lipid metabolism [ ] [ ] [ ] [ ] , acute phase regulators of insulin sensitivity [ ] , cellular division [ ] , dna [ ] and tissue repair [ ] , extracellular matrix organization [ , [ ] [ ] [ ] , immune function [ ] cellular adhesion and migration [ , , ] , signal transduction [ ] , p signaling [ ] and cancer progression [ ] among others ( figure ). conversely, genes modulating mitochondrial function [ ] oxidative, glucose, fatty acid and aminoacid or protein metabolism [ , - , , ] as well as insulin signaling and transcription factors [ , ] are downregulated. and underexpression of pzp mrnas [ ] . hepatic transcriptomes of nafld are distinct from those of normal and healthy obese subjects, yet a -gene signature, with extracellular matrix remodeling and immune system genes as its main components, distinguishes nash from simple steatosis [ ] . more specifically, the transcriptional snapshot of nash is defined by upregulation of pdgf, stat, hnf- and smad- pathway-related genes [ ] , as well as downregulation of bnip , igfbp [ ] , slc a , c orf [ ] sdc , atf , various inflammatory suppressors [ ] and amino acid-metabolizing and reactive oxygen species (ros) scavenger genes [ ] . interleukin- (il- ) was recently ascertained as the most significantly upregulated transcript in advanced nafld and nash (fold-change = . vs non-nash), correlating with lipid accumulation and disease severity [ ] . additionally, several cancerrelated genes, such as akr b , krt and gpc , are enhanced in nash compared with nafl or controls, by up to -fold and -fold respectively [ ] . concerning fibrosis, cross-sectional transcriptome analysis of hepatic cells defines and differentially expressed transcripts for the inflammatory and fibrotic states, respectively, and further underlines fibrosis-defining genetic routes, pertaining to cytokine and extracellular matrix receptor interaction, focal adhesion and the pi k-akt signaling pathway [ ] . differentially expressed microarray signals, such as ube v , bnip l, rp - n . , linked to oxidative stress, inflammation, apoptosis and fibrogenesis, are upregulated in nas ≥ and fibrosis stages - [ ] . j o u r n a l p r e -p r o o f likewise, transcriptomics pinpoint dermatopontin, a propagator of extracellular matrix remodeling, which is robustly expressed (by -fold) in murine stellate cells, enhanced in human fibrotic livers and implicated in nafld fibrogenesis [ ] . minor molecules such as micro-rnas (mirna) along with long noncoding rnas (lncrna) perform a variety of epigenetic and post-transcriptional functions, thus significantly affecting transcriptional activity in numerous diseases and among them also nafld [ ] . for this reason, they are also considered important epigenetic modifiers. these processes are thoroughly delineated through large panels of differential mirna expression [ ] [ ] [ ] while the distinct nafld-related roles of mirna subspecies were recently reviewed [ , ] . of note, microrna- (mir- ), deriving from chromosome , represents % of hepatic mirna and has been implicated in cholesterol metabolism and liver cancer [ ] . cheung et al. first mapped a % underexpression of liver mir- in nash patients compared with controls, through an evaluation of a -mirna panel that identified differentially expressed mirna species and their corresponding targets [ ] . mir- was subsequently associated with steatosis severity [ ] . the (a,b), mir- b (associated with metabolic syndrome, atherogenesis and circulation) [ ] as well as mir- a, mir- (associated with cancer) [ ] , mir- and mir- [ ] are upregulated in both nafld and nash. moreover, mir- a, mir- a and mir- alter lipid and carbohydrate j o u r n a l p r e -p r o o f metabolism, reflect nafld severity and are implicated in hepatocellular carcinomas [ ] . expression levels of lesser non-coding molecules have also been investigated [ , , ] . global hepatic rna profiling has identified lncrna and mrna species overexpressed in nafld, whereas lncrna and mrna species are underexpressed. these enriched rnas chiefly belong to small molecule and organic acid metabolic pathways [ ] . functional transcriptome research of lncrna species by atanasovka et al. pinpoints elevated levels of hepatic lncrna rp - n . in nash, and robustly associates it with nash grade, lobular inflammation and nas, while its knockdown in vitro aggravates hepatocellular growth and viability [ ] . identified and validated through whole serum transcriptome analysis, lncrna rp - n . has been linked to nash diagnosis and is upregulated in nas ≥ [ ] . furthermore, transport rna (trna) profiling pinpoints the differential expression levels of several anticodons for lysine, aspartate and glutamate in cirrhosis, in addition to various mitochondrial and amino acid trnas in cirrhosis versus nash and nash cirrhosis versus normal respectively [ ] . the same study has also compared the abundance of several small rna molecules (ribosomal, small nuclear and nucleolar) between healthy, nash and cirrhotic patients [ ] . various predictive modalities involving gene expression parameters, targeted measurements and mirna panels with increased functionality are based on transcriptomics. in general, the reported accuracies for many of these tests j o u r n a l p r e -p r o o f have been high for diagnosing nafld or nafl from healthy status but rather low for differentiating nafl from nash or for diagnosing advanced fibrosis. additionally, even the most promising studies, have not been further tested or have performed poorly in other cohorts. among them, predictive models based on mirnas have in theory higher chances to be robust and reproducible, since mirnas can withstand numerous freeze-thaw cycles and long-term storage before degrading [ ] . several studies have identified mir- as a potential diagnostic biomarker. most of them have shown that mir- alone or in combination with other mirnas (e.g. mir- , mir- , mir- , mir- , mir- a) can accurately predict the presence of nafld or nafl, but they all perform inadequately when trying to differentiate nafl from nash [ , , [ ] [ ] [ ] (table ) . with other proteins in their algorithm. among them, nis tm , which includes mir- a, alpha- -macroglobulin, chitinase- -like protein , has been recently tested in a population with type diabetes (n= ), showing an auroc performance of . for diagnosing advanced nash (nas≥ ) and significant fibrosis (f≥ ), which was also higher than in other tests, such as fib ( . ), elf ( . ) or fibrometer ( . ) but probably still suboptimal [ ] . additionally, the same test demonstrated % sensitivity and specificity in detecting nash patients at risk of cirrhosis [ ] . another model integrating mir- , mir- , mir- and ck- fragments, can differentiate nash from nafl (auroc = . ) [ ] (table ) . new frontiers, such as circular rna (circrna) transcriptomics, are being investigated in animals. researchers have already mapped and correlated j o u r n a l p r e -p r o o f circrna species and their corresponding transcriptional pathways in mouse models of nash, showcasing their associations with metabolism [ ] and stellate cell activation [ ] . yet whether these results could yield meaningful conclusions that can be extrapolated in human cohorts remains unclear. other prospective research questions in diagnostic nafld transcriptomics concern alternative rna splicing [ ] and the scrutiny of unexplored transcriptional components, such as cd [ ] . curative and bedside implications of transcriptomics in nafld are considerably limited. from an interspecies aspect, the human and murine nafld transcriptomes are largely dissimilar, yet their likelihood increases in murine models following high-fat diets [ ] . hence the transcriptional signatures of nafld or nash as well as the organic transcriptomic responses to relevant treatments for liver disease can be evaluated by numerous available pre-clinical in vitro prototypes and animal models [ ] . for instance, mapping of the murine nash transcriptome demonstrated an increase in the levels of tsukushi, a novel adipose-related thermogenetic and metabolic hepatokine. subsequent silencing of tsukushi improved nash and attenuated the hepatic transcriptional alterations associated with it [ ] . transcriptomics were also implemented in recent murine models assessing the nafld-related role of poly rc binding protein , a cytosolic iron chaperone, whose deficiency exacerbates steatosis and inflammation, increases lipid biosynthesis and oxidative stress pathway expression levels, and is treated via reduced iron intake and vitamin e supplementation [ ] . lastly, in view of the covid- pandemic, the expression levels of sars-j o u r n a l p r e -p r o o f cov- entry proteins were assessed by liver transcriptomics in human and murine nafld and nash cohorts, with no significant changes being detected [ ] . future transcriptomic modalities should approach all different phases and components of transcription, kindle research for new remedies, and investigate the associations of liver disease with various pathologies precipitated by its metabolic milieu. transcriptomics have been widely used and have contributed significantly to our understanding of the pathophysiology of nafld. transcriptomics, with the exception of mirnas, do not add significant value in non-invasive diagnostic scores, since their hepatic profile is not adequately reflected in peripheral blood. with the rapid improvement of available technologies and data analysis, transcriptomics are now routinely used in order to identify potential mechanistic pathways affected by drug candidates for the treatment of nafld. proteomics refers to the investigation of "proteome", thus to all proteins expressed by a cell. the proteome, similar to transcriptome and epigenome, is highly dynamic. cell activation, trafficking, differentiation or transformation as well as tissue type have a major impact on the proteome. additionally, posttranslational modifications (phosphorylation, acetylation, methylation, glycosylation etc.) as well as alternative splicing render proteome much more j o u r n a l p r e -p r o o f complex than transcriptome, with more than , proteins being expressed by a human cell. finally, the proteome is downstream of genome and transcriptome, and thus it is stronger associated with the final phenotype [ ] . proteomic analyses have been performed for cell line -characterization or for protein investigation and identification in tissues under specific conditions or treatments, but their use has been in general rather limited, especially for biomarker or drug discovery [ ] . there are several reasons for this. first, significant technical limitations exist that restrict the number of proteins that can be detected and can be identified with certainty. the first efforts involved two-dimensional gel electrophoresis and were able to detect some few dozens of spots that were subsequently matched to specific proteins with the use of ms. later approaches managed to detect more protein peaks without though being able to identify the corresponding proteins. advances in methods have enabled in the last years the identification of some thousands of proteins but still the largest part of proteome cannot be investigated with certainty with the available technologies. second, reliable quantification of the levels of proteins, especially in blood has been very challenging. in plasma, three classes of proteins mainly exist: a) the highly abundant proteins, that include human serum albumin (approximately % of protein mass), apolipoproteins, coagulation markers and acute phase proteins of the immune system, that are measured in mg to ug/ml level, b) the organ-tissue secreted proteins without functional role in the circulation such as alt, ast or troponins, measured in ug to ng/ml, c) the signaling molecules, such as cytokines or insulin, measured at ng to pg/ml level. this extreme range while keeping in mind the above limitations, we next present the efforts to identify pathophysiological pathways and develop diagnostic models for nafld with the use of proteomics. several studies have investigated hepatic proteome alone or in combination with blood proteome, either in animal models or in humans with nafld, aiming to answer fundamental pathophysiological questions [ ] [ ] [ ] . in one recent study, the levels and cellular distributions of , liver proteins and , phosphopeptides have been assessed in the liver of mice developing hepatic steatosis due to high fat diet (hfd). this provided important j o u r n a l p r e -p r o o f fundamental information about the reorganization of organelles, lipid accumulation and cellular dysfunction that occurs with nutrient overload [ ] . in another study, proteomics in liver biopsies identified almost proteins that their levels significantly differ in patients with nafld compared to obese metabolically healthy individuals. the proteins that their levels were increased in nafld were involved in ppar-signaling and extracellular matrix-receptor interactions whereas the ones that their levels were reduced, were mainly localized in mitochondria and participated in oxidative phosphorylation [ ] . in a combined hepatic phosphoproteome and serum proteome analysis of biopsy-proven nafld subjects, the ask -mapk pathway that is activated by il- was recognized as important for liver fibrosis, indicated by its strong association with higher % of hepatic collagen. in serum, alpha- macroglobulin and coagulation factor v were strongly associated with hepatic collagen [ ] . it has thus been suggested that these pathways can potentially serve as therapeutic targets. in another approach, proteomic differences in liver tissues of obese patients with vs without t dm were investigated. the analysis identified proteins, from which were significantly different between the two groups. levels of proteins involved in methionine metabolism and especially of glutathione were reduced whereas levels of other proteins involved in oxidative stress were increased in the t dm group [ ] . expanding on complications of the disease, the proteome of liver tissues with nafld from mice developing hcc has been assessed. differences in tumor suppressor genes and oncogenes were observed, which were further investigated in human samples with nafld and hcc. among them s a , which is secreted by cancer cells and stimulates cell j o u r n a l p r e -p r o o f proliferation and migration, was identified and associated with high-grade hcc and poor prognosis [ ] . a small number of studies has aimed to develop diagnostic models with the use of proteomics (table ) . when evaluating the results of these studies, we have to keep in mind the limitations and challenges related to such procedures, that are described below ( . conclusions -challenges -perspectives). in one of the early efforts, combined genomic/proteomic analysis involving liver tissues for assessing gene expression and serum samples for assessing proteins was performed in patients with different stages of nafld. a seldi-tof mass spectrometry (ms) identified approximately protein peaks with of them being significantly different between groups. at that timepoint, it was not possible to identify in which proteins these peaks corresponded to but their masses were compared with the masses of , serum proteins, which led to the identification of fibrinogen γ as one possible biomarker [ ] . in another study including eighty morbidly obese patients, seldi-tof ms has identified in serum three protein peaks that their intensity significantly increased with the severity of nafld, i.e. when moving from steatosis to nash. none of these peaks correlated with liver function tests or metabolic parameters and all of them returned to normal after bariatric surgery. with immunoseldi assay these peaks were recognized as alpha-and beta-hemoglobin subunits [ ] . one of the most comprehensive studies so far was able to identify , serum proteins in a population with nafld. among them were significantly different between nalf and nash with fibrosis stage or (f /f ) and between nash vs j o u r n a l p r e -p r o o f nash f /f group. these proteins were involved in immune system regulation and inflammation, in coagulation cascades (including fibrinogen γ demonstrated by previous studies), in cell growth and proliferation (including igfbp , igfbp , also shown in targeted approaches to be relevant [ ] ) and many of them were apolipoproteins (demonstrated also in [ ] ) and blood carrier proteins. six (fibrinogen β chain, retinol binding protein , serum amyloid p component, lumican, transgelin , and cd antigen-like) were selected to create a panel for the non-differential diagnosis of nafld and its stages, ranging from healthy liver status to nafl, nash or nash with f /f showing good to very good diagnostic accuracy (aurocs . , . , . , . , respectively). this was though a study with small sample size and its findings have not been replicated so far [ ] . in another approach, diagnostic models for differentiating between nafld vs healthy controls among the obese population undergoing bariatric surgery were developed. these models combined genomic information and especially the pnpla genotype, phenomic data (i.e. selected biochemical measurements such as glucose, insulin, lipid profile, alt) and proteomic data. specifically, a proteomic analysis identified proteins with of them being significantly different between the two groups. among them was aminoacylase- , sex hormonebinding globulin, galectin- , antithrombin iii and the hepatocyte growth factor. logistic regression models for differentiating between nafld and healthy status were trained by using each type of data individually (i.e. genomic, however, the utility of such algorithms is questionable given that ultrasound or other simpler algorithms (e.g. fatty liver index, etc.) show similar or higher sensitivity and specificity. additionally, diagnosing simply nafld does not initiate treatment decisions [ ] . other studies performed first a proteomic analysis in a relevant condition or in another species and then tested their findings in patients with nafld. for example, in one study, an hcc pig model was used and a proteomic analysis in serum was performed. the subsequent analysis identified the serum interalpha-trypsin inhibitor heavy chain as the most important protein corresponding with nafld progression and hcc development. this was further tested in human serum samples and showed that the levels of the proteins were higher in nafld patients with hcc compared to those without [ ] . similarly, in another study a proteomic analysis in plasma of patients with liver cirrhosis vs healthy ones was performed. this identified significantly different proteins which were involved in immune system regulation, inflammation, coagulation and fibrinolysis. several of these proteins were subsequently tested in nafld patients and were significantly different in people with nafld and normal glucose tolerance compared to nafld with t dm [ ] . proteomic studies have rather limited contribution to drug development against nafld so far. most of the treatment targets have been identified by animal studies supported by human physiology or epidemiological studies. proteomics have not been widely used in nafld research. the proteins and signaling pathways involved in the pathophysiology of the disease have been almost exclusively identified by targeted "hypothesis-driven" approaches based on data from mechanistic animal, observational human and in some cases large epidemiological studies. this is probably due to methodological challenges and technical limitations related to proteomics. we expect in the near future proteomics to be much more frequently applied in nafld studies. we expect that the main contribution of proteomics will not be in direct development of non-invasive diagnostic scores but rather in the identification of novel molecules and/or pathways with important role in the pathophysiology of the disease and with diagnostic and therapeutic potentials. metabolomics refers to the investigation of small molecules and metabolic products, such as aminoacids, fatty acids and carbohydrates. lipidomics are j o u r n a l p r e -p r o o f considered part of metabolomics and refer to the investigation of cellular lipids. numerous studies have investigated the metabolome and lipidome in mouse models or patients with nash. the evidence linking bile acid homeostasis as well as gut microbiome with the development and progress of nafld have been recently reviewed elsewhere [ , ] . in this section, we will focus particularly on the robust and extensive changes that are observed both in the hepatic (figure ) as well as in the circulating lipidome, which have led to the development of numerous diagnostic models for nafld as well as to the identification of novel therapeutic targets. the development of nafld is strongly associated with obesity and is characterized by increased lipid accumulation due to higher uptake of elevated ffa by unrestricted adipose tissue lipolysis and due to increased [ ] [ ] [ ] . in non-parenchymal liver cells, sfa stimulate the production and secretion of proinflammatory and profibrotic cytokines from kupffer cells and induce proinflammatory m polarization of macrophages [ , ] . additionally, they stimulate the secretion of chemokines from hepatic stellate cells that recruit more macrophages in the liver [ , ] . similar to sfa, lysophosphatidylcholines (lpc) have unfavorable hepatic effects. specifically, they stimulate er stress, cause mitochondrial dysfunction and increase apoptosis [ ] . additionally, they stimulate the release of hepatic extracellular vesicles from hepatocytes that trigger inflammatory procedures [ ] . moreover, the increased activity of the enzyme phospholipase a (pla ) that catalyzes the formation of lpc from pc, leads to the rapid depletion of pc which affects hepatocyte membrane integrity and results in hepatocyte apoptosis, high release of lipotoxic lipids and increased inflammation [ ] . additionally, pc deficiency reduces vldl secretion resulting in higher intrahepatic lipid degradation and formation of toxic intermediates. furthermore, the low pc/pe ratio affects lipid droplet stability, leading to the formation of larger lipid droplets. finally, lpc is metabolized by the enzyme autotaxin to phospholipid lysophosphatidic acid which stimulates liver fibrosis and development of hcc [ ] . ceramides are a type of sphingolipids whose hepatic levels are increased in nafld and correlate positively with disease severity [ , [ ] [ ] [ ] [ ] [ ] . in hepatocytes, ceramide promotes insulin resistance by inhibiting akt-mediated insulin signaling, impairs beta-oxidation, induces ros production, er stress and proinflammatory cytokine secretion, enhances de novo lipogenesis, cholesterol synthesis and triglyceride accumulation and induces apoptosis [ , [ ] [ ] [ ] [ ] . finally, ceramide stimulates fibrogenesis and angiogenesis by increasing extracellular matrix deposition and secretion of pro-angiogenic factors by hepatic stellate cells [ ] . [ , , ] . the increased ω- concentrations lead to the synthesis of eicosanoids by their enzymatic oxidation (especially of aa but also of epa and dihomo-γ-linolenic acid) with proinflammatory properties such as prostanglandines, thromboxanes and leukotrienes which induce hepatic inflammation. this occurs in expense of the synthesis of spms that mainly act to restore normal cell function and thus reduce chronic inflammation and fibrosis [ , ] . finally, the high ω- to ω- ratio is associated with an impaired fads activity that can affect cell membrane phospholipid composition resulting in membrane deficiency, cell necrosis and extracellular deposition of lipotoxic lipids that can aggravate hepatic injury [ , ] . many studies have reported several diagnostic models based on metabolomics, lipidomics alone or combined with other biochemical and clinical parameters for the diagnosis and staging of nafld (table ). the best studies use populations of biopsy-proven nafld to create non-invasive algorithms based on blood measurements. the models aim to diagnose advanced fibrosis (≥ ) (in most studies), differentiate nafld from healthy status, distinguish between nash and nafl or nash vs non-nash (in many studies), or detect the presence of fibrosis independently of its stage (in few studies). challenges related to the development of these models are [ ] . based on the above findings and methodology, the owliver® test was created, which includes triglycerides and aims to discriminate nash from nafl patients. the test, which was initially trained in white patients without t dm, performed poorly in a multiethnic cohort of patients with t dm (auroc= . ). this shows that patient heterogeneity is an important factor and that tests developed in non-diabetic populations may not be as accurate in patients with t dm [ ] . in another study, information from metabolomics and lipidomics were combined with biochemical and genotypic measurements to create an algorithm for differentiating nash from non-nash (i.e. combined healthy and nafl). subjects were used to train the algorithm and to validate it. the final model consisted of ast, insulin, pnpla genotype, glutamate, two aminoacids (glycine and isoleucine) and two lipids (lysopc : and pe : ) and was able to differentiate nash from non-nash with % sensitivity and % specificity [ ] .however, the non-nash population consisted both of healthy subjects ( %) and of nafl ( %). it is though important to be able to distinguish accurately nafl from healthy status. additionally, sensitivity and specificity in most tests is high when healthy subjects are included, but the same tests may not perform as well when nafl is directly compared with nash. in a smaller proof-of-concept study a different approach was followed. a lipidomics, glycomics and targeted hormonal analysis was performed. results were analyzed with different machine learning methods and different diagnostic models were reported for classifying the subjects simultaneously to healthy or nafl or nash group. the models consisted of varying number of j o u r n a l p r e -p r o o f variables (from up to ) and demonstrated high sensitivities and specificities [ ] . the variables included in the models were mainly lipids and often adiponectin, which its protective role against steatosis, inflammation and liver fibrosis is well established [ , , ] . a validation cohort did not exist, but / of the study population was used to develop the models and / to validate it, with this procedure being repeated times with several slices of data. this approach helps to control against random results but it still does not address population heterogeneity. nevertheless, the ability to diagnose between three and not two conditions (healthy, nafl, nash) can be attractive for large screenings of high-risk population in primary care setting and without the need for any additional imaging [ ] and should be further evaluated in future studies. several studies have attempted to develop non-invasive algorithms for differentiating between advanced (stage - ) vs not advanced liver fibrosis (stage - ). in one of the most comprehensive studies so far, a cohort of subjects was initially used to develop an algorithm after a metabolomic and lipidomic analysis, which finally consisted of metabolites/lipids. among the parameters, six were cholesterol-derived precursors of steroid hormones which were significantly reduced in advanced fibrosis, one was the primary conjugated bile acid, glycopholate, that was increased in advanced fibrosis and one the aminoacid taurine that is associated with bile acid conjugation and its levels were decreased in fibrosis. finally, the other two parameters were palmitoleate acid and fucose, which were both higher in advanced fibrosis. increased fucosylation, as described below (s. glycomics) is one of the main findings of studies that investigated alterations in circulating glycome sensitivity and % specificity for detecting advanced fibrosis, which was higher compared to fib- index ( % specificity) and nafld fibrosis score ( % specificity) [ ] . finally, very few studies have tried to develop such models in pediatric population. the most comprehensive one so far included children and adolescent with biopsy-or mri-proven nafld and without the disease. metabolomics identified metabolites of interest which together with waist circumference, whole-body insulin sensitivity index and triglycerides could detect the presence of nafld with % specificity and % sensitivity [ ] . nafld is considered an unmet clinical need. several treatments that are currently in the pipeline restore many of the abnormalities observed in the lipidome. specifically, agonists of farnesoid x receptor (fxr) reduce lipotoxicity by promoting mitochondrial beta-oxidation, decreasing de novo lipogenesis and stimulating cholesterol excretion [ ] . in phase clinical trials and in an interim analysis of an ongoing phase clinical trial, obeticholic acid reduced hepatic steatosis, inflammation and fibrosis compared to placebo [ , [ ] [ ] [ ] . dual pparα and pparδ agonists (e.g. elafibranor) also stimulate mitochondrial and peroxisome β-oxidation as well as omega-oxidation [ - j o u r n a l p r e -p r o o f ]. pparγ agonists reduce circulating ffa and consequently the flow of sfa to the liver, they improve insulin sensitivity and guide macrophage and kupffer cell polarization from the proinflammatory m phenotype towards to the anti-inflammatory m one [ , ] . various selective pparγ modulators, dual pparα/γ agonists (e.g. saroglitazar) or pan-ppar agonists (e.g. lanifibranor) have shown promising results in human studies and are currently under evaluation in phase or phase clinical trials [ , [ ] [ ] [ ] . in this context, it was recently announced, that lanifibranor met the primary endpoint of a reduction in steatosis activity fibrosis score, including nash resolution without worsening of fibrosis in a phase ii clinical trial, thus being the first study that met both fda and ema regulatory endpoints for accelerated approval [ ] acc inhibitors (e.g. firsocostat) attenuate the function of acetyl-coa carboxylase (acc) that catalyzes the conversion of acetyl-coa to malonyl-coa. consequently, malonyl-coa is reduced and hepatic de novo lipogenesis as well as mitochondrial fa beta oxidation are downregulated [ , ] . in humans, acc inhibitors reduce hepatic steatosis and on-going clinical trials will evaluate their effect also on inflammation and liver fibrosis [ , ] . regarding lpc accumulation, animal studies have shown that interruption of the generation of lpc from pc with inhibition of pla results in normalization of pc levels, prevention of nafld and reversal of nash and fibrosis [ , ] . several treatments are targeting also ceramide levels by aiming to reduce ceramide synthesis (de novo or through hydrolysis from sphingomyelin) or j o u r n a l p r e -p r o o f increase ceramide degradation. the reduction of ceramide synthesis can be achieved by inhibiting relevant enzymes (i.e. palmitoyltransferase) but it should be limited only to hepatic ceramides, since global reduction of ceramides can negatively affect nervous system [ ] . alternatively, lower ceramide synthesis can be achieved by blocking intestinal fxr activation [ ] . however, in that case, fxr blocking should be limited to the intestine and not affect fxr activation in the liver which acts beneficially. in other approaches, reduction of the accumulation of specific ceramides (i.e. c : ceramide) is targeted with the use of several inhibitors (e.g. des- ) or degradation of ceramides is stimulated by enzymes (e.g. acid ceraminidase). these have till now been tested only in animal studies and not in humans. many treatments have targeted to restore the increased ω- /ω- ratio by ω- supplementation. although the findings from animal studies had been very promising, and the first results from human studies had shown an improvement in liver steatosis, later studies could not demonstrate any benefit in steatosis, necro-inflammation or fibrosis in patients with nash [ , [ ] [ ] [ ] . newer experimental strategies are focusing not on the supplementation of ω but on restoration of the ω- /ω- ratio with the use of ω- desaturase that leads to the production of ω- from ω- pufas, showing beneficial effects in steatosis and necro-inflammation in animal studies [ ] . finally, different experimental approaches are aiming to decrease proinflammatory eicosanoids and increase the concentrations of spms. specifically, some of them are targeting the inhibition of pla α enzyme that catalyzes the cascade leading to aa and lysophospholipids generation, showing promising results in animal studies [ , ] . others are targeting j o u r n a l p r e -p r o o f the production of proinflammatory lipids by inhibiting the enzyme -lox [ , ] which catalyzes the lipooxygenation of aa to leukotriene lipids or they aim to block the binding of leukotrienes to their receptors [ ] . the -lox inhibitor tipelukast has been approved by fda for a phase iia clinical trial. finally, other approaches are aiming to increase the spms rvd , rve , protectin dx and maresin- leading to robust improvement of fibrosis in animal models of nafld [ , , ] . given that spms are rapidly inactivated by oxidoreductases and that spm receptors are downregulated in obesity, recent efforts are also focusing on improving the pharmacologic properties of them by creating either resistant to deactivation analogues [ ] or by enhancing their delivery on their site of function. altogether, most drugs in on-going clinical trials are targeting, among other mechanisms, hepatic lipidome in order to improve nafld. additionally, there is a significant number of medications that have shown beneficial effects on animal models of nafld by improving hepatic metabolome/lipidome and remain to be further clinically tested. metabolomics/lipidomics are one of the most investigated omics in nafld. glycomics refers to the comprehensive investigation of glycome, i.e. of glycan structures that circulate either as free glycans or they are bound to proteins (glycoproteins), lipids (glycolipids) or phospholipids (glycophospholipids). glycome is highly dynamic as it is affected by transcriptome, proteome, environmental factors (nutrition) and cellular secretory machinery [ , ] . glycans can affect protein morphology and interaction with other proteins as well as regulate nutrient storage and sequestration [ ] . additionally, they can protect cell stability and facilitate cell to cell interactions [ ] . glycosylation is the process of the formation of the glycoconjugates (glycoproteins or glycolipids) [ ] which can happen both intra-and extracellularly. in the extracellular glycosylation, enzymes secreted mainly j o u r n a l p r e -p r o o f from liver hepatocytes and platelets (i.e. glycosyltransferases) are involved [ ] . changes in glycan profile have been observed in numerous inflammatory diseases and in different types of cancer and have been often linked causally with the pathogenesis and progression of these diseases [ ] [ ] [ ] . in nafld, few glycomic studies have been performed so far (table ). these have either aimed to assess the circulating glycome in untargeted approaches or they have focused on the identification of glycans related to specific proteins (i.e. haptoglobin, transferrin, igg , iga , alpha- antitrypsin, ceruloplasmin). the findings of these studies suggest that in nafld and during its progression from nafl to nash and liver fibrosis higher concentrations in fucosylated, sialylated and agalactosylated glycans are observed. sialic acids in glycolipids or glycoproteins have diverse functions, including formation of a protective cell surface barrier, involvement in interactions of white blood cells with endothelial lining of blood vessels, recognition by pathogens and toxins and facilitation of cell migration by some cancers [ ] . circulating sialic acid levels have been positively associated with metabolic syndrome and with nafld [ , ] . fucosylated glycans are also involved in a variety of physiological and pathological procedures, including cell adhesion and migration, angiogenesis, malignancy, tumor metastasis as well as immune cell development and regulation [ ] . consequently changes in fucosylation have been reported in numerous inflammatory conditions, such as in rheumatoid arthritis, chronic pancreatitis, sickle cell disease and crohn disease [ ] . in the liver, fucosylation serves as a signal for the secretion of fucosylated glycoproteins from normal j o u r n a l p r e -p r o o f hepatocytes into bile [ ] . in ballooning hepatocytes, which are observed in nash, the fucosylation-related sorting machinery is dysfunctional which may result in the secretion of the fucosylated glycoproteins in the sera instead of bile [ ] . similarly, hypogalactosylation (lack of galactose in the formed glycoprotein or glycolipid) especially of igg has been associated with inflammatory response and with a number of autoimmune diseases [ ] . most of the glycomic studies in nafld have tried to identify glycans or glycoproteins that can serve as blood biomarkers for differentiating between nafl and nash or for detection of the presence of liver fibrosis and its stage ( table ). the diagnostic accuracy of most of these tests has been limited, with some reporting higher accuracy for diagnosing nash and others for diagnosing advanced fibrosis. this suggests that although changes in circulating glycome/glycoproteins are observed in nafld, these are not sufficient in order to be used alone for the development of diagnostic models of the disease and thus combination with other clinical or biochemical parameters are needed. in this context, fucosylated-haptoglobin showed below % accuracy at differentiating nafl from nash, but - % when combined with mac bp [ ] . similarly, in our study, which detected with the use of mass-spectrometry the highest number of glycans so far, the concentrations of glycans in serum were able to differentiate between the presence of liver fibrosis or not with % sensitivity and % specificity [ ] . glycans alone could poorly differentiate between nafl and nash but when combined with lipid species in models of variables ( lipid species and j o u r n a l p r e -p r o o f glycans) the sensitivity and specificity for discriminating between nash, nafl or healthy liver status increases significantly. regarding potential treatments, no study thus far has investigated whether targeting glycome can be a therapeutic option in nafld. treatments aiming to decrease fucosylation are currently under evaluation in autoimmune diseases and cancer. in this context, inhibition of fucosylation with a -deoxyd-galactose, a fucosylation inhibitor, reduces inflammation in rheumatoid arthritis by decreasing inflammatory macrophages and th cells in the lymph nodes as well as by reducing the levels of tnfα, interleukin- (il- ), and antibodies to type ii collagen in the serum [ ] . similarly, inhibition of fucosylation by -fluorofucose suppresses the proliferation, migration and tumor formation of hepg liver cells [ ] . whether similar interventions can be beneficial in nafld by reducing inflammation, fibrosis or hcc formation should be addressed in future studies. glycomics are one of the least investigated "omics" in nafld. given the strong relation between glycoprotein-glycolipid formation and liver function, more studies assessing the potentials of glycans in biomarker discovery and drug development are needed. performed. thus, these models often do not account for population heterogeneity, which is crucial for the accuracy all non-invasive diagnostic panels. this has been repeatedly shown in studies that focused on patients with overweight, obesity and especially with t dm. several and diverse biomarker panels (including steatotest, actitest, nashtest- , cytokeratin- , fibrotest, owliver tests) were evaluated but none of them demonstrated optimal performance for diagnosing nash or liver fibrosis especially in patients with t dm [ , , , , ] . third, very few tests have been evaluated prospectively. this is a very important step, which even other simpler algorithms or imaging modalities have failed to pass [ ] . additionally, depending on their mechanism of function, medications may have different levels of impact on the metabolic and lipid profile in the liver which may affect circulating metabolome/lipidome and its ability to reflect the stage of the disease through a diagnostic algorithm originally designed in untreated patients. altogether, diagnostic models based on omics have yet to reach exceptional levels of performance, while satisfactory study outcomes need to be cautiously scrutinized and repeatedly validated into large and diverse patient cohorts both cross-sectionally as well as prospectively. to this direction, fda has recently approved the parallel evaluation of diagnostic biomarker panels [ ] metab j o u r n a l p r e -p r o o f (rs ) impairs pufa transfer from dags to pcs, thus increasing pufa in tg and dag. tm sf e k (rs ) impairs pufa synthesis, increases polyunsaturated ffas and prevents pufa incorporation into tgs and pcs. both mechanisms lead to impaired vldl synthesis and lipid droplet hydrolysis. gckr p l (rs ) incites glycolysis, glycogen deposition and de novo lipogenesis by disinhibiting glucokinase. epigenetic modifications characteristic of nafld progression include cpg site hypermethylation, thus reduced expression, of genes pertaining to lipid and aminoacid metabolism and stellate cell inhibition. hypomethylation, thus increased expression, of genes pertaining to tissue repair, inflammation, carcinogenesis and fibrogenesis, increases insulin resistance and further propagates the disease. methylation levels of cytoskeletal, transcriptional, proliferation-related and metabolic genes are affected by age, fasting glucose levels and body weight. at the histone level, depletion of sirtuins and and hdac may propagate nash and increase susceptibility to mets, insulin resistance and hyperlipidemia. on the other hand, the glucose-activated hat p activates chrebp and thus precipitates stellate cell activation, elevates lipogenic gene expression and expedites steatosis, though these effects can be attenuated by tannic acid. finally, the nafld transcriptome is characterized by overexpression of lipid metabolism, cellular stress, division and adhesion, extracellular matrix production and repair, cancer progression and immunomodulatory genes, whereas several pro-metabolic and insulin signaling genes are downregulated. mirnas, especially mir- , mir- and mir- a, are linked to steatosis, cholesterol metabolism, liver cancer, atherogenesis and mets, whereas other noncoding molecules, such as lncrnas, are indicators of nash grade and hepatocellular viability. the 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individual participant data pooled analysis key: cord- - me nopu authors: rabinowicz, shira; leshem, eyal; pessach, itai m. title: covid- in the pediatric population—review and current evidence date: - - journal: curr infect dis rep doi: . /s - - - sha: doc_id: cord_uid: me nopu purpose of review: coronavirus disease (covid- ) pandemic has major health and economic impacts. we review disease characteristics in children. recent findings: children comprise – % of the diagnosed cases, and typically suffer mild disease. the median age of infected children is . – years, and male/female ratio is . – . . common symptoms in children include upper respiratory symptoms ( – %), cough ( – %), fever ( – %), and gastrointestinal ( – %) symptoms. substantial proportion ( – %) are asymptomatic. death rates are up to . %. risk factors associated with severe disease are neonatal age group, male gender, lower respiratory tract disease, and pre-existing medical conditions. vertical transmission was reported. multisystem inflammatory syndrome (mis), characterized by fever, multisystem organ involvement, and laboratory markers of inflammation, causes critical illness in > % of cases and is increasingly reported from endemic countries. indirect effects of the coronavirus epidemic include higher rates of psychiatric morbidities, education loss, unhealthy lifestyle changes, and increased child neglect. vaccines are in clinical trials and immunogenicity has not yet been shown in children. summary: overall, covid- has lower incidence and causes milder disease in children compared with adult patients. mis is a rare severe complication more common in children. more data on the efficacy and safety of antivirals in children are needed. emerging in late , coronavirus disease (covid - ) has been spreading worldwide, with major health and economic impacts. by mid-august , the world health organization reported over million confirmed cases of infection with sars-cov- (severe acute respiratory syndrome coronavirus ), resulting in more than , death worldwide [ ] . according to current data, children show lower incidence of symptomatic disease and develop a milder course [ ] [ ] [ ] [ ] . we review the current evidence of epidemiology, clinical presentation, treatment, and indirect health consequences of sars-cov- on children. (pids) for references. the search was restricted to english language publications during january-august . pathophysiology sars-cov- is an enveloped, positive sense single-stranded rna virus with a glycoprotein spike (s) on the surface. cell entry requires binding of the s protein to the cellular receptor ace- (angiotensin-converting-enzyme- ) and priming of the s glycoprotein by the host cell serine protease tmprss [ ] . the milder morbidity in children, despite similar or higher viral loads compared with adults [ , ] , is the focus of multiple studies but has yet to be fully understood. the differences may be partly explained by several characteristics of the pediatric immune system. according to the hypothesis by carsetti et al., the immune system of children is highly prepared to novel pathogens, due to high levels of innate igm antibodies and the ability to rapidly produce natural antibodies with broad reactivity, in addition to the production of the antiinflammatory interleukin (il)- by neonatal b cells [ ] . additional suggested explanations are alterations in t cell populations in adults due to continuous antigen stimulation and thymic involution, varied levels of ace- expression in children, and the simultaneous presence of other viruses in the respiratory mucosa of children, competing with sars-cov- [ ] . furthermore, children have fewer comorbidities and a stronger pulmonary regenerative potential than adults [ ] . disease burden of covid- in children is difficult to determine because case definitions for screening, testing, and disease severity in children are not universal and the proportion of asymptomatic infected children is high. in addition, young children attending daycare may contract several febrile and respiratory illnesses in a course of a few months [ ] , and it is plausible that sars-cov- test is not routinely performed. in reports from countries that were severely affected early in course of the pandemic, children comprise - % the diagnosed covid- cases, underrepresented compared with other age groups [ , [ ] [ ] [ ] . the median age of the diagnosed children ranges from . - years in different reports, and data shows that children younger than year are disproportionally represented [ , , [ ] [ ] [ ] . like in the adult population, there is a male predominance [ , , [ ] [ ] [ ] ( table ) . the contribution of children in spreading the virus through the community is a field of uncertainty, mainly due to the high rates of asymptomatic infection at younger age groups. a recent report found that in only % of households of sick children, the child was the suspected index case. in the reminder, the child developed the symptoms following or together with a sick adult [ ] . the same finding was seen in a cohort of sick children from china [ ] . however, these cohorts may have been evaluated when educational institutes were closed, so children were less likely to contract the disease outside the house. in addition, recent reports show outbreaks in a high school and a summer camp [ , ] . this is an issue of concern considering recent evidence of equivalent or higher amounts of viral nucleic acid in children < years with mild to moderate disease, compared with older children and adults [ ] . as schools worldwide are set to reopen, proposed adjustments of the education system include universal masking, breaking classes into capsules, attendance on alternate days, outdoors classrooms, online lessons, temperature checks, and reconfiguration of ventilation and air conditioning systems [ , ] . the rate of asymptomatic children, ranging from . - % of confirmed cases, is higher than reported in adults and most probably represents a significant underestimation as many asymptomatic children are not screened [ , , , ] . the clinical presentation in adults ranges from mild illness to severe pneumonia. severe cases may suffer complications including acute respiratory distress syndrome (ards), acute cardiac injury, and thromboembolic complications. patients with severe disease have evidence of hyperimmune response with persistent fevers, elevated inflammatory markers (d-dimer, ferritin), and elevated proinflammatory cytokines [ , ] . in children, respiratory symptoms are the most common, followed by fever and gastrointestinal symptoms [ , [ ] [ ] [ ] (table ) . anosmia and ageusia are commonly described in adults [ ] but may be more difficult to elicit in young children and thus underreported [ ] . the rate of children with critical illness ranges from . - % of confirmed cases, probably reflecting population bias since some reports include mainly patients diagnosed in hospitals [ , , [ ] [ ] [ ] . data from the usa indicates a hospitalization rate of per , population in children < years, much lower than . per , in adults. however, a third of the hospitalized children required admission to the intensive care unit (icu) [ ] . the analysis of critical patients may indicate which children are at higher risk. in various reports, half of the children admitted to the icu had an underlying medical condition [ - , , ] . factors associated with icu admissions were neonatal age group, male gender, lower respiratory tract disease, and pre-existing medical conditions [ ] . infants aged < months comprised % of the hospitalized children in a recent report from the usa. however, this may have been due to the diagnosis of neonatal fever and not due to disease severity [ ] . because these data are driven from potentially biased datasets with over representation of symptomatic children, the association of young age and severity needs to be further investigated. death ensued in - . % of diagnosed children [ , , [ ] [ ] [ ] . in the european cohort of children aged - years, four children died, all were older than years, two had no pre-existing medical conditions, one had undergone human stem cell transplant (hsct) months earlier, and the other patient's condition was not specified [ ] . in a multinational study from north america on children hospitalized in the icu, two patients, aged and years, died; both had unspecified pre-existing comorbidities and had also prior gram-negative sepsis [ ] . in a cohort from new york, one patient with metastatic malignancy died [ ] . two cohorts from china reported one death each-a -month-old baby with intussusception [ ] and a -year-old boy with no further details [ ] . because mortality rates in pediatric cohorts are low, it is difficult to define risk factors and disease course leading to fatal result in pediatric population. a more distinct population among the pediatric population are neonates. early reports showed that infants of mothers infected in the last trimester have normal course and that amniotic fluid, umbilical cord blood, throat swab, and breast milk were negative to the virus [ , ] . however, more recent reports show clinical infections in neonates and suggest prevention procedures, such as isolation of the infant and physical barriers after the delivery [ ] [ ] [ ] . recently, vertical transmission with high placental viral load and neonatal compromise requiring resuscitation was documented [ ] . when screening neonates, a possible contamination of the neonatal swab by maternal sars-cov- should be taken into account [ ] , in addition to transmission of an undiagnosed maternal infection after birth. in a review of cases in the neonatal period, most of them were asymptomatic ( %) or had mild ( %) and moderate ( %) signs of clinical infection. the rate of severely ill patients was higher compared with older children ( % vs. up to % in the general pediatric population aged < years). dyspnea was the most common sign ( %), followed by fever ( %) and feeding intolerance ( %) [ ] . since may , several highly endemic countries reported an exceptional high incidence of multisystem inflammatory syndrome (mis) in children [ ] [ ] [ ] [ ] [ ] [ ] . several case definitions were proposed, all include fever, elevated inflammatory markers, and organ dysfunction not attributed to another infectious cause ( table ) . a minority of patients had been symptomatic prior to onset of mis onset, and the median interval from covid- symptom onset to mis onset is days [ ] . the higher rate of positive serologic tests compared with nasopharyngeal reverse transcription-polymerase chain reaction (rt-pcr) is suggestive of a late complication of the disease (table ) [ , , , , ] . according to the centers for disease control and prevention (cdc) report, in % of the patients, both were positive. similar to the acute covid- [ ] , obesity is a risk factor for mis, present in a quarter of the patients in the cdc report [ ] . suggested mechanisms for mis include viral mimicry, formation of immune complexes, and host immune cell activation due to viral superantigen sequences [ ] . besides fever, the most common presentations of mis are gastrointestinal (diarrhea, vomiting, abdominal pain), cardiovascular, mucocutaneous (rash, mucus membrane changes, conjunctival injection), respiratory (including sore throat), headache, and limb and periorbital edema [ , , ] . associated laboratory findings are elevated inflammation markers (neutrophilia, c-reactive protein, ferritin, erythrocyte sedimentation rate), thrombocytopenia, lymphopenia, elevated troponin and n-terminal pro-b-type natriuretic peptide (nt-probnp), hypertriglyceridemia, and elevated d-dimer and fibrinogen. some patients meet the criteria for macrophage activation syndrome (mas). the disease course is typically severe, with high rates of icu admissions, mechanical ventilation, and death (table ) . severe course is characterized by shock, and coronary aneurysms [ , , , , , ] . in addition to supportive care, children diagnosed with mis were treated with intravenous immune globulin ± aspirin, glucocorticoids, il- receptor antagonist, il- receptor antagonist, and tnf-α antagonist [ ] . in the larger cohorts, from the usa, % of the patients died, one of them reported that half of them had underlying medical conditions [ , ] . the clinical presentation was compared with that of kawasaki disease. however, children diagnosed with mis were older [ , , , ] , showed a greater elevation of inflammatory markers [ ] , and more frequent cardiovascular hemodynamic involvement [ , , ] . treatment in children infected with sars-cov- consists mainly on supportive care, including oxygen and advanced respiratory support, hydration, and antipyretics [ , ] . metered dose inhalers are preferred over nebulizers due to the decreased risk of virus dissemination [ , ] . the inclusion of children in early phase clinical trials of novel agents is usually delayed; hence, trial data regarding efficacy and safety are scarce [ ] . antiviral and antiinflammatory drugs may be considered in severely ill children and those at higher risk for severe disease, preferably as part of a clinical trial. severe disease is often associated with hyperinflammation and cytokine storm that may lead to acute respiratory distress syndrome [ , ] . hence, medications targeted to the immune system were suggested, in addition to antivirals. several medications were used in hospitalized children [ , ] , but not in the context of a clinical trial, making it difficult to accurately assess their outcome. the antiviral agent remdesivir was suggested as the preferred agent for treating covid- in children [ ] . this is an adenine nucleoside analogue that interferes with the virus' rna-dependent rna polymerase. remdesivir was used in children suffering ebola infection, but pediatric safety data were not separately reported [ ] . studies in adults showed it may have some benefit [ ] [ ] [ ] , and clinical trials including children are ongoing. currently, the nih recommends using remdesivir in adults with severe disease [ ] . the drug is available through the us food and drug administration emergency use authorization and compassionate use requests are reviewed by the manufacturer [ ] . a clinical trial is currently evaluating the pharmacokinetics in children [ ] . dexamethasone was found in the randomized-controlled uk-based recovery trial to reduce mortality in patients who require respiratory support [ ] . despite the enrolment of children, it is not clear if the analysis included children, so further data is still needed. the nih guidelines state that dexamethasone may be beneficial in pediatric patients who require mechanical ventilation and suggest treatment according to individual considerations in milder cases [ ] . another potential adjunctive therapy for covid- is convalescent plasma, with only scarce experience in adults [ , , ] . shekerdemian et al. reported the use of convalescent plasma in a child, but the results were not discussed [ ] . currently, there are insufficient data to recommend either for or against the use of convalescent plasma for the treatment of covid- . clinical trials of covid- convalescent plasma in children are ongoing [ ] . other drugs were initially suggested for the treatment of covid- and their use in infected children was reported [ , ] . however, current recommendations are against their use due to questionable safety and efficacy [ ] . the antiviral lopinavir/ritonavir (kaletra) is a protease inhibitor used for treatment of hiv infection, including young infants. its suggested mechanism of action is inhibition of the sars-cov- proteinases papain-like proteinase and c-like proteinase, which are key enzymes in polyprotein processing [ ] . the nih recommends against its use outside of clinical trials in covid- due to lack of proven efficacy and concerns on its pharmacodynamics [ ] . hydroxychloroquine was suggested as another potential treatment. it was previously shown to inhibit sars-cov- entry into cells and interfere with the glycosylation of the ace- receptor (virus' binding site) and inhibit its spread [ , ] , and has additional host immunomodulatory effects [ ] . there is no solid evidence for its efficacy in adults [ ] . the drug is available and was previously used to treat children in other indications. due to substantial risk of qt prolongation, it is not recommended combining hydroxychloroquine with azithromycin. patients with known g pd deficiencies should be monitored for hemolysis [ ] . shekerdemian et al. reported its use in almost half of a cohort of children hospitalized in intensive care units in north america, but there is no analysis of the outcome of the specific treatment [ ] . the nih recommends against its use except for clinical trials [ ] . tocilizumab, an il- receptor antagonist, was used in adults with cytokine storm and hyperinflammation due to sars-cov- with conflicting results [ , [ ] [ ] [ ] , and in a small number of children admitted to icu, but the outcome was not specified [ ] . it is fda approved to treat cytokine release syndrome in children years of age and older, and in the recovery trial used in children > year [ , ] . screening and monitoring infectious complications especially latent tuberculosis should be performed prior and during therapy [ ] . the nih recently recommended against the use of il- inhibitors for the treatment of covid- , except for clinical trials [ ] . however, reports suggest its use in children who develop multisystem inflammatory syndrome (discussed later). other potential treatments are currently under evaluation, including antiil- (anakinra), interferon-beta, and ivermectin [ , , ] . following the concern regarding the consequences of sars-cov- infection in children with chronic diseases, several guidelines were published. a statement endorsed by the us pediatric infectious diseases society has recently proposed that children with severe immunocompromise, severe cardiac, or severe pulmonary diseases may be more likely to experience severe covid- disease. obesity and diabetes should be also taken into account, especially with comorbidities [ ] . the european academy of allergy and clinical immunology recommends treating children with allergic asthma, allergic rhinitis, or other allergy conditions according to usual guidelines. one exception to this is the advice to withhold biologics (antiil- rα, il- rα, and omalizumab) during acute covid- disease, since they are directed towards type response, which may counteract the "cytokine storm" seen in severe covid- [ , ] . the global initiative for asthma (gina) recommends continuation of inhaled asthma treatment and treatment with biologic therapies if needed. treatment with oral corticosteroids should be administered in the lowest possible dose in patients at risk of severe attacks [ ] . patients with immunodeficiency, either primary, secondary to other diseases or medical treatments, are advised to strictly follow national precaution recommendations and in case of a suspected infection be in touch with their physician [ ] . clear data regarding the severity of the disease in immunocompromised children are lacking. previously, immunocompromised children showed increased risk for severe lower respiratory tract disease due to seasonal coronaviruses [ • ]. on the contrary, a report from bergamo, italy, stated that children who underwent liver transplant did not develop clinical pulmonary disease during the outbreak [ ] . in adults, patients with malignancy and solid organ transplant recipients may be at increased risk of severe covid- disease and death. evidence regarding other types of immunocompromise is scarce [ ] . according to the guidance endorsed by the pediatric infectious diseases experts, patients with mild to moderate immunodeficiency were not proven to be at increased risk, and those severely immunocompromised (e.g., severe combined immunodeficiency, < days post-allogenic-hsct, hiv infection with cd count < % or < /mm , treatment with costimulation inhibitors like belatacept or abatacept, highdose corticosteroids, and more conditions) should be considered for antiviral treatment. the panel suggests reducing t cell immunosuppression in infected children [ ] . beyond the physiological manifestation of covid- , other pediatric health issues during this pandemic bear mention. data show that lockdown, combined with intense fear of covid- contagion, led to a dramatic decrease in patients seeking medical care for other emergent issues [ ] [ ] [ ] . in addition, ambulatory and screening services were postponed, including routine immunizations given to infants and children [ ] . lower immunization rates may diminish herd immunity for some vaccine preventable diseases and lead to the reemergence of other infectious diseases in children. this trend may wane as the epidemic continues and routine health seeking behaviors resume. masking of the medical staff poses another barrier for the routine medical care of children, making communication with pediatric patients challenging [ ] . school closure was a major step of infection control in many countries, affecting over . billion learners [ ] . the consequences to the child's well-being of these steps are numerous: learning loss, (especially for those in low-income settings), lack of access to school-provided social assistance, reduced physical activity, and a significant harm to social life. in low-and middle-income countries, where access to education may be limited, some children may drop out as a result of the indirect impact of the outbreak [ , ] . following school reopening, frontal teaching is partially replaced by remote online lessons. despite its innovative nature, this mode of studying is impossible to children affected by a lack of resources and requires extreme effort from children dealing with attention deficit hyperactivity disorder (adhd) [ ] . a study from china found that children's adhd behaviors significantly worsened during covid- outbreak in comparison with their normal state [ ] . the european adhd guidance group (eagg) adjusted its protocol, and frontal cardiovascular exam is no longer needed to initiate drug therapy, given normal personal and familial cardiac history and normal blood pressure and heart rate [ ] . additional effects of the epidemic on mental health include anxiety and depression [ ] [ ] [ ] . a survey among chinese school-aged children during lockdown revealed higher rates of anxiety and depression than usual [ ] . safe, secure, and supportive domestic environment for children requires engaged parenting. however, during these times, parents are challenged by unemployment, remote work, economic instability, home confinement, health worries, and home-learning of their children [ , ] . thus, children are at higher risk than usual to neglect abuse and domestic violence [ ] [ ] [ ] . despite increased incidence of child abuse and neglect during covid- pandemic [ , ] , the number of official reports to maltreatment lines in a few us states decreased sharply, raising a concern of under-reporting due to decreased contact with the insulted children. spotting signs for abuse and assessing home safety through distance learning should be practiced [ ] . the economic impact of the pandemic is likely to deepen unemployment and poverty worldwide. the resultant food insecurity and malnutrition are concerning [ , ] , particularly in young children who are the most vulnerable to its consequences [ ] . on the other hand, in wealthier countries, quarantine, social distancing, and parental difficulties led to unhealthy lifestyle modifications among adolescents with increased consumption of unhealthy foods and reduction in physical activity that may lead to obesity and sleep disorders [ ] [ ] [ ] [ ] [ ] . suggested steps to encourage physical activity during this period include incorporating physical activity into children's daily routine, using electronic devices for engaging children to physical activity, encouraging family members to join ongoing activities, and avoiding extended sitting [ ] . the standard precautions face masks, hand hygiene, and social distancing are extremely difficult to implement in young children. alcohol-based hand sanitizers contain above % ethanol, and according to the cdc should be used with adult supervision in children under years of age. the use of masks may be cumbersome in children. the minimal proposed age for mask use is years old. in younger ages, the smaller airways may interfere with breathing and the child may be unable to remove the mask on his own. in older children, size fitted mask and education on appropriate mask removal are needed [ ] . over sars-cov- vaccine candidates are currently evaluated, including nucleic acid-based, viral vector vaccines, and inactivated or recombinant protein vaccines. most of them focus on immunity against the viral spike (s) glycoprotein [ , ] . results of three vaccine trials were recently published: a phase trial of an mrna vaccine that encodes the s glycoprotein [ ] , a phase trial of a recombinant adenovirus type- vectored expressing the s glycoprotein [ ] , and a phase / trial of a chimpanzee adenovirus-vectored vaccine expressing the s glycoprotein (chadox ncov- ) [ ] . all showed both humoral and cellular immunogenicity to the spike glycoprotein. the most common reported side effects include fatigue, headache, and fever, with higher rates compared with other vaccines [ ] [ ] [ ] . none of the trials included children. in summary, children at any age may be infected with sars-cov- , with reduced frequency and severity compared with adults, although clear epidemiologic data is still missing. in addition, the recently identified mis may pose an additional threat. data on the outcome of antiviral treatments, the safety and immunogenicity of vaccinations, and better specification of high-risk patients in the pediatric population are still needed. as the pandemic continues to evolve, it is still hard to fully assess or forecast the mid-and long-term effects 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report of a phase / , single-blind publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -audh qa authors: novick, tessa k.; rizzolo, katherine; cervantes, lilia title: covid- and kidney disease disparities in the united states date: - - journal: adv chronic kidney dis doi: . /j.ackd. . . sha: doc_id: cord_uid: audh qa abstract racial, ethnic, socioeconomic, age, and sex-related health disparities in kidney disease are prominent in the united states. the coronavirus disease (covid- ) pandemic has disproportionately affected marginalized populations. older adults, people experiencing unstable housing, racial and ethnic minorities and immigrants are potentially at increased risk for infection and severe complications from covid- . the direct and societal effects of the pandemic may increase risk of incident kidney disease and lead to worse outcomes for those with kidney disease. the rapid transition to telemedicine potentially limits access to care for older adults, immigrants, and people experiencing unstable housing. the economic impact of the pandemic has had a disproportionate effect on women, minorities and immigrants, which may limit their ability to manage kidney disease, and lead to complications or kidney disease progression. we describe the impact of covid- on marginalized populations and highlight how the pandemic may exacerbate existing disparities in kidney disease. racial and ethnic minorities experience a disproportionate and complex burden of medical, mental health, and social needs. the coronavirus disease (covid- ) pandemic has exacerbated a particularly critical one-disparities in marginalized patients with kidney disease. latinx, blacks, and native americans/pacific islanders already make up nearly % of the population with kidney failure and compared to non-latino whites, these same groups are also more heavily impacted with covid- . further, many with covid- develop multiorgan failure, including kidney failure, and subsequently require kidney replacement therapy. in the context of the viral pandemic, here we describe many disparities and additional struggles of patient populations with kidney disease, including those faced by the aging and homeless, and among racial and ethnic minorities, immigrants, and refugees. the impacts from covid- on these populations as well as recommendations are summarized in table . older adults have been disproportionately affected by covid- , with greater risk for infection, , severe complications, and death. , among patients with covid- in new york city, the median age was years, and older, compared with younger patients, were more likely to require invasive mechanical ventilation, kidney replacement therapy, and die. this pattern is consistent worldwide. for example, among patients with covid- admitted to intensive care units in italy, the median age was years, and older versus younger patients experienced more severe acute respiratory distress syndrome and higher mortality ( % versus %). drivers of the variability in infection risk and severity with age are poorly understood and likely multifactorial. older adults are more likely to have underlying medical conditions like chronic kidney disease, which have been associated with covid- infection and complications. an estimated % of adults over age have chronic kidney disease, compared to . % of adults aged - years. among hospitalized patients with covid- in wuhan, older adults were more likely to have elevated creatinine on presentation, which was associated with times higher risk for in-hospital death. the relationship between age, immune response to sars-cov- infection, and chronic diseases remains poorly understood. the physical environment and limitations in the ability to self-isolate may also impact covid- risks for older adults. many older adults live in long-term care facilities, which are high-risk settings for covid- outbreaks. a confirmed covid- case was identified on february , at a skilled nursing facility in king county, washington. even if older adults live alone, many rely on visiting caregivers. many older adults cannot drive and rely on public transportation, which was theorized to be a primary driver in covid- propagation early in the pandemic. the impact of covid- on frailty and depression among older adults with kidney disease has not been studied. however, shelter-in-place rules have forced older adults to stay inside and remain inactive, and higher levels of inactivity exacerbate frailty and depression. frailty has been associated with increased risk of hospitalization and mortality among people with chronic kidney disease and kidney failure, and allograft loss among transplant recipients. a survey of older adults with cognitive impairment found that individuals who live alone were more likely to experience negative feelings. depression may have long-term effects on medication adherence and treatment engagement, and in people with kidney disease has been associated with lower quality of life and higher mortality. while nephrology practices are using telemedicine to limit sars-cov- transmission in clinical settings, older adults may also not have access to required technology for videoconferencing. further, hearing loss and low health-literacy may compound the already poor communication during telephone visits, or might lead to skipped visits altogether due to the elderly being unable to participate. however, the impact of telemedicine on older adults with kidney disease has not been studied, and research is needed to understand how to effectively deliver telemedicine to this population. the impact of covid- is especially prominent among unstably housed populations. living in shelters and frequent moves makes social distancing impossible, and limits the ability to comply with hand washing and other hygiene measures to prevent infection. , sars-cov- transmission in shelters has led to clusters of covid- in large cities. people experiencing homelessness are more likely to have chronic medical conditions than the general population, and if infected, are at increased risk for severe complications. mortality among unsheltered homeless adults is times higher than the general population, and whether covid- will inflate relative risks over time is unknown. having kidney disease may intensify risk for covid- infection for people experiencing unstable housing. a study in san francisco found that chronic kidney disease patients experiencing homelessness have higher acute care utilization than stably housed counterparts, and greater exposure to hospitals increases covid- exposure. in boston, out of asymptomatic people tested at a homeless shelter were positive for covid- , indicating high rates of asymptomatic transmission. similarly, homeless dialysis patients might have increased risk for exposure and infection due to inability to attend dialysis and increased reliance on emergency departments for treatment. , how the covid- pandemic will impact kidney disease outcomes for patients experiencing unstable housing is unknown. prior to the covid- pandemic, a study in baltimore found that chronic kidney disease patients experiencing housing insecurity were % more likely to post-pone needed medical care. this population may not be able to participate in needed telemedicine visits due to lack of required technology, and be even more likely to postpone needed treatment during covid- than before. untreated chronic kidney disease and comorbidities may lead to progression, and ultimately exacerbate existing socioeconomic disparities in kidney failure. , sex differences clinical and socioeconomic gender-specific consequences of covid- will be likely to have ripple effects across the kidney failure community. though the prevalence of chronic kidney disease is more common in women, men have higher rates of kidney failure progression and mortality. early covid- data shows a similar trend: in new york city, . men per , have died, compared with . per , women, with almost twice the number of hospitalizations among men compared with women with similar trends observed in china and italy. a recent meta-analysis of reports representing , cases demonstrated no differences in cases between men and women, but did show an increase in intensive treatment and mortality in men compared with women . though the etiology explaining this difference still remained unclear, differences in immune response may play a role. male patients with kidney failure are thus at a unique disadvantage given increased risk of morbidity and mortality both from kidney failure and covid- . further research will be needed to demonstrate sex as a determinant of adverse sequelae from covid- . conversely, the socioeconomic effects of the covid- pandemic will have a inequitable effect on women. one in three jobs held by women are essential, with the highest proportion in the healthcare industry. as such, women account for % of us health care worker covid- infections. the employment losses related to social distancing are in predominantly female employment sectors, in addition to the pressing need of child care at home largely falling to women. incidence of domestic abuse cases may rise, as additional time at home means more time around an abusive partner. further, complications with pregnancy, or unintended pregnancy, may be a consequence of restricted access to care. women with chronic kidney disease are more likely to belong to a racial or ethnic minority group, be a single mother, and have a low income and low-level education , placing this community at higher risk of exposure and economic challenges due to lost employment and childcare needs. as such, existing and future community services and social policies should continue to support women throughout the pandemic-especially those in disadvantaged and resource-poor communities. the covid- pandemic exposes the systemic interplay of race, social determinants of health, and comorbidities. the incidence of kidney failure is . times higher for blacks, and . times greater for latinx, with higher rates of progression to kidney failure in both communities. the etiology for this disturbing finding is multifactorial in the setting of structural racism and individual factors, including undertreated metabolic conditions and cardiovascular disease as well as poor access to care, and socioeconomic factors such as living conditions, education, and health literacy. notably, these same factors are exposed in the covid- pandemic. though data collection by race and ethnicity on the covid- pandemic is sparse and incomplete, limited data shows black, latinx, and native americans make up a disproportionate number of covid- related deaths and cases across the united states. , in new york city, the age adjusted mortality rate from covid- for blacks is double that of white and asian residents, and age adjusted case rate is highest in the latinx and black community. among the navajo nation, the infection rate has surpassed the state of new york. health disparities are primarily driven by structural racism, defined as "the ways in which historical and contemporary racial inequities in outcomes are perpetuated by social, economic, and political systems, including mutually reinforcing systems of health care, education, housing, employment, the media, and criminal justice." racial and ethnic minorities, for example, are more likely to live in crowded living conditions such as densely populated urban areas, and multigenerational households. they are over represented in homeless shelters and prisons, and are more likely to live in poverty. blacks make up a majority of those living in poverty in urban areas. . furthermore, racial and ethnic minorities are disproportionately essential workers: % of latinx or black workers are employed in service industry jobs, compared with % of whites. further, access to care may be out of reach in states that have not expanded medicaid; it should be of no surprise these areas are associated with higher mortality and lower life expectancies. in the covid- era, there will likely be further evidence of racial disparities in health care access due to loss of insurance, poor quality of care, and disproportionate distribution of testing and resources. moreover, language barriers in some minority communities limits contact tracing and preventative outreach. thus, the aggregate of kidney failure and covid- in minorities portends a potentially lethal combination. in the wave of the pandemic, myriad health disparities will disproportionately affect minorities with kidney failure and therefore must be fully considered when evaluating actionable prevention strategies. the impact of covid- on immigrant populations has been profound. undocumented immigrants are disproportionately essential workers, risking daily exposure to many immigrants work low-wage jobs in the service and hotel industries and are among the millions in america out of work. the $ trillion economic relief package will not reach the undocumented population as they face unemployment and mounting financial resource strain. many support large families, and will have little to spend on medications, healthy food, and other health-related social needs. increasing financial resource strain increases risk for incident albuminuria and rapid kidney function decline, and the economic impact of covid- alone may result in a higher prevalence of kidney disease and kidney disease progression in this population. undocumented immigrants are not eligible for the affordable care act, and there are an estimated . million without health insurance. without insurance, undocumented immigrants do not have consistent access to primary care, and undiagnosed or poorly controlled chronic conditions increase their risk for severe complications from covid- if infected. without primary care undocumented immigrants do not have a place to call for guidance or testing if they experience symptoms, and many rely instead on emergency services. increased reliance on acute care increases their changes for exposure to covid- , and unnecessarily contributes to overwhelmed hospital systems if they could otherwise be managed at home. the rapid transition to telemedicine during covid- has potentially increased the number of immigrants without nephrology care, and led to suboptimal care for those connected to nephrology. undocumented immigrants may not seek care or testing due to fear of the public charge rule and repercussions from the u.s. immigration and customs enforcement agency. this rule, which went into effect in february , states that "aliens are inadmissible if they are unable to care for themselves," and discourages them from seeking assistance. this population often faces fear and mistrust of the medical system, which might be worse if they cannot see the clinical staff or environment. , for those who do seek care, the technology needed for telemedicine visits complicates clinical encounters that are already threatened by low health literacy and language barriers. undocumented immigrants attempting to establish care might not know where to call, or if they will be understood when they call. additionally, providers are forced to have nuanced discussions over the phone, with translators, and without assistance from facial cues, about complex kidney management issues and dialysis planning. pre-dialysis nephrology care is an established predictor of early mortality among incident dialysis patients. there are . million latinx in the us as of , and that number is expected to double in the next twenty years. the prevalence of kidney failure among latinx populations is -fold higher than whites. research is needed to determine whether the lack of, or flawed, nephrology encounters during covid- will exacerbate existing disparities in kidney failure among latinx populations. there are an estimated , - , uninsured immigrants with kidney failure in the u.s. in most states, this population relies on hospitals for emergency-only dialysis. the emergency medical treatment and active labor act (emtala) requires hospitals to treat anyone who enters with an emergency medical condition. these individuals do not have funding for outpatient dialysis, and emtala enables them to receive treatment when they present with emergency indications for dialysis. emergency-only dialysis is associated with worse morbidity and mortality , for patients, promotes burnout and emotional exhaustion for providers, and is extremely costly to the medical system. individuals who receive emergency-only dialysis have high acute care utilization and spend less time in the outpatient setting than those who receive standard dialysis. in one study, patients receiving emergency-only dialysis presented to the emergency room times in a -day period, on average. this population is at increased risk for severe complications and death if they are infected with sars-cov- , but they repeatedly risk exposure for themselves and their families every time they present for dialysis. fear of exposure to covid- may also promote waiting longer to present for dialysis and increase risk of out-of-hospital death. they are also unnecessarily utilizing emergency, inpatient and dialysis resources that could otherwise be used for patients with covid- during a time when resources are limited. the covid- pandemic is re-exposing an underlying and long-standing set of critical weaknesses in our nation's health care system, especially relevant to the stark burdens already faced by many underserved populations. numerous complications and struggles with kidney disease have unfortunately intensified in patient groups, and have risen to the forefront during a time of great patient need and distress. the risk for the elderly, the homeless, differences between sexes, and overwhelming threats to racial, ethnic, and underserved minorities and immigrants are compounding. all together, the broad and complex set of inter-connected disparities, both nationally and globally, will require a similarly broad and complex set of solutions. as the nation pulls together during these times of great uncertainty, it will be our continued responsibility to move the needle forward on the inequities faced by marginalized populations that have been unheeded far too long. spread of sars-cov- in the icelandic population early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region clinical characteristics of patients who died of coronavirus disease in china age, complexity, and crisis -a prescription for progress in pandemic clinical characteristics of covid- in new york city kidney disease is associated with in-hospital death of patients 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clinicians' perspectives on providing emergency-only hemodialysis to undocumented immigrants: a qualitative study association of scheduled vs emergency-only dialysis with health outcomes and costs in undocumented immigrants with end-stage renal disease • males have higher risk of progression of kidney failure and mortality from kidney failure • women with ckd are more likely to be a minority, low education and low income • higher burden of caregiving on women key: cord- -no mbg d authors: vandegrift, kurt j.; wale, nina; epstein, jonathan h. title: an ecological and conservation perspective on advances in the applied virology of zoonoses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: no mbg d the aim of this manuscript is to describe how modern advances in our knowledge of viruses and viral evolution can be applied to the fields of disease ecology and conservation. we review recent progress in virology and provide examples of how it is informing both empirical research in field ecology and applied conservation. we include a discussion of needed breakthroughs and ways to bridge communication gaps between the field and the lab. in an effort to foster this interdisciplinary effort, we have also included a table that lists the definitions of key terms. the importance of understanding the dynamics of zoonotic pathogens in their reservoir hosts is emphasized as a tool to both assess risk factors for spillover and to test hypotheses related to treatment and/or intervention strategies. in conclusion, we highlight the need for smart surveillance, viral discovery efforts and predictive modeling. a shift towards a predictive approach is necessary in today’s globalized society because, as the h n pandemic demonstrated, identification post-emergence is often too late to prevent global spread. integrating molecular virology and ecological techniques will allow for earlier recognition of potentially dangerous pathogens, ideally before they jump from wildlife reservoirs into human or livestock populations and cause serious public health or conservation issues. generated concern for the environment and thrust ecologists into a new political field where preserving the integrity of our global ecosystems was the priority [ ] . even so, the society for conservation biology was not established until [ ] . as a part of this transition, ecology shifted from a descriptive science to one of prediction, reflecting the hope that ecologists might mitigate changes which can have negative impacts upon the ecosystem. ecologists have branched out into the study of parasites and disease as it has become increasingly apparent that parasites are inextricably linked to the ecology of their hosts and environments, to the point where they have been a driving force in the evolution of sexual reproduction and in the shaping of biodiversity [ , ] . over the past years, disease ecologists have developed the study of parasites and pathogens in the wild. this knowledge has been synthesized into mathematical models which describe the dynamic properties of ecosystems and predict how parasites and pathogens flow through them. [ , ] . these models are becoming more commonly integrated into epidemiological studies that seek to predict outbreaks or periods of time when cross-species spillover risk is highest. parallel to this progress, the field of virology, particularly the subfields of molecular virology and viral evolution, have also been burgeoning, largely due to advances in technology that have made molecular assays and genetic sequencing more accessible to a greater number of scientists. the development of high-throughput sequencing has greatly increased our ability to efficiently detect known viruses as well as to discover new types of viruses, thereby improving our understanding of viral diversity, pathology and evolution. this increased capacity has spawned the development of new fields of study. for example, phylodynamics allows researchers to determine the origin of circulating viruses in space and time. mutations among viral strains can be used to investigate interactions among host species as well as long-range host movement via corridors and flyways. phylodynamic analyses can also inform livestock management practices, as was the case with foot and mouth disease in the united kingdom [ ] . conducting viral surveillance in animal reservoirs and invertebrate vectors can help explain circulation within host species; observed patterns of zoonotic transmission; and even allow for the prediction of periods of increased risk of zoonotic transmission (e.g., rift valley fever and rainfall [ ] ; west nile virus (wnv) and american robin (turdus turdus) migration [ ] ; as well as hantavirus in mice [ , ] ). understanding viral ecology in wildlife reservoirs and identifying high-risk human-wildlife interfaces is especially critical in the context of ever increasing globalization, whereby transportation networks facilitate rapid spread of pathogens well beyond bounds where traditional epidemiological methods can be effective [ ] [ ] [ ] . the influenza a (h n ) pandemic spread from the presumptive point of emergence in la gloria mexico to new zealand in just under a month [ ] while sars radiated from guangdong, china to different countries within several months [ ] . the negative impacts of emerging infectious diseases are not limited to humans. indeed, wildlife conservationists have documented several mass mortality events in other animal species. western lowland gorillas (gorilla gorilla gorilla) have been decimated by ebola virus [ ] and an especially virulent calicivirus, rabbit hemorrhagic disease virus, spread through both domestic and wild rabbit populations, resulting in tens of millions of deaths [ ] . in some instances the viruses have attenuated, while in others the animal populations have been brought to the brink of extinction. importantly, the risk from disease to humans and animals should not be separated. the global transportation network facilitated the introduction of infected vectors (e.g., mosquitoes) into new york and wnv caused both avian and human mortality, and this virus has subsequently spread across the united states [ ] . table . definitions of key terms used in the text. the formation of a hybrid population through the mixing of two ancestral, or long-separated, populations. a method for estimating historical population dynamics from a sample of sequences without assuming a predefined demographic model. coalescent theory a mathematical framework which describes the distribution of gene trees in populations. it provides mathematical methods for connecting demographic or ecological models with a phylogenetic tree. demography is the statistical study of populations. in the field of ecology, demography encompasses the study of the size, structure and distribution of populations, and spatial and/or temporal changes in them in response to birth, migration, aging and death. however, here we use a more rigid definition of demography-as the pattern and rate of population growth. the number of breeding individuals in an idealized population that would show the same amount of dispersion of allele frequencies under random genetic drift, or the same amount of inbreeding, as the natural population under consideration. the analogue of ne for viruses, the ‗effective number of infections' is related to the number of infected host individuals and to the number of new transmission events [ ] . a discipline which uses next-generation sequencing technologies to characterize the entirety of genomic material found in environmental samples. the molecular clock is derived from the hypothesis that sequence evolution, while random, occurs at stable rate such that the time since the divergence of two or more sequences can be estimated. recent ‗relaxed molecular clock' analysis can account for variation in the rate of sequence evolution through time or between lineages. the relations among a set of sequences showing which shares a most recent common ancestor with other sequences. the study of the principles and processes governing the geographical distribution of genealogical lineages. in the field of population genetics, population structure is defined as the absence of random mating within a population. this is the definition used here. in ecology, population structure is defined by several key parameters including number of individuals in a population, age distribution of individuals, probabilities of survival (or mortality), and rates of fecundity. a process that occurs in segmented viruses by which one or more segments ‗swap' to create a new viral genome. this drives the process of antigenic shift in influenza a viruses. the process by which new genotypes are created by the combination of distinct lineages. in sexual organisms it occurs during meiotic division, by the exchange of dna between different chromosomes or ‗crossing-over'. viral recombination occurs during viral replication and is an important factor in viral evolution (for more details see worobey and holmes, [ ] ). mutation of a virus such that it changes ‗back' to its wild-type state. the timing of an organism's schedule of reproduction and death. species with long life histories, also known as ‗k' strategists, tend to have low reproductive rates, stable populations, long generation times and long lifespans. where the parasite population is not randomly distributed among hosts, such that the variance is greater than the mean. the macroparasites in a host population are often best described by the negative binomial distribution such that a minority of hosts possess the majority of the parasites. r (basic reproduction number) in the case of viruses and other microparasites, r is the average number of secondary infections which an infection produces. as such it is a measure of parasite fitness. a host species that can independently maintain a disease and act as a source of infection to other host species. infection in reservoirs is usually more persistent and less harmful than that of other hosts. zoonotic disease a disease transmissible from animals to humans or vice versa. globalization, host ecology, host-virus dynamics, climate change, and anthropogenic landscape changes all contribute to the complexity of zoonotic viral emergence and disease, and create significant conservation and public health challenges. comprehensive and collaborative scientific approaches that transcend disciplinary boundaries are necessary to address these challenges. it is the goal of this paper to review new methods for understanding viral dynamics and illustrate how and when these techniques can be used by not only public health officials, but also disease ecologists and conservation biologists. the phylodynamic paradigm, established in [ ] , exemplifies the power of a multidisciplinary approach. it unites the ecological and evolutionary study of viruses and builds upon advances in sequencing technologies and coalescent theory, by which gene genealogies are reconstructed backward in time [ ] . the analysis of phylogenetic trees enables researchers to address many of the primary questions posed by disease ecologists (figure ). in some cases this approach can provide an estimate of a virus's basic reproductive number (r ), which is a measure of parasite fitness [ ] . phylodynamics has far-reaching applications for the control of viruses in both human and animal populations, in addition to being vital to our understanding of the interconnectedness between them. phylodynamic studies can be used to identify reservoir species as well as defining the spatial and temporal origin of emerging infectious diseases [ ] . they can also help to elucidate how these viruses spread following their emergence [ ] . firstly, chains of viral transmission can be extrapolated from the branching topology of phylogenetic trees. one example of the utility of this approach is with rabies virus. rabies causes thousands of human deaths a year in africa and has been implicated in the decline and local extinction of several populations of african wild dog (lycaon pictus) [ , ] . lembo et al. analyzed sequences of rabies virus from the serengeti, revealing that domestic dogs were the reservoir of the virus and that they had transmitted it to other resident carnivore populations on repeated occasions [ ] . this work has applicability in that it can be used to design efficient and effective vaccination strategies, both to alleviate current distress and prevent future outbreaks [ ] . secondly, by mapping the geographical origin of each sequence onto the nodes of phylogenetic trees, the geographical origin of a virus might be identified. wallace et al. ( ) [ ] used this phylogeographic approach to identify guangdong province, china as the most parsimonious origin of highly pathogenic h n strain of avian influenza and to delineate the most likely pathways of viral spread [ ] . however, a recent bayesian analysis of this data did not support the conclusion that h n had dispersed from guangdong to indonesia [ ] . instead, the bayesian analysis suggested it had spread to indonesia from guangxi or hunan in china. this example demonstrates that different statistical techniques may yield different conclusions. as yet, neither the bayesian nor the frequentist method is universally considered to be superior and there is much room for improvement as statistical phylogeography develops as a field. phylogeographic tools have also been applied by walsh et al. ( ) [ ] to locate the putative origin of the zaire strain of ebola virus. in an attempt to resolve the controversy over the time of emergence and spreading trajectory of ebola in the congo basin, they then used spatial data and two different tests for the impact of selection on the virus genome [ ] . where a virus is expanding in range, as the zaire strain of ebola virus appears to be, using a ‗landscape genetics' approach may help identify geographical barriers to viral spread and help identify vulnerable human or wildlife populations lying in the path of infection [ , ] . these phylogenies may reflect transmission chains, however sampling must be sufficient for them to do so, while recombination may obscure ‗true' relationships between viral sequences. (b) simple molecular clock theory, predicated on the neutral theory of molecular evolution [ ] assumes that mutation occurs at a constant rate over time, thus the time that has elapsed since a pair of virus strains diverged from a common ancestor may be quantified. methods that account for differences in the evolutionary rates of different strains, and for variation in these rates through time, have been recently developed [ ] . here variants represented by thick lines evolve much faster than those represented by thin lines. (c) using a phylogeographic approach, the location at which a sequence was sampled may be mapped onto the viral phylogeny and the likely spreading trajectory of the virus inferred. while parsimony approaches have been popular, powerful bayesian methods that account for uncertainty of dispersal process and historical phylogeny have been developed to reconstruct viral dispersal events [ ] . crosses on the phylogeny represent such viral dispersal events, in this example. (d) coalescent theory provides the basis for many phylodynamic approaches. here, circles on the same row represent temporally simultaneous infections. working back from sampled infections (red circles), lineages can be traced back to the most recent common ancestor (black circle) via hypothetical, unsampled ancestors (grey circles). the time it takes for sampled lineages to coalesce is dependent on a variety of variables (i.e., viral effective population size, population structure, selection, stochastic infection die-out and recombination). a variety of methods are available to test for selection and recombination. phylodynamic analyses are not without limitations. dense and representative sampling at a scale equivalent to epidemiological surveys is required to fulfill the potential of phylodynamics for understanding epidemics of rapidly-evolving viruses [ ] . the construction of phylogenetic trees from these viral sequences may be complicated by recombination and, in the case of segmented viruses, reassortment of viral genomes [ ] . as a result of these processes, the genes on a single viral sequence may have very different origins (see the discussion of the different origins of the hemagglutinin and neuraminidase segments of h n avian influenza in lemey et al. [ ] ). therefore, concatenated analysis of multiple genes may be confounded. the ability to construct phylogenies is further limited by the total viral genetic information available. genbank is a vast public database that contains records of genetic sequences; however, its usefulness is dependent upon the willingness and/or ability of individuals and organizations to submit viral sequences. governments and industrial institutions may be reluctant to report sequences of economically important viruses (i.e., avian influenza) due to the potential negative economic impacts that may ensue. although phylodynamics is currently encumbered by the aforementioned factors, there is hope for progress. advancements in coalescent theory will help us to deal with the phylogeny construction problems caused by recombination and reassortment. they will also facilitate better utilization of genomic and spatial data, provided these advancements are also accompanied by a simultaneous increase in computing power, which is also currently limiting. the utility of phylodynamics is not limited to questions of interest to virologists and disease ecologists. this approach may also inform investigations of host population biology and, in so doing, aid in the development of conservation policy. host molecular markers (e.g., microsatellites, mitochondrial dna) are used by conservation biologists and ecologists to infer population structure, historical demography and other critical features of wildlife populations [ ] and have proved particularly powerful when analyzed in combination (i.e., [ , ] ). recently, it has been demonstrated that the pathogens of host populations might also be useful to this end. research using helminths and bacteria has revealed patterns of ancient human migration and dispersal [ , ] identified ancient refuges of rodent and bird taxa [ , ] and shown that there was past contact between contemporary non-sympatric bat species [ ] . however, there have been few attempts to utilize viruses to this end (save [ , ] ). it is surprising that viruses have not been used more for the inference of host population biology since some of their characteristics make them ideal for doing so. most viruses have large population sizes and short generation times, and many replicate using a highly error-prone rna-dependent rna polymerase, causing them to accumulate many more mutations (nucleotide changes) per unit time than the host genomes [ , ] . consequently, viruses may provide information about host demographics on a shorter timescale than molecular markers of the host. one of the signature tools of phylodynamics, the bayesian skyline plot, might also be utilized to infer changes in historical population size of the host. these plots incorporate the use of a molecular clock and coalescent theory to infer historical changes in virus population sizes without assuming a predefined demographic model [ ] . it is important, however, that the timescale over which the evolutionary dynamics of the virus population can be reliably reconstructed is appropriate for the parameters of interest in the host population. unfortunately, the very characteristics that make viruses useful for estimating host population structure and demography may also impede the analyses. multiple substitutions can occur quickly in the viral genome and this will obscure the host population's actual evolutionary history. meanwhile, variations in the transmission mechanisms of viruses (vertical vs. horizontal) can alter the ability to accurately infer a virus' relationship to a host population. cross-species transmission is also problematic in that it can cause pathogen phylogenies to inaccurately reflect the history of their hosts [ ] . therefore, before the genetic information contained within a virus population can be used to infer the population structure and demography of the host, it is critical to test for congruence in the evolutionary history of the host and virus populations. this is accomplished by statistically comparing the respective phylogenies within the relevant timescale. viruses with high host specificity have a greater likelihood of exhibiting such congruence. feline immunodeficiency virus (fiv) is known for high host specificity and is, thus far, the only virus to have been used to elucidate changes in host population structure and size. from a phylogenetic analysis of fivpco, the fiv type specific to the cougar (puma concolor), biek, drummond, and poss ( ) [ ] inferred that the north american population of cougars became subdivided during the last century but subsequently expanded in both size and range. subsequently, antunes et al. ( ) [ ] used the distribution of fiv ple subtypes in the serengeti to infer that recent admixture has occurred between the region's lion (panthera leo) populations. these recent changes in felid population size and structure could not have been inferred from host genetic data. there is great potential for the further use of this technique by conservation biologists and ecologists, and for it to complement existing methods which utilize host genetic data. host genetic markers are used to define management units for conservation purposes [ ] . many ‗flagship' endangered species have long life histories [ ] , a feature that correlates with both extinction risk in certain regions [ ] and difficulty in reconstructing recent demographic history from molecular markers. because of the latter, the use of viral genetics to define management units may be an important avenue of exploration. in addition to aiding the definition of management units, viral data could be used to analyze the consequences of management activities and other environmental changes on target species. where viral genetic diversity exists in a spatially heterogeneous distribution, viral movement patterns could be used to study the migratory behavior of animals (as macroparasites have been [ , ] ). as such, researchers could monitor the use of wildlife corridors and the efficacy of control measures aimed at limiting the range of a host species [ , ] . where viruses can be readily amplified from non-invasively collected samples (see [ ] ), the above objectives could be achieved in a cost effective manner with minimal disturbance of the study species. viruses with specific transmission routes may also serve as proxies for behaviors related to transmission (i.e., sexually transmitted diseases). similarly, where cross-species transmission occurs, viruses might be indicative of types of sustained, direct contact between different, sympatric taxa which facilitate such transmission, for example predator-prey interactions [ ] . at the broader ecosystem level, inferences about long-term evolutionary processes might also be made by examining the phylogeographic structure of numerous host and virus populations of a region (see [ ] ). a major hurdle for both virologists and ecologists is defining the biodiversity of life. at present, scientists do not know the actual number of mammal species, much less the diversity of viruses they harbor [ ] . indeed, the diversity of viruses known to infect the house mouse (mus musculus), a staple in biomedical research, is not yet completely known. recent advances in genetic sequencing, including high-throughput sequencing and other -next-generation sequencing‖ techniques, as well as masstag pcr and microarray multiplex assays [ , ] have made the study of microbial diversity feasible [ ] . these technologies have facilitated a movement from classical virology, where the focus was on disease etiology, toward a broader discipline that considers the rest of the viral diversity or -the virosphere‖. metagenomic studies have used next-generation sequencing to study biodiversity in substrates such as ocean water and soil [ , ] . metagenomics has also been used to screen human and animal clinical samples in order to determine etiologic agents of disease or to describe the microbial flora normally present in a vertebrate host-in many cases the result has been the discovery both of novel pathogens and novel associations between clinical disease and agent [ ] [ ] [ ] [ ] . as technology becomes more affordable, and thus accessible, there will be increasing opportunities to ask large-scale questions such as: how does the virosphere vary across space and time? how does it vary across species? can we use this to define risk of cross-species transmission or to inform conservation efforts? and how might co-infections with these undiscovered viruses influence the dynamics of the more well known viruses? the paucity of information about viral diversity within a host poses problems for research progress. it is difficult to understand viral pathogenesis and transmission without completely understanding the dynamics of co-infections. indeed, it is currently difficult to ascribe a host's symptoms to an individual virus with any certainty because a virus' actions, and even its ability to infect the host, could be a function of another (possibly undetected) co-habitant of the host. evidence of interactions between co-infecting species has been clearly demonstrated [ , ] and it will be critically important to elucidate the interactions that occur between multiple pathogens as well as the combined effects they may have on a host's immune system. broadening our understanding of the diversity of pathogens that exist in human and animal hosts through wildlife and domestic animal surveillance will significantly improve our ability to recognize novel zoonotic agents in the context of a disease outbreak. phylogenetic information obtained from comparative sequence analyses can improve our understanding of the impact of sequence mutation on virulence, as well as inform decisions about vaccine development. a final noteworthy benefit of viral discovery efforts is that these techniques should be important for identifying candidates for future vaccines as a virus's most worthy competitor is often another virus. from a health perspective, vaccination is arguably the most important technology that has arisen from the study of viruses. vaccination offers a direct means of intervening in a host-pathogen system and it has become routine in many parts of the world. efforts to this end have resulted in the eradication and/or control of smallpox, polio, mumps, measles, rubella and most recently, rinderpest. vaccines take several forms including live-attenuated viruses; inactivated whole viruses; inactivated toxins and viral protein subunits, and these are often delivered in combination. while live-attenuated vaccines have been predominant, a new generation of techniques including gene delivery and nano-technologies are being used to develop highly-efficacious and safer vaccines, that have less risk of reversion [ ] . new types of administration methods are also being developed with oral, aerosolized and nasal vaccines currently on the market. these less invasive administration techniques decrease labor costs associated with administration and offer increased capacity for mass-dispersal of vaccines to both humans and free-ranging wildlife [ ] . vaccination campaigns aimed at both protecting threatened species and decreasing public health risks via animal vaccination have taken place. swiss health officials were pioneers in this field, using oral vaccines to control rabies in wild red foxes (vulpes vulpes) [ , ] . these vaccines were inserted into chicken heads which were distributed in the wild beginning in [ ] . as can be observed from the supplemental movie (video s ), their initial barrier approach evolved into a large-scale treatment of infected areas and resulted in rabies being successfully pushed back to and then eliminated from the swiss alps [ , ] . following the success of these trials, campaigns were conducted in western europe [ ] and canada [ ] with similar results, though the situation in the united states has proven more challenging. other successful vaccination examples include canine distemper virus in black-footed ferrets (mustela nigripes; [ ] ) and ethiopian wolves (canis simensis; [ ] ), as well as rabies in florida panthers (felis concolor coryi; [ ] ) and african wild dogs [ , ] . a caveat to this success is that there is growing evidence that vaccination against a specific strain of pathogen can result in inadvertent selection for related co-infecting strains. thus vaccination can influence the dynamics of a pathogen [ ] . the possibility of inadvertent viral strain selection highlights the importance of understanding the long-term evolutionary and ecological consequences of vaccination. indeed, where threatened or endangered animals are concerned, mishaps may prove disastrous. attenuated canine distemper vaccines did not provide immunity to critically endangered black-footed ferrets, while the use of a live canine distemper virus vaccine resulted in clinical distemper arising in one of the few remaining populations [ ] . ideally, long-term clinical trials with suitable animal models might avert these problems. these trials should also be used to provide an a priori understanding of how vaccination might shape future evolutionary processes. in contrast to the ferret experience, efforts with the endangered ethiopian wolf serve as an example of a successful vaccination program. wolf populations were suffering severe mortality due to rabies and distemper acquired from the wild dogs that shared their home range [ ] . on the basis of a spatially explicit individual-based model, which indicated rabies could be controlled in dogs given just over % coverage [ ] , knobel et al. executed an intensive vaccination plan [ ] . both the extent and duration of outbreaks in the treated areas were limited and, although monitoring and continued vaccination are required, the situation appeared to be under control in [ ] . wildlife vaccination campaigns are also being investigated as tools to limit public health risks. tsao et al. vaccinated mice in an effort to break the cycle of lyme disease and reduce the risk of emergence in human populations, in which it causes tens of thousands of deaths per year in the us [ , ] . in the same vein, griffing et al. [ ] have tested the efficacy of vaccinating american robins to interrupt the wnv transmission cycle. this species can absorb up to % of the potentially infective mosquito bites in early spring and is thus a key host in the wnv system [ ] . targeted vaccination of this single species could potentially result in herd immunity and reduce the risk of human infection as well as decreasing wildlife mortality. these works exemplify how ecological knowledge can be used to identify and exploit some of the heterogeneities which so often dominate the dynamics of pathogens. while the lasting efficacy of wildlife vaccination efforts has yet to be demonstrated with either endangered species or in breaking the transmission cycle of human pathogens, an increasing number of researchers are drawing attention to systems where it seems feasible [ , ] ; demonstrating that intricate knowledge of host and virus ecology can greatly reduce the amount of vaccine coverage that is necessary to control these viruses. the problems entailed by the sheer number of viruses, viral resistance, the explosive potential for spread, and the economic burden, make it clear that currently available vaccination methods do not provide a sustainable solution for either human or animal disease. the unambiguous indication is that researchers need to work towards the goal of developing a predictive framework where risk can be defined for different scenarios and not only to rank pathogens, and species, but also, places and times of year that can be identified as more or less precarious for global health. pending questions include: which geographic areas will experience more disease and conservation problems? which areas pose the highest risk for pandemic spread of pathogens? what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission events? and what are the relative roles of genetic relatedness and contact rate for transmission? some modeling work and reviews of historic data have been informative [ , ] , but novel uses of phylogenies of both viruses and hosts (as discussed above) provide promise for progress to this end, especially when coupled with high quality surveillance data. once we have this information, scientists will be able to design -smart surveillance‖ strategies whereby valuable vaccine resources can be efficiently targeted and efficiently distributed. ecological studies can effectively inform conservation as well as public health policy. gaining knowledge of reservoir host ecology can be critical for the development of eradication strategies. most viral disease systems are dominated by heterogeneities and identifying and understanding these can be crucially important when trying to interrupt the chain of events that leads to persistence. ecological studies of wnv have shown how forest fragmentation and decreased biodiversity can alter transmission among avian hosts as well as to humans [ ] . likewise, researchers have used satellite imagery to identify habitat characteristics that accurately predict the prevalence of sin nombre virus [ , ] , a hantavirus that uses the deer mouse (peromyscus maniculatus) as a reservoir host and it occasionally infects and kills humans [ ] . these studies epitomize the type of effort scientists will need to successfully fight viral pathogens in the future. however, piecing together emerging disease and conservation problems ex posto facto is only of limited value. increased pathogen surveillance and ecosystem process monitoring may provide the insight necessary to mitigate problems before they become serious human health or conservation concerns. this is especially the case for zoonotic viral pathogens where the reservoir hosts are known and a targeted approach is feasible. rodents rank as the number one reservoir of emerging and re-emerging zoonotic viruses [ ] . conveniently, these small mammals also present a manageable system for studying disease dynamics [ ] [ ] [ ] . individuals can be marked and sampled individually through time. their locations as well as their contacts with other individuals can be measured. as such, wild populations of rodents can be valuable as model disease systems to address relevant questions like: are there key hosts for transmission? how does prevalence vary seasonally or over time? what is the contact rate between the reservoir and humans? how do these pathogens flow through populations? the answers are of critical importance because they provide an indication of when and where there is increased risk of a zoonotic event whereby a human becomes infected, or when a species becomes at genuine risk of extinction. by monitoring and manipulating wild populations, one might also be able to identify factors that may increase a pathogens chance of emerging. for instance, what characteristics of hosts and viruses make them more or less likely to be involved in cross-species transmission? and what are the relative roles of genetic relatedness and contact rate for transmission? long-term monitoring and surveillance in reservoirs will also enlighten us to the kind of aggregations and other heterogeneities that exist through time and that and can be exploited with efficient vaccination campaigns. we are experiencing a global increase in the rate of emerging viral zoonoses, which are primarily driven by anthropogenic activities such as land-use change, agricultural intensification, and driven by global travel and trade [ ] . in order to adequately understand, predict and ultimately interrupt the processes by which zoonoses cross the species barrier from their natural reservoirs to humans, and then become established as human pathogens, comprehensive scientific studies that use the tools of ecology, virology, microbiology, and epidemiology are needed [ ] . the study of disease ecology has become an established discipline with advances in both the formulation of new theory as well as the integration of molecular virological techniques that provide important information about epidemiology, ecology and viral evolution, all of which has been applied to both health and conservation [ ] [ ] [ ] . because ecological systems are rife with heterogeneities and often have non-intuitive processes underlying their dynamics, it is critically important for scientists to use a comprehensive approach to understanding the population processes of an ecosystem before successful intervention strategies can be developed or implemented. admittedly this is a daunting task and it is often the case that scientists need to operate with less than complete information. where this is the case, a modeling approach is necessary to identify key processes that allow successful interventions. technological advances in molecular virology and genetics, as well as the expanded use of mathematical models in epidemiology and disease ecology have dramatically changed our ability to manage both conservation and health. finally, it is only with this type of interdisciplinary approach that considers free-ranging wildlife, domestic animals and humans as inextricable components of a single disease system, that progress can be made that will satisfy both conservation and public health needs. this is the essence of conservation medicine [ ] and indeed we believe a -one health‖ approach to infectious disease is necessarily the way forward. supplemental movie (video s ). are we in the midst of the sixth mass extinction? a view from the world of amphibians how many species? philos the future of biodiversity the impact of infection and disease on animal populations: implications for conservation biology disease and conservation emerging viruses emerging infectious diseases of wildlife-threats to biodiversity and human health risk factors for human disease emergence host range and emerging and reemerging pathogens rates of evolutionary change in viruses: patterns and determinants years of hiv the re-emergence 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endangered species an ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the lyme disease cycle mosquito landing rates on nesting american robins (turdus migratorius). vector borne zoonotic dis vaccination of wildlife to control zoonotic disease: west nile virus as a case study transmission dynamics and prospects for the elimination of canine rabies global trends in emerging infectious diseases ability to replicate in the cytoplasm predicts zoonotic transmission of livestock viruses land use and west nile virus seroprevalence in wild mammals satellite imagery characterizes local animal reservoir populations of sin nombre virus in the southwestern united states ecology of hantaviruses and their hosts in north america. vector borne zoonotic dis chain reactions linking acorns to gypsy moth outbreaks and lyme disease risk could parasites destabilize mouse populations? the potential role of pterygodermatites peromysci in the population dynamics of free-living mice, peromyscus leucopus predicting the emergence of human hantavirus disease using a combination of viral dynamics and rodent demographic patterns anthropogenic environmental change and the emergence of infectious diseases in wildlife collaborative research approaches to the role of wildlife in zoonotic disease emergence ecology of infectious diseases in natural populations the ecology of wildlife diseases conservation medicine and a new agenda for emerging diseases this work was supported in part by nih/nsf -ecology of infectious diseases‖ awards from the john e. fogarty international center (grant r -tw - s ) and an nih award (k ai ) from the national institute of allergy and infectious diseases. we thank c. j. wale, b. wale and h. ewing for their help in proofing the manuscript. key: cord- - ao rq authors: cossart, yvonne e title: the rise and fall of infectious diseases: australian perspectives, ‐ date: - - journal: med j aust doi: . /mja . sha: doc_id: cord_uid: ao rq australia has been fortunate in its experience with infectious diseases over the past century. by the s, many communicable diseases were controlled through a combination of high living standards, progressive adoption of vaccines and antimicrobial treatment. australian medical scientists have made substantial contributions to the understanding of many historically significant communicable diseases and global initiatives for control. new challenges have emerged as previously unrecognised viral infections have emerged, and microbial resistance to antibiotics has developed in many old pathogens. ongoing evolutionary forces, both environmental and social, change the balance between humans and microbes. the effects of these forces are most sorely felt in poor countries and communities. i n , when the british medical association launched the medical journal of australia, the medical profession and the general public believed that infectious diseases would soon be conquered. acrimonious th century disputes between the contagionists and the sanitarians had given way to an alliance which was steadily improving health. rising living standards reduced infant deaths from gastroenteritis, through better food and water hygiene, and reduced deaths from "consumption" (tuberculosis), because of better nutrition. the success of rat extermination in controlling plague in sydney provided a triumphant validation of new microbiological theories; joseph lister's carbolic spray was adopted by local surgeons; emil von behring's antitoxin treatment reduced mortality from diphtheria; and paul ehrlich's vision of a magic bullet to cure all infections was given credibility by the effi cacy of salvarsan (arsphenamine, an arsenic-containing compound) against syphilis. maritime quarantine provided a signifi cant, if not impregnable, barrier to the introduction of epidemic diseases. the mja refl ected the progressive mentality of australian doctors through reports on new international discoveries and leading articles. in reality though, infection still caused at least a quarter of all deaths, % due to tuberculosis alone. many of the victims were children or young adults. eradication of bovine tuberculosis produced a welcome fall in infant cases, but about cases continued to be notifi ed annually between and . the fi ne sanatorium buildings in "healthy" locations such as the blue mountains are reminders of the desperate attempts to combine sanitary and microbiological principles by isolating patients to prevent spread of the disease while they were treated with rest and diet. , , during world war ii (wwii), intensive screening of australian troops by miniature x-ray was followed up with bacteriological testing to identify patients with active infection, for whom treatment was compulsory. the success of this program prompted a postwar attempt to eradicate the disease from the civilian population, and the advent of streptomycin and sickness benefi t payments made compulsory treatment acceptable to the community. tuberculosis has again become resurgent in many countries. the high expense and long duration of triple therapy test the resources of the poorest countries. undertreatment allows emergence of multidrug-resistant strains for which treatment reverts to pre-antibiotic options. in australia, such strains are, mercifully, still uncommon. syphilis also imposed a high burden of chronic disease on society. in , about % of admissions to mental institutions and % of cases of aortic aneurysm in victoria were due to tertiary syphilis, while % of pregnant women had positive wassermann test results. the social stigma of sexually transmitted diseases and the heroic nature of combined mercurial and salvarsan therapy deterred many asymptomatic patients from seeking treatment. notifi cation of acute cases and compulsory treatment were not successfully implemented, even in the army. in civilian settings, policies of testing only female prostitutes doomed any hope of eradication. many acute infections also remained endemic. there were dedicated wards for patients with typhoid in general hospitals, and the prevalence and mortality of typhoid remained high until the advent of antibiotics. bedside vigils to await the crisis of pneumonia were all too familiar, and childhood infections claimed many young lives. in , gastroenteritis, diphtheria, scarlet fever, whooping cough and measles between them killed one of every live-born children. about as many more died from pneumonia and meningitis. in the interwar decades, diphtheria and pertussis vaccines were produced in the newly established commonwealth serum laboratories and school-based vaccination began. but in , public confi dence was shaken by the bundaberg tragedy, in which a multidose vial of diphtheria toxin-antitoxin became contaminated with staphylococci from the skin of a vaccinee. twelve other children died. this galvanised regulation of vaccine manufacture, safety testing and surveillance for adverse effects. however, almost a century on, the antivaccination movement still opposes mass vaccination of children in early life. during its fi rst few years of publication, the mja reported on mortality due to infection among wwi troops, which exceeded combat deaths. but the battlefi elds were also a clinical laboratory where the effi cacy of tetanus antitoxin was proven and typhoid vaccination was introduced. the mja also reported episodes which, with hindsight, showed that evolutionary forces were altering the balance between microbes and their human hosts. in , infl uenza killed more europeans than had perished in the war. the source of the pandemic strain remains obscure, but there is no doubt about the role of returning troops in its global spread. australia's isolation and its quarantine system protected it for some time, but eventually the country experienced a catastrophic outbreak. australia became an active participant in the subsequent international efforts to maintain surveillance and produce effective infl uenza vaccines. pivotal infl uenza studies led by frank macfarlane burnet at the walter and eliza hall institute were conducted during this period, and the institute's interests soon broadened into basic scientifi c research on many important infections. in the postwar period, the establishment of the australian national university strengthened the national capability in infectious diseases research. poliomyelitis was on the increase in the most "hygienic" countries of scandinavia, north america and australasia: a paradox for an infection spread by the faecal-oral route. this was the penalty for the delay in the average age when infection occurred, which was a consequence of improved hygiene. this in turn increased the numbers of clinical cases because the chance of neurological involvement increased spectacularly with age. the crippling legacy of infection made the sight of children with leg braces all too familiar in schools nationwide, while the invention of the iron lung enabled many patients with respiratory paralysis to survive. norman gregg's pivotal discovery of the role of rubella in causing congenital defects altered scientifi c attitudes to infections during pregnancy. it was almost two decades before cell culture techniques paved the way for vaccines against polio, and then other childhood infections. the growing list of vaccines demanded new combinations to reduce the number of injections needed, and the cost became an issue even in wealthy countries where the plummeting prevalence of vaccine-preventable diseases justifi ed the investment. in the second half of the th century, even the basic triple antigen (diphtheria, tetanus, pertussis) was unaffordable in many developing countries, but international philanthropy plus political support have progressively been mobilised, resulting in signifi cant reduction of vaccine-preventable disease. money is not the only problem. the projected global eradication of polio has stalled because confl icts in africa and pakistan have disrupted infrastructure and fanned ideological doubts about the political motivations of governments and charities. in the optimistic political climate of the post-wwii years, the world health organization undertook an unprecedented program to achieve global eradication of smallpox. frank fenner was chairman of the project's management commission. using edward jenner's th century vaccination technique and an international army of fi eld workers, smallpox eradication was achieved in . it remains the only example of intentional eradication of a human infectious disease. not all microbial evolution resulted in populations of organisms with increased virulence for humans. scarlet fever, after causing devastating epidemics in the late th century, declined in terms of incidence and mortality by the s. this was attributed to the loss of toxin-encoding genes from streptococcus pyogenes. conversely, recent resurgence of severe streptococcal infections underlines the mutability of "old" pathogens. antimicrobial drugs sulfonamides made their spectacular entry into medicine just before wwii and cut the mortality from pneumonia and puerperal fever dramatically. prontosil, a forerunner of all sulfonamide drugs, was not patentable, and manufacturers fl ooded the market with sulfonamide-like drugs. penicillin soon followed -with australian scientist howard florey being a key fi gure in its development -and was quickly adopted in military medicine. the antibiotic era had begun; as discovery followed discovery, it seemed that no bacterial infection would remain untreatable. osteomyelitis, empyema, rheumatic fever and subacute bacterial endocarditis disappeared from the wards, and gonorrhoea and syphilis notifi cations plummeted. patients expected to recover from septicaemia, pneumonia and even meningitis; but it did not take long for the fi rst drug-resistant organisms to appear. sophisticated medical technology has offered unprecedented opportunities to microorganisms. drug resistance has rapidly developed owing to selective pressure resulting from profl igate use of antibiotics in medicine and agriculture. australian hospitals experienced some of the earliest outbreaks of antibiotic-resistant staphylococci, which led to radical improvements in infection control. although most of the resistant organisms were no more (and often less) virulent than the original susceptible strains, their competitive advantage meant that acute urinary infections could no longer be reliably treated with ampicillin, nor gonorrhoea with penicillin, by the mid s. discovery of new antibiotics could not keep pace, and regulatory attempts to restrict their use have proved diffi cult. transfusion-associated hepatitis b infection was discovered during wwii. after the virus was identifi ed in , it was found that about . % of most western populations were chronically infected, but carrier rates of % or more were common in asia and africa. moreover, these rates diptheria* patients expected to recover from septicaemia, pneumonia and even meningitis; but it did not take long for the fi rst drug-resistant organisms to appear were maintained by silent transmission -from mother to infant during delivery, rather than by intravenous drug use or blood transfusion. before blood banks started screening donated blood for hepatitis b, transfusion was a major route of transmission of acute hepatitis b infection in western countries. the need for mass screening prompted development of commercial testing kits, which have revolutionised laboratory diagnostic services. the most concerning feature of hepatitis b infection was the recognition during the s that adult carriers often developed liver cancer, which was then the leading cause of cancer deaths in asia. rather than a rarity, hepatitis b was a major global health problem; this justifi ed universal infant vaccination, which was made possible by recombinant dna technology in the s. the hepatitis b vaccine was the fi rst human recombinant dna vaccine and the fi rst human cancer vaccine. other agents soon emerged from obscurity, often in response to changes in human activity. aids fi rst appeared in the gay communities of "world cities" in the early s -the downside of sexual liberation. the inexorable rise in prevalence and apparently inevitable mortality of hiv infection spurred public health initiatives and scientifi c investigation. in australia, safe-sex campaigns had an almost immediate effect in reducing numbers of new cases, as did controversial needle-exchange and harm-minimisation strategies for injecting drug users. global investment in research led to effective antiviral drugs and greatly extended the healthy lifespan of infected individuals in western countries. however, in the populous countries of africa, south america and asia, heterosexual transmission dominated and led to epidemics of neonatal infection via transplacental transmission. this social and economic catastrophe reawakened respect for infection, and also fuelled fear of nosocomial transmission of bloodborne viruses. the shadowy entity of non-a, non-b hepatitis unexpectedly proved to cause both liver cirrhosis and cancer. acute hepatitis c infection causes only minor symptoms, but the hepatitis c virus often establishes chronic infection with sinister consequences. tests were developed to screen donated blood and it soon became apparent that injecting drug use had silently amplifi ed prevalence of hepatitis c infection in young people in western countries. hepatitis c infection became the commonest indication for liver transplant in australia, and health authorities struggled to fi nd an effective control strategy. treatment was protracted and beset by the adverse effects of interferon, a key component of drug regimens that were initially used. newer drugs achieve high cure rates, but cost puts them out of reach for patients in poor countries, where the reservoir of infection remains high. human papillomavirus was the fourth "new" infection to engage late th century society. genital warts were well known to the ancient romans, and for centuries they were regarded as an embarrassing but harmless sexually transmissible disease. this attitude changed dramatically when their association with cervical cancer was established in the s. the high-risk types of papillomavirus produce "fl at" penile warts which are easily ignored, but results of cervical cytology testing made the cancer connection -it was noted that cells with telltale warty changes were often seen in cervical smears which had malignant or premalignant changes. in australia, preventive human papillomavirus vaccination was pioneered by ian frazer and viral dna detection has been added to screening by pap smear, but neither of these approaches are affordable in poor countries, where death rates from cervical cancer are highest. the challenges of demographic and environmental change in , old infections still lurk, while human and environmental changes create new opportunities. malaria -which depends on humans as a reservoir for the parasite and mosquitoes for transmission -was vulnerable to a combination of treating patients with drugs and spraying the environment with dichlorodiphenyltrichloroethane (ddt) to kill mosquitoes. large areas of the temperate zone and even the tropics became malaria free but, because mosquito eradication measures were inconsistently applied, drug-resistant parasites and ddt-resistant mosquitoes soon emerged. in the absence of an effective vaccine, prevention now relies on avoidance of mosquito bites by use of repellents, protective clothing and screens, plus an ever-diminishing number of effective prophylactic drugs. malaria has been holding its own in many countries, aided by global warming. military deployment of troops in exotic areas and largescale movement of refugees are associated with outbreaks of communicable diseases. cholera has been a recurrent problem when people seek shelter from war or natural disaster, most recently in haiti. hantaan virus caused over cases of korean haemorrhagic fever and almost deaths among american troops during the korean war. the soldiers were exposed through inhalation of aerosolised rodent faeces when camped in wilderness areas. other haemorrhagic fevers -such as ebola virus disease and lassa fever, for which native wildlife act as reservoirs -have caused human outbreaks with high mortality, particularly in sub-saharan africa. australia has several indigenous arboviruses, including murray valley encephalitis, and ross river and barmah forest viruses. as global population pressure drives clearance of forested areas for agriculture, humans have become targets for many infections carried by wild animals. in australia, outbreaks of hendra virus infection and the emergence of the australian lyssavirus have been linked to contact with fl ying foxes. severe acute respiratory syndrome (sars), which caused a deadly outbreak of respiratory disease centred in southern but was transmitted to humans via infected palm civets, which were often for sale in chinese markets. control of sars was achieved through international cooperation in identifying the new coronavirus and applying strict isolation procedures. even so, the high mortality brought home to the world the potential threat of contagious disease. memories of the infl uenza pandemic lent renewed vigour to the who surveillance system for respiratory infections that emerge from reservoirs of infl uenza viruses in pigs, horses, poultry and wild birds (the latter two in particular). alas, the ability to identify novel strains has not been matched by the ability to predict infectivity and severity of disease. intensive agricultural production also provides new routes of infection. mad cow disease resulted from the use of inadequately rendered animal-based food supplements for cattle which allowed the variant creutzfeldt-jakob disease agent to survive. infected cattle often reached maturity and were slaughtered for human consumption before they developed clinical disease, and then humans became infected. the ramifi cations of this outbreak were economic and social. more new infections will undoubtedly emerge as humans change their environment. these pressures also affect old infections such as tuberculosis, malaria, cholera and even plague. the lessons of the past should not be forgotten. for a hundred years, the mja has reported on the overall decline of most infections in our "lucky country" -the result of our high standard of living combined with rational treatment and control measures. aboriginal australians who endure poverty and limited access to medical resources have not shared this luck. this mirrors the disparity in communicable diseases mortality between industrialised and developing countries. abolishing this gap is the immediate priority for the forthcoming century. provenance: commissioned; externally peer reviewed anticontagionism between and the australian mortality decline: all-cause mortality - on the epidemiology of plague the beginning of antiseptic surgery in australia the diphtheria prophylactic of e. von behring sex, disease and society: a comparative history of sexually transmitted diseases and hiv/aids in asia and the pacifi c australian quarantine service. maritime quarantine administration. melbourne: arthur j mullett, government printer tuberculosis in new south wales: a statistical analysis of the mortality from tubercular diseases during the last thirty-three years consumption: report of a conference of principal medical offi cers on uniform measures for the control of consumption in the states of australia tuberculosis: its nature, prevention, and treatment, with special reference to the open air treatment of phthisis the epidemiology, mortality and morbidity of tuberculosis in australia: - health and disease in australia: a history the mortality in australia from measles, scarlatina and diphtheria the hazards of immunization the australian army medical services in the war of - . volume ii. canberra: australian war memorial natural history of infectious disease congenital cataract following german measles in the mother immunisation: a public health success progress toward global polio eradication -africa a successful eradication campaign. global eradication of smallpox severe streptococcal infections in historical perspective history of staphylococcal infection in australia control of fl uoroquinolone resistance through successful regulation jaundice occurring one to four months after transfusion of blood or plasma recent advances in the study of the epidemiology of hepatitis b vertical transmission of hepatitis b antigen in taiwan hepatitis b virus. the major etiology of hepatocellular carcinoma clinical and immunologic sequelae of aids retrovirus infection in and out of africa epidemiology of hepatitis c virus infection among injecting drug users in australia liver transplantation for hepatitis c-associated cirrhosis in a single australian centre: referral patterns and transplant outcomes papillomaviruses and cancer: from basic studies to clinical application hpv immunisation: a signifi cant advance in cancer control global malaria mortality between and : a systematic analysis cholera surveillance during the haiti epidemic -the fi rst years hemorrhagic fever with renal syndrome in korea emerging viral infections in australia bats are natural reservoirs of sars-like coronaviruses world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome an overview of bovine spongiform encephalopathy (bse) in britain indigenous disparities in disease-specifi c mortality, a cross country comparison: new zealand, australia, canada, and the united states key: cord- -duxm u v authors: sweileh, waleed m. title: bibliometric analysis of peer-reviewed literature on climate change and human health with an emphasis on infectious diseases date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: duxm u v background: assessing research activity is important for planning future protective and adaptive policies. the objective of the current study was to assess research activity on climate change and health with an emphasis on infectious diseases. method: a bibliometric method was applied using sciverse scopus. documents on climate change and human health were called “health-related literature” while documents on climate change and infectious diseases were called “infection-related literature”. the study period was from to . results: the search query found documents in the health-related literature and in the infection-related literature. the growth of publications showed a steep increase after . there were four research themes in the health-related literature: ( ) climate change and infectious diseases; ( ) climate change, public health and food security; ( ) heat waves, mortality, and non-communicable diseases; and ( ) climate change, air pollution, allergy, and respiratory health. the most frequently encountered pathogens/infectious diseases in the infection-related literature were malaria and dengue. documents in infection-related literature had a higher h-index than documents in the health-related literature. the top-cited documents in the health-related literature focused on food security, public health, and infectious diseases while those in infection-related literature focused on water-, vector-, and mosquito-borne diseases. the european region had the highest contribution in health-related literature (n = ; . %) and infection-related literature (n = ; . %). the usa led with ( . %) documents in health-related literature and ( . %) documents in infection-related literature. the australian national university ranked first in the health-related literature while the london school of hygiene & tropical medicine ranked first in the infection-related literature. international research collaboration was inadequate. documents published in the environmental health perspectives journal received the highest citations per document. a total of ( . %) documents in the health-related literature were funded while ( . %) documents in the infection-related literature were funded. conclusion: research on climate change and human health is on the rise with research on infection-related issues making a good share. international research collaboration should be funded and supported. future research needs to focus on the impact of climate change on psychosocial, mental, innovations, policies, and preparedness of health systems. climate change refers to long-term statistical shifts of the earth's climate system that result in new climate patterns [ ] . over the past century, industrial activities have led to long-term changes in the climate system that included global warming, flooding, and drought [ ] . the paris agreement, an agreement within the united nations framework convention on climate change (unfccc) signed in , represents an opportunity for all countries to implement measures to reduce, combat, and adapt to climate change [ ] [ ] [ ] [ ] . combating and reducing climate change is an important goal of the sustainable development goals (sdgs) which states "take urgent action to combat climate change and its impacts" [ ] . the implications of climate change on human health have led the world health organization (who) to declare climate change as one of the top ten global health threats in [ ] . climate change is negatively affecting human lives by changing the quality of air, water, and food supply [ ] [ ] [ ] . it is estimated that between and , climate change will cause approximately , additional deaths per year and - billion usd loss per year by [ ] . these devastating economic and health consequences require national and international planning to slow down climate change and to build resilient health systems that can tackle these changes [ ] . the effects of climate change are global and diverse [ , , ] . however, the impact on developing countries with limited resources and weak health systems will be more obvious [ ] [ ] [ ] [ ] [ ] . climate change has affected the epidemiology and pattern of both communicable and non-communicable diseases [ ] . for example, changes in temperature have serious adverse effects on the pattern and incidence of infectious diseases [ ] . global warming favors the survival and transmission of causative pathogens or vectors of the causative agent [ ] [ ] [ ] [ ] [ ] . climate change influences the dynamics of vector-borne, water-borne, foodborne, rodent-borne, and air-borne infectious diseases [ , ] . furthermore, a recent study predicted that climate change might worsen antimicrobial resistance [ ] . the study indicated that a spike in temperature of c was linked with a . % increase in antibiotic resistance to e.coli, which can trigger serious food poisoning; a . % increase in staphylococcus aureus, which can cause skin infections and food poisoning; and a . % increase in klebsiella pneumoniae, which can cause pneumonia. the spread of antimicrobial resistance is believed to have a serious negative global impact on human health [ ] . a study predicted that if antimicrobial resistance is not addressed, then by , million people will die because of antimicrobial resistance [ ] . many recent studies predicted that serious and emerging infectious diseases could appear or get worsened by climate change [ , ] . it is expected that the epidemiology and geography of many infectious diseases will change due to climate variability [ ] . for example, climate change will be an important factor for the spread of lassa virus in western africa [ ] . droughts are expected to increase the epidemics of west nile virus globally [ ] . higher incidence of cases of chikungunya and zika virus infections in brazil have been attributed to areas with more frequent rainfall and severe droughts [ ] . climate change and increased global temperatures have been associated with an increase in the probability of rift valley fever, cholera and malaria [ ] . the expected rapid spread of infectious diseases with climate change in the presence of antimicrobial resistance might cause global mass fatalities [ ] [ ] [ ] . the who considers climate change as a new threat to global health. this threat is compounded by globalization and modernization which can allow novel diseases to travel rapidly as what happened in the case of covid- [ ] . actually, the emergence of entirely novel diseases, like covid- , reintroduced the discussion of the impact of climate change on infectious diseases carried by wild animals or mosquitos and transmitted to humans. assessing research activity on climate change helps identify the national and international contribution to this field, the hot themes discussed by researchers, and research gaps in the field. climate change is a broad scientific topic and assessing research activity on climate change, in general, might not be very helpful. therefore, in the current study, the research activity of climate change on human health with an emphasis on infectious diseases was investigated. emphasis on infections was made due to suspected serious global outbreaks of infectious diseases such as dengue, ebola, and others [ ] [ ] [ ] [ ] [ ] . second, investigating research activity on climate change will help understand the type of infections mostly affected by climate change. third, research on climate change helps in developing appropriate protective measures and preparedness plans for certain infectious diseases in certain geographical areas. fourth, research on climate change comes as a response to calls made by international organizations such as the who on the importance of the impact of climate change on health and infectious diseases. based on the argument mentioned above and based on calls for papers made by certain specialized and prestigious journals in the field of public health and infectious diseases, the current study was undertaken to analyze the research aspects and research activity of climate change and human health with an emphasis on infectious diseases. the method used to display the research pattern and research activity on a certain topic is the bibliometric analysis which has been commonly used recently in various health topics [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . climate change has diverse effects that include aquatic organisms, forests, animals, and humans. the use of a bibliometric analysis is a suitable methodology to identify the volume and growth pattern of literature focusing on humans for further analysis related to health and infectious diseases. furthermore, bibliometric analysis is a suitable methodology to spot important research themes and active researchers and research institutions for future funding and planning. a literature search using well-known databases and search engines such as scopus database and google scholar revealed that there were at least ten bibliometric studies on climate change and its effects on various aspects on ecology or agriculture or adaptation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, no bibliometric research papers were published on climate change and health or climate change and infectious diseases. therefore, the current study will establish the first baseline data on this topic for future comparisons and for policymakers to draw plans on climate change and human health with an emphasis on infectious diseases. the first step in any bibliometric study is to decide on the appropriate database to be used to retrieve the relevant documents. in the current study, sciverse scopus was used to accomplish the objective of the study. scopus is larger than web of science and has more than , indexed journals in all scientific fields [ ] . scopus is % inclusive of medline and therefore, it is far better than medline. furthermore, the export of data from scopus to other programs is easy to perform. scopus offers two methods of search; a basic and an advanced search in which complex and long search queries can be made to accomplish the objective with high validity. scopus allows for search using terms in titles or titles/abstracts or name of the journal or name of the author or affiliation. the second challenge in any bibliometric study is to build a valid search query that will retrieve as many documents as possible but with minimum irrelevant (false-positive) results. in the field of climate change, many keywords could be used. however, in the current study, the authors reviewed many articles published as "systematic reviews" or "bibliometric analyses" to build a search query for climate change [ , [ ] [ ] [ ] [ ] [ ] . the keywords used included, but not restricted to, the followings "climat* chang*" or "greenhouse effect" or "changing climate" or "global warming" or "extreme weather" or "climate variability" or "greenhouse gas" or "rising temperature" or "heat waves". other non-specific keywords were also used but under certain constraints. for example, keywords such as flood, drought, temperature, warm*, rain*, and "air pollution" were used under the condition that a phrase related to "climat* chang*" was also present in the title/abstract of the same document. the keyword "air pollution/air pollutant*" was used with restrictions because air pollution and climate change are closely related but are not the same. therefore, documents on air pollution within the context of climate change were included [ ] . actually, in systematic review studies on climate change, the keyword air pollution was not included [ , ] . similarly, in previously published blibliometric studies on climate change, air pollution was not included in the search terms [ ] . in scopus, the quotation marks were used to retrieve the exact words while the asterisk was used as a wild card. in the current study, the authors developed an extensive and comprehensive search query to retrieve all potential documents focusing on climate change and human health. the keywords used to retrieve healthrelated documents included, but not restricted to, health, respirat*, mood, cardiac, heart, hunger, "food *security", pregnancy, asthma, infect*, "infectious", "vector-borne disease", "water-borne disease", and many others. the search query was built mainly on title search to make sure that the retrieved documents are obviously and directly related to human health. additional file included all keywords and steps used to retrieve documents on climate change and health. documents retrieved from the search query on human health and climate change were called "health-related literature". for documents related to infectious diseases, the authors used the same search query stated above but with all possible keywords related to infection/infectious diseases, pathogens, and vectors transmitting pathogens to humans. documents retrieved for infectious diseases were called "infection-related document". details on the search query are shown in additional file . validation of the search queries was based on two approaches. in the first approach, the top cited documents in the health-and infection-related literature were reviewed to make sure that they fit within the scope of climate change and health or climate change and infectious diseases. this approach was adopted to eliminate false-positive results by excluding documents focusing on the impact of climate change on certain plants or animals or any document irrelevant to human health. the second approach was based on comparing the actual number of articles for each author, obtained from his/her personal scopus profile, with the number of articles obtained by the search query for active authors. the comparison was made using the pearson correlation test. a significant and strong correlation is indicative of a high validity of the search query and the absence of missing results. this approach was previously used in several bibliometric studies [ ] . data in the retrieved literature was exported to microsoft excel. the exported data included annual growth of publications, types of documents, languages, countries, authors, institutions, journals, citations, and funding agencies. the retrieved literature was also exported to vosviewer program [ ] to create network visualization maps. the strength of international research collaboration was presented as total link strength (tls) which is automatically given by vosviewer upon mapping research activity of selected countries. the tls is proportional to the extent of international research collaboration where higher tls value indicates greater collaboration. bibliometric indicators were presented as top ten active ones. for annual growth, statistical package for social sciences (spss statistics for windows, version . . armonk, ny: ibm corp.) was used to draw the annual growth of publications. for geographical distribution of documents, the who regional classification was used: the region of the americas (amro), the european region (euro), the western pacific region (wpro), the eastern mediterranean region (emro), the south-eastern asia region (searo), and african region (afro). the quality of publications was measured by the number of citations and h-index [ ] while the quality or impact of the journal was measured using the quartile ranking of journals obtained from scimago journal rank [ ] . journals in the q rank are considered to have the highest impact. the study period was from to . all citation analysis and data export were carried out on the same day (april , ) to avoid misinterpretation. the search query found documents on healthrelated literature and documents on infectionrelated literature. therefore, infection-related literature constituted . % of the health-related literature. retrieved documents were of different types (table ). research articles constituted . % (n = ) of the health-related literature and % (n = ) of the infection-related literature. there was a larger percentage of editorials (n = ; . %) in the health-related literature compared with that in the infection-related literature (n = ; . %). the annual growth of publications in the health-related literature was low in the s and s but showed a steep increase after . the annual growth pattern of documents in the infection-related literature followed the same pattern. however, the annual growth of documents in infectionrelated literature was relatively faster than that of the health-related literature. figure shows the growth of publications in the health-and infection-related literature depicted in dual-axis for easy comparison. mapping the most frequent terms in title/abstract fields of documents in the health-related literature with a minimum occurrence of gave terms distributed in four clusters representing four main research themes ( fig. ): . the first cluster (red) included items and focused on the following topics arranged alphabetically: adaptation, climate change, food production, food security, public health, health policy, healthcare system, and psychology. analysis of author keywords in infection-related literature indicated that malaria ( occurrences), dengue ( occurrences), and arboviruses (arthropod-borne viruses) ( occurrences) were the most frequent infectious diseases/pathogens encountered ( (table ). there was a significant and strong correlation between the percentage contribution of each region to health-and infection-related literature (p < . , r = . ). the top ten cited documents in health-related literature focused on the impact of climate change on food security, public health, and infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the infection-related literature, the top ten cited documents discussed water-, vector-, and mosquito-borne diseases as well as general effects of climate change on infectious diseases, particularly malaria and dengue [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the list of top active institutions/organizations for the health-related literature was dominated by australian and american institutions with australian national university ranking first with ( . %) documents ( for the health-related literature, international journal of environmental research and public health was the leading journal with ( . %) documents. in the list of active journals, two journals were in the field of infection while the remaining were in the field of public health, environment, and general medicine ( table ) . table shows the top ten active authors of health-and infection-related literature. researchers from europe, north america, australia, and china dominated both lists. the top active author for the health-related literature was from the usa while the top active author for the infection-related literature was from china. in total, author names participated in publishing the infection-related literature, an average of . authors per cholera salmonella west nile virus plasmodium falciparum influenza tick-borne encephalitis ross river virus zika rift valley fever leishmaniasis japanese encephalitis campylobacter cercariae hantaviruses a this list is not % inclusive of all pathogens or infections present in the retrieved literature. please see result section and fig. for more details document. one hundred and thirty-nine ( . %) documents were single-authored. for health-related literature, a total of ( . %) documents were funded. the national natural science foundation in china was the most active funding agency (n = ; . %) followed by the national institutes of health (nih; usa) and national science foundation (nsf; usa) ( table ) . for the infection-related literature, analysis showed that ( . %) documents were funded. the nih (usa) was the most active in funding (n = ; . %) followed by nsf (usa) (n = ; . %). the who was listed as one of the top ten active funding agencies infection-related literature. the current study was carried out to give a snap shot of research on climate change on human health with an emphasis on infectious diseases. the current study showed an increasing number of publications on climate change and health in the past decade. the gradual increase in the number of publications was parallel to international warning signals since the early s about the impact of climate change on human health. the first major international conference on the greenhouse effect at villach, austria, warned that greenhouse gases will cause a rise of global mean temperature which is greater than any in man's history [ ] . in , the united nations (un) created the intergovernmental panel on climate change (ipcc) to analyze and report on scientific findings. in , climate change convention, agrees to [ ] . in , the kyoto protocol calls for cutting emissions from industrialized nations [ ] . however, due to political and economic reasons in industrialized nations, the koyoto protocol did not come into force until . the ipcc fourth report issued in called for all countries to take adaptive measures to face climate changes [ ] . the steep rise in the number of publications on climate change and human health coincided with the release of the fourth ipcc report which blamed humankind activities for the irreversible climate changes. in the face of increasing evidence of the impact of climate change on human health, the who took an active role in developing policies to minimize the impact of climate change on health. in , the who executive board endorsed a new work plan on climate change and health that included raising awareness, endorse science and research on climate change, and support public health adaptive measures for climate change [ ] . the timeline history, debate, negotiations, and conventions at the international levels affected both the volume and pattern of research on climate change and its impact on human health. the current study indicated that there were four research themes on climate change and human health. these research themes were closely related. of particular interest in the current study was the research theme focusing on climate change and infectious disease. however, there was a small cluster representing the interplay of climate change and air pollution in the context of human health. air pollution is a complex subject and has fundamental effects on human health. in the current study, we focused on air pollution within the context of climate change and the resultant effect on human health. that is why the air pollution research theme was the smallest research theme as shown in the map. both climate change and air pollution are global environmental problems that are closely related and considered as twins but they are not the same thing. climate change is the global variation of the earth's climate which is accelerated by greenhouse gases caused by human activity. carbon dioxide is the main gas contributing to climate change, but it is not harmful to human health. air pollution is defined as the presence, in the air, of substances or particles that imply danger, damage or disturbance for humans, flora or fauna. the main sources of atmospheric contamination are gases that result mainly from emissions caused by the burning of fossil fuels emissions generated by transport, industrial processes, burning of forests, aerosol use, and radiation. both climate change and air pollution are worsened by the burning of fuel, increasing the co emissions which cause global warming. meanwhile, the generation of other pollutants, such as nitrogen oxides (no and no ), sulfur oxides (so and so ) and particulate matter, is the main reason the air is contaminated [ , ] . climate variations affect air quality; air pollution can worsen climate change and both can directly or indirectly affect health [ ] . the major and obvious health effect of climate change and air pollution is on respiratory health where both can exacerbate allergies and bronchial asthma [ ] . the complex interactions between climate change and air quality is a new area of research that requires further investigation [ , ] . the current study indicated that infections constituted a major theme of research on climate change and human health. climate change and temperature rise affect the transmission and spread of many pathogens [ ] . the current study showed that documents about malaria and dengue were among the top ten cited documents. malaria was the most frequently encountered infectious disease affected by climate change. malaria is a vectorborne disease that is sensitive to long-term climate change. for example, malaria epidemic risk increases around five-fold in the year after an el niño event in india [ ] . researchers have developed mathematical models to forecast future climatic influences on infectious diseases. the model aims to apply the statistical equations to future climate scenarios in order to predict the actual distribution of the disease. these models have been applied to malaria and dengue fever [ , , [ ] [ ] [ ] [ ] [ ] [ ] . the case incidence of dengue fever has multiplied fold since the s [ ] . according to the who estimation, . - . % of the world's population ( . - . billion) is living in areas where dengue viruses can be transmitted [ ] . in ranking, two equally active journals were given similar ranks and one position in the rank was skipped. c/d = number of citations per document. q = quartile infection-related literature: documents on climate change and infectious diseases. health-related literature: documents on climate change and health the current study showed that more than two-thirds of the global publications came from the amro and euro regions. there are many reasons for the leading role of these two regions. the presence of the us cdc, euro cdc, and many other governmental and nongovernmental research and academic institutions the field of public health and infectious diseases helped these two regions to make this tremendous and significant contribution. second, the main funding agencies are located in these two regions. third, infections have no borders and pathogens could travel with human migration waves which made europe and north american regions in a critical geographic position to any infectious disease outbreak. fourth, both regions have a great responsibility toward climate change since many of these changes were made by industrial activities. the current study showed that china was among the top ten active countries. the contribution of china might be underestimated because it is possible that most publications from china were published in national chinese journals that are not indexed in scopus. the same argument could be applied to other regions and countries with a limited number of peer-reviewed journals indexed in scopus such as russia or certain countries in south america. the current study showed that afro region made a greater contribution than either emro or searo region. a possible reason for the relatively higher contribution of the afro region is the strong research collaboration between certain african countries and the usa and the uk. climate change in the afro region increased the number of people in africa who are at risk of malaria [ , ] . the increase in the number of mosquitoes increased the opportunity for both plasmodium falciparum and plasmodium vivax parasites to proliferate and place more people at risk of contracting malaria [ , ] . aside from malaria, the afro region is expected to suffer from hunger and food insecurity due to climate change [ , ] . the climate change in the afro region is worsened by the weak economies, lack of resilient health systems, and lack of political stability in certain african regions. the current study showed that the emro region had the least contribution despite that the region is expected to suffer from serious climate variations [ ] . a systematic review on climate change and health in the emro region identified many knowledge and research gaps with research scarcity in this field [ ] . the authors of the systematic review concluded that the impact of climate change on health is not recognized as a priority area by health researchers, health professionals and policymakers in the emro region. international research collaboration and funding are important for countries in the emro, afro, searo regions where effects of climate change are expected to be beyond their economic and research capabilities. the current study showed that retrieved publications received a high number of citations suggestive of a large number of researchers who are interested in the topic. this could be attributed to the following reasons: first, the topic of climate change has caught the attention of scientists all over the world since the early s with many national and international warning reports about the climate change. second, the climate change is a multidisciplinary subject and therefore, researchers and scientists in public health, infectious diseases, nutrition, environmental health, ecology, and others were highly keen to investigate the subject and to have an input in this evolving topic. third, the fact that the top ten active journals in publishing documents were influential in their field gave credibility and attracted a larger number of citations. fourth, the leading role of the who as an international health agency played a positive role in raising the number of citations. finally, the number of authors played a positive role in increasing the number of citations [ ] . the current study indicated that infection-related documents received a higher number of citations and a higher h-index than documents in the health-related literature. this finding suggests that of the diverse health effects of climate change, its impact on the epidemiology and emerging infections receives the greatest scientific attention. this is due to the high and immediate risks of emerging and re-merging infections on global health. the h-index of the infectionrelated literature was higher than that reported for strongyloidiasis literature [ ] , epidermal parasitic skin diseases [ ] , antimalarial drug resistance [ ] , but lower than that on campylobacter or carbapenem resistance [ , ] . the current study emphasizes the importance of certain future research directions in the field of climate change and human health. the current study has a few limitations. the literature investigated has been retrieved from journals indexed in scopus while grey literature and publications in nonindexed journals have not been analyzed. therefore, journals from non-english speaking countries might be underestimated. this has further consequences on the top ten active countries, institutions and authors. the second limitation was the method for counting the number of documents for each country or author or institution. scopus makes all analysis based on the number of different affiliations in the documents. therefore, a document with several authors having the same country affiliation was counted once for that country. however, a document with two authors having two different country affiliations were counted once for each country. this has increased the research output of certain countries with greater international research collaboration even if the authors from that country was not the main or corresponding author. the citation analysis did not take into consideration the self-citations which could create a bias in the number of citations for countries, journals, and authors. finally, the search query was built to focus on climate change and human health. the definition and scope of human health and climate change are broad and complex. therefore, it is difficult to ensure a % inclusion of literature on both topics. however, the author did his best to include all relevant literature with minimum irrelevant documents. the final point is the inclusion of air pollution and air pollutants in the search query. the author included these keywords with restrictions to keep the manuscript focused on climate change. the purpose of including these terms was to retrieve documents discussing air pollution and health within the context of climate change. therefore, the number of documents retrieved in this topic was presented by research theme (cluster ) which was the smallest cluster. inclusion of air pollution in the search query without restriction will retrieve large volume of irrelevant documents on pollution that were irrelevant to climate change. this was the first bibliometric study on climate change and health or infection-related literature. key players, research themes, and research gaps were identified. the current study provided researchers and policymakers with baseline data in this field. the current study emphasized the importance of climate change on the epidemiology and geography of infectious diseases. adaptive national and international measures to combat climate change should include plans to contain the expected increase in vector-borne diseases particularly malaria and dengue. the current study showed inadequate international research collaboration which is highly needed for countries in emro, afro, and searo regions. finally, national and international health organizations should encourage and fund researchers to do continuous assessment and research on the impact of climate change on various health aspects and on various types of infections. united nations framework convention on climate change (unfcc): fact sheet: climate change science -the status of climate change science today accessed climate change-causes, impacts, mitigation: a review the paris 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institutional affiliations the authors would like to thank an-najah national university for giving us the opportunity to access most recent information sources. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . search strategy and keywords for documents on climate change and health (health-related literature). the authors declare that they have no competing interests.received: january accepted: may key: cord- -q imoe k authors: zhong, shaobo; huang, quanyi; song, dunjiang title: simulation of the spread of infectious diseases in a geographical environment date: - - journal: sci doi: . /s - - - sha: doc_id: cord_uid: q imoe k the study of mathematical models for the spread of infectious diseases is an important issue in epidemiology. given the fact that most existing models cannot comprehensively depict heterogeneities (e.g., the population heterogeneity and the distribution heterogeneity) and complex contagion patterns (which are mostly caused by the human interaction induced by modern transportation) in the real world, a theoretical model of the spread of infectious diseases is proposed. it employs geo-entity based cellular automata to simulate the spread of infectious diseases in a geographical environment. in the model, physical geographical regions are defined as cells. the population within each cell is divided into three classes: susceptible, infective, and recovered, which are further divided into some subclasses by states of individuals. the transition rules, which determine the changes of proportions of those subclasses and reciprocal transformation formulas among them, are provided. through defining suitable spatial weighting functions, the model is applied to simulate the spread of the infectious diseases with not only local contagion but also global contagion. with some cases of simulation, it has been shown that the results are reasonably consistent with the spread of infectious diseases in the real world. the model is supposed to model dynamics of infectious diseases on complex networks, which is nearly impossible to be achieved with differential equations because of the complexity of the problem. the cases of simulation also demonstrate that efforts of all kinds of interventions can be visualized and explored, and then the model is able to provide decision-making support for prevention and control of infectious diseases. infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi, and can spread, directly or indirectly, from one person (or animal) to another. a serious infectious disease might cause mass deaths, social panics, or even a national crisis. some examples of infectious diseases are the black death during the middle fourteenth century, the spanish flu pandemic in , the severe acute respiratory syndrome (sars) in , and more recently, the highly pathogenic avian influenza (hpai) [ ] . in order to prevent and control infectious diseases, the study of mathematical models for the spread of infectious diseases is an important issue in epidemiology. through scientifically modeling the spread of an infectious dis-ease, some key factors that disclose the mechanism of the spread of the disease can be found out and the effect of some interventions (e.g., therapy, isolation, immunization) on the spread of the disease can be evaluated. modeling infectious diseases using differential equations has been a popular approach. in differential equation models, the population is generally divided into three classes: susceptible, infective, and recovered. the susceptible refers to those healthy individuals who are susceptible to a certain infectious disease and are likely to be infected with the disease. the infective refers to the individuals under infection. the recovered refers to those who are recovered from infection with the disease and are contemporarily or permanently immune to the disease. to characterize the varying infection force of an infected individual, the infection period of a disease is further divided into three phases: incubation, when the individual is already infected and infectious but presents no symptoms; proper infection, when the individual is infectious and shows the symptoms; and latency, when the infected individual is not infectious anymore but still has the symptoms [ ] . two basic differential equation models were extensively studied. one is sir model for modeling the infectious diseases that confer immunity to the infected individuals after recovery (e.g., measles, chickenpox, mumps, hiv, and poliomyelitis). the other is sis model for modeling the infectious diseases that do not confer immunity to the infected individuals after recovery (e.g. meningitis, plague, venereal diseases, and malaria). some other differential equation models are more or less adapted from sir or sis, such as sirs, seirs and so on [ ] . these models all assume that the population under study is homogeneous and the interaction among individuals is equiprobable. these assumptions are unrealistic, which are also the intrinsic drawbacks of these differential equation models, because the local characters are neglected in these models [ ] . they do not include variable susceptibility of individuals, and cannot handle the complicated boundary and initial conditions [ ] . furthermore, these models are all adimensional, i.e., they can depict the changes of the number of all classes of individuals in time, but not in space. some types of methods are developed to deal with the heterogeneous characteristics. spatial infectious disease models and cellular automata (ca) models are two outstanding types of them. in essence, spatial infectious disease models [ ] are composed of a system of differential equations and understandably have the common shortcomings as differential equation models. however, ca models are a new kind of computational techniques for simulating the real world, introduced first by von neumann [ ] . at first, they were mainly used for simulation of physics systems. in recent years, many researchers from all kinds of fields have had an increasing interest in them thanks to the development of the computer techniques. some authors have applied ca to model infectious diseases [ , , , , ] . they basically construct the ca based on differential equation models. by that way, some epidemiological features of an infectious disease can be expressed more directly. moreover, ca simulation can present visual results that enable us to ascertain some key features of the spread of an infectious disease through experiment and observation. in most existing studies, ca models use cellular space that is generated by equably dividing the research area into regular lattices. and in those studies, the classic neighbor patterns, such as moore and von neumann neighbors, are applied and the transition rules of the ca are derived from the relevant differential equation models. these, however, have some shortcomings. first of all, the classic neighbor patterns are not suitable for some special diseases while they are suitable for characterizing the spread of some infectious diseases. sars is one of the typical examples. the spread of sars shows the small world characteristic [ ] which is completely different from the behavior expressed by the classic neighbor patterns. on the other hand, the real background space where diseases spread comprises irregular geographical regions (which generally have different sizes and shapes), so it is also not suitable to impersonate physical geographical regions using regular lattices. as mentioned above, the classic ca model is too simple for modeling the dynamics in a geographical environment. it has some limitations with respect to the cellular shape, the neighbor pattern, and the transition rule, which constrains its ability to simulate the real world. to overcome these drawbacks, some researchers have studied the theories and applications of geographical ca [ , ] . in this paper, we employ a geo-entity based ca model [ , ] , which defines physical geographical regions as cells. in addition to the consideration of the epidemiological characteristics of infectious diseases (e.g., effective contact rate, recovery rate, and the varying infection force), we pay more attention to the influence of contagion patterns on the spread of infectious diseases. two typical kinds of contagion patterns are explored. one is the local contagion good for portraying local diffusion and the other is the global contagion, which is potentially likely to express the contact behavior of infectious diseases on complex networks [ ] [ ] [ ] [ ] [ ] [ ] . the cases of simulation for the two situations are also presented and analyzed. it has been shown that the results are reasonably consistent with the spread of infectious diseases in the real world. the model is supposed to model dynamics of infectious diseases in complex networks, which is nearly impossible to be achieved with differential equations because of the complexity of the problem. the additional cases of simulation also demonstrate that efforts of all kinds of interventions can be visualized and examined, and then the model is able to provide decision-making support for prevention and control of infectious diseases. the main features of an infectious disease and the environment where it is spreading are as follows. (i) s(t), i(t) and r(t) denote the proportions of susceptible, infective and recovered. the population within the study area is constant. there are no death and birth during the epidemic, i.e., s(t)+i(t)+r(t)= . (ii) the effective contact rate is constant λ, i.e., the average number of susceptible infected by each infected individual in a given time (e.g., one day) is constantly proportioned to the number of the current susceptible. (iii) the cured rate is constant ν, i.e., the number of infective cured in a given time is constantly proportioned to the number of the current infective. the cured infective are contemporarily immune to the disease. (iv) the recovery rate is constant γ, i.e., the number of recovered losing immunity in a given time is constantly proportioned to the number of the current recovered. after losing immunity, recovered are susceptible to the disease again. according to the above assumptions, the sirs model can be formulated using the following differential equations: . eq. ( ) can be reduced to the following ones: it is difficult to calculate the analytical solution of eq. ( ). a phase plane method is generally employed to analyze some characteristics of the equations [ ] . bidimensional ca are discrete dynamical systems formed by a finite number of row×col identical objects called cells, which are arranged uniformly in a two dimensional cellular space. each cell is endowed with a state (from a finite state set q) that changes at every step of time according to a local transition rule. the state of a cell at the step of time t depends on the states of the cell and its neighbors. generally, ca can be defined by a -uplet (c, q, v, f), where c={(i,j), ≤i≤row, ≤j≤ col} is the cellular space; q is the finite state set whose elements are the all possible states of the cells; v={(α k , β k ), ≤k≤n}⊂z×z, is the finite set of indices defining the neighborhood of each cell, such that the neighborhood of the cell (i, j) is v ij ={(i+α , j+β ), (i+α , j+β ), … , (i+α n ,j+β n )}; finally, the function f is the local transition function: where t ij s stands for the state of the cell (i, j) at the step of time t. we consider a geographical environment composed of k geographical regions. these regions can be any type of geo-entity, such as administrative region, community, building. label these geo-entities with , , , …, k in arbitrary order. first, we define the following notations: n i , the total amount of the population in region i. a i , the area of region i; s i now, we consider the geographical area under study as the cellular space, and look on a geo-entity (e.g., jurisdiction, community, residence) in the area as a cell. use a set m={m i , i= , ,…, k} to denote the cellular space. each cell m i comprises three classes of individuals, i.e., susceptible, infective and recovered. every cell is further divided into subclasses by the states of individuals. the state of every subclass will dynamically change with time. here we assume, for a certain disease, its phases' average times are fixed. t i , t p , t l , and t r denote the average times of the incubation, proper infection, latency, and immunity respectively (unit: day). thus, we define the state set which each subclass may go through as p={ , , ,…, t i +t p +t l +t r }. the subclass with state consists of the individuals susceptible to the disease. the subclass with state consists of the individuals on the first day of incubation, state t i + on the first day of proper infection,…, and state t i +t p +t l + on the first day of immune. when the state of a subclass is t i +t p +t l +t r , which means that the individuals with the state are on the last day of the immune phase, the state of the subclass should be changed to at the next step of time. every subclass will go through each state in the state set orderly except that they are cured during the infection period. if the subclass is cured during the infection period, it will directly enter into the first day of the immune phase, i.e. its state should be changed to t i +t p +t l + . figure illustrates the process each subclass may go through. in order to describe the transition rules of our ca, we further define the following notations: s i t , the proportion of susceptible with state in region i at the step of time t; i i t (q), the proportion of infective with state q in region i at the step of time t, q∈{ , ,…, now, let us examine the state set of the ca (please note that the state set of a ca is different from the state set of a subclass described above). we define the state set of a cell according to proportions of all subclasses in it. set , then, the state of the ca used in the model is the subclasses of all cells of the cellular space interact with each another. the interaction of every cell involves two portions: internal and external. internal refers to reciprocity of the subclasses inside the same cell, whereas external refers to reciprocity of subclasses from different cells. based on the above definitions and concepts, we formulate the following transition rules: t t t t t t t t t eq. ( a) depicts that the state of recovered who do not reach the end of the immune phase increases one. eq. ( b) reflects that susceptible will lose some individuals due to the internal interaction and the external interaction (the first summation and the second summation) while they gain some individuals since some recovered reach the end of the immune phase. the first summation depicts susceptible infected by infective inside the same cell at the step of time t. the second summation depicts susceptible infected by infective from the neighbor cells at the step of time t. λ(q), q∈ { , …, t i +t p +t l } denote the effective contact rates under a benchmark density of individual distribution ρ (e.g. person/km ). we call them benchmark effective contact rate (becr). the becr may be different for each state, which is a function of state q. in eq. ( b), here, we assume the effective contact rate between susceptible and infective is positively proportioned to the density of susceptible distribution. c ij is the correlation factor, which ranges from to . the greater the distance between two cells is, the smaller the c ij is. r t i (t r ) means recovered who become susceptible to the disease again because their immune phases end at the step of time t. eq. ( c) expresses that the new infective are equal to the loss of susceptible. eq. ( d) means that some infective will be cured, and the cured rate γ (q) may be different for each state, which is a function of state q. eq. ( e) expresses that some infective at step of time t will enter into the immune phase. these individuals come from two parts: one is infective reaching the end of the infection period, and the other is infective cured. in the above model, the correlation factor c ij reflects the force of the external interaction between region i and region j. quantifying the variable reasonably is crucial for effective simulation of an infectious disease. in this paper, we will quantify c ij based on the spatial weighting function. the tobler's first law of geography points out that everything is related to everything else, but near things are more related than distant things [ ] . the law illustrates the universal correlation. in classic geography, the spatial weighting function is used for quantifying the spatial autocorrelation. a spatial weighting function is a set of rules that assign values or "weights" to every pair of locations in a study area [ ] . in essence, the aim of defining a spatial weighting function is to represent nearness and distance as consistently as possible for a set of irregular regions or irregularly spaced points. the simplest weighting function for regional data is the binary weight. in that case, if two regions have shared boundaries, then the weight equals to , or else the weight equals to . this function assigns the same values to pairs of regions with very short boundaries and pairs with very long boundaries. an improved approach is to define the weight of pairs of regions considering their shared boundary length. cliff and ord [ ] proposed a weighting function as follows where z ij is the length of the boundaries of region i that are shared with region j, and l i is the perimeter of region i. this approach gives greater weight to regions that share long boundaries. whereas the weights calculated by this means are unsymmetrical, i.e., w ij ≠w ji , so we modify eq. ( ) as follows: any set of regions can also be represented by points located at their centers so that weighting functions defined for point data can also be applied to regional data. tobler listed a series of weighting functions based on distance relation for point data [ ] . there are also some other definitions which are not limited to distance relation, such as travel and individual contact, journey-to-work, journey-to-school, and journey-to-hospital [ ] . in order to model an infectious disease, first we select a suitable spatial weighting function given the epidemiological characteristics of the infectious disease. then standardize the resulting weights. the standardization process is to transform the weights to meet the following conditions: the correlation factors approach zero for small weight but do not become negative and they approach one for large weight but are not greater than one, i.e., the weights and the correlation factors meet the following mapping relationship ( , ). the following is an alternative of the transformation functions: if an infectious disease is locally contagious, in which case it would spread mainly around nearby regions, the weighting functions based on distance relation may be appropriate alternatives. however, while an infectious disease is not limited to local contagion, the weighting functions based on global contagion patterns, such as travel and individual contact, journey-to-work, journey-to-school, and journey-to-hospital [ ] , would be preferred. in the cases of simulation, we demonstrate the state of each cell by means of pie charts. the sectors filled with colors white, grey, black represent the proportions of susceptible, infective and recovered respectively. t i , t p , t l , t r are assigned with the following artificially chosen parameters: , , , days, and λ(q)= . , γ (q)= . , q∈ { , , …, t i +t p +t l } (except explicitly claimed). the steps of time of the cases of simulation are all days. figure illustrates the sketch map of the geographical environment of simulation. the environment comprises tens of geographical regions and several transportation lines. there are entries/exits distributed unevenly along these lines through which people can get up and off vehicles. in the following cases of simulation, we will examine the spatial dynamics of infectious diseases with the local contagion and the global contagion, and the effect of two typical interventions (isolation and quarantine) on the spread of infectious diseases. the first case we present is the situation with local contagion. here the spatial weighting function defined by eq. ( ) is employed. since the calculated weights are within ( , ), we define c ij =w ij . four configurations considering only the local contagion are shown in figure : those at the steps of time t= , , , days. the curves that depict the numbers of susceptible, infective and recovered change according to the steps of time are shown in figure . as seen, the spread will arrive at a global stationary state, i.e., the numbers of the individuals of every subclass will not change any more after a long period. furthermore, by comparing figure (c) with figure (d), we found that the local stationary state is also reached (the proportions of the individuals of each subclass are nearly same in the two configurations). through running the simulation program with different values of λ(q) and initial configurations, we also found that a threshold λ exists. when λ(q)>λ , the prevalence can hold and arrive at a stationary state finally. however, when λ(q)<λ , the prevalence cannot persist. the final state (whether stationary or not) has nothing to do with the initial configuration. in this case of simulation, the spread presents a typical character of local diffusion. the second case of simulation considers not only the local contagion but also the global contagion. the weights are calculated according to the proximityδ ij ( , not adjacent; , adjacent) and the number of road links n ij between two regions (eq. ( ) figure . figures and show the change curves of the numbers with different becr under the local and the global contagions respectively. comparing the two figures, we found that a threshold also exists but the threshold is slightly lower when considering the global contagion. with same becr, the spread with the global contagion is faster and the number of infective under the stationary state is more. in fact, with more and more complex road links, correspondingly the threshold is getting progressively lower. with the global contagion, the spread shows aggression along transportation lines. in the third case of simulation, we consider the isolation of individuals after they are infected. the operation of isolation is equivalent to lowering the density of infective distribution in regions. at every step of time, the individuals meeting the conditions of isolation are removed from infective in a region and the density of infective distribution is calculated dynamically. the curves with different isolation times (those on th, th, th days after infected) are drawn in figure . as seen, the earlier isolation is carried out, the better control effort can be obtained. in the forth case, we will simulate the effect of quarantine which prevents infective from moving among the weighting function in eq. ( ) into the following form: figure illustrates the curves of the number of infective with different ω. the curves show that when the quarantine strength ω is getting higher and higher, the number of infective under stationarity is getting lower and lower. nevertheless, as ω increases, the decrease of the number of final infected individuals is not remarkable. the reason is that the weights assigned for region pairs without road links between them are comparatively high. and yet the quarantine for this situation is not considered in this case of simulation. imaginably, given this kind of quarantine or equivalent (e.g., refrain the interaction between two adjacent regions), the number under stationarity will decrease greatly. in the following study, we use the epidemic data of sars in beijing of china to validate the proposed model in the above sections. sars is a new infectious disease first reported in november in the guangdong province of china (www.who.int/sarsarchive/ - - /en). in beijing, the first sars case was confirmed on march , . from then on, sars spread rapidly over the city since people have hardly recognized sars and some interventions for prevention and control of the disease have not been carried out in the early phase of the epidemic. here, we will compare the execution results of the proposed model with the real observed disease data. in order to reduce the bias caused by interventions, the disease data from march to april are selected (these data are supposed to be affected hardly due to little interventions such as isolation, quarantine and so on) and they are compared with the simulating results from the model. the map in figure demonstrates the political areas of beijing, which are looked upon as cells. and we calculate the distribution of beijing of by means of yue-smpd [ ] . given the fact that the whole city is connected with roads in length and breadth, eq. ( ) is used to calculate the connection distance between two areas i and j: where x i , y i ; x j , y j are the coordinates of the centroids of areas i, j respectively. thus the connection factor between areas i and j can be quantified according to eq. where the unit for d ij is km. based on the existing research [ ] [ ] [ ] [ ] [ ] [ ] , t i , t p , t l , t r are assigned with the following parameters: , , , days (this means the recovered are permanently immune to sars, which is based on the fact that nobody is re-infected with sars when recovered from the disease). γ(q), q∈{ , , …, t i +t p +t l } is fixed to (in the early phase of sars, little interventions are carried out). the simulation time is days (from march to april ). we change the model parameters λ to run the model and the results of model simulation are drawn in figure figure the political area map of beijing. when λ(q), q∈{ , , …, t i +t p +t l } is assigned with . . figure shows that the simulated data have good consistence with the real disease data before march . however, from march on, the curve for the simulated data has remarkable rise while that for the real data are still slight increase. the reason for this phenomenon is likely that the interventions have been augmented with increasing respect with the disease. furthermore, the real road network is quite complex so that it is very difficult to precisely calculate the connection factors without detailed road data. but imaginably, given perfect road data and well defined road network topology, the more practical connection factors can be calculated and the model will achieve higher accuracy. since the sir model was first published, it has being widely used in modeling transmission of contagious diseases. it suffers from the assumption of mass action, or the homogenous interaction between populations. a lot of efforts are required to relax the assumption. the paper proposes to embed ca model under sir framework; a new effort has been made to revise sir model and the study on the simulation and real-world cases shows that work. in this paper, a theoretical model of infectious diseases is introduced. it incorporates two main heterogeneities: population heterogeneity and distribution heterogeneity which relax the assumption of homogeneity. it employs geo-entity based ca considering physical geographical regions as cells. the model incorporates two categories of parameters (the becr and the cured rate) that may express man's interventions, through which the evaluation of the control effect (e.g., therapy, isolation, immunization) is executable. in the proposed model, the parameter correlation factor c ij reflects the force of the external interaction between region i and region j. quantifying the variable reasonably is crucial for effective simulation of an infectious disease. a spatial weighting function is a set of rules that assign values or "weights" to every pair of locations in a study area and it is appropriate for quantifying the correlation factors. based on spatial weighting functions, some new neighbor patterns can be applied in the model, which are different from those used in the classic ca and supposed to more reasonably depict the spatial interaction of infectious diseases. consequently, the model is applicable for not only the diseases with local contagion but also ones with global contagion (e.g., sars, foot and mouth disease and avian influenza). the model has some advantages over the classic ca. it can couple cells with geographical phenomena closely. thus, the incorporation of region related parameters, such as various attributes for geographical regions, spatial weighting functions and so on, becomes more direct and convenient. according to the model, a simulation program is developed in gis. for two typical contagion patterns: the local contagion and the global contagion, some results of simulation are obtained. and the results show that the model is adaptable for modeling the complicated phenomena of the spread of diseases in real world, which is nearly impossible to be achieved with differential equations because of the complexity of the problem. fur-thermore, the model couples visualization with complexity and is supposed to effectively simulate the dynamics of infectious diseases on complex networks which is unreachable with either the classic ca or analytical complex networks. in this paper, the cases of simulation considering isolation and quarantine are also presented, which demonstrate several sensible uses of the model in control and prevention of infectious diseases. the case study of beijing sars also shows that the simulation results of the model are in good agreement with the real disease data, which in a sense explains the applicability of the proposed model. through visualizing and examining efforts of all kinds of interventions, the model can provide decision-making support for prevention and control of infectious diseases. for his critical reading of the manuscript and li b. t. for his careful copy-editing of it. the anonymous reviewers are thanked for their constructive reviews modeling epidemics using cellular automata cellular automata and epidemiological models with spatial dependence infectious diseases of humans: dynamics and control on modeling epidemics including latency, incubation and variable susceptibility a cellular automaton model for the effects of population movement and vaccination on epidemic propagation spatial analysis theory of self-reproducing automata cellular automata modelling of seirs modeling infectious diseases using global stochastic cellular automata epidemiological modeling and public health policy assessments discovery of transition rules for geographical cellular automata by using ant colony optimization do irregular grids make a difference? relaxing the spatial regularity assumption in cellular models of social dynamics an vector-based geographic cellular automata model allowing 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international diffusion of severe acute respiratory syndrome (sars) the effect of global travel on the spread of sars data-driven exploration of "spatial pattern-time process-driving forces" associations of sars epidemic in beijing key: cord- -v lahyw authors: van seventer, jean maguire; hochberg, natasha s. title: principles of infectious diseases: transmission, diagnosis, prevention, and control date: - - journal: international encyclopedia of public health doi: . /b - - - - . - sha: doc_id: cord_uid: v lahyw infectious disease control and prevention relies on a thorough understanding of the factors determining transmission. this article summarizes the fundamental principles of infectious disease transmission while highlighting many of the agent, host, and environmental determinants of these diseases that are of particular import to public health professionals. basic principles of infectious disease diagnosis, control, and prevention are also reviewed. an infectious disease can be defined as an illness due to a pathogen or its toxic product, which arises through transmission from an infected person, an infected animal, or a contaminated inanimate object to a susceptible host. infectious diseases are responsible for an immense global burden of disease that impacts public health systems and economies worldwide, disproportionately affecting vulnerable populations. in , infectious diseases resulted in over million years lost due to disability and over million deaths (naghavi et al., ) . lower respiratory tract infections, diarrheal diseases, hiv/ aids, malaria, and tuberculosis (tb) are among the top causes of overall global mortality (vos et al., ) . infectious diseases also include emerging infectious diseases; diseases that have newly appeared (e.g., middle east respiratory syndrome) or have existed but are rapidly increasing in incidence or geographic range (e.g., extensively drug-resistant tuberculosis (xdr tb) and zika virus (morse, ) . infectious disease control and prevention relies on a thorough understanding of the factors determining transmission. this article summarizes some of the fundamental principles of infectious disease transmission while highlighting many of the agent, host, and environmental determinants of these diseases that are of particular import to public health professionals. a classic model of infectious disease causation, the epidemiological triad (snieszko, ) , envisions that an infectious disease results from a combination of agent (pathogen), host, and environmental factors ( figure ). infectious agents may be living parasites (helminths or protozoa), fungi, or bacteria, or nonliving viruses or prions. environmental factors determine if a host will become exposed to one of these agents, and subsequent interactions between the agent and host will determine the exposure outcome. agent and host interactions occur in a cascade of stages that include infection, disease, and recovery or death (figure (a) ). following exposure, the first step is often colonization, the adherence and initial multiplication of a disease agent at a portal of entry such as the skin or the mucous membranes of the respiratory, digestive, or urogenital tract. colonization, for example, with methicillin-resistant staphylococcus aureus in the nasal mucosa, does not cause disease in itself. for disease to occur, a pathogen must infect (invade and establish within) host tissues. infection will always cause some disruption within a host, but it does not always result in disease. disease indicates a level of disruption and damage to a host that results in subjective symptoms and objective signs of illness. for example, latent tb infection is only infectionevidenced by a positive tuberculin skin test or interferon gamma release assaybut with a lack of symptoms (e.g., cough or night sweats) or signs (e.g., rales on auscultation of the chest) of disease. this is in contrast to active pulmonary tb (disease), which is accompanied by disease symptoms and signs. recovery from infection can be either complete (elimination of the agent) or incomplete. incomplete recovery can result in both chronic infections and latent infections. chronic infections are characterized by the continued detectable presence of an infectious agent. in contrast, latent infections are distinguished by an agent which can remain quiescent in host cells and can later undergo reactivation. for example, varicella zoster virus, the agent causing chicken pox, may reactivate many years after a primary infection to cause shingles. from a public health standpoint, latent infections are significant in that they represent silent reservoirs of infectious agent for future transmission. when a potential host is exposed to an infectious agent, the outcome of that exposure is dependent upon the dynamic relationship between agent determinants of infectivity, pathogenicity, and virulence, and intrinsic host determinants of susceptibility to infection and to disease (figure (b) ). environmental factors, both physical and social behavioral, are extrinsic determinants of host vulnerability to exposure. environment disease figure the epidemiological triad model of infectious disease causation. the triad consists of an agent (pathogen), a susceptible host, and an environment (physical, social, behavioral, cultural, political, and economic factors) that brings the agent and host together, causing infection and disease to occur in the host. infectivity is the likelihood that an agent will infect a host, given that the host is exposed to the agent. pathogenicity refers to the ability of an agent to cause disease, given infection, and virulence is the likelihood of causing severe disease among those with disease. virulence reflects structural and/or biochemical properties of an infectious agent. notably, the virulence of some infectious agents is due to the production of toxins (endotoxins and/or exotoxins) such as the cholera toxin that induces a profuse watery diarrhea. some exotoxins cause disease independent of infection, as for example, the staphylococcal enterotoxins that can cause foodborne diseases. agent characteristics can be measured in various ways. infectivity is often quantified in terms of the infectious dose (id ), the amount of agent required to infect % of a specified host population. id varies widely, from organisms for shigella dysenteriae to - for vibrio cholerae (gama et al., ; fda, ) . infectivity and pathogenicity can be measured by the attack rate, the number of exposed individuals who develop disease (as it may be difficult to determine if someone has been infected if they do not have outward manifestations of disease). virulence is often measured by the case fatality rate or proportion of diseased individuals who die from the disease. the outcome of exposure to an infectious agent depends, in part, upon multiple host factors that determine individual susceptibility to infection and disease. susceptibility refers to the ability of an exposed individual (or group of individuals) to resist infection or limit disease as a result of their biological makeup. factors influencing susceptibility include both innate, genetic factors and acquired factors such as the specific immunity that develops following exposure or vaccination. the malaria resistance afforded carriers of the sickle cell trait exemplifies how genetics can influence susceptibility to infectious disease (aidoo et al., ) . susceptibility is also affected by extremes of age, stress, pregnancy, nutritional status, and underlying diseases. these latter factors can impact immunity to infection, as illustrated by immunologically naïve infant populations, aging populations experiencing immune senescence, and immunocompromised hiv/aids patients. mechanical and chemical surface barriers such as the skin, the flushing action of tears, and the trapping action of mucus are the first host obstacles to infection. for example, wound infection and secondary sepsis are serious complications of severe burns which remove the skin barrier to microbial entry. lysozyme, secreted in saliva, tears, milk, sweat, and mucus, and gastric acid have bactericidal properties, and vaginal acid is microbicidal for many agents of sexually transmitted infections (stis). microbiome-resident bacteria (a.k.a. commensal bacteria, normal flora) can also confer host protection by using available nutrients and space to prevent pathogenic bacteria from taking up residence. the innate and adaptive immune responses are critical components of the host response to infectious agents ( table ) . each of these responses is carried out by cells of a distinct hematopoietic stem cell lineage: the myeloid lineage gives rise to innate immune cells (e.g., neutrophils, macrophages, dendritic cells) and the lymphoid lineage gives rise to adaptive immune cells (e.g., t cells, b cells). the innate immune response is an immediate, nonspecific response to broad groups of pathogens. by contrast, the adaptive immune response is initially generated over a period of - days, it recognizes specific pathogens, and it consists of two main branches: ( ) t cell-mediated immunity (a.k.a. cell-mediated immunity) and ( ) b cellmediated immunity (a.k.a. humoral or antibody-mediated immunity). the innate and adaptive responses also differ in table comparison of innate and adaptive immunity innate immune response adaptive immune response immediate response; initiated within seconds gradual response; initially generated over - days (primary response) targets groups of pathogens targets-specific pathogens no memory memory progression from one stage to the next is dependent upon both agent properties of infectivity, pathogenicity, and virulence, and host susceptibility to infection and disease, which is in large part due to both protective and adverse effects of the host immune response. credit: modification of original by barbara mahon, md, mph. that the latter has memory, whereas the former does not. as a consequence of adaptive immune memory, if an infectious agent makes a second attempt to infect a host, pathogenspecific memory t cells, memory b cells, and antibodies will mount a secondary immune response that is much more rapid and intense than the initial, primary response and, thus, better able to inhibit infection and disease. immune memory is the basis for the use of vaccines that are given in an attempt to stimulate an individual's adaptive immune system to generate pathogen-specific immune memory. of note, in some cases the response of the immune system to an infectious agent can contribute to disease progress. for example, immunopathology is thought to be responsible for the severe acute disease that can occur following infection with a dengue virus that is serotypically distinct from that causing initial dengue infection (screaton et al., ) . an immune host is someone protected against a specific pathogen (because of previous infection or vaccination) such that subsequent infection will not take place or, if infection does occur, the severity of disease is diminished. the duration and efficacy of immunity following immunization by natural infection or vaccination varies depending upon the infecting agent, quality of the vaccine, type of vaccine (i.e., live or inactivated virus, subunit, etc.), and ability of the host to generate an immune response. for example, a single yellow fever vaccination appears to confer lifelong immunity, whereas immune protection against tetanus requires repeat vaccination every years (staples et al., ; broder et al., ) . in malariaendemic areas, natural immunity to malaria usually develops by years of age and, while protective from severe disease and death, it is incomplete and short-lived (langhorne et al., ) . functionally, there are two basic types of immunization, active and passive. active immunization refers to the generation of immune protection by a host's own immune response. in contrast, passive immunization is conferred by transfer of immune effectors, most commonly antibody (a.k.a. immunoglobulin, antisera), from a donor animal or human. for example, after exposure to a dog bite, an individual who seeks medical care will receive both active and passive postexposure immune prophylaxis consisting of rabies vaccine (to induce the host immune response) and rabies immune globulin (to provide immediate passive protection against rabies). an example of natural passive immunization is the transfer of immunity from mother to infant during breastfeeding. vaccination does not always result in active immunization; failure of vaccination can be due to either host or vaccine issues. individuals who are immunosuppressed as, for example, a result of hiv infection, malnutrition, immunosuppressive therapy, or immune senescence might not mount a sufficient response after vaccination so as to be adequately immunized (protected). similarly, vaccination with an inadequate amount of vaccine or a vaccine of poor quality (e.g., due to break in cold chain delivery) might prevent even a healthy individual from becoming immunized. environmental determinants of vulnerability to infectious diseases include physical, social, behavioral, cultural, political, and economic factors. in some cases, environmental influences increase risk of exposure to an infectious agent. for example, following an earthquake, environmental disruption can increase the risk of exposure to clostridium tetani and result in host traumatic injuries that provide portals of entry for the bacterium. environmental factors promoting vulnerability can also lead to an increase in susceptibility to infection by inducing physiological changes in an individual. for example, a child living in a resource-poor setting and vulnerable to malnutrition may be at increased risk of infection due to malnutritioninduced immunosuppression. table provides examples of some of the many environmental factors that can facilitate the emergence and/or spread of specific infectious diseases. a unique characteristic of many infectious diseases is that exposure to certain infectious agents can have consequences for other individuals, because an infected person can act as a source of exposure. some pathogens (e.g., sti agents) are directly transmitted to other people, while others (e.g., vectorborne disease (vbd) agents) are transmitted indirectly. from a public health standpoint, it is useful to define stages of an infectious disease with respect to both clinical disease and potential for transmission ( figure ). with respect to disease, the incubation period is defined as the time from exposure to an infectious agent until the time of first signs or symptoms of disease. the incubation period is followed by the period of clinical illness which is the duration between first and last disease signs or symptoms. with respect to transmission of an infectious agent, the latent (preinfectious) period is the duration of time between exposure to an agent and the onset of infectiousness. it is followed by the infectious period (a.k.a. period of communicability) which is the time period when an infected person can transmit an infectious agent to other individuals. in parasitic infections, the latent and infectious periods are commonly referred to as the prepatent period and patent period, respectively. the duration of disease stages is unique for each type of infection and it can vary widely for a given type of infection depending upon agent, host, and environmental factors that affect, for example, dose of the inoculated agent, route of exposure, host susceptibility, and agent infectivity and virulence. knowledge of the timing of disease stages is of key importance in the design of appropriate control and prevention strategies to prevent the spread of an infectious disease. for example, efforts to control the recent ebola west africa outbreak through contact tracing and quarantine were based on knowledge that the infectious period for ebola does not begin until the start of the period of clinical illness, which occurs up to days following exposure (figure (a) ; pandey et al., ) . a carrier is, by definition, an infectious individual who is not showing clinical evidence of disease and, thus, might unknowingly facilitate the spread of an infectious agent through a population. incubatory carriers exist when the incubation period overlaps with the infectious period, as can occur in some cases of chicken pox (figure (b) ). convalescent carriers occur when the period of infectiousness extends beyond the period of clinical illness (figure (c) ). carriers of this type can be a significant issue in promoting the spread of certain enteric infections, such as those caused by the bacterium, v. cholerae. healthy carriers, infected individuals that remain asymptomatic but are capable of transmitting an infectious agent, occur commonly with many infectious diseases (e.g., meningococcal meningitis and typhoid fever) and are also significant challenges to disease control ( figure (d)). a variety of terms are used to describe the occurrence of an infectious disease within a specific geographic area or population. sporadic diseases occur occasionally and unpredictably, while endemic diseases occur with predictable regularity. levels of endemicity can be classified as holoendemic, hyperendemic, mesoendemic, or hypoendemic depending upon whether a disease occurs with, respectively, extreme, high, moderate, or low frequency. for some infectious diseases, such as malaria, levels of endemicity are well defined and used as parameters for identifying disease risk and implementing control activities. malaria endemicity is quantified based upon rates of palpable enlarged spleens in a defined (usually pediatric) age group: holoendemic > %, hyperendemic - %, mesoendemic - %, and hypoendemic < % (hay et al., ). an epidemic refers to an, often acute, increase in disease cases above the baseline level. an epidemic may reflect an escalation in the occurrence of an endemic disease or the appearance of a disease that did not previously exist in a population. the term outbreak is often used synonymously with epidemic but can occasionally refer to an epidemic occurring in a more limited geographical area; for example, a foodborne illness associated with a group gathering. by contrast, a pandemic is an epidemic that has spread over a large geographic region, encompassing multiple countries or continents, or extending worldwide. influenza commonly occurs as a seasonal epidemic, but periodically it gives rise to a global pandemic, as was the case with h n influenza. two fundamental measures of disease frequency are prevalence and incidence. prevalence is an indicator of the number of existing cases in a population as it describes the proportion of individuals who have a particular disease, measured either at a given point in time (point prevalence) or during a specified time period (period prevalence). in contrast, incidence (a.k.a. incidence rate) is a measurement of the rate at which new cases of a disease occur (or are detected) in a population over a given time period. usually measured as a proportion (number infected/number exposed), attack rates are often calculated during an outbreak. in some circumstances, a secondary attack rate is calculated to quantify the spread of disease to susceptible exposed persons from an index case (the case first introducing an agent into a setting) in a circumscribed population, such as in a household or hospital. during the sars epidemic, secondary attack rates in toronto hospitals were high but varied from % to % depending upon the hospital ward (cdc, b) . the basic reproductive number (basic reproductive ratio; r ) is a measure of the potential for an infectious disease to spread through an immunologically naïve population. it is defined as the average number of secondary cases generated by a single, infectious case in a completely susceptible population. in reality, for most infectious diseases entering into a community, some proportion of the population is usually immune (and nonsusceptible) due to previous infection and/or immunization. thus, a more accurate reflection of the potential for community disease spread is the effective reproductive number (r) which measures the average number of new infections due to a single infection. in general, for an epidemic to occur in a population, r must be > so that the number of cases continues to increase. herd immunity (a.k.a. community immunity) refers to population-level resistance to an infectious disease that occurs when there are enough immune individuals to block the chain of infection/transmission. as a result of herd immunity, susceptible individuals who are not immune themselves are indirectly protected from infection ( figure ). vaccine hesitancy, the choice of individuals or their caregivers to delay or decline vaccination, can lead to overall lower levels of herd immunity. outbreaks of measles in the united states, including a large measles outbreak at an amusement park in california, highlight the phenomena of vaccine refusal and associated increased risk for vaccine-preventable diseases among both nonvaccinated and fully vaccinated (but not fully protected) individuals (phadke et al., ) . an important public health consequence of herd immunity is that immunization coverage does not need to be % for immunization programs to be successful. the equation r ¼ r ( À x) (where x equals the immune portion of the population) indicates the level of immunization required to prevent the spread of an infectious disease through a population. the proportion that needs to be immunized depends on the pathogen (table ) . when the proportion immunized (x) reaches a level such that r < , a chain of infection cannot be sustained. thus, ro and r can be used to calculate the target immunization coverage needed for the success of vaccination programs. proper diagnosis of infectious illnesses is essential for both appropriate treatment of patients and carrying out prevention and control surveillance activities. two important properties that should be considered for any diagnostic test utilized are sensitivity and specificity. sensitivity refers to the ability of the test to correctly identify individuals infected with an agent ('positive in disease'). a test that is very sensitive is more likely to pick up individuals with the disease (and possibly some without the disease); a very sensitive test will have few false negatives. specificity is the ability of the test to correctly identify individuals not infected by a particular agent ('negative in health'); high specificity implies few false positives. often, screening tests are highly sensitive (to capture any possible cases), and confirmatory tests are more specific (to rule out false-positive screening tests). broadly, laboratory diagnosis of infectious diseases is based on tests that either directly identify an infectious agent or provide evidence that infection has occurred by documenting agent-specific immunity in the host ( figure ). identification of an infecting agent involves either direct examination of host specimens (e.g., blood, tissue, urine) or environmental specimens, or examination following agent culture and isolation from such specimens. the main categories of analyses used in pathogen identification can be classified as phenotypic, revealing properties of the intact agent, nucleic acid-based, determining agent nucleic acid (dna or rna) characteristics and composition, and immunologic, detecting microbial antigen or evidence of immune response to an agent ( figure ). direct phenotypic analyses include both macroscopic and/or microscopic examination of specimens to determine agent morphology and staining properties. cultured material containing large quantities of agent can undergo analyses to determine characteristics, such as biochemical enzymatic activity (enzymatic profile) and antimicrobial sensitivity, and to perform phage typing, a technique which differentiates bacterial strains according to the infectivity of strain-specific bacterial viruses (a.k.a. bacteriophages). nucleic acid-based tests often make use of the polymerase chain reaction (pcr) to amplify agent dna or complementary dna (cdna) synthesized from messenger rna (mrna). the ability of pathogen-specific pcr primers to generate an amplification product can confirm or rule out involvement of a specific pathogen. sequencing of amplified dna fragments can also assist with pathogen identification. restriction fragment analysis, as by pulse-field gel electrophoresis of restriction enzyme-digested genomic dna isolated from cultured material, can yield distinct 'dna fingerprints' that can be used for comparing the identities of bacteria. the cdc pulsenet surveillance program uses dna fingerprinting as the basis for detecting and defining foodborne disease outbreaks that can sometimes be quite widely dispersed (cdc, ) . most recently, next-generation sequencing technologies have made whole-genome sequencing a realistic subtyping method for use in foodborne outbreak investigation and surveillance (deng et al., ) . the objective of immunologic analysis of specimens is to reveal evidence of an agent through detection of its antigenic components with agent-specific antibodies. serotyping refers to the grouping of variants of species of bacteria or viruses based on shared surface antigens that are figure herd immunity occurs when one is protected from infection by immunization occurring in the community. using influenza as an example, the top box shows a population with a few infected individuals (shown in red) and the rest healthy but unimmunized (shown in blue); influenza spreads easily through the population. the middle box depicts the same population but with a small number who are immunized (shown in yellow); those who are immunized do not become infected, but a large proportion of the population becomes infected. in the bottom box, a large proportion of the population is immunized; this prevents significant transmission, including to those who are unimmunized. source: national institute of allergy and infectious diseases (niaid). identified using immunologic methodologies such as enzymelinked immunosorbent assay (elisa) and western blotting. immunologic assays are also used to look for evidence that an agent-specific immune response has occurred in an exposed or potentially exposed individual. serologic tests detect pathogen-specific b cell-secreted antibodies in serum or other body fluids. some serologic assays simply detect the ability of host antibodies to bind to killed pathogen or components of pathogen (e.g., elisa). others rely on the ability of antibodies to actually neutralize the activity of live microbes; as, for example, the plaque reduction neutralization test which determines the ability of serum antibodies to neutralize virus. antibody titer measures the amount of a specific antibody present in serum or other fluid, expressed as the greatest dilution of serum that still gives a positive test in whatever assay is being employed. intradermal tests for identification of t cell-mediated immediate type (type i) hypersensitivity or delayed type (type iv) hypersensitivity responses to microbial antigen can be used to diagnose or support the diagnosis of some bacterial, fungal, and parasitic infections, such as, the mantoux (tuberculin) test for tb. based on the classic model of leavell and clark ( ) , infectious disease prevention activities can be categorized as primary, secondary, or tertiary. primary prevention occurs at the predisease phase and aims to protect populations, so that infection and disease never occur. for example, measles immunization campaigns aim to decrease susceptibility following exposure. the goal of secondary prevention is to halt the progress of an infection during its early, often asymptomatic stages so as to prevent disease development or limit its severity; steps important for not only improving the prognosis of individual cases but also preventing infectious agent transmission. for example, interventions for secondary prevention of hepatitis c in injection drug user populations include early diagnosis and treatment by active surveillance and screening (miller and dillon, ) . tertiary prevention focuses on diseased individuals with the objective of limiting impact through, for example, interventions that decrease disease progression, increase functionality, and maximize quality of life. broadly, public health efforts to control infectious diseases focus on primary and secondary prevention activities that reduce the potential for exposure to an infectious agent and increase host resistance to infection. the objective of these activities can extend beyond disease control, as defined by the dahlem workshop on the eradication of infectious diseases, to reach objectives of elimination and eradication (dowdle, ; box ). as noted earlier, the causation and spread of an infectious disease is determined by the interplay between agent, host, and environmental factors. for any infectious disease, this interplay requires a specific linked sequence of events termed the chain of infection or chain of transmission ( figure ). the chain starts with the infectious agent residing and multiplying in some natural reservoir; a human, animal, or part of the environment such as soil or water that supports the existence of the infectious agent in nature. the infectious agent leaves the reservoir via a portal of exit and, using some mode of transmission, moves to reach a portal of entry into a susceptible host. a thorough understanding of the chain of infection is crucial for the prevention and control of any infectious disease, as breaking a link anywhere along the chain will stop transmission of the infectious agent. often more than one intervention can be effective in controlling a disease, and the approach selected will depend on multiple factors such as economics and ease with which an intervention can be executed in a given setting. it is important to realize that the potential for rapid and far-reaching movement of infectious agents that has accompanied globalization means that coordination of intervention activities within and between nations is required for optimal prevention and control of certain diseases. the cause of any infectious disease is the infectious agent. as discussed earlier, many types of agents exist, and each can be characterized by its traits of infectivity, pathogenicity, and virulence. a reservoir is often, but not always, the source from which the agent is transferred to a susceptible host. for example, bats are both the reservoir for marburg virus and a source of infection for humans and bush animals including african gorillas. however, because morbidity and mortality due to marburg infection is significant among these bush animals, they cannot act as a reservoir to sustain the virus in nature (they die too quickly), although they can act as a source to transmit marburg to humans. infectious agents can exist in more than one type of reservoir. the number and types of reservoirs are important determinants of how easily an infectious disease can be prevented, controlled, and, in some cases, eliminated or eradicated. animal, particularly wild animal, reservoirs, and environmental reservoirs in nature can be difficult to manage and, thus, can pose significant challenges to public health control efforts. in contrast, infectious agents that only occur in human reservoirs are among the dahlem workshop on the eradication of infectious diseases defined a continuum of outcomes due to public health interventions targeting infectious diseases: " ) control, the reduction of disease incidence, prevalence, morbidity or mortality to a locally acceptable level as a result of deliberate efforts; continued intervention measures are required to maintain the reduction (e.g. diarrheal diseases), ) elimination of disease, reduction to zero of the incidence of a specified disease in a defined geographical area as a result of deliberate efforts; continued intervention measures are required (e.g. neonatal tetanus), ) elimination of infections, reduction to zero of the incidence of infection caused by a specific agent in a defined geographical area as a result of deliberate efforts; continued measures to prevent re-establishment of transmission are required (e.g. measles, poliomyelitis), ) eradication, permanent reduction to zero of the worldwide incidence of infection caused by a specific agent as a result of deliberate efforts; intervention measures are no longer needed (e.g. smallpox), and ) extinction, the specific infectious agent no longer exists in nature or in the laboratory (e.g. none)" (dowdle, ) . the chain of infection (a.k.a. chain of transmission). one way to visualize the transmission of an infectious agent though a population is through the interconnectedness of six elements linked in a chain. public health control and prevention efforts focus on breaking one or more links of the chain in order to stop disease spread. those most easily targeted, as illustrated by the success of smallpox eradication. humans are the reservoir for many common infectious diseases including stis (e.g., hiv, syphilis) and respiratory diseases (e.g., influenza). humans also serve as a reservoir, although not always a primary reservoir, for many neglected tropical diseases (ntds) as, for example, dracunculiasis (a.k.a. guinea worm). from a public health standpoint, an important feature of human reservoirs is that they might not show signs of illness and, thus, can potentially act as unrecognized carriers of disease within communities. the classic example of a human reservoir is the cook mary mallon (typhoid mary); an asymptomatic chronic carrier of salmonella enterica serovar typhi who was linked to at least cases of typhoid fever (soper, ) . animals are a reservoir for many human infectious diseases. zoonosis is the term used to describe any infectious disease that is naturally transmissible from animals to humans. these diseases make up approximately % of all infectious diseases, and an estimated % of recently emerging infectious diseases (burke et al., ) . zoonotic reservoirs and sources of human disease agents include both domestic (companion and production) animals (e.g., dogs and cows) and wildlife. control and prevention of zoonotic diseases requires the concerted efforts of professionals of multiple disciplines and is the basis for what has become known as the one health approach (gibbs, ) . this approach emphasizes the interconnectedness of human health, animal health, and the environment and recognizes the necessity of multidisciplinary collaboration in order to prevent and respond to public health threats. inanimate matter in the environment, such as soil and water, can also act as a reservoir of human infectious disease agents. the causative agents of tetanus and botulism (clostridium tetani and c. botulinum) are examples of environmental pathogens that can survive for years within soil and still remain infectious to humans. legionella pneumophila, the etiologic agent of legionnaires' disease, is part of the natural flora of freshwater rivers, streams, and other bodies. however, the pathogen particularly thrives in engineered aquatic reservoirs such as cooling towers, fountains, and central air conditioning systems, which provide conditions that promote bacterial multiplication and are frequently linked to outbreaks. soil and water are also sources of infection for several protozoa and helminth species which, when excreted by a human reservoir host, can often survive for weeks to months. outbreaks of both cryptosporidiosis and giardiasis commonly occur during summer months as a result of contact with contaminated recreational water. soil containing roundworm (ascaris lumbricoides) eggs is an important source of soil-transmitted helminth infections in children. early steps in preventing exposure to an infectious agent include interventions to control or eliminate the agent within its reservoir, to neutralize or destroy the reservoir, and/or to stop the agent from exiting its reservoir. central to these interventions are surveillance activities that routinely identify disease agents within reservoirs. when humans are the reservoir, or source, of an infectious agent, early and rapid diagnosis and treatment are key to decreasing the duration of infection and risk of transmission. both active surveillance and passive surveillance are used to detect infected cases and carriers. some readily communicable diseases, such as ebola, can require isolation of infected individuals to minimize the risk of transmission. as part of the global effort to eradicate dracunculiasis, several endemic countries have established case containment centers to provide treatment and support to patients with emerging guinea worms to keep them from contaminating water sources and, thereby, exposing others (hochberg et al., ) . contact tracing and quarantine are other activities employed in the control of infections originating from a human reservoir or source. during the west africa ebola outbreak, key control efforts focused on the tracing and daily follow-up of healthy individuals who had come in contact with ebola patients and were potentially infected with the virus (pandey et al., ) . one health emphasizes the importance of surveillance and monitoring for zoonotic pathogens in animal populations. for some diseases (e.g., rift valley fever) epizootics (analogous to epidemics, but in animal populations) can actually serve as sentinel events for forecasting impending human epidemics. once animal reservoirs (and sources) of infection are identified, approaches to prevention and control include reservoir elimination and prevention of reservoir infection. zoonotic diseases exist in nature in predictably regular, enzootic cycles and/or epizootic cycles and are transmitted to humans via distinct pathways. the focus of prevention and control activities for these diseases reflects the extent to which a zoonotic pathogen has evolved to become established in human populations (wolfe et al., ) . for some zoonotic diseases (e.g. anthrax, nipah, rabies), primary transmission always occurs from animals, with humans acting as incidental (dead end) hosts; control of these diseases thus concentrates on preventing animal-to-animal and, ultimately, animal-to-human transmission. currently, most human cases of avian influenza are the result of human infection from birds; human-to-human transmission is extremely rare. thus, reservoir elimination by culling infected poultry flocks is a recommended measure for controlling avian influenza in birds and preventing sporadic infection of humans (cdc, ) . other zoonotic diseases demonstrate varying degrees of secondary human-to-human transmission following primary transmission (a.k.a. spillover) from animals. both rates of spillover and the ability to sustain human-tohuman transmission can vary widely between zoonoses and, in consequence, control strategies can also be quite different. for example, outbreaks of ebola arise following an initial bush animal-to-human transmission event, and subsequent human-to-human transmission is often limited (feldmann and geisbert, ) . in contrast, the four dengue viruses originally emerged from a sylvatic cycle between non-human primates and mosquitoes, and are now sustained by a continuous human-mosquito-human cycle of transmission with outbreaks occurring as a result of infected individuals entering into naïve populations (vasilakis et al., ) . thus, while ebola outbreak prevention efforts would include limiting contact with bush animals, such efforts would not be useful for prevention of dengue outbreaks. hiv is an example of a virus that emerged from an ancestral animal virus, simian immunodeficiency virus, but has evolved so that it is now hiv is an example of a virus that emerged from an ancestral animal virus, simian immunodeficiency virus, but has evolved so that it is now only transmitted human to human (faria et al., ) . infectious agents exit human and animal reservoirs and sources via one of several routes which often reflect the primary location of disease; respiratory disease agents (e.g., influenza virus) usually exit within respiratory secretions, whereas gastrointestinal disease agents (e.g., rotavirus, cryptosporidium spp.) commonly exit in feces. other portals of exits include sites from which urine, blood, breast milk, and semen leave the host. for some infectious diseases, infection can naturally occur as a result of contact with more than one type of bodily fluid, each of which uses a different portal of exit. while infection with the sars virus most frequently occurred via contact with respiratory secretions, a large community outbreak was caused by the spread of virus in a plume of diarrhea (yu et al., ) . control interventions targeting portals of exit and entry are discussed below. there are a variety of ways in which infectious agents move from a natural reservoir to a susceptible host, and several different classification schemes are used. the scheme below categorizes transmission as direct transmission, if the infective form of the agent is transferred directly from a reservoir to an infected host, and indirect transmission, if transfer takes place via a live or inanimate intermediary (box ). direct physical contact between the skin or mucosa of an infected person and that of a susceptible individual allows direct transfer of infectious agents. this is a mode of transmission for most stis and many other infectious agents, such as bacterial and viral conjunctivitis (a.k.a. pink eye) and ebola virus disease. direct droplet transmission occurs after sneezing, coughing, or talking projects a spray of agent-containing droplets that are too large to remain airborne over large distances or for prolonged periods of time. the infectious droplets traverse a space of generally less than m to come in contact with the skin or mucosa of a susceptible host. many febrile childhood diseases, including the common cold, are transferred this way. diseases spread by direct contact and droplet transmission require close proximity of infected and susceptible individuals and, thus, commonly occur in settings such as households, schools, institutions of incarceration, and refugee/displaced person camps. infectious agents spread exclusively in this manner are often unable to survive for long periods outside of a host; direct transmission helps to ensure transfer of a large infective dose. direct contact to an agent in the environment is a means of exposure to infectious agents maintained in environmental reservoirs. diseases commonly transmitted in this manner include those in which the infectious agent enters a susceptible host via inhalation (e.g., histoplasmosis) or through breaks in the skin following a traumatic event (e.g., tetanus). animal bites are another way in which some infectious agents are directly transferred, through broken skin. this is the most common means of infection with rabies virus. transplacental (a.k.a. congenital, vertical) and perinatal transmissions occur during pregnancy and delivery or breastfeeding, respectively. classic examples include mother-to-child transmission of the protozoa toxoplasma gondii during pregnancy, hiv during pregnancy, delivery, or breastfeeding, and zika virus during pregnancy (rasmussen et al., ) . case finding and contact tracing are public health prevention and control activities aimed at stopping the spread of infectious diseases transmitted by either direct contact or direct spread of droplets. once identified, further activities to limit transmission to susceptible individuals can involve definitive diagnosis, treatment, and, possibly, isolation of active cases and carriers, and observation, possible quarantine, or prophylactic vaccination or treatment of contacts. patient education is an important feature of any communicable infectious disease control effort. environmental changes, such as decreasing overcrowded areas and increasing ventilation, can also contribute to limiting the spread of some infectious diseases, particularly respiratory diseases. central to prevention of transplacental and perinatal infectious disease transmission is avoidance of maternal infection and provision of early diagnosis and treatment of infected women prior to or during pregnancy. for example, public health efforts targeting congenital toxoplasmosis focus on preventing pregnant women from consuming undercooked meat or contacting cat feces that may be contaminated. current who guidelines for prevention of mother-to-child hiv transmission recommend that hiv-infected pregnant and breastfeeding women should be maintained on antiretrovirals (who, ) . there are three main categories of indirect transmission: biological, mechanical, and airborne. box provides definitions of the different types of hosts, vectors, and vehicles involved in the life cycle of agents that are transmitted indirectly. biological transmission occurs when multiplication and/or development of a pathogenic agent within a vector (e.g., biological vector or intermediate host) is required for the agent to become infectious to humans. the time that is necessary for these events to occur is known as the extrinsic incubation period; in contrast to the intrinsic incubation period which is the time required for an exposed human host to become infectious. indirect transmission by mosquito vectors is the primary mode of transmission of a large number of viruses (arthropod-borne viruses or arboviruses) of public health concern (e.g., west nile, zika). a number of ntds are also transmitted by biological vectors including lymphatic filariasis (a.k.a. elephantiasis) by mosquitoes. ticks are biological vectors for many bacterial etiological agents (e.g., lyme disease and ehrlichiosis), and the parasitic agent causing babesiosis. the infectious agent of the helminthic ntds, schistosomiasis, and dracunculiasis are transmitted indirectly via intermediate freshwater snail and copepod hosts, respectively. mechanical transmission does not require pathogen multiplication or development within a living organism. it occurs when an infectious agent is physically transferred by a live entity (mechanical vector) or inanimate object (vehicle) to a susceptible host. classic examples of diseases spread by mechanical vector transmission are shigellosis (transmission of shigella spp. on the appendages of flies) and plague (transmission of yersinia pestis by fleas). many diarrheal diseases are transmitted by the fecal-oral route with food and water often acting as vehicles (figure ) . other types of vehicles for infectious disease agents are biologic products (e.g., blood, organs for transplant) and fomites (inanimate objects such as needles, surgical instruments, door handles, and bedding). transfusion-related protozoal infection resulting in chagas disease has been of increasing concern to the us blood banks that have instituted screening measures (cdc, ) . airborne transmission involves aerosolized suspensions of residue (less than five microns in size, from evaporated aerosol droplets) or particles containing agents that can be transported over time and long distance and still remain infective. tb is a classic example of an infectious disease often spread by airborne transmission. vbds comprise approximately % of the global burden of infectious diseases (townson et al., ) . for some diseases (e.g., dengue, zika, chagas), chemoprophylaxis and immunoprophylaxis are not prevention and control options, leaving vector control as the primary means of preventing disease transmission. integrated vector management is defined by the who as, "a rational decision-making process to optimize the use of resources for vector control" (who, ) . there are four major categories of ivm vector control strategies: biological, chemical, environmental, and mechanical. ivm interventions are chosen from these categories based upon available resources, local patterns of disease transmission, and ecology of local disease vectors. two key elements of ivm are collaboration within the health sector and with other sectors (e.g., agriculture, engineering) to plan and carry out vector control activities, and community engagement to promote program sustainability. another core element is the integrated approach which often permits concurrent targeting of multiple vbds, as some diseases are transmitted by a single, common vector, and some vector control interventions can target several different vectors. in addition, combining interventions serves not only to reduce reliance on any single intervention, but also to reduce the selection pressure for insecticide and drug resistance. table , adapted from the who handbook for ivm, illustrates some of the many types of ivm activities and provides examples of vbds that might be controlled by such interventions (who, ) . diarrheal diseases primarily result from oral contact with water, food, or other vehicles contaminated with pathogenic agents originating from human or animal feces. most ($ %) of diarrhea-associated deaths are attributable to unsafe drinking water, inadequate sanitation, and insufficient hygiene (black et al., ; prüss-Üstün et al., ) . interruption of fecaloral transmission through provision of safe water and adequate sanitation, and promotion of personal and domestic hygiene are fundamental to diarrhea prevention and control. fecaloral transmission of a diarrheal agent can occur via one of several routes. in , wagner and lanoix developed a model of major transmission depicted in what has become known as the 'f-diagram,' based on steps within the fecal-oral flow of transmission starting with the letter 'f': fluids (drinking water), definitive host: a host in which a parasite reproduces sexually. humans are definitive hosts for roundworms. by strict definition, mosquitoes are the definitive host of malaria as they are the organism in which sexual reproduction of the agent protozoa, plasmodium spp., occurs. reservoir host: a host that serves to sustain an infectious pathogen as a potential source of infection for transmission to humans. note that a reservoir host will not succumb to infection. lowland gorillas and chimpanzees can be infected by ebola virus, but they are not a reservoir host as they suffer devastating losses from infection. bats are a suspected reservoir for ebola virus. intermediate dead-end host: a host from which infectious agents cannot be transmitted to other susceptible hosts. humans are a dead-end host for west nile virus which normally circulates between mosquitoes and certain avian species. vector: a generic term for a living organism (e.g., biological vector or intermediate host) involved in the indirect transmission of an infectious agent from a reservoir or infected host to a susceptible host. biological vector: a vector (often arthropod) in which an infectious organism must develop or multiply before the vector can transmit the organism to a susceptible host. aedes spp. mosquitoes are a biological vector for dengue, chikungunya, and zika. mechanical vector: a vector (often arthropod) that transmits an infectious organism from one host to another but is not essential to the life cycle of the organism. the house fly is a mechanical vector in the diarrheal disease shigellosis as it carries feces contaminated with the shigella spp. bacterium to a susceptible person. vehicles: inanimate objects that serve as an intermediate in the indirect transmission of a pathogen from a reservoir or infected host to a susceptible host. these include food, water, and fomites such as doorknobs, surgical instruments, and used needles. fingers, flies, fields (crops and soil), floods (representative of surface water in general), and food (wagner and lanoix, ; figure ). other f's that can be considered include facilities (e.g., settings where transmission is likely to occur such as daycare centers) and fornication. the f-diagram is useful for depicting where water, sanitation, and hygiene (wash) interventions act as barriers in the fecal-oral flow of diarrheal pathogens. safe excreta disposal and handling act as primary barriers to transmission by preventing fecal pathogens from entering the environment. once the environment has become contaminated with pathogen-containing feces, secondary and tertiary barriers to transmission include water treatment, safe transport and storage of water, provision of sewage systems to control flooding, fly control, and good figure the 'f-diagram' illustrates major direct and indirect pathways of fecal-oral pathogen transmission and depicts the roles of water, sanitation, and hygiene interventions in providing barriers to transmission. primary barriers prevent contact with feces, and secondary barriers prevent ingestion of feces. source: water, engineering and development centre (wedc), loughborough university. personal and domestic hygiene (e.g., food hygiene) practices (requiring adequate water quantity) ( figure ) . as with ivm, the control of diarrheal diseases increases with integration of control measures to achieve multiple barriers to fecal-oral transmission. the basic approach to preventing transmission of an infectious agent from a contaminated vehicle is to prevent contamination of, decontaminate, or eliminate the vehicle. food is a common vehicle for infectious agents, and it can potentially become contaminated at any step along the food production chain of production, processing, distribution, and preparation. production refers to the growing of plants for harvest and raising animals for food. an example of contamination at this step includes the use of fecally contaminated water for crop irrigation. processing refers to steps such as the chopping, grinding, or pasteurizing of food to convert it into a consumer product; if the external surface of a melon is contaminated, chopping it into pieces for sale can result in contamination of the fruit. distribution, in which food is transferred from the place where it was produced and/or processed to the consumer, can result in contamination if, for example, the transportation vehicle is not clean. finally, preparation is the step in which food is made ready to eat; not cleaning a cutting board after cutting raw chicken can result in microbial pathogen crosscontamination of other food items. food hygiene is the term used to describe the conditions and activities employed to prevent or limit microbial contamination of food in order to ensure food safety. decontamination includes sterilization, the destruction of all microbial agents, and disinfection, the destruction of specific agents. control of airborne diseases focuses on regulating environmental airflow and air quality to minimize contact with infectious droplet nuclei. in health-care settings, negative pressure isolation rooms and exhaust vents can be used to manipulate airflow. recirculating, potentially infectious air can undergo high-efficiency particulate air (hepa) filtration and/or be mixed with 'clean' (noncontaminated) air to remove or dilute the concentration of infectious particle to below the infectious dose. health-care workers should use n masks. on commercial aircraft, airborne pathogen transmission is minimized by methods including regulating airflow to prevent widespread dispersal of airborne microbes throughout the cabin, hepa filtering recirculating air, and mixing recirculating air with fresh air (considered sterile) (dowdall et al., ) . the portal of entry refers to the site at which the infectious agent enters a susceptible host and gains access to host tissues. many portals of entry are the same as portals of exit and include the gastrointestinal, genitourinary, and respiratory tracts, as well as compromised skin and mucous membrane surfaces. some infectious agents can naturally enter a susceptible host by more than one portal. for example, the three forms of human anthrax can be distinguished according to the route of agent entry: cutaneous anthrax due to entry through the skin, gastrointestinal anthrax resulting from ingestion of spores, and pulmonary anthrax following inhalation of spores. standard infection control practices target portals of exit (and entry) of infectious agents from human reservoirs and sources. cdc guidelines suggest two levels of precautions to stop transmission of infectious agents: standard precautions and transmission-based precautions (siegel et al., ) . standard precautions prevent transmission of infectious agents that can be acquired by contact with blood, body fluids, nonintact skin, and mucous membranes. they can be used to prevent transmission in both health-care and non-health-care settings, regardless of whether infection is suspected or confirmed. hand hygiene is a major component of these precautions, along with use of personal protective equipment (ppe). common ppe include gloves, gowns, face protection (e.g., eye-protecting face shields), and respiratory protection using n masks to prevent inhalation of airborne infectious particles (e.g., from mycobacterium tuberculosis). of note, depending on the circumstance, ppe can be used to prevent dispersal of infectious agents from their source by providing a barrier to the portal of exit, or to protect a susceptible individual by placing a barrier to a portal of entry. respiratory hygiene/cough etiquette is used to prevent spread of infection by respiratory droplets. main elements of respiratory hygiene/cough etiquette include covering the nose and mouth area with one's elbow during coughing or sneezing or using a surgical mask to limit dissemination of infectious respiratory secretions, and hand hygiene after contact with respiratory secretions. other components of standard precautions include needle stick and sharp injury prevention, safe injection practices, cleaning and disinfection of potentially contaminated equipment and other objects, and safe waste management. a susceptible host is an individual who is at risk of infection and disease following exposure to an infectious agent. as discussed previously, there are many determinants of host susceptibility, including both innate factors determined by the genetic makeup of the host and, acquired factors such as agent-specific immunity and malnutrition. important prevention and control interventions that target the susceptible host include both those that address determinants of susceptibility in the host (e.g., immunoprophylaxis, provision of adequate nutrition, treatment of underlying diseases) and those that target an infecting agent (e.g., chemoprophylaxis). immunoprophylaxis encompasses both active immunization by vaccination and passive immunization through provision of pathogen-specific immunoglobulin. malnutrition is a strong risk factor for morbidity and mortality due to diarrheal disease, and a vicious cycle exists between infectious diarrheal disease leading to malnutrition and impaired immune function which, in turn, promotes increased susceptibility to infection (keusch et al., ) . consequently, breastfeeding and safe complementary feeding play crucial roles in protecting infants and young children from infectious diseases, particularly in resource-poor settings. micronutrients are required for normal immune function, and vitamin a and zinc supplementations have been shown to decrease some types of infections in children deficient in these micronutrients (mayo-wilson et al., ; imdad et al., ) . in certain circumstances, chemoprophylaxis is employed to protect a susceptible host in anticipation of, or following exposure to an infectious agent. antimalarial drugs are routinely used in combination with personal protective measures to prevent malaria in travelers and established guidelines exist for antibiotic prophylaxis prior to surgery. another important element in the prevention and control of infections is the recognition and management of patients with underlying diseases and conditions that can weaken host barriers to infection. for example, tb is the leading opportunistic infection in hivinfected individuals, and antiretroviral therapy reduces risk of developing tb and mortality due to tb disease. infectious complications are a major cause of morbidity and mortality in cancer and transplant patients, often resulting from immunosuppression that can be primary or related to drug and/or radiation therapy. infectious disease control is also critical in individuals with 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and preventing hiv infection large outbreak of cryptosporidium hominis infection transmitted through the public water supply origins of major human infectious diseases impact of deforestation and agricultural development on anopheline ecology and malaria epidemiology evidence of airborne transmission of the severe acute respiratory syndrome virus key: cord- -bxm yxjm authors: zeng, yawen; pu, xiaoying; du, juan; yang, xiaomeng; li, xia; mandal, md. siddikun nabi; yang, tao; yang, jiazhen title: molecular mechanism of functional ingredients in barley to combat human chronic diseases date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: bxm yxjm barley plays an important role in health and civilization of human migration from africa to asia, later to eurasia. we demonstrated the systematic mechanism of functional ingredients in barley to combat chronic diseases, based on pubmed, cnki, and isi web of science databases from to . barley and its extracts are rich in ingredients to combat more than chronic diseases, which include the similar and different chronic diseases between grains and grass, due to the major molecular mechanism of six functional ingredients of barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch). the antioxidant activity of barley grass and grain has the same and different functional components. these results support findings that barley grain and its grass are the best functional food, promoting ancient babylonian and egyptian civilizations, and further show the depending functional ingredients for diet from pliocene hominids in africa and neanderthals in europe to modern humans in the world. this review paper not only reveals the formation and action mechanism of barley diet overcoming human chronic diseases, but also provides scientific basis for the development of health products and drugs for the prevention and treatment of human chronic diseases. global cost of five chronic diseases (diabetes, cardiovascular disease, mental illness, chronic respiratory disease, and cancer) treatment to reach $ trillion from to [ ] . the intake of high sodium with low whole grains and fruits was the top most dietary risk factors for deaths and disability-adjusted life years globally and in many countries [ ] . diabetes in production regions of polished rice with high glycemic index (gi ≥ ) caused the biggest reduction in health-adjusted life expectancy at birth in regions in countries from to [ ] . the micronutrients deficiencies at the highest risk are fe, zn, and vitamins (v b , v b , v b , and v c ) [ ] . the outbreak of human chronic disease is due to taste pursuit that changes a healthy diet, i.e., the ancients switched from brown rice (gi ≤ , high k and high micronutrients) and barley (gi ≤ ) or its grass flour (k/na ≥ ) as staple foods to modern polished rice (gi ≥ ) and wheat white flour (gi ≥ ) with low and low micronutrients as staple foods [ , ] . barley grass is not only the best functional food for cell nutrition and detoxification in human body but also the most abundant bioactive ingredients for lots of health-promoting effects [ , ] . it can combat more than chronic diseases due to gaba, flavonoids, sod, k-ca, vitamins, and trypto-phan mechanism in barley grass ( figure ) [ ] . the sustaining major foods+barley grass powder can achieve the who's intake target of low sodium (< g) with high potassium (> . g) every day [ ] . more than functional ingredients in barley grass can combat over chronic diseases, and functional ingredients in barley grains may prevent chronic diseases [ ] . barley enhanced the sterols accumulation through ltp gene action that take part in the abiotic stress reaction of mediating intracellular lipid transport [ ] . barley straw ( . ~ . g/l) for phenolic acid in degradation inhibited the alga (m. aeruginosa) blooms of aquatic eutrophication by cell shrinkage of metabolic activity and chlorophyll a fluorescence decay [ , ] . therefore, barley grass powder plays an important role for solving human chronic diseases. barley grains have the highest functional value (low gi with high β-glucans and resistant starch) and antioxidant properties among cereal crops. the soluble fiber β-glucans is a group of polysaccharides found in barley, oats, mushrooms, yeasts, and seaweed [ ] . hulless barley variety zangqing has a . -gb sequence with , genes in seven chromosomes [ ] ; three hvcslf genes take part in ( , ; , )-β-glucan synthesis [ ] . qingke (hulless barley) is a major food for tibetan people and an important livestock feed in the qinghai-tibetan plateau, which has lots of gene family related to stress reactions [ ] , especially different antioxidant capacities due to some polysaccharide and phytochemical compositions [ ] . regular daily consumption of whole barley flour can prevent chronic diseases, especially diabetes, colonic cancer, hyperlipidemia, high blood pressure, and gallstones [ ] . although barley grains have played an important role in health effects of human being, health contribution and different major mechanisms from barley grass for preventive human chronic diseases and functional ingredients in barley grains are unclear. [ , , ] resistant starch (%) whole grains : ± : . ~ . [ ] [ ] [ ] arabinoxylan (%) endosperms : ± : . ~ . [ ] barley bran : ± : . ~ . [ , ] grains flour : ± : . ~ . [ , ] polyphenols (mg/ g) whole grains : ± : . ~ . [ , ] barley bran : ± : . ~ . [ ] grains flour : ± : . ~ . [ ] phenolic acids (mg/ g) whole grains : ± : . ~ . [ ] total flavones (mg/ g) whole grains : ± : . ~ . [ , , ] flavonoids (mg/ g) whole grains : ± : . ~ . [ ] catechin (mg/ g) whole grains : ± : . ~ . [ , ] quercetin (mg/ g) purple grains : ± : . ~ . [ , ] kaempferol (mg/ g) whole grains : ± : . ~ . [ , ] myricetin (mg/ g) whole grains : ± : ~ . [ ] total alkaloid (mg/ g) whole grains : ± : . ~ . [ ] total anthocyanin (mg/ g) whole grains : ± : . ~ . [ ] barley bran : ± : ~ . [ ] refined flours : ± : . ~ . [ ] proanthocyanidin (mg/ g) whole grains : ± : . ~ . [ ] total tocols (mg/ g) whole grains : ± : . ~ . [ , , , ] antioxidant activity (%) whole grains : ± : . ~ . [ ] gaba (mg/ g) whole grains : ± : . ~ . [ ] protein % whole grains : ± : . ~ . [ ] folates (mg/ g) whole grains : ± : . ~ . [ , ] phytosterols (mg/ g) whole grains : ± : . ~ . [ ] p (mg/kg) whole grains , : ± , : ~ [ ] k (mg/kg) whole grains , : ± , : ~ [ ] ca (mg/kg) whole grains : ± : . ~ . [ ] mg (mg/kg) whole grains , : ± : . ~ . [ ] fe (mg/kg) whole grains : ± : . ~ . [ ] zn (mg/kg) whole grains : ± : . ~ . [ ] cu (mg/kg) whole grains : ± : . ~ . [ ] mn (mg/kg) whole grains : ± : . ~ . [ ] na (mg/kg) whole grains : ± : . ~ . [ ] s (mg/kg) whole grains , : ± : . ~ . [ ] oxidative medicine and cellular longevity and metal chelating activity . - . %) than that of plains ( ~ m altitude), but the soluble β-glucan and arabinoxylan content ranged from . % to . % and . % to . %, respectively [ ] . . . β-glucan. β-glucan in barley is the most abundant group of polysaccharides in cell wall. the molecular weight of β-d-glucan in hulless barley grains is . kda, which composes of ( → )-and ( → )-glucopyranosyl residues, especially its trisaccharide and tetrasaccharide accounted for . % of total cellulosyl units [ ] . β-glucan content (%) in naked, malt, black, waxy-naked, and blue barley is . , . , . , . , and . , respectively, especially waxy-naked barley flour has the highest extraction rate ( . %); however, gi in vitro starch digestibility was lowered by adding β-glucan [ ] . the fatty acid derived flavouring substance (dodecanoic acid, octyl butanoate, ethyl decanoate, and decyl acetate) in beer has important role in the aggregation behavior of barley β-glucan [ ] . the β-glucan concentrations in the six hulless barley grains varied from . % to . %, among shorts ( . ~ . %)>bran ( . ~ . %)>flour ( . ~ . %) [ ] . the total β-glucan contents in the nine hulless barley are . ~ . % in bran and > . ~ . % in refined flour [ ] . barley. phenolic compounds have the antioxidant, anti-inflammatory, and antitumor potentials [ ] . there are the most abundant polyphenols in barley grains, especially p-hydroxybenzoic ( . %), pcoumaric ( . %), and ferulic acids ( . %) [ ] . the most abundant polyphenol in barley extract include free polyphenols ( : ± : mg/ g) and bound polyphenols ( : ± : mg/ g), especially ferulic acid ( . mg/ g) and >procyanidin b ( . mg/ g) [ ] . phenolic acids such as ferulic acid and p-coumaric acid were . mg/ g and . mg/ g in barley grains and . mg/ g and . mg/ g in malt, respectively [ ] . the extraction polyphenols yield of barley lactobacillus fermented solution of % ethanol concentration was . %, main components (mg/ g) including rutin ( . ), vanillic acid ( . ), ferulic acid ( . ), coumaric acid ( . ), gallic acid ( . ), protocatechuic acid ( . ), and p-coumaric acid ( . ) [ ] . compared to the raw barley extract, the protein, total phenols, and βglucan of fermented barley extract with lactobacillus plantarum dy- can significantly increase to . %, . mg/g, and . %, respectively [ ] . there were larger genetic variations in the contents of total polyphenol ( : ± : mg/ g), total flavonoid content ( : ± : mg/ g), and antioxidant activity ( : ± : %) among the barley genotypes; however, major qtls between bpb- and bpb- control phenolic compounds in tibetan wild barley, especially the udp-glycosyltransferase gene with biosynthesis of flavonoid glycosides was colocated with bpb- [ ] . barley. nacl stress increased the phenolic compounds (vanillic acid, p-coumaric acid, ferulic acid, and sinapic acid) accumulation and synthesis by upregulating the gene expression of phenylalanine ammonia lyase, cinnamic acid -hydroxylase, -coumarate coenzyme a ligase, p-coumaric acid -hdroxylase, and caffeic acid/ -hydroxyferulic acid o-methyltransferase of germinated hulless barley [ ] . the blue hulless barley grains have larger variation on phenolic compounds and antioxidant activity, such as the free, bound, and total phenolic acids varied between . ~ . , . ~ . , and . ~ . mg/ g, respectively, where the major phenolic compounds include quercetin, rutin, naringenin, hesperidin, (+)-catechin, gallic acid, benzoic acid, syringic acid, and -coumaric acid [ ] . the anthocyanin and total phenolic contents in hulless barley grains are higher for high altitude, and the contents in its refined flours were . ~ . mg/ g and . ~ . mg fae/ g and in its bran were . ~ . mg/ g and . ~ . mg fae/ g, respectively [ ] . whole grain hulless barley had high contents of total phenolic ( . mg/ g), total pentosan ( . g/ g), and orac values ( : ± : mol/ g) [ ] . the bran extract of hulless barley rich in phenolic acids on the nε-carboxymethyllysine formation during processing biscuits, which can reduce glycation and benefiting health [ ] . total polyphenols ( . mg/ g) and proanthocyanidins ( . anthocyanin for human health belongs to flavonoids, which is a secondary metabolite that plants adapt to harsh environments. ant gene ( hl) with a bhlh domain control purple grain and ant gene control red leaf sheath and pericarp in barley, r r -myb (ant )+bhlh (ant ) complex promotes the synthesis by affecting expression of the anthocyanin biosynthesis structural genes (f ′ h) and ans genes [ ] . the hl alleles from barley purple pericarp synthesis the peonidin- glucoside [ ] . the anthocyanin synthesis hvmyc gene is the major variant factor for blue aleurone of barley [ ] . barley anthocyanins take part in the amino acid biosynthesis, carbon metabolism, phenylpropanoid biosynthesis, and metabolic pathways [ ] . flavonoid ′ -hydroxylase (f ′ h) and flavonoid ′ , ′ -hydroxylase (f ′ ′ h)-coding genes take part in anthocyanin synthesis in barley [ ] . the anthocyanin bran-rich fractions of yellow ( . mg/ g, anthocyanins) and purple barley ( . mg/ g, anthocyanins) are times higher than that of the whole grain flours ( . and . mg/ g), especially cyanidin -glucoside, delphinidin -glucoside, petunidin -glucoside, delphinidin -rutinoside, and cyanidin chloride [ ] . arabinoxylan in barley is the second highest cell wall polysaccharide [ ] . arabinoxylan in barley plays an important role in quality traits of malt and beer product [ ] ; however, arabinoxylan arabinofuranohydrolase i can be used as novel enzyme products in the beer industry [ ] . the starch degradation for seedling relies on cell wall degradation, where the iminosugar , dideoxy- , -imino-l-arabinitol inhibits dextrinase and arabinoxylan arabinofuranohydrolase but permits rapid diffusion of αand β-amylase [ ] . arabinoxylan contents in barley grains range from . % to . % [ ] where it ranged from . mg/ g to . mg/ g at an average value of . mg/ g in barley endosperm in spring -row barley; its two qtls include glycosyltransferases and glycoside hydrolases [ ] . arabinoxylan accounts for % of total polysaccharide [ ] and ~ % of total monosaccharide in barley husk [ ] . arabinoxylan contents in hulless barley are in bran . %>in shorts . %>in flour . % [ ] which differ from the report of moza et al. [ ] (in bran . ~ . %>in flour . ~ . %). . . phytosterols. phytosterols in plant membrane are similar in structure to cholesterol [ ] . higher phytosterols are found in the outer layers of barley grains and ranged between . mg/ g and . mg/ g, among which β-sitosterol is : ± : mg/ g and campesterol is : ± : mg/ g. the other phytosterols include stigmasterol ( . mg/ g), brassicasterol, δ -avenasterol, stigmastanol, stigmastadienol, and other minor sterols (δ -and δ -avenasterols, δ -stigmastenol, and stigmastadienol: : ± : mg/ g) [ ] . . vitamin e is the major lipid-soluble antioxidant for human health, which has eight different stereoisomers [ , ] by three chiral centers in tocopherols from barley. spring barley has higher α-tocotrienol content in four tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, and α-tocopherol) [ ] . [ ] . a tocochromanol in barley grains ranged from . to . mg/ g, but which is much higher than in oat ( . mg/ g) and triticum ( . mg/ g) [ ] . tocochromanols content in barley is % in pericarp, > % in endosperm, and > % in germ; about % of the tocochromanols were tocotrienols, and tocopherols in germ ( %) was higher than that in pericarp ( %) [ ] . the hulless barley especially with waxy, double waxy and tercel cultivars have the highest tocols content. tocol in whole grain was . mg/ g to . mg/ g, and in pearling flour was . mg/ g to . mg/ g; however, the ratios of total tocotrienols to total tocopherols ranged from . to . [ ] . the highest content of tocols ( . ~ . mg/ g) and vitamin e concentrations ( . ~ . mg/ g) was found in the waxy barley, especially in the hulless waxy washonubet (tocols . mg/ g and α-tocotrienols isomer . mg/ g) [ ] . . . resistant starch. resistant starch (rs) can prevent dietrelated chronic diseases such as diabetes and colon cancer. rs in hulless barley grains is related with b-type granules and the amylopectin f-iii fraction; however, sequential rate of enzymatic hydrolysis in diets is waxy>normal>high amylose barley [ ] . rs of spring barley cultivars approved and popularized during the past years in oxidative medicine and cellular longevity europe, in which rs content ranged from < % to > % [ ] . rs content of unprocessed grains of high-amylose, normal, and waxy barley is : ± : %, : ± : %, and : ± : %, respectively, but slowly digestible starch in unprocessed grains of normal ( : ± : %)>high-amylose ( : ± : %)>waxy barley ( : ± : %) and rapidly digestible starch in normal ( : ± : %)<high-amylose ( : ± : %)<waxy barley ( : ± : %) [ ] . the rs content (mg/ g) of accessions barley grains was : ± : %, with a highest content up to . % [ ] . snps (i.e., th exon g( )-to-t and fifth exon c( )-to-t) in three exons play different roles on the expression of the waxy transcript, granule-bound starch synthase i (gbss i), protein, amylose, and starch properties of hulless barley [ ] . the chronology of rs contents in different barley diets is ground pearled barley ( . %)>pearled barley flakes ( . %)>whole pearled barley ( . %)>cut barley . . gaba and linoleic acid. gaba increased α-amylase gene expression by treating barley aleurone with exogenous gaba, especially α-amylase activity began to rise after about h and reached a peak at h [ ] . the gaba content (mg/ g) in accessions of barley grains is : ± : mg/ g, the highest up to : : ± : mg/ g [ ] . the gaba has a very important role in mediating nacl stress phenolic compounds accumulation in germinated hulless barley [ ] . linoleic acid content increased from . % to . % and oil from . to . %, while oleic content decreased from . % to . % and palmitic acid from . % to . % during barley malting process [ ] . . . phytases. hydrolyze phytate in barley associated the bioavailable nutrient elements (p, fe, and zn), which exists as a single gene (paphy_a) in barley, but as two or three homeologous copies in wheat [ ] . the improvement of hvpaphy_a transformed barley showed phytase activity increases up to -fold in green leaves, -fold in grains, and -fold in dry straw [ ] . barley grains for preventive chronic diseases . . . antidiabetic properties. diabetes is a chronic metabolic disease with high mortality rates; therefore, search for novel natural inhibitors has gained much attention [ ] . major antidiabetic elements in barley are β-glucan, phenolic compounds (phenolic acids and flavonoids), phytosterols, tocols, arabinoxylan, and resistant starch ( table ) . oxidative stress not only leads to insulin resistance, impaired glucose tolerance, b-cell dysfunction, ultimately diabetes but also can treat diabetes and obesity by phytochemicals (phenolic acids, flavonoids, phytosterols, and tocols) in barley [ ] . chronic consumption of foods with high β-glucans in barley can improve insulin resistance and lower the postprandial glucose response and increase satiety [ ] . the β-glucan in hulless barley reduced the insulin resistance, arterial sclerosis, serum glucose, and serum lipid in high-fat mouse [ ] . high phenolic content ( . mg/ g) and low rapidly digested starch ( . %) make barley muffin to modulate glycemic response [ ] . the hypoglycemic effect of ethanol extract polysaccharide from barley malt is better for decreased fasting plasma glucose of the diabetes mice than that of water extract [ ] . the boiled barley kernels evening meal can facilitate glucose regulation, increase the release of glucagon-like peptide- , and reduce energy intake and fasting serum free fatty acids, mediated through gut microbial fermentation of the indigestible carbohydrates [ ] . the glycemic index (gi = : ) of all-wheat bread is higher than that (gi = : ) of % wheat+ % barley flour ( . % β-glucan) [ ] . gi for barley with . % β-glucan and oat tempe are and , respectively [ ] . the hulless barley can reduce postprandial glucose and improved insulin sensitivity by amino acid and biogenic amine profiles [ ] . . . . antiobesity. major antiobesity components in barley are β-glucan, resistant starch, polyphenols, dietary fiber, arabinoxylan, tocols, and phytosterols ( table ) . β-glucan in barley significantly treats obesity that reduced low-density lipoprotein, total cholesterol, and serum p-cresyl sulfate levels and increased flow-mediated dilation [ , ] . barley β-glucan can prevent visceral fat (≥ cm ) obesity and increase faecal scores, but decreased nutrient digestibility and antiobesity [ , ] . rs and β-glucans as well as soluble arabinoxylan were utilized mainly in the caecum, especially rs shifted the utilization of other polysaccharides to more distal parts of the colon of pigs [ ] . obesity and insulin resistance associated with bile acid changes and lower dietary fiber (β-glucan) in barley diet [ ] . the aqueous extract of fermented barley has antiobesity effects due to β-glucan and phenolic acids (vanillic acid and ferulic acid) [ ] . β-glucans in black and blue hulless barley for preventive obesity were very higher than that of white one, based on its molecular weights, particle sizes, viscosities, binding capacities (fat, cholesterol, and bile acid), and inhibiting activities on pancreatic lipase [ ] . the polyphenols extracted in black hulless barley show notable decreases in total cholesterol, low-density lipoprotein cholesterol, and atherosclerosis, but significant increase in high-density lipoprotein cholesterol levels [ ] . barley β-glucan can reduce low-density lipoprotein cholesterol and non-highdensity lipoprotein cholesterol as well as alters the gut microbiota for preventing cardiovascular disease (see table ) [ ] . oxidative stress and inflammation are two important factors of atherosclerosis, and polysaccharide extracts with antioxidation and anti-inflammation of hulless barley prevent cardiovascular diseases [ ] . some other functional components of barley have been associated with oxidative medicine and cellular longevity cardiovascular health, such as polyphenols, phytosterols, lignans, tocols, and folate [ ] . major anticancer elements in barley are β-glucan, phenolics, arabinoxylan, phytosterols, lignan, and resistant starch ( table ). functional ingredients of barley with antioxidative and immunomodulatory activities are associated with anticancer effects [ ] . barley with high dietary fiber (β-glucan) has an important role for the prevention of colon cancer and cardiovascular diseases [ ] ; low molecular weight β-d-glucan can enhance antioxidant and antiproliferative activities [ ] . aqueous extract of fermented barley can induce subcutaneous transplantation tumor apoptosis that can be used for a nutrient supplement in the treatment of human colon cancer [ ] . β-glucans in hulless barley has anticancer activities in vitro, but its anti-inflammatory activities increased as their molecular weights decreased [ ] . the bound phenolics in dehulled hulless barley have excellent antioxidant and antiproliferative effects to human liver cancer cells [ ] . a water soluble polysaccharide (glucose : xylose : arabinose : rhamnose = . : . : . : . ) from hulless barley can inhibit colon cancer, which induce ht- apoptosis through ros-jnk and nf-κb-regulated caspase pathways [ ] . . . . antioxidation. antioxidants are compounds that remove reactive oxygen species from cells, which play a dual role in aggravating and preventing diseases [ ] . major antioxidants in barley are phenolic compounds (phenolic acids, flavonoids, and anthocyanin), tocols (vitamin e), polysaccharide (arabinoxylan), dietary fiber, and phytic acid ( table ) . antioxidant effects of polyphenols in barley are flavanols>flavonols (quercetin)>hydroxycinnamic acids (ferulic, caffeic, and coumaric acids) [ ] . malt has higher phenolic content (sinapinic acid and epicatechin) than its barley grains, which plays a key role on antioxidant stability of beer [ ] . the antioxidant activity for anthocyanin in barley bran was markedly higher than that of whole grains flour [ ] . five of the seven associations in barley were with markers near genes associated with the tocochromanol (vitamin e with the most powerful antioxidants) pathway [ ] . overexpression of homogentisate geranylgeranyl transferase for barley enhanced the tocotrienol levels (δ-, β-, and γ-tocotrienol - %) and antioxidant capacity (radical scavenging activity - %) in barley seeds [ ] . the antioxidant effects of dietary fiber in hulless barley bran were associated with total phenolic concentration, which had the dpph ( , -diphenyl- -picrylhydrazyl radical , -diphenyl- -( , , -trinitrophenyl)-hydrazyl) radicalscavenging activity and ferric-reducing antioxidant power oxidative medicine and cellular longevity [ ] . gaba induces the accumulation of proline and total phenolics and enhances the antioxidant system in germinated hulless barley under nacl stress [ ] . the chapatti quality score reduced by % and its phenolic concentration increased from . to . mg/ g, while biscuit spread factor reduced by % and its β-glucan concentration increased from . to . % as well as phenolic content increased from . to . mg/ g after blending of % hulless barley flour, especially markedly increased antioxidant activity [ ] . . . . anti-inflammation. major anti-inflammatory ingredients in barley are β-glucans, lignans, vanillic acid, arabinoxylan, and so on ( table ) . endothelial cell adhesion molecules were identified as an early step in inflammation and atherogenesis; barley β-glucans not only have a maximum anti-inflammatory activity at mw~ : × , especially the inhibition of tnf-α-induced expression of vascular cell adhesion molecule was stronger than that of oat β-glucans, but also have a higher ratio of -o-β-cellobiosyl-d-glucose to -o-β-cellotriosyl-d-glucose oligomers in the polymeric chains [ ] . the molecular weights of β-glucans in hulless barley increased that add inhibitory abilities on α-amylase and pancreatic lipase, but the antiinflammatory abilities decreased, especially the low intrinsic viscosity and high solubility of β-glucans acid hydrolysis for min might contribute to its higher anti-inflammatory activity, which significantly affected their bioactivities (e.g., anticancer), which was beneficial for a better understanding of their structure-function relationships [ ] . the fermented barley extracts (vanillic acid) downregulate glucose consumption and reducing proinflammatory cytokine secretion [ ] . the anti-inflammatory property of malt and wholegrain barley is due to the formation of short chain fatty acid (scfa) and changes in microbiota composition [ ] . . . . immunomodulation. major immunomodulatory substances in barley are β-glucans, arabinoxylan, and so on ( table ). the immunomodulatory activity of barley β-glucans insolubility associated with its particle size, granule conformation, and particulate homogeneity. all β-glucan fractions can induce more cytokines in bone marrowderived dendritic cells than their oat equivalents; however, the insolubility of β-glucan affects its immunomodulatory activity, which is related to its particle size, particle configuration, and particle uniformity [ ] . a water-soluble polysaccharide (bp- , molecular weight : × da) from hulless barley can improve the immune ability of immunosuppressive mice through increasing the serum levels of il- , tnfα, and ifn-γ, such as bp- ( mg/kg and mg/kg) can not only significantly increase the number of bone marrow cells and peripheral blood white blood cells, as well as enhance the production of il- , tnf-α, ifn-γ, igg, and igm in the spleen and serum levels for improving the immune function, but also promote the proliferation and phagocytosis activity of macrophages as well as repair the damage induced by ctx in the spleen cells of immunosuppressive mice [ ] . table ), which shows a % survival rate after cardiac ischemia ( min)/reperfusion ( min) injury, reduces left ventricular anion superoxide production ( %) and infarct size ( %), and increases the capillary ( %) and arteriolar density ( %) and vegf expression ( . %) of hearts in mice [ ] . the products of barley grains can reduce the cardiometabolic risk and regulate the blood glucose and appetite hormones in - h after intake; however, its mechanisms are gut fermentation of indigestible carbohydrates [ ] . . . . hypocholesterolaemic effects. cholesterol is a synthesis lipid in the body [ ] . dietary β-glucan of hulless barley reduces the plasma ldl cholesterol content (see table ) by promoting the excretion of faecal lipids and regulating the activities of -hydroxy- -methyl glutaryl-coenzyme a oxidative medicine and cellular longevity reductase and cholesterol -αhydroxylase in hypercholesterolaemic rats [ ] . barley bran % and % in diet to the hypercholesterolaemic rats improved the level of lipids, lactate dehydrogenase, liver enzymes, and creatine kinase-mb [ ] . whole grain hulless barley has hypocholesterolaemic effects by promoting bile acid synthesis and reabsorption, controlling cholesterol synthesis and accumulation in peripheral tissue, decreasing the expression of -hydroxy- methylglutaryl coenzyme a reductase, while increasing the hepatic expressions of amp-activated protein kinase α, cholesterol α-hydroxylase, ldl receptor, liver x receptor, and pparα [ ] . . . . blood pressure regulation. higher consumption of barley β-glucan is associated with lower systolic and diastolic blood pressure (see table ), i.e., diets rich in β-glucans reduce systolic blood pressure by . mmhg ( % ci . to . mmhg) and diastolic blood pressure by . mmhg ( % ci . to . mmhg) for a median difference in β-glucans of g [ ] . the consumption of high molecular weight barley β-glucan can reduce blood pressure [ ] . . . . bowel health improvement. gastrointestinal tract disease is a major global health problem. β-glucan in hulless barley has protective effects to the gastrointestinal tract (see table ) [ ] . the dietary fiber in hulless barley improved indices of bowel health compared with refined cereal foods, especially himalaya possesses high amylose and resistant starch due to lacking activity of a key enzyme responsible for starch synthesis; however, consumption of himalaya foods resulted in % higher faecal weight, a lowering of faecal ph from . to . , a % higher faecal concentration, a % higher excretion of butyrate, a % higher faecal total scfa excretion, and a % lower faecal p-cresol concentration [ ] . fermented barley extract ( mg/ g) can act as a promising laxative agent to cure spastic constipation [ ] . butyric acid for improving the colonic health is produced by degradation of barley dietary fiber by microbiota [ ] . . . . gastroprotective effects. β-glucan from hulless barley can mitigate the gastric lesions and gastric mucosal damage as well as gastric oxidative stress injury through decreasing the level of malondialdehyde [ ] . the oral administration of fermented barley extract had strong gastroprotective effects through strengthening antioxidant defense system and anti-inflammatory effects, as well as decreasing lipid peroxidation and cat activity by increasing the gsh levels and sod activity in the body, and the mg/kg dose of fermented barley extract was similar gastroprotective as the mg/kg dose of omeprazole, which indicates that this dosage can be used for patients suffering from different levels of gastric damages [ ] . . . . reduce chronic kidney disease. barley β-glucans is associated with a saccharolytic shift in the gut microbiota metabolism by a reduction of pcs toxin blood levels and an increase of scfa production at colonic site, which can reduce the microbial-derived uremic toxin and cardiovascular complications in end-stage renal disease (see table ), especially chronic kidney disease [ ] . the total cholesterol and triglycerides were reduced, and hdl cholesterol increased in % and % barley intervention in breakfast diet; however, barley in the diet of stage chronic kidney disease patients has significantly improved the nutritional status and renal functions [ ] . . . . improve metabolic syndrome. barley β-glucan can improve postprandial glucose response and cholesterol levels as well as the metabolic syndrome based on individual gut microbiota composition (see table ) [ ] . tibetan hulless barley can reduce insulin resistance, dyslipidemia, and body weight gain, which can diminish the prevalence of metabolic syndrome induced by high-fat-sucrose diets, i.e., rats fed with tibetan hulless barley can increase the assessment of insulin resistance scores (body weight, abdominal fat deposition, liver weight, liver fat deposition, triglyceride, fasting blood glucose, and serum fasting insulin) and decrease lowdensity lipoprotein cholesterol levels compared to rats fed with a basal diet [ ] . . . . hepatoprotective effect. β-glucans in barley can decrease fatty liver in diabetes with obesity (see table ) [ ] . the free phenolic extract in barley added the hepatic levels of antioxidant enzymes [ ] . whole grain hulless barley had significantly lower liver lipid levels (total phenolic and pentosan) [ ] . barley sprout extract protects liver cells under oxidative stress by activating nrf and adding glutathione synthesis, especially against alcohol-induced liver injury, as it inhibits glutathione depletion and hepatic lipid accumulation, reduces serum biochemical markers of liver injury, and inhibits inflammatory responses [ ] . . . . wound healing acceleration. nowadays, β-glucans represent effective topical agents for the treatment of chronic wounds and burns due to the activation of the immune and cutaneous cells (see table ), which increase wound repair by enhancing the infiltration of macrophages and promote tissue granulation, collagen deposition, and reepithelialization based on inducing the proliferation and migration of keratinocytes and fibroblasts through specific receptors such as dectin- , cr , or tlrs [ ] . barley β-glucan in vivo promotes the wound closure in mouse skin by promoting the migration and proliferation of human dermis fibroblasts [ ] . . . . heart failure prevention. barley β-d-glucan is a natural activator of mnsod expression, which can prevent heart failure (see table ) [ ] . the food and drug administration has made a health claim between β-glucan and reduced risk of coronary heart disease, diabetes, and heart-related problems [ ] . barley products can prevent and reduce the risk of coronary heart disease, which is associated with the constituents like β-glucan, phenolics, tocols, linoleic acid, and folate [ , ] . . . . atopic dermatitis alleviation. the allergen produced by barley and the protein expressed in insect cells induce the same amount of ifn-γ and il- in pbmc from vaccinated horses (see table ) [ ] . fermented barley extract oxidative medicine and cellular longevity p reduced skin lesions by inhibiting inflammatory cytokines [ ] , but gaba alleviated atopic dermatitis by suppressing serum immunoglobulin e and splenocyte interleukin production [ ] . barley intake reduces the risk of total stroke by significantly increasing the medium and small particle sizes of high-density lipoprotein cholesterol (see table ) [ ] . increasing the expression of antioxidant genes in the liver and the regulation of nrf played a role in the regulation of metabolic diseases in stroke-prone spontaneously hypertensive rats consuming a fermented barley extract p diet [ ] . . . . allergic rhinitis alleviation. ma-al-shaeer formulation based on barley can treat for allergic rhinitis, especially reduced the nasal congestion, post nasal drip, and headache (see table ) [ ] . fermented barley extract alleviated allergic rhinitis in ova-sensitized mice by regulating cytokines (ifn-γ or il- ) related to chronic inflammation [ ] . in addition, the common edible whole-barley flour can reduce the risk of hyperlipidemia and cholelithiasis [ ] , antiaging [ ] as well as increase body strength and increase the content of linoleic acid and linolenic acid in pigs, cattle, sheep, and geese. in a word, there are more than kinds health effects for barley grain preventive chronic diseases according to the summary of current retrieval literature. the descriptions of some literatures for barley preventive chronic diseases (antidiabetes, antiobesity, anticancer, antioxidation, anti-inflammation, hypocholesterolaemic effects, blood pressure regulation, cardioprotection, immunomodulation, improve gastrointestinal, hepatoprotection, bowel health, cardiovascular disease prevention, atopic dermatitis alleviation, wound healing acceleration, heart failure prevention, and so on) are relatively sufficient, but some literatures for barley preventive chronic diseases (antiaging, reduce cholelithiasis, and increase body strength) are relatively less. these results fully demonstrate the health contribution relationship between barley functional food and human chronic disease prevention. the efficacy of preventing chronic diseases is related to barley genotype and its location, composition, extraction, and compatibility, and with the in-depth study of barley grain in prevention and treatment of human chronic diseases, the novel mechanisms for the prevention of chronic diseases may be ascertained as well as its putative role, either at major or minor level, would be further validated. grains for preventive chronic disease . . . β-glucans mechanism. β-glucans can be used as candidates for the medication in the treatment of human chronic diseases [ ] . β-glucans have many bioactivities including antidiabetes; anticancer; antiobesity; anti-inflammation; immunomodulation; cardioprotection; lower cholesterol and lower blood pressure; improve bowel health, gastroprotection, and hepatoprotection; reduce chronic kidney disease and metabolic syndrome; prevent the risk of heart and cardiovascular diseases; and accelerate wound healing activities (figure , tables and ) [ , , , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the major mechanisms of β-glucans of barley involve in the prevention of chronic diseases are as follows: β-glucans can interact with intestinal lipids and bile salt to reduce cholesterol levels and subsequently prevent diabetes, hypertension, cardiovascular disease, and metabolic syndrome [ ] . barley β-glucans not only control appetite and improve insulin sensitivity by gut hormone secretion via microbiota produced scfa [ ] due to its high molecular weight and high viscosity but also increase their antigen ability and enhances the proinflammatory cytokines, which can be degraded by macrophages and natural killer cells mediating its cellular cytotoxicity opsonized tumor cells [ ] . β-glucans of barley are the major regulators of adipogenesis, especially markedly downregulated the target genes in the adipose tissue including adipocyte fatty acid-binding protein, lipoprotein lipase, uncoupling protein- , and glucose transporter in t -l cells [ ] and also have the effect of inhibiting the α-amylase and pancreatic lipase [ ] . barley β-glucan can reduce blood pressure and cardiovascular diseases that alters the composition of gut microbiota, decrease body mass index, waist circumference, and triglyceride levels [ ] and also reduce the systemic inflammatory profile, prevent alveolar bone loss, and improve βcell function in diabetic animals [ ] . barley β-glucans can not only regulate immune responses and connect innate and adaptive immunity [ ] but also have cardioprotective mechanism of promoted angiogenesis through endothelial upregulation of the vascular growth factor [ ] . the hypocholesterolaemic effect of β-glucan in barley is due to the increased bile acid synthesis [ ] and improved bowel health by inhibited feed intake and increased cecal fermentation [ ] . barley β-glucan not only has the gastroprotective effects by increasing the sod and cat activity, decreasing the gastric ulcer index, and increasing prostaglandin e and nitric oxide in laboratory rodents [ ] but also affects lipid metabolism and scfa production, lowering microbes in patients with metabolic syndrome [ ] and improving chronic kidney disease by reducing the microbial-derived uremic toxin. chronic consumption of barley β-glucans can decrease fatty liver by increasing small intestinal contents viscosity and improving glucose, lower glycated hemoglobin and relative kidney weights [ ] , strengthen the angiogenic ability of ros-exposed endothelial cells for preventive heart disease [ ] , and accelerate the wound closure by promoting the migration and proliferation of human dermal fibroblasts [ ] . barley polyphenols have lots of bioactivities including antidiabetes, antiobesity, anticancer, antioxidant, anti-inflammation, hepatoprotection, and prevention of cardiovascular and heart diseases ( figure , tables and ). barley lignan as natural polyphenols has anticancer, antioxidant, anti-inflammation properties, and preventive role for cardiovascular diseases. anthocyanin belongs to flavonoids, and flavonoids belongs to polyphenols. black, purple, and blue barley grains have gained much attention recently because their anthocyanins have anticancer, glycemic and body weight regulation, antioxidation, anti-inflammation, neuroprotection, hypolipidemia, retinal protection, hepatoprotection, and antiaging effects [ , ] . several mechanisms of barley polyphenols for preventing chronic diseases have been documented so far. zhang et al. [ ] has verified the molecular mechanism of insulin resistance by fermented barley extract vanillic acid through regulating mir- expression. the polyphenols with antiobesity from black hulless barley has strong superoxide radical, hydroxyl radical and , -diphenyl- -picrylhydrazyl radicalscavenging activity, ferric reducing antioxidant power, and moderate metal ion-chelating activity [ ] . an efficacious antiproliferation capacity in caco- cells of black barley malt free extract was predicted due to its phenolic constituents which have cellular antioxidant and oxygen radical absorbance as well as peroxyl radical scavenging activities, dpph, and abts radical scavenging assays [ ] . black barley sprouting stimulates the phenolic biosynthesis by upregulating proline-associated pentose phosphate pathway to support structure of sprouts with antioxidant capacity [ ] . total phenolic and pentosan in hulless barley grains have antioxidant activity by downregulated expression of heat shock protein and phosphatidylethanolamine binding protein but upregulated expression of enoyl-coenzyme a hydratase and peroxiredoxin [ ] . the total polyphenol (flavonoid) content and the , -diphenyl- -picrylhydrazyl and abts radical scavenging abilities increased as the barley added to the food mixture [ ] . lignan (-)- (s)-hydroxymatairesinol inhibited tumor necrosis factor-α stimulated endothelial inflammation by inhibiting nf-κb activation and upregulating nrf antioxidant element signaling pathway [ ] . lignans in barley have high anti-inflammatory abilities in endothelial cells by reducing nuclear factor-κb and extracellular signal as well as regulating kinase phosphorylation [ ] . the polyphenols such as (+)-catechin, protocatechuate, and quercetin in barley not only have hepatoprotective effect [ ] but also prevent coronary heart disease by reducing oxidativeinduced tissue damage through modulating intracellular signaling pathways [ ] . polyphenols play an important role in alleviating cardiovascular diseases due to their antiradical scavenging abilities [ ] . barley arabinoxylan has a lot of health benefits, which include antidiabetes, antiobesity, anticancer, lowering cholesterol, immunomodulation, antioxidant, cardiovascular diseases prevention, and so on (figure , tables and ). arabinoxylan in barley is the most abundant polysaccharide that has the capacity of lowering cholesterol and glucose as well as antioxidant activities [ ] . the major mechanisms of barley arabinoxylan for preventing chronic diseases are as follows: arabinoxylan can improve urinary metabolites associated with diabetes by improvement of carbohydrate and lipid as well as amino acid metabolism [ ] . the antioxidant and antiobesity as well as immunomodulation of arabinoxylans associated with prebiotic effects and short-chain fatty acids production by interaction of gut microbiota and arabinoxylans [ ] . arabinoxylan rice bran can increase anticancer effects in the older population by increased nk activity [ ] . the arabinoxylan in barley with the immunomodulatory activity consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and -o-methylglucuronic acid [ ] . the arabinoxylan diet led to a lower postprandial blood for glucose-dependent insulinotropic polypeptide response, especially fat oxidation has an important role in the antiobesity and in the prevention of cardiovascular diseases [ ] . barley polysaccharide prevent cardiovascular diseases by the vasodilatory effect of controlling angiotensin-converting enzyme production [ ] . tables and ). barley grains contain phytosterols that can esterify to fatty acids, phenolic acids, steryl glucosides, or acylated steryl glycosides [ ] . phytosterols significantly inhibited the ability of oxysterols to activate the liver x receptors transcription in modulating cancer cell behavior [ ] , which are thought to influence multiple processes related to cancer, such as carcinogen production, cancercell growth, angiogenesis, invasion, metasis, and cancer-cell apoptosis [ ] . for the prevention of cardiovascular diseases, barley phytosterols can compete with cholesterol for micelle formation, inhibiting cholesterol absorption in intestine and lowering cholesterol in central nervous system of the brain [ , ] . the effect of plant sterols on neurodegenerative diseases is due to its passage through the blood-brain barrier, modulating cholesterol metabolism and inflammation in the central nervous system process in the brain, which involve low-density-lipoprotein, apolipoprotein e, and scavenger receptor class b type [ ] . the lowering of cholesterol and prevention of cardiovascular disease are due to the fact that the phytosterol structure in the barley membrane is similar to the configuration of different cholesterol [ ] , but functionally similar to precursors in phytohormone synthesis, but lowering the blood concentration of cholesterol is beneficial to reduce the risk of cardiovascular disease. extremely rich in content in barley embryos. barley tocols are the best in cereals due to a high concentration and favorable distribution of eight active vitamers [ ] . barley tocols have anticancer, antiobesity, and antioxidant effects and can lower cholesterol level, reduce the risk of stroke, and prevent cardiovascular and heart diseases ( figure , tables and ) [ , ] . tocotrienols can suppress various cancers (breast, lung, ovary, prostate, liver, brain, colon, myeloma, and pancreas) by its molecular mechanisms of cellular proliferation, apoptosis, angiogenesis, metastasis, and inflammation [ ] , which are associated with human intake of whole grains, especially barley and rye. tocotrienol is a functional food of obesity and diabetes by regulating adipogenesis and increase apoptosis of adipocytes and improve glucose homeostasis through oxidative medicine and cellular longevity suppression of inflammation and oxidative stress [ ] . tocol is one of the most powerful antioxidants that has the ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species, thus protecting fatty acids from lipid peroxidation [ ] . all barley pitas had the greatest antioxidant and vitamin e levels from barley malt flour [ ] . the antioxidant properties of barley tocols due to its ability to inhibit lipid peroxidation in biological membranes induce the immune system, promote apoptosis induction, and reduce the risk factors of cardiovascular diseases and stroke by atherosclerotic blockages in the carotid artery [ ] . barley rs has many immune properties like antidiabetes, antiobesity, anticancer, and so on ( figure ,table ). ceramide can promote lipid storage, impaired glucose utilization, and inhibited enzyme dihydroceramide desaturase , which can treat hepatic steatosis and metabolic disorders [ ] . antidiabetes of rs can be increased by suppressing amylopectin synthesis through silencing of starch branching enzymes in barley [ ] . t cell development and gut iga production suppress host lipid absorption by modulating cd expression [ ] to achieve the effect of antiobesity of barley rs. gbss i is mainly responsible for amylose synthesis whereas sss i and sbe ii for amylopectin synthesis in amyloplasts [ ] . barley with high β-glucan and moderate rs may benefit hyperglycemia-impaired lipid metabolism [ ] . the blending of barley starch citrate with resistant starch iv up to % can produce noodles of acceptable quality and numerous health benefits [ ] . early hominids used fruits/vegetables and leaves rich in polyphenols and k-ca as well as vitamins as staple foods to increase the dependence of the human body on these functional ingredients (see figure ). diet played an important role in early hominids evolution [ ] , but no reports have been delivered to date for diet in coevolution of human chronic diseases. miocene ( . ~ . ma) apes had a variety of foods that included folivory, soft-fruit eating, and hard-object feeding [ ] . the diet of pliocene ( . ~ . ma) and early pleistocene ( . ~ . ma) hominids in africa was mainly fruits and leaves of c plants (trees, bushes, shrubs; . ~ . ma) which was gradually transformed into grass (c plant/tropical grasses and sedges) and hard-object (seeds and nuts) ( . ~ . ma) [ ] [ ] [ ] , which was due to low availability of fruits in dry and active glacier ( . ~ . ma) as well as migration to warm grasslands. ethiopia's pliocene lucy is one of the oldest and most complete fossils in hominid bones, her death due to fall out vertically and live on tall tree [ ] . early hominids and australopithecines inhabited forests and savannas for collinearity oxidative medicine and cellular longevity found between tasty fruits (fructose/sucrose, quinine, and tannins) and primate sensory perception, which offered evidence of the two-direction evolutionary trend determining taste sensitivity [ ] . our ape ancestors possessed a digestive dehydrogenase enzyme capable of metabolizing ethanol about ma that they began using fruits fermentation from the forest floor [ ] . there were differences in the proportion of meat and vegetables between the early hominids australopithecus and paranthropus; paranthropus ate more hard food than australopithecus [ ] . early hominid australopithecus africanus, like chimpanzees, are dominated by fruit, leaves, and carbon- -enriched foods about ma [ ] . the worldwide daily consumption of fruits and vegetables as well as tea has become the main tool for prevention of cardiovascular disease, stroke, cancer and diabetes due to their polyphenols modulate tau hyperphosphorylation and beta amyloid aggregation [ ] . the anthocyanins and polyphenols for major functional ingredients in blueberry played a key role in preventing chronic diseases [ ] . the organopolysulfides and quercetin for major functional ingredients in allium genus played a key role in preventing chronic diseases [ ] . baobab was cultivated from seeds from countries in east and west africa, its leaves had the highest vitamin b content ( : ± : mg/ g) from senegal, adult leaves provided the highest ca content ( . %) and young leaves with the highest ca and k content of nankoun in burkina faso [ ] . the diets of early hominids related with five center of crop origin (mediterranean, middle east, central asia, indo-burma, and china-korea); however, the rich food structure maintained the survival and development of early hominids who lacked survival competition and migration could not improve their intelligence. therefore, polyphenols and k-ca as well as vitamins ingredients in fruits/vegetables and leaves as well as grass/seeds (such as gramineae and its ancestor species of barley) for preventive chronic disease are the results of long-term dependence for diet from pliocene hominids in africa to modern human beings ( figure ). ingredients for neanderthals. neanderthals used mushrooms and nuts rich in polysaccharide and phytosterols as well as linoleic acid as staple foods to increase the dependence of the human body on these functional ingredients (see figure ). neanderthals as well as early homo sapiens show high dietary variability in mediterranean evergreen habitats, but less diet in high latitude steppe or coniferous forests [ ] . the steppe-like neanderthals of belgium feed on the meat of the woolly rhinoceros and wild sheep, while the neanderthals of the spanish forest feature root feed on mushrooms and pine nuts [ ] ; neanderthals in northern spain roasted vegetables and used medicinal plants about . ~ . ma [ ] . neanderthals ate meat (high chloroprostol) and plants ( β-sitosterol) as staple foods [ ] . plant foods of neanderthals from iraq and belgium had the typical modern human diets, which include palms, date, legumes, and grass seeds [ ] . medicinal functions (major polysaccharide) in medicinal mushrooms and fungi can prevent and treat more than chronic diseases [ ] ; however, β-glucans in barley preventive chronic diseases. the total polysaccharide content in morchella sp. reached up to . % of dried biomass in a mixture of : of wheat grains and potato peels [ ] . β-glucans are group of polysaccharides found in mushrooms, yeasts, seaweed, barley, and oats [ ] . macrofungal β-glucans are major β- , and β- , -glycosidic bonds, which have immunomodulatory, anticancer, and antioxidant properties; total β-glucan content varied from . % in a. bisporus to . % in t. rutilans [ ] . phytosterols are diet ingredients found in an array of nuts, seeds, and vegetables which have anticancer activities via their interactions with the plasma cell membrane [ ] . oil palm (elaeis guineensis jacq.) is one of the highest oil-yield crops in the world; however, palm oil is the largest variety of plant oil produced, consumed ( %), and internationally traded in the world, rich in linoleic acid ( %) that is associated with egfad gene [ ] . ~ % carbohydrate diets (especially whole-grain breads, vegetables, and nuts) had minimal risk of mortality [ ] . fruits, vegetables, and whole grains were indispensable among four top diets (mediterranean diet with five best ranked first, dash diet with lower blood pressure, flexitarian diet with lose weight and mind diet with brain health). the diets of neanderthals was not correlated with the complete center of crop origin, poor diet (meats, mushrooms, and pine nuts) and lack of migration lead to extinction ( figure ). these results reveal the importance of whole grain and vegetables or fruits in human health; however, lack of three categories of food suggesting the cause of the neanderthal destruction. therefore, polysaccharide (βglucan) and phytosterols as well as linoleic acid in mushrooms and nuts as well as palm oil for preventive chronic disease are the results of long-term dependence for diet from neanderthals in europe to modern human beings ( figure ). ingredients for homo sapiens. homo sapiens not only used grass and seeds rich in gaba and enzymes as well as resistant starch as staple foods to increase the unique dependence of the human body on these functional ingredients (see figure ) but also inherited the staple foods of early hominids and neanderthals. feeding and diet played key roles in human evolution, especially homo sapiens have a relative masticatory structure similar to that of other primates [ ] . homo sapiens moved from africa into the middle east about ka, according to fossils at skhul and qafzeh caves in israel [ ] . the aba-mirna- in belladonna with highly homologous to homo sapiens mirna can target and downregulate human brain-enriched transcription factor (znf- ) and gene expression in the human central nervous system [ ] . for homo sapiens, foragers have greater complexity than farmers or pastoralists; meanwhile, the old world foragers had significantly higher anisotropy values than new world foragers, but similarity between hard food foragers and hard food farmers [ ] . the meat diet abuse by a herbivorous homo sapiens can lead to atherosclerosis [ ] . the diets of african homo sapiens associated with center of crop origin in ethiopia, their migration along eight center of crop origin changed the fate of mankind [ ] . variation in cranial robusticity from geographical homo sapiens associated with cranial shape, size, climate, and neutral genetic distances; however, cranial robusticity may be an adaptation to cold and harsh environments as well as masticatory differences in diet [ ] , especially higher gaba (such as barley grass . mg/ g) grass diets in crop suiting environmental extremes improved intelligence [ ] . gaba contents in picked tea leaves under anoxic treatments at h and h are : ± : and : ± : mg/kg (fresh weight) [ ] . gaba a receptors modulate vigilance, emotions, cognition, and muscle tension, and they are the targets of anxiety-reducing and sedative-hypnotic benzodiazepines and some general anesthetics [ ] . shisa regulates gaba ar trafficking, function, and pharmacology, especially modulates benzodiazepine action in the brain [ ] . barley can be grown in four seasons (spring, summer, autumn, and winter) at , - , m in yunnan province of china which may be associated with harboring enzymes in barley grass [ ] . the diet high in sodium and low four diets (whole grains, fruits, vegetables, nuts, and seeds) were major dietary risk factors for deaths and disability-adjusted life-years globally and in many countries [ ] ; however, the whole grain is the manifestation of resistant starch type i surrounded by protein matrix and bran layer for making the starch unavailable for enzymes. the least chronic disease of ancient humans due to replacing the salt with seasoning crops, such as onions, ginger, garlic, coriander, pepper, chili, and so on [ ] . the coevolution of the preventive human chronic diseases are related to major diets of vavilov's eight crop origin centers (ethiopia, mediterranean, middle east, central asia, indo-burma, china-korea, mexico-guatemala, and peru-ecuador-bolivia) [ ] . human chronic diseases are related with six dietary structures (fruits/vegetables, young grass/barley grass, carnivorous, cereals crop, polished rice/wheat flour, and polished rice/-wheat+grass powder), but polished rice/wheat+barley grass powder is the most major healthy dietary guidelines for modern humans; therefore, it is necessary to unravel coevolutionary mechanism between preventive chronic diseases and human diet for functional foods [ ] . these results support homo sapiens used grass/seeds (rich in gaba and enzymes as well as resistant starch), fruits/vegetables and leaves (rich in polyphenols and k-ca as well as vitamins), mushrooms, and nuts (rich in polysaccharide and phytosterols as well as linoleic acid) as staple foods to increase the dependence of the human body on these functional ingredients. barley is the oldest and more important cereal crop with the utmost dietary fiber in the world; its malt, as a functional food, is not only the largest beer raw material in the world but also one of the most commonly used chinese herbal medicines [ ] . our review point out that barley grass has antidiabetic, anticancer, antidepressant, antioxidant, fatigue, anti-inflammatory, hypolipidemic, antigout, calcium supplementary, and antiacne/detoxifying effects; promotes sleep; regulates blood pressure; enhances immunity; protects liver; reduces hyperuricemia; alleviates atopic dermatitis; improves cognition, constipation, gastrointestinal function; and prevents hypoxia, cardiovascular diseases, and so on [ ] . barley grass powder is known to play a pivotal role in prevention of chronic diseases that involves six molecular mechanism of gaba, flavonoids, sod, k-ca, vitamins, and tryptophan [ ] ; however, barley grains play key roles in prevention of chronic diseases that involves six molecular mechanism of β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch. modern humans had originated in the progeniture of african homo sapiens with cognitive hominin [ ] . the staple foods of modern human are the synthesis of homo sapiens that inherited early hominids and neanderthals, which carry the neanderthal dna due to interbreeding between homo sapiens and neanderthal took place in the middle east. human flt ligand isolated from transgenic barley seeds is a glycoprotein including α( , )-fucose and α( , )xylose, which showed expression of human growth factor in barley grains with active protein [ ] . the peptide ll- is a component of the human innate immune system, it accumulated . mg/kg in the barley grains [ ] . human fgf- gene was fused with barley α-amylase signal peptide dna sequence and expressed in transgenic salvia miltiorrhiza plants driven by s promoter; however, recombinant fgf- in leaves was ng/ fresh weight [ ] . therefore, functional ingredients in barley grass and grains are essential for the health contribution of modern human (homo sapiens), neanderthals, and early hominids staple food to prevent and treat human chronic diseases. barley has health beneficial properties and was part of the modern hominid diet; thus, it has evolved in its functional ingredients and contributed to a reduced risk of diseases. it is amazing that barley grains and malt as well as grass powder can prevent or treat more than human chronic diseases. we think the major scientific basis for that is as follows: first, barley prevent over humans chronic diseases which associated with the similar origin and evolution center of barley and human beings: ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley ( figure ) [ , , ] . ethiopia, morocco, fertile crescent, and tibet of china have been proposed as centers of barley origin and the primary habitat of wild barley (figure ) [ ] . wild barley is a selfing annual grass of predominantly morocco and irano-turanian and israel-jordan in arid desert or salt environments, the cold region in tibet of china and ethiopia, which has accumulated abundant functional ingredients for drought, salt, and cold resistances. the earliest modern human originate from ethiopia and morocco are dated to~ ka and~ ka (figure ) , respectively [ ] . the earliest human occupied high-altitude habitats in the andes and the tibetan plateau, especially late pleistocene humans adapted to the severe environments of these glaciated above , -meter elevation oxidative medicine and cellular longevity in the bale mountains of ethiopia ( figure ) [ ] . neanderthals in modern-day iraq and belgium ate grasses, cooked barley grains, and others. second, barley grass powder plays a key role in the promotion of human intelligence in the early stage: the incremental evolution of globular braincase associated with diets of brain health from ethiopia and middle east as well as from mediterranean center of crop origin, especially the highest gaba in crop diets suiting environmental extremes improved intelligence. brain development is a self-reinforcing process in which brain cells proliferate, differentiate, migrate, and connect functional neural circuit, especially primate-specific features of gabaergic interneuron development [ ] on the basis of gaba content in diet. survival depends on the selection of behaviors adaptive for environment; however, stimulation of dorsal raphe gaba neurons promoted movement in negative but not positive environments to promote environment-specific adaptive behaviors of serotonin [ ] . barley is a major crop in many developed countries [ ] ; gaba in barley grass suiting environmental extremes (cold, arid, and salt) can significantly increase, which can improve cognition and prevent chronic diseases [ ] . the average content of gaba in cultivars that we bred is . mg/ g which is . fold higher than that of other barley crops around the world [ ] . third, healthy effects of functional ingredients of barley grass and grains are the sum of staple foods for early hominids and neanderthals as well as homo sapiens: early hominids used fruits/vegetables and leaves rich in polyphenols (flavonoids) and k-ca as well as vitamins (tocols); neanderthals used mushrooms and nuts rich in polysaccharide (β-glucans and arabinoxylan) and phytosterols as well as linoleic acid; homo sapiens used grass and seeds rich in gaba and enzymes (sod) as well as resistant starch; modern human used barley grass rich in gaba, flavonoids, sod, k-ca, vitamins and tryptophan; however, barley grains rich in β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, resistant starch, and so on. therefore, barley played an important role in solving the problem of depending functional ingredients of homo sapiens and hominids as well as neanderthals, especially food safety in the process of migration and evolution from ancient humans to modern people. migration. food shortages and survival struggles caused by climate change were the causes of early human evolution of suiting environmental extremes from africa to asia and later to eurasia (figure ) [ , ] . barley is not only the most widely used cereal crop with comprehensive utilization of forage, materials for intoxicating liquor, functional food, stable food, ornamental weaving, and chinese medicines but also is the crop with the strongest resistance to stress (drought, cold, and salt) for the highest content of functional components, especially the growth period of barley varies from to days, which can be grown in four seasons in the world or at , ~ , m in yunnan province of china. these excellent characteristics become the best food for human migration. interestingly, there is a striking similarity between the human migration route and barley translocation/evolution route (see figure ). in [ ] . the spread of farming peoples of eurasia from the near east ( . ka), with movements both westward and eastward, especially ancestor of modern south asians is a mixture between early holocene populations of iran and south asia; however, yamnaya in the bronze age of europe moved both westward and eastward from north of the black sea [ ] . discovery of different crushing apparatuses in mountains of iran revealed that people were grinding wheat and barley about , years ago [ ] . these results support the healthy food contribution between human migration and barley translocation/evolution, especially climate change increased functional ingredients in barley for preventive chronic diseases. civilization. this point of view reveals the evolution of human skull morphology on the basis of the hybridization between h. sapiens and other hominin species in morocco at , years ago, all of which are descendants of the african homo sapiens population [ ] . all living people in europe and asia carry the same amount of neanderthal dna due to the interbreeding between homo sapiens and neanderthal that took place in the middle east [ ] . fertile crescent is the concentrated area of wild barley [ ] and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam ( figure ). barley is one of the oldest crops used by ancient farmers, its cultivation has been optimized by modern humans in the ancient era for less shattering, higher yield and better grains; however, the sharp awn of barley has become an important guarantee for the most resistant birds trouble and largescale farming civilization in cereal crops. climate change stimulated agricultural innovation and exchange across asia between , and , years ago; sorghum and millet made their way from china to central asia; wheat and barley moved from central asia to the far east and became a staple food in the north of china at . ka, which exchanges across central and high-altitude asia coalesced to form the silk road ( . ka~ . ka) and grand canal for traffic great artery of north-south in ancient china ( . ka) [ ] . tibetan barley (qingke) is derived from eastern domesticated barley, north pakistan, india, and nepal between , and , years ago, which supports a feral or hybridization origin for tibetan weedy barley [ ] . the rise of barley against stress (drought, cold, salt, and bird) to staple food has increased functional ingredients (especially gaba) in diet to prevent chronic diseases and promoted human civilization. gaba-mediated inhibitory interneurons control memory-encoding ca neurons; nucleus incertus (ni) establishes gabaergic inhibitory synapses on interneurons; ni gabaergic cells can regulate hippocampusdependent episodic memory formation bidirectionally, and its dysfunction may contribute to anxiety-like syndromes [ ] . cities and words and metallurgy as well as complex ceremonial buildings are the four standards of world civilization; fertile crescent for one of centers of barley origin is closely related to ancient babylonian and ancient egyptian civilizations, especially ancient babylonian civilizations have the earliest human civilization (agriculture ka, cities ~ ka, metallurgy ~ ka, words . ka, calendar ka, and systematic religion ka) in the world; however, ancient egyptian civilizations has the earliest empire (mathematics . ka, geometry ka, and writing tool ka). the ancestral blocks of the domesticated barley genomes were descended from all over the fertile crescent, especially levantine (western) and zagros (eastern) clusters of the origin of agriculture for nine wild barley populations, i.e., carmel and galilee, golan heights, hula valley and galilee, judean desert and jordan valley, lower mesopotamia, negev mountains, north levant, sharon, coastal plain and judean lowlands, and upper mesopotamia [ ] . the neolithic crops facilitated the early agricultural establishment; the barley evolution followed the agricultural development in the near east [ ] . for humans from hunter gathering to agriculture about ka of the levant in near east, barley was a founder crop for converting the brittle floral axis of the wild-type into a tough and nonbrittle spike, which made a major contribution to the emergence of early agrarian societies [ ] . for ancient indian civilization, the earliest known farming cultures in south asia emerged in the hills of balochistan in pakistan about . ka; however, seminomadic peoples domesticated wheat, barley, sheep, goat, and cattle [ ] . for ancient chinese civilization, the earliest beer recipe in china included broomcorn, millet, barley, job's tears, and tubers around , y ago, which may have motivated the initial translocation of barley from the western eurasia into the central plain of china [ ] . diseases. western medicine solves the issue of human organs, and traditional chinese medicine solves the issue of human body system; however, functional food is to address the problem of human cells. barley grains and its grass not only are the best functional food that provides nutrition and eliminates toxins from cells in human beings, which are rich in ingredients to combat more than chronic diseases but also have all the nutrients needed for cell nutrition and detoxification, which is the result of the long-term coevolution of the dietary structure of ancient apes for plants and early homo sapiens with the staple food of barley. interestingly, the types of prevention and treatment of human chronic diseases by key functional components in barley grain were in order: β-glucans ( )>polyphenols ( )>arabinoxylan ( ) = tocols ( )>phytosterols ( )>resistant starch ( ), but gaba ( )>flavonoids ( )>sod ( )>k-ca ( ) = vitamins ( )>tryptophan ( ) in barley grains. the key functional components of barley grains and its grass not only play an important role in the prevention and treatment of more than chronic human diseases but also interact with other nutritional functional components to provide the possibility to solve hundreds of human diseases caused by cell undernutrition and their detoxification disorders. the unique theory and practice system of the whole regulation improve the immunity of chinese medicine to prevent and treat human diseases. with advances in science and technology, barley will also find many behavioral mechanisms that combat human diseases, such as barley malt has been used in many prescriptions for the prevention and treatment of covid- in lots of provinces in china, which includes jiangxi, guangdong, gansu, guizhou, and jiangsu province in china [ ] . dietary restriction is an activator of prolongevity molecular pathways based on an escape from costs incurred under nutrient-rich conditions [ ] . chronic disease deaths due to heredity, environment, and lifestyle are as high as million, accounting for more than % of all deaths worldwide [ ] . these results support that human has only new cell disease theory, which are made up of sixty million cells, more than , diseases are due to cell nutritional deficiencies and detoxification disorder caused by the disease [ ] . ingredients of barley were preserved in the chinese crop genebank. the detection of functional ingredients in grains and grass powder of more than , accessions barley gremplasm especially combinated with high-density snp markers can not only reveal the molecular mechanism of functional ingredients and their molecular breeding and gene editing techniques but also make great contributions for improving human health by increasing functional ingredients in barley grains and grass powder. ingredients in barley. some excellent barley varieties have very high functional ingredients both in grains and grass powder, which are the result of adaptation to high and low temperature, drought and waterlogging, long and short sunshine, and day and night changes caused by altitude, latitude, and seasonal differences. first of all is the breeding of high functional ingredients in barley grains. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (β-glucan > . %, resistant starch > %, arabinoxylan > %, or polyphenols > . %) and cultivated barley at low temperature and drought as well as high altitude, such as yunke and zangqing with high β-glucan, yungongmai , and yungongmai with high vitamin c. secondly is the breeding of high functional ingredients in barley grass. we have bred some functional barley by crossbreeding between the highest functional ingredients germplasm (gaba > . %, k > . %) and cultivated barley at low temperature and drought as well as high altitude. the average content of gaba in cultivars that we bred is . mg/ g which is . fold higher than that of other barley crops around the world, especially barley grass powder of yungong brand contains times gaba ( . mg/ g) and times ca as well as times k than those of polished rice [ ] . in addition, the functional ingredients of barley grain and grass powder can also be greatly improved by gene editing technology, which needs to be further studied and enriched in the future. functional ingredients of barley. functional ingredients of barley grain and its grass powder have larger variation due to latitude, altitude, season, light, temperature, water, day, and night. all the high-altitude ( , ~ , m) hulless barley can increase higher functional ingredient content than that of plains ( ~ m altitude) [ ] . therefore, it is necessary to promote the functional components of the barley grain and its grass powder under the best ecological conditions, and we also make a useful exploration of the functional components at different altitudes and in different seasons. in addition, the functional ingredients of different parts of barley grain (see table ) and the functional ingredients of different grass cutting stages were different. barley is the oldest and the richest functional food among global cereals. its grains are rich in β-glucan; polyphenols (phenolic acids, flavonoids, and anthocyanins), polysaccharide (arabinoxylan), phytosterols (β-sitosterol, campesterol), tocols (β-tocotrienol, α-tocotrienol, β-tocopherol, α-tocopherol), resistant starch, alkaloid, gaba, folates, linoleic acid, phytate, and so on. this review paper summarizes the obvious efficacy of barley grains that includes antidiabetes, antiobesity, anticancer, antioxidants, anti-inflammation, immunomodulation, cardioprotection, gastroprotection, and hepatoprotection properties, and also, barley grains can lower blood pressure; prevent cardiovascular diseases; optimize cholesterol; improve bowel health and metabolic syndrome; prevent heart disease; reduce chronic kidney disease; decrease stroke; alleviate allergic rhinitis and atopic dermatitis; and accelerate wound healing activities. barley grains, grass, straw, husk, bran, and fine powder are rich in ingredients and food structure to defeat chronic diseases during human migration, especially molecular mechanisms of six functional ingredients barley grass (gaba, flavonoids, sod, k-ca, vitamins, and tryptophan) and grains (β-glucans, polyphenols, arabinoxylan, phytosterols, tocols, and resistant starch) involve to combat more than chronic diseases. these results suggest that barley plays an important role in a healthy diet and in the promotion of early human intelligence. in particular, the healthy effects of functional components of barley grains and grass are the result of longterm continuous evolution of early hominids (fruits/vegetables and leaves rich in polyphenols, k-ca, and vitamins), neanderthals (mushrooms and nuts rich in polysaccharides, phytosterols, and linoleic acids), and homo sapiens (grasses and seeds rich in gaba, enzymes, and resistant starch), which associate with modern humans originating in the progenitor of african homo sapiens with cognitive hominin, especially after interbreeding between homo sapiens and neanderthals that took place in the middle east. the migration route from africa to asia and then to eurasia is basically consistent with the origin and spread of barley and its domestication path, which indirectly supports that barley against stress (drought, cold, and salt) enriched with functional ingredients prevented chronic disease from ancient humans to modern people. fertile crescent is the concentrated area of wild barley and the distribution area of ancient babylonian civilization and ancient egyptian civilization, among which jerusalem is the holy place of judaism, christianity, and islam. these results indirectly support this great contribution of barley for promoting world civilization. the polyphenols in fruits/leaves and polysaccharide in mushrooms/nuts as well as gaba in grass/seeds for prevention of chronic disease are associated with depending functional ingredients for diet from pliocene hominids in africa to modern humans. ethiopia and morocco in africa are top choices for cradle of modern humans homo sapiens and miocene hominoids as well as are the centers of origin for functional barley. food shortages and survival struggles caused by climate change were the causes of early human evolution associated with gaba in the barley grass increased oxidative medicine and cellular longevity sharply under environmental extremes from africa to asia and later to eurasia, especially gaba in crop diets suiting environmental extremes improved intelligence. these results support findings that barley and its grass may be the best functional food crop, especially barley prevents over human chronic diseases based on six functional ingredients of barley grass and grains due to three aspects of the scientific basis, i.e., the similar origin and evolution center of barley and human, the promotion of human intelligence in the early stage, and the sum of staple foods for early hominids and neanderthals as well as homo sapiens. we put forward the strategy of increasing the functional ingredients of barley grain and its grass powder that is as follows: ( ) exploration of excellent germplasm with high functional ingredients in barley; ( ) breeding excellent cultivars with high functional ingredients in barley; ( ) optimization of ecological conditions for high functional ingredients of barley. in addition, the functional ingredients of different parts of barley grain and the functional ingredients of different grass cutting stages are different. although therapeutic mechanisms of functional ingredients in barley grains and grass powder for prevention of human chronic diseases seem a very complicated task, and functional food for therapeutic interventions opens up new ways, it is necessary to find further scientific evidence that demonstrates the health effects of functional ingredients of barley and their extracts from barley on the treatment of chronic diseases. barley is one of the most exciting potential natural sources for the development of functional foods and new drugs with improved efficiency and safety. although we have found some relationship of origin and migration between human and barley, especially preventive role of barley for chronic diseases of human beings, it is necessary to conduct more systemic studies to unravel coevolutionary interconnection mechanism between chronic diseases prevention and human diet for barley functional foods. unfortunately, so far there, is no evidence provided of barley evolving as part of evolutionary consumption. barley plays an important role in promoting the development of functional food and has a potential underlying molecular mechanism and formation as well as action mechanism, which is worthy of further study. this review can be used as a starting point for novel nutraceuticals and functional foods and drugs for barley to improve the prognosis of chronic diseases. the authors declare that they have no conflict of interests whatsoever to declare. the global economic burden of non-communicable diseases health effects of dietary risks in countries, - : a 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analysis of functional ingredients in barley grains from different regions between southwest china and icarda nutrition analysis and food process of barley the research and publication of this article was funded by china agriculture research system (cars- - a, cars- - b) and yunnan provincial expert grass-roots scientific research workstation. key: cord- -ndzgxk k authors: kassa, semu m.; njagarah, john b.h.; terefe, yibeltal a. title: analysis of the mitigation strategies for covid- : from mathematical modelling perspective date: - - journal: chaos solitons fractals doi: . /j.chaos. . sha: doc_id: cord_uid: ndzgxk k in this article, a mathematical model for the transmission of covid- disease is formulated and analysed. it is shown that the model exhibits a backward bifurcation at [formula: see text] when recovered individuals do not develop a permanent immunity for the disease. in the absence of reinfection, it is proved that the model is without backward bifurcation and the disease free equilibrium is globally asymptotically stable for [formula: see text]. by using available data, the model is validated and parameter values are estimated. the sensitivity of the value of [formula: see text] to changes in any of the parameter values involved in its formula is analysed. moreover, various mitigation strategies are investigated using the proposed model and it is observed that the asymptomatic infectious group of individuals may play the major role in the re-emergence of the disease in the future. therefore, it is recommended that in the absence of vaccination, countries need to develop capacities to detect and isolate at least % of the asymptomatic infectious group of individuals while treating in isolation at least % of symptomatic patients to control the disease. a novel coronavirus, named a severe acute respiratory syndrome coronavirus (sars-cov- ; previously known as -ncov), was identified as the infectious agent causing an outbreak of viral pneumonia in wuhan, china, in december [ ] . the world health organization (who) medical team codenamed the new outbreak caused by sars-cov- as "coronavirus disease (covid- ) ". the infection is in the same category as the severe acute respiratory syndrome (sars) which emerged in southern china in , spreading to up to countries, with a total of , cases and claiming lives [ ] . covid- is also in the same category as the middle east respiratory syndrome (mers) which was first identified in saudi arabia in , and ended up spreading to countries around the world, reaching a total of , cases confirmed and claiming up to lives [ ] . since january , an increasing number of cases confirmed to be infected with covid- have been detected outside wuhan, and currently the infection has spread all over the world. as of may , , ( : gmt), covid- had affected all continents including island nations ( countries and territories as well as international conveyances), with the total number of cumulative infections globally standing at , , cases and , deaths [ ] , and the numbers still increasing. the major portal of entry of the virus into the body is the tissue lining the t-zones of the face (including the nose, eyes and mouth). the infection is characterised by loss of the sense of smell (a condition referred to as hyposmia/anosmia), taste and poor appetite. although, such conditions have been observed in covid- patients, many carriers of the infection may not show any severe symptoms like fever and cough but have hyposmia, loss of taste and loss of appetite. whereas knowledge of the virus dynamics and host response are essential for formulating strategies for antiviral treatment, vaccination, and epidemiological control of covid- , estimation of changes in transmission over time can provide insights into the epidemiological situation and help to identify whether public health control measures are having a measurable effect [ , ] . the analysis from mathematical models may assist decision makers to estimate the risk and forecast the potential spread of the disease in the population. understanding the transmission dynamics of the infection is crucial in designing alternative intervention strategies [ ] . in general, by approaching infectious kassa) mechanisms and adherence to the advice given by public health agencies. in particular, the people in some parts of asia have fully embraced the measures while many in other parts of the world were very much hesitant to use them. for example, wearing a face mask everyday in public appearances is like a ritual in most of the countries in southeast asia, while the same is considered as a bad gesture in many of the other parts of the world [ ] (even if it is now becoming a "new normal" also everywhere in the globe). one key difference between these societies and the people in the west is that, the communities in the southeast asia have experienced similar disease outbreaks before and the memories are still fresh and painful [ ] . that means, recent history of a similar event plays a role in behavioural change of the population especially at the beginning of the outbreak in addition to the perceived threat from the disease. therefore, in this paper, we consider a mathematical model that takes into account . the transmission dynamics of covid- similar to the sirs model, . the contribution of the asymptomatic infectious individuals in the transmission dynamics of the disease in the population, . the effect of indirect transmission of the disease through the environment, . behavioural change of individuals in the society to apply self-protective measures, and . the intensity of historical events from recent similar outbreaks. by analysing the proposed basic mathematical model, the effect of each of these factors is investigated in terms of their contribution to the control strategies of the disease. moreover, the use of isolation or quarantining and strict social distancing measures are also considered as mitigation strategies, and a comparative study is made for different scenarios. the layout of this paper is as follows: the model is described and formulated in the next section. its qualitative analysis is presented in section . estimation of the parameters and the sensitivity analysis of the reproduction number of the model with respect to involved parameters are discussed in section . numerical simulations of the model with some assumed intervention scenarios are also presented and analysed in this same section. concluding remarks of the study are given in sections . in this section, we present a mathematical model for the transmission dynamics of covid- which spreads in a population. the susceptible individuals can be infected through either direct contact with infectious individuals or indirect contact with the novel coronavirus infected environment. the population under consideration is grouped into disjoint compartments. individuals who are susceptible to the disease and without formal awareness about the prevention mechanisms or who did not decide to use any protective mechanisms are grouped in the class. individuals who are susceptible but are aware of and decide to apply any of the existing protective mechanisms after receiving public health information on how to protect themselves from the novel coronavirus infection are placed in the class. covid- infected individuals who are asymptomatic and symptomatic are grouped in classes and , respectively. some studies consider the asymptomatic class as the "exposed" class (see for instance [ ] ). but since the individuals in this group are known to be infectious and some of them also recover from the disease without going through the group [ ] , we used a name "carrier" to avoid confusion. the class contains the recovered individuals from covid- . finally, denotes the amount of the novel coronavirus pathogen that contaminates the environment due to shedding by covid- infectious individuals. in the analysis of the model, we intentionally excluded the actual exposed class for mathematical simplicity. however, a days incubation period is taken into consideration in the numerical simulation part of this paper. by combining the direct and indirect ways of transmission, the force of infection will have the form where = + + + + and is the concentration of the novel coronavirus in the environment which increases % chance of triggering the disease transmission. the proposed flow diagram for the transmission dynamics of covid- is depicted in figure while the description of each of the state variables is given in table . pathogen concentration on contaminated surfaces or objects in the environment. table description of the model variables the dynamics of the pandemic is described by using the following system of differential equations (see table for the description of the involved parameters): where with is the value of the force of infection corresponding to the threshold infectivity in which individuals start reacting swiftly (that means, the point at which the behaviour change function changes its concavity). we append the following nonnegative initial conditions to the system ( ): ( ) = , ( ) = , ( ) = , ( ) = , ( ) = , and ( ) = . rate of disease transmission from the environment the pathogen concentration in the environment that yields % of chance for a susceptible individual to catch the viral infection from the environment modification parameter (transmission of relative to ) rate of loosing immunity after recovery natural death rate fraction of recovered individuals moving into the class after loosing immunity table description of the model parameters in this section, we study the quantitative and qualitative analysis of the model system eq. ( ). we begin by determining the biologically feasible set for the model ( ) . the following theorem implies that the solutions of ( ) are nonnegative and bounded from above, provided that the initial conditions are nonnegative. proof: the proof of this theorem is outlined in appendix a. to determine the equilibrium solutions, we set the right-hand-side of eq. ( ) equal to zero and obtain then, the disease-free equilibrium (dfe) is found to be the basic reproduction number, which is very important for the qualitative analysis of the model, is determined here below by using the method of the next generation matrix used in [ , ] . for the model under consideration, using the notation = ( , , ), we have the vector functions with = + + and = + + represent the rates at which the disease compartments increase and decrease in size due to the infection, respectively. then the next generation matrix is where the basic reproduction number denoted by  is defined as the average number of secondary cases produced in a completely susceptible population by a typical infected individual during its entire period of being infectious [ , ] . mathematically,  is the spectral radius of in eq. ( ) and after further simplification, we obtain the next result is a direct application of theorem in [ ] . ( ) is locally asymptotically stable whenever  < and unstable if  > . the epidemiological implication of theorem . is that the transmission of covid- can be controlled by forcing the dynamics through its parameter values so that  < if the initial total numbers in each of the subpopulation involved in eq.( ) are in the basin of attraction of  . to ensure that elimination of the disease is independent of the initial size of the subpopulation, the disease-free equilibrium must be globally asymptotically stable when  < . this is what we present here below. where = the proof of theorem . is carried out using the center manifold theory in [ ] and is given in appendix b. in the above theorem (theorem . ), if the parameter describing the waning of immunity, , is zero, we can observe that both and are negative. hence, eq. ( ) fails to be true. in this case, we give below a theorem which asserts the global stability of the dfe of the model. the disease-free equilibrium of system ( ) in the case when = is globally asymptotically stable for  < . to prove the theorem for the case = , we use kamgang-sallet stability theorem stated in [ ] . let = ( , ) with = ( , , ) ∈ r and = ( , , ) ∈ r . then the system ( ) can be written aṡ and analysis of the mitigation strategies for covid- we show that the five sufficient conditions of kamgang-sallet theorem (in [ ] ) are satisfied as follows. . the system ( ) is a dynamical system on Ω. this is proved in theorem . . . the equilibrium * is gas for the subsysteṁ = ( , )( − * ). this is obvious from the structure of the involved matrix. . the matrix ( ) is metzler (i.e., all the off-diagonal elements are nonnegative) and irreducible for any given ∈ Ω. again this is straight forward from the formulation of matrix ( ). . there exists an upper-bound matrix̄ for the set this can also be verified by checking the eigenvalues of̄ under the condition of  < . hence, by the kamgang-sallet stability theorem [ ] , the disease-free equilibrium is globally asymptotically stable for  < . □ the reality behind theorem . is that, if immunity is permanent ( = ), coronavirus will be effectively controlled in the community by reducing  effectively to a value less than unity. the novel coronavirus being a new strain of coronaviruses, information about the dynamics of the infection is still evolving. biological studies of parameter values describing the vital dynamics of the infection are still ongoing as more laboratory tests become available. although some studies have been done on the early dynamics of the disease most especially on data from wuhan, extensive reading reveals that some of the disease dynamics parameters are highly variable and some processes are not fully explored. in this work, we use new cases data from hubei province of china extracted from who situation reports - [ ] , i.e. for the period january , to march , . we ought to fit the proposed model to the extracted data and estimate the unknown parameters. the total population of hubei province was estimated as . million. the life expectancy of hubei province varies depending on the area of dwelling (i.e urban or rural) as well as gender [ ] . for urban dwellers, the average life expectancy is estimated to be . years (with an average being . years for men and . years for women). the life expectancy of china of the year was estimated to be . years whereas that for the year is estimated as . years [ ] . owing to the negligible difference in the provincial and countrywide value, we use the country life expectancy for the year which gives an average mortality rate of = . × − per day. the recruitment rate is thus given as = × , where is taken to be the total population size, . million. the average time period taken for symptoms to appear after exposure is observed to vary considerably, with ranges between - days [ ] , - days [ ] , and some outliers going to up to days. the observed median incubation period was nearly days [ ] . the time to recovery from the onset of symptoms varies depending on the seriousness of the infection. individuals presenting mild illness were observed to recover in an average period of weeks while those presenting serious/critical illness recovering in about to weeks. for our parameter estimation and simulations, we consider a nominal value of . day − (corresponding to weeks) estimated from an interval ( . , . ). we note that a patient is considered recovered: ( ) if two swab tests taken in a time interval of at least hours both test negative, ( ) if the time taken after the end of respiratory symptoms and fever is at least hours. the waning of the immunity after recovery is estimated to range between months to year, which gives an interval for as ( . , . ) day − . for our simulation, we consider a nominal value of = . day − (approximately one year). we propose that at the end of the epidemic, at least − % of the recovering population will learn from the experiences during the infection and even when the acquired immunity wanes, such individuals will become susceptible individuals with past history/knowledge of the disease. the rate of recovery for the symptomatic individuals ( ) in wuhan varied considerably but majority of individuals who recovered from the virus were discharged from hospital after weeks [ ] . however, the patients in wenzhou-china stayed in hospital for days ( . per day) on average. in the model fitted on the early trends data from wuhan-china [ ] , the recovery rate obtained for symptomatic cases was . per day (accounting for days to recovery). the rate of recovery ( ) for carrier individuals is expected to be higher [ ] . according to the who situation report [ ] , it is estimated that up to % of covid- cases are asymptomatic or show mild symptoms, % show severe symptoms and up-to % end up with critical infection and require oxygen or a ventilator. the proportion of individuals who do not show symptoms or have mild symptoms can be as high as % [ ] . for our model fitting, we use a range of ( . , . ) for both and with selected initial values within the prescribed interval. although hubei province was put on a lockdown on january , , the first major decline in the number of new confirmed cases was only observed on february , (situation report [ ] ), approximately month after the lockdown was imposed. from february , , the method of identification of new cases was revised to include both cases confirmed through laboratory tests and clinical observations. as such, there was an observed spike in the number of new cases on february , to compared to cases (reported on february , ) and cases (reported february , ). applying the above described set of assumptions in the bounds for some of the parameters, we optimized the model output to fit the daily new cases data reported from the hubei province, china. the parameter values for which the model best fits to the incidence data are given in table . figure shows the plot of the reported new-cases data together with the incidence of the disease obtained from the model. as we can observe from the graph, the model analysis of the mitigation strategies for covid- slightly overestimates the reported data except for the two highest points. in addition, since our model does not assume any control measure at this stage while the reported data after the st day may represent the effect of the strict lockdown measure taken by the authorities, the parameters estimated give a good result. we note however that, in some recent work [ , ] , the use of fractional-order calculus is recommended to get better data fit. when we calculate the value of  from eq. ( ) using the estimated parameters given in table , we obtain  ≈ . , which is within the range of values reported in [ , ] . we examine the sensitivity of  to variations in parameter values and establish the significance of the sensitivity indices. we used the latin hypercube sampling (lhs) scheme, which is an efficient stratified monte carlo sampling that allows for simultaneous sampling of the multi-dimensional parameter space [ ] . for each run, simulations were done and partial rank correlation coefficients (prccs) [ ] calculated between each of the selected input parameters and the disease threshold. the prccs indicate the degree of effect each parameter has on the outcome, which in this case is the disease threshold. the sign of the prcc identifies the specific qualitative relationship between the input parameter and the output variable. the positive value of the prcc of the variables implies that when the value of the input parameter increases, the future number of cases will also increase. on the other hand, processes underlying the parameters with negative prccs have a potential to contain of the number of cases when enhanced. the results of sensitivity analysis are indicated in figure (a) and the box plot (figure (b) ) gives the five-number summary for the computed disease threshold value from the sampled parameter space. the processes described by parameters , , and with the greatest positive prccs have the greatest potential of worsening pandemic when increased. on the other hand, parameters (ℎ and ) with negative prccs have the greatest potential in helping contain the infection when maximised. in this respect, we note that increasing social/physical distancing directly reduces as this lowers the likelihood of a susceptible individual getting in contact with a potentially infected individual. in addition, practising good hygiene (such as regularly washing hands, using sanitisers to disinfect the infected environment and avoiding touching the t-zones of the face) is associated with lowering the likelihood of contracting the virus from infected surfaces. anything contrary to the above increases the likelihood of getting the infection through the two aforementioned routes. we further note that practising good hygiene also involves the infected individuals reducing the shedding of the virus into the environment. it is evident from the results in figure table . the processes underlying the parameters , , and have the greatest potential of making the epidemic worse if increased, whereas processes described by and ℎ have the greatest potential of containing the epidemic when enhanced. table that reducing the rate at which the virus is shed into the environment is significant in reducing the severity of the problem. from the five number summary of the results in figure (b), the lower quartile of the computed values of  is about , the median around . and the upper quartile of about . the obtained value of  is within the range of . ( % , . − . ) obtained in the early studies in [ ] . we note that for a selected combination of underlying processes much higher values of  can be obtained, which is an indication of possible worsening of the situation. in a similar way, we observe that for particular underlying processes (a selected combination of parameters) the value of  can be reduced to values below one. as indicated in [ ] , we note that although some parameters in the model may have very small magnitudes of prccs (non-monotonically related to the disease threshold output), they may still produce sizeable changes in the disease burden. to identify the most important parameters in containing or aggravating the epidemic, we computed p-values for the simulated parameters using fisher's transformation [ ] . we note that the computed prccs are bounded between the interval [− , ]. for this matter, some sampling distribution of variables that are highly correlated is skewed. the fisher's transformation ( ) = . log + − is used to transform the skew distribution to a normal distribution and then compute p-values for each of the parameters based on the prccs [ ] . the prccs for the parameters together with their corresponding p-values are indicated in table . we carry out pairwise comparison of the significant parameters (whose p-values are less than . , see table ) to ascertain whether the processes described by the compared parameters are different. we computed the p-values for the different pairs of significant parameters while accounting for the false discovery rate (fdr) adjustment and the results are given in table . the major question posed at this point is: are the different pairs of significant parameters different after fdr adjustment? based on the fdr adjusted p-values in table , the compared pair of parameters are rendered to be different if their p-value is less than . and not different otherwise. we summarise our results in table , where "true" indicates that the compared parameters are significantly different and "false" indicating otherwise. table are the parameters different after fdr adjustment? we observe that the more sensitive parameters are also significantly different (see table ) except for the − pair which may not necessarily be related. we examine effect of variation of the sensitive parameters on the reproduction number ( ). the results of the variation of parameters with more negative prccs are indicated in the bar graphs in figure ( ) . from figure , it is evident that the decay of the virus from the environment (figure (b) ) which can be accelerated by disinfecting surfaces reduces the value of  and consequently the disease burden. in addition, we observe that an increased proportion of individuals with knowledge of similar infections from the past that are practising selfprotection and preventive measures (see figure (a)) is important in slowing down the infection at the initial stage. such proportions of individuals would normally have knowledge about prevention and control mechanisms of the infection just at the onset of the disease. we observe in figure that the increase in person-to-person contact, (figure (a) ), poor personal hygiene, ( figure (b) ), and the rate of shedding of the virus into the environment by both carriers (figure (c) ) and symptomatic individuals ( figure (d) ) increase the value of  and therefore the disease burden. it is evident that the most effective way of containing the infection is by minimizing contact, which is why in most cases imposing a lockdown becomes an effective way of slowing the spread of the infection. in addition, good hygiene practices by all individuals are twofold: ( ) avoiding touching surfaces, always washing hands with soap and water, or using alcohol based hand sanitizer, which reduced the likelihood of contracting the pathogen from the environment; ( ) those who are sick with symptoms like cough and flu, ought to use masks, when they cough or sneeze, must do so in a sanitary tissue which must then be properly disposed off. we also note that hygienic practices without social/physical distancing may not significantly slow down the infection. in summary, we observe that it is possible to reduce the value of  to a value less than unity by reducing only the value of below . (see figure (a) ). this observation is in direct agreement with mitigation approaches that are aimed at minimising human-to-human contact (such as social distancing and imposing a lockdown). therefore, the table paremeter is more influential in the model and can also play a significant role in eradication of the disease. the other parameters (see figures , (b) , (c) and (d)) may reduce the value of  significantly when applied in combination but not as independent mitigation processes. there are various intervention mechanisms for covid- that are being implemented in different parts of the world. the strategies differ from country to country depending on the scientific information available to decision makers. to investigate the outcomes of the mitigation strategies, we include the isolated and/or quarantined classes to the model system ( ) . we assume that the individuals in the asymptomatic class ( ) are detected at a rate of and placed in isolation class, while the individuals in the symptomatic class ( ) are identified at a rate of and quarantined in class. moreover, the system is formulated as a mixed-delay system of differential equations, where the time delay is assumed to account for the incubation period of the disease. then the system becomes where the force of infection is now modified to with representing the average percentage of contacts reduced due to the social distancing measures and representing the total average rate (or percentage) of disinfecting the environment. in the modified model system ( ) , the parameters analysis of the mitigation strategies for covid- table . for the simulation purpose of this study, we considered five different cases or scenarios of how to apply the interventions. the strategies described in each of the cases below are in addition to the awareness creation for voluntary self-protective mechanisms which are widely communicated through various media outlets. here, we assume that the average effectiveness of the self-protective measures is % (as estimated from the data and reported in table ) , and the individuals who decided to use any one of them are strict in following the appropriate rules. here below, we consider each of the scenarios for mitigation strategies case by case. in this case, we assume that about % of the symptomatic infectious individuals and only % of the asymptomatic infectious individuals are detected and quarantined. this scenario is based on the assumption that among the people in the class only about % show "above mild" symptoms and hence visit health care facilities, while the remaining individuals in this class (nearly % of them) remain at home or at large in the society. then, through contact tracing mechanisms corresponding the hospitalized individuals, some people will be traced and tested, thereby about % of the total asymptomatic individuals can be detected. a similar scenario is being applied currently in some sub-saharan african countries. the time profile in figure shows the situation described in case . from this graph we can observe that the infection stabilizes around its endemic equilibrium, which is nearly at cases. (this number depends on the initial conditions and the demographic variables of the population under study.) this shows that the disease persists in the population. analysis of the mitigation strategies for covid- figure : dynamics of the disease with no additional intervention is applied. in this case, we assume that strict and longer time ( weeks) of social distancing rules are enforced by the government nearly weeks after the first positive case of covid- is reported in the community. we assume for simulation purpose that the implementation of the intervention strategy is divided into the following time phases. phase : the first phase in this case, is days long (measured starting from the time the first positive case of covid- is reported). during this phase, because of lack of information and the nature of the infection, we assume (as in case ) that only % of symptomatic infectious individuals and % of the asymptomatic infectious individuals are detected and quarantined. phase : the second phase is assumed to last for weeks ( days). during this period, it is also presumed that; * % of the symptomatic class and % of the asymptomatic class are detected and quarantined, * a mandatory strict social distancing rule is imposed, which is assumed to have a % reduction of effective contacts of individuals in the society, * environmental disinfection is widely carried out, which is assumed to result in a % reduction in the rate of infection from the environment, and to contribute about the same percent impact in increasing the rate of decay of the pathogen from the environment. phase : the third phase is assumed to be weeks ( days) long, and is characterised by the partial lifting of the 'lockdown' imposed in phase . during this period, it is assumed further that; * % of the symptomatic class and % of the asymptomatic class are detected and quarantined, * a relaxed social distancing rule is exercised, which is assumed to have a % reduction of effective contacts of individuals in the society, * environmental disinfection is partially carried out, which is assumed to have an impact of reducing the rate of infection from the environment by % and increasing the rate of decay of the pathogen from the environment by the same %. phase : the last and fourth phase is the time when the social distancing rule is fully lifted. due to the lesson learnt from the previous phases, we assume that the following interventions will continue during this period as well; * % of the class and % of the class are detected and quarantined, analysis of the mitigation strategies for covid- possible to significantly reduce the infection to a level where it cannot be a public threat. otherwise, any lower proportion of this effort will imply the resurgence of a second wave of the infection in the community. therefore, to contain covid- in every given community, public health authorities need to work more on the detection and quarantining of the asymptomatic infectious individuals. in this case, we assume that there is no lockdown imposed but only a large number of testing is applied to detect and quarantine a larger proportion of infected cases. if it is possible to intensify the effort of tracing the asymptomatic infectious individuals and be able to quarantine at least % of them continuously and effectively, our simulation shows that there is a possibility for the disease to be contained without imposing the strict lockdown rule on the total population. the plot in figure shows the time profile of the count of the infected groups while about % of the individuals from class are effectively quarantined (for example inside appropriate health facilities). we can observe that this intervention strategy can also produce the required result in containing the outbreak as some countries (like south korea) are currently following this pattern. in this case, we assume that the length of the lockdown period is nearly twice to the scenario in case . however, the effort in detecting the asymptomatic infectious individuals is kept at the minimum level. this scenario is more applicable in highly resource constrained countries as the current cost of testing is high. in this case, it is assumed that the length of the duration of each phase (except for phase ) is the same as that given in case . more still, it is supposed that; . the conditions in phase remain the same, . phase lasts weeks with % of the symptomatic individuals and % of the asymptomatic individuals detected and quarantined. moreover, strict social distancing rules are still in place with an effect of reducing % of human contacts and % of environmental variables, . phase lasts weeks (the same as in case ), but with % of individuals in the class and % of individuals in the class detected and quarantined, while partial social distancing rules are in place with an effect of reducing % of human contacts and % of environmental variables, . phase continues with detecting and quarantining % of members in the class and % of members in the class, while the other mandatory intervention are lifted. the time profile of the infection following the scenario in case is plotted in figure . the simulation for this scenario shows that even if we increase the length of lockdown period to weeks (like it was practised in the hubei province, china) the disease may re-emerge after some period of time. however, the heights of the peaks in the subsequent waves of the disease are much lower than the first peaks. therefore, once again, unless the authorities apply some kind of strict contact tracing mechanism and conduct enough testing to detect and isolate up to % of the asymptomatic infectious individuals, the disease persists in the community with multiple subsequent waves. in general, from the simulations, we can observe that in all of the above scenarios a transition from one phase to the other intervention phase is characterised by a surge in new cases. however, the number will eventually go down if the intervention in the immediate next phase is effective, or else the disease re-emerges in the population. analysis of the mitigation strategies for covid- figure : dynamics of the disease with at most % of the population in the class and at least % of the class are detected and quarantined just after phase period, with strict social distancing rule imposed for weeks. we presented a mathematical model for the dynamics of covid- whose first cases were reported in december in wuhan-china. the model incorporates a behaviour change function to account for the proportion of individuals who decided to use any of the self-protective measures and adhered to them. in addition, it also considers a proportion of individuals with a history/knowledge of similar infections from the past and practice necessary protective measures right from the onset of the epidemic. the model also accounts for asymptomatic carriers of the infection as well as the concentration of the pathogen within the environment. the basic properties of the model including well-posedness, the disease free equilibrium and its stability, model basic reproduction number as well as the existence of backward bifurcation were examined. to estimate the parameter values, the model was fitted to the data on daily new cases reported in who situation reports - [ ] , which accounts for the period from january , to march , . from the nominal values from the data fitting, we obtained a reproduction number,  ≈ . ( . - ) which compares well with the values of  obtained in other researches, for instance, ( . − . ) [ ] and . ( %ci, . − . ) [ ] . from our sensitivity analysis simulations, we observed that for some given parameter combinations, the value of  can be reduced to below , and similarly for values much higher than . we observed that if the recovering individuals do so with permanent immunity ( = ), then reducing the reproduction number to a value below unity is enough to contain the infection. on the other hand, if recovering individuals do so with temporary immunity ( ≠ ), the proposed model exhibits backward bifurcation, which implies that reducing the value of  below is not enough to contain the infection. by applying the latin hypercube sampling scheme, we observed that if the disease is to be easily contained, measures such as; physical/social distancing (which reduces the rate of disease transmission directly from human to human), improved personal hygiene (which reduces the rate of disease transmission from the environment to humans), and minimal shedding of the pathogen into the environment by both asymptomatic and symptomatic individuals, have the greatest potential of slowing the epidemic when enhanced. we further observed that increased decay of the pathogen from the environment (achieved by disinfecting surfaces) alone is less significant in reducing/curbing the number of cases. we further observed that having high numbers of people with knowledge from previous similar infections, that are practising the prescribed self-protective measures can delay/slow down the otherwise potentially explosive outbreak. consequently, the daily number of cases is kept at low manageable levels. in addition, increasing the average effectiveness of the self-protective measures and adherence to such measures is vital in realising low peaks of the number of cases. furthermore, due to the absence of vaccination or any approved medication, developing capacity to detect carrier groups is very important. from our results, it is recommended that countries should develop capacities to identify and quarantine at least % of carriers as well as at least % of symptomatic cases if the infection is to be controlled. our model predicts a possible resurgence of the number of cases, if the asymptomatic cases are still many by the time disease spread curbs/lockdown measures are lifted. in addition, we observe from simulations that although disease spread curbs (such as a lockdown measure) may be imposed, their real impact on the number of new cases may only be realised after approximately days, and the reduction (when it appears) could be sharp in the case of a strict lockdown measure with high impact in reducing effective contact between individuals in the population. when providing the mitigation strategies, we did not account for the delay between the actual incidence and the point when cases have been confirmed since the actual parameters describing such a delay are not known. in health systems where testing of suspected cases is done after individuals show symptoms or on demand, it is likely to have a big gap between the actual incidence and confirmation of new cases. the impact of the delay between actual incidence and confirmation of case can be explored in future work. in addition, we assumed that all individuals who recover, do so with the same level of immunity. however, this may not necessarily be the case since immunity of individuals is affected by a number of factors, including age, cortisol levels and nutrition among others. the impact of differentiated levels of immunity on the disease dynamics and potential resurgence of the epidemic can be explored in the future when relevant data becomes available. our model did not include the possibility of vaccination or treatment. we, however acknowledge their importance in controlling the infection. therefore, optimal control of the infection in the presence of these mitigation strategies can be explored in future works when relevant data becomes available. since the disease has been observed to affect age groups differently, it is plausible to consider age-structured models to better understand the effect of the disease in the respective age groups. likewise, from the fourth equation of ( ), we obtain ′ = ( − ) + ( − )( − ) + − ( + + ) ≥ −( + + ) . solving ( ) similarly, using the last two equations of eq. ( ), we have and because ( ), ( ), ( ), and ( ) are nonnegative for ≥ . solving eqs. ( ) and ( ) gives and respectively. thus, any solution of eq. ( ) is nonnegative for ≥ and any initial condition in Ω. finally, the total number of the population ( ) at time is governed by thus, for the initial data ≤ ( ) ≤ , by gronwall inequality, we obtain moreover, for the environmental variable , we have because ( ) and ( ) are less than for all ≥ . applying again the gronwall inequality, for ≤ ( ) ≤ ( + ) , leads into combining the above two steps and theorem . . in [ ] for the existence of unique bounded solution, we infer that any solution of eq. ( ) is nonnegative and bounded. hence, eq. 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coronavirus incubation period covid- coronavirus pandemic nowcasting and forecasting the potential domestic and international spread of the -ncov outbreak originating in wuhan, china: a modelling study duration of antibody responses after severe acute respiratory syndrome preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak the analysis of the mitigation strategies for covid- figure : dynamics of the disease with a mandatory -weeks lockdown and a -weeks of partial social distancing is imposed as described in case * environmental disinfection is partially carried out, which is assumed to have an impact of reducing the rate of infection from the environment by % and increasing the rate of decay of the pathogen from the environment by the same %.the time profile of the disease dynamics after implementing the above described interventions strategy is plotted in figure . the figure shows that the count of the infected individuals decreases down to nearly zero in phases and , but the disease resurges back into the society soon after. however, the peak of the second wave looks to be much smaller than the first one. that means, the intervention mechanisms described in the above phases of this case are not enough to contain the disease, and unless some additional intervention mechanisms are developed the disease persists in the society. in this case, we assume that early action with shorter time social distancing rule is applied. in this scenario, it is assumed that the interventions described in case started half way through the time that phase was implemented in case . that means, the implementation of the interventions described in the four phases of case is assumed to be followed, but the length of the time in phases and is reduced as described below. otherwise, all the details of the interventions in case are kept the same. the time profile for this set of interventions is given in figure . the general behaviour of the graph in figure is the same as that of figure . however, this strategy has an advantage in significantly reducing the height of the first peak. the height of the subsequent peaks are found to be the same unless some additional measures are taken after phase .unfortunately, the strategies in both of the above two scenarios (case and case ) do not help to fully contain the disease once it spreads in the population. as it can be seen from figures and , another wave of outbreaks of the disease emerge at a later stage. here, we can see that the asymptomatic infectious individuals play the greater role in becoming the major source for the second wave. therefore, if there is a possibility to track and detect people with asymptomatic infection, and if they can be effectively quarantined for the required period of time, then there is a possibility for the disease to be contained. as it can be observed from figure , if we can increase the rate of detecting and quarantining the asymptomatic individuals to a proportion of about %, it is appendix a. proof of theorem . the proof of theorem . is outlined here below based on the following two steps. first, we show that all solutions of eq.( ) are nonnegative as required in [ , ] . to show that the state variables and of the model are positive for all ≥ , we use proof by contradiction. we suppose that a trajectory crosses one of the positive cones at times or such that:using the first equation of eq. ( ), the first assumption leads towhich contradicts the first assumption that ′ ( ) < . thus, ( ) remains positive for all ≥ . here, is chosen so that our point to be on the positive axis of ( ) so that ( ) is positive.using the second equation of eq. ( ), ′ ( ) = ℎ + + > , which also contradicts the assumption ′ ( ) < . hence, ( ) remains positive for all ≥ . based on the third equation of eq. ( ),analysis of the mitigation strategies for covid- proof: the theorem is the direct application of theorem . in [ ] . to check the existence of backward bifurcation of the model eq. ( ) at  = , we use the center manifold theorem [ ] . for this purpose, we introduce the following change of variables.so that = + + + + , = ( + ) + + , and ( ) = + .moreover, by using the vector notation = ( , , , , , ) , the model eq.( ) can be written in the form ′ ( ) = = ( , , , , , ) as follows: where, = + + , = + + , and = + . when  = and is considered as a bifurcation parameter, from ( ) we get further more, < * if and only if  < and > * whenever  > . the jacobian of the system ( ) at the associated ( ) isthe transformed system eq. ( ), with = * , has a non-hyperbolic equilibrium point such that the linear system has a simple eigenvalue with zero real part and all other eigenvalues have negative real parts. hence, the centre manifold theory [ ] can be used to analyse the dynamics of the model eq. ( ) near = * . by using the notation in [ ] , the following computations are carried out.the right-eigenvectoranalysis of the mitigation strategies for covid- associated with the zero eigenvalue of ( ) such that ( ). = at = * is given byof ( * ) such that the right-eigenvector and the left-eigenvector need to satisfy the condition . = .the bifurcation coefficient at the dfe ( ) is given by analysis of the mitigation strategies for covid- thus, the bifurcation coefficient , can be positive for the right choice of the parametric values that satisfy the condition in eq. ( ) .the second bifurcation coefficient is given by clearly, > because and are positive. when = , and in ( ) are negative and in ( ) is negative as well. hence, by theorem . in [ ] , the model will not exhibit a backward bifurcation at  = . ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: semu mitiku kassa, hatson j. b. njagarah, yibeltal a. terefe. key: cord- -fan sf authors: thacker, stephen b.; stroup, donna f.; sencer, david j. title: epidemic assistance by the centers for disease control and prevention: role of the epidemic intelligence service, – date: - - journal: am j epidemiol doi: . /aje/kwr sha: doc_id: cord_uid: fan sf since , the centers for disease control and prevention has responded to urgent requests from us states, federal agencies, and international organizations through epidemic-assistance investigations (epi-aids). the authors describe the first years of epi-aids, breadth of problems addressed, evolution of methodologies, scope of activities, and impact of investigations on population health. they reviewed epi-aid reports and eis bulletins, contacted current and former epidemic intelligence service staff, and systematically searched the pubmed and web of science databases. they abstracted information on dates, location, staff involved, health problems, methods, and impacts of investigations according to a preplanned protocol. they assessed the methods presented as well as the quality of reports. during – , a total of , investigations of health events were initiated by , epidemic intelligence service officers. in the early years, the majority were in response to infectious agents, although environmental problems emerged. investigations in subsequent years focused on occupational conditions, birth defects, reproductive health, tobacco use, cancer, violence, legal debate, and terrorism. these epi-aids heralded expansion of the agency's mission and presented new methods in statistics and epidemiology. recommendations from epi-aids led to policy implementation, evaluation, or modification. epi-aids provide the centers for disease control and prevention with the agility to respond rapidly to public health crises. in , the us congress established the communicable disease center (cdc) to support state health departments in controlling communicable diseases ( ) . a critical part of this support was technical assistance, initially in the form of laboratory expertise and vector control necessary to support the agency's initial mission to combat malaria in the southeastern united states. by , under the guidance of alexander d. langmuir, md, epidemiologic support became the critical consultative tool, and epidemic-assistance investigations (epi-aids) became the most visible program of the new agency. the system of epidemiologic support was formalized by langmuir in with establishment of the epidemic intelligence service (eis) program ( ) . although the cdc (now the centers for disease control and prevention) works with health agencies throughout the world in multiple ways, the term epi-aid refers to investigations of serious and urgent public health problems in response to formal requests for rapid assistance from states, federal agencies (e.g., the indian health service, the national institutes of health, the food and drug administration, and the us department of defense), international organizations (e.g., the world health organization), and ministries of health from other countries. certainly, extensive service is provided by eis officers (eisos) and other cdc staff outside the formal epi-aid mechanism, including the majority of the work performed by officers and staff assigned to states and international settings. for example, the investigation of polio vaccine contaminated with live virus involved essentially all eisos and was conducted both in the field and at cdc headquarters, yet it was never classified as an official epi-aid ( ) . two other studies illustrate such service in later years. a study led by an eiso using data from a multicenter casecontrol study documented the high risk of pelvic inflammatory disease for women using the dalkon shield intrauterine device and recommended that all women using the device have it removed ( ) . an investigation led by an eiso assigned to the state health department documented the transmission of cryptosporidium infection through the public water supply in milwaukee, wisconsin, that led to an outbreak of disease affecting more than , residents and subsequent modifications of water quality standards ( ) . nevertheless, this critical mechanism of response remains a symbol of the cdc and eis: rapid response to a public health need whenever and wherever it is detected. as inaugurated in , epi-aid responses still are documented by an initial announcement, the epi- , followed by a report of the investigation, the epi- (now called the epi aid trip report), and sometimes later reports (epi- s and epi- s), all of which are addressed to the cdc director and relevant staff at the requesting agency and at the site of the investigation ( ) . these documents are labeled for administrative use, limited distribution and not for publication because results are preliminary and might contain identifying information about patients, providers, and institutions. meanwhile, epi-aids provide a rich chronicle of the practice of field epidemiology at the cdc since and often are key events that marked the evolution of the agency and public health practice (refer to appendix table ). use of the epi-aid mechanism expanded dramatically after establishment of the eis program, which provided a pool of field epidemiologists in training who were available to depart immediately for an investigation anywhere in the world. the success of the eis program, in part enriched by the availability of the epi-aid mechanism, plus the expansion of the cdc mission over time has resulted in more than , epidemiologists being trained ( ) . the initial class of men included physicians and a sanitary engineer. in contrast, the majority of the and eis classes of - members are women and include at least foreign citizens. approximately % of the us citizens are members of racial/ethnic minorities, and % are physicians. the majority hold a master's or doctorate degree, and the others hold a mixture of doctorates in multiple disciplines or are veterinarians, dentists, and nurses. in this report, we describe the first years of epi-aids. we highlight the breadth of health problems as the cdc's mission expanded; the evolution of the epidemiologic, statistical, and information display tools; the national and global scope of activity; and the impact of these investigations on the health of communities. we reviewed bound copies of all epi-aid reports maintained by the cdc's public health library. in addition, we reviewed available copies of eis bulletins and contacted current and former eis supervisors and officers in an attempt to locate published reports based on the epidemiologic investigations. we also identified related publications through online searches of the pubmed and web of science databases (updated january , ). we abstracted information according to a preplanned protocol and included relevant dates, location, staff involved, and suspected or confirmed health problems (table ). in addition, we reviewed laboratory, statistical, and data presentation methods as well as recommendations and impacts on health resulting from the investigations. to assess the statistical and other methodological characteristics of eis investigations, we adapted a rubric for assessing published statistical practice ( ) . we used this rubric to code all reports on epi-aids according to statistical and other methods presented. types and frequencies of applied methods were classified systematically into categories ( table ). reports containing analyses beyond descriptive ones were classified as basic, intermediate, or advanced according to the sophistication of applied statistical techniques. reports using stratification, nonparametric methods, one-way analysis of variance, simple correlation, and regression were counted as intermediate analyses. reports containing any method of multivariate analysis, statistical modeling, advanced contingency table analysis, or survival analysis were categorized as advanced analysis. for this report, we did not implement a rigorous assessment of quality of statistical methods applied; however, we do comment on different aspects of methodological quality. during the years from through , eisos and their colleagues initiated investigations of , health problems in every state and globally (figures - ). during the early years, the geographic distribution of epi-aids was a function of location of the cdc facilities as well as population. since , the cdc facilities outside atlanta, georgia, have been located in alaska (current), arizona, california, colorado (current), kansas, maryland (current), montana, new mexico, north carolina (current), ohio (current), pennsylvania (current), puerto rico (current), texas, washington (current), west virginia (current), and washington, dc. the district of columbia and states did not invite the cdc for epi-aids during the first decade, including new york (the most populous state at the time), massachusetts, connecticut, rhode island, wisconsin, and south dakota, but all states and the district of columbia had one or more requests during each of the subsequent decades. the first response to a health concern outside the united states was after a flood in canada in . as the cdc's international presence became known, international requests for assistance followed from world regions ( figure ). first decade. during the initial decade, - , a total of of ( %) epidemic assistance requests were conducted after the eis program was established in july , and all but were conducted in response to infectious agents. interestingly, a investigation of dysentery in table . classification and number of health problems addressed in epi-aids, - - - - - - abbreviations: epi-aids, epidemic-assistance investigations; hiv, human immunodeficiency virus. a the numbers in parentheses are not included in the virus total. b these numbers also include epi-aids that were discontinued or consolidated into one investigation. year - - - - - - early epi-aids provided the foundation for later methodological developments to address emerging health problems and organizational decisions. in , a response to hepatitis in georgia included an innovative analysis of racial disparities. a investigation of diphtheria in montana resulted in a recommendation that a full-time health department be established in that state. we identified the first peerreviewed publication that resulted from a investigation of western equine encephalitis in colorado ( ). recommendations from an epi-aid to investigate impetigo in a laundry associated with a neonatal ward in provided the foundations for the cdc's hospital infection prevention activities. the first example of a health communication professional participating in an investigation occurred during a epi-aid addressing polio in ohio; this investigation subsequently was covered in life magazine ( ) . during that period, more diseases were identified as the cdc added programmatic responsibilities and increased laboratory capacity, and environmental problems began to be reported. for example, a epi-aid documents the death of a child at the mayo clinic in rochester, minnesota. the cause was ini-tially thought to be trichloroethylene exposure but later was determined to be caused by agranulocytosis from an unknown contaminant in raw milk. second decade. the number of epi-aids increased to during - . occupational conditions appeared in epi-aid files in , when workers in a garment factory suffered from heat exhaustion and anxiety. in , we have the first epi-aid concerning birth defects; in that same year, an outbreak initially reported as fatty degeneration syndrome was probably among the first reported cases of reye syndrome. forty-three outbreaks of suspected polio were confirmed in investigations during the decade after vaccine availability, often among vaccinated populations. the cdc responded with stockpiled oral polio vaccine. indeed, a epi-aid documented the highest recorded incidence of polio in west germany, probably attributable to live vaccine. in , the eis investigation of the first adverse reaction to live virus polio vaccine was reviewed by a blue-ribbon panel that included albert sabin. in , epi-aids involved eisos in the first isolation of the virus causing st. louis encephalitis from a bird in nature, the first outbreak of turkey ornithosis in which human cases occurred among persons not involved in processing work, and the first outbreak of hepatitis in the united states attributable to a municipal water supply. in , an epi-aid resulted in the first report of inhalation anthrax in modern world literature. the first identification of type e botulinum toxin came from an epi-aid in michigan in , and, in that same year, an epi-aid investigation of the first reported localized outbreak of neisseria meningitidis group b at a year - - - - - - - - - - - - california naval base demonstrated failure of antimicrobial prophylaxis. throughout the second decade, epi-aids thrust eisos into the midst of legal debate. for example, a investigation of hepatitis caused by drug toxicity was postponed because of legal concerns. in , an investigation of illness associated with an industrial cafeteria resulted in litigation brought by the employees against management, and an outbreak of foodborne illness resulted in a lawsuit against the grocery chain distributing the product. of note is that, as commissioned officers of the us public health service, eisos need permission from the us surgeon general to testify in a court of law, which limits their availability for testimony. third decade. during - , eisos and their colleagues investigated , reported health problems (figure ), an increase of more than % over the previous -year period. botulism was suspected in epi-aids; of these, were confirmed food botulism, were wound botulism, was a laboratory incident, and the other suspect cases were found to have multiple etiologies (e.g., guillain-barré syndrome). in of the botulism investigations, the father and mother of a family of moving from oklahoma to colorado were found dead in their car, and their -year-old daughter was comatose for days before recovery. apparently, the family had eaten contaminated meatloaf brought on the journey. other investigations of suspected botulism among people in a car in and ultimately were determined to have been caused by carbon monoxide poisoning; the investigations documented increased susceptibility to carbon monoxide among smokers. in a investigation, an outbreak of pneumonia at a washington, dc, hospital seemed to be caused by exposure to the hospital grounds, but no etiologic agent was identified. a investigation of an outbreak of febrile myalgia was determined to be related to the air-conditioning in the pontiac, michigan, health department, which also led to illness among the eisos; again, however, the etiologic agent was undetermined. (in , an epi-aid in pennsylvania led to identification of legionella pneumophila by the cdc laboratorians as the causal agent in all outbreaks, as well as a epi-aid in a minnesota meat-packing plant.) the cdc's role in the global smallpox eradication program had a profound and lasting effect on the agency. during - , domestic and international epi-aids were initiated for smallpox or cowpox; however, only in the international epi-aids was smallpox confirmed or surveillance activities initiated where disease was known to be prevalent. typically, the cause of the other cases was chickenpox or insect bites. smallpox eradication campaigns involved eisos in ghana, india, and yugoslavia. other international epi-aids required eisos to investigate emerging hemorrhagic fevers in germany (marburg virus, ) and sierra leone (lassa fever, ) . development of the cdc's chronic disease program evolved from a investigation of acute lymphocytic leukemia in illinois. interest in chronic disease persisted throughout the decade- cancer clusters were investigated (the vast majority were leukemia), as well as cardiomyopathy and diabetes. attention was directed toward broader aspects of reproductive health and family planning beginning in . investigations related to maternal death from abortion and increased incidence of abortion led to an evaluation of family planning services in new york in . during this decade, investigations involved aspects of reproductive health; others involved birth defects. also during the third decade, eisos moved into other fields of public health (e.g., the epidemiology of illicit drug use and violence, infectious complications of needle sharing by intravenous drug users ( and ) , and heroinrelated overdose deaths in georgia ( )). in , an eiso investigated homicides in atlanta. fourth decade. during - , eisos and their colleagues investigated a total of , reported health problems, a number comparable to that in the previous -year period, but with the size and complexity of the investigations reaching new levels. new infectious problems initially investigated as epi-aids included legionnaires disease in and ebola hemorrhagic fever in zaire and sudan in . in , an investigation of kaposi's sarcoma and pneumocystis carinii pneumonia represented the initial investigation of what would become known as acquired immunodeficiency syndrome, and, in , an epi-aid was conducted to study a cluster of deaths related to conjunctivitis among children, later termed brazilian purpuric fever. attention to social and behavioral aspects of health also evolved during this decade. for example, in , the asso-ciation of binge drinking with bacterial infections was noted among american indians. a analysis of health effects of drought in haiti included income as well as more innovative measures of socioeconomic status, and the investigator recommended that a refugee unit be established at the cdc. in the first epi-aid regarding unintentional injuries caused by motor vehicles in , increased mortality was related to -wheeled all-terrain vehicles in alaska. testimony before the consumer product safety commission led to a nationwide ban on these vehicles. eisos were dispatched to solve problems involving mysterious circumstances, including a investigation of cardiac deaths associated with a popular liquid protein diet, unexplained sudden deaths among hmong refugees in , and infant deaths from digoxin overdose associated with a hospital nurse in . fifth decade. the epi-aids conducted during - involved substantial and increasingly complex health problems. for example, a epi-aid involved more than , reported cholera cases in peru, the first time the disease had been discovered in that country during the th century and an event marking the beginning of a new cholera pandemic. also in , an epi-aid documented the first cholera outbreak in the united states associated with a commercial product, a coconut milk topping. during a decade highlighted by emerging infectious diseases, epi-aid investigations uncovered the first human case of salmonella enterica serotype newport associated with antibiotic use in animals ( ); the first occurrence of meat as a vehicle for listeria monocytogenes ( ); the first confirmed case of human ehrlichiosis ( ); and the first outbreak caused by escherichia coli o :h , a non-o , shiga-like toxin-producing e. coli ( ). investigation of a epidemic of cholera in guinea bissau confirmed that those who had eaten rice and fishmeal prepared by the same persons who had prepared an epidemic victim for burial were at increased risk. in , eisos discovered that apple cider can carry e. coli o :h , and, years later, an epi-aid identified hundreds of confirmed cases of bloody diarrhea and dozens of cases of hemolytic uremic syndrome caused by e. coli o :h associated with consumption of undercooked hamburgers sold at the jack-in-the-box (san diego, california) fast-food chain. in addition to these advances in infectious conditions, the percentage of epi-aids related to disabilities, injuries, environmental exposures, cardiovascular disease, and other chronic diseases increased. the first physical-activity-related epi-aid in assessed the impact of a community-based exercise program; a survey of tobacco outlets revealed that the majority of them sold to minors despite laws forbidding such sales. hurricane andrew stimulated an acute response in , with long-term follow-up the next year. an epi-aid in philadelphia, pennsylvania, confirmed more than deaths resulting from heat exposure in philadelphia in ; a year later, an investigation of heat-related deaths in states uncovered deaths in chicago, illinois, and in milwaukee during days. in , an epi-aid in georgia noted that . % of children aged years or younger were confirmed victims of child abuse or neglect, and, during an investigation of school violence in , approximately % of students reported having access to handguns and % of students favored using metal detectors in schools. increasingly, recommendations from epi-aids led to federal policy implementation, evaluation, or modification. a investigation of an epidemic of diarrhea linked to s. enterica serotype enteritidis contamination of commercial pasta led to a labeling change. the food and drug administration recalled l-tryptophan when a series of investigations in - linked it to eosinophilia-myalgia syndrome. a epi-aid evaluated a policy prohibiting tobacco sales to minors; in , another evaluated the effectiveness of anonymous human immunodeficiency virus testing and counseling. sixth decade. during - , fewer epi-aids (n ¼ ) were requested, probably reflecting increased state and local health department capacity as well as more field-based eisos, but an increase in multistate and nationwide investigations was also evident. two infectious disease events might have highlighted any other decade had they not been overshadowed by terrorism and disasters. the first was the introduction of west nile virus in new york city in and its subsequent systematic march across the country in ensuing years. the threat posed by this virus was anticipated in a epi-aid that identified more than suspect west nile virus cases in romania. during - , a total of west nile virusrelated epi-aids were conducted in new york, louisiana, mississippi, and arkansas. the second event, the devastation wrought by the newly identified coronavirus that causes severe acute respiratory syndrome led to epi-aids in the united states and in asia-vietnam, taiwan, and thailand. three multistate investigations confirmed the strong association (odds ratio ¼ ) between rotavirus vaccine and intussusception ( ), discovered the association between development of meningitis infection and cochlear implants ( ), and documented transmission of monkeypox from exotic animals sold as pets by a single distributor ( ) . the events of september , , were not the only criminal activities that involved eiso investigations. a epi-aid documented acute renal failure attributable to contaminated glycerin being added to acetaminophen syrup in haiti, resulting in deaths. that same year, epidemiologic data from an epi-aid supported a criminal investigation of abscesses in of patients treated with intramuscular adrenal cortex injections by a single physician. a investigation exposed intentional poisoning of texas hospital employees who had consumed tainted pastries in a break room, leading to shigellosis among persons. in , epi-aids were conducted in response to anthrax threats directed at california clinics performing abortions and in pennsylvania in response to a cluster of serratia marcescens cases associated with a respiratory technician tampering with equipment. food and waterborne disease investigations were more frequently national and international in scope, no doubt reflecting broader societal changes in both the sources of food and dietary preferences. a epi-aid linked cases of gastroenteritis to raspberries from guatemala contaminated with cyclospora and traced it to birds in the berry fields. a multistate outbreak investigation for the first time associated salmonella with cold cereal, and the largest outbreak of e. coli ever documented in the united states was investigated among an estimated , persons who became ill after eating at catered events. an investigation in of a multistate outbreak of cases of s. enterica serotype bareilly from contaminated bottled water convinced the food and drug administration to recall the product. in , eisos documented the first norovirus outbreak attributed to commercially prepared food (delicatessen-type meat). by this time in public health, diet, fitness, and recreation were well documented as being necessary for a healthy lifestyle, but eisos demonstrated that these efforts are not without risk. health consequences included herpes infection among wrestlers, methicillin-resistant staphylococcus aureus among football players, and cryptosporidiosis among swimmers. persons from countries and us states who participated in ecochallenge contracted leptospirosis, and campylobacter jejuni was identified among bike tour participants from states and canada in . in addition, military trainees suffered from muscular overuse injuries, and hockey players experienced carbon monoxide and nitrogen dioxide poisoning from incorrect use of an ice resurfacing machine. efforts to maintain a healthy diet were complicated by widespread salmonella epidemics associated with presliced tomatoes in and and with unpasteurized orange juice in , and by the largest hepatitis a foodborne outbreak in us history in , which was associated with green onions. an internet hoax claiming that necrotizing fasciitis was associated with bananas was disproven by a epi-aid. the first transmission of nipah virus from pigs to humans was reported in malaysia in , recurring in bangladesh in . in , the ebola hemorrhagic fever investigation team diagnosed cases and deaths in sudan and uganda, the largest documented outbreak of this disease and the first with an onsite laboratory. in , investigations of cases and deaths in kenya showed that they were related to hepatotoxicity caused by aflatoxin-contaminated maize. environmental hazards continued to challenge eisos as they evaluated allergic reactions to tattoo products in multiple states and assessed access to sidewalks, safety, and ease of walking in west virginia as part of their obesity prevention activities. epi-aids evaluated the effect of president clinton's welfare reform on access to prenatal care in new york city ( ) and the impact of legislation granting oregon adoptees aged years or older access to their birth records ( ) . the investigation of migrant deaths (most commonly from heat stroke) within years at the us-mexico border documented a serious consequence of illegal immigration. a epi-aid studied the health and behavioral repercussions of the electricity blackout in ohio, michigan, and new york. during early epi-aids, reported statistical methods were primarily calculations of rates and measures of central tendency (percentages, means, and modes). data display most often consisted of -or -way tables. the first use of a frequency distribution occurred in a epi-aid concerning suspected measles. also in that year, a report on hepatitis in kentucky contained the first mention of an analysis using punch cards; this report was also the first to use a map. a epi-aid report on encephalitis among florida horses provided an early cost-benefit analysis, noting in the recommendations that the price of a horse was less than the price of one dose of the recommended vaccine. a social network analysis was used in to investigate rabies among dogs. by the end of , we began to see sporadic, if not routine, use of epidemic curves. during - , a total of of ( %) reports included no statistical results. of the remainder, the most frequent statistical methods were summary measures of central tendency (e.g., mean, median, and percentage) or attack rates. of the epi-aids that reported any statistical methods, ( %) collected data through convenience survey methods or used existing surveillance data. rarely were measures of variability or confidence intervals reported. we located only one analysis that used stratification and no advanced methods. still, change was evident during the s. a statistician eiso introduced an innovative measurement of socioeconomic status as a risk factor for illness, and the first anthropologist in the eis used anthropologic methods in an investigation of polio in arizona in . perhaps as a result of increasing diversity of disciplines in eis, epidemiologic methods advanced as well. the first dose-response analysis occurred during a investigation of hepatitis in a texas housing project. a report of the association of breastfeeding with illness from an investigation of staphylococcus in a newborn nursery was the first to use exclusion criteria, a chi-square statistic, and a p value; the fatality rate was the highest reported to date in the united states. we begin to observe use of odds ratios ( ), stratified analysis ( ), and computers (the ibm (international business machines corporation, armonk, new york) method, ). randomization methods were reported first in as the cdc randomization technique. inferential methods appeared as the first use of an epidemic threshold ( ), and the first use of the t test in an epi-aid was reported in . in that same year, a pie chart was used for the first time in a report of an outbreak of polio in the marshall islands. influenced by langmuir, epi-aids often were initiated after review of surveillance data; this was documented in of , ( %) of all epi-aids conducted during [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . only ( %) provided no statistical methods compared with % during the previous decade. the most frequent statistical methods continued to be summary measures of central tendency or attack rates. however, during the third decade, data collection methodology in epi-aids shifted from convenience survey methods or case-series to case-control or cohort studies. we note the emergence of multivariate methods in regression ( ) in part, the increasing sophistication of statistical methods was a result of the increasing use of computers and software packages, as well as coverage of these topics in the eis introductory course. indeed, the first mention of sas (sas institute, inc., cary, north carolina) and spss (spss, inc., chicago, illinois) software in an epi-aid was in in an investigation of injuries during a prison riot. quality control for keypunching was reported first in a suicide study. the first use of a deterministic mathematical disease model in an epi-aid, the reed-frost model, was reported in a investigation of a measles outbreak. despite this sophistication in methods, we continue to observe reporting of personal identifiers as late as , the lack of statistical methods in ( . %) of , investigations during - , and a tendency to report p values without accompanying statistical methods in ( %) reports. new methods continued to be used in epi-aids during - , including time-series analysis to study food poisoning in peru ( ), capture-recapture methods to study school-related deaths ( ), record matching to study motor vehicle injuries among older drivers ( ), and generalized estimating equations to study dengue in palau ( ). case-control studies became the method of choice during this decade; epi-aid reports often used matching techniques. frequently, both risk ratios and odds ratios were reported. as computer software became more widely used, logistic regression began to replace mantel-haenszel adjustment for risk estimates. unfortunately, power calculations were reported rarely, and still p values often were given without explanation of methods. during - , eisos increasingly incorporated economic data into their investigations of disasters ( ), coxsackievirus infection ( ), hepatitis a ( ), meningitis ( ), program evaluation ( ), measles ( ), and coccidioidomycosis ( ) . again, during this decade, use of surveillance methods was prevalent; ( %) of epi-aids were initiated through review of surveillance data or used surveillance data for analysis. only ( %) epi-aids included no statistical methods in the report. eisos continued to explore new methods of analysis: a logic model ( ), walter's method ( ) for cluster analysis ( ) a method for repeated measures ( ) ( ) , calculations for an overcrowding rate ( ), and a time/ space method ( ). as during previous decades, the method for randomization was reported rarely. the data and stories included in the , epi-aids describe a fascinating history, a history elaborated in scientific publications, in multiple books, and frequently in the popular press, the last most notably in the pages of the new yorker magazine by berton roueché ( , ) . the importance of this history to the eis program and of the , eisos to the cdc and to public health is, however, the reason for summarizing the epi-aid story in this report. the reason for absence of epi-aids in some us states during certain years was not always clear. certainly, throughout the years, both state and local health departments developed increased epidemiology and laboratory science capacity. sometimes the reasons might have been based on local politics or legal considerations. at other times, the absence of epi-aids might be attributed to personality clashes or a perception that eisos or their supervisors did not respect the local needs and constraints. for example, no epi-aids occurred in new york state during - , and the state health officer, herman hilleboe, an assistant surgeon general temporarily assigned to the state, is reported to have said, ''i don't want any of langmuir's storm troopers in new york.'' more often, the resistance was less explicit and more passive. nonetheless, the legacy is one primarily of collaboration and mutual respect, and the data support this conclusion. during the years reported on here, , epi-aids were initiated by , eisos (many eisos participating in multiple investigations). the leveling off in the number of investigations since paralleled enhanced capacity of public health departments across the united states and increased complexity of investigations conducted by eisos (figure ). during this time, , epi-aids were completed in the united states and puerto rico ( figure ); were conducted in countries and territories ( figure ) . naturally, as the communicable disease center until , the cdc focused almost exclusively on infectious disease; only limited numbers of epi-aids were not directed to infections (table ) . sometimes, outbreaks of unclear cause led to new or unexpected etiologies. the pursuit of an infectious cause of cancers led to more than investigations of clusters during the s and s. no infectious causes were identified, but the effort formed the basis for development of a chronic disease program and, in , formation of the national center for chronic disease prevention and health promotion. similar efforts to apply epidemiology and surveillance to birth defects and to population concerns were highlighted by epi-aids, and programs in women's health and the cdc's national center for birth defects and developmental disabilities were established in . today, approximately % of the cdc's staff and budget remains devoted to combating infectious diseases around the world, but an increased proportion of the work and of the eis classes is focused on prevention and control of chronic diseases, injuries, environmental health exposures, and noninfectious conditions affecting women and children. the number of epi-aids directed to bacterial disease declined from a peak in the decade ending in but has remained at approximately since then. the number devoted to viral diseases declined at the same time and stabilized at approximately during the past decades, with a similar pattern for parasitic diseases. in contrast, epi-aids for mycobacteria, particularly tuberculosis, have increased during the past decades, as have those for chlamydia and fungal infections. the epi-aid mechanism has also been increasingly used for urgent program evaluations in those years. other than investigations of cancer clusters and problems related to women's and children's health, the epi-aid process has had limited application to chronic disease. similar to investigations of infectious diseases, urgent program evaluation using the tools of an epidemiologist has been adopted some in the past decades, particularly to evaluate laws and policies. in contrast, epi-aids for environmental health and injury events have been used more extensively, starting with the flood disaster in winnipeg, ontario; langmuir was the consulting epidemiologist. these investigations were often in response to natural and human-made disasters and led to development of standard practices that are now routine in response to floods, heat waves, hurricanes, tornadoes, and other widespread health events. similar epidemiologic and surveillance methods have been applied to injuries, both intentional and unintentional, with limited but increasing frequency during the past decades. the evolution of statistical methods in the acute setting of the epi-aid reflects a similar pattern in other public health settings ( table ). especially notable is the increased use of multivariate modeling beginning in the late s, paralleling advances in computer hardware, especially the laptop, and advances in computer software, most notable cdcsponsored epi info, an open-source software package developed in the s for practicing epidemiologists and now translated into other languages ( ) . epi-aids provide the cdc with a unique ability to respond immediately to public health crises throughout the united states and internationally. to many people, the cdc's capacity for rapid response symbolizes its, and more particularly the eis program's, special role in protecting the public's health. the scope and impact of the eis program since its inception in is reflected dramatically by these investigations. such investigations are only a part of what the agency and eisos do; nonetheless, they contribute considerably to the trust in the cdc expressed by the us public and the international community ( ) . the ability to adapt to new challenges in often uncertain and sometimes hazardous settings is chronicled in these reports. continuing success of the eis program and the cdc depends on the ability to retain that agility and anticipate new challenges. to do so, the cdc must continue to prepare its workforce adequately for future public health needs and to base all its work on the best science available. retired from the centers for disease control and prevention, atlanta, georgia (david j. sencer). sentinel for health: a history of the centers for disease control fifty years of epidemiology at the centers for disease control and prevention: significant and consequential the cutter incident. poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the united states during the spring of . i. background type of intrauterine device and the risk of pelvic inflammatory disease a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply epidemiologic field investigations by the centers for disease control and epidemic intelligence service epidemic intelligence service of the centers for disease control and prevention: years of training and service in applied epidemiology the use of statistics in medical research: a comparison of the new england journal of medicine and nature medicine ecology of western equine and st. louis encephalitis viruses; a summary of field investigations in weld county, colorado, to health squad traces polio in ohio mapping mortality and morbidity patterns: an international comparison application of gee procedures for sample size calculations in repeated measures experiments the medical detectives ga: us department of health and human services usda get the highest ratings of thirteen federal government agencies. the harris poll author affiliations: office of surveillance, epidemiology, and laboratory services, centers for disease control and prevention, atlanta, georgia (stephen b. thacker); data for solutions, inc., decatur, georgia (donna f. stroup); and the authors are indebted to alexander d. langmuir for his contributions to the practice of field epidemiology and to the establishment of the epidemic intelligence service. they thank lisa pealer and meghan spall for their tireless efforts in collecting and organizing historical information about epi-aids and the eis program.the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention.conflict of interest: none declared. key: cord- - qib authors: nan title: the th annual meeting of the european society for blood and marrow transplantation: physicians – poster session date: - - journal: bone marrow transplant doi: . /s - - - sha: doc_id: cord_uid: qib nan background: allogeneic hematopoietic stem cell transplantation is routinely offered to patients with high-risk or advanced all in the hopes of improving outcomes. use of truly non-myeloablative (nma) conditioning reduces toxicity in other contexts but outcome data for all patients after nma transplants is lacking. we report the outcomes of patients with all transplanted using a nma conditioning without t cell depletion. methods: first transplant patients between october and june were reviewed. these were consecutive patients until then only those considered unfit for fmc conditioning as per the ukall protocol. all patients were conditioned with fludarabine mg/m /day for days and cyclophosphamide g/m /day for days. short course mtx and ciclosporin were used for gvhd prophylaxis. standard supportive care was employed. thirty-one patients with a median age of ( - ) met the criteria for this case review. had b-all and were philadelphia chromosome positive. patients ( %) had high risk disease by standard diagnostic criteria. ( %) were in first complete remission (cr ). matched sibling donors were used in instances with the remaining being fully matched unrelated donors. % of patients had a hct-ci score of , % a score of or with patients having a score of or higher. median cd dose was . x /kg ( . - . ) with a median cd dose of . x / kg ( . - . ) results: trm was low at % at year and % at and years respectively. no factors included in a univariate analysis (which included age, diagnosis, disease status, hct-ci, donor type, cmv risk and cell dose) significantly impacted trm. the incidence of classical acute (a) gvhd grade - and - was % and % by day and % and % by day if late onset agvhd is included. out of eligible patients developed chronic gvhd of any stage. relapse incidence was low ( % at years in all patients, % in cr patients) and was not impacted by any pre-transplant factors including positive mrd post phase induction (present in patients). notably, in univariate analysis relapse was significantly lower in patients who developed chronic gvhd. background: allogeneic stem cell transplantation (allosct) is the treatment of choice for many patients (pts) suffering from acute myeloid leukemia (aml). the graft vs. leukemia effect (gvl), applied by immunocompetent cells of donor origin, is the most important effector mechanism for the eradication of leukemia, the presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (allosct or donor-lymphocyte infusion (dli)). the purpose was to test, whether the ability of malignant blasts to differentiate in vitro towards dendritic cells of leukemic origin (dc leu ) is associated with response to allosct or outcome after immunotherapy (second allosct or dli) for post-transplant relapse in aml. methods: leukemic blasts were isolated from peripheral blood (pb) or bone marrow (bm) samples of aml patients before allosct (n= ) or at relapse after allosct (n= ). a panel of different assays was used to generate dc leu in vitro ( of them containing gm-csf). finally, in vitro results were correlated with clinical characteristics and outcome of patients treated with donor lymphocyte infusion and/or allosct. results: dc leu could be generated in vitro from all samples. when correlating proportions of dc-subtypes generated ex vivo with clinical data, significantly higher mean proportions of dc leu in the dc-fraction were found in responders vs. non-responders to immunotherapy ( . % vs . %,p= . , range: %- %). vice versa, the chance for response to immunotherapy was significantly higher, if a dc leu /dc ratio of >= % could be reached in vivo (p= . ). those patientswere characterized by a longer time to relapse (p= . ) and by a higher probability for leukemia-free survival (p= . ). similarly, generation of higher amounts (> %, p= . ) of dc leu in the mnc-fraction, and generation of more mature dc (> % cd +, p= . using the best gm-csf containing assay) were associated with a longer time to relapse in the respective patients. moreover, overall survival was improved, if > % dc leu /dc could be generated with the best gm-csf containing assay (p= . ). conclusions: in vitro generation of dc/dc leu from leukemic blasts obtained in active stages of aml before allosct or at relapse post transplant were associated with clinical outcome. this observation supports a role of antigen presentation by leukemic cells for an allogeneic immune response in aml. disclosure: nothing to declare background: the role of autologous hematopoetic cell transplantation (hct) in the treatment of aml is not clear. trials in the past have shown that autologous hct consolidation lowers the risk of relapse, however the magnitude of this effect is limited . autologous hct is advocated in patients with aml with lower genetic risk in cr .many of these patients will eventually relapse and will undergo reinduction followed by allogeneic hct in cr . methods: the aims of this study is to analyze outcome of allogeneic hct performed in cr comparing patients with prior consolidation by autologous hct vs. patients with chemotherapy consolidation. primary outcome is non relapse mortality (nrm) of allogeneic hct in cr in patients with, or without prior autologous hct in cr . secondary outcomes include leukemia free survival (lfs), relapse rate (ri), graft versus host disease free relapse free survival (grfs), overall survival (os), and treatment related toxicities. results: adult patients reigstered with the alwp of the ebmt with de novo aml were included, receiving a first allogeneic hct in cr , in cr , in - or without (n= ) prior autologous hct. patient and transplant characteristics are shown in the table. patient groups were not entirely comparable, patients with prior autologous hct were younger, had less often a favorable cytogenetic profile, had more commonly donors other than matched siblings and more often received reduced intensity conditioning (ric) as compared to mac conditioning. univariate outcomes are shown in the table with slightly higher nrm risks in patients with prior autologous hct consolidation. in multivariate analysis nrm risks in patients with prior autologous hct were . ( . - . ), p= . after adjustment for patient age, cytogenetic risk category, year of transplant, donor type, conditioning intensity, sex matching, time from diagnosis to relapse and time from relapse to allogeneic hct as compared to patients with chemotherapy consolidation. similarly, risks of events in lfs and grfs were higher with prior autologous hct, . ( . - . ), p= . and . ( . - . ) p= . , respectively, risk of death was also higher . ( . - . ) p= . but this was not statistically significant. conclusions: we may conclude that some of the advantages of potentially higher anti-leukemic activity of high dose chemotherapy and autologous hct when given to patients with aml in cr (as was shown in a randomized trial by vellenga e et al with lower relapse and higher lfs by approximately % but no significant differences in overall survival) may be lost by higher toxicity of allogeneic hct in cr in case of subsequent relapse. background: although relapse is a major cause of mortality in patients receiving allogeneic hematopoietic cell transplantation (hct) for acute leukemia, limited and conflicting data exist on extramedullary relapse (emr). we aimed to describe the incidence, risk factors, outcomes and prognosis in relapsed hct recipients. methods: we retrospectively reviewed charts of consecutive allogeneic hct recipients transplanted in our center with the indication of acute leukemia ( / - / ). we recorded: age, gender, disease, previous extramedullary involvement, phase at transplant, type of transplant, donor, conditioning, graft-versus-host-disease (gvhd), infections, treatment-related mortality and relapse mortality. in patients with extramedullary relapses, additional data on clinical manifestations, imaging, cerebrospinal fluid testing, histopathology and management were additionally documented. incidence of isolated emr (iemr) and bone marrow relapse (bmr) was calculated using cumulative incidence (ci) analysis, with each and treatment-related mortality considered a competing risk. results: among allohct recipients followed for . ( . - . ) years, ( %) patients presented with emr. the majority of emrs involved the central nervous system (cns, %). isolated emr was observed in patients at . ( . - . ) months. -year cumulative incidence (ci) of . % for iemr was associated only with pre-transplant advanced disease phase (p< . ). bmr was observed in patients at ( . - months), with a -year ci of . %. in the multivariate analysis, bmr ci was independently associated with fungal infections (p< . ), pre-transplant disease phase (p< . ) and lines of treatment (p= . ). -year trm of our whole cohort was . %. the majority of iemr and bmr ( % and %, respectively) patients received systemic treatment combined with local radiation for iemr ( %) and donor lymphocyte infusions (dlis, % and % respectively) when feasible. extensive chronic gvhd was recorded in % of iemr and % of bmr patients. outcomes were poor in iemr, with -year overall survival (os) of . %. favorable os in iemr was associated only with sibling donors (p= . ) and not with other factors, such as treatment with dlis or presence of chronic gvhd. similarly poor outcomes ( year os of . %) were observed in bmr. favorable os was independently associated only with the diagnosis of aml (p= . ) and absence of bacterial infections (p= . ). in the whole cohort, both iemr and bmr were independent unfavorable predictors of os (p< . ) along with extensive chronic gvhd (p= . ). conclusions: in a large population with long-term follow-up, incidence of iemr was relatively high, developed at the late post-transplant period and associated only with disease phase at transplant. furthermore, iemr and bmr conferred similarly poor outcomes despite systemic treatment or extensive chronic gvhd. these independent predictors of survival highlight the unmet clinical need of novel approaches either as maintenance or treatment to reduce extramedullary or systemic relapse post allohct for acute leukemia. disclosure: no competing financial interest. impact of t-cell depletion on outcome in patients undergoing allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia background: after a diagnosis of acute myeloid leukemia (aml) the majority of patients (pts) who achieve complete remission (cr) eventually relapse, with only approximately % of pts maintaining cr for years or longer. late relapses (after years in cr) occur rarely ( - %) in pts receiving hsct in cr and late effects are followed up by routine surveillance as well as preventative measures. the purpose of this study was to investigate long-term outcomes in pts with diagnosis of aml undergoing hsct at our institution in cr . methods: a standardized follow-up of hsct-survivors is applied at our center. we analyzed adult pts with aml in cr consecutively transplanted between january and december at our institution. a written consent was given for the use of medical records for research. a landmark analysis was adopted for patients in cr at -y after hsct (ltcr -long-term cr). results: ltcr was achieved after hsct in / patients (male , female ) transplanted in cr . the median follow-up was years and the median age at transplant years (r - ). the selected donor was a family haploidentical relative in cases, an hla identical relative in , a match unrelated donor in and a cordblood in . in this cohort of ltcr, the -year overall survival was % ( % ci - ). cumulative incidence of relapseevaluated in competing risk with transplant related mortality (trm) -and trm -evaluated in competing risk with relapse -were respectively % ( % ci - ) and % for the cr cohort. the event-free-survival (efs) was % ( % ci - ). the causes of death were relapse ( / pts), second cancer ( / pts) and sepsis ( / pts). the -year incidence of dyslipidemia -defined as cholesterol >/= mg/dl, and/or ldl >/= mg/dl, and/or triglycerides >/= mg/dl or need for specific treatment -was %. the -year incidence of osteopenia / osteoporosisdefined as t-score lower than - and greater than - . and t-score lower than . respectively -was %. the -year incidence of second cancer was %: nonmelanoma skin cancer, lung carcinoma, cervical intraepithelial neoplasm, thyroid cancer, gastric cancer and colon cancer. the -year incidence of chronic moderate-severe gvhd was % ( % ci - ), with the latest diagnosis performed on day . of note, / pts are still on active treatment at last follow-up. conclusions: relapse incidence is low for patient that reached ltcr: patients in cr at transplant can obtain excellent os and efs once reached the target of ltcr. a proactive long-term follow-up and strategy of counseling are essential to keep at best quality the survival advantage offered by hsct in patients with aml in cr . disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. background: relapse, graft-versus-host disease (gvhd) and gvhd-associated mortality are major obstacles to success of transplantation from unrelated (mud) donors in children with acute leukemia (al). negative depletion of αβ t cells and cd + b lymphocytes, conserves the mature donor-derived natural killer cells and γδ t cells in the graft, may improve gvhd control, immune reconstitution and prevent the relapse. we present a retrospect analyses of a cohort of pts with al in cr transplanted from mud with depletion. methods: a total of children with acute leukemia ( aml, all, female, male, median age , y) underwent allo hsct from matched unrelated donor between june and july . all pts were in complete remission (cr = , cr = , cr> = ). all pts, except one, received treosulfan-based conditioning. either melphalan (n= ) or thiophosphamide (n= ) or etoposide (n= ) were added as a second agent. fludarabine was used in all pts. two types of gvhd prophylaxis were used: type (n= ): hatg mg/kg and post-hsct tacro/mtx (n= ) or without prophylaxis (n= ); type (n= ): thymoglobulin(ratg) mg/kg, rituximab mg/ m with either bortezomib on days + , + (n= ) or tacro/ mtx (n= ) . aβ t cell depletion with clinimacs was used in all cases. the median dose of cd + cells was x / kg, aβ t cells - x /kg. median time of follow-up for survivors was , years (range, , - , ) . results: primary engraftment was achieved in % pts., the median time to neutrophil and platelet recovery was and days, respectively. all evaluable pts achieved sustained complete donor chimerism by day + . early ( day) mortality was , % ( pt -bacterial sepsis, pt -adv fulminant hepatitis), -years overall ptrm at years was , % ( %ci: - ). six late trm events were due to: viral infection in pts (cmv= , adv+cmv= ), bacterial sepsis in pts and pts had bacterial and viral infection, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv was % ( % ci: - ), acute gvhd grades iii-iv , % ( % ci : , - , ) . ci of cgvhd was %( %ci: - ). regimen was more effective in prevention of agvhd ii-iv in comparison with regimen : % ( % ci: , - ) vs , %, respectively, p= , . all events with acute gvhd grades iii-iv had pts with regimen . ratg was also effective in prevention of cgvhd: ci at years after hsct was , % vs. %, respectively, p= , . cumulative incidence of relapse was % ( %ci: - ) without difference between ratg and hatg. event-free survival (efs) (event=death or relapse) at years was % ( %ci: - ), overall survival %( % ci: - ), there were no difference between age and diagnosis. conclusions: we confirm that the depletion of tcrαβ +/cd + t lymphocytes from the graft ensures high engraftment rate. transplant-related mortality is caused by infections, mostly associated with cases of chronic gvhd. gvhd prophylaxis including ratg/rituximab/ bortezomib improves gvhd control in recipients of tcrαβ+/cd +depleted grafts in comparison to hatg/ tacro/mtx apparently without loss of anti-leukemic activity. disclosure results: at baseline, r/r all with emd and lbl were diagnosed in and ino patients and and sc patients. median (range) age of the ino and sc patients was . ( - ) and . ( - ) years, with / ( . %) and / ( . %) males, respectively. the rate of cr/cri was significantly higher in the ino group ( / [ . %] , % confidence interval [ci] : . - . ) compared with sc ( / [ . %], % ci: . - . ; p= . ) (table) . allogeneic hematopoietic stem cell transplantation was carried out in / ( . %) ino and / ( . %) sc patients prior to any post-study induction therapy. the pfs hazard ratio [hr] was . ( . % ci: . - . ; p= . ), with median pfs of . ( % ci: . - . ) months among ino and . ( % ci: . - . ) months in sc patients. the os hr was . ( . % ci: . - . ; p= . ), with median os of . ( % ci: . - . ) months in ino versus . ( % ci: . - . ) months in sc patients (figure) . all patients had adverse events (aes). serious aes occurred in / ( . %) ino and / ( . %) sc patients; ( . %) ino and sc patients had grade ae. one ( / , . %) patient in the ino group died from veno occlusive disease. conclusions: among r/r all patients with emd and lbl, improvement in remission rates, transplant rates, and progression free survival was shown in the ino group versus the sc group. although patient numbers were small and limited the ability for a robust comparison, these results support the use of ino in patients in this difficult to treat population with r/r all and emd or lbl. background: bcr-abl-targeted tyrosine kinase inhibitors (tki) revolutionized the outcome of patients inflicted with ph+ b-all. moreover, addition of tki may be relevant strategy for ph-like all patients. methods: we hypothesized that overcoming the bm microenvironment-mediated protection of all cells from tki-mediated apoptosis may further enhance the responsiveness to tki therapy. results: in vitro treatment of bcr-abl-positive all cell lines nalm and nalm ) with dasatinib resulted in significant dose-dependent cell growth inhibition, with ic of - nm (p< . ). furthermore, dasatinib exhibited significant growth suppression of bcr-abl -negative all cells (nalm and reh), with ic of nm and nm, respectively. however, when cocultured with bone marrow stromal cells (bmscs), dasatinib-mediated effect was abrogated in both ph-and ph+ all cells. furthermore, dasatinib treatment promoted significant upregulation of chemokine receptor cxcr , on both mrna and cell surface levels. elevated cxcr expression was accompanied by increased responsiveness of all cells to cxcl stimulation, resulting in strong and sustained phosphorylation of erk / and akt and increased adhesion capacity to bmscs. therefore, dasatinib-induced upregulation of cxcr promotes stroma-mediated survival advantage of all cells upon tki therapy. next, in order to overcome the cxcr -mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete cxcr in aml cells. single-agent treatment with panobinostat demonstrated significant inhibition of ph-and ph+ all cell growth at low nanomolar concentrations (p< . ). importantly, combination of panobinostat with dasatinib synergized (ci< . ), effectively overcoming the protection provided by bmscs and inducing the apoptosis of ph-and ph+ all cells, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization and dna fragmentation. furthermore, combining panobinostat with dasatinib significantly reduced cxcr surface levels in ph-and ph+ all cells. accordingly, cxcl mediated responses, including erk / and akt activation and adhesion to bmscs were significantly reduced upon combined panobinostat/dasatinib treatment. these data indicate that panobinostat effectively suppresses both basal and dasatinib-induced cxcr expression and function in all cells overcoming stroma-mediated resistance to dasatinib. to determine the molecular mechanism, we performed gene and protein expression analysis. panobinostat, alone or in combination with dasatinib, significantly down-regulated the protein levels of calcineurin, a serine-threonine protein phosphatase previously implicated in t-all and b-all pathogenesis, as well as of nfatc , a critical effector of the calcineurin signaling cascade, and nfatc -regulated target genes. it was previously found that calcineurin signaling positively regulates cxcr expression in t lymphocytes. additionally, cyclosporin a (csa) decreased both basal and dasatinib-induced cxcr surface levels in all cells, overcoming the protection of the bmscs which result in potentiation of the cytotoxic effect of dasatininb and panobinostat. combining csa with panobinostat resulted in deeper suppression of nfatc -regulated target genes. we thus link the effect of panobinostat with calcineurin-dependent downregulation of cxcr , blocking the ability of the leukemic cells to respond to cxcl mediated stromal support. conclusions: taken together, our results identify calcineurin signaling pathway as a novel target of panobinostat in all cells and indicate that hdac inhibition with panobinostat may be effective strategy for facilitating the anti-leukemic activity of tki therapy. disclosure: nothing to disclose background: the treatment of relapsed/refractory acute lymphoblastic leukemia (rr-all) remains a clinical challenge with a generally dismal prognosis. allo-sct using a sequential conditioning ("flamsa"-like regimen) has shown promising results in relapsed/refractory aml, but little is known about the efficacy of this procedure in rr-all. methods: we identified adult patients ( % females; median age: y; range, - ) with all in primary refractory phase ( %) or in relapse ( %), allografted between and from a matched sibling ( %), matched unrelated ( %) or haploidentical donor ( %) at ebmt participating centers. almost half ( %) of the patients had t-all and % had a positive philadelphia chromosome. six patients ( %) underwent a previous autotransplant. karnofsky score was above in % of patients. conditioning was myeloablative (mac) with high dose tbi in % of patients, reduced intensity (ric) including low dose tbi in %, or with chemotherapy alone in %. in vivo t cell depletion was performed in cases ( %). most patients ( %) and about half of the donors ( %) were cmv positive. % of patients were males who received a graft from a female donor. the median follow-up was (range, - ) months. results: overall, patients ( %) failed to engraft, ( %) died within days after allo-sct without relapse, and ( %) could achieve complete remission. at day , the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were % and %, respectively. the year cumulative incidences of chronic and extensive chronic gvhd were % and %, respectively. the -year relapse incidence (ri) and non-relapse mortality (nrm) were % and %, respectively. the -year leukemia free survival (lfs), overall survival (os) and gvhd relapsefree survival (grfs) were %, % and %, respectively. in a multivariable cox analysis, karnofosky score below negatively affected ri, lfs, os and grfs. also, conditioning with chemotherapy alone, compared to tbibased conditioning, negatively affected relapse rates (hr= . ; p= . ), lfs (hr= . ; p= . ) and os (hr= . ; p= . ). conclusions: allo-sct using a sequential conditioning regimen is proposed by different teams in rr-all, and could be an option, especially when considering a tbibased regimen. however, the overall -year lfs of % suggests that these patients still face extremely dismal outcomes, highlighting that other therapies (e.g. bite antibodies, inotuzumab, car t cells) need to be combined prior and/or after allo-sct in order to further improve outcome. disclosure: no conflict of interest, no funding received chemotherapy courses, only pts were not treated: pts for the worsening of the general status and the other for invasive fungal infection. results: forty-three pts ( %) were in complete remission (cr) and negative minimal residual disease (mrd) at the time of hsct; pts were in active disease ( %), and ( %) showed a morphological cr with positive mrd. pts ( %) developed chronic graft versus-host disease (cgvhd) as followed: pts ( %) mild, pts ( %) moderate, and only sever grade respectfully. only patient developed cgvhd after dli. the overall leukemia free survival (lfs) time was months, the absence of cgvhd (hazard ratio -hr: , ; p = , ) and the pre-hsct disease status (hr , ; p = , ) were the most important factors on lfs. all pts treated with chemo-based regimens died due to progression or infective complications. patient of aza/dli group is still alive with a extramedullary relapse; pts treated with bl/dli are in cr. os was better for the dli group compared to the chemotherapy group ( vs months respectfully; p < , ). conclusions: dli after allo-hsct has exhibited definite anti-leukemic effects in post-transplant patients. bl and aza were reported to increase dli's graft vs-leukemia (gvl) effect. although cgvhd could be the most important protective factor against the relapse but it remains the main cause of morbidity. maximising the gvl effect without putting the patient at risk of gvhd still represents an unmet need. our data show that the combination of either bl or aza with dli infusion is safe and might represent an improvement in disease control in the early phase of relapse. disclosure: nothing to declare p increased detection of (leukemiaspecific) adaptive and innate immune-reactive cells under treatment of amldiseased rats and one therapy-refractory aml-patient with blastmodulating, clinically approved response modifiers (pg-e ,kit-k) or +pge (kit-m),patent ) convert myeloid blasts into dendritic cells of leukemic origin (dc leu ). after stimulation with dc leu , antileukemic tcells can be generated ex vivo. the compounds are approved for clinical use and are therefore attractive tools for immunotherapy in myeloid leukemia. methods: dc/dcleu-culture from rats'/patients' wholeblood (wb) with kits, mixed lymphocyte culture (mlc) of tcells with kit-treated blood, functional blast-cytotoxicity and leukemia-specificity assays (csa/elispot/degranulation/intracellular cytokine-assays). in addition flowcytometric evaluations of cellular and (leukemia-specific) lymphocyte compositions were performed from rats'/pts' blood in the course of the disease. results: ) aml-diseased rats: each rats were treated with "i", "k" or "m" or were untreated (controls). a significant increase of dcleu could be detected in spleen/pb in kit-(esp. m) treated compared to untreated animals without induction of blasts' proliferation (ki positivity): a significant reduction of blasts was seen with "m" (p= . / . in spleen/pb) and "i", but not "k". successful treatment correlated with an increase of cd l+tcells, most likely representing tmem-cells, (p= . ) and a reduction of cd +treg (p= . ). ) therapy-refractory aml-patients (during the course of decitabine/ld-aractreatment): kit-m was shown to ex vivo generate dcleu, activate immunereactive cells and mediate leukemia-specific/antileukemic response. activated or leukemia-specific lymphocytes were monitored in low proportions in active stages of the disease as well as of two patients during the further course of persisting disease. one of these patients ( yo male), was offered an individual systemic salvage-treatment (kit-m, applied as continuous infusions) for refractory leukemia. after approval from the local ethical commitee,extensive information of the patient about the experimental nature of the treatment and obtaining his written informed consent. clinically the treatment was well tolerated and the patient improved clinically. neutrophils in wbc increased from % to %, thrombocytes reached g/l after days. after weeks of treatment, the patient was discharged in good clinical conditions. days later, progression of aml was seen with high blast counts in pb and bm. the patient developed severe sepsis and died few days later. immune monitoring showed (other than before treatment and in the patients without kit-m-treatment) a continuous increase of proliferating and non-naïve tcells, nk, cikand nkt-, th cells, bmem-cells and dc in pb. the production of ifnƔ producing t-, cik and nkt-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. immune stimulatory effects decreased after discontinuation of therapy. conclusions: treatment of wb as well as leukemically diseased organisms with blast-modulating kits (especially gm-csf and pge ) was well tolerated and induced clinical and immunological improvement (adaptive and innate immune system), whereas low counts of (leukemiaspecific) activated immune-reactive cells were found in non-kit-treated organisms. disclosure: nothing to declare p long-term outcomes after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia with non-myeloablative and myeloablative conditioning: a single-center cohort study of consecutive patients lars klingen gjaerde, niels smedegaard andersen , lone smidstrup friis , brian thomas kornblit , søren lykke petersen , ida schjødt , henrik sengeløv rigshospitalet, copenhagen, denmark background: since , we have at our institution used a non-myeloablative (nma) conditioning regimen for older (> years) or significantly comorbid younger patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) for acute myeloid leukemia (aml). we aimed to compare the long-term outcomes of nma conditioned patients with myeloablative (ma) conditioned patients. methods: we studied nma and ma conditioned adult (> years) consecutive patients receiving their first allo-hsct for aml from to at rigshospitalet. nma conditioning consisted mainly of gy total body irradiation (tbi) and fludarabine mg/m ( % of cases). ma conditioning consisted mainly of cyclophosphamide mg/ kg and either gy tbi ( % of cases) or busulfan . mg/ kg ( % of cases), or fludarabine mg/m and treosulfan mg/m ( % of cases). five percent and % of nma and ma conditioned patients, respectively, received anti-thymocyte globulin. patients were followed until death or end-of-followup on october st , . cumulative incidences with % confidence intervals (ci) of acute graft-versus-host disease (agvhd) grade ii-iv, chronic graft-versus-host disease (cgvhd), relapse and non-relapse mortality (nrm) were calculated and compared between nma and ma conditioned patients using gray's test with death as a competing risk (or relapse when comparing nrm). overall survival (os) was estimated by the kaplan-meier method. results: nma and ma conditioned patients were comparable when regarding sex ( % and % female, respectively) and donor (matched related donor in % and %, respectively), but differed, as expected by indication, with regards to age (median of versus years, respectively) and karnofsky score (< in % and %, respectively). nma conditioned patients had generally a lower aml stage at transplant ( st complete remission in % versus % of ma conditioned patients) and a lower aml cytogenetic risk (adverse risk in % versus % of ma conditioned patients). patients were followed for a total of person-years (median follow-up in surviving patients was . years). agvhd grade ii-iv occurred less frequently in nma conditioned patients ( % [ci: %- %] versus % [ci: %- %] in ma conditioned patients, p < . ), while cgvhd occurred in similar rates ( % [ci: %- %] in nma conditioned patients and % [ci: %- %] in ma conditioned patients, p = . ). there was a trend towards a higher relapse rate in nma conditioned patients ( % [ci: %- %] versus % [ci: %- %] in ma conditioned patients, p = . ), and nma conditioned patients had, however not with statistical significance, lower nrm ( % [ci: %- %] versus % in ma conditioned patients, p = . ). os ( figure) was comparable, with -year os rates of % (ci: %- %) in nma conditioned patients and % (ci: %- %) in ma conditioned patients. conclusions: patients with aml undergoing allo-hsct with nma conditioning at our institution were older and frailer than ma conditioned patients, but their overall survival after transplantation was comparable. this might be explained by a generally lower aml stage and cytogenetic risk at transplant in nma conditioned patients. jedlickova , saskia güller , rosa toenges , juliane steinmann , hans martin , hubert serve , gesine bug background: allogeneic hsct is urgently indicated in patients with aml in first complete hematologic remission (chr) after intensive chemotherapy with increasing or recurrent minimal residual disease (mrd). these patients are at high risk of hematologic relapse (hr) during preparation of their transplant and hsct with active aml was found associated with poor outcome. azacitidine has recently been shown to substantially delay or even prevent hr in > % of patients (relaza trial, platzbecker et al., lancet oncology ) . we here present the outcome of a small cohort of consecutive patients with mrd-positive aml who received low dose cytarabine (ldarac) as bridging therapy prior to hsct. methods: mrd was assessed by quantitative polymerase chain reaction (qpcr) using mutated npm (n= ), runx -runx t (n= ), cbfb-myh (n= ) or kmt a-ptd (n= ). mrd negativity was defined as ratio of oncogene to control gene (abl ) ≤ , % while increased or recurrent mrd required a ratio > % (shayegi et al., blood ) . primary endpoint of our retrospective analysis was progression to hr (≥ % bone marrow blasts or extramedullary disease); secondary endpoints were achievement of molecular remission prior to hsct, neutropenia g according to ctcae, thrombocytopenia g , anemia ≥g , admission to hospital, os and rfs. os and rfs were calculated from the first dose of ldarac. ldarac was self-administered subcutaneously by the patients at home at a flat dose of mg bid over days and repeated after weeks if necessary. results: between / and / , nine patients (median age , range, - years) with low (n= ), intermediate (n= ) or high-risk cytogenetics (n= ) according to eln criteria were treated in continuous chr for increasing (n= ) or recurrent mrd (n= ) starting at a median of (range, - ) days after the last consolidation therapy, i.e., duration of chr was > months in all pts. patients received one (n= ), two (n= ) or three cycles (n= ) of ldarac prior to hsct. in three patients, neutropenia g occurred and one patient needed platelet transfusion. all patients were managed in the outpatient setting. in eight out of nine patients ( %), hr was successfully prevented and patients ( %) even became mrd negative prior to hsct. one patient (runx -runx t positive aml) progressed to hr after one cycle of ldarac and received salvage therapy with high-dose arac and mitoxantrone (ham) prior to hsct. all patients proceeded to hsct from a matched related (n= ), unrelated (n= ) or haploidentical donor (n= ) and are still alive (median follow-up of days). conditioning regimens included fludarabine (flu)/melphalan (mel)/tbi (n= ), flu/mel (n= ), flu/tbi (n= ), flu/busulfan (bu) (n= ) and thiotepa/bu /flu (n= ). after hsct, only the ldarac-refractory patient relapsed, resulting in a probability of rfs of % at years. conclusions: our data suggest that a bridging therapy with up to three cycles of ldarac prior to hsct is feasible and was associated with favorable outcomes in patients with npm -mutated or core binding factor aml and molecular relapse > months after achieving a first chr. the treatment has low costs, can be administered on an outpatient basis and is very well tolerated. clinical background: allogenic hematopoietic stem cell transplant (hsct) is the only curative treatment for all the patients with aml. high risk disease qualifies for upfront hsct irrespective of the presence of matched sibling donor (msd). in the absence of msd, haploidentical stem cell transplant is easier option with success rates as high as msd in a high volume transplant centre. we present our experience from a single centre. methods: we analyzed retrospective data of aml patients who have undergone hsct at our centre between january- and august- . for msd transplant we used fludarabine + busulfan or fludarabine + melphalan conditioning regimen, in matched unrelated donor transplant (mud) regime used was fludarabine + busulfan + atg. we followed john hopkins's protocol for haploidentical hsct. cyclosporine + methotrexate was used as gvhd prophylaxis in msd and unrelated donor group and cyclophosphamide + tacrolimus + mycophenolate was used for haploidentical post-transplant. day survival, overall survival (os), incidence of gvhd and cmv reactivation was computed. results: a total of aml patients underwent hsct during the study period, the basic and clinical characteristics of the study patients are presented in table . conditioning regime did not have significant impact on os. survival at day was %. the os function and relapse free survival (rfs) function did not significantly differ between msd and haploidentical transplantation ( . % vs . %; p= . ) and ( . % vs . %; p= . ) (graph ). disease status at latest follow up showed that % were in remission and % had relapsed. overall one year survival and five year survival in the entire cohort was % and % respectively. the average cost of msd transplant at our centre is inr , , (€ - ), haploidentical transplant is inr , , (€ - ) and mud transplant is inr , , (€ , + for stem cell procurement). conclusions: our study showed comparable outcomes in msd and haploidentical transplant with respect to day survival, os, and rate of gvhd. in a developing country like india where patients are not covered under state health insurance, the additional cost of procurement of stem cells in a mud transplant would add to the financial burden to the patients. haploidentical transplant is a feasible option in case of non-availability of msd, due to ease of donor availability and strong motivation from the family donor to donate the stem cells. background: allogeneic stem cell transplantation (allo-hsct) is not indicated as consolidation of first complete remission (cr ) in favorable-risk acute myeloid leukemia (aml) bearing mutations in nucleophosmin (npm ) in the absence of flt internal tandem duplication (flt -itd). nevertheless, a substantial proportion of patients eventually proceed to allo-hsct beyond cr or for chemoresistant minimal residual disease (mrd) while in cr , which might compromise transplantation outcomes. the study aimed at examining the characteristics and results of allo-hsct in aml cases with mutated npm and wild-type flt (npm mut/flt wt), with special focus on molecular monitoring of mrd following transplantation. methods: from / until / , patients (women/men, / ) underwent allo-hsct for npm mut/ flt wt aml. at transplant, median age of patients was . years (range, - ) , and disease phase was cr (n= ), cr (n= ), or primary refractory (n= ). among the patients who were transplanted in cr and had available molecular mrd assessments, had detectable mutant npm transcripts by real-time quantitative pcr (rq-rcr). also, patients fulfilled criteria of molecular relapse (increasing levels of npm -mutated transcripts in two successive bone marrow samples), with mutant npm load of - , transcripts/ , abl transcripts). the conditioning regimen was myeloablative in the majority of cases (n= ) or reduced-intensity (n= ). the type of donor varied, namely hla-identical sibling (n= ), matched unrelated (n= ), haploidentical relative (n= ), or double umbilical cord blood (n= ). results: engraftment was achieved in all cases, with a median time to absolute neutrophil count > /ul of days (range, - ) . among the patients with posttransplant monitoring of mrd by rq-pcr, exhibited a stable molecular remission whereas a rising level of npm mutated transcripts was observed in cases due to either hematologic (n= ) or molecular (n= ) relapse of disease. the cumulative incidences (cin) of hematologic relapse and non-relapse mortality (nrm) were . % and % at months, respectively. no events of relapse or nrm were encountered beyond months from allo-hsct. out of patients with hematologic relapse post transplant, died of disease whereas one achieved a stable complete remission after withdrawal of immunosuppression. at a median follow-up time of months (range, - ), / patients continue to be alive in cr. the estimated disease-free background: cpx- (vyxeos®) is an advanced liposomal encapsulation of cytarabine/daunorubicin at a synergistic : molar ratio. cpx- is approved by the us fda and ema for the treatment of adults with newly diagnosed, therapy-related aml or aml with myelodysplasia-related changes. methods: safety data were pooled from studies of cpx- in adults aged - years with newly diagnosed or relapsed/refractory aml. cpx- induction consisted of units/m (cytarabine mg/m + daunorubicin mg/m ) on days , , and (second induction: days and ) . cpx- consolidation consisted of or units/m (varying by study) on days and . cpx- was evaluated against standard-of-care controls. results: baseline characteristics were generally balanced between cpx- (n= ) and controls (n= ); the majority of patients were aged ≥ years ( %; %) and had secondary aml ( %; %). controls included + (n= ) and salvage therapy with mitoxantrone/etoposide/ cytarabine (n= ), idarubicin/cytarabine (n= ), other cytarabine-based chemotherapy (n= ), and mitoxantrone/ etoposide (n= ). the treatment-emergent adverse event (teae) profile of cpx- units/m was comparable to induction controls, but associated with a greater proportion of patients with teaes, grade ≥ teaes, and serious teaes during consolidation (table) . therefore, the cpx- consolidation dose was reduced to units/m in latter studies; this dose demonstrated an improved teae profile similar to consolidation controls. the most frequent system organ class was gastrointestinal disorders for both cpx- and controls; a lower incidence was reported for cpx- ( %) versus controls ( %), with this difference driven by the lower incidence of diarrhea for cpx- ( %) versus controls ( %). the most frequently reported grade ≥ teaes were febrile neutropenia (cpx- : %; controls: %), pneumonia ( %; %), hypoxia ( %; %), and bacteremia ( %; %). early mortality rates, both overall and by treatment period, appeared lower with cpx- versus controls at day and day ( table) ; the majority of early deaths were attributable to teaes. conclusions: across the studies comprising the cpx- clinical development program, cpx- demonstrated a safety profile comparable to conventional chemotherapy in adults with newly diagnosed or relapsed/refractory aml. background: haploidentical hematopoietic stem cell transplantation (hsct) with post-transplantation cyclophosphamide (pgcy) marked improved clinical outcome. recent studies comparing allogeneic hsct using unrelated donors versus haplo donors in patients with acute leukemia have suggested equivalent outcomes. the depletion of tcells with pgcy was subsequently applied for unrelated hsct setting for patients with unrelated donor. methods: we performed a retrospective study on patients with acute leukemia in order to compare the outcome after hla haploidentical (n= ) and unrelated hsct (n= ) with pgcy. the main characteristics of patients were similar in both groups. baseline disease were: aml ( %) and all ( %) for haplo group and aml ( %) and all ( %) for unrelated group. disease state at time of haplo and unrelated-hsct were following: and patients in cr ( % and %) and and non cr ( % and %). for aml recipients mainly received thiotepa, busulfan and fludarabine and for all recipients received tbi and etoposide conditioning. all patients who received pbsc graft were treated with rabbit antithymocyte globulin (atg) on days - and - . results: at the time of analysis, the os and dfs did not differ between the haplo and unrelated groups ( % vs %, and % vs %). incidence of severe (grade [ ] [ ] acute gvhd was the same in two groups ( % versus %). recipients of haplo-hsct transplant were statistical significance less likely to experience disease relapse ( % vs %) and chronic gvhd ( % vs , %). however, gvhd free relapse free survival (grfs) rate was slightly higher after haplo-hsct ( % vs %). addition, cumulative incidence of trm rate was higher after haplo-hsct ( % vs %).for haplo and unrelated groups who underwent hsct in cr , the os were % and % versus % and % for those in non cr . for aml, the os was same in two groups (haplo % versus unrelated %). however patients with all, the os was higher in haplo group compared with unrelated group ( % versus %). the impact of pretransplant disease state have a more powerfull effect on survival in the haplo-hsct setting (for aml cr % versus non cr % and for all cr % versus non cr %). viral reactivations were significant concern in both groups. conclusions: our retrospective analysis suggests largerly similar os and dfs with haplo versus unrelated transplants with pgcy for acute leukemia. our data indicate that haplo-hsct results in a lower incidence relapse and of chronic gvhd and higher grfs compared with unrelated hsct. in addtion, the pretransplant disease state have the important effect on the outcomes in both groups. allo-pbsc with atg can be used safely and effective as graft source in haplo-hsct with acceptable post-transplant outcomes and replaced bm in this settings. more statistical data for transplant related characteristics will be provided at the presentation.we emphasize that use the same pgcy gvhd prophylaxis for all types of allogeneic transplant. based on our results, we recommend haplo-hsct with pgcy against unrelated transplant for patients with acute leukemia. disclosure: disclosure of conflict of interest: none. excellent efficacy and tolerability of inotuzumab ozogamicin in b-cell all relapsed after allo-hsct background: donor lymphocyte infusion (dli) could be used prophylactically to reduce relapse after allogeneic hematopoietic stem cell transplantation for very high-risk leukemia/lymphoma without effective targeted therapy. to compare the safety and efficacy of prophylactic dli for prevention of relapse after allogeneic peripheral blood stem cell transplantation from haploidentical donors (hid-sct) and matched-sibling donors (msd-sct) in patients with very high-risk acute myeloid leukemia (aml), we performed a retrospective, observational cohort study enrolled in hid-sct and msd-sct recipients. methods: the very high-risk features were defined as: (i) in the non-remission (nr) state prior to transplantation, including primary induction failure, relapse untreated or refractory to reinduction chemotherapy, or untreated aml evolution from mds; (ii) achieving complete remission with ≥ cycles of induction of chemotherapy; (iii) carrying tp , dnmt a, tet or flt -itd gene mutation. the scheduled time of the prophylactic dli was + - days after transplantation for msd-sct recipients and + - days for hid-sct recipients. the g-csfmobilized peripheral blood stem cells were infused to the recipient at a dose of × cd + cells/kg. csa was given at mg/kg b.i.d from day - to day + (hid-sct) or to day + (msd-sct), and then tapered at % per month to be discontinued on day + - (hid-sct) or on day + - (msd-sct) unless graft-versus-host disease (gvhd) developed. if the patients received dli before day + (hid-sct) or day + (msd-sct), csa was given weeks after dli in hid group and weeks in msd group at a though concentration of - ng/ml for dliassociated gvhd prophylaxis, and then tapered and discontinued within weeks unless gvhd developed. if gvhd occurred before the scheduled time of prophylactic dli, it would be delayed for weeks when gvhd was well controlled. results: prophylactic dli was administered at a median of ( - ) days for hid-sct recipients and ( - ) days for msd-sct recipients (p= . ), and both groups displayed similar baseline characteristics except for donor's gender distribution (table ) . grade - acute graft-versushost disease (gvhd) at -day post-dli was higher in hid-sct group than that in msd-sct group ( . % vs. . %, p= . ). grade - acute gvhd ( . % vs. . %), -year chronic gvhd ( . % vs. . %) and severe chronic gvhd ( . % vs. . %) were similar between two groups (p> . ). one-year non-relapse mortality was higher in hid-sct group than that in msd-sct group with marginal significance ( . % vs. . %, p= . ). one-year relapse rate was similar between hid-sct group and msd-sct group ( . % vs. . %, p> . ). estimated -year overall survival (os, . % vs. . %) and relapsefree survival (rfs, . % vs. . %) rates were both similar between hid-sct group and msd-sct group (p> . ). in multivariate analyses, non-remission status prior to transplant, poor-risk gene mutations and donor's age ≥ years predicted a higher risk of relapse after dli. nonremission status prior to transplant predicted inferior os and rfs. patient's age ≥ years also predicted an inferior os. conclusions: prophylactic dli after hid-sct demonstrated similar tolerance and efficacy for reducing relapse compared to that after msd-sct for very high-risk aml. disclosure: the authors declare no conflict of interest. prognostic impact of pre-transplant tim levels on transplant outcome in acute leukemia patients background: t cell immunoglobulin and mucin domaincontaining protein- (tim ), a negative regulator of t cells, is expressed on a variety of tumors including hematological malignancies like acute myeloid leukemia (aml) and some lymphoma types in which it was shown to be associated with an adverse prognosis. the aim of this study is to identify the prognostic impact of pre-transplant tim levels on early and late transplant related complications as well as post-transplant relapse and survival methods: a total of hematopoietic stem cell transplantation (hsct) recipients with an initial diagnosis of acute leukemia [median age: ( - ) years; male/ female: / ] were included in the study. aml was the initial diagnosis in patients ( . %), acute lymphoblastic leukemia (all) in patients ( . %), mixed phenotype acute leukemia in patients ( . %) and blastic plasmacytoid dendritic cell neoplasm in patient ( . %). soluble tim- levels in pre-transplant serum samples were measured with enzyme linked immunosorbent assay (elisa). results: median pre-transplant tim level was . ( . - . ) pg/ml in the whole cohort. pre-transplant tim levels were significantly higher in aml patients when compared to all [ . ( . - . ) vs . ( . - . ); p= . ]. tim levels were significantly lower in patients with abnormal cytogenetics when compared to normal karyotype (p= . ). cytogenetic abnormalities, including mainly a complex karyotype or chromosome abnormalities, were more frequent in patients with low tim levels (p= . ). pre-transplant tim levels were significantly higher in patients who developed post-transplant viral hemorrhagic cystitis (p= . ). a positive correlation was demonstrated between tim levels and acute graft versus host disease (gvhd) grade (p= . ; r= . ). at a median follow-up of . ( . - . ) months, overall survival (os) was found to be better in low-tim group when compared to high-tim group, without statistical significance (% . vs % . ; p> . ) ( figure ). probability of os was relatively better in both aml ( . % vs . %; p> . ) and all patients ( . % vs %; p> . ) representing low pretransplant tim levels in the subgroup analysis conclusions: in this study, elevated levels of pretransplant tim levels in aml patients were compatible with the previous reports which had underlined an increased tim expression on aml stem cells. the possible association of tim expression with cytogenetic features should be confirmed with further studies as there is no adequate data except its relationship with flt -itd mutational status. tim- is also expressed on exhausted t cells in patients with viral infections, including human immunodeficiency virus, hepatitis b and hepatitis c virus. it plays an essential role in the regulation of antiviral and antitumor immune responses which may be an explanation for the increased frequency of hemorrhagic cystitis in patients with higher tim- levels. the adverse prognostic impact of tim on gvhd and os was confirmed without statistical significance which may be related to small sample size. as tim has a wide spectrum of action in the tumor microenvironment including stimulatory and inhibitory activities, further clues are required to define the exact role of this molecule in the clinical course of allogeneic hsct in order to develop targeted therapeutic strategies clinical trial registry: n/a disclosure: nothing to declare p homozygous hla-c is associated with increased risk of relapse after hla-matched transplantation in recipients with acute lymphoid leukemia: a japanese national registry study background: after hematopoietic stem cell transplantation (hsct), the role of natural killer (nk) cells which express killer-cells immunoglobulin-like receptors (kirs) and recognize hla-class ligands is important. kir dl recognizes not hla-c asp (c ), but hla-c lys (c ) and has polymorphism based on the th amino acid of the transmembrane domain. low frequency of c and high frequency of strong kir dl are characteristics observed in japanese. by using large transplant database, we reported that homozygous hla-c (c /c ) recipients displayed lower relapse rates than did c /c recipients after hla-matched hsct for acute myeloid leukemia (aml; hr = . , p = . ) or chronic myeloid leukemia (cml; hr = . , p = . ). this effect seemed to be independent of acute graft-versus-host disease (agvhd) or cytomegalovirus reactivation occurrence (arima n et al bbmt ) . methods: relapse rates of japanese recipients who first underwent hla-matched hsct between and for the treatment of acute lymphoid leukemia (all) were compared between c /c pairs and c /c pairs, using data from japanese data center for hematopoietic cell transplantation and adjusting for transplant characteristics. cord blood transplantation was excluded. multivariable competing risk regression analyses were performed to evaluate relapses and relapse-free survival (rfs) was estimated using kaplan-meier method. results: after recipients who did not achieve remission or experienced graft failure and recipients not-expressing c were excluded, resting recipients aged - years (median, . years) were analyzed. the median follow-up period for survivors was . years. there were recipients expressing c /c and recipients expressing c /c , respectively. after hla-matched hsct, c /c recipients had higher relapse rates than c / c recipients (hr = . , p = . ), resulting in worse rfs among c /c recipients (hr = . , p = . ). the frequent relapse in c /c recipients than in c /c was noticeable among recipients with agvhd (hr = . , p = . ), those without cytomegalovirus reactivation (hr = . , p = . ), and those with ph-negative all (hr = . , p = . ). conclusions: kir dl -positive nk cells may promote graft-versus-leukemia (gvl) in c /c recipients with aml or cml but suppress gvl in c /c recipients with all. one interpretation is that transplant-activated nk cells impair antigen-presenting cells or deprive cytotoxic tlymphocytes of their gvl effects on all cells. this hypothesis may be explained by the fact that agvhd was necessary for the recessive relapse in c /c recipients with all. furthermore, ph-positive all cells sometimes mimic aml cells in terms of their frequent myeloid antigen expression and might be directly targeted by nk cells. it would be necessary to further clarify in vitro the character of nk cell-affecting in the transplant immunity against residual leukemia cells. disclosure: authors have nothing to declare. hematopoietic stem cell transplantation with sequential conditioning for children with relapsed/refractory acute leukemia nao yoshida , kazuki matsumoto , daiki yamashita , yiqing zhu , daichi sajiki , ryo maemura , hirotoshi sakaguchi , asahito hama children's medical center, japanese red cross nagoya first hospital, nagoya, japan background: patients with acute leukemia who fail to achieve complete remission show a dismal prognosis even with allogeneic hematopoietic stem cell transplantation (hsct). this study evaluated whether sequential conditioning approach that is cytoreductive chemotherapy applied shortly prior to the main conditioning followed by hsct can improve prognosis in such high-risk patients. methods: we retrospectively analyzed the outcomes of children (median , range - years old) with primary refractory (n = ) or refractory relapsed (n = ) acute leukemia (aml n = , all n = ) who received hsct in our department between and . the stem cell source was related peripheral blood (pb) in patients, related bone marrow in , unrelated bone marrow in , or unrelated cord blood in . the grafts were hla serologically matched (n = ) or mismatched (n = ) with the recipient. in total, patients received the sequential conditioning approach. as cytoreductive chemotherapy, fludarabine/cytarabine/idarubicin/g-csf (flag-ida) was used in patients, mitoxantrone or daunorubicin/cytarabin in , or other regimens in , and of them were combined with gemtuzumab ozogamicin. without waiting for hematological recovery, the patients promptly underwent hsct; therefore, the median interval between cytoreductive chemotherapy and main conditioning was days. the main conditioning regimens were total body irradiation-based myeloablative (n = ), busulfan-based myeloablative (n = ), or reduced intensity (n = ). results: in children with relapsed/refractory acute leukemia, the -year overall survival (os), leukemia-free survival (lfs), cumulative incidence of relapse (ri), and transplantation-related mortality (trm) were %, %, %, and %, respectively. in multivariate analysis, the use of sequential conditioning was identified as the most favorable factor for lfs (hazard ratio [hr] . ; p = . ), although there were no differences in the outcomes according to the types of cytoreductive chemotherapy or the main conditioning regimen. hla-matched donor (hr . ; p = . ) and pb blasts-negative at the beginning of conditioning (hr . ; p = . ) were also independently associated with better lfs. with sequential conditioning, leukemia burden prior to the hsct was significantly reduced; pb blasts became undetectable at the beginning of conditioning in % patients given the approach, while in % patients without the approach (p = . ). notably, the outcomes in the patients without pb blasts at the beginning of conditioning who received sequential conditioning were promising; the -year os and lfs reached % and % and the -year ri and trm were % and %, respectively. conclusions: our study reveals that hsct with sequential conditioning can be an effective and tolerable treatment option for children with relapsed/refractory acute leukemia. the treatment strategies that focus on the reduction of leukemia burden immediately prior to hsct may contribute to the induction of long-term remissions in patients with high-risk acute leukemia. disclosure: this research was funded by japanese red cross, nagoya st. hospital research grant nfrch - . use of blinatumomab to achieve remission and consolidation with haploidentical transplant with cyclophosphamide post for the treatment of children with refractory acute lymphoblastic leukemia (all) background: most of patients with all in relapse or refractory to conventional treatment have only % possibilities to achieve long term remission. this report refers to the therapeutic efficacy and adverse events from the blinatumomab to achieve molecular remission in patients with pre-b cd + which lead to haploidentical with cyclophosphamide post transplant as a consolidation. methods: a pilot study was conducted in children with refractory all preb-cd +. as a strategy to achieved remission blinatumomab was used at a dose μg/m for continous infusion of hours, increasing the dose to μg/m during days, patients with a mrd of < . log, after cycles received an haploidentical bone marrow transplant as a consolidation, the conditioning regimen was with total body irradiation scheme at cgy/day/ days, cyclophosphamide and etoposide. receiving prophylaxis for gvhd with cyclophosphamide. results: a total of patients were included, seven of them achieved complete remission after cycles of blinatumomab, one with partial remission (table ) , these seven patients, six received an haploidentic transplant achieving graft in of the transplanted patients. one patient had a bone marrow relapse in the first months of the follow-up and patients are free of disease with a follow-up to months (figure ). as a acute complication the patients presented cytokine release syndrome, during the infusion of blinatumumab patients presented tachycardia (table ) and the patients presented agvhd after hsct ( grade i-ii and i grade iv). conclusions: allogeneic bone marrow transplant constitutes a treatment option on those patients that relapse or become refractory to treatment, one of the major problem is basically to identify a hla-identical donor, the alternative is an haploidentical donor. the most important factor to get these results is the disease status before transplant. the use of blinatumomab has proven to be effective in achieving remission in relapse acute linfoblastic leukemia pre-b cd + or refractory to treatment. characteristics nº % male % median age at diagnosis, (range), years . ( - ) status of disease o + relapse % refractory to primary or salvage therapy % complete remission after blinatumomab % partial remission after blinatumomab % active disease % [[p table] . table n° . demographic characteristics of patients undergoing blinatumomab (n= )] disclosure: a. olaya-vargas, r. rivera-luna, y. melchor-vidal, h. salazar-rosales, g. lopez-hernandez, n. ramirez-uribe. we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. sequential high-dose chemotherapy reinduction followed by myeloablative allogeneic transplant for active acute myeloid leukemias methods: at our center, relapsed/refractory aml patients were transplanted during chemo-induced neutropenia after high-dose salvage chemotherapy. median age at transplant was years (range - ). patients suffered from de novo (n= / , %) or secondary aml (n= / , %). genetic risk stratification was reported using stardardized groups proposed by the european leukemia net (eln) in . favorable, intermediate i and ii and adverse risk category at diagnosis was observed in / ( %), / ( %), / patients ( %) respectively. all patients had active disease at the time of sequential therapy and median marrow blast count was % (range - %). patients received a high-dose cytarabine based (mec in / , %) regimen as salvage therapy. donors were haploidentical relatives for / ( %) patients, identical siblings and matched-unrelated for / patients ( %) and / ( %), respectively. a myeloablative conditioning was used to further implement anti-leukemic effects. conditioning, thiotepa-busulfan-fludarabine in % patients, was started at a median of days (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] after the last day of chemotherapy. bone marrow and peripheral blood stem cells were used as graft source in / ( %) and / ( %) patients. graft-versus-host disease (gvhd) prophylaxis and supportive care were administered accordingly to each hsct platform. results: all patients engrafted. median day of neutrophil recovery was day + (range - ). median follow-up of survivors was months (range - ). non relapse mortality and relapse incidences (nrm, ri) were % and % at year and % and % at years, respectively. overall cumulative incidences of acute and chronic gvhd were % and % at day + and + . one and year overall survival (os) were % and %, while and year event-free survival (efs) were % and %. significant better os and efs were observed in patients with favorable-intermediate i-ii versus adverse risk score ( - years os % and % vs % and % p= . ; - years efs % and % vs % and % p= . ). adverse risk had a significant impact on os (hr . , p= . ) and efs (hr . , p= . ) by univariate analysis and on ri (sdhr . , p= . ) by fine and gray test. conclusions: though small the patient cohort, our findings suggest that sequential therapy with a myeloablative hsct is feasible in treating relapsed/refractory aml. transplant-related toxicity was low ( %) and relapse was the major treatment-failure. however, even with this approach, patients with adverse cytogenetic features have a very dismal prognosis. for these patients, the use of new drugs before hsct and/or maintenance therapy after transplant is highly encouraged to improve outcomes. disclosure: alessandro busca: honoraria from gilead sciences, merck, pfizer pharmaceuticals and jazz background: in spite of satisfactory results of overall survival (os) after allohsct in st and nd cr aml, relapse free survival (rfs) and graft-versus-host-disease free/relapse free survival (grfs) require further improvement. the detection of mrd is one of the factors which influence on the outcome of allohsct in aml is unclear but identification is important to improve risk-adapted relapse prophylactic treatment after allohsct. aim. to evaluate outcomes of allohcst in st and nd cr pediatric aml depending on the level of mrd status before myeloablative (mac) or reduced intensity conditioning regimens (ric). methods: the data of children with aml in st and nd cr underwent allohsct between and were analyzed. median age at the moment of allohcst was years old ( - ). mrd negative status had ( %) patients, ( %) were mrd positive by flow cytometry. mac based on busulfan ( mg/b.w.) received ( %) patients, on treosulfan - ( %) patients. ric based on melphalan received ( %) patients, based on busulfan ( mg/b.w.) - ( %) patients. patients received prophylaxis of agvhd by atg ( %) or ptcy - ( %) patients plus csa - ( %) or tacrolimus ± sirolimus - ( %) patients that depended on source of transplant (related, unrelated or haplo donor) . results: at the median follow up years in the cohort of mrd positive patients os is % vs % in mrd negative (p> , ). rfs is % vs % accordingly (p= , ). graft-versus-host-disease free/relapse free survival (grfs) in mrd positive patients is % vs % in mrd negative (p> , ). os, rfs, grfs in mrd positive patients after mac is %, %, % vs %, %, % after ric accordingly (p> , ). os, rfs, grfs in mrd negative patients after mac is %, %, % vs %, %, % after ric accordingly (p> , ). os, rfs in mrd negative patients with/without ptcy is %, % vs %, % (p> , ); grfs is % vs % accordingly (p= , ). os, rfs, grfs in mrd positive patients with/without ptcy is %, %, % vs %, %, % (p> , ). conclusions: mrd status does not statistically significant affect on os that can be related to different approaches to the treatment of relapse after allohsct. mrd positive status statistically significant decreases rfs that underline the necessity of posttransplant therapy improvement. ric vs mac in all patients in first and second remission do not show statistically significant impact on os, rfs, grfs. ptcy significantly improves grfs in mrd negative patients. disclosure: none of the authors has anything to disclose. background: with increasing overall-survival (os) of lymphoma patients, higher incidences of therapy-related clonal bone marrow diseases, such as acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) are occuring. generally, the outcome is considered poor. allogeneic hematopoietic stem cell transplantation (allo hsct) often remains the only potentially curative treatment option. nonetheless, there is only little data available concerning this patient group. methods: we retrospectively collected data from patients with therapy-related aml (taml) and mds (tmds) after treatment for hodgkin's lymphoma (hl; n= and n= ) or non-hodgkin's lymphoma (nhl; n= and n= ), who received an allo hsct between and . median follow-up of surviving patients was . years (range . months- . years). background: the prognosis of relapsed/refractory acute leukemia (r/r al) is poor and the treatment is challenging. in this setting, allogeneic stem cell transplantation (allo-sct) constitutes the only curative option although the high relapse rate and non-relapse mortality (nrm). the sequential conditioning regimen followed by allo-sct has been used for persistent disease and aims to improve disease control by intensified chemotherapy, thus conceding more time for the presumed graft-versus-leukemia effect to occur. methods: the clinical outcome of r/r al with the sequential conditioning regimen combining a chemotherapy rescue followed by ric allo-sct in our center is described. patients who underwent a sequential allo-sct from to are included. the primary endpoint was progression free survival (pfs) and overall survival (os) that were estimated by the kaplan-meier method. secondary endpoints were non-relapse mortality (nrm). background: recommendations of the european leukemia net (eln) for favorable-risk genetics (frg) acute myeloid leukemia (aml) favor consolidation over transplantation, although reviews suggest advantage of autologous stem cell transplant (asct) in event free survival. our objective was to compare the progression free survival (pfs) and overall survival (os) of normal karyotype npm mutated without flt itd or allelic ratio < . (npm +) aml patients treated with consolidation chemotherapy alone (cc), asct or allogeneic stem cell transplant (allosct). methods: retrospective review of npm + frg-aml patients, treated in one institution ( to ) with the following induction regimens: cytarabine (ara-c) and vp- with daunorubicin (ade) or mitoxantrone (mice). consolidation regimens were ara-c with daunorubicin (ac-d), idarubicin, vp- and ara-c (mini-ice) or highdose ara-c (hidac). in asct, conditioning regimens were bucy or bvac and in allosct were bucy or flubu. pfs and os were calculated from the start of the last consolidation or stem cell infusion. results: a total of patients were evaluated, with a median age of years (y) ( - y), % female, % with ecog performance status (ps) - and % with ageadjusted charlson comorbidity index (aacii) ≥ at diagnosis. patients were treated with cc in % (n= ), asct in % (n= ) and allosct in % (n= ) of cases. there were no differences between groups for age, aacii, ps, leucocytes at diagnosis or extra-medullary disease. flt -itd was more frequent in allosct group ( %) than cc ( %) or asct ( %; p= . ). at induction, ade was used in % and mice in % of patients, with a complete remission (cr) rate of %. there were no differences between groups for induction regimen or cr. in cc group, consolidation regimens were cycle ( %) and cycles ac-d ( %) and cycles mini-ice ( %). asct patients received consolidation with - cycles ac-d ( %) and cycle mini-ice ( %), while allosct patients received - cycles ac-d ( %), cycles hidac ( %) and no consolidation in %. [[p image] . figure . pfs at y in cc, asct and allosct groups.] median follow-up was months, pfs at y was % and os at y was %. pfs at y for asct group was superior then cc and allosct groups ( %, % and %, respectively; figure ), although not statistically significant. os at y was statistically similar between groups, although inferior in allosct comparing to cc and asct ( %, % and %, respectively). conclusions: in this historical cohort review, although there was no advantage in os for asct in npm + frg aml, our data suggests that there might be a pfs improvement in asct over cc, which needs to be further addressed in prospective studies. disclosure: nothing to declare background: acute myeloid leukemia is a hematological malignant disease that motivates the persistent struggle in the scientific world to provide effective cure that can establish acceptable survival rates in this group of patients. autologous stem cell transplantation with myeloablative conditioning is still a powerful weapon that can be used against this entity methods: we have evaluated retrospectively patients with aml where autologous stem cell transplantation was performed in the period from till . our group consisted of patients; male patients ( . %), female patients ( . %). median age at diagnosis was years ( - ). the average period from time of diagnosis to autologous sct was . months. results: in the majority of our group, we used myeloablative conditioning regimen with busulphan-cyclophosphamide, patients ( . %), in patients ( . %) we have added melphalan to bu-cy conditioning, in ( . %) patients we used beam conditioning and in the rest, patients ( . %) we used bam conditioning regimen. as auto graft we used peripheral blood stem cells (pbsc) in patients ( . %), and in patients ( . %) we used bone marrow. the main mobilising regimen for pbsc was g-csf + etoposide and it was performed in patients ( . %), and in the remaining patients ( . %) mobilising of pbsc was performed only with g-csf. the mean number od apheresis procedures done in our group was . , and the mean number of collected mononuclear cells was . x /kg tt. the mean time to engraftment was . days ( - ). the transplant related mortality (trm) was . %. the year overall survival of our patients was . patients. the main reason for death was relapse of the primary disease( %). patients ( %)were treated with salvage chemotherapy regimen (flag-ida) because with the standard induction regimen + there was absence of adequate therapeutic response, or predominantly no complete remission was achieved. all patients were transplanted in complete remission conclusions: autologous stem cell transplantation could be an acceptable therapeutic solution for patients with aml as a consolidation therapy, where neither suitable compatible donor is available nor allogeneic stem cell transplantation could not be performed from various reasons depending on the bone marrow transplant unit disclosure: nothing to declare p prophylaxis dli alone may not prevent relapse of flt -itd positive aml after allogeneic hct background: one of the most potent prognostic factors affecting outcomes in aml is the presence of cytogenetic and molecular markers which can guide the selection of post-remission therapies. recently, favorable outcomes of npm wt /flt -itd neg /non-cebpa dm group after allogeneic hematopoietic cell transplantation (allo-hct) have been reported, that is similar to those of favorable risk by the eln risk classification. however, the role of allo-hct compared to consolidation chemotherapy has not yet been elucidated. methods: the data of patients who were diagnosed with aml and received intensive induction therapy from march to july were included in the current study. to address the time dependence of the allo-hct, the simon and makuch method was used in the graphical representation and the mantel-byar test and andersen and gill methods for identifying risk factors for long-term survival. results: median age of the patients were years (range - ), and patients ( %) were male. npm mutation was detected in patients ( %), and flt -itd were none, low, and high ratio in patients ( %), ( %), and ( %), respectively. the eln risk classification divided the patients into favorable, intermediate, and adverse risk group in patients ( %), ( %), and ( %), respectively. npn and flt -itd both negative group included patients ( %). allo-hct was performed in patients ( %). overall, complete response (cr) after induction therapy achieved in patients ( %), and patients ( %) were primary refractory disease. cr rates did not differ between npm wt /flt -itd negative group (n= / , . %) and other intermediate risk group (n= / , . %; p= . ) . with median follow-up duration of . months (range . - . months), one-year os rate were %, . ± . %, . ± . % in favorable, intermediate, and adverse risk group (p < . ). among intermediate risk group, os rate of npm wt /flt -itd negative group was similar to other intermediate risk (p= . ). allo-hct was performed in patients of npm wt /flt -itd negative group. one-year os rates did not differ between npm wt /flt -itd negative and other for allogenic hematopoietic stem cell transplant (allo-hsct), as a strategy to prolong survival. methods: data from aml pts over years, who underwent ric allo-hsct in our institution between september and september , was retrospectively collected from clinical files to evaluate the overall survival (os) up to november . we calculated the os using kaplan-meyer curves. results: we identified pts, median age y.o. ( - ) and median htc-i score . the median follow-up was months. one patient (pt) had cml blast crisis and was on first major molecular remission. of the remaining aml pts, were in st complete remission (cr), in nd cr and with progressive disease (pd); the other pts could be classified as mds according to who diagnostic criteria and were in cr . donors (d) were: matched unrelated (mud), mismatched unrelated (mmud - / ), matched siblings and haploidentical. thirteen pts were infused with peripheral blood hsc and with bone marrow. conditionings were: flubcnumel in unrelated donor (ud) pts and siblings, flumel in ud pt and sibling, flubu in ud pts and flutbi gy in sibling and in the haploidentical. graft versus host disease (gvhd) prophylaxis was tacrolimus (tac) + mmf in ud pts and sibling, tac + mtx in ud pts and cyclosporine (cya) + mmf in ud pt and siblings. all mmud pts had atg. ptcy was done in the haploidentical setting with tac + mmf. the median time to neutrophil and platelet engraftment for the whole cohort was and days, respectively. one pt with secondary engraftment failure required re-infusion of selected cd + cells. ten pts presented with mild acute skin gvhd. eleven pts had chronic gvhd, classified as severe; required systemic therapy, of those beyond year. the median time on immunosuppressants was days. at years the os was . %. there were deaths: disease-related ( relapses at and months and pd at d+ ), infection complications ( septic shock) and to secondary neoplasia. other relevant complications were hypoxemic pneumonia in pts, urinary sepsis, cmv and ebv reactivation respectively in and pts; pulmonary and renal toxicity either in pts. at end of follow-up, pts were in remission, without negative measurable residual disease (mrd), the other mrd negative pt died of septic shock and severe intestinal gvhd. conclusions: in this small cohort, chronic gvhd and infectious complications were major causes of morbidity but there were no treatment related deaths before d+ . pts maintaining or achieving mrd negativity after transplant had better survival. although with only pts, these results suggest that allo-hsct is feasible as consolidation strategy in selected aml pts over years. [[p image] . overall survival] disclosure: nothing to declare. background: hematopoietic stem cell transplantation (hsct) is the only curative option for fanconi anaemia (fa); an inherited disorder characterized by congenital anomalies, progressive bone marrow failure (bmf) and a predisposition to develop malignancies. methods: we retrospectively analysed the data of consecutive patients that underwent hsct at this centre from till june . the data was analysed for variables affecting the outcome in terms of overall survival (os). results: median age at diagnosis was years ( - years). median age at transplant was . years ( - yrs). all patients at transplant were in aplastic phase. male to female ratio was . : . twenty-four ( . %) patients had congenital anomalies along with bmf while were phenotypically normal. twenty-three ( . %) patients were / hla matched with siblings, with parents and with cousins. eleven ( . %) patients had gender mismatch transplant. three patients had major and had minor abo mismatch. background: paroxysmal nocturnal hemoglobinuria (pnh) is a rare clonal non-neoplastic hematopoietic stem cell disease whose incidence is . - . cases/million of individuals worldwide. disease characteristics and natural history have been mostly analyzed by multicenter, retrospective studies, with the limit of heterogeneous approaches. herein we report the incidence of severe complications and outcome in a real life setting scenario of pnh patients consecutively diagnosed and managed at our pnh referral center between january and june . methods: patients received a homogeneous diagnostic and treatment approach according to the period of observation (availability of diagnostic tests and eculizumab). all patients treated with eculizumab received vaccination with conjugated anti-meningococcus acwyserotypes and, since , conjugated anti-meningococcus b-serotype. in the event of any complication, patients could refer to dedicated hematology emergency rooms (er) hours daily. the occurrence of renal failure and pulmonary hypertension was specifically evaluated. the renal function was studied according to the cockcroft-gault formula and the lung function was prospectively monitored by daytime-on exertion, nocturnal pulsoximetric profiles and complete spirometric tests, with dlco measurement. results: overall, pnh patients, median age years (range - ), were analyzed. at diagnosis, patients had classic pnh, aplastic pnh and an intermediate form. the cumulative incidences (ci) of thrombosis, and clonal hematologic neoplasm were %, and %, respectively. ci of pancytopenia in the patients with classic pnh was %. one patient showed a spontaneous disappearance of the pnh clone. since , eculizumab was administered in patients. after eculizumab treatment % and % of patients reached hemoglobin level > g/dl and > < g/ dl without transfusion, respectively, while % were nonresponsive. during eculizumab treatment no thrombotic event was observed while two severe infectious episodes (respiratory tract and urinary tract infection) were observed in only one of the patients. extravascular hemolysis was demonstrated in % of patients. no patient showed a significant reduction of the renal function.out of patients prospectively monitored for lung function no pathological alteration in any diurnal or nocturnal pulseoximetric test was observed. no patient showed obstructive or restrictive ventilatory deficiency, nor reduced dlco values. -years overall survival (os) was % ( patients who died for non-pnh related reasons were censored at the last follow-up).a better os, even if not statistically significant,was associated to the absence of thrombotic events ( %vs %), and the period of diagnosis ( % in - , % in - , % in - ) . conclusions: our study reports a better os and lower rate of severe complications in pnh compared to previous experiences. although renal failure and lung hypertension have been reported by other groups, we did not observe these complications along a prolonged follow-up. we can assume that the availability of a dedicated er service enabled an early diagnosis and prompt treatment in case of hemoglobinuria crises (reducing the risk or organ damage) or other complications. the use of eculizumab, together with improved supportive approaches, presumably accounts for the trend towards a better survival witnessed over the last decade in the management of pnh patients. disclosure: nothing to declare haploidentical and unrelated allogeneic stem cell transplantation in aplastic anemia:single center experience zafer gulbas , elif birtas atesoglu , meral sengezer , İmran dora , cigdem eren , suat celik , demet cekdemir background: aplastic anemia is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. allogeneic stem cell transplantation from hlamatched sibling is performed in the firstline setting in young aplastic anemia patients and in elderly patients who are refractory to immunosuppressive treatment. but if the patient does not have a hla-matched sibling, allogeneic stem cell transplantation is performed from unrelated and haploidentical donors. in this study, we analyzaed and compared the results of aplastic anemia patients who had undergone allogeneic stem cell transplantation either from matched unrelated or haploidentical donors. methods: we collected and analyzed data of aplastic anemia patients who had undergone allogeneic stem cell transplantation from matched unrelated or haploidentical donor between and . results: there were patients who had received allogeneic stem cell transplantation from unrelated donors and there were patients who had undergone haploidentical transplantation. but in patients who had undergone haploidentical transplantation, engraftment failure had occurred and they were transplanted from different haploidentical donors fort he second time. so a total of unrelated and haploidentical transplants were performed. the median age of patients who had undergone unrelated transplantation was ( - ) and the median age of patients who had undergone unrelated transplantation was ( - ) . the results of the haploidentical and unrelated transplantations are shown in table . the neutrophil and platelet engraftment times were significantly longer in haploidentical transplantations (p= , and p= , , respectively). however, vod, gvhd and day mortality rates were not different in the groups. similarly overall survival (os) of the patients who had undergone haploidentical or unrelated transplantation were not significantly different (p= , ) ( figure ) . conclusions: although the number of patients are low in this study, we can conclude that urelated and haploidentical transplantation in aplastic anemia have comparable toxicity and efficacy. background: autologous stem cells transplantation (ahsct) is an effective treatment for very aggressive autoimmune diseases such as multiple sclerosis (ms). however, toxicity remains the major concern to a wide application of this approach. post transplant viral reactivations may induce severe complications and a rigorous monitoring of peripheral blood viral load for a prompt and effective therapy is required. a higher rate of ebv and cmv reactivation has been observed in patients affected by ms and conditioned with beam plus atg compared with a controlled group of lymphoma patients without atg in the conditioning regimen [ ] . we report here the policy of our center about both monitoring and treatment of such side effect. methods: a series of consecutive patients with ms, transplanted between and is included in this analysis. all patients were mobilized with cyclophosphamide g/sqm + g-csf and conditioned with beam plus rabbit atg (thymoglobulin©, . mg/kg). monitoring of cmv/ebv dna on whole blood by quantitative pcr was performed after the engraftment, weekly for at least one month, then at longer intervals. pre-emptive treatment with valgancyclovir was started in case of cmv viral load ³ x ^ copies/ml. in case of ebv assay between x ^ and x ^ copies/ml further determinations were performed and rituximab-based treatment was started if the viral copies exceeded x copies. patients received treatment in case of symptomatic disease for any value of the pcr of both viruses or ebv titer ³ x ^ copies/ml. results: detectable dna for cmv was observed in / ( , %) patients at a median time from transplant of days (range - ) and / ( %) required pre-emptive treatment. all patients promptly responded to treatment within weeks. ebv viral load was detectable in / patients ( , %) at a median time of days (range - ). one patient out of started the treatment on first determination for high viral load (> x ^ /ml); nine presented an ebv viral load over x ^ copies/ml, three of them were treated thereafter for the persistent increase of the viral load (> /ml). six patients spontaneously recovered the ebv reactivation. previous treatments were not predictive of any higher risk of viral reactivation. no impact on engraftment related to the reactivation was observed. conclusions: this policy shows that, despite a high rate of cmv and ebv reactivation, no grade iii-iv adverse events were observed, suggesting the key role of viral monitoring in these patients and the efficacy of the preemptive treatment. ebv reactivation at low titers should be monitored to identify those cases that could achieve a spontaneous resolution and avoid the induction of further immunosuppression by rituximab. these data confirm that patients diagnosed with ad undergoing autologous hsct need a more intense pattern of care than hematological patients. background: autoimmune diseases are chronic serious conditions that are often refractory to standard therapies. since , autologous haematopoietic stem cell transplantation (hsct) has been a very promising alternative that has shown satisfactory long-term results. the aim of this study is to evaluate immune reconstitution and mortality following hsct in patients with autoimmune disease. methods: a retrospective study was conducted on patients with diagnosis of autoimmune diseases that underwent autologous hsct between july and january at a tertiary referral center in colombia, south america. descriptive statistics were used to analyze patient's demographic and clinic characteristics. results: seven patients were included, with a mean age of years (range - ). five patients were female ( %). the indications for hsct were systemic sclerosis (n= ) , multiple sclerosis (n= ), and myasthenia gravis (n= ). the conditioning regimen administered in patients with systemic sclerosis was cyclophosphamide + human anti-t lymphocyte immunoglobulin, beam (carmustine, etoposide, cytarabine (ara-c), and melphalan) + human anti-t lymphocyte immunoglobulin in patients with multiple sclerosis, and cyclophosphamide + human anti-t lymphocyte immunoglobulin in myasthenia gravis. median time to myeloid engraftment (neutrophils> . × /l) was days post-transplantation, and platelet engraftment, defined as > , platelets/mm untransfused, was days post-hsct. median time of hospitalization was days (range - ); longer in-patient management was due to infectious complications. infectious complications included bacteremia caused by e. coli and pneumocystis pneumonia that resulted in septic shock and acute respiratory failure, respectively. evaluating t-cell immune reconstitution, none of the patients had reached normal cd + cell value after one year of hsct follow-up. four patients ( . %) reached normal cd + cells value at months post-hsct. regarding bcell immune reconstitution, . % of the patients ( / ) had reached both igg and iga normal levels at one-month post-hsct, and four patients ( . %) had achieved normal igm background: multiple sclerosis(ms) is an inflammatory disease caused by autoimmune reactivity of t cells against myelin. there is accumulating evidence of the efficacy of highdose chemotherapy followed by autologous haematopoietic stem cell transplantation(ahsct) in ms patients who failed response to standard immunotherapy, despite a variability in eligibility criteria, conditioning regimens and outcome. methods: we retrospectively reviewed ms patients submitted to ahsct in our centre ( ) ( ) ( ) ( ) ( ) . patient eligibility criteria were active relapsing remitting(rrms) or secondary-progressive ms (spms), with prior failure to treatment with disease-modifying therapies and evidence of disease activity (clinical relapse or new active lesions in magnetic resonance [mr] ). mobilization of cd +cells to peripheral blood was performed with granulocyte colony-stimulating factor(g-csf μg/kg/day) and conditioning regimen according to beam protocol. the severity of ms disability was classified according to the expanded disability-status scale (edss) and ahsct toxicity was evaluated by ctcaev . . results: seven ms patients had undergone ahsct ( female/ male), with a median age at ahsct of . years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . median age at ms diagnosis was . years( - ) and median time from diagnosis until ahsct was . years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . four patients ( . %) had spms and ( . %) had rrms, with ( . %) having mr active lesions. pre-ahsct relapse rate per year was ( ) ( ) ( ) ( ) . median baseline edss was . ( . - . ). median number of previous dmts was ( ) ( ) ( ) ( ) ( ) ( ) ( ) . all patients had been treated with corticosteroids and copaxone, ( . %) received rituximab, ( . %) natalizumab, ( . %) alemtuzumab, ( . %) fampridine and ( . %) fingolimod. all patients collected enough cd + cells in a single apheresis session. median number of cd + cells infused was . ± . x ^ /kg, for a mean dmso volume of . ± . ml. median inpatient stay during ahsct was days( - ). all patients developed febrile neutropenia, one was admitted to intensive care unit due to sepsis. one patient developed an anaphylactic reaction to transfusion and another a self-limited encephalopathy. two ( . %) patients had grade≥ oral mucositis, and all had gastrointestinal toxicity (grade - ). median time until neutrophils> /μl was ( - ) days, to platelets> , /μl was days( - ), and to engraftment was days ( ) ( ) ( ) . patients were transfused with a median of ( - ) unit for erythrocytes and ( - ) for platelets. there was no treatment-related mortality and no long term side effects have been observed so far. for a median post-ahsct follow-up of . months, no patient developed new lesions in mr, but ( . %) experienced symptoms consistent with a clinical relapse, at a median time of . ( - ) months, effectively rescued with corticosteroids. the absence of evidence of disease activity at -months was . %. although there was no variation concerning edss punctuation, ( . %) patients self-reported significant benefits, especially concerning limb strength and sphincter continence improving. conclusions: our real life results claim a stabilization effect of ms patients with highly active/progressive disease after ahsct, with no significant toxicity. the failure in reporting benefits in edss punctuation is probably due to a small sample size and short follow-up. more studies are needed to establish the best patient selection criteria and define the ideal time to include these patients in transplantation programs, as well as to evaluate its long term outcome. disclosure: nothing to declare. elena poponina , elena butina , anna yovdiy , galina zaytseva , natalia minaeva , igor paramonov background: reactivation of epstein-barr virus (ebv) represents a potentially life-threatening condition in approximately % of patients after allogeneic stem cell transplantation, with no specific treatment available. methods: we have previously developed a manufacturing protocol for the expansion of cytomegalovirus (cmv) and ebv-specific t cells by stimulation of g-csfmobilized stem cell grafts with defined peptide pools (gary, ) . this advanced therapeutic medicinal product is currently investigated in an ongoing phase i/iia trial (eudract number: - - ) . however, the expansion of virus-specific t cells relies on a pre-existing virusspecific memory compartment in the stem cell donor. in virus-seronegative donors, no expansion can be achieved. we therefore aim to identify ebv peptide-specific t cell receptors (tcrs) that can be translated into off-the-shelf cell products for the treatment and prophylaxis of ebv infection and ebv-associated malignancies. leftover cells from five allogeneic stem cell grafts were expanded in vitro in the presence of hla-b * -restricted peptides (hpvgeadyfey from ebna , eplpqgql-tay from bzlf ) associated with latent and lytic ebv infection. after expansion, single ebv-specific t cells were facs sorted using peptide-mhc (pmhc) tetramers, and individual tcr αand β-chain pairs were determined with single cell sequencing (han , penter . to confirm peptide specificity, dominant tcr pairs were transfected into a murine αβreporter t hybridoma cell line with nfat-driven gfp expression (siewert, ) . functional tcrαβ candidates were transduced into human peripheral background: lymphoid and myeloid acute leukemia are the most frequent cause of cancer related death in children. interactions between nkg d receptor, expressed in cytotoxic immune cells, and its ligands (nkg dl), that are upregulated in many types of tumor cells including leukemic blasts, are important for anti-tumor immune surveillance. nevertheless, tumor cells may develop immune scape strategies like ligand shedding, which reduces nkg dl expression and may cause nkg d receptor downregulation. engineering t lymphocytes with nkg d car may overcome immune evasion and become an effective therapeutic strategy. methods: cd ra -t cells were obtained by depletion of non-mobilized apheresis with cd ra magnetic beads using clinimacs. nkg d-car t cells were generated by lentiviral (nkg d- bb-cd z) transduction of cd ra -t cells with moi= . the expression of nkg d ligands was analyzed in peripheral blood or bone marrow samples from a total of leukemia patients (aml= , b-all= and t-all= ), at different status of the disease (diagnosis, remission, relapse/refractory), and in different leukemia cell lines by qpcr and flow cytometry. cytotoxicity of nkg d-car t cells against leukemia cells was evaluated by performing conventional- hours europium-tda assays. soluble nkg dl (snkg dl) concentration was measured in the sera of leukemia patients by elisa. to evaluate the effect of snkg dl on nkg d-car t cells, those were cultured in the presence or absence of different concentrations of snkg dl for days. one week later, cell proliferation and car downregulation were measured by flow cytometry using cell trace violet and nkg d labeling, respectively. the production of ifn-g and tnf-a was measured in the supernatants by elisa. the effect on cytotoxicity was evaluated in a hoursdegranulation assay by co-culturing snkg dl pretreated nkg d-car t cells against k cell line. results: nkg d ligands were expressed in leukemia cell lines and leukemic blasts. nkg dl expression changed with disease status with a trend to decrease at diagnosis and relapse/refractory compared to remission. nkg d-car t cells were cytotoxic against / leukemia cell lines with a percentage of specific lysis over %. myeloid and t-all cell lines were more susceptible to nkg d-car t cells (specific lysis ranging from - %) compared to b-all cell lines ( - %). physiological concentrations of snkg dl caused an increase in nkg d-car expression. however, supra-physiological levels of snkg dl decreased nkg d-car expression up to times and increased cell proliferation up to times. cd + subpopulation was more affected by downregulation, while proliferation had more impact on cd + subset. the effects of snkg dl were dose-dependent and attenuated by il- . conclusions: nkg d-car t cells are cytotoxic against leukemia cells, specially aml and t-all, and thus could be a novel therapeutic approach for non-b leukemia, or those b-all that relapse with undetectable cd after cd -car treatment. nkg d-car expression may be downregulated only by supra-physiological levels of snkg dl, although antitumor activity is not affected. il- softens the negative effects of snkg dl inducing nkg d expression, cell proliferation and cytokines production. the changes observed in nkg dl surface expression at the different stages of the disease could be related to ligands release and immune escape. disclosure: nothing to declare denis-claude roy , , ines adassi , , céline leboeuf , , vibhuti p. dave , hôpital maisonneuve-rosemont research center, montréal, canada, university of montréal, montréal, canada background: for patients with high-risk leukaemia, allogeneic haematopoietic stem cell transplantation is the only curative treatment. the presence of alloreactive t cells in the donor graft, however, leads to a high probability of developing graft-versus-host disease (gvhd) . t-cell depletion minimises the presence of gvhd-causing alloreactive cells, but often results in an increased incidence of infections and disease relapse. photodepletion treatment (pdt) can specifically deplete activated alloreactive t cells while conserving resting t cells. pdt-treated cells have been utilised after t-cell-depleted haploidentical transplant to help reduce infection and relapse. the efficacy and safety of such pdt-treated cells is currently under clinical investigation in a phase iii trial (hatcy, nct ; kiadis pharma). here the reactivity of pdt-treated donor t cells was assessed toward tumour-associated and viral antigenic peptides derived from wilm's tumour protein (wt p), preferentially expressed antigen in melanoma (pramep), and from cytomegalovirus and epstein-barr virus (cmv/ ebvp). methods: healthy donor (hla-a* ) peripheral blood mononuclear cells (pbmcs) were co-cultured with irradiated pbmcs from another mismatched donor ( : ) in a -day mixed lymphocyte reaction. th , a photoactive rhodamine derivative, was added and cells were exposed to visible light to deplete the th -containing activated alloreactive cells. elimination of alloreactive cells post-pdt was assessed using cd and hla-dr as activation markers. an ex vivo expansion protocol was exploited to evaluate the impact of pdt on reactivity to tumour and viral antigenic peptides. post-pdt t cells were co-cultured with irradiated autologous monocyte-derived dendritic cells ( : ) pulsed with wt p, pramep or cmv/ebvp. antigen-specific t cells were re-stimulated on days and with wt p-or pramep-pulsed autologous pbmcs or with cmv/ebvp added directly to the culture. mhctetramer staining was performed on days and ; ifn-γ elispot was conducted on day . [[p image] . functional wt -specific and viralspecific t cells can be expanded post-pdt] results: pdt resulted in a drastic decrease of cd and/ or hla-dr activation marker-expressing cd + and cd + t cells. pdt-treated cells showed a significant increase in background: acute lymphoblastic leukemia (all) is the most common childhood cancer and relapsed or refractory all is still difficult to treat. engineered t cells equipped with a synthetic chimeric antigen receptor (car) targeting cd have demonstrated remarkable efficacy to treat all. however natural killer (nk) cells are known for their target-independent cytotoxic potential without induction of cytokine release syndrome (crs) or graft-versus-hostdisease (gvhd), car-nk cells can overcome the persisting problem in the therapy with car t cells. as the use of viral vector generated car nk cells is limited by theire genotoxicity, cost and regulatory demands, we are developing an innovative protocol using non-viral sleeping beauty (sb) transposition of third party nk cells as a source to produce 'off the shelf' car-engineered cell products. methods: nk cells are isolated from peripheral blood mononuclear cells (pbmcs) using cd selection kits. they are successfully expanded ex vivo with il- cytokine stimulation under feeder-cell free conditions. after few days of expansion nk cells are electroporated using pmaxgfp. transfection efficiency and percent of living cells after electroporation is analyzed by flow cytometry. transposition based nucleofection using an sb x mrna and a minicircles (mc) dna vector is performed at different time points after nk cell isolation. the transient mc-venus longtime expansion and the viability after sb x based nucleofection is measured over two weeks. furthermore, α-retroviral (α-rv) cd -car transduction of nk cells with different viral amounts (moi) is conducted and the cytotoxicity of the engineered cd -car-nk cells against the cd positiv cell line supb is addressed. results: for an α-rv cd -car transduction of maximal x nk cells we could show transduction efficiency of , % for moi . the α-rv cd -car modified nk cells had a high killing activity against cd positiv supb cells (e:t ration : , %) compared to cd negativ k cell lines (e:t ration : , %) and the non-transduced nk cells (e:t ratio : , %). in first experiments with pmaxgfp vector based nucleofection, we could show an increasing efficiency of , % h post electroporation with a only slightly decreas of living cells ( , %) comparing to the non-electroporated nk cell viability. using sb x mrna with mc-venus dna we electrotransfected x nk cells after fews days of cultivation and we reached , % of transfected nk cells h post electroporation and a transient expression of mc-venus positive nk cells up to , % efficiency with an increasing rate of live cells over days after electroporation. conclusions: the sleeping beauty based nucleofection of nk cells is a very promising non-viral method to generate more easy, safer and higher amounts of genetically modified third party nk cells for therapy of all and has also a broad range of clinical applications. disclosure: winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. axel schambach is an inventor on a patent describing alpharetroviral sin vectors. michael hudecek and zoltan ivics are inventors on patents related to sleeping beaut gene transfer technology. the remaining authors have nothing to disclose. background: mature immune cells from the stem cell graft are essential for the graft-versus-tumor (gvt) effect to eliminate residual malignant cells after hematopoietic stem cell transplantation (hsct), but donor cells are also involved in complications such as graft-versus-host disease (gvhd). methods: we performed a prospective study of the detailed graft composition in recipients of peripheral blood stem cells (pbsc) or bone marrow (bm) in order to identify correlations to clinical outcomes, table . grafts were characterized with concentrations of t cells and nk cells together with a multi-color flow cytometry panel with staining for tcrαβ, tcrγδ, vδ , vδ , cd , cd , cd , hla-dr, cd , cd ro, cd ra, cd , cd , cd and cd for detailed immune phenotyping. cell contents in stem cell grafts were analyzed both as fractions of cd positive lymphocytes and as absolute concentrations converted to transplanted cells/ kg. fractions were evaluated in patients receiving both bm and pbsc (n= ), while concentrations (cells/kg) were only analyzed in patients transplanted with pbsc (n= ). table] . table ] [[p image] . figure ] results: we found, that patients transplanted with graft nk cell doses above the median of x /kg and fractions of nk cells out of lymphocytes above the median of . % had significantly increased relapse-free-survival compared to patients transplanted with grafts containing nk cell doses below these values, figure ; results stayed significant in multivariate analyses. relapse incidence was significantly lower in uni-and multivariate analyses in patients receiving grafts with high nk cell fractions compared with low fractions, p= . , with -year relapse rates of % versus % in patients transplanted with high versus low fractions of nk cells, p= . . peripheral blood concentrations of nk cells obtained from samples from pbsc donors before g-csf mobilization were significantly correlated to graft concentrations-and fractions of nk cells.. analyses of graft contents of nkt cells showed that the incidence of grade ii-iv acute gvhd were significantly lower in patients background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory acute gvhd, but the mechanism of action is only partially understood. there is no clear relationship between the ecp-treated mononuclear cells (mnc) or lymphocyte numbers and response to ecp. the objective of the study was to analyse the relationship between the infused subpopulation cellularity and response. methods: patients from different centers with a total of ecp procedures were retrospectively analized. ecp procedures were performed from january- to june- . all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day of ecp. finally, the response and survival impact of infusing a number of cells above or below the median and in different tertiles was assessed until the median number of procedures needed to achieve a response. [[p image] . results: the median number of procedures until response was . we observed a trend towards a higher median number of monocytes per procedure and cumulative infused monocytes in responding patients (median number infused . vs . x /kg p= . , cumulative infused median number . vs x /kg p= . ) that was lost in the day of treatment. there was also a trend toward higher median infused mnc until response for responders ( vs x /kg p= . ). we observed no differences in the number of lymphocytes infused, but patients who received a number of lymphocytes per procedure over the first tertile ( x /kg) presented higher response rates ( % vs %, p= . ). none of the other analysed parameters showed a significant impact in overall survival. conclusions: patients with acute gvhd who responded to ecp received higher numbers of monocytes and mnc in the early phase of the treatment (a median of the first processes). also the patients who received higher numbers of lymphocytes in the first procedures achieved a higher response rate. these findings suggest the possibility that higher number of treated and infused cells could influence the response to ecp, but specifically designed prospective studies are need to asses this possibility. disclosure: nothing to declare background: the field of kidney transplantation has made enormous progress over the last decades towards being a standard treatment for patients with end-stage renal disease. however, administration of immunosuppressive drugs is still one of the major limitations of long-term allograft survival. therefore, strategies for induction of donorspecific tolerance are highly desirable. to this aim, a clinical phase i study with donor-derived modulated immune cells (mics) was conducted. methods: donor-derived mics were manufactured under gmp conditions. potency of mics was tested by different in vitro bio-assays. mics were administered to patients with an escalation from . x mics/kg on day - (n= , group a), to . x mics/kg on day - (n= , group b) or on day - (n= , group c) before kidney transplantation accompanied by standard immunosuppressive medication post-transplantation. frequency of adverse events (ae) was assessed from day until day post-transplant. dynamic changes of various lymphocyte subsets in patients after mic therapy were detected by multicolor flow cytometry. donor-specific immunosuppression was assessed by measuring anti-donor antibodies and mixed lymphocyte reaction (mlr) against donor and thirdparty cells. results: in all kidney transplant recipients, we observed a median serum creatinine of . mg/dl at day which remained stable until day (median creatinine of . mg/ dl) without significant proteinuria. none of patients experienced rejection episode. aes were observed while three aes being severe. most importantly, none of them was associated with mics transfusion. besides two infectious complications, no post-transplant positive cross match results against the donor or titers of de novo donorspecific antibodies were recorded. notably, immunosuppressive therapy could be reduced without signs of rejection in group c. after infusion, we observed a dramatic increase of cd + b cells up to a median of cells/μl until day , followed by a reduction to cells/μl on day in group c. notably, regulatory b cells significantly increased from a median of % on day to % on day . in parallel, the plasma il- /tnf-α ratio increased from a median of . before cell therapy to . on day . after mic cell therapy recipient lymphocytes showed no or only minimal reactivity against irradiated donor pbmcs in vitro, while reactivity against rd -party-donor pbmcs was not impaired. moreover, in vitro mics product demonstrated their immunomodulatory potency by inducing tolerogenic dendritic cells (tdcs) characterized by low expression of costimulatory (cd , cd ) and maturation (cd ) as well as high expression of inhibitory marker cd . functionally, tdcs could inhibit not only the release of ifn-γ but also the proliferation of cmv specific cd + t cells. moreover, mic-induced tdcs showed the capacity to inhibit donor-specific allo-reactive cd + and cd + t cell proliferation. conclusions: mic cell therapy modulates the immune system of kidney transplant recipients by increasing the ratio of regulatory b cells and facilitates the reduction of conventional immunosuppressive therapy without allograft injury or rejection episodes. therefore, mic cell therapy represents a promising strategy in transplantation medicine. we currently prepare a phase ii trial with mic cell therapy. disclosure: nothing to declare p genome editing of graft-derived t cells for post- background: immunotherapy using car t cells has shown promising results to fight cancer. however, car-t cell production requires specialized infrastructure and operators, which implies high cost and centralized production. automated production of car-t cells in clinimacs prodigy device allows clinical-grade manufacturing of car t cells. methods: million cd ramemory t cells from healthy donors were cultured in texmacs supplemented with iu/ml il- . at day cells were activated with t cell transact for h. at day one, activated cd ramemory t cells were transduced with nkg d-cd tm- bb-cd z lentiviral vector at moi = . then, nkg d-car t cells were expanded for - days. nkg d-car t cell products were next harvested, counted and analyzed for viability, nkg d-car expression and anti-tumor cytotoxicity. different quality tests including sterility, vector copy number, genetic stability, quantification of viral particles in the supernatant, myc/tert expression and endotoxin detection were performed. spare cells were cryopreserved either in autologous plasma and % dmso, m % albumin and %dmso or hypothermosol. after months, cryopreserved nkg d-car t cell products were analyzed for viability, nkg d-car expression and cytotoxicity. results: nkg d-car memory t cells expanded up to ± million with , ± % nkg d-car expression and ± % viability. harvested car t cells showed ± % of specific lysis against jurkat cells and ± % against mii osteosarcoma cell line. no microbiological contamination was observed in final car t cells products. vector copy number was ≤ in all validations except for one. cgh and karyotype showed no genetic alterations. free viral particles were undetectable in the supernatants. no overexpression of myc/tert was found except for one validation. endotoxins were ≤ . eu/ml. all cryopreserved nkg d-car t cell products kept nkg d-car expression one year after freezing. however, viability and cytotoxicity was best preserved using autologous plasma %dmso. conclusions: automated production of large-scale clinical-grade nkg d-car t cells using clinimacs prodigy is feasible and reproducible, allowing a decentralized protocol to generate car t cells for clinical use. background: immune reconstitution (ir) is essential to control severe infections after hematopoietic stem cell transplantation (hsct). reconstitution of adaptive immunity may take up to years to recover t-lymphocytes (lt). delay in early lt recovery increases the risk of relapse, viral infections and transplant related mortality. adoptive transfer of selected t cell subset with low alloreactivity potential is emerging as a strategy to improve ir. methods: depletion of cd ra+ naive t cells, preserving cd ro+ memory t cells could provide functional lymphocytes to protect against infection and leukemia relapse with low risk of graft versus host disease (gvhd). we present our experience with high-dose donor cd ro + memory t cell as donor lymphocyte infusions (dli) to assess safety and outcome. a total of dli of cd ro+ after hsct was performed in cases of cmv/ebv reactivation ( %), mixed chimerism ( %), persistent lymphopenia ( , %), graft rejection ( , %) , relapse ( %) or to boost ir ( %). dli product was obtained performing a cd ra depletion on donor leukapheresis product using the clinimacs® device. results: twenty-two pediatric patients, median age years (range - ), with malignant (n= ) and nonmalignant diseases ( ), received cd ra+ (n= ), tcr alpha/beta (n= ) depleted grafts from haploidentical and cd ra+ depleted grafts from match unrelated (n= ) and match related (n= ) donors. at a median of days (range - ) after transplantation, patients received a total of dli of cd ro+ cells, median (range - ), containing a median of . x /kg (range . x - x /kg), cd +cd ro+ . x /kg (range . x - . x /kg) and cd ra+ cells x /kg (range - . x /kg). all infusions were well-tolerated and did not develop or worsen gvhd. a total of / episodes of cmv/ebv viral reactivations decreased viral load, / cleared viral load and / showed a clinical improvement. a total of / patients with persistent lymphopenia there was a slightly increase in total lymphocyte count, but not to normal levels. prophylactic dli of cd ro+ to boost ir increased lymphocyte count in of cases. none of the dli administered in cases of mixed chimerism, graft failure or relapse were effective in reverting those situations. conclusions: our preliminary data suggest that infusions of high dose cd ro+ memory t cells are a safe adoptive immunotherapy strategy. efficacy has been observed in patients with lymphopenia and cmv/ebv reactivation, with no positive results in patients with mixed chimerism, graft failure and relapse. however, to determine the real efficacy of this strategy, prospective studies are required. disclosure: nothing to declare. background: increasing clinical trials have confirmed that chimeric antigen receptor t cells (car-t) targeting cd antigen (car-t- ) is a promising effective approach for the treatment of relapsed/refractory(r/r) b-cell lineage malignancies. considering cd is frequently expressed in large part of t( ; ) acute myeloid leukemia (aml) cells, we suppose that car-t- may be used as an approach to rescuing r/r t( ; ) aml patients. methods: both patients received lymphodepletion chemotherapy with decitabine mg/m × d, fludarabine mg/m × d and cyclophosphamide mg/m × d (dac +fc). two days after chemotherapy, autologous/allogeneic cart- cells provided by the unicar-therapy biomedicine technology co.(shanghai, china) at a total dose of - × cells per kilogram(kg) were infused dose escalation within to days. the research protocol was approved by the institutional review boards of the first affiliated hospital of soochow university and both patients gave written informed consent. results: both cases responded well with transient and reversible toxicities. case presented with grade cytokine release syndrome (crs), manifested by intermittent fever and chill from day after car-t- infusion for half months associated with neutropenia. car-t cells expansion were observed in blood without obvious increase of cytokines. after infusion, case achieved and maintained molecular complete remission (cr) for more than months. case presented with grade crs manifested by continuous high fever, hypotension and grade liver disfunction from day after car-t- cell infusion for week. obvious cytokines releasing (peak il- serum concentration . pg/ml, peak crp serum concentration pg/ml) were detected which were associated with car-t- cell expansion in blood and no severe off-tumor effect was observed. after infusion, case achieved hematological cr and cytogenetic cr and got months disease free survival. conclusions: our report implicates that car-t- is a safe and promising approach to managing r/r t( ; )aml with cd expression, and may provide a salvage treatment approach for all aml patients with cd expression and benefit a certain population with aml besides b-linage malignancies. clinical trial registry: na disclosure: nothing to declare. this work was supported by research grants from the national key r&d program of china ( yfc ), national natural science foundation of china ( , , , ) background: chimeric antigen receptor engineered t (car-t) cells have emerged as a powerful cellular therapy to treat malignant disease, which is currently revolutionizing field of cancer immunotherapy. a cryopreservation step postmanufacture is not only a logistical necessity for large scale cell manufacturing processes but also a mandatory request by regulatory authorities. in case relapse after st car-t cell transplantation, a second application, maybe at a higher dose constitutes a therapeutical option. however, data concerning clinical grade car-t cell stability and functionality after months of cryopreservation have not been released by companies so far. to investigate the effect of cryopreservation on car-t cells, we performed this study. methods: different batches of cd car-t cells were manufactured according to gmp requirements at our institution. final car-t products were frozen at concentrations of x cells/ml (high batch) and x cells/ml (low batch) by a controlled freezing process with the biofreeze bv device and stored in liquid nitrogen tanks below - °c until release. quality control tests for sterility, endotoxin and mycoplasma were performed for each batch according to european pharmacopoeia and united states pharmacopoeia guidelines. stability of cd car-t cells in terms of viability, recovery, transduction efficiency and functional capacity were determined by microscopy, multi-parametric flow cytometry as well as chromium- release tests following our sops. results: all the results of quality controls fully met the requirements of the regulatory authorities. stability results were highly robust and reproducible over time for all our gmp car-t batches. duration of cryopreservation (up to days) had no negative influence on cell viability, recovery of viable cd car-t cells and transduction efficiency. however, the cell concentration for cryopreservation has a significant impact on the post-thawing viability (low batches vs. high batches: . ± . vs. . ± . , p < . ) and recovery (low batches vs. high batches: . ± . vs. . ± . , p < . ) of cryopreserved cd car-t cells, but not the transduction efficiency. moreover, we observed four transient side-effects of cryopreservation on the amount of cytokines released by car-t cells, the cytokine release on a per-cell basis, the multifunctionality of car-t cells and the killing capacity. of note, functional capacity of cryopreserved car-t cells after overnight resting was comparable or even enhanced for inf-γ and tnf-α release by cd + and cd + cd car-t cells when compared to fresh car-t cells. the multi-functionality of car-t cells could be preserved. furthermore, the killing capacity of cryopreserved cd car-t cells after overnight resting could reach the level of non-cryopreserved/fresh car-t cells. conclusions: cryopreservation up to days has no harmful effect on transduction efficiency and functionalities of car-t cells. however, the cell number per milliliter freezing medium matters. dose over x cells/ml should be avoided. for the conduction of in vitro bio-assays to determine the function of car-t cells, an overnight resting process could mimic the situation after clinical application and eliminate the transient side-effects of cryopreservation to fully regain the functional potency of car-t cells. disclosure: nothing to declare background: dc and specific t-cells are important mediators of ctl-responses. we could already show that allogeneic donor-or autologous t-cells obtained from amlpatients can be stimulated by dc leu , resulting in a very efficient lysis of naive blasts. methods: chemokine-release (cxcl , - , - , ccl , - , and il- ) was analysed by cytometric bead array in serum of aml/mds-pts as well as in supernatants from different dc-generating-methods and correlated with pts' clinical course, dc-and t-cell-interactions as well as specific t-cell-reactions. the lytic activity of dcleu/blast -stimulated t-cells in mlc against naive blasts was quantified in a cytotoxicity assay. results: minimal differences in median chemokine-levels in pts' serum subdivided in subtypes were seen, but higher release of cxcl , - , - and lower release of ccl and - tendentially correlated with more favourable subtypes (< years of age, < % blasts in pb). in persisting disease, a higher serum-release of ccl and at relapse a significantly higher ccl -release were found compared to first diagnosis -pointing to a change of 'disease activity' on a chemokine level. whereas chemokine-levels in dc-culture supernatants compared to serum were variable, clear correlations with lateron (after stimulating t-cells with dcleu in mlc) improved antileukemic t-cell activity were seen: higher values of all chemokines in dc-culture supernatants always correlated with improved t-cells' antileukemic activity (compared to stimulation with blast-containing mnc as control) -whereas with respect to the corresponding serum values higher release of cxcl , - , and - but lower values of ccl and - correlated with higher probabilities to improve antileukemic activity of dcleu-stimulated (vs. blaststimulated) t-cells. predictive significant cut-off-values could be evaluated separating the groups compared. moreover, correlations with lateron achieved response to immunotherapy and occurrence of gvhd were seen: higher serum values of cxcl , - , - and ccl and lower values of ccl correlated with achieved response to immunotherapy. predictive cut-off-values could be evaluated separating the groups compared in 'responders' and 'non-responders'. higher levels of ccl and - but lower levels of cxcl , - , - correlated with occurrence of gvhd. conclusions: we conclude, that in aml-pts' serum higher values of cxcl , - , - and lower values of ccl and in part of ccl correlate with more favorable subtypes and improved antitumor'-reactive function. since in dcculture supernatants higher values of all chemokines correlated with improved antileukemic t-cell reactivity we conclude a change of functionality of ccl and - from an 'inflammatory' or 'tumor-promoting' to an 'antitumor'reactive function. this knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune-responses. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) is a curative treatment option for patients suffering from hematologic malignancies. infusion of donor lymphocytes (dlis) can induce sustained remission in case of minimal residual disease or relapse through potent graftversus-leukemia (gvl) effects, although graft-versus-host disease (gvhd) represents a common dose-limiting toxicity. as invariant natural killer t (inkt) cells are known to prevent gvhd while promoting beneficial anti-tumor effects, we investigated the role of inkt cells for successful dlis. methods: we analyzed dli samples by flow cytometry. inkt cells were identified by staining with pbs -loaded cd d tetramers. culture-expanded and purified dli-inkts were then tested against tumor cell lines and primary leukemia cells in an ex vivo tumor control model. tumor cell viability after coincubation with dli-inkts was measured by flow cytometry using -aad. results: inkt cells represent . % (range . - . %) of donor lymphocytes and can be expanded fold following a two-week protocol with a preferential expansion of cd + inkt cells. tumor cell lines such as jurkat were efficiently lysed after coincubation with dli-inkts. cd a as a marker of degranulation was significantly upregulated on dli-inkts after stimulation by jurkat. in addition, increased concentrations of tnfα, ifn-γ, sfasl and perforin were measured after coincubation of dli-inkts with jurkat. we observed that tumor cell lysis correlated with the expression of the mhc-i-like molecule cd d. consequently, adding a cd d antibody to the coculture abrogated the dli-inktmediated kill of tumor cells. dli-inkts also efficiently lysed primary leukemia cells such as aml blasts: expression of cd d on these aml blasts significantly correlated with dli-inkt-mediated tumor cell lysis (r = . , p= . ). conclusions: ex vivo expansion of dli-inkts and subsequent dli enrichment is an immunotherapeutic approach that could improve leukemia control and thus, prevent relapse after allo-hct without exacerbating gvhd. disclosure: nothing to declare. generation of antigen-specific cytotoxic t lymphocytes targeting wt using activated b cells sun ok yun , kyung won baek , hee young shin , hyoung jin kang seoul national university, seoul, korea, republic of background: the wilms tumor antigen (wt ) is highly expressed in many malignancies including leukemia and targeting wt as a tumor associated antigen (taa) in cancer immunotherapy is attractive. in this study, we generated wt -specific cytotoxic t lymphocytes to confirm if activated b cells can act as a cancer antigen presenting cell and induce ctls. methods: for the induction of ctls against wt , activated b cells were used as an antigen presenting cells. b cells were isolated from pbmcs of normal healthy donors and activated with α-galactosylceramide (α-galcer) and nucleofected with wt -coding plasmid dna. activated b cells were the cultured with pbmcs for days in vitro and harvested for assay. results: cells expanded about times after days of culture. we examined characteristic of wt -specific ctls by their surface markers. wt -specific ctls had more than % cd + marker, and ratio of cd to cd was . - . . we also examined nkt cell markers to see if nkt cells were activated by il- , a cytokine used in the induction of ctls, and the portion of nkt cells was about %. the ctls showed a decrease in naïve cell (cd l+cd ra +) and an increase in effector memory (cd l+cd ra-) and central memory (cd l-cd ra-) compared with non-stimulated pbmcs. subsequently, the ifn-γ elispot (enzyme-linked immunospot) assay was performed to confirm the response of the induced wt -specific ctls to the wt antigen. when wt -specific ctls encounters a target that does not have a wt antigen, it did not produce ifn-γ, but when it encounters a target cells loaded wt antigen, it responded to secrete ifn-γ. killing assays were also performed to determine the immunogenicity of induced ctls. the induced wt ctls was found to be killing more than % when the e:t ratio was : when the autologous pbmc met the target with wt pepmix. in addition, we found that wt ctls has killing activity when it encounters leukemia cell lines that express wt and matched hla-a* . conclusions: in this study, we can induce antigenspecific ctls that specifically react to wt using activated b cells as antigen-presenting cells. these observations confirmed that b cells activated by α-galcer can act as a taa presenting cell to induce taa specific ctls as viral antigen, such as pp and ie , and consequently wt specific ctls could be induced. moreover, ctls induced activated b cells had ability to recognize and kill the target cells expressing wt correctly. our results demonstrate that these in vitro expanded wt -specific ctls using activated b cells can be a promising candidate for adoptive immunotherapy against cancer. disclosure: nothing to declare judith böhringer , michael schumm , christiane braun , marina schmidt , patrick schlegel , christian seitz , murat aktas , georg rauser , sandra karitzky , peter lang , rupert handgretinger university children's hospital tübingen, tübingen, germany, miltenyi biotec gmbh, bergisch gladbach, germany background: t cells with chimeric antigen receptors (cars) on their surface facilitate to target specific surface expressed antigens. research and clinical trials with cd -car t cells show impressive remission induction rates and increased survival in heavily pretreated patients. therefore, car t cells are introduced as new potent cellular therapeutics in the clinical routine. in order to establish the manufacture of cd -car t cells, validation runs with the fully automated clinimacs prodigy t cell transduction process have been performed using the miltenyi anti-cd -car lentiviral vector. methods: unmobilized leukaphereses from donors ( x healthy, x all) were used for the clinimacs prodigy t cell transduction process. leukocytes undergo a cd + / cd + t cell enrichment via magnetic beads, followed by stimulation with macs ® gmp t cell transact™, transduction with an anti-cd -car lentiviral vector, expansion with il and il , and final formulation to the cellular product. during and after the manufacture, facs analyses were performed as well as cytotoxicity assays after cd -car t cell production. results: total volumes of leukaphereses were between and ml with . - . x total mononuclear cells. after enrichment x cd + / cd + t cells were transduced with anti-cd -car lentiviral vector and were further expanded. cells were harvested on day . the final cell counts of the cellular products were . , . and . x mononuclear cells from two healthy volunteers and the all-patient, respectively. the transduction efficiency of the cd -car t cells was . %, . % and . % among viable cd + cells. the final count of car t cells was therefore . , . and . x cells. the final products exerted excellent cytolytic activity against cd + bcp-all cell line nalm- . importantly, cd -car t cells generated from the all patient demonstrated complete eradication of autologous blasts at . to e:t ratio after hours incubation. conclusions: the clinimacs prodigy t cell transduction process has been shown to run a fully-automated manufacturing process over days without any deviations in a clean room environment on a single device. the user interaction was reduced to activities at only days to set up the system and provide fresh medium and reagents. the transduction process yielded a high number of t cells with a high frequency of cd -car t transduced cells. the results were comparable for both unmobilized leukaphereses from healthy donors and showed expected slightly lower results in the patient. finally, these results demonstrate that the clinimacs prodigy t cell transduction process is well suited to provide the clinical mb-cart . r/r cd + bcm study with appropriate investigational medical products. disclosure background: allogeneic hematopoietic stem cell transplantation (allohct) is an effective strategy in the long term control of several hematologic diseases, however, patients could experience complications, as graft versus host disease (gvhd) and disease relapse. recently, the introduction of post-transplant cyclophosphamide (ptcy) allowed to significantly reduce gvhd, but disease relapse remains an important issue. donor-lymphocyte infusion (dli) is an established adoptive cell therapy for disease relapse after allohsct, but, in order to be efficient and safe, patients have to be off immunosuppression treatments and gvdh-free. here we report our data about efficacy and safety of dli infusion as treatment for disease relapse in patients who received peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related plus ptcy as gvdh prophylaxis in our clinical trial (nct ). methods: we collected data from patients, treated with ptcy ( mg/kg/die, days + + ), mofetil mycophenolate (mmf) and tacrolimus (t) as gvhd prophylaxis after allo-pbsct, who received dli infusions. they were treated between january and october . we report data about overall response rate (orr), disease control rate (dcr), and dli-related mortality and morbidity. diagnosis were as follow: had multiple myeloma, had acute myeloid leukemia, had acute lymphoblastic leukemia and had lymphomas. all patients but one, who had chimerism loss, received dli because of disease relapse. results: median time between transplant and dli was (range - ) months. median number of dli infusions was (range - ). patients ( %) received cyclophosphamide mg/m preparative regimen the day before the cryopreserved dli infusions, while in the other cases dli were associated with lenalidomide, ponatinib and -azacitidine. the overall response rate (orr) was %, while disease control rate (dcr) was achieved in %. the patient who received dli because of loss of chimerism converted it in full donor after infusions. after dli treatment the incidence of acute gvhd grade i-iii was %, while was % for grade ii-iii and patients were started on short course of systemic immunosuppression treatments . none of these patients died because of dli adverse events. estimated -year overall survival was % with a limited follow-up length ( months). conclusions: the infusion of non-manipulated lymphocytes from allogeneic donors is a valuable and safe strategy of treatment for patients relapsing after allopbsct with ptcy. ptcy showed high efficacy in gvhd prevention, allowing early discontinuation of immunosuppression drugs. because of this, we can reach the goal to transform transplant in a platform where we could add early dli infusions as a new strategy for disease control. clinical trial registry: nct disclosure: nothing to declare p extracorporeal photopheresis in the treatment of refractory chronic gvhd: analysis of mononuclear cell infusion gillen oarbeascoa , maria luisa lozano , , , luisa maria guerra , cristina amunarriz , nuria revilla , , , pastora iniesta , , , cynthia acosta fleitas , jose luis arroyo , eva martinez revuelta , andrea galego , dolores hernandez-maraver , mi kwon , , aurora viejo , jose maria garcia gala , concepcion andon saavedra , jose luis diez-martin , , background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory chronic gvhd, but the mechanism of action is only partially understood. in some studies, a correlation has been suggested between treated mononuclear cells (mnc) or lymphocytes and response to ecp. the objective of the study was to analyze the relationship between the infused cellularity and response in chronic gvhd. methods: patients from different centers with a total of ecp procedures were retrospectively analyzed. ecp procedures were performed from january- to june- . all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day of ecp. finally, the response and survival impact of infusing a number of cells over or below the median and in different tertiles (t , t and t ) was assessed until the median number of procedures needed to achieve a response. results: the median number of procedures until response was . we observed no differences in the median number of lymphocytes, monocytes or mncs infused until response or until day between responding and non-responding patients. there were no differences in response if patients received lymphocytes or monocytes above or below the median number. nevertheless, patients that received a total absolute number of mncs above the median ( x cells) showed a trend towards a higher response rate ( % vs %, p= . ). the patients that received a cumulative number of lymphocytes in the first ecp procedures above the median showed improved overall survival (os) ( y os % vs %, p= . ). patients that received a number of monocytes above the median showed a trend towards better survival (p= . ), that was significant when the number of monocytes infused surpassed the first tertile ( y os % for t , % for t , % for t , p= . ). finally, the patients that received a cumulative number of mncs above the first tertile also showed improved survival ( y os % for t , % for t , % for t , p= . ). conclusions: there were no differences in the infused cellularity between responding and non-responding patients with chronic gvhd. at the same time, we found that except for a trend toward better response with higher mncs infused, there was no relationship between lymphocytes and monocytes with the response rate as other previous studies have suggested. however, even if there is no relationship with the response rate, the patients receiving the highest numbers of lymphocytes, monocytes and mncs in the cohort showed an improved survival, suggesting that larger quantities of cells could exhibit a protective effect. nevertheless, prospective studies that address this relationship are needed. disclosure: nothing to declare comparative analysis of the cytotoxic potential of cytokine-induced killer and natural killer cells for neuroblastoma therapy annekathrin heinze , beatrice grebe , eva mudry , jochen früh , bushra rais , claudia cappel , sabine hünecke , eva rettinger , thomas klingebiel , peter bader , evelyn ullrich , university hospital frankfurt, frankfurt, germany, german cancer consortium (dktk) partner site:, frankfurt, germany background: neuroblastoma (nb) is the most common solid extracranial tumor in childhood. despite therapeutic progress, prognosis for high-risk nb is poor and innovative therapies are of medical need. therefore, we investigated the cytotoxic potential of interleukin (il)-activated natural killer (nk) cells compared to activated cytokine-induced killer (cik) cells against different human nb cell lines in vitro. methods: nk cells were isolated from peripheral blood mononuclear cells (pbmcs) using cd enrichment or cd /cd depletion kits. they were successfully expanded ex vivo with different cytokine combinations such as il- , il- , il- and/or il- under feeder-cell free conditions. in contrast, cik cells were generated from pbmcs by ex vivo stimulation with interferon-γ, il- , okt- and il- . a comparative analysis of expansion rate, purity, phenotype and cytotoxic activity against different nb cell lines following different culturing protocols was performed. results: cd enriched nk cells showed a median expansion rate of . -fold after to days in culture with a final frequency up to . % nk cells and a median frequency of . % cd + cd -t cells. in contrast, the starting cell product after cd /cd depletion consisted of a median frequency of . % nk cells that expanded significantly faster with . -fold and also reached up to . % purity without any relevant t cell contamination. cik cells expanded with a median rate of . -fold and contained . % nk, . % t and . % nk-like t cells. interestingly, nk cells, particularly after cd /cd , showed a significantly higher median cytotoxic capacity against nb cells depletion ( . % for cd enrichment, . % for cd /cd depletion) compared to cik cells that induced . % killing of nb cells with e:t ratio : in a hours' co-incubation assay. interestingly, prolonging the ex vivo stimulation after cd /cd depletion to days enhanced the median expansion rate to . -fold with a slightly reduced cytotoxic potential ( . % for days' ex vivo expansion, . % for days' ex vivo expansion, comparison of the same donors). the addition of an il boost prior harvesting increased the expansion rate to median . -fold (compared to . -fold for the same donors) with an improved cytotoxicity of . % (compared to . %) . fortunately, all nk cell products showed a high viability and no relevant t or b cell contamination (median < . %). interestingly, further optimization of the culturing procedure with use of another cell culture medium led to an improved median . -fold (compared to . -fold) nk cell expansion rate in days, also resulting in comparable cytotoxicity of . %. conclusions: nk and cik cell products may offer an innovative immune therapeutic option for patients with high-risk nb after allogenic stem cell transplantation. our study revealed that nk cells have a significantly higher cytotoxic potential to combat nb. interestingly, the use of il- expanded and il- activated nk cells developed from a cd / depleted apheresis product is highly promising as additional immunotherapy in combination with haploidentical stem cell transplantation of children with nb. disclosure: nothing to declare. quantitative determination of donor allo-reactive t-cells in haploidentical donor-recipient pairs by enzymelinked immunospot (elispot) and mixed lymphocyte culture (mlc) assays background: t-cell alloreactivity is responsible not only for graft versus host disease and morbidity, associated with hematopoietic stem cell transplantation (hsct) but also for graft-versus-leukemia (gvl) activity. in this regard, monitoring and quantitation of alloreactive t-cells (allo-t) may potentially provide valuable information for individualized clinical management of transplant recipients. the aim of this study was the optimization of allo-Т detection and comparison of the elispot and mlc assays. methods: allo-t were determined in haploidentical donor-recipient pairs before hsct. donor mononuclear cells (mnc) served as effector cells (ec) . patient cd depleted mnc were used as stimulatory cells (sc).the ratio ec:sc were : and : . the frequency of allo-t in donor peripheral blood was tested in elispot assay and mlc. elispot provides the detection and quantitation of activated t-cells on the basis of cytokine secreted by each cell. the co-incubation time was h for ifn-gamma and h for il- detection. in mcl assay donor mnc were labeled with cfse and allo-t, proliferating in response to stimulation with alloantigens, were determined by flow cytometry on day . results: the median number of ifn-gamma producing allo-t per donor mnc was , ( - ; ec:sc ratio - : ) and , ( - ; ec:sc ratio - : ). the median frequency of allo-t was , % ( , - , ; ec:sc ratio - : ) and , % ( , - , ; ec:sc ratio - : ) among lymphocytes. il- -producing allo-t were less frequent in donor mnc in comparison with ifngamma-producing allo-t. the median number per mnc was , ( , - ; ec:sc ratio - : ) and , ( - ; ec:sc ratio - : ). the median frequency of il- -allo-t was , % ( , - , ; ec:sc ratio - : ) and , % ( - , ; ec:sc ratio - : ) among lymphocytes. the ec:sc ratio : is enough for stimulation of il- producing by mnc in elispot assay, but for optimal stimulation of ifn-gamma producing cells ec:sc ratio : is preferable. this suggests that allo-t are predominantly ifn-gamma producing cells. alloreactive proliferating t-clones were detected in mlc in of donor-recipient pairs on day of cocultivation. median percentage of proliferating t-clones were , % ( , - , ; ec:sc ratio - : ) and , % ( , - , ; ec:sc ratio - : ) among lymphocytes. however, mlc assay only permit a qualitative analysis that confirmed the presence of alloreactive t-clones, giving no information on their frequency within the culture. results of elispot and mcl assay directly correlated. conclusions: allo-t were detected in , % of assayed haploidentical donor-recipient pairs by elispot and only in , % by mlc. this difference in detection is due to the fact that elispot allows to detect single cytokine secreting cell whereas mlc can reveal proliferating аllo-t clones. the analysis of allo-t in haploidentical donor-recipient pairs may provide rationale to manipulate the allo-immune response and to exploit the powerful ability of allo-t to control hematologic malignancies. disclosure: nothing to declare allogeneic mesenchymal stromal cell as rescue therapy in an infant with life-threatening respiratory syndrome due to a filamin a mutation background: cell-based therapy has gained attention in the respiratory system diseases and encouraging results are reported following mesenchymal stromal cells (mscs) administration. due to their capacity to produce and secrete a variety of paracrine factors and bioactive macromolecules, mscs became a key player in lung tissue injuries and function, reducing fibrosis, promoting the normal development of alveoli and pulmonary vessels. for the first time we used the msc infusions as rescue therapy in a pediatric patient with flna gene mutation and life-threatening respiratory syndrome. methods: a child with a new pathogenic variant of the flna gene c. _ del; (p.val alafster ) at the age of months, due to the serious and irreversible chronic respiratory failure and dismal prognosis, was treated with intravenous infusions of allogeneic bone marrow (bm)-mscs at the dose of × mscs/kg body weight. bm-mscs were produced at "cell factory", fondazione irccs policlinico s. matteo, pavia,isolated and expanded ex vivo from healthy donor bm, following a previously reported protocol. premedication with antistaminic drug, min before every infusion to avoid any potential reaction was performed. the evolution of the respiratory condition was detected. peripheral blood were collected before each msc treatment for treg and th monitoring. treg, defined as cd + cd neg cd + cells expressing the forkhead box p (foxp ) transcription factor, and th , defined as cd + cells expressing intracellular il- , evaluation was performed by flow cytometry (facscanto; bd biosciences, san diego, ca) as previously reported, following standard procedures. results: no acute adverse events related to mscs infusion was recorded. during follow-up, patient maintained a good general condition and showed a regular growth. no systemic or respiratory infections occurred. after the second infusion, the child experienced a progressive improvement of his clinical respiratory condition, with a good adaptation to mechanical ventilation, in the absence of episodes of respiratory exacerbations. the baby maintains adequate volumes of exchange with substantial reduction of the inspiratory support. a reduction of trigger sensitivity was also obtained. thorax ct scan showed a recovery of the basal parenchyma bilaterally and the improvement of the anatomical-functional alignment and aerial penetration. after the first msc administration, an enrichment of treg and th percentage in peripheral blood, was observed. while, after the second msc infusion a significant increase in treg/th ratio was noted. conclusions: this report suggest that msc serial infusions are a promising therapy in aiding the respiratory failure, even in a pediatric patient with flna mutation. intravenous administrations of allogeneic mscs are feasible and safe without toxicity. our results suggest that to mitigate lung injury, mscs may act as regulators of treg and th balance. further investigations are upcoming to establish the useful of this therapeutic proposal in interstitial lung diseases in children. disclosure: nothig to declare p feasibility of il- stimulated donor nk cells manufacturing for early infusion in patients with high risk acute myeloid leukemia undergoing haploidentical transplantation background: nk cells provide a potent antitumor effect in the setting of manipulated haploidentical hematopoietic stem cell transplant (haplo-hsct). we propose a novel strategy to enhance the antitumor effect of allogeneic transplant through the infusion of nk cells stimulated with il- exvivo in adult high-risk acute myeloid leukemia (aml) patients undergoing unmanipulated haplo-hsct. the objective of this study was to provide efficiency and productivity data obtained in the manufactured cellular products infused. methods: selection criteria included patients with highrisk aml undergoing unmanipulated haplo-hsct. lymphoapheresis of the haploidentical donor was performed using spectra optia (terumo® bct) on days + and + after transplant. from the obtained product a double immunomagnetic cellular selection with clinimacs system (miltenyi biotec®) was performed in two steps: cd + depletion followed by positive cd + selection. the obtained an enriched cellular product of cd -cd + nk cells was incubated with il- ( ng/ml) between and hours at ºc and % co in gmp conditions. quality and microbiological controls were performed at the end of each manufacturing step. dxh cellular counters (beckman coulter®) and multiparametric flow cytometry were used for lymphocyte subpopulations and viability analysis (navios cytometer; beckman coulter®, conjugated monoclonal antibodies; miltenyi biotec®). the final product was infused intravenously to the patient on days + and + if manufacturing conditions were met (range of . - x nk/kg, purity ≥ %, viability≥ % and < x cd + cells/kg). if not, it was discarded. nk cell activation in the product was measured by the expression of cd and cd . results: between november and april , patients were included in this ongoing trial. two products were manufactured for of the patients, and only one for the first patient, due to transplant complications between first and second infusion. one product did not meet minimum viability criteria and was discarded. in the infused final products mean and sem of nk cell purity, recovery and viability were . %± . , . %± and . %± . , respectively. log cd + depletion ranged between - . and - . . median infused doses of nk cells and cd + cells per kg were . x ( . x - . x ) and ( - ). complete manufacturing data of all procedures are shown in table background: cytokine-induced killer (cik) cells are a promising immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (hsct) for acute leukemia or myelodysplastic syndrome. to show safety and efficacy, a multicenter clinical study with pediatric and adult patients including up to eight cik cell applications with escalating doses is ongoing. methods: we favor single large scale cik cell generation with the aim to apply fresh cik cells and cryopreserve ready-for-use doses according to the study protocol in contrast to recurrent manufacturing. therefore cryopreserved cik cells were tested against freshly generated cik cells to approve equivalence. furthermore, an alternative medium supplement for cik cell culturing was investigated to avoid supply bottlenecks in ab-serum. results: fresh frozen plasma (ffp), platelet lysate (pl) and ab-serum in cik cell culture showed median expansion rates of -fold, whereas cultivation without medium additive resulted in significantly lower proliferation (p< . ). cik cell composition including t cells, nk like t cells and a minor part of nk cells was not significantly influenced by changing the medium additive. moreover, neither cytotoxicity against thp- cells nor cd expression on nk like t cells were significantly influenced by the different medium additives. for cik cell generation either ficollized peripheral blood (pb) or unstimulated leukapheresis (lp) products were utilized. with regard to repeated manufacturing within the clinical study, also cryopreserved lp and pbsc as starting material came into the focus of interest. comparing cik cell expansion rates, no significant differences for the entire cik cells and the subgroup of t cells were detected between the four starting materials. cryopreservation of cik cells had no significant effect on cik cell composition, cytotoxicity and cd expression on nk like t cells. a small, albeit not significant effect of cryopreservation on viability was detected, which was . % before and . % after freezing and thawing. conclusions: the challenge was an efficient time-, personal-and cost saving production of cik cells within the clinical study. introducing ffp enabled cik cell manufacturing for an increased patient cohort by avoiding supply bottlenecks in ab-serum. furthermore, cryopreservation allows the storage of ready-for-use cik cell doses fulfilling the demands of the clinical study. clinical trial registry: eudract number - - disclosure: nothing to declare. automated generation of cd ra depleted donor lymphocyte infusion (dli) with the clinimacs prodigy® cd ra system , methods: the current clinimacs cd ra system was developed for graft engineering. up to x e magnetically labeled cd ra+ cells from leukapheresis products can be depleted from up to x e white blood cells (wbc). we developed a new clinimacs prodigy® process in order to ease the procedure for routine-use, to reduce the specifications according to reported cell numbers for dli applications, and to enable the use of peripheral blood products with high amounts of red blood cells (rbc). the new system was tested by performance runs. an new fluorescent flow analysis protocol was developed. results: the resulting clinimacs prodigy pb- ra system is an automated procedure with integrated labeling and washing steps. the new application software pb- ra depletion enables to deplete up to . x e cd ra + cells from up to x e total wbc from peripheral blood products. a major difference of this process is the rbc removal option based on an integrated camera for cell pellet detection. the final cell product is provided in physiologic saline. verification runs with peripheral blood products (n= in total, n= with whole blood, n= with leukapheresis products) resulted in a mean depletion of . log (range . - . ) for cd ra + t cells in the cd ra depleted product. viability of the target products was always above %, and mean wbc recovery was %. the mean process time was h min (range h to h min) without including the manual steps, i.e. tubing set installation and downstream analysis of blood products by flow cytometry. this data were in line with preceding evaluation runs (n= ), and results obtained in cooperation with an external beta test site. the performance results were furthermore in line with results obtained on clinimacs plus instrument runs. for quality control of cd ra depleted products we developed a flow cytometric analysis strategy for fast, accurate, and convenient analysis of even rare counts of remaining unwanted cells. it allows to determine naïve t cells at two different levels of subset staining. the minimum requirement for the flow cytometric analysis includes colors to define viable cd +cd ra+ cells. for further evaluation of the naïve t cell subsets additional colors are used to define viable cd +cd ro-cd -cd l+cd + cells. conclusions: the automated clinimacs prodigy pb- ra system process is capable to deplete cd ra+ cells efficiently from peripheral blood products within hours. the new process is a fast, convenient, and regulatory compliant method for the preparation of ready-to-use cd ra-depleted cell products for clinical applications. the submission to an european notified body for ce certification is an important next step. myeloablative conditioning regimen was preferred for patients out of . gvhd prophylaxis regimens are csa +mtx: (% ), csa+mmf: (% ), csa only: (% ). atg was given patients. despite been given gvhd prophylaxis (% , ) patients out of transplanted patients had gvhd features. of patients, had experienced steroid resistant gvhd after transplantation, including (% ) grade and (% ) grade . ecp treatment was started mean days after diagnosis of steroid resistant gvhd and (% ) patients had complete response while (% ) patients had partial and (% ) patients had no response to ecp treatment on day . sixteen out of patients had also received mesenchymal stem cell therapy as salvage therapy. only one patient had experienced hypocalcemic tetany, a complication of ecp procedure. thirteen patients had died and were directly related with steroid resistant gvhd. other conditions like relapse of primary disease or pres syndrome also played role in death. conclusions: extracorporeal photopheresis is a reliable and effective second line treatment modality in steroid resistant gvhd. starting ecp sessions as soon as gvhd symptoms occur increases its effectivity. mesenchymal stem cell administration with ecp for (% ) patients limits our study to reach o conclusion for efficacy of ecp itself. need for hemodialysis catheters, the prolonged sessions while adequate flow is not possible and catheter related infections are the lmitations for feasibility of ecp. disclosure: nothing to declare donor lymphocyte infusion administrations after allogeneic stem cell transplantations in pediatrics: a single center experience background: loss of chimerism is one of the major problems after allogeneic stem cell transplantation(sct). donor-lymphocyte infusions(dli) are used as a treatment after taper or stopping immunosuppression. in this study, dli experience in patients with loss of chimerism after sct due to various benign and malign hematological diseases was presented. methods: between july -august , twenty patients, detected chimerism loss and received dli after sct were evaluated retrospectively. patients received myeloablative or reduced intensity conditioning, atg, cyclosporine a and methotrexate for gvhd prophylaxis. chimerism analyses were performed with short tandem repeat(str) method from peripheral blood. results below % were considered as mixed chimeric and below % were nonchimeric. when patients considered as mixed chimeric, immunosuppression therapy was ceased immediately and treated with dli. donor lymphocyte infusions were performed at two-week intervals with chimerism follow-up. student t, mann whitney u, ki kare tests and kaplan-meier analysis were used. results: between - ages (median ), female, male patients were evaluated. the initial diagnoses were thalassemia major( ), aplastic anemia ( ),all( ), aml ( ) . dli initiation time was . +- . days after sct, total number of dli administrations were . +- . . dose of dli was x - . x /kg (mean . x /kg). nine patients' chimerism out of , fell below % at first month after transplant; patients were nonchimeric, of them were complet chimeric and were mixed chimeric. eleven patients´chimerism were below % between - months after sct, patients were nonchimeric and were mixed chimeric. early mixed chimerism was found relevant with graft rejections (p= . ). patients were followed up for - days. eight patients' chimerism increased after dli infusion and continued to decrease in patients. after dli, acute gvhd has been seen in both group.the group with decreased chimerism after dli, dose was mean x ± x /kg while the group with increased chimerism had dli dose mean x ± x /kg. although the difference was not statistically significant, numerical value revealed significantly different. eventually patients out of were mixed chimeric, patients were complete chimeric and were none. in thalassemic patients, patients with thalassemia-trait donor were mixed chimeric, in patients whose donors were normal, of them were complete chimeric and one of them was nonchimeric.the difference was significant (p= . ). the cd infusion doses revealed mean . ± . x /kg in mixed chimeric patients, . ± . x /kg in complete chimeric patients and . ± . x /kg in the patients with loss of chimerism. cd amount was seen high as numerical value in complete chimerics but no statistical significance was found. overall survival was %, disease-free survival was %. conclusions: we evaluated the efficacy of dl for patients with mixed chimerism in our patient group. we concluded that chimerism loss in patients with early decreased chimerism is similar to those in literature in spite if dli practices. dose and application frequency were greater in patients with increased chimerism. the small number and the heterogeneity of the patients limited our study. in this regard, studies with larger series and homogeneous groups are acquired. disclosure: nothing to declare phase i clinical trial of repeated administrations of bone-marrow derived mesenchymal stem cells in steroid-refractory chronic graft-versus-host disease patients nayoun kim , young-woo jeon , jae-deog jang , keon-il im , nak-gyun chung , young-sun nam , yunejin song , jun-seok lee , seok-goo cho (cghvd) is the most common long-term complication of allogenic hematopoietic stem cell transplantation which is associated with poor quality of life and increased risk of morbidity and mortality. currently, there is no standardized treatment available for patients who do not respond to steroids. as an alternative to immunosuppressive drugs, mesenchymal stem cells (mscs) have been used to treat and prevent steroidrefractory acute gvhd patients. these studies and reports have also provided a basis for using mscs in steroid refractory cgvhd patients. methods: to evaluate the safety and efficacy of repeatedinfusions of mscs, we enrolled ten severe steroid-refractory cgvhds patients. steroid refractory was defined as either no response to steroids lasting at least weeks or progression of disease during treatment or tapering lasting at least weeks. patients were intravenously administered with mscs produced from third-party bone marrow donors at a -week interval for a total of four doses. each dose contained x cells per kg body weight and all four doses consisted of mscs from the same donor and same passage. results: we enrolled ten patients ( female/ male, with a median age of . (range - ). median of cgvhd affected organs was (range - ) including the skin (n= ), eyes (n= ), oral cavity (n= ), lung (n= ), liver (n= ) and joints (n= ). all ten patients received their planned four doses of mscs, administering a total of infusions. median time from initial cgvhd diagnosis to first msc treatment was days (range - ). msc infusions were well tolerated with no immediate or delayed toxicities. after weeks of the first msc infusion, all ten patients showed partial response showing alleviation in clinical symptoms and increased quality of life. organ responses were seen in skin (n= ), eyes (n= ), oral cavity (n= ), liver (n= ), and joint(n= ). however, one patient died of progressive gvhd and one patient relapsed from primary disease. conclusions: repeated infusions of mscs was feasible and safe and may be an effective salvage therapy in patients with steroid-refractory cgvhd. further large-scale clinical studies with long-term follow up is needed in the future to determine the role of mscs in cgvhd. background: the majority of pregnant polish women ( %) have heard of cord blood banking. however, most doctors do not have sufficient knowledge about the possibility of using cord blood in order to respond to their potential concerns. only . % of healthcare professionals were aware that cord blood could be used to treat haematological diseases. in order to make doctors aware of this issue and provide patients with the information they expect, we would like to present data on the use of cord blood stored in our blood bank for haematological and nonhaematological therapies. methods: the table presented below has been created using data from the general database of the polish stem cell bank, warsaw, poland. no data regarding umbilical cord blood data have been excluded. all patients were planned to be assessed on day , day , on discharge, days after transplantation and , , , , and years after transplantation, but in some cases, patients were lost from follow-up due to a persistent lack of reports from transplantation centres. results: in cases, the therapeutic use of cb was transplantation (replacement of patients' own tissue); in cases it was administration (infusion without destruction of patients' own tissue). thirty-three were administered as standard therapy and as experimental therapy. conclusions: the survey study cited above, indicated low awareness of cord blood use among healthcare professionals. on the other hand, one study indicated that the expectations placed in cord blood banking may be unreasonable. as a private cord blood bank, we support recommendations which underline the importance of patient education. in poland, cord blood has been approved as standard therapy in approx. diseases; most of them are rare, but polish law allows the use of cord blood for the siblings, parents and grandparents of a donor. additionally, active and planned clinical trials throughout the world evaluate the therapeutic efficacy of cord blood in such areas as haematology, neurology, cardiology, diabetology, congenital paediatric disorders, ophthalmology, dermatology, gastroenterology, hiv infection, and the quality of life during aging. therefore, further indications may be expected in the future. background: cytokine release syndrome (crs) has been identified as a clinically significant, on target, off tumour side effect of the chimeric antigen receptor (car) t-cell therapies. it is clinically increased in interkeukin and elevations in other cytokines, lactate dehydrogenase (ldh), c-reactive protein (crp), and ferritin. these side effects can include fever, fatigue, headache, encephalopathy, hypertension, tachycardia, coagulopathy, nausea, capillary leak and multi organ dysfunction. crs symptoms can appear as early as one day after infusion and can resolve quickly or last for weeks. it´s severity to be related to the disease burden prior to car t-cell therapy. methods: the bristol oncology and haematology centre will be providing car t-cell therapy to patients in early . on collating from the leading consultant on the ward and fellow nursing team members it was apparent that an effective way at managing patients post car t-cell therapy side effects is to provide an educational and informative poster depicting a flow chart that will aid the practitioner to recognise and effectively treat/manage a patient with crs symptoms. results: none as of yet as this is a prospective tool ready for our first patient in early conclusions: through continuing reading and study days prior to the ward receiving its first car t-cell patient it is increasingly important that the entire multi disciplinary team recognise crs and understand the importance of early detection, careful monitoring and early intervention. background: allogeneic stem cell transplantation (allosct) is the only curative procedure for primary and secondary myelofibrosis (pmf, smf). elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. the introduction of reduced-intensity conditioning (ric) made allosct feasible and effective for old patients. nevertheless, the incidence of pmf and smf is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. very few data are available about the efficacy of ric-allosct in the particular setting of young-aged mf patients. methods: this study includes myelofibrosis young patients (age < y) who received allosct between and at the university hospital hamburg/germany. four patients were previously splenectomized. patients mostly fall into intermediate risk groups according to dipss. four patients belonged to the high-risk triple-negative category (jak /calr/mpl-). asxl -mut was tested in patients (pos: ). in % graft source was pbsc, patients received bmsc. only % of patients had a / hla-matched sibling, the others were transplanted from fully-matched ( %) or partially-matched ( %) unrelated donor. all transplants were conditioned according the ebmt protocol with busulfan ( mg/kg po or mg/kg iv), fludarabine ( mg/m ), atlg (grafalon® neovii, germany) administered in days at a dose of mg/kg die for mud, mg/kg die for mrd transplants, followed by cylosporina, and mycophenolate in the first days. results: engraftment rate was %, median neutrophil engraftment time days. platelet engraftment was reached by patients ( %, median days). four patients ( %) developed poor graft function, successfully treated with cd + selected pbsc-boost. after a median follow up of . years, estimated y-pfs and os were % and % respectively. dipss-risk and donor hla-matching resulted the only significant impacting factors on os. neither cytogenetic nor molecular abnormalities were significantly related to os. twenty-five patients ( %) experienced agvhd grade > . c-gvhd was observed in patients ( %), mostly ( %) beginning in the first days after transplantation. cumulative incidence of trm was % at year, with a plateau after the first year ( y trm = %). trm was observed only in patients with maximal grade ( ) of marrow fibrosis. furthermore, trm never occurred in previously splenectomized patients (p= . ), but no significant impact from splenectomy on os was observed (p= . ). after transplant, patients ( %) relapsed: died without any treatment because of infection, received dli ( durable cr), patients ( after dli) underwent a second allosct, with long-term survival in cases. conclusions: ric followed by allogeneic sct is a curative treatment approach for younger patients with myelofibrosis with a low nrm. the most important outcome-determining factor is donor hla-matching. interestingly, marrow high grade fibrosis showed to significantly impact trm. biological markers such as asxl mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from allosct. further data collection, and a prospective randomized trial are needed to confirm our conclusion. disclosure: nothing to declare p abstract already published. splenectomy as a risk factor for relapse after allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis -retrospective cohort study background: splenectomy is a common procedure in patients (pts) with myelofibrosis (mf) performed to achieve improvement in blood cell counts and reduce b-symptoms. however, it has also been shown that splenectomy may adversely predispose to leukemic transformation in this setting. aim: to evaluate in a single-center retrospective analysis the long-term impact of pre-or post-transplant splenectomy on transplant outcome regarding overall survival and relapse risk. methods: this retrospective analysis comprises the data of pts ( male and female) with primary (n= ) or secondary (n= ) mf after allo-hsct from hla-matched sibling (n= ) or unrelated (n= ) donors in our center between and . the median age was years (range, to years). a myeloablative conditioning regimen was performed in pts, while pts where treated with a reduced intensity conditioning. peripheral blood stem cells (n= ) or bone marrow (n= ) with a median of . x cd + cells/kg bodyweight (bw) (range, . to ) were transplanted. splenectomy was performed in of pts: pts were splenectomized prior to and pts after allo-hsct. relapse was diagnosed in ( %) of pts. the median duration to relapse after transplantion was months (range, - months). results: the median duration of follow-up of this cohort was months (range, - months), the -year overall survival (os) was %. pts died, including pts who relapsed and pts who died of treatment related causes. the observed probability of relapse was significantly higher in splenectomized pts than in non-splenectomized pts: % versus % (relative risk (rr) . , % ci, . - . , p= . ). at years, the os was % in nonsplenectomised and % in splenectomised pts (p= . ) (fig. ) . the relapse rate in splenectomised pts was independent of pre-( of pts, %) or post-transplant ( of pts, %) treatment (rr . , % ci, p= . ) . conclusions: on the basis of our cohort, we could assert that pre-and post-allo-hsct splenectomy was equally and significantly associated with an increased relapse ratio in patients with mf, which also tends to negatively affect overall survival. [[p image] . figure : the overall survival after allo-hsct in patients with myelofibrosis.] background: b-cell prolymphocytic leukemia (b-pll) is a very rare lymphoproliferative disorder. although allogeneic stem cell transplantation (allosct) could be a curative option, patients often do not qualify for this consolidation treatment due to an aggressive course of disease. in this case study, we report on three patients who failed ibrutinib therapy but achieved complete remission and even mrd negativity after treatment with the chimeric cd -antibody rituximab, enabling them to undergo allosct. methods: clinical data and follow-up data were collected by chart review. results: all three patients (pt# : male, years; pt# : female, years; pt# : female, years) were referred with b-pll harboring highly complex aberrant karyotypes, including p abnormalities in pt# and pt# . a tp mutation could be detected in pt# and pt# . all three patients had symptomatic disease with rapidly increasing hyperleukocytosis and massive splenomegaly. two of them were treatment-naive and one relapsed after chemoimmunotherapy. all patients were put on ibrutinib mg. despite initial response to treatment, two patients developed progressive disease after (pt# ) and months (pt# ) on ibrutinib, whereas pt# remained in partial remission with persisting leukocytosis, precluding consolidating allosct as originally intended. in pt# , ibrutinib was replaced by venetoclax, but without response. in order to control rapid lymphoproliferation, rituximab was added to venetoclax. grade infusion reaction / tumor lysis syndrome (tls) (fever, tachycardia and hypotonia requiring intravenous vasopressors) followed each rituximab administration despite fractionating rituximab to small doses. however, continuation of rituximab ( mg/d over d) led to complete and durable clearance of hyperleukocytosis (from /nl to mrd negativity) despite venetoclax cessation. a similar pattern was observed in pt# , who received rituximab while showing rapidly increasing leukocytosis upon ibrutinib. again, complete clearance of b-pll cells in the peripheral blood (from /nl to mrd negativity) occurred after initial grade tls despite only modest cd expression on tumor cells in this patient. also, pt# achieved profound b-pll cell depletion (from /nl to a mrd rate of . %) upon addition of rituximab to ibrutinib (without tls in this case). subsequently, all three patients were able to undergo allosct after conditioning with fludarabine and total body irradiation with gy. pt# received stem cells from a hla-ident sibling donor, whereas pt# and pt# had unrelated donors (hla-ident and hla-matched respectively). with follow-up times of and months post-transplant, pt# and pt# are currently in ongoing mrd-negative remission. pt# developed an acute graft-versus-host disease (gvhd) of the liver (grade ), nevertheless the clinical course was well controlled by immunosuppression. in pt# a chronic gvhd of the skin occurred. pt# , who achieved mrd negativity after allosct, developed acute and chronic steroidrefractory gvhd of the skin and gastrointestinal tract. nine months post-transplant, gvhd deteriorated and after further complications the patient died of pneumonia months post-transplant. conclusions: supplementary treatment with rituximab can induce deep remissions in patients with ibrutinibresistant, genetically poor-risk b-pll, thereby enabling them to undergo successful consolidation with allosct. a high risk of life-threatening infusion reactions / tls associated with the addition of rituximab has to be taken into account. background: there is little experience on the use of the newer targeted therapies in cll patients relapsed after allogeneic stem cell transplantation (allo-sct). against this background, we evaluate the safety and efficacy of the bcr inhibitors (bcri), ibrutinib and idelalisib, administered after allo-sct for the purpose of treating the cll relapse. methods: data from cll pts who relapsed after sct, and were subsequently treated with ibrutinib (n= ), idelalisib (n= ) or both (n= ),were retrospectively collected in collaboration with the spanish group of cll (gellc) and the spanish group of stem cell transplantation (geth). results: transplant characteristics are summarized in table . eight patients received the bcri as the first salvage treatment after sct relapse, whereas patients had received ≥ prior lines of treatment. at the time of the onset of the bcri, patients had rai stage and patients had a lymph node size ≥ cm. del p was present in patients and del q and complex karyotype in patients, respectively. tp gene mutation was detected in patients (all with del p ). median time from sct to bcri therapy was . months, being shorter in patients treated with ibrutinib (n= , median months) than in those treated with idealisib (n= , median months). median time on ibrutinib and on idelalisib was . months ( . - . ) and months ( . - . ), respectively. the best overall response rate (orr) obtained with ibrutinib was % ( cr, pr, pr+l) whereas it was of % for patients receiving idelalisib ( cr, pr+l). among the patients treated with ibrutinib, ( . %) presented an adverse event (ae), being diarrhea (n= ), asthenia (n= ) and infections (n= ) the most frequent. hypertension was seen in patient and none patient developed atrial fibrillation. five patients stopped ibrutinib treatment, due to toxicity (n= ) or progression (n= ). after ibrutinib discontinuation, patients were newly treated with idelalisib (n= ) or venetoclax (n= ). all patients treated with idelalisib developed at least one ae, being diarrhea (n= ), pneumonitis (n= ) and neutropenia (n= ) the most common. four patients discontinued idelalisib because progression (n= ) or toxicity (n= ). venetoclax was given after idelalisib in patients. although acute and/or chronic gvhd before bcri was documented in ( . %) and ( . %) patients, respectively, only one patient (treated with idelalisib) reactivated a mild chronic gvhd. none patient received infusion lymphocyte from donor after bcri and one patient underwent a second sct. with a median follow-up of . months ( . - . ) after the onset of the bcri treatment, patients had died, all of them due to cll progression ( richter´s transformation), whereas patients remained in response ( cr, pr). the overall survival probability of the whole series at months was . % ± . %. conclusions: in our study, ibrutinib and idelalisib, administered in cll patients relapsed after sct did not increase the risk of gvhd reactivation but they show high incidence of adverse events. nevertheless, bcri offers a possibility of disease control in these patients with poor prognosis. further studies are needed to confirm these data. background: prior to the introduction of tyrosine kinase inhibitors (tki), median survival of chronic phase chronic myeloid leukemia (cp-cml) patients was approximately months and the standard treatment with interferon-alpha resulted in complete cytogenetic responses in about % of the patients. autologous stem cell transplantation (auto-sct) was first attempted for patients in transformation in order to restore a second cp and was introduced secondarily in cp to try to prolong the response. the main rational for autografting in cp resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation. nevertheless, auto-sct alone was not able to maintain a long-term remission. nowadays, tkis represent the state-of-the-art therapy for cml and the concept of auto-sct has only little interest while long-term follow-up and outcome in this setting are currently unknown. the aim of our study is to evaluate at a first time the longterm outcome of cml patients who received auto-sct in chronic phase, and to evaluate at a second time in a subgroup analysis, the outcome of those who received tki after having been auto-transplanted, mainly for disease progression/loss of response and/or to enhance disease response. methods: we found a total of patients who received auto-sct for cp-cml in europe between years and , ( %) were males, median age at auto-sct was years (range: - ), the median time between diagnosis and auto-sct was months, stem cells source was peripheral blood in % of patients, most frequent conditioning regimen was busulfan mg/kg/day days + day of melphalan mg/m² one day prior to the cells reinfusion. information about receiving tki post auto-sct was available only for patients, first tki was imatinib for ( %) patients, dasatinib for ( %), nilotinib for ( %) and ponatinib for one ( %) patient. results: after a median follow-up of . years (range: - ) from time of auto-sct for the whole population, the probability of overall survival (os) at years was % ( % ci: - ); there was ( %) patients who relapsed after a median time of months after auto-sct. there was a total of patients transplanted before the tki era and survived until the availability of tkis. when we performed a landmark analysis evaluating the outcome of patients who received auto-sct, survived until the tki era and received tki (n= ), the years os probability of these patients from tki treatment was % ( % ci: - ). additional data requests will be sent to centers querying about prognosis, molecular responses, treatment and disease details. conclusions: we demonstrate here with these preliminary results that the introduction of tki has improved survival of cml patients. in addition, patients who received auto-sct, survived until the tki era and also received tki, had encouraging rates of long-term survival. an extensive analysis will be performed when additional data will be available and the study will be updated with more results. disclosure: nothing to declare a year single center transplant experience in chronic myeloid leukemia background: allogeneic hematopoietic stem cell transplantation (hsct) has been considered for decades the only curative approach for patients with chronic myeloid leukemia (cml). in the tyrosine kinase inhibitors (tkis) era, hsct for cml has been reserved only to patients not achieving a cytogenetic remission or showing progressive disease after multiple tki treatment lines. however, a progressive improvement in the long-term survival has been obtained in the overall hsct population. the present study aimed at evaluating whether in cml patients transplanted at our center over a long time period -from to -the outcome improved over time. methods: consecutive patients who underwent a transplant between and were compared to patients who received the transplant between and . overall survival (os), leukemia-free survival (lfs) and graft-leukemia-free survival (glfs) were estimated using the kaplan-meier method and the log-rank test was used to compare risk factors categories. results: of the patients [median age years (range - )], ( . %) were in st or nd chronic phase, ( . %) in accelerated phase and six ( . %) in blast crisis. matched related donors and alternative donors (matched unrelated donors, cord blood or mismatched related donors) were used in and cases, respectively. as stem cell source, bone marrow was used in patients, peripheral blood in and umbilical cord blood in . tbibased conditioning regimens were used in patients, while in the other cases irradiation-free conditioning regimens were used. both in univariate and multivariate analysis, irradiationfree conditioning regimens (hr . ; %ci . - . , p=. ) and transplants performed in st chronic phase (accelerate phase hr . ; %ci . - . , p=. - nd chronic phase hr . ; %ci . - . , p=. -blast crisis hr . ; %ci . - . , p< . ) were associated with a better os. patients transplanted before had a worse os (hr . ; %ci . - . , p < . ) and dfs (hr . ; %ci . - . , p=. ). a trend for a worse glfs was observed in univariate analysis (hr . ; %ci . - . , p= . ), in the first period of observation. conclusions: our single center experience confirms that higher os, dfs and glfs are observed in cml patients allografted in more recent years. improvement of conditioning regimens, use of tbi-free conditioning regimens and supportive therapy, have presumably contributed to these results, together with the more recent strategy of close monitoring of minimal residual disease, and prompt use of tki or donor lymphocyte infusion in case of relapse. hsct is nowadays a safer therapeutic procedure in cml patients that should be considered timely in tki-resistant patients to avoid progression into a more advanced disease phase. disclosure: the authors declare no conflict of interest. reduced-intensity transplantation (rit) in patients with high-risk or advanced chronic lymphocytic leukemia in last years: improvement of transplant outcomessingle centre experience . hct-ci ≥ was in % of pts. source of stem cells was peripheral blood in % and bone marrow in % of pts. the median of infused cd + cells was , x ^ / kg. the conditioning regimen consisted of fludarabine and melphalan (+atg in unrelated donor). gvhd prophylaxis were cyclosporine and methotrexate. results: all pts engrafted. none of pts in cr before rit progressed at day + after rit and among pts beyond cr before rit all of them achieved at least pr at day + after rit. pts ( %) developed acute gvhd ( pts grade iii-iv) and among evaluable pts ( %) of them developed chronic gvhd ( mild, moderate, severe). with median follow-up months (range - months) pts ( %) are alive in cr. pts ( %) relapsed or progressed , and months after rit and of them died. last relapsed patient achieved next cr after ibrutinib. pts ( %) died due to nrm. nrm till day + after rit was %. the estimated probabilities of -years cgrfs, pfs and os are %, % and %. conclusions: in spite of relatively small number of evaluated pts and retrospective type of analysis our data show that rit in pts with high-risk or advanced cll has achieved promising results ( -and -years pfs and os % and % resp. and %) in recent years and these results are better than outcomes of our historical patient cohort from period - ( -and -years pfs % and % resp. os % and %, p= . ) or ebmt published data of pts transplanted for cll in period - ( and -years pfs % and % resp. os % and %). current results of transplantation should be taken into account in our future decision-making process on indications for transplantation in pts with high-risk cll, of course also in the context of new or updated results of targeted cll treatment and its complications. disclosure methods: retrospective data and treatment outcomes were collected from the singapore childhood cancer registry (sccr). most children with cancer in singapore receive therapy at one of the two public paediatric cancer centers (kkh or nuh). a total of thirty two cases were diagnosed with cml and received treatment in either of these centers over a twenty year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: the age at diagnosis of the thirty two children ranged from to years (median . years). six patients in the pre-tki era were treated with an upfront hsct. the remainder twenty six patients were initially started on a tki. of these / ( %) had a hsct at a median period of . months from diagnosis (range - months). the reason for hsct in ten out of the twelve children was due to high risk features i.e. accelerated/blastic phase/ no ccr/no cmr. the remaining two patients had a hsct due to parent and patient preference for attempt at upfront cure rather than the use of life-long and expensive tki therapy. non-compliance to tki therapy was a major finding in our teenage cohort. eleven of the eighteen transplants used a matched sibling donor. three patients had cord blood as their stem cell source. one patient had a single antigen mismatched related donor and three patients had a mismatched unrelated donor for their hsct. all patients except one had myeloablative conditioning with busulfan and cyclophosphamide. atg was added according to physician preference. one patient had cy/tbi conditioning because of pre-transplant lymphoid blast crisis. anti gvhd medications included cyclosporine/ methotrexate or tacrolimus and methylprednisolone in the cord transplant patients. six of the eighteen ( %) patients who had a hsct died. four died due to treatment related mortality ( infections, acute gvhd and pulmonary fibrosis). one patient died due to an early relapse and one had a late relapse related mortality. for the pre-tki era, hsct related and year os was % and % respectively. post-tki era and year os was %. for the entire cohort, the year os was %. conclusions: the post-tki era transplant outcomes from our two centers is comparable to leading centers in the world. outcomes for patients with mismatched unrelated donors was poor in our cohort. we recommend a haploidentical related donor transplant or an unrelated cord blood stem cell source for patients when a matched sibling or unrelated donor is not available. clinical trial registry: na disclosure: we have nothing to disclose. fludarabine, busulfan, and thiotepa may be a promising conditioning regimen for myelofibrosis patients undergoing allogeneic stem cell transplantation background: allogeneic stem-cell transplantation (sct) is a curative therapy for patients with myelofibrosis. however, recurrent disease and non-relapse mortality (nrm) are frequent causes of treatment failure. the optimal conditioning regimen for sct in this disease has not been defined. methods: we retrospectively analyzed transplantation outcomes of all adult patients given sct for myelofibrosis between and at a single large academic medical center. patients (n= ) were treated with several conditioning regimens that were grouped according to conditioning intensity. myeloablative conditioning (mac) included busulfan . mg/kg and cyclophosphamide mg/kg (bucy, n= ), fludarabine and busulfan . mg/kg (flu/ bu , n= ) and fludarabine and treosulfan - g/m (flu/ treo, n= ). reduced-intensity conditioning included fludarabine and busulfan . - . mg/kg (flu/bu , n= ). more recently we adopted the tbf regimen including fludarabine, busulfan . - . mg/kg and thiotepa - mg/ m (n= ). all patients were also given anti-thymocyte globulin during conditioning, irrespective of donor source. results: the median age was years (interquartile range [iqr] - ). the majority of patients had documented splenomegaly ( %) and were not previously exposed to ruxolitinib ( %). donor type was an hla-matched sibling ( %), / ( %) or / ( %) matched unrelated donor. the dipps+ score distribution was intermediate- ( %), intermediate- ( %), or high (n= %). the median followup was . years since the success of tyrosine kinase inhibitors (tkis), transplant-related mortality is considered too high to justify allogeneic hematopoietic stem cell transplantation (allohsct) as first-line treatment for chronic myeloid leukemia (cml) patients in chronic phase (cp). allohsct is currently considered for patients failing to at least tkis or with disease in advanced phase. nevertheless, the optimal timing for transplant referral is still not well defined. methods: we performed a retrospective analysis on consecutive patients with cml in cp receiving first transplants from an hla-identical sibling donor with partially t-cell depleted grafts from to at our center. partial t-cell depletion (ptd) consisted of in vitro alemtuzumab incubation of a part of the graft for infusion at day while the rest, containing x cd + cells/kg was given as a t-cell add-back at day . donor lymphocyte infusions (dlis) were provided, in the absence of gvhd, in case of disease relapse or mixed chimerism. molecular monitoring was performed by -month bcr-abl rt-qpcr testing in peripheral blood during at least a -year period after hsct. thereafter, -month testing schedule was maintained where possible, or followed by a -month one. kaplan-meier method was employed to determine the probability of overall survival (os) and leukemia free survival (lfs) since allohsct. results: median age at hsct was years (range, - ). all patients were in first cp but one who was in second cp. twelve patients were tki-naïve at hsct ( hsct ( - , patients had presented suboptimal response or/ and intolerance to imatinib ( - period) , while the last seven patients had presented suboptimal response or/ and intolerance to imatinib, dasatinib and nilotinib ( - period) . the time interval from diagnosis to transplant was < months in / ( %) patients. ( %) patients had an ebmt risk score of - , while ( %) patients of - . the conditioning regimen was myeloablative for all but one patients. the stem cell source was pbsc for patients and bone marrow for one. all patients engrafted. patients presented molecular relapse and one patient hematological relapse with a median interval from transplant to relapse of months (range, - ) . patients received dlis ( for relapse and for mixed chimerism), while patients in relapse also received tki. without prior administration of dli, ( %) patients presented grade ii agvhd and patients moderate cgvhd. after dli, agvhd occurred in and cgvhd in patients. one patient died of disease progression years after hsct and one of myocardial infarction years after hsct. with a median follow-up of . years (range . - . ), -year os and lfs were %. at the time of the analysis / patients were alive and in major molecular response. conclusions: these results of excellent long-term survival and no transplant-related mortality suggest that ptcd improves the outcome of cp-cml patients transplanted from an identical sibling donor and they can be useful for deciding risk-adapted strategies. we believe that ptcd could allow earlier transplant referral of patients failing tkis and having an identical sibling donor. disclosure: nothing to declare single tertiary centre experience in allogeneic haematopoietic stem cell transplantation (allo-hsct) for primary and secondary myelofibrosis (mf) the only curative option for fit patients is allo-hsct. novel therapy is emerging but current recommendation is that eligible patients with life expectancy less than years should be considered for allografting. methods: we retrospectively looked at the clinical features and outcomes of all allo-hsct for mf performed in our centre since . results: patients ( male, female) aged between - years old (median age ) with intermediate- or high-risk mf as per the international prognostic scoring system (ipss) or dynamic ipps (dipps) were transplanted in our centre since . of them ( %) were diagnosed with pmf and the remaining % with secondary mf; post-et and post-pv mf. / of our patient group received a sibling allograft and / a matched unrelated donor allograft ( % received a / human leukocyte antigen (hla)-matched transplant, % a / hlamatched and % a / hla-matched graft). all patients received a reduced intensity conditioning (ric); / patients with fludarabine/ melphalan/ campath (fmc), / fludarabine/ busulphan/ atg (fbatg) and fludarabine/ ara-c/ campath (flag/ campath); all received peripheral blood as source of hsc. engraftment occurred between day - , with a median of d+ . one late graft rejection occurred. all patients were alive at d+ . patients are currently alive; overall survival (os) is %. transplant related mortality (trm) was . % at year, % at years. patient died of graft versus host disease (gvhd) and patients of septicaemia leading to multiorgan failure. acute gvhd grade ii skin occurred in patients, grade iii and above in patients. patients have limited chronic gvhd. / patients received donor lymphocyte infusion (dli) for mixed chimerism (one of which had nd graft failure). out of these patients developed acute grade gvhd and died. response rate: / alive patients i.e . % exhibit no fibrosis in trephine biopsies, / alive patients had residual fibrosis but % donor chimerism, / alive patients had residual fibrosis with mixed donor chimerism, other patient non-assessable. conclusions: allo-hsct remains the only potentially curable option for myelofibrosis. in our centre which serves . million population, with new cases per year, patients were transplanted since . our data suggest that close collaboration between mpn-treating haematologists and transplant physicians is required so that all suitable patients have a transplant assessment early in their disease course. novel molecular prognostic systems are likely to identify those best placed to benefit in future but this series currently supports allo-hsct survival and cure. (range, - ) . dynamic international prognostic scoring system (dipss) score at the time of hct was intermediate- or high risk in patients ( %), intermedate- in patient. molecular evaluation was available in out of : jak v f mutation was detectable in patients, mpl-w k in patient, carl in patient. patients were "triple negative" for driver mutations. cytogenetics information was available for out of ; among which patients had complex karyotype, trisomy and trisomy . patients underwent splenectomy before hct. ruxolitinib was administered in patients before hct. ( %) patients received stem cells from an hla identical sibling, ( %) from a matched unrelated donor and ( %) from an haploidentical sibling. graft source was bone marrow in patients ( %) and peripheral blood in ( %). conditioning was myeloablative in patients ( %), reduced intensity in ( %). all patients engrafted. acute graft versus host disease was absent in patients ( %), grade i-ii in ( %), grade iii-iv in ( %). in evaluable patients chronic graft versus host disease was limited in ( %), extensive in ( %) and absent in ( %). transplantrelated mortality at days was %. main causes of death were: acute gvhd in patients, chronic gvhd in , pancreatitis in , pulmonary aspergillosis in . relapse occurred in patients and was the main cause of death in of them. notably, patients experienced late relapse after . and . years after hct. both of them are living while receiving ruxolitinib therapy. after a median follow up of days (range, - ), out of patients are alive. of them ( %) are disease-free and are living. the kaplan-meyer overall survival and disease-free survival at years was % and %, respectively. conclusions: our experience confirms that hct is a valid option to achieve cure in one third of mf patients. two patients experienced very late (> years) recurrence of mf. the rarity of this condition limits the amount of data and cases available for evaluation and study. life-long follow-up of all mf transplanted patients is warranted to better understand this rare event. disclosure: nothing to declare methods: А -years old female was diagnosed with jak v f-positive pmf, xx, ipss low risk, dipssplus intermediate - risk, subacute budd-chiari syndrome and portal vein thrombosis four years before allohsct. to reduce the splenomegaly and constitutional symptoms we performed pre-transplant ruxolitinib therapy mg daily. after three months of therapy the patient achieved clinical improvement (eln criteria). contrast-enhanced computer tomography and magnetic resonance imaging showed enlarged intrahepatic collateral vessels and signs of portal vein thrombosis with cavernous transformation and multiple dilated collateral veins. gastroscopy documented enlarged esophageal veins. allogeneic stem cell transplantation was performed from / -hla matched unrelated donor with peripheral stem cells ( . x Сd + cells/kg). conditioning regimen consisted of fludarabine ( mg/ m ), busulfan ( mg/kg p.o.). post-transplant cyclophosphamide was administered at mg/kg at day + , + , and ruxolitinib mg was used from d+ till d+ as graft versus host disease prophylaxis. results: starting d+ the patient experienced eight episodes of ebv some of them with severe blood loss. to treat the bleeding episodes blackmore tube was placed six times with temporary effect. to place blackmore tube the patient was two times intubated and required mechanical ventilation. at d+ leukocyte and neutrophil engraftment, full donor chimerism and molecular remission were achieved. platelet engraftment was documented only at d + and poor graft function was present due to cytomegalovirus reactivation (d+ ) and parvovirus b reactivation (d+ ). evb was stopped at d+ only after two esophageal veins ligations, and two procedures of gastric veins sclerotherapy. soon (d+ ) the patient achieved complete platelet recovery (more than x /l) and became red blood cells transfusion independent. at day + complete remission was confirmed by splenomegaly resolution, regression of bone marrow fibrosis, full donor chimerism, jak v f-negative molecular status. cbc showed hb g/l, platelets x /l, leucocytes , x /l. ultrasound examination after transplant documented portal vein thrombosis recanalization. at day + she developed mild (nih) chronic graft versus host disease with eyes and mouth involvement, which was managed with topical steroids. at d+ after transplant the patient is alive in complete remission and has no recurrent bleedings. conclusions: splanchnic vein thrombosis can significantly complicate the course of allohsct in pmf. easy access to surgical, intensive care unit and endoscopic teams is required to make allohsct more feasible in this group of patients. disclosure all patients received treosulfan-based mac regimens, treosulfan(total dose, - gms/m ) was given in combination with different conditioning drugs. the most commonly used regimen was treosulfan, fludarabine ( mgs/m ) and thiotepa( mgs/kg) referred to as ftt that was used in %(n= ). serotherapy was given in % of patients(n= ), as either alemtuzumab or antithymocyte globulin in %(n= ) and %(n= ), respectively. post-transplant graft-versus-host disease (gvhd) prophylaxis was given in all patients, based mostly on ciclosporin. patients( %) received the transplant from identicalrelated donors, patients( %) received the transplant from matched-unrelated donors, and two patients( %) had haploidentical transplants. % of the patients(n= ) were fully hla-matched. all stem cell sources were used as bone marrow in %(n= ), peripheral blood stem cells in %(n= ), and umbilical cord blood in %(n= ). this treosulfan-based conditioning was given as the st transplant in %(n= ), and as the nd transplant after the failure of a first procedure in %(n= ). two patients received treosulfan-based conditioned transplant twice. results: neutrophil engraftment and platelet engraftment occurred at a median of days and days respectively. chimerism was full donor in %(n= ), high donor in %(n= ), and mixed donor in %(n= ). gvhd developed in % of patients(n= ), with acute gvhd grade i/ii and grade iii/iv developed in %(n= ) and %(n= ), respectively. chronic gvhd grade i/ii and grade iii/iv developed in %(n= ) and %(n= ), respectively. all chronic gvhd were mild, limited, non-extensive, and resolved completely. none of our patients had persistent gvhd necessitating long-term systemic immunosuppression. mild vod occurred in %(n= ), and severe vod occurred in %(n= ). one of them died but was believed to be related to the underlying disease (wolman syndrome). viral reactivation occurred in % of patients(n= ), with cmv, ebv, and adenovirus reactivation was found in %, %, and %, respectively. five patients had invasive adenoviraemia that contributed to death in two of them. primary graft failure happened in two patients( %) due to adenoviraemia. seven patients( %) had secondary graft failure with autologous reconstitution. graft failure was significantly lower (p . ) in the ftt group than other conditioning groups. at a median follow-up of months (range, two- months), eleven patients( . %) died, with overall survival of . %, and event-free survival of . %. five patients died due to complications related to their original disease, while six patients died due to transplant-related causes (transplant-related mortality . %). immune reconstitution in alive patients was achieved at a median of eight months. this time was significantly longer (p . ) in ftt group. conclusions: this study demonstrates that treosulfan is a safe and effective conditioning drug that can achieve engraftment, with low rates of graft failure, transplantrelated mortality and morbidity, even if it is used twice in the same patient. disclosure: nothing to declare background: high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the treatment of choice for the patients with relapsed or high risk nhl. although the high-dose conditioning regimens commonly used in patients with non-hodgkin lymphoma (nhl) are beam (bcnu, etoposide, cytarabine, and melphalan), beac (bcnu, etoposide, cytarabine, and cyclophosphamide), survival of patients with nhl received above high-dose chemotherapy followed by asct was still unsatisfactory. methods: we prospectively evaluated the efficacy and toxicity of busulfan, etoposide, cytarabine and melphalan (bueam) including iv busulfan instead of bcnu of standard beam as a conditioning for asct in patients with nhl. the high-dose chemotherapy consisted of bu ( . mg/kg i.v. q.d. from day - to day - ), e ( mg/m i.v. b.i.d. on day - and day - ) a ( g/m i.v. q.d. on day - and day - ) and m ( mg/m i.v. q.d. on day - ) at centers in korea. results: two hundred five patients were enrolled onto the study. main subgroup was diffuse large b cell lymphoma (n= , . %), t cell lymphomas (n= , . %), and nk/t cell lymphoma (n= , . %). upfront asct was performed in patients ( . %), and salvage asct in patients ( . %). the disease status of the patients before hdt/asct consisted of patients ( . %) with complete response and patients ( . %) with partial response. treatment related toxicities included nausea in patients ( . %), diarrhea in patients ( . %), anorexia in patients ( . %) and stomatitis in patients ( . %), which were grade i or ii in the majority of cases. the common grade iii toxicities were stomatitis ( . %), diarrhea ( . %), and anorexia ( . %). there were no vod, and transplant-related mortality occurred in patients ( . %), due to infection. one hundred fifty three patients ( . %) achieved a complete response and patients ( . %) after asct, while patients ( . %) showed progressive disease. at a median follow-up duration of . months, the estimated -year overall survival and progression free survival for all patients was . % and . %, respectively. conclusions: the conditioning regimen of bueam for asct was well tolerated and seemed to be effective in patients with relapsed or high risk nhl. disclosure: none of declare background: allogeneic hematopoietic cell transplantation (hct) is potentially curative for high risk acute myeloid leukemia (aml) and myelodysplastic syndrome (mds), however both gvhd and disease relapse remain major challenges. we recently introduced a combination of posttransplant cyclophosphamide (ptcy) and atg ( . mg/kg) as graft-versus-host disease (gvhd) prophylaxis. the purpose of our study was to compare outcomes between ptcy/ atg and other gvhd prophylaxis regimens for high risk aml and mds. methods: we retrospectively investigated outcomes of patients that underwent allogeneic hct between january and july for high risk aml (n= , %) and mds (n= , %). gvhd prophylaxis regimens were compared for overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) in univariate and multivariable analysis. high risk aml was defined as secondary aml, therapy related aml, high risk cytogenetics (eln criteria) in cr , good/ intermediate cytogenetic risk aml in cr and primary induction failure; high risk mds was defined as high/very high risk wpss score. results: median age of patients was years (range - years). donors were matched related in ( %) patients, matched unrelated in ( %) patients and haploidentical in ( %) patients. graft source was peripheral blood stem cells in patients ( %). myeloablative conditioning was used in patients ( %), reduced intensity regimens in ( %) patients. ptcy combined with atg was used in ( %) patients, other gvhd prophylaxis regimens were used in ( %) patients. both donor and recipient were cmv negative in ( %) patients. median follow-up of survivors was months (range - months). univariate analysis demonstrated os of the entire cohort at years was % ( %ci - %), cir at years was % ( %ci - %) and nrm at years was % ( %ci - %). concerning gvhd prophylaxis regimen, -year os for ptcy/atg versus others was % ( %ci - %) versus % ( %ci - %) (p= . , figure) , -year cir for ptcy/atg versus other was % ( %ci - %) versus % ( %ci - %) (p= . ) and -year nrm for ptcy versus other was % ( %ci - %) versus % ( %ci - %) (p= . ). grade ii-iv acute gvhd was seen in % of ptcy/atg patients versus % using other regimens (p< . ). chronic gvhd was observed in % of ptcy/atg patients versus % using other regimens (p= . ). multivariable analysis for os confirmed that the gvhd prophylaxis regimen has no influence (p= . ), while the predominant predictor of survival was age at hct (hr . , %ci . - . , p= . ). for cir, the ptcy/atg combination had no influence compared to other gvhd prophylaxis regimens (p= . ), while ric conditioning was the predominant predictor of relapse (hr . for ric, % p= . ) . for nrm, the atg with ptcy combination demonstrated no significant difference (p= . ), while age at hct was the predominant predictor (hr= . , %ci . - . , p= . ). conclusions: the ptcy/atg combination for gvhd prophylaxis has demonstrated on multivariable analysis similar os, cir and nrm with other previously used regimens at our center. a decrease in atg dose may potentially decrease the relapse rate while retaining the advantage of decreased gvhd. [ background: the combination of fludarabine with myeloablative doses of busulfan (fb ) represents a standard of care conditioning regimen before allogeneic transplantation in patients with myeloid malignancies (giralt, s.: the lancet oncology ). fb has potent antileukemic activity and is associated with low transplantrelated mortality and acute gvhd. however, early after transplantation (days - ), a proportion of patients may not convert to a full donor haemopoietic chimerism, particularly if anti-t lymphocyte globulin (atg) is used as gvhd prophylaxis (rambaldi a, et al.: the lancet oncology ) methods: we retrospective analyzed patients who underwent an allogeneic stem cell transplantation after fb conditioning regimen at our hospital, from november to august . the median age was years (range - ) and diagnoses were aml %, mds % cml % mfi %). the disease status at transplantation was: cr in %, cr in % and active disease in % of patients. the stem cell source was represented by pbsc in more than % of cases and anti-t lymphocyte globulin (atg) was part of the conditioning regimen in more than % of cases at a dose of mg/kg. the donor was a hla identical sibling ( %), a matched unrelated ( %) or mismatched (one allele or one antigen mismatched) unrelated, %. hematopoietic chimerism was molecularly evaluated by variable number of tandem repeats (vntr) on bone marrow (bm) mononuclear cells or peripheral blood (pb) t lymphocytes, purified by immunomagnetic positive selection (miltenyi, biotec). the analysis was performed at day , , , and after transplantation results: after , and days from transplantation, the proportion of patients with a full bm chimerism was %, % and %, respectively. at the same time points, the pb t cell chimerism was %, % and %. before day , patients required the infusion of dli to treat a pending or overt hematologic relapse and patients to convert the lymphoid chimerism from mixed (median %, range - ), to complete (successfully in cases). after day , additional patients required dli to treat disease relapse or progression and patients to improve the chimeric status or the immune reconstitution. at years, the overall survival is %, with a relapse and non-relapse mortality of % and %, respectively ( figure ). by uni and multivariable analysis, aml diagnosis and a mixed bm chimerism before day were associated with relapse and overall survival while age > was the only factor significantly associated with nrm. a mixed pb t-lymphoid chimerism before day does not adversely impact on non-relapse mortality, cumulative incidence of relapse, leukemia-free and overall survival. conclusions: after fb and atg, a progressive increase of pb lymphoid donor chimerism develops gradually after transplantation, in most of cases without the need of dli. early mixed lymphoid chimerism does not compromise the main long-term clinical outcomes and may at least partially explain the low non-relapse mortality. an incomplete bm chimerism within the first months strongly correlates with early disease progression or relapse. background: busulfan (bu) is widely used as a component of myeloablative conditioning regimen before hematopoietic stem cell transplantation (hsct) in children. bu has a narrow cumulative exposure window. the relation of bu exposure with toxicity is well established, but the link between the exposure and efs is not clear due to conflicting reports especially in pediatric patients. obtaining the ratio of bu to its metabolite i.e. metabolic ratio (mr) may serve as an indicator of bu gsh conjugating capacity of an individual, thus cumulative exposure of bu for a particular day that could be used along with auc as a marker to predict efs. the present investigation is aimed at evaluating the utility of bu mr to predict efs in children undergoing allogeneic hsct. methods: two different cohorts with children receiving bu in four times daily (qid, n= ) and once daily doses (qd, n= ) at st. justine's hospital, montreal were studied. bu and su levels were measured on day of the conditioning regimen at the end of infusion (dose in qid or dose in qd dosing). efs was defined from the time of transplant until death, relapse, or rejection, whichever occurred first. a receiver-operator characteristic curve (roc) for bu mrs measured was plotted to show the trade-off in sensitivity vs. -specificity rates for efs, as the cut-off of the test was shifted from low to high. cutoff values were defined based on the youden´s j statistic (i.e. sensitivity+specificty- ). results: twenty-two males and females aged from . to . years (mean±sd: . ± . ) from bu qid cohort had the mean mr of . (sd: . ). a cut off value of . in mr was chosen in roc analysis in this cohort, with better sensitivity ( %) and specificity ( %) for efs prediction (p= . , auc= . ( % ci= . - . ). in qd cohort nine females, and four males aged between . and . years ( . ± . ) had the mean mr of . (sd: . ). in roc analysis, a cut off value of . was chosen with better sensitivity ( %) and specificity ( %) for efs prediction (p= . ; auc= . ). conclusions background: treosulfan is an alkylating agent increasingly used prior to hematopoietic stem cell transplantation (hsct). the main objective of this study was to develop a population pharmacokinetic model of treosulfan in pediatric hsct recipients and to explore the effect of different covariates on treosulfan pharmacokinetics (pk). also, a limited sampling model (lsm) was developed. methods: in this multicentre study, patients, receiving a dose of , or g/m treosulfan a day, administered during consecutive days, were enrolled. a population pharmacokinetic model was developed using nonlinear mixed effect modelling (nonmem version . . , using psn toolkit . . and piraña version . . as modelling environment). demographic factors, as well as laboratory parameters, were included as covariates. results: treosulfan pk was best described by a twocompartment model. a bodyweight-based allometric model improved the model more than a model incorporating body surface area (bsa). clearance (cl) and intercompartmental clearance parameters were . l/h/ . kg ( %ci . - . ) and . l/h ( %ci . - . ). typical volumes of distribution of the central and peripheral compartments were . l/ . kg ( %ci . - . ) and . l ( %ci . - . ). a model-based dosing table based on bodyweight is created to achieve a target exposure of mg*hr/l (table ) , which was the median exposure of our population. estimated glomerular filtration rate (egfr) was shown to be the only parameter that significantly reduced interpatient variability in cl from . % to . %. a limited sampling model with samples (taken at . , and hours after start of infusion) accurately estimated pharmacokinetic parameters of treosulfan. conclusions: to the best of our knowledge, this is the largest cohort of pediatric patients treated with treosulfan used for a population pharmacokinetic study. we developed a two-compartment model with weight and egfr as covariates influencing treosulfan pk. recently we showed a relationship between treosulfan exposure and early toxicity. patients with an exposure > mg*hr/l have an increased risk of developing grade or higher mucositis and skin toxicity. another study in pediatric patients with thalassemia major reported an association between treosulfan clearance (< . l/h/m ) and poor overall survival. our model, together with the limited sampling strategy, can be used to adjust the dose, prior to or during treosulfan administration. ongoing studies conducted in different disease settings will determine if treosulfan exposure can influence patient outcome. subsequently, the optimal target exposure can then be established. background: autologous stem cell transplant (asct) is an effective treatment method for non-hodgkin lymphoma (nhl). until recently, carmustine, etoposide, cytarabine and melphalan (beam) was the most commonly used conditioning regimen. despite acceptable efficacy with beam, carmustine is associated with major pulmonary toxicity. for this reason, the aim of this study was to investigate the safety and efficacy of beb conditioning regimen for asct in nhl. methods: we conducted a prospective, multicenter, phase ii study for beb conditioning regimen for asct in nhl patients. a total of patients were enrolled from centers. they underwent asct with beb conditioning regimen (busulfan . mg/kg for days, etoposide mg/ m for days, bendamustine mg/m for days) between and . [[p image] . two year progression-free survival and overall survival.] results: the median age was years (range - ) and patients ( . %) were men. the most common type was diffuse large b cell lymphoma (n= , . %) and more than half of patients (n= , . %) were classified as ipi score or . eight patients ( . %) had a history of relapse and patients ( . %) received more than lines of chemotherapy before asct. most patients (n= , . %) were complete remission (cr) state at asct. a median number of . x /kg cd cells were infused (range . - . ). all patients engrafted after a median time of days (range - ). twelve patients ( . %) experienced neutropenic fever and patients ( . %) had grade toxicities during asct. however, no one had a documented infection, veno-occlusive disease, or treatment-related death. three months cr rate was . %. during a median follow-up period of . month, patients ( . %) exhibited relapse or progression, while patient ( . %) died of the disease. the estimated -year pfs and os rate were . % and . %, respectively ( figure ). conclusions: the beb conditioning regimens for asct is a feasible with tolerable toxicity in patients with nhl. disclosure: nothing to declare long-term report of total marrow or total lymphoid imrt in advanced leukemia, myeloma and lymphoma background: during the last three decades, total body irradiation (tbi) continues to play an important role in the conditioning regimens for patients undergoing stem-cell transplant (sct) for a wide variety of advanced hematological malignancies. however, tbi showed boundaries in dose limits for toxicity in allogenic and moreover in autologous stem cell transplantation. currently, the choice of conditioning regimen is based on the use of the least-toxic regimen to achieve the optimal therapeutic result. this report aims to assess the feasibility of a conditioning strategy based on high dose chemotherapy and whole-body radiotherapy focused on selective extensive tumor burden irradiation, both in allogeneic and autologous stem cell transplantation. methods: since december , sixty-two patients (pts) have been irradiated by helical tomotherapy (ht) to extensive target before allogeneic or autologous transplantation. selected total marrow irradiation (tmi) schedules were planned to treat patients with high risk acute leukemia (all or aml) or multiple myeloma (mm) as a part of conditioning regimen. total lymphoid irradiation (tli) was planned for patients with refractory or relapsed (r/r) hodgkin (hd) or non-hodgkin lymphomas (nhl). results: tmi and tli allowed delivering therapeutic dose over extensive selected targets with wide reduction of toxicity to all the organs at risk (oars). the higher radiation doses rate to the oars is reduced from % to %. allogenic conditioning regimen was tli ( gy x fx) than fludarabine + endoxan for patients with hd ( pts). tmi ( gy x fx) + fludarabine + melphalan for patients with mm ( pts). tmi ( gy x fx) + thiotepa + fludarabine + busulfan for advanced lam patients ( pts). tmi as the boost ( - gy) after conventional tbi was ( gy in bi-fractionated doses) by cyclophosphamide ( pts). autologous preparation to sct consisted of tli ( gyx fx) followed by high-dose bendamustine and melphalan for patients older than years and conventional feam (fotemustine, etoposide, cytarabine, and melphalan) for younger patients, in hd e nhl ( pts). while tmi ( gy x fx) plus melphalan was delivered for autologous sct in mm and lam ( pts). no unexpected acute toxicity was found. in the allogenic setting, all the patients' engraftment was achieved in all patients. no acute graft versus host disease increasing was detected. within the autologous setting, only % developed grade / mucositis. none experienced grade / extra-hematological toxicity. outcomes of the specific disease will be reported. conclusions: the current report describes the clinical feasibility of using ht to deliver tmi or tli in the setting of autologous transplantation or during allogenic stem cell conditioning regimen, to allow all patients (old, fragile or with high tumor burden) to achieve an ablative regimen before sct. to our knowledge, this single institution experience describes data from one of the largest cohort of patients treated in europe since the development of this irradiation techniques. disclosure induction therapy in both groups of patients was based in polychemotherapy without the use of new drugs. case matching was performed according to age, clinical stage at diagnosis, and response to induction therapy. conditioning regimen consisted of iv bu at a dose of . mg/ kg once a day on days - to - followed by mel at a dose of - mg/m on day - in the bumel group versus mel in the control group. maintenance therapy after transplant consisted of interferon and steroids in the majority of patients. results: the cut-off date for this update was june , . after a median follow-up of and months in the bumel and mel groups respectively, patients had relapsed in the bumel group and patients in the control group. median pfs was ( % ci, . - . ) months in the bumel and ( % ci, . - . ) months in the mel group (p = . ) ( figure ). in this update, patients in the bumel group are in maintained response and of them are in continuous cr (two with negative status for minimal residual disease) between and years after transplantation. ten-year os was not significantly different between both groups, being ( % ci - ) months in the bumel and ( % ci - ) months in the control group.transplant-related mortality was similar in both groups of patients ( % in the bumel and % in the mel group). regarding toxicity, bumel was associated with a higher incidence of mucositis and liver toxicity than the melphalan-only approach but no patient in our series developed sinusoidal occlusive syndrome and the hepatic toxicity observed was only grade i/ii. finally, no long-term side effects have been reported among bumel recipients. conclusions: this long-term follow-up analysis confirms that a therapeutic strategy including bumel as conditioning regimen beforeasct in patients with newly diagnosed mm is highly active and safe in these patients. [[p image] . figure . progression free survival in the bumel (____) and control group (…… frequency of acute gvhd grade iii-iv [cc: %; ct: %; tt: %, p= . ], and transplant-related mortality was higher in tt-carriers (cc: %; ct: %; tt: %, p= . cc&ct vs tt) . ta-tma, cmv infection/reactivation and cgvhd were also not different according to donor genotypes. fungal infections occurred more frequently as causes of death in carriers (cc: . % vs. ct: . % vs tt: . %, p= . ). conclusions: our results suggest that donor tgfb - c>t may exert an adverse influence on the outcome of myeloablative conditioning. our finding might be explained by the combination therapy of calcineurin and mtor inhibition in gvhd prophylaxis in myeloablative conditioning. disclosure: nothing to declare. treosulfan-based reduced intensity conditioning in hla-haploidentical transplantation using ptcy as gvhd prophylaxis in high-risk mds /aml of the elderly background: standard conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (allo-hsct) are often associated with a considerable risk of severe adverse events, especially in elderly patients suffering from high-risk (hr) mds/aml. previous clinical studies have demonstrated feasibility of treosulfan-based reduced-intensity conditioning (ric) by stable engraftment, low non-relapse mortality (nrm), and favorable survival in elderly patients undergoing hla-matched related or unrelated allo-hsct (beelen et al, ash # ). however, data for treosulfan-based conditioning in the t-cell-replete hlahaploidentical (haplo-hsct) setting in high-risk aml/mds patients are rare. here we report on the outcome of eleven patients treated with a treosulfan-based conditioning undergoing haplo-hsct using exclusively post-transplantation cyclophosphamide (ptcy) as gvhd prophylaxis. methods: eleven patients with high-risk (hr) aml (n= )/mds (n= ) who underwent haplo-hsct using treosulfan for reduced intensity conditioning (ric) and ptcy as gvhd prophylaxis were retrospectively analyzed with respect to outcome and toxicity. all patients were > years old and transplanted between january and february at our institution. the majority of the patients ( / ) suffered from active disease at time of treatment initiation, only two patients presented in cr. all but one received sequential conditioning with cytoreductive chemotherapy using flamsa applied shortly prior to treosulfan-based ric ( g/m over days). a bone marrow graft was used in / patients. post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf. national cancer institute common terminology criteria for adverse events version . were used for nonhematologic toxicity assessment starting from sequential therapy initiation or conditioning until day + . results: median age of the entire cohort was years (range: - ). the hct-ci was ≥ in eight pts (median hct-ci= , range: - ). no graft rejection occurred. neutrophil and platelet engraftment were achieved in % and % of the patients at a median of ( - ) and . ( - ) days, respectively. acute gvhd grade ii-iv occurred in % of the patients, exclusively involving the skin. no one developed severe (°iii-iv) acute gvhd. no patient died prior to haplo-hsct. severe nonhematologic regimen-related toxicities (°iii-iv) occurred in / patients, predominately affecting the gastrointestinal tract. no patient suffered from ≥two iii-iv°toxicities. all patients developed fever during treatment course, four with positive blood cultures. cmv reactivated in / patients at risk. no ebv reactivation or ptld occurred. six patients had clinical and radiological signs of pneumonia (probable invasive aspergillosis) without detection of aspergillus/antigen in the bronchoalveolar lavage. ci of nrm at day + was %. four patients relapsed within the first year after haplo-hsct, with two of them dying due to relapse. at last follow-up (dec ) / patients were alive. with a median follow-up of months ( - ) estimated -year os and dfs were % and %, respectively. conclusions: treosulfan-based unmanipulated hlahaploidentical allo-grafting using ptcy as gvhd prophylaxis in hr mds and aml patients aged over years is safe and well tolerated resulting in stable engraftment and a favorable toxicity profile. our preliminary data further show promising outcome with low nrm, no severe acute gvhd and favorable survival offering an attractive alternative in ric for haplo-hsct of the elderly. disclosure: nothing to declare comparison of outcomes of total body irradiation (tbi) vs non-tbi conditioning regimens in acute lymphoblastic leukemia for allogeneic transplantation background: in adult patients diagnosed acute lymphoblastic leukemia (all) long-term results are poor with intensive chemotherapy. allogeneic stem stem cell transplantation is the potential treatment that provides cure for these patients. myeloablative preparation regimens include total body irradiation (tbİ)+ cyclophosphamide(cy) and busulfan + cyclophosphamide.in adult all patients wbi/cy widely used, but the toxicity rate is higher. the aim of this study is to compare the result and effect of the tbi/cy and busulfan/cy regimens in allogenic bone marrow transplantation in all patients. methods: between - there were all patients who underwent transplantation using myeloablative preparation regimen with or without addition tbi in the adult bone marrow transplantation units of medipol medical faculty, istanbul university istanbul medical faculty, sisli florence nightingale hospital, atakent acıbadem hospital adult bone marrow units . we analyzed overall survival (os), progression free survival (pfs), veno occlusive disease, acute and chronic graft versus disease development rates in these patients. results: demographic characteristics of patients summarized in table - there was no significant difference between groups in donor age, gender, stem cell source. it was observed that the relapse rate was not statistically significant in both group.there was no statistically significant difference between the patients who underwent myeloablative regimen and myeloablative regimen with tbi in relaps,death, os, pfs. (figure- ) [[p image] . figure ] in terms of transplant complications there was also no respectable difference in development of vod and acute and chronic graft versus disease but vod was more common in the group that did not use tbi (p: . ) ( conclusions: although there are contradictory data in the literature, in our multicentre study, it was revealed that the addition of tbi in the myeloablative preparation regimen compared with myeloablative preparation regimen alone did not have a positive or negative effect on overall survival.we think that if we can prepare a good vod prophylaxis approches, we can give up tbi in future. disclosure (n= ) . for gvhd prophylaxis, cyclosporine a was given either alone (n= ), with mmf (n= ) or with methotrexate (n= ). the graft source was bone marrow (bm) in most cases (n= ), pbsc in seven cases, matched sibling cord +bm in two cases and one matched related cord. twentyfive of the donors were family donors and ten were unrelated. twenty-nine of the donors were / hla matched, six were / mismatched and one haploidentical. four patients had engraftment failure and required a second transplant, two of them were re-transplanted with cyclophosphamide and tbi, one with fludarabine, busulfan and campath, and one with no conditioning. thirty of the patients are alive ( %). four patients died of transplant complications and one died of metastatic squamous cell carcinoma. eight survivors are mixed chimeras ( %- % donor) and are all doing well, none of them developed any gvhd. nine patients developed acute gvhd, four of them with grade - . seven of these patients later developed chronic gvhd, two of them have extensive disease. conclusions: our results show a high survival rate of %, with a low rate of engraftment failure and reasonable rates of gvhd. only one of our patients died of late effects of hsct for fa. mixed chimerism does not seem to present a problem. we conclude that reduced intensity fludarabine based conditioning regimens are a good treatment option for patients with fanconi anemia undergoing hsct. disclosure: nothing to declare total marrow irradiation + bendamustine as reducedtoxicity myeloablative conditioning prior to allohsct for younger patients with multiple myeloma background: the prognosis of patients with multiple myeloma (mm) has improved markedly over the last two decades. despite that, allohsct remains the only treatment option with curative potential. however, its use is limited due to high incidence of non-relapse mortality (nrm) after myeloablative conditioning while insufficient efficacy of reduced-intensity regimens. we developed a new protocol characterized by reduced toxicity while preserved myeloablative potential, based on the use of total marrow irradiation (tmi) in combination with bendamustine. the aim of this study was to evaluate its safety and efficacy in a singlecenter experience. methods: between years - , mm patients below years old were offered tandem auto-allohsct as part of first-line therapy. the decision was based on individual patient preferences after detailed description of potential risks. autohsct was preceded by melphalan mg/m iv. the conditioning prior to allohsct consisted of tmi performed using helical tomotherapy at the dose of gy/d on days - , - , - (total gy) and bendamustine - mg/m /d iv. on days - , - (total - mg/m ). the immunosuppressive therapy consisted of cyclosporine + methotrexate +/-atg. peripheral blood was used as a source of stem cells. results: the analysis included patients (women - , men - ). the median follow-up was ( - ) months. the median age at allohsct was ( - ) years. the disease stage before allohsct was as follows: cr- , vgpr- , pr- . patients were treated with hsct from either hlamatched siblings (n= ) or unrelated donors (n= ). the interval between autohsct and allohsct was ( - ) months. all patients engrafted after allohsct with median time of neutrophil and platelet recovery of and days, respectively. one patient ( %) experienced grade acute gvhd, while there were no cases of grade - acute gvhd. the incidence of mild, moderate and severe chronic gvhd was %, % and %, respectively. the rate of grade non-hematological toxicities was %. one patient died of late bacterial infection. the incidence of trm was %. grade adverse events were not reported. disease status months after allohsct was: cr- , vgpr- , pr- . the probability of os and pfs after months was % (+/- %) and % (+/- %), respectively. the incidence of progression and trm was % and %, respectively. conclusions: allohsct using tmi gy + bendamustine conditioning protocol is characterized by good tolerance and low risk of gvhd. it may be used for younger patients with mm as part of tandem auto-allohsct strategy. encouraging results reported in this study should be confirmed in prospective clinical trials. disclosure: nothing to declare p comparison between two reduced intensity conditioning regimens in patients with a myeloid malignancy: a single center experience comparing fb with flumel background: hematopoietic stem cell transplantation (hsct) remains the only curative option for high-risk myeloid neoplasms. the optimal reduced-intensity conditioning (ric) is still debated. methods: a single-center retrospective analysis was conducted at our institution to compare two different ric regimens in adult patients transplanted for myeloid malignancy from to . a total of patients were analysed, of them treated with busulfan-based (fludarabine mg/m , busulfan . mg/kg, fb ) and with melphalan-based conditioning regimen (fludarabine mg/m ,melphalan mg/m , flumel). antithymocyte globulin (atg) was administered in all patients while no one received tbi. partial in vitro t-cell depletion was performed using alemtuzumab for low risk patients. results: the two groups were well balanced with a median age of and years in the fb and flumel group, respectively, and a median follow up of months. the most frequent indication for transplant in both groups was aml ( . and . % for fb group and flumel group, respectively) and the stem cell source was peripheral blood in . and . % of patients. more patients in the first group had near to significant worst karnosfky status (< ) at transplant compared to second ( . vs %, p= . ) and more patients received a t-partial depleted graft ( . vs . %, p= . ). the neutrophil engraftment was significantly shorter after flumel ( vs days, p < . ). the -year overall survival (os) and disease-free survival (dfs) were of . and . %, respectively, after fb and . and . % after flumel, respectively, and were not significantly different (p=. for os and . for dfs), with a karnofsky >= being the only factor significantly associated in univariate analysis with better os and dfs (p=. for both). the cumulative incidence (ci) of grade to acute graft-versus-host disease (agvhd) was . % after fb and . % after flumel (p< . ) and was associated in multivariate analysis with both t depletion and ric type (p< . and . , respectively). the ci of chronic gvhd at years was . % in fb and . % in flumel group (p=. ) . the ci of non-relapse mortality at years was . % after fb and . % after flumel (p=. ). the ci of relapse at years was . % for the first and . % for the second group (p< . ) and was associated with conditioning regimen in multivariate analysis (p=. ). no difference in -years gvhd-free/ relapse-free survival (grfs) was observed between the two group ( . % for fb and . % for flumel, p=. ). conclusions: when comparing two ric regimens for myeloid neoplasms, we observed a higher incidence of agvhd after flumel whereas no statistical difference was noted for the cgvhd occurrence. while the toxicity appears to be higher after flumel, this result is counterbalanced by a higher proportion of relapse after fb , accounting for no difference in os, dfs and grfs between the two groups. these findings could be partially explained by a larger proportion of patients receiving a partial t-depletion after fb ric, but a larger trial is needed to clarify this issue. disclosure: nothing to declare. once-daily vs -times daily intravenous busilvex in conditioning regimen before allogeneic stem cell transplantation for patients with myeloid malignancies: safety and efficacy background: busilvex (bu) is part of standard conditioning regimen before allogeneic stem cell transplantation (asct) for patients with myeloid malignancies and usually administered as an intravenous (iv) infusion -times daily. this study aimed to compare the saftey and efficacy of this schedule to a once-daily iv bu. we conducted a retrospective study in adult patients (≥ years) with myeloid malignancies who received asct from hla-identical sibling donors between january and june following iv bu-based preparative regimens. graft-versus host disease (gvhd) prophylaxis consited of cyclosporine and short course of methotrexate. intravenous bu was administered -times daily ( . mg/kg every hours x to doses) or oncedaily in a -hour infusion ( . mg/kg x to days) since june . results: ninty-nine patients were enrolled ( men and women). median age was years (range, - y). the median time from diagnosis to asct was months (range, days - years). diagnosis were acute myeloid leukemia (n= , %), chronic myeloid leukemia (n= , %), myelodysplasic syndrome (n= , %), primitive myelofibrosis (n= , %) and chronic myelomonocytic leukemia (n= , %). thirty-seven ( . %) patients had ebmt-score ≥ . sixty-five ( . %) patients were transplanted in cr , ( %) beyond cr and ( . %) had active disease. conditioning regimens consisted of bu/cyclophosphamide in patients ( . %), bu/fludarabine in patients ( . %). four-times daily bu was given to patients ( . %, groupe ) and once-daily bu to patients ( . %, groupe ). stem cell source were bm in patients ( . %) and pbsc in patients ( . %). globally, patients characteristics were well balanced between the two groups. the rates of severe complications were similar between the two groups with no statistically significant differences except oral mucositis (table ). non-relapse mortality (nrm) was comparable in the two groups ( % and % in groups and , respectively, p= . ). the relapse rate was % and %, respectively (p= . ). after a median follow-up of years (range, days - years), the os was not significantly different between groups and : % vs % (p= . ). however, the rfs was significantly better in the groupe : % vs % (p= . ). conclusions: once-daily iv bu regimen seems to be an efficient and safe alternative to the -times daily protocol. however, results should be interpreted with caution because the historical comparison and lack of bu pharmacokinetics studies. disclosure background: standard therapy of the most patients with juvenile myelomonocytic leukemia (jmml) is allogeneic hematopoietic stem cell transplantation (ahsct). the choice of optimal conditioning regimen for patients with jmml is crucial as well as long-term observation. we aimed to estimate the long-term follow-up and survival rates of patients with jmml after ahsct with the help of busulfan or treosulfan-based conditioning regimens. methods: thirty eight patients with jmml underwent ahsct in - . we compared equal groups of patients received busulfan (n= ) and treosulfan-based (n= ) conditioning regimen. m:f= : . median of age at hsct was . ( . - ). donor type: hla-related / - % (n= ), hla-related / - . % (n= ), hlaunrelated / - . % (n= ), hla-unrelated / - . % (n= ), and haploidentical - . % (n= ). stem cell source: bm - . % (n= ), pbsc - . % (n= ), ucb - , % (n= ), and ucb+bm - . % (n= ) . disease status on hsct: cr - . % (n= ), refractory - . % (n= ). results: median follow-up . months ( - months) . the estimated -year overall survival (os) probability in patients received busulfan-based conditioning was , ± , % in comparison with , ± , % in patients with treosulfan-based regimen (р= , ). event-free survival (efs) was , ± , % in group with busulfan-based regimen and , ± , % in patients with treosulfanbased conditioning (р= , ). background: post-transplant relapse remains the leading cause of treatment failure in high risk (hr) acute myeloid leukemia (aml), myelodysplastic syndrome (mds), myeloproliferative neoplasia (mpns) receiving allogeneic hematopoietic cell transplantation (allo-hct), especially for patients with relapsed or refractory aml. recently, a sequential transplant approach, as developed by the munich group, comprising of intensive cytoreductive chemotherapy flamsa (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, has been successfully used for high-risk (hr) aml/mds with promising results. methods: we studied patients (median age years, range - ) with hr aml (n= ), as defined by refractory, relapsed disease, secondary leukemia, or high/ very risk disease risk index risk, and hr mds (n= ) according to ipss-r, undergoing allo-hct using the sequential transplant approach in institutions between january and october . the sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either flamsa (n= ), flag +/-ida (fludarabine/cytarabine/granulocyte colony stimulating factor /idarubicin) (n= ), or clo-arac (clofarabine/cytarabine) (n= ), followed by reduced (ric) (n= ) or myeloablative (mac) (n= ) conditioning regimen. all patients received peripheral blood stem cell from matched related donors (n= ) matched unrelated donors (n= ), or mismatched unrelated donors (n= ). post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. thymoglobulin was added for gvhd prophylaxis for unrelated donor transplant. results: the median time to neutrophil > /μl was days (range, - ) . with a median follow-up of . months (range, . to . months), the kaplan-meier estimate of leukemia-free (lfs) and overall survival (os) at years were % ( % ci, - ), % ( % ci, - ), respectively. patients receiving flag or clo-arac based sequential regimen showed a trend towards more favourable overall survival (os) as compared to patients given flamsa ( year os: % vs %; p= . ). at years, the cumulative incidences of relapse and non-relapse mortality (nrm) were % ( % ci, - %) and % ( % ci, - %), respectively. in multivariate analysis, the type of sequential conditioning regimen did not show any significant impact on lfs, os, nrm or relapse. conclusions: sequential transplant conditioning with flamsa, flag or clo-arac followed by allo-hct is an effective strategy in overcoming the dismal prognosis of hr aml and mds, and enabling long-term disease free survival. more studies on effective strategies such as posttransplant maintenance therapy of prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. disclosure: nothing to declare optimization of the blood sampling procedure for busulfan therapeutic drug monitoring (tdm) to optimize our sampling scheme ( minutes, , , , , , and hours after the end of a -hour infusion), we reduced the number of blood samples collected, reducing nursing and laboratory staff time and increasing patient convenience. this study aims to show the performance of a simplified sampling protocol which includes the first samples from the original protocol. methods: individual pk parameters were retrospectively estimated using samples (simplified protocol) and were compared with those obtained after samples (original protocol). individual pk parameter values for a one compartment model were estimated using a maximum likelihood estimation modelling algorithm (adapt . ) and the statistical analysis of the results was performed (statgraphics centurion xv). results based on the approved dosage recommendations, mean (sd) initial dose was . ( . ) mg. after tdm, mean (sd) calculated dose at day for the remaining days (to achieve the defined target cumulative auc) was , ( , ) mg obtained from the original protocol. according to the simplified protocol the result would be , ( , ) mg. the median and the mean variation of the calculated dose were % and % ( - %) between protocols. a strong relationship between the cl of the day - obtained from the original protocol and the simplified protocol is observed (r = . ). this high correlation is also observed for patients with busulfan t / > h (r = . ), a population were the reduction of sampling could be more problematic. anova test for the log cl with the factors: patient, day of busulfan and type of sampling protocol was performed. sampling protocol was determined as non-statistically significant (p = . ). conclusions: results suggest that both protocols are equivalent concerning to the busulfan cl estimation and calculated auc. variation between protocols regarding the calculated dose at day for the remaining days to achieve the defined target cumulative auc is considered acceptable. we verified a strong relationship between busulfan cl obtained from both protocols and sampling protocol doesn't influence cl statistically. a reduced sampling collection of determinations until h after the end of the infusion is shown to be sufficient for the tdm of busulfan, so this was implemented in our centre in line with published data. disclosure: nothing to declare p impact of anti-thymocyte globulin doses in unrelated hematopoietic stem cell transplantation for patients with myeloid neoplasm background: anti-thymocyte globulin (atg) is widely used for the prophylaxis of graft-versus-host disease (gvhd) in hematopoietic stem cell transplantation (hsct). however, there is still controversy regarding the optimal dose of atg. therefore, we analyzed the impact of atg doses in unrelated hsct for patients with myeloid neoplasm. methods: this was a retrospective multi-center study that assessed the impact of atg doses on clinical outcomes in patients with acute myeloid leukemia (aml) or myelodysplastic syndrome (mds) undergoing an unrelated hsct. the patients who received peripheral blood stem cells (pbsc) transplantation after conditioning regimens containing i.v. busulfan (bu), fludarabine and rabbit atg between and were included in this study. results: a total of patents, median age years, with aml (n= ) or mds (n= ) were included in our analyses. patients ( %) received a myeloablative regimen (i.v. bu> . mg/kg). high-atg (atg mg/kg), intermediate-atg (atg . - mg/kg) and low-atg (atg mg/kg) were given in , and patients, respectively. after a median follow-up of months, the cumulative incidence of extensive chronic gvhd was . % in the high-atg group, . % in the intermediate-atg group and . % in the low-atg group (p= . ). conclusions: our study shows that the incidence of extensive chronic gvhd was similar regardless of the doses of atg after transplantation of pbsc from unrelated donor for patients with aml or mds. however, the rate of relapsefree survival and the rate of a composite end point chronic gvhd-free and relapse-free survival were significantly higher in the intermediate dose ( methods: we retrospectively retrieved data from the electronic medical records for consecutive patients aged and older, who underwent an asct for lymphoma over the last years at our institution. results: forty four patients ≥ years old underwent asct between aug and aug . twenty eight of them received a reduced-dose conditioning (median %, range %- % dose reduction). the dose was reduced for % of patients ≥ years old and for % of patients aged - . the outcomes of the following three groups of patients were compared: a) age ≥ ; without dose reduction, b) age - ; with dose reduction and c) age ≥ ; with dose reduction (table ). only one patient aged received full-dose conditioning. there was no significant difference between the groups in the number of previous chemotherapy cycles (median , range - ). however, significantly more patients at the age of - were in complete remission (cr) pre-transplant in both full and reduced-dose conditioning groups (a and b). no significant intergroup differences were observed in the occurrence of complications (mucositis and infections), day transplantrelated mortality (trm) or engraftment day. similarly, no significant differences were found either in the -year progression-free survival (pfs), which was %, % and %, or -year non-relapse mortality (nrm), which was %, % and %, respectively for groups a, b and c. the -year overall survival (os) tended to be higher in group b ( %), compared to groups a ( %) and c ( %). conclusions: beam/beac conditioning dose reduction was not found to adversely affect -year pfs and os rates. despite the fact that / of the patients in the age group ≥ underwent asct in partial remission and had dose reduction, theier achieved trm, pfs and os rates were similar to those of patients aged - . beam/beac conditioning at a %dose may be a suitable option for patients in their seventh decade requiring asct. this strategy should be further evaluated in prospective clinical trials. background: the transplant related mortality in autologous transplants for lymphoma and multiple myeloma, reported worldwide ranges from - %. from - , the trm at our center for these two diseases was approximately %. we introduced changes in mobilization schedule, conditioning regimens and drug dosages to determine whether these changes affected the transplant related mortality and overall survival. methods: from april -december , we used beam (bcnu: mg/m on day - ; etoposide mg/ m on days - to - , cytarabine mg/m on days - to - and melphalan mg/m on day - as conditioning chemotherapy for patients admitted in transplant unit for autologous transplants in hodgkin's and non-hodgkin's lymphoma. in patients with multiple myeloma high dose melphalan ( mg/m ) was used. the mobilization protocol consisted of cyclophosphamide . gm/m followed by gcsf μgm/kg twice daily till stem cell collection was completed. from january , we changed the beam protocol to bendaeam with dose modifications that included: bendamustine mg/m on days - and - , cytarabine mg/m on days - to - , etoposide mg/m on days - to - and melphalan mg/m on day - . for multiple myeloma melphalan was reduced to mg/m . we used only gcsf for mobilization of stem cells, which was continued till stem cell harvest was complete. response to treatment was evaluated by comparing trm and overall survival for two time periods: - and from till date. results: from april till december , n= autologous transplants were performed. the male:female ratio was . : . fifty seven patients underwent transplant for lymphomas, n= for multiple myeloma and n= for other diagnosis. median age was ± . ( - years). the mean mnc was . × ± . /kg. engraftment was achieved in % of patients. the transplant related mortality was . % and overall survival was % (follow up: months). since january till march we have performed n= autologous transplants of which n= were males. fifteen transplants were performed for lymphomas (nhl: , hd: ) and n= for multiple myeloma. median age was ± ( - years). the mean mononuclear cell count was . x /kg and the mean cd count was . x /kg. engraftment was achieved in all patients. the transplant related mortality was % and the overall survival was % (follow up months). conclusions: we were able to reduce the autologous transplant related mortality to % by decreasing dosages of conditioning chemotherapy and changing the mobilization protocol. long follow-up is needed to determine late mortality and late relapse in comparison to standard chemotherapy dosages disclosure: nothing to declare risk and benefit of thiotepa based conditioning followed by autologous stem cell transplantation in high risk lymphomas years ( - ) . stage (ann-arbor) at diagnosis of hd/dlbcl/pcnsl: stage ie n= / / ( / / %), stage ii n= / / ( / / %), stage iii n= / / ( / / %), stage iv n= / / ( / / %). median time from diagnosis to asct hd/dlbcl/pcnsl: / / month ( - ). induction treatment in hd patients was abvd, in most dlbcl patients r-chop and in pcnsl patients high dose methotrexate and cytosin arabinoside. tumor status at asct hd/dlbcl/pcnsl: complete metabolic remission (cmr) n= / / ( / / %) and from pcnsl patient's n= ( %) were in first complete remission (cr ). type of stem cell graft was periferial blood stem cell in all case. conditioning: thiotepa ( mg/ m on days - to - , busulphan , mg/kg on days - to - and cyclophosphamide mg/kg on days - to - plus rituximab mg/m on day - in dlbcl and pcnsl. median follow up from asct days . tumor stage at asct was defined with computer tomography with positron emission tomography (pet-ct). results: median time of engraftment was days ( - ). thiotepa caused toxicoderma appeared at ( %) patients. cytomegalovirus (cmv) reactivation was seen in ( %) cases with low dna content ( , , copies/ml) and responded completely to oral valgancyclovir therapy. transplantation related mortality hd/dlbcl/pcnsl n= / / ( / / %), in cases bacterial sepsis and one systemic mycoses and one pulmonary fibrosis. incidence of long-lasting grade iii-iv thrombocytopenia and anaemia: n= ( , %) and n= ( %), median time of duration from transplantation days ( - ) and days ( background: this study evaluated the efficacy and toxicity of intravenous busulfan and thiotepa as a conditioning regimen for autologous stem cell transplantation (asct) in patients with multiple myeloma (mm). methods: we retrospectively analyzed the data of patients with mm who received the intravenous busulfan and thiotepa conditioning for asct between november and april in korea. results: the median time to transplant was . months, and patients ( . %) underwent asct within months of the diagnosis. the overall response rate after asct was . %, including . % with complete response, . % with very good partial response, and . % with partial response. the most common severe non-hematologic toxicity (grade - ) was infection ( . %). three patients ( . %) developed venous-occlusive disease. one patient ( . %) died due to severe pneumonia after asct. after a median follow-up of . months, the median progression-free survival (pfs) and overall survival (os) were not reached. conclusions: in conclusion, a conditioning regimen of intravenous busulfan and thiotepa was effective and tolerable. clinical trial registry: not applicable disclosure: the authors have declared no conflicts of interest. myeloablative haploidentical bone marrow transplantation with post-transplant cyclophosphamide in paediatric patients with haematological malignancies santanu sen , sameer tulpule background: haploidentical transplants have been shown to be safe and effective in treating haematological malignancies in the paediatric population. we have previously reported on our experience of using reduced intensity conditioning with post transplant cyclophosphamide in haploidentical patients. we herein report our experience of using a tbi based myeloablative conditioning to treat our first patients with haematological malignancies. methods: patients were enrolled in the study, with relapsed acute lymphoblastic leukemia (all) and with relapsed/resistant acute myeloid leukemia (aml). all aml patients had genetic markers of high risk disease and all all patients had very early relapses (either on therapy or within months of stopping therapy). all patients were conditioned with an identical protocol using tbi-based myeloablative preparative regimen (fludarabine mg/m /d × d and tbi cgy bid on d − to − [total dose cgy]) followed by an infusion of unmanipulated peripheral blood stem cells from a haploidentical family donor. postgraft immunosuppression consisted of cyclophosphamide mg/kg/day on days and , mycophenolate mofetil through day , and tacrolimus through day . results: median time of neutrophil and platelet engraftment was and days, respectively. all patients achieved sustained complete donor chimerism by day + . acute gvhd, grades ii-iv and iii-iv, was seen in % and %, respectively. disease progression occurred in patients: & months after transplant and there was one death due to severe fungal infection. estimated twoyear survival and relapse were % and %, respectively. patients had severe bk viremia and cmv reactivation occurred in patients. all patients were successfully managed with appropriate supportive and antiviral therapy. conclusions: we report good outcome with a myeloablative conditioning in haploidentical transplants with excellent engraftment and hopefully a longer life expectancy. with small number of patients, it is difficult to state whether using a myeloablative conditioning would lead to better long term outcomes in this cohort of patients with very haematological malignancies, but we certainly showed that it is possible to achieve excellent early results. disclosure: nothing to declare fludarabine in combination with melphalan and atg can be the best conditioning for hematopoietic stem cell transplant of children with hemophagocytic lymphohistiocytosis methods: in this prospective study, we analyzed the outcome of two pediatric patients with hlh who had received hsct, using reduced-intensity conditioning (ric) regimen. they received the same ric regimen based on the use of fludarabine ( mg/m /day for days) in combination with melphalan ( mg/m /day for days) and horse antithymocyte globulin (atg mg/kg/d for days). cyclosporine and methotrexate were used as graft-vs.-host disease (gvhd) prophylaxis. results: a months boy with primary hlh (fhl ) was transplanted from his mother and a years girl with secondary hlh was transplanted from her brother. both of donors were hla match with their recipients. they were received x /kg and x /kg cd + cells from the harvested peripheral blood stem cells, respectively. they achieved full neutrophil and platelet recovery. the time to neutrophil recovery was and days, respectively. full chimerism was achieved for both of them. in addition, they was developed grade and of acute gvhd, respectively. gvhd was completely controlled with prednisolone. they are alive and in complete remission without any significant complications after and months, respectively. conclusions: it appears that fludarabine in combination with melphalan and atg may be the best conditioning regimen for hematopoietic stem cell transplant of children with hlh. due to a few number cases of this study, a study with sufficient sample size is required. disclosure background: hematopoietic cell transplant (hct) recipients often report depression and impaired quality of life (qol) before transplant. mixed evidence suggests depression may be a risk factor for greater mortality and worse qol. inconsistent findings may be due to the fact that previous studies have not evaluated antidepressant use. the aim of the study was to compare pre-transplant patientreported physical functioning and post-transplant overall survival (os) between four groups of hct recipients: ) non-depressed/taking antidepressant (treated depression), ) depressed/taking antidepressant (undertreated depression), ) depressed/not taking an antidepressant (untreated depression), and ) not depressed/not taking an antidepressant (control). it was hypothesized that physical functioning and os would be worse among patients with untreated and undertreated depression relative to those with treated depression and controls. methods: this retrospective case-control study included patients completing depression (phq- ) and quality of life (sf- ) questionnaires at pre-transplant. analyses were conducted separately for allogeneic and autologous recipients. results: participants (n= , ) were % men, mean age years ( - ), % allogeneic recipients. regarding depression and antidepressant use, ( %) allogeneic patients were characterized as having treated depression, ( %) as untreated depression, ( %) as undertreated depression, and ( %) as controls. hierarchical linear regression models indicated that after adjusting for significant univariate factors (performance status, disease status, and regimen intensity), allogeneic patients with treated depression (b=- . , % ci=- . , - . ) reported better physical functioning than patients with undertreated depression (b=- . , % ci=- . , - . ) and untreated depression ) but worse physical functioning than controls (p values < . ). cox regression models indicated depression/antidepressant usage was not associated with os among allogeneic patients (p values> . ).among autologous patients, ( . %) were characterized as having treated depression, ( . %) as untreated depression, ( . %) as undertreated depression, and ( . %) as controls. hierarchical linear regression models indicated that after controlling for significant univariate factors (gender, performance status, diagnosis, and disease status), autologous patients with treated depression (b=- . , % ci=- . , - . ) reported better physical functioning than patients with undertreated depression (b=- . , % ci=- . , - . ) and untreated depression (b=- . , % ci= . , - . ), but worse physical functioning than controls (p values < . ). cox regression models showed depression/antidepressant usage was associated with os (p values < . ), with patients with treated depression demonstrating significantly worse os than other groups (p= . ), but this association was no longer significant in multivariate analyses controlling for diagnosis and disease status (p= . ). conclusions: patients with untreated or undertreated depression pre-transplant may benefit from depression screening and treatment to improve physical functioning. disclosure: hslj: consultant for redhill biopharma and janssen scientific affairs p eltrombopag (epag) induces a high percentage of responses in patients with post allo-hsct poor graft function (pgf) and no active gvhd lourdes aguirre , aitziber lizardi , pilar bachiller , brigida esteban , carmen gonzález , nagore argoitia , maría araiz , aranzazu aguirre , anunciación urquía , carlos vallejo background: persistent cytopenia is a life-threating complication after hsct. several causes can lead to this situation (viruses, gvhd, drugs, etc) . a specific entity is the one called "poor graft function (pgf)", which is diagnosed in pts with ≥ cytopenias after day + , in the presence of donor chimerism and the absence of gvhd or relapse. pgf is more frequent after alternative allo-hscts, such a haplo-identical, mismatched, or ucb. several therapeutic approaches for pgf, with poor results, have been tested. recently, epag has been shown to improve platelet counts in the post-allo-hsct setting. in this study, we analysed the efficacy of epag in pts with post-transplant persistent cytopenias. methods: the population analyzed includes all pts who underwent allo-hsct from june through may in our unit. median age was years ( - ). were male ( . %) and female ( . %). baseline diseases were: aml, lpd, all, mds, mpd, mm, and bmf. donor was unrelated in ( . %) and was family in ( . %) (including haplo-identical). conditioning was ric in ( . %) and intensive in ( . %). sc source was pb in ( . %) and bm in ( . %). median followup was months ( - ). epag was initiated at some point during the first -month post-hsct period in pts ( . % of the series) due to thrombocytopenia (< /mcl) plus, at least, one other cytopenia. patients characteristics shown in table . epag was started at mg/day and escalated each weeks to , and mg/day if platelet count was < /mcl. global response was considered when, after epag, the patient needed no transfusions and reached the three of the following: platelets > /mcl, hgb > g/dl, and anc > /mcl. epag was tapered off in responders and discontinued if no response was reached after weeks. results: at epag initiation, all the pts had thrombocytopenia (< /mcl), had anemia (hgb < g/dl), and had neutropenia (anc < /mcl). counts pre and post and response to epag are shown in table . among the responders, all but one (who relapsed from thrombocytopenia and died from bleeding) were alive at analysis close ( . %). among the non-responders, three pts had gvhd-associated cytopenias, and finally died from infectious complications; the other patient relapsed from her aml, reached a new cr after treatment, and is alive and well months afterwards. epag was tapered off and discontinued in / pts who responded; / responders are still on epag. epag was discontinued in the / pts who did not respond. rest of treatment details shown in table . conclusions: ) epag worked striking well in subjects with pgf, an otherwise a life-threatening situation for patients. ) epag induced impressive responses in platelets, but strong bilinear and trilinear responses were also seen. ) epag did not improve gvhd-associated cytopenias. ) to confirm these innovative and transcendent results, we have just initiated a multicenter prospective study on the role of epag for treatment of post-hsct pgf. * five out of the six urdt were mismatched ** the donor was a woman in the six cases: three sisters and three daughters background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with > % mortality. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged > month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the us. this analysis provides an overview of the safety results from studies of patients with vod/sos, with or without mod, who received defibrotide mg/kg/day. methods: safety data were pooled from patients with vod/sos post-hsct treated with defibrotide in a phase trial (n= ) and a phase , randomized dose-finding trial (n= receiving mg/kg/day). safety data for historical controls (hc) from the phase study (n= ) also are provided. reported separately, due to differences in patient population and data monitoring protocol, are aes from the expanded-access program (t-ind) in patients with vod/ sos with and without mod (n= post-hsct). vod/ sos was diagnosed by baltimore criteria/biopsy for the phase / studies; diagnosis by baltimore or modified seattle criteria was permitted in the t-ind. results: median patient age at hsct for the phase / studies was . years, . years for the hc, and . years for the t-ind. in the phase / studies defibrotide-treated group (n= ), ( . %) experienced aes; most common (> %) were hypotension ( . %), diarrhea ( . %), and multi-organ failure ( . %). treatment-related aes were at least possibly related to defibrotide (table) . any hemorrhage (an ae of special interest) occurred in patients ( . %); most commonly epistaxis ( . %), gastrointestinal and pulmonary alveolar hemorrhage and hematuria ( . % each), and conjunctival hemorrhage ( . %). all hc experienced an ae; most common (> %) were hypotension ( . %), tachycardia ( . %), diarrhea ( . %), nausea ( . %), and pyrexia, agitation, and petechiae ( . % each). any hemorrhage occurred in patients ( . %): most common (> %) were petechiae ( . %); hematuria, epistaxis, and pulmonary alveolar hemorrhage ( . % each); and lip hemorrhage ( . %). in the t-ind (n= ), / patients with mod ( . %) and / patients without mod ( . %) had an ae; other than vod/sos and mod, most commonly (> % in either subgroup) hypotension ( . % and . %, respectively). traes occurred in patients ( . %) ( table) . any treatment-emergent hemorrhage occurred in patients with mod ( . %) and patients without mod ( . %); most commonly (> % in either subgroup) pulmonary hemorrhage ( . % and . %, respectively) and gastrointestinal hemorrhage ( . % and . %, respectively). conclusions: the incidence and type of aes were as expected in these critically ill patients. of the pooled patients, % had aes; . % had a hemorrhage. all hcs had an ae, with . % having a hemorrhage. in the t-ind, patients with mod had higher rates of aes. support: jazz pharmaceuticals event, n(%) phase / studies (n= ) disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p ca ; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. defibrotide for treatment of adults with hepatic vod/ sos with or without multiorgan failure after hematopoietic cell transplantation: results of a systematic review/meta-analysis background: although hematopoietic cell transplantation (hct), autologous or allogeneic, is potentially curable in various hematologic malignancies, the procedure is associated with serious and potentially life-threatening complications, among them veno-occlusive disease/sinusoidal obstructive syndrome (vod/sos) of the liver. several studies, prospective or retrospective, have reported outcomes of defibrotide, when used as prophylaxis or treatment, in a mixed population of adult and pediatric patients. in this systematic review/meta-analysis, we analyze outcomes of defibrotide when specifically used for treatment of adult patients with hepatic vod/sos with or without multiorgan failure. methods: a comprehensive search of large databases (medline/pubmed, cochrane and embase) on november , identified publications. analysis was restricted only to adult patients (defined as median age older than years) who received defibrotide for treatment of vod/sos and were reported in prospective or retrospective (which included ≥ patients) studies published in full manuscript form. there were no limitations based on language. data were extracted in relation to benefits [complete remission (cr) rate and overall survival (os)] and harms (hemorrhage, any site or organ-specific). a total of studies (prospective= ; retrospective= ) with patients met inclusion criteria. results: the median year of publication of prospective studies was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and for retrospective ones ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the prescribed starting dose of defibrotide varied among studies ranging from . mg/kg/day to mg/kg/day, mostly for a -day course. the pooled cr rate was % ( %ci= - %) for prospective and % ( %ci= - %) for retrospective studies. the pooled day + os rates were % ( %ci= - %) and % ( % ci= - %) for prospective and retrospective studies, respectively. the pooled rates of hemorrhage (any site) were % ( %ci= - %) for prospective and % ( % ci= - %) for retrospective studies. when analyzing organ-specific hemorrhage, prospective studies (n= patients) reported pooled rates of pulmonary alveolar (pa) hemorrhage of % ( %ci= - %) and of % ( %ci= - %) for gastrointestinal (gi) hemorrhage. only one retrospective study (n= patients) reported an incidence of pa hemorrhage of % ( %ci= - %) and a different study (n= patients) reported an incidence of gi hemorrhage of % ( %ci= - %). none of the studies reported cerebral hemorrhage as a complication of defibrotide therapy. conclusions: this systematic review/meta-analysis confirms the efficacy of defibrotide for treatment of vod/sos with or without multiorgan failure, yielding cr rates of - % and day + os rates of - %. the purportedly higher pooled cr and os rates observed with retrospective (vs. prospective) studies are likely due to assignment-bias inherent to observational studies. moreover, although the pooled hemorrhage (any site) rates of - % is considered proportionally significant, the pooled rates of pa and gi hemorrhage were ≤ %, in prospective studies. clinical trial registry: not applicable disclosure: m.a.k-d: consultancy for pharmacyclics m.m: received lectures honoraria and research support from jazz pharma efficacy and safety of defibrotide in the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome following hematopoietic stem cell transplantation: interim results from the defifrance study background: hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially lifethreatening complication of conditioning for hematopoietic stem cell transplant (hsct) but may occur after nontransplant chemotherapy alone. vod/sos with multi-organ dysfunction (mod) may be associated with > % mortality with supportive care alone. diagnosis of vod/sos was traditionally based on baltimore or modified seattle criteria; however, the ebmt recently published separate diagnostic criteria for adults and children. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged > month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the usa. the goal of the defifrance study, requested by the french health authorities, is to collect real-world data on safety and efficacy in a broader patient population in france, including all indications. this is the first interim analysis of the largest current evaluation of defibrotide for the treatment of vod/sos in europe. methods: defifrance is an observational, multicenter, post-marketing study that includes any patient treated with defibrotide from hsct centers in france. this interim analysis is based on all patients treated with defibrotide, including those with severe and very severe post-hsct vod/sos. vod/sos was diagnosed using traditional criteria. day+ survival, complete remission (cr; total serum bilirubin < mg/dl and resolution of mod), and safety profile are reported. results: a total of patients treated with defibrotide were included retrospectively and prospectively between july and october from table] disclosure: mohamad mohty: has received honoraria and research funding from jazz pharmaceuticals, delphine lebon: nothing to disclose, ann berceanu: none, charlotte jubert: has received funding from jazz pharmaceuticals, ibrahim yakoub-agha: has received honoraria from jazz pharmaceuticals, stéphane girault: none, marie detrait: has received research funding from jazz pharmaceuticals, cécile pochon: none, fanny rialland: none, virginie gandemer: none, jean-hugues dalle: has received honoraria from jazz pharmaceuticals, régis peffault de latour: has received research grant / honoraria / board from pfizer, novartis, alexion; research grant amgen; and honoraria from jazz pharmaceuticals, david michonneau: has received honoraria from jazz pharmaceuticals, myriam labopin: has received honoraria from jazz pharmaceuticals, floriane delaval: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, gerard michel: none, anne sirvent: none, laurence clement: none anne-lise menard: none, anne huynh: has received honoraria from jazz pharmaceuticals, virginie bouvatier: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, raj hanvesakul: employee of jazz pharmaceuticals and holds stock and/ or stock options in jazz pharmaceuticals plc, zakaria medeghri: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc p incidence and predictors of severe cardiotoxicity in patients with severe aplastic anemia after haploidentical hematopoietic stem cell transplantation zheng-li xu , lan-ping xu , yuan-yuan zhang , yi-fei cheng , xiao-dong mo , feng-rong wang , yu-hong chen , wei han , chen-hua yan , yu-qian sun , ting-ting han , yu wang , xiao-hui zhang , xiao-jun huang peking university institute of hematology, peking university people's hospital, beijing, china background: severe cardiotoxicity after hematopoietic stem cell transplantation (hsct) is a rare but fatal complication. the aim of this study was to evaluate the frequency of severe cardiac complications and to assess the ability of various factors to predict these complications in patients with aplastic anemia after haploidentical transplantation., this is the first study evaluating the values of both clinical and imaging factors in the prediction of severe cardiotoxicity among saa patients after haploidentical transplantation. methods: a retrospective study was conducted in consecutive aplastic anemia patients who received haploidentical transplantation from to . all patients received a unified regimen including busulfan, cyclophosphamide (ctx) and antithymocyte globulin at our single center. results: a total of ( . %) patients developed grade iii or iv cardiac toxicity. patients with cardiotoxicity had significantly poorer overall survival (os) than those without cardiotoxicity ( . % vs. . %, p< . ). our multivariable model identified four independent adverse predictors of severe cardiotoxicity, including pre-transplant ecog score (≥ ), abnormal st-t wave on -lead electrocardiogram (ecg), hyperlipemia and recalculated ctx dose (≥ . g/m /d). a predictive risk model was refined as low risk ( - factor), intermediate risk ( factors) and high risk ( - factors) . the respective incidences of severe cardiotoxicity were . %, . %, and . % in the high-, intermediate-and low-risk groups (p< . ). the corresponding os rates were . %, . %, and . % in the three groups (p< . ) at the last follow-up. conclusions: patients with high risk scores had the poorest outcomes and should be monitored closely. a reduced intensity conditioning might be recommended for these patients. disclosure: there are no conflicts of interest to declare. background: allogeneic stem-cell transplantation (allo-sct) is associated with significant transplant-related mortality (trm). acute renal failure (arf) is a frequent complication and usually presents early after the procedure, compromising its feasibility. the aim of this study is to analyse the incidence of arf, its risk factors and its potential impact on trm after allo-sct. methods: patients were included ( males [ %]; median age years, range - ) treated with allo-sct consecutively between january and april in a single institution. patient characteristics are detailed in table . median follow-up was . years (range, . - . ). renal function was evaluated using creatinine and data was collected pre-transplant (baseline) and at the point when arf was developed after allo-sct. arf was evaluated using akin criteria, being akin- an increase . -to . -fold from baseline, akin- an increase . -to . -fold and akin- an increase ≥ -fold. chronic renal disease was evaluated one year after the date of arf using kdigo criteria. results: cumulative incidence of arf at year was % (akin- , %; akin- , %; akin- , %). in the multivariate analysis, arf (akin- / ) was associated with: non-use of antithymocyte globulin in conditioning chemotherapy, p= . (hr . , . to . ) and development of severe agvhd, p= . (hr= . , to . ). in patients with arf akin- , the most important variables in the multivariate analysis were: use of methotrexate (mtx) plus cyclosporine vs mycophenolate mofetil plus cyclosporine as gvhd prophylaxis, p= . (hr= . , . to . ); myeloablative conditioning vs reduced intensity, p= . (hr= . , to . ) and use of total irradiation therapy in conditioning, p= . (hr= . , . to . ). trm at year increased significantly according to akin: akin- , %; akin , %; akin , %; p= , ; hr= . . overall survival at years according to akin was: akin , %, akin , % and akin , %; p= , (figure ). the incidence of chronic renal disease at year after allo-sct according to arf was: no arf ( %), akin- ( %), akin- ( %) and akin- ( %); p= . . conclusions: arf is a frequent complication during the first year after allo-sct and is associated with several factors. arf akin- was associated with more intensive strategies received during conditioning, meanwhile akin- / were related to development of gvhd. there is an association of arf (akin- , or ) with development of chronic renal disease. background: the introduction of cellular therapies such as car-t and modalities of gvhd-prophylaxis with posttransplant/cyclophosphamide (ptcy) that increase the number of admission days have boosted the pressure of available beds in the bm-units. in this sense, our centre started an at-home allogeneic stem cell transplantation (allo-sct) program to follow aplasia from the d+ until independent ambulatory patient. to evaluate the feasibility and safety of allosct, we compared two groups: allohsct/athome (ah-group) vs. allohsct/in-patient (ip-group). methods: we included patients receiving allosct (january -november ) in a single centre: patients, ah-group and , ip-group. all patients received conditioning at the hospital. gvhd-prophylaxis consisted in tacrolimus (tk) plus mycophenolate (mpm) or methotrexate, or ptcy (d+ , d+ ) plus tk (d+ ). all patients received prophylaxis with levofloxacin, fluconazole and acyclovir. besides that, ah-group patients received prophylaxis with ceftriaxone g/ h iv or ertapenem g/ h iv, and aspergillus-prophylaxis with inhaled liposomal amphotericin-b or posaconazole during neutropenia. patients of ah-group since d+ or d+ (in ptcyprophylaxis) received a nurse visit at-home once daily. the visits by the physician were performed at the hospital and only during complication events. first-line therapy of neutropenic fever was meropenem g/ h in both groups, using a portable infusion pump in ah-group. in this group, the absence of focal infection or signs of severe sepsis allowed returning home after the initiation of antibiotics. the platelets support was performed at-home and the red blood support at hospital. results: the median (range) age (years) of the series was . the median follow-up of the series has been not achieved. the source of the sct was peripheral blood in all cases. we didn't find statistical differences between two groups (ah vs ip) in terms of age, diagnosis, type of donor, intensity of conditioning, gvhd-prophylaxis, toxicity (mucositis, acute renal injury, neutropenia and thrombocytopenia), agvhd, aspergilosis and trm. interestingly, a significant reduction of neutropenic fever was observed resulting the lower use of meropenem in the ah-group than ip-group. the admission median days were similar in the both groups and it represented - days the reduction in the total economic cost of the ah-group. the whole analysis of the results are detailed in table: in-patient group, conclusions: in our experience, at home allosct, including ptcy-gvhd prophylaxis, is a feasible and safe procedure reflected in similar trm and aspergillosis incidence. at-home allo-sct is associated with a significant lower risk of neutropenic fever than in-patient group, as well as a very low readmission rate. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical and janssen. laura rosiñol: honoraria from takeda, janssen, amgen and celgene. the others author do not have any disclosures to declare. background: renal complications in sickle cell disease (scd) include episodes of acute kidney injury (aki), progressive chronic kidney disease (ckd) and hyperfiltration, defined by abnormally high glomerular filtration rates (gfrs). hematopoietic stem cell transplant (hsct) from an hla identical sibling donor is a well-established curative treatment for scd, but traditional myeloablative conditioning (mac) regimens pose risks of kidney injury due to intensive use of chemotherapeutic agents, infectious risks, and use of calcineurin inhibitors (cnis). aki and subsequent fluid overload (fo) are common in pediatric hsct with reported aki incidence of %- % (kyung-nam koh et. al., ). we report renal outcomes in pediatric patients with scd who received hsct following a non-myeloablative conditioning (nma) regimen without cni exposure. methods: retrospective chart review describing renal outcomes in pediatric patients ( years of age or younger) with scd (hbss) who underwent nma hsct in alberta, canada from july to february . the nma regimen is illustrated in figure . reported renal outcomes: ) measured gfr (dtpa) pre-hsct, ) aki (kdigo definition) post-hsct by reviewing all serum creatinine levels from pre-hsct to one month post-hsct, ) %fo calculated: (max post hsct weight -baseline weight)/ baseline weight x for the two first weeks post-hsct, and ) estimated gfr (egfr) using the pediatric schwartz formula at last follow-up post-hsct, ckd defined as egfr < ml/min/ . m , mildly reduced gfr: - ml/min/ . m , and hyperfiltration: gfr ≥ ml/ min/ . m . [[p image] . results: eighteen patients ( % male, - years old at transplant) were included. most common pre-morbid events: vaso-occlusive crisis (n= ), acute chest syndrome (n= ), splenic sequestration (n= ), and cholelithiasis (n= ). median follow-up time: months (range: - months). all patients engrafted successfully with no acute or chronic gvhd. baseline measured gfrs were all > ml/min/ . m (range: - ) with mildly reduced gfr and hyperfiltration seen in one ( . %) and ( . %) patients respectively. at baseline (pre-hsct), the only aki event was one transplant related aki secondary to delayed hemolytic reaction after exchange transfusion in preparation for transplant. post-hsct, there were no aki events. additionally, no substantial %fo post-hsct was observed. average %fo week one post-hsct: + . % (range: - . % -+ . %) and week two post-hsct: + . % (range: - . % -+ . %). post-hsct egfr remained > ml/min/ . m at last follow-up in all patients. hyperfiltration was present in ( . %) of the patients. conclusions: this is the first study describing stable kidney function in children with scd after the present nma hsct regimen with alemtuzumab/ cgy total body irradiation (tbi) with prolonged post-hsct sirolimus. no episodes of aki or significant fluid overload were observed during the first month post-hsct, and no patient developed ckd during follow-up. further prospective studies are needed to confirm our findings and to determine if stable renal function persists during longer-term followup. disclosure: nothing to declare. lung microbiota in patients with idiopathic pneumonia syndrome (ips) after hct background: idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication after hematopoietic cell transplantation (hct) and the etiology remains unknown. recent studies have reported that various diseases are associated with changes of microbiota. the aim of this study was to evaluate the lung microbiota in hct recipients with ips and identify microorganisms potentially associated with ips. methods: frozen bronchoalveolar lavage (bal) samples from hct recipients with ips (n= ) and research bal samples from asymptomatic hct recipients as controls (n= ) were retrospectively analyzed. all samples were negative for common viruses by quantitative pcr. sequencing libraries were made with ng of input dna per sample (nextera xt, illumina). samples were pooled and sequenced by hiseq to obtain -bp paired end data. sequence data analysis and read classification were performed with sunbeam and the quality control and read classification were performed using komplexity and kraken, which classifies bacterial, archeal, and viral genomes. we used sequence data of bronchoscope prewashes from a separate cohort as controls for environmental sources (n= ). bray-curtiss dissimilarity among samples was calculated using the vegan r packages. permanova and a two-sided wilcoxon rank sum test were used to compare between the study groups. results: bal samples started at a median of x raw read pairs per sample and reduced to x reads assignable to microbial taxa following quality control. the bacterial phyla proteobacteria and firmicutes were most abundant followed by bacteroidetes and actinobacteria in both bal and bronchoscope prewash samples. separation of bal and prewash microbiota using bray-curtiss dissimilarity plots showed that bal samples were distinguished by sequences assigned to staphylococcus, acidovorax, and bradyrhizobium species, while prewash samples were distinguished mostly by pseudomonas and elizabethkingia species, consistent with environmental sources (figure) . within bal samples, staphylococcus species were the main drivers of separation between ips cases and the controls (p= . , permanova, figure) . consistent with this, a linear discriminant analysis to identify taxa best distinguishing cases and controls identified staphylococcus, especially s. epidermidis, in ips cases with lactobacillus and streptococcus species in controls. we then compared relative abundances of s. epidermidis between all study groups. ips case samples were significantly enriched in s. epidermidis compared to control (p< . , two-sided wilcoxon rank sum test) and prewash samples (p< . ). viruses were classified by category as human pathogens, non-human pathogens, and bacteriophages. torque teno viruses (ttv) was the most commonly detected virus among viruses that replicate on human cells, and there was a trend towards higher abundance in ips case samples than controls. conclusions: lung microbial sequences in hct recipients predominantly consisted of proteobacteria and firmicutes, and had considerable overlap with environmental background. patients with ips had significantly more staphylococcus sequences detected than asymptomatic hct patients. these results suggest that patients with acute lung injury post-hct show distinct patterns of lung microbiota, although heterogeneity of sample collection and processing cannot be excluded and no singular organism was uniquely associated with ips. a prospective study is required to confirm these findings and define the clinical significance of differences in abundance patterns. disclosure: nothing to declare p abstract withdrawn. romiplostim for the treatment of thrombocytopenia after allogeneic stem cell transplantation background: thrombocytopenia is a common complication after allogeneic stem cell transplantation (allo-hct). with variable possible causes, such as drug side effects, infections, poor graft function, graft vs host disease (gvhd) and immune mediated. the purpose of this study was to evaluate the efficacy of romiplostim, a thrombopoietin receptor agonist, in patients with prolonged thrombocytopenia with no obvious cause after allogeneic transplantation. methods: retrospective analysis of allo-hct patients who received romiplostim at a single bmt unit between november and november . romiplostim was given because of prolonged (> weeks) thrombocytopenia (< , μl) that couldn't be explained by obvious causes such as administration of drugs (antibiotics/antivirals), infection or gvhd. all patients were in complete remission and had complete chimerism. response to romiplostim treatment was considered transfusion independence or plt> . /μl. results: in total, patients (median years, - ) received romiplostim. patients ( male, females) had aml ( pts), all ( ), mds ( ) or hodgkin ( ), received a myeloblative (busiphex-based: , tbi-based: ) or ric ( ) conditioning and were transplanted from a sibling ( ), vud ( ) or haploidentical ( ) donor with pbsc ( ) or bm ( ) . all patients revealed primary neutrophil (median days, range - ) and > . /μl platelet ( days, - ) engraftment. romiplostim was started at median day + (range - ) with a median dose μg/kg ( ) ( ) ( ) ( ) ( ) . the median platelet count before commencement of treatment with romiplostim was . /μl (range . - . ) and them ( %) were transfusion-dependent. in total / ( %) patients responded to romiplostim treatment. eight out of the ( %) transfusion dependent patients responded to the administration of romiplostim. six out of the patients ( %) who were transfusion independent at romiplostin initiation (plt median . /μl, range . - . ) responded. the median duration of treatment was days ( - ) and the median follow up from the commencement of romiplostim was days ( - ). three out of ( %) patients experienced relapse of thrombocytopenia after discontinuation of romiplostim and re-initiation of romiplostim was commenced in all of them, of which responded and didn't. the administration of romiplostim was done on an external basis and was well tolerated by the patients. two patients experienced gvhd during romiplostim treatment (both patients transplanted from / unrelated donor, and days after initiation treatment with romiplostim). / patients interrupted romiplostim due to disease relapse. / patients receiving romiplostim are alive in complete remission and died ( due to relapse, and due to trm). conclusions: we present high response rates to romiplostim in patients with prolonged thrombocytopenia after allogeneic transplantation. in this retrospective study there were no side effects from the administration of romiplostim. however, the administration of romiplostim after allo-hct should be controlled in prospective trials. disclosure we report a single-center analysis of adult patients (median age years, range - , m/f / ), receiving tpo agonists for isolated severe thrombocytopenia (n= ) and spgf (n= ) after allo-hsct. primary diagnoses were aml ( ), all ( ), mds ( ), pmf ( ), mds/mpn ( ), saa ( ), cml ( ), nhl ( ) . severe pgf was defined as cytopenia in ≥ lineages (platelet < × /l, anc < . × /l, hemoglobin < g/l any time after sustained engraftment), full or stable mixed donor chimerism > % and no signs of relapse. median dose of romiplostim was (range, - ) mcg/kg weekly, eltrombopag - (range, - ) mg/day. overall response (or) included cr (platelet ≥ × /l, anc ≥ . × /l, and hemoglobin ≥ g/l) and pr (platelet > × /l, anc ≥ , × /l, hemoglobin > g/l). results: median time from pgf diagnosis to treatment with tpo agonists was days ( - ), median treatment duration was weeks ( - ). tpo agonists were well tolerated with no cases of grade iii-iv toxicity. tpo agonists were combined with rituximab (n= ), rituximab and dli (n= ) and hsc boost (n= ) in ( %) patients. a total of ( %) patients met criteria of response (cr: n= , %; pr: n= , %). combination therapy showed no difference in or compared to tpo agonists alone. or was not depended on the tpo agonist used nor the time to therapy initiation. median increase in anc in responders was . × /l ( . - . ), in platelet count - × /l ( - ). a total of patients died due to relapse (n= ), gvhd iii-iv grade (n= ) and infection (n= ). two-year os from the start of tpo agonist therapy was % ( % ci, - ) with a significant difference between responders and non-responders: % ( % ci, - ) vs. % ( % ci, - ) (p= , ). conclusions: this study showed promising results of tpo agonists for management of spgf. further studies are warranted to specify optimal timing and dosing regimen, predictors of response. [[p image] . two-year os in responders and nonresponders to tpo agonist therapy] disclosure: there are conflicts of interest to disclose p cytomegalovirus reactivation kinetics and peak titers as novel predictors of survival and relapse after allogeneic cell transplantation for hematologic malignancies saskia leserer , evren bayraktar , nikolaos tsachakis-mück , michael koldehoff , lara kasperidus , esteban arrieta-bolanos , mirko trilling , katharina fleischhauer , dietrich w. beelen , amin t. turki university hospital essen, essen, germany, background: after allogeneic hematopoietic cell transplantation (hct), human cytomegalovirus (cmv) reactivation associates with non-relapse mortality (nrm) but also with reduced relapse in patients with leukemia, as shown by numerous studies that evaluated cmv reactivation as a qualitative yes/no parameter in the first months posttransplant. we hypothesized that longitudinal quantitative assessment of cmv reactivation kinetics and virus loads might improve patient-specific clinical outcome associations. methods: this retrospective study included patients with hct for hematologic malignancies treated between / and / at university hospital essen, germany. cmv titers were monitored weekly by quantitative pcr (qpcr); cmv reactivation was defined by a cutoff of > genome copies per ml. patients were included for analysis, if at least measurements were available during the first days after hct. in total, , samples were analyzed. subgroup analyses were performed according to the time of cmv reactivation (before/after + d) or the cmv viremia titer (> , , , - , and - , copies/ml). results: cmv reactivation was detected in (median age years; range - years) out of patients. baseline characteristics (age, gender, underlying disease, transplant) of patients without cmv reactivation were comparable. cmv reactivation kinetics followed a gaussian normal distribution with a median first reactivation at + d and peak titers at + d. all except patient reactivated before d, % before + d. overall survival (os) of the cmv reactivation group as a whole did not significantly differ from the non-reactivation group ( vs. months). however, in subgroup analyses os was significantly reduced in patients with very early (< + d) compared to later reactivation ( vs. months, p= . ). moreover and importantly, os was significantly reduced in patients with cmv reactivation at high titers of > , copies/ml compared to those with lower titers (( vs. months) p< . ).cox regression analyses confirmed significantly reduced os for patients with cmv reactivation > , copies/ml and < day + as compared to the other cohorts (hr . , %ci . - . , p< . and hr . , % ci . - . , p= . ) respectively). the nrm was consistently higher (hr . ; %ci, . - . , p< . ) for patients with cmv copies > , /ml. the risk of hematologic relapse was exclusively reduced in patients with a peak cmv viremia between , and , copies/ml (hr . , % ci . - . ; p= . ) as compared to patients without cmv reactivation. for other levels of cmv reactivation this effect was not observed. conclusions: our data showed that cmv reactivations before + d or with high titers of > , copies/ml associated with significantly reduced os, while cmv reactivations at intermediate titers between , and , copies/ml had a positive impact on relapse incidence. these findings underline the complexity of cmv reactivations after hct outcome, and support longitudinal evaluation of cmv titers and individualized quantitative kinetics models for risk assessment after hct to distinguish the advantageous from the detrimental aspects of cmv reactivation. disclosure: att has received lecture fees from jazz pharmaceuticals and travel subsidies from neovii biotech outside the submitted work. the other authors declare no competing financial interests within the submitted work. association of serum ferritin levels before start of conditioning with mortality after allosct -a prospective, non-interventional study of the ebmt transplant complication working party background: elevated serum ferritin levels occur due to iron overload or during inflammation and macrophage activation. a correlation of high serum ferritin levels with increased mortality after allosct has been suggested by several retrospective analyses as well as by two smaller prospective studies. methods: this international multicentric study aimed to study the association of ferritin serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditioning. data were prospectively collected between / and / . a comparison of outcomes between patients with high and low ferritin level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from european countries reported data on allosct recipients. patient characteristics are given in table . the ferritin cut off point was determined at μg/l (median of measured ferritin levels). overall survival of allosct recipients with ferritin levels above cut off measured before start of conditioning was significantly shorter ( figure a , univariate hr= . ci= . - . p= . ; multivariate hr= . , ci= . - . , p= . ). progression-free survival was also shorter ( figure b , univariate hr= . ci= . - . p= . ; multivariate hr= . , ci= . - . , p< . ). excess mortality in the high ferritin group was due to both higher relapse incidence (univariate hr= . ci= - . p= . ; multivariate hr= . , ci= . - . , p= . ) and increased non-relapse mortality (univariate hr= . ci= . - . p= . ; multivariate hr= . , ci= . - . , p= . ). non-relapse mortality was driven by significantly higher infection-related mortality in the high ferritin group (univariate hr= . ci= . - . p = . ; multivariate hr = . , ci = . - . p = . ). acute and chronic gvhd incidence or severity were not associated to serum ferritin levels. conclusions: ferritin levels before start of conditioning can serve as routine laboratory biomarker to predict mortality after allosct. disclosure: the authors declare no confict of interest related to this study p prediction of reduced lung function and acute gvhd by surfactant protein d in allogeneic stem cell transplantation transplantation (hsct) and may progress to bronchiolitis obliterans that has a high mortality rate. surfactant protein d (sp-d) is an innate defense molecule involved in immune regulation at the epithelial surfaces, particularly in the lungs, and elevated levels have been associated with exacerbation of chronic obstructive pulmonary disease (copd). the aim of this study was to investigate, whether sp-d plasma levels and variants in the gene encoding sp-d may predict the development of reduced lung function after allogenic hsct. methods: we performed a population-based, singlecenter study of children (aged - years) treated with allogeneic hsct. the study consisted of ) a prospective study of serial plasma sp-d levels and rs genotypes in patients during the first months after hsct, and ) a retrospective study of rs genotypes within the sp-d gene in patients transplanted between - . pulmonary function tests were performed regularly as part of the clinical monitoring. results: at the day of graft infusion (day ) sp-d levels were reduced compared to levels before start of treatment with conditioning chemotherapy, defined as baseline ( ng/ml (quartiles - ) at day vs ng/ml ( - ) at baseline, p< . ). from day + sp-d levels increased and remained increased during the whole study period ( ng/ml ( - ) at baseline vs ng/ml ( - ) at months, p< . ). acute gvhd (agvhd) occurred in patients, of those patients with grade - . high sp-d levels at day + were associated with the development of agvhd ( ng/ml ( - ) vs ng/ml ( - ), p< . ) ( fig. ). the c/c genotype was associated with generally low sp-d levels and low fev /fvc at all time intervals compared to the other genotypes, significantly - months post-hsct (p= . ). there was no overall correlation between sp-d levels and lung function, but stratifying for genotype, high baseline sp-d levels were predictive for reduced fev /fvc at - months in cc and tt homozygous individuals. conclusions: patients with a genotype causing low capacity for sp-d production are at increased risk of developing pulmonary impairment after hsct. in addition, our data lend support to other studies indicating that spd production may increase during inflammatory pulmonary disease, acting as a reactive, protective mechanism. further research is warranted to define the role of sp-d levels and genotypes as a prognostic tool for lung function and agvhd. [[p image] . background: allogeneic hematopoietic cell transplantation (allohct) means a long period of restricted mobility and a range of therapy related side effects on muscle function. in this context patients demonstrated a huge decline of physical capacity and muscle mass in particular, accompanied with a decrease of quality of life (qol). resistance training could maintain muscle mass but is limited by patientsb lood values (platelet-count) and well-being. whole body vibration (wbv) was shown to maintain muscle mass during bed rest and has less impact on blood pressure than conventional resistance exercises. furthermore it was also shown to be feasible in patients during high dose chemotherapy. therefore the aim of our study was to examine the effects of wbv during allohct on patients physical and functional performance as well as qol. methods: patients receiving allohct were randomly allocated to either a wbv exercise group (ig) or an active control group (cg) doing stretching and mobilization. both groups exercised during the whole time of hospitalization for times per week and underwent pre-, post-and followup-assessment. physical capacity was determined by maximum oxygen consumption (vo peak ) and maximum power (p max ) during cardiorespiratory exercise test and by maximum strength of the knee extensors and flexors (ex max , flex max ) during isokinetic strength test. functional performance was assessed by jumping height during counter movement jump (cmj) and time of chair rising test (crt) as well as power output during both tests. qol was assessed by questionnaires of the eortc. results: during allohsct vo peak and p max decreased in both groups but till follow-up an increase is seen in the ig (p= . ; p= . ). at day + /follow-up a vo peak group difference is seen (p= . ). ex max (p= . ) and flex max (p= . ) were only reduced in the cg during hospitalization. jumping height and power output decreased in the cg during hospitalization (p= . , p= . ) and a difference between groups were seen in changes of jumping height from pre-to follow-up-assessment (p= . ): increase in the ig and decrease in the cg. the ig showed a decrease in time from baseline to follow-up (p= . ) in the crt and an increase of power output (p= . ). qol decreased only in the cg during hospitalization (p= . ) while during follow-up qol increased in both groups (ig: p= . ; cg: p= . ). in the cg physical functioning decreased during intervention (p= . ) whereas an increase was seen in the ig from pre-to follow-upassessment (p= . ). body image was significant worse in the cg compared to the ig at hospital discharge (p= . ) as well as at follow-up measurement (p= . ) where it got worse over time (p= . ). conclusions: wbv was shown to maintain maximum strength, jumping performance and qol during allohct. although cardiorespiratory fitness could not be maintained by wbv during hospitalization, it seems in the follow up period till day + that recovery of the cardiorespiratory system is enhanced by wbv carried out during allohst. nevertheless reasons for this changes in recovery have to be analyzed in further studies as well as treatment effects of wbv compared to conventional resistance training. disclosure: supported by a grant of the faculty of medicine and comprehensive cancer center freiburg respiratory virus infection within year after of allo-sct is the significant risk factor of obstructive ventilatory disturbance kosei kageyama , michiho ebihara , mitsuhiro yuasa , daisuke kaji , aya nishida , shinsuke takagi , hisashi yamamoto , go yamamoto , yuki asano-mori , naoyuki uchida , atsushi wake , akiko yoneyama , shigeyoshi makino , shuichi taniguchi toranomon hospital, hematology, tokyo, japan, background: obstructive ventilatory disturbance (ovd) is one of the major life-threading complication at the chronic phase of allogeneic stem cell transplantation (allo-sct). bronchiolitis obliterans has been the most established etiology as a part of chronic graft-versus-host disease and major cause of late non-relapse mortality of allo-sct. but other etiologies impact on respiratory function after allo-sct and risk factor of ovd have not been well understood. methods: to address these issues, we retrospectively reviewed the medical record of consecutive patients who first allo-sct at toranomon hospital between and . to detect ovd, forced expiratory volume in second (fev . ) showed less than % of predicted in spirometry test was defined as positive. in the recipients who showed fev . less than % in pre-transplant test, more than % reduction of fev . was regarded as positive. nasopharyngeal swab of those who had upper respiratory tract symptoms were tested for the presence of respiratory viral antigens (adv, piv, and rsv). patients with ecog performance status of , had active infection at transplant were excluded from this analysis. the cases of early death or relapse before days post-transplant, and the cases of graft failure were also excluded. results: the median age was years (range, - ). underlying diseases were aml in , mds/mpd in , cml in , all in , atl in , hl in , nhl in , and others in . five hundred twenty-nine ( %) were not in remission at the time of transplant. five hundred eightythree patients ( %) were conditioned with myeloablative regimens, whereas patients received reduced-intensity regimens. donor sources consisted of related peripheral blood /bone marrow (bm) (n= ), unrelated bm ( ) forty-six developed ovd on median of ( - ) days post-transplant. cumulative incidence of ovd was . % in total population. in recipients those who could spirometry, overall survival at years was . % in patients who developed ovd and was comparable with those who did not develop it ( . %, p= . ). in univariate analysis, disease status (cr/aa or noncr), recipient age (age< or ≥ ), prior autologous stem cell transplantation (yes or no), intensity of conditioning regimen (mac or ric), tbi dose (< gy or ≥ gy), busulfan dose (< . mg/kg or ≥ . mg/kg), donor source (cord blood or non-cord) had no impact on the incidence of ovd. patients who developed respiratory virus infection showed significantly higher incidence of ovd compared to those who did not developed it ( . % vs . %, p< . ). in multivariate analysis, respiratory virus infection was the only significant risk factor for the development of ovd (hr= . , % ci . - . , p< . ). conclusions: respiratory virus infection within year after allo-sct is the significant risk factor of ovd. disclosure: nothing to declare. background: metabolic syndrome (mets) is related to increased risk of cardiovascular disease and type- diabetes (dm- ) and usually seen in overweight individuals in the general population. we investigated mets and clinical risk factors two decades after hsct. methods: all male survivors treated with myeloablative allo-hsct during childhood (< years) between - in denmark were invited to a follow-up study. mets was defined as the presence of at least three ncep atp iii criteria: fasting plasma triglyceride (tg) ≥ . mmol/l, high density lipoprotein (hdl) < . mmol/l or medical treatment of hyperlipidemia; fasting plasma glucose (fpg) ≥ . mmol/l; abdominal circumference (ac) > cm; bp ≥ mmhg (systolic) / ≥ mmhg (diastolic) or medical treatment for hypertension. patients with overt dm- were included into the mets group. furthermore, patients were examined for chronic graft-versus-host disease (cgvhd) by the nih-criteria at the time of follow-up and high sensitivity c-reactive protein (hscrp) was measured. the prevalence of mets was compared to a nordic reference group (hildrum et al. ) . results: we included out of eligible males (participation rate %) aged - years, median years. median (range) follow-up was ( - ) years. of these males, % had a malignant diagnosis and % were treated with tbi-based conditioning. donors were matched siblings (n= ), matched relatives (n= ) or matched unrelated donors (n= ). mets was more prevalent ( %) in the young adult survivors compared to the prevalence reported for - year-olds in the nordic reference ( %). instead the prevalence was comparable to that reported for the - year-olds ( %). of the components of mets, elevated tg ( %), hypertension ( %), and decreased hdl ( %) were frequent, while fpg was elevated in %. importantly, only % of those with mets had increased ac and mean bmi ( . kg/m ) of the hsct survivors was within normal range in contrast to features of mets observed in the background population. having mets was significantly associated with tbi (rr = . , %ci ( . - . ), p= . ) as was the following single components of mets (mean in tbi group vs. mean in non-tbi group): elevated tg ( . mmol/l vs. . mmol/ l, p= . ), lower hdl ( . mmol/l vs. . mmol/l, p= . ) and higher diastolic bp ( mmhg vs. mmhg, p= . ). mets was only demonstrated in one patient who received non-tbi based conditioning. sixteen of patients had cgvhd of which nine were moderate to severe cases, but cgvhd was not associated with mets. however, low-grade inflammation measured by hscrp was related to increased ac (rho= . , p= . ) and tg (rho= . , p= . ). conclusions: our results indicate that male long-term survivors of allo-hsct during childhood have a high risk of mets at an earlier age than the general population. the presence of mets despite normal bmi in several patients suggests unconventional etiologies like the effect of tbi and low-grade inflammation. disclosure: nothing to declare. results: this survey was completed by transplant directors ( %), transplant consultants ( %), nonconsultant grade physicians ( %), hsct clinical nurses specialists (cns) ( %) and other ( %) from centres in countries. % of the centres are adult-only, % paediatric-only and % treat adult and paediatric patients (mixed centres). % are located higher than degrees latitude (northern countries) and % lower than this latitude (southern countries). at the time of the survey % were members of the european union (eu). measurement of serum vd is routinely performed in % of the centres prior and in % after allogeneic hsct. the main clinical indications are known osteopaenia/osteoporosis ( %), previous fracture ( %), treatment with steroids ( %), premature menopause ( %) and established menopause ( %). monitoring occurs every months ( %), every months ( %), once a year ( %) or at other time-points ( %). in this regard, seasonality is not taken into account in the majority of the centres ( %). local and national/international guidelines (nice) are only followed by % and % of the centres, respectively. the most common cut-off value of serum vd for commencing on replacement is nmol/l ( %). northern countries tend to use values of ≥ nmol/l whereas southern countries ≤ nmol/l. % do not use cut-off values. following hsct, % of centres prescribe vd supplements to maintain calcium metabolism and bone health ( %), enhance immune reconstitution post-hsct ( %), gvhd prevention ( %), enhance immune-suppression to treat gvhd ( %), treat depression/fatigue ( %) and reduce relapse risk %. a "loading" dose is administrated in % ( % adult, % mixed and % paediatric), with a mean duration of weeks . the median daily loading dose is , iu ( - , ). the median "maintenance" daily dose is iu ( - , ). there are not remarkable differences between adult and paediatric centres or northern and southern countries. vd replacement is prescribed by transplant physicians ( %), family physicians ( %), endocrinologists ( %), cns ( %), others ( %) and in % of the centres, patients are advised to buy it over-the-counter. vd is prescribed combined with calcium carbonate in % and alone in % of the centres. it is eventually discontinued by % of the centres when therapeutical levels of vd are reached ( %), dexa scan returns to normal ( %) and symptomatic improvement ( %). conclusions: this survey has demonstrated discrepancies in monitoring and replacement of vd across ebmt allogeneic hsct programmes. although awareness has arisen over the last decade, there is still lack of evidence about the optimal levels of vd required for immunemodulation post-hsct. this survey emphasises the need for specific guidelines to harmonise the current management of vd deficiency in adult and paediatric hsct setting. disclosure background: the use of unmanipulated haploidentical sct (haplo-sct) with post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis has widely extended. primary and secondary graft failure are relatively uncommon complications. however, poor graft function (pgf) after haplo-sct with pt-cy has not been described thoroughly. the objective of this study is to describe characteristics, treatments and outcomes of patients with pgf after haplo-sct with pt-cy. methods: we retrospectively analyzed haplo-sct with pt-cy consecutively performed between and in our centre. pgf was defined as either occurring after initial engraftment: persistent neutropenia (anc < /ul) with the need of at least doses of g-csf and/or thrombocytopenia (platelets < . /ul) with platelet transfusion dependence, with complete donor chimerism and without concurrent severe gvhd or disease relapse. results: nineteen patients were excluded from the analysis due to early mortality (death before day + ), primary graft failure (absence of neutrophil engraftment by day + , with mixed chimerism) or secondary graft failure (development of severe cytopenias and mixed chimerism after initial achievement of neutrophil engraftment). thirty one patients ( , %) were diagnosed with pgf. main characteristics of these patients are summarized in table . twenty six patients ( %) presented with neutropenia and were treated with g-csf, while patients ( %) only developed severe thrombocytopenia without neutropenia, and were treated only with platelet transfusion. twenty four patients ( , %) had at least cmv reactivation, patients ( %) had or more cmv reactivations and patients ( %) received valganciclovir for cmv reactivation treatment. although most patients achieved adequate peripheral blood counts (pbc) with initial salvage therapy, patients ( %) had persistent cytopenias in spite of g-csf, platelet transfusion, cmv reactivation resolution and myelotoxic drugs withdrawal. four of them were treated with a boost of cd + selected peripheral blood donor cells at a median of days after . median cd + cells infused was , x /kg. these patients achieved adequate pbc after salvage therapy and two developed gvhd. the other patients were treated with increasing doses of thrombopoietin (tpo) receptor agonist (tra) eltrombopag. one patient started treatment days after hsct with mg daily and increased dose to mg daily, with complete recovery of pbc months after initiating tra. the second patient started treatment days after hsct with mg daily and increased dose to mg daily, with complete recovery of pbc months after initiating tra. twenty one patients ( %) with pgf diagnosis had long term survival. conclusions: poor graft function is a frequent complication after haplo-sct with cy-post. cmv reactivation and myelotoxic drugs could be the most relevant factors associated with development of this entity. although most patients recover pbc without specific therapies beyond g-csf and platelets transfusion, there is a small group of patients with persistent cytopenias. boost of cd + selected cells is effective in reverting this condition, with gvhd as main complication of this procedure. use of tra seems to be an interesting option for these patients, although more experience is needed to draw definitive conclusions. disclosure: nothing to declare. were also frequently observed. the high risk patients for anxiety (hads-a score ≥ ) and depression (hads-d score ≥ ) was found in . % and . %, respectively. . % of patients was in high distress status (nccn dt score ≥ ). we found that younger age (< years) was significantly associated with poor quality of life score (fact-bmt) (p= . ) and high risk of fatigue (p= . ), anxiety (hads-a) (p= . ), and depression (hads-d) (p= . ). female sex was significantly related to lower physical well-being score and higher distress score (p= . and p= . , respectively). acute lymphoblast leukemia (all) survivors after allo-hct showed significantly worse quality of life score (fact-bmt) (p= . ) and higher depression score (hads-d) (p= . ) compared to those with other disease. chronic graft versus host disease (gvhd) and continuous immunosuppressant usage also have significant adverse impact on lower fact-bmt score (p= . and p= . , respectively) and higher hads-d score (p= . and p= . , respectively). but there was no significant difference in fact bmt, hads-a, hads-d, nccn dt according to donor type, conditioning intensity, anti-thymocyte globulin use, acute gvhd. smoking and alcohol drinking was continued in . % and . % of allo-hct survivors. . % of survivors did not exercise regularly. regular health screening tests have been done only in patients ( . %). conclusions: allo-hct survivors over years following allo-hct still have many physical and psychological symptoms. younger patients (< years), female, all, chronic gvhd, and sustained use of immunosuppressant were significant risk factors for poor quality of life and anxiety. we need to build more active survivorship care plan after allo-hct especially for those patients. disclosure: all authors have nothing to declare. evaluation of the new ebmt criteria for the diagnosis of vod/sos in consecutive transplant patients using an electronic patient record analysis system asha aggarwal , nicola gray , oliver lomas , katalin balassa , nadjoua maouche , robert danby , , andy peniket , grant vallance background: veno-occlusive disease (vod), or sinusoidal obstruction syndrome (sos), is a recognised complication of haematopoietic stem cell transplantation. hepatic vasculature endothelial cells are damaged by conditioning chemotherapy, leading to venous occlusion and centrilobar necrosis. the ebmt criteria for diagnosis of vod are bilirubin >= with two of painful hepatomegaly, > % weight gain and ascites. vod is often under-diagnosed, and as a result, treatment may be delayed. integrated electronic patient record (epr) systems are now widely used, and provide an opportunity to retrospectively audit practice to identify patients in whom vod may have been un-diagnosed or in whom treatment was delayed. in addition these systems have potential for alerting clinicians to the potential diagnosis of vod. methods: we have developed software to analyse the data downloaded from epr to identify patients in whom vod was a possible diagnosis according to the new ebmt criteria. in order to identify patients who may have had vod we first screened for patients with a bilirubin of >= mmol/l (which is an absolute requirement for the clinical diagnosis of vod) within the first days of transplantation. epr data was then used to assess whether patients had > % weight gain. radiology reports were reviewed for patients who had bilirubin >= mmol/l to ascertain if they revealed ascites or painful hepatomegaly. results: patients underwent transplant procedures (january st to july st ). of all transplant patients ( . %) were found to have a bilirubin of >= mmol/l. of ( . %) autograft patients and of ( . %) allograft patients had an elevated bilirubin at this level. these patients were assessed for evidence of % weight gain. this was the case in patients overall- % of autograft patients, . % of allograft patients. seven patients ( autograft and allograft) had radiological evidence of ascites. two patients had a recording of painful hepatomegaly (both post allograft). overall our analysis identified patients ( . % overall) fulfilling the ebmt diagnostic criteria for classic early vod all of whom received defibrotide. all patients had received allogeneic transplants. we failed to identify any cases of late onset vod or any undiagnosed patients over this period. conclusions: this analysis enabled us to efficiently perform a complete audit of our practice to identify patients with vod. we would recommend using electronic patient records to retrospectively audit practice in this way. the tool that we have created for this analysis will be made freely available for public use and the details will be presented at the ebmt meeting. we now plan to extend the function of our epr system to provide alerts to clinicians when vod is a possible diagnosis and may lead to more rapid treatment of these patients. our data suggests that elevation of bilirubin and weight gain of > % will be the most frequently occurring criteria on which to base these alerts. disclosure: g.vallance has performed consultancy work for jazz pharmaceuticals. endothelial activation and stress index in predicting outcome of allogeneic stem cell transplantation-a retrospective cohort analysis zinaida peric , tomislav taborsak , nadira durakovic , lana desnica , alen ostojic , ranka serventi-seiwerth , radovan vrhovac university hospital centre zagreb, zagreb, croatia background: endothelial dysfunction is a common pathophysiology of major complications after allo-sct, such as graft-versus-host disease, veno-occlusive disease, thrombotic microangiopathy and sepsis. endothelial activation and stress index (easix) is a simple score comprised of standard laboratory parameters (creatinine, ldh and thrombocytes) developed as a potential tool to predict allo-sct mortality by luft and colleagues. a recent validation of easix included three retrospective cohorts and showed that easix taken before start of conditioning can be used as an independent predictor of survival after allo-sct. methods: the aim of our study was to retrospectively evaluate pre-transplant easix in our cohort of consecutive patients who underwent allo-sct in the university hospital centre zagreb between and . with the use of a cut-off used in the validation cohorts, we compared two groups of patients for overall survival (os) and transplantrelated mortality (trm). group comparisons were done using the log-rank test or gray test for competing risks outcomes. a multivariate analysis evaluated the association of os with relevant variables by using a cox's proportionalhazard regression model. results: our study group included patients and comprised males ( %) and females ( %, with a median age of years (range, to years) at the time of transplantation. the most frequent malignancies in our population were acute leukemia ( patients, %) and myelodysplastic/myeloproliferative neoplasm ( patients; %). the donor was an identical sibling for patients ( %), matched unrelated donor for patients ( %) and haploidentical for patients ( %). patients ( %) received a myeloablative conditioning regimen while patients ( %) received a reduced-intensity conditioning regimen. with a median follow-up of months (range, - ) for the whole study group, the os at months was %, ( %ci - ) in the group of patients with low easix score and % ( % ci - ) in the group of patients with high easix score (p= . ). this difference was mainly attributed to higher trm in the group with high easix score ( %, %ci - at months) compared to the group with low easix score ( %, %ci - at months) (p= . ). in the multivariate analysis which included easix, patients' age, intensity of conditioning, diagnosis (lymphoid vs myeloid), status of the disease at transplant and type of the donor, worse os was independently associated only with older age of patients (hr . ; % ci, . - . , p= . ) and high easix score (hr . ; % ci, . - . , p= . ). conclusions: our retrospective data support previous data and suggest that easix could potentially serve as a valid tool for prediction of allo-sct outcomes. as a simple biomarker panel, easix could easily be implemented in clinical decision making in the field of allo-sct. these retrospective data need validation in a prospective study which is currently being conducted. clinical background: veno-occlusive disease (vod) is a potentially devastating complication that can occur after hematopoietic stem cell transplant (hsct) and in severe cases can lead to multi-organ failure. (mohty ) defibrotide has been proven to be effective to prevent and treat vod, and it is critical that clinicians are aware of how to diagnose and treat this serious complication of hsct. this study was conducted to determine if an online, simulation-based continuing medical education (cme) intervention could improve performance of hematologists/oncologists (hem/ onc) and advanced practice providers (nurse practitioners and physician assistants, apps) in the diagnosis and treatment of patients with vod. ( methods: a cme certified virtual patient simulation (vps) was made available via a website dedicated to continuous professional development. the vps consisted of cases presented in a platform that allows clinicians to assess the patients and make diagnostic and therapeutic decisions supported by an extensive database of diagnostic and treatment possibilities, matching the scope and depth of actual practice. clinical decisions were analyzed using a sophisticated decision engine, and tailored clinical guidance (cg) employing up-to-date evidence-based and faculty recommendations was provided after each decision. one case was about vod and the other case was about acute myeloid leukemia (aml). decisions were collected post-cg and compared with each user's baseline (pre-cg) decisions using a -tailed paired t-test to determine p-values (p < . indicates significance). data were collected between / / and / / . results: at the time of assessment, hem/oncs and apps had fulfilled the participation criteria for completing the vod case simulation. conclusions: this study demonstrates that vps that immersed and engaged clinicians in an authentic and practical learning experience improved evidence-based clinical decisions related to the management of vod. this vps increased the percentage of clinicians who utilized standardized criteria to diagnose vod and who ordered defibrotide and iv fluids for vod management. however, further education is needed to increase the competence and performance of clinicians, particularly apps, in these areas in order to positively impact patients. disclosure: nothing to declare. a nationwide retrospective study of hematopoietic stem cell transplantation in solid organ transplant recipients: on behalf of jshct, transplant complications working group background: the outcome of hematopoietic stem cell transplantation (hsct) in solid organ transplant remain unclear. to address this issue, we conducted a retrospective survey of the japan society for hematopoietic stem cell transplantation centers. methods: to address this issue, we conducted a nationwide retrospective survey of the japan society for hematopoietic stem cell transplantation (jshct) centers. a first questionnaire was emailed to jshct centers requesting information on cases of hsct in sot recipient. patients' data about sot were collected by sending a second questionnaire to the centers with the patient. based on these reports, patients' data about hsct was identified in the japan transplant outcomes registry database by the transplant registry unified management program (trump), confirmed in . results: of the jshct centers, responded to the survey ( . %). of the responding centers reported a total of patients who had undergone sot from living donor, and subsequent hsct. they consist of three autologous hsct (auto-hsct) and allogeneic hsct (allo-hsct). in auto-hsct, all patients had received liver transplant for hapatoblastoma. they achieved neutrophil engraftment at days after hsct, and two of three patients were alive at one year after hsct. in allo-hsct (n= ), seven patients had received liver transplants, and nine patients had received kidney transplants. five patients received hsct from unrelated donor, and patients received hsct from related donor; two donors were identical in sot. their stem cell sources were seven peripheral blood stem cell, six bone marrow, and three cord blood. all but one patients achieved neutrophil engraftment at days after hsct. five-year overall survival ( yos) was . %. while yos in patients with bone marrow failure (n= ) was %, that in patients with malignant disease (n= ) was . %; all but one patients with malignant disease received allo-hsct in non-remission. seven of nine kidney-transplant recipients experienced dialysis, and three patients experienced renal rejection after hsct. on the contrary, no liver-transplant recipient experienced hepatic rejection. conclusions: in sot recipients, the outcome of allo-hsct for malignant disease was poor, partly due to disease status before allo-hsct. severe renal complications were common in kidney-transplant recipients, suggesting renal care with caution during and after allo-hsct. disclosure: this work was supported in part by the practical research project for allergic diseases and immunology (research technology of medical transplantation) from japan agency for medical research and development, amed. high incidence but low mortality of ebv related ptld after t-cell replete allo-peripheral blood hct with aggressive monitoring and without pre-emptive rituximab background: the aim of the study is to report the incidence and outcome of post-transplant lymphoproliferative disorder (ptld) in the setting of allogeneic peripheral blood hematopoietic stem cell transplantation (allo-hsct) combining post-transplant cyclophosphamide (ptcy) and anti-thymocyte globulin (atg) as graft versus host disease (gvhd) prophylaxis. methods: between october and may , adult patients diagnosed with hematological malignancies underwent a first t-cell replete allo-hsct in our center. all patients received a reduced intensity conditioning regimen with fludarabine, busulfan, and cgy of total body irradiation, combined with rabbit-atg, ptcy and cyclosporine (csa). ebv titres were monitored weekly by quantitative pcr in plasma samples. the cut-off value for test positivity was > copies of ebv dna/ml of plasma. last follow up was november . median follow up for patients known to be alive was months (range - ). results: patient information is summarized in table . ebv reactivation was documented in ( %) patients. median time to ebv reactivation and the diagnosis of presumed/proven (p/p)-ptld were ( - ) days and ( - ) days [ ( - ) months], respectively. median time between first ebv reactivation to p/p-ptld was ( - ) days. seventeen ( %) of the patients developed p/p-ptld. median age was years . two ( %) received mrd, ( %) / mud, ( %) / mud, and ( %) haploidentical donor grafts. twelve ( %) were on therapeutic cyclosporine at diagnosis. pre-emptive therapy was not given to any case and only probable or proven ptld were given rituximab. treatment was based on reduction of the immunosuppression in patients and with the addition of weekly rituximab mg/ m in cases. fifteen ( %) achieved complete clinical responses with pcr negativity. two ( %) patients died secondary to ptld. conclusions: atg based conditioning is associated with increased viral reactivations. frequent ebv monitoring and pre-emptive treatment may lead to rapid disease control. further research is required to optimize monitoring and management strategies in allo-hsct recipients. disclosure: nothing to declare p acoustically enriched extracellular vesicles as potential markers for allogeneic hematopoietic stem cell transplantation complications hooi-ching lim , robert palmason , stig lenhoff , thomas laurell , stefan scheding , background: extracellular vesicles (evs) contain a number of condition-specific proteins, dna and rna types and might therefore be used for the early detection of posttransplant complications. however, traditional ev isolation (ultracentrifugation) is time consuming and requires large sample volumes thus making it difficult to perform longitudinal studies on larger patient cohorts. we therefore investigated whether recently-developed acoustic trapping could be applied to isolate evs from patient plasma for biomarker development. methods: plasma samples were collected from consecutive patients before and up to months after allogeneic hematopoietic stem cell transplantation. patients (age: - years) with high-risk or refractory/relapsed diseases were transplanted with mobilized pbsc from related (n= ) and unrelated donors (n= ) after standard conditioning. gvhd prophylaxis was cyclosporine and methotrexate. plasma samples were frozen and thawed for ev enrichment using a novel acoustofluidic-based technology (acoustic trapping). acoustic trapping uses ultrasound as a local λ/ acoustic standing wave produced by a piezoelectric transducer over a capillary. first, μm polystyrene beads are captured which serve as seeding particles. after washing, target particles (evs) are then captured ("trapped") in the acoustic field. a semi-automatic trapping device (acoutrap) was used to isolate evs from diluted plasma ( : in pbs). the number of evs and size distribution were analyzed by nanoparticle tracking analysis. mirna analysis was performed by qpcr.evs were enriched in duplicate from μl and μl of diluted plasma for nanoparticle tracking analysis and qpcr analysis, respectively. results: evs were successfully isolated from all plasma samples. a total of plasma samples were processed. numbers of trapped evs ranged from . x - . x before conditioning to . x - . x per μl diluted plasma after transplantation. the maximum change in ev numbers in individual patients compared to pretransplantation values ranged from -fold to -fold. most patients showed slight increases in ev size after transplantation. eight of the patients showed signs of infection and received i.v. antibiotics. increased levels of evs (> -fold) were recorded in three patients during these episodes. furthermore, increased ev numbers were observed in a patient who required i.v. antiviral therapy for cmv reactivation. acute grade i gvhd was observed in five patients of which two had increased ev numbers (> -fold). one patient developed grade iv gvhd which was accompanied by a -fold increase in ev numbers. interestingly, progressively increasing ev numbers preceded the detection of early relapse in a pre-b all patient by three weeks. rna isolation from trapped evs yielded sufficient material for mirna profiling. here, first mirna profiling data demonstrated that mirnas were detected in ev samples (mir- a, - a, - c, - and - a) , and that acoustically enriched evs were not affected by hemolysis in contrast to the corresponding whole plasma samples (dcq of mir- a and mir- a). conclusions: acoustic trapping allows for efficient and rapid enrichment of evs from small volume plasma samples. trapped ev samples contain sufficient amounts of mirna for downstream analysis and are thus promising candidates for biomarker development in transplantation. disclosure: laurell and scheding are founders and board members of acousort ab, a lund-based biotech sme that develops particle and cell sorting methods based on ultrasound. the incidence, risk factors and outcomes of primary poor graft function after allogeneic hematopoietic stem cell transplantation fei gao , , jimin shi , , yi luo , , yamin tan , , xiaoyu lai , , jian yu , , he huang , , yanmin zhao , background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative therapy for both hematologic malignancy and many other blood disease. while, primary poor graft function (pgf) is still a severe complication following hsct which lead to poor prognosis. up to now, the incidence and risk factors of pgf have not been totally revealed. methods: from january to december , a total of patients who received allo-hsct in our center were analyzed retrospectively. there were males ( . %) and females ( . %) with a median age of . years ( - years) . pgf was defined as persistent neutropenia (≤ . × /l), thrombocytopenia (platelets≤ × /l), and/ or hemoglobin≤ g/l after engraftment with hypocellular bone marrow and full donor chimerism, without concurrent graft-versus-host disease or disease relapse. incidence was calculated from all patients. of the total patients, nineteen ( . %) developed primary pgf. a : ratio of nested case control study using the good graft function (ggf) subjects transplanted in the same year with the same sex and age of ± years was carried out. results: data was analyzed by univariate and multivariate logistic regression, and univariate analysis identified disease species, the time from diagnosis to transplantation, disease states, myelofibrosis, splenomegaly, serum ferritin (sf) level, cmv infection, mononuclear and cd + cells in graft as potential risk factors (p < . ) for pgf. multivariate analysis identified elements as the independent risk factors (p < . ), including cd + cells < × / background: transplant survivors affected by cgvhd usually take one or more immunosuppressants, as well as prophylactic antimicrobials; use of multiple medication classes concurrently poses a risk for drug-drug interactions or amplified side-effects. the use of medications other than cgvhd-direct immunosuppressive therapies has not been well-characterized. this study aims to evaluate patterns of opioid analgesic use in a cohort of patients severely affected by cgvhd. methods: patients (n= ) with cgvhd were consecutively enrolled in a cross-sectional natural history study (nct ) from / - / at the nih. participants underwent a comprehensive evaluation including a detailed history and physical examination (including current medications), multidisciplinary evaluations, and laboratory and diagnostic testing. for this analysis, respondents were classified as receiving or not receiving an opioid analgesic. following the initial screening by univariate methods (n= ), multivariable logistic regression analysis (mlr) was used to identify a set of factors which could jointly impact opioid use. for mlr data were divided into a training (n= patients) and a validation set (n= ). results: study participants´median age was . years ( - ), % were female, % had severe cgvhd per nih scoring criteria, and % were currently receiving high or moderate levels of systemic immunosuppression. approximately one third ( %) were taking opioid analgesics (oa). based on the univariate screening results (p< = . ), a set of parameters was evaluated by univariate logistic regression in the -patient training set, and the following parameters retained their significance and were included in the mlr model: nih average score per organ, total lss, patient impression of severity, nih cgvhd severity, presence of skin erythema, karnofsky performance score (kps,) clinician's therapeutic intent, nih joint score, and with the presence of several cgvhd symptoms including rashes, mouth sores, avoidance of food, vomiting, weight loss, joint and muscle aches, joint limitation, energy loss, need for naps, fevers, anxiety. multivariable logistic regression identified kps < % as predictive of oa use, or . , % ci . - . . in the training set . % of pts using opioids were correctly identified, . % of those not taking opioids were identified, an overall fraction of correctly identified pts was . % ( % ci . - . %), while in the testing set, . % of those using opioids were correctly identified, and . % of those not taking opioids were correctly identified, with overall . % ( % ci: . - . %) classification accuracy. conclusions: this study showed the burden of oa in this cgvhd cohort. lower kps was significantly associated with oa use, as well as self-reported symptoms and a more severe cgvhd disease, which could be of interest in the development of non-pharmaceutical interventions in this patient population. additional, prospective studies are needed to explore the indications for and effectiveness of oa in this population of survivors. disclosure: no conflict of interest to declare. rcts that tested an internet-based program and patientcentered survivorship care plans for hct survivors. patient and caregiver input is essential to inform the design and features for the mobile app platform so that it is usable and engaging for those it targets. methods: using a qualitative research design, we conducted telephone focus groups of adult patients and caregivers in the united states. adult (age > years at the time of study entry) hct recipients had to be at least oneyear post-hct to participate. participants had to be able to communicate in english, and could have received a hct for any diagnosis, and from any donor source or stem cell type. those who had multiple transplants were included. participants were asked to review printed and online visual presentations of the mobile app before the focus groups so they were prepared to discuss their responses to the materials during the call. focus groups were conducted to saturation, when no new qualitative content was offered. results: three focus groups were conducted with total participants ( patients, two caregivers/patient advocates). all patients received an allogeneic hct; average time since hct was years (range: - years).the majority of participants were female ( . %). participants had differing perspectives on the usefulness of the app to track follow-up appointments, lab values, and other health care plans. there was high interest in having the app tailored to meet specific needs of patients, including tracking information over time (e.g. test results, medications), and having health information available specific to their needs. to minimize duplication of information and data entry, participants recommended syncing the app with their calendars and online patient portals they already use. reasons provided for not using the app included perception that the materials repeated information already received, side effects such as graft-versus-host disease that restricted vision or motor skills, and lack of comfort with apps for some older participants. conclusions: many health technology and mobile apps are being created to improve patients' health and survivorship care. in this study, hct survivors and caregivers identified a variety of features that they would want in an app or website, in particular, features tailored to individual needs. health technologies provide an opportunity to improve survivorship care, but patients and caregivers should be engaged in the process of developing these tools to assure the technology fits their needs and will be used. given the effort required to maintain these technologies, they require testing for health benefits in rigorous clinical trials. clinical background: thanks to allogeneic stem cell transplantation (allo-hsct) patients suffering from hematologic malignancies have seen an increase in there life duration expectancy, but they are many side effets including decreasing in physical performance and in quality of life. the intensity of physical performance decrease is variable between patients, and today we did not know why. the aim of our study was first to characterize the physical performance of subjects less than year following allo-hsct by the use of a cardiopulmonary exercise testing (cpet), and then to determine the predictive factors of exercise performance. methods: we did a retrospective analysis from patients who had an allo-hsct at hematology department of toulouse-oncopole and cpet from / to / . the cpet was performed using a cycle ergometer with o and co analyzer breath by breath, (masterscreen cpx carefusion, san diego, usa), a continuous -lead electrocardiogram, and a blood pressure monitoring. the protocol included a -min rest period, a -min warm-up of w pedaling followed by a w/min incremental phase, up to exhaustion, then a -min active recovery of w pedaling, then a -min passive recovery. three exercise markers were analysed: the peak of oxygen uptake (peak vo ), the ve/vco slope and the first ventilatory threshold (vt ). data relative to conditioning regimen, short-term complications, impairment at cpet day, and physical activity since allo-hsct were gathered. results: after allo-hsct, nearly over patients reported fatigue, a half reported dyspnea, and over or more reported pain, muscular, neurological or psychological impairment. more than % of patients suffer from moderate or severe physical intolerance, particularly when myeloablative conditioning regimen was used. only % of patients followed rehabilitation sessions supervised by a physiotherapist, and non-supervised physical activity has been performed by % of patients. despite normal lung function tests and echocardiography findings in most patients, % had exercise intolerance (ei), % exercise deconditioning, and % had abnormal ventilatory efficiency. patients with moderate and severe impaired exercise capacity were significantly younger at diagnosis and at allo-hsct, such as patients with severe deconditioning conclusions: based on a retrospective study, we reported for the first time complete results from cpet and detailed clinical evaluation concerning deficiency and disability following first year after allo-hsct. these results confirm that exercise impairment is very frequent with more than a half of patients suffering from alterations of one or more of the three performance markers, despite being active. disclosure: nothing to declare demyelinating disorders: a paradigm of immunity disorders after hematopoietic stem cell transplantation background: neurologic complications are a major problem in patients who undergone hematopoietic stem cell transplantation (hsct). given the higher survival of transplanted patients, the burden of neurological complications is increasing in the last years. a significant reduction in overall survival was demonstrated in patients who developed neurological complication after hsct, irrespectively of the hematopoietic stem cell (hsc) source. neurologic disorders in transplanting setting comprise a wide variety of ethiologies including demyelinating disease, which are caused by immune and non-immune mechanisms. here, we analyzed the clinical presentation and the underlie ethiologies of patients developing hsct-related demyelinating disorders in order to give diagnostic and prognostic clues useful to manage these severe but treatable complications in the transplant setting. methods: a total of patients of our department which developed neurological complications after hsct were consecutively collected and ( %) of them, namely those having a diagnosis of a demyelinating disorder, were grouped and described according to the ethiologies of their neurological disorder. results: in / ( %) patients, an immune-mediated process was found, while / ( %) were diagnosed as having an infective etiology and / ( %) were supposed to have a demyelinating disorder caused by toxic exposition. a definitive etiologic diagnosis was not formulated in the remaining / ( %) patients. when patients who developed an immune-mediated demyelinating disorder ( / ) were compared to those in which a clear immune pathogenic mechanism was not detected ( / ), a higher incidence of acute graft-versus-host disease (agvhd) was detected in the former than in the latter ( % vs %). moreover, comparison of these two groups revealed that those with no evidence of immune-mediated process have a slight higher prevalence of t-cell depleted hsct thanthose with an immune-mediated demyelinating disorder ( % vs %). finally, a lymphoproliferative disorder pre-existing the hsct was detected in / ( %) patients with immune-mediated demyelinating disorder but only in / ( %) of those without evidence of immune-mediated processes. conclusions: demyelinating disorders may be responsible of near % of neurologic complications in the posttransplant setting and, among them, an immune-mediated process is likely to be involved in more than % of cases. our results suggest that the immune mechanism that underliesthe agvhd may also be involved in developing demyelinating disease in transplanted patients. it also may be possible that the lymphoproliferative disorder preexisting the hsct is a risk factor able to increase the risk to develop an immune-mediated demyelinating disorder in the post-transplanting setting. using a t-cell depleted hsct can increase the risk of immune-mediated disorders in at least a small fraction of transplanted patients. despite our results should be validated on a larger cohort of patients, we can speculate on the possible connections between the wide range of complex and still poorly defined immunity disorders which can influence the prognosis and course of transplanted patients. disclosure background: injury to the mucosal barrier and subsequent development of oral mucositis (om) is among the most common toxicities of allogeneic stem cell transplantation (sct). despite the high prevalence of om and its debilitating nature, prospective studies evaluating determinants of om are scarce. we therefore prospectively evaluated the occurrence of om following sct. risk factors for om and its implications short and long-term outcomes were assessed. methods: om was prospectively evaluated on a weekly basis in patients undergoing allogeneic hsct. the grade of om was determined based on the national cancer institute common toxicity criteria for adverse events (ctcae) scale (v. . ). severe om was defined as grade ii to iv. conditioning regimens were evaluated individually and according to intensity; myeloablative (mac), reduced intensity (ric) or reduced toxicity (rtc). the latter category included only patients receiving fludarabine and treosulfan at dose of - g/m (flu/treo). risk factors for the development of severe om were initially identified by a univariate analysis and then analyzed in a multivariate logistic regression model. association of om with peritransplant infectious complications, iv morphine consumption, hospitalization length, neutrophil engraftment, acute and chronic graft-versus-host disease (gvhd), non-relapse mortality (nrm) and overall survival were assessed in a univariate analysis. competing events were considered in analyzing engraftment, gvhd, and nrm. results: patients who underwent an allogeneic sct between and were included. median follow-up was days. leading indications for transplantation were acute myeloid leukemia ( %), lymphoma ( %), and myelodysplastic syndrome ( %). the majority of patients received an allograft from a matched sibling or unrelated donor ( %) and methotrexate gvhd prophylaxis ( %). the median time to om onset was (interquartile range [iqr] - ) days. prevalence of grade ii-iv om was %. the median duration om was [ - ] days, and iv morphine was administrated for a median of [ - ] days for patients with grades iii-iv om ( %). in a univariate analysis a younger age (p= . ), lower bmi (p= . ), recent smoking history (p= . ), recent antibiotics exposure (p= . ), mac (p< . ), and use of methotrexate (p= . ) were associated with an increased risk for grade ii-iv om. in a multivariable model the risk for grade ii-iv om was lower with rtc (i.e., flu/treo) vs. mac (odds ratio [or] . ; p< . ) and rtc vs ric (or . ; p= . ), mycophenolate mofetil vs. methotrexate (or . ; p= . ) and recent smoking (or . ; p= . ). compared to lower grades, grade ii-iv om was associated with a longer hospitalization duration (median days vs. days; p= . ), delayed neutrophil engraftment (median vs. days; p=- . ), and more gastrointestinal related infections ( % vs. %; p= . ). grade ii-iv om was not associated with increased risk of bloodstream infections, acute or chronic gvhd, non-relapse mortality, and increased mortality. conclusions: oral mucositis is prevalent among allogeneic-sct recipients. importantly, fludarabine-treosulfan, which is considered a myeloablative is associated with a markedly reduced risk for om. consequences of om include prolongation of hospitalization, delay in neutrophil engraftment, and a tendency for gastrointestinal infections, but does not increase the risk for gvhd and mortality. disclosure: nothing to declare background: the advent of recent diagnostic techniques for the assessment of iron overload (t *-mri) and their systematic use as screening tools in the setting of secondary hemochromatosis have led to an increased awareness that focal nodular hyperplasia (fnh) represents a possible incidental finding after hematopoietic stem cell transplantation (hsct). methods: clinical and radiological features of patients undergoing hsct in a single pediatric institution have been retrospectively reviewed for fnh. in order to provide an estimate of the prevalence of fnh after hsct, we analysed all the t *-mri scans performed during the last years in our centre and recorded the number of patients with fnh (group a). in addition, data about patients incidentally diagnosed with fnh at abdominal imaging performed for different clinical indications have been collected (group b). results: eight out of ( %) transplanted patients who underwent at least one t *-mri scan from september to september were incidentally diagnosed with fnh. group b included subjects with fnh incidentally found at ultrasound or non-t * mri scans performed before . overall, transplanted patients ( males, %), transplanted for al ( cases) or bone marrow failure ( cases) at a median age of . ± . years, were diagnosed with fnh between . and . years after hsct, namely . ± . years in group a and . ± . years in group b. a variable degree of iron overload was demonstrated in all patient (lic: - ± microg/g; baseline serum ferritin: - ng/ml). the potential risk factors for fnh are reported in table . in / patients, the radiological finding was pathognomonic; in / the diagnosis of fnh was confirmed histologically, while / subjects were labelled as "fnhlike", although a potential diagnosis of hepatic adenoma could not be ruled out. in / patients, fnh presented with an isolated lesion, while / had to more than hepatic nodules at diagnosis. the size of nodules at diagnosis ranged from to mm. in unenhanced mri scans, lesions were predominantly hyperintense on both t -and t weighted sequences. in dynamic studies with contrast medium, all lesions strongly enhanced during the arterial phase, with a variable degree of wash-out in the late venous scans. hepatic function tests were normal in all the enrolled patients at diagnosis of fnh. among the / patients for whom at least a follow-up scan was available, presented a complete regression, a reduction and an increase in the size and/or number of lesions, while in patients the nodules remained substantially unchanged after a mean radiological follow-up of . ± . years. no malignant transformations were observed. conclusions: fnh represents a relatively frequent incidental finding after hsct. although a malignant transformation is rare, given the demonstrated variable evolution of the hepatic nodules, a radiological follow-up is highly recommended. disclosure: nothing to disclose. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic transplantation: retrospective multicenter study of turkish hematology research and education group (threg), updated data methods: ten centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january and december . a baltimore criterion was used for assessment of hsos. four hundred twenty six ( . %) of patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide ( - mg/kg/day). results: the study included patients ( males/ females) with median age of ( - ) years. the demographic and clinical characteristics of patients were summarized in table seventy-three ( . %) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was . ( - ) days. thirty-seven ( %) of patients with hsos recovered completely and forty-nine ( %) of them died as a result of multi organ failure. the incidence of hsos-related mortality in allo-hsct cohort was found to be . %. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsos-associated mortality (p= . ). conclusions: hsos still remains a serious lifethreatening complication of allo-sct. although the incidence is low, hsos is associated with increased -day non-relapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. disclosure: nothing to declare prophylaxis with defibrotide in adults at very high risk of veno-occlusive disease: results in patients background: hepatic sinusoidal obstruction syndrome/ veno-occlusive disease (sos/vod) is a life threatening complication that can occur after hematopoietic stem cell transplantation (hsct). severe sos/vod rapidly evolves in multiple organ dysfunction syndrome (mods), associated with a mortality rate exceding %. precocity of defibrotide (df) treatment is the leading factor for efficacy. prophylactic use of df is recommended in children, but its value has not been validated in the adult population, although factors for individual risk assessment for vod are debated. we here present a real-world experience of df prophylaxis in adult patients at very high risk of sos/vod receiving allogeneic hsct. methods: from to we treated with prophylactic defibrotide and ursodeoxycholic acid (udca) patients, median age years (range - ). nine patients received allogeneic hsct for acute lymphoblastic leukemia ( b-all and t-all), one patient for severe aplastic anemia, one patient for primary myelofibrosis. they were all at high risk for sos/vod because of previous hepatotoxicity ( patients), previous hsct ( patients), double alkylating agent ( patients) or previous treatment with inotuzomab ozogamicin (io; patients). of the patients treated with io, received cycles of io, and received cycle, with the last io dose administered a median . days before hsct (range - d). defibrotide was administered in daily doses for a total dose of mg/ kg per day and udca at the dose of mg twice per day, starting from day - prior transplant. all patients received treosulfan-fludarabine based conditioning. in patients thiotepa was added to the conditioning and in patients a low dose gy tbi. gvhd prophylaxis included posttransplant cyclophosphamide, rapamycin and mycophenolate in all patients, except one patient with aplastic anemia receiving atg, rapamycin and mycophenolate. donor source was pbsc in all cases. seven patients received family haploidentical (mmrd) transplant, patient a mrd transplant and patients a mud transplant. results: the median duration of defibrotide therapy was days (range - days). documented non-severe gastrointestinal bleeding occurred in patients requiring defibrotide temporarily discontinuation, no other significant bleedings were experienced. four patients developed grade ii-iv acute gvhd and no transplant-associated thrombotic microangiopathy were diagnosed. overall, sos/vod occurred in / cases within days after hsct (days , and ) and no late-onset sos/vod were diagnosed. sos/vod was very severe, causing mods and death in all cases. all patients were characterized by a common pattern of very high risk factors for sos/vod by prior hsct and salvage treatment for b-all with cycles of io close to hsct. furthermore, they all received a fully myeloablative conditioning regimen with treosulfan and thiotepa and a mmrd transplant. conclusions: defibrotide prophylaxis was safe and well tolerated with no severe related complications. sos/vod occurred despite continuous df prophylaxis in / patients treated with inotuzomab ozogamicin close before undergoing nd transplant. to reduce the incidence of severe vod, pre allo-hsct treatment with inotuzomab ozogamicin should prompt avoidance of other cumulative risk factors for vod, such as use of double alkylating agents. disclosure background: busulfan is the backbone of many preparative regimens administered to children undergoing allogeneic and autologous hematopoietic stem cell transplantation (hsct). among its many long-term adverse effects, busulfan can cause various degrees of pulmonary injury. although well described in adults, there are few large series exploring pulmonary toxicity of busulfan in children. we describe long-term pulmonary follow-up in a large group of children treated at a single center who had received high-dose busulfan and examine the relationship of systemic drug exposure and lung function over time. methods: all surviving children who had received highdose busulfan between - in the context of hsct at the schneider children´s medical center, were referred for serial pulmonary function monitoring (including spirometry, plethysmography and diffusing capacity for carbon monoxide [dlco] . pre-transplant testing was available for children who were old enough to perform the procedure. spirometry results were adjusted according to the revised global lung initiative formulas for age, gender, and height. pulmonary injury was defined as a z score below - . for spirometry, or < % of predicted for the other parameters. busulfan levels were monitored following the second drug dose. all patients received busulfan in four daily doses. area under the curve (auc) calculations were performed by bayesian calculations. results: between - , patients aged - years were diagnosed with malignant or non-malignant diseases and treated with high-dose busulfan. of shortterm survivors, had at least one post-transplant pulmonary function evaluation. the mean age at treatment with busulfan was . years (range, . - years). of these children, children had undergone autologous transplantation and children had an allogeneic transplant. of these patients eventually relapsed and died. children had one or more pulmonary risk factors before hsct -chest or upper abdomen radiation ( ), chest wall tumors or lung metastasis ( ), chest surgery ( ), prior administration of pulmonary-toxic drug ( ) or asthma ( ). during follow-up (up to years, median . years), fev and fvc spirometry tests both decreased significantly (p= . and . , respectively), while the decrease in dlco was not statistically significant. % of patients had abnormal pulmonary function tests and seven children had symptomatic disease which in two may have been manifestations of gvhd. interestingly, no correlation was found between busulfan auc, busulfan peak levels, the number of busulfan doses administered, the type of transplantation (autologous vs. allogeneic) or primary disease to pulmonary injury. even after censoring of children with pre-transplant pulmonary risk factors we noted a decrease of fev and fvc. conclusions: as in adults, pulmonary injury is observed in children treated with high-dose busulfan prior to hsct. no correlation was observed between busulfan auc and pulmonary injury. follow-up of children who receive this drug should include regular pulmonary monitoring, referral to a pulmonologist when subclinical pulmonary compromise is found, and counseling regarding measures that might prevent or ameliorate pulmonary damage. continued follow-up of this cohort of patients should inform our pretransplant patient information sessions, and the future use of busulfan in children. disclosure: nothing to declare background: transplant-associated thrombotic microangiopathy (ta-tma) is a specific complication of allogeneic hematopoietic stem cell transplantation (hsct). post-hsct tma has been attributed to the vascular endothelial damage caused by high-dose chemotherapy, calcineurin inhibitors (cnis), graft-versus-host disease (gvhd), infections. there is a little evidence published regarding the efficacy and factors influencing the outcome of withdrawal of cnis. methods: the analysis comprised a total of patients, with diagnosed hematologic malignancy (aml ( ), all ( ), mds ( ), hodgkin lymphoma ( ), cml ( ) and neuroblastoma ( ) received allo-sct, from a matched related, unrelated or haploidentical donor between and . patients were diagnosed with ta-tma based on cho criteria. the median age of patients was ( adults, children). gvhd prophylaxis was performed with tacrolimus (tac) in , cyclosporine a(csa) in , combination tacrolimus+sirolimus (sir) in . patients received atg and ptcy. withdrawal of cnis was accompanied by administration of systemic steroids ( patients) or substitution with sir after reaching levels of csa< ng/ml or tac < ng/ml in . the target concentration of sir was - ng/ml. in pediatric patients who received combination tac+sir, the tac was discontinued in one step while sir continued. median time to development tma was , days after allo-sct (range - ). median follow-up of surviving patients was days. the primary outcome was overall survival (os) up to years after development of ta-tma. results: the following significant predictors of -year os were identified: tac replacement with sir (p< , ), ptcy in prophylaxis (p< , ), acute gvhd (agvhd) grade - (p= , ), previous sepsis (p= , ), level of ldh in debut (p= , ), combination sir+tac in prophylaxis (p= , ), major ab -mismatch (p= , ), severity of cns symptoms (p< , ). there was no significant difference in os according to patients' age, sex, "salvage" disease status at transplantation, previous vod, viral (hhv , , , cmv, ebv) reactivations, count of cd + cells transfused, ldh level, shizocytes and creatinine in the debut of ta-tma. in the multivariate analysis replacement of cnis with sir (hr . , %ci . - . , p= . ) and baseline ldh level (hr . , %hr . - . , p= . ) were associated with survival differences. the cut off for ldh was xunl. agvhd grade - (hr . , p= , ) and use of ptcy (hr . , p= . ) were not significant in the multivariate analysis (figure ). ta-tma cases after ptcy were significantly less frequently associated with clinically significant agvhd ( % vs %, p< , ). the survival was higher after ptcy ( % vs %), but not significant due to sample size and other ta-tma factors. leading causes of death were: gvhd progression ( %), bacterial infection ( %), tma ( %) and other ( %) . conclusions: replacing tac by sir is an effective therapeutic strategy in a group of patients with debut of ta-tma at least after ptcy, where it is less likely to be associated with agvhd. there is a significant overlap of populations with ptcy prophylaxis and substitution with sir, thus the study is not powered to provide guidance for patients on conventional prophylaxis with ta-tma. [[p image] . disclosure: none of the authors has anything to disclose. donor-recipient ab mismatch effect on the allogeneic hematopoietic stem cell transplantation outcome: a single-center retrospective study background: because transmission of major histocompatibility complex and blood group system genes is independent from each other, approximately - % of all allogeneic hematopoietic stem cell transplantations (allo-hsct) are realized crosswise the ab -blood group boundary. however, due to the widespread expression of ab antigens on a variety of human tissues other than erythrocytes, ab incompatibility may have an impact on the outcome of allogeneic hsct that goes beyond the wellknown immune-hematological complications such as immediate hemolysis due to the presence of isoagglutinins and delayed hemolysis due to passenger b lymphocytes. here we aimed to assess the donor-recipient ab mismatch effect on the allo-hsct outcome, comprising non-relapse mortality (nrm), overall and relapse-free survival, posttransplant prc transfusion requirement, as well as relapse rate, incidence of graft-failure and acute gvhd. methods: clinical and laboratory data from consecutive patients undergoing allogeneic hsct between / and / at the fondazione irccs ca' granda maggiore policlinico hospital in milan, italy, were retrospectively collected. kaplan meier estimates were used for the analysis of survival outcomes while nrm, relapse and acute gvhd cumulative incidences were investigated by competing risk analysis. results: the patient series included ab -match, major ab -mismatch, minor ab -mismatched and bidirectionally ab -mismatch transplants. indication for allo-hsct were mainly aml/mds ( pts), all ( pts) and t-nhl/ctcl ( pts). mean overall survival for groups of patients undergoing ab -identical, major ab mismatch and minor ab mismatch hsct were months ( % ci [ ; ]), months ( % ci [ ; ) and months ( % ci [ ; ]), respectively. nrm in the three groups were significantly different, with point estimates of %, % and % at years, respectively, whereas no significant differences were observed for relapse rate and graft failure incidence. although not statistically different, incidence of acute grade iii-iv gvhd was twice as high in patients transplanted from minor ab mismatched donors than in the ab identical group ( % vs %). following transplantation, prc transfusion requirement was significantly higher in the major ab mismatch then in the ab -match transplanted patients (median vs , p= . ), with a marginal positive correlation between the anti-donor a/b igg titers measured prior hsct and the total number of prc transfused during the first year following transplantation. we observed only one case of prca occurring in a year-old + woman who was transplanted from a -yearold male a+ hla-identical sibling using peripheral blood as the stem cell source following a myeloablative conditioning for aml in first complete remission. anti-a igg isoagglutinin titers prior to transplantation were : . during the first year post transplantation, the patient required a total of prc transfusions, with gradual resolution occurring only after introduction of danazole treatment. conclusions: in our patient cohort, both major and minor ab mismatch associated to a significantly higher nrm. major ab mismatch associated to a higher prc transfusion requirement. a more frequently occurring severe acute gvhd was also suggested in minor ab -mismatch transplants. altogether, our results suggest that allo-hsct outcome may be significantly affected by ab blood group mismatch. disclosure: nothing to declare background: at-tma is a severe endothelial injury complication and it may involve the intestinal vasculature. intestinal tma could be fatal and missdiagnosed. clinical and pathological criteria to differentiate from intestinal gvhd are needed. the aim of this study was to analyze the incidence and histological characteristics of intestinal tma in patients diagnosed of systemic tma. methods: we analyzed the incidence of tma in patients who underwent allo-hsct in our institution between january -august . tma diagnosis was based on ho criteria. we do a pathological review in biopsies from out of patients in whom an endoscopy have been performed days before and days after the diagnosis of tma for suspicious of gvhd. review was performed by a pathologist expert in gvhd, who examined the biopsies in search of hystopathological features of gvhd, tma or viral infection. diagnosis of gastrointestinal gvhd was stablished according to mcdonald and sales criteria, while intestinal tma diagnosis was stablished by warren et al criteria. results: out of patients ( , %) were diagnosed of tma. transplant characteristics and tma data of patients with systemic tma are shown in image. out of patients with tma ( %) had been diagnosed with prior/ simultaneous acute gvhd, of them grade iii-iv, and % with gastrointestinal gvhd. intestinal tma have been reported only in out of patients ( %) at diagnosis, whereas when review based on warren criteria was performed, in patients ( %) the pathologist found at least of the criteria of endothelial damage and % of the patients or more warren criteria were founded. the most frequent features were endothelial cell swelling (n= , %) and perivascular mucosal hemorrhage (n= , %). review hystological features of biopsies are shown in table of the image. regarding gvhd, it was found in patients ( %) at diagnosis and in ( %) at pathological review. with a median follow-up of months ( - ) patients of the with systemic tma ( %) are dead. of the deaths ( %) were related to tma ( tma, tma +gvhd, and tma+infection). patients with or more warren criteria in pathological review had poor outcome compared with patients less than criteria ( % alive vs % at months, p= . ). conclusions: intestinal tma is a life-threatening underdiagnosed entity. only patients of patients were diagnosed of intestinal tma. we found that most of our patients had endothelial damage in the gastrointestinal biopsy pathological reviews. gvhd histological criteria were present in most of the patients, mainly histological grade i-ii. prognosis of these patients is poor and pathologist effords in diagnosed the entity is guarranted. disclosure: nothing to disclosure p strategies to reduce neutropenic fever and hospital readmission in multiple myeloma patients managed at home after autologous stem cell transplantation background: neutropenic fever (nf) is the most frequent cause of readmission in the outpatient autologous stem cell transplantation (asct) programs. in our at home model for multiple myeloma patients, we added primary prophylaxis with ceftriaxone, decreasing the incidence of fever during aplasia phase from % to . %. the aim of this study was to analyze the addition of two strategies to reduce the non-infectious nf: withdrawal of g-csf and the addition of primary prophylaxis for engraftment syndrome with corticosteroids after asct. methods: between january and august myeloma patients were managed at-home since day + of asct. all were conditioned with mel . all patients received prophylaxis with quinolone, fluconazole, aerolized pentamidine, low-dose acyclovir (hvs+), and ceftriaxone (since day + ). the patients were classified into groups: group a (n= ; g-csf since day + without corticosteroid), group b (n= ; no g-csf and no corticosteroid), group c (n= ; no g-csf with prednisone . mg/kg/day since day + until granulocyte recovery). first-line therapy at home of nf was piperacillin-tazobactam . g/ h i.v. using a portable intermittent infusion pump. fever was an indication of immediate medical consultation and those patients presenting signs of focal infection or severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver, uncontrolled nausea, vomiting or diarrhoea, and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients and outcomes are shown in table . there were no differences between groups regarding age, gender, immunological subtype, response before asct, hct-ci, and cd cell dose infused. there were more patients with advanced disease (iss iii) in group c compared to group a ( . % vs. . %; p= . ). the duration of neutropenia was longer in those groups that did not receive g-csf (a: days, b: days, c: days; p< . ). comparing group a with group c, we observed that the incidence of nf and the readmissions rates were lower in group c (nf: . % vs. . %; p= . ; relative risk reduction: . , and number needed to treat . ; readmissions: . % vs. . %; p= . , respectively). the -day cumulative incidence of nf were . % in group a, . % in group b, and . % in group c; p= . . the non-administration of g-csf with the addition of prophylactic corticosteroid did not modify the incidence and grade of mucositis, the first day and duration of fever, nor the number of bacterial infections documented. in the multivariate analysis, this combination (no g-csf with corticosteroid) maintained its protective effect for the development of nf and hospital readmission (or . ; p= . and or . ; p= . , respectively). conclusions: the non-use of g-csf and the addition of prophylactic corticosteroid in mm patients managed at home after asct minimize the incidence of non-infectious fever and optimize hospital resources by reducing hospital readmissions. disclosure: nothing to declare. background: antibody titers to vaccine-preventable diseases decline during the - years after allogeneic hematopoietic stem cell transplantation (hsct) if the recipient is not revaccinated. it is therefore considered best practice to try to offer hsct recipients the same level of protection against all vaccine preventable diseases as the general population. few data in the literature are available concerning vaccine-related problems in hsct recipients. we performed a farmacovigilance evaluation in a cohort of allotransplanted patients followed in our clinic during a year period. methods: from october to november we administered a list of recommended vaccines to hsct recipients attending our routine out patient clinic who fulfilled the following criteria: cd t cells> /μl, cd b cells> /μl, anti-cd antibody infusion> months, ivig therapy> months, no active and severe graft-versus-host-disease (gvhd), no chemotherapy or biological therapeutic agents on going. vaccines suggested were influenza, pneumococcal conjugate (pcv ), polio (inactivated polio vaccine), diphteria, tetanus, acellular pertussis, hepatitis b, hepatitis a, haemophilus influenzae type b, meningococcal quadrivalent (mcv ), human papillomavirus, meningococcal b, measles-mumps-rubella (mmr), varicella. live vaccines (mmr and varicella) were not recommended before years after hsct and in patients with chronic gvhd. all the patients were asked to take the list to the local health facilities in order to have the vaccines injected and a vaccination table arranged with the doses already received and those to receive. we checked the vaccination tables at each visit and monitored potential side effects and gvhd status at , , and months after the first vaccine injection. results: twenty-nine out of patients were evaluable (table ), without gvhd and with chronic gvhd ( mild, moderate, severe). median time after hsct was months ( - ). median number of vaccines received was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . as regards patients without chronic gvhd, out of experienced fever after vaccine injections; out of developed transient mild reduction of platelet count; patient reported headache and otalgia after vaccine injection, while another one transient joint pain; out of patients presented signs of mouth chronic gvhd (score nih) and transaminase increase (grade according to world health organization toxicity scale) months after the first vaccine dose, so that cyclosporine dose had to be augmented. as regards patients with chronic gvhd, out of experienced fever after vaccine injections; patients with mild chronic gvhd of the mouth presented hepatic flare two and three months after the first vaccine dose, respectively. in both cases a new increase of cyclosporin and methylprednisolone doses determined progressive normalization of liver enzymes. conclusions: these data show that vaccines were globally well tolerated in hsct recipients, even when they suffered from chronic gvhd. however, close monitoring is warranted in order to better evaluate possible vaccine side effects in this setting of patients. background: allogeneic hsct improves survival for aml patients over the age of years of age when compared to chemotherapy alone. the haematopoietic stem cell transplantation comorbidity index (hct-ci) and ebmt score predict for non-relapse mortality and overall survival, yet little is known about whether qol is preserved in this patient group and whether hct-ci and other performance scores pre-bmt correlate with qol post allo-hsct. methods: we conducted a retrospective analysis of patients years and older who underwent ric allo-hsct at the university hospital of wales, cardiff between september and december (n= ). hct-ci, karnofsky performance score (kps) and ebmt scores were calculated prior to transplant and qol measured using the fact bmt (version ) questionnaire, which was completed at , and months post transplant. patients were grouped at the -, -and -month time points for each of the different performance indices, allowing group comparison against compound sub scores using the mann-whitney u test. results: patients were included in this study, with median age years (range - ). patient characteristics, including conditioning, donor type, pre-transplant hct-ci and kps scores are summarised in table . the year and year overall survival (os) for the patient cohort was . % and . % respectively. hct-ci of ≥ vs was significantly associated with poorer bmt-related qol domains at months (p= . ) and general qol domains at months (p= . ) post-transplant. while ebmt score showed no correlation with qol parameters, patients with kps of vs ≤ showed significant differences in both general (p= . ) and bmt-related qol (p= . ) at months and in all qol domains at months (symptomrelated qol p= . , general qol p= . , bmt-related qol p= . ). importantly neither the hct-ci nor the kps pre-transplant predicted for qol at months post transplant. conclusions: patient selection is key to ensuring maximum benefit from allo-hsct both in terms of overall survival but also with regards to qol and survivorship. we note that while patients with hct-ci ³ or kps ≤ had significantly poorer qol at months post allo-hsct, qol was recovered by months post transplant, with this significant difference no longer seen. our data shows that in selected aml patients over the age of years with good performance status and low comorbidity index, a favourable outcome can be achieved with good qol maintained throughout the post transplant period. background: advances in allosct technology, supportive care, and use of reduced intensity conditioning regimens for older patients have led to significant improvements in longterm survival after transplant. the survivors have an elevated probability of late morbidity and mortality, including abnormalities in phosphocalcic metabolism and bone disease. rapid and progressive bone loss occurs within the first - months after transplant, and this is followed by a slow process of recovery, with bone loss persisting for to months. bone fractures can worsen the quality of life of allosct survivors, but the real burden of the disease is unknown. the objective of the study is to ascertain the prevalence of bone pathology and vertebral fractures early after transplant in our center. methods: this is a retrospective and observational study. forty-nine patients ( male/ female, median age y, range - ) that underwent allosct were included in the study in the period of to months after transplant (may -december ). pre-and post-transplant risk factors associated with bone disease were recorded: age > years, female sex, menopause, hormone replacement therapy, previous treatment with steroids, previous fractures, weight < kg, bmi < - , low physical activity, low calcium intake, smoking, alcohol intake, and history of femoral fractures in parents. in all patients laboratory data (including serum calcium, -hydroxyvitamin d, and pth), lumbar and femoral bmd (dxa), and spinal x-ray were also evaluated. a vertebral fracture was defined as a reduction of > % in the anterior, middle or posterior high of the vertebral body. results: we identified vertebral fractures in ( %) patients. five patients had fractures prior to transplantation, and patients presented "de novo" vertebral fractures following transplantation; therefore, the prevalence of "de novo" postransplant fractures was / ( %). most ( %) of these fractures were asymptomatic at the time of diagnosis. most patients ( %) with vertebral fractures had > pre-sct risk factors (median risk factors pre-sct , range - ), the most frequent being low calcium intake, steroid exposure, presence of previous fractures, and menopause. those patients with fractures and less than risk factors pre-tph, added new risk factors after transplant, mainly steroid treatment. forty-four patients ( %) had vitamin d insufficiency (< ng/ml), ( %) had osteopenia and ( %) had osteoporosis. vitamin d insufficiency and bone disease were more frequent in women than in men ( % vs. % for vitamin d, % vs. % for osteopenia, % vs. % for osteoporosis, and % vs. % for vertebral fractures, respectively). conclusions: the prevalence of post-transplant bone disease and vertebral fractures in our series is high. most fractures appearing "de novo" after allosct were asymptomatic and were diagnosed by x-ray. patients who presented vertebral fractures frequently had more than risk factors identified pre-sct. patients undergoing allosct should have their bone health assessed early in their treatment and, if indicated, should start preventative therapy to avoid bone loss and fractures. other measures such as physical exercise, vitamin d and calcium supplementation, and dxa and spinal x-ray at baseline and following transplantation are also highly recommended. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare the use of g-csf in selected patients after autologous stem cell transplantation is associated with low incidence of engraftment syndrome background: the use of g-csf after autologous stem cell transplantation (asct) accelerates neutrophil recovery, however it has been related to an increased risk of engraftment syndrome (es) development in some studies. for this reason, we do not routinely prescribe g-csf after asct and we only use it in patients with significant complications (enterocolitis, severe sepsis, atrial fibrillation) after stem cell infusion. the main objective of this study is to evaluate the incidence of es in patients who receive asct for monoclonal gammopathies (mg), non-hodgkin lymphoma (nhl) and hodgkin lymphoma (hl) and receive g-csf only if needed. as secondary objectives we evaluate differences in the engraftment day as well as the length of inpatient stay. methods: we retrospectively analyzed patients with mg or lymphoma, who underwent asct conditioned with high dose melphalan ( - mg/m ) or beam, respectively, between and in our center. specific clinical features for es according to spitzer and maiolino criteria were evaluated between days before and days after the engraftment. statistical analysis was performed with spss v. . . results: thirty-one patients with mg and patients with lymphoma were analyzed. median age at transplant was . years ( . - . ) and patients ( . %) were male. median prior lines of treatment in patients with gm or lymphoma were ( - ) and ( - ), respectively. table shows patients´characteristics. mobilization with g-csf ± plerixafor was performed in patients ( %) and chemotherapy + g-csf ± plerixafor in patients ( %). median cd x /kg cells infused was . ( . - . ). eleven patients ( . %) received g-csf, due to infection ( enterocolitis, listeriosis, acute hepatitis, septic shock) and because of atrial fibrillation or fibrilloflutter. median time from sct to first day of g-csf was days ( - ) and median time on g-csf treatment was days ( - ). patients who received g-csf showed a short time to neutrophil engraftment (≥ . x /l), days vs. days, p< . but longer duration of hospitalization, days vs. days, p = . . non-relapse mortality at day + , + and + was %. es was diagnosed in ( . %) patients, amyloidosis, multiple myeloma and plasmablastic lymphoma. there was not statistical difference in the incidence of es between patients who received g-csf ( . %) and patients who did not ( . %), p= . . analyzed by disease, es appeared in of patients who received g-csf in the lymphoma group ( . %) but none of the patients with mg that received g-csf developed it. we did not find statistical differences between patients who developed es and those who did not in age ( years vs. years, p= . ), length of hospitalization ( days vs. days, p= . ) and the number of cd x /kg cells infused ( . vs. . , p= . ) . conclusions: the use of g-csf in selected patients is associated with low incidence of es. our study confirms that the use of g-csf accelerates neutrophil recovery but it is unclear if it can increase the incidence of es, especially in patients with lymphoma. [[p image] . background: graft failure is one of the top- problems of allo-hsct (after gvhd and relapse). the problem of graft failure becomes more significant due to increasing number of allo-hsct with ric conditioning regimen from haploidentical and hla-mismatched unrelated donors. role of t cells in graft failure is well known. here we report an impact of t-memory cell subsets count before antithymocyte globulin (atg) administration on primary graft failure after allo-hsct. methods: sixteen patients with acute leukemia transplanted in national research center for hematology were included on this prospective study. all patients received horse atg at dose mg/kg/day from day - to - before allo-hsct as gvhd prophylaxis and were balanced by other factors that could affect engraftment. detailed patients characteristics are listed in table . peripheral blood samples were collected on day - before allo-hsct (before atg injection) in edta-tubes. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+scm) -cd r -ccr +cd +; t-central memory (tcm) -cd r +ccr +cd +; t-transitional memory (ttm) -cd r +ccr -cd +; t-effector memory (tem) -cd r +ccr -cd -; t-terminal effector (tte) -cd r -ccr -cd -, among cd + and cd + t-cells . sysmex xe- was used to calculate absolute count of different t-cell subsets. mann-whitney u test was used for nonparametric data analysis between two groups. fisher's exact test was used for x tables. p-value less than . was considered statistically significant. results: an influence of t-memory cell subsets count before atg administration on primary graft failure is shown in figure . according to our data high absolute number of cd +ttm and cd +tte is associated with primary graft failure. conclusions: based on these findings high absolute number of cd +ttm and cd +tte could be one of the prognostic factors of primary graft failure after allo-hsct. optimizing atg dose due to recipient absolute t-memory cell subsets count before atg administering may prevent graft failure and improve posttransplant results. background: upper gastrointestinal graft-versus-host disease (gi gvhd) has been an increasingly recognised entity following allogeneic stem cell transplantation (sct). budesonide, widely used in inflammatory bowel conditions, has also been found beneficial in gi gvhd. the major benefit of budesonide is attributable to its poor absorption and extensive first-pass metabolism via cytochrome p (cyp) a , which translates to less systemic steroid-related effects. however, transplant patients are often exposed to multiple drugs, among which some agents act as cyp a inhibitors and therefore can increase budesonide bioavailability and might lead to systemic toxicity. azole antifungal drugs are probably the most common concomitantly used cyp a inhibitors in transplant recipients. methods: we reviewed allogeneic sct records for patients treated with oral budesonide for gi gvhd at our transplant centre between and retrospectively. the aim of the work was to assess the development of adrenal suppression with or without clinical features of iatrogenic cushing`s syndrome. the standard dose of budesonide was mg three times a day. patients receiving prednisolone or other glucocorticosteroids and those with no available serum cortisol level measurements were excluded. results: our analyses identified four allogeneic sct patients in whom adrenal suppression was diagnosed with undetectable serum cortisol levels during oral budesonide treatment. of these patients two developed iatrogenic cushing`s syndrome and both patients were treated with cyp a inhibitors concomitantly: . clarithromycin and fluconazole; . clarithomycin and voriconazole. the development was rapid (within and weeks). symptoms included morphological features such as moon face, high blood pressure, weight gain, peripheral oedema and proximal myopathy. symptoms resolved gradually following cessation of azole antifungal agents and on gradual weaning of budesonide. conclusions: although single agent budesonide treatment given for gi gvhd is rarely associated with systemic side effects, patients on azole antifungal drugs and macrolide antibiotics are at higher risk of systemic toxicity due to drug interactions. patients who are allergic to penicillin and receive macrolide-based prophylaxis can be especially vulnerable. to our knowledge the number of cases reported in literature about systemic effects of oral budesonide in transplant recipients is less than . our observation supports previous reports on the potential of oral budesonide to induce systemic effects. we therefore advise careful monitoring of patients treated with budesonide in combination with cyp a inhibitors, including antimicrobial agents routinely used in sct. disclosure: none implemented strategies to overcome barriers in the establishment of a consolidated hematopoietic stem cell transplantation program in a developing country background: the national institute of medical sciences and nutrition "salvador zubiran" is a national health institute located in mexico city. although mexico is considered an upper-middle income country, more than % of the population lives in poverty without health care coverage and patients within this social stratum are referred to our institution. the first hematopoietic stem cell transplantation (hsct) in mexico was performed at our institution in . from this year until , hsct were sporadically performed (n= ), showing a poor overall survival (os) and high non-relapse mortality (nrm). these outcomes resulted from an unstructured hsct program, limitedresources, patient low socioeconomic status, and paucity of population-adapted procedures. in , according to these results, a decision to establish a hsct program was made. therefore, in order to set up a successful hsct program, implementation of financial and medical strategies were necessary. the objectives of this study were to describe the barriers and implemented strategies for the establishment of a hsct program in mexico along with the outcomes of patients undergoing this procedure throughout the reorganization of the program. methods: this study is a health services research. barriers were detected based on the results of the hsct program from - (not shown). table shows the financial, medical, and research strategies that were implemented for each barrier. results: from november to november , hsct have been performed in patients at our institution. most hsct were autologous (n= , %). forty one patients underwent hsct. from the patients, most were males (n= , %) and the median age was . years (range, - ). the most frequent underlying diseases for auto-hsct were lymphomas (n= , %), non-seminomatous germ cell tumors (n= , %), and multiple myeloma (n= , %). acute leukemias (n= , %), aplastic anemia (n= , %), and myelodysplastic syndromes (n= , %) were the most frequent diagnosis for patients undergoing allo-hsct; and acute leukemia was the most frequent diagnosis for patients undergoing haploidentical hsct (n= , %). acute and chronic gvhd were present in % (grades i-ii %) and % (limited %), respectively. for allo-hsct, , day, and -year nrm was . %, %, and %, respectively; and -day nrm in auto-hsct was . %; year os was % and % for auto and allo-hsct, respectively. conclusions: future perspectives of the hsct program include the acquisition of funds for unrelated donors; to improve outcomes of patients undergoing haploidentical hsct, and to increase the number of in-patient rooms. we conclude that despite paucity of resources and other limitations, the implementation of financial, medical, and research strategies have shown that barriers can be effectively overcome in a developing country in order to establish a consolidated and nationally renowned hsct program, providing good outcomes for patients. disclosure: none of the authors have any conflict of interest to disclose. the effect of protective buffering on daily stress and relationship quality in dyads following hematopoietic stem cell transplantation: results from daily process methodology malgorzata sobczyk-kruszelnicka , aleksandra kroemeke , zuzanna kwissa-gajewska , sebastian giebel background: cancer-related support communication (e.g., protective buffering) may impact the risk for psychological and relationship distress in patients following hematopoietic stem cell transplantation (hsct) and their caregivers. previous studies have revealed that protective buffering (i.e., hiding one's concerns and denying one's worries) has mixed effects: is beneficial (for "protected" person), costly (especially for the person using it), or unrelated to dyadic wellbeing. there has been, however, little evidence linking dyadic protective buffering with distress using daily process methodology. we assessed ( ) the relationship between daily protective buffering, and same-and next-day stress and relationship quality in patient-caregiver dyads following hsct and ( ) whether similarity or complementarity in protective buffering between dyads is adaptive. methods: two hundred patients (after first autologous or allogeneic hsct) and their caregivers (spouse or another relative) independently completed measures of daily protective buffering, daily relationship quality, and daily stress for consecutive evenings after patients´hospital discharge. actor-partner-interdependence model (i.e., both partners' and caregivers' reports regarding support communication and distress were studied) was used to test study hypotheses. results: for both patients and caregivers, multilevel structural equation modeling showed a significant positive relationship between daily protective buffering and sameday relationship quality. association of protective buffering with same-day stress level was negative. in next-day analyses, patient-reported protective buffering was related to patient's higher relationship quality, whereas caregiverreported protective buffering increased patient's daily stress. complementarity in protective buffering was related to higher immediate same-day relationship quality for both patients and caregivers, while benefits from similarity have delayed effects, although only in patients. conclusions: contrary to previous studies, protective buffering rather has a beneficial effect in dyads following hsct. protection of the partner and relationship against revealing negative emotions and powerlessness was not related to costs in both parties. the findings suggest that the effect of daily protective buffering in dyads following hsct depends on support timing (same-or next-day effect) and differs for both parties. patients seem to benefit the most from the similarity in protective buffering, while caregivers from complementarity. the "fit" between patient and caregiver in support communication ought to be taken into consideration in the practical approach. disclosure: nothing to declare. virus reactivation and low dose of cd + cell were associatied with secondary poor graft function within the first days after allogeneic stem cell transplantation yuqian sun , xiao-jun huang background: secondary poof graft function (spgf) was defined as the secondary cytopenia after initial engraftment of hsct. it was shown to be associated with poor prognosis, however there are very few reports on the incidence, risk factors and outcomes of spgf. methods: patients who received transplantation from peking university people's hospitial during january, to december, were retrospectively reviewed if they fulfilled the following conditions: ( ) diagnosed with acute leukemia or myelodysplastic syndrome; ( ) received allo-sct from either matched sibling donor (msd) or haploidentical related donor (hid). pgf was defined as persistent neutropenia (≤ . × /l), thrombocytopenia (platelets ≤ × /l), and/or hemoglobin ≤ g/l for at least consecutive days, transfusion-dependence, associated with hypoplastic-aplastic bone marrow (bm), and complete donor chimerism without concurrent graftversus-host disease (gvhd) or disease relapse. primary pgf was defined as the failure to achieve initial engraftment by days after transplantation, while secondary pgf was defined as the fulfillment of the criteria after initial engraftment hsct. results: during january, to december, , patients who received transplantation from peking university people's hospitial were retrospectively reviewed. among the patients who achieved initial engraftment, patients developed spgf. the cumulative incidence of spgf on day was . %. the median time of secondary pgf was . ( - ) days after transplantation. low (< median) cd + cell dose (p= . , hr . ( %ci, . - . )), ebv reactivation (p= . , hr . ( %ci, . - . )) and cmv reactivation (p= . , hr . ( %ci, . - . )) were identified as independent risk factors with spgf. there is no significant difference of pgf incidence in msd group and hid patients (p= . ). the overall survival of patients with spgf at year after transplantation was significantly poor than patients with ggf ( . % versus . %, p< . ). conclusions: in conclusion, spgf develop in . % patients after allo-sct, especially in patients with cmv, ebv reactivation or infused with low dose of cd + cell. the prognosis of spgf is still poor due to lack of standard treatment. disclosure: there is no conflict of interet thiotepa with treosulfan and busulfan based conditioning are significantly more gonadotoxic than treosulfan previous studies suggest that busulfan results in long-term gonadal toxicity. no previous studies have compared gonadal toxicity outcomes after treatment with busulfan with treosulfan, a newer agent with similar marrow toxicity to busulfan but with reduced non-marrow toxcitiy. our aim was to determine whether there are differences in pubertal and fertility outcomes in paediatric patients treated with treosulfan compared with busulfan. methods: inclusion criteria were patients who had received either busulfan or treosulfan or treosulfan with thiotepa, only one hct and were aged years and above in august . eligible patients were reviewed in clinic as part of their routine follow-up, thus research ethical approval was not required. follice stimulating hormone, luteinising hormone, oestradiol, and pubertal history were noted. ovarian reserve was estimated in female patients by measuring serum anti-mullerian hormone (amh). male patients had serum testosterone measured and were also offered semen analysis. results: thirty-five patients met the inclusion criteria, of which twenty-five wanted to be reviewed ( %); seventeen females and eight males. mean age at hct was years, mean age at review was years and mean years since hct was years. female patients treated with busulfan or treosulfan with thiotepa (n= ) had minimal amh and none of these patients were having regular periods. females treated with treosulfan (n= ) had normal amh and regular periods without needing hormone replacement. only four male patients opted for a semen analysis and all had significantly reduced sperm counts. conclusions: our results suggest that females treated with treosulfan have minimal (if any) reduction in ovarian reserve compared to other conditioning regimens which casue significant compromise. although this was a small study, and thus not suitable for statistical analysis, the clinical findings are marked. future studies should further investigate optimal doses of treosulfan that could be used to achieve bone marrow engraftment and limit long-term effects on fertility. disclosure background: autologous and allogenic hematopoietic stem cell transplantation (hsct) are potentially curative treatments for hematological malignancies. patients with related complications may need admission to the intensive care unit (icu) for specific therapy and organ support. mortality risk factors, supportive care and principal causes of admission in icu are described in our cohort of patients (pts). methods: we retrospectively studied pts, male, with a median age of , years (range: - ) who underwent allo-hsct in our center between july and october . two hundred and twenty-seven( , %) pts received autologous hsct (auto-hct) and ( , %) allogenic hsct (allo-hsct); from unrelated donor, from identical sibling, and the remainder, mismatched related donor . twenty-three ( , %) out of pts were admitted in the icu in the transplant procedure admission. results: fifteen ( , %) out of pts were male with a median age of years (range: - ). patients' baseline diseases were: multiple myeloma ( , %), non-hodgkin´s lymphoma ( , %), hodgkin´s lymphoma ( , %), acute lymphoblastic leukemia ( , %), myelodisplasic syndrome ( , %), solid tumor ( , ) and acute myeloblastic leukemia ( , %). fifteen ( , %) pts received auto-hsct, ( , %) allo-hsct from unrelated donor, ( , %) allo-hsct from identical sibling, and the remainder haploidentical hsct ( ) ( , %). so, , % of auto-hsct pts and % of allo-hsct were admitted in the icu. the median stay in the icu was days (range: - ) and reasons for admission were: respiratory insufficiency ( , %), septic shock ( , %), renal insufficiency ( , %) and multi-organic failure ( , %). twenty-one ( . %) pts required respiratory support with: nasal cannula or oxygen mask (c/m) ( %), non-invasive mechanical ventilation (nimv) ( , %) and invasive mechanical ventilation (imv) ( , %). fourteen ( %) pts needed inotropic agents for shock treatment. finally, ( , %) pts required substitutive renal therapy with hemodialysis or haemofiltration (hd/hf). eleven ( , %) out of pts died, ( , %) were male with a median age of years (range: - ). ten of them ( , %) needed imv and were treated with inotropic agents. all patients who required hd/hf (n= ) died. imv and treatment with inotropic agents were associated with icu mortality (or , ; p= , , or ; p= , ; respectively). conclusions: in our series of pts, , % needed admission in the icu, presenting a mortality rate of % approximately. there were no differences in the prevalence of icu admission regarding hsct donor. main reason for admission was respiratory failure with imv requirement in , % of pts. imv and treatment with inotropic agents were associated with icu mortality. an early identification of pts at risk of icu admission could have a beneficial impact on survival improvement disclosure: nothing to declare is there any association between thrombotic risk factors and veno-oclusive disease in childhood allogeneic hematopoietic stem cell transplantation? background: veno-oclusive disease (vod) is a major complication of hematopoietic stem cell transplantation (hsct). in some studies levels of fibrinolytic factors especially plasminogen activator inhibitor- (pai- ) level were found associated with vod. however, little is known about the relationship between thrombophilia risk factors and vod. in this study we aimed to investigate association of major thrombophilic gene mutations on vod in pediatric hsct patients. methods: we reviewed retrospectively patients with vod who underwent hsct between - in ankara pediatrics and pediatric hematology-oncology training and education hospital, bone marrow transplantation unit, turkey. fifty-one patients who did not develop vod and transplanted during the study period were accepted as control group. we evaluated plasma homocysteine and lipoprotein a level, protein s and c activity and antigen levels and factor v g a mutation, prothrombin g a mutation, methylenetetrahydrofolatereductase (mthfr) c t and a c mutations, plasminogen activator inhibitor- - g/ g polymorphism before hsct. we also evaluated the patients' hospital files and noted the demographic values and complications of hsct. statistical investigations were done with spss statistics . for windows and p< . has been accepted as significant. results: there was no difference between control and vod groups as regard to age, sex, diagnosis, donor type, conditioning regimen, hsc source, and hla typing . there was no difference between the groups according to homocysteine, lipoprotein a, protein s and c activity and antigen levels. we did not find any relation between the genetic variations of thrombophilia and vod (table ). in vod group there were patients ( . %) with acute graft versus host disease (agvhd) and in control group there were ( . %) patients with agvhd (p= . ). febrile episodes were more frequent in vod group compared to the controls (respectively; n= , . % vs. n= , . %, p= . ). -year overall survival was % . in vod group and % in control group (p= . ). disease free survival was also different between vod and control groups (respectively; . % vs. . %, p= . ). conclusions: in literature there are recent studies showing higher pai- levels in patients with vod. however, in our study we did not find any relationship between congenital thrombophilia factors and vod. new studies with larger sample groups is necessary to better evaluate the association of congenital thrombophilia factors and vod. disclosure: nothing to declare p different strategies of chemotherapy-induced nausea and vomiting (cinv) prevention in hematological patients receiving an autologous hematopoietic stem cell transplantation: a single center experience ilaria cutini , riccardo boncompagni , chiara nozzoli , antonella gozzini , stefano guidi , chiara innocenti , massimo di gioia , lorenzo tofani , riccardo saccardi background: despite the improvements of pharmacological control, cinv still represents a major problem in patient undergoing hematopoietic stem cell transplantation (hsct). we present here a comparison of two pharmacological strategies for preventing cinv in multiple myeloma (mm), hodgkin (hl), and non-hodgkin lymphoma (nhl) patients who received an autologous hsct in our institution. methods: from january to july , we retrospectively analyzed consecutive patients, median age years ( - yo) , diagnosed with mm, hl, and nhl, who underwent an autologous hsct following a melphalan mg/sqm and beam/feam condition regimens, respectively. the first patients received cinv prophylaxis with palonosetron i.v and dexamethasone mg die (regimen a), whilst the following were administered with fosaprepitant iv, ondansetron iv and dexamethasone mg die (regimen b) both cinv prophylaxis was administered the day of melphalan infusion (day - form transplant). emesis breakthroughs were treated with alizapride and metoclopramide. nausea and vomiting were assessed through the ctcae . score system. categorical variables were compared with pearson chi-square test. results: the overall incidence of nausea was %, ( % grade , % grade , and % grade , respectively). in regimen a was shown to be %, ( % grade , % grade , and % grade , respectively) while in regimen b was % ( % grade , % grade , and % grade , respectively). pearson chi-square test did not show any differences between the groups (p= . ). the overall observed vomit was % ( % grade , % grade , and % grade ). in regimen a it was ( % ( % grade % grade , and % grade ), and % in regimen b ( % grade and % grade ). conditioning regimens didn't' have any significant impact on either nausea or vomit. patientsyounger then median ( yrs), were reported to have higher incidence of both nausea, (p= . ) not related to cinv treatment, and vomit ( % vs %, p= . ). in multivariate analysis the overall incidence of nausea is related to age (younger patients have higher probability to develop nausea (or , ; p= , ) whilst the higher incidence of vomit is related to: regimen a (or . ; p< , ), previously reported nausea (or , ; p< , ), and no smoking habits (or , ; p= , ). conclusions: both regimens are equally effective for nausea control however regimen b evidenced a better vomiting control. this finding is particularly relevant when the center policies include an early discharge program, therefore improving both patient's quality of life and procedure cost-effectiveness. clinical background: patients who underwent an allogeneic hematopoietic cell transplantation (hct) are challenged by medical, psychological and social complications. support groups might help hct-survivors to cope with these challenges. however, the existing literature about post-hct support groups is scarce. moreover, data on professionallyfacilitated support groups do not exist. the aim of this project was ( ) to establish a professionally-facilitated support group and ( ) to assess the discussed topics. methods: from / until / all patients who received an allogeneic hct at the adult stem cell transplantation program of the university clinic mannheim were invited to participate in a professionally-facilitated support group. additionally, spouses and life partners were invited. a theologian who is also a physician served as facilitator. he had no further function within the transplant team. the format of the group was unstructured without any rules regarding regular attendance. the facilitator did not provide topics or a curriculum. during the first year the group met every days followed by a monthly schedule. from the fifth until the th meeting the attendance and the discussed topics were minuted by the facilitator. the content of the minutes was analysed by a combination of an inductive and a deductive approach. all participants provided their informed consent for the study. results: altogether patients (female: n= ; male: n= ) and spouses/life partners (female: n= ; male: n= ) participated. patients ( %) and spouses ( %) attended more than one meeting. among those who participated in ≥ meetings the median time of participation was months. the median count of participations was eight. % of the participants attended the meetings longer than one year, % longer than three years. there was no sex difference with respect to the frequency and the duration of participation. however, the frequency of participation decreased significantly the longer a participant was attending the meetings. during group meetings the facilitator recorded thematically different contributions to the discussions divided in distinct topics. these topics were grouped into main categories [(a) medical topics, (b) private life and environment, (c) human relationships, (d) physical and mental condition and (e) the support group itself] and eight further categories [( ) compliance, ( ) economic issues, ( ) religion, ( ) sexuality, ( ) death and dying, ( ) support and coping, ( ) objectives and needs and ( ) not otherwise specified issues] which could not be grouped in one of the main categories. the most frequent issues were medical topics ( %), human relationships ( %), physical and mental condition ( %), private life and environment ( %), financial issues ( %), the support group itself ( %), support and coping ( %) and objectives and needs ( %). noteworthy, death and dying ( . %) were rare topics and sexuality was never mentioned. conclusions: to our knowledge, this is the first prospective and systematic analysis of a professionallyfacilitated support group for hct-survivors. these data might help to establish support groups and to identify psychosocial needs of patients and targets for specific support. disclosure: nothing to declare background: endothelial damage is associated with inflammatory complications that appear early after hsct, such as sinusoidal obstruction syndrome or acute gvhd. engraftment syndrome (es) is an inflammatory condition diagnosed by maiolino clinical score. potentially, es can exhibit high morbidity and mortality, especially after autologous-hsct in multiple myeloma (mm) patients since the introduction of new drugs such proteasome inhibitors and immunomodulatory drugs (imids). the objective of the present study was to evaluate if es is associated with endothelial dysfunction in patients with mm who underwent auto-hsct. methods: we included six patients with mm who received induction treatment including new drugs and consolidated their response with an autologous-hsct. we analysed comparatively the effect of incubating endothelial cells in vitro with serum samples from patients with es vs. no es. serum samples were collected before (pre), and after , , and days from the transplant. an additional sample was collected at the es onset and at the discharge day (no es group). endothelial cells (hmec) in culture were exposed to media containing % of serum from each patient for h. cell growth was controlled morphologically. expression of the adhesion receptor icam- on the cell surface was analysed by immunofluorescence, and activation of the inflammation related p- mapk signalling pathway was evaluated by sds-page and western blot. results: exposure of hmec monolayers to sera from patients who developed es (onset day, n= ) resulted in an increased icam- expression on the cell surface, higher that the observed with sera from patients who did not develop es (discharge day, n= ) ( . % of labelled area vs. . %, respectively). in addition, in experiments with sera from patients not developing es, icam- expression on cells exposed to sera from day + was reduced with respect to the observed with sera from day + , probably due to the corticosteroid used as a prophylaxis in our centre. this reduction was not observed in es patients. regarding phosphorylation of p- , it was significantly higher in cells exposed to sera from es patients than in response to sera from patients who did not develop es. conclusions: the increase in the expression of the adhesion receptor icam- on the surface and the intracellular activation of p mapk in endothelial cells exposed to sera from patients developing es indicates the existence of endothelial activation in association with es. interestingly, the prophylaxis of es with corticosteroid seems to be less effective in patients who developed es than in patients who did not develop this complication. these results need to be validated in a higher number of patients and modifications in additional markers of endothelial dysfunction should be investigated. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical the other authors do not have any disclosure to comment. p association between uric acid levels before and after allogeneic haematopoetic stem cell transplant and transplant outcomes: a single centre experience background: uric acid (ua) is a known endogenous danger signal which activates the nod-like receptor protein (nlrp) inflasome.ua is released from injured cells during conditioning in allogeneic stem cell transplantation (hsct). a pre-clinical study has demonstrated that nlrp inflasome-mediated il- production regulates graft-versushost disease (gvhd). the ua role in inflammation and gvhd is unclear. there are discordant reports in the literature about a potential protective role of ua on gvhd after a hsct. methods: we performed a retrospective study to assess the association between serum ua levels pre-and post- table] . table ] results: the characteristics of the patients are shown in table . median age was years (range - ), and patients ( %) were male. twenty-seven patients ( %) received low doses atg as part of gvhd prophylaxis. allopurinol was from the day before start of conditioning therapy until day . the median levels of ua were , mg/ dl before conditioning, , mg/dl at day , , mg/dl at day + and , mg/dl at day + . there was no impact between the ua levels and os at any time of the hsct. ua levels at day + were associated with a higher ci relapse at years ( % [ % ci, - %] for ua level > , mg/dl, and % [ % ci, %- %] for ua level ≤ , mg/dl [p= , ]). there was a trend for a higher ci of grade ii-iv agvhd for the subgroup of patients not treated with atg with ua < , mg/dl ( % vs %; p= , ) on day - and a higher nrm with ua < , on day ( % vs %; p= , ). conclusions: in our study the ua levels showed no impact on os, and only a tendency for ci of grades ii-iv agvhd grades ii-iv and nrm for the subgroup of patients not treated with atg. surprisingly, high levels of ua at day + of hsct were associated with a significant higher incidence of relapse. disclosure: dkms foundation, pi / (instituto carlos iii) and sgr (grc), generalitat de catalunya. background: veno-occlusive disease (vod) is an early, uncommon but serious complication of stem cell transplantation (sct) that is associated with high morbidity and mortality. defibrotide is the only licensed treatment for vod, and time to start of treatment (tst) affects outcomes. minor differences exist between the seattle, baltimore and classical ebmt ( ) criteria, which may trigger different start points for treatment. late onset vod (> days) is less recognised and we hypothesize, may have worse outcomes with longer time to diagnosis, and more limited treatment options across different healthcare systems. methods: electronic patient records from sept. -oct. at king´s bmt centre and pharmacy databases were reviewed, timepoint to clinical and bio-chemical manifestation of vod, diagnosis, tst, survival and longterm outcomes were analysed. results: of the patients( . %) who underwent an allogeneic sct, developed vod, including paediatric cases. none of the autologous sct patients developed vod. the paediatric and autologous sct patients were not analysed any further. adult patients (male= ; . %) developed vod at a median age of years(range - ), of whom developed < days and patients had late-onset vod as per ebmt criteria(range - days). cases classed as severe and as moderate vod. patients received defibrotide at diagnosis, patients within days, patients between - days, and patients received treatment after days. overall mortality for this cohort was %( / ). / ( . %) of patients with early onset vod and / ( . %) patients with late-onset vod died. of the deaths, died of liver failure and a further patients had vod as a likely contributing factor in their deaths. patient died with subarachnoid haemorrhage and with relapsed disease. patients that received defibrotide after days, / patients( . %) died, as compared to / ( %) for treatments between - days, / ( . %) for treatments within days. the lone surviving patient who received treatment after days has severe chronic liver disease and it's complications. of the patients who fit seattle criteria for early-onset vod, only fit the baltimore or ebmt criteria for classical vod. of these patients met the baltimore criteria later than the seattle criteria were met(range = - days). conclusions: vod carries high morbidity and mortality, and beyond the known risk factors and with the caveat of limited numbers in this study, we strongly suspect this is further increased when time to definitive treatment with defibrotide is delayed, particularly beyond days. nearly a quarter of cases with vod are late-onset as per classical ebmt criteria. however contrary to our hypothesis, their overall outcomes and mortality do not appear worse, with time to treatment again emerging as a strong predictive factor. conditioning treatment related factors, which play a stronger role in endothelial dysfunction in the hepato-portal circulation, may not be as much at play, perhaps for late-onset disease. uniformity in the use of diagnostic criteria, and high degree of vigilance, even beyond days, leading to early treatments may improve outcomes in vod. disclosure: nothing to declare background: hsct-associated thrombotic microangiopathy (ta-tma) affects - % of patients receiving an allogenic sct, with a high mortality up to - % in severe cases. endothelial injury mediated by complement activation has been atribuited a major role in the pathogenesis, and blockade of c with eculizumab offers promising results. methods: we present our experience with pediatric cases of ta-tma treated with eculizumab. the diagnosis of ta-tma was stablished attending to jodele et al criteria. clinical data were collected retrospectively from medical records. results: all cases were diagnosed between august and april , with a median age of years ( . - ) at time of diagnosis. primary disease was acute leukemia in cases ( all and aml), severe aplastic anemia in , and primary immunodeficiency in . they received their first sct in all cases, from mud and from mmrd (cd ra+ depleted haploidentical grafts), with mac regimen in cases, and ric in cases. of them received calcineurin inhibitors (cyclosporine) as gvhd prophylaxis. all patients developed agvhd (grade or higher in cases). and patients presented viral reactivation. hypertension was present in cases at tma diagnosis, requiring or more antihypertensive drugs in of them. all patients had renal injury consisting of less-than-normal glomerular filtration rate (median of ( - )) and proteinuria, with urine protein-to-creatinine ratio higher tan mg/mg in cases (data not available in patients). serum haptoglobin was decreased in just cases at diagnosis, and schistocytes were detected in patients. cutaneos signs were present in all cases, digestive symptoms in , neurological affection in , and notoriously all of them developed polyserositis. c and c were normal in all cases, with sc b higher than ng/ml in patients and lower in (data not available in cases). all patients received defibrotide as treatment, and cases received also rituximab, associated to therapeutical plasma exchange in . all of them received eculizumab, as first line in cases (median of days between diagnosis and eculizumab start). treatment was correctly monitorized with ch levels in cases (not available quick enough in other ). median number of doses needed in induction therapy was , and median interval between doses was days. patients required reduced interval and higher doses to maintain ch supressed. patients did not respond, and died because of tma. patients had hematological response, with chronic renal injury in of them and resolution of acute renal failure in case. nevertheless patient responding to eculizumab died because of tma related complications, and because of an invasive fungal infection. patients are alive, with a median follow up of months from treatment start. conclusions: our experience supports promising results of eculizumab based treatment for ta-tma, highlighting the importance of an early treatment and a careful therapy monitoring by ch supression. prospective studies are needed to achieve a better knowledge of this pathology and its treatment. disclosure: nothing to declare background: approximately - % of allogeneic hematopoietic stem cell transplant (allo-hsct) are made with some sort of abo blood group system incompatibility. an hsct abo donor-recipient incompatibility implies risks of complications during the process of infusion as acute hemolytic anemia (ah), delayed graft and other later complications due to the presence of isohemaglutinins (pure red cell aplasia or passenger lymphocyte syndrome). also, abo incompatibility could impact on graft versus host disease (gvhd) incidence, and could be associated with not relapse mortality (nrm) and overall survival (os). there are not concluded evidence about the abo incompatibility impact, so the aim of this study was to identify complications and response associated with abo incompatibility in patients undergoing allogeneic hematopoietic stem cell transplantation. methods: a retrospective study was performed on patients who receive an allo-hsct between january and august . two groups were performed according to the presences or not of abo incompatibility. demographic and clinical information was collected from physical and electronic medical records, and information was analyzed in spss v results: sixty-eight patients were enrolled in the study, % male, the median age was years ( - ) with the following diagnoses: acute lymphoblastic leukemia %, acute myeloblastic leukemia . %, granulocytic chronic leukemia . %, myelodysplastic syndrome . %, dendritic cell neoplasm . %, aplastic anemia . %. ninety-one percent of the patients received a transplant from an identical hla donor and . % received a haploidentical transplant. fifty-two patients ( %) were abocompatibility (g ) and patients ( %) had aboincompatibility (g ). none patient with aboincompatibility received a haploidentical transplant. the contrast between groups didn't show differences in fever, infections, bacterial isolation, presence and degree of acute or chronic gvhd and relapse of the disease. graft failure was %(g ) vs %(g ) (p= . ), intermediate risk cmv serostatus %(g ) vs (g ) (p= . ). the most relevant characteristics and complications are described in table . contrast analysis between g vs g showed that within the whole group there were deaths ( % vs % respectively) (p= . ), the overall survival -year was % vs % (p= . ) with a median of vs months respectively; mortality associated with relapse was % vs % respectively, and mortality related with transplantation was % vs % respectively. conclusions: abo incompatibility did not show association with complications related with the infusion, but there was a higher tendency of graft failure in the abo incompatibility group. it has no statistical significance, but it is important to expand its study. disclosure: none declared methods: retrospective data for nhl patients who underwent asct between and was analysed. patients were identified using the swbmt database and data on mets was collected using paper and electronic hospital records. forty-eight patients were excluded due to loss of follow-up, inaccessible/incomplete records, or death. cause of death was not determined. the ncep-atpiii definition of mets was used. this requires ≥ of criteria to be met. a bmi of ≥ kg/m and hba c of ≥ mmol/l were used to replace central obesity and impaired fasting glucose, respectively. other criteria include triglycerides (tgs) ≥ . mmol/l or treatment, high density lipoprotein cholesterol (hdl-c) < . mmol/l (male), < . mmol/l (female) or treatment and blood pressure > mmhg systolic or > mmhg diastolic, or treatment. results: the prevalence of mets in the cohort was % (n= ). eighty-two percent of patients (n= ) met one or more criterion for mets. twenty-seven percent (n= ) fulfilled only one criterion, % (n= ) fulfilled two criteria, % (n= ) three criteria, % (n= ) four criteria, and % (n= ) five criteria. the greatest prevalence of mets was in the + age group, accounting for out of ( %) patients with mets. overall prevalence decreased with declining age ( table ). the number of patients aged < years was too small to make any judgement on risk. raised triglycerides was the criterion most frequently met ( / patients), followed by hypertension ( ), raised bmi ( ), low hdl-c ( ) and an increased hba c ( ). conclusions: the prevalence of mets in our cohort ( %) was higher than the estimated worldwide prevalence of %, with the majority in the + age category. this is in keeping with other post-transplant studies, which show an increase in prevalence of mets after transplantation. moreover, the overall prevalence of mets was greater in the older population, which could be associated with the cumulative effect of ageing on the decline of normal metabolic homeostatic mechanisms. background: acute renal failure (arf) is a frequent complication in the early post-allogeneic hematopoietic stem cell transplant (allohsct) period with either myeloablative (ma) or non-myeloablative (nma) conditioning regimens. the aim of this study was to compare the incidence of arf in both types of hsct and to evaluate its impact on overall survival (os) and non-relapse mortality (nrm). methods: all allosct performed in one center between and were included in this study. allohsct from cord blood and from haploidentical donors were excluded. the renal function and the incidence of the main complications after allosct from day to day + were evaluated. arf was defined according to kdigo (kidney disease improving global outcomes) classification; the relative increase of serum creatinine levels was considered a marker of kidney damage. results: seventy-seven patients received a ma allohsct and a nma allohsct. recipients of nma allohsct had a higher median age ( years [range: - ] vs. years , p< . ), higher frequency of arterial hypertension ( % vs. %, p< . ) and showed most frequently active disease at allosct ( % vs. %, p= . ). in both groups the most frequent graft-versushost disease (gvhd) prophylaxis regimen was cyclosporine a and methotrexate. the median follow-up time was . years for the nma group and . years for the ma group. patients from the ma group had higher incidence of grade - mucositis ( % vs. %, p< . ) and acute gvhd of any grade ( % vs. %, p= . ) than patients from the nma allohsct. the incidence of arf was similar in both groups ( % in nma and % in ma). in the nma group arterial hypertension (hr . , p= . ), obesity (hr . , p< . ) and prior pneumonia (hr . , < . ) were predisposing factors for arf by multivariate analysis, whereas any factor was identified in the ma group. arf had no impact on -year os in both groups ( % vs. % p= . for the nma group and % vs. % p= . for the ma group). however, worse os were observed in patients with grade - arf in the nma group ( % vs. %, p= . ) and in patients with grade arf in the ma group ( % vs. %, p= . ). in turn, arf had no influence on nrm in the ma group but was associated with a trend for higher nrm in the nma group ( % vs. %, p= . ). conclusions: arf is a frequent complication in patients receiving allohsct irrespective of the intensity of the conditioning regimen. moderate and severe arf had negative impact on os. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc biga), instituto carlos iii (pi / fi), -sgr (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. treatment and risk factors of hepatic veno-occlusive disease after pediatric hematopoietic stem cell transplantation: a single-center experience barbaros sahin karagün , ilgen sasmaz , ali bülent antmen background: defibrotide emerged as a promising treatment option for hepatic veno-occlusive disease, a significant cause of mortality in recipients of hsct. as vod diagnosis is quite difficult even with the recently introduced ebmt criteria, studies which report treatment outcomes and response to prophylaxis are required. our aim was to evaluate the efficacy of defibrotide prophylaxis in hsct recipients at our center. methods: a total of transplants in patients from january to july were included in this study. all patients had factors that increased the risk of vod and all received mg/kg/day prophylaxis. patients' coagulation, renal and liver function test were monitored daily and all clinical findings and complaints were recorded. diagnoses were made via the ebmt vod criteria and patients who developed vod received treatment with increased df dose ( mg/kg/day) and supportive interventions. after complete remission of vod findings, patients were returned to the prophylaxis dose. close follow-up of patients was performed until days. results: in total, patients developed vod ( . %), none of the cases were severe ( mild, moderate). median age was . years and the most common clinical findings were weight increase, hepatomegaly, right upper quadrant pain and ascites development. in those with vod, treatment with mg/kg/day df was initiated and average duration of treatment with this dosage was . ( - ) days. no adverse events were reported in any of the patients. conclusions: our findings are consistent with previous studies on this topic, and we believe that the use of df as a prophylactic agent for vod is beneficial for pediatric patients with risk factors. disclosure: the authors report no conflicts of interest in this work. background: several factors might influence outcome of allo-hsct. analysis of the impact of donor-receptor blood group-incompatibility have been performed in different series not always showing the same results. as a consequence, its clinical impact remains controversial. minormismatch is characterized by the ability of donor b lymphocytes to produce anti-recipient antibodies. in majormismatch cases, antibodies against donor antigens are present in the recipient. methods: pts underwent allo-hsct between may and august in our center. median age was years (range: - ). pts were male ( . %) and female ( . %). baseline diseases were: aml, lpd, mds, all, mpd, mm, and bmf. donor was unrelated in , and related in cases (including haplo-identical). donor-recipient abo compatibility was as follows: ( . %) majormismatched (including bidirectional), and ( . %) nonmajor-mismatched (including minormismatched and matched). donor-recipient rh compatibility was as follows: ( . %) major-mismatched, and ( . %) nonmajor-mismatched (including minor-mismatched and matched). the impact of donor-recipient abo and rh compatibility on transfusion needs (prbc and platelet concentrates) and survival by day + was analyzed. results: for the global series the median number transfusions by day + was: ( - ) prbc and ( - ) platelets concentrates. day + overall mortality was . %. rh-incompatible and nonmajor abo incompatible cases showed no different results. however, major abomismatched cases needed more prbc transfusions (median: ; range: - ) and more platelet transfusions (median: ; range: - ), and had higher day + mortality ( . %) (p < . ) (see table) . conclusions: our analysis showed: ) donor-recipient rh-incompatibility, as well as minor aboincompatibility had no impact on prbc and platelet concentrates transfusion needs nor on -day mortality; ) contrarily, donor-recipient major abo-incompatibility had a significant adverse impact on prbc and platelet concentrates transfusion needs and -day mortality. ) donor-recipient rh-incompatibility and minor aboincompatibility.might be considered of marginal importance at the time to choose a potential donor. ) donorrecipient major abo-incompatibility should probably be a factor to be considered, along with other features, to choose the best donor background: survivors of haematopoietic stem cell transplantation (hsct) are at significant risk of developing treatment-related complications, including cardiovascular risk factors such as arterial hypertension, that could eventually lead to cardiovascular disease. the aim of this study is to evaluate the incidence and risk factors of hypertension following hsct in a colombian population. methods: a retrospective cohort study was conducted to assess the incidence and risk factors of hypertension in consecutive adult hsct recipients who underwent transplantation between and at a tertiary referral center in colombia, south america. blood pressure data, from two different measures, were collected at time points: day of mobilization for autologous hsct and day before infusion for allogeneic transplantation, day , and months , , and post-transplantation. hypertension was defined as having a systolic blood pressure >= mmhg and/or a diastolic blood pressure >= mmhg. patients with history of arterial hypertension were excluded. results: one hundred and seventy-five patients were included, with a mean age of years (range - ). ninety-one patients ( %) were male. one hundred and sixteen patients ( . %) underwent autologous hsct and ( . %) allogeneic hsct. the most common indication for hsct was acute leukemia ( . %), followed by non-hodgkin lymphoma ( . %) and multiple myeloma ( . %). twelve patients ( . %) had medical history of type diabetes mellitus (dm), ( . %) dyslipidemia, ( . %) alcohol consumption, and ( . %) tobacco smoking. only two of the patients with history of tobacco smoking were active smokers at time of transplantation. twenty-four patients ( . %) had developed hypertension by the end of the first year post-hsct follow-up. two patients ( . %) had systolic and diastolic, ( %) had only systolic, and ( . %) had only diastolic hypertension. only one patient was hypertensive at more than two time points. the incidences of hypertension at each time point were . % on day post-hsct, . % at first month, . % at three months, . % at months, and . % at one-year post-transplantation. allogeneic hsct (p< . ), therapy with calcineurin inhibitors (p< . ), pre-hsct fasting glucose levels (p< . ), acute gvhd (p< . ), chronic gvhd (p< . ), and media of diastolic blood pressure (p< . ) were significantly associated with the development of arterial hypertension. however, age, history of type dm, history of tobacco consumption, volume of infusion, prophylactic treatment for gvhd with mycophenolate, chronic gvhd, serum creatinine level on day of hsct, and being overweight or obese at time of transplantation were not significantly associated with the development of hypertension. conclusions: arterial hypertension is a fairly common complication in hsct recipients. similar to findings reported in previous studies, association between allogeneic stem cell transplantation, therapy with calcineurin inhibitors, and acute and chronic gvhd, and post-hsct hypertension was found in the present cohort. further studies are needed to assess the link between hsct and developing long-term cardiovascular complications. disclosure: nothing to declare tramadol-based pain management of oral and esophageal mucositis in pediatric hsct recipients background: mucositis is one of the most common early hsct complications seen in about % transplant recipients with % of patients developing gr iii-iv mucositis. mucositis is characterized by painful gastrointestinal mucosa lesions impairing the solid and liquid foods intake and increased risk of infections, bleeding, and intestinal paresis. thus, it greatly decreases the quality of life of a transplant recipient. according to who recommendations, the moderate pain control in pediatric patient is based on the use of low-dose morphine. however, there are some factors such as genetic polymorphisms causing variable morphine pharmacokinetics in children, side effects, and social factors (caregivers' general unwillingness to use narcotic analgesics), which cause the need for alternative pain relief options in pediatric practice. tramadol, which has both opioid and non-opioid mechanisms of action, may be a feasible option in mild to moderate pain. it may be delivered via patient-controlled analgesia (pca), although there is no consensus on its optimal parameters in pediatric practice. methods: a total of pediatric patients with a median age of (range to ) years receiving an autologous or allogeneic hsct in our clinic as part of the treatment regimen for solid tumor (n= ), leukemia (n= ), acquired aplastic anemia (n= ) or inherited condition (n= ) were included. conditioning regimens were myeloablative (mac) in and reduced-intensity (ric) in patients. all patients had oral and/or esophageal mucositis accompanied by moderate pain. the pain severity was assessed using the scales corresponding to patient's age and varied from to points. the pain control was based on intravenous tramadol administration using patientcontrolled analgesia (pca) approach. the following pca parameters were used: loading dose of . mg / kg (not exceeding mg), basal infusion rate of . mg / kg (not exceeding . mg), a bolus of . mg / kg (not exceeding . mg), lockout interval of min. the maximal daily dose was mg/kg/day. the pain control was considered adequate if a patient was satisfied or the basic and breakthrough pain score values were not higher than and, accordingly. in case of inadequate pain control nsaids were added. non-responders were switched to morphine. all patients were divided into groups based on conditioning regimen intensity. results: as a whole, % of patients did not require pain control measures escalation. the tramadol pain control rate was slightly higher for ric (n= , %) compared to mac (n= , %) recipients. in most cases the inadequate pain control was due to progressive mucosal lesions. the pca regimen used was characterized by very few complications. drowsiness was observed in ( %) of patients, in all cases the patients also had anemia. there was only ( %) patients with severe nausea requiring switching to morphine. conclusions: tramadol is an effective pain control option in transplant recipients with mild to moderate pain due to oral and esophageal mucositis without progressive mucosal lesions. the pca allows achieving a very low complication rate. therefore, this option may be considered for both mac and ric recipients. disclosure: no immune reconstitution of lymphocyte subsets after allogenic stem cell transplant (sct) and vaccination background: infectious diseases are a major cause of morbidity and mortality after allogenic stem cell transplant (sct). vaccines constitute an effective strategy to prevent infections but the optimal timing to start vaccinating is not well stablished. in order to individualize the early vaccination schedule, we studied the lymphocyte subsets involved in generating enough response to produce protective serological levels. methods: we studied retrospectively patients that had undergone allogenic sct at our hospital. patient distribution -age range: - years-old; diagnosis: acute leukaemia/myelodysplastic syndrome/ chronic myeloid leukemia ( patients), lymphoma ( patients). analytic parameters: tcd +, tcd +, nk, total b and functional b lymphocyte subsets (naïve igd+cd -, memory igd+cd + and igd-cd +, and effectors cd ++cd ++). immunoglobulin levels (igg, iga, igm) and specific igg for pneumococcus, tetanus, hbv, chickenpox, measles, rubella and mumps. clinical parameters were collected from medical records. results: we distributed patients in two groups, based on the timing of lymphocyte analyses: -less than months since sct ( patients) no patient showed complete immune reconstitution, although had enough t and functional b lymphocytes to generate response to vaccination. in these patients, vaccination for pneumococcus was completed and they generated sufficient protection antibody levels, despite being under immunosuppressive treatment. -more than months since sct ( patients) before the beginning of vaccination, we collected specific antibodies of patients. we compared the serological status before and after sct and observed that protection against tetanus was the most frequently preserved ( patients) and hbv the least frequent ( patients). other than one patient treated with alemtuzumab, all patients in this group had minimum absolute count of tcd + (> cells/microl), tcd + (> cells/microl), nk (> cells/microl) and b cells (> cells/microl). we also observed presence of b effector and b memory cells, with predominance of igd-cd + memory cells. immunoglobulin levels were within the normal range. in this group, we registered vaccination in patients. all of them were vaccinated against flu, and against pneumococcus and hbv. the rest of vaccines administered were heterogeneous in type and timing. patients were under immunosuppressive treatment at the time of vaccination and were able to generate enough specific antibodies for pneumococcus. conclusions: immune reconstitution was not completed months after sct, although minimal immunological reconstitution was observed tcd + and no-switching memory b lymphocytes were the last ones to reach minimum normal values according to patient age. some patients maintain serological protection after allogenic sct. immunoglobulin levels were normal, suggesting no need for immunoglobulin administration to prevent infections. flu, pneumococcus and hbv vaccines were the most frequently administered. pneumococcus vaccination generated a much larger serological response than hbv. this seroconversion occurred in patients under immunosuppressive treatment. the analysis of lymphocyte t, nk, b total and b functional subsets could be useful when programming an early vaccination schedule after sct. completion of the vaccination schedule was heterogeneous despite giving specific indications. therefore a more rigorous supervision of the process may be required. background: the significant advances that have been achieved in the allogeneic transplantation (allohct) field, have resulted in better post-transplant outcome and therefore complications other than the graft vs. host disease (gvhd) or disease recurrence become increasingly important. the post transplant metabolic syndrome (pt-ms), which caused by several factors (i.e. immunosuppressive agents, chemo-radiotherapy, anti-viral, and biologic therapies) is a well known post transplant complication in pediatric allografted long-term survivors however, only few studies have evaluated the prevalence of the pt-ms in adults. in this retrospective study, we sought to evaluate the incidence, the risk factors and the impact of the pt-ms on the allosct outcome. methods: since , patients ( males and females) with adequate clinical and laboratory data and a minimum follow-up of months were included in the study. their median age was . ( - ) years and after a myeloablative (n= ) or a reduced intensity (n= ) regimen they received either mobilized peripheral blood stem cells (n= ) or marrow graft (n= ), originated from full-matched siblings (n= ) or haploidentical donors (n= ). calcineurin inhibitors plus either short-term methotrexate or mycophenolate mofetil were given as gvhd prophylaxis. the diagnosis of pt-ms was based on the ncep-atpiii criteria; for patients with unknown data for abdominal circumference the body mass index (bmi) ≥ kg/m was consider as a criterion for pt-ms diagnosis. the independent t-test, logistic regression analysis and logrank tests were used for the statistical analysis. results: twenty ( . %) patients ( males, females) assessed to have pt-ms within the first months following the allograft. seventeen diagnosed after the st trimester post allosct and additional patients after nd trimester. sixteen out of patients had elevated glucose and bmi> kg/m , / elevated triglycerides levels, / low hdl levels and / hypertension. four ( %) had already known history of ms before allosct (for patients no data were available for ms diagnosis before allosct). interestingly, for / ( %) patients who had diagnosed with pt-ms either in the st or in the nd trimester the syndrome was reversible and did not fulfill the criteria for pt-ms beyond months post allosct. patients' gender, age, bmi, the type of conditioning regimen and gvhd co-existence evaluated as potential predisposing factors for pt-ms diagnosis. in univariate and multivariate analysis only the: bmi> kg/m and age> years were detected as significant risk factors (p< . ). the pt-ms did not affected negatively the survival or the nrm incidence post allosct conclusions: in our study, in agreement with other publications, we demonstrated that the pt-ms is not an uncommon complication post in the early post transplant period however, for a significant number of patients the syndrome was a reversible. for patients with high risk features (bmi> kg/m , age> years, known history of diabetes-mellitus, dyslipidemia, hypertension) apart of close monitoring, specific diet and encouragement for adequate exercise might help to reduce the incidence and the severity of pt-ms. nevertheless, prospective and well design trials are warranted to determine the accurate incidence, severity and the impact of pt-ms on the allosct outcome. disclosure: no conflict ofinterest experience of a single center in the humanization of the hospitalization process: technology and team training impact on the qol of the patient and family maria claudia moreira , marcia rejane , marcia garnica , andrea ribeiro , paulo cesar dias , ilza fellows background: hematopoietic stem cell transplantation (hsct) is one of the most aggressive therapeutic modalities of internal medicine, making it a highly stressful experience for the patient and his family. the duration of hospitalization can be prolonged by several intercurrences, frequently generating anxiety in the patient and their caregiver, which may lead to confinement and reactive depression. interventions in the hospital environment, in addition to the continuous training of the multidisciplinary team, can have a positive impact in this process with improvement in the process of discharge and quality of life of the patient and his / her family. methods: the objective of this research was to evaluate the impact of a reformulation in the unit, completed in may , which modified the facilities with availability of hermetic balconies in each room, with a view of an internal garden. there was also the addition of a screen in the corridor of the floor with images -technology known as videoowall, interconnected to motion sensors (kinects) that allow interaction between patients and families, besides facilitating physiotherapy and physical exercise. there was re-training of the multidisciplinary team with emphasis on the practice of humanization. the methodology consisted in the application of questionnaires of satisfaction to patients and their families during the period of hospitalization in a bone marrow transplant unit in the third quarter of . the items evaluated ranged from the quality of the information provided by the medical team and nursing, to the cordiality and agility with which the patient and his patient were treated by the global team. the results were compared with a similar period of the same unit in the previous year and with the indices collected simultaneously in another unit of the same hospital (cardio-intensive). results: overall and segmental satisfaction scores in the various items surveyed were higher when compared to the previous period of the same unit and were also higher in those obtained in a high complexity unit of the same hospital, composed of patients submitted to mental and psychological stress similar to onco-hematologicos.a reports of "free speech" were also obtained anonymously, in order to guarantee the authenticity and free expression of the subjects analyzed. conclusions: the results obtained allowed the validation of the technical and professional team initiatives, bringing indicators that will allow better monitoring and support of these patients and their relatives in this difficult time of treatment. they served as an initial tool in the continuous process of humanization and stimulated the multidisciplinary team to continuously improve this process. disclosure background: pure red cell anemia (prca) is a rare complication of abo-incopatible hematopoetic stem cell transplantation characterized by anemia, reticulocytopenia and absence of erythroid precursors in patient's bone marrow. most patients with prca resolve spontaneously within months, however a small number of patients requires continued red blood cell (rbc) transfusions. the treatment of this complication is difficult and not standardized. different approaches has been used such as rituximab, donor lymphocytes, plasma exchange with different outcome. recently, a remarkable response to treatment with bortezomib has been described in a case of prca. methods: we reviewed patients who received an allogeneic hematopoetic stem cell transplant (hct) between januar and august at our institution. sixty eight patients received a major abo-mismached hct. prca was defined as a completely absence of erythroid precursors on day + bone marrow puncture, with absence of donor red cells and the recipient requiring rbc transfusion. results: only one patient developed prca ( . %). a years old male received a myeloablative hla-matched abomismatched sibling donor transplant (brother, years) for acute myeloid leukemia (aml), with t( ; ) cr ,mrd positive (runx -runx t ). the donor was blood type a rh positive and the patient rh positive. the patient had no complication after transplant. the day + bone marrow puncture has shown only few erythroid precursors and day + puncture and biopsy no erythroid precursors, he had transfusion dependent anemia requiring a rbc transfusion every two weeks and retukulocytopenia. parvo virus and cytomegalovirus were negative. due to very high ferritin level (> . u/l) and increased luiver enzymes without signs of gvhd, the treatment with deferasirox has been started. the patient has achived cr , mrd negative, and has evidence of complete chimerism. high titers of anti-a and anti-b issohemagglutinin was present.we started the treatment with rituximab mg/m weekly, weeks, however without response. the pathogenesis of the prca is thought to be due to the recipients plasma cells, bortezomib, a proteasome inhibitor inducing apoptosis of plasma cells has been given s. c. , mg/m two times weekly, for two weeks. the patient responded to the treatment two weeks later with increase in hb, which was , g/dl and increase in retikulocyte number. the patient has continued to be well at the last control. conclusions: prca aplasia is a rare but serious complication after abo-incompatible hct. bortezomib is an effective treatment for this complication if mediated by residual host isohemeagglutinins after hct and should be recommended as standard of care. clinical methods: this work is retrospective, observational, cross-sectional and analytical. it included all patients who received hsct at stem cell transplantation unit (utmo, by its spanish acronym) at solca-guayaquil, between the years - .we use the kaplan-meier method to analyze the survival rate between the autologous and allogeneic transplant. the information collected for this study was obtained from the database of the solcay institute and the review of the files of the patients included. results: at least, patients have been undergoing to hsct between - years. according to the type of hsct, . % received an autologous transplant and . % received an allogeneic transplant, from which . % were from a related donor. the main source of transplant was peripheral blood in . %, followed by % obtained from umbilical cord blood and . % by bone marrow aspiration. the most frequently reported pathologies were acute lymphoblastic leukemia (all) ( %), multiple myeloma (mm) ( %) and acute myeloid leukemia (aml) ( . %). the overall survival was % (ic: %). the . % of patients that were undergoing to autologous transplant have survive, meanwhile the patients that were undergoing allogeneic transplant only the . % have survived (p< . ). the highest death rate occurred during the first year after hsct, and decreased considerably after that period. the main cause of mortality related to transplant (mrt) was the graft-versus-host disease (gvhd) ( %); however, the main cause of mortality in the study population (n= ) was relapse in . % of the patients, presented more frequently in all. conclusions: the results showed that % of patients undergoing to hsct have survived. a high rate of deceased patients in this study, have died in the first year before the transplant ( . %%), due to relapse. the main cause of deceased in the study is not related to hsct, and was the relapse in % of patients, in compare the gvhd was the main cause of mrt ( %). we consider that hsct is a technique that is still under development in ecuador, but despite the short time it has been taking and the institutional and medical limitations present in the health field, has presented excellent results comparable to studies conducted in developed countries. [ background: pigmented epithelioid melanocytoma (pem, early known as 'Аnimal type' melanoma) is a rare tumor with unpredictable clinical behavior and metastatic potential. pem generally has favorable prognosis. involvement of regional lymph nodes is not rare. extranodal and distant nodal metastases are extremely rare. we report about patient with fanconi anemia (fa) and pem with developed distant metastases in the early term after allogeneic hematopoietic stem cell transplantation (hsct). methods: -years old boy with fa was hospitalized for hsct. the blue-black painless nodulus х mm was noted on the left cheek. this lesion was observed from early childhood and during life only slightly increased in size. there were no distant and regional metastases on computerized tomography (ct) and scintigraphy with m tc. the nodulus and regional lymph nodes were radically removed before hsct. the resection margin was within the normal tissue. microscopically the derma and subcutaneous fat were infiltrated with epithelioid and spindle cells with total expression of s , melana, mhb , cyclind . ki- expression level was - %. histological structure was specific for pem. hsct with tcrαβ+/cd + graft depletion from match unrelated donor was performed. the conditioning regimen included total lymphoid irradiation gy, fludarabin mg/ m , cyclophosphamide mg/kg, rabbit atg mg/kg and rituximab mg/m . results: at + day after hsct was detected the tumor on the left cheek and parotid region with a histological structure identical to the primary lesion. on ct in s segment of the left lung was detected focus x mm with a cavity. invasive aspergillosis was suspected and empirical antifungal treatment was started. but in days the lung lesion increased in size to x x mm and penetrated in the bronchus. after bronchoscopy with biopsy, pem metastasis was histologically confirmed. moreover, the tumor on the face continued to grow. therapy with cobimetinib and vemurafenib was not effective and patient died from progression of pem on + day after hsct. conclusions: pem was early described as indolent tumor with rare distant metastasis and favorable prognosis. we suspect that pem may acquire an aggressive course in the absence of immunological control, especially in high immunocompromised patients after hsct. disclosure: nothing to declare p abstract withdrawn lidia gartcheva , antoaneta mihova , penka ganeva , margarita guenova , branimir spassov background: the main objective of the study is to assess the dynamics of quantitative and qualitative changes in the parameters of the b cell population and the production of immunoglobulins in patients after autologous transplantation of hematopoietic stem cells in the course of recovery of the immune system. methods: patients with hematological neoplasms undergoing autologous transplantation were included in the study: women and men, with an average age of years. patients were diagnosed with lymphoma (n = ), multiple myeloma (n= ), leukemia (n = ) and solid tumors (n = ). at the time of transplantation, patients were in complete clinical remission or at least with very good partial response, patients were in partial remission and patients -with progression. all patients were evaluated in nine time points through examinations by clinical-laboratory, flow cytometric and immunochemical methods. results: the percentage of cd (+) b cells reached the minimum values one month after transplantation then began to increase in the second month reaching a plateau around the mean values in the period - months after transplantation. the absolute number remained low during the entire period of observation. the amounts of igg and igm serum immunoglobulins gradually increased within the reference range throughout the entire period, while the iga level varied around the lower reference range. conclusions: implementation of an adequate humoral immune response is hampered by the reduction of circulating b cells, suppressed proliferative potential and functional deficits. restoration of b-cell function occurs over a period of months to years after autologous transplantation. clinical trial registry: no clinical trials disclosure: nothing to declare justyna background: allogeneic hematopoietic stem cells transplantation (allo-hsct) is a life-saving and well established therapy for wide range of diseases. however, it is still uncommon treatment for infants less than months of age. the data about indications and outcome of allo-hsct in the youngest group of patients is sparse. the primary objective of this study was to assess the incidence, indications, post-hsct complications and general outcome of allo-hsct among infants not older than months. latter sequelae of hsct such as physical and cognitive development were secondary aim of this study. methods: we retrospectively analyzed data of patients who underwent allo-hsct before year of age in department of pediatric hematology, oncology and bone marrow transplantation in wrocław during years - . clinical and epidemiological features as well as indications for transplantation, early and late complications and general outcome were assessed. results: infants who underwent hsct in our department comprise . % of all patients undergoing hsct in analyzed period of time. thirty-one ( . %) patients received stem cells from matched unrelated donor (mud), ( . %) from mismatched (haploidentical) related donor (mmrd) and ( . %) from a sibling donor (msd). non-malignant disorders were indication for transplant in ( . %) patients and malignant diseases in ( . %) . acute graft versus host disease (agvhd) occurred in ( %) infants, chronic graft versus host disease (cgvhd) in ( %). majority of graft rejections were seen in infants transplanted from mmrd ( . %), whereas the rest ( . %) was associated with mud. median follow-up in study cohort was days, days for alive patients (range days- . yrs) and days for those deceased (range days- days). overall survival (os) in study cohort was . and transplant related mortality (trm) was . . in children with malignancy ( . %) patients died comparing to ( . %) patients in non-malignant group respectively. main cause of death in analyzed group of infants was infection ( %). conclusions: . allo-hsct is rarely performed in children less than months of age. . majority of those patients receive stem cells due to non-malignant disorder. . among youngest hsct recipients, haploidentical transplant are more common than in general pediatric transplant population. . graft rejection is a significant problem in infants transplanted from mmrd. disclosure: nothing to declare unusual non-infectious lung complication after allogeneic haematopoietic stem cell transplantation claudia lucia sossa melo , , manuel rosales , francisco fernando naranjo junoy , , sara inés jiménez , , luis antonio salazar , , angela maría peña , , maría angélica chacón manosalva , maria luna-gonzález , claudia marcela chalela , manuel ardila-báez jirovecii infections, viral infections or nocardia. we describe the case of a patient with acute lymphoblastic leukemia (all) diagnosis with pap associated to a hsct and pulmonary pneumocystis. methods: a -year-old colombian female patient diagnosed with b-precursor all of high-risk in january , positive philadelphia chromosome, positive bcr / abl in february , infiltration to the central nervous system (cns), . % of lymphoblasts, and karyotype without legible metaphases. refractory to induction according to the pethema protocol (vincristine, daunorubicin, prednisone, l-asparaginase) with presence of . % blasts at the end of the induction. re-induction was performed with the flag-ida protocol (idarubicin, fludarabine, cytarabine) achieving complete remission, obtaining minimum residual disease (mrd) < . . dasatinib was initiated by bcr / abl expression and cns involvement at the time of diagnosis. an allogeneic hsct was performed, from a male brother donor, with low intensity conditioning tt buflu and prophylaxis of graft-versus-host disease (gvhd) with tacrolimus and sirolimus. patient showed early posttransplant complications, given the reactivation of cytomegalovirus and hemorrhagic cystitis grade i due to adenovirus. late complications such us gvhd at the cutaneous level and subsequent hepatic and gastrointestinal involvement were seen too, for which immunosuppressive therapy was administered with high doses of systemic corticosteroid. results: patient was hospitalized on day + posttransplantation due to febrile neutropenia and respiratory symptoms, with normal chest ct, and ct of paranasal sinuses with acute pansinusitis, for which she received meropenem gr intravenously every hours plus vancomycin gr intravenously every hours during days with symptom resolution. she remained hospitalized for cytopenias with normal bone marrow and % chimerism. on day + posttransplant she presented fever and leukocytosis, with acute respiratory failure with chest ct that showed bilateral alveolar occupation, "crazy-paving" pattern and frosted glass (see image), so diagnostic fibro-bronchoscopy was performed, reporting postoperatively for pneumocystis jirovecii. she received days of trimethoprim-sulfamethoxazole, with a torpid evolution requiring mechanical ventilation and tracheostomy, persisting with hypoxemia. the report of cultures for fungi, mycobacteria, and respiratory panel of filmarray were negative. a pathology report was obtained with % neutrophils, as well as pas staining with acellular pink material and elevated serum ldh, with a diagnosis of secondary pap. the patient continued with poor general condition, refractory hypoxemia, high ventilatory parameters and hemodynamic instability, due to which she was not able to be a candidate for treatment with total pulmonary lavage; leading to multi-organ failure and later death. [[p image] . high resolution chest ct. sample opacification in frosted glass (a) and pattern ''crazypaving'' (b)] conclusions: the importance of considering the diagnosis of pap as a noninfectious pulmonary complication in patients with allogenic hsct despite its low incidence is recognized. disclosure: nothing to declare methods: once the project was approved by the clinical trials and ethics committee, pairs of blood samples were drawn ( from picc line and from venepuncture) from voluntary allo-hsct recipients who were receiving continuous infusion tacrolimus from february through august . the pts had inserted a double-lumen polyurethane picc. tacrolimus was always administered through the red line, and the blood draw always performed through the purple line. all of the patients signed the informed consent. were male and women. median age was years ( - ). of the venepunctures were carried out in the arm where the picc was set, and the other from the contralateral arm. a limited group of nurses performed the extractions of the samples. results: as shown in the table, tacrolimus trough levels determined in blood from venepuncture were similar to those in blood drawn through the picc (median: . vs . ng/ml). when comparing one by one in the individual patients, the differences were not significant, and changed the dosing prescription in no cases. conclusions: in our experience, there are not significant differences in tacrolimus levels draw from the picc line, compared with a peripheral site. so, in our opinion, if the line for tacrolimus infusion is properly identified and the one used for the sample draw is the alternative one, venepunctures to obtain sample from peripheral sites are not justified for tacrolimus levels measurements. background: patients undergoing a hsct may require icu admission due to transplant-related toxicities. the aim of this study was to analyse a single centre experience with hsct patients requiring icu admission and the factors affecting outcome. methods: we included all adult patients (age >= ) who had an allogeneic or autologous hsct during (d between - - to - - ) at st. george's hospital. data was retrospectively collected from patients' notes. icu outcome and -day survival were analysed. for those patients who were admitted to icu more than once, outcome was analysed from their last icu admission. results: allograft patients were included. were male, with a median age years (range - years). were female, with median age years (range - years). diagnosis n (%) includes all ( %), aml ( %), acml ( %), cmml ( %), hl ( %), mds ( %), mds/mpn ( %), fl ( %), scd ( %). sixteen ( %) patients received their first transplant, ( %) received second transplant. eight ( %) patients had sibling donor, patients ( %) had unrelated donor. sixteen ( %) patients had / matched donor, ( %) patients had / matched donor, ( %) patients had / matched donor. nineteen ( %) received reduced intensity conditioning (ric), one ( %) received myeloablative (ma) conditioning. majority of ric allo-hsct patients were conditioned with fludarabine, mephalan, campath (fmc). a small number were conditioned with busulfan, fludarabine and atg. the ma allo-hsct patient was conditioned with tbi, cyclophosphamide. gvhd prophylaxis was ciclosporin alone starting on day - with a target level of - ug/l for all ric and ciclosporin and methotrexate for the ma patients. two ( %) allograft patients were admitted to icu on three occasions. both patients were male, and years old. one had mmud allograft for mds/mpn. the other had nd mud allograft for relapsed aml. the reasons for icu admission include sepsis, cardiac arrest and respiratory failure. the median duration of icu admission was days (range - ). there were deaths within days of transplant. one patient died on day + during his second icu admission with multi organ failure (mof). one patient died after icu discharge on day + with relapsed disease, bronchopneumonia with disseminated fungal infection. icu mortality rate was %, and -day mortality rate was %. nineteen autologous patients were included (median age (range - years)), ( %) were myeloma patients who were conditioned with melphalan, ( %) were lymphoma patients who were conditioned with beam. the icu admission was %. the -day mortality rate was %. conclusions: our centre's icu admission rate, icu mortality rate, cause of icu admission in allo-hsct patients and autologous patients is comparable to literature reports. autologous transplant is safe with no deaths and icu admissions despite an older age. the mortality rate for allo-hsct patients requiring icu admission remain high. all patients were appropriately referred to icu and there was no one who was denied icu admission. this analysis is being extended to preceding years. disclosure: nothing to declare liposomal doxorubicin for the treatment of iatrogenic kaposi sarcoma following hematopoietic stem cell transplantation background: iatrogenic kaposi's sarcoma (iks) represent a rare complication after hematopoietic stem cell transplantation (hsct), related to hhv- infection in hivnegative immunocompromised patients (pts). methods: we describe a case of iks occurred after an allogeneic hsct and we provide a review of the literature using pub med. results: a -year-old man, hiv-negative, received full hla-matched related hsct after a reduced intensity conditioning regimen for relapsed aml. gvhd prophylaxis was based on atg (fresenius mg/kg), cyclosporine (cya) and methotrexate. no severe complication occurred in the first days after transplant. shortly after cya withdrawal, he developed grade i acute gvhd. gvhd resolved after restarting cya. at fifth month after transplant, the patient developed several red and purple angiomatous plaque and nodules involving the skin of both lower limbs, right arm and the nose (figure ). skin biopsy revealed multiple localizations of iks and positive hhv- viremia was detected in the peripheral blood. a visceral involvement was excluded. patient was treated with cya tapering and nine courses of liposomal doxorubicin mg/m every days, obtaining a negativity of hhv- viremia and partial response of the skin lesions. at last follow up, at months after transplant, the patient was in complete remission (cr) for aml, cya-free without signs of gvhd recurrence and with his single stable residual iks lesion on his left limb, currently waiting for local radiotherapy. we found additional iks published cases after hsct. most of post-hsct iks were secondary to an allogeneic-hsct ( out of , . %) and occurred in adult ( , %) and male ( , %) pts. median age at the time of iks diagnosis was . years (range - ). thirteen pts ( . %) had mediterranean origin. the most frequent underlying disease was aml ( . %). gvhd prophylaxis was primary based on calcineurin inhibitor. half of the pts developed gvhd and were treated with steroid and other immune suppressive drugs. median time between the hsct and the occurrence of iks was . months (range . cutaneous iks was the prevalent form of manifestation, however visceral involvement was reported in pts ( . %). in four cases ( . %) an hhv- associated bm failure was report. immune suppression drugs tapering ( . %) and chemotherapy ( . %) were the most frequent actions taken after the diagnosis of iks. in most cases, liposomal doxorubicin was used as chemotherapy. cr rate was high, . %, whereas progression disease occurred in out pts ( . %), all of which had visceral involvement. in pts ( . %), iks was the cause of death. conclusions: withdrawn of immune suppression drugs and anthracycline based chemotherapy can represent a feasible treatment option for pts with iks after hsct. clinical background: acquired haemophilia a (aha) is an autoimmune disease caused by the spontaneous production of neutralizing immunoglobulin g (igg) autoantibodies (inhibitors) targeting endogenous fviii. treatment of these inhibitors presents additional challenges in a hematopoietic stem cell transplantation (hsct) recipient, because preservation of the graft that restores a normal hematopoiesis is critical. here we describe the management of a case of aha in an acute myeloid leukemia patient following hsct. methods: the clinical, laboratory and molecular aspects of a -year-old italian male who developed aha after allogenic bone marrow transplantation were collected and presented in order to show how we diagnose and manage this severe but rare complication within the special setting of hsct. results: a -years-old man with a flt- itd, npm- , runx -runx t and cbfb-myh negative, not differentiated, chromosomally normal acute myeloid leukemia (aml) in third complete remission (cr) was submitted to a hematopoietic stem cell transplantation (hsct) from his haploidentical son. the conditioning regimen consisted of oncothiotepa, busulfan and fludarabine and was followed by the infusion of a t-cell depleted bone marrow graft. gvhd prophylaxis consisted of cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day + . recipient's autoimmunity was negative. at months post-transplantation the patient received an antipneumococcal vaccination. fifteen days post-vaccination the patient was admitted to our in-patient ward due to general malaise, diffuse muscle and joint pains, cutaneous bleedings, oedemas, hyperchromic urines and constipation. physical examination revealed diffuse ecchymosis, swelling of deep muscles with a progressive functional disability due to hematomas and hemorrhagic suffusions of the tongue frenulum. and anti-factor viii inhibitors . bu/ml (high titers > bu/ml). thus, a diagnosis of acquired autoimmune haemophilia a was made and treatment with feiba combined with prednisone was started. patient's clinical conditions dramatically improved as he referred an improvement of movements and the resolution of joint and muscle pains despite the persistence of deep hematomas just after one day of treatment that had determined an increase of fviii:c value to . % and an improvement of aptt to . seconds. on the following medical checks physical examination showed the progressive disappearance of deep muscle hematomas, and normal values of fviii:c. conclusions: aha is a rare but severe complication following hsct and it could appear years afterengraftment. a prompt diagnosis and an early treatment with feiba and corticosteroid are necessary to avoid life-threatening sequelae. the inclusion of the coagulation panel in the laboratory exams performed during the follow-up is advisable in order to early detect this life-threatening complication. disclosure: nothing to declare background: splanchnic thrombosis is an uncommon complication of myelofibrosis and a controindication to proceed to hematopoietic stem cell transplantation (hsct) due to the risk of additional vascular and endothelial complications. we present a patient with myelofibrosis (mf) that proceeded to hsct from an unrelated donor, despite splanchnic thrombosis unresolved after heparin treatment and unable to proceed to surgical treatment due to severe thrombocytemia. methods: a -year woman with mf secondary to essential thrombocythemia, with intermediate- score according dynamic international prognostic staging system (dipss) and with extreme splenomegaly (maximum diameter cm), refractory to ruxolitinib, showed an extensive thrombosis of the portal and splenic veins, unresolved after -week heparin therapy, at the time of availability of an hla ( / ) and abo matched unrelated donor. she received a conditioning regimen including fludarabine and thiotepa and a gvhd prophylaxis with atg thymoglobuline, cyclosporine and methotrexate, followed by the reinfusion of . x /kg cd + pbsc. at the time of transplant we were aware of an high risk of developing sos, on the basis of the older age of the recipient, the unrelated donor, the advanced stage of myelofibrosis and the ferritin serum level of . ng/mg. results: on day + after hsct sos complicated the aplasia phase, characterized by jaundice, ascites, weight gain, progressive increase in creatinine and bilirubin serum levels. an ultrasound of abdomen confirmed an unchanged thrombosis extension and the development of ascites. on day + the patient was categorized as very severe sos stage, according to ebmt severity criteria, because of doubling of bilirubin serum level in hours and a % increase in comparison with her baseline weight. therefore, defibrotide was promptly started in association with diuretic therapy. the treatment was continued for weeks and allowed gradual restoration of the water balance and normalization of bilirubin serum level. at the last follow-up, months after hsct, the patient shows the persistence of a non-transfusion dependent anemia, platelets . x ^ /ul, palpable spleen cm below the rib, > % allogeneic chimerism in the granulocytic compartment and % in the t lymphocyte compartment. splanchnic thrombosis is partially recanalized and replaced by collateral circles with cavernous aspects. the patient is on treatment with fondaparinux and has shown neither significant infectious episodes or acute or chronic gvhd. conclusions: we conclude that defibrotide treatment allowed to perform a successfull allogeneic transplant in a patient with mf associated with an overt picture of splanchnic thrombosis. background: hematopoietic stem cell transplantation (hsct) is associated with an increased incidence of secondary malignancies including skin cancer. squamous cell carcinoma (scc) is the most common type in patients who are receiving immunosuppressive therapy and chronic graft-versus-host disease (cgvhd) appears to be an important risk factor for its development. recent studies describe voriconazole exposure as an independent factor that may contribute to this increased risk as well. in our best knowledge, no cases of scc have been reported in pediatric allogeneic hsct to date. methods: we present a case report of a year-old boy who developed a scc with high-risk features six years after undergoing hematopopoietic stem cell transplant. results: a year-old boy with acute lymphoblastic leukemia (all) underwent a matched unrelated bone marrow transplant years ago. he developed grade iv agvhd followed by extensive cgvhd with generalized scleroderma. he required intensive and continued immunosuppressive therapy and was on prolonged antifungal prophylaxis with voriconazole. in march , he developed scc involving left temporal region that was completely excised. two months later, more lesions in scalp and nose were noted and intralesion treatment with methotrexate was started. however, an unfavorable evolution was noted and he was put on systemic treatment including cisplatin and cetuximab receiving the whole scheme from january to march and continuing only with cetuximab, ten doses in total, until may, for unaceptable and severe tubulopathy that required admission at the hospital in several ocassions. he achieved a very good partial response but progression was noted shortly in follow up. at this point, non curative therapeutic options were found and he was put on intralesion methotrexate and photodynamic theraphy in a weekly basis with palliative intention. unfortunately, tumor growth was fast and patient passed away in august , fifteen months after squamous cell carcinoma diagnosis, due to tumoral progression. conclusions: ) scc is a rare, non-previously described, secondary malignancy in children undergoing hsct. ) high-risk features scc constitutes an aggresive disease with a median overall survival below year. ) cgvhd appears to be an important risk factor for its development. ) voriconazole induced-photosensitivity might have played a role. ) cisplatin based regimens +/-cetuximab are a therapeutic option in disseminated and/or high risk cases. as outcomes are unsatisfactory in these cases, alternative therapeutic options need to be explored. disclosure background: pregnancy is a rare event after allogeneic stem cell transplantation (sct) for acute leukemia. here we report, to the best of our knowledge, for the first time on a successful pregnancy after treosulfan-based conditioning. methods: a -year old woman was diagnosed with acute myeloid leukemia (aml) secondary to chronic myelomonocytic leukemia in july . ovarian preservation was performed by leuprolide acetate depot injection prior to cytostatic chemotherapy. of note, no cryopreservation of oocytes or ovarian tissue was conducted. she received two cycles of chemotherapy consisting of idarubicine ( mg/m² on day - ) and cytarabine ( mg/m² b.i.d. on days , , and ). due to secondary origin of aml sct was performed in first complete remission of aml after conditioning with treosulfan ( g/ m² days - ) and fludarabine ( mg/m² days - ). she received . × cd -positive cells per kilogram body weight from a hla-matched unrelated donor. results: follow-up bone marrow aspirates showed continuous complete remission of aml. seven months after sct she became pregnant, but decided for induced abortion. in january , months after hsct she became pregnant again and desired the child. medical examinations were performed monthly on an outpatient basis in stringent cooperation with the maternity clinic. the course of pregnancy was unremarkable, although she was hospitalized due to premature labor in the th week of pregnancy. however, gynecological examination showed no clinical significant findings, so that section was planned and she could be discharged again. in the th week of pregnancy she gave birth to a healthy girl ( cm, g) by cesarean section. peripartum she developed hypoethesia of the left body half. neurological examination showed no abnormalities and she recovered immediately. there were no other postpartum complications. breastfeeding was established but additional food was necessary for a sufficient nutrition of the child. conclusions: this case of successful pregnancy following sct demonstrates that fertility can recover after treosulfan-based conditioning. however, detailed studies of ovarian function and fertility are necessary to gain more insight into the risk of premature ovarian failure. disclosure: nothing to declare. experimental stem cell transplantation p cd -cart therapy before allo-hsct in children and adolescents patients who diagnosed r/r b-all with e a-pbx background: b-all with e a-pbx in children and adolescents is described with favourable prognosis. but there are more than % patients with e a-pbx diagnosed as relapsed or refractory. the results of allo-hsct in children and adolescents with this group leukemia in our center was analyzed in order to understand the therapeutic effect of cd -cart on the patients. methods: retrospective analysis, from june st, to july , , all children and adolescents diagnosed relapse or refractory b-all with e a-pbx who received allo-hsct, total cases. all patients was divided into two groups depending on whether or not accepted cd -cart before allo-hsct. according to fcm-mrd and e a-pbx level before allo-hsct, os lfs and cumulative recurrence rate were analyzed. r . . was used as statistical analysis software. results conclusions: . for r/r b-all with e a-pbx in children and adolescents, fcm-mrd pre-transplant hasn't obvious effect on the outcome of allo-hsct, while the level of e a-pbx has obvious effect. the out come of e a-pbx negative group was obviously better than positive group. . cd -cart can obviously improve the os and lfs, it is mainly because of cd -cart can makes more patients fusion to zero. . for r/r b-all with e a-pbx in children and adolescents, if chemotherapy can't make the fusion to zero. it is suggested to accept cd -cart therapy to make the fusion zero. it can improve the outcome of os and lfs. disclosure background: currently, hematopoietic stem cell transplantation (hsct) represents the only curative treatment for numerous hematopoietic malignancies like leukemias, immune deficiencies or metabolic diseases. cd serves a quality marker for stem cell grafts, which is not solely expressed on stem cells but also on a variety of progenitors. the role and the impact of these subpopulations remains unknown. we made use of our genetic barcode system to analyze the influence and contribution during reconstitution on a clonal level. methods: fluorescence activated cell sorting (facs) was used to sort hematopoietic stem and progenitor populations, namely hscs, mpps, cmps and clps, which were lentivirally transduced with our previously established bc barcoding system. after mixing the marked cells with bone marrow support, lethally irradiated recipient animals were and transplanted and monitored over weeks. we focused on bone marrow, blood, spleen and thymus, on chosen endpoints ( w, w, w, w) and samples were used to analyze the contribution of the subpopulations during the reconstitution process based on fluorescent protein (fp) expression. to investigate the clonal contribution in different organs, we performed next generation sequencing (ngs) and frequencies of unique barcodes in a sample were analyzed by bioinformatical approaches. results: a maximum of % of cells expressed the encoded fps, which were mostly derived from the hscs and mpps. cmp-derived cells were only detected week after transplantation in the myeloid compartment. cells derived from the clps were not detected at any time point. we analyzed the barcode content of the differently marked cells after next-generation-sequencing. in accordance with the facs data, the majority of the clones during the weeks of observation are derived from hscs and mpps. cmp-derived clones were only contributing during the first weeks and clp-derived clones are barely detectable. we did not observe any major differences with regard to age of donor or recipient, despite the total number of clones is higher in the group, which received the "aged" graft, independently from the transduced cell population. conclusions: here we show the suitability of our highly complex multi-color barcode system to study the clonal contribution of hscs and three progenitor populations after hsct. our results will contribute to a better understanding how these different populations interact to support the establishment of a new hematopoietic system. emphasized by the variability in data of graft and recipient age, this comprehensive analysis gives rise to an impression to the necessity of personalized graft composition, by which treatment success could be influenced. disclosure: nothing to declare survival and fate of adipose derived mesenchymal stem cells in a rat brain injury model background: mesenchymal stem cells have been identified as promising candidates in the treatment of central nervous system (cns) injury through neurotrophic support and immunomodulation. adipose tissue is an attractive source of mesenchymal stromal/stem cells (ascs) for regenerative therapeutic applications because they can be harvested from autologous donors with minimally invasive methods, can be rapidly expanded ex vivo, show low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. the present study examines the fate and effects of intracerebroventricularly (icv) transplanted ascs in a traumatic brain injury (tbi) model. methods: ascs were isolated from inguinal fat pad of adult wistar rats under sterile conditions and cultured according to standard procedures. ascs at passage ( x cells) were seeded and transfected with sleeping beauty transposase and pt venus-neo r plasmids. selection with g antibiotic resulted in the generation of a homogeneous asc population which expressed fluorescent venus protein for several passages, phenotypic characterization showed that these cells were . % double positive for cd and cd stem cell markers, verifying their mesenchymal origin. tbi was induced by stereotactic surgery under deep anaesthesia and subsequently icv transplantation of venus+ ascs was performed on adult wistar rats. normal ascs-transplanted and tbi-saline transplanted rats were used as controls. the proliferation, migration, survival and fate of transplanted ascs and their effect on injury restoration were examined six weeks post transplantation (pt). results: six weeks pt ascs expressed the fluorescence venus protein and therefore were identified in brain parenchyma. their presence into brain was also confirmed by masson trichrome staining, which revealed their collagen depositions. ascs were found in lesser numbers compared to those transplanted and exhibited no proliferative activity. ascs were found scattered distributed in brain as individual cells, and there were no aggregates of ascs or mass formation into lateral ventricles. extensive migration of ascs was mainly performed through white matter tracks in the corpus callosum and fimbria of hippocampus. six weeks pt ascs retained the characteristics of mesenchymal cells and did not differentiate into cells of neural lineage. ascs exhibited limited long-term survival, which is restricted in perivascular areas probably contributing to vascular formation. homing of ascs into peri-injured area was detected in half of the animals and achieved through the corpus callosum, as revealed by the collagen depositions, in this white matter track. transplanted ascs reduced the area of tbi cavity and did not enhance the astroglial scarring in peri-injured area. in tbi +ascs transplanted animals, the cortical injury site, showed a significantly smaller volume and lower % tissue loss compared to that of tbi+vehicle animals ( . ± . mm and . ± . % respectively, versus . ± . mm and . ± . %, p= . and p= . respectively). conclusions: considering the effects of ascs on inflammation and regeneration, we suggest that their transplantation after brain injury may promote host brain repair mechanisms. ascs transplantation may be beneficial in tbi, however some of its effects need careful and indepth evaluation. disclosure: nothing to declare xie-na cao , yuan kong , zhong-shi lyu , , qi wen , min-min shi , , qian-yu sun , yu-hong chen , yu wang , lan-ping xu , xiao-hui zhang , xiao-jun huang , background: poor graft function (pgf) remains a serious complication after allogeneic hematopoietic stem cell transplantation (allo-hsct). our previous work reported that abnormal bone marrow (bm) endothelial cells (ecs) were involved in the pathogenesis of pgf patients after allo-hsct (bbmt ; bmt ; blood ), but the explicit mechanism requires further clarification. autophagy is a self-degradative process responsible for the elimination of cytosolic components including proteins and damaged organelles. recent findings demonstrated that stimulation of autophagy could reduce oxidative status and angiogenic potential in ecsafter high-glucose exposure, from diabetic patients.however, little is known regarding the autophagy of bm ecs in pgf patients. therefore, the current study was performed to evaluate whether autophagy in bm ecs play a role in the pathogenesis of pgf. moreover, to investigate the effects of autophagic regulation on ecs and thereby regulating hematopoietic stem cell (hscs). methods: in the prospective case-control study, the autophagy levels were compared in bm ecs from pgf patients, and their matched good graft function (ggf) patients.the expression levels of autophagy-related markers (lc , beclin , and p ), and intracellular autophagosomes were detected by immunohistochemical staining, flow cytometry, western blot and transmission electron microscopy. subsequently, rapamycin (the autophagy activators) or hydroxychloroquine (hcq, the autophagy inhibitor) were administrated tothe -day cultivated bm ecs and human umbilical vein endothelial cells (huvecs), respectively.the autophagic vacuoleswere detected by monodansylcadaverine (mdc) staining assay. the bm ecsand huvecs were evaluated by cell counting, dii-ac-ldl and fitc-lectin-uea- double staining, migration, cell proliferation, and levels of reactive oxygen species (ros). to explore whether autophagy would affect the ability of bm ecs to support hscs in vitro, bm cd + cells from healthy donors were co-cultured with cultivated bm ecs and huvecs. colony-forming unit (cfu) and the apoptosis of co-cultured hscs were analyzed. results: the defective autophagy in bm ecs, characterized by decreased intracellular autophagosomes and autophagic vacuoles, decreased expression of lc -ii and beclin , and high level of p , were observed in pgf patients compared with ggf patients. moreover, the coculture of bm cd + cells with bm ecs showed significant deficient cfu plating efficiency, and increased apoptosis of cd + cells in pgf patients. in vitro upregulation of autophagy by rapamycin quantitatively and functionally improved bm ecsand huvecs, which manifested as more dii-ac-ldl and fitc-lectin-uea- double stained cells, increased capacities of migration, lower levels of ros and apoptosis via regulating beclin pathway, whereas inhibition of autophagy by hcq aggravated the huvecs and bm ecs from pgf patients. furthermore, in vitro upregulation of autophagy by rapamycin significant improved cfu plating efficiency, and decreased apoptosis in bm hscs co-cultured with huvecs and bm ecs from pgf patients. conclusions: these findings suggest that defective autophagy in bm ecs may be involved in the pathogenesis of pgf. the effect of rapamycin in pgfpatients is potentially mediated by improving the dysfunctional bm ecsto support hscs. therefore, it would be of value to investigate whether upregulating of cytoprotective autophagy of bm ecs may ameliorate pgf, thereby providing a novel clinical intervention for pgf in the future. clinical background: heparanase (hpse) in an endoβ-glucuronidase that specifically cleaves the saccaride chains of heparan sulphate proteoglycans (hs), leading to a loss of integrity of the extracellular matrix and to release of hs-bound cytokines, chemokines, angiogenic and growth factors. hpse gene is polymorphic and includes approximately snps. the combination of two snps, rs and rs , are involved in the regulation of hpse expression with an inverse correlation between mrna expression and protein levels: gg-cc, gg-ct, gg-tt, ga-cc (low group) expressed high hpse concentration; ga-ct and ga-tt (median group) expressed intermediate hpse levels; aa-tt and aa-ct expressed low hpse concentration (high group). we studied hpse snps in the allogeneic stem cell transplantation (hsct) setting to evaluate a possible association with post-hsct outcomes. methods: we enrolled patients submitted to hsct in our department since to . for each couple recipient-donor, rs snp was genotyped using restriction fragment lenght polymorphism assay, whereas for rs snp an allele-specific polimerase chain reaction was applied. hpse genotype distribution was compared in different groups according to post-hsct outcome: graft-versus-host disease (gvhd), transplantrelated mortality (trm), overall survival (os), infectious complication and disease-free survival (dfs). statistical analysis was performed using ncss . results: distribution of rs snp was as follows: gg . %, ga . % and aa . % among recipients and . %, . % and . % among donors, respectively. hardy-weinberg equilibrium (hwe) was respected. distribution of rs snp was as follows: cc . %, ct . % and tt . % among recipients and . %, % and . % among donors, respectively. rs snp distribution did not respect the hwe. an association was found between recipient rs snp and the cumulative incidence of agvhd among patients submitted to a reduced intensity conditioning (ric): . % for tt genotype and % for ct or cc genotype (p= . ). on the other hand, an association was identified between donor rs /rs snps combination and the cumulative incidence of agvhd: . % for low group donor, % for median group donor and . % for high group donor (p= . ). conversely, aa genotype for donor rs resulted independent risk factor for cgvhd de novo development (p= . , od . ) together to donor-recipient sex mismatch (female donor to male recipient vs. others: p= . , od . ) . considering cmv reactivation rate after hsct, an association was observed according to recipient rs snp: % for cc genotype, . % for ct genotype and . % for tt genotype (p= . ). multivariate analysis confirmed recipient rs snp as independent risk factor for cmv reactivation after hsct (p= . , od . ) together with recipient cmv serostatus at transplant (positive vs. negative: p< . , od . ). conclusions: hpse role was widely studied in the setting of inflammation, autoimmune diseases, hematological disease and tumor. however, it still remains debated the inducing or protective activity of hpse in the setting of gvhd. obviously, our results need to be confirmed in a validation cohort. clinical trial registry: na disclosure: nothing to declare novel protocol for autologous hsct in patients with high risk of complications: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) is standard of treatment in many patients with high risk of complications: dialysed patients, patients with heart and kidney amyloidosis or patients with systemic sclerosis. we introduced recently a novel protocol for ahsct: combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. this protocol allowed us to reduce the transplant risk in various patient groups traditionally connected with high risk of complications. in this work we summarize the experience in such high risk patients. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells and who fulfilled at least one study inclusion criteria: a) dependence on dialysis b) amyloidosis c) systemic sclerosis d) disqualification from transplantation at other centre due to the high risk of complications. there were together patients selected for this analysis - with amyloidosis ( with ≥ organs involved), dialysed, with systemic sclerosis, unfit at other centre. the database included prospectively recorded serious adverse events during the mobilization and transplantation. results: there were transplantations performed in this group of patients. mortality was % at days. all patients underwent successful ambulatory mobilization. all patients received mephalan conditioning with single infusion with median dose of mg/m (min , max ). mean engraftment was . days for white blood cells and . days for plt over g/l. the rate of complications was low with cases of neutropenic fewer, single bacterial culture with staphylococcus epidermidis without clinical signs of infection, median mucositis grade of . and without patients on parenteral nutrition. the median time of hospitalization was days (min , max ). conclusions: we present here novel protocol of transplantation combining chemomobilization and ahsct with fresh cells with excellent safety profile among most severely ill patients allowing for safe and efficient transplants. with this protocol we were able to overcome multiple risk factors and perform full intensity transplantation in very fragile patients. disclosure: nothing to declare single umbilical cord blood transplantation provides durable disease remission of advanced hematological malignancies in elderly patients background: although allogeneic hematopoietic stem cell transplantation (allo hsct) is potentially curative therapy in a variety of hematological malignancies, little has been reported of the outcome for elderly patients who are not in remission at transplantation. but it has been pointed out that recipient age alone can not be regarded as contraindication for allo hsct in the literature recently, supported by suitable donor, conditioning regimens and appropriate management of complications. we conducted a retrospective study of elderly patients who had advanced hematologic malignancies to elucidate the outcome of single umbilical cord blood transplantation (sucbt) in toranomon hospital kajigaya, japan. methods: we retrospectively investigated the outcomes of patients aged over who underwent their first ucbt from june to december in our medical center. results: diseases included acute myelogenous leukemia (n= ), myelodysplastic syndrome (n= ), adult t-cell leukemia/lymphoma (n= ), myelofibrosis (n= ) and chronic lymphocytic leukemia (n= ). the median age at transplantation was years (range, - ) and follow-up for survivor post transplantation was day (range, - ). all patients were not in complete remission (cr) at the time of transplantation. reduced intensity conditioning (ric) regimens were used in patients. all patients received tacrolimus and mycophenolate mofetil as graftversus-host disease (gvhd) prophylaxis. all cases except early death achieved neutrophil recovery at median days (range, - ). at year, overall survival (os) rate and disease free survival (dfs) were , % ( % confidence interval (ci), . - . ). we performed univariate analysis to identify the factor that influenced os at year, but no statistical significance was demonstrated at the age of transplantation (aged to vs. ≧ , . % ( % ci, . - . ) vs. . % ( % ci, . - . ), p= . ). the cumulative incidence of non-relapse mortality (nrm) at days was . % ( % ci, . - . %) and relapse at year was . % ( % ci, . - . %). only two patients developed acute gvhd(ii-iv) and one developed severe gvhd at days after transplantation. the main causes of death was infection (n= ), including sepsis (n= ) and viral encephalitis (n= ), followed by idiopathic pneumonia syndrome (n= ) and thrombotic microangiopathy (n= ) during the early phase of transplantation. in contrast, no patients died of recurrence. conclusions: although our report consisted relapsed/ refractory disease of elderly patients at the time of sucbt, durable remission and lower incidence of gvhd could be noteworthy compared with previous reports. further strategies to reduce the rate of nrm and longer duration of follow up would be warranted. disclosure background: pearson syndrome and kearns-sayre syndrome are metabolic disorders caused by a de-novo deletion in the mitochondrial dna (mtdna). allogeneic stem cell transplantation has shown to improve metabolic function in distal organs in several metabolic disorders, but bears significant morbidity and mortality, especially for patients with mitochondrial disorders. novel gene therapies may correct diseases rising from genomic dna mutations, but targeting the mitochondrial dna is complex. mitochondria are able to transfer into cells and between cells, as seen in preclinical models of mitochondrial and other metabolic disorders. here, we introduce a novel concept of mitochondrial augmentation therapy (mat) of autologous cd + cells in children with mitochondrial deletion syndromes. methods: patients were treated under a compassionateuse program, approved by the sheba medical center irb and the israeli ministry of health. briefly, mobilization was performed using gcsf alone (n= ) or in addition to plerixafor (n= ) . cd + cells were isolated via miltenyi clinimacs system and co-cultured with maternal mitochondria, drawn from peripheral blood and confirmed nondeleted, for hours, and re-infused to the patient without any conditioning. patients were followed for clinical and metabolic parameters. results: all four patients presented with different deletions in mitochondrial dna, and different baseline characteristics, and were treated at the age of . , , and years. despite normal cbc, significant bone marrow hypocellularity was seen in evaluated patients ( %, % and % cellularity at age , and ), which correlated with low colony forming unit capacity of patients and low yield of cd + mobilization in the leukapheresis product. patients received on average x enriched cells/kg (range, . - . ), and the median enrichment of cd + cells was % (range, - %). no infusion reactions occurred, and the only severe adverse events of this cellular therapy were leukapheresis-related anemia, hypokalemia, hypocalcemia and alkalosis, all resolved promptly with proper supplementation. follow-up duration is variable, ranging - months. we were able to show improvement in mitochondrial heteroplasmy (proportion of deleted mtdna of total mitochondrial dna) and in normal mtdna content, starting - months from cell therapy, which correlated with improved atp production in peripheral blood derived mononuclear cells. clinically, patients showed improvement in aerobic function and endurance (measured by the half-bruce protocol, sit-to-stand test and -minute walk test), muscle strength (hand-held dynamometry), and in quality of life, measured by the international pediatric metabolic disability scale. no metabolic crises occurred following cell infusion. conclusions: patients with deletion in mtdna have metabolic dysfunction, including poor bone marrow cellularity and function. hematopoietic stem cells in patients with mtdna deletions can be enriched with normal mitochondria, via mat, as first shown in our patients. this novel process is safe and results in increase in the normal mtdna in peripheral blood of patients, and in improved metabolic and clinical function. clinical trial registry: clinicaltrials.gov nct disclosure: moria blumkin, noa sher and natalie yivgi ohana -minovia therapeutics, employment p high cytotoxic efficiency of alpharetrovirally engineered cd -specific chimeric antigen receptor natural killer cells for treatment of acute lymphoblastic leukemia stephan müller , tobias bexte , annekathrin heinze , franziska schenk , axel schambach , winfried s. wels , , ute modlich , evelyn ullrich , background: autologous chimeric antigen receptormodified (car) t cells with specificity for cd showed potent antitumor efficacy in clinical trials regarding relapsed and refractory acute lymphoblastic leukemia (all). natural killer (nk) cells are cytotoxic lymphocytes that are capable to kill their targets in a non-specific manner and additionally do not cause gvhd. therefore, using cd -car-nk cells exhibits several advantages, such as safety in clinical use, possible allogenic settings and the potential to also attack heterologous leukemia cells which lost cd . previous approaches used cd -car-nk cells pre-stimulated by feeder cells, bearing potential risks. thus, we focused on the optimization of generating cd -car-nk cells by viral transduction under feeder-cell free conditions. methods: human nk cells were isolated from healthy donor peripheral blood mononuclear cells via cd negative selection. after a feeder-cell free expansion phase with interleukin , transductions were performed with an egfp or a cd -car encoding vector at different multiplicities of infection (moi). to optimize gene modification different transduction enhancers (retronectin and vectofusin- ) and viral vector systems (lentiviral and alpharetroviral) were compared. finally, generated cd -car-nk cells were tested in their ability to kill cd positive and cd -negative cell lines. results: nk cells transduced with a lentiviral egfp encoding vector or a lentiviral cd -car vector using retronectin and vectofusin- showed similar transduction efficiencies for both transduction enhancers (egfp: retronectin moi : . %; vectofusin- moi : . %; cd -car: retronectin moi : . %, moi : . %; vectofusin- moi : . %, moi : . %). the generated cd -car-nk cells showed increased cytotoxic capacity against cd -positive cells compared to nontransduced (nt) nk cells ( . % vs. . %, effector to target (e:t) ratio : ). both nk cell populations were equally efficient in killing cd -negative cells ( . % vs. . %). alpharetroviral transduction of nk cells with an egfp encoding vector showed higher transduction rates with vectofusin- than with retronectin (retronectin moi : . %, moi : . %; vectofusin- moi : . %, moi : . %). further using vectofusin- , similar transduction efficiencies could be achieved with an alpharetroviral cd -car encoding vector (moi : . %, moi : . %, moi : . %), outperforming the efficiencies of lentivirally generated cd -car-nk cells in the same experiments (moi : . %, moi : . %, moi : . %). additionally, alpharetroviral cd -car-nk cells showed a higher cell killing activity against cd -positive cells than lentiviral cd -car-nk cells or nt-nk cells ( . % vs. . % vs. %, e:t ratio : ). interestingly, similar killing activities were achieved with an e:t ratio of . : ( . % vs. . % vs. . %) and alpharetroviral cd -car-nk cells remained a stable cytotoxicity level at lower cell concentrations down to an e:t ratio of . : . all three nk cell populations were equally efficient in killing cd negative cells ( . % vs. . % vs. . %, e:t ratio : ). conclusions: cd -car-nk cells can be successfully generated under feeder-cell free conditions using different transduction enhancers and viral vector systems. these data suggest the usage of vectofusin- in combination with alpharetroviral vectors to genetically modify nk cells to achieve sufficient amounts of transduced cells. these cd -car-nk cells mediate high cytotoxicity and therefore may offer a new therapeutic option in the treatment of all. disclosure: axel schambach is an inventor on a patent describing alpharetroviral sin vectors. winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. the remaining authors have nothing to disclose. graft-versus-host diseaseclinical walter spindelböck , bianca huber-krassnitzer , barbara uhl , gregor gorkiewicz , hildegard greinix , christoph högenauer , peter neumeister background: steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictor of mortality. methods: the fecal microbiota transplantation (fmt) procedures were performed according to a protocol approved by the local ethical committee ( - ex / ) after obtaining informed consent. donors were healthy adult subjects screened for potential infections by serologic and microbiologic tests according to local standards. donor stool was diluted with saline and homogenized to a volume of~ ml fecal solution for instillation into the terminal ileum and caecum via colonoscope. microbiota sequencing analysis of s rdna was performed before fmts and afterwards at predefined timepoints. results: we report the outcome of nine patients refractory to - lines of immunosuppressive therapies with lower gi-stage iii (n= ) or iv (n= ) agvhd following repetitive fmts from a single donor. all patients had received an allo-hsct for mds (n= ) , aml (n= ), pmf (n= ) and mm (n= ) following a reduced intensity (n= ) or mac (n= ) conditioning regimen using pbsc as stem cell source. after an onset of lower gi agvhd between - days after allo-hsct, nine patients refractory to several lines of immunosuppressive therapies received - fmts ( patients were treated with more than fmts, in patients fmt was only administered once or twice) mostly in weekly intervals. five patients achieved a clinical complete response with resolved diarrhea and no gastrointestinal complaints, and four of these could be discharged without gvhd symptoms. two patients (pr, nc) were discontinued after or fmts in pr or nc due to concomitant infections (metapneumoviral pneumonia, cmv gastroenteritis), the other non-responders succumbed to gvhdrelated infectious complications. the establishment of donors' microbiota with the emergence of new taxa, an increase in bacterial richness/diversity, and the disappearance of the "enterococcus signature" were associated with disease control and response to fmt. except the possible transmission of adenovirus by fmt in one patient, no other immediate procedure-related infections or other side effects were observed. conclusions: restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for a subset of patients with refractory lower gi-agvhd. vigorous donor screening for infectious disease is mandatory. clinical background: migration of allo-activated donor effector tcells from lymphoid tissues to target organs is an important step in acute graft versus host disease (gvhd). the sphingosine- -phosphate- (s p ) receptor plays a crucial role in lymphocyte trafficking. data from animal models suggest that pharmacological modulation of the s p receptor reduces gvhd and improves mortality. we investigated this mode of action by using the secondgeneration s p modulator krp for the prophylaxis of gvhd in a pilot clinical trial in patients undergoing allogeneic hsct. methods: a multi-centric, phase b, prospective, open label, two-part study was conducted to evaluate the safety, tolerability and pharmacokinetics of krp in patients undergoing allogeneic hsct for hematological malignancies. primary endpoint was safety. initial efficacy was explored based on the incidence of gvhd, mortality and relapse. part was a single arm open label study to investigate the safety of mg/day krp added to standard of care gvhd prophylaxis (csa/mtx) in patients. part was a randomized two-arm open label study to compare the safety, efficacy and pk of mg/day of krp in combination with tacrolimus/mtx to mg/day of krp in combination with csa/mtx in patients. in both parts, treatment with krp was initiated days before hsct and continued for an additional days. patients were followed up for up to years. results: patients were included in the study. of patients completed the -day treatment with krp at the assigned doses. median duration of follow-up was days (range to days). krp was safe and well tolerated. serious adverse events (saes) suspected to be related to krp were observed. macular edema (n= ) and peripheral edema (n= ) as s p related adverse events occurred and resolved without sequelae. of note, the incidence of macular edema in hsct recipients is unknown. neutrophil engraftment was confirmed in all patients with a median of days (range to days). of patients presented with grade iii or iv acute gvhd (on days , , , and ) . no gvhd or infection related death occurred during the first days. -day survival was %, with no death occurring during krp treatment. death occurred on study day due to lymphoma relapse. a second death occurred on study day due to liver gvhd. four patients died in the follow-up period due to gastrointestinal gvhd (day ), aspiration pneumonia (day ) and relapse (day and day ). the kaplan-meier estimate of overall survival at year was . . when comparing the data from the two dose groups ( and mg krp ), no major differences in safety, engraftment, gvhd rate or mortality were observed. conclusions: this clinical trial was the first to test s p modulation in this population. our data suggest that krp had no negative impact on engraftment and overall, was safe, and well tolerated. based on exploratory data, when comparing to matched historical mortality data, krp may have favorable effects on overall survival ( figure ). background: uric acid is a danger signal contributing to inflammation. relevance to allosct has been demonstrated in preclinical models: the depletion of uric acid led to improved survival and reduced gvhd (j exp med. sep ; ( ): - ). results of a clinical pilot trial suggested that peri-transplant uric acid depletion reduce acute gvhd incidence (bbmt may; ( ): - ). methods: this international multicentric study aimed to study the association of uric acid serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditionning. data were prospectively collected between / and / . a comparison of outcomes between patients with high and low uric acid level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from european countries reported data on allosct recipients. patient characteristics are given in table . the uric acid cut off point was determined at . mg/dl (median of measured uric acid levels). overall survival (os) and progression free survival (pfs) of allosct recipients with uric acid levels above cut off measured before start of conditioning were significantly shorter ( figure a , os univariate hr= . ci= . - . p< . ; multivariate hr= . , ci= . - . , p< . ) ( figure b , pfs univariate hr= ci= . - . p= . ; multivariate hr= . , ci= . - , p= . ). nonrelapse mortality was significantly increased in allosct recipients with high uric acid levels prior to start of conditioning (univariate hr= ci= . - . p= . ; multivariate hr= . , ci= . - . , p= . ). in addition, there was a non-significant trend towards higher acute gvhd incidence (gvhd grades ii-iv univariate hr= . ci= . - . p= . ; multivariate hr= . ci= - . , p= . ) in allosct recipients with uric acid levels above cut off before transplantation. finally, the incidence of relapse after allosct was moderately increased in the cohort with higher uric acid levels (univariate hr= . ci= - . p= . ; multivariate hr= . , ci= . - . , p= . ). conclusions: high uric acid levels before start of conditioning correlate with high mortality after allosct. our results can serve as rationale for clinical trials on depletion of uric acid during allosct. results: we found significant correlation between donors' ctla- + a>g polymorphism and hsct outcome. genotype aa was present in donors, ag in donors and donors was homozygous for g allele. recipients who received graft from g allele carrier donors showed significantly increased cumulative incidence of relapse (at months aa: . %, ag: . % and gg: . %; p= . ). on contrary, the frequency of the acute gvhd grades iii-iv and cytomegalovirus (cmv) reactivation/disease decreased according to the presence of the g allele in the donor ctla- genotype [agvhd: aa: %, ag: %, gg: %; p= . ; cmv: aa: %, ag: %, gg: %; p= . ]. cumulative incidence of agvhd was also markedly decreased among patients with g allele carrier donors (at days aa: . %, ag: . %, gg: . %; p= . ). donor genotype similarly influenced hsct outcome in mud donor and mac conditioning subgroups. overall survival (os) was not different in patient subgroups according to donor genotypes [os at months: aa: . ± . %, ag: . ± . %, gg: . ± . %; p= . ]. we did not find any correlation between recipients' ctla- + a>g polymorphism and hsct outcome. conclusions: several ctla- snps have previously been described to be associated with relapse rate, incidence of agvhd and os, but results are often contradictory in the publications. in our study, ctla- + a>g polymorphism of hsct donors influenced risk of relapse, agvhd, cmv and cause of death, but not overall survival. the genotyping of ctla- + a>g polymorphism in donors may help in the risk assessment process and the choice of personalised therapy. disclosure: nothing to declare. background: although steroids remain first-line therapy for the treatment of acute graft versus host disease (agvhd), response rates in patients with grade iii-iv disease are poor, with no apparent improvement in survival over the past years. we performed a prospective, multicenter trial to assess the efficacy and safety of the combination of ruxolitinib and etanercept as a novel approach to treat grades iii-iv sr-agvhd . methods: forty malignant hematologic disease patients with grades iii-iv sr-agvhd after allo-sct from three centers in east china were enrolled from january to june . ruxolitinib was initiated at a dose of - mg bid for months, and then tapered gradually for another one month. etanercept was administrated at mg biw for - weeks. results: the median age of patients was (range - ) years. at day after the combination treatment, the overall response rate (orr) was % including crs ( %) and prs ( %). the median time to the optimal response was (range - ) days. the incidences of cr per organ were . %, . %, and % for skin, liver, and gut, respectively. the agvhd relapse rate was analyzed for the patients who had achieved cr or pr and survived beyond days. relapses in agvhd occurred in . % ( / ) of responsive patients. the patients who received ruxolitinib within days after agvhd onset have a significant higher cr rate that those with delayed ruxolitinib therapy ( . % vs. . %, p= . ). and the patients without gut infections have a significant higher cr rate than infected cohort ( . % vs. . %, p= . ). by logistic regression analysis, the time from agvhd to ruxolitinib (rr= . , p= . ) and gut infection (rr= . , p= . ) were independent predictors for incomplete response. thirteen patients ( / , . %) suffered from at least infectious episode after the start of the combination therapy, and pulmonary infectious diseases was a frequent complication ( / , . %). iii-iv cytopenia and cmvreactivation were observed in % and . % of patients. the -year overall survival (os) after initiation of the combination therapy were . %. the -year nrm and relapse incidence was . % and . %, respectively. patients with complete response on day had significantly higher os probability than non-cr patients ( -year os: . % vs . %, p= . ). compared with the historical cohort of basiliximab and etanercept for sr-agvhd in our center (n= ), no significant difference was found on the baseline. although the orr in patients treated with ruxolitinib and etanercept is identical with the historical cohort, ruxolitinib group achieved rapider remissions in liver agvhd and gut agvhd than the historical cohort (gut agvhd: days vs. days, p= . ; liver agvhd: days vs. days, p= . ), thus, with regard to hospital stay after agvhd onset, the ruxolitinib cohort stayed shorter (median: days vs. days, p= . ) than basiliximab cohort. conclusions: combined treatment with ruxolitinib and etanercept resulted in a rapid cr to visceral agvhd and meanwhile reserve graft anti-leukemia (gvl) effect as the relapse rate of primary disease is relatively lower. the various infection complications associated with ruxolitinib merit more attention. disclosure: nothing to declare background: graft-versus-host disease (gvhd) remains one of the main life-threatening complications after allo-hsct, especially in patients with non-malignant diseases. the standard gvhd prophylaxis strategy is mostly based on the use of calcineurin inhibitors alone or in combination with other immunosuppressive (is) post-transplant cyclophosphamide (ptcy) is effective gvhd prophylaxis optiont for adult patients (pts), but has limited data in children. methods: the study aim was to evaluate ptcy as gvhd prophylaxis in pediatric pts with inherited disorders undergoing allo-hsct. pts, the most of them are pediatric age (median age - y.o., range month - y.o.) with different types inherited disorders (β-thalassemia - , bone marrow failure syndromes - , storage diseases - , primary immunodeficiencydisorders - ) were inrolled in retrospective study. donor type was: matched/mismatched unrelated (mud/mmud) - , matched related donor (mrd)- , haploidentical (haplo) - . conditioning regimen was: myeloablative (mac) - , reduce-intensity (ric) - . graft sourse was: bone marrow (bm) - , peripheral blood stem cells (pbsc) - , combintions bm +pbsc/bm+cord blood - . ptcy mg/kg days + , + based gvhd prophylaxis recived pts., standart gvhd prophylaxis based on calcineurin inhibitors - pts. results: cumulative incidence (ci) of agvhd was %. grade - , - agvhd were % and % respectively. ptcy based gvhd prophylaxis reduced ci of agvhd ( % vs %, p= , ). another reduce ci of agvhd factors were mac ( % vs % in ric pts group, p= , ), mrd ( % vs % in haplo group vs % in mud/mmud group, p= , ), bm as a transplant source ( % vs % in pbsc group, p= , ). in a multivariate analysis mac (hr , %ci , - ,, p= , ), time from diagnosis to allo-hsct less then month (hr , %ci , - , , p= , ) were predictive for reducing ci agvhd. for agvhd - st. significant factor increase ci was female donor both in univariate ( % vs %, p= , ) and multivariate analysis (hr , %ci , - , , p= , ). years overall survival (os) was %. improving os factors were: transplant age younger then y.o. ( % vs %, p= , ), time from diagnosis to allo-hsct less then month ( % vs %, p= , ), engraftment ( % vs %, p= , ). in a multivariate analysis only transplant age younger then y.o. (hr , %ci , ( ) ( ) ( ) ( ) ( ) p= , ) and engraftment (hr , %ci , - , , p= , ) were predictive for os. conclusions: ptcy-based gvhd prophylaxis can be effective options for reduce risk of acute gvhd. using unrelated donors, bone marrow as transplant source and mac can reduce ci of gvhd. performing allo-hscr earlier from diagnos and in earlier age can improve os patients with inherited disorders background: diarrhea is a frequent complication after allo-sct. at onset it is often difficult to differentiate gi gvhd from other causes of enterocolitis. recently, non-invasive tests, such as fecal calprotectin (fc), have been validated as markers of gut inflammation in patients with inflammatory bowel disease, but only a few studies have been published regarding its use as a diagnostic marker in gi gvhd. methods: our aim in this study was to explore the levels of fc in allo-sct recipients with new-onset diarrhea. so far we have included allo-sct recipients who developed acute diarrhea ≥ stage - at a median of days (range: - ) post allo-sct. stool samples were analyzed as soon as possible after the onset of diarrhea. fc levels were determined in addition to an extensive microbiological panel for infectious enterocolitis (including norovirus pcr and c. difficile associated diarrhea). endoscopies for histologic analysis were performed according to the treating physicians' discretion (n= ). results: patients characteristics are summarized in table . median follow-up for survivors was days (range: - ). twenty-eight patients ( %) were diagnosed of gi-gvhd. the additional causes of diarrhea were: drug-related enterotoxicity (n= ), viral enteritis (n= ), food intolerance (n= ), c.jejuni-enteritis (n= ), and non-specific causes (n= ). the concentration of fc was higher in patients with gi gvhd vs. other causes of diarrhea ( μg/g +/- vs. μg/g +/- , p= . ). patients who did not develop severe enterocolitis had normal to slightly raised calprotectin at the onset of diarrhea [< - in out of ( %) cases], including % ( / ) of patients with enterotoxic drug-related diarrhea. among the patients with gi-gvhd, ( . %) were later found to be steroid-resistant. as shown in figure , we found a significant association between high fc (≥ μg/g) and severe-refractory gvhd (hr . , p= . ). of note, high values of fc were also found in patients with severe infectious enteritis (norovirus, adenovirus and c.jejuni infections), with baseline fc> μg/g, respectively. overall survival was % (ic %: - ) at months. hypoalbuminemia and thrombocytopenia were the only variables linked to -yr os in univariate analysis, regardless of the cause of enterocolitis. conclusions: in the absence of standarized (and expensive) biomarker panels for analyzing and predicting gvhd onset and outcomes, the fc test may be an useful tool in the allo-sct setting. our initial results show that fc is helpful in predicting mild causes of diarrhea and to identify patients with a high probability of developing severe (and potentially steroid-refractory) gi gvhd, although high levels are also found in severe infectious enteritis. background: there is an urgent need for effective therapy for severe acute gvhd. results of gvhd therapies beyond months are rarely reported. we here report a median follow-up of years. we introduced mesenchymal stromal cells as therapy for severe acute gvhd, with a dramatic response in some, but not all patients. the placenta protects the fetus from the mothers haploidentical immune system during pregnancy. we found that maternal stromal cells from the fetal membrane, so called decidua stromal cells (dscs) were more immunosuppressive than other sources of stromal cells. methods: we treated patients, median years of age (range . - ) for severe acute gvhd. all had biopsy proven gastro-intestinal gvhd. all were steroid refractory, after > days or with progression and after > days. we used an improved protocol where dscs were thawed and infused in a buffer with % albumin. dscs were given at a median dose of . ( . - . ) x cells/kg and ( - ) doses, given one week apart. viability of frozen and thawed dscs was % ( - ) and cell passage was ( - ). results: complete resolution of gvhd was seen in patients and had a partial response. the cumulative incidence of chronic gvhd was %. six had mild, moderate and one severe nih overall gvhd severity scoring. nine patients died, from relapse, acute gvhd and septicemia, zygomycetes infection, liver insufficiency, cerebral hemorrhage, multiorgan failure and chronic gvhd with obstructive bronchiolitis. four years transplant related mortalliy was . % and overall survival was %. survival was not significantly worse (p= . ) than % for all patients undergoing allogeneic hematopoietic cell transplantation during the same period - . conclusions: to conclude, dscs seems to be a promising therapy for severe acute gvhd. randomized trials are under way. disclosure: nothing to declare p anti-apoptotic protein bcl- is upregulated in graftversus-host disease stem cell transplantation (allo-hsct) with - % developing either acute or chronic gvhd. recently, bcl- inhibitor venetoclax was approved for treatment of chronic lymphocytic leukemia. induction of apoptosis and depletion of lymphocyte subpopulations e.g. follicular b-cells or cd + and cd + t-cells led to further exploration in autoimmune disease. methods: to establish expression levels of genes in the bcl- pathway, low-input rna sequencing was performed on t cells isolated from non-inflamed skin and peripheral blood of hsct recipients at different time points before until year after transplantation. furthermore, we analyzed blood, lung, gut and skin samples of patients post allo-hsct with and without previously untreated acute or chronic gvhd by rt-pcr, flow cytometry and tissue immunofluorescence. [[p image] . bcl- is up-regulated in t and b lymphocytes of acute and chronic gvhd lesions.] results: rna-sequencing revealed that t cells upregulated bcl- upon conditioning treatment (day ) and cells of patients who later developed gvhd failed to downregulate bcl- after transplantation (day+ , day+ ). bcl- protein levels were elevated in overall leukocytes and pathogenic cell subsets including monocytes, cd + t lymphocytes and nkt cells showed significantly higher expression of bcl- in peripheral blood of gvhd patients as compared to healthy controls. these results could be recapitulated in tissue samples, where disease-promoting lymphocytes (t, b, nk, nkt) were numerically expanded and expressed bcl- in acute and chronic gvhd skin lesions. notably, non-pathogenic cell types such as keratinocytes did not exhibit increased bcl- expression compared to control samples from hsct recipients and healthy donors. while bcl- rna expression did not depend on type of conditioning (mac vs. ric) or gvhd grade, it correlated to disease severity and was significantly elevated in biopsies of patients with steroidrefractory gvhd. conclusions: we could show exclusive upregulation of bcl- in gvhd-mediating cell types in peripheral blood and tissue samples affected by gvhd, correlating to gvhd severity and response to first-line therapy. thus, bcl- inhibition may present a novel and urgently needed targeted therapy in treatment of steroid-refractory acute and chronic gvhd. disclosure: supported by a docmed fellowship od the austrain academy of sciences background: graft-versus-host disease (gvhd) represents a major contributor to morbidity and mortality in recipients of allogeneic hematopoietic cell transplants (hct). several therapeutic strategies exist for gvhd prophylaxis and include post-transplant cyclophosphamide (ptcy) and antithymocyte globulin (atg). while several groups have described the use of ptcy in younger patients, there is a paucity of data about the efficacy of ptcy in older individuals, particularly when combined with atg. we investigated the combined effect of ptcy with atg on transplant outcomes in older patients at princess margaret cancer centre, toronto, canada. methods: this retrospective study included all patients age ≥ who underwent allogeneic hct for any indication at our centre between december and july . overall survival (os) was calculated using kaplan-meier analysis and multivariable cox proportional hazards regression. cumulative incidence of relapse (cir) and non-relapse mortality (nrm) were calculated using competing risk regression (fine and gray method). incidences of acute (agvhd) and chronic (cgvhd) were compared using the fisher's exact test. results: of patients, ( %) were male. median age was (range - ) and median follow-up among survivors was months (range - ). acute myeloid leukaemia (aml) was the most common indication for hct ( patients, %), followed by myelodysplastic syndrome ( patients, %) and myelofibrosis ( patients, %). eightyfour ( %) patients had a matched unrelated donor, ( %) had a matched related donor and ( %) had a haploidentical donor. one hundred twenty-five ( %) patients received reduced intensity conditioning. sixty-two ( %) patients received ptcy combined with atg ( . mg/kg) while ( %) received other forms of gvhd prophylaxis. os at years was % ( % confidence interval (ci) - ) in the entire cohort. patients who received ptcy with atg had a superior -year os compared with other gvhd prophylaxis regimens ( figure a ): % ( % ci - ) vs. % ( % ci - ), respectively (hr= . , % ci . - . , p= . ). the -year nrm for the entire cohort was % ( % ci, - ). patients who received ptcy with atg had a lower -year nrm compared to those who did not ( figure b ): % ( % ci - ) vs. % ( % ci - ), respectively (hr= . , % ci . - . , p= . ). the -year cir in the whole group was % ( % ci - ). use of ptcy with atg was associated with a modest increase in cir at two years ( figure c ): % ( % ci - ) vs. % ( % - ), respectively (hr= . , % ci . - . , p= . ). there was a trend toward lower incidence of grade ii-iv agvhd among patients who received ptcy with atg compared to those who did not: % vs. % (p= . ). the incidence of grade ii-iv cgvhd was lower in individuals who received ptcy with atg compared to those who did not: % vs. % (p= . ). conclusions: in older hct recipients, use of ptcy combined with atg is associated with improved os, lower nrm, decreased risk of both agvhd and cgvhd and a modest increase in relapse risk. therefore the ptcy with atg combination represents an effective strategy for gvhd prophylaxis in older allogeneic hct recipients. disclosure: the authors have no conflict of interest to declare. outcome of severe graft versus host disease in pediatric patients with nonmalignant diseases after allogeneic bone marrow transplantation. a single center experience irina zaidman , sigal grisariu , batia avni , ehud even-or , bella shadur , adeeb nasereddin , polina stepensky background: hematopoietic stem cell transplantation (hsct) remained the only curative option for many nonmalignant diseases in pediatric patients. survival after hsct has improved the last few years due to significant advancement in human leukocyte antigens (hla) typing techniques, less toxic conditioning regimens and better supportive care and resulted to % survival and cure in some non malignant diseases. graft-versus-host disease (gvhd) remains a major complication of hsct and leading cause of morbidity and mortality. prognosis of patients with high grade gvhd is dismal and survival rate varies between % to % in pediatric patients. methods: the retrospective study included patients with non malignant diseases who underwent allogeneic hsct at hadassah medical center from to . the collected data included patient´s clinical data and transplant characteristics. the study was approved by the institutional helsinki committee. results: children with nonmalignant diseases underwent allogeneic bone marrow transplantations in hadassah university hospital during ten years period. fifty seven patients ( %) developed agvhd grade - , twenty five of them ( . %) grade - . median age was . (range . - . ), most patients were males ( males, females). patients underwent bmt from fully matched family members, children were transplanted from matched unrelated donors and from mismatched donors. twenty one of patients with severe gvhd ( %) survived. four patients ( %) died from severe gvhd and complications of immunosuppressive treatment. of deceased patients were transplanted from mismatched donor, in of cases the age of donor was advanced, of patients developed severe gvhd and died after second hsct. all patients were refractory to different treatment modalities. three of patients died in and one in , it was no death from severe gvhd in patients that were transplanted and developed high grade gvhd after . conclusions: the results of this study show a high survival rate of % in pediatric patients with non malignant diseases and severe gvhd. significant risk factors for mortality in our group included mismatched donor, advanced age of donor and second transplant. trend to better survival was observed after . additional multicentral studies analyzed the outcomes of agvhd in pediatric patients with nonmalignant diseases are urgently required. background: chronic graft-versus-host disease (cgvhd) is a serious late complication after allogeneic hematopoietic stem cell transplantation (allohsct) with heterogeneous presentation and still poorly understood pathophysiology including inflammation and endothelial dysfunction. factor viii (fviii) and von willebrand factor (vwf) are coagulation factors but also known indicators of endothelial dysfunction and inflammation in different settings, and therefore could serve as interesting candidate biomarkers of cgvhd. methods: since patients after allohsct were assessed by the multidisciplinary cgvhd team at the university hospital center zagreb, croatia, using established nih cgvhd-related measurements. an extensive history, physical and laboratory evaluations were performed, including fviii, vwf:ag and vwf:ac analysis. descriptive statistic and non-parametric analyses were performed. variables that showed significant univariate correlations were used in multivariate logistic regression (mlr) to identify the most predictive for fviii, vwf:ag and vwf:ac in cgvhd patients. results: cgvhd patients and controls (subjects after allohsct without cgvhd) were analysed. median age of cgvhd patients was ( - ) years, % females, . % underwent allohsct for hematologic malignancies, . % had myeloablative conditioning and . % matched related donor. median time from hsct to study was . days and from cgvhd diagnosis to study days. there were no demographic neither transplant related significant differences between cgvhd patients and controls beside stem cell source (peripheral blood . % vs . %, p= . ) and history of acute gvhd ( . % vs . %, p< . ). majority of patients had moderate ( . %) or severe ( . %) nih global cgvhd score, . % active cgvhd by clinician´s impression. median number of organs involved by cgvhd was ( - ), and the most frequently involved organs were mouth, skin and eyes ( . % each). cgvhd patients compared to controls had higher fviii levels (median ( - )% vs ( - )%, p= . , reference range - %) and higher vwf:ag (median . ( . - )% vs . ( . - )%, p= . , reference range - %), while vwf: ac showed a trend toward higher levels among patients (median . ( - )% vs ( . - )%, p= . , reference range - %). patients had higher ggt (p= . ), lower anticardiolipin igg (p= . ) and igm (p= . ), and lower albumin (p= . ) than controls, without differences between other laboratory parameters. univariate analysis showed that among cgvhd patients higher fviii was associated with worse karnofsky score (ks) (p= . ) and performance score (ps) (p= . ), higher leukocytes (p= . ), cholesterol (p= . ), triglycerides, ast, alt, ggt, ldh, and lower albumin. higher vwf:ag and vwf:ac in cgvhd patients were associated with worse ks and ps (p< . ), with more active cgvhd (p< . ), worse nih cgvhd liver (p= . ; p= . ) and nih cgvhd mouth (p= . ; p= . ), higher total nih score (p= . ; p= . ), higher number organs involved (p= . ; p= . ), higher esr, monocytes, ddimers, ast, alt, ggt, ldh, triglycerides, β- -microglobulin, ferritin, total proteins, iga and lower albumin. mlr analysis showed leukocytes (p= . ) and cholesterol (p= . ) as the strongest predictor of fviii (r = . %; p< . ), while strongest predictor of vwf: ac was number of organs involved by cgvhd (r = . %; p= . ). conclusions: results of this study detected high fviii and vwf levels in cgvhd patients with possible reflections to cgvhd manifestations, what needs to be further confirmed in larger longitudinal studies. disclosure: this work was supported, in part, by the unity through knowledge fund project entitled "clinical and biological factors determining severity and activity of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation", and also, in part, by the croatian science foundation project entitled "new biomarkers for chronic graft-versus-host disease". antonela samardzic -work financed by the croatian science fondations`"young researchers`career development project -training of doctoral students" background: thrombotic microangiopathy (tma) is a severe complication of allogeneic hematopoietic cell transplantation (hct) with multisystem involvement. a few recent reports have recognized evidence of tma in the intestinal vasculature (intestinal tma/itma) of patients with graft-versus-host disease (gvhd) with or without tma. we aimed to identify patients with itma and describe histological, clinical and prognostic features. methods: we prospectively evaluated available endoscopic samples (stomach and/or colon) from consecutive adult hct recipients for previously described histopathologic signs of itma (january -september ). systemic tma was diagnosed according to the international working group criteria. we compared findings among clinical groups: gvhd/systemic tma, gvhd/no systemic tma and no gvhd/no tma. results: we studied patients, classified as gvhd/ systemic tma, gvhd/no systemic tma and no gvhd/no tma. baseline transplant characteristics (age, donor, hla matching, conditioning) did not differ significantly among groups. histological features of itma, including loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells, intraluminal fibrin, intraluminal microthrombi and mucosal hemorrhage were found in patients. previously described features of intraluminal schistocytes were not observed in our patients. interestingly, loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells were also found in patients with gvhd and no itma, suggesting that these features are not pathognomonic of itma. among itma patients, two patients were classified in the clinical group of acute gvhd/systemic tma, while the other patients had clinical and histopathological features of itma and severe grade iii-iv steroid-refractory acute gvhd ( patients) or extensive chronic gvhd ( patient) but no evidence of systemic tma. in the majority of patients ( / ), itma occurred during the early posttransplant period at . ( . - ) months. clinical features (gastrointestinal bleeding, diarrhea, pain, nausea) presented no differences between patients with or without itma. prognosis was poor for patients with itma who suffered from a significantly higher mortality rate of % compared to the rest patient population (p= . ). with a median follow-up of . ( . - . ) months, year overall survival probability (os) was . for itma, % for gvhd and % for systemic tma. unfavorable predictive factors for os were itma (p= . ), hla mismatched donors (p= . ) and gastro-intestinal bleeding (p= . ). conclusions: intestinal tma has emerged as a novel distinct entity in patients with gvhd and/or systemic tma. distinct histological features may be useful in differential diagnosis of these severe hct complications. mortality rates higher than those of systemic tma highlight the need of proper recognition of itma that needs to be further studied in terms of diagnostic and therapeutic potential. disclosure: e.g. was supported by the european hematology association clinical research grant. the remaining authors declare no competing financial interest. the beneficial effects of thrombomodulin gene polymorphisms after hematopoietic stem cell transplantation background: chronic graft-versus-host disease (cgvhd) remains the major cause of late morbidity and mortality after allogeneic blood and marrow transplantation. treatment options for cgvhd, particularly its sclerotic forms remain limited. active hedgehog (hh) signaling was shown as a therapeutic target in both mouse and human cgvhd, with limited efficacy and significant toxicities described in a published clinical trial (defilipp, ). methods: adult patients with steroid refractory sclerodermatous cgvhd, defined as requiring > . mg/kg/day of prednisone dose equivalent (pde), or need for second-or third-line therapy beyond corticosteroids and calcineurin inhibitors or sirolimus were eligible for this open label study of vismodegib, a first generation hh pathway inhibitor. primary endpoint was failure free survival, defined as absence of non-relapse mortality, no recurrent malignancy, steroid dose at months =< . mg/kg/day of pde, and no addition of new systemic treatment. vismodegib was administered orally for - months, with dose reductions at development of toxicities. peripheral blood mononuclear cells were isolated from samples collected at treatment initiation and every three months thereafter. the immune profile of circulating b cells was analyzed by flow cytometry and t helper polarization by qrt-pcr of sortpurified cd + t cells. results: at the time of interim analysis, patients were evaluated. patients completed months of treatment and five patients completed months of treatment. therapy was discontinued in patients prior to months due to treatment-related (n= ) and unrelated (n= ) side effects. most patients experienced grade toxicities (muscle cramps and dysgeusia), with only a single grade toxicity (weight loss). patients who completed months of therapy demonstrated partial response, and overall, the primary endpoint was reached in % ( / ) of patients. in patients who discontinued vismodegib, cgvhd worsened acutely after discontinuation. correlative analysis of immune cellular subsets in peripheral blood in paired samples (pre-treatment and month of therapy) documented modulation of b cell subsets pathogenic in cgvhd (pregerminal center and plasmablast-like b cells) and diminished t helper polarization in cd t cells. conclusions: overall, use of vismodegib was associated with potential clinical efficacy in sclerodermatous cgvhd with possible mechanistic evidence arising in correlative studies. while side effects were common, further studies of hh inhibition in cgvhd are warranted. future studies should employ adjusted dosing regimens, along with supportive care interventions to offset side effects, and testing of novel hh inhibitors with enhanced safety profiles. clinical background: graft-versus-host disease (gvhd) results from recognition of host antigens by donor t cells following allogeneic hematopoietic stem cell transplantation (sct). we tested the hypothesis that somatic neomutations occurring after sct from donor and/or recipient dna may trigger gvhd. methods: we longitudinally analyzed both constitutive and somatic mutations by whole exome sequencing (wes) in patients who received sct from a sex-matched hlaidentical sibling for npm mutated acute myeloid leukemia (pt# ) and jak v f mutated primary myelofibrosis (pt# ). both patients were initially refractory to alloreactivity, i.e. not displaying any signs of gvhd, even after several donor lymphocyte infusions. acute gut gvhd finally occurred after a further dli preceded by a lymphodepleting chemotherapy. in pt# , gvhd correlated with a graft-versus-tumor effect. wes was performed on dna from recipient saliva and donor pbmcs (germline samples) and from sequential post-sct pbmcs samples on a hiseq illumina with x bp paired-end reads at a mean depth of coverage of - x. germline and somatic mutations were determined using in-house bioinformatic pipelines (named ewok from the curie institute and smaug from the henri mondor hospital), using briefly gatk as variant caller for germline samples, and a combination of variant callers for matched normaltumor pairs. we adjusted parameters to detect somatic mutations at a minimal variant allelic frequency (vaf) of % compared to recipient and donor germline for all variations (minimal coverage = x for germline and x for tumor sample). results: wes allowed detecting somatic driver mutations explaining aml and pmf for both patients in the initial timepoint and all these driver mutations disappeared at the following timepoints. as expected, the somatic variant rate was x higher in pt# with aml than in pt# with pmf at each timepoint, except for the final gvhd timepoint. indeed, at this final point, the somatic variant rate dramatically decreased by % as compared to previous timepoints. by subtracting variants detected pre-and post-sct from those identified at the ultimate time-point of gvhd occurrence, we created sets of and variants respectively for each patient (keeping only variants with at least reads of mutated dna). these variants can be classified in categories: (i) those with only with a slight increase at time of gvhd, i.e. ≤ -fold compared to highest previous vaf (lrrc , or u , or g , alpp, frg , frg b and lilrb genes), and (ii) those with a significant increase at that time, i.e. > -fold compared to highest previous vaf (phf , smpd , ercc and krtap - genes). none of the variants or genes involved was common between the patients. ontology classification of mutated genes showed the implication of some of them in cell death, regulation of map kinase activity, mrna splicing and immune system process, making them good candidates for further studies. identification of variants appearing pre-gvh and turning off at time of gvhd is ongoing to unveil putative neoantigens that could trigger the alloreactive response. conclusions: using a comprehensive, pre-and post-sct, wes of donor/recipient pairs, we identified several neomutations from donor and/or recipient dna correlating with gvh/gvt effect development. disclosure results: a total of patients experienced cgvhd, and mild, moderate, and severe cgvhd were observed in , , and patients, respectively. the -year cumulative incidence of total cgvhd was . % ( % ci, . - . %), and the -year cumulative incidence of moderate to severe and severe cgvhd was . % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. the patients who had loci mismatched had a higher -year cumulative incidence of total cgvhd ( . % vs. . %, p= . ) and moderate to severe cgvhd ( . % vs. . %, p= . ) compared to those of the patients who had - loci mismatched. the patients who had maternal donors had a higher -year cumulative incidence of moderate to severe cgvhd ( . % vs. . %, p= . ) compared to that of the patients who had other donors. the patients who had grade iii to iv acute graft-versus-host (agvhd) had a higher -year cumulative incidence of total cgvhd ( . % vs. . %, p< . ) and moderate to severe cgvhd ( . % vs. . %, p< . ) compared to those of the patients without agvhd. in multivariate analysis, grade iii to iv agvhd was the only independent risk factor for total cgvhd (hr= . , %ci, . - . ; p< . ) and moderate to severe cgvhd (hr= . , %ci, . - . ; p< . ). in the model excluding agvhd, maternal donor was the risk factor for moderate to severe cgvhd (hr= . , %ci, . - . ; p= . ). conclusions: we observe that severe agvhd was the most important risk factors for cgvhd after haplo-hsct, and further interventions should be considered in these patients to prevent severe cgvhd. disclosure: none of the authors have any potential financial conflict of interest related to this manuscript. background: extracorporeal photopheresis (ecp) has been successfully used for the treatment of graft-versus-host disease (gvhd). ecp therapy might restore the balance between effector and regulatory cells which is severely impaired in gvhd. nk cells are the first lymphocyte subset to be reconstituted after allogeneic hematopoietic stem cell transplantation (allo-hsct). as an important innate immune cell population, nk cells can temporally bridge the transient period of t-cell deficiency post allo-hsct, by protection from opportunistic infections and prevention of leukemic relapse by graft-versus-leukemia (gvl) effect. nk cells not only preserve homeostasis through targeted killing of allo-reactive t cells and thereby control gvhd but also enhance inflammation by secretion of tnf-α and ifn-γ and thereby promote gvhd. therefore, we investigated here the role of nk cells in gvhd patients under ecp therapy. methods: thirty four patients with steroid-refractory/ resistant agvhd ≥ ii°and moderate to severe cgvhd received ecp therapy which performed according to the guidelines. glucksberg and nih criteria were used for clinical staging of agvhd and cgvhd under ecp therapy, respectively. the comprehensive phenotypical analysis of nk cells was evaluated by multicolor flow cytometry. nk activity in terms of killing function, cytokine release capacity and proliferation function was monitored by chromium- release assay, intracellular cytokine staining and cfse staining, respectively. results: five different nk cell subsets were defined based on cd and cd expression. cd bri nk cells displayed an immature and activation profile with high expression of cd l and nkg d. agvhd patients had a higher frequency of cd bri nk cells when compared with hds and cgvhd patients, who were characterized by significant increase of the cd dim cd + and cd -cd + nk cell subsets with high expression of differentiation markers cd b and cd . of note, cd bri cd -nk cells could serve as a novel predictive biomarker for the response of agvhd patients to ecp treatment. in responding agvhd patients, an increase of cd bri nk cells was observed already during the early ecp treatment phase, suggesting immune reconstitution. after priming of the progenitors, ecp could differentiate immature cd bri nk cells into mature cd dim nk cells with reduction of cd l on cd bri nk cells. moreover, cd dim nk cells could further be matured through upregulation of cd expression by ecp. notably, ecp therapy could shift the nk cells from a cytotoxic to a regulatory phenotype within the cd bri nk cells. in spite the immunomodulatory effect of ecp on nk cells, nk activity could be kept intact under ecp therapy. the killing activity of nk cells was stable as confirmed by a cr release assay. ecp therapy had no negative effect on the quantity and quality of cytokine release by nk cells upon k stimulation. especially, the polyfunctionality of nk cells was not altered significantly by the ecp therapy. conclusions: nk cells play an important role in gvhd and could serve as a predictive cell population for the clinical response to ecp therapy. in the current study, ecp influenced the differentiation, maturation and education of nk cells ameliorating gvhd without comprising the antiviral immune defense and gvl effect. disclosure: the authors declare no competing financial interests, except the following: therakos mallinckrodt gave a financial support to as and ms for the documentation of the clinical course and for the analysis of immune cells of the patients, pw has honoraria and membership on advisory boards for sanofi-aventis. abstract withdrawn. cyclosporine levels > µg/l on day post-transplant was associated with significantly reduced acute graftversus-host disease following allogeneic hematopoietic stem cell transplantation monica bianchi , dominik heim , claudia lengerke , martina kleber , dimitrios tsakiris , jakob passweg , alexandar tzankov , michael medinger background: acute graft-versus-host disease (agvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients, especially with steroid-refractory agvhd, have a very poor prognosis. prophylaxis with cyclosporine a (csa) is the backbone of gvhd prevention in most conditioning regimens. methods: in a retrospective analysis of patients treated with allo-hsct, we correlated csa levels at the day of transplantation (day ) and day + with the incidence of acute and chronic gvhd. we postulate that higher target csa levels > μg/l will result in a lower incidence rate especially of agvhd after allo-hsct. results: we assessed patients with either aml n= , lymphoma/myeloma n= , mds/mpn n= , all n= , cll n= , cml n= , or bone marrow failure n= . in patients with clinically relevant agvhd grade ≥ , mean csa levels was lower on day and day + ( ± μg/l; and ± μg/l; respectively) compared to patients without agvhd ( ± μg/l; and ± μg/l; respectively; day : p= . ; day + : p= . x - ). in patients with csa level < μg/l, the incidence of agvhd was significantly more frequent compared to patients with csa levels > μg/l [( / ; %) versus / ( %); p= . x - ]. in patients with cgvhd, there was no significant difference between csa levels < μg/l ( / ) compared to csa levels > μg/l ( / ; p= . ). the optimal csa cut-off level for the prevention (i.e. roughly % incidence reduction) of agvhd was > μg/l at day and > μg/l at day + ( figure ) in a competing risk analysis, time to agvhd grade ≥ (using death of other causes as competing risk) was associated with csa levels > μg/l on day and on day , unrelated donors, myeloablative conditioning (mac), and for the diagnosis lymphoma/myeloma. conclusions: our data support close monitoring with active adjustments of csa dosing to maintain therapeutic csa levels above μg/l in the first days of allo-hcst to reduce agvhd. disclosure: noting to declare. liposomal cyclosporine a for inhalation (l-csa-i) to treat bronchiolitis obliterans syndrome: novel formulation with therapeutic potential for patients with bos following allo-hsct noreen roth henig , emilie hofstetter , dominik kappeler , gerhard boerner background: bronchiolitis obliterans syndrome (bos) is a rapidly progressive lung disease caused by t-cell mediated inflammation that leads to blockage of bronchioles, leading to respiratory failure and death shortly after diagnosis. approximately % to % of patients who undergo allogeneic hematopoietic stem cell transplant (allo-hsct) will develop bos, with - % developing bos as a respiratory form chronic graft-vs-host disease (cgvhd) in addition to other signs of cgvhd. mean time to bos diagnosis ranges from to days post-transplant. the histopathology of bos after allo-hsct and lung transplantation is identical. early studies of l-csa-i for the prevention of bos in lung transplant recipients demonstrated therapeutic benefit. l-csa-i is a novel, liposomal formulation of cyclosporine administered via a pari investigational eflow â nebulizer which delivers a potent immunosuppressant to the site of disease. pharmacokinetics and tolerability of l-csa-i is presented. methods: retrospective review of two clinical studies of l-csa-i (isotonic, mg/ml) for bos associated with lung transplantation. both studies had a control arm and results reported here are for patients who received l-csa-i. subjects received mg (single lung transplant) and mg (double lung transplant) bid via inhalation. blood samples for pharmacokinetic analysis of cyclosporine a concentrations were collected before inhalation, immediately after inhalation, and thereafter in intervals of , , min and , , and hours. local and general tolerability of l-csa-i was investigated. results: between the two studies, subjects received either or mg bid of l-csa-i. pharmacokinetic models predict a constant drug level in the lung. maximum serum cyclosporine a concentration after inhalation was . ± . ng/ml. trough levels for up to -years of daily administration was - ng/ml with no evidence of accumulation following repeated exposure. tolerability data was assessed from patient-month exposure to l-csa-i. reported symptoms were: pharyngeal soreness %; cough %; dyspnoea %; and wheezing %. no subject discontinued due to intolerability. inhalation time is on average - min. conclusions: l-csa-i provides high and constant concentrations to the airways of the lungs and the site of bos. l-csa-i is well tolerated in lung transplant patients. use of l-csa-i instead of augmentation of systemic csa reduces the total drug exposure. a multicentre phase safety and exploratory efficacy trial for the treatment of bos in allo-hsct recipients is underway. disclosure background: there are a number of biomarkers that predict non-relapse mortality (nrm), graft-versus-host disease (gvhd) and relapse incidence (ri) after conventional gvhd prophylaxis based on calcineurin inhibitors with or without antithymocyte globulin. currently there is limited data whether the conventional predictive biomarkers work with posttransplantation cyclophosphamide (ptcy) prophylaxis. methods: prospective single-center study in - enrolled adult patients with acute leukemia in cr ( % with all, % with aml). received matched related bone marrow (bm) graft with single-agent ptcy and received unrelated peripheral blood stem cell graft (pbsc) with ptcy, tacrolimus and mmf. the grafts were studied by flow cytometry (facs aria ii, antibodies by miltenyi biotec). the following populations were analyzed: cd , cd , cd , cd cd , nkt, inkt, treg, double-positive t-cells, double-negative t-cells, tcralpha/beta, tcr v memory cells. the crypreserved plasma from were analysed by elisa (commercial kits by ebioscience and critical diagnostics) for vegf a soluble tnf receptor (stnfr), il- , il- , soluble il- receptor, st , il- and stnfr. the above mentioned biomarkers were tested in logistic regression with roc analysis, assays with auc> . were selected for analysis in fyne-gray regression with competing risks. cut off levels were determined for significant parameters. results: median follow-up was months (range - ). in the whole group overall survival (os) was %, eventfree survival (efs) %, grade ii-iv acute gvhd %, moderate and severe (m&s) chronic gvhd %, nrm %, mortality in patients with gvhd %, ri %. there was no difference between bm/related and pbsc/unrelated grafts in the incidence of gvhd, nrm and ri (p> . ). the only significant predictor of acute gvhd were low levels of il- level on day+ (p= . , % vs % with the cut off pg/ml). m&s chronic gvhd was predicted only by the high percentage of inkt cells in the graft (p= . , % vs % with the cut off . %). there was a correlation between il- levels and number of nk cells in the graft (p= . ). nrm was related to infectious complications, nonetheless high levels of vegf a on day (p= . , % vs % with the cut off ng/ml), st on day+ (p= . , % vs % % with the cut off ng/ml) and low percentage of cd +cd -cells in the graft (p= . , % vs % with the cut off . %). the identified biomarkers of nrm had no association with the pre-transplant crp and ferritin levels (p> . ). the only significant parameter for ri was the level of cd cells in the graft (p= . ). none of the identified biomarkers significantly predicted overall survival (p> . ). conclusions: in the related and unrelated grafts with ptcy the study of biomarkers has low clinical utility due to very low gvhd-related mortality. however st and vegf a can predict infection-related mortality. also the study verified previous observations that high level of il- is associated with reduced gvhd incidence after ptcy and identified the importance of nk and inkt cells in the induction of tolerance with ptcy. references background: hematopoietic cell transplantation (hct) is the only curative approach for many hematological malignancies but life-threatening toxicities, such as graft-versushost disease (gvhd) and infections, still limit its fullpotential impact on the disease. strategies for keeping allohsct more effective and safe are needed in order to reduce morbidity while improving its immunological effect to control disease relapse. post-transplant cyclophosphamide (ptcy) has been demonstrated to improve acute gvhd (agvhd) and chronic gvhd (cgvhd) control in allogeneic bone-marrow hct from identical and haploidentical donor. the use of ptcy, after peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related donors, has been investigated by our group in a clinical trial (nct ) and preliminary results were published last year. here we report updated efficacy and safety data about the expanded cohort of patients treated with ptcy followed by tacrolimus and mycophenolate mofetil (t/mmf). methods: we analysed data about consecutive patients with high-risk hematologic malignancies received allopbsct from hla-matched unrelated/related donors between march and august . gvhd prophylaxis was ptcy mg/kg (days + + ), tacrolimus from day + and mmf from day + to day + . primary objectives were cumulative incidence of agvhd and cgvhd. secondary objectives were event-free survival (efs), cgvhd-efs, overall survival (os) and non-relapse mortality (nrm). results: patients median age at transplant was (range - ) years. ( %) patients were transplanted in first complete response (cr), ( %) patients in second/third cr, the others in disease control. a median dose of . (range - ) x ^ cd /kg was infused. primary graft failure was observed in one patient. all patients were off mmf on day + , the median day of tacrolimus discontinuation was (range - ). eight out of ( %) patients developed agvhd, ( %) of them were grade ii-iii; median day of onset was day (range . no grade iv was observed. no cases of late-onset agvhd were reported. cumulative incidence of cgvhd was % ( / ), median day of onset was (range - ). systemic treatments were required, but all patients were able to discontinue immunosuppression (is). with a median follow-up of (range - ) months, efs was %, cgvhd-efs was % and os was %. non-relapse mortality (nrm) was % ( / ): patients died because of multidrug resistant bacteria septicemia. nowadays patients are alive with no evidence of disease, being continuously off is and completely reintegrated in their normal daily life activities. conclusions: the updated reported results confirm, in a larger cohort of patients with a longer follow-up, that ptcy after pbsc-hct is highly active in agvhd and cgvhd prevention with extremely limited nrm. this strategy, not only allowed earlier discontinuation of immunosuppression, but also reduced the overall time of exposure to is for most of the patients. all these features might contribute, in the future, to transform hct into a safe immunologic platform that may be combined with advanced form of cellular therapies (car-tcells), aiming to increase safely the graftversus-tumor effect. clinical methods: pediatric patients ( - years) with nmd undergoing unrelated hct were eligible for this single center, phase i trial. following reduced intensity conditioning, abatacept ( mg/kg iv on days - , + , + , + ) was added to standard gvhd prophylaxis (cyclosporine, mycophenolate mofetil [mmf]). patients were followed for years for standard hct outcomes. [[p image] . figure results: since june , patients have been enrolled and transplanted (table , excluding # ). donor source was bone marrow in all. with median follow-up of . years, of patients survive without disease. initial engraftment was successful in , at a median of and days, for neutrophils and platelets respectively. one patient ( ) had secondary graft rejection in the setting of viral reactivation (cmv/ebv), with successful engraftment following a nd unrelated hct. in engrafted patients, myeloid (cd ) chimerism was % at all timepoints; t-lymphoid (cd ) chimerism was mixed but reached >/= % (figure ). one patient ( ) with saa had primary graft rejection in the setting of inadequate tnc dose ( . x /kg) and died from marrow aplasia/infection despite nd hct. a second death from wilms' tumor occurred months post successful hct, in a patient ( ) with dba and constitutional chromosome abnormality. except patient , all patients received doses of abatacept, which was well tolerated, with all severe adverse events expected for a hct population. cmv and ebv reactivation occurred in patients each, with resolution using standard anti-viral therapy. one patient ( ) was diagnosed with ebv-driven post-transplant lymphoproliferative disease, which responded to rituximab and immune suppression withdrawal. no patients developed severe acute (grade iii-iv) or chronic gvhd (table ) , and no patients required systemic immune suppression at > year. conclusions: these preliminary data suggest that abatacept can be safely added to cyclosporine and mmf gvhd prophylaxis in pediatric patients with bone marrow failure undergoing unrelated donor hct, with encouraging rates of gvhd despite half of patients having a mismatched ( / ) donor. given the higher risk of graft rejection in this non-malignant cohort, rejection (in addition to gvhd) will be a primary focus in our subsequent multi-center, phase trial. clinical trial registry: clinicaltrials. gvhd and may to have be separated from from toxicity to infectious complications in the early phase after allohsct. methods: from our files we identified patients which had upper gastrointestinal tract endoscopy after allohsct in with biopsies were taken from the esophagus, stomach and duodenum simultaneously. of these patients were excluded because of infection, reflux disease or drug toxity and the remaining patients were included in our study. we evaluated the routine stained esophageal biopsies, applied a grading scheme and compared the histological findings with those within the stomach and duodenum, the endoscopic findings and the clinical course. results: in of biopsy samples of the esophagus, we identified histological features of acute gvhd, ranging from vacuolar degeneration (grade ) and single-cell apoptosis (grade ) to the formation of clefts (grade ) and mucosa denudation in advanced cases (grade ), resembling epithelial lesions in acute gvhd of the skin. these findings correlated with gvhd involving the stomach and duodenum and the clinical manifestations of gvhd in other organs. endoscopically patients with gvhd revealed signs of inflammation, ranging from erythema to ulceration in the more advanced cases, sometimes reminiscent of reflux or infection. clinically these patients had abdominal discomfort ranging from inappetence to nausea, accompanied by emesis or diarrhea and weight loss. conclusions: we have shown that acute esophageal gvhd occurs after allohsct and is correlated with acute gvhd in stomach and duodenum. it could be diagnosed and graded histologically. the endoscopic findings are signs of inflammation. our results may help to establish the histological diagnosis of acute gvhd using endoscopic biopsies from the esophagus and to explain the alterations observed in the esophageal mucosa in patients after allohsct. [ background: intestinal acute graft versus host disease (agvhd) is a major thread after allogenic hematological stem cell transplantation (allohsct), with a high mortality in patients which were refractory to steroid treatment in particular. recent papers point to a correlation of histological grading of intestinal gvhd and prognosis in patient after allohsct. however a comparison with clinical scores has not been performed so far. methods: in this analysis, retrospective data from patients who underwent endoscopy due to clinical signs of agvhd (day + to + after allohsct) were evaluated. of each patient least biopsies from different sites of the colon which were taken simultaneously. of each biopsy series the maximum histological grad of agvhd according to the lerner scheme was obtained and compared with the glucksberg stage of the lower gastro intestinal tract (gslgi) and the overall glucksberg grade (ogg). these three grades were compared for non-relaps related mortality using the log-rank test and for sensitivity to steroid treatment applying the receiver operating characteristic for the patients who received steroid treatment. for these patients the non-relaps related mortality for responder and non-responder were calculated using also log-rank test. results: the histological grade strongly correlated with the survival (p= . ). a statistical significant correlation was also found for the gslgi (p= . ), whereas the ogg revealed no significant correlation (p= . ). non-relaps related mortality was mainly related to infection or sepsis (in / patients who died). -eighty-one of the patients received steroid therapy. the sensitivity to the steroid therapy correlated with each of the three scores (p< . ) but was the strongest for gslgi (area under the curve (auc) . ), compared to ogg (auc . ) and the histological score (auc . ). the survival of the patients, which were sensitive to steroid treatment was significantly better than those of steroid refractory patients (p= . ). conclusions: we found that histological and clinical grading in patients after allohsct with intestinal gvhd was correlated with survival and respond to steroid treatment. histological scoring may predict survival more precisely than ogg and gslgi but did not add substantial information to the prediction of treatment response. [ emerging evidences suggest that regulatory b cells (bregs) play essential roles in inflammation, autoimmune diseases and tumors. few data exist about the role of bregs in the contest of hematopoietic allogeneic stem cell transplantation (hsct). some authors have observed that bregs from patients with chronic graft-versus-host disease (cgvhd) were less frequent and less likely to produce il- than bregs as compared to healthy donors or patients without cgvhd. these findings suggest that bregs may be involved in cgvhd pathogenesis. the purpose of our study was to evaluate a possible role of b cell subsets on gvhd occurrence. methods: lymphocyte subset enumeration was performed by aquios cl flow cytometer (beckman coulter), a quantitative automated analyzer that performs two diagnostic panels: tetra- cd -fitc/cd -rd / cd -ecd/cd -pc and tetra- cd -fitc/cd +cd -rd /cd -ecd/cd -pc . b cell subsets (memory, mature and transitional b cells) on peripheral blood samples were analyzed by aquios designer software, a tool for the creation of user-defined applications. panel- cd -fitc/cd -pe/cd -ecd/cd -pc /cd -pc and panel- cd -fitc/cd -pe/cd -ecd/cd -pc / cd -pc were specifically designed by beckman coulter for our center. the flow cytometric analysis was performed as follows: in donors and patients at basal level; on graft products and in patients at days + , + , + , + after hsct. statistical significance was assessed with prism software (graphpad) by mann withney test. p < . was considered statistically significant. results: actually we enrolled patients submitted to hsct in our center from november . a preliminary statistical analysis was performed on patients. stem cells source was peripheral blood (pb) in cases and bone marrow (bm) in the others . the conditioning regimen was myeloablative in patients and ric in patients. agvhd was diagnosed in patients ( %). no associations were found between b cell subsets in donors and patients at baseline and the occurrence of agvhd. however we found a higher median percentage of transitional b cells in graft products in patients without agvhd ( . %, . - . ) compared to patients with agvhd ( . %, . - . ) (p= . , fig a) . in addition, patients without agvhd showed a lower median percentage of memory b cells ( . %, range . - ) in graft product as compared to patients with agvhd ( . %, range . - . ) (p = . , fig. b ). finally in the subgroup of patients receiving pb as stem cell source we observed a higher percentage of cd + lymphocytes in graft product in patients with agvhd ( %; range - ) compared to patients without agvhd ( %; range - ) (p= . ). in the monitoring of b cells reconstitution we observed that cd + events did not appear before day + after hsct and these were b transitional immature events predominantly. conclusions: our data suggest a possible protective link between transitional b cells and agvhd development. these results data need to be confirmed in a larger cohort of patients. moreover, it will be interesting to evaluate the relationship between transitional b cells at day + and the occurrence of cgvhd. clinical background: agvhd is a major complication of allogeneic hematopoietic stem cell transplant (hsct) and a risk factor for post-hsct mortality. the objective of this analysis is to describe patients with agvhd who had a suboptimal response to corticosteroids. methods: patients who developed ibmtr severity index ii-iv agvhd after first hsct between / / to / / were included in an ongoing chart review at centers in the united states. patients who had ever participated in a gvhd prophylaxis trial or used jak inhibitors were excluded from the study. suboptimal response to corticosteroids was defined as use of additional systemic anti-gvhd therapy, inability to taper high-dose steroids (≥ mg/ kg) by ≥ %, or tapered corticosteroids by ≥ % but not to < mg/day. results: the analysis included patients with suboptimal response to corticosteroids. mean age was years; % were male. median time from transplant to agvhd diagnosis was days. at the time of maximum agvhd grade, % of patients were grade ii and % were grade iii-iv; % had lower gi involvement, and % had ≥ organs involved. from time of diagnosis to maximum agvhd grade, % of patients had new organ involvement or an increase in agvhd grade. median time from diagnosis to maximum grade was . days, and was . days for patients with lower gi involvement. systemic corticosteroids were initiated on the day of diagnosis for % of patients. average starting daily dose was mg ( . mg/kg) for prednisone and mg ( . mg/kg) for methylprednisolone. steroid dose was increased for % of patients during follow-up; % were unable to taper below mg/day. among patients who received additional systemic anti-gvhd therapy (n= ), % increased their corticosteroid dose before initiation of additional anti-gvhd therapy. median time from initiation of corticosteroids to additional therapy was . days. frequently used therapies were mycophenyalate mofetil ( %), atg ( %), extracorporeal photophoresis ( %), tocilizumab ( %), etanercept ( %), and sirolimus ( %). agvhd recurred in % of patients and was managed by increasing corticosteroid dose in % of patients. % had any infection within first days post-hsct. forty patients ( %) required hospital readmission(s); % had ≥ readmissions within days post-hsct, with a mean inpatient lengthof-stay of days. relapse of underlying malignancy was reported for ( %) patients. two-thirds ( %, n= ) patients died at a median of (interquartile range (iqr): - ) days from agvhd diagnosis; a higher proportion ( %) of patients with maximum grade iii-iv agvhd died at a median of (iqr: . - . ) days; majority ( %) of patients with lower gi agvhd died at a median of . (iqr: - ) days. conclusions: a majority of patients with agvhd who had suboptimal response to systemic corticosteroids had severe and rapidly progressing disease and resulted in a high mortality rate ( %); progression was more rapid and mortality increased for patients with lower gi involvement. most patients required readmission to the hospital with extended length-of-stay. an urgent need exists for effective and tolerable therapies that quickly resolve life-threatening agvhd in early stages of disease. disclosure results: median time to onset of bo from allohct was . months (range . - . ). previous acute gvhd in . % (n = ) [grades iii-iv . % (n = )]. in . % (n = ) cgvhd had exclusive lung involvement, while the other patients ( %) had other organs affected. at diagnosis of bo, . % (n = ) were under immunosuppressive treatment. . % (n= ) of patients with bo received ecp as second-line treatment. median duration of treatment was months ( . - . ) and time to response . months ( . - . ). median of sessions was ( - ). evaluation of response was based on the evolution of fev measurement: . % (n = ) complete response; % (n = ) partial response and % (n = ) stable disease. one patient did not get any response and another was not evaluable. . % of patients (n = ) could reduce immunosuppression, and in one case it was completely discontinued. there is a trend for early separation between survival curves in favor of ecp ( figure ). one patient had sepsis secondary to central venous catheter infection as complication related to ecp. conclusions: ecp has emerged as a promising treatment for bo after allohct. in our experience, ecp was effective to stabilize or improve the disease in many patients and allowed to taper esteroids with minimal associated complications. however, prospective studies and longer follow-up are needed to support these findings. disclosure: nothing to declare background: the key role of il- signaling in acute graft vs. host disease (agvhd) and cytokine release syndrome (crs) has evoked growing use of tocilizumab, an anti-il receptor (il -r) antibody, in these settings. apart from regulation of t-and b-cell differentiation, immune cells migration to inflammatory sites and t-cell recruitment, il- complex with il -r through gp upregulates production of fibrinogen (fg) and other acute phase proteins, including c-reactive protein (crp). methods: we retrospectively analyzed data of patients treated with tocilizumab ( mg/kg) due to steroid-refractory (sr) agvhd and patients because of crs. median age was and years, respectively. seven patients were transplanted from unrelated donors (mud/mmud) and from sibling donors. eight patients received myeloablative and reduced intensity conditioning regimen. analyzed data included concentrations of fg, crp, an incidence of infections at tocilizumab administration and in weeks following the infusion. results: stage ii agvhd was diagnosed in patient, stage iii in , and stage iv in patients. involvement of the gastrointestinal tract (gi) was observed in % of cases. the median fg concentration before tocilizumab administration was . g/l (range, . - ) and crp mg/dl (range, - ) and % of patients had an active infection. after infusion of the antibody, we observed a decline of fg and crp levels. the median level of fg was . g/l (range, . - . ) - days after the tocilizumab infusion with no severe bleeding complications. a median crp value was . mg/dl (range, . - ) despite confirmed infectious complications. three weeks after infusion of tocilizumab fg raised to the normal range in % of patients (fig ) . five patients with sr agvhd achieved a complete response, and had a partial response after tocilizumab therapy. [[p image] . fibrinogen levels in gvhd patients following tocilizumab infusion.] a group treated with tocilizumab due to crs had higher initial levels of fg . g/l (range, . - . ) and crp mg/ dl (range, - ) before administration of the drug. reduced fg and crp levels from a baseline value were also observed in this group. however, concentrations were higher than in gvhd patients: fg . g/l (range . - . ) and crp . mg/dl (range . - ). in all patients, a differential diagnosis of disseminated intravascular coagulation was excluded. conclusions: . fibrinogen declines after tocilizumab therapy due to its cytokine-regulated production in the liver. coagulation monitoring should be performed during the first weeks after administration of the antibody to avoid serious bleeding complications. . crp concentrations remain low despite the presence of active infections following infusion of tocilizumab. crp fails as a marker of infection during weeks following the therapy. . tocilizumab is an effective therapy in patients with agvhd, especially with the gi involvement. disclosure: nothing to declare vanishing bile ducts after allogenic hsct: is it really gvhd? antonio grasso , lorenzo d'antiga , aurelio sonzogni , massimo gregori , alessandra maestro , roberto simeone , natalia maximova background: evaluation of liver gvhd was historically based by elevation of bilirubin levels and by reduction and degeneration of small bile ducts on histological samples of post-transplant liver biopsy. however, there is a lack of studies that compared histological finding of ductopenia between post-autologous hsct and post-allogenic hsct. studying severity of ductopenia following allogenic hsct, we aimed to demonstrate lack of correlation between ductopenia and clinical signs of liver gvhd. methods: we retrospectively collected a series of allogeneic hsct performed from to in the institute burlo garofolo. all patients undergo percutaneous liver biopsy in most cases at three months, one year and three or more years after hsct. indications for biopsy were alteration noted at weekly follow-up assessments of at least one clinical or laboratory marker of liver impairment or cholestasis. ductopenia was defined by number of portal tracts with no interlobular bile duct divided by the total number (severe if the ratio was less than . ). clinical gvhd was defined by nih consensus criteria results: our population involved % males and % females with oncological ( %) and non-oncological underlying disease ( %). clinical signs of liver gvhd were present in % of the patients (n= ), % with contextual intestinal involvement, % with cutaneous and intestinal involvement. patients underwent biopsy at a mean time of +/- days after hsct, patients underwent a biopsy at months after hsct and patients after three or more years from hsct. results of biopsies are showed in table . no difference in incidence of ductopenia were found between liver gvhd group and no gvhd. table] . table : incidence of ductopenia - months, months and or more years after hsct in total population, gvhd group and no-gvhd group] the group that not received chemotherapy prior the hsct had an overall incidence of ductopenia of % (severe ductopenia of %) statistically significative in comparison with the oncological underlying disease group ( % of ductopenia and % of severe ductopenia). furthermore, a little sub-group of patients extrapolated from our population received liver biopsy before hsct for diagnostic assessments: of the with an oncological underlying disease % already showed ductopenia, while no signs of ductopenia were found in the others with a nononcological disease. conclusions: there is no correlation between incidence of gvhd and histologically finding of ductopenia on liver biopsy. ductopenia may be caused in the first place by chemotherapy treatment received before hsct and myeloablative conditioning for hsct and it's not related with gvhd. this hypothesis is strengthened by the subgroup analysis of pre-hsct biopsy. background: second and third line therapies for steroid refractory acute graft versus host disease (agvhd) after allogeneic stem cell transplantation (asct) are still lacking. ruxolitinib, a selective januskinase / inhibitor could show high efficacy in agvhd, as well as extracorporeal photopheresis (ecp). here we report a single center experience of combining both therapeutic approaches in severe steroid refractory agvhd with additional analysis of immune status of these patients to elucidate direct effects of this treatment on immune response. methods: from june to february , patients ( . % male, . % female, median age: . years, r: - ) with steroid refractory agvhd of lower gi-tract after asct were treated with ruxolitinib and extracorporeal photopheresis as third, fourth or fifth line therapy. some patients showed additional agvhd of skin (n= ), liver (n= ) or upper gi-tract (n= ). all patients had an overall grade iii ( %) or iv agvhd ( %). steroid refractoriness was defined as no improvement in days or aggravation after days of steroid treatment.median start of ruxolitinib or ecp was day . after asct (r: - ). medianduration of ruxolitinib therapy was . days (r: - ) with a median start dosage of mg per day ( x mg; r: - mg). all patients started with ecp treatments per week with an individual reduction of treatment frequency. median number of ecp treatments was . (r: - ) with a median frequency of ecp therapy once a week (r: . - . ). cytomegalovirus (cmv) status of all patients and immune status of ten patients (lymphocyte count with cd + t helper lymphocyte and regulatory t cell count) were collected previously, after four weeks of starting combined treatment and four weeks after stopping the treatment. results: one-year estimated overall survival (os) of all patients was % with a median estimated os of days. patients died because of relapse of underlying disease, one of severe therapy refractory agvhd of lower gi tract and due to infection complications in agvhd refractory setting. overall response was . % (complete remission rate: . %, partial remission rate: . %). . % (n= ) of the patients had cytopenia ctc i-iii during the treatment, no grade iv cytopenia was reported. cmv reactivation during ruxolitinib occured in . % of cases (n= ). tapering of steroids could be performed rapidly with a medium reduction time of . days for reducing to half of the dosage.remarkably, regulatory t cells significantly increased during combined ruxolitinib/ecp treatment compared to regulatory t cell count before treatment (p= . ) and after stopping treatment, regulatory t cell count decreased again (p= . , see figure) . significant changes in whole lymphocyte count or in cd + t helper cell count were not observed. conclusions: treatment of severe steroid refractory agvhd with ruxolitinib plus ecp could show a high complete remission rate of . % with an one year os of %. detecting increased regulatory t cell count during the treatment underlines its direct effects on immune response and encourages to pursue this promising therapeutic approach. [ background: due to increased immunosuppression infections remain the main cause of death followed by higher risk of relapse in patients treated for acute graft versus host disease (agvhd) after allogeneic stem cell transplant (sct). here we report a single-centre experience with extracorporeal photopheresis (ecp) for acute gvhd that was introduced in order to reduce steroid treatment. comparison of overall survival (os) for patients on ecp and patients that received standard first line therapy for agvhd was performed. methods: we retrospectively analysed patients ( %) with acute gvhd grade ii-iv treated from january to october out of total allogeneic sct in that period. all patients received calcineurin inhibitors or sirolimus while receiving steroid treatment for agvhd. twenty-five patients ( %) received ecp with steroid lowering intent. we defined response as ( ) reduction of steroid dose for at least % from baseline while not adding another immunosuppresive agent and ( ) not repeating second steroid treatment if the ecp was started after lowering of steroids to prevent agvhd flare. we checked separately patient responsive and refractory/dependent to steroids. on average patients received ecp procedure once weekly. results: tapering of immunosuppressive therapy as defined was successful in ( %) out of patients in ecp group. in a group of patients without ecp ( %) patients had steroid refractory or steroid dependent agvhd compared to ( %) patients in ecp group. four ( %) patients with steroid refractory or dependent agvhd showed improvement in ecp group compared to only one ( , %) in non ecp group. twenty ( %) patients died due to infectious complication and ( %) due to relapse in non ecp cohort. in ecp cohort ( %) patients died due to infection and ( %) due to relapse. median os was months in non ecp group (r., - ) compared to months (r., - ) in ecp group and os of % at years in non ecp compared to % in ecp cohort was observed. patients with agvhd treated with ecp and faster steroid tapering had longer os compared to patients without ecp (p= , ). conclusions: ecp enables successful tapering or withdrawal of steroid therapy in many patients, even in those who are steroid refractory or steroid dependent. reduction of immunosuppression leads to reduced incidence of infection and relapse which translates into a better overall survival. background: the curative potential of allogenic stem cell transplantation is hampered by graft-versus-host disease (gvhd). pre-clinical study showed an efficacity of jak / inhibitor, ruxolitinib, in treatment of steroidrefractory gvhd. methods: we reported in this monocentric retrospective study, ruxolitinib response and follow up of cases of chronic gvhd (cgvhd) not improved with standard immunosuppressive therapy. complete organ response (cr) was defined as the resolution of clinical manifestations of cgvhd in a specific organ. very good response partial (vgpr) was defined as an improvement of clinical manifestations of cgvhd with more than % decrease of corticosteroid, while a partial response (pr) was associated with less than % decrease of corticosteroid. treatment failure was defined by the absence of improvement of cgvhd, deterioration of cgvhd in any organ by at least one stage, the development of cgvhd manifestations in a previously unaffected organ, and the use of any additional agents to control the disease. results: median age at transplant was years (range, - ). % of patients presented an acute myeloid leukemia. donor type was sibling (n= ), unrelated (n= ) or haploidentical (n= ).two patients benefited a cord blood transplant. patients received either myeloablative ( %) or reduced intensity ( %) conditioning regimens. stem cell source was peripheral blood for % of patients. patients presented mild (n= ), moderate (n= ) or severe (n= ) cgvhd according to nih score. median number of regimens prior to ruxolitinib was (range, - ), among those corticosteroids (n= ). median follow-up after ruxolitinib was months (range, - ). overall responses rate (orr) at month was % with % cr, % vgpr and % pr ( figure ). % of patients failed at month after introduction of ruxolitinib. the rate of cr increased with time : % at months (n= ), % at months (n= ) and % at months (n= ). but vgpr rate was rather stable at months ( %), at months ( %) and at months ( %) vs % at month. among the patients under steroids, ( %) patients discontinued steroids. the -months overall survival (os) and diseasefree survival (dfs) after ruxolitinib was % ( %- %, % ci) and % ( %- %, % ci), respectively. severe cytopenia (grade and ) was observed in patients. after introduction of ruxolitinib, patients presented bacterial infections, patients presented an invasive pulmonary aspergillosis and patient developped a pneumocystis. cytomegalovirus reactivation requiring preemptive treatment was observed in patients. no toxicities required withdrawal of ruxolitinib. [[p image] . figure and partial response to mesenchymal stem cells (msc), as second-line therapy, varies from % to % in acute gvhd patients. we report our experience using mscs to treat refractory agvhd. methods: the study was a retrospective single center study. all data were collected from patients' files. twenty patients were enrolled (age ranging from months to years) between april and april . results: five of these patients received reduced intensity conditioning and patients received myeloablative regimens before hsct. one haploidentical, autologous, cord blood, mud, msd transplantations were performed. the patients were eligible if they developed grades ii-iv agvhd. all patients were treated with standard first-line treatment with corticosteroids and at least one second-line therapy. the definition of steroid resistant agvhd considered as either no response to steroid treatment lasting at least days or progression during treatment of at least one grade within the first hours. prophylactic treatment with calcineurin inhibitors continued at therapeutic dose level. totally, doses of mscs were infused. the median dose of msc was . × cells per kg body weight. the median duration between the diagnosis of agvhd and initiation of mscs therapy was days (range: - ). the received msc doses ranged from one to seven. none of our patients had severe side-effects during infusions of mscs. overall, complete response (ocr) was obtained in patients, partial response in patients and no response (nr) was documented in patients. in our study group, the complete response rates in liver, gastrointestinal, skin agvhd were %, %, % respectively. four patients ( %) died in days after using mscs from complications of agvhd. eleven of patients ( %) were still alive with a median follow-up of days (range: - days) after first mscs infusion. one year estimated probability of overall survival for patients achieving ocr and partial remission/no remission in th day of mscs were . % and %, respectively. conclusions: in conclusion, mscs appears to be a safe and effective treatment option for pediatric patients with steroid refractory agvhd. disclosure: nothing to declare effect of extracorporeal photopheresis on production of serum elafin in chronic graft versus host disease arun alfred , charlotte burton , kathryn goddard , nichloas matthews background: extracorporeal photopheresis (ecp) is a second line therapy for steroid refractory, dependent or intolerant chronic gvhd (cgvhd). in order to guide ecp there is an unmet need for predictive and diagnostic biomarkers. elafin is a serine-protease inhibitor primarily produced by epithelial cells, particularly keratinocytes in inflammatory skin diseases and plasma and epidermal elafin have been identified as biomarkers of skin gvhd ( , ) . since skin cgvhd is noted for a particularly high response rate to ecp, we conducted a study to investigate whether ecp affects the production of elafin. methods: serum samples were collected from cgvhd patients ( male / female; age range: - ) and age-matched healthy controls ( male / female) before ecp and at month intervals up to year. patients had gvhd affecting skin ( / ), mucosal membranes ( / ), liver ( / ), joints ( / ), gut ( / ), eye ( / ), genital ( / ), and respiratory involvement ( / ). serum elafin was assessed by elisa (r&d systems). data were analysed using graphpad prism . statistical tests performed include -tailed mann-whitney, pearson's correlation test, and -way anova with repeat measures, as appropriate. results: chronic gvhd patients presenting for ecp had significantly elevated serum levels of elafin (p= . ; median of ng/ml, iqr - ng/ml) compared to healthy controls (median of ng/ml, iqr . - ng/ml).while % of patients had skin involvement, only % had elafin levels above the iqr of healthy controls. where disease scores were available (n= ) there were no significant correlations with modified rodnans (r= . ) or nih bsa scores (r= . ).sub-analysis was performed by grouping cgvhd patients according to quartiles of serum elafin at pre-ecp baseline. retrospective analysis of patients after months of ecp (n= ) revealed that those with serum elafin levels in the upper quartile (elafin hi ) pre-ecp (min-max: - ng/ml), showed a significant reduction after months of therapy (p< . ; mean +/-sd : ng/ml +/- ng/ml vs ng/ml +/- ng/ml, respectively), which was sustained up to months of ecp (p< . ; mean +/-sd: ng/ ml +/- ng/ml). in contrast, patients with elafin levels below the upper quartile (elafin lo ) showed no significant change (mean +/-sd: ng/ml +/- ng/ml vs ng/ml +/- ng/ml, respectively). of note, pre-ecp patients with elafin below the median received significantly more corticosteroid (cs) than those above, (p< . ; mean +-sd: +/- mg/d vs +/- mg/d, respectively), which was significantly reduced after months of ecp (p< . ; to +/- mg/d), while cs dose was not significantly changed in elafin hi patients until months (p< . ; mean +/-sd: mg/d +/- mg/d vs mg/d +/- . mg/d, respectively). conclusions: consistent with recent data, we found that serum elafin is significantly elevated in a subset of cgvhd patients compared to healthy controls, but did not correlate with skin disease scores. ecp administration was associated with a reduction in serum elafin in the elafin hi subset. further, elafin lo and elafin hi patients tolerated different rates of ecp-mediated tapering of cs immunosuppression suggests pre-ecp elafin measurements may have predictive value. references : background: allogenic hematopoietic stem cell transplantation (hsct) is a potential curative treatment for many malignant and no malignant hematologic diseases, primary immunodeficiencies and some metabolic and deposit diseases in children. graft versus host disease (gvhd) is a major cause of morbidity and a leading cause of non-relapse mortality. corticosteroids are the standard first-line systemic treatment for both acute and chronic gvhd, whereas no second line option for corticosteroid-refractory patients is standardised. ruxolitinib is a potent inhibitor of jak / showing significant responses in refractory gvhd patients in recent reports. methods: we present two centres experience with ruxolitinib for gvhd treatment in pediatric patients. the study was conducted in two spanish pediatric hsct centres, hospital vall d'hebron (barcelona) and hospital universitario la paz (madrid). all patients receiving ruxolitinib since the drug was available were included for retrospective analysis. results: between march and december pediatric patients with acute or chronic gvhd with refractoriness to corticosteroids were treated with ruxolitinib, in different episodes (one patient received it in different moments, and one patient received it in ). patient's sex at birth was female in and male in cases. median age at hsct was , years ( , - , ). primary disease was malignant in patients and non malignant in . median time of gvhd diagnosis was , days ( - ). all gvhd episodes were treated with corticosteroids as first line, with maximum doses between - mg/kg/day (the main dose used was mg/kg/day, / episodes). patients received a median number of , ( - ) previous lines of treatment including steroids before starting ruxolinib; they were extracorporeal photopheresis ( / episodes), sirolimus ( / ), mesenchymal cells ( / ) ruxolitinib initiation was indicated for acute gut refractory/steroid dependant gvhd in episodes and chronic multisystemic in episodes. other indications were chronic lung ( / episodes), chronic skin ( / ) and acute skin gvhd ( / ) . median post-hsct time of ruxolitinib start was days. doses ranged between , - mg/ h depending on age, weight, and tolerance (hematologic and liver toxicities). average duration of treatment was days ( - ). complete response (cr) rate was , %, global partial response (pr) , %, and no response (nr) % (progression in one patient and recent treatment start in other patient). mean time to maximum response was weeks. treatment stop cause was cr in cases, infection in , liver toxicity in . no severe side effects directly related to ruxolitinib treatment were described. conclusions: ruxolitinib has been recently introduced as second line strategy for rescuing corticosteroid-refractory gvhd in pediatric patients. while results of randomized trials are lacking, we present our experience (two centres). the main indications for starting treatment were acute gut and chronic multisystemic gvhd. most patients achieved some grade of response (partial or complete), allowing stopping or tapering corticosteroids. toxicity profile appears to be acceptable. disclosure: nothing to declare. stability of tacrolimus concentration early after allogeneic hematopoietic stem cell transplantation reduces the risk of acute gvhd background: tacrolimus is used as an immunosuppressive drug after allogeneic hematopoietic stem cell transplantation (allo-hsct). it is well known that early concentration level of tacrolimus is correlated with the risk of acute graft versus host disease (agvhd), however, whether range of standard derivation (sd) of early tacrolimus concentration after allo-hsct also affect to the risk of agvhd still remains unknown. here, we investigate the correlation between the range of sd of early tacrolimus concentration after donor hematopoietic cells engraftment and the development of agvhd. methods: we retrospectively assessed patients who underwent allo-hsct in our hospital from - . all patients received standard gvhd prophylaxis by continuous intravenous (iv) tac with starting dose of . mg/ kg/day from day before allo-hsct (day - ) and iv methotrexate on day , , at dose of mg/m , mg/m , mg/m , respectively. tac dosage was adjusted to target the serum concentration of - ng/ml until at least day and then tapered. to evaluate the sd of weekly tacrolimus concentration, the range of sd of tacrolimus concentration at day - (week- ), day - (week- ), day - (week- ) and day - (week- ) were calculated. the difference of the range of sd between the groups that develop or did not develop agvhd was compared by using mann-whitney u test. multivariate analysis was performed by using multiple logistic regression analysis. patients had given written consent allowing the use of medical records for research, in accordance with the declaration of helsinki, and the institutional review board approved the study. results: there were males and females and the median age was years (range, to years). the risks of disease were low-standard in and high in pts. the number of donors were in hla-identical sibling, in hla-mismatched related donor, in hla-matched unrelated donor and in hla-mismatched unrelated donor. thirty-seven patients developed agvhd (grade i-ii; , gradeiii-iv; patients). as a result, the wide range of sd at week- significantly increased the risk of agvhd (agvhd-group; . ± . ng/ml, agvhd+ group; . ± . ng/ml, p= . ). multivariate analysis demonstrated that narrow range of sd of tacrolimus concentration at week- reduce the risk of agvhd (or= . ; % ci: . - . ; p= . ). there were no correlation between gender, age, disease status, hla with the development of agvhd. conclusions: the range of sd at week- , an engraftment phase of donor hematopoietic cells, was significantly correlated with the development of agvhd. fine tuning of early tacrolimus concentration with narrow range of sd reduces the risk of agvhd, resulting in improvement of the overall survival after allo-hsct. disclosure: nothing to declair p ruxolitinib treatment for steroid-refractory graftversus-host disease han-seung park , je-hwan lee , jung-hee lee , eun-ji choi , miee seol , young-shin lee , young-ah kang , mijin jeon , kyoo-hyung lee background: steroid-refractory graft versus-host disease (gvhd) is one of the most lethal complications after allogeneic hematopoietic cell transplantation. recent studies have shown that ruxolitinib, a janus kinase / inhibitor, is effective in patients suffering from gvhd. here, we report a retrospective result of ruxolitinib treatment for steroid-refractory gvhd. methods: all patients had received cyclosporine and a short course of methotrexate as gvhd prophylaxis. antithymocyte globulin was added for unrelated or mismatched familial donor hct. ruxolitinib mg twice daily was added to immunosuppressive treatment in patients with steroid-refractory gvhd. results: a total of patients with gvhd (acute, , including patients with donor lymphocyte infusion [dli]related; and chronic, ) were included in the analysis. all patients had grade / acute gvhd or severe chronic gvhd at the time of ruxolitinib treatment. six ( . %) of patients with acute gvhd responded to ruxolitinib, including with complete response (cr). the median time to response was . days (range, - ) . nineteen patients received ruxolitinib for severe chronic gvhd, with the median of involved organs (range - ). fourteen patients ( . %) showed response to ruxolitinib, including crs. the median time to response was days (range, - ). five responders discontinued ruxolitinib and patients are still on the agent. after a median follow-up duration of . months, died ( from relapse of disease, from infection). the -year survival probability was . %. eleven of responders discontinued ruxolitinib. gvhd relapsed in of patients at , , and days after ruxolitinib discontinuation. thrombocytopenia ( / , grade / ; ) was the most common adverse event of ruxolitinib. during treatment, with grade / infectious adverse events occurred; pneumonias, brain abscess, and liver abscess. conclusions: ruxolitinib treatment seems to be effective for the treatment of steroid-refractory gvhd including long-standing chronic gvhd. the agent was well tolerated and relatively safe. disclosure: nothing to declare. il -receptor antibody tocilizumab as salvage therapy in the treatment of severe chronic gvhd after stem cell transplantation: a retrospective analysis background: severe chronic graft-versus-host disease (cgvhd) remains the most relevant factor affecting survival and long-term quality of life after allogeneic hematopoietic stem cell transplantation (hct). besides corticosteroids there is no established therapy for cgvhd and many of the used immunosuppressive agents may lead to significant toxicity incl. infectious complications. tocilizumab (an il -receptor antibody) has shown efficacy in acute gvhd and cgvhd. we retrospectively analyzed the efficacy and safety of patients having received tocilizumab for treatment of advanced cgvhd at our center between the years and . methods: patients with severe steroid refractory cgvhd and a median age of years (range: - yrs) having received at least two prior lines of therapy for cgvhd (range: - regimens) were treated with tocilizumab for at least one cycle (q w, dosage: mg/kg iv, maximum: mg) with a median number of cycles (range: . nih consensus criteria grading for cgvhd and the immunosuppressive regimen were noted at the time of the first tocilizumab administration and after , and months of therapy. all patients received additional concomitant immunosuppressive agents already given at least weeks without response before start of tocilizumab. no new immunosuppression (is) was added in parallel to tocilizumab and response assessment was stopped at start of any additional new is. all patients had received peripheral stem cell allografts. gvhd prophylaxis consisted of a calcineurin inhibitor in combination with methotrexate or mycophenolate and in case of unrelated donors atg was added. / patients had quiescent onset of cgvhd, one patient developed de novo cgvhd. the median number of days between hct and onset of cgvhd was ( - ). the median number of days between hct and initiation of tocilizumab therapy was ( - ) days. at cgvhd onset, / patients had mild cgvhd and / patients had moderate cgvhd. the thrombocyte count was < /nl in / patients. organs involved at initiation of tocilizumab therapy were skin ( %, all grade ), eyes ( %), mouth ( %), fascia ( %), lungs ( %) and genitals ( %). / patients are still receiving tocilizumab at the time of analysis. results: as tocilizumab was given fairly recently in most patients, -and -month follow-up was only reached in / patients ( %). at three-month follow-up after initiation of tocilizumab therapy, / patients ( %) showed partial remission, / patients stable disease ( %), and / patients progressive disease ( %) of cgvhd. maximal response was partial remission ( %), stable disease ( %) and progressive disease ( %). patients required subsequent new immunosuppressive treatment. one patient has not yet reached -month follow-up. during tocilizumab therapy none of the patients suffered recurrence of underlying malignancy. two patients developed significant respiratory infection and one patient developed soft tissue infection, all requiring antibiotic treatment and pausing of tocilizumab administration, hospital admission was not required. the os and rfs was % with median follow up of . months (range - months). conclusions: tocilizumab appears to be a promising treatment option in advanced cgvhd but further evaluation within a phase ii trial is required. disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (hsct) is for many patients suffering from aml the only curative treatment option. one major complication is graft versus host disease (gvhd), caused by donor immune cells attacking healthy tissue. regulatory t cells (treg) have been getting huge attention during the past years because of their important role in maintaining immune balance. here we collected peripheral blood samples from patients at different time points after hsct to investigate immune-reconstitution of treg as predictive marker for the development of gvhd. methods: we collected blood samples from patients in the course of allogeneic hsct prospectively once a week from d+ up to d+ . all patients received conditioning regimen with fludarabine and melphalan, combined with alemtuzumab for t cell depletion. patients developed acute gvhd in the later course. after isolation of pbmc`s we performed facs multicolor staining of t cell and nk cells. treg were identified as cd + cd + cd ++ foxp + , nk cells were characterized as cd neg cd + cd + and divided in nk cell subpopulation due to their expression of cd dim or cd high . results: . cd neg t cells: all patients developing acute gvhd in the later course showed significant elevated levels of cd + cd neg t cells, especially cd neg treg at d+ . . cd neg treg / cd + cd + t cells: one patient not developing acute gvhd showed lots of cd neg treg but missed cd + cd + effector t cells. we recently showed that cd + cd neg effector t cells are of impaired effector function. these data suggest that cd neg treg are only of relevance combined with functional cd + cd + effector t cells in the development of agvhd. . t cell marker: patients without agvhd showed elevated expression of garp on treg. garp was significantly higher expressed on cd + treg, indicating a better suppressive capacity of cd + treg. this was detected throughout from d+ until d+ . tigit and ilt showed a heterogeneous expression profile without significant differences between the two groups. . nk cells: we detected a higher ratio of cd ++ /cd dim nk-cell population in patients without. we could also show that tigit is mainly expressed on cd dim nk cells. conclusions: we and others showed reconstitution of cd neg t cell subsets after alemtuzumab mediated t cell depletion -our data on effector t cells showed an impaired effector function for cd neg cd and cd t cells. recently we presented data on impaired suppressive capacity of cd neg treg and the association with acute gvhd retrospectively (wölfinger ebmt , ash ). here we provide prospective data on patients after the use of alemtuzumab in the context of hsct: our preliminary data suggest that the total amount of cd neg-treg and the ratio of cd neg treg to cd + cd + treg on d+ after allogenic hsct could predict agvhd. this data may be a basis for immune monitoring of patients at d+ to evaluate their risk for agvhd and could lead to the use of prophylactic treg dli in the context of alemtuzumab mediated t cell depletion. disclosure: medac -travel support, novartis -consultancy fee, pfizer -consultancy fee, shireconsultancy fee background: multiple factors such as disease activity and severity, therapy and/or dietary habits can cause changes in nutritional status independently or by interacting with each other. presence of malnutrition or significant weight loss in chronic gvhd (cgvhd) patients was reported in literature up to %. the aim of this cross-sectional study was to identify factors that affect nutritional status in cgvhd patients. methods: nutritional status in patients with cgvhd treated at the university hospital center zagreb, croatia from to was assessed. anthropometric measurements (height, body weight (bw), body mass index (bmi)) and clinical validated tool patient-generated subjective global assessment (pg-sga) (where patients were categorized as well-nourished (pg-sga a), moderately malnourished (pg-sga b) or severely malnourished (pg-sga c)) were used. all patients were evaluated according to nih criteria for cgvhd diagnosis. descriptive and correlation analysis were preformed. results: in total, adult cgvhd patients were included in the study, women ( . %), median age ( - ) years, with mild cgvhd in ( . %), moderate in ( . %) and severe in ( . %) patients. according to the pg-sga rating ( . %) patients had pg-sga a, ( . %) pg-sga b and ( . %) had pg-sga c, giving a total malnutrition or risk of malnutrition prevalence of . %. the mean bmi was . ± . kg/m with correlation to pg-sga rating (r= . , p= . ). malnutrition according to the bmi (defined as bmi< kg/m ) was found in patients ( . %). bw changes ( % or more in months) were significant in patients ( . %). according to the pg-sga assessment tool, oral symptoms reported by patient ( . %) and decreased appetite reported by patients ( . %) were associated with oral cgvhd nih score (r= . , p= . ; r= . , p= . ) but not with bw or bmi. gastrointestinal (gi) symptoms assessed with sga, were generally mild with no correlation to gi cgvhd nih score. no significant association was found between nutritional status and other nih cgvhd scores. corticosteroid therapy present in ( . %) correlated with pg-sga rating (r= . , p= . ) but not with bw, bmi or appetite changes. in patients ( . %) with altered pg-sga rating, bmi, appetite and body weight changes, dietary counseling and oral nutritional supplementation were initiated. conclusions: oral symptoms, decreased appetite and corticosteroid therapy in our cgvhd patients were associated with altered nutritional status according to the pg-sga, but not with bmi. therefore, pg-sga might be a more sensitive tool in assessment of changes of the nutritional status and detection of patients at risk of malnutrition than bmi since it includes different factors like physical examination, presence of gi symptoms and corticosteroid therapy in its scoring system. nutritional counseling and support are important in cgvhd patients especially in presence of oral symptoms. disclosure: nothing to declare. background: sclerotic skin changes are common features in chronic graft versus host disease (cgvhd). one of the most challenging aspects in the diagnosis and management of sclerodermoid cgvhd (scgvhd) is the differentiation between reversible symptoms related to active cgvhd and nonreversible symptoms related to residual permanent damage such as long-standing fibrosis. although several candidate biomarkers of cgvhd inflammatory activity have been proposed, none of them are currently validated. therefore, there is a need for the development of more quantifiable and reproducible measurements tools to guide clinical decisions. we report our experience evaluating the usefulness of high-frequency ultrasonography (hfus) plus doppler ultrasound (doppler-us) and serum fibrosis biomarkers to determine the inflammatory activity of scgvhd. methods: we report patients with scgvhd. hfus plus doppler-us were performed at diagnosis of scgvhd and at different time-points after treatment initiation. serum hyaluronic acid and pro-colagen-iii were measured as fibrosis biomarkers simultaneously with hfus and doppler-us. nih cgvhd consensus conference diagnosis criteria, scoring system, and response criteria were used to assess global and organ-specific cgvhd, and to measure overall response to therapy. abnormal ultrasound findings were defined as the presence of ≥ of the following: hypoechogenic dermis, dermo-epidermal junction effacement, hypoechogenicity of septa and/or hyperechogenicity of lobules in hypodermis, hypoechogenic fascia, or myositis, for hfus; and, vessels thicker than mm in dermis and/or hypodermis, systolic pressure > cm/sec, and index of vascular resistance > . , for doppler-us. inflammatory activity was classified as mild, moderate and severe according to the severity of doppler-us findings. results: hfsu showed abnormal findings in all patients at diagnosis with no changes except in two patients along the treatment follow-up. inflammatory activity by doppler-us was observed in / patients at diagnosis ( mild, moderate, severe). four patients responded to treatment ( complete responses, cr, and partial responses, pr), one presented clinical improvement less than pr, and one, progressive disease. all patients with clinical response had also a p-rom improvement or normalization. all patients achieving a response showed normalization (n= ) or improvement (n= ) of doppler-us findings. the patient with clinical improvement less than pr and the patient with progressive disease showed persistence of inflammatory doppler-us findings. most patients had normal or light increase of pro-collagen levels at diagnosis and no significant changes were observed during follow-up. levels of hyaluronic acid tended to be very high in patients with progressive scgvhd (patients and ) and tended to decrease or normalize in those who responded to therapy (patients , , and ). conclusions: in this exploratory study, hfsu was a reliable method for evaluating sclerotic skin changes in scgvhd. doppler-us showed a good correlation with disease activity and response to treatment. serum hyaluronic acid levels might be a biomarker of disease activity that deserves further investigation. hfsu plus doppler-us is a useful, non-invasive, repeatable device in monitoring patients suffering from scgvhd. according to our results, doppler-us may be a more sensitive parameter than hfsu in assessment inflammatory activity of scgvhd. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare post-transplant cyclophosphamide versus antithymocyte-globulin in hla-matched unrelated and haploidentical transplantation for hematologic malignancies background: post-transplant cyclophosphamide(ptcy) and antithymocyte-globulin(atg) are the most commonly used regimens for the prophylaxis of graft-versus host disease(gvhd). we compared these two regimens in hlamatched unrelated (mud) and haploidentical transplantation for hematologic malignancies. methods: we retrospectively analyzed the consecutive adult patients with hematologic malignancies who received mud and haploidentical transplantation at chungnam national university hospital between january and january . patients who received second transplantation and had refractory disease were excluded. results: this study included patients with median age of (range, - ) years: ( . %) patients received mud transplant ( and patients in ptcy and atg group, respectively), and ( . %) patients received haploidentical transplant ( and patients in ptcy and atg group). graft source was peripheral blood stem cell in all patients. median follow-up duration was . months (range, . - . ). in mud transplant, the estimated -months survival rate were . % in ptcy vs. . % in atg (p= . ), the -months relapse rate were . % in ptcy vs. . % in atg (p= . ), the cumulative incidence of grade to acute gvhd were . % in ptcy vs. . % in atg (p= . ), and the estimated -month extensive chronic gvhd rate were . % in ptcy vs. . % in atg (p= . ). in haploidentical transplant, the estimated -months survival rate were . % in ptcy vs. . % in atg (p= . ), the -months relapse rate were . % in ptcy vs. . % in atg (p= . ), the cumulative incidence of grade to acute gvhd rate were . % in ptcy vs. . % in atg (p= . ), and the estimated month extensive chronic gvhd rate were . % in ptcy vs. . % in atg (p= . ). patients receiving ptcy had significantly longer neutrophil engraftment time than those receiving atg in haploidentical transplant [median(range); . ( . - . ) days vs. . ( . - . ) days, p= . ]. conclusions: ptcy might be a good option for the prophylaxis of gvhd in hla-matched unrelated transplant as well as haplo-identical transplant. disclosure: nothing to declare early fam therapy for post allo-hsct bronchiolitis obliterans syndrome background: bronchiolitis obliterans syndrome (bos) is a potential major complication after allogeneic hematopoietic stem cell transplantation (hsct). attributed to an allo-immune reaction against the small airways, bo is considered a pulmonary manifestation of chronic gvhd. reported incidence of bos ranges from to %, and bos-attributed mortality as high as %- %. a few years ago, a new therapeutic approach with fluticasone, azithromycin, and montelukast (fam) was described (norman bc, et al. bmt ) . our aim was to analyze the outcomes of pts who developed bos and were precociously treated with the fam scheme. methods: all the allo-hsct performed in our center from january and july were included in the analysis. baseline diseases were: aml, lpd, mds, all, mpd, mm, and bmf. day + and day + overall mortality were , % and , %, respectively. rest of characteristics of the series are shown in table. fam therapy was systematically started when any patient was first diagnosed with bo. results: eleven patients ( , %) were diagnosed with bos. at diagnosis of bos, the pts exhibited a fev % of predicted (median fev : %; range; - %) and/or a decline > % from pre-hsct . at day + , pts had already the syndrome. two of them died before the end of the first year: one due to invasive zygomycosis (cns plus pulmonary) and the other to baseline disease progression. at day + , more pts had bos. two more pts with bos died at and months post-hsct due to baseline disease progression. at the close of the analysis, of the pts were alive. so, with a median follow-up of months (range: - ), mortality and bos-attributable mortality of the pts with the complication were , % and , %, respectively. conclusions: ) bos is an infrequent but very severe complication of allo-hsct; ) bos seems to be less frequent in pts with prophylactic pre-transplant ratg or post-transplant cyclophosphamide, as well as in pts undergoing transplantation with bm (compared to pbsc). ) early diagnosis and therapy are critical to minimize the bos-attributable mortality. disclosure background: donor lymphocyte infusion (dli) is an established treatment for patients with hematological malignancies relapsed after allogeneic hematopoietic stem cell transplantation (hsct). however, it is associated with an increased risk of graft-versus-host disease (gvhd) and modest anti-tumor activity. compared to the infusion of nonmobilized lymphocytes, granulocyte colony-stimulating factor (g-csf)-primed dli might induce a stronger anti-tumor effect and reduce the risk of infusion-induced gvhd. due to the limited experience of g-csf primed dli in patients relapsed after haploidentical hsct, we conducted a retrospective study of all patients at our hospital who received dli for the relapsed hematological diseases following related hla-matched or hla-haploidentical hsct. methods: the institutional research board approved the study. we identified patients with hematological malignancies receiving dli following related allo-hsct at national taiwan university hospital between and aug. the infusate was obtained from the cryopreserved specimen, which had been collected and stored at multiple aliquots at the same time as the initial haploidentical peripheral stem cell graft. patients received dli for either hematological relapse, preemptive or prophylactic treatment. univariate and multivariate analysis was performed using cox proportional hazard regression model. results: for the patients following related hlamatched and the patients following hla-haploidentical hsct received and doses of dli, respectively. in comparison, the median cd + cell dosage of haplo-dli is significantly lower (p = . ) than that of dli from sibling donors, with median cell dosage . × /kg (range, . - × /kg) and . × /kg (range, . - . × /kg), respectively. the median time to dli from initial sibling hsct and haplo-hsct was days (range, - days) and days (range, - days), respectively. overall, ( %) of the patients following sibling hsct developed grade - acute gvhd after dli, whereas ( %) of the patients receiving haplo-hsct developed grade - acute gvhd after dli (p= . ). importantly, for patients receiving dli with cd + cell dosage less than × /kg, there is no difference in the risk of developing grade - acute gvhd between patients receiving dli from sibling or haplo donors ( figure a) . interestingly, for patients receiving dli with cd + cell dosage more than or equal to × /kg, ( %) of the patients following haplo-hsct developed grade - acute gvhd after dli, significantly more than ( %) of the patients following sibling hsct developed grade - acute gvhd after dli ( figure b) . the cumulative incidence of grade - acute gvhd at day after haplo-hsct and sibling hsct were % ( % ci: . - . ) and . % ( % ci: . - . ), respectively ( figure b , p = . ). [[p image] . conclusions: our study shows that the administration of g-csf mobilized dli is feasible after haploidentical hsct for relapsed hematological malignancies. however, dli with cd + cell dosage more than or equal to × /kg in patients receiving haplo-hsct is associated with significantly higher risk of developing acute gvhd than dli from the sibling donors. disclosure: the authors declare no competing financial interests. background: the fresenius phelix is a uva irradiation device used to photoactivate mnc collected on the amicus. the system is closed, utilizing a special mnc kit and modified instrument software. the preliminary results of a phase i safety trial involving three patients ( treatments) with chronic graft vs. host disease are presented. methods: reasons for transplantation for the patients ages , and years were: acute myelogenous leukemia, myelodysplastic syndrome, and myelodysplastic syndrome with pnh. stem cell source was peripheral blood with a / match for all. each developed chronic skin gvhd. inclusion criteria included wbc and plt counts > and x /l, gfr > ml/min/bsa, and ast - unit/l. exclusion criteria included active gi bleeding, nyha cardiac disease greater than grade iii, and the presence of light-sensitive diseases. amicus software . and phelix software . were used. settings included: ml/min max draw rate, ml fixed cycle volume, . mg/kg/min citrate infusion rate, and : acd-a ratio. venous access was peripheral or subcutaneous port. target uva dose was . j/cm and -methoxypsoralen dose was . ml. results: the following mean + sd procedure results were obtained: , + ml whole blood with acd-a drawn, + ml acd-a used, + ml saline used, + minutes procedure time, and , + ml total blood volume. minor alarms (n= ) on the amicus and no alarms on the phelix were encountered. all -day aerobic and anaerobic cultures were negative and mean endotoxin levels were . + . eu/ml. mean pre/post cbc and plasma hemoglobin levels were: . / . wbc, . / . neutrophils, . / . basophils, . / . eosinophils, . / . lymphocytes, . / . monocytes, / platelets x /l, / % hct, . / . g/dl hgb, and . / . mg/dl plasma hemoglobin. plasma hemoglobin delta in the product was . + . grams and the subject was - . + . grams. collected product hct. mean . + . %. yields are in the table. adverse events included one each: acute respiratory failure, respiratory failure, muscular weakness, musculoskeletal discomfort, and peripheral swelling. three of four events occurred in one patient two weeks after the study procedure. none of the adverse events were considered related to the procedure or investigational product. the patient who experienced acute respiratory failure was removed from the study because of death due to pneumonia, felt to be unrelated to the procedure. conclusions: results indicate the new closed photopheresis system is capable of collecting sufficient mnc and irradiating the cells producing high lymphocyte apoptosis, with minimal alarms and adverse reactions. ( . %)). ( . %) of the patients also had acute gvhd of the skin or liver. patients ( . %) could be treated and controlled with methyl-prednislone monotherapy, patients had steroid refractory gvhd of whom patients ( . %) could be salvaged with additional drugs (infliximab: ; tacrolimus: ); patients ( . %) had refractory acute gut gvhd and could not be salvaged despite more than three lines of therapy. at the time of reporting, patients ( . %) of the are alive. patients died due to transplant related mortality, while patients developed relapsed disease. on binary logistic regression analysis, no baseline clinical or treatment related predictor (disease indication, disease status at transplant, transplant type, graft source, type of conditioning) could be identified for developing acute gvhd of the gastrointestinal system. conclusions: acute gvhd of the gastro-intestinal system is a significant cause for morbidity in allo-hct patients at our centre. further studies are warranted in our cohort, and a prospective analysis of gut microbiome analysis, faecal multi-drug resistance organism surveillance, conditioning related toxicity and antibiotic usage is ongoing. clinical trial registry: not applicable disclosure: the authors declare no potential conflicts of interest benefits and precautions of ruxolitinib in steroidrefractory acute gvhd background: corticosteroids are the standard first-line treatment option for patients with acute graft-versus host disease (gvhd), but approximately half of patients become refractory to steroids and require second-line treatment. ruxolitinib has the potential to treat gvhd in steroidrefractory (sr) patients based on retrospective clinical data. the ongoing prospective trials are currently enrolling patients to evaluate the therapeutic potential of ruxolitinib for gvhd. methods: we analyzed retrospectively clinical experience with ruxolitinib in patients (n= ) with grade ~ steroid-refractory acute gvhd patients compared with the control group not receiving ruxolitinib. in addition, immune status was evaluated about weeks~ weeks after the administration of ruxolitinib using flow-cytometry. ruxolitinib was used as a third option for sr gvhd, combined with previously used immunosuppressive drugs. and steroids were gradually decreased according to the symptoms and discontinued. patients received ruxolitinib mg twice daily (bid), with increase to mg bid if hematologic parameters are stable and no treatmentrelated toxicities. results: fifteen patients all were assessable for response. seven patients achieved a complete response, had a partial response, and had no response at weeks after the first ruxolitinib dose. overall response rate was %. three were treatment failures. most adverse effects were manageable, except infectious complications. infectious complications were occurred in about % patients (n = ), resulting in two deaths. common cause of infectious events included cytomegalovirus (n = ), herpes-zoster (n= ), epstein-barr virus (n= ), fungal infection (n = ), pneumocystis jiroveci (n = ), bacterial infections (n = ), and pneumonia of unknown origin (n = ). t cell counts tended to decreased in the group with ruxolitinib compared with the control group, especially cd cell counts. conclusions: ruxolitinib is effective in controlling sr gvhd and can lead to clinical benefits. however, we need to be aware of the infectious complications because ruxolitinib may lead to increased risk of opportunistic infections or reactivation of latent infections. in addition, common infectious complications are presumed to involve t cell dysfunction. clinical background: graft versus host disease (gvhd), being one of most common life-threatening complication post hsct, contributes significantly to morbidity and mortality. when affecting gastrointestinal tract (gi) it is the major cause of death in early period post hsct. due to widespread tissue involvement in most patients diagnosed with gi gvhd, surgical treatment is rarely considered. methods: among allo-hsct performed in department of pediatric hematology, oncology and bone marrow transplantation in wroclaw, poland during years - , ( , %) cases were diagnosed with gi gvhd. in this study we present cases ( %) which were referred to and benefit from surgical approach. results: . male, years old underwent hsct from matched unrelated donor (mud) due to chronic myelogenous leukemia (cml) and subsequent molecular relapse succesfully treated with donor lymphocyte infusion, followed by agvhd (skin and gut involvement, grade iv). extensive immunosuppression (steroids, mycofenolate mofetile, atg, okt ) resulted in significant resolution of agvhd symptoms. however aggravating severe abdominal pain and lack of gut movement suggesting bowel obstruction. due to presence of acute abdomen patient was immediately directed for laparotomy. resection of constricted bowel segment followed by subsequent laparotomies for secondary obstruction provided complete resolution of abdominal symptoms. after years of follow-up patient is alive and well. . eleven years old male was diagnosed with skin and gut grade iv agvhd on day + post mud-hsct performed due to acute myelogenous leukemia (aml). he received pronlonged immunosuppressive treatment including steroids, antibodies, msc and ecp which led to resolving of skin leasions and diarhoea. nevertheless patient was suffering from severe paroxysmal abdominal pain and incidentally vomiting. ct enterography showed partial small bowel constriction. after numerous surgical consultations, eventually on day+ patient underwent laparotomy with constricted bowel resection. histopatological examination of resected tissue revealed moderate gvhd. immunosuppersion was tapered to low dose of steroids with ecp. for now, years post hsct patient is alive, rarely experiencing mild abdominal cramps . fourteen years old female developed severe abdominal pain and high volume diarhoea on day + post mud-hsct performed for severe anaplastic anemia (saa). despite extensive immunosuppression (steroids, anti-tnf, anti-il antibodies) patient condition did not improved. through consistent stomach pain, suspected subileus confirmed by ct enterography, laparotomy was performed (day+ ). resection of inflamated and obstructed bowel was made. microscopic evaluation confirmed prior gvhd diagnosis therefore immunosuppression including csa and tapered doses of steroids was continued. complete resolution of abdominal symptoms was almost immediately achieved post-surgery, however months after recurrent abdominal cramps were observed and are now well controlled by pain killers. conclusions: commonly gi gvhd is diffused inflammatory process. however in some cases it may be localized and may lead to partial bowel constriction. in case of severe and prolonged stomach pain, despite of partial resolving of other gvhd symptoms, ileus should be considered. ct enterography may be useful for diagnosis confirmation. in those patients, surgical intervention may improve quality of life or even be a salvage approach. disclosure: nothing to disclose is there any impact of the uric acid levels during the preand early post-graft infusion period, on the gvhd occurence and allotransplant outcome? . ( . - . ) years, who underwent allogeneic stem cell transplantation (allosct) from full-matched sibling donors for acute leukemia (n= ), very severe aplastic anemia/pnh (n= ), lymphoma (n= ), myelodysplastic/ myeloproliferative syndrome (n= ) . thirty-two patients were in remission at the time of allosct (cr : , cr : , beyond cr : ). for a better and more accurate assessment of the ua levels on the agvhd incidence, unlike to the other published studies which evaluated the ua levels only at day , we evaluated the ua levels in different time points during the the peri-transplant period (at the conditioning regimen initiation, and at days , + and + ). because the majority of our patients developed agvhd within the - days post-transplant, we did not incorporated in the study the of ua levels beyond the + day. we also investigated the effect of the ua on survival and the non-relapse mortality (nrm). the vast majority of patients received allopurinol from the st day of conditioning regimen till day - . the independent t-test, kaplan-meir method and logrank test were used in the statistical analysis. results: the median ua levels were . , . , . and . mg/dl at days - , , + and + respectively. for the statistical analysis purposes, we grouped our patients as low-ua if they had values < mg/dl or high-ua if they had > mg/dl. this threshold was chosen based on the ua values from all the collected samples (n= ). finally / ( %) patients developed agvhd; ( %) were assessed as gr ≥ii, while ( %) as gr iii-iv. the incidence of the agvhd gr ≥ii was similar (ranged from - %) in both groups of patients (low-ua and high-ua) and for all the estimated time points (days - , , + , + ). we noticed a better -years overall survival for patients with low-ua ( % vs. %) however without any statistical significance. ten patients succumbed to nrm causes; / deaths attributed to gvhd complications. the nrm was assessed higher in the high-ua group ( % vs. %) but also this difference was not statistically significant. conclusions: though our study bears the limitations of the small number of patients and the retrospective origin, at least to our knowledge is the first which evaluates the impact of ua levels at different time points in the peritransplant period, on the agvhd incidence. in our study the ua levels did not influence the incidence or the severity of agvhd. the higher nrm rates for patients with ua> . mg/dl merits further evaluation. definitely, the role of ua on the allosct outcome will be clarified through well designed prospective trials. disclosure results: five male patients ( %) had genital cgvhd manifestations presented by urethral stricture in / patients and phimosis requiring surgical treatment in one patient. all five patients had simultaneously cutaneous, oral, and/or ocular cgvhd manifestations. the first patient underwent urethroplasty of bulbomembranous part of urethra with termino-terminal anastomosis and urethroplasty of penile part of urethra with buccal mucosa autograft -bmg (dorsal onlay) that resulted in significant improvement of symptoms and normal miction afterwards. biopsy of the urethra showed mononuclear infiltration in lamina propria consistent with cgvhd. biopsy of the buccal mucosa was done prior to surgery and was negative for cgvhd involvement. the second patient underwent urethrotomy due to circular strictures, but symptoms reappeared again and he is now candidate for bmg. in two patients urethral dilatation was done, and the fifth patient presented with phimosis requiring circumcision, resulted in significant improvement of symptoms. conclusions: male genital cgvhd is an underrecognized and under-reported manifestation. patients after allo-hsct need to be actively asked about their genital symptoms and sexual function, especially if they are diagnosed with other mucocutaneous or ocular cgvhd. multidisciplinary approach, early recognition and frequent follow-up is necessary for timely start of treatment. new methods, such as bmg for cgvhd patients with urethral stricture seem promising and should be further investigated. disclosure: nothing to declare. p abstract withdrawn. heracles: a phase ii single-arm prospective study to assess the efficacy of fecal microbiota transfer in the treatment of steroid refractory gastro-intestinal agvhd post allo-hsct background: steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with an % mortality rate and reduced quality of life (qol). so far, there is no approved standard of care for agvhd second-line treatment. there is an urgent need to identify effective therapy for sr-agvhd to improve patients' outcomes. fecal microbiota transfer (fmt) might be beneficial to substantially improve the prognosis. higher gut microbial diversity is strongly associated with increased survival in gvhd patients. recent studies reported promising results of sr-agvhd patients treated with fmt. further evaluation to confirm the efficacy and safety of fmt for agvhd is warranted. the ongoing phase study (heracles) investigates the efficacy of allogeneic fmt in the treatment of patients with sr-agvhd. heracles was launched after the odyssee study showed promising results in the reconstruction of gut microbiota diversity after induction chemotherapy with fmt in acute myeloid leukemia patients. we expect that fmt-based biotherapeutic drugs could be effective treatments to contain sr-agvhd, and thereby reduce the risk of life-threatening complications after allogeneic hsct. methods: heracles is a single-arm, multicenter prospective trial in european countries. patients aged ≥ years-old, who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct) and developed a first episode of stage or agvhd with gut predominance resistant to a first-line steroid therapy are eligible for inclusion. main exclusion criteria comprise the use of other second-line gvhd therapy, patients with grade iv hyperacute gvhd, late onset agvhd, and overlap chronic gvhd and agvhd after donor lymphocyte infusion. patients receive a first maat enema within days after sr diagnosis (v ) and additional ones week apart (v / v ) from each other. maat is a highly-diverse, microbiome-rich enema formulation obtained from pooled, rigorously screened faeces from healthy donors, manufactured with a standardized process using the signature maat microbiome restoration biotherapeutic (mmrb) platform. at inclusion (v ), before each dosing (v , ), and days post inclusion (v ), patients' faeces and blood are collected. safety monitoring will be performed with corresponding blood analyses. exploratory measures on faeces include characterization of gut microbiota composition and evolution, impact of maat on metabolism, and gut inflammation. immune system phenotyping will be performed by flow cytometry on peripheral blood mononuclear cells, and by elisa assay on plasma. patients' qol will be assessed using a standard, eq- d- l questionnaire. the primary objective is to assess the efficacy of maat by evaluating complete response (cr, according to modified glucksberg criteria) and very good partial response (vgpr, defined by martin et al., bbmt, ) days post-inclusion (primary follow-up). secondary objectives include fmt safety assessment and evaluation of fmt impact on several endpoints, such as overall, relapsefree or gvhd-free survival and chronic gvhd evaluation, as well as multi-drug resistant bacteria carriage. patients will be followed-up until year after inclusion. overall, patients are planned to be enrolled and treated, to assess overall response rates and maat 's safety profile. results background: anti-programmed cell death protein (pd ) monoclonal antibodies can be used as "bridge to" a subsequent allogeneic hematopoietic stem cell transplantation (hsct) in patients with relapsed/refractory hodgkin´s lymphoma (hl). this strategy has been reported to be effective, but a frequent onset of steroid-refractory graft versus host disease (gvhd) was also reported. we report clinical cases of patients affected by hl undergoing allogeneic hsct after having been treated with nivolumab. methods: the patients of , and years respectively had advanced hl and had relapsed after a previous autologous ( ) or allogeneic ( ) hsct. they underwent a rescue therapy with , , nivolumab cycles respectively, depending on the time of partial response achievement and the availability of a donor. two patients received a thiotepa-fludarabinecyclophosphamide conditioning, atg-based prophylaxis and pb cells from unrelated donors. the third patient received bm cells from an haploidentical donor using the "baltimora" nonmieloablative platform. results: at a follow-up of , , months after hsct, respectively, all patients achieved and maintained a complete remission by pet-ct scans. all the patients developed acute gvhd on day + , + and + , respectively. patient progressed to grade iv acute gvhd with hepatic and intestinal involvement unresponsive to first line mg/kg steroid therapy and second line etanercept plus extracorporeal photopheresis (ecp). third line therapy with ruxolitinib partially controlled the gvhd. gvhd onset in patients and was preceeded by a prolonged fever without microbiological findings. patient developed hepatic grade ii gvhd with high transaminase levels, initially responsive to steroid therapy, then it progressed to gut requiring second line therapy with etanercept. patient progressed to severe chronic gvhd with skin involvement and resulted unresponsive to steroids and ecp and it was partially controlled by ruxolitinib. immune reconstitution was delayed in all patients: at months post transplantation cd levels were /μl, / μl and /μl and cd levels were /μl, /μl and / μl respectively. only patient , that underwent haploidentical transplant and received post-trasplant cyclophosphamide (pt-cy), is off of immunosuppressive treatment at months after hsct, without evidence of gvhd and no history of infections. out of the patients receiving pbsc from unrelated donors and atg prophylaxis, patient developed a disseminated fusariosis on day + and died of cns fusarium localization year after hsct, despite targeted antifungal therapy. patient had pulmonary aspergillosis, sepsis by multidrug resistant psuedomonas aeruginosa and otomastoiditis: at + months after hsct, he is on ruxolitinib treatment with skin clinical partial response. conclusions: this case series confirms that nivolumab as "bridge to transplant" is effective in appropriately selected patients. however, risk of acute gvhd and delayed immune reconstitution may require a careful consideration at the moment of planning the transplant. a possible advantage of pt-cy gvhd platform and haploidentical donors should be addressed in larger studies. background: acute graft-versus-host disease (agvhd) is the most important complication after an allogeneic hematopoietic stem cell transplant (hsct). no standard secondline treatment has been established for the corticosteroid refractory agvhd. the anti-tnfα agents are a good option of treatment for these patients, especially when lower gi tract is involved. methods: from april to july we reviewed the outcome of patients with steroid-refractory (sr) agvhd treated with etanercept as at least, second line treatment. etanercept dose was mg twice a week for the first weeks, followed by weekly doses. results: median age was years (range - years), and patients ( %) were male. fourteen patients ( %) had a non-advanced disease status at hstc. eleven patients ( %) received a myeloablative conditioning, and the stem cell source was peripheral blood in patients ( %). sixteen patients ( %) were / hla matched. the characteristics of the patients, their agvhd stage previous to rescue treatment with etanercept and their outcome are shown in table . seventeen patients ( %) had a classic agvhd while had a late-onset agvhd. etanercept was given as a nd , rd and th line in ( %), ( %) and ( %) patients respectively. the median doses of etanercept administered were (range - ), and just patients ( %) completed the doses planned treatment, of whom were alive at , and months from the onset of rescue treatment. complications during etanercept treatment were: infection (n= [ %]: gram negative bacilli [n= ]), grade - neutropenia (n= ) and grade - thrombocytopenia (n= ). etanercept was indicated as a rescue treatment due to: progression after days of agvhd treatment (n= ), no response after days of treatment (n= ), no complete remission after days of treatment (n= ) and relapse due to decrease corticosteroid doses (n= ). at the end of treatment patient achieved a complete response and patients a partial response, all of them are alive. these patients received etanercept as a nd (n= ) and th line (n= ) treatment, all of them had lower gi agvhd without any other organ significantly involved. causes of death were: agvhd with or without infection in patients ( %) and leukemia relapse in patient. conclusions: although if etanercept is an option for treatment of sr agvhd in some patients, their prognostic remains poor and more effective alternative strategies are needed. a prompt initiation of etanercept as a rescue treatment for sr agvhd is crucial to improve the prognosis. ( ) ( ) background: although both cyclosporine (csa) and tacrolimus are calcineurin inhibitors, csa is more widely used in pediatric hematopoetic stem cell transplantation (hsct) as a prophylactic drug for acute graft versus host disease (agvhd). there are some clinical experience but very few data about the clinical efficacy of conversion to tacrolimus. here, we present our single center data on this arguable topic. methods: this study involves the data of pediatric hsct patients in medical park göztepe hospital between - . all patients had prophylactic csa therapy and for various reasons csa was converted to tacrolimus therapy. most of the patients had this conversion due to agvhd. as steroid is the first line therapy for agvhd, conversion to tacrolimus is done concurrently at the start of steroid therapy (within hours after the start of steroid). and also, patients who had any other immunosupressive therapy for agvhd are excluded. response is defined as resolution of symptoms within days after conversion. results: mean age of the study population is months ( - months), male/female ratio is , ( / ), donor types are mud patients ( %), mfd patients ( %), haplo patients ( %) and mean conversion time is days ( - days) . the rationales for conversion are agvhd for patients, unproper csa plasma levels for patients, allergic reaction for patients, nephrotoxicity for patients, hepatotoxicity for patients, severe headache for patients, high arterial blood pressure for patients and one each for refractory vomiting, autoimmune thyroiditis and visual disturbance. the subgroup analysis of agvhd patients reveals that mean conversion time for agvhd is days ( - days) and there are only responders whose agvhd resolve completely (% ) after conversion. all of the patients had proper tacrolimus levels after conversion due to unproper csa levels and also patients in allegic reaction, severe headache, visiual disturbance and refractory vomiting group responded to conversion completely but only one of the patients in nephrotoxicity group responded and also of the patients in hepatoxicity group responded. the only one patient suffered from autoimmune thyroiditis did not respond to conversion. conclusions: in this study, it is obvious that there are response to conversion for some specific adverse effects of csa and tacrolimus is a good alternative for the patients who have unproper csa levels. conversely, the high percentage (% ) of non-responders shows that it is not feasible to make a conversion to tacrolimus for acute gvhd. disclosure: nothing to declare background: capillary leak syndrome is caused by the dysfunction of the vascular endothelial cells,and is characterized by weight gain,generalized edemas,unresponsive to diuretic treatment,and hypotension.it usually develops in the first days post hsct.and it is of great difficuty to distinguish from other complications which are occured post the allo-hsct. to diagnose this complication at the early stage,it is very difficulty. methods: a -year-old man was admitted to ningbo first hospital for its abnormal in the peripheral blood .he was diagnosed with aml-m by the classical morphology and immunophenotype.cytogenetic evaluation showed a normal , xy( ).the patient achieved cr with induction therapy including idarubicin, cytarabine and etoposide. after consolidation therapy,an allo-hsct from hla identical related dornor( -year-old male, donorrecipient matched by high resolution hla typing at hlaa, -b, -c, drb , and dqb , / matches) was performed.the recipient received conditioning with busulfan, mg/kg/day injection for days; cyclophosphamide, mg/kg/day injection for days; cytarabine, g/m /day injection for day; semustine, mg/m /day orally for day; donor peripheral blood stem cells (pbsc:mnc: . × /l, cd +: . × /l) were mobilized, pheresed and administered to the recipient. gvhd prophylaxis consisted of traditional cyclosporine, short-course methotrexate ( mg/ m at day + , mg/m at days + , + , and + ) and cyclosporin a injection mg/kg qer day was mot reduced untill the hematopoietic reconstitute sucessfully . on day + , complete donor chimerism was acheieved. the csa was gradually reduced and tapered.on day + ,the patients was manifested with increasing in the time and volume of the faeces, he was diagnosed with ii°gvhd (gut).the standarded does of immunosuppressive drug including methylprednisolone and cyclosporin a was administrated. the immunosuppressive drug was gradually reduced when the gvhd was controlled.on day+ ,the patient felt distress and the distress was not related to with the exercise,the temperature was normal,and he did not gain weight.there was no edema in the body.laboratory test including routine blood test,c-reactive protein,procalcitonin,blood gas analysis,cmvdna,ebvdna was normal. the ct scan shows that the pleural is filled up with water, and could not be enlarged promptly, there is pericardial effusion in the body.pulmonary function test shows that reduced function in ventilation and diffusion fuction.the laboratory test of the pleural effusion was normal,the blast cell was not detected in the pleural effusion,the cd + cell count was below the dectable level,the next generation sequencing for minimal residual disease shows that there was no gene mutation .thus, post capillary leak syndrome was considered .sirolimus was adopted and taken the place of cyclosporin a,immunoglobulin was adminstrated to reduce the edema. results: taking together comprehensively,the effusion in the pleural and cardiac was absorbed well. conclusions: occurance of capillary leak syndrome is rare,there is limited data about capillary leak syndrome. comprehensively,the mechanism of cls has not been totally identified.and there is no standard treatment to treat the complication.at present,the cls of this patient was absorbed well by administrating sirolimus,closely followup is needed. disclosure: nothing to declare graft-versus-host diseasepreclinical and animal models p short-term krp and posttransplant cyclophosphamide for graft-versus-host disease prophylaxis emi yokoyama , daigo hashimoto , takahide ara , eko hayase , takanori teshima hokkaido university faculty of medicine, hematology, sapporo, japan background: post-transplant high-dose cyclophosphamide (ptcy) in combination with other immunosuppressants such as calcineurin-inhibitors (cis) has been increasingly used as gvhd prophylaxis after hla-haploidentical or matched hematopoietic stem cell transplantation (hsct). however,cis could hamper reconstitution of regulatory t cells (tregs) and tolerance induction after hsct, facilitating us to develop novel ci-free/ptcy-based gvhd prophylaxis. in the current study, we developed a novel gvhd prophylaxis in which ptcy was combined with short-term administration of krp , a selective agonist of sphingosine- -phosphate receptor type (s pr ), using murine models of mhc haploidentical bone marrow transplantation (bmt). methods: b d f (h- b/d ) recipients were lethally irradiated and transplanted with bone marrow cells and splenocytes from allogeneic b (h- b ) donors. cy at a dose of mg/kg was intraperitoneally injected into the recipients on day + , and krp at a dose of . mg/kg was orally administrated daily from day to day + after bmt. donor t cells in the target organs and secondary lymphoid organs were evaluated by flow cytometric analysis. plasma levels of tnf-α were determined using cytometric beads array. to evaluate graft-versus-leukemia (gvl) effects, recipient mice were intravenously injected with luciferase-transduced p cells (p -luc) on day , and in vivo bioluminescence imagingwas conducted weekly after bmt. results: severe gvhd was developed in allogeneic recipients and all mice died by day after bmt.ptcy alone at a dose of mg/kg significantly ameliorated gvhd and % of ptcy-treated allogeneic recipients survived. oral administration of krp alone enhanced contraction of donor t cells in the lymph nodes and also ameliorated gvhd as has been previously shown with multi-s pr agonist, fingolimod. next, we tested if shortterm krp on days to + added to ptcy enhances anti-gvhd effects of ptcy. we found that survivals of ptcy+krp group were significantly prolonged compared to those of ptcy-alone group ( figure a) . plasma levels of tnf-a, clinical gvhd scores ( figure b) , and donor t-cell infiltration into the target organs such as the gut and skin were also significantly reduced in ptcy +krp group compared to ptcy-alone group (figure c and d) . unlike cis, addition of krp to ptcy promoted treg reconstitution after bmt. finally, bioluminescence imaging demonstrated that proliferation of p -luc injected on day was significantly delayed in ptcy +krp -treated allogeneic recipients compared to control mice transplanted only with t-cell depleted bone marrow cells, suggesting that significant gvl effects persisted in ptcy+krp -treated recipients. conclusions: a combination of short-term krp and ptcy is a promising novel calcineurin-free gvhd prophylaxis in mhc-haploidentical sct. we recently showed that donor inkt cells can be expanded ex vivo and that they are able to prevent activation and proliferation of alloreactive donor t cells while promoting efficient graft-versus-leukemia effects (schmid et al. ). however, the underlying mechanisms how human inkt cells induce immune tolerance after allogeneic hct are not fully understood. methods: monocyte-derived dendritic cells (dcs) were cultured in a mixed lymphocyte reaction with mhcmismatched t cells and culture-expanded inkt cells. tcell activation and proliferation was analyzed by multiparametric flow cytometry and released cytokines were measured via multiplex analysis. transwell assays and imaging flow cytometry were performed to elucidate cellcell interactions. bead-controlled flow cytometry-based cytotoxicity assays were used to evaluate dc apoptosis. apoptotic dcs were then purified by fluorescence-activated cell sorting to investigate their tolerogenic potential to prime regulatory t cells (tregs). results: the addition of inkt cells to mixed lymphocyte reactions resulted in a significantly reduced activation and proliferation of mhc-mismatched t cells. transwell assays and imaging flow cytometry revealed a cell contactdependent mechanism between inkt cells and dcs leading to apoptosis with increasing dna fragmentation of dcs over time. interestingly, various fluorescence-activated single cell sorted inkt-cell subsets were all able to induce apoptosis of host dcs. multiplex analysis revealed that dcs triggered inkt-cell release of cytotoxic factors like perforin, granzyme b and granulysin. blocking the inktcell receptor engagement with a cd d antibody prevented inkt-cell degranulation as well as the subsequent induction of host dc apoptosis. inhibition of cytotoxic factors also abrogated apoptosis of dcs. in turn, sorted apoptotic dcs induced tolerogenic dcs characterized by a high expression of pd-l in mixed lymphocyte reactions. such tolerogenic dcs promoted the expansion of cd + cd + foxp + tregs and prevented activation and proliferation of mhcmismatched t cells. conclusions: we propose a novel mechanism how culture-expanded human inkt cells prevent gvhd after allogeneic hct. host dc apoptosis through donor inkt cells induces a tolerogenic immunoenvironment characterized by pd-l high dcs and expanding donor tregs inhibiting activation and expansion of alloreactive donor t cells. our findings pave the avenue for clinical translation of adoptively transferred culture-expanded inkt cells in humans. disclosure: nothing to declare results: vip-ko mice transplanted with allogeneic tcd bm alone had increased graft rejection with lower levels of donor chimerism and % day survival compared with % survival of wt recipients. transplanting tcd bm plus × e donor t cells from b .br or balb/c donors in vip kio mice led to > % donor chimerism and significantly increased gvhd-mortality compared with wt recipients, with % vs % survival in the b .br-->b model (p< . ), and % vs % survival in the balb/c-->b model (p< . ). donor-derived t cells in vip-ko recipients had significantly higher th and th polarization, with higher rorγt in both cd + (p< . ) and cd + (p< . ) t cells, and higher frequencies of ifn-γ (p< . ), tnf-α (p< . ), and il (p< . ) in cd + and cd + t cells compared to wt recipients. b .br-->b second allogeneic transplantation of radiation chimeras caused lethal gvhd mortality in vip-ko-->vip-ko and wt-->vip-ko mice, but not in wt-->wt or vip-ko-->wt b mice, demonstrating the protective effect of vip was due to synthesis by non-hematopoietic recipient cells. immunofluorescent imaging of allo-bmt recipients showed marked up-regulation of vip in lungs post-transplant and high vip production within neurons innervating the lungs. finally, we demonstrated that short-term administration of vip ( mcg/day) from day to day prevented gvhdmortality in vip-ko recipients transplanted with b .br-->b mhc donor bm & t cells. conclusions: the absence of vip in recipient cells led to increased graft rejection in the absence of donor t cells and increased lethal gvhd when donor t cells were transplanted, indicating vip induced post-transplant regulates allo-reactivity of host graft-rejecting lymphocytes and donor gvhd-causing t cells. the protective effect of parenteral vip administration suggests vip-mimetics represent a novel approach to prevent and treat gvhd. these data also suggest a mechanism of action for the mitigation of gvhd by alpha- anti-trypsin (aat) whereby aat inhibits the proteolytic inactivation of endogenous vip. disclosure: dr. waller reports personal fees and other support from cambium medical technologies, grants from celldex, personal fees from kalytera, grants and personal fees from novartis, grants and non-financial support from pharmacyclics, and equity ownership in cerus corporation and chimerix outside the submitted work. in addition, dr. waller has intellectual property related to vip signaling that has been licensed to cambium oncology in which he holds equity. low-density neutrophils expansion is associated with acute graft versus host disease in allogeneic hematopoietic stem cell transplant patients background: low-density neutrophils (ldns) are distinguished from normal-density neutrophils (ndns) by their anomalous sedimentation within the mononuclear cell fraction after density gradient centrifugation of peripheral blood (pb). by analysing ldns and ndns from g-csfstimulated donors or lymphoma patients, we have previously demonstrated that, depending on physiopathological conditions, immature cd b + cd -ldns can promote t cell survival and ifn-γ production, while mature cd b + cd + ldns can exert immunosuppressive proprieties. aim of this study was to establish the frequency of cd b + cd and/or cd b + cd + ldns in pb of allogeneic hematopoietic stem cell transplant (hsct) patients throughout immune reconstitution, and verify their potential correlation with acute graft versus host disease (agvhd). methods: patients undergoing hsct in our institution between december and june were prospectively enrolled in the study upon informed consent and after institutional board approval. criteria of inclusion were age ≥ years and absence of rheumatologic or viral diseases. pb samples were collected at day + , + , + , + and + after hsct and any time within day + in case of gvhd, before first-line therapy. eight healthy donors (hds) were enrolled as control. mononuclear, polymorphonuclear, and whole blood cells were analysed by flow cytometry after cd vioblue, cd apc-cy , cd b pe-cy , cd pe, cd b fitc staining. cd b + ldns were expressed as percentage of cd + pb mononuclear cells (pbmcs) or cd + whole blood cells and were further characterized based on cd expression. cd b + ndns, expressed as percentage of cd + whole blood cells, were also analysed for cd staining. results: patients (m/f / , median age ) were enrolled in the study. patients received hsct from hlaidentical ( ) or haploidentical ( ) related and from hlaidentical unrelated ( ) donors. after a median time of ( - ) days, patients developed grade ii-iv agvhd. no patients were receiving g-csf at agvhd onset. the scheduled assessments were interrupted in agvhd patients at the beginning of first-line treatment and in patients relapsed of their primary malignancy. no patients developed de novo late-acute or chronic gvhd. starting from day + the frequency of ldns within cd + pbmcs was higher in all patients as compared to hds. the patients that did not develop agvhd showed a decreasing frequency of cd b + cd -ldns, with a progressive increase of cd b + cd + ldns, from day + to + . interestingly, patients with agvhd showed a significantly higher frequency of cd b + cd -ldns as compared to patients without agvhd throughout the same time lapse (i.e. from day + to + ) ( . vs . , p= . ). consistently, patients with agvhd had a significantly lower frequency of cd b + cd + ldns ( . vs . , p= . ). the frequency of mature cd b + cd + ndns was normal in all patients since day + . conclusions: ldns are more represented in hsct patients than in hds, with a significant expansion of the cd b + cd subpopulation (with a parallel decrease of the cd b + cd + subpopulation) in patients with agvhd as compared to those without agvhd. according to the previously demonstrated t cell activating function of cd b + cd -ldns, it is tempting to speculate that the expansion of this subpopulation may contribute to agvhd development. disclosure: nothing to declare background: acute graft-versus-host disease (agvhd) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-hsct) which has negative impact on the morbidity and mortality of the patients. accumulating evidences suggest that abnormalities of foxp + regulatory t (treg) cells contributed to the pathogenesis of gvhd, but the underlying molecular mechanisms still remain largely unknown. methods: in this study, we enrolled all the patients treated with allogeneic hsct at the institute of hematology, chinese academy of medical sciences between and ,as well as age-matched healthy adults as control samples. the ratio of tregs in pb and bm of healthy controls (hcs) and patients with and without agvhd was determined by flow cytometry. the transcription profile between tregs from patients with or without acute gvhd was measured,the pathway enrichment analyses were performed by the kyoto encyclopedia of genes and genomes (kegg) pathway database and geneset enrichment analysis (gsea).the expression of lkb at transcript levels and protein levels was measured by realtime pcr and analyzed by the nanopro tm system. a series of functional assays in vitro were performed to assess the function and stability of tregs from patients with and without agvhd.meanwhile, to assume the affect of lkb on gvhd outcome, we established a murine transplant model,which recipient balb/c animals were transplanted with the same amount of mixture made by bm, cd +cd -tcon cells from c bl/ and cd + foxp yfp+ tregs from either foxp crelkb f/f or foxp cre mice. results: in this study, we demonstrated that bm had decreased frequencies of tregs, accompanied with a reversed lower ratio of tregs frequencies between bm and pb in agvhd patients. meanwhile, the number and function of tregs in bone marrow also affected hematopoietic reconstitution. futhermore,to elucidate these mechanisms which regulate tregs homeostasis, we examined the role of lkb on tregs in patients with agvhd and in agvhd murine model. studies demonstrated that lkb deficient tregs lost foxp expression and weaken suppressor function during agvhd. transcriptional profiling and pathway analysis revealed that nf-kb signaling activation and the impairment of a wide spectrum of immunosuppressive genes in agvhd tregs. further mice experiments suggested that cns methylation might lead to the instability of tregs in agvhd group. transplantation with marrow grafts from foxp crelkb f/fmice exacerbates gvhd lethality. conclusions: these studies indicate that lkb is a critical homeostatic regulator for tregs during agvhd. targeting of lkb therefore represents a novel therapeutic strategy that promote immune tolerance to mitigates the severity of agvhd. disclosure: national program on key basic research project ( program) role of aryl hydrocarbon receptor in intestine after allogeneic hsct in mice won-sik lee , soung-min lee , sj-kil seo inje university, busan paik hospital, hemato-oncology, busan, korea, republic of background: aryl hydrocarbon receptor (ahr) is a ligandactivated transcription factor that is activated by various small molecules from the diet, microorganisms, host metabolism, and xenobiotic toxic chemicals. the function of ahr has been demonstrated as a crucial regulator in intestinal homeostasis. here, we investigated the regulatory role of ahr in intestine of recipients after allogeneic hematopoietic cell transplantation in mice. methods: wild-type (wt) b (h- b ), ido -/-(h- b ) and ahr -/-(h- b ) mice were lethally irradiated and transplanted with x tcd-bm plus x t cells from balb/c donor mice. ahr activation in colon tissue of recipients was determined by the ahr target genes cyp a and cyp b expression using real-time pcr. the recipient mice were monitored every other day for survival and clinical score. histopathology and pathogenic effector cytokine levels in colon tissue were analyzed for evaluating ahr function. results: we observed that cyp a was constitutively expressed in the colon tissue of naïve recipient mice. although the expression levels were increased by tbi conditioning, the additive up-regulation of its levels with donor t cell alloreactivity was not observed. in contrast, cyp b expression was markedly induced in the colon tissue by donor t cell alloreactivity. we further observed that the cyp b expression was significantly decreased in the colon of ido-/-recipients with donor t cell alloreactivity, but cyp a was not changed. ido-/-and ahr-/recipient mice showed higher histopathological score for intestinal gvhd and increasing pathogenic cytokine levels in the colon compared with wt mice. conclusions: our results demonstrate that ahr-induced target gene profiles might be differently induced in intestine by ligand dependent manner after hsct, which affect intestinal gvhd. disclosure: nothing to declare. abstract already published. abstract withdrawn. in vitro platelet activation evaluation in allogeneic hematopoetic stem cell transplanted patients in response to haemostatic stimulation and cytomegalovirus stimulation (gvhd), complication of which one of the risk factor is cmv reactivation. the resultant inflammatory platelet response during the high-risk period of gvhd after allogeneic hsct remains unknown. our study aimed to characterize spontaneous platelet activation during the d and d months after allogeneic hsct, and in response to haemostatic stimulation and cmv stimulation. methods: we compared a group of healthy volunteers to a group of allogeneic hsct patients followed between the th and the th days after hsct. platelet activation was determined by the platelet surface expression of cd p and cd using flow cytometer after stimulation by an haemostatic agent, thrombin-receptor activating peptid (trap) and after stimulation by cmv glycoprotein b. the inflammatory response was determined by the detection of immune mediators, rantes, cd ps, pf , cd l and ccl , using the elisa technique in the stimulated platelet supernatants. results: no platelet activation or molecules release were observed after stimulation by cmv glycoprotein b in both groups. rantes and cd ps baseline levels are spontaneously higher in allogeneic hsc patients than in healthy volunteers. platelets from allogeneic hsct patients can be activated after haemostatic stimulation and release cd ps and rantes. in this situation, platelets release more cd ps, rantes and pf than platelets from healthy volunteers. conclusions: although no platelet activation was detected in response to cmv glycoprotein b stimulation, our study revealed a chronic platelet activation condition during the d and d months after allogeneic hsct with an haemostatic inducible hyper-responsiveness. this leads to the release of molecules with immune-modulating properties involved in the pathophysiology of gvhd. as we move further away from the hsct, that phenomenon seems to gradually weaken. clinical trial registry: nct , fipalloc https://clinicaltrials.gov/ct /show/nct disclosure: nothing to declare p efficient process and characteristics of umbilical cordderived mesenchymal stromal cells as a feasible source for anti-inflammatory therapy background: recently, umbilical cord (uc) has become attracted source of mesenchymal stromal cells (msc), because of abundant sources and ease of collection of fetal origin without invasive process for the donor and low immunogenicity with immunosuppressive ability and tissue repair potency. objectives of this study were to explorer the efficient and safe products and to evaluate the antiinflammatory potency of uc-mscs for the application of acute graft versus host disease (gvhd). methods: informed consent was obtained from mothers planning to have cesarean sections. uc tissue was cut and once cryopreserved. the safety assessment including infections and baby's health and development were done after months of birth, and performed small-scale quality test of the frozen uc. then we initiated to isolate master uc-mscs from frozen-thawed uc by an improved explant method, which was passed for quality test. the master uc-mscs were cryopreserved once and thawed and expanded until p . product cells were cryopreserved in original serum-free cryoprotectant dba-d solution. mixed lymphocyte reaction (mlr) assay co-cultured with uc-mscs was carried out using responder mononuclear cells (mnc) stained with cfse, and proliferation and cytokine secretion were analyzed by flowcytometry. results: uc-msc cultured showed significantly higher proliferation ability compared with those from bone marrow-derived mscs, and positive for cd , cd , cd , and negative for cd , hla-dr. cd , and cd were negative even in the high concentration of ifn-γ, while bm-mscs became positive for hla-dr. pd-l was constitutively expressed in uc-msc, while pd-l was induced by the addition of ifn-γ. in mlr, responder t cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by rd party derived uc-mscs, in which was induced ido, pge , hgf, and tgf-β analyzed by rt-pcr, and inhibited ifn-γ and tnf-α in the supernatant by cytokine beads array. uc-mscs migrated toward the tnf-α treated mnc and increased regulatory t cells incidence in peripheral mononuclear cells by the coculture. conclusions: these results demonstrated that cryopreserved uc are feasible and efficient source of mscs and frozen-thawed uc-mscs have high anti-inflammatory background: a new protocol is under development on the amicus separator that enables the device to perform ecp procedures. the amicus separator is used with a photoactivation device, disposable kit and -mop to provide ecp therapy in a closed system. the objective of this study was to evaluate the safety and performance of the investigational amicus ecp system in healthy human subjects. methods: an irb-approved written informed consent was obtained from subjects ( male, female). the amicus ecp system processed either , or ml whole blood (n ≥ per arm) using double-needle access and acd-a anticoagulation at a : wb:ac ratio. after mnc collection was completed, the subject was disconnected from the device. -mop ( . ml, μg/ml) was injected directly into the collected mnc product and saline (approximately ml total), which was photoactivated with - j/cm uva light. post photoactivation, the amicus separator reinfused the treated mncs into a transfer pack. subject laboratory and safety parameters were evaluated; in vitro evaluations were performed on subject whole blood, collected mncs, treated mncs, and reinfused cells. lymphocyte and monocyte analysis were performed on samples purified using density gradient separation and cultured for up to days post treatment. results: in procedures, median (range) wb processed was . ( - ) ml using . ( - ) ml of acd-a. procedure time was . ( - ) minutes, including photoactivation. no adverse events were reported. subjects' vital signs and hematology values were unremarkable and within expected values. the wbc count of the collected mncs was . ( . - . ) x /μl, comprised of . ( . - . ) % lymphocytes, . ( . - . ) % monocytes and . ( . - . ) % granulocytes and platelet count was . ( - ) background: transfusion of white blood cells (wbc) causes a number of transfusion reactions and complications, for example transfusion-associated graft versus host disease (tagvht), which still does not have effective treatment and is a fatal complication of transfusions. the only effective method of preventing tagvht is irradiation of blood components with ionizing radiation (x-ray or gamma radiation). but the use of ionizing radiation sources has a number of technical and material difficulties. the emergence of pathogen reduction technologies (prt) in blood components targeted by nucleic acids has opened the possibility of using these technologies as an alternative to irradiating of blood components. several prt demonstrated effective inactivation of wbc in platelet concentrates and blood plasma. so, determination of the influence of prt based on the combined effect of riboflavin (rf) and ultraviolet (uv) on the viability and proliferating potential of lymphocytes in whole blood is important. methods: samples of whole blood were obtained in healthy volunteers. each sample was divided into three unequal parts: untreated control, gamma irradiated, and treated by rf and uv prt (mirasol, terumo bct inc.). mononuclear cells (mnc) were cfse stained, viability and proliferating activity were tested at intervals of hours for consecutive days by flow cytometry. statistical analysis was performed with xlstat . . levels of significance were calculated by mann-whitney test, expressed as p-values (p< , ). results: the median viability of mnc after application of both methods of treatment was over , % on day and decreased to day -median percentage of viable mnc were , % (control group), , % (after gamma irradiation) and , % (rf/uv prt). the median of spontaneous proliferative activity on day of untreated and gamma irradiated mnc did not differ ( , % and , % respectively, p< , ). phytohemaglutenin (pha) induced proliferation on day in gamma-irradiated samples was significantly lower in comparison with control group ( , % and , % respectively, p< , ). in samples treated with rf/uv, spontaneous and stimulated proliferating cells was not detected. median percentage of proliferating mnc was less than , %. the use of this prt on whole blood, as well as gamma irradiation, significantly reduces the viability of lymphocytes during storage for days. conclusions: inactivation of wbc using rf/uv prt is a useful and very necessary bonus for a number of reasons. in one procedure two effects are achieved: infectious and immunological safety. the use of prt on whole blood gives the potential for obtaining pathogen-reduced and immunological safety components of blood, which reduces their material cost and staff loading. the use of rf/uv system does not have such complex security requirements and difficulties in servicing as the use of sources of ionizing radiation. the results demonstrate a promising potential for using this technology as an alternative to irradiation disclosure: nothing to declare p influence of patients´serum after allogeneic stem cell transplantation on t cell proliferation and treg function background: acute or chronic graft versus host disease (a/ cgvhd) is one of the major complications after allogeneic hematopoietic stem cell transplantation (ahsct). application of regulatory t cells (treg) as "immunosuppressive dli" to prevent or treat gvhd is investigated in clinical trials. here we ask the question, if there could be clinical conditions (e.g. cytokines or drug effects) limiting the efficacy of this approach. to face this problem we tested the influence of patients´serum on t cell proliferation and treg function. methods: lymphocytes from healthy donors were incubated with t cell medium ( % aim v + % serum + il /okt ) containing serum from healthy donors or serum derived from patients after ahsct with or without gvhd (n= ). next we evaluated the suppressive function of treg by performing treg suppression assays, also comparing serum from patients suffering from gvhd versus serum obtained from healthy donors (n= ). proliferation of cfse stained t cells was measured after days. to test the effect of immunosuppressive drugs on treg we performed treg suppression assays after incubation of treg with corticosteroids or tacrolimus or the combination of both drugs. results: serum of patients with acute or chronic gvhd had a negative effect on t cell proliferation. to avoid bias tests were performed with samples from patients without or only with low levels of immunosuppressive drugs. incubation with serum of patients without gvhd or with serum of healthy individuals showed no differences in t cell proliferation. treg from healthy donors showed a stronger antiproliferative capacity when incubated with serum derived from patients with gvhd. treg previously incubated with immunosuppressive drugs showed no decreased suppressive capacity. conclusions: components of serum from gvhd patients seem to have an antiproliferative effect on t lymphocytes itself. this fact might influence the clinical course of gvhd, but should not be a limiting factor for therapeutic application of treg dli. even the systemic treatment with immunosuppressive drugs e.g. corticosteroids or calcineurin-inhibitors should not diminish the treg application. in a next step we will analyze serum components responsible for this immunosuppressive effect with multi cytokine assays and proteomic analysis. the aim of our project is to develop new strategies to avoid gvhd and to optimize clinical settings for treg dli. disclosure background: hypercalcaemia can be very severe following stem cell transplant (sct) in some osteopetrosis patients. denosumab is a fully human monoclonal antibody that binds the cytokine rankl (receptor activator of nfκb ligand), an essential factor initiating bone turnover. rankl inhibition blocks osteoclast maturation, function and survival, thus reducing bone resorption. we describe the effective management of hypercalcaemia in a patient with rank mutation osteopetrosis who received a haploidentical sct. methods: our patient was diagnosed with osteopetrosis at year of age with a defect in the tnfrsf a gene which codes for rank and received a maternal haploidentical sct aged years. the patients calcium levels were monitored regularly post sct. denosumab was administered for hypercalcaemia as per laboratory reports or clinical symptoms. the drug was diluted with water for injection to make mg/ml solution to facilitate subcutaneous administration. results: significant hypercalcaemia emerged on day + with a level of mmol/l and treated with hyper-hydration and diuretics. this was ineffective in reducing the hypercalcaemia; therefore denosumab was initiated on day + post-transplant. initial dosing was determined using the only available paediatric case report at . mg/kg. a repeated larger dose of . mg/kg was given days later due to an inadequate response (calcium decreased from . mmol/l to . mmol/l). the calcium decreased to . mmol/l after this dose. four weeks later a third dose was required at . mg/kg as the calcium level had increased to . mmol/l. the dose was further increased to . mg/kg for another four doses and then further increased to . mg/kg for another doses and repeated every weeks. normalisation, but not excessive drop in calcium was achieved with these larger doses. over the month follow up post-transplant there were three admissions lasting less than hours for symptoms of hypercalcaemia. these were managed with denosumab administration and hyper-hydration. the remaining doses were given in an outpatient setting. conclusions: denosumab can be safely used as a first line agent in treating post stem cell transplant hypercalcemia in patients with osteopetrosis. a dose of . mg/kg is required as an initial starting dose in order to control hypercalcemia. this is a new higher dose than previously suggested by the original report. denosumab can be effective even after dilution and safely given in children weighing less than kg. disclosure: nothing to declare methods: the clinical, laboratory and molecular aspects of this italian male patient who developed such a complication were collected and presented in order to discuss the origin, clinical outcome and management of this very rare post-transplant event. results: a -years-old man affected by a high-risk chromosomally abnormal, ph -, mll-pro-b (egil b-i) all relapsed during maintenance treatment, nonresponsive to re-induction chemotherapy, in second complete remission (ii cr) after blinatumumab treatment received a female cb transplant. according to sorror's and ebmt scores he was considered a high-risk transplant. the patient and the cb unit were sex-mismatched, shared the same blood groups and were both cmv+/ebv+. he received a tbf conditioning regimen that was followed by the infusion of . x /kg cd + cb cells. gvhd prophylaxis consisted of rabbit atg, cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day + , whereas platelets were never > . /μl. on day + a cm bulged area became apparent on the left parietal region of the skull. an echotomography showed that the lesion adhered to the bone without infiltrating it and lacked blood vessels and suggested that it may be either a site of disease relapse or an area of infection. at the same time a bone marrow (bm) aspiration showed morphological cr confirmed by immune-phenotypic studies and x-y fish a complete chimera. since the patient was still febrile no biopsy was performed, but on day + the axial diameter of the lesion that on a ct scan showed the same appearance revealed by the previous echo-tomography increased to cm. thus, the lesion was surgically removed and histological examination showed cd +, cd +/-, cd +/-, cd /lca+/-, cd -, cd -, cd -, cd -, and s -neoplastic cells whose phenotype suggested a granulocytic sarcoma rather than a histiocytic sarcoma. immuno-chemistry confirmed this suggestion by showing a nuclear npm positivity. fish studies demonstrated that these neoplastic cells were of recipient's origin. a novel bm aspiration showed cr confirmed by immune-phenotypic studies and fish revealed a complete chimera. since the patient was still pancytopenic due to anti-cmv treatment, radiotherapy with gy in nine fractions were given and the lesion completely resolved. conclusions: a granulocytic sarcoma of recipient's origin occurring three months after a cb transplant is a very rare and unusual event. in order to explain such a complication we suggest that granulocytic sarcoma cells were dormant but already present at the time of pro-b all diagnosis and survived not only the initial all treatment but also the cb transplant conditioning regimen. we can't exclude that immune-suppressive treatments given early post-transplant might have promoted the outgrowth of these neoplastic cell population. disclosure: nothing to declare haemoglobinopathy and inborn errors of metabolism p abstract already published. addition of fludarabine on to anti-thymocyte globulin, busulfan and cyclophosphamide conditioning improves outcomes in low-risk matched-related bone marrow transplantation in children with severe thalassaemia flu-atg-bucy. atg dose was mg/kg in all patients except patients with splenomegaly > cm from costal margin and/or sex-mismatched/maternal donor in whom atg was increased to mg/kg. all patients were younger than years and had no hepatomegaly (liver ≤ cm from costal margin) at bmt. results: actuarial overall survival (os) in the atg-bucy and flu-atg-bucy groups is % and %, thalassemia-free survival (tfs) % and %, gvhdfree and thalassaemia-free survival (gtfs) at a median follow up of . and . months was . % and . % months respectively, which is a significantly improved outcome by log-rank statistics (p= . ) in the flu-atg-bucy group. there was no significant difference between the groups in pre-transplant characteristics and posttransplant complications except for the following: median cell dose more in nd group with total nucleated cell dose of . vs . x cell/kg with p< . ; csa taper started later in the new protocol ( day vs. p= . ); median age at bmt ( . vs. . years, p= . ); number of pre-bmt transfusions (p= . ) and ferritin at bmt ( . vs. . ng/ml, p= . ) were higher in the second group; day and chimerisms were also significantly higher in new protocol (p= . and . respectively). there was a trend towards increased incidence of veno-occulsive disease (vod) and posterior reversible encephalopathy syndrome (pres) on the second group but this difference did not reach statistical significance. conclusions: adding fludarabine and targeted dose increase of atg in the standard bucy context seems to significantly improve outcomes of thalassaemia transplants without contributing to excessive gvhd or infectious complications. this protocol can be easily administered in low resource setting without major additional costs. clinical is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. however, there are still few reports on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in patients with pnh compared to paroxysmal nocturnal hemoglobinuria-aplastic anemia (pnh-aa) syndrome. our study aimed to compare the outcomes of allo-hsct for pnh with pnh-aa syndrome. methods: the clinical data of pnh patients received allo-hsct (pnh = , pnh-aa = ) in our center from july to june were analyzed retrospectively to compare the outcomes of pnh group with pnh-aa group. the clinical data including male patients and female patients, the median age was years (range - ). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to hsct was months (range - ). the conditioning regimen was modified bu/cybased regimen in haploidentical donors and unrelated donors, csa, mycophenolate mofetil (mmf) and shortterm methotrexate (mtx) were administered for graftversus host disease (gvhd) prophylaxis. patients with matched sibling donors were treated with the flu/cybased regimen and csa were administered for gvhd prophylaxis. results: there were no differences of baseline between the groups (p> . ) except gender and haploidentical donors. the median values of absolute nucleated cell counts were . ( . - . ) × /kg in the pnh group and . ( . - . ) × /kg in the pnh-aa group (p = . ). the median doses of cd + cells infused were . ( . - . )× /kg and . ( . - . )× /kg (p = . ), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were (range, - ) days in the pnh group and (range, - ) days in the pnh-aa group (p = . ). the median time for platelet engraftment were (range, - ) days and (range, - ) days (p = . ), respectively. with a median follow-up of ( - ) months in the pnh group and ( - ) months in the pnh-aa group (p = . ). in pnh and pnh-aa groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were . % and . % (p = . ), grade ii-iv agvhd were . % and . % (p = . ); chronic gvhd were . % and . % (p = . ), moderatesevere chronic gvhd were . % and . % (p = . ). in haplo-hsct and msd groups the incidences of infection were . % ( / ) and . % ( / ) (p = . ). no patient occurred early death and relapse. -year estimated overall survival (os) of pnh and pnh-aa groups were . % ± . % and . % ± . % (p = . ), gvhd-free and failure-free survival (gffs) were . % ± . %、 . % ± . % (p = . ). conclusions: the preliminary results indicated that allo-hsct is a feasible choice for pnh with favorable outcomes, time for myeloid and platelet engraftment in pnh group were faster than pnh-aa group. there were no differences in os and gffs between pnh group and pnh-aa group. disclosure: no disclosure pattern of calcineurin inhibitor-associated neurotoxicity in sickle cell disease patients receiving a stem cell transplantation background: allogeneic hsct with a msd represents currently the only curative option for sickle cell disease (scd), limited by a donor availability < %. neurotoxicity (nt) contributes significantly to hsct-associated morbidity and mortality. calcineurin-inhibitor (cni) associated nt ranges from . %- . % (severe nt %- %). the elevated incidence of nt in scd (around %) might be triggered by the systemic vasculopathy of scd, with the brain being the primary target. although both cyclosporine a (csa) and tacrolimus (fk ) have a proinflammatory effect, it is more pronounced in csa. infusion modalities also might impact ( . % after bolus injections versus . % after continuous infusion). methods: in a pilot study, we compared t-cell depleted haploidentical hsct (t-haplo hsct) with msd hsct in patients (pts) with advanced stage scd, using almost identical conditioning regimens. pts ( - years; yrs) with homozygous scd or hbs /+ ß-thal were treated between and . nine pts received a msd bone marrow graft, pts received t-haplo-hsct ( second t-haplo due to graft rejection). immunosuppression consisted of either csa ( msd, t-haplo) or fk ( msd, , in combination with mycophenolate mofetil (mmf). fk was administered as a -hours continuous infusion, csa as -hours bolus injections; both target level adjusted (csa: - ng/ml; fk : - ng/ ml). duration of immunosuppression was > months in thaplo-sct and < months in msd, depending on chimerism. results: cni-related nt was observed in . %, severe nt (pres, visual disturbance, aphasia) in . %. nt was more prevalent in msd (n= , . %) than in t-haplo (n= , . %). the incidence of nt was identical under csa ( / ; . %) and fk ( / ; . %), however the majority of severe nt (all pres) occurred with csa. complete recovery of nt was achieved in all pts either spontaneously or after switching to fk /everolimus or withdrawal of fk . moreover, . % of pts with nt were > yrs, and . % > yrs, suggesting an increased risk with age. only . % of pts with pre-existing cerebrovascular disease experienced post-hsct nt. of note, . % of pts with severe nt also developed mild acute gvhd. the overall (os) and disease-free survival (dfs) with a median follow-up of months in t-haplo-hsct and months in msd hsct was % vs. %, respectively. conclusions: our data confirm an elevated nt risk in scd pts following allo-hsct. importantly, the incidence of nt seems to be related to age ( % of pts with nt were > yrs), donor source (msd . % vs. t-haplo . %) and type of cni inhibitor where almost all severe nt ( . %, particularly all pres) was observed under csa. continuous infusion of fk vs. bolus injections of csa might have levelled concentration peaks. the nt observed with csa could be the consequence of predominantly csarelated vascular toxicity inflicting pre-damaged vessels in scd. the mechanism of action could be related to other systemic endotheliopathies such as vod, tam and agvhd, which was observed in . % of pts with severe nt, compared to an overall agvhd rate of %. disclosure: nothing to declare background: matched-related bone marrow transplantation (bmt) may cure over % of low-riskchildren with severe thalassemia (st) defined as a thalassemia syndrome with inability to keep a spontaneous hemoglobin > g/dl. it is well known that patient status at the time of transplant is critical in predicting transplant outcome. liver size > cm is an established adverse prognostic factor in terms of transplant-related mortality and, in our own experience,a spleen size > cm from costal marginis associated with increase rejection rates (blood vol. no. suppl ) . optimising liver and spleen size prior to transplant is likely to improve transplant outcomes. methods: we retrospectively reviewed the effectiveness of our strategy to reduce liver and spleen size pre-transplant using hydroxyurea, super-transfusion and intensive iron chelation. we considered liver size < cm and spleen size less than cm below costal margins as good risk features. liver biopsies were not performed thus pesaro risk classification could not be assigned. all transplant candidates were started on hydroxyurea for a minimum of months and pre-transfusion haemoglobin was maintained > gm/dl while on hydroxyurea. if the child had hepatospenomegaly at enrollmentand no improvement in liver and spleen size after an adequate trial of hydroxyurea (minimum of months of treatment achieving maximum dose of mg/kg day or tolerable haematological toxicity, i.e. neutrophil count between and /μl and/or platelet count between . and . /μl) patients were given a trial of supertransfusion maintaining haemoglobin above g/dl) for a minimum of months prior to declaring the patient as having failed downstaging. results: out of transplants across collaborating centers in india, patients had no hepatosplenomegaly at enrolment and hence were not actively downstaged. twelve patients were excluded due to inadequate information on their records. all of the remaining patients with enlarged liver and/or spleen were downstaged to low-risk features. all patients received adequate hydroxyurea trial among which seven ( %) patients required super transfusion in addition to maximal hydroxyurea. out of the patients ( %) were successfully down-staged with the above strategy and proceeded to transplant as low-risk patients. among the remaining ( %) patients had liver > cm and one had a spleen > cm only. there was significant improvement in liver and spleen size from the time of enrollment to transplant (p value . and . respectively by wilcoxon test for paired samples -two tailed) with median duration of downstaging of months (range - months). there was no significant difference in overall survival (os) and disease-free survival (dfs) by log rank test between the downstaged group and those who did not have hepatosplenomegaly at enrollment (p value . . respectively). conclusions: in the majority of children with thalassaemia and high transplant risk features liver and spleen size can be reduced pre-transplant using hydroxyurea and supertransfusions thereby decreasing transplant risk. disclosure: nothing to declare p abstract already published. abstract withdrawn. longitudinal analysis of the effect of hematopoietic cell transplantation on ocular disease in children with mucopolysaccharidosis i shows ongoing disease progression background: corneal clouding is seen in nearly all patients with mucopolysaccharidosis- (mps- ) causing visual impairment. hematopoietic cell transplantation (hct) is able to stabilize disease in many organs including the brain. however, residual disease in peripheral tissues is often described. therefore, the aim of this study was to determine the long-term effect of hct on ocular disease in mps- patients. methods: corneal clouding (grade - ) and visual acuity (decimal scale) were prospectively collected from all consecutive mps- patients treated with hct between and at the umc utrecht. the primary outcomes of interest, the effect of time on corneal clouding and visual acuity, were analyzed using a linear mixed model. the correlation between corneal clouding and visual acuity was analyzed with pearson's rho. other parameters studied were clinical phenotype, age at time of transplantation and hematological enzyme level after transplantation. other outcomes of interest analyzed included intra-ocular pressure, refraction, and macula and lens abnormalities. [[p image] . results: successfully engrafted mps- patients were included ( % with > % chimerism and normal enzyme levels after hct). corneal clouding stabilized during the first years after hct, but increased rapidly beyond three years (figure ). other predictors for increased corneal clouding were age at time of transplantation ( . , %ci . : . ; p= . ) and clinical phenotype (- . , %ci - . :- . ; p= . ). visual acuity also worsened significantly over time (- . , %ci - . :- . ; p= . ). corneal clouding was strongly negatively correlated with visual acuity (ρ - . , p = . e- ). conclusions: after initial stabilization, ongoing ocular disease is seen in mps- patients despite successful hct. this hallmarks the shortcomings of current standard therapies. new therapies that overcome the weak spots of current therapies are necessary to improve the late outcomes of these patients. clinical trial registry: n.a. disclosure: b.t.a.v.d.b. was supported by a research grant from the sylvia toth charity foundation, the hague, the netherlands, while working on this study. the sponsors of this study are public or nonprofit organizations that support science in general. they had no role in gathering, analyzing, or interpreting the data. all authors would like to thank all parents and patients for participating in this study. all authors state they have no competitive (financial) interests in this study. background: paroxysmal nocturnal hemoglobinuria (pnh) is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. haploidentical donor hematopoietic stem cell transplantation (haplo-hsct) is now increasingly applied as a curative therapy for patients with hematologic diseases. however, there are still few reports on the use of haplo-hsct for the treatment of pnh. our study aimed to compare the outcomes of haplo-hsct with matched-sibling donor transplantation (msd-hsct) for pnh. methods: the clinical data of pnh patients received hsct (haplo-hsct = , msd-hsct = ) in our center from july to may were analyzed retrospectively to compare the outcomes of haplo-hsct group with msd-hsct group. the clinical data including male patients and female patients, classical pnh and pnh-aa syndrome, the median age was years (range - ). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to sct was months (range - ). the conditioning regimen was modified bucybased regimen in haplo-hsct group, csa, mycophenolate mofetil (mmf) and short-term methotrexate (mtx) were administered for graft-versus host disease (gvhd) prophylaxis. patients with msd-hsct were treated with the flucy-based regimen and csa were administered for gvhd prophylaxis. results: there were no differences of gender, age, patients of pnh-aa and median time from diagnosis to transplantation between the groups (på . ). the median values of absolute nucleated cell counts were . ( . - . ) × /kg in the haplo-hsct group and . ( . - . ) × /kg in the msd-hsct group (p = . ). the median doses of cd + cells infused were . ( . - . ) × /kg and . ( . - . ) × /kg (p = . ), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were (range, - ) days in the haplo-hsct group and (range, - ) days in the msd-hsct group (p = . ). the median time for platelet engraftment were (range, - ) days and (range, - ) days (p = . ), respectively. with a median followup of ( - ) months in the haplo-hsct group and ( - ) months in the msd-hsct group (p = . ). in haplo-hsct and msd-hsct groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were . % and . % (p = . ), grade ii-iv agvhd were . %、 . % (p = . ). chronic gvhd were . % and . % (p = . ), moderate-severe chronic gvhd were . % and . % (p = . ). in haplo-hsct and msd groups the incidences of infection were . % ( / ) and . % ( / ) (p = . ). no patient occurred early death and relapse. -year estimated overall survival (os) of haplo-hsct and msd-hsct groups were . % ± . % and . % ± . % (p = . ), gvhd-free and failure-free survival (gffs) were . % ± . % and . % ± . % (p = . ). conclusions: the preliminary results indicated that haplo-hsct is a feasible choice for pnh with favorable outcomes, haplo-hsct and msd-hsct had similar therapeutic efficacy. disclosure: no disclosure p pres in bmt for thalassemia major in india: lower incidence and limited impact background: posterior reversible encephalopathy syndrome (pres) is a relatively common complication seen after blood or marrow transplantation (bmt) for hemoglobinopathies with a reported frequency of - %. pres has also been associated with poorer survival rates. severe hemoglobinopathies are one of the most frequent indications for bmt in the developing world, particularly in india. given the risk of rejection in multiply transfused patients and the need to minimize gvhd risk, immunosuppression post-bmt for these non-malignant conditions can be particularly intense and prolonged. we sought to measure the incidence and impact of pres in developing countries. methods: we analysed successive transplants for thalassemia using protocol (atg-bucy+csa/mmf or csa/mtx) maintaining cyclosporine a (csa) blood levels - ng/ml for patients and protocol (flu-atg-bucy+csa/mtx) maintaining higher csa levels post, i.e. - ng/ml for patients from fully matched donors with g-csf-primed bone marrow. for patients this was the second transplant from a different matched related donor. pres was confirmed with brain ct/mri for all patients. results: all recipients who had pres had sibling donors, males and females. age median . (iqr . - years). the frequency of pres was . %; disease free survival for patients who had pres was %. pres resolved completely in all. csa was switched to mmf in patients who had received mtx and were on csa only at the time of pres occurrence, while csa was stopped but mmf continued in patients taking csa/mmf combination and csa was continued for patient. three patients with pres had grade acute gvhd, had grade gvhd and none developed chronic gvhd. csa levels at the time of pres were a median of ng/ml (iqr: to ) with patient having ng/ml. three patients had pres while they were thrombocytopenic. hypertension stage was observed in four patients, stage in one patient, one patient was not hypertensive and in one patient blood pressure values were not available. two patients were on methylprednisolone and . mg/kg/day and one was on dexamethasone mg/m /day. one patient was started on csa again after the pres episode and within weeks had another one while on csa (level ng(ml), methylprednisolone . mg/kg/day and ruxolitinib for gvhd. protocol had statistically significant improvement in disease free survival from % to % (p< . ) with probability of occurrence of pres increasing from . % to . % (p = . , see figure ), yet had a benign course in all patients. conclusions: not stopping immunosuppression may have been the key factor which could explain why we have better outcomes with pres than what is reported. intensifying immunosuppression pre-bmt did lead to more pres, albeit not significantly, and yet it was quite manageable. even with addition of fludarabine our pres incidence is lower than previously reported. [[p image] . background: sickle-cell diseases (scd) are a group of genetic hemoglobin disorders marked by brain vasculopathy. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option able to stop vascular disease progression. diffusion-tensor imaging (dti) is a magnetic resonance imaging (mri) technique sensitive to the brownian motion of water molecules and cellular environment. this microscopic quantitative technique is able to detect white matter (wm) alterations before a conventional mri. the aim of this study was to use dti to evaluate axonal damage and structural connectivity in the brain of patients with scd submitted to hla-identical sibling allogeneic hsct. methods: sixteen scd patients with no extensive vasculopathy detected by conventional mri ( male, age range: - years) and age-matched healthy controls ( male, age range: - years) participated in this prospective study. mri acquisitions were performed in a t scanner two times for patients (before and - years after hsct) and at a single moment for controls. from dti acquisitions, fractional anisotropy (fa), mean (md), radial (rd) and axial diffusibility (ad) were calculated in the wm of the whole brain. structural connectivity was also analyzed, based on graph theory, obtaining efficiency, length path and clustering coefficients of the brain network. an anova test was applied to analyze fa differences among controls and patients, before and after hsct. a paired two-tailed t-test was used to determine statistical significance of changes in the fa, diffusivity mean values and network parameters before and after hsct. results: mean fa was lower in patients before hsct than controls (p = , ) and increased after hsct being not statistically different when compared to controls (controls = , ; patients before hsct = , ; patients after hsct = , ; post hoc dunnett's test -error , ; anova test). when patients were compared before and after hsct, md and rd decrease after hsct (p = , and , , respectively). on the other hand, fa increased (p = , ). after hsct, efficiency was higher (p = , ) and path length index was lower (p= , ) than at study entry (table ) . conclusions: this study indicates that, before hsct, patients with scd present axonal damage not detectable by conventional mri, when compared to healthy controls. we also suggest that hsct is able to promote axonal recovery and reorganization. partial diffusivity recovery could be associate to a still unidentified mechanism of myelin regeneration. in the future, longer follow up and comparisons with other forms of treatment are required. background: bmt is a well-established treatment modality for haemoglobinopathies, limited by the availability of related donors. unrelated transplantation has historically shown variable outcomes driven by gvhd and toxicity, and usually restricted to / matches, but the impact of reduced toxicity conditioning regimens is yet to be known. methods: from to twenty-five consecutive unrelated bone marrow transplants were conditioned with fludarabine mg/m , treosulfan g/m , thiotepa mg/ kg and atg (thymoglobulin) . mg/kg if the source of stem cells was marrow (n = ) or ptcy if pbsc (n = ). endogenous haemopoiesis was suppressed pretransplantation for a minimum of weeks. gvhd prophylaxis was provided with ciclosporin/sirolimus and mmf. thirteen patients were transplanted for b thalassaemia major, one of a thalassaemia major and sickle cell disease. the median age was years ( - ). ten patients were / matched ( thalassemia and sickle) and patients had a / match ( thalassaemia and sickle). the median cell dose was . x tnc/kg (range . - . ) and . x cd +/kg (range . - . ). the median survival was . months ( . - . ). patients with thalassaemia were pesaro class i or ii (pesaro class iii patients were intensively chelated pretransplantation to return to class i or ii). patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. results: all patients engrafted and achieved evidence of donor haemopoiesis on day + and achieved transfusionindependence and donor haematological values, but subsequently one / patient with thalassaemia suffered secondary graft failure on day + after macrophage activation syndrome. median neutrophil engraftment was days (range to ) and days ( - ) for / and / patients respectively. patient with sickle cell disease had the platelet count maintained > x /l at all times. the median platelet engraftment > x /l was days (range to ) and days (range to ) / and / patients respectively. there were three deaths, all in the / matched group: two with thalassaemia (day + due to idiopathic pneumonia syndrome and day + due to mas) and one with scd (day + due to ips). there were different trends of complications seen by degree of matching that did not segregate otherwise by disease. conclusions: in conclusion, unrelated bmt for haemoglobinopathies with reduced toxicity regimens is feasible. whilst gvhd caused significant morbidity during the transplant period, other alloreactive/endothelial complications (vod, macrophage activation syndrome, idiopathic pneumonia syndrome) were only seen in the / transplants. disease-free survival, dependent on transplantrelated mortality, and lack of long-term toxicity, including chronic gvhd, are determined by the degree of matching. / matched transplants have excellent long-term outcomes with no chronic gvhd > months and can be considered for patients without a related donor; whereas / transplant have significant toxicity and mortality, warranting a haploidentical approach. disclosure: no conflict. long-term safety and efficacy of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia following completion of the phase / northstar study patients with transfusion-dependent β-thalassemia (tdt) may benefit from gene therapy involving β-globin gene addition to hematopoietic stem cells (hscs) enabling production of functional hemoglobin (hb). lentiglobin gene therapy contains autologous cd + hscs transduced ex vivo with the bb lentiviral vector encoding β-globin with a t q substitution under transcriptional control of the encoding β-globin locus control region. the safety and efficacy of lentiglobin was evaluated in adults and adolescents with tdt in the -year phase / northstar study (hgb- ; nct ). methods: patients with tdt (≥ ml/kg/year of red blood cells [rbcs] or ≥ rbc transfusions/year) received g-csf and plerixafor for hsc mobilization. to generate drug product (dp), cd + hscs were transduced with the bb lentiviral vector. patients underwent single-agent, myeloablative busulfan conditioning, were infused with the dp, and were followed for safety and efficacy. results: eighteen patients have been treated in the completed northstar study. as of september , patients had a median follow-up of . (min -max: . - . ) months. the median age at consent was (min -max: - ) years including patients ≥ years old. patients received a median cell dose of . (min -max: . - . ) cd + cells x /kg with a median dp vector copy number (vcn) of . (min -max: . - . ) vector copies/ diploid genome. the median liver iron content (lic) at baseline was . (min -max: . - . ) mg fe/g dw. outcomes by age and baseline iron status will be presented. the median time to neutrophil and platelet engraftment was . (min -max: - ) and . (min -max: - ) days, respectively. four patients had platelet engraftment ≥ day and four patients had platelet counts of ≤ x /l at month . none of these patients had ≥ grade bleeding events post-lentiglobin infusion. transfusion independence (ti, defined as weighted average hb ≥ g/dl without rbc transfusions for ≥ months) was achieved in / patients with non-β /β genotypes and / patients with β /β genotypes. in patients who achieved ti, total hb at last visit was . - . g/dl. lic increased from baseline in patients who achieved ti by a median of . % and . % at month and then decreased from baseline by a median of . % and . % at month and , respectively. non-hematologic grade ≥ adverse events post-infusion in ≥ patients included stomatitis, febrile neutropenia, pharyngeal inflammation, and irregular menstruation. there was no transplant-related mortality, vector-mediated replication competent lentivirus, or clonal dominance. two patients experienced grade serious veno-occlusive liver disease (table ) . events resolved following treatment with defibrotide and were attributed to myeloablative conditioning. conclusions: in the northstar study, % of patients with tdt and non-β /β genotypes and % of patients with β / β genotypes achieved transfusion independence. the safety profile of lentiglobin remains consistent with myeloablative busulfan conditioning. longer time to platelet engraftment was observed in some patients, but no graft failure was reported. clinical background: sickle cell disease (scd) is an inherited hemoglobin disorder associated with high morbidity and mortality. currently, allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy for scd. transplant outcomes with thiotepa, treosulfan and fludarabine (ttf) preparative regimen are encouraging but this regimen has not been directly compared to other preparative regimens in scd. we therefore planned to compare the event free probability for death, rejection and high grade acute graft versus host disease (agvhd) between ttf and busulfan and fludarabine (bf) regimens. methods: in this retrospectively cohort study, we included all patients with scd who received allogeneic hsct at our center or who were transplanted in other centers and referred to ours for follow up before day . patients were transplanted between july and december . we used kaplan-meier curve to estimate the event free probability for death, rejection and high grade agvhd (grades - ). cox regression was used to assess the impact of the preparative regimen on these outcomes. results: a total of patients were included with a median age of years (interquartile range [iqr]: - ) and a median hemoglobin of g/dl (iqr: - ). sixtytwo percent were males. the proportion of patients who had splenectomy, stroke and acute chest syndrome was %, % and % respectively. all patients received peripherally collected hematopoietic stem cells from a matched sibling donor with a median stem cell dose of x /kg (iqr: . - . ). most patients, %, received cyclosporine or tacrolimus based agvhd prophylaxis. most patients received ttf ( %) or bf ( %) preparative regimens. all patients in the bf group received atg. the median follow-up time was months (range: - ). four patients died during the follow-up period with an os of % ( % confidence interval [ci]: %- %) at years. the os was not different (hr . , p = . ) between the ttf ( %) and the bf ( %) regimens. the probability of high grade agvhd free survival at day was % ( % ci: - ) for all patients. this probability was % in the ttf group and % in the bf group and the difference was not statistically significant (hr . , p = . ). the rejection free survival at months was % ( % ci: - ) for all patients. no patients in the ttf group rejected while the rejection free survival at months for the bf group was %. this was not statistically significant (p = . ). conclusions: in patients with scd undergoing allogeneic hsct from a matched sibling donor, the ttf preparative regimen is not associated with improved os, rejection free or high grade free agvhd survival when compared to the bf preparative regimen. larger studies are needed to confirm these findings. disclosure: nothing to declare. novel strategy for haploidentical hematopoietic stem cell transplant in sickle cell disease methods: consecutive patients suffering from scd who underwent hhsct between jan till date were enrolled in the study. all underwent autologous backup (target dose> x /kg) followed by pre-transplant immune suppression (ptis) cycles at weekly intervals using fludarabine @ mg/m /day(d -d ) + cyclophosphami-de@ mg/m /day(d ) + dexamethasone@ mg/m / day(d -d ) along with hypertransfusion (target hb - gm/dl), hydroxyurea ( mg/kg/day) and azathioprine ( mg/kg/day) from day - . the graft was mobilized using gcsf@ mcg/kg/day(d -d ) + plerixafor@ . mg/kg s/ c on d - hours before the pbsch. conditioning included thiotepa mg/kg in two divided doses (d- ), fludarabine mg/m (d- to d- ), cyclophosphamide . mg/kg (d- , d- ), tbi gy with thymic shielding (d- ), ratg (genzyme thymoglobulin . mg/kg (d- to d- ). gvhd prophylaxis included ptcy mg/kg/day on d and , sirolimus (target levels - ng/ml) (till - months post hsct) and mmf (till d ) starting from d . results: the median age of patient's was years (range - years). before transplantation all patients had repeated episodes of one or other complication warranting a transplant, non-responsive to hydroxyurea. six had maternal donors, paternal and sibling. median age of the donor was years (range - years). all were dsa negative with a cutoff mfi of > iu. all patients received x /kg cd cells irrespective of harvested dose which ranged from ( . - . x /kg). median cd dose was . x /kg (range . - . x /kg). all patients engrafted with median time to neutrophil engraftment days (range - days) and median time to platelet engraftment days (range - days). median duration of hospital stay was days (range - days). one patient had cytokine release syndrome needing tocilizumab. five had engraftment syndrome treated with short course of steroids. two had cmv reactivation needing treatment with ganciclovir/valganciclovir. acute gvhd grade ii was seen in one patient. till date of analysis none had features compatible with chronic gvhd. of the patients, are alive without sickle cell disease with lansky/ karnofsky scores of . at median follow up of days (range - ) the probabilities of survival, sca-free survival, and transplant-related mortality after transplant were . %, . %, and . %, respectively. one patient died due to mdr klebsiella sepsis after being discharged initially while he was receiving iv ganciclovir on day care basis. he had full donor chimerism. none of the patient had primary or secondary graft failure. conclusions: pre-transplant immune suppression and upfront use of plerixafor for graft mobilization decreases the risk of graft failure and graft versus host disease leading to overall better survival in hhsct for sickle cell disease. disclosure: none. combined haematopoietic stem cell transplant and enzyme replacement therapy in wolman disease: outcomes and challenges jane kinsella , denise bonney , helen campbell , robert wynn , simon jones background: infantile lysosomal acid lipase deficiencymore commonly known as wolman disease -is an autosomal recessive lysosomal storage disease, characterised by storage of cholesterol esters in the liver, spleen and gastrointestinal tract. these children present under the age of months and traditionally had a poor prognosis, with almost all being dead by the age of months. bone marrow transplant has been used to correct disease manifestations, but limited by high procedure-related mortality with the significant co-morbidities. the survival has changed over the past few years due to pharmacological enzyme replacement therapy but still presents challenges for these patients and their clinicians. in these children haematopoietic stem cell transplant we have offered bmt with enzyme replacement therapy, in certain specific circumstances. methods: four children with wolman disease being treated with enzyme replacement therapy, limited by alloantibody, or poor venous access, received treosulfan-based, myeloablative conditioning with serotherapy followed by a matched haematopoietic stem cell transplant: two family donors, one sibling donor and one unrelated donor. results: three of the four children survived transplant. they have continued to receive enzyme replacement therapy but at reduced dose and frequency with improved tolerability. they have continues to grow and develop. growth and gastrointestinal histology is improved for children having received transplant compared to those receiving enzyme replacement alone. monitoring of peripheral blood chimerism has shown a disease-associated engraftment defect, with mixed chimerism in the surviving patient. conclusions: haemopoietic stem cell transplant is a suitable treatment option in children with wolman disease in whom receiving enzyme replacement therapy is not possible because of venous access, sensitisation or cost reasons. it improves their tolerability of the enzyme treatment and allows for a reduction in enzyme dose and frequency. however, the results of engraftment are not as good as expected for a transplant with myeloablative conditioning and a matched donor. an engraftment defect has been observed in lysosomal acid lipase deficient animal models. a further understanding of this poor engraftment in children with wolman disease is required as to determine whether the risks of transplant is beneficial in these patients and for the consideration of future treatment options including gene therapy. background: thalassemia major is the most common transfusion dependent hemolytic anemia in the world. the absent or reduced production of the β-chain of hemoglobin causes severe ineffective erythropoiesis, massive erythroid hyperplasia in the bone marrow and extramedullary hematopoesis occurs. patients require regular transfusion therapy lifelong. currently, the only proven curative treatment of thalassemia is allogeneic stem cell transplantation (sct). methods: we evaluated the immune reconstitution results of patients at year after hematopoetic stem cell transplantation at our pediatric bone marrow transplantation center between january and december . all patients were not receiving any immunosuppressive treatment at least for months and they have normal lymphocyte counts, immunoglobulin levels and transfusion independent. lymphocyte subtypes and chimerism percentages and the relationship with the donor type were evaluated at year of transplantation. results: ages of transplantation was ranged between - years (median: years). seven ( %) of them was male. matched unrelated donor type was chosen in patients while others ( patients) were transplanted from family matched donor (matched sibling: patients, matched family: patients). all patients received myeloablative conditioning regimen containing busulfan/treosulfan, cyclophosphamide, thiotepa and fludarabine. follow up time was between - months (mean: ± months). in patients, whole bone marrow product was used while peripheral stem cell harvest in remaining patients. cd levels were found low in only patients, in normal patients mean was % ± %. cd levels were severely low in patients while cd in only patient. cd levels were increased in total patients in as compensatory. cd /cd ratios were very low in all patients (range: . - . ). b cells (cd +) were low in patients while immunoglobulin levels were normal. chimerism values between - % (mean: ± %). donor and product types did not differ in cd + lymphocyte reconstitution at year (p= . , p= respectively). all patients were alive and well at year after transplantation. conclusions: after year of transplantation, although patients are in well condition regarding to infection frequency and transfussion dependency, it was seen that their lymphocyte subtypes reconstitution could not be achieved enough as in normal children. we can conclude that low cd + cell levels were an expected finding in almost all patients. so, these patients may have a tendency to suffer serious bacterial and viral infections, and close follow up be required in terms of infections as long as cd levels continue to be low. immunoglobulin replacement therapy did not required even in patients with low b cell levels. disclosure: nothing to declare p phase international, multicentre trial to assess haploidentical aß t-cell depleted stem cell transplantation in patients with sickle cell disease with no available sibling donor background: sickle cell disease (scd) is an inherited disorder with an estimate of , affected newborns per year worldwide. allogeneic hematopoietic stem cell transplantation (hsct) with a matched sibling donor (msd) is currently the curative standard of care for scd patients (pts). however, msd availability is < %. a t-cell depleted haploidentical hsct (t-haplo-hsct) from a relative, mostly a parent, expands the donor availability while exhibiting low gvhd rates and thus could offer cure to the remaining % of scd patients. in a pilot study, comparing t-haplo-hsct with msd hsct in advanced stage scd, using almost identical transplant regimens for both. the overall (os) and disease-free survival (dfs) was % vs. %, respectively. methods: these results led to the design of a clinical trial to assess tcd-haplo-hsct prospectively which aims to demonstrate that a hsct from a haploidentical relative is not inferior to a msd hsct with regard to major outcome parameter. this phase , prospective, stratified, open-label study is targeting enrollment of patients aged - years with homozygous hbs disease or heterozygous hbsc or hbs /+ ß-thal suffering from severe or moderate scd related complications. inclusion criteria are clinically significant scd related complications such as stroke, silent crisis, pathological angio-mri, transcranial doppler (tcd) velocity > cm/s, or more episodes of acute chest syndrome (acs) in a lifetime, chronic transfusion dependency, transfusion-refractory allo-immunization and others. pts fulfilling inclusion criteria will be stratified according to donor availability. pts with a msd will receive a bone marrow graft, pts requiring an alternative donor will be transplanted with an aß/cd depleted graft from a haploidentical family donor. the conditioning regimen for both groups will be identical with the exception that antithymoglobulin (atg-neovii ® ) is given upfront in thaplo-hsct versus day - to - in msd. chemotherapy consists of thiotepa, fludarabine and treosulfan. posttransplant immunosuppression will consist of mofetil mycophenolate and tacrolimus for a duration > months in t-haplo-hsct and < months in msd, depending on chimerism. (eudract number: - - ) results: primary efficacy endpoint: event free survival (efs). event is defined as incidence of acute gvhd, grade iii -iv, chronic gvhd, rejection (graft failure) or death (for any reason). key secondary endpoint(s) are os, dfs, graft failure, hematological and immunological reconstitution, quality of life (qol) assessment and fertility. the primary null hypothesis is: efs of scd patients treated with t-haplo-hsct is non-relevantly inferior to efs in the msd arm. conclusions: results will help to determine if an a/ß depleted t-haplo-hsct can be considered equivalent to msd hsct with regard to dfs, adverse events and safety, in order to offer this form of cure to the majority of patients with scd. disclosure: nothing to declare hit three birds with one stone: successful stem cell transplantation from one family donor to three siblings methods: in august , three thalassemic siblings were admitted to hospital for stem cell transplantation from a full match donor, their years old sister. the patients' general health conditions and specific health issues due to thalassemia were checked extensively. it was decided to perform first transplant to older sister whom the disease and transplant complications are expected more intense due to prolonged transfusion and chelation therapy. the oldest daughter of family, healthy, was planned to accompany her sisters in transplantation unit so parents can take care the others and organize this period for whole family. results: the years old sibling was first admitted to bone marrow transplantation unit in july . the conditioning regimen was busulfan, fludarabine, cyclophosphamide and thiotepa with antithymocyteglobulin(atg) and defibrotide prophylaxis was given. the healthy donor was admitted to hospital and received g-csf for continuous days before harvesting. stem cells were collected peripherally on day and viable cd + cells were /ul. patient received , x e /kg stem cell and the other cell products were divided into parts according to other recipients´weight. no infusion problems were recorded in stem cell transfusion. gvhd prophylaxis was given with cyclosporin and methotrexate. severe sinusoidal obstruction syndrome was observed and successfully managed with supportive therapy. neutrophils were engrafted + . day, and platelets were on day . full blood chimerism results were % in day , % in day and % in day consecutively. after months from first transplant the years old sister was admitted to hospital on october . same conditioning with defibrotide prophylaxis and gvhd prophylaxis were given and , x e /kg peripherally derived and previously frost stem cell was infused without any complications. mild sinusoidal obstruction syndrome was observed and managed with supportive therapy successfully. neutrophils were engrafted + . day, and platelets were on day . full blood chimerism results were % in day , % in day and % in day consecutively. the third transplant was performed on january with the same conditioning and prophylaxis regimen. although defibrotide was used mild sos was observed and treated with supportive therapy with success. neutrophils were engrafted + . day, and platelets were on day . full blood chimerism results were % in day , % in day and % in day consecutively. conclusions: the patients are being followed for over a year after first transplat, neither adverse nor gvhd symptoms were observed. we presented this case for being a unique example for match family donor transplant and the first successful example from one donor to three recipients. disclosure: nothing to declare results: our female patient admitted for anemia at rd month of birth and was transfused every - months from th months to . years of age. since investigations directed towards hemoglobinopathies or membrane defects like hereditary spherocytosis were unremarkable, she was not transfused for years after the age of . -years because hemoglobin level was constant over g/dl. her bm examination showed erythroid hyperplasia and feature of dyserythropoiesis with a few binucleated erythroblasts. it was decided to follow-up the patient with a diagnosis of cda ii. after the age of -years, the need for transfusion started again for every to months which led the parents of our patient to request for bone marrow transplantation, however, the diagnosis was not definite, and because of the insufficient data for the transplantations for cda ii patients, it was decided to go on to follow-up. nevertheless, after years, the frequency of transfusion gradually increased to every - weeks, and bone marrow transplantation was brought into question again. at that time, genetic examination was started and sec b gene was analyzed by direct sequencing. hsct decision from her hla / matched brother, carrying sec b mutation in heterozygous state, was taken. in the preparation regimen, busulfan (bu) at a myeloablative weight adjusted dose ( days), mg/kg cyclophosphamide (cy) ( days), and mg/kg antithymocyte globulin (atg fresenius) were used. graftversus-host disease (gvhd) prophylaxis was with cyclosporin a started on day - and short-term methotrexate on day + ,+ and + . she was transplanted with bm with a dose of total nucleated cells= . x /kg and cd = x / kg. neutrophile and platelet engraftment were achieved at + and + , respectively. indeed, grade hemorrhagic cystitis due to bk virus and a moderate veno-occlusive disease prolonged platelet transfusion days which concealed the exact engraftment day of platelet. the patient was discharged on day with no more need for any transfusion and followed up as a complete chimeric with no type of gvhd since then. now, she is years old, under regular surveillance at our transplant centre without any symptoms. conclusions: hsct data in cda ii patients is still insufficient, however based on data from tm patients with similar treatment approaches in td cda ii patients, it is seen that the hsct is reliable and effective. disclosure: nothing to declare hematopoietic stem cells background: the prognosis after frontline therapy in b-all patients have improved due to monoclonal antibodies (cd , cd , cd ) and approximately % of patients achieve complete remission. in relapsed and refractory (r/ r) b-all and also in mrd + outcomes are relatively poor. disease-free survival (dfs) in this cohort is - %. in this cohort allo-hsct is indicated and complete remission before transplantation is crucial for prognosis. conventional chemotherapy is associated with high failure rate and significant toxicity. immunotherapy with monoclonal antibodies and car-t are more promising approaches. the aim was to evaluate the efficacy (frequency of responses, os, dfs) and toxicity, especially neurotoxicity and cytokinerelease syndrome, of a bispecific monoclonal antibody blinatumomab in patients both children and adults with persistence of minimal residual disease (mrd + ) or r/r b-all as a bridge to allo-hsct. methods: this study included patients with high risk b-all blinatumomab treated in - , among them pts ( %) with t( ; ), ( %) with t( ; ), with mll ( %), pts ( %) who were refractory to previous chemotherapy, ( %) after allo-hsct from deferent type of donors. median age was y.o. (range m- y.o), children - y.o. ( %) and adults > y.o. ( %). r/r all had pts ( %), mrd + - pts ( %), median days of follow up were ( - ). blinatumomab was applied as -day cycles followed by a -day off-period before the start of the following cycle. majority pts received one cycle (n= , %). in r/r all group dose was of mcg/d during the first days and afterwards mcg/d. patients with weight less than kg received mcg/m /d and mkg/m /d accordingly. in mrd group dose was mcg/m /d. results: the frequency of responses to blinatumomab was higher in mrd + pts in comparison r/r all pts ( % vs % p= . ). in mrd + pts cr mrdwas achieved in pts ( . %), pts ( . %) were mrd+ after blinatumomab. two-year os in this group was %. twenty pts ( %) received allo-hsct. in rr all pts cr mrdwas achieved in pts ( %), pts ( %) were mrd+ after blinatumomab, pts ( %) had no hematological response . two-year os in r/r all was %. fifteen pts ( %) received allo-hsct. os in cr mrdpatients who received allo-hsct was not significantly different in comparison with patients who received blinatumomab as a monotherapy ( % vs %, p= . ). no significant differences in dfs were observed at two years in cr mrdpts depending status of the disease before therapy-mrd vs r/r ( % vs %). of the reported adverse events, febrile fever was the most common pts ( %), neutropenia ( %), thrombocytopenia ( %), infection ( %), neurotoxicity ( %), cytokine-release syndrome ( %). all complications were reversible. conclusions: blinatumomab is effective option in patients with high risk b-all especially in the group with mrd persistence after previous chemotherapy and facilitates effective bridging to hsct. blinatumomab therapy is generally well tolerated. disclosure here we address the transcriptional regulation of differentiated cells from human embryonic stem cells (escs) using self-assembling peptide hydrogel without stromal cells, and compare with embryoid body (eb) culture system. methods: esc differentiation was induced in eb culture system or three-dimensional ( d) hydrogel culture system. the engraftment potential of differentiated cells was evaluated by flow cytometry. cd + cells from mobilized peripheral mononuclear blood cells (mpbmcs) or differentiated from escs at different times (day , day , day ) were purified by fluorescent-activated cell sorting. sorted cells were captured on medium-sized microfluidic chips using the fluidigm c single cell auto prep system. sequencing was performed by hiseq x ten. results: self-assembling peptide hydrogel formed a d scaffold for cell culture, the pore diameter of which ranged from to nm. compared to eb culture system, escs in d culture system differentiated more potently. the differentiated cells from d system were short-term engrafted in the nog mice, and myeloid cells, b cells and t cells could all be detected in peripheral blood after transplantation. however, the engraftment was not obtained in differentiated cells from eb culture system. we obtained and analyzed escs, cd + cells from eb culture system, cd + cells from d culture system, and cd + cells from mpbmcs. the cells were divided into cluters ( figure a ). in both differentiation systems, the cd + cells from day were more heterogeneous than cd + cells from day and day ( figure b) . however, cd + cells from mpbmcs were more homogeneous, probably because the differentiated cd + cells contained several cell lineages, including hematopoietic cells, endothelial cells and mesenchymal cells. there is transcriptional overlap between individual cd + cells from eb and d culture systems. however, we found that cluster , which is composed mainly of cd + cells from d at day and day , expressed similar level of several hematopoietic regulator as hsc, such as tal , lmo , erg ( figure c ). the cluster , which is almost the cd + cells from d at day , also expressed the highest gata among the clusters from differentiated cells ( figure c) . conclusions: our study demonstrates that d hydrogel culture system facilitates hematopoietic specification of escs. disclosure: nothing to declare higher cd + cell dose increases overall survival in the setting of dual t-lymphocyte suppression with atg and ptcy in matched related and unrelated donor allosct background: there is no consensus on the cd + donor cell numbers required for optimal outcomes in allogeneic stem cell transplant (allosct). there is controversy on the benefits or harm in higher cell dose for allosct. this study aims to evaluate the impact of cd + cell dose in allosct patients receiving reduced intensity conditioning (ric) combined with anti-thymoglobulin (atg) and posttransplant cyclophosphamide (ptcy) using related (mrd) and / and / matched unrelated donors (mud). methods: this is a single-centre retrospective analysis of adult patients who received allosct for hematologic malignancies between october and may . all received ric using fludarabine ( mg/m /day: day - to - ), busulfan ( . kg/m /day: day - and - ) and total body irradiation ( cgy: day - ). all patients also received rabbit-atg ( . mg/kg: day - to - ), ptcy ( mg/kg/day: day + ,+ ) and cyclosporine (from day + ). unmanipulated peripheral blood stem cells were infused on day . analyses were done using thresholds: ( ) an arbitrary cd + cell dose of x /kg (as this was our target dose) and ( ) cell dose according to quartiles (< . , . - . , . - . and ≥ . x /kg). results: median cd + cell dose was . x /kg. median follow up was months (range - ). median neutrophil engraftment was (range - ) days and platelet engraftment was (range - ) days. a cell dose greater than x /kg was associated with an increased overall survival (os) at year ( . %; % ci, . - . vs . %; % ci . - . ; p= . , figure ). the higher dose was also associated with shorter platelet engraftment time (p= . , figure ). there was no significant difference in neutrophil engraftment, nonrelapse mortality (nrm), relapse free survival (rfs), grade ii-iv acute graft versus host disease (agvhd) and moderate to severe chronic graft versus host disease (cgvhd), (table ) . analyses using quartile cell dose thresholds showed a trend towards decreased os with a cell dose of < . x ^ /kg, however this was not statistically significant ( figure ). higher cd + cell doses were associated with shorter platelet engraftment time (p= . , figure ). there was no significant difference in neutrophil engraftment, nrm, rfs, agvhd and cgvhd (table ) . conclusions: cd + cell dose greater than x /kg significantly increases overall survival in the setting of ric and dual t-lymphocyte suppression with atg and ptcy in mrd and mud allohsct. further studies in a larger number of patients and longer follow up are recommended to validate these findings. disclosure methods: fifty two adult patients were included. median cd + cells requested for infusion were x ^ /kg. all patients received the same ric regimen including fludarabine ( mg/m /day day - to - ), busulfan ( . kg/m /day day - and - ), and total body irradiation ( cgy) (day - ) combined with rabbit-atg ( . mg/kg: day - to - ), ptcy ( mg/kg/day: day + ,+ ), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last followup was november . median follow-up was months (range - ). median cell dose count infused was . cd +/kg. we arbitrarily divided the cohort in two groups with cd + dose of > x ^ cd /kg as cut-off point. results: findings are summarized in figure . the infusion of more than x ^ cd /kg dose had a significant worse impact on overall survival (os) (p= . ), relapse-free survival (rfs) (p= . ) and cumulative incidence of acute gvhd (p= . ). chronic gvhd could not be compared between the two cohorts due to the different median follow-up. conclusions: the infusion of a cd + cell dose count higher than x ^ cells/kg had a significant adverse impact in overall survival and grade ii-iv acute gvhd in the setting of ric and dual t-lymphocyte suppression with atg and ptcy for haplohsct. disclosure: nothing to declare p long-term thymic activity and immune-reconstitution after haplo-identical allografting with post-transplant cyclophosphamide background: the use of post-transplant cyclophosphamide (ptcy) has expanded the application of t repleted haploidentical stem cell transplantation (haplo-hsct). in this setting, to investigate thymus role in longterm clinical outcomes, evaluation of immune reconstitution kinetics was performed. methods: twenty-nine patients (median age ) were enrolled. blood samples were collected before conditioning and at , , , , , months after haplo-hsct. analyses of cd and cd t-cell subsets by flow-cytometry were correlated by generalized linear models with real-time pcr (rt-pcr) quantification of signal joint t-cell receptor excision dna circles (sjtrecs), specific marker of naive t-cells thymopoiesis. a) naive; b) central; c) memory; and d) revertant cd and cd t-cells were defined as follows: a) cd ra+cd l+; b) cd ro +cd l+; c) cd ro+cd -; and d) cd ra+/ ro +, respectively. sjtrecs rt.pcr was performed on genomic dna ( ng) extracted from sorted cd and cd t-cells. results: a gradual increase in absolute numbers of all cd and cd t cell subsets and of sjtrecs copies from the first month up to years post-transplant was observed ( figure ) . however, at years, cd and cd t-cell levels and sjtrecs levels were lower than those observed in healthy donors. sjtrecs kinetics was associated with the increase in cd naive t-cells (overall, p < . ). this correlation suggests that most of cd naive t-cells derives from thymic re-education of donor precursor stem cells, whereas cd naive t-cells undergo peripheral expansion after thymic production. furthermore, an increase in cd revertant memory t-cells was also significantly correlated with sjtrecs kinetic (p , ). central and effector memory t-cells showed a faster thymic-independent expansion in both cd and cd tcells. interestingly, sjtrecs levels and thymic dependent immune-reconstitution were higher in a cohort of patients undergoing hsct from hla identical donors (manuscript in preparation). clinical outcomes and thymic function were correlated starting at months after hsct. lower thymic output was significantly associated by multivariate analysis with low pre-transplant trecs values (p , and p < , in cd and cd , respectively), moderate-severe chronic graft-versus-host disease (gvhd; p < , in cd ), and age (≥ years, p , in cd ). conclusions: the thymus, despite age-dependent involution, substantially contributes to t-cell reconstitution after haplo-hsct. chronic gvhd and older age were significantly correlated with reduced thymic function. overall, lower production of sjtrecs after haplo-hsct as compared after hla identical sibling hsct may partly be due to a higher degree of "mismatching" of mhc molecules during thymic re-education. [[p image] . figure ] background: the use of allogenic hematopoietic stem cell transplantation (hsct) in the treatment of adolescents and young adults (aya) with philadelphia negative all is decreasing with the adoption of pediatric inspired protocols to treat this age group and the incorporation of minimal residual disease assessment in the routine care of all patients. previously, its use was defined mainly by disease risk features at presentation. methods: a study on aya (age - years), who underwent allogenic hsct at our institute for philadelphia negative all, between february and december . all the studied patients received calgb based adult chemotherapy protocol for induction, and underwent a matched related donor (mrd) transplant with cy/tbi conditioning and mtx/csa as gvhd prophylaxis. the patients were eligible for allogeneic hsct, if they have a mrd plus one or more of the following risk factors: ( ) age ˃ years, ( ) high presenting wbc count (> for b-all, > for t-all), ( ) high risk immuno-phenotyping (pro-b, pro-t, early t, and mature t), ( ) bulky splenomegaly or bulky lymphadenopathy, ( ) high risk cytogenetics ( ; , ; , low hypodiploidy/near triploidy, complex), ( ) cns involvement, ( ) relapsed or refractory disease at d of induction. in this study, we investigated the impact of those different risk factors on the long term outcome of allogeneic hsct. results: the median os of our studied patients was not reached at . years, with a median dfs of . years (figure ). in a univariate analysis, relapsed or refractory disease prior to transplant was the only independent risk factor for os and dfs (p-value= . , and . respectively) (figure ). in addition, patients who had or more risk factors ( , . %) prior to transplant had a significantly lower long term outcome compared to patients, who had one ( , . %) or two risk factors ( , . %) with a median os of months, and a median dfs of only months (p-value= . , and . respectively) ( figure ) . conclusions: our results show that the long term outcomes of hsct in aya with philadelphia negative all treated on an adult type chemotherapy regimen, were significantly better in patients who showed a good response to initial therapy and a limited poor prognostic factors at presentation, with worsening of dfs as the number of poor prognostic features increase. we can conclude that, using this risk score can be helpful in predicting the outcome of allogenic hsct in aya with philadelphia negative all treated with adult type chemotherapy protocol. disclosure: no conflict of interest a prospective single center survey on donor-specific anti-hla antibodies and desensitization strategy in patients undergoing an allogeneic stem cell transplant background: in the setting of hematopoietic stem cell transplantation (hsct), considering the risk of poor engraftment or graft failure (gf), the detection of antibodies (ab) directed against donor specific hla loci (dsa) represents a contraindication to proceed with the same donor, suggesting the search of other donors. in many cases, there is not sufficient time to search for alternative donors and it is necessary to plan an immunosuppressive strategy to decrease the dsa level, thus reducing the risk of gf. to date, there is no consensus on desensitization standards to manage dsas in hsct. the aim of this study was to determine the incidence of anti-hla ab and dsas in hematologic patients candidate to an allogeneic hsct, and the efficacy of our desensitization protocol. here, we present an update of the results obtained with our strategy. methods: between august and september , we prospectively screened for dsa consecutive patients candidates to an allogeneic hsct. anti-hla ab research was carried out using the luminex bead assay (lifecode screen and lsa i/ii-immucor). the results were expressed as mean fluorescence intensity (mfi); mfi > was considered positive. in case of a mismatched related donor, a flow cytometric crossmatch test (fcxm) was performed. if the patient had dsas and only one available donor, a desensitization strategy was employed, scheduled with rituximab on day - , single-volume plasmapheresis procedures (pp), usually on day - and - , intravenous immunoglobulins on day - , infusion of hla selected platelets for dsa absorption in case of persistent antibodies directed against class i hla antigens. the aim of this schedule was to avoid interferences with chemotherapy and anti-t-cell globulins, infused during condition regimen results: since august , patients have been prospectively screened. thirty-three patients ( . %) showed anti-hla ab and of them ( . %) had dsas: were treated with the desensitization strategy, applied according to the mfi score and the fcxm result, and all of them obtained an engraftment; in cases, an alternative donor was selected and in case the research for an alternative donor is still underway. dsa detection was performed every days after hsct for the first month and , and days following hsct. neither a dsa rebound nor other complications were observed during the follow-up. conclusions: our prospective analysis underlines the high frequency of anti-hla antibodies detection in hematologic patients, confirming the necessity to routinely evaluate the presence of dsas before an allogeneic mismatched hsct. our desensitization schedules based on the combination of pp, rituximab, ivig and platelet absorption proved successful in reducing dsas. we confirm the necessity of a prospective multicenter collaboration to better define the role of dsas against each hla locus and the critical mfi cut-off level associated with a higher risk of gf. transplant and transfusion specialists should joint to define a consensus for a standard desensitization strategy. disclosure the most frequent technique used for counterbalance partial incompatible hsct is cd + selection that is associated with sustained engraftment and effective reduction of t cells that minimizes gvhd. on the other hand, this approach could delay immune reconstitution and increase risk of viral and fungal infection. in mud setting the use of pbsc is the procedure that most centers have recently adopted. this implies the infusion of a relevant higher number of t cells to times more as compared with bone marrow (bm). since in our centre most part of our patients are primary immunodeficiencies, we applied a procedure to minimize the risk of severe gvhd infusing a controlled number of cd positive cells. methods: we report data about paediatric patients who received mud hsct ( patient received hsct) between and in the bmt unit of the children's hospital of brescia. patients received conditioning, according to the european group for bone marrow transplantation (ebmt) and the european society for immunodeficiencies (esid) guidelines. cd + selection has been realized by a milteny column with an ideal addback of cd positive cells of x /kg. stem cell source was bm in cases and pbsc in cases. results: median patients age at transplant was years (range . months- years). the mean number of infused cells were: x /kg cd + and x /kg cd + in bm product, while x /kg cd + and x /kg cd + in pbsc. mean time for engraftment was day post-hsct. as concerns acute gvhd overall incidence . % ( / ) of the children presented this complication, but only % ( / ) presented gvhd grade iii and none gvhd grade iv, while chronic gvhd presented in . % ( limited, extensive/ ). while acute gvhd incidence and severity weren't significantly different between bm recipients and pbsc recipients, the cases of chronic gvhd were prevalently in the latters. no major infections presented in the post-transplant period and immunological reconstitution both cellular and humoral was completed by months. overall survival at years is % ( / ). the results obtained show how it is possible control severity of gvhd if an addback of a controlled number of cd + lymphocytes. acute gvhd wasn't severe and only few children presented with limited chronic gvhd. the method allows to graft primary immunodeficiencies patients even with pbsc without infusing too many t cells. in fact, especially in very young children, the number could be excessive and risky. nevertheless in case of an oncohaematological patient, gvl effect is preserved. disclosure background: dc is a rare genetic disorder that results from a defective telomere length maintenance and is characterized by mucocutaneous features, bone marrow failure (bmf) and a high predisposition to cancer and pulmonary fibrosis. bmf remains the major cause of mortality and the hsct is the only definitive treatment to restore hematopoiesis but is limited by a high incidence of treatmentrelated mortality. methods: a retrospective analysis of patients (pts) with dc who underwent hsct at the bone marrow transplantation unit in the clinical hospital of federal university of paraná, brazil, between july- and november- . results: boys and girls, with a median age of y ( - y) received a hsct from a mds (n= ), mud (n= ) or mmrd (haploidentical, n= ). pts received bone marrow (bm) and pt received a cord blood unit (cbu). the median of tnc infused was , x /kg (range , - , x /kg) and in the cbu was , x / kg. two pts received a myeloablative preparatory regimen with busulfan (bu) mg/kg + cyclophosphamide (cy) mg/kg or fludarabine (flu) mg/m + antithymocyte globulin (atg). the remaining pts received a ric regimen with cy mg/kg (n= ), flu mg/m + cy mg/kg + atg mg/kg (n= ), and flu mg/m + cy + tbi rads (n= , haplo). graft versus host disease (gvhd) prophylaxis consisted of cyclosporin (csa) and methotrexate or steroids (cbu) and post-transplant cy + csa + mycophenolate mofetil in the haploidentical transplants. of evaluable pts engrafted with a median time to neutrophil recovery of days (range: - days). one patient experienced primary graft failure (haplo) while second graft failure occurred in other pts. all these pts went a second hsct and survived. acute gvhd grade ii-iv occurred in of pts at risk. moderate to severe chronic gvhd occurred in pts with cases occurring in pts who had previously presented acute gvhd. overall survival (os) was , % at a median follow-up of y. the y os was slightly better in msd transplants compared to the others ( , % x , % p= , ). causes of early death include adenovirus sepsis (n= ), toxicity to preparatory regimen and sepsis (n= ), primary graft failure (n= ). pts remain alive between - y after hsct with a median fu of y. among them only pt has developed organ involvement by the underlying disease: hepatopulmonary syndrome (hps). pts died due to pulmonary fibrosis (n= ), liver fibrosis(n= ), gi bleeding(n= ), hps (n= ); cgvhd and sepsis(n= ), infection (n= ), and pts were lost to fu. conclusions: early mortality from bmf can be reduced by hsct, but late outcomes remain a consequence of the underlying disease. long term fu is essential in order to detect late complications related to the hsct procedure or the underlying disease. disclosure: nothing to declare single intra-bone cord-blood transplantation with a treosulfan-based regimen, atg-free and sirolimusbased gvhd prophylaxis: fast hematopoietic engraftment and immune-reconstitution in patients background: cord blood transplants (cbt) require less stringent hla-matching, compared to peripheral blood stem cell or bone marrow. however, cbt has been associated with delayed engraftment and immune reconstitution, especially if in vivo t-cell depletion, such as antithymoglobulin (atg), is used. methods: from to , patients with high-risk diseases received intra-bone infusion of unwashed single cb unit with an atg-free gvhd prophylaxis; were in active disease at cbt and had received prior allogeneic stem cell transplantation. median age was y [range (r) . conditioning regimen was myeloablative, with treosulfan and fludarabine in all, intensified with melphalan in or with gy tbi in . hla matches was / , / , / in , and cases, respectively. gvhd prophylaxis included sirolimus and mycophenolic acid (mmf). results: after thawing, median cd + cells was . x /kg [r . - . ], median cd + cells . x /kg [r . - . ], and median cd + cells . x /kg [r . - . ]. of the evaluable patients all engrafted with a sustained full donor chimerism at day . median time to neutrophils ( / , anc> /μl for consecutive days) and platelet engraftment ( / immune-reconstitution of cbt patients (tables a-b ) was compared with two cohorts of patients transplanted at our center from any adult donor with ( ) or without ( patients, including post-transplant cyclophosphamide cohort) atg in association with sirolimus and mmf. profiles of immune-reconstitution at day - - showed a better cd + recovery at any time-point in both cbt and no-atg versus atg cohort, with no statistic significant difference in the first cohorts. moreover, cd +/cd + ratio at any time point was better in the cbt cohort vs the no-atg cohort. b cell recovery was faster in the cbt cohort; immunoglobulin recovery was superimposable across different platforms. focusing on late events (> days from cbt), / pts experienced ebv reactivation, median time days [ - ] treated with rituximab, and experienced late hhv and cmv reactivation, both solved at last visit. sirolimus was withdrawn after a median of days [r - ]. only patient developed severe chronic gvhd, solved at last visit. overall, after a median follow-up of days [r - ], pts are alive and well. conclusions: our data confirm that intra-bone cbt without in-vivo t-cell depletion is associated with fast hematopoietic engraftment and immune-reconstitution, with very low rate of chronic gvhd and late infective events. background: a promising improvement of hematopoietic stem cell transplantation (hsct) may lie in the transplantation of high numbers of pluripotent stem cells to minimize the time span between transplantation and immunological reconstitution. hence, an ex vivo platform is needed that supports hsc proliferation before application and, at the same time, the maintenance of pluripotency by diminishing hsc differentiation into lineage-specific progenitor cells. methods: to artificially model the natural hsc niche in vitro, we used d bone marrow (bm)-like scaffolds made of polydimethylsiloxane (pdms). these structures are based on a human long bone cross section as a representative of the bm. human cryoconserved hscs were cultured in distinct cultivation systems for days under different conditions. cell counting and facs analyses at day were conducted. for characterization of the cultivated hscs, we used antibodies against cd alone or in combination with antibodies against cd , cd , cd ra and cd f. results: for optimization of culture conditions for human hscs, a commercially available medium was supplemented with a panel of cytokines and valproic acid. we found a significant increase in the number of cd + hscs by simultaneously increasing their vitality using the d system compared with conventional d culturing. a further improvement was achieved by introducing a silicon oxidecovering of the d pdms structures, suggesting that hydrophilic surface properties offer superior attachment for semi-adherent hscs. for a more precise characterization of the cultivated hscs, we introduced a panel of facs markers reflecting the immaturity of the amplified hsc. surprisingly, with increasing immaturity of the cultivated hsc, non-covered d pdms revealed to be best suited for amplification: cell number of vital immature hscs was increased after cultivation on non-covered d pdms compared with silicon oxide-covered d pdms and the d system. conclusions: by establishing a d scaffold according to the human bm, we found a platform mimicking the natural niche of human hsc which is suitable to amplify human hscs in vitro and support their vitality, pluripotency and ability for self-renewal. [[p image] . with the introduction of sion covering of d pdms structures the maintenance of cd + hscs could be further improved. despite the better conservation of cd + hscs by using silicon oxide-covered d pdms, we found that immature human hscs obviously prefer more hydrophobic conditions found on non-covered d pdms. disclosure: nothing to declare. abstract already published. improvements in neutrophil engraftment following changes in freezing method background: in the setting of autologous haematopoietic progenitor cell (hpc) transplants for haematological disorders, peripheral blood stem cells are routinely collected via apheresis and cryopreserved. leicester royal infirmary had been using a controlled rate freezer (crf) to cryopreserve cellular therapy products up until . in a literature review of cryopreservation techniques was undertaken, since the crf required replacement. this review found consistent evidence that cryopreservation using minus o c is comparable to crf, and engraftment times should not be negatively affected by changing to a more simplified method of freezing. there would also be cost saving benefits from switching from a crf to minus o c freezers. methods: as a result two minus o c freezers were purchased following the acceptance of a preparation process dosier (ppd) which was prepared for the human tissue authority. validation was carried out, and from january stem cell laboratory at the lri switched from the crf method to minus o c for cryopreservation of cellular therapy products. briefly, cells are frozen using % dmso (wak-chemie) in g/l human albumin solution (grifols) in cyrobags (origen biomedical, inc). the cells are transferred on cold packs to the minus o c freezer. cells are packaged in between stainless steel heattransfer plates. the plates are placed within a bubble wrap bag, and are placed in a rack within the minus o c freezer, which allows air to circulate freely around each bag. in one plate a el-usb-tc thermocouple data logger (thermosense) is inserted between the bag and stainless steel plate, to record the freezing profile. this data is downloaded after each run. after an overnight freeze at minus o c, the cells are subsequently removed from the bubble wrap and plates, and are transferred to minus o c freezers the following morning. results: a total of patients have had autologous stem cells frozen using this method so far this year. in addition to engraftment data for neutrophils, post-freeze trypan blue viabilities were also compared to the previous year. during a total of patients, who had all their cells cryopreserved, underwent collections. the post freeze median viability was % ( - %). a total of median neutrophil engraftment was . days, with a median cd dose infused of . x /kg. during so far, patients have undergone collections. median viability is . % ( - %). subsequent median neutrophil engraftment is days, with a median cd dose infused of . x /kg. conclusions: ongoing savings of approximately £ per annum have been made by changing our procedure. the benefit of changing to a simplified method of freezing has also resulted in a reduction in staff working overtime. more importantly, this simplified cryopreservation method has resulted in an improvement in neutrophil engraftment times since changing the cryopreservation technique from the previous method using crf to mechanical freezing using a minus o c freezer. disclosure: nothing to declare low doses of granulocytes in the apheresis product predict a better outcome of autologous hematopoietic stem cell transplantation in multiple myeloma patients background: high-dose chemotherapy with autologous stem-cell transplantation (asct) remains the standard consolidation therapy for multiple myeloma (mm). peripheral blood stem cell collection may be contaminated with large quantities of granulocytes and its consequences on the outcome of asct are still unclear. on the other hand, the effect of performing apheresis with high levels of monoclonal component (mc) on outcome is unknown. the objective of this study was to analyze the effect of total nucleated cells (tnc) and granulocytes count (considered as contaminating components of apheresis products) as well as the influence of mc in the apheresis product on outcome of asct in mm. methods: eighty-two patients diagnosed with mm were mobilized with filgrastim μg/kg/day (plus plerixafor if insufficient mobilization of cd + cells on day ). apheresis collection was performed with cmnc program by spectra optia cell separator. cd + count was carried out according to ishage protocol (target: ≥ x e cd +/kg). subsequent cryopreservation were performed according to the local protocol. results: the medians (range) of collected cd +, tnc and granulocytes were . x /kg ( . - . ), . x / kg ( . - . ) and . x /kg ( . - . ), respectively. the medians (range) of infused cd +, tnc and granulocytes were . x /kg ( . - . ), . x /kg ( . - . ) and . x /kg ( . - . ), respectively. a successful collection after first line therapy was performed in % of patients. treatment for mm was continued after carrying out apheresis in % of patients, as per protocol. a significant reduction of mc was observed prior to asct, indicating a further improvement in responses after apheresis (p= . ). an optimal response (cr or vgpr) at the time of apheresis was achieved in % of patients and a suboptimal response (partial or minimal response) was observed in the remaining %. undergoing apheresis in optimal response did not result in lower number of tnc or granulocytes in the harvest. the subtype of mc did not influence on the number of tnc and granulocytes in the product of apheresis. no differences in collected tnc and granulocytes were observed when plerixafor was used as mobilizing agent. the type of chemotherapy given prior the apheresis did not have influence on the characteristics of the apheresis product. a significant improvement in overall survival (os) probability ( %ci) was observed when low tnc (< . x figure a ). lower incidence of relapse (p= . ) ( figure b ) and non-relapse mortality (p= . ) was observed in patients who received low granulocyte count in the graft. no significant correlation was observed between the time of engraftment and the number of tnc or granulocytes infused. similarly, no increase in the frequency of the engraftment syndrome was observed when higher number of tnc or granulocytes were infused. conclusions: in our series, low doses of granulocytes in the product of apheresis predicted a better outcome of asct in mm patients. the amount of mc at the time of apheresis did not have influence in the characteristics of the harvest. efforts for avoiding contamination in grafts are important for its impact on outcome of asct in mm patients. disclosure: dkms foundation, pi / (instituto carlos iii) and sgr (grc), generalitat de catalunya. background: our initial experience (from to years) with hematopoietic stem cell transplantation (hsct) from mud with tcrαβ+/cd + graft depletion in patients with cgd showed high rate of secondary graft dysfunction, the incident of graft rejection was %. to improve the outcome, we hypothesized that the use of plerixafor and g-csf as additional agents in conditioning regimen would offers advantages and better outcomes. this trial was registered at www.clinicaltrials.gov (nct ) methods: between april and september , patients with cgd underwent allogeneic hscts from mached unrelated donors with tcrαβ + / cd + graft. the conditioning regimen included -treosulfan g / m , fludarabine mg / m , thiotepa mg / kg, g-csf mcg / kg and plerixafor mcg / kg. all patients received rabbit atg -timoglobulin® ("genzyme europe b.v.", the netherlands) mg/kg for serotherapy. in all cases, tcrab +/cd + graft depletion was used with the immunemagnetic method (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instructions. stem cell source was g-csf mobilized peripheral blood stem cells in all cases. posttransplant gvhd prophylaxis was not performed. minimal follow up was days after hsct. results: neutrophil and platelet engraftment occurred at - days post-hsct in all patients. patients had full donor chimerism in whole blood and in cases we observed predominantly donor mixed chimerism at last follow up. all patients had full donor chimerism in cd + compartment and mixed chimerism in cd + lineage, but it stable without any sign of graft dysfunction. acute gvhd is verified in of cases and was limited to skin grade . all patients are alive for periods from to months post-hsct with good graft function without severe clinical problems. conclusions: we presented first experience of g-csf and plerixafor addition to conditioning regimen before hsct with tcrαβ+/cd + graft depletion in cgd patients . we suppose, the preliminary results are encouraging, as the frequency of primary and secondary graft dysfunction in patients from this group is not observed today, there are no significant toxic complications, as well as clinically severe manifestations of gvhd. currently, the recruitment of patients is continuing, and estimation of the rates of immune reconstitution and a more detail analyses will be evaluated later. background: introduction -it is believed that aboincompatibility is of minor importance in allogeneic stem cell transplantation (allo-sct) and that the clinical outcome is equivalent to abo-compatible sct. therefore, we performed a single center retrospective study to characterize the impact of abo-incompatibility on the outcome of haploidentical stem cell transplantation (haplo-sct). methods: this analysis included consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october and may . we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table . all of the patients had advanced hematological disease with a high risk of relapse ( patients with acute leukemia). out of patients, early transplant-related mortality was seen in this cohort of patients. the remaining patients were followed in and months. donor type abo group switch was observed in a median of days ( - days) after transplant. we were not able to show any statistical difference in terms of blood group switch between minor and major abo incompatible transplant. the median red blood cell (rbc) transfusions in the first days for the abo compatible and incompatible transplants were median units (range, - ) and median units (range, - ) (p= . ). no statistical difference was also encountered for the rbc transfusion need for stem cell source, peripheral blood vs bone marrow. a total of patients were followed up for reticulocyte engraftment. the median time for reticulocyte engraftment was days (range, - ) for all patients. reticulocyte engraftment was tended to be faster in minor abo-mismatched group (p= . ) than major or abo-compatible ones. nineteen patients achieved independence from rbc support after a median time of days (range, - days) in abocompatible patients, days (range, - days) in minor abo-incompatibilityand days (range, - days) in major abo-incompatibilitygroup, respectively (p> . ). the engraftman kinetics due to major and minor aboincompatibilitytransplants were presented in table- . pure red cell aplasia was not developed in our cohort. conclusions: the present single center study provides new evidence for the importance of the abo system for erythrocyte recovery in haploidentic stem cell transplantation. it's important to note that, randomize prospective and larger studies are warranted. disclosure: nothing to declare low dose of anti-t lymphocyte globulin protects against severe forms of graft versus host disease in patients undergoing allogenic stem cell transplantation background: graft versus host disease (gvhd) is the most important complication after allogeneic stem cell transplantation (allosct). optimal dose of different anti-tlymphocyte globulin (atg) formulations in this setting has not been established yet. the aim of this study was to analyze the impact of a low dose of atg-fresenius (atg-f) in allosct outcomes. methods: we analyzed adult patients who received an allosct for hematologic malignancies from october to march . the gvhd prophylaxis included a total dose of mg/kg ( mg/kg on day - , - and - ) of atg-f for patients who received a graft from peripheral blood, an unrelated donor; with a mismatch, and/or were older than years; associated to a calcineurin inhibitor and mycophenolate mofetil/short course-methotrexate. statistical analysis was performed using spss v. and r software. results: median age was years ( - ) and . % of patients were males. seventy-four percent of patients underwent myeloablative conditioning. the stem cell source was peripheral blood in patients ( . %), % were from unrelated donors ( % mismatched). seventeen ( . %) patients had high risk cmv status (d-/r+) (see image b). engraftment was observed in patients ( . %). primary graft failure occurred in patients ( myelofibrosis, aml). twenty ( . %) out of evaluable patients developed grade - acute gvhd. the cumulative incidence of severe agvhd and moderatesevere chronic gvhd were . % ( % ci, . - . %) and . % ( % ci . - . %), only ( . %) patients developed severe cgvhd. twenty-nine patients ( . %) discontinued immunosuppression before the first year of transplant. the median duration of immunosuppression for patients with moderate-severe cgvhd was days ( - ). at years non-relapse mortality (nrm) was . % ( % ci, . - . %). thirty-nine ( %) patients developed relevant infectious complications. two ( %) patients died within the first days due to gram negative blood stream infection. eleven ( . %) had at least two episodes of cytomegalovirus (cmv) reactivation between day and . three ( %) patients developed cmv gastrointestinal disease, ( %) had probable invasive fungal infection and ( . %), post-transplant lymphoproliferative disorder associated to epstein barr virus. with a median follow up of months for alive patients , the gvhd and relapse free survival (grfs) at one year, overall survival (os) and progression free survival (pfs) at two years were . % ( % ci, . - . %), % ( % ci, . - . %) and % ( % ci, . - . %), respectively. the relapse incidence at two years was . % ( % ci . - . %). complete remission at transplant was associated with better long term survival ( % at years, p < . ). hla disparity did not affect os (see image a). conclusions: the use of low doses of atg-f is protective against severe forms of acute and chronic gvhd in a cohort with high prevalence of unrelated donors and a high median age. this strategy showed good results in grfs, os and pfs in a population at high risk for developing gvhd or relapse. disclosure: nothing to declare performance parameters of a ngs-product for chimerism monitoring -applicable in patients after hematopoietic stem cell transplantation methods: for this purpose, samples from patients with mixed chimerism (mc) with increasing amounts of recipient dna were analyzed and compared using realtime pcr of insertions/deletions (indels), fragment analysis of short-tandem repeats (str) and ngs of indels. results: whereas real-time pcr displayed excellent sensitivity down to , % mc, but poor precision above %, fragment analysis exhibited good precision with limited sensitivity (> , %). in contrast, ngs chimerism demonstrated good sensitivity, with a limit of detection (lod) of , % mc, and precision throughout the whole spectrum of patient/donor mixed chimerism. the ngs chimerism product (devyser chimerism) exhibited at least three (average eight) and at least two (average ) informative genetic markers (indels), suitable for monitoring mixed chimerism of patients with their corresponding matched unrelated ( ) or related ( ) donor samples. in order to establish the performance of the separate techniques for determination of mixed chimerism on retrospective patient samples, a cohort of patient monitoring samples ( - weeks post-hsct) with low (< %), intermediate or high mixed chimerism (> %) were included and analyzed. dna from all monitoring samples was extracted from sorted cell fractions. the results show that although all evaluated techniques are suitable for monitoring patient/donor chimerism after allogeneic hematopoietic stem cell transplantation (hsct), only the ngs chimerism product exhibits high sensitivity (lod , %) and a broad dynamic range (detection range , - %) with good precision and accuracy throughout the whole spectrum of mixed chimerism (% patient/donor). in addition, the ngs chimerism product employ non-population dependent highly informative genetic markers providing stable resolution power and thus suitable for monitoring mixed chimerism. disclosure: dan hauzenberger is medical adviser at devyser ab and shareholder in devyser holding gender distribution: male - % (n= ), female - % (n= ). age median - , years old ( months - ). stem cell source: bone marrow - % (n= ), peripheral blood stem cells - % (n= ). patients ( %) received / matched unrelated donors hematopoetic stem cells transplantation and patients ( %) - / matched unrelated donors hematopoetic stem cells transplantation. differences in the antigen blood system: single group % (n= ), minor % (n= ), major % (n= ), mixed % (n= ). age of donor: - years old - % (n= ), - - % (n= ), - - % (n= ), and more % (n= ). gender differences in donor/recipient: male/female % (n= ), female/male % (n= ), one sex % (n= ). we also took into account the impact of gender difference and cytomegalovirus serostatus in the donor/recipient pair. results: in / group the estimated probability of overall -years survive was % and in the / group -years survive was %. the increase in donor age of years reduces the -years survive by - % (p= , ), however, the -years survive from donors over years old was %. we have found no difference between -years survive in transplants from donors that are compatible/ incompatible with the antigen blood system, cytomegalovirus serostatus, or the gender differences in donor/ recipient. in the study of donor-related factors, we found the negative impact of an human leucincompatibility ( / ) on the incidence of chronic gvhd - % (p = . ). the combination of cytomegalovirus positive serostatus of the donor and the negative status of the recipient increases the risk of primary graft rejection up to %, in comparison with others (p = . ). conclusions: our study showed the role of genetic matching on the hla system between the patient and the unrelated donor, and the donors age value. / transplants have better outcome and lower incidence of severe a. gvhd and ch. gvhd. younger donor increases -years survive, but there is a significant increase in -years survive if the donor is over years old. disclosure: nothing to declare allogeneic stem cell transplantation in chronic myelomonocytic leukemia. a single center experience nine patients ( %) relapsed with a median of , months ( - ) with different strategies at this point: in all cases we modulated immunosuppression, in cases as the unique strategy, in cases with donor lymphocyte infusion (dli), in cases we employed hypometilating agents (hma) and in cases with intensive chemotherapy, reaching cr only in two patients, one of them after dli and the other one after hma and consolidation with a second asct. eleven patients ( %) died being the relapse the main cause ( %). transplant related mortality (trm) at + were % and global trm were %. in the last follow-up, patients ( %) are still alive, ( %) in cr and ( %) in relapse situation. with a median follow-up of months ( - ), the event free survival (efs) were months ( - ) and the overall survival (os) were months ( - ). we observed advantage in terms of os in those patients that reach cr at + post asct and in those who develop chronic gvchd (p= . and p= . respectively) conclusions: asct is still the only curative option despite the high relapse rates. to reach cr at + post asct and the development of chronic gvhd seems that they confer advantage in terms of os. the importance of knowing the molecular profile of the entities that we consider for asct. disclosure this study documents a first experience of a cell processing lab seeking to integrate process automation technology to wash and volume reduce products which can account for the initial material source volume variability, product characteristics, and number of bags. methods: here we report the pre-clinical assessment of the lab's initial work with the lovo cell processing system for a product experience over days with machine. this study used products intended for destruction. the workflow used parallel and sequential processing schedule. after water-bath thawing, bags were sampled, weighed to determine volume, and subsequently connected to lovo or pooled into a transfer pack and then connected to lovo. the bags were then diluted : at ml/min with lovo at + - c using % hydroxyethylstarch / . (voluven, fresenius kabi) and processed using a cycle procedure. after processing the bags were weighed for volume, sampled, and stored in a - c refrigerator in their lovo final product bags. samples were assessed from t= to t= hours. results: cd + viability and absolute counts were determined using flow cytometry. processing duration and solution volume consumed was determined by the lovo's sensors and confirmed by the operator. data is presented as a percentage relative to the post-thaw values. note, the values presented are not total process yields. the results focus on the lovo processing step. conclusions: the operators easily integrated into the software to drive the machine. the machine demonstrated it's flexibility with a wide-range of volumes, cell-inputs, and number of bags. the lovo produced products which meet our specifications in a quick and reliable manner. further work on this platform will be performed to validate and qualify this system for production use. properties. the aim of this study was to evaluate prospectively the efficacy of a fbm protocol for the prevention of om in patients undergoing a hsct. methods: all patients consecutively who underwent a hsct at our center from x onwards received five weekly fbm sessions with a bidiodic laser (lumix ®, prodent, italy), which simultaneously emitted at nm and nm with a power of mw and energy of j per point. the procedure started the day before the beginning of the conditioning regimen up to the tenth day post-transplant. the laser was applied in a defocused mode on each of the mucosal surfaces ( areas). at each session, the morphine dose, the om level (according to the who scale) and pain through a numerical rating scale (nrs) were recorded. results: consecutive patients ( male/ female) submitted to a hsct were analyzed. the median age was years (range - ). eighteen patients had acute leukemia, myelodysplastic syndromes, lymphoproliferative diseases. the median number of treatment lines before hsct was (range - ). at transplant, patients had advanced disease. the myeloablative conditioning regimen mac (thyotepa, busulphan, fludarabine) was employed in patients; the same conditioning, with a reduced dose of busulphan (ric), was infused in patients. seven patients ( %) had no evidence of om. the incidence of grade ii-iv om was % in the group of patients receiving mac and the median duration days (range - ); grade om was observed, for day, in patient. in the ric group the incidence of om was %, the median duration days (range - ); no patient had evidence of grade iv om. in the whole population, the maximum nrs value was . morphine administration was required in patients, due to the occurrence of non-oral complications. conclusions: in our experience, prophylaxis with fbm to prevent or reduce om was safe and effective, compared to results of previous experiences reported in the literature, which used no prevention against this complication that negatively affects the quality of life of transplanted patients. further studies on a large series of are necessary to confirm our results. disclosure: nothing to declare background: cytogenetic abnormalities are an essential part of prognostic systems in myeloid malignancies before hematopoietic stem cell transplantation (hsct), however, their role in posttransplantation prognosis is unknown. the aim of this study was to assess the prognostic impact of genetic risk stratification of aml and mds patients on posttransplantation course, which could be an additional tool in making decisions regarding preemptive therapy. methods: a retrospective analysis covering patients treated with allo-hsct between and . cytogenetic studies included karyotyping (c-and gbanding) and fluorescence in situ hybridization (fish). the number of analyzed cells exceeded european cytogenetics association guidelines (for each fish at least interphase nuclei were analyzed). cytogenetic risk group in aml was assessed based on the eln criteria. patients with mds were stratified into three groups; favorable (good and very good prognostic score), intermediate, and adverse (poor and very poor) prognostic score according to ipss-r . interestingly, the poorest survival was in patients with monosomy of chromosome , which was present in patients of whom succumbed to refractory disease, while all patients who had deletion of long arm of chromosome (del q)-are alive at the time of writing of this report after a median follow-up of months ( - ). relapse was diagnosed in patients ( %), including; ( %) with adverse, ( %) with intermediate and ( %) with favorable cytogenetic risk. among patients with a complex karyotype and/or cytogenetic evolution prior hct: patients ( %)relapsed, including ( %)-who died. follow-up cytogenetic studies in relapse after transplantation were performed for patients; of them ( %) had clonal evolutions of the original karyotype with additional abnormalities-( % died) and ( %) had new aberrations in cells without primary changes (all died). in patients ( %) ( unfavorable, intermediate group) cytogenetic relapse was diagnosed by fish analysis and they were treated with azacitidine (+/-dli) achieving cr (n= , %), stabilization-(n-= , %), transient response (n= ), while deceased). conclusions: cytogenetic studies in patients after transplantation may facilitate assessment of mortality. karyotype may undergo cytogenetic evolution after allo-hsct. patients with monosomy of chromosome seem to have a particularly poor prognosis. transplanted patients are vulnerable to new cytogenetic alterations. disclosure: nothing to declare methods: the primary end-points were the rate of complete response (cr), defined as no emesis and no nausea without rescue medications, for both acute (cr- ) and delayed (cr - ) cinv and rate of post-transplant complications until discharge. we prospectively analyzed patients undergoing autologous ( %) and allogeneic ( %) stem cell transplantation and receiving cinv prophylaxis with nepa and dexamethasone (schedules shown in fig. ). in our series, patients ( %) were female. patients median age was years ( - ). the most frequent diagnosis were myeloma ( %) and lymphoma ( %), while % of patients were diagnosed with aml or mds. myeloma patients received one day hd-ct with melphalan ( % mel / % mel ). lymphoma patients were conditioned with feam ( , %) or tt_flu_edx ( , %) hd-ct. busulfan-based mds-ct regimen was offered to aml/mds patients. results: the incidence of cr- and cr - observed was % ( / ) and , % ( / ), respectively. more than grade nausea and vomiting (according to ctae- ), was reported in % ( / ) and % ( / ) of patients, respectively. female sex was associated with an increased risk of acute (hr , ; p , % ci . - . ) but not delayed (hr , ; p , % ci - . ) cinv. similar rate of cr and cr - was observed in one-day hd-ct ( % and %) compared to mds-ct ( % and , %) group (pns). median lenght of stay was days ( - ). no case of cardiotoxicity and no exitus was observed. the incidence of febrile neutropenia was % ( % fuo; % sepsis; % pneumonia). only one patient experienced an agvhd on day + . neutrophil (> /mcl) and platelet (> /mcl) recovery occurred in median on day ( - ) and on day ( - ) respectively. conclusions: nepa seems to be safe and effective in preventing acute and delayed cinv in patients receiving both one day hd-ct and mds-ct as conditioning regimen for hsct. more studies are needed to define the better -ht ra and nk- ra combination and the better schedule in transplant setting. disclosure: "nothing to declare background: vod and ta-tma represent two early endothelial complications occurring after allogeneic stem cell transplantation (sct) sharing many pre-transplant risk factors. the aim of our study is to evaluate the impact of donor graft composition, engraftment kinetics and infections on the development of these endothelial complications (ec). methods: we retrospectively reviewed consecutive sct recipients at our institu-tion between january and june . the median age was years (range - ). acute leukemia was diagnosed in patients ( %). complete remission was documented in % of patients at transplant. donor source was from hla mismatched donor in % and from unrelated donor in % of the patients. hbv positive patients were % of the sample. conditioning regimen was busulfan based in % of patients. ursodeoxycholic acid and unfractionated heparin were given to all patients as vod prophylaxis. cyclosporine was used as gvhd prophylaxis. lymphocytic subpopulation analysis (cd +, cd +, cd + and cd +/cd +) and cd + cells count on the donor graft were performed using bd facs cantoll. all patients had routine monitoring for ebv and cmv pcr, hemolysis tests, creatinine and electrolyte panels, proteinuria, complete blood count, blood pressure and schistocytes by direct examination until day + . the fisher's exact test was used to compare categorical variables, while continuous variables were analyzed with anova test. diagnosis of vod and ta-tma were carried out by using ebmt and cho criteria respectively. results: the incidence of very severe vod and tma was % ( / ) and , % ( / ) respectively. cmv reactivations with viral load over . cv/ ml was % and % in patients with and without ec, respectively (p , ; hr , p , %ci , - , ). the median day to neutrophils (ns) engraftment ( /ml and /ml) was vs and , vs in vod/tma group vs control group (p , and , , respectively). more rapid neutrophils engraftment (ns > /ml and ns> /ml within days) was related to a higher risk of ec with a hr of , (p , ; %ci , - , ) and , (p , ; % ci , - , ). patients with ec received a donor graft with a higher median numbers (x e /kg) of cd + and cd + (p> , ) and a lower numbers (x e /kg) of nk cells (p> , ). patients who received a cd + cells count > x e /kg and nk cells count < , x e /kg presented a relative risk of ec of , (p , ; % ci , - , ) and , (p , ; % ci , - , ), respectively. there were no differences with respect to the other analyzed variables between patients who developed vod/tma compared with those who did not. (pic_ ) conclusions: cmv viremia, early neutrophils engraftment and donor nk and cd + cells infused are associated with the risk of vod and tma. very few studies evaluated the link between these variables and the risk of developing such two complications. it could be interesting to investigate these relationships on larger series. clinical trial registry: not applicable disclosure: nothing to declare p when the last hope turns out to be just as good as best: haplosct following tbf conditioning, pt cyclophosphamide and tacrolimus as gvhd prophylaxis background: hematopoietic stem cell transplantation is an effective therapy for a variety of severe hematological diseases. in last decades, haploidentical sct (haplosct) followed by ptcy as gvhd prophylaxis has been reported as a valid alternative for patients who lack an hla matched donor. we therefore analysed outcomes with this. methods: patients without hla-matched donor received a haplosct between / and / . thiotepa mg/kg ( days), fludarabine mg/m ( days) and oral busulfan mg/kg/ h ( days,with pkd dose adjustments) was used as conditioning regiment; in patients > years busulfan administration was limited to two days. gvhd prophylaxis consisted of cyclophosphamide mg/kg on day + and + , and tacrolimus as a continuous iv infusion from day + . all patients received pbsc as the stem cell source. outcomes analysed were overall survival (os), progression free survival (pfs); cumulative incidences (ci) of gvhd, relapse and non-relapsed mortality (nrm). results: median ages was (range - ). % male and % female. diagnoses were: aml ( %), mds ( %), all ( %), hl and nhl ( %), and mm ( %). % (n ) were transplanted in early disease status, while most cases ( %) were in advanced status, including, second/third cr ( %, n ), one ( %) in aplasia without progressive disease and % (n ) had active/progression disease, % (n ) had stable disease; four cases were second alosct. thus, % of patients had a high or very high rdri and % had intermediate. ebmt score was ≤ was in % of patients (n ) . the donor was as son/ daughter in %, % a sibling and % a patient's mother. median time to neutrophil ( . x e /l) and platelet (> x e /l) recoveries were + and + days, respectively (g-csf was not used). only one patient had a primary graft failure attributed to anti-hla donor specific antibodies. median follow up in survivor is months (range - ). overall survival is ± . % (at months) and ± % (at months). pfs is ± % and ± % respectively. the cumulative incidence of relapse was % and %, respectively, while nrm is % at months. day + , grade - acute gvhd were %, while mild/ moderate and severe chronic gvhd were % and % respectively. ebmt ≤ and first alosct were the only variables to clearly impact -months os in univariate analysis. a combined covariate of ebmt ≤ -no prior alosct vs other patients showed a month os ± % vs ± % (p . ), pfs ± % vs ± % (p . ) and nrm % vs % (p . ) but without impact on relapse % vs % (p . ). conclusions: haplosct with an age-adapted tbf conditioning regimen, pbsc and ptcy followed by tacrolimus, led to very encouraging results, mainly in patients with a low ebmt score and as a first alosct. although formerly considered as a last alosct strategy, we now agree that the time has come to compare this strategy with hla mud (and even elderly sibling donors) in ongoing prospective randomized multinational trials. disclosure: nothing to disclosure background: autologous hematopoietic stem cell transplantation (autosct) for acute myeloid leukaemia (aml) is increasing becoming a viable option for an increasing number of patients due to limited availability of matched sibling or unrelated donor for allogeneic hematopoietic stem cell transplantation (allosct). we examined the relevant long-term outcomes in our local patient cohort. methods: we retrospectively reviewed the data for all autosct done for aml in our centre over a -years period between st january until st dec from our electronic record. patients with acute promyelocytic leukaemia (apml) were excluded from this analysis. patients were further stratified based on the number of high risk features present; not achieving complete remission (cr) following induction chemotherapy, high presenting total white cell count (wbc > x /ml, adverse cytogenetics (example: complex cytogenetics) and adverse molecular mutations (example: flt -itd & mll gene arrangement). outcome data including mortality (overall survival (os) and non-relapse mortality (nrm)) and morbidity (leukaemia free survival (lfs)) were recorded and analysed. results: a total of patients were identified. median age at diagnosis is -years old. the cohort comprised of males and females. the overall median os and median lfs is . years and . years respectively. the nrm is . % ( / ). there was no difference in the median os and median lfs for the patients achieving cr following induction chemotherapy and those not in cr following induction chemotherapy; . years versus . years (log-rank, p= . ) and . years versus . years (log-rank, p= . ) respectively. the median os were statistically significant for patients with zero versus one and two and more high risk features present; . years versus . years versus . years (log-rank, p= . ) respectively. however, the median lfs were not statistically significant for these three patient cohorts; . years versus . years versus . years (log-rank, p= . ) respectively. conclusions: in our patient cohort, autosct appeared to be a feasible option for patents with aml without matched sibling or unrelated donor available. disclosure: none to declare methods: between - , thalassemic patientsunderwent hisct. the median age of patients was ( - )years with male preponderance (n= , . %). across the gender and abo mismatch transplants were done in . % and % of patients. stem cell source was bone marrow in ( %) while peripheral blood in ( %) of patients. mean stem cell dose was . ± . x cells / kg and mean volume of product was ± . ml. preparative regimen included anti-thymocyte globulin, busulfan, fludarabine and cyclophosphamide.graft versus host disease (gvhd) prophylaxis comprised of posttransplant cyclophosphamide on day + & + followed by tacrolimus and mycophenolate mofetil. patients were observed for hematopoietic recovery (neutrophil and platelet engraftment) and transplant related mortality including acute and chronic gvhd for skin, gut, liverand lungs, primary and secondary graft failure and infectious complications. results: nine( . %) of sixteenpatients were engrafted with full donor chimerism. twelve ( %) patients belonged to pesaro class i and ( %) toclass ii patients. median time to neutrophil and plateletengraftment were ( - ) and ( - )days respectively. average number of packed red cell and platelet transfusions were . ± . and . ± . respectively.primary graft failure was observed in ( %) and secondary graft failure was observed in ( %) patients. two patients received a second dose of stem cells and they engrafted at and days of infusion respectively. of patients with primary graft failure died, one with sepsis (day + ) and the other because of intracranial bleeding (day + ).acute gvhdof gut and skin (grade ii-iii) was observed in patients each, within first days post-transplant. none of the patients had grade iv gvhd. cytomegalovirus reactivation occurred in % of patients, all of them received pre-emptive therapy with intravenous ganciclovir. none of them developed cmv disease. invasive fungal infection was not observed in any of the patient. culture proven bacterialinfection was documented in % of patients requiring intravenous antibiotics during first days post-transplant.overall survival and relapse free survival were . % and . % over a median follow-up of ( - ) days. conclusions: haploidentical transplant is a suitable modality for thalassemic patients lacking a full matched donor in pakistan. in view of our results, we suggest that thalassemia patients should be offered hisctas an option for cure. clinical background: gemtuzumab ozogamicin (go) is an anti-cd monoclonal antibody with significant activity in de novo and relapsed/refractory (r/r) acute myeloid leukemia (aml). a relevant side effect consists of hepatotoxicity and especially sinusoidal obstruction syndrome (sos). the objective of this study was to analyze tolerability of go during the induction and reinduction therapy in patients with aml, and its possible impact on subsequent hematopoietic stem cell transplantation (hsct). methods: from to , patients who had received go in three hospitals were collected and their medical records were retrospectively reviewed. results: fourteen patients diagnosed with de novo aml received go ( mg/m ) on day + in combination with standard chemotherapy (idarubicin and cytarabine, x schedule) as induction therapy. hyperbilirubinemia (bilirubin > . unl) was detected in patients and increase of aspartate aminotransferase (ast) (> . unl) in . twelve patients achieved complete remission (cr) and one was refractory ( not evaluated). in the r/r setting, patients diagnosed with aml (n= ), biphenotypic acute leukemia (n= ) and acute promyelocytic leukemia (n= ) received go as rd or subsequent rescue therapy either as monotherapy (n= ) or in combination with cytotoxic chemotherapy (n= ). prior hsct was performed in patients (autologous [n= ], allogeneic [n= ] ). rescue therapy was indicated for refractoriness (n= ), relapse (n= ), partial response (n= ) or absence of donor (n= ). four patients received doses of go ( mg/m ) and patients, one dose. hyperbilirubinemia (> . unl) was observed in patient and increase in ast (> . unl) in patients. seven patients achieved cr, was refractory, obtained partial response and died early during induction). thirteen patients received subsequent hsct (autologous [n= ], allogeneic [n= ]) after go therapy ( in the de novo aml and in the r/r group). the reasons for not performing hsct in the remaining patients were: low cytogenetic risk (n= ), active chronic graft versus host disease (gvhd) in previous hsct (n= ), early death during treatment (n= ), relapse (n= ), severe complications in rescue treatments (n= ), and unknown (n= ). the conditioning regimen was myeloablative (n= ), non-myeloablative (n= ) and sequential (n= ), and the donors were matched sibling (n= ) or unrelated (n= ) . cyclosporine, methotrexate and thymoglobulin were administered as gvhd prophylaxis in patients and cyclosporine, mycophenolate and thymoglobulin in . hyperbilirubinemia was observed in patients belonging to the de novo aml group. death after hsct occurred in patients due to infection (n= ), relapse (n= ), gvhd (n= ) and traffic accident (n= ). three patients are currently alive in remission. no sos was observed in any patient. conclusions: in both de novo and r/r aml the administration of low dose go is feasible and does not have impact on subsequent hsct outcome. although some degree of hepatotoxicity was observed, no cases of sos were observed, either before or after hsct. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc biga), instituto carlos iii (pi / fi), -sgr (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. outcome of allogeneic hematopoietic stem cell transplantation in patients with benign hematological disorders saqib ansari , tahir shamsi , uzma zaidi , saima siddiqui , tasneem farzana background: allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for hematological disorders. we evaluated the outcome of patients suffering from benign hematological disorders, including aplastic anemia, fanconi's anemia and thalassemia after matched related allogeneic transplantation. methods: all patients having hematological disorders including aplastic anemia (aa), beta thalassemia (btm), fanconi's anemia (fa) and severe combined immune deficiency disorder (scid) with hla identical related donors who underwent allogeneic transplantation were included. donors were given g-csf at a dose of μg/ kg/day daily for four days prior to harvest. the conditioning regimens for thalassemia included cyclophosphamide (cy) + busulfan (bu) in ( %), bu + cy + thiotepa in ( %) and bu + cy + antithymocyte globulin (atg) in ( %). conditioning regimens for aplastic anemia included, fludarabine (flu) + cy in ( %), flu + atg in ( %) and cy in ( %), cy+ atg in ( %) patients. for fanconi's anemia flu + atg in ( %), flu in ( %), cy + atg in ( %) and flu+ cy + atg in ( %). flu + atg in ( %) and cy given in ( %) and no conditioning regimen was offered to ( %) patients with scid. results: a total of allogeneic transplants were performed for benign hematological disorders including aa (n= ), btm (n= ), fa (n= ) and scid (n= ) from to july . median age was . years (range . - ). across the gender and abo blood group transplants were ( . %) and ( %). the median time to neutrophil and platelet recovery was days (range: - ) and (range: - ). primary and secondary graft failure was observed in ( . %) and ( . %). overall survival in aplastic anemia ( / , %), beta thalassemia ( / , %),fanconi's anemia ( / , %) and severe combined immune deficiency disorder ( / , %). eighty four patients expired ( . %) among them patients expired within days post transplant main cause of deaths included sepsis ( %), multi organ failure ( %) and gut gvhd ( %) conclusions: in developing world scenario where non malignant disorders are leading cause of morbidity and mortality. bone marrow transplantation has been successfully implemented with better long term diseases free survival and quality of life. clinical background: hematopoietic cell transplantation (hct) remains the only curative therapy for many diseases, yet transplant survivors carry an unusually high burden of morbidities, primarily because of exposure to intense chemotherapy, radiation and /or gvhd. this study aimed to evaluate the burden of chronic diseases at the end of life after allogeneic-hct and to identify the disability-adjusted life years (daly). methods: the pubmed, medline, and ovid databases were queried utilizing specific mesh terminology (post, allo stem cell, hematopoietic, bone marrow, transplantation).we collected data on the impact of the hct on the variables affecting survivor's health in all aspects .the rates of late complications were compared to the risks in the general population (united states). results: a total of studies fulfilled the selection criteria totaling to patients (table ) . median os at -year mark varied widely between studies from % to %. majority of the patients at -year mark were found to have new comorbidities thereby indicating a huge burden of late effects at the end of life, though exact dalys could not be calculated due to incomplete data. hct survivors were found to have higher risk of premature arterial disease (pad) at . % compared to the general population, however gvhd or the addition of tbi to the conditioning regimen were not found to be significantly associated with pad. regarding the risk of new cancers, the cumulative incidence of their development at and years was . , and . , respectively. increased risks compared with the general population were seen for some solid cancer including cancers of the lip: p= . , tonsils: p= . , oropharynx: p< . , bone: p< . , soft tissue: p< . , and vulva: p= . . with respect to mental health, depression was prevalent in ( . %) survivors, in whom ( . %) were still on antidepressants at the last follow-up. cognitive impairment and other psychiatric disorders were found in ( . %) and ( . %) survivors, respectively. the most common cause of nrm in the first years was gvhd. however, after years, the leading cause of death in those conditioned with mac regimen was secondary cancer, but in the ric group, new cancers and gvhd contributed equally. conclusions: hct survivors remain at risk of significant complications which lead to premature death and their burden of comorbidities at the end of life is significantly more than that of general population. background: late onset hemorrhagic cystitis (hc) is a common complication of hematopoietic stem cell transplantation (hsct) frequently associated with reactivation of bk virus (bk-hc).there is no consensus as to the best therapy for bk-hc, and many different treatments have been reported. hyper baric oxygen therapy (hbot) is used as primary or adjuvant therapy in diverse clinical situations involving hypoxic injury to tissues and has been explored as a useful tool in treating bk -hc. we report our experience with hbot in combination with non-invasive supportive care in children and adolescents suffering from bk-hc following allogeneic hsct. methods: the computerized database of schneider children´s medical center of israel was reviewed for all patients aged to years who underwent hsct between january and june and developed bk-hc. hbot therapy consisted of hours sessions at atmospheres, with patients breathing oxygen by mask. parents accompanied patients during the treatment. results: fourteen patients with a variety of underlying diseases received (hbot) for treatment of bk-hc following hsct. the initial treatment for children with bk-hc at our center prior to included continuous bladder irrigation and intravesicular instillation of various medications. beginning in , we adopted a non-invasive strategy that included the administration of oral anticholinergics (oxybutinin), systemic pain management, hyperhydration and the administration of weekly cidofovir with probenecid. hbot was administered to patients who failed the above regimen. with this protocol, the average time of starting hbot dropped from (prior to ) to . days. the median onset of hc was days post hsct. all patients were receiving immunosuppressive treatment at the onset of bk-hc. all patients suffered from macrohematuria with blood clots (grade iii cystitis), ( . %) experienced severe dysuria and ( . %) urgency. bk viruria was present in all patients, and concurrent bk viremia was detected in % of those who were tested. patients reported symptomatic improvement at a mean of . days following the initiation of hbot. no patient experienced serious adverse effects due to hbot, but two patients required insertion of tympanic ventilation tubes. eleven of our patients ( . %) experienced complete remission of bk-hc following hbot, with an overall response rate of . % ( / patients).eight of our patients ( . %) eventually succumbed due to either hsct complications or disease relapse. conclusions: hyperbaric oxygen therapy is a safe, effective, non-invasive and well tolerated treatment modality for bk -hc and should be considered for first line therapy for this complication of hsct. clinical background: every year, almost one thousand cases of hematological malignancies in pediatric population are reported in peru. allogeneic hematopoietic stem cell transplantation (allo-hsct) is an alternative strategy in many of these cases. only between - % of the pediatric population that requires a hsct has a compatible human leukocyte antigen (hla) donor. the remaining % have to access international donor registries, extending the awaiting time and conditioning the progress of the disease. allo-hsct has the potential to help children with several hematological disorders with non-compatible hla donor. hla genotypically identical sibling donors are the best option when pursuing an hsct. nevertheless, patients' alternative sources of stem cells could be obtained from an haploidentical donor like one of their parents. the haploidentical transplantation program with macs was implemented in peru in to reduce the risk of graft-versus-host disease (gvhd), support the immune system reconstitution and to expand pool of donors. it allows patients to access a treatment that is efficient and safe, as shown in the depletion of positive tcrα/β+ and b cells procedures for allo-hsct. methods: the mobilized leukapheresis products (n= ) of haploidentical healthy donor was washed to remove platelets and preparations were performed according to miltenyi's clinimacs® manual for tcrα/β+ and cd + cell depletion. analysis of the initial leukapheresis product and tcrα/β + and cd + depleted graft (target and non-target product) was performed using flow cytometer. cells were analyzed for cd +, cd +, -aad, cd +, tcrα/β+, tcrγ/δ +, cd + and cd + with fluorochrome-labeled antibodies from miltenyi biotec using a novocyte cytometer. the results obtained with the ex vivo t-cell depleted allo-hsct procedures show an overall survival (os) over % with an ic % at the end of the first year with low incidence of gvhd. macs of tcrα/β+ and cd + cells are effective (logp . and logp . , respectively) obtaining minimum levels of depleted lymphocytes within clinical established parameters for diverse pathologies. in addition, tcrα/β+ and cd + cell depletion does not significantly affect hematopoietic stem cell populations such as cd + and cd + cells or tcrγ/δ+ cell population. cd + and tcr γ/δ cells are highly recovered ( . % and . , respectively), which contributes with a better engraftment after allo-hsct. conclusions: peruvian results oscillated within european ranges with an os over %. our data suggests that the macs method is an efficient, effective and safe strategy for haploidentical hsct which has a remarkable cost-benefit ratio and makes it viable in countries of the latin american region with peruvian socio-economic characteristics. evaluation of genoresistance for viral reactivation treatment has been implemented as a strategy to improve os. better results are achieved in patients after allo-hsct with the validation of these tests in peru. preliminary pharmacoeconomic evaluations allow us to establish magnetic activated cell sorting (macs) as a promissory strategy compared to other alternatives for haploidentical hsct. it is necessary to increase the number of procedures in order to confirm efficacy and safety of macs in a larger population. disclosure: all authors have no conflicts of interest. abstract already published. micro-costing study of hematopoietic stem cell transplantation in two hospital institutions from southern of brazil background: hematopoietic stem cell transplantation (hsct) is a potentially curative treatment indicated for patients with onco-hematological, hereditary and immunological diseases. considering the increase of patients indicated to the hsct and the lack of knowledge about the costs resulting from this treatment, is important to identify and detail the resources consumed in each phase of hsct and provide knowledge to the public health brazilian system. we aimed measure the total cost of related hsct, based on micro-costing study of patients assisted in two hospitals in the south region of brazil. methods: hsct costs were estimated using the timedriven activity based costing method (tdabc), which measured cost of services / products based on actual consumption of resources. we collected data from medical records of patients submitted to allogeneic hsct in from public and private (philanthropic) hospitals. we interviewed professionals involved in the tcth activities, we performed chrono-analysis and, we consulted financial and administrative systems reports of hospitals. in order to compare costs according to clinical complications observed in patients, we grouped into two ranges of complexity: low/ medium and high. the study was divided into stages: hsct processes mapping; costs measurement; and analysis of results. finally, the costs were compared: by activity, by resource and by hospital. this study was financed by psid-uhs, by an agreement signed between ministry of health and moinhos de vento hospital, through adjustment term number: / , and approved by research ethics committees. results: from the hsct processes mapping, the following steps were defined: (i) hospitalization; (ii) conditioning; (iii) transplantation; (iv) period of aplasia; (v) engraftment; (vi) observation; (vii) pre-and medical discharge. seven patients were classified in low / medium complexity level, with hospitalization median time of days and an median cost of usd , . , whereas the other five patients, classified as high complexity, presented median time of days and median cost of usd , . . the hsct costs evaluation identified that steps ii and iv presented greatest cost in high complexity patients. lower complexity patients presented, in steps ii and iv, median costs of usd , . while in higher complexity usd , . . in addition, median costs of materials and drugs were usd , . and usd , . in lower and higher complexity patients. conclusions: tdabc method allowed the identification of the moment when patients consume the most resources. of all the hsct stages, periods of conditioning and aplasia presented higher costs, representing . % of the total hospitalization value. in these stages, higher complexity patients presented three times higher the median cost. the resources that had the greatest impact were medicines and medical materials, costing times more than lower complexity patients. conclusion: this study allowed a detailed identification of the hsct costs in patients with different complexity ranges in two hospitals from southern brazil. therefore, the identification of service demand regarding the clinical complexity, allows the generation of important information for the management of the best care in the health service. disclosure background: immunodeficiency due to lrba deficiency is characterized by hypogammaglobulinemia and autoimmunity. hemolytic anemia, lymphadenopathy, autoimmune hepatitis and, above all, autoimmune enteropathy are the fundamental characteristics of these patients together with the history of recurrent invasive infections.the therapy includes immunosuppressants, endovenous immunoglobulins. bone marrow transplantation is the final therapy of these patients, especially in the most serious cases and in recent years, also on the light of increasingly targeted conditioning regimes, it is associated with ever better prognosis. methods: we present the case of caterina, an -year-old patient with lrba deficiency, diagnosed at years of age for a history of hypogammaglobulinemia, recurring invasive, bacterial and fungal infections and a picture of autoimmunity represented by ahia, myelitis (c -c ), autoimmune hepatitis and enteropathy in treatment with abatacept and sirolimus. it also presents leptin deficiency lipodystrophy.she presented to our observation for ostemielitis in multiple outbreaks (tibia, femur and left knee) secondary to sepsis from mrsa. broad spectrum antibiotic therapy and curettage surgery were performed during hospitalization.after months of broad-spectrum antibiotic therapy (daptomycin, rifampicin, ceftaroline, cotrimoxazole, levofloxacin, dalbavancin) there was resolution of the infections.because of the seriousness of the disease it is therefore decided to subject catherine to hematopoietic stem cell transplantation. results: therefore were performed bone marrow transplant from mud after conditioning at reduced intensity, delayed in days, with treosulfan, fludarabine and thiotepa. prophylaxis for gvhd was performed with atg ( mg / kg / day), sirolimus (already practiced for enteropathy) and mmf. because of the hepatic picture, we also performed prophylaxis for vod with defibrotide for days. transplantation was performed by peripheral stem cells with x ^ cd / kg and x ^ cd / kg.the patient had always presented good general conditions with engrafment of the pmn to the d + and the plt to the d + . the chimerism at d + was % donor both on pmn and on pbl. prophylaxis for gvhd was changed on d + by replacing the sirolimus with tacrolimus for the appearance of grade i cutaneous gvhd . conclusions: we have successfully performed bone marrow transplantation in patients with lrba deficiency. the new antibiotic molecules, used to induce infectious remission, the new low-intensity regimens, the prevention of the most fearful complications (vod) have been the key to success in such complicated case. the high number of cd cells infused with a controlled number of cd were the key then of rapid engraftment with minimal gvhd readily controlled by the immunosuppressant. disclosure background: in the absence of hla-matched related donor, allogeneic stem cell transplantation from haploidentical donors are potential alternatives for patients with hematological malignencies with an indication to allogeneic stem cell transplantation. herein, we retrospectively assessed the outcome of haplo-sct for patients with refractory hematological malignancies. methods: this analysis included consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october and may . we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table . all of the patients had advanced disease with a high risk of relapse. the majority of patients underwent haplo-sct from their parents. out of patients, early transplant-related mortality was seen in this cohort of patients. four patients treated with second haplo-sct and recovered hematopoiesis after second transplant. the remaining patients were followed in a median of months. donor type abo group switch was observed in a median of days ( - days) after transplant. the median time for engraftment was days (range, - ) for all patients. after the first transplant, patients developed acute gvhd ( . %) with patients having grade ii-iii acute gvhd. five ( . %) had chronic gvhd, none of them with extensive manifestation. the prepative regimen was relatively well tolerated with limited regimen-related toxicity. cmv reactivation occurred in patients ( . %) during the follow-up of the study. eight patients ( . %) relapsed after a median of days post transplant (range, - days). cr was achieved in ( %) patients after haplo-sct. mean estimated -year os and pfs are . %± . % and . %± . %, respectively. conclusions: given the growing data on the similarity of outcomes after hla-matched and haploidentical sct, further studies are required to determine whether factors may be more important for donor selection than hlamatching. clinical trial registry: -disclosure: nothing to declare outcome of allogeneic stem cell transplantation for hodgkin and non-hodgkin lymphoma: single center experince from turkey ayşe uysal , hale bülbül , nur akad soyer , mahmut tobu , murat tombuloglu , guray saydam , filiz vural background: allogeneic sct (allosct) is generally optionally treatment choice for young and fit patients with relapsed/refractory lymphoma who were heavily pre-treated and after the failure of autologous stem cell transplantation (asct). relapse after asct is associated with a poor prognosis and allosct is a potentially curative therapy for lymphomas which have relapsed after asct. methods: in this study, we evaluated patients with hl and nhl who had treated with allo-hsct between november and december in ege university adult hematology transplantation unit. results: patients, disease and transplant characteristics were illustrated in table. histologic subtype of nhl was evaluated as t cell lymphoma (n= ; , %), mantle cell (n= ; , %), diffuse large b-cell lymphoma (n= ; , %) and b-cell lymphoma, unclassifiable, with features intermediate between dlbcl and classical hodgkin lymphoma (n= ; , %). all histologic subtype of hl was determined as nodular sclerosing. the median number of prior treatments before allo-hsct was (range, - ). twelve ( , %) patients had refractory disease, ( , %) patients were in complete remission and ( , %) patients were in partial remission before allo-hsct. the median time from diagnosis to allosct was (range, - ) months. peripheral stem cell was used for stem cell source in all of them. total body irritation plus fludarabine plus cyclophosphamide and busulfan plus cyclophosphamide were preferred most frequently for conditioning as non-myeloablative and myeloablative, respectively. neutrophil engraftment was occurred median of (range, - ) days. graft versus host disease (gvhd) prophylaxis was applied all of them and cyclosporine plus methotrexate was preferred most frequently (n= ; , %). gvhd was occurred in , % of them ( , % acute gvhd, , % chronic gvhd and , % both). veno-occlusive disease (vod) was occurred in ( , %) patients. transplant related death was observed in ( , %) patients. overall survival (os) and disease-free-survival (dfs) were evaluated as median (range, - ) and (range, - ) months, respectively. analyze of os and dfs was illustrated in figure. six patients are alive without disease. after a rapid tapering of immunosuopressive therapy was underwent a therapy with ponatinibat dose of mg/die results: afther a month of therapy we observed a rapid decrease in minimal residual disease on molecular assessment with an mmr of p -bcr-abl/abl non detectable confirmed by bone marrow revaluations at days + , + nd + after the salvage therapy. the patient has not had experienced of graft-versus-host disease, ponatinib treatment was well tolerated and considered safe with easily manageable side. conclusions: maybe in the era of tyrosine kinase inhibitors (tkis), philadelphia chromosome positive acute lymphoblastic leukemia (ph+ all) it could benefit from a combined treatment between transpalnt and tkis however more studies are needed to confirm these hypotheses. disclosure: nothing to declare immunodeficiency diseases and macrophage background: although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the largest spanish experience of hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. methods: we retrospectively reviewed hiv-positive patients who received allogeneic hsct between and in spanish centers within geth (grupo español de trasplante hematopoyético y terapia celular). results: baseline and transplant characteristics of patients are shown in table . median age was years and % of the patients were men. the most frequent underlying malignancies were non-hodgkin lymphoma ( , %) and aml ( , %). in half of the patients an hlaidentical sibling was the donor; and in the other half, an alternative donor was used. peripheral blood was used as graft source in % of the transplants. at the time of hsct, all patients had been receiving suppressive cart for a median of years and only of them showed detectable plasma hiv rna, one of them because of poor adherence to cart together with the accumulation of multiple resistance mutations; and the other patient had detectable hiv rna at low levels (< copies/ml). all patients received cart throughout the transplant procedure, being temporally stopped in two patients due to significant mucositis. after a median follow-up of months ( - ), -year overall survival (os) and event-free survival (efs) were %. nrm was % at months and relapse was % at months. grade ii-iv agvhd rate was %, and moderate/severe cgvhd rate was % at months. a significant proportion of patients ( %) showed infectious complications with viral infections as the most frequent cause. two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death included infections ( %), relapse ( %) and toxicity ( %). among the patients who died due to infections, had severe chronic gvhd and were under immunosuppressive therapy. two patients showed severe toxicity related to drug interaction with anti-retroviral therapy. all survivors except one showed undetectable hiv load at last follow-up after hsct. conclusions: allogeneic hsct is an effective therapy for high-risk hematological malignancies in patients with hiv infection, providing long-term disease free survival together to long-term hiv suppression with cart. however, drug interactions with anti-retroviral agents, occurrence of gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematological malignancies should be considered for allo-hsct when indicated, in experienced centers, with a multidisciplinary care. disclosure background: primary immunodeficiencies (pid) are rare diseases often associated with genetic defects in the immune system, predisposing individuals to recurrent infections and increased risk of allergy, autoimmunity and malignancy. allogeneic haematopoietic stem cell transplantation (hsct) has been successfully used as a curative therapy for most severe forms of pid. because pid is a genetic disease, < % of these children will have a healthy, human leukocyte antigen (hla) matched sibling donor available, and umbilical cord blood grafts from unrelated donors are a suitable alternative cell source. we report the results of umbilical cord blood transplantation (ucbt) performed in patients with pid between and at children's hospital of fudan university in china. methods: patients included chronic granulomatous disease (cgd, n= ), severe combined immunodeficiency (scid, n= ), interleukin- receptor-a deficiency (il- rad, n= ), wiskott-aldrich syndrome (was, n= ), leukocyte adhesion deficiency (lad, n= ), severe congenital neutropaenia (scn, n= ) and other immunodeficiencies (n= ). all patients were assessed by clinical immunologist to confirm clinical phenotype and genetic diagnosis. median age of patients was months (range, to months), and median body weight was . kg (range, . to kg). all patients received a ≤ / hla alleles-mismatched cord blood unit, were hla fully matched, were / matched, were / matched and were / matched. median nucleated cells of the cord blood were . x /kg (range, . to . x /kg), and median cd + cells were . x /kg (range, . to . x /kg). results: median follow-up time was months (range, to months), the overall survival rate at year for all patients was . %, and was . %, . % and % for cgd, scid and il- rad, respectively. patients died, most deaths ( / , . %) occurred in + days after transplantation, the main cause of death was infection ( / , . %). / ( . %) patients engrafted, median time of neutrophil engraftment was days (range, to d), and median time of platelet engraftment was days (range, to d). the cumulative incidence of grade - acute gvhd was . %, and that of chronic gvhd was . %. conclusions: unrelated ucbt should be considered for pid patients without an hla -matched sibling donor. effective control of infection before and after transplantation is important for improving survival. disclosure background: dedicator of cytokinesis (dock ) deficiency causes a combined immune deficiency characterised by recurrent bacterial infections, susceptibility to viral infection, eczema, food allergies, vasculitis and increased risk of malignancy. due to the high morbidity and mortality of the disease hsct has been increasingly offered to patients as a potentially curative therapy . methods: we retrospectively reviewed the outcomes of hsct for patients with dock deficiency at great north children's hospital newcastle upon tyne between and ( in published reference). results: ten patients with dock deficiency were treated with hsct (median age . y range . - . y). median duration of follow up was . years (range . - . y). there were a range of donor sources ( msd, mud and tcr ab/cd + depleted haploidentical), conditioning regimens ( treo-flu, treo-flu-thiotepa) and serotherapy ( alemtuzumab, atg+rituximab, none). one patient who received a cd + tcr alpha beta/cd + depleted haploidentical transplant received add back t-cells with caspacide molecular safety switch (bellicum pharmaceuticals). skin only agvhd occurred in / patients ( x stage , x stage ). no patients had cgvhd. overall survival was % ( / ). survival was comparable regardless of donor source. all deaths occurred within months of transplant. the patients who died had significant burden of disease pre-transplant: patient had chronic liver failure secondary to cryptosporidial sclerosing cholangitis, had a cirrhotic liver secondary to cryptosporidium, cerebral vasculitis, an axillary aneurysm and aortic vasculitis requiring grafting of an ascending aortic aneurysm, was pn dependent for failure to thrive with a history of cryptosporidium infection and had candida in a bal pre-transplant. causes of death in these patients were: respiratory failure (n= ), progressive encephalopathy (n= ), multi-organ failure with septic shock and encephalopathy (n= ) and multiorgan failure and septic shock after treatment for tma (n= ). two of these patients had reactivation of cryptosporidium prior to their death. pretransplant cryptosporidium was associated with mortality (graph ). one patient who survived had suffered from stroke pretransplant. one suffered from a basilar artery aneurysm years post-transplant at yo. at the time of latest follow up donor chimerism was % in / survivors and high level mixed in the other( % cd , % cd and % cd ). conclusions: this single centre study of hsct for patients is consistent with literature indicating that hsct is a potentially curative therapy for patients with dock deficiency. the increased morbidity associated with cryptosporidial infection is likely to be a consequence of overall disease burden rather than an infection specific effect. this does however highlight the improved outcomes of transplant prior to development of multiple comorbidities and suggests that hsct should be considered early. it is unclear whether the late occurrence of vascular complications after transplant were caused by a manifestation of disease which is not corrected by transplant or a result of vascular injury sustained pre-transplant. reference methods: referred infants underwent testing for: immune phenotype (ab, gd, naïve and memory t cell, b cell and nk cell numbers); functional activity of t and nk cells; maternal engraftment; adenosine deaminase (ada) and purine nucleoside phosphorylase (pnp) enzyme activity; and genetic testing. those with a confirmed diagnosis of scid underwent either allogeneic hematopoietic stem cell transplant (hct) or (if eligible) gene therapy (gt). infants identified as having ada deficiency as the etiology of their scid received enzyme replacement therapy prior to proceeding to definitive therapy. results: twenty-three ( %) infants were confirmed to have scid. three ( %) of these infants had a family history of scid but would not have been identified without nbs. in addition, one infant, born prematurely at weeks, was diagnosed as having pnp deficiency only after developing infections. this infant was identified by nbs but repeat testing at weeks gestation was normal likely due to support of transient t cell production from exogenous enzyme provided by red cell transfusion. twelve infants with confirmed low trecs had a non-scid diagnosis: with transient lymphopenia of infancy who normalized trec, immune phenotype and function, with prenatal exposure to -mecaptopurine ( -mp), genetically confirmed with digeorge syndrome, and with prolonged lymphopenia. of the three with prolonged lymphopenia, two had recurrent infections: one ultimately diagnosed with ataxia telangiectasia and one with absent trec but near normal number of t cells, normal pha but no specific antigen responses, and absent b cells who will be undergoing transplant in the near future. the third continues with absent trec, short telomeres, low numbers of a/b t cells, presence of g/d t cells, vaccine responses and freedom from infection with no identified genetic etiology. in summary, % of the patients referred to msk with confirmed abnormal nbs for scid have a non-scid diagnosis. there is no uniform collection of data for these infants and the threshold trigger for repeat testing varies from state to state, so the incidence of significant non-scid disorders identified will also likely vary from state to state. although our institution specific experience is biased, as most infants had confirmation of a low number of trec prior to referral, the significant number of disorders in the non-scid cohort emphasizes the importance of full evaluations and follow-up for these infants. disclosure: none of these relate to the work being presented. susan prockop -research funding mesoblast and atara biotherapeutics. nancy kernan -research funding jaz pharmaceuticals. richard o´reilly research funding and royalties atara biotherapeutics. kevin curran consulting juno pharmaceuticals, novartis. j.j. boelens avrobio, magenta, chimerix and bluebird bio background: post-transplant autoimmune cytopenia (aic) is challenging and associated with substantial morbidity and mortality. we aimed to study the cumulative incidence (ci) of post-hct autoimmune cytopenia (aic) and its predictors in a cohort of children with primary immunodeficiency (pid). methods: in this retrospective study, we included children with pid who underwent their first hsct with fludarabine(f)-treosulfan(t)±thiotepa(thio) at great north children's hospital from - . main outcomes of interest were the ci of aic and its predictors. fine-and-grey regression models were used to analyse predictors of aic, considering death as a competing event. variables included were age at transplant (< . years vs > . years), gender, diagnosis (scid vs immune-dysregulatory disorders vs other pids), pre-transplant aic, pre-and posttransplant respiratory virus, donor (mfd vs mud vs mmfd/mmud vs haploidentical donor), abo incompatibility, conditioning (ft vs ftthio), serotherapy (none vs alemtuzumab . - . mg/kg vs alemtuzumab . - . mg/kg vs atg), stem cell source (marrow vs pbsc vs cord vs exvivo t depleted pbsc), infused stem cell doses (tnc, cd and cd ), agvhd (none vs any agvhd), cgvhd (none vs any cgvhd), viral infections (cmv/adenovirus/ ebv/hhv viraemia), chimerism (full vs mixed chimerism (wb < %) within first year post-hct). impact of thymopoiesis using naïve t cell recovery was studied. results: median age at transplant was . years (range, . - . years). primary diagnoses were scid ( %), immune-dysregulatory disorders ( %) and other pids ( %). donors were mfd ( %), mud ( %), mmfd/ mmud ( %) and haploidentical parents ( %). stem cell sources were marrow ( %), unmanipulated pbsc ( %), ex-vivo t-depleted pbsc ( %) and cb ( %). % received additional thiotepa and % had csa/mmf as gvhd prophylaxis. median duration of follow-up of survivors was . years (range . to . years). -year os for the entire cohort was %. -month and -year ci of aic were % and %. of developed aic, ( %) had aiha, ( %) had aiha±itp and ( %) had aiha ±itp±ain. median onset of aic was . months post-hct (range . - . months). patients were treated with a median of treatment modalities (range, - ). one ( %) had steroid, ( %) had steroid+high-dose-ivig, ( %) had steroid+high-dose-ivig+rituximab, ( %) had steroid +high-dose-ivig+sirolimus and ( %) had steroid +high-dose-ivig+rituximab+sirolimus. the median time to resolution in ( %) who achieved remission after first aic was . months (range . - . months). had one relapse and had two relapses. died after development of aic ( aspergillus pneumonia; multi-organ failure). of ( %) surviving patients after aic, had on-going aiha at median of follow-up . years post-hct (range . - . years). on univariate completing-risk analysis, age at transplant > . years (p= . ) and pre-transplant aic (p= . ) were associated with higher incidence of aic (figure a -ic). on fine-and-grey models, only age at transplant (hr . , %ci . - . , p= . ) was independently associated with aic. of with complete immune reconstitution data, naïve t cells > cells/ml at months post-hct was associated lower incidence of aic (hr . , %ci . - . , p= . ) (figure d) conclusions: younger age and thymopoiesis were associated lower incidence of aic in children with pid after hct clinical background: human heme-oxygenase- (ho- ) deficiency has been reported to present with tetrad of anemia, nephritis, inflammation and asplenia and is fatal if not treated. its an auto-inflammatory disorder. macrophages/ monocytes express ho- and are engaged in recycling of red cells. human ho- deficiency results in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. transplantation of either healthy wild type macrophages or new macrophages produced by sct from healthy donor has been proven to be curative for ho- deficiency in mice. in , we had reported first successful allogeneic sct for human ho- deficiency. here we report second successful non-myeloablative msd hsct for a child with ho- deficiency. methods: a -yr-old-girl presented with complaints of fever, anemia and severe hypertension. in the past, at the age of years she was admitted for high fever for month and needed blood transfusion for the first time for severe anemia and had high platelets and high ferritin. she was treated as macrophage activation syndrome with prednisolone alone and later cyclosporine was added. she had short stature, abnormal facies but normal development. hemoglobin g/dl, urine for haemoglobin was positive, platelets , /ul, ferritin mcg/l and urine albumin + and urine rbc - /hpf. ultrasound and ct scan abdomen showed asplenia. a diagnosis of ho- deficiency was suspected. mutation analysis showed homozygous missense mutations in exon (r x) on chromosome q , which would result in the absence of the functional ho- protein. both parents were carriers of this mutation. we managed her over next -years with prednisolone, hydroxyurea and mycophenolate mofetil (mmf). however she remained steroid dependent. hla-typing confirmed her healthy unaffected -year-old brother to be a fully matched donor. at the age of years she was taken up for msd sct after taking informed consent. she weighed just kg. we conditioned her with alemtuzumab- . mg/kg, fludarabine- mg/m , cyclophospamide- mg/kg and total body irradiation gray. we infused million/kg peripheral blood stem cells from her brother. graft-vs.-host disease (gvhd) prophylaxis consisted of tacrolimus & mmf. results: she tolerated procedure very well. her entire hospital stay was uneventful and lowest platelet count recorded was , /ul. her neutrophils engrafted on day + and she was discharged on day+ . his urine albumin was nil by day+ . she had no gvhd. her chimerism on day+ showed % donor cells, on day+ was % and on day+ was % donor. now he is day+ post-sct and doing well. she has no evidence of hemolysis, proteinuria, hypertension, fever. she has normal ferritin and platelets. she has gained cm height and kg weight in last months. she had no viral reactivation and her immune recovery at months post sct is good. conclusions: non-myeoablative allogeneic msd sct is a curative treatment option for human ho- deficiency. disclosure: nil two decades of excellent transplant survival in children with chronic granulomatous disease: a report from a supraregional immunology transplant centre in europe background: haematopoietic stem cell transplantation (hsct) confers life-long curative therapy for chronic granulomatous disease (cgd). the ability of donor-derived neutrophils to replace recipient's defective neutrophils makes hsct a superior therapy compared to conventional standard of care using antimicrobial therapy. methods: we examined the outcome of children with cgd who received a first hsct at great north children's hospital from to . outcomes included overall survival (os), event-free survival (es), toxicity endpoints, autoimmune disease, long-term survival and graft function. cox proportional-hazard models were used to analyse predictors of os and es. variables included for predictor analysis were age at transplant, donor, stem cell source, stem cell doses and conditioning. results: = children were included in this analysis. median age of transplant was . years (range, . - . years). ( %) had x-linked and ( %) autosomal recessive cgd. twenty ( %) had matched family donor, ( %) had unrelated donor and ( %) had parental haploidentical donor. prior to , various conditioning regimens were used, with ( %) patients undergoing conditioning with pharmacokinetic guided intravenous (iv) busulfan (bu) and iv cyclophosphamide with or without serotherapy. from , the conditioning regimen was switched to flu-treosulfan-alemtuzumab with gvhd prophylaxis using ciclosporin (csa) and mycophenolate mofetil (mmf) for family and unrelated donors (n= , %). flu-treosulfan-thiotepa-atg-rituximab was used for cd tcr alpha-beta cd depleted haploidentical grafts (n= , %). ten ( %) patients had grade ii-iv acute gvhd while had ( %) had grade iii-iv acute gvhd. none had chronic gvhd. the -year os for the entire cohort was % ( % ci, - %) (figure ). analysis by age at transplant revealed a -year os of % for children transplanted at < = years of age and % ( %ci, - %) for the children > years of age (p< . ) (figure ) . the os was comparable between match family donor ( %, % ci, - %) and unrelated donor transplant ( %, - %) ( figure ). all four haploidentical transplants were successful. the -year es for the entire cohort was % ( % ci - %). none of the variables was associated with es. all seven patients with slipping chimerism received a successful second transplant. the five deaths were all due to transplantrelated complications ( multi-organ failures; pulmonary haemorrhage; graft iv acute gvhd; post-transplant lymphoproliferative disease). the median age at transplant of deceased patients was . years (range . to years). the -year and -year cumulative incidence of autoimmune diseases were % and % respectively. three ( %) had immune cytopenia while ( %) had autoimmune endocrinopathy ( thyroid dysfunction; type diabetes mellitus). the median age of long-term survivors was years (range, to years) with the median duration of follow-up of . years (range, . to . years). there was no late death in the entire cohort. the median donor myeloid chimerism was % (range to %) conclusions: despite the limitations of a single centre study, our findings confirm that hsct is a safe and longlasting curative therapy for children with cgd disclosure: none non -medical challenges in the diagnosis and transplantation of patients with primary immune deficiency: an experience from a tertiary care center in india sagar bhattad , stalin ramprakash , raghuram cp , chetan ginigeri , fulvio porta , background: primary immune deficiencies (pid) are increasingly being recognized in several parts of india. despite being diagnosed, many patients fail to receive optimal care due to financial and social constraints. methods: case records of patients diagnosed and treated (including hematopoietic stem cell transplants) for pid diseases during feb -nov at aster cmi hospital, bangalore, india were analysed. factors leading to deferred or suboptimal care were assessed in detail. results: patients with various pids were diagnosed during the study period (details in table). of them warranted a hematopoietic stem cell transplant (hsct) as definitive curative treatment. a total of children received hsct. of them died while of them are alive and well. children ( with severe combined immune deficiency) died before a hsct could be carried out. of them were critically ill at presentation, while were stable but deferred further treatment citing financial and social constraints. children needing transplant continue to remain on follow-up and have not been transplanted to date ( of them have significant financial constraints, families are not convinced about the need for transplant and of them are being prepared for transplant). table: (scid -severe combined immune deficiency, vodi-veno-occlusive disease with immunodeficiency, cgd -chronic granulomatous disease, hlh -hemophagolymphohistiocytosis, was -wiskott aldrich syndrome, xlt -x linked thrombocytopenia, lad-leukocyte adhesion deficiency, msmd -mendelian susceptibility to mycobacterial disease, xla -x linked agammaglobulinemia, cvid -common variable immune deficiency, apeced -autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, cmcc -chronic mucocutaneous candidiasis, at -ataxia telangiectasia). conclusions: we present our experience from a developing country and discuss non-medical factors leading to suboptimal care in children with pid. only % children warranting hsct could be transplanted in our cohort. among those where hsct is potentially curative % of children died before hsct could be offered. transplants in developing countries pose unique challenges due to the absence of government funding and/or universal insurance coverage. in addition to delay in diagnosis and critical state of patients at admission, financial and social factors significantly contributed to poor outcome. disclosure: none the outcome of hematopoietic stem cell transplantation (hsct) in pediatric patients with hemophagocytic lymphohistiocytosis (hlh) in korea methods: the korea histiocytosis working party retrospectively collected nation-wide data from the patients diagnosed with hlh and underwent allogeneic hsct between and . the clinical characteristics and treatment outcomes of the patients were analyzed. results: a total of patients were enrolled. there were patients with fhl ( fhl , fhl , and fhl ), infection associated hlh, and secondary hlh of unknown cause. all the patients were treated with hlh- protocol, and patients achieved complete response (cr) after treatment for weeks, while did not. the main reasons for receiving transplantation were fhl in , reactivation in , and refractory disease in . the conditioning regimens were busulfan-based in patients, fludarabine-based in , treosulfan-based in , and busulfan/fludarabine-based in . stem cell sources used for hsct were from peripheral blood in patient, cord blood in , and bone marrow in . the donor types of hsct were unrelated donor in patients and related in ( matched sibling donor, haploidentical donor, partially matched donor). the causes of death of patients were disease reactivation/ progression in , acute gvhd with/without vod in , and graft failure in . five year overall survival rates were . %, respectively. the disease status at the time of hsct was cr in patients, and non-cr in . the -year survival rate of patients who received hsct in cr was % and % for patients transplanted while in non-cr status (p= . ). patients who received hsct using peripheral blood stem cells had a better -year survival rate of % compared to % of patients who received cord blood stem cells, significantly. the presence of neurologic symptoms, disease status after intial week therapy, conditioning regimen, and cd positive cell count did not have statistically significant impact on survival. conclusions: hsct improved the survival of patients who had familial, or relapsed, or refractory hlh in the korean nation-wide hlh registry. these results are similar to other reports in the literature. the disease status at the time of hsct and the stem cell source of the transplant were the important prognostic factors that affected the survivals of the hlh patients who underwent hsct. clinical trial registry: no registry number. disclosure: to the best of our knowledge, the named authors have no conflict of interest, financial or otherwise p hematopoietic cell transplantation with reduced intensity conditioning regimen using fludarabine/ busulfan and fludarabine/melphalan for primary immunodeficiency diseases background: primary immunodeficiency disease (pid) is congenital disorders of innate or acquired immune system. hematopoietic cell transplantation (hct) was a treatment option for pids with life-threatening infections or immune dysregulations. reduced intensity conditioning (ric) was increasingly used to prevent complications in hct, but optimized regimens have not been established. we performed hct for pids with ric using fludarabine and busulfan (flubu) or melphalan (flumel) according to the guidelines of european society for immunodeficiencies (esid) / european society for blood and marrow transplantation (ebmt), and assessed the efficacy and safety of these ric. methods: from april to december , pid patients underwent ric-hct using flubu or flumel in tmdu were analyzed retrospectively. the auc of bu was set to mg*hour/l for severe combined immunodeficiency disease (scid) and mg*hour/l for non-scid. overall survival (os) was analyzed. results: the median age at hct was . ( . - ) years old ( male and female patients). flubu was used for patients ( scid, combined immunodeficiency disease (cid), ectodermal dysplasia (eda), and severe congenital neutropenia (scn)) and flumel was used for patients ( scid, cid, xiap deficiency, and eda). anti-thymocyte globulin was used in patients of flubu group and patients of flumel group. cord blood in and bone marrow in was used for donor sources. matched donor was used for and patients in flubu and flumel groups ( . % and . %), respectively. median follow up period was . years. two years-os of all patients, flubu group patients and flumel group patients was . %, . % and . %, respectively. neutrophil engraftment was . %, . % and . % (all patients, flubu group and flumel group). in scid, all patients in flubu group achieved engraftment and survived. seven out of in flumel group achieved engraftment, but patient had secondary graft failure and patients died. in non-scid, out of in flubu group achieved engraftment, but patients had secondary graft failure. all non-scid patients in flumel group were engrafted and survived. two and patients in flubu group and flumel group suffered from severe acute graft-versus-host disease (grade iii-iv). ten patients had hemophagocytic lymphohistiocytosis (hlh). viral reactivation or infection was observed in patients, and resolved in all but one patient. conclusions: the ric-hct using flubu or flumel was advantagous for neutrophil engraftment, and flubu for scid and flumel for cid with immune dysregulation may be an effective opinion. flubu regimen needs to be improved for secondary graft failure in non-scid. prevention of hlh after transplantation using dexamethasone palmitate will be considered. disclosure: nothing to declare. survival after hematopoietic stem cell transplantation with tcrαβ/cd graft depletion in older children with primary immunodeficiencies background: tcrαβ/cd depletion is a graft engineering method that proved valuable in increasing the survival rate after hematopoietic stem cell transplantation (hsct) in patients with primary immunodeficiencies (pid). decreased survival rate in older patients with pid was previously reported after transplantations with nonmanipulated grafts. methods: patients with various pid (excluding classic scid) who received allogenic hsct with tcrαβ/ cd graft depletion from to september in our center were analyzed. the median age at hsct was , years (range , - , ). patients were divided into age groups: - years - patients, - years - , - years - . patients received hsct from matched unrelated, -haploidentical donors, -siblings. conditioning regimens with - alkylating agents and antithymocyte globulin were used in all patients. patient received short courses of various posttransplant immunosuppressants. median follow up after hsct is , years (range , - , years). results: overall survival (os) in patients was , ( % ci , - , ). we observed similar os in the younger age groups: , ( % ci , - , ) in - years and , ( % ci , - , ) in - years of age. seven of patients in older group ( - years of age) died, the os was only , ( % ci , - , ), p= , . all patients from older age group who died had combined pid: -wiscott-aldrich syndrome, -undefined pid, -il rg deficiency, -dclre c deficiency, -nijmegen breakage syndrome (nbs), -kabuki syndrome, -icf syndrome. the median time of death after hsct was , years (range , - , ). six of those had transplant-related mortality (trm). five patients had hsct-associated viral infections: -cmv pneumonias, -adv infections ( -fulminant hepatitis, -multiorgan). interestingly, of them had prolonged history of disseminated viral infections (adv), with the reduction of viral load in blood and other fluids and tissues upon treatment. one patient with kabuki syndrome after hsct developed hhv associated kaposi sarcoma, was successfully treated. eventually all patients with reduction of viral infection and sarcoma symptoms developed multiorgan failure with some clinical and laboratory evidences of endothelium cell damage syndrome. one patient with nbs died of high grade lymphoma progression. conclusions: hsct with tcrαβ/cd depletion demonstrates high survival rate in patients with various pid. in our group patients' age older than years predisposes to decreased posttransplant survival. patients with combined pid are at higher risks of posttransplant mortality. we conclude that at least in some of our patients with prolonged history of viral infections after hsct the cause of death could be multiorgan failure due to endothelium cell damage syndrome resulting from persistent inflammation and drug toxicity effects. disclosure background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) caused by a mutation in of the subunits of the nicotinamide dinucleotide phosphate (nadph) oxidase, which leads to a reduction in the microbicidal activity of phagocytic cell. starting at an early age, cgd patients suffer from severe recurrent infections, as well as inflammatory events. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for cgd in patients with insufficient benefit from supportive care and prophylactic antibiotics. we reported a series of patients with cgd who underwent unrelated umbilical cord blood transplantation (ucbt) at our center. methods: in this retrospective study, we observed a series of consecutive ucbt performed at our center in children with cgd between and .median age at transplantation was months (range, to months), median body weight was kg (range, to kg). / patients received a myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf, / patients received another myeloablative conditioning regimen consisting of busulfan, fludarabine, cyclophosphamide and atg. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. results: engraftment occurred in / ( . %) patients. / ( . %) patients occurred graft failure, all of them received a myeloablative conditioning regimen without atg. median time to neutrophil and platelet engraftment were (rang, - ) and . (rang, - ) days. / ( . %) patients developed acute gvhd, with / ( . %) episodes of grade iii-iv agvhd. chronic gvhd occurred in / patients ( . %). at a median follow-up of months (rang, to months), the overall survival rate was . %, and event-free survival rate was . %. conclusions: unrelated ucbt should be considered as potential curative methods in children with cgd. cgd patients who used myeloablative conditioning regimen with atg shows better graft and survival. disclosure: nothing to declare background: invasive fungal infections (ifi) remain a major cause of treatment-related morbidity and mortality in aml patients. although not uncommon, the presentation of unusually severe clinical features might be indicative for an underlying immunodeficiency. caspase-associated recruitment domain (card ) is recognized to have a crucial role in effective antifungal response, leading to th and th differentiation and to the initiation of the inflammatory cytokine cascade. particularly interferon-gamma (ifng) increases macrophage activity. patients with homozygous card mutations are known to have a significantly increased susceptibility to life-threatening systemic candidiasis. however, some sequence variants may lead to increased ifi-susceptibility even in heterozygosity, e.g. under immunosuppression. ifn-γ has been described as an additive treatment option because of its immune stimulating effect on the leukocyte immune response in a situation of immunological "blindness". methods: here, we report the case of an -year old male with aml m with a severe systemic candida tropicalis infection, unresponsive to triple-antimycotic regimen, leading to multi-organ failure. he was discovered to bear a heterozygous card mutation, and ifn-γ immunotherapy leaded to complete response of all disseminated infections. results: the patient developed septic fever immediately after the first chemotherapy cycle. unexpectedly, candida tropicalis was confirmed in the blood culture within hours. liposomal amphotericin b (ambisome ® ) was started immediately, however candida rapidly disseminated to lungs, liver, spleen, kidneys and cns despite extended antimycotic therapy with caspofungin, voriconazole and fluconazole. the patient was splenectomized due to massive infiltration ( figure ). genetic testing for mycosis predisposition revealed a heterozygous mutation in the card gene, inherited from the father (c. a>t(p.glu val)). ifn-γ treatment was started ( μg subcutaneously, times per week), leading to an almost complete response of disseminated infections. due to the severe infection, chemotherapy had to be interrupted after one course. however, bone marrow remained in complete remission for almost one year. the patient experienced altogether two relapses requiring an unrelated allogeneic and a haploidentical hsct. under combined ifn-γ and ambisome/ fluconazole prophylaxis no further mycosis was observed despite extensive and prolonged immunosuppression. conclusions: ifi in aml patients are common, however an unusual presentation in presumably immune competent individuals should raise the suspicion for immunodeficiencies. in our case, an unexpected early candida-sepsis was completely unresponsive to an adequate multi-agent treatment. while ifn-γ is used in adults as an immune stimulatory cytokine, little data are available for children. to our knowledge, this is the first case of successful ifn-γ treatment of a pediatric aml patient with disseminated candida sepsis, bearing a card mutation. given the elevated mortality risk for ifi, and the apparently safe and well-tolerated application of ifn-γ, an adjuvant immunotherapy might be considered. further studies are needed to define the indication and duration of this kind of adjunctive immunotherapy. moreover, considering the wide heterogeneity of genetic mutations involved in ifi-susceptibility, genome-wide expression profiling might be useful for pediatric cancer patients, as the identification of specific immune pathways might help to identify individual ifisusceptibility in order to improve the outcome of those high-risk patients. [[p image] . background: chronic granulomatous disease (cgd) is curable by allogeneic hematopoetic stem cell transplant (hsct). recent reports of haploidentical donor hsct with with post transplant cyclophosphamide (ptcy) from family donors in pediatric primary immune deficiencies have shown encouraging results. however, it has not been reported in cgd. here we describe successful haploidentical hsct in a child with cgd with myeloablative conditioning and ptcy. a year-old, male child diagnosed with cgd showed oxidative activity . % by dihydrorhodamine (dhr) test. he had no matched related or unrelated donor available so underwent haploidentical hsct after taking informed consent of the parents in may . donor was his / hla matched healthy elder sister (oxidative activity % by dhr). he has had multiple admissions for recurrent pneumonia prior to hsct. the conditioning was with rituximab mg/m iv on day - , thiotepa mg/ kg/dose intravenous (iv) for day (day- ), busulfan . mg/kg/dose daily iv for days (day - to - ) and fludarabine mg/m /dose daily iv for days (day - to - ) and rabbit anti-thymoglubulin (thymoglobulin) . mg/ kg/dose daily for days (day- to - ). peripheral blood stem cells ( million/kg cd + cells) were harvested from his sister and transfused to the patient on day . graft vs. host disease (gvhd) prophylaxis was with ptcy mg/ kg on day+ & , intravenous cyclosporine from day- (targeting levels - ng/ml) and mmf from day+ . results: his neutrophils engrafted on day+ and platelets on day+ . chimerism on day+ , and months was fully donor. he developed no acute or chronic gvhd. at months his lymphocyte counts showed cd - /ul, cd - /ul, cd - /ul and cd / - /ul. his had no viral reactivation. he is disease free and gvhd free on day+ post hsct and is on tapering doses of cyclosporine. his dhr test showed oxidative activity of % on day + . background: primary immune deficiencies (pid) are a functional disorder of inheritance immune system that increase predisposition to infectious disease in number and severity. the incidence is : , birth live; its immunological dysregulation may increase the predisposition of autoimmune diseases and malignancy, the latter being more frequent ( - %). at present, the only curative treatment is hematopoietic stem cells transplant (hsct). methods: we describe all patients transplanted with primary immune deficiencies at instituto nacional de pediatria. the conditioning regimen depended on the type donor and pathology: myeloablative ( . %), reduced intensity ( . %) and non myeloablative ( . %) without modification statistically significantly in overall survival. results: a total of patients were included from to january/ . severe combined immunodeficiency (scid) is the pathology most frequently transplanted ( figure ) . seventy three percent have molecular diagnosis, and . % have cases of family pid. the most used sources were umbilical cord blood (ucb) with . % and peripheral blood ( . %), however the trend of the source of obtaining it has been modified a few years ago (figure ) . the median graft was days for ucb, days for bone marrow (bm) and days for peripheral blood (pb) (figure ) the main complications are infectious (bacterial . % and viral . %) and non-infectious as pre-graft syndrome ( . %). conclusions: the overall survival was . % survival according to pathology was: % chediak higashi syndrome, % scid, . % griselli syndrome, . % hyper igm syndrome, % was, % cgd, % hemophagocytic lymphohistiocytosis. disclosure: ramírez-uribe rosa maria nideshda, salazar-rosales haydeé, olaya-vargas alberto, lópez-hernández gerardo, del campo-martínez maria de los Àngeles we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. long-term outcome following hematopoietic stem cell transplantation of wiskott-aldrich syndrome in a single institute mamoru honda , , yukayo terashita , minako sugiyama , yuko cho , akihiro iguchi background: wiskott-aldrich syndrome (was) is an xlinked disorder of hematopoietic cells, characterized by thrombocytopenia with small platelets, eczema, and immunodeficiency. hematopoietic stem cell transplantation (hsct) is the only curative treatment, and it is recommended to be performed as soon as was is diagnosed. myeloablative conditioning before hsct is recommended because there is a high risk of development of autoimmune disease in patients with mixed chimera after hsct. however, there are few reports about late complications such as pubertal development and eruption of teeth in patients with was receiving hsct. thus, we evaluated late complications in patients with was receiving hsct at hokkaido university hospital. methods: we reviewed medical records of male patients with was who received hsct between and . results: mean age at hsct was . (range, . - . ) years, and median follow-up time after hsct was . (range . - . ) years. conditioning regimen in all patients comprised busulfan at mg/kg for days and cyclophosphamide at mg/kg for days or mg/kg for days. additionally, anti-thymocyte globulin at . mg/kg/day for - days was administered in patients. engraftment, normal platelet count, and complete chimera were confirmed in all patients. no patients showed complications such as severe chronic graft-versus-host disease, autoimmune disease, short stature (≤ - . sd) and second malignancy. however, high ige level was observed in patients. pubertal development has been confirmed in patients. lack of complete eruption of permanent teeth has been observed in patients who received hsct at age of < years. conclusions: although this was a small-cohort study in a single institute, complete chimera has been achieved in all patients who received hsct with busulfan-based myeloablative conditioning. however, late complications such as male infertility and incomplete eruption of permanent teeth remain major problems. disclosure: nothing to declare methods: we performed unrelated umbilical cord blood transplantation (ucbt) in consecutive children with lad-i. median age of children was months (range, to months), and median body weight was kg (range, to . kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. all patients received a ≤ hla alleles-mismatched cord unit, was hla fully matched, were / matched, was / matched. median nucleated cells of the cord blood were . x /kg (range, . to . x /kg), and median cd + cells were . x / kg (range, . to . x /kg). results: all patients engrafted, median time of neutrophil engraftment was days (range, to d), and median time of platelet engraftment was days (range, to d). median follow-up time was months (range, to months), all patients were alive with continuous completely donor engraftment, and achieved complete clinical remissions. / patients developed grade ii/iii acute graft-versus-host disease (gvhd), and / patients developed chronic gvhd with skin. conclusions: it is the first successful unrelated ubct for lad-i children in china. our data shows ucbt provided excellent outcome for patients with lad-i. disclosure: nothing to declare p excellent outcome using 'nktm' enriched hematopoietic stem cell transplants for patients with inborn errors of immunity results: majority of patients in the cohorts had significant infective co-morbidities at the time of hsct with patients in the later cohort entering hsct earlier. patients in the later cohort were sicker at hsct. final engraftment occurred in all except patient who received a hla mis-matched cord blood hsct. graft failures occurred in patients ( in earlier and in later cohort); of these patients received unmanipulated hscts from hla mis-matched unrelated donors ( cb, bm). second hsct were with same donors in and different donors in patients. no grade ii to iv acute gvhd or extensive gvhd occurred. one patient (cbt) died of infections/ non-engraftment. all patients in the later cohort compared to of patients in earlier cohort are alive, engrafted and cured. performance status were % in all alive patients. of the patients in the later cohort, ( hla mis-matched related and hla matched related donors) received 'nktm' enriched hsct. in the hla mis-matched 'nktm' enriched hscts, patients received high cd +, cd +cd ro+ and nk cell doses, with median of . (range, . - . ), . (range, . - . ) and . (range, - ) x /kg, respectively. no invasive infections occurred in these patients and immune reconstitution in t, b, nk compartments were complete at year after hsct with cd > by months and tcrαb > by year after hsct. background: viral infections contribute to significant morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-hsct), increasing both the human and the financial cost. antiviral agents are often ineffective or/ and associated with toxicity. methods: in view of t-cell anti-viral immunotherapy in greece, we evaluated the actual cost of conventional pharmacotherapy for cmv, ebv and bkv reactivations after allo-hsct, by calculating the costs of (i) the antiviral agents, (ii) the treatment (excluding transfusions) of antiviral drug primary toxicity (e.g. graft failure, cytopenias, renal or hepatic dysfunction) and secondary toxicity (e.g. leukopenia-associated bacterial infections), iii) the treatment (excluding transfusions, ie for bk cystitis) of infectionrelated complications, iv) the transfusions due to treatmentrelated toxicity (ie cytopenias) or infection-related complications (ie, bk cystitis), v) the inpatient or outpatient daily care. notwithstanding that blood and its products, as a common natural good, are provided free in our country, the costs related to blood and platelet collection, processing, storage, laboratory testing and infusions were included in our model. results: the treatment cost of cmv, ebv and bkv reactivations/infections for the first six months post allo-hsct was evaluated in / patients who reactivated viruses and were transplanted between / - / from matched related ( / ), matched unrelated ( / ), mismatched unrelated ( / ), haploidentical ( / ) and mismatched related donors ( / ). we detected cmv, ebv and bkv infections/reactivations in , and patients respectively, with a mean of ± . infection per patient from all three viruses ( - /patient). of note, / patients experienced reactivations from more than one virus, requiring repeated treatments with antiviral agents and/or rituximab. the cost of antiviral agents for all cmv, ebv and bkv reactivations/infections was , €, , € and , € respectively ( , €, , € and , €/patient, respectively). the treatment cost of toxicity related to antiviral drugs and infection-related complications was , € ( , €/patient) excluding transfusions and , € ( , €/patient) including transfusions. in particular, the cost of transfusions for bkv hemorrhagic cystitis reached , €/patient. repeated ( - ) and/or prolonged (Δm d, range - d) hospitalizations were needed, up to a total of and days of inpatient hospitalization and short-term outpatient treatment, respectively. hospitalizations further increased the cost of inpatient and outpatient post-transplant care by , € and , € respectively ( , € and €/patient, respectively), onthe basis of a, rather underestimating the true cost, fixed, unified hospitalization fee ( €/day and €/day). conclusions: overall, in a six-month study period, the treatment of cmv, ebv and bkv infections substantially increased the cost of post-transplant care by , € ( , €/patient). the actual cost is undoubtedly higher as the hospitalization fee for transplant recipients is largely underestimated in greece. considering not only the hematopoietic but also the solid organ transplant recipients, the financial burden of antiviral treatment for national economies is enormous. given that antiviral pharmacotherapy is often associated with suboptimal efficacy, toxicity, development of drug resistance, reactivation recurrences and repeated hospitalizations, it is expected that a one-time treatment with multi-virus-specific t cells, able to expand in vivo and provide a long-lasting protection without significant toxicity, will serve as a powerful and costeffective treatment over conventional pharmacotherapy. disclosure: nothing to declare methods: this is a single-centre retrospective analysis of consecutive patients who underwent tcd allo-hscts for myeloid malignancies between january -june . ebv-dna was monitored frequently on whole blood samples with standardised quantitative real-time pcr. serum protein electrophoresis was routinely tested with immunoglobulin subclasses identified by immunofixation electrophoresis. histological confirmation of ptld was based on standard who diagnostic criteria ('proven'), while those without biopsy were classed as 'probable' based on clinical & radiological criteria as defined by ecil- guidelines. results: majority of patients had aml(n- / ) and mds(n- / ) with a median age of years(range . median follow up of survivors was months(range - ). majority of patients(n- / ; %) developed ebv-r with a median time of days[inter quartile range(iqr) - days] &higher cumulative incidence with atg(n- ) versus alemtuzumab(n- )(p< . ). figure- a shows schematic representation of ebv and ptld events (cumulative incidence of . %( %ci- . %- . %) at months). significantly higher peak ebv dna viral load(evl) were noted in patients with ptld(p-< . ). development of post-hsct mg was observed in %(n- / ). roc curve identified peak blood evl> , copies/ml significantly correlated with risk of developing ptld (sensitivity- . %,specificity- . %;auc- . ,p< . ). based on these estimates, subgroup of patients with no ebv-r(n- / ), peak evl < , (< k)copies/ ml(n- / ) & > , (> k)copies/ml(n- / ) were categorised in groups along patients with/without mg accordingly (groups - ;figure- b). patients with ebv-r had significantly better os [ -year os of % vs %(no ebv-r);log-rank p< . ],with this survival benefit mainly driven by subgroup of patients with lower evl(< k)(p< . ). ptld patients had trend towards inferior -year os( % vs %;p- . ). patients with mg had a significantly better os irrespective of degree of evl (group - ,p< . ).we report a 'sweet spot' of low evl & presence of mg in these patients, with a clear survival advantage compared to those with no ebv-r and/or no mprotein (group- -year os % vs % in group- ; hr- . ; %ci: . - . ;p< . ;figure b). overall cumulative incidence of relapse (cir) was %( %ci: - ) and non-relapse-mortality(nrm) of %( %ci: . - ) at years. multivariate analysis(mva) revealed absence of m-protein,high evl (> k copies/ml) or no ebv-r and absence of any gvhd as significant factors for high cir. similarly, high evl or no ebv-r, absence of m-protein and itu admission were significant predictors of high nrm. conclusions: this study adds to our understanding of role of ebv viraemia & associated mg in tcd-hscts while highlighting its significant impact on risk of ptld, os, nrm & cir. low ebv burden and development of mg is protective with significantly better survival outcomes and we recommend pre-emptive approach of using rituximab for ebv-r /ptld is best employed at higher ebv burden (e.g. > k copies/ml dna) in high risk patients and be prospectively evaluated in future studies. clinical trial registry: n/a disclosure: nothing to declare p impact of early candidemia on the long-term outcome of allogeneic hematopoietic stem cell transplant in non leukemic patients: an outcome analysis on behalf of idwp background: to assess the incidence of, and risk factors for, candida infection in the first days post-allogeneic hematopoietic stem cell transplantation (hsct) and the impact on long-term survival. methods: outcome analysis of , patients, % male, median age years (range - ), with diagnosis of hemoglobinopathies in ( . %), bone marrow failure in ( . %), lymphoma in ( . %) and myelodysplastic/myeloproliferative disesases in ( . %) patients who underwent hsct from to : with candidemia by day + , and , without candidemia. the incidence of -day candidemia was estimated by using the cumulative incidence method. the univariate and multivariate risk factor analysis for -day candidemia was performed with the cause-specific cox regression model. the occurrence of candidemia was analyzed as a timedependent covariate. the overall survival and non-relapse mortality after day + were assessed in a land-mark setting, this analysis was restricted to patients surviving to day + post transplant. [[p image] . figure a - b] results: the incidence of candidemia by day + was . % ( % c.i. . - . ) ( / , ) and occurred at a median of days post-hsct (range - - ). considering the candidemia within -day from hsct as a time dependent covariate, a higher -day non-relapsemortality (nrm) (hr . ( . - . ), p < . ), and a lower -day overall-survival (os) (hr . , % ci . - . ), p< . ) were obtained from the cox model for patients with candidemia. factors significantly associated with candidemia occurrence in the multivariate analysis were: gender female, increased age at hsct, bone marrow failure, lymphoma or myelodysplastic/myeloproliferative diagnosis, bone marrow or cord blood stem cell source, t-cell depletion, less recent year of hsct. among patients alive at day + , the -year nrm and os with and without candidemia were . % vs. . %, p < . , and . % vs. . %, p< . , respectively, after a median follow-up of . years ( % ci . - . ) (figure ). in multivariate analysis, the occurrence of a candidemia episode within day + was an independent risk factor for higher nrm, hr . ( . - . ), p= . , and lower os, hr . ( . - . ), p= . . conclusions: despite the general improvements in prophylaxis and treatment, the occurrence of early post-hsct candidemia had a negative impact on transplant outcome as showed previously in leukemic patients. abstract already published. carbapenem-resistant enterobacteriaceae colonizationimportance in the risk of cre bacteremia and mortality in stem cell transplant (hsct) and acute leukemia patients marcia garnica , , marco a f bellizze , priscila g a de jesus , rafaela r c gomes , filipe m akamine , alan j marçal , luzinete co rangel , andreia assis , marcia rejane valentim , angelo maiolino background: spread of infections due to carbapenemresistant enterobacteriaceae (cre) is a worldwide phenomenon and has been associated with high mortality and clinical complications. gut translocation is the most important portal of entry of bacteria during neutropenia, and cre gut colonization is a possible risk factor for bacteremia during neutropenia. goals: in the present study, we describe the frequencies of cre colonization and analyzed its relationship with development of cre bacteremia and mortality in two different scenarios: stem cell transplant patients (hsct) and leukemia patients. methods: prospective cohorts of hsct (from to ) and leukemia patients (from to ). hsct patients were analyzed from conditioning until discharge (pre-engraphment phase) and leukemia patients from first induction chemotherapy until last intensification. if an hsct was performed in leukemia patient the patient was censored in leukemia cohort and included in hsct cohort. all patients had rectal swabs performed weekly during hospitalization for the identification of cre colonization. patients with at least one positive swab (cre colonization group) were compared to patients with no documentation of colonization (controls). the outcomes analyzed were bacteremia due to cre, and overall mortality. results: there were hsct performed during the study ( [ %] autologous and [ %] allogeneic). multiple myeloma and non-hodgkin lymphoma were the most frequent baseline diseases (n= ; %, and n = ; %), respectively. cre colonization was documented in % (n= ), and it was more frequent among allogeneic hsct and leukemia patients (p< . for both). cre colonized patients had longer hospitalization ( vs. days, p< , ), higher frequency of cre bacteremia ( % vs. . %; p= . ), and mortality ( % vs. . %, p< , ) compared to non-colonized hsct. negative and positive predicted values for cre bacteremia were % and %, respectively. thirty-one patients were analyzed in leukemia cohort, accounting to hospitalizations (median hospitalizations per patient, ranging from to ). the median age was years, and % aml vs. % all. cre colonization was documented in eight ( %), with a median time from leukemia diagnosis and colonization of days ( - days). cre bacteremia was documented only in colonized patients ( % vs. zero; p= , ). all eight colonized patients were submitted to other cycles of chemotherapy after colonization, in one of them cre bacteremia relapsed. conclusions: a routine surveillance of cre colonization showed colonization frequencies from % to % in hsct and leukemia patients respectively and was effective to stratify cre bacteremia risk as the predictive negative value was over %. colonization had association with cre bacteremia and overall mortality. efforts to minimize risks for colonization and mortality are necessary. the information of surveillance can be a tool to improve adequacy in empirical febrile neutropenia therapy in hsct and leukemia patients. disclosure: nothing to declare background: the incidence of hepatitis b virus infection is high to . % in asian population, so there is more and more attention to the risk of hepatitis b virus(hbv) reactivation in the hepatitis b core antibody positive patients during chemotherapy, anti-cd monoclonal antibody, hsct, or other intense immunosuppressive drug therapy (isdt). hepatitis b core antibody is associated with a significant risk of hbv reactivation in patients undergoing hsct. however, there are remain uncertain that the effect of anti-hbsag antibodies in hepatitis b virus reactivation among the hepatitis b core antibody positive patients undergo hsct. we aim to investigate the role of the anti-hbs and the necessity of anti virus in hepatitis b surface antigen(hbsag) negative, hepatitis b core antibody positive patients during hsct. methods: we enrolled hematological malignant patients received hsct in our center from to . we classified hbsag negative and undetectable hbv dna patients into groups as anti-hbc(-)anti-hbs(-) (n= ), anti-hbc(-)anti-hbs(+) (n= ), anti-hbc(+) anti-hbs(-) (n= ), and anti-hbc(+)anti-hbs(+) (n= ). results: hbv reactivation was identified in patients ( . %) after hsct. there was a significant difference in hbv reactivation rate in anti-hbc(+)anti-hbs(-) ( . %) vs anti-hbc(+)anti-hbs(+) ( . %) (p= . ) and anti-hbc (+)anti-hbs(-) ( . %) vs anti-hbc(-)anti-hbs(-) ( . %) (p= . ), anti-hbc(+)anti-hbs(-) ( . %) vs anti-hbc(-) anti-hbs(+) ( . %) (p= . ), but not among anti-hbc(+) anti-hbs(+) ( . %) and anti-hbc(-)anti-hbs(-) ( %) and anti-hbc(-)anti-hbs(+) ( . %). whereas there were no difference according to the donor viral profile(p= . ). the median time of hbv reactivation in hbsag negative patients accepted hsct was ( - ) days after hsct. all of the patients with hbv reactivation have been controlled with nucleos(t)ide analogues drugs, and of them achieved reverse seroconversion which detect persistent anti-hbsag antibodies in their bodies. conclusions: the anti-hbsag antibodies negative and anti-hbc positive patients have the highest risk of hbv reactivation after hsct in resolved hbv patients. the anti-hbsag antibodies play a protective role in resolved hbv patients receiving hsct. we recommend not prophylactic anti hepatitis b virus in hbsag negativity and anti-hbsag antibodies positive patients following hematopoietic stem cell transplantation. disclosure: nothing to declare methods: allo-hscts performed between and for acquired bone marrow failure ( . %) or hemoglobinopathies ( . %), with bm±cb ( . %) or pb±cb+bm ( . %) as a stem cell source were included in this retrospective registry megafile idwp ebmt study. results: demographics: the median age of recipient was . years (range: . - ), and . % were children. . % recipients and . % donors were ebv-seropositive. . % had hsct from a matched family donor, . % from a mismatched family donor, and . % from an unrelated donor. t-cell depletion was performed in vivo in . %, and ex vivo in . % patients. conditioning regimen was myeloablative in . %, ric in . %. median follow-up was . years ( % c.i. . - . ). transplant out-comes: ebv-seropositive recipients in comparison to ebv-seronegative recipients had lower os ( . % vs . %, p= . ), and higher nrm ( . % vs . %, p= . ). no other significant differences were found for: ri, rfs, and acute or chronic gvhd with respect to ebv pretransplant serostatus donor and/or recipient. multi-variate analysis: ebv serostatus as a risk factor did not reach significance, while a trend towards higher risk of development of cgvhd (hr= . ; %ci . - . ; p= . ) and better survival (hr= . ; %ci . - . ; p= . ) in allo-hsct from ebv-seropositive donors. allo-hsct in ebv-seropositive recipients had a trend towards lower risk of development of cgvhd (hr= . ; %ci . - . ; p= . ). when subgroups (r-/d-, r-/ d+, r+/d-, r+/d+ ebv serology) were analyzed, the ebv serostatus had no significant impact on os, rfs, ri, trm and development of acute or chronic gvhd. conclusions: allo-hsct from ebv-seropositive vs ebv-seronegative donors are at % higher risk of chronic gvhd in patients with non-malignant hematological disorders undergoing allo-hsct, however this difference is non-significant in multivariate analysis. disclosure: nothing to declare. results: twenty-eight ( %) pts ( male, female) were tested positive (group ) for subtype a (n= , %), b (n= , %) or a (h n ) (n= , %) while ( %) pts ( male, female) were negative (group ). vaccination rate in group ( %) was significantly lower compared to group ( %, p= . ). the median time after transplantation ( vs days), t-cell counts ( vs / μl), bcell counts ( vs / μl), igg-level ( , vs , g/ l), proportion of immunosuppressed pts ( % vs %), male/ female ratio was not significantly different between groups and . within group influenza subtypes were similarly distributed in vaccinated and not vaccinated pts (a % vs %, b % vs %, a (h n ) % vs %). pts. with subtype b infection had higher levels of t-( vs / μl) and b-cells ( vs / μl) and a longer follow up from sct ( vs days) compared to subtype a / a (h n ) infection but differences were not significant. conclusions: influenza could be proven in one third of all tested pts. dominance of b and a (h n ) pdm e subtype occurrence corresponded to the flu epidemic dissemination in the german population. the most important protective factor for outpatient sct recipients was influenza vaccination. disclosure: nothing to declare background: cmv infection is one of the most frequent complications after haplo. some risk factors are well known but the best strategy (prophylactic or preemptive treatment) to mitigate this complication is not still well defined. the primary endpoint in our study is to describe incidence and risk factors to develop cmv infection or disease in haplo. as secondary objective we analyzed efficacytoxicity of treatment and cmv related mortality. methods: we analyzed patients who underwent haplo in our center between may and may . all of them received ptcy (d+ and d+ ), tacrolimus and mycophenolate as graft versus host disease prophylaxis. a preemptive therapy based on viral load was applied. treatment was started when > ui/ml of cmv were detected in one determination or > ui/ml in two consecutive determinations. cmv analyses were made in plasma using cobas pcr technique® and positive viral load cut-off point was ui/ml. the cmv viremia was determined weekly until d+ and then every two weeks until immune reconstitution. results: the cmv infection and disease incidence at d + was . % ( episodes) and . % ( episodes), respectively. cmv disease was digestive (n= ), pulmonar (n= ), neurologic (n= ) and disseminated (n= ). the median time to first cmv infection was . days ( - ). thirty-six patients had at least one episode of cmv infection: of them ( . %) had one episode, ( . %) had two episodes and ( . %) had or more episodes, respectively. only pre-transplantation cmv status was significantly associated with cmv infection (p< . ). risk factors are shown in image . the median viral load in first cmv infection and disease was ui/ml ( - ) and ui/ml ( - ), respectively (p= . ).the median counts of cd lymphocytes at d+ in cmv infection and disease were /mm and /mm , respectively (p= . ). preemptive therapy for the first episodes of cmv infections (n= ) was valganciclovir ( , %), ganciclovir ( . %) or foscarnet ( . %), reaching a complete viral load clearance in %, with a median time to response of . days ( - ) and a median treatment duration of days ( - ). grade iii-iv toxicity (mainly hematologic) was observed in . % (n= ), % (n= ) and . % (n= ), respectively. three patients had an ul mutation, one of them with clinical and microbiological resistance to the mentioned drugs. three patients ( %) had a graft failure secondary to cmv infections. five patients ( . %) died as consequence of cmv infection: before d+ secondary to cmv disease ( pulmonar, disseminated) and after d+ due to graft failure and infectious complications. with a median follow up of . months, overall survival at months for patients who had cmv infection was . % compared to . % for those who had no infection (p= . ). conclusions: a high incidence of cmv infection in haplo with ptcy was shown in our series and it contributed to mortality in . % of patients. only cmv status (d-/r+ and d+/r+) was significantly associated with higher risk of infection. identification of high risk patients and new prophylactic and treatment strategies may improve these results. disclosure: nothing to declare. methods: consecutive patients admitted at the sct unit between january- to november- were reviewed. only first admission was analysed. screening consisted of rectal and perineal swap on admission and weekly until discharge. in case of detection of mdro, patients were isolated and infection control strategies were applied. results: patients were analysed, median age years ( - ). % were male (n= ). median duration of hospitalization was days( - ). swabs were performed, with a median of swaps/patient ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . patient characteristics are shown in table . patients ( %) spiked fever in a median of days after admission . % (n= ) had previous documented mdro colonization. median neutrophil engraftment was days ( %ci - ), in % patients (n= ) of patients had a positive screen: in ( %) patients at baseline and in ( %) patients were detected for the first time beyond baseline screen. cumulative incidence of colonization at days was . % ( %ci . - . ), at days % ( %ci . - . ), and at days . % ( . - . %) (figure ). mdro identified were: with extended-spectrum beta-lactamases producing e. coli (esbl-ec), multidrug-resistant pseudomonas aeruginosa (mr-ps), vancomycin resistant enterococci (vre) and patient with carbapenemaseproducing (cp) citrobacter freundii. / colonized patients developed mdro infection ( %): patients mr-ps, site of infection was urinary tract infection (uti), urethritis, genital ulcer. two patients were treated with ceftolozane/ tazobactam, with meropenem+amikacin; patients esbl-ec both uti treated with meropenem; patient cpcitrobacter freundii uti treated with ceftazidime/avibactam. in % patients ( / ) antibiotic treatment at febrile episode was guided by positive screening. no mdro related icu admission or mortality was observed. in % patients (n= ) background: hepatitis e virus (hev) can cause chronic infection and liver cirrhosis in immunocompromised individuals. there is limited data on hev infections in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). the aim of this study was to investigate the frequency and clinical importance of hev in a swedish cohort of hsct recipients. methods: we analyzed serum samples from hsct patients ( adults and children), collected months after hsct. hev igg and igm were detected by elisa (dia.pro®), hev rna by reverse transcriptase pcr, and quantification of hev rna was performed by digital pcr. in all patients, who were positive for hev-rna and/or serology at months, also samples collected at the time of hsct from both the patients and their donors were analyzed. in the hev rna positive patients, additional samples were analyzed to determine the duration of viremia. three hev rna negative controls were selected for each case of hev infection, matched for age, diagnosis, conditioning regimen and donor type. results: hev rna was detected in / ( . %) patients. in three of the patients hev rna was positive during a period of - months, and two of these patients were infected already at the time of hsct. in five patients hev-rna was positive, at a low level, only at months. / ( . %) patients had detectable hev igg and/or igm, whereof eight patients were hev rna negative. in / ( %) patients with hev infection (hev rna positive) alanine aminotransferase (alt) was > upper limit of normal (uln), in / ( . %) patients > . uln, and in / ( . %) patients alt was normal, at months after hsct. bilirubin was elevated > . uln in / ( . %) patients, and > uln in no patient at months after hsct. two patients died with ongoing signs of hepatitis and hev rna detected in blood. one of them developed acute liver failure, at the time interpreted as drug toxicity, and died of multi-organ failure. the other patient died of unrelated causes. the remaining six patients had cleared the infection at - (median . ) months after hsct. active gvhd was present at months after hsct in / ( . %) patients with hev infection, involving the liver in of these patients. corticosteroid treatment was ongoing in / ( %) patients; the mean dose during the preceding days was > . mg/kg in / ( . %) patient, . - . mg/ kg in / ( . %) patients, and < . mg/kg in / ( %) patients. hev infection correlated to elevated alt > . uln, or . p= . ) and > uln, p= . ) at months, but not at months, after hsct, compared to hev rna negative controls. conclusions: hev infection was detected in . % of patients tested at months after hsct and was correlated to abnormal alt. spontaneous clearance was common but one patient died in acute liver failure, where hev may have contributed. hev infection is a differential diagnosis in patients with elevated alt months after hsct. disclosure: nothing to declare monitoring of t-cell responses to viral-coded antigens in pediatric patients receiving tcrαβ-depleted haplo-hsct followed by bpx- cell administration background: αβ t-cell-depleted haplo-hsct is an effective option for children with hematological disorders in need of an allograft. however, recovery of adaptive immunity is impaired in these patients. thus, in order to accelerate immune reconstitution, we developed a novel approach based on post-transplant infusion of a titrated number of donor t cells, transduced with the suicide gene inducible-caspase- , ic (bpx- cells, sponsor bellicum pharmaceuticals®; nct ). we previously reported on immune recovery of children transplanted at our institution, showing that bpx- cells infused after αβ t-cell-depleted haplo-hsct expand in-vivo and persist over time, contributing to fasten adaptive immunity recovery (merli, ash ). here, we report the results of lymphoproliferation assay to viral-encoded antigens to assess tcell function in patients transplanted with this approach. methods: we evaluated children, male and female. median age at transplant was . years (range . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . patients had either malignant ( children) and nonmalignant ( ) disorders. no patient was given any posttransplant graft-versus-host disease prophylaxis. nine children were enrolled in the phase i portion of the trial consisting of cohorts receiving escalating doses of bpx- cells. the remaining patients (phase ii portion) received the recommended dose of x bpx- cells/kg identified in phase i. bpx- cells were infused at a median of days post-hsct (range - ). antigendriven activation of peripheral mononuclear cells was evaluated by lymphoproliferation assay with h-thymidine pulsing at d+ and harvesting at d+ . stimuli included pha or cmv, ebv and adv whole viral lysate. results are given as stimulation indexes (si, cpm stimulated sample/cpm unstimulated control). thresholds for positive response were arbitrarily set at si> for viral-encoded antigens and at si> for mitogenic stimulation with pha. fractions of responders are indicated in the figure. results: patients were analyzed from d+ to d+ post-hsct. pha responders (a) increased to %, while cmv (b), ebv (c) and adv (d) responders were %, % and % at years after haplo-hsct. responses to ebv and adv antigens were slightly delayed but improved over time. responses to pha and to cmv (e,f) were analyzed in the cmv-reactivating and cmv-non reactivating groups (cmv-yes/cmv-no). significant differences in pha response were observed at d+ and d+ . moreover, increased cmv responses were observed in cmvreactivators at d+ , d+ and d+ , with approximately % of responders at d+ , as opposed to cmvnon reactivators which comprised % responders. neither primary disease, age nor tbi during the conditioning regimen influenced proliferative capacity of the two subgroups (not shown). conclusions: we showed a rapid recovery over time of t-cell function after αβ t-cell-depleted haplo-hsct followed by bpx- cells administration. when patients were grouped according to cmv reactivation (previously demonstrated as a strong driver of immune reconstitution), a significant difference in the number of responders among the patients experiencing viral reactivation was observed using the cmv lysate only but not the immunodominant pp protein (not shown), suggesting that other viral antigens account for increased t-cell responses. results of t-cell function after bpx administration complements the phenotypic data we already reported. clinical trial registry: nct disclosure: nothing to declare. background: cytomegalovirus (cmv) is associated with significant morbidity and mortality in allogeneic hematopoietic cell transplantation (allo-hct) patients (pts). cumulative incidence of cmv infection in high-risk patients such as cd -selected or haploidentical hct have been reported as high as . - . % and - %, respectively. letermovir (ltv) was approved in / for prophylaxis (ppx) in cmv-seropositive recipients (r+) of allo-hct. since / , ltv ppx was implemented at our center for both primary and secondary ppx. we report our real-world experience. methods: adult cmv r+ allo-hct pts who initiated ltv as primary and/or secondary prophylaxis were identified between / / and / / . cord blood transplants were excluded. the primary outcome was the incidence of clinically significant cmv infection (cmv viremia requiring preemptive treatment or cmv disease). pts were followed through / . results: pts initiated ltv. . % pts were at high risk for cmv reactivation and disease (primarily ex vivo t-cell depleted hct [n = ; %] or haploidentical t-replete hct [n = ; . %]). the most common indication for hct was acute myeloid leukemia (n = ; . %) and the majority of patients received myeloablative conditioning (n = ; . %). pts ( . %) received ltv as primary ppx after hct, with a median day of ltv initiation of d+ (range d+ ─d+ ). at ltv initiation, pts had an undetectable cmv dna, and had cmv < iu/ml. clinically significant cmv infection requiring preemptive treatment occurred in of pts ( . %). one patient was treated with valganciclovir (vgv) for persistent cmv < iu/ml and received ltv as secondary ppx. a nd patient developed persistently detectable cmv (< iu/ ml) and breakthrough cmv viremia with a mutation in ul at site c yltv successfully treated with vgv. the median duration of primary ltv ppx was days ( - ), with primary ppx continuing beyond weeks post hct in pts. the median additional follow-up in patients who discontinued ltv was days ( - ), without clinically significant cmv infection to date. an additional pts ( pts overall; . %) received ltv as secondary ppx after cmv pre-emptive therapy. the median duration of secondary ltv ppx was days ( - ), with no reactivation. ltv was not discontinued due to toxicity or intolerance in any patient. cmv outcomes are summarized in figure . all-cause mortality for the pts over the observational period was . %. conclusions: primary ltv ppx significantly reduced cmv reactivation, and high-risk patients may benefit from extended prophylaxis. in patients who received preemptive therapy for cmv, use of secondary ppx showed no recurrent cmv reactivation. ltv is well tolerated. additional studies are needed to determine optimal ppx duration and to clarify role of secondary cmv ppx in high-risk allo-hct. the future standard of care will likely include extended primary ppx and secondary ppx and result in decreased morbidity and mortality associated with cmv. disclosure: andrew lin -nothing to declare, molly a. maloy -nothing to declare, valkal bhatt -nothing to declare, lauren derespiris -nothing to declare, meagan griffin -nothing to declare carmen lau -nothing to declare, anthony j. proli -nothing to declare, juliet barker -angiocrine bioscience , letermovir primary prophylaxis (pp) has been shown to reduce clinically significant cmv infection with a favorable safety profile. letermovir pp will improve the outcome of seropositive patients. however, patients who did not benefit from pp and experienced > cmv episode (infection or disease) after hct may be candidate to secondary prophylaxis (sp). indeed half of them will have > recurrent episode after pre-emptive treatment (pet). letermovir is available since november as part of the french early access program for pp and sp. we report the outcome of patients who benefited from letermovir sp in the context of this program. methods: letermovir is granted, in a restrictive manner, by the french drug agency (ansm) on a case-by-case basis for prophylaxis of cmv episode, in cmv-seropositive adult allogeneic hct recipients. sp patients should have a negative baseline cmv pcr, have already experienced > cmv episode, in the context of a potentially harmful pet according to physicians. planned letermovir daily dose was mg in case of concomitant cyclosporine and mg otherwise. all patients were routinely screened by blood or plasma cmv pcr. results: between november -july , patients received letermovir in the early access program, for pp, and for sp. among the sp patients, had previous cmv disease (gut: ; cns: ). mean age was ± years, m/f ratio was / . the sp cohort included one cord blood and haplo-identical hct. main diagnoses were acute leukemia ( %) and myelodysplastic syndrome ( %). the conditioning regimen was myeloablative in % and included atg in %. based on available data ( missing data, md), previous gvhd was present in ( %) patients, and active at letermovir initiation in ( %). thirty two ( %) patients were planned to receive immunosuppressants. donor's cmv serology was negative in / ( %) ( md). at baseline, cmv pcr was detectable in / patients. letermovir was initiated a median of days (iqr: - ) after transplant for a mean duration of ± days. only one ( %) patient developed cmv breakthrough. the median follow-up from letermovir initiation was days. among the patients exposed to letermovir prophylaxis, two patients permanently discontinued because of letermovir-related adverse events (acute gvhd and nephropathy for one, loss of appetite, pruritus, diarrhoea and weight loss for the other); two deaths occurred with no causal relationship to letermovir. data were consistent with the known safety profile of letermovir. conclusions: letermovir is or will be soonly available in most european countries for cmv prophylaxis in hct recipients. pending its routine use, letermovir used as sp was well tolerated and effective, with only / patients developing a breakthrough infection. in this high-risk population for cmv recurrence, letermovir may provide a safe bridge between pet and specific immune reconstitution, pending tapering or discontinuation of immunosuppressants. whether sp may improve survival deserves further studies. disclosure: thierry allavoine is a former employee of msd france, nathalie benard and amir guidoum are employees of msd france, marion masure is an employee of icta pm, sophie alain and catherine cordonnier have participated in advisory boards and have been members of the speaker bureau of msd. ibrahim yacoub-agha has received honoraria from msd, other authors: nothing to declare p real-world data on letermovir prophylaxis for cytomegalovirus reactivation after allogeneic hematopoietic cell transplantation: a single center experience patrick derigs , maria-luisa schubert , paul schnitzler , carsten müller-tidow , peter dreger , michael schmitt heidelberg university hospital, heidelberg, germany, background: reactivation of cytomegalovirus (cmv) still contributes substantially to morbidity and mortality after allogeneic hematopoietic cell transplantation (allohct). recently, letermovir became available as the first drug approved in europe for prophylaxis of cmv reactivation in seropositive patients who have undergone allohct. letermovir is neither myelo-nor nephrotoxic, and significantly reduced the incidence of cmv reactivation in a pivotal phase iii trial (nejm ; : ) . therefore we adopted letermovir prophylaxis according to the label as standard policy in our institution: in seropositive recipients letermovir was initiated after engraftment and continued until day + or cmv reactivation. the aim of the present study was to investigate if the favorable trial results could be reproduced under real-world conditions. methods: the study cohort consisted of the first seropositive patients who received letermovir prophylaxis at our institution (between march and august ). these were compared with a control cohort transplanted between august and march before the advent of letermovir. study and control cohorts were matched for cmv donor/recipient sero-status, underlying disease and donor type source of stem cells and application of atg. cmv viremia was monitored by a quantitative pcr twice a week during the inpatient period and weekly thereafter. patients reactivating cmv prior to engraftment were not considered as event in both groups. results: no major side effects of letermovir intake were observed. with altogether reactivation events, the cumulative rate of cmv reactivation on day + was % ( %ci - %) in the letermovir cohort and thus significantly lower than in the control group ( events, % ( %ci - %); hr . ( . - . ); p= . ). the median time to reactivation was days for the control group and not reached for the letermovir group. the cumulative number of days on valganciclovir before d + was d for the letermovir patients vs d for the control patients. there were no hospitalizations for foscavir administration in the letermovir group compared to hospitalizations in the control group. there were deaths before d + in the letermovir group (one pd, one nrm) and deaths in the control group (all pd). conclusions: this observational study confirms the safety and efficacy of letermovir for the prophylaxis of cmv reactivation in seropositive patients after allohct in a real-world setting. our results are in good concordance with the phase iii trial. although letermovir appeared to reduce the need for therapeutic valganciclovir and foscavir tremendously, larger samples with longer follow-up are needed to assess the impact of letermovir prophylaxis on non-relapse and overall mortality as well as on resource consumption. background: cmv viremia occurs in %- % of cmv r + hct recipients. pet use has reduced the risk of cmv end-organ disease (eod) and associated mortality; however, pet use may lead to substantial antiviral use and healthcare resource utilization. limited real-world data are available on the outcomes of pet. therefore, we aimed to examine cmv outcomes (eod, resistance), cmv-related mortality by day (d) and healthcare resource utilization between pet and no-pet groups among cmv r+ recipients undergoing first hct. methods: we conducted a retrospective cohort study of adults, cmv r+ recipients of first peripheral blood or marrow allograft at mskcc identified from march through december . data was extracted from electronic medical records and hct databases. cmv+ recipients were monitored weekly by quantitative pcr assay starting on d through d post hct. use of antiviral therapy for cmv viremia defined pet. high cmv risk (hr) comprised t-cell depleted (tcd) hct by cd +-selection regardless of donor hla match or conventional hct from mismatched or haploidentical donors; low risk (lr) included conventional hct from matched related donors. cmv eod was scored by standard criteria. cmv resistance mutations were confirmed by sequencing (viracor-eurofins). length of stay (los) for hct admissions and readmissions were identified through d . stratified analyses were performed to examine outcomes by pet use and cmv risk. background: in a phase iii randomized, double-blind, placebo-controlled study of cmv-seropositive post-hsct recipients, letermovir prophylaxis significantly reduced the incidence of clinically significant cmv infection through week . the objective of this research was to assess the impact of cmv prophylaxis on rates of rehospitalization in adult cmv seropositive allogeneic hsct recipients from the letermovir phase clinical trial. methods: rehospitalization was recorded as an exploratory endpoint in the clinical trial at end of treatment (week ), time of primary endpoint (week ) and through an extended follow-up period (week ). cmv-related rehospitalization was assessed in the trial. prespecified analyses describe the observed rates of rehospitalization for the letermovir and placebo groups at the specified times. fine-gray cumulative incidence function(cif) regression models were used to explore the rate of all-cause, and cmv-related rehospitalization accounting for the competing risk of mortality. a multiple linear regression model was used to describe the cumulative length of stay (los) for all-cause rehospitalizations that occurred through week (excluding time of initial transplant stay). results: observed rates of all-cause rehospitalization were lower for the letermovir group compared to placebo at end of treatment ( . %vs. conclusions: letermovir was shown to significantly reduce the rate of clinically significant cmv infection in a placebo-controlled randomized clinical trial. these analyses suggest that there is also a reduction in the rate and cumulative days of rehospitalization. this trial was not sufficiently powered to detect differences in this exploratory endpoint. nonetheless, these data provide valuable insights into the economic burden of cmv. real world data and findings from future clinical trials are needed to better understand the nature of the association between cmv and rehospitalizations. clinical methods: all consecutive patients with hematologic disorders who received hsct at our center between january and august were included. among the evaluable patients, received levofloxacin as antibacterial prophylaxis (group a) while did not receive any fq prophylaxis (group b). baseline characteristics were similar in the two groups, except for the number of patients with advanced disease ( % in group a and % in group b, p , ). median duration of neutropenia was days (range - ) in group a and days (range - ) in group b. a positive rectal swab for carbapenem-resistant enterobacteriaceae (cre) was detected in patients in group a and patients in group b. results: overall, bsi was detected in patients ( , %), ( , %) in group a and ( , %) in group b (p= , ). the median onset of bsi was days post transplant (range - ), without significant differences between the two groups. in univariate analysis, fq prophylaxis (or , ; % ic , - , ) and bone marrow stem cell source (or , ; % ic , - , ) were significant factors associated with the risk of bsi. gramnegative bacteria accounted for , % (n= ) of bsi in group a and , % (n= ) in group b, and gram-positive bacteria for , % (n= ) of bsi in group a versus , % (n= ) in group b, without statistically significant differences (p = , ). polymicrobic bsi were , % (n= ) in group a and , % (n= ) in group b. mdrgram negative bsi were detected in patients ( %) in group a and in patients ( , %) in group b (overall, cre, esbl producing enterobacteriaceae and mdr-pseudomonas). death attributable to bsi occurred in of patients ( , %); of these patients did not receive fq prophylaxis, but of them had both a pre transplant kpc colonization and active disease at transplant. neither antibacterial prophylaxis (p = , ) nor bsi (p = , ) had a significant impact on overall survival (os). conclusions: the preliminary data of our study show that fq prophylaxis is associated with a reduced incidence of bsi, in particular gram-negative infections, with no impact on os. the limitations of our study may be the different group sizes and the retrospective nauture of the study. whether antibacterial prophylaxis should be avoided in the pre-engraftment period in still a matter of debate and needs to be evaluated in larger prospective studies. disclosure: nothing to disclose. gillen oarbeascoa , nieves dorado , , laura solan , , rebeca bailen , , pascual balsalobre , , carolina martinez-laperche , , ismael buño , , javier anguita , , jose luis diez-martin , , , mi kwon , hospital general universitario gregorio marañón, hematology, madrid, spain, instituto de investigación sanitaria gregorio marañón, madrid, spain, universidad complutense de madrid, madrid, spain background: incidence and outcome of invasive fungal infection (ifi) are not well characterized in the setting of peripheral blood, non-manipulated haploidentical stem cell transplantation with postransplant cyclophosphamide (haplosct). the aim of the study was to analyze incidence and risk factors of ifi in patients who underwent haplosct at our institution. methods: consecutive patients who underwent peripheral blood haplosct with postransplant cyclophosphamide between and at our centre were reviewed. ifi was classified according to eortc definitions. proven and probable ifi were included. results: patients´characteristics are shown in table . primary antifungal prophylaxis was performed with micafungin from day - until oral intake, followed by posaconazole until day + . patients on steroid treatment for gvhd received prophylaxis with micafungin or posaconazole. % of patients obtained neutrophil engraftment. twenty-two episodes of ifi were observed in patients: proven and probable, with a cumulative incidence of ifi of % at days. most commonly isolated organism was aspergillus spp (n= ), followed by candida spp (n= : c. kruseii and c. parapsilosis), and fusarium spp (n= ). isolated cases of inonotus spp, mucor spp and trichosporon ashii were observed. pulmonary involvement was the most frequent clinical presentation (n= ), followed by fungemia (n= : candidemia, trichosporon ashii) and skin-pulmonary involvement (n= ). among patients with lung involvement, showed probable ifi: with elevated serum galactomannan and positive galactomannan in bronchoalveolar lavage (bal). there were patients without galactomannan, one with a positive bal culture for penicillum spp and the other with an aspergillus spp. median time to ifi diagnosis was days. thirteen cases were diagnosed in the pre-engraftment period, after engraftment and cases after day + . among patients with late ifi, median time to development was days. all of them were associated with gvhd ( grade iii-iv acute gvhd and moderate/severe chronic gvhd). ifi outcome was favorable in out of the ifi. treatment was liposomal amphotericin b in cases, voriconazole in and combined treatment (amphotericin b and azole) in . there were ifi related deaths, with a cumulative incidence of ifi related death of . %. prior transplant (or . , p< . ), particularly allohsct was associated to ifi development (or . , p< . ). patients with previous allohsct presented ifi mainly from molds: aspergillus, fusarium, inonotus, trichosporon and mucor. there were also candidemia episodes. no other factors were significantly associated to ifi occurrence. conclusions: in our experience, cumulative incidence of ifi in the setting of haplosct with posttransplant cyclophosphamide was similar than observed in previous studies in allosct. having received a previous sct, especially allosct, was the most significant factor related to ifi development. this high risk population should be closely monitored and could benefit from prophylaxis with azoles. disclosure: nothing to declare. methods: rsv infection was diagnosed in nasal wash (nw) or bronchoalveolar fluid (bal) by dfa (millipore, usa) or pcr (seeplex, seegene, kor). urti and lrti were defined according to ecil- guidelines. death from all causes was assessed within days after rsv infection and was attributed to rsv if the patient had persistent or progressive rsv infection with respiratory failure at the time of death. neutropenia and lymphocytopenia were defined as an absolute neutrophil count (anc) < /ul and absolute lymphocyte count (alc) < /ul, respectively. results: median number of confirmed rsv infections per year was , ranging from to . an outbreak of rsv was detected in , possibly due to a lack of compliance with contact precautions in the unit. median patients' age was years and time to rsv infection was day (- to ). twenty-three patients (pts) had received an autologous transplantation ( . %) and were allogeneic hsct recipients ( , %). median time to engraftment was days, ranging from to days. at rsv diagnosis, pts presented with urti ( . %) and with lrti ( . ) . surprisingly, around % of the auto hsct recipients had rsv pneumonia at diagnosis. variables significantly associated with lrti at diagnosis were mud hsct (no/ yes, or . ; ci . - . ); anc < /ul (or . ; ci . - . ); alc < /ul (or . ; ci . - . ); and recent or pre-engraftment hsct (no/yes, or . ci . - . ). among the pts with urti at diagnosis, progressed to lrti ( . %). forty-four of the pts died ( . %) and mortality rate was significantly higher in pts with lrti in comparison with pts with urti ( . % versus . %, p= . ). death was attributed to rsv in of the pts who died ( %). conclusions: autologous hsct recipients are also at risk of lrti caused by rsv. risk of rsv lrti is higher in mud hsct, infection acquired pre-engraftment or early after hsct, and low neutrophil and lymphocyte counts. continued education is necessary to sustain compliance to contact precautions in hsct units. disclosure background: measles is a life-threatening infection after allogeneic hct. due to the decreased coverage of vaccination in many countries, the disease reappears, increasing the risk of outbreaks worldwide. allogeneic hct recipients have been shown to be seropositive for measles in roughly - % of the cases years after transplant. however, these data were obtained before the 's from hct populations mainly conditioned with myeloablative (ma) regimens. our aim was to assess measles immunity before considering vaccination in a cohort of hct survivors including patients conditioned with reduced intensity (ric) or non-ma regimens. methods: allogeneic hct adult recipients who had not been vaccinated for measles since hct were routinely screened for measles immunity. measles igg titers were determined with a chemiluminescence immunoassay (liaison measles igg kit, liaison xl analyser, diasorin, italy). patients were considered to be seropositive if the igg titer was > . ua/ml. risk factors for seropositivity were analyzed. qualitative variables were described as numbers (%) and compared using the chi- test or fisher exact test as appropriate. quantitative variables were described as median or mean (range) and compared using the kruskall-wallis test. ors were estimated separately for factor yielding a p-value < . in the univariate analysis using logistic regression models. results: eighty-six patients, transplanted . to years (mean: , years) ago, were included. the mean age was years (range: - ), the sex ratio m/f: , . the underlying diseases were acute leukemia: ( %), myelodysplastic syndrome: ( %), lymphoproliferative diseases: ( . %), myeloproliferative neoplasms: ( %) and non-malignant diseases : ( . %). the hct was performed from an hla-identical donor in , an unrelated donor in , and a cord-blood in . conditioning regimen were ma in ( %), ric in ( %) and non-ma in ( %) patients no patient had experienced measles or had received measles vaccination since transplant. fifty-seven of the ( %) patients were seropositive for measles. measles seropositivity was not associated with conditioning regimen, patient age at transplant, patient age at time of assessment, donor age at transplant, lymphocyte count or gammaglobulin levels, or type of transplant (hlaid. vs others) measles vaccination before transplant or previous measles before transplant. the only parameters significantly associated to seropositivity were absence of previous gvhd (any type or severity, p= , or , [ , - , ]), and absence of previous extensive chronic gvhd (or , [ , ] p , ). conclusions: sixty-seven percent of allogeneic hct are seropositive for measles at a median of years after hct before vaccination. the only risk factor strongly associated with seronegativity is extensive chronic gvhd. in patients background: cytomegalovirus (cmv) reactivation is a frequent complication after hematopoietic stem cell transplantation (hsct). extracellular vesicles (evs) have emerged as a promising new category of biological biomarkers in different scenarios, including inflammation, tissue damage, cancer and viral infections. we recently reported on the potential use of serum evs as biomarkers of agvhd (lia g. et al. leukemia ( ) , ) . here, we investigated the potential correlation of cmv reactivation with plasma evs in post-transplant cyclophosphamide (ptcy) haploidentical-hsct (haplo-hsct). methods: plasma samples were collected after mononuclear cell separation at given time-points (pre-transplant, on day , , , , , , , , , and after haplo-hsct) and evs were extracted by a protamine-based precipitation method and their concentration and dimension were characterized by nano-tracking particle analysis (nanosight). after extraction, evs were analyzed by flowcytometry (guava easycyte flow cytometer) with a panel of antibodies (cd , cd , cd , krt , cd a, cd , cd , cd , cd , cd , cd , cd , cd , and cd a). results: thirty-two patients with hematological malignancies underwent haplo-hsct between and . cmv reactivation was observed in / ( , %) and occurred at a median of (range: - ) days after transplant. preliminary analysis ( / patients) showed that cd a fluorescence (platelet-derived growth factor receptor-α or pdgfr-α), cd fluorescence (ki- antigen) and cd fluorescence (ve-cadherin) were associated with an increased risk of cmv reactivation (or . p= . ; or . p= . ; or . p= . ), whereas cd (platelet endothelial cell adhesion molecule, pecam- ) concentration level was associated with a decreased risk of cmv reactivation (or . , p= . ). all these biomarkers showed a signal change before cmv reactivation (an increase with cd a, cd and cd , a reduction with cd ). (figure ). conclusions: we observed a potential association of evs membrane proteins with cmv reactivation: cd a, cd , cd and cd . these proteins are crucial for endothelium and immune cells interaction. cmv can infect different cell types including endothelial cells (bentz gl. pnas ( ) ). moreover, cd a (pdgfr-α) has been shown to function as an entry receptor for cmv expressing gh/gl/go complex (wu y. et al. plos pathog ( ) ). we plan to implement our analysis characterizing evs contents (mirnas) and will be applied to investigate other viral reactivations (e.g. epstein barr virus and human herpes virus ). [[p image] . methods: to explore the value of cmv dna extracted from gi tissue for the diagnosis of cmv gastroenteritis, we retrospectively evaluated patients, aged - (median . years) who received allo-hct from sibling( ), matched unrelated( ) or haploidentical donors( ), after receiving myeloablative ( ) or reduced intensity conditioning( ). they all underwent endoscopy for gastrointestinal symptoms between - . cmv dna from tissue samples and parallel blood samples were measured by q-pcr. positive cmv dna on the tissue was considered cmv gi infection.cmv gi disease was proven with the identification of cmv inclusion bodies or positive immunehistochemical staining using anti-cmv antibodies. results: overall, endoscopic tests were performed ( gastro-, colonoscopies) at a median of days (iqr: ) post transplantation. symptoms included nausea, vomiting, diarrhea, abdominal pain and weight loss. cmv dna was positive in / tissue samples: median copies/ml, range: - x ^ . only half patients ( / ) had concurrent cmv viremia (plasma viral load> c/ml). cmv gi infection was not correlated to the type of transplant, acute or chronic gvhd. gi cmv disease was documented by biopsy in patients. cmv dna of the tissue, but not the plasma viral load, was a predictor of biopsy positivity (or: . , %ci: . - . , p= . ). thirty-six out of cmv dna positive patients received specific treatment for at least days. symptoms resolved in / patients ( %) and the gi viral load was not a significant factor to predict cure. gi gvhd was diagnosed in / patients, among which %( / ) with cmv dna positivity. median os was days ( %ci: - ) for patients with cmv infection, similar to those without (median os: , %ci: - days, p=ns). we studied separately endoscopies of the upper ( / ) or lower gi tract ( / ) . there was no significant relationship between cmv gastritis proven by biopsy and cmv dna levels in gastric tissue. however, the viral load of the colon was a predictor of cmv enteritis (or: . , %ci: . - , p= . ). the auroc of the q-pcr was . ( %ci: . to ), the sensitivity was . % and the specificity was . % with a cutoff value of copies/ml dna. conclusions: pathognomonic findings in the biopsy remain the gold standard for the diagnosis, especially for the upper gi tract. however, when the lower gi tract is involved, quantification of cmv viral load in the tissue may be a valuable tool to support the diagnosis. positivity of cmv dna of the gi tissue, in linearity to the cmv viremia, may guide to preemptive treatment for prevention of cmv disease . disclosure: nothing to declare background: clostridium difficile infection (cdi) is caused by cd overgrowth in antibiotic-disturbed intestinal microbiota. antibiotics targeting unselectively beneficial for t-regulatory cell formation strains of clostridiales may increase pro-inflammatory processes in the guts promoting or augmenting the development of graft vs. host disease (gvhd). the efficacy of cdi treatment has impact on the persistence of inflammation which might influence the alloreactive reactions. methods: we retrospectively and, from , prospectively analyzed the data from transplant centers concerning cdi occurrence, treatment efficacy, and gvhd development. the study included patients with hematological malignancies who underwent allogeneic hematopoietic cell transplantation (allohct) between - . results: median time to cdi was days post-allohct with detection of both toxins a and b in % of cases. disturbance of intestinal microbiome was confirmed by a % rate of colonization with multidrug-resistant bacteria (mdrb). the cdi symptoms resolved with the negative toxins after the first line treatment in . % of patients. the median time to remission and therapy duration was and days, respectively. fifteen therapeutic failures were observed after treatment with metronidazole ( ), vancomycin ( ) and a combination therapy ( ) . eleven patients responded to second line treatment. thirty-seven ( %) patients died due to infections ( ), relapses ( ) and gvhd/infections ( ). we noted recurrent cdi in cases. eight patients died with active cdi. we observed occurrence or exacerbation of gvhd in ( %) patients following cdi, including cases with gut involvement (gi-gvhd). treatment with metronidazole and failure of the first line therapy increased the development or escalation of gi-gvhd (p= . and p< . , respectively). the duration of cdi exceeding days also had impact on the gi-gvhd incidence (p= . ). conclusions: . patients colonized with mdrb are at high risk of cdi. . high mortality due to infections and/or gvhd in patients with cdi. . due to lower efficacy and harmful immunomodulatory impact, metronidazole should not be the first line treatment in cdi post-hct. . emphasis must be put on fast cdi resolution to interrupt a vicious circle of the intestinal inflammatory processes. disclosure: nothing to declare establishing optimal preemptive cytomegalovirus therapy threshold post allogeneic hct in a patient population with high prevalence of seropositive status background: preemptive therapy (pet) for cytomegalovirus (cmv) reactivation post allogeneic hematopoietic stem cell transplantation (hct) was shown to decrease the incidence of cmv disease. however, the optimal pet threshold is unknown and there are significant toxicities associated with anti-cmv therapy. at our institution, we initiate pet at cmv dna titer above copies/ml ( iu/ml). our aim was to examine the efficacy of this approach including the incidence of spontaneous clearance in a population with high prevalence of cmv seropositive status. methods: after due irb approval, patients that underwent allogeneic hct were identified and records retrospectively extracted.cmv reactivation was defined as the first detectable viral titer post hct from plasma samples whereas clearance of viremia as the first date of two negative pcr values obtained at least week apart. cmv monitoring was initiated post hct performed at least weekly during the first days and every - weeks thereafter. a high sensitivity assay abbott realtime cmv was used with detection threshold of copies/ml ( . iu/ml). analysis was computed using jmp v. . . results: a. baseline characteristics: a total of patients were identified and included with a median follow up of . ( . - . ) months. median age was ( - ) years and % were male. indication for hct was for a malignant disorder in % of cases. the majority had a matched related donor ( %) and cmv igg was positive in both donor and recipient in % of cases. myeloablative conditioning was given to ( %) and ( %) received tbi. in vivo t-cell depletion was given to ( %); atg in ( %) and alemtuzumab in ( %). b. cmv reactivation and pet: a total of ( %) patients had a positive cmv pcr with median days to reactivation post hct of ; ( %) patients had peak cmv titer < copies/ml (low titer) whereas the remaining ( %) had a peak titer ≥ copies/ml (high titer). patients with high titer were more likely to be older (p = . ), have malignant disease (p = . ), haploidentical or unrelated donor (p < . ) and higher incidence of agvhd grade ii-iv (p = . ) as shown in the table. median peak titers for the low and high groups were vs. , respectively (p < . ). ( %) patients with low titers cleared spontaneously with median time to clearance of days ( - ), ( %) received anti cmv therapy and the remaining died with active viremia (range - copies /ml) with active disease. one patient in the high titer group developed cmv disease. -year os and ci-nrm was . % vs. . % (p = . ) and % vs. . % (p = . ) in the low and high titer groups, respectively. conclusions: cmv reactivation was high in this cohort however of low titer viremia in over %. a pet threshold of copies/ml ( iu/ml) appears desirable as it was associated with spontaneous clearance in almost all patients while minimizing treatment related toxicity. validation of these observations is warranted. background: the risk of pneumocystis pneumonia often warrants antifungal prophylaxis for recipients of blood and marrow or solid organ transplantation. however, complications such as myelosuppression, nephrotoxicity, and intolerance with the existing standard, trimethoprim/sulfamethoxazole (tmp/smx), may hinder or interrupt prophylaxis. rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal disease caused by candida, aspergillus, and pneumocystis species in blood and marrow transplant patients. rzf has a favorable safety and tolerability profile and a low risk of drug-drug interactions. furthermore, the stability and pharmacokinetics of rzf allow for once-weekly dosing and broad distribution to the lung and other target organs. rzf was shown to prevent in vitro pneumocystis biofilm formation and to reduce the viability of mature biofilms. a previous prophylactic study was conducted using a broader range of rzf doses. in the current study, the efficacy of rzf was evaluated to better understand the minimum doses necessary to prevent pneumocystis growth in a mouse model. methods: c h/hen mice were immunosuppressed (dexamethasone mg/l in acidified drinking water) and then infected intranasally with p. murina ( x / μl). given the slow growth of p. murina, test agents were administered at the same time mice were inoculated to test for prophylactic efficacy. mice received intraperitoneal injections of either vehicle (control/steroid [c/s]), tmp/ smx / mg/kg/ x/week (wk), caspofungin mg/kg once daily, or rzf mg/kg or . mg/kg once daily, x, or x/wk. after a -week dosing period, mice were sacrificed and lung homogenates were processed for analysis to quantify the nuclei (trophic) and asci (cyst) forms of pneumocystis. prophylaxis efficacy was based on reduction of organism burden compared with c/s. nuclei and asci counts were log transformed and analyzed by anova; individual groups were compared by the student-newman-keuls t test. survival rates were compared using graphpad prism v . results: all mice in the rzf groups had significantly reduced nuclei and asci burdens compared with the c/s group, and all but the lowest doses of rzf ( . mg/kg x or x/wk) worked as well as tmp/smx at reducing nuclei levels. similarly, all rzf groups except for the . mg/kg x/wk group showed reductions in asci levels comparable to that of tmp/smx. the survival rates were not statistically different between treatment groups. conclusions: rzf demonstrated potent in vivo efficacy for prophylaxis against pneumocystis in an in vivo mouse infection model at dose regimens much lower than the human equivalent phase regimen. these data support the development of rzf for the prevention of invasive fungal infections including pneumocystis pneumonia. disclosure: melanie t. cushion: research funding (cidara therapeutics) taylor sandison: employee, stockholder (cidara therapeutics) alan ashbaugh: nothing to declare. yuhua ru , , ziling zhu , , yang xu , , suning chen , , xiaowen tang background: immunocompromising period following allogeneic hematopoietic stem cell transplantation (allo-hsct) may allow opportunistic pathogens to thrive and result in fatal complications. epstein-barr virus (ebv) infects more than % of chinese population, and its reactivation after hsct is one of the major concerns due to the increased risk of ebv diseases and post-transplant lymphoproliferative disease. with the development of infection prophylaxis and supportive care after hsct, demographic data on ebv reactivation post-hsct needs to be updated. methods: we retrospectively analyzed the data of patients who received allo-hsct between july and july in the first affiliated hospital of soochow university. quantitative pcr (q-pcr) was used to monitor ebv-dna load in peripheral blood dynamically. ganciclovir (pre-hsct) followed by acyclovir was given as viral prophylaxis. the treatment protocol for ebv reactivation consisted of tapering of immunosuppressive agents, antiviral agents (including ganciclovir and sodium phosphonatel), and rituximab for persistent positive patients. results: totally cases from most of the provinces in china were enrolled (characterized in table ), among whom ebv reactivation developed in recipients. most reactivation events ( . %) occurred in the first year post-hsct, with a peak of . incidence rates per personyears at the second month. besides, more episodes of lateonset reactivation occurred in patients receiving grafts from haploidientical donors ( figure a ) . multivariate analyses revealed that the major impactors of ebv reactivation included atg as gvhd prophylaxis (p< . ), hlamismatched donor (p= . ) and the appearance of chornic gvhd (p= . ). cumulative incidence of ebv reactivation was low ( . %) among patients with no major risk factors, but increased to . %, . % or . % with , , and major risk factors, respectively ( figure b) . there was no statistical difference of overall survival between people with or without ebv reactivation (p= . ). conclusions: we concluded that there are similar ebv reactivation impactors in chinese population compared to literatures, including atg use, hla-mismatched donor and the appearance of chronic gvhd. additionally, incidences of ebv reactivation increased significantly with the accumulation of risk factors. however, ebv reactivation had no impact on overall survival in current virus management protocol. disclosure: nothing to declare background: several studies have shown loss of diversity of the gut microbiome in association with significant gut injury following hematopoietic stem cell transplantation (hsct). prolonged broad spectrum antibiotic use further promotes loss of microbiome diversity and increases the risk of intestinal colonization by multi-drug-resistant (mdr) bacteria. aims of this study were to prospectively evaluate the overall changes in gut microbiome composition after hsct and differences in patients colonized by mdr bacteria and treated with carbapenems. methods: we performed a prospective observational study evaluating the gut microbiota of hematological patients undergoing hsct, from admission (t ) through day + (t ). fecal microbiota was assessed by s amplicon-based sequencing. clinical, and microbiological data as well as fecal samples were collected every th day from admission. results: one-hundred fecal samples were analyzed. overall, we found a progressive decrease of bacterial richness from t to t , with a significant reduction of blautia, ruminococcus and dorea species, which are strictly associated with the production of short chain fatty acids (sca) (fig. ) . moreover, in the % (no. ) of patients who were colonized by esbl bacteria, we observed a significant reduction of clostridium spp and bifidobacterium species. as for antibiotic therapies, carbapenems were used as second line treatment of febrile neutropenia in % (no ) of cases, usually associated with aminoglycosides. in patients treated with meropenem, a strong decline of blautia and ruminococcus species was observed. this finding suggests a correlation between carbapenem regimens and increase of pro-inflammatory bacterial strains in the gut. conclusions: our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids may increase dysbiosis. moreover, for the first time we report significant and progressive alterations in the composition of blautia, ruminococcus and bifidobacterium species in patients treated with meropenem and colonized by esbl bacteria, respectively. our findings offer potential modifiable targets to reduce risk of colonization by mdr bacteria and to promote a carbapenem-sparing approach in the hsct setting. clinical background: cmv is associated with significant morbidity after allogeneic hematopoietic stem cell transplantation. strategies to prevent cmv-related complications include universal prophylaxis and preemptive therapy, more widely spread. antivirals used for cmv reactivation (cmv-r) produces major toxicities and costs. rate and characterization of cmv-r after haploidentical transplantation with post-transplant cyclophosphamide (haplo pt-cy) is scarce. our goal was to analyze cmv-r rate after haplo pt-cy, outcome, complications associated to therapy, and to identify risk factors. methods: one hundred haplo pt-cy transplants using peripheral blood as stem cell source performed between and in our center have been retrospectively reviewed. gvhd prophylaxis consisted of pt-cy mg/ kg/day on days + and + , mmf and csa from day + for all cases. cmv pcr was performed in a biweekly basis during admission for transplant and treatment, and weekly thereafter. cmv-r was considered with any cmv dna level by pcr assay above copies/ml. prior four consecutive negative weekly pcrs were needed to consider a new reactivation episode. preemptive strategy was applied in all cases. data collected in relation to cmv-r included: cmv serostatus of donor/recipient (d/r), number of cmv reactivations, length of each reactivation, antiviral treatment used, need for admission to receive treatment and adverse events related to cmv reactivation and/or antiviral treatment. results: patients characteristics are summarized in table . among patients, of them with positive cmv serology, episodes of cmv-r were detected. seventysix patients ( %) had at least one cmv-r in a median of days after transplant. none of them had cmv disease or die as a consequence of cmv-r. median duration was days ( - ). valganciclovir or ganciclovir was used in episodes ( %). foscarnet was used in episodes ( %). six of the episodes occurred after initial discharge, and required re-admission for treatment, with a median length of hospitalization of days ( - ). cytopenias requiring transfusion or g-csf support occurred in episodes ( %) treated with ganciclovir or valganciclovir. three of them needed further cd + cells booster for graft rescue. mild acute renal failure and genital ulcers were found in ( %) and ( , %) events treated with foscarnet, respectively. no cases of severe renal failure were observed. serological status different than negative/negative (n/n) (p . ) and older age ( vs years, p . ) were significantly associated with cmv-r. no relationship was observed with gender, disease, donor relationship, conditioning, gvhd or cells infused. more than reactivations were more frequent among patients with grade ii-iv acute gvhd (agvhd) and moderate-severe chronic gvhd (cgvhd). conclusions: in our experience, rate of cmv-r after unmanipulated haplo pt-cy, using pbsc as stem cell source, is considerably high. a significant proportion of patients presented complications associated with cmv-r and its treatment. cmv serological status other than n/n and older age are associated with high risk of cmv-r. patients with grade ii-iv agvhd are at higher risk of multiple reactivations. this population could be benefited from primary prophylaxis, in order to decrease treatment´s complications, re-admissions and costs. disclosure: nothing to declare. impact of infectious events occurring during the first hundred days after hsct for hematological malignancy: a monocentric retrospective study over a five-year period marie-pierre ledoux , célestine simand , , karin bilger , annegret laplace , bruno lioure background: patients undergoing hematopoietic stem cells transplantation (hsct) for hematological malignancy often present with infectious events in the early stages of the procedure, some of which having a documented impact on the outcome of the graft. for instance, cytomegalovirus (cmv) has been shown by some authors to have a protecting effect against relapse, whose features remain to be elucidated. we conducted a retrospective monocentric study regarding the outcome in terms of graft versus host (gvhd), relapse and survival of consecutive patients over a period of years, whether they presented or not with an infectious event by day among the following: cmv viremia, epstein-barr virus (ebv) viremia, human herpes virus (hhv ) viremia, bk virus (bkv) viruria, bacterial bloodstream infection (bsi) or invasive fungal infection. results: a high proportion of cmv seropositive recipients underwent a viral reactivation of cmv by day of the hsct: % if the donor is seronegative and % if the donor is seropositive. we observed that cmv wasn't associated with a lower relapse rate in our cohort, and data weren't sufficient to conclude firmly, but showed a trend towards a worse acute gvhd (hazard ratio hr . , pvalue . ). no significant correlation was found for ebv viremia. occurring in % of our patients and mostly with an early timing, hhv strongly correlated with worse acute gvhd (hr . , p-value < . ) but its impact on survival was not significant. bkv ( % of our patients) and bsi ( % of our patients) both correlated with poorer outcome in terms of overall survival (logrank < . and . respectively) although not significantly associated with relapse or acute gvhd. fungal infections were too rare events to draw any conclusion. conclusions: thus, contrary to many studies, we found no protection against relapse induced by cmv, although the trend for worse acute gvhd was obvious. the mechanisms behind this discordance could include early treatment, but remain to be studied. whether hhv is a cause rather than a consequence of acute gvhd or its treatment is debated, but the correlation is strong and the sequence of events suggests hhv might act as a trigger for gvhd. the association between bkv viruria and a higher mortality is in contrast with previous observations, and the lack for association with gvhd and relapse could suggest bkv is a surrogate for poor immune recovery and therefore other causes of non-relapse mortality. in addition to the direct lethal risk of bacteriemia, bsi also are a promoter of late non-relapse mortality through indirect toxicity. through the expansion of immune effectors they promote, one could assume that infectious events play a role in gvhd and gvl, and therefore have an interference with relapse. however, the association between each infectious event and outcome remains to be clarified to guide our prophylactic and therapeutic choices by a better understanding of the bright and dark sides of infectious events. disclosure background: rezafungin (rzf) is a novel echinocandin in phase development for treatment of candidaemia and invasive candidiasis and for antifungal prophylaxis against invasive fungal diseases caused by candida, aspergillus, and pneumocystis in blood and marrow transplant patients. rzf is differentiated by stable, prolonged pharmacokinetics (pk) that allow for once-weekly dosing and a pk-pharmacodynamic (pd) profile correlating with efficacy. clinical in vivo evaluations of drug interaction potential were performed proactively to assess the risk of drug-drug interactions (ddis) with respect to the phase dose of mg once weekly and known pk exposure in healthy individuals. methods: this open-label study of healthy inpatients assessed ddis between rzf (as perpetrator) and drugs known to have interactions with cyp enzymes and transporters (probe drugs): repaglinide (cyp c ), metformin (oct/mate), rosuvastatin (bcrp/oatp), pitavastatin (oatp), caffeine (cyp a ), efavirenz (cyp b ), midazolam (cyp a ), and digoxin (p-gp), as well as tacrolimus, a drug likely to be coadministered with rzf. an initial dose of rzf mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly -mg doses, followed by once-weekly -mg doses on days and . probe drug cocktails containing ≥ drugs were administered, once before and once after rzf administration, on a schedule designed to allow for washout between doses and to limit interactions with other probe and test drugs. samples were analysed to determine respective drug concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the pk profile of each analyte. area under curve (auc) and maximum concentration (c max ) were calculated from the plasma/blood concentration-time profiles by noncompartmental analysis. ln-transformed pk parameters were statistically analysed using an analysis of variance model. the ratio of geometric least squared means between each substrate drug when administered with and without rzf and corresponding % confidence intervals (cis) were calculated for lntransformed c max and auc. results: when rzf was given concomitantly with the probe drugs, six of nine substrates (metformin, pitavastatin, caffeine, efavirenz, midazolam, and digoxin) statistically demonstrated the absence of drug-drug interaction, as their % ci were all included within the default - % noeffect boundary. three substrates had the upper (repaglinide and rosuvastatin) or lower (tacrolimus) bounds of their ci falling just outside of this range (figure ), and these changes are considered unlikely to be clinically significant. conclusions: no meaningful pk interactions were observed between rzf and drugs known to have ddis and/or likely to be coadministered with rzf. these findings provide evidence that no dose adjustment is expected when rzf is co-administered with these commonly used drugs, which stand in contrast with the ddi complications widely associated with azole antifungals. disclosure: voon ong: employee, stockholder (cidara therapeutics), michael boily: employee (altasciences), hong wong: employee (altasciences), taylor sandison: employee, stockholder (cidara therapeutics), shawn flanagan: employee, stockholder (cidara therapeutics) abstract withdrawn. background: cytomegalovirus (cmv) continues to cause morbidity following allogeneic hematopoietic stem cell transplantation (hsct). letermovir is a newly approved drug for cmv prophylaxis in cmv-seropositive allogeneic hsct recipients. however, there is a paucity of data for its efficacy in patients receiving in-vivo t-cell depletion (tcd). at weill cornell medical center, we perform in-vivo tcd with alemtuzumab for related and hla-identical unrelated transplants, and anti-thymocyte globulin for umbilical cord blood transplant supported by third party accessory cells (haplo-cord transplant).although these drugs reduce the frequency of graft-versus-host-disease (gvhd), they significantly delay t-cell immune reconstitution post hsct, and may cause higher rates of cmv reactivation. our historical rate of cmv reactivation in cmv seropositive recipients receiving high dose valacyclovir prophylaxis is approximately %. therefore, we implemented letermovir for cmv prophylaxis in february . the primary aim of this study is to determine the incidence of cmv infection (defined as cmv viremia warranting treatment or development of end-organ disease) in tcd cmv seropositive allogeneic hsct patients who received letermovir prophylaxis. methods: this is a single center, retrospective cohort study to determine the incidence of cmv infection in adult, cmv-seropositive recipients receiving letermovir prophylaxis after in vivo tcd hsct with atg or alemtuzumab for gvhd prophylaxis. all included subjects were at least days post-transplant. results: allogeneic hsct transplant recipients met inclusion criteria. median age was years, iqr [ , ] and % were male. eight ( %) had a matched related donor, six ( %) had a matched unrelated donor, and ( %) were haplo-cord transplants. their underlying malignancy and conditioning regimens are summarized in table . ( %) received atg and ( %) received alemtuzumab for gvhd prophylaxis. median follow up time for survivors is days, iqr [ , ] . the incidence of cmv infection in the first days post-transplant was % as only one patient reactivated with detectable cmv viremia. this same patient developed cmv pneumonitis with documented ul resistance, and was successfully treated with ganciclovir. the incidence of cmv infection within the first days post-transplant was % ( / patients) . six patients ( %) developed acute gvhd in the first days, and one ( %) had relapse of their malignancy. five patients ( %) died within days post-transplant, but none of these deaths were cmv related. background: infectious complications caused by endogenous adenovirus (adv) are common and associated with morbidity and mortality rates in patients after hematopoietic stem cell transplantation (hsct). adv infections occur in about % to % of hsct recipients, with significantly higher rates in pediatric patients. a better understanding of adenoviral-specific t-cells (advt) response in donors can serve as a basis to develop more effective strategies for antiviral therapy. methods: frequencies of cytomegalovirus (cmv)-and adv-specific t cells were determined by enzyme-linked immunospot (elispot) assays with adv hexon and cmvpp respectively in health donors. we used x of mononuclear cells (mnc) per well in elispot assays. all donors were divided into groups according to the number of spots per well (spw) as follows: high responders (hr) (≥ spots; n= ), low responders (lr) (> and < spots; n= ), nonresponders (nr) (≤ spots; n= ). the average spot area of adv-and cmv-specific lymphocytes was calculated by immunospot® multiplate autocount™. cd ra+ and cd ro+ t-cells were generated by immunomagnetic negative selection. hla typing for class i and ii was performed by sequence specific oligonucleotides technology. statistical analysis was performed using graphpad prism v . software. levels of significance were calculated by mann-whitney rank-sum test, expressed as p-values (p< . ). results: the median frequency per well of advts were in hr group, in lr group, in nr group. the median spw of cmv-specific t cells in donors mnc were and didn´t differ between groups. antiviral activity may depend not only on the amount of advt but also on their ability to produce ifnγ. the average spot area for advt did not differ between hr, lr and nr groups and were , , , and , mm respectively. the median of the average spot area for anti-cmv t-lymphocytes was equal to , mm . thus, the frequency of advt was lower than cmv-specific t-cells, but advt have the ability to produce more ifnγ per cell (p< . ). in order to evaluate the distribution of the advt between naive and memory t cell compartments, we evaluated response to adv in preselected cd ra+ and cd ro+ fractions of t-cells in a group of donors. the median frequency of advt in unfractionated mnc was ; the median frequency of advt in cd ra and cd ro fractions were and , respectively. the amounts of cd ra and cd ro tcells were normalized to their amounts in mnc. we evaluated the impact of hla-alleles on the anti-adv response of t-cells in different groups and found association: hla-a* with hr group (p-value= , ; rr= , ; % ci: , to , ) and hla-a* with lr group ; rr= , ; % ci: , to , ) . conclusions: in this study the frequency of donors with advt is , % which corresponds to the reported frequency of adv-seropositive people ( %) in population of russia. advt are exclusively cd ro-positive cells. the analysis of advt in potential hsct donors will allow to determine more accurately the amounts and functional activity of specific antiviral t-lymphocytes administered to patient and optimize antiviral therapy. disclosure: nothing to declare p abstract withdrawn. chemotaxis and exhaustion of γδ t cells in the allografts are associated with cmv reactivation after hematopoietic transplantation background: cytomegalovirus (cmv) reactivation and its related diseases remain the most common and serious complications in patients who underwentallogeneic hematopoietic stem cell transplantation (allohsct). we previously reported that the incidences of total and refractory cmv reactivation reached approximately % and % after haploidentical hsct. while majority of studies in the literatures focused on the adaptive cd + αβ t cellsand nk cells in anti-cmv immunity, increasing evidences highlighted the important role of γδt cells in this context. a progressive and prolonged expansion of vδ + t cells in response to cmv reactivation was observed after allohsct. the effect of vδ + t cells associated with cmv clearance has been reported in vitro and in vivo. in contrast to the reconstituted γδt cells post transplantation, whether the phenotypes of γδt subsets in allografts correlate to cmv reactivation in hsct recipients have not been documented. methods: the proportions and phenotypes of γδ t cells were detected inallografts those were unmanipulated g-csf-mobilized bone marrow (bm) and peripheral blood (pb) harvests from donors for haplohsct. bm grafts were collected by aspiration on the fourth day of g-csf treatment (filgrastim, μg/kg/day), and pb grafts were obtained on the fifth day by leukapheresis. immunophenotyping for γδ t-cell subpopulations, including the expression of cd , cd , cd , tcrγδ, tcrvδ , tcrvδ , hla-dr, nkg d, cxcr , ccr , pd , ki , ifnγ, tnfα, and il- , was performed using flow cytometry. for detection of the intracellular cytokines, bm and pb grafts were pre-stimulated with x cell stimulation cocktail ( x, ebioscience). cmv dna in the peripheral blood of recipients was routinely monitored by quantitative pcr. the association of γδ t-cell contents in allografts with cmv reactivation in haplohsct recipients was analyzed using the mann-whitney u test and spearman test. all calculations were performed using spss . statistical software. results: we found that the proportions of total γδ t cells, and vδ and vδ subsets in both bm and pb grafts for cmv+ and cmv-recipients were comparable. neither the expression of hla-dr nor nkg d in the allografts were significantly different in correlation to cmv reactivation after hsct. the productions of intracellular cytokines of γδ t subsets did not varied in bm and pb grafts for cmv+ and cmv-recipients. interestingly, the proportions of cxcr +vδ and ccr +vδ cells in bm grafts for cmv + recipients were significantly higher than those for cmvrecipients (p = . and . , respectively). meanwhile, pma-stimulated ki +vδ cells in bm grafts for cmv+ recipients were less than those for cmv-recipients (p = . ). in parallel, the concentration of pd +vδ cells in pb grafts for cmv+ recipients were significantly higher than those for cmv-recipients (p = . ). conclusions: this study is the first to connect the chemotaxis and exhaustion of γδ t cells in grafts to the risk of cmv infection after allogeneic hsct. future studies should explore how the expressions of chemokines and exhaustion marker on the effector γδ t cells in allografts facilitate cmv replication and/or dissemination in the setting of hematopoietic transplantation. disclosure: all authors do not have conflicts of interest. this study is supported by the national natural science foundation of china (grants no. and no. ) results: incidence of ic was , %: allo-hsct - % (n= ), auto-hsct - , % (n= ). the etiology: c. parapsilosis %, c. albicans %, c. krusei %, candida tropicalis %, candida dubliniensis %. the most frequent underlying diseases was acute leukemia - % (n= ). the median age was y.o. [ month - years] . the median day of onset of ic after allo-hsct was , auto-hsct - [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . febrile fever was the main clinical symptom; septic syndrome develops in % cases. antifungal therapy was with echinocandins - %, lipid ampho b - %, azoles (fluconazole, voriconazole) - %, without therapy (the early mortality) - %. overall survival (os) at days from diagnosis of invasive candidiasis was %. the central venous catheter (cvc) removal was the only factor significantly improved os ( % vs %, p= , ). conclusions: incidence of invasive candidiasis in children after hematopoietic stem cell transplantation was . %. the main etiology agent was c. parapsilosis. invasive candidiasis infections most often affect leukemia patients, developed later after allo-hsct than auto-hsct. overall survival at days from the diagnosis was %. removing of cvc improved overall survival in children with invasive candida infections after hsct. disclosure: nothing to declare background: graft versus host disease (gvhd) and virusassociated enteropathy in allogeneic hematopoietic stem cell transplantation (allo-hsct) may cause severe quantitative and qualitative composition changes of intestinal microbiota, leading to the development of small intestinal bacterial overgrowth (sibo) on the background of immunodeficiency, which can have a negative impact on treatment effectiveness. the gold standard for diagnosis and the main criterion for sibo is the detection in the jejunum aspirate > /ml bacteria and/or the appearance of colonlike microbiota in small intestine. it is also acceptable to use an alternative non-invasive technique -hydrogen breath test, which could be especially important in patients with severe mucositis, grade iii-iv and thrombocytopenia grade iv. sibo diagnosis in the setting of the gastrointestinal tract damage and dysfunction in patients treated with allo-hsct is insufficiently studied. methods: the study included patients with acute myeloid leukemia (n= ), acute lymphoblastic leukemia (n= ), myelodysplastic syndrome (n= ), non-hodgkin´s lymphoma (n= ), hurler syndrome (n= ), who underwent allo-hsct from an unrelated (n= ) and haploidentical donor (n= ) , and which were complicated by enteropathy development. in cases, the enteropathy reason was a combination of intestinal gvhd and viral colitis (hhv- ), in cases -viral colitis (hhv- ). all patients had esophagogastroduodenoscopy with species aspiration from descending part of the duodenum and feces collection, with further bacteria pcr identification. hydrogen breath test was performed also in which patients were treated with oral lactose g/kg with subsequent hydrogen assessment after and minutes. the study was performed in the period from to days after allo-hsct. results: according to feces analysis data, colon microbiota composition significantly differed from the reference values. at the same time total bacterial mass of the duodenum was less in comparison with colon microbiota: e+ ( e+ / e+ ) and e+ ( e+ / e+ ), respectively (p< . ). quantitative composition of the duodenal microbiota was comparable to that of colon: lactobacillus spp. e+ ( e+ / e+ ) > . e+ ( e+ / e+ ), (p= . ); bifidobacterium spp. e+ ( e+ / e+ ) < . e+ ( e+ / e+ ), (p= . ); escherichia coli e+ ( e+ / e+ ) > e+ ( e+ / e+ ), (p= . ); bacteroides fragilis group e+ ( e+ / e+ ) > e+ ( e+ / e + ), (p= . ); faecalibacterium prausnitzii e+ ( e+ / e+ ) < . e+ ( e+ / e+ ), (p= . ), indicating the presence of sibo. in this case, the hydrogen breath test was completely uninformative: basal values - . ( . / . ) ppm, hydrogen concentration in minutes - . ( . / . ) ppm, in minutes - . ( . / . ) ppm, which is less than in healthy volunteers. conclusions: quantitative composition of the duodenal and colon microbiota is similar in the case of intestinal gvhd and/or virus-associated enteropathy in allo-hsct patients, which may be of diagnostic value for sibo confirmation. the hydrogen breath test is an uninformative method for sibo identification in patients after allo-hsct. disclosure: nothing to declare johannes schulte , patrick hundsdörfer , sebastian voigt background: adenovirus (adv) infections or reactivations frequently occur in the pediatric hematopoietic stem cell transplant (sct) setting and these infections contribute to increased morbidity and mortality. the nucleotide analog cidofovir might be effective in reducing adv load, however, nephrotoxicity is a considerable side effect. the new antiviral compound brincidofovir (bcv, cmx- ), a lipid-conjugate nucleotide analog with broad-spectrum antiviral activity in vitro, has been reported to be effective in cases where cidofovir treatment was unsuccessful. methods: data of eight pediatric patients undergoing sct for malignant and nonmalignant indications were analyzed. all patients were weekly monitored for adv viremia by pcr. in case two consecutive positive adv pcr results indicating a viral copy number > /ml were documented, patients received a weekly dose of cidofovir. if no reduction of adv load was seen within two weeks after the commencement of treatment or side effects demanded cidofovir discontinuation, bcv was obtained through an emergency expanded access programme. results: eight pediatric patients developed adv viremia with maximum viral loads ranging between and copies/ml. six patients had c type adv and two patients had non c type adv infections. five patients had viral co-infections: two had an additional cmv infection, one had an epstein-barr virus (ebv) and herpes simplex virus co-infection, one patient had an ebv co-infection and one patient had a bk virus co-infection. all eight patients initially received cidofovir, however, a substantial decrease in adv load could not be observed in any patient after a two-week administration course. except in one patient who had extensive intestinal graft-versus host disease (gvhd), adv infection was cleared in all patients within three weeks after the beginning of bcv treatment. in addition, all coinfections were cleared. no nephrotoxicity or other side effects were observed. conclusions: bcv was effective in all but one patient. oral bcv might not be effective in advanced upper gut gvhd, especially when applied via a gastric tube, yet this was observed in only one patient. eventually, without nephrotoxic side effects, bcv could be an useful alternative to cidofovir. disclosure: nothing to declare. background: the use of post-transplant high-dose cyclophosphamide (ptcy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. however, reconstitution of cellular immunity may be delayed even after t cell replete haploidentical stem cell transplantation (haplo-sct) with ptcy. the study of the incidence and severity of viral reactivation is therefore relevant to the outcomes of haplo-sct with ptcy. methods: our study enrolled patients (women/men, / ), who underwent t cell replete haplo-sct from / to / and achieved hematopoietic engraftment. median age at transplant was . years (range, - ) . the underlying disease was aml (n= ), all (n= ), mds (n= ), myelofibrosis (n= ), cml (n= ), or cll (n= ). the conditioning regimen was myeloablative (n= ), reduced-intensity (n= ) or non-myeloablative (n= ). peripheral blood was the graft source in the majority of cases (n= ) and bone marrow in the remaining (n= ). recipient/donor cytomegalovirus (cmv) serostatus was -/-(n= ), -/+ (n= ), +/-(n= ), or +/+ (n= ). the combination of tacrolimus and mycophenolate mofetil was administered in addition to ptcy for prevention of graftversus-host disease. cmv, epstein-barr virus (ebv), and human herpesvirus- (hhv- ) reactivation was monitored by real-time quantitative pcr (rq-pcr) in blood twice weekly post haplo-sct. bk virus (bkv) reactivation was assessed by rq-pcr in urine and/or blood specimens in cases with symptoms suggestive of bkv-associated hemorrhagic cystitis (hc). results: with a median follow-up time of months (range, , the cumulative incidences (cin) of relapse and non-relapse mortality (nrm) were . % ( % ci, . - . %) and . % ( % ci, . - . %) at years, respectively. median disease-free (dfs) and overall survival (os) were . % ( % ci, . - . %) and . % ( % ci, . - . %) at years, respectively. the cin of cmv reactivation/infection (> copies/ml) reached . % ( % ci, . - . %) at months. cmv infection developed in out of patients who were at risk, whereas recurrent cmv reactivation was observed in patients with a median number of episodes (range, - ) per patient. the median total duration of antiviral therapy for cmv infection was days (range, - ) . cmv disease (pneumonia) was documented in patients. the cin of ebv reactivation (> , copies/ml) was . % ( % ci, . - . %) at months. no case of ebv-related post-transplant lymphoproliferative disorder was observed, however preemptive therapy with rituximab was required in patients with rapidly increasing ebv viral load. hhv- reactivation (> , copies/ml) was observed in patients (cin, . % at months; % ci, . - . %), with none of them requiring specific therapy. bkv-related hc occurred at a cin of . % ( % ci, . - . %) at months. cystoscopy for bladder hemostasis was required in / and nephrostomy in / patients with hc. conclusions: despite preservation of non-alloreactive memory t cells, haplo-sct with ptcy is associated with substantial rates of viral reactivation (especially cmv and bkv) resulting in the need for prolonged antiviral therapy and considerable morbidity as well. therefore, strategies to prevent viral reactivation and disease are still warranted in haploidentical stem cell transplantation. disclosure: nothing to declare. background: adenovirus(adv) infections are a wellrecognised cause of morbidity and mortality in children and adults receiving an allogenic stem cell transplant(hsct).the reported incidence of adv infection is higher( %- %) in paediatric hsct than in adults( - %),but we currently lack accurate data of adv infection burden among adults.cidofovir has been extensively used as a pre-emptive anti-adenoviral therapy and is current standard of care.we present our single centre experience of adv incidence and outcomes with pre-emptive approach in adult patients receiving t-cell depleted(tcd) hscts for myeloid disorders. methods: this is a single-centre retrospective analysis of consecutive hsct patients for myeloid disorders including aml, mds, mpn & aplastic anaemia between january -june using atg or alemtuzumab based tcd.adv screening was performed in all patients with standardised real time quantitative pcr on weekly basis during standard risk period. figure a - b] results: baseline characteristics (table ) of patients were similar across both cohorts with or without adv infection. overall . %(n- / ) patients were positive for adv dna on atleast one of the sanctuary sites(upper respiratory airway,blood,faeces,urine) and %(n- / ) of these experienced disseminated infection(defined by adv in ≥ sanctuary sites or rising adv dna copies in blood), while developed typical adenoviral disease (pulmonary). among patients with disseminated infection,majority had adv in gastro-intestinal( %), . % in genitourinary and % as both sanctuary site of infection,in addition to blood viraemia( % of all cases).cumulative incidence of adv infection was . %( %ci: . - . %) at months with median time of days(iqr: - days) to detect adv-dna post hsct.overall survival(os) at years for whole cohort was %( %ci: - ;median os- months) with no statistical difference between patients with disseminated adv infection vs those with none(log rank; p- . ; fig- a ). overall cumulative incidence of non-relapse mortality (nrm) was %( %ci: - %) and relapse(cir) was . %( %ci: - %) at years,but no statistical difference noted between patients with disseminated adv infection & those with none(nrm:p- . ;cir: p- . ;gray test).pre-emptive therapy with cidofovir ( mg/kg weekly iv infusion for weeks and fortnightly thereafter until infection free) was required in %( / ) of symptomatic adv infection patients and %( / ) with disseminated infections.one patient required brincidofovir therapy for refractory disease,but one patient died due to severe sepsis, before adv specific therapy could be given.remaining patients were monitored and all self-recovered on cessation of immunosuppression.all patients treated with cidofovir developed renal impairment(defined by atleast > % increase in baseline creatinine),however majority( %) recovered their renal function near their baseline (fig- b) . conclusions: adv infection remains a significant cause of morbidity in adult hsct patients, however pre-emptive management with cidofovir has improved os and nrm despite use of tcd conditioning.renal toxicity remains common with cidofovir but with use of intermediate doses, majority do recover their renal functions. clinical trial registry: n/a disclosure: nothing to declare background: publications on invasive fungal disease (ifd) in lymphoma patients are limited especially after allo-hsct. there are no data on outcome of allo-hsct in lymphoma patients with prior ifd. this study focuses on epidemiology of ifd before and after allo-hsct in children and adults with hodgkin's lymphoma (hl). methods: single center prospective study included patients with classical r/r hl who received allo-hsct from to . the median age was ( - ) y.o., children (< yo) - %. allo-hsct from mud was performed in , % (n= ), mrd - , % (n= ), mmud - , % (n= ), haplo - , % (n= ), with ric ( %) and predominantly ptcy-based gvhd prophylaxis ( %). primary antifungal prophylaxis was fluconazole in %, secondary -voriconazole ( %). eortc/msg criteria for diagnosis and bronchoscopy before allo-hsct in pts with ct-scan lung lesions were used. "active ifd" means ifd diagnosed just before hsct. median follow-up time was months . results: incidence of ifd before allo-hsct was , % (n= ). ifd prior to hsct were invasive aspergillosis (ia) with lungs involvement. antifungal therapy before allo-hsct was used in , % pts with median duration - months. complete response to antifungal therapy was in , % pts, partial response or stabilization - , %, and , % pts had an "active ifd". after allo-hsct all pts received voriconazole as an antifungal therapy or secondary prophylaxis. cumulative incidence of relapse or progression of ia after allo-hsct was , % with the median day after hsct, which were successfully treated with voriconazole in post hsct period. incidence of ifd after allo-hsct for naïve patients was , % (n= / ). etiology of ifd after allo-hsct was ia - %, invasive candidiasis (ic) - %, mucormycosis - % and % combined ifd caused by aspergillus fumigatus + rhizopus stolonifer. the median day of onset of ifd after allo-hsct was day+ and was associated with post-hsct relapse of hl (p= , ). the main site of infection were lungs ( %), the main clinical symptom -febrile fever ( %). antifungal therapy was used in all patients: voriconazole - %, micafungin - %, posaconazole - %, lipid amphotericin b - % and combination lipid amphotericin b with caspofungin - %. overall survival (os) at weeks from the diagnosis of ifd after allo-hsct was %. the -year os in children and adult with hl after allo-hsct was , %. development of ifd after allo-hsct do not decrease the -year os rate ( , % vs %, p= , ). the impact of prior ifd on -year os in allo-hsct recipients was not statistically significant in all group ( , % vs , %, p= , ) , and separately in children and adults. conclusions: incidence of ifd in children and adults with hodgkin's lymphoma before allo-hsct was , %. incidence of ifd after allo-hsct in patients with hodgkin's lymphoma was , %. the major etiology agents as before as after allo-hsct were aspergillus spp. ifd was a late complication after allo-hsct and associated with post-hsct relapse. despite the high incidence ifd before or after allo-hsct didn't influence the outcome in children and adults with hodgkin lymphoma. disclosure: nothing to declare our community has high cmv positive serostatus, which is a known risk for cmv infection or reactivation. we conducted a study to explore the incidence and outcome of cmv infection among post-hsct children. methods: medical records of pediatric patients (age ≤ years) undergoing single allogeneic hsct from january to december , at king faisal specialist hospital and research centre, riyadh, saudi arabia, were reviewed. all patients with active cmv infection or disease before and during transplant were excluded. a total of patients were included in the study; were female. median age at hsct was years. recipient cmv serostatus was positive in patients before hsct, and donors were cmvpositive. the recipient-donor (r/d) serology was . % r +/d+, . % r+/d-, . % r-/d+, and . % r-/d-. indication for hsct was immune disorders . %, hemoglobinopathies . %, bone marrow failure . %, malignant disorders . %, histiocytic . %, and metabolic disorders . %. source of stem cells was bone marrow in , cord blood in and peripheral blood stem cell in cases. donor was matched related among , unrelated matched/mismatched in , haploidentical , and related with -antigen mismatch in . total body irradiation (tbi) based conditioning was used for patients, while atg was used in patients. results: out of a total of patients, patients developed cmv infection post-hsct ( . %). incidence in female recipients was high ( . % versus . %, p-value . ). both recipient and donor cmv serology positive ( . %) developed cmv infection (p-value < . ). however, no cmv infection in both recipient and donor negative group (r-/d-). the incidence of cmv infection post-hsct was high in patients received tbi based conditioning ( out of , . %, p-value . ), and in haploidentical transplant with . % (p-value . ). source of stem cells, myeloablative versus nonmyeloablative conditioning, atg use in conditioning and agvhd, did not exhibit significant association with cmv infection. in multivariable setting, when adjusted for primary indication for transplant, donor hla type, tbi based conditioning and recipient and donor cmv serology at transplant, haploidentical donor (odds ratio: . , p-value: . ) and donor-recipient cmv sero-positivity (odds ratio: . , p-value: . ) were found to be significant risk factors. cmv infection resolution rate was . % ( ). with a median follow-up time of . ± . months from infusion, five-year overall survival of cmv infected group was lower ( . ± . ) as compared to non-cmv infected ( . ± . , p-value: . ). conclusions: incidence of cmv infection post-hsct in our center is comparable to other centers. our data suggest that donor-recipient cmv positive serostatus, haploidentical donor, and use of tbi based conditioning necessitate close attention and surveillance. background: toxoplasmosis is a rare and underestimated complication following allogeneic stem cell transplantation (allo-sct) with an often fatal course. this is in part due to limited diagnostics relying mainly on imaging and detection of parasite dna by pcr. we present here eleven cases of toxoplasma disease following allo-sct. methods: we retrospectively analyzed consecutive adult patients who received an allo-sct in our bone marrow transplant unit between july and july . eleven ( %) of these patients were diagnosed of toxoplasma disease. the main characteristics of the patients are shown in table . all patients, except two cord blood, have received peripheral blood stem cells. fludarabine-based conditioning regimes were used in all patients. only the two cord blood patients received thymoglobulin in the conditioning. graft-versus-host disease (gvhd) prophylaxis consisted on tacrolimus plus mycophenolate mofetil in ( %) patients and post-transplant cyclophosphamide followed by tacrolimus in ( %). before the allo-sct was performed the igg/igm toxoplasma serology of the recipient and donor. we reviewed the absolute lymphocyte count (alc) and cd + lymphocyte count within four weeks prior to the diagnosis of toxoplasmosis, and if the patients took effective primary prophylaxis for this parasite. toxoplasma disease was defined as the presence of toxoplasma infection plus clinical, radiological or pathological evidence. toxoplasma disease was considered the main cause of death when no other major life-threatening infection or other potential fatal complication occurred immediately before death. results: median (range) age (years) of the eleven patients diagnosed with toxoplasma disease was ( - ). for pancytopenia, no patient received trimethoprimsufmethoxazole (tmp-smz) but pentamidine for pneumocystis jirovecii-pneumonia (pcp) prophylaxis in cases and atovaquone in one. toxoplasma serology pretransplant was positive (igg+/igm-) in ten of the eleven patients. all donors were seronegative (igg-/igm-) except two. toxoplasmosis was diagnosed a median (range) of days ( - ) post allo-stc. the clinical presentations were as cerebral-encephalitis (n= ), chorioretinitis (n= ), pneumonitis (n= ) and disseminated toxoplasmosis (n= ). one case, patient and donor seronegative pre-transplant, was presented as a primary infection in form of chorioretinitis. all three patients with chorioretinitis were diagnosed after day + of allo-sct. at the time of toxoplasma disease, of ( %) of patients had an alc < cells/μl and all of them with immunosuppressive therapy and corticosteroids for acute or chronic gvhd. we had cd + lymphocyte count only in four patients and in three of them was < cells/μl. eight of the eleven ( %) patients died, with a median (range) of days ( - ) since diagnosis of toxoplasmosis, and in of them the toxoplasma disease was the main cause of death. conclusions: in our series, the incidence of toxoplasma disease after allo-sct is low and is related to high mortality, in accordance with what has been reported by other groups. positive pre-transplant serology and gvhd and its treatment were factors strongly related with toxoplasmosis. we encourage the use of tmp-smz instead of pentamidine for pcp-pneumonia prophylaxis in patients seropositive for toxoplasma gondii pre-transplant. clinical trial registry: data about its epidemiology in children are scarce. we retrospectively analyzed the incidence, the severity and the risk factors that contribute to the manifestation of this complication in a pediatric population. methods: during a -year period (january -june ) we performed in our center allogeneic transplantations, for malignant hematological diseases and for non-malignant. the majority of our patients received myeloablative conditioning regimens. diagnostic criteria of hemorrhagic cystitis were the detection of the virus with pcr in urine samples and/or in blood samples, in combination with hematuria and lower urinary tract symptoms (dysuria, urinary frequency, urgency, suprapubic pain) that couldn't be attributed to any other reason. we defined the hemorrhagic cystitis as severe when one of the following factors was present: formation of clots and continuous bladder irrigation, obstructive uropathy with creatinine elevation or need for urological intervention. results: a total of patients with median age , years ( , - ) were studied. children ( %, % ci, , - , ) manifested bk virus associated hemorrhagic cystitis with median age , years ( , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . onset of cystitis occurred at a median time of days (day +-day + ) after transplantation. in children cystitis was severe. the median duration of symptoms was days ( - ). the median time of hospitalization for children with severe cystitis was days ( - ) whereas for those who didn't manifest cystitis was ( - ) . in of the patients we examined the presence of the virus not only in urine but also in blood samples. in of them the test was positive and almost half of them ( ) manifested severe cystitis. the risk factors that were examined were age, administration of antithymocyte globulin, type of disease, graft source, type of donor and the presence of acute graft versus host disease (agvhd). in multivariable analysis, independent risk factors for the manifestation of hemorrhagic cystitis were age > years old (hr: , , % ci, , - , p< , ), transplantation for malignant disease (hr: , , % ci, , - , , p= , ) and the presence of agvhd (hr: , , % ci, , - , , p< , ). the overall survival of children with hemorrhagic cystitis was , % vs , % of those who didn't manifest this complication, but in multivariable analysis for survival cystitis wasn't a statistically significant risk factor. conclusions: according to our results, stem cell transplantation in children > years old who suffer from a malignant disease and the presence of agvhd consist independent risk factors for the manifestation of bk virus associated hemorrhagic cystitis. the identification of the risk factors of this serious complication will contribute to better management of transplanted patients. further research through prospective trials can contribute to the better understanding of the pathophysiology of hemorrhagic cystitis and to the establishment of appropriate diagnostic and therapeutic guidelines. disclosure: nothing to declare p impact of natural killer cell reconstitution on outcomes in patients with early cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation background: cytomegalovirus (cmv) reactivation influences survival after allogeneic hematopoietic stem cell transplantation (sct) and induces natural killer (nk) cell expansion. we evaluated nk cell reconstitution and clinical outcomes following early cmv reactivation after sct. methods: lymphocyte subsets were measured by flow cytometry on day in patients with hematologic malignancies undergoing sct between january and december at kanagawa cancer center, excluding patients with graft failure or death within days. cmv reactivation was defined as initiation of preemptive cmv therapy following pp antigenemia surveillance. results: the subjects were males and females with a median age of years (range: - years). the median follow-up period for survivors was . years (range: . - . years). there were patients with acute myeloid leukemia, with acute lymphoblastic leukemia, with myelodysplastic syndromes, and with other diseases. at transplantation, patients were standard risk and were high risk. myeloablative conditioning and reduced-intensity conditioning were employed in and patients, respectively. bone marrow transplantation, peripheral blood stem cell transplantation, and cord blood transplantation was performed in , , and patients, respectively. cmv reactivation occurred in patients ( %) at a median of days (range: - days) after sct. grade ii-iv acute gvhd and chronic gvhd affected patients ( %) and patients ( %), respectively. among all patients, -year overall survival (os), cumulative nonrelapse mortality (nrm), and cumulative relapse (cir) rates were %, %, and %, respectively. in patients without cmv reactivation (cmvr-) versus patients with cmv reactivation (cmvr+), -year os, nrm, and cir were % vs. % (p < . ), % vs. % (p = . ), and % vs. % (p = . ), respectively. among all patients, the median level of cd -cd + cells, cd +cd cells, and cd +cd + cells on day was /μl (range: - /μl), /μl ( - /μl), and /μl ( - /μl), respectively. nk cell subsets showed no significant differences between cmvr-and cmvr+ patients. when patients were divided into low and high groups at the median level of each nk cell subset, cmvr+ patients with high cd -cd +, cd +cd -, or cd +cd + cells showed significantly better -year os than those with low cells ( % vs. %, p < . ; % vs. %, p < . ; % vs. %, p = . , respectively). high cd -cd + cells were significantly associated with lower nrm ( % vs. %, p = . ), while high cd +cd -cells were significantly associated with lower cir ( % vs. %, p = . ). multivariate analysis confirmed these nk cell subsets as prognostic factors in cmvr+ patients. conclusions: nk cell reconstitution may contribute to improved transplantation outcomes in subgroups of cmvr + patients. disclosure: nothing to declare background: rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal infections caused by candida, aspergillus, and pneumocystis spp. in patients at high risk of infection. rzf has demonstrated in vivo prophylaxis efficacy and low risk of drug-drug interactions. furthermore, the stability and pk profile of rzf allow for once-weekly dosing. rzf is also in development for treatment of candidemia and invasive candidiasis using a dosing regimen of rzf mg followed by mg once-weekly, which achieved > % target attainment against candida. while lower doses might be useful to prevent candida and pneumocystis, invasive aspergillosis is a different challenge. we evaluated rzf dosing for prophylaxis against aspergillus fumigatus in blood and marrow transplant (bmt) patients using pk/pd simulations of the treatment dosing regimen. methods: a previous population pk model was refined using data from phase and phase trials of iv rzf (nonmem vers . ). stepwise forward selection (α = . ) and backward elimination (α = . ) were used to assess for relationships between interindividual pk variability and covariates, such as age, sex, bsa, albumin, liver and renal function markers, and infection status. the final model was validated by comparing model-based predictions to observed data. the model and demographic data from bmt recipients at stanford medical center were used for monte carlo simulation (n= , ) of expected rzf concentrationtime profiles in bmt patients receiving iv rzf mg on week followed by mg weekly x . of the patients included in the demographic dataset, were female (mean values at baseline: age, years [ - years]; weight, . kg [ - kg] ). the median (range) bsa in the demographic dataset was . m ( . - . ), and albumin was . g/dl ( . - . g/dl) . free-drug concentration-time profiles were evaluated ( . % human protein-binding) relative to the a. fumigatus minimal effective concentration required to inhibit % of isolates tested (mec ; jmi - sentry international surveillance data). results: the population pk model was a linear, compartment model with zero order iv input. albumin, sex, infection status, and body surface area were statistically significant predictors of interindividual variability; clinical significance of these factors was not determined. the model provided precise, unbiased fits to the observed data (r = . observed vs individual-predicted concentrations). rzf plasma free-drug concentrations at weeks , , and were above the a. fumigatus mec ( . mg/l) for the entire dosing interval in . %, . %, and . % of simulated patients, respectively, and in ≥ . % for all weeks based on the mec ( . mg/l). conclusions: these data modelled from bmt patients support the rzf dosing regimen of mg iv followed by mg once-weekly for prophylaxis against a. fumigatus. current antifungal prophylaxis may be limited by toxicity, ddis, or patient factors such as mucositis. the pk of rzf and its spectrum, safety, tolerability, and lack of ddis may address current unmet needs in ifi prophylaxis for bmt and other immunocompromised patients. disclosure: janice brown: research funding, cidara therapeutics, elizabeth lakota: research funding, cidara therapeutics, shawn flanagan: employment, cidara therapeutics, taylor sandison: employment, cidara therapeutics, voon ong: employment, cidara therapeutics, christopher rubino: research funding, cidara therapeutics p is fungal prophylaxis necessary in non myeloablative peripheral blood stem cell allogeneic transplantation in the pre-engrafment period? julien vaidie , jean-baptiste woillard , stéphane girault , marie-laure dardé , arnaud jaccard , daniel ajzenberg , bernard bouteille , pascal turlure background: non myeloablative peripheral blood stem cell transplantation (pbsc), by limiting toxicity, can be proposed to elderly patients or patients with comorbidities. however, fungal infections remain a key issue that can negatively impact outcome, and increase duration and cost of hospitalization. systematic fungal prophylaxis have demonstrated benefits in outcome in the context of myeloablative conditioning but are not currently in reduced intensity conditioning allograft with pbsc. fluconazole prophylaxis is currently recommended in this situation (ecil). methods: primary objective of this retrospective study was to evaluate fungal infection incidence after allograft procedure in patients who received a non myeloablative allograft with pbsc in limoges university hospital between june and june . patients received fludarabine mg/m /day between d- and d- before allograft and busulfan . mg/kg/day at d- and d- . gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of . mg/kg at d- and d- , and ciclosporin at the beginning dose of mg/kg per os twice a day. mycophenolate mofetil was adding for patients with hla-matched or mismatched unrelated donors. patients did not systematically receive antifungal prophylaxis during the neutropenic pre-engraftment period. when patients had fever during more than hours, an empirical fungal treatment (caspofungine) was added to empirical antibiotics. as soon as neutropenic recovered and in the case of apyrexia without microbiologic documentation, antimicrobial treatments were stopped while in the case of microbiologic documentation, treatments were adjusted to germ in term of dosing and time of administration following recommendations. however, some patients received antifungal azole prophylaxis during the neutropenic pre-engraftment period in case of history of previous invasive aspergillosis (ia), or a nasal colonization by aspergillus. in post-engraftment period, posaconasole prophylaxis was administered for patients with systemic corticotherapy for acute graft-versus-host disease. results: patients were evaluated (median [min-max] age of [ - ] years). % of patients received an hlaidentical related donor, % an hla-matched related donor and % an hla-mismatched unrelated donor. the five years overall survival and survival without relapse or gvhd were % ic [ %- %] and % respectively ic [ %- %]. the median time for neutrophil recovery was days. patients did not receive prophylaxis and only patients received systematic fungal azole prophylaxis in the pre-engraftment period. two patients received an empirical treatment by caspofungine. only ifi was documented during the neutropenic period : candida krusei in blood culture. in the post engraftment period, patients with acute gvhd treated by corticotherapy received an antifungal prophylaxis by posaconazole and only patient had a probable ia at day despite prophylaxis by posaconasole. conclusions: except for patients with previous history of ifi, our results provide additional arguments against systematic fungal prophylaxis after reduced intensity conditioning with pbsc allogenic transplantation in the pre-engraftment period with a very low incidence of invasive fungal infections. in post-engrafment period, posaconazole prophylaxis is required for patient with gvhd treated by corticotherapy. disclosure methods: a simple, rapid and sensitive method using hplc with a diode-array detector (dad) was developed and validated for the quantification of letermovir in human serum using sorafenib as internal standard. after pretreating serum samples by liquid-liquid extraction with tert-butyl methyl ether, separation was achieved on a x-terra rp- column (dimension x . mm, μm) at c using gradient elution with a mobile phase of mm ammonium bicarbonate ph . (mobile phase solvent-a) and acetonitrile: mm ammonium bicarbonate ph . (mobile phase solvent-b). samples were eluted at a flow rate of . ml / min throughout the -minute run. uv wavelength mode was used, detection was at nm. results: the calibration curve was linear (r > . ) in a concentration range of - ng / ml for letermovir. the hplc assay established for letermovir determination showed a high rate of accuracy and precision with an intraday variability of - . to % (accuracy) and . to . % (precision) and an interday variability of - . to . % (accuracy) and . to . % (precision), respectively. letermovir serum concentrations of patients ( male / female, mean age . years) were determined in daily clinical practice. the mean concentration was ng / ml (median ng / ml, standard deviation ng / ml, range - ng / ml). conclusions: the newly developed hplc method is useful for the determination of letermovir concentrations. patient samples analyzed in a routine clinical setting demonstrated considerable interindividual variability. all measured concentrations were above the ec of letermovir. monitoring the concentration of letermovir could help to prevent over-or underexposure, especially in patients with polypharmacy which is frequent in allogeneic hematopoietic stem cell transplant recipients. disclosure background: the use of preemptive strategy (pet) has lowered the incidence of cmv disease in allo-sct to - %. nonetheless the use of this strategy implies that more than % of seropositive patients will replicate cmv. several studies have shown that cmv replication is detrimental for patient survival although the viral load related to this bad outcome variates among studies. objective: to analyse thel impact of cmv replication in overall survival (os) in allo-hct patients. methods: to analyse the impact of cmv replication in os we perform a unicentric, retrospective study on consecutive first allo-hsct patients transplanted between jan- and oct- with a median follow-up of days ( - ). all patients were monitored post-hct with real time pcr cobas-taqman® /cobas ® (rtpcr) in plasma. the cut-off for inception of pet was iu/ml. cmv mutations (ul /ul gene), were studied in plasma samples by sanger sequencing, median cmv viral load iu/ml ( - ). results: patients ( ): women/men ( %/ %), median age was years (range - ). identical allogeneic scts ( %), haploidentical scts ( %). donors were related in cases ( %) and ( %) unrelated. progenitors source was % peripheral blood and % bone marrow. cmv status was (d+/r+) in %(n= ), (d+/r-) in %(n= ), (d-/r+) in % (n= ) and (d-/r-) in % (n= ), unknown cases. positive pcrs were detected in patients: one episode in ( %); episodes in ( %) and to episodes in patients ( %). fifty-five patients ( %) received preemptive therapy. fourteen episodes ( %) were refractory/ probable refractory cmv infections (according to the criteria of chemaly r. cid ). a resistant mutation (ul gene) was detected in one patient with refractory infection patients that developed cmv infection had an inferior non-significant os at years ( , % vs , % log-rank p , ). those patients that received pet for cmv had a significant inferior os compared with those that replicate cmv but didn't receive preemptive therapy ( , % vs %, log rank p= . ). os of patients that received pet was inferior compared with those without pet (with or without infection) ( , % vs , %, logrank p , ). no difference in survival was found for those patients treated pre-emptively that were refractory vs no refractory ( % vs , %, log-rank p , ). conclusions: patients that received preemptive therapy had a significant inferior overall survival compared with those that didn´t replicate and those that replicate cmv but didn't receive preemptive therapy. this reinforce the relevance of prophylactic strategies for cmv with drugs with good safety profile like letermovir that in a randomised trial proved to decrease the need for preemptive therapy. disclosure: rafael, de la camara: has received grants from astellas, gilead, janssen, merck, novartis and pfizer clinical evaluation of stenotrophomonas maltophilia infection in allogeneic hematopoietic stem cell transplant recipients -retrospective single-center data analysis negative bacillus that causes severe infections associated with high morbidity and mortality in immunocompromised patients. the aim of our study was to determine incidence, characteristics and outcome of s. maltophilia infection in patients (pts) who underwent allogeneic hematopoietic stem cell transplantations (allo-hsct) in institute of hematology and transfusion medicine between october and november . methods: we retrospectively evaluated incidence, clinical features and outcome of s. maltophilia infections in consecutive patients with median age- years (range - ), who underwent allo-hsct from unrelated donors - ( . %), matched sibling donors - ( . %) and haploidentical donors - ( . %) in our center. s. maltophilia was detected by culture-based microbiological tests. invasive infection was defined by isolation s. maltophilia from cultures in the presence of both clinical symptoms and signs of infection -blood stream infection (bsi), pneumonia with or without pulmonary haemorrhage. the only colonization status was defined as s. maltophilia culture-positive samples in the absence of infection symptoms. in vitro susceptibility tests to antibiotics were performed. results: pts ( . %) with median age- years (range - ) with s.maltophilia culture positive samples were identified. ( . %) underwent allo-hsct from unrelated donors, -from matched sibling donor and -from haploidentical donor. among them bsi developed in pts ( . %), pneumonia in pts ( %) -with fulminant and fatal pulmonary hemorrhage in pts ( . %). all patients with pneumonia demonstrated bsi. positive sputum cultures were detected in pts, in pts hemoptysis was observed. the rest of isolated strains were identified as colonization (throat -in pts, stool -in pts). all patients with invasive s. maltophilia infection before pathogen identification demonstrated persistent fever despite of the use of broadspectrum antibiotics (carbapenems, glycopeptides, aminoglycosides, colistin), prophylactic antifungals and antivirals. all of them received fluoroquinolone (ciprofloxacin) as a standard antibacterial prophylaxis before neutropenic fever occurred. all patients ( %) with bsi, pneumonia and pulmonary hemorrhage died before engraftment (anc - . g/l) - of them during - hours from the onset of a positive blood culture for s. maltophilia. the c-reactive protein (crp) concentration before identification of s. maltophilia invasive infection was > x- x upper normal limits (unl). susceptibility to antibiotics of isolated strains from blood and sputum was respectively: % and % for ceftazidime, % and % for trimethoprim-sulfamethoxazole, % and % for levofloxacin; while % and % strains were resistant to ciprofloxacin. -year overal survival (os) and -y os for this group was . % and . % respectively compared with . % and . % for group without s. maltophilia infection. conclusions: s. maltophilia invasive infections are associated with high morbidity and mortality in allo-hsct recipients especially in the period from conditioning therapy to engraftment. an exposure to broad-spectrum antibiotics in the treatment of neutropenic fever or confirmed bacteremia of other etiology is one of risk factors of breakthrough s. maltophilia infections. empiric therapy against s. maltophilia in selected patients in risk of such infection before pathogen identification may be lifesaving procedure. disclosure: nothing to declare. role of cmv reactivation following allogeneic stem cell transplantation in preventing relapses in patients with acute myeloid leukemia background: cytomegalovirus(cmv) reactivation is common in patients undergoing allogeneic stem cell transplantation. it has been shown recently that cmv reactivation is associated with reduced risks of relapse in patients undergoing allogeneic stem cell transplantation for aml. however the analysis of cibmtr data did not show any effect of cmv reactivation on relapse. with this background we conducted an analysis of patients suffering from aml who are undergoing allo-sct for their long term disease free survival with respect to cmv reactivation. methods: after obtaining permission from hospital medical records committee, we retrospectively analysed data from electronic medical records of patients undergoing allo-sct for aml at our center between january to august . patients who underwent matched sibling, matched unrelated and partially matched allo sct were included. all patients underwent cmv monitoring with weekly pcr starting from the time of engraftment till d+ following allo sct. value of ≥ copies/mcl was considered as cut off for initiation of treatment in matched sibling donor transplant but in unrelated donor or partially matched donor transplants, ≥ copies/mcl was used as cut off for initiation of pre emptive therapy. results: total of patients were included in study. median age was . ± . years ( - yrs). ( . %), ( . %) and ( . %) patients underwent matched sibling, haplo (partially matched) and mud transplantation respectively. median follow up was months( - months). (table ) acute gvhd (grade - ) was observed in ( . %) of patients. cmv reactivation occurred in ( . %) of patients. overall survival at last follow up was . % ( / patients). ( . %) patients relapsed during follow up. relapse free survival at at last follow up was . %. ( . %) of patients who had cmv reactivation didń t relapse, whereas ( %) of patients who didn´t have cmv reactivation relapsed which was statistically strongly significant p < . . (figure ) similar results were seen in recently published paper from japanese society for hematopoietic cell transplantation (jshct) transplantation-related complication working group. conclusions: . cmv reactivation following allo sct had beneficial effect on preventing relapse in patients with aml. . probable immune activation resulting due to cmv reactivation may result in better graft versus leukemia effect preventing subsequent relapses. [ background: human herpesvirus (hhv- ) causes lifethreating central nervous system disorders such as encephalitis after allogeneic hematopoietic stem cell transplantation (hsct). recent studies showed that cd , a member of the tumor necrosis factor receptor superfamily, has been implicated as a specific receptor of hhv- b, and that its expression levels in cd -positive t cells after hsct could be related to the reactivation of hhv- . real-time quantitative polymerase chain reaction analysis (qpcr) is the most commonly used method for detecting and evaluating hhv- reactivation after hsct, but more sensitive detection method is required. we recently developed a new monitoring method for hhv- reactivation using digital pcr (dpcr) which provides high sensitivity of detecting hhv- dna in clinical samples. in this prospective study, we evaluated the relationship between hhv- reactivation monitored by dpcr and expression of cd on cd + t cells before and after allogeneic hsct. methods: thirty-four patients who underwent allogeneic hsct for hematological diseases at keio university hospital (tokyo, japan) between january and march were consecutively enrolled into this study. peripheral blood samples of the patients were obtained before the conditioning (pre), the day of transplant (day ), and weekly during the first month after transplantation (days , , , and ) . hhv- viral load in plasma was quantitatively measured by dpcr. the primers and a probe of dpcr for hhv- b were selected from immediate-early (ie- ) protein transactivator region (u ). we evaluated the relationship between hhv- reactivation and the serial expression rates of cd in cd + t cells (cd /cd ratio) measured by flow cytometry before and after hsct. results: median age of the patients was . years. onethird of patients received cord blood as a stem cell source. hhv- reactivation was detected in patients ( %) with dpcr. a comparison of cd /cd ratio between the patients with and without hhv- reactivation after hsct revealed that cd /cd ratio was significantly higher in patients with hhv- reactivation than those without before conditioning ( in contrast, there was no such significant difference after transplant (days to ) . in multivariate analysis, higher cd /cd ratio before conditioning (odds ratio (or) = . , % confidence interval (ci): . - . , p = . ) and stem cell source from human leukocyte antigen mismatched donor (including all cord blood transplantation cases) (or = . , %ci: . - . , p = . ) remained to be significantly associated with the incidence of hhv- reactivation. conclusions: higher cd expression rate in cd + t cells before hsct was associated with higher risk of hhv- reactivation, which could be a promising marker for predicting hhv- reactivation after allogeneic hsct. careful observation and monitoring may be needed in cd highly expressed patients. it is a subject of further research to clarify the role of cd + cd + t cell in hhv- reactivation. disclosure: nothing to declare. methods: criteria for the administration of ici (vistide) were grade iii-iv (clinically significant hematuria with clots) bk-related hemorrhagic cystitis after allo-hct which showed no improvement after symptomatic therapy with hyperhydration and bladder irrigation. cidofovir was diluted in ml of normal saline and installed via a foley catheter which was blocked for hour. not knowing the level of absorption of the drug we decided to give probenecid prophylaxis in all patients. ici was repeated weekly according to severity of symptoms. urine and plasma bkv viral loads were quantified by rq-pcr results: six patients (median years, - ) received ici after allo-hct. patients had haematological malignancies (aml , all , mds ), received busilfex-based myeloablative conditioning and a graft (pbsc , bm ) from a / hla-matched ( pts), / ( pt) or haploidentical ( pt) donor. median time for the onset of bkv-hc after allo-hct were . days (range - ). all patients were under standard cyclosporine prophylaxis and none of the patients had any signs of acute gvhd at the time of onset of hc. the median pcr-bkv viral load at the onset of bkv-hc in urine and plasma were . x (range . x - x ) and . (range - ), respectively. the median maximum pcr-bkv viral load in urine and plasma were . x (range . x - x ) and . (range - . ), respectively. five patients had impaired renal function (median egfr ml/min, range - ) at first ici which was probably multifactorial. the median dose of intravesical cidofovir was mg/kg (range . - mg/kg) and a median number of . instillations (range - ) were given. in / cases symptoms of cystitis improved dramatically and hematuria resolved. virological response (at least log reduction) was observed in all cases. two patients experienced relapse of hemorrhagic cystitis and were retreated with ici which resulted in resolution of the symptoms and the hematuria. no deterioration of renal function of other systemic adverse effects were observed. after a median follow up of . days after transplantation (range - ), / patients are alive without cystitis symptomatology and died ( due to relapse and due to trm). conclusions: in this retrospective study we propose that local therapy of bkv-hc with ici is safe and has high clinical and virological response rates. the administration of ici after allo-hct should be controlled in prospective randomized trials. disclosure: nothing to declare background: since cmv-preemptive therapy approach was implemented, cmv disease frequency is very low. however, cmv reactivation and the need of using nephrotoxic plus/less myelotoxic drugs is very frequent. in addition to the toxicity of the medications to avoid cmv disease, other potential adverse effects of cmv have been mentioned in medical literature. in this study, we wanted to estimate how recipient/donor serologic status influences the outcome of allo-hsct in our most recent series of patients. methods: the population analyzed for this report is the all patients who underwent allo-hsct during the -year period from october september in our unit. median age at transplant was years (range: - ). one hundred and thirty were male ( %) and were female ( %). baseline diseases were: aml, lpd, all, mds, mpd, mm, and bmf. donor was unrelated in transplants ( , %) and was family in ( , %) (including haplo-identical). conditioning regimen was ric in procedures ( %) and intensive in ( %). stem cell source was pb in ( , %) and bm in cases ( , %). median follow-up was months (range: - ). patient's and donor's cmv igg were positive in ( , %) and ( , %), respectively. recipient/donor serology was +/-(risk group ) in ( , %), +/+ (risk group ) in ( , %) , -/+ (risk group ) in ( , %) y -/-(risk group ) in ( , %). results: two pts underwent a second transplant before day + due to graft failure. overall mortalities (om) at days + and + of the rest of the series ( pts) are shown in table. the highest risk group (recipient cmv + / donor cmv -) exhibited more than double om at day + and more than four times om at day + , when compared with pts at lowest risk (recipient cmv -). those striking differences were mainly due to nrm. om for risk group ii (recipient cmv + / donor cmv +) was intermediate. conclusions: in our studied population, mainly adult patients, the combination of cmv-seropositive patient with a cmv-seronegative donor had a very clear adverse impact on hsct outcome. as a result, we considered that the election of a cmv-positive donor for a cmv-positive patient continues to be strongly advisable, whenever is possible. on the other hand, once letermovir has proved to be efficient and well-tolerated and has been licensed for prophylaxis of cmv in high risk recipients, this approach appears to be very attractive to try to avoid the adverse impact of recipient cmv-seropositivity, particularly when finally chosen donor is cmv negative. disclosure: nothing to declare an active surveillance and an early and individualized management is critical to avoid mortality from respiratory viral infections in allo-hsct recipients background: respiratory viral infections (rvis) are frequent among the general population. in transplant recipients, rvis are known to cause an important morbidity and potential mortality. for this reason and several others, as the need of preventing other pts from contagious or avoiding misdiagnosis with other infections processes, a high index of suspicion of vris is necessary. during the last few years, we have implemented an active and systematic surveillance policy orientedto early detection and management of rvis in the hsct recipients. methods: the population analyzed for this report is the patients who underwent allo-hsct from january through march in our unit. median age at transplant was years (range: - ). one hundred and four were male ( . %) and were female ( , %). baseline diseases were: aml, lpd, all, mds, mpd, mm, and bmf. donor was unrelated in transplants ( . %) and was family in ( . %) (including haplo-identical). conditioning regimen was reduced in procedures ( %) and intensive in ( %).stem cell source was pb in ( . %) and bm in pts ( . %).median follow-up was months (range: - ); at the close of the analysis, majority of the series ( . %) had a follow-up superior to one year from hsct. a throat swab(ts) was taken from every patient with any, even minor, respiratory symptoms. the respiratorysamples were tested whith a complete pcr panel of human respiratory viruses: rhinovirus (rv), influenza a and b virus (iv-a, iv-b), parainfluenza virus (pivs - ), respiratory syncytial virus (rsv), metapneumovirus (mpv), coronavirus (cov), adenovirus (adv), and bocavirus (bov). results: day + overall mortality of the series was , %. day + overall mortality was , % ( , % nonrelapse mortality -nrm-, and , % progression/relapse mortality). causes of nrm reflected in table . no patients died due to rvis. from st july through th june (a -month period), ts samples were obtained from pts ( , %).the median number of samples/patient was (range: - ).a total of ( - ) rvis episodes were diagnosed in pts ( , %).the median presentation of the first rvi was at the day + ( - ) post-hsct. the viral distribution was: rv ( . %), iv ( . %), piv ( . %), rsv ( . %), mpv ( . %), cov ( . %), adv ( . %), and bov ( . %).there were mixed (two or more viruses) rvi episodes. the temporary distribution of vri episodes is shown in figure . conclusions: ) symptomatic infections due to respiratory viruses are very frequent among the allo-hsct recipients. ) a high level of suspicion, as well as an early and systematic screening and management policy, are critical to avoid potential attributable mortality and the nosocomial spread of rvis among the transplant recipients. ) in our series, rhinovirus, parainfluenza and adenovirus might be detected at any moment of the year; the rest of the viruses showed a clear seasonal pattern (november to april). [[p image] . background: trimethoprim-sulfamethoxazole (tmp-smx) is the most suitable drug for prophylaxis against pneumocystis pneumonia and infections with toxoplasma after allogeneic haematopoietic stem cell transplantation (allo-hsct). allergic reactions or hypersensitivities, mainly exanthemas, occur in about - % of the patients, usually resulting in the use of alternative prophylactic drugs (e.g. pentamidine or atovaquone). it has been hypothesised that allergies might be cured with allo-hsct. methods: we conducted a retrospective chart review of patients with tmp-smx re-exposition after allo-hsct from december to september . follow-up is current as of december . results: six patients (f/m: / , median age: years, range: - years) with a history of tmp-smx hypersensitivity prior to allo-hsct were re-exposed to tmp-smx after engraftment of a matched related (mrd, n= ) or matched unrelated (mud, n= ) donor. median time to re-exposition was . (range: - ) days after allo-hsct with one oral dose of tmp-smx. in four patients, tmp-smx was tolerated without any signs of hypersensitivity reactions and has been continued for a median of days (range - ) until last followup. one patient (mud, re-exposition at d+ ) experienced pruritus and erythema some hours after tablet intake. another patient (mud, re-exposition at d+ ) developed an exanthema one day after re-exposition which was later diagnosed as a cutaneous gvhd. conclusions: re-exposition of tmp-smx in patients with prior hypersensitivity is feasible after allo-hsct. after successful re-exposition, patients can be treated with the best-studied drug for prophylaxis of infections with pneumocystis and toxoplasma. disclosure: nothing to declare brincidofovir for adenoviremia in paediatric hsct for primary immune deficiency background: reactivation of adenovirus is a severe complication of hsct associated with significant morbidity and mortality, particularly for children with primary immune deficiency (pid). the only drug currently licensed to treat adenovirus infection is cidofovir. brincidofovir is a lipidlinked derivative of cidofovir which has been shown to be a safe and effective alternative treatment to cidofovir. there is limited data describing the use of brincidofovir in patients undergoing hsct for primary immune deficiency. we reviewed all patients who received brincidofovir after undergoing hsct for primary immune deficiencies between and at the great north children's hospital, newcastle upon tyne, uk. results: of patients transplanted for pid, developed significant adenoviraemia ( %). all were treated with cidofovir initially but were switched to brincidofovir because of a failure to respond or because of renal toxicity. of these, resolved their adenoviraemia within days of commencing treatment (figure ). donor sources were tcr alpha/beta/cd depleted haplo-identical (n= ), tcr alpha/beta/cd depleted mmud (n= ) and / mud (n= ). patients were conditioned with treosulphan/fludarabine/thiotepa/atg/ rituximab (n= ), treosulphan/fludarabine/atg/rituximab (n= ) or treosulphan/fludarabine/alemtuzumab/ gcsf/plerixafor (n= ). occurrence of agvhd and treatment of agvhd are outlined in table . patient died + days post-transplant of multi-organ failure, severe thrombotic microangiopathy and sepsis. although patient initially responded to brincidofovir, reactivation occurred after cessation of treatment; severe diarrhoea precluded the reintroduction of brincidofovir and the adenoviraemia persisted with poor immune reconstitution. treatment with addback t cells was attempted however the patient died days post-transplant after a cerebral haemorrhage. patient had long-standing chronic diarrhoea which was thought not severe enough to warrant cessation of brincidofovir treatment. conclusions: the complete resolution of adenoviraemia in / patients who had previously failed to respond to prior therapy with cidofovir suggests that brincidofovir may be an effective treatment option for adenoviral reactivation post-hsct for pid. however, resolution of adenoviraemia is influenced by many other factors, including the adequacy of immune reconstitution, the degree of induced immune suppression and the presence of comorbidities such as gvhd. due to the small sample size it was difficult to assess the relative importance of these factors in this cohort. brincidofovir was well tolerated however its effectiveness may have been limited by poor gastrointestinal function in one patient (patient ) and could not be used after a viral reactivation in another for the same reason. further studies of the use of brincidofovir in this specific cohort are needed to clarify the role and effectiveness of this treatment. background: there is a high prevalence of cmv seropositivity in algerian population. because of high morbidity and mortality in pts who underwent allo sct with cmv reactivation, effective surveillance and timely treatment using anti-viral therapy s required. the risk of cmv reactivation depends on the type of stem cell source, immunosuppression (is) and serological status of the donor/ recipient pair. methods: over a months period (from / / to / / ), pts underwent allo-hsct for malignant or non-malignant hematology diseases of which pts are evaluated for this study. cmv reactivation was observed in pts ( . %) (aml: pts, all: pts, cml: pts, multiple myeloma: pt, nhl skin: pt, primary myelofibrosis: pt, aplastic anemia: pts, fanconi anemia: pts, β-thalassemia: pt), with a median age of years ( - ), sex ratio (m/f) of . . allo-hsct done with sibling donors: pts, haplo-identical donors: pts and pheno-identical donor: pt. all pts were treated by chemotherapy alone with myéloablative conditioning (mac) in pts and reduced intensity (ric) in pts. all pts received peripheral blood stem cells with an average rate of cd + cells: , . /kg ( . - . ). additional bone marrow graft was used in pts that received a haploidentical graft without pt-cy. gvh prophylaxis associated cyclosporine (csa) and methotrexate (sibling and phenoidentical); csa-mtx-mmf or csa-mtx-cyclophosphamid (haplo-identical). before transplantation, donor/recipient pair is at high risk reactivation in pts ( . %). detection of cmv reactivation done by antigenaemia pp or by quantitative pcr weekly for the first months and during an is treatment for acute or chronic gvhd. pre-emptive therapy is initiated by ganciclovir as soon as positivity of antigenaemia or increased viral load in pcr. results: a first reactivation occurred on average day ( - ) in pts ( . %) of which pts under corticotherapy for acute gvhd ( pts), thrombotic micro-angiopathy ( pt) and renal failure ( pts) or due to a reinforced is for haplo-identical transplantation ( pts). one pt with chronic gvhd presented a late reactivation months after transplant. twenty pts presented a nd reactivation on average day ( - ) and pts a rd reactivation on average day ( - ). pre-emptive treatment is introduced in the first episode by a viral dna polymerase inhibitor (ganciclovir: pts; valganciclovir: pts, foscarnet: pt). the negativity of antigenaemia is observed on average at days of treatment ( ) ( ) ( ) ( ) ( ) . second line treatment was required in pts ( %) due to resistance ( pts), severe cytopenia ( pt) or renal failure ( pt). the onset of severe cytopenia imposed a dose reduction ( pts) or a therapeutic stop ( pts) before days. two pts received additional maintenance treatment for negativation delay. three pts ( . %) died from cmv infections resistant to antiviral treatment (pneumonia: , colitis: ). conclusions: cmv infection is a serious complication after allo-hsct. in the absence of vaccination, the systematic monitoring for cmv reactivation is strongly recommended for the establishment of a rapid and effective preemptive treatment. disclosure: nothing to declare p abstract withdrawn. results: in transplanted group, episodes of bkv reactivation occurred in patients ( %). in cases only urine colonization (c) found before hsct. in this group in patients ( %) virus was transmitted from urine to the blood (b) . dysuria and/or hc were observed in / ( %) patients . all of them ( %) had urine and serum involvement. in cases bkv replication was found after hsct ( -cases detected in urine, cases-bothserum and urine). dysuric syndromes and/or hc were found in / of cases ( %)-all in patients with serum and urine involvement. urinary tract was always first location of the virus. there was no case of isolated serum reactivation. the incidence of bk infection was higher in patients older than > yrs (p< . ), transplanted from family donor (msd) (p< . ). mud recipients had more often both serum and urine reactivation (p< . ) than isolated urine involvement. sex, day of neutrophil recovery, conditioning regimen, or use of total body irradiation were not significant risk factors for bkv infection, or hc . six patients were treated with cidofovir (range - doses) with good response. there was no death due to evident bkv infection. conclusions: bkv reactivation remains one of the most frequent infectious complication in children undergoing allogeneic hsct. most of patients experienced mild infection and age < years was the positive prognostic factor influencing its incidence. bkv monitoring and prompt treatment of hc resulted in excellent outcome. we observed surprisingly high rate of new bkv replication after hsct. disclosure: nothing to declare background: high-dose chemotherapy (hd-ct) and auto pbsct have been the standard therapy for multiple myeloma (mm) for more than two decades, despite a wide range of new therapeutic options. recurrent/refractory malignant lymphomas and recurrent/metastatic germ cell tumors (gct) also benefit from this intensive therapy. in comparison to allogeneic transplantation, this treatment is known for lower complication rates, e.g. infections. however previous studies have schown that treatment related toxicity may not be underestimated and depending on the conditioning regimen used. methods: we retrospectively analyzed patients ( cases) who underwent hd-ct plus auto pbsct between and in a single-center study. to anlyze the incidence of infections depending on the conditioning regimen, we formed the following categories based on the agiho: no infections, neutropenic fever, sepsis and severe sepsis. results: the median age in this analysis was years; . % were male. the most frequent diagnosis was mm ( . %) receiving high dose melphalan (mel), followed by malignant lymphoma ( . %) receiving beam (bcnu, etoposide cytarabine, melphalan) and relapsed/metastatic germ cell tumours (gct) ( . %) receiving high dose carboplatin/etoposide (ce). % of all patients developed severe sepsis, patients had to be ventilated and patients died. sepsis was documented in . % of all cases ( cases). the majority of patients ( . %, cases) developed neutropenic fever and . % ( cases) didn´t have any infection complications. the beam conditioning regimen showed the highest tendency to result in a septic course ( . %), followed by ce ( . %) and mel ( . %). the most commonly documented pathogen in blood cultures was s. epidermidis ( . %), followed by e. coli ( . %) and s. mitis ( . %). only in one blood culture we detected a multi-resistant pathogen ( mrgn e. coli). p. aeruginosa was detected in blood cultures ( . %), l. monocytogenes in ( . %) and s. aureus in ( %). . % of all patients developed diarrhea, only in . % of these cases we could detect c. difficile. the conditioning regimen shows no significant effect on the incidence of c. difficile. the mean neutropenic period was . days in malignant lymphoma patients, followed by . in mm patients and . days in gct patients. the hospital discharge, calculated from the day of transplantation, was significantly different: for malignant lymphoma the mean was . days, for mm . days and for gct . days. conclusions: our data correspond to former published results by many groups. the beam regimen shows the highest infectious complication rate followed by ce and mel. the duration of neutropenia and hospital stay depends on the conditioning regimen. the type of infectious complication doesn't effect the progression free-and overall survival in our analysis. disclosure: nothing to declare. impact of donor and recipient cytomegalovirus serostatus on outcomes of unrelated allogeneic haematopoietic stem cell transplantation background: cytomegalovirus (cmv) is an important cause of morbidity and mortality in allogeneic haematopoietic stem cell transplant (hsct) patients. the aim of our study is to evaluate the outcomes of our cmv seropositive recipients who received grafts from seropositive unrelated donors (d+r+) compared with grafts from seronegative unrelated donors (d-r+). methods: this is a retrospective single center study on a series of cmv seropositive recipients who underwent hsct from unrelated donors between febuary to july . a total of patients were analyzed. their clinical course and laboratory results were reviewed for evidence of cmv reactivation and/or cmv disease. we defined cmv infection as detection of cmv reactivation or primary infection by antigenaemia or polymerase chain reaction (pcr) assays, but was not accompanied by signs and/or symptoms suggestive of a systemic disease. cmv disease occurred when cmv was isolated from any site in association with organspecific signs and/or symptoms. monitoring for cmv infection commenced upon engraftment (approximately day + ). peripheral blood samples were sent twice a week for cmv antigenaemia or cmv quantitative pcr. the duration of twice weekly monitoring was at least about days. longer monitoring was performed in patients who experienced cmv infection after hsct. results: all patients received graft-versus-host-disease (gvhd) prophylaxis using anti-thymocyte globulin (atg) at . mg/kg in addition to cyclosporin or tacrolimus. among the entire cohort of patients, ( %) had cmv infection, including ( . %) out of patients from the d-r+ group and ( %) out of patients from the d+r + group. patients ( . %) from the d-r+ group and patients ( . %) from the d+r+ group had ≧ cmv reactivation above the threshold for preemptive therapy respectively; p= . . patients developed cmv disease, ( . %) from the d-r+ group and ( . %) from the d +r+ group. cmv resistance to both foscarnet and ganciclovir was detected in patients ( . %) from the d-r+ group but none from the d+r+ group. patients died due to cmv disease, both were from d-r+ group. year overall survival (os) were % versus % for d-r+ group and d+r+ group respectively; p= . . median survival was not reached at years. year non-relapse mortality (nrm) were % for d-r+ group and % for d +r+ group respectively; p= . . conclusions: the incidence of recurrent cmv infection was higher in the d-r+ group compared to the d+r+ group. there were no statistically significant differences between the groups in terms of os and nrm. however, there was a trend towards higher nrm in the d-r+ group compared to d+r+ group. our findings suggest that for matched unrelated hsct, it may still be important to select a seropositive donor for a seropositive recipient. disclosure: none background: it´s known that some patients submitted to allogeneic stem cell transplantation (asct) could present a greater susceptibility to infection even when they are in long term complete remission or potentially cured. this fact is related to the dynamic of immunological recovery that is variable in every single patients and it is dependent from many factors: the haematological disease, the conditioning regimen, the age of patient and donor, the number of stem cell and lymphocytes infused, the anti-gvhd prophylaxis, the use of anti-thimoglobulins and others. in clinical practise we can observe patients who are potentially cured, who tapered and stopped the immunosuppressive treatment months or years ago and who are suddenly graved from opportunistic infections. the largest part of these infections is represented from varicella-zoster virus (vzv) cutaneous eruption. methods: in this report we retrospectively analysed a monocentric cohort of patients submitted to asct for haematological malignancies from a median time of months. all of them were free of disease. they stopped the immuno-suppressive treatment in a median time of days after asct (range: - ) and did not present later chronic gvhd needing treatment neither other moderate or severe chronic post transplant complications nor other diseases. prophylactic treatment with anti viral agents (acyclovir or valacyclovir) has been conducted simultaneously to immuno-suppressive treatment and for a period ranging between to months after its suspension. in this cohort of patients we considered the incidence of vzv eruption occurred after the suspension of the immunosuppressive treatment, and we analysed the immunological recovery in terms of lymphocytes sub-population after , , and months from asct. results: of these patients considered, developed at least one vzv manifestation. all the vzv presentation were cutaneous, we did not observe neurological, ophthalmic or visceral presentation. all the vzv manifestation occurred in patients who ended the anti-viral prophylaxis. median time of presentation was days after asct (range: - ) the remaining patients did not present vzv manifestation nor other kind of opportunistic infection despite the absence of anti-viral prophylaxis. the analysis of lymphocyte sub-population after - - and months did not show a significant difference in b, t, t , t and nk lymphocytes in the different post transplant period. conclusions: vzv reactivation seems not to be correlated with the number of the different lymphocyte subpopulations in the post transplant period. actually it is not possible to distinguish patients more suitable of vzv reactivation on the basis of lymphocyte sub-populations analysis, so anti-viral prophylaxis should be prolonged for a medium period after suspension of immuno-suppressive drugs. in absence of anti viral prophylaxis a careful clinical surveillance should be performed in order to treat early eventual vzv manifestations. disclosure background: infection and disease cytomegalovirus (cmv) are common problems in patients undergoing hematopoietic stem cell transplantation (hsct). cmv infection has a high overall seroprevalence, therefore, during the first days post-hsct, it is important to prevent reactivation of cmv. the international clinical recommendation is the use of ganciclovir as prophylaxis in hsct patients; however, the cost of this treatment is not accessible for our population. in this respect it has been used as an alternative valganciclovir because of its lower cost and oral administration. our study´s aim was to assess the response and safety of valganciclovir in comparison with ganciclovir to prevent viremia and cytomegalovirus disease in patients undergoing allogeneic hsct methods: a retrospective study was performed on patients who receive an hsct-allo between january and august . participants were enrolled in two groups according to prophylaxis treatment: (a) ganciclovir mg/k once daily and (b) valganciclovir mg twice daily for days pretransplant, at day + ; viremia was measured by pcr. demographic and clinical information was collected from medical records and furthermore analyzed in spss v . results: sixty-eight patients were enrolled in the study, % male, the median age was years ( - ) with the following diagnoses: acute lymphoblastic leukemia %, acute myeloblastic leukemia . %, granulocytic chronic leukemia . %, myelodysplastic syndrome . %, dendritic cell neoplasia . %, and aplastic anemia . %. ninety-one percet of the patients received a transplant from an identical hla donor and . % received a haploidentical transplant. thirty-four patients received ganciclovir (g ) and thirtyfour valganciclovir (g ). median age was vs years (p= . ), intermediate risk cmv % vs (p= . ), associated bacterial infections was %vs % (p= . ), and fungal infections % vs % respectively (p= . ). the reactivation by cmv was presented in % vs % respectively (p= . ). there were no significant differences in fever, bacterial isolation, dysfunction or graft failure, presence and degree of acute or chronic gvhd and relapse of the disease. the most relevant characteristics and complications are described in table . within the whole group there were deaths, % in the ganciclovir group and % in valganciclovir group (p= . ), overall survival -year was % vs % (p= . ) respectively; in both groups % was associated with relapse and % associated with transplantation. conclusions: ganciclovir and valganciclovir were effective in preventing the reactivation of cmv, the only statistically significant difference was that the presentation of the disease appeared earlier in the valganciclovir group. no difference in toxicity between the groups was identified. disclosure: none declared background: invasive pulmonary aspergillosis (ipa) is a severe and serious complication that occurs in the immediate post-transplant period due to severe neutropenia or late usually following prolonged corticosteroid therapy during treatment of graft-versus-host disease (gvhd). the objective of this study is to analyze the epidemiological, diagnostic and evolutionary characteristics of this major complication over a period of years. methods: from january to december , patients (pts) received an allogeneic hematopoietic stem cell transplantation (allo hsct) for malignant and nonmalignant haematological diseases. during the transplant procedure, anti-infectious prophylaxis consisted of pts isolation, digestive decontamination, fluconazole and aciclovir. secondary prophylaxis done for pts with prior history aspergillosis. during the follow-up, a standard chest x-ray is performed systematically at each control or in case of clinical signs a thoracic ct scan is requested from suspicion. the diagnosis of ipa is made according to the criteria of the eortc-msg based on the predisposing criteria of the host and clinico-radiological criteria (possible infection). galactomannan antigen and histopathology criteria are not common practice. results: a total of ipa episodes ( %) were identified in pts (aml: , aa: , all , cml , mm ) of median age ( - ) , sex ratio: . . all of them had transplantation from a family donor (geno-identical: , haplo-identical: ) with conditioning by chemotherapy alone and a graft of csp ( pts) and peripheral stem cells-bone marrow ( pts). all pts had at least one predisposing risk factor: antecedent of aspergillosis ( pts), prolonged neutropenia> d ( pts), acute gvhd ( pts), chronic gvhd ( pts), prolonged corticosteroid therapy ≥ , mg /kg/day exceeding days ( pts). the diagnosis of api was possible on average at j ( - ) after appearance of clinical signs (in all cases) and evocative radiological in cases (in cases, the standard chest x-ray was normal). at the time of thoracic ct scan, pts ( %) had characteristic signs: halo sign ( pts), crescent sign ( pt) and cavity ( pts). other minor radiological signs are found in the other pts. empirical first-line antifungal therapy was started as monotherapy in pts (voriconazole: , caspofungin: pts) or in combination in pts. a secondline treatment was required in pts for failure after an average duration of days . three pts presented a second episode after an average delay of months ( ) ( ) ( ) ( ) with a favorable evolution of resumption of thetreatment. fourteen pts ( %) are alive with complete resolution after a median treatment time of months ( - ). twelve pts ( %) died rapidly on average days after diagnosis (ipa , relapse of his disease: ) conclusions: ipa occurring after an allograft of allo-hsct is a severe complication with high mortality. it is essential, in each case, to identify the pts with risk factors, perform a thoracic ct-scan, send serum serology for apergillus galactomannan antigenand start specific treatment as soon as possible while waiting to be able to reinforce the diagnosis by direct examination or sputum or brochoalveolar lavage with aspiration. disclosure: nothing to declare background: cmv (cytomegalovirus) has a prevalence varying between - %. its pathogenicity is relatively low in the general population, usually resulting in a selflimiting viral illness. in an immunosuppressed host, infection can lead to life threatening illness. disseminated cmv infection can manifest in a number of organs and is diagnosed using internationally accepted criteria. in the post solid organ and stem cell transplant (sct) setting, it is postulated that it is viral reactivation, rather than primary reinfection that leads to cmv viraemia. prevention of reactivation requires the presence of a competent immune system, mediated by t-cells. this accounts for the increased incidence in intensive and t-cell depleting sct conditioning regimens. despite improved outcomes following the introduction of cmv monitoring by pcr and pre-emptive treatments (current uk guidance), cmv pneumonitis still carries a high mortality. the use of cmv specific immunoglobulins (cmvig) for the treatment of this complication is generally not recommended post chemotherapy or sct in haematological cancers due to lack of evidence. however, cmvigs are widely used in the setting of cmv reactivation post solid organ transplants. we report the use of cmvig in patients with suspected cmv pneumonitis at a single uk centre. the aims of this retrospective study were to establish safety and review efficacy in this highly immunocompromised group of patients. methods: data was collected retrospectively on the use of cmvig in patients with haematological cancers post sct or chemotherapy alone between and at manchester royal infirmary, uk. all patients included had cmv positive pcr in blood (and or from bronchoscopy), as well as high resolution ct imaging evidence of cmv infection. the data was sourced from pharmacy database and crossreferenced with a departmental list. for each patient identified, case notes and prescriptions were sourced. data collected included patient baseline characteristics, timing of treatment, number of doses of cmvig and outcome. results: eight patients received cmvig for suspected cmv pneumonitis. seven patients were post sct and one patient was severely immunosuppressed with chemotherapy alone. median age was years (range - ). the cmvig regimen used was ml/kg of cytotect ® on days , , and , followed by ml/kg every four days until resolution of symptoms. there were no infusion related reactions observed. patients received a median of doses of cmvig. four out of patients responded to the treatment and showed full recovery but only are alive and well to date. conclusions: this study shows that the use of cmvig is safe in the post-sct setting of acutely unwell patients with multi-organ failure. despite limitations of retrospective studies, there appears to be benefit for the use of cmvig in our patient population, with % of patients showing a full recovery from that episode. allogeneic sct plays a confounding role in the outcome of patients although the numbers in our study are small. there is clearly a need for better treatments of cmv pneumonitis. cmvig is a promising treatment but further studies are needed to identify the optimal dosing regimen and provide evidence of efficacy. disclosure: biotest-honaria p abstract withdrawn. background: the risk of fungal infection related to allogeneic transplantation is a well-known cause of morbidity and mortality. the main agents implicated are yeast during the neutropenic period and filamentous fungi after this period. methods: we decided to evaluate the effectiveness of a prophylactic regimen containing fluconazole since day - . after discharge fluconazole was kept until day or or switched to posaconazole in high-risk patients. patients with gvhd under steroids were kept under prophylaxis.the group of high risk patients was defined by one of the following variables: -non related donors -atg, campath or fludarabine in the conditioning -presence of gvhd with need of steroids above . mg/kg we have analyzed the patients submitted to allobmt during and . all patients were first admitted to an isolation room with hepa filters.patients under secondary prophylaxis were excluded. breakthrough fungal infections during the first year and toxicity leading to discontinuation was evaluated. results: sixty six patients were included with transplants. male/female ratio was / . the age range was . - yo with a median of . malignant ( ) and nonmalignant ( )diagnosis were included. donor type was related ( ) haploidentical ( ) and non-related ( ). the conditioning regimen includes atg in , campath in and fludarabine in . fourteen patients were treated after discharge with fluconazole and with posaconazole. three patients fluconazole were switched to micafungin for hepatic toxicity, two cases to amphotericin due to persistent fever and in one case to caspofungin for a proven fungal infection (candida parapsilosis in blood stream in day + ). after discharge and during the first year of follow-up a single case of possible fungal infection was diagnosed, in a patient with gvhd with a lung nodule. conclusions: during the neutropenic period after transplantation the main risk of fungal infection is associated with candidiasis. the greatest risk of aspergillosis occurs later and have a significant relation with gvhd. except for candida parapsilosis the main source of yeasts are the gi tract. the main source of aspergillus are aerosolized particles retained by hepa filters. in patients without a previous episode of fungal infection the main risk of filamentous fungi occurs only after discharge. we conclude that fluconazole alone or followed by posaconazole in high risk patients is a feasible and effective regimen for primary prophylaxis, in allogeneic transplantation. disclosure background: bk virus-associated hemorrhagic cystitis (bkv-hc) has emerged as a serious infection after hematopoietic stem cell transplantation (hsct). it is characterized by painful hematuria due to hemorrhagic inflammation of the urinary bladder mucosa, this causes significant morbidity, prolonged hospital care with extensive nursing requirements and increases in healthcare costs. the purpose of this study is to determine the incidence, risk factors, and duration of treatment in our center. methods: we performed a retrospective review of hsct patients at luis calvo mackenna children´s hospital in santiago, chile diagnosed with bkv-hc, from st january to th november . we investigated the incidence, risk factors and duration of treatment of bkv-hc in paediatric patients undergoing hsct over a months period. bkv-hc was defined as bk virus (bkv) detection in urine by pcr testing in association with clinical symptoms and hematuria grade or higher. sixty-seven patients were trasplanted during this period. results: eleven patients were diagnosed with bkv-hc at our institution, only one with bk viremia. the cumulative incidence of bkv-hc in our series was %. all of them were treated with cidofovir. the median age at diagnosis was years old (range: - y.o.). the median time from hsct to hemorrhagic cystitis (hc) was days (range: - days), the median length of treatment was weeks (range: - ). all patients received myeloablative conditioning regimens and used cyclophosphamide ( %); ten ( %) were unrelated cord blood transplant recipients and nine ( %) used antithymocyte globulin. a concomitant viral reactivation (cmv/vh ) was demonstrated in six ( %) patients. no patient died due to bkv-hc or its complications, but in the follow up three patients died, one in relapse and two of other post transplant´s complications. conclusions: bkv-hc is the result of a complex interaction between patient characteristics, donor type and conditioning regimen intensity. these patients experienced significant morbidity and prolonged treatment. in our cohort bkv-hc of all patients but one were transplanted with an unrelated umbilical cord blood unit, all of them received myeloablative conditioning regimen with cyclophosphamide and most of them received anti-thymocyte globulin. we also observed frequently co-existence of viral infections from herpes family as cmv and vh . the main limitations of this work are its retrospective nature and it´s from a single center. more studies are necessary to better understand the epidemiology and risk factor associated with bkv-hc and the morbidities associated with its treatment. disclosure: nothing to declare how we manage hhv- reactivation in the posttransplant setting oscar borsani , anna amelia colombo , daniela caldera , paolo bernasconi university of pavia, san matteo hospital, pavia, italy background: hhv- encephalitis is a life-threatening complication in the post-transplant setting and it develops in about % of patients receiving traditional hsct. several risk factors were described. a differential diagnosis between hhv- encephalitis and other neurological complications is extremely important but often not-easy to achieve because of the highly heterogeneous clinical and radiological features and complexity of interpretation, especially in transplanted patients. here we described vignettes that represent and highlight distinct problems in the diagnosis and management of transplanted patients with suspected hhv- reactivation. methods: we collected the clinical, laboratory and radiological (electroencephalogram, brain mri and brain ct) data of transplanted patients who developed a neurological syndrome suspected for hhv- reactivation. hhv- was detected on serum and csf using rt-qpcr. results: ) a -years-old patient developed a diffuse erythema and subsequent encephalitic syndrome following hsct. the brain mri revealed clear signs of limbic encephalitic and searching for hhv- on serum and csf revealed . copies/ml and . copies/ml respectively. an antiviral therapy was started but no clinical benefit was achieved. ) a -years-old patient developed a typical neurological syndrome without brain mri findings of encephalitis and with no evidence of skin involvement. the lumbar puncture and csf analysis showed a total of . hhv- dna copies/ml. antiviral therapy with ganciclovir and foscarnet was promptly started with clinical improvement and a drastically reduction of hhv- dna on both csf and serum. a new brain mri revealed an acute limbic encephalitis. ) a slight neurological syndrome consisting of confusion and amnesia developed in a -years-old-patient. brain mri findings were compatible with a wernicke syndrome, but no improvement of neurologic symptoms were obtained with thiamine supplementation. csf analysis did not revealed hhv- dna, which was detected at low copies number on serum analysis. a second brain mri was conclusive for limbic encephalitis, so an antiviral therapy with foscavir was started and radiological but not clinical improvement was noted. the patient died after few days. ) in the last case we present a -years-old patient who developed a clinical picture of encephalopathy (i.e. amnesia, ataxia, drowsiness, weakness, depression) with rapid progression to coma after seventy-eight days from hsct. a brain mri showed a slight contrast enhancement in parietal-occipital regions. during the recovery phase from conditioning-induced cytopenia, an increasing in serum hhv- dna was detected. searching for hhv- dna on donor's follicles showed a chromosomally integrated hhv- (cihhv- ). cyclosporin a (csa) was interrupted and neurological improvement was observed in the following hours: a diagnosis of pres was made. conclusions: hhv- encephalitis should be suspected in transplanted patients with a clinical syndrome of encephalopathy. pcr detection of hhv- dna in csf associated with either typical brain mri abnormalities or a clinical diagnosis of nonspecific encephalopathy must lead to the urgent initiation of systemic antiviral treatment. if an increase of both serum hhv- dna and wbc is detected, a cihhv- should be confirmed. pres is an important differential diagnosis in transplanted patients which developed an encephalitic syndrome. disclosure: nothing to declare background: cytomegalovirus (cmv) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (hsct). it causes end-organ disease, multi-organ dysfunction syndrome, graft failure, increased susceptibility to infections and gvhd. greatest risk of cmv infection in a seropositive host is the reactivation of latent virus. methods: a prospective descriptive study performed at armed forces bone marrow transplant centre, rawalpindi, pakistan from dec to sep . hundred consecutive patients who underwent hsct were followed with weekly cmv dna quantitative pcr from engraftment till day for cmv reactivation. patients in whom cmv pcr showed more than copies/ml were treated with antiviral therapy. factors associated with cmv reactivation, outcome of antiviral therapy and effect of cmv on transplant outcome is studied. results: out of cases, were hla matched siblings, were matched family donors and were haploidentical transplants there were males and females. mean age was . ± . years. fourty-two transplants were done in thalassemia, in aplasia, in leukemias and in other hematological disorders and immune deficiencies. ninety-eight recipients and all the donors were cmv seropositive before hsct. cmv reactivation was seen in patients and of them had cmv viral load more than copies/ml and patients had cmv viral load less than copies/ml. nineteen patients had no cmv reactivation. mean time to reactivation since transplant was ± days. valganciclovir was given in patients due to ease of administration and six patients were treated with ganciclovir during their hospital stay. only one patient had resistant disease. mean time to clear viremia was ± . days. the patients having viral load less than copies/ml, subsequently cleared cmv without any treatment. antiviral agents; ganciclovir and valganciclovir were equally effective for treating cmv infection with % efficacy, however, more adverse effects were seen with ganciclovir. myelosuppression i-iii was seen in % patients treated with valganciclovir and in % treated with valganciclovir. renal impairment i-ii was seen in % of valganciclovir and % of ganciclovir treated patients. steroid administration was strongly associated with cmv reactivation (p = . ). no statistically significant association was found with the use of atg, gvhd, underlying disease, abo or gender mismatch. os was . % and . % in with and without cmv reactivation (p= . ) and dfs was . % and . % in with and without cmv reactivation (p= . ) conclusions: cmv reactivation was seen in % of the transplant recipients, this is higher compared to the western world due to high cmv seropositivity is this region. steroids administration in post-transplant period significantly increase the risk of cmv reactivation. preemptive therapy with valganciclovir effectively treats cmv reactivation with acceptable side effects. viral threshold for treatment should be decided considering the regional endemicity. cmv adversely affects the transplant outcome in terms of dfs and os. disclosure: no conflict of interest. acute nephritis requiring nephrectomy caused by adenovirus (hadv) and human polyomavirus bk (bkpyv) following allogeneic hematopoietic stem-cell transplantation in a patient with ph+ all background: adenovirus infection represents an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct), with no established therapy. although different organs may be affected by disseminated hadv infections, kidney involvement has been rarely reported. co-infection of hadv and bkpyv are common complication in patients undergoing allo-hsct, but recent studies demonstrate that bkpyv may facilitate the replication of hadv and lead to elevated viremia with increased virulence and serious clinical consequences. here we report a case of an adult patient who required a monolateral nephrectomy due to hadv pyelonephritis as an early complication of allo-hsct for philadelphia-positive acute lymphoblastic leukemia (ph+ all). methods: in september , an ethiopian gentleman was diagnosed with ph+ all at the age of years. he was treated with polychemotherapy in association with the tyrosin kinase inhibitor imatinib mesylate achieving a complete remission (cr). one year later, due to disease relapse with cns involvement, he was started on vincristine and dexamethasone plus imatinib treatment and in april he was referred to our bmt center from ethiopia. upon confirmation of the p ph+ b-all diagnosis, therapy with the scr/abl dual inhibitor dasatinib associated to intrathecal chemotherapy was started and a salvage treatment with inotuzumab ozogamicin followed by an allogeneic hsct from a hla-identical brother was planned. having achieved a documented molecular cr disease status, in june the patient underwent allo-hsct following the fludarabine-melphalan reduced-intensity conditioning regimen. graft-versus-host prophylaxis included anti-thymocyte globulin, cyclosporine and mycophenolate mofetil results: on day + post-transplantation the patient developed macro-hematuria due to hemorrhagic cystitis and a ct scan unveiled a left pyelonephritis with marked kidney enlargement. kidney microbial investigations were all negative. at the same time, hadv viremia with very high copy number (> cp/ml) was documented and also elevated bkpyv (> cp/ml) viruria and viremia ( cp/ml). the genotyping of hadv evidenced serotype b mainly involved in infections of the urinary tract. treatment with cidofovir was immediately started; nonetheless, due to rapid clinical worsening despite maximal antibiotic therapy, on day + a left nephrectomy was performed, which led to a subsequent progressive resolution of the clinical symptoms and negativization of hadv and bkpyv viremia and viruria. pcr real time performed on the kidney tissue unveiled very high concentration of hadv copy number. conclusions: acute pyelonephritis due to disseminated hadv infection may represent a possible cause of severe complication following allo-hsct. monitoring of hadv copy number is helpful to evaluate infection severity and response to treatment. co-infection of hadv and bkpyv in immunocompromised patients should be always considered likely to worsen clinical course and outcome. disclosure: nothing to declare background: infection is a major cause of morbidity and mortality in patients (pts) receiving an allo-hsct. its severity is related primarily to the depth and duration of neutropenia. febrile neutropenia (fn) is defined as a neutrophil count below cells/mm and a fever ≥ . °c at a single measurement or≥ °c times at one hour intervals. the objective of our study is to analyze the epidemiological, clinical, biological characteristics of febrile episodes (fe) occurred in pts who benefited an allo-csh over a period of years. methods: from january to december , allo-hsct were performed in pts including sibling-hla identical, haplo-identical and phenoidentical for essentially acute leukemia ( pts, %), acquired and congenital aplasia ( pts, %). the median age is years ( - ) and sex-ratio (m/f): . . prophylaxis consisted on isolation sterile room with laminar flow, digestive decontamination, fluconazole and aciclovir. nine pts ( . %) were infected at the time of hospitalization (cellulitis , pneumoniae , bacterial angina , veinitis , bronchial pneumonia , furuncle cutaneous ) requiring treatment with antibiotics. conditioning regimen is myeloablative in all pts. anti-thymocyte globulin is used in pts ( . %). peripheral blood stem cells (pbsc) are used in pts ( %) with an average level of cd + cells: , . /kg ( . - . ) , bone marrow (bm) in pts with a mean level of nucleated cells: . x /kg ( . - . ) and the association of pbsc-bm in pts (haplo-identical). at each fe, are practiced: chest x-ray, procalcitonin test, blood culture, microbiological study of urine and stool (if diarrhea). results: all pts showed aplasia with an average duration of days ( - ), neutrophil engraftment was observed at day ( - ). one hundred and twenty-nine pts ( . %) presented fe with an average of . per pt. eleven pts ( %) had fe or more. forty nine ( , %) fe are clinically documented (digestive: , skin: , pulmonary: , urinary: , oto-rhino-laryngology: ). the blood cultures are made at fe, fe are microbiologically documented ( %): gram-positive bacteremia in % (mainly coagulase negative staphylococci) and gramnegative bacilli in % of cases. procalcitonin test performed during fe: normal ( cases), probable infection ( cases), probable sepsis ( cases), severe sepsis ( cases) and septic shock (one case). empirical double antibiotic therapy is initiated in pts without waiting for the results of the microbiological study. this association was sufficient in pts ( %). the transition to a second line was needed in pts ( . %) and third line in pts ( %). antifungal is added in cases ( %). eight pts benefited from g-csf. the evolution is favorable in fe ( . %), apyrexia obtained after an average of . days . three pts died ( %) by severe sepsis on a durable aplasia, of which had a cellulitis before the conditioning. conclusions: fe increase morbidity and mortality in allo-hsct so prophylactic measures are essential. empirical antibiotics treatment has to be instituted very quickly in the absence of documentation. disclosure: nothing to declare p abstract withdrawn. atsushi satake , masaaki hotta , ryo saito , akiko konishi , hideaki yoshimura , takahisa nakanishi , shinya fujita , tomoki ito , kazuyoshi ishii , shosaku nomura kansai medical university, osaka, japan background: cytomegalovirus (cmv) infection remains a common complication after allogeneic hematopoietic stem cell transplantation (ahsct), which results in increased morbidity and mortality. letermovir is a novel anti cmv drug that inhibits the cmv-terminase complex. the purpose of this retrospective study is to elucidate the efficacy and safety of cmv prophylaxis with letermovir early after ahsct in clinical practice. methods: we retrospectively analyzed the incidence of cmv infection, cmv disease, preemptive therapy, adverse events through week after ahsct, the rates of engraftment and overall survival. all patients underwent ahsct in our institution for hematopoietic malignancies between may and nov . data collected in this study included patient's characteristics such as age, sex, disease status, donor source and cmv disease risk. cmv infection was evaluated by cmv antigenemia. this study was approved by the research ethics committee of the faculty of medicine, kansai medical university. results: thirteen patients (male , female ) underwent ahsct and received cmv prophylaxis with letermovir. the median age was years (range, - years). overall, of patients ( . %) were considered to be at high risk for cmv, including patients ( . %) with haploidentical donors, and ( . %)with mismatched, unrelated donors. all patients began letermovir from day after ahsct, and achieved engraftment (median , - days). no patient developed cmv disease and required preemptive therapy. one patient died of treatment-related mortality, and patients died of acute gvhd. although one patient discontinued letermovir before day after ahsct because letermovir was suspected to be a cause of persistent nausea, severe adverse events were not observed. conclusions: it is still unknown whether cmv prophylaxis with letermovir improves os and reduces trm; however, our data suggests that cmv infection is considerably inhibited by administration of letermovir early after ahsct. clinical trial registry: not applicable. disclosure: the authors declare noconflicts of interest for this study. background: cytomegalovirus (cmv) is cause of increased morbidity and mortality after transplantation of hematopoietic cells. the pathogenesis of cmv disease or infection is complex with multiple interactions with the immune system, mainly in acute and chronic graft-versus-host disease (gvhd). the aim of this study is to analyze the risk factors for the reactivation of cmv in patients undergoing allogeneic hematopoietic cell transplantation (hct). methods: prospective descriptive study of the risk factors for the reactivation of cmv in the described population. univariate and multivariate analysis of the predisposing factors were performed: donor graft, treatment with corticosteroids, use of antithymoglobin, serologic status, conditioning regimen and the presence of gvhd. results: during the period between august until january , patients were evaluated. . % (n: ) had reactivation of cmv. average reactivation was days post transplant. both (recipient and the donor) had positive cmv igg in . %. in the univariate analysis, the reactivation of cmv was associated with haploidentical transplantation (p: < . ), with the use of corticosteroids (p: < . ) and gvhd (p: < . ). in the multivariate analysis, the haploidentical transplant maintained its statistical significance in comparison with the related allogeneic transplant (p: . , or: . ; ic %: . - . ) as well as the use of corticosteroids (p: . , or: . ; ic %: . - . ). % of patients receiving corticosteroid treatment had grade ii / iii gvhd. the serologicac status, myeloablative conditioning regimen and the use of atg showed no statistically significant association. conclusions: in patients undergoing allogeneic transplantation, were found as risk factor to reactivation, those who received haploidentic transplantation and treatment with corticosteroids. another risk factor that showed greater reactivation was the presence of gvhd. disclosure: nothing to declare methods: a y/o male was referred for allogeneic transplant following cycles of induction therapy for aml with complex karyotype and axsl mutation having achieved complete remission following the first cycle of chemotherapy. his first induction cycle was complicated by a perianal myeloid sarcoma which became infected and required surgical drainage and formation of a defunctioning colostomy. results: following allogeneic transplants, the first complicated by secondary graft failure and the second by primary graft failure he presented with two skin lesions, with a third lesion adjacent to his stoma developing shortly after admission. all lesions were erythematous with central necrosis and progressed rapidly in size over hours. biopsy of the skin and para-stomal lesions revealed fungal mycelia, with culture subsequently identifying rhizopus oryzae. initial treatment was with liposomal amphotericin b mg/kg/day followed by dose escalation to mg/kg/day due to the development of new skin lesions. the patient had been taking posaconazole (tablet) prophylaxis since his first allogeneic transplant and peripheral blood drug levels checked at the time of admission were therapeutic confirming that this was a breakthrough fungal infection. consequently posaconazole was stopped and isavuconazole added to the treatment regimen. surgical assessment was undertaken but surgery was deferred on the basis of high risk due to the extent of the infection and the patient´s profound pancytopenia. the organism was tested for in vitro susceptibilitiy and found to be resistant to posaconazole (mic > mg/l), with borderline resistance to isavuconazole (mic mg/l) and sensitive to amphotericin b (mic . mg/l) (phe mycology reference laboratory, england). isavuconazole was therefore stopped and the patient was managed with liposomal amphotericin b along with daily granulocyte infusions. he underwent a third allogeneic transplant using a different unrelated donor and stable engraftment was achieved. post transplant there was initially an increase in the size of the para-stomal lesion, but no new skin lesions developed. following engraftment he underwent resection of the stomal lesion, with primary closure and re-siting of his stoma. amphotericin b was replaced by isavuconazole prophylaxis on discharge and he continues to make an excellent recovery. conclusions: whilst aspergillus species remain the most common cause of invasive fungal infections in allogeneic transplant patients, other species including the mucorales are seen, and generally associated with poorer outcomes. whilst there are standardised methodologies for susceptibility testing, fungi specific cut offs based on clinical outcomes are only available for a limited number of species/ antifungal agents. in this case, susceptibility testing demonstrated resistance to posaconazole which was consistent with the clinical presentation of invasive infection despite therapeutic levels of posaconazole. it is also worth noting that an estimated % of r. oryzae isolates in the uk are resistant to posaconazole. treatment with high dose amphotericin b resulted in improvement in small skin lesions with stabilisation of the larger stomal lesion until count recovery allowed surgical resection. background: total depletion of innate and adaptive immune cell populations occurs after intensive chemotherapy and hematopoietic stem cell transplantation (hsct). both t and b lymphocyte pools are restored slower that myelomonocytic populations. hsct patients are at high risk for bacterial and viral infections at early terms (< days) post-transplant. the reconstitution of the immune system depends on the time required for stem cell recruitment, differentiation, expansion, maturation and release into the bloodstream. restoration terms for myeloid cells after hsct are usually defined as the st day with neutrophil count of ≥ . x ^ /l with mean recovery terms of to days. high occurrence of cytomegalovirus (cmv) in hsct patients mostly result from reactivation of a latent virus acquired in early childhood. however, delayed immune reconstitution and subsequent infections such as cmv, adenovirus (adv) or herpes (hhv- ) diseases are not unusual and still constitute a major cause of death in peru. methods: peruvian pediatric patients (n= ) diagnosed with aplastic anemia, mds, aml or all underwent a haploidentical hsct performed with the clinimacs device. patients treated were separated in two groups. the group of patients who received viral prophylaxis (ganciclovir) was compared to the group that did not receive any prophylaxis treatment. viral reactivation was confirmed by pcr test twice a week and clinical signs within days after hsct. results: in the group that didn´t received prophylactic treatment, engraftment occurred close to day post haplo-hsct and none of the patients developed gvhd (graft versus host disease). nevertheless, incidences of cmv, hhv- and bkv infections before day post haplo-hsct were still high. an overall survival (os) over % with an ic % was reached at the end of the first year. on the other hand, the group of patients that received prophylaxis with ganciclovir did not developed gvhd and reached the engraftment close to day with a very low viremia incidence after the first month post haplo-hsct. all viral reactivations were caused by cmv and the os was over % with an ic % at the end of the first year. previous prophylaxis to both the donor and the receptor with ganciclovir ( mg/kg) every hours before and during the conditioning regimen has allowed a better control of viral reactivation. conclusions: the attempts to improve immune function and reduce nonrelapse mortality from infectious complications without increasing gvhd have focused on a partial t cell depleted graft, such as t cell depletion (tcr α/β). this graft retains a large numbers of effector cells, such as tcr γ/δ and natural killer cells. however, delayed immune reconstitution and subsequent infections are a big issue. a novel partial t cell depletion strategy such as depleted naïve t cells (cd ra+ t cells) could enhance the recovery of immune function after haplo-hsct because donor pathogen memory t cells from the donor are retained. it is necessary to increase the studies and the database to set the scheme of previous prophylaxis to the recipient to contain the viral reactivation and to help a rapid immune reconstitution. disclosure: no conflict of interest is declared information was recovered from the medical records. results: thirty-four patients were included, of them with the following diagnoses: acute leukemia ( ), granulocytic chronic leukemia ( ), dendritic cell neoplasia ( ), aplastic anemia ( ). % of the patients received a transplant from an identical hla donor and % received a haploidentical transplant. mean age's patients was years ( - ). prophylaxis with posaconazole was performed on % of the patients with identical hla and % on haploidentical group; the rest of the patients received fluconazole. the posaconazole group presented: fever %, mucositis gi-ii %, gastrointestinal toxicity gi-ii % (p= . ), hepatic toxicity %, kidney toxicity %, oral candidiasis %. during this period none of the patients presented invasive fungal infection in any group. there were deceases, one on each group and none related to a fungal infection. the overall survival was of the % versus % on the posaconazole group and the fluconazole group respectively. conclusions: the prophylaxis with posaconazole and fluconazole is effective on the prevention of invasive fungal infection on the first days. the toxicity was similar on both groups. posaconazole can be effective on the prevention of the haploidentical type. is necessary to continue following the patients with infection risk on a long-term period associated with the chronic gvhd. disclosure: none declared lymphoma these results open the question whether allo-sct should still be offered to these patients. methods: we aimed to define the role of allo-sct in refractory or relapsing after two lines de novo or transformed dlbcl patients, and its comparison with zuma- car-t cells trial (neelapu et al nejm ). we analyse long-term allo-sct results in de novo (n= ) or transformed dlbcl (n= ) out of the allo-sct performed in our institution between october to october . results: patients and transplant characteristics are summarized in table . complete response (cr) at days was , % and % of them remain in cr at months. with a median follow-up of months, -year progression-free survival (pfs) was % and -year overall survival (os) %, with a -year transplant-related mortality of %. refractoriness at the time of the transplant was associated with a poorer prognosis, with only out of refractory patients being long term survivors (figure ). similar results were reported for zuma- trial, with a best response of % cr retained in % of them at months. with a median follow-up of months, -months pfs was % and -months os %. patients characteristics did not differ in our series and zuma- , except that all the patients in zuma- were refractory prior to therapy (table ) . conclusions: although very few patients with de novo or transformed dlbcl are offering an allo-sct ( % of all allo-sct), this is a curative option in chemosensitive patients and with more mature data and longer follow-up than with car-t therapy; for these reasons, it should still be offer to these poor prognosis patients. moreover, almost all patients have now available donor, better graft-versus-host disease prophylaxis will decrease trm and morbidity, and new therapies will make more patients in sensitive disease before allo-sct. therefore, allo-sct and car-t cells are strategies to be discussed in every young patient with available donor. disclosure: honoraria as advisor or speaker from gilead ( methods: consecutive patients transplanted for hgbl (excluding burkitts lymphoma) between - in the ebmt database were included. data collected included age, sex, pathology subtype (hgbl (including subtypes), tfl, dhl), disease status at sct, conditioning (ma vs beam cam vs flu-mel-cam/atg), engraftment, day outcome, trm, os and pfs and eligibility for emea licensed indication of car-t therapy. results: fifty patients ( m, f) with a median age of at diagnosis and at sct were included. the subtypes included hgbl (n= ), tfl (n= ) and dhl (n= ). indications for sct were: primary refractory (n= ), relapse < months after primary treatment (n= ), previous autologous-sct (n= ) and dhl (n= ). the median lines of therapy was (range to ). conditioning used was cytbi n= , bu/cy n= , etop/tbi n= , flubucy n= , beamcam n= , fmc/t, n= . all patients engrafted with neutrophil > . /l at median days and platelets > /l at median days. the day mortality was % (progressive disease %, nrm %) with a year os of % and mortality due to progressive disease % and nrm %. disease subtype influenced outcome with an os for primary refractory hgbl, relapsed hgbl, tfl and dhl respectively of %, %, % and %. patients were eligible for a licensed car-t product. conclusions: the outcome of these high risk hgbl patients have an acceptable os of %, with relapsed disease being the commonest cause of mortality. patients with dhl have a particularly good outcome in this series; recent evidence indicates that some of these patients with a non-immunoglobulin gene associated myc translocation could be managed more conservatively (ash sehn). the outcomes achieved with allogeneic-sct in this series will provide a baseline for outcome assessment with a cart program. disclosure: nothing to declare background: immune checkpoint inhibitors (ici) allow to achieve a durable remission in patients with resistant or refractory (r/r) classical hodgkin lymphoma. however, the information about optimal duration of therapy and the prognosis of the patients after ici cessation is limited (manson, blood ). therefore, the optimal role of hematopoietic sct (hsct) in this patient group is not defined. our aim was to determine remission duration in patients who discontinued ici monotherapy after achieving complete remission (cr). methods: this analysis included patients ( male/ female) aged to (median years) with r/r classical hodgkin lymphoma who were treated with nivolumab ( mg/kg every days) and achieved cr. response was assessed by positron-emission tomography/computed tomography (pet/ct) using lyric criteria every month. after nivolumab therapy had been stopped the patients received no other treatment and disease was assessed every months by pet/ct. median follow-up after therapy discontinuation was ( - ) months. results: at the moment of therapy initiation ( %) patients had stage disease, ( %) patients had progressive disease (pd), ( %) patients had stable disease, ( %) patients had partial remission and ( %)complete remission; ( %) patients had b-symptoms and ecog score > . the median number of previous therapy lines was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . before nivolumab initiation high dose chemotherapy with autologous sct was performed in patients ( %) and ( %) received brentuximab vedotin. the median number of nivolumab cycles was ( - ). cr was achieved after median of ( - ) cycles. the median duration of therapy after achievement of cr was ( - ) months. at the time of analysis, all patients were alive, ( %) out of patients relapsed after therapy discontinuation. the median progression-free survival (pfs) for the total group was not achieved. among patients with relapse, the median time before pd was ( - ) months. after relapse all patients were retreated with nivolumab monotherapy or with chemotherapy combination. one patient achieved complete remission; -partial remission; -indeterminate response type . other patients are continuing the therapy and their response has not yet been evaluated. conclusions: while complete response was maintained in some patients at median follow up of months after nivolumab therapy cessation, the pfs plateau was not reached. we report that patients with relapse after nivolumab discontinuation sustained sensitivity to nivolumab and achieved a response during retreatment with nivolumab monotherapy or with chemotherapy combination. in patients with unsatisfactory response to nivolumab retreatment, hsct option should be considered. disclosure: nothing to declare high dose chemotherapy with autologous stem cell transplantation in primary central nervous system lymphoma: data from the japan society for hematopoietic cell transplantation (jshct) registry center hospital, tokyo, japan, national cancer institute, bethesda, md, united states, okayama university hospital, okayama, japan, kanazawa medical university, uchinada, japan, kyoto university, kyoto, japan, aomori prefectural central hospital, aomori, japan, yamagata unversity school of medicine, yamagata, japan, tenri hospital, tenri, japan, hiroshima university, hiroshima, japan, japanese data center for hematopoietic cell transplantation, nagoya, japan, nagoya university graduate school of medicine, nagoya, japan, shimane university hospital, izumo, japan background: high-dose chemotherapy (hdt) with autologous stem cell transplantation (asct) has been shown to improve prognosis of patients with central nervous system (cns) lymphoma. whereas the common regimen of hdt for pcnsl in the europe and the us is thiotepa-based regimen, e.g. bcnu-thiotepa, tbc (thiotepa-busulfan-cyclophosphamide), thiotepa-based regimen was only available before discontinuation of thiotepa in in japan. we report the results of asct for pcnsl from the japan society for hematopoietic cell transplantation (jshct) registry. methods: data from the jshct registry were retrospectively analyzed. patients with pcnsl who received first hdt/asct between and were evaluated. distribution differences of clinical characteristics between groups were analyzed with fisher´s exact or mann-whitney u tests. overall survival (os) and progression free survival (pfs) were calculated using kaplan-meier method. two-group analysis of the cumulative incidence of relapse was conducted using the grey test. factors were analyzed in univariable analysis, and all factors with p≤. were retained in the multivariable model. all p values were sided, and values were regarded statistically significant if p< . . results: median age was months (range - ) with patients over years of age; males and females. ecog-performance status (ps) at diagnosis was better (ps - ) in patients and poor (ps - ) in patients. serum lactate dehydrogenase (ldh) levels at diagnosis were elevated in patients. karnofsky ps and cerebrospinal fluid (csf) protein concentration at diagnosis were not collected in the registry. patients were in complete remission (cr), patients were in partial response (pr), and patients were stable disease (sd) or progressive disease (pd) at the time of hdt/asct. after hdt/asct, additional patients achieved cr. with median follow-up period of months, the -year os and pfs were . % and . %, respectively. the was no significant difference in os and pfs between upfront and salvage hdt/asct. since thiotepa, a key agent in hdt/asct for pcnsl, has been unavailable after the discontinuation in japan, the hdt regimens used were not uniform. thiotepa-containing hdt was received by out of patients before , but by out of patients after . thiotepa-containing hdt showed improved pfs (p=. ), lower relapse (p=. ) and a trend toward a survival benefit. in the multivariate analysis, non-complete remission at hdt/asct was an independent predictor for os (hr= . , %ci: . - . , p=. ) and thiotepacontaining hdt remained significant for pfs (hr= . , %ci: . - . , p=. ). [[p image] . os(a),pfs(b) in all patients (n= ) and cumulative incidence of relapse in cr patients (c; n= )] conclusions: our results confirm the activity of thiotepacontaining regimen for hdt/asct in pcnsl patients. currently a pharmaceutical company re-develops thiotepa for new approval of hdt/asct in pediatric solid cancer and adult lymphoma in japan (japiccti- ). further evaluation with the thiotepa by prospective clinical trials is warranted. disclosure background: t-cell non-hodgkin lymphomas (t-nhl) are rare diseases and they are associated with worse prognosis when compared to their b-cell counterparts. allogeneic stem cell transplantation (allo-sct) may have a curative potential for these patients due to the graft versus lymphoma effect. however, data is limited on the efficacy of allo-sct for these diseases. methods: we identified patients ( % females; median age: y; range, - ) with t-nhl that underwent allo-sct at university hospital eppendorf between and . twenty-one patients (underwent allo-sct from a matched sibling donor (msd) and ( %) from a matched unrelated donor (mud). sixteen patients had ptcl ( %), n= ( %) anaplastic large-cell lymphoma (alcl), n= ( %) angioimmunoblastic large cell lymphoma, n= ( %) adult t-cell leukemia/lymphoma, n= ( %) hepatosplenic gamma/delta t-cell lymphoma, n= ( %) enteropathy associated t-cell lymphoma, n= ( %) tcell-prolymphocytic leukemia, and n= ( %) each extranodal t/nk-cell lymphoma, cutaneous t-cell lymphoma as underlying diagnosis. the median ann arbour stage at diagnosis was (range, - ). ten patients ( %) had bone marrow involvement at diagnosis. all patients were heavily pretreated, ( %) patients relapsed post autologous stem cell transplant (apsct) and one patient post allo-sct. fifteen patients ( %) were transplanted in complete remission (cr) ( in st cr, in nd cr), n= ( %) in partial remission (pr), and n= ( %) with advanced disease. most of the patients received myeloablative conditioning ( %). thirty-eight ( %) patients received total body irradiation based regimens and ( %) received chemotherapy based regimens. twenty patients ( %) received anti-t-lymphocyte globulin (atlg neovii), and most patients ( %) received g-csf mobilized peripheral stem cells. results: overall, patients ( %) had neutrophil engraftment (median days: ; range, - ) . at day , the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were % and %, respectively. after a median follow up of months (range, - ) the cumulative incidences of chronic gvhd was % distributed evenly between limited and extensive. twenty nine patients ( %) achieved cr after allo-sct. median overall survival (os) and disease free (pfs) survival were months and months respectively. the year os and pfs were % and % respectively. fourteen ( %; % ci [ . - . ]) deaths were due to non relapse mortality (nrm) and patients ( %; % ci [ . - . ]) died due to disease progression. patients with a male donor had improved os compared to those with a female donor ( year os male %, female %; p= . ). patient gender, disease subtype, bone marrow involvement, type of allo-sct, donor, patient cmv status, abo incompatibility, disease stage at diagnosis, previous transplant, disease status at transplant, conditioning regimen, atg and stem cell source had no effect on os, pfs, nrm, and post transplant complications. conclusions: acknowledging the retrospective nature, our study shows that allo-sct induces high rates of complete remission, and may have a curative potential even in diseases relapsing post asct. however our findings need to be confirmed in larger prospective studies. disclosure: no funding, no conflict of interest p abstract already published. at-home autologous stem cell transplantation in lymphoma patients: clinical impact of non-g-csf administration post-transplant background: severe neutropenia remains the main cause of morbidity and mortality after autologous stem cell transplantation (asct). g-csf administration after asct is a common practice, performed to reduce the duration of neutropenia and its complications. in a previous work in patients with multiple myeloma managed at home after asct, we did not observe a deleterious clinical impact in those patients that did not receive g-csf post-transplant (martinez-cibrian n. et al, bmt ) . despite the fact that lymphoma patients receive a more intensive conditioning regimen that multiple myeloma patients, we hypothesized that the use of g-csf in lymphoma patients managed at home during the aplasia phase of asct does not provide a significant clinical benefit. methods: lymphoma patients were managed at-home since day + of asct. between february and july , patients received at-home g-csf μg/kg per day since day + until their anc reached x /l (g-csf group) and, since august , patients did not receive g-csf (non-g-csf group). all patients were conditioned with beam and received prophylaxis with a quinolone, fluconazole, aerosolized pentamidine and low-dose acyclovir (hvs+). in all cases we added primary prophylaxis with piperacillin-tazobactam . g/ h i.v., using a portable intermittent infusion pump (iip), from an absolute neutrophil count (anc) < . x /l until the first day of fever or until attaining an anc of x /l. first-line therapy at home of neutropenic fever (nf) was refrigerated meropenem g/ h i.v using a portable iip. fever was an indication of immediate visit to the hospital, and those patients presenting with focal infection or signs of severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhea and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients are shown in table . there were no differences between groups with respect to gender, diagnosis, stage of disease, comorbidity index (hct-ci), source of stem cells (peripheral blood) and cd + cell dose infused. the median (range) age (years) was ( - ) in g-csf group and in non-g-csf group (p= . ). duration of neutropenia less than . x /l was significantly longer in non-g-csf group, with a median of days (range - ), compared with (range - ) in g-csf group (p < . ). severe neutropenia, less than . x /l, was also longer in the non-g-csf group ( days ( - ) vs. ( - ); p= . ). no differences were observed in the time to platelet engraftment. g-csf post-transplant avoidance did not influence the incidence of neutropenic fever, the first day and duration of fever, the incidence and severity of oral mucositis, bacterial infections documented and number of readmissions. the median duration of the whole procedure at-home was day shorter in the g-csf group ( vs. days; p= . ). conclusions: the policy of not administering g-csf post-asct in our home-based program for lymphoma patients, that include intensive bacterial prophylaxis, did not have a deleterious impact on the main results reviewed, which suggests that elimination of its use can be achieved. disclosure the aim of this study was to analyze the spanish experience with patients diagnosed of nhl who received haplosct with pt-cy. methods: sixty patients who received haplosct with pt-cy in spanish centers from to were analyzed. patients were followed-up until . gvhd prophylaxis consisted in cyclophosphamide mg/kg/d on days + and + , and mmf and a calcineurin inhibitor from day + . results: patients' characteristics are summarized on table . median age of patients was , % male, and diagnosed from t cell lymphoma ( %). most of them didn´t achieve complete response prior to transplant ( %), but only % with active disease. up to % of patients had received previous transplant, from which % was an allogeneic transplantation. source of stem cells was mainly peripheral blood ( %), and reduced intensity conditioning was the preferred ( %) regimen. donors were % siblings ( ), % offspring ( ), and % parents ( ). median neutrophil and platelet engraftment was ( - ) and ( - ) days, respectively. acute gvhd grade ii-iv rate was %, with only patients developing grade iii-iv ( %). chronic gvhd rate was %, and only in ( %) was extensive. median follow-up was months. the -year overall survival and event free survival was % and %, respectively. the -year cumulative incidence of relapse was % and -year cumulative incidence of nrm was %. conclusions: relapsed/refractory nhl are aggressive entities with a fatal course in a short period of time. haplosct with pt-cy permit a new treatment option among these patients, with acceptable outcomes. more studies are needed with a larger cohort of patients and longer follow-up to confirm these results. disclosure: nothing to disclose. higher suv at pre-transplant and day posttransplant pet scan both independently predict inferior survival in patients with diffuse large b cell lymphoma background: autologous stem cell transplant (auto-hct) can cure some patients with relapsed diffuse large b-cell lymphoma (dlbcl) but relapse occurs in about % of patients. while our center and others utilize routine surveillance imaging post-transplant, the utility in this setting is unclear. imaging is costly and exposes patients to radiation. novel interventions are now available for patients relapsing after auto-hct making early disease recognition crucial to intervene prior to clinical progression. hence, we studied impact of post-auto-hct surveillance ( )f-fdg-pet ct at day on transplant outcomes. methods: we analyzed a cohort of consecutive auto-hct recipients with relapsed/refractory dlbcl who then underwent pre-transplant pet/ct and surveillance pet ct at day (interquartile range (iqr): - days) post-hct at the university of minnesota medical center. univariate analysis was performed to analyze pet parameters including deauville score (d), standardized uptake values (suv), total lesion glycolysis (tlg) and total metabolic tumor volume (tmtv) as predictors of relapse and survival after auto-hct. in addition, we assessed outcomes of patients with clinically versus radiographically detected relapsed dlbcl after auto-hct. other pre-hct factors analyzed included age, gender, conditioning regimen, performance status, consolidation radiation therapy, tmtv, suv, tlg. results: five-year cumulative incidence of relapse after auto-hct was % ( %ci to ) and overall survival (os) was % ( % ci to ). twelve ( %) relapsed prior to day . d-score for patients with d pet/ct were d ( %), d ( %), d ( %), d ( %), d ( %) with median survival in years for d , d , d and d of . , . , . , and . , respectively. mean suv varied from . (d ) to . (d ). suv was predictive of relapse and os. risk of relapse increased with doubling of suv; -fold higher suv increased hr by . ( %ci . - . ; p= . ). mortality increased with doubling of suv in both pre-hct ( -fold increase in suv associated with hr . [ % ci . to . ]; p= . ) as well as post-hct pet (hr . [ % ci . to . ]; p= ) irrespective of the bulk of tumor. in addition, risk of death was times higher in d patients relative to d (hr . [ % ci = . to . ]; p≤ . ). patients with d (n= ) had higher tmtv ( cm ) compared to d (n= , tmtv . cm ). the hazard ratio for death following relapse was -fold higher (hr . [ % ci . to . ]; p= . ) if relapse was detected clinically versus only radiographically over a median follow-up time period of . years. other pretransplant patient and disease characteristics did not significantly influenced the outcomes. conclusions: in patients with r/r dlbcl undergoing auto-hct, surveillance pet/ct at day identified patients with poor survival~ year. higher suv in both pre-transplant as well as post-hct pet was predictive of increased mortality. these patients may benefit from novel treatments. [ there are concerns about the risks of nivolumab treatment before and after allo-hsct, due to the risk of heavy gvhd, thus the place of immune checkpoints inhibitors is not yet defined. this report include analysis of our center experience of nivolumab treatment in patients with r/r hl before and after allohsct. methods: we retrospectively evaluated the results of allohsct in patients with r/r chl who had undergone transplant from to . the analysis included patients received the flube conditioning and ptcy gvhd prophylaxis. in group a patients (n= ) received bridge therapy with nivоlumab and in group b patients (n= ) received bridge therapy with brentuximab vedotin or chemotherapy-based bridges. time from the last nivolumab administration to allohsct was at least months. results: at the time of analysis, median follow-up was ( - ) months for group a, and ( - ) months for group b. there was no difference in two-year os (p= , ) with significantly better efs (p= , ) for group a versus group b: % and % vs , % and % respectively. relapse incidence was % for group a versus , % in group b (p= , ). cumulative incidence of non-relapse mortality at years was , % and , % in group a and group b, respectively (p= , ). there was no difference in grade ii-iv ( % vs %, p= . ) and grade iii-iv ( % vs %, p= . ) agvhd, as well as extensive chronic gvhd ( % vs %, p= , ) in groups a and b, respectively. ten patients with relapse after allohsct were treated with different doses ( , - mg/kg) of nivolumab in cic center. at the median follow up of mo ( , - ) all patients remain alive. the objective response to therapy was assessed in patients noted in all patients ( %), disregard the dose of the nivolumab: cr in %, and pr in %. the response was lost in four patients, which required nivolumab retreatment. none of the patients developed gvhd after nivolumab administration. in this analysis, there was also no correlation between dose of nivolumab and incidence and severity of adverse events. conclusions: allohsct in combination with immune checkpoints inhibitors is a good option for patients with r/r chl. consideration for immune-mediated toxicities and the potential for increased graft-versus-host disease remain important. early data suggest that nivolumab may be an efficient therapy in patients with r/r chl relapse after allo-hsct. further research needed. disclosure: the authors declare no conflicts of interest. background: transformation to diffuse large b-cell lymphoma (dlbcl) is considered to be one of the most unfavourable events of lymphoma natural history with poorer outcome as compared to de novo dlbcl (alonso-Álvarez et al, bjh ). in patients suitable for salvage therapy, hematopoietic stem-cell transplantation (sct) could be an option, although its role is not well stablished. we analyse indication and outcome after transplant in transformed dlbcl at a single reference transplant unit. methods: out of total of transplants performed at our unit between and - autologous and allogeneic- were dlbcl transformed from an indolent nhl. of them, received an autologous sct (asct) and an allogeneic sct (allo-sct). results: median age was years old (range - ) and (range - ) for patients receiving asct and allo-sct, respectively. all asct received beam as a conditioning regimen and most of the patients in the allo-sct group received a fludarabine/melphalan combination ( %). gvhd prophylaxis consisted on tacrolimus/sirolimus combination in % and calcioneurin plus methotrexate in %. regarding transplant disease status, ( %) of the asct patients were transplanted in complete response (cr). in the allo-sct group, ( %) patients had received three or more treatment lines before transplant and patients ( %) had received a previous asct, being ( %) in cr, in partial response (pr) and in progressive disease. transplant related mortality (trm) was . % in the asct and % in the allo-sct group. overall survival (os) and progression-free-survival (pfs) at months were % (os), % (pfs) for patients receiving asct and % (os) and % (pfs) for allo-sct. with a median follow up of months for patients receiving an asct, ( %) remain in cr. in the allo-sct group median follow up is months for the whole group and months for alive patients; patients are alive and disease free and have died, due to trm ( %). regarding progression, ( %) have progressed after autologous transplant and after allo-sct. conclusions: indication for hematopoietic sct in transformed dlbcl is stablished in few patients. only % of the patients in our transplant unit receive a transplant due to transformed lymphoma, corresponding to a . % of autologous activity and . % of allogeneic activity. according to our results transplant should be considered a curative option. most of our patients were transplanted in cr, so new agents trying to reach best response before transplant should be considered. [[p image] . eva konirova , antonin vitek , marta krejci , edgar faber , katerina steinerova , david belada , jan novak , juraj duras , petr sedlacek , veronika valkova , andrea janikova , ludek raida , pavel jindra , pavel zak , tomas kozak , marie trnkova , michal karas , marek trneny management. however, differences in patient's characteristics as well as frequency of hsct indication in different lymphoma subtypes have been observed in the last decade. the aim of this study was retrospective analysis of hsct for lymphomas in czech republic. methods: data of adult patients transplanted between years - were retrospectively analyzed using ebmt database. results: between and , autologous hsct (asct) were performed in patients ( men, %) with different lymphoma subtypes. the median age was years (range - ). out of these, ( %) were patients with non-hodgkin lymphoma (nhl), ( %) with hodgkin lymphoma (hl). the nhl group comprised of diffuse large b-cell lymphoma (dlbcl, %), follicular lymphoma (fl, %), mantle cell lymphoma (mcl, %) and t-nhl ( %). the frequency of asct in lymphomas increased from to and has been constant since ( - transplants per year). differences in frequency of asct were observed among lymphoma subtypes -decreasing numbers of dlbcl and fl and increasing numbers of t-nhl and mcl, with asct as part of the induction therapy. between and a total of allogeneic hsct (allosct) were performed in patients ( men, %). median age was years (range - ). out of these ( %) were patients with nhl, ( %) hl. the most common nhl subtypes were fl ( %), mcl ( %), t-nhl ( %) and dlbcl ( %). in the last years the number of allosct for lymphoma is fluctuating around per year. the median age at asct was significantly higher in the years - vs - [ . ( . - . ) vs. . ( . - . ), p < . , fig ] , while the increase at allosct [ . ( . - . ) vs . ( . - . )] did not reach statistical significance (p= . ). with median follow up for allosct, y probability os for patient transplanted in the later period - was in relapsed dlbcl . %, in fl . %, in hl . % and in mcl . %, y os for asct as part of first line therapy in the same period was in mcl . % and in t-nhl . %. os was significantly better in all patients who underwent asct in the years - vs - ( .% vs. . %, p < . ) and there was a trend towards better os in patients after allosct (with . % vs . %, p= . ) (fig ) . conclusions: hsct remains important treatment modality for lymphomas in the era of targeted antibody and molecular therapy and we can transplant older patients due to better supportive treatment. acknowledgment: progress q - uk from the czech ministry of education youth and sports disclosure: nothing to declare background: disease chemosensitivity to salvage treatment has been proven to be a major predictive factor for a favorable outcome after autologous stem cell transplantation (asct) for patients with refractory lymphomas. therefore the importance of effective and safe salvageregimens is indisputable. methods: we retrospectively compared the outcomes in terms of safety and efficacy, in (hl: , nhl: ) patients, with a median age of . ( - ) years, who received as st salvage either dicep [cyclophoshamide ( mg/m ), etoposide ( mg/m ), cisplatin ( mg/ m ), days - , (n= )] or the widely used regimen eshap (n= ). rituximab was additionally given to all cd- positive lymphoma patients. the statistical analysis based on the independent t-test, kaplan meir method and logrank test. results: the reason for salvage treatment was primary induction failure (pif, n= ), early relapse (< months post induction-remission therapy n= ) and late relapsed disease (n= ). more specifically, / patients ( %) in the dicep-group, and / patients ( %) in the eshapgroup were assessed with pif or early relapsed disease, however this difference was not statistically significant. both regimens were well tolerated and no major organ toxicities were noticed. eleven patients ( %) from the dicep-group, while only ( %) from the eshap-group developed febrile infections. all patients were successfully managed with the appropriate treatment and only one, from the eshap-group, required for short period admission to the intensive care unit. after cycle of dicep and cycles of eshap the disease response was re-assessed by pet/ct scan. the overall response rate (> % tumor reduction) was significantly superior for the dicep-regimen, reaching % ( / patients) vs. % ( / patients) for eshapregimen (p= , ). eleven patients ( %) from the dicep-group and ( %) from the eshap-group achieved complete metabolic remission according to pet/ ct criteria (p=ns). the median hospitalization period was ( - ) days for the dicep-group compared to ( - ) days for the eshap-group. however, for the eshapgroup, an additional median of ( - ) hospitalization days were required, since of the non-responders patients received a nd salvage before asct. the mobilization and stem cell collection was successful for both groups, though significant higher number of cd + cells were collected in the dicep-group ( . x /kg vs. . x /kg, p= , ). all but two patients (due to refractory disease) underwent asct. noticeably, the median period from st salvage treatment to asct was significantly shorter for the dicepgroup ( vs. days, p= , ), apparently because non-responders patients from eshap-group treated with a nd salvage. the -years overall and progression free survival were similar for dicep-and eshap-groups ( % vs. % and % vs % respectively). two heavily pretreated patients from the eshap-group developed secondary myelodysplastic syndrome post asct conclusions: in our series of patients both regimens proved to be safe. interestingly, despite the fact that more patients in dicep-group had poor risk disease the dicepregiment was significantly more effective, resulting thus in an earlier asct, less exposure to chemotherapeutic agents, that might led in less long-term toxicity. nevertheless, prospective trials with large series of patients are needed to define the role of dicep in the salvage treatment setting. disclosure: no conflict of interest background: although autologous hematopoietic stem cell transplantation (auto-hsct) is one of the best curative strategies for patients with chemosensitive t-cell lymphoma, major limitation remains a tumor contaminated graft-related relapse or residual disease after chemotherapy. several purging methods were introduced in auto-hsct for these limitations, however there are few studies of ex vivo purging of the autograft in lymphomas, especially t-cell lymphoma. therefore, we retrospectively analyzed consecutive t-cell lymphoma patients receiving auto-hsct with/without ex vivo purging. methods: among them, patients underwent autograft manipulation with ex vivo purging by cd + cells selection using a clinimacs device. results: with median follow-up duration of months (range, - months), -year overall survival (os; . % vs. . %, p= . ) and -year progression-free survival (pfs; . % vs. . %, p= . ) in a purged and unpurged group, respectively. transplant-related mortality was observed in both groups ( patients of a purged group and patient of an unpurged group). neutrophil ( vs. days, p= . ) and platelet ( vs. days, p= . ) recovery were similar in both group and there was no engraftment failure. on subgroup analysis according to upfront and salvage auto-hsct, while survival outcomes were improved by stem cell purging in the upfront auto-hsct (os with p= . and pfs with p= . ), there were no different survival outcomes in salvage auto-hsct. the unmanageable late-infectious complications were few in both groups except for predominantly cytomegalovirus reactivation in a purged group ( vs. patient). conclusions: although cohort was a small number, ex vivo graft-purging method was feasible and safe in t-cell lymphomas. and this purging strategy observed the more favorable survival outcomes in the upfront auto-hsct than salvage setting. therefore, further randomized studies are needed to determine the firm efficacy of cd + purification with the large number of patients in auto-hsct for t cell-lymphomas. disclosure: nothing to declare nivolumab-based regimens in relapsed or refractory non hodgkin lymphomas: the role of hematopoietic stem cells transplantation methods: we analyzed data of patients with r/r nhl, among them n with diffuse large b-cell lymphoma (dlbcl), n with primary mediastinal b-cell lymphoma (pmbcl), n with gray zone lymphoma (gzl) and n with gamma-delta peripheral t-cell lymphoma (ptcl), who received nivolumab-based regimens. the median age was years (range, - years). most of the patients n ( %) had a primary chemoresistant disease, the rest patients n ( %) had a relapse. the median of lines of prior therapy was lines (range, - lines). all sixteen patients with dlbcl and pmbcl received - cycles of nivolumab in combination with bendamustine, gemcitabine and rituximab (begern). the patient with gzl received cycles of nivolumab in combination with brentuximab vedotin and epoch. and the patient with ptcl received cycles of nivolumab monotherapy. results: at median follow up months ( - ) objective response (or) after nivolumab-based regimens was noted in n ( %) patients, complete response (cr) and partial response (pr) in n ( %) and n ( %) patients, respectively. cr observed in n patients with dlbcl, n with pmbcl, n with gzl, n with ptcl. and pr observed in patient with dlbcl. two responding patients with dlbcl underwent auto-hsct. and four responding patients (n dlbcl, n pmbcl, n gzl, n ptcl) received allogeneic hematopoietic stem cells transplantation (allo-hsct). the median duration of response for all n patients with or was (range: - +) months. among n patients who achieved or without hsct, only n remain in cr. two patients who received auto-hsct had a relapse. one patient with dlbcl improved the response after allo-hsct from pr to cr, and all four patients with allo-hsct remain in cr. the probabilities of -year os and pfs rates were % and %, respectively. conclusions: nivolumab-based regimens can lead to an objective response in % patients with r/r nhl. however, the durability of response to therapy is not long. nivolumab-based regimens can be used as bridge to allo-hsct disclosure: there are no conflicts of interest to disclose background: patients with aggressive non-hodgkin lymphoma (nhl) who relapse after autologous stem cell transplantation have a dismal outcome and could benefit from radiotherapy, allogeneic stem cell transplantation or experimental treatments. systemic inflammatory parameters at diagnosis have demonstrated to be useful to predict lymphoma evolution. methods: we conducted a retrospective review of patients with aggressive nhl who underwent autologous stem cell transplantation (astc) to evaluate the relationship between ldh, β -microglobulin, inflammatory parameters (lymphocyte (alc) and monocyte count (amc), ferritin or c-reactive protein) and imaging techniques before and on day + post-astc and progression free survival (pfs), as well as the role of residual disease directed radiotherapy (rt). results: one hundred and sixty patients with aggressive nhl received asct as consolidation treatment in our center between and . the most common diagnosis was diffuse large b-cell lymphoma (dlbcl). one hundred and nine patients received upfront asct for high risk dlbcl (defined as age-adjusted ipi - )(n= ) or for having received two or more lines to obtain first complete remission (n= ), for t-cell lymphoma (n= ) and for mantle cell lymphoma (n= ). the rest was performed in relapsed lymphomas. forty-seven patients ( %) relapsed and pfs was months. pretransplant response was evaluated with ct scan in patients ( of this with partial remission (ct-pr) and patients were evaluated with fdgpet/ct ( were pretransplant positive (pet ); of these, patients maintained positivity at day after astc (pet ). pfs in patients with ct-pr was months, in pet positive ones months and in pet positive ones months. univariate analysis showed pet positivity as the most accurate predictor of relapse (hr , , p= , ) followed by amc at day + (hr , , p= , ), albumin at day + (hr , , p= , ), ldh at day + (hr , , p< , ) and pretransplant alc/amc ratio (hr , p= , ). multivariate analysis only demonstrated an association with pet positivity (hr , ) p< and ldh in day + (hr , ) p= , with pfs. five and ten years overall survival were % and % in pet negative patients vs and % in pet positive ones (p< , ). eight out of patients with pet positivity did not relapse. salvage radiation therapy was used in patients with positive residual mass and of them did not relapse. two patients relapsed: one patient had residual mass and another had remote affectation from primary site and could be considered as progression before day + . conclusions: post asct fdgpet/ ct is superior to conventional ct in predicting outcome in aggressive lymphoma after astc. pre and post asct systemic inflammatory parameters didn't help to improve the relapse risk prediction. addition of consolidative rt after astc has demonstrated improvement in pfs in patients with pet positivity. it would be neccesary to develop randomized trials to assess the role of rt in residual disease in advanced aggressive nhl with insufficient response to systemic treatment with pet response evaluation. disclosure: nothing to declare long term outcome of patients with lymphoid malignancy who underwent high dose chemotherapy followed autologous hematopoietic cell transplantation at a single institution over years joanna romejko-jarosinska , ewa paszkiewicz-kozik , lukasz targonski , lidia popławska , jan walewski background: high dose chemotherapy (hdt) and autologous hematopoietic cell transplantation (auto-hct) is a standard of care for relapsed/refractory lymphoma patients (pts) or it is used as a consolidation for myeloma and high risk lymphoma patients in first line treatment. we retrospectively evaluated long-term outcome including late effects and risk factors in patients with lymphoid malignancy who underwent auto-hct. methods: we collected data from consecutive patients with hodgkin lymphoma (hl) (n= ), aggressive b lymphoma (dlbcl) (n= ), myeloma (n= ), indolent lymphoma (n= ), mantle cell lymphoma (n= ) and peripheral t cell lymphoma (n= ) who underwent auto-hct at our institution between and . at transplant median (range) age was ( - ) years, clinical stage iii/iv was found in of lymphoma pts, complete remission, partial remission and stable/ progressive disease occurred in ( %), ( %), ( %) pts, respectively. beam regimen was used in pts ( %), mel in pts ( %) and other myeloablative regimens in pts ( %). results %) ], respectively. partial remission or stable disease at transplant, clinical stage iii or iv, and age more than , were identified as risk factors associated with inferior os and pfs in univariate and multivariate analysis. histopathologic diagnosis was not a risk factor for os and pfs (p=ns). the outcome of patients who underwent auto-hct between - was inferior to the outcome of patients treated in - or - . - year os was %, %, %(p< . ) and year pfs was %, %, % (p< . ), respectively. we recorded ( %) cases of second primary cancer ( solid tumors and hematologic cancers). acute cardiotoxicity occurred in patients from to years after transplant, and required heart transplant in patients. patients ( %) died. the main causes of death were progressive disease in pts ( %), second primary malignancy in pts ( . %) treatment related mortality was . % ( pts), and mortality within days was ( , %). [[p image] . pfs and os in patients underwent hdt and auto-hct - , - , - conclusions: more than % of patients who underwent hdt and auto-hct had long term survival without progressive disease. older age, non-complete remission at transplant, advanced stage are associated with poor outcome. patients recently transplanted had a better outcome than patients transplanted before . disclosure: nothing to declare outcomes after haploidentical and matched related hsct in lymphoma do not differ significantly: a single center study nadira durakovic , , zinaida perić , , lana desnica , ranka serventi-seiwerth , sandra bašić kinda , ivo radman-livaja , alen ostojić , ante vulić , dražen pulanić , , pavle rončević , zorana grubić , igor aurer , , radovan vrhovac , university of zagreb, school of medicine, internal medicine, zagreb, croatia, uhc zagreb, zagreb, croatia background: allogeneic hsct still offers patients with relapsed/refractory lymphoma the best chance of long-term survival. in most such patients timing of hsct is crucial, therefore a related donor is preferred. we analyzed acute and chronic gvhd incidence, relapse and overall survival, but also time to immunosuppression (is) discontinuation and hematopoietic recovery comparing transplantation using haploidentical (haplo) and matched related donors (mrd) in single center in this indication. methods: in the time period between / and / at uhc zagreb, croatia, mrd and haplo transplantations in lymphoma were done, for hodgkin and for nhl. all patients transplanted from haploidentical donors received ptcy. data were computed using the r package. the probability of gvhd was calculated using the cumulative incidence method and subgroups were compared using the gray test. results: median age was ( - ) years; ( - ) in haplo and ( - ) in mrd group. four patients were in pr and in cr in haplo group, while in mrd group patients were in cr and in pr. in haplo group patients ( %) received bone marrow (bm) and only ( %) peripheral blood stem cells (pbsc). in mrd group all patients received pbsc. all patients in haplo group received nma ("baltimore") conditioning with ptcy while in mrd group patients ( %) received flu-bu atg, and only one received flutbi as conditioning protocol. in haplo group % patients were previously treated with autologus transplantation, % in mrd group. there was no significant difference in time to is discontinuation, and days in haplo and mrd group, respectively. patients after haplo recovered slower, recovering anc after . days ( % ci, . - . ) and . ( % ci, . - ) (p= . ) and recovering platelets after . days ( % ci, . - . ) and . ( % ci, . - . ) (p< . ) in haplo and mrd group. with a median follow up of days, overall survival was % ( % ci, - ) in haplo and % ( % ci, - ) in mrd group. trm was % in haplo and % in mrd group. cumulative incidence of agvhd ii-iv was % ( % ci, - ) and % ( % ci, in haplo and mrd group, respectively (p= . ). cumulative incidence of cgvhd requiring treatment was % ( % ci, and mrd % ( % ci, - ) in haplo and mrd group, respectively (p= . ). all cases of cgvhd developed after dli. cumulative incidence of relapse was % ( % ci, - ) and % ( % ci, - ) for haplo and mrd group, respectively (p= . ). conclusions: we found no significant difference in overall survival, relapse incidence, agvhd and cgvhd incidence between these two groups. hematopoietic recovery was slower after haploidentical transplantation, but it did not influence trm as it was higher after mrd. even though limited in number, this data contribute to the growing body of evidence that use of haploidentical donors, particularly in lymphoma setting, is as worthy as using matched related donors and should be at least second choice in donor selection, and in older patients (with older donors) probably the first one. disclosure: nothing to declare. adjuvant involved field radiotherapy post autologous stem cell transplantation for refractory/relapsed lymphomas results in favorable outcome with low toxicity: a single center experience background: involved field radiotherapy (ifrt) to previous bulky or localized residual disease, is a widely used treatment approach to minimize the risk of relapse post autologous stem cell transplantation (asct). however, the proper time for irradiation treatment remains controversial. adjuvant ifrt (adj-ifrt) in pre-asct period could cause undesirable toxicity which might delays or even cancel the asct resulting in increased risk of relapse, or could affect the marrow environmental and marrow niche resulting thus in impaired engraftment. on the other hand, the ajd-iftr in the early post-asct period, upon marrow recovery, offers a potential advantage by delivering irradiation after sufficient disease response, without affecting the engraftment. in this retrospective study we evaluated the safety and efficacy of the ifrt as adjuvant treatment in patients who had previously treated with asct for relapsed or refractory lymphomas. methods: twenty-three patients (hodgkin= , non-hodgkin= ), aged of ( - ) years, underwent asct, for primary refractory (n= ) or relapsed (n= ) disease. patients who had bulky disease at the time of relapse or those with residual mass post salvage treatment, were considered as candidates for adj-ifrt, early (within - months) after documentation of autologous stem cells engraftment. all patients proceeded to asct with chemosensitive disease after a median of lines of salvage therapy. at the time of asct patients ( %) had residual disease while ( %) evaluated to be in complete remission. the preparative regimens were: single-agent melphalan (n= ), busulfan-etoposide-melphalan (n= ), beam (n= ) and bendamustin-etoposide-cytarabine-melphalan (n= ). filgrastim was given till neutrophills recovery, while prophylaxis against bacteria, fungus, viruses and pcp were administered till the completion of adj-ifrt. results: all patients engrafted promptly and successfully. no patient experienced any severe toxicity or active infection before adj-iftr. though our plan was to proceed with adj-ifrt within months post asct, finally it was delivered after a median of . ( - ) months; the median radiation dose was ( - ) gy. ten patients received radiotherapy in the mediastinum, in the abdomen/pelvis/ inguinal area in the neck, and in the left leg. the adj-ifrt was well tolerated. no patient experienced toxicity grade > and none required hospitalization. currently, after a median follow-up of ( - ) years, / patients are alive and well; the -years overall and progression free survival rates are % and % respectively. four patients died; due to relapsed disease and heavily pretreated patients due to secondary myelodyspalstic syndrome conclusions: in our study, the adj-ifrt in the early post transplant period demonstrated a safe and well-tolerated profile. taking into consideration the poor risk status of our patients (residual disease post salvage regimen or bulky disease at the time of relapse), the promising overall and progression free survival rates suggested that adj-ifrt post asct is also an effective approach. well designed trials are needed to clarify the role and the appropriate time of radiotherapy in the asct setting disclosure: no conflict of interest adverse prognostic impact of pre-transplant neutrophil/ lymphocyte ratio in lymphoproliferative disorders background: brentuximabvedotin(bv) is a chimeric anti cd igg antibody, conjugated to synthetic antitubulinmomomethylauristatin. bv is approved for the treatment of classical hodgkin lymphoma (hl) in relapse either after autologous stem cell transplantation (asct) or after two lines of combination chemotherapy in transplant ineligible patients. the aethera trial revealed increased pfs when bv is used as maintenance therapy for cycles in high risk patients after asct. however, this schedule is associated with a high cost and significant toxicity particularly in term of peripheral neuropathy. our primary objective is to assess the efficacy of cycles brentuximab as consolidation therapy after asct for relapsed/refractory (r/r) hl. secondary objectives include side effects, progression free survival (pfs), and overall survival (os). methods: this is a retrospective single center analysis approved by the irb of the american university of beirut medical center. we included in this study consecutive patients with r/r hl who underwent asct between and , and received bv consolidation post-asct. results: we identified consecutive adult patients with r/r hl treated with bv . mg/kg iv every weeks as consolidation therapy after asct. the indications for bv consolidation was primary refractory disease in patients ( %), early relapse in patients ( %) (after a median time of months; range, - ) andextranodalinvolvement in one patient ( %). the median number of lines of therapy pre-asct was (range, - ). the median time to bv initiation post-asct was days (range, - ). patients received a median of cycles (range, - ) of bv post-asct. after a median follow up of months (range, - ), five ( %) patients relapsed after asct. the median time to relapse was months (range, - ). median pfs and os were not reached. we did not observe any significant toxicities during or after therapy. conclusions: cycles of bv consolidation after asct seem to be safe and effective in preventing relapse, however our findings need to be confirmed with larger prospective studies. chemotherapy or who progress after autohsct is poor. despite introduction of novel agents like brentuximab vedotin (bv) or nivolumab, allohsct appears the most effective treatment option with curative potential. the goal of this study was to evaluate efficacy of allohsct for hl, including patients pre-treated with novel agents. methods: between years - , patients (including males) with hl were treated with allohsct in msc institute of oncology in gliwice, poland. median age was ( - ) years. median lines of preceding chemotherapy was ( - ); ( %) patients had been pre-treated with autohsct, ( %) with radiotherapy, ( %) with bv, ( %) -with nivolumab. disease status at allohsct was as follows: cr- , pr- , nr- . patients were treated with hsct from either hla-matched sibling donor (msd, n= ), unrelated donor (urd, n= ) or haploidentical donor (n= ). conditioning was myeloablative in ( %) cases. peripheral blood was used as a source of stem cells. results: all but one patient engrafted with median time of neutrophil recovery of ( - ) days. the incidence of grade - and grade - acute gvhd was % and %, respectively, while the incidence of chronic gvhd was %. the probabilities of os and pfs at years were % (+/- %) and % (+/- %), respectively. the incidences of progression and transplant-related mortality were % and %, respectively. the y pfs rates were % for msd, % for urd and % for haploidentical donors. in a univariate analysis pfs was affected by recipient gender (female - %, male - %, p= . ) and disease status at allohsct (cr - %, pr - %, nr - %, p= . ). in a multivariate model the disease status other than cr was the only factor associated with increased risk of treatment failure (reverse pfs) -hr= . , %ci . - . , p= . . neither donor type nor conditioning affected long-term outcome. conclusions: results of allohsct for patients with relapsed/refractory hl are determined by disease status at transplantation. efforts should be done to reduce tumour burden before transplantation, optimally to achieve cr. disclosure: nothing to declare background: brentuximab vedotin (bv), nivolumab and pembrolizumab have been assigned to chemorefractory hodgkin lymphoma treatment. impact of these agents on disease-free-survival after autologous stem cell transplantation (asct) remains under investigation. aim of the study is to compare bv-and nivolumab-treated patients with a control group. methods: clinical characteristics and outcomes of chemo refractory hodgkin lymphoma patients who underwent asct during - . results: a total of patients ( men; women, median age years old, - ) were treated with bv: pre-transplant, post-transplant and pre-and posttransplant. pre-transplant bv patients had primary refractory disease or early relapse in the majority ( %). post-transplant treatment occurred in the context of relapsed/refractory disease in patients; ( %) had an allogeneic stem cell transplant. among them, had additional chemotherapy and nivolumab, gaining a complete metabolic response. in the rest of patients, change of treatment due to eventual bv failure occurred. bv was administered as a maintenance treatment in patients. in six of them bv had already been administered pre-transplant as well. out of maintenance treatment patients, relapsed and subsequently received nivolumab. two patients died due to prior chemotherapy complications, whereas are currently on nivolumab treatment. pet-based response was available in patients, having a complete metabolic response (cmr) and a partial metabolic response. stable disease was achieved by ctbased response in the rest patients. no major toxicities were observed. one patient presented with grade asymptomatic hypothyroidism and one with grade anemia attributed to non-inflammatory upper gastrointestinal blood loss. in total, patients received anti-pd treatment, all post bv failure. with a median follow-up of . ( . - . ) months, -year overall survival (os) was . % in patients treated only with bv compared to . % in patients treated with additional anti-pd treatment (p= . , figure) . median os for patients treated only with bv was . months, whereas median os has not been reached for patients that received anti-pd treatment. conclusions: bv pre or post-transplant and anti-pd treatment post-transplant after bv failure have outstanding results in chemo refractory lymphoma patients. treatment sequence in allogeneic transplantation eligible patients remains to be further studied. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) with reduced-intensity conditioning (ric) has been used in heavily pretreated lymphoma patients with the promise of decreased treatment-related mortality. despite improvements in outcomes of patients with lymphoid neoplasms, several new agents emerge as potential therapies. therefore, we aimed to describe our long-term experience in patients with hodgkin (hl), non-hodgkin lymphomas (nhl) and chronic lymphocytic leukemia (cll) post allo-hct. methods: in this retrospective study, we enrolled consecutive patients who underwent allo-hct for lymphoid neoplasms in our institution ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: in total, patients (male:female= : ) aged ( - ) years, underwent allo-hct for hl (n= ), nhl (n= ) and cll (n= ). the majority of patients were diagnosed at stage iv ( %); % had bone marrow involvement and % had undergone autologous hct. most patients were heavily pretreated (median lines= , range - ), of them had received more than treatment lines and at the time of transplantation only had complete response, while had partial response and were refractory. according to disease-risk index (dri), patients were stratified at low (n= , . %), intermediate (n= , . %), high (n= , . %) or very high (n= , . %) category. among patients with hodgkin lymphoma, brentuximab vedotin was administrated in and of them were effectively bridged to allo-hct. all patients received ric, mainly fludarabine ( mg/ m )-cyclophosphamide ( g/ m ) in cll and nhl and thiotepa ( mg/kg)-fludarabine ( mg/m )-cyclophosphamide ( mg/kg) in hl from matched sibling (n= ), matched (n= ) or mismatched unrelated (n= ) donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine or tacrolimus and mycophenolate mofetil or short-term methotrexate and additional low-dose antithymocyte globulin ( mg/kg) in unrelated donors. peripheral blood was the main cell source ( / ) and median number of cd + cells infused was . x /kg ( . - . ) . two patients succumbed to advanced underlying disease before engraftment; while the other engrafted successfully. median time until neutrophil and platelet engraftment was and days respectively. eighteen patients ( . %) developed acute gvhd (grade iii-iv, n= ), steroid sensitive in ( . %) and relapsed. one-year cumulative incidence (ci) of extensive chronic gvhd was . %, and patients required more than one additional line of immunosuppression (range - ). ten patients presented cmv reactivation successfully treated with antiviral medication and patient died from hsv encephalitis. with a median follow of years ( - years), -year os was . %, -year non-relapse mortality ci . % and year dfs %. there was no difference in survival according to original disease ( -year os for nhl= . %, hl= . %, cll= %%, p= . ). multivariate analysis revealed high and very high dri as the single predicting factor of os (hr . , ci . - . , p= . ), when assessing impact of disease, dri, prior treatment lines, gender and bone marrow infiltration at diagnosis. conclusions: our data suggest that ric allo-hct provides encouraging survival rates, potentially offering the chance of cure, with acceptable long-term mortality in selected high-risk patients with lymphoid neoplasms. dri that is mainly associated with disease stage at transplant independently affects survival. therefore, continued efforts are necessary for clinical application of novel agents aiming to lower disease stage pre-transplant. disclosure: nothing to declare results: six pts were identified, with a median age of years at diagnosis: five with hl nodular sclerosis and with lymphocyte depletion. the median number of therapeutic lines prior to allo-hsct was [ - ]; four pts were previously treated with brentuximaband two pts had been submitted to high dose chemotherapy with autologous bone marrow support. at the time of allo-hsct, pts had progressive disease (dp), was in partial response and in complete response (cr). five allo-hsct were performed with a related donor, of wich were haploidentical ( parents, sibling and descendant) and with an unrelated donor ( / ). prophylaxis for gvhd was performed with tacrolimus and mycophenolate mofetil (with post-transplant cyclophosphamide in haploidentical allo-hsct). on day + evaluations, pts had a cr and patient (pt) had dp. the median time to relapse after allo-hsct was of months. at the time of initiation of nivolumab, pts were under steroid therapy for disease control, without other immunosuppressive therapy. the median time between allo-hsct and the beginning of nivolumab was months. the initial dose was mg / kg (associated with corticosteroid therapy), escalated up to mg/kg biweekly, according to patient's tolerance. after the start of nivolumab, patients, with previous gvhd manifestations, presented a worsening of the cutaneous gvhd, which required an escalation of immunosuppressive therapy. as toxicity, pt had a grade pneumonitis, pt had a grade encephalitis/hypophysitis, pt had a grade pancreatitis, pts had headache (grade and ), pts had a grade - cutaneous reation. with a median follow-up of months since nivolumab treatment, the overall response rate was %: pt obtained cr and pts partial remission. nevertheless, there were deaths after the onset of nivolumab: pt at monts with dp and another one due to acute myocardial infarction at months. at the time of analysis, pts maintained response under nivolumab treatment (median cycles ) and pt had therapy suspended because of toxicity. conclusions: these results demonstrate the high probability of achieving response with nivolumab treatment in patients with rr-hl relapsing after allo-hsct, but adverse events of grade were frequent in this small group, and the treatment toxicity was significant. disclosure: nothing to declare background: intravascular large b-cell lymphoma (ivlbcl) is a rare form of large b-cell lymphoma with pathological findings of intravascular proliferation and/or sinusoidal involvement of lymphoma cells. according to their geographic distribution, ivlbcl could be dichotomized into asian and western variants. compared with the western variant, where skin involvement was common, the asian variant was reported to involve more frequently the liver, spleen and bone marrow, and hemophagocytic lymphohistiocytosis is more common in asian variant. diagnosis of ivlbl is still difficult because of the lack of overt lymphadenopathy and peripheral blood involvement. thus, timely diagnosis and immediate treatment remain as a challenge to improve outcomes for patients with the asian variant. therefore, we analyzed the clinical features and treatment outcomes of patients with the asian variant of ivlbcl. methods: we analyzed patients who were diagnosed with ivlbcl between and . all patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop). results: forty-six patients were diagnosed with ivlbcl, and their median age at diagnosis was years (range: - years). male patients predominated (n= , %), and b symptoms were present in patients ( %). hepatomegaly and/or splenomegaly were observed in patients ( %), whereas lymphadenopathy was less common (n = , %). bone marrow and liver were the most commonly involved extranodal organs ( %, and %, respectively) and were the most common sites of biopsy for diagnosis in this study. all patients received r-chop as a first-line treatment after diagnosis with a median number of six cycles (range one to eight). at the end of treatment, patients achieved a complete response (cr), whereas eight patients showed progression. six patients died after the first or second cycle of r-chop, and the causes of death were treatment-related adverse events including cytopenia, infectious complications, and pulmonary hemorrhage. upfront asct was done for two patients including one patient with cns involvement at diagnosis, and these patients were still alive at the time of analysis without evidence of relapse. on the other hand, the outcome of six patients undergoing salvage asct after relapse was poor; thus, only one patient was alive. likewise, patients with disease progression at the end of treatment with r-chop showed dismal prognoses even after salvage chemotherapy except for one. at a median follow-up of . months ( % confidence interval, ci . - . ), the median overall survival was . months ( % ci . - . ). the treatment outcome of patients with the asian variant of ivlbcl is still not satisfactory. although upfront autologous stem cell transplantation might be effective for selected patients at high-risk of relapse, its role is still not clear, either. thus, further study should be warranted to develop more effective strategies for diagnosis and treatment. clinical trial registry: not applicable disclosure: nothing to disclare background: peripheral t-cell lymphomas (ptcls) are about % of non-hodgkin´s lymphomas usually with an aggressive clinical course and unfavorable prognosis.given their heterogeneity, consensus on the best first-line treatment and the role of autologous/allogeneic (asct/allosct) stem cell transplantation as consolidation is controversial. methods: we evaluated the overall survival (os), progression-free survival (pfs) and toxicities of a cohort of patients with ptcls submitted to asct/allosct intensification at our institution between january and july . os was calculated from the date of diagnosis until death. pfs was measured from transplant until relapse, progressive disease or last follow-up. os and pfs rates were estimated using the kaplan-meier method and compared with the log-rank test. results: twenty-six patients were identified, female ( %), median age was years (range: to ). ninetytwo percent of patients presented with advanced-stage disease at diagnosis (ann arbor stage iii or iv), % with b symptoms. according to the who classification, histologic ptcl subtypes included angioimmunoblastic tcell lymphoma (n = ); ptcl not otherwise specified (n = ); anaplastic large cell lymphoma, alk-negative (n = ); anaplastic large cell lymphoma, alk-positive (n = ); nodal peripheral t-cell lymphoma with tfh phenotype (n = ). extranodal nk/t-cell lymphoma, nasal type and primary cutaneous subtypes were excluded. the ageadjusted ipi (aaipi) was low/intermediate low in patients ( %) and intermediate high/high in patients ( %). twenty-seven transplants were performed ( asct, allosct); were consolidation in st response ( asct and allosct) with in complete remission (cr) and in partial remission (pr). nine transplants were performed as consolidation of nd response ( asct and allosct) with in cr and pr. in patient allosct was performed after asct, due to early relapse (< months). beam regimen was used in asct as conditioning and flumel in allosct. all patients engrafted, the median time to leukocyte recovery > , /μl was days (range, to ). four of the pts ( %), submitted to allosct had chronic graft versus host disease which was the most relevant complication of this analysis. considering the whole cohort, the median follow-up was . months (range, to ). the estimated -year os and pfs were % and %, respectively. seven patients relapsed ( early) all after asct, there were no relapses after allosct, however, the results were not statistically significant between the allosct and asct groups; the -year os rates were % and % (p = , ) and the year pfs rates were % and % (p = , ) respectively. for the all patients treatment-related mortality (trm) was , %; patients died, with progressive disease (asct) and for hepatic toxicity (allosct) before d+ . conclusions: the results of this retrospective study, taking into account the adverse risk profile of the population, suggest that autologous/allogeneic stem cell transplantation as an effective and safe option for the consolidation of patients with ptcls. these results need to be validated in prospective studies, including a larger number of patients. disclosure background: autologous stem cell transplantation is used as consolidation therapy in relapsed lymphoma patients. however, outcome of lymphoma patients relapsing after autologous stem cell transplantation is poor and allogeneic stem cell transplantation which can be curative is used in the transplant eligible patients in this setting. besides, allogeneic stem cell transplantation can be an option before autologous stem cell transplantation in some high risk patients. in this study, we aimed to compare the survival rates of lymphoma patients older than years of age and patients aged - who had undergone allogeneic transplantation in our center. methods: we collected the data of lymphoma patients older than years of age who had undergone allogeneic transplantation in our center and analyzed the results by grouping them into , namely the ones between - years of age and the ones over years of age. [[p image] . figure results: there were patients over the age of who had undergone allogeneic stem cell trasplantation with the diagnosis of lmphoma between and . of these patients were over years of age. patients had non-hodgkin lymphoma and patients had hodgkin lymphoma. the characteristics of the patients are summarized in table . patients' comorbidity indexes were calculated with augmented hct-ci which includes patients' pretransplant ferritin, albümin and thrombocyte counts as a variable. no difference could be found between groups regarding neutrophil and platelet engraftment times and comorbidity indexes. however, acute graft versus rate and documented bacterial infection rate during the hospitalization period were higher in the - years age group (p= , ). day mortality rate and non-relapse mortality rate were not different between groups. more importantly, progression free survival(pfs) and overall survival(os) of patients in the - years age group and over years of age group were not different (p= , ) (figure ) conclusions: in the present study, although the number of patients is low, we showed that lymphoma patients over years of age have similar outcomes and transplant related toxicity as the patients between to years of age. pfs and os were very close in this study. we think that this may be due to low relapse rate in the patients and high mortality rate in relapsing patients. in conclusion, allogeneic stem cell transplantation which has a curative potential may be employed in transplant eligible elderly lymphoma patients disclosure: nothing to declare background: follicular lymphoma (fl) histologic transformation consist on the development of an aggressive lymphoma, usually a diffuse large b cell lymphoma (dlbcl). histological transformation has been considered to have poor prognosis. in pre-rituximab era median os ranged between and years, however, in recent series of patients treated with chemotherapy plus rituximab, the outcome of transformed fl has improved, especially in those that receive autologous stem cell transplantation (asct), who reach -year os up to % in some series. methods: we have retrospectively studied consecutive patients undergoing asct for transformed fl between and in a tertiary center in the basque country, spain. patients were considered to have a transformed fl if they were diagnosed of a dlbcl and they have previous history of fl or histological evidence of a fl in another location. these patients were compared to a retrospective cohort of dlbcl patients with high ipi or stage that received asct in first remission according to our institution strategy. pfs and os were calculated from the time of the asct. in the case of transformed fl, both relapses of the aggressive or indolent lymphoma were considered. survival analysis was performed with kaplan-meyer estimator results: a total of transformed fl and dlbcl patients were studied, with a median follow up of . and . months respectively. patient characteristics are described in table . -year pfs was % in transformed fl and % in dlbcl, and -year os was % and %, respectively (picture ). there were no significant differences in pfs or os between this two groups (p = . ). in both groups all relapses occurred in the first three years after asct. among the patients with transformed fl relapses were observed. five of them ( %) were aggressive relapses, while only one patient presented relapse as an indolent lymphoma (fl histological grade a with an aggressive clinical course). [[p image] . image : transformed fl and dlbcl pfs after asct] conclusions: in our experience, asct in transformed fl offers good results, similar to those in dlbcl. fl presents a natural course akin to that of dlbcl, with relapses occurring early and survival reaching a plateau. this data suggests that some patients with transformed fl can be cured after asct. disclosure: nothing to declare. safety and efficacy of intensive preconditioning regimen containing cladribine in autologous peripheral blood stem cell transplantation of refractory and relapsed young highly invasive lymphoma background: autologous peripheral blood stem cell transplantation (apbsct) is one of the main treatments for patients with non-hodgkin's lymphoma (nhl). effective and safe conditioning regimens can improve the cure rate of nhl. beam is the most common pretreatment scheme, but for refractory and relapsed young highly invasive lymphoma, especially for dual-expression dlbcl, pretreatment needs to be strengthened. studies have shown that the cladribine (clad)+gemcitabine (gem)+busulfan (bu) combination provides synergistic cytotoxicity in lymphoma cell lines.we evaluated the the safety and short-term efficacy of intensive preconditioning regimen containing cladribine (clad+gem+bu) for refractory and relapsed young highly invasive lymphoma undergoing apbsct. methods: ten patients with nhl received apbsct. ca)ctx+ ara-c) therapy followed by g-csf was used for pbsc mobilization. sevenr patients received conditioning regimens of beam(beam group): bcnu mg/ m ·d - × d (- d), vp mg/m · q h× d (- d-- d), ara-c mg/m ·q h× d (- d-- d), mel mg/m ·d - × d (- d). three patients received intensive preconditioning regimen containing cladribine (clgb group): clad mg/m ·d - × d (- d-- d), gem mg/m ·d - × d (- d, - d), bu . mg/kg q h× d (- d-- d). follow-up date expires on december , . results: the age of patients in clgb group was , and years, respectively. two patients were diagnosed as diffuse large b-cell lymphoma with double expression and one was diffuse large b-cell lymphoma with two recurrences. the patients of beam group were all high-risk, relapsed and refractory nhl.all patients were successfully engrafted after infusing apbsc. the average lowest leukocyte in clgb group and beam group were ( . ± . ) × /l vs ( . ± . )× /l, respectively. the average lowest leukocyte in clgb group was lower than that in beam group. the average time to anc < . × /l in clgb group and beam group were . d ± . d vs . d± . d. the average time to anc≥ . × /l in clgb group and beam group were . d± . d vs . d± . d; the average time to plt≥ × /l of clgb group was not different to that of beam group ( . d± . d vs . d± . d) the average time of neutropenia wasn't significantly different in two groups ( . d± . d vs . d± . d). the adverse reactions of gastrointestinal tract and oral mucosa were close in tow groups.vod, hemorrhagic cystitis, pretreatment-related interstitial pneumonia, liver and kidney dysfunction were not happened in tow groups. the rate of infectious fever was close in two groups ( / vs / ). the median followup period in beam group was ( ~ ) months. in the beam group, a patient died days after transplantation, because he was diagnosed with recurrent nkt cell lymphoma and intracranial infection caused by severe sinus infection. another case of beam group was diagnosed as double-expressed dlbcl, which relapsed months after transplantation. the remaining patients in ebmt group survived disease-free. the follow-up time of patients in clgb group were months, months and months respectively. all patients survived without disease.however, the follow-up time is short and needs long-term follow-up. conclusions: the treatment of intensive preconditioning regimen containing cladribine (clgb) for refractory and relapsed young highly invasive lymphoma undergoing apbsct is safe. the time of hematopoietic reconstruction is short, and the adverse effects is tolerable for patients with refractory and relapsed young highly invasive lymphoma. the current short-term outcome is good, but the long-term effect need a longer time to follow-up. disclosure: this work was supported by national nature sciences found of china ( ). there is no disclosure of conflict of interest.the all authors name: xiang-li chen, yu-zhu zang, wen-hui zhang, yin zhang, zhong-wen liu, ping-chong lei, jing yang, yu-qing chen, kai sun. background: small part of children with hodgkin disease (hd) demonstrate initial resistance to the standard and even "salvage" chemotherapy and need innovative drugs for the treatment. methods: a -year girl was diagnosed with classical hd (nodular sclerosis)corresponding to stage ii e b (fever > °c) in april .after two cycles of oepa (vincristine, etoposide, prednisone and doxorubicin) and next two cycles copp (cyclophosphamide, vincristine, prednisone and procarbazine) the patient again had progressive disease. as the patient achieved a partial response (pr) after "salvage"therapy with two cycles of igev (ifosfamide, gemcitabine, vinorelbine, and prednisone), she received auto-sct in february (patient status before auto-sct was pr). we used ccnu-containing conditioning regimen cem: lomustine (ccnu) mg/m + etoposide mg/m + melphalan mg/m . at day + after auto-sct, the patient again demonstrated progression of the disease: pet/ct-examination showed mediastinal tumor mass enlargement with increased left lung nodule simultaneously to short metabolic activity. patient was under observation. at day + the disease had relapsed and progressed -examination by pet/ct demonstrated multifocal progressive disease with multiple pulmonary lesions and increased metabolism in comparison with the previous pet/ct scan. in july-october , the patient had salvage chemotherapy with a combination of brentuximab vedotin (bv) (bv dhap (dexamethasone, cytarabine, cisplatin) + bv (without chemotherapy due to suspected invasive mycosis) + bv dhap), however, only partial pet-positive remission was achieved. because of many times relapsed and progressed disease pembrolizumab therapy was started in october : mg / kg every three weeks four cycles totally. toxic effects and serious complications during and after therapy by pembrolizumab were not observed. in february , after pembrolizumab # , the patient showed complete metabolic remission of the disease by control pet-ct. in april , the patient received haplo-sct with post-transplant hd-cyclophosphamide. we used conditioning modes with reduced toxicity (fludarabin mg/m + treosulfan g/m ), high doses of cy ( mg / kg) on days + and + . tacrolimus and mycophenolate mofetil started on day + . mmf was terminated on day , tacrolimus -on day . patient did not have acute and chronic gvhd. results: at the moment the patient is alive and still in pet-negative cr with duration more than mo. conclusions: pembrolizumab has demonstrated high activity against resistant hd even after post-auto-sct progression with good tolerability for the sick child. disclosure: nothing to declare p high dose chemotherapy followed by autolougous peripheral blood stem cell transplantation (asct) in diffuse large b cell lymphoma (dlbcl) median age is , years ( to ) and sex ratio (m/f) . ; ann arbor stage iii-iv: pts. before hdt induction chemotherapy (chop, c h opa) was instituted and associated with rituximab in pts ( , %), pts ( , %) received more than treatment lines and pts ( , %) received complementary radiotherapy. transplant disease status before hdt was complete remission (cr) in pts, partial remission in pts (rp) and disease progression in pts. the delay from diagnosis to hdt is , months ( - ). the hdt protocols used are: tutshka : pts, tutshka+vp : pts, bam (busulfan +cytarabine+melphalan) : pts et beam : pts. all pts received, after thawing, mobilized pbsc obtained by g-csf mobilization ( μg/kg/d, days) alone and froze in liquid nitrogen. the median rate cd + cells infused is , x /kg ( . - . ) . the median follow-up at / / is months . results: the median time to graft (pnc > . x /l) was days ( - ). ten early deaths were observed including infection (trm: , %) and in disease progression at months. after months of hdt pts are assessable including pts in cr ( , %) and pts in pr ( , %). relapse was observed in pts ( . %) and it was earlier relapse in a period of months in pts ( %). deaths were among / pts ( %). persistent cr was achieved in / pts ( , %) including / ( , %) mlcl and / ( , %) others dlbcl. the overall survival (os) and event free survival (efs) at years are respectively % and %. the os and efs are better in patients who received rituximab in initial therapy : os ( % vs %; p< , ) et efs ( % vs %; p< , ). herein, we present one patient with refractory mcl, who were insensitive to chemotherapy and then experienced a dramatic improvement with ibrutinib mono-therapy but later developed ibrutinib resistance,ultimately resulting in the deterioration of disease and death. methods: we give the patient several examinations including ultrasound, bone marrow biopsy, lymph node biopsy, exome sequencing, sanger sequencing, and so on. for the treatment of lymphoma, the patient received chemotherapy, including course of chop(cyclophosphamide . g day , doxorubicin mg day ,vinorelbine mg day , and dexamethasone mg from day to ) and course of r-dhap (rituximab mg day , cytarabine g q day , cisplatin mg day ,dexamethasone mg from day to )in succession.because of the failure to control disease progression, ibrutinib mg qd was used until the patient died. results: the -year-old man initially referred to our hospital for complaints of abdominal pain and distention over months. ultrasound showed splenomegaly and multiple enlarged retroperitoneal lymph nodes.excisional biopsy conducted on the right neck lymph node revealed the presence of malignant cells.immunohistochemically, the neoplastic cells were positive for bcl , bcl ,cd , cd , cd a, cd , ki- ( %), sox , cd (fdc) and cyclin d and negative for cd , cd and cd ; fluorescence in situ hybridization(fish) showed igh/ ccnd ,t( ; ) %.thus a diagnosis of mcl was confirmed. course of therapeutic chemotherapy were applied to the patient but he did not respond well.he suffered recurrent fever, thrombocytopenia, left abdominal pain, splenomegaly and multiple enlarged lymph nodes. then he received ibrutinib mono-therapy, and experienced a dramatic improvement as his body temperature was controlled, his hemogram became normal and his spleen and lymph node tapered.after about months response of ibrutinib, the disease deteriorated rapidly and he died very soon. exome sequencing from the patient peripheral blood at this time detected one missense mutation in exon of tp at nucleotide g>a, resulting in an argnine to histidine change at amino acid (p.arg his). but sanger sequencing of the patient bone marrow ffpe sample at the time of original diagnosis did not detect this mutation. conclusions: thus, our study reported a tp r h mutation mcl patient who developed ibrutinib resistance and progressed aggresively, which may open new insight for future effort for alternative therapeutic strategies in ibrutinib-refractory mcl. disclosure: nothing to declare. minimal residual disease, tolerance, chimerism and immune reconstitution peripheral blood samples were obtained for routine analysis at several time points after hsct. all available blood samples between . and years were used in the analysis. to assess changes in the cd +b and cd +cd +memory b cell counts over time while accounting for the correlation between the repeated measurements of each patient, we used linear mixed-effects models. wilcoxon rank test, kruskal-wallis test and linear regression were used for univariate analysis. results: at one year after hsct, univariate analysis showed that patients transplanted with a cb graft compared to bm and pbsc had a significantly higher absolute number of b cells (median bm= , median cb= , median pbsc= cells/μl, p= . e- ) and memory b cells (median bm= , median cb= , median pbsc= cells/μl, . recipients with age under years had significantly higher absolute numbers of b (median= , median= cells/μl, p= . e- ) and memory b cells (median= , median= cells/μl, p= . e- ) than above years. increase in donor age was associated with a decreasing effect on b cell (r = . , p= . e- ) and memory b cell (r = . , p= . e- ) reconstitution as determined in regression analysis. following univariate analysis, we analysed these factors in a mixed effects model to assess the relation with differences in b cell or memory b cell numbers . - years after hsct. in our analysis we found significant decreasing b cell and memory b cell numbers with increasing donor age corrected for recipient age and source (both p< . ). increasing recipient age also showed a significant decrease in b cell and memory b cell numbers (both p< . ) but there was no significant influence of donor source ( figure ) . conclusions: b cell and memory b cell numbers after hsct are influenced by donor and recipient age but not by donor source. older donors and recipients show a decrease in b cells and memory b cells numbers . - years after hsct. [[p image] . figure . b cell development and donor age. green shows cb, red bm, blue pbsc at mean donor age.] copenhagen university hospital rigshospitalet, copenhagen, denmark, leiden university medical center, leiden, netherlands background: the outcome of allogeneic hsct is challenged by a delayed and long-lasting imbalanced t-cell reconstitution increasing the risk of acute gvhd, infections and disease progression. although the role of differentially and functionally distinct t-cell subsets in the development of complications has been addressed, little is known about the factors controlling their recovery. in this study, we investigated the impact of immuneregulating and homeostatic cytokines on the reconstitution of functionally distinct t-cell subsets and associated clinical outcomes. methods: we included children undergoing allogeneic hsct for all (n= ) or aml (n= ) with a median age of . years (range: . - . ). donors were either mrd (n= ), mud (n= ) or mmud (n= ). bm (n= ) or pb (n= ) were used as stem cell source. conditioning regimens were based on tbi (n= ) or highdose chemotherapy alone (n= ) and included atg in patients. thirty age-matched healthy children were included as controls. cytokines (il- , il- , il- , scf, il- , il- and tnfα) and active atg in plasma were longitudinally measured from before conditioning until months after hsct (n= ) along with an extended phenotyping of t-cell maturation and differentiation by flow cytometry (n= ). results: the homeostatic cytokines il- and il- increased from pre-conditioning to peak - weeks post-hsct and gradually declined thereafter. il- levels were shortly elevated, while il- and scf remained relatively stable, and il- and tnf-α levels were below threshold of detection at all time points. the early rise of il- and il- was strongly associated with the degree of t-cell depletion by atg, while il- also correlated with markers of systemic inflammation. il- and il- levels were significantly higher in children treated with atg (p< . ) and correlated with both longer exposure to atg (p< . ) and increased levels of active atg (day + : il- : r= . , p< . ; il- : r= . , p< . ), indicating that high levels of these cytokines reflected more pronounced t-cell depletion during lymphopenia. higher circulating levels of il- and il- were associated with a slow recovery of cd +, cd + and cd + t-cell counts at day + and + post-hsct (p< . ), while the remaining cytokines did not correlate with immune reconstitution. looking into t-cell subpopulations, increased levels of il- and il- during the first month post-transplant were associated with lower numbers of naïve t cells and correlated with an increased proportion of cd + and cd + effector memory cells ( figure) . no differential effect of cytokines on polarization of cd + t cells into th , th , th cells or treg cells was found. in atg-treated patients, il- and il- levels at day + were significantly lower in patients developing acute gvhd grade ii-iv (p= . and p= . , respectively). in the total cohort, increased il- levels were associated with a reactivation of ebv (p= . ). conclusions: these findings suggest that quantification of il- and il- can be indicative for the degree of t-cell depletion during the first weeks after hsct and predictive of complications. overall, these results indicate that the lymphopenia-induced elevation of il- and il- is a major driver of the initial expansion of donor t-cells. background: mathematical kinetic models were adopted to study immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-hsct). the associations between acute graft-versus-host disease (agvhd), relapse and the immune cell reconstitution kinetic models were explored. methods: from june , to may , , sixty-five patients with hematological malignancies after allo-hsct were recruited. peripheral blood was collected on + day, + day, + day and in + month, + month, + month, + month, + month, + month, + month. lymphocyte subsets were determined by flow cytometry, including in total t lymphocytes (cd + ), helper t cells (cd + cd + ), cytotoxic t cells (cd + cd + ), cd /cd ratio, nature killer (nk) cells (cd -cd + ), nkt cells (cd + cd + ), b lymphocyte (cd + ), naive t cells (cd + hla-dr + ), static t cells (cd + hla-dr -), and regulatory t cells (cd + cd high foxp + ). mathematical kinetic models were calculated for immune cell reconstitution with spss. results: after allo-hsct, a logarithmic curve model was observed for cd + t cells. cubic curve models were observed for cd + cd + t cells, cd + cd high+ foxp + t cell, cd + hla-dr -t cells, cd + cd + nkt cells, cd + b cells. cd + cd + t cells, cd + hla-dr + t cells, and cd -cd + nk cells showed s type curve models. considering t cells were the major mediators for agvhd and graft-versusleukemia effect after allo-hsct. with established immune cell kinetic models, we found that different curve models were observed between patients with and without agvhd after allo-hsct. although the kinetic models were almost the same for leukemia-free and relapsed patients in the first months after allo-hsct, significantly different kinetic curves could be observed thereafter. conclusions: the immune cell reconstitution showed different mathematical curve models after allo-hsct. kinetic reconstitution model of certain immune cell was associated with agvhd and relapse. hence, mathematical kinetic models of immune cell reconstitution may be potential indictor for predicting agvhd and relapse after allo-hsct. disclosure: nothing to declare lineage specific chimerism analysis in pediatric patients following allogeneic hematopoietic cell transplantation (hct background: the outcome of allogeneic hct is dependent on several variables that include patient age, disease and stage, cytoreduction, graft, graft manipulation, and graft versus host disease (gvhd) prophylaxis. one aspect of hct that remains poorly defined and studied is the donor/ host (d/h) chimerism post hct. since , we followed patients with d/h lineage specific chimerism post hct. analyses were performed by short tandem repeat (str) polymorphism analysis at the american red cross blood services (philadelphia, pa). studies were performed on blood total leukocytes, myeloid/neutrophil cells, t-cells, bcells, and nk-cells. methods: in this retrospective study, the charts of consecutive patients who underwent allogeneic hct between january to june on the pediatric bone marrow transplant service at mskcc were retrospectively reviewed. lineage specific donor chimerism post hct was studied including d/h chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and t-cell depletion (tcd). preliminary analyzes performed on this cohort included wilcoxon rank test and cox proportional hazard analyses. results: patients were selected based on the number of analyses. the median age was . years. patients had hematologic malignancies (n= ) or non-malignant hematologic diseases (n= ), or immune disorders (n= ). cytoreduction included tbi-(n= ), or chemotherapybased regiments (n= ). patients were recipients of t-cell depleted marrow or peripheral blood grafts (n= ), unmodified marrow or peripheral blood grafts (n= ) or cord blood grafts (n= ). full donor chimerism of myeloid cells, b-cells and nkcells, but not t-cells occurred early post-transplant. there was no difference in the percentage of total donor leukocytes at months vs. months post hsct (n= ), while the median of donor t-cell chimerism was % at months and % at months post hsct (p< . , n= ). figure shows the impact of different factors including: (a) the use of tbi-or chemotherapy-based regimens, (b) age (< or > years), and (c) type of graft (t-cell depleted vs unmodified vs cord blood). donor total leukocytes chimerism was significantly lower at months as compared to months for patients < years of age (p= . ). for most grafts, full donor chimerism of t-cells occurred early, while for t-cell depleted transplants, it took up to one year to complete. cord blood grafts were associated with high t-cell donor chimerism throughout the post-transplant period. there was a significant difference in the % donor t-cells at and months post hct when comparing t-cell depleted and unmodified grafts (p= . ). conclusions: this preliminary analysis of lineage specific chimerism post-transplant showed that donor tcells may take one year to fully recover post-transplant, mostly following t-cell depleted grafts, without intervention. cord blood grafts were associated with high donor chimerism throughout the post-transplant period. lastly, factors associated with loss of donor chimerism posttransplant were younger age and non-malignant disorders. more in-depth analyses are being performed including the relation of chimerism and hct outcome. disclosure: eileen nicoletti -employee rocket pharmaceuticals, susan prockop -investigator atara biotherapeutics -research funding, susan prockop -mesoblast -research funding, nancy kernan -gentium -support; jazz pharmaceuticals -support, richard o´reilly -atara biotherapeutics -royalty, consultancy and research, jaap jan boelens -bluebird bio -consultancy, avrobio -consultancy; jaap jan boelens -chimerix -consultancy; magenta -consultancy background: the success of hematopoietic stem cell transplantation (hsct) lies with the ability of the engrafted immune system to remove residual leukemia cells via a graft-versus-leukemia effect. despite this, relapse remains the major cause of mortality among patients receiving hsct. one of the immune evasion mechanisms of leukemic cells to escape from donor t cell recognition in haplo-hsct is the genomic loss of the patient specific hla. it has been described in - % of acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) relapses after haplo-hsct. the aim of this study was to analyze hla loss in a large cohort of patients who relapsed after t-cell replete haploidentical transplantation with posttransplant cyclophosphamide. methods: from december to september , patients with hematological malignancies who received a haplo-hsct were recruited. among them, patients presented a relapse after haplo-hsct. hla typing was performed by real-time pcr using hla-kmr kit (gendx, netherlands).nine patients were excluded from the analysis because the kit employed did not include the recipientspecific hla. thus, a total of relapse cases were analyzed. the analysis of chimerism was carried out using short tandem repeat pcr amplification (ampflstr sgm plus, thermo fisher, usa) with a sensitivity of %. results: genomic loss of the patient hla occurred in / patients ( %) ( table ) . these patients presented different hematological neoplasms. interestingly, patients presented lymphoid neoplasm ( acute lymphoblastic leukemia (all-t), dentritic cell leukemia (dcl) and hodking´s lymphoma (hl)). hla loss relapses occurred later than classical relapses ( vs. days). regarding the treatment received (table ) , four patients were studied retrospectively. three of them were treated with donor lymphocyte infusions (dlis) + chemotherapy or other drugs at the time of the relapse. the other patient did not receive any treatment. in the end, all patients died from disease progression. prospectively, we detected hla loss at relapse in other two patients. at the moment of relapse, the first case received brentuximab + haplo-hsct from alternative donor and the other case received daratumumab + haplo-hsct (pending). both patients are alive, the first one in complete remission (cr) and the second one in partial remission (pr). conclusions: the frequency of hla loss at relapse is similar in our cohort to what is described in the literature. hla loss has been identified in patients with lymphoid neoplasms, while this mechanism has not been previously described in such diseases. the analysis of this immune evasion mechanism should be implemented in the routine screening of patients transplanted from haploidentical donors in order to design effective rescue strategies. these treatments should not be based on dlis or second transplantation with the same donor, instead, alternative donors should be used. background: an adequate immune reconstitution (ir) is crucial to reduce transplant toxicity, relapse rate and mortality after allogeneic stem cell transplantation (allohsct). the aim of this, single center retrospective study was to investigate the correlation between the recovery of different lymphocyte subpopulations with the main transplant outcomes, including overall survival (os), disease free survival (dfs) and non-relapse mortality (nrm). methods: we analyzed the ir of adult patients (aml n= , all n= , mds n= , nhl n= , hd n= , cll n= , cml n= , mm, n= , mpn n= ) who underwent (allohsct) between january and march . median age at transplant was years (range . - . ) with male/female ratio of %. donors were hlaidentical siblings (n= , %), family haploidentical (n= , %), matched unrelated ( , %), mismatched unrelated (n= , %) and cord blood units (n= , %). the stem cell source was the bone marrow (bm) in patients ( %), the cord blood in ( %) and g-csf mobilized peripheral blood in ( %). the conditioning regimen was myeloablative in ( %) transplant, reduced intensity in ( %) and immunosuppressive in ( %). gvhd prophylaxis was based on calcineurin inhibitors in combination with methotrexate or mofetil mycophenolate. antilymphocytes immunoglobulins was used in patients ( %) (anti thymocytes globulin, atg sanofi-genzyme in or anti t-lymphocyte globulin, atlg -neovii biotech, in ). the peripheral blood lymphocyte subsets (cd +, cd +cd +, cd +cd +, cd + (b cells) and cd +cd + (nk) were analyzed by flow cytometry at , , , , and months after hsct. post-transplant engraftment was molecularly determined by vntr analysis. results: as detailed in table the proportion of full donor chimerism analyzed in the peripheral blood t lymphocytes improved progressively after transplantation and the same pattern was observed when the chimeric status was measured in bone marrow mononuclear cells. to favor the achievement of a full donor chimerism, dli were performed in patients starting at the median of days after transplant (range: - ). with a median follow-up observation of months (range - ), the one year os and nrm was % and %, respectively. at months after allohsct, the achievement of values higher than , and /μl for cd +, cd + and nk cells, respectively was significantly associated to a better os (figure ), dfs (p = . ), and to a lower nrm (p< . for cd + and cd +, p= . for nk). a better lymphoid reconstitution was observed after the use of either a sibling or a haplo donor than a matched unrelated or cord blood donors. the use of atg was significantly associated with a delayed cd + recovery but with a faster nk cells reconstitution. conclusions: at six months after allohsct, recovery of cd + and nk cells predicts survival. monitoring of immune recovery may help to guide pre and post-transplant treatment strategies. days infections and disease control. several groups have demonstrated the independent prognostic value of different lymphocyte subpopulations in hsct outcomes. posttransplant cyclophosphamide (pt-cy) effectively prevents gvhd after hla-haploidentical hematopoietic stem cell transplantation (haplo). the use of pt-cy in hla matched related (mrd) or unrelated (mud) donors hsct, although less explored, has also been introduced. the aim of this study was to compare the early immune reconstitution after allogeneic hsct from haploidentical and hla-matched donors using pt-cy. methods: one hundred and sixty-four hsct performed in our center were analyzed: haplo performed between and and hsct from hla-identical donors ( consecutive mrd sct performed with pt-cy between and and mud sct with pt-cy between and ). pt-cy was administered at mg/kg/d in days + and + postransplant, followed by mmf mg/kg/ d and a calcineurin inhibitor (ciclosporina a or tacrolimus) from day + ahead. we retrospectively compared early immune reconstitution at day + and day + among these three populations. early ir was assessed through the analysis of lymphocyte subpopulations including total t lymphocytes cd +, cd + and cd +subpopulations, nk cd -cd + cells, cd + bright immature subpopulation and total b cd + lymphocytes.. lymphocytes subpopulations were determined by multiparametric flow cytometry (fc and navios, beckman coulter®). results: all patients, but mud and haplo, received pb as stem cell source. patients received prior transplant in haplo group. patient´s characteristics are shown in table . patients who received hsct from mrd showed the fastest ir, with statistically significant differences compared to haplo in almost all lymphocyte populations at day + (cd +, cd +, cd + and nk cells), and also in cd +, cd + and b lymphocytes at day + . comparison between haplo and mud hsct showed better ir among haplo, demonstrated by higher counts in cd +,cd +, cd + and nk cell counts at day + . no differences were seen at day + . (figure ). percentage of immature cd bright nk cells was higher in mud hsct at + , with no differences between haplo and mrd hsct. conclusions: in our cohort of patients with pt-cy based gvhd prophylaxis regimen, those who received hsct from mrd showed the earliest immune reconstitution compared to haplo and mud at day + and + . haplo showed better ir compared to mud at day + . nk maturation at day + was a little better among haplo and mrd hsct recipients than mud hsct patients. in our experience, using mostly pbsc as graft source, type of donor influenced early ir in pt-cy based hsct, background: cell-free dna (cfdna) isolated from plasma or serum has received increasing interest for diagnostic applications. however, the reported clinical usefulness of cfdna in patients undergoing allogeneic cell transplantation (hsct) is scarce. methods: the chimeric status both in peripheral blood and in cfdna obtained from plasma was investigated in patients undergoing hsct. dna and rna were isolated from plasma within four hours of blood draw. patients were evaluated for chimerism at day + , + and + post-transplant. a panel of seven microsatellites was amplified by pcr for chimerism detection and pcr products were analysed by capillary electrophoresis. for further cfdna characterization the micro rna (mirna) c was analysed using digital pcr. mutations frequently used for minimal residual disease assessment such as flt -itd, npm and jak were also investigated in cfdna. results: the mean cfdna concentration in transplanted patients was ng/ml, while in healthy donors used as control group (n= ) was ng/ml. the mean cfdna concentration difference between both groups reached statistical significance (p= . ). when analysing cfdna from transplanted patients and in the control group we could not detect dna fragments larger than bp and the size range of the analysed fragments was between and bp. in out of patients a mixture of donor and recipient cfdna (mc) was detected. with the exception of three patients relapsing after transplant in which mc was detected both in peripheral blood and plasma in the rest of the patients (n= ) mc was detected only in plasma. the mean percentage of recipient cfdna in the plasma samples was % (range: - %). all the patients with acute gvhd (agvhd) (grade: i-iv) (n= ) showed mc in plasma at least in one of the time-point tested. no significant difference was found in the mean recipient cfdna percentage in patients with agvhd grade i-ii when compared with grade iii-iv. meanwhile in the group of patients with chronic gvhd (n= ) mc in plasma was detected in patients. in those patients with clinical improvement of agvhd (n= ) a decrease in the percentage of recipient cfdna was observed during treatment. in patients without improvement or even agvhd worsening (n= ) stable or increasing recipient cfdna percentage was detected. since recipient cfdna can be detected in patients without transplant-related complications we analysed the mirna c expression in all patients with recipient cfdna. a significant difference was found in the mirna c expression in patients with agvhd (mean mirna c: . mirna c copies/ u copies) when compared with patients without gvhd (mean mirna c: . mirna copies/ u copies). in those patients with extramedullary aml relapse (n= ) frequent mutations (flt -itd, npm ) were only detected in the cfdna fraction. conclusions: longitudinal analysis of cfdna represents a useful complementary tool in particular for those patients with clinical complications after hsct. disclosure: nothing to declare comparison of the impact of atg/pt-cy-based and tcr αβ-depletion as gvhd prophylaxis regimens on the recovery of memory t-cell compartment background: over recent years haploidentical and hlamismatched donors have been increasingly adopted as a valid donor source. modern graft-versus-host disease (gvhd) prophylaxis regimens such as drug-based (antithymocyte globulin (atg), post-transplant cyclophosphamide (pt-cy)) or graft-manipulated (tcr αβ-depletion) demonstrate effective prevention of gvhd. here we report our data about an influence of different gvhd prophylaxis regimens after allo-hsct with pbsc as a graft source on cd + memory t-cells recovery. methods: our study comprised leukemia patients who underwent allo-hsct with pbsc as a graft source in national research center for hematology, moscow, russia. detailed patients characteristics are presented in table . peripheral blood samples were collected on day + , + and + after allo-hsct. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+tscm) -cd +cd r -ccr +cd +; t-central memory (tcm) -cd +cd r +ccr +cd +; t-transitional memory (ttm) -cd +cd r +ccr -cd +; t-effector memory (tem) -cd +cd r +ccr -cd -; t-terminal effector (tte) -cd +cd r -ccr -cd -. sysmex xe- was used to calculate absolute count of different t-memory cell subsets. mann-whitney u test was used for nonparametric data analysis. a p-value less than . was considered as significant. results: results of mann-whitney u test (calculated pvalues) to compare absolute number of t-memory cell subsets in terms of different gvhd prophylaxis regimens are presented in figure . during all follow-up period the number of tnv+scm and tcm remains significantly reduced after atg+pt-cy or tcr αβ-depletion compared to atg-based immunosuppressive regimen. on day + we observe no difference in the number of tnv+scm and tcm cells after atg+pt-cy or tcr αβ-depletion. terminally differentiated cd + cells (ttm, tem, tte) count is significantly lowered in tcr αβ-depletion patients group in comparison to atg+pt-cy. nevertheless recovery of tnv+scm and tcm after pt-cy is faster than after tcr αβ-depletion. conclusions: according to our data the mechanism of pt-cy is seems to be more selective compared to tcr αβ-depletion due to its transient impact just on tnv+scm and tcm without affecting on the effector pool. through this it may lead to delayed reconstitution of adaptive immunity after tcr αβ-depletion compared to using pt-cy. clinical relevance of the quantitative characteristics of immune recovery in the context of different approaches to gvhd prevention remains to be established. background: immune effector cells, belonging to either innate or acquired immunity, play a key role on preventing disease relapse after allogeneic haematopoietic stem cell transplantation (hsct). most of known immune effector are cd + cd + t-cells and cd -cd + natural killer lymphocytes, while cd + cd + cells act as modulatory and regulatory cells. the early post-hsct ratio between these cellular subsets may be an indicator of graft vs-tumor (gvt) effect. methods: we retrospectively revised the immune recovery of allogeneic hsct performed at our institution from to , analysed on peripheral blood by multiparametric flow cytometry lymphocyte subpopulations panel. diagnosis were acute leukemias ( %), chronic myeloproliferative neoplasms ( %), lymphomas ( %), myelodysplastic syndromes ( %), multiple myeloma ( %) and severe aplastic anemia ( %). we established early time-points of evaluation, and days from the graft infusion, to analyse the differences in disease free survival (dfs) and overall survival (os) between patients according to the cd + cd + x cd -cd + / cd + cd + ratio. results: median ratio at + days was of , . at this time point, patients who showed the ratio higher than the median had both a better dfs (median dfs time not reached vs months; p = , ) ( figure ) and os (median os time not reached vs months; p = , ). likewise, ratio at + showed an advantage on dfs (p = , ), and not on os (p = , ). other factors possibly affecting both dfs and os were analysed in univariate analysis, such as the use of antithymocyte globulin (atg), conditioning regimen intensity, graft source, hla-matching and disease status at hsct, the latter being the only variable with a significantly detrimental impact on both os and dfs. disease status was confirmed an independent valriable associated with both dfs and os as well as + ratio both on dfs (hazard ratio [hr] - , ; p = , ) and os (hr , ; p = , ). conclusions: our data show that cd + cd + x cd -cd + / cd + cd + ratio assessed at + is and independent predictor of transplant outcome, possibly representing a row indicator of anti-leukemic immune surveillance. the integration of this index with other known outcome predictors may help in improving the management of post-transplant phase. [[p image] . figure background: allogeneic stem cell transplantation (alo-hsct) is a curative treatment but it is associated with lifethreatening complications. most deaths are due to relapse, graft versus host disease (gvhd) and infection. the pattern and quality of the immune reconstitution (ir) after transplantation may affect these outcomes. however, there are limited data on the association of the quality of the ir and either the development of gvhd and survival. methods: eighty-five patients who received a non t-cell depleted alo-hsct in our center from to were prospectively studied. most patients received hla-identical grafts. total cd + and cd + t cells, ccr +cd + and ccr +cd + (which include both naïve and central memory t cells) and naïve ccr +cd l+ t lymphocytes were quantified by flow cytometry. data were collected at days + , + , + , + and + after alo-hsct. the association between ir and the gvhd was studied through an anova. for the multivariate analysis, a logistic regression was performed including those confusing clinical variables that were significant in the univariate analysis (p≤ . ). the study of overall survival (os) versus ir was performed with a cox regression model. results: total cd + t lymphocytes reached normal numbers within the first two months. median t cd + count was cells/ul after one month, which is within the normal range. conversely, it took nearly one year to get normal counts of cd + t cells ( cells/ul). the only two clinical parameters conditioning a worse recovery of the cd + t cells were the previous alosensitization of the donor and the sex, being female donor and male recipient the worst combination for the ir. no parameters influenced the quality of the reconstitution of cd + t cells. of note, the age or the hla status did not influence the quality of the ir. when the patients were divided into gvhd and no gvhd, we found no differences in the recovery of either the proportion or absolute count of every t cell subpopulation, including total t cells as well as naïve/central memory t cells, both cd + and cd +. finally, a multivariant analysis confirmed that the absolute counts of cd +ccr + t cells at day + as well as the absolute counts of both cd +ccr + t cells and naïve cd +ccr +cd l+ at day + were associated with better os. conclusions: in conclusion, neither the development of gvhd nor other relevant parameters seem to play a determinant role in the quality of the ir. to our knowledge this is the first study which demonstrate a clear association between the recovery of naïve cd + t cells measured by flow cytometry and the os. disclosure: nothing to declare p abstract already published. azacitidine (aza) for prophylaxis or pre-emptive therapy for myeloid neoplasms after allogeneic stem cell transplantation whom were treated prophylactically and preemptively. median age was years [range, - ] and all patients had a diagnosis of aml or high-risk mds. prophylactic treatment consisted of aza mg/m for days in cycles of days. in the pre-emptive setting, patients received mg/m for days per cycle and patients mg/m for days per cycle. a median of cycles [range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] was administered in the prophylactic group and of cycles [range, - ] in the pre-emptive group. ten patients also received at least one dli after the third aza cycle: patients in the prophylactic group and patients in the pre-emptive one. results: during follow-up, patients had significant delays in treatment plan due to transitory mild complications. however, % of patients (n= ) presented infectious complications requiring hospitalisation and % of patients (n= in the prophylaxis group and in the pre-emptive group) presented some form of gvhd. in patients who developed gvhd, had to discontinue treatment (all in the prophylaxis group); also patients discontinued treatment due to disease progression. the overall drop-out rate was . % (n= ). survival was analysed from initiation of treatment with aza and median follow-up was months. one-year efs was % in the prophylaxis group, with only one patient relapsing and no deaths. in the pre-emptive group, the -year efs was % and the median efs was months; -year os was % and median os was months. conclusions: we conclude that post-transplant aza treatment is a well-tolerated therapy, but the incidence of side effects remains discordant in the literature. results in the prophylaxis group are excellent, but patients with positive minimal residual disease treated pre-emptively had a lower outcome with only stabilisation of the disease. randomised prospective trials are needed to define patients who would benefit the most from this treatment and at what timing, dosage and duration of treatment. disclosure: nothing to declare. abstract already published. results: all subjects experienced hematopoietic engraftment at a median of days (range - ) and demonstrated full donor myeloid chimerism. m-mdsc and pmn-mdsc recovery peaked at a median of days posttransplant. the median peak absolute m-mdsc count was , cell/ml (range , - , /ml) representing a range of . % to . % of pbmcs. the pmn-mdsc peak was more robust, with a median absolute peak of , cells/ml (range , - , /ml) representing a median of . % of pbmcs (range . - . %) . of note, the one patient who developed severe, life-threatening gvhd had the lowest absolute and relative pmn-mdsc recovery ( , cells/ml and . % of total pbmcs). recovery of m-and pmn-mdscs occurred at a similar tempo and magnitude in two recipients of standard gvhd prophylaxis (tacrolimus/methotrexate). however, while mdscs isolated from ptcy recipients exhibited clear t-cell suppressive capacity, those from the comparison patients did not (see figure) . conclusions: mdscs recover rapidly and robustly after allohct using ptcy as gvhd prophylaxis, and may play a role in mitigating gvhd risk by mediating t-cell suppression. this may be a mechanism by which ptcy results in donor-recipient tolerance. background: high dose chemotherapy followed by autologous stem cell transplantation (asct) offers a cure in the upfront and relapsed setting in both hodgkin (hl) and non-hodgkin lymphoma (nhl). asct also remains standard of care in previously untreated multiple myeloma (mm) patients after induction therapy, if eligible. the availability of new cellular or other immune therapies that can be used after asct underscores the potential importance of monitoring immune reconstitution after asct. methods: immune reconstitution panels (irp) were evaluated retrospectively in all lymphoma and mm patients over a -year span ( - ) whom underwent asct at our institution. patients were included if they had a pre-asct measured within days of asct and two other irp at any of the following timepoints ( ) day - , ( ) day - , and ( ) at -year post-asct. patients in the lymphoma cohort had their irp excluded if they had additional treatment within the first year post-asct (other than maintenance rituximab). mononuclear cells from peripheral blood were analyzed by flow cytometry for assessment of lymphocyte phenotype and numbers. absolute values were compared using the mann-whitney u test. results: the data on patients were available for analysis ( mm, nhl, hl) . all lymphoma patients were conditioned with beam. all mm patients were conditioned with a standard high dose melphalan regimen. the median pre-asct absolute cd counts in the lymphoma cohort were significantly lower than the mm cohort at cells/μl vs cells/μl, respectively (p= . ). however, the mm cohort exhibited a greater percent reduction in cd cells on day at . % vs . %, respectively which continued through day at . % vs . %, respectively. this led to nonsignificant changes in absolute cd count by day at cells/μl vs cells/μl, respectively (p= . ) (figure ). the median absolute cd count pre-asct for mm and lymphoma cohorts were cells/μl and cells/μl, respectively (p= . ). similarly, a greater percent reduction in cd cells led to comparable absolute counts on day at cells/μl vs cells/μl, respectively (p= . ). the failure of post-asct cd reconstitution to pre-asct levels was driven by lack of cd + cell recovery, namely cd +cd ra+ cells with a median of cells/μl and cells/μl in the mm and lymphoma cohorts, respectively at day (figures and ). this led to markedly diminished cd :cd ratios through day (figure ). [[p image] . conclusions: impaired t-cell reconstitution in both lymphoma and mm continues through -year post-asct. as shown, a larger percent reduction in median cd and cd counts through day was appreciated in mm compared to lymphoma leading to the nonsignificant differences in the post-asct absolute counts despite significantly higher pre-asct counts in the mm cohort. impaired recovery of cd t-cells may increase the risk of opportunistic infections, decrease the response to vaccination and lead to ineffective anti-tumor response. further prospective and larger retrospective studies like this should continue in the modern-era as they may help predict responses to further interventions requiring a robust t-cell repertoire for maximal efficacy such as car-t cell and bite therapies. disclosure: nothing to declare p peri-transplant detection of measurable residual disease by multicolor flow cytometry is highly predictive for relapse in acute myeloid leukemia patients background: presence of measurable residual disease (mrd) prior to allo-sct has been shown to be predictive for survival in patients in hematological cr of aml. in this study we analyzed the impact of mrd in such patients measured by -color multiparameter flow cytometry (mfc) prior to and on day + post-transplant. methods: the bone marrow samples immediately prior to allo-sct and on day + post-transplant were retrospectively analyzed. mrd evaluation was carried out with antibodies against: ( ) results: a number of aml patients (male, n= ) with median age of years ( - ) in hematological cr prior to allo-sct were enrolled in the study. we observed lower survival in patients with mrd by mfc pre-transplant ( y os: % ( - %) vs. % ( - %), p= . ) due to increased relapse incidence ( % ( - %) vs. % ( - %), p= . ). in multivariate analysis, mrd pos prior to allo-sct has strong significant impact on os (hr . ( . - ) , p= . ). of patients, a sample both before and on day + after transplantation was available in patients. of those patients, ( %) were mrd negative prior to transplant and on day + (mrd neg/neg ); ( %) patients were mrd positive prior to transplant and negative at day + (mrd pos/neg ); and ( %) patients were mrd positive at both timepoints (mrd pos/pos ). dfs and os for these three groups were as follow: y dfs: mrd neg/neg : % ( - %), mrd pos/neg : % ( - %); mrd pos/pos : % ( - %, p= . ); y os: mrd neg/neg : %; mrd pos/neg : % ( - %); mrd pos/pos : % ( - %, p< . ). upon multivariate analysis, the mrd status prior to transplant and on day + showed strong significant impact on dfs (hr . ( . - . ), p= . ) and os ), p= . ). we did not observe any significant impact of other factors included in the multivariate analysis (patient's age, patient's sex, and recipient/ donor sex constellation). conclusions: mrd positivity prior to allotransplant and at day + by mfc is highly predictive for survival after allo-sct. disclosure: nothing to declaire background: immune reconstitution is a critical factor for risk assessment of life threatening infections and long-term survival in patients undergoing allogeneic cell transplantation (hsct). methods: immune cell subsets (cd , cd , cd , cd , cd +cd -) were quantified by flow cytometry. trec and krec were quantified simultaneously using droplet digital pcr (dpcr). a total of patients were evaluated. mean age at transplant was years (range: - years) samples were obtained before hsct and at day , and after hsct. results: absolute numbers of cd and cd cells remained below pre-transplant levels until day , increasing further and eventually reaching pre-transplant levels one year after hsct. absolute counts of cd and cd +cd -cells remained below pre-transplant levels beyond one year after hsct. cd cells were characterized by fast reconstitution kinetics, reaching pre-transplant levels already at day . b cells correlated with krec levels at all time-points tested, whereas t cells correlated with trec levels only one year after transplantation. when we compared cell subsets, trec, krec levels and the reconstitution kinetics thereof between patients with reduced intensity conditioning (n= ) or full conditioning (n= ) no significant differences were observed. patients with pre-transplant trec levels above the mean ( trec copies/ml blood) showed higher trec levels and a faster t-cell reconstitution after hsct suggesting that tcell reconstitution can be predicted by analysing thymic functionality before transplantation. indeed, in patients with a pre-transplant trec above trec copies/ml blood, the positive predictive value for an efficient t-cell reconstitution was . (p= . ). we analysed the recovery kinetics of the cell subsets, trec and krec levels in patients with and without transplant-related complications. patients with either acute graft-versus-host disease or severe infections showed a slower trec reconstitution when compared with patients without complications. conclusions: our data suggest that the analysis of immune cell subsets together with trec and krec quantification can be used to evaluate the immune reconstitution process after hsct. pre-transplant trec levels allow t-cell reconstitution efficiency prediction after hsct. disclosure: nothing to declare background: falling donor / mixed chimerism after allogeneic haematopoetic stem cell transplant (sct) is associated with an increased risk of relapse and the potential for graft rejection. donor lymphocyte infusions (dli) are often administered in patients with mixed chimerism to achieve full donor chimerism but there is little data on long term outcomes for dli given for persistent mixed chimerism. methods: a retrospective analysis of all patients administered dli for mixed chimerism between to january was performed. all patients were transplanted at the university hospital of wales within the south wales blood and marrow transplant (swbmt) programme. patients were identified by the swbmt database and additional outcome data gathered by review of patients' medical records. results: patients were treated with donor lymphocyte infusions between and january . thirty one patients treated for relapse (with or without mixed chimerism) were excluded as was a further patient with a mismatched donor. the rest were / match. twenty six patients received a total of donor lymphocyte infusions for mixed chimerism alone. the median age was years (range: - ) with % women. fourteen ( %) of the patients had sibling donor transplants and twelve ( %) from matched unrelated donors. indications for transplant were: for aml or saml (n= ), myelofibrosis (n= ), mds (n= ), hodgkin lymphoma (n= ), non-hodgkin lymphoma (n= ) and all (n= ). escalating doses of donor cd + t cells were administered commencing at × /kg to × /kg then increased at half log increments according to chimerism results until full donor chimerism was achieved. the median number of doses administered was (range - ). the median interval was days (range - ). the median dose was × / kg (range × - × ). seventeen patients ( %) achieved full donor chimerism and remained so until most recent follow up (median months, range - ). one patient continued to receive dli after the study period and later reverted to full donor. two patients had ongoing mixed chimerism with no evidence of relapse. two patients relapsed; one of whom later achieved remission. there were six cases of gvhd; acute gvhd (grade ii n= , grade iii n= ) and cases of chronic extensive gvhd. one patient had gvhd features consistent with overlap syndrome. a total of five patients died, four due to infection (one in a patient with gvhd) and one due to cardiac toxicity from previous treatment (confirmed on post-mortem). conclusions: the results of our single centre study help reinforce the evidence for dli in establishing full donor chimerism when mixed chimerism is detected in the absence of relapse. incremental dli dosing is an effective strategy and associated with a low relapse rate. caution should still be given to the risk of gvhd following dli, however the risk appears to be low in this study. larger prospective studies are ongoing to address the optimal dosing strategy for dli post-transplant. disclosure: nothing to declare hypomethylating agents for the treatment of relapsed acute myeloid leukemia after allogeneic blood stem cell transplantation: a single center experience mariarita sciume , giorgia saporiti , elena tagliaferri , nicola fracchiolla , federica grifoni , giorgia levati , luca baldini , francesco onida the post-transplant period with well-balanced profile of good efficacy and moderate toxicity. we retrospectively evaluated the safety and efficacy of hma +/-dli in a reallife cohort of aml patients relapsing after allo-sct. methods: data from all patients with aml who underwent allo-sct at our institution in the last years and subsequently received hma as a salvage treatment for disease recurrence or preemptively for loss of complete donor chimerism were collected. results: eleven patients with a median age of years (range - ) were identified; median time between allo-sct and time to hma therapy was months (range - ). according to eln genetic risk stratification, patients were classified in the favorable group, in the intermediate-i, in the intermediate-ii and in the adverse one. six patients were treated with aza, whereas the remaining patients with dac. the cycles were repeated every days. ten patients ( %) started hma for morphological aml relapse, while one patient received aza as a sequential treatment after dli administered for loss of complete donor chimerism. median number of cycles was (range - ). treatment strategy included combination with dli in patients ( in the dac cohort, in the aza cohort), while in one case of flt -itd + aml sorafenib was also associated to dac and dli. no grade / toxicities and no acute gvhd occurred. a clinically significant response was observed in four patients ( %), all receiving at least cycles of hma therapy; in particular, a complete remission (cr) was achieved in / patients treated for morphological relapse, including the one who received the dac/dli/sorafenib combination and one (favorable eln risk) who received aza alone (not eligible for dli due to a concomitant lateonset cutaneous grade gvhd). of interest, the latter patient also displayed a resolution of the cutaneous gvhd. full donor chimerism recovery with no gvhd was observed in the patient who received aza for the progressive donor chimerism loss not responding to dli alone. with a median follow-up of months (range - ), the median os from hma treatment in responding patients was months (range - ); at the time of data collection responses were maintained in all four patients. seven patients had died, six from aml progression and one for severe intestinal gvhd occurring after failure of dli+aza and a following salvage induction chemotherapy treatment. conclusions: although arising from a limited number of patients, our real-life experience of treatment with hmas +/-dli in aml patients relapsing after allo-sct showed a general very good safety profile and promising antileukemic activity, altogether suggesting a facilitation of the graftversus-leukemia effect (gvl) associated to a possible suppression of the gvh reaction. disclosure: nothing to declare conclusions: in this study, cd -positive cell count and igg value had recovered about months after sct. in our institute, we have achieved a low incidence of infection by education and medication for patients until recovery of cd -positive cell count and igg. however, we found a higher incidence of infection after recovery of cd -positive cell count and igg. at - months after sct, administration of prophylactic medications such as sulfamethoxazole-trimethoprim were terminated and social comeback such as return to school or work were achieved in most patients. it is possible that the high incidence of community-acquired infection was associated with their comeback. thus, we should consider additional prevention of infection for patients in this period and further evaluation of immunological markers is needed. disclosure: no potential conflicts of interest were disclosed. effect of minimal residual disease before transplantation on the outcome of haplo-identical hematopoietic stem cell transplantation for high-risk acute lymphoblastic leukemia yehui tan , sujun gao , xiaoliang liu , long su , wei han , yu liu , yangzhi zhao background: to analyze the effect of haploid hematopoietic stem cell transplantation (hid-hsct) on high-risk acute lymphoblastic leukemia (all), and to explore the effect of minimal residual disease (mrd) before transplant on the prognosis. methods: a retrospective analysis was made on high risk all patients accepted hid-hsct in our hospital from january to january . the clinical features, stem cell implantation, complications, survival and recurrence were compared between pre-transplant mrd + and mrdpatients. results: all the patients got successfully implanted. the overall survival (os) was . %, the disease free survival (dfs) was . %, the incidence of acute graft versus host disease (agvhd) was . %, including . % ii~iv degree agvhd and . % iii~iv degree agvhd. there was no significant difference in stem cell implantation, gvhd, cytomegalovirus and hemorrhagic cystitis between mrd + and mrdpatients. dfs and os in mrd + patients were significantly lower than those in mrd -patients, and the cumulative rr rate increased significantly, there was no significant difference in cumulative trm. conclusions: hid-hsct was an effective method to treat high risk all, but mrd + patients had high recurrence rate and poor prognosis. strategy adjustment should be considered to reduce tumor residual and the transplantation strategy should be optimized for these kind of high risk patients, so as to improve survival rate. disclosure: nothing to declare background: lymphocytes are responsible for the cellular and humoral immunity and, consequently, its recovery after allo-hsct might be linked with the survival after the procedure. the aim of this study was to analyze this hypothesis in our series of patients. methods: all the allo-hsct performed in our center from january through july were included in the analysis. median age was years (range: - ). pts were male ( , %) and were female ( , %). baseline diseases were: aml, lpd, mds, all, mpd, mm, and bmf. donor was unrelated in ( , %), and was family in cases ( , %) (including haplo-identical). stem cell source was pb in ( , %) and bm in pts ( , %). conditioning regimen was reduced in procedures ( , %) and intensive in ( , %) (including just one non-myeloablative). overall mortalities at days + and + (the latter in patients with follow-up superior to year) were , % and , %, respectively. median follow-up was months (range: - ). evolution of absolute lymphocyte counts (alc) and subpopulations at pre-hsct and during the first year after allo-hsct were analyzed. results: as shown in table , alc and cd + lymphocytes decreased after conditioning therapy, and recovered progressively during the post-hsct period. at day + , majority of patients had > alc/mcl, clearly improved compared to admission values. cd + lymphocytes at day + was still very low, but at day + around half of the series had - /mcl. we found a strong link between alc, cd + lymphocytes, and cd + lymphocytes at days + and day + with overall survival at day + of the series (table ) . conclusions: in our series, immunity recovery was a late event for majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc, cd +, and cd + cells recovery was clearly linked with long-term survival. background: the reconstitution of t and natural killer (nk) cells after hematopoietic stem cell transplantation (hsct) strongly influences the outcome of hsct including viral infection and graft versus-host disease (gvhd). the purpose of this study was to investigate the clinical efficacy of immune reconstitution including t and nk cells after hsct in children. methods: we reviewed the records of patients who undergoing allogeneic hsct in department of pediatrics, pusan national university children's hospital, from january to july . the counts of t lymphocyte subsets and nk cells was monitored in peripheral blood by flow cytometric technique during , , , and months post-hsct. blood samples for cytomegalovirus (cmv) and epstein-barr virus (ebv) monitoring were tested by real-time pcr assay. results: for total of patients, the mean age was . years (range, months- years), of the patients were boys and was girl. out of a total patients without pre-hsct cmv viremia or cmv infection, ( . %) recipients experienced cmv infection. the number of cd + t cells in and months post-hsct was significantly higher in patients with cmv reactivation compared to patients without (median . /μl vs. . /μl, p= . , and . /μl vs. . /μl, p= . ) . in ( %) recipients presented acute gvhd, the number of cd + t cells in and months post-hsct was significantly lower in patients with acute gvhd compared to patients without (median . /μl vs. . /μl, p= . , and . /μl vs. . /μl, p= . ) . the number of nk cells in months post-hsct was significantly lower in patients with cmv reactivation and acute gvhd compared to patients without ( . /μl vs. . /μl, p= . , and . /μl vs. . /μl, p= . , respectively) . in multivariable analysis, acute gvhd was shown to be the decisive factor influencing total t cells (p= . ) and cmv reactivation was independently associated with cd + t cells (p= . ). the cd + t cells counts were associated with prior hsct history and acute gvhd (p= . and p= . ), and the cd + t cells counts were also significantly associated with donor type (p= . ). conclusions: overall, our study documents that immune reconstitution of cd + , cd + t cells and nk cells is strongly associated with cmv reactivation and acute gvhd. additionally, we show that acute gvhd is influenced by lack of sufficient numbers of nk cells as well as cd + t cells early after sct. cd + t cells, on the other hand, significantly increase after cmv-reactivation and most likely play an important role in reactivation. disclosure: nothing to declare background: curative effect of allogeneic hematopoietic stem cell transplantation (allo-hsct) depends on the alloreactive t-cell immune response toward residual malignant cells -graft-versus-leukemia reaction. however, alloreactive population has not been phenotypically defined. recent studies suggest that alloreactive t cells express both costimulatory and inhibitory receptors simultaneously. exhaustion caused by the inhibitory signaling dampens tcell functionality, which could lead to the disease relapse. here we aimed to investigate the expression of costimulatory and inhibitory receptors on antigen-experienced t cells after transplantation, to isolate subpopulation specific for allo-hsct patients and analyze their t-cell receptor (tcr) repertoire. methods: expression of coinhibitory and costimulatory molecules on pbmcs patients at various time points after allo-hsct was analyzed for expression of: cd , cd , cd , cd ra, ccr , cd , cd , cd , klrg , tigit, pd , cd and ox by flow cytometry and compared to healthy donors. cd +cd +cd -cd +cd +pd +tigit+ fraction and cd + cd + control fractions were separated on facs aria ii cell sorter. double barcoded cdna libraries of tcr beta-chains for both fractions were prepared and analyzed by sequencing on illumina platform. sequencing results were processed by migec, mixcr and vdjtools software. enriched clones were identified by fisher's exact test (p> - ). results: we did not find any significant differences between patients after allo-hsct and healthy donors in single marker's expression, but, when considering coexpression of co-stimulatory and inhibitory molecules on t cells we found that cd +cd +cd -cd +cd +pd +tigit+ subpopulation was significantly increased in allo-hsct patients. moreover it increased with the time since the transplantation (fig. ). this population was isolated by cell sorting and alongside with total cd + fraction subjected to tcr beta-chain repertoire sequencing. the population contained clones significantly enriched compared with cd + fraction representing potentially alloreactive cells. this hypothesis is further supported by the notion that the level of expression of cd and cd co-stimulatory molecules is lower in the group of patients who subsequently relapsed, compared with the patients with complete remission, while the expression of inhibitory receptors was high in both groups. conclusions: according to our data patients after allo-hsct have a phenotypically distinct t-cell population characterized by simultaneous expression of costimulatory and inhibitory markers. this population contains specifically enriched clones, which may be specific for alloantigens. further functional assays are needed to confirm the alloreactive potential of this subpopulation. besides low expression of costimulatory molecules combined with high expression of inhibitory receptors on antigen-experienced t-cells of patients after allo-hsct might be associated with a disease relapse. fondazione mbbm, monza, italy, ospedale san gerardo, laboratorio stefano verri, monza, italy background: poor graft function (pgf) is a severe complication after hsct, with a high risk of morbidity and mortality, mainly due to infections. donor cd + scb seems to offer high chances of haematological recovery, not jeopardized by gvhd. however, pediatric reports remain scarce. methods: out of patients undergoing transplantation in our pediatric unit from to have been retrospectively evaluated for at least line persistent cytopenia (hb< . g/dl, plt< /mmc, n< / mmc) and/or transfusion-dependency beyond months after hsct in the presence of full donor chimerism. bone marrow cellularity was evaluated through biopsy as further indicator of pgf. ( / ) to donate or medical decision ( / ). bone marrow cellularity was < % in % of the patients who underwent scb for which the histology was available ( cases), and % in those who have not been treated ( / ). at days after scb / ( %) patients had hematological response, which was complete in % and partial in % of the patients. only patient had no response. the infusion was always well tolerated with no adverse events, and no worsening of gvhd. haematological recovery occurred spontaneously at days after bone marrow biopsy in a significantly lower proportion of patients ( / , %, p< . ) within the non-scb group. in two cases platelets engraftment was significantly delayed, up to one year after bone marrow biopsy and in one case thrombocytopenia persists and the patient is still receiving thrombopoietin agonists and red blood cells transfusions at months after bone marrow biopsy. conclusions: a stem cell boost matched the goal to yield count recovery in our cohort. viral infections and gvhd may be possible risk factors for pgf.bilinear or trilinear cytopenia with transfusion dependency and bom cellularity < % and full donor chimerism are good indications for scb, that can provide a significantly earlier hematological reconstitution, without risks of gvhd. due to the proved early efficacy and safety of cd + stem cell infusion, we suggest that this procedure should be taken in consideration in children with severe bone marrow hypoplasia and persistent cytopenia after hsct. disclosure background: as allogeneic hematopoietic stem cell transplantation (hsct) is sometimes performed despite erythrocyte's antigens incompatibility and mismatch, it is essential to carefully track patients' genotypes after it. methods: for the study we used erythrocytes (n= ) and dna (n= ) from patients undergoing abo-or rhesus-mismatch hsct and their donors. we used posttransplant no transfused patients on the periods according transplant protocol by hemagglutination methods in plate and tube using monoclonal antibodies to abo and rhesus antigens (hematolog, russia). we extracted dna with dna kit (bag, germany) and conducted pcr-ssp with kits abo-type, rh-type (bag, germany). chimerism was assessed by the str-pcr analysis with cordis plus multiplex kit for amplification of polymorphic strmarkers and amelogenin loci. the fragment analysis was performed on a genetic analyzer. informative loci were chosen by comparison of pretransplant patient's and donor's dna. the percentage of donor chimerism was calculated using standard formula. precise rhce and abo genotypes were determined by direct sanger sequencing. we revealed patients with unexpected erythrocyte abo, rhesus phenotypes and genotypes after hsct on + ( patients) and + days (all patients). chimerism analyses on str showed in a.e.kh. and g.l.v. patients % of donor's dna and less than % of recipient's one. b.n.a. patient was relapsed and chimerism analysis revealed % of recipient's dna and % of donor's one. using serological methods and pcr-ssp we revealed genotypes abo * a b ; rhd+; rhce * ccee in patient a. e.kh. before hsct, abo * a o ; rhd+; rhce * ccee in her donor, and abo * a a ; rhd+; rhce * ccee on + d after hsct. genotype a a was no recipient's neither donor's origin. direct sequencing did not prove this genotype, but revealed donor's one.on + d serological methods and pcr-ssp also revealed donor's genotype in this patient. patient b.n.a. had genotypes abo * o /o ; rhd+; rhce * c w cee before hsct, her donor -abo * b o ; rhd +; rhce * ccee. on + d this patient relapsed, but rhesus genotype has been detected as rhd+; rhce * c wcee (lack e gene). direct sequencing revealed gene rhce*ee. abo genotype was recipient's origin -o o . in patient g.l.v. using serological and pcr-ssp methods we determined genotypes abo * o /o ; rhd +; rhce * ccee genotype before hsct, and abo * a / o ; rhd+; rhce * ccee genotype in her hsc donor. on + d patient had unexpected genotype abo * o /o (a lack of a antigen); rhce * ccee (a lack of e antigen). in order to explain unexpected patient's genotypes after hsct we sequenced her rhce and abo genes and found donor's genotype ccee; a o that was in agreement with results of str analysis. to resolve discrepancies between serological, pcr-ssp and sequencing analysis data we sequenced patient's rhce cdna and observed only ce allele. at present time the molecular basis of selective inactivation one of the two rhce alleles is not clear. on + d patient had donor's genotype. conclusions: what kind of mechanisms led to discrepancies between results obtained by different laboratory methods are still not clear. an interesting case of expression of only one rhce allele in patient g.l.v. allows us to suggest involvement of some epigenetic mechanisms like dna methylation or histone modification in this process. clinical background: in relapsed patients with acute b -lymphoblastic leukemia (all-b) who achieved complete remission (cr) after re-induction therapy, minimal residual disease (mrd; ≥ - all-b cells/ul) is often detected. according to available data, such condition varies from % to even % of cases, as assessed by pcr or flow cytometry (fc), while the presence of mrd is the most important risk factor for all recurrence. in this abstract, we describe our experience with bridging therapy using blinatumomab infusion after re-induction regimens and before the planned allogeneic stem cell transplantation (allo-sct). the procedure was performed in three young men suffering from relapsed ph (-) all-b at the age of , and years. in the first case ( yo), relapse with previous mrd accounting for . % occurred months after cr mrd neg . in the next patient ( yo) the second relapse with central nervous system (cns) involvement occurred months after allo-sct performed in cr ( months after cr , mrd neg ), while in the third patient ( yo), recurrence with cns and testis involvement occurred years after cr (mrd neg ). all patients underwent chemotherapy (flam, hypercvad and dnr/vcr/pegasp/dexa regimens respectively) followed by one cycle of blinatumomab (at a dose of mcg/d on days - , followed by mcg/d on days - in a continuous infusion) and allo-sct (using eto/cy/tbi/atg/ conditioning regimen for ist and iiird patient and bucy for iind patient; using matched unrelated donor (mud, ist and iiird patient) or matched related donor (iind patient)). mrd status was assessed after each cycle of blinatumomab by fc. results: all patients achieved cr mrd pos after reinduction therapy followed by clearance of mrd after blinatumomab course (tab. ). the second patient, due to positive mrd months after allo-sct received donor lymphocyte infusions additionally. during the administration of blinatumomab, no adverse events (aes) were observed in grade or . one patient developed cytokine release syndrome in grade . the progression free survival, time to positive mrd and follow up are presented in tab . conclusions: the use of blinatumomab as a bridging therapy between re-induction regimens and allo-sct in patients with all-b and mrd pos appears to be safe and leads to the clearance of mrd which may be crucial in os and pfs prolongation after following allo-sct. future studies on larger groups of patients are necessary to confirm this thesis. background: haploidentical hematopoietic stem cell transplantation (hsct) is considered an alternative treatment for hematologic malignancies in patients who do not have an hla-identical sibling donor [ ] . since infections and disease relapse resulting from delayed immune reconstitution (ir) are the most common causes of mortality among patients undergoing haploidentical-hsct [ ], timely ir is essential in the recovery and survival of these patients. the aim of this study is to describe the evolution of ir after haploidentical-hsct and to estimate survival rates in patients with delayed vs. adequate reconstitution in a single center in colombia, south america. methods: a retrospective cohort study was conducted on consecutive adult haploidentical-hsct recipients at a tertiary referral center. cd +cells, cd +cells, cd +cells, and immunoglobulins levels were monitored before hsct, at first month, and then every three months for the first two years post-transplantation. descriptive statistics were used to analyze patient's clinical characteristics. the kaplan-meier method was used to assess overall survival (os) and relapse-free survival (rfs) rates. results: twenty-six patients were included ( % were male), with a median age of . years (range - ). the most common indication for haploidentical hsct was acute lymphoblastic leukemia (n= , . %), followed by non-hodgkin lymphoma (n= , . %) and myelodysplastic syndrome (n= , . %). all patients received gvhd prophylaxis therapy with cyclophosphamide, tacrolimus, and mycophenolate mofetil. fifteen patients ( . %) presented cytomegalovirus reactivation ( / at risk), patients ( . %) epstein-barr virus reactivation, and patients ( . %) developed adenovirus infection. median time to neutrophil engraftment (neutrophils> . × /l) was days (range - ) for the patients recipients of peripheral blood progenitor cells (pbpcs) and days (range - ) for the three remaining bone marrow recipients. platelet engraftment, defined as > , platelets/ mm background: daratumumab is a human monoclonal antibody directed against the glycoprotein cd that is overexpressed on the surface of plasma cells in multiple myeloma patients. it is approved as second line therapy either as single agent therapy or in combination with lenalidomide or bortezomib for the treatment of patients with relapsed/refractory multiple myeloma. despite the curative potential of an allo-sct, the high relapse rate remains a clinical problem. data addressing the choice of an optimal salvage therapy regime for these heavily pre-treated patients is missing. methods: from april till november a total of patients (male, n= ) with the median age of years ( - ) received daratumumab as a salvage therapy for relapse of multiple myeloma after allo-sct at the university of hamburg. prior to allo-sct all but one patient had received an autograft, patients even ≥ autografts and patients also a . allograft. the median number of salvage lines post-transplant and prior to first daratumumab infusion was ( - ). these salvage regimens included cyclophosphamide, etoposide, bortezomib, lenalidomide, pomalidomide and carfilzomib. daratumumab was started at a median of months ( - ) after relapse/ progress and initiated as single agent therapy in all patients. concomitantly, patients received either an immunomodulatory drug (lenalidomid, n= ; pomalidomid, n= ) or a proteasome inhibitor (bortezomib, n= ) during a later course of daratumumab infusions. combination therapy was initiated when a slow rise of paraprotein and/or free light chains or no response to monotherapy was observed (median at the th infusion). results: the median number of infusions was . twenty adverse reactions were observed in of ( %) patients: dyspnea (n= ), bronchospasm (n= ) shivering (n= ) , cough (n= ), musculoskeletal pain (n= ), acute coronary syndrome (n= ), skin rush (n= ), facial edema (n= ), pressure on eyes (n= ). all adverse reactions appeared during the first infusion and were mostly mild or moderate (ctc - , n= ). tolerance of the following infusions improved and in none of the cases therapy had to be stopped due to adverse events. three patients developed late onset infections (pneumonia, n= ; urinary tract infection, n= ) followed by temporarily therapy interruption. with a median follow-up of months after the first administration of patients remain alive . %). one patient died due to progress of myeloma and another died due to severe infection/sepsis. of patients responded ( %; pr, n= ; vgpr, n= ; cr, n= ) to the therapy with daratumumab. the responses (decrease of paraprotein and/or free light chains ≥ %) occurred at a median of days ( - ) after the first administration and lasted for . months ( . - . ). conclusions: daratumumab shows an encouraging efficacy and acceptable toxicity profile in patients with relapsed/refractory myeloma after allo-sct. further studies are needed to investigate the role of the combination therapy with immunomodulatory drugs or proteasome inhibitors in this setting. disclosure: nothing to declare p clonal plasma cell detection by high sensitive flow cytometry in aphaeresis product is poor prognostic and not increased by use of plerixafor alone background: in an earlier from our center we have demonstrated that residual clonal plasma cells (cpc) decrease both overall survival (os) and disease free survival (dfs) (ash ).plerixafor is a selective antagonist of cxc chemokine receptor (cxcr ) and able to mobilize human peripheral blood stem cell (pbscs) by acting synergistically with g-csf.the purpose of this study was to evaluate the safety and efficacy of plerixafor in myeloma patients who were proven poor mobilizers and specifically to assess the flow cytometric measurement of residual clonal plasma cells in the apheresis products. methods: patients with a diagnosis of mm who underwent auto hsct at our center between january -november were retrospectively analyzed.out of patients, patients received plerixafor as mobilization regimen due to poor mobilization with g-csf.pbsc grafts were tested for the presence of clonal pcs (cpc) and the number of normal pcs (npc) by multi-parameter flow cytometry (fcm).the acquisition of the cells was performed using the navios flow cytometer beckmancoulter) .upon the daily checks of the instrument, x cells for each sample were acquired and the collected data was analyzed using the kaluza software (beckmancoulter,usa). results: patient demographics are shown in table .the majority of patients were male and median age was years in the plerixafor group.the median interval from time of diagnosis to mobilization and follow-up from mobilization were . months and . months in plerixafor group, respectively. cpc contamination in the pbsc grafts was detectable in and patients with counts ranging between - . x - and - . x - in g-csf alone and g-csf+plerixafor groups, respectively (p= . ).there were no significant differences in the proportion of the patients with graft contamination between subtypes of mm in both groups. one hundred (gcsf/plerixafor; / ) patients had pre-asct pet-ct imaging done with (gcsf/plerixafor; / ) have active lesion at the time of mobilization. statistically significant association could not be demonstrated between the disease < cr status at mobilization and the number of apc in the apheresis product in both groups (p> . ).twelve of patients from plerixafor treatment arm proceeded to transplantation within median . months.the best overall response to induction treatment is shown in table .thirtyfour patients from the g-csf alone arm and patients from the g-csf+plerixafor arm died during the follow-up (p= . ).disease progression was seen in patients from g-csf alone group and patients from g-csf+plerixafor group of the study(p= . ).estimated mean os was better among patients w/o apc contamination in plerixafor group, respectively ( . ± . mos vs . mos; p= . ). conclusions: our results on and few plerixafor used patients show that clonal plasma cells are detectable by multiparametric flow more frequently when patients are poor mobilizers and require plerixafor.the clonal pc contamination can be attributed to the myeloma biology as manifested by higher number of lines induction regimens and pet positivity among the plerixafor-required patients. the overall and disease survival was impaired by residual clonal pcs in the graft but not by plerixafor per se. neither was the content of clonal pcs differed from others.thus the cxcr shared by hsc and myeloma cells do not cause a myeloma mobilization. clinical trial registry: -disclosure: nothing to declare prognostic factors for overall survival after allogeneic hematopoietic cell transplantation in multiple myeloma patients all factors with significant influence on pts survival were included multivariate analysis (cox regression model) but only re-admission in the first days demonstrated impact on os (hr , ; p= , ) . conclusions: we analyzed risk factors for survival in mm pts who received allo-hct. our study identified disease-related risk factors like iss and transplantationrelated factors such as hct-ci and pam, hospital readmission, days of hospitalization and cmv reactivation that were associated with worse long-term survival. in our series, the most frequent death and re-admission cause was infection, so focusing the efforts in reduction of infection could have a beneficial impact on improvement of survival in mm undergoing allo-hct. [[p image] . figure ] disclosure: there is no disclosure. novel protocol for autologous hsct in multiple myeloma: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) after melphalan conditioning is still a part of standard treatment of multiple myeloma patients. traditional transplantation of frozen stem cells poses additional risk for the patients connected with dmso and central venous catheter. the transplantation of fresh cells is an option -however, most mobilization protocols are either low-efficient (g-csf), expensive (g-csf + plerixafor) or toxic (standard dose chemomobilization) to directly proceed to transplantation in this fragile group of patients. we describe here the novel combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells (fc) and compared with control group of consecutive patients transplanted with standard protocol (sp) (transplantation with frozen cells) between july and october . protocol of ambulatory mobilization was: mg/m² of arac on days + and + and g-csf at the dose μg/kg/day from day + and escalated to μg/kg/day split into two doses + to + , apheresis started on day + (or later) and finished when at least . x e cd + positive cells were collected. the collected cells were split in three equal parts: ) for use as fresh transplant ) frozen for possible nd transplant ) frozen as backup. results: there were transplantations with fresh cells and transplantations with frozen cells compared. both groups had same mobilization protocol -ambulatory low dose ara-c. the median age and number of transplanted cells was similar in both groups ( vs , p= . ; . vs . cd +/kg, p= . conclusions: we present novel approach that allows direct ahsct after chemo mobilization in all patients who are treated with melphalan. we show that it is not only feasible to do ahsct directly after chemomobilization but also the results may favour this approach when compared with current standard. disclosure: nothing to declare background: high dose chemotherapy followed by autologous hematopoietic cell transplantation (hsct) is considered, since the nineties, the standard of care for patients aged less than - years old with newly diagnosed multiple myeloma (mm). however, the optimal induction treatment prior to hsct to reduce the tumor burden has changed during the last few years. improved regimens have shown to be able to increase the quality of the pre-hsct response, which might subsequently impact on the post-hsct response, which has been proved to be associated with a longer pfs. we recently changed the induction therapy for pts candidates to hsct. in this analysis, we aimed to check if newer regimens impacted on pretransplant responses, and how auto-hsct changed the pre-hsct status. methods: all the auto-hsct for mm patients performed in our center from january through august were included in the analysis. median age was years (range: - ). pts were male and were female. durie-salmon stage was distributed as follows: i ( . %), ii ( . %) and iii ( %); % had creatinine > mg/dl. iss was: ( %), ( . %), and ( . %). type of monoclonal component was: igg ( . %), light chains ( . %), iga ( . %) , and non-secretory ( . %). . % had bence jones proteinuria. conditioning regimen was melphalan mg/m in ( . %), melphalan - mg/m in ( . %), and other in ( . %). results: pre-transplant therapy was: vcd in (mostly in - ), vtd/vrd/krd in (mostly in - ), and others in cases. status of the disease at transplant was: cr/vgpr in , pr in , and sd in . distribution of pretransplant response based on the type of induction is shown in table . peri-transplant mortality was %. day + mortality was . % ( pts), due to progressive disease. as shown in table , all patients ( / ) who obtained cr pre-hsct, maintained the response at day + post-hsct. among the patients in vgpr at hsct, ( . %) became into cr, and ( . %) maintained the response. the cr rate at post-hsct increased % compared to pre-hsct ( versus pts). altogether, after hsct pts ( . %) improved and ( . %) maintained the pre-hsct response. during the last period of time, pts started on post-hsct maintenance/consolidation, mainly with lenalidomide. conclusions: ) with the new chemotherapeutic schemes, . % of patients underwent hsct in cr or vgpr; ) majority of pts ( . %) consolidated or improved the pre-hsct response; ) cr increased substantially ( . times) after transplant; ) optimized induction regimens, along with auto-hsct followed by the recently licensed use of maintenance therapy with lenalidomide, might result in a better pfs of patients with mm. background: autologous stem cell transplantation (asct) is commonly used in treatment of patients over years with multiple myeloma (mm), however the safety and efficacy of this procedure is debatable. methods: we conducted a retrospective review of mm patients who underwent asct from to at our institution. the purpose of this retrospective study was to compare the -day mortality, time to engraftment, and incidence of grade - toxicities in elderly mm patients with younger patients taking into account comorbidity information. other secondary end points measured were overall survival (os) and progression-free survival (pfs). results: a total of patients were analysed and categorized by age as young patients ( to y; n= ) or elderly ( to y; n= ). the compared groups did not differ in terms of gender, ecog, hct-specific comorbidity index (hct-ci), and disease status at asct. melphalan in a dose of mg/m was used as preparative regimen in % of younger patients, and in % of the elderly (p= . ). the remaining patients received mg/m of melphalan or lower dose (range, - mg/m ) due to hct-ci > or age, on the physician discretion. in the whole study group there were no transplant related deaths within the first days of asct. stratifying by age, there was no statistically significant difference concerning febrile neutropenia (fn) incidence, which was observed in % of younger patients, and % of elderly. in contrast, fn was observed more frequently in patients with hct-ci > ( % vs %, p= . ). grade - infections were more frequent in older patients ( % vs %, p= . ), but no difference was found in grade - infections incidence rate such as pneumonia, uroinfections and neutropenic enterocolitis ( % vs %, p= . ), nor grade - and - noninfectious toxicities ( % vs %, p= . , and % vs %, p= . , respectively) . the median time to granulocyte engraftment was days (range, - days) in elderly and was comparable with younger patients. the time to platelet recovery was also similar. after the median follow-up of months for survivors, os at months was % in both groups. pfs at moths was % for younger patients, and % for elderly (p= . ).however, the association between pfs and the dose of melphalan used in conditioning was observed. pfs probability at months for patients conditioned with the dose of mg/m , mg/m and mg/m was %, % and %, respectively (p= . ). conclusions: our data show that asct in transplant eligible mm patients ≥ years of age is safe and provides similar outcomes as seen in younger patients. disclosure: nothing to declare is mobilization with chemotherapy effect response in the multiple myeloma? background: high dose melphalan therapy with autologous stem cell support is a standart approach in symptomatic multiple myeloma patients. response rates increased with the novel anti myeloma agents and the use of chemotherapy for stem cell mobilization should be questioned. the purpose of this study is to determine the effect of cyclophosphamide used during stem cell collection on disease response and transplantation results. methods: we retrospectively collect data from myeloma patients who underwent autologous stem cell transplantation (asct) in ankara university medicine faculty, blood and bone marrow transplantation unit between january and november . patients who received cyclophosphamide protocol for stem cell mobilization were included in the study. disease response were determined according to international myeloma study group (imwg) criteria before and after the cyclophosphamide. transplant responses and their effects on survival were also indicated. results: after the diagnosis of mm, patients (male/ female: / ; median age: years (between - years)) with median follow-up of . months (between , - , months) underwent asct at a mean of , ± , months.. forty-one patients were evaluated before and after cyclophosphamide (table ). in % of the patients, the disease response was not altered by cyclophosphamide therapy, and % of the patients improved their response status. post-transplant response rates of patients who underwent stem cell mobilization with cyclophosphamide are also shown in table- . the mean survival of the patients was , ± , months. when patients were grouped according to changes in response status before and after cyclophosphamide; there was no statistical difference between mean calculated survival (improved response, disease progression and stable disease; , ± , months, , ± , months and , ± , months respectively, p= . ) (figure- ) . the rates of -year and -year overall survival (os) of the patients with no response to cyclophosphamide treatment were as follows; , %± , % and , %± , % respectively. thirteen patients who were followed up median months after transplantation died at an average of , ± , months; of these deaths were caused by the infection after transplantation. conclusions: in our study, it was observed that the use of cyclophosphamide for cd + stem cell mobilization did not change the disease response rates by %. the posttransplant survival rates of mm patients who had progressive disease after cyclophosphamide use were lower. however, these results warranted confirmed by randomized controlled trials. clinical trial registry: -disclosure: nothing to declare results of a single center experience: an attempt to augment conditioning regimen in first autologous stem cell transplantation treatment of multiple myeloma (mm) continues to evolve in the era of novel agents. the addition of bortezomib to highdose melphalan (bor-hdm) has been reported by several groups, and it has been outcome and toxicity profile is comparable to high dose melphalan (hdm) alone. the aim of this retrospective study was to evaluate the outcome of the bor-hdm conditioning regimen on overall response for patients with mm undergoing first single asct at our institution. methods: this retrospective single center study reviewed consecutive myeloma patients who had received the first asct either with bor-hdm (n= , m/f= / ) or single agent hdm (n= , m/f= / ) conditioning regimen. in the single agent hdm conditioning regimen, melphalan was administered intravenously at a total dose of mg/m on day - and - and stem cells were infused on day . in the bor-hdm group, melphalan mg/m was administered on day - . bortezomib was administered intravenously at a dose of mg/m on day's - , - , + , and + as described in a phase study by intergroupe francophone du myeĺome (ifm). results: all consecutive patients who underwent single asct from january to march using bor-hdm as conditioning or hdm were evaluated. conditioning regimen was hdm in patients and bor-hdm in patients. median age was significantly lower in bor-hdm conditioned asct compared to hdm group ( years vs years, p= ). there was no significant difference for mm subtype, iss stage at diagnosis, prior treatment line among hdm vs bor-hdm cohorts (p> . ). after a median of cycles of induction chemotherapy, patients in the bor-hd exhibited ≥vgpr of . % (n= ) compared to . % (n= ) in the hdm group (p= p> . ). pre-asct immune response (if (-)) was reported in . % of patients treated with hdm, higher than that seen in the bor-hdm group ( . %) (p= . ). nine ( . %) patients achieved post-asct immune response (if (-)) ≥vgpr compared to ( . %) in the hdm group. at the time of this analysis, ten patients in the bor-hdm group and in the hdm group had already died, respectively (p> . ). a total of ( . %) patients in the bor-hdm group and ( . %) patients in hdm group had already progressed (p> . ). estimated mean os and pfs was shorter for group treated with bor-hdm; . ± . mos and . ± . mos vs. . ± . mos and . ± . mos, respectively (p> . ) (figure- ) . we could not demonstrate the impact of pre or post transplant ≥vgpr immune response on survival and disease free survival. there was no engraftment failure observed on either treatment group and no worsening peripheral neuropathy was developed in the bortezomib arm. conclusions: this single center experience on a small patient pool was able to repeat the prospective randomized study results of ifm. further studies are warranted to explore this regimen, especially when induction treatment with novel agents are employed, with special emphasis on the high-risk myeloma patients where response rates are good but sustainability remains an issue. disclosure: nothing to disclosure the efficacy and safety of bortezomib plus busulfan/ melphalan as conditioning regimen in multiple myeloma undergoing autologous stem cell transplantation: phase / study background: bortezomib have a powerful antimyeloma activity and was regarded as backbone of therapy in the past decade but its safety and efficacy as a part of busulfan/ melphalan conditioning regimen of autologous stem cell transplantation is yet to be shown. methods: a phase / trial to explore the safety and activity of a bortezomib on days - , - , and + added to a conditioning regimen with busulfan and melphalan (bumel, . mg/kg/day and busulfan during day - and - , mg/ m /day of melphalan on the day - ), in multiple myeloma (mm) patients who received autologous stem cell transplantation following bortezomib-based induction chemotherapy. in phase , escalating doses ( . , . , and . mg/m ) of bortezomib with bumel were administered in each group with three patients. with determined maximum tolerated dose of bortzomib at a . mg/m /day, cohort with patients were analyzed for phase . results: in phase , no dose limiting toxicity was observed at a . mg/m /day of bortezomib. in phase , overall responses at months was shown as % of very good partial response (vgpr) or better and % of complete response (cr), whereas post-transplant overall best response included % of vgpr or better, and % of cr, respectively. with median follow-up duration of . months, median progression-free survival (pfs) was . months. the probabilities of years-pfs and overall survival (os) were . % and not estimable, respectively. especially, high-risk cytogenetics were associated adverse survival outcome compared to standard-risk cytogenetics, respectively (pfs, . vs. . months, p= . ; os, . vs. . months, p= . ) . with median days and days for neutrophil and platelet engraftments, any graft failure or delayed engraft was not observed. the common grade or severe non-hematological adverse events included neutropenic fever ( . %) and stomatitis ( . %). except three cases with transplant-related mortality due to sepsis, other adverse events were manageable. conclusions: these results demonstrate that bortezomib is safe and can be a part of conditioning regimen in combination with bumel, for patients with transplanteligible multiple myeloma. clinical background: allogeneic stem-cell transplantation (allo-sct) is one of treatment option for patients with multiple myeloma (mm) refractory to novel agents. the reports on allo-sct for mm are limited and it is an important issue to argue appropriate conditioning regimens and stem-cell sources, and patient population who will benefit from allo-sct. methods: we retrospectively analyzed consecutive patients who received allo-sct for relapsed and refractory multiple myeloma (rrmm) between oct and july at japanese red cross medical center. characteristics of patients, progression-free survival (pfs), and overall survival (os) were analyzed. results: median age at allo-sct was (range - ). twelve patients were male and were female. myeloma type were igg: , iga: , igd: , and bence-jones: . stem-cell sources were peripheral blood from hlamatched related donor (rpbsct): , bone mallow from hla-matched unrelated donor (mud): , bone marrow from hla-mismatched donor (mmud): , and cord blood (cb): . twenty-three of patients received flu/mel-base, one patient received bu/mel-based, and one patient received etoposide/cyclophosphamide-based conditioning regimens. twenty-two patients who transplanted after received gy of total body irradiation (tbi). responses before allo-sct were cr: , vgpr: , pr: , sd: . five-year pfs was % ( %ci: - ) and -year os was % ( %ci: - ). ten patients died during observation period and causes of death were primary disease: and treatment-related mortality: . patients with vgpr or better before allo-sct showed significantly better pfs (p= . ) and os (p= . ) as compared with others. female recipients showed significantly better pfs (p= . ) and os (p= . ) as compared with male recipients. recipients of mmud showed significantly better pfs (p= . ). among patients surviving, patients received treatments including maintenance therapy. conclusions: the reason for better pfs and os in female recipients is unknown. it is interesting that recipients of mmud showed better pfs, suggesting graft-versusmyeloma effects. allo-sct can be an effective treatment option if patients and stem-cell sources are appropriately selected. disclosure: authors declare that there are no conflicts of interest. second autologous hematopoietic stem cell tranpslant versus chemoimmunotherapy in relapsed multiple myeloma after first transplantation: single center data background: combination therapy, mostly triple, followed by autologous hematopoietic stem cell transplantation (auto-hct) is widely accepted as the first-line standard therapy for multiple myeloma (mm). despite the availability of agents such as new immunomodulatory drugs (imids), proteasome inhibitors (pis), histone-deacetylase inhibitors and antibodies, it is still possible to achieve longer and deeper responses, however, multiple myeloma is still not cured and relapse is inevitable. the availability of these novel agents has increased questions for determining optimal treatment of patients with relapse after the first auto-hct. methods: we retrospectively analyzed patients who relapsed according to international myeloma working group (imwg) criteria after st auto-hct. first group [salvage chemotherapy(ct)] (n= ) was treated with only chemoimmunotherapy because of early relapse or refractory first auto-sct (within months), ineligible to second transplantation because of co-morbidity, unwillingness to transplant. second group (n= ) (salvage transplantion) was treated with second auto-hct as a salvage therapy. consolidation and long term maintenance treatments were used in both groups. results: there was no difference in sex and age between salvage ct and auto-sct groups [female/male: vs / vs ; ]. the best response after salvage auto-sct was complete remission (cr) in , %, partial remission (pr) in , % patients, while cr in %, pr in , % patients treated with salvage ct. progression free survival (pfs) were significantly better in second transplant group (pfs; % on the first year; , % on the second year after transplant vs % on the first year; % on the second year after the salvage therapy in chemotherapy patients)[p: , ]. overall survival (os) in salvage auto-sct group was longer than salvage ct ( , % . %), although it did not reach a statistical significance (p> . ). time to achieving the best response after salvage auto-sct and salvage ct was ( - ) month versus , ( - ) months [p: , ]. grade or nonhematological toxicities were similar (auto-sct %, salvage ct %) in both groups. conclusions: salvage auto-hct may provides longer progression free survival with similar toxicity profile according to chemoimmunotherapy especially in patients with sensitive to first auto-sct. it is suggested that earlier and better responses, long-term progression free survival can be achieved with salvage auto-sct. we believe that there will be statistical significance in os such as pfs by increasing the number of patients. the authors believe that large scale randomized clinical trials are needed for optimal treatment of relapsing multiple myeloma after first auto-sct. disclosure: nothing to declare background: one of the conditions for successful transplantation of autologous hematopoietic stem cells (auto-hsct) in patients with multiple myeloma (mm) is the timely recovery of hematopoiesis, which is associated with the quantitative and qualitative characteristics of the graft. one of the key indicators is the content of cd + cells in the autograft, which depends on many factors. some of them are due to previous treatment, others are directly related to the patient: age, stage of the disease, features of the hematopoietic stem cells (hsc) microenvironment. the aim of the study was to assess the influence of the immune response genes on the autograft cellularity in patients with mm. methods: А retrospective analysis of the genotyping results was performed. evaluation of loci in genes immune response and harvesting of autologous hsc in patients with mm has been made. hematopoietic stem cell mobilization regimen included cyclophosphamide g/m with granulocyte colony-stimulating factor. genotyping of the immune response genes polymorphic regions was carried out by the polymerase chain reaction with allelespecific primers. the number of cd + cells was counted on a -color facs canto ii flow cytometer. results: according to the results of the autologous transplant harvesting, two groups of patients were identified. first included patients with an autograft cellularity of more than × /kg body weight. the second group consisted of patients examined with the number of cd + cells in the autograft ≤ × /kg of the patient's body weight. comparing the identified haplotypes of the immune response genes with the cellularity of the transplantation material, it was found that the presence of the mutant allele in the homo-and heterozygous haplotypes of the il β gene (t- c) increased the chances of harvesting cellular material with a higher content of cd + cells in times (χ = . , p= . ), and the carriage of the wild type allele in the homo-and heterozygous state of the tlr (arg gln) gene is more than in times (χ = . , p= . ). currently, it has been shown that single nucleotide or amino acid substitutions in genes can lead to changes in the expression pattern of their final products: increased secretion of interleukin β (il- β) or changes in the spatial configuration and functionality of the receptors (tlr ). thus, in the presence of mutations in the il β gene, the enhanced synthesis of il- β influences on fibroblasts, immunocompetent, endothelial, epithelial and other cells, by activating hemopoiesis. in turn, the mutational status of the arg gln locus located within the tir domain of the tlr receptor in the cytosol, determines the spatial configuration of the tlr acting as a co-stimulatory receptor of cd + cells, which ensure the engraftment of the graft. conclusions: identified haplotypical features of the il β and tlr genes in patients with mm may act as predictors of the response effectiveness to mobilization of hscs in their carriers, which may contribute to the mobilization regimen optimization and will contribute to harvesting the optimal cellularity of an autologous graft. clinical trial registry: none. disclosure: authors declare no conflict of interest. differentiating diffuse from focal pattern on computed tomography: added values of a radiomics approach background: focal pattern in multiple myeloma (mm) seems to be related to poorer survival and differentiation from diffuse to focal pattern on computed tomography (ct) has inter-reader variability. therefore the purpose of this study is to assess if a radiomic approach could help radiologists in differentiating diffuse from focal patterns. methods: we retrospectively reviewed imaging data of patients with mm between january and september of whom ( men and women; mean age . ± . ) with ct, pet-ct or mri available before bone marrow transplant. two general radiologist evaluated in consensus only ct images to define a focal (at least one lytic lesion > mm in diameter) or a diffuse (lesions < mm, not osteoporosis) pattern. radiomic analysis on ct thinslice images was then applied with regions of interest (rois) done by one researcher not expert in medical imaging or mm blindly to the condition of the patients. the reference standard to differentiate diffuse from focal pattern was done by radiological evaluation of two expert musculosketal radiologists blinded to the clinical data reviewing ct, mri and pet-ct images. n= radiomics features were extracted and evaluated with an open source software. mann-whitney u test for unpaired data with bootstraps samples was used to compare radiomics features of diffuse and focal patterns and then feature reduction was done to avoid over-fitting. receiver operator characteristic (roc) analysis with area under the curve was done to compare radiologists and radiomics evaluation against reference standard. reading time to perform radiomic analysis was also estimated. results: the pathological group included: diffuse and focal patterns. after feature reduction, features were different (p< . ) in the diffuse and focal patterns (n= / features were shape-based: majoraxislength and sphericity; n= / were gray level run length matrix (glrlm)). mg/kg). a number of eleven patients did not receive any additional immunosuppression except of post-cy. results: after a median follow up of . months (range . - . ) patients were alive. the -year probabilities of pfs and os were % ( - %) and % ( - %).the cumulative incidences (cis) of relapse and nrm at years were % ( - %) and % ( - %), respectively. lower serum albumin level at transplantation (≤ g/dl) was associated with increased relapses (hr . ( . - . ), p= . ) and nrm (hr . ( - ), p= . ) and resulted in poorer pfs ), p= . ) and os ), p= . ). mmud and haploidentical donors were associated with poorer nrm (hr . ( . - . ), p= . ), and resulted in decreased pfs ), p= . ). the high-risk cytogenetic at diagnosis showed no impact on survival. the cis of acute (grade ii-iv) at day + and chronic gvhd at years were % ( - %) and % ( - %), respectively. absence of immunosuppressive medication beside post-cy was associated with poorer os ), p= . ). conclusions: the conditioning with bu, tt and post-cy leads to a favorable pfs and os due to low incidences of relapse and nrm for patients with multiple myeloma relapsing after autografting. disclosure: nothing to declare methods: between january and may , we included patients with mm who underwent asct and received bortezomib/lenalidomide/dexamethasone (vrd) consolidation and maintenance therapy, mainly lenalidomide(r) mg/day for days every days. results: the median age at transplant was years ( - ). forty-six ( %) of patients received r maintenance, patients received vrd maintenance for higher risk features. median duration of r maintenance was months . r dose was changed for toxicity (grade i-ii) in ( %) patients. twenty-nine ( %) patients relapsed: ( %) patients were shifted to different treatment protocols (treatment change). patients ( %) were kept on the same r maintenance (observation group) and ( %) patients had increased lenalidomide dose with dexamethasone (r/ d group). patients ( %) of the last groups required change of treatment later. the median follow up was months . median tnt was months ( - ). at years, the estimated pfs and os were % and . % respectively. the median os and pfs (from change of therapy) were and months for patients in the observation group, versus and months in the r/d group, and and months with treatment change, respectively. no statistically significant difference was noted. conclusions: our small monocentric study is limited by its retrospective design and small sample size. however, it suggests that increasing lenalidomide dose as well as adding dexamethasone in selected patients can postpone change to different lines of treatment without affecting survival. disclosure: nothing to declare can the drugs used before autologous hematopoietic stem cell transplantation have impact on cmv reactivation that results in decreased os in myeloma patients after asct? more intensive treatment regimens, such as proteasome inhibitors (pi) and/or immunomodulatory (imid) agents. we performed a retrospective, single center study to evaluate the incidence, risk factors, and outcomes of cmv infection in patients with mm who underwent asct with a high-dose melphalan-based regimen. methods: this study involved a retrospective review of all patients with who underwent asct between january and november at our stem cell transplantation center. a total of consecutive adult patients with a diagnosis of mm (median age at diagnosis: , range: - ) underwent asct following induction treatment with novel agents (pis and/or imids). all patients received antiviral prophylaxis with acyclovir mg/day (n= ) or valaganciclovir mg/day (n= ). results: baseline patient characteristics, according to induction treatment, are summarized in table- . one hundred-five of the patients ( . %) were cmv iggpositive before asct. overall, . % (n= ) of cmvseropositive patients developed at least one episode of cmv viremia (cmv dna > copies/ml) after a median months (range; - mos) follow-up. persistent cmv viremia (detectable cmv dna load in more than sequential plasma specimens) occurred in . % ( of ) of the seropositive asct recipients and all of them were preventive treated with ganciclovir (n= ) or valganciclovir (n= ). the time from stem cell infusion to the development of cmv viremia ranged from days to days. none of the patients with untreated viremia developed identifiable cmv sequelae. no case of primary infection in seronegative patients at transplant was observed. adding to that none of the patients developed cmv disease post asct. if we analyzed the subgroups of patients according to induction therapy (pi-based, imids, pi+imid), the incidence of post-asct cmv reactivation was higher but not statistically significant, in patients who received only pi vs pi+imid ( ( . %) vs ( . %); p= . ). in univariate analysis, we could not demonstrate the importance of induction therapy with novel agents the occurrence of a post-asct cmv reactivation requiring antiviral treatment. however, statistically significant association found between the disease < vgpr status at asct and cmv reactivation ( . % vs. . %; p= . ). after a median follow-up . months (range; - . months), there was no significant impact on pfs, however there was significant decrease in estimated mean os who had cmv reactivation when compared to those without cmv reactivation ( . ± . vs. . ± . ; p= . ) (figure- ) . conclusions: cmv establishes lifelong latency within host cells and in the setting of impaired cellular immunity; cmv may reactivate from latency, disseminate, and directly cause life-threatening disease. our data suggests that mm patients treated with pi-based induction regimens and immunological response < vgpr at time of asct seem to have higher risk of developing symptomatic cmv reactivation. however, further studies on a large number of patients are warranted to clarify these findings. clinical background: high-dose therapy followed by autologous stem cell transplantation (asct) has been shown to prolong survival in patients with multiple myeloma (mm) in randomized trials. however, these trials only include patients aged < years. data regarding safety and outcomes in this patient population is lacking. methods: the aim of this study was to compare safety profile and outcomes in mm patients younger and older than years-old who underwent asct in our unit from july to october . patient's demographics, clinical characteristics, transplant related variables and probability of admission to the intensive care unit (icu) were analyzed. patients aged < and ≥ years-old would be called m and m , respectively, from now on. sorror index was used to estimate risk of mortality in the two cohorts. results: a hundred and eleven patients with mm underwent asct in the study period. median age was . years-old (range - ) and . % were male. thirtythree ( , %) patients were ≥ years. the probability of having a high risk comorbidity index was similar in both groups (m , vsm , %). the median cells obtained in the apheresis procedure was . x ( , - . ) in m compared to . x ( . - - ) in m . there were no differences in median admission lenght between the cohorts (m : days vs m : days). median days for neutrophil recovery above was days in both groups with a wider range in m ( - ) compared to m ( - ) . no differences were found in platelet recovery above . (m days vs m days). median packed red blood cells and platelets transfusions were ( - ) and ( - ), respectively, in m . in m cohort, they were ( - ) and ( - ), respectively. the incidence of grade - mucositis in m and m was . % and . %, respectively. there were no statistically significant differences in terms of using morphine for pain control between the two cohorts (m , , % vsm , , %). none patient requiered total parenteral nutrition (tpn) in group m and only one in group m . the incidence of icu admission was . times higher in patients aged ≥ than in patients < years-old , % vs , %), but differences were not statistically significant (p = . ). there were no deaths during the transplant procedure in any of the cohorts conclusions: ) in our series, high-dose therapy followed by autologous hematopoietic cell transplantation in mm patients aged ≥ was feasible. ) transplant procedure in older patients was as safe as in patients < years-old. ) no differences were found in terms of graft, transfusion support, transplant related complications and length of admission. ) age should not be a limiting factor in considering the modality of asct in this patient population disclosure: nothing to declare the correlation between the kinetics of peripheral blood counts and the response to treatment after high-dose melphalan with stem cell support in multiple myeloma patients background: the long-term survival of mm patients has dramatically increased in the last years, particularly for younger patients. this is attributable in part to the introduction and development of high dose chemotherapy with melphalan with stem cell support (hdm-asct). currently, frontline asct is still considered the standard of care for all eligible patients. many prognostic factors pre and post transplantation have been identified, e.g.: age, comorbidities, cytogenitcs, response to treatment and disease status prior to and post transplantation. to our knowledge there is no data correlating between kinetics of counts response to melphalan and prognosis. our aim was to assess the prognostic significance of the neutrophil and platelets decaying counts after high dose melphalan. methods -we retrospectively analyzed our cohort of multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years - . the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. methods: we retrospectively analyzed our cohort of multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years - . the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. results: factors associated with prolonged os in univariate analysis were: iss stage (p= . ), ≤ lines of treatment prior to asct(p< . ), favorable cytogenetics(p= . ), response to treatment (pr or better, p= . ) and rapid linear neutrophil decay (p = . ). in multivariate analysis, only ≤ lines of treatment before hdm-asct and rapid linear neutrophils count decay remained statistically significant for os prolongation. no predictive threshold value of the neutrophil decay incline was found. improved pfs was associated with ≤ lines of treatment prior to asct, and the response status after hdm-asct (p= . , p= . ). additionally, toxicity evaluation showed prolonged neutropenia to be associated with inferior os (hr = . , p= . ) and rapid exponential decay of neutrophil counts to correlate with higher incidence of mucositis (p = . ). fast platelet decay was associated with delayed platelet engraftment (p< . ) conclusions: we have shown that rapid linear decay in neutrophil counts predicts better os without a significant benefit in pfs in mm patients undergoing hdm-asct. this discrepancy might reflect the problematic estimation in a retrospective analysis of pfs. rapid decrease in neutrophils and platelet counts was associated with more toxicity: higher mucositis rate and delayed engraftment, respectively. therefore a rapid decay of blood counts after hdm-asct appears to be an in-vivo phamacodynamic marker of higher efficacy and toxicity of melphalan. disclosure: nothing to declare p do we need to freeze hematopoietic cells for autotransplants in patients with myeloma conditioned with melphalan? daniel garcia belmonte , beatriz aguado bueno , miguel herrero coderch , rafael de la camara background: multiple myeloma (mm) is the most frequent indication of auto-hsct, representing % of all auto-hsct in (passweg jr. bmt ; : - ) . nearly all are performed with peripheral blood progenitor cells (pbpc), and melphalan mg/m is considered the gold standard conditioning regimen. the standard procedure consists in obtaining progenitors, cryopreserved with dimethyl sulfoxide (dmso) and stored and subsequently thawed and re-infused in the patient on day . the procedure of cryopreservation is expensive and has some inherent toxicities (dmso) and loss of cells during the procedure. several groups have used non-cryopreserved progenitors showing some benefits compared with cryopreserved transplants, mainly a faster engraftment and a shorter length of hospitalization. objective: to compare noncryopreserved vs cryopreserved auto-hsct in mm methods: we perform an unicentric, retrospective study on consecutive first auto-hsct mm patients transplanted with pbpc between nov- and oct- , and conditioned with high dose melphalan ( mg/m ). the median follow-up was days (range: - ). patients received non-cryopreserved and cryopreserved auto-hsct. patients characteristics, without differences between non-cryopreserved vs cryopreserved: women/men ( / ); median age was years (range - ); in the majority auto-hsct was done as consolidation after first line therapy ( %); year of transplant ≤ ( %), ≥ ( %). the number of infused cd cells were not different: median . x /kg (range . - . ) in noncryopreserved patients and . x /kg (range . - ) in cryopreserved patients. results: we didn´t observe significant differences in the day of engraftment between non-cryopreserved vs cryopreserved although always was a little bit faster in the noncryopreserved group with a tendency to faster platelet engraftment (> /mm ): > platelets/mm (median day: vs . , p . ), > platelets/mm (median: vs days, p . ); > neutrophils/mm (median: vs . days, p . ). the media of days of hospitalization was shorter in non-cryopreserved patients ( vs days) although not statistically significant (p . ). transplantrelated mortality at day + was % in both groups. overall survival at years was not different: . % in in non-cryopreserved vs . % in cryopreserved patients (kaplan-meier, log-rank p . ). the accumulative incidence of relapse at the median follow up ( days) was similar: . % in non-cryopreserved vs . % in cryopreserved patients. conclusions: in our short experience, auto-hsct with non-cryopreserved pbpc in myeloma patients conditioned with high dose melphalan obtain similar results to those performed with classical cryopreserved pbpc and might has a faster platelet engraftment and shorter length of hospitalization. if no advantages are associated with cryopreservation, the simplicity of using fresh product is appealing. disclosure: nothing to declare p abstract withdrawn. . results: a early death is observed in one pt (group ) and pts(group ). the median delay of aplasia is days ( - ) and days ( - ) respectively. in group , among the evaluable pts, / ( %) are in cr, pts in pr and refractory. in group , cr: / ( %), pr: and refractory. a relapse is observed in pts/ ( , %) in group and pts/ ( %) in group with a frequency of % and % respectively in the first months. at months: pts/ ( %) in group and pts/ ( %) in group are dead. at months: pts ( %) and pts ( %). at months: pts ( , %) and pts ( , %). the overall survival (os) of the group and group pts were % and % at months; . % and . % at months; % and , % at months respectively (without significant difference). the event free survival (efs) of group and pts were % and , % at months, % and % at months and % and % at months respectively (without significant difference). conclusions: these protocols with equivalent toxicity allow obtaining of long-term equivalent results on the response rate early transplant, on the rate of relapse and on the overall survival. these results are identical to those of fermand ( ) . disclosure: nothing to declare background: dimethylsulfoxide (dmso) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. it is toxic to both cells and patients at temperatures above o c. reduction of this effect is achieved by either washing of cells after thawing or by reduction of dmso during freezing and storage. the latter requires addition of extracellular cryoprotectants to the freezing media. we assessed the effect of low dmso concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma. methods: cells were non-programmed frozen and stored at - o c in a cryoprotectant solution achieving final concentrations of % dmso, . % of hydroxyethyl starch (hes, weight average molecular weight da) and % of human serum albumin. the cell concentration in the frozen product for the first patients ( transplantations) varied between x and x cells/ml. in an attempt to reduce the amount of dmso infused, for the rest of the patients (n= ; transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma. the average age of the transplanted patients was ( - ). the cells were bedside thawed at o c water bath. the average cell dose was , x /kg ( , - , x /kg). results: viability of the stem cells following thawing assessed by trypan blue exclusion was , % . the hematocrit of the frozen cells had no effect on cell viability ( , %(low) vs , %(high)). the major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning. the average time for hematological recovery was , days (between and ) for the neutrophils, and , (between and ) days for the platelets. there was no significant difference in viability and hematologic recovery ( , and , vs , and , ) between patients receiving cells frozen with low or high hematocrit. conclusions: dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero c and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. to reduce those side effects we use lower dmso concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of dmso to be infused to the patients. this does not affect the cell viability or the hematologic recovery of patients after transplantation. our easily performed method for unprogrammed freezing of stem cells with final dmso concentration % at - o c is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of dmso. disclosure: nothing to disclose the conditioning regimen consisted of melphalan for most of the patients. the average age at the time of transplantation was years ( - ). patients were transplanted with an average cell dose of , x /kg ( , - , x /kg) for the first transplantation and , x /kg ( , - , x /kg) for the second one (every patient received the same cell dose as for the first) with average cell viability , % ( - %), with little difference between first and second transplantation. results: the average time for hematological recovery was , (between and ) days for the neutrophils, and . (between and ) days for the platelets. we found no correlation between the cell dose and the hematological recovery. there was no difference in the hematopoietic recovery between the first and the second transplantation in the patients, who underwent tandem or two transplantations. conclusions: recovery time is considered by some to be a function of the effective stem cell number. we did not find such correlation, probably because in the analyzed group all the patients, except four of them, received a dose greater than x /kg cell, which is accepted as a safe dose for autologous stem cell transplantation. disclosure: nothing to disclose p plerixafor-mobilized patients have a high risk of noninfectious fever during engraftment after autologous peripheral blood stem cell transplantation background: plerixafor enables rapid and efficient mobilization of hematopoietic stem cells. however, its impact on adverse clinical events after autologous peripheral blood stem cell transplantation (pbsct) is not fully understood. fever is one of the major complications in the preengraftment phase of pbsct. in this research, we focused on non-infectious fever around the time of bone marrow recovery and investigated whether plerixafor as mobilization therapy plays a role in engraftment fever. methods: we reviewed autologous pbscts for treatment of multiple myeloma at the japanese red cross medical center between - . non-infectious fever was defined as temperature ≥ °c with onset between two days prior to and two days after engraftment without clinical or microbiological documentation of infection. results: patients were mobilized by cyclophosphamide and filgrastim in . % (n = ) and filgrastim and plerixafor in . % (n = ). the median number of transfused cd + cells were . × /kg and . × / kg, respectively (p= . ). patients transfused with plerixafor-mobilized grafts had a higher risk of noninfectious fever ( . % vs . %, p< . ). cd + cell number or cyclophosphamide pretreatment had no relationship to non-infectious fever. the recovery of lymphocytes was more rapid in plerixafor-mobilized patients (p= . ). however, the number of lymphocytes was not associated with non-infectious fever. conclusions: combination of filgrastim and plerixafor as mobilization therapy resulted in an increased risk of noninfectious fever during engraftment comparing to mobilization with cyclophosphamide and filgrastim. while the mechanism remains unclear and requires further studies, plerixafor-mobilized grafts may result in an unintended increase in engraftment fever. clinicians should be aware of this possibility if patients are transplanted with those grafts. disclosure: ks received honorarium outside the submitted work from janssen, novartis, celgene, ono pharmaceuticals, takeda, fujimoto pharmaceuticals and srl. ti received honorarium outside the submitted work from janssen, celgene, ono pharmaceuticals and takeda. we assessed the efficacy of a new conditioning regimen consisted of decitabine (dec), busulfan (bu), cyclophosphamide (cy), fludarabine (flud) and cytarabine (ara-c) for allo-hsct in patients with mds and mds/ mpn. fifty patients were enrolled, including with mds and with cmml. patients received dec mg/m /day on days - to - , combining bu/cy/ flu/ ara-c modified preparative regimen. results: at a median follow-up of ( - ) days, the overall survival (os) was %, disease-free survival (dfs) was %, and relapse incidence was %. the incidence of severe acute (grade iii/iv) graft-versus-host disease (gvhd) was %, and that of (predominantly mild) chronic gvhd was %. os at years was % for mds patients with high risk, % for mds patients with very high risk, respectively. the survival was delightful in patients with poor-risk mutations, such as tp and asxl , ( %) and with three or more gene mutations ( %). among the total patients with poor-risk mutations in our research, only one patient ( %) with tp relapsed and one ( %) with asxl and tet died. result of continuous observation after transplantation, the percentage of nk cells in the peripheral blood of all patients who had received dec/flu/bu/cy/ara-c conditioning increased at day , which may essentially contribute to disease control post-transplantation. conclusions: in summary, the addition of a -day schedule of decitabine to a flu/bu/cy/ara-c conditioning regimen has proven feasible, with a low level of toxicity and promising early disease control especially in patients with high risk mds. disclosure: there are no conflicts of interest. the sfgm-tc mds score at day is associated with post-transplant outcomes in patients with myelodysplastic syndrome who underwent cd + selected allogeneic stem cell transplant conclusions: in patients with mds undergoing tcd-hct, the sfgm-tc score at day is significantly associated with survival. the lower incidence of acute gvhd in recipients of cd -selected transplants and the use of myeloablative condition regimens, with lower relapse, may explain the difference with the original finding that the sfgm-tc was predictive at day in unmodified grafts. disclosure the most frequent grade , toxicities were thromobocytopenia and neutropenia. infections developed in patients ( . %), neutropenic fever in ( . %). five patients ( . %) either developed or experienced exacerbation of acute graft versus host disease (gvhd), nonechronic gvhd. conclusions: azacitidine use is associated with only modest activity in patients who relapse after allo-hsct. however, in patients who respond to treatment it may allow for a durable disease control. disclosure: the authors declare no competing conflicts of interest background: somatic mutations in mds patients are closely related with clinical phenotypes and prognosis in mds patients. but whether mutations are prognostic for outcomes after allogeneic hematopoietic stem-cell transplantation (allo-hsct) remains to be elaborated. methods: targeted mutational analysis were performed on samples obtained before transplantation from patients underwent hsct. we analyzed the relationship of mutations and clinical outcomes. results: all patients carried more than one somatic mutations, most frequently in kmt d( . %), arid b ( . %), ccdc ( . %), pclo( . %), asxl / ( . %), srcap( . %), u af ( . %), dnah ( . %), ush a( . %) and tet ( . %). tp mutations were associated with higher ipss-r risk, complex karyotype and monosomal karyotype. dnah were more frequent in pediatric patients. in univariable analyses, tp mutations were related with decreased disease-free survival (p= . ); dnah mutations were related with increased disease-free survival (dfs) (p= . ). in multivariable analysis including ipss-r stratification, gvhd, hct-ci and candidate genes, dnah mutations were independently associated with better dfs(p= . ). conclusions: dnah mutations is independently associated with better outcomes in mds patients treated with allo-hsct while tp may predict unfavorable outcomes. accounting for these somatic mutations may help better selection of candidates for allo-hsct among mds patients. disclosure background: there is a controversy among experts if and how patients with mds and saml should receive cytoreductive therapy before transplant. while aiming to reduce disease burden in order to lower the risk of relapse after transplant cytoreductive therapy is associated with several drawbacks. besides a considerable risk for toxicity and mortality preventing patients to proceed to transplant cytoreductive therapy may also favour the selection of resistant clones which may be difficult to treat at relapse. methods: to address this hypothesis we retrospectively analysed the response and survival following salvage therapy in patients with mds and saml who had relapsed in median . months ( to months) after allo-sct according to their pre-transplant strategy (upfront transplantation n= %; induction chemotherapy [ctx] n= %; hypomethylating agents [hma] n= %). results: the majority of these patients received salvage therapy with hma (n= , %; aza n= , dac n= ) mostly in combination with dli, while the remaining received other salvage treatments (intensive chemotherapy n= , dli alone n= , nd transplant n= , bsc n= , miscellaneous n= , missing information n= ). when focussing on those patients treated with hma and dli it became apparent that a significantly higher proportion of patients in the upfront group ( %) achieved cr after salvage therapy in comparison to pre-treated patients ( % cr, p= . ; ctx group % cr; hma group % cr). accordingly, overall survival (os) calculated from the time of relapse was significantly longer in patients in the upfront group than in the group of pre-treated patients ( -year os % vs. %, p= . ). conclusions: overall, these findings imply that pretransplant therapy may favour the iatrogenic selection of resistant clones, which poorly respond to salvage therapy with hma and dli in case of relapse after allo-sct. furthermore, the results support the concept that an upfront transplant strategy is a promising alternative for patients with mds and saml that can be augmented by salvage therapy with hma and dli. disclosure: ts and gk received travel support, lecture fees and research funding from celgene gmbh conclusions: in our country, this procedure has shown to be feasible and we hope to improve it, with better infection control and by acquiring more experience related to the management of these patients. background: extramedullar relapse of mds is a rare complication after allogeneic stem cell transplantation. we present the case of a -year-old woman who was admitted into hospital because of insecure walking. paresis of both legs, hypaesthesia of the inner thighs, increased effort at urinating, reduced sphincter tonus, central paresis of the right arm and discreet paresis of the right facial nerve were documented at neurological exam. mri showed a large tumour of the dorsal thorax that immured the adjacent ribs and spine, affected the processus transversus of t - and invaded the spinal canal. the patient had undergone ric allogeneic stem cell transplantation five years ago for mds-eb with complex aberrant karyotype. following an uneventful course and no signs of gvhd, she had been off immunosuppression since , years. at the time of the admission the patient had slightly lowered wbc ( , gpt/l) and plt ( gpt/l) and clearly increased ldh ( u/l). methods: histology of a ct-based biopsy of the paravertebral tumour showed an infiltration of the muscles by blastous cells that were cd -, cd -, pax -positive, tdt and cd a were questionably positive. provisonal diagnosis therefore was lymphoblastic lymphoma, pox tested negative. the bone marrow was hypocellular with increased numbers of mature lymphocytes, but no definite signs of malignancy. cerebrospinal fluid revealed cells/μl with % blasts. immunotype was cd , cd , cd , cd , hla-dr positive, pox and lymphatic markers were negative. because of this we finally suspected meningeosis leucaemica. we completed the diagnostic workup with genetical and chimaerism tests and compared the result to the patients' mds before allogeneic stem cell transplantation. [[p image] . mri scan of the large thoracic tumour] results: cerebrospinal fluid (csf) cells consisted of % recipient cells, whereas peripheral blood cells were % donor. high risk mds at transplant displayed a complex caryotype including trisomy and tetrasomy , now % of the cells in csf showed trisomy and % tetrasomy . chimerism and fish of the solid tumour could not be performed, coexpression of myeloid markers within the tumour is pending. conclusions: in conclusion the patient has meningeosis as a result of exclusively extramedullary relapse of myeloid blasts originating from the initial high risk mds with blast excess and complex aberrant caryotype. the evolution of a trisomy clone to tetrasomy clone in relapse is linked to extramedullar manifestations. whether the solid tumour represents myeloid sarcoma with coexpression of lymphoid markers, extramedullary relapse of mds with lymphoid differentiation or, less likely, a separate lymphobastic lymphoma, is not yet clear. disclosure background: adoptive t cell therapy with genetically engineered t cells is a potent innovative immunotherapeutic approach for cancer treatment. unfortunately, the use of t cells redirected against tumor antigens, is severely limited by ) the difficulty in identifying appropriate cell surface antigens, that could be targeted by car t cells and ) the paucity of tumor-specific t cell receptors (tcrs) against shared, oncogenic antigens. methods: focusing on wilms´tumor (wt ), a tumorassociated antigen overexpressed by acute myeloid leukemia and several solid tumors, we designed and implemented an innovative protocol for the rapid isolation of wt -specific t cells and for the generation and characterization of a library of wt -specific tcrs displaying different human leukocyte antigen (hla) restrictions, to be exploited by tcr gene transfer and tcr gene editing. to this aim, we repetitively stimulated t cells with autologous antigen-presenting cells, including immortalized b cells, pulsed with overlapping peptides spanning the entire wt protein. t cell recognition was assessed by flow cytometry in terms of cd a expression and ifnγ production. recognized peptides were mapped by a deconvoluting grid and t cell clonotypes were longitudinally tracked by tcrαβ sequencing. results: we successfully expanded tumor-specific t cells from consecutive healthy donors, in an average of rounds of in vitro stimulations. the ability of wt specific t cells to recognize naturally processed epitopes and their on-target specificity was demonstrated upon coculture with antigen-expressing targets including primary leukemic blasts. tracking of the tcrαβ repertoire during culture led to the identification of clonotypes that recognize several tumor-associated peptides and are restricted by more than hla alleles, including hla-a* : . tcrs were then expressed via genome editing. briefly, simultaneous editing of endogenous tcr α and β chain genes was achieved using crispr/cas technology (efficiency > %), followed by transduction of t cells with lentiviral vectors encoding wt -specific tcrs (efficiency > % of cd + t cells). phenotypic characterization of edited t lymphocytes showed a major enrichment of cells harboring t stem cell memory properties. functional validation of the edited t cells is currently ongoing. preliminary results of a hours coculture experiment show that tcr edited t cells kill fresh wt + leukemic blasts, harvested from hla-matched patients, with an efficiency up to % at an effector to target ratio of to , while no killing of controls is observed. conclusions: we set up a protocol enabling consistent and efficient hunting for tumor-specific tcrs with no need for labor intensive t cell cloning. tcr genes can be easily and rapidly used to redirect t cell specificity against cancer cells by tcr gene editing. disclosure: chiara bonini: research funding from intellia therapeutics p car t cell therapy targeting relapsed or refractory cd + lymphoid disease with third-generation vector rv-sfg.cd .cd . - bbzeta maria-luisa schubert , anita schmitt , leopold sellner , , brigitte neuber , angela hückelhoven-krauss , kunz alexander , lei wang , gern ulrike , birgit michels , susanne hofmann , carsten mueller-tidow , , dreger peter , , michael schmitt , background: t cells genetically engineered to express chimeric antigen receptors (carts) directed against cd have demonstrated significant efficacy in patients with iymphoid malignancies including relapsed or refractory (r/r) b-lineage acute lymphoblastic leukemia (all) or r/r b-cell non-hodgkin's lymphoma (nhl). access to cart treatment for patients in europe has been limited so far given that the vast majority of cart trials have been performed in the united states and the p. r. of china. here we present the preliminary results of the first investigator-initiated trial (iit) cart trial in germany. hd-car- (eudract no. - - ; nct ) is a phase i/ii trial with in-house cart manufacturing which was initiated in september at the university hospital heidelberg. methods: adult as well as pediatric patients with r/r all and patients with chronic lymphocytic leukemia (cll) or nhl including diffuse large b-cell lymphoma (dlbcl), follicular lymphoma (fl) or mantle cell lymphoma (mcl) are treated with autologous t lymphocytes transduced with a third-generation car retroviral vector (rv-sfg.cd .cd . - bbzeta) targeting cd . the main purpose of hd-car- is to evaluate safety and feasibility of escalating third-generation car t cell doses ( - × transduced cells/m ) after lymphodepletion with fludarabine and cyclophosphamide. patients are monitored for cytokine release syndrome (crs), car-t-cell related encephalopathy syndrome (cres) and/or other toxicities. in vivo function, survival and anti-tumor efficacy of carts are assessed. results: to date, three patients (cll, dlbcl and mcl, respectively) have been enrolled and subjected to leukapheresis. high numbers of transduced carts were harvested on day of culture ( - x carts). transduction efficiency ranged between and %. cart products were sterile and free from mycoplasms and endotoxins. no production failure occurred and all patients received the cart product. no signs of crs or cres > grade have been observed. assessments of clinical responses are pending and will be presented at the conference along with updated technical results. conclusions: for hd-car- , gmp-conform leukapheresis as well as cart manufacturing was effective. administration, patient monitoring and follow-up were performed in-house providing independency from transport or production sites outside the university hospital heidelberg, altogether suggesting that academic cart iits are feasible in germany. clinical background: the prognosis of adult patients (pts) with relapsed/refractory (r/r) precursor b-acute lymphoblastic leukemia (all) is dismal, including with allogeneic hematopoietic stem cell transplantation (allo-hsct). blinatumomab, a bispecific cd -directed cd t-cell engager and inotuzumab ozogamycin (io), a cd -directed antibody-drug conjugate revolutionized the field, improving their outcomes. anti-cd chimeric antigen receptor t (cart) cell therapy has led to further progress and improved outcome (jacoby e; am j hematol, ). nowadays, patients with r/r b-all can be offered both therapies, but there are limited data on the safety and efficacy of cart -cell therapy post antibody treatment. we detailed our single center experience in this regard. methods: this report is a part of a single center, phase b/ study on therapy of b-cell malignancies with locally produced cart-cells (nct ). the approach uses autologous t cells with car construct that is composed of an anti-cd single-chain fv, cd co-stimulatory and cd -zeta intracellular domains. cd expression on the blasts was documented prior treatment in all pts by flow cytometry. all pts received x /kg cart-cells after lymphodepletion with fludarabin and cyclophosphamide. results: six pts ( males and females) with r/r b-all were enrolled, including one with ph-positive b-all. the median age was years ( - ). median number of prior therapy was ( ) ( ) ( ) . five pts had prior allo-hsct. four pts were given antibodies as the last therapy prior to cart cells. two pts received blinatumomab resulting in pr in one of them. two additional pts received io ( after failing blinatumomab) achieving mrdpositive cr. cytokine release syndrome occurred in all pts and was severe in only one patient who required tocilizumab treatment. this patient was also the only patient who experienced neurotoxicity (grade ), and was treated with dexamethasone. this patient eventually died days post infusion of cart cells due to severe pseudomembranous colitis, toxic megacolon and sepsis. all pts had prolonged neutropenia for a median of days ( - ) after the infusion of cart cells. at day after infusion of cart-cells the cr for the entire cohort was %: three pts with mrd-negative and one with mrd-positive response. among the four pts who received antibodies prior the cart-cells, one patient had mrd-positive and two pts had mrd-negative response. the patient with ph positive b-all had progressive disease during the treatment. two pts were referred to second allo-hsct from other donors. one patient with mrd-negative response relapsed after the second transplant and was treated by salvage therapy. the second patient with mrdnegative response demonstrated prolonged remission ( months) even without second transplantation. with a median follow-up of months ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) the median progression-free and overall survival for the entire cohort were . and months, respectively. conclusions: autologous anti-cd car t-cell therapy after debulking treatment with antibodies, including blinatumomab and/or io, is feasible and results in high response rates in pts with r/r b-all. patients may respond to anti-cd car t-cell therapy even after failure to their last salvage therapy with blinatumomab, which demonstrates similar mechanism of action. clinical background: genetically engineered t cells expressing a chimeric antigen receptor (car-t) targeting specific antigens present on acute lymphoblastic leukemia (all) blasts have generated promising results in children and adults with relapsed and refractory disease. the below report provides an insight of lineage switch occuring as a result of intense immunological selection after car-t cell therapy, even with a tumor clone that has no potential for this switch. methods: an eight year old caucasian male with precursor b (pb) cell lymphocytic leukemia was treated with cd directed car-t cell therapy in third remission, and after relapse after previous bone marrow transplantation (bmt) . he was diagnosed with t( ; ) pb cell all at years of age and treated with bfm protocol. he relapsed months after completion of maintenance therapy, and had a / mmud bone marrow transplant after etoposide, tbi and alemtuzumab conditioning therapy. he had cutaneous acute and chronic gvhd but months post-transplant, he relapsed again with pb cell all, with the same cytogenetic and immunophenotypic disease characteristics.. he was treated with lymphodepleting chemotherapy with fludarabine, cyclophosphamide and alemtuzumab followed by infusion of cd directed car t cells. he developed cytokine release syndrome of grade severity manifested as persistent fever, associated with car t cell expansion in the blood. after car-t infusion,there was no detectable b cell all clone in the marrow by pcr and the cytogenetics were negative for t( : ) translocation. months after the car t cell therapy, he was found to have a mrd positive disease which was monitored closely. results: we document clonal evolution from cd negative, mrd positive disease to aml, with the same ig rearrangement (the same clonal disease) but with complete myeloid phenotype mpo, cd , cd positive disease. there was cytogenetic evolution of the underlying clone but the original t( ; ) was retained within the evolved karyotype. sadly, our patient developed fludarabineneurotoxcity during an attempt to induce aml remission, and further curative-intent chemotherapy was not possible. conclusions: there are two case reports of mll rearranged b-all acquiring a clonally related myeloid phenotype associated with cd -negative escape after cd directed car t cell therapy,so far. but, this is the first post car-t cell therapy transformation of all to aml with etv -runx mutation, which is not recognised to have such lineage-switch potential. unlike mll, etv -runx translocation in the pathogenesis of acute myeloid leukemia is not been reported in the literature. the theory behind such transformation is an intense immunological selection of the tumor, driving it to myeloid differentiation with additional clonal cytogenetic events. disclosure: nothing to declare p ivac-all- : interim analysis of a phase i/ii clinical study on personalized peptide vaccination based on patient-individual tumor-specific variants in relapsed pediatric acute lymphoblastic leukemia armin rabsteyn , , christopher mohr , olaf witt , , roland meisel , , cristiane chen-santel , tobias feuchtinger , , christopher schroeder , jakob matthes , background: acute lymphoblastic leukemia (all) is the most common pediatric malignancy. standard chemotherapy is a successful treatment in % of patients, only about % develop a relapse, however these patients have a dismal prognosis. prevention of relapse after firstline chemotherapy or stem cell transplantation (sct) is therefore an urgent clinical need. we established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient hla types. we started clinical translation of this approach by starting a phase i/ii clinical trial in (nct ). besides feasibility and toxicity assessments, we aim to assess the capability of the peptide vaccination to induce neoantigen-specific t cell responses in high-risk all patients to target residual tumor cells and prevent leukemic relapses. methods: key inclusion criteria are: pediatric patients with all who suffered from second relapse after standard therapy or first relapse after sct. hematological remission has to be reached prior to vaccination. nonsynonymous mutations are identified by whole exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. hla binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms syfpeithi, netmhc and netmhcpan for the patients' individual hla type. vaccine cocktails consisting of - individual peptides are produced and formulated under gmp conditions. the vaccination schedule is vaccinations over weeks using gm-csf and imiquimod as adjuvants. response to the vaccination is monitored by detection of t cells recognizing the vaccinated peptides occurring over time in peripheral blood of patients by prestimulation and intracellular cytokine staining. results: until now, patients were recruited, for of those, whole exome sequencing was performed to identify all-specific snvs using a comparative bioinformatics pipeline. we found an average of . mutations per patient on dna level. based on these data, an average of hla binders derived from neoantigens could be predicted per patient. an average expression of . % of mutations was assessed by rna sequencing. in all cases validated mutations could be identified and cocktail design was feasible. until now, patients received vaccinations. the vaccine was generally well tolerated and no or only mild side effects were observed. immune monitoring was performed for patients until now. in the first patient, we observed a transient cd + response against one vaccinated mhc class ii ligand and a sustained cd + response against the included wildtype control peptide derived from the antigen survivin. in the second patient, immune monitoring was performed for the first vaccination timepoints, a t cell response was not measurable at this timepoint of vaccination. conclusions: whole exome sequencing of pediatric all patients is feasible and yields small amounts of expressed, tumor-specific mutations. these few mutations are sufficient to predict hla-binding peptides that can be used to formulate individualized peptide vaccine cocktails. we currently conduct a clinical phase i/ii trial in a small cohort of high-risk all patients to assess safety, toxicity and immunogenicity. clinical background: chimeric antigen receptor t cells (cart) are considered as gene therapy medicinal products (gtmp) and genetically modified organisms (gmo). hence, carts manufacturing for clinical application is strictly regulated. appropriate methods assessing car transgene copy number (cn) in a cart product and definition of the frequency of carts in treated cart patient are mandatory. although quantitative real-time pcr-based (qpcr) analysis has been used for this purpose, no standardized procedure to minimize systematic errors and enable comparability has been established yet. here, we report on a single copy genebased (scg) duplex (dp) pcr (scg-dp-pcr) for the determination of the vector copy number (vcn) in cart products as well as patient samples following cart administration. scg-dp-pcr was validated and compared to the broadly used absolute copy number qpcr (acn) approach within the framework of a clinical trial treating patients with good manufacturing practice (gmp)-grade carts (hd-car- ). methods: for conventional acn, primers and probe targeting the car vector rv-sfg.cd .cd . - bbzeta were designed. standard curves were established via serial dilutions of the sfg.car plasmid. amplification of the standard curve as well as target genomic dna for vcndetermination was performed as singleplex (sp) pcr (sp-car) (method a). on the same qpcr plate, duplex (dp) qpcr reactions were carried out. additionally to the components comprised within method a, the experimental setup contained haploid human genomes as well as primers and probe targeting ribonuclease (rnase) p as human scg. the amplifications for the sfg.car plasmid (dp-car) and the rnasep gene (dp-rnasep) were performed simultaneously (scg-dp-pcr; method b). scg-dp-pcr was performed for standard curves and target samples. target-sample dna was extracted from carts prepared from leukapheresis products of three healthy donors (hd). results: for method validation, efficiency and linearity (correlation coefficient) of the qpcr reactions of method a (sp-car) and method b (dp-car, dp-rnasep) were assessed by linear regression of the pcr signal to the reference standard curve. overall, standard curves were only considered valid if a correlation coefficient (r ) of above . and efficiencies of % ± % were achieved. vcns applying method a and b to the same target sample were compared. sp-car pcr reaction displayed efficiency of . ± . %; . % ± . % and . ± . % efficiencies were achieved for dp-car and dp-rnase pcr reaction, respectively (table ) . applying scg-dp-pcr using formula for relative cn assessment ( -Δct (dp-car -dp-rnasep) ) on hd samples resulted in an average of . ± . increased cn when compared to method a (table ) . conclusions: in terms of efficiency and linearity by linear regression qpcr reactions were comparable. validation of scg-dp-pcr was achieved and represents an exact and less error-prone method to fulfil regulatory safety release criteria of cart products. besides of accurately assessing vcn of transduced cells, scg-dp-pcr is also a highly robust method to follow-up carts in treated patients. applying this approach, no standard curve is needed, hence significantly economizing required material as well as time. disclosure: nothing to declare background: t-cells' antileukemic responses in aml-pts need to be improved. dc leu effectively activate t-cells against leukemic blasts, resulting in blast-lysis ex-vivo. factors influencing these activities are not known. methods: we generated dc/dc leu from aml-blastcontaining mononuclear cells (n= ) using standard methods (mcm-mimic/ca-ionophore/picibanil/ifn-α, "mnc-methods") and from blast-containing heparinized whole blood (n= ) using modulatory kits (various combinations of - clinically approved response-modifiers, "wb-kits", patent ) and correlated statistically (t-/u-test, pearsons correlation, # means significant) proportions of dc-/t-cell-subtypes/cytokine-profiles with t-cells' antileukemic cytotoxicity (ctx), achieved after mixed lymphocyte culture (mlc) with/without mncmethod-("t*dc mnc "/"t*bla mnc ") or wb-kit-treated cultures ("t*dc wb "/t*bla wb "). ctx was given as proportions of cases with achieved or "improved" blast-lysis compared to control and as frequencies of viable blasts (bla via ) after effector-cell-influence. pooled data and data obtained with single methods in different cohorts are given. results: . generation of dc/dc leu : with a) mncmethods: Ø ± % dc and Ø ± % dc leu and b) wb-kits: Ø ± % dc and Ø ± % dc leu without induction of blasts' proliferation. t-cell-proliferation increased (vs uncultured t-cells) after mlc with a) mnc-methods: Ø ± % vs Ø ± % and b) wb-kits: Ø ± % vs Ø ± %. . antileukemic reactivity (t-effector-cell-cytotoxicity after mlc): pooling all data: a) mnc-methods ("t*dc mnc " vs "t*bla mnc "): we found a ) blast-cytotoxicity in Ø %(vs %) of cases with Ø %(vs %) bla via , a ) blast-cytotoxicity was improved (vs control) in % with Ø decrease of bla via of %; b) wb-kits ("t*dc wb " vs "t*bla wb "): we found b ) blastcytotoxicity in Ø %(vs %) of cases with Ø %(vs %) bla via , b ) blast-cytotoxicity was improved (vs control) in % of cases with Ø decrease of bla via of %. in general, these results could be confirmed with single methods: best mnc-methods were picibanil and mcm-mimic, best wbkits were kits containing gm-csf+picibanil or prostaglandins. . correlations: pooling all data: cases with "improved" lysis (vs "not improved" lysis) were characterized by a) mnc-methods: increased proportions of mature dc/cells (Ø ± % vs Ø ± %), dc leu /cells (Ø ± % vs Ø ± %) and proliferating t-cells (Ø ± % vs Ø ± %), b) wb-methods: dc/cells (r= . # ), dc leu /cells (r= . # ), dc leu /bla (Ø ± % vs Ø ± %), dc leu /dc (Ø ± % vs Ø ± %), cd + t-cells (Ø ± % vs Ø ± %), ifn-γ (r= . #) , mcp- ( ± vs ± pg/ml). conclusions: blasts are regularly converted to dc leu in the presence of mnc-methods and wb-kits (simulating the in vivo microenvironment). t-cells' coculture with dc/ dc leu after mlc induces and improves antileukemic t-cell activation compared to controls. blast-cytotoxicity correlates with proportions of dc/dc leu -and t-cell subtypes and released cytokines. these data support a role of antigen presentation by leukemic cells (dc leu ) for the stimulation of an immune response in aml in vitro and possibly in vivo. disclosure: nothing to declare evaluation year after the launch of the motion comic immuno-t, explaining patients and their caregivers how immunotherapy strategies work background: one year ago, the first version of immuno-t, a motion comic explaining to patients and caregivers how immunotherapy strategies work, was released. people were informed on and inspired to use the application during (inter)national meetings and events for the general public. meanwhile, the motion comic was further refined and adapted into a second version, based on the evaluations we've collected on the first version. adaptations included a multi-language tool (currently languages), increased user friendliness, and a new supporting musical score. also, a website was launched from which the second version could be downloaded on tablet or smartphone (both android and apple) and a new online evaluation form could be filled in. in months, people have evaluated the motion comic online, and these results are presented here, as well as our future plans within the immuno-t program. methods: through an online questionnaire, participants from belgium (n= ) and the netherlands (n= ) have evaluated the dutch version, and belgian participants evaluated the french version of the motion comic. results: the total group (n= ) consisted of patients (n= ) and their families (n= ), general public (n= ), students (n= ), health care professionals (n= ), researchers (n= ) and kindergarten teachers (n= ). participants' age ranged from to years, with an even distribution amongst the different generations. the majority of the evaluators ( , %, n= ) thought the motion comic is a good way to explain immunotherapy to patients. individuals ( , %) felt mainly interested after watching immuno-t, and a total of participants ( , %) felt hopeful or motivated. focussing on the patient group (n= ), all of the responders think the immuno-t motion comic is a good tool to use in a patient-doctor consultation. patients ( , %) felt hopeful and/or motivated after watching the whole motion comic, while of them ( %) felt combative and ( , %) felt gripped and intrigued. as for the new musical score, participants ( , %) think the music is suitable for the app, while evaluators ( , %) think the new music is not or not at all fitted to support the motion comic. conclusions: the detailed evaluations allow us to further improve immuno-t, and aid us in the development of other motion comics we plan to release under the cancer-t in motion umbrella. with the current version of immuno-t, a single-center pilot study is being set up, to test the efficacy and usability of immuno-t, based on qualitative research during the experience of the tool, and using validated questionnaires. with this study we want to evaluate the impact of immuno-t on patient empowerment, and the decision making process. the study protocol will be presented at ebmt. disclosure: the development of immuno-t was partly financially supported by celyad, calgene, novartis, roche, amgen, bms, but these companies did not by any means influence the contents and development of the motion comic. a therapeutic strategy to trespass the blood brain barrier for adoptive nk cell therapy in glioblastoma multiforme induced rat: a preclinical study background: glioblastoma multiforme (gbm) is among the most common and aggressive primary brain tumors with very poor prognosis. according to the central brain tumor registry of the united states, central nervous system (cns) tumors in pediatric patients (ages between - years old) are the second most common malignancies after blood-born malignancies, and the first amongst solid tumors, and known the most common cause of cancer-related deaths. although hematopoietic stem cell transplantation has been exploited to treat many kinds of malignancies, currently its success rate in gbm is limited. therefore, the gbm treatment paradigm needs shifting towards more effective treatments such as immune cell therapy. natural killer (nk) cells have been recognized as potential anti-cancer effector cells, as they can recognize and target tumor cells. since a small percentage of blood cells are differentiated as nk cells, the number of this group of cells is hardly enough to fight tumors, and so their multiplication and activation would be a potential effective cancer treatment. methods: this preclinical study was focused on setting up an optimal protocol for expansion and activation of naïve nk cells and assessing their efficacy towards induced gbm in rat models. ex-vivo expanded and interlukin- (il- )and heat shock protein- (hsp- )-treated nk cells have been exploited. after in vitro study and confirming the efficacy of treated cells through cytotoxicity assays, we induced gbm in male wistar rats (weighted - gr) using c tumor cells injection in rat brain through stereotactic surgery. the tumor formation was proven by mri imaging. following tumor establishment, we analyzed the effect of single injection of il- -and hsp- -treated nk cells compared with single injection of non-treated nk cells in two groups of rats. results: systemic intravenous delivery of il- -and hsp- -treated nk cells through tail's vein resulted in tumor shrinkage in different time intervals and complete remission in the first group of gbm-induced rat models, whereas in the other group of gbm rats receiving untreated nk cells, the tumor progressed. therapeutic efficacy of the treated nk cells was ascertained compared with non-treated nk cells considering tumor shrinkage observed in mri and survival rates between the two model groups. conclusions: the amelioration of tumor which has been confirmed by mri, proved the migration of activated nk cells through blood brain barrier and homing to cns, and finally targeting gbm tumor cells. our data suggest that nk-cells treated with il /hsp may be a promising immune cell-based therapeutic approach towards treating the fatal gbm. disclosure: nothing to declare p abstract withdrawn. long term sorafenib response for extramedullary flt + aml relapse after allogeneic stem cell transplantation since june , sorafenib dose has been tapered to mg/day, due to mild skin and gi toxicity. after years of treatment, she maintains cr at medullary and extramedulary levels, with no evidence of a disease that had escaped the mechanisms of action of chemo, hsct and dli. conclusions: in our patient, treatment with sorafenib has provided long-term control of this refractory extramedullary disease, even at adjusted doses. further studies are needed to confirm the efficacy of flt inhibitors in the control of relapses after allo-hsct, extramedullary disease and its potential role as maintenance agent. disclosure: nothing to declare background: although chemotherapeutic(ct) agents that used in the treatment of acute lymphoblastic leukemia (all) increase survival, the results are still weak. longterm survival with ct's in relapse all cases is difficult and the prognosis is very weak. inotuzumab ozogamicin is an anti-cd monoclonal antibody and it has the potential to reduce the overall toxicity of intensive regimens for all, as well as to possibly increase the number of patients who may achieve a state of minimal residual disease. methods: -year-old male patient was diagnosed with b-cell all in december .after the hoelzer kt protocol was started, maintenance treatment was continued. in the fifth month of treatment,flag ct protocol was started cause of recurrence was seen on % blast detection in peripheral blood smear. in august , inotuzumab ozogamicin treatment was started and six cures were completed because the patient was not in remission. in september , he had gone haploidentical bone marrow transplantation from his sibling donor( / )with defibrotid prophylaxis for veno-occlusive disease(vod)s. he engrafted succesfully and chimerizm was . % in th days of transplantation. he is th day of transplantation and in a remission. results: bone marrow transplantation cannot be performed since the complete response cannot be achieved in patients with relapse and resistant b-all.in these patients, new therapies targeting malignant lymphoblasts are needed. inotuzumab ozogamicinis a monoclonal antibody drug conjugate that targets cd antigen on malignant lymphoblasts.in many studies, it has been shown that inotuzumab ozogamycin is effective and reliable anti-tumor activity in adults with recurrent and resistant cd positive all. however, monoclonal antibody drug conjugates have been shown to be associated with vod's.for this purpose, we used defibrotid to protect our patient from vod. conclusions: treatment with combination ct regimens in b-all is suboptimal and long-term survival is achieved in only - % of patients.targeted molecular therapy and new regimens are needed in relapse and resistant patients.at this point, inotuzumab ozogamycin is an anti-cd- monoclonal antibody, as in our case, it provides remission in recurrent and resistant b-all patients and allows patients to complete their treatment with an allogeneic transplant from a fully compatible donor. disclosure: nothing to declare background: mesenchymal stem cells (mscs) are an attractive consideration for therapeutic cures of many difficult diseases on the cellular-level. due to the trophic effects of the cytokines and chemokines that they produce, mscs have shown multiple beneficial properties in the field of oncology. in this study, we will be investigating the effect of mscs derived from human bone marrow (bm), adipose tissue (at), and umbilical cord derived mscs (uc-mscs) on ovarian cancer. to differentiate the mscs, we performed a comparative analysis between the various sources for proliferative capacity, surface antigen expression, differentiation ability, tumor marker and paracrine activity, and their influence on ovarian cancer cell proliferation. methods: measurements of ovarian tumor marker proteins were computed by elisa. proliferative effects, immunomodulatory effects, and apoptosis of the mscs were measured by the culture and counting of colony formations. flow cytometry (fcm) was used to measure the variation of the immunophenotyping and cytokine secretions in co-culture, as well as gene expression. results: cells noticeably proliferated without any modifications to their immunophenotype during the third subculture. the colony-forming unit fibroblast (cfu-f) test showed a proliferation of the mscs along with healthy cells and cancer cell lines with no changes in their phenotype. the supernatant of mscs showed an increase in cellular death of the ovcar in ovarian cancer cell lines. a reduction in the level of ca- ( - %; p= . ) with ovcar in co-culture, and a decline of ldh ( - %; p= . ) and beta-hcg ( - %; p= . ) were observed in co-culture in caov , skov and igrov cell lines. a decrease in cd of the cancer cell lines in co-culture with the msc supernatant showed a reduction of the cancer tumorigenicity and aggressiveness, while the rate of the cd and cd asserted their stem state. msc supernatant decreased cell proliferation and mmp- , mmp- , and ca- mrna expression, while increasing timp , , and . this suggests that mscs have a role in cell death and inhibition of ovarian cancer cell proliferation. an increase of anti-inflammatory il- and il- cytokines, and a decrease in growth factor gm-csf along with their proinflammatory inf-a, tnf-a, il- , and il- a cytokines were also noted. conclusions: the gene and cytokine activity indicate a potential therapeutic anti-inflammatory and antiproliferative role of mscs on ovarian cancer despite their sources. the reduction of ca- , ldh, and beta-hcg in co-culture, along with the decrease in cd and amplified cellular apoptosis demonstrate the beneficial effects of stem cells in ovarian cancer cell lines. disclosure background: hematopoietic stem cell transplant (hsct) is the only cure in sickle cell disease (scd) so far. because of the risk of toxicity, its indication in france is restricted to severe patients with match sibling donor. this study compares the incidence of severe acute toxicity after hsct, between children aged less than years and teenagers aged to years old, treated for scd. methods: all patients suffering from scd, aged less than years at transplant, who received hsct in chu robert debré and necker, between / / and / / , were included. severe acute toxicity, defined by onset of severe acute gvhd, organ toxicity or infection, was compared between the two groups of age. results: patients ( children and teenagers) were included. all patients received a myeloablative conditioning regimen. bone marrow from a sibling donor was the main stem cell source (n= ; %). neither death nor rejection was observed with a median follow-up of . months (range, . the incidence of grade iii-iv acute gvhd was . % and was similar between the two groups; no risk factor was identified in univariate analysis. teenagers had more frequently acute skin toxicity ( . % vs %, p= . ). in univariate analysis, patients presenting severe organ toxicity were significantly older than others ( . vs . years old, p= . ). teenagers were more frequently treated for bacterial ( . % vs . %, p= . ) or bk virus ( . % vs . %, p= . ) infections. in univariate analysis, patients who developed infection were also significantly older at time of transplant (respectively . vs . years old, p= . ). no severe sinusoidal obstruction syndrome was observed. regarding long-term toxicity, patients presented an extensive chronic gvhd, they were both aged less than years old. no cut-off of age could have been defined. conclusions: this study confirms the excellent results of hsct in scd, with a -year event-free survival and overall survival of %. an older age at transplant seems to be associated with more frequent severe acute toxicity. these results are consistent with previous studies and suggest that hsct should be performed as soon as possible, without any defined "best age". prospective studies are needed, in order to define the place of each therapeutic in scd, with the aim of reducing treatment-related toxicity and developing alternative strategies for patients without match sibling donor. disclosure: nothing to declare p new insights into risk factors for transplant-associated thrombotic microangiopathy (ta-tma) in paediatric hsct (n= ) was associated with ta-tma in % vs . % vs . % respectively (p= . ). the presence of comorbidities at d (n= ) was significantly associated with an increased risk of ta-tma . % vs . % in absence of co-morbidity (n= ); p= . . use of csa/tac-based gvhd prophylaxis was associated with less ta-tma with an incidence of % vs % if these agents were not included (p= . ). in univariate analysis ta-tma was significantly higher among patients with agvhd grade ii-iv ( / ; . %) vs grade -i ( / ; . %) (p= . ). pres was recorded among cases and % of them developed ta-tma. two out of the patients with ta-tma had pathological gene mutations in their complement pathway. on multivariate analysis the presence of active comorbidity was a risk factor for ta-tma (or: . ; % ci: . - . ; p= . ) while the use of csa/tac-based gvhd prophylaxis did not increase the risk for ta-tma (or: . ; ci: . - . ; p= . ). in the presence of comorbidities the use of defibrotide as prophylaxis or therapy for vod (n= ) was associated with a drop in the incidence of ta-tma from % ( / ) in absence of defibrotide to % ( / ). -year overall survival was significantly lower among ta-tma cases ( %) in comparison to . % in absence of ta-tma (p= . ) (figure ). conclusions: active co-morbidity is a significant risk factor for ta-tma. use of defibrotide prophylaxis in patients with co-morbidities at the time of hsct might offer protection against ta-tma. surprisingly the use of csa/tac based gvhd prophylaxis is not a risk factor for ta-tma probably through limiting the development of high grades agvhd. the association between pres and ta-tma suggests a common pathway of endothelial damage background: gonadal impairment is a severe late effect of myeloablative conditioning regimes with significant impact on quality of life of cancer survivors. the aim of this study was to analyze gonadal function after busulfan (bu) or treosulfan (treo) containing regimens with regard to pubertal stage. methods: this was a retrospective, multicenter study involving patients treated in pediatric ebmt centers between - . patients receiving myeloablative doses of bu or treo as part of hsct conditioning were eligible for inclusion. analysis was conducted in two groups according to pubertal status at time of hsct. results: patients (pts) were treated in pediatric ebmt with bu or treo before allogeneic hsct. the median age at transplant was . years (range - ); / ( %) were males (m), / ( %) were females (f). / ( %) pts were pre-pubertal at hsct (f= ;m= ) and / ( %) were post pubertal (f= ;m= ). / ( %) patients received bu (f= ;m= ), / ( %) were pre-pubertal. / ( %)(f= ;m= ) received treo, / ( %) were pre-pubertal ( figure ). females who received treo in pre-pubertal stage (n= / ) reached more often spontaneous puberty ( % vs %; p= . ) compared to pre-pubertal bu group (n= / ) and occurrence of menarche was higher in treo group (p< . ) hormonal replacement therapy was given in / ( %) females transplanted in pre-pubertal stage and in / ( %) of those transplanted in post-pubertal stage. / ( %) males were pubertal at last follow-up and of them ( %) performed sperm analysis ( oligo-azoospermic, unknown). three pregnancies were reported in the population group, all received bu. regarding the evaluation of hormonal levels in pubertal patients at time of hormonal dosage (median . yrs) ( bu and treo), males treated with treo had significant lower lh levels (p = . ) compared to bu group. females treated with treo had significant lower levels of lh and fsh (p= . and p= . respectively). conclusions: gonadal damage related to treo was significantly lower compared to bu. we observed that: females transplanted during pre-pubertal period had spontaneous puberty more frequently after treo compared to bu and that hypogonadism hypergonadotropic was more frequent after bu than treo. these results must be further confirmed on a larger population. background: viral infections significantly contribute to both morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. traditional antiviral therapy is associated with lack of efficacy, potential toxicity, prolonged hospitalization and increased patient costs. viral specific t cells can be manufactured from donor blood to treat viral infections post-transplant, and are associated with increased clinical efficacy and low toxicity. we postulated that direct costs of vst therapy are lower compared to traditional anti-viral medications methods: vsts were manufactured according to local protocols and fda requirements. total drug cost (as per institutional charges per drug) was calculated for patients who required treatment for viral infections post-hsct. manufacturing costs of vsts are fixed per fda requirements. patients who were treated with investigational antiviral medications (including brincidofovir) were excluded from analysis. patients treated with vsts +/anti-viral medications over a year period were compared to patients treated only using traditional anti-viral medications, including cidofovir, ganciclovir, valganciclovir, foscarnet and rituximab. results: demographics are shown in table . there were no major differences between the two groups treated. the number of anti-viral medications used in the vst group was lower compared to the anti-viral treatment group. median cost of vst treatment was significantly lower compared to those threated with traditional anti-viral therapy ($ , vs $ , , p-value= . ) . conclusions: treatment with vsts post-hsct for viral infections was lower in cost compared to anti-viral medical therapy. it is likely that overall costs are further reduced with vsts due to reduced inpatient hospital time, less monitoring of labs associated with anti-viral medication side-effects and reduced ancillary costs including nursing and pharmacy. more studies are needed to examine these indirect costs further. background: dock deficiency is an autosomal recessive primary immunodeficiency (pid) disease caused by loss-offunction mutations in the dock gene ( ) . patients with dock deficiency present with multiple abnormalities of the immune system, including defective t cell function and impaired production of antigen-specific antibodies. these lead to persistent viral infections of the skin, mucocutaneous candidiasis, recurrent sinopulmonary infections, atopic dermatitis, and other allergic disease, malignancies, and sometimes autoimmunity ( ). hematopoietic stem cell transplantation (hsct) is currently the only curative treatment option available ( ) . methods: we retrospectively evaluated our patients who underwent allogeneic hematopoietic stem cell transplantation due to dock deficiency in ege university pediatric stem cell transplantation unit between and . results: we identified patients transplanted at a median age of . years (range: - . years). of patients; (% ) received hsct from matched sibling, (% ) from unrelated donors and patient from haploidentical donor. we used busulfan-based myeloablative conditioning regimen to patients (% ), reduced toxicity myeloablative regimen with treosulfan to patients (% . ) and nonmyeloablative regimen to patients (% . ). eight of the recipients received bone marrow, of the patients received peripheral blood stem cells, of the recipients received cord blood as stem cell source. fifteen of patients (% ) had achieved engraftment and median follow-up of patients was months ( - ). grade iii-iv acute graft versus host disease (gvhd) occurred in % of patients and chronic graft versus host disease was seen % of patients. one patient received cord blood from unrelated donor did not engraft and died from septic shock. four patients died from transplant related toxicity. our patient's survival was % ; / patients alive. conclusions: hsct is the only curative treatment option for dock deficiency. in particular, patients with high comorbidity scores have a high risk of toxicity and toxic death. therefore, reduced toxicity conditioning regimens should be used for these patients. references : background: eltrombopag, a low-molecular-weight synthetic nonpeptide thrombopoietin receptor agonist (tpo-r), is a second-generation tpo. it is an oral thrombopoietin mimetic licensed in chronic immune thrombocytopenic purpura that induces platelet maturation and release by binding to c-mpl receptors on megakaryocytes. in a recent study; for patients with refractory saa, eltrombopag induced a response (at weeks) in at least one hematologic lineage in % patients and % no longer required platelet transfusions. and also % patients became rbc transfusion independent and % had a neutrophil response. trilineage responses were seen in % of patients; although surprising, this might indicate stimulation of c-mpl receptors on remaining stem cells. delayed recovery from thrombocytopenia is common after stem cell transplantation. in a study including adult patients, eltrombopag was used to enhance platelet recovery for post-hsct thrombocytopenia. it is well tolerated and efficacious offering transfusion independence. methods: in our retrospective study, eltrombopag ( mg/day) was started in pediatric patients (age ranging from to years with a median age . years) for posthematopoietic stem cell transplantation (hsct) thrombocytopenia. all patients fulfilled the following criteria: ( ) undergone hematopoietic stem cell transplantation (hsct), ( ) had improved total leucocyte counts after leucocyte engraftment, ( ) had prolonged thrombocytopenia (< . ) needing platelet transfusion. results: four of the patients have received ric while patients ma conditioning regimens before hsct. two haploidentic, autologous, mud, msd transplantations were performed. et ( mg/day) was started in patients who had thrombocytopenia despite neutrophil engraftment on the + th day of hsct a reduction in platelet transfusions and a platelet count of more than , were the primary endpoints. before et treatment, bone marrow biopsy was checked in / patients, / patients had decreased number of megacocyocytes. none of the patients had active bleeding at the start of eltrombopag but they were all at high risk of bleeding. according to the platelet monitoring, patients had a dose increase starting from the second week. the number of patients in need of platelet transfusions was at the end of the first month; and only at the end of the nd month. all patients had a thrombocyte count of more than . in the third month. in patients, et was discontinued after - months. no dose limiting toxicities have been observed. conclusions: as a conclusion, et was found highly effective for posthsct thrombocytopenia, with no drug related adverse effects. there was a gradual increase in platelet count, and none of the patients had any complication due to thrombocytopenia. disclosure background: isavuconazole (isa) is a new triazole approved for ifi treatment in the adult population. advantages are activity against both moulds and yeasts spp, excellent bioavailability after oral administration without relevant food or gastric ph effect, a water-soluble prodrug developed to facilitate intravenous administration without nephrotoxic excipients such as β-cyclodextrin, potentially poor drug-drug interactions. isa does not currently appear to require tdm. isa safety and efficacy have not been yet established in children and, in particular, no data are available in the pediatric hsct setting. methods: italian association pediatric hematology oncology (aieop) multicentric analysis of a cohort of allogeneic hsct pediatric patients who received isa as ifi treatment or prophylaxis. due to the lack of recommended dosing in pediatric patients and a clear target isa plasma trough-level range, the therapeutic monitoring (tdm) of isa concentrations was applied by a validated liquid chromatography-tandem mass spectrometry (hlpc-ms/ ms) assay technique. isa trough plasma concentrations (c ) and hours after drug intake (c h) were measured. results: a total of allo-hsct recipients were included, (m/f / ); median age: , years, range - , median body-weight , kg (range - ). isa was used as ifi treatment in cases and as prophylaxis in patients. donors were haploidentical in patients, matched-sibling in , allogenic-unrelated in cases. according to eortc criteria, ifi was proven in patients ( penicilum, mucor, aspergillus fumigatus), probable in and possible in patients. lungs were the main localization ( cases), associated with cns involvement in cases and paranasal sinuses in ; patient had possible hepatic candidiasis. all patients, but one, received isa as rescue treatment for previous therapeutic failure ( ambisome, voriconazole, combination therapy, posaconazole). seven patients received only iv isa, received only oral isa whereas patients received both iv and oral isa. patients under kg body weight received half isa dose ( mg tid loading dose on days and , mg/die manteinance). the others received adult schedule; only patients received loading dose. isa was administered for a median of days (range: - ). in patients isa was administered in combination with caspofungin. tdm was applied to patients including patient with severe vod and with renal failure secondary to ta-tma. the median isa concentrations were . ( . - . ) mg/l and . ( . - . ) mg/l for c and c h, respectively. ifi complete remission was achieved in cases, partial remission in ; treatment failure was experienced by patients. in cases fungal lesions remained stable. ctae grade ii-iii toxicity was observed during treatment in patients, with increased transaminase and/or creatinine levels which resolved after temporary isa withdrawal. no drug-drug interactions were observed in patients receiving csa as gvhd prophylaxis and no modification of csa daily dose was needed. conclusions: isavuconazole use may be considered in the pediatric population, even in the hsct setting, for its safety, efficacy, tolerability, no drug-drug interaction. of course these data deserve further evaluation. disclosure: nothing to declare p new treosulfan-based conditioning regimens including epigenetic agents in patients with very high-risk neuroblastoma background: the pts aged mos. or older with disseminated nb involving bone and bone marrow constitute a group of pts with very poor prognosis. although the majority of them are responsive to intensive conventional chemotherapy, most eventually relapse with efs at years of < %. at the beginning of the year we came up with a protocol for this very unfavorable group including epigenetic therapy ( -azacitidine) in the phases of consolidation. methods: seven pts with a median age of ( , - ) years completed the protocol and received hdct with autologous sct as a consolidation. hdct included two different epigenetic agent containing regimes according to tumor response to the induction therapy assessed by i-mibg and mri (ct-scan). three pts revealing large active residual tumor assessed by i-mibg scan or multiple active bone metastases received a conditioning regimen (regimen a) including i-mibg therapy at a dose . - . mbe/kg on d- , treosulfan mg/m /d, on d- ,- and - (total mg/m ), melphalan mg/m /d, on d- ,- (total mg/m ), -azacitidine mg/m /d on d- to d- (total mg/m ). four pts with cr or vgpr received a «split» conditioning regimen (regimen b) including treosulfan mg/m /d, on d and - , and on d- and d- (total mg/m ), melphalan mg/m /d, on d- and - (total mg/m ), and -azacitidine mg/m /d, on d - to d - and on d- to -d- (total mg/m ). a median number of . ( . - ) cd +/kg was infused on d . results: the median recovery times to wbc> . x /l and to an unsupported plt> x /l were ( - ) and ( - ) days, respectively. all pts experienced grade hematological as well as infectious toxicity assessed by nci-ctc score. there were episodes of severe organ toxicity of grade occurring in pts. in cases we observed a severe mucositis, in cases gi toxicity and episode of the erythema multiforme occurred. one pt revealed a lifethreatening episode of hypotension of grade . no transplant-related death occurred. the median number of transfused rbc and plt doses was ( - ) and ( - ), respectively. all pts are alive and well without signs of disease progression in complete hematological recovery with a limited follow-up of . ( - ) mos. from day of hdct. conclusions: although it is rather early to evaluate the efficacy of the epigenetic agent's inclusion in the induction and/or consolidation phases of a very high-risk nb treatment, we can assume that, first, the hdct combining mibg i and/or high dose of treosulfan with epigenetic agent such a -azacitidine was feasible and had an acceptable toxicity. second, the "split" modality of the treosulfan use in conditioning regimen would permit to increase the total dose of the alkylating agent with no inacceptable toxicity. disclosure: nothing to declare pre-and-post magnetic resonance imaging of hips and knees for detecting osteonecrosis in children undergoing hematopoietic cell transplantation: in whom is it necessary? ali y suliman , sue c kaste , ying li , dinesh keerthi , guolian kang , brandon m triplett , ashok srinivasan between april and august at the university medical center hamburg-eppendorf, germany, were included. total and active ratg plasma levels were analyzed by elisa and flow cytometry, respectively. primary endpoint of the study was exposure to ratg. secondary endpoints included transplant-related mortality, incidence of acute and chronic gvhd, immune reconstitution, chimerism, rejection and viral infections. patients were monitored at least days post transplantation. statistical analyses were performed using ibm spss statistics software, or graphpad prism software. results: median total grafalon™ and thymoglobuline™ peak plasma levels were . μg/ml and . μg/ml, respectively; median active grafalon™ and thymoglobu-line™ peak plasma levels (appl) were . μg/ml and . μg/ml, respectively. active thymoglobuline™ plasma levels showed highly variable pharmacokinetics compared to grafalon™. neither grafalon™ nor thymoglobuline™ exposure correlated with lymphocyte count prior to transplantation, cell count in the graft (wbc, mnc, t cells), age, body weight or body surface area (bsa). this is indicative for a saturation effect in both groups. to correlate high or low ratg exposure with clinical outcome parameters, we built two groups within each patient cohort by median appl. the incidence of gvhd was not dependent on high or low ratg exposure. until day post hsct, viral infections or reactivations (ebv, cmv, adv, hhv , bkv) occurred in the patients. interestingly, adv infections affected only children with high ratg exposure. the median time to leukocyte engraftment was not significantly longer in the high ratg groups compared to the low ratg groups ( to days for grafalon™, and to days for thymoglobuline™). there was a decreased and/or delayed recovery of cd + , cd + and cd + t cell reconstitution, but not of b cells and nk cells in the high thymoglobuline™ exposure group compared to the low thymoglobuline™ exposure group. overall survival was not statistically significant with % in the grafalon™ and . % in the thymoglobuline™ group without influence of ratg exposure. conclusions: high and low exposure to grafalon™ or thymoglobuline™ did not result in significant differences in outcome parameters as incidence of survival, agvhd, cgvhd, rejection, or mixed chimerism in this limited cohort. delayed and decreased immune reconstitution in the high ratg exposure groups did not translate into different clinical outcome parameters. adv infections only occurred in the high ratg exposure group. grafalon™ and thymoglobuline™ showed distinct pharmacological and immunological differences in children and larger cohorts are needed to detect clinically significant differences and adjust dosing regimens individually. disclosure: nothing to declare background: the optimal conditioning regimen for allogeneic hematopoietic cell transplantation (allohct) in children with myeloid malignancies remains undefined, particularly when reduced-intensity conditioning (ric) regimens are utilized. methods: we performed a retrospective review of children undergoing allohct for acute myeloid leukemia (aml) and myelodysplasia-related aml (mdr-aml) over a year period ( - ) at our institution, comparing the outcomes of those who received either a busulfan (bu)-or melphalan/thiotepa (mel/thio)-based conditioning regimen. results: a total of patients were analyzed. twentyone received fludarabine/melphalan/thiotepa and received myeloablative busulfan-based conditioning, either in combination with cyclophosphamide (n= ) or fludarabine (n= ). atg was used in patients depending on donor. recipients of mel/thio were selected for ric regimens due to pre-transplant comorbidities (cardiac dysfunction, n= , requiring peri-transplant milrinone), transplant during chemotherapy-induced aplasia (n= ), underlying diagnosis of treatment-related aml (t-aml) and significant pre-allohct chemotherapy exposure (n= ). proportions of patients with de novo aml (mel/ thio, %; bu, %) and mdr-aml ( % and %) were similar between groups; however, recipients of mel/thio were more likely to have t-aml ( % vs %). cytogenetic and molecular risk factors were similar between groups. the majority of patients were transplanted in cr (mel/thio, % vs bu, %) or cr ( % vs %). more recipients of bu conditioning ( % vs %) were mrd-negative at the time of allohct; both groups had comparable proportions of patients with ≥m marrow (~ %). donor types and stem cell sources were similar between groups, except unrelated umbilical cord blood which was more common in bu recipients ( % vs %). there were no graft failures in mel/thio recipients, compared to % (n= ) in those receiving bu-based regimens. engraftment kinetics and immune reconstitution were similar. overall acute and chronic gvhd incidence was higher in recipients of mel/ thio compared to bu ( % vs %, and % vs %, respectively), but rates of grade iii-iv acute or extensive chronic gvhd were comparable. vod requiring treatment was diagnosed in ( %) recipients of bu conditioning and no mel/thio recipients. median duration of follow-up was months (range - ) in the mel/thio group, and months (range - ) in the bu group. transplantrelated mortality (trm) was similar in both groups ( patient), occurring before day . relapse incidence was comparable (mel/thio, % vs bu, %); however, relapse occurred at a later time in mel/thio recipients (median d + vs d+ ). overall survival at and years was superior in mel/thio recipients ( % vs %, and % vs %, respectively). conclusions: in our single institution experience, use of a melphalan/thiotepa-based ric regimen was associated with similar outcomes compared to full-intensity bu-based conditioning, despite higher risk patient and disease characteristics. the majority of recipients of mel/thio conditioning had significant pre-transplant comorbidities, which did not translate into higher trm. while mel/thio recipients had less optimal leukemia control at the time of transplant and high-risk leukemia features (e.g. t-aml), relapse was similar between groups, occurring later in mel/ thio recipients, which may have contributed to better overall survival. disclosure: nothing to disclose methods: pubertal development and biological gonadal parameters were assessed in a retrospective monocentric cohort of pre-pubertal patients who underwent hsct after myeloablative conditioning with total body irradiation (tbi) or busulfan between and . results: seventy-four patients ( girls and boys) were included. no spontaneous pubertal development was found in % of girls and % of boys (p < . ) and delayed puberty or no spontaneous pubertal development was found in % of girls and % of boys (p= . ). hormone replacement therapy was used in % of girls and % of boys (p < . ). in univariate analysis, tbi conditioning (p= . ), female sex (p < . ), acute gvhd (p= . ), extensive chronic gvhd (p= . ), steroid treatment > months (p= . ), and malignant diseases (p= . ) were associated with no spontaneous pubertal development, whereas tbi conditioning (p= . ) and extensive chronic gvhd (p= . ) were associated with delayed puberty. in multivariate analysis, factors independently associated with no spontaneous puberty onset were female sex (p= . ) and age > years (p= . ). factors independently associated with delayed puberty were extensive chronic gvhd (p= . ) and age > years (p= . ). tbi was not an independent risk factor for pubertal complications. conclusions: this study confirms the toxicity of myeloablative conditioning on pubertal development and the role of older age and female sex in increased pubertal issues, and suggests a possible role of gvhd in delayed puberty. disclosure: nothing to declare p abstract already published. neutrophil elastase activity may serve as a marker for neutrophil extracellular traps formation following stem cell transplantation ronit elhasid , sivan berger-achituv , hila rosenfeld-keidar , szilvia baron tel aviv sourasky medical center -tel aviv university, tel aviv, israel background: post-transplant infections rise dramatically in patients with quantitative or qualitative neutrophil defects and constitute a major source of morbidity and mortality following hematopoietic stem cell transplantation (hsct). neutrophils protect the host from microorganisms via multiple processes including phagocytosis and formation of neutrophil extracellular traps (nets). although reactive oxygen species (ros) production seems to be essential for nets formation, the key enzymes of the process are neutrophil elastase (ne) and myeloperoxidase (mpo). methods: ne and mpo activity as well as nets formation were investigated following hsct in patients at week to and after neutrophil engraftment. neutrophils were isolated using easysep direct human neutrophil isolation kit (stemcell technologies inc.) by immunomagnetic negative selection. enzymatic activity of ne and mpo were measured using colorimetric assays. nets formation of phorbol -myristate -acetate (pma)activated neutrophils was investigated by confocal fluorescence microscopy. all results were compared to those of healthy volunteers. statistical significance was calculated using one way-anova with bonferroni post hoc test. results: patients (median age of . years [range - years]) were investigated, following allogeneic hsct ( acute lymphoblastic leukemia, acute myeloblastic leukemia, epidermolysis bullosa, rhabdomyosarcoma) and following autologous hsct ( ewing sarcoma, desmoplastic small round cell tumor). all patients experienced fever and neutropenia. at engraftment, average ne activity was significantly decreased compared to the average value of healthy individuals. ne activity improved week by week in patients, reached the lower reference range at weeks following transplantation (fig. a) and continued to increase. the enzymatic activity of mpo was comparable to the average value of healthy individuals (fig. b) and showed no significant difference between the distinct time points. at neutrophil engraftment, nets formation was absent and comparable to those of non-activated neutrophils (fig. c) . although nets formation increased week by week, it did not reach the average of normal controls during the monitored time period. also linear correlation between ne activity and nets formation (r = . ) was demonstrated. conclusions: impaired ne activity following hsct corresponds to decreased nets formation and could serve as a marker for netosis. strategies to accelerate the recovery of ne function post transplantation might improve nets formation and thereby induce better infection control. a) the average of ne activity (n= ) during weeks following hsct. reference range was measured and calculated from measurements of healthy volunteers using. b) the average of mpo activity (n= ) during weeks following hsct. reference ranges were measured and calculated from measurements of healthy volunteers using the quartile method. c) the average of netosis activity after nm pma activation for h (n= background: to have a better understanding of incidence, treatment, outcome and risk factors of immune cytopenia in children after allogeneic hsct. methods: between january and september , pediatric allogeneic hsct have been performed in patients at the ghent university hospital (ghent, belgium). autoimmune hemolytic anemia was defined by a positive direct agglutinin test (dat). dat was performed at moment of engraftment and in case of hemolysis or unexplained anemia. platelets antibodies were evaluated in case of no otherwise explained thrombocytopenia. results: the cumulative incidence of post allo sct autoimmune cytopenia is . % ( / ). in cases there were positive antibodies against red blood cells, and one patient against had antibodies against platelets. of these cases, only ( . %) were clinically relevant and needed treatment. the median observation period post sct for the whole cohort was months ( - ) . the clinically significant immune cytopenia started at a median time of day+ and day + in the group without symptoms. the patient who presented the autoimmune thrombopenia developed antibodies against anti-gpiib/iiia, this was resolved after days, the treatment consisted intravenous immunoglobulins (ivig). two of the patients with autoimmune hemolytic anemia had igg mediated antibodies, and had complementmediated dat. these patients were treated with ivig, steroids, rapamune and rituximab. one patient has still dat positive after months, but clinical stable. the other two are also dat positive and have some hemolysis, but the follow up is much shorter ( months). treosulfan-contained conditioning regimens were more frequently used in patients with significant immune cytopenia. conclusions: immune cytopenia is an infrequent complication after allogeneic hsct. however, its treatment can be challenging, and the hemolysis can persist for years. the association of rapamune and rituximab was adequate to treat this problem in our patients. background: approaches to the management of refractory and relapsed classical hodgkin´s lymphoma (r-r chl) are changing and become more effective. the role of anti-cd targeted immunochemotherapy with brentuximab vedotin (bv) has been extensively investigated in adults with r-r chl and is only to be elucidated in children. the study included children and adolescents with r-r chl that were sucessfully treated with bv-based therapy prior to hematopoetic stem cell transplantation (hsct). median age of patients was years ( - ), main histological variant -nodular sclerosis ( %, n= ), advanced stage at diagnosis - % (n= ). most were heavily pre-treated (median number of previous therapies - ) and progression after autologous hsct was documented in ( %). refractory disease was diagnosed in ( %) and relapsed in ( %). among relapsed patients ( %) were with multiple episodes, ( . %) -early and ( . %) -late relapse. treatment regimens consisted of bv in monotherapy . mg/kg triweekly (n= ) or combination of bv . mg/kg on day with bendamustine - mg/ m on days and of -week cycles (n= ) or combination of bv . mg/kg on day with dhap (n= ). median number of bv infusions was . ( - ). all selected patients achieved complete (n= , %) or partial remission (n= , %) prior to hsct. consolidation with autologous hsct was performed in ( %) and with allogeneic hsct -in ( %). primary end points were overall (os) and progression free survival (pfs). response to bv was not assessed in the study as only responders to the bv-based treatment were included. results: with median follow-up of days ( - ) os and pfs for all patients are % and %, respectively. pfs after autologous hsct and allogeneic hsct are % and %, respectively (p= . ) at present moment ( %) patients are alive and are in remission. three patients died ( %): disease progression (n= ), postransplant idiopathic pneumonia syndrome (n= ) and posttransplant pneumonia (n= ). bv was generally well tolerated with only mild polyneuropathy in patients ( %) as the main reversable documented adverse event. conclusions: in prognostically unfavourable heavily pretreated children and adolescents with r-r chl achievement of response to bv-based therapy prior to hsct is assosiated with promising rates of os and pfs. disclosure provides a treatment by restoring thymidine phosphorylase function and improving disease manifestations. here we report the outcomes of affected siblings who underwent transplantation using an unaffected sibling donor to highlight important experiences in the transplant of such a rare condition. methods: four siblings of consanguineous pakistani descent aged , , and years underwent myeloablative hsct using fully hla-matched ( / ) peripheral blood stem cells harvested in a single apheresis from an unaffected year old sibling. the oldest sibling, a year old male, first presented in having emigrated from pakistan with a history of growth failure and abnormal movements. biochemical, nerve conduction and imaging studies confirmed a diagnosis of mngie. testing on three other siblings identified similar biochemical abnormalities, though the youngest children had minimal clinical manifestations of the disease. based on the progressive nature of the disease and the availability of a fully matched donor, a decision was made to pursue transplant for all affected siblings. results: due to the severity of their disease, the oldest siblings were transplanted first using a myeloablative conditioning regime of fludarabine, thiotepa and treosulfan with alemtuzumab. neutrophil engraftment occurred on day + for both, with % donor chimerism achieved. there were no significant transplant related complications. the post-transplant course of the year old sibling was complicated by a major stroke-like event characterised by dramatic imaging changes and requiring ventilation, though no cause was identified and the patient's neurologic deficits have since resolved. gastrointestinal symptoms have persisted and both remain tpn dependent, though symptomatically have shown gradual improvement. following the neurologic complications in their older sibling, the younger siblings were conditioned with auc-targeted busulfan and fludarabine plus alemtuzumab. neutrophil engraftment occurred on day + , with full donor chimerism achieved. progression to enteral feeding has been much more rapid, with nutrition now fully enteral for both. there were no significant transplant related complications. conclusions: stem cell transplantation represents the only curative option for mngie. due to its rarity and relative infancy as a condition, little is known of the expected course following transplant or the best approach to transplantation itself. despite previous challenges with graft failure in mngie recipients, we were able to gain rapid and sustained donor engraftment using different myeloablative conditioning regimes with minimal transplant-related morbidity and no mortality. in keeping with previous reports, resolution of established gastrointestinal symptoms has been slow, though the siblings transplanted earlier in their disease course have shown more rapid improvement supporting the role of early recognition and access to transplant. it is essential moving forward that specialised transplantation centres collaborate so as to guide clinicians in the management of such a challenging condition. disclosure: there are no conflicts of interest to disclose. g pc congenital neutropenia -biology of inflammatory colitis associated with gcsf use, and disease response to allogeneic transplant, a report of cases background: an autosomal recessive disease, glucose- phosphatase catalytic subunit (g pc ) deficiency is a relatively recently identified cause of chronic severe neutropenia. there can be a spectrum to the disease and patients may also present with non-haematological features including prominent chest veins, cardiac, endocrine or urogenital abnormalities. we describe in our patient cohort a response to gcsf but an inflammatory, incapacitating, biopsyproven colitis associated with that g-csf response. we have transplanted children with such colitis, and describe a similar colitis with intestinal failure in a fourth. methods: we investigate the biology of the neutropenic colitis, and demonstrate necrosis of the stimulated neutrophils. in vitro studies demonstrated that unstimulated neutrophils from patients with g pc d exhibited significantly increased production of il , reactive oxygen species (ros) and neutrophil extracellular traps (nets) alongside significantly higher expression of cd b, cd b and cd . in contrast, neutrophils from patients with g pc d produced significantly less ros, mmp- , neutrophil elastase and nets upon stimulation. neutrophils from patients with g pc d also exhibited significantly accelerated apoptosis and secondary necrosis which was exaggerated upon stimulation with live escherichia coli bacteria but could only be partially rescued with supplemental exogenous glucose. results: patients have undergone hsct for g pc neutropenic enterocolitis ( unrelated donor and msd) after fludarabine treosulfan and thiotepa conditioning therapy. alemtuzumab was given as as serotherapy. all patients are alive and well, immune suppression has been discotniuned and there is no gvhd with normal organ function, and resolution of colitis. we describe a th patient with no good donor who has continuing intestinal failure with g-csf use. conclusions: we describe the aetiology of intestinal inflammation and failure with an extensive study of neutrophil biology in this metabolic neutropenia. we describe a novel indication for hsct in this "g-csfresponsive neutropenia". disclosure: nothing to declare p does body mass index (bmi) pose a risk to outcome for pediatric non-infantile patients undergoing hematopoietic cell transplantation (hsct)? mona al-saleh , khawar siddiqui , amal al-seraihy , abdullah al-jefri , ali al-ahmari , hawazen al-saedi , awatif al-anazi , mouhab ayas , ibrahim al-ghemlas with no evidence of toxicity. as benefits of stoss therapy in hsct remain unknown, and safety has yet to have been studied extensively in the pediatric population, we hypothesize that stoss therapy is an effective and safe method to reach and attain sufficient levels of vd in pediatric patients undergoing hsct. methods: this is an ongoing prospective, randomized clinical control trial at phoenix children's hospital that commenced december st , . following consent, subjects are randomized to the intervention (stoss) or control arm prior to hsct. stoss therapy consists of a single oral dose of vd (ranging , iu - , iu), given based on baseline -hydroxyvitamin d [ (oh)d] level and age, followed by standard weekly supplementation. subjects enrolled on the control arm receive standard of care based on endocrine society guidelines of weekly vd supplementation. data collection includes demographics, (oh)d levels at baseline, day + , and day + , vd toxicity (hyperphosphatemia, hyperkalemia and renal calculi), as well comorbidities were collected. at each time point and for each trial arm, the mean (oh)d level and changes from baseline were computed with corresponding % confidence intervals (cis) to indicate variability. results: presently, subjects have completed baseline assessment, with day + and day + follow-up completed for and of these, respectively. at baseline, the mean ( % ci) (oh)d was . ng/dl ( . , . ) among stoss patients and . ng/dl ( . results: total hrqol scores of transplanted patients were significantly improved compared to those on supportive care and also compared to healthy siblings (p < . and . respectively), the same was true for physical (p < . and . respectively) and emotional functioning (p < . and . respectively). social and school functioning of transplanted children were not different from healthy siblings (p . and . respectively) while were very significantly improved compared to children with st on supportive care (p < . in both cases). conclusions: bmt in a lower-middle income setting may be even more impactful compared to high-income regions. our analysis clearly indicates normalization of hrqol in all major areas of children transplanted for st. a possible resilience effect was noted for physical and emotional scores which were improved compared to healthy sibling controls. we could not however quantify the effect of longer-term issues like fertility impairment after bmt which may eventually adversely impact hrqol, particularly in the indian culture. disclosure: none allogeneic hematopoietic stem cell transplantation in ataxia telangiectasia patients without malignancy background: ataxia telangiectasia (a-t) is a primary immunodeficiency with mutations in atm-gene. besides a slowly progressive neurodegenerative course, a-t leads to increased susceptibility to malignancies which affects % of patient (median: . years) with a high mortality mainly due tocomplications of conventional radio-chemotherapy. the incidence of cancer correlates with the extent of immunodeficiency. patients often develop severe progressive granulomatous skin disease with evidence of vaccine-strain rubella-virus in the lesions. prolonged survival, neurologic improvement and malignancy prevention was observed in atm-deficient mice after treatment by syngeneic hsct. nevertheless, pre-emptive hsct is not routinely performed in a-t patients due to concerns about neurodegeneration and toxicity. methods: we present three a-t patients with severe immunodeficiency phenotype, undergoing successful hsct as an individual treatment strategy intending to restore immunodeficiency for long-term malignancyprevention (patient- ) and to treat progressive skin/joint granulomas (patients- and - ). results: patient- underwent a reduced intensity conditioning (ric) regimen at years of age including fludarabine ( mg/m ), cyclophosphamide ( mg/kg), and atg-fresenius ( mg/kg/d) which was tolerated well. hematopoietic engraftment occurred by day + . there was an expansion of naïve and memory cd + t-cells and cd + cells. while initially a mixed donor chimerism in patient's pbmcs ( - % donor) was observed, patient's tcells (cd + ) reached over % of donor origin over time. at last follow-up ( years) he is well, without signs of gvhd and organ toxicity, off immunosuppression with normal levels of atm-protein; his granulomas resolved. patient- is a year-old male who was transplanted from his hla-identical sibling, conditioned with fludarabine ( mg/m²), cyclophosphamide ( mg/kg), and atg-fresenius ( mg/kg). hematopoietic engraftment was observed by day + . t-cell reconstitution started by day + with > μl cd + t-cells. his mixed chimerism rapidly turned to donor origin ( % donor cd +) over time. there was no acute toxicity, however, he developed lumbosacral pain episodes with evidence of urine bk-virus with spontaneous remission. an intermittent metapneumovirus associated pulmonary hypertension was observed with pericardial effusion. treatment included sildenafil and oxygen. at last follow-up ( months) patient is well without immunosuppression. patient- suffered from recurrent chest infections, failure to thrive and progressive and debilitating rubella positive progressive granulomas of the skin. she received allohsct from an hla-identical family donor at years of age. conditioning included busulfan ( . mg/kg), fludarabine ( mg/m²), cyclophosphamide ( mg/kg), and alemtuzumab ( x mg/m², x mg/m²). hsct was complicated by intermittent acute renal failure, cmv reactivation and tma. hematopoietic recovery was observed by day + . t-cell chimerism increased rapidly over time (> % donor). at last follow-up ( months) patient is well, off immunosuppression and ivig. her skin granuloma resolved with scarring residues. conclusions: pre-emptive allohsct is feasible in a-t when reduced intensity conditioning is used and can correct the immunodeficiency. it might be a treatment option for some a-t patients at high risk of hematological malignancy and severe granulomatous skin disease. to what extent the restored immune system and the increase of atm-protein in these patients could prevent the development of other malignancies needs to be evaluated further. disclosure: nothing to disclose p abstract withdrawn. hematopoietic stem cell transplantation in diamond blackfan anemia: brazilian experience background: diamond-blackfan anemia (dba) is a rare inherited red cell aplasia caused by an intrinsic defect of erythropoietic progenitors. the main therapeutic approach is based on repeated red blood cell transfusions and/or corticosteroid therapy. hematopoietic stem cell transplantation (hsct), a potentially curative treatment for dba, is indicated for patients that do not respond to first-line therapy. methods: the aim of our retrospective study is to report the outcomes of brazilian dba patients transplanted between and in bmt centers. the median age of the patients was ys (range - ) and % were male. seventeen patients ( %) were transplanted with matched related donors (mrd) and thirteen ( %) from matched and mismatched unrelated donors (mud/mmud). in the mrd group all patients received bone marrow as hsc source, while in the mud/mmud, eight patients received bone marrow and five received cord blood. all patients with incompatibilities (mismatched) were ucb ( / ). nineteen recipients were conditioned with busulfan plus cyclophosphamide, while the remaining received fludarabine and busulfan, which has been the preferred regimen in brazil in the recent years. after transplant, most (n= ) of the mrd and mud recipients received cyclosporine and short course methotrexate as graft versus host disease (gvhd) prophylaxis. results: twenty-two out of the patients were alive and disease-free at a median follow-up of months (range to months). the -year overall survival (os) was % (ci - %) (fig ) . similar results have been demonstrated in studies from europe and from the united states. when analyzed according to donor type, os was % (ci - %) and % (ci - %) in mrd and mud/mmud respectively (fig ) . three out of the patients who were transplanted with ucb died. these results are in agreement with those of previously published data showing worse results in unrelated ucb transplants. twenty-nine out of the patients engrafted successfully. in of the evaluated patients, the median time to neutrophil engraftment was days (range - ). one patient experienced an early death from hemorrhagic shock on day , before neutrophil recovery, and another two patients experienced primary graft failure. post-transplant chimerism was available for patients. sixteen had complete chimera (> % chimerism), while patients presented with mixed chimerism. acute gvhd was observed in patients ( %), of which classified as grade iv. five patients developed chronic gvhd, considered severe in three of them. eight patients died at a mean of days (range - days) after hsct and the main causes of death were infections and hemorrhagic disorders. conclusions: hsct is a potentially curative treatment option for dba. in the present study, we report the outcomes of patients with dba transplanted in brazil with a os of %, with better results in mrd compared to mud, as expected. despite the small numbers, we observed lower survival after mud/mmud ucb transplantation. since dba is a rare disease, international collaborative studies are essential to better understand the benefits of the hsct in the treatment of these patients. disclosure: nothing to declare p treatment of the obliterant bronchiolitis in pediatric allogeneic recipients: two periods compared results: in group , the therapies administered for bo included prolonged treatment with steroids in all patients, anti-tnf in , azatioprine in ; while in the group , all patients received ima, montelukast and azitromicin, and received i.v. mpd. the median duration of imatinib therapy was years ( . - . years). after a median follow-up of . years (range . - . yrs), / patients of group ( %) died with bo in progress for transplant-related causes. while in the group , / ( %) died in presence of worsening bo. the estimated os at year after hsct was % ( % ci; - ) in group and % ( % ci, - ) in group (p= . ) (figure ), while the os after year decreased at % ( % ci; - ) in the group while remained stable in the group . conclusions: this experience shows a relevant improving in prognosis of children with bo with the use of this protocol including ima, since the significant improving of survival obtained, confirming as reported in adult populations. disclosure results: we presented patients with pres, age ranging from months to years with a average of . years. there were ten patients with thalassemia major, two patient with acute lymphoblastic leukemia, three patients with sickle cell disease and one patient with myelodysplastic syndrome, one patient with immune deficiency, two patients with acute miyeloid leukemia, one patient with aplastic anemia. ten patients were males, ten were female. all patients were treated with csa or tacrolimus and metilprednisolone for the prophylasix of gvhd. pres occurred at a median of days (range - ). clinical findings at onset of leukoencephalopathy were hypertension, headache, seizures, visual disturbance, and altered mental function. eighteen patients alive with normal neurological status. mri showed abnormalities in all patients including patchy bilateral cortical and subcortical lesions, especially in parieto-occipital lobes. conclusions: bmt is associated with several neurological complications that may be underlying diseases, bmt procedure, and severe immunosupression. pres is an uncommon but serious complication after bmt. we report cases of pres who received allogeneic bmt for thalassemia major to emphasize the importance of early recognition and institution of appropriate management of pres during bmt. disclosure: nothing to declare p continuous complete molecular remission using three different monoclonal antibodies followed by allogeneic bone marrow transplantation in an infant with chemotherapy-refractory acute lymphoblastic leukemia bernd gruhn , susan wittig , thomas ernst , jana ernst university of jena, jena, germany, background: a -week-old infant was diagnosed with very immature acute lymphoblastic leukemia (all) with myeloid markers in a foreign university hospital. at the end of induction therapy according to the current lal/shop protocol % leukemic cells were detectable in the bone marrow. treatment was changed to fludarabine, cytarabine and granulocyte colony-stimulating factor (flag) in combination with liposomal doxorubicin. after this re-induction still % leukemic cells were detected in bone marrow, so the bispecific t-cell engager antibody blinatumomab was given. due to an increasing portion of leukemic cells during the continuous infusion, antibody therapy was stopped and a cycle of clofarabine, cyclophosphamide and etoposide was administered. unfortunately, still % leukemic cells were detectable afterwards. because of chemotherapy-refractory leukemia a palliative oral treatment with mercaptopurine was started. however, the parents did not accept the palliative situation and searched for alternative therapeutic options in other university hospitals in europe. after plenty of refusals the infant was admitted to our hospital five months after diagnosis. methods: for molecular characterization genomic dna was isolated from leukemic cells. a mll-mllt /af rearrangement as a consequence of the translocation t( ; ) (p ;q ) was detected and used as a marker for minimal residual disease. for further molecular characterization targeted deep next-generation sequencing was performed for a panel of leukemia-associated genes. interestingly, no mutation was found. to allow precise immunophenotyping of the leukemic cells treatment with mercaptopurine was stopped. results: as in the first immunophenotyping the cd antigen was found, we administered the anti-cd monoclonal antibody gemtuzumab ozogamicin twice within two weeks. because of the detection of cd + leukemic cells after infusion of gemtuzumab ozogamicin, the anti-cd antibody daratumumab was given alternating twice within two weeks. unfortunately afterwards, leukemic cells reappeared being negative for cd und cd , but positive for cd . therefore, we administered the third antibody, the anti-cd monoclonal antibody inotuzumab ozogamicin, whereupon our patient developed a tumor lysis syndrome and a severe bone marrow aplasia. shortly after, allogeneic bone marrow transplantation from an unrelated donor using a special conditioning regimen consisting of thymoglobulin, busulfan, fludarabine and clofarabine was conducted. clofarabine was added because an additional antileukemic effect especially in infant all with mll rearrangement was described. after transplantation the patient suffered from a severe hepatic sinusoidal obstruction syndrome with massive ascites, renal and pulmonary dysfunction, but finally the patient recovered completely. the first bone marrow examination days after transplantation revealed a donor chimerism of % and a complete molecular remission using the mll-mllt /af rearrangement as marker for minimal residual disease. in all follow-up bone marrow samples we observed a complete donor chimerism and a complete molecular remission. currently, eight months after transplantation the patient is in a very good physical condition with normal development according to the age. background: paediatric chronic graft versus host disease (cgvhd) is a debilitating condition associated with substantial morbidity and mortality. to date, there are no approved therapies for paediatric patients with cgvhd, and current treatments often lack sufficient efficacy or lead to severe/life-threatening toxicities that limit their effectiveness. ibrutinib, a first-in-class, once-daily inhibitor of bruton's tyrosine kinase (btk), is approved in the us for the treatment of adult patients with cgvhd after failure of ≥ lines of systemic therapy. this phase / study will evaluate the use of ibrutinib in paediatric patients with moderate or severe cgvhd. methods: this open-label, multicenter, international phase / study (pcyc- ) includes patients with moderate or severe cgvhd as defined by the nih consensus development project criteria. it is divided into two parts: part a will determine the recommended paediatric equivalent dose (rped) of ibrutinib in patients aged ≥ to < years, and part b aims to evaluate the safety and efficacy of ibrutinib in patients age ≥ to < years. for part a, patients with cgvhd aged ≥ to < years who have failed ≥ lines of systemic therapy will receive once daily oral ibrutinib at a starting dose of mg/m to be escalated up to mg/m after days, if no grade ≥ toxicities occur, until the rped is determined. for part b, patients aged ≥ to < years with cgvhd who have failed ≥ lines of systemic therapy or have newly diagnosed cgvhd will receive once daily ibrutinib ( mg) until one of the following criteria is met: treatment is no longer required; new systemic treatment for cgvhd is initiated; progression of cgvhd; recurrence of underlying disease; or unacceptable toxicity. patients with newly diagnosed cgvhd will receive ibrutinib in addition to daily corticosteroids ( . - mg/kg prednisone). patients < years of age may be enrolled in part b and treated at the rped after it is determined in part a. key exclusion criteria include uncontrolled active systemic infection or active infection requiring systemic treatment; progressive underlying malignant disease or any post-transplant lymphoproliferative disease; or active hepatitis c/hepatitis b virus. patients must have adequate renal, hepatic, and hematologic function to be enrolled. the primary endpoint of part a is the rped of ibrutinib, as based on pharmacokinetic (pk) data; secondary endpoints include safety and pharmacodynamics (btk occupancy). the primary endpoints of part b are pk and safety of ibrutinib in paediatric patients with cgvhd. secondary endpoints for part b include response rate at weeks as defined by the nih consensus development project criteria; duration of response; overall survival; and late effects on growth, development, and immune reconstitution. results: this global study is currently enrolling. conclusions: this phase / study will explore the use of ibrutinib in paediatric patients with cgvhd to potentially meet the high unmet need for proven effective therapies for this population. disclosure enzyme replacement therapy (ert) is the treatment of choice in non-neuropathic hunter syndrome, but as the recombinant enzyme does not cross the blood brain barrier and neuropathic hunter syndrome is left untreated. hematopoietic stem cell transplantation (hsct) is the standard of care in patients with severe mucopolysaccharidosis (mps) type i (mpsih, hurler syndrome) as early transplantation halts cognitive decline in these patients and significantly improves survival. only few case studies have been published on the potential benefit of hsct in mps ii and mostly used busulfan-based conditioning regimens. in one comparative non-randomised multicenter study, hsct might to be superior compared to ert. here, we present our experience in hsct in three children with hunter syndrome using a treosulfan-based conditioning regimen. methods: a retrospective chart review was carried out in patients, who underwent hsct for hunter syndrome. the conditioning chemotherapy regimen included fludarabine, treosulfan, thiotepa and atg. all patients received bone marrow of either related and or matched unrelated donors. gvhd prophylaxis was performed with csa and methotrexat. results: three patients with hunter syndrome were transplanted in our department in . the age was six months, two years and four years, respectively. bone marrow donors were related in one patient and matched unrelated in two patients. the conditioning therapy was generally well tolerated. major complications were fever of unknown origin with need for antibiotic therapy and a mucositis. one patient developed a cmv reactivation. all patients engrafted successfully and recovered well from the hsct. there was no case of acute or chronic gvhd. in all three patients are alive. donor chimerism is complete in one patient; two patients have a mixed donor chimerism. after application of donor lymphocyte infusions in one patient, donor chimerism is stable at a low level of %. the donor chimerism of the other patient still slowly declines to currently %. after stem cell transplantation, two patients did not show further progression of the disease and even achieved psycho-motor improvements. interestingly, one of these patients is the one with the low donor chimerism of %. one patient suffers from a further progression of the underlying disease with psycho-motoric agitation, aggressive behavior and loss of speech, that occurred within the first year following hsct, but neurocognition stabilized thereafter. conclusions: we found a beneficial effect of hsct on the neuropsychological outcome or at least stabilization of neurocognitive function in our patients with a follow-up of eight years. despite low toxicity of the conditioning regimen, increased donor chimerism may further improve the neurological outcome. disclosure: nothing to declare. tandem sct in pediatric solid tumors, other than brain tumors, has no advantage in terms of efs over single procedure-single center experience , germ-cell tumour (gct), ewing sarcoma (es), nefroblastoma. patients were divided into groups according to the number of procedures: st group-single sct procedure, nd group-multiple procedures. regimens used for stem cell mobilisation were: topo-cy for nbl and epi-tax for gct, followed by g-csf±plerixafor. conditioning regimens: bu-mel and thiotepa-cy for pts with nbl, thio-tax and ice for pts with gct. patients received antibiotic, antiviral and antifungal prophylaxis, parenteral nutrition and supportive treatment. patients received consolidation treatment, followed up monthly in the first year, then yearly. patients were evaluated for residual disease by imaging tests. parents signed informed consent forms. results: we performed sct procedures to patients: . % nbl, . % es, . % gct and . % nefroblastoma. for this study only patients with nbl and gct were considered. in st group were % of pts, % in nd group. patients were diagnosed, staged and treated according to international protocols. sex ratio was f/ m. age distribution was - y % ( pts), - y % ( pts), > y % ( pts). peripheral stem cell (pbsc) mobilisation was more difficult in patients with multiple courses of chemotherapy±radiotherapy. we found no difference in the period of engraftment following a nd or rd procedure. hospitalization and supportive measures increased in nd and rd procedures ( to days). patients with multiple courses of chemotherapy and multiple hospitalizations had increased infectious risk and during the nd or rd procedures developed various infectious complications.incidence of severe oral mucositis after the first hsct was %, after tandem hsct was %. nbl patients : st group- / patients alive and efs, / receives anti g treatment; nd group- / patients-alive, / patients-not reached timepoint for mibg scan; / patients-mibg negative at first, relapsed after mo; / patient deceased due to pulmonary toxicity. gct patients: st group- / patients alive and ef, / high values in afp levels and receives metronomic therapy, / patients deceased due to progressive disease, but only had sct. only / patient had one procedure and died due to progressive disease. conclusions: in our study, tandem hsct in children with solid tumours lead to an increase in survival rates, at least in the first months after sct. most patients ( %) had progressive or relapsed/refractory disease when referred to our department. multiple procedures require a higher number of cd cells, very hard to achieve in patients with multiple courses of chemo± radiotherapy. new approaches have to be considered in these diseases, especially in high risk group. disclosure: nothing to declare background: antimicrobial prophylaxis for prolonged neutropenia occurred during the pre-engraftment period is a common practice in allogeneic hsct recipients. data on its effectiveness are few and generally from cases series and not from randomized clinical trials, especially in children. methods: all clinical records of allo-hsct performed from january st to november th at hsct-unit of istituto g.gaslini, genoa-italy, were retrospectively reviewed. collected data were underlying diseases, type of donor, antibiotic prophylaxis administration and type, development of fever and pathogen isolated from blood culture, if any, during pre-engraftment neutropenia. antibiotic prophylaxis, usually starting together with the conditioning regimen, was categorized in "standard" (with amoxicillin/clavulanate or ampicillin/sulbactam) or "tailored" (when based on previous bacterial isolations or colonizations). results: allo-hscts were performed in pediatric patients ( % males) with a median age at hsct of years (iqr: - ; range: - ). hscts were performed from alternative donor (ad) in % patients, from relative donor (rd) in %, and from haploidentical donor in %. table shows the pre-engraftment febrile neutropenia episodes according to type of antibiotic prophylaxis. ( %) hscts received standard prophylaxis, while ( %) the tailored one; only in ( %) did not receive any prophylaxis. fever occurred in ( %) of episodes in patients receiving standard prophylaxis, in ( %) of those treated with tailored prophylaxis and ( %) in the group without prophylaxis; only % of patients who received prophylaxis did not develop fever.in % of patients, the febrile episodes were diagnosed as bloodstream infections: staphylococcus aureus in %; cons in %; enterococcus spp in %; enterobacteriaceae in %; pseudomonas aeruginosa in %; other non-fermenting gram negatives in % and fungi in %. conclusions: the occurrence of fever in patients who received antibiotic prophylaxis suggested that it could not be effective in prevention of fever related to neutropenia after allo-hsct. the personalization of prophylaxis could be a possible path to follow these patients. disclosure methods: a total of patients with leukemia or neuroblastoma were included in the study. patients' mothers signed an informed consent for participation in the study. six of study participants were boys and girls, all aged to years. the control group consisted of healthy preschool children ( groups of children aged to years), boys and girls. results: in most of games the role of a doctor was played by a child. only one child declined to impersonate both a patient and a doctor. younger children mostly agreed to have for a "patient" a toy (proposed by psychologist or one of child's own), child's mother or a medical psychologist. the game lasted for - minutes. most patients preferred using real medical consumables and instruments (syringes, adhesive tape, winged infusion sets or, more rarely, pills). most often a syringe or an adhesive tape was chosen. as known from their mothers, among medical manipulations most unpleasant for children are injections and changing implanted catheter dressing. also, most healthy preschool children preferred using real medical instruments over toy ones. group more often used a syringe, a winged infusion set, adhesive tape, gauze or pills. group most often chose syringe or gauze. among medical instruments both groups more often chose a phonendoscope or thermometer.one patient refused to cause pain to a "toy patient". other children sympathized with a "toy patient", stroke injection or dressing location sites or used soothing terms ("wait a little", "it's going to be all right"), wished prompt recovery and hugged their "patients". one child was angry over his "patient" wishing him to "get hurt too". first preschool group children were mostly scolding a toy "patient" for "being guilty of getting sick". second group children were mostly compassionate, encouraged a "toy patient" telling that "all the procedures are needed to get healthy". from children's schoolmasters we know that all first group children received vaccination about a week before a test. children from second group had no injections. overall attitude towards toy "patients" was more mild in the second group. conclusions: . during a play children mostly use the medical devices which cause them most discomfort and/or pain. . manipulating the items children illustrate their own impression of medical procedures, which are most unpleasant. . children may express their negative emotions directed towards medical manipulations via their play actions, these negative reactions may be suppressed in different ways by parents or medical staff. . the intensity of child's own traumatic experience and an attitude of nearby adults may influence the child's attitude towards other patients. . the mother's wish for a child to tolerate all medical procedures with ease exceeds real capabilities of a small child. disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type iiia (sanfilippo): a case series methods: allogeneic sct was performed at the ages of , and years, respectively. all three patients received intrathecal enzyme replacement therapy within a clinical trial setting prior to hsct. the conditioning regimen consisted of treosulfan, fludarabine, thiotepa and thymoglobuline. gvhd-prophylaxis was carried out with csa and mtx in two patients and csa and mmf in one patient. stem cell source was bone marrow in two patients and peripheral blood stem cells in one patient. results: the conditioning regimen was well tolerated and all three patients successfully engrafted. two of three patients had an uncomplicated course without occurrence of acute or chronic graft-versus-host-disease (gvhd). at last follow-up and months after hsct, both patients are in good condition and show constant progress of psychomotor development. the third patient experienced severe steroid refractory acute gvhd of intestines (stage ) and skin (stage ), which resolved under intensive immunosuppression with cyclosporine, mycophenolate and ruxolitinib. around day after hsct, this patient showed clinical and biochemical signs of transplant-associated microangiopathy (tma) with cerebral seizures and acute renal failure. the cerebral mri showed progressive cerebral atrophy and leukoencephalopathy, also consistent with a progress of the mps iiia. at last follow-up months after hsct, this patient had recovered from tma and was in a stable clinical condition. conclusions: in consideration of the small case number and the short follow-up period in our cohort, allogeneic hsct might be considered as a salvage therapy for patients with mps iiia if other therapeutic options are unavailable for children with this otherwise unfavourable prognosis. however, the early psychoneurological course after transplant seems promising compared to the literature and hsct could become a treatment option for this rare disease. disclosure: nothing to declare methods: for the identification of underlying molecular mechanisms leading to the increased sensibility of rms cells, the activation status of different nf-kb signaling pathways were analyzed using western blot analysis and quantitative real time pcr (qpcr). further, flow cytometry was used to analyze the surface expression of death receptors on either sm treated or untreated rms cells. the overall effect on cell death induction was measured by pi/hoechst staining using a fluorescent microscope. results: treatment with sm led to the suspected degradation of iaps. followed by the activation of both the canonical nf-κb signaling pathway, indicated by the phosphorylation of iκbα and p , and the non-canonical nf-κb signaling pathway, as indicated by the accumulation of nik and the degradation of p to p . determination of selected target gene transcription revealed an upregulation of the inhibitor iκbα, nik, p , il- and at later time points the death receptors trail-r and trail-r . analysis of gene transcription also led to the finding of neither up-nor downregulation of ciap and p . to evaluate the involvement of trail-r and trail-r in the sm induced sensitization towards nk cell-mediated killing, surface expression of both death receptors was analyzed. treatment with sm led not only to an induced transcription of trail-r and trail-r , but also to an increased surface presentation of trail-r . subsequent ligation of trail-r by either wt-trail or a specific agonistic antibody (etr- ) resulted in a significant increase in cell death induction. the aforementioned analysis of gene transcription hints towards a bimodal feedback mechanism regulating both, the canonical and non-canonical nf-κb signaling pathway. on the one side, the canonical pathway is negatively regulated by the induced transcription of the inhibitor iκbα. on the other side, the induced transcription of nik, p and relb points towards a positive feedback loop of the non-canonical pathway. one mechanism of the increased rms cell sensitivity might be the induced transcription and surface presentation of the death receptor trail-r . the involvement of trail receptors is further validated by the cytotoxicity data, illustrating a sm mediated sensitization towards a trail induced cell death induction. this mode of cell death fits to the previous research, were trail transcription could be induced in nk cells by sm treatment. the graphical abstract shows the transcriptional upregulation of target genes leading to a putative bimodal nf-kb regulation and increased surface presentation of trail-r by treatment with smac mimetics. aim: to investigate the outcome of ucb transplantation in pediatric patients with malignant and non malignant diseases methods: data from patients underwent first allogeneic bone marrow transplantation with ucb from / until / were retrospectively analyzed. eighteen had malignant disease (md), of whom in complete hematologic response, and non malignant disease (nmd) (scid , chronic granulomatous disease , severe aplastic anemia , s.kostmann , osteopetrosis , wiskott-aldrich , amegakaryocytic thrombocytopenia ). the majority of the patients were male, for md and nmd, as well (m: /f: , m: /f: , respectively), of median age . years (range . - . years) and . years (range . - . years), respectively results: all patients but one, received ucb unit. hla compatibility in antigen/allele level was at least / and only in patients with md was / . conditioning regimens were myeloablative and tbi gy was given in / . gvhd prophylaxis consisted of cyclosporine and atg was given in all patients pre-transplantation. median value of nucleated cells for md was . χ /kg (range - . χ / kg) and for nmd was . χ /kg (range - . χ /kg). neutrophil and platelet engraftment was achieved in / and / patients with md respectively, in a median time of days (range - ) και days (range - ). in patients with nmd, neutrophil and platelet engraftment was achieved in / and / with median day of engraftment days (range - ) και days (range respectively. acute gvhd grade ΙΙ-Ιv presented in / patients with md and / with nmd, although none had cgvhd. the incidence of viral infections was cases in patients with md and cases in patients with nmd. disease relapse occured in / patients with md. after a median time of years follow up, overall survival (os) and event free survival (efs) for children with md were % and . % respectively, while for nmd, os and efs were %.treatment related mortality at d+ was % for md and % for nmd. among patients with md, are still alive, while the rest died from relapse (n: ), viral infections (n: ), septicemia (n: ) and agvhd(n: ). among patients with nmd, are alive, while the rest died from viral infections (n: ), septicemia (n: ) and multiple organ failure (n= ). the median time of hospitalization for patients with md was days (range - ), whilst for nmd was days (range - ). conclusions: transplantation of unrelated ucb in our unit was combined with high trm in children with nmd and higher probability of relapse for md. disclosure: nothing to declare p serum levels of -s cysteinyldopa is associated with stem cell transplantation related complications yukayo terashita , mamoru honda , minako sugiyama , yuko cho , akihiro iguchi hokkaido university hospital, pediatrics, sapporo, japan background: diffuse hyperpigmentation is common in patients who received chemotherapy or stem cell transplantation (sct). however, there are few reports of the relationship between skin reaction such as pigmentation and the other complications. pigmentation of the skin is thought to be the result of melanin stagnating in the dermic layer due to increased synthesis of melanin and destruction of the basement membrane due to inflammation induced chemoradiotherapy. melanin pigments are classified into two types: brown to black eumelanin and yellow to reddishbrown pheomelanin. -s cysteinyldopa ( scd) is precursors of pheomelanin, and its serum level has been used specific biochemical marker for malignant melanoma. here, we examined serially scd during the course of sct to determine association with sct related complications, because visual evaluation of skin color is difficult, and there have been no reports about scd as sct related biomarker. methods: we prospectively analyzed ( males, females) patients who received sct between may and march in hokkaido university hospital. the median age at transplantation of the patients was . years (range, - ). indication for sct were acute myelogenous leukemia in patients, acute lymphoblastic leukemia in patients, and other disease in patients; juvenile myelomonocytic leukemia( ), malignant solid tumor( ), immunodeficiency( ), anaplastic anemia( ), and diamond blackfan anemia ( ) . patients received allogeneic sct and received autologous sct. myeloablative conditioning (mac) was used for patients and reduced intensity conditioning (ric) was used for patients. sera were obtained from patients before conditioning therapy, on day , + , + , + , + and + . all blood samples were centrifuged at , rpm for min, and stored at - ˚c until used. we also examined sct related complications such as graft-versus-host disease (gvhd), viral infection, and pre-engraft syndrome (pes). statistical analyses were completed using the mann-whitney u test for unpaired samples, and kruskal-wallis test for three samples. each test was performed with a % level of significance. results: the average value of scd reached two peaks, day ( . nmol/l) and day ( . nmol/l), regardless of stem cell source and intensity of conditioning. in all patients, we found that the level of scd on day was associated with viral reactivation (p= . ), scd on day was associated with pes (p= . ), and scd on day was associated with malignant disease (p= . ). similarly, in patient who received allogeneic sct (n= ), the level of scd on day was associated with viral reactivation (p= . ), scd on day was was associated with pes (p= . ), scd on day was associated with malignant disease (p= . ). in addition, the level of scd on day was associated with gvhd of skin (p= . ), the peak level of scd was associated with acute gvhd (p= . ). conclusions: we found that scd can be a biomarker for sct-related complications such as aute gvhd. it is presumed that the production of pheomelanin could be induced by inflammatory procedure in sct. disclosure: nothing to declare p hsct in children with bone marrow failure: outcomes from a single singapore centre prasad iyer , michaela seng , vijayakumari k , ah moy tan , mei yoke chan , rajat bhattacharyya kk women's and children's hospital, paediatric haematology-oncology, singapore, singapore background: children presenting with pancytopenia often present a challenge to the paediatric haematologist. the underlying diagnosis can be hard to establish as many of the inherited bone marrow failure syndromes (ibmfs) can present with protean manifestations. the large majority of patients with bone marrow failure are often diagnosed with idiopathic severe aplastic anaemia (saa) despite extensive testing. we report our experience of hsct in patients treated with primary and acquired bone marrow failure. methods: we reviewed case notes of all the children who underwent hsct for bone marrow failure in our centre. results: a total of fifteen patients underwent eighteen stem cell transplants in our centre between and . three patients were diagnosed with fanconi anaemia, one with hoyeraal-hreidarsson syndrome, one with paroxysmal nocturnal haemoglobinuria and the remaining ten children had idiopathic saa. eight children had matched sibling donor transplant, had a matched related donor, had a matched unrelated donor, had umbilical cord blood transplants and the remainder were haploidentical transplants. four of the haploidentical transplants were t-cell depleted and one was t-cell replete. one child with fanconi anaemia had primary graft rejection with cord blood transplant and was successfully rescued with a haploidentical transplant. one child with saa had primary graft rejection twice (t-cell depleted graft) and then was rescued with an alternate haploidentical donor with a t-cell replete graft. of the two patients who died, one had a fatal fungal infection ten months after transplant, and the other died due to a severe influenza pneumonitis three and a half years after bmt. conclusions: haematopoietic stem cell transplant outcomes from our centre are comparable to leading centres in the world. the understanding of underlying conditions that present with bone marrow failure has improved our approach and the way we treat bone marrow failure syndromes. clinical trial registry: not applicable. disclosure: nothing to declare. methods: a retrospective study was performed in children treated with hsct who received pos or flu during early neutropenic period until engraftment from january to december at siriraj hospital in thailand. the efficacy, safety and tolerability of pos were compared to flu. results: there were hsct recipients (allo-hsct . %, auto-hsct . %) with mean age of . + . years. most of the patients were thalassemia ( . %) followed by hematologic malignancy ( . %) and solid tumor ( . %). seventeen and cases received pos and flu, respectively. all of patients in pos group were allo-hsct whereas . % in flu group were allo-hsct. in pos group, cases were diagnosed with suspected ifi and cases were probable ifi with total cases ( . %). in flu group, cases were diagnosed with suspected ifi and cases were probable ifi with total cases ( . %) which compared groups were not statistically significant (p= . ). no possible and proven ifi in both groups. in flu group patients received empirical antifungal treatment more than pos group but no statistical significance ( . % vs. . %, p= . ). both groups had similar rate of elevated liver function test (p= . ). no early discontinuation of antifungal prophylaxis for intolerance was found in both groups. only . % of patients achieved pos target trough level of . mg/l after days of treatment with started dose mg/kg three times a day. conclusions: pos and flu are comparably effective, safety and tolerability in ifi prophylaxis in neutropenic children treated with hsct. defining dose recommendation of pos in this setting requires larger studies. disclosure background: severe congenital neutropenia (scn) is typically characterized by anc of < /μl, maturation arrest of bone marrow myeloid precursors at the promyelocyte-myelocyte stage, and susceptibility to lethal pyogenic bacterial and fungal infections. scn is a rare group of disorders resulting from intrinsic defects in myeloid cell proliferation and maturation caused by mutations in several genes; elane, hax , gfi , was, and g pc are among the most common ones. almost % of patients are refractory to g-csf, and the only definitive curative approach for such patients is allogeneic hsct. the current absolute indications for hsct is failure to respond to g-csf treatment, or the development of mds/leukemia in patients with scn. here, we present the result of children with scn who received allogeneic hsct . methods: we retrospectively assessed allogeneic hsct in children with severe congenital neutropenia. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted dose. in addition, all patients received fludarabine mg/m in five days or cyclophosphamide mg/kg in days and atg mg/kg in days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in either bone marrow or peripheral blood on days + , + and + . results: the median transplantation age of the patients was months (range - months). six of them are male. two of the donors were matched siblings and were unrelated two of which were ag ag mismatched. stem cell source was bone marrow in patients, peripheral blood in and cord blood in patients. all patients engrafted. the median time of neutrophil and platelet engratment to was ( - ) days and ( - ) days, respectively. graft rejection was experienced in patients, one of them had received unrelated cord blood. all patients are alive, eight of which are with full donor chimerism (between - %) without any complication (no infection, no gvhd) with a median months (range - months) follow up. probability of disease free and overall survival were found % and %, respectively. conclusions: we concluded that hsct is a useful treatment for scn patients, especially those who are unresponsive to gcsf treatment and at high risk for leukemic transformation. however, a larger number of scn patients and longer follow-up are necessary to identify appropriate conditioning regimens and long-term prognosis. disclosure: nothing to declare background: prolonged thrombocytopenia (pt) or secondary failure of platelet recovery (sfpr) are a lifethreatening complications that occurs in - % and - % respectively of the patients following allogeneic hematopoietic stem cell transplantation (allo-hsct). management strategies, including the use of growth factors, cd +-selected stem cell boost, mesenchymal stem cell (msc) transfusion, and second allo-hsct, are not effective or possible for all patients. eltrombopag, is an oral non-peptide thrombopoietin receptor agonist, that leads to signal transduction and results in promoting the proliferation and differentiation of megakaryocytes. some recently studies show that also can promote haematopoiesis along all three lineages. methods: we described our experience in paediatric patients with poor graft function or secondary failure of platelet recovery after allogeneic stem cell transplantation treated with eltrombopag. results: patients characteristics are detailed in table . all the patients received and allo-hst. the median dose of cd + cells infusion was . x e /kg ( . - . ). neutrophils engraftment occurred in + day ( - d) and platelets in + day ( - d). all the patients had an hypoplastic bone marrow with complete chimerism. the median duration from transplantation to spcf diagnosis was months ( . - m) . one of the patients received a stem cell boost prior to eltrombopag, without response. the time onset from spgf/sfpr diagnosis to initiating eltrombopag was days ( - d). eltrombopag was started at a dose of mg/kg/d, requiring an increase dose in all cases. the median dose was mg/d ( - mg). the overall response rate was % ( / ). two patients achieved complete response (cr), as defined by platelet ≥ × /l. both patients already got neutrophil ≥ . × /l without g-csf. the time from eltrombopag initiation to achieving cr was ( - d) days. the treatment was given for a median of days ( - ). it was discontinued after and days respectively in the two responder patients. both patients maintain stable blood counts after discontinuing the treatment. the non-responders patients had to stop the treatment because of other reasons not related to eltrombopag. patient had to be rescued with a cd + cells boost with a good response. two patients that were in treatment with voriconazole for a fungal infection developed hyperbilirubinemia. there were no grade - toxicities related to eltrombopag. conclusions: in our experience, according to recently published studies, eltrombopag is a safe and efficacy drug in the treatment of secondary failure of platelet recovery post-hsct. it may be used successfully in children. sometimes higher doses may be considered if no response is achieved. further prospective trials are needed to increase the level of evidence and to identify predictors of response. disclosure: nothing to declare very slow clearance of busulfan in a child with infant leukemia background: busulfan is a drug with a high interindividual variability between dose and exposition. therefore, it is recommended to perform therapeutic drug monitoring (tdm) in the context of myeloablative conditioning, especially in children. methods: we report on a -month old boy ( . kg, cm) of caucasian decent born to non-consanguine parents with mll-rearranged prob-lymphoblastic leukemia. diagnosis was established one month after birth from peripheral blood and csf tap showed cns involvement. primary chemotherapy was commenced according to the interfant- protocol. however, mrd remained positive two months under treatment, leading to an indication for allogeneic stem cell transplantation. in the interfant- protocol, we opted for a conditioning regimen comprised of fludarabine ( . mg/kg for days), busulfan and thiotepa ( x mg/kg). in our institution, busulfan is applied once daily with a target auc of - h*mg/l in this very high risk situation. according to body weight, busulfan was given with . mg/ kg as a three-hour infusion on the first day. busulfan concentrations in plasma were measured with gas chromatography-mass spectrometry (gc-ms) and auc was calculated using bayesian curve fitting. results: exact busulfan quantification was not possible after the first dose due to technical reasons. as the levels were estimated to be very high, we decided to reduce the second dose of busulfan by %. this resulted in a very high auc of h*mg/l for the second dose, so that busulfan was discontinued after two days, because it was calculated that the patient already received busulfan with a cumulative auc of h*mg/ml. trough levels after the first and second dose were and μg/l, respectively. the patient showed a very slow clearance of . l/h/sqm, while the volume of distribution was in the usual range ( . l/kg). bilirubin and liver transaminases were in the normal range at the time of conditioning, while albumin and quick were decreased on day + after transplantation the patient developed clinical und biochemical signs of venoocclusive disease (vod). vod symptoms completely resolved under therapy with defibrotide. leukocyte engraftment was established on day + . unfortunately, the patients suffered from an early relapse of the leukemia from day + . attempts to induce a second remission with blinatumomab failed. the patient is currently under palliative chemotherapy. conclusions: busulfan tdm is very important especially in infants receiving myeloablative doses of busulfan to prevent under-or over-exposure. there is evidence that high busulfan trough levels contribute to the development of vod, but anti-leukemic activity of busulfan and cns permeability make it a valuable drug for very high risk patients in childhood leukemia. larger patient cohorts are needed to assess the exposure dependent risks of toxicity versus relapse in infants and toddlers. disclosure: nothing to declare blood (ucb) obtained at delivery from three children who received a diagnosis of cerebral palsy. methods: immunophenotyping of the ucb leukocyte fraction was performed using multicolor flow cytometry. the procedure was performed according to the protocol by shatorje and colleagues ( ) . briefly, the ucb samples were labeled with specific antibodies and incubated in the dark for minutes. afterwards, the samples were treated with ml of bd facs lysing solution for minutes to preserve the leukocyte fraction only. the cells were washed using pbs (roche) and then centrifuged twice ( rpm, °c, minutes). the results were analyzed using the facsdiva software (becton dickinson). results: we found an increased white blood cell (wbc) count, lymphocyte count, and cd :cd ratio in all ucb samples. one patient had a low nk cell count and percentage, and another had a low b-cell count and percentage. one sample displayed high t (cd +) and th cell (cd +) counts, but with percentages within the limits of the reference values. conclusions: we detected elevated wbc and lymphocyte counts in all ucb samples, despite a lack of intrauterine infection symptoms. many authors have described the pathogenesis of hypoxic-ischemic encephalopathy. briefly, after an acute hypoxia-ischemia insult, activated resting microglia show macrophage-like activity. this leads to a break-down of the blood-brain barrier, infiltration by peripheral leukocytes, and brain exposure, which further exacerbates inflammation. the role of systemic inflammation is being evaluated in the animal model. it is known that systemic inflammation plays a role in traumatic brain injury (tbi) and is an independent risk factor for poor outcome in isolated tbi patients. on the other hand, m -phenotype microglia inhibit inflammation and protect neurons from secondary damage and death. however, anti-inflammatory mechanisms in neonates are immature and expose them to extremely intensive inflammation. therefore, anti-inflammatory agents, including stem cells, may be beneficial in these patients. disclosure: three of four authors are employees of the polish stem cell bank, warsaw, poland reference: background: under the hypothesis that early natural killer cell infusion (nki) following haploidentical stem cell transplantation (haplo-sct) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of nki following haplo-sct in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous sct. methods: we used the high-dose i-metaiodo benzylguanidine and cyclophosphamide/fludarabine/antithymocyte globulin regimen for conditioning and infused × /kg of ex-vivo expanded nk cells derived from a haploidentical parent donor on days , , and posttransplant. results: seven children received a total of nkis, and nki-related acute toxicities were fever (n = ) followed by chills (n = ) and hypertension (n = ); all toxicities were tolerable. grade ≥ii acute gvhd and chronic gvhd developed in two and five patients, respectively. higher amount of nk cell population were detected in peripheral blood until days post-transplant compared with reference cohort. cytomegalovirus and bk virus reactivation occurred in all patients and epstein-barr virus in six patients. six patients died of relapse/progression (n = ) or treatment-related mortality (n = ), and one patient remained alive. conclusions: nki following haplo-sct was relatively safe and feasible in patients with recurrent neuroblastoma. further studies to enhance the graft-versus-tumor effect without increasing gvhd are needed. disclosure: nothing to declare regenerative medicine p repeated administration of g-csf using stem cell mobilization protocol could induce improvement of cognitive functions of children with cerebral palsy: phase ii randomized placebo-controlled study background: we performed phase ii randomized placebocontrolled clinical study to reveal the safety and feasibility of repeated granulocyte colony-stimulating factor (g-csf) administration for improvement of cognitive functions of children with cerebral palsy. methods: forty-four children with non-severe type of cerebral palsy were enrolled, and their age were - years old. g-csf ( μg/kg) was administered for days subcutaneously every months during months. we compared their cognitive functions with the magnetic resonance imaging (mri) findings and the following tools between before and months after treatment; zoo location and picture memory as working memory index (wmi) in wechsler preschool and primary scale of intelligence (wpssi), receptive and expressive vocabulary test (revt), and visual motor integration (vmi)/visual perception (vp) test. mobilized stem cell count and cytokine levels were measured before (d+ ) and after (d+ ) g-csf administration for days every months. results: no significant findings in demography was noticed between g-csf (g-) and placebo (p-) groups. no serious adverse events were observed during the whole study period. the non-severe adverse events such as urticaria (n= ), itching sense (n= ), bone pain (n= ), headache (n= ), fever (n= ), and stomatitis (n= ) were tolerable. the parents felt the clinical improvements of cognition in cases of g-group and cases of p-group (p= . ), of language in cases of g-group and cases of p-group (p= . ). in zoo location test, we can not find out the significant score (expressed as age equivalent) differences between g-and p-groups. however, in picture memory test, there were significant improvement of age equivalent of months ( . ± . → . ± . ) during months of study period in g-group compared to months in p-group (p= . ). in revt, there were significant improvement of months of age equivalent in expressive tests of g-group ( . ± . → . ± . , p= . ) compared to months in p-group. no significant findings were noted in receptive test. vmi test showed the increasing tendency of months of age equivalent in g-group ( . ± . → . ± . , p= . ) compared to p-group. the increment of cd + cell counts in peripheral blood were significant in ggroup compared to p-group. the changed levels of interleukin (il)- , il- , vascular endothelial growth factor (vegf) as well as g-csf were noted in g-group. we also observed the correlation of cognitive function tests and white matter connectoms of several networks using functionally-defined white matter atlases. conclusions: the repeated administration of g-csf using stem cell mobilization protocol is safe and feasible to improve the language and cognitive functions in children with cerebral palsy. further studies for cellular and paracrine effect of g-csf and/or mobilized peripheral blood stem cells would be needed. background: while high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) is an integral part of multimodal therapy for highrisk neuroblastoma (hr nb), there are still subgroups, in which the results are extremely poor. for these patients allografting (allo-hsct) may offer some hope. methods: we summarize the experience of consecutive hr nb patients receiving therapy in our pediatric transplant department in - . the median age was years ( months to years). a total of auto-hscts and allo-hscts were performed. all auto-hsct recipients were characterized by one or several high-risk features: age of more than months at disease (onset n= ), primary disseminated disease (n= ), unfavorable biologic variant (n= ), poor st -line therapy response (n= ) or systemic relapse (n= ). most patients (n= ) received bu-mel hdct (in younger patients oral busulfan was replaced by busilvex), in primary resistant cases a d/ d regimen was used. a total of patients with st (n= ) or nd (n= ) chemosensitive relapse, resistant relapse (n= ) or poor mobilizers with locally advanced resistant tumor (n= ) received allo-hsct from haploidentical donor with fludarabine-based ric. in cases the transplant was modified via immunomagnetic positive or negative selection, patients received post-transplant cyclophosphamide (post-cy)-based gvhd prophylaxis. gvhd prophylaxis also consisted of calceneurin inhibitors and sirolimus. thirteen of allo-hsct recipients received posttransplant immunoadoptive (n= ) or targeted (n= ) therapy. results: the -year os and efs in auto-hsct recipients was % and %, accordingly. all but one patient engrafted with a median time of ( - ) days. bu-mel regimen was characterized by acceptable toxicity with most common toxicities being oral mucositis and infectious complications. the vod/sos incidence was only %. four patients dies due to infection (n= ), cns hemorrhage (n= ), and secondary leukemia (n= ). according to multivariate analysis the most important prognostic factors were response to st line therapy and post auto-hsct mibg scan results. the prognosis in initially resistant patients with good response to nd or rd -line therapy was still very poor (all patients relapsed with the median efs of months). all patients receiving a second auto-hsct after relapse died due to disease progression. with a median follow up of ( - ) months allo-hsct recipients are alive, of them with no signs of disease progression. all long-term responders received post-transplant therapy. one patient died due to transplant complications, other deaths were caused by disease progression. there was no obvious difference between outcomes in post-cy based and transplant modification-based transplantations. agvhd more often developed in modified transplant recipients ( patients vs in post-cy group, of these cases gr iii-iv), patients in post-cy group had grade iii-iv hemorrhagic cystitis. the median time to engraftment was longer for ptcm group compared to transplant modification group (d + vs. d+ , accordingly). conclusions: while single hsct with auto-hsct is a golden standard in hr nb patients, the relapse rate is still high and the prognosis in relapsed/refractory patients is dismal. the allografting has some limited effectiveness in these cases and post-transplant therapy has a potential for further improvement. disclosure: nothing to declare p abstract already published. veno-occlusive liver disease (vod) is frequent but well treatable with early defibrotide administration in children with neuroblastoma receiving high-dose busulfan and melphalan background: using high-dose intravenous busulfan and melphalan (bumel) prior to autologous stem cell transplantation (sct) in children with high-risk neuroblastoma, seems to decrease toxicity of the myeloablative regimen, except for vod. in this multicenter retrospective study we aimed to assess the outcome of bumel-associated vod with early defibrotide treatment intervention. methods: we retrospectively analyzed children with high-risk neuroblastoma who underwent autologous sct with i.v. bumel regimen in slovakia and prague, czech republic in the period / - / . busulfan was administered in q hour schedule, with therapeutic drug level monitoring in % of patients. all vod patients except one were treated with defibrotide starting at a standard dose of mg/kg/day, given in doses per day. patient was treated with supportive therapy only. ursodeoxycholic acid was used as prophylaxis in all patients. vod was established using the modified seattle clinical criteria (corbacioglu, lancet ). results: the incidence of vod was % ( / ) in patients treated with intravenous busulfan and melphalan. there was no significant difference in busulfan total dose/kg between patients with ( . mg/kg (sd= , )) and without ( . mg/kg (sd= , )) vod manifestation. vod developed at a median of days after sct (range - days). anicteric forms of vod were documented, although % patients with vod ( / ) presented with increased bilirubin. % patients with vod ( / ) developed ascites but only patients ( %) required ascites drainage. no vod patient received renal replacement therapy and only one needed mechanical ventilation. importantly, we successfully treated vod in all patients. relapse or progression of neuroblastoma was the cause of death in vod patients ( %) who died. conclusions: despite targeting busulfan levels to decrease toxicity of the regimen, vod is common (we observed vod incidence exactly in the range of the siopen hr nbl- multicenter study (ladenstein, ) ). early recognition and early treatment with defibrotide seems to be effective in vod associated with bumel regimen -none of our patients died due to vod. disclosure: nothing to declare results of high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) in the treatment of ewing sarcoma family tumors (esft) background: while current dose-intense treatment protocols allow achieving - % survival in localized esft patients, the long-term survival in high-risk cases is still unsatisfactory. although there is a considerable body of data on high-dose consolidation the real effectiveness and optimal indications for this option are still not completely clarified. therefore, a large prospective cohort analysis may still yield useful data. methods: the whole cohort includes consecutive highrisk esft patients with median age of (range - ) years receiving hdct with auto-hsct in to after obtaining st (n= ) or nd (n= ) cr, pr (n= ), or stable disease (n= ). the high-risk features included lung (n= ), bone (n= ) or bone marrow (n= ) involvement, inadequate local control in primary axial tumors (n= ), large lesions volume or poor treatment response (n= ), and chemosensitive relapse (n= ). most patients had several risk factors. disseminated disease patients were also evaluated according to prognostic score by r.ladenstein et al. highdose busulfan-melphalan followed by autologous stem-cell transplantation (hdt/sct) was used. results: the median observation time was (range - ) months. the -year overall (os) and event-free (efs) survival were % and %, accordingly. most important outcome predictors were inadequate local control in chemoresistant cases, a primary tumor volume more than ml, more than one bone metastatic site, bone marrow involvement and additional lung metastases. according to prognostic risk score in disseminated disease esft patients identified three groups with -year os rates of % for score ≤ ( patients), % for score to ( patients), and % for score ≥ ( patients), (p< . ). conclusions: while bu-mel hdct with auto-hsct may still be a feasible option with acceptable toxicity for chemosensitive patients with inadequate local control and some primarily disseminated cases it is ineffective in primary resistant or very high-risk metastatic patients. disclosure: nothing to declare efficacy of tandem high-dose chemotherapy with autologous hematopoietic stem cell transplantation in the treatment of infant embryonal brain tumors day + (range, - ), after the second auto-hsct was day + (range, - ). two-year overall survival (os) was % and disease free survival (dfs) was %. dfs was significantly better among patients with mb ( %) and pnet ( %) in compared to children with etmr ( %), pb ( %) and atrt ( %), (p= , ). dfs in patients who received tandem auto-hsct was % in compare to infants who received only one auto-hsct ( %), (p= , ). complications grade (according to common toxicity criteria ) were observed in % of cases. conclusions: employment of tandem hdct with auto-hsct in primary infant embryonal brain tumors may be a feasible option for patients after induction treatment. both conditioning regimens had acceptable toxicity. all patients who had tandem hdct with auto-hsct had better os ( %) in compare with single hdct ( %). patients with mb and pnet had better prognosis with os % and %, respectively, in compare with other embryonal tumors. disclosure: nothing to declare background: metastatic extra ocular retinoblastoma is carrying a poor prognosis. therapeutic intensification with high-dose, marrow-ablative chemotherapy and autologous hsct has been explored, but its role is not yet clear. this study aimed to evaluate the survival outcome of patients with extraocular retinoblastoma post autologous stem cell transplant, treated at single center methods: this is a retrospective study included all patients with metastatic extraocular retinoblastoma (stages a and b) that underwent autologous hsct at the children cancer hospital egypt (cche) from november to july , the treatment protocol was adopted from cog protocol (aret ) as all patients received cycles induction chemotherapy followed by consolidation myloablative conditioning, cem (vp . mg/m x , melphalan: mg/m x , carboplatin: mg/m x ) and stem cell rescue. patients data including initial disease characteristics, transplant data, and survival outcomes were collected and analyzed results: a total of cases were included with median age of . years, and male to female ratio . . nine patients ( %) were initially presented by extra ocular disease, while patients were presented by intra ocular disease and progressed to metastatic disease. according to cog staging of extra ocular disease, patients had stage a, and were stage b ( of them had trilateral disease). after induction therapy, ( %) showed complete response and ( %) had ≥ partial response. with average cd count of x / kg, the median time to anc and platelet engraftment were days and days respectively, and there was no transplant related mortality. post-transplant radiotherapy was given only to patients. with median duration of follow up of months, the overall and event free survival rates of whole patients were . % and . % respectively conclusions: high dose chemotherapy and stem cell transplantation is a promising potential curative option for patients with metastatic extra ocular only two primary gf ( . %) occurred, both without dsa. patients developed a primary pgf ( %). -years os, years pfs and -year nrm were analyzed according to the presence of dsa in comparison with negative population. no statistically difference was found. no impact of the presence of dsa on the risk of developing gf and pgf was revealed. major outcomes of transplant was analyzed separately in patients with pgf and good graft function (ggf). -years os, -years pfs and year-nrm in ggf and primary pgf populations were % vs % (p< . ); % vs % (p< . ), % vs % (p= . ), respectively. conclusions: the presence of low level of dsa in the absence of desensitization doesn't correlate with the risk of developing gf and pgf. patients who experienced a pgf had worse outcomes in comparison with patients with ggf. disclosure: nothing to declare. the impact of hla-dpb mismatch in t-cell replete unrelated donor allogeneic stem cell transplantation background: high resolution matching of donor-recipient hla improves outcome in allogeneic stem cell transplants. matching for hla-a, -b, -c, -drb and -dq is mandatory in our transplant centre, to identify / or / matched unrelated donors. high resolution matching for dpb has been added over the last - years. however, the role of dpb matching is not yet clearly defined. methods: in this study, we retrospectively analyzed the impact of hla-dpb matching on the outcome of t-cell replete allogeneic hematopoietic stem cell transplants with cya/mtx-and without atg as gvhd prophylaxis in patients with hematological malignancies at oslo university hospital between and . patients with an unrelated donor fully matched ( / ) at hla-a, -b, -c, -drb and -dqb loci were included. further, patientrecipient pairs were also fully matched on dpb ( / ); had permissive and had non-permissive mimatches of one or two dpb alleles. the three groups were comparable with respect to diagnosis, gender, age, cytomegalovirus serostatus and conditioning regimen. results: cumulative incidence of relapse at years were significantly higher in the dpb matched pairs compared with the permissive and non-permissive mimatched ones, at % vs % and % (p< . ) respectively. relpase free survival and overall survival were superior in the nonpermissive and permissive dbp mismatched groups vs the fully matched, at % and % vs % (p= . ) and % and % vs % (p= . ) respectively. no difference in frequency of acute gvhd grade ii-iv between the three groups were found; dp match %, permissive mismatch % and non-permissive mismatched % (p= . ). neither was there a difference seen in gvhd grade iii-iv; % vs % vs %, respectively. finally, there were similar outcomes between the three groups regarding chronic gvhd and trm. in corrected multivariate analysis, only dp matching had significant influence on mortality and survival. conclusions: our results show a favorable relapse free and overall survival following a mud allotransplant with a dpb permissive or non-permissive mismatched donor compared to a fully dpb matched. this is likely due to an increased gvl-effect in dpb mismatched groups without the counterbalance of increased acute gvhd and trm. disclosure: nothing to declare p a haploidentical may be a better choice than a female genoidentical donor to transplant a patient with high risk acute myelogenous leukemia in first remission norbert gorin , myriam labopin , didier blaise , goda choi , gerard socie , jean henri bourhis , fabio ciceri , emmanuelle polge , arnon nagler , mohamad mohty china, the first affiliated hospital of soochow university, hematology, suzhou, china background: despite the incidence of leukemia increases with age, currently the geriatric population is poorly represented in the standards of care concerning that older adults undergoing hematopoietic cell transplant (hct) may experience higher transplant-related mortality (trm). previous studies have demonstrated that donor age is vital for older patients by affecting trm and survival. accordingly a relevant question is whether outcomes can be improved with a younger hla-haploidentical offspring donor rather than an older hla-matched sibling (msd). in our previous multi-center report under atg+g-csf based protocol for haplo hct, offspring donor is correlated with lower trm and higher leukemia free survival (lfs) as compared with older msd in subgroup analysis for recipients > years although it did not reach statistical significance. on the contrary, in a recent report from ebmt and cibmtr under ptcy modality for haplo hct, among patients aged to years, despite lower chronic graft-versus-host-disease (gvhd), graft failure, trm, and overall mortality were higher after transplant from offspring compared with an msd although there were differences in transplant platforms between the groups. methods: we extended our multi-center dataset and a matched pair analysis was performed. outcomes of acute leukemia patients (>= years) transplanted in cr / cr who received hct from offspring (n= ) or msd (n= ) between jan, and june, present in the multi-center database were analyzed. because the patient population was small, a : ratio matched pair analysis was implemented with the following matching factors: underlying disease (acute myeloid leukemia, acute lymphoblastic leukemia), disease status (cr /cr ), age and sex of patients, year of transplant, blood group incompatibilities, and sex of donor. results: we were able to match offspring with msd patients. the two matched groups were comparable in baseline characteristics except for donor age due to the family relationship. all patients achieved myeloid recovery with a median time of d and d for msd cohort and offspring group (p= . ). the d platelet recovery rate was % in both groups. the cumulative incidence of grade ii-iv acute gvhd in msd cohort was significantly lower than in offspring group ( % vs %, p= . ) while the incidence of chronic gvhd in msd cohort was significantly higher than in offspring group ( % vs %, p= . ). the -year trm ( % vs %, p= . ) were significantly lower in offspring-hct compared with in msd-hct and relapse incidence was comparable ( % vs %, p= . ). as a result, the -year overall survival ( % vs %, p= . ) and lfs ( % vs %, p= . ) ( figure ) were significantly higher in offspring-hct compared with in msd-hct. in a multivariate analysis, msd-hct remained a significant factor for decreased overall survival (hr . ( . - . ), p= . ) by increased trm ), p= . ) in comparison with offspring-hct. conclusions: these data favor a young offspring over an older msd in patients > years. the current analyses confirm non-hla donor characteristics, rather than hla disparity, predominantly influence survival in older acute leukemia patients. validation of these findings requires a prospective trial wherein the transplant platforms can be closely matched. [[p image] . figure . lfs in offspring-hct compared with in msd-hct ( % vs %, p= . )] disclosure: nothing to declare. impact of sibling donor-recipient sex combinations on rejection after hla-matched bone marrow transplantation for severe thalassemia cure children foundation, florence, italy, sankalp india foundation, bangalore, india, people tree hospitals, bangalore, india, south east asia institute for thalassemia, jaipur, india, pakistan institute of medical sciences, islamabad, pakistan, central asiri hospital, colombo, sri lanka, nawaloka hospital, colombo, sri lanka, kokilaben dhirubhani ambani hospital, mumbai, india background: severe thalassemia (st), i.e. a thalassemia syndrome with inability to keep spontaneous hemoglobin > g/dl, is a common indication for bone marrow transplantation (bmt) in children in the middle east and south east asia. sex mismatch has been associated with increased risk of solid organ rejection but is not generally considered an important transplant-associated risk factor in the context of fully matched sibling bmt for st. methods: a total of consecutive sibling bone marrow transplants carried out between january and april after conditioning with busulfan ( mg/kg oral, not adjusted to serum levels) and cyclophosphamide ( mg/kg) ( patients) in addition to either thiotepa ( mg/kg) ( patients), or anti-thymocyte globulin (genzyme mg/ kg or fresenius mg/kg on days - to - ) ( patients) and fludarabine mg/m ( patients) were analysed. all cases received cyclosporine and methotrexate or mycophenolate mofetil as gvhd/rejection prophylaxis. in the thiotepa group methylprednisolone at . mg/kg/day was also used during the first days after bmt (lucarelli protocol i). bone marrow was the source of hematopoietic stem cells in all cases, in the atg group it was g-csfprimed ( μg/kg/dose twice daily for to days prior to harvest). all patients were considered low risk based on liver size < cm from costal margin and age less than years (median . years, range . to . ), all sibling pairs where hla-compatible. results: [[p image] . sibling donor-recipient sex combinations.] the lowest rejection rate ( %) was observed in the sister to sister (s s) group of cases, followed by brother to brother (b b) group of cases with %. in the sister to brother (s b) group of cases, rejection rate was %, and % in the brother to sister (b s) group of cases. on univariate analysis the only significant difference at the p . level by log rank test was b s vs. s s groups (rejection proportions of % and % respectively). interestingly, all patients with rejection and persistent pancytopenia were female recipients of male grafts. conclusions: even though several preparative regimens were employed over an -year period, our data suggests that sex mismatch among compatible siblings should be considered as a relevant variable related to bmt decisionmaking. we also recommend to consider autologous back up hematopoietic stem cell collection and storage in sibling sex mismatched transplants, particularly in brother to sister bmts. same-sex fully matched related bmt for severe thalassemia might be the best scenario in which reducedintensity preparation strategies aiming at maximizing fertility preservation might be explored. disclosure: nothing to declare. outcomes of t-cell replete hematopoietic cell transplantation from mismatched related or unrelated donors using high dose post-transplant cyclophosphamide based gvhd prophylaxis background: high dose post-transplant cyclophosphamide (ptcy) based gvhd prophylaxis overcomes immunological barriers in hla mismatched donor transplantation. ptcy has been adopted in many centers as de facto standard for hct from haploidentical donors (haplo hct). it's use in mismatched unrelated donor transplant (mmud hct) is less well established. methods: we analyzed retrospectively outcomes of contemporary cohorts of patients who underwent haplo hct or mmud hct using ptcy + cyclosporine (csa) and mycophenolate mofetil (mmf) at our center. we compared these outcomes with outcomes of cohorts of patients who underwent hct from matched unrelated donors (mud) using atg based gvhd prophylaxis or matched sibling donor (msd) with csa and mmf. patients and donors were considered matched if they background: hla-alloantibodies are a major risk factor for engraftment failure in allogeneic hematopoietic stem cell transplantation (hsct). particularly, complement fixing, donor specific antibodies were shown to be associated with early engraftment failure. prospective antibody-screening, although not currently required for donor search, could permit early identification of high risk patients for positive crossmatch. aim of this study is to set the basis for future applicability of antibody-screening-based definition of acceptable mismatches in donor selection, by creating a large prospective antibody-screening database of patients due to receive an hla-mismatched allogeneic hsct. methods: patients (n= ) diagnosed with mds/mps, nhl, mm, cll, cml, anaemia (aplastic anemia, hemoglobinopathies, pnh) and hl were prospectively screened for hla-antibodies whenever initial donor search indicated that no completely matched donor would be available. screening was performed with an elisa class i +ii screening assay. all positive screening cases were tested for antigen-specific antibody identification with luminex sab, and acceptable mismatches were defined. the results were subsequently considered in donor search and selection. we now report the frequencies of alloimmunization observed in these patients. results: the highest rate of alloimmunization was observed in patients from the anaemia disease group (overall . %) followed by those from the mds/mpn group (overall . %). the lowest immunization rates were observed in cll (overall . %) and hl ( . %) patients. alloimmunization rates for hla-class i antigens (p< . ) were significantly higher compared to hla-class ii antigens. overall hla-class i immunization rates ranged from . % to . %. hla-class ii immunization rates ranged from . % to . % (table ) . conclusions: our findings suggest that patients with high transfusion burden like anaemia and mds/mpn patients have the highest risk of hla-alloimmunization with . % and . % anti-hla prevalence rates, respectively. analysis of follow-up data, will enable us to confirm whether prospective definition and consideration of acceptable mismatches in donor selection may lead to similar engraftment failure rates between immunized and non-immunized patients undergoing hlamismatched hsct. background: mothers displaying a persistent fetal microchimerism (fm) proved to be the most suitable donor in t cell-depleted haploidentical stem cell transplantation (hhsct) in children. we presumed that fetal cells leave an imprint in the mothers' immune system which positively affects recognition and elimination of malignant cells in the child by maternal effector cells. distinct killer cell immunoglobulin-like receptors (kir)/hla constellations are not only associated with reduced relapse rates after hsct in children, but also supposedly influence the establishment of an fm. methods: after approval by the local irb and obtaining informed consent, we initiated a protocol to elucidate the factors that influence the establishment, persistence and effect of fm. we established a digital droplet pcr (ddpcr) protocol to determine the fetal microchimerism. for differentiation between maternal and fetal cells, biallelic short insertion/deletion polymorphisms were used. kir and hla-c genotyping was performed by ssp-pcr. parental nk cell alloreactivity against the respective leukemic blasts and kir phenotyping were analyzed by flow cytometry. results: we analyzed parents, whose children were treated for hematological diseases at the university medical center hamburg-eppendorf. a fetal microchimerism was detected in % of the mothers. the amount of fetal cells varies between individuals ( x - - x - ). we observed a positive correlation between a persisting fm and hla-c homo-and heterozygous mothers along with a maternal cen a/b and cen b/b genotype. additionally, fm positive mothers showed a higher surface expression of the hla-c respective receptors kir dl /s . the percentage of alloreactive maternal nk cells against fetal cells was higher compared to paternal nk cells; while alloreactivity of fm positive maternal nk cells was similar to nk cells from fm negative mothers. conclusions: persistence of fm was more frequent in mothers carrying at least one hla-c allele and a centromeric b/x motif. phenotypically, fm positive mothers had higher expression of kir dl /s indicating a role of these receptors on the persistence of an fm. in vitro, maternal nk cells showed a higher alloreactivity compared to paternal nk cells. there was no difference in alloreactivity whether the mothers were fm positive or negative, suggesting other mechanisms are responsible for the superior outcome in transplantation from fm positive mothers. disclosure: nothing to declare background: although there have been significant improvements with conventional therapies in beta thalassemia major, hematopoietic stem cell transplantation is only curative therapy. related donors are preferred to diminish transplant risks. in lack of identical related donor, identical unrelated donors are second best choice. in this study, thalassemia major patients transplanted from unrelated donors (mud) were compared with thalassemic patients transplanted from relative donor (mrd) retrospectively. methods: patients who were transplanted between june and december in bahçelievler medical park hospital pediatric bone marrow transplantation unit were evaluated retrospectively. all patients were classified according to pesaro risk classification. thirty four of received busulfan, fludarabine, cyclophosphamide, thioteopa for conditioning, patients received myeloablative preparation regimen with treosulfan, fludarabine, thiotepa, cyclophosphamide. all patients were given atg, cyclosporine and methoteraxate for gvhd prophylaxis. the patients were compared in terms of acute complications in first days, engraftment, chimerism, acute and chronic gvhd after transplantation. results were evaluated with ibm spss statistics (ibm spss) program. results: a total of patients, ( . %) male and ( . %) female, aged between and years (median years) were evaluated. patients were evaluated in two groups as "mud" (n = ) and "mrd" (n = ) groups. there was no difference between groups about given stem cells (mud , ± , x /kg and mrd , ± , x / kg). neither significant difference between different pesaro risk groups in terms of developing acute and chronic gvhd and nor decreased chimerism were detected. neutrophil engraftment time ( , days) in mrd group was significantly longer than mud group ( , days) (p = . ) but no difference between platelet engraftments were observed. gvhd ratio was . % in mud donor group and . % in mrd group and no statistically significant difference was found(p> . ). the incidence of engraftment loss in mud group was . % and . % in the mrd group, and there was no statistically significant difference (p> . ). the rate of decreased chimerism was found to be significantly higher in the mrd group ( %) than in the mud group ( . %) (p: . ; p< . ). the survival rate was . % in the mud group and . % in the mrd group. the disease-free survival rate was . % in the mud group and % in the mrd group. the disease-free survival of mud group was significantly higher than mrd group (p: . ). conclusions: in our study, transplant related complications and success of transplantation with both mud and mrds were found to be similar. it is promising for mud transplantations to found lower decreased chimerism and similar os and dfss. based on these results, it was concluded that hsct from non-family donors, especially for patients incompatible with chelation therapy and had organ damage, transplantation from unrelated identical donors can be a good choice. although the results of our study seem promising, larger patient groups and prospective clinical trials are required. disclosure: nothing to declare background: use of g-csf stimulation of bone marrow (bm) donors is beneficial in many aspects; it can enhance tnc yield, but also have an immunomodulatory effect on donor t cell function, particularly invariant natural killer t (inkt) cells expansion as well as apcs. we analyzed outcomes of consecutive patients receiving bone marrow from hla-haploidentical donors that were stimulated with g-csf prior to harvest. methods: in the time period between / and / , patients received bone marrow from donors stimulated with ug/kg bw of g-csf on days - , - and day of bm collection. four patients ( %) received myeloablative (bucy) conditioning, one ( %) received tec ric conditioning while ( %) received nma ("baltimore") conditioning. all patients received posttransplantation cyclophosphamide (ptcy) on days + and + , tacrolimus and mmf were started on day + . for patients donors were fathers, mothers, siblings and children. results: median age was years ( - ), there were female and male patients. twelve patients had aml, hodgkin lymphoma, all, mds, nhl and cml. median number of infused tnc in graft was . x /kg bw ( . - . ) and cd + cells . x /kg bw ( - . ) . after median follow up of days (range - ), overall survival was %, with median survival of months. engraftment was established in ( %) patients, ( %) had primary rejection and patients ( %) died in sepsis prior to engraftment. of patients that engrafted, further ( %) patients had secondary rejection, two of them were transplanted again from a haploidentical donor, both using pbsc as a source of graft. median time to neutrophil recovery (anc> ) was days ( - ), while median time to platelet recovery (plt> x /l) was days ( - ) in evaluable patients. cumulative incidence of agvhd ii-iv was . % ( % ci, - );of note is that of patients that developed agvhd only one had grade iii, while remaining patients had grade ii. cumulative incidence of cgvhd requiring treatment was . % ( % ci, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cumulative incidence of relapse was . % ( % ci, - ) and trm was . % ( % ci, - ). conclusions: the use of g-csf mobilized bm graft in the hla-haploidentical setting with ptcy has proven to be useful to us, not only in terms of tnc yield which was more than satisfactory and contributed to adequate hematological recovery, but also in the excellent control of both acute and chronic gvhd, with most patients developing agvhd of grade ii and only one grade iii (actually developed only after dli given for decreasing chimerism). comparative studies are of course warranted to prove benefit, but this data contributes to the growing body of evidence that indeed donor stem cell stimulation with g-csf has potentially powerful immunomodulatory effect. disclosure: nothing to disclose p other-relative donors as a reliable bank for allogeneic hsct in countries with culturally accepted cousin-cousin marriages: a two-year report from a pediatric center in iran background: although the optimal donors for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) are fully-matched siblings, the cousincousin (consanguineous) marriages in some countries have extended the chance to find a matched donor for the hsctrecipient. in this study, an outcome analysis of transplanted patients receiving stem cells from their relatives other than siblings (other-relatives or non-sibling donors) is provided. methods: in this retrospective cross-sectional study, a two-year report of patients who received allo-hsct from their other-relative donors during september to september at the department of stem cell transplantation of children's medical center in tehran, iran is presented. the patients were followed up until st december . results: during this time period, patients underwent hsct (both autologous and allogeneic) at this center, of which cases received allo-hsct. out of allo-hsct recipients, the donors of stem cells for cases ( . %) were their other-relatives. the median (range) age at hsct was ( - ) years and the majority of patients were boys ( / , . %). according to disease class, the patients were most commonly involved with non-malignant hematologic diseases ( / patients, . %) (figure) . the source of hscs for most patients ( cases, . %) was peripheral blood and for only patients the source was bone marrow. the donors for patients were fully matched and only one patient received the hscs from a one-locus mismatched donor. hsct was successful in patients with most of them achieving full chimerism ( patients, . %) followed by those developing mixed chimerism ( patients, . %) and only one patient ( %) experienced graft failure. post-hsct complications included cmv infection in patients ( . %), other infections in ( . %), hemorrhagic cystitis in ( . %) and pres in ( . %). acute gvhd occurred in patients ( . %) and chronic gvhd in ( . %). death occurred in cases and of them were transplant-related, while was due to disease relapse and due to graft failure. the median of overall survival was ( - ) days. conclusions: the likelihood of receiving hscs from an hla-matched other-relative donor in one-thirds of children undergoing allo-hsct, with comparable outcomes to sibling and unrelated donors (as evidenced in this study compared with other studies), introduces family bank as a reliable source for pediatric allo-hsct in countries with culturally accepted cousin-cousin marriages. hence, for transplant physicians, parental consanguinity would be an indication of an extended search for a potential matched donor among the patient's family. [[p image] . distribution of patients according to disease and disease class] disclosure: nothing to declare. abstract already published. update on the hla frequency distribution of the portuguese bone marrow donor registry eduardo espada , dário ligeiro , hélder trindade , joão forjaz de lacerda frequency distribution varied throughout the country, allowing for analyses of molecular variance and generation of relatively geographically accurate graphical representations of genetic distances between regions and districts. conclusions: with the most recent hla analysis of the portuguese bone marrow donor registry we were able to extrapolate high-resolution haplotype frequencies from the most common low-resolution hla-a/-b/-drb haplotypes (corresponding to % of the estimated haplotypes at that level), which will lead to an optimization of its use, hopefully limiting the time between donor search and allogeneic hematopoietic stem cell transplant. disclosure: nothing to declare. abstract already published. abstract already published. unmanipulated haploidentical donor transplantation compared to identical sibling donor had better antileukemia effect for refractory/relapsed acute myeloid leukemia in not remission status background: patients diagnosed with saa with no sibling donors and who are refractory to immunosupression are candidates to hematopoietic stem cell transplant using alternative donors. haploidentical donor transplants has been reported using cyclophosphamide (cy) post stem cell infusion as immunephrophylaxis. the present study has the objective of evaluating overall survival and engraftment rates after haploidentical stem cell transplant for saa in a reference center. methods: saa adult patients (≥ yo) received hsct from haploidentical donors from de january/ to august/ . median age was y ( - ); donor was the father in six, mother in five and a brother in four cases. stem cell source was marrow in cases ( %). conditioning: patients ( %) received cy mg/kg, fludarabine mg/m² e tbi cgy. the remaining received the same drugs but radiotherapy dose varied from - cgy, all them received immunephrophylaxis with post transplant cy mg/kg, cyclosporine and mmf. median of infused cells (tcn) was , x /kg ( , - , ). results: eight patients engrafted ( %). among seven graft failures four received a second haploidentical transplant and one received an unrelated donor transplant as salvage regimen. two patients were successfully rescued after the second haplo and the others died from infectious complications. three years overall survival was %. death causes included: five infections and two lung hemorrhage. median survival was days ( - ). no patient had acute graft-versus-host-disease (gvhd) and one patient had mild c-gvhd. conclusions: haploidentical transplant was feasible as therapy for saa refractory to immunessupression with an overall survival of % in this cohort. graft failure however is still a problem to be addressed in this setting. disclosure: no disclosure stem cell mobilization, collection and engineering p abstract already published. key performance and quality indicators for a successful bone marrow collection marco sampaio , , ana salselas , fátima amado , filipa bordalo , sérgio lopes , catarina pinho , susana roncon and one from ecc (staphylococcus spp.)presented positive microbiological results. conclusions: bm collection is a challenging strategy because it is a one-time procedure and manually operatordependent technique; simultaneously it is more difficult to control the final cellular content of the bm, which is a risk for donor volume depletion. bm collection is feasible even with donor and recipient weight difference. poorer performance may be found when higher tnc are requested. we respond efficaciously when the request is between and * tnc but we fail to accomplish higher tnc values. we must emphasise that icc tnc demanded was generally lower than ecc. deciding the appropriate tnc for each patient remains a dare and an art. disclosure: nothing to declare. impact of adding plerixafor to mobilization protocol in the immune reconstitution of vδt cells after autologous hematopoietic stem cell transplantation efrat luttwak , , yael chava cohen , , odelia amit , , irit avivi , , svetlana trestman , , esti rom , , rinat eshel , , ram ron , tel aviv medical center, tel aviv, israel, sackler faculty of medicine, tel aviv university, tel aviv, israel background: multiple myeloma has remained an incurable disease even in the era of novel therapies. front line treatment typically comprises of induction chemotherapy with - cycles of a bortezomib-based regimen, stem cell mobilization, and harvesting of peripheral blood stem cells (pbsc) by apharesis, followed by high dose melphalan with hct. while brotezomib-based induction regimens have demonstrated no adverse impact on hematopoietic cell harvest number and quality, no study analyzed the impact of timing of the last brotezomib dose prior to collection. in this study we aimed to determine the effect of the timing of the last dose of brotezomib before hematopoietic cell collection and the collection yield. methods: this was a single center historical prospective study, including all sequential newly diagnosed patients with myeloma between and that were given a bortezomib-based induction therapy (≤ cycles) followed by pbsc collection. we excluded patients who either received st line vtd-pace or lenalidomide-containing regimens. peripheral blood cd + cells were measured on the day of collection. patients with cd + levels of > cells/ microliter started collection on the same day, while those with lower levels were given plerixafor. we performed regression analyses to analyze the impact of a variety of precollection factors, including days from last bortezomib therapy on the collection yield. results: we identified patients who fulfilled the inclusion criteria, table. median time from last dose of brotezomib to first leukapheresis was (range, - ) days. a statistically significant correlation was found between the days from last dose of brotezomib and both the first collection day-cd + cells/kg (r= . , p< . ), and the total collected cd + cells/kg(r= . , p= . ), figure. the optimal cutoff point as indicated by the roc curve was . days according to collection success with sensitivity of % and specificity of %, youden´s index . . in multivariate analysis included other factors affecting collection yield (age, gender, status of disease at collection, and prior radiation) -timing of last dose of brotizomib remained significantly associated with the total collected cd + cells/kg (p= . ). increasing age, female gender, and prior radiation were associated with lower collection yield (p= . , . , . , respectively). based on this, we developed a model to predict the total collected cd pos cells = . + . (timing in days of last dose of brotezomib) - . (age) - . (if female) - . (≥pr) - . (if prior radation). conclusions: timing of last dose of brotezomib is an important factor for predicting a successful collection. a washout period of days is associated with a better collection yield. these results should be further validated in a prospective study. age (median, range) ( - ) gender -male (%) ( ) prior radiation treatment (%) ( ) disease status at collection (%) ≥pr - ( disclosure: nothing to declare p mobilization with plerixafor in "poor mobilizer" related and unrelated donors of hpc-a in case of failed mobilization with g-csf background: in the allogeneic hpc transplantation, both from related and unrelated donors, the most commonly used source is peripheral blood after mobilization with g-csf. it is however known that about % of donors are "poor mobilizers"; the rescue strategies are: a third apheretic collection; bone marrow donation. methods: in - in italy a procedure to be adopted in case of failed mobilization of peripheral blood stem cells has been defined and shared between ibmdr, cnt (transplant national center) and cns (blood national center) and the scientific societies simti, sidem and gitmo, using plerixafor, a selective reversible antagonist of the cxcr receptor with its binder (the stromal derived factor sdf- ), in combination with standard g-csf dose. moreover since , in accordance with this protocol, the competent authority (aifa) has extended the registration of plerixafor (law no. / ), also for the mobilization in "poor mobilizer" healthy donors. finally, in the protocol was extended to "poor mobilizer" family donors, making management equivalent in related and unrelated donors. at the time of the donor´s informed consent for the donation of hpc, the hypothesis of the lack of mobilization or inadequate collection was illustrated and the possible actions proposed as "back-up donation" were anticipated. failed mobilization of cse has been defined as the presence of one of the two criteria: a number of cd + circulating on peripheral blood lower than /μl on the th day of stimulation ( d), or the collection of cd + < . x /kg weight of the recipient at first apheresis. in these cases, a single dose of plerixafor is administered subcutaneously by health professionals under medical supervision, . mg/kg of body weight, - hours before the start of apheresis. in case of use plerixafor due to failed mobilization of hpc-a or collection of an inappropriate number of cd +, the notification is made by the collection center to ibmdr (for both family and non-family donors), and to the recipient transplant center : both donor and recipient express their consent; finally once the collection is completed, the collection center informs ibmdr, which in turn notifies cnt/cns/simti/sidem/gitmo. any adverse reactions/events are notified in real time, based on the sop specifications and current regulations. results: since the introduction of the national protocol, donors ( unrelated donors and related donors) were treated, presenting at least one of the two inclusion criteria (cd < /μl at d or cd < x e /kg after first collection)after use of plerixafor in all donors, the required dose of cd was obtained to ensure successful transplantation, with a sufficient increase in the cd + cells. no side effects or adverse reactions related to the administration of plerixafor occurred. conclusions: in cases of failed mobilization in the related and unrelated donor, the use of plerixafor according to the methods described in the shared protocol between ibmdr, cns, cnt, simti, sidem, gitmo, proved to be safe and effective. this protocol emphasizes the great value of the sharing of procedures between the register, institutions and scientific societies, ensuring the supervision of the process and the protection of the donor and recipient. disclosure: nothing to declare background: autologous stem cells transplantation (asct) is an effective treatment option for young patients with multiple myeloma (mm). a minority of patients may still experience untoward toxicity due to delayed engraftment. thus, the current policy in many centers is aimed to increase the target dose of collected cd + cells up to an "optimal" level of x /kg per procedure. therefore, an ideal mobilization, aimed to collect to cd + cells/kg in one apheresis, should achieve a number of circulating cd + cells > /mcl (very good mobilizers). plerixafor may help to maximize the cd + collection but its use is limited by high cost. we carried out a retrospective analysis aimed to predict the quality of mobilization and develop an algorithm to optimize both timing of collection and use of plerixafor. methods: we retrospectively collected data from mobilization procedure performed in our center between and for mm. all received the same mobilization protocol with cyclophosphamide (range - gr/sqm) and g-csf mcg/kg from + . cd + cell count was started when white blood cells (wbc) count exceeded x /l. patients were excluded from this analysis if ) showed a cd + count > /mcl (target achieved at first day count) and/or ) cd + count on second day was missing and/or ) plerixafor was administered on first day according to previous policies. sixty-eight patients were evaluable for the study. univariate and multivariate logistic regression analysis to study ccd + kinetics and assess predictors impact on mobilization was carried out. ratio cd +/wbc in first day count, gender, disease category and time from mobilization chemotherapy were also included results: among the patients included in the analysis, the threshold of cd +/mcl cells on the second day was reached by ( , %) of patients (groupa) whilst the remaining ( , %) failed the target (groupb). median (range) wbc x /l and cd + /mcl counts in group a and b were , ( - , ) and , ( , - , ), , ( ) ( ) ( ) ( ) ( ) ( ) ) and , ( , - , ) respectively, with a statistically significant differences among group (mann-whitney p= , and p= , respectively). only cd + /wbc ratio and cd + /mcl on first day count had an impact on kinetics and optimal mobilization. logistic regression model highlight cd +/mcl (or= , ; % ci: , - , ) on first count as an independent predictor of second day optimal mobilizer, with auc of . % ( , ) in roc analysis. two cd + thresholds were then calculated: < , /mcl (ppv , ; npv , ) that identified poor mobilizer, and ≥ , /mcl (ppv , ; npv , ) that exclude probability to fail on second day. for those with a cd + count between , - , the cd +/wbc ratio (or= , , % ci: , - , ) was a predictor of optimal mobilization (auc , ; , - , ); cut-off value was , (sensibility , ; specificity , ) conclusions: assessment of circulating wbc, cd + and their ratio at wbc recovery in a chemo-based mobilization is a valid tool to manage the collection strategy and the on-demand use of plerixafor. we have developed an algorithm aimed to the use of plerixafor to both rescue poor mobilizers and boost cd + count in intermediate mobilizers. background: successful autologous stem cell transplantation (asct) requires the infusion of a sufficient number of hematopoietic stem cells (hscs). peripheral blood (pb) is the most commonly used source of hscs, therefore, it is important to optimize methods used to mobilize the hscs. the most clinically used chemotherapeutic agents for effective mobilization are cyclophosphamide and etoposide. recent published studies suggest that etoposide has a better mobilization effect than cyclophosphamide even at lower doses, but it is not clear why this difference occurs. in this study, we tried to determine whether there is a difference in the mechanism of mobilization between cyclophosphamide and etoposide. methods: first, in order to confirm the clinical data for efficacy and toxicity of mobilization, we retrospectively analyzed the data of patients who were diagnosed with lymphoma and performed mobilization using cyclophosphamide or etoposide from january to december . second, mesenchymal stem cells (msc) were primarily cultured from the healthy controls, then treated cyclophosphamide or etoposide at a concentration of % inhibition of cell growth, and cytokine analysis was performed to identify cytokines known to be associated with mobilization. third, mobilization mouse model using cyclophosphamide or etoposide was generated, total blood was collected at the time of hscs collection, and cytokine and network analysis (using ingenuity pathway analysis) was performed. results: the mobilization yields for cyclophosphamide or etoposide were analyzed. etoposide miblized a significantly higher median number of cd +cells than cyclophosphamide. the rate of successful or adequate mobilization was also significanctly higher for etoposide in univariate and multivariate analysis (table ). in the analysis of toxicity during mobilization, the incidence of neutropenic fever was higher in the cyclophosphamide group (p = . ). during mobilization, cyclophosphamide maintained lower wbc counts than etoposide and showed a large increase in wbc counts at the start of collection ( figure ). the cumulative dose of cyclophosphamide or etoposide in patients who underwent autologous stem cell transplantation did not affect leukocyte (anc> /microl or platelet (plt > k/microl) engraftment. in msc treated with etoposide at a concentration of % inhibition of cell growth, il- , which is a cytokine that promotes hematopoietic stem cell mobilization, were shown a statistically significant increase (figure ). in the mouse model of mobilization (figure ), the levels of kc, one of the il- homologues in mice, had significantly increased in the etoposide-treated group compared with the levels in the cyclophosphamide-treated group. the levels of other il- homologues, mip- and lix, also showed increases in the etoposide-treated group compared with those in the cyclophosphamide-treated group; these differences, however, were not statistically significant (figure ). network analysis based on in vivo cytokine results identified that etoposide could promote mobilization in association with matrix metalloproteinase as compared to cyclophosphamide ( figure ) . conclusions: etoposide has a higher mobilization efficacy when compared to cyclophosphamide, which could due to the different mechanisms of mobilization through the elevation of il and the activation of matrix metalloproteinase associated therewith. background: high-dose chemotherapy followed by autologous blood stem cell transplantation (asct) is a standard therapy for wide range of hematologic and solid malignancies. although various methods have been introduced to improve the peripheral blood stem cell (pbsc) mobilization, autologous stem cell collection (ascc) is not successful in every patient. furthermore, even if the ascc is complete, not all of them lead to asct. we evaluated the result of ascc and actual use of pbsc grafts in current practical setting. methods: we retrospectively reviewed the all consecutive ascc procedures performed at the department of oncology in asan medical center, seoul, korea, between january and october . the targeted number of background: fanconi anemia (fa) is a rare inherited genetic bone marrow (bm) failure syndrome. while abnormal bm cells production occurs very early in life, the usual age of diagnosis is - years old. gene therapy (gt) might be an alternative to hematopoietic stem cells (hsc) transplantation, but harvest a large number of autologous hsc remains a challenge. we started a mobilization assay, fancomob, to evaluate the safety and the efficacy of fa patients' mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. this study is part of the fa's european gt project "eurofancolen". methods: four patients with fanca mutations following the inclusion criteria were selected before pancytopenia. to note, fa was diagnosed before clinical manifestation through family screening. they received subcutaneous injection of g-csf ( μg/kg twice a day) from d- and plerixafor ( . mg/kg/day) from d- . the collection protocol targeted x e /kg of cd + cells, based on a predicted future weight in years. cd + cells and white blood cells (wbc) blood count were monitored tightly along the mobilization. patients with more than cd +/μl or between and cd /μl with a clustered aspect detected by flow cytometry after plerixafor injection underwent apheresis. cd + cells were immunoselected from the collection with clinimacs purification system (miltenyi) and cryopreserved for further gt manipulation. results: the mobilization target was not achieved for the first two included patients (fa years old and fa years old). the minimum value of cd +/μl required wasn't obtained for fa and the flow cytometry cd + aspect was not clustered for fa . cd + cells were mobilized quickly but transitionally after plerixafor injection for the last two patients, fa and fa , and years old respectively. both patients underwent apheresis procedures. no cd + cell rebound was observed after the apheresis was stopped.collection target was not achieved after four days of collection for fa . it was obtained the first day for fa (figure ). back-up for hsc transplantation could not be cryopreserved because of the limited number of cd + cells collected in patients fa and fa . no short-term adverse events were observed. following cd + immunoselection, cd + cell purity and recovery were poor but in the normal range described in the literature for fanconi patients ( - %)( table i) . one month after the collection hemograms were unchanged. conclusions: our clinical study offer new data showing that mobilization of fa patients with g-csf and plerixafor is safe and more efficient for younger patients, especially before clinical manifestations of bm failure. further efforts are required to establish an effective technic to purify the cd + cells after harvesting. basal cd + cell and platelets count are a strong predictor for mobilized peripheral blood stem cells on the th day of g-csf treatment in donors cryopreserved pbscs. this was associated with considerable efforts for the patients and caused additional treatment costs. on the one hand, having the therapeutic option of an autologous transplantation in the future may represent a clinically relevant advantage. however, a huge number of stem cell products are kept in storage for many years without ever been used for transplantation. our study provides cause for a careful reevaluation of the current clinical practice, which may help to focus more precisely on patients who actually benefit from a cryostored autologous stem cell graft. [[p image] . fig. : absolute numbers and relative distribution of stem cell grafts] disclosure: the authors confirm that there are no potential conflicts of interest to disclose, except the following: katharina kriegsmann: research funding from bms, celgene, and sanofi. patrick wuchter: membership in advisory boards for sanofi-aventis. reduction of dimethylsulfoxide (dmso) concentration from % to % in criopreservation of stem cells. influence on the kinetics of engraftment and tolerance to infusion patricia lopez-pereira , beatriz aguado , elena sola , carmen cámara , isabel vicuña , lorena vega , adrian alegre hospital universitario de la princesa, madrid, spain background: dmso is the cryoprotectant most used in the cryopreservation of stem cells. it is associated with adverse effects during the infusion of the product, its toxicity being proportional to the volume infused. the most common concentration used has been %, although recent publications report that reducing it to % leads to lower rate of side effects without impact on the product or the graft. we retrospectively analyzed patients recipients of autologous peripheral blood stem cell transplantation (hct) in our hospital from january to september . they are divided into two groups according to the concentration of dmso used in the freezing ( % until september or % since october ). the baseline characteristics of the patients, the infused product and the graft are shown in table . the cd count was performed by flow cytometry. all freezings were performed with a biological freezer for programmed controlled rate cryopreservation and stored in ultrafreezers at - ºc. results: both population groups are homogeneous. the t-test was used for statistical analysis. regarding the cd + variable, no statistically significant differences were observed (p = . ). neither for the variables leukocyte recovery and platelet recovery (p = . , p = . respectively). the difference in the variable viability is . units (ci %: [ . - . ]) and is statistically significant (p = . ) in favor of dmso %. regarding adverse effects, % (n = ) of the serious adverse reactions occurred in the % dmso group (hypotension and seizures). the mild and moderate ones were similar in both groups, most were mild nausea, vomiting and flushing. overall, no statistically significant differences were observed due to the low rate of adverse effects found. patients starting with until october , total of patients attempted collection of autologous pbscs, and poor mobilizers recieved plerixafor during first mobilization cycle. in total, patients required repeated mobilization cycles ( , %) of which were from the plerixafor group. in total patients recieved pleriksafor; females and males, median age ( - ) with following diagnoses: nhl, mh, multiple myeloma, neuroblastoma, nephroblastoma, sarcoma ewing and seminoma. of repeated mobilizations with plerixafor, patients ( , %) still failed to collect adequate transplant. in this period we had altogether unsuccessful mobilizations ( , % in repeated cycles, , % in total). this group of patients consisted of male and female patients, median age ( - ), diagnosis of nhl and failure to collect after leukapheresis procedures each. median number of leukapheresis needed for adequate collection was with preemtive plerixafor use, and in repeated mobilizations. conclusions: our expirience shows that preemtive use of plerixafor in poor mobilizers is efficient and has enhached success of the pbsc collections. due to drug high cost each institution needs to develop its own algorythm in management of poor mobilizers. the factors contributing to plerixafor mobilization failure still need to be elucidated. disclosure: nothing to declare. platelets recovered from mobilized leukapheresis units obtained from hla-haploidentical donors fulfill the criteria of a conventional hemocomponent and can be used for transfusion background: central venous catheter (cvc) related complications may lead to high morbidity and mortality. unlike cvc, peripheral cannulation offers a quick and inexpensive method for safe and non-traumatic vascular access (va) thus its utilization is strongly recommended whenever possible. the ultrasound (us) guidance for acquiring peripheral va is a useful tool for reduction or elimination of the need of using cvc for stem cells collection. we have made an attempt to introduce us method in our apheresis unit having no previous experience with us devices. the aim of the study was to measure the decrease of cvc insertions after introducing us and evaluate the quality of va by comparing average flow rate and confirming that the desired blood volume could be processed. methods: the theoretical education involved a free elearning course in peripheral ultrasound-guided va (pugva, usabcd, aarhus, denmark). subsequently, the personnel have implemented knowledge in practical training on gelatine and silicone phantoms and healthy volunteers. the practical activities also included a fiveday course in an apheresis centre with us-guided cannulation experience. the details concerning va were recorded, including va site, cannula size, average inlet flow rate, number of inlet pressure alarms reported by the apheresis device. the procedure details where traditional approach was applied i.e. palpable cannulation and cvcs have been collected. similarly, the necessary data for procedures where veins were assessed with ultrasound prior to apheresis were recorded. results: before introducing ultrasonography, stem cell collections have been performed in patients. of all these procedures, were accomplished with cvc ( %) and with peripheral va ( %). median cubital vein was the vessel of choice. out of the peripheral va procedures, ( %) were problematic, with or more inlet pressure alarms during every procedure. after the training stage, collection procedures were performed in patients. after introducing us we have observed a significant reduction of the number of cvc insertion required for successful apheresis from % to % (p= . ; chi-square test with fisher's exact). thirty one procedures were completed with peripheral va ( %). ultrasound device enabled cannulation not only the superficial veins but also for the deeper veins. cannulation sites included upper arm cephalic vein ( %), median cubital vein ( %), upper arm basilic vein ( %), median antebrachial vein ( %). out of the collections, were considered problematic ( %). no difference in an average flow rate was observed between procedures performed peripherally with and without ultrasound usage (p= . ; u mann-whitney test). conclusions: despite no previous experience in us guidance, we have successfully managed to introduce the new method in our apheresis unit. within months, we have reduced cvc usage threefold and as the personnel is gaining more experience, we suppose that the cvc usage may be reduced to episodic cases. despite slightly higher number of pressure alarms, all procedures with ultrasound guidance were completed as planned. ultrasound guidance is the most important tool for significant increase in peripheral va usage and may become the only option for patients with difficult va. disclosure: nothing to declare. abstract already published. donor blood management in healthy bone marrow donors: a retrospective single institution analysis background: over the last two decades mobilized peripheral blood stem cells (pbsc) have been established as the main source of stem cells because of improved engraftment and no necessity for hospitalization for the donors. nevertheless, due to the introduction of promising new transplant regimens, especially in the haploidentical transplantation setting bone marrow (bm) donations are regaining importance. although for both donation methods severe side effects are rarely described, bm collection is associated with considerable blood loss and hence symptoms of acute blood loss are commonly observed. therefore autologous blood are collected routinely in some institutions before donation. since the collected bone marrow amount depends on the target dose, the wbc yield in the product influences the required bone marrow volume. therefore we sought to investigate the relationship between collection volume, rbc volume removal, drop in hb and indications for blood transfusion. furthermore, we assessed wbc and cd +yields in relationship to various donor parameters and to product volume, in order to find prediction tools for collection volumes. methods: allogeneic bone marrow harvests from adult donors were performed at our institution and retrospectively analyzed. complete blood counts, serum iron and ferritin were assessed at work-up and weeks after donation. the bone marrow product quality including wbc, hematocrit (hct) and cd + cells were assessed by automatic hemocytometry and single-platform flow cytometry with ishage gating. results: besides local pain most of the side effects were related to blood loss. none of the donors received blood transfusions. the mean reduction of hemoglobin levels was . g/dl with a minimum hemoglobin level of . g/dl and a persistent anemia according to who criteria after weeks in . % and pathologically low ferritin levels in %. no donor presented symptoms with indication for blood transfusion. the median wbc concentration of the bm product was . /nl ( - % percentile: . - . / nl) the cd +cell concentration . /μl ( - % percentile: . - . /μl). in the linear regression analysis leukocyte counts of the donor before donation correlated significantly with wbc concentration in the product. thus in order to collect with % certainty the mio wbc which are a typical per-kg dose for an allogeneic recipient, . ml of bone marrow must be collected. collection volume did not systematically affect wbc or cd + cell concentration. conclusions: achieving high wbc yields in the bone marrow product allowed for collection of relatively modest bm volumes, thus protecting donors from excessive blood loss. acute adverse events were acceptable. optimization of perioperative management in healthy bone marrow donors may be achieved by good collection technique and reevaluation of wbc yields of each institution to calculate required bone marrow amount. the collection of autologous blood is not indicated. furthermore stringent pre-and postoperative hemoglobin management is predicted to limit adverse effects. disclosure: nothing to declare. donor-recipient weight ratio predicts successful stem cell mobilization on day four of gcsf mobilization results: in group median age of donor was years (range to years). in group median age of donor was years (range to years). table] . table ] elaborates other parameters analyzed between the two groups. one patient in group developed grade ii acute gvhd whereas patients in group developed acute gvhd grade ii-iv. at the last follow up no ( / ) patient in group has any symptoms of chronic gvhd whereas ( / ) patients in group have features of chronic gvhd (one extensive, one limited). conclusions: our observation suggests that upfront use of plerixafor in combination with gcsf modifies the graft favorably decreasing the risk of graft failure and graft versus host disease both acute and chronic. it also helps the donor by decreasing the total volume processed, amount of acd exposure and the duration of harvest. disclosure: none. impact of vitamin d levels on peripheral stem cell mobilization in autologous hematopoietic stem cell transplant recipients ferda can , zeynep arzu yegin , zubeyde nur ozkurt , orhun akdogan , lale aydın kaynar and total product cd + cell count [ . ( . - ) vs . ( . - . ); p= . ] were significantly higher in patients receiving chemotherapy+g-csf than g-csf only. the study group was divided into two groups based on peripheral cd (cut-off level: x /kg) as well as product cd levels (cut-off level: x /kg). vitamin d levels were found to be similar among these groups (p> . ). total product cd + cell count was found to be relatively lower in patients with vitamin d levels below μg/l [ . ( . - ) vs . ( . - . ); p= , ]. (figure ) conclusions: based on its effect on stem cells in in vitro studies, it may be considered that vitamin d may have a favourable impact on stem cell mobilization. statistically insignificant but relatively lower total product cd + cell count in patients who had lower vitamin d levels, which may indicate a role for vitamin d in stem cell mobilization, needs to be confirmed with larger studies. considering the high prevalence of vitamin d deficiency in the general population, the possible role of vitamin d in hematopoietic stem cell mobilization deserves further consideration. disclosure: nothing to declare background: one of the factors, affecting efficiency of autologous hematopoietic stem cell transplantation (autohsct) in hodgkin lymphoma (hl) patients is early recovery of graft, depending on cd + cell count and conditions of cell product cryopreservation and storage. it is well known, that dimethylsulfoxide (dmso), used for cryopreservation, can be cardiotoxic and cause diverse gastrointestinal, pulmonary, kidney, liver side effects and acute hemolysis. lethal for animals dose - mg/kg leads to life threatening arrhythmias and respiratory arrest. in order to improve dmso toxicity different ways of alternative cryoconservation modes are studied -lower dmso concentration ( % vs %), temperature - ˚c instead of ultra-low and washing of cell product. aim of the study is to evaluate the influence of dmso washing on hematopoietic recovery after autohsct. methods: retrospective analysis of hematopoietic recovery of relapse/refractory hl patients after autohsct was performed. mobilization regimen included second line chemotherapy for hl (dhap, begev, igev, ice) with consecutive g-csf administration. cd + cells were assessed, using -colour flow cytometer facs canto ii while cell collection, thawing and washing. cells with % dmso were stored at - ˚and washed in cases of transplantation with human albumin-dextran (reopolyglukin) and centrifugation. statistical data processing was performed by the χ method -pearson criterion; p -the level of significance of differences. results: patient groups had no difference in age, disease stage, gender, time from treatment start to autohsct and cd + cell count (p> , ). time to wbc recovery > х /л was - (median , ) days vs - (median , ) days, time to platelet> х /л recovery was - (median , ) days vs - (median , ) days in groups without and with cell washing respectively (p= , ). no difference in blood component consumption was observed (p= , ). in out of ( %) patients during cell reinfusion without washing nausea, vomiting, arterial hypertension was observed, no reactions were detected after cell washing (p = , ). conclusions: washing autologous mononuclear cells from cryopreservant dmso does not lead to low hematopoietic recovery rate after autohsct and can avoid toxicity, thus making autohsct more safe. disclosure: authors declare no conflict of interests. quality assesment of hematopoietic stem cells autografts after cryostorage, harvested using plerixafor background: the introduction of high-dose chemotherapy followed by transplantation of autologous hemopoietic stem cells (hscs) into the treatment program for multiple myeloma (mm) has significantly increased the frequency of achieving complete remissions and overall survival in patients. to obtain a sufficient amount of hscs, hematopoiesis is stimulated with granulocyte-macrophage factors (gm-csf) both in mono mode and after the administration of cytostatics followed by cytapheresis sessions (alone or after the cytostatics followed by cytapheresis sessions) . cryopreservation protocols are used to preserve cells in a viable state, followed by long-term storage of transplants in liquid nitrogen. however, in some patients it is not possible to obtain the necessary amount of hscs. the inclusion of plerixafor in standard mobilization schemes allows you to prepare the sufficient quantity of hscs in most patients with mm. methods: the study included samples of autografts from patients with mm from to (median . ± . ). hscs mobilization was performed on the background of unstable blood formation after high doses of cyclophosphamide g/m with the subsequent administration of g-csf at a dose of - μg / kg ( samples from patients) and with the addition of plerixaphor at a dose of . μg / kg ( samples from patients). immunophenotype viability of hscs in autotransplants after cryopreservation were determined by flow cytometry using the ishage protocol on a flow cytometer (facs cantoii, becton dickinson) by expressing surface markers of antibodies against cd , cd , cd , cd and staining with aminoactinomycin ( -aad). the colony-forming activity of hscs (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) was evaluated in methylcellulose (methocult h , stemcell technologies, canada) for x transplanted cells for days. results: the viability of hscs in autografts (cd + / cd + / add-) after cryopreservation in both groups was ± . %. in the group of samples using plerixaphor, a higher content of primitive hemopoiesis precursors (primitive cells) (cd + cd + cd -) was detected compared with the control group ( . ± . % and . ± %, respectively). the cfu count (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) in the plerixafor group was ± . per x explanted cells, in the control group - ± . ( figure a-d) . conclusions: the use of plerixafor against the background of standard protocols for the mobilization of hscs allows to obtain high-quality graft with a higher content of primitive cells and proliferative activity. disclosure: no conflict of interest. nothing to declare. comparison of effectiveness of plerixafor plus g-csf in poor and very poor-movilizers: efficacy of the combination of plerixafor and g-csf in poor-movilizer background: healthy donors ocassionally show a poor response to mobilization agents. plerixafor+g-csf can be a salvage strategy in poor mobilizers. some series describe the use of plerixafor to collect greater doses of cd + cells in hematopoietic stem cell transplantation (hsct) with tcell depletion. plerixafor use in the mobilization protocol could help collecting higher cd + dose in indirect t-cell depletion (cd + selection) for ex-vivo manipulated haploidentical transplantation, with less number of apheresis and a rapid engraftment. methods: data of fourteen healthy peripheral-blood donors was retrospectively collected. they received days mcg/kg/day g-csf and , mg/kg/day plerixafor on º day as mobilization treatment. fourteen pediatric patients (median age years, range - ) diagnosed with malignant and no malignant hematological diseases received haploidentical hsct with cd + selection and cd ra+ depletion between february and july . results: one leukoapheresis procedure was performed in all cases. median processed volume was liters (range - ). median of cd + cells obtained was , x /kg (range , - , ) . after positive selection, > x /kg cd + cells were infused in all cases (figure ). neutrophil engraftment was achieved after a median of days (range - ). few donors presented only plerixafor mild secondary effects. conclusions: our experience showed that a mobilization protocol using g-csf and standard dose of plerixafor (compasive use) is a safe strategy that allows collecting great cd + dose in one apheresis procedure. this could be useful for haploidentical transplantation with ex-vivo t depletion, especially if there´s a weight disproportion between donor and patient. background: mesenchymal stem cells (mscs) are selfrenewing multipotent progenitor cells with wide differentiation potential. their ease of isolation and expansion in vitro as well as their unique regenerative therapeutic properties suggest the use of msc as an approach for treating several disorders. extra-embryonic tissues as placenta have been proposed as potential sources of mscs due to the absence of ethical problems neither risks for the patients. furthermore, only protocols using fresh placental tissue have been described so far. a protocol for isolating mscs from delayed-manipulated tissue was designed and tested in order to optimize the use of placental mscs (mscs-p) in an advanced therapies context. methods: full term placentas (n= ) were obtained from healthy mothers in hospital universitario central de asturias (spain). informed consent was obtained from each mother prior to delivery. after dissection of gr decidual tissue it was washing with saline (b. braun, germany) and cut into small pieces. these biopsies were conserved hours in dmem media with % antibiotic solution x (gibco, usausa) until processing. the day after, tissue was mechanically minced and then enzimatically digested with a combination of ui/ml dnase i (sigma aldrich, usa) and . % tripsin-edta solution (w/v) (biochrom, germany) at ºc for hour. then, the mixture was filtered with μm cell strainer (bd bioscience, usa) and centrifuge at xg for minutes. finally, cells were resuspended in ml of dmem media suplemented with % fbs and antibiotic, seeded in -cm flask and incubated in forma stericult co incubator (thermo fisher scientific, usa) at ºc, %co . culture-expanded mscs cells were phenotipically characterized by flow cytometry (facs aria iiu, bd) with antibodies against cd , cd , cd , cd , cd , cd , cd , cd , hla-dr cd and cd using mesenchymal cell kit (immunostep, spain). afterwards, these cells were differentiated to adipogenic, osteogenic and chondrogenic lineages using stemmacs adipodiff media, stemmacs osteodiff media and nh chondrodiff medium (miltenyi biotec, germany) respectively. after three weeks of differentiation cells were fixed in % paraformaldehide (merck, usa) and analyzed. adipogenic, osteogenic and chondrogenic differentiation was visualized after staining with oil red o, alkaline phosphatase and hematoxilin-eosin (sigma-aldrich, usa). results: mscs-p isolated cells were characterized according to the isct criteria for mesenchymal stem cells. they were positive for cd , cd , cd , cd and cd and negative for cd , cd , cd and hla-dr, indicative of a typical msc phenotype ( figure ). all the markers showed a high percentage of expression between . and . %, meaning that msc population obtained with the designed method was very homogenous. similarly, staining for the three studied lineages was positive ( figure ). conclusions: the described protocol allows us to obtain mscs from decidual placental tissue stored and processed hours after the biopsy extraction using a unique enzymatic digestion. this circumstance permits to take advantage of placentas that are discarded after delivery giving us the option to obtain mesenchymal cells that could be used in clinical trials. disclosure: nothing to declare outcomes of umbilical cord transplant in high risk relapsed or refractory acute myeloid leukaemia background: high-risk relapsed/refractory acute myeloid leukaemia (aml) is a fatal disease. allogeneic haematopoietic stem cell transplantation represents the only chance of cure. as the transplant relies on the graft-versusleukaemia (gvl) effect, and if different donors exert different gvl effects, then choosing the right donor assumes great importance. in manchester, a large bmt centre in the north of england, our practice in such aml has been to choose unrelated cord blood (ucbt), without serotherapy in the conditioning therapy, as our preferred donor cell source. methods: we report the results of unrelated ucbt in patients (five boys and ten girls) with high-risk aml, defined as relapsed or refractory disease. thirteen patients ( %) received this as a st transplant, two patients ( %) received this as a nd transplant for relapsed aml post matched unrelated donor transplant, and one ( %) received ucbt twice, once in cr and once in cr . nine patients ( %) had mismatched ucbt, and the rest were fully matched at class-i (hla-a, -b, and-c) and class-ii (hla-drb ). conditioning was given as treosulfan, fludarabine and thiotepa in half of the patients (n = ), other treosulfanbased regimens were used in two patients ( %), and busulfan-based regimens were used in six patients ( %). no serotherapy was given. results: the median age at transplant was years (range, months - years). neutrophil and platelet engraftment were achieved in and patients at a median of and days, respectively. patients ( %) had engraftment syndrome. all engrafted patients achieved % donor chimerism, except one patient who had mixed lymphoid chimerism initially, that was corrected spontaneously to % at three months after transplant. acute gvhd grade i-ii developed in six patients ( %), and grade iii-iv developed in three patients ( %). all cases resolved, except two patients where acute gvhd evolved into chronic gvhd (one with grade i skin gvhd which fully resolved, and one with grade iii gvhd gut colitis who was parenteral nutrition dependent till death). two more patients developed chronic grade i skin gvhd and resolved (chronic gvhd developed in % in total). three patients ( %) developed veno-occlusive disease (vod), that completely resolved with defibrotide treatment and necessitated ascitic drainage in one of them. viral reactivations occurred in five patients ( %) and were successfully treated. at a median follow-up of months (range, seven months -four years), eight patients ( %) died at a median of (range, to days), with a transplantrelated mortality of % and relapse-related mortality of %. five patients ( %) relapsed post-ucbt; four died and one had a successful second ucbt (event-free survival was %). immune reconstitution in alive patients was achieved at a median of eight months. conclusions: very high-risk patients treated with ucbt with good overall survival and event-free survival, similar to aml treatment rate with low-risk disease. disclosure: nothing to declare in haploidentical transplants is the incidence of acute and chronic gvhd strictly related to the stem cell source? results: the odds ratio was . with a % confidence interval of . - . (p= . ). conclusions: the risk of infection of the uc is not related to the microbiological status of the ucb. a possible explanation for this is the presence of antibiotics in the medium used for uc, but not ucb, transport. this means that cryopreservation of ucs from which contaminated cord blood has been obtained is justified. comparison of turkish stem cell coordination center (turkok) with istanbul university bone marrow bank (tris); a single center experience in match unrelated donors azize mergen , selime aydoğdu , başak aksoy , yunus emre savcı , gürcan dikme , funda Çipe , ceyhun bozkurt , tunç fışgın older patients are increasingly being transplanted, thanks to improvement in allogeneic hematopoietic stem cell transplantation (allo-hsct) techniques. increasing donor age is associated with greater risk for mortality and graftversus-host disease (gvhd). since sibling donors are of similar age to recipients, we hypothesized that, in older patients, a young matched unrelated donor (mud) would be comparable to an hla-matched sibling donor (msd). methods: we retrospectively compared outcomes of allo-hsct from msd (n= ) and / hla mud (n= ) in patients aged ≥ years with hematological malignancies transplanted between - . all patients received reduced-intensity conditioning and graft source was peripheral blood. the primary outcome was overall survival. msds served as the reference category and were compared to muds split into three age groups (≤ [n= ], - [n= ], > [n= ] years) using univariable analyses and multivariable cox regression models adjusted for patient, disease, and transplantation features. results: the median age of hsct recipients was years and was similar across groups. median donor age for msd was years and , , and for the mud age groups ≤ , - , and > years. acute leukemia was the leading transplant indication ( %) followed by myelodysplastic syndrome, myeloproliferative neoplasms and indolent non-hodgkin lymphoma. disease risk distribution was similar across donor groups (low [ %], intermediate [ %] , and high [ %] in the complete population; p= . ). time from diagnosis to hsct was longer with mud compared to msd and increased with an older age of mud. in a univariate analysis, overall survival was % (msd), % (mud≤ ), % (mud - ), and % (mud≥ , p= . ). corresponding non-relapse mortality (nrm) cumulative incidence was %, %., %, and . % (p< . ) (figure) . gvhd-relapse-free (grfs) was %, %, %, and % (p= . ). in a multivariable cox model, young mud (≤ ) had a similar risk for mortality compared to msd (hr . , p= . ), while a monotonic increase in risk was observed with an older donor age (mud - y: hr . ,p= . ; mud≥ y: hr . , p= . ) (table) . findings were confirmed in a propensity score analysis, matched for key covariates. nrm and grade - acute gvhd were consistently higher with mud, with the greatest risk associated with older muds. the hazard for grfs was higher with mud aged or higher compared to msd; risk was not higher with younger mud. conclusions: in older patients receiving reduced intensity conditioning, msd remain the optimal choice. however, when not available, young mud provide comparable results. disclosure: nothing to declare background: there is growing evidence that community acquired respiratory virus (carv) increase the risk of pulmonary invasive fungal disease (ifd) in recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct). to date, there is a lack of knowledge regarding the rate of ifd, risk factors (rfs) as well as the most critical period for the development of a later ifd after carv infections in allo-hsct recipients. methods: in this prospective observational study, we retrospectively analyzed the effect of carv on the development of a later ifd in a consecutive cohort of allo-hsct adult recipients who developed carv infectious episodes from december to december . respiratory virus in upper and/or lower respiratory tract specimens were tested using multiplex pcr panel assays. results: overall, out of allo-hsct recipients ( %) developed ifd within months after a carv episode at median of days (range - days) from the day of carv detection. all the ifds involved the lungs and in cases ( %) the diagnostic was ia accomplishing criteria of probable (n= ) or proven (n= ). of note, out of ifd ( %) occurred within the first year after transplantation. the overall rate of ifd after carv episodes was % whereas this rate was higher in recipients developing carv during the first year of transplant ( %). ifd was diagnosed in out of with carv lower respiratory tract disease (lrtd) episodes ( %) compared to out of carv upper respiratory tract disease (urtd) ( %) (p= . ). twenty-three out of carv episodes involving the lrtd during the first year after transplant ( %) developed ifd. we did not found differences in ifd rates according to the type of carv identified. multivariate analysis identified rfs for ifd: the use of atg as a part of conditioning [odds ratio (or) . , % confidence interval (c.i.) . - . , p= . ], carv lrtd (or . , % c.i. . - , p= . ), carv infection during the first year of transplant (or . , p natural killer cell alloreactive haploidentical stem cell transplantation for multiple myeloma patients catharina elssen , lotte wieten , peter von dem borne , ellen meijer , gerard bos maastricht university medical center, maastricht, netherlands, leiden university medical center, leiden, netherlands, amsterdam university medical center, location vumc, cancer center, amsterdam, netherlands background: in the past years many new drugs for multiple myeloma (mm) have been developed and are responsible for a increase in survival. notwithstanding such progress, mm remains incurable. results from allogeneic stem cell transplantation (sct), including haploidentical transplantation, in mm has shown clinical results. however, these responses are only observed in a minority of patients. we hypothesize that this observation might be due to differences in natural killer (nk) cell alloreacitvity, since we have shown in in vivo and in vitro models that mismatched alloreactive nk cells hold the capacity to kill mm cells. the aim of this prospective phase study is to evaluate if kir-ligand mismatched haploindentical bone marrow transplantation (bmt) with post-transplant cyclophosphamide will improve progression free survival (pfs) in poor risk mm patients. methods: poor risk mm patients, aged < years were enrolled if they were responsive to their last line of therapy. poor risk was defined as, high-risk cytogenetics, or relapse within a year after autologous sct, or treated with three or more previous lines of therapy. a prerequisite of enrolment was the possibility of an nk cell mismatch and availability of a mismatched family donor. patients were excluded if donor-specific hla-antibodies were present. patients received a haploidentical bmt with a non-myeloablative conditioning regimen and post-transplant cyclophosphamide. primary endpoint is pfs at , years. secondary endpoints are engraftment, bone marrow reconstitution, nk cell reconstitution and repertoire, graft versus host disease (gvhd), infections and non-relapse mortality (nrm) at , years. results: in total poor risk patients were included in the study of which could be evaluated for the primary end point. graft failure and disease progression before transplant rendered the remaining two patients not evaluable. at this interim analysis patients have already reached the , years of follow up, relapsed within , years and died due to treatment related infections, without showing progression of disease ( % nrm). average time of progression is days ( - days). two of the remaining patients at follow up, still show responsive disease (days en ). the average time to neutrophil reconstitution is days ( - days). all evaluated patients ( / ) show nk cell reconstitution with a mature phenotype in the bone marrow and peripheral blood by day . three patients developed acute gvhd ( %) of which / grade i-ii agvhd and / patient showed a grade iv agvhd. treatment related mortality was / ( %), which was in all cases due to infectious disease. conclusions: our interim analysis of mismatched haploidentical bmt in mm showed that the treatment is feasible and forms a possible platform for immunotherapeutic strategies. the majority of patients showed an early disease progression. we predefined that with a pfs of % at , years we would qualify this treatment option successful. with only two patients still in remission this goal will not be achieved and we hypothesize that the late nk cell reconstitution (day ) is responsible for the lack of response. clinical background: mscs are known to have immune modulatory capacity and may be effective in the treatment of patients with acute gvhd. however clinical studies yielded inconclusive results which was in part due to the great heterogeneity of the msc used. the off-the-shelf msc preparation "msc-ffm", generated by a proprietary pooling process, selection by plastic-adherence, expansion for an aggregate four weeks followed by cryopreservation until use, is available in germany through a national marketing authorization. "msc-ffm" is indicated in steroidrefractory agvhd, dosed at - x /kg bw i.v. in four doses one week apart. methods: we report seven consecutive pediatric patients (median age . y), who received "msc-ffm" from unrelated hla disparate donors between december and november in our institution. we gave msc infusions to patients with steroid-refractory grades iii-iv agvhd and one patient who had therapy-refractory background: regulatory t cells (treg) are known for their immunosuppressive function and have proven successful as graft-versus-host disease (gvhd) prophylaxis after allogeneic bone marrow transplantation in a number of preclinical as well as first clinical studies without compromising graft-versus leukemia (gvl) effects. in murine models of acute gvhd lymph node homing capacity via cd l (l-selectin) proved to be essential for disease prevention. yet, treg recruitment from lymph nodes to peripheral sites of ongoing gvhd also seems necessary to achieve maximum protective as well as therapeutic effects. the chemokine receptor ccr directs activated t cells to sites of inflammation, thus high ccr expression should facilitate treg homing to affected gvhd target organs. with this project we lay the foundation for future in vivo studies of treg therapy for gvhd by upregulation of ccr expression. methods: we performed systematic ex vivo analysis of ccr expression on murine naive and memory conventional (tconv) and regulatory t cells isolated from spleen, blood, bone marrow, lymph nodes, liver and lung. cells were stained for characteristic surface and intracellular markers and characterized by multiparametric flowcytometric analysis. ccr expression kinetics following stimulation were analysed in tconv and treg isolated from murine splenocytes by facs and polyclonally activated by anti-cd /cd -coated beads in the presence of exogenous il- . expression was monitored by daily flow cytometric analysis. ccr overexpression was induced by transduction of expanded treg with ccr mrna via electroporation. expression kinetics were monitored by facs, receptor function was tested in transwell migration assays using ccr ligands ccl- and ccl- . results: systematic analyses showed higher ccr expression on memory treg than on their naive counterpart in all examined organs with bone marrow samples displaying the greatest disparity. memory treg showed higher ccr expression than memory tconv in all analysed organs, except for lymph nodes where both memory populations revealed equal expression levels. stimulation of in vitro expanded treg and tconv lead to a strong increase in ccr expression with maximum levels on d and d respectively, whereas restimulation (d ) resulted in no further relevant ccr expression on treg. we performed systematic optimization of stimulation and mrna-electroporation conditions to reliably achieve highlevel short-term ccr expression. transduction of treg on d of in vitro expansion resulted in a strong ccr expression, with maximum levels h after electroporation and strong ccr expression being detectable for at least h. transwell migration assays showed enhanced migrational properties of mrna-electroporated treg towards ccr ligands. analyses performed h and h after electroporation showed persistent migration even though measured ccr surface expression had already declined significantly. conclusions: we showed that high ccr expression can be detected on memory treg in all analysed organs. since in vitro stimulation of murine treg did not reliably induce ccr expression, we established a protocol for ccr mrna-electroporation. electroporated cells showed stable short-term ccr expression and enhanced migrational properties towards ccr ligands in vitro. future studies will show whether the induction of short-term ccr expression will facilitate in vivo homing of adoptively transferred treg to sites of ongoing gvhd and thus mediate long-term inflammation suppression. disclosure: the authors have no conflict to disclose. survival and immune reconstitution of syngeneic, haploidentical and allogeneic hematopoietic stem cell transplantation in atm-deficient mice ruth pia duecker , patrick c. baer , stefan zielen , ralf schubert from allo-hsct. it´s not necessary to do chemotherapy before transplantation for patients with bone marrow blast cells more than % at the time of diagnosis. [[p image] . figure the hci-ct of patients before transplantation and occurance of grade iii-iv agvhd on overall surviv] background: . allogeneic haematopoietic stem cell transplantation (sct) offers the chance of cure for patients with transfusion-dependent thalassemia (tdt). based on the non-neoplastic nature of this condition sct approaches urgently require to prove both efficacious and safe. methods: . we report on children, adolescents and young adults (median age: years; range - years) with tdt receiving sct from an hla-matched donor (mud n= , msd n= , mfd n= ) in our center from - . all patients received the same treosulfan-based conditioning regimen (treosulfan x g/m , fludarabine x mg/m , thiotepa x mg/kg). gvhd prophylaxis was based on atg-fresenius™ ( x mg/kg, if mud or mfd as donor), csa (with taper from day + ) as well as mtx (day , , , ) in / and mmf in / patients with mtx toxicity, respectively. stem cell source was bone marrow in , peripheral blood stem cells in and cord blood in patient. prior to transplantation children received cytoreductive treatment with azathioprine, hydroxycarbamide and intensified erythrocyte transfusion. iron elimination therapy was carried out in / children with deferasirox. among the patients with available ferris-can™ analysis patients showed substantial liver iron overload (liver iron > mg/g) despite intensive chelation prior to sct. results: . all patients achieved leukocyte engraftment at median day + (range - ), however two patients required a cd -selected pbsc boost on day + and day + based on delayed platelet and/or erythrocyte engraftment. nine patients exhibited full donor chimerism in the bm at day + , the other showed mixed chimerism with < % autologous cells. on day + peripheral blood chimerism was complete in / patients with the remaining patient exhibiting stable split chimerism with % donor-derived erythrocytes and - % autologous myeloid cells. acute gvhd was observed in three patients (grade : n= , grade : n= ). however, all patients responded to immunosuppressive therapy with steroids ± ecp and re-initiation of cni (n= ). one patient suffered background: patients with relapsed or refractory acute myeloid leukaemia (aml) have a poor prognosis. allogeneic hematopoietic stem cell transplantation is the only curative option. however, allogeneic transplantation with active leukemia failed to improve significantly the longterm outcome. to improve the outcome of allo-hsct in such high-risk and refractory patients, sequential schedule of cytoreduction therapy followed by nonmyeloablative conditioning has been developed. methods: to evaluate the outcome of sequential intensified conditioning regimen followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for refractory acute myeloid leukemia (aml). results: a total of patients with primary or secondary refractory aml transplanted between june to july were included. refractoriness was defined as primary induction failure, relapse within months from induction/ consolidation chemotherapy or second relapse. median age is years ( to ). the salvage chemotherapy administered was flag-ida. two patients did not receive intensive chemotherapy because of no recovery after induction chemotherapy. seven days after the end of flag-ida, a reduced intensity conditioning consisting of fludarabine, mg/m , thiotepa, mg/kg and busulfan , mg/kg i.v. (n= ) for haploidentical donors or fludarabine plus busulfan (n= ) for hla identical sibling or unrelated donors was administered. graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus and mycophenolate mofetil. the mycophenolate was withdrawn at day + post-transplantation and tacrolimus at day + . donor lymphocytes (dli) were infused in patients without agvhd at day + post-transplantation. seventeen patients achieved complete donor chimerism, patients progressed early and patient died before engraftment. one of the patients which recovery was with persistent leukemia reached donor chimerism after immunosupression discontinuation. ten patients are alive in complete remission. median follow-up of survivors is months (range: - ). five patients died of leukemic progression, as result of gvhd and suffered intracranial hemorrhage. five patients received prophylactic dli. the incidence of acute moderate-severe gvhd and moderate-severe chronic gvhd were % (n= ) and % (n= ), respectively. the non-relapse mortality was % (n= ), mainly due to acute gvhd (n= ) . the -year cumulative incidence of relapse posttransplantation was . %. the probability of relapse was %± %. the -year os and dfs were % ± % and % ± % conclusions: the strategy of sequential chemotherapy followed by allohsct ± prophylactic dli has an acceptable toxicity profile and improves both the relapse rate and the survival for refractory aml patients. disclosure: nothing to declare background: the concept of immunological intervention to prevent relapse after hematopoietic stem cell transplantation is associated with the assessment of the chimerism status. distinguishing patient and donor hematopoiesis is usually performed by str-pcr, a powerful method developed for forensic purposes. however, this method shows restrictions with respect to detection limit, preciseness, and the possibility of automated read out. digital pcr could circumvent some of these limitations. methods: recently, validated for the bio-rad droplet digital platforms, the biotype mentype digitalquant assay was released. the assay uses indel polymorphisms on chromosomal dna to distinguish patient and donor hematopoiesis on a fret hydrolysis assay basis ("taqman assays"). thus the assay in principle is applicable on the chamber based d digital pcr system (thermo fisher). due to different reaction chemistry and physical properties of thermal transfer between the digital pcr platforms protocols are reasonably not fully compatible which would lead to lower fluorescence intensities and poor signal resolution on the solid chip based thermo fisher d platform. an adjusted pcr protocol was established and optimized using representative markers, followed by determination of tolerable and optimal amount of input dna. specificity, sensitivity and reproducibility testing with artificial mixed samples preceded the extensive verification by comparative measurement of clinical samples (n= ) and ring-trial samples (n= ). source to allogenic bm or pbsc. ucb units are immediately available for transplantation as they are frozen and banked with defined hla typing and it has an advantage for patients who need urgent transplantation. in addition, a higher degree of hla mismatch appears to be acceptable with a comparatively lower risk of acute and chronic gvhd. meanwhile, a higher incidence of engraftment failure, delayed neutrophil and platelet recovery, and posttransplant immune disorders including pre-engraftment immune reactions (pir) are major problems in unrelated ucbt. methods: in our institute, gvhd prophylaxis in ucbt was changed after march . between january and march , thirty-two patients received tacrolimus plus methylprednisolone (tac/mpsl) and between april and january , thirty-one patients received tac plus methotrexate (tac/mtx) for gvhd prophylaxis. to investigate better gvhd prophylaxis after ucbt, we compared transplant outcomes after ucbt using gvhd prophylaxis with tac/mpsl (n= ) and tac/mtx (n= ) in single-pediatric transplantation center. results: the cumulative incidence of neutrophil engraftment at day in tac/mpsl group was . % and . % in tac/mtx group (p= . ). median time of neutrophil engraftment was days earlier in tac/mtx group ( days) than tac/mpsl group ( days). according to pir, and acute gvhd, tac/mtx group showed superior outcomes; the incidence of pir (p= . ) and the cumulative incidences of acute gvhd at day ( . vs . %, p = . for grade ii-iv, . vs . %, p= . for grade iii-iv) was significantly lower in tac/mtx group than in tac/mpsl. however, the incidences of relapse (p= . ) and cytomegalovirus viremia (p= . ), and estimated overall survival (p= . ) and event-free survival (p= . ) were comparable between two groups. conclusions: our results indicated that gvhd prophylaxis with tac/mtx had favorable effects; reduced incidence of rip and acute gvhd after ucbt without any negative influences. disclosure: nothing to declare transfer of donor regulatory t-cells after atg reconditioning cures severe refractory gvhd and leads to long term persistence of regulatory t-cells in the recipient cells on a clinimacs® plus device (miltenyi biotec). the cell product contained % foxp + t-cells. the patient received , x /kg t reg on day + . subsequently intestinal gvhd decreased and finally resolved. three months after the first t reg transfer the patient got a second t reg transfer ( , x /kg) on day + due to decreasing t reg levels. thereafter t reg persisted and there was no recurrence of gvhd. the patient is well with low dose sirolimus and prednisone as the only immunosuppressants and is particularly recovering intestinal function. conclusions: this case illustrates an unusually severe acute gvhd after matched sibling sct. transfer of donorderived t reg was able to cure severe and refractory gvhd after t-cell ablation by atg. transferred t reg persisted in the recipient for a long period and did not lead to any adverse events. disclosure: no disclosures to declaim allogeneic hsct for patients with transfusion dependent anemia from matched and mismatched donors julia fekadu , andrea jarisch , jan sörensen , emilia salzmann , eva rettinger , andré willasch , shahrzad bakhtiar , thomas klingebiel , peter bader after first asct. the mean harvest for patients receiving dhap was , x cd (+) cells/kg, , x cd (+) cells/kg for cy, . x cd (+) cells/kg for igev, , x cd (+) cells/kg for ice, , x cd (+) cells/kg for choep. the patient mobilized with vtd-pace achieved , x cd (+) cells/kg after apheresis. of the patients achieved the target number of > x /kg cd + cells after apheresis, after two, and after three apheresis. the median time to apheresis was days ( - ) without significant difference between the regimens. the mean wbc count at the time of apheresis was , x /l after dhap, , x /l after cy, , x /l after igev, , x /l after ice, , x /l after choep. there was correlation between wbc and cd harvested cells (p= . ). grate - thrombocytopenia was found in patient ( dhap, ice, igev, vtd-pace). grate - anemia was registered in patients ( dhap and vtd-pace). no correlation was found between the cd + harvest and the age, number of previous lines chemotherapy, the response before mobilization, the type of the lymphoma and the clinical stage. conclusions: our results demonstrate that the chemo-g-csf protocols have comparable effectiveness with accep- background: cytomegalovirus (cmv) may cause severe complications in recipients of allogeneic haematologic stem cell transplantation (allohsct). letermovir (ltv, / mg daily without/with co-administration of cyclosporine) was recently licenced only for cmv prophylaxis in adult allohsct-recipients. paediatric data as well as data on cmv therapy are missing so far. methods: we administered letermovir mg orally once daily (with no co-administration of cyclosporine a) to paediatric patients after allohsct. edta-plasma were occasionally obtained at different time points and frozen for determination of letermovir levels using liquid chromatography/mass spectrometry (lc-ms/ms). results: for details on patients, treatment and cmv load see table . in short periods of letermovir administration, cmv blood levels became negative in both patients. considering the lacking safety data in paediatric patients, we stopped letermovir treatment in both patients, when liver parameters increased. in patient hepatopathy turned out to represent histologically proven graft versus host disease (gvhd). in patient liver parameters further increased despite withdrawal for another weeks, however, hepatopathy was only mild and self-limiting. both patients additionally received other possibly hepatotoxic substances (mycophenolate mofetil and trimethoprim/ sulfamethoxazole). letermovir plasma levels were . ng/ml ( h), . ng/ml ( h), . - . ng/ml (median . ng/ ml, n= , h) and . ng/ml ( h after administration). conclusions: during short letermovir treatment, we observed fast resolution of cmv viraemia as well as rising liver parameters in both patients. while elevated liver parameters represented gvhd in patient, a causal relationship with letermovir might be considered in the other patient. letermovir peak levels after administration of mg were within ranges reported in adults after administration of mg while trough levels were higher indicating differences in pharmacokinetics in terms of delayed clearance. inguinal lymphadenomegaly. after failure to respond to seven conventional treatment lines: methotrexate, cop (cyclophosphamide, vincristin and prednisone); gemcitabine; puva; interferon; acitretin and extracorporeal photopheresis), allogeneic hsct from an identical hla male donor was indicated. the non-myeloablative conditioning consisted of fludarabine ( mg / m ), cyclophosphamide ( mg / kg) and total body irradiation(tbi) ( cgy). prophylaxis of graft versus host disease (gvhd) was performed with cyclosporine ( mg / kg) and mycophenolate mofetil ( mg/kg). after conditioning, there was improvement of pruritus and involution of the skin. bone marrow infusion occurred on / / (d ). on d + he presented recurrence of skin lesions of fmf. donor lymphocyte infusion (dli) was performed ( x cd + cells / kg / recipient). he presented oral lichen and diarrhea respectively as manifestations of gvhd on d + and d + . as infectious intercurrence, hemorrhagic cystitis occurred by bk virus months after the first dli and he received conservative treatment and remained without systemic immunosuppression. nine months after hsct, a second dli ( x cd + cells / kg / receptor) was performed and at this time the patient is without clinical manifestations of fmf or gvhd. conclusions: the clinical response of the presented case confirms what has been reported in the literature. ctcls appear to be particularly susceptible to gvl effect, which makes hsct a potential cure for advanced ctcls in eligible patients. the timing to perform hsct in the clinical course of the disease remains a matter to be settled clinical trial registry: not applicable disclosure: no conflict of interest p abstract already published. methods: we applied next generation sequencing (pgm, ion torrent/ fischer lifetechnologies) to an unselected cohort of patients ( female, male, median age ( - ) years) who had been referred for allogeneic stem cell transplantation due to the presence of a high-risk myeloid disorder dnmt a r h ( . %) . ), cebpa ( . %; ceb-pap s ( . %) vus) kras ( . %; krasr r ( . %) vus), and kit ( . %; kitm l ( . %) . ). patients displayed a median of sequence variants (aml, mpn and cmml patients each ; mds, saa and patients with other, non-malignant hematologic diseases each sequence variants found most frequently in aml were cebpap s ( . % of all sequence variants in patients with aml, p< - , chi² test) in patients suffering cmml, dnmt ar h was particularly frequent ( . % of all sequence variants in cmml, p=. ). asxl e d ( . % of all sequence variants in other, non-malignant hematologic diseases, p=. ), idh r q ( . %, p=. ) and krasr r ( . %, p=. ) were frequent in other, non-malignant hematologic diseases. in saa, nrasg d ( % of all sequence variants in saa patients; p=. ) was frequently found, as were dnmt ak fs* ( %, p=. ), tet l w ( %, p=. ) and tet i v ( %, p=. ). conclusions: taken together, these data show that vus occur with high abundancy in this high-risk cohort of patients, and that they differ in frequency between various myeloid disorders methods: retrospective analysis of patientswho experienced either hematological relapse or progressed to aml after allo-hsct and were treated with azacitidine for this indication at hematological centers in poland. the primary end-point was overall survival (os), the secondary -response rate. results: patients, males ( . %), median age (range, - ), were enrolled. the primary indication for allo-hsct was aml median time from allo-( %ci: . - ); with -year os of . % ( %ci: . - . ). for patients stratified according to ebmt aza relapse prognostic score -year os was: prophylaxis of agvhd: with atg -atgam mg/b.w.- pts( , %), posttransplant cyclophosphomide (ptcy) mg/b.w. on d+ ;d+ - pts ( , %) conclusions: unmanipulated haplo-hsct in st - nd cr in children and adolescents with high risk al allows achieving the long-term survival in , %. the use of g-csf stimulated unmanipulated haplo-bm is associated with a satisfactory rate of engraftment. the main cause of death in our study was relapse after allo-hsct. the frequency of acute and chronic gvhd was acceptable, -years grfs rate of , % in st - nd cr represents good quality of life following unmanipulated haplo-hsct and therefore may be recommended as option for use in children and adolescents with high-risk al. disclosure: nothing to declare background: b -h (cd ) is thought to act as an immune checkpoint and regulates t and nk cell responses. it is highly overexpressed on many solid tumors while on healthy tissue protein expression is limited. this makes b -h an interesting target for cancer immunotherapy. in highrisk neuroblastoma patients, targeting disialoganglioside gd with the recently approved monoclonal antibody (mab) ch . after autologous or allogeneic sct, significantly improves survival. however, gd expression is heterogeneous and ch . causes severe adverse effects. thus, we evaluated b -h as an alternative or additional target antigen. we investigated different anti-b -h mab constructs and mab-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependent cellular cytotoxicity (adcc) using expanded γ/δ t cells of healthy donors, chex lf-il was confirmed to be the most effective mab construct. interestingly, chek -il showed comparable target cell lysis -however, lysis was only transient while chex lf-il mediated permanent target cell lysis. using patient pbmcs after receiving allogeneic sct, chex lf-il and ch . mediated comparable lysis. calculated specific lysis of lan- (after hrs.; in ascending order): targets + effectors w/o mab ( %) conclusions: b -h is a suitable target antigen in case gd expression is low or absent. immunocytokines and fcoptimized mabs targeting b -h might increase the efficacy of immunotherapy in gd -negative tumors and in combinatory approaches. until now, the low-fucose immunocytokine chex lf-il seems to be the most promising anti-b great ormond street hospital, nhs foundation trust petersburg/ raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, rehabilitation medicine, saint petersburg, russian federation, first i. pavlov state medical university of st. petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, bone marrow transplantation for pediatric solid tumor petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, pediatric hsct outpatients, saint petersburg, russian federation university hospital carl gustav carus james`s hospital diagnosis was aml in %, all in % and mds in % of patients. % of patients received pbsc grafts, % received unmanipulated bone marrow grafts. os at years was % in msd/mud-atg, % in haplo-ptcy, % in mmud-ptcy and % in mmud-atg groups (p= . ). in a multivariate cox model non-relapse mortality was %, %, . % and % in msd/mud-atg, haplo-ptcy, mmud-ptcy and mmud-atg groups, respectively. cumulative incidence of acute gvhd grade or was %, . %, % and % after in msd/mud-atg, haplo-ptcy cumulative incidence of chronic gvhd was % in the msd/mud-atg group uwe platzbecker , verena wais republic of china background: there are two most noteworthy strategies of haploidentical stem cell transplantations (haplo-hsct), the baltimore post-transplantation cyclophosphamide (ptcy) with or without anti-thymoglobulin (atg), and the beijing g-csf primed bone marrow (bm) plus peripheral blood stem cells (pbsc) (giac). however, the comparison of these two modalities is scarce. in this study, we aim to compare these two approaches for hematological malignancies based on the taiwan blood and marrow transplantation registry (tbmtr) with the comparable cd infusion amounts, the neutrophil engraftment time were statistically distinct among these three groups [d+ (group ) vs. d+ (group ) vs. d+ (group ), respectively as to the graft-versus-host disease (gvhd), the patients in group had more grade ii-iv but similar grade iii-iv acute gvhd compared with others (grade ii-iv: . % vs. . % vs. . %, respectively conclusions: haplo-hsct with different strategies is a feasible treatment modality for hematologic malignancies in taiwan. regarding the retrospective nature and limited patient numbers of this study republic of china background: we previously presented a low-resolution hla analysis of cedace, the voluntary portuguese bone marrow donor registry (ebmt , poster p ) and, more recently, its epidemiological characterization (ebmt , poster b ). currently, cedace is one of the largest bone marrow donor registries in the world, including nearly % of the country's population, twice the number of donors present in . the current work is an update on the most common hla haplotypes found in cedace results: of the donors in the cedace registry, . % were typed in at least loci (hla-a/-b/-drb ), . % in (including hla-cw), and . % in (including hla-dqb )the , and most common haplotypes accounted for, respectively, . %, . % and % of the haplotypes found in the entire registry. the five most common haplotypes at the low-resolution at the loci, low-resolution level, out of donors, individual genotypes were identified, leading to an hla matching probability at this level of . % hid-hsct) have a stronger anti-leukemia effect compared to identical sibling donor hsct(isd-hsct) in high-risk features .but in refractory/relapsed(r/r) aml patients who not in remission status, it is unclear whether it also augments the gvl effect antithymocyte globulin was used in haploidentical hsct. unmanipulated bone marrow and peripheral blood stem cells for all patients. cyclosporine, short-term methotrexate were employed for gvhd prophylaxis. mycophenolate mofetil included in hid-hsct. performed multivariate analysis for all patients of pretransplantation variables and developed a predictive scoring system for survival according to adverse factors. results: the total survivor median period of follow up was ( - ) months. hid -cohort had higher -year actuarial of os multivariate analysis showed isd-hsct,standard conditioning regimen and less than % proportional reduction in blast percentage pre-≥ . conclusions: haploidentical donor compared to identical sibling donor had better anti-leukemia effect in allo-hsct for r/r aml in nr status conditioning protocol was melphalan mg/m for mm and beam for nhl. the quantification and characterization of γδt cells in peripheral blood samples were performed by flow cytometry based on the expression of cd /cd /vδ /vδ /vγ /cd at , and days after ahsct. percentage (%) of γδt cells represented the proportion of these cells among all t cells. results: median age at ahsct was ( - ) years, % male. median time from diagnosis to mobilization was ( - ) months, after a median number of therapeutic lines of ( - ); pts ( . %) received radiotherapy. seventeen pts ( . %) were re-mobilized with plerixafor±g-csf ( % mm vs % nhl there was no difference in febrile neutropenia incidence (p= . ), timeto-engraftment (p= . ), time-to-neutrophils> /μl (p= . ) or erythrocyte transfusions (p= . ). however, there was more time-to-platelets> , /μl ( vs days; subpopulations did not affected pfs. conclusions: our results showed that pts mobilized with plerixafor need more collection volume (less cd +cells, higher dmso). plerixafor negatively affected platelet recovery, with similar hematologic and immune recovery for the remaining variables. its use was associated with higher %vδ +, suggesting that it induces an antineoplastic phenotype. more studies with larger samples and follow-up period are needed to evaluate plerixafor results: total ascc procedures were carried out in patients over years, once in patients, twice in patients, three times in patients, and four times in patient. non-hodgkin lymphoma (nhl) comprised . % of all cases (n = ) ), total number of chemotherapy cycles before ascc (or . , % ci . - . , p = . ), failure to achieve at least partial response before ascc (or . , % ci . - . , p = . ), total number of days receiving g-csf for mobilization (or . , % ci . - . , p = . ), and salvage use of plerixafor (or . , % ci . - . , p < . ) were found to be independent factors associated with failure of ascc. at the end of the study period, . % of successful collections (n = / ) were used for asct, . % (n= / ) were in storage awaiting transplantation hôpital necker-enfants malades melf methods: a total ahsct candidates [median age: ( - ) years; male/female: / ] were included in this study. twenty-seven patients ( . %) were diagnosed as non hodgkin's lymphoma, patients ( . %) hodgkin's lymphoma, patients ( . %) multiple myeloma, patients ( . %) acute myeloid leukemia, patients ( . %) plasmocytoma and patient ( %) testis cancer. premobilization serum -hydroxy vitamin d ( -oh d) levels were measured with immunoassay method peripheral cd + cell count background: some published data suggest a positive effect of iron chelators on the risk of post-transplant relapse, with an improvement in overall survival after allogeneic hematopoietic stem cell transplantation (hsct) conditioning regimen consisted to intravenous bu mg/m and, flu mg/m d- to d- . gvhd prophylaxis included atg . mg/kg on d- and d- , ciclosporin and methotrexate. all patients received peripheral blood stem cell transplant from an identical hla-related donor. iron chelation consisted on deferasirox (exjade ® ) - mg/kg/day, started at day , if serum ferritin level ≥ - ug/l and stopped when the level decreased below ug/l or normalized at day after transplant, / patients were evaluable (g = pts), (g = pts). patients were abo compatible ( %), had major incompatibility ( %), and % had minor incompatibility. median serum ferritin level at day , were ug/l ( - ) and ug/l ( - ) in g and g respectively with a median follow-up of months, ( - ), disease relapse incidence was higher in patients who did not received iron chelation treatment (g : . %) versus those who received oral deferasirox (g : . %) (figure ), but the difference was not statistically significant conclusions: these results deserve more investigation choep (n= ), and patient received vtd-pace μg/kg depending on the protocol. the aim was to collect at least x cd (+) cells/kg body weight. results: forty patients all patients were stage iii and iv at diagnosis. of the patients were mobilized after one line of treatment, six after two lines of treatment and five after and more lines of treatment alexandra martínez-roca , gerardo rodriguez-lobato , gonzalo gutierrez-garcia , , maria suárez-lledó background: hematopoietic stem cell transplantation (hsct) is an established procedure in lymphoma background: the role of inflammatory cascade in tumor microenvironment has been demonstrated in several studies. as a part of this issue, elevated neutrophil/lymphocyte ratio (nlr) was shown to be associated with an adverse prognosis, particularly in solid tumors. the aim of this study is to determine the impact of pre-transplant nlr on early transplant complications, as well as post-transplant relapse and survival.methods: a total of lymphoma patients [median age: ( - ) years; male/female: / ] who underwent autologous hematopoietic stem cell transplantation (hsct) were included in this retrospective study.results: the initial diagnosis was hodgkin lymphoma (hl) in ( . %), b-cell non-hodgkin lymphoma (nhl) in ( . %) and t-cell nhl in ( . %) patients. of patients who were evaluated for pretransplant disease status, patients ( . %) were in complete remission, patients ( . %) were in partial remission and ( . %) patients had refractory disease. median pre-transplant nlr was found to be . ( - ) . when the study population was divided into two subgroups as "low-" and "high-nlr", based on median nlr value, number of febrile days were found to be relatively higher in the low-nlr group (p= . ). a positive correlation was demonstrated between nlr and lactate dehydrogenase levels (r= . ; p= . ); and nlr and ferritin levels (r= . ; p= . ). at a median follow-up of ( - ) months, overall survival (os) was found to be better in the low-nlr group without statistical significance [ vs ( - ) months; p= . ]. in univariate analysis, pre-transplant nlr represented a significant impact on os (p= . ). other prognostic factors were age (p= . ), platelet engraftment (p= . ), post-transplant relapse (p= . ) and pre-transplant ferritin level (p< . ). the permanent impact of ferritin on os was confirmed in multivariate analysis (p= . ).conclusions: in this study, an adverse impact of elevated pre-transplant nlr on os was demonstrated in autologous hsct recipients with lymphoma. as a predictor of prognosis, nlr may be considered as a safe and cost-effective parameter. further studies are required in order to use this predictor in routine clinical practice.background: high cure rates in childhood diseases have been achieved by stem cell transplantation (sct). however, there is little knowledge concerning recovery of the immune system and community-acquired infection after sct. here we studied the long-term reconstitution of the immune system and incidences of community-acquired infection after sct.methods: we reviewed medical records for patients (m/f: / , median age: years (range: - years) who were treated in the department of pediatrics, hokkaido university hospital. we analyzed cd -positive cell counts, serum immunoglobulin g (igg) levels, and incidences of community-acquired infection until years after sct. indications for sct were all in patients, aml in , aa in , nb in , rms in , jmml in , nhl in , cgd in , was in , xscid in , apds in , cd ld in , and other solid tumor in patients. stem cell sources were autologous pb/bm in , allogenic bm in ( related and unrelated) and allogenic cb in patients. in this study, we excluded patients who relapsed after sct.results: the duration of cd -positive cell counts < / ml after sct was . ± . months in all patients. the durations were . ± . months in patients with hematologic malignancies, . ± . months in patients with hematologic disorders such as aplastic anemia and pid, . ± . months in patients with solid tumor, . ± . months in patients who received autologous sct, . ± . months in patients who received related bmt/ pbsct, . ± . months in patients who received unrelated bmt, and . ± . months in patients who received cbt. the durations of igg < mg/dl after sct were . ± . months in all patients, . ± . months in patients with hematologic malignancies, . ± . months in patients with hematologic disorders, . ± . months in patients with solid tumor, . ± . months in patients who received autologous sct, . ± . months in patients who received related bmt/pbsct, . ± . months in patients who received unrelated bmt, and . ± . months in patients who received cbt. there was a significantly higher incidence of community-acquired infection from months after sct. there were significant differences in the incidence of community-acquired infections between patients with cd -positive cell counts of < /ml and background: allogeneic hematopoietic cell transplantation (allo-hct) has the potential to cure subgroups of patients with multiple myeloma (mm) but its role is controversial due to high transplant-related mortality. while autologous hct is well established as consolidation after induction therapy using novel agents, allo-hct is still considered experimental due to excessive early toxicity.methods: we retrospectively studied mm patients (pts) ; ( , %) were males with a median age of years (range: - ), who underwent allo-hsct in our center between and .median time between diagnosis and allo-hct was months (range: - ).results: the international staging system (iss) was iss i ( , %), iss ii ( , %), and iss iii ( , %). twentyeight ( , %) pts received cells from unrelated donor and pts from identical siblings. stem cell source were: peripheral blood (n= ) and bone marrow (n= ). grouprisk distribution using hct-ci and pam index can be seen in figure . response was evaluated at day + : cr ( , %), vgpr ( , %), pr ( , %) and sd ( , %); response was not evaluated in pts. regarding hospitalization, ( , %) pts and ( , %) pts needed readmission in the first days and days post-allohct, respectively, and median days of hospitalization was days (range: - ). reasons for first re-admission were: infection ( , %), gvhd ( , %) and renal insufficiency ( , %). twenty-seven ( %) died and death causes were: infection ( , %), progression ( , %), secondary tumor ( , %) and the remainder, gvhd and intracranial hemorrhage. in addition, pts ( , %) suffered cytomegalovirus (cmv) reactivation.median overall survival (os) was months (range: - ). univariate analysis showed that re-admission in first days (hr , ; p= , ) , re-admission in first days (hr , ; p= , ) , cmv reactivation (hr , ; p= , ), iss (iss-i vs iss-ii and iii; hr , ; p= , ), days of hospitalization in the first days and response at + day (cr plus vgpr vs the remainder; hr , ; p= , ) were predictor factors for os. other factors like karyotype, response before allo-hct, hla-mismatch or baseline cmv serostatus, among others, do not impact on os.median os in pts with low hct-ci were months (range: - ) vs months in intermediate-high hct-ci background: allo-sct is a potentially curative therapy for patients with multiple myeloma as it provides a graftversus-myeloma effect and a myeloma-free graft. due to increased nrm and unclear os benefit the recent guidelines suggested allo-sct to be used in context of clinical trials focusing on the high-risk patients and those who relapsed early after autograft. reduced-intensity conditioning regimens may improve rate of nrm; however, optimal conditioning regimen is still to be determined. here we studied conditioning regimen with alkylating agents consisting of thiotepa (tt), busulfan (bu) and gvhd prophylaxis with post-transplant cyclophosphamide (post-cy) in high risk myeloma patients relapsing after autograft.methods: a total of patients (m, n= ) with median age of years (range - ) underwent an allo-sct (mud, n= ; mrd, n= ; mmud, n= , haploidentical, n= ) during a period from to in university of hamburg. the majority of patients had advanced disease (stage iiiab, %) and high-risk cytogenetics ( %). the median response durartion after autograft was . years (range . - . ). the conditioning included tt (cum. dosage mg/kg), bu (median cum. dosage . mg/kg i.v. or . mg/kg in elderly patients) and post-cy (cum. dosage mg/kg, day + and + ). eight patients (all of them received allografts from mmud or haploidentical donors) received additionally fludarabine (flu, cum. dosage of background: the overall incidence of active cmv infection in patients with multiple myeloma (mm) receiving background: several scoring systems have been developed to estimate outcomes in mds patients who undergo allohct. however, none of them have been specifically validated in t cell depleted grafts. the aim of this study is to investigate the prognostic ability of a recently published scoring system (sfgm-tc) in a cohort of patients with mds who underwent tcd transplants.methods: patients underwent a first tcd allohct for mds from to . the sfgm-tc score (caulier et al. curr res transl med. ) is performed at day and ranges from - , discriminating low ( ), intermediate ( ) ( ) ( ) , and high risk ( - ). additional analyses were performed at day and day . a landmark analysis was done at each time point for the day , , and analyses, respectively. background: hematopoietic stem cell transplantation (hsct) is the only curative procedure for the treatment of myelodysplastic syndrome (mds), but among several limiting factors for its accomplishment, such as the patient´s performance status, is a very relevant issue, i.e., the availability of a compatible hla donor and, when available, very often the donor´s age and comorbidities also constitute factors that hinder this medical conduct. considering this scenario, the possibility of a haploidentical transplantation (ht) has emerged as an option. in latin america, ht has been included as a treatment option since . since then, these patients have been included in the latin american registry of transplantation in myelodysplastic syndrome, making it possible to analyze the viability and results of these transplants.methods: from october to october , seventeen ( ) patients were transplanted with a haploidentical donor and included in the latin american registry. none of these patients had an identical hla ( / match) related or unrelated donor. data were obtained from the latin american registry of hsct in mds. the statistical analyses were performed using the software spss, version . and graphpad prism version . , with significance being set at p < . .results: table shows the patients and their characteristics. all donors were haploidentical. there was a predominance of reduced intensity conditioning, which was performed in patients ( . %), whereas the others received the myeloablative conditioning. cell source was peripheral blood in ( . %) and bone marrow in ( . %) of the patients. graft-versus-host disease (gvhd) prophylaxis in post-hsct was carried out with cyclophosphamide mg / kg on d+ and d+ , cyclosporin from d and mycophenolate from d+ . complete hematologic recovery was achieved in ( . %) patients. the incidence of grade ii-iv acute gvhd was . %, whereas chronic gvhd was . %. one death occurred due to graft failure and none of the patients showed autologous recovery. three other patients died. one on d+ due to a fungal infection, the second on d+ due to sinusoidal obstruction syndrome and a third on d+ due to pneumonia caused by pseudomonas. regarding overall complications, there was a predominance of mucositis ( %), overall infections ( . %) and reactivated cmv in . % of cases. of the total number of living patients, ( %) achieved complete remission and ( . %) showed disease relapse. the mean follow-up was months (ranging from to months). the lowest probability of disease-free survival at years was % ( % ci: . - . ).background: relapse is the most important cause of failure after allogeneic hematopoietic stem cell transplantation (hsct) for flt -itd-positive acute myeloid leukemia (aml). treatment with flt tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy.methods: a year-old woman diagnosed with aml secondary to myelodisplastic syndrome (mds) with npm mutation and internal tandem duplications of the flt gene (flt -itd) in october . after achieving complete remission (cr) with conventional chemotherapy, she received a hla sibling allogenic-hsct in february , with bucy. four months later, aml relapsed only at medullary level (flt ratio: , %), treated with chemotherapy and donor lymphocytes infusions (dli). she achieved nd cr and developed limited chronic graft-versushost disease (cgvhd). nine months later (april ), she suffered the first extramedullary relapse only, breast and skin. disappearance of the lesions at all levels was achieved with chemo and radiotherapy. she always had full hematologic donor chimerism.in december , she referred atypical precordial pain irradiated to the back. cardiac mri was performed and several masses were visualized in the pericardial sac, up to cm in diameter. bm remained in cr with full donor chimerism.pericardial fluid showed massive infiltration by leukemic-flt positive cells (ratio: , %). she was not considered background: ta-tma is a severe complication that can reduce survival after hsct. risk factors have been variably reported in adults although data on children remains scarce. we aimed to identify a risk profile for development of ta-tma in children undergoing hsct.methods: we retrospectively reviewed clinical charts of children who underwent hscts between - : at great ormond street hospital (gosh) and the great north children's hospital (gnch). ta-tma was defined according to revised criteria (jodele et al. ) . risk factors were categorized into patient derived [age, gender, active co-morbidity at d of hsct (uncontrolled viral/ bacterial or fungal infection, pulmonary, cardiovascular instability, steroid therapy > . mg/kg beyond d , bcgiosis or autoimmune disease), transplant related factors (conditioning intensity, stem cell source, hla-matching, use of ciclosporin a (csa) or tacrolimus (tac), cd + dose, ex-vivo t cell depletion, use of defibrotide) and post-hsct factors (agvhd, post-hsct viral reactivation, venoocclusive disease (vod) and occurrence of posterior reversible leukoencephalopathy (pres).results: at a median of months post-hsct, ta-tma occurred among / transplants ( . %). there was no reported centre variation ( . % vs . % in gosh vs gnch; p= . ). gender, underlying disease -primary immune deficiency (pid) (n= ) vs haematological disease (n= ), use of myeloablative (n= ) vs reducedor minimal intensity conditioning (n= ), use of serotherapy or mega doses of cd ≥ x * /kg did not influence the development of ta-tma. donor type: msd/ mfd(n= ) vs mud (n= ) vs mmud/haplo-hsct background: we evaluated the outcome of haploidentical hct (hhct) using ex vivo αβ t cell-depleted (tcd) grafts after reduced-intensity conditioning (ric) containing low-dose tbi (ld-tbi) in pediatric patients with acute leukemia (al) in complete remission (cr).methods: between may and october , patients with acute leukemia ( all and aml) in cr received haploidentical hematopoietic cell transplantation (hhct) using tcrαβ-depleted graft at asan medical center children's hospital. eighteen patients received hhct between and (earlier time period) and the remaining between and (recent study period). the conditioning regimens, the dose of αβ+ t cells and gvhd prophylaxis are summarized in table. results: all patients achieved a sustained neutrophil engraftment at a median of days (range, - ) . of patients, patients ( all & aml) relapsed at a median of months (range, - ) after transplant. of the patients, patients died of disease. one patient died of disseminated tuberculosis at months after transplant, leading to the trm of % at year. as of december , of the patients survive free of disease at a median follow-up of months (range, . at a median followup of months (range, - ), efs and os at years for all patients were % and %, respectively. outcome of hhct in the recent study cohort was significantly better than that in the earlier study period (efs of % vs efs of %, p= . ). among the patients with all, the efs of patients, who received hhct in early time period after conditioning with tbi of cgy, was significantly worse than that of patients, who received in recent study period after a higher dose of tbi at cgy ( % vs %, p< . ). the efss of aml were similar between the two study groups ( % for earlier cohort vs % for recent study, p> . ).conclusions: in pediatric patients with acute leukemia in cr, our current haploidentical hct using ex vivo αβ tcd graft after ric containing ld-tbi without gvhd prophylaxis is feasible approach with a low trm. the background: anti-hla antibodies (ahab) have been recently recognized as an important risk factor for graft failure (gf), especially in hla-haploidentical stem cell transplantation (haplo-hsct). although, recently, ebmt consensus guidelines have been published [ciurea, bone marrow transplant ] , experience in pediatric t-cell depleted (tcd, another well-known risk factor for gf) haplo-hsct is lacking. in the present study, we report our experience on the use of a desensitization approach based on ebmt guidelines.methods: between june and august , all patients affected by non-malignant diseases and scheduled for a transplant from an hla-haploidentical donor after negative depletion of αβ t and b cells as previously described [li pira, biol blood marrow transplant. ], were tested for ahab with luminex® solid-phase immunoassay (one lambda, thermo fisher scientific) month before the hsct. all patients with a mfi higher than , which is considered a cutoff predicting for gf, were treated with a desensitization protocol based on the use of anti-cd rituximab ( mg/m before and immediately after the end of plasma-exchange cycle) and plasma-exchange (pe) ± infusion of irradiated buffy coat (bc) (if after pe ahab mfi was still > mfi). this latter was obtained by the non-target fraction of the αβ t-cell/b-cell-depletion procedure and consisted of * irradiated nucleated cells/kg of the recipient; this was infused - hours before the infusion of the tcd graft. pe was performed with miltenyi life tm apheresis unit (miltenyi, bergish-gladback) .results: in the study period, patients received αβ tand b-cell depleted haplo-hsct. eighteen ( %) resulted positive for ahab (mfi> ); ( %) of them had an mfi > for either anti-class i or ii ahab. these patients (see table i background: osteonecrosis (on) is a debilitating complication in survivors of allogeneic hematopoietic cell transplantation (hct). limited data is available on its course post-transplantation in children. the purpose of our study was to identify recipients of hct in whom pre-and post-magnetic resonance imaging (mri) is indicated.methods: the retrospective cohort consisted of patients who underwent first allogeneic hct from - , and prospectively underwent a total of pre-and post-transplant mri studies of the hips and knees done annually for years regardless of symptoms. surviving patients were followed for a median time of . (range . - . ) years. cases of on were compared to controls matched for age, sex, transplant type, and follow up in a : ratio for the following variables: ethnicity, underlying disease, on pre-hct, conditioning regimen, graft source, bone mineral density z-scores, body mass index, presence or absence of graft-versus-host disease, steroid use and dosage, and survival status.results: thirty ( . %) patients had mri findings confirming on post-hct. all patients with on except one were more than years of age. twenty ( %) patients were male. on pre-hct (p < . ) was the only factor associated with presence of on post-hct. epiphyseal on was seen in ( %) patients pre-hct, and ( %) post-hct. eighteen ( %) patients had involvement of more than % of articular surface, and were more likely to undergo surgery (p = . ).conclusions: the incidence of on in this large pediatric cohort was %. the only risk factor for on post-hct was pre-existing on. mri evaluation for on pre-hct is indicated in all patients. mri evaluation for on post-hct is only indicated for patients with on pre-hct and symptomatic patients. this will entail cost savings of usd , per surviving allogeneic hct patients per year. patients with more than % involvement of the articular surface need close follow up.clinical trial registry: none disclosure: none impact of rabbit anti thymocyte globulin exposure on immune reconstitution and outcome after stem cell transplantation in children background: rabbit anti thymocyte globulin (ratg) has been frequently used for many years as gvhd prophylaxis in pediatric stem cell transplantation. precise dosing regimens are crucial but remain challenging due to several pharmacological characteristics in children.methods: ratg levels were measured in pediatric patients undergoing allogeneic stem cell transplantation after obtaining approval by the local irb and informed consent by legal guardians. pediatric patients who received either thymoglobuline™ (n= ) or grafalon™ (atg-f) (n= ) as part of their conditioning regimen background: obesity among children is a growing health problem. malnutrition or being over-weight can be of prognostic impact among children who need hsct.scientific literature shows a lot of controversy in terms of effect of bmi at the time of infusion on the outcome of hsct.methods: we reviewed data of patients who underwent hsct at king faisal specialist hospital & research centre between - to correlate bmi with the outcome and complications of hsct. transplant naïve recipients with age at infusion between - years who received hsct from matched related donor or autologous hsct, were included in the dataset for analysis. a total of patients' profiles were reviewed of whom . % were boys. median age at transplant was . years. primary indication of disease was malignancy in . % followed by hemoglobinopathies . %, bone marrow failures . % and immune disorders . %. solid tumors accounted for . % among malignant disorders. myeloablative conditioning was used in . % transplants with . % regimens containing total body irradiation. majority of the patients . % underwent allogeneic transplantation using bm as the source in . % and pbsc in the remaining . % cases. donor was hla-identical sibling in %, parents in . % and others in the remaining . % patients. median tnc dose was . x ^ and cd was . x ^ per kg of the body weight at the time of infusion. age and gender specific bmi percentiles were obtained and classified according to the definition of centers for disease control and prevention (cdc); . % ( ) recipients were categorized as under-weight, . % ( ) normal, . % ( ) over-weight and remaining % ( ) as obese.results: based on chimeric studies at day- , our engraftment rate was . % ( ) out of evaluable cases. median time to neutrophil recovery was -days from infusion and -days for platelets. no statistically significant difference was found for engraftment rate on d- as it was % ( ) among . % ( ) in normal, % ( ) in under-weight and . % ( ) in the obese (p-value: . ). median time to neutrophil recovery from the infusion date was -days in over-weight patients and in the remaining three groups (p-value: . ). acute graft vs. host disease (agvhd) of any grade at day- was recorded in . % ( ). any-grade agvhd was more common in over-weight % ( ), followed by obese with . % ( ), % ( ) in under-weight and . % ( ) in normal bmi-category (p-value: . ). chronic gvhd was more frequent in over-weight ( . %, ), compared to ) background: hematopoietic stem cell transplantation (hsct) is the standard treatment for children with severe aplastic anemia (saa) who have hla-identical related donor. there is no standard conditioning regimen for children with saa secondary to non-fanconi anemia (fa) constitutional bone marrow failure syndromes such as telomeropathies. we report the outcome of a consistent reduced intensity conditioning regimen in patients with idiopathic saa or inherited bone marrow failure syndromes other than fa who underwent hla matched related hsct methods: children with saa underwent hsct using the following conditioning regimen: fludarabine mg/m , cyclophosphamide mg/m , and atg (thymoglobulin) ( mg/kg) . gvhd prophylaxis included cyclosporin and mycophenolate mofetil. donors were all matched related and bone marrow was the stem cell source. all patients had normal chromosomal fragility testresults: a total of children with saa underwent hsct, females and males. average age was . (range . - . years). all nine patients who were tested for telomere length had short telomeres. pathogenic or likely pathogenic mutations were reported in patients ( ercc l , ankrd , tinf , lztfl ). all donors had normal physical examination, normal cbc, and negative genetic testing if patient mutation is known. all patients engrafted successfully, median time to neutrophil engraftment was (range, - days) and platelet engraftment (range, - days). median infused nucleated cell dose was . (range, . - . x /kg) and cd cell dose was . (range, . - . x /kg). none of our patients had acute gvhd and one patient had mild classic chronic gvhd of the skin that was controlled with topical therapy for a short period. three patients had secondary graft failure in the first-year post transplant. first patient had pancytopenia with loss of donor chimerism and underwent successful second transplant using fludarabine, atg, and melphalan. the second patient had a nonfunctioning graft despite full donor chimerism suggesting that the related donor might be affected and had silent phenotype. the third patient had homozygous ercc l mutation and developed progressive cytopenia with myelodysplastic features few months post-transplant and subsequently underwent myeloablative matched unrelated transplant using busulfan, fludarabine, thiotepa and atg. however, the patient progressed to have acute myeloid leukemia six months post hsct. fifteen patients ( %) have normal cbc and stable donor chimerism. median follow-up duration of days (range - days). one patient with lztfl mutation developed chronic renal impairment five years post hsct.conclusions: hsct using lower dose cyclophosphamide ( mg/kg) as part of fludarabine based regimen was safe and effective in saa patients with shorter telomeres and described genetic abnormalities. optimal conditioning regimen in ercc l -associated bone marrow failure needs to be defined. larger study is needed to confirm our results.clinical trial registry: not applicable disclosure: nothing to declare late effects in patients with hemophagocytic lymphohistiocytosis treated with hematopoietic stem cell transplantation: a review of the literature background:hemophagocytic lymphohistiocytosis (hlh) is an inherited or acquired disorder of immunedysregulation. early diagnosis and immunosuppressive treatment can prevent significant organ-failure. the inherited forms, and some acquired cases can only be cured by hematopoietic stem cell transplantation (hsct). with modern transplant practices, a significant number of patients survive. the purpose of this literature review was to collect data on the frequency and type of late effects in hlh patients surviving after hsct and to examine the association with pre-existing hlh conditions (eg. involvement of the central nervous system (cns) before transplant) and with the pre-transplant conditioning regimens.methods: the medline, embase, web of science and pubmed databases were searched, by two librarians at the karolinska institutet, between may and september according to the preferred reporting items for systematic review and meta-analysis (prisma) statement. the search terms included "hlh", "fhl", "mas", multiple terms for "hsct" and late-effect conditions. inclusion criteria were publications in english that included children between january and may . authors of this review screened all the abstracts of studies against the inclusion criteria.results: only nine papers published between and , with information on late effects in hlh patients who had undergone hsct, were identified. three reports include only small numbers of hlh patients. the remaining papers contain data on long-term survivors with a median follow-up of . years. five papers address neurological sequelae with a reported incidence from - %. the highest incidence was found after a thorough neurological assessment of hlh patients compared to matched sibling controls. however, the association with cns disease before transplant and age at transplant was not clear. patients with ebvassociated hlh seem to have fewer long-term neurological problems. non-neurological late effects are described in papers only, with endocrinological problems, namely short stature, being the most frequent. one paper specifically analyzed poor growth, thyroid dysfunction and vitamin d deficiency in a cohort of patients with non-malignant disorders including hlh who had undergone hsct after a reduced intensity conditioning regimen and found significant abnormalities in all groups.conclusions: data on late effects in hlh patients is scarce and is mostly based on the retrospective evaluation of small national cohorts. the available information indicates that a significant number of patients suffers from problems which affect their daily life, but lack of information does not allow to analyze the association between pre-transplant conditions and long-term sequelae. therefore, a retrospective comprehensive analysis of patients registered in the ebmt and cibmtr registries is currently performed. it will be crucial to better define the frequency and type of late effects in a large cohort. this knowledge will aid counselling prior to hsct, provide guidance for long-term monitoring of these patients, and potentially identify specific risk factors for late effects in this rare patient population.disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type ii (morbus hunter)bernd hartz , nicole muschol , matthias bleeke , johanna schrum , ingo müller background: the transplantation of hematopoietic stem cells (hsct) is one of the leading methods of treatment in patients with blood system diseases, primary immunodeficiency syndromes and genetic diseases. at the same time, the quality of life in patients in the long-term after hsct significantly differs from the quality of life of healthy people of the same age. deformations in psychosexual development including problems in the gender identity formation cause social isolation of adolescents, which makes their sexual selfrealization impossible and significantly reduces the quality of their life. the purpose of our study was an assessment of the level of gender identity formation of adolescents and psychosexual development correlation to the normal adolescents of the same age.methods: in a prospective single-center study in , on the base of the department of rehabilitation medicine raisa gorbacheva memorial institute of children´s oncology, hematology and transplantation, we conducted a study of families. the respondents were: ) parents / guardians of patients accompanying them in the process of examination; ) adolescents who underwent hsct treatment and undergo planned examinations at the clinic in the posttransplant period (after d + ), (n = , of which girls and boys, age - years, from the date of hsct - years).the following methods were used to assess gender identity: specially developed questionnaires for teenagers and parents; questionnaire by sandra l. bem (sandra l. bem, ) ; projective techniques "the human picture", "the non-existent animal"; max lüscher´s color choices test.results: the traditional type of gender identity, which characterizes high masculinity among male respondents and high female gender indicators in % of cases, was not revealed. both among girls and among boys, the androgynous type prevails with a tendency toward femininity.on average, adolescents see themselves as a bit more courageous than their mothers, with rare exceptions, regardless of gender. this confirms the thesis that we received in a previous study that parents tend to see and encourage complacency of adolescents of both sexes, passivity instead of leadership, dedication and independence. all % of adolescents who participated in the test demonstrate a shift in the theme of aggression, % have some signs of preventing sexual self-determination, abandoning their body, gender, and age. % of patients do not communicate with their peers. in % of them, negative emotions prevail over positive ones. one third of the test participants demonstrate strong support for rest and minimizing their efforts.conclusions: the characteristics of family upbringing of adolescents who have undergone hsct often contribute significantly to limiting their social experience and lead to specific deformities of individuality, including in the sphere of gender identity. we consider advisable to introduce thematic group counseling of parents within the framework of the "patient's school" in psychological treatment support in the clinic. early diagnosis of the personal aspects of the psychosexual development of adolescents after hsct allows for timely identification of individual problems in this area and identification of general trends in the long term after hsct.disclosure: all authors -nothing to disclose. abnormalities in the morphology of the umbilical cord blood obtained at delivery from children who received a diagnosis of cerebral palsy maciej boruczkowski , izabela zdolińska-malinowska , maciej rojek , dariusz boruczkowski background: embryonal brain tumors are the most common malignancies in infants less than months of age. histologically characterized as undifferentiated small round cell tumors, all are similarly aggressive, have a tendency to disseminate throughout central nervous system and very poor prognosis. we tried to assess the effectiveness of tandem highdose chemotherapy (hdct) with autologous hematopoietic stem-cell transplantation (auto-hsct) in this patient group. methods: from to , infants under months with different primary embryonal brain tumors such as medulloblastoma (n= ), different pnet nos (n= ), pineoblastoma (n= ), atypical teratoid rhabdoid tumor (n= ), etmr (n= ) after surgical resection and induction chemotherapy were planned to receive tandem hdct with auto-hsct. nine patients were conducted only single transplantation because of the development of lifethreatening complications after the first hdct (n= ) or the emergence of early disease progression (n= ). at the moment of hdct patients were in complete remission (cr), patients were in partial remission (pr) and patient had stable disease (sd). the conditioning regimen for tandem auto-hsct were: the first hdct was carboplatin and etoposide, the second was thiotepa and cyclophosphamide, both with intraventricular methotrexate.results: the median follow-up is months (range, - ). the median time to engraftment after the first auto-hsct was background: a series of findings suggest that optimizing natural killer (nk) cell reactivity could further improve outcome after allogeneic hematopoietic cell transplantation (allohct). this could be achieved by killer cell immunoglobulin-like receptor (kir) genotype informed donor selection. an enhanced receptor-ligand model which used kir ds and kir dl donor genotype information to augment nk cell activation and minimize inhibition demonstrated improved survival in one large aml study (boudreau et al, jco ) . likewise, a second model built on the classification of centromeric and telomeric kir haplotype motifs, also predicted mortality after allohct for aml (cooley et al, blood ) . this joint ebmt and cibmtr study aimed at validating the two approaches in an independent cohort of patients with mds or secondary aml.methods: donor samples were retrieved from the collaborative biobank (dresden, germany) and mapped to patient outcome data extracted from the ebmt and cibmtr. genotyping of all kir genes by sequencing exons , , , , , and was performed by high resolution amplicon-based next generation sequencing. the impact of the classifiers on time-to-event outcomes was tested in cause-specific cox regression models adjusted for patient age, a modified disease risk index, performance status, donor age, hla-match, sex match, cmv match, conditioning intensity, type of t-cell depletion and graft type.results: clinical data from patients and corresponding donor genotype information were analyzed. the median age at allohct was . years (range, . to . years). the indication for allohct was mds for % and saml for % of patients. disease risk was low/intermediate and high/very high in % and %, respectively. donors were / matched for % of patients. myeloablative, reducedintensity and non-myeloablative conditioning regimens were used in %, %, and % of patients, respectively. peripheral blood stem cells were the predominant graft source ( % of patients). atg was administered in % and alemtuzumab in % of patients. during follow-up after allohct patients died. in univariable and multivariable analyses of the whole cohort, overall survival and the cumulative incidence of relapse of patients with kiradvantageous versus disadvantageous donors were not statistically significantly different. we could not replicate the pattern of outcomes predicted by the kir dl / conclusions: relapse incidence and overall survival after unrelated donor allohct could not be predicted using the kir dl /kir ds -receptor-ligand model and centromeric/telomeric kir-motif model in this large cohort of patients with mds or secondary aml. this points at the possibility of interactions between nk-cell mediated alloreactivity and disease type or procedural variations of allohct. available information on kir-genes, which have been sequenced but not yet analysed, will be investigated in exploratory analyses.disclosure: the authors have nothing to disclose in the absence of an alternative donor, it is recommended that patients undergo desensitization therapy, especially with high dsa levels (> , mfi). the aim of this study is to analyze the impact of dsa on risk of gf and poor graft function (pgf), and on major outcomes in a consecutive cohort of patients who were systematically screened for dsa before haplo-sct. methods: consecutive patients were candidates for unmanipulated haplo-sct with post-transplant cyclophosphamide (pt-cy) at our center from january to january and were analyzed for the presence of hla antibodies.results: patients underwent haplo-sct. hla antibodies were detected in patients, of them were dsa, while were non-dsa (ndsa). patients out of with dsa were transplanted using the same donor; underwent a desensitization program before transplant.background: a recent study from ebmt comparing matched sibling (msd) versus haploidentical donors transplantations, showed better outcome with msd in adult patients with intermediate risk aml in first remission (cr ). however, a female donor to a male recipient transplant combination is a poor prognostic factor and this study did not address the question whether in this situation, a haploidentical donor transplant might do better. the present study compared the outcomes of allografted male patients according to whether they received stem cells from a female msd or a haploidentical donor, in the intermediate and high risk cytogenetics groups (mrc classification).methods: the study included male patients with cytogenetics transplanted between january and june and reported to ebmt. received stem cells from a msd female donor and from a haploidentical donor ( male and female). the follow up was months ( - ). we studied separately intermediate and high risk patients. multivariate analysis was adjusted on factors differing significantly between the groups.results: -intermediate risk group: male patients received a female msd and a haploidentical transplant. the distribution of group characteristics was even except that in the haploidentical transplant group, donors were younger ( y versus ; p< . ), marrow was more frequently used ( % versus %, p< . ) and the interval from diagnosis to transplant was longer ( . versus . months, p< . ). by univariate analysis at two years post transplant, cumulative incidence (ci) of nrm post haplo was higher ( % versus %, p= . ) and ci of extensive chronic gvh lower ( % versus %; p= . ). lfs post msd and post haplo were % and % (p= . ), os % and % (ns), grfs ( % and %). by multivariate analyses the only significant poor risk factors were the haplo-identical transplant for nrm (hr: . ( . - - )) and the patient age for os (hr: . ( . - . ; p= . ). haploidentical transplantation resulted in less chronic gvhd (hr: . ( . - . ); p < - ), but a lower lfs (hr: . ( . - . ); p= . ). -high risk group: male patients received a female msd and a haploidentical transplant. in the haploidentical group, donors were younger ( y versus ; p< . ), marrow was more frequently used ( % versus %, p< . ) and the interval from diagnosis to transplant was longer ( . versus . months, p= . ). by multivariate analysis, haploidentical transplants were associated with a lower relapse incidence (hr: , ( . - . ; p = . ),a better lfs (hr: , ( . - . ; p = . ),os (hr: , ( . - . ; p = . ), and grfs (hr: , ( . - . ; p = . )(see figure) . the only other significant prognostic factor was patient age.conclusions: this study shows that in a male patient with intermediate risk aml, a genoidentical sister donor remains associated with a better lfs. in contrast, in a male patient with high risk aml in cr , a haploidentical donor may be a better choice than an hla genoidentical sister.disclosure: nothing to declare p abstract already published. hematopoietic transplant for older acute leukemia patients: improved survival with offspring donor in comparison with older-aged matched siblingsyu wang , sheng-ye lu , qi-fa liu , de-pei wu , xiao-jun huang background: post-transplant relapse remains the major cause of death of treatment failure. therapeutic options for relapse after first allogeneic stem cell transplant ( st hsct) include chemotherapy followed by donor lymphocyte infusion or second allo-hsct ( nd hsct) from the original donor or change to another donor. however, there is unclear outcome for different treatment approach. in this retrospective cohort study, we aim to compare the clinical outcome after different treatment strategy for relapse after first allo-hsct.methods: between jan and oct, consecutive patients receiving st hsct registered to the bmt database in national taiwan university hospital were analyzed. among them, cases had relapsed after first allo-hsct. one hundred and three patients who received no treatment after relapse or with incomplete data were excluded. their transplant data was collected following the ebmt registry data collection forms and manuals. overall survival rate and progression free survival rate were performed by the kaplan-meier method. univariate and multivariate analysis were performed using cox proportional hazard regression model.results: of the patients who experienced relapse after st hsct, total patients ( %) received chemotherapy followed by dli or nd hsct from the same donor (no change group), patient ( %) received chemotherapy followed by nd hsct from different donors (change group), and ( %) had conventional chemotherapy alone. the patients in "change group" were younger (median age vs , p = . ), and had more patients achieving complete remission (cr) prior to nd hsct ( % vs %, p = < . ) than patients in "no change group". after the nd hsct, the cr was % for "no change group" and % for "change group". the progression-free survival at -year and -year were . % and . % (fig a, p = . %), respectively, for "no change group" and . % and . %, respectively, for "change group". while the overall survival (os) at -year and -year were . % and % (fig b, p = . %), respectively, for "no change group" and . % and %, respectively, for "change group". those who achieved cr prior to nd hsct had a trend of better os than those without cr ( . % vs . % at -year; . % vs . % at year, p =. )( fig c) . there were cases survived for more than years in "change donor group" and cases survived more than years in "no change group". only one had developed relapse after nd hsct but achieved subsequent remission again.conclusions: our study shows that change donor had similar poor outcome comparing to those using the same donor after the st hsct. patients who achieved cr before nd hsct had a trend of better os than those without remission and the long-term survivors were only those who achieved cr prior to nd hsct. novel therapy for cr induction would be warrant for this poor prognostic population.disclosure: nothing to declare functional relevance of fetal microchimerism in nk cell cytotoxicity against leukemic blasts in children: a role for hla-c and kir dl /s ?background: allogeneic stem cell transplantation (allo-sct) remains the most effective curative intent therapy for patients with unfavorable risk acute leukemia. various donor options are available for the patient who lacks an hla-matched sibling donor, such as unrelated donors (urd) and hla-mismatched family (haploidentical) donors. in order to discover the exact role of transplantation type, there are many retrospective analysis, which compared these donor sources, have been reported. recent studies showed some promising results of haploidentical donor transplantation (hidt) using post-transplant cyclophosphamide in comparison with unrelated donor. the goal of this study was to compare the outcome of allo-sct from haploidentical versus matched unrelated (mud / ) or mismatched unrelated donor at a single hla-locus (mmud / ) for patients with acute leukemia in remission.methods: ninety-six adult ( - years) patients with acute leukemia in first or second remission who underwent allogeneic transplantation with a minimum days follow-up at florence nightingale hospital hematopoietic stem cell transplantation center between and were included in this study. patient characteristics and medical records of all patients were reviewed retrospectively. thirty-eight patients who received haploidentical donor transplantation were compared with patients receiving a mud / and receiving a mmud / . patients who completed minimum days post-transplantation follow-up were identified as eligible for survival analysis.results: the characteristics of the patients and transplant donors in this study are summarized in table . median age of patients was . ± years. proportion of male patients was . %, . % and . % for mud / , mmud / and hidt groups, respectively, which is significantly different (p= . ). the other baseline factors were similar, including patient age, donor age, recipient cytomegalovirus (cmv) status, donor cmv status, graft versus host disease incidence, median neutrophil and platelet engraftment times and disease status at post-transplant th day. no significant difference was identified in survival analysis among the mud / , mmud / and hidt groups, even if they were classified according to primary disease (aml vs all) and pre-transplant disease status (cr vs cr ). also, donor cmv status (cmv igg positivity or negativity) was not an important factor on survival analysis when compared between these three groups (p= . ).conclusions: in our study population, clinical outcomes of hidt patients were inferior to mud / and mmud / groups. when choosing an alternative donor for patients without an available hla-matched sibling, urgency of transplantation and host/donor features should be considered. we believe that hidt might be a feasible alternative choice in this subset of patients.disclosure: nothing to declare p g-csf primed bone marrow in hla-haploidentical transplantation using post-transplantation cyclophosphamide (ptcy) could promote tolerance and further reduce risk of gvhd nadira durakovic , , zinaida perić , , lana desnica , ranka serventi-seiwerth , mirta mikulić , brian melamed , alen ostojić , dražen pulanić , , pavle rončević , zorana grubić , radovan vrhovac , implementation, development, and coordination of unique quality management systems with evaluation audits, intrahospital and international accreditation and certification processes. quality of health care is a major focus for providers, patients, and accreditors; so, in this study, we aim to compare the quality of bm harvested at ipo-collection centre (icc) with the quality of bm received from external collection centres (ecc) during these last years.methods: this retrospective evaluation included the number of total nucleated cells (tnc) requested by the transplant centre, the tnc collected, and the results of bm microbiological analysis performed; donor age, weight and infectious disease markers (idm); patient demographics and diagnosis. bm collection technique in use at icc was validated according to jacie standards.we consider successful a collection (sc) with tnc between and % of the requested value, unsuccessful (uc) if lower than % and outstanding (oc) if over %.results: a total of bm was collected, for allogenic ( unrelated) and seven for autologous transplant; unrelated bm were received from ecc (nine from germany, seven from usa and five from portugal). patient main diagnosis were severe aplastic anaemia (n= ), acute myeloblastic leukaemia (n= ), and acute lymphoblastic leukaemia (n= ). donors idm were all negative and nonreactive.mean age (±standard deviation, sd) was (± . ) and (± . ) years for icc and ecc donors, respectively. at icc, we were asked to collect an average (±sd) of . * (± . ) tnc while ecc were asked for . * (± . ). we collected . * (± . ) and received . * (± . ) tnc. correlation between requested and collected tnc was . at icc and . for ecc.we had . % sc and . % oc meaning an accomplishment of . %. we failed to collect required tnc in . %. although % of received bm fulfil tnc requirements, bm processing lowered this value to % due to erythrocyte removal (seven patients with major abo incompatibility) and plasma reduction (two patients with abo minor incompatibility). these steps reduce final tnc available for transplant. weight difference between donor and patient had no significant impact on final tnc collection performance.sixteen bm from icc (seven staphylococcus spp., five propionibacterium acnesand fourcorynebacterium spp.) background: success of peripheral blood stem cell (pbsc) collections depends on patient biological parameters and stable apheresis device performance. peripheral blood cd + cell enumeration is the most reliable predictive factor of apheresis yield however, there are some unexpectedly poor cd + cell harvests despite successful mobilization. the aim of the study was assess total collections cd + yields and factors influencing main apheresis procedure outcomes including collection efficiency (ce).methods: of consecutive donors covering the period - - to - - were analyzed for the following parameters: pre cd count, cd yield per procedure, total cd dose collected per patient, cd collection targets requested by clinical teams. the efficiency of pbsc procedures was determined by calculating the ce and the correlation coefficient between pre cd count and yield per procedure. ce was correlated to preprocedure wbc, platelet count, pre cd count and blood volume processed. all pbsc collections were performed by optia spectra across units in uk.results: of the donors, were autologous and allogeneic. the autologous donors underwent in total procedures. the median cd target dose for these donors was x /kg. ( %) achieved the target dose with procedure and ( %) with procedures. the median pre cd count was /μl. the median cd yield per procedure was . x /kg and the median total cd dose collected per donor was . x /kg. ( . %) of autologous donors collected a total cd dose < x /kg, of those ( . %) had a pre cd count < /μl and ( %) > /μl.the allogeneic donors underwent in total procedures. the median cd target dose for these donors was x / kg. allogeneic donors ( %) achieved the target dose with procedure and ( %) with procedures. the median pre cd count was /μl. the median cd yield per procedure was . x /kg and the median of total cd dose collected per donor was . x /kg. ( . ) % of allogeneic donors collected a total cd dose < x / kg, of those ( . %) had a pre cd count < /μl and ( . %) > /μl. the median ce for autologous donors was % (range - ) and for allogeneic donors was % (range - ). the ce was negatively correlated to wbc (r= - . and - . ) and platelet count (r=- . and - . ) for auto and allogeneic donors respectively, but did not correlate to the pre cd and blood volume processed. the correlation coefficient between pre cd count and cd yield per procedure was r = . for the autologous and r = . for the allogeneic collections.conclusions: the majority of autologous and allogeneic donors achieved the target cd dose with one procedure. . % of autologous and . % allogeneic donors collected a transplantable cd dose of > x /kg. % of autologous and . % of allogeneic donors did not collect a transplantable dose despite a precd count of > /μl indicating suboptimal procedure performance. the ce was variable and was negatively correlated to the preprocedure wbc and platelet count. the ce and correlation coefficient are lower in allogeneic donors compared to autologous donors.disclosure: nothing to declare the outcome of autologous blood stem cell collection and its actual use in real world: the st century experiencekyoungmin lee , jung yong hong , dok hyun yoon , jae-lyun lee , shin kim , kyoung min lee , jung sun park , cheolwon suh background: mobilized peripheral blood stem cells (pbscs) have largely replaced bone marrow as the graft source for allogeneic stem cell transplantation. pbscs mobilization with g-csf is highly effective even on the th day in order to collect enough number of stem cells. a longitudinal, prospective, observational, single-center, cohort study on healthy donors (hds) was designed to identify predictors of cd + cells on the th day. methods: as potential predictors of mobilization, age, sex, body weight, height, blood volume as well as white blood cell count, peripheral blood (pb) mononuclear cells, platelet count, hematocrit, and hemoglobin levels were considered. two different evaluations of cd + cell counts were determined for each donor: baseline (before granulocyte colony-stimulating factor [g-csf] administration) and in pb after g-csf administration on day . a total of consecutive hds with a median age of . years were enrolled.results: the median value of cd + on day was cells/μl (iq - ). basal wbc, plt and basal cd +, are significantly higher for group with cd + on the th day over the median than below. a multivariate quartile regression analysis, adjusted by gender, age, basal cd + and basal plt, shows a, progressively steeper, relationship between baseline cd +, basal plt and cd + on the th day. the basal cd + cut-off for prevision of cd + on the th day was < = cells/μl and >= cells/μl whereas basal platelets count was < = x /l and >= x /l.conclusions: g-csf can be highly effective in hds on the th day in order to collect enough number of stem cells and we have developed a model for predicting the probability to perform pbsc collection after a short course of g-csf.disclosure: nothing to declare p pre-apheresis peripheral blood cd + cell counts highly correlates to actual stem cells collected background: prediction of stem cell yield on the basis of pre-apheresis cd + cell count and the processed blood volume is essential for the planning and executing of the apheresis process.methods: data analyzed included donor weight, complete blood count and cd + count on day of collection, total processed blood volume, cd + cell dose collected in the apheresis product and the number of aphereses performed. using the method described by pierelli et al, predicted cd + yields were calculated: predicted cd + yield x /kg = (benchmark ce x volume of blood to be processed x peripheral cd + count per μl) / (patient's weight in kg x metric conversion factor).results: in we established the method described by pierelli to predict the cd + cell yield. allogenic aphereses were performed in with this approach. mean processed volume was . liters. the mean cd + peripheral count before apheresis was /ul, the mean collected cd +count per kg bodyweight recipient was . . pearson´s correlation coefficient (r) between predicted yield using pre-apheresis cd +count and actually collected cd + cells per kg bodyweight recipient was . . the mean difference between predicted and collected cell dose was + . %.with knowledge of the predicted stem cell count, we were able to adjust apheresis procedure. in case of marginal predicted yield compared to the requested cell dose, we increased the blood volume to be processed. this proceeding led to a significant reduction of second day donations in by % compared to . in only cases we saw more than - % lower cd + doses collected than initially predicted. all donors showed mild iron deficiency with rbc microcytosis, a factor known to affect apheresis procedure.conclusions: pierellis method of calculating the stem cell yield shows a good correlation between pre-apheresis cd + count and actual collected stem cells, making planning and adjusting of the apheresis procedure more feasible and reliable. this proceeding led to significant reduction of second day donations. attention should be paid to iron deficiency anemia, leading to lower than estimated cd + dose.[ background: for more than a decade many transplant centers routinely collect and cryopreserve two or more peripheral blood stem cell (pbsc) grafts for a tandem and/ or salvage autologous blood stem cell transplantation (absct) in patients with hemato-oncological diseases. however, subsequent high-dose chemotherapy (hd-cht) and absct is in many cases not performed for a variety of reasons, specifically in patients with aml, all, mpn and burkitt lymphoma. data about the actual utilization rate of the cryostored stem cell products are lacking.methods: we retrospectively analyzed the collection, storage and disposal practices of pbsc products from a large cohort of patients who were treated at the university hospital heidelberg or at the university medical center mannheim during a -year period. disease entities included acute myeloid leukemia (aml, n= ), acute lymphoblastic leukemia (all, n= ), mpn (n= ; primary myelofibrosis [pmf], n= ; chronic myeloid leukemia [cml], n= ; secondary fibrosis/essential thrombocythemia [et], n= ; not specified, n= ) and burkitt lymphoma (n= ). patients between and were included and followed until .results: an adequate stem cell graft was defined as ≥ . x exp cd + cells /kg body weight. % of the patients were able to collect at least one stem cell graft and the median number of grafts per patient was (range - ). we could demonstrate that only % of all patients who had collected sufficient pbscs for transplant subsequently underwent an absct. among the disease entities the actual use of the stored pbsc grafts varied considerably from % to % (figure ) .conclusions: we could identify striking discrepancies between the collection/storage and actual utilization of background: biosimilars (bio) of granulocyte colony stimulating factors (gcsf) were approved several years ago on the basis of some studies that indicated similar efficacy to the already patented gcsf (neupogen®, neu) both in terms of shortening the neutropenic period after chemotherapy as well as peripheral blood stem cell mobilization in patients with lymphoma and multiple myeloma (mm) treated with autologous stem cell transplantation (auto-hct). however, all these studies are retrospective and there are still concerns about the real efficacy of bio and even more about the real benefit on final costs.methods: we have retrospectively compared the characteristics of the mobilization procedure in both patients with mm and lymphomas, and healthy donors that received either neu or bio (with no chemotherapy) in university hospitals in catalunya from december / to november . bio replaced neu in june in all institutions. primary objectives were the mobilization rate (defined as the percentage of patients that achieved ≥ x /ml cd + cells in peripheral blood on day ) and the use of plerixafor (plex) in each group as pre-emptive strategy. a multivariable analysis of risk factors influencing the use of plex and mobilization failure (defined as collection of < x /kg cd + cells) was also performed.results: we treated patients ( lymphomas and mm) and healthy donors. both groups of patients ( neu and bio) and donors ( neu and bio) were comparable regarding pre-mobilization general characteristics. there was a trend for a lower median cd + peak on day for bio patients ( vs , p value = . ). a total of patients received plex, although of them ( . %) out of strict theoretical indication, cd + cells > x /ml (range . - . ) and were removed for further analysis (n = , in the neu group and in the bio group). median number of cd + cells on day was significantly lower in the group bio who needed plex ( . vs . for neu+plex, p= ), as well as cd + cells finally harvested ( . vs . x /kg, p= . ). mobilization failure rate was higher in bio group ( vs %, p= . ). regarding no plex patients, median number of cd +cells on day was also significantly lower for bio patients ( . vs . , p= . ). risk factors for plex use were age, basal disease (lymphoma) and number of prior mobilization therapies. the use of bio was the only risk factor for mobilization failure patients receiving plex [hr . ( %ci . - . ), p= . ]. with respect to healthy donor mobilization, none of them needed plex but cases from the bio group ( %) needed more than one apheresis procedures ( and , respectively).conclusions: we found a lower efficacy of bio in the setting of stem cell mobilization of patients with only gcsf both in terms of a lower cd + cells peak on day and a lower number of cd + cells in final apheresis product. bio gcsf also seems to be less effective in healthy donors.disclosure: no conflict of interest to be declaredbackground: auto-sct is a common treatment in patients with mm or nhl. the aim of the prospective multicenter goa (graft and outcome in autologous transplantation) study was to investigate the impact of mobilization method used on the cellular composition of collected blood grafts and eventually hematological and immune recovery as well as long-term outcome post-transplant. altogether patients with mm or nhl transplanted between / and / at four university hospitals were included. the long-term goal of the study is to evaluate characteristics of optimal blood grafts in regard to post-transplant recovery and outcome. methods: altogether patients with mm undergoing first auto-sct were compared with patients with nhl. all nhl patients were mobilized with chemotherapy + g-csf, whereas % of mm patients were mobilized with g-csf only (p < . ). mobilization data, graft cellular composition including cd + cell subsets and lymphocyte subsets of the blood grafts, post-transplant hematological recovery and outcome were evaluated. the median followup time was months in mm patients and months in nhl patients.results: mm patients mobilized cd + cells better (median peak blood cd + vs. x /l, p < . ). the median number of aphereses was in both groups (p = . ). altogether % of the nhl patients received plerixafor compared to % in mm patients (p = . ). the median number of cd + cells collected was higher in mm patients ( . vs. . x /kg, p < . ).the median amount of cd + cells (with -aminoactinomycin) in the infused graft was . x /kg in mm group and . x /kg in nhl group (p = . ). the grafts contained more nk cells (median . vs. . x /kg, p = . ) and cd + cells (median . vs. . x /kg, p < . ) in mm patients. neutropenic fever tended to be more common in nhl patients ( % vs. %, p = . ) but mm patients had significantly more bloodstream infections ( % vs. %, p = . ). the median duration of hospitalization was longer in the nhl patients ( d vs. d, p < . ) and the nhl patients had significantly more often icu admissions ( % vs. %, p = . ).post-transplant neutrophil engraftment was faster in the nhl group (median d vs. d, p < . ). the median time to platelet engraftment was days in both groups. platelet count was higher in the mm group from day until year after auto-sct. there were significantly more early deaths (< d from the graft infusion) ( % vs. %, p = . ) and non-relapse deaths ( % vs. %, p = . ) in the nhl group.conclusions: mm and nhl patients differ in terms of cd + cell mobilization, graft cellular composition and post-transplant recovery as well as risk of non-relapse death. thus, the optimal graft may be different in nhl and mm patients.disclosure: the study was supported by vtr fund from north savo hospital district and study grant from sanofi. abstract already published. single-center experience in use of plerixafor for autologous stem cell mobilization: change in practice over years background: plerixafor has been proven to mobilize human periferal blood stem cells (pbscs) alone or acting synergistically with granulocyte-colony stimulating factor (g-csf). it has mainly been used for rescue mobilization after failed regimen of chemotherapy plus g-csf, but lately preemptive use in poor mobilizers has been established as cost-effective. we aim to show ten years of experience and change in practice with plerixafor use in our center.methods: we retrospectively evaluated the outcome of mobilization procedures and leukapheresis collections in our center in the period from january to october . practice from the first years, when plerixafor was mainly used as rescue agent after failed attempt, was compared to period from till present when preemptive use in poor mobilizers (defined as cd + cell counts < x /l blood) was established.results: in the period from to , total of patients underwent collection of autologous pbscs, and patients required repeated mobilization cycles ( , %). g-csf alone was used in patients and patients ( %) recieved combination g-csf with plerixafor. this cohort consisted of males and females with non-hodgkin (nhl) and hodgkin lymphoma (mh); and respectively. we noted unsuccessful mobilizations ( , % in repeated mobilization, , % in total) of which one patient was from plerixafor group ( , %).background: transplantation of hla-haploidentical hematopoietic stem cells (haplo-hsct) is an established procedure for the treatment of several different hematological diseases. one possible strategy to reduce the risk of graft-versus-host disease is represented by the selective depletion of ab t-lymphocytes (coupled with the depletion of cd + b-cells in order to reduce the risk of ptld) using the clinimacs device (miltenyi, bergish-gladback). before depletion, leukapheresis units are washed by lowspeed centrifugation, resulting in a platelet (plt) rich supernatant (prs) as a by-product generally discarded. we studied the possibility of recovering plt from prs of the haplo donor for transfusion to the recipient during the aplasia period occurring after hsct.methods: hsc donors were mobilized with g-csf (plus plerixafor in out of donors) as previously described [locatelli et al, blood ] . leukapheresis units, obtained with a spectra optia device, were washed twice (producing prs bags) at low speed to remove plt before starting the ab t-cell/b-cell depletion. the two prs were leukodepleted by filtration, centrifuged, resuspended and pooled in a total volume ranging from to ml intersol (is-plt) for overnight incubation at °c with agitation ( cycles/min). the is-plt samples were analyzed for the criteria established by the italian transfusion law. is-plt bags were examined for the following parameters: volume > ml, plt after leukodepletion > x , residual leukocytes < x , ph > . at °c at the end of the -day storage period. the sterility was tested using bd bactec culture vials. the evaluation of the residual leukocytes was performed with the bd leucocount kit. the absolute plt counts were determined using hemocytometer sysmex xn .results: prs bags from donors were processed to produce is-plt units. median resuspension volume in intersol was ml (range - ). the absolute mean value of plt counts measured at the end of the storage period was . x (range . - . x ). this value was found below the threshold fixed by italian regulation in cases ( . %). mean value of residual leukocytes was . x (range - x ); the ph value was always > . . sterility was observed in all cases. according to the work of slichter et al. conclusions: we demonstrated that plts recovered from leukapheresis bags can be accepted as a conventional hemocomponent according to the parameters fixed by italian transfusion law and thus can be administered to the haplo-hsct recipients early after transplantation. this strategy carries several advantages. indeed, apart from the obvious advantages in terms of reduced costs, is-plt can be used to desensitize the recipient by absorption of anti-hla class i antibodies, if present in the recipient. moreover, this strategy can avoid the risk of sensitizing the transplanted patients with hla alleles that differ from the donor's ones. finally, the is-plt unit is readily available. a clinical study aimed at testing the use of is-plt units in transplant recipients will be performed to confirm the clinical efficacy of the approach.disclosure: nothing to declare p ultrasound guidance as a powerful tool in increasing background: peripheral blood stem cells are generally the preferred graft source for allogeneic stem cell transplantation for malignant disease. in most centers first apheresis is performed on day after to doses of granulocyte colony stimulating factor (gcsf) up to ug/kg twice daily. the dose of gcsf and the number of apheresis procedures required contribute to symptom, travel and time burden donors are put through during the process. we hypothesized that taking donor-recipient weight differences into consideration may help reduce this burden methods: a total of healthy donors who donated peripheral blood stem cells on day of gcsf mobilization in the period between january and august at the university medical center hamburg-eppendorf were included in this quality control evaluation. the donors were divided into two cohorts. the impact of donorrecipient weight ratio on stem cell harvest was tested in the training cohort ( - ) and validated in the second cohort ( ) ( ) . for the training cohort, donors were grouped according to donor-recipient weight ratio < . vs. . - . vs. > . . for the purpose of this analysis a stem cells dose of x cd +/kg recipient weight was set for successful apheresis.results: in the training cohort including donors, ( %), ( %) and ( %) had a donor-recipient weight ratio of < . , . - . and > . respectively. the target stem cells count of x cd +/kg recipient weight was achieved in of ( %), of ( %) and of ( %) donors with donor:recipient weight ratio < . , . - . and > . respectively. the cut-off for the validation cohort was therefore set at a weight ratio of . .in der validation cohort including donors, ( %) had a weight ratio > . while ( %) had a weight ratio ≤ . . overall in this cohort target cell count of x cd +/kg recipient weight was reached in ( %) cases. this target was reached in of ( %) of donors with weight ratio ≤ . and in of ( %) donors with weight ratio > . , p = . .conclusions: a donor-recipient weight ratio of > . is seen in about % of peripheral blood stem cell donations for allogeneic stem cell transplantation. in these cases apheresis on day after doses of gcsf is reasonable. donors with lower weight ratios should preferentially donate on day after to doses of gcsf.disclosure: all authors declare no conflicts of interest background: the effect of a second mobilization and collection of peripheral blood stem cells (pbsc) on the cell yield is low, as we previously demonstrated. however, donor safety has been poorly addressed with no changes in the clinical practices.methods: second donations of unrelated and related donors performed between and were evaluated (n= ), including pbsc+pbsc (n= ), bone marrow (bm)+pbsc (n= ) and pbsc+bm (n= ). analytical parameters including leukocyte, lymphocyte, hemoglobin and ldh quantification, obtained on the pre-harvest evaluation of first and second donation, were retrospectively analyzed and compared for all donors. the portuguese bone marrow donors registry (cedace) recommends a time between donations no lesser than months. it also states that in very urgent situations like graft failure, donor should be clinical and analytical cleared and its safety ensured.in order to evaluate the impact of time between donations, donor population was divided in groups: < months, - months, > months; to determine the influence of donor age, donors were divided in groups: < and ≥ years.results: among the total of donors, were volunteer donors of cedace and were familiar. fifteen second donations were performed because of recipient graft failure and due to disease progression or relapse. at the time of second collection, median donor age was years (range - ). the median delay between both collections was days ( - ). time between donations did not seem to substantially impact the analytical donor evaluation: leukocytes, lymphocytes, hemoglobin and ldh results are kept within the reference values. however, donors with less than months between donations showed a slight decrease on leukocyte counts ( %) and hemoglobin values ( %), from the first to the second pre-harvest evaluation. donor age showed no significant influence on the analytical evaluation. nevertheless, when considering only the pbsc+pbsc donations, donors with ≥ years showed a small decrease on lymphocyte counts ( %) .conclusions: this study demonstrated that the analytical parameters, chosen based on literature, had no significant changes between first and second donation. however, particular attention should be paid when time between donations is lesser than months or donor age is ≥ years.as we concluded that no significant changes were observed in the group of - months, it is our opinion that the minimum of - months established by the registries can be shortened to months ensuring donor safety. an accurate donor risk assessment with a larger population should be accomplished in order to strengthen this recommendation.disclosure: nothing to declare. gaurav kharya , atish bakane , pratibha dhiman , anil khetrapal , vikrant bhar background: t cell replete haploidentical stem cell transplant (hhsct) is complicated mainly by increased risk of graft failure (gf) and graft versus host disease (gvhd). conventionally gcsf has been used to mobilize hematopoietic stem cells (hsc). in tcr hhsct gcsf mobilized graft with megadose of cd + cells expose the patient to higher doses of alloreactive t cells increasing the risk of gvhd. plerixafor based mobilization gives an advantage of giving high cd cell dose limiting exposure to high alloreactive t cell dose. we share our experience of gcsf + plerixafor based mobilization for tcr hhsct. methods: consecutive patients suffering from scd who underwent hhsct between jan till date along with the respective donors were enrolled in the study (group ). all underwent pre-transplant immune suppression (ptis) cycles at weekly intervals using fludarabine+cyclophosphamide+dexamethasone.the graft was mobilized using gcsf@ mcg/kg/day(d -d )+plerixafor@ . mg/kg(d ) - hours before the pbsch. gvhd prophylaxis included ptcy mg/kg/ day on d and , sirolimus and mmf starting from d . group included historical controls where graft was mobilized using gcsf@ mcg/kg/day(d -d ). various parameters pertaining to mobilization, harvest, engraftment, gf and gvhd were assessed between the two groups.background: poor mobilizers (pm) defined as those with a peripheral blood cd + count ≤ cells/μl on day+ is a significant risk factor for mobilization failure. within these, patients with < cells/μl are considered as very poor mobilizers (vpm). use of plerixafor in vpm patient is controversial. the aim of our study is to compare mobilizing and engraftment between pm and vpm who received plerixafor plus g-csf (p+g-csf).methods: in our center, mobilization with g-csf at dose of μg/kg/day was used in all pts. apheresis were scheduled on day+ . plerixafor ( . mg/kg) was added if the number of cd + cells on day + was < /ul for x cd + /kg requested (or < /ul for x cd + /kg), or if the number of cd + cells collected in the first apheresis was < % of cd + cells requested.between january and september , out of pts ( , %) received plerixafor for mobilization. we retrospectively studied pts who mobilized with p+g-csfdue to the number of cd + cells on day + was < /ul.results: twelve out of pts were pm, were females, median age , years (range: - ). patients' baseline diseases were: non-hodgkin lymphoma (nhl) ( , %), multiple myeloma (mm) and hodgkin lymphoma. median cd + cell count on day + was / ul (range: - ).there was no mobilization failure. eighteen out of pts ( %) were vpm, were females, median age , years (range: - ). patients' baseline diseases were: nhl ( , %), mm and solid tumor. median cd + cell count on day+ was , /ul (range: - ). two out of pts ( , %) were considered mobilization failure, in of them did not realized apheresis due to cd + cell count on day + was /ul. no difference was seen between both groups regarding gender, age, patients baseline disease or median cd + cells count on day + .vpm needed more apheresis sessions, / pts required sessions against / pts in pm (p=not significant (ns). we obtained enough cells to carry asct in % pts, although mean number of cd + cells obtained in vpm was lower than in pm ( , x /kg vs , x /kg, respectively) (p=ns).twenty-six pts underwent asct and mean number of cd + cells infused were , x /kg in vpm vs have been the only route used for chpc administration. the appearance of other catheters types made us to reconsider the exclusive use of the cvc for the infusion of chpc. we analyzed the use of a peripheral iv cannula (pivc) as an alternative to cvc for the infusion of chpc in patients with cardiovascular diseases.methods: medical records of patients who received an asct for hematological malignant diseases at the hospital clínic of barcelona from january to february were reviewed. of those, eight were infused through a pivc due to cardiac impairment related to previous treatments, ischemic cardiomyopathy or amyloid deposition.hpc were obtained from peripheral blood by apheresis after mobilization with g-csf using acd-a as an anticoagulant. cryopreservation was performed with autologous plasma and dmso % by mechanical means and stored in liquid nitrogen. analytical controls were performed including hematocrit, total nucleated cells, total polymorphonuclear neutrophils and platelets using the advia analyzer. the cd + / cd + population was analyzed by flow cytometry following the ishage single-platform protocol. viability of total nucleated cellularity was carried out by vital staining with acridine orange and the specific viability of the cd + population through the technique of -aminoactinomycin d. thawing was performed bag to bag by immersion in a water bath at ºc and transferred to the bedside of the patient for gravity infusion using an infusion set without filter through pivc of gauche. vital sings monitoring performed before, during and the end of the every infusion bag including: blood pressure, heart rate, oxygen saturation, body temperature and central venous pressure. other aspects assessed during the infusion were pain, cold sensation and signs of extravasation in the area of pivc insertion. after the infusion, the recovery time of the granulocyte series and platelet were evaluated.results: median volume and bags administered was ( - ) ml and ( ) ( ) ( ) . the median of total nucleated cells, total nucleated cells / ml and total cd + cells/kg was . x ( . - . ), . x ( . - . ) and . ( . - . ) respectively. vital signs were within the normal range and allowed to perform the infusion in an average of - minutes/bag. no patient required stopping the infusion due to pain in the area of peripheral catheter insertion and no extravasations were detected. all patients referred some cold sensation in the insertion vein and its path. median hematopoietic recovery was ( - ) days for neutrophils and ( - ) days for platelets, similar to the recovery experienced from patients who received chpc through cvc.conclusions: based on our data, we conclude that the administration of chpc, through pivc and by gravity is safe for the product and for the patient, being the preferred choice for patients suffering from some type of cardiovascular disease.disclosure: nothing to declare background: by selective depletion of potentially alloreactive cd ra + cells, t memory cells might be retained in the graft and could mediate pathogen specific immunity. however, cd ra expression is not restricted to naïve t cells, but also available on b cells, nk cells and cd + stem cells to some extent. methods: within this project we aim to analyze cd ra expression on stem-and nk cells by flow cytometric analysis to estimate the eventual loss of these cells during cd ra-depletion. furthermore, clinimacs depletion following a one-step approach of direct cd radepletion and a two-step approach with primary cd selection followed by cd ra-depletion of the negative fraction was investigated.results: cd ra expression on cd + stem cells was in median . %. with a median of . % cd ra expression was measurable on nearly all b cells, which obviates depletion via cd . a comparably high cd ra expression of in median . % was detected on nk cells. unfortunately, the amount of nk cells in the cd ra-depleted product was . %. clinimacs depletion following one-step approach resulted in a stem cell recovery of . %. memory t cell recovery was . % following one-step and . % applying two-step approach. depletion quality measured by log-depletion was . and . for cd ra + t cells and . and . for cd + b cells for one-and two-step approaches, respectively.conclusions: with regard to stem cell recovery, a previous cd -selection before cd ra-depletion is recommendable.background: current clinical practice of routine use of filters for infusion of autologous hematopoietic cell transplantation (ahct) at bone marrow transplant centers across north america and europe is not known. the use of "y" administration tubing without a filter could possibly increase the risk of infusion of macro-aggregates and cellular debris, which may result in increased side effects.methods: we carried out a retrospective chart review of patients (pts) at spectrum health who underwent ahct. group a (gp a) pts received ahct using a "y" administration tubing with -micron filter from / - / . these patients were compared to a control group (gp b) that received ahct without filter administration tubing from / - / .this change in clinical practice occurred due to a change in policy at our transplant center as a result of inorganic particles noticed during cryopreservation. we compared the neutrophil and platelet engraftment duration between these groups. we also studied the length of hospital stay and the effect of filter use on any immediate side effects after infusion. due to the retrospective nature of the study it was not feasible to evaluate the difference in duration of infusion between these groups results: the two groups were similar in their age, gender, primary disease distribution and median number of cd stem cells infused (table) . there was no difference in median neutrophil ( vs days) or platelet engraftment ( vs days) duration for the filter group and the nonfilter group respectively. the median length of hospital stay was also comparable ( days). there was no statistically significant difference in the immediate side effects (fever, cough, dyspnea, fluid overload, flushing, nausea, vomiting, hypertension, hypotension and anaphylaxis) or confirmed post-transplant infections (viral, bacterial, fungal) experienced by these two groups.conclusions: our results show that the routine use of filter does not prolong hospital stay, and neutrophil/ platelet engraftment duration, thereby, suggesting that viable stem cells are not affected. on the other hand, filter use failed to demonstrate any appreciable decline in the immediate side effects experienced after ahct. gp background: allo-hsct from related haplo-identical donors (haplo-hsct) with post-transplant high-dose cyclophosphamide is increasingly employed in patients who lack a matched related or unrelated donor. the current standard is to use bone marrow grafts (bm) as peripheral blood stem cell grafts (pbsc) have been associated with an increased risk of acute and chronic gvhd. thus, the aim of our study was to compare the main transplant outcomes and especially the incidence of acute and chronic gvhd in recipients of bm and pbsc grafts. methods: thirty-five unselected patients with hematologic malignancy who underwent an haploidentical transplant at our unit between and and received bm (n = ) or pbsc (n = ) grafts after the same tbf conditioning regimen were analysed in order to assess differences in transplant outcomes.our gvhd prophylaxis consisted in cyclosporine a (csa) from day - to + , a methotrexate "short course" and mycophenolate mofetil (mmf) from day + to + .results: no statistically-significant differences were observed between patients who received bm grafts and those who received pbsc grafts. at transplant fourteen patients were in first complete remission (cr), twelve in advanced cr and had active disease. according to sorror's risk, nine patients were low-risk, nine intermediate-risk and seventeen high-risk. twenty-eight cmv+ patients received the graft from twenty-three cmv+ and five cmv-donors, seven cmv-patients received the graft from five cmv+ and two cmvdonors. mean age at transplant was years (range - ), mean donor's age years (range - ) and mean follow-up . months (range . - . ). median cd + cell dose was . x /kg (range . - . ), . x /kg (range . - . ) in bm recipients and . x /kg (range . - . ) in pbsc recipients. median time to neutrophil recovery (> /μl) was days (range - ) posttransplant, days (range - ) for bm recipients and (range - ) for pbsc recipients. platelet recovery (> . /μl) occurred in all patients except one at a median of days (range - ) post-transplant, at a median of days ( - ) post-transplant for bm recipients and at a median of days (range ( - ) for pbsc recipients. seven patients never reached platelets > . /μl. three patients developed a poor graft function. acute and chronic gvhd incidence was . % and . % and the risks of acute (hazard ratio [hr], . ; p = . ) and chronic (hr, . ; p = . ) graft-versus-host disease were similar in the two patient groups. in addition, there were no differences in relapse risks post-transplant (hr, . ; p = . ); relapse-free survival was better with pbsc grafts but this difference did not reach any statistical significance. finally, no significant differences were noted in overall mortality by graft type (hr, . ; p = . ).conclusions: despite in haplo-hsct the incidence of acute and chronic gvhd is reported to be higher with pbsc than with bm our small patient series does not confirm this assumption that should be clarified by additional studies. instead, our data suggest that pbsc background: since , cord blood (cbu) has become an alternative source of stem cells for transplantation, with approximately , procedures currently performed. with a -year gs of %.objective: analyze the outcomes from all the patients transplanted with cbu in our hospital unit.methods: retrospective, longitudinal study. all patients transplanted with cbu in our hospital between and were included. we analyzed patients with ages from months to years.results: two of them received doubled cord transplantation the ratio male: female was . : . the transplanted pathologies were: bone marrow failure %, immunodeficiency %, aml %, all . %, osteopetrosis . %. ric regimens were used in patients with bone marrow failure and immunodeficiency and myeloablative conditioning regimens were used in patients with malignant hematology diseases. antithymocyte rabbit globulin (atg) based serotherapy was used. one case received cbu from a related donor (sister), the rest received unrelated cbu obtained from centro nacional de la trasfusión sanguínea. the infusion of cd + was in a range of . to of . x /kg with and average of . x / kg. compatibility was / in %, / in % and / in %. post-thawing cellularity was not measured. the hla-c was not analyzed. forty two point five percent of the patients had a successful engraftment; the average time of engraftment was days. primary graft failure was detected in . % and secondary graft failure in %, for a total success of . %. gvhd was detected in % of patients, of which % was grade i-ii and % grade iii-iv. the overall mortality was . %. causes of death were: infection % relapse %, hemorrhage % and gvhd %. the cbsct continues to be an essential alternative in our patients who required transplantation knowing that this stem cell source allows the procedure to be done with less histocompatibility requirements and it is available immediately, which facilitates the process considering the great diversity that exists within our population. however, in our experience, the cbsct has shown a higher mortality risk, which can be improved by analyzing the hla c, choosing in this way the units with better compatibility, and improving cellular dosage since this is key in success.disclosure: nothing to declare the risk of infection of the umbilical cord is not related to the microbiological status of the umbilical cord blood methods: the statistical analysis was carried out on data obtained from samples taken in poland between -jan- and -dec- . the samples were collected in hospitals by external midwifes and sent to the pbkm in accordance with the requirements of the american association of blood banks. after arrival in the laboratory, the blood samples were cultured and the ucs were assessed immediately for visual signs of infection, such as odor, altered color, or visible bloom. the status of both kinds of samples was introduced into the pbkm general database. for the purpose of this analysis, the ucs were considered as microbiologically pure if stored, destroyed after storage, or handed over to the pbkm. samples marked as infected or disqualified for unknown reasons (other than termination of the contract with the customer, viral infection of the mother, and lack of cell growth) were considered as infected. at the time of the statistical analysis, the samples of unknown ucb microbiological culture status were removed from the generated report. the data was summarized as percentages and the odds ratio was calculated. statistical significance was considered at p˂ . .background: match family donors are the preferable options in allogenic stem cell transplant. however, in the absence of donor relatives match unrelated donors have been an option. in this study, the donor screening, transplant preparation phases of turkish stem cell coordination center (turkok) and the İstanbul university bone marrow bank (tris), were compared.methods: the unrelated donor scanning data between march and november in pediatric stem cell transplantation unit of altınbas university bahcelievler medical park hospital were evaluated. unrelated transplants were performed in total. % of these transplants (n= ) were included from the donors of turkok registration system and , % of these transplants (n= ) were included by means of tris from donors outside of turkey. patients ( tris, turkok) were excluded from the study in consequence of screening update and postpone of transplantation. the statistics were carried out on a total of patients, , % of whom were in turkok (n= ) and , % were transplanted via tris (n= ). the day of application to stem cell transplantation unit, reply dates and the transplantation dates were examined for the transplant patients.results: in the current study, the average response time of turkok was found as , ± , day (median: ), the average transplant time after receiving a reply was found as , ± , (median: ) day, the average number of days from date of application to date of transplantation of patients was found as , ± , (median: ). the average response time of tris was , ± , (median: ) day, the average transplant time after receiving a reply from tris was , ± , (median: ) day, average number of days from date of application of tris to date of transplantation of patients , ± , (median: ) day.the average response time of turkok, the average transplant time after receiving a reply from turkok and the average number of days from date of application of turkok to date of transplantation of patients was shorter than tris. the difference between them was found statistically significant (p< . ).conclusions: in this study, it was determined that the transplantation processes with turkok were progressing more rapidly. the rapid progress of the process was attributed to the fact that all donor hla tissue groups in the turkok database were studied in high resolution. in international scans carried out through tris, it was thought that the examination of the donor castings coming from bone marrow banks and the time differences between the countries prolong the process. it was thought that hla tests of the registered donors in the tris database and some international bone marrow banks were studied in low resolution but not studied the all hla loci, the centers wanted high-resolution hla, and therefore the involvement of social security institutions and payment procedures were among the factors extending this process.disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (ahsct) is being performed for a group of hematologic diseases with a curative intent. outcomes after ahsct are influenced by the type of donor used. haploidentical transplantation is an emerging option when a fullmatched donor is unavailable. methods: we retrospectively analyzed our transplants performed between january and november , investigating outcomes and complications among haploidentical stem cell recipients.results: one hundred and nineteen patients underwent ahsct, of them ( . %) were recipient of a haploidentical stem cell and included in this study. one patient diagnosed with acute lymphoblastic leukemia (all) were performed a haploidentical ahsct for two times due to relapse. among those transplants, of them were diagnosed with acute myeloid leukemia, with all, with chronic lymphocytic leukemia, with myelodysplastic syndrome and with hodgkin lymphoma. the mean age of group was . ± . years. three patients ( aml, all) were in remission at the time of transplantation. patients were given a conditioning regimen based mostly on busulfan, fludarabin and total body irradiation with a myeloablative intent. patients were also given a various combinations of post-transplant cyclophosphamide, calcineurin inhibitors, mycophenolate mofetil and antithymocyte globulin for graft versus host disease (gvhd) prophylaxis; post-transplant cyclophosphamide administered on ( %) of those transplantations. peripheral blood was the source of stem cells in all patients. patients were infused with mean . ± . x /kg of cd + cells. hematological recovery was achieved with neutrophil engraftment at a mean of . ± . days and platelet engraftment at a mean of . ± . days. after a median month ( . - . months) follow up, the cumulative rates of grade - gvhd, relapse and non-relapse mortality were %, % (n= ) and %, respectively. one patient died due to relapse, at the end of the follow up two were still alive with remission. only one patient has died due to chronic gvhd affecting serosa and resulting with a fatal tracheoesophageal fistula. the mean overall survival was . ± . months in our study.conclusions: haploidentical transplant is a feasible option in hematologic malignancies with novel gvhd prophylaxis approaches, especially post-transplantation cyclophosphamide. however, these results need to be supported with further investigations with a larger patient group.disclosure: nothing to declare results: a total of patients between and years (median age: years). transplant was done for following disease: acute leukemia (n= , %), aplastic anemia (n= , %), lymphoma (n= , %), myelofibrosis (n= , %), myelodisplastic syndrome (n= , %), chronic myeloid leukemia (n= , %). ten donors were from turkey and fifteen donors were from different countries of europe and america. two of donors were / and the other was / hla matched. the conditioning regimen was mostly non myeloablative (n= , %) while eight patients were treated with myeloablative regimen. other than two patients who took tacrolimus and mycophenolate mofetile all of them got cyclophosphamide and methotrexate for graft versus host disease (gvhd) prophylaxis. the median time of neutrophil and platelets engrafman were days (range - ) and , days (range - ) respectively. acute gvhd was seen nearly half of the patients ( , %).overall survival was % for all patients and of patiens ( %) died within first month to months (median months). the mortality rate was more higher for the recipients who had donor source from countries other than turkey ( % vs % p= , ). transplant related mortality was the most common reason of mortality (n= / , , %) and other reasons were gvhd ( , %), infections and cirrhosis respectively.conclusions: we found the mortality rate more higher in the patients whose donors were from out of our country. however, we need to further multicentric and prospective investigations to confirm our hypothesis, it would be related with impact of ethnicity.disclosure: nothing to disclose late-breaking abstracts p targeted twice daily busulfan-based ric-conditioning for allogeneic hematopoietic stem cell transplantation in pediatric patients with chronic granulomatous disease: a -year experience with the zurich protocol matthias felber , mathias hauri-hohl , ulrike zeilhofer , federica achini , jana pachlopnik-schmid , janine reichenbach , seraina prader , tayfun güngör background: chronic granulomatous disease (cgd) needs sufficient myeloablation to avoid graft failure. for this purpose the ebmt inborn errors working party currently recommends treosulfan or busulfan-based conditioning regimens for cgd-sct. we analyzed the last years of targeted busulfan-based ric-conditioning including engraftment, gvhd rates, chimerism and late term effects in our pediatric sct center in zurich. methods: between and , n= consecutive pediatric cgd patients (median age years, range - years, n= female, n= autosomal recessive inheritance) have been transplanted. all patients received therapeutic drug monitoring of twice daily administered iv busulfan ( or hour infusions; d- to -d ) to achieve a targeted cumulative auc of - mg/l*h. fludarabine ( mg/ sqm; d- to -d ) and serotherapy (thymoglobuline . mg/ kg total, d- to d- ) or alemtuzumab ( . - . mg/kg total; d- -to d- ) were used for immunoablation. donors were matched unrelated ( / hla; n= ), mismatched unrelated ( / hla; n= ), mismatched unrelated (hla / ; n= ), matched sibling (hla / ; n= ) and haploidentical parental (hla / ; n= ). for patients with haploidentical donor post-transplant cyclophosphamide (d- and d- with mg/kg iv each) and upfront atg-grafalon ( mg/kg - to-d- ) was used. stem cell sources were bm (n= ) and pbsc (n= ). gvhdprophylaxis included iv csa (d- ; continuous infusion) and iv. mmf (d- , in - doses).results: follow-up was to months. good overall engraftment was noted, with n= secondary graft failure followed by successful retransplantation. in one patient a stem cell boost/dli was necessary due to decreasing myeloid donor chimerism during ebv reactivation, resulting in rapid myeloid donor reconstitution after intervention. low rates of gvhd were documented with n= agvhd grade iv and n= mild/limited cgvhd (nih criteria). with exception of one patient, myeloid donor chimerism at last follow-up was over %, mostly over %. overall survival was / ( %). deaths were due to gi-gvhd (n= ), autoimmune hemolytic anemia/sepsis (n= ) and thrombotic microangiopathy (n= ).conclusions: precision dosing of iv busulfan in combination with fludarabine and serotherapy results in excellent outcome of hsct for pediatric cgd-patients with good engraftment, low overall cgvhd rates and stable, mostly excellent donor chimerism. graft failure rate was as low as %. low dose alemtuzumab prevented gvhd in the majority of patients. this analysis demonstrates that targeted busulfan-based conditioning is a valid option for pediatric cgd-patients. serum alemtuzumab or atg monitoring could further improve gf and gvhd rates in the future.disclosure: the authors declare no conflict of interest. abstract already published. young hla-matched unrelated donors are comparable to matched sibling donors in elderly patients receiving reduced-intensity conditioning: an analysis on behalf of the ebmt scientific council % c.i. . - . , p= . ) and voriconazol prophylaxis during carv (or . , % c.i. . - . , p= . ). conclusions: we provide evidence that ifd after carv infection. allo-hsct recipients developing a carv lrtd during the first year after transplant may benefit from an adequate antifungal prophylaxis and a close monitoring for the development of a later ifd.disclosure: jose luis piñana has received both, advisory for preclinical/clinical research and financial support to assist to the spanish society of hematology annual meeting from msd. favorable outcome and engraftment following reducedintensity conditioned allo-hsct in children with primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) laura m. moser, emilia salzmann-manrique, andrea jarisch, jan sörensen, shahrzad bakhtiar, peter bader background: primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) represent two major entities of childhood histiocytoses, which are -although only of rare occurrence -severe in their clinical manifestations. patients present with multisystemic uncontrolled inflammation and multi-organ involvement requiring diverse courses of immunosuppressive and chemotherapy regimens. allogeneic haematopoietic stem cell transplantation (allohsct) is the only available curative option; however, the cumulative treatment toxicity and the underlying inflammatory disease often result in high organ toxicity and inflammatory complications of transplantation, such as graft versus host disease (gvhd) and/or graft failure. especially patients with unrelated donors often deal with high transplant-related mortality (trm) in the setting of conventional intensity conditioning.herein, we present the clinical course of the disease and transplant outcome of children diagnosed with primary hlh (n= ) and high-risk lch (n= ) who underwent allohsct at our centre from / to / .methods: the hlh cohort consisted of cases of familial hlh (fhlh), cases of griscelli syndrome, one xiap-deficient patient and one hlh-patient with inconclusive genetic testing. all hlh patients had developed clinical symptoms prior to transplantation and had been treated according to hlh-protocols , hlh-protocols , or median age at transplantation was months ( to months). stem cells were derived from hla-matched siblings (msd, n= ), matched unrelated donors (mud, n = ) or haploidentical donors (n= ).the majority of patients ( / ) received a ric regimen containing fludarabine, melphalan and thiotepa (n= ) and fludarabine plus cyclophosphamide (n= ). myeloablative treatment ( / ) included a treosulfan-based regimen (n= ) and busulfan-containing treatment (n= ). the entire cohort received serotherapy using either muromonab (n= ), atg-fresenius® (n= ) or alemtuzumab (n= ).results: the overall survival of the entire cohort was . % ( / ) on a median follow-up of . years ( figure a+b) .all lch patients, being treated with fludarabine, melphalan and thiotepa, survived transplantation and showed complete remission ( / ) . within the hlh cohort the overall survival was . % ( / ). fatalities (n= ) included two patients from the myeloablative group and one ric-treated patient. the cause of death were progressive disease activity during the conditioning phase, leading to multi-organ failure on day + despite immunosuppressive treatment (n= ) and complicated cerebral seizures followed by lung haemorrhage, possibly due to aspiration pneumonia with evidence of enterococcus faecium, resulting in septic multi-organ failure on day + (n= ). a third hlh patient developed a sudden cerebral edema and ensuing respiratory insufficiency on day + . whether this was caused by acute neurotoxic damage by fludarabine or a consequence of relapsed hlh could not be conclusively specified. none of our patients suffered from transplant failure or nonengraftment. there was neither severe acute gvhd (iii-iv) nor chronic gvhd observed in this cohort.conclusions: primary hlh and high-risk lch are lifethreatening medical conditions needing rapid allohsct. ric regimens are well-tolerated and sufficient for proper engraftment and disease clearance. disclosure: the authors have no conflicts of interest to disclose. abstract withdrawn. thrombocytopenia following allo-sct concomitant to stem cell boosts. steroid refractoriness was defined as: progression after three days or no response after days of steroid treatment. the median time from sct to the onset of agvhd was . d (range - d), and d (range - d) from the onset of agvhd to the first msc infusion, respectively.the majority of our patients (n= ) suffered from a malignant disease and received a graft from a matched unrelated donor (n= ), while one patient had a haploidentical donor. gvhd prophylaxis was performed in all patients except the patient with the haplo-identical graft. all patients with agvhd were treated with steroids and the patient with thrombocytopenia required regularly transfusions and romiplostin therapy. the median msc dose was . x cells/kg bw (minimum . x ; maximum x ). three patients received msc doses, two patients , one patient and another doses.results: at the time point of agvhd manifestation and msc application, two patients had cmv reactivation, one patient adenovirus infection and one patient ebvreactivation. by day , / agvhd patients responded to msc administration: with complete response and with partial response. at the last follow-up (median: . months, range , - . months), of patients were alive without acute or chronic gvhd. one patient died soon after msc treatment with no obvious response in the course of a systemic hyperinflammation syndrome. the other patient although complete responder to msc-ffm developed fatal adenovirus sepsis. this based on the profound tcell depletion induced by concomitant application of steroids. the overall survival probability at six month was . %. no acute side effects occurred after msc infusions. the previously mentioned patient suffering from thrombocytopenia did not need any further transfusions after receiving doses mscs combined with stem cell boosts while continuing romiplostin application.conclusions: our data confirm excellent tolerability and high efficacy of the licensed off-the-shelf msc preparation "msc-ffm" in pediatric steroid-refractory agvhd. in our center, current treatment algorithms have escalated "msc-ffm" to the second line, i.e. immediately after steroid refractoriness has been established. besides immunoregulatory properties, this product might facilitate hematopoietic stem cell engraftment.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) expression of ccr modulates migrational properties of in vitro expanded murine regulatory t cells laura m. moser , , ulrike tischler , christin riegel , julia minderjahn , rüdiger eder , jaqueline dirmeier , isabel zimmermann , evelyn röseler , petra hoffmann , , matthias edinger , background: hematopoietic stem cell transplantation (hsct) as it is carried out successfully at other genetic instability syndromes seems to be an encouraging opportunity for a curative therapy to restore immunity and prevent the development of hematologic malignancies in ataxiatelangiectasia (a-t). however, experience in the conditioning regimen is limited and no transplantation strategy for a-t patients exists, especially in an allogeneic setting. conditioning regimen and donor selection are critical factors in the clinical setting of hsct and incur substantial risks, especially in a-t. the aim of this study was ( ) to evaluate whether different approaches of hsct including allogeneic hematopoietic hsct are feasible in regard to graft versus host response (gvhd) and sufficient concerning immune reconstitution ( ) and to de-escalate the toxic effects of the conditioning regimen by reducing the dose of cyclophosphamide (cp).methods: t cells from syngeneic, allogeneic and haploidentical donor mice were used to determine gvhd induced t cell proliferation in a mixed lymphocyte reaction (mlr). atm-deficient mice were treated with cp or reduced cp in combination with fludarabine (flu) and transplanted with x cd . depleted bone marrow donor cells from /svev gfp-transfected wildtype mice (syngeneic) or from mice of the f generation of /svev wildtype mice and c bl/ mice (haploidentical), or from c bl/ mice (allogeneic). tracking of gfp-positive donor derived cells was performed using flow cytometry and atm pcr. oxidative stress and damage were detected by a rt profiler pcr array and -hydroxy- ′deoxyguanosine.results: mlr resulted in an increased proliferation of allogeneic donor t cells compared to syngeneic and haploidentical donor cells. response was lower on dendritic cells isolated from atm-deficient mice compared to wildtype controls. in vivo results showed the restoration of t cells in atm-deficient mice accompanied by a prolonged life span and through reduction of thymic tumors. however, allogeneic stem cell transplantation was accompanied with a higher mortality rate, compared to the haploidentical and syngeneic setting. decreased antioxidative capacity and a higher dna-damage were seen in cp treated atmdeficient mice.conclusions: haploidentical hsct seems to be a feasible strategy for a-t. our data provided further evidence for the high sensitivity against ros-inducing agents in a-t and this fact needs to be taken into consideration in the choice of the host-conditioning strategy.disclosure: nothing to declare this research received funding from action for a-t charity ( gou ) background: prognosis of pediatric patients and young adults suffering from refractory or high-risk soft tissue sarcomas remains poor with limited improvement over the last decades despite multimodal treatment strategies. replacing the immune system by an allogeneic hematopoietic stem cell transplantation (hsct) in refractory solid malignancies has been proposed as a potentially curative therapy due to its presumable graft versus tumor effect. based on this concept we additionally performed consecutive donor-derived lymphocyte infusions in allogeneic hsct-patients with refractory or relapsed solid malignancy to further increase anti-tumor efficacy post-transplant.methods: pediatric patients with relapsed and/or refractory cancers or with delayed responses to the respective induction therapies were offered donorderived cellular therapies after immune system replacement by an allogeneic hsct. cellular immunotherapies comprised of donor lymphocyte infusions (dli), natural killer (nk) cell or cytokine-induced killer (cik) cell infusions generated from the original stem cell donors. allogeneic nk cells were generated from unstimulated leukapheresis by a two-step purification procedure using immunomagnetic cd t cell depletion, followed by nk cell enrichment (cd +) with or without in vitro il- stimulation and expansion for - days. for cik cell generation peripheral blood mononuclear cells were isolated and activated by in vitro cytokine stimulation (inf-γ, anti-cd , il- and il- ) an expanded over - days. expanded cik cells represented a heterogeneous population of polyclonal t cells with in part shared phenotypic and functional properties of nk cells.results: between october st and january st a total of patients (eight patients with rhabdomyosarcoma, one patient with synovial sarcoma, two patients with ewing sarcoma, five patients with neuroblastoma, one patient with hepatoblastoma, and one patient with nasopharynx carcinoma) were enrolled. seven of ( %) patients in this study had achieved complete remission (cr) before hsct while another of ( %) patients had obtained at least very good partial or partial response (vgpr or pr). dli was applied in patients, nk cell treatment was offered to another patients, while cik cell therapy was given to patients. . -year probabilities of overall survival (os) and progression-free survival (pfs) were . % and . % for all patients with a median follow up of . months (range, . - . months). patients in cr at the time of immune cell therapy (it) showed estimated . -year os and pfs of . % and . %, respectively. the majority of patients relapsed and ultimately succumbed to their diseases with two of ( %) patients still being alive . and . years after it. cumulative incidence of relapse was . % at . years. t cell engraftment and immune reconstitution (ir) was improved by it, and correlated with treatment response. however, two of heavily pretreated patients ( %) died due to cumulative treatment-related mortality (trm). furthermore, acute graft-versus-host-disease (agvhd) occurred in of patients ( %) with agvhd grade i-ii observed in ( %) and agvhd grade iii seen in three ( %) patients.conclusions: altogether, the results of this study indicate that allogeneic donor-derived cellular therapy at its current state offers curative benefit in selected refractory childhood cancers but warrants further improvement. background: allogeneic stem cell transplantation (allo-sct) is the only curative treatment option for a variety of nonmalignant diseases. the success of allo-sct is strongly associated with rapid and sustained immune reconstitution (ir). we analyzed the ir in patients who received an allo-sct for nonmalignant diseases.ir was assessed on days + , + , + , + and + after allo-sct analyzing leukocytes, lymphocytes, monocytes, cd + t cells, cd + cd + t helper cells, cd + cd + cytotoxic t cells, cd -cd + natural killer (nk) cells and cd + b cells.methods: we analyzed ir-data of consecutive patients receiving allo-scts between september and november . indications of allo-sct were hereditary anemias (thalassemia, sickle cell disease, diamond blackfan anemia; ha, n= , %), inherited bone marrow failure syndrome (fanconi anemia, severe aplastic anemia, others; bmfs, n= , %), hemophagocytic lymphohistiocytosis (hlh, n= , %), immunodeficiency (id, n= , %) and metabolic disorders (n= , %). the median age at allo-sct was years (range, . - ) and at diagnosis . years (range, - . - . ).patients received st allo-sct from msd/mfd (n= , %), mud (n= , %), haploidentical mismatch family donors (n= , %) and mmud (n= , %). conditioning regimens were busulphan-based (n= , %), treosulphan-based (n= , %), flu-mel-thio (n= , %) or others (n= , %). graft sources were bm (n= , %) and pbsc (n= , %).in order to consider the age-dependency of ir we normalized each absolute cell count with its corresponding age-specific expected mean values. (huenecke et al.; eur j haematol; ) results: the ir pattern was similar between the ha and bmfs groups. the cd + t cells were recovering slightly faster in ha patients compared to the recovery of bmfs patients.monocytes and nk cells proliferate very fast. at day + half of the patients already reached their respective monocytes reference value except for id patients, who reached % of the reference value at the end of the first year.cd + cd + cytotoxic t cells recovered significantly faster in patients with hematologic diseases compared to patients with hlh (p< . ) and id (p= . ). half of the patients reached the reference value of cytotoxic t cells in the hematologic diseases group at day + . by far inferior was the ir for the hlh patients. in this group only % of the patients reached the th percentile of the healthy age-matched reference. in the id group % of all patients reached the th percentile of the age-matched reference group at day + .b cells are profoundly decreased at day + in all groups. however, the longitudinal expansion of b cells was significantly lower both in the id group and hlh group compared to the hematologic diseases group. at day + fifty percent of patients with id, hlh and hematologic diseases reached the th percentile, th percentile and the th percentile, respectively (p< . ; p= . ).conclusions: allo-sct is the only curative option for patients with nonmalignant diseases. ir is dynamic and revealed a complex diversity pattern with regard to the original disease. to investigate factors influencing ir after allo-sct is crucial to improve outcome of these patients.disclosure: nothing to declare. allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome of relatively high-risk groups: curative effect analysis and optimal timing selection results: among the patients, patients were transplanted successfully. the -year overall survival (os) rate and disease-free survival (dfs) rate was . % ± . % and . %± . % respectively. the -year cumulative relapse rate (rr) and the non-relapse mortality (nrm) rate was . %± . % and . %± . % respectively. the incidence of grade ii-iv acute graft versus host disease (agvhd) was . %± . %. for the patients who survived more than days after allo-hct, the years cumulative incidence of chronic graft versus host disease (cgvhd) was . %± . %. univariate analysis showed that the hematopoietic cell transplantation comorbidity index(hct-ci) and grade iii-iv agvhd are the high risk factors for os( . ± . % vs . ± %, p= . and . ± . % vs . ± . %,p < . ). multivariate analysis demonstrated that grade iii-iv agvhd and hct-ci are independent risk factors for os(hr= . , p < . , % ci: . ~ . and hr= . ,p= . , %ci: . ~ . ). chemotherapy before transplantation did not improve os or dfs for patients with bone marrow blast cells more than % at the time of diagnosis.conclusions: allo-hsct is an effective treatment for mds patients of relatively high-risk groups. the physical condition of the patients and occurrence of agvhd are independent risk factors. for intermediate and high risk ipss-r mds patients, transplantation before the disease progressed into very high risk can achieve better prognosis, high-risk group can still benefit from rebound gvhd after cni tapering which was promptly responsive to treatment steroids, fk and ecp. aa one year after sct / patients were without gvhd and off all immunosuppression while one single patient was still on taper of immunosuppressant after rebound acute gvhd. no chronic gvhd occurred. sctrelated toxicity was common with mucositis in all patients (who grade : n= ), elevated liver enzymes (≥grade : n= ) and impaired renal function (gfr - ml/min: n= ). five patients developed neurologic symptoms (seizures n= , pres n= , pseudotumor cerebri n= ) which all resolved without sequelae. overall survival and transfusion-free survival was % with a median observation time of ( - ) years.conclusions: . treosulfan-based conditioning followed by sct from hla-matched related or unrelated donors represents a highly efficacious treatment approach for children, adolescents and young adults with tdt and exhibits an acceptable but not negligible safety profile. an individual risk-benefit assessment incorporating hazards such as secondary graft failure, gvhd and long-term toxicity including infertility and nd malignancy has to be executed in the informed consent process for every patient and his/her guardians.disclosure: "nothing to declare" p abstract already published. early iron chelation with deferasirox might prevent relapse after busulfan plus fludarabine and atg as a myeloablative conditioning for hla-identical sibling allogeneic hct in aml results: we show an excellent concordance between chimerism assessment on bio-rad and d platforms over the complete range of mixture ratios (r > , ) and proof the lower detection limit ( , %) compared to str-pcr.conclusions: our results promote the transfer of the established mentype assay to a more diverse instrument portfolio. that will allow to implement the analysis of patient and donor hematopoiesis by digital pcr methods in our lab.disclosure background: with the immense progress in therapeutic regimens in pediatric oncology and stem cell transplantation the survivor rates increased up to %. at the same time the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation. therefore it is of great importance to implement fertility counseling and fertility preserving (fp) procedures for patients facing gonadotoxic therapy. in the report on the expert meeting of the paediatric diseases working party (pdwp) of the ebmt in counseling related to fp opportunities should be offered to each patient receiving stem cell transplantation (sct), as part of the pre-sct workup by a dedicated and trained team. yet there many medical, ethical, structural and financial issues to consider and overcome. we describe the setting up process to enable fertility counseling for all children with newly diagnosed cancer or those facing stem cell transplantation for malignant and nonmalignant diseases in our department of pediatric oncology and immunology/stem cell transplantation.methods: at our tertiary care center we assembled a multidisciplinary team involved in fertility preservation (pediatric hematology/oncology, pediatric immunology/ stem cell transplantation, reproductive medicine, andrology, psychology and pediatric surgery). we developed an internal grading system for recommendations regarding fertility preservation based on the current recommendations for fertility preservation of leading societies in this field like ebmt, gpoh and dggg. it is important to find a consensus within the team for the counseling to ensure reliable counseling. the third step is to implement structures needed for fertility counseling and performance of invasive procedures including legal aspects (amg). a detailed description of this process is given.results: after setting up structures for the counseling process we counseled oncology and stem cell transplant patients ( - years) between january and may . we analysed data of the patients including age subgroups and disease entities and the results of the counseling process. for those patients undergoing stem cell transplantation the risk of gonadotoxicity is very high, therefore even the very young children underwent fertility preserving procedures in alignment with our recommendations if they suffered from a nonmalignant disease. currently we discourage tissue preserving in malignant systemic disease due to possible contamination with malignant cells. postpubertal female patients were more likely to undergo invasive procedures such as ovarian tissue cryopreservation in the case of oncological diseases, while sperm cryopreservation was recommended in all postpubertal male patients. overall a high percentage of the patients and their family followed our recommendations.conclusions: fertility preservation should be considered as a very important part of the treatment plan for newly diagnosed children and young adults with cancer and those facing stem cell transplantation. unfortunately there is still a great need for setting up structures in institutions taking care of these patients. in addition fertility preservation sadly lacks funding by health insurance in some countries. with the presentation of our experience and data we want to facilitate incorporation of fertility counseling in other pediatric care centers to provide counseling for pediatric patients in need for fertility preservation.disclosure: no conflict of interest regulatory t-cells (t reg ) have been shown to play a role not only in autoimmune diseases and solid organ transplantation but also in gvhd. several mouse models showed a decrease of gvhd incidence after t reg administration. the few clinical trials regarding the application of t reg for the treatment of gvhd are encouraging, however the data is limited. methods: patient: a -year-old boy underwent allogeneic sct for chronic myeloid leukemia refractory to imatinib, dasatinib and nilotinib treatment from his / hla identical brother. freshly derived unmanipulated bone marrow was transplanted after conditioning with of fludarabine ( mg/m²/d, day - to - ), thiotepa ( x mg/ kg, day - ) and melphalan ( mg/m², day - ). cyclosporin a (csa) and mycophenolatmofetil (mmf) were used for gvhd prophylaxis. leukocyte regeneration (> /μl) was seen on day + , granulocyte regeneration (> /μl) on day + and thrombocyte regeneration (> . /μl) on day + . on day + after sct he developed acute intestinal gvhd that exacerbated to grade iv°(bloody diarrhea, ileus) and did neither respond to steroids, nor to different immunosuppressive drugs such as cyclosporin, tacrolimus, sirolimus, mycophenolatemofetil and ruxolitinib. extracorporal photopheresis and the administration of immunmodulatory antibodies (adalimumab and tocilizumab) did not succeed either.results: by administration of low-dose interleukin- (il- ) in vivo induction of t reg was expected but did not succeed. finally antithymocyte globuline (atg, mg/kg/ d) was administered on day + to + to eliminate the gvhd-triggering cells. hence, the gvhd declined to grade iii. finally, a decision was made to manufacture t reg from his stem cell donor. from an unstimulated leukapheresis t reg were selected by magnetic depletion of cd + t-cells and cd + b-cells followed by positive selection of cd + background: treatment of patients with transfusion dependent anemia like thalassemia major (tm), sickle cell disease (scd) and diamond-blackfan anemia (dba) has improved over the last decades. for the vast majority of patients, allogeneic hematopoietic stem cell transplantation (hsct) is the only available curative therapy. for a long time, hsct has only been performed from hla-identical sibling donors (msd) or matched family donors (mfd). however, approximately only - % of affected patients do have a matched sibling donor, therefore hsct from / (mud) and even / (mmud) matched unrelated donors has gained importance in recent years.methods: patients (age range: - years) with scd (n= ), dba (n= ) or tm (n= ), receiving hsct from a msd, mfd, mud or mmud between and were included in our analysis. patients received transplants from msd/mfd, patients from mud/ mmud. patients were identical for hla-a, b, cw, drb and dqb , patients shared only / genes. we analyzed extended haplotypes including drb , drb , drb and dpb for all patients with thalassemia. pairs showed non-permissive dpb mismatch and pair mismatch for dpb and drb .results: median time for granulocyte recovery was days in patients transplanted from msd/mfd and days in patients transplanted from mud/mmud. platelet recovery was reached after days after hsct from msd/mfd and days after hsct from mud/ mmud. / ( %) patients showed complete donor chimerism in all controls. / ( %) patients showed low level mixed chimerism up to % during follow up. patient died shortly after hsct, patient showed slowly increasing mixed chimerism and finally developed autologous recovery and one patient rejected the graft.cumulative incidence of grade ii-iv acute graft-versushost disease (agvhd) of mud/mmud was , %, whereas only cases of agvhd grade i occurred in patients transplanted from msd/mfd. as patient rejected the graft from a hla-identical parent, patient transplanted from a hla-identical grandparent developed autologous recovery after year and patient transplanted from a mud lost the graft due to hemophagocytosis, the probability of event-free survival was , % after hsct from msd/mfd and , % from mud/mmud.altogether / patients ( , %) are alive and transfusion-independent with complete donor chimerism two years after hsct; resulting in an overall survival probability of , %. in contrast, overall survival probability was % in the group of patients transplanted from msd/mfd and , % in patients transplanted from mud/ mmud after years.there were patients with thalassemia ( , %) who died from transplantation-related causes. the first patient died days after hsct from a mmud due to candida sepsis with pulseless electrical activity resulting from cardiac iron overload. the second patient died months after hsct from a mud due to graft failure.conclusions: hsct from mud and mmud is a feasible therapy option for patients with transfusion dependent anemia. nevertheless, it should be noted that iron overload can cause severe complications; therefore, measurement of liver and heart iron concentration through mri prior to hsct as well as phlebotomy after transplantation are advisable.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) background: the therapeutic options for patients with hodgkin´s disease who relapse after first high-dose chemotherapy with autologous stem cell ( st asct) support are limited. allogeneic stem cell transplantation in this setting is associated with a high level of transplant-dependent mortality rates in excess of - %. new agent, such as brentuximab vedotin, have been approved for the treatment of these patients, however, their efficacy to provide longterm control or cure is still unknown. a second autologous stem cell ( nd asct) has historically been considered as an option only in a small group of patients so the published experience is scarce. we report our institution´s experience with second autologous transplants in this patient population.methods: we evaluated the outcome of adult patients ( ( %) female and ( %) male), who received an nd asct between / and / . planned tandem asct were excluded. the median age at nd asct was years (range - ), ( %) patients had a karnofsky performance score ≥ %. ( %) patients were in complete remission (cr) and ( %) patients were in partial remission (pr) at day after st asct. seven ( %) relapses within months after st asct. patients received a median of ( - ) treatment lines between st asct and nd asct. only ( %) patients received brentuximab vedotin and none of the patients in our series received checkpoint inhibitors as salvage after st asct. the median interval from st asct to relapse/progression was , months (range , - , ). the median interval from relapse/progression to nd asct was , months (range , - , ). all patients received beam as the conditioning regimen for st asct, and beeam as the conditioning regimen for nd asct.results: the median time to neutrophil recovery (> . x /l) after nd asct were days (range - ). best response at day following nd asct included cr in ( %) patients and pr in ( %); ( %) had stable disease. ( %) patients received brentuximab vedotin and none of the patients received checkpoint inhibitors after nd asct. ( , %) patients are currently alive, with a median follow-up , months (range , - , ). patient died after nd asct. causes of death were hl progression. the -year overall survival was %.conclusions: the second asct in patients with a longterm response after the first asct may be the optimal therapeutic option, the effectiveness of which can be enhanced by using new drugs, such as brentuximab vedotin, at all stages of treatment.disclosure: nothing to declare effectiveness of chemo-g-csf protocols for mobilization of peripheral stem cells in patients with non-hodgkin lymphomas and hodgkin disease-single center experienceilina micheva , stela dimitrova , vladimir gerov , trifon chervenkov , liana gercheva , igor reznik background: high-dose chemotherapy and autologous stem cell transplantation (asct) play an important role in achieving long-term remission in patients with non-hodgkin lymphoma (nhl) and hodgkin disease (hd). granulocyte colony stimulating factor (g-csf) combined with high-dose chemotherapy is a frequently used mobilization approach; however, the optimal mobilization strategy has not been determined.the objective of the study was to analyze the mobilizing potential of different regimens used for the collection of peripheral stem cells in patients with relapsed or refractory (r/r) nhl and hd. methods: we retrospectively analyzed patients with r/r nhl and hd undergoing stem cell collection after chemo-mobilization in the transplant unit at the university hospital, varna. patients were mobilized after dhap letermovir is promising, even as a therapeutic agent. more paediatric data are urgently needed.disclosure: nothing to declare p development of paroxysmal nocturnal hemoglobinuria in a patient after mudallohsct due to jak v fpositive myelofibrosis-a case of successful treatment with the second transplantation from another donor agnieszka tomaszewska , barbara nasiłowska-adamska , iwona solarska , kazimierz hałaburda background: paroxysmal nocturnal hemoglobinuria (pnh) is a very rare disease associated with pig-a gene mutations in hematopoietic stem cells. there are only single case reports on evolving myeloproliferative diseases to pnh in the literature. there is no data concerning development of pnh de novo after allogeneic hematopoietic stem cell transplantation. in our report we describe a patient with recurrence of jak v -positive myelofibrosis years after matched unrelated donor allogeneic hematopoietic stem cell transplantation (mudallohsct) with simultaneous development of clinically significant pnh. a year-old-man with a history of mudal-lohsct in may due to jak v -positive myelofibrosis secondary to essential thrombocythemia was admitted to our department years later with mild anemia (hb- . g/dl) and elevated lactate dehydrogenase ( u/l). during last years he remained in complete remission of myelofibrosis with jak v mutation negativisation and % donor chimerism. suspecting disease recurrence we performed trephine biopsy confirming myelofibrosis (mf /mf ) with heterozygous jak v mutation and in flow cytometry analysis of bone marrow we identified cell membrane defect in myeloid line (loss of cd c). we decided to perform detailed diagnostic tests on pnh -multiparametric flow cytometry of peripheral blood revealed % granulocytes and % red blood cells with loss of gpi-anchored proteins -pnh clone. these results corresponded with donor chimerism -it was only % of donor dna in bone marrow and % in blood tests. molecular analysis didn't revealed any mutations in genes: calr, asxl and mpl. finally the diagnosis of myelofibrosis recurrence after mudallohsct with presence of pnh clone was established. the therapy with eculizumab was unreachable. so the second allohsct from another matched unrelated donor after fludarabinemelphalan-thymoglobuline-tbi cgy conditioning was performed on . . . we didn't observe any complications of this procedure, engraftment was slightly delayed: anc> . g/l on the day and plt> g/l on the day.results: at present, more than years after the second mudallopbsct, the patient remains in a very good condition with % of the second donor chimerism and without any features of pnh (clone is undetectable) and myelofibrosis.conclusions: presented case is the first in the literature well documented myelofibrosis recurrence after mudal-lohsct with concurrently development of clinically significant paroxysmal nocturnal hemoglobinuria. the second mudallohsct from another donor was safe and successful treatment strategy in this situation.disclosure: nothing to declare. abstract already published. cutaneous refractory t-cell lymphoma treated with allogeneic hematopoietic stem cell transplantationmarcia silva , ercole orlando , maria claudia moreira , simone lermontov , simone maradei , yung gonzaga , leonardo arcuri , renato araujo , decio lerner instituto nacional de cancer, cemo, rio de janeiro, brazilbackground: folliculotropic mycosis fungoides (fmf) is an aggressive clinical course variant of cutaneous t-cell lymphoma (ctcl) -classic mycosis fungoides (mf) , with distinct clinical and pathological characteristics, and it is less responsive to skin-directed therapies. for diseases in advanced stages, chemotherapy, autologous hematopoietic stem cell transplantation (hsct) or immunomodulator drugs may provide remissions with limited duration and the treatment remains substantially palliative , . these dismal results have induced to explore the therapeutical approach with allogeneic hematopoietic stem cell transplantation (hsct) in such patients. early studies have shown encouraging results also in patients with advanced disease, suggesting a major therapeutical role played by the graft versus lymphoma (gvl) effect , , . methods: this is a case report of the use of allogeneic hsct as a potential cure for cutaneous refractory t-cell lymphoma type folliculotropic mycosis fungoides .results: case presentation : a -year-old male patient with refractory subtype b fmf t-cell lymphoma , diagnosed in , clinically characterized by exfoliative erythroderma, widespread plaques on the trunk and limbs, solitary tumor on the right shoulder, pruritus and bilateral